Uncovering bacterial diversity on and below the surface of

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Uncovering bacterial diversity on and below thesurface of a hyper-arid environment, the AtacamaDesert, ChileDanielle Rene BagaleyLouisiana State University and Agricultural and Mechanical College

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UNCOVERING BACTERIAL DIVERSITY ON AND BELOW THE SURFACE OF A HYPER-ARID ENVIRONMENT, THE ATACAMA DESERT, CHILE

A Thesis

Submitted to the Graduate Faculty of the Louisiana State University and

Agricultural and Mechanical College in partial fulfillment of the

requirements for the degree of Master of Science

in

The Department of Biological Sciences

by Danielle Rene Bagaley

B.S., Louisiana State University, 2002 May 2006

ii

DEDICATION

I dedicate this composition to my parents Robbie and Daniel Bagaley. Without their

unconditional love and support this work would never have been possible. I also would like to

dedicate this work to my fellow thesis writing buddy, Jennifer Movassaghi. Her companionship

during this experience helped make this accomplishment more enjoyable.

iii

ACKNOWLEDGEMENTS

During my Master of Science study at Louisiana State University I have significantly

advanced in skill and knowledge as a result of determination, diligence, and immeasurable

attributions from professors and fellow graduate students. For that reason, I would first like to

give my utmost thanks to my major professor Dr. Fred A. Rainey for all of his patience, advice,

assistance, and support. I would also like to express my gratitude to the other members of my

graduate committee, Dr. Greg Pettis and Dr. Annette S. Engel.

I also am very appreciative of a Ryan P. Callegan, a fellow graduate student, and Brian

A. Rash, a postdoctoral researcher. They have both contributed considerable amounts of their

time and knowledge to questions I had concerning this research. Current and former student

workers of the RaineyLab were very helpful throughout the course of my research. These people

include Monica Morgan, Mallory Durel, Claire Ponson, and Jeff Cutrera. I also thank Abhishek

Satyendranath who created the RaineyLab webpage and the isolate database for this study.

I am very grateful to Dr. Mark A. Batzer and Dr. Randy Garber, a postdoctoral researcher

in the Batzer laboratory, for their aid in the process of 16S rRNA gene sequencing.

I could never have even begun this research if it were not for support from the NASA-

Headquarters (NASA-Ames/LSU Cooperative Agreement NCC 2-5469 and NCC 2-5528) and

the NASA - Ames Research Center, especially Chris Mckay. I also am grateful to those who

helped to collect the numerous soil samples, mainly the subsurface samples, from the Atacama

Desert.

I am thankful for our collaboration with the National Autonomous University of Mexico,

in particularly Dr. Rafael Navarro-González, who preformed the organic analyses for the

Atacama Desert soil samples.

iv

I appreciate Dr. Bob Gambrell of the Wetland Biogeochemistry Institute within the

Department of Oceanography and Coastal Sciences at Louisiana State University for his

contribution of the Atacama Desert soil elemental analysis. Also, I would like to express my

gratitude to Sarah Jones of the Institute for Ecological Infrastructure Engineering and Water

Quality Laboratory at the Louisiana State University College of Engineering who performed

anion analysis on all Atacama Desert soil samples.

I must thank the Socolofsky Microscopy Center, particularly Cindy Henk, for all of the

hours that she spent assisting me with fluorescence microscopy, scanning electron microscopy,

and many other microscopy related issues.

Lastly, I express thanks to the Department of Biological Sciences and the Office of

Graduate Studies, Priscilla M. Milligan, Dr. Thomas S. Moore, and Chimene Boyd for their time,

efforts, and direction throughout my graduate school career.

v

TABLE OF CONTENTS

DEDICATION………………………………………………………………………….....ii ACKNOWLEDGEMENTS…………………………………………………………….....iii

LIST OF TABLES……………………………………………………………..................vii

LIST OF FIGURES…………………………………………………………………….…viii

LIST OF ABBREVIATIONS………………………………………………………….….x

ABSTRACT…………………………………………………………………………….…xiv

CHAPTER 1 INTRODUCTION……………………………………………………………..1

The Driest Place on Earth, the Atacama Desert of Chile………………….1 Methods for Microbial Community Analysis……………………………...4

2 MATERIALS AND METHODS………………………………………….......14 Samples Collection………………………………………………………...14 Naming of Sites, Samples, and Isolates……………………………………14

Culture Media………………………………………………………….......15 Soil Dilution Plating………………………………………………….........15 Enrichment Cultures…………………………………………………….....16 Plate Counting and Colony Forming Unit Determination………………....16 Isolation of Pure Cultures………………………………………………….17 Preservation………………………………………………………………...17

DNA Extraction…………………………………………………………....18 Polymerase Chain Reaction (PCR) and PCR Purification………………...18 Sequencing, Sequence Purification, and Data Analysis…………………...19 Isolate Database…………………………………………………………....20 Direct Counts Using Fluorescence Microscopy…………………………...20 Determining Limit of Direct Counts on Atacama Desert Soils…..….….....22 Lake Water Sample Preparations for DAPI Staining Control……………..25 DAPI Staining Preparations for Control Soil Sample……………………...26 Determination of Salinity in Atacama Desert Soil Samples……………….27 pH Determination…………………………………………………………..27 Detection of Carbonate Presence in Selected Samples…………………….28 Elemental Assay on Atacama Desert Soils………………………………...28 Inorganic Anion Analysis………………………………………………….29

3 QUANTIFYING AND IDENTIFYING CULTURABLE HETEROTROPHIC BACTERIA IN THE SURFACE SOILS OF THE ATACAMA DESERT…...31

Results……………………………………………………………………...31

vi

Colony Forming Units……………………...……………………...31 Diversity of Culturable Bacteria………………...…………………39

Discussion………………………………………………………………….42 4 HETEROTROPHIC BACTERIA BENEATH THE SURFACE……………...48

Results……………………………………………………………………...48 Colony Forming Units...……………………………………………48

Diversity of Culturable Heterotrophic Bacteria Isolated from the Soil Pits…………………………………………………………......52

Discussion…………………………………………………………………..61

5 DIRECT CELL COUNTS USING FLUORESCENCE MICROSCOPY……..67 Results……………………………………………………………………...67

DAPI Direct Count Detection Limit ………………………………67 DAPI Direct Cell Count Determination for AT04-166…………….75 Sample Sites Having Cell Totals below DAPI Direct Count Detection Limit…………………………………………………….80

Discussion………………………………………………………………….85

6 ASSOCIATION BETWEEN VALUES OF CFUS AND SOIL CHEMICAL COMPOSITION…………………………………………………97

Results…………………………………………………… ………………...97 Elemental Cation Analysis of Soil Samples….……..……...……….97

Inorganic Anion Analysis of Soil Samples………………….……..104 Detection of Carbonates in Atacama Desert Soils…………………109

Discussion………………………………………………………………….110 7 CONCLUSIONS……………………………………………………................112

REFERENCES………………………………………………………………….................118 APPENDIX

A MEDIA INSTRUCTIONS …………………………………………..……….123 B TABLE OF SURFACE AND SOIL PIT ISOLATES ..………………...….....126

C TABLE OF PHOSPHATE, NITRITE, AND BROMIDE CONCENTRATIONS (MG/L)……………………………………………….156 D MEASUREMENTS OF SALINTY, TDS, AND CONDUCTIVITY IN TABLE OF SOLUBLE SALTS WITHIN SOILS…..………………………..157

VITA………………………………………………………………………………………158

vii

LIST OF TABLES

Table 3.1: Degree minute decimal coordinates for surface sites within the core arid region of Yungay…………………………….……………......33

Table 3.2: Surface sample pH values and CFUs/g of soil on five media after 20

days incubation…………..…………………………………….…..35 Table 3.3: Values of CFU/g recovered on various culture media for the 33 sites

sampled…………………………………………………………….37 Table 4.1: CFUs/g of soil for all four soil pits sampled on five media………………..50

Table 4.2: Values of CFU/g recovered on various culture media for the AT04-159 southern soil pit…………………………………………………….53

Table 5.1: Direct count detection limit trials displaying cell numbers per field,

average cells per field, and known versus calculated cells/g for stock and 1/10 diluted cell suspension after overnight and one month drying experiment……………………….………….………73

Table 5.2: Direct count trials displaying cell numbers per field, average cells per

field, and calculated cells/g for each tested sample site…….……...84 Table 6.1: Cation concentrations (mg/L) for Atacama Desert soils……………...........98 Table 6.2: Major anion concentrations (mg/L) and CFU/g values for Atacama

Desert soils………………………………………………………....105

viii

LIST OF FIGURES

Figure 3.1: Scatter plot illustrating proximity of surface sites in core arid region and distribution of CFUs…………………….…………………….34 Figure 3.2: CFUs/g of soil for each of the 33 surface samples on five media………....36 Figure 3.3: Diversity at the phyla level within surface samples…………………….....40

Figure 3.4: Closest relatives level diversity within surface sites………………….…...41

Figure 3.5: Photomicrographs depicting cell morphology of Blastococcus aggregatus……………………………………………….…….…..45 Figure 4.1: CFUs/g of soil for layers within each soil pit on five media…………..….51 Figure 4.2: Diversity at the phyla and closest relative levels in soil pit AT04-150

and AT04-150B enrichment cultures……………………………...54

Figure 4.3: Diversity at the phyla and closest relative levels in soil pit AT04-152 and AT04-152B enrichment cultures………………….…………..55

Figure 4.4: Diversity at the phyla and closest relative levels in soil pit AT04-153 and AT04-153B enrichment cultures……………………………...56

Figure 4.5: Diversity at the phyla and closest relative levels in soil pit AT04-159 (the Altamira soil pit)……………………………………………...58

Figure 5.1: Photomicrographs of AT04-170 spiked with cultured bacterial cells and dried overnight to determine DAPI direct count detection limit….70

Figure 5.2: Detection limit photomicrographs of AT04-170 spiked with cultured bacterial cells and dried for one month……….…………………..74

Figure 5.3: Photomicrographs from the fluorescence direct count method

employing DAPI in surface site AT04-166……………..………...76 Figure 5.4: Photomicrographs of AT04-165 soils unable to be truly quantified by

DAPI direct counts………………………………………………..81 Figure 5.5: Photomicrographs of AT04-170 soils unable to be quantified by DAPI

direct counts……………………………………………………....86 Figure 5.6: Comparison photomicrographs of cornfield soil sample……………….....94

ix

Figure 5.7: Comparison photomicrographs of LSU lake water sample…..…………...95 Figure 6.1: CFU/g values and cation concentrations for Yungay surface soils….…....102 Figure 6.2: Charts displaying relationship between CFU/g values and cation

concentrations for Atacama Desert soil pits………………………103 Figure 6.3: CFU/g values anion concentrations for Yungay surface soils....……….....107 Figure 6.4: CFU/g values and anion concentrations at depth for Atacama Desert

soil pits……………………………………………..………..….....108

x

LIST OF ABBREVIATIONS

1X IO 1 times Instant Ocean®



Al aluminum

APTs agar precipitation tests

As arsenic

ATP adenosine triphosphate

BCECF 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein

BLAST basic local alignments search tool

bp base pairs

Ca calcium

CaCO3 calcium carbonate

Cd cadmium

CFU colony forming unit

Cl chloride

cm centimeters

Cr chromium

Cu copper

DAPI 4'-6-Diamidino-2-phenylindole

DIC differential interference contrast

DNA deoxyribonucleic acid

DVC direct microscopic viable count

xi

ECe Electrical Conductivity of the extract EDTA ethylenediaminetetraacetic acid ELISA enzyme-linked immunosorbent assay

eV electron volt F fluoride

FC flow cytometry FDA fluorogenic ester fluorescein diacetate Fe Iron

FISH fluorescence in situ hybridization

FS filter sterilized

g grams

GCMS gas chromatograph/mass spectrometer

HCl hydrochloric acid

I.D. inner diameter

IF immunofluorescence

IMC immunomagnetic capture

K potassium

m meters

M Molarity

MA Marine Agar

MB Marine Broth

MA+IO Marine Agar + Instant Ocean®

MB+IO Marine Broth + Instant Ocean®

xii

MER Mars Exploration Rovers

Mg magnesium

min. minutes

µL microliter

mL milliliter

mm millimeter

mM millimolar

µm micrometer

Mn manganese

MPN most probable number

mS/g milliSiemens per gram m/z mass-to-charge ratio Na sodium

NA Nutrient Agar NB Nutrient Broth ng nanogram Ni nickel

NO3 nitrate

P phosphorous

Pb lead

PBS phosphate buffered saline PCA Plate Count Agar PCB Plate Count Broth

xiii

PCR polymerase chain reaction ppt parts per trillion pyr-GC-MS pyrolysis-gas chromatography-mass spectrometry RFLP restriction fragment length polymorphism RNA ribonucleic acid rRNA ribosomal RNA SDS sodium dodecyl (lauryl) sulfate sec. seconds SEM scanning electron microscopy Si silicon

spp. species

SO4 sulfate

TDS total dissolved solids Z zinc

xiv

ABSTRACT

The Atacama Desert in Chile extends from latitudes 17º S to 28º S between longitudes

69º W and 71º W. It has been reported that surface soils in the hyper-arid region contain low

numbers of culturable heterotrophic bacteria. These soils are considered Mars-like and offer an

ideal setting to investigate the application of life detection systems. Thirty-three surface sites

were sampled to examine further the extent of the hyper-arid region. We also excavated four soil

pits, 40 cm to 90 cm in depth, to explore subsurface microbial communities. One pit was dug in

a southern region of the desert compared to the location of the pits in the hyper-arid region.

Samples were examined using culture-dependent techniques, including serial dilution plating

methods on five media for the cultivation of heterotrophic bacteria. Using 16S rRNA gene

sequence comparisons, 1,260 organisms have been recovered and identified. Fifty-four percent

of the samples obtained from within the hyper-arid region show numbers of culturable bacteria

above the detection limit, yet there is evidence of microbial patchiness in surface and subsurface

soils. In some samples, no bacterial colonies were retrieved; in the majority, less than ten were

recovered. One pit showed an increase in CFUs/g at 40 cm then dropped back to levels near and

below the detection limit. A second pit showed an increase at 10 cm with numbers near and

below the detection limit at further depths. The southern soil pit had CFUs/g up to four orders of

magnitude greater than those in the hyper-arid region. Direct count methods employing DAPI

epifluorescence microscopy were applied to samples, but proved suitable only for a sample

having the highest CFUs/g of soil (7.4 x 105) due to the determined detection limit for the

technique utilized on the minimal-life containing soils. A chemical composition analysis was

performed on all soil samples and showed that elevated ion concentrations may correlate with

low numbers of culturable bacteria. The data obtained for the desert samples point to the

xv

importance of developing surface and subsurface sampling protocols for future missions to Mars

searching for evidence of past or present life.

1

CHAPTER 1 INTRODUCTION

THE DRIEST PLACE ON EARTH, THE ATACAMA DESERT OF CHILE

The Atacama Desert of northern Chile is a sparsely populated, virtually rainless plateau,

extending from the Pacific Ocean to the Andes Mountains and encompassing an area from

latitudes 17º to 28º S and 69º to 71º W. The northern desert region is considered to be one of the

driest places on Earth. The Pacific Coastal Range, the mountains on the western side of the

desert, has an average elevation of approximately 1,000 m (Hickman 1998). In the core of the

desert, rain is measured in millimeters (mm) per decade. There are some areas where rain has

never been recorded, at least as long as humans have measured it (Hickman 1998).

The collective effects of five different factors result in the hyper-aridity the desert: (1)

precipitation is reduced by a high-pressure system that produces descending stable air because of

the Hadley circulation, (2) the cold north-flowing Peruvian Current prevents the ability of

onshore winds and generates a constant layer of warm air that prevents the rise of cooling air and

traps moisture beneath it, (3) the oceanic cloud barrier effect of the coastal range, (4) the

rainshadow effect of the Andes Mountains to the east prevents moisture transfer from the east,

(5) and the substantial distance (≤ 2000 km) from the Amazon basin and Atlantic Ocean

moisture supply (Houston and Hartley 2003).

The Atacama Desert is also a unique source for nitrate (NO3) minerals, which are

traditionally used in agricultural production systems as a fertilizer (NOSB TAP 2002). Chilean

NO3 is a naturally occurring inorganic mineral salt that is derived from caliche ore, which is a

crude mineral conglomerate of salts comprised of: NO3, SO4, Cl of Na, Ca, K, Mg, and various

micronutrients (Ericksen 1983). Darwin (1871) put forward that the NO3 deposits may have

2

formed at the inland border of the oceanic expansion. It was proposed by Ericksen (1983),

however, that the NO3 can be accredited to nitrogen fixing and nitrifying microorganisms in the

soils or in nearby Andean salars, which are present due to preexisting salt-water lakes, 10-15

million years ago. The NO3 is proposed to make its way into the Atacama Desert via wind or

groundwater (Ericksen 1983). Other possibilities for the origin of the NO3 deposits include the

nitrification of ammonia from seaweed, or vegetation in salt lakes, or bird guano (Ericksen

1983). Atmospheric NO3 accumulation from sea spray, ammonia deposition, or nitric acid

deposition may also contribute to the NO3 mineral abundance observed in the Atacama Desert

(Ericksen 1981). Chong (1994) states that the NO3 deposition was caused by magmatic

processes that produced NO3 through unknown catalytic reactions. The real reason for the NO3

and other soluble salts that exist in the Atacama Desert has yet to be agreed upon by researchers;

most attribute these characteristics to a combination of sources (Michalski et al. 2004).

The Atacama Desert provides excellent testing grounds for Mars research due to three

similarities that exist between the Atacama Desert and Mars; the similarities between the two

environments are compared and contrasted throughout the remainder of this section.

The existence of Mars-like soils in the core hyper-arid region, the area known as Yungay,

of the Atacama Desert has been described (Navarro-González et al. 2003). The Yungay region is

located around 24º S and 69º W. This region consists of surface-soils containing organic

materials at trace concentrations and extremely low levels of culturable bacteria (Navarro-

González et al. 2003). In the Navarro-González et al. (2003) study the organic analysis was

determined for surface soils from the Atacama Desert using pyrolysis-gas chromatography-mass

spectrometry (pyr-GC-MS). In addition, numbers of colony forming unit (CFU) of heterotrophic

bacteria below detection limits of the dilution plating technique were observed. In a number of

3

cases no bacterial colonies formed from the lowest dilution. The aim of this study was to

investigate further the CFUs/g and diversity of heterotrophic bacteria in additional soils collected

from the Yungay region.

The 1976 Viking missions to Mars carried pyrolysis gas chromatograph/mass

spectrometers (GCMS), and provided evidence that the Martian soils were depleted in organic

compounds, at only a few parts per billion, in the upper 10 cm of surface soil. This suggested

that there was an absence of Martian life (Glavin et al. 2001). In the Yungay region of the

Atacama Desert, high ratios of formic acid/benzene were discovered from of pyrolysis of organic

matter, suggesting that this region is depleted in organic matter due to highly oxidizing

characteristics (Navarro-González et al. 2003). Martian soils and Atacama Desert soils appear to

be similar because both appear to be depleted of organic matter due to strong soil oxidants which

result in the fast isolation of extinct or existing life forms. The Atacama Desert is considered to

be a Mars analog because it has attributes similar to those found on Mars, in terms of chemistry,

mineralogy, geology, topography, etc.

The Atacama Desert is a Mars analog for other reasons. The findings of Navarro-

González et al. (2003), initially demonstrated that the Atacama Desert is similar to Mars, which

is considered to be a hyper-arid environment. The data obtained by Mars Exploration Rovers

(MER), however, suggest that at one time water may have been present on Mars (Baldridge and

Calvin 2003, Herkenhoff et al. 2004, Haskin et al. 2005). The Atacama Desert is one of the

oldest and driest deserts in the world; it has been said to be at least 15 million years old (Eriksen

1981). At the end of the last glacial maximum (approximately 12,000 years ago) the Atacama

existed in a less arid state than today (Bortman 2003). The salars of the Atacama Desert were

once saline lakes that eventually dried out up over time. Any life that existed in the Atacama

4

Desert prior to its hyper-aridity either died or adapted to the hyper-arid environment. If the MER

are accurate in revealing that Mars was wetter in the past, then the Atacama Desert is similar to

Mars in the sense that a previously wetter environment once existed where today exists a hyper-

arid environment; therefore, the Atacama Desert may reveal possibilities of life on Mars.

The core hyper-arid region of the Atacama Desert provides an ideal setting to investigate

the survival of microorganisms under extremely arid conditions. Areas with little or no

culturable bacteria provide sites for the testing of life detection systems as well as for the

development of planetary exploration protocols. In order to search for evidence of water and

oxidants at some depths, NASA’s Mars Science Laboratory is preparing to send a Lander/Rover

that is capable of drilling and retrieving soil samples at Martian shallow subsurface in 2009

(NASA 2003 Strategic Plan). Preparations for a 2018 Deep Drill mission are also underway to

search 3 to 10 m of Mars’ subsurface for evidence of past or present life, although the landing

site depends on outcomes of earlier expeditions (Mars Science Program Synthesis Group 2003).

With regards to the 2009 and 2018 missions, this thesis not only focuses on the surface soil, but

also the subsurface soil in the hyper-arid core region of the Atacama Desert. This study uses

both culture-dependent and culture-independent methods to analyze bacterial communities that

exist above and below the surface of the Atacama Desert.

METHODS FOR MICROBIAL COMMUNITY ANALYSIS

Culture-dependent and culture-independent approaches are used to study prokaryotic

populations existing in environmental samples. The culture-dependent approach employs

artificial media in order to culture or enrich for groups of microorganisms inhabiting

environmental samples. This approach is also used simply to isolate single colonies. Until

recently, this method was considered standard and was carried out in most microbial ecology

5

studies. In recent years it has been discovered that the majority of microorganisms in the

environment are not culturable by traditional plate culture methods. With this, the culture-

independent approach is essential to evaluate microbial diversity in various types of

environments (Amann et al. 1995). With the culture-independent approach, scientists have been

able to identify unculturable prokaryotes in situ (Brock 1987). This method commonly

concentrates on the ability to extract total nucleic acids from cells occupying a sample and

employing biomarker-based techniques, such as DNA analysis (reviewed in Head et al. 1998).

The use of ribosomal gene sequences as indicators of bacterial diversity is most common,

although other genes, including protein-coding genes, have also been used (reviewed in

Wintzingerode et al. 1997). Although the 16S rRNA gene research is more widely utilized, there

are various culture-independent techniques that target other genes that are responsible for a

number of metabolic actions. Culture-independent approaches using the 16S rRNA gene as a

marker neglect to consider any detail regarding the differences in cellular metabolic activity, or

to ensure that ecologically significant microbes are analyzed and not microbial cells existing in

quiescent forms that do not add to the function of the environmental system (Ellis et al. 2003).

By incorporating both of these analytical strategies, it is possible to target only those cells that

are actively undergoing the specific metabolic function coded for by the targeted gene.

In culture-dependent methods, viable culturable cells can be recovered with the plate

count method, which is thoroughly described in chapter 2. After incubation, colonies that form

are assumed to result from a single cell, thus total CFUs per volume of sample can be calculated.

The most probable number (MPN) technique is similar to the plate count method, except that the

method depends on detection of certain qualitative characteristics of the organism of interest, in

particular on growth of the target organisms in liquid medium. Microorganisms capable of being

6

cultured are preserved in laboratory culture and strain collections. These organisms are

classified by phenotypic and genotypic characteristics and are ultimately assigned to a specific

taxonomic group.

Although culture-dependent techniques provide an excellent understanding of the

physiological potential of isolated organisms, they do not necessarily offer insight into the

complete makeup of microbial communities (Horner-Devine 2003). Culture-dependent methods

are biased in that a microorganism can only be cultivated after its physiological niche is evident

and experimentally reproduced. The biases that culture-dependent analyses demonstrate are

evident in the inconsistencies that exist between total cells viewed microscopically versus total

CFUs determined for environmental samples (Staley and Konopka 1985). It is widely accepted

that culture-dependent studies can only assess between 0.001% and 15% of the actual microbial

communities in a sample (Amann et al. 1995). Most of the viable cells that can be viewed

microscopically, but are not considered culturable, can be classified into one of two groups. The

first of which are known species that have entered an unculturable state or for which the

cultivation conditions utilized are not adequate. The second group consists of unknown species

that as of now have never been able to be cultured due to a lack of appropriate processes (Amann

et al. 1995).

The culture-dependent method when applied to soil microbiology is relevant because it

offers an overall fraction of the microbes residing in a specific soil. Although the research

executed for this study focuses mainly on these culture-dependent techniques, and in turn is

influenced by the biases mentioned above, it is possible that in environments having extreme

conditions, such as the Atacama Desert, culturable organisms tell more of the community than in

less extreme environments. Culture-dependent methods provide an undeniable knowledge of

7

any previously undescribed environments, and with regards to this study, it is necessary to

perform the culture-dependent studies, because it provides a solid backbone to future research

that applies culture-independent protocols.

Culture-independent methods were developed to understand better the complete

microbial populations existing in natural habitats. The culture-independent approach involves

the extraction of total environmental nucleic acids, but the extraction method employed depends

on the type of environment being studied. Soil samples require a harsh extraction method due to

the tight bonds that can usually exist between soil particles and inhabiting microorganisms

(Priemé et al. 1996). In order to lyse microbial cell walls, extraction methods can utilize both

physical and chemical processes. Physical methods include freeze/thaw protocols and/or bead

beating; chemical procedures usually entail lysozyme and sodium dodecyl (lauryl) sulfate.

Extraction of total nucleic acids from soil samples yields better results when chemical and

physical methods are combined due to the strong attachment of microbial cells to soil particles.

Often, extraction kits designed specifically for soil join together bead beating and chemical lysis

methods. Once the microbial cell has been disrupted and the DNA/RNA has been extracted, the

nucleic acids must be purified, usually with proteinase K and phenol/chloroform washes.

Because nucleic acids carry a fixed negative charge per unit length of molecule, for purification

purposes nucleic acids are collected using binding material, which is positively charged, and

washed to discard of unnecessary compounds.

Gene clone library construction is a widely used procedure that employs purified nucleic

acids from an environmental sample and amplifies a specific sequence on the various genomes

present using Polymerase Chain Reaction (PCR), for which oligonucleotide primers specific to

the preferred amplicon gene sequence can be designed. The amplified gene sequences are then

8

inserted into engineered plasmids containing an antibiotic resistant gene, resulting in

recombinant plasmids, which can then be taken up by competent Escherichia coli host cells. The

cells are then grown on nutrient media containing the antibiotic, thus only cells that have the

plasmid survive and replicate. Resulting colonies, or clones, contain copies of the same

recombinant plasmid with its fragment of foreign DNA. A variety of assay methods can be used

on the bacterial clones to determine which colonies contain particular DNA sequences.

The gene sequence most commonly used for the construction of gene clone libraries in

microbial ecology studies is the 16S rRNA gene because it is highly conserved in all prokaryotes

(Hillis and Dixon 1991). The 16S rRNA gene is conservative because it is the slowest gene

sequence to evolve, indicating that research based on the 16S rRNA gene can be used to study

ancient evolutionary events, also the 16S rRNA gene sequences are all similar in length

(approximately 1500 nucleotides). The slow rate of change that has been observed in the 16S

rRNA gene allows for the construction of universal primers (Hillis and Dixon 1991). The first

study that used the 16S rRNA gene for the construction of gene clone libraries was done by

Liesack and Stackebrandt (1992). The research was performed on an Australian soil sample to

determine the total microbial diversity from the cloned and retrieved 16S rRNA gene sequences.

A total of 116 clones were created, which significantly revealed diversity in nature that had never

before been discovered. The technique opened new doors in microbiology, especially soil

microbiology, and has since been used extensively, although it is expensive and extremely time-

consuming. Therefore, most 16S rRNA gene clone libraries usually consist of between 100 and

300 clones. Gene clone libraries having considerable more clones have an overall higher degree

of retrieved diversity, in terms of community composition, richness, and structure (Dunbar et al.

2002).

9

An inexpensive technique that is based on the PCR amplification of a mixture of genes

representing different microorganisms from environmental nucleic acid extracts is Restriction

Fragment Length Polymorphism (RFLP). This method employs the digestion of amplicons by

restriction enzymes that nick DNA at specific sites, and forms an assortment of DNA fragments

(Lee et al. 1993; Gundersen et al. 1994; Muyzer et al. 1995). The fragments are separated via gel

electrophoresis; the ultra-pure agarose used is capable of resolving fragments that vary by < 25

bp. The gel is stained with ethidium bromide and viewed under ultra-violet illumination; only

the fluorescently labeled restriction fragments are visualized. It is useful to apply multiple

restriction enzymes because ordinarily this will allow for better resolution since it will increase

the number of different sized DNA fragments.

Denaturing or temperature gradient gel electrophoresis (DGGE/TGGE) is a culture-

independent technique first developed by Myers et al. (1987) that involves separation of DNA in

solution by various melting properties. DNA molecules melt in distinct segments, known as

melting domains, when the temperature or denaturant (usually urea) concentration is increased.

The melting temperature (Tm) of a melting domain depends on the nucleotide sequence. As

DNA fragments are separated in an electrical field through a linear gradient of increasing

denaturing concentration or increased temperature, the separation of double-stranded DNA into

single-stranded segments increases causing the DNA segments to form a less uniform three-

dimensional structure that moves through a polyacrylamide matrix at a slower rate (Muyzer et al.

1993). Profiles that are produced are also known as fingerprints, which can be compared and

evaluated to match up any similar banding patterns across environmental samples. Unique

banding patterns can be cut out of the gel and sequenced for additional testing. One

disadvantage to this method is that DGGE and TGGE techniques cannot provide any quantitative

10

data (van Hannen et al. 1999). Another downside to this method is that as the complexity of a

community increases, it may be more difficult to excise individual bands for sequence analysis.

Thus, this technique could limit the extent of the environment’s diversity.

Although the extraction of total nucleic acids from environmental samples is widely used,

there are many other conventional methods that do not involve these extraction techniques and

are considered culture-independent. One commonly used approach includes DNA-binding

fluorochromes such as 4’,6-diamidino-2-phenylindole (DAPI) and acridine orange (3,6-

bis[dimethylamino] acridinium chloride. Direct counts using acridine orange or DAPI to

examine microbial populations in most natural environments usually surpass counts derived from

plate counts by some order of magnitudes because not only culturable microbes are revealed, but

viable unculturable and nonviable microbes are also uncovered (Hartmann et al. 1997).

The direct microscopic viable count (DVC) method determines viable microbes in

environmental samples by preventing cells to replicate using a DNA gyrase inhibitor, nalidixic

acid, but promoting cell development with yeast extract (Kogure et al. 1979). Epifluorescence

microscopy employing DNA-binding fluorochromes immediately ensues for quantification

purposes of active cells (Hartmann et al. 1997).

Fluorescence nucleic acid hybridization probes are rRNA directed oligonucleotides that

can be made and used to label targeted microorganisms for in situ detection. This technique is

able to detect a single targeted cell and can give some clue to an organism’s specific genera or

higher taxonomic classification (Hartmann et al. 1997). By using probes that target regions of

rRNA that have been conserved, measurements of total rRNA in an environmental sample can be

determined.

11

Flow cytometry is a culture-independent technique that can also be used to study

microbial cells (Melamed et al. 1990). The advantages of using flow cytometry (FC) are

because it is possible to analyze cells that stream across a laser beam, according to several

parameters at the same time, for example, most flow cytometers feature light scatterers and

fluorescent detectors. Universal oligonucleotide probes can be combined with FC to investigate

diverse prokaryotic communities. The FC method could possibly generate exceptional results

for microbial studies if the method was further developed because it can analyze masses of cells

in a short amount of time, but the problem at hand is that the size of microbes is generally too

close to the detection limit of these instruments (Davey 1994).

There are culture-independent approaches that utilize fluorescent dyes to specifically find

active microorganisms in natural environments; some are described at the Invitrogen webpage.

Cell viability can be assessed using fluorogenic esterase substrates, such as fluorescein diacetate

(FDA), calcein AM, 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) AM and

various other FDA derivatives. Membrane integrity and activity are good indicators of viability

and can be evaluated by the cell-permeant esterase substrates. This method is commonly used in

conjunction with FC (Diaper and Edwards 1994). Flourescein and fluorescein derivatives easily

diffuse into viable microbial cells and most respiring cells can break down fluorogenic ester to

create the fluorescein molecule (Breeuwer et al. 1995). This method does not detect all active

cells because some microbes are not able to capable of taking up FDA or FDA derivatives

efficiently, therefore it does not necessarily reflect the growth potential of microbial cells within

the given natural environment.

Enzymatic activity of microorganisms can also be revealed and measured by using other

artificial substrates (Gazenko et al. 1998). Special enzymes, such as lipases, phosphatases, and

12

dehydrogenases, of live microbial cells attach directly with fluorogenic substrates through

biochemical reactions and turn to fluorescent substances either intracellular or extracellular. The

amount of the resulting fluorescence depends on the enzymatic activity of the cell (Gazenko et

al. 1998). This method, which has the same disadvantage as mentioned above, does not recover

all active cells because some microbes cannot take up the substrate-enzyme complex

proficiently. Fluorescent redox probes are also useful in direct fluorescence microscopy because

they allow for the visualization of metabolically active bacteria that are competent to reduce the

redox dye (Rodriguez et al. 1992).

Immunological techniques are culture-independent methods that include

immunofluorescence (IF) and ELISA (enzyme-linked immunosorbent assay). IF staining uses

epifluorescence microscopy and involves antibodies either directly joined with a fluorescence

marker or marked with a secondary antibody. The disadvantage to IF is that the antibodies

produced are specific for distinct taxonomic groups and cannot detect entire microbial

population; however, IF can be used in soil extracts for in situ research or in PCR for locating

target microbes (Hartmann et al. 1997). ELISA, a technique based on enzyme-linked antibodies,

remains the most widely used immunological method for detecting microbes in soil (Lee et al.

1990). The primary specific antibody is added first and then the secondary antibody, which

carries one enzyme molecule per antibody, is added. ELISA is an effective method because

fairly low numbers of microbial cells in soil can be detected (Hartmann et al. 1997).

Immunomagnetic capture (IMC) is an alternative procedure that involves microscopic

magnetic beads that are coated with monoclonal or polyclonal antibodies specific for the

prokaryotes of importance. Both direct and indirect IMC methods have been successful in

recovering organisms from soil (Wellington et al. 1997).

13

Microbial biomass can be also be approximated using culture-independent methods such

as microbial respiration, metabolic heat production, adenylate energy content, adenosine

triphosphate (ATP) levels, or soil fumigation techniques. These methods only provide data for

the active organisms in the community, and therefore cannot provide an accurate microbial

biomass enumeration (Hartmann et al. 1997).

Culture-dependent and culture-independent microbiological studies of the Atacama

Desert’s core hyper-arid region of Yungay were the focus for this thesis. The first study was of

surface soils collected from within the Yungay region, and the second study was of soils sampled

from subsurface soil pits that were excavated to a depth of up to one meter. CFUs were

determined for all soil samples using five nutrient media. Isolates recovered from the soil

samples were identified by 16S rRNA gene sequencing in order to examine the diversity of

culturable heterotrophic bacteria isolated from these soils. The direct count technique using

DAPI epifluorescence microscopy was done to enumerate the unculturable fraction of the

microbial population. The results of these studies are presented in chapters 3, 4 and 5 that deal

with surface CFUs and diversity, subsurface CFUs and diversity and direct counts approaches

using DAPI epifluorescence microscopy.

14

CHAPTER 2 MATERIALS AND METHODS

SAMPLES COLLECTION

Soil samples were collected from the Atacama Desert in 2003 and 2004 from 33

accessible surface sites within the core hyper-arid region and from four soil pits that were

excavated and sampled. Three pits were excavated within the core arid region, while one soil pit

was dug at a site further south and was considered a control pit. The four soil pits ranged in

depth from 50 to 90 cm. The coordinates of each sample site were recorded using a Global

Positioning System (GPS) device. The top 3 cm were collected for surface samples using sterile

plastic scoops and sterile Whirl-Pak® bags. A surface sample for all four pits was acquired

before pit excavation. Pits were excavated using standard shovels and after construction was

complete, the pit wall was cleaned by scraping soil from the wall using a plastic Sterileware®

scoop (Bel-Art Products®). Samples were taken every 10 cm, beginning with the deepest layer,

and were extracted using a Sterileware® sampling spatula (Bel-Art Products®). To prevent

contamination of the soil samples in the laboratory, approximately 10 g of each soil were

transferred to a clean sterile 50 mL centrifuge tube to be used as a stock sample.

NAMING OF SITES, SAMPLES, AND ISOLATES

Soil pits and surface samples were labeled according the year and the collection site,

beginning with the letters AT for Atacama Desert. The numbers that follow denote the

collection year and the last digits represent the sample site number. Isolate names were derived

from the soil sample designation from which the original microbial colony was cultured. Each

colony isolated from a given soil sample was numbered consecutively.

15

CULTURE MEDIA

Five nutrient media were used to culture heterotrophic microbes from each soil sample,

including (all Difco products) Plate Count Agar (PCA), Marine Agar (MA), Nutrient Agar (NA),

1/10 strength Plate Count Agar (1/10 PCA), and 1/100 strength Plate Count Agar (1/100 PCA).

Each culture medium was also used in the liquid form without agar added for dilution

preparation. In addition soils from sites AT04-158, AT04-163, and AT04-164 were plated on

MA and 1/10 PCA that had been adjusted to pH 5.0. Soils from sites AT03-35 and AT04-170

were also plated on media varying in marine salt concentrations at pH 8.0 using a combination of

Marine Agar and Instant Ocean®, 2X Instant Ocean®, and 3X Instant Ocean®. Recipes for all

culture media are provided in Appendix A.

SOIL DILUTION PLATING

A serial dilution plating method was used to plate each soil sample on the five nutrient

media, as well as selected soil samples on media with varying marine salt concentrations or

media prepared at pH 5.0. Serial dilutions were prepared by adding 9 mL of each nutrient

medium broth to individual 15 mL dilution tubes. Using a Sterileware® sampling spatula (Bel-

Art Products®), 1 g of soil was weighed into the first dilution tube. The first dilution tubes were

vortexed at maximum speed for 30 sec. One milliliter was transferred from the first dilution tube

to the second dilution tube containing 9 mL of a culture medium broth. The second dilution was

vortexed at maximum speed and 1 mL was transferred to the third dilution tube, and so forth.

After dilutions were prepared, an aliquot of 150 µL was transferred onto the corresponding

dilution agar plate and distributed over the surface of the agar using a sterile plastic 60 mm cell

spreader. Plating was performed in triplicate. Plates were sealed with Parafilm®, inverted and

incubated at 28ºC for 20 days. Plates on the media containing varying salt concentrations were

16

incubated for 35 days. Sites AT03-35 and AT04-170 were plated an additional time using the

serial dilution plating method on the five media and incubated for 45 days to determine whether

slow-growing organisms would appear after the 20th day.

ENRICHMENT CULTURES

The tubes that were used for the serial dilutions (except AT04-159) were incubated at

28ºC for 20 days for the purpose of creating enrichment cultures. After incubation, the

enrichment cultures were plated in the same manner as the dilution plating method. Enrichment

tubes were vortexed at maximum speed for 30 sec. An aliquot of 150 µL was transferred onto

the corresponding dilution agar plate. Plates were sealed with Parafilm®, inverted and incubated

at 28ºC for 20 days. Microbial colonies grown from the enrichment cultures were isolated,

preserved, and treated in the same way as isolates retrieved in the dilution plating method.

Isolates recovered from enrichment cultures have been named similarly to dilution plating

isolates except that a capital B precedes the isolate number; for example, AT04-150-B11.

PLATE COUNTING AND COLONY FORMING UNIT DETERMINATION

CFUs/g of soil on plates were counted after incubation times of 5, 10, and 20 days. The

second plating of AT03-35 and AT04-170 were counted after the incubation days of 5, 10, 20,

35, and 45. Plates on the media containing varying salt concentrations were counted after 5, 10,

20, and 35 days. Plate counts were recorded along with a description of all visible colonies.

CFUs/g of soil were calculated from the countable dilution plate (30 to 300 colonies) by

multiplying the number of colonies by the dilution factor and 6.67 to account for the 150 µL

aliquot.

17

ISOLATION OF PURE CULTURES

Colonies were transferred to agar culture media from dilution plates on the five media

after the 20 day counts were recorded. Each colony was transferred to the same nutrient medium

on which it was originally isolated. The streak plate method was used to isolate individual

colonies. Colonies were selected from all plates of the triplicate set. If there were multiple

colonies with similar morphological characteristics on a dilution plate, two or three were selected

for isolation. In an attempt to isolate sufficient representatives of the culturable heterotrophic

bacteria from all sample sites, all unique colonies from each dilution plate for each medium were

transferred. Records for each isolate included a colony description, medium type, source

dilution, and depth (for soil pits). Each transfer plate was inverted and incubated at 28ºC until

pure cell growth was observed. Colonies on transfer plates was divided into three to four

sections; cell material from two to three were used for preservation and the remaining cells were

used for DNA extraction.

PRESERVATION

All five nutrient media were prepared as preservation solutions by adding 15% (v/v)

glycerol and 750 µL was aliquoted into three sterile Eppendorf® 1.5 mL Safe Lock Tubes.

Saline EDTA was prepared for the DNA extractions; 300 µL of saline EDTA was added to one

clean sterile Eppendorf® 1.5 mL Safe Lock Tube for each isolate. Cell material was transferred

to saline EDTA, and to tubes containing 750 µL of the culture medium plus 15% (v/v) glycerol.

The tubes with saline EDTA plus bacterial cell material were labeled and stored in the freezer (-

20ºC) for future DNA extraction. The tubes with the bacterial cell material plus 15% (v/v)

glycerol media were labeled and preserved in a Thermo Forma® ultra low temperature freezer at

-80ºC.

18

DNA EXTRACTION

DNA was extracted and purified from isolated cells using the UltraClean™ Microbial

DNA Isolation Kit (Mo Bio Laboratories, Inc.). The first five steps of the manufacturer’s

instructions were slightly modified as provided. The samples were thawed and the tubes were

centrifuged at 16,100 x g for 5 min. to pellet cells. The supernatant, (approximately 300 µL),

was removed and discarded using a pipette. The MicroBead solution was added (300 µL) to

each extraction tube following disaggregation of the pellet. The entire contents of each tube

were transferred to a MicroBead tube and 50 µL of solution MD1 was added to each MicroBead

tube. The tubes were placed on the Mo Bio vortex adapter for 5 min at maximum speed,

removed from the vortex and incubated in a 65ºC water bath for 10 min. After incubation the

MicroBead tubes, they were placed on the vortex adapter and vortexed at maximum speed for an

additional 5 min. The manufacturer’s methods were applied from this point forward. The DNA

containing solution was transferred to a clean sterile Eppendorf® 1.5 mL Safe Lock Tube for

storage and to prevent evaporation. DNA was verified by gel electrophoresis prior to PCR

amplification of 16S rRNA genes.

POLYMERASE CHAIN REACTION (PCR) AND PCR PURIFICATION

The 16S rRNA genes from the isolates were amplified by PCR from 20 to 200 ng of

DNA in reaction mixtures, totaling 50 μl, containing (as final concentrations): 1X PCR Buffer

(Applied Biosystems), 50 mM each deoxynucleoside triphosphate, 200 nM each forward and

reverse primer, and 33.3 μl sterile distilled water. Reaction mixtures for universal primers were

incubated in a BioRad iCycler® at 98°C for six min. (for initial denaturation). After the

temperature was reduced to 90°C, 0.2 µL of Taq polymerase (Roche) was added; followed by 28

cycles at 52°C for 30 sec., 54°C for one minute, and 94°C for 30 sec. A final extension period of

19

7 min. at 72°C was done. 16S rRNA genes were amplified with the forward primer 27F (specific

for Bacteria) (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-

GGTTACCTTGTTACGACTT-3’). PCR products were confirmed by gel electrophoresis prior

to PCR purification. PCR fragments were purified with the Invitek Invisorb® Spin PCRapid Kit,

using manufacturer’s guidelines.

SEQUENCING, SEQUENCE PURIFICATION, AND DATA ANALYSIS

16S rRNA gene PCR products were sequenced using the BigDye Terminator reagent

(Applied Biosystems) and the 27F primer. Total reaction volumes were 10 μl and comprised: 0.5

μl 16S rRNA cleaned gene PCR product, 2.5 μl 27F primer (25 ng/μl), 2.5 μl BigDye Terminator

reagent and 4.5 μl sterile distilled water. Reaction mixtures were incubated in a BioRad

iCycler® at 94°C for 30 sec., followed by 25 cycles at 96°C for 10 sec., 50°C for 5 sec, and 60°C

for 4 min.

The sequence reactions were purified by ethanol precipitation after completion of the

sequencing cycles. Sequence reaction tubes were removed from a BioRad iCycler® and 10 µL

of sterile distilled water were added to each sequence reaction tube, that was transferred to a

clean sterile 0.6 mL tube containing three µL of sodium acetate (3M pH 5.0) and 50 µL of 200

proof absolute ethanol. Tubes were placed on ice for 10 min. The tubes were centrifuged for 30

min. at 16,100 x g. Approximately 75 µL of the supernatant was removed by pipette from each

tube and discarded. Next, 250 µL of 70% (v/v) ethanol was added to each tube and centrifuged

for 10 min. at 16,100 x g. The supernatant was removed and discarded. The tubes were than

placed in a drying oven set at 60ºC for roughly 20 min. or until tubes were completely dry.

Purified DNA was stored at -20ºC until ready for resuspension. The purified DNA was

20

resuspended in HiDi Formamide (Applied Biosystems) and analyzed using an ABI Prism 3100

Genetic Analyzer.

The sequences were compiled using the Bioedit™ program version 7.0.0. The sequences

were subjected to BLAST searches in GenBank®, the National Institutes of Health genetic

sequence database, and the closest phylogenetic relative was determined. The BLAST program

was described by Altschul et al. (1990). Appendix B lists the isolate name, colony description,

medium type, source dilution, depth, BLAST result, sequence length, similarity, and accession

number for each identified isolate.

ISOLATE DATABASE

The following data were recorded in the database: isolate numbers, dilutions from which

isolates were recovered, media from which isolates were recovered, depths of sample from

which isolates were recovered, description of colonies isolated, lengths of sequences (bp)

imported into the BLAST interface of GenBank®, GenBank® BLAST results, nucleotide

identity similarities, sample sites from which isolates were recovered, year sites were sampled,

phylogenetic groups of closest relatives as identified by BLAST results, latitudes and longitudes

of sample sites, closest relatives as identified by BLAST results, growth temperature of isolates,

and sequences imported into the BLAST interface of GenBank® have been placed in an online

database. The Atacama Desert isolate database, which was designed by Abhishek

Satyendranath, is accessible at:

http://www.biology.lsu.edu/webfac/frainey/raineylab/databases/ATISOL/searchdata.php

DIRECT COUNTS USING FLUORESCENCE MICROSCOPY

Fluorescence microscopy was applied to three surface soils from the Atacama Desert

(AT04-165, AT04-166, and AT04-170) for direct counts of microbial cells within each sample.

21

DAPI, 4'-6-Diamidino-2-phenylindole, forms a fluorescent complex with DNA and can be used

as a vital tool when quantifying cells. A combined modified version of the total cell counts

DAPI protocol from Carman (1993), Janssen et al. (2002), was Glavin et al. (2004), was applied

to this study. For each sample, 2 g of soil were fixed in 2 mL of cold filter sterilized (FS)

phosphate-buffered saline (PBS) (0.13 M NaCl, 7 mM Na2HP04, 3 mM NaH2PO4; pH 7.2)

containing 4% (w/v) paraformaldehyde for at least 3 hours at 4ºC in a sterile 15 mL centrifuge

tube. The soil and fixing solution were agitated using a Sonifier® ultrasonic cell disruptor

(Branson Sonic Power Co). The sonication was carried out on ice with the probe tip at two

thirds of the liquid depth for 1 min. at 50% power (pulsing in 30 sec. intervals). The samples

were centrifuged at 2,350 x g for 10 min. and the supernatant (fixing solution) was removed and

discarded. Soil pellets were washed with 2 mL of FS PBS and gently vortexed. Samples were

centrifuged for 10 min at 2,350 x g and the supernatants removed and discarded. An additional 2

mL of PBS was added to the soil pellets and gently vortexed. Tubes were centrifuged again at

650 x g for 1 min to spin down larger soil particulates, but to allow the cells to remain suspended

in the supernatant (Carman 1993). The supernatant from each tube was split into 15 mL

centrifuge tubes containing 1 mL, 500 µL, 250 µL, 100 µL, and 50 µL. For each tube, a final

DAPI concentration of 5 µg/mL (stock concentration of 1000 µg/mL) was added and incubated

for at least 3 hours in the dark. The extra 100 µL of supernatant (not stained with DAPI) from

each sample was used as a control for autofluorescing particulates and treated in the same

manner as the DAPI stained samples. The liquid extract in each 15 mL sample tube was

increased to 5 mL with the addition of FS PBS. Each 5 mL solution was filtered onto a Millipore

Isopore™ Membrane Filter (0.2 µm 25 mm GTBP). A 5 mL aliquot of FS PBS was passed

through each filter to ensure affixing of all cells to the filter. The filters were mounted on clean

22

glass slides with Citifluor™, an anti-fade solution, and coverslips (22 by 22 mm) were placed on

each mounted filter. The preparations were examined under UV illumination (excitation filter

BP 365nm) at 800x magnification with a Nikon Microphot-FXA microscope.

At least 20 fields, evenly distributed over the area beneath the coverslip, were examined

for each of the samples and autofluorescing controls. Fluorescing cells in each of the 20 fields

were counted and an average number of cells per field determined. Calculations were made

using the area of the filter (490.65 mm2) and microscopic field measurements at 800x

magnification (88 µm x 117 µm). It was determined that at this magnification there are 47,652

fields per polycarbonate filter. The average was multiplied by the number of fields per filter and

then again to determine the number of cells per 1 g of soil. To account for the amount of soil the

supernatant was extracted from, the 1 mL extract calculations were multiplied by one, the 500

µL extract calculations were multiplied by two, the 250 µL extract calculations were multiplied

by four, the 100 µL extract calculations were multiplied by ten, and the 50 µL extract

calculations were multiplied by 20.

DETERMINING LIMIT OF DIRECT COUNTS ON ATACAMA DESERT SOILS

The Atacama Desert soil isolate strain AT03-37-10 grown on Nutrient Agar, had the

closest relative being Blastococcus aggregatus (AJ430193). In a sterile 15 mL tube, a loop full

of bacterial growth was transferred to 9 mL of Nutrient Broth. Differential Interference Contrast

(DIC) and fluorescence photomicrographs of the strain AT03-37-10 cells were captured in order

to observe the cell size and shape. Scanning electron microscope (SEM) photomicrographs were

also taken of strain AT03-36-10 (preparations given below), whose closest relative is also

Blastococcus aggregatus (AJ430193), to observe the cell size and shape. A 1/10 dilution of the

cell suspension was prepared (500 µL into 4.5 mL FS PBS) and filtered onto a Millipore

23

Isopore™ Membrane Filter (0.2 µm 25 mm GTBP). A 5 mL aliquot of FS PBS was passed

through each filter to ensure affixing of all cells to the filter. The filters were mounted on clean


each mounted filter. The preparations were examined under UV illumination (excitation filter

BP 365nm) at 800x magnification with a Nikon Microphot-FXA microscope for the DAPI

fluorescence microscopy and DIC images were captured at 1000x magnification with a Nikon

Microphot-FXA microscope. DIC and fluorescence photomicrographs were acquired from the

same filter and can be viewed in Figure 3.5.

Preparations for the SEM photomicrographs were done by Cindy Henk of the Socolofsky

Microscopy Center, Department of Biological Sciences. The Atacama Desert isolate strain

AT03-36-10 was grown on 1/10 strength PCA. In a sterile 15 mL tube, a loop full of bacterial

growth was transferred to 5 mL of 1/10 strength PCB. The cell culture was fixed in an equal

volume of 4% (v/v) glutaraldehyde, 0.1 M sodium cacodylate buffer (pH 7.0), and half strength

culture medium for 30 min. then collected on a 0.22 µm polycarbonate filter. The filter was

transferred to 4% (v/v) glutaraldehyde in 0.1 M buffer for 5 min. and rinsed three times in 0.1 M

buffer. The filter was dehydrated in ethanol, critical-point dried, mounted on an aluminum

specimen stub, and coated with gold:palladium 60:40 in an Edwards S150 sputter coater. The

filter was viewed using a Cambridge S-260 SEM. A representative SEM photomicrograph can

be viewed in Figure 3.5.

The strain AT03-37-10 cell suspension was serially diluted to 1/1000 in Nutrient Broth.

The optical densities of each of the cell suspensions were determined using SmartSpec™ Plus

Spectrophotometer (BioRad). The cell suspension dilution series was plated to determine the

number of viable cells in the stock culture. Using these data a standard curve plotting turbidity

24

versus total viable cell number was created. Either 1 mL of the stock culture, each dilution, or

the control (Nutrient Broth) was added to 1 g of soil from site AT04-170, a soil previously found

to have zero CFUs on the five culture media, and spread evenly over the base of an empty Petri

dish. Soils were air dried overnight. The soils were transferred to a sterile 15 mL tube and fixed

at 4ºC for at least 3 hours in 2 mL of cold FS PBS containing 4% (w/v) paraformaldehyde. The

tubes were then centrifuged at 2,350 x g for 10 min. and the supernatant (fixing solution) was

discarded. Two milliliters of FS PBS was added to the soil pellets, gently vortexed, and

centrifuged at 2,350 x g for 10 min. The supernatants were discarded and 2 mL of FS PBS was

added to the soil pellets. The tubes were gently vortexed and centrifuged for 1 min. at 650 x g to

spin down larger soil particulates, but keep bacterial cells suspended in the supernatants (Carman

1993). The 2 mL supernatants were split in half; 1 mL was transferred to two clean sterile 1.5

mL tubes. In one of the tubes for each sample 5 µL of FS DAPI (stock concentration of 1000

µg/mL and final concentration of 5 µg/mL) was added and the tubes were incubated for at least 3

hours at room temperature in the dark. The tube for each sample without DAPI was used as a

control for autofluorescing particulates and treated in the same exact manner as the DAPI stained

samples. From each DAPI treated sample (and each autofluorescing control) 1 mL was

transferred to 4 mL of FS PBS. Each 5 mL solution was filtered onto a Millipore Isopore™

Membrane Filter 0.2 µm, 25 mm GTBP. Five milliliters of FS PBS was passed through each

filter to ensure affixing of all bacterial cells to the filters. The filters were mounted on clean


each mounted filter. The preparations were examined under at a magnification of 400x with a

Nikon Microphot-FXA microscope under UV illumination (excitation filter BP 365nm). At least

20 fields, evenly distributed over the area beneath the coverslip, were examined for each sample,

25

and the blue fluorescing cells were counted. Focusing at different planes was done for each

field; this was especially necessary when counting cells in small clumps. Calculations of cell

numbers were made using the area of the filter (490.65 mm2) and microscopic field

measurements at 400x magnification (176 µm x 235 µm). At the magnification used, each filter

contains 11,913.61 fields, thus the average cell number per field was multiplied by 11,913.61.

The method used only accounts for 0.5 g of soil, thus the value was multiplied by two to convert

the value to cells per gram of soil. The computed results determine the quantity of cells

recovered from those added to 1 g of AT04-170 soil and consecutively serve to determine the

detection limit per gram of soil for the DAPI direct count procedure on the Atacama Desert soils

examined in this study.

The procedure above was repeated, but instead of allowing the AT04-170 soils to dry

overnight after the cell suspension or control (Nutrient Broth) was added, the soils were allowed

to dry for a month in an empty sterile Parafilmed® Petri dish on the countertop. The purpose of

this trial was to determine the effects of the Atacama Desert soil and extended desiccation on the

added cultured microbial cells and what differences were noticed (if any) in the ability for DAPI

to bind to these cells, consequently possibly changing the established detection limit.

LAKE WATER SAMPLE PREPARATIONS FOR DAPI STAINING CONTROL

Approximately 30 mL of water was collected from a Louisiana State University lake with

a clean sterile 50 mL centrifuge tube. Immediately after sample collection, the lake water was

brought back to the lab and 5 mL was transferred to a clean sterile 15 mL centrifuge tube with a

10 mL serological pipette in a clean bench environment. A 1/1 ratio of FS PBS containing 4%

(w/v) paraformaldehyde (5 mL) was transferred to the 5 mL of lake water within the 15 mL

centrifuge tube for at least 3 hours at 4ºC. Fifty microliters of FS DAPI (stock concentration of

26

1000 µg/mL and final concentration of 5 µg/mL) was added to the total 10 mL of cold filter FS

PBS containing 4% (w/v) paraformaldehyde and lake water. The solution was allowed to DAPI

stain for at least 3 hours at 4ºC. In three new clean sterile 15 mL tubes, the solution was split

into 7 mL, 2 mL, and 1 mL, which equated to roughly 3.5 mL of lake water, 1 mL of lake water,

and 0.5 mL of lake water. The solutions in each of the three 15 mL sample tubes were increased

to 5 mL with the addition of FS PBS. Each 5 mL solution was filtered onto a Millipore

Isopore™ Membrane Filter 0.2 µm, 25 mm GTBP. Five milliliters of FS PBS was passed

through each filter to ensure affixing of all bacterial cells to the filters. The filters were mounted

on clean glass slides with Citifluor™, an anti-fade solution, and coverslips (22 by 22 mm) were

placed on each mounted filter. The mounted filters were examined under at a magnification of

800x with a Nikon Microphot-FXA microscope under UV illumination (excitation filter BP

365nm).

DAPI STAINING PREPARATIONS FOR CONTROL SOIL SAMPLE

A soil sample was collected from a cornfield in close proximity to the Louisiana State

University campus. A Global Positioning System (GPS) device was used to record the

coordinates of the sample site. The soil sample was retrieved using a sterile plastic scoop and

sterile Nasco Whirl-Pak® bag. One gram of soil was fixed in 1 mL of cold FS PBS containing

4% (w/v) paraformaldehyde for at least 3 hours at 4ºC in a sterile 15 mL centrifuge tube. The

samples were centrifuged at 2,350 x g for 10 min. and the supernatant (fixing solution) removed

and discarded. The soil pellet was washed with 1 mL of FS PBS and vortexed gently. The

sample was centrifuged for 10 min. at 2,350 x g and the supernatant was removed and discarded.

An additional one mL of PBS was added to the soil pellet and vortexed gently. The tube was

centrifuged again at 650 x g for 1 min. to spin down larger soil particulates, but allow the cells to

27

remain suspended in the supernatant (Carman 1993). A volume of 250 µL of the supernatant

was transferred to a 15 mL centrifuge tube and 1.25 µL of DAPI (stock concentration of 1000

µg/mL and final concentration of 5 µg/mL) was added and allowed to incubate for at least 3

hours in the dark. The liquid extract in the 15 mL sample tube was increased to 5 mL with the

addition of 4.75 mL of FS PBS. The 5 mL solution was filtered onto a Millipore Isopore™

Membrane Filter (0.2 µm 25 mm GTBP). A 5 mL aliquot of FS PBS was passed through each

filter to ensure affixing of all bacterial cells to the filter. The filter was mounted on clean glass

slides with Citifluor™, an anti-fade solution, and a coverslip (22 by 22 mm) was placed on the

mounted filter. The mounted filter was examined under UV illumination (excitation filter BP

365nm) at 800x magnification with a Nikon Microphot-FXA microscope.

DETERMINATION OF SALINITY IN ATACAMA DESERT SOIL SAMPLES

All Atacama Desert samples and the control cornfield soil sample were tested for soil

salinity, which is a measure of the total amount of soluble salt in soil. One gram of soil for each

site was weighed into a clean sterile 50 mL centrifuge tube and 20 mL of water from a Millipore

Milli-Q® Ultrapure Water Purification System were added to each tube containing soil with a

sterile 10 mL serological pipette. Tubes were shaken for an hour at room temperature at

approximately 180 rpms. The 20 mL of water from each sample tube was transferred to a

sample solution cup and the salinity was measured with an Extech EC400 Waterproof ExStik

Conductivity/TDS/Salinity Meter. The salinity, TDS (total dissolved solids), and conductivity

for all soil samples were recorded and are displayed in Appendix D.

PH DETERMINATION

One gram of each soil sample was weighed into a clean sterile 15 mL centrifuge tube and

1 mL of water from a Millipore Milli-Q® Ultrapure Water Purification System was added to

28

each tube, and then shaken for 10 min. An IQ Scientific Instruments model 170 isfet pH probe

(non-glass & temp corrected) was placed into the centrifuge tube to obtain a pH reading for each

soil sample. Duplicate readings were acquired for samples AT03-34, AT03-36, AT03-44, AT04-

169, AT04-150 10 cm, AT04-150 70 cm, AT04-152 20 cm, AT04-152 70 cm, AT04-152 80 cm,

AT04-153 10 cm, AT04-153 30 cm, AT04-153 40 cm, AT04-153 80 cm, AT04-159 surface, and

AT04-159 40 cm to determine if there were significant differences between each reading. After

24 hours, pH readings for samples AT04-163, AT04-166, AT04-150 80 cm, and AT04-152 80

cm were taken again to determine if there was a significant difference between readings taken

the previous day. Also, to detect any variance between pH readings taken immediately after

addition of distilled water and readings taken after samples (including distilled water) had shaken

for 10 min., pH readings for samples AT04-163, AT04-166, and AT04-150 80 cm were acquired

as distilled water was added.

DETECTION OF CARBONATE PRESENCE IN SELECTED SAMPLES

Approximately five drops of dilute hydrochloric acid (HCl) (approximately a 1/10

dilution) were added to approximately 0.5 g of dry samples AT04-167, AT04-152 70 cm, AT04-

153 30 cm to detect carbonation (bubbles) indicating presence of carbonates.

ELEMENTAL ASSAY ON ATACAMA DESERT SOILS

Dr. Robert Gambrell and associates of his laboratory completed metal analyses for every

surface and subsurface soil sample of Al, As, Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, Pb, Si,

and Z (mg/L). The protocol used was on approximately 1 g of soil material that was weighed

into a 100 mL Pyrex test tube with aggregates broken up with a glass rod, if necessary. Five

milliliters of concentrated, trace metals grade nitric acid was added. A small glass funnel was

placed in the top of each tube to facilitate reflux action of the acid during the heating step to keep

29

the acid from evaporating. The tubes were put on a block digester at approximately 120º C for

about 8 hours. The next day, the glass funnels were removed and the block temperature

increased to 120º C again to evaporate the acid down to an approximate volume of 1.5 to 2 mL.

At this point, the tubes were removed from the block, allowed to cool, then the volume brought

to 50 mL with de-ionized water and the tubes were capped with parafilm. The contents

(acidified water and extracted soil solids) were mixed vigorously, then allowed to settle

overnight. The solids settled to the bottom and a clear supernatant appeared above the settled

solids. Part of the clear supernatant was poured into approximately 15 mL plastic tubes and

placed on the autosampler for a Varian MPX Inductively Coupled Plasma (ICP) Emission

Spectrometer. Also analyzed were two reagent blanks (diluted acid) and replicate extractions of

approximately 20% of the samples to demonstrate reproducibility. For metals present in

concentrations greater than the linear range of the instrument, dilutions were made and the

samples reanalyzed. The results were reported in mg/g, and were determined by multiplying the

extract concentration by 50 mL and by any applicable dilution factor, then dividing by the initial

soil sample weight. Table 6.1 displays the concentrations (mg/L) of the elemental components

for each soil sample.

INORGANIC ANION ANALYSIS

Sarah Jones at the Institute for Ecological Infrastructure Engineering and Water Quality

Laboratory at the College of Engineering at Louisiana State University performed anion analysis

on all Atacama Desert and cornfield soil samples. Samples were given to Sarah Jones in the

manner of 1 g of soil weighed into a clean sterile 50 mL centrifuge tube and 50 mL of water

from a Millipore Milli-Q® Ultrapure Water Purification System added to each tube, then shaken

overnight at room temperature at approximately 180 rpms. A Dionex IC25 Ion Chromatograph

30

and LC20 Chromatography Enclosure with an AS14A column (these two instruments make up

the DX 320 IC process analyzer) were used for the analysis. An AS14A eluent (purchased from

Dionex) was prepared with water filtered through a 0.2 µm membrane according to the directions

on the concentrate bottle: 8 mM sodium carbonate, 1 mM sodium bicarbonate, and 10 mL

Dionex AS14A. Samples were added to Dionex 5 mL autosampler vials, inserted into the filter

caps, and placed in the autosampler cassette. The eluent along with 25 µL of each sample was

run for 18 min. The latest five point standard curve for each anion (PO4, F, Br, Cl, NO3, SO4,

and NO2) was used to calculate the anion concentration. The anions ion chromatography

procedure is based on the EPA Method 300.0 (Determination of Inorganic Anions by Ion

Chromatography). Table 6.2 (chloride, fluoride, nitrate, and sulfate) and Appendix C

(phosphate, nitrite, and bromide) display the concentrations (mg/L) of analyzed anions present in

each soil sample.

31

CHAPTER 3 QUANTIFYING AND IDENTIFYING CULTURABLE HETEROTROPHIC BACTERIA

IN THE SURFACE SOILS OF THE ATACAMA DESERT

It was shown in a previous study of a limited number of samples that the surface soils

within this hyper-arid region of the Atacama Desert contained low numbers of or no culturable

heterotrophic bacteria (Narvarro-González et al. 2003). In order to examine the extent of these

soils containing low numbers of culturable heterotrophic bacteria, 33 additional samples in the

Yungay region were studied. It was hypothesized that the distribution, in terms of abundance

and diversity, of culturable bacteria in the surface soils would not be uniform and that patchiness

would be observed, similar to what has been observed for plant species in desert soils that

support plant growth (Rietkerk et al. 2004).

RESULTS

Colony Forming Units

Thirty-three accessible surface sites within the Yungay region were studied (Table 3.1).

Using the culture-dependent technique of dilution plating, a wide range of CFU/g values were

observed for the samples. Figure 3.1 shows the distribution of surface sites and the CFU values

determined for each site.

The surface site CFU data (Table 3.2 and Figure 3.2) showed that for all media, the

values from range 0 to 7.4 x105 CFUs/g of soil. Of the 33 sites, 22 showed no CFUs/g for at

least one of the media. Four sites showed no CFUs/g on all five media. Twenty of the 33 sites

had values for all five media of < 1 x 103 CFUs/g. Seven of the 33 sites had CFU/g values of > 1

x 104 and one of these (AT04-166) had CFU/g values of >105 (Table 3.3). The largest number of

samples showing no detectable CFUs/g were those plated on PCA (16 samples), while the lowest

number of samples with no detectable CFUs/g were those plated on 1/100 PCA (10 samples).

32

Samples plated on PCA consistently had lower CFU values (Table 3.3). For 1/100 PCA, higher

CFU values were found overall, with less samples showing CFU/g of 0 to 103 than those plated

on other culture media (Table 3.3). The highest CFU/g value of all samples that of site AT04-166

and was obtained on 1/10 PCA (Table 3.3).

Soil samples AT03-35 and AT04-170 were chosen for the experimental plating on media

differing in salt concentrations. AT03-35 represented sites with very low counts (i.e., below the

detection limit) and AT04-170 represented sites with 0 CFUs/g on all five principal media. After

35 days of incubation, AT04-170 showed 0 CFUs/g of soil on all media varying in marine salt

concentrations. After 35 days of incubation, AT03-35 had no CFUs/gram of soil on MAIO, 3.17

x 103 CFUs/gram of soil on 1X IO, 44.4 CFUs/gram of soil on 2X IO, and no CFUs/gram of soil

on 3X IO. More cells in sample AT03-35 were capable of being cultured on media prepared

with 1X IO than any other of the five key media and all other media containing varying salt

concentrations. The highest CFU value for AT03-35 on the five primary media occurred on the

culture medium MA with 4.44 x 102 CFUs/gram of soil. Colonies that appeared on the culture

media with 1X IO were all very similar in morphology to colonies observed on MA.

Soils from sites AT03-35 and AT04-170 were also plated onto the five media used in this

study (MA, NA, PCA, 1/10 PCA, 1/100 PCA) for a second time to determine if additional

colonies would grow after 20 days of incubation, i.e. to recover any slow-growing culturable

heterotrophic prokaryotes. Plates were allowed to incubate for 25 days beyond the original

dilution plates that were only allowed to incubate for a total of 20 days before colonies were

isolated. After 45 days of incubation, no further colonies had grown past 20 days.

Soils from sites AT04-163, AT04-158, and AT04-164 were plated on MA and 1/10

strength PCA media at pH 5.0 to determine whether a more acidic culture media would improve

33

Table 3.1: Degree minute decimal coordinates for surface sites within the core arid region of Yungay

Surface Sample Site West (minutes) South (minutes)

AT03-33 69º W 51.840′ 24º S 4.112′ AT03-34 69º W 51.806′ 24º S 4.103′ AT03-35 69º W 51.828′ 24º S 4.008′ AT03-36 69º W 51.854′ 24º S 3.836′ AT03-37 69º W 51.891′ 24º S 3.734′ AT03-38 69º W 52.088′ 24º S 3.645′ AT03-39 69º W 52.187′ 24º S 3.548′ AT03-40 69º W 52.451′ 24º S 3.637′ AT03-41 69º W 53.091′ 24º S 3.666′ AT03-42 69º W 54.649′ 24º S 3.445′ AT03-43 69º W 54.637′ 24º S 3.445′ AT03-44 69º W 54.496′ 24º S 3.695′ AT03-45 69º W 51.854′ 24º S 4.151′ AT03-46 69º W 51.897′ 24º S 4.151′ AT03-48 69º W 52.194′ 24º S 4.875′ AT03-49 69º W 52.632′ 24º S 4.545′ AT03-50 69º W 52.926′ 24º S 4.452′ AT04-151 69º W 51.858′ 24º S 3.720′ AT04-154 69º W 51.415′ 24º S 3.613′ AT04-155 69º W 51.445′ 24º S 3.728′ AT04-156 69º W 51.438′ 24º S 3.655′ AT04-157 69º W 51.495′ 24º S 3.548′ AT04-158 69º W 51.453′ 24º S 3.415′ AT04-161 69º W 54.065′ 24º S 4.440′ AT04-162 69º W 53.465′ 24º S 4.200′ AT04-163 69º W 52.925′ 24º S 3.900′ AT04-164 69º W 52.560′ 24º S 3.660′ AT04-165 69º W 52.680′ 24º S 4.140′ AT04-166 69º W 52.260′ 24º S 4.200′ AT04-167 69º W 51.480′ 24º S 4.800′ AT04-168 69º W 51.660′ 24º S 4.500′ AT04-169 69º W 53.580′ 24º S 3.900′ AT04-170 69º W 54.300′ 24º S 4.320′

34

AT03

-48

AT03

-33

AT03

-34*

AT03

-35

AT03

-36*

AT03

-37*

AT03

-38*

AT03

-39*

AT03

-40

AT03

-41

AT03

-42*

AT03

-43*

AT03

-44*

AT03

-45

AT03

-46

AT03

-49*

AT03

-50

AT04

-151

*

AT04

-154

*

AT04

-155

*

AT04

-156

*

AT04

-157

*

AT04

-158

*

AT04

-161

AT04

-162

*

AT04

-163

AT04

-164 AT

04-1

65 AT04

-166

AT04

-167

*

AT04

-168

AT04

-169

AT04

-170

3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0

50.0

50.5

51.0

51.5

52.0

52.5

53.0

53.5

54.0

54.5

55.0

Long

itiud

e - 69

º W (m

in.)

Latit

ude -

24

º S (m

in.)

Figu

re 3

.1:

Scat

ter p

lot i

llust

ratin

g pr

oxim

ity o

f sur

face

site

s in

core

arid

regi

on a

nd d

istri

butio

n of

CFU

s.

The

red

circ

les r

epre

sent

site

s with

no

(zer

o C

FUs/

g on

all

med

ia) h

eter

otro

phic

cul

tura

ble

bact

eria

l gro

wth

. Th

e pi

nk sq

uare

s re

pres

ent t

he si

tes w

ith lo

w c

ount

s bel

ow th

e de

tect

ion

limit

(CFU

s/g ≤

102 on

all

med

ia),

and

blue

squa

res r

epre

sent

site

s with

co

lony

num

bers

nea

r or s

light

ly a

bove

the

dete

ctio

n lim

it (C

FUs/

g be

twee

n 10

2 and

104

on a

t lea

st o

ne m

ediu

m).

The

gre

en

trian

gles

repr

esen

t the

site

s with

hig

h ba

cter

ial C

FUs (

CFU

s/g

of 1

04 or g

reat

er, o

n at

leas

t one

med

ium

).

* si

tes h

avin

g ze

ro c

olon

ies o

n at

leas

t one

med

ium

.

35

Table 3.2: Surface sample pH values and CFUs/g of soil on five media after 20 days incubation * average of duplicate pH

Media Surface Sites pH MA 1/10 PCA 1/100 PCA PCA NA

AT03-33 7.30 1.39 x 104 1.17 x 104 2.45 x 103 2.22 x 102 1.06 x 104 AT03-34 6.76* 67 0 22 1.11 x 102 0 AT03-35 7.22 4.44 x 102 3.78 x 102 2.22 x 102 67 89 AT03-36 6.48* 44 3.33 x 102 22 0 67 AT03-37 7.22 22 0 1.56 x 102 0 67 AT03-38 7.11 0 0 1.78 x 102 44 0 AT03-39 7.61 5.78 x 102 3.20 x 103 7.78 x 102 0 67 AT03-40 7.24 6.49 x 104 1.97 x 104 4.47 x 104 67 1.09 x 104 AT03-41 7.91 1.19 x 104 3.27 x 103 8.73 x 103 2.67 x 102 5.53 x 103 AT03-42 7.08 22 0 0 22 0 AT03-43 7.54 6.67 x 102 2.00 x 102 3.33 x 102 0 2.00 x 102 AT03-44 7.01* 0 22 0 0 0 AT03-45 7.35 2.47 x 103 2.89 x 102 7.78 x 102 22 9.33 x 102 AT03-46 7.09 1.11 x 104 4.85 x 103 1.43 x 104 8.87 x 103 7.40 x 103 AT03-48 7.56 1.76 x 103 1.40 x 103 1.04 x 103 0 1.33 x 102 AT03-49 7.11 0 0 0 22 0 AT03-50 7.42 3.11 x 102 1.56 x 102 67 1.11 x 102 44 AT04-151 7.16 3.56 x 102 67 3.11 x 102 0 0 AT04-154 7.58 0 0 1.62 x 103 0 0 AT04-155 7.58 0 0 8.44 x 102 22 0 AT04-156 7.22 22 0 7.33 x 102 0 0 AT04-157 7.79 0 22 0 0 22 AT04-158 7.49 67 67 67 0 22 AT04-161 7.97 1.77 x 104 1.70 x 104 1.78 x 104 1.49 x 103 5.15 x 103 AT04-162 8.46 3.91 x 103 2.66 x 102 0 22 2.67 x 102 AT04-163 8.21 0 0 0 0 0 AT04-164 7.55 1.22 x 103 2.18 x 103 8.44 x 102 3.33 x 102 2.02 x 103 AT04-165 8.02 6.49 x 104 8.20 x 104 5.07 x 104 5.82 x 103 4.20 x 104 AT04-166 7.38 6.80 x 105 7.40 x 105 4.67 x 105 1.91 x 104 6.65 x 105 AT04-167 8.23 0 0 0 0 22 AT04-168 7.37 0 0 0 0 0 AT04-169 8.15* 0 0 0 0 0 AT04-170 8.21 0 0 0 0 0

36

1.E+0

1

1.E+0

2

1.E+0

3

1.E+0

4

1.E+0

5

CFUs

/Gr

am of

Soil

AT03-33AT03-34AT03-35AT03-36AT03-37AT03-38AT03-39AT03-40AT03-41AT03-42AT03-43AT03-44AT03-45AT03-46AT03-48AT03-49AT03-50AT04-151AT04-154AT04-155AT04-156AT04-157AT04-158AT04-161AT04-162AT04-163AT04-164AT04-165AT04-166AT04-167AT04-168AT04-169AT04-170

Samp

le Sit

es

PCA

1/10 P

CA

1/100

PCA

MA

NA

Figu

re 3

.2:

CFU

s/g

of so

il fo

r eac

h of

the

33 su

rfac

e sa

mpl

es o

n fiv

e m

edia

37

Table 3.3: Values of CFU/g recovered on various culture media for the 33 sites sampled

Culture Media CFU/g of soil / number of sites MA 1/10 PCA 1/100 PCA PCA NA

0 11 13 10 16 13 <102 17 17 14 24 21 <103 22 23 24 29 25 <104 26 28 28 32 29 <105 32 32 32 33 32 >105 1 1 1 0 1

38

organism cultivation because Richard Quinn at the NASA Ames Research Center reported that

Yungay soils have an acidic pH 5. AT04-163 represented sites with zero CFUs on all principal

media, AT04-158 represented sites with very low counts, below the detection limit (CFUs below

103 on all five major media), AT04-164 represented sites with CFUs near or slightly above the

detection limit (CFUs between 103 and 104 on at least one medium). After 20 days of incubation,

all three representative surface sample sites showed zero CFUs/g of soil on the acidic media.

Regarding the results of this experiment, which suggest that organisms from the Yungay region

soils prefer conditions having a more neutral pH, I chose to determine the pH of all surface and

soil pit samples (soil pit pH readings in Table 4.1). The pH range for the surface samples was

found to be from 6.30 to 8.46, with a mean pH 7.5. Therefore, the Yungay surface soils were

found to have a more neutral pH than originally reported (Table 3.2). Selected sample pH

readings were obtained in duplicates to confirm accuracy of pH readings: AT03-34 (6.65 and

6.87), AT03-36 (6.30 and 6.66), AT03-44 (6.90 and 7.12), AT04-169 (8.15 and 8.15). The pH

for selected samples was determined again 24 hours after the first reading. The sample AT04-

163 had pH 8.21, and 24 hours later, the pH reading was 8.22. Sample AT04-166 had pH 7.38,

and 24 hours later, the pH reading was 7.45. Thus, there was no significant difference in the pH

readings obtained over a 24 hour period. Also, pH values as distilled water was added to the

chosen soil samples were recorded. The sample AT04-163 had pH between 8.25 and 8.17 at the

time water was added; the sample AT04-166 had pH between 7.65 and 7.26 at the time water

was added. Hence, the pH readings for the 33 surface samples were concluded to be consistent

despite if a reading was obtained as water was added to soil, 10 min. after water was added, or 24

hours after water was added (pH readings for soil pit samples were also consistent regardless if a

39

reading was obtained as water was added to soil, 10 min. after water was added, or 24 hours after

water was added).

Diversity of Culturable Bacteria

From each of the 33 sites, isolates were recovered and identified to examine the diversity

of culturable heterotrophic bacteria in the soils. The identity of the isolates was based on

comparison of partial 16S rRNA gene sequences (400 to 1100 nucleotide positions) with the

GenBank database using the BLAST tool. A total of 859 isolates were recovered from the 33

soil samples studied (Figure 3.3). The majority of the isolates (833) recovered from the 33

surface soils belong to the Actinobacteria phylum. The remaining 26 isolates were divided

between the two phyla, Proteobacteria (5 isolates) and Firmicutes (19 isolates). The total number

of isolates recovered from each soil sample and identified to the genus/species level was related

to the CFU count of that sample, as well as the successful subculturing of the initial isolate.

Therefore, in some cases only a single isolate was recovered and maintained for some of the soil

samples that had very low CFU values. The list of isolates, colony characteristics and 16S rRNA

gene sequence based identity are provided in Appendix B. The summary of the identities (Figure

3.4) is based on closest relatives provided by the BLAST search and are for the most part

identities at the genus level although in many cases the closest species identity is provided.

Figure 3.4 only includes isolates whose closest relatives were identified to at least genus level

according to BLAST result from GenBank® database.

The majority of the isolates recovered from the surface soils are strains of the genus

Blastococcus. These 623 isolates identified as members of the genus Blastococcus came from 24

of the 28 soils analyzed. In addition to Blastococcus spp., 11 of the 33 soil samples contained

species of the genera Geodermatophilus and Modestobacter, which are taxonomically close

40

483

81

122

161

84

112

159

198

11

13012689

211

213

15311914010

376

227

152

31

0%20

%40

%60

%80

%10

0%

Perc

entag

e

AT03

-33AT

03-34

AT03

-35AT

03-36

AT03

-37AT

03-38

AT03

-39AT

03-40

AT03

-41AT

03-42

AT03

-43AT

03-44

AT03

-45AT

03-46

AT03

-48AT

03-49

AT03

-50AT

04-15

1AT

04-15

4AT

04-15

5AT

04-15

6AT

04-15

7AT

04-15

8AT

04-16

1AT

04-16

2AT

04-16

3AT

04-16

4AT

04-16

5AT

04-16

6AT

04-16

7AT

04-16

8AT

04-16

9AT

04-17

0

Surfa

ce

Samp

le Si

te

Actin

obac

teria

Firmi

cutes

Prote

obac

teria

Figu

re 3

.3:

Div

ersi

ty a

t the

phy

la le

vel w

ithin

surf

ace

sam

ples

Id

entit

y of

the

clos

est r

elat

ives

is b

ased

on

com

paris

on o

f par

tial 1

6S rR

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gen

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ces (

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to 1

100

nucl

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sitio

ns)

with

the

Gen

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k da

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se u

sing

the

BLA

ST to

ol.

The

num

bers

with

in e

ach

bar r

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sent

the

num

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f iso

late

s sel

ecte

d an

d id

entif

ied

to p

hylu

m le

vel f

rom

that

par

ticul

ar si

te.

41

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21

15

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AT03

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42

relatives of Blastococcus. The remaining actinobacterial species were representatives of a

number of taxa including species of the genera Arthrobacter, Cellulomonas, Frankia, Georgenia,

Kocuria, Micrococcus, Mycobacterium, Nocardioides, Promicromonospora, Rothia,

Streptomyces, and Williamsia. The 21 species of the low G+C Gram positive phylum (the

Firmicutes) represent a range of genera that fall within the order Bacilliales, including species of

the genera Bacillus, Brevibacillus, Exigubacterium, Halobacillus, Ornithinicoccus,

Paenibacillus, and Staphylococcus. Representatives of only four genera within the

Proteobacteria were isolated from the surface soil samples, namely Acinetobacter, Oxalobacter,

Pantoea, and Sphingomonas represented a total of five isolates (Figure 3.4).

DISCUSSION

Patchiness in the distribution of CFUs/g is clearly shown in Figure 3.1. There is no

obvious explanation for the observed patchiness throughout the surface of the Yungay region

with respect to the proximity of one site to another. Microbes obtain nutrients from the

environment in which they inhabit; thus, it can be supposed that microbial populations are

located in the core arid region directly as a result of the availability of resources throughout the

soils. It has been reported that the crucial requirement for self-organized patchiness to develop is

that organisms are positively correlated with resource abundance at short spatial range, but

negatively correlated at long spatial range (Rietkerk et al. 2004). This idea supports the short

range patchiness seen in the 15.3 km2 of the core arid region of the Atacama Desert.

Wind currents are known it move across the Atacama Desert from the Pacific Ocean. It

can be considered that marine salts could be carried by these winds and deposited in the inland

desert region. The two samples that were plated onto media containing different salt

concentrations were AT03-35, representing sites with low counts below the detection limit

43

(CFUs <103 on all five major media), and AT04-170, representing sites with zero CFUs on all

five principal media. The sample AT04-170 showed zero colonies on all media including media

varying in marine salt concentrations. The sample AT03-35 showed a higher CFU value on

media containing 1X Instant Ocean® than on any other culture media. The colonies viewed on

the 1X IO medium had the same morphology as colonies observed on all other media; the only

apparent difference was the number of colonies. These finding may indicate that recoverable

heterotrophic organisms that exist in Yungay soils prefer to mature in an environment having

slightly high marine salt concentrations due to in situ conditions of rare marine salt moisture

events.

In order to recover any slow-growing culturable heterotrophic bacteria two surface

samples (AT03-35 and AT04-170) were plated and incubated for 45 days. No additional

colonies were observed after 20 days of incubation indicating that the culturable heterotrophic

bacteria from the collected Yungay surface samples are capable of becoming active and

replicating within 20 days on the five culture media selected for this study.

According to Richard Quinn of the NASA Ames Research Center in Moffett Field,

California, the Atacama Desert soil within the core arid region had a pH value of 5.0. Three

surface soils were plated onto 1/10 PCA (a dilute nutrient medium) and MA (a nutrient rich

marine medium) at a pH 5.0 in order to consider whether or not culturable bacteria from this soil

would grow better at acidic pH. Regarding the findings, organisms existing within the hyper-

arid soils grow better under neutral pH conditions. In order to provide substantiating evidence

for the results, I performed pH analyses for all soil samples (surface and subsurface). The results

are consistent for the surface and soil pit samples; they contradict the initial pH report and

44

indicate that the hyper-arid soils have a neutral pH. Also, the results suggest that the

heterotrophic culturable bacteria inhabiting the soils are neutrophilic organisms.

For each of the 33 sites, isolates were recovered and identified to compare every site’s

bacterial diversity (Figures 3.3 and 3.4). The number of isolates from each sample in the phyla

and closest relative charts may not match exactly due to the fact that the BLAST results do not

always give a closest relative down to genus, the results are sometimes only defined to phylum

or family. In the closest relative charts diagramed in this study, only isolates that are identified

particular to genera are included.

There is low diversity among the culturable heterotrophic bacteria recovered from surface

soils (Figures 3.3 and 3.4). The majority of the culturable bacteria from surface samples

belonged to the genus Blastococcus, a member of the Actinobateria phylum. This group is the

most prevalent of the recoverable microorganisms from the arid regions of the Atacama Desert

using the dilution plating method. Blastococcus is a member of the family

Geodermatophilaceae, with the other genera Geodermatophilus and Modestobacter, which were

also recovered Yungay surface samples (Geodermatophilus was also found in Yungay

subsurface samples). Blastococcus is a Gram-positive organism whose name in Greek means

“sprouting berry”. Strains showing the highest similarity to Blastococcus aggregatus was found

in nine surface samples, along with strains related to Blastococcus sp. strains BC448, which is

the most abundant strain observed throughout this study, BC412, BC521, and BC512. Colonies

of the genus Blastococcus are various shades of pink or orange, are round, vary in size depending

on the media, and are smooth to rough in surface texture. To determine cell morphology,

photomicrographs of Blastococcus sp. strains AT03-36-10 and AT03-37-10 were taken using

DIC microscopy, DAPI epifluorescence microscopy, and SEM (Figure 3.5). Cells occurred

45

Figure 3.5: Photomicrographs depicting cell morphology of Blastococcus aggregatus DAPI-stained B. aggregatus (A), DIC photomicrograph of B. aggregatus (B), and scanning electron micrographs of B. aggregatus (C and D).

B

A

budding cell

46

Figure 3.5: (Continued)

C

D dividing cell

detached bud

47

singly, in pairs, tetrads, and often formed aggregates (Figure 3.5). Cells ranged from

approximately 0.5 to 2.0 µm in diameter, which is very close to the diameter range for the genus

Blastococcus reported in Urzì et al. (2004) of 0.5-1.7 µm. Under the conditions used, bud

formation was observed, motile spores were produced, elongated cells formed, and germ tubes

and long filaments were produced. Urzì et al. (2004) describe that strains BC412 and BC521

also bud, form elongated cells, and produce germ tubes and long filaments. Blastococcus

aggregatus was first discovered by Ahrens and Moll (1970) in a sample recovered from the

Baltic Sea. Members of this genus have mostly been isolated from arid environments.

The increase in the numbers of culturable bacteria among sites did not always correlate to

an increase in diversity. The sample site AT04-166, which had the highest CFU value and from

which the highest number of isolates could be recovered, shows more diversity than any of the

other sites, but about 50% of the culturable heterotrophic bacteria recovered from AT04-166 are

members of the genus Blastococcus. Sites with higher CFU values did not have more isolates

identified, as isolates were recovered based on the different colony morphologies. If a site had

elevated CFU values, but all colonies were identical in morphology, then fewer colonies were

selected for isolation and identification.

Surface samples studied in the Yungay region showed overall low numbers of

recoverable prokaryotes, yet patchiness in value for CFUs/g of soil was observed. Low diversity

among the culturable heterotrophic bacteria was detected. Also, the abundance of recoverable

cells occurred erratically throughout the surface samples. These observations give new insights

to what could have occurred or may currently take place in Martian soils, thus Mars’ surface

explorations should sample a vast area at as many possible sites before any conclusions are made

regarding the microbiology past or present of the surface layers of soils on Mars.

48

CHAPTER 4 HETEROTROPHIC BACTERIA BENEATH THE SURFACE

NASA intends to send several missions to Mars in the upcoming years. At least two of

these missions will focus on subsurface Martian soils; hence, it is also important to explore the

subsurface soils of environments analogous to Mars. For this reason, the subsurface soils in the

hyper-arid core region, the Yungay region, of the Atacama Desert were investigated. Four soil

pits were excavated with depths of 40 cm to 90 cm to examine the subsurface microbial

communities. Three of these soil pits were excavated in the hyper-arid region of the Atacama

Desert. An additional soil pit was constructed in a more southern region of the Atacama Desert,

known as Altamira, as a comparison with the pits in the Yungay region. Samples were collected

at the surface and at 10 cm intervals from the surface. All samples were examined using the

previously described culture-dependent techniques. The identity of pure-culture isolates,

selected on the basis of their availability or novel colony morphology, was determined by 16S

rRNA gene sequence determination.

RESULTS

Colony Forming Units

Three soil pits (AT04-150, AT04-152, and AT04-153) were excavated in the Yungay

region of the Atacama Desert. Soil pit AT04-150 was located at 69º W 51.847′ and 24º S 3.672′

to a total depth of 90 cm. Soil pit AT04-152 was located at 69º W 51.858′ and 24º S 3.745′ to a

total depth of 80 cm. The soil pit designated AT04-153 at 69º W 51.423′ and 24º S 3.585′ to a

total depth of 80 cm. The fourth soil pit, which was constructed at a southern site with sparse

vegetation, was constructed at 70º W 11.758′ and 25º S 45.543′ to a depth of 40 cm.

49

The hyper-arid region subsurface CFU/g data are shown in Table 4.1 and Figure 4.1. The

CFU/g values for all media range from 0 to 8.07 x 103. Of the 28 layers from the three hyper-

arid pits, 27 showed no CFUs/g on at least one of the media used for detection of culturable

bacteria. There were seven layers that showed no CFUs/g on all five media (Table 4.1).

Twenty-six layers from the three Yungay pits had CFU/g values for all five culture media of < 1

x 103/g of soil and none of the 28 layers had CFU/g values of > 1 x 104. The largest number of

layers showing no detectable CFU/g were those plated on 1/100 PCA (25 layers) while the

lowest number of layers showing no detectable CFU/g were those plated on MA (11 samples).

Most CFU values were below the detection limit of the dilution plating method, but two high

values were observed (Table 4.1). The soil pit AT04-150 had 8 x 103 CFUs/g occurring at a

depth of 40 cm, while the soil pit AT04-152 has a slightly higher culturable population at 4 x 103

CFU/g at 10 cm below the surface; both of these were observed on MA culture medium (Table

4.1).

The CFUs/g of subsurface layers in the southern pit AT04-159, range from 6.4 x 103 to

1.86 x 106 for all media (Table 4.1 and Figure 4.1). Of the five layers of pit AT04-159, only the

surface layer displayed < 1 x 104 CFU/g on the culture medium PCA. The depths of 30 cm and

40 cm have CFU/g values of > 1 x 106 per gram of soil on at least one of the five culture media

used for detecting culturable bacteria. On MA culture medium, two layers (surface and 10 cm)

show CFU/g of soil > 1 x 104 and < 1 x 105, one layer (20 cm) shows CFU/g of soil > 1 x 105 and

< 1 x 106, and two layers (30 cm and 40 cm) show CFU/g of soil > 1 x 106 and < 1 x 107. On NA

culture medium, three layers (surface, 10 cm, and 20 cm) show CFU/g of soil > 1 x 104 and < 1 x

105, one layer (30 cm) shows CFU/g of soil > 1 x 105 and < 1 x 106, and one layer (40 cm) shows

CFU/g of soil > 1 x 106 and < 1 x 107. On PCA culture medium, one layer (surface) shows

50

Table 4.1: CFUs/g of soil for all four soil pits sampled on five media * average of duplicate pH

Media Soil Pit Depth (cm) pH PCA 1/10 PCA 1/100 PCA MA NA

AT04-150 0 7.25 0 0 0 0 0 10 7.76* 0 0 0 22 44 20 7.71 0 0 0 89 0 30 7.84 0 0 0 1.78 x 102 0 40 7.91 44 0 0 8.07 x 103 0 50 7.96 0 0 0 1.56 x 102 0 60 8.02 0 0 0 0 0 70 7.95* 1.56 x 102 0 0 0 0 80 8.08 89 0 0 0 0 90 8.20 0 0 0 0 0

AT04-152 0 7.16 22 0 0 0 22 10 7.80 1.18 x 103 4.44 x 102 6.00 x 102 3.96 x 103 4.22 x 102 20 7.86* 0 0 22 1.78 x 102 22 30 7.67 22 0 0 3.78 x 102 0 40 8.01 0 1.11 x 102 0 67 0 50 7.93 0 0 0 0 0 60 8.03 0 0 0 44 0 70 7.87* 0 0 0 22 0 80 7.77* 0 0 0 0 0

AT04-153 0 7.52 0 3.11 x 102 1.11 x 102 5.11 x 102 67 10 7.94* 0 0 0 44 0 20 7.88 1.56 x 102 0 0 22 0 30 7.79* 0 0 0 67 0 40 8.27* 0 0 0 0 0 50 8.09 22 0 0 0 0 60 8.14 0 0 0 0 0 70 8.02 0 22 0 67 22 80 7.86* 22 22 0 22 0

AT04-159 0 7.99* 6.40 x 103 5.51 x 104 1.08 x 105 6.80 x 104 4.09 x 104 10 7.60 6.47 x 104 1.04 x 105 1.50 x 105 8.95 x 104 8.98 x 104 20 7.54 9.96 x 104 1.53 x 105 1.16 x 105 4.33 x 105 9.96 x 104 30 7.70 4.87 x 105 1.53 x 106 5.56 x 105 1.60 x 106 3.91 x 105 40 7.85* 1.09 x 106 1.53 x 106 1.80 x 106 1.54 x 106 1.29 x 106

51

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

CFUs/gram of soil

0 10 20 30 40 50 60 70 80 90

Depth below surface (cm)

Soil Pit AT04-150 CFUs/g

PCA1/10 PCA1/100 PCAMANA

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

CFUs/gram of soil

0 10 20 30 40 50 60 70 80




1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

CFUs/gram of soil

0 10 20 30 40 50 60 70 80




1.00E+001.00E+011.00E+02

1.00E+031.00E+041.00E+051.00E+061.00E+07

CFUs/gram of soil

0 10 20 30 40


Soil Pit AT04-159 (Altamira Soil Pit) CFUs/g


Figure 4.1: CFUs/g of soil for layers within each soil pit on five media

52

CFUS/g of soil > 1 x103 and < 1 x 104, two layers (10 cm and 20 cm) show CFU/g of soil > 1 x

104 and < 1 x 105, one layer (30 cm) shows CFU/g of soil > 1 x 105 and < 1 x 106, and one layer

(40 cm) shows CFU/g of soil > 1 x 106 and < 1 x 107. On 1/10 PCA culture medium, one layer

(surface) shows CFU/g of soil > 1 x 104 and < 1 x 105, three layers (10 cm, 20 cm, and 30 cm)

show CFU/g of soil > 1 x 105 and < 1 x 106, and one layer (40 cm) shows CFU/g of soil > 1 x 106

and < 1 x 107. On 1/100 PCA culture medium, four layers (surface, 10 cm, 20 cm, and 30 cm)

show CFU/g of soil > 1 x 105 and < 1 x 106 and one layer (40 cm) shows CFU/g of soil > 1 x 106

and < 1 x 107. This outlined data is also exhibited in Table 4.2.

Diversity of Culturable Heterotrophic Bacteria Isolated from the Soil Pits

Isolates were selected based on the criterion of their availability and colony morphology

uniqueness. Isolates were identified using the 16S rRNA gene sequence. Of the three Yungay

pits, 3 Firmicutes, 3 Proteobacteria, and 25 Actinobacteria were recovered from AT04-150; 2

Firmicutes, 4 Proteobacteria, and 98 Actinobacteria were recovered from AT04-152; and 1

Firmicutes, 1 Proteobacteria, and 27 Actinobacteria were recovered from AT04-153 (Figures 4.2,

4.3, and 4.4). Based on these data, there was not variation in the diversity among individual

layers. The most commonly observed taxa recovered from the Yungay pits belonged to the

genera Actinospora, Amycolatopsis, Arthrobacter, Bacillus, Blastococcus, Brevibacillus,

Cellulomonas, Chelatococcus, Curtobacterium, Frankia, Geodermatophilus, Micrococcus,

Mycobacterium, Nocardioides, Pseudomonas, Pseudonocardia, Sphingomonas, and

Streptomyces. Even in the two layers that have elevated CFUs, more of the same kinds of

organisms are found.

The most dominant cultured organism from the hyper-arid soil pit isolates based on its

closest relative is Pseudonocardia saturnea. P. saturnea is the closest relative of isolates

53

Table 4.2: Values of CFU/g recovered on various culture media for the AT04-159 southern soil pit

Culture Media CFU/g of soil / number for AT04-159 layers MA NA PCA 1/10 PCA 1/100 PCA

<104 0 0 1 0 0 >104 and <105 2 3 2 1 0 >105 and <106 1 1 1 3 4

>106 2 1 1 1 1

54

2

1 2 1

3

7 2

8

2

2 1

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Percentage

90 cm80 cm70 cm60 cm50 cm40 cm30 cm20 cm10 cm

surface

Depth

Soil Pit AT04-150 - Diversity at the Phlya Level

ActinobacteriaFirmicutesProteobacteria

2

1 2 1

3

6 2 1

2 2 1 1 1

21 1 1

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Percentage


surface

Depth

Soil Pit AT04-150 - Diversity at the Closest Relative Level

Blastococcus sp.Bacillus sp.Pseudonocardia saturneaChelatococcus asaccharovoransNocardioides sp.Mycobacterium sp.Nostocoida aromativoraGeodermatophilus sp.Micrococcus sedentariusStaphylococcus sp.Methylobacterium mesophilicum Curtobacterium flaccumfaciensMicrococcus luteus

4 2

8 13

1 1

4 3 1

4

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Percentage


surface

Depth

Soil Pit AT04-150B Enrichment - Diversity at the Phlya Level


1 2 1 2

6 1 1

31

2 1 1 32 1 1

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Percentage


surface

Depth

Soil Pit AT04-150B Enrichment - Diversity at the Closest Relative Level

Blastococcus sp.Pseudonocardia saturneaStreptomyces sp.Brevibacillus agriHerbaspirillum seropedicaeStreptomyces californicus

Figure 4.2: Diversity at the phyla and closest relative levels in soil pit AT04-150 and AT04-150B enrichment cultures

55

1

2

2 4

4 1

8

80

1 1

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Percentage

80 cm

70 cm

60 cm50 cm

40 cm

30 cm

20 cm

10 cmsurface

Depth



1

1 1

3 1 1 1

4 1

5 3

2 12 12 3 1

1

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Percentage

80 cm

70 cm

60 cm

50 cm

40 cm

30 cm

20 cm

10 cm

surface

Depth

Soil Pit AT04-152 - Diversity at the Closest Relative LevelBlastococcus sp.Pseudonocardia saturneaNocardioides sp.Mycobacterium sp.Streptomyces sp.Pseudonocardia petroleophilaStaphylococcus epidermidis Actinobispora xinjiangensisSphingomonas sp.Amycolatopsis sp.Micrococcus sp.Pseudomonas stutzeri

1

2

1

2 1

5

6 3

11 1

7

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Percentage

80 cm

70 cm

60 cm

50 cm

40 cm

30 cm

20 cm

10 cm

surface

Depth


ActinobacteriaFirmicutes

1

1 1

1

1 1 1

3 1

2 2 1 1 1

3 4 1 1

5 2

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Percentage

80 cm70 cm60 cm50 cm40 cm30 cm20 cm10 cm

surface

Depth


Blastococcus sp.Pseudonocardia saturneaStreptomyces sp.Brevibacillus agriStreptomyces californicusBlastococcus aggregatusBacillus cereusAmycolatopsis sp.Streptomyces neyagawaensis


56

3

4

1

4 1

1 1

14

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Percentage

80 cm

70 cm

60 cm

50 cm

40 cm

30 cm

20 cm

10 cm

surface

Depth



2 1

1 1 1 1

1

1 1 1

1

12 2

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Percentage

80 cm

70 cm

60 cm50 cm

40 cm

30 cm

20 cm

10 cmsurface

Depth

Soil Pit AT04-153 - Diversity at the Closest Relative Level

Blastococcus sp.Curtobacterium flaccumfaciensBlastococcus aggregatusBrevibacillus agriGeodermatophilus obscurusMicrococcus luteusCellulomonas cartaeFrankia sp.

7

11

6

1

7

6

3

4

9 6

0% 20% 40% 60% 80% 100%

Percentage

80 cm

70 cm60 cm

50 cm40 cm

30 cm20 cm

10 cmsurface

Depth


ActinobacteriaFirmicutes

7

6 2 3

5

1

4 1 2

5 1

2

2 2

8 1 5 1

0% 20% 40% 60% 80% 100%

Percentage

80 cm70 cm

60 cm

50 cm40 cm

30 cm20 cm

10 cm

surface

Depth


Blastococcus sp.Pseudonocardia saturneaStreptomyces sp.Brevibacillus agriStreptomyces californicusBlastococcus aggregatusBacillus cereusMicrococcus luteusGeodermatophilus obscurus


57

recovered from soil pit AT04-150 in layers 10 cm (1 isolate out of 3), 20 cm (2 isolates out of 2),

30 cm (2 isolates out of 7), 40 cm (6 isolates out of 9), and 50 cm (3 isolates out of 3); this

organism was also recovered from soil pit AT04-152 in sample layers 10 cm (12 isolates out of

30), 20 cm (5 isolates out of 8), and 30 cm (4 isolates out of 5). However none of the isolates

from pit AT04-153 were identified as a closest relative to P. saturnea. The other dominant

organisms isolated from soil pit AT04-152 have closest relatives in species of the genus

Streptomyces.

Isolates whose closest relatives are members of the genus Streptomyces were recovered in

pit AT04-152 in sample layers 10 cm (12 isolates recovered out of 30) and 20 cm (3 isolates

recovered out of 8); as with P. saturnea, members of the genus Streptomyces were not detected

in the dilution plating method in soil pit AT04-153. In contrast, the isolates from soil pit AT04-

153 are dominated by organisms most closely related to species of the genus Blastococcus,

similar to what was found in the surface samples. Members of the genus Blastococcus were the

closest relatives of isolates recovered from soil pit AT04-153 on the surface layer (12 isolates out

of 14), 30 cm below the surface (1 isolate out of 1), 70 cm below the surface (2 isolates out of 4),

and 80 cm below the surface (2 isolates out of 3).

Isolates were selected and identified from the southern soil pit at Altamira, AT04-159, on

the basis of their availability and novel morphologies. All isolates were identified based on 16S

rRNA gene sequence determination. A few morphology types (approximately six) undeniably

dominated the solid culture media (both nutrient-rich and nutrient-dilute) (Figure 4.5).

Increased diversity in AT04-159 was observed when compared with isolate diversity from the

three core arid region pits (Figure 4.5). In the southern-most soil pit, about 42 different bacterial

taxa were recovered. The most commonly observed isolates recovered from the Altamira soil pit

58

8 1 5

12 1

22 9 6

16 6 1

76 11 3

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Percentage

40 cm

30 cm

20 cm

10 cm

surface

Depth

Soil Pit AT04-159 (Altimira Soil Pit) - Diversity at the Phyla Level

ActinobacteriaFirmicutesProteobacteriaBacteroidetes

8 1

9 1 1

5 2 6 3 1 1 1 1 2 1 1 1 1 1 1 1 2 1 1

5 1 3 1 1 1 1 1 2 1 1 1

10 1 4 7 21 18 10 3 21 3 8 21 3 111

0% 20% 40% 60% 80% 100%

Percentage

40 cm

30 cm

20 cm

10 cm

surface

Depth

Soil Pit AT04-159 (Altimira Soil Pit) - Diversity at the Closest Relative LevelBlastococcus sp. Bacillus sp.Chelatococcus asaccharovorans Nocardioides sp.Geodermatophilus sp. Arthrobacter sp.Streptomyces sp. Pseudonocardia petroleophilaActinobispora xinjiangensis Sphingomonas sp.Blastococcus aggregatus Geodermatophilus obscurusHymenobacter sp Actinobispora alaniniphilaArthrobacter agilis Cellulomonas cellasea Modestobacter sp. Modestobacter multiseptatus Paenibacillus sp. Cellulosimicrobium funkei Saccharothrix sp. Cellulomonas septicaCellulomonas uda Hongia sp. Streptomyces albidochromogenes Actinobispora yunnanensisBacillus niacini Subtercola pratensisPseudonocardia zijingensis Mesorhizobium sp.Streptomyces plemorphus Streptomyces carpinensisPromicromonospora aerolata Mycobacterium celatum Cellulomonas sp. Actinomadura spBacillus litoralis Rhizobium spOxalophagus oxalicus Streptomyces rutgersensis Bacillus krulwichiae

Figure 4.5: Diversity at the phyla and closest relative levels in soil pit AT04-159 (the Altamira soil pit)

59

have closest relatives in the genera Arthrobacter, Bacillus, Blastococcus, Geodermatophilus,

Modestobacter, and Streptomyces. Although the CFUs/g increased with depth in the Altamira

pit, the diversity decreased with depth. A greater number of the Altamira soil pit isolates were

recovered from layers at or near surface because most of the colonies growing on the dilution

plates from these depths were morphologically unique; the bacterial colonies from deeper layers

were more numerous, but less diverse both in their morphology and phylogenetic diversity.

Enrichment cultures were set up for the three pits constructed within the Yungay region

of the Atacama Desert. Isolates from the enrichment cultures were recovered just as the dilution

plate isolates were selected on the basis of their availability and morphological uniqueness and

identified based on their 16S rRNA gene sequence. Of the enrichment isolates recovered from

soil pit AT04-150 and sequenced, 6 belonged to the Firmicutes, 2 to the Proteobacteria, and 24 to

the Actinobacteria; from pit AT04-152, 5 isolates belonged to the Firmicutes and 35 to the

Actinobacteria; from pit AT04-153, 6 isolates belonged to the Firmicutes, and 54 Actinobacteria

strains were recovered (Figures 4.2, 4.3, and 4.4 (the third chart in each figure)). The enrichment

approach did not result in the recovery of addition bacterial diversity from the soil pits of hyper-

arid region and the closest relatives to these isolates were species of the genera Amycolatopsis,

Bacillus, Blastococcus, Brevibacillus, Geodermatophilus, Herbaspirillum, Micrococcus,

Pseudonocardia, and Streptomyces (Figures 4.2, 4.3, and 4.4 (the fourth chart in each figure)).

The most dominant isolate among the Yungay enrichment isolates has P. saturnea as their

closest relative. P. saturnea was the closest relative of enrichment isolates recovered in pit

AT04-150 in the surface layer (1 isolate out of 4), the layer at 10 cm below surface (1 isolate out

of 7), 20 cm below the surface (1 isolate out of 1), 30 cm below the surface (3 isolates out of 3),

40 cm below the surface (6 isolates out of 8), and 50 cm below the surface (2 isolates out of 6).

60

This organism was also found at AT04-152 on the surface (2 isolates out of 7), 10 cm below the

surface (3 isolates out of 9), 20 cm below the surface (2 isolates out of 7), 30 cm below the

surface (3 isolates out of 4), 70 cm below the surface (1 isolate out of 2), and 80 cm below the

surface (1 isolate out of 1). From pit AT04-153, only isolates from the layer at 40 cm below the

surface (1 isolate out of 7) and 70 cm below the surface (2 isolates out of 11) were identified as

closest relatives to P. saturnea.

Other dominant organisms were detected in the Yungay pits enrichment cultures were

those whose closest relatives were related to the genus Streptomyces and these were recovered

from enrichment cultures of soil pit AT04-150. From the surface layer (1 isolate out of 4), 10 cm

below the surface (1 isolate out of 7), 40 cm below the surface (1 isolate out of 8), and 50 cm

below the surface (1 isolates out of 6). Enrichment isolates identified by partial 16S rRNA gene

sequences as Streptomyces spp. were recovered from soil pit AT04-152 in the layers 10 cm

below the surface (5 isolates out of 9), 20 cm below the surface (1 isolate out of 7), and 30 cm

below the surface (1 isolate out of 4). Also, enrichment culture isolates from soil pit AT04-153

were found to be similar to members of the genus Streptomyces at 10 cm below the surface (2

isolates out of 4) and at 30 cm below the surface (1 isolate out of 6).

Another dominant species among the Yungay soil pit enrichment isolates are

Blastococcus spp. This organism is much more dominant in pit AT04-153; 43 out of the 58

enrichment isolates recovered from this pit were identified as close relatives of Blastococcus spp.

Enrichment isolates with relatives most similar to those of the genus Blastococcus were

recovered from soil pit AT04-150 in the surface layer (2 isolates out of 4), 10 cm below surface

(2 isolates out of 7), and 50 cm below the surface (1 isolate out of 6); in pit AT04-152 the

surface layer (5 isolates out of 7), 20 cm below the surface (2 isolates out of 7), 40 cm below the

61

surface (1 isolate out of 3), and 70 cm below the surface (1 isolate out of 2). Also, most of the

enrichment isolates recovered from soil pit AT04-153 were identified as members of the genus

Blastococcus. Members of this genus were detected in every layer of soil pit AT04-153; surface

(8 isolates out of 15), 10 cm below the surface (2 isolates out of 4), 20 cm below the surface (2

isolates out of 2), 30 cm below the surface (5 isolates out of 6), 40 cm below the surface (4

isolates out of 7), 50 cm below the surface (1 isolate out of 1), 60 cm below the surface (5

isolates out of 5), 70 cm below the surface (9 isolates out of 11), and 80 cm below the surface (7

isolates out of 7).

The genus Brevibacillus is also uniformly distributed throughout the enrichment isolates

recovered from layers within all three of the hyper-arid region soil pits. In soil pit AT04-150,

isolates whose closest relatives were Brevibacillus spp. were observed at 10 cm below the

surface (3 isolates out of 7) and 50 cm below the surface (2 isolates out of 6); in soil pit AT04-

152, isolates whose closest relatives were Brevibacillus spp. were recovered at 10 cm below the

surface (1 isolate out of 9) and 20 cm below the surface (1 isolate out of 7); also in soil pit AT04-

153 at the surface layer (1 isolate out of 15).

DISCUSSION

The three Yungay pits show very low CFU values overall throughout the surface and

subsurface layers sampled and investigated. These findings further contribute to the idea that the

hyper-arid region of the Atacama Desert is an analog of Mars both on the surface and in the

subsurface. The majority of the subsurface layers sampled and investigated show zero CFUs on

all culture media utilized for this study. Even in the Atacama Desert at sites outside the core

hyper-arid region, such that at Altamira, the CFU/g of soil is as high 1.08 x 105 at surface layers

and 1.80 x 106 at subsurface depths on at least one of the five culture media employed for this

62

study. The two CFU spikes observed in pits AT04-150 and AT04-152 (core hyper-arid region

pits) are not comparable to the CFUs/g from the Altamira soil pit samples, nevertheless these

elevated CFUs/g are indicative of the need for multiple site surface and subsurface sampling on

Mars where similar microbial patchiness with depth may exist.

The fact that the largest number of colonies from the Yungay soil pit samples was

observed on the Marine Agar (MA) culture media suggests that recoverable heterotrophic

bacteria existing in hyper-arid Atacama soils may be of marine origin and brought there by

deposition from winds from the coastal region to the west.

The CFUs/g found for Altamira pit samples (especially near the surface) in this study

coincide with the Navarro-González et al. (2003) reports of surface CFU levels at the latitude 25º

S 45′ to be between 104 and 105 CFUs/g. The higher CFUs/g at this site are to be expected when

comparing this region to Yungay CFUs/g due to the increase of culturable bacterial numbers

along the north-to-south precipitation gradient (Navarro-González et al. 2003). The Altamira

site, a more southern site, accumulates more moisture throughout the year than the hyper-arid

Yungay region, thus the area is more accommodating to microbial life.

The diversity of culturable heterotrophic bacteria isolated from each of the three pits

within the hyper-arid core area is much less than that recovered in the Altamira pit. The isolates

from three Yungay pits fall within three phyla, whereas the Altamira pit was determined to

contain representatives of at least four phyla. The most prevalent of the isolates belong to the

phylum Actinobacteria, which is the phylum that such organisms as Arthrobacter spp.,

Blastococcus spp., Cellulomonas spp., Frankia spp., Geodermatophilus spp., Hymenobacter

spp., Kocuria spp., Micrococcus spp., Modestobacter spp., Nocardioides spp., Pseudonocardia

spp., and Streptomyces spp. belong. The isolates falling into the phylum Firmicutes were

63

Bacillus spp., Brevibacillus spp., and Paenibacillus spp. Finally, the isolates belonging to the

phylum Proteobacteria were Chelatococcus spp., Herbaspirillum spp., Pseudomonas spp., and

Sphingomonas spp.

There is not a great deal of diversity observed between individual layers among the three

hyper-arid region pits. Even in the two pits that exhibit elevated CFUs in two layers, we find

more of the same types of bacteria. This is consistent with the patchiness in the surface

diversity, and may indicate spatial patchiness vertically as well as horizontally. Isolates

recovered from the surface of each pit are in many cases identical to the bacteria found at lower

depths of the soil pit. When comparing isolates recovered from the surface samples (Chapter 3)

and Yungay pit samples, it is apparent that the diversity is slightly greater in the surface samples.

Nevertheless, many overlapping organisms were found in surface samples and in samples taken

at some depths. Because the diversity of the species found throughout the core region samples is

very low, this overlap relates surface soils to subsurface soils and may signify similar resource

abundance at short spatial range (Rietkerk et al. 2004).

An interesting finding revealed from the comparison of the Yungay surface and Yungay

pit samples was that the large number of Blastococcus sp. cultured from surface samples were

not found in the subsurface soil pit samples, especially at sites AT04-150 and AT04-152. Also,

Pseudonocardia spp. were recovered only from the soil pit samples and was not observed among

the organisms recovered from surface sites. Of the three core soil pits, AT04-150 and AT04-152

were constructed on the top of a hill with an altitude of about 1070 m (nearly 50 m above the

valley), whereas AT04-153 was dug in the valley adjacent to the hill. Most of the 33 surface

sites showing patchiness were also sampled in a valley. As shown in the surface study (Chapter

3) and in the soil pit located in the valley (AT04-153), the culturable bacteria that dominate were

64

members of the genus Blastococcus. In the two soil pits constructed on the hill (AT04-150 and

AT04-152), less than 2% of the culturable bacteria isolated were members of the genus

Blastococcus, instead the dominating genera were Pseudonocardia and Streptomyces. Of the

total culturable heterotrophic bacteria recovered from the soil pit AT04-150, about 30% were

members of the genus Pseudonocardia, and from soil pit AT04-152 about 20% were members of

the genus Pseudonocardia and 25% were of the genus Streptomyces. This suggests that the

difference in the bacterial populations may be attributed to the differences between the types of

soils found in low lying valleys and those found at elevated sites.

As anticipated, the soil pit AT04-159 in the less arid, more southern region has CFUs up

to four magnitudes greater than those in the core arid region. As formerly discussed, the

Altamira soil pit was found to contain about 42 different bacterial taxa, which supports the

theory that further south, outside of the hyper-arid region of the Atacama Desert, moisture levels

increase in soils, therefore they have a greater diversity of culturable heterotrophic bacteria and

much higher CFUs especially in the subsurface layers (Navarro-González 2003). While the CFU

values increase at greater depths in the Altamira pit, the diversity is reduced with depth. At

layers near the surface, fewer bacterial colonies were observed using the dilution plating method

than at deeper subsurface layers, yet a larger number of the Altamira pit isolates were recovered

from layers at nearer the surface because most colonies growing on the dilution plates at these

depths were morphologically different. Although bacterial colonies from deeper subsurface

layers within the Altamira pit were more numerous, they were less diverse in morphological

characteristics; therefore, less diversity of isolates were recovered. A detail apparent throughout

this study is that samples having elevated CFUs do not necessarily contain more recovered

isolates if indeed the high numbers of colonies all appear to be similar in colony morphology.

65

Enrichment cultures were prepared for each of the pits sampled in the core arid region.

Although they cannot be used for quantification purposes, they are useful in uncovering

organisms that grow rapidly in favorable nutrient conditions. A fog event, which is a rare

moisture occurrence in the hyper-arid region, could be one source of a natural enrichment

environment in which organisms generally found at low numbers can rapidly flourish.

For the three soil pits sampled in the Yungay region, the diversity in the enrichment

isolates is similar to that of the dilution plating isolates since both have low diversity of

organisms, and the organisms recovered during the enrichment are generally the same as the

isolates from the dilution plating experiment. The enrichment isolates from soil pits AT04-150

and AT04-152 like the dilution plating isolates, are dominated by the P. saturnea like strains.

These P. saturnea related strains are important to this study because they have been found to be

abundant in the Yungay soil pit samples (both from dilution plating and enrichment methods),

but were never recovered from surface samples. As previously mentioned, this may be attributed

to the fact that the soil pits from which this particular organism was recovered were located on

the elevated area in Yungay.

Strains of the genera Brevibacillus and Streptomyces were not only consistently identified

from the enrichment isolates that were recovered from the three Yungay pits, but they were also

identified from isolates recovered throughout this study within the hyper-arid region (both

surface and subsurface dilution plate isolates). These taxa along with other overlapping

microorganisms (mentioned earlier in this chapter), belong to the culturable heterotrophic

bacterial community surviving in the core arid region of the Atacama Desert.

Blastococcus spp. have also been found to be abundant among the enrichment isolates,

especially those from soil pit AT04-153. This is to be expected since the majority of the surface

66

study revealed the genus Blastococcus to be the principal recoverable microorganism inhabiting

the hyper-arid region. The fact that more enrichment isolates from soil pit AT04-153 (the

Yungay valley pit) were identified as closest relatives of Blastococcus sp. than enrichment

isolates from the two pits constructed at elevated area also implies that the difference in the

bacterial inhabitants may be accredited to distinctions between the soils found in low lying

valleys and those found at elevated sites. Since bacteria obtain nutrients from the environment in

which they inhabit, bacterial populations in the core arid region may be distributed directly as a

result of the availability of resources throughout the soils.

The studies described in this and the previous chapter, highlight the need for multiple site

selection for either surface or subsurface sampling of Martian soils where similar patterns of

patchiness as uncovered in the Atacama Desert might exist.

67

CHAPTER 5

DIRECT CELL COUNTS USING FLUORESCENCE MICROSCOPY

DAPI, 4'-6-Diamidino-2-phenylindole, is a fluorochrome that binds with DNA to form a

fluorescent complex that will fluoresce blue under ultraviolet exposure and is popularly used in

direct count protocols to quantify both viable and nonviable bacterial cells in environmental

samples. In order to investigate the actual bacterial cell concentrations in Yungay soil samples, a

direct count method was attempted using DAPI (4'-6-Diamidino-2-phenylindole) fluorescence

microscopy. However, this method, which is described in chapter 2, proved to be problematic in

conjunction with the Atacama Desert soils due to low bacterial cell numbers and non-specific

binding of DAPI with soil particles from the Yungay region. The complications that were

recognized by applying the DAPI direct count method for Atacama Desert hyper-arid soils lead

to an inquiry to determine the limit of detection of these particular soils in conjunction with the

DAPI direct count protocol applied.

RESULTS

DAPI Direct Count Detection Limit

Known cell concentrations (determined by dilution plating technique) of a suspension of

an Atacama Desert isolate whose closest relative was identified to be Blastococcus aggregatus

were added to AT04-170 soils (CFU value of zero as described in Chapter 3). This strain was

selected for the experiment given that the genus Blastococcus is the predominate organism found

in Yungay soils. The organism was added to the soil as a stock suspension and a 1/10 dilution of

the stock solution and allowed to dry overnight (another experiment discussed later allowed the

soil and cell suspension to dry for one month). The liquid extract from the AT04-170 soils along

with the added cells was stained with DAPI as described in Chapter 2 to determine the detection

68

limit for the DAPI method used in this study. The cell concentration of the Blastococcus strain

AT03-37-10 stock solution was determined to be 1.05 x 106 CFUs per mL. Figure 5.1 shows

three epifluorescence photomicrographs, two photomicrographs at varying bacterial cell

concentrations added to soil and dried overnight and one representing a control of DAPI stained

soil lacking additional cells, that represent the detection limit of the direct count method

performed on Yungay soils.

Twenty fields were counted and averaged from the filter with the stock cell suspension

added to soil and from the filter with 1/10 diluted stock suspension added to soil. Table 5.1 gives

the results of counted cells per field, averages, and known versus calculated cells per gram of soil

for both the stock and 1/10 diluted cell suspension. Each field counted has an area of 41,184

µm2 at the magnification used (400x), and since the filters that the cells were captured on have

an area of 490.625 mm2, there are 11,913 fields per filter.

The number of cells per field range from 20 to 101 for the filter containing the stock cell

suspension and AT04-170 soil, and the average cell number is 45.5 Blastococcus spp. strain

AT03-37-10 cells per field. Fields 3 and 16 have unusually high cell numbers (101 and 92) due

to cells aggregating amid clusters of soil particles; Figure 5.1 (A) demonstrates this arrangement

in the upper left-hand portion of the image. The average (45.5) was multiplied by 11,913.61 and

then again by 2 to account for the dilution within this procedure. The final cell concentration

was calculated to be 1.08 x 106 cells per gram (or cells per mL since 1 mL of cell suspension was

added to 1 gram of soil), which is almost exactly the known concentration added to the soil.

Therefore, it can be assumed that for a Yungay soil with a cell concentration above 1 x 106 cells

per gram the numbers of cells present can accurately be detected using the DAPI staining

69

procedure used in this study. Figure 5.1 (A) shows a photomicrograph of field 12 from the filter

with the stock suspension (1.05 x 106 cells per mL) added to AT04-170 soil.

A 1/10 dilution of the cell suspension (1.05 x 105 cells per mL) was prepared and handled

exactly as the stock solution. The number of cells per field range from 0 to 4. Fields 3, 8, 10,

and 11 zero fluorescing bacterial cells were observed, for fields 5, 9, 12, 14, and 17 one cell was

observed, for fields 1, 4, 6, 7, 13, 18, and 19 two cells were observed, for fields 2, 15, and 20

three cells were observed, and for field 16 four cells were observed. The average number of

cells observed from the 20 evenly distributed fields is 1.6. Calculations were performed in the

same manner as above and the final cell concentration was determined to be 3.81 x 104 cells per

gram of Yungay soil. Figure 5.1 (B) shows field 4 from the polycarbonate filter prepared with 1

x 105 cells per mL added to AT04-170 soil. The known cell concentration and direct count cell

concentration (3.81 x 104 cells per gram of soil) contradict one another. Therefore, the detection

limit of the DAPI staining method used on Yungay soils is concluded to exist at some point

between 1 x 105 and 1 x 106 cells per gram of soil.

Figure 5.1 (C) shows the control epifluorescence photomicrograph of the soil AT04-170

without the addition of a cultured cell suspension. This figure demonstrates the non-specific

binding of DAPI to the Atacama Desert soil (the green fluorescing particles).

The experiment above was repeated, but the soil was allowed to dry for one month after

the cells (stock cell suspension and 1/10 dilution) were added to the AT04-170 soils instead of

overnight in order to compare the appearance of added cells after an extended time period in

AT04-170 soil. The filters were viewed under a higher magnification (800x); therefore, the

calculations to convert average cells per field to total cells per gram of soil vary from the above

experiment. Twenty fields were counted and averaged. Table 5.1 gives the counted cell

70

Figure 5.1: Photomicrographs of AT04-170 spiked with cultured bacterial cells and dried overnight to determine DAPI direct count detection limit Stock + AT04-170 soil: truly detected (gold arrow shows aggregate cells where cells are distinguishable; red arrow shows cells difficult to tell apart since amongst fluorescing soil) (A), 1/10 dilution + soil: below detection limit (arrows show two bacterial cells) (B).

A

B

71

Figure 5.1: (Continued) AT04-170 soil stained with DAPI without Blastococcus strain AT03-37-10 cells added (C).

C

72

numbers, averages, and known versus calculated cells per gram of soil for the one month drying

experiment and the overnight drying experiment for both the stock and 1/10 diluted cell

suspension. Each field counted has an area of 10,296 µm2 at the magnification used (800x), and

since the filter that the cells were captured on has area of 490.625 mm2, there are 47,652 fields

per slide. The average cell number was multiplied by 47,652 and then again by 2 to account for

the dilution within this procedure.

Figure 5.2 shows two representative epifluorescence photomicrographs at varying

concentrations of the bacterial cell stock added to soil and dried for one month. As Figure 5.2

indicates, the cells appeared less bright blue than the cells that dried on AT04-170 soil overnight.

Also the cells somewhat lost the characteristic coccid shape that was apparent in Figure 5.1.

Regarding the one month drying experiment, the number of cells per field range from 3 to 23

and the average cell number on the stock cell suspension plus AT04-170 soil filter is 10.9 cells

per field. After the calculations mentioned in the previous paragraph were performed, the final

cell concentration was calculated to be 1.04 x 106 cells per gram of soil (or per mL since one mL

was added to one gram of soil), which is practically the known concentration that was added to

the soil just as the experiment where the cell suspension and soil were dried overnight.

Therefore, it can be still be assumed that a Yungay soil with a cell concentration above 1 x 106

cells per gram can accurately be detected using the DAPI staining procedure used in this study.

Figure 5.2 (A) shows a photomicrograph taken of field 1 from filter containing 1.05 x 106 cells

per mL added to one gram of AT04-170 soil and dried for one month. The number of cells

observed per field from the 1/10 dilution filter (1.05 x 105 cells per mL) range from zero to three.

For fields 2, 3, 8, 12, 13, 14, 15, 16, 17, and 19 zero fluorescing bacterial cells were observed,

for fields 5, 18, and 20 one cell was observed, and for fields 1, 4, 6, 7, 10, and 11 two cells were

73

Table 5.1: Direct count detection limit trials displaying cell numbers per field, average cells per field, and known versus calculated cells/g for stock and 1/10 diluted cell suspension after overnight and one month drying experiment

Number of Cells per Field in DAPI Detection Limit Experiment

Stock 400x (overnight)

1/10 dilution 400x (overnight)

Stock 800x (one month)

1/10 dilution 800x (one month)

Field Number

Field 1 34 2 12 2 Field 2 36 3 8 0 Field 3 101 0 9 0 Field 4 49 2 15 2 Field 5 25 1 10 1 Field 6 66 2 7 2 Field 7 26 2 5 2 Field 8 72 0 11 0 Field 9 40 1 9 3 Field 10 39 0 6 2 Field 11 36 0 14 2 Field 12 30 1 23 0 Field 13 42 2 8 0 Field 14 20 1 16 0 Field 15 28 3 3 0 Field 16 92 4 10 0 Field 17 24 1 5 0 Field 18 51 2 19 1 Field 19 29 2 13 0 Field 20 70 3 15 1 Average Cells/Field 45.5 1.6 10.9 0.9 Known Cells/Gram 1.05 x 106 1.05 x 105 1.05 x 106 1.05 x 105 Calculated Cells/Gram 1.08 x 106 3.81 x 104 1.04 x 106 8.58 x 104

74

Figure 5.2: Detection limit photomicrographs of AT04-170 spiked with cultured bacterial cells and dried for one month 1.05 x 106 cell/mL plus AT04-170 soil: accurately detected (arrows indicate counted fluorescing cells (A), 1.05 x 105 cell/mL plus soil: detection limit for this study (arrow indicates one counted fluorescing bacterial cell) (B).

A

B

75

observed. The average number of cells observed was 0.9; the average was multiplied by 47,652

and then again by 2 for a final cell concentration of 8.58 x 104 cells per gram of Yungay soil.

Figure 5.2 (B) shows a photomicrograph taken of field 5 from the filter containing 1 x 105 cells

per mL added to AT04-170 soil. The known cell concentration (1 x 105 cells per mL) and direct

count cell concentration (8.58 x 104 cells per gram of soil) disagree with one another just as the

experiment where the cell suspension and soil were dried overnight. Therefore, the detection

limit of the DAPI staining method used on Yungay soils can still be assumed to exist somewhere

between 1 x 105 and 1 x 106 cells per gram of soil.

DAPI Direct Cell Count Determination for AT04-166

On the basis the detection limit stated above, only the soil sample having the highest CFU

value of 7.4 x 105 per gram of soil on 1/10 PCA culture medium, AT04-166, was able to be

counted using the fluorescence direct count procedure described in Chapter 2. Extraction fluid

from five different amounts of soil (1.0 gram, 0.5 grams, 0.25 grams, 0.1 grams, and 0.05 grams)

was stained with DAPI and used to determine a soil amount that would achieve countable

bacteria under an epifluorescence microscope without serious interference from fluorescing soil

particles. The enumeration of total bacterial cells was accomplished by counting and averaging

bacterial cells in 20 countable fields evenly distributed over the area beneath the coverslip.

Figure 5.3 shows representative epifluorescence photomicrographs of the DAPI direct count

method performed on the extraction supernatants from all five amounts of AT04-166 soil.

Regarding the AT04-166 soil, the filter containing extraction fluids that account for 0.25

grams of soil attested to be the countable filter; the number of cells counted per field, the average

cell number per field, and calculated total cells per gram of soil is displayed in Table 5.2. The

number of cells per field for sample AT04-166 range from 17 to 51 and the average cell number

76

Figure 5.3: Photomicrographs from the fluorescence direct count method employing DAPI in surface site AT04-166 Photomicrograph represents 1.0 g of soil: overly concentrated with fluorescing cells and soil particles (A), represents 0.5 g of soil: still too concentrated with fluorescing cells and soil particles (B).

A

B

77

Figure 5.3: (Continued) Photomicrograph represents 0.25 g of soil: countable (arrows indicate all fluorescing cells counted in this field) (C), represents 0.1 g of soil: too few cells; not countable (arrows indicate all fluorescing cells) (D).

C

D

78

Figure 5.3: (Continued) Photomicrograph represents 0.05 g of soil: no fluorescing cells in this field (E).

E

79

per 20 evenly distributed fields on the countable filter is 34.2. Each field counted has an area of

10,296 µm2, and since the filter that the cells were captured on has an area of 490.625 mm2, there

are 47,652 fields per slide. The average cell count per field (34.2) was multiplied by 47,652

fields. Since the method used for this experiment accounts for 0.25 grams of soil, the value was

then multiplied by four to convert the value to cells per gram of soil. It was resolved that the

total number of bacterial cells (viable and nonviable) per gram of AT04-166 soil is 6.52 x 106.

The concluded direct count value is above the determined detection limit for the utilized method

and soils, thus the total cell count for AT04-166 can be assumed to be an accurate enumeration.

On the filter containing extraction fluids that account for 0.1 grams of AT04-166 soil less

than or equal to five cells in eight fields were observed, but the remaining fields contained zero

countable cells, therefore, was not selected as the soil concentration containing a countable total

bacterial cell number. Figure 5.3 (D) shows one of the eight fields (out of 20) on the filter

containing extracts from 0.1 grams of soil in which fluorescing bacterial cells could be observed;

for fields 9, 14, and 19 one fluorescing bacterial cell was observed, for fields 2, 8, and 11 two

cells were observed, for field 5 three cells were observed, and for field 17 (image shown in

Figure 5.3 (D)) five cells. Liquid extract from 1.0 and 0.5 grams of soil contained high

concentrations of fluorescing soil particles which prevented the ability to view any bacterial

cells. The filter prepared with 0.05 grams of soil showed zero fluorescing bacterial cells in all 20

evenly distributed fields.

With respect to the appearance of the cells in Figure 5.3, it was demonstrated in the one

month drying experiment that fluorescing cells became dimmer and lost characteristic bacterial

forms after extended time periods in the Yungay desert soil. This is also true for bacteria that

permanently inhabit environmental hyper-arid soils from within the Yungay region; thus, the

80

fluorescing bacterial cells viewed in AT04-166 soils were difficult to count. The fluorescing

cells were also difficult to differentiate from fluorescing soil debris, a problem encountered

throughout this experiment because DAPI non-specifically binds to Atacama Desert soil

particles.

Sample Sites Having Cell Totals below DAPI Direct Count Detection Limit

AT04-165, a sample having the second highest CFUs/g (8.20 x 104) on 1/10 PCA culture

medium, and AT04-170, a sample having a 0 CFUs/g on all five primary culture media, were

prepared and observed using the DAPI direct count method to determine if the remaining

Yungay samples are adequate candidates for DAPI direct count methods based on the detection

limit. These two surface samples were not able to be accurately quantified due to the constraints

of the determined detection limit per gram of Yungay region soil. Extraction supernatant from

five soil amounts (1.0 g, 0.5 g, 0.25 g, 0.1 g, and 0.05 g) was stained with DAPI from both

AT04-165 and AT04-170 soils and filtered onto a black polycarbonate filter to determine a soil

amount that would generate countable bacteria using epifluorescence microscopy without

significant obstruction from fluorescing soil particles. Figure 5.4 shows one representative

photomicrograph from each of the five AT04-165 soil sample amount trials. The filter prepared

using 0.5 g of soil is the only amount at which fluorescing cells can be viewed because the

extract from 1.0 g contains a high concentration of fluorescing soil particles which obstruct the

view of any bacterial cells, and the other three filters that were prepared with smaller amounts of

soil (0.25 g, 0.1 g, and 0.05 g) show a decrease in fluorescing soil, but zero fluorescing bacterial

cells are observed in all 20 fields. All 20 evenly distributed countable fields for sample AT04-

165 are presented in Table 5.2 as number of fluorescing cells/field, average cells per field, and

calculated total cells per gram of soil. The average cells per field on the filter prepared from 0.5

81

Figure 5.4: Photomicrographs of AT04-165 soils unable to be truly quantified by DAPI direct counts Photomicrograph represents 1.0 g of soil: overly concentrated with fluorescing soil particles to count (A), photomicrograph represents 0.5 g of soil (arrows indicate counted fluorescing cells) (B).

B

A

82

Figure 5.4: (Continued) Photomicrograph represents 0.25 g of soil: no fluorescing cells (C), represents 0.1 g of soil: no fluorescing cells (D).

C

D

83

Figure 5.4: (Continued) Photomicrograph represents 0.05 g of soil: no fluorescing cells in this field (E).

E

84

Table 5.2: Direct count trials displaying cell numbers per field, average cells per field, and calculated cells/g for each tested sample site

Number of Cells per Field in Soil Sample AT04-165 (800x) AT04-166 (800x) AT04-170 (800x) Field Number

Field 1 2 44 0 Field 2 4 23 0 Field 3 0 17 0 Field 4 2 27 0 Field 5 0 36 0 Field 6 0 40 0 Field 7 1 47 0 Field 8 1 37 0 Field 9 0 34 0 Field 10 2 28 0 Field 11 0 21 0 Field 12 0 36 0 Field 13 6 44 0 Field 14 5 41 0 Field 15 0 30 0 Field 16 0 29 0 Field 17 0 26 0 Field 18 1 33 0 Field 19 3 51 0 Field 20 0 40 0 Average Cells/Field 1.35 34.2 0 Calculated Cells/Gram 1.29 x 105 6.52 x 106 0 < or > Detection Limit < detection limit > detection limit < detection limit

85

grams of AT04-165 soil was found to be 1.35. The representative photomicrograph labeled B in

Figure 5.4 portrays field 14 (5 fluorescing cells). For sample AT04-165, fields 3, 5, 6, 9, 11, 12,

15, 16, 17, and 20 contain zero fluorescing bacterial cells and fields 7, 8, and 18 contain one

fluorescing cell. Also, fields 1, 4, and 10 have two fluorescing bacterial cells, field 19 has three

cells, field 2 has four cells, and field 13 has six cells. The average (1.35) was multiplied by

47,652 fields per filter and then again by two to convert the value to cells per gram of soil. The

total number of bacterial cells (viable and nonviable) per gram of AT04-165 soil was found to be

1.29 x 105, but because this number falls below the determined detection limit, it cannot be

presumed an accurate total bacterial cell count.

Sample AT04-170 was prepared in the same manner as AT04-165 and AT04-166. Figure

5.5 shows one representative photomicrograph from each of the five soil amount trials from

sample AT04-170. All 20 evenly dispersed fields for all five soil amounts from sample AT04-

170 have zero fluorescing microbial cells; thus, the average number of fluorescing cells per field

is zero. Because zero cells were detected at all attempted soil concentrations, it is understood

that sample AT04-170 contains no viable or non-viable bacterial cells.

DISCUSSION

The widely used polycarbonate filters contain thousands of fields (depending on the

magnification) capable of quantification purposes. Therefore, evenly distributed sample fields

are counted and averaged to compute a representative number of cells per field. For this reason,

DAPI direct counts should only be performed on filters consisting of microbial cells uniformly

distributed over the entire surface of the filter. When preparing filters from soil samples, it is

important to prepare an appropriate soil dilution in order to obtain a countable microbial cell

concentration, and to find a dilution that is not so overly concentrated with soil that cells cannot

86

Figure 5.5: Photomicrographs of AT04-170 soils unable to be quantified by DAPI direct counts Photomicrograph represents 1.0 g of soil: overly concentrated with fluorescing soil particles (A), photomicrograph represents 0.5 g of soil: no apparent fluorescing bacterial cells (B).

A

B

87

Figure 5.5: (Continued) Photomicrograph represents 0.25 g of soil: no apparent fluorescing bacterial cells (C), photomicrograph represents 0.1 g of soil: no apparent fluorescing bacterial cells (D).

C

D

88

Figure 5.5: (Continued) Photomicrograph represents 0.05 g of soil: no apparent fluorescing bacterial cells (E).

E

89

be distinguished from fluorescing soil particles. Also, too many cells per field could result in

cell clumping resulting in inefficient cell totals. Conversely, cells at extremely low

concentrations can result in inaccurate counts because every cell at low concentrations is

imperative for reliable findings. Given that the Yungay samples have such low microbial cell

numbers, more soil must be used for direct count methods to increase cell concentrations in order

to enumerate cells more accurately. Unfortunately increasing soil volumes to concentrate

microbial cell volumes creates a downside for DAPI direct count procedures because DAPI

shows evidence of nonspecific binding to Atacama Desert soil. The nonspecific binding of

DAPI to mineral particles makes it difficult to distinguish microbial cells from mineral particles

although the mineral particles fluoresce green instead of blue.

Another disadvantage of direct count procedures occurs when microbial cells are grouped

together, making it challenging to distinguish single cells from one another. This is particularly

troublesome in this study because the genus Blastococcus dominates the culture-dependent

studies in all surface samples from the core arid region.

In spite of this problem, I attempted to count blue fluorescing cells ranging from

diameters 0.2 µm and 5.0 µm (the typical bacterial cell size range). Only one soil sample, AT04-

166, was able to be counted, although number of cells in sample AT04-166 was still difficult to

quantify for the reasons stated above. Cells that have survived in the extreme environment of the

Yungay region in the Atacama Desert appear differently under DAPI epifluorescence

microscopy than cells that have been thriving in a nutrient rich environment such as culture

media. This is apparent when comparing the differences between the appearance of cells dried

overnight within Yungay soil (Figure 5.1) and cells dried over a month within Yungay soil

90

(Figure 5.2). This is yet another disadvantage to quantifying cells in Atacama Desert hyper-arid

soils even when cells are able to be observed via DAPI staining, such as in sample AT04-166.

Quantifying microbial cells with DAPI staining in samples with low CFU/g, except

AT04-166, proved impossible with the DAPI direct count method. This dilemma, along with the

obstacles mentioned above, lead to an investigation of the detection limit of Yungay soil in

conjunction with the DAPI direct count protocol applied. Even samples that have obvious

fluorescing cells in certain fields, such as sample AT04-165, may not be adequate for

quantification purposes if the final calculated total cells per gram of soil is below the determined

detection limit for the method and soils used.

Wagner et al. (2003) states that the detection limit of fluorescence in situ hybridization

(FISH) is about 1 x 103 cells/mL. Another study done by the Ocean Drilling Program reports at

that the detection limit of the DAPI direct count procedure used by microbiologists researching

ocean drilling samples is approximately 1 × 105 cells/cm3 (Asada et al. 2002). Considering these

reported detection limits, I explored the detection limit for the DAPI method used for this study.

Known cell concentrations (determined by dilution plating) of microbial cells, particularly

Blastococcus sp. strain AT03-37-10, were added to soil AT04-170 (CFU value and detected total

cell number per gram of soil is zero), stained with DAPI, and counted to find the detection limit

for the DAPI technique used in this study. The detection limit was found to exist at someplace

between 1 x 105 and 1 x 106 cells/g.

AT04-166 is the only sample in this study (except the control pit) that had CFU values

for culturable organisms greater than 1 x 105 cells/g of soil. AT04-166 was also the only Yungay

tested soil to have total cell counts above the determined detection limit. The CFU value per

gram of soil for AT04-166 was as high as 7.40 x 105 on 1/10 PCA medium and the total cell

91

count is found to be 6.52 x 106 cells/g of soil. The sample site having the next highest CFU

value per gram of soil (8.20 x 104) on 1/10 PCA medium is AT04-165, but the calculated total

cell count (1.29 x 105) is below the detection limit; therefore, all other samples fall below the

detection limit of the direct count method of this study and cannot be accurately calculated.

For AT04-166, the total cell count per gram of soil is higher than the CFU value found

using the dilution plating technique and is to be expected because the dilution plating technique

only includes viable culturable heterotrophic bacteria. In non-extreme environments the

difference would be greater; the culturable cell concentrations would be much lower than actual

cell numbers since culturable bacteria only account for 0.001-15% of the total bacterial cell

numbers in most environmental samples (Amann et al. 1995). The extent to how inaccurate the

culturable portion of microorganisms is, when compared to total microbial cell numbers, depends

greatly on the soil under study. The Atacama Desert soils in the Yungay region is known to be

unique in their hyper-aridity and as this study indicates the culturable versus actual microbial cell

concentrations in Yungay region desert soils may not vary significantly. A study by Hartmann et

al. (1997) states that the culturable fraction of prokaryotes underestimates the inhabiting

population of active and dormant microbes by at least one to two orders of magnitude. With

regards to this statement, this thesis focuses on the least amount of variance (one order of

magnitude) between the culturable and total cells. Culturable cell concentrations and total cell

concentration for sample AT04-166 differ only by one order of magnitude. The results from the

direct counts on AT04-165 soils cannot be declared completely valid due to the detection limit,

but the difference between the values of CFUs per gram of soil and total cells per gram of soil is

less than one order of magnitude indicating that some Yungay soils may be exceptions to the “at

least one to two magnitudes” (Hartmann et al. 1997). Organisms cultured from certain Yungay

92

samples may in fact only underestimate the total resident population by less than one order of

magnitude due to low nutrient availability and low diversity among the inhabiting organisms.

For this reason, samples researched in this thesis, other than AT04-166, all contain total cell

counts below the detection limit of this study; thus, DAPI direct count procedures are not

suitable for quantifying most Atacama Desert soils from within the hyper-arid region of Yungay.

Control samples of lake water and soil were also prepared using DAPI epifluorescence

microscopy. A water sample from a Louisiana State University campus lake was fixed, DAPI

stained (a final concentration of 5 µg/µL), and filtered onto a black polycarbonate filter. Also, a

soil sample was retrieved from a cornfield near the Louisiana State University campus and

prepared in a similar manner to the other soil samples tested in this study. The only difference in

this sample preparation was that the cornfield sample was not sonicated due to the likely delicate

nature of some cells thriving in this fertile environment.

The cornfield soil is extremely organic in comparison to the Yungay soils researched in

this study. The purpose of the cornfield experiment was not to quantify, but to determine

whether the problems of non-specific DAPI binding to Yungay soil particles is a drawback when

handling other soils, in particular, soils from non-extreme environments. Figure 5.6 shows four

photomicrographs that were captured from a filter prepared with an extract from 0.25 grams of

cornfield soil. These images reveal that non-specific binding of DAPI to the cornfield soil

particles also is a setback in soils other than Atacama Desert soils.

The water sample was collected in order to compare necessary DAPI direct count

methods on soil samples with necessary methods on water samples. Excessive preparations that

are required for soil samples were not necessary for the water sample; the water was simply

fixed, stained, and filtered. When handling water, the DAPI direct count problems that are

93

obvious with soil samples (discussed throughout this chapter) are no longer concerns. All

volumes showed numerous various types of microorganisms in every field; the number of

fluorescing cells decreased consecutively from the 3.5 mL volume to the 0.5 mL volume. Figure

5.7 shows four images of each water volume filtered. Compared to all other figures throughout

this chapter (including the cornfield figures), Figure 5.7 indicates the substantial variation

between soil and water samples. This control study indicates the simplicity of obtaining direct

counts from water samples by DAPI epifluorescence and makes the case that DAPI direct count

methods are more efficient when employed on water environmental samples as opposed to soil

samples.

94

Figure 5.6: Comparison photomicrographs of cornfield soil sample Four photomicrographs represent 0.25 g of cornfield soil sample.

95

Figure 5.7: Comparison photomicrographs of LSU lake water sample Four photomicrographs represent 3.5 mL of lake water (A), four photomicrographs represent 1.0 mL of lake water (B).

A

B

96

Figure 5.7: (Continued) Four photomicrographs represent 0.5 mL of lake water (C).

C

97

CHAPTER 6 ASSOCIATION BETWEEN VALUES OF CFUS AND

SOIL CHEMICAL COMPOSITION

To examine the potential influence of the chemical composition of the soils studied on

heterotrophs in the Atacama Desert soils, major cation and anion concentrations were measured.

Also, a simple check for carbonate (CO32-) was done using dilute HCl in selected hyper-arid

soils.

RESULTS

Elemental Cation Analysis of Soil Samples

Table 6.1 displays all cation concentrations for each Atacama Desert soil sample studied.

There appears to be no relationship between metal concentrations and CFUs/g (Figures 6.1 and

6.2), except possibly for Na concentrations for surface and subsurface sites sampled within the

Yungay area. Specifically, three of the four sites that had no CFUs/g of soil on all media (AT04-

168, AT04-169, and AT04-170) and another site (AT04-167) that had < 102 (below detection

limit) CFUs/g on one culture medium and 0 CFUs/g on the other four media all had increased Na

levels (> 400 mMol/L) compared to the other soil samples. Conversely, some samples that

contained between 102 and 106 CFUs/g on at least one culture medium had lower Na

concentrations (< 50 mMol/L) (Figure 6.1). For some soils (AT03-42, AT03-44, and AT03-49)

with CFU/g values of < 102 on all five culture media, however, there were also low Na

concentrations measured. For these soils, however, Al concentrations were elevated (Table 6.1

and Figure 6.1).

The concentration of most of the major cations did not vary significantly with depth for

the hyper-arid and southern soil pits (Figure 6.2). However, Na concentrations did vary,

generally increasing with depth, and Ca concentrations decreased in two of the four pits. The

98

Table 6.1: Cation concentrations (mg/L) for Atacama Desert soils All values are reported in mg/L. ND refers to no data obtained.

Sample Site Al As Ca Cd Cr Cu Fe K

AT03-33 17311.48 12.63 18796.51 1.79 15.33 43.80 21767.50 3224.65AT03-34 20831.21 17.38 37856.49 2.51 18.15 75.49 26597.38 3992.88AT03-35 21778.58 11.98 25810.72 2.66 21.20 60.78 29910.39 4324.80AT03-36 14781.87 12.30 21006.71 1.65 13.73 53.78 19443.31 1907.50AT03-37 23982.85 15.45 27847.81 2.49 18.12 73.48 27517.94 4611.27AT03-38 21206.19 18.16 19903.36 2.01 14.33 63.18 22234.04 3866.73AT03-39 15033.08 9.07 17961.55 1.88 16.24 46.47 23865.70 2484.88AT03-40 11544.42 12.96 24908.00 1.35 11.89 93.79 17621.40 2177.02AT03-41 11469.02 9.72 19190.00 1.63 15.23 46.20 21389.48 2101.73AT03-42 24362.06 17.17 8771.11 2.22 16.28 57.84 25040.13 4627.96AT03-43 19016.96 14.09 9033.38 1.85 15.03 50.60 22018.89 3691.51AT03-44 22450.92 15.76 25668.55 2.45 18.39 66.09 24850.20 4582.33AT03-45 18788.49 14.57 17164.40 1.90 15.54 57.64 22298.79 3531.13AT03-46 18530.35 13.31 18892.83 1.85 15.76 63.51 21065.35 3255.45AT03-48 23838.18 17.30 36127.54 2.51 20.59 81.81 26571.73 4953.20AT03-49 16699.24 16.23 26468.40 1.70 13.63 64.10 18928.56 4041.59AT03-50 14903.95 11.60 13745.10 1.35 12.16 43.92 17857.14 3133.42AT04-151 19418.26 14.96 20170.94 1.91 17.00 55.29 23324.81 3502.57AT04-154 15400.95 10.79 19441.67 1.59 13.84 35.68 21227.53 2371.29AT04-155 15301.79 11.07 15913.07 2.11 19.26 42.93 27682.66 2337.01AT04-156 17863.81 11.62 15235.26 1.98 17.37 47.98 25970.93 2726.07AT04-157 16820.66 8.47 7203.69 1.85 17.45 37.96 24700.99 2747.49AT04-158 23654.97 14.93 14402.50 1.90 15.96 59.30 23308.34 3844.11AT04-161 15948.41 8.32 31796.12 2.24 20.37 43.92 26812.20 3829.01AT04-162 8465.73 5.24 34724.80 1.12 13.55 20.24 16933.68 2029.26AT04-163 12136.24 5.97 25315.87 2.04 26.05 35.91 29227.86 2053.89AT04-164 11350.73 3.25 16284.94 1.03 8.36 156.10 15750.05 1723.10AT04-165 8656.57 8.02 28230.61 0.97 9.25 32.65 12966.62 2065.61AT04-166 23181.98 14.78 17149.21 2.41 20.32 50.35 27383.14 5208.40AT04-167 9777.78 7.72 30791.76 0.96 10.44 20.77 12811.04 4225.21AT04-168 12996.20 10.55 23162.22 1.13 15.43 30.79 13211.95 5588.61AT04-169 18988.05 8.09 23170.60 1.56 16.19 42.85 22211.38 2833.86AT04-170 11404.57 6.57 32332.01 1.86 18.85 25.27 24219.15 2330.26

99

Table 6.1: (Continued) Sample Site Mg Mn Na Ni P Pb Si Zn

AT03-33 6897.46 327.19 1006.57 12.09 455.76 10.95 97.12 49.67 AT03-34 8587.15 382.83 1064.93 15.31 510.79 21.89 230.20 73.73 AT03-35 8672.53 364.64 1125.51 16.00 475.01 17.24 117.89 77.28 AT03-36 6430.00 312.57 876.24 10.63 379.56 13.86 203.19 41.71 AT03-37 10376.88 412.76 1153.73 18.94 599.39 17.50 81.79 84.21 AT03-38 8553.22 361.74 1037.73 14.41 537.68 16.01 124.26 65.47 AT03-39 5010.76 277.73 1284.55 9.90 368.22 10.42 153.49 42.41 AT03-40 5234.75 238.02 1120.22 7.66 321.26 9.91 330.25 35.62 AT03-41 4591.66 239.08 1057.89 8.34 399.84 9.76 207.77 34.51 AT03-42 11462.47 430.11 993.53 17.89 662.94 12.67 235.48 72.50 AT03-43 9394.18 369.04 731.49 15.25 576.18 10.59 154.85 57.12 AT03-44 9562.77 415.47 1118.10 16.79 590.74 15.02 148.79 82.48 AT03-45 8441.62 349.24 999.27 14.16 539.01 13.40 65.94 58.48 AT03-46 9122.64 357.49 935.30 14.71 508.72 15.61 93.88 60.66 AT03-48 13063.53 458.89 1086.36 18.49 473.19 21.32 133.52 79.91 AT03-49 8061.01 332.13 1240.64 11.52 524.83 16.03 88.55 57.57 AT03-50 7378.50 312.85 774.80 9.20 416.95 10.32 191.42 44.62 AT04-151 7559.75 344.09 1252.54 13.87 510.04 12.86 91.92 57.30 AT04-154 5160.14 316.71 1150.89 10.02 426.76 9.75 123.56 39.12 AT04-155 5263.43 308.24 1204.55 11.74 466.24 11.66 57.05 46.62 AT04-156 5588.04 334.33 1283.15 11.79 457.57 12.27 108.68 47.61 AT04-157 5596.40 309.68 2402.64 11.44 450.47 8.82 170.25 43.69 AT04-158 9729.95 379.21 2628.14 15.69 584.57 12.27 77.61 60.15 AT04-161 7885.17 301.09 1524.06 13.69 401.85 13.21 104.64 49.97 AT04-162 5027.07 195.83 3573.49 6.07 216.12 5.25 341.22 26.07 AT04-163 4149.75 205.72 3645.65 8.58 359.29 6.76 282.56 33.01 AT04-164 5199.71 264.96 794.13 5.78 264.44 6.60 42.65 28.24 AT04-165 4534.41 221.09 3064.48 5.43 314.84 8.92 183.23 27.75 AT04-166 11247.46 405.25 1110.29 17.67 534.48 12.72 125.31 63.05 AT04-167 4020.94 235.51 41635.13 10.26 291.11 7.61 201.92 42.68 AT04-168 8781.30 212.86 65840.75 7.70 272.90 9.15 179.91 41.88 AT04-169 6366.58 232.46 13682.44 9.50 352.46 6.22 158.59 33.35 AT04-170 5043.64 180.27 9935.88 7.97 291.31 7.41 160.93 32.27

100

Table 6.1: (Continued) Sample Site Al As Ca Cd Cr Cu Fe K

AT04-150 0cm 18015.36 14.48 18130.15 2.33 21.23 50.01 27841.65 3136.59 AT04-150 10cm 10777.60 10.17 46252.91 1.18 11.89 12.01 15969.52 2135.61 AT04-150 20cm 11984.70 8.81 37054.24 1.35 11.83 14.27 17948.12 2611.72 AT04-150 30cm 13227.53 17.66 55166.27 1.36 14.06 15.11 17762.63 3452.11 AT04-150 40cm 11925.56 6.89 39526.98 1.84 17.23 15.14 23497.39 2480.83 AT04-150 50cm 13695.33 18.92 43350.73 1.62 14.76 16.89 20446.43 3502.28 AT04-150 60cm 12298.46 10.18 27273.39 1.05 11.11 14.82 14142.91 3202.53 AT04-150 70cm 10852.27 19.11 37620.61 0.98 11.08 12.97 13463.13 3500.42 AT04-150 80cm 10451.96 24.27 31122.99 0.61 9.82 14.15 8238.36 5406.99 AT04-150 90cm 10699.02 15.56 23732.78 0.90 11.03 15.69 12617.48 3848.15 AT04-152 0cm 21575.38 14.42 19384.57 2.11 17.04 53.87 24068.80 3795.26 AT04-152 10cm 7448.39 5.96 71929.80 0.71 7.00 9.85 9700.99 2005.43 AT04-152 20cm 11665.19 8.16 58427.04 1.20 11.97 14.90 15630.08 3054.93 AT04-152 30cm 13148.50 9.14 59981.19 1.12 11.59 17.55 15371.47 3814.85 AT04-152 40cm 12164.52 8.36 40271.38 1.15 10.44 16.72 14962.68 3339.63 AT04-152 50cm 12409.81 9.41 35310.10 1.14 12.50 15.51 15650.16 3400.24 AT04-152 60cm 13855.61 10.33 16353.51 1.07 10.58 16.67 15210.93 2813.29 AT04-152 70cm 13151.05 12.18 29201.90 1.05 12.20 16.41 14569.32 4307.67 AT04-152 80cm 12268.11 12.81 27100.99 1.07 12.06 15.36 15257.03 3778.53 AT04-153 0cm 16145.22 8.93 7877.56 1.57 15.25 26.47 19832.51 2270.56 AT04-153 10cm 8245.30 8.55 78082.17 0.60 6.54 10.30 8578.90 2936.92 AT04-153 20cm 10649.00 8.55 55835.20 0.94 8.82 12.46 12712.52 2629.64 AT04-153 30cm 9377.53 8.36 49228.73 0.86 7.75 12.49 11807.43 2540.22 AT04-153 40cm 11484.18 14.10 53403.54 1.07 8.32 14.44 11790.34 3514.62 AT04-153 50cm 14194.64 23.23 35192.94 1.41 11.68 18.09 15734.38 3836.49 AT04-153 60cm 10968.92 12.46 23399.33 0.55 9.41 14.34 7780.04 3722.38 AT04-153 70cm 12502.35 14.08 24909.71 0.89 9.08 16.98 10845.91 4533.28 AT04-153 80cm 14160.91 14.99 21563.66 0.90 11.06 18.51 11960.97 4841.45 AT04-159 0cm 14322.63 4.29 15925.79 1.97 11.67 43.48 25592.00 2184.88 AT04-159 10cm 11340.49 3.30 16261.76 1.12 6.34 25.37 15854.48 1669.81 AT04-159 20cm 9500.60 4.64 43184.61 0.94 6.45 20.80 13820.81 1964.10 AT04-159 30cm 15935.83 6.68 17007.05 1.69 12.31 33.12 24147.68 3067.44 AT04-159 40cm 17978.00 9.71 16585.50 1.58 12.62 36.30 22778.00 3979.00

Cornfield ND ND ND ND ND ND ND ND

101

Table 6.1: (Continued) Sample Site Mg Mn Na Ni P Pb Si Zn

AT04-150 0cm 6725.49 338.18 1096.98 12.77 459.40 12.25 107.46 52.18 AT04-150 10cm 4936.55 204.68 998.52 4.84 256.77 3.97 19.18 21.55 AT04-150 20cm 5952.84 203.80 959.19 6.92 282.12 3.57 22.54 26.38 AT04-150 30cm 6894.31 224.61 1133.69 6.12 310.69 3.97 37.29 27.49 AT04-150 40cm 5394.75 231.96 1138.73 8.02 313.76 4.62 63.42 30.83 AT04-150 50cm 7140.27 252.41 2642.80 7.48 328.18 5.49 42.67 29.72 AT04-150 60cm 6212.97 246.34 22664.09 6.98 78.75 4.12 33.75 27.11 AT04-150 70cm 6453.35 192.58 22531.65 5.23 205.93 3.22 63.54 21.66 AT04-150 80cm 7107.77 172.75 48175.61 4.92 118.09 2.57 33.80 20.30 AT04-150 90cm 6076.92 226.44 24619.80 5.98 126.61 3.97 51.41 23.98 AT04-152 0cm 9336.82 383.56 1302.48 15.18 509.83 11.74 68.67 58.70 AT04-152 10cm 3955.04 127.36 524.71 3.45 189.43 2.20 34.81 16.83 AT04-152 20cm 6231.26 198.67 997.70 6.99 291.94 3.68 46.03 27.09 AT04-152 30cm 6812.13 215.69 951.29 7.38 305.07 4.11 41.54 30.31 AT04-152 40cm 6484.66 225.01 3228.97 6.82 309.84 4.44 32.11 27.20 AT04-152 50cm 6124.77 224.14 6262.57 6.71 287.34 4.48 47.65 26.84 AT04-152 60cm 6574.17 289.55 4337.63 7.46 304.64 5.33 30.64 25.98 AT04-152 70cm 8773.74 214.75 3319.58 6.30 260.65 3.88 44.90 27.76 AT04-152 80cm 8412.82 217.85 3322.14 5.81 255.59 3.96 32.38 26.55 AT04-153 0cm 4916.17 296.94 1273.21 9.68 399.84 7.61 254.59 35.01 AT04-153 10cm 5224.92 116.24 750.52 3.28 183.24 2.44 30.63 16.97 AT04-153 20cm 4618.08 176.24 1073.49 4.41 260.12 3.68 33.85 20.46 AT04-153 30cm 4877.63 164.02 925.76 4.52 259.33 3.36 23.39 19.72 AT04-153 40cm 6313.82 182.28 3142.76 5.05 270.87 3.70 72.32 22.16 AT04-153 50cm 7589.97 224.15 4860.07 7.29 328.61 5.05 67.93 28.04 AT04-153 60cm 6074.91 167.49 48792.51 5.63 122.79 3.01 25.28 22.05 AT04-153 70cm 9480.13 197.90 28408.63 6.53 218.70 3.86 24.05 26.70 AT04-153 80cm 10301.81 206.39 22670.00 7.07 207.42 4.03 33.98 29.15 AT04-159 0cm 7375.20 380.27 580.96 8.12 279.74 8.36 29.81 42.69 AT04-159 10cm 4029.76 253.83 322.15 4.66 202.97 3.32 32.53 26.38 AT04-159 20cm 3754.48 201.95 292.16 3.68 169.51 2.50 26.82 20.97 AT04-159 30cm 6450.76 315.16 536.71 6.69 226.82 5.08 80.96 36.76 AT04-159 40cm 9064.00 363.50 659.00 7.13 274.00 5.98 44.15 40.10

Cornfield ND ND ND ND ND ND ND ND

102

010

020

030

040

050

060

070

080

090

010

0011

0012

0013

0014

0015

0016

0017

0018

0019

0020

0021

0022

0023

0024

0025

0026

0027

0028

0029

0030

00

Sam

ple

Site

s

mMol/L

110100

1000

1000

0

1000

00

1000

000 CFU

Na

(mM

ol/L

)K

(mM

ol/L

)C

a (m

Mol

/L)

Mg

(mM

ol/L

)Fe

(mM

ol/L

)M

n (m

Mol

/L)

Pb (m

Mol

/L)

Zn (m

Mol

/L)

As (

mM

ol/L

)C

d (m

Mol

/L)

Cr (

mM

ol/L

)C

u (m

Mol

/L)

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l (m

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Si (m

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CFU

AT04-170 AT04-169 AT04-168 AT04-167 AT04-166 AT04-165 AT04-164 AT04-163 AT04-162 AT04-161 AT04-158 AT04-157 AT04-156 AT04-155 AT04-154 AT04-151 AT03-50 AT03-49 AT03-48 AT03-46 AT03-45 AT03-44 AT03-43 AT03-42 AT03-41 AT03-40 AT03-39 AT03-38 AT03-37 AT03-36 AT03-35 AT03-34 AT03-33

Figu

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.1:

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alue

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unga

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twee

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CFU

104

three soil pits sampled from within the Yungay hyper-arid region had no CFUs/g, other than the

two CFU/g peaks at 10 cm in soil pit AT04-152 and at 40 cm in soil pit AT04-150; therefore, it

was difficult to determine if there was any correlation among the CFU/g values and cation

concentrations (Figure 6.2). But, some layers from the three core soil pits with the highest Na

concentrations did have 0 or < 102 CFUs/g on all five culture media (Figure 6.2). For soil pit

AT04-152, CFU/g values decreased with depth. Ca concentrations also decreased with depth, as

Na concentrations. At 10 cm, consistently high CFU/g values between 102 and 104 were

measured on all five culture media, and this layer also had a low Na concentration. However, at

50 cm depth, the Na concentration was the highest in the pit and there was 0 CFUs/g measured

on all five culture media. Although this Na concentration was not higher than Na concentrations

for other soil samples (Table 6.1), it was higher than Na concentrations measured at sites having

higher culturable heterotrophic bacteria abundances (Figure 6.1). Because Ca concentrations

also decreased with depth, there could be a correlation with CFU/g. For the southern soil pit

(AT04-159), all of the five layers had > 105 CFUs/g on at least one of the five culture media.

This site, as discussed in Chapter 4, was considered a control soil pit because the region is less

arid than the Yungay region; the increased CFUs/g may reflect the increased amounts of

precipitation and a larger organic component in this soil. The concentration of Na was low

throughout the soil pit, with values much lower than the Yungay soils (Table 6.1 and Figure 6.2).

Inorganic Anion Analysis of Soil Samples

Table 6.2 lists major anion concentrations for each surface and subsurface sample along

with their CFU/g range. Of the 33 Yungay surface sites, only seven sites had Cl anion

concentrations > 1.0 mMol/L (Figure 6.3). Of these seven soils, samples AT04-162 and AT04-

165 had CFUs/g of soil > 103 on at least one culture medium, while the remaining soils had 0 or

105

Table 6.2: Major anion concentrations (mg/L) and CFU/g values for Atacama Desert soils NA represents the anion value was below detection (< 0.01 mg/L). ND refers to no data was obtained.

Sample Site Cl F NO3 SO4 CFUs/g AT03-33 0.88 0.60 0.51 1657.97 < 105 AT03-34 0.53 NA 0.51 2013.20 < 103 AT03-35 0.78 0.51 0.72 1397.27 < 103 AT03-36 0.46 0.84 0.55 2126.90 < 103 AT03-37 0.73 0.48 0.63 2059.77 < 103 AT03-38 0.66 0.27 0.65 1747.73 < 103 AT03-39 0.42 NA 0.32 979.20 < 104 AT03-40 0.81 0.47 0.41 2358.29 < 105 AT03-41 0.84 0.52 0.46 1485.75 < 105 AT03-42 1.12 0.28 0.71 93.22 < 102 AT03-43 0.66 0.48 0.59 158.78 < 103 AT03-44 1.01 0.48 0.91 2362.98 < 102 AT03-45 0.44 0.44 0.30 1344.27 < 104 AT03-46 NA NA 0.48 875.42 < 105 AT03-48 0.61 NA 0.47 1923.91 < 104 AT03-49 0.52 NA 0.61 1940.37 < 102 AT03-50 0.32 0.88 0.36 633.87 < 103 AT04-151 1.11 NA 1.33 2279.62 < 103 AT04-154 0.89 NA 0.54 1827.93 < 104 AT04-155 0.57 0.50 0.53 754.53 < 103 AT04-156 0.56 0.41 0.57 989.19 < 103 AT04-157 21.1 NA 12.54 27.78 < 102 AT04-158 16.00 NA 5.36 71.87 < 102 AT04-161 1.38 NA 0.64 1554.82 < 105 AT04-162 124.62 NA 4.27 712.50 < 104 AT04-163 53.38 0.61 4.61 2843.06 0 AT04-164 4.07 0.92 0.29 1788.43 < 104 AT04-165 40.75 0.60 7.92 2671.57 < 105 AT04-166 0.35 NA 0.21 514.02 < 106 AT04-167 65.11 1.75 253.30 1877.13 < 102 AT04-168 46.63 3.75 99.43 3141.63 0 AT04-169 207.61 1.29 8.30 1217.13 0 AT04-170 580.14 4.71 2.67 1851.42 0

106

Table 6.2: (Continued)

Sample Site Cl F NO3 SO4 CFUs/g AT04-150 0cm 0.39 NA 0.52 1659.12 0 AT04-150 10cm 0.42 NA NA 2077.94 < 102 AT04-150 20cm 1.26 0.21 0.14 2305.36 < 102 AT04-150 30cm 0.73 0.33 0.08 2318.13 < 103 AT04-150 40cm 1.05 0.27 0.09 2322.96 < 104 AT04-150 50cm 11.95 0.78 10.65 2780.12 < 103 AT04-150 60cm 64.59 1.14 3.41 3391.57 0 AT04-150 70cm 52.72 1.46 9.83 3189.31 < 103 AT04-150 80cm 295.55 4.36 32.07 4969.68 < 102 AT04-150 90cm 71.08 3.40 21.19 3527.04 0 AT04-152 0cm 0.69 0.30 0.41 1483.39 < 102 AT04-152 10cm 0.40 NA 0.02 2289.31 < 104 AT04-152 20cm 0.58 NA 0.06 2316.73 < 103 AT04-152 30cm 0.68 0.32 0.07 2304.91 < 103 AT04-152 40cm 27.74 NA 1.21 2703.80 < 103 AT04-152 50cm 77.62 NA 10.24 2559.13 0 AT04-152 60cm 45.89 0.55 12.51 1834.08 < 102 AT04-152 70cm 53.63 1.45 24.80 2831.09 < 102 AT04-152 80cm NA NA 21.99 2791.28 0 AT04-153 0cm 0.19 0.12 0.16 31.26 < 103 AT04-153 10cm 0.24 NA NA 2319.46 < 102 AT04-153 20cm 0.32 0.28 NA 2326.12 < 103 AT04-153 30cm 2.98 0.2118 0.66 2299.37 < 102 AT04-153 40cm 51.23 1.29 3.07 2652.08 0 AT04-153 50cm 83.24 2.72 3.20 2883.70 < 102 AT04-153 60cm 72.90 1.01 11.86 4873.15 0 AT04-153 70cm 138.90 1.82 15.82 3279.26 < 102 AT04-153 80cm 653.09 1.58 13.56 2822.31 < 102 AT04-159 0cm 0.38 NA 0.03 9.34 < 106 AT04-159 10cm 0.60 NA NA 1406.09 < 106 AT04-159 20cm 2.47 NA NA 2323.30 < 106 AT04-159 30cm 3.21 NA NA 1209.20 > 106 AT04-159 40cm 2.76 NA NA 1380.97 > 106

Cornfield 0.34 NA 0.03 0.62 ND

107

0246810121416182022242628303234

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Figu

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.3: C

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108

Figu

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109

near 0 CFUs/g on all five culture media. Moreover, soil from AT04-169 and AT04-170 both

contained 0 CFUs/g and had the two highest Cl concentrations of all the analyses. Therefore, the

elevated Cl values may be correlated to no recoverable bacteria per gram of soil and soils having

elevated CFU/g values also had relatively low Cl concentrations (Figure 6.3).

Compared to Cl concentration, there was no apparent relationship with the other anion

concentrations and CFU/g for the surface soils (Table 6.2 and Figure 6.3). For instance, of all 33

surface samples from within the Yungay region, only two (AT04-167 and AT04-168) had NO3

levels > 1.0 mMol/L, with most of the NO3 concentrations < 0.2 mMol/L (Table 6.2 and Figure

6.3). Both of the soils with high NO3 concentrations had low CFUs/g on all five culture media

(Figure 6.3). Sixteen soils with NO3 concentrations between 0.003 mMol/L and 0.01 mMol/L

had > 102 CFUs/g on at least one medium. The concentration of SO4 in all of the Yungay

surface soils were very high, and yet there was no correlation to high or low CFUs/g values .

For the soil pits, Cl and SO4 concentrations increased with depth, which did not

necessarily correlate to CFU/g values (Figure 6.4). The other anions did not seem to relate to

CFU/g values, either. As with the surface soils, although it appeared that low CFU/g values

were associated with high Cl levels, the 80 cm layer from soil pit AT04-152 had 0 CFUs/g and

yet it had a low Cl concentration. SO4 levels were just as elevated as were measured in surface

soil samples and SO4 concentrations did not correlate to the CFU/g levels in the soil pits.

Detection of Carbonates in Atacama Desert Soils

Carbonate anions (e.g., HCO3-, CO3

2-) were not measured with the other anions, but to

check if carbonate was present in the soils, dilute HCl was added to dry soil. Sample AT04-167,

representing the surface samples from the Yungay region, and sample AT04-153 at 30 cm,

representing a subsurface layer from the hyper-arid region, both tested positive for carbonates.

110

Soil from AT04-152 at 70 cm, representing Yungay region subsurface soils at deep layers, had

negative results for carbonates.

DISCUSSION

The chemical composition of the soils was determined in this study to establish if there

were any possible correlations between soil chemical composition and CFU/g values in Atacama

Desert soils. The results of the chemical analyses described provide some insight into the results

previously discussed in Chapters 3 and 4. The similarities observed between the ion analyses

and the CFU/g values relate to the idea that a requisite for self-organized patchiness at short

spatial range is a positive connection between organisms and nutrient availability (Rietkerk et al.

2004). Therefore, excess soluble salts in soils can shape bacterial distributions because elevated

salinity concentrations can cause nutrient discrepancies that will result in the accumulation of

elements that might be harmful to prokaryotic cells (Kotuby-Amacher et al. 1997).

“Chilean nitrate” is the trade name for the naturally occurring mined inorganic mineral

salt that is found in high abundance within the soils of the Atacama Desert because of a lack of

precipitation (NOSB TAP 2002). The substance quickly ionizes into Na and NO3 in water, but it

can also contain Cl, NO3, and SO4 (NOSB TAP 2002). As a result, these anions may build up in

the soil and could account for the correlations observed between soils with high concentrations

of anions and little to no CFUs/g of soil. Specifically, Na is locally persistent in soil systems in

that it is relatively immobile. Soil has a net negative charge; thus, it binds Na strongly and Na

tends to accumulate in arid environments (NOSB TAP 2002). As indicated by Kotuby-Amacher

et al. (1997), high soil salinity can cause nutrient imbalances that result in a buildup of elements,

which can be harmful to cells; this reduces water infiltration if the amount of the salt element,

Na, is elevated. For these reasons, it was expected that soils containing high concentrations of

111

Na, namely soils from the studied hyper-arid region, would not result in high values of

heterotroph CFUs/g.

The results from the elemental analyses of the Atacama Desert soils support the

suggestion that elevated concentrations for some elements and compounds (e.g., Na, Cl, NO3)

may contribute to decreased abundances of bacteria (Figures 6.1 and 6.3). For instance, higher

Na levels in the majority of the soils correlated to low CFU/g values. Regarding the depth at

which the increased levels were observed, Na may accumulate over time more efficiently in

subsurface layers because of its persistent nature (Figure 6.2). By comparison, the control soil

pit at Altamira contained considerably higher amounts of culturable heterotrophic bacteria and

exhibited Na concentrations less than most of the Yungay soil samples. Elevated SO4

concentrations were ubiquitous throughout Atacama Desert samples (Yungay and Altamira soil

pit samples). Although there was no direct correlation between levels of culturable heterotrophic

bacteria within individual samples and SO4 concentrations, the relatively low levels of CFUs/g

observed throughout the Atacama Desert samples, in comparison with soils from non-arid

environments, may have been influenced by the accumulation of SO4 in the soils. Carbonate

detected in the Yungay region of the Atacama Desert at surface and near surface layers, but not

in more subterranean layers, could be in the mineral form of CaCO3 in the soils. The absence of

precipitation probably results in the accumulation of CaCO3 in the surface soils of the hyper-arid

region; this mineral can have a neutralizing effect on pH and may be one of the reasons why the

soil pH for most of the desert soils was pH 7 or above.

112

CHAPTER 7 CONCLUSIONS

The purpose of this thesis was to study the microbiology of the hyper-arid core region of

the Atacama Desert, Chile. The research on these hyper-arid soils involved the use of culture-

dependent methods, epifluorescence direct count techniques, and chemical composition analyses.

The findings confirm the Mars like nature of the soils within the hyper-arid region and

demonstrate the need for extensive and comprehensive sampling strategies on any future Mars

exploration missions that involve surface or subsurface sampling for life detection.

Chapter 2 presents a thorough description of the techniques applied to Atacama Desert

soil samples throughout this research to better understand microbiological aspects of the “Mars-

like” soils. The essential components of this study include culturable bacteria quantification and

identification methods which were employed on 33 surface soil sites as well as four soil pits

from which subsurface layers were sampled. Serial dilution plating procedures were applied to

each surface and subsurface sample using five culture media, three nutrient rich and two nutrient

low for the determination of CFUs/g of soil. Isolation methods of pure cultures, preservation

procedures, DNA extraction methods, PCR and PCR purification protocols, and sequencing

techniques are described within Chapter 2. Sequencing amplified 16S rRNA genes from pure

isolates permitted identification of the isolates to the closest phylogenetic relatives by using the

BLAST interface and the GenBank database. The methodologies for DAPI epifluorescence

studies are described in Chapter 2. Also, procedures to perform pH, salinity, elemental,

inorganic anion, and carbonate analyses are outlined.

Chapter 3 represents the culture-dependent findings for surface samples acquired from

the hyper-arid core region of the Atacama Desert. The chapter investigates the hypothesis that

113

the distribution of culturable heterotrophic bacteria in terms of quantity and diversity in surface

soils would not be homogeneous because organisms acquire nutrients from their environment

and are spread across desert regions according to resource accessibility throughout the soils

(Rietkerk et al. 2004). Patchiness was anticipated and later confirmed to exist throughout the

surface of the hyper-arid region. Evidence that at short spatial range, for self-arranged

patchiness to take place, organisms must be directly linked to resource abundance from Rietkerk

et al. 2004, supports the short range patchiness found in the 15.3 km2 core arid region of the

Atacama Desert, Yungay. The patchiness in terms of numbers is apparent in the CFUs

determined for the 33 surface sites since the range was found to be between 0 and 7.4 x105

CFUs/g of soil.

Isolates belonging to three phyla, Acintobacteria, Firmicutes, and Proteobacteria were

found within the culturable heterotrophic bacteria inhabiting the surface sites. Members of the

phylum Actinobacteria are prevalent throughout the sampled surface sites. One family included

in the phylum Actinobacteira, Geodermatophilaceae, was found to dominate the majority of the

surface sites, especially species of the genus Blastococcus. Chapter 3 also discusses

characteristics of species in the genus Blastococcus.

Chapter 4 describes the examination of the subsurface samples via culture-dependent

methods. Other than two CFU value peaks that barely exceed 1 x 103 CFUs/g of soil on at least

one of the five culture media used, the three soil pits in the core hyper arid region demonstrate

very low CFU values in all layers sampled. The southern site at Altamira, where a control pit

was excavated, had higher CFU values per gram of soil than soils samples from within the

hyper-arid region. The pit at Altamira, AT04-159, was found to have CFUs/g of soil of

approximately 1.0 x 105 on at least one culture media at layers near the surface, and > 1.0 x 106 at

114

the deepest subsurface layer on all culture media employed for this study. The diversity of

culturable heterotrophic bacteria retrieved from each of the three pits within the hyper-arid core

region is greatly reduced as compared to the diversity recovered in the Altamira pit. The isolates

from these three Yungay soil pits consist of members of the phyla, Acintobacteria, Firmicutes,

and Proteobacteria, while the Altamira pit was found to have representatives of at least four

phyla including the three listed for the core region soil pits and an additional phylum,

Bacteroidetes. The most abundant of the three phyla are representatives of the phylum

Actinobacteria, precisely as was found in the surface samples. The diversity recovered from the

individual layers of the three hyper-arid region soil pits is low. Even in the two pits that exhibit

spiked CFU levels, larger amounts of the same types of bacteria that were recovered from the

surface layers were detected. Bacteria able to be cultured from the surface of each of the three

pits are in many instances the same bacteria found at some depths. Comparative evaluations of

isolates recovered from the surface samples and isolates obtained from the Yungay soil pit layers

show that the diversity is slightly greater in the surface samples; although, many transverse

organisms were discovered.

The amount of Blastococcus sp. recovered from surface samples is greatly reduced in the

Yungay soil pit samples, specifically in soil pits AT04-150 and AT04-152. Pseudonocardia sp.

are found to be dominate the culturable isolates within pits AT04-150 and AT04-152 and are

only detected in Yungay pit samples. Of the three hyper-arid region soil pits, AT04-150 and

AT04-152 were excavated on the top of a hill, while soil pit AT04-153 was constructed in the

valley adjacent that hill. The majority of the surface sites showing spatial patchiness in terms of

CFU levels and diversity were also sampled in a valley at Yungay. The culturable bacteria that

dominate surface samples and the valley sampled soil pit, AT04-153, are members of the genus

115

Blastococcus. Soil pits AT04-150 and AT04-152, the two soil pits that were dug on the hilltop

have less than 2% of the recoverable bacteria identified as members of the genus Blastococcus,

and the dominate organisms are members of the genera Pseudonocardia and Streptomyces. This

suggests some differences in the nature of the soils found in Yungay low lying valley and those

soils found at elevated sites in the Yungay region that may result in the observed variation within

the bacterial communities. The findings reported and discussed in Chapters 3 and 4 suggest the

existence of spatial patchiness in terms of numbers of recoverable bacteria and diversity on a

vertical as well as on a horizontal plane.

The soil pit in the less arid, more southern region at Altamira has CFU/g values up to four

magnitudes greater than those in the hyper-arid region. The Altamira soil pit was found to

contain approximately 42 different prokaryotic taxa. Vegetation increases along a moisture

gradient in southern regions of the Atacama Desert (Navarro-González et al. 2003). For this

reason, it was projected that CFU levels and bacterial community diversity of organisms

recovered from the Altamira soil pit increase in comparison with the CFUs and diversity within

the hyper-arid region.

Enrichment cultures for each of the Yungay pits proved useful in uncovering organisms

that grow rapidly in favorable conditions; although, they cannot function as enumeration studies.

A fog event, which is a rare moisture occurrence in the hyper-arid region, could be one source of

a natural enrichment process wherein organisms normally found at small concentrations could

multiply quickly. In the three Yungay pits, the diversity among the recovered enrichment

isolates was found to be similar to that of the dilution plating isolates in terms that both contain

low diversity of organisms, and the organisms recovered were commonly the same.

116

Chapter 5 describes the direct count research carried out in an attempt to quantify

bacterial cells through DAPI epifluorescence microscopy. DAPI staining for enumeration

purposes in low CFU value samples, which includes all samples from the Yungay region besides

AT04-166, proved undoable with the DAPI direct count method used for this study. An

investigation of the detection limit for the Yungay soils with the applied DAPI direct count

procedure was carried out. Blastococcus sp. strain AT03-37-10 cells were added to soils

containing zero culturable bacteria, stained with DAPI, and counted to find the detection limit for

the DAPI technique used in this study. The detection limit was found to exist between 1 x 105

and 1 x 106 cells per gram of soil.

AT04-166 was the only sample in this study (besides the Altamira pit samples) that has

CFU values greater than 1 x 105 cells per gram of soil. AT04-166 was also the only Yungay soil

found to have total cell counts above the determined detection limit. The CFU value per gram of

soil for AT04-166 is as high as 7.40 x 105 on one of the culture media utilized and the total cell

count was found to be 6.52 x 106 cells per gram of soil. Direct count procedures performed on

the sample site, AT04-165, which has the second highest CFU/g value of 8.20 x 104, were

unsuccessful because the computed total cell count of 1.29 x 105 was beneath the determined

detection limit. Because other soil samples from the Yungay region were found to contain lower

CFU numbers than AT04-165, the results obtained from the direct cell count attempt on AT04-

165 suggest that other samples would show results that also fall below the detection limit of the

direct count method and therefore not be able to accurately be calculated by the method

employed in this study.

In chapter 6 elevated concentrations of the metal, Na, and inorganic anionic molecules

Cl, F, SO4, and NO3 within Yungay soils, was shown, somewhat, to correlate with low numbers

117

of culturable bacterial cells; the inverse was also observed. This parallel may be related to the

idea that claims a necessity for self-organized patchiness at short spatial range is an established

positive association linking organisms and nutrient availability (Rietkerk et al. 2004). Thus, an

abundance of soluble salts in soils can affect bacterial distributions as well since high soil

salinity can cause nutrient imbalances that result in an accrual of elements that is lethal to cells

(Kotuby-Amacher et al. 1997).

This thesis adds to previous reports by Navarro-González et al. 2003 characterizing this

region as having soils considered analogous to Martian soils. Discoveries made as a result of

this study not only are beneficial to future Atacama Desert research, but can also be applied to

preparations concerning upcoming Mars exploration. The increased CFU values at surface sites

and subsurface layers from the Yungay region signify the need for numerous site surface and

subsurface sampling on Mars where similar patchiness of life over the surface of the planet and

with depth at subsurface layers may exist.

118

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123

APPENDIX A MEDIA INSTRUCTIONS

The media recipes listed are for solid and liquid given that this study uses culture media in both agar and broth forms. Broth forms are identical to solid media except for they do not contain agar, which is the solidifying agent.

For the experiment involving acidic media (pH 5), the MA and MB and 1/10 PCA and 1/10 PCB were made exactly as stated here except that HCl was added to the media before being autoclaved in order to drop the pH to 5.

Plate Count Agar (PCA)

5.0 grams Bacto™ Tryptone (Pancreatic Digest of Casein) 2.5 grams Bacto™ Yeast Extract 1.0 grams Dextrose GR from EM® Science 15.0 grams Bacto™ Agar Fill to 1 liter with distilled H20

Plate Count Broth (PCB)

5.0 grams Bacto™ Tryptone (Pancreatic Digest of Casein) 2.5 grams Bacto™ Yeast Extract 1.0 grams Dextrose GR from EM® Science Fill to 1 liter with distilled H20

1/10 Plate Count Agar (1/10 PCA)

0.5 grams Bacto™ Tryptone (Pancreatic Digest of Casein) 0.25 grams Bacto™ Yeast Extract 0.1 grams Dextrose GR from EM® Science 15.0 grams Bacto™ Agar Fill to 1 liter with distilled H20

1/10 Plate Count Broth (1/10 PCB)

0.5 grams Bacto™ Tryptone (Pancreatic Digest of Casein) 0.25 grams Bacto™ Yeast Extract 0.1 grams Dextrose GR from EM® Science Fill to 1 liter with distilled H20

1/100 Plate Count Agar (1/100 PCA)

0.05 grams Bacto™ Tryptone (Pancreatic Digest of Casein) 0.025 grams Bacto ™Yeast Extract 0.01 grams Dextrose GR from EM® Science 15.0 grams Bacto™ Agar Fill to 1 liter with distilled H20

1/100 Plate Count Broth (1/100 PCB)

124

0.05 grams Bacto™ Tryptone (Pancreatic Digest of Casein) 0.025 grams Bacto ™Yeast Extract 0.01 grams Dextrose GR from EM® Science Fill to 1 liter with distilled H20

Nutrient Agar (NA) 3.0 grams Bacto™ Beef Extract 5.0 grams Bacto™ Peptone (Enzymatic Digest of Protein) 15.0 grams Bacto™ Agar Fill to 1 liter with distilled H20 Nutrient Broth (NB) 3.0 grams Bacto™ Beef Extract 5.0 grams Bacto™ Peptone (Enzymatic Digest of Protein) Fill to 1 liter with distilled H20

Marine Agar (MA)

55.1 grams Difco™ Marine Agar Fill to 1 liter with distilled H20 Marine Broth (MB)

37.4 grams Difco™ Marine Broth Fill to 1 liter with distilled H20 Marine Agar + Instant Ocean® (MA+IO) 55.1 grams Difco™ Marine Agar 35.79 grams Instant Ocean® Fill to 1 liter with distilled H20 pH to 8 Marine Broth + Instant Ocean® (MB+IO) 37.4 grams Difco™ Marine Broth 35.79 grams Instant Ocean® Fill to 1 liter with distilled H20 pH to 8 1 times Instant Ocean® (1xIO) 35.79 grams Instant Ocean®

5.0 grams of Bacto™ Tryptone (Pancreatic Digest of Casein)

125

1.0 gram of Dextrose GR from EM® Science Fill to 1 liter with distilled H20

pH to 8 (for agar - 15.0 grams Bacto™ Agar was added before H20) 2 times Instant Ocean® (2xIO)

71.58 grams Instant Ocean® 5.0 grams of Bacto™ Tryptone (Pancreatic Digest of Casein) 1.0 gram of Dextrose GR from EM® Science Fill to 1 liter with distilled H20

pH to 8 (for agar - 15.0 grams Bacto™ Agar was added before H20) 3 times Instant Ocean® (3xIO)

107.37 grams Instant Ocean® 5.0 grams of Bacto™ Tryptone (Pancreatic Digest of Casein) 1.0 gram of Dextrose GR from EM® Science Fill to 1 liter with distilled H20

pH to 8 (for agar - 15.0 grams Bacto™ Agar was added before H20)

126

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sy c

onve

x 93

3 Bl

asto

cocc

us sp

. BC

448

0.99

A

J316

571

AT0

3-33

-30

-1

1/10

0 PC

A s

urfa

ce l

t pk

~tra

ns g

loss

y co

nvex

83

8 Bl

asto

cocc

us sp

. BC

448

0.99

A

J316

571

AT0

3-33

-33

-1

1/10

0 PC

A s

urfa

ce t

an d

ull ~

opaq

ue fl

at

809

Mod

esto

bact

er m

ultis

epta

tus

0.98

A

J871

304

AT0

3-33

-34

-1

1/10

0 PC

A s

urfa

ce !

lt pk

~tra

ns g

loss

y co

nvex

85

8 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

33-3

5 -2

1/

10 P

CA

su

rfac

e or

ange

~tra

ns g

loss

y co

nvex

86

6 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

33-3

8 -2

1/

10 P

CA

su

rfac

e or

ange

/pea

ch ~

trans

glo

ssy

conv

ex

861

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-33

-39

-2

1/10

PC

A

surf

ace

pk/ta

n tra

ns g

loss

y co

nvex

65

3 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

33-4

0 -2

1/

10 P

CA

su

rfac

e of

fwhi

te ~

trans

glo

ssy

conv

ex

923

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-33

-41

-2

1/10

PC

A

surf

ace

hot o

rang

e/ho

t pk

~glo

ssy

opaq

ue c

onve

x 91

8 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

33-4

3 -2

1/

10 P

CA

su

rfac

e ho

t pk/

hot p

each

~du

ll co

nvex

opa

q 92

3 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

33-4

4 -2

1/

10 P

CA

su

rfac

e lt

pk tr

ans g

loss

y co

nvex

91

0 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

33-4

7 -2

1/

10 P

CA

su

rfac

e lt

pk d

ull ~

roug

h co

nvex

~op

aque

59

9 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

33-4

8 -1

1/

10 P

CA

su

rfac

e da

rker

ora

nge

glos

sy c

onve

x ~o

paqu

e 88

6 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

33-4

9 -1

1/

10 P

CA

su

rfac

e lt

oran

ge/ta

n gl

ossy

con

vex

opaq

ue

924

Mod

esto

bact

er m

ultis

epta

tus

0.99

A

J871

304

AT0

3-33

-50

-1

1/10

PC

A

surf

ace

crea

m/ta

n (d

arke

r in

cent

er) o

paqu

e gl

ossy

con

vex

811

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-33

-53

-1

NA

su

rfac

e of

fwhi

te/y

ello

w c

ream

y bi

g co

nvex

glo

ssy

opaq

ue

911

Mic

roco

ccus

sp. R

G-6

4

0.99

A

Y56

1623

APP

EN

DIX

B

TA

BL

E O

F SU

RFA

CE

AN

D S

OIL

PIT

ISO

LA

TE

S Th

e ta

ble

belo

w sh

ows i

sola

tes r

ecov

ered

from

surf

ace

and

pit s

ampl

es, t

he d

ilutio

n, m

edia

, and

dep

th th

at th

e is

olat

e w

as re

cove

red

from

, col

ony

mor

phol

ogy,

leng

th o

f th

e se

quen

ce (b

p) im

porte

d in

to th

e B

LAST

inte

rfac

e of

Gen

Ban

k®, c

lose

st re

lativ

e as

iden

tifie

d by

BLA

ST re

sult,

nuc

leot

ide

iden

tity

sim

ilarit

y, a

nd G

enB

ank®

ac

cess

ion

num

ber.

127

(Tab

le c

ontin

ued:

Sur

face

isol

ates

) Su

rfac

e Is

olat

e

D

ilutio

n M

edia

D

epth

C

olon

y M

orph

olog

y

S

eque

nce

Len

gth

B

LA

ST R

esul

t

Si

mila

rity

Acc

essi

on n

o.

AT0

3-33

-54

-1

NA

su

rfac

e ta

nger

ine

big

~fla

t rou

gh b

umpy

ring

s ~op

aque

~gl

ossy

91

5 M

odes

toba

cter

mul

tisep

tatu

s 0.

99

AJ8

7130

4 A

T03-

33-5

6 -1

N

A

surf

ace

oran

ge p

ink

~opa

que

glos

sy c

onve

x m

ed

989

Baci

llus s

p. H

SCC

165

1

0.95

A

B04

5098

A

T03-

33-5

6A

-1

NA

su

rfac

e sm

all t

ange

rine

opaq

ue g

loss

y co

nvex

81

6 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

33-5

7 -1

N

A

surf

ace

trans

glo

ssy

conv

ex

980

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-33

-58

-1

NA

su

rfac

e pk

bum

py ir

regu

lar ~

trans

dul

l con

cave

cen

ter

751

Blas

toco

ccus

sp. B

C41

2

0.98

A

J316

574

AT0

3-33

-59

-1

NA

su

rfac

e ta

n/m

auve

glo

ssy

irreg

ular

~tra

ns ra

ised

86

1 Bl

asto

cocc

us sp

. BC

412

0.

99

AJ3

1657

1 A

T03-

33-6

0 -1

N

A

surf

ace

mau

ve g

loss

y co

nvex

~tra

ns

864

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

3-33

-61

-1

NA

su

rfac

e or

ange

rais

ed g

loss

y irr

egul

ar tr

ans d

arke

r spo

t in

cent

er

845

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-33

-63

-1

NA

su

rfac

e lt

pk o

paqu

e gl

ossy

con

vex

854

Mod

esto

bact

er m

ultis

epta

tus

0.98

A

J871

304

AT0

3-34

-1

-2

PCA

su

rfac

e ta

n no

n-gl

ossy

~co

nvex

(mor

e in

cen

ter)

opa

que

text

ured

(bum

py)

787

Baci

llus f

ortis

stra

in R

-651

4

0.97

A

Y44

3038

A

T03-

34-2

-3

PC

A

surf

ace

pk g

loss

y co

nvex

~tra

ns ~

irreg

ular

79

2 M

odes

toba

cter

mul

tisep

tatu

s 0.

98

AJ8

7130

4 A

T03-

34-3

-1

PC

A

surf

ace

hot p

k/or

ange

rigi

d bu

mpy

roug

h op

aque

irre

gula

r ~du

ll 92

7 W

illia

msi

a m

ural

is is

olat

e 50

0/04

0.

99

AY

9867

34

AT0

3-34

-4

-1

PCA

su

rfac

e ho

t pk

huge

irre

gula

r ~co

nvex

opa

que

glos

sy

833

Will

iam

sia

mur

alis

isol

ate

500/

04

0.99

A

Y98

6734

A

T03-

34-6

-1

PC

A

surf

ace

yello

w (m

usta

rd) o

paqu

e gl

ossy

con

vex

92

0 N

ocar

dioi

des s

p. V

4.M

E.19

0.

97

AJ2

4465

6 A

T03-

34-7

-1

M

A

surf

ace

hot p

k ~i

rreg

ular

opa

que

~glo

ssy

rais

ed b

umpy

smoo

th

925

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-34

-8

-1

MA

su

rfac

e ho

t ora

nge/

hot p

k ~i

rreg

ular

glo

ssy

~rou

gh ra

ised

92

5 M

odes

toba

cter

mul

tisep

tatu

s 0.

99

AJ8

7130

4 A

T03-

34-9

-1

M

A

surf

ace

pk ~

glos

sy c

onve

x op

aque

96

8 Bl

asto

cocc

us sp

. BC

412

0.

98

AJ3

1657

4 A

T03-

34-1

0 -1

M

A

surf

ace

dark

er p

k ~d

ull c

onve

x op

aque

89

7 Bl

asto

cocc

us sp

. BC

412

0.

98

AJ3

1657

4 A

T03-

35-1

-1

1/

10 P

CA

su

rfac

e or

ange

/pin

k gl

ossy

con

vex

opaq

ue

960

Blas

toco

ccus

sp. B

C41

2

0.97

A

J316

574

AT0

3-35

-2

-1

1/10

PC

A

surf

ace

lt ta

n gl

ossy

~fla

t with

con

vex

cent

er ~

trans

80

8 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

35-6

-1

1/

100

PCA

sur

face

lt t

an g

loss

y ~f

lat w

ith c

onve

x ce

nter

~tra

ns

711

Unc

ult e

arth

wor

m c

ast b

acte

rium

clo

ne c

250

0.96

A

Y15

4604

A

T03-

35-7

-1

1/

100

PCA

sur

face

lt t

an g

loss

y ~f

lat w

ith c

onve

x ce

nter

~tra

ns

575

Bac

teriu

m E

llin6

023

0.

98

AY

2346

75

AT0

3-35

-11

-1

MA

su

rfac

e pe

ach/

lt pk

~co

nvex

glo

ssy

~bum

py su

rfac

e op

aque

51

3 Bl

asto

cocc

us sp

. BC

448

0.

96

AJ3

1657

1 A

T03-

35-1

2 -1

M

A

surf

ace

lt pk

(lik

e br

ain)

ripp

led

glos

sy ra

ised

~irr

egul

ar m

argi

ns o

paqu

e 77

3 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T03-

35-1

3 -1

M

A

surf

ace

oran

ge/p

k op

aque

con

vex

glos

sy

745

Blas

toco

ccus

sp. B

C41

2

0.97

A

J316

574

AT0

3-35

-18

-1

PCA

su

rfac

e lt

yello

w b

ig ir

regu

lar r

ough

with

bum

ps ri

gid

opaq

ue ~

dull

90

8 M

icro

cocc

us sp

. RG

-64

1.

00

AY

5616

23

AT0

3-35

-19

-2

1/10

0 PC

A s

urfa

ce l

t pk

glos

sy c

onve

x ~t

rans

82

9 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

35-2

0 -1

1/

100

PCA

sur

face

lt p

k gl

ossy

con

vex

~opa

que

908

Blas

toco

ccus

agg

rega

tus

0.97

A

J430

193

AT0

3-35

-21

-1

1/10

0 PC

A s

urfa

ce t

an ~

trans

glo

ssy

conv

ex

718

Blas

toco

ccus

agg

rega

tus

0.98

A

J430

193

AT0

3-35

-22

-2

1/10

0 PC

A s

urfa

ce p

k gl

ossy

con

vex

~tra

ns

831

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-36

-2

-1

1/10

0 PC

A s

urfa

ce l

t pk

~tra

ns ir

regu

lar f

lat g

loss

y 79

6 B

acte

rium

Elli

n602

3

0.98

A

Y23

4675

A

T03-

36-3

-1

PC

A

surf

ace

dark

ora

nge/

pink

irre

gula

r rai

sed

roug

h bi

g gl

ossy

but

dul

l 83

0 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

36-4

-1

1/

100

PCA

sur

face

lt p

ink

glos

sy ir

regu

lar ~

flat ~

trans

76

3 Ex

iguo

bact

eriu

m sp

.BTA

H1

0.98

A

Y20

5564

A

T03-

36-5

-1

1/

100

PCA

sur

face

lt p

ink/

lt pe

ach

~tra

ns ~

flat g

loss

y da

rker

in c

ente

r 66

4 Bl

asto

cocc

us a

ggre

gatu

s 0.

97

AJ4

3019

3 A

T03-

36-6

-1

1/

100

PCA

sur

face

lt p

ink

~tra

ns ~

flat g

loss

y da

rker

in c

ente

r 40

9 Bl

asto

cocc

us sp

. BC

521

0.

98

AJ3

1657

3 A

T03-

36-7

-1

1/

100

PCA

sur

face

lt p

ink

non-

glos

sy d

ull f

lat ~

trans

dar

ker i

n ce

nter

81

2 Bl

asto

cocc

us sp

. BC

521

0.

97

AJ3

1657

3 A

T03-

36-1

0 -1

1/

10 P

CA

su

rfac

e lt

oran

ge c

onve

x ~g

loss

y ~t

rans

mor

e op

aque

rais

ed d

ot in

cen

ter

961

Blas

toco

ccus

agg

rega

tus

0.98

A

J430

193

AT0

3-36

-11

-1

1/10

PC

A

surf

ace

pk fl

at ~

opaq

ue ro

ugh

rigid

~du

ll 86

9 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

36-1

2 -1

1/

10 P

CA

su

rfac

e ta

n/cr

eam

~op

aque

glo

ssy

conv

ex

941

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-36

-13

-1

1/10

PC

A

surf

ace

pk c

onve

x op

aque

~du

ll 73

7 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

36-1

4 -1

1/

10 P

CA

su

rfac

e pk

con

vex

opaq

ue ~

dull

830

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-36

-15

-1

1/10

PC

A

surf

ace

hot o

rang

e gl

ossy

con

vex

~tra

ns

924

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-36

-16

-1

NA

su

rfac

e ta

n ~t

rans

glo

ssy

conv

ex

949

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-36

-17

-1

NA

su

rfac

e or

ange

/pk

~tra

ns g

loss

y co

nvex

92

4 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

36-1

8 -1

M

A

surf

ace

pk sm

all o

paqu

e ~d

ull c

onve

x 98

1 Bl

asto

cocc

us sp

. BC

412

0.

98

AJ3

1657

4 A

T03-

36-1

9 -1

M

A

surf

ace

lt pk

opa

que

~dul

l con

vex

965

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-36

-20

-1

1/10

0 PC

A s

urfa

ce p

k tra

ns g

loss

y co

nvex

85

0 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1

128

(Tab

le c

ontin

ued:

Sur

face

isol

ates

) Su

rfac

e Is

olat

e

D

ilutio

n M

edia

D

epth

C

olon

y M

orph

olog

y

S

eque

nce

Len

gth

B

LA

ST R

esul

t

Si

mila

rity

Acc

essi

on n

o.

AT0

3-37

-1

-1

1/10

0 PC

A s

urfa

ce l

t pk/

lt pe

ach

~tra

ns g

loss

y co

nvex

66

2 B

acte

rium

Elli

n502

4

0.95

A

Y23

4441

A

T03-

37-2

-1

1/

100

PCA

sur

face

lt p

k ~t

rans

glo

ssy

conv

ex

839

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-37

-4

-1

1/10

0 PC

A s

urfa

ce l

t pk/

lt pe

ach

~tra

ns g

loss

y co

nvex

~irr

egul

ar

664

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-37

-6

-1

MA

su

rfac

e pk

~bu

mpy

surf

ace

rais

ed o

paqu

e gl

ossy

86

6 Bl

asto

cocc

us a

ggre

gatu

s 0.

98

AJ4

3019

3 A

T03-

37-7

-1

M

A

surf

ace

pk ~

bum

py su

rfac

e ra

ised

opa

que

glos

sy

700

Blas

toco

ccus

agg

rega

tus

0.96

A

J430

193

AT0

3-37

-8

-1

MA

su

rfac

e da

rker

pk

~opa

que

~glo

ssy

rais

ed e

dges

~irr

egul

ar su

rfac

e 82

8 Bl

asto

cocc

us a

ggre

gatu

s 0.

97

AJ4

3019

3 A

T03-

37-9

-2

M

A

surf

ace

~lt p

k op

aque

~irr

egul

ar &

~bu

mpy

surf

ace

glos

sy ra

ised

! 63

0 Bl

asto

cocc

us a

ggre

gatu

s 0.

96

AJ4

3019

3 A

T03-

37-1

0 -1

N

A

surf

ace

pk ~

opaq

ue d

ull c

onve

x 10

03

Blas

toco

ccus

agg

rega

tus

0.98

A

J430

193

AT0

3-38

-1

-3

PCA

su

rfac

e ta

nger

ine

oran

ge v

ery

conv

ex g

loss

y op

aque

82

2 B

acte

rium

Elli

n500

4

0.94

A

Y23

4421

A

T03-

38-2

-2

1/

10 P

CA

su

rfac

e w

hite

smal

l fla

t irr

egul

ar o

paqu

e 81

7 Ro

thia

sp. C

CU

G 2

5688

0.

99

AJ1

3112

2 A

T03-

38-4

-2

1/

10 P

CA

su

rfac

e sm

all w

hite

glo

ssy

conv

ex ~

trans

82

7 M

ycob

acte

rium

sp. E

SD

0.98

A

F284

430

AT0

3-38

-5

-2

1/10

PC

A

surf

ace

trans

whi

te ir

regu

lar m

ore

opaq

ue a

roun

d ed

ge w

hite

spec

s tra

ns in

cen

ter

835

Stap

hylo

cocc

us a

ureu

s 0.

99

BX

5718

57

AT0

3-38

-8

-2

1/10

0 PC

A s

urfa

ce p

k !~

trans

glo

ssy

conv

ex w

ith b

row

n sp

ec in

cen

ter

577

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-39

-1

-2

1/10

PC

A

surf

ace

smal

l whi

te ~

trans

glo

ssy

conv

ex

808

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-39

-2

-2

1/10

PC

A

surf

ace

whi

te fu

ngi l

ooki

ng b

ut n

ot a

hai

ry su

rfac

e fla

t glo

ssy

grow

th o

n su

rfac

e 64

7 Ba

cillu

s fir

mus

0.

99

D16

268

AT0

3-39

-3

-1

1/10

PC

A

surf

ace

smal

l lt p

urpl

e gl

ossy

con

vex

light

er a

roun

d ed

ge ~

trans

76

7 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

39-4

-1

1/

10 P

CA

su

rfac

e sm

all l

t pur

ple

glos

sy c

onve

x lig

hter

aro

und

edge

~tra

ns

784

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-39

-5

-1

1/10

PC

A

surf

ace

lt pi

nk sm

all g

loss

y co

nvex

~tra

ns

838

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-39

-6

-1

1/10

0 PC

A s

urfa

ce l

t pin

k sm

all g

loss

y co

nvex

~tra

ns

704

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-39

-7

-1

1/10

0 PC

A s

urfa

ce w

hite

/lt p

ink

smal

l glo

ssy

conv

ex

770

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-39

-11

-1

1/10

PC

A

surf

ace

smal

l lt p

urpl

e gl

ossy

con

vex

light

er a

roun

d ed

ge ~

trans

80

2 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

39-1

2 -1

1/

10 P

CA

su

rfac

e lt

pink

smal

l glo

ssy

conv

ex ~

trans

84

6 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

39-1

3 -1

1/

10 P

CA

su

rfac

e lt

purp

le sm

all g

loss

y co

nvex

ligh

ter a

roun

d ed

ge ~

trans

70

7 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

39-1

4 -1

1/

10 P

CA

su

rfac

e lt

pink

~tra

ns sm

all g

loss

y co

nvex

83

8 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

39-1

5 -2

1/

100

PCA

sur

face

lt p

ink

trans

glo

ssy

~fla

t sm

all

800

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

3-39

-16

-1

NA

su

rfac

e ta

nger

ine/

peac

h ~t

rans

glo

ssy

conv

ex

842

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-40

-1

-3

1/10

PC

A

surf

ace

lt pe

ach/

pink

mor

e da

rk in

cen

ter g

loss

y co

nvex

77

1 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

40-2

-3

1/

10 P

CA

su

rfac

e lt

peac

h fla

t glo

ssy

781

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-40

-3

-3

1/10

PC

A

surf

ace

lt pe

ach/

pink

irre

gula

r glo

ssy

flat

830

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

3-40

-4

-3

1/10

0 PC

A s

urfa

ce !

lt pi

nk f

lat i

rreg

ular

glo

ssy

smal

l col

ony

arou

nd b

lob

741

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

3-40

-5

-2

1/10

0 PC

A s

urfa

ce !

lt pi

nk (a

lmos

t whi

te) ~

trans

flat

glo

ssy

smal

l 87

0 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T03-

40-6

-2

1/

100

PCA

sur

face

!lt

pink

(alm

ost w

hite

) ~tra

ns fl

at g

loss

y sm

all

932

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-40

-7

-1

1/10

PC

A

surf

ace

smal

l irr

egul

ar lt

pin

k/pe

ach

conv

ex ~

trans

83

9 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

40-8

-1

1/

10 P

CA

su

rfac

e lt

pink

smal

l con

vex

~tra

ns g

loss

y 78

4 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

40-1

1 -1

PC

A

surf

ace

dark

er o

rang

e co

nvex

big

glo

ssy

~tra

ns

458

Blas

toco

ccus

sp. B

C52

1

0.99

A

J316

573

AT0

3-40

-12

-1

PCA

su

rfac

e lt

pink

/lt o

rang

e co

nvex

~gl

ossy

tran

s bum

p at

cen

ter t

op

737

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-40

-13

-1

PCA

su

rfac

e hu

ge d

arke

r. pi

nk ri

gid

~tra

ns n

on-g

loss

y irr

egul

ar

747

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-40

-14

-1

PCA

su

rfac

e lt

pink

con

vex

non-

glos

sy ~

opaq

ue

785

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-40

-15

-1

PCA

su

rfac

e bi

g lt

pink

/lt ta

n gl

ossy

con

vex

~tra

ns

805

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-40

-17

-1

PCA

su

rfac

e lt

pink

tran

s irr

egul

ar n

on-g

loss

y ra

ised

roug

h 77

1 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

40-1

9 -2

1/

100

PCA

sur

face

tra

nslu

cent

~fla

t glo

ssy

rais

ed sp

ot in

cen

ter

950

Orn

ithin

icoc

cus h

orte

nsis

0.

94

Y17

869

AT0

3-40

-20

-2

1/10

0 PC

A s

urfa

ce v

ery

lt pi

nk (a

lmos

t whi

te) g

loss

y co

nvex

~tra

ns

947

Orn

ithin

icoc

cus h

orte

nsis

0.

94

Y17

869

AT0

3-40

-21

-2

1/10

0 PC

A s

urfa

ce l

t pin

k gl

ossy

smal

l con

vex

~tra

ns li

ghte

r aro

und

edge

88

1 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

40-2

2 -1

1/

100

PCA

sur

face

tra

nslu

cent

~fla

t glo

ssy

rais

ed sp

ot in

cen

ter

822

Orn

ithin

icoc

cus h

orte

nsis

0.

94

Y17

869

AT0

3-40

-23

-1

1/10

0 PC

A s

urfa

ce v

ery

lt pi

nk (a

lmos

t tra

ns) c

onve

x gl

ossy

74

6 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

40-2

4 -1

1/

100

PCA

sur

face

lt p

ink

~tra

ns g

loss

y co

nvex

ligh

ter a

roun

d ed

ge

825

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-40

-25

-1

1/10

0 PC

A s

urfa

ce t

rans

luce

nt ~

flat g

loss

y ra

ised

spot

in c

ente

r 94

0 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1

129

(Tab

le c

ontin

ued:

Sur

face

isol

ates

) Su

rfac

e Is

olat

e

D

ilutio

n M

edia

D

epth

C

olon

y M

orph

olog

y

Se

quen

ce L

engt

h

BL

AST

Res

ult

Sim

ilari

ty A

cces

sion

no.

A

T03-

40-2

6 -1

1/

100

PCA

sur

face

tin

y lt

pk g

loss

y co

nvex

~op

aque

81

2 Bl

asto

cocc

us sp

. BC

448

0.

96

AJ3

1657

1 A

T03-

40-2

8 -1

1/

100

PCA

sur

face

tra

nslu

cent

ver

y lt

pink

con

vex

glos

sy

851

Orn

ithin

icoc

cus h

orte

nsis

0.

94

Y17

869

AT0

3-40

-29

-1

1/10

PC

A

surf

ace

crea

my

lt pi

nk ~

trans

con

vex

glos

sy

811

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-40

-30

-1

1/10

PC

A

surf

ace

lt pi

nk/lt

pea

ch ir

regu

lar r

ough

~tra

ns n

on-g

loss

y 83

4 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

40-3

1 -1

1/

10 P

CA

su

rfac

e cr

eam

y lt

pink

~op

aque

con

vex

glos

sy

780

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-40

-32

-1

1/10

PC

A

surf

ace

dark

pin

k gl

ossy

~op

aque

con

vex

777

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-40

-33

-1

1/10

PC

A

surf

ace

dark

pin

k gl

ossy

~op

aque

con

vex

833

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

3-40

-34

-1

1/10

PC

A

surf

ace

crea

my

lt ta

n/lt

peac

h co

nvex

glo

ssy

~tra

ns li

ghte

r aro

und

edge

83

9 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T03-

40-3

5 -1

1/

10 P

CA

su

rfac

e da

rk p

ink

glos

sy ~

opaq

ue c

onve

x 77

7 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

40-3

6 -1

1/

10 P

CA

su

rfac

e lt

peac

h gl

ossy

con

vex

~tra

ns

851

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

3-40

-39

-2

1/10

0 PC

A s

urfa

ce p

inki

sh ta

n ~t

rans

~fla

t irre

gula

r glo

ssy

rais

ed sp

ot in

cen

ter

842

Orn

ithin

icoc

cus h

orte

nsis

0.

94

Y17

869

AT0

3-40

-40

-2

1/10

0 PC

A s

urfa

ce l

t pin

k tra

ns g

loss

y co

nvex

83

2 Bl

asto

cocc

us sp

. BC

521

0.

97

AJ3

1657

3 A

T03-

40-4

2 -2

1/

100

PCA

sur

face

cre

amy

very

lt p

ink

(alm

ost w

hite

) ~op

aque

glo

ssy

conv

ex

845

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-40

-43

-2

1/10

PC

A

surf

ace

lt pe

ach

trans

glo

ssy

conv

ex

817

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-40

-44

-2

1/10

PC

A

surf

ace

lt pe

ach

trans

glo

ssy

conv

ex

464

Blas

toco

ccus

sp. B

C52

1

0.97

A

J316

573

AT0

3-40

-45

-3

1/10

0 PC

A s

urfa

ce l

t tan

/lt p

ink

glos

sy ~

conv

ex ~

trans

84

5 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

40-4

6 -3

1/

10 P

CA

su

rfac

e ta

n lt

pink

~co

nvex

glo

ssy

~tra

ns

854

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-40

-47

-3

1/10

PC

A

surf

ace

tan

lt pi

nk ~

conv

ex g

loss

y ~t

rans

irre

gula

r clu

ster

ed

790

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

3-40

-48

-2

1/10

PC

A

surf

ace

lt ta

n da

rker

in c

ente

r lig

ht e

dges

~gl

ossy

~tra

ns c

onve

x 81

2 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

40-4

9 -2

1/

10 P

CA

su

rfac

e lt

tan

dark

er c

ente

r mor

e tra

ns e

dge

very

glo

ssy

~tra

ns c

onve

x ce

nter

82

0 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

40-5

0 -3

1/

10 P

CA

su

rfac

e ta

n/or

ange

dar

ker i

n ce

nter

ver

y lig

ht a

roun

d ed

ge v

ery

glos

sy ~

conv

ex

818

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-40

-51

-2

1/10

PC

A

surf

ace

tan

lt pi

nk ~

conv

ex ~

glos

sy ~

trans

79

2 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

40-5

2 -1

PC

A

surf

ace

dark

er o

rang

e ve

ry c

onve

x irr

egul

ar o

paqu

e du

ll no

n-gl

ossy

75

0 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

40-5

3 -1

PC

A

surf

ace

lt or

ange

ver

y co

nvex

~tra

ns g

loss

y 82

4 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

40-5

4 -1

PC

A

surf

ace

oran

ge ~

glos

sy c

onve

x m

ore

conv

ex d

ome

in c

ente

r opa

que

711

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

3-40

-55

-1

PCA

su

rfac

e or

ange

~gl

ossy

!con

vex

mor

e co

nvex

smal

l dom

e of

fsid

e of

cen

ter ~

trans

76

7 St

aphy

loco

ccus

sp. A

I-22

0.

98

AY

4376

31

AT0

3-40

-56

-1

PCA

su

rfac

e da

rker

ora

nge

conv

ex w

ith h

ole

in c

ente

r opa

que

dull

non-

glos

sy

819

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-40

-57

-1

PCA

su

rfac

e lt

oran

ge c

onve

x gl

ossy

~op

aque

97

2 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

40-5

8 -1

N

A

surf

ace

lt pk

con

cave

cen

ter ~

dull

~opa

que

roun

d ra

ised

edg

es

729

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

3-40

-59

-1

NA

su

rfac

e ta

n tra

ns g

loss

y co

nvex

79

7 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T03-

40-6

0 -1

N

A

surf

ace

tan

trans

glo

ssy

conv

ex

927

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-40

-61

-1

MA

su

rfac

e lt

pk o

paqu

e gl

ossy

con

vex

768

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

3-40

-62

-1

MA

su

rfac

e lt

pk o

paqu

e gl

ossy

con

vex

705

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

3-40

-63

-2

MA

su

rfac

e lt

pk c

onve

x op

aque

~gl

ossy

87

4 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

40-6

4 -2

M

A

surf

ace

!lt p

k (a

lmos

t whi

te) i

rreg

ular

roug

h rig

id d

ull r

aise

d op

aque

81

2 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T03-

40-6

6 -2

N

A

surf

ace

lt pk

tran

s glo

ssy

conv

ex

902

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-40

-67

-1

NA

su

rfac

e ta

n tra

ns g

loss

y co

nvex

67

0 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

40-6

8 -1

N

A

surf

ace

lt pk

irre

gula

r con

cave

cen

ter ~

glos

sy ra

ised

bum

py

902

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

3-40

-69

-1

NA

su

rfac

e lt

pk g

loss

y ra

ised

with

~co

ncav

e ce

nter

opa

que

622

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-1

-2

PCA

su

rfac

e or

ange

ver

y co

nvex

glo

ssy

spec

s in

colo

ny ~

trans

74

1 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

41-2

-2

PC

A

surf

ace

dark

ora

nge/

pink

irre

gula

r rou

gh ra

ised

non

-glo

ssy

614

Fran

kia

sp. (

stra

in A

VN

17s)

0.

93

L406

13

AT0

3-41

-3

-2

1/10

PC

A

surf

ace

pink

/ora

nge

big

~glo

ssy

opaq

ue ~

flat

781

Blas

toco

ccus

sp. B

C41

2

0.97

A

J316

574

AT0

3-41

-4

-3

1/10

0 PC

A s

urfa

ce l

t pin

k gl

ossy

flat

irre

gula

r dar

ker i

n ce

nter

tran

s aro

und

edge

s 85

0 Bl

asto

cocc

us a

ggre

gatu

s 0.

97

AJ4

3019

3 A

T03-

41-5

-1

PC

A

surf

ace

brow

n no

n-gl

ossy

irre

gula

r bum

py o

paqu

e 81

6 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

41-6

-1

PC

A

surf

ace

whi

te n

on-g

loss

y op

aque

con

vex

with

who

le in

cen

ter p

aste

y 82

9 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

41-7

-1

PC

A

surf

ace

big

purp

le/p

ink

conv

ex n

on-g

loss

y op

aque

ring

s in

colo

ny

841

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-8

-1

PCA

su

rfac

e irr

egul

ar b

umpy

ora

nge

~tra

ns g

loss

y ed

ges &

dar

ker ~

glos

sy o

paqu

e ce

nter

76

9 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1

130

(Tab

le c

ontin

ued:

Sur

face

isol

ates

) Su

rfac

e Is

olat

e

D

ilutio

n M

edia

D

epth

C

olon

y M

orph

olog

y

S

eque

nce

Len

gth

B

LA

ST R

esul

t

Si

mila

rity

Acc

essi

on n

o.

AT0

3-41

-9

-1

PCA

su

rfac

e sm

all p

ink/

oran

ge g

loss

y tra

ns c

onve

x 86

2 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

41-1

0 -1

PC

A

surf

ace

dark

er p

ink

glos

sy o

paqu

e co

nvex

but

con

cave

in c

ente

r 82

4 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

41-1

1 -1

PC

A

surf

ace

smal

l lt p

ink/

oran

ge ~

trans

con

vex

~glo

ssy

roug

h 81

6 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T03-

41-1

2 -1

PC

A

surf

ace

smal

l bro

wn

conv

ex n

on-g

loss

y ha

rd d

arke

r in

cent

er o

paqu

e 70

3 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

41-1

3 -1

PC

A

surf

ace

dark

er. p

ink

bum

py ro

ugh

opaq

ue c

onve

x no

n-gl

ossy

84

1 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

41-1

5 -2

1/

10 P

CA

su

rfac

e bi

g lt

pink

flat

non

-glo

ssy

opaq

ue c

onve

x do

t in

cent

er

827

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

3-41

-17

-1

1/10

PC

A

surf

ace

opaq

ue lt

pur

ple/

pink

~co

nvex

glo

ssy

arou

nd e

dge

dull

in c

ente

r 85

8 B

acte

rium

Elli

n500

4

0.98

A

Y23

4421

A

T03-

41-1

8 -1

1/

10 P

CA

su

rfac

e lt

tan

glos

sy ~

trans

edg

es &

dar

ker o

rang

e ~c

onve

x gl

ossy

opa

que

cent

er

810

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-41

-20

-1

1/10

PC

A

surf

ace

whi

te/ta

n ~t

rans

glo

ssy

arou

nd e

dges

& ta

n gl

ossy

~tra

ns c

onve

x ce

nter

71

5 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

41-2

1 -1

1/

10 P

CA

su

rfac

e tra

nslu

cent

flat

non

-glo

ssy

850

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-22

-1

1/10

PC

A

surf

ace

trans

~co

nvex

glo

ssy

ring

in c

ente

r 83

6 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

41-2

3 -1

1/

10 P

CA

su

rfac

e lt

pink

irre

gula

r con

vex

dull

non-

glos

sy ro

ugh

~tra

ns

800

Geo

rgen

ia sp

. 221

6.35

.28

0.

93

AB

0944

66

AT0

3-41

-24

-2

1/10

0 PC

A s

urfa

ce l

t tan

glo

ssy

~con

vex

~tra

ns

830

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-41

-26

-1

1/10

0 PC

A s

urfa

ce w

hite

tran

s glo

ssy

flat w

ith ra

ised

cen

ter s

mal

l 80

6 Bl

asto

cocc

us sp

. BC

521

0.

98

AJ3

1657

3 A

T03-

41-2

7 -1

1/

100

PCA

sur

face

lt p

ink

irreg

ular

glo

ssy

~con

vex

~tra

ns

837

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-28

-1

1/10

0 PC

A s

urfa

ce l

t pin

k irr

egul

ar n

on-g

loss

y ~c

onve

x ~t

rans

81

6 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

41-2

9 -1

1/

100

PCA

sur

face

lt p

k tra

ns e

dge

glos

sy ra

ised

cen

ter d

arke

r pin

k &

mor

e op

aque

cen

ter

622

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-30

-1

PCA

su

rfac

e lt

pink

/lt b

row

n gl

ossy

smal

l con

vex

~tra

ns

894

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-31

-2

1/10

PC

A

surf

ace

~lt p

ink

~fla

t dul

l non

-glo

ssy

big

past

ey o

paqu

e 84

2 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T03-

41-3

2 -2

1/

10 P

CA

su

rfac

e da

rker

pin

k ~f

lat g

loss

y ~d

ull b

ig p

aste

y op

aque

82

3 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

41-3

3 -2

1/

100

PCA

sur

face

whi

te ~

opaq

ue g

loss

y co

nvex

70

1 Bl

asto

cocc

us a

ggre

gatu

s 0.

97

AJ4

3019

3 A

T03-

41-3

5 -2

PC

A

surf

ace

crea

my

brig

ht p

k op

aque

glo

ssy

conv

ex

827

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-36

-1

1/10

PC

A

surf

ace

dark

er o

rang

e gl

ossy

~tra

ns c

onve

x 60

4 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

41-3

7 -1

1/

10 P

CA

su

rfac

e cr

eam

y lt

pk/ta

n ~o

paqu

e gl

ossy

con

vex

825

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-38

-1

1/10

PC

A

surf

ace

purp

le ~

trans

glo

ssy

conv

ex

825

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-41

-39

-1

1/10

PC

A

surf

ace

crea

my

dark

er p

k/or

ange

~op

aque

con

vex

glos

sy

834

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-40

-1

1/10

PC

A

surf

ace

crea

my

dark

er o

rang

e/da

rker

pk

opaq

ue c

onve

x gl

ossy

77

3 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

41-4

1 -1

1/

10 P

CA

su

rfac

e cr

eam

y ta

n ~o

paqu

e co

nvex

glo

ssy

729

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-41

-42

-1

1/10

PC

A

surf

ace

lt pk

non

-glo

ssy

dull

conv

ex o

paqu

e te

xtur

ed

847

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-43

-1

1/10

PC

A

surf

ace

brig

ht p

ink

tiny

opaq

ue c

onve

x ~g

loss

y 86

5 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

41-4

4 -1

1/

10 P

CA

su

rfac

e da

rker

red

tiny

trans

glo

ssy

conv

ex

783

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-45

-1

1/10

PC

A

surf

ace

yello

w c

ream

tran

s glo

ssy

~con

vex

788

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-41

-46

-1

1/10

PC

A

surf

ace

dark

er tr

ans g

loss

y co

nvex

77

7 Bl

asto

cocc

us a

ggre

gatu

s 0.

98

AJ4

3019

3 A

T03-

41-4

7 -1

1/

10 P

CA

su

rfac

e br

ight

pin

k op

aque

glo

ssy

conv

ex

840

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-48

-1

1/10

PC

A

surf

ace

crea

my

tan

~opa

que

conv

ex g

loss

y 81

2 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

41-4

9 -1

1/

10 P

CA

su

rfac

e lt

pk n

on-g

loss

y du

ll co

nvex

opa

que

text

ured

72

2 Bl

asto

cocc

us a

ggre

gatu

s 0.

98

AJ4

3019

3 A

T03-

41-5

0 -1

1/

10 P

CA

su

rfac

e lt

pk/ta

n gl

ossy

con

vex

~tra

ns

759

Blas

toco

ccus

sp. B

C44

8

0.96

A

J316

571

AT0

3-41

-51

-1

1/10

PC

A

surf

ace

dark

er p

k/lt

oran

ge g

loss

y co

nvex

~op

aque

80

8 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

41-5

2 -1

1/

10 P

CA

su

rfac

e ye

llow

cre

am tr

ans g

loss

y ~c

onve

x 73

3 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

41-5

3 -2

1/

10 P

CA

su

rfac

e w

hite

tran

s glo

ssy

conv

ex d

ark

spot

in c

ente

r 79

4 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

41-5

4 -2

1/

10 P

CA

su

rfac

e lt

pk n

on-g

loss

y du

ll co

nvex

opa

que

text

ured

77

7 Bl

asto

cocc

us sp

. BC

448

0.

95

AJ3

1657

1 A

T03-

41-5

5 -2

1/

10 P

CA

su

rfac

e cr

eam

y lt

pk ~

trans

con

vex

glos

sy

805

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-41

-56

-1

PCA

su

rfac

e br

ight

pin

k op

aque

irre

gula

r rai

sed

smoo

th (l

ooks

like

a n

erd)

81

1 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

41-5

7 -1

PC

A

surf

ace

lt pk

/tan

~tra

ns c

onve

x gl

ossy

83

9 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

41-5

8 -1

PC

A

surf

ace

brig

ht p

k op

aque

smal

l con

vex

~glo

ssy

767

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-41

-59

-1

1/10

0 PC

A s

urfa

ce w

hite

tran

s glo

ssy

conv

ex

816

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-60

-1

1/10

0 PC

A s

urfa

ce t

rans

glo

ssy

~con

vex

746

Blas

toco

ccus

sp. B

C52

1

0.97

A

J316

573

131

(Tab

le c

ontin

ued:

Sur

face

isol

ates

) Su

rfac

e Is

olat

e

D

ilutio

n M

edia

D

epth

C

olon

y M

orph

olog

y

Se

quen

ce L

engt

h

BL

AST

Res

ult

Sim

ilari

ty A

cces

sion

no.

A

T03-

41-6

1 -1

1/

100

PCA

sur

face

cre

amy

lt pk

glo

ssy

conv

ex ~

trans

73

8 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

41-6

2 -1

1/

100

PCA

sur

face

tra

ns g

loss

y ~c

onve

x 81

0 Pr

omic

rom

onos

pora

ent

erop

hila

0.

95

X83

807

AT0

3-41

-63

-1

1/10

0 PC

A s

urfa

ce y

ello

w fl

at g

loss

y tra

ns ir

regu

lar

731

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-64

-1

1/10

0 PC

A s

urfa

ce l

t pk

glos

sy c

onve

x ~t

rans

79

1 C

ellu

lom

onas

car

tae

MSD

201

06

0.95

X

7945

6 A

T03-

41-6

5 -1

1/

100

PCA

sur

face

cre

amy

lt pk

glo

ssy

conv

ex ~

trans

82

6 O

xalo

bact

er sp

. Es2

-1

0.98

A

Y36

7029

A

T03-

41-6

7 -2

1/

100

PCA

sur

face

cre

amy

very

lt p

k gl

ossy

con

vex

~opa

que

810

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-68

-1

1/10

0 PC

A s

urfa

ce c

ream

y ve

ry lt

pk

glos

sy c

onve

x ~o

paqu

e 61

2 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T03-

41-6

9 -1

1/

100

PCA

sur

face

tra

ns g

loss

y co

nvex

tiny

46

3 Bl

asto

cocc

us sp

. BC

521

0.

98

AJ3

1657

3 A

T03-

41-7

0 -1

1/

100

PCA

sur

face

tra

ns lt

pk

glos

sy c

onve

x 60

1 Bl

asto

cocc

us sp

. BC

521

0.

98

AJ3

1657

3 A

T03-

41-7

1 -1

1/

100

PCA

sur

face

lt p

k gl

ossy

con

vex

~tra

ns

807

Cel

lulo

mon

as c

arta

e M

SD 2

0106

0.

95

X79

456

AT0

3-41

-73

-1

1/10

0 PC

A s

urfa

ce c

ream

y tra

ns g

loss

y co

nvex

82

3 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

41-7

4 -2

1/

100

PCA

sur

face

lt p

k gl

ossy

con

vex

~opa

que

659

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

3-41

-76

-1

MA

su

rfac

e lt

pk g

loss

y co

nvex

opa

que

826

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-41

-77

-1

MA

su

rfac

e of

fwhi

te g

loss

y co

nvex

opa

que

762

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-78

-1

MA

su

rfac

e of

fwhi

te ~

trans

glo

ssy

conv

ex

765

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-79

-1

MA

su

rfac

e da

rker

pea

ch/o

rang

e gl

ossy

rais

ed o

paqu

e bu

mpy

surf

ace

660

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

3-41

-80

-1

MA

su

rfac

e of

fwhi

te ir

regu

lar r

igid

roug

h su

rfac

e du

ll ra

ised

opa

que

481

Cel

lulo

mon

as c

arta

e M

SD 2

0106

0.

94

X79

456

AT0

3-41

-81

-1

MA

su

rfac

e da

rker

pea

ch/o

rang

e irr

egul

ar ri

gid

roug

h su

rfac

e du

ll ra

ised

opa

que

705

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-82

-1

MA

su

rfac

e lt

pk ~

conv

ex g

loss

y ~t

rans

75

0 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T03-

41-8

3 -1

M

A

surf

ace

trans

~of

fwhi

te g

loss

y co

nvex

75

1 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

41-8

4 -2

M

A

surf

ace

~yel

low

tran

s glo

ssy

conv

ex

706

Mod

esto

bact

er m

ultis

epta

tus

0.98

A

J871

304

AT0

3-41

-85

-2

MA

su

rfac

e pk

~irr

egul

ar ~

dull

rais

ed b

umpy

rigi

d su

rfac

e 75

1 Pr

omic

rom

onos

pora

ent

erop

hila

0.

95

X83

807

AT0

3-41

-86

-2

MA

su

rfac

e ye

llow

~tra

ns g

loss

y co

nvex

74

2 Pr

omic

rom

onos

pora

ent

erop

hila

0.

95

X83

807

AT0

3-41

-87

-2

MA

su

rfac

e m

ed lt

pk

with

dar

ker c

ente

r con

vex

(con

vex

bum

p in

cen

ter)

glo

ssy

opaq

ue

779

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-88

-2

NA

su

rfac

e pe

ach/

pk c

onve

x op

aque

glo

ssy

764

Cel

lulo

mon

as c

ella

sea

0.

98

X83

804

AT0

3-41

-89

-1

NA

su

rfac

e or

ange

~op

aque

con

vex

glos

sy

606

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-90

-1

NA

su

rfac

e of

fwhi

te o

paqu

e ~f

lat g

loss

y 80

8 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

41-9

1 -1

N

A

surf

ace

trans

~pu

rple

/pk

glos

sy c

onve

x 79

1 Bl

asto

cocc

us a

ggre

gatu

s 0.

97

AJ4

3019

3 A

T03-

41-9

2 -1

N

A

surf

ace

offw

hite

/tan

dark

er c

ente

r glo

ssy

conv

ex ~

trans

77

2 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

41-9

3 -1

N

A

surf

ace

brig

ht o

rang

e irr

egul

ar ra

ised

opa

que

dull

rigid

roug

h 77

0 Bl

asto

cocc

us a

ggre

gatu

s 0.

97

AJ4

3019

3 A

T03-

41-9

4 -1

N

A

surf

ace

lt pk

opa

que

~dul

l ~irr

egul

ar ra

ised

78

0 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

41-9

5 -1

N

A

surf

ace

lt pk

opa

que

~glo

ssy

conv

ex

775

Blas

toco

ccus

agg

rega

tus

0.97

A

J430

193

AT0

3-41

-96

-1

NA

su

rfac

e pe

ach

crea

msa

ver g

loss

y ra

ised

~op

aque

77

6 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T03-

41-9

7 -1

N

A

surf

ace

pk g

loss

y co

nvex

opa

que

702

Blas

toco

ccus

agg

rega

tus

0.97

A

J430

193

AT0

3-41

-98

-2

NA

su

rfac

e bi

g/m

ed lt

pk/

peac

h ~c

onve

x gl

ossy

opa

que

781

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-99

-1

NA

su

rfac

e tra

ns g

loss

y co

nvex

~ta

n/pu

rple

78

2 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

41-1

00

-1

NA

su

rfac

e ra

ised

ora

nge/

pk ir

regu

lar o

paqu

e ~g

loss

y 76

1 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

41-1

01

-1

NA

su

rfac

e pe

ach/

pk g

loss

y ~o

paqu

e co

nvex

80

9 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

41-1

03

-2

NA

su

rfac

e ho

t pk

conv

ex o

paqu

e ~d

ull

630

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-41

-104

-2

N

A

surf

ace

trans

tan

glos

sy c

onve

x 96

1 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

41-1

05

-2

NA

su

rfac

e ho

t pk

conv

ex c

ente

r opa

que

~dul

l rai

sed

edge

s 10

27

Blas

toco

ccus

agg

rega

tus

0.98

A

J430

193

AT0

3-41

-106

-2

N

A

surf

ace

trans

tan

glos

sy c

onve

x 92

5 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

41-1

07

-2

NA

su

rfac

e ho

t pk

conc

ave

cent

er o

paqu

e ~d

ull r

aise

d ed

ges

821

Blas

toco

ccus

sp. B

C41

2

0.98

A

J316

574

AT0

3-41

-108

-2

N

A

surf

ace

~lt p

k gl

ossy

con

vex

~opa

que

854

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-42

-1

-2

PCA

su

rfac

e bi

g w

hite

irre

gula

r ~co

nvex

opa

que

~glo

ssy

849

Baci

llus c

ereu

s 0.

99

AJ5

7728

1 A

T03-

42-2

-2

M

A

surf

ace

roun

d ye

llow

glo

ssy

flat o

paqu

e 80

4 N

ocar

dioi

des s

p. M

WH

-CaK

6

0.99

A

J565

419

AT0

3-43

-1

-1

1/10

0 PC

A s

urfa

ce p

each

/pin

k irr

egul

ar n

on-g

loss

y ~c

onve

x ro

ugh

827

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

132

(Tab

le c

ontin

ued:

Sur

face

isol

ates

) Su

rfac

e Is

olat

e

D

ilutio

n M

edia

D

epth

C

olon

y M

orph

olog

y

S

eque

nce

Len

gth

B

LA

ST R

esul

t

Si

mila

rity

Acc

essi

on n

o.

AT0

3-43

-3

-1

1/10

PC

A

surf

ace

oran

ge/p

ink

conv

ex g

loss

y op

aque

75

9 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

43-4

-2

PC

A

surf

ace

dark

er o

rang

e ve

ry c

onve

x ~d

ull ~

glos

sy o

paqu

e 82

6 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

43-5

-2

1/

10 P

CA

su

rfac

e lt

pink

~fla

t ~tra

ns ~

glos

sy

783

Blas

toco

ccus

sp. B

C52

1

0.97

A

J316

573

AT0

3-43

-6

-1

PCA

su

rfac

e da

rker

ora

nge

very

con

vex

very

glo

ssy

opaq

ue

543

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-43

-7

-1

MA

su

rfac

e lt

oran

ge ~

irreg

ular

~fla

t opa

que

glos

sy ~

rigid

/roug

h 10

36

Kin

eoco

ccus

-like

bac

teriu

m A

S313

8

0.99

A

F060

686

AT0

3-43

-8

-1

MA

su

rfac

e ho

t pk/

oran

ge o

paqu

e ra

ised

roug

h/rig

id b

umpy

irre

gula

r 10

52

Cel

lulo

mon

as c

ella

sea

0.

98

X83

804

AT0

3-43

-9

-1

MA

su

rfac

e lt

oran

ge ~

irreg

ular

rais

ed g

loss

y ~u

neve

n su

rface

~op

aque

10

03

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-43

-10

-1

MA

su

rfac

e ho

t ora

nge

big

opaq

ue g

loss

y co

ncav

e irr

egul

ar u

neve

n ce

nter

edg

es ra

ised

10

17

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-43

-11

-1

MA

su

rfac

e irr

egul

ar m

ed lt

ora

nge/

hot y

ello

w ra

ised

bum

py ri

gid

roug

h ~t

rans

10

42

Kin

eoco

ccus

-like

bac

teriu

m A

S313

8

0.98

A

F060

686

AT0

3-43

-12

-1

MA

su

rfac

e pk

irre

gula

r rai

sed

opaq

ue b

umpy

rigi

d ro

ugh

1043

C

ellu

lom

onas

cel

lase

a

0.98

X

8380

4 A

T03-

43-1

3 -2

N

A

surf

ace

med

ora

nge

opaq

ue c

ente

r glo

ssy

conv

ex ~

trans

off

whi

te e

dges

10

44

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-43

-14

-1

NA

su

rfac

e pk

/ora

nge

opaq

ue ~

umbo

nate

glo

ssy

1034

Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

43-1

5 -1

N

A

surf

ace

pk/o

rang

e gl

ossy

~op

aque

con

vex

1044

Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

43-1

6 -1

N

A

surf

ace

pk/o

rang

e gl

ossy

um

bona

te o

paqu

e 90

9 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

43-1

7 -2

1/

100

PCA

sur

face

pk

glos

sy ~

conv

ex ~

opaq

ue

1000

K

ineo

cocc

us-li

ke b

acte

rium

AS3

138

0.

98

AF0

6068

6 A

T03-

43-1

8 -1

1/

100

PCA

sur

face

pk/

purp

le g

loss

y ~o

paqu

e ~c

onve

x ~i

rreg

ular

10

45

Blas

toco

ccus

sp. B

C41

2

0.98

A

J316

574

AT0

3-43

-19

-1

1/10

0 PC

A s

urfa

ce l

t pk

~tra

ns g

loss

y ~c

onve

x ~d

arke

r in

cent

er

966

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-43

-22

-1

1/10

0 PC

A s

urfa

ce h

ot o

rang

e/ho

t pin

k ~i

rreg

ular

glo

ssy

~tra

ns ~

conv

ex

1023

Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

43-2

3 -1

1/

100

PCA

sur

face

pk

trans

glo

ssy

irreg

ular

~ra

ised

85

0 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

43-2

5 -1

1/

100

PCA

sur

face

lt p

k ~i

rreg

ular

~ra

ised

glo

ssy

~tra

ns

932

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-43

-26

-1

1/10

PC

A

surf

ace

pk/o

rang

e ~c

onve

x op

aque

glo

ssy

roun

d 92

1 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

43-2

7 -1

1/

10 P

CA

su

rfac

e pk

/ora

nge

conv

ex g

loss

y ro

und

~opa

que

980

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-43

-28

-1

1/10

PC

A

surf

ace

brow

nish

ora

nge

~tra

ns g

loss

y co

nvex

84

7 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

43-2

9 -2

M

A

surf

ace

dark

er b

right

ora

nge

roug

h rig

id b

umpy

irre

gula

r opa

que

glos

sy

1071

K

ineo

cocc

us-li

ke b

acte

rium

AS3

138

0.

98

AF0

6068

6 A

T03-

43-3

0 -2

M

A

surf

ace

gold

en y

ello

w) ~

irreg

ular

rais

ed g

loss

y ~u

neve

n su

rfac

e ~t

rans

10

03

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-43

-31

-2

MA

su

rfac

e bi

g or

ange

/pk

opaq

ue ~

glos

sy c

onca

ve u

neve

n ce

nter

edg

es ra

ised

roun

d 95

9 K

ocur

ia e

ryth

rom

yxa

0.

99

Y11

330

AT0

3-43

-32

-1

1/10

PC

A

surf

ace

med

pk/

oran

ge ro

und

~con

vex

glos

sy ~

trans

94

4 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

43-3

3 -1

1/

10 P

CA

su

rfac

e sm

all l

t tan

/lt p

k gl

ossy

con

vex

~tra

ns

961

Koc

uria

ery

thro

myx

a

0.99

Y

1133

0 A

T03-

43-3

4 -1

1/

10 P

CA

su

rfac

e da

rker

brig

ht o

rang

e ro

und

conv

ex g

loss

y op

aque

96

5 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

44-1

-2

1/

10 P

CA

su

rfac

e lt

peac

h co

nvex

ver

y gl

ossy

~op

aque

in c

ente

r tra

ns e

dges

84

4 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

45-1

-1

PC

A

surf

ace

lt pi

nk ir

regu

lar ~

glos

sy ra

ised

~op

aque

85

9 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

45-2

-1

1/

100

PCA

sur

face

lt p

ink

glos

sy ~

conv

ex ~

irreg

ular

~tra

ns

851

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-45

-3

-1

1/10

0 PC

A s

urfa

ce l

t pin

k gl

ossy

~co

nvex

~irr

egul

ar ~

trans

76

1 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

45-4

-1

1/

100

PCA

sur

face

dar

ker p

k gl

ossy

con

vex

~tra

ns

809

Unc

ult a

ctin

obac

teriu

m c

lone

AC

TIN

O2B

0.

96

AY

4946

41

AT0

3-45

-5

-1

1/10

0 PC

A s

urfa

ce d

arke

r pk

glos

sy c

onve

x ~t

rans

84

8 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

45-7

-1

1/

100

PCA

sur

face

lt p

ink

glos

sy c

onve

x ~t

rans

72

0 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T03-

45-9

-1

1/

10 P

CA

su

rfac

e cr

eam

y lt

pk g

loss

y co

nvex

~tra

ns

812

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-45

-10

-1

1/10

PC

A

surf

ace

dark

er p

k gl

ossy

con

vex

~tra

ns

839

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-45

-11

-1

1/10

PC

A

surf

ace

tan/

lt pk

glo

ssy

conv

ex ~

trans

77

6 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

45-1

2 -1

1/

10 P

CA

su

rfac

e cr

eam

y lt

pk g

loss

y co

nvex

~tra

ns

858

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-45

-13

-1

1/10

PC

A

surf

ace

trans

~co

nvex

glo

ssy

945

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-45

-14

-1

1/10

PC

A

surf

ace

crea

my

lt pe

ach/

pk g

loss

y co

nvex

~tra

ns

852

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-45

-15

-2

1/10

PC

A

surf

ace

trans

ver

y lt

pk g

loss

y co

nvex

84

9 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

45-1

6 -1

N

A

surf

ace

roun

d pi

nk o

paqu

e gl

ossy

con

vex

837

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-45

-17

-2

MA

su

rfac

e sm

all r

ound

yel

low

opa

que

glos

sy c

onve

x 84

3 Pr

omic

rom

onos

pora

ent

erop

hila

0.

95

X83

807

AT0

3-45

-18

-2

MA

su

rfac

e sm

all p

ink

irreg

ular

gro

wth

con

vex

~tra

ns

834

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-45

-19

-1

MA

su

rfac

e w

hite

glo

ssy

opaq

ue c

onve

x 93

3 Pr

omic

rom

onos

pora

ent

erop

hila

0.

96

X83

807

133

(Tab

le c

ontin

ued:

Sur

face

isol

ates

) Su

rfac

e Is

olat

e

D

ilutio

n M

edia

D

epth

C

olon

y M

orph

olog

y

S

eque

nce

Len

gth

B

LA

ST R

esul

t

Sim

ilari

ty A

cces

sion

no.

A

T03-

45-2

0 -1

M

A

surf

ace

lt pk

glo

ssy

conv

ex o

paqu

e 93

2 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

45-2

2 -1

M

A

surf

ace

lt pk

smoo

th ~

irreg

ular

~gl

ossy

rais

ed o

paqu

e 89

7 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

45-2

3 -1

M

A

surf

ace

hot o

rang

e ~c

onve

x op

aque

~gl

ossy

65

6 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T03-

45-2

4 -1

M

A

surf

ace

brig

ht y

ello

w ~

flat g

loss

y op

aque

~ro

ugh

962

Act

inom

ycet

acea

e

0.99

X

8731

0 A

T03-

45-2

5 -1

M

A

surf

ace

yello

w c

onve

x ro

und

opaq

ue ~

glos

sy ri

ngs

872

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-45

-26

-1

MA

su

rfac

e ho

t ora

nge/

pk g

loss

y co

nvex

opa

que

924

Mod

esto

bact

er m

ultis

epta

tus

0.99

A

J871

304

AT0

3-45

-27

-1

MA

su

rfac

e of

fwhi

te g

loss

y ~c

onve

x op

aque

89

5 Pr

omic

rom

onos

pora

ent

erop

hila

0.

95

X83

807

AT0

3-45

-28

-1

NA

su

rfac

e ho

t pea

ch g

loss

y ~c

onve

x op

aque

94

5 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

45-2

9 -1

N

A

surf

ace

trans

tan

glos

sy c

onve

x 80

1 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

46-2

-1

1/

100

PCA

sur

face

cre

amy

lt pk

~tra

ns g

loss

y co

nvex

smal

l 97

6 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

46-3

-1

1/

100

PCA

sur

face

dar

ker p

k tra

ns g

loss

y co

nvex

smal

l 85

0 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

46-4

-1

1/

100

PCA

sur

face

tra

ns g

loss

y ~c

onve

x sm

all

792

Cel

lulo

mon

as c

ella

sea

0.

96

X83

804

AT0

3-46

-7

-1

1/10

0 PC

A s

urfa

ce w

hite

tran

s glo

ssy

conv

ex sm

all

813

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-46

-8

-1

1/10

0 PC

A s

urfa

ce c

ream

y lt

pk ~

trans

glo

ssy

conv

ex sm

all

852

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-46

-9

-1

1/10

0 PC

A s

urfa

ce c

ream

y lt

pk ~

trans

glo

ssy

conv

ex sm

all

800

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-46

-10

-1

1/10

0 PC

A s

urfa

ce c

ream

y lt

pk ~

trans

glo

ssy

conv

ex sm

all

860

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-46

-11

-1

1/10

0 PC

A s

urfa

ce w

hite

tran

s glo

ssy

conv

ex sm

all

770

Prom

icro

mon

ospo

ra e

nter

ophi

la

0.93

X

8380

7 A

T03-

46-1

2 -1

1/

100

PCA

sur

face

dar

ker p

k tra

ns g

loss

y co

nvex

smal

l 82

4 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

46-1

4 -2

1/

100

PCA

sur

face

dar

ker p

k/pe

ach

glos

sy c

onve

x ~t

rans

84

4 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

46-1

5 -2

1/

100

PCA

sur

face

dar

ker r

ed/o

rang

e gl

ossy

con

vex

trans

59

0 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T03-

46-1

6 -2

1/

100

PCA

sur

face

cre

amy

lt pk

~tra

ns g

loss

y co

nvex

78

9 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T03-

46-1

8 -2

1/

100

PCA

sur

face

whi

te tr

ans g

loss

y co

nvex

smal

l 70

9 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T03-

46-1

9 -2

1/

100

PCA

sur

face

dar

ker p

k/pu

rple

tran

s glo

ssy

conv

ex sm

all

859

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-46

-20

-2

1/10

0 PC

A s

urfa

ce w

hite

/lt p

k gl

ossy

con

vex

~opa

que

846

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

3-46

-21

-2

1/10

0 PC

A s

urfa

ce c

ream

y lt

pk g

loss

y co

nvex

~tra

ns

856

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-22

-2

1/10

0 PC

A s

urfa

ce d

arke

r pk/

oran

ge g

loss

y co

nvex

tran

s 73

2 Bl

asto

cocc

us sp

. BC

448

0.

96

AJ3

1657

1 A

T03-

46-2

3 -2

1/

100

PCA

sur

face

pk/

peac

h tra

ns g

loss

y co

nvex

85

4 C

ellu

lom

onas

car

tae

MSD

201

06

0.94

X

7945

6 A

T03-

46-2

4 -2

1/

100

PCA

sur

face

pk/

peac

h tra

ns g

loss

y co

nvex

mor

e co

nvex

poi

nt in

cen

ter

805

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-25

-2

1/10

0 PC

A s

urfa

ce w

hite

glo

ssy

conv

ex ~

opaq

ue

828

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-46

-26

-2

1/10

0 PC

A s

urfa

ce p

urpl

e tra

ns g

loss

y co

nvex

84

3 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

46-2

7 -1

PC

A

surf

ace

crea

m n

on-g

loss

y co

nvex

pas

tey

~opa

que

852

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-28

-1

PCA

su

rfac

e lt

pk/c

ream

glo

ssy

conv

ex ~

opaq

ue

769

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-29

-1

PCA

su

rfac

e br

ight

pk

rais

ed ir

regu

lar s

moo

th o

paqu

e (lo

oks l

ike

a ne

rd)

792

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-30

-1

PCA

su

rfac

e lt

pk/ta

n ~g

loss

y co

nvex

~op

aque

83

3 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

46-3

1 -1

PC

A

surf

ace

oran

ge/ta

n du

ll no

n-gl

ossy

smoo

th c

onve

x ~o

paqu

e 64

8 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

46-3

2 -1

PC

A

surf

ace

oran

ge/p

k ~g

loss

y co

nvex

opa

que

727

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-33

-1

PCA

su

rfac

e tra

ns ta

n co

nvex

glo

ssy

844

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-46

-34

-1

PCA

su

rfac

e tra

ns c

ream

con

vex

glos

sy

810

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-46

-36

-1

PCA

su

rfac

e cr

eam

non

-glo

ssy

conv

ex p

aste

y ~o

paqu

e 86

5 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

46-3

7 -1

PC

A

surf

ace

lt pk

/tan

~glo

ssy

conv

ex ~

opaq

ue

748

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-38

-1

PCA

su

rfac

e or

ange

/dar

ker r

ed g

loss

y ~t

rans

con

vex

811

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-46

-39

-1

PCA

su

rfac

e cr

eam

~op

aque

non

-glo

ssy

conv

ex p

aste

y 82

9 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

46-4

0 -1

PC

A

surf

ace

tan

~glo

ssy

conv

ex ~

opaq

ue so

ft 81

3 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

46-4

1 -1

PC

A

surf

ace

crea

my

brig

ht p

k/or

ange

opa

que

glos

sy c

onve

x 77

5 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

46-4

3 -1

PC

A

surf

ace

dark

er o

rang

e gl

ossy

~tra

ns c

onve

x 71

5 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

46-4

4 -1

PC

A

surf

ace

tan

~glo

ssy

conv

ex ~

opaq

ue so

ft 80

6 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

46-4

5 -1

PC

A

surf

ace

dark

er o

rang

e ~g

loss

y co

nvex

~op

aque

95

2 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1

134

(Tab

le c

ontin

ued:

Sur

face

isol

ates

) Su

rfac

e Is

olat

e

D

ilutio

n M

edia

D

epth

C

olon

y M

orph

olog

y

S

eque

nce

Len

gth

B

LA

ST R

esul

t

Si

mila

rity

Acc

essi

on n

o.

AT0

3-46

-46

-1

PCA

su

rfac

e br

ight

dar

ker p

k ve

ry g

loss

y co

nvex

opa

que

606

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-47

-1

1/10

PC

A

surf

ace

dark

er p

k/pu

rple

big

tran

s edg

es c

olor

cen

ter ~

trans

cen

ter g

loss

y co

nvex

83

3 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

46-4

8 -1

1/

10 P

CA

su

rfac

e da

rker

pk/

purp

le ir

regu

lar t

rans

glo

ssy

flat

775

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-49

-1

1/10

PC

A

surf

ace

lt pu

rple

tran

s glo

ssy

conv

ex

862

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-46

-50

-1

1/10

PC

A

surf

ace

trans

tan

glos

sy c

onve

x 78

5 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

46-5

1 -1

1/

10 P

CA

su

rfac

e tra

ns lt

pk

glos

sy ~

conv

ex

818

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-52

-1

1/10

PC

A

surf

ace

big

lt pk

/ora

nge

smal

l con

vex

~opa

que

~glo

ssy

841

Blas

toco

ccus

sp. B

C41

2

0.98

A

J316

574

AT0

3-46

-53

-1

1/10

PC

A

surf

ace

pink

irre

gula

r ~co

nvex

~op

aque

glo

ssy

772

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-46

-54

-1

1/10

PC

A

surf

ace

purp

le/ta

n tra

ns ~

flat g

loss

y 82

3 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

46-5

5 -1

1/

10 P

CA

su

rfac

e cr

eam

y lt

pk/p

each

glo

ssy

conv

ex ~

opaq

ue

560

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-46

-56

-1

1/10

PC

A

surf

ace

tang

erin

e ~o

paqu

e gl

ossy

con

vex

815

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-57

-1

1/10

PC

A

surf

ace

crea

my

trans

glo

ssy

conv

ex

862

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-46

-58

-1

1/10

PC

A

surf

ace

brig

ht p

k/pe

ach

trans

edg

e m

ore

colo

r cen

ter ~

tarn

s cen

ter g

loss

y co

nvex

83

4 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

46-5

9 -1

1/

10 P

CA

su

rfac

e tra

ns g

loss

y ~c

onve

x 78

5 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

46-6

2 -1

1/

10 P

CA

su

rfac

e

839

Cel

lulo

mon

as c

arta

e M

SD 2

0106

0.

94

X79

456

AT0

3-46

-63

-1

1/10

PC

A

surf

ace

crea

my

lt pe

ach/

lt pk

glo

ssy

conv

ex ~

opaq

ue

830

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-46

-64

-1

1/10

PC

A

surf

ace

dark

er o

rang

e/da

rker

red

trans

glo

ssy

conv

ex

766

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-65

-1

1/10

PC

A

surf

ace

brig

ht p

k/pe

ach

trans

edg

e m

ore

colo

r cen

ter ~

trans

cen

ter g

loss

y co

nvex

85

6 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

46-6

6 -1

1/

10 P

CA

su

rfac

e br

ight

ora

nge

irreg

ular

text

ured

~op

aque

flat

aro

und

edge

~co

nvex

cen

ter

800

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-67

-1

1/10

PC

A

surf

ace

crea

my

lt pe

ach/

lt pk

glo

ssy

conv

ex ~

opaq

ue

837

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-68

-1

1/10

PC

A

surf

ace

trans

dar

ker p

urpl

e/da

rker

pk

glos

sy c

onve

x 65

5 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T03-

46-6

9 -1

1/

10 P

CA

su

rfac

e da

rker

ora

nge/

dark

er re

d tra

ns g

loss

y co

nvex

85

2 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

46-7

1 -1

1/

10 P

CA

su

rfac

e tra

ns lt

pk

glos

sy c

onve

x 70

9 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T03-

46-7

2 -1

1/

10 P

CA

su

rfac

e

844

Cel

lulo

sim

icro

bium

cel

lula

ns

0.95

A

B16

6888

A

T03-

46-7

3 -1

1/

10 P

CA

su

rfac

e lt

pk ~

glos

sy c

onve

x ~o

paqu

e 80

4 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T03-

46-7

4 -1

1/

10 P

CA

su

rfac

e cr

eam

y lt

pk g

loss

y co

nvex

~tra

ns

858

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-75

-1

NA

su

rfac

e cr

eam

y/w

hite

~tra

ns g

loss

y co

nvex

80

1 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

46-7

6 -1

N

A

surf

ace

lt pk

~op

aque

glo

ssy

conv

ex

545

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

3-46

-79

-1

NA

su

rfac

e pk

opa

que

conv

ex ~

glos

sy fr

ayed

edg

es

897

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-80

-1

NA

su

rfac

e pk

~op

aque

con

cave

irre

gula

r ~du

ll 80

7 Bl

asto

cocc

us sp

. BC

412

0.

98

AJ3

1657

4 A

T03-

46-8

1 -1

N

A

surf

ace

crea

m e

dges

con

vex

glos

sy ~

trans

dar

ker (

brow

n/ta

n) c

ente

r 95

7 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

46-8

2 -1

N

A

surf

ace

very

lt ta

n/pk

glo

ssy

conv

ex ~

trans

89

5 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

46-8

3 -1

N

A

surf

ace

irreg

ular

tran

s cle

ar g

loss

y ed

ges &

ora

nge

cent

er ~

conv

ex

848

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-84

-1

NA

su

rfac

e tra

ns g

loss

y co

nvex

10

30

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-85

-1

NA

su

rfac

e tra

ns g

loss

y co

nvex

fray

ed e

dges

ora

nge

trans

cen

ter

701

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-86

-1

NA

su

rfac

e pe

ach/

oran

ge ~

opaq

ue c

onve

x gl

ossy

96

8 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

46-8

9 -1

M

A

surf

ace

brig

ht o

rang

e/ho

t pk

irreg

ular

bum

py !r

aise

d op

aque

~gl

ossy

86

4 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

46-9

0 -1

M

A

surf

ace

!lt p

k gl

ossy

con

vex

opaq

ue

982

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-46

-91

-1

MA

su

rfac

e tra

ns fl

at g

loss

y irr

egul

ar

866

Prom

icro

mon

ospo

ra e

nter

ophi

la

0.96

X

8380

7 A

T03-

46-9

2 -1

M

A

surf

ace

lt pk

dul

l irr

egul

ar ro

ugh

rigid

bum

py ra

ised

opa

que

978

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-93

-1

MA

su

rfac

e pk

~op

aque

irre

gula

r glo

ssy

fray

ed e

dges

rais

ed

948

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-94

-1

MA

su

rfac

e pk

con

vex

glos

sy ~

opaq

ue sm

all b

umps

on

surf

ace

in c

ente

r 94

3 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

46-9

5 -2

M

A

surf

ace

lt ye

llow

opa

que

glos

sy c

onve

x 85

5 Pr

omic

rom

onos

pora

ent

erop

hila

0.

96

X83

807

AT0

3-46

-96

-2

MA

su

rfac

e lt

pk o

paqu

e ~g

loss

y co

nvex

94

2 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

46-9

7 -2

M

A

surf

ace

hot p

k irr

egul

ar ra

ised

bum

py sm

ooth

opa

que

~dul

l 94

4 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

46-9

8 -2

M

A

surf

ace

hot p

k irr

egul

ar d

ull r

aise

d ro

ugh

rigid

opa

que

961

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-46

-100

-2

N

A

surf

ace

trans

ora

nge

glos

sy c

onve

x 97

9 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1

135

(Tab

le c

ontin

ued:

Sur

face

isol

ates

) Su

rfac

e Is

olat

e

D

ilutio

n M

edia

D

epth

Col

ony

Mor

phol

ogy

Seq

uenc

e L

engt

h

BL

AST

Res

ult

Sim

ilari

ty A

cces

sion

no.

A

T03-

46-1

01

-2

NA

su

rfac

e tra

ns !~

oran

ge g

loss

y co

nvex

80

1 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

46-1

02

-2

NA

su

rfac

e pk

irre

gula

r hol

e in

cen

ter o

paqu

e ra

ised

smoo

th ~

glos

sy

953

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-46

-103

-2

N

A

surf

ace

flat o

ffw

hite

opa

que

oran

ge c

ente

r ~du

ll cu

rled

up e

dges

95

4 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

46-1

04

-2

NA

su

rfac

e tra

ns ~

pk/p

each

glo

ssy

conv

ex

911

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-48

-1

-1

PCA

su

rfac

e pe

ach

non-

glos

sy o

paqu

e ve

ry c

onve

x 74

4 Bl

asto

cocc

us a

ggre

gatu

s 0.

98

AJ4

3019

3 A

T03-

48-2

-2

1/

10 P

CA

su

rfac

e fla

t pur

ple/

pink

glo

ssy

~tra

ns

848

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-48

-4

-1

1/10

PC

A

surf

ace

tan

glos

sy c

onve

x ~t

rans

dar

ker s

pot i

n ce

nter

74

5 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

48-5

-1

1/

10 P

CA

su

rfac

e pi

nk n

on-g

loss

y du

ll irr

egul

ar c

onve

x ~o

paqu

e 86

4 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

48-6

-1

1/

10 P

CA

su

rfac

e pi

nk n

on-g

loss

y du

ll co

nvex

~op

aque

97

9 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

48-7

-2

N

A

surf

ace

smal

l rou

nd p

each

smoo

th c

onve

x op

aque

67

0 Bl

asto

cocc

us sp

. BC

448

0.

95

AJ3

1657

1 A

T03-

48-8

-1

N

A

surf

ace

larg

e ro

und

crea

m sm

ooth

flat

~tra

ns

795

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-48

-9

-1

NA

su

rfac

e ro

und

crea

m sm

ooth

con

vex

~tra

ns

759

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-48

-10

-1

NA

su

rfac

e ro

und

pink

tran

s con

vex

smoo

th

701

Blas

toco

ccus

agg

rega

tus

0.97

A

J430

193

AT0

3-48

-11

-2

1/10

PC

A

surf

ace

roun

d ye

llow

flat

~tra

ns sm

ooth

76

7 St

aphy

loco

ccus

sp. 1

6b-5

A

0.99

A

Y56

1556

A

T03-

48-1

2 -2

M

A

surf

ace

roun

d pi

nk ~

trans

kno

blik

e pr

otub

eran

ce

698

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-48

-13

-1

MA

su

rfac

e lt

pk w

ith b

lack

spec

kles

on

surf

ace

opaq

ue ~

glos

sy c

onve

x 95

5 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

48-1

4 -1

M

A

surf

ace

lt pk

opa

que

umbo

nate

~gl

ossy

91

0 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

48-1

5 -1

M

A

surf

ace

lt pk

irre

gula

r rig

id ro

ugh

opaq

ue c

onca

ve c

ente

r dul

l 91

1 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

48-1

8 -1

1/

10 P

CA

su

rfac

e br

owni

sh o

rang

e ~t

rans

glo

ssy

conv

ex

957

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-48

-19

-1

1/10

PC

A

surf

ace

brow

nish

ora

nge

opaq

ue ~

dull

conv

ex

858

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-48

-20

-1

1/10

PC

A

surf

ace

brow

nish

ora

nge

~irr

egul

ar g

loss

y fr

ayed

edg

es ~

trans

rais

ed

954

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-48

-21

-1

1/10

PC

A

surf

ace

dark

er o

rang

e ~o

paqu

e gl

ossy

con

vex

951

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-48

-22

-1

1/10

PC

A

surf

ace

dark

er o

rang

e ~t

rans

glo

ssy

conv

ex (s

ome

are

umbo

nate

) 95

8 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

48-2

4 -1

1/

10 P

CA

su

rfac

e !lt

pk

~con

vex

opaq

ue g

loss

y 97

9 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

48-2

7 -2

1/

10 P

CA

su

rfac

e tra

ns !l

t pk

glos

sy c

onve

x 92

1 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T03-

48-2

8 -2

1/

10 P

CA

su

rfac

e of

fwhi

te/!l

t pk

~tra

ns g

loss

y co

nvex

98

0 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

49-2

-2

1/

100

PCA

sur

face

lt y

ello

w ~

flat g

loss

y irr

egul

ar ~

trans

82

1 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

49-3

-1

PC

A

surf

ace

big

yello

w c

onve

x no

n-gl

ossy

text

ured

(bum

py) o

paqu

e 80

0 M

icro

cocc

us lu

teus

0.

99

AJ4

0909

6 A

T03-

50-1

-1

PC

A

surf

ace

dark

er o

rang

e ve

ry c

onve

x gl

ossy

big

opa

que

797

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-50

-2

-2

1/10

PC

A

surf

ace

peac

h gl

ossy

tran

s rai

sed

in c

ente

r mor

e op

aque

in c

ente

r 84

5 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T03-

50-3

-2

1/

100

PCA

sur

face

lt p

k ~

conv

ex n

on-g

loss

y ro

ugh

838

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-50

-4

-1

1/10

PC

A

surf

ace

lt pk

flat

glo

ssy

~opa

que

837

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-50

-5

-1

PCA

su

rfac

e da

rk o

rang

e co

nvex

glo

ssy

~opa

que

871

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-50

-6

-1

PCA

su

rfac

e w

hite

opa

que

over

grow

th o

ver 1

/2 p

late

irre

gula

r ~fla

t 78

3 Pa

ntoe

a an

anat

is st

rain

BD

390

0.

96

AY

5307

95

AT0

3-50

-8

-1

NA

su

rfac

e sm

all r

ound

pin

k co

nvex

~tra

ns

793

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

3-50

-9

-2

MA

su

rfac

e sm

all r

ound

bro

wn

opaq

ue c

onve

x 57

0 G

eode

rmat

ophi

lus o

bscu

rus o

bscu

rus

0.95

L4

0620

A

T03-

50-1

0 -2

M

A

surf

ace

smal

l rou

nd b

row

n op

aque

con

vex

768

Geo

derm

atop

hilu

s obs

curu

s obs

curu

s 0.

98

L406

20

AT0

3-50

-11

-1

MA

su

rfac

e sm

all r

ound

pin

k co

nvex

~tra

ns

928

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-50

-12

-1

MA

su

rfac

e sm

all r

ound

bro

wn

opaq

ue c

onve

x 52

4 G

eode

rmat

ophi

lus o

bscu

rus o

bscu

rus

0.96

L4

0620

A

T03-

50-1

3 -1

M

A

surf

ace

lt pk

with

bla

ck sp

eckl

ed su

rfac

e ~o

paqu

e ~d

ull c

onve

x 86

9 G

eode

rmat

ophi

lus o

bscu

rus o

bscu

rus

0.98

L4

0620

A

T03-

50-1

4 -1

M

A

surf

ace

hot o

rang

e op

aque

~gl

ossy

con

vex

867

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

3-50

-15

-1

MA

su

rfac

e ho

t ora

nge

opaq

ue ~

glos

sy c

onve

x 76

3 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

151-

1 -1

M

A

surf

ace

smal

l rou

nd p

ink

smoo

th c

onca

ve ~

trans

79

1 Bl

asto

cocc

us sp

. BC

521

0.

98

AJ3

1657

3 A

T04-

151-

2 -1

M

A

surf

ace

smal

l rou

nd p

ink

conv

ex o

paqu

e 83

3 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

151-

3 -2

1/

10 P

CA

su

rfac

e sm

all r

ound

pin

k co

nvex

~tra

ns

802

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-15

1-4

-1

1/10

PC

A

surf

ace

smal

l rou

nd p

ink

conv

ex ~

trans

45

6 Bl

asto

cocc

us sp

. BC

521

0.

98

AJ3

1657

3 A

T04-

151-

5 -1

1/

100

PCA

sur

face

off

whi

te ~

opaq

ue c

onve

x fr

ayed

edg

es

889

Blas

toco

ccus

sp. B

C41

2

0.98

A

J316

574

136

(Tab

le c

ontin

ued:

Sur

face

isol

ates

) Su

rfac

e Is

olat

e

D

ilutio

n M

edia

D

epth

Col

ony

Mor

phol

ogy

Seq

uenc

e L

engt

h

BL

AST

Res

ult

Sim

ilari

ty A

cces

sion

no.

A

T04-

154-

1 -1

M

A

surf

ace

smal

l rou

nd g

rey

trans

con

vex

862

Geo

derm

atop

hilu

s obs

curu

s obs

curu

s 0.

97

L406

20

AT0

4-15

4-4

-1

1/10

0 PC

A s

urfa

ce l

t pk

tiny

trans

glo

ssy

conv

ex

780

Blas

toco

ccus

sp. B

C42

1

0.96

A

J316

574

AT0

4-15

6-2

-1

1/10

0 PC

A s

urfa

ce p

k ~i

rreg

ular

~op

aque

glo

ssy

rais

ed

852

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-15

7-3

-2

1/10

PC

A

surf

ace

dark

er b

row

n du

ll op

aque

in a

gar m

aybe

fung

us

959

Stre

ptom

yces

sp. X

13

0.99

A

F060

793

AT0

4-15

8-1

-1

NA

su

rfac

e pe

ach

roun

d ~t

rans

con

vex

unev

en e

dges

83

3 Bl

asto

cocc

us a

ggre

gatu

s 0.

97

AJ4

3019

3 A

T04-

158-

2 -1

N

A

surf

ace

smal

l pea

ch ro

und

~tra

ns

735

Kin

eoco

ccus

-like

bac

teriu

m A

S297

8

0.98

A

F060

676

AT0

4-15

8-3

-1

1/10

PC

A

surf

ace

smal

l pin

k ro

und

conv

ex o

paqu

e 97

7 Bl

asto

cocc

us a

ggre

gatu

s 0.

98

AJ4

3019

3 A

T04-

158-

4 -1

M

A

surf

ace

smal

l pin

k ro

und

rais

ed ~

trans

con

vex

797

Blas

toco

ccus

agg

rega

tus

0.97

A

J430

193

AT0

4-15

8-6

-1

MA

su

rfac

e ro

und

pink

con

vex

bum

py

786

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-15

8-7

-1

MA

su

rfac

e ro

und

yello

w fl

at o

paqu

e 75

9 C

ellu

lom

onas

cel

lase

a

0.98

X

8380

4 A

T04-

158-

8 -1

1/

100

PCA

sur

face

hot

pk

glos

sy c

onve

x ~o

paqu

e 82

9 Bl

asto

cocc

us sp

. BC

412

0.

97

AJ3

1657

4 A

T04-

158-

9 -1

1/

100

PCA

sur

face

pea

ch ~

trans

glo

ssy

conv

ex (~

dark

er in

cen

ter)

67

3 Bl

asto

cocc

us a

ggre

gatu

s 0.

97

AJ4

3019

3 A

T04-

158-

10

-2

1/10

0 PC

A s

urfa

ce t

ange

rine

glos

sy c

onve

x ~t

rans

66

3 B

acte

rium

Elli

n602

3

0.99

A

Y23

4675

A

T04-

161-

3 -1

M

A

surf

ace

whi

te ~

opaq

ue ~

irreg

ular

bum

py ri

gid

roug

h ra

ised

84

5 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

4 -1

M

A

surf

ace

brow

n/ta

n gl

ossy

~tra

ns c

onve

x 75

4 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T04-

161-

5 -1

M

A

surf

ace

lt pk

/pea

ch ~

opaq

ue g

loss

y co

nvex

95

3 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

161-

6 -1

M

A

surf

ace

whi

te o

paqu

e gl

ossy

con

vex

860

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-7

-1

MA

su

rfac

e lt

pk/p

each

~op

aque

~irr

egul

ar b

umpy

rigi

d ro

ugh

rais

ed

965

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-8

-1

MA

su

rfac

e lt

pk ~

opaq

ue c

onve

x gl

ossy

87

8 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

9 -1

M

A

surf

ace

lt ye

llow

~tra

ns g

loss

y co

nvex

87

6 Pr

omic

rom

onos

pora

ent

erop

hila

0.

95

X83

807

AT0

4-16

1-10

-1

M

A

surf

ace

pk/o

rang

e gl

ossy

con

vex

~opa

que

463

Bac

teriu

m E

llin6

023

0.

98

AY

2346

75

AT0

4-16

1-11

-1

M

A

surf

ace

trans

tan

glos

sy c

onve

x 75

8 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

12

-1

MA

su

rfac

e of

fwhi

te/lt

pk

glos

sy ~

opaq

ue c

onve

x (m

ore

conv

ex c

ente

r)

1003

Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T04-

161-

13

-1

MA

su

rfac

e w

hite

glo

ssy

~opa

que

conv

ex

1003

Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

14

-1

MA

su

rfac

e pk

/ora

nge

~tra

ns g

loss

y co

nvex

83

6 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

161-

15

-1

MA

su

rfac

e of

fwhi

te ~

trans

~irr

egul

ar b

umpy

rigi

d ro

ugh

rais

ed

850

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-16

-1

M

A

surf

ace

oran

ge g

loss

y ~o

paqu

e co

nvex

88

4 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

161-

17

-1

MA

su

rfac

e or

ange

~tra

ns g

loss

y co

nvex

(mor

e co

nvex

in c

ente

r)

774

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-18

-1

M

A

surf

ace

yello

w/ta

n tra

ns g

loss

y co

nvex

70

4 C

ellu

lom

onas

car

tae

MSD

201

06

0.95

X

7945

6 A

T04-

161-

19

-1

MA

su

rfac

e of

fwhi

te g

loss

y co

nvex

~op

aque

75

7 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

20

-1

MA

su

rfac

e lt

pk ~

opaq

ue c

onve

x gl

ossy

85

1 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

21

-1

MA

su

rfac

e lt

pk ~

opaq

ue c

onve

x gl

ossy

83

1 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

22

-1

MA

su

rfac

e ve

ry lt

pk

~tra

ns ~

irreg

ular

bum

py ri

gid

roug

h ra

ised

98

7 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

23

-1

MA

su

rfac

e w

hite

~op

aque

glo

ssy

conv

ex

751

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-25

-1

M

A

surf

ace

tan/

brow

n ~o

paqu

e gl

ossy

con

vex

774

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-26

-3

M

A

surf

ace

crea

m c

onve

x op

aque

~gl

ossy

72

1 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T04-

161-

27

-3

MA

su

rfac

e cr

eam

con

vex

wrin

kly

clum

py ~

trans

dul

l 77

0 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

28

-3

MA

su

rfac

e lt

peac

h co

nvex

clu

mpy

~tra

ns d

ull

694

Blas

toco

ccus

agg

rega

tus

0.98

A

J430

193

AT0

4-16

1-29

-3

M

A

surf

ace

peac

h/lt

pk c

onve

x du

ll op

aque

clu

mpy

76

2 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

30

-1

MA

su

rfac

e or

ange

con

vex

opaq

ue g

loss

y sm

all d

ip in

cen

ter

736

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

1-31

-1

M

A

surf

ace

lt or

ange

/pea

ch c

onve

x gl

ossy

opa

que

945

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

1-32

-1

M

A

surf

ace

offw

hite

glo

ssy

~con

vex

~opa

que

635

Cel

lulo

sim

icro

bium

funk

ei st

rain

CD

C#J

B40

83ot

0.

92

AY

5237

89

AT0

4-16

1-33

-1

M

A

surf

ace

crea

m o

paqu

e co

nvex

glo

ssy

691

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

4-16

1-34

-1

M

A

surf

ace

crea

m c

onve

x op

aque

~du

ll ~c

lum

py

854

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-35

-1

M

A

surf

ace

lt pk

/pea

ch o

paqu

e co

nvex

~gl

ossy

84

9 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

36

-1

MA

su

rfac

e lt

yello

w g

loss

y ~c

onve

x ~t

rans

76

9 Pr

omic

rom

onos

pora

ent

erop

hila

0.

95

X83

807

AT0

4-16

1-37

-1

M

A

surf

ace

peac

h gl

ossy

opa

que

~fla

t 77

5 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1

137

(Tab

le c

ontin

ued:

Sur

face

isol

ates

) Su

rfac

e Is

olat

e

D

ilutio

n M

edia

D

epth

Col

ony

Mor

phol

ogy

Seq

uenc

e L

engt

h

BL

AST

Res

ult

Sim

ilari

ty A

cces

sion

no.

A

T04-

161-

38

-1

MA

su

rfac

e lt

pk ~

dull

conv

ex ~

trans

~cl

umpy

10

39

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

1-39

-3

M

A

surf

ace

flat y

ello

w g

loss

y ~o

paqu

e 80

3 Pr

omic

rom

onos

pora

ent

erop

hila

0.

94

X83

807

AT0

4-16

1-40

-3

M

A

surf

ace

grey

con

vex

opaq

ue ~

glos

sy m

ore

conv

ex in

cen

ter t

op (l

ike

crea

msa

ver)

75

8 Bl

asto

cocc

us sp

. BC

412

0.

97

AJ3

1657

4 A

T04-

161-

41

-3

MA

su

rfac

e of

fwhi

te c

onve

x cl

umpy

~du

ll op

aque

94

3 Bl

asto

cocc

us a

ggre

gatu

s 0.

98

AJ4

3019

3 A

T04-

161-

42

-3

MA

su

rfac

e pe

ach

wrin

kly

conv

ex o

paqu

e ~g

loss

y 64

4 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

43

-2

MA

su

rfac

e pk

/ora

nge

clum

py c

onve

x ~d

ull ~

opaq

ue

520

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

4-16

1-44

-2

M

A

surf

ace

crea

m/y

ello

w ~

conv

ex ~

glos

sy o

paqu

e 64

9 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

161-

45

-2

MA

su

rfac

e of

fwhi

te c

lum

py c

onve

x du

ll ~o

paqu

e 76

5 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

46

-2

MA

su

rfac

e lt

pk ~

conv

ex o

paqu

e ~g

loss

y 78

0 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

47

-2

MA

su

rfac

e or

ange

flat

with

rais

ed ri

ng in

cen

ter o

paqu

e ~g

loss

y 93

2 Bl

asto

cocc

us a

ggre

gatu

s 0.

99

AJ4

3019

3 A

T04-

161-

48

-2

MA

su

rfac

e lt

pk c

lum

py o

paqu

e du

ll 10

20

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-49

-2

M

A

surf

ace

lt or

ange

flat

opa

que

~dul

l 61

1 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

161-

50

-2

MA

su

rfac

e or

ange

con

vex

opaq

ue g

loss

y

614

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

4-16

1-51

-2

M

A

surf

ace

offw

hite

glo

ssy

opaq

ue c

onve

x (m

ore

conv

ex in

cen

ter l

ike

crea

msa

ver)

63

1 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

52

-2

MA

su

rfac

e ye

llow

glo

ssy

~con

vex

trans

84

1 Pr

omic

rom

onos

pora

ent

erop

hila

0.

94

X83

807

AT0

4-16

1-53

-2

M

A

surf

ace

pk ~

opaq

ue ~

irreg

ular

bum

py ri

gid

roug

h ra

ised

86

2 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

161-

54

-2

MA

su

rfac

e of

fwhi

te g

loss

y op

aque

con

vex

(mor

e co

nvex

in c

ente

r lik

e cr

eam

save

r)

836

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-55

-2

M

A

surf

ace

pk/p

each

con

vex

glos

sy o

paqu

e 98

2 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

161-

56

-2

MA

su

rfac

e lt

pk ~

trans

~co

nvex

glo

ssy

unsm

ooth

edg

es

631

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

4-16

1-57

-2

M

A

surf

ace

lt pk

glo

ssy

conv

ex o

paqu

e (li

ke c

ream

life

save

r)

695

Blas

toco

ccus

sp. B

C41

2

0.98

A

J316

574

AT0

4-16

1-58

-2

M

A

surf

ace

pk w

ith d

arke

r spe

cs g

loss

y co

nvex

~op

aque

75

2 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T04-

161-

59

-2

MA

su

rfac

e w

hite

opa

que

glos

sy c

onve

x 77

0 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

60

-2

MA

su

rfac

e lt

pk tr

ans g

loss

y co

nvex

64

1 Bl

asto

cocc

us a

ggre

gatu

s 0.

97

AJ4

3019

3 A

T04-

161-

61

-2

MA

su

rfac

e lt

pk g

loss

y op

aque

con

vex

(mor

e co

nvex

in c

ente

r lik

e cr

eam

life

save

r)

739

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-62

-2

M

A

surf

ace

lt pk

clu

mpy

~op

aque

irre

gula

r bum

py ri

gid

roug

h ra

ised

55

1 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T04-

161-

63

-2

MA

su

rfac

e da

rker

ora

nge

(~re

d) g

loss

y co

nvex

~op

aque

73

2 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

64

-2

MA

su

rfac

e lt

pk ~

glos

sy c

onve

x ~o

paqu

e 69

9 Bl

asto

cocc

us sp

. BC

412

0.

97

AJ3

1657

4 A

T04-

161-

65

-2

MA

su

rfac

e bu

bble

gum

pk

opaq

ue g

loss

y co

nvex

75

1 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

161-

67

-2

MA

su

rfac

e of

fwhi

te g

loss

y op

aque

con

vex

(mor

e co

nvex

in c

ente

r lik

e cr

eam

save

r)

677

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-68

-2

M

A

surf

ace

lt pk

/off

whi

te w

ith d

arke

r spe

cs o

n su

rfac

e co

nvex

glo

ssy

~opa

que

731

Blas

toco

ccus

sp. B

C41

2

0.96

A

J316

574

AT0

4-16

1-69

-2

M

A

surf

ace

lt pk

with

dar

k (r

ust)

cent

er g

loss

y co

nvex

~op

aque

78

1 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

70

-2

MA

su

rfac

e lt

pk g

loss

y co

nvex

~op

aque

bum

py su

rfac

e 78

3 Bl

asto

cocc

us sp

. BC

448

0.

96

AJ3

1657

1 A

T04-

161-

71

-2

MA

su

rfac

e pk

/pea

ch ir

regu

lar ~

opaq

ue ri

gid

roug

h ra

ised

~du

ll 75

7 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

72

-2

MA

su

rfac

e of

fwhi

te o

paqu

e gl

ossy

con

vex

(dip

in c

ente

r)

783

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-73

-1

1/

10 P

CA

su

rfac

e pk

/pur

ple

glos

sy c

onve

x tra

ns

794

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-74

-1

1/

10 P

CA

su

rfac

e lt

pk g

loss

y co

nvex

~tra

ns

715

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-75

-1

1/

10 P

CA

su

rfac

e da

rker

ora

nge

~irr

egul

ar in

aga

r opa

que

~glo

ssy

672

Blas

toco

ccus

sp. B

C44

8

0.96

A

J316

571

AT0

4-16

1-76

-1

1/

10 P

CA

su

rfac

e or

ange

/pk

glos

sy c

onve

x ~t

rans

55

0 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

161-

78

-1

1/10

PC

A

surf

ace

lt pk

~tra

ns in

aga

r irr

egul

ar b

umpy

rigi

d ro

ugh

rais

ed

760

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-79

-1

1/

10 P

CA

su

rfac

e ve

ry lt

pk

trans

glo

ssy

conv

ex

761

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

1-81

-1

1/

10 P

CA

su

rfac

e of

fwhi

te/ta

n gl

ossy

con

vex

~tra

ns

692

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-82

-1

1/

10 P

CA

su

rfac

e pe

ach/

pk ~

trans

glo

ssy

conv

ex

799

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-83

-1

1/

10 P

CA

su

rfac

e ho

t pk/

oran

ge ~

opaq

ue g

loss

y co

nvex

73

1 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

84

-1

1/10

PC

A

surf

ace

lt pk

/pea

ch g

loss

y co

nvex

~tra

ns

951

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

1-85

-1

1/

10 P

CA

su

rfac

e tra

ns ~

whi

te g

loss

y ~c

onve

x (c

onve

x ce

nter

/edg

es ~

flat)

754

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-86

-1

1/

10 P

CA

su

rfac

e pe

ach/

tan

glos

sy c

onve

x ~t

rans

74

4 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

161-

87

-1

1/10

PC

A

surf

ace

hot p

k/or

ange

~op

aque

glo

ssy

conv

ex

614

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

138

(Tab

le c

ontin

ued:

Sur

face

isol

ates

) Su

rfac

e Is

olat

e

D

ilutio

n M

edia

D

epth

Col

ony

Mor

phol

ogy

Seq

uenc

e L

engt

h

BL

AST

Res

ult

Sim

ilari

ty A

cces

sion

no.

A

T04-

161-

88

-1

1/10

PC

A

surf

ace

very

lt p

k ~t

rans

~irr

egul

ar g

loss

y ra

ised

bum

py su

rfac

e 73

3 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

89

-1

1/10

PC

A

surf

ace

pk/p

each

~du

ll co

nvex

~op

aque

uns

moo

th e

dges

73

7 Bl

asto

cocc

us a

ggre

gatu

s 0.

97

AJ4

3019

3 A

T04-

161-

90

-1

1/10

PC

A

surf

ace

pk/p

each

tran

s glo

ssy

conv

ex

517

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

1-91

-1

1/

10 P

CA

su

rfac

e ru

st g

loss

y co

nvex

~tra

ns

740

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

1-92

-1

1/

10 P

CA

su

rfac

e lt

pk g

loss

y co

nvex

~tra

ns

670

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-93

-1

1/

10 P

CA

su

rfac

e ve

ry lt

pk/

offw

hite

glo

ssy

conv

ex ~

trans

79

5 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

94

-1

1/10

PC

A

surf

ace

lt pk

glo

ssy

conv

ex tr

ans

680

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

4-16

1-95

-1

1/

10 P

CA

su

rfac

e br

own/

purp

le tr

ans g

loss

y co

nvex

40

9 B

acte

rium

Elli

n602

3

0.98

A

Y23

4675

A

T04-

161-

96

-1

1/10

PC

A

surf

ace

purp

le/ta

n tra

ns g

loss

y co

nvex

80

1 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

161-

97

-1

1/10

PC

A

surf

ace

lt pk

~op

aque

con

vex

glos

sy

653

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-98

-1

1/

10 P

CA

su

rfac

e w

hite

~op

aque

glo

ssy

conv

ex

765

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

4-16

1-99

-1

1/

10 P

CA

su

rfac

e pk

glo

ssy

conv

ex ~

opaq

ue (u

nsm

ooth

edg

es)

689

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

1-10

0 -1

1/

10 P

CA

su

rfac

e lt

pk/p

each

~du

ll co

nvex

~op

aque

98

4 Bl

asto

cocc

us a

ggre

gatu

s 0.

98

AJ4

3019

3 A

T04-

161-

101

-1

1/10

PC

A

surf

ace

lt pk

~tra

ns g

loss

y co

nvex

61

3 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

161-

103

-1

1/10

PC

A

surf

ace

very

lt p

k/of

fwhi

te ~

dull

~fla

t ~op

aque

in a

gar

793

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-10

4 -1

1/

10 P

CA

su

rfac

e lt

pk/ta

n gl

ossy

con

vex

~tra

ns

701

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

4-16

1-10

5 -1

1/

10 P

CA

su

rfac

e or

ange

~tra

ns ~

irreg

ular

~du

ll ra

ised

rigi

d bu

mpy

79

7 Bl

asto

cocc

us a

ggre

gatu

s 0.

97

AJ4

3019

3 A

T04-

161-

106

-1

1/10

PC

A

surf

ace

whi

te tr

ans ~

conv

ex g

loss

y (c

onve

x ce

nter

/edg

es ~

flat)

731

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-10

7 -1

1/

10 P

CA

su

rfac

e pu

rple

/bro

wn

trans

glo

ssy

conv

ex

681

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-10

8 -1

1/

10 P

CA

su

rfac

e or

ange

glo

ssy

conv

ex ~

opaq

ue

649

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-10

9 -1

1/

10 P

CA

su

rfac

e pk

glo

ssy

conv

ex o

paqu

e 61

6 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

161-

110

-1

1/10

PC

A

surf

ace

lt pk

/pea

ch g

loss

y co

nvex

~op

aque

65

9 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

111

-1

1/10

PC

A

surf

ace

lt pk

tran

s glo

ssy

conv

ex

682

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-11

2 -1

1/

10 P

CA

su

rfac

e lt

oran

ge/ta

n tra

ns g

loss

y co

nvex

65

5 Bl

asto

cocc

us sp

. BC

448

0.

96

AJ3

1657

1 A

T04-

161-

113

-1

1/10

PC

A

surf

ace

lt pk

~du

ll ~o

paqu

e co

nvex

57

1 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T04-

161-

114

-1

1/10

PC

A

surf

ace

offw

hite

/lt p

k w

ith d

ark

spot

in c

ente

r con

vex

glos

sy ~

opaq

ue

785

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-11

5 -3

1/

10 P

CA

su

rfac

e or

ange

glo

ssy

conv

ex ~

opaq

ue

630

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

1-11

6 -3

1/

10 P

CA

su

rfac

e ta

n ~t

rans

glo

ssy

conv

ex

748

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-11

7 -3

1/

10 P

CA

su

rfac

e lt

oran

ge ~

trans

glo

ssy

conv

ex

802

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-11

8 -3

1/

10 P

CA

su

rfac

e lt

pk ~

trans

glo

ssy

conv

ex

770

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

1-11

9 -3

1/

10 P

CA

su

rfac

e or

ange

/hot

pk

~opa

que

glos

sy c

onve

x 75

3 G

eode

rmat

ophi

lus s

p. B

C51

8

0.99

A

J296

064

AT0

4-16

1-12

0 -3

1/

10 P

CA

su

rfac

e pk

/pea

ch ~

opaq

ue g

loss

y co

nvex

73

5 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T04-

161-

121

-3

1/10

PC

A

surf

ace

tan

/ora

nge

~tra

ns g

loss

y co

nvex

80

4 G

eode

rmat

ophi

lus s

p. B

C51

8

0.98

A

J296

064

AT0

4-16

1-12

2 -3

1/

10 P

CA

su

rfac

e lt

yello

w/ta

n ~i

rreg

ular

big

glo

ssy

rippl

ed b

umpy

opa

que

905

Mic

roco

ccus

sp. R

G-6

4

0.99

A

Y56

1623

A

T04-

161-

123

-3

1/10

PC

A

surf

ace

lt or

ange

/pk

~tra

ns g

loss

y co

nvex

63

7 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T04-

161-

124

-3

1/10

PC

A

surf

ace

tan

with

dar

ker t

an in

cen

ter g

loss

y ~t

rans

con

vex

821

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-12

5 -2

1/

10 P

CA

su

rfac

e or

ange

/tan

glos

sy ~

trans

con

vex

838

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-12

6 -2

1/

10 P

CA

su

rfac

e or

ange

/pk

opaq

ue g

loss

y co

nvex

74

6 Bl

asto

cocc

us sp

. BC

412

0.

97

AJ3

1657

4 A

T04-

161-

127

-2

1/10

PC

A

surf

ace

pk/p

each

~tra

ns g

loss

y co

nvex

76

7 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

129

-2

1/10

PC

A

surf

ace

pk/p

each

glo

ssy

opaq

ue c

onve

x ce

nter

uns

moo

th fl

at e

dges

76

7 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

130

-2

1/10

PC

A

surf

ace

oran

ge/p

k ~t

rans

glo

ssy

~con

vex

!~irr

egul

ar

751

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

1-13

1 -2

1/

10 P

CA

su

rfac

e sm

all l

t pk

opaq

ue ~

glos

sy c

onve

x 79

5 Bl

asto

cocc

us sp

. BC

412

0.

98

AJ3

1657

4 A

T04-

161-

133

-2

1/10

PC

A

surf

ace

peac

h/pk

~tra

ns g

loss

y co

nvex

68

3 G

eorg

enia

sp. T

04-0

4

0.95

A

Y88

0044

A

T04-

161-

134

-2

1/10

PC

A

surf

ace

trans

off

whi

te/ta

n gl

ossy

con

vex

633

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

4-16

1-13

5 -2

1/

10 P

CA

su

rfac

e da

rker

ora

nge

glos

sy m

ore

opaq

ue a

nd c

onve

x ce

nter

(~tra

ns fl

at e

dges

) 74

9 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

137

-2

1/10

PC

A

surf

ace

trans

glo

ssy

conv

ex

700

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-13

8 -2

1/

10 P

CA

su

rfac

e pk

/pea

ch ~

trans

glo

ssy

conv

ex (m

ore

conv

ex in

cen

ter)

79

8 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1

139

(Tab

le c

ontin

ued:

Sur

face

isol

ates

) Su

rfac

e Is

olat

e

D

ilutio

n M

edia

D

epth

Col

ony

Mor

phol

ogy

Seq

uenc

e L

engt

h

BL

AST

Res

ult

Sim

ilari

ty A

cces

sion

no.

A

T04-

161-

140

-2

1/10

PC

A

surf

ace

peac

h/pk

~tra

ns g

loss

y co

nvex

74

0 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

141

-2

1/10

PC

A

surf

ace

yello

w /o

rang

e ~t

rans

glo

ssy

conv

ex

794

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

4-16

1-14

2 -2

1/

10 P

CA

su

rfac

e or

ange

/pk

~fla

t glo

ssy

~tra

ns

772

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

1-14

3 -2

1/

10 P

CA

su

rfac

e lt

pk ~

trans

glo

ssy

conv

ex (h

ard)

60

0 Bl

asto

cocc

us sp

. BC

448

0.

96

AJ3

1657

1 A

T04-

161-

144

-2

1/10

PC

A

surf

ace

oran

ge/h

ot p

k op

aque

~gl

ossy

rais

ed ir

regu

lar r

igid

bum

py ro

ugh

738

Blas

toco

ccus

agg

rega

tus

0.97

A

J430

193

AT0

4-16

1-14

5 -2

1/

10 P

CA

su

rfac

e bu

rnt o

rang

e ~c

onve

x gl

ossy

~op

aque

83

6 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

146

-2

1/10

PC

A

surf

ace

pk o

paqu

e co

nvex

glo

ssy

979

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

1-14

7 -2

1/

10 P

CA

su

rfac

e of

fwhi

te fl

at b

ut ra

ised

mid

dle

opaq

ue g

loss

y 63

2 Bl

asto

cocc

us sp

. BC

448

0.

95

AJ3

1657

1 A

T04-

161-

148

-2

1/10

PC

A

surf

ace

lt or

ange

opa

que

conv

ex g

loss

y 73

0 Bl

asto

cocc

us sp

. BC

448

0.

96

AJ3

1657

1 A

T04-

161-

149

-2

1/10

PC

A

surf

ace

pink

opa

que

conv

ex g

loss

y 97

8 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

161-

150

-2

1/10

PC

A

surf

ace

peac

h ~c

onve

x gl

ossy

~op

aque

78

9 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

161-

151

-2

1/10

0 PC

A s

urfa

ce l

t pk

~tra

ns g

loss

y co

nvex

79

8 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

162-

1 -1

1/

10 P

CA

su

rfac

e lt

pk ~

trans

~irr

egul

ar b

umpy

rigi

d ro

ugh

rais

ed

769

Geo

derm

atop

hilu

s obs

curu

s obs

curu

s 0.

97

L406

20

AT0

4-16

2-3

-1

MA

su

rfac

e bl

ack

~tra

ns ~

irreg

ular

bum

py ri

gid

roug

h ra

ised

66

0 G

eode

rmat

ophi

lus o

bscu

rus o

bscu

rus

0.93

L4

0620

A

T04-

162-

5 -1

M

A

surf

ace

blac

k ~t

rans

~irr

egul

ar b

umpy

rigi

d ro

ugh

rais

ed

569

Geo

derm

atop

hilu

s obs

curu

s obs

curu

s 0.

96

L406

20

AT0

4-16

2-6

-1

MA

su

rfac

e bl

ack

~tra

ns ~

irreg

ular

bum

py ri

gid

roug

h ra

ised

77

6 G

eode

rmat

ophi

lus o

bscu

rus o

bscu

rus

0.97

L4

0620

A

T04-

162-

7 -2

M

A

surf

ace

blac

k ~t

rans

~irr

egul

ar b

umpy

rigi

d ro

ugh

rais

ed

750

Geo

derm

atop

hilu

s obs

curu

s obs

curu

s 0.

97

L406

20

AT0

4-16

2-8

-2

MA

su

rfac

e bl

ack

~tra

ns ~

irreg

ular

bum

py ri

gid

roug

h ra

ised

78

2 G

eode

rmat

ophi

lus o

bscu

rus o

bscu

rus

0.97

L4

0620

A

T04-

162-

9 -1

M

A

surf

ace

blac

k ~t

rans

~irr

egul

ar b

umpy

rigi

d ro

ugh

rais

ed

744

Geo

derm

atop

hilu

s obs

curu

s obs

curu

s 0.

96

L406

20

AT0

4-16

2-10

-2

M

A

surf

ace

blac

k ~t

rans

~irr

egul

ar b

umpy

rigi

d ro

ugh

rais

ed

776

Geo

derm

atop

hilu

s obs

curu

s obs

curu

s 0.

97

L406

20

AT0

4-16

2-12

-1

PC

A

surf

ace

brig

ht y

ello

w ~

irreg

ular

opa

que

glos

sy b

ig c

onve

x 98

3 M

icro

cocc

us lu

teus

0.

99

AJ4

0909

6 A

T04-

162-

13

-2

PCA

su

rfac

e lt

yello

w ~

glos

sy ~

irreg

ular

opa

que

conv

ex b

ig

959

Mic

roco

ccus

lute

us

0.99

A

J409

096

AT0

4-16

4-1

-2

MA

su

rfac

e gl

ossy

flat

opa

que

crea

m y

ello

w

764

Hal

obac

illus

sp. M

O22

0.

96

AY

5531

13

AT0

4-16

4-2

-1

MA

su

rfac

e gl

ossy

con

vex

opaq

ue p

ink

749

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

4-3

-1

MA

su

rfac

e gl

ossy

con

vex

opaq

ue c

ream

78

5 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

164-

5 -1

M

A

surf

ace

dull

clum

py o

ff-w

hite

con

vex

opaq

ue

720

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

4-6

-1

MA

su

rfac

e ~d

ull c

lum

py c

onve

x lt

pink

ish

crea

m

767

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

4-7

-1

MA

su

rfac

e gl

ossy

flat

tran

s cre

am c

olor

ed

749

Bac

teriu

m K

2-24

0.

96

AY

3454

29

AT0

4-16

4-8

-1

MA

su

rfac

e ~g

loss

y ~o

paqu

e ~c

onve

x lt

pk

762

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

4-9

-1

MA

su

rfac

e gl

ossy

offw

hite

flat

~op

aque

77

0 G

laci

al ic

e ba

cter

ium

G50

0K-1

9

0.98

A

F479

330

AT0

4-16

4-10

-1

M

A

surf

ace

smal

l ~co

nvex

lt p

ink

glos

sy o

paqu

e 72

2 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

164-

11

-1

MA

su

rfac

e fla

t glo

ssy

lt ye

llow

ish

opaq

ue

570

Bac

teriu

m K

2-24

0.

96

AY

3454

29

AT0

4-16

4-12

-1

M

A

surf

ace

flat g

loss

y op

aque

off

whi

te p

inki

sh

735

Hal

obac

illus

sp. M

O22

0.

97

AY

5531

13

AT0

4-16

4-13

-2

1/

10 P

CA

su

rfac

e gl

ossy

lt p

ink

conv

ex ~

opaq

ue

640

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

4-14

-1

1/

10 P

CA

su

rfac

e gl

ossy

lt p

ink

conv

ex o

paqu

e 77

7 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

164-

16

-1

1/10

PC

A

surf

ace

glos

sy ~

conv

ex o

rang

e/pi

nk o

paqu

e 75

8 M

odes

toba

cter

sp. E

llin1

65

0.98

A

F409

007

AT0

4-16

4-17

-1

1/

10 P

CA

su

rfac

e gl

ossy

flat

opa

que

whi

te

624

Gla

cial

ice

bact

eriu

m G

500K

-9

0.93

A

F479

338

AT0

4-16

4-18

-1

1/

10 P

CA

su

rfac

e gl

ossy

~co

nvex

lt p

ink

opaq

ue

747

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

4-19

-1

1/

10 P

CA

su

rfac

e du

ll ye

llow

flat

opa

que

grow

s in

agar

74

8 St

rept

omyc

es c

hung

whe

nsis

stra

in A

A-9

8

0.99

A

Y38

2292

A

T04-

164-

20

-1

1/10

PC

A

surf

ace

brow

nish

opa

que

flat g

row

ing

arou

nd b

ubbl

e in

aga

r 78

2 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

164-

21

-3

MA

su

rfac

e of

f whi

te fl

at o

paqu

e gl

ossy

76

6 St

aphy

loco

ccus

epi

derm

idis

0.

99

AY

0303

42

AT0

4-16

4-22

-3

M

A

surf

ace

peac

h gl

ossy

con

vex

~clu

mpy

opa

que

1005

Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

164-

23

-2

MA

su

rfac

e lig

ht o

rang

e/pi

nk c

onve

x op

aque

glo

ssy

768

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

4-24

-2

M

A

surf

ace

light

yel

low

ish

peac

h co

nvex

clu

mpy

~gl

ossy

opa

que

786

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

4-25

-1

M

A

surf

ace

~con

vex

with

dip

in th

e ce

nter

lt p

ink

opaq

ue g

loss

y 78

0 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

164-

26

-1

MA

su

rfac

e fla

t opa

que

off w

hite

/yel

low

glo

ssy

964

Hal

obac

illus

sp. M

O22

0.

97

AY

5531

13

AT0

4-16

4-27

-1

M

A

surf

ace

light

pea

ch o

paqu

e cl

umpy

con

vex

760

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

140

(Tab

le c

ontin

ued:

Sur

face

isol

ates

) Su

rfac

e Is

olat

e

D

ilutio

n M

edia

D

epth

Col

ony

Mor

phol

ogy

Seq

uenc

e L

engt

h

BL

AST

Res

ult

Sim

ilari

ty A

cces

sion

no.

A

T04-

164-

28

-1

MA

su

rfac

e lig

ht p

ink

opaq

ue c

lum

py c

onve

x 97

4 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

164-

29

-1

MA

su

rfac

e of

f whi

te ~

dull

conv

ex g

reen

ish

cent

er o

paqu

e 77

3 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

164-

30

-2

MA

su

rfac

e fla

t opa

que

crea

m g

loss

y 62

5 Ba

cillu

s lito

ralis

0.

97

AY

6086

05

AT0

4-16

4-31

-2

M

A

surf

ace

peac

h co

nvex

opa

que

clum

py

754

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

4-32

-2

M

A

surf

ace

light

pin

k co

nvex

glo

ssy

opaq

ue

986

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

4-34

-2

M

A

surf

ace

light

pin

k co

nvex

glo

ssy

opaq

ue

763

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

4-35

-2

M

A

surf

ace

flesh

col

ored

clu

mpy

~gl

ossy

opa

que

conv

ex

739

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

4-36

-1

M

A

surf

ace

peac

h co

nvex

clu

mpy

opa

que

~glo

ssy

771

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

4-37

-1

M

A

surf

ace

light

pin

k co

nvex

glo

ssy

opaq

ue

987

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

4-38

-1

M

A

surf

ace

peac

h cl

umpy

~du

ll co

nvex

opa

que

772

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

4-39

-1

M

A

surf

ace

flat g

loss

y ~t

rans

yel

low

77

1 B

acte

rium

K2-

24

0.97

A

Y34

5429

A

T04-

164-

40

-1

MA

su

rfac

e pi

nk c

onve

x op

aque

glo

ssy

774

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

4-41

-1

1/

10 P

CA

su

rfac

e pi

nk g

loss

y op

aque

con

vex

792

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

4-42

-1

1/

10 P

CA

su

rfac

e pi

nk g

loss

y op

aque

~co

nvex

79

4 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

164-

43

-1

1/10

PC

A

surf

ace

yello

w g

loss

y op

aque

~co

nvex

77

1 K

ocur

ia e

ryth

rom

yxa

0.

98

Y11

330

AT0

4-16

4-44

-3

1/

10 P

CA

su

rfac

e fla

t whi

te tr

ans g

loss

y 73

7 Sp

hing

omon

as sp

. SIA

181-

1A1

0.

97

AF3

9503

2 A

T04-

164-

45

-2

1/10

PC

A

surf

ace

oran

ge fl

at o

paqu

e gl

ossy

61

1 Sp

hing

omon

as sp

. SIA

181-

1A1

0.

97

AF3

9503

2 A

T04-

164-

46

-2

1/10

PC

A

surf

ace

light

pin

k gl

ossy

opa

que

~con

vex

843

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

4-47

-2

1/

10 P

CA

su

rfac

e lig

ht p

ink

glos

sy o

paqu

e ~c

onve

x 77

4 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

164-

48

-4

1/10

PC

A

surf

ace

whi

te tr

ans f

lat

795

Stap

hylo

cocc

us e

pide

rmid

is

0.99

A

Y03

0342

A

T04-

164-

49

-4

1/10

PC

A

surf

ace

pink

glo

ssy

conv

ex o

paqu

e 80

9 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

164-

50

-1

1/10

PC

A

surf

ace

pink

glo

ssy

opaq

ue ~

conv

ex

774

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

4-51

-2

1/

100

PCA

sur

face

lt p

k ~t

rans

glo

ssy

conv

ex

764

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

4-52

-2

N

A

surf

ace

pk/o

rang

e tra

ns g

loss

y co

nvex

98

6 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

164-

53

-2

NA

su

rfac

e or

ange

/tang

erin

e tra

ns g

loss

y co

nvex

68

2 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

165-

1 -1

M

A

surf

ace

glos

sy o

paqu

e lt

pk c

onve

x 77

2 G

eode

rmat

ophi

lus s

p. B

C51

8

0.99

A

J296

064

AT0

4-16

5-2

-1

MA

su

rfac

e gl

ossy

opa

que

lt ye

llow

ish

crea

m c

onve

x 77

1 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T04-

165-

3 -1

M

A

surf

ace

glos

sy o

paqu

e of

f-w

hite

~co

nvex

74

8 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

165-

4 -2

M

A

surf

ace

glos

sy c

onve

x lt

pk o

paqu

e 72

9 G

eode

rmat

ophi

lus s

p. B

C51

8

0.98

A

J296

064

AT0

4-16

5-6

-1

MA

su

rfac

e gl

ossy

~co

nvex

opa

que

lt pi

nk

616

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

5-7

-1

MA

su

rfac

e gl

ossy

~co

nvex

opa

que

crea

m

716

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

5-8

-2

MA

su

rfac

e gl

ossy

con

vex

opaq

ue p

inki

sh o

rang

e 64

5 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

165-

9 -2

M

A

surf

ace

glos

sy c

onve

x op

aque

off

-whi

te c

ream

55

0 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

165-

10

-1

MA

su

rfac

e gl

ossy

con

vex

opaq

ue lt

pk

709

Geo

derm

atop

hilu

s sp.

BC

518

0.

99

AJ2

9606

4 A

T04-

165-

11

-1

MA

su

rfac

e gl

ossy

opa

que

conv

ex o

ff-w

hite

79

9 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

165-

12

-2

MA

su

rfac

e gl

ossy

opa

que

conv

ex p

ink

798

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

5-13

-2

M

A

surf

ace

glos

sy o

paqu

e co

nvex

off

-whi

te

779

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

5-14

-2

M

A

surf

ace

glos

sy o

paqu

e co

nvex

lt y

ello

w

766

Cel

lulo

mon

as c

ella

sea

0.

96

X83

804

AT0

4-16

5-15

-2

1/

10 P

CA

su

rfac

e gl

ossy

opa

que

conv

ex lt

pk

784

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

5-16

-2

1/

10 P

CA

su

rfac

e gl

ossy

opa

que

conv

ex lt

pin

k 77

2 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

165-

17

-2

1/10

PC

A

surf

ace

glos

sy o

paqu

e co

nvex

off

-whi

te

773

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

4-16

5-18

-1

1/

10 P

CA

su

rfac

e gl

ossy

opa

que

conv

ex c

ream

ish

687

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

5-19

-2

1/

10 P

CA

su

rfac

e gl

ossy

opa

que

conv

ex lt

pin

kish

75

5 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

165-

20

-1

1/10

PC

A

surf

ace

glos

sy o

paqu

e ~c

onve

x lt

pink

ish

oran

ge

802

Geo

derm

atop

hilu

s sp.

BC

518

0.

98

AJ2

9606

4 A

T04-

165-

21

-1

1/10

PC

A

surf

ace

glos

sy o

paqu

e ~c

onve

x lt

pk

777

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

5-22

-1

1/

10 P

CA

su

rfac

e ~g

loss

y op

aque

con

vex

brow

n 65

3 M

odes

toba

cter

sp. E

llin1

64

0.97

A

F409

006

AT0

4-16

5-23

-1

PC

A

surf

ace

tang

erin

e op

aque

con

vex

glos

sy

958

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

141

(Tab

le c

ontin

ued:

Sur

face

isol

ates

) Su

rfac

e Is

olat

e

D

ilutio

n M

edia

D

epth

Col

ony

Mor

phol

ogy

Seq

uenc

e L

engt

h

BL

AST

Res

ult

Sim

ilari

ty A

cces

sion

no.

A

T04-

165-

25

-1

PCA

su

rfac

e ta

nger

ine

opaq

ue c

onve

x gl

ossy

10

23

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

5-27

-2

N

A

surf

ace

tang

erin

e/ta

n op

aque

con

vex

glos

sy

1028

Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

165-

29

-1

NA

su

rfac

e lt

pk/ta

n op

aque

glo

ssy

conv

ex

882

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

5-31

-2

N

A

surf

ace

tan/

peac

h gl

ossy

con

vex

opaq

ue

963

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

5-32

-2

1/

100

PCA

sur

face

lt p

k gl

ossy

con

vex

~tra

ns

783

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

6-1

-2

MA

su

rfac

e lt

pk g

loss

y bi

g co

nvex

opa

que

751

Koc

uria

sp. 2

216.

35.3

1

0.99

A

B09

4467

A

T04-

166-

2 -2

M

A

surf

ace

smal

l lt p

k gl

ossy

con

vex

~opa

que

708

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

6-3

-2

MA

su

rfac

e bi

g bu

bble

gum

pk

~irr

egul

ar g

loss

y ~c

onve

x ~o

paqu

e 73

6 K

ocur

ia sp

. 221

6.35

.31

0.

98

AB

0944

67

AT0

4-16

6-4

-2

MA

su

rfac

e ye

llow

irre

gula

r glo

ssy

med

~op

aque

~co

nvex

72

3 K

ocur

ia e

ryth

rom

yxa

0.

98

Y11

330

AT0

4-16

6-5

-2

MA

su

rfac

e w

hite

~tra

ns g

loss

y co

nvex

77

5 C

ellu

lom

onas

car

tae

MSD

201

06

0.96

X

7945

6 A

T04-

166-

6 -2

M

A

surf

ace

flat s

mal

l pk

glos

sy tr

ans d

arke

r cen

ter

773

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

6-7

-2

MA

su

rfac

e lt

pk v

ery

~irr

egul

ar ~

dull

rais

ed ~

rigid

opa

que

781

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

6-8

-2

MA

su

rfac

e pk

irre

gula

r rai

sed

rigid

bum

py ~

trans

~gl

ossy

76

8 G

eorg

enia

sp. 2

216.

35.2

8

0.94

A

B09

4466

A

T04-

166-

9 -2

M

A

surf

ace

lt pk

opa

que

~irr

egul

ar ~

glos

sy c

onve

x bu

t mor

e co

nvex

in c

ente

r 77

0 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

166-

10

-2

MA

su

rfac

e w

hite

tran

s glo

ssy

conv

ex

775

Cel

lulo

mon

as sp

. CJ1

0597

0.

95

AF5

0020

8 A

T04-

166-

11

-2

MA

su

rfac

e da

rker

pk

glos

sy c

onve

x ~t

rans

73

8 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

166-

13

-2

MA

su

rfac

e pk

~op

aque

glo

ssy

conv

ex

773

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

6-14

-2

M

A

surf

ace

lt pk

opa

que

glos

sy c

onve

x 87

5 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T04-

166-

15

-2

MA

su

rfac

e gl

ossy

flat

opa

que

brig

ht y

ello

wis

h w

ith ta

n 85

8 K

ocur

ia e

ryth

rom

yxa

0.

97

Y11

330

AT0

4-16

6-17

-2

M

A

surf

ace

opaq

ue c

ream

ish

clum

py c

onve

x ~d

ull

862

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

6-19

-2

M

A

surf

ace

glos

sy c

onve

x op

aque

cre

amis

h 86

4 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

166-

20

-2

MA

su

rfac

e or

ange

/tan

~dul

l ~cl

umpy

~tra

ns c

onve

x 86

1 G

eorg

enia

sp. 2

216.

35.2

8

0.93

A

B09

4466

A

T04-

166-

21

-2

MA

su

rfac

e fla

t whi

te g

loss

y op

aque

86

3 Ar

thro

bact

er sp

. R-2

3173

0.

99

AJ7

8682

1

AT0

4-16

6-22

-2

M

A

surf

ace

hot p

k co

nvex

glo

ssy

~tra

ns

739

Arth

roba

cter

sp. F

a21

0.

99

AY

1312

25

AT0

4-16

6-23

-2

M

A

surf

ace

whi

te ~

opaq

ue g

loss

y co

nvex

86

0 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

166-

24

-2

MA

su

rfac

e lt

pk tr

ans i

rreg

ular

rig

id ra

ised

bum

py

863

Geo

rgen

ia sp

. 221

6.35

.28

0.

93

AB

0944

66

AT0

4-16

6-25

-1

M

A

surf

ace

dark

er p

k/pe

ach

glos

sy c

onve

x da

rker

~tra

ns c

ente

r mor

e tra

ns e

dges

75

8 Bl

asto

cocc

us sp

. BC

521

0.

98

AJ3

1657

3 A

T04-

166-

26

-1

MA

su

rfac

e gl

ossy

flat

but

~co

nvex

hot

salm

on (p

inki

sh) o

paqu

e 77

8 K

ocur

ia e

ryth

rom

yxa

0.

97

Y11

330

AT0

4-16

6-27

-1

M

A

surf

ace

glos

sy fl

at p

each

col

ored

opa

que

761

Koc

uria

ery

thro

myx

a

0.97

Y

1133

0 A

T04-

166-

28

-1

MA

su

rfac

e gl

ossy

~co

nvex

brig

ht y

ello

w o

paqu

e 84

5 K

ocur

ia e

ryth

rom

yxa

0.

97

Y11

330

AT0

4-16

6-31

-1

M

A

surf

ace

flat ~

rais

ed o

ff-w

hite

/cre

am g

loss

y op

aque

circ

le

782

Baci

llus f

irm

us

0.99

D

1626

8 A

T04-

166-

32

-1

MA

su

rfac

e fla

t off

-whi

te/c

ream

glo

ssy

irreg

ular

73

7 B

acill

us fi

rmus

0.

99

D16

268

AT0

4-16

6-33

-1

M

A

surf

ace

flat g

loss

y op

aque

lt o

rang

e go

ld c

olor

77

6 K

ocur

ia e

ryth

rom

yxa

0.

98

Y11

330

AT0

4-16

6-34

-1

M

A

surf

ace

brig

ht sa

lmon

pin

kish

glo

ssy

opaq

ue fl

at c

ircle

s 80

5 K

ocur

ia sp

. 221

6.35

.31

0.

99

AB

0944

67

AT0

4-16

6-35

-1

M

A

surf

ace

peac

h co

lore

d gl

ossy

flat

opa

que

circ

les

729

Koc

uria

sp. 2

216.

35.3

1 0.

99

AB

0944

67

AT0

4-16

6-36

-1

M

A

surf

ace

~glo

ssy

~con

vex

gold

ora

nge

blob

opa

que

790

Arth

roba

cter

sp. R

-231

73

0.99

A

J786

821

A

T04-

166-

37

-1

MA

su

rfac

e fla

t opa

que

glos

sy c

ream

col

ored

74

9 Ar

thro

bact

er sp

. R-2

3173

0.

99

AJ7

8682

1

AT0

4-16

6-38

-1

M

A

surf

ace

thin

lt o

rang

e ~g

loss

y la

yer o

ver e

ntire

pla

te

708

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

6-39

-1

M

A

surf

ace

flat o

paqu

e gl

ossy

brig

ht y

ello

w

404

Koc

uria

ery

thro

myx

a

0.97

Y

1133

0 A

T04-

166-

40

-1

MA

su

rfac

e du

ll co

nvex

opa

que

whi

te d

ots

805

Koc

uria

sp. 2

216.

35.3

1

0.99

A

B09

4467

A

T04-

166-

41

-1

MA

su

rfac

e ~o

paqu

e ~c

lum

py c

ream

ish

conv

ex

786

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

6-42

-1

M

A

surf

ace

tiny

glos

sy ~

conv

ex lt

pin

k ci

rcle

s 77

7 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

166-

43

-1

MA

su

rfac

e gl

ossy

flat

and

rais

ed p

each

col

ored

opa

que

770

Koc

uria

sp. 2

216.

35.3

1

0.98

A

B09

4467

A

T04-

166-

44

-1

MA

su

rfac

e gl

ossy

flat

and

rais

ed b

right

salm

on p

ink

opaq

ue

761

Koc

uria

sp. 2

216.

35.3

1

0.99

A

B09

4467

A

T04-

166-

45

-1

MA

su

rfac

e du

ll w

hite

opa

que

conv

ex

771

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

6-46

-1

M

A

surf

ace

flat g

loss

y op

aque

brig

ht y

ello

w

532

Koc

uria

ery

thro

myx

a

0.97

Y

1133

0 A

T04-

166-

47

-1

MA

su

rfac

e br

owni

sh ta

n th

in la

yer

801

Koc

uria

sp. 2

216.

35.3

1

0.98

A

B09

4467

142

(Tab

le c

ontin

ued:

Sur

face

isol

ates

) Su

rfac

e Is

olat

e

D

ilutio

n M

edia

D

epth

Col

ony

Mor

phol

ogy

Seq

uenc

e L

engt

h

BL

AST

Res

ult

Sim

ilari

ty A

cces

sion

no.

A

T04-

166-

48

-1

MA

su

rfac

e sm

all g

loss

y ~o

paqu

e co

nvex

lt p

inki

sh

749

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

6-49

-2

M

A

surf

ace

brig

ht p

ink

glos

sy fl

at o

paqu

e 76

4 K

ocur

ia sp

. 221

6.35

.31

0.

99

AB

0944

67

AT0

4-16

6-50

-2

M

A

surf

ace

brig

ht y

ello

w fl

at g

loss

y op

aque

83

8 K

ocur

ia e

ryth

rom

yxa

0.

98

Y11

330

AT0

4-16

6-51

-2

M

A

surf

ace

smal

l cre

amis

h lt

pink

circ

les ~

opaq

ue g

loss

y co

nvex

81

1 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

166-

52

-1

1/10

PC

A

surf

ace

brig

ht p

ink

glos

sy o

paqu

e ~c

onve

x/fla

t 80

6 K

ocur

ia sp

. 221

6.35

.31

0.

99

AB

0944

67

AT0

4-16

6-53

-1

1/

10 P

CA

su

rfac

e lt

peac

h gl

ossy

~op

aque

/flat

opa

que

770

Koc

uria

ery

thro

myx

a

0.97

Y

1133

0 A

T04-

166-

54

-1

1/10

PC

A

surf

ace

dull

~clu

mpy

opa

que

conv

ex w

hite

74

8 K

ocur

ia sp

. 221

6.35

.31

0.

99

AB

0944

67

AT0

4-16

6-55

-1

1/

10 P

CA

su

rfac

e or

ange

thin

laye

r cov

erin

g 1/

2 of

pla

te

756

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

6-56

-1

1/

10 P

CA

su

rfac

e gl

ossy

flat

yel

low

opa

que

772

Koc

uria

sp. 2

216.

35.3

1

0.99

A

B09

4467

A

T04-

166-

57

-1

1/10

PC

A

surf

ace

dull

~fuz

zy o

paqu

e co

nvex

whi

te

801

Koc

uria

sp. 2

216.

35.3

1

0.98

A

B09

4467

A

T04-

166-

59

-1

1/10

PC

A

surf

ace

glos

sy ~

conv

ex/fl

at p

each

opa

que

753

Koc

uria

sp. 2

216.

35.3

1

0.99

A

B09

4467

A

T04-

166-

60

-1

1/10

PC

A

surf

ace

glos

sy c

onve

x op

aque

cre

am/o

ffw

hite

76

7 K

ocur

ia sp

. 221

6.35

.31

0.

99

AB

0944

67

AT0

4-16

6-61

-1

1/

10 P

CA

su

rfac

e gl

ossy

flat

/~co

nvex

brig

ht p

ink

opaq

ue

740

Koc

uria

sp. 2

216.

35.3

1

0.99

A

B09

4467

A

T04-

166-

62

-2

1/10

PC

A

surf

ace

glos

sy c

onve

x op

aque

brig

ht p

ink

753

Koc

uria

sp. 2

216.

35.3

1

0.99

A

B09

4467

A

T04-

166-

63

-2

1/10

PC

A

surf

ace

glos

sy c

onve

x ~t

rans

lt p

ink

76

8 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

166-

64

-2

1/10

PC

A

surf

ace

glos

sy c

onve

x op

aque

yel

low

74

1 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

166-

65

-2

1/10

PC

A

surf

ace

glos

sy c

onve

x br

ight

pin

k op

aque

75

6 K

ocur

ia sp

. 221

6.35

.31

0.

99

AB

0944

67

AT0

4-16

6-66

-2

1/

10 P

CA

su

rfac

e gl

ossy

con

vex

peac

h op

aque

81

4 K

ocur

ia e

ryth

rom

yxa

0.

98

Y11

330

AT0

4-16

6-67

-2

1/

10 P

CA

su

rfac

e lt

pink

con

vex

~opa

que

glos

sy

761

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

6-68

-2

1/

10 P

CA

su

rfac

e lt

pink

con

vex

~opa

que

glos

sy

749

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

6-69

-2

1/

10 P

CA

su

rfac

e gl

ossy

opa

que

conv

ex b

right

yel

low

52

3 K

ocur

ia e

ryth

rom

yxa

0.

97

Y11

330

AT0

4-16

6-70

-1

1/

10 P

CA

su

rfac

e du

ll w

hite

con

vex

opaq

ue

746

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

6-71

-1

1/

10 P

CA

su

rfac

e br

ight

pin

k gl

ossy

flat

~co

nvex

opa

que

770

Koc

uria

sp. 2

216.

35.3

1

0.99

A

B09

4467

A

T04-

166-

72

-1

1/10

PC

A

surf

ace

peac

h gl

ossy

~co

nvex

/flat

opa

que

77

8 K

ocur

ia sp

. 221

6.35

.31

0.

99

AB

0944

67

AT0

4-16

6-73

-1

1/

10 P

CA

su

rfac

e lt

oran

ge th

in la

yer c

over

ing

plat

e

771

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

6-74

-2

1/

10 P

CA

su

rfac

e sa

lmon

opa

que

~fla

t glo

ssy

769

Koc

uria

sp. 2

216.

35.3

1

0.99

A

B09

4467

A

T04-

166-

75

-2

1/10

PC

A

surf

ace

pink

opa

que

~con

vex

glos

sy

991

Arth

roba

cter

agi

lis

0.99

A

J577

725

AT0

4-16

6-77

-2

1/

10 P

CA

su

rfac

e lt

pink

opa

que

conv

ex g

loss

y 75

9 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

166-

78

-2

1/10

PC

A

surf

ace

brig

ht y

ello

w o

paqu

e co

nvex

glo

ssy

970

Koc

uria

sp. S

26-8

0.

98

DQ

0603

77

AT0

4-16

6-79

-4

1/

10 P

CA

su

rfac

e lig

ht p

ink

glos

sy o

paqu

e ~c

onve

x 76

4 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

166-

80

-4

1/10

PC

A

surf

ace

light

pin

k gl

ossy

opa

que

~con

vex

776

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

6-81

-4

1/

10 P

CA

su

rfac

e lig

ht p

ink

glos

sy o

paqu

e ~c

onve

x 72

3 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

166-

82

-2

1/10

PC

A

surf

ace

pink

glo

ssy

opaq

ue c

onve

x 62

2 B

acte

rium

K2-

25

0.99

A

Y34

5428

A

T04-

166-

83

-2

1/10

PC

A

surf

ace

yello

w g

loss

y op

aque

con

vex

776

Koc

uria

sp. 2

216.

35.3

1

0.99

A

B09

4467

A

T04-

166-

84

-1

1/10

PC

A

surf

ace

pink

glo

ssy

opaq

ue ~

conv

ex

818

Koc

uria

sp. 2

216.

35.3

1

0.99

A

B09

4467

A

T04-

166-

85

-1

1/10

PC

A

surf

ace

gold

glo

ssy

opaq

ue ~

conv

ex

863

Koc

uria

aeg

yptia

stra

in Y

IM 7

0003

0.

96

DQ

0596

17

AT0

4-16

6-87

-3

1/

10 P

CA

su

rfac

e lig

ht p

ink

glos

sy o

paqu

e ~c

onve

x 61

0 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

166-

88

-3

1/10

PC

A

surf

ace

yello

w o

paqu

e gl

ossy

~co

nvex

78

7 N

ocar

dioi

des k

ribb

ensi

s stra

in K

SL-6

0.

93

AY

8359

26

AT0

4-16

6-89

-3

1/

10 P

CA

su

rfac

e lig

ht p

ink

glos

sy o

paqu

e ~c

onve

x 51

0 Bl

asto

cocc

us sp

. BC

521

0.

84

AJ3

1657

3 A

T04-

166-

90

-3

1/10

PC

A

surf

ace

light

pin

k gl

ossy

opa

que

~con

vex

764

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

6-91

-3

1/

10 P

CA

su

rfac

e lig

ht y

ello

w ~

conv

ex o

paqu

e gl

ossy

61

1 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T04-

166-

92

-1

1/10

PC

A

surf

ace

peac

h gl

ossy

~co

nvex

opa

que

739

Koc

uria

sp. 2

216.

35.3

1

0.99

A

B09

4467

A

T04-

166-

93

-1

1/10

PC

A

surf

ace

pink

glo

ssy

~con

vex

opaq

ue

702

Arth

roba

cter

sp. M

uzt-C

11

0.99

A

Y52

6639

A

T04-

166-

95

-1

1/10

PC

A

surf

ace

pink

glo

ssy

~con

vex

opaq

ue

823

Koc

uria

sp. 2

216.

35.3

1

0.99

A

B09

4467

A

T04-

166-

96

-1

1/10

PC

A

surf

ace

oran

ge ~

dull

flat

773

Geo

derm

atop

hilu

s sp.

BC

509

0.

99

AJ2

9606

3 A

T04-

166-

97

-1

1/10

PC

A

surf

ace

gold

glo

ssy

opaq

ue ~

conv

ex

980

Koc

uria

sp. 2

216.

35.3

1

0.99

A

B09

4467

A

T04-

166-

98

-3

MA

su

rfac

e br

ight

pin

k gl

ossy

opa

que

conv

ex

773

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

143

(Tab

le c

ontin

ued:

Sur

face

isol

ates

) Su

rfac

e Is

olat

e

D

ilutio

n M

edia

D

epth

Col

ony

Mor

phol

ogy

Seq

uenc

e L

engt

h

BL

AST

Res

ult

Sim

ilari

ty A

cces

sion

no.

A

T04-

166-

99

-3

MA

su

rfac

e lig

ht p

each

/pin

k op

aque

con

vex

glos

sy

885

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

6-10

0 -3

M

A

surf

ace

flesh

col

ored

~cl

umpy

con

vex

opaq

ue g

loss

y 97

6 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

166-

101

-3

MA

su

rfac

e lig

ht p

ink

conv

ex o

paqu

e gl

ossy

75

4 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

166-

102

-3

MA

su

rfac

e br

ight

yel

low

con

vex

opaq

ue g

loss

y 73

9 C

ellu

lom

onas

car

tae

MSD

201

06

0.

96

X79

456

AT0

4-16

6-10

3 -3

M

A

surf

ace

brig

ht p

ink

conv

ex o

paqu

e gl

ossy

78

5 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

166-

104

-3

MA

su

rfac

e fle

sh c

lum

py o

paqu

e co

nvex

glo

ssy

766

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

6-10

5 -4

M

A

surf

ace

pink

/ora

nge

conv

ex o

paqu

e ~g

loss

y 76

3 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

166-

107

-4

MA

su

rfac

e pe

ach

conv

ex o

paqu

e gl

ossy

76

3 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

166-

108

-4

MA

su

rfac

e of

f whi

te c

onve

x op

aque

glo

ssy

755

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

6-10

9 -4

M

A

surf

ace

off w

hite

con

vex

opaq

ue d

ull ~

clum

py

979

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

6-11

0 -2

M

A

surf

ace

flat b

right

pin

k gl

ossy

opa

que

731

Koc

uria

sp. 2

216.

35.3

1

0.98

A

B09

4467

A

T04-

166-

111

-2

MA

su

rfac

e fla

t brig

ht y

ello

w g

loss

y op

aque

98

4 K

ocur

ia e

ryth

rom

yxa

0.

98

Y11

330

AT0

4-16

6-11

2 -2

M

A

surf

ace

off w

hite

flat

glo

ssy

opaq

ue

749

Arth

roba

cter

sp. R

-231

73

0.99

A

J786

821

AT0

4-16

6-11

3 -2

M

A

surf

ace

peac

h fla

t glo

ssy

opaq

ue

707

Koc

uria

sp. 2

216.

35.3

1

0.98

A

B09

4467

A

T04-

166-

114

-2

MA

su

rfac

e w

hite

opa

que

dull

conv

ex

707

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

4-16

6-11

6 -2

M

A

surf

ace

brig

ht p

ink

flat o

paqu

e gl

ossy

73

5 K

ocur

ia sp

. 221

6.35

.31

0.

99

AB

0944

67

AT0

4-16

6-11

7 -2

M

A

surf

ace

off w

hite

flat

opa

que

glos

sy

577

Arth

roba

cter

sp. R

-231

73

0.96

A

J786

821

A

T04-

166-

118

-2

MA

su

rfac

e ye

llow

flat

opa

que

glos

sy

760

Koc

uria

ery

thro

myx

a

0.98

Y

1133

0 A

T04-

166-

119

-2

MA

su

rfac

e lig

ht p

ink

glos

sy c

onve

x op

aque

76

4 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

166-

120

-2

MA

su

rfac

e lig

ht p

ink

glos

sy c

onve

x op

aque

79

9 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

166-

121

-2

MA

su

rfac

e w

hite

dul

l con

vex

opaq

ue

734

Exig

uoba

cter

ium

sp. B

TAH

1

0.99

A

Y20

5564

A

T04-

166-

122

-2

MA

su

rfac

e br

ight

pin

k fla

t glo

ssy

753

Koc

uria

sp. 2

216.

35.3

1

0.99

A

B09

4467

A

T04-

166-

123

-2

MA

su

rfac

e pe

ach

flat g

loss

y 80

6 K

ocur

ia sp

. 221

6.35

.31

0.

99

AB

0944

67

AT0

4-16

6-12

4 -2

M

A

surf

ace

off w

hite

flat

glo

ssy

933

Arth

roba

cter

sp. R

-231

73

0.99

A

J786

821

AT0

4-16

6-12

5 -1

M

A

surf

ace

brig

ht p

ink

glos

sy o

paqu

e ~c

onve

x 87

0 K

ocur

ia sp

. 221

6.35

.31

0.

99

AB

0944

67

AT0

4-16

6-12

6 -1

M

A

surf

ace

brig

ht y

ello

w g

loss

y op

aque

~co

nvex

78

9 K

ocur

ia e

ryth

rom

yxa

0.

98

Y11

330

AT0

4-16

6-12

7 -1

M

A

surf

ace

light

pin

k gl

ossy

opa

que

~con

vex

986

Koc

uria

sp. 2

216.

35.3

1

0.99

A

B09

4467

A

T04-

166-

128

-1

MA

su

rfac

e of

f whi

te g

loss

y op

aque

con

vex

910

Arth

roba

cter

sp. R

-231

73

0.99

A

J786

821

AT0

4-16

6-12

9 -1

M

A

surf

ace

oran

ge ~

glos

sy o

paqu

e ~c

onve

x 75

3 K

ocur

ia sp

. 221

6.35

.31

0.

99

AB

0944

67

AT0

4-16

6-13

0 -3

M

A

surf

ace

pink

con

vex

opaq

ue g

loss

y 77

5 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

166-

132

-3

MA

su

rfac

e or

ange

pin

k co

nvex

opa

que

glos

sy

790

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

6-13

3 -3

M

A

surf

ace

flesh

con

vex

clum

py o

paqu

e ~g

loss

y 71

6 Bl

asto

cocc

us sp

. BC

448

0.

95

AJ3

1657

1 A

T04-

166-

134

-3

MA

su

rfac

e lig

ht y

ello

w c

onve

x cl

umpy

~tra

ns ~

dull

800

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

4-16

6-13

5 -4

M

A

surf

ace

light

pin

k co

nvex

opa

que

glos

sy

765

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

6-13

6 -4

M

A

surf

ace

pink

con

vex

opaq

ue g

loss

y 80

5 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

166-

137

-4

MA

su

rfac

e cr

eam

dul

l con

vex

opaq

ue

603

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

6-13

8 -4

M

A

surf

ace

light

yel

low

opa

que

conv

ex g

loss

y 77

0 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

166-

139

-1

MA

su

rfac

e pi

nk fl

at g

loss

y op

aque

98

2 K

ocur

ia sp

. 221

6.35

.31

0.

99

AB

0944

67

AT0

4-16

6-14

1 -1

M

A

surf

ace

peac

h fla

t glo

ssy

opaq

ue

803

Koc

uria

sp. 2

216.

35.3

1

0.99

A

B09

4467

A

T04-

166-

142

-1

MA

su

rfac

e tin

y lig

ht p

ink

~con

vex

~glo

ssy

753

Koc

uria

sp. 2

216.

35.3

1

0.99

A

B09

4467

A

T04-

166-

144

-4

MA

su

rfac

e lig

ht p

ink

conv

ex g

loss

y op

aque

10

09

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

6-14

5 -1

M

A

surf

ace

yello

w fl

at g

loss

y op

aque

76

3 K

ocur

ia e

ryth

rom

yxa

0.

97

Y11

330

AT0

4-16

6-14

6 -1

M

A

surf

ace

pink

flat

glo

ssy

opaq

ue

774

Koc

uria

sp. 2

216.

35.3

1

0.99

A

B09

4467

A

T04-

166-

147

-1

MA

su

rfac

e pe

ach

flat g

loss

y op

aque

75

4 K

ocur

ia sp

. 221

6.35

.31

0.

99

AB

0944

67

AT0

4-16

6-14

8 -4

N

A

surf

ace

peac

h op

aque

glo

ssy

conv

ex

774

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

6-14

9 -4

N

A

surf

ace

pk/p

each

opa

que

glos

sy c

onve

x 78

2 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

166-

150

-3

NA

su

rfac

e tra

ns p

each

/bro

wn

glos

sy ~

conv

ex

732

Blas

toco

ccus

sp. B

C52

1

0.97

A

J316

573

144

(Tab

le c

ontin

ued:

Sur

face

isol

ates

) Su

rfac

e Is

olat

e

D

ilutio

n M

edia

D

epth

Col

ony

Mor

phol

ogy

Seq

uenc

e L

engt

h

BL

AST

Res

ult

Sim

ilari

ty A

cces

sion

no.

A

T04-

166-

151

-3

NA

su

rfac

e pe

ach

dark

er c

ente

r opa

que

~glo

ssy

conv

ex li

ghte

r edg

es

787

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

6-15

2 -3

N

A

surf

ace

trans

glo

ssy

conv

ex

496

Blas

toco

ccus

agg

rega

tus

0.97

A

J430

193

AT0

4-16

6-15

3 -3

N

A

surf

ace

crea

m o

ffw

hite

~tra

ns g

loss

y co

nvex

83

4 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

166-

154

-3

NA

su

rfac

e or

ange

~op

aque

tiny

~gl

ossy

~co

ncav

e 69

7 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

166-

156

-3

NA

su

rfac

e pe

ach/

pk ~

opaq

ue ~

glos

sy c

onve

x 77

2 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

166-

157

-3

NA

su

rfac

e pe

ach

~opa

que

~glo

ssy

conv

ex

704

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

4-16

6-15

8 -3

N

A

surf

ace

oran

ge ~

opaq

ue ~

glos

sy ~

conc

ave

765

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

6-15

9 -3

N

A

surf

ace

oran

ge g

loss

y co

nvex

~tra

ns

842

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

6-16

0 -3

N

A

surf

ace

lt pk

tran

s glo

ssy

conv

ex

787

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

6-16

2 -4

1/

100

PCA

sur

face

pk

opaq

ue ~

conv

ex ~

glos

sy

488

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

4-16

6-16

3 -4

1/

100

PCA

sur

face

lt p

k tra

ns g

loss

y co

nvex

(dar

ker i

n ce

nter

tran

s edg

es)

588

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

6-16

4 -4

1/

100

PCA

sur

face

tra

ns g

loss

y ~l

t pk/

offw

hite

con

vex

641

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

6-16

5 -4

1/

100

PCA

sur

face

pk

opaq

ue ~

conv

ex ~

glos

sy

819

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

6-16

6 -4

1/

100

PCA

sur

face

tra

ns g

loss

y ~l

t pk/

offw

hite

con

vex

848

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-16

6-16

7 -3

1/

10 P

CA

su

rfac

e ta

n/or

ange

~op

aque

~gl

ossy

con

vex

roun

d 94

3 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

166-

168

-3

1/10

PC

A

surf

ace

trans

~fla

t glo

ssy

955

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

6-16

9 -4

1/

10 P

CA

su

rfac

e bi

g br

ight

ora

nge

glos

sy o

paqu

e !~

irreg

ular

~co

nvex

94

0 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

166-

170

-4

1/10

PC

A

surf

ace

trans

bum

py ro

ugh

rigid

irre

gula

r glo

ssy

952

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

6-17

1 -3

1/

100

PCA

sur

face

sm

all o

rang

e/pk

tran

s glo

ssy

conv

ex

957

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-16

6-18

5 -1

N

A

surf

ace

hot o

rang

e ~c

onve

x !g

loss

y bi

g op

aque

92

3 K

ocur

ia sp

. 221

6.35

.31

0.

98

AB

0944

67

AT0

4-16

7-1

-1

NA

su

rfac

e irr

egul

ar fl

at ~

trans

cre

amy

offw

hite

glo

ssy

(may

be

cont

amin

atio

n)

1020

Ac

inet

obac

ter r

adio

resi

sten

s 0.

99

X81

666

145

(Tab

le c

ontin

ued:

Pit

isol

ates

) P

it Is

olat

e

D

ilutio

n M

edia

D

epth

Col

ony

Mor

phol

ogy

Seq

uenc

e L

engt

h

BL

AST

Res

ult

Sim

ilari

ty A

cces

sion

no.

A

T04-

150-

1 -1

M

A

50 c

m

whi

te c

halk

y su

rfac

e op

aque

non

glos

sy ~

irreg

ular

rais

ed ~

bum

py

804

Pseu

dono

card

ia sa

turn

ea

0.97

A

J252

829

AT0

4-15

0-2

-1

MA

50

cm

w

hite

cha

lky

surf

ace

opaq

ue n

ongl

ossy

~irr

egul

ar ra

ised

~bu

mpy

80

4 Ps

eudo

noca

rdia

satu

rnea

0.

97

AJ2

5282

9 A

T04-

150-

3 -1

M

A

20 c

m

whi

te c

halk

y su

rfac

e op

aque

non

glos

sy ~

irreg

ular

rais

ed ~

bum

py

828

Pseu

dono

card

ia sa

turn

ea

0.98

A

J252

829

AT0

4-15

0-4

-1

MA

50

cm

w

hite

cha

lky

surf

ace

opaq

ue n

ongl

ossy

~irr

egul

ar ra

ised

~bu

mpy

99

5 Ps

eudo

noca

rdia

satu

rnea

0.

98

AJ2

5282

9 A

T04-

150-

5 -1

M

A

40 c

m

whi

te c

halk

y su

rfac

e op

aque

non

glos

sy ~

irreg

ular

rais

ed ~

bum

py

772

Pseu

dono

card

ia sa

turn

ea

0.98

A

J252

829

AT0

4-15

0-6

-1

MA

40

cm

of

fwhi

te/ta

n ~g

loss

y ra

ised

~op

aque

bum

py ri

gid

hard

94

1 Ps

eudo

noca

rdia

satu

rnea

0.

98

AJ2

5282

9 A

T04-

150-

7 -1

M

A

20 c

m

whi

te c

halk

y su

rfac

e op

aque

non

glos

sy ~

irreg

ular

rais

ed ~

bum

py

736

Pseu

dono

card

ia sa

turn

ea

0.98

A

J252

829

AT0

4-15

0-8

-1

MA

40

cm

br

ight

yel

low

opa

que

glos

sy ~

conv

ex

1020

M

icro

cocc

us lu

teus

0.

99

AJ4

0909

6 A

T04-

150-

9 -1

M

A

30 c

m

whi

te c

halk

y op

aque

dul

l irr

egul

ar ra

ised

bum

py ri

gid

tan

base

gro

ws i

nto

agar

88

3 Ps

eudo

noca

rdia

satu

rnea

0.

97

AJ2

5282

9 A

T04-

150-

10

-1

MA

30

cm

br

ight

mus

tard

yel

low

with

bro

wni

sh c

ente

r glo

ssy

conv

ex o

paqu

e 69

9 N

ocar

dioi

des s

p. G

WS-

BW

-H31

1M

0.98

A

Y33

2121

A

T04-

150-

11

-1

MA

30

cm

w

hite

opa

que

conv

ex g

loss

y 86

1 N

ocar

dioi

des s

p. 4

P1-A

0.

96

AY

0275

87

AT0

4-15

0-12

-1

M

A

30 c

m

oran

ge o

paqu

e ~g

loss

y co

nvex

but

cen

ter o

f col

ony

is !c

onve

x (li

ke d

ome)

77

9 M

ycob

acte

rium

sp. P

X3.

15

0.99

A

Y33

7605

A

T04-

150-

13

-1

MA

30

cm

w

hite

opa

que

conv

ex g

loss

y 81

7 N

ocar

dioi

dace

ae st

r. El

lin10

1 0.

96

AF4

0894

3 A

T04-

150-

14

-1

MA

30

cm

br

ight

yel

low

glo

ssy

conv

ex o

paqu

e 80

8 N

osto

coid

a ar

omat

ivor

a 0.

96

AY

6823

81

AT0

4-15

0-15

-2

M

A

40 c

m

whi

te c

halk

y du

ll irr

egul

ar ra

ised

opa

que

bum

py ri

gid

tan

base

in a

gar

855

Pseu

dono

card

ia sa

turn

ea

0.98

A

J252

829

AT0

4-15

0-16

-1

M

A

40 c

m

whi

te c

halk

y du

ll irr

egul

ar ra

ised

opa

que

bum

py ri

gid

tan

base

in a

gar

997

Pseu

dono

card

ia sa

turn

ea

0.97

A

J252

829

AT0

4-15

0-17

-1

M

A

40 c

m

offw

hite

/tan

~glo

ssy

rais

ed ~

opaq

ue b

umpy

rigi

d ha

rd b

ase

in a

gar

890

Pseu

dono

card

ia sa

turn

ea

0.98

A

J252

829

AT0

4-15

0-18

-2

M

A

40 c

m

tan

dull

rais

ed ~

opaq

ue w

hite

cha

lky

spot

s on

surf

ace

bum

py h

ard

in a

gar

877

Pseu

dono

card

ia sa

turn

ea

0.97

A

J252

829

AT0

4-15

0-19

-1

M

A

30 c

m

whi

te c

halk

y su

rfac

e du

ll irr

egul

ar ra

ised

opa

que

rigid

tan

base

in a

gar

882

Pseu

dono

card

ia sa

turn

ea

0.98

A

J252

829

AT0

4-15

0-21

-1

M

A

10 c

m

whi

te c

halk

y su

rfac

e du

ll irr

egul

ar ra

ised

opa

que

rigid

tan

base

in a

gar

966

Pseu

dono

card

ia sa

turn

ea

0.98

A

J252

829

AT0

4-15

0-24

-2

1/

10 P

CA

30

cm

pk

/ora

nge

~tra

ns g

loss

y co

nvex

87

9 G

eode

rmat

ophi

lus s

p.

0.98

X

9235

8 A

T04-

150-

25

-2

1/10

PC

A

30 c

m

oran

ge v

ery

conv

ex g

loss

y co

nvex

87

8 U

ncul

t soi

l bac

teriu

m c

lone

0.

96

AF4

2327

7 A

T04-

150-

27

-1

PCA

80

cm

pk

con

vex

~glo

ssy

roug

h sm

all b

umps

on

colo

ny ~

opaq

ue

873

Cur

toba

cter

ium

flac

cum

faci

ens

0.99

A

Y27

3210

A

T04-

150-

28

-1

PCA

80

cm

br

ight

mus

tard

yel

low

glo

ssy

~opa

que

flat i

rreg

ular

88

1 C

urto

bact

eriu

m fl

accu

mfa

cien

s 0.

98

AY

2732

10

AT0

4-15

0-29

-1

PC

A

70 c

m

brig

ht su

nshi

ne y

ello

w ~

dull

opaq

ue v

ery

conv

ex

869

Mic

roco

ccus

sede

ntar

ius

0.99

X

8775

5 A

T04-

150-

30

-1

PCA

70

cm

ta

n ~i

rreg

ular

opa

que

edge

s rin

g ar

ound

tran

s fla

t glo

ssy

cent

er w

ith sp

ots

414

Stap

hylo

cocc

us sp

. Tf 2

54

0.97

A

Y46

1700

A

T04-

150-

31

-1

PCA

70

cm

ta

n ~i

rreg

ular

opa

que

edge

s rin

g ar

ound

tran

s fla

t glo

ssy

cent

er w

ith sp

ots

727

Stap

hylo

cocc

us sp

. 16b

-5A

0.

99

AY

5615

56

AT0

4-15

0-32

-1

PC

A

70 c

m

tan

~irr

egul

ar o

paqu

e ed

ges r

ing

arou

nd tr

ans f

lat g

loss

y ce

nter

with

spot

s 73

4 M

ethy

loba

cter

ium

mes

ophi

licum

0.

98

AJ4

0091

9 A

T04-

150-

33

-1

PCA

40

cm

lt

pk ~

trans

glo

ssy

smal

l bum

ps o

n co

lony

~co

nvex

89

8 C

hela

toco

ccus

asa

ccha

rovo

rans

0.

98

AY

1678

39

AT0

4-15

0-34

-1

PC

A

40 c

m

lt pk

~tra

ns g

loss

y sm

all b

umps

on

colo

ny ~

conv

ex

900

Che

lato

cocc

us a

sacc

haro

vora

ns

0.97

A

Y16

7839

A

T04-

150-

37

-1

NA

10

cm

lt

pk/ta

n ve

ry g

loss

y co

nvex

~op

aque

91

0 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T04-

150-

38

-2

NA

10

cm

bi

g irr

egul

ar ri

gid

~opa

que

glos

sy ~

flat y

ello

wis

h irr

egul

ar c

ente

r ~ta

n ed

ges

889

Baci

llus s

p. F

a29

16S

0.

98

AY

1312

22

AT0

4-15

2-1

-1

MA

10

cm

of

fwhi

te/ta

n ra

ised

~gl

ossy

~op

aque

~bu

mpy

~irr

egul

ar

895

Pseu

dono

card

ia sa

turn

ea

0.98

A

J252

829

AT0

4-15

2-2

-1

MA

10

cm

of

fwhi

te ~

trans

flat

dul

l edg

es p

urpl

e/br

own

near

ripp

led

whi

te fu

zzy

cent

er

785

Stre

ptom

yces

sp. I

M-7

082

0.

97

AF1

3154

9 A

T04-

152-

3 -1

M

A

10 c

m

chal

ky/w

hite

opa

que

dull

rais

ed ri

gid

surf

ace

offw

hite

bas

e gr

ows i

n ag

ar

792

Stre

ptom

ycet

acea

e

0.97

X

8732

0 A

T04-

152-

4 -1

M

A

10 c

m

whi

te c

halk

y su

rfac

e op

aque

~bu

mpy

rais

ed d

ull o

ffw

hite

bas

e gr

ows i

n ag

ar

777

Pseu

dono

card

ia sa

turn

ea

0.97

A

J252

829

AT0

4-15

2-5

-1

MA

10

cm

of

fwhi

te ~

trans

flat

dul

l edg

es p

urpl

e/br

own

near

ripp

led

whi

te fu

zzy

cent

er

800

Stre

ptom

yces

sp. I

M-7

082

0.97

A

F131

549

AT0

4-15

2-6

-1

MA

10

cm

of

f whi

te/ta

n ra

ised

~gl

ossy

~op

aque

~bu

mpy

~irr

egul

ar

889

Pseu

dono

card

ia sa

turn

ea

0.97

A

J252

829

AT0

4-15

2-7

-1

MA

10

cm

ch

alky

/whi

te o

paqu

e bu

mpy

dul

l rai

sed

cent

er ta

n op

aque

dul

l fla

t bas

e in

aga

r 79

0 St

rept

omyc

etac

eae

0.

95

X87

320

AT0

4-15

2-8

-1

MA

30

cm

ch

alky

/whi

te ~

irreg

ular

dul

l opa

que

bum

py o

ffw

hite

/tan

base

gro

ws i

n ag

ar

797

Pseu

dono

card

ia sa

turn

ea

0.98

A

J252

829

AT0

4-15

2-9

-1

MA

30

cm

of

f whi

te/ta

n ra

ised

~gl

ossy

~op

aque

smal

l. ~i

rreg

ular

88

0 Ps

eudo

noca

rdia

satu

rnea

0.

97

AJ2

5282

9 A

T04-

152-

10

-3

MA

10

cm

ch

alky

/whi

te d

ull o

paqu

e ~c

onve

x ~b

umpy

cen

ter t

an e

dges

bas

e in

aga

r 52

7 St

rept

omyc

es sp

. IM

-703

4

0.89

A

F131

546

AT0

4-15

2-11

-3

M

A

10 c

m

81

0 St

rept

omyc

etac

eae

0.

97

X87

320

AT0

4-15

2-12

-3

M

A

10 c

m

~cha

lky/

whi

te d

ull ~

opaq

ue ra

ised

~irr

egul

ar ri

gid

tan

~dul

l bas

e in

aga

r 79

6 Ps

eudo

noca

rdia

satu

rnea

0.

98

AJ2

5282

9 A

T04-

152-

13

-3

MA

10

cm

of

fwhi

te/ta

n ra

ised

dul

l ~op

aque

~bu

mpy

~irr

egul

ar w

ith w

hite

cha

lky

in c

ente

r 79

9 Ps

eudo

noca

rdia

satu

rnea

0.

98

AJ2

5282

9 A

T04-

152-

14

-3

MA

10

cm

of

f whi

te/ta

n ra

ised

~gl

ossy

~op

aque

~bu

mpy

~irr

egul

ar

794

Pseu

dono

card

ia sa

turn

ea

0.98

A

J252

829

AT0

4-15

2-15

-1

M

A

20 c

m

chal

ky/w

hite

dul

l opa

que

surf

ace

rais

ed ~

irreg

ular

bum

py ta

n ba

se in

aga

r 77

9 Ps

eudo

noca

rdia

satu

rnea

0.

98

AJ2

5282

9

146

(Tab

le c

ontin

ued:

Pit

isol

ates

) P

it Is

olat

e

D

ilutio

n M

edia

D

epth

Col

ony

Mor

phol

ogy

Seq

uenc

e L

engt

h

BL

AST

Res

ult

Sim

ilari

ty A

cces

sion

no.

A

T04-

152-

16

-1

MA

20

cm

ch

alky

/whi

te d

ull o

paqu

e su

rfac

e ra

ised

~irr

egul

ar b

umpy

tan

base

in a

gar

785

Pseu

dono

card

ia sa

turn

ea

0.98

A

J252

829

AT0

4-15

2-17

-2

M

A

10 c

m

offw

hite

~tra

ns fl

at e

dges

pur

ple/

brow

n ne

ar ri

pple

d du

ll ~f

uzzy

rais

ed c

ente

r 77

7 St

rept

omyc

es sp

. IM

-708

2

0.97

A

F131

549

AT0

4-15

2-18

-2

M

A

10 c

m

chal

ky/w

hite

dul

l opa

que

~rai

sed

rigid

surfa

ce &

tan

edge

s bas

e in

aga

r 77

7 St

rept

omyc

etac

eae

0.

97

X87

320

AT0

4-15

2-19

-1

M

A

10 c

m

tan

~tra

ns fl

at e

dges

pur

ple/

brow

n ne

ar ri

pple

d du

ll w

hite

fuzz

y ra

ised

cen

ter

767

Stre

ptom

yces

sp. I

M-7

082

0.

97

AF1

3154

9 A

T04-

152-

21

-1

MA

10

cm

w

hite

/cha

lky

~rai

sed

dull

opaq

ue ro

ugh

cent

er &

tan

~dul

l edg

es in

aga

r 75

0 St

rept

omyc

etac

eae

0.

97

X87

320

AT0

4-15

2-22

-1

M

A

10 c

m

tan

~dul

l opa

que

irreg

ular

rais

ed (s

ome

rippl

es c

halk

y w

hite

opa

que

dull)

76

3 St

rept

omyc

etac

eae

0.

97

X87

320

AT0

4-15

2-23

-1

M

A

10 c

m

offw

hite

/tan

conv

ex ~

glos

sy ~

trans

~bu

mpy

~irr

egul

ar

1004

Ps

eudo

noca

rdia

satu

rnea

0.

98

AJ2

5282

9 A

T04-

152-

24

-2

MA

10

cm

ta

n ra

ised

edg

es c

onca

ve c

ente

r ~gl

ossy

~tra

ns ~

bum

py e

dges

~irr

egul

ar

762

Pseu

dono

card

ia sa

turn

ea

0.98

A

J252

829

AT0

4-15

2-25

-2

M

A

10 c

m

whi

te/c

halk

y op

aque

dul

l bum

py e

dges

con

cave

irre

gula

r tan

bas

e in

aga

r 76

6 Ps

eudo

noca

rdia

satu

rnea

0.

98

AJ2

5282

9 A

T04-

152-

26

-2

MA

10

cm

ta

n ~t

rans

flat

dul

l edg

e pu

rple

/bro

wn

near

opa

que

rippl

ed w

hite

cha

lky

cent

er

748

Stre

ptom

yces

late

ritiu

s 0.

97

AF4

5476

4 A

T04-

152-

27

-2

MA

10

cm

ta

n ba

se ~

irreg

ular

~op

aque

dul

l in

agar

with

cha

lky

whi

te o

n so

me

of su

rfac

e

753

Pseu

dono

card

ia sa

turn

ea

0.98

A

J252

829

AT0

4-15

2-28

-2

M

A

10 c

m

offw

hite

/tan

rais

ed ~

conv

ex ~

glos

sy ~

opaq

ue ~

bum

py ~

irreg

ular

75

3 Ps

eudo

noca

rdia

satu

rnea

0.

98

AJ2

5282

9 A

T04-

152-

29

-2

MA

10

cm

w

hite

/cha

lky

opaq

ue d

ull r

ippl

ed ra

ised

irre

gula

r rou

gh ta

n ~t

rans

bas

e in

aga

r 10

35

Stre

ptom

yces

sp. 8

0134

0.

98

AY

9968

29

AT0

4-15

2-30

-1

M

A

60 c

m

oran

ge ~

trans

glo

ssy

conv

ex

878

Myc

obac

teri

um sa

crum

BN

315

1

0.97

A

Y23

5429

A

T04-

152-

31

-1

MA

60

cm

ye

llow

~op

aque

glo

ssy

conv

ex

762

Mic

roco

ccus

sp. E

llin1

49

0.99

A

F408

991

AT0

4-15

2-32

-2

M

A

70 c

m

crea

m ta

n/br

own

~con

vex

glos

sy ~

opaq

ue d

arke

r in

cent

er

875

Noc

ardi

oide

s OS4

0.

97

U61

298

AT0

4-15

2-33

-1

M

A

20 c

m

whi

te/c

halk

y op

aque

irre

gula

r dul

l bum

py ra

ised

gre

en/b

row

n ba

se in

aga

r 88

6 Ps

eudo

noca

rdia

satu

rnea

0.

96

AJ2

5282

9 A

T04-

152-

34

-1

MA

30

cm

w

hite

/cha

lky

opaq

ue ir

regu

lar d

ull b

umpy

rais

ed e

dges

con

cave

bas

e in

aga

r 89

6 Ps

eudo

noca

rdia

satu

rnea

0.

97

AJ2

5282

9 A

T04-

152-

35

-1

MA

30

cm

w

hite

/cha

lky

opaq

ue ~

irreg

ular

roug

h du

ll bu

mpy

rais

ed w

ith ta

n ba

se in

aga

r 88

3 Ps

eudo

noca

rdia

satu

rnea

0.

98

AJ2

5282

9 A

T04-

152-

36

-1

MA

20

cm

w

hite

opa

que

chal

ky ir

regu

lar r

igid

dul

l rai

sed

edge

s con

cave

tan

base

in a

gar

885

Pseu

dono

card

ia sa

turn

ea

0.97

A

J252

829

AT0

4-15

2-37

-1

M

A

20 c

m

tan

base

in a

gar f

uzzy

whi

te o

paqu

e irr

egul

ar d

ull r

igid

gre

en/b

row

n be

low

74

6 St

rept

omyc

es sp

. AS

4.11

82

0.98

A

Y11

4179

A

T04-

152-

38

-1

MA

20

cm

w

hite

opa

que

chal

ky ~

irreg

ular

dul

l bum

py ra

ised

with

tan

base

in a

gar

879

Pseu

dono

card

ia sa

turn

ea

0.97

A

J252

829

AT0

4-15

2-39

-1

M

A

40 c

m

tan/

offw

hite

~gl

ossy

rais

ed ~

opaq

ue b

umpy

rigi

d ha

rd ir

regu

lar b

ase

in a

gar

877

Amyc

olat

opsi

s sp.

1B

dz

0.97

A

F479

268

AT0

4-15

2-41

-2

1/

10 P

CA

10

cm

ch

alky

~irr

egul

ar ri

ngs t

owar

d ce

nter

~fla

t opa

que

dull

trans

whi

te b

ase

in a

gar

897

Stre

ptom

ycet

acea

e

0.96

X

8732

0 A

T04-

152-

42

-2

1/10

PC

A

10 c

m

chal

ky ~

irreg

ular

ring

s tow

ard

cent

er ~

flat o

paqu

e du

ll tra

ns w

hite

bas

e in

aga

r 91

3 St

rept

omyc

etac

eae

0.

96

X87

320

AT0

4-15

2-43

-1

1/

10 P

CA

10

cm

ch

alky

~irr

egul

ar ri

ngs t

owar

d ce

nter

~fla

t opa

que

dull

trans

whi

te b

ase

in a

gar

901

Stre

ptom

ycet

acea

e

0.96

X

8732

0 A

T04-

152-

44

-1

1/10

PC

A

10 c

m

tan

trans

irre

gula

r edg

e in

aga

r gre

en/b

row

n ce

nter

cha

lky

whi

te v

eins

cen

ter

901

Stre

ptom

yces

sp. L

K13

23.3

0.

96

AY

4653

12

AT0

4-15

2-45

-1

1/

10 P

CA

10

cm

of

fwhi

te tr

ans ~

irreg

ular

edg

es in

aga

r tan

cha

lky

cent

er o

paqu

e du

ll 83

3 A

ctin

obac

teriu

m R

G-5

1

0.97

A

Y56

1610

A

T04-

152-

46

-1

1/10

PC

A

10 c

m

offw

hite

/cre

am sm

all g

loss

y ~o

paqu

e co

nvex

89

9 St

rept

omyc

etac

eae

0.

96

X87

320

AT0

4-15

2-47

-1

1/

10 P

CA

10

cm

tra

ns o

ffw

hite

~irr

egul

ar e

dges

gro

wn

in a

gar w

hite

cha

lky

cent

er o

paqu

e du

ll 88

7 St

rept

omyc

etac

eae

0.

97

X87

320

AT0

4-15

2-48

-1

1/

10 P

CA

10

cm

sm

all t

rans

off

whi

te ~

irreg

ular

edg

es in

aga

r whi

te c

halk

y ce

nter

opa

que

dull

90

7 A

ctin

obac

teriu

m R

G-5

1

0.97

A

Y56

1610

A

T04-

152-

49

-1

1/10

PC

A

10 c

m

tan

base

in a

gar d

ull i

rreg

ular

rais

ed ri

gid

opaq

ue w

hite

cha

lky

spot

at c

ente

r 87

9 Ps

eudo

noca

rdia

pet

role

ophi

la

0.96

A

J252

828

AT0

4-15

2-50

-1

1/

10 P

CA

10

cm

ch

alky

~irr

egul

ar ri

ngs t

owar

d ce

nter

flat

opa

que

dull

trans

whi

te b

ase

in a

gar

876

Stre

ptom

ycet

acea

e

0.96

X

8732

0 A

T04-

152-

51

-1

1/10

PC

A

10 c

m

lt pk

/pea

ch ~

opaq

ue g

loss

y co

nvex

84

6 Bl

asto

cocc

us sp

. BC

448

0.

92

AJ3

1657

1 A

T04-

152-

52

-1

1/10

PC

A

10 c

m

offw

hite

smal

l ~op

aque

glo

ssy

conv

ex

876

Blas

toco

ccus

sp. B

C44

8

0.86

A

J316

571

AT0

4-15

2-53

-1

1/

10 P

CA

10

cm

sm

all w

hite

cha

lky

opaq

ue d

ull s

urfa

ce ~

irreg

ular

col

onie

s tan

bas

e in

aga

r 88

1 Ps

eudo

noca

rdia

pet

role

ophi

la

0.97

A

J252

828

AT0

4-15

2-54

-1

1/

10 P

CA

10

cm

bi

g tra

ns e

dges

in a

gar w

ith lt

bro

wn

~tra

ns c

ente

r (ce

nter

in a

gar a

lso)

84

0 A

ctin

obac

teriu

m R

G-5

1

0.95

A

Y56

1610

A

T04-

152-

55

-1

1/10

PC

A

40 c

m

brig

ht d

ark

oran

ge ~

opaq

ue ~

irreg

ular

glo

ssy

conv

ex ~

bum

py

877

Sphi

ngom

onas

sp. S

AFR

-028

0.

98

AY

1678

33

AT0

4-15

2-56

-1

1/

10 P

CA

40

cm

br

ight

dar

k or

ange

~op

aque

~irr

egul

ar g

loss

y co

nvex

~bu

mpy

88

8 Sp

hing

omon

as sp

. SA

FR-0

28

0.97

A

Y16

7833

A

T04-

152-

57

-1

1/10

PC

A

40 c

m

brig

ht d

ark

oran

ge ~

opaq

ue ~

irreg

ular

glo

ssy

conv

ex ~

bum

py

899

Sphi

ngom

onas

sp. S

AFR

-028

0.

98

AY

1678

33

AT0

4-15

2-58

-1

1/

10 P

CA

10

cm

w

hite

/cha

lky

~irr

egul

ar ri

ngs t

owar

d ce

nter

flat

opa

que

dull

~tra

ns b

ase

in a

gar

910

Stre

ptom

ycet

acea

e

0.96

X

8732

0 A

T04-

152-

59

-1

1/10

PC

A

10 c

m

whi

te c

halk

y ce

nter

rais

ed ~

trans

bas

e in

aga

r 86

2 St

rept

omyc

etac

eae

0.

96

X87

320

AT0

4-15

2-60

-1

1/

10 P

CA

10

cm

ta

n sm

all ~

irreg

ular

rais

ed ~

dull

~opa

que

782

Actin

obis

pora

xin

jiang

ensi

s 0.

98

AF3

2572

8 A

T04-

152-

62

-1

PCA

10

cm

of

fwhi

te ~

opaq

ue v

einy

~du

ll irr

egul

ar b

ase

in a

gar &

"vie

ns"

abov

e su

rfac

e 89

8 A

ctin

obac

teriu

m R

G-5

1

0.96

A

Y56

1610

A

T04-

152-

69

-1

PCA

10

cm

tra

ns ~

dull

in a

gar c

rack

ed in

x e

dges

off

whi

te d

ull o

paqu

e bu

mps

aro

und

899

Stre

ptom

ycet

acea

e

0.95

X

8732

0 A

T04-

152-

70

-1

PCA

10

cm

tra

ns ~

glos

sy in

aga

r cra

cked

in x

edg

es o

ffwhi

te ~

dull

opaq

ue b

umps

aro

und

854

Act

inob

acte

rium

RG

-51

0.

97

AY

5616

10

AT0

4-15

2-72

-1

PC

A

10 c

m

big

irreg

ular

tran

s tan

rigi

d ~g

loss

y co

ncav

e an

d ba

se in

aga

r 89

5 A

ctin

obac

teriu

m R

G-5

1

0.97

A

Y56

1610

A

T04-

152-

73

-2

PCA

10

cm

of

fwhi

te ~

trans

~du

ll bu

mpy

edg

e ce

nter

whi

te c

halk

y op

aque

dul

l rai

sed

ridge

s 76

8 St

rept

omyc

etac

eae

0.

96

X87

320

147

(Tab

le c

ontin

ued:

Pit

isol

ates

) Pi

t Iso

late

D

ilutio

n M

edia

D

epth

Col

ony

Mor

phol

ogy

Seq

uenc

e L

engt

h

BL

AST

Res

ult

Sim

ilari

ty A

cces

sion

no.

A

T04-

152-

74

-1

PCA

10

cm

tra

ns ~

glos

sy c

rack

ed in

x e

dges

off

whi

te ~

dull

opaq

ue b

umps

in a

gar

909

Stre

ptom

ycet

acea

e

0.96

X

8732

0 A

T04-

152-

75

-1

PCA

10

cm

bi

g irr

egul

ar ~

opaq

ue ta

n rig

id ~

dull

in a

gar r

aise

d on

ridg

es o

r "ve

ins"

86

8 A

ctin

obac

teriu

m R

G-5

1

0.97

A

Y56

1610

A

T04-

152-

76

-2

PCA

10

cm

tra

ns ~

irreg

ular

rigi

d du

ll ed

ges r

aise

d w

hite

cha

lky

vein

y op

aque

cen

ter

888

Act

inob

acte

rium

RG

-51

0.

96

AY

5616

10

AT0

4-15

2-77

-2

PC

A

10 c

m

trans

~irr

egul

ar ri

gid

dull

edge

s rai

sed

whi

te c

halk

y ve

iny

opaq

ue c

ente

r 89

3 St

rept

omyc

etac

eae

0.

97

X87

320

AT0

4-15

2-78

-2

PC

A

10 c

m

brow

n ve

ins ~

dull

rais

ed o

paqu

e bu

mps

on

surf

ace

~tra

ns ~

irreg

ular

in a

gar

817

Act

inob

acte

rium

RG

-51

0.

98

AY

5616

10

AT0

4-15

2-79

-2

PC

A

10 c

m

offw

hite

vei

ns (m

ore

in c

ente

r) ~

dull

rais

ed o

paqu

e gr

ows i

n ag

ar ~

irreg

ular

83

4 A

ctin

obac

teriu

m R

G-5

1

0.98

A

Y56

1610

A

T04-

152-

80

-2

PCA

10

cm

tra

ns c

onca

ve ~

dull

rigid

irre

gula

r cha

lky

yello

w v

eins

opa

que

bum

ps a

roun

d 79

5 St

rept

omyc

etac

eae

0.

96

X87

320

AT0

4-15

2-81

-2

PC

A

30 c

m

flat t

rans

off

-whi

te ir

regu

lar g

loss

y 86

6 St

aphy

loco

ccus

epi

derm

idis

0.

98

AY

0303

42

AT0

4-15

2-82

-2

PC

A

surf

ace

brig

ht y

ello

w b

ig g

loss

y co

nvex

~op

aque

87

9 U

ncul

t act

inob

acte

rium

clo

ne

0.98

A

Y36

0679

A

T04-

152-

83

-1

PCA

10

cm

of

fwhi

te o

paqu

e rip

pled

(vei

ny) ~

dull

irreg

ular

bas

e in

aga

r "ve

ins"

on

surf

ace

901

Act

inob

acte

rium

RG

-51

0.

97

AY

5616

10

AT0

4-15

2-85

-1

PC

A

10 c

m

offw

hite

tran

s ~gl

ossy

dig

s all

the

way

into

aga

r cen

ter i

s cra

cked

in a

n x

902

Stre

ptom

ycet

acea

e

0.97

X

8732

0 A

T04-

152-

88

-2

1/10

0 PC

A 4

0 cm

or

ange

/yel

low

irre

gula

r glo

ssy

conv

ex ~

trans

& lt

ora

nge

!irre

gula

r edg

es fl

at

881

Pseu

dom

onas

stut

zeri

0.

99

X98

607

AT0

4-15

2-89

-2

1/

100

PCA

40

cm

lt ye

llow

opa

que

conv

ex d

ull m

ed

884

Mic

roco

ccus

sp. E

llin1

49

0.99

A

F408

991

AT0

4-15

2-90

-2

1/

100

PCA

10

cm

whi

te tr

ans i

n ag

ar w

hite

cha

lky/

fuzz

y op

aque

dul

l spo

kes r

adia

te fr

om c

ente

r 83

9 A

ctin

omyc

etal

es b

acte

rium

HPA

66

0.97

D

Q14

4230

A

T04-

152-

91

-2

1/10

0 PC

A 1

0 cm

w

hite

tran

s in

agar

with

few

spot

s of c

halk

y/fu

zzy

opaq

ue d

ull o

n su

rfac

e

787

Act

inob

acte

rium

RG

-51

0.

97

AY

5616

10

AT0

4-15

2-93

-1

1/

100

PCA

20

cm

whi

te tr

ans i

n ag

ar w

ith o

ffw

hite

cha

lky

in c

ente

r of c

olon

y in

shap

e of

a st

ar

586

Stre

ptom

yces

sp. A

S 4.

1182

0.

97

AY

1141

79

AT0

4-15

2-10

0 -1

1/

100

PCA

10

cm

whi

te tr

ans i

n ag

ar w

hite

cha

lky/

fuzz

y op

aque

dul

l spo

kes r

adia

te fr

om c

ente

r 81

3 St

rept

omyc

etac

eae

0.

97

X87

320

AT0

4-15

2-10

1 -1

1/

100

PCA

10

cm

whi

te tr

ans i

n ag

ar w

ith w

hite

cha

lky

opaq

ue d

ull r

ing

arou

nd c

ente

r on

agar

60

0 St

rept

omyc

es sp

. 337

F08

0.

98

AB

1243

65

AT0

4-15

2-10

2 -1

1/

100

PCA

10

cm

offw

hite

/tan

base

gro

wn

into

aga

r sm

all w

hite

opa

que

chal

ky/fu

zzy

surf

ace

720

Pseu

dono

card

ia p

etro

leop

hila

0.

97

AJ2

5282

8 A

T04-

152-

103

-1

1/10

0 PC

A 1

0 cm

w

hite

tran

s w

ith sp

ots o

f fuz

zy g

reen

/off

whi

te o

paqu

e du

ll on

surf

ace

806

Act

inob

acte

rium

RG

-51

0.

97

AY

5616

10

AT0

4-15

2-10

4 -1

1/

100

PCA

10

cm

whi

te tr

ans i

n ag

ar c

halk

y op

aque

dul

l rin

gs &

spok

es ra

diat

e fro

m c

ente

r 78

8 St

rept

omyc

etac

eae

0.

97

X87

320

AT0

4-15

2-10

5 -1

1/

100

PCA

10

cm

whi

te tr

ans g

row

n in

aga

r sur

face

has

spot

s of w

hite

fuzz

y op

aque

dul

l 90

2 A

ctin

obac

teriu

m R

G-5

1

0.97

A

Y56

1610

A

T04-

152-

106

-1

1/10

0 PC

A 1

0 cm

in

aga

r sur

face

nea

r cen

ter i

rreg

ular

off

whi

te fu

zzy/

chal

ky o

paqu

e du

ll 85

7 A

ctin

obac

teriu

m R

G-5

1

0.97

A

Y56

1610

A

T04-

152-

107

-1

1/10

0 PC

A 1

0 cm

w

hite

cha

lky

opaq

ue d

ull r

ing

arou

nd c

ente

r & w

hite

cha

lky

outs

ide

ring

89

2 St

rept

omyc

etac

eae

0.

96

X87

320

AT0

4-15

2-10

8 -2

1/

100

PCA

10

cm

~tra

ns in

aga

r whi

te c

halk

y sp

ecks

opa

que

dull

(con

vex

big

spec

k in

cen

ter)

75

8 St

rept

omyc

etac

eae

0.

97

X87

320

AT0

4-15

2-10

9 -2

N

A

surf

ace

whi

te ~

opaq

ue g

loss

y fla

t ~irr

egul

ar

794

Stap

hylo

cocc

us e

pide

rmid

is

0.99

A

Y03

0342

A

T04-

152-

111

-1

NA

20

cm

du

ll ra

ised

bum

py w

hite

cha

lky

opaq

ue e

dges

bro

wn

opaq

ue c

ente

r in

agar

75

7 St

rept

omyc

es sp

. AS

4.11

82

0.97

A

Y11

4179

A

T04-

152-

112

-2

NA

10

cm

bi

g ta

n tra

ns ir

regu

lar ~

flat ~

dull

digs

into

aga

r bro

wn

in c

ente

r in

shap

e of

x

739

Act

inob

acte

rium

RG

-51

0.

97

AY

5616

10

AT0

4-15

2-11

3 -2

N

A

10 c

m

rais

ed ~

trans

roug

h rig

id b

umpy

off

whi

te ~

dull

~irr

egul

ar

826

Pseu

dono

card

ia sa

turn

ea

0.98

A

J252

829

AT0

4-15

2-11

4 -2

N

A

10 c

m

tan

irreg

ular

opa

que

ring

arou

nd c

onca

ve c

ente

r out

side

ring

cha

lky

dull

895

Stre

ptom

yces

sp. A

S 4.

1182

0.

97

AY

1141

79

AT0

4-15

2-11

7 -1

N

A

10 c

m

~irr

egul

ar tr

ans f

lat ~

glos

sy e

dges

whi

te c

halk

y irr

egul

ar ~

rais

ed o

paqu

e du

ll 89

3 St

rept

omyc

etac

eae

0.

96

X87

320

AT0

4-15

2-11

8 -1

N

A

10 c

m

trans

tan

irreg

ular

flat

roug

h rig

id g

loss

y 90

8 A

ctin

obac

teriu

m R

G-5

1

0.96

A

Y56

1610

A

T04-

152-

120

-2

NA

10

cm

irr

egul

ar fl

at o

ffw

hite

spot

s in

agar

& o

n co

lony

cha

lky

opaq

ue d

ull c

ente

r 83

2 St

rept

omyc

es sp

. MS-

037

0.

95

AY

6459

01

AT0

4-15

2-12

1 -2

N

A

10 c

m

irreg

ular

flat

off

whi

te sp

ots i

n ag

ar &

on

colo

ny c

halk

y op

aque

dul

l cen

ter

874

Stre

ptom

ycet

acea

e

0.96

X

8732

0 A

T04-

152-

122

-2

NA

10

cm

br

own

vein

y rip

pled

in x

glo

ssy

opaq

ue in

aga

r irr

egul

ar w

hite

bum

py e

dges

88

5 A

ctin

obac

teriu

m R

G-5

1

0.96

A

Y56

1610

A

T04-

152-

123

-1

NA

10

cm

~o

paqu

e ta

n ~g

loss

y rig

id c

ente

r edg

es v

einy

& ri

pple

d ~f

lat d

igs i

n ag

ar

870

Act

inob

acte

rium

RG

-51

0.

97

AY

5616

10

AT0

4-15

2-12

4 -1

N

A

10 c

m

trans

lt b

row

n/ta

n di

g in

to a

gar ~

glos

sy c

rack

ed in

cen

ter

877

Stre

ptom

yces

sp. L

K12

24.4

0.

96

AY

4652

68

AT0

4-15

2-12

5 -1

N

A

10 c

m

lt ye

llow

tran

s cra

cked

& c

aved

in c

ente

r dig

s in

agar

~gl

ossy

88

4 St

rept

omyc

etac

eae

0.

96

X87

320

AT0

4-15

2-12

6 -1

N

A

10 c

m

~opa

que

tan

~glo

ssy

bum

py ri

gid

digs

in a

gar

872

Act

inob

acte

rium

RG

-51

0.

96

AY

5616

10

AT0

4-15

2-12

7 -1

N

A

10 c

m

whi

te c

halk

y op

aque

dul

l rig

id ~

rais

ed c

ente

r tra

ns e

dges

dig

s in

agar

87

1 St

rept

omyc

etac

eae

0.

96

X87

320

AT0

4-15

2-12

9 -1

N

A

10 c

m

brow

n/ta

n tra

ns ~

irreg

ular

rigi

d gl

ossy

~fla

t dig

s in

agar

but

is e

asy

to g

et u

p 88

8 A

ctin

obac

teriu

m R

G-5

1

0.97

A

Y56

1610

A

T04-

153-

1 -2

N

A

10 c

m

flat y

ello

w g

loss

y op

aque

74

9 Br

evib

acill

us a

gri

0.

99

AJ5

8638

8 A

T04-

153-

2 -1

N

A

surf

ace

crea

m c

onve

x gl

ossy

tran

s 76

8 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

153-

3 -1

N

A

surf

ace

light

pin

k co

nvex

glo

ssy

~tra

ns

798

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-15

3-4

-1

NA

70

cm

pe

ach

conv

ex g

loss

y op

aque

79

9 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

153-

5 -1

PC

A

20 c

m

dark

pin

k w

rinkl

y co

nvex

opa

que

glos

sy

643

Unc

ult M

ethy

loba

cter

iace

ae b

acte

rium

clo

ne 1

0-3B

a02

0.97

A

Y36

0526

A

T04-

153-

6 -1

PC

A

20 c

m

oran

ge c

onve

x op

aque

glo

ssy

814

Unc

ult A

ctin

omyc

etal

es b

acte

rium

clo

ne E

B10

74

0.96

A

Y39

5393

148

(Tab

le c

ontin

ued:

Pit

isol

ates

) Pi

t Iso

late

D

ilutio

n M

edia

D

epth

Col

ony

Mor

phol

ogy

Seq

uenc

e L

engt

h

BL

AST

Res

ult

Sim

ilari

ty A

cces

sion

no.

A

T04-

153-

7 -1

PC

A

20 c

m

brig

ht y

ello

w fl

at g

loss

y op

aque

47

6 C

urto

bact

eriu

m fl

accu

mfa

cien

s pv.

bas

ella

e

0.99

A

Y27

3210

A

T04-

153-

8 -1

PC

A

20 c

m

oran

ge o

paqu

e co

nvex

glo

ssy

~clu

mpy

65

9 Fr

anki

a sp

. (st

rain

AV

N17

s)

0.94

L4

0613

A

T04-

153-

9 -1

PC

A

80 c

m

light

yel

low

opa

que

conv

ex g

loss

y 85

1 M

icro

cocc

us lu

teus

0.

98

AJ4

0909

6 A

T04-

153-

10

-1

1/10

PC

A

surf

ace

whi

te/c

ream

~tra

ns c

lum

py c

onve

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97

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99

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97

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98

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98

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4 A

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98

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obac

teriu

m c

lone

AK

YG

1666

0.

95

AY

9217

86

AT0

4-15

9-56

-3

1/

10 P

CA

40

cm

tra

ns o

ffw

hite

glo

ssy

conv

ex

829

Unc

ult a

lpha

pro

teob

acte

rium

clo

ne A

KY

G16

66

0.95

A

Y92

1786

A

T04-

159-

57

-3

1/10

PC

A

20 c

m

gree

n ch

alky

opa

que

bum

py c

onve

x 51

2 St

rept

omyc

es sp

. ATT

508

0.

99

AY

7581

94

AT0

4-15

9-58

-3

1/

10 P

CA

20

cm

of

fwhi

te o

paqu

e gr

ows i

n ag

ar ti

ny p

art g

row

ing

on to

p of

aga

r ~co

nvex

81

8 Pr

omic

rom

onos

pora

aer

olat

a

0.98

A

J487

303

AT0

4-15

9-59

-3

1/

10 P

CA

20

cm

ye

llow

opa

que

conv

ex c

halk

y/~f

uzzy

68

6 M

ycob

acte

rium

cel

atum

0.

92

AJ5

3604

0 A

T04-

159-

60

-3

1/10

PC

A

20 c

m

whi

te o

paqu

e co

nvex

cha

lky

base

in a

gar

744

Stre

ptom

yces

sp. 1

413

0.

95

AY

8769

43

AT0

4-15

9-61

-3

1/

10 P

CA

20

cm

ta

n/ye

llow

flat

in a

gar t

rans

84

9 St

rept

omyc

es sp

. NR

RL

5799

0.

98

AJ3

9181

4 A

T04-

159-

62

-3

1/10

PC

A

20 c

m

crea

m tr

ans g

loss

y co

nvex

66

2 U

ncul

t alp

ha p

rote

obac

teriu

m

0.95

A

J318

115

AT0

4-15

9-63

-3

1/

10 P

CA

20

cm

cr

eam

bas

e in

aga

r with

whi

te o

paqu

e ch

alky

circ

le o

n to

p 61

4 C

ellu

lom

onas

sp. X

7

0.98

A

F060

791

AT0

4-15

9-64

-3

1/

10 P

CA

20

cm

gr

een

chal

ky o

paqu

e ~c

onve

x 82

6 St

rept

omyc

es c

arpi

nens

is st

rain

NR

RL

B-1

6921

0.

98

AY

9999

08

AT0

4-15

9-65

-3

1/

10 P

CA

20

cm

of

fwhi

te c

halk

y ho

les o

n to

p ~c

onve

x op

aque

66

1 A

ctin

obac

teriu

m R

G-5

1

0.98

A

Y56

1610

A

T04-

159-

66

-3

1/10

PC

A

20 c

m

crea

m ~

glos

sy tr

ans f

lat g

row

s in

agar

81

0 St

rept

omyc

es a

lbid

ochr

omog

enes

stra

in A

S 4.

1863

0.

99

AY

9998

84

AT0

4-15

9-67

-3

1/

10 P

CA

su

rfac

e lt

pk ~

opaq

ue g

loss

y co

nvex

84

5 M

odes

toba

cter

sp. E

llin1

65

0.99

A

F409

007

AT0

4-15

9-69

-4

1/

10 P

CA

20

cm

gr

ey ir

regu

lar l

umpy

con

vex

opaq

ue

544

Stre

ptom

yces

sp. A

TT50

8

0.98

A

Y75

8194

A

T04-

159-

71

-4

1/10

PC

A

20 c

m

smal

l whi

te o

paqu

e fla

t rou

nd

627

Stre

ptom

yces

sp. A

TT50

8

0.98

A

Y75

8194

A

T04-

159-

72

-3

1/10

PC

A

surf

ace

pink

~tra

ns g

loss

y co

nvex

roun

d 53

1 Ar

thro

bact

er sp

. Fa2

1

0.97

A

Y13

1225

A

T04-

159-

73

-3

1/10

PC

A

surf

ace

smal

l ora

nge

roun

d gl

ossy

con

vex

opaq

ue

423

Blas

toco

ccus

sp. B

C52

1

0.97

A

J316

573

AT0

4-15

9-74

-3

1/

10 P

CA

su

rfac

e la

rge

brow

n irr

egul

ar fl

at ri

dges

aro

und

edge

63

0 St

rept

omyc

es sp

. AS

4.11

82

0.96

A

Y11

4179

A

T04-

159-

75

-3

1/10

PC

A

surf

ace

tiny

whi

te tr

ans c

onve

x gl

ossy

roun

d 74

7 St

rept

omyc

es sp

. AS

4.11

82

0.97

A

Y11

4179

A

T04-

159-

77

-4

1/10

PC

A

40 c

m

smal

l rou

nd w

hite

tran

s fla

t 53

6 U

ncul

t alp

ha p

rote

obac

teriu

m

0.94

A

J318

115

150

(Tab

le c

ontin

ued:

Pit

isol

ates

) Pi

t Iso

late

D

ilutio

n M

edia

D

epth

Col

ony

Mor

phol

ogy

Seq

uenc

e L

engt

h

BL

AST

Res

ult

Sim

ilari

ty A

cces

sion

no.

A

T04-

159-

78

-4

1/10

PC

A

40 c

m

smal

l rou

nd w

hite

tran

s ~fla

t 93

6 U

ncul

t alp

ha p

rote

obac

teriu

m c

lone

AK

YG

1666

0.

95

AY

9217

86

AT0

4-15

9-79

-3

1/

10 P

CA

10

cm

w

hite

cha

lky

text

ure

like

surf

ace

of m

oon

opaq

ue ir

regu

lar r

aise

d du

ll 61

4 A

ctin

obac

teriu

m R

G-5

1

0.98

A

Y56

1610

A

T04-

159-

81

-5

1/10

PC

A

30 c

m

whi

te ~

opaq

ue v

ery

cent

er c

onve

x th

e re

st is

~fla

t ~irr

egul

ar ~

glos

sy

705

Stre

ptom

ycet

acea

e ba

cter

ium

CN

Q71

9

0.98

A

Y46

4544

A

T04-

159-

82

-4

1/10

PC

A

30 c

m

gree

n fu

zzy

opaq

ue d

ull r

aise

d ce

nter

flat

spar

ce e

dges

61

1 St

rept

omyc

es sp

. NR

RL

5799

0.

98

AJ3

9181

4 A

T04-

159-

83

-4

1/10

PC

A

40 c

m

in a

gar t

rans

~cr

eam

y gl

ossy

73

0 St

rept

omyc

es sp

. IM

-714

0

0.95

A

F131

554

AT0

4-15

9-84

-4

1/

10 P

CA

40

cm

gr

een

fuzz

y/ch

alky

opa

que

dull

rais

ed

611

Stre

ptom

yces

sp. N

RR

L 57

99

0.98

A

J391

814

AT0

4-15

9-85

-4

1/

10 P

CA

40

cm

tin

y tra

ns c

ream

y co

nvex

roun

d 84

8 U

ncul

t alp

ha p

rote

obac

teriu

m c

lone

AK

YG

1666

0.

94

AY

9217

86

AT0

4-15

9-86

-3

1/

10 P

CA

40

cm

gr

een

fuzz

y/ch

alky

opa

que

dull

rais

ed

658

Stre

ptom

yces

sp. N

RR

L 57

99

0.97

A

J391

814

AT0

4-15

9-87

-3

1/

10 P

CA

40

cm

sm

all t

rans

~cr

eam

y co

nvex

roun

d 64

7 St

rept

omyc

es sp

. NR

RL

5799

0.

98

AJ3

9181

4 A

T04-

159-

88

-4

1/10

PC

A

20 c

m

big

in a

gar r

ust c

ente

r is ~

rais

ed fr

om a

gar ~

trans

roun

d ~g

loss

y 65

4 Sa

ccha

roth

rix

sp. A

S 4.

1731

0.

96

AY

1356

93

AT0

4-15

9-89

-4

1/

10 P

CA

20

cm

of

fwhi

te in

aga

r mak

es a

gar c

onve

x op

aque

glo

ssy

big

806

Noc

ardi

opsi

s sp.

98-

08-2

90A

0.

97

AF3

2840

8 A

T04-

159-

90

-4

1/10

PC

A

20 c

m

offw

hite

smal

l har

d in

aga

r rou

nd c

onve

x op

aque

85

3 Ac

tinom

adur

a sp

. IM

-622

6

0.97

A

F131

308

AT0

4-15

9-92

-4

1/

10 P

CA

20

cm

of

fwhi

te b

ase

dull

in a

gar w

hite

surf

ace

roug

h rig

id b

umpy

73

4 N

ocar

dioi

dace

ae st

r. IM

-775

7

0.96

A

F131

634

AT0

4-15

9-92

B

-4

1/10

0 PC

A 2

0 cm

536

Unc

ult S

phin

gom

onas

sp. c

lone

BL0

04B

95

0.98

A

Y80

6057

A

T04-

159-

93

-3

1/10

PC

A

30 c

m

trans

glo

ssy

roun

d sm

all c

onve

x ce

nter

is w

hite

84

0 U

ncul

t alp

ha p

rote

obac

teriu

m c

lone

AK

YG

1666

0.

95

AY

9217

86

AT0

4-15

9-94

-4

1/

10 P

CA

20

cm

tra

ns ta

n gl

ossy

con

vex

1057

Sp

hing

omon

as sp

. Alp

ha4-

5

0.93

A

Y77

1798

A

T04-

159-

97

-3

1/10

PC

A

30 c

m

whi

te ti

ny c

halk

y rin

g ar

ound

cen

ter i

n ag

ar d

ull o

paqu

e ro

und

490

Stre

ptom

yces

sp. 4

31E1

1

0.98

A

B12

3477

A

T04-

159-

100

-3

1/10

PC

A

20 c

m

flat ~

trans

off

whi

te g

loss

y 56

3 Ba

cillu

s sp.

IDA

0663

0.

97

AJ5

6353

3 A

T04-

159-

102

-3

1/10

PC

A

20 c

m

med

off

whi

te ~

trans

glo

ssy

852

Rhiz

obiu

m sp

. H-4

0.

97

AF2

7988

9 A

T04-

159-

104

-3

1/10

PC

A

20 c

m

~irr

egul

ar d

ull c

ente

r is w

hite

cha

lky

~opa

que

712

Noc

ardi

oida

ceae

str.

IM-7

757

0.

97

AF1

3163

4 A

T04-

159-

106

-5

NA

su

rfac

e lt

pk/ta

n m

ed o

paqu

e co

nvex

glo

ssy

667

Blas

toco

ccus

agg

rega

tus

0.97

A

J430

193

AT0

4-15

9-10

7 -5

N

A

20 c

m

offw

hite

rais

ed o

paqu

e ~i

rreg

ular

not

smoo

th ~

glos

sy

707

Actin

obis

pora

yun

nane

nsis

0.

95

D85

472

AT0

4-15

9-11

2 -4

N

A

surf

ace

whi

te ~

trans

glo

ssy

conv

ex c

ente

r is b

umpy

66

1 M

odes

toba

cter

sp. E

llin1

65

0.97

A

F409

007

AT0

4-15

9-11

3 -4

N

A

surf

ace

lt pk

/tan

~opa

que

glos

sy c

onve

x 88

0 Bl

asto

cocc

us a

ggre

gatu

s 0.

97

AJ4

3019

3 A

T04-

159-

114

-4

NA

su

rfac

e bi

g ho

t pk/

hot p

each

opa

que

~con

vex

text

ured

bum

py &

rigi

d ~i

rreg

ular

68

6 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T04-

159-

116

-4

NA

10

cm

!ir

regu

lar w

hite

cha

lky

surf

ace

base

in a

gar d

ull o

paqu

e 68

2 Ac

tinob

acte

rium

RG

-51

0.

98

AY

5616

10

AT0

4-15

9-11

8 -4

N

A

surf

ace

yello

w ~

opaq

ue g

loss

y !c

onve

x 64

7 C

ellu

losi

mic

robi

um fu

nkei

isol

ate

W6

0.

93

AY

7299

60

AT0

4-15

9-11

9 -4

N

A

surf

ace

hot p

k bi

g ~i

rreg

ular

glo

ssy

~con

vex

~opa

que

850

Arth

roba

cter

agi

lis

0.99

A

J577

725

AT0

4-15

9-12

0 -4

N

A

surf

ace

pk/p

each

~du

ll bu

mpy

rigi

d ro

ugh

~opa

que

conv

ex

610

Mod

esto

bact

er m

ultis

epta

tus

0.97

A

J871

304

AT0

4-15

9-12

1 -4

N

A

surf

ace

oran

ge ~

trans

glo

ssy

conv

ex

665

Blas

toco

ccus

agg

rega

tus

0.97

A

J430

193

AT0

4-15

9-12

2 -4

N

A

surf

ace

big

pk/p

each

~op

aque

glo

ssy

conv

ex

665

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-15

9-12

3 -4

N

A

surf

ace

peac

h tra

ns ri

dges

~op

aque

cen

ter g

loss

y co

nvex

44

3 Bl

asto

cocc

us sp

. BC

521

0.

97

AJ3

1657

3 A

T04-

159-

124

-4

NA

su

rfac

e w

hite

glo

ssy

conv

ex ~

trans

bum

py su

rfac

e in

cen

ter

697

Mod

esto

bact

er sp

. Elli

n165

0.

98

AF4

0900

7 A

T04-

159-

125

-4

NA

su

rfac

e ta

n/lt

pk g

loss

y co

nvex

~op

aque

67

0 Bl

asto

cocc

us a

ggre

gatu

s 0.

96

AJ4

3019

3 A

T04-

159-

126

-4

NA

su

rfac

e 67

7 Bl

asto

cocc

us a

ggre

gatu

s 0.

97

AJ4

3019

3 A

T04-

159-

127

-4

NA

su

rfac

e tra

ns g

loss

y co

nvex

65

0 G

eode

rmat

ophi

lus o

bscu

rus o

bscu

rus

0.97

L4

0620

A

T04-

159-

128

-4

NA

su

rfac

e w

hite

rais

ed d

ull o

paqu

e bu

mpy

roug

h rig

id ~

irreg

ular

88

0 G

eode

rmat

ophi

lus s

p.

0.98

X

9235

8 A

T04-

159-

129

-4

NA

su

rfac

e tra

ns g

loss

y co

nvex

with

whi

te ~

irreg

ular

opa

que

bum

py ro

ugh

in c

ente

r 67

1 Bl

asto

cocc

us a

ggre

gatu

s 0.

97

AJ4

3019

3 A

T04-

159-

132

-4

NA

10

cm

tra

ns b

row

n rip

pled

rigi

d ra

ised

in c

ente

r & o

n rip

ples

glo

ssy

540

Stre

ptom

yces

sp. 3

37F0

8

0.99

A

B12

4365

A

T04-

159-

133

-4

NA

10

cm

of

fwhi

te ~

trans

ripp

led

rigid

rais

ed o

n rip

ples

~gl

ossy

65

5 Sa

ccha

roth

rix

sp. A

S 4.

1731

0.

97

AY

1356

93

AT0

4-15

9-14

1 -4

N

A

20 c

m

whi

te g

loss

y fla

t mor

e op

aque

in c

ente

r ~tra

ns e

dges

68

5 Pa

enib

acill

us sp

. DSM

135

2

0.95

A

J345

017

AT0

4-15

9-14

8 -3

N

A

surf

ace

brow

nish

yel

low

rais

ed ro

ugh

rippl

ed ri

gid

opaq

ue ~

irreg

ular

74

8 Sa

ccha

roth

rix

sp. A

S 4.

1731

0.

98

AY

1356

93

AT0

4-15

9-14

9 -3

N

A

surf

ace

whi

te ~

irreg

ular

rais

ed ~

dull

roug

h bu

mpy

~op

aque

con

cave

cen

ter

801

Actin

obis

pora

ala

nini

phila

0.

98

AF3

2572

6 A

T04-

159-

150

-3

NA

10

cm

bi

g lt

yello

w tr

ans i

n ag

ar li

ke v

eins

~gl

ossy

ripp

led

rigid

rais

ed o

n rip

ples

81

7 St

rept

omyc

es a

lbid

ochr

omog

enes

stra

in A

S 4.

1863

0.

99

AY

9998

84

AT0

4-15

9-15

1 -3

N

A

10 c

m

yello

w/g

reen

opa

que

~dul

l rai

sed

rigid

pun

ctifo

rm tr

ans y

ello

w sp

ots a

roun

d

893

Unc

ult S

trep

tom

yces

sp. c

lone

clo

RD

L+29

0.

98

AY

8343

79

AT0

4-15

9-15

2 -5

M

A

surf

ace

smal

l pin

k op

aque

con

vex

dull

654

Blas

toco

ccus

sp. B

C41

2

0.95

A

J316

574

AT0

4-15

9-15

3 -3

M

A

10 c

m

larg

e irr

egul

ar fl

at c

ream

glo

ssy

trans

on

edge

s opa

que

in c

ente

r 65

0 Ba

cillu

s sp.

LM

G 2

0243

0.

9 A

J316

317

151

(Tab

le c

ontin

ued:

Pit

isol

ates

) Pi

t Iso

late

D

ilutio

n M

edia

D

epth

Col

ony

Mor

phol

ogy

Seq

uenc

e L

engt

h

BL

AST

Res

ult

Sim

ilari

ty A

cces

sion

no.

A

T04-

159-

154

-3

MA

10

cm

la

rge

crea

m fl

at w

hite

on

edge

s opa

que

glos

sy ir

regu

lar b

umps

on

edge

s 63

1 Ar

thro

bact

er sp

. 1c-

1

0.98

A

Y56

1525

A

T04-

159-

155

-3

MA

10

cm

w

hite

roun

d op

aque

glo

ssy

flat d

arke

r in

cent

er

615

Unc

ult B

acill

us sp

.

0.97

A

J640

178

AT0

4-15

9-15

6 -3

M

A

10 c

m

brow

n tra

ns fl

at ri

dges

on

edge

s dar

ker i

n ce

nter

cle

ar a

rea

arou

nd it

58

7 N

ocar

diop

sis s

p. 9

8-08

-290

A

0.94

A

F328

408

AT0

4-15

9-15

7 -3

M

A

10 c

m

whi

te ir

regu

lar o

paqu

e du

ll ro

und

in c

ente

r with

2 p

oint

s com

ing

out f

lat

564

Baci

llus s

p. V

AN

14

0.94

A

F286

482

AT0

4-15

9-15

8 -3

M

A

10 c

m

smal

l yel

low

con

vex

bum

py o

paqu

e 61

6 Ps

eudo

noca

rdia

pet

role

ophi

la

0.96

A

J252

828

AT0

4-15

9-16

0 -3

M

A

10 c

m

smal

l rou

nd w

hite

tran

s fla

t dul

l 59

8 Ba

cillu

s sp.

JL-3

4

0.95

A

Y74

5866

A

T04-

159-

161

-4

MA

su

rfac

e pi

nk ro

und

opaq

ue fl

at g

loss

y 54

7 M

odes

toba

cter

sp. E

llin1

65

0.99

A

F409

007

AT0

4-15

9-16

2 -4

M

A

surf

ace

pink

roun

d op

aque

con

vex

non-

glos

sy "d

onut

-like

" w

ith h

ole

in c

ente

r 60

8 Bl

asto

cocc

us sp

. BC

521

0.

95

AJ3

1657

3 A

T04-

159-

163

-4

MA

su

rfac

e ye

llow

irre

gula

r sha

pe c

onve

x op

aque

non

-glo

ssy

"bum

py"

642

Cel

lulo

mon

as c

ella

sea

0.

95

X83

804

AT0

4-15

9-16

4 -4

M

A

surf

ace

crea

m ro

und

conv

ex g

loss

y op

aque

66

1 C

ellu

losi

mic

robi

um fu

nkei

isol

ate

W6

0.

94

AY

7299

60

AT0

4-15

9-16

5 -4

M

A

surf

ace

smal

l pin

k ro

und

conv

ex d

ull o

paqu

e 98

2 G

eode

rmat

ophi

lus s

p.

0.98

X

9235

8 A

T04-

159-

170

-4

MA

30

cm

sm

all r

ound

cre

am ~

trans

con

vex

glos

sy

773

Stre

ptom

yces

sp. N

RR

L 57

99

0.98

A

J391

814

AT0

4-15

9-17

3 -5

M

A

surf

ace

yello

w ro

ugh

rigid

bum

py ir

regu

lar ~

glos

sy ra

ised

opa

que

802

Cel

lulo

mon

as c

ella

sea

0.

96

X83

804

AT0

4-15

9-17

5 -4

M

A

surf

ace

offw

hite

opa

que

glos

sy c

onve

x ~r

ound

81

9 Ba

cter

ium

K2-

12

0.94

A

Y34

5437

A

T04-

159-

176

-4

MA

su

rfac

e pi

nk o

paqu

e rig

id ro

ugh

bum

py ir

regu

lar ~

glos

sy

815

Blas

toco

ccus

sp. B

C44

8

0.95

A

J316

571

AT0

4-15

9-17

7 -4

M

A

surf

ace

yello

wis

h/ta

n gl

ossy

roun

d co

nvex

~tra

ns

838

Mic

roba

cter

iace

ae st

r. El

lin16

6

0.96

A

F409

008

AT0

4-15

9-17

8 -4

M

A

surf

ace

lt pk

opa

que

glos

sy c

onve

x ro

und

753

Bac

teriu

m E

llin6

023

0.

98

AY

2346

75

AT0

4-15

9-18

0 -4

M

A

10 c

m

brow

n in

aga

r opa

que

flat d

ull ~

roun

d 80

7 St

rept

omyc

es sp

. IM

-736

2

0.99

A

F131

591

A

T04-

159-

182

-5

MA

20

cm

cr

eam

/off

whi

te ir

regu

lar g

loss

y ra

ised

~op

aque

66

8 O

xalo

phag

us o

xalic

us

0.95

Y

1458

1 A

T04-

159-

183

-5

MA

40

cm

tra

ns o

ffw

hite

~irr

egul

ar g

loss

y ~c

onve

x 61

2 Ba

cillu

s kru

lwic

hiae

0.

96

AB

0868

97

AT0

4-15

9-18

4 -3

M

A

surf

ace

~irr

egul

ar !h

ot p

ink

~fla

t glo

ssy

opaq

ue m

ed

708

Arth

roba

cter

sp. F

a21

0.

97

AY

1312

25

AT0

4-15

9-18

5 -3

M

A

surf

ace

!hot

pin

k ~f

lat g

loss

y op

aque

med

roun

d 66

9 Ar

thro

bact

er sp

. Muz

t-F95

0.

94

AY

5266

77

AT0

4-15

9-18

6 -3

M

A

surf

ace

whi

te/c

ream

opa

que

glos

sy c

onve

x 61

7 C

ellu

lom

onas

sept

ica

stra

in W

7388

0.

94

AY

6557

33

AT0

4-15

9-18

7 -3

M

A

surf

ace

lt pk

glo

ssy

conv

ex o

paqu

e (li

ke c

ream

save

r)

846

Blas

toco

ccus

sp. B

C44

8

0.95

A

J316

571

AT0

4-15

9-18

8 -3

M

A

surf

ace

yello

w ~

glos

sy o

paqu

e irr

egul

ar ra

ised

but

in a

gar r

igid

fray

ed e

dges

81

7 C

ellu

losi

mic

robi

um fu

nkei

isol

ate

W6

0.

91

AY

7299

60

AT0

4-15

9-18

9 -3

M

A

surf

ace

lt ye

llow

~gl

ossy

opa

que

irreg

ular

rais

ed b

ut in

aga

r rig

id fr

ayed

edg

es

775

Cel

lulo

mon

as u

da

0.96

X

8380

1 A

T04-

159-

190

-3

MA

su

rfac

e pk

~op

aque

glo

ssy

rais

ed ro

und

807

Arth

roba

cter

sp. F

a21

0.

97

AY

1312

25

AT0

4-15

9-19

1 -3

M

A

surf

ace

smal

l ora

nge

~irr

egul

ar d

ull o

paqu

e ro

ugh

787

Geo

derm

atop

hilu

s obs

curu

s obs

curu

s 0.

98

L406

20

AT0

4-15

9-19

2 -3

M

A

surf

ace

lt pk

~tra

ns ~

irreg

ular

flat

dul

l 60

2 G

eode

rmat

ophi

lus o

bscu

rus o

bscu

rus

0.98

L4

0620

A

T04-

159-

193

-3

MA

su

rfac

e tin

y ~t

rans

yel

low

edg

es b

row

n ce

nter

glo

ssy

conv

ex

171

Unc

ult C

ellu

lom

onad

acea

e ba

cter

ium

clo

ne m

M3

0.

97

AY

7314

68

A

T04-

159-

194

-3

MA

su

rfac

e ho

t yel

low

har

d irr

egul

ar d

ull o

paqu

e ra

ised

roug

h 80

0 Ac

tinob

ispo

ra a

lani

niph

ila

0.98

A

F325

726

AT0

4-15

9-19

5 -3

M

A

10 c

m

whi

te c

halk

y op

aque

cen

ter f

lat i

n ag

ar b

ig ro

und

trans

dul

l edg

es

815

Act

inob

acte

rium

RG

-51

0.

97

AY

5616

10

AT0

4-15

9-19

7 -3

M

A

10 c

m

offw

hite

opa

que

fray

ed e

dges

glo

ssy

~rou

nd ~

conv

ex

863

Unc

ult B

acill

us sp

.

0.95

A

J640

178

AT0

4-15

9-19

8 -3

M

A

10 c

m

trans

off

whi

te ro

und

glos

sy fl

at

757

Subt

erco

la p

rate

nsis

0.

97

AJ3

1041

2 A

T04-

159-

200

-5

MA

30

cm

ta

n ed

ges b

row

n ce

nter

opa

que

smoo

th ro

und

glos

sy in

aga

r ~co

nvex

83

6 St

rept

omyc

es sp

. EF-

76

0.96

A

F112

173

AT0

4-15

9-20

1 -4

1/

100

PCA

sur

face

lt p

k ro

und

opaq

ue fl

at g

loss

y 81

7 M

odes

toba

cter

sp. E

llin1

65

0.99

A

F409

007

AT0

4-15

9-20

2 -4

1/

100

PCA

sur

face

ora

nge

irreg

ular

opa

que

flat d

ull

984

Mod

esto

bact

er sp

. Elli

n165

0.

99

AF4

0900

7 A

T04-

159-

203

-4

1/10

0 PC

A s

urfa

ce c

ream

roun

d fla

t dul

l ~tra

ns

658

Geo

derm

atop

hilu

s obs

curu

s obs

curu

s 0.

96

L406

20

AT0

4-15

9-20

5 -5

1/

100

PCA

30

cm

very

larg

e cr

eam

dar

ker i

n ce

nter

roun

d ~t

rans

flat

dul

l 67

3 St

rept

omyc

es sp

. NR

RL

5799

0.

98

AJ3

9181

4 A

T04-

159-

207

-4

1/10

0 PC

A 4

0 cm

gr

ey ro

und

flat d

ull o

paqu

e 71

5 St

rept

omyc

es sp

. NR

RL

5799

0.

97

AJ3

9181

4 A

T04-

159-

209

-3

1/10

0 PC

A 3

0 cm

gr

ey ro

und

flat d

ull ~

trans

70

8 St

rept

omyc

es sp

. NR

RL

5799

0.

98

AJ3

9181

4 A

T04-

159-

214

-3

MA

20

cm

la

rge

beig

e ro

und

trans

glo

ssy

flat

797

Baci

llus s

p. L

MG

202

43

0.98

A

J316

317

AT0

4-15

9-21

5 -3

M

A

20 c

m

smal

l bro

wn

roun

d fla

t dul

l opa

que

794

Baci

llus s

p. L

MG

202

43

0.99

A

J316

317

AT0

4-15

9-21

7 -3

M

A

20 c

m

crea

m fl

at g

loss

y op

aque

roun

d 88

7 Ba

cillu

s lito

ralis

0.

98

AY

6086

05

AT0

4-15

9-21

8 -3

M

A

20 c

m

brow

n co

nvex

dul

l ~tra

ns ro

und

spot

ted

with

bro

wn

690

Noc

ardi

opsi

s sp.

98-

08-2

90A

0.

97

AF3

2840

8 A

T04-

159-

220

-3

MA

20

cm

op

aque

bei

ge c

ream

mar

gin

trans

out

side

flat

dul

l whi

te in

cen

ter i

rreg

ular

78

7 Ba

cillu

s sp.

VA

N14

0.

95

AF2

8648

2 A

T04-

159-

223A

-3

M

A

20 c

m

crea

m g

loss

y irr

egul

ar o

paqu

e fla

t 68

4 Ps

eudo

noca

rdia

zijin

gens

is

0.96

A

F325

725

152

(Tab

le c

ontin

ued:

Pit

isol

ates

) Pi

t Iso

late

D

ilutio

n M

edia

D

epth

Col

ony

Mor

phol

ogy

Seq

uenc

e L

engt

h

BL

AST

Res

ult

Sim

ilari

ty A

cces

sion

no.

A

T04-

159-

223B

-3

M

A

20 c

m

71

4 Ba

cillu

s lito

ralis

0.

95

AY

6086

05

AT0

4-15

9-22

4 -3

M

A

20 c

m

crea

m d

ull f

lat r

ound

smal

l ~tra

ns

761

Baci

llus s

p. L

MG

202

43

0.99

A

J316

317

AT0

4-15

9-22

5 -4

1/

10 P

CA

10

cm

ta

n irr

egul

ar w

ith o

paqu

e da

rk ta

n sp

ots t

rans

on

1 si

de o

paqu

e ot

her f

lat d

ull

782

Baci

llus n

iaci

ni

0.99

A

F468

221

AT0

4-15

9-22

8 -3

1/

10 P

CA

30

cm

da

rk g

rey

roun

d op

aque

dul

l bum

py c

onve

x 74

0 St

rept

omyc

es sp

. NR

RL

5799

0.

97

AJ3

9181

4 A

T04-

159-

229

-3

1/10

PC

A

30 c

m

tan

roun

d op

aque

flat

dul

l 44

9 St

rept

omyc

es sp

. 434

F11

0.

97

AB

1235

50

153

(Tab

le c

ontin

ued:

Enr

ichm

ent i

sola

tes)

E

nric

hmen

t Iso

late

Dilu

tion

Med

ia

Dep

th

Col

ony

Mor

phol

ogy

Seq

uenc

e L

engt

h

BL

AST

Res

ult

S

imila

rity

Acc

essi

on n

o.

AT0

4-15

0-B

6 -1

1/

100

PCA

50

cm

whi

te o

paqu

e ~c

onve

x 79

9 St

rept

omyc

es sp

. AS

4.11

82

0.98

A

Y11

4179

A

T04-

150-

B8

-1

1/10

0 PC

A 1

0 cm

tin

y w

hite

opa

que

82

2 Br

evib

acill

us a

gri

0.99

A

J586

388

AT0

4-15

0-B

9 -2

1/

100

PCA

40

cm

whi

te o

paqu

e ~f

uzzy

con

vex

yello

wis

h in

side

78

8 Ps

eudo

noca

rdia

satu

rnea

0.

98

AJ2

5282

9 A

T04-

150-

B10

-2

PC

A

40 c

m

lt ye

llow

ish

clum

py c

onve

x ~t

rans

57

6 Ps

eudo

noca

rdia

satu

rnea

0.

98

AJ2

5282

9 A

T04-

150-

B11

-1

PC

A

10 c

m

lt ye

llow

clu

mpy

con

vex

~tra

ns

816

uncu

lt al

pha

prot

eoba

cter

ium

0.

96

AJ3

1196

2 A

T04-

150-

B12

-1

PC

A

10 c

m

yello

w b

lob

glos

sy o

paqu

e fla

t 80

7 Br

evib

acill

us a

gri

0.

99

AJ5

8638

8 A

T04-

150-

B13

-1

PC

A

10 c

m

offw

hite

glo

ssy

opaq

ue ~

conv

ex

973

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-15

0-B

14

-1

PCA

40

cm

ye

llow

ish

clum

ps ir

regu

lar c

onve

x ~t

rans

69

7 St

rept

omyc

es sp

. AS

4.11

82

0.97

A

Y11

4179

A

T04-

150-

B15

-2

N

A

40 c

m

lt ye

llow

clu

mpy

con

vex

~tra

ns

805

Pseu

dono

card

ia sa

turn

ea

0.97

A

J252

829

AT0

4-15

0-B

19

-1

NA

10

cm

cr

eam

glo

ssy

opaq

ue c

onve

x cr

eam

ish

pk

812

Brev

ibac

illus

agr

i

0.99

A

J586

388

AT0

4-15

0-B

20

-1

NA

50

cm

w

hite

clu

mpy

opa

que

conv

ex y

ello

w in

side

98

3 Br

evib

acill

us a

gri

0.

99

AJ5

8638

8 A

T04-

150-

B21

-1

1/

10 P

CA

50

cm

w

hite

opa

que

conv

ex y

ello

w in

side

that

gro

ws i

n ag

ar

869

Pseu

dono

card

ia sa

turn

ea

0.97

A

J252

829

AT0

4-15

0-B

22

-2

1/10

PC

A

40 c

m

whi

te o

paqu

e cl

umpy

yel

low

insi

de c

onve

x 10

20

Her

basp

irill

um se

rope

dica

e st

rain

X8

0.

96

AF1

6406

5 A

T04-

150-

B23

-2

1/

10 P

CA

40

cm

gl

ossy

ora

nge

opaq

ue c

onve

x 84

2 Ps

eudo

noca

rdia

satu

rnea

0.

97

AJ2

5282

9 A

T04-

150-

B24

-2

1/

10 P

CA

50

cm

op

aque

whi

te c

onve

x cl

umpy

yel

low

insi

de

786

Brev

ibac

illus

agr

i

0.99

A

J586

388

AT0

4-15

0-B

25

-2

1/10

PC

A

surf

ace

lt p

k ~g

loss

y th

in la

yer c

over

ing

part

of p

late

83

9 St

rept

omyc

es c

alifo

rnic

us st

rain

DSM

400

58

0.98

A

Y99

9845

A

T04-

150-

B26

-1

1/

10 P

CA

10

cm

th

in lt

pk

laye

r cov

erin

g pl

ate

736

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glos

sy o

paqu

e 82

1 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

153-

B23

-2

M

A

20 c

m

lt pk

opa

que

glos

sy ~

conv

ex/~

flat

757

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-15

3-B

24

-2

MA

10

cm

cr

eam

glo

ssy

opaq

ue fl

at g

row

s in

agar

61

3 St

rept

omyc

es sp

. AS

4.11

82

0.97

A

Y11

4179

155

(Tab

le c

ontin

ued:

Enr

ichm

ent i

sola

tes)

E

nric

hmen

t Iso

late

Dilu

tion

Med

ia

Dep

th

Col

ony

Mor

phol

ogy

Seq

uenc

e L

engt

h

BL

AST

Res

ult

S

imila

rity

Acc

essi

on n

o.

AT0

4-15

3-B

25

-1

MA

10

cm

lt

oran

ge/p

k op

aque

con

vex

glos

sy

822

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-15

3-B

26

-1

MA

su

rfac

e lt

pk c

onve

x gl

ossy

opa

que

tiny

597

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

4-15

3-B

27

-1

MA

su

rfac

e or

ange

con

vex

glos

sy o

paqu

e 85

2 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

153-

B29

-1

M

A

30 c

m

lt pe

ach

conv

ex g

loss

y op

aque

70

5 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T04-

153-

B30

-2

1/

10 P

CA

40

cm

lt

oran

ge c

onve

x op

aque

~du

ll 70

3 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

153-

B31

-1

1/

10 P

CA

70

cm

tin

y of

fwhi

te tr

ans ~

conv

ex g

loss

y 59

3 Bl

asto

cocc

us a

ggre

gatu

s 0.

97

AJ4

3019

3 A

T04-

153-

B32

-1

1/

10 P

CA

70

cm

lt

oran

ge fl

at th

in la

yer c

over

ing

plat

e 84

7 Bl

asto

cocc

us a

ggre

gatu

s 0.

97

AJ4

3019

3 A

T04-

153-

B33

-1

1/

10 P

CA

60

cm

lt

pk g

loss

y tra

ns ~

conv

ex/~

flat

661

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-15

3-B

34

-2

1/10

PC

A

30 c

m

pale

pk

trans

glo

ssy

conv

ex

683

Blas

toco

ccus

sp. B

C44

8

0.97

A

J316

571

AT0

4-15

3-B

35

-2

1/10

PC

A

80 c

m

clea

r lay

er c

over

ing

plat

e - ~

lt pk

69

4 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

153-

B36

-1

1/

10 P

CA

30

cm

cr

eam

tran

s lay

er c

over

ing

plat

e 68

8 Bl

asto

cocc

us sp

. BC

448

0.

97

AJ3

1657

1 A

T04-

153-

B37

-1

1/

10 P

CA

20

cm

lt

pk c

onve

x tra

ns g

loss

y 64

9 Bl

asto

cocc

us sp

. BC

448

0.

96

AJ3

1657

1 A

T04-

153-

B38

-1

1/

10 P

CA

40

cm

w

hite

con

vex

glos

sy ~

trans

94

5 Ps

eudo

noca

rdia

satu

rnea

0.

98

AJ2

5282

9 A

T04-

153-

B39

-1

1/

10 P

CA

40

cm

lt

oran

ge tr

ans l

ayer

cov

erin

g pl

ate

619

Blas

toco

ccus

sp. B

C44

8

0.96

A

J316

571

AT0

4-15

3-B

40

-1

1/10

PC

A

80 c

m

lt or

ange

tran

s lay

er c

over

ing

plat

e w

ith w

hite

spot

s 84

0 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

153-

B42

-1

PC

A

80 c

m

peac

h ~c

onve

x gl

ossy

~op

aque

70

7 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

153-

B43

-1

PC

A

surf

ace

brig

ht o

rang

e op

aque

con

vex

~glo

ssy

716

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-15

3-B

44

-1

PCA

60

cm

lt

peac

h bu

nch

of ti

ny d

ots m

akin

g th

in o

paqu

e la

yer

704

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-15

3-B

45

-1

PCA

70

cm

lt

oran

ge/c

lear

glo

ssy

laye

r 67

1 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

153-

B47

-2

PC

A

70 c

m

peac

h gl

ossy

opa

que

conv

ex

716

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-15

3-B

48

-2

PCA

su

rfac

e lt

yello

w g

loss

y op

aque

con

vex

681

Baci

llus c

ereu

s stra

in M

SU13

10

0.99

A

Y64

7292

A

T04-

153-

B49

-2

PC

A

surf

ace

flat o

paqu

e gl

ossy

off

whi

te

707

Baci

llus c

ereu

s stra

in M

SU13

10

0.99

A

Y64

7292

A

T04-

153-

B50

-2

PC

A

surf

ace

brow

n op

aque

gro

win

g in

aga

r 70

0 Ba

cillu

s cer

eus s

train

MSU

1310

0.

98

AY

6472

92

AT0

4-15

3-B

51

-2

PCA

su

rfac

e ta

n op

aque

dul

l ~fu

zzy

conv

ex

659

Baci

llus c

ereu

s stra

in M

SU13

10

0.98

A

Y64

7292

A

T04-

153-

B52

-2

PC

A

surf

ace

oran

ge tr

ans g

loss

y co

nvex

69

1 Ba

cillu

s cer

eus s

train

J-1

0.

99

AY

3052

75

AT0

4-15

3-B

53

-1

NA

10

cm

tra

ns y

ello

w g

row

ing

in a

gar

692

Stre

ptom

yces

sp. A

S 4.

1182

0.

98

AY

1141

79

AT0

4-15

3-B

54

-2

MA

40

cm

m

auve

opa

que

conv

ex ~

clum

py g

loss

y 65

8 G

eode

rmat

ophi

lus o

bscu

rus o

bscu

rus

0.97

L4

0620

A

T04-

153-

B56

-2

1/

10 P

CA

70

cm

tra

ns fl

at la

yer c

over

ing

plat

e 69

1 Bl

asto

cocc

us a

ggre

gatu

s 0.

98

AJ4

3019

3 A

T04-

153-

B57

-1

1/

10 P

CA

su

rfac

e fa

intly

pk

trans

laye

r cov

erin

g pl

ate

732

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-15

3-B

58

-2

1/10

PC

A

surf

ace

yello

w g

loss

y op

aque

con

vex

704

Mic

roco

ccus

lute

us

0.95

A

J409

096

AT0

4-15

3-B

59

-1

1/10

0 PC

A 1

0 cm

tin

y w

hite

tran

s con

vex

glos

sy

687

Blas

toco

ccus

sp. B

C44

8

0.98

A

J316

571

AT0

4-15

3-B

61

-1

1/10

0 PC

A s

urfa

ce t

hin

trans

laye

r cov

erin

g pl

ate

(may

just

be

soil)

61

9 Bl

asto

cocc

us sp

. BC

448

0.

98

AJ3

1657

1 A

T04-

153-

B62

-2

1/

100

PCA

60

cm

tiny

trans

lt p

k gl

ossy

con

vex

686

Rhi

zosp

here

soil

bact

eriu

m is

olat

e R

SI-2

1 0.

95

AJ2

5258

8 A

T04-

153-

B63

-2

1/

100

PCA

sur

face

thi

n tra

ns la

yer y

ello

wis

h 56

3 Br

evib

acill

us a

gri s

train

NC

HU

1002

0.

98

AY

3193

01

AT0

4-15

3-B

64

-1

1/10

0 PC

A 7

0 cm

th

in tr

ans p

each

laye

r cov

erin

g pl

ate

877

Pseu

dono

card

ia sa

turn

ea

0.97

A

J252

829

AT0

4-15

3-B

65

-1

1/10

0 PC

A 8

0 cm

th

in tr

ans p

each

laye

r cov

erin

g pl

ate

693

Blas

toco

ccus

sp. B

C44

8

0.96

A

J316

571

AT0

4-15

3-B

66

-1

1/10

0 PC

A 4

0 cm

lt

pk ~

conv

ex g

loss

y op

aque

88

5 Bl

asto

cocc

us sp

. BC

448

0.

99

AJ3

1657

1 A

T04-

153-

B68

-1

1/

100

PCA

30

cm

tiny

whi

te o

paqu

e gl

ossy

con

vex

678

Blas

toco

ccus

sp. B

C44

8

0.99

A

J316

571

AT0

4-15

3-B

69

-1

1/10

0 PC

A 6

0 cm

tin

y w

hite

opa

que

glos

sy c

onve

x 95

0 A

ctin

omyc

etal

es b

acte

rium

HPA

66

0.97

D

Q14

4230

A

bbre

viat

ions

in c

olon

y m

orph

olog

y co

lum

n ar

e as

list

ed: l

t - li

ght,

pk –

pin

k, tr

ans –

tran

sluc

ent,

~ - s

light

ly

156

APPENDIX C TABLE OF PHOSPHATE, NITRITE, AND BROMIDE CONCENTRATIONS (MG/L)

“NA” means that nitrate was not detected (detection limit is 0.012 mg/L)

Surface Site Phosphate Nitrite Bromide Soil Pit Sample Phosphate Nitrite BromideAT03-33 0.2540 NA NA AT04-150 surface NA NA NA AT03-34 NA NA NA AT04-150 10cm NA NA NA AT03-35 0.2920 NA NA AT04-150 20cm NA NA NA AT03-36 0.2840 NA NA AT04-150 30cm NA NA NA AT03-37 0.0800 NA NA AT04-150 40cm NA NA NA AT03-38 NA NA NA AT04-150 50cm NA NA NA AT03-39 0.5700 NA NA AT04-150 60cm NA NA NA AT03-40 NA NA NA AT04-150 70cm 0.1260 NA NA AT03-41 NA NA NA AT04-150 80cm 0.4000 NA NA AT03-42 0.2920 NA NA AT04-150 90cm 0.2080 NA NA AT03-43 0.1080 NA NA AT04-152 surface 0.2360 NA NA AT03-44 0.3600 NA NA AT04-152 10cm NA NA NA AT03-45 0.0800 NA NA AT04-152 20cm NA NA NA AT03-46 0.2700 NA NA AT04-152 30cm NA NA NA AT03-48 NA NA NA AT04-152 40cm NA NA NA AT03-49 NA NA NA AT04-152 50cm NA NA NA AT03-50 NA NA NA AT04-152 60cm NA NA NA AT04-151 NA NA NA AT04-152 70cm NA NA NA AT04-154 NA NA NA AT04-152 80cm 0.4960 NA NA AT04-155 NA NA NA AT04-153 surface NA NA NA AT04-156 0.3120 NA NA AT04-153 10cm NA NA NA AT04-157 0.1740 NA NA AT04-153 20cm NA NA NA AT04-158 NA NA NA AT04-153 30cm NA NA NA AT04-161 NA NA NA AT04-153 40cm NA NA NA AT04-162 NA NA NA AT04-153 50cm NA NA NA AT04-163 NA NA NA AT04-153 60cm NA NA NA AT04-164 NA NA NA AT04-153 70cm NA NA NA AT04-165 NA NA NA AT04-153 80cm 0.6040 NA NA AT04-166 NA NA NA AT04-159 surface NA NA NA AT04-167 0.3980 NA NA AT04-159 10cm NA NA NA AT04-168 NA NA NA AT04-159 20cm NA NA NA AT04-169 NA NA NA AT04-159 30cm NA NA NA AT04-170 NA NA NA AT04-159 40cm NA NA NA Cornfield NA NA NA

157

APP

EN

DIX

D

ME

ASU

RE

ME

NT

S O

F SA

LIN

TY

, TD

S, A

ND

CO

ND

UC

TIV

ITY

IN T

AB

LE

OF

SOL

UB

LE

SA

LT

S W

ITH

IN S

OIL

S

Surf

ace

Sam

ple

Salin

ity (p

pt)

Con

duct

ivity

(mS)

T

DS

(ppt

) Pi

t Sam

ple

Salin

ity (p

pt)

Con

duct

ivity

(mS)

T

DS

(ppt

)

A

T03

-33

1.10

00

2.20

00

1.54

00

AT

04-1

50 su

rfac

e 1.

1000

2.

2100

1.

5400

A

T03

-34

1.16

00

2.33

00

1.63

00

AT

04-1

50 1

0cm

1.

0500

2.

1000

1.

4700

A

T03

-35

1.16

00

2.33

00

1.63

00

AT

04-1

50 2

0cm

1.

1000

2.

2100

1.

5400

A

T03

-36

1.14

00

2.29

00

1.60

00

AT

04-1

50 3

0cm

1.

1000

2.

2000

1.

5400

A

T03

-37

1.20

00

2.40

00

1.68

00

AT

04-1

50 4

0cm

1.

0900

2.

1800

1.

5200

A

T03

-38

1.09

00

2.19

00

1.53

00

AT

04-1

50 5

0cm

1.

3000

2.

6100

1.

8200

A

T03

-39

1.17

00

2.34

00

1.63

00

AT

04-1

50 6

0cm

2.

1600

4.

3200

3.

0200

A

T03

-40

1.24

00

2.49

00

1.74

00

AT

04-1

50 7

0cm

2.

6000

5.

2000

3.

6300

A

T03

-41

1.15

00

2.30

00

1.61

00

AT

04-1

50 8

0cm

5.

2900

10

.630

0 7.

4300

A

T03

-42

0.29

60

0.59

50

0.41

70

AT

04-1

50 9

0cm

4.

1600

8.

3500

5.

8400

A

T03

-43

0.31

00

0.62

00

0.43

30

AT

04-1

52 su

rfac

e 1.

1000

2.

2100

1.

5400

A

T03

-44

1.12

00

2.25

00

1.57

00

AT

04-1

52 1

0cm

0.

9980

1.

9990

1.

4000

A

T03

-45

1.07

00

2.14

00

1.49

00

AT

04-1

52 2

0cm

1.

1000

2.

2100

1.

5400

A

T03

-46

1.08

00

2.17

00

1.51

00

AT

04-1

52 3

0cm

1.

0700

2.

1400

1.

4900

A

T03

-48

1.16

00

2.33

00

1.63

00

AT

04-1

52 4

0cm

1.

3300

2.

6600

1.

8600

A

T03

-49

1.18

00

2.36

00

1.65

00

AT

04-1

52 5

0cm

1.

5600

3.

1300

2.

1900

A

T03

-50

0.85

90

1.71

90

1.20

30

AT

04-1

52 6

0cm

1.

2000

2.

4000

1.

6800

A

T04

-151

1.

1800

2.

3600

1.

6500

A

T04

-152

70c

m

2.15

00

4.30

00

3.01

00

AT

04-1

54

1.10

00

2.20

00

1.54

00

AT

04-1

52 8

0cm

1.

4700

2.

9500

2.

0600

A

T04

-155

1.

0200

2.

0400

1.

4200

A

T04

-153

surf

ace

0.09

50

0.18

10

0.12

67

AT

04-1

56

1.14

00

2.29

00

1.60

00

AT

04-1

53 1

0cm

1.

0900

2.

1800

1.

5100

A

T04

-157

0.

2980

0.

5970

0.

4170

A

T04

-153

20c

m

1.06

00

2.12

00

1.48

00

AT

04-1

58

0.31

50

0.63

00

0.44

10

AT

04-1

53 3

0cm

1.

0900

2.

1800

1.

5200

A

T04

-161

1.

2000

2.

4000

1.

6800

A

T04

-153

40c

m

1.42

00

2.83

00

1.98

00

AT

04-1

62

1.87

00

3.74

00

2.61

00

AT

04-1

53 5

0cm

1.

5500

3.

1000

2.

1700

A

T04

-163

1.

3600

2.

7300

1.

9100

A

T04

-153

60c

m

4.94

00

9.88

00

6.92

00

AT

04-1

64

0.90

80

1.81

40

1.27

20

AT

04-1

53 7

0cm

2.

8000

5.

6200

3.

9200

A

T04

-165

1.

3800

2.

7600

1.

9300

A

T04

-153

80c

m

2.88

00

5.77

00

4.03

00

AT

04-1

66

0.76

80

1.53

40

1.07

50

AT

04-1

59 su

rfac

e 0.

0225

0.

0451

0.

0316

A

T04

-167

5.

6700

11

.330

0 7.

9000

A

T04

-159

10c

m

0.90

40

1.80

80

1.26

60

AT

04-1

68

8.86

00

17.7

900

12.4

500

AT

04-1

59 2

0cm

1.

0000

2.

0200

1.

4200

A

T04

-169

2.

1300

4.

2700

2.

9800

A

T04

-159

30c

m

0.90

50

1.81

30

1.27

00

AT

04-1

70

3.48

00

6.95

00

4.86

00

AT

04-1

59 4

0cm

0.

8960

1.

8000

1.

2600

C

orn

Fiel

d 0.

0128

0.

0257

0.

0179

158

VITA

Danielle Bagaley was born in Baton Rouge, Louisiana, on May 6, 1979. She is the

daughter of Daniel and Robbie Bagaley and the sister of Jonathan Bagaley. She attended St.

Aloysius Catholic School in Baton Rouge from kindergarten until eighth grade and acquired her

high school degree in August of 1997 from Baton Rouge Magnet High School. Danielle began

her college career at Louisiana State University in 1997 and obtained a Bachelor of Science

degree in August of 2002. She participated in the National Student Exchange program and

attended the University of Georgia at Athens during the 1999/2000 school year. In her final

semester at LSU she enrolled in an undergraduate laboratory research course conducted by Dr.

Fred Rainey. She gained great interest in studies that the RaineyLab was undergoing at the time,

mainly a survey taking place in the Atacama Desert located in Chile. After graduating with her

bachelor degree she was hired as a research associate in the RaineyLab. After working for a year

and a half, Danielle decided to pursue a Master of Science degree from Louisiana State

University. She studied the extent of “Mars-like” soils in the Atacama Desert including

subsurface as well as surface samples. After graduating with a Master of Science degree,

Danielle Bagaley will presumably pursue a career with either a pharmaceutical corporation in

validation and quality control/assurance divisions, a biomedical research company, or with a

scientific research supply business as a sales representative.

Documents

Uncovering bacterial diversity on and below the surface of