Bacterial reduction of hexavalent molybdenum to molybdenum blue

ORIGINAL PAPER

Bacterial reduction of hexavalent molybdenumto molybdenum blue

M. Y. Shukor Æ M. F. Rahman Æ Z. Suhaili ÆN. A. Shamaan Æ M. A. Syed

Received: 3 October 2008 / Accepted: 19 February 2009 / Published online: 12 March 2009

� Springer Science+Business Media B.V. 2009

Abstract A bacterium that was able to tolerate and reduce

as high as 50 mM of sodium molybdate to molybdenum

blue has been isolated from a metal recycling ground. The

isolate was tentatively identified as Serratia sp. strain Dr.Y8

based on the carbon utilization profiles using Biolog GN

plates and partial 16S rDNA molecular phylogeny.

ANOVA analysis showed that isolate Dr.Y8 produced

significantly higher (P \ 0.05) amount of Mo-blue with

3, 5.1 and 11.3 times more molybdenum blue than previ-

ously isolated molybdenum reducers such as Serratia

marcescens strain Dr.Y6, E. coli K12 and E. cloacae strain

48, respectively. Its molybdate reduction characteristics

were studied in this work. Electron donor sources such as

sucrose, mannitol, fructose, glucose and starch supported

molybdate reduction. The optimum phosphate, pH and

temperature that supported molybdate reduction were

5 mM, pH 6.0 and 37�C, respectively. The molybdenum

blue produced from cellular reduction exhibited a unique

absorption spectrum with a maximum peak at 865 nm and a

shoulder at 700 nm. Metal ions such as chromium, silver,

copper and mercury resulted in approximately 61, 57, 80,

and 69% inhibition of the molybdenum-reducing activity at

1 mM, respectively. The reduction characteristics of strain

Dr.Y8 suggest that it would be useful in future molybdenum

bioremediation.

Keywords Serratia sp. � Molybdate-reduction �Molybdenum blue

Abbreviations

EC 48 Enterobacter cloacae strain 48

Mo-blue Molybdenum blue

LPM Low phosphate molybdate

LPP Laboratory-prepared phosphomolybdate

Introduction

Heavy metals pollution in Malaysia has emerged as a

health threat due to indiscriminate dumping and illegal

discharge. Documented sources show that heavy metals

pollution comes from industrial complex and scrap metal

yards (Shukor et al. 2006, 2008a; Yin et al. 2007). A more

serious source of pollution is scheduled waste. Some of

these wastes include oil and hydrocarbon, metal sludge,

mineral sludge, ink and paint and spent catalyst amounting

to 200,000 metric tonne (DOE 2007). One of the heavy

metals known to be predominantly found in these types of

wastes is molybdenum (King et al. 1992). Molybdenum

can be found in the discharged effluents from these

industries (Shineldecker 1992). In the Tokyo Bay and the

Black Sea, molybdenum level is in the range of hundreds

of ppm (parts per million) (Davis 1991). In Tyrol, Austria,

molybdenum pollution is caused by industrial effluents,

and has contaminated large pasture areas, reaching as high

as 200 ppm causing scouring in ruminants (Neunhauserer

et al. 2001). In Malaysia, a ruptured pipe and leaching of

metals from a copper and molybdenum mining area in

Sabah have contaminated 2,000 acres of paddy fields and

the Ranau River (Yong 2000). This and coupled with the

fact that a significant amount of the scheduled waste in

Malaysia have been found to be illegally discharged and

dumped (Mokhtar et al. 2003) make molybdenum pollution

a serious emerging threat. Resistance of microbes towards

M. Y. Shukor (&) � M. F. Rahman � Z. Suhaili �N. A. Shamaan � M. A. Syed

Department of Biochemistry, Faculty of Biotechnology

and Biomolecular Sciences, University Putra Malaysia,

43400 Serdang, Selangor, Malaysia

e-mail: [email protected]

123

World J Microbiol Biotechnol (2009) 25:1225–1234

DOI 10.1007/s11274-009-0006-6

heavy metals can be used for their bioremediation of toxic

metal ions (Liu et al. 2007; Rehman et al. 2007; Malek-

zadeh et al. 2007; Zhu et al. 2008). Bioremediation to treat

molybdenum pollution was first carried out in Tyrol,

Austria. Ghani et al. (1993) were the first to suggest the

possibility of using molybdenum-reducing bacterium to

remediate molybdenum pollution. The reduced product is

molybdenum blue and it forms a precipitable mass together

with the bacteria; this could easily aid removal of molyb-

denum (Ghani et al. 1993).

Molybdate reduction by microbes has been reported

since the last 100 years. The first detailed study on moly-

bate reduction to Mo-blue was carried out by Campbell

et al. (1985) in E. coli K12. This is followed by T. ferro-

oxidans in 1988 (Sugio et al. 1988), Enterobacter cloacae

strain 48 (EC 48) in 1993 (Ghani et al. 1993) and Serratia

marcescens strain Dr.Y6 (Shukor et al. 2008a, b, c, d). The

mechanism of enzymatic reduction is originally proposed

to involve enzymatic molybdate (Mo6?) reduction to Mo5?

by molybdenum reductase (Mo-reducing enzyme) prior to

the joining of phosphate anions forming Mo-blue (Ghani

et al. 1993; Ariff et al. 1997). However, the very similar

spectra of the molybdenum blue produced from bacterial

reduction to that of the phosphate determination method

hinted on the possible formation of a phosphomolybdate

intermediate (Shukor et al. 2000, 2003, 2007, 2008a, b, c,

d). This mechanism was proposed because under acidic

conditions such as those found in anaerobic or static

growth, E. coli, EC 48 and many fermentative heterotrophs

will undergo fermentation yielding organic acids that

would transform molybdate ions to phosphomolybdate

since phosphate is abundance in the medium. The aim of

this work is to search for a better molybdenum reducer

from metal contaminated soils. In this work, we report on

the reduction characteristics of such bacterium.

Materials and methods

Reagents

All of the metal ions used in this work are of analytical-grade.

Cr6? (K2Cr2O7), Fe3? (FeCl3 � 6H2O), Fe2? (Fe(NH4)2

(SO4)2 � 6H2O), Zn2? (ZnCl2), Mg2? (MgCl2), Co2?

(CoCl2 � 6H2O), Ni2? (NiCl2 � 6H2O) and Sn2?(SnCl2 �2H2O) were purchased from BDH (British Drug House),

Poole, UK. Ag? (AgNO3), Mn2? (MnCl2 � 4H2O), Cu2?

(CuSO4 � 5H2O), Hg2? (HgCl2), Cd2? (CdCl2 � H2O) and

Pb2? (PbCl2) were purchased from JT Baker, John Town-

send Baker, Phillipsburg, NJ, USA. All other organic

chemicals were of analytical grade. Biolog GN MicroPlate�

and the Biolog� system were purchased from Biolog, Hay-

ward, CA, USA.

Isolation and maintenance of molybdenum-reducing

bacterium

Soil samples from the scrap metal recycling ground in

Serdang, Malaysia, were taken at the depth of 5 cm from

topsoil in July 2004. Five grams of a well-mixed soil sample

were suspended in 45 ml of 0.9% saline solution. About

0.1 ml from four serial dilutions (102–105) of soil suspen-

sion was each spread plated onto an agar of low phosphate

molybdate (LPM) medium pH 7.0 (Ghani et al. 1993)

containing glucose (1%), (NH4)2SO4 (0.3%), MgSO4 �7H2O (0.05%), NaCl (0.5%), yeast extract (0.05%),

Na2MoO4 � 2H2O (10 mM) and Na2HPO4 (2.9 mM). Glu-

cose was autoclaved separately. Blue colonies indicate

molybdate reduction activity. One single colony exhibiting

the strongest blue intensity observable by eyes was inocu-

lated into 50 ml of low phosphate media and incubated at

30�C for 24 h. This bacterium is kept in the Department’s

bacterial culture collection in the Bioremediation and Bio-

assay Laboratory (Lab 204). Identification at species level

was performed by using Biolog GN MicroPlate (Biolog,

Hayward, CA, USA) according to the manufacturer’s

instructions and molecular phylogenetics studies. A pure

culture of bacterium was grown on a Biolog Universal

Growth agar plate. The bacteria were swabbed from the

surface of the agar plate, and suspended to a specified

density in GN Inoculating Fluid. About 150 ll of a bacterial

suspension was pipetted into each well of the MicroPlate.

The MicroPlate was incubated at 30�C for 24 h according to

manufacturer’s specification. The MicroPlate was then read

with the Biolog MicroStationTM System and compared to

database. Enterobacter cloacae strain 48 and S. marcescens

strain Dr.Y6 were obtained from our culture collection.

Enterobacter cloacae strain 48, S. marcescens strain Dr.Y6

and E. coli K12 (American Type Culture Collection,

Rockville, USA) were periodically streaked on agar plates

containing the low phosphate media. For comparing

molybdenum-reducing property, one single colony from

each bacterium was inoculated statically into 50 ml of low

phosphate media and incubated at 30�C for 24 h.

16S rDNA gene sequencing and multiple alignments

A single Serratia sp. strain Dr.Y8 colony grown on Nutrient

agar (Oxoid) was suspended in 1 ml of physiological saline.

Genomic DNA was extracted from this suspension by

alkaline lysis. PCR amplification was performed using a

Biometra T Gradient PCR (Montreal Biotech Inc., Kirk-

land, QC). The PCR mixture contained 0.5 pM of each

primer, 200 lM of each deoxynucleotide triphosphate, 19

reaction buffer, 2.5 U of Taq DNA polymerase (Promega)

to achieve a final volume of 50 l1. The 16S rDNA gene

from the genomic DNA was amplified by PCR using the

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123

following primers; 50-AGAGTTTGATCCTGGCTCAG-30

and 50-AAGGAGGTGATCCAGCCGCA-30 corresponding

to the forward and reverse primers of 16S rDNA, respec-

tively (Devereux and Wilkinson 2004). PCR was performed

under the following conditions: initial denaturation at 94�C

for 3 min; 25 cycles of 94�C for 1 min, 50�C for 1 min, and

72�C for 2 min; and a final extension at 72�C for 10 min.

Cycle sequencing was subsequently performed with the Big

Dye terminator kit (Perkin-Elmer Applied Biosystems) as

recommended by the manufacturer. Sequence data were

initially recorded and edited using CHROMAS Version

1.45. The resultant 1,282 bases were compared with the

GenBank database using the Blast server at NCBI

(http://www.ncbi.nlm.nih.gov/BLAST/). This analysis

showed this sequence to be closely related to rrs from

Gammaproteobacteria. The 16S rRNA ribosomal gene

sequence for this isolate has been deposited in GenBank

under the following accession number DQ226209.

Phylogenetic analysis

Multiple alignment of 18 16S rRNA gene sequences clo-

sely matched strain Dr.Y8 were retrieved from GenBank

and were aligned using clustal_W (Thompson et al. 1994)

with the PHYLIP output option. The alignment was

checked by eye for any obvious missed-alignments, and

alignment positions with gaps were excluded from the

calculations. A phylogenetic tree was constructed by using

PHYLIP, version 3.573 (J. Q. Felsenstein, PHYLIP—

phylogeny inference package, version 3.573, Department

of Genetics, University of Washington, Seattle, WA

[http://evolution.genetics.washington.edu/phylip.html]), with

Bacillus subtilis as the outgroup in the phylogram.

Evolutionary distance matrices for the neighbor-joining/

UPGMA method were computed using the DNADIST

algorithm program. The model of nucleotide substitution is

from Jukes and Cantor (1969). Phylogenetic tree was

inferred by using the neighbor-joining method of Saitou

and Nei (1987) and with each algorithm, confidence levels

for individual branches within the tree were checked by

repeating the PHYLIP analysis with 1,000 bootstraps

(Felsenstein 1985). Majority rule (50%) consensus trees

were constructed for the topologies found using a family of

consensus tree methods called the Ml methods (Margush

and Mc Morris 1981) using the CONSENSE program and

the tree was viewed using TreeView (Page 1996).

Method to ascertain whether molybdate reduction

in strain Dr.Y8 is catalyzed by enzyme

This experiment was carried out to study whether the

reduction of molybdate to Mo-blue in this bacterium is

chemically or enzymatically mediated. Strain Dr.Y8 was

grown in 250 ml of high phosphate media overnight, and

was shaked at 150 rpm at room temperature (28–30�C).

Cells were harvested by centrifugation at 15,000g for

10 min and the pellet was resuspended in low phosphate

solution (pH 7.0) containing (w/v) (NH4)2SO4 (0.3%),

MgSO4 � 7H2O (0.05%), NaCl (0.5%), yeast extract

(0.05%) and Na2HPO4 (0.05%). About 10 ml of this sus-

pension was then placed in a dialysis tubing previously

boiled for 10 min (1,000 molecular weight cut-off) and

immersed in 100 ml of sterile (LPM) media. Aliquots

(1 ml) of the media were taken at the beginning of the

experiment and after a static incubation period of 4 h at

room temperature (28–30�C), and then read at 865 nm. At

the same time, 1 ml was taken out from the content of the

dialysis tubing and centrifuged at 15,000g for 10 min. The

supernatant was then read at 865 nm. Experiments were

carried out in triplicate.

Assay for molybdenum-reducing enzyme

The molybdenum-reducing enzyme was assayed using

laboratory-prepared phosphomolybdate (LPP) (Shukor

et al. 2008c). The LPM was prepared by mixing 600 mM

molybdate (Na2MoO4 � 2H2O) with 240 mM phosphate

(Na2HPO4 � 2H2O), or 10:4 molybdate to phosphate ratio,

to make 60 mM stock solution. Final pH adjustment of the

LPP solution to pH 5.0 was carried out using 1 M HCl.

Into 1 ml of reaction mixture containing 15 mM (final

concentration) of LPP in 50 mM citrate-phosphate buffer

pH 5.0 at room temperature (28–30�C), 100 ll of NADH

(80 mM stock) was added to a final concentration of

8 mM. Fifty microliters of crude Mo-reducing enzyme was

added to start the reaction. The absorbance increased in

1 min was read at the wavelength of 865 nm. One unit of

Mo-reducing activity is defined as the amount of enzyme

that produces 1 nmole Mo-blue per minute at room tem-

perature (28–30�C). The specific extinction coefficient at

865 nm for the product, Mo-blue, is 16.7 mM-1cm-1

(Shukor et al. 2000). An increase in absorbance at 865 nm

of 1.00 unit absorbance per minute per mg protein would

yield 60 nmole of 12-phoshomolybdate or 60 units of

enzyme activity in a 1 ml assay mixture.

Preparation of crude enzyme

Bacteria were grown in 1 l of media containing high

phosphate at 30�C for 24 h on orbital shaker (100 rpm).

Although the high phosphate inhibits molybdate reduction

to Mo-blue, the cells contained active enzymes (Ghani

et al. 1993). Growth on low phosphate resulted in a blue

sticky culture that complicated the preparation of crude

enzyme and enzyme assay. The following experiment was

carried out at 4�C unless stated otherwise. Cells were

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harvested through centrifugation at 10,000g for 10 min.

Cells were washed at least once with distilled water,

resuspended and recentrifuged. The pellet was reconsti-

tuted with 10 ml of 50 mM Tris–HCl buffer pH 7.5 (Tris

buffer prepared at 4�C) containing 0.5 mM Dithiothreitol

and 0.1 mM PMSF (phenylmethylsulfonyl fluoride). Cells

were sonicated for 1 min in ice bath with 4 min cooling

until a total sonication time of at least 20 min was

achieved. The sonicated fraction was centrifuged at

10,000g for 20 min and the supernatant consisting of the

crude enzyme fraction was taken.

The effect of electron donor sources

Electron donor sources such as acetate, formate, glycerol,

citric acid, lactose, fructose, glucose, mannitol, tartarate,

maltose, sucrose, and starch (all from Sigma, St Loius, USA)

were prepared as 10% (w/v) sterile stock solution in

deionized water. Starch was prepared as a 1% (w/v) solution.

The electron donors were added into 50 ml conical flasks

containing low phosphate media to the final concentration of

0.2% (w/v). Serratia sp. strain Dr.Y8 was grown in 100 ml

of high phosphate media in a 250 ml conical flask at 30�C

for 24 h on an orbital shaker (100 rpm). About 0.1 ml of this

bacterial solution was added into each of the bottles con-

taining the low phosphate media and electron donor. The

final volume of the media was 20 ml. The increase in

absorbance at 865 nm was measured after a 24-h growth

period at 30�C under static conditions.

The effects of metal ions and respiratory inhibitors

Metal ions were dissolved in 20 mM Tris–HCl buffer

pH 7.0. Antimycin A, sodium azide, potassium cyanide

and rotenone stock solutions were dissolved in acetone or

deionized water and were added into the enzyme assay

mixture to the final concentrations of 1.2, 10, 10 and

0.2 mM, respectively. Inhibitors and metal ions were pre-

incubated with 100 ll of enzyme in the reaction mixture at

4�C for 10 min minus NADH. The incubation mixture was

then warmed to room temperature (28–30�C) and NADH

was added to start the reaction. Deionized water was added

so that the total reaction mixture was 1.0 ml. As a control,

50 ll of acetone was added in the reaction mixture without

inhibitors. The increase in absorbance at 865 nm was

measured after a period of 5 min. Experiments were carried

out in triplicate.

Statistical analysis

Values are means ± SE. All data were analyzed using

Graphpad Prism version 3.0 and Graphpad InStat version 3.05

available from www.graphpad.com. Comparison between

groups was performed using a Student’s t-test or a one-way

analysis of variance with post hoc analysis by Tukey’s

test. P \ 0.05 was considered statistically significant.

Results and discussions

Comparison of Mo-blue production among

molybdenum-reducing isolates

The newly isolated molybdenum-reducing bacterium is a

gram negative and oxidase negative. Table 1 shows the

amount of Mo-blue from a 24-h culture of Serratia sp.

strain Dr.Y8, E. cloacae strain 48 and E. coli K12.

ANOVA showed that isolate Dr.Y8 produced significantly

higher (P \ 0.05) amount of Mo-blue with 3, 5.1 and 11.3

times more Mo-blue than Serratia marcescens strain

Dr.Y6, E. coli K12 and E. cloacae strain 48, respectively.

The effect of electron donors on molybdate reduction

Previous works on microbial molybdate reduction have

shown that a variety of electron donors could support

molybdate reduction, and two electron donors; glucose and

sucrose, are the best electron donors (Campbell et al. 1985;

Ghani et al. 1993; Shukor et al. 2008b). All of the electron

donors supported cellular growth, but only sucrose, malt-

ose, glucose, fructose and glycerol supported molybdate

reduction with sucrose giving the significantly highest

amount of Mo-blue after 24 h of incubation (P \ 0.05;

Fig. 1). Optimum concentration of sucrose for molybdate

reduction was 5% (w/v) after 24 h of static incubation (data

not shown). In S. marcescens strain Dr.Y6, sucrose is the

best electron donor followed by maltose, glucose and

glycerol (Shukor et al. 2008a). In EC 48 it was found that

sucrose gives the highest rate of molybdate reduction fol-

lowed by glucose, fructose, galactose, mannose, maltose,

lactose, raffinose and sorbitol while 2-ketoglutarate, citrate,

pyruvate, xylose, arabinose and ribose do not support

molybdate reduction (Ghani et al. 1993). Campbell et al.

Table 1 Amount of Mo-blue produced from a 24-h static culture

Bacterial strains Micromole Mo blue produced

after 24 h of static incubation

(± SD, n = 3)

Serratia sp. strain Dr.Y8 10.53 ± 0.47a

Serratia marcescens strain Dr.Y6 3.45 ± 0.53b

E. cloacae strain 48 2.04 ± 0.63b

E. coli K12 0.93 ± 0.34c

Experiments were carried out in triplicate. Value with the same letter

(b) is not significantly different (P \ 0.05). Values are mean ± SE of

three independent experiments

1228 World J Microbiol Biotechnol (2009) 25:1225–1234

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(1985) reported that the best electron donor that supported

molybdate reduction in E. coli K12 is glucose while for-

mate, succinate, glycerol and ethanol did not support

reduction. Both glucose and sucrose could produce reduc-

ing equivalents such as NADH and NADPH more easily

than other electron donors through metabolic pathways

such as the glycolytic and pentose phosphate, and both

reducing equivalents are known electron donating sub-

strates for the molybdenum-reducing enzyme (Ghani et al.

1993; Shukor et al. 2003, 2008c).

The effect of molybdate and phosphate concentrations

on molybdate reduction

Both phosphate and molybdate ions are tetrahedral

oxyanions and thus, they are similar to each other. Phos-

phomolybdate anions contain both anions with molybdate

ions at a higher number than phosphate anions (Lee 1977).

This is probably the reason why previous studies have shown

that higher phosphate concentrations than molybdate could

inhibit molybdate reduction in bacteria (Campbell et al.

1985; Ghani et al. 1993). This study is very important to find

the optimum concentrations for both anions in supporting

molybdate reduction in this bacterium. The results showed

that at a fixed phosphate concentration of 2.9 mM, molyb-

date reduction increased linearly as molybdate

concentrations were increased from 0 to 50 mM and reached

optimum at 50 mM. Higher concentrations inhibited

molybdenum reduction (Fig. 2). We found that the optimum

concentration of phosphate for molybdate reduction when

molybdate concentration was fixed at 50 mM was 5 mM and

molybdate reduction decreased rapidly at much higher

phosphate concentration and was totally inhibited at

100 mM phosphate (Fig. 3). Phosphate and molybdate

concentrations at suboptimal ratios have been previously

reported to inhibit molybdate reduction. Thus it is very

important to ascertain the effects of phosphate and molyb-

date ions in this bacterium to molybdate reduction. The

optimum ratio of 5 mM phosphate to 50 mM molybdate in

this work was lower than that of E. coli K12. The latter

shows an optimum ratio of 5 mM phosphate to 80 mM

molybdate (Campbell et al. 1985). However, it is reported

that phosphate concentrations greater than 0.5 mM inhibit

molybdate reduction in EC 48. In addition, although high

phosphate (100 mM) prevents molybdate reduction, cells

contain active enzymes Ghani et al. (1993). Complete

inhibition of Mo-blue production at 100 mM phosphate was

observed in all of the other bacteria studied above (Campbell

Fig. 1 Molybdate reduction using various electron donors at the final

concentration of 0.2% (w/v). Error bars represent mean ± standard

error (n = 3)

Fig. 2 The effect of molybdate concentrations on molybdate reduc-

tion by strain Dr.Y8. Error bars represent mean ± standard error

(n = 3)

Fig. 3 The effect of phosphate concentrations in the media on

molybdate reduction by strain Dr.Y8. Error bars represent

mean ± standard error (n = 3)

World J Microbiol Biotechnol (2009) 25:1225–1234 1229

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et al. 1985; Ghani et al. 1993; Shukor et al. 2008b). It is

hypothesized that high phosphate inhibits molybdate

reduction by maintaining the pH at neutral; a pH that is

undesirable for the formation and stability of phosphomo-

lybdate (Lee 1977; Sidgwick 1984). A lower range of

molybdenum concentration (between 15 and 25 mM) sup-

porting molybdate reduction is reported in S. marcescens

strain Dr.Y6 (Shukor et al. 2008b) suggesting that this strain

is a much better candidate for molybdenum bioremediation

compared to all of the local isolates to date.

The effect of temperature and pH on molybdate

reduction

Molybdate reduction increased steadily as the temperature

was raised culminating with a sharp optimum at 37�C.

Molybdate reduction was completely abolished at 50�C

(data not shown). The effects of pH was carried out using an

overlapping buffer system consisting of 25 mM acetate

buffer spanning the pH range from 4.0 to 6.0 and 25 mM

Bis-Tris propane buffer to span the range from 6.0 to 8.0.

Molybdate reduction in this bacterium was optimum at

pH 6.0 (data not shown). The optimum temperature sup-

porting molybdate reduction reported in this work is quite

similar to S. marcescens strain Dr.Y6, with the latter opti-

mum at 35�C (Shukor et al. 2008b). In this work we

discovered that the pH optimum was clearly acidic at

pH 6.0 while it was observed that growth of S. marcescens

strain Dr.Y6 under static conditions resulted in the pH ini-

tially adjusted to pH 7.0 to drop to pH 5.0 before

molybdenum reduction commences. The conditions

employed in S. marcescens strain Dr.Y6 is different from

this work since the low phosphate media (LPM) cannot

maintain the initial pH of 7.0 as the buffering species is

phosphate at 2.9 mM. Thus the study of pH optima cannot

be carried out. In this work we employed an overlapping

buffer system to prevent drifting of pH during reduction to

find the optimum pH. The results obtained so far implies the

requirement of acidity for molybdate reduction. Ghani et al.

(1993) grew EC 48 at 30�C and at pH 7.0 using the same

unbuffered LPM media while the optimum temperature and

pH ranges for molybdate reduction in E. coli K12 are from

30 to 36�C and pH 5.0–6.0, respectively (Campbell et al.

1985). This isolate is a suitable indigenous bacterium that

could be employed in local bioremediation since soils in

Malaysia could reach temperatures as high as 35�C and are

generally acidic (Sinnakkannu et al. 2004).

Mo-blue absorption spectrum

During the progress of molybdate reduction the Mo-blue

intensity increased as well as the overall absorption spectra

of the culture media, especially at the peak maximum at

865 nm and the shoulder at 700 nm. This unique finger-

print was preserved as molybdate reduction progressed

(Fig. 4). Other recently isolated molybdenum-reducing

bacteria also exhibit similar Mo-blue absorption spectra to

strain Dr.Y8, EC 48, S. marcescens strain Dr.Y6, and the

spectra appears not to be similar to the absorption spectra

of other Mo-blue products such as silicomolybdate and

sulfomolybdate (Shukor et al. 2007). The spectra supported

the hypothesis that the Mo-blue produced is a reduced form

of phosphomolybdate. The involvement of phosphomo-

lybdate in molybdate reduction has been suggested by

Campbell et al. (1985). The idea that phosphomolybdate is

an important intermediate between molybdate and Mo-blue

is probably plausible since phosphomolybdate is known to

form when molybdate and phosphate are mixed under

acidic conditions, and phosphomolybdate, but not molyb-

date, is easily reduced by many reducing agents including

enzymes (Glenn and Crane 1956; Clesceri et al. 1989). The

requirement for an intermediate species in molybdate

reduction is also seen in related works on biological

chromate reduction. In at least two bacteria; Shewanella

putrefaciens (now S. oneidensis) and Pseudomonas amb-

igua, the reduction of Cr6? to Cr3? goes through an

intermediate species, Cr5? (Suzuki et al. 1992; Myers et al.

2000).

Effect of electron transport system inhibitors

on molybdenum reduction

In this work it was found that none of the respiratory

inhibitors tested showed any inhibition of more than 10%

to the Mo-reducing activity in this bacterium (data not

shown). The inhibitors tested inhibited the transfer of

electrons at certain sites of the electron transport chain.

Rotenone is an inhibitor to NADH dehydrogenase while

Fig. 4 Scanning spectra of Mo-blue after 14, 16, 18, 20 and 24 h of

static incubation labelled A, B, C, D, and E, respectively

1230 World J Microbiol Biotechnol (2009) 25:1225–1234

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sodium azide and cyanide are inhibitors to the terminal

cytochrome oxidase while antimycin A inhibits cyto-

chrome b (Dawson et al. 1969). In the beginning, it was

thought that the use of phosphomolybdate employed in the

new assay system might affect the outcome of the results.

Thus, we employed the original assay using molybdate as

the electron acceptor substrate. We observed no inhibition

on the molybdenum-reducing activity (data not shown). In

EC 48, the molybdenum-reducing activity is suggested to

be located at the electron transport chain downstream from

cytochrome b based on the inhibition of the Mo-reducing

enzyme by cyanide (Ghani et al. 1993). The results with the

inhibitors in this work suggest that the electron transport

chain of this bacterium is not the site of molybdate

reduction. However, the effects of respiratory inhibitors

were not carried out in E. coli K12 (Campbell et al. 1985)

and S. marcescens strain Dr.Y6 (Shukor et al. 2008b), thus

no comparison can be made. Our current works include the

study of the effect of respiratory inhibitors to all of the

other strains.

Effect of metal ions on molybdate reduction

Among the metal ions tested, ferrous and stannous ions

markedly increased the activity of molybdenum-reducing

activity in this bacterium (Table 2). Other metal ions such

as chromium, silver, copper and mercury resulted in

approximately 61, 57, 80, and 69% inhibition of the

molybdenum-reducing activity, respectively. The rest of the

metal ions did not have any effect on the molybdenum-

reducing activity from this bacterium. In EC 48 it was found

that ferric, stannous, nickel, zinc, cobalt, ferrous and silver

enhanced the activity of the molybdenum-reducing enzyme

14-, 12.5-, 5.5-, 5-, 4-, 2.5- and 2-fold, respectively. On the

other hand, cupric and chromium ions strongly inhibited the

activity of the enzyme (Ghani et al. 1993). Chromium ions

also strongly inhibited Mo-blue production in E. coli K12

(Campbell et al. 1985) while the effects of metal ions were

not carried out in S. marcescens strain Dr.Y6 (Shukor et al.

2008b). Surprisingly, it was found that molybdate reduction

could be mediated by ferrous and stannous ions without the

presence of enzyme, and this can only be seen when the

molybdate stock solution was adjusted to pH 7.0 prior to

addition of metal ions to the reaction mixture. If the

molybdate stock solution is not adjusted to neutrality, no

chemical reduction is seen. In the original assay, only the

salt solution is adjusted to pH 7.0 with buffering capacity

provided by phosphate at 5 mM. The electron accepting

substrate, molybdate, was not adjusted to pH 7.0 and was

added to the final concentration of 100 mM. At this con-

centration, when we measured the pH of the reaction

mixture we found the pH to be highly alkaline ([pH 9.0).

Thus, there is a high likelihood that the effect seen is

mediated chemically. These two ions; ferrous and stannous,

have been used as reducing agents for the conversion of

molybdate to Mo-blue in the phosphate determination

method (Clesceri et al. 1989), and it is anticipated that their

use will mask enzymatic reduction of molybdate by bacteria

in general and precautions should be taken to address this

problem. This brought out the role of ferrous ion in the

reduction of molybdate to Mo-blue T. ferrooxidans (Yong

et al. 1997). Due to this, we have devised a testing tool

utilizing phosphomolybdate reduced with ascorbic acid to

determine the effects of interfering substances, and dis-

covered that mercury was found to be a physiological

inhibitor towards molybdate reduction in EC 48 while

phosphate and arsenate included in the growth did not

inhibit the molybdenum-reducing activity (Shukor et al.

2008d). The mechanism of inhibition is suggested to be

through the destabilization of phosphomolybdate, a key

component of molybdate reduction although as previously

discussed, the strong buffering capacity of these ions

probably contributed towards the instability of phospho-

molybdate and Mo-blue, its reduced product (Glenn and

Crane 1956; Lee 1977; Sidgwick 1984). The inhibitory

effect of some heavy metals also means that molybdenum

bioremediation using this bacterium would be challenging

if the concentrations of these metal ions are elevated in the

environment. Metal reduction by enzymes has been known

to be inhibited by metal ions and this is not surprising since

enzymes in general have long been known to be inhibited by

metal ions, especially so by heavy metals ions. For instance,

copper and mercury are both known inhibitors to chromate

reductase. The target of inhibition has been suggested as the

thiol group (Rege et al. 1997; Elangovan et al. 2006). Thus,

Table 2 Effect of metal ions on

molybdate reduction

Values are mean ± SE of three

independent experiments

Metal ions

(1 mM)

Molybdenum blue

produced (nmole/

min/mg)

Control 30.21 ± 0.51

Cr6? 11.67 ± 0.16

Fe3? 30.28 ± 1.52

Fe2? 59.76 ± 0.76

Zn2? 30.10 ± 0.59

Mg2? 30.57 ± 0.69

Co2? 30.67 ± 0.63

Ni2? 29.99 ± 0.68

Cd2? 28.12 ± 1.26

Ag? 12.85 ± 0.61

Mn2? 29.22 ± 0.63

Cu2? 6.16 ± 0.35

Hg2? 9.27 ± 0.02

Pb2? 28.24 ± 0.63

Sn2? 60.90 ± 1.28

World J Microbiol Biotechnol (2009) 25:1225–1234 1231

123

it would be important to screen and isolate bacteria with as

many metal resistance capability as possible since sites with

metal pollution usually contain a variety of toxic heavy

metals and the success of a bacterium to remediate a target

metal would depend upon its resistance to other toxic non

target metal ions presence in the site. However, the enor-

mous amount of energy required for multi toxic metal

resistance suggests that it would be difficult to isolate

bacteria with such a capability.

Dialysis tubing experiment

Munch and Ottow (1983) proved that ferric reduction is an

enzymatic process by relying on the colloidal property of

Mo-blue. Like all colloids, enclosure in dialysis tubing

would trap the colloid but not permanently as it would

diffuse very slowly (Sidgwick 1984). We hypothesize that

if the reduction is mediated by chemical reductants pro-

duced biotically by the bacterium, Mo-blue would be

observed in the inside and outside of the dialysis tubing at

approximately of equal concentration. If the reduction is

mediated by enzyme(s) either extracellular or intracellular,

reduction would only be observed inside of the tubings.

The overwhelming presence of Mo-blue only in the dial-

ysis tubing indicates that the reduction of molybdate by this

bacterium requires the presence of cells or mediated

enzymatically. In this method molybdate-reducing bacteria

were enclosed in dialysis tubing and allowed to reduce

molybdate which is present in the outside and inside of the

dialysis tubing. It was observed that molybdate reduction to

Mo-blue predominantly occurs in the dialysis tubings. We

observed a faint blue on the outside of the dialysis tubing

only at the end of the incubation period. This is due to a

small percentage of the Mo-blue leaking through the dial-

ysis tubing (Shukor et al. 2002). By measuring the blue

intensity at 865 nm we found that 95.4% of Mo-blue was

found inside of the dialysis tubing after the incubation

period has ended. In our previous works in EC 48, it was

found that almost 90% of the Mo-blue produced was

entrapped in the dialysis tubing (Shukor et al. 2002) and

the same conclusion was achieved.

Identification of the isolate

A low bootstrap value (\50%) was seen when strain Dr.Y8

was associated to Serratia marcescens and Serratia sp.

(Fig. 5) indicating that the phylogenetic relationship of this

isolate to a particular species cannot be done. Together

with the Biolog identification system which gave the

closest ID to Serratia sp. with low probability, the isolate

was tentatively assigned as Serratia sp. strain Dr.Y8. This

bacterium is the third molybdenum-reducing bacterium

from the genus Serratia isolated so far indicating the

importance of this genus in molybdate reduction. The first

bacterium from this genus was reported by Jan (1939)

while Serratia marcescens strain Dr.Y6 (2008b) is the

second.

Conclusions

In this work, a local molybdenum-reducing bacterium has

been isolated and characterized. Optimal conditions for

molybdate reduction were obtained, and the results would be

useful in future bioremediation works. The bacterium’s

ability to form a precipitous mass together with the reduced

molybdenum product is an interesting avenue to pursue since

this would facilitate molybdenum removal from aqueous

solution. The recurring similarity of the molybdenum blue

Fig. 5 A phylogram (neighbor-joining method) showing genetic

relationship between strain Dr.Y8 and other related reference

microorganisms based on the 16S rRNA gene sequence analysis.

Species names are followed by the accession numbers of their 16S

rRNA sequences. The numbers at branching points or nodes refer to

bootstrap values, based on 1,000 re-samplings. Scale bar represents

100 nucleotide substitutions. The branch lengths in the phylogram are

not to scale

1232 World J Microbiol Biotechnol (2009) 25:1225–1234

123

spectra of this bacterium to others suggests a universal

involvement of phosphomolybdate as an intermediate during

molybdate reduction to Mo-blue. The effect of metal ions

suggests that this molybdate reduction is quite tolerant to

several toxic heavy metals, and this would be an important

advantage during future remediation works. The inability of

several respiratory inhibitors to inhibit reduction means that

the identity of the molybdenum-reducing enzyme is still a

mystery, and we have to wait sequencing results for confir-

mation of its novelty. The dialysis tubing experiment has

shown that the reduction is enzymatically mediated, and

work is underway to purify the molybdenum-reducing

enzyme from this bacterium.

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