Bacterial reduction of hexavalent molybdenum to molybdenum blue.

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 previously 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

Development of an inhibitive enzyme assay for copper

In this work the development of an inhibitive assay for copper using the molybdenum-reducing enzyme assay is presented. The enzyme is assayed using 12-molybdophosphoric acid at pH 5.0 as an electron acceptor substrate and NADH as the electron donor substrate. The enzyme converts the yellowish solution into a deep blue solution. The assay is based on the ability of copper to inhibit the molybdenum-reducing enzyme from the molybdate-reducing Serratia sp. Strain DRY5. Other heavy metals tested did not inhibit the enzyme at 10 mg l(-1). The best model with high regression coefficient to measure copper inhibition is one-phase binding. The calculated IC50 (concentration causing 50% inhibition) is 0.099 mg l(-1) and the regression coefficient is 0.98. The comparative LC50, EC50 and IC50 data for copper in different toxicity tests show that the IC50 value for copper in this study is lower than those for immobilized urease, bromelain, Rainbow trout, R. meliloti, Baker's Yeast dehydrogenase activity Spirillum volutans, P. fluorescens, Aeromonas hydrophilia and synthetic activated sludge assays. However the IC50 value is higher than those for Ulva pertusa and papain assays, but within the reported range for Daphnia magna and Microtox assays

Isolation and Characterization of a Molybdenum Reducing Enzyme in Enterobacter cloacae Strain 48

Molybdenum reducing enzyme was isolated from Enterobacter cloacae Strain 48 by ammonium sulphate fractionation, DE-cellulose ion-exchange chromatography and Sephacryl S-200 gel filtration. SDS-PAGE of the concentrated Sephacryl S-200 gel filtration eluates revealed the presence of 3 protein subunits of molecular weight 80, 90 and 100 kDa. The active concentrated fraction from the Sephacryl S-200 gel filtration step was then characterized for molybdenum reducing activity with 12-molybdophosphate (12-MoP) as a substrate. The optimum pH and temperature of the reaction was 5.0 and 28-33°C, respectively. ADH was a better reducing agent in the reaction than NADPH; the double reciprocal plot of activity against ADH and NADPH revealed apparent Km and V""", values of 1.65 mM, 6.28 nmole molybdenum blue produced/min/mg and 2.13 mM and 4.10 nmole molybdenum blue produced/min/mg, respectively. The double reciprocal plot of activity against 12-MoP and 20-molybdodiphosphate revealed apparent K m values of 0.3 mM and 0.4 mM, respectively. The apparent Vmax values are similar for both substrates at 6 nmole molybdenum blue produced/min. The assay method for molybdenum reducing activity using 12-MoP was found to be easier and more rapid than the present method of using molybdate as a substrat

An improved enzyme assay for molybdenum-reducing activity in bacteria.

Molybdenum-reducing activity in the heterotrophic bacteria is a phenomenon that has been reported for more than 100 years. In the presence of molybdenum in the growth media, bacterial colonies turn to blue. The enzyme(s) responsible for the reduction of molybdenum to molybdenum blue in these bacteria has never been purified. In our quest to purify the molybdenum-reducing enzyme, we have devised a better substrate for the enzyme activity using laboratory-prepared phosphomolybdate instead of the commercial 12-phosphomolybdate we developed previously. Using laboratory-prepared phosphomolybdate, the highest activity is given by 10:4-phosphomolybdate. The apparent Michaelis constant, K m for the laboratory-prepared 10:4-phosphomolybdate is 2.56 ± 0.25 mM (arbitrary concentration), whereas the apparent V max is 99.4 ± 2.85 nmol Mo-blue min−1 mg−1 protein. The apparent Michaelis constant or K m for NADH as the electron donor is 1.38 ± 0.09 mM, whereas the apparent V max is 102.6 ± 1.73 nmol Mo-blue min−1 mg−1 protein. The apparent K m and V max for another electron donor, NADPH, is 1.43 ± 0.10 mM and 57.16 ± 1.01 nmol Mo-blue min−1 mg−1 protein, respectively, using the same batch of molybdenum-reducing enzyme. The apparent V max obtained for NADH and 10:4-phosphomolybdate is approximately 13 times better than 12-phoshomolybdate using the same batch of enzyme, and hence, the laboratory-prepared phosphomolybdate is a much better substrate than 12-phoshomolybdate. In addition, 10:4-phosphomolybdate can be routinely prepared from phosphate and molybdate, two common chemicals in the laboratory

Enhanced caffeine degradation by immobilised cells of Leifsonia sp. strain SIU

In a previous study, we isolated Leifsonia sp. strain SIU, a new bacterium from agricultured soil. The bacterium was tested for its ability to degrade caffeine. The isolate was encapsulated in gellan gum and its ability to degrade caffeine was compared with the free cells. The optimal caffeine degradation was attained at a gellan gum concentration of 0.75% (w/v), a bead size of 4 mm diameter, and 250 beads per 100 mL of medium. At a caffeine concentration of 0.1 g/L, immobilised cells of the strain SIU degraded caffeine within 9 h, which is faster when compared to the case of free cells, in which it took 12 h to degrade. The immobilised cells degraded caffeine completely within 39 and 78 h at 0.5 and 1.0 g/L, while the free cells took 72 and 148 h at 0.5 and 1.0 g/L, respectively. At higher caffeine concentrations, immobilised cells exhibited a higher caffeine degradation rate. At concentrations of 1.5 and 2.0 g/L, caffeine-degrading activities of both immobilised and free cells were inhibited. The immobilised cells showed no loss in caffeine-degrading activity after being used repeatedly for nine 24-h cycles. The effect of heavy metals on immobilised cells was also tested. This study showed an increase in caffeine degradation efficiency when the cells were encapsulated in gellan gum

Hexavalent molybdenum reduction to molybdenum blue by S. marcescens Strain Dr. Y6

A molybdate-reducing bacterium has been locally isolated. The bacterium reduces molybdate or Mo(6+) to molybdenum blue (molybdate oxidation states of between 5+ and 6+). Different carbon sources such as acetate, formate, glycerol, citric acid, lactose, fructose, glucose, mannitol, tartarate, maltose, sucrose, and starch were used at an initial concentration of 0.2% (w/v) in low phosphate media to study their effect on the molybdate reduction efficiency of bacterium. All of the carbon sources supported cellular growth, but only sucrose, maltose, glucose, and glycerol (in decreasing order) supported molybdate reduction after 24 h of incubation. Optimum concentration of sucrose for molybdate reduction is 1.0% (w/v) after 24 h of static incubation. Ammonium sulfate, ammonium chloride, valine, OH-proline, glutamic acid, and alanine (in the order of decreasing efficiency) supported molybdate reduction with ammonium sulfate giving the highest amount of molybdenum blue after 24 h of incubation at 0.3% (w/v). The optimum molybdate concentration that supports molybdate reduction is between 15 and 25 mM. Molybdate reduction is optimum at 35 degrees C. Phosphate at concentrations higher than 5 mM strongly inhibits molybdate reduction. The molybdenum blue produced from cellular reduction exhibits a unique absorption spectrum with a maximum peak at 865 nm and a shoulder at 700 nm. The isolate was tentatively identified as Serratia marcescens Strain Dr.Y6 based on carbon utilization profiles using Biolog GN plates and partial 16s rDNA molecular phylogeny

An alternative bioassay using Anabas testudineus (climbing perch) colinesterase for metal ions detection

Climbing Perch or its scientific name, Anabas testudineus is one of the freshwater fish belonging to the family of Anabantidae. It is widely distributed in ponds, swamps and estuaries in Asia. In this study, cholinesterase (ChE) was partially purified from the liver of A. testudineus through ion exchange chromatography. This purification method provided a recovery yield of 5.36% with a purification fold of 6.6. The optimum conditions for ChE assay were identified to be 2.5 mM of butyrylthiocholine iodide (BTC) with pH 8.0 in Tris-HCl buffer at 40°C. Substrate specificity profile also indicated that ChE favours BTC as substrate because it records the highest catalytic efficiency (Vmax/Km). Protein analysis through Native-PAGE showed that ion exchange chromatography is an effective method to partially purify ChE. Metal ion inhibition tests were conducted and mercury (Hg) was found to show the highest inhibition effect (87.30%) whereas lead (Pb) shows the lowest inhibition effect (28.01%). All these findings showed that partially purified ChE from the liver of A. testudineus is suitable to be used as a bioindicator to detect the presence of metal ions

Optimisation of biodegradation conditions for cyanide removal by Serratia marcescens strain AQ07 using one-factor-at-a-time technique and response surface methodology

Gold mining companies are known to use cyanide to extract gold from minerals. The indiscriminate use of cyanide presents a major environmental issue. Serratia marcescens strain AQ07 was found to have cyanide-degrading ability. Optimisation of biodegradation condition was carried out utilising one factor at a time and response surface methodology. Cyanide degradation corresponded with growth rate with a maximum growth rate of 16.14 log cfu/mL on day 3 of incubation. Glucose and yeast extract are suitable carbon and nitrogen sources. Six parameters including carbon and nitrogen sources, pH, temperature, inoculum size and cyanide concentration were optimised. In line with the central composite design of response surface methodology, cyanide degradation was optimum at glucose concentration 5.5 g/L, yeast extract 0.55 g/L, pH 6, temperature 32.5 °C, inoculum size 20 % and cyanide concentration 200 mg/L. It was able to stand cyanide toxicity of up to 700 mg/L, which makes it an important candidate for bioremediation of cyanide. The bacterium was observed to degrade 95.6 % of 200 mg/L KCN under the optimised condition. Bacteria are reported to degrade cyanide into ammonia, formamide or formate and carbon dioxide, which are less toxic by-products. These bacteria illustrate good cyanide degradation potential that can be harnessed in cyanide remediation