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Isolation and phylogenic identificationof soil haloalkaliphilic strains in theformer Texcoco LakeMarisela Y. Soto-Padilla a , César Valenzuela-Encinas b , LucDendooven b , Rodolfo Marsch b , Pablo Gortáres-Moroyoqui a &María Isabel Estrada-Alvarado aa Biotecnología y Ciencias Alimentarias , Instituto Tecnológico deSonora , Obregón , Mexicob Cinvestav, Biotecnología y Bioingeniería , Distrito Federal ,MexicoPublished online: 19 Jun 2013.

To cite this article: Marisela Y. Soto-Padilla , César Valenzuela-Encinas , Luc Dendooven , RodolfoMarsch , Pablo Gortáres-Moroyoqui & María Isabel Estrada-Alvarado (2013): Isolation and phylogenicidentification of soil haloalkaliphilic strains in the former Texcoco Lake, International Journal ofEnvironmental Health Research, DOI:10.1080/09603123.2013.800957

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Isolation and phylogenic identification of soil haloalkaliphilic strainsin the former Texcoco Lake

Marisela Y. Soto-Padillaa, César Valenzuela-Encinasb, Luc Dendoovenb,Rodolfo Marschb, Pablo Gortáres-Moroyoquia and María Isabel Estrada-Alvaradoa*

aBiotecnología y Ciencias Alimentarias, Instituto Tecnológico de Sonora, Obregón, Mexico;bCinvestav, Biotecnología y Bioingeniería, Distrito Federal, Mexico

(Received 11 June 2012; final version received 19 March 2013)

A wide diversity of organisms exists in soil. Well-adapted groups can be found inextreme environments. A great economic and metabolic potential for extremozymesproduced by organisms living at extreme environments has been reported. Extremecharacteristics such as high salt content and high pH level make the soil of theformer Texcoco Lake a unique place which has not been exploited. Therefore, inthis study, 66 strains from soil of the former Texcoco Lake were isolated andphylogenetically analyzed using universal oligonucleotide primers. Different generasuch as Kocuria, Micrococcus, Nesterenkonia, Halomonas, Salinicoccus, Kurthia,Gracilibacillus, and Bacillus were found. However, only 22 from all isolated strainswere identified at specie level.

Keywords: extremophile; haloalkaliphilic

Introduction

Organisms in the soil contribute to a wide range of essential services for sustainablefunctioning of all ecosystems. Soil is an appropriate environment for the developmentof both eukaryotes (algae, fungi, protozoa) and prokaryotes organisms (bacteria andarchaea). Also, it is possible to find viruses and bacteriophages (Nogales 2005).Although environmental conditions such as pH, temperature, and salinity concentrationsare extremely high or low on extreme environments, there are groups of organisms spe-cifically adapted to these particular conditions. These types of extreme micro-organismsare usually known as alkaliphiles, halophiles, thermophiles, and acidophiles, reflectingthe particular type of extreme environment they inhabit (Ulukanli & Digrak 2002). Theuse of ribosomal RNA (rRNA) sequence-based analysis to characterize microbial popu-lations (mainly bacterial and archaeal populations) has increased significantly since thedecade of 1980. The 16S rRNA gene analysis has been used to study diverse bacterialand archaeal communities in extreme environments. These environments are interestingto carry out studies such as microbial diversity novel micro-organisms identification,and understanding ecosystem dynamic (Lizama et al. 2001).

Research has been conducted on the isolation of new extremophile micro-organ-isms from the soil of extreme conditions (Kristjansson & Hreggvidsson 1995); (Rossiet al. 2001) to get their extremozymes, (Madigan & Marrs 1997; Rothschild &

*Corresponding author. Email: maria.estrada@itson.edu.mx

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Mancinelli 2001; Satyanarayana et al. 2005; Podar & Reysenbach 2006; Ferrer et al.2007). These enzymes offer new opportunities for biocatalysis and biotransformationsas a result of their extreme stability. From recent works (Satyanarayana et al. 2005;Podar & Reysenbach 2006) major approaches to extend the range of applications ofextremozymes have emerged. Both the discovery of new extremophilic species andthe determination of genome sequences provide a route to new enzymes, with possi-ble novel applications (Hough & Danson 1999). The soil of the former Texcoco Lakeis saline-sodic with a pH between 8.5 and 10.5, electrolytic conductivity in saturatedextracts between 0.04 and 0.7 Sm�1 (Siemens per meter), and high exchangeablesodium percentage between 60 and 80 (Beltrán-Hernández et al. 1999); (Luna-Guidoand Dendooven 2001); (Luna-Guido et al.(2002); (Valenzuela 2005). Simulating thedynamics of glucose and NH4

+ in alkaline saline. These features make it an extremeenvironment for the growth of haloalkaliphiles micro-organisms. Several micro-organ-isms adapted to extreme conditions have been identified from the soil of the formerTexcoco Lake (Luna-Guido et al. (2002); (Valenzuela-Encinas et al. 2008). Recently,Ruiz-Romero et al. (2013) reported a haloalkaliphilicarchaeon isolated from the soilof the former Texcoco Lake; also the isolation of two cellulolytic micro-organisms(Ruiz-Romero et al. 2010).

Halophilic micro-organisms are salt-loving organisms that inhabit hypersalineenvironments worldwide. Many of these organisms are found in natural hypersalinebrines in arid soils, coastal zones, and even deepsea locations, as well as in artificialsalterns used to mine salts from the sea. They include mainly prokaryotic andeukaryotic micro-organisms with the capacity to balance the osmotic pressure of theenvironment and to resist denaturing effects of salts. Different groups of halophilicmicro-organisms such as Cyanobacteria, Archea, gram-negative, and gram-positivebacteria are found among the prokaryotic ones (Dassarma & Arora 2001). Halophilicmicro-organisms can be loosely classified as slightly, moderately, or extremelyhalophilic, depending on their requirement for NaCl. Halophilic micro-organisms areconsidered as an extremophilus group with biotechnology potential (Ventosa et al.1998); (Margesin & Schinner 2001). Micro-organisms developed in alkaline conditionscan be classified into two groups: alkaliphiles and alkalitolerants. The term alkaliphilesapplies to micro-organisms requiring alkaline conditions for growing; their optimumgrowth rate is observed in at least two pH units above neutrality, nevertheless theseorganisms are capable of growing in pH values greater than 9 or 10. In contrast,micro-organisms with an optimum growth around neutral pH are known as alkalitoler-ant (Ulukanli & Digrak 2002). This study is aimed at the isolation and phylogeneticidentification of haloalkaliphilic micro-organism from the soil of the former TexcocoLake.

Material and methods

Site description and soil sampling

Sampling site is located at the former Texcoco Lake (Northern Latitude 19° 30′52″Western Longitude 98°59′24″) in the state of Mexico, Mexico (Valenzuela-Encinaset al. 2008). An area of 1m2 was delimited, the first 2 cm of soil were discarded, andthe next 10 cm of soil was sampled with a hand spade and taken to the laboratory in ablack polyethylene bag. Samples were 5mm sieved under aseptic conditions, divided in250 g sub-samples and stored at �80 °C until the analysis was performed.

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Media and growth conditions

Three types of media were tested for the growth of micro-organisms. A preincubation of20 g of soil in 17.74ml of liquid medium (A, B, and C) in Erlenmeyer flasks at 37 °C andaerobic conditions was carried out. The medium A had the following composition: NaCl(97.5 g/l), MgCl2.6H2O (32.5 g/l), MgSO4.7H2O (50.8 g/l), CaCl2 (0.8 g/l), KCl (5.0 g/l),NaHCO3 (0.16 g/l), NaBr (0.6 g/l), Na2CO3 (17.4 g/l), yeast extract (5.0 g/l), and traceelements solution (0.4ml/l) (Ochsenreiter et al. 2002). pH in medium A was adjusted to10 with Na2CO3. The medium B had the following composition: NaCl (100 g/l),MgSO4.7H2O (0.24 g/l), CaSO4.2H2O (0.17 g/l), KCl (1.0 g/l), Na2CO3 (5.0 g/l), yeastextract (5.0 g/l), casamino acids (5.0 g/l), and trace elements solution (1.0ml/l). Themedium C had the following composition: stock solution 30%: NaCl (240 g/l),MgCl2.6H2O (30.0 g/l), MgSO4.7H2O (35.0 g/l), KCl (7.0 g/l), NaHCO3 (0.2 g/l), NaBr(0.8 g/l) (400ml/l), distilled water (567ml/l), peptone (5.0 g/l), yeast extract (1.0 g/l). pHin medium B and medium C was adjusted to 9. Media and growth conditions were asdescribed by Valenzuela et al. (2008). Dilutions were performed from 10�1 to 10�6 of thesamples in preincubation taken from 3,7,14, and 21 days of incubation. Pure cultures wereobtained by picking and restreaking individual colonies grown in Petri plates solidifiedwith 2% agar at a temperature of 37 °C for 48 h.

DNA extraction

Genomic DNA was extracted according to Guo et al. (1997) with modifications as fol-lows: pure colonies were transferred from solid medium to TE 80/20 solution (Tris-HCl1M – EDTA 0.5M) centrifuging and resuspending the organic material in lysis buffer(Tris-HCl pH 8, 20mM, EDTA 2mM, Triton X-100, 20mg/ml lysozyme). After incu-bation of cells at 37 °C during 60–120min, both proteinase-K (0.2mg/ml) and 20 μlSDS 10% were added. Then the sample was incubated at 56 °C for 60–120min; subse-quently a solution of TE and NaCl 5M (10/1, V/V) was added stirring carefully. Thelysate was extracted with buffered phenol, followed by an extraction with chloroform:isoamyl alcohol (24:1), and washed with ether. Genome DNA was precipitated with iso-propanol (98%) at room temperature and washed with ethanol 70% (w/v). Finally,genomic DNA was resuspended in 40 μl of sterile water.

PCR amplification of rDNA genes

Purified DNA was used as template for the amplification of bacteria 16S rRNA genesvia PCR. The reaction mixture (25 μl) contained 1μlof genomic DNA; the appropriateprimers (27F and 1,492R) at 0.5 μM each one; dATP, dCTP, dGTP, and dTTat 10mMeach one; 50 mMMgCl; and 1 U of Taq DNA polymerase in the PCR buffer providedby the manufacturer (Invitrogen). The specific primers were 27F (5′-AGA GTT TGATCM TGG CTC AG-3′) and 1,492R (5′-TAC GGY TAC CTT GTT ACG ACT T-3′)(Todd et al. 2007).Amplification conditions included denaturation of the sample for10min at 94 °C followed by 1min of annealing at 57 °C and elongation during 2min at72 °C for a total number of 35 cycles (Valenzuela-Encinas et al. 2009). The amplifica-tion was performed in a Touchgene Gradient thermal cycler FTGRAD2D (TECHNEDUXFORT, Cambridge, UK).

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DNA purification and sequencing of 16S rDNA

PCR products were purified by PCR Purification Kit (250) QIAquick. Sequencing of16S rDNA was carried out in Macrogen In., Korea.

Phylogenetic analyses

Sequences were subjected to BLAST search (Altschul et al. 1997) and RDPAnalysisTools of Ribosomal Database Project-II Release 9 (http://rdp.cme.msu.edu/index.jsp) todetermine taxonomic hierarchy. Multiple alignment analyses were performed withCLUSTAL X (Thompson et al. 1997) selecting related sequences from the NCBITaxonomy Homepage (TaxBrowser) and Ribosomal Database Project-II. Only common16S rDNA regions and parsimony informative sites were included in the analysis. Allalignment gaps were treated as missing data. The transversion/transition weighting usingthe Tamura–Nei model (Tamura & Nei 1993) and the number of bases substitution 355between each pair of sequences were estimated using MEGA v. 4.0 (Kumar et al.2001). Phylogenetic trees were constructed using the Neighbor-joining method; andTamura-Nei model of distance analysis and 500 Bootstrap replications were assessed tosupport internal branches. Sequences that differed by less than 3% were considered tobelong to the same phylotype (Stackebrandt & Goebel 1994). Sequences with similitudepercentages below 95% were assigned to the closest family (Rossello-Mora & Amman2001).

Access numbers of the nucleotide sequences (http://www.ncbi.nlm.nih.gov)

Genus Nesterenkonia: AY226510, EF680886, AJ290397, EF601724, X80747,AY226508, AY928901, AY588278.Genus Micrococcus: KLH-29, DQ660308, FJ423763, AM158920, AB247644,NR037113, HQ285773.Genus Kocuria: HQ018931, AY881237, DQ979377, AY987383, AJ278867,DQ059617, HQ331530, HM209734, EU660350.Genus Halomonas: DQ289060, AF054286, AM229317, EF613114, EF613110,EF622233, AF212218, AM229315, EF144148, HVU85871, AF212204, AY245449,AJ564880, AB354933, AF212202.Genus Salinicoccus: DQ989633, EF177692, EF590121, DQ471329, EF397606,AY028927, GU397409, AF237976, AY328901, DQ352839.Genus Kurthia: GU586148, AB271741, AB271740.Genus Gracilibacillus: AB101591, EU709020, EU784646, AF036922.Genus Bacillus: HQ893539, DQ026060, AB043851, AB018595, AB086897, NR-036,889, AB043846, AJ605772, Z48306, AJ493660.

Results and discussion

In this study, 66 strains from the former Texcoco Lake were isolated and analyzed byweb-based phylogenetic tools; the analysis provided the classification of the isolatedstrains from soil samples in the following eight genera: Kocuria, Micrococcus,Nesterenkonia, Halomonas, Salinicoccus, Kurthia, Gracilibacillus, and Bacillus(Figure 1). Table 1 shows micro-organism isolated using different media. Medium B waswhere more strains were isolated (75%), which shows this micro-organism does not

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H33(111)N32

HJ021(1)HJ01

Nesterenkonia xinjiangensis AY22651OH3(22)HJ02

N31Nesterenkonia flava EF680886Nesterenkonia lacusekhoensis AJ290397

Nesterenkonia terrae EF601724Nesterenkonia halobia X80747H51(05)

Nesterenkonia halotolerans AY226508Nesterenkonia jeotgali AY928901H222(11)Nesterenkonia lutea AY588278

Micrococcus antarticus KLH-29

Micrococcus chenggongenseDQ660308Micrococcus terreus FJ423763

Micrococcus indicus AM158920Micrococcus thailandicus AB247644H261(29)Micrococcus luteusNR037113Micrococcus yunnanensisHQ285773N34

Kocuria assamensisHQ018931Kocuria palustris AY881237

Kocuria himachalensisAY987383Kocuria halotoleransDQ979377

Kocuria polarisAJ278868Kocuria aegyptia DQ059617Kocuria flava HQ331530

HJ014Kocuria turfanensis HM209734Kocuria rosea EU660350

Halomonas campisalisDQ289060Halomonas campisalis

H222(16)Halomonas denitrificansAM229317

HJ016(2)HJ016(1)HJ017(1)

Halomonas nitroreducensEF613114HJ012(2)HJ010Halomonas cerina EF613110HJ04

H3(16)Halomonas andensisEF622233Halomonas hydrothermalis AF212218

Halomonas janggokensisAM229315

Halomonas variabilisHVU85871Halomonas subterraneaEF144148

Halomonas sulfidaerisAF212204N33Halomonas boliviensisAY245449H444(18)Halomonas alkantarcticaAJ564880Halomonas taeheungiiAB354933Halomonas neptunia AF212202

A52(08)Salinicoccus halodurans

Salinicoccus luteus

Salinicoccus albus

Salinicoccus siamensisEF397606

Salinicoccus halophilusSalinicoccus jeotgali

Salinicoccus hispanicusAY028927HJ023Salinicoccus alkaliphilusHJ026(121)

Salinicoccus roseus AF237976N131N244

Salinicoccus marinus

N132Kurthia gibsonii GU586148Kurthia sibirica AB271741

A53(08)Kurthia zopfii AB271740

Bacillus firmus HQ893539

Bacillus okhensisDQ026060Bacillus wakoensisAB043851

Bacillus alcaliinulinusAB018595Bacillus krulwichiae AB086897Bacillus alcalophilusNR_036889

Bacillus hemicellulosilyticusAB043846

HJ019

H2312(14)

HJ012(1)H431(25)

H223(16)H2311(11)H133(16)H22

Bacillus saliphilusAJ493660

Bacillus vedderi Z48306Bacillus aurantiacusAJ605772

Gracilibacillus dipsosauri

Gracilibacillus ureilyticus EU709020Gracilibacillus saliphilusGracilibacillus halotoleransAF036922

Actinobacteria

Firmicutes

Acidianus convivatorAJ634763

HJ09

Proteobacteria

2724

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H33(111)N32

HJ021(1)HJ01

AY22651OH3(22)HJ02

N31Nesterenkonia flava

Nesterenkonia terrae EF601724Nesterenkonia halobia X80747H51(05)

Nesterenkonia halotolerans AY226508Nesterenkonia jeotgali AY928901H222(11)Nesterenkonia lutea AY588278

Micrococcus antarticus KLH-29

Micrococcus chenggongenseMicrococcus terreus FJ423763

Micrococcus indicus AM158920Micrococcus thailandicus AB247644H261(29)Micrococcus luteusNR037113Micrococcus yunnanensisN34

Kocuria assamensisKocuria palustris AY881237

Kocuria himachalensisAY987383Kocuria halotoleransDQ979377

Kocuria polarisAJ278868Kocuria aegyptia DQ059617Kocuria flava HQ331530

HJ014Kocuria turfanensis HM209734Kocuria rosea EU660350

Halomonas campisalisHalomonas campisalis AF054286

H222(16)Halomonas denitrificansAM229317

HJ016(2)HJ016(1)HJ017(1)

Halomonas nitroreducensEF613114HJ012(2)HJ010Halomonas cerina EF613110HJ04

H3(16)Halomonas andensisEF622233Halomonas hydrothermalis AF212218

Halomonas janggokensisAM229315

Halomonas variabilisHVU85871EF144148

Halomonas sulfidaerisAF212204N33Halomonas boliviensisAY245449H444(18)Halomonas alkantarcticaAJ564880Halomonas taeheungiiAB354933Halomonas neptunia AF212202

A52(08)Salinicoccus halodurans DQ989633

Salinicoccus luteus DQ352839

Salinicoccus albus EF177692

Salinicoccus siamensis

Salinicoccus halophilus EF590121Salinicoccus jeotgali DQ471329

Salinicoccus hispanicusHJ023Salinicoccus alkaliphilus GU397409HJ026(121)

Salinicoccus roseus AF237976N131N244

Salinicoccus marinus AY328901

N132Kurthia gibsonii GU586148Kurthia sibirica AB271741

A53(08)Kurthia zopfii AB271740

Bacillus firmus HQ893539

Bacillus okhensisDQ026060Bacillus wakoensisAB043851

Bacillus alcaliinulinusAB018595Bacillus krulwichiae AB086897Bacillus alcalophilusNR_036889

Bacillus hemicellulosilyticusAB043846

HJ019

H2312(14)

HJ012(1)H431(25)

H223(16)H2311(11)H133(16)H22

Bacillus saliphilusAJ493660

Bacillus vedderi Z48306Bacillus aurantiacusAJ605772

Gracilibacillus dipsosauri AB101591

Gracilibacillus ureilyticusGracilibacillus saliphilus EU784646Gracilibacillus halotoleransAF036922

Actinobacteria

Firmicutes

Acidianus convivatorAJ634763

HJ09

Proteobacteria

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Figure 1. Phyhlogenetic relation between the 16S rDNA sequences obtained from the soil of theformer Texcoco Lake. The tree was constructed with related sequences obtained from NCBIdatabase by using the neighbor-joining algorithm. Acidianus convivator (Accession numberAJ634763) was used as the out group.

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require specific nutrient. Table 2 shows the allocation of 22 out of 66 haloalkaliphilic iso-lated strains which came from the species assignment methodology according to(Rosello-Mora & Amman 2001).

Strains HJ019 and A53(08) corresponded to Gracibacillus and Kurtia, respectively.These strains may represent new species in these genera. Therefore, additional analysessuch as biochemical and chemotaxonomic characterization would continue in order toconfirm the latter. There are diverse applications of micro-organisms in human activi-ties, therefore it is important to discover new species of organisms that can be studiedand classified for potential industrial uses.

Research has been conducted on the isolation of new extremophile micro-organismsfrom soil of extreme conditions in order to get their extremozymes, which can beapplied to industry. For example, halophilic bacterial diversity was comprehensivelyreviewed by Grant et. al. (1998), Aerobic Gram-negative organotrophic bacteria thatare abundant in saline medium are species of Acinetobacter, Alteromonas, Deleya,Flavobacterium, Marinomonas, Pseudomonas, and Vibrio. Species of generaMarinococcus, Sporosarcina, Salinicoccus, and Bacillus have been isolated from salinesoils and salterns (Quesada et al. 1982); (Rodriguez-Valera 1988); (Dassarma & Arora2001). Recently, Ramirez et al. (2006) demonstrated the presence of halophilicactinomycetes in different geographical regions of Mexico; with reference to speciesalkaliphilic and halophilic bacteria of the genera Bacillus and Halomonas. Valenzuela-Encinas et al. (2009) reported bacterial populations of highly alkaline saline soil of theformer Texcoco Lake such as Halomonas, organisms that belong to the actinomycetalesand firmicutes order. Recent developments in the isolation and sequencing of environ-mental DNA samples have indicated that only a small fraction, probably between 0.1

Table 1. Isolated micro-organisms using different media.

Medium Micro-organism

A N34N31N131N244

B HJ014H261(29)H222(11)H3(22)HJ02HJ026(122)H444(18)H3(16)H222(16)HJ017HJ016(2)H431(25)H2311(11)H133(16)HJ09HJ026(121)HJ019HJ023

C A52(08)A53 (08)

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and 1%, of organisms from the environment can be obtained in pure culture. The acti-nomycetes included the genera Nesterenkonia, Micrococcus, and Kocuria, which coin-cides with the results obtained in this study.

The interest in obtaining extremozymes has been recently aroused due to theirpotential applications in industries such as paper, food, detergents, waste, and petro-leum. Halophilic extremozymes such as amylases and xylanases showed stability athigh salt concentration (Sanchez-Porro et al. 2003); (Gomes & Steiner 2003). Amy-lases, xylanases, lipases, and cellulases have been obtained from micro-organisms ofthe genera isolated in this study (Ilori et al. 1997; Bakhtiar et al. 2003; Gupta et al.2003; Rohban et al. 2009; Poosarla et al. 2010). The haloalkaliphilic micro-organismsrepresent a potential source to obtain extremozymes.

Staphylococcaceae, Planococcaeae, and Bacillaceae families belong to the orderof the Bacillales (Skerman et al. 1980), where the following genera can be found:Salinicoccus, Kurthia, Bacillus, and Gracilibacillus. The genus Halomonas belongs tothe family of Halomonadaceae, which was comprehensively reviewed by Arahal andVentosa (Arahal & Ventosa 2006). In conclusion, this work shows that there is a widevariety of novel micro-organisms in extreme environments such as that from the formerTexcoco Lake. The latter represents an open field for studies on metabolites of interestat the industrial level.

ReferencesAltschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped

BLAST and PSI-BLAST: a new generation of protein database search programs. NucleicAcids Res. 25:3389–3402.

Table 2. Species assigned for isolated strains based on % of similarity.

Micro-organism Species comparison % similarity

HJ014 Kocuria turfanensis 99.2H261(29) Micrococcus yunnanensis 99.9N34 Micrococcus yunnanensis 99.6H222(11) Nesterenkonia sandarakina 99.6H3(22) Nesterenkonia xinjiangensis 99.4N31 Nesterenkonia xinjiangensis 99.0HJ02 Nesterenkonia xinjiangensis 98.4HJ026(122) Nesterenkonia halobia 97.9H444(18) Halomona taeheungii 99.6H3(16) Halomona frigidi 98.7A52(08) Halomona venusta 98.7H222(16) Halomona nitritophilus 99.1HJ017 Halomona pantelleriense 98.2HJ016(2) Halomona pantelleriense 98.2H431(25) Bacillus saliphilus 97.8H2311(11) Bacillus saliphilus 97.6H133(16) Bacillus saliphilus 97.7HJ09 Bacillus firmus 98.9HJ026(121) Salinicoccus alkaliphilus 98.9N131 Salinicoccus roseus 99.8N244 Salinicoccus roseus 99.4HJ023 Salinicoccus hispanicus 99.6

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