Arsenic is a toxic metalloid which is widely distributed in nature. It is normally present as arsenate under oxic conditions while arsenite is predominant under reducing condition. The major discharges of arsenic in the environment are mainly due to natural sources such as aquifers and anthropogenic sources. It is known that arsenite salts are more toxic than arsenate as it binds with vicinal thiols in pyruvate dehydrogenase while arsenate inhibits the oxidative phosphorylation process. The common mechanisms for arsenic detoxification are uptaken by phosphate transporters, aquaglyceroporins, and active extrusion system and reduced by arsenate reductases via dissimilatory reduction mechanism. Some species of autotrophic and heterotrophic microorganisms use arsenic oxyanions for their regeneration of energy. Certain species of microorganisms are able to use arsenate as their nutrient in respiratory process. Detoxification operons are a common form of arsenic resistance in microorganisms. Hence, the use of bioremediation could be an effective and economic way to reduce this pollutant from the environment.
A novel molybdate-reducing bacterium, tentatively identified as Klebsiella sp. strain hkeem and based on partial 16s rDNA gene sequencing and phylogenetic analysis, has been isolated. Strain hkeem produced 3 times more molybdenum blue than Serratia sp. strain Dr.Y8; the most potent Mo-reducing bacterium isolated to date. Molybdate was optimally reduced to molybdenum blue using 4.5 mM phosphate, 80 mM molybdate and using 1% (w/v) fructose as a carbon source. Molybdate reduction was optimum at 30 °C and at pH 7.3. The molybdenum blue produced from cellular reduction exhibited absorption spectrum with a maximum peak at 865 nm and a shoulder at 700 nm. Inhibitors of electron transport system such as antimycin A, rotenone, sodium azide, and potassium cyanide did not inhibit the molybdenum-reducing enzyme. Mercury, silver, and copper at 1 ppm inhibited molybdenum blue formation in whole cells of strain hkeem.
Heavy metals with high chemical activity from sludge and waste release, agriculture, and
mining activity are a major concern. They should be carefully managed before reaching the
main water bodies. Excessive exposure to heavy metal may cause toxic effect to any types of
organism from the biomolecular to the physiological level, and ultimately cause death. Monitoring is the best technique to ensure the safety of our environment before a rehabilitation is
needed. Nowadays, enzyme-based biosensors are utilised in biomonitoring programmes as
this technique allows for a real-time detection and rapid result. It is also inexpensive and easy
to handle. Enzyme-based biosensors are an alternative for the preliminary screening of
contamination before a secondary screening is performed using high-performance technology.
This review highlights the current knowledge on enzyme-based biosensors, focusing on
cholinesterase for toxic metal detection in the environment.
An inhibitive assay of insecticides using Acetylcholinesterase (AChE) from the local fish Clarias batrachus is reported. AChE was assayed according to the modified method of Ellman. Screening of insecticide and heavy metals showed that carbofuran and carbaryl strongly inhibited C. batrachus AChE. The inhibition concentration (IC) IC50 values (and the 95% confidence interval) for both carbofuran and carbaryl inhibition on C. batrachus AChE at 6.66 (5.97-7.52) and 130.00 (119.3-142.5) microg l(-1), respectively was within the IC50 range of Electrophorus electricus at 6.20 (6.03-6.39) and 133.01 (122.40-145.50) microg l(-1), respectively and were much lower than bovine AChE at 20.94 (19.53-22.58) and 418.80 (390.60-451.60) microg l(-1), respectively. The results showed that C. batrachus have the potential to be used as a cheaper and more readily available source of AChE than other more commercially available sources.
A stab-culture method was adapted to screen for azo dyes-decolorizing bacteria from soil and water samples. Decolorized azo dye in the lower portion of the solid media indicates the presence of anaerobic azo dyes-decolorizing bacteria, while aerobic decolorizing bacteria decolorizes the surface portion of the solid media. Of twenty soil samples tested, one soil sample shows positive results for the decolourisation of two azo dyes; Biebrich scarlet (BS) and Direct blue 71 (DB) under anaerobic conditions. A gram negative and oxidase negative bacterial isolate was found to be the principal azo dyes degrader The isolate was identified by using the Biolog identification system as Serratia marcescens.
In this work, we report on the isolation of a phenol-degrading Rhodococcus sp. with a high tolerance towards phenol. The isolate was identified as Rhodococcus sp. strain AQ5NOL 2, based on 16S rDNA analysis. The strain degraded phenol using the meta pathway, a trait shared by many phenol-degraders. In addition to phenol biodegradation, the strain was also capable of degrading diesel. Strain AQ5NOL 2 exhibited a broad optimum temperature for growth on phenol at between 20 °C and 35 °C. The best nitrogen sources were ammonium sulphate, glycine or phenylalanine, followed by proline, nitrate, leucine, and alanine (in decreasing efficiency). Strain AQ5NOL 2 showed a high tolerance and degradation capacity of phenol, for it was able to register growth in the presence of 2000 mg l(-1) phenol. The growth of this strain on phenol as sole carbon and energy source were modeled using Haldane kinetics with a maximal specific growth rate (μ(max)) of 0.1102 hr(-1), a half-saturation constant (K(s) ) of 99.03 mg l(-1) or 1.05 mmol l(-1), and a substrate inhibition constant (K(i)) of 354 mg l(-1) or 3.76 mmol l(-1). Aside from phenol, the strain could utilize diesel, 2,4-dinitrophenol and ρ-cresol as carbon sources for growth. Strain AQ5NOL 2 exhibited inhibition of phenol degradation by Zn(2+), Cu(2+), Cr(6+), Ag(+) and Hg(2+) at 1 mg l(-1).
As well as for chemical and environmental reasons, acrylamide is widely used in many industrial applications. Due to its carcinogenicity and toxicity, its discharge into the environment causes adverse effects on humans and ecology alike. In this study, a novel acrylamide-degrading yeast has been isolated. The isolate was identified as Rhodotorula sp. strain MBH23 using ITS rRNA analysis. The results showed that the best carbon source for growth was glucose at 1.0% (w/v). The optimum acrylamide concentration, being a nitrogen source for cellular growth, was at 500 mg l(-1). The highest tolerable concentration of acrylamide was 1500 mg l(-1) whereas growth was completely inhibited at 2000 mg l(-1). At 500 mg l(-1), the strain MBH completely degraded acrylamide on day 5. Acrylic acid as a metabolite was detected in the media. Strain MBH23 grew well between pH 6.0 and 8.0 and between 27 and 30 °C. Amides such as 2-chloroacetamide, methacrylamide, nicotinamide, acrylamide, acetamide, and propionamide supported growth. Toxic heavy metals such as mercury, chromium, and cadmium inhibited growth on acrylamide.
Extensive use of metals in various industrial applications has caused substantial environmental pollution. Molybdenum-reducing bacteria isolated from soils can be used to remove molybdenum from contaminated environments. In this work we have isolated a local bacterium with the capability to reduce soluble molybdate to the insoluble molybdenum blue. We studied several factors that would optimize molybdate reduction. Electron donor sources such as glucose, sucrose, lactose, maltose and fructose (in decreasing efficiency) supported molybdate reduction after 24 h of incubation with optimum glucose concentration for molybdate reduction at 1.5% (w/v). The optimum pH, phosphate and molybdate concentrations, and temperature for molybdate reduction were pH 6.5, 5.0, 25 to 50 mM and 37 degrees C, respectively. The Mo-blue produced by 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, cadmium, copper, silver and mercury caused approximately 73, 71, 81, 77 and 78% inhibition of the molybdenum-reducing activity, respectively. All of the respiratory inhibitors tested namely rotenone, azide, cyanide and antimycin A did not show any inhibition to the molybdenum-reducing activity suggesting components of the electron transport system are not responsible for the reducing activity. The isolate was tentatively identified as Enterobacter sp. strain Dr.Y13 based on carbon utilization profiles using Biolog GN plates and partial 16S rDNA molecular phylogeny.
A local molybdenum-reducing bacterium was isolated and tentatively identified as Acinetobacter calcoaceticus strain Dr.Y12 based on carbon utilization profiles using Biolog GN plates and 16S rDNA comparative analysis. Molybdate reduction was optimized under conditions of low dissolved oxygen (37 degrees C and pH 6.5). Of the electron donors tested, glucose, fructose, maltose and sucrose supported molybdate reduction after 1 d of incubation, glucose and fructose supporting the highest Mo-blue production. Optimum Mo-blue production was reached at 20 mmol/L molybdate and 5 mmol/L phosphate; increasing the phosphate concentrations inhibited the production. An increase in an overall absorption profiles, especially at peak maximum at 865 nm and the shoulder at 700 nm, was observed in direct correlation with the increased in Mo-blue amounts. Metal ions, such as chromium, cadmium, copper, mercury and lead (2 mmol/L final concentration) caused approximately 88, 53, 80, 100, and 20 % inhibition, respectively. Respiratory inhibitors, such as antimycin A, rotenone, sodium azide and cyanide showed in this bacterium no inhibition of the Mo-blue production, suggesting that the electron transport system is not a site of molybdate reduction.
Luminescence-based assays for toxicants such as Microtox, ToxAlert, and Biotox have been used extensively worldwide. However, the use of these assays in near real time conditions is limited due to nonoptimal assay temperature for the tropical climate. An isolate that exhibits a high luminescence activity in a broad range of temperatures was successfully isolated from the mackerel, Rastrelliger kanagurta. This isolate was tentatively identified as Photobacterium sp. strain MIE, based on partial 16S rDNA molecular phylogeny. Optimum conditions that support high bioluminescence activity occurred between 24 and 30°C, with pH 5.5 to 7.5, 10 to 20 g/L of sodium chloride, 30 to 50 g/L of tryptone, and 4 g/L of glycerol as the carbon source. Assessment of near real time capability of this bacterial system, Xenoassay light to monitor heavy metals from a contaminated river running through the Juru River Basin shows near real time capability with assaying time of less than 30 minutes per samples. Samples returned to the lab were tested with a standard Microtox assay using Vibrio fishceri. Similar results were obtained to Xenoassay light that show temporal variation of copper concentration. Thus, this strain is suitable for near real time river monitoring of toxicants especially in the tropics.
The first purification of the Mo-reducing enzyme from Serratia sp. strain DRY5 that is responsible for molybdenum reduction to molybdenum blue in the bacterium is reported. The monomeric enzyme has an apparent molecular weight of 105 kDalton. The isoelectric point of this enzyme was 7.55. The enzyme has an optimum pH of 6.0 and maximum activity between 25 and 35°C. The Mo-reducing enzyme was extremely sensitive to temperatures above 50°C (between 54 and 70°C). A plot of initial rates against substrate concentrations at 15 mM 12-MP registered a V max for NADH at 12.0 nmole Mo blue/min/mg protein. The apparent K m for NADH was 0.79 mM. At 5 mM NADH, the apparent V max and apparent K m values for 12-MP of 12.05 nmole/min/mg protein and 3.87 mM, respectively, were obtained. The catalytic efficiency (k cat/K m ) of the Mo-reducing enzyme was 5.47 M(-1) s(-1). The purification of this enzyme could probably help to solve the phenomenon of molybdenum reduction to molybdenum blue first reported in 1896 and would be useful for the understanding of the underlying mechanism in molybdenum bioremediation involving bioreduction.
A diesel-degrading bacterium was isolated from a diesel-contaminated site in Selangor, Malaysia. The isolate was tentatively identified as Acinetobacter sp. strain DRY12 based on partial 16S rDNA molecular phylogeny and Biolog GN microplate panels and Microlog database. Optimum growth occurred from 3 to 5% diesel and the strain was able to tolerate as high as 8% diesel. The optimal pH that supported growth of the bacterium was between pH 7.5 to 8.0. The isolate exhibited optimal growth in between 30 and 35 degrees C. The best nitrogen source was potassium nitrate (between 0.6 and 0.9% (w/v)) followed by ammonium chloride, sodium nitrite and ammonium sulphate in descending order. An almost complete removal of diesel components was seen from the reduction in hydrocarbon peaks observed using Solid Phase Microextraction Gas Chromatography analysis after 10 days of incubation. The best growth kinetic model to fit experimental data was the Haldane model of substrate inhibiting growth with a correlation coefficient value of 0.97. The maximum growth rate- micromax was 0.039 hr(-1) while the saturation constant or half velocity constant Ks and inhibition constant Ki, were 0.387% and 4.46%, respectively. MATH assays showed that 75% of the bacterium was found in the hexadecane phase indicating that the bacterium was hydrophobic. The characteristics of this bacterium make it useful for bioremediation works in the Tropics.
A bacterium capable of biodegrading surfactant sodium dodecyl sulphate (SDS) was isolated from Antarctic soil. The isolate was tentatively identified as Pseudomonas sp. strain DRY15 based on carbon utilization profiles using Biolog GN plates and partial 16S rDNA molecular phylogeny. Growth characteristic studies showed that the bacterium grew optimally at 10 degrees C, 7.25 pH, 1 g l(-1) SDS as a sole carbon source and 2 g l(-1) ammonium sulphate as nitrogen source. Growth was completely inhibited at 5 g l(-1) SDS. At a tolerable initial concentration of 2 g l(-1), approximately 90% of SDS was degraded after an incubation period of eight days. The best growth kinetic model to fit experimental data was the Haldane model of substrate inhibition with a correlation coefficient value of 0.97. The maximum growth rate was 0.372 hr(-1) while the saturation constant or half velocity constant (Ks) and inhibition constant (Ki), were 0.094% and 11.212 % SDS, respectively. Other detergent tested as carbon sources at 1 g l(-1) was Tergitol NP9, Tergitol 15S9, Witconol 2301 (methyl oleate), sodium dodecylbenzene sulfonate (SDBS), benzethonium chloride, and benzalkonium chloride showed Tergitol NP9, Tergitol 15S9, Witconol 2301 and the anionic SDBS supported growth with the highest growth exhibited by SDBS.
Near-real-ime assay is anassay method that the whole process from sampling until results could be obtained in approximately Iess than one hour. The ElIman assay for acetyl cholinesterase (AChE) has near real-time potential due to its simplicity and fast assay time. The commercial acetylcholinesterase from Electrophorus electricus is well known for its uses in insecticides detection. A lesser known fact is AChE is also sensitive to heavy metals. A near real-time inhibitive assay for heavy metals using AChE from this source showed promising results. Several heavy metals such as copper, silver and mercury could be etected with IC50 values of1.212, 0.1185 and 0.097 mg I-1, respectively. The Limits of Detection (LOD) for copper, silver and mercury were 0.01, 0.015 and 0.01 mg I-1, respectively. TheLimits of quantitation (LOQ) or copper, silver and mercury were 0.196, 0.112 and 0.025 mg I-1, respectively. The LOQvalues for copper, silver and mercury were well below the maximum permissible limit for these metal ions as outlined by Malaysian Department of Environment. A polluted location demonstrated near real-time applicability of the assay with variation oftemporal levels of heavy metals detected. The results show that AChE from Electrophorus electricus has the potential to be used as a near real-time biomonitoring tool for heavy
A diesel-degrading bacterium has been isolated from a diesel-polluted site. The isolate was tentatively identified as Staphylococcus aureus strain DRY11 based on partial 16S rDNA molecular phylogeny and Biolog GP microplate panels and Microlog database. Isolate 11 showed an almost linear increase in cellular growth with respect to diesel concentrations with optimum growth occurring at 4% (v/v) diesel concentration. Optimization studies using different nitrogen sources showed that the best nitrogen source was potassium nitrite. Sodium nitrite was optimum at 1.2 g l(-1) and higher concentrations were strongly inhibitory to cellular growth. The optimal pH that supported growth of the bacterium was between 7.5 to 8.0 and the isolate exhibited optimal broad temperature supporting growth on diesel from 27 to 37 degrees C. An almost complete removal of diesel components was seen from the reduction in hydrocarbon peaks observed using Solid Phase Microextraction Gas Chromatography analysis after 5 days of incubation. The characteristics of this bacterium suggest that it is suitable for bioremediation of diesel spills and pollutions in the tropics.
Sodium dodecyl sulfate (SDS) is one of the main components in the detergent and cosmetic industries. Its bioremediation by suitable microorganism has begun to receive greater attention as the amount of SDS usage increases to a point where treatment plants would not be able to cope with the increasing amount of SDS in wastewater. The purpose of this work was to isolate local SDS-degrading bacteria. Screening was carried out by the conventional enrichment-culture technique. Six SDS-degrading bacteria were isolated. Of these isolates, isolate S14 showed the highest degradation of SDS with 90% degradation after three days of incubation. Isolate S14 was tentatively identified as Klebsiella oxytoca strain DRY14 based on carbon utilization profiles using Biolog GN plates and partial 16S rDNA molecular phylogeny. SDS degradation by the bacterium was optimum at 37 degrees 0. Ammonium sulphate; at 2.0 g l(-1), was found to be the best nitrogen source for the growth of strain DRY14. Maximum growth on SDS was observed at pH 7.25. The strain exhibited optimum growth at SDS concentration of 2.0 g l(-1) and was completely inhibited at 10 g l(-1) SDS. At the tolerable initial concentration of 2.0 g l(-1), almost 80% of 2.0 g l(-1) SDS was degraded after 4 days of incubation concomitant with increase in cellular growth. The K(m(app) and V(max(app)) values calculated for the alkylsulfatase from this bacterium were 0.1 mM SDS and 1.07 micromol min(-1) mg(-1) protein, respectively.
The presence of acrylamide in the environment poses a threat due to its well known neurotoxic, carcinogenic and teratogenic properties. Human activities in various geographical areas are the main anthropogenic source of acrylamide pollution. In this work, an acrylamide-degrading bacterium was isolated from Antarctic soil. The physiological characteristics and optimum growth conditions of the acrylamide-degrading bacteria were investigated. The isolate was tentatively identified as Pseudomonas sp. strain DRYJ7 based on carbon utilization profiles using Biolog GN plates and partial 16S rDNA molecular phylogeny. The results showed that the best carbon sources for growth was glucose and sucrose with no significant difference in terms of cellular growth between the two carbon sources (p>0.05). This was followed by fructose and maltose with fructose giving significantly higher cellular growth compared to maltose (p<0.05). Lactose and citric acid did not support growth. The optimum acrylamide concentration as a nitrogen source for cellular growth was at 500 mgl(-1). At this concentration, bacterial growth showed a 2-day lag phase before degradation took place concomitant with an increase in cellular growth. The isolate exhibited optimum growth in between pH 7.5 and 8.5. The effect of incubation temperature on the growth of this isolate showed an optimum growth at 15 degrees C. The characteristics of this isolate suggest that it would be useful in the bioremediation of acrylamide.
The need to isolate efficient heavy metal reducers for cost effective bioremediation strategy have resulted in the isolation of a potent molybdenum-reducing bacterium. The isolate was tentatively identified as Serratia sp. strain DRY5 based on the Biolog GN carbon utilization profiles and partial 16S rDNA molecular phylogeny. Strain DRY5 produced 2.3 times the amount of Mo-blue than S. marcescens strain Dr.Y6, 23 times more than E. coli K12 and 7 times more than E. cloacae strain 48. Strain DRY5 required 37 degrees C and pH 7.0 for optimum molybdenum reduction. Carbon sources such as sucrose, maltose, glucose and glycerol, supported cellular growth and molybdate reduction after 24 hr of static incubation. The most optimum carbon source that supported reduction was sucrose at 1.0% (w/v). Ammonium sulphate, ammonium chloride, glutamic acid, cysteine, and valine supported growth and molybdate reduction with ammonium sulphate as the optimum nitrogen source at 0. 2% (w/v). Molybdate reduction was optimally supported by 30 mM molybdate. The optimum concentration of phosphate for molybdate reduction was 5 mM when molybdate concentration was fixed at 30 mM and molybdate reduction was totally inhibited at 100 mM phosphate. Mo-blue produced by this strain shows a unique characteristic absorption profile with a maximum peak at 865 nm and a shoulder at 700 nm, Dialysis tubing experiment showed that 95.42% of Mo-blue was found in the dialysis tubing suggesting that the molybdate reduction seen in this bacterium was catalyzed by enzyme(s). The characteristics of isolate DRY5 suggest that it would be useful in the bioremediation ofmolybdenum-containing waste.
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.