Screening of three phenol-degrading bacteria for copper bioremoval of copper

Aim: Potential microbial isolates for bioremoval of copper were collected from phenolic and heavy metal-contaminated soils and screened in copper-containing medium for determining the maximum tolerance level for copper. Methodology: Bioremoval of copper was assessed using sodium diethyl dithiocarbamate assay. Physical and cultural conditions influencing copper bioremoval such as initial concentration, biomass dosage (inoculum volume), temperature and pH were optimised via one-factor-at-a-time. Results: The highest maximum tolerance level was displayed by Serratia sp. AQ5-03 at 600 mg l-1, while for Alcaligenes sp. AQ5-02 and Pseudomonas sp. AQ5-04 it was 500 mg l-1. The highest bioremoval for Alcaligenes sp. AQ5-02, Serratia sp. AQ5-03 and Pseudomonas sp. AQ5-04 was achieved at 20, 50 and 75 mg l-1, respectively. The optimum biomass dosage (inoculum volume) for both Serratia sp. AQ5-03 and Pseudomonas sp. AQ5-04 were 15%, whereas it was 10% for Alcaligenes sp. AQ5-02. The results also revealed that maximum bioremoval was achieved at pH 6 at an optimum temperature of 20°C for both Alcaligenes sp. AQ5-02 and Pseudomonas sp. AQ5-04, while Serratia sp. AQ5-03 showed optimum removal at pH 7 and at 30°C temperature. The maximum bioremoval efficiency for Alcaligenes sp. AQ5-02, Serratia sp. AQ5-03 and Pseudomonas sp were found to be 62, 57 and 70%, respectively. Interpretation: The results indicate that Alcaligenes sp. AQ5-02, Serratia sp. AQ5-03, Pseudomonas sp. AQ5-04 can be utilised as bioremoval agent since it possessed the highest bioremoval efficiency for copper remediation.

Histopathological and cholinesterase changes in the gills of Clarias gariepinus as a result of cadmium exposure

Aim: The cholinesterase (ChE) based inhibition and histopathological studies from fish were investigated and represented in this study to develop as one of the great potential biomarkers for heavy metals monitoring. Methodology: In this study, the histopathological study of gills were observed a under microscope. The capability of ChE extracted from the gills of Clarias gariepinus was assessed for declining Cd. ChE was purified through affinity chromatography and continued with the optimisation and inhibition study (IC50) of cholinesterase. Results: Histopathological study of gills was carried out and several changes such as aneurysm, necrosis and lamella fusion were noted. Purification fold obtained from purified enzyme was 1.15 with 30% a yield specific activity 20.726. The optimum temperature for purified AChE was 35°C along with acetylthiocholine iodide (ATC) as a preferable substrate that had the highest Vmax value of 0.5452 U mg-1 and the lowest Km value of 0.0311 mM. The optimum pH was observed to be 10 of Tris-HCl as a medium. Meanwhile, the IC50 of cadmium was 6.808 mg l-1 with R2 value of 0.9532. Interpretation: The result of the study can be used as a tool for further developing a biomarker for the detection of heavy metals in aquatic ecosystems. In addition, the baseline data provided can also be used for designing a kit, which would give rapid and accurate result.

Characterisation of cholinesterase and histopathological features of brain of Clarias gariepinus following exposure to cadmium

Aim: Cholinesterase (ChE) inhibition and histopathological features of brain tissues of Clarias gariepinus were investigated with a purpose to develop biomarkers for monitoring heavy metals. Methodology: The effects of cadmium toxicity on C. gariepinus were assessed by ChE inhibition and characterisation of ChE by affinity chromatography and histological variations in brain tissues were studied by H&E staining under light microscope. Results: The purified enzyme gave 10.58 fold purification with the recovery of 12% with specific activity of 159.729 U mg-1. The Michaelis-Menten constant (Km) and Vmax value obtained was 1.985 mM and 0.4479 µmol min-1 mg-1, respectively. The enzyme showed the ability to hydrolyse acetylthiocholine iodide (ATC) at a faster rate compared to other two synthetic substrates, propionylcholine iodide (PTC) and butyrylthiocholine iodide (BTC). ChE showed highest activity at 35°C in Tris-HCl pH 7.5. The metal inhibition study also found the maximal inhibitory concentration (IC50) value for cadmium to be 0.6517 mg l-1. Histological study revealed decrease in the number of cells, degeneration of Purkinje cells, increased aggregation area and cells necrosis. Interpretation: Inhibition of cholinesterase activity and degeneration of Purkinje cells observed in the present study can be used as a tool for further developing a biomarker for detecting heavy metals in the aquatic ecosystems.

Growth of probiotic bacteria in trypticase phytone yeast medium supplemented with crude polysaccharides from Ganoderma lucidum

Ganoderma lucidum is a fungus usually used in traditional Chinese medicine. The high value of G. lucidum is related to its polysaccharides content. Crude polysaccharides from G. lucidum (GLCP) were obtained using hot water extraction method. There is about 0.57 g of GLCP in 1 g crude of G. lucidum. The prebiotic potential of GLCP was tested against probiotic bacteria namely: Bifidobacterium longum BB536, Bifidobacterium pseudocatenulatum G4, Lactobacillus acidophilus and Lactobacillus casei Shirota. The prebiotic potentials were studied in 10 mL basal Trypticase Phytone Yeast (abbreviated as bTPY) medium (without glucose) supplemented with various concentrations of GLCP (abbreviated as bTPYglcp) (0.5%, 1.0%, 1.5% and 2.0%). bTPY medium supplemented with glucose (abbreviated as bTPYglu) and inulin (abbreviated bTPYinu) were used as comparison. Viable cell counts of the bacteria and the pH of the medium were determined during anaerobic incubation period of 0 h, 12 h, 24 h and 48 h at 37 °C. In the presence of carbohydrate source, cultures showed various degree of growth increment. With regards to the growth supporting property: bTPYglu, bTPYglu+glcp, bTPYglcp and bTPYinu were ranked first, second, third and fourth respectively. Interestingly, in bTPYglcp medium, bacterial growth increased with increasing GLCP concentrations when incubated until 24 h. B. longum BB536 was ranked first (10.53 log cfu/mL) in term of their growth in this medium. Growth of B.pseudocatenulatum G4 was ranked second with 10.40 log cfu/mL. This study shows that, GLCP could support the growth of the bacteria tested.

Characterization of a diesel-degrading strain isolated from a hydrocarbon-contaminated site.

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 oC. 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.

Assessment of Clarias batrachus as a source of acetylcholinesterase (AChE) for the detection of insecticides.

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) μg 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) μg l-1, respectively, and were much lower than bovine AChE at 20.94 (19.53-22.58) and 418.80 (390.60-451.60) μg 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.

Isolation and characterization of an SDS-degrading Klebsiella oxytoca.

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 37oC. 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 Km (app) and Vmax (app) values calculated for the alkylsulfatase from this bacterium were 0.1 mM SDS and 1.07 >mol min-1 mg-1 protein, respectively.

Isolation and characterization of an acrylamide-degrading Antarctic bacterium.

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°C. The characteristics of this isolate suggest that it would be useful in the bioremediation of acrylamide.

A simple method to screen for azo-dye-degrading bacteria.

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 BiologTM identification system as Serratia marcescens.

Reduction of Mo(VI) by the bacterium Serratia sp. strain DRY5.

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 oC 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 of molybdenum-containing waste.

Isolation and characterization of an acrylamide-degrading Bacillus cereus.

Several local acrylamide-degrading bacteria have been isolated. One of the isolate that exhibited the highest growth on acrylamide as a nitrogen source was then further characterized. The isolate was tentatively identified as Bacillus cereus strain DRY135 based on carbon utilization profiles using Biolog GP plates and partial 16S rDNA molecular phylogeny. The isolate grew optimally in between the temperatures of 25 and 30oC and within the pH range of 6.8 to 7.0. Glucose, fructose, lactose, maltose, mannitol, citric acid and sucrose supported growth with glucose being the best carbon source. Different concentrations of acrylamide ranging from 100 to 4000 mg l-1 incorporated into the growth media shows that the highest growth was obtained at acrylamide concentrations of between 500 to 1500 mg l-1. At 1000 mg l-1 of acrylamide, degradation was 90% completed after ten days of incubation with concomitant cell growth. The metabolite acrylic acid was detected in the media during degradation. Other amides such as methacrylamide, nicotinamide, acetamide, propionamide and urea supported growth with the highest growth supported by acetamide, propionamide and urea. Strain DRY135, however, was not able to assimilate 2-chloroacetamide. The characteristics of this isolate suggest that it would be useful in the bioremediation of acrylamide.

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.

The development of an inhibitive determination method for zinc using a serine protease.

A new inhibitive heavy metals determination method using trypsin has been developed. The enzyme was assayed using the casein- Coomassie-dye-binding method. In the absence of inhibitors, casein was hydrolysed to completion and the Coomassie-dye was unable to stain the protein and the solution became brown. In the presence of metals, the hydrolysis of casein was inhibited and the solution remained blue. The bioassay was able to detect zinc and mercury with IC50 (concentration causing 50% inhibition) values of 5.78 and 16.38 mg l-1 respectively. The limits of detection (LOD), for zinc and mercury were 0.06 mg l-1 (0.05-0.07, 95% confidence interval) and 1.06 mg l-1 (1.017-1.102, 95% confidence interval), respectively. The limits of quantitation (LOQ) for zinc and mercury were 0.61 mgl-1 (0.51-0.74 at a 95% confidence interval) and 1.35 mg l-1 (1.29-1.40 at a 95% confidence interval), respectively. The IC50 value for zinc was much higher than the IC50 values for papain and Rainbow trout, but was within the range of Daphnia magna and MicrotoxTM. The IC50 value for zinc was only lower than those for immobilized urease. Other toxic heavy metals, such as lead, silver, arsenic, copper and cadmium, did not inhibit the enzyme at 20 mg l-1. Using this assay, we managed to detect elevated zinc concentrations in several environmental samples. Pesticides, such as carbaryl, flucythrinate, metolachlor, glyphosate, diuron, diazinon, endosulfan sulphate, atrazine, coumaphos, imidacloprid, dicamba and paraquat, showed no effect on the activity of trypsin relative to control (One-way ANOVA, F12, 26 = 0.3527, p> 0.05). Of the 17 xenobiotics tested, only (sodium dodecyl sulphate) SDS gave positive interference with 150 % activity higher than that of the control at 0.25% (v/v).

Isolation and characterization of a Pseudomonas diesel-degrading strain from Antarctica.

A diesel-degrading bacterium from Antarctica has been isolated. The isolate was tentatively identified as Pseudomonas sp. strain DRYJ3 based on partial 16S rDNA molecular phylogeny and Biolog® GN microplate panels and Microlog® database. Growth on diesel was supported optimally by ammonium sulphate, nitrate and nitrite. The bacterium grew optimally in between 10 and 15 oC, pH 7.0 and 3.5% (v/v) diesel. The biodegradation of diesel oil by the strain increased in efficiency from the second to the sixth day of incubation from 1.4 to 18.8% before levelling off on the eighth day. n-alkane oxidizing and aldehyde reductase activities were detected in the crude enzyme preparation suggesting the existence of terminal n-alkane oxidizing activity in this bacterium.