perchlorate-reducingbacteriafromhypersalinesoilsofthe

Research ArticlePerchlorate-Reducing Bacteria from Hypersaline Soils of theColombian Caribbean

Rosa Acevedo-Barrios12 Angela Bertel-Sevilla1 Jose Alonso-Molina 3

and Jesus Olivero-Verbel 1

1Environmental and Computational Chemistry Group School of Pharmaceutical Sciences Zaragocilla CampusUniversity of Cartagena Cartagena 130015 Colombia2Biological and Chemical Studies Group School of Basic Sciences Technological University of Bolıvar Cartagena 130010Colombia3Research Institute of Water and Environmental Engineering Universitat Politecnica de Valencia Valencia 46022 Spain

Correspondence should be addressed to Jesus Olivero-Verbel joliverovunicartagenaeduco

Received 24 September 2018 Revised 24 November 2018 Accepted 11 December 2018 Published 17 February 2019

Academic Editor Hidetoshi Urakawa

Copyright copy 2019 Rosa Acevedo-Barrios et al 0is is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Perchlorate (ClO4minus) has several industrial applications and is frequently detected in environmental matrices at relevant con-

centrations to human health Currently perchlorate-degrading bacteria are promising strategies for bioremediation in pollutedsites 0e aim of this study was to isolate and characterize halophilic bacteria with the potential for perchlorate reduction Tenbacterial strains were isolated from soils of Galerazamba-Bolivar Manaure-Guajira and Salamanca Island-Magdalena ColombiaIsolates grew at concentrations up to 30 sodium chloride 0e isolates tolerated pH variations ranging from 65 to 120 andperchlorate concentrations up to 10000mgL Perchlorate was degraded by these bacteria on percentages between 25 and 10 16SrRNA gene sequence analysis indicated that the strains were phylogenetically related to Vibrio Bacillus Salinovibrio Staphy-lococcus and Nesiotobacter genera In conclusion halophilic-isolated bacteria from hypersaline soils of the Colombian Caribbeanare promising resources for the bioremediation of perchlorate contamination

1 Introduction

Perchlorate pollution is a problem of global impact becauseit has a negative effect on ecosystems with a loss of envi-ronmental quality which increases with anthropogenicactivity Perchlorate is an ubiquitous emerging contaminantproduced from both natural and anthropogenic sources [1]particularly present in areas associated with the use andmanufacture of rockets and ammunition 0is compound isa potent endocrine disruptor that mainly affects the fixationof iodine by the thyroid gland responsible for regulatingmetabolism growth and development [2 3] thus beingdangerous to infants and young children [4] Acute short-term exposure has been shown to affect the nervous re-spiratory immune and reproductive systems [5] It has also

been related to thyroid cancer and teratogenesis during thefirst trimester of pregnancy [6]

Perchlorate is highly distributed in ecosystems and or-ganisms thus it is frequently found in several matricesincluding breast milk fertilizers plants food and humantissues 0is scenario has led to the prioritization of studiesthat allows for the removal of this contaminant from pol-luted sites as it is extremely toxic and persistent thereforeefficient treatments for its degradation are needed in order tomaintain the quality of soils from biodiversity hotspots0ere are different physicochemical techniques commonlyused for the environmental removal of this anion such asion exchange but it is not selective and the process usuallyonly separates the perchlorate from contaminated sources[7] also generating by-products which requires subsequent

HindawiInternational Journal of MicrobiologyVolume 2019 Article ID 6981865 13 pageshttpsdoiorg10115520196981865

treatment [7] Perchlorate is persistent but possesses bio-degradability [8] However enzymes such as the perchloratereductase and superoxide chlorite carry out the reduction orelimination of perchlorate A reductase enzyme is re-sponsible for reducing perchlorate to chlorate and chlorateto chlorite while the enzyme superoxide chlorite changeschlorite to chloride and molecular oxygen Biological re-duction through the use of bacteria completely degrades theperchlorate ions into Clminus and O2 (equation (1)) [9ndash11] 0eperchlorate degradation pathway is as follows

ClO4minus(perchlorate)⟶ ClO3

minus( 1113857(chlorate)

⟶ ClO2minus

( 1113857(chlorite)⟶ Clminus(chloride) + O2(1)

Marine soils usually contain bacterial species with bio-chemical versatility and ability to tolerate salt being aninteresting target for researchers due to the potential re-duction of environmental perchlorate [10] 0e reason forselecting this type of environment is that degradation ofperchlorate may be carried out using salt-tolerant bacteria[12] although this perchlorate-reduction process could beimpaired with increasing salinity [11 13] Moreover theseorganisms are available in diverse environments fromAntarctica saline lakes and hot springs even in hyper-thermophilic and hypersaline soils [10 11] Additionallyperchlorate deposits in these environments may be formedby chemical reactions between sodium chloride from land orsea and ozone [14]

Pilot testing of biotechnologies using perchlorate-reducing bacteria has been studied and tuned in suspen-sion fixed-bed fluidized and biofilm reactors [15 16]evaluating the effectiveness of treatments on soil and watercontaminated with perchlorate [15] Nowadays the use ofintegrated systems combining physicochemical treatmentsand perchlorate-reducing halophilic bacteria is beingstudied to increase the efficiency of water treatmentallowing the handling of residual flows with high salinity andlarge perchlorate concentrations simultaneously solving thewaste disposal problem [17]

Colombia has a variety of ecosystems with differentclimatic and biogeochemical features which facilitates thedevelopment of different halophilic bacteria 0e aim of thisstudy was the characterization of perchlorate-reducingbacteria recovered from hypersaline soils of the Colom-bian Caribbean

2 Materials and Methods

21 Study Area and Sample Collection Soil samples wereobtained from salt mines of Colombia specifically fromGalerazamba-Bolivar (10deg47prime21PrimeN 75deg15prime41PrimeW) Manaure-Guajira (11deg46prime30PrimeN 72deg26prime40PrimeW) and saline soils locatedin Salamanca Island-Magdalena (10deg56prime00PrimeN 75deg15prime00PrimeW)(Figure 1) A sterile spatula was employed to collect ap-proximately 100 g of soil from the upper 1ndash10 cm layer inMay 2015 All samples were collected in 50mL Falcon tubeskept refrigerated at 4degC and taken to the laboratory forprocessing Salinity and pH were recorded for each sampleaccording to Nozawa-Inoue et al [18]

22 Isolation of Strains and Culture Conditions Isolationpurification and preservation techniques were used as de-scribed by Shimkets and Rafiee [19] Samples were treatedwith amphotericin B (025mgmL) for 3 h until inoculation(50 microL) in the isolation media Subsequently samples wereincubated in Petri dishes containing modified sterilized agarLuria-Bertani in seawater (LB NaCl) [20] and incubated at37degC for 24 h under aerobic conditions [21] Bacterial growthwas monitored by observation of colonies For preservationa bacterial colony was transferred to modified LB broth andincubated at 37degC aerobically during 12 h adjusting the celldensity to 05 Mc-Farland turbidity standard 0en 720 microLof each culture was transferred to a Cryovial with 80 microLglycerol and stored at minus80degC

23 Molecular Identification

231 16S rRNA Gene Sequencing and Phylogenetic AnalysisFor 16S rRNA gene amplification genomic DNA wasextracted using a QIAampreg DNA Mini Kit (Qiagen CAUSA) as described by the manufacturer 0e 16S rRNAgene was amplified from the total genomic DNA of thebacterial strains by PCR using the forward primer PF (5prime-AGAGTTTGATCCTGGCTCAG-3prime) and the reverse primer1492R (5prime-ACCTTGTTACGACTT-3prime) [22 23] PCR wasperformed with an AmpliTaq Goldreg 360 Master Mix(Applied Biosystems) according to the manufacturerrsquos in-structions Each 25 μL reaction mixture contained 04 μM ofeach primer and sim100 ng of template DNA 0e amplifi-cation was performed as follows initial denaturation for10min at 95degC 25 cycles of denaturation for 1min at 94degCannealing for 1min at 43degC and extension for 15min at72degC and a final extension for 55min at 72degC All PCRproducts were checked by electrophoresis on 12 (wv)agarose gels stained with ethidium bromide (10mgmL) andanalyzed using a gel documentation system (IngGenius 3System-Syngene)

0e PCR product was purified with a QIAquick PCRpurification kit (Qiagen CA USA) following the manu-facturerrsquos instructions Automated DNA sequencing wasperformed by the National Center for Genomic Sequencing(CNSG) (Medellin-Colombia) using PF and 1492Rprimers Sequence readings were edited and assembledwith the CAP3 software [24] 0e resulting 16S rRNA genesequences were compared to reference strains with validlypublished names using the EzTaxon-e server (httpwwwezbiocloudneteztaxon) After multiple alignments ofthe data via CLUSTAL_W four methods includingneighbor joining (NJ) [25] maximum likelihood (ML)[26] minimum evolution (ME) and maximum parsimony(MP) [27] were used to perform phylogenetic analysisPhylogenetic trees were constructed using MEGA version6 [28] Distances were calculated using the Kimura cor-rection in a pair-wise deletion manner [29] 0e topologiesof the phylogenetic tree were evaluated by the bootstrapresampling method described by Felsenstein [30] with1000 replicates 0e GenBankEMBLDDBJ accessionnumbers for the 16S rRNA gene sequences of isolated

2 International Journal of Microbiology

strains BBCOL-031 BBCOL-023 BBCOL-024 BBCOL-025 BBCOL-026 BBCOL-027 BBCOL-028 BBCOL-029BBCOL-032 and BBCOL-033 are KX821664ndashKX821673respectively

24 Morphological Characterization Strains were incubatedon LB NaCl agar and both growth and morphogenesis wereobserved under light microscopic examination (Olympusmicroscope BX41) Gram staining was conducted followingBergeyrsquos Manual Taxonomic Key [31] and Konemanrsquos Mi-crobiologic Atlas [32] Samples were further processed bymeans of scanning electron microscopy (SEM) visualizationas previously described [33]

25 Biochemical Characterization Biochemical character-istics were investigated using the BBL Crystaltrade Kit (BectonDickinson Microbiology Systems Cockeysville USA) asdescribed by the manufacturer Catalase and oxidase testswere performed according to the reported methods [21]

26 Chloride Susceptibility Assay All strains were assayedfor perchlorate susceptibility in LB broth in the presence ofNaCl (35 50 75 and 30 wv) [20 34] e experimentsstarted adding 20 microL of cell suspension OD 06 into 5mLLB broth e cultures were incubated at 37degC for 24 h andafter that strains showing turbidity [35] were identied asresistant to NaCl e experiments were carried out bytriplicates and performed three times

27 Perchlorate Susceptibility Assay All strains were assayedfor perchlorate susceptibility in LB broth in the presenceof perchlorate at concentrations of 100 250 500 750 10001250 1500 2000 3000 5000 7500 and 10000mgL[20 36 37] e experiments were carried out as de-scribed for the chloride susceptibility assay After the in-cubation time (24 h) a culture of each isolate was tested on

LB agar at their corresponding KClO4 concentrations toconrm cell viability and purity of each of the bacterialstrains

28 Evaluation of Perchlorate Reduction by Isolates eexperiments were carried out using concentration of10000mgL KClO4 in LB with 35 NaCl inoculatingwith the isolates as described in the chloride susceptibilityassay and incubating for a 24-hour time period at 37degCAfter incubation time the nal KClO4 concentration wasmeasured with a ermo Scientic Orion 93 perchlorateelectrode (ermo Fisher Scientic Inc Beverly MA) usedaccording to the manufacturerrsquos instructions e diiexclerencein concentration after and before the incubation wasemployed to calculate the perchlorate reduction percentageelicited by each strain

3 Results and Discussion

31 Molecular Identication Almost-complete 16S rRNAgene sequences were obtained from strains BBCOL-023to BBCOL-033 (GenBank accession numbers KX821664ndashKX821673) e results of the phylogenetic analysis showedthat these strains belonged to members of Bacillus VibrioSalinivibrio Nesiotobacter and Staphylococcus StrainBBCOL-023 presented 99 similarity with Nesiotobacterstrains BBCOL-024 BBCOL-028 BBCOL-029 and BBCOL-033 had 99 homology with Bacillus species and strainsBBCOL-025 BBCOL-026 BBCOL-027 and BBCOL-031had 99 homology with members of the Vibrionaceaefamily particularly Vibrio and Salinivibrio species StrainBBCOL-032 presented 99 similarity to Staphylococcus spp

e 16S rRNA gene sequence had similar values betweenstrains BBCOL-024 BBCOL-028 BBCOL-029 and BBCOL-033 and validly named type strains of Bacillus were cal-culated by using the EzTaxon-e server Strains BBCOL-024and BBCOL-028 showed high 16S rRNA gene sequence

Manure

Salamanca island

GalerazambaLongitude

72deg 26prime 40Prime W



11deg 4

6prime 3

0Prime N

10deg 5

6prime 0

0Prime N

Latti

ude

10deg 4

7prime 2

1Prime N

Figure 1 Map of the Caribbean region of Colombia showing the geographic location of the sampling sites

International Journal of Microbiology 3

similarities with Bacillus vallismortis DV1-F-3(T) (996 and995 respectively) Bacillus subtilis subsp inaquosorumKCTC 13429(T) (996 and 994 respectively) and Bacillussubtilis subsp spizizenii NRRL B-23049(T) (996 and 994respectively)

Levels of 16S rRNA gene sequence similarity betweenstrains BBCOL-024 and BBCOL-028 and other currentmembers of the genus Bacillus were below 990 In theneighbor-joining (Figure 2) and the minimum-evolutionphylogenetic dendrograms based on 16S rRNA gene se-quences strains BBCOL-024 and BBCOL-028 were placed ina cluster in Bacillus and were shown to be closely related to Bvallismortis B subtilis subsp spizizenii TU-B-10 B vanilleaand B atrophaeus

Strain BBCOL-029 shares high-sequence similaritywith B oryzaecorticis R1 (T) B haikouensis C-89(T) Baquimaris TF-12(T) and B vietnamensis 15-1(T) with 982979 979 and 979 respectively and nucleotide differencesof 20 29 30 and 29 nucleotides respectively 0e com-parative analysis of 16S rRNA gene sequences and phylo-genetic relationships showed that the BBCOL-029 strain liesin a subclade in the tree with B marisflavi B aquimaris andB vietnamensis (supported by a bootstrap value of 77(Figure 2)) with which it shares the highest 16S rRNAgene sequence similarity 0e affiliation of strain BBCOL-029 and its closest neighbors was also supported by themaximum parsimony and maximum likelihood algorithmswith bootstrap values above 70 EzTaxon-e server searchanalysis revealed that the BBCOL-033 strain is closely relatedto B flexus FO 15715(T) (997 16S rRNA gene sequencesimilarity) B paraflexus RC2 (T) (99) B megateriumNBRC 15308ATCC 14581(T) (985) and other bacilli(lt97) 0e sequence similarities of strain BBCOL-033 withB flexus using different clustering algorithms (100 in NJtree 100 in ME tree and 100 in ML tree) along with theEzTaxon-e server analysis consistently indicated that Bflexus is the closest relative

For strain BBCOL-023 1329 nt of the 16S rRNA genesequence was determined Comparative 16S rRNA genesequence analysis showed that strain BBCOL-023 wasmost closely related to members of the genus NesiotobacterStrain BBCOL-023 shares the highest sequence similaritywith Nesiotobacter exalbescens LA33B (T) Roseibiumaquae DSG-S4-2 (T) and Pseudovibrio hongkongensisUST20140214-015B (T) with 998 959 and 957 andnucleotide differences of 3 55 and 57 respectively 0e 16SrRNA gene sequence similarities to the type strains of othermembers of the family Rhodobacteraceae with validlypublished names were below 94 In the phylogenetic treebased on the NJ algorithm (Figure 3) strain BBCOL-023formed a single clade with Nesiotobacter exalbescens (sup-ported by a bootstrap value of 100 (Figure 3)) with whichit shares the highest 16S rRNA gene sequence similarity 0eaffiliation of strain BBCOL-023 and its closest neighbors wasalso supported by the MP and ML algorithms with above90 bootstrap values

0e 16S rDNA sequences of strains BBCOL-025BBCOL-026 BBCOL-027 and BBCOL-031 determinedin this study comprised 1402 1361 1399 and 1389 nt

respectively representing approximately 90 of theEscherichia coli 16S rRNA sequence 0e results of phylo-genetic analysis of the 16S rRNA gene sequences revealedthat the isolated strains were related phylogenetically tomembers of the Vibrionaceae family and belong within thephyletic group classically defined as the genus Salinivibrioand Vibrio (Figure 4) Strain BBCOL-025 shows high 16SrRNA gene sequence similarities with Salinivibrio costicolasubsp vallismortis DSM 8285(T) (984) Salinivibrio cos-ticola subsp costicola ATCC 33508(T) (978) and Sali-nivibrio proteolyticus AF-2004(T) (977) Levels of 16SrRNA gene sequence similarity between strain BBCOL-025and the other current members of the genus Salinivibrio arebelow 97 In the neighbor joining (Figure 4) and theminimum evolution phylogenetic dendrograms based on16S rRNA gene sequences strain BBCOL-025 was placed ina cluster in the Salinivibrio genus and was shown to beclosely related to S budaii and S costicola subsp alcaliphilus(supported by a bootstrap value of 75) 16S rRNA genesequence comparison between the BBCOL-026 strain andother members from the Vibrionaceae family by using theEzTaxon-e server indicated that the strain was closely relatedto members of Vibrio genus showing 992 gene sequencesimilarity to V antiquarius Ex25 (T) 99 to V alginolyticusNBRC 15630(T) 98 to V neocaledonicus NC470 (T) and989 to V natriegens DSM 759(T) Likewise strainsBBCOL-027 and BBCOL-031 show high 16S rRNA genesequence similarities with V alginolyticus NBRC 15630(T)(998 and 989 respectively) and V antiquarius Ex25(T)(995 and 989 respectively) A phylogenetic tree gener-ated from the neighbor joining algorithm showed thatstrains BBCOL-026 and BBCOL-031 both fell within theradiation of the cluster comprising Vibrio species andformed a coherent cluster that is supported by a bootstrapanalysis at a confidence level of 98 (Figure 4) 0is clusterjoins the phylogenetic clade comprising V alginolyticusand V parahaemolyticus which is supported by a 71bootstrap value 0is topology was also found in treesgenerated with the ML and MP algorithms (not shown)0e NJ and ME phylogenetic trees based on 16S rRNA genesequence data showed that the strain BBCOL-027 forms acoherent cluster with Vibrio alginolyticus (a bootstrap valueof 72)

For strain BBCOL-032 1416 nt of the 16S rRNA genesequence was determined Comparative 16S rRNA genesequence analysis showed that strain BBCOL-032 was moreclosely related to the Staphylococcus species Strain BBCOL-032 shares highest sequence similarity with Staphylococcushaemolyticus ATCC 29970(T) (999) Staphylococcus pet-rasii subsp pragensis NRLSt 12356(T) and Staphylococcuspetrasii subsp jettensis SEQ110 (T) (992) 0e 16S rRNAgene sequence similarities to strains from other members ofthe Staphylococcaceae family were below 99 In thephylogenetic tree based on the NJ algorithm (Figure 5)strain BBCOL-032 fell within a coherent cluster comprisingS haemolyticus S petrasii subsp pragensis S petrasii subspjettensis and S lugdunensis 0e sequence similarities ofstrain BBCOL-032 with S haemolyticus using differentclustering algorithms (100 in NJ tree 99 in ME tree and


100 in ML tree) along with the EzTaxon-e server analysisconsistently indicated that S haemolyticus is the closestrelative

32 Microscopic and Biochemical Characterization 0ecolonies of strains (BBCOL-023 to BBCOL-033) on LB agar

were circular convex and smooth Cells were facultativeanaerobic with an optimal growth at pH 65 to 75 Mor-phological features are observed by SEM (Figure 6) andbiochemical characteristics of isolated bacteria strainsare shown in Table 1 Morphological and biochemicalcharacteristics detected in the isolated strain BBCOL-023correspond to the species Nesiotobacter as previously

Bacillus vanillea (KF986320)Bacillus aerius (AJ831843)

Bacillus vietnamensis (AB099708)Bacillus aquimaris (AF483625)

Bacillus marisflavi (AF483624)BBCOL 029 (KX821672)

Bacillus seohaeanensis (AY667495)Bacillus shackletonii (AJ250318)

Bacillus ginsengihumi (AB245378)Bacillus sporothermodurans (U49078)

Bacillus acidicola (AF547209)Bacillus firmus (X60616)

Bacillus flexus (AB021185)BBCOL 033 (KX821667)

Bacillus simplex (AB363738)Bacillus kochii (FN995265)

Bacillus hemicellulosilyticus (AB043846)Bacillus trypoxylicola (AB434284)

Bacillus pseudalcaliphilus (X76449)Bacillus alcalophilus (X76436)Bacillus bogoriensis (AY376312)

Bacillus krulwichiae (AB086897)Bacillus wakoensis (AB043851)

Bacillus okhensis (DQ026060)Bacillus akibai (HM768321)

Bacillus alkalisediminis (AM051268)Bacillus nanhaiisediminis (GQ292773)

Bacillus alkalinitrilicus (EF422411)Bacillus plakortidis (AJ880003)

Bacillus gibsonii (X76446)BacillusBacillus clausii (X76440)

Bacillus halodurans (AJ302709)Bacillus okuhidensis (AB047684)

Bacillus marmarensis (EU621902)Bacillus pseudofirmus (X76439)

Bacillus berkeleyi (JN187498)Bacillus decolorationis (AJ315075)

100

100

100

99

100

95

5498

71

58

89

76

99

100

42

42

54

30

25

31

69

44

92

7877

3757

67

45

88

32

31

49

5889

Bacillus subtilis subsp spizizenii TU-B-10 (CP002905)BBCOL 028 (KX821671)

Bacillus hwajinpoensis (AF541966)

89100

100

BBCOL 024 (KX821666)

Bacillus algicola (AY228462)Geobacillus stearothermophilus (AB021196)

955399

Bacillus atrophaeus (AB021181)Bacillus vallismortis (AB021198)88

001

Figure 2 Neighbor joining tree based on 16S rRNA gene sequences showing the phylogenetic position of strains BBCOL-024 BBCOL-028BBCOL-029 and BBCOL-033 compared to the most closely related members of the genus Bacillus Bootstrap values based on 1000replications are listed as percentages at the branching points Accession numbers are given in parentheses Bar 001 nucleotide substitutionsper nucleotide position


Vibrio alginolyticus (AB680916)BBCOL027 (KX821670)

Vibrio parahaemolyticus (AB497066)BBCOL031 (KX821664)

BBCOL026 (KX821669)Vibrio diabolicus (X99762)

Vibrio navarrensis (X74715)Vibrio orientalis (X74719)

Vibrio splendidus (AJ515220)Vibrio crosai (JQ434120)

Vibrio proteolyticus (X74723)Vibrio tubiashii (X74725)

Vibriofluvialis (AB681959)Vibrio brasiliensis (AJ316172)Vibrio nereis (X74716)

Vibrio vulnificus (X56582)Vibrio vulnificus (AB680930)

Vibrio neptunius (AJ316171)Vibrio coralliilyticus (AJ440005)

Vibrio halioticoli (AB000391)Vibrio cholerae (X76337)

Grimontia hollisae (X56583)Photobacterium phosphoreum (AJ746358)

Photobacterium halotolerans (AY551089)Salinivibrio siamensis (AB285018)

Salinivibrio proteolyticus (DQ092443)Salinivibrio costicola subsp vallismortis (AF057016)

Salinivibrio costicola (X95527)Salinivibrio costicola subsp alcaliphilus (AJ640132)

Salinivibrio budaii (AB617564)BBCOL025 (KX821668)

Pseudoalteromonas haloplanktis (X67024)Halomonas elongata (AJ295147)

100

7564

5785

100

94 66

86

100

93

81

98

98

48

63

727146

92

5627

36

61

22

33

6

401626

001

Figure 4 Neighbor joining tree based on 16S rRNA gene sequences showing the phylogenetic position of strains BBCOL-025 BBCOL-026BBCOL-027 and BBCOL-031 compared to the most closely related members of the Vibrionaceae family Bootstrap values based on 1000replications are listed as percentages at the branching points Accession numbers are given in parentheses Bar 001 nucleotide substitutionsper nucleotide position

Nesiotobacter sp H02E (GU213180)BBCOL 023 (KX821665)

Nesiotobacter exalbescens (AF513441)Roseibium denhamense (D85832)Roseibium hamelinense (D85836)Stappia indica (GU593615)

Stappia stellulata (D88525)Stappia taiwanensis (FR828537)

Rhodobium orientis (D30792)Phyllobacterium myrsinacearum (AB681130)

Defluvibacter sp QDZ-C (HQ890470)Aquamicrobium defluvii (Y15403)

Albidovulum inexpectatum (AF465833)Roseobacter litoralis (X78312)

Erythrobacter longus (AF465835)Vibrio furnissii (X76336)

100

67100

86100

98

99

99

93

99

87 69

53

001

Figure 3 Neighbor joining phylogenetic tree based on 16S rRNA gene sequences showing the relationships of strain BBCOL-023 andrelated taxa Percentage bootstrap values based on 1000 replications are given at branch points 0e gammaproteobacterium Vibrio furnissii(X76336) was used as the outgroup Bar 001 substitutions per nucleotide position


reported [38] 0e strains were able to utilize lactosemannose galactose sorbitol and sucrose as carbon sourcesNitrate was reduced to nitrite 0e strains were ONPG- andH2S-negative

Microscopic morphological results showed that isolatesBBCOL-024 BBCOL-028 BBCOL-029 and BBCOL-033correspond to the genus Bacillus [39] When grown at 37degCduring 24 h in LB the cells were Gram-positive bacilli witha size between 06-07 μm and 16-17 μm Endospores wereellipsoidal Cells were motile aerobic facultative anaero-bic and oxidase- and catalase-positive Optimal growthconditions of BBCOL-024 were at pH 60ndash65 and 37degC0ese isolates showed VogesndashProskauer and nitrate re-duction activity However they were ONPG- and H2S-negative

0e cells of strain BBCOL-025 Salinovibrio costicolawere Gram-negative motile nonsporulating and curvedpresenting an average bacterial size of 14times 07 microm In ad-dition these were motile facultative anaerobic and oxidase-and catalase-positive Optimal growth conditions forBBCOL-025 were at pH 60ndash65 and 37degC 0e isolatesshowed morphological differences regarding both curvedsize and growth 0ese differences may arise depending onenvironmental conditions at the sampling moment labo-ratory procedures and conservation techniques amongothers [40 41] 0ese strains presented positive activity forVogesndashProskeuer and nitrate reduction and negative activityagainst ONPG and H2S production

0e isolates BBCOL-026 BBCOL-027 and BBCOL-031 shared the main properties of the genus Vibrio 0ey

were motile curved facultative Gram-negative andoxidase-positive and were able to reduce nitrate to nitrite0ese bacteria also formed yellow colonies as reported byseveral authors [42 43] Optimal growth conditions forBBCOL-025 were at pH 60ndash65 and 37degC 0e isolatesshowed high phenotypical homogeneity although variablereactions were observed for aesculin hydrolysis N-acetyl-glucosaminidase activity and fermentation of galactoseand lactose

0e cells of strain BBCOL-032 Staphylococcus spp wereGram-positive motile and nonsporulated presenting anaverage bacterial size of 128times 068 microm Cells were facultativeand oxidase- and catalase-positive Optimal growth condi-tions for BBCOL-025 were at pH 60ndash65 and 37degC Isolatedstrains presented positive activity for the VogesndashProskauertest and nitrate reduction and showed negative activityagainst ONPG and H2S production as previously reported[44 45]

33 Sodium Chloride and Perchlorate Susceptibility AssayAll isolates showed growth in the culture medium with highconcentrations of NaCl reaching tolerance up to 30Strains BBCOL-023 to BBCOL-033 presented characteristicsof moderately halophilic bacteria according to what wasreported by Acevedo-Barrios [20]

0e ability of isolates to grow and tolerate concentrationsof KClO4 between 100 and 10000mgL is presented inTable 2 All isolates showed biofilm formation at the highestconcentration 0is barrier allows the bacteria to generate a

Staphylococcus saprophyticus (L37596)Staphylococcus saprophyticus subsp saprophyticus (AB681788)Staphylococcus xylosus (AB626129)

Staphylococcus gallinarum (AB726090)Staphylococcus succinus subsp succinus (JQ988944)

Staphylococcus kloosii (AB009940)Staphylococcus arlettae (AB009933)

Staphylococcus equorum (AB009939)Staphylococcus cohnii subsp cohnii (D83361)

Staphylococcus cohnii subsp urealyticus (AB009936)Staphylococcus nepalensis (AJ517414)Staphylococcus nepalensis (AB697719)

Staphylococcus lugdunensis (AB009941)Staphylococcus haemolyticus (AB626124)

BBCOL032 (KX821673)Staphylococcus petrasii subsp pragensis (KM873669)

Staphylococcus petrasii subsp jettensis (JN092111)Staphylococcus epidermidis (L37605)Staphylococcus aureus (D83356)

Staphylococcus simiae (AY727530)Staphylococcus felis (D83364)

Staphylococcus lutrae (AB233333)Staphylococcus schleiferi subsp coagulans (AB009945)

Staphylococcus delphini (AB009938)Bacillus subtilis (AB016721)

67

7194

9492

99

100

99

40

40

45

84

9290

41

1512

9545

2511

26

001

Figure 5 Neighbor joining phylogenetic tree based on 16S rRNA gene sequences showing the relationships of strain BBCOL-032 andrelated taxa Percentage bootstrap values based on 1000 replications are given at branch points Bacillus subtilis (AB016721) was used as theoutgroup Bar 001 substitutions per nucleotide position


concentration gradient as a means of protection against thetoxicity of this chemical [2]

Other aspects evidenced in these strains are associationsbetween NaCl and KClO4 tolerance 0ese isolates havebecome interesting targets for this research given the needto identify native bacteria with potential biotechnologicaland biochemical versatility capable of degrading environ-mental contaminants such as KClO4

34 Evaluation of Perchlorate Reduction by IsolatesPerchlorate-reducing bacteria are phylogenetically diverseand these include Alphaproteobacteria Betaproteobac-teria Gammaproteobacteria and Deltaproteobacteriaclasses with Betaproteobacteria being the most commonlydetected class [46 47] In this work bacterial strainsBBCOL-023 to BBCOL-033 (Figure 7) showed biological

capacity to reduce concentrations of KClO4 on percentagesbetween 10 and 25

0e genera Nesiotobacter and Salinivibrio showed thehighest percentage (25) of perchlorate reduction while thegenera Vibrio Bacillus and Staphylococcus presented alowest proportion of KClO4 reduction with 14 12 and 10respectively Recent studies have shown that the amount ofperchlorate reduced may be inversely proportional to in-creased salinity [13 17] Future studies should be carried outto describe the role of salinity on perchlorate reduction bythese strains

0e ability of bacteria to grow in perchlorate pollutedareas is determined by their degrading enzymes [48 49]0egeneral metabolic reduction pathway widely accepted byresearchers [9 10] involves the reductase enzyme as this isresponsible to reduce perchlorate to chlorate and chlorate tochlorite while the superoxide chlorite enzyme changes

Figure 6 Cell morphology studied by SEM Bacteria isolated from hypersaline soils (a) BBCOL-023 (b) BBCOL-024 (c) BBCOL-025 (e)BBCOL-027 (f ) BBCOL-028 (g) BBCOL-029 (h) BBCOL-031 (i) BBCOL-032 (j) BBCOL-033 (SEM at 10000x)


Tabl

e1

Morph

ological

andbiochemical

characteristicsof

isolatedstrains

Characteristics

BBCOL-

023

BBCOL-

024

BBCOL-

025

BBCOL-

026

BBCOL-

027

BBCOL-

028

BBCOL-

029

BBCOL-

031

BBCOL-

032

BBCOL-

033

Molecular

identifi

catio

nNesiotobacter

sp

Bacillu

svallism

ortis

Salin

ivibrio

costicola

Vibrio

algino

lyticus

Vh

arveyi

Bcohn

iiBa

cillu

sspp

Vibrio

sp

Staphylococcus

spp

Bflexu

s

Color

ofcolony

Beige

Yello

wYe

llow

Yello

wYe

llow

Yello

wYe

llow

Yello

wYe

llow

Yello

w

Morph

ology

Rodshaped

Rodshaped

Vibrioshaped

Vibrioshaped

Vibrio

shaped

Rod

shaped

Rod

shaped

Coco

shaped

Rodshaped

Rod

shaped

Leng

th(μm)

156

160

145

086

015

153

390

123

133

307

0ickn

ess(μm)

07

065

068

076

062

06

08

07

128

079

Motility

minusminus

+minus

minus+

++

minus+

Gram

straining

minus+

minusminus

minus+

minusminus

++

Endo

spore

minus+

minusminus

minus+

minusminus

minus+

Sporepo

sition

++

++

++

++

minusminus

Oxidase

++

++

W+

++

++

Catalase

minus+

minusminus

minus+

+minus

++

Arabino

seminus

++

++

++

++

+Manno

se+

minusminus

minusminus

minusminus

minusminus

minusSu

crose

++

++

++

++

++

Melibiose

++

++

++

++

++

Rham

nose

++

++

++

++

++

Mannitol

minusminus

++

++

++

++

Ado

nitol

minusminus

minusminus

minusminus

minusminus

minusminus

Galactose

++

++

++

++

++

Inosito

lminus

minusminus

minusminus

minusminus

minusminus

minusp-n-p-Ph

osph

ate

minusminus

+minus

+minus

minusminus

+minus

p-n-pa-szlig-Glucosid

eV

minusminus

minus+

minusV

minusminus

minusp-n-p-szlig-Galactosid

e+

++

++

minus+

++

+Prolinenitroanilid

eminus

minusminus

minus+

minusminus

minusminus

minusp-n-pbis-Ph

osph

ate

minusminus

minusminus

minusminus

minusminus

minusminus

p-n-p-Xyilosid

e+

++

++

minus+

++

+p-n-p-a-Arabino

side

++

++

+minus

++

++

p-n-p-Ph

osph

orylcholine

minusminus

minusminus

minusminus

minusminus

minusminus

p-n-p-szlig-Glucuronide

minusminus

minusminus

minusminus

minusminus

minusminus

p-n-p-N-A

cetylglucosamide

minusminus

minus+

+minus

minus+

minusminus

c-L-G

lutamyl-p-nitroanilid

eminus

minusminus

minusminus

minusminus

minusminus

minusAesculin

++

++

++

++

++

p-Nitro-DL-ph

enylalanine

minusminus

minusminus

minusminus

minusminus

minusminus

Urea

minusminus

minusminus

minusminus

minusminus

minusminus

Glycine

minusminus

minusminus

minusminus

minusminus

minusminus

Citrate

Vminus

W+

+minus

Vminus

W+

Malon

icacid

++

++

++

++

++

Tripheny

ltetrazoliumchloride

++

++

++

++

++

Lactose

++

++

++

++

++

Bacteriolytic

capacity

++

++

++

++

++

Cellulolytic

capacity

minusminus

minusminus

minusminus

minusminus

minusminus


Tabl

e1

Con

tinued

Characteristics

BBCOL-

023

BBCOL-

024

BBCOL-

025

BBCOL-

026

BBCOL-

027

BBCOL-

028

BBCOL-

029

BBCOL-

031

BBCOL-

032

BBCOL-

033

Nitrateredu

ction

++

++

++

++

++

Indo

leminus

minusminus

minusminus

minusminus

minusminus

minusONPG

minusminus

minusminus

minusminus

minusminus

minusminus

Ornith

ineutilizatio

nminus

minusminus

minusminus

minusminus

minusminus

minusH

2Sprod

uctio

nminus

minusminus

minusminus

minusminus

minusminus

minusVogesndashP

roskauerrsquostest

minus+

+minus

minusminus

minusminus

minusminus

Methylred

minusminus

minusminus

minusminus

minusminus

minusminus

Sorbito

l+

minusminus

minusminus

minusminus

minusminus

minus+

positivereactio

nminus

negativ

ereactio

nWw

eaklypo

sitivereactio

nVv

ariablereactio

n


chlorite to chloride and molecular oxygen [9 46 50] 0eoptimal temperature range for perchlorate reduction is28ndash37degC [46 51 52]

Perchlorate-reducing bacteria are usually anaerobic andsome facultative despite being the molecular oxygen pro-duced as an intermediate of the microbial perchlorate re-duction [46 53] in a process that exudes nitrate [46] In ourstudy isolated perchlorate-reducing bacteria were also an-aerobic but facultative 0erefore although these may un-dergo degradation processes in a wide range of environmentalconditions it is also probable that some critical anaerobicstrains were missed during the aerobic treatment In con-sequence additional experiments should be carried out underanaerobic conditions just to enrich some active microor-ganisms that may improve perchlorate degradation

Perchlorate is an ubiquitous and persistent pollutant inthe environment causing toxic effects in biota and humans0erefore different technologies have been developed toremove and eliminate this chemical One of the mostpromising effective and economic ones is the use of bacteriain biotechnological systems that are capable of reducing andeliminating perchlorate 0e rates of perchlorate reductionobtained in this study were comparable to those reported byWang et al [54] suggesting their potential application inbioremediation of perchlorate contaminated areas

4 Conclusions

0e strains isolated from Galerazamba-Bolivar Manaure-Guajira and Salamanca Island-Magdalena Colombia werehalotolerant organisms belonging to the Vibrio Bacillus

Salinovibrio Staphylococcus and Nesiotobacter genera0ese strains could reduce KClO4 levels in aqueous solutionsfrom 10 up to 25 Bacteria-mediated remediation ofperchlorate is a suitable process to control pollution by thistoxic chemical

Abbreviations

LB NaCl Luria-Bertani in seawaterML Maximum likelihoodMP Maximum parsimonyME Minimum evolutionNJ Neighbor joiningSEM Scanning electron microscopyOD Optical density

Data Availability

0e datasets generated andor analyzed during the currentstudy are available from the corresponding author onrequest

Conflicts of Interest

0e authors declare that they have no conflicts of interest

Acknowledgments

0e authors thank the Institute of Water and EnvironmentalEngineering Polytechnic University of Valencia (Spain)0is research received support from the Vice Presidency of

Table 2 Bacterial strains BBCOL-023 to BBCOL-033 exposed to different NaCl and KClO4 concentrations and pH changes

Characteristics BBCOL-023

BBCOL-024

BBCOL-025

BBCOL-026

BBCOL-027

BBCOL-028

BBCOL-029

BBCOL-031

BBCOL-032

BBCOL-033

Maximum NaCltolerance () 30 30 30 10 10 10 10 10 15 10

Maximum KClO4tolerance (mgL) 10000 10000 10000 10000 10000 7500 10000 10000 10000 7500

pH range tolerance 65ndash12 65ndash12 65ndash12 65ndash12 65ndash12 65ndash12 65ndash12 65ndash12 65ndash12 65ndash12

0 10 20 30

BBCOL-023BBCOL-024BBCOL-025BBCOL-026BBCOL-027BBCOL-028BBCOL-029BBCOL-030BBCOL-031BBCOL-032BBCOL-033

KClO4 concentration reduction ()

Isol

ated

bac

teria

Figure 7 Percentage of KClO4 concentration reduction of bacteria BBCOL-023 to BBCOL-033 from saline environments in the ColombianCaribbean Effect of the 48 h contact time optical density at OD 600 and optimal pH (70plusmn 05)


Research University of Cartagena and Colciencias-Uni-versity of Cartagena (Grant RC-758-20111107-521-29360)

References

[1] J Cole-Dai K M Peterson J A Kennedy T S Cox andD G Ferris ldquoEvidence of influence of human activities andvolcanic eruptions on environmental perchlorate from a 300-year Greenland ice core recordrdquo Environmental Science ampTechnology vol 52 no 15 pp 8373ndash8380 2018

[2] R Acevedo-Barrios C Sabater-Marco and J Olivero-VerbelldquoEcotoxicological assessment of perchlorate using in vitro andin vivo assaysrdquo Environmental Science and Pollution Researchvol 25 no 14 pp 13697ndash13708 2018

[3] M V Maffini L Trasande and T G Neltner ldquoPerchlorateand diet human exposures risks and mitigation strategiesrdquoCurrent Environmental Health Reports vol 3 no 2pp 107ndash117 2016

[4] B A Knight B M Shields X He et al ldquoEffect of perchlorateand thiocyanate exposure on thyroid function of pregnantwomen from South-West England a cohort studyrdquo yroidResearch vol 11 no 1 p 9 2018

[5] P N Smith e Ecotoxicology of Perchlorate in the Envi-ronment BT-Perchlorate Environmental Occurrence In-teractions and Treatment B Gu and J D Coates EdsSpringer US Boston MA USA 2006

[6] C Steinmaus M Pearl M Kharrazi et al ldquo0yroid hormonesand moderate exposure to perchlorate during pregnancy inwomen in southern Californiardquo Environmental Health Per-spectives vol 124 no 6 pp 861ndash867 2016

[7] A Ghosh K Pakshirajan P K Ghosh and N K SahooldquoPerchlorate degradation using an indigenous microbialconsortium predominantly Burkholderia sprdquo Journal ofHazardous Materials vol 187 no 1ndash3 pp 133ndash139 2011

[8] R Nerenberg B E Rittmann and I Najm ldquoPerchloratereduction in a hydrogen-based membrane-biofilm reactorrdquoJournal-American Water Works Association vol 94 no 11pp 103ndash114 2002

[9] J Xu and B E Logan ldquoMeasurement of chlorite dismutaseactivities in perchlorate respiring bacteriardquo Journal of Mi-crobiological Methods vol 54 no 2 pp 239ndash247 2003

[10] B E Logan J Wu and R F Unz ldquoBiological perchloratereduction in high-salinity solutionsrdquoWater Research vol 35no 12 pp 3034ndash3038 2001

[11] T Matsubara K Fujishima C W Saltikov S Nakamura andL J Rothschild ldquoEarth analogues for past and future life onMars isolation of perchlorate resistant halophiles from BigSoda Lakerdquo International Journal of Astrobiology vol 16no 3 pp 218ndash228 2016

[12] B C Okeke T Giblin and W T Frankenberger ldquoReductionof perchlorate and nitrate by salt tolerant bacteriardquo Envi-ronmental Pollution vol 118 no 3 pp 357ndash363 2002

[13] A Vijaya Nadaraja P G P Veetil and K BhaskaranldquoPerchlorate reduction by an isolated Serratia marcescensstrain under high salt and extreme pHrdquo FEMS MicrobiologyLetters vol 339 no 2 pp 117ndash121 2012

[14] C W Murray and P M Bolger ldquoEnvironmental contami-nants perchloraterdquo in Encyclopedia of Food SafetyY Motarjemi Ed pp 337ndash341 Academic Press WalthamMA USA 2014

[15] J Xu Y Song B Min L Steinberg and B E Logan ldquoMi-crobial degradation of perchlorate principles and applica-tionsrdquo Environmental Engineering Science vol 20 no 5pp 405ndash422 2003

[16] O Wang and D J Coates ldquoBiotechnological applications ofmicrobial (per)chlorate reductionrdquo Microorganisms vol 5no 4 2017

[17] Y Xiao and D J Roberts ldquoKinetics analysis of a salt-tolerantperchlorate-reducing bacterium effects of sodium magne-sium and nitraterdquo Environmental Science amp Technologyvol 47 no 15 pp 8666ndash8673 2013

[18] M Nozawa-Inoue K M Scow and D E Rolston ldquoReductionof perchlorate and nitrate by microbial communities in va-dose soilrdquo Applied and Environmental Microbiology vol 71no 7 pp 3928ndash3934 2005

[19] L J Shimkets and H Rafiee ldquoCsgA an extracellular proteinessential for Myxococcus xanthus developmentrdquo Journal ofBacteriology vol 172 no 9 pp 5299ndash5306 1990

[20] R Acevedo-Barrios A Bertel-Sevilla J Alonso-Molina andJ Olivero-Verbel ldquoPerchlorate tolerant bacteria from salineenvironments at the Caribbean region of Colombiardquo Toxi-cology Letters vol 259 p S103 2016

[21] D R Boone R W Castenholz G M Garrity D J BrennerN R Krieg and J T Staley Bergeyrsquos Manualreg of SystematicBacteriology Springer Science amp Business Media BostonMA USA 2005 httplinkspringercom1010070-387-29298-5

[22] T Iizuka M Tokura Y Jojima A Hiraishi S Yamanakaand R Fudou ldquoEnrichment and phylogenetic analysisof moderately thermophilic myxobacteria from hot springsin Japanrdquo Microbes and Environments vol 21 no 3pp 189ndash199 2006

[23] Z-H Wu D-M Jiang P Li and Y-Z Li ldquoExploring thediversity of myxobacteria in a soil niche by myxobacteria-specific primers and probesrdquo Environmental Microbiologyvol 7 no 10 pp 1602ndash1610 2005

[24] X Huang and A Madan ldquoCAP3 a DNA sequence assemblyprogramrdquo Genome Research vol 9 no 9 pp 868ndash877 1999

[25] N Saitou and M Nei ldquo0e neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo MolecularBiology and Evolution vol 4 pp 406ndash425 1987

[26] J Felsenstein ldquoEvolutionary trees from DNA sequences amaximum likelihood approachrdquo Journal of Molecular Evo-lution vol 17 no 6 pp 368ndash376 1981

[27] W M Fitch ldquoToward defining the course of evolutionminimum change for a specific tree topologyrdquo SystematicBiology vol 20 no 4 pp 406ndash416 1971

[28] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 molecular evolutionary genetics analysis version60rdquo Molecular Biology and Evolution vol 30 no 12pp 2725ndash2729 2013

[29] M Kimura ldquoA simple method for estimating evolutionaryrates of base substitutions through comparative studies ofnucleotide sequencesrdquo Journal of Molecular Evolution vol 16no 2 pp 111ndash120 1980

[30] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 no 4 pp 783ndash7912017

[31] R S Breed E G D Murray and N R Smith BergeyrsquosManual of Determ Bacteriol Williams Wilkins CompanyPhiladelphia PA USA 7th edition 1957

[32] E W Koneman S D Allen W M JandaP C Schreckenberger and W C Winn Jr MicrobiologicalDiagnosis Text and Color Atlas Guanabara Koogan Rio deJaneiro Brazil 6th edition 2008

[33] J L Alonso G Cuesta G W Ramırez J J MorenillaI Bernacer and R M Lloret Manual de Tecnicas Avan-zadas Para la Identificacion y Control de Bacterias


Filamentosas EPSAR-Generalitat Valencia ValenciaSpain 2009

[34] L Bahamdain F Fahmy S Lari and M Aly ldquoCharacter-ization of some Bacillus strains obtained frommarine habitatsusing different taxonomical methodsrdquo Life Science Journalvol 12 no 4 2015

[35] L Albuquerque I Tiago M Taborda M F NobreA Verissimo andM S da Costa ldquoBacillus isabeliae sp nov ahalophilic bacterium isolated from a sea salt evaporationpondrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 58 no 1 pp 226ndash230 2008

[36] L A Fernandez P Zalba M A Gomez and M A SagardoyldquoBacterias solubilizadoras de fosfato inorganico aisladas desuelos de la region sojerardquo Ciencia Del Suelo vol 23 no 1pp 31ndash37 2005

[37] F Gholamian M A Sheikh-Mohseni and M Salavati-Nia-sari ldquoHighly selective determination of perchlorate by a novelpotentiometric sensor based on a synthesized complex ofcopperrdquo Materials Science and Engineering C vol 31 no 8pp 1688ndash1691 2011

[38] S P Donachie ldquoNesiotobacter exalbescens gen nov sp nova moderately thermophilic alphaproteobacterium from anHawaiian hypersaline lakerdquo International Journal of Sys-tematic and Evolutionary Microbiology vol 56 no 3pp 563ndash567 2006

[39] J Ling G Zhang H Sun Y Fan J Ju and C ZhangldquoIsolation and characterization of a novel pyrene-degradingBacillus vallismortis strain JY3Ardquo Science of the Total Envi-ronment vol 409 no 10 pp 1994ndash2000 2011

[40] I Romano A Gambacorta L Lama B Nicolaus andA Giordano ldquoSalinivibrio costicola subsp alcaliphilus subspnov a haloalkaliphilic aerobe fromCampania Region (Italy)rdquoSystematic and Applied Microbiology vol 28 no 1 pp 34ndash422005

[41] M Ali Amoozegar A Zahra Fatemi H Reza Karbalaei-Heidari and M Reza Razavi ldquoProduction of an extracellularalkaline metalloprotease from a newly isolated moderatelyhalophile Salinivibrio sp strain AF-2004rdquo MicrobiologicalResearch vol 162 no 4 pp 369ndash377 2007

[42] J Dubert J L Romalde S Prado and J L Barja ldquoVibriobivalvicida sp nov a novel larval pathogen for bivalvemolluscs reared in a hatcheryrdquo Systematic and Applied Mi-crobiology vol 39 no 1 pp 8ndash13 2016

[43] J Paek J H Shin Y Shin et al ldquoVibrio injenensis sp novisolated from human clinical specimensrdquo Antonie VanLeeuwenhoek vol 110 no 1 pp 145ndash152 2016

[44] W E Kloos and K H Schleifer ldquoSimplified scheme forroutine identification of human Staphylococcus speciesrdquoJournal of Clinical Microbiology vol 1 no 1 pp 82ndash88 1975

[45] P S Kumar M G Paulraj S Ignacimuthu N A Al-Dhabiand D Sukumaran ldquoIn vitro antagonistic activity of soilStreptomyces collinus Dpr20 against bacterial pathogensrdquoJournal of Microbiology Biotechnology and Food Sciencesvol 7 no 3 pp 317ndash324 2017

[46] R A Bruce L A Achenbach and J D Coates ldquoReduction of(per)chlorate by a novel organism isolated from paper millwasterdquo Environmental Microbiology vol 1 no 4 pp 319ndash3291999

[47] A S Waller E E Cox and E A Edwards ldquoPerchlorate-reducing microorganisms isolated from contaminated sitesrdquoEnvironmental Microbiology vol 6 no 5 pp 517ndash527 2004

[48] S K Chaudhuri S M OrsquoConnor R L GustavsonL A Achenbach and J D Coates ldquoEnvironmental factorsthat control microbial perchlorate reductionrdquo Applied and

Environmental Microbiology vol 68 no 9 pp 4425ndash44302002

[49] M G Liebensteiner M J Oosterkamp and A J M StamsldquoMicrobial respiration with chlorine oxyanions diversity andphysiological and biochemical properties of chlorate- andperchlorate-reducing microorganismsrdquo Annals of the NewYork Academy of Sciences vol 1365 no 1 pp 59ndash72 2015

[50] K Sellers K Weeks W R Alsop et al Perchlorate Envi-ronmental problems and solutions CRC Press Boca Raton FLUSA 2006

[51] Y Zhu N Gao W Chu S Wang and J Xu ldquoBacterialreduction of highly concentrated perchlorate kinetics andinfluence of co-existing electron acceptors temperature pHand electron donorsrdquo Chemosphere vol 148 pp 188ndash1942016

[52] T Giblin and W T Frankenberger ldquoPerchlorate and nitratereductase activity in the perchlorate-respiring bacteriumperclacerdquo Microbiological Research vol 156 no 4pp 311ndash315 2001

[53] S Sevda T R Sreekishnan N Pous S Puig and D PantldquoBioelectroremediation of perchlorate and nitrate contami-nated water a reviewrdquo Bioresource Technology vol 255pp 331ndash339 2018

[54] C Wang L Lippincott and X Meng ldquoKinetics of biologicalperchlorate reduction and pH effectrdquo Journal of HazardousMaterials vol 153 no 1-2 pp 663ndash669 2008


Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



Journal of Parasitology Research

GenomicsInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018


BioinformaticsAdvances in

Marine BiologyJournal of



Neuroscience Journal


BioMed Research International

Cell BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018


Genetics Research International


Advances in

Virolog y Stem Cells International



Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

treatment [7] Perchlorate is persistent but possesses bio-degradability [8] However enzymes such as the perchloratereductase and superoxide chlorite carry out the reduction orelimination of perchlorate A reductase enzyme is re-sponsible for reducing perchlorate to chlorate and chlorateto chlorite while the enzyme superoxide chlorite changeschlorite to chloride and molecular oxygen Biological re-duction through the use of bacteria completely degrades theperchlorate ions into Clminus and O2 (equation (1)) [9ndash11] 0eperchlorate degradation pathway is as follows

ClO4minus(perchlorate)⟶ ClO3

minus( 1113857(chlorate)

⟶ ClO2minus

( 1113857(chlorite)⟶ Clminus(chloride) + O2(1)

Marine soils usually contain bacterial species with bio-chemical versatility and ability to tolerate salt being aninteresting target for researchers due to the potential re-duction of environmental perchlorate [10] 0e reason forselecting this type of environment is that degradation ofperchlorate may be carried out using salt-tolerant bacteria[12] although this perchlorate-reduction process could beimpaired with increasing salinity [11 13] Moreover theseorganisms are available in diverse environments fromAntarctica saline lakes and hot springs even in hyper-thermophilic and hypersaline soils [10 11] Additionallyperchlorate deposits in these environments may be formedby chemical reactions between sodium chloride from land orsea and ozone [14]

Pilot testing of biotechnologies using perchlorate-reducing bacteria has been studied and tuned in suspen-sion fixed-bed fluidized and biofilm reactors [15 16]evaluating the effectiveness of treatments on soil and watercontaminated with perchlorate [15] Nowadays the use ofintegrated systems combining physicochemical treatmentsand perchlorate-reducing halophilic bacteria is beingstudied to increase the efficiency of water treatmentallowing the handling of residual flows with high salinity andlarge perchlorate concentrations simultaneously solving thewaste disposal problem [17]

Colombia has a variety of ecosystems with differentclimatic and biogeochemical features which facilitates thedevelopment of different halophilic bacteria 0e aim of thisstudy was the characterization of perchlorate-reducingbacteria recovered from hypersaline soils of the Colom-bian Caribbean

2 Materials and Methods

21 Study Area and Sample Collection Soil samples wereobtained from salt mines of Colombia specifically fromGalerazamba-Bolivar (10deg47prime21PrimeN 75deg15prime41PrimeW) Manaure-Guajira (11deg46prime30PrimeN 72deg26prime40PrimeW) and saline soils locatedin Salamanca Island-Magdalena (10deg56prime00PrimeN 75deg15prime00PrimeW)(Figure 1) A sterile spatula was employed to collect ap-proximately 100 g of soil from the upper 1ndash10 cm layer inMay 2015 All samples were collected in 50mL Falcon tubeskept refrigerated at 4degC and taken to the laboratory forprocessing Salinity and pH were recorded for each sampleaccording to Nozawa-Inoue et al [18]

22 Isolation of Strains and Culture Conditions Isolationpurification and preservation techniques were used as de-scribed by Shimkets and Rafiee [19] Samples were treatedwith amphotericin B (025mgmL) for 3 h until inoculation(50 microL) in the isolation media Subsequently samples wereincubated in Petri dishes containing modified sterilized agarLuria-Bertani in seawater (LB NaCl) [20] and incubated at37degC for 24 h under aerobic conditions [21] Bacterial growthwas monitored by observation of colonies For preservationa bacterial colony was transferred to modified LB broth andincubated at 37degC aerobically during 12 h adjusting the celldensity to 05 Mc-Farland turbidity standard 0en 720 microLof each culture was transferred to a Cryovial with 80 microLglycerol and stored at minus80degC

23 Molecular Identification

231 16S rRNA Gene Sequencing and Phylogenetic AnalysisFor 16S rRNA gene amplification genomic DNA wasextracted using a QIAampreg DNA Mini Kit (Qiagen CAUSA) as described by the manufacturer 0e 16S rRNAgene was amplified from the total genomic DNA of thebacterial strains by PCR using the forward primer PF (5prime-AGAGTTTGATCCTGGCTCAG-3prime) and the reverse primer1492R (5prime-ACCTTGTTACGACTT-3prime) [22 23] PCR wasperformed with an AmpliTaq Goldreg 360 Master Mix(Applied Biosystems) according to the manufacturerrsquos in-structions Each 25 μL reaction mixture contained 04 μM ofeach primer and sim100 ng of template DNA 0e amplifi-cation was performed as follows initial denaturation for10min at 95degC 25 cycles of denaturation for 1min at 94degCannealing for 1min at 43degC and extension for 15min at72degC and a final extension for 55min at 72degC All PCRproducts were checked by electrophoresis on 12 (wv)agarose gels stained with ethidium bromide (10mgmL) andanalyzed using a gel documentation system (IngGenius 3System-Syngene)

0e PCR product was purified with a QIAquick PCRpurification kit (Qiagen CA USA) following the manu-facturerrsquos instructions Automated DNA sequencing wasperformed by the National Center for Genomic Sequencing(CNSG) (Medellin-Colombia) using PF and 1492Rprimers Sequence readings were edited and assembledwith the CAP3 software [24] 0e resulting 16S rRNA genesequences were compared to reference strains with validlypublished names using the EzTaxon-e server (httpwwwezbiocloudneteztaxon) After multiple alignments ofthe data via CLUSTAL_W four methods includingneighbor joining (NJ) [25] maximum likelihood (ML)[26] minimum evolution (ME) and maximum parsimony(MP) [27] were used to perform phylogenetic analysisPhylogenetic trees were constructed using MEGA version6 [28] Distances were calculated using the Kimura cor-rection in a pair-wise deletion manner [29] 0e topologiesof the phylogenetic tree were evaluated by the bootstrapresampling method described by Felsenstein [30] with1000 replicates 0e GenBankEMBLDDBJ accessionnumbers for the 16S rRNA gene sequences of isolated












Manure

Salamanca island





11deg 4

6prime 3

0Prime N

10deg 5

6prime 0

0Prime N

Latti

ude

10deg 4

7prime 2

1Prime N
































100

100

100

99

100

95

5498

71

58

89

76

99

100

42

42

54

30

25

31

69

44

92

7877

3757

67

45

88

32

31

49

5889



89100

100

BBCOL 024 (KX821666)


955399


001



















100

7564

5785

100

94 66

86

100

93

81

98

98

48

63

727146

92

5627

36

61

22

33

6

401626

001









100

67100

86100

98

99

99

93

99

87 69

53

001






















67

7194

9492

99

100

99

40

40

45

84

9290

41

1512

9545

2511

26

001











Tabl

e1

Morph

ological

andbiochemical

characteristicsof

isolatedstrains

Characteristics

BBCOL-

023

BBCOL-

024

BBCOL-

025

BBCOL-

026

BBCOL-

027

BBCOL-

028

BBCOL-

029

BBCOL-

031

BBCOL-

032

BBCOL-

033

Molecular

identifi

catio

nNesiotobacter

sp

Bacillu

svallism

ortis

Salin

ivibrio

costicola

Vibrio

algino

lyticus

Vh

arveyi

Bcohn

iiBa

cillu

sspp

Vibrio

sp

Staphylococcus

spp

Bflexu

s

Color

ofcolony

Beige

Yello

wYe

llow

Yello

wYe

llow

Yello

wYe

llow

Yello

wYe

llow

Yello

w

Morph

ology

Rodshaped

Rodshaped

Vibrioshaped

Vibrioshaped

Vibrio

shaped

Rod

shaped

Rod

shaped

Coco

shaped

Rodshaped

Rod

shaped

Leng

th(μm)

156

160

145

086

015

153

390

123

133

307

0ickn

ess(μm)

07

065

068

076

062

06

08

07

128

079

Motility

minusminus

+minus

minus+

++

minus+

Gram

straining

minus+

minusminus

minus+

minusminus

++

Endo

spore

minus+

minusminus

minus+

minusminus

minus+

Sporepo

sition

++

++

++

++

minusminus

Oxidase

++

++

W+

++

++

Catalase

minus+

minusminus

minus+

+minus

++

Arabino

seminus

++

++

++

++

+Manno

se+

minusminus

minusminus

minusminus

minusminus

minusSu

crose

++

++

++

++

++

Melibiose

++

++

++

++

++

Rham

nose

++

++

++

++

++

Mannitol

minusminus

++

++

++

++

Ado

nitol

minusminus

minusminus

minusminus

minusminus

minusminus

Galactose

++

++

++

++

++

Inosito

lminus

minusminus

minusminus

minusminus

minusminus

minusp-n-p-Ph

osph

ate

minusminus

+minus

+minus

minusminus

+minus


eV

minusminus

minus+

minusV

minusminus


e+

++

++

minus+

++

+Prolinenitroanilid

eminus

minusminus

minus+

minusminus

minusminus

minusp-n-pbis-Ph

osph

ate

minusminus

minusminus

minusminus

minusminus

minusminus

p-n-p-Xyilosid

e+

++

++

minus+

++

+p-n-p-a-Arabino

side

++

++

+minus

++

++

p-n-p-Ph

osph

orylcholine

minusminus

minusminus

minusminus

minusminus

minusminus


minusminus

minusminus

minusminus

minusminus

minusminus

p-n-p-N-A

cetylglucosamide

minusminus

minus+

+minus

minus+

minusminus

c-L-G


eminus

minusminus

minusminus

minusminus

minusminus

minusAesculin

++

++

++

++

++

p-Nitro-DL-ph

enylalanine

minusminus

minusminus

minusminus

minusminus

minusminus

Urea

minusminus

minusminus

minusminus

minusminus

minusminus

Glycine

minusminus

minusminus

minusminus

minusminus

minusminus

Citrate

Vminus

W+

+minus

Vminus

W+

Malon

icacid

++

++

++

++

++

Tripheny


++

++

++

++

++

Lactose

++

++

++

++

++

Bacteriolytic

capacity

++

++

++

++

++

Cellulolytic

capacity

minusminus

minusminus

minusminus

minusminus

minusminus


Tabl

e1

Con

tinued

Characteristics

BBCOL-

023

BBCOL-

024

BBCOL-

025

BBCOL-

026

BBCOL-

027

BBCOL-

028

BBCOL-

029

BBCOL-

031

BBCOL-

032

BBCOL-

033

Nitrateredu

ction

++

++

++

++

++

Indo

leminus

minusminus

minusminus

minusminus

minusminus

minusONPG

minusminus

minusminus

minusminus

minusminus

minusminus

Ornith

ineutilizatio

nminus

minusminus

minusminus

minusminus

minusminus

minusH

2Sprod

uctio

nminus

minusminus

minusminus

minusminus

minusminus

minusVogesndashP

roskauerrsquostest

minus+

+minus

minusminus

minusminus

minusminus

Methylred

minusminus

minusminus

minusminus

minusminus

minusminus

Sorbito

l+

minusminus

minusminus

minusminus

minusminus

minus+

positivereactio

nminus

negativ

ereactio

nWw

eaklypo

sitivereactio

nVv

ariablereactio

n





4 Conclusions



Abbreviations


Data Availability




Acknowledgments




BBCOL-024

BBCOL-025

BBCOL-026

BBCOL-027

BBCOL-028

BBCOL-029

BBCOL-031

BBCOL-032

BBCOL-033




0 10 20 30



Isol

ated

bac

teria




References





























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018












Manure

Salamanca island





11deg 4

6prime 3

0Prime N

10deg 5

6prime 0

0Prime N

Latti

ude

10deg 4

7prime 2

1Prime N
































100

100

100

99

100

95

5498

71

58

89

76

99

100

42

42

54

30

25

31

69

44

92

7877

3757

67

45

88

32

31

49

5889



89100

100

BBCOL 024 (KX821666)


955399


001



















100

7564

5785

100

94 66

86

100

93

81

98

98

48

63

727146

92

5627

36

61

22

33

6

401626

001









100

67100

86100

98

99

99

93

99

87 69

53

001






















67

7194

9492

99

100

99

40

40

45

84

9290

41

1512

9545

2511

26

001











Tabl

e1

Morph

ological

andbiochemical

characteristicsof

isolatedstrains

Characteristics

BBCOL-

023

BBCOL-

024

BBCOL-

025

BBCOL-

026

BBCOL-

027

BBCOL-

028

BBCOL-

029

BBCOL-

031

BBCOL-

032

BBCOL-

033

Molecular

identifi

catio

nNesiotobacter

sp

Bacillu

svallism

ortis

Salin

ivibrio

costicola

Vibrio

algino

lyticus

Vh

arveyi

Bcohn

iiBa

cillu

sspp

Vibrio

sp

Staphylococcus

spp

Bflexu

s

Color

ofcolony

Beige

Yello

wYe

llow

Yello

wYe

llow

Yello

wYe

llow

Yello

wYe

llow

Yello

w

Morph

ology

Rodshaped

Rodshaped

Vibrioshaped

Vibrioshaped

Vibrio

shaped

Rod

shaped

Rod

shaped

Coco

shaped

Rodshaped

Rod

shaped

Leng

th(μm)

156

160

145

086

015

153

390

123

133

307

0ickn

ess(μm)

07

065

068

076

062

06

08

07

128

079

Motility

minusminus

+minus

minus+

++

minus+

Gram

straining

minus+

minusminus

minus+

minusminus

++

Endo

spore

minus+

minusminus

minus+

minusminus

minus+

Sporepo

sition

++

++

++

++

minusminus

Oxidase

++

++

W+

++

++

Catalase

minus+

minusminus

minus+

+minus

++

Arabino

seminus

++

++

++

++

+Manno

se+

minusminus

minusminus

minusminus

minusminus

minusSu

crose

++

++

++

++

++

Melibiose

++

++

++

++

++

Rham

nose

++

++

++

++

++

Mannitol

minusminus

++

++

++

++

Ado

nitol

minusminus

minusminus

minusminus

minusminus

minusminus

Galactose

++

++

++

++

++

Inosito

lminus

minusminus

minusminus

minusminus

minusminus

minusp-n-p-Ph

osph

ate

minusminus

+minus

+minus

minusminus

+minus


eV

minusminus

minus+

minusV

minusminus


e+

++

++

minus+

++

+Prolinenitroanilid

eminus

minusminus

minus+

minusminus

minusminus

minusp-n-pbis-Ph

osph

ate

minusminus

minusminus

minusminus

minusminus

minusminus

p-n-p-Xyilosid

e+

++

++

minus+

++

+p-n-p-a-Arabino

side

++

++

+minus

++

++

p-n-p-Ph

osph

orylcholine

minusminus

minusminus

minusminus

minusminus

minusminus


minusminus

minusminus

minusminus

minusminus

minusminus

p-n-p-N-A

cetylglucosamide

minusminus

minus+

+minus

minus+

minusminus

c-L-G


eminus

minusminus

minusminus

minusminus

minusminus

minusAesculin

++

++

++

++

++

p-Nitro-DL-ph

enylalanine

minusminus

minusminus

minusminus

minusminus

minusminus

Urea

minusminus

minusminus

minusminus

minusminus

minusminus

Glycine

minusminus

minusminus

minusminus

minusminus

minusminus

Citrate

Vminus

W+

+minus

Vminus

W+

Malon

icacid

++

++

++

++

++

Tripheny


++

++

++

++

++

Lactose

++

++

++

++

++

Bacteriolytic

capacity

++

++

++

++

++

Cellulolytic

capacity

minusminus

minusminus

minusminus

minusminus

minusminus


Tabl

e1

Con

tinued

Characteristics

BBCOL-

023

BBCOL-

024

BBCOL-

025

BBCOL-

026

BBCOL-

027

BBCOL-

028

BBCOL-

029

BBCOL-

031

BBCOL-

032

BBCOL-

033

Nitrateredu

ction

++

++

++

++

++

Indo

leminus

minusminus

minusminus

minusminus

minusminus

minusONPG

minusminus

minusminus

minusminus

minusminus

minusminus

Ornith

ineutilizatio

nminus

minusminus

minusminus

minusminus

minusminus

minusH

2Sprod

uctio

nminus

minusminus

minusminus

minusminus

minusminus

minusVogesndashP

roskauerrsquostest

minus+

+minus

minusminus

minusminus

minusminus

Methylred

minusminus

minusminus

minusminus

minusminus

minusminus

Sorbito

l+

minusminus

minusminus

minusminus

minusminus

minus+

positivereactio

nminus

negativ

ereactio

nWw

eaklypo

sitivereactio

nVv

ariablereactio

n





4 Conclusions



Abbreviations


Data Availability




Acknowledgments




BBCOL-024

BBCOL-025

BBCOL-026

BBCOL-027

BBCOL-028

BBCOL-029

BBCOL-031

BBCOL-032

BBCOL-033




0 10 20 30



Isol

ated

bac

teria




References





























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018































100

100

100

99

100

95

5498

71

58

89

76

99

100

42

42

54

30

25

31

69

44

92

7877

3757

67

45

88

32

31

49

5889



89100

100

BBCOL 024 (KX821666)


955399


001



















100

7564

5785

100

94 66

86

100

93

81

98

98

48

63

727146

92

5627

36

61

22

33

6

401626

001









100

67100

86100

98

99

99

93

99

87 69

53

001






















67

7194

9492

99

100

99

40

40

45

84

9290

41

1512

9545

2511

26

001











Tabl

e1

Morph

ological

andbiochemical

characteristicsof

isolatedstrains

Characteristics

BBCOL-

023

BBCOL-

024

BBCOL-

025

BBCOL-

026

BBCOL-

027

BBCOL-

028

BBCOL-

029

BBCOL-

031

BBCOL-

032

BBCOL-

033

Molecular

identifi

catio

nNesiotobacter

sp

Bacillu

svallism

ortis

Salin

ivibrio

costicola

Vibrio

algino

lyticus

Vh

arveyi

Bcohn

iiBa

cillu

sspp

Vibrio

sp

Staphylococcus

spp

Bflexu

s

Color

ofcolony

Beige

Yello

wYe

llow

Yello

wYe

llow

Yello

wYe

llow

Yello

wYe

llow

Yello

w

Morph

ology

Rodshaped

Rodshaped

Vibrioshaped

Vibrioshaped

Vibrio

shaped

Rod

shaped

Rod

shaped

Coco

shaped

Rodshaped

Rod

shaped

Leng

th(μm)

156

160

145

086

015

153

390

123

133

307

0ickn

ess(μm)

07

065

068

076

062

06

08

07

128

079

Motility

minusminus

+minus

minus+

++

minus+

Gram

straining

minus+

minusminus

minus+

minusminus

++

Endo

spore

minus+

minusminus

minus+

minusminus

minus+

Sporepo

sition

++

++

++

++

minusminus

Oxidase

++

++

W+

++

++

Catalase

minus+

minusminus

minus+

+minus

++

Arabino

seminus

++

++

++

++

+Manno

se+

minusminus

minusminus

minusminus

minusminus

minusSu

crose

++

++

++

++

++

Melibiose

++

++

++

++

++

Rham

nose

++

++

++

++

++

Mannitol

minusminus

++

++

++

++

Ado

nitol

minusminus

minusminus

minusminus

minusminus

minusminus

Galactose

++

++

++

++

++

Inosito

lminus

minusminus

minusminus

minusminus

minusminus

minusp-n-p-Ph

osph

ate

minusminus

+minus

+minus

minusminus

+minus


eV

minusminus

minus+

minusV

minusminus


e+

++

++

minus+

++

+Prolinenitroanilid

eminus

minusminus

minus+

minusminus

minusminus

minusp-n-pbis-Ph

osph

ate

minusminus

minusminus

minusminus

minusminus

minusminus

p-n-p-Xyilosid

e+

++

++

minus+

++

+p-n-p-a-Arabino

side

++

++

+minus

++

++

p-n-p-Ph

osph

orylcholine

minusminus

minusminus

minusminus

minusminus

minusminus


minusminus

minusminus

minusminus

minusminus

minusminus

p-n-p-N-A

cetylglucosamide

minusminus

minus+

+minus

minus+

minusminus

c-L-G


eminus

minusminus

minusminus

minusminus

minusminus

minusAesculin

++

++

++

++

++

p-Nitro-DL-ph

enylalanine

minusminus

minusminus

minusminus

minusminus

minusminus

Urea

minusminus

minusminus

minusminus

minusminus

minusminus

Glycine

minusminus

minusminus

minusminus

minusminus

minusminus

Citrate

Vminus

W+

+minus

Vminus

W+

Malon

icacid

++

++

++

++

++

Tripheny


++

++

++

++

++

Lactose

++

++

++

++

++

Bacteriolytic

capacity

++

++

++

++

++

Cellulolytic

capacity

minusminus

minusminus

minusminus

minusminus

minusminus


Tabl

e1

Con

tinued

Characteristics

BBCOL-

023

BBCOL-

024

BBCOL-

025

BBCOL-

026

BBCOL-

027

BBCOL-

028

BBCOL-

029

BBCOL-

031

BBCOL-

032

BBCOL-

033

Nitrateredu

ction

++

++

++

++

++

Indo

leminus

minusminus

minusminus

minusminus

minusminus

minusONPG

minusminus

minusminus

minusminus

minusminus

minusminus

Ornith

ineutilizatio

nminus

minusminus

minusminus

minusminus

minusminus

minusH

2Sprod

uctio

nminus

minusminus

minusminus

minusminus

minusminus

minusVogesndashP

roskauerrsquostest

minus+

+minus

minusminus

minusminus

minusminus

Methylred

minusminus

minusminus

minusminus

minusminus

minusminus

Sorbito

l+

minusminus

minusminus

minusminus

minusminus

minus+

positivereactio

nminus

negativ

ereactio

nWw

eaklypo

sitivereactio

nVv

ariablereactio

n





4 Conclusions



Abbreviations


Data Availability




Acknowledgments




BBCOL-024

BBCOL-025

BBCOL-026

BBCOL-027

BBCOL-028

BBCOL-029

BBCOL-031

BBCOL-032

BBCOL-033




0 10 20 30



Isol

ated

bac

teria




References





























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


















100

7564

5785

100

94 66

86

100

93

81

98

98

48

63

727146

92

5627

36

61

22

33

6

401626

001









100

67100

86100

98

99

99

93

99

87 69

53

001






















67

7194

9492

99

100

99

40

40

45

84

9290

41

1512

9545

2511

26

001











Tabl

e1

Morph

ological

andbiochemical

characteristicsof

isolatedstrains

Characteristics

BBCOL-

023

BBCOL-

024

BBCOL-

025

BBCOL-

026

BBCOL-

027

BBCOL-

028

BBCOL-

029

BBCOL-

031

BBCOL-

032

BBCOL-

033

Molecular

identifi

catio

nNesiotobacter

sp

Bacillu

svallism

ortis

Salin

ivibrio

costicola

Vibrio

algino

lyticus

Vh

arveyi

Bcohn

iiBa

cillu

sspp

Vibrio

sp

Staphylococcus

spp

Bflexu

s

Color

ofcolony

Beige

Yello

wYe

llow

Yello

wYe

llow

Yello

wYe

llow

Yello

wYe

llow

Yello

w

Morph

ology

Rodshaped

Rodshaped

Vibrioshaped

Vibrioshaped

Vibrio

shaped

Rod

shaped

Rod

shaped

Coco

shaped

Rodshaped

Rod

shaped

Leng

th(μm)

156

160

145

086

015

153

390

123

133

307

0ickn

ess(μm)

07

065

068

076

062

06

08

07

128

079

Motility

minusminus

+minus

minus+

++

minus+

Gram

straining

minus+

minusminus

minus+

minusminus

++

Endo

spore

minus+

minusminus

minus+

minusminus

minus+

Sporepo

sition

++

++

++

++

minusminus

Oxidase

++

++

W+

++

++

Catalase

minus+

minusminus

minus+

+minus

++

Arabino

seminus

++

++

++

++

+Manno

se+

minusminus

minusminus

minusminus

minusminus

minusSu

crose

++

++

++

++

++

Melibiose

++

++

++

++

++

Rham

nose

++

++

++

++

++

Mannitol

minusminus

++

++

++

++

Ado

nitol

minusminus

minusminus

minusminus

minusminus

minusminus

Galactose

++

++

++

++

++

Inosito

lminus

minusminus

minusminus

minusminus

minusminus

minusp-n-p-Ph

osph

ate

minusminus

+minus

+minus

minusminus

+minus


eV

minusminus

minus+

minusV

minusminus


e+

++

++

minus+

++

+Prolinenitroanilid

eminus

minusminus

minus+

minusminus

minusminus

minusp-n-pbis-Ph

osph

ate

minusminus

minusminus

minusminus

minusminus

minusminus

p-n-p-Xyilosid

e+

++

++

minus+

++

+p-n-p-a-Arabino

side

++

++

+minus

++

++

p-n-p-Ph

osph

orylcholine

minusminus

minusminus

minusminus

minusminus

minusminus


minusminus

minusminus

minusminus

minusminus

minusminus

p-n-p-N-A

cetylglucosamide

minusminus

minus+

+minus

minus+

minusminus

c-L-G


eminus

minusminus

minusminus

minusminus

minusminus

minusAesculin

++

++

++

++

++

p-Nitro-DL-ph

enylalanine

minusminus

minusminus

minusminus

minusminus

minusminus

Urea

minusminus

minusminus

minusminus

minusminus

minusminus

Glycine

minusminus

minusminus

minusminus

minusminus

minusminus

Citrate

Vminus

W+

+minus

Vminus

W+

Malon

icacid

++

++

++

++

++

Tripheny


++

++

++

++

++

Lactose

++

++

++

++

++

Bacteriolytic

capacity

++

++

++

++

++

Cellulolytic

capacity

minusminus

minusminus

minusminus

minusminus

minusminus


Tabl

e1

Con

tinued

Characteristics

BBCOL-

023

BBCOL-

024

BBCOL-

025

BBCOL-

026

BBCOL-

027

BBCOL-

028

BBCOL-

029

BBCOL-

031

BBCOL-

032

BBCOL-

033

Nitrateredu

ction

++

++

++

++

++

Indo

leminus

minusminus

minusminus

minusminus

minusminus

minusONPG

minusminus

minusminus

minusminus

minusminus

minusminus

Ornith

ineutilizatio

nminus

minusminus

minusminus

minusminus

minusminus

minusH

2Sprod

uctio

nminus

minusminus

minusminus

minusminus

minusminus

minusVogesndashP

roskauerrsquostest

minus+

+minus

minusminus

minusminus

minusminus

Methylred

minusminus

minusminus

minusminus

minusminus

minusminus

Sorbito

l+

minusminus

minusminus

minusminus

minusminus

minus+

positivereactio

nminus

negativ

ereactio

nWw

eaklypo

sitivereactio

nVv

ariablereactio

n





4 Conclusions



Abbreviations


Data Availability




Acknowledgments




BBCOL-024

BBCOL-025

BBCOL-026

BBCOL-027

BBCOL-028

BBCOL-029

BBCOL-031

BBCOL-032

BBCOL-033




0 10 20 30



Isol

ated

bac

teria




References





























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





















67

7194

9492

99

100

99

40

40

45

84

9290

41

1512

9545

2511

26

001











Tabl

e1

Morph

ological

andbiochemical

characteristicsof

isolatedstrains

Characteristics

BBCOL-

023

BBCOL-

024

BBCOL-

025

BBCOL-

026

BBCOL-

027

BBCOL-

028

BBCOL-

029

BBCOL-

031

BBCOL-

032

BBCOL-

033

Molecular

identifi

catio

nNesiotobacter

sp

Bacillu

svallism

ortis

Salin

ivibrio

costicola

Vibrio

algino

lyticus

Vh

arveyi

Bcohn

iiBa

cillu

sspp

Vibrio

sp

Staphylococcus

spp

Bflexu

s

Color

ofcolony

Beige

Yello

wYe

llow

Yello

wYe

llow

Yello

wYe

llow

Yello

wYe

llow

Yello

w

Morph

ology

Rodshaped

Rodshaped

Vibrioshaped

Vibrioshaped

Vibrio

shaped

Rod

shaped

Rod

shaped

Coco

shaped

Rodshaped

Rod

shaped

Leng

th(μm)

156

160

145

086

015

153

390

123

133

307

0ickn

ess(μm)

07

065

068

076

062

06

08

07

128

079

Motility

minusminus

+minus

minus+

++

minus+

Gram

straining

minus+

minusminus

minus+

minusminus

++

Endo

spore

minus+

minusminus

minus+

minusminus

minus+

Sporepo

sition

++

++

++

++

minusminus

Oxidase

++

++

W+

++

++

Catalase

minus+

minusminus

minus+

+minus

++

Arabino

seminus

++

++

++

++

+Manno

se+

minusminus

minusminus

minusminus

minusminus

minusSu

crose

++

++

++

++

++

Melibiose

++

++

++

++

++

Rham

nose

++

++

++

++

++

Mannitol

minusminus

++

++

++

++

Ado

nitol

minusminus

minusminus

minusminus

minusminus

minusminus

Galactose

++

++

++

++

++

Inosito

lminus

minusminus

minusminus

minusminus

minusminus

minusp-n-p-Ph

osph

ate

minusminus

+minus

+minus

minusminus

+minus


eV

minusminus

minus+

minusV

minusminus


e+

++

++

minus+

++

+Prolinenitroanilid

eminus

minusminus

minus+

minusminus

minusminus

minusp-n-pbis-Ph

osph

ate

minusminus

minusminus

minusminus

minusminus

minusminus

p-n-p-Xyilosid

e+

++

++

minus+

++

+p-n-p-a-Arabino

side

++

++

+minus

++

++

p-n-p-Ph

osph

orylcholine

minusminus

minusminus

minusminus

minusminus

minusminus


minusminus

minusminus

minusminus

minusminus

minusminus

p-n-p-N-A

cetylglucosamide

minusminus

minus+

+minus

minus+

minusminus

c-L-G


eminus

minusminus

minusminus

minusminus

minusminus

minusAesculin

++

++

++

++

++

p-Nitro-DL-ph

enylalanine

minusminus

minusminus

minusminus

minusminus

minusminus

Urea

minusminus

minusminus

minusminus

minusminus

minusminus

Glycine

minusminus

minusminus

minusminus

minusminus

minusminus

Citrate

Vminus

W+

+minus

Vminus

W+

Malon

icacid

++

++

++

++

++

Tripheny


++

++

++

++

++

Lactose

++

++

++

++

++

Bacteriolytic

capacity

++

++

++

++

++

Cellulolytic

capacity

minusminus

minusminus

minusminus

minusminus

minusminus


Tabl

e1

Con

tinued

Characteristics

BBCOL-

023

BBCOL-

024

BBCOL-

025

BBCOL-

026

BBCOL-

027

BBCOL-

028

BBCOL-

029

BBCOL-

031

BBCOL-

032

BBCOL-

033

Nitrateredu

ction

++

++

++

++

++

Indo

leminus

minusminus

minusminus

minusminus

minusminus

minusONPG

minusminus

minusminus

minusminus

minusminus

minusminus

Ornith

ineutilizatio

nminus

minusminus

minusminus

minusminus

minusminus

minusH

2Sprod

uctio

nminus

minusminus

minusminus

minusminus

minusminus

minusVogesndashP

roskauerrsquostest

minus+

+minus

minusminus

minusminus

minusminus

Methylred

minusminus

minusminus

minusminus

minusminus

minusminus

Sorbito

l+

minusminus

minusminus

minusminus

minusminus

minus+

positivereactio

nminus

negativ

ereactio

nWw

eaklypo

sitivereactio

nVv

ariablereactio

n





4 Conclusions



Abbreviations


Data Availability




Acknowledgments




BBCOL-024

BBCOL-025

BBCOL-026

BBCOL-027

BBCOL-028

BBCOL-029

BBCOL-031

BBCOL-032

BBCOL-033




0 10 20 30



Isol

ated

bac

teria




References





























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018










Tabl

e1

Morph

ological

andbiochemical

characteristicsof

isolatedstrains

Characteristics

BBCOL-

023

BBCOL-

024

BBCOL-

025

BBCOL-

026

BBCOL-

027

BBCOL-

028

BBCOL-

029

BBCOL-

031

BBCOL-

032

BBCOL-

033

Molecular

identifi

catio

nNesiotobacter

sp

Bacillu

svallism

ortis

Salin

ivibrio

costicola

Vibrio

algino

lyticus

Vh

arveyi

Bcohn

iiBa

cillu

sspp

Vibrio

sp

Staphylococcus

spp

Bflexu

s

Color

ofcolony

Beige

Yello

wYe

llow

Yello

wYe

llow

Yello

wYe

llow

Yello

wYe

llow

Yello

w

Morph

ology

Rodshaped

Rodshaped

Vibrioshaped

Vibrioshaped

Vibrio

shaped

Rod

shaped

Rod

shaped

Coco

shaped

Rodshaped

Rod

shaped

Leng

th(μm)

156

160

145

086

015

153

390

123

133

307

0ickn

ess(μm)

07

065

068

076

062

06

08

07

128

079

Motility

minusminus

+minus

minus+

++

minus+

Gram

straining

minus+

minusminus

minus+

minusminus

++

Endo

spore

minus+

minusminus

minus+

minusminus

minus+

Sporepo

sition

++

++

++

++

minusminus

Oxidase

++

++

W+

++

++

Catalase

minus+

minusminus

minus+

+minus

++

Arabino

seminus

++

++

++

++

+Manno

se+

minusminus

minusminus

minusminus

minusminus

minusSu

crose

++

++

++

++

++

Melibiose

++

++

++

++

++

Rham

nose

++

++

++

++

++

Mannitol

minusminus

++

++

++

++

Ado

nitol

minusminus

minusminus

minusminus

minusminus

minusminus

Galactose

++

++

++

++

++

Inosito

lminus

minusminus

minusminus

minusminus

minusminus

minusp-n-p-Ph

osph

ate

minusminus

+minus

+minus

minusminus

+minus


eV

minusminus

minus+

minusV

minusminus


e+

++

++

minus+

++

+Prolinenitroanilid

eminus

minusminus

minus+

minusminus

minusminus

minusp-n-pbis-Ph

osph

ate

minusminus

minusminus

minusminus

minusminus

minusminus

p-n-p-Xyilosid

e+

++

++

minus+

++

+p-n-p-a-Arabino

side

++

++

+minus

++

++

p-n-p-Ph

osph

orylcholine

minusminus

minusminus

minusminus

minusminus

minusminus


minusminus

minusminus

minusminus

minusminus

minusminus

p-n-p-N-A

cetylglucosamide

minusminus

minus+

+minus

minus+

minusminus

c-L-G


eminus

minusminus

minusminus

minusminus

minusminus

minusAesculin

++

++

++

++

++

p-Nitro-DL-ph

enylalanine

minusminus

minusminus

minusminus

minusminus

minusminus

Urea

minusminus

minusminus

minusminus

minusminus

minusminus

Glycine

minusminus

minusminus

minusminus

minusminus

minusminus

Citrate

Vminus

W+

+minus

Vminus

W+

Malon

icacid

++

++

++

++

++

Tripheny


++

++

++

++

++

Lactose

++

++

++

++

++

Bacteriolytic

capacity

++

++

++

++

++

Cellulolytic

capacity

minusminus

minusminus

minusminus

minusminus

minusminus


Tabl

e1

Con

tinued

Characteristics

BBCOL-

023

BBCOL-

024

BBCOL-

025

BBCOL-

026

BBCOL-

027

BBCOL-

028

BBCOL-

029

BBCOL-

031

BBCOL-

032

BBCOL-

033

Nitrateredu

ction

++

++

++

++

++

Indo

leminus

minusminus

minusminus

minusminus

minusminus

minusONPG

minusminus

minusminus

minusminus

minusminus

minusminus

Ornith

ineutilizatio

nminus

minusminus

minusminus

minusminus

minusminus

minusH

2Sprod

uctio

nminus

minusminus

minusminus

minusminus

minusminus

minusVogesndashP

roskauerrsquostest

minus+

+minus

minusminus

minusminus

minusminus

Methylred

minusminus

minusminus

minusminus

minusminus

minusminus

Sorbito

l+

minusminus

minusminus

minusminus

minusminus

minus+

positivereactio

nminus

negativ

ereactio

nWw

eaklypo

sitivereactio

nVv

ariablereactio

n





4 Conclusions



Abbreviations


Data Availability




Acknowledgments




BBCOL-024

BBCOL-025

BBCOL-026

BBCOL-027

BBCOL-028

BBCOL-029

BBCOL-031

BBCOL-032

BBCOL-033




0 10 20 30



Isol

ated

bac

teria




References





























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Tabl

e1

Con

tinued

Characteristics

BBCOL-

023

BBCOL-

024

BBCOL-

025

BBCOL-

026

BBCOL-

027

BBCOL-

028

BBCOL-

029

BBCOL-

031

BBCOL-

032

BBCOL-

033

Nitrateredu

ction

++

++

++

++

++

Indo

leminus

minusminus

minusminus

minusminus

minusminus

minusONPG

minusminus

minusminus

minusminus

minusminus

minusminus

Ornith

ineutilizatio

nminus

minusminus

minusminus

minusminus

minusminus

minusH

2Sprod

uctio

nminus

minusminus

minusminus

minusminus

minusminus

minusVogesndashP

roskauerrsquostest

minus+

+minus

minusminus

minusminus

minusminus

Methylred

minusminus

minusminus

minusminus

minusminus

minusminus

Sorbito

l+

minusminus

minusminus

minusminus

minusminus

minus+

positivereactio

nminus

negativ

ereactio

nWw

eaklypo

sitivereactio

nVv

ariablereactio

n





4 Conclusions



Abbreviations


Data Availability




Acknowledgments




BBCOL-024

BBCOL-025

BBCOL-026

BBCOL-027

BBCOL-028

BBCOL-029

BBCOL-031

BBCOL-032

BBCOL-033




0 10 20 30



Isol

ated

bac

teria




References





























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





4 Conclusions



Abbreviations


Data Availability




Acknowledgments




BBCOL-024

BBCOL-025

BBCOL-026

BBCOL-027

BBCOL-028

BBCOL-029

BBCOL-031

BBCOL-032

BBCOL-033




0 10 20 30



Isol

ated

bac

teria




References





























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



References





























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




























Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


perchlorate-reducingbacteriafromhypersalinesoilsofthe

Documents