Bioremediation of Mercury by Vibrio fluvialis Screened...

Research ArticleBioremediation of Mercury by Vibrio fluvialis Screenedfrom Industrial Effluents

Kailasam Saranya1 Arumugam Sundaramanickam1 Sudhanshu Shekhar1

Sankaran Swaminathan2 and Thangavel Balasubramanian1

1CAS in Marine Biology Faculty of Marine Sciences Annamalai University Parangipettai 608 502 India2AVC College of Arts and Science Mannampandal Mayiladuthurai 609 305 India

Correspondence should be addressed to Arumugam Sundaramanickam fish laryahoocom

Received 11 February 2017 Revised 19 March 2017 Accepted 19 April 2017 Published 25 May 2017

Academic Editor Raluca M Hlihor

Copyright copy 2017 Kailasam Saranya et al This 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 is properlycited

Thirty-one mercury-resistant bacterial strains were isolated from the effluent discharge sites of the SIPCOT industrial area Amongthem only one strain (CASKS5)was selected for further investigation due to its highminimum inhibitory concentration ofmercuryand low antibiotic susceptibility In accordance with 16S ribosomal RNA gene sequences the strain CASKS5 was identified asVibriofluvialis The mercury-removal capacity of V fluvialis was analyzed at four different concentrations (100 150 200 and 250 120583gml)Efficient bioremediation was observed at a level of 250 120583gml with the removal of 60 of mercury ions The interesting outcome ofthis study was that the strain V fluvialis had a high bioremediation efficiency but had a low antibiotic resistance Hence V fluvialiscould be successfully used as a strain for the ecofriendly removal of mercury

1 Introduction

Mercury is one of the most hazardous heavy metals which isconsidered as a significant contaminant of the environmentMercury contamination and its threat to the environmentand living organisms is a worldwide problem [1] It isreleased into the environment in twoways natural events andhuman activities When compared to the natural processeshuman activities have been discharging excessive amountsof mercury into the environment [2] A primary sourceof mercury pollution is chloralkali plants paper pulpsamalgamation industries fungicides and paints [3] Thepotential of mercury toxicity is only based on its combinationwith for example sulfide oxide hydroxide chloride andmethyl groups After the incident of Minamata Bay in Japanmercury poisoning to human health has become evident [4]A small amount of mercury can have dangerous effects formonths in human beings animals and plants and even affectthe growth of bacteria in microorganisms although somebacteria are capable of surviving and growing in mercury-contaminated sites [5] Most of the mercury-refinement

processes follow common physical and chemical meth-ods which are highly expensive and have some limita-tions whereas biological methods are cost-effective viableand friendly to the environment [6] The use of microor-ganisms for the removal of metals from contaminatedeffluents and mining and industrial wastes is consideredto be effective because of its efficiency and ecofriendlynature [7] Recently the utilization of bacterial biomassunder either live or dead conditions for bioremediationhas emerged as an efficient ecofriendly and cost-effectivealternative for the elimination of low concentrations of heavymetals

The heavy metal and antibiotic-resistant bacteria werefound in normal and polluted environmentswhich is aworld-wide problem [8] An array of heavy metals and antibioticsat concentrations found in different polluted environmentshave the potential to coselect both metal-antibiotic-resistantstrains and their plasmids [9] The bacterial agent associatedwith coselectivemechanism ofmetal-antibiotics is significantat higher threats Hence in the present investigation thestrain was selected based on the mercury resistance and

HindawiBioMed Research InternationalVolume 2017 Article ID 6509648 6 pageshttpsdoiorg10115520176509648

2 BioMed Research International

Table 1 Similarity of minimum inhibitory mercury concentration value against that observed in the present strain Vibrio fluvialis to thosereported elsewhere

Strain MIC (120583gml) Location ReferenceVibrio fluvialis 1 Tagus Estuary (Portugal) Figueiredo et al 2016Vibrio natriegens 20 Coastal sediments Bushehr Iran Jafari and Cheraghi 2014Vibrio sp 12ndash16 Chesapeake Bay Walker and Colwell 1974Vibrio sp 271 Mai Po Nature Reserve Hong Kong Zhang et al 2006Vibrio parahaemolyticus 45 Coastal sediments Bushehr Iran Jafari and Cheraghi 2014Vibrio fluvialis 100 Parangipettai coast (India) Present study

antibiotic susceptible characteristics Subsequently its biore-mediation capability was investigated

2 Methods and Materials

21 Sample Collection Sediment sampleswere collected fromthe common effluent discharge point of the State IndustriesPromotion Corporation of Tamil Nadu Limited (SIPCOT)industrial area located in the banks of the Uppanar estu-ary Tamil Nadu southeast coast of India The geographiccoordinates of the station are 11∘411015840450010158401015840N latitude and79∘461015840050010158401015840E longitude Surface sediments were collectedaseptically in triplicate kept in an insulated box at 4∘C andimmediately transferred to the laboratory

22 Enrichment and Primary Screening Sediment sampleswere added to a 250ml Erlenmeyer flask containing 100ml ofZobell Marine Broth (ZMB) at pH 71 plusmn 01 incubated for 24 hand centrifuged at 160 rpm at 35∘C in a conventional rotaryshaker incubator The bacterial inocula were transferred toa 100ml ZMB with a supplement of HgCl

2and kept in an

orbital shaker at 200 rpm for 5 days

23 Antibiotic Sensitivity Test Antibiotic sensitivity test wasperformed using the disc diffusion method on MH agarwith antibiotic disks by following the methods of CLSI(2013) [10] Multiple antibiotic profiles of V fluvialis werechecked at the following antibiotic concentrations amoxi-cillin (10mcgdisc) bacitracin (10mcgdisc) erythromycin(15mcgdisc) amoxicillin (10mcgdisc) bacitracin (10mcgdisc) erythromycin (15mcgdisc) oxytetracycline (30mcgdisc) novobiocin (30mcgdisc) cephalothin (30mcgdisc)vancomycin (30mcgdisc) and amikacin (10mcgdisc) Bac-terial cultures swabbed on nutrient agar plates and the above-mentioned antibiotics discs were placed in the plates andincubated at 37∘C for 24 hrs

24 Growth Assessment of CASKS5 with Mercury Theselected V fluvialis overnight culture was inoculated into thenutrient broth which was supplemented at different concen-trations of HgCl

2such as 100 120583gml 150120583gml 200120583gml

and 250 120583gml in triplicate and kept in a shaking incubator at37∘C for 48 hrs Growth curves ofV fluvialiswere observed at

periodic time intervals that is 2 4 8 16 24 and 48 hrs usinga spectrophotometer (SHIMADZUUV 1800) at 600 nmOD

25 Bioremediation Capacity of CASKS5 After the comple-tion of the incubation period the cultures were centrifugedfor 20mins at 10000 rpm pellets removed and supernatantscollected and digested with nitric and sulfuric acids Theresidualmercury in themediumwas analyzed by a cold vapormercury analyzer (Model MA 5840)

3 Results and Discussion

Mercury-resistant bacterial strains were initially screenedusing the Luria Bertani (LB) medium in the presence of20 120583gml HgCl

2from sediment samples collected at effluent

discharge sites

31 Isolation Screening and Selection of Mercury-ResistantBacteria The high mercury-resistant strains were isolatedfrom sediments of industrial discharge sites using severalscreening techniques Prolonged exposure to mercury con-tamination can generate resistance mechanisms in bacteria[11] During preliminary findings 31 bacterial isolates exhib-ited high resistance to mercury Secondary screening wasperformed to estimate MIC The MIC results observed forall the 31 isolates confirmed that the strain CASKS5 hadthe highest mercury tolerance (100 120583gml concentration) asshown in Table 1 which is several times greater than thatobtained in an earlier study done on the same species byFigueiredo et al (2016) [12]

32 Biochemical Characterization and Molecular Identifica-tion of CASKS5 The selected strain CASKS5 was morpho-logically and biochemically characterized as a gram-negativecurved rod-shaped bacterium and tentatively identified asVibrio sp (Table 2) The 16S rDNA sequence was carried outfor CASKS5 and submitted to GenBank (NCBI 2013) forsearching similar published sequencesThe accessionnumberof the strain CASKS5 is KM186606

BLAST analysis revealed that the partial 16S rDNA ofCASKS5 hadmore than 99 similarity to that ofVibrio fluvi-alis strain in NCBI A phylogenetic tree based on 16S rDNAwas constructed using the MEGA 60 software to determinethe relationship between CASKS5 and V fluvialis (Figure 1)

BioMed Research International 3

Vibrio fluvialis (NR114218)

CASKS5 (KM186605)

Vibrio fumissii (NR102977)

Vibrio tubiashii (NR113791)

Vibrio hepatarius (NR025491)

Vibrio brasiliensis (NR117887)

Vibrio neptunius (NR118243)

Vibrio caribbeanicus (NR108732)

Vibrio variabilis (NR117550)

Vibrio vulnicus (NR074889)

Marinobacter sp (NR044509)

6798

39

45

75 58

47

002

Figure 1 Phylogenetic tree constructed from the 16S rRNA gene sequence of Vibrio fluvialis (KM186605) (GenBank accession numberKM186605) and closely related organisms using NCBI BLAST The scale bar represents 002 substitutions per nucleotide position

Table 2 Biochemical characteristics of strain mercury resistantbacterial strain CASKS5

Tests ResultsMorphology

Gram reaction minusveShape Rod

Biochemical reactionsCitrate utilisation +Indole +Methyl red minus

Nitrate reduction +Oxidase +Catalase +Voges Proskauer minus

Gelatin +Carbohydrate utilisation

Glucose +Arabinose minus

Sucrose +

Based on the above characterization strain CASKS5 wasidentified as V fluvialis

33 Antibiotic Profile of V fluvialis Earlier investigationsdealt with the heavy metal resistance of bacteria frommarine environments having high resistance to most of theantibiotics [13 14] Nakahara et al [15] stated that antibiotic-and metal-resistance ability is created by the same plasmid ofthe bacteria However in the present investigation the resultsof the antibiotic sensitivity test with eight different antibioticsindicate that the strain CASKS5 was found to be suscep-tible to the majority of antibiotics for example amikacinerythromycin novobiocin oxytetracycline and vancomycin

minus010

010203040506

Zone

of i

nhib

ition

(cm

)Antibiotic susceptibility test

Am

ikac

in

Am

oxyc

illin

Baci

trac

in

Cep

halo

thin

Eryt

hrom

ycin

Nov

obio

cin

Oxy

tetr

acyc

line

Vanc

omyc

in

Figure 2 Antimicrobial susceptibility test profile for mercury-resistant bacteria isolate Vibrio fluvialis (KM186605)

although it was resistant to only three antibiotics namelyamoxicillin bacitracin and cephalothin (Figure 2) Muchcontroversy exists within the scientific community overwhether metal-resistant bacteria from polluted areas can alsohave antibiotic resistance In the present investigation theselected strain proved to have high resistance to mercuryalthough with little resistance to antibiotics The results ofFigueiredo et al (2016) [12] specify that out of 10 bacterialstrains isolated from mercury-contaminated regions of theTagus estuary only three have multidrug resistance Theyalso reported that V fluvialis has resistance only to nalidixicacid among the six antibiotics tested Hence the presence ofthese antibiotic- and mercury-resistant genetic elements inthe same gene is again highly contentious

34 Effect of Mercury on the Growth of V fluvialis Basedon the MIC results 100 120583gml was taken as an initial metalconcentration for growth curve studies A significant growthrate of V fluvialis was observed after 24 hrs of incubation in


minus020

02040608

11214

Initial 2 hrs 4 hrs 8 hrs 16 hrs 24 hrs 48 hrs

Control100

150

200

250

Gro

wth

OD

at 6

00nm

120583gml120583gml

120583gml120583gml

Figure 3 Growth kinetics of Vibrio fluvialis (KM186605) in HgCl2

(100 150 200 and 250 120583gml) containing medium Control culturesdid not contain any metal ions

control as well as in culture broth-containing metal whichindicated that gram-negative V fluvialis has developed apotential resistance to mercury An earlier study by Aramet al [16] also states that gram-negative bacteria isolatedfrom the Maharloo River Iran have higher resistance whencompared to gram-positive bacteria Bioremediation resultsshow that enhanced growthwas observed in control as well assolutions with mercury at a lower concentration (100120583gml)as compared to higher concentrations (150 120583gml 200120583gmland 250 120583gml) which indicates an increase in the con-centration of mercury and a decrease in the growth rateof cells (Figure 3) The present observation corroboratesan earlier study described by Zeng et al (2009) [17] fromChina

35 Mercury-Removal Capacity of V fluvialis Bacteria area valuable tool to treat mercury because they have vitalreactive interfaces for the adsorption of nutrients and foreigncontaminants on their cell surface particularly bacterialmembranes act as sites of uptake and exudation and pro-vide plenty of enzymatic actions [18] In the case of metalcontaminants some bacterial cells uptake metals for theirrequirements some of them chelate with metals and someeither reduce or oxidize them [19] Mercury-remediationcapacity of V fluvialis was observed by growth carve atdifferent concentrations of mercury chloride The highestmercury-remediation rate (60) was found at a lowermercury concentration of 100 120583gml after 42 h of incuba-tion At higher concentrations of 150 200 and 250 120583gmlthe mercury-removal percentages were 40 2533 and 19respectively (Figure 4) Some of the previous works ofvarious researchers on mercury bioremediation by bacterialstrains are given in Table 3 At a mercury concentration of10 120583gml 8947 of the mercury was removed by a speciesunder the same genus Vibrio parahaemolyticus over 40 hof incubation [20] The results of other bacterial speciesare as follows Bacillus sp 681 [21] Bacillus thuringiensis427 [22] Pseudomonas aeruginosa 80 [23] Brevibac-terium casei 70 [23] Tetrahymena rostrate 40 [24]

Mercury removal capacity of Vibriouvialis

0

20

40

60

80

100

o

f mer

cury

rem

oval

100 150 200 250

(ppm)

Figure 4 Bioremediation efficiency by Vibrio fluvialis (KM186605)with different initial concentration HgCl2 (100 150 200 and 250120583gml)

Pseudomonas sp 65 [25] Pseudomonas fluorescens 3430[26] and 427 [22] Pseudomonas aeruginosa 80 [23]Brevibacterium casei 70 [23] Tetrahymena rostrate 40[24] Pseudomonas sp 65 [25] Pseudomonas fluorescens3430 [26] Pseudomonas aeruginosa 25 [27] Klebsiellapneumoniae 15 [27] and the Enterobacter cloacae efficiencywas below the detectable limit [28] Some of them observeda better removal of mercury than in the present studyHowever the concentration level used in other studies waslower than that in our present investigationwhich is shown inTable 3

4 Conclusion

The results demonstrate that V fluvialis has strong abilityto detoxify mercury from mobile solutions In additionmercury and antibiotic resistance were appraised in detailfor the selected strain Generally metal-resistant strainsexhibit a strong antibiotic resistance nevertheless the presentfindings specify that the isolate CASKS5 has resistanceto only a few antibiotics Hence the strain CASKS5 canbe utilized as a good chelating agent for the removal ofmercury from contaminated effluents because of its highefficiency Further studies have to be performed to findout the mechanism behind the removal of mercury by Vfluvialis and also to examine the preference for heavy metaluptake by this bacterium in the presence of other heavymetals

Disclosure

The paper does not discuss policy issues and the conclusionsdrawn in the paper are based on interpretation of results bythe authors and in noway reflect the viewpoint of the fundingagency

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper


Table3Biorem

ediatio

neffi

ciency

ofmercury

byVibrioflu

vialis(C

ASK

S5)com

paredwith

different

bacterialspecies

isolated

from

elsew

here

Locatio

nSpecies

Con

centratio

nof

mercury

used

forthe

experim

ent

120583gml

Biorem

ediatio

neffi

ciency

(inpercentage)

References

Bushehr(Iran)coast

Vibrioparahaem

olyticu

s10

gt8947

Jafarietal2015

Urıa

sestu

arySinaloaMexico

Bacillussp

10681

Green-Ruiz2006

Bacillusthu

ringiensis

427

Hassenetal1998

Sialkot(Pakista

n)po

ndBrevibacteriu

mcasei

5070

Rehm

anetal2007

Sialkot(Pakista

n)po

ndPseudomonas

aeruginosa

5080

Rehm

anetal2007

Kasur(Pakista

n)po

nds

Tetra

hymenarostrata

100

40Mun

eere

tal2013

Sialkot(Pakista

n)po

nds

Pseudomonas

sp

100

65Re

hman

etal2008

SouthKo

ream

unicipalwaste

water

treatmentp

lant

Pseudomonas

fluorescens

30343

Noghabietal2007

King

Abdu

l-AzizM

edicalCity

Jedd

ahSaudi

Arabia

Pseudomonas

aeruginosa

150

25Al-G

arni

etal2010

King

Abdu

l-AzizM

edicalCity

Jedd

ahSaudi

Arabia

Klebsiella

pneumoniae

100

15Al-G

arni

etal2010

WestC

oastof

India

Enterobacterclo

acae

100

0Iyer

etal2005

Cudd

alorec

oast(In

dia)

Vibrioflu

vialis

100

60Presentstudy


Acknowledgments

Theauthors gratefully acknowledge the financial support pro-vided by the ICMAM-PD Ministry of Earth Sciences Gov-ernment of India Grant no ICMAM-PDSWQMCASMB352012 to conduct this research

References

[1] S A Jafari and S Cheraghi ldquoMercury removal from aqueoussolution by dried biomass of indigenous Vibrio parahaemolyti-cus PG02 kinetic equilibrium and thermodynamic studiesrdquoInternational Biodeterioration and Biodegradation vol 92 pp12ndash19 2014

[2] H F Canstein Y Li A Felske and I Wagner-Dobler ldquoLong-term stability of mercury-reducing microbial biofilm commu-nities analyzed by 16S-23S rDNA interspacer region polymor-phismrdquoMicrobial Ecology vol 42 no 4 pp 624ndash634 2001

[3] P Li X B Feng G L Qiu L H Shang and Z G Li ldquoMercurypollution in Asia a review of the contaminated sitesrdquo Journal ofHazardous Materials vol 168 no 2-3 pp 591ndash601 2009

[4] Social Scientific StudyGroup onMinamataDisease In theHopeof Avoiding Repetition of Tragedy of Minamata Disease NationalInstitute for Minamata Disease Minamata Japan 1999

[5] S H Afrasayab A Yasmin and S H Hasnain ldquoCharacter-ization of some indigenous mercury resistant bacteria frompolluted environmentrdquo Journal of Biological Science vol 5 no7 pp 792ndash797 2007

[6] I Wagner-Dobler ldquoPilot plant for bioremediation of mercury-containing industrial wastewaterrdquo Applied Microbiology andBiotechnology vol 62 no 2-3 pp 124ndash133 2003

[7] A Sinha K K Pant and S K Khare ldquoStudies onmercury biore-mediation by alginate immobilized mercury tolerant Bacilluscereus cellsrdquo International Biodeterioration and Biodegradationvol 71 pp 1ndash8 2012

[8] F Matyar ldquoAntibiotic and heavy metal resistance in bacteriaisolated from the Eastern Mediterranean Sea coastrdquo Bulletin ofEnvironmental Contamination and Toxicology vol 89 no 3 pp551ndash556 2012

[9] E Gullberg L M Albrecht C Karlsson L Sandegren and DI Andersson ldquoSelection of a multidrug resistance plasmid bysublethal levels of antibiotics and heavy metalsrdquo mBio vol 5no 5 Article ID e01918-14 2014

[10] Clinical and Laboratory Standards Institute CLSI M100-S23Clinical and Laboratory Standards Clinical and LaboratoryStandards Institute Wayne 2013

[11] V V Umrania ldquoBioremediation of toxic heavy metals usingacidothermophilic autotrophesrdquoBioresource Technology vol 97no 10 pp 1237ndash1242 2006

[12] N L Figueiredo J Canario N J OrsquoDriscoll A Duarte andC Carvalho ldquoAerobic Mercury-resistant bacteria alter Mercuryspeciation and retention in the Tagus Estuary (Portugal)rdquoEcotoxicology and Environmental Safety vol 124 pp 60ndash672016

[13] S H AHassan R NN Abskharon SM F Gad El-Rab andAA M Shoreit ldquoIsolation characterization of heavy metal resis-tant strain of Pseudomonas aeruginosa isolated from pollutedsites in Assiut city Egyptrdquo Journal of Basic Microbiology vol 48no 3 pp 168ndash176 2008

[14] J Selvin S Shanmugha Priya G Seghal Kiran T Thangaveluand N Sapna Bai ldquoSponge-associated marine bacteria as indi-cators of heavy metal pollutionrdquo Microbiological Research vol164 no 3 pp 352ndash363 2009

[15] Y Nakahara Y Nakahara Y Ito and T Ogawa ldquoTotal synthesisof B-chain of human 1205722HS glycoproteinrdquo Tetrahedron Lettersvol 38 no 41 pp 7211ndash7214 1997

[16] M Aram A Sharifi F Kafeelzadeh M Naghmachi and EYasari ldquoIsolating Mercury-resistant bacteria from Lake Mahar-loordquo International Journal of Biology vol 4 no 3 pp 63ndash712012

[17] X X Zeng J X Tang X D Liu and P Jiang ldquoIsolationidentification and characterization of cadmium-resistant-Pseudomonas aeruginosa strain E1rdquo Journal of Central SouthUniversity of Technology vol 16 no 3 pp 416ndash421 2009

[18] J Claessens T Behrends and P Van Cappellen ldquoWhat do acid-base titrations of live bacteria tell usApreliminary assessmentrdquoAquatic Sciences vol 66 no 1 pp 19ndash26 2004

[19] F M M Morel and N M Price ldquoThe biogeochemical cycles oftrace metals in the oceansrdquo Science vol 300 no 5621 pp 944ndash947 2003

[20] S A Jafari S Cheraghi M Mirbakhsh R Mirza and AMaryamabadi ldquoEmploying response surface methodology foroptimization of mercury bioremediation by Vibrio para-haemolyticus PG02 in coastal sediments of Bushehr IranrdquoCleanmdashSoil Air Water vol 43 no 1 pp 118ndash126 2015

[21] C Green-Ruiz ldquoMercury(II) removal from aqueous solutionsby nonviable Bacillus sp from a tropical estuaryrdquo BioresourceTechnology vol 97 no 15 pp 1907ndash1911 2006

[22] A Hassen N Saidi M Cherif and A Boudabous ldquoResistanceof environmental bacteria to heavy metalsrdquo Bioresource Tech-nology vol 64 no 1 pp 7ndash15 1998

[23] A Rehman A Ali B Muneer and A R Shakoori ldquoResistanceand biosorption of mercury by bacteria isolated from industrialeffluentsrdquo Pakistan Journal of Zoology vol 39 no 3 pp 137ndash1462007

[24] B Muneer M J Iqbal F R Shakoori and A R ShakoorildquoTolerance and biosorption of mercury by microbial consortiapotential use in bioremediation of wastewaterrdquo Pakistan Journalof Zoology vol 45 no 1 pp 247ndash254 2013

[25] A Rehman A Ali andA R Shakoori ldquoBiosorption ofmercuryby bacteria isolated from industrial effluents potential use inbioremediation of wastewaterrdquo Pakistan Journal of Zoology vol40 no 2 pp 115ndash122 2008

[26] K A Noghabi H S Zahiri A S Lotfi J Raheb S Nasri andS C Yoon ldquoMercury absorption by Pseudomonas fluorescensBM07 grown at two different temperaturesrdquo Polish Journal ofMicrobiology vol 56 no 2 pp 111ndash117 2007

[27] S M Al-Garni KM Ghanem and A S Ibrahim ldquoBiosorptionof mercury by capsulated and slime layerforming Gram -vebacilli from an aqueous solutionrdquo African Journal of Biotechnol-ogy vol 9 no 38 pp 6413ndash6421 2010

[28] A Iyer K Mody and B Jha ldquoBiosorption of heavy metals by amarine bacteriumrdquoMarine Pollution Bulletin vol 50 no 3 pp340ndash343 2005

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 Similarity of minimum inhibitory mercury concentration value against that observed in the present strain Vibrio fluvialis to thosereported elsewhere

Strain MIC (120583gml) Location ReferenceVibrio fluvialis 1 Tagus Estuary (Portugal) Figueiredo et al 2016Vibrio natriegens 20 Coastal sediments Bushehr Iran Jafari and Cheraghi 2014Vibrio sp 12ndash16 Chesapeake Bay Walker and Colwell 1974Vibrio sp 271 Mai Po Nature Reserve Hong Kong Zhang et al 2006Vibrio parahaemolyticus 45 Coastal sediments Bushehr Iran Jafari and Cheraghi 2014Vibrio fluvialis 100 Parangipettai coast (India) Present study

antibiotic susceptible characteristics Subsequently its biore-mediation capability was investigated

2 Methods and Materials

21 Sample Collection Sediment sampleswere collected fromthe common effluent discharge point of the State IndustriesPromotion Corporation of Tamil Nadu Limited (SIPCOT)industrial area located in the banks of the Uppanar estu-ary Tamil Nadu southeast coast of India The geographiccoordinates of the station are 11∘411015840450010158401015840N latitude and79∘461015840050010158401015840E longitude Surface sediments were collectedaseptically in triplicate kept in an insulated box at 4∘C andimmediately transferred to the laboratory

22 Enrichment and Primary Screening Sediment sampleswere added to a 250ml Erlenmeyer flask containing 100ml ofZobell Marine Broth (ZMB) at pH 71 plusmn 01 incubated for 24 hand centrifuged at 160 rpm at 35∘C in a conventional rotaryshaker incubator The bacterial inocula were transferred toa 100ml ZMB with a supplement of HgCl

2and kept in an

orbital shaker at 200 rpm for 5 days

23 Antibiotic Sensitivity Test Antibiotic sensitivity test wasperformed using the disc diffusion method on MH agarwith antibiotic disks by following the methods of CLSI(2013) [10] Multiple antibiotic profiles of V fluvialis werechecked at the following antibiotic concentrations amoxi-cillin (10mcgdisc) bacitracin (10mcgdisc) erythromycin(15mcgdisc) amoxicillin (10mcgdisc) bacitracin (10mcgdisc) erythromycin (15mcgdisc) oxytetracycline (30mcgdisc) novobiocin (30mcgdisc) cephalothin (30mcgdisc)vancomycin (30mcgdisc) and amikacin (10mcgdisc) Bac-terial cultures swabbed on nutrient agar plates and the above-mentioned antibiotics discs were placed in the plates andincubated at 37∘C for 24 hrs

24 Growth Assessment of CASKS5 with Mercury Theselected V fluvialis overnight culture was inoculated into thenutrient broth which was supplemented at different concen-trations of HgCl

2such as 100 120583gml 150120583gml 200120583gml

and 250 120583gml in triplicate and kept in a shaking incubator at37∘C for 48 hrs Growth curves ofV fluvialiswere observed at

periodic time intervals that is 2 4 8 16 24 and 48 hrs usinga spectrophotometer (SHIMADZUUV 1800) at 600 nmOD

25 Bioremediation Capacity of CASKS5 After the comple-tion of the incubation period the cultures were centrifugedfor 20mins at 10000 rpm pellets removed and supernatantscollected and digested with nitric and sulfuric acids Theresidualmercury in themediumwas analyzed by a cold vapormercury analyzer (Model MA 5840)

3 Results and Discussion

Mercury-resistant bacterial strains were initially screenedusing the Luria Bertani (LB) medium in the presence of20 120583gml HgCl

2from sediment samples collected at effluent

discharge sites

31 Isolation Screening and Selection of Mercury-ResistantBacteria The high mercury-resistant strains were isolatedfrom sediments of industrial discharge sites using severalscreening techniques Prolonged exposure to mercury con-tamination can generate resistance mechanisms in bacteria[11] During preliminary findings 31 bacterial isolates exhib-ited high resistance to mercury Secondary screening wasperformed to estimate MIC The MIC results observed forall the 31 isolates confirmed that the strain CASKS5 hadthe highest mercury tolerance (100 120583gml concentration) asshown in Table 1 which is several times greater than thatobtained in an earlier study done on the same species byFigueiredo et al (2016) [12]

32 Biochemical Characterization and Molecular Identifica-tion of CASKS5 The selected strain CASKS5 was morpho-logically and biochemically characterized as a gram-negativecurved rod-shaped bacterium and tentatively identified asVibrio sp (Table 2) The 16S rDNA sequence was carried outfor CASKS5 and submitted to GenBank (NCBI 2013) forsearching similar published sequencesThe accessionnumberof the strain CASKS5 is KM186606

BLAST analysis revealed that the partial 16S rDNA ofCASKS5 hadmore than 99 similarity to that ofVibrio fluvi-alis strain in NCBI A phylogenetic tree based on 16S rDNAwas constructed using the MEGA 60 software to determinethe relationship between CASKS5 and V fluvialis (Figure 1)



CASKS5 (KM186605)










6798

39

45

75 58

47

002









Sucrose +



minus010

010203040506

Zone

of i

nhib

ition

(cm


Am

ikac

in

Am

oxyc

illin

Baci

trac

in

Cep

halo

thin

Eryt

hrom

ycin

Nov

obio

cin

Oxy

tetr

acyc

line

Vanc

omyc

in





minus020

02040608

11214


Control100

150

200

250

Gro

wth

OD

at 6

00nm

120583gml120583gml

120583gml120583gml






0

20

40

60

80

100

o

f mer

cury

rem

oval

100 150 200 250

(ppm)



4 Conclusion


Disclosure





Table3Biorem

ediatio

neffi

ciency

ofmercury

byVibrioflu

vialis(C

ASK

S5)com

paredwith

different

bacterialspecies

isolated

from

elsew

here

Locatio

nSpecies

Con

centratio

nof

mercury

used

forthe

experim

ent

120583gml

Biorem

ediatio

neffi

ciency

(inpercentage)

References

Bushehr(Iran)coast

Vibrioparahaem

olyticu

s10

gt8947

Jafarietal2015

Urıa

sestu

arySinaloaMexico

Bacillussp

10681

Green-Ruiz2006

Bacillusthu

ringiensis

427

Hassenetal1998

Sialkot(Pakista

n)po

ndBrevibacteriu

mcasei

5070

Rehm

anetal2007

Sialkot(Pakista

n)po

ndPseudomonas

aeruginosa

5080

Rehm

anetal2007

Kasur(Pakista

n)po

nds

Tetra

hymenarostrata

100

40Mun

eere

tal2013

Sialkot(Pakista

n)po

nds

Pseudomonas

sp

100

65Re

hman

etal2008

SouthKo

ream

unicipalwaste

water

treatmentp

lant

Pseudomonas

fluorescens

30343

Noghabietal2007

King

Abdu

l-AzizM

edicalCity

Jedd

ahSaudi

Arabia

Pseudomonas

aeruginosa

150

25Al-G

arni

etal2010

King

Abdu

l-AzizM

edicalCity

Jedd

ahSaudi

Arabia

Klebsiella

pneumoniae

100

15Al-G

arni

etal2010

WestC

oastof

India

Enterobacterclo

acae

100

0Iyer

etal2005

Cudd

alorec

oast(In

dia)

Vibrioflu

vialis

100

60Presentstudy


Acknowledgments


References




































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



CASKS5 (KM186605)










6798

39

45

75 58

47

002









Sucrose +



minus010

010203040506

Zone

of i

nhib

ition

(cm


Am

ikac

in

Am

oxyc

illin

Baci

trac

in

Cep

halo

thin

Eryt

hrom

ycin

Nov

obio

cin

Oxy

tetr

acyc

line

Vanc

omyc

in





minus020

02040608

11214


Control100

150

200

250

Gro

wth

OD

at 6

00nm

120583gml120583gml

120583gml120583gml






0

20

40

60

80

100

o

f mer

cury

rem

oval

100 150 200 250

(ppm)



4 Conclusion


Disclosure





Table3Biorem

ediatio

neffi

ciency

ofmercury

byVibrioflu

vialis(C

ASK

S5)com

paredwith

different

bacterialspecies

isolated

from

elsew

here

Locatio

nSpecies

Con

centratio

nof

mercury

used

forthe

experim

ent

120583gml

Biorem

ediatio

neffi

ciency

(inpercentage)

References

Bushehr(Iran)coast

Vibrioparahaem

olyticu

s10

gt8947

Jafarietal2015

Urıa

sestu

arySinaloaMexico

Bacillussp

10681

Green-Ruiz2006

Bacillusthu

ringiensis

427

Hassenetal1998

Sialkot(Pakista

n)po

ndBrevibacteriu

mcasei

5070

Rehm

anetal2007

Sialkot(Pakista

n)po

ndPseudomonas

aeruginosa

5080

Rehm

anetal2007

Kasur(Pakista

n)po

nds

Tetra

hymenarostrata

100

40Mun

eere

tal2013

Sialkot(Pakista

n)po

nds

Pseudomonas

sp

100

65Re

hman

etal2008

SouthKo

ream

unicipalwaste

water

treatmentp

lant

Pseudomonas

fluorescens

30343

Noghabietal2007

King

Abdu

l-AzizM

edicalCity

Jedd

ahSaudi

Arabia

Pseudomonas

aeruginosa

150

25Al-G

arni

etal2010

King

Abdu

l-AzizM

edicalCity

Jedd

ahSaudi

Arabia

Klebsiella

pneumoniae

100

15Al-G

arni

etal2010

WestC

oastof

India

Enterobacterclo

acae

100

0Iyer

etal2005

Cudd

alorec

oast(In

dia)

Vibrioflu

vialis

100

60Presentstudy


Acknowledgments


References




































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


minus020

02040608

11214


Control100

150

200

250

Gro

wth

OD

at 6

00nm

120583gml120583gml

120583gml120583gml






0

20

40

60

80

100

o

f mer

cury

rem

oval

100 150 200 250

(ppm)



4 Conclusion


Disclosure





Table3Biorem

ediatio

neffi

ciency

ofmercury

byVibrioflu

vialis(C

ASK

S5)com

paredwith

different

bacterialspecies

isolated

from

elsew

here

Locatio

nSpecies

Con

centratio

nof

mercury

used

forthe

experim

ent

120583gml

Biorem

ediatio

neffi

ciency

(inpercentage)

References

Bushehr(Iran)coast

Vibrioparahaem

olyticu

s10

gt8947

Jafarietal2015

Urıa

sestu

arySinaloaMexico

Bacillussp

10681

Green-Ruiz2006

Bacillusthu

ringiensis

427

Hassenetal1998

Sialkot(Pakista

n)po

ndBrevibacteriu

mcasei

5070

Rehm

anetal2007

Sialkot(Pakista

n)po

ndPseudomonas

aeruginosa

5080

Rehm

anetal2007

Kasur(Pakista

n)po

nds

Tetra

hymenarostrata

100

40Mun

eere

tal2013

Sialkot(Pakista

n)po

nds

Pseudomonas

sp

100

65Re

hman

etal2008

SouthKo

ream

unicipalwaste

water

treatmentp

lant

Pseudomonas

fluorescens

30343

Noghabietal2007

King

Abdu

l-AzizM

edicalCity

Jedd

ahSaudi

Arabia

Pseudomonas

aeruginosa

150

25Al-G

arni

etal2010

King

Abdu

l-AzizM

edicalCity

Jedd

ahSaudi

Arabia

Klebsiella

pneumoniae

100

15Al-G

arni

etal2010

WestC

oastof

India

Enterobacterclo

acae

100

0Iyer

etal2005

Cudd

alorec

oast(In

dia)

Vibrioflu

vialis

100

60Presentstudy


Acknowledgments


References




































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table3Biorem

ediatio

neffi

ciency

ofmercury

byVibrioflu

vialis(C

ASK

S5)com

paredwith

different

bacterialspecies

isolated

from

elsew

here

Locatio

nSpecies

Con

centratio

nof

mercury

used

forthe

experim

ent

120583gml

Biorem

ediatio

neffi

ciency

(inpercentage)

References

Bushehr(Iran)coast

Vibrioparahaem

olyticu

s10

gt8947

Jafarietal2015

Urıa

sestu

arySinaloaMexico

Bacillussp

10681

Green-Ruiz2006

Bacillusthu

ringiensis

427

Hassenetal1998

Sialkot(Pakista

n)po

ndBrevibacteriu

mcasei

5070

Rehm

anetal2007

Sialkot(Pakista

n)po

ndPseudomonas

aeruginosa

5080

Rehm

anetal2007

Kasur(Pakista

n)po

nds

Tetra

hymenarostrata

100

40Mun

eere

tal2013

Sialkot(Pakista

n)po

nds

Pseudomonas

sp

100

65Re

hman

etal2008

SouthKo

ream

unicipalwaste

water

treatmentp

lant

Pseudomonas

fluorescens

30343

Noghabietal2007

King

Abdu

l-AzizM

edicalCity

Jedd

ahSaudi

Arabia

Pseudomonas

aeruginosa

150

25Al-G

arni

etal2010

King

Abdu

l-AzizM

edicalCity

Jedd

ahSaudi

Arabia

Klebsiella

pneumoniae

100

15Al-G

arni

etal2010

WestC

oastof

India

Enterobacterclo

acae

100

0Iyer

etal2005

Cudd

alorec

oast(In

dia)

Vibrioflu

vialis

100

60Presentstudy


Acknowledgments


References




































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Acknowledgments


References




































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Bioremediation of Mercury by Vibrio fluvialis Screened...

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