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Is Soil Microbial Diversity Impacted by Distance from Water in Prince Edward County?
Matthew Bowman
BIOL 250-50
Dr. Amorette Barber
April 16, 2017
Is Soil Microbial Diversity Impacted by Distance from Water in Prince Edward County? 2
Introduction
The environmental microbiome is being studied in various locations around the world
and in the United States; however, there are still various locations we have yet to study.
Scientists can formulate new medications, discover diverse sets of bacteria, and identify the
microbes in that specific location because of studying the environmental microbiome. Because of
studying different microbiomes, we are not only able to better the human microbiome, but evolve
environmental genomics. Not only are these microbes affecting the human microbiome, but the
environment we live in. Microbial communities are controlled solely by environmental factors
such as wind dispersion, runoff, amount of sunlight, and temperature (Heino et al. 2014).
These environmental factors can transfer microbes between distant environments
(Hooper et al. 2008). These microbes are effecting wildlife, especially the botanical life in distant
environments. The microbes are establishing new communities, embedding in the soil, and are
driving the classic plant diversity (Schnitzer 2011). Ecosystem productivity helps increase
asymptotically with plant species diversity, and determining these microbes are responsible in
determining species loss (Schnitzer 2011). Traces of certain microbial communities are being
found in soil due to water sources such as streams, rivers, and creeks. These water sources are
inputting high levels of nitrogen into the soil but low amounts of carbon (Heffernam and Fisher
2012). Microbes transport high levels of nitrogen, allowing plant roots to intake high levels of
nitrogen; however, plants are not receiving the amount of carbon required to grow properly
(Heffernan and Fisher 2012). Plant roots are now microengineering their environment and
habitats by altering the porosity and clustering properties of the soil pores (Feeney et al. 2006).
Is Soil Microbial Diversity Impacted by Distance from Water in Prince Edward County? 3
These studies sparked an interest for the microbiomes surrounding plant roots, and how
the microbes directly affect a plant’s water source. In this study, our objective is to determine
how microbial diversity changes the further a root system is from a water source. We assume
that plant roots close to a particular water source is benefitting from that microbial community,
while the plant roots further away from a particular water source is not benefitting from that
microbial community.
Materials and Methods
Environmental Sampling & Site Description
Samples of water and soil from a pond and creek was collected in February of 2017 at the
Environmental Education Center (EEC) at Lancer Park at Longwood University. Pond and creek
water was collected on the banks of the water. The soil was collected 5 and 10 meters away from
the creek and pond. The samples were then returned to the ECC and plated on the corresponding
plate at the following dilutions: 100μl of water and soil, 100μl of 1:10 dilution of water and soil
sample, and 100μl of 1:100 of water and soil sample. The water sample that was collected was
used for the direct count plate. 90μl of sterile nutrient broth was placed into the 1:10 and 1:100
tubes. 10μl of the water sample from the original tube was transferred to the 90μl of nutrient
broth in the 1:10 tube. 10μl of sample from the 1:10 dilution tube was also transferred to the 90μl
of nutrient broth in the 1:100 tube. Once samples were diluted and plated on corresponding
plates, the agar plates were incubated at room temperature.
Bacterial Growth & Characterization
Plate were checked 18-24 hours, 48 hours, and 7 days after sampling. Each day, pictures
were taken of the plates for data records. Any plate which had more than 200 colonies was
designated as “too many to count,” plates with fewer than 30 colonies were not taken into
Is Soil Microbial Diversity Impacted by Distance from Water in Prince Edward County? 4
consideration for analyzation. Plates were analyzed through number of colonies, color, size,
shape, texture, form, elevation, and margin.
DNA Isolation from Water & Soil Samples
One colony from each environmental sample was used to perform DNA analysis.
Microcentrifuge tubes were labeled for each colony and 300μl of microbead solution was added
to each tube and vortexed. The bacteria cells were then transferred to the labeled microbead tube
along with 50μl of solution MD1. The microbead tubes were heated to 65 degrees Celsius for 10
minutes and then transferred to a clean 2 mL collection tube.
900μl of solution MD3 was added to the collection tube and vortexed. 700μl was loaded
into the spin filter and centrifuged at 10,000 x g for 30 seconds and room temperature. 300μl of
solution MD4 was then transferred to the collection tube and centrifuged. A new spin filter was
added in a new 2 mL collection tube, while adding 50μl of solution MD5 to release the bound
DNA. The collection tube was then centrifuged at 10,000 x g for 30 seconds at room
temperature. Analyzation of purity and concentration of DNA was then completed using a
Nanodrop.
PCR- 16srRNA – Amplification
6μl of DNA and 50μl of PCR reaction mix (1μl of Forward Primer, 1μl of Reverse
Primer, 25μl of OneTaq 2X Master Mix, and 17μl of Nuclease-free water) were added to the
PCR tube, the PCR was added to the tube first to simplify the process. The reactions were gently
mixed by pipetting up and down. 3μl of the genomic DNA that was isolated was inserted into the
PCR tubes. One PCR tube received 3μl of DNA from one of the targets of a single molecule of
DNA, and the second PCR tube received 3μl of DNA from the other target of a single molecule
Is Soil Microbial Diversity Impacted by Distance from Water in Prince Edward County? 5
of DNA. The PCR tubes were then transferred to a PCR machine to begin thermocycling. The
PCR tubes were exposed to initial denaturation, 30 cycles, final extension, and hold. The 16S
rDNA was then expressed through universal primers: Y (C, T), M (A, C), N (G, T, A, C) and R
(A, G). The universal primers that were used have the following sequences:
Forward – 5’ GAGTTTGATYMTGGCTC 3’
Reverse – 5’ NRGYTACCTTGTTACGACTT 3’
PCR Cleanup – Restriction Enzyme Digestion – Gel Electrophoresis
Restriction Enzyme Digestion was performed to cleave a functional piece of DNA. PCR
clean-up had to be performed before restriction enzyme digestion could occur. 100μl of a
reaction was transferred to a 1.5mL microcentrifuge tube along with 5 volumes of DF Buffer.
The microcentrifuge tube was then vortexed. A DF column was then placed in a 2mL collection
tube. The mixture was then transferred to the DF column and centrifuged, discarding the flow
through. 600μl of wash buffer was then placed into the center of the DF column and centrifuged
for 30 seconds, again discarding the flow through. The dried DF column was then transferred to
a new microcentrifuge tube, adding 25μl of Electron Buffer and centrifuging for two minutes.
Once the PCR products were cleaned, a tube of MspI premixed with the buffer was
retrieved to being the restriction enzyme digestion. For each PCR product, 5μl of PCR product
and 10μl of MspI was setup for each PCR product. The samples were then mixed by pipetting up
and down with the pipettor set to 10μl and incubated for 45 minutes at 37°C. A 1.5% agarose gel
electrophoresis was then casted. 10μl of each sample was then placed into separate wells in the
gel chamber. The gel was then developed at 120 V for 30 minutes and placed under a UV camera
for visualization.
Is Soil Microbial Diversity Impacted by Distance from Water in Prince Edward County? 6
DNA Sequencing
DNA was mixed with 3μl of deionized water, 5μl of clean PCR product, and 4μl of the
sequencing primer. The sequencing primer used follows: 5’-GAGTTTGATCCTGGCTCAG-3’.
Once the DNA was sequenced, SnapGene Viewer was used to analyze the sequences. Once the
DNA sequences were edited, a BLAST – Targeted Loci (16s ribosomal RNA) search was used to
identify the prokaryote.
Results
Number of Colonies
The number of colonies for each site were counted twice, the average was then taken
between the two numbers. The number of colonies from each sampling site did vary, as
represented in Figure 1. The tree 5 meters from Buffalo creek showed the highest growth rate of
colonies, while the pond at Lancer Park showed the slowest growth rate of colonies. Once the
tree 5 meters from the pond at Lancer Park reached the 48-hour growth mark, it stabilized and
did not show a dramatic increase nor a decrease of bacteria colonies.
Is Soil Microbial Diversity Impacted by Distance from Water in Prince Edward County? 7
Figure 1. Total Number of Colonies Present on Plates after 48 hours of growth.The number of colonies on each plate were counted at 24 hours and 48 hours. Plates that were counted were multiplied by their dilution factor.
Characterization
To correctly identify the bacteria isolated from our collections sites, the bacteria had to be
characterized. First, the colors present in our samples were yellow, purple, orange, and a white
color. The number of white colonies were dominant over all the other colors present at all sites
(Fig. 2). Figure 3 represents the shapes of the bacteria that were collected. Each site showed a
variety of shapes: punctiform, spindle, circular, filamentous, irregular, and rhizoid. The Lancer
Park pond showed a high amount of irregular and punctiform bacteria; however, Buffalo Creek
showed a mixture of all shapes. Plate sample pictures from Lancer Park are represented in Figure
4, Buffalo Creek samples are represented in Figure 5.
0 24 480
500
1000
1500
2000
2500
Buffalo Creek Buffalo Creek Tree (5m) Buffalo Creek Tree (10m) Lancer Park PondLancer Park Tree (5m) Lancer Park Tree (10m)
Hours
Num
ber o
f Col
onie
s
Is Soil Microbial Diversity Impacted by Distance from Water in Prince Edward County? 8
Figure 2. Characterization of bacteria colonies through color.Color of bacteria colonies were recorded. Colonies observed were light yellow, purple, orange, and white.
Figure 3. Shape of Colonies from Collection Sites. Six different shapes were observed and recorded. Plates chosen were those that had DNA isolated from them. Diluted plates were accounted for through multiplying by the dilution factor.
punctiform spindle circular filamentous irregular Rhizoid0
200400600800
1000120014001600
Lancer Park Pond Lancer Park Tree 5m Lancer Park Tree 10mBuffalo Creek Buffalo Creek Tree 5m Buffalo Creek Tree 10m
Num
ber o
f Col
onie
s
Lancer Park Pond
Lancer Park Tree 5m
Lancer Park Tree 10m
Buffalo Creek Buffalo Creek Tree 5m
Buffalo Creek Tree 10m
0
20
40
60
80
100
120
Yellow white purple orange
Num
ber o
f Col
onie
s
Is Soil Microbial Diversity Impacted by Distance from Water in Prince Edward County? 9
Figure 4. Colonies taken from Lancer Park pond and nearby treesTop row: Pseudomonas koreensis, Flavobacterium Sp. R30-9,Bottom row: Bacillus cereus, Pseudomonas frederiksbergensis
Figure 5. Colonies take from the Buffalo Creek and nearby trees1st: Janthinobacterium lividum2nd: Pseudomonas trivialis 3rd: Pseudomonas extremoustralis
DNA Purity/Gel Electrophoresis
DNA was extracted from our isolated colonies. To send the DNA for testing we were
required to have substantial concentrations of DNA and relativity good quality DNA, which
ranges from A260 and A280. The purity of DNA collected in this study ranged from 1.71 and 2.23,
with an overall average of 1.94 (Table 1). The second pond water had a significant difference in
the concentration of DNA (26.7 ng/mL) compared to the other locations. For example, pond
Is Soil Microbial Diversity Impacted by Distance from Water in Prince Edward County? 10
water two indicated a 26.7 ng/mL concentration of DNA while Buffalo creek two indicated a 7.8
ng/mL concentration of DNA.
The sequenced PCR product for the 16s rRNA gene generated 857 bases of high-quality
base pairs that were used to identify the genus and species of the bacteria colonies. This allowed
us to separate and analyze the DNA and their fragments, based on their size and charge. Figure 6
shows the nucleic acid molecules separated by bands. The shorter molecules moved faster and
traveled farther than the longer ones because the shorter molecules traveled through the pores of
the gel. As you can clearly see in Figure 6 bands lining up, which represents a protein that
compromises that band is highly abundant.
Location Concentration of DNA (ng/mL) Purity of DNA (260/280)Pond Water 1 22.8 1.98Pond Water 2 26.7 1.95
Pond Tree, 5 meters 17.2 2.23Pond Tree, 10 meters 14.3 1.71
Buffalo Creek Water 1 15.1 1.96Buffalo Creek Water 2 7.8 1.98
Buffalo Creek Tree, 10 meters 12.3 1.76
Table 1. Concentrations and Purity of DNA Genomic DNA extraction efficiencies of the eight sampling sites. The sites were assessed by transferring up to 100 μl of a reaction produce to a 1.5 microcentrifuge tube.
Is Soil Microbial Diversity Impacted by Distance from Water in Prince Edward County? 11
Figure 6. Gel Electrophoresis results of PCR amplification of Msp1 digestion. The digested gels are 3, 5, 7, and 9 with the top being Buffalo Creek tree at 5m, tree at 10m, and the two water samples. Each had a digested and undigested form. The bottom was the Lancer Park Pond tree at 5m, tree at 10m, and the two water samples also with a digested and undigested form of each.
Genus and species classification
The prokaryotes were then identified based on the sequence of their 16s rDNA, as shown in table
two. The first set of samples came from Buffalo Creek. The prokaryote collected from creek one
was identified as Janthinobacterium lividium. This prokaryote showed a 99% identity with 863/865
bases matching and 1/865 gaps in alignment. Janthinobacterium lividium is primarily isolated from
the soils of maritime Antarctica/the Peninsula of Antarctica. The prokaryote collected from creek
two was identified as Pseudomonas trivialis. This prokaryote had a 99% identity with 954/961
bases matching and 1/961 gaps in alignment. Pseudomonas trivialis is thought to be isolated from
the rhizosphere of Hippophae rhamnoides. The final prokaryote identified at the Buffalo Creek site
was collected at a tree located 10 meters from the creek, which was identified as Pseudomonas
Is Soil Microbial Diversity Impacted by Distance from Water in Prince Edward County? 12
extremoustralis. This prokaryote had a 99% identity with 897/905 bases matching and 2/905 gaps
in alignment. Pseudomonas extremoustralis is also thought to be isolated from cold environments,
such as the Antarctica.
The second set of samples came from the pond at Lancer Park. The prokaryote collected
from pond one was identified as Flavobacterium sacchorophilum. This prokaryote had a 99%
identity with 984/993 bases matching and 1/993 gaps in alignment. Flavobacterium
sacchorophilum is commonly isolated from the artic sea waters and cold environments. The
prokaryote collected from pond two was classified as Pseudomonas koreensis. This prokaryote
had a 99% identity with 942/949 bases matching and 2/905 gaps in alignment. Pseudomonas
koreensis is native to Korea and usually found in colder environments. The third sample site at
Lancer Park was a tree 5 meters from the pond and the prokaryote found here was identified as
Pseudomonas frederiksbergensis. This prokaryote had a 100% identity with 862/862 bases
matching and 1/862 gaps in alignment. This prokaryote has only been isolated from a coal site
located in Fredericksburg, VA. The final prokaryote identified was from the fourth sampling site
at Lancer Park, the tree 10 meters from the pond. The prokaryote identified here is known as
Bacillus cereus. This prokaryote showed a 100% identity with 887/887 bases matching and
0/887 gaps in alignment. This prokaryote is a soil bacterium found near plant roots causing food
poisoning (Table 2).
Sample Site Closest BLAST % identity Gaps in Environment
Is Soil Microbial Diversity Impacted by Distance from Water in Prince Edward County? 13
Match Alignment
Buffalo Creek 1
Janthinobacterium lividum
863/865(99%) 1/865
Isolated from the soils of maritime
Antarctica/the Peninsula of Antarctica
Buffalo Creek 2
Pseudomonastrivialis
954/961(99%) 1/961
Isolated from the rhizosphere of
Hippophae rhamnoides
growing in the Lahaul-Spiti district, India
Lancer Park Pond 1
Flavobacterium sacchorophilum
984/993(99%) 1/993
Found in arctic sea water and
cold environment
Lancer Park Pond 2
Pseudomonas koreensis
942/949(99%) 1/949
Native to Korea and found in
generally colder environments
Buffalo Creek Tree (10 m)
Pseudomonas extremoustralis 897/905 (99%) 2/905
Isolated from a temporary pond
in Antarctica/cold environments
Lancer Park Pond Tree (5
m)
Pseudomonas frederiksbergensis
862/862(100%) 1/862
Isolated from a coal site in
Fredericksburg
Lancer Park Pond Tree (10
m) Bacillus cereus887/887(100%) 0/887
Soil bacterium found near roots and can cause food poisoning
Table 2. Genus and species classification using 16S rRNA sequencing The taxanomic affiliation of representative isolates was determined using 16S rRNA gene sequencing and is classified through the bacteria’s percent identity and gaps in the sequencing alignment.
Is Soil Microbial Diversity Impacted by Distance from Water in Prince Edward County? 14
Discussion
In this study, our objective was to determine how microbial diversity changes the farther
a root system is from a water source. We assumed that plant roots close to a particular water
source is benefitting from that microbial community, while the plant roots further away from a
particular water source is not benefitting from that microbial community.
The samples collected from both sites did show similarities. All the bacteria showing a
light-yellow color came from the genus, Pseudomonas. There were multiple types of this
bacteria, and each site contained at least one type of Pseudomonas. This shade of pseudomonas
was also present in other studies that have been conducted (Tribelli et al. 2015). Also, our study
did align with other studies supporting the environment of Janthinobacterium lividum (Valdes et
al. 2015). Valdes et al. 2015 found that Janthinobacterium lividum was isolated from the soils of
maritime Antarctica/the Peninsula of Antarctica, and we were also able to isolate this bacterium
from the soil. Based on this finding, we can conclude that there are microbes establishing new
communities, embedding in the soil, and are driving the classic plant diversity (Schnitzer 2011).
Also, according the Feeney et al. plants are now micro engineering their environment and
habitats by altering the porosity and clustering properties of the soils pore. We can support that
through our finding of the Bacillus cereus. This prokaryote is found near plant roots and can
cause food poison or even poison the plant. We can assume that the plants are micro engineering
their selves to be able to support this bacterium and live in a habitat with this bacteria present.
Pseudomonas trivialis has been isolated from the rhizosphere of Hippophae rhamnoides
(Mejri, Gamalero, & Souissi et al. 2012). The rhizosphere is a soil bacterium, and we also found
this bacterium in the soil; which can also support our hypothesis of species diversity (Schnitzer
2011). This bacterium also consumes nitrogen, which is a macronutrient. This supports the
Is Soil Microbial Diversity Impacted by Distance from Water in Prince Edward County? 15
theory of microbes transporting high levels of nitrogen, allowing plant roots to intake high levels
of nitrogen; however, plants are not receiving the amount of nitrogen/carbon needed to grow
properly (Heffernan and Fisher 2012).
From the bacterium, we isolated in this study, four of the seven were found to thrive in a
colder environment. When we collected our samples, and isolated them, the environment was
cold. If we had collected our samples when the environment was warm we may not have found
Flavobacterium sacchorophilum, which is a prokaryote that thrives in artic sea water and cold
environments.
Is this study, we can now conclude the microbial diversity does change the farther a root
system is from a water source. We can also conclude that some plant roots are intaking the
required number of nutrients needed to survive due to certain prokaryotes. This study does need
to be extended to fully conclude our hypothesis and rule out some of the possibilities. For
example, bacteria samples should be isolated during each season of the year. This will allow us
to see if diversity of bacteria occurs due to the season or not. Also, other sites should be sampled
as well. Sites should be extended away from each other, within a few miles’ radius. By doing
this, we will also be able to see if certain bacteria are found in other areas and see if that
influences the environment.
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