Comparative studies on the metabolism of aniline and chloroanilines by Pseudomonas multivorans strain An 1

Summary Pseudomonas multivorans strain An 1 used aniline but not chloroanilines as the sole source of carbon and energy for growth. The aniline-adapted cells, however, were able to oxygenate chloroanilines. Relative oxygenation rates for aniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline, and 3,4-dichloroaniline were 100, 46, 66, 20, and 3%, respectively.The first intermediates in the metabolism of chloroanilines were chlorocatechols. 3-Chlorocatechol accumulated during growth of the organism in the presence of 2-chloroaniline, whereas 4-chlorocatechol was an intermediate metabolite of 3-chloroaniline and 4-chloroaniline.Chloroanilines were able to induce synthesis of the aniline oxygenating enzyme system of Pseudomonas multivorans strain An 1. In continuous culture experiments, induction of this enzyme system appeared to depend on cell density, concentration, toxicity, and pK-values of aniline or chloroanilines.Studies with ¹⁴C-labelled 3-chloroaniline and 4-chloroaniline showed that the turnover of chloroanilines did not cease with the formation of chlorocatechols, because radioactivity was detected in the CO₂ released and in bacterial cell components. The results suggest that the turnover of chloroanilines is due to metabolism rather than to cometabolism.     

Degradation of aniline and monochlorinated anilines by soil-born Pseudomonas acidovorans strains

Four bacterial strains (CA26, CA28, CA37, and CA45), which all were able to use aniline, 3-chloroaniline (3-CA), and 4-chloroaniline (4-CA) as sole sources of carbon, nitrogen and energy, were isolated after enrichment in aerated soil columns and identified as Pseudomonas acidovorans strains. In addition strains CA26 and CA45 were able to degrade 2-chloroaniline (2-CA) at very low rates. At 25°C strain CA28 was grown on aniline and 3-CA with generation times of 3.0 and 7.7 h, respectively, and exhibited complete mineralization of these substrates in degradation rates of 2.25 mmol aniline and 1.63 mmol 3-CA g^-1 of biomass per hour, respectively. Degradation of 4-CA occurred at 1.54 mmol 4-CA g^-1 of biomass per hour and a generation time of 18.7 h but, in contrast, was not complete due to formation of minor amounts of chlorohydroxymuconic semialdehyde, a meta-cleavage product of 4-chlorocatechol. The initial attack on the substrate, the formation of corresponding chlorocatechols from 3-CA and 4-CA, was found to be the rate-limiting degradation step. Evidence for two different aniline-oxygenase systems in strain CA28 with distinct activity pattern on chlorinated and nonsubstituted anilines was demonstrated by oxygen uptake rate experiments with aniline and chloroaniline pregrown cells. Further degradation was shown to be initialized by catechol dioxygenases.Non-standard abbreviations CA chloroaniline - DCA dichloroaniline - ECM enrichment and cultivation medium - CFU colony forming unit     

Microbial mineralization of ring-substituted anilines through an ortho-cleavage pathway

Moraxella sp. strain G is able to utilize as sole source of carbon and nitrogen aniline, 4-fluoroaniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline (PCA), and 4-bromoaniline but not 4-iodoaniline, 4-methylaniline, 4-methoxyaniline, or 3,4-dichloroaniline. The generation time on PCA was 6 h. The pathway for the degradation of PCA was investigated by analysis of catabolic intermediates and enzyme activities. Mutants of strain G were isolated to enhance the accumulation of specific pathway intermediates. PCA was converted by an aniline oxygenase to 4-chlorocatechol, which in turn was degraded via a modified ortho-cleavage pathway. Synthesis of the aniline oxygenase was inducible by various anilines. This enzyme exhibited a broad substrate specificity. Its specific activity towards substituted anilines seemed to be correlated more with the size than with the electron-withdrawing effect of the substituent and was very low towards anilines having substituents larger than iodine or a methyl group. The initial enzyme of the modified ortho-cleavage pathway, catechol 1,2-dioxygenase, had similar characteristics to those of corresponding enzymes of pathways for the degradation of chlorobenzoic acid and chlorophenol, that is, a broad substrate specificity and high activity towards chlorinated and methylated catechols.     

Microbial mineralization of ring-substituted anilines through an ortho-cleavage pathway.

Moraxella sp. strain G is able to utilize as sole source of carbon and nitrogen aniline, 4-fluoroaniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline (PCA), and 4-bromoaniline but not 4-iodoaniline, 4-methylaniline, 4-methoxyaniline, or 3,4-dichloroaniline. The generation time on PCA was 6 h. The pathway for the degradation of PCA was investigated by analysis of catabolic intermediates and enzyme activities. Mutants of strain G were isolated to enhance the accumulation of specific pathway intermediates. PCA was converted by an aniline oxygenase to 4-chlorocatechol, which in turn was degraded via a modified ortho-cleavage pathway. Synthesis of the aniline oxygenase was inducible by various anilines. This enzyme exhibited a broad substrate specificity. Its specific activity towards substituted anilines seemed to be correlated more with the size than with the electron-withdrawing effect of the substituent and was very low towards anilines having substituents larger than iodine or a methyl group. The initial enzyme of the modified ortho-cleavage pathway, catechol 1,2-dioxygenase, had similar characteristics to those of corresponding enzymes of pathways for the degradation of chlorobenzoic acid and chlorophenol, that is, a broad substrate specificity and high activity towards chlorinated and methylated catechols.     

Bioreduction of para-chloronitrobenzene in a hydrogen-based hollow-fiber membrane biofilm reactor: effects of nitrate and sulfate

A continuous-stirred, hydrogen-based, hollow-fiber membrane biofilm reactor (HFMBfR) that was active in nitrate and sulfate reductions was shown to be effective for degradation or detoxification of para-chloronitrobenzene (p-CNB) in water by biotransforming it first to para-chloroaniline (nitro-reduction) and then to aniline (reductive dechlorination) with hydrogen (H₂) as an electron donor. A series of short-term experiments examined the effects of nitrate and sulfate on p-CNB bioreduction. The results obtained showed both higher nitrate and sulfate concentration declined the p-CNB bioreduction in the biofilm, and this suggests the competition for H₂ caused less H₂ available for the p-CNB bioreduction when the H₂ demand for the reductions was larger. Denitrification and sulfate reduction intermediates were thought to be potential factors inhibiting the p-CNB bioreduction. Analysis of electron-equivalent fluxes and reaction orders in the biofilm further demonstrated both denitrification and sulfate reduction competed more strongly for H₂ availability than p-CNB bioreduction. These findings have significant implications for the HFMBfR used for degrading p-CNB under denitrifying and/or sulfate reducing conditions.     

Biofilm model calibration and microbial diversity study using Monte Carlo simulations

Mathematical models are useful tools for studying and exploring biological conversion processes as well as microbial competition in biological treatment processes. A single‐species biofilm model was used to describe biofilm reactor operation at three different hydraulic retention times (HRT). The single‐species biofilm model was calibrated with sparse experimental data using the Monte Carlo filtering method. This calibrated single‐species biofilm model was then extended to a multi‐species model considering 10 different heterotrophic bacteria. The aim was to study microbial diversity in bulk phase biomass and biofilm, as well as the competition between suspended and attached biomass. At steady state and independently of the HRT, Monte Carlo simulations resulted only in one unique dominating bacterial species for suspended and attached biomass. The dominating bacterial species was determined by the highest specific substrate affinity (ratio of µ/K_S). At a short HRT of 20 min, the structure of the microbial community in the bulk liquid was determined by biomass detached from the biofilm. At a long HRT of 8 h, both biofilm detachment and microbial growth in the bulk liquid influenced the microbial community distribution. Biotechnol. Bioeng. 2013; 110: 1323–1332. © 2012 Wiley Periodicals, Inc.     

Biodegradation of chlorpropham and its major products by <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NKC-1

Chlorpropham [isopropyl N-(3-chlorophenyl) carbamate] (CIPC), an important phenyl carbamate herbicide, has been used as a plant growth regulator and potato sprout suppressant (Solanum tuberosum L) during long-term storage. A bacterium capable of utilizing the residual herbicide CIPC as a sole source of carbon and energy was isolated from herbicide-contaminated soil samples employing selective enrichment method. The isolated bacterial strain was identified as Bacillus licheniformis NKC-1 on the basis of its morphological, cultural, biochemical characteristics and also by phylogenetic analysis based on 16S rRNA gene sequences. The organism degraded CIPC through its initial hydrolysis by CIPC hydrolase enzyme to yield 3-chloroaniline (3-CA) as a major metabolic product. An inducible 3-CA dioxygenase not only catalyzes the incorporation of molecular oxygen but also removes the amino group by the deamination yielding a monochlorinated catechol. Further, degradation of 4-chlorocatechol proceeded via ortho- ring cleavage through the maleylacetate process. 3-Chloroaniline and 4-chlorocatechol are the intermediates in the CIPC degradation which suggested that dechlorination had occurred after the aromatic ring cleavage. The presence of these metabolites has been confirmed by using ultra-violet (UV), high-performance liquid chromatography (HPLC), thin layer chromatography (TLC), Fourier transmission-infrared (FT-IR), proton nuclear magnetic resonance (¹H NMR) and gas chromatography-mass (GC-MS) spectral analysis. Enzyme activities of CIPC hydrolase, 3-CA dioxygenase and chlorocatechol 1, 2-dioxygenase were detected in the cell-free-extract of the CIPC culture and are induced by cells of NKC-1 strain. These results demonstrate the biodegradation pathways of herbicide CIPC and promote the potential use of NKC-1 strain to bioremediate CIPC-contaminated environment with subsequent release of ammonia, chloride ions and carbon dioxide.     

Microbiological Community Structure of the Biofilm of a Methanol-Fed,Marine Denitrification System,and Identification of the Methanol-Utilizing Microorganisms

We demonstrated in a previous study that the biofilm of the methanol-fed fluidized marine denitrification reactor at the Montreal Biodome was composed of at least 15 bacterial phylotypes. Among those were 16S ribosomal RNA (rDNA) gene sequences affiliated to Hyphomicrobium spp., and Methylophaga spp.; the latter made up 70% of a clone library. By using fluorescent in situ hybridization (FISH), we investigated the structure of the biofilm during the colonization process in the denitrification reactor by targeting most of the bacterial families that the 16S rDNA gene library suggested would occur in the biofilm. Our results revealed that gamma-Proteobacteria (mostly Methylophaga spp.) accounted for up to 79% of the bacterial population, confirming the abundance of Methylophaga spp. within the biofilm. alpha-Proteobacteria represented 27–57% of the population, which included Hyphomicrobium spp. that appeared after 20 days of colonization and represented 7–8% of the population. We noticed a great abundance and diversity of eukaryotic cells, which made up 20% of the biomass at the beginning of the colonization but decreased to 3–5% in the mature biofilm. We then used FISH combined with microautoradiography (MAR–FISH) to identify the methylotrophs in the biofilm. The results showed that alpha-Proteobacteria used ¹⁴C methanol in the presence of nitrate, suggesting their involvement in denitrification. Despite their abundance, Methylophaga spp. did not assimilate methanol under those conditions.     

Anaerobic treatment of highly concentrated aniline wastewater using packed-bed biofilm reactor

The performance of packed-bed biofilm reactor (PBBR) with self-floating bio-carriers was investigated to treat highly concentrated organic nitrogenous aniline wastewater with a COD value as high as 24,000 mg/L. With 45 vol% of carrier charge inside the reactor, the aniline wastewater can be effectively treated with 94% of COD removal efficiency at a low organic loading rate (OLR) of 0.9 kg COD/(m³ d). The removal efficiency decreased gradually down to 75% when OLR increased to 12.27 kg COD/(m³ d) that corresponded to 1 day of HRT. Separate tests with biofilm alone showed that the conversion contribution of the biofilm was about half of the overall COD conversion by the biofilm plus sludge system at the same OLRs of 3–4 kg COD/(m³ d), and that the biofilm had higher activity than suspended sludge. Ammonium released from decomposed aniline was increased gradually from 500 to 1700 mg/L with the OLR increase from 0.9 to 12.27 kg COD/(m³ d), which resulted in inhibitory effect to the microorganism due to the toxicity of free ammonia. Batch anaerobic toxicity tests showed that the biofilm was less sensitive to toxic compounds than suspended sludge and could tolerate higher concentration of free ammonia.     

Influence of Hydraulic Retention Time on Extent of PCE Dechlorination and Preliminary Characterization of the Enrichment Culture

The extent of tetrachloroethene (PCE) dechlorination in two chemostats was evaluated as a function of hydraulic retention time (HRT). The inoculum of these chemostats was from an upflow anaerobic sludge blanket (UASB) reactor that rapidly converts PCE to vinyl chloride (VC) and ethene. When the HRT was 2.9 days, PCE was converted only to cis-dichloroethene (cDCE). When the HRT was 11 days, the end products were VC and ethene. Further studies showed that the dechlorinating microbial community in the UASB reactor contained two distinct populations, one of which converted PCE to cDCE and the other cDCE to VC and ethene. Methanogenic activity was very low in these cultures. The cDCE dechlorinating culture apparently has a lower growth rate than the PCE dechlorinating culture, and as a result, at a shorter HRT, the cDCE dechlorinating culture was washed out from the system leading to incomplete dechlorination of PCE. Both enrichment cultures used pyruvate or hydrogen as electron donors for dechlorination. Acetate was the carbon source (but not energy source) when hydrogen was used. Both cultures had undefined nutrient requirements and needed supplements of cell extract obtained from the mixed culture in the UASB reactor. However, the two cultures were different in their response to the addition of an inhibitor of methanogenesis (2-bromoethanesulfonate [BES]). BES inhibited the dechlorinating activity of the enriched cDCE dechlorinating culture, but had no influence on the PCE dechlorinating culture. Preliminary studies on BES inhibition are presented.     

Analysis and comparison of the microbial community structures of two enrichment cultures capable of reductively dechlorinating TCE and cis-DCE

In order to study the effect of different chloroethenes (electron acceptors) on the bacterial composition of dechlorinating communities, two reductive dechlorinating enrichment cultures were developed that were able to reduce trichloroethene (TCE) and cis-1,2-dichloroethene (cis-DCE) to ethene using hydrogen as electron donor, respectively. The inoculum for the cultures was material from a methanogenic fluidized bed reactor (FBR), which was originally seeded with digester sludge and showed a stable capacity for tetrachloroethene (PCE) reduction to ethene for over six years. Molecular methods were used to determine and compare the microbial communities of these two enrichment cultures. A clone library of bacterial 16S rRNA genes was generated for each enrichment. The clones were screened into different groups by restriction fragment length polymorphism (RFLP) analysis using two different four base pair recognition restriction enzymes. A total of 12 sequence types were identified by phylogenetic analysis of nearly complete 16S rDNA sequences ( approximately 1450 bp). The sequences were affiliated with six recognized phyla of the domain Bacteria: Firmicutes (low G+C Gram-positives), Chloroflexi (green non-sulphur bacteria), Actinobacteria (high G+C Gram-positives), Bacteroidetes (Cytophaga-Flexibacter-Bacteroides), Nitrospira and Spirochaetes. The results led to the identification of an organism closely related to Dehalococcoides ethenogenes to be the presumptive dechlorinator in both enrichments. Different electron acceptors affected the bacterial diversity and the community profiles of the two enrichments. Most of the sequences identified in our dechlorinating enrichments shared high similarities with sequences previously obtained from other enriched dechlorinating cultures and chlorinated-compound-contaminated sediments or aquifers, suggesting these bacteria may have direct or indirect roles in reductive dechlorination.     

Population diversity in model potable water biofilms receiving chlorine or chloramine residual

Most water utilities use chlorine or chloramine to produce potable water. These disinfecting agents react with water to produce residual oxidants within a water distribution system (WDS) to control bacterial growth. While monochloramine is considered more stable than chlorine, little is known about the effect it has on WDS biofilms. Community structure of 10-week old WDS biofilms exposed to disinfectants was assessed after developing model biofilms from unamended distribution water. Four biofilm types were developed on polycarbonate slides within annular reactors while receiving chlorine, chloramine, or inactivated disinfectant residual. Eubacteria were identified through 16S rDNA sequence analysis. The model WDS biofilm exposed to chloramine mainly contained Mycobacterium and Dechloromonas sequences, while a variety of alpha- and additional beta-proteobacteria dominated the 16S rDNA clone libraries in the other three biofilms. Additionally, bacterial clones distantly related to Legionella were found in one of the biofilms receiving water with inactivated chlorine residual. The biofilm reactor receiving chloraminated water required increasing amounts of disinfectant after 2 weeks to maintain chlorine residual. In contrast, free chlorine residual remained steady in the reactor that received chlorinated water. The differences in bacterial populations of potable water biofilms suggest that disinfecting agents can influence biofilm development. These results also suggest that biofilm communities in distribution systems are capable of changing in response to disinfection practices.     

Biodegradation of 4-chloroaniline by bacteria enriched from soil

4-Chloroaniline has been released into the environment due to extensive use in chemical industries and intensive agriculture; hence, it becomes one of the hazardous pollutants in the priority pollutant list. In this study, three gram-negative bacteria were enriched and isolated from agricultural soil as 4-chloroaniline-degrading bacteria. They were identified as Acinetobacter baumannii CA2, Pseudomonas putida CA16 and Klebsiella sp. CA17. They were able to utilize 4-chloroaniline as a sole carbon and nitrogen source without stimulation or cocultivation with aniline or another cosubstrate. The biodegradation in these bacteria was occurred via a modified ortho-cleavage pathway of which the activity of chlorocatechol 1, 2-dioxygenase was markedly induced. They grew well on 0.2-mM 4-chloroaniline exhibiting a 60-75% degradation efficiency and equimolar liberation of chloride. The isolates were able to survive in the presence of 4-chloroaniline at higher concentrations (up to 1.2 mM). 2-Chloroaniline, 3-chloroaniline and aniline, but not 3, 4-dichloroaniline, were also growth substrates for these isolates. The results of cosubstrate supplementation illustrated the suitable conditions of each isolate to improve growth rate and 4-chloroaniline biodegradation efficiency. These results suggest that these isolates have a potential use for bioremediation of the site contaminated with 4-chloroaniline.     

Microbial sulfate reduction with acetate: process performance and composition of the bacterial communities in the reactor at different salinity levels

Microbial sulfate reduction with acetate as carbon source and electron donor was investigated at salinity levels between 0.53 and 1.48%. The experiment was carried out in a 2.3-1 upflow anaerobic sludge blanket reactor inoculated with granular methanogenic sludge. A pH of 8.3, a temperature of 32 +/- 1 degrees C and a chemical oxygen demand (COD)/SO4(2-)-S ratio of 2 were maintained in the reactor throughout the experiment. Sulfate reduction and the composition of the dominant bacterial communities in the reactor were monitored. The results showed that a maximal conversion rate for SO4(2-)-S of 14 g l(-1) day(-1) and a conversion efficiency of more than 90% were obtained at a salinity level of 1.26-1.39%. A further increase in the salinity level led to reactor instability. Denaturant gradient gel electrophoresis of 16S rDNA fragments amplified by PCR from total bacterial DNA extracted from the inoculum and reactor sludge showed that salinity level had an impact on the composition of the bacterial communities in the reactor. However, no clear relationship was found between reactor performance and the composition of the dominant bacterial communities in the reactor.     

Characterization of sulfate-reducing bacteria dominated surface communities during start-up of a down-flow fluidized bed reactor

An anaerobic down-flow fluidized bed reactor was inoculated with granular sludge and started-up with sulfate containing synthetic wastewater to promote the formation of a biofilm enriched in sulfate-reducing bacteria (SRB), to produce biogenic sulfide. The start-up was done in two stages operating the reactor in batch for 45 days followed by 85 days of continuous operation. Low-density polyethylene was used as support. The biofilm formation was followed up by biochemical and electron microscopy analyses and the composition of the community was examined by 16S rDNA sequence analysis. Maximum immobilized volatile solids (1.2 g IVS/L_support) were obtained after 14 days in batch regime. During the 85 days of continuous operation, the reactor removed up to 80% of chemical oxygen demand (COD), up to 28% of the supplied sulfate and acetate was present in the effluent. Sulfate-reducing activity determined in the biofilm with ethanol or lactate as substrate was 11.7 and 15.3 g COD/g IVS per day, respectively. These results suggested the immobilization of sulfate reducers that incompletely oxidize the substrate to acetate; the phylogenetic analysis of the cloned 16S rDNA gene sequences showed high identity to the genus Desulfovibrio that oxidizes the substrates incompletely. In contrast, in the granular sludge used as inoculum a considerable number of clones showed homology to Methanobacterium and just few clones were close to SRB. The starting-up approach allowed the enrichment of SRB within the diverse community developed over the polyethylene support.     

Continuous dechlorination of tetrachloroethene in an upflow anaerobic sludge blanket reactor

Influences of hydraulic retention time (HRT) on dechlorination of tetrachloroethene (PCE) were investigated in an upflow anaerobic sludge blanket (UASB) reactor inoculated with anaerobic granular sludge non-pre-exposed to chlorinated compounds. PCE was introduced into the reactor at a loading rate of 3 mg/l d. PCE removal increased from 51 +/- 5% to 87 +/- 3% when HRT increased from 1 to 4 d, corresponding to an increase in the PCE biotransformation rate from 10.5 +/- 2.3 to 21.3 +/- 3.7 mumol/d. A higher ethene production rate, 0.9 +/- 0.2 mumol/d, was attained without accumulation of dichloroethenes at the HRT of 4 d. Dehalococcoides-like species were detected in sludge granules by fluorescence in situ hybridization, with signal strength in proportion to the extent of PCE dechlorination.     

Population diversity in model potable water biofilms receiving chlorine or chloramine residual

Abstract

Most water utilities use chlorine or chloramine to produce potable water. These disinfecting agents react with water to produce residual oxidants within a water distribution system (WDS) to control bacterial growth. While monochloramine is considered more stable than chlorine, little is known about the effect it has on WDS biofilms. Community structure of 10-week old WDS biofilms exposed to disinfectants was assessed after developing model biofilms from unamended distribution water. Four biofilm types were developed on polycarbonate slides within annular reactors while receiving chlorine, chloramine, or inactivated disinfectant residual. Eubacteria were identified through 16S rDNA sequence analysis. The model WDS biofilm exposed to chloramine mainly contained Mycobacterium and Dechloromonas sequences, while a variety of alpha- and additional beta-proteobacteria dominated the 16S rDNA clone libraries in the other three biofilms. Additionally, bacterial clones distantly related to Legionella were found in one of the biofilms receiving water with inactivated chlorine residual. The biofilm reactor receiving chloraminated water required increasing amounts of disinfectant after 2 weeks to maintain chlorine residual. In contrast, free chlorine residual remained steady in the reactor that received chlorinated water. The differences in bacterial populations of potable water biofilms suggest that disinfecting agents can influence biofilm development. These results also suggest that biofilm communities in distribution systems are capable of changing in response to disinfection practices.     

Populations implicated in anaerobic reductive dechlorination of 1,2-dichloropropane in highly enriched bacterial communities

1,2-Dichloropropane (1,2-D), a widespread groundwater contaminant, can be reductively dechlorinated to propene by anaerobic bacteria. To shed light on the populations involved in the detoxification process, a comprehensive 16S rRNA gene-based bacterial community analysis of two enrichment cultures derived from geographically distinct locations was performed. Analysis of terminal restriction fragments, amplicons obtained with dechlorinator-specific PCR primers, and enumeration with quantitative real-time PCR as well as screening clone libraries all implied that Dehalococcoides populations were involved in 1,2-D dechlorination in both enrichment cultures. Physiological traits (e.g., dechlorination in the presence of ampicillin and a requirement for hydrogen as the electron donor) supported the involvement of Dehalococcoides populations in the dechlorination process. These findings expand the spectrum of chloroorganic compounds used by Dehalococcoides species as growth-supporting electron acceptors. The combined molecular approach allowed a comparison between different 16S rRNA gene-based approaches for the detection of Dehalococcoides populations.     

Degradation of aniline and monochloroanilines by Rhodococcus sp. An 117 and a pseudomonad: a comparative study

Two newly isolated aniline-degrading bacterial strains were characterized with regard to their enzyme systems responsible for aniline catabolism. One of them identified as a Rhodococcus sp. metabolized aniline exclusively via the beta-ketoadipate pathway by means of inducible enzymes. The aniline-degrading enzyme system of the second isolate, presumably a pseudomonad, was shown to consist of an inducible aniline-converting enzyme and constitutive meta-pathway enzymes. Both isolates failed to metabolize monochlorinated anilines in the absence of additional carbon sources. To explain this the ring-cleaving enzymes of both isolates were examined for their substrate specificities. Furthermore, the effect of 4-chlorocatechol on the enzymes catalyzing aniline conversion and catechol oxygenation was investigated.     

Enhanced biodegradation of hexachlorocyclohexane in upflow anaerobic sludge blanket reactor using methanol as an electron donor

Anaerobic dechlorination of technical grade hexachlorocyclohexane (THCH) was studied in a continuous upflow anaerobic sludge blanket (UASB) reactor with methanol as a supplementary substrate and electron donor. A reactor without methanol served as the experimental control. The inlet feed concentration of THCH in both the experimental and the control UASB reactor was 100 mg l(-1). After 60 days of continuous operation, the removal of THCH was >99% in the methanol-supplemented reactor as compared to 20-35% in the control reactor. THCH was completely dechlorinated in the methanol fed reactor at 48 h HRT after 2 months of continuous operation. This period was also accompanied by increase in biomass in the reactor, which was not observed in the experimental control. Batch studies using other supplementary substrates as well as electron donors namely acetate, butyrate, formate and ethanol showed lower % dechlorination (<85%) and dechlorination rates (<3 mg g(-1)d(-1)) as compared to methanol (98%, 5 mg g(-1)d(-1)). The optimum concentration of methanol required, for stable dechlorination of THCH (100 mg l(-1)) in the UASB reactor, was found to be 500 mg l(-1). Results indicate that addition of methanol as electron donor enhances dechlorination of THCH at high inlet concentration, and is also required for stable UASB reactor performance.