Research Article Molybdate Reduction to Molybdenum Blue by ...downloads.hindawi.com/journals/bmri/2013/871941.pdf · metals to terrestrial Antarctic organisms and precautionary steps

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 871941 10 pageshttpdxdoiorg1011552013871941

Research ArticleMolybdate Reduction to Molybdenum Blueby an Antarctic Bacterium

S A Ahmad1 M Y Shukor1 N A Shamaan2 W P Mac Cormack3 and M A Syed1

1 Department of Biochemistry Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia43400 Serdang Selangor Malaysia

2 Faculty of Medicine and Health Sciences Universiti Sains Islam Malaysia 13th Floor Menara B Persiaran MPAJJalan Pandan Utama Pandan Indah 55100 Kuala Lumpur Malaysia

3 lnstituto Antartico Argentino Cerrito 1248 (1010) Buenos Aires Argentina

Correspondence should be addressed to M Y Shukor mohdyunusupmedumy

Received 24 July 2013 Revised 22 October 2013 Accepted 9 November 2013

Academic Editor Marcelo A Soares

Copyright copy 2013 S A Ahmad et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A molybdenum-reducing bacterium from Antarctica has been isolated The bacterium converts sodium molybdate or Mo6+ tomolybdenum blue (Mo-blue) Electron donors such as glucose sucrose fructose and lactose supported molybdate reductionAmmonium sulphate was the best nitrogen source for molybdate reduction Optimal conditions for molybdate reduction werebetween 30 and 50mMmolybdate between 15 and 20∘C and initial pH between 65 and 75 The Mo-blue produced had a uniqueabsorption spectrumwith a peakmaximumat 865 nmand a shoulder at 710 nm Respiratory inhibitors such as antimycinA sodiumazide potassium cyanide and rotenone failed to inhibit the reducing activityTheMo-reducing enzyme was partially purified usingion exchange and gel filtration chromatography The partially purified enzyme showed optimal pH and temperature for activityat 60 and 20∘C respectively Metal ions such as cadmium chromium copper silver lead and mercury caused more than 95inhibition of the molybdenum-reducing activity at 01mM The isolate was tentatively identified as Pseudomonas sp strain DRY1based on partial 16s rDNA molecular phylogenetic assessment and the Biolog microbial identification system The characteristicsof this strain would make it very useful in bioremediation works in the polar and temperate countries

1 Introduction

Heavy metals pollution is a global problem Remediationof heavy metals contaminated sites by bacteria is becomingmore andmore importantMicrobes have the amazing abilityto resist the toxicity of heavy metals and this property isuseful for bioremediation purposes [1ndash4] It is very toxicto ruminants and its bioremediation has been reported [5]Traces of heavy metals due to anthropogenic sources havebeen found in soils in Antarctic and toxicity effects on fishin the surrounding area have been reported to occur [6ndash8]Few studies have been carried out to study the possibility ofmicrobes for the remediation of heavymetals in cold regionsLow temperature microbial reduction of chromium [9] andmercury [10] has been reported

Molybdenum is a ubiquitous metal and often pollution ofthis metal comes frommining operations and anthropogenic

sources [11]Molybdenum is fast becoming a global pollutantIts pollution ranging from several hundreds to thousands ofpart per million has been documented in water and soils allaround the world [11 12] It is not toxic to human but deadlyto ruminants at several parts per million [13] Molybdenumdeposits have been identified in Antarctica [14] and althoughthese deposits have been banned frommining by the Madridprotocol the ban is up for review in 2041 [15] Even withoutthe presence of mining activities molybdenum has beenfound as a pollutant in Antarctica due to anthropogenicsources [8]There is very limited study on the effects of heavymetals to terrestrial Antarctic organisms and precautionarysteps in removing heavy metals pollution should be taken

Bioremediation using microbes may be the only cheapand workable technology to remediate metal contaminantsin Antarctica and other cold regionsThus it is vital that cold-tolerant molybdenum-reducing microbes are isolated from

2 BioMed Research International

polar regions as a preparative step for future bioremediationworks It is also anticipated that other cold region areas wouldbenefit from this research In this work we report on theisolation and characterization of a psychrotolerant bacteriumwith the ability to transform molybdenum to the insolublemolybdenum blue This is the first molybdenum-reducingbacterium isolated from cold region This bacterium couldbe used in future remediation works of molybdenum inAntarctica and cold climate regions

2 Experimental

21 Isolation of Molybdenum-Reducing Bacterium Sampleswere collected from jubany Station an Argentinean Baseon King George Island South Shetlands Islands Antarctica(615∘S 5455∘W) in the austral summer 2002-2003 expedi-tion Soils were collected 15ndash20 centimetres (cm) beneath thesurface and were placed in sterile screw-capped vials Thesamples were immediately placed in a freezer and stored atminus20∘C until returned to the laboratory for further examina-tion About five grams of soil sample that was well-mixedwere suspended in 45mL of 09 saline solution A suitableserial dilution of soil suspension was then spread platedonto an agar of low phosphate medium (LPM) (28mMphosphate) (pH 70) The medium (wv) consisted of glu-cose (1) MgSO

4sdot7H2O (005) (NH

4)2SO4(03) NaCl

(05) Na2MoO4sdot2H2O (0242) yeast extract (005) and

Na2HPO4sdot2H2O (005) to isolate molybdenum-reducing

bacteria [16] After 72 hours of incubation at 10∘C severalwhite and blue colonies appeared The colony exhibiting thestrongest blue intensity was then inoculated into 50mLof lowphosphate medium and incubated at 10∘C for 72 hours stati-cally Incubation under aerobic conditions gave lower amountof Mo-blue compared to static incubation It was observedthat during cellular reduction of molybdate cells precipitatedtogether with the molybdenum blue product preventingcellular growth A portion of the growth medium containingMo-blue was centrifuged at 10000timesg for 10min and thesupernatant was scanned from 400 to 980 nm using a Cintra5 spectrophotometer A freshly prepared low phosphatemedium was used for baseline correction This bacteriumwas kept in the Bioremediation and Bioassay Laboratory (Lab204) The production of molybdenum blue in the culturesupernatant was monitored at 865 nm Identification of thebacterium was performed by using molecular phylogeneticsstudies and Biolog GN MicroPlate (Biolog Hayward CAUSA) according to the manufacturerrsquos instructions

22 Optimization of Molybdenum Reduction Optimizationof several parameters that support molybdenum reductionto molybdenum blue such as temperature pH the effectof carbon sources as electron donors the effect of nitrogensources sodium molybdate and phosphate concentrationswere studied using the LPM in Section 21 as the basalmedium for 48 hours statically

23 16s rDNA Gene Sequencing and Phylogenetic AnalysisExtraction of genomic DNA was performed using the alka-line lysis method [17] PCR amplification was carried out

on a Biometra T Gradient PCR (Montreal Biotech IncKirkland QC Canada) The PCR mixture consisted of 05pM of each primer 1x reaction buffer 25U of Taq DNApolymerase (Promega) and 200120583Mof each deoxynucleotidetriphosphate at the final volume of 50120583L Amplificationof the 16s rDNA gene was carried out using the follow-ing primers 51015840-AGAGTTTGATCCTGGCTCAG-31015840 and 51015840-AAGGAGGTGATCCAGCCGCA-31015840 corresponding to theforward and reverse primers of 16s rDNA respectively[18] PCR was carried out under the following conditionsinitial denaturation at 94∘C for 3min 25 cycles of 94∘C for1min 50∘C for 1min and 72∘C for 2min a final extensionat 72∘C for 10min Cycle sequencing was subsequentlyperformed with the Big Dye terminator kit (Perkin-ElmerApplied Biosystems) as recommended by the manufacturerSequence data were initially recorded and edited usingCHROMAS version 145 The resultant 1452 bases werecompared with the GenBank database using the Blast serverat NCBI (httpwwwncbinlmnihgovBLAST) Analysisshowed that this sequence to be closely related to rrsfrom Gammaproteobacteria The 16s rRNA ribosomal genesequence for this isolate has been deposited in GenBankunder the accession number DQ226202

24 Phylogenetic Analysis Twenty-three 16S rRNA genesequences closely matched to isolate DRY1 were re-trieved from GenBank and a multiple alignment of thesequences was carried out using clustal W [19] A phylo-genetic tree was constructed by using PHYLIP version3573 (J Q Felsenstein PHYLIPmdashphylogeny inference pack-age version 3573 Department of Genetics Univer-sity of Washington Seattle WA USA (httpevolutiongeneticswashingtoneduphyliphtml)) [20] with Serratiamarcescens as the outgroup in the cladogram Evolutionarydistance matrices for the neighbour-joiningUPGMAmethod were computed using the DNADIST algorithmprogram Phylogenetic tree was inferred by using theneighbour-joining method of Saitou and Nei [21] With eachalgorithm confidence levels for individual branches withinthe tree were checked by repeating the PHYLIP analysiswith 1000 bootstraps by the SEQBOOT program in thePHYLIP package Majority rule (50) consensus trees wereconstructed using the Ml methods [22] and the tree wasviewed using TreeView [23]

25 Crude Enzyme Preparation Crude enzyme was pre-pared from a 2 L culture grown at 20∘C for 72 hours onan orbital shaker at 150 rpm on a modified high phos-phate medium (HPM) consisting ofMgSO

4sdot7H2O (05 gLminus1)

(NH4)2SO4(3 gLminus1) yeast extract (1 gLminus1) NaCl (5 gLminus1)

NaMoO4sdot2H2O) (121 gLminus1 or 50mM) glucose (10 gLminus1) as

the source of electron donor and Na2HPO4sdot2H2O (100mM)

at pH 73 Growth at high phosphate under aerobic con-ditions prevents Mo-blue production but cells containedhigh enzyme activity Experiments were carried out at 4∘Cunless stated otherwise Bacterial cells were first harvestedat 10 000timesg for 20min at 4∘C The pellet was then reconsti-tuted in 15mL of 50mM TrissdotCl buffer (pH 70) containing

BioMed Research International 3

1mMphenylmethanesulphonylfluoride (PMSF) as a proteaseinhibitor and 2mM of DTT The cells were then sonicatedon a Biosonik 111 sonicator on an ice bath and then ultra-centrifuged at 105000timesg for 90min at 4∘C The supernatantcontaining the crude enzyme was collected The enzyme hadan optimum temperature at 20∘C (data not shown)

26 Enzyme Assay Enzymewas assayed at 20∘C according tothe method of Shukor et al [24]The reaction mixture (1mL)contained 3mM of 12-molybdophosphate (electron acceptorsubstrate) in 50mM citrate phosphate buffer pH 50 at roomtemperature and 100 120583L of NADH at the final concentrationof 3mM Fifty microlitres of enzyme fraction containingabout 1mg protein were added to start the reaction Theabsorbance increase in one minute was read at 865 nm Oneunit of Mo-reducing activity is defined as the amount ofenzyme that produces 1 nmole Mo-blue per minute at 20∘CThe specific extinction coefficient for the product Mo-blue at865 nm is 167mMminus1sdotcmminus1 [25]

27 Ion Exchange and Gel Filtration ChromatographyAmmonium sulphate fractionation was omitted as it gavevery low enzyme yield The experiment was carried out at4∘C unless stated otherwise Crude fraction (cytoplasmicfraction after ultracentrifuge) was directly applied tothe strong anion exchange matrix Mono-Q (AmershamPharmacia) The column was first equilibrated with 50mLof 50mM TrissdotCl pH 75 (buffer A) containing 01mM DTTAbout 40 milligrams of the crude enzyme in 2mL of volumewas injected into the Mono-Q column at a flow rate of 1mLper minute and then washed with 20mL of the same bufferEnzyme was eluted from the column with a linear gradientof 0ndash05MNaCl in buffer A at a flow rate of 1mLminminus1The protein elution profile was monitored at 280 nm Thisprocess was repeated several times until the entire crudefraction was loaded into the column

The fractions with enzyme activity were pooled anddialyzed against 5 L of buffer A overnight The dialyzedenzyme was then concentrated using a cellulose triacetate fil-termembranewith amolecular weight cut-off point of 10 kDain an Amicon ultrafiltration cell at 4∘C to a final volume of05mL One hundredmicrolitres of sample containing 12mgof protein was applied to Zorbax GFC-250 column (250 times94mm) and eluted using buffer A containing 02MKCL ata flow rate of 05mLminminus1 Protein was quantified accordingto the method of Bradford [26] using BSA as the standardMichaelis Menten kinetics constants were determined usingGraphPad prism nonlinear regression analysis available fromhttpwwwGraphpadcom

28 The Effects of Respiratory Inhibitors and Metal IonsRespiratory inhibitors such as antimycin A sodium azidepotassium cyanide and rotenone were dissolved in acetoneor deionised water and were added into enzyme assaymixture to the final concentrations of 12 10 10 and02mM Metal ions such as Cr6+ (K

2Cr2O7 BDH) Fe3+

(FeCl3sdot6H2O BDH) Zn2+ (ZnCl

2 BDH) Mg2+ (MgCl

2

BDH) Co2+ (CoCl2sdot6H2O BDH) Ni2+ (NiCl

2sdot6H2O BDH)

Cd2+ (CdCl2sdotH2O SparkChem) Ag+ (AgNO

3 JT Baker)

Mn2+ (MnCl2sdot4H2O JT Baker) Cu2+ (CuSO

4sdot5H2O JT

Baker) Hg2+ (HgCl2 JT Baker) and Pb2+ (PbCl

2 JT Baker)

were dissolved in 20mM TrissdotCl buffer pH 70 and wereadded into enzyme assay mixture to the final concentrationof 01mM Inhibitors and metal ions were then preincubatedwith 100 120583L of enzyme in the reaction mixture at 4∘C for10 minutes minus NADH The incubation mixture was thenwarmed to 20∘C and 100 120583L of NADH at the final concentra-tion of 3mMwas added to start the reaction Deionised waterwas added so that the total reaction mixture was 10mL As acontrol 50120583L of acetone was added in the reaction mixturewithout inhibitors

29 Dialysis Tubing Experiment Briefly the bacterium wasgrown in 250mL high phosphate medium with shaking at150 rpm at 20∘C for 72 hours Cells were harvested by cen-trifugation at 15000 g for 10 minutes The pellet was washedseveral times with distilled water and then resuspended in50mL of low phosphate solution (pH 70) at 20∘C contain-ing (wv) MgSO

4sdot7H2O (005) NaCl (05) (NH

4)2SO4

(03) yeast extract (005) and Na2HPO4sdot2H2O (005)

with sodiummolybdate omitted About 10mL of this suspen-sion was transferred into dialysis tubing that was previouslyboiled for ten minutes (12 kDa molecular weight cutoff) Thetube was then immersed in sterile 50mL of low phosphatemedium (pH 70) for 10 hours at 20∘C statically as describedin Section 21 Periodically aliquots (1mL) of the externalmedium were taken at the start of the experiment and afteran incubation period of 10 hours and then the molybdenumblue produced was monitored at 865 nm At the same time1mL was taken out from the dialysis tubing and centrifugedat 15000 g for 10 minutes The supernatant was then read at865 nm Experiments were carried out in triplicate

210 Statistical Analysis Values aremeansplusmn SE All data wereanalyzed using Graphpad Prism version 30 and GraphpadInStat version 305 available at wwwgraphpadcom Compar-ison between groups was performed using a Studentrsquos 119905-testor a one-way analysis of variance with post hoc analysis byTukeyrsquos test 119875 lt 005 was considered statistically significant

3 Results

31 Identification of the Isolate A low bootstrap value (lt50)was seen associating isolate DRY1 to several Pseudomonasspecies such asP panacisP fulgidaP gessardiiPmucidolensP azotoformans and P reactants indicating that the tyingup of isolated DRY1 to any Pseudomonas species cannot bedone at thismomentThe identifications performed by BiologGN also gave no conclusive identification to the species levelwith the closest identification to several Pseudomonas specieswith very low probability For now isolate DRY1 is assignedtentatively as Pseudomonas sp strain DRY1 (Figure 1)

32The Effect of ElectronDonor onMolybdate Reduction Theelectron donor glucose sucrose fructose and lactose sup-ported molybdate reduction in decreasing order of efficiency


100

856483

626

902

622

995

372

356

997593

192

245

591502

871

816986

971

842892

433

602

Bacillus subtilis strain KT1003 [AB115959]

Pseudomonas syringae isolate Lz4 [WAJ576247]

Pseudomonas migulae [AF074383]

Pseudomonas panacis strain CG20106 [AY787208]

Pseudomonas fulgida type strain DSM 14938T [AJ492830]

Pseudomonas gessardii [AF074384]

Pseudomonas mucidolens [D84017]

Pseudomonas azotoformans [D84009]

Pseudomonas reactants [AY747594]

Uncultured bacterium KFGS-JG36-13 [AJ295653]

Strain DrY1

Pseudomonas migulae [AY047218]

Pseudomonas sp isolate EK1 [AJ237965]

Pseudomonas sp An1 isolate An1 [AJ551142]


Pseudomonas tolaasii [D84028]

Pseudomonas fluorescens [D84013]

Pseudomonas veronii strain INA06 [AB056120]

Pseudomonas borealis [AJ012712]

Pseudomonas cannabina type strain CFBP 2341T [AJ492827]

Pseudomonas tremae type strain CFBP 6111T [AJ492826]

Pseudomonas caricapapayae [D84010]

Pseudomonas corrugata [D84012]

Pseudomonas kilonensis strain 520-20 [AJ292426]

Pseudomonas fluorescens strain F113 [AJ278814]

Figure 1 A phylogram (neighbour-joining method) showinggenetic relationship between strainDRY1 and other related referencemicroorganisms based on the 16S rRNA gene sequence analysisSpecies names are followed by the accession numbers of their 16SrRNA sequences The numbers at branching points or nodes referto bootstrap values based on 1000 resamplings Scale bar represents100 nucleotide substitutions B subtilis is the outgroup

with glucose and sucrose exhibiting more than four timesthe amount of Mo-blue compared to lactose and fructose(Figure 2) Optimum molybdate reduction was achieved at1 (wv) glucose after 72 hours (data not shown) Theexploitation of the Biologrsquos carbon sources as electron donorsformolybdate reductionwas not possible since we discoveredthat the purplish color of the reduced formazan was toointense and masked the appearance of molybdenum blueThe Biolog system utilizes 95 carbon sources as a metabolicfingerprint for microbial identification

33 The Effect of Nitrogen Sources on Molybdate ReductionThe effect of nitrogen sources on molybdate reduction

00

10

20

30

40

50

60

70

Citr

ic ac

id

Con

trol

Form

ate

Fruc

tose

Glu

cose

Lact

ose

Lact

ose

Man

nito

l

Star

ch

Sucr

ose

Mo-

blue

(120583m

ole)

Figure 2 Molybdate reduction using various electron donorsIsolate DRY1 was grown at 10∘C for 72 hours in low phosphateliquid medium (pH 70) containing (wv) MgSO

4sdot7H2O (005)

(NH4)2SO4(03) NaCl (05) Na

2MoO4sdot2H2O (0242) yeast

extract (005) and Na2HPO4sdot2H2O (005) and various electron

donors at the final concentration of 02 Molybdate reduction isconsidered negligible if the absorbance at 865 nm is below 0020Error bars represent mean plusmn standard error (119899 = 3)

was studied using ammonium formate ammonium sul-phate ammonium chloride sodium nitrate sodium nitriteoxaloacetate and the amino acids alanine asparagine aspar-tic acid valine cysteine glutamic acid glycine histidineleucine and OH-proline Ammonium sulphate was found tobe the most effective supplement for supporting molybdate(Figure 3) Concentrations of ammonium sulphate givingoptimummolybdate reduction were between 02 and 03Further increase in ammonium sulphate concentration showsa strong inhibitory effect on molybdate reduction (data notshown)

34 The Effect of Temperature and Initial pH on MolybdateReduction Theeffect of temperature onmolybdate reductionwas carried out at temperatures from 0 to 40∘CThe optimumtemperature for molybdate reduction was in between 10and 20∘C (Figure 4) with ANOVA analysis showing thatthere was no significant difference (119875 gt 005) in terms ofmolybdate production at these temperatures The optimuminitial pH for reductionwas between pH65 and 75 (Figure 5)with ANOVA analysis showing that there was no significantdifference (119875 gt 005) in terms of molybdate production atthese pHs

35The Effects of Molybdate and Phosphate Concentrations onMolybdate Reduction Molybdate reduction was discoveredto increase linearly asmolybdate concentrationwas increasedfrom 0 to 40mM and reached a plateau in between theconcentrations of 30 and 50mM Concentrations higher than50mMwere strongly inhibitory (Figure 6) Whenmolybdate


00

10

20

30

40

50

60

70

Am

m f

orm

ate

L-hi

stidi

ne

L-as

part

ate

L-le

ucin

e

Am

m c

hlor

ide

L-ph

enyl

alan

ine

Am

m s

ulph

ate

Sodi

um n

itrat

e

Gly

cine

Hyd

roxy

-L-p

rolin

e

L-cy

stine

Con

trol

Mo-

blue

(120583m

ole)

Figure 3 Molybdate reduction using various nitrogen sourcesIsolate DRY1 was grown at 10∘C for 72 hours in low phosphate liquidmedium (pH 70) containing (wv) glucose (1) MgSO

4sdot7H2O

(005) NaCl (05) Na2MoO4sdot2H2O (0242) yeast extract

(005) Na2HPO4sdot2H2O (005) and various nitrogen sources at

the final concentration of 02 Molybdate reduction is considerednegligible if the absorbance at 865 nm is below 0020 Error bars rep-resent the standard error of themean between three determinations

00

20

40

60

80

100

0 10 20 30 40Temperature (∘C)

Mo-

blue

(120583m

ole)

Figure 4 The effect of temperature on molybdate reduction byisolate DRY1 Isolate DRY1 was grown statically at various tempera-tures for 72 hours in 50mL low phosphate liquid medium (pH 70)containing (wv) glucose (1) MgSO

4sdot7H2O (005) NaCl (05)

Na2MoO4sdot2H2O (0242) yeast extract (005) Na

2HPO4sdot2H2O

(005) and ammonium sulphate (02) Error bars representmeanplusmn standard error (119899 = 3)

concentration was fixed at 45mM the optimum concen-tration of phosphate for molybdate reduction was 5mMMolybdate reductionwas inhibited atmuchhigher phosphateconcentrations and total inhibition occurred at 100mMphosphate (Figure 7)

65

7

75

8

85

9

5 55 6 65 7 75 8 85pH

Mo-

blue

(120583m

ole)

Figure 5 The effect of initial pH on molybdate reduction byisolate DRY1 Isolate DRY1 was grown statically at 20∘C for 72hours in 50mL low phosphate liquid medium at various pHscontaining (wv) glucose (1) MgSO



2HPO4sdot2H2O


00

20

40

60

80

100

0 20 40 60 80 100 120Molybdate (mM)

Mo-

blue

(120583m

ole)

Figure 6 The effects of molybdate concentrations on molybdatereduction by isolate DRY1 Isolate DRY1 was grown at 20∘C for 72hours in low phosphate liquid medium (pH 70) containing (wv)glucose (1) MgSO

4sdot7H2O (005) NaCl (05) yeast extract

(005) Na2HPO4sdot2H2O (005) and ammonium sulphate (02)

Error bars represent mean plusmn standard error (119899 = 3)

36 Molybdenum Blue Absorption Spectrum The molybde-num blue produced from this isolate showed a spectrumwith an absorption profile that exhibited a maximum peakat 865 nm and a shoulder at 710 nm This unique profileincreases proportionately to the blue intensity as molybdatereduction progressed (Figure 8)


0

2

4

6

8

10

0 20 40 60 80 100 120Phosphate (mM)

Mo-

blue

(mm

ole)

Figure 7 The effects of phosphate concentrations on molybdatereduction by isolate DRY1 Isolate DRY1 was grown statically at 20∘Cfor 72 hours in 50mL liquid medium (pH 70) containing (wv)glucose (1) MgSO


(005) ammonium sulphate (02) Na2MoO4sdot2H2O (121) and

various phosphate (Na2HPO4sdot2H2O) concentrations Error bars

represent mean plusmn standard error (119899 = 3)

0

1

2

3

4

5

400 500 600 700 800 900 1000Wavelength (nm)

Abs

A

B

C

D

E

Figure 8 Scanning spectra of molybdenum blue after 28 32 36 48and 72 hours of static incubation at 20∘C labeled (A) (B) (C) (D)and (E) respectively

37 Characterization of the Molybdate Reduction ReactionUsing the Dialysis Tubing Method The result showed that95 of the amount ofMo-blue formed (13775plusmn0395 120583mole)was found in the dialysis tube while only 5 (1225 plusmn0025 120583mole) was found at the outside of the tube

38 Partial Purification of Mo-Reducing Activity A five-foldpurification was achieved after gel filtration but the yieldwas too low to incorporate another chromatographic step

Table 1 Partial purification scheme of Mo-reducing enzyme fromPseudomonas sp strain DRY1

FractionTotalprotein(mg)

Specificactivity

(Unitsmgprotein)

Totalactivity(Units)

Yield

Foldpurification

Crude 400 3 1200 1000 10Mono Q 36 90 324 27 30Zorbax GFC-250 24 152 3648 304 506

Table 2 Effect of respiratory inhibitors on molybdate reductionData represent mean plusmn standard error (119899 = 3)

Respiratory inhibitors Mo-reducing enzyme activityControl 10029 plusmn 1013

Cyanide (12mM) 10241 plusmn 882

Antimycin A (10mM) 10571 plusmn 735

Rotenone (10mM) 9150 plusmn 581

Azide (02mM) 9260 plusmn 1073

Native-PAGE results showed that multiple bands indicatingpurification were not successful (data not shown) Table 1shows that ion exchange and gel filtration chromatographyremoved much of the enzyme yield

39 Kinetics of the Mo-Reducing Enzyme The partially puri-fied enzyme had an optimum pH for activity at 60 andoptimum temperature for activity at 20∘C Preliminary resultsshow that 20mM phosphomolybdate (electron acceptor sub-strate) was saturating A plot of initial rates against electrondonor substrate concentrations at 15mM phosphomolybdateregistered a119881max for NADH at 2698 nmole Mo blueminmgprotein and an apparent 119870

119898of 468mM The 119881max for

NADPH was 2904 nmole Mo blueminmg protein and theapparent119870

119898for NADPH was 739mM

At 25mM NADH (saturating NADH concentration offive times119870

119898) the apparent119881max and apparent119870119898 values for

phosphomolybdate were 2348 nmoleminmg protein and352mM respectivelyWhen the electron donor substratewasNADPH The apparent 119881max and 119870

119898values for phospho-

molybdate were 2775 nmoleminmg protein and 38mMrespectively

310 Effect of Respiratory Inhibitors and Metal Ions on Molyb-date Reduction In this work it was discovered that noneof the respiratory inhibitors tested showed any appreciableinhibition of more than 10 to the Mo-reducing activity(Table 2) Metal ions such as chromium copper cadmiumlead silver and mercury cause more than 95 inhibition ofthe molybdenum-reducing activity (Table 3) The remainingmetal ions tested showed no effect on the molybdenum-reducing activity


Table 3 Effect ofmetal ions onmolybdate reductionData representmean plusmn standard error (119899 = 3)

Metal ions (01mM) Molybdenum blue produced(nmoleminmg)

Control1000 plusmn 017

Cr6+005 plusmn 002

Fe3+944 plusmn 051

Zn2+939 plusmn 020

Mg2+953 plusmn 023

Co2+957 plusmn 021

Ni2+935 plusmn 023

Cd2+046 plusmn 002

Ag+023 plusmn 087

Mn2+911 plusmn 021

Cu2+000 plusmn 012

Hg2+010 plusmn 003

Pb2+050 plusmn 011

4 Discussions

Microbial molybdate reduction to molybdenum blue isan interesting phenomenon and is important not only asa bioremediation tool but also for advancing knowledgeon microbial metal reduction According to Levine [27]molybdate-reducing property was first reported in E coli byCapaldi and Proskauer in 1896 [28] The first detailed studyon this phenomenon was reported by Campbell et al [29]This is followed by Sugio et al [30] and Ghani et al [16]From the year 2000 onwards works on microbial molybdatereduction to molybdenum blue were exclusively carried outon local Malaysian isolates [24 25 31ndash40]

In this work we report on the first isolation of a Mo-reducing bacterium from Antarctica and a second from theMo-reducing bacterium from the Pseudomonas genera [40]Further identification of the bacterium to the species levelcan be done by adding several more polyphasic identificationmethods such as 16S rRNA gene sequencing determinationof genomic DNA G+C content DNA-DNA hybridizationand fatty acid profile [41]

Previous works have shown that easily assimilable car-bon sources such as sucrose glucose and fructose are thepreferred electron donor source for molybdate reductionIn this work glucose is the best electron donor or carbonsource for supporting molybdate reduction similar to severalother Mo-reducing bacteria previously reported [16 29 3739 40] Other Mo-reducing bacteria require sucrose [35 3638] Ammonium sulphate as the best nitrogen source forsupportingmolybdate reduction in this bacterium reflects thepreference of the bacterium for an easily assimilable nitrogensource and this is also shared by many of the Mo-reducingbacteria isolated so far [16 31ndash40] Since molybdenumreduction is growth associated [16 31ndash40] the use of glucoseand ammonium sulphate as the carbon and nitrogen sourcesof choice respectively reflect a simple growth-associatedprocess of molybdenum reduction in this bacterium The

requirement of static growth for optimal molybdate reduc-tion may reflects a facultative anaerobic property of thisbacterium Many Pseudomonas spp are obligate aerobes butsome have been reported to be facultative anaerobes [42 43]

The optimum temperature in between 10 and 20∘Creflects a psychrotolerant property concurrent with its geo-graphical polar origin This strain would be very useful inbioremediation works in the polar and temperate countriesOther recently isolated molybdenum reducers exhibit higheroptimal reduction in between 30 and 40∘C [24 25 31ndash40]Optimum pH that supported Mo-reduction was within therange of optimum pH of other Mo-reducing bacteria thatvaries from pH 6 to 8 [24 25 31ndash40]

Both Mo-blue the detoxification product of molybdateand molybdate itself are nontoxic with reported productionofMo-blue at 80mMmolybdate inE coliThis bacteriumwasable to produce comparable Mo-blue at molybdate concen-trations similar to other previously isolated strains [24 25 31ndash40] Furthermore it is able to reducemolybdate toMo-blue ata high initial concentration of sodiummolybdate at 100mMSince molybdenum pollution has been reported to reachas high as 2000 ppm (208mM molybdate) [44] toleranceand reduction at concentrations higher than 20mM are anadvantage for a microbe At this concentration molybdate islethal to ruminant [45]

Phosphate is a known inhibitor towardsMo-blue produc-tion with themechanism of inhibition involving destabilizingthe phosphomolybdate intermediate involved in the reduc-tion of molybdate [24 25 31ndash40] In this work the optimalconcentration of phosphate supporting molybdate reductionat 5mM is shared by many Mo-reducing bacteria [24 31ndash40] The optimum ratio of phosphate to molybdate concen-trations was similar to the results obtained from Serratia spstrain DrY8 [36] Enterobacter sp strain DrY13 [37] and Smarcescens strain DrY9 [38]This ratio is important since theformation of the proposed intermediate phosphomolybdatein molybdate reduction [31ndash40] is dependent on exact ratiofor optimal formation [46 47]

The dialysis tubing experiment showed similar resultto previous works [35 39 40] indicating the prominentrole of enzyme(s) in molybdate reduction The remainingmolybdenum blue found in the outside of the tubing is dueto a slow diffusion of the molybdenum blue as we discoveredin previous works

Amongst the metal ions molybdenum is unique inits formation of large heteropoly structure in acidic con-ditions [47] We previously suggest the formation of thephosphomolybdate as an intermediate compound based ona unique absorption spectra of the reduced product thatshows close similarity to Mo-blue produced in the phosphatedetermination method using ascorbic acid as the reducingagent [25 48 49]

The telltale Mo-blue spectra with a peak increase at865 nm and a shoulder at 710 nm is a signature spectrum forreduced phosphomolybdate species [48 49] and is seen ina majority of the Mo-reducing bacteria to date [31ndash40] Theunique spectrum indicating a common species is involvedFurther identification of this specieswould reveal its structureand once the purified enzyme structure is solved software


such as AutoDock can be used to model the site of thereduction It is interesting to note that in enzymatic chromatereduction an intermediate species dichromate with a highnegative oxidation state has been observed to dock at thelysine residues and heme centres at the chromate reductaseenzyme active site responsible for dichromate stabilizationand proton and electron transfer activities This leads toreduction to the insoluble oxidation state [50] Due to asimilar high negative charge of the phosphomolybdate albeitwith a very large structure themechanism of reduction couldbe similar

The ion exchange stage though vital as a purification stepremoves much of enzyme activity This was also observedduring the purification of the enzyme from EC 48 [33] Prob-ably a soluble coenzyme was removed during adsorption tothe exchanger or this enzyme is extremely heat-labile in strainDRY1 To date the enzyme responsible for the Mo-reducingactivity in EC 48 or any other bacterium has never beenpurified Ariff et al manage to partially purify Mo-reducingenzyme from the bacterium Enterobacter cloacae strain 48 orEC 48 up to the stage of ammonium sulphate fractionation[51] Further purification using gel filtration on SephadexG 200 was not successful since no active peak containingMo-reducing enzyme activity was detectedThe apparent119870

119898

for phosphomolybdate NADH and NADPH were highercompared to 032 165 and 213mM respectively for EC48 [33] suggesting lower affinity of the electron acceptorand donors to the Mo-reducing enzyme from this bacteriumcompared to EC 48 NADH is themore preferred substrate toNADPH due to the lower apparent119870

119898values obtained in EC

48 [33] and in this workThe only known enzyme which could reduce molyb-

date is molybdate reductase [29] The enzyme catalyses thereduction of Mo(6+) to the (4+) oxidation state before itsintegration with the sulphur atoms of a pterin derivativenamed molybdopterin cofactor of molybdoenzymes Thepurification of the Mo-reducing enzyme would ultimatelysolve a century-old phenomenon

The inhibition by toxic metal ions in this study is seenin all of the Mo-reducing bacteria [25 30ndash40] and in otherheavy-metal reducing bacteria as well [52ndash54]The inhibitionofmetal-reducing activity by heavymetals generally indicatesan enzymatic origin and not abiotic [42ndash44]

In contrast to previous results in EC 48 [16] our resultssuggest that the electron transport chain (ETC) of thisbacterium is not the site of molybdate reduction In EC 48the role of the electron transport chain is concluded basedon the inhibition of molybdate reduction by cyanide [16]We recently demonstrated that cyanide causes the reactionmixture to become very alkaline and this was the reason forthe inhibition When the reaction mixture was neutralizedupon the addition of cyanide no inhibition of molybdatereduction to Mo-blue was seen [31] Similar negative resultsfor cyanide and other respiratory inhibitors have beenobtained with recently isolatedMo-reducing bacteria [31ndash40]confirming that the electron transport chain is not involvedin molybdate reduction The respiratory inhibitors used inthis work inhibit certain sites of the ETC Rotenone is aninhibitor to NADH dehydrogenase whilst sodium azide and

cyanide are inhibitors to the terminal cytochrome d oxidaseAntimycin A is an inhibitor to cytochrome b [55] Theuse of respiratory to probe the location or identity of othermetal-reducing enzymes in various studies has shown mixedresults Respiratory inhibitors tested in this work such asazide rotenone and cyanide had failed to inhibit chromatereduction in E coli [54] and in Pseudomonas mendocina [56]while cyanide and azide inhibit the reduction of chromate inBacillus subtilis [53] and oxidation of arsenite in Alcaligensesp [52]

5 Conclusion

In conclusion we have isolated and characterized amolybdenum-reducing bacterium from Antarctic soil Thelow optimal temperature requirement for molybdenumreduction confirms the psychrotolerant property of thisorganism To our knowledge this is the first report ofan Antarctic bacterium able to reduce molybdate tomolybdenum blue We have studied the effect of nitrogensource and electron donors temperature phosphate andmolybdate on the reduction of molybdate to molybdenumblue in this bacterium Only a partial purification ofthe enzyme was achieved due to the fact that the lowenzyme yield of the final step prevents further addition ofa chromatographic step The similarities and differencesseen in this work reflect the unique characteristics of Mo-reducing enzyme from different bacteria The inability ofthe respiratory inhibitors to abolish Mo-reducing enzymeactivity indicates that the electron transport chain is not thesite of reduction The information obtained in this work isnot only important for contributing to the understanding offundamental mechanism of molybdate reduction but will beextremely important in future works on the bioremediationof molybdenum in polar environments

Abbreviations

LPM Low phosphate mediumHPM High phosphate medium

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the research grants from theMinister of Science Technology and Innovation (MOSTI)and the Academy Sciences of Malaysia (ASM) The authorswould like to thank the Argentinean Institute of Antarctica(IAA) for providing logistics support during the expeditionto Antarctica


References

[1] D H Nies ldquoHeavy metal-resistant bacteria as extremophilesmolecular physiology and biotechnological use of Ralstonia spCH34rdquo Extremophiles vol 4 no 2 pp 77ndash82 2000

[2] R K Upreti V Sinha R Mishra and A Kannan ldquoIn vitrodevelopment of resistance to arsenite and chromium-VI in Lac-tobacilli strains as perspective attenuation of gastrointestinaldisorderrdquo Journal of Environmental Biology vol 32 no 3 pp325ndash332 2011

[3] R A Wuana and F E Okieimen ldquoHeavy metals in contam-inated soils a review of sources chemistry risks and bestavailable strategies for remediationrdquo ISRN Ecology vol 2011Article ID 402647 20 pages 2011

[4] A Branzini and M S Zubillaga ldquoComparative use of soilorganic and inorganic amendments in heavy metals stabiliza-tionrdquo Applied and Environmental Soil Science vol 2012 ArticleID 721032 7 pages 2012

[5] C Neunhauserer M Berreck and H Insam ldquoRemediation ofsoils contaminated with molybdenum using soil amendmentsand phytoremediationrdquo Water Air and Soil Pollution vol 128no 1-2 pp 85ndash96 2001

[6] G G Claridge I B Campbell H K Powell Z H Amin andM R Balks ldquoHeavy metal contamination in some soils of theMcMurdo Sound region Antarcticardquo Antarctic Science vol 7no 1 pp 9ndash14 1995

[7] A B Crockett and G J White ldquoMapping sediment contami-nation and toxicity in Winter Quarters Bay Mcmurdo StationAntarcticardquo Environmental Monitoring and Assessment vol 85no 3 pp 257ndash275 2003

[8] I R Santos E V Silva-Filho C E G R Schaefer M RAlbuquerque-Filho and L S Campos ldquoHeavy metal contami-nation in coastal sediments and soils near the Brazilian Antarc-tic Station King George Islandrdquo Marine Pollution Bulletin vol50 no 2 pp 185ndash194 2005

[9] R N Horton W A Apel V S Thompson and P P Sheri-dan ldquoLow temperature reduction of hexavalent chromiumby a microbial enrichment consortium and a novel strain ofArthrobacter aurescensrdquo BMC microbiology vol 6 no 5 2006

[10] A J Poulain S M Nı Chadhain P A Ariya et al ldquoPotential formercury reduction bymicrobes in the high ArcticrdquoApplied andEnvironmental Microbiology vol 73 no 7 pp 2230ndash2238 2007

[11] G K Davis ldquoMolybdenumrdquo inMetals andTheir Compounds inthe Environment Occurrence Analysis and Biological RelevanceE Merian Ed pp 1089ndash1100 VCHWeinheim New York NYUSA 1991

[12] D D Runnells D S Kaback and E M Thurman ldquoGeochem-istry and Sampling ofMolybdenum in Sediments Soils Plants inColoradordquo in Molybdenum in the Environment W R Chappeland K K Peterson Eds Marcel and Dekker New York NYUSA 1976

[13] E J Underwood ldquoEnvironmental sources of heavy metals andtheir toxicity toman and animalsrdquoProgress inWater Technologyvol 11 no 4-5 pp 33ndash45 1979

[14] P D Rowley P L Williams and D E Pride ldquoMineraloccurrences of antarctica petroleum and mineral resources ofantarcticardquo in US Geological Survey Circular J C BehrendtEd pp 25ndash49 1983

[15] K R Wood ldquoThe uncertain fate of the Madrid Protocol to theAntarctic Treaty in the maritime areardquo Ocean Development andInternational Law vol 34 no 2 pp 139ndash159 2003

[16] B Ghani M Takai N Z Hisham et al ldquoIsolation andcharacterization of a Mo6+-reducing bacteriumrdquo Applied andEnvironmental Microbiology vol 59 no 4 pp 1176ndash1180 1993

[17] H C Bimboim and J Doly ldquoA rapid alkaline extractionprocedure for screening recombinant plasmid DNArdquo NucleicAcids Research vol 7 no 6 pp 1513ndash1523 1979

[18] R Devereux and S S Wilkinson ldquoAmplification of riboso-mal RNA sequencesrdquo in Molecular Microbial Ecology ManualNetherland A D L Akkermans J D Van Elsas and F J DeBruijn Eds pp 1ndash17 KluwerAcademic Publishing 2nd edition2004

[19] J D Thompson D G Higgins and T J Gibson ldquoCLUSTALW improving the sensitivity of progressive multiple sequencealignment through sequence weighting position-specific gappenalties and weight matrix choicerdquoNucleic Acids Research vol22 no 22 pp 4673ndash4680 1994

[20] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985

[21] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular biol-ogy and evolution vol 4 no 4 pp 406ndash425 1987

[22] TMargush and F RMcMorris ldquoConsensus n-treesrdquo Bulletin ofMathematical Biology vol 43 no 2 pp 239ndash244 1981

[23] RDM Page ldquoTreeView an application to display phylogenetictrees on personal computersrdquo Computer Applications in theBiosciences vol 12 no 4 pp 357ndash358 1996

[24] M Y Shukor M F A Rahman N A Shamaan C H LeeM I A Karim and M A Syed ldquoAn improved enzymeassay for molybdenum-reducing activity in bacteriardquo AppliedBiochemistry and Biotechnology vol 144 no 3 pp 293ndash3002008

[25] M Y Shukor N A Shamaan M A Syed C H Lee and M IA Karim ldquoCharacterization and quantification ofmolybdenumblue production in Enterobacter cloacae Strain 48 using 12-molybdophosphate as the reference compoundrdquo Asia-PacificJournal of Molecular Biology and Biotechnology vol 8 pp 166ndash172 2000

[26] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[27] V E Levine ldquoThe reducing properties of microorganismswith special reference to selenium compoundsrdquo Journal ofBacteriology vol 10 pp 217ndash263 1925

[28] A Capaldi and B Proskauer ldquoBeitrage zur Kenntnis derSiurebildung bei Typhusbacillen undBacterium colirdquoZeitschriftfur Hygiene und Infectionskrankheiten vol 23 no 3 pp 452ndash474 1896

[29] A M Campbell A Del Campillo-Campbell and D B VillaretldquoMolybdate reduction by Escherichia coli K-12 and its chlmutantsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 82 no 1 pp 227ndash231 1985

[30] T Sugio Y Tsujita T Katagiri K Inagaki and T Tano ldquoReduc-tion ofMo6+ with elemental sulfur byThiobacillus ferrooxidansrdquoJournal of Bacteriology vol 170 no 12 pp 5956ndash5959 1988

[31] M Y Shukor M A Syed C H Lee M I A Karim and NA Shamaan ldquoA method to distinguish between chemical andenzymatic reduction of molybdenum in Enterobacter cloacaeStrain 48rdquo Malaysian Journal of Biochemistry vol 7 pp 71ndash722002


[32] M F A Rahman M Y Shukor Z Suhaili S Mustafa NA Shamaan and M A Syed ldquoReduction of Mo(VI) by thebacterium Serratia sp strain DRY5rdquo Journal of EnvironmentalBiology vol 30 no 1 pp 65ndash72 2009

[33] M Y Shukor C H Lee I Omar M I A Karim and MA Syed ldquoIsolation and characterization of a molybdenum-reducing enzyme in Enterobacter cloacae strain 48rdquo PertanikaJournal of Science amp Technology vol 11 no 2 pp 261ndash272 2003

[34] Y Shukor H Adam K Ithnin I Yunus N A Shamaan and ASyed ldquoMolybdate reduction to molybdenum blue in microbeproceeds via a phosphomolybdate intermediaterdquo Journal ofBiological Sciences vol 7 no 8 pp 1448ndash1452 2007

[35] M Y Shukor S H M Habib M F A Rahman et alldquoHexavalent molybdenum reduction tomolybdenum blue by SMarcescens strain Dr Y6rdquo Applied Biochemistry and Biotechnol-ogy vol 149 no 1 pp 33ndash43 2008

[36] M Y Shukor M F Rahman Z Suhaili N A Shamaan andM A Syed ldquoBacterial reduction of hexavalent molybdenumto molybdenum bluerdquo World Journal of Microbiology andBiotechnology vol 25 no 7 pp 1225ndash1234 2009

[37] M Y Shukor M F Rahman N A Shamaan and M S SyedldquoReduction of molybdate to molybdenum blue by Enterobactersp strain DrY13rdquo Journal of Basic Microbiology vol 49 no 1pp S43ndashS54 2009

[38] S M Yunus H M Hamim O M Anas S N Aripin and SM Arif ldquoMo (VI) reduction to molybdenum blue by Serratiamarcescens strain Dr Y9rdquo Polish Journal ofMicrobiology vol 58no 2 pp 141ndash147 2009

[39] M Y Shukor M F Rahman Z Suhaili N A Shamaan andM A Syed ldquoHexavalent molybdenum reduction toMo-blue byAcinetobacter calcoaceticusrdquo Folia Microbiologica vol 55 no 2pp 137ndash143 2010

[40] M Y Shukor S A Ahmad M M M Nadzir M P AbdullahN A Shamaan and M A Syed ldquoMolybdate reduction byPseudomonas sp strain DRY2rdquo Journal of Applied Microbiologyvol 108 no 6 pp 2050ndash2058 2010

[41] Y Nogi H Takami and K Horikoshi ldquoCharacterization ofalkaliphilic Bacillus strains used in industry proposal of fivenovel speciesrdquo International Journal of Systematic andEvolution-aryMicrobiology vol 55 no 6 Article ID 63649 pp 2309ndash23152005

[42] B Philipp H Erdbrink M J-F Suter and B Schink ldquoDegra-dation of and sensitivity to cholate in Pseudomonas sp strainChol1rdquo Archives of Microbiology vol 185 no 3 pp 192ndash2012006

[43] J C Comolli and T J Donohue ldquoPseudomonas aeruginosaRoxR a response regulator related to Rhodobacter sphaeroidesPrrA activates expression of the cyanide-insensitive terminaloxidaserdquo Molecular Microbiology vol 45 no 3 pp 755ndash7682002

[44] S Sinnakkannu A R Abdullah N M Tahir and M R AbasldquoDegradation of metsulfuron methyl in selected malaysianagricultural soilsrdquo Fresenius Environmental Bulletin vol 13 no3 pp 258ndash261 2004

[45] AMHassan BMHaroun A A Amara andA Ehab ldquoSerourproduction and characterization of keratinolytic protease fromnew wool-degrading Bacillus species isolated from Egyptianecosystemrdquo BioMed Research International vol 2013 Article ID175012 14 pages 2013

[46] J L Glenn and F L Crane ldquoStudies on metalloflavoproteins VThe action of silicomolybdate in the reduction of cytochrome c

by aldehyde oxidaserdquo Biochimica Et Biophysica Acta vol 22 no1 pp 111ndash115 1956

[47] J D LeeConcise Inorganic Chemistry Van Reinhold NewYorkNY USA 1977

[48] L P Kazansky and M A Fedotov ldquoPhosphorus-31 andoxygen-17 NMR evidence of trapped electrons in reduced 18-molybdodiphosphate(V) P

2Mo18O28minus

6 rdquo Journal of the ChemicalSociety Chemical Communications no 14 pp 644ndash646 1980

[49] L S Clesceri A E Greenberg and R R Trussel Eds StandardMethods For the Examination of Wastewater American PublicHealth Association Port City Press Baltimore Md USA 17thedition 1989

[50] M Sundararajan A J Campbell and I H Hillier ldquoHow doenzymes reduce metals The mechanism of the reduction ofCr(VI) in chromate by cytochrome c7 proteins proposed fromDFT calculationsrdquo Faraday discussions vol 148 pp 195ndash2282011

[51] A B Ariff M Rosfarizan B Ghani T Sugio and M I AKarim ldquoMo-reducing enzyme inEnterobacter cloacae strain 48rdquoWorld Journal of Microbiology and Biotechnology vol 13 no 6pp 643ndash647 1997

[52] F H Osborne and H L Ehrlich ldquoOxidation of arsenite by a soilisolate of Alcaligenesrdquo Journal of Applied Bacteriology vol 41no 2 pp 295ndash305 1976

[53] C Garbisu I Alkorta M J Llama and J L Serra ldquoAerobicchromate reduction by Bacillus subtilisrdquo Biodegradation vol 9no 2 pp 133ndash141 1998

[54] H Shen and Y-T Wang ldquoCharacterization of enzymatic reduc-tion of hexavalent chromium by Escherichia coli ATCC 33456rdquoApplied and Environmental Microbiology vol 59 no 11 pp3771ndash3777 1993

[55] R M C Dawson D C Elliott W H Elliott and K M JonesEds Data For Biochemical Research Clarendon Press OxfordUK 1969

[56] J M Rajwade P B Salunke and K M Pknikar ldquoBiochemicalbasis of chromate reduction by Pseudomonas mendocina Bio-hydrometallurgy and the Environmentrdquo in Proceedings of theInternational Biohydrometallurgy Symposium R Amils and ABallester Eds pp 105ndash114 Elsevier New York NY USA 1999

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


polar regions as a preparative step for future bioremediationworks It is also anticipated that other cold region areas wouldbenefit from this research In this work we report on theisolation and characterization of a psychrotolerant bacteriumwith the ability to transform molybdenum to the insolublemolybdenum blue This is the first molybdenum-reducingbacterium isolated from cold region This bacterium couldbe used in future remediation works of molybdenum inAntarctica and cold climate regions

2 Experimental

21 Isolation of Molybdenum-Reducing Bacterium Sampleswere collected from jubany Station an Argentinean Baseon King George Island South Shetlands Islands Antarctica(615∘S 5455∘W) in the austral summer 2002-2003 expedi-tion Soils were collected 15ndash20 centimetres (cm) beneath thesurface and were placed in sterile screw-capped vials Thesamples were immediately placed in a freezer and stored atminus20∘C until returned to the laboratory for further examina-tion About five grams of soil sample that was well-mixedwere suspended in 45mL of 09 saline solution A suitableserial dilution of soil suspension was then spread platedonto an agar of low phosphate medium (LPM) (28mMphosphate) (pH 70) The medium (wv) consisted of glu-cose (1) MgSO

4sdot7H2O (005) (NH

4)2SO4(03) NaCl

(05) Na2MoO4sdot2H2O (0242) yeast extract (005) and

Na2HPO4sdot2H2O (005) to isolate molybdenum-reducing

bacteria [16] After 72 hours of incubation at 10∘C severalwhite and blue colonies appeared The colony exhibiting thestrongest blue intensity was then inoculated into 50mLof lowphosphate medium and incubated at 10∘C for 72 hours stati-cally Incubation under aerobic conditions gave lower amountof Mo-blue compared to static incubation It was observedthat during cellular reduction of molybdate cells precipitatedtogether with the molybdenum blue product preventingcellular growth A portion of the growth medium containingMo-blue was centrifuged at 10000timesg for 10min and thesupernatant was scanned from 400 to 980 nm using a Cintra5 spectrophotometer A freshly prepared low phosphatemedium was used for baseline correction This bacteriumwas kept in the Bioremediation and Bioassay Laboratory (Lab204) The production of molybdenum blue in the culturesupernatant was monitored at 865 nm Identification of thebacterium was performed by using molecular phylogeneticsstudies and Biolog GN MicroPlate (Biolog Hayward CAUSA) according to the manufacturerrsquos instructions

22 Optimization of Molybdenum Reduction Optimizationof several parameters that support molybdenum reductionto molybdenum blue such as temperature pH the effectof carbon sources as electron donors the effect of nitrogensources sodium molybdate and phosphate concentrationswere studied using the LPM in Section 21 as the basalmedium for 48 hours statically

23 16s rDNA Gene Sequencing and Phylogenetic AnalysisExtraction of genomic DNA was performed using the alka-line lysis method [17] PCR amplification was carried out

on a Biometra T Gradient PCR (Montreal Biotech IncKirkland QC Canada) The PCR mixture consisted of 05pM of each primer 1x reaction buffer 25U of Taq DNApolymerase (Promega) and 200120583Mof each deoxynucleotidetriphosphate at the final volume of 50120583L Amplificationof the 16s rDNA gene was carried out using the follow-ing primers 51015840-AGAGTTTGATCCTGGCTCAG-31015840 and 51015840-AAGGAGGTGATCCAGCCGCA-31015840 corresponding to theforward and reverse primers of 16s rDNA respectively[18] PCR was carried out under the following conditionsinitial denaturation at 94∘C for 3min 25 cycles of 94∘C for1min 50∘C for 1min and 72∘C for 2min a final extensionat 72∘C for 10min Cycle sequencing was subsequentlyperformed with the Big Dye terminator kit (Perkin-ElmerApplied Biosystems) as recommended by the manufacturerSequence data were initially recorded and edited usingCHROMAS version 145 The resultant 1452 bases werecompared with the GenBank database using the Blast serverat NCBI (httpwwwncbinlmnihgovBLAST) Analysisshowed that this sequence to be closely related to rrsfrom Gammaproteobacteria The 16s rRNA ribosomal genesequence for this isolate has been deposited in GenBankunder the accession number DQ226202

24 Phylogenetic Analysis Twenty-three 16S rRNA genesequences closely matched to isolate DRY1 were re-trieved from GenBank and a multiple alignment of thesequences was carried out using clustal W [19] A phylo-genetic tree was constructed by using PHYLIP version3573 (J Q Felsenstein PHYLIPmdashphylogeny inference pack-age version 3573 Department of Genetics Univer-sity of Washington Seattle WA USA (httpevolutiongeneticswashingtoneduphyliphtml)) [20] with Serratiamarcescens as the outgroup in the cladogram Evolutionarydistance matrices for the neighbour-joiningUPGMAmethod were computed using the DNADIST algorithmprogram Phylogenetic tree was inferred by using theneighbour-joining method of Saitou and Nei [21] With eachalgorithm confidence levels for individual branches withinthe tree were checked by repeating the PHYLIP analysiswith 1000 bootstraps by the SEQBOOT program in thePHYLIP package Majority rule (50) consensus trees wereconstructed using the Ml methods [22] and the tree wasviewed using TreeView [23]

25 Crude Enzyme Preparation Crude enzyme was pre-pared from a 2 L culture grown at 20∘C for 72 hours onan orbital shaker at 150 rpm on a modified high phos-phate medium (HPM) consisting ofMgSO

4sdot7H2O (05 gLminus1)

(NH4)2SO4(3 gLminus1) yeast extract (1 gLminus1) NaCl (5 gLminus1)

NaMoO4sdot2H2O) (121 gLminus1 or 50mM) glucose (10 gLminus1) as

the source of electron donor and Na2HPO4sdot2H2O (100mM)

at pH 73 Growth at high phosphate under aerobic con-ditions prevents Mo-blue production but cells containedhigh enzyme activity Experiments were carried out at 4∘Cunless stated otherwise Bacterial cells were first harvestedat 10 000timesg for 20min at 4∘C The pellet was then reconsti-tuted in 15mL of 50mM TrissdotCl buffer (pH 70) containing







2Cr2O7 BDH) Fe3+


2 BDH) Mg2+ (MgCl

2


2sdot6H2O BDH)


3 JT Baker)


4sdot5H2O JT


2 JT Baker)




4)2SO4




3 Results




100

856483

626

902

622

995

372

356

997593

192

245

591502

871

816986

971

842892

433

602











Strain DrY1


















00

10

20

30

40

50

60

70

Citr

ic ac

id

Con

trol

Form

ate

Fruc

tose

Glu

cose

Lact

ose

Lact

ose

Man

nito

l

Star

ch

Sucr

ose

Mo-

blue

(120583m

ole)


4sdot7H2O (005)

(NH4)2SO4(03) NaCl (05) Na








00

10

20

30

40

50

60

70

Am

m f

orm

ate

L-hi

stidi

ne

L-as

part

ate

L-le

ucin

e

Am

m c

hlor

ide

L-ph

enyl

alan

ine

Am

m s

ulph

ate

Sodi

um n

itrat

e

Gly

cine

Hyd

roxy

-L-p

rolin

e

L-cy

stine

Con

trol

Mo-

blue

(120583m

ole)


4sdot7H2O




00

20

40

60

80

100


Mo-

blue

(120583m

ole)




2HPO4sdot2H2O



65

7

75

8

85

9

5 55 6 65 7 75 8 85pH

Mo-

blue

(120583m

ole)




2HPO4sdot2H2O


00

20

40

60

80

100

0 20 40 60 80 100 120Molybdate (mM)

Mo-

blue

(120583m

ole)







0

2

4

6

8

10

0 20 40 60 80 100 120Phosphate (mM)

Mo-

blue

(mm

ole)






0

1

2

3

4

5

400 500 600 700 800 900 1000Wavelength (nm)

Abs

A

B

C

D

E






Specificactivity

(Unitsmgprotein)


Yield

Foldpurification























Cr6+005 plusmn 002

Fe3+944 plusmn 051

Zn2+939 plusmn 020

Mg2+953 plusmn 023

Co2+957 plusmn 021

Ni2+935 plusmn 023

Cd2+046 plusmn 002

Ag+023 plusmn 087

Mn2+911 plusmn 021

Cu2+000 plusmn 012

Hg2+010 plusmn 003

Pb2+050 plusmn 011

4 Discussions














119898








5 Conclusion


Abbreviations




Acknowledgments



References



















































2Mo18O28minus

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







2Cr2O7 BDH) Fe3+


2 BDH) Mg2+ (MgCl

2


2sdot6H2O BDH)


3 JT Baker)


4sdot5H2O JT


2 JT Baker)




4)2SO4




3 Results




100

856483

626

902

622

995

372

356

997593

192

245

591502

871

816986

971

842892

433

602











Strain DrY1


















00

10

20

30

40

50

60

70

Citr

ic ac

id

Con

trol

Form

ate

Fruc

tose

Glu

cose

Lact

ose

Lact

ose

Man

nito

l

Star

ch

Sucr

ose

Mo-

blue

(120583m

ole)


4sdot7H2O (005)

(NH4)2SO4(03) NaCl (05) Na








00

10

20

30

40

50

60

70

Am

m f

orm

ate

L-hi

stidi

ne

L-as

part

ate

L-le

ucin

e

Am

m c

hlor

ide

L-ph

enyl

alan

ine

Am

m s

ulph

ate

Sodi

um n

itrat

e

Gly

cine

Hyd

roxy

-L-p

rolin

e

L-cy

stine

Con

trol

Mo-

blue

(120583m

ole)


4sdot7H2O




00

20

40

60

80

100


Mo-

blue

(120583m

ole)




2HPO4sdot2H2O



65

7

75

8

85

9

5 55 6 65 7 75 8 85pH

Mo-

blue

(120583m

ole)




2HPO4sdot2H2O


00

20

40

60

80

100

0 20 40 60 80 100 120Molybdate (mM)

Mo-

blue

(120583m

ole)







0

2

4

6

8

10

0 20 40 60 80 100 120Phosphate (mM)

Mo-

blue

(mm

ole)






0

1

2

3

4

5

400 500 600 700 800 900 1000Wavelength (nm)

Abs

A

B

C

D

E






Specificactivity

(Unitsmgprotein)


Yield

Foldpurification























Cr6+005 plusmn 002

Fe3+944 plusmn 051

Zn2+939 plusmn 020

Mg2+953 plusmn 023

Co2+957 plusmn 021

Ni2+935 plusmn 023

Cd2+046 plusmn 002

Ag+023 plusmn 087

Mn2+911 plusmn 021

Cu2+000 plusmn 012

Hg2+010 plusmn 003

Pb2+050 plusmn 011

4 Discussions














119898








5 Conclusion


Abbreviations




Acknowledgments



References



















































2Mo18O28minus

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


100

856483

626

902

622

995

372

356

997593

192

245

591502

871

816986

971

842892

433

602











Strain DrY1


















00

10

20

30

40

50

60

70

Citr

ic ac

id

Con

trol

Form

ate

Fruc

tose

Glu

cose

Lact

ose

Lact

ose

Man

nito

l

Star

ch

Sucr

ose

Mo-

blue

(120583m

ole)


4sdot7H2O (005)

(NH4)2SO4(03) NaCl (05) Na








00

10

20

30

40

50

60

70

Am

m f

orm

ate

L-hi

stidi

ne

L-as

part

ate

L-le

ucin

e

Am

m c

hlor

ide

L-ph

enyl

alan

ine

Am

m s

ulph

ate

Sodi

um n

itrat

e

Gly

cine

Hyd

roxy

-L-p

rolin

e

L-cy

stine

Con

trol

Mo-

blue

(120583m

ole)


4sdot7H2O




00

20

40

60

80

100


Mo-

blue

(120583m

ole)




2HPO4sdot2H2O



65

7

75

8

85

9

5 55 6 65 7 75 8 85pH

Mo-

blue

(120583m

ole)




2HPO4sdot2H2O


00

20

40

60

80

100

0 20 40 60 80 100 120Molybdate (mM)

Mo-

blue

(120583m

ole)







0

2

4

6

8

10

0 20 40 60 80 100 120Phosphate (mM)

Mo-

blue

(mm

ole)






0

1

2

3

4

5

400 500 600 700 800 900 1000Wavelength (nm)

Abs

A

B

C

D

E






Specificactivity

(Unitsmgprotein)


Yield

Foldpurification























Cr6+005 plusmn 002

Fe3+944 plusmn 051

Zn2+939 plusmn 020

Mg2+953 plusmn 023

Co2+957 plusmn 021

Ni2+935 plusmn 023

Cd2+046 plusmn 002

Ag+023 plusmn 087

Mn2+911 plusmn 021

Cu2+000 plusmn 012

Hg2+010 plusmn 003

Pb2+050 plusmn 011

4 Discussions














119898








5 Conclusion


Abbreviations




Acknowledgments



References



















































2Mo18O28minus

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


00

10

20

30

40

50

60

70

Am

m f

orm

ate

L-hi

stidi

ne

L-as

part

ate

L-le

ucin

e

Am

m c

hlor

ide

L-ph

enyl

alan

ine

Am

m s

ulph

ate

Sodi

um n

itrat

e

Gly

cine

Hyd

roxy

-L-p

rolin

e

L-cy

stine

Con

trol

Mo-

blue

(120583m

ole)


4sdot7H2O




00

20

40

60

80

100


Mo-

blue

(120583m

ole)




2HPO4sdot2H2O



65

7

75

8

85

9

5 55 6 65 7 75 8 85pH

Mo-

blue

(120583m

ole)




2HPO4sdot2H2O


00

20

40

60

80

100

0 20 40 60 80 100 120Molybdate (mM)

Mo-

blue

(120583m

ole)







0

2

4

6

8

10

0 20 40 60 80 100 120Phosphate (mM)

Mo-

blue

(mm

ole)






0

1

2

3

4

5

400 500 600 700 800 900 1000Wavelength (nm)

Abs

A

B

C

D

E






Specificactivity

(Unitsmgprotein)


Yield

Foldpurification























Cr6+005 plusmn 002

Fe3+944 plusmn 051

Zn2+939 plusmn 020

Mg2+953 plusmn 023

Co2+957 plusmn 021

Ni2+935 plusmn 023

Cd2+046 plusmn 002

Ag+023 plusmn 087

Mn2+911 plusmn 021

Cu2+000 plusmn 012

Hg2+010 plusmn 003

Pb2+050 plusmn 011

4 Discussions














119898








5 Conclusion


Abbreviations




Acknowledgments



References



















































2Mo18O28minus

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

2

4

6

8

10

0 20 40 60 80 100 120Phosphate (mM)

Mo-

blue

(mm

ole)






0

1

2

3

4

5

400 500 600 700 800 900 1000Wavelength (nm)

Abs

A

B

C

D

E






Specificactivity

(Unitsmgprotein)


Yield

Foldpurification























Cr6+005 plusmn 002

Fe3+944 plusmn 051

Zn2+939 plusmn 020

Mg2+953 plusmn 023

Co2+957 plusmn 021

Ni2+935 plusmn 023

Cd2+046 plusmn 002

Ag+023 plusmn 087

Mn2+911 plusmn 021

Cu2+000 plusmn 012

Hg2+010 plusmn 003

Pb2+050 plusmn 011

4 Discussions














119898








5 Conclusion


Abbreviations




Acknowledgments



References



















































2Mo18O28minus

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Cr6+005 plusmn 002

Fe3+944 plusmn 051

Zn2+939 plusmn 020

Mg2+953 plusmn 023

Co2+957 plusmn 021

Ni2+935 plusmn 023

Cd2+046 plusmn 002

Ag+023 plusmn 087

Mn2+911 plusmn 021

Cu2+000 plusmn 012

Hg2+010 plusmn 003

Pb2+050 plusmn 011

4 Discussions














119898








5 Conclusion


Abbreviations




Acknowledgments



References



















































2Mo18O28minus

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




119898








5 Conclusion


Abbreviations




Acknowledgments



References



















































2Mo18O28minus

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


References



















































2Mo18O28minus

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















2Mo18O28minus

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Molybdate Reduction to Molybdenum Blue by ...downloads.hindawi.com/journals/bmri/2013/871941.pdf · metals to terrestrial Antarctic organisms and precautionary steps