Enrichment and Isolation of a Mixed Bacterial Culture for Complete Mineralization of Endosulfan

In the present study, we isolated three novel bacterial species, namely, Staphylococcus sp., Bacillus circulans–I, and Bacillus circulans–II, from contaminated soil collected from the premises of a pesticide manufacturing industry. Batch experiments were conducted using both mixed and pure cultures to assess their potential for the degradation of aqueous endosulfan in aerobic and facultative anaerobic condition. The influence of supplementary carbon (dextrose) source on endosulfan degradation was also examined. After four weeks of incubation, mixed bacterial culture was able to degrade 71.82 ± 0.2% and 76.04 ± 0.2% of endosulfan in aerobic and facultative anaerobic conditions, respectively, with an initial endosulfan concentration of 50 mg l^?1. Addition of dextrose to the system amplified the endosulfan degradation efficiency by 13.36 ± 0.6% in aerobic system and 12.33 ± 0.6% in facultative anaerobic system. Pure culture studies were carried out to quantify the degradation potential of these individual species. Among the three species, Staphylococcus sp. utilized more beta endosulfan compared to alpha endosulfan in facultative anaerobic system, whereas Bacillus circulans–I and Bacillus circulans–II utilized more alpha endosulfan compared to beta endosulfan in aerobic system. In any of these degradation studies no known intermediate metabolites of endosulfan were observed.     

Biodegradation of endosulfan-contaminated soil in a pilot-scale reactor-bioaugmented with mixed bacterial culture

A novel mixed bacterial culture was enriched from an endosulfan (6, 7, 8, 9, 10, 10 - hexachloro-1, 5, 5a, 6, 9, 9a-hexahydro-6, 9-methano-2, 3, 4-benzo (e) dioxathiepin-3-oxide) processing industrial surface soil. The cultures were successful in the degradation of aqueous phase endosulfan in both aerobic and anaerobic conditions. Using the cultures, endosulfan degradation in silty gravel with sand (GM) was examined via pilot scale reactor at an endosulfan concentration of 0.78 +/- 0.01 mg g(- 1) of soil, and optimized moisture content of 40 +/- 1%. During operation, vertical spatial variability in endosulfan degradation was observed within the reactor. At the end of 56 days, maximum endosulfan degradation efficiency of 78 +/- 0.2% and 86.91 +/- 0.2% was observed in the top and bottom portion of the reactor, respectively. Both aerobic and anaerobic conditions were observed within the reactor. However, endosulfan degradation was predominant in anaerobic condition and the total protein concentration in the reactor was declined progressively down the soil depth. Throughout the study, no known intermediate metabolites of endosulfan reported by previous researchers were observed.     

Biodegradation of lindane, methyl parathion and carbofuran by various enriched bacterial isolates

In the present study, lindane (1,2,3,4,5,6-hexachlorocyclohexane), methyl parathion (O-dimethylO-(4-nitro-phenyl) phosphorothioate) and carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) degradation potential of different enriched bacterial cultures were evaluated under various environmental conditions. Enriched cultures behaved differently with different pesticides. Degradation was more in a facultative anaerobic condition as compared to that in aerobic condition. A specific pesticide enriched culture showed maximum degradation of that pesticide irrespective of pesticides and environmental conditions. Lindane and endosulfan enriched cultures behaved almost similarly. Degradation of lindane by lindane enriched cultures was 75 +/- 3% in aerobic co-metabolic process whereas 78 +/- 5% of lindane degradation occurred in anaerobic co-metabolic process. Degradation of methyl parathion by methyl parathion enriched culture was 87 +/- 1% in facultative anaerobic condition. In almost all the cases, many intermediate metabolites were observed. However, many of these metabolites disappeared after 4-6 weeks of incubation. Mixed pesticide-enriched culture degraded all the three pesticides more effectively as compared to specific pesticide- enriched cultures. It can be inferred from the results that a bacterial consortium enriched with a mixture of all the possible pesticides that are present in the site seems to be a better option for the effective bioremediation of multi-pesticide contaminated site.     

Biodegradation of endosulfan-contaminated soil in a pilot-scale reactor-bioaugmented with mixed bacterial culture

A novel mixed bacterial culture was enriched from an endosulfan (6, 7, 8, 9, 10, 10 – hexachloro-1, 5, 5a, 6, 9, 9a-hexahydro-6, 9-methano-2, 3, 4-benzo (e) dioxathiepin-3-oxide) processing industrial surface soil. The cultures were successful in the degradation of aqueous phase endosulfan in both aerobic and anaerobic conditions. Using the cultures, endosulfan degradation in silty gravel with sand (GM) was examined via pilot scale reactor at an endosulfan concentration of 0.78 ± 0.01 mg g^{? 1} of soil, and optimized moisture content of 40 ± 1%. During operation, vertical spatial variability in endosulfan degradation was observed within the reactor. At the end of 56 days, maximum endosulfan degradation efficiency of 78 ± 0.2% and 86.91 ± 0.2% was observed in the top and bottom portion of the reactor, respectively. Both aerobic and anaerobic conditions were observed within the reactor. However, endosulfan degradation was predominant in anaerobic condition and the total protein concentration in the reactor was declined progressively down the soil depth. Throughout the study, no known intermediate metabolites of endosulfan reported by previous researchers were observed.     

Kinetics of the biodegradation pathway of endosulfan in the aerobic and anaerobic environments

The enriched mixed culture aerobic and anaerobic bacteria from agricultural soils were used to study the degradation of endosulfan (ES) in aqueous and soil slurry environments. The extent of biodegradation was ∼95% in aqueous and ∼65% in soil slurry during 15 d in aerobic studies and, ∼80% in aqueous and ∼60% in soil slurry during 60 d in anaerobic studies. The pathways of aerobic and anaerobic degradation of ES were modeled using combination of Monod no growth model and first order kinetics. The rate of biodegradation of β-isomer was faster compared to α-isomer. Conversion of ES to endosulfan sulfate (ESS) and endosulfan diol (ESD) were the rate limiting steps in aerobic medium and, the hydrolysis of ES to ESD was the rate limiting step in anaerobic medium. The mass balance indicated further degradation of endosulfan ether (ESE) and endosulfan lactone (ESL), but no end-products were identified. In the soil slurries, the rates of degradation of sorbed contaminants were slower. As a result, net rate of degradation reduced, increasing the persistence of the compounds. The soil phase degradation rate of β-isomer was slowed down more compared with α-isomer, which was attributed to its higher partition coefficient on the soil.     

Enrichment and isolation of endosulfan degrading and detoxifying bacteria

In the present study, degradation of endosulfan by a mixed culture isolated from a pesticide-contaminated soil was studied in batch experiments. After two weeks of incubation, the mixed culture was able to degrade 73% and 81% of alpha and beta endosulfan respectively. Endodiol was identified by GC/MS as degradation intermediate. The toxicity studies of endosulfan before and after degradation were carried out using micronucleus assay on human polymorphonuclear cells. The findings suggested that the metabolism of endosulfan isomers by the mixed culture was accompanied by significant reduction in the toxicity. Studies were also carried out to quantify the degradation potential of the individual species in the mixed bacterial culture. Two cultures identified by 16S rRNA as Stenotrophomonas maltophilia and Rhodococcus erythropolis were found to be responsible for majority of the degradation by the mixed culture. S. maltophilia showed better degradation efficiency compared to that by R. erythropolis. This is the first report of endosulfan degradation using the above-mentioned organisms.     

Destruction of lindane and atrazine using stabilized iron nanoparticles under aerobic and anaerobic conditions: effects of catalyst and stabilizer

Highly stable Fe-Pd bimetallic nanoparticles were prepared with 0.2% (w/w) of sodium carboxylmethylcellulose (CMC) as a stabilizer. The effectiveness of the stabilized Fe-Pd nanoparticles was studied for degradation of two chlorinated pesticides (lindane and atrazine) under aerobic and anaerobic conditions. Batch kinetic tests showed that under anaerobic condition the nanoparticles can serve as strong electron donors and completely reduce 1 mgl(-1) of lindane at an iron dose of 0.5 gl(-1) or 1mg l(-1) of atrazine with 0.05 gl(-1) iron with a trace amount (0.05-0.8% of Fe) of Pd as a catalyst. In contrast, under aerobic condition, the nanoparticles can facilitate Fenton-like reactions, which lead to oxidation of 65% of lindane under otherwise identical conditions. Under aerobic condition, the presence of CMC reduced the level of hydroxyl radicals generated from the nanoparticels by nearly 50%, and thus, inhibited the oxidation of the contaminants. While the particle stabilization greatly enhanced the anaerobic degradation, it did not appear to be beneficial under aerobic condition. The degradation rate was progressively enhanced as the Pd content increased from 0.05% to 0.8% of Fe, and the catalytic effect of Pd was more significant under anaerobic condition. Under anaerobic condition, lindane is degraded via dihaloelimination and dehydrohalogenation, whereas atrazine is by reductive dechlorination followed by subsequent reductive dealkylation. Under aerobic condition, reactive oxygen species and hydroxyl radicals from the iron nanoparticles are responsible for oxidizing the pesticides. Lindane is oxidized via dechlorination/dehydrohalogenation, whereas atrazine is destroyed through dealkylation of the alkylamino side chain.     

Kinetic studies of endosulfan photochemical degradations by ultraviolet light irradiation in aqueous medium

Kinetic studies of endosulfan photochemical degradation in controlled aqueous systems were carried out by ultraviolet light irradiation at lambda = 254 nm. The photolysis of (alpha + beta: 2 + 1) endosulfan, alpha-endosulfan and beta-endosulfan were first-order kinetics. The observed rate constants obtained from linear least-squares analysis of the data were 1 x 10(-4) s(-1); 1 x 10(-4) s(-1); and 2 x 10(-5) s(-1), respectively, and the calculated quantum yields (phi) were 1, 1 and 1.6, respectively. Preliminary differential pulse polarographic (DPP) analysis allowed to observe the possible endosulfan photochemical degradation pathway. This degradation route involves the formation of the endosulfan diol, its transformation to endosulfan ether and finally the ether's complete degradation by observing the potential shifts.     

Biodegradation of mixed pesticides by mixed pesticide enriched cultures

This paper discusses the degradation kinetics of mixed (lindane, methyl parathion and carbofuran) pesticides by mixed pesticide enriched cultures (MEC) under various environmental conditions. The bacterial strains isolated from the mixed microbial consortium were identified as Pseudomonas aeruginosa (MTCC 9236), Bacillus sp. (MTCC 9235) and Chryseobacterium joostei (MTCC 9237). Batch studies were conducted to estimate the biokinetic parameters like the maximum specific growth rate (μ_max), Yield Coefficient (Y_T), half saturation concentration (K_s) and inhibition concentration (Ki) for individual and mixed pesticide enriched cultures. The cultures enriched in a particular pollutant always showed high growth rate and low inhibition in that particular pollutant compared to MEC. After seven weeks of incubation, mixed pesticide enriched cultures were able to degrade 72% lindane, 95% carbofuran and 100% of methyl parathion in facultative co-metabolic conditions. In aerobic systems, degradation efficiencies of lindane methyl parathion and carbofuran were increased by the addition of 2g L^{? 1} of dextrose. Though many metabolic compounds of mixed pesticides were observed at different time intervals, none of the metabolites were persistent. Based on the observed metabolites, a degradation pathway was postulated for different pesticides under various environmental conditions.     

The fate of endosulfan in aquatic ecosystems

Endosulfan, one of the major pesticides used in cotton-growing, is of environmental concern because of its toxicity to fish and its apparent persistence in the environment. This study examines the distribution and degradation pathways for endosulfan in an aquatic system and the processes by which it is removed. In the alkaline waters of the cotton region, hydrolysis is the dominant degradation process. By this mechanism alone, the expected half-lives for the alpha- and beta-endosulfan isomers were found to be 3.6 days and 1.7 days, respectively. Partitioning studies showed, however, that the major proportion of endosulfan would associate with the sediments (log Koc(alpha) 3.6 and log Koc(beta) 4.3). Field studies confirmed the presence of high concentrations in sediments. Microcosm experiments showed that loss of endosulfan was slower than predicted from hydrolysis rates. Models are presented to explain how desorption from sediment limits the loss of endosulfan from a system.     

Adsorption and desorption characteristics of hydrophobic pesticide endosulfan in four Indian soils

Adsorption and desorption characteristics of endosulfan in four Indian soils were studied extensively. The soils used were clayey soil (CL--lean clay with sand), red soil (GM--silty gravel with sand), sandy soil (SM--silty sand with gravel) and composted soil (PT--peat) as per ASTM (American Society for Testing and Materials) standards. Adsorption and desorption rates were calculated from kinetic studies. These values varied for alpha and beta endosulfan depending on the soil type. Maximum specific adsorption capacities (qmax) for different soils were calculated by Langmuir model. The values varied from 0.1 to 0.45 mg g(-1) for alpha endosulfan and 0.0942-0.2722 mg g(-1) for beta endosulfan. Maximum adsorption took place in clay soil followed by composted soil and red soil. Adsorptions of alpha and beta endosulfan were negligible in sand. The binding characteristics of various functional groups were calculated using Scatchard plot. Effect of functional groups was more predominant in clayey soil. Organic matter also played a significant role in adsorption and desorption of endosulfan. Endosulfan adsorption decreased drastically in clay soil when the pH was reduced. Desorption was higher at both acidic and alkaline pH ranges compared to neutral pH. Results indicated that alpha endosulfan is more mobile compared to beta endosulfan and mobility of endosulfan is maximum in sandy soil followed by red soil. It can be inferred that crystal lattice of the clay soil plays a significant role in endosulfan adsorption and desorption. Immobilization of endosulfan is more advisable in clay soil whereas biological and or chemical process can be applied effectively for the remediation of other soil types.     

A rapid, direct measurement of bacterial growth rate in anaerobic wastewater treatment.

Reliable design and operation of biological wastewater treatment systems demand robust models of biological degradation processes. However, methods to directly measure key bacterial growth kinetics have not been readily available. Those methods that are available rely on the classic measurement of aerobic respiration using oxygen uptake take rates. This paper shows how the thymidine assay can be used as a rapid and direct measurement of bacterial specific growth rates (mu) in situ for an anaerobic treatment process, independent of aerobic respiration. A filtration-based assay is applied and evaluated a dispersed-phase high-rate anaerobic treatment process, with results obtained in less than an hour. The chemical oxygen demand (COD) biomass in the reactor was 0.52 kg COD m(-3) and the specific growth rate of these anaerobic bacteria was 0.8 +/- 0.2 d(-1). It took the bacterial populations 21.6 hours to double. This is an important advancement from existing methods that use aerobic respiration as a pseudo measurement of bacterial specific growth rates. The method allows rapid and direct measures of microbial growth rates for anaerobic treatment processes.     

The anaerobic degradation of endosulfan by indigenous microorganisms from low-oxygen soils and sediments

Indigenous mixed populations of anaerobic microorganisms from an irrigation tailwater drain and submerged agricultural chemical waste pit readily biodegraded the major isomer of endosulfan (endosulfan I). Endosulfan I was biodegraded to endosulfan diol, a low toxicity degradation product, in the presence of organic carbon sources under anaerobic, methanogenic conditions. While there was extensive degradation (>85%) over the 30 days, there was no significant enhancement of degradation from enriched inocula. This study demonstrates that endosulfan I has the potential to be biodegraded in sediments, in the absence of enriched microorganisms. This is of particular importance since such sediments are prevalent in cotton-growing areas and are typically contaminated with endosulfan residues. The importance of minimizing non-biological losses has also been highlighted as a critical issue in determining anaerobic biodegradation potential. Seals for such incubation vessels must be both oxygen and pollutant impermeable. Teflon-lined butyl rubber provides such a seal because of its resistance to the absorption of volatiles and in preventing volatilization. Moreover, including a 100 mM phosphate buffer in the anaerobic media has reduced non-biological losses from chemical hydrolysis, allowing biodegradation to be assessed.     

Biodegradation of endosulfan by Mortieralla sp. strain W8 in soil: Influence of different substrates on biodegradation

To examine the bioremediation potential of Mortierella sp. strain W8 in endosulfan contaminated soil, the fungus was inoculated into sterilized and unsterilized soil spiked with endosulfan. Wheat bran and cane molasses were used as substrates to understand the influence of different organic materials on the degradation of endosulfan in soil. Strain W8 degraded α- and β-endosulfan in both sterilized and unsterilized soil. In unsterilized soil with wheat bran+W8, α- and β- endosulfan were degraded by approximately 80% and 50%, respectively after 28 d incubation against the initial endosulfan concentration (3 mg kg(-1) dw). The corresponding values for α- and β-endosulfan degradation with wheat bran only were 50% and 3%. Endosulfan diol metabolite was detected after 14 d incubation in wheat bran+W8 whereas it was not found with wheat bran only. Production of endosulfan sulfate, the main metabolite of endosulfan, was suppressed with wheat bran+W8 treatment compared with wheat bran only. It was demonstrated that wheat bran is a more suitable substrate for strain W8 than cane molasses. Wheat bran+W8 is a superior fungus and substrate mix for bioremediation in soil contaminated with endosulfan.     

Biodegradation of malathion, α- and β-endosulfan by bacterial strains isolated from agricultural soil in Veracruz,Mexico

The objective of this study was to evaluate the capacity of two bacterial strains isolated, cultivated, and purified from agricultural soils of Veracruz, Mexico, for biodegradation and mineralisation of malathion (diethyl 2-(dimethoxyphosphorothioyl) succinate) and α- and β-endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6-9-methano-2,4,3-benzodioxathiepine-3-oxide). The isolated bacterial strains were identified using biochemical and morphological characterization and the analysis of their 16S rDNA gene, as Enterobacter cloacae strain PMM16 (E1) and E. amnigenus strain XGL214 (M1). The E1 strain was able to degrade endosulfan, whereas the M1 strain was capable of degrading both pesticides. The E1 strain degraded 71.32% of α-endosulfan and 100% of β-endosulfan within 24 days. The absence of metabolites, such as endosulfan sulfate, endosulfan lactone, or endosulfan diol, would suggest degradation of endosulfan isomers through non-oxidative pathways. Malathion was completely eliminated by the M1 strain. The major metabolite was butanedioic acid. There was a time-dependent increase in bacterial biomass, typical of bacterial growth, correlated with the decrease in pesticide concentration. The CO₂ production also increased significantly with the addition of pesticides to the bacterial growth media, demonstrating that, under aerobic conditions, the bacteria utilized endosulfan and malathion as a carbon source. Here, two bacterial strains are shown to metabolize two toxic pesticides into non-toxic intermediates.     

Combined anaerobic/aerobic digestion: effect of aerobic retention time on nitrogen and solids removal

A combined anaerobic/aerobic sludge digestion system was studied to determine the effect of aerobic solids retention time (SRT) on its solids and nitrogen removal efficiencies. After the anaerobic digester reached steady state, effluent from the anaerobic digester was fed to aerobic digesters that were operated at 2- to 5-day SRTs. The anaerobic system was fed with a mixture of primary and secondary sludge from a local municipal wastewater treatment plant. Both systems were fed once per a day. The aerobic reactor was continuously aerated with ambient air, maintaining dissolved oxygen level at 1.1 +/- 0.3 mg/L. At a 4-day or longer SRT, more than 11% additional volatile solids and 90% or greater ammonia were removed in the aerobic digester, while 32.8 mg-N/L or more nitrite/nitrate also was measured. Most total Kjeldahl nitrogen removal was via ammonia removal, while little organic nitrogen was removed in the aerobic digester.     

Pesticide residues in Nile tilapia (Oreochromis niloticus) and Nile perch (Lates niloticus) from Southern Lake Victoria, Tanzania

Nile tilapia (Oreochromis niloticus) and Nile perch (Lates niloticus) samples were collected from fish landing stations in nine riparian districts on the Tanzanian side of Lake Victoria and screened for residues of 64 organochlorine, organophosphorus, carbamate, and pyrethroid pesticides. The residue levels in the fish fillet were up to 0.003, 0.03 and 0.2 mg/kg fresh weight (0.7, 3.8 and 42 mg/kg lipid weight) of fenitrothion, DDT and endosulfan, respectively. Mean levels within sites were up to 0.002, 0.02 and 0.1 mg/kg fresh weight (0.5, 0.5 and 16 mg/kg lipid weight), respectively. The detection of higher levels of p,p'-DDT than the degradation products (p,p'-DDD and p,p'-DDE), and higher levels of endosulfan isomers (alpha and beta) than the sulphate, in fish samples, implied recent exposure of fish to DDT and endosulfan, respectively. Generally, most of the fish samples had residue levels above the average method detection limits (MDLs), but were within the calculated ADI.     

Biodegradation of alpha and beta endosulfan in soil as influenced by application of different organic materials

A laboratory pot experiment was conducted to study the effect of amending soil with four different sources of organic matter on the degradation rate of alpha and beta endosulfan isomers. Poultry by-product meal, poultry manure, dairy manure, and municipal solid waste compost were cured, dried, ground (<1 mm) and thoroughly mixed with a calcareous soil at a rate of 2% and placed in plastic pots. Endosulfan was added at the rate of 20 mg kg(-1). The moisture level was kept near field capacity and the pots were kept at room temperature. Soil sub-samples, 100 g each, were collected from every pot at days 1, 8, 15, 22, 29, 43, and 57 for the measurement of endosulfan isomers. Endosulfan residues were extracted from the soil samples with acetone. The supernatant was filtered through anhydrous sodium sulphate, 5 mL aliquot was diluted to 25 mL with hexane, mixed well, and then two sub-samples from the filtrates were analyzed for alpha and beta endosulfan isomers by gas chromatography. The results indicated that the half-life (T(1/2)) of alpha-endosulfan in the poultry by-product meal treatment was 15 days compared to about 22 days in the other treatments. The T(1/2) of beta-endosulfan was 22 days in the poultry by-product meal treatment and followed a bi-phasic pattern, 57 days in the municipal solid waste compost treatment and the extrapolated T(1/2) was about 115 days for the other three treatments.     

Assessment of the impact of pesticide residues on microbiological and biochemical parameters of tea garden soils in India

The main aim of this study was to assess the impact of pesticidal residues on soil microbial and biochemical parameters of the tea garden soils. The microbial biomass carbon (MBC), basal (BSR) and substrate induced respirations (SIR), beta-glucosidase activity and fluorescein diacetate hydrolyzing activity (FDHA) of six tea garden soils, along with two adjacent forest soils (control) in West Bengal, India were measured. The biomass and its activities and biochemical parameters were generally lower in the tea garden soils than the control soils. The MBC of the soils ranged from 295.5 to 767.5 micro g g(- 1). The BSR and SIR ranged from 1.65 to 3.08 mu g CO2-C g(- 1) soil h(- 1) and 3.08 to 10.76 micro g CO2-C g(- 1)h(- 1) respectively. The beta-glucosidase and FDHA of the soils varied from 33.3 and 76.3 micro g para-nitrophenol g(- 1) soil h(- 1) and 60.5 to 173.5 micro g fluorescein g(- 1)h(- 1)respectively. The tea garden soils contained variable residues of organophosphorus and organochlorine pesticides, which negatively affected the MBC, BSR, SIR, FDHA and beta -glucosidase activity. Ethion and chlorpyriphos pesticide residues in all the tea garden soils varied from 5.00 to 527.8 ppb and 17.6 to 478.1 ppb respectively. The alpha endosulfan, beta endosulfan and endosulfan sulfate pesticide residues in the tea garden soils ranged from 7.40 to 81.40 ppb, 8.50 to 256.1 ppb and 55 to 95.9 ppb respectively. Canonical correlation analysis shows that 93% of the total variation was associated with the negative impact of chlorpyriphos, beta and alpha endosulfan and endosulfan sulfate on MBC, BSR and FDHA. At the same time ethion had negative impact on SIR and beta-glucosidase. Data demonstrated that the pesticide residues had a strong impact on the microbial and biochemical components of soil quality.     

Characterisation and optimisation of three potential aerobic bacterial strains for kraft lignin degradation from pulp paper waste

Eight aerobic bacterial strains were isolated from pulp paper mill effluent sludge. Out of eight through nutrient enrichment technique three potential aerobic bacterial strains ITRC S(6), ITRC S(7) and ITRC S(8) were found capable to effectively degrade the kraft lignin (KL), a major byproduct of the chemical pulping process and main contributor to the colour and toxicity of effluent. Further, these potential strains (ITRC S(6), ITRC S(7) and ITRC S(8)) were biochemically characterised as Gram variable small rod, Gram negative rod and Gram positive rod respectively. Subsequently, 16S rRNA sequencing showed 95% base sequence homology and it was identified as Paenibacillus sp. (AY952466), Aneurinibacillus aneurinilyticus (AY856831), Bacillus sp. (AY952465) for ITRC S(6), IITRC S(7) and ITRC S(8), respectively. In batch decolourization experiments Bacillus sp. ITRC S(8) reduced the colour of lignin amended mineral salt medium, pH 7.6 by 65% after 6th d, at 30 degrees C, A. aneurinilyticus ITRC S(7) by 56% and Paenibacillus ITRC S(6) 43%. Under these conditions the three strains degraded the KL by 37%, 33% and 30%, respectively while the mixed culture of these three bacteria reduced colour by 69%, lignin by 40% and total substrate by 50% under same conditions. Biodegradation of the KL was not affected by low (<0.2 mg l(-1)) dissolved oxygen content; thus oxygen inhibition is more likely to be a metabolism-dependent event. Initially with 48 h incubation the decolourization was slow with decreased pH. Further incubation there was rapid decolourization with slight increase in pH at 6d compared with initial pH by increasing culture optical density. The lignin analysis from medium with HPLC indicated complete degradation rather than biotransformation with complete loss of absorbance peak at 280 nm.