Development of four-stage moving bed biofilm reactor train with a pre-denitrification configuration for the removal of thiocyanate and cyanate.

Two trains (A and B) of four-stage moving bed biofilm reactors (MBBRs) were developed for the degradation of thiocyanate (SCN(-)), cyanate (OCN(-)) and ammonia (NH3). A pre-denitrification configuration was established in the first-stage reactor of the B train using SCN(-) and OCN(-) as the sole carbon source. SCN(-), OCN(-) and NH3 were completely removed in both trains. The highest removal of total nitrogen equivalent (total-N) occurred at a loading rate of 5.6 mg-N L(-1) h(-1). The pre-denitrification configuration resulted in increased total-N removal in the B train (62.6%) compared to the A train (38.5%). Thiobacillus spp. were the predominant bacteria in all MBBRs. Bacteria related to bioprocesses involving anaerobic ammonium oxidation were present in the B train, suggesting that part of nitrogen removal occurs via this pathway. Our results showed that the pre-denitrification configuration increases the efficiency of removal of total-N compounds in the SCN(-)/OCN(-)-degrading MBBR process.

Biodegradation of endocrine disruptors in solid-liquid two-phase partitioning systems by enrichment cultures.

Naturally occurring and synthetic estrogens and other molecules from industrial sources strongly contribute to the endocrine disruption of urban wastewater. Because of the presence of these molecules in low but effective concentrations in wastewaters, these endocrine disruptors (EDs) are only partially removed after most wastewater treatments, reflecting the presence of these molecules in rivers in urban areas. The development of a two-phase partitioning bioreactor (TPPB) might be an effective strategy for the removal of EDs from wastewater plant effluents. Here, we describe the establishment of three ED-degrading microbial enrichment cultures adapted to a solid-liquid two-phase partitioning system using Hytrel as the immiscible water phase and loaded with estrone, estradiol, estriol, ethynylestradiol, nonylphenol, and bisphenol A. All molecules except ethynylestradiol were degraded in the enrichment cultures. The bacterial composition of the three enrichment cultures was determined using 16S rRNA gene sequencing and showed sequences affiliated with bacteria associated with the degradation of these compounds, such as Sphingomonadales. One Rhodococcus isolate capable of degrading estrone, estradiol, and estriol was isolated from one enrichment culture. These results highlight the great potential for the development of TPPB for the degradation of highly diluted EDs in water effluents.

Isolation of estrogen-degrading bacteria from an activated sludge bioreactor treating swine waste, including a strain that converts estrone to ß-estradiol.

An estrogen-degrading bacterial consortium from a swine wastewater biotreatment was enriched in the presence of low concentrations (1 mg/L) of estrone (E1), 17ß-estradiol (ßE2), and equol (EQO) as sole carbon sources. The consortium removed 99% ± 1% of these three estrogens in 48 h. Estrogen removal occurred even in the presence of an ammonia monooxygenase inhibitor, suggesting that nitrifiers are not involved. Five strains showing estrogen-metabolizing activity were isolated from the consortium on mineral agar medium with estrogens as sole carbon source. They are related to four genera ( Methylobacterium (strain MI6.1R), Ochrobactrum (strains MI6.1B and MI9.3), Pseudomonas (strain MI14.1), and Mycobacterium (strain MI21.2)) distributed among three classes (Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria). Depending on the culture medium, strains MI6.1B, MI9.3, MI14.1, and MI21.2 partially transform ßE2 into E1, whereas Methylobacterium sp. strain MI6.1R reduces E1 into ßE2 under aerobic conditions, in contrast with the usually observed conversion of ßE2 into E1. Since ßE2 is a more potent endocrine disruptor than E1, it means that the presence of Methylobacterium sp. strain MI6.1R (or other bacteria with the same E1-reducing activity) in a treatment could transiently increase the estrogenicity of the effluent. MI6.1R can also reduce the ketone group of 16-ketoestradiol, a hydroxylated analog of E1. All ßE2 and E1 transformation activities were constitutive, and many of them are favoured in a rich medium than a medium containing no other carbon source. None of the isolated strains could degrade EQO.

Construction and functional screening of a metagenomic library using a T7 RNA polymerase-based expression cosmid vector.

The metagenomic approach has greatly accelerated the discovery of new enzymes by giving access to the genetic potential of microorganisms from various environments. Function-based screening depends on adequate expression of the foreign genes in the heterologous host, which can be challenging in large-insert libraries. In this study, the shuttle cosmid vector pFX583 was used for the construction and screening of a metagenomic library. This vector allows T7 RNA polymerase-directed transcription of the cloned DNA and can be used in Escherichia coli and Streptomyces lividans. The DNA used for the library construction was obtained from an enriched biomass. The library was screened for lipolytic and proteolytic activities using E. coli and S. lividans as hosts. Numerous E. coli clones with lipolytic activity were detected. Unfortunately, proteases could not be detected in both hosts. From the lipolytic activity screen, a gene coding for a new lipase was isolated, and partial characterization was conducted.

Impact of an aerobic thermophilic sequencing batch reactor on antibiotic-resistant anaerobic bacteria in swine waste.

The introduction of antibiotics to animal feed has contributed to the selection of antibiotic-resistant bacteria in concentrated animal feeding operations. The aim of this work was to characterize the impact of an aerobic thermophilic biotreatment on anaerobic antibiotic-resistant bacteria in swine waste. Despite 162- to 6,166-fold reduction in antibiotic-resistant populations enumerated in the swine waste at 25 degrees C and 37 degrees C, resistant populations remained significant (10(4) to 10(5) most probable number per milliliter) in the treated swine waste. Five resistance genes were detected before [tet(LMOS) erm(B)], and six resistance genes were detected after [tet(LMOSY) erm(B)] biotreatment. However, the biotreatment decreased the frequency of detection of resistance genes by 57%. Analysis by denaturing gradient gel electrophoresis of polymerase chain reaction-amplified 16 S ribosomal DNA (rDNA) fragments showed that the biotreatment reduced the bacterial diversity of resistant populations enumerated at 37 degrees C. Cloning and sequencing of the 16 S rDNA of these populations revealed that most clones in the treated swine waste were closely similar to some of the clones retrieved from the untreated swine waste. This study revealed that the aerobic thermophilic biotreatment developed in our laboratory does not prevent the introduction of facultatively anaerobic antibiotic-resistant bacteria and their resistance genes into agricultural ecosystems. Horizontal transfer of ecologically advantageous genes within microbial communities are likely to prevent thermophilic biotreatments from completely eliminating antibiotic-resistant bacteria and their resistance genes in animal wastes.

Isolation and characterization of a new alkali-thermostable lipase cloned from a metagenomic library.

The construction of a cosmid library from the biomass produced in an enriched Sequencing Fed-Batch Reactor allowed the isolation of a new lipase by functional screening. The open reading frame of 928 bp encoded a polypeptide of 308 amino acids with a molecular mass of 32.6 kDa. The amino acid sequence analysis revealed the presence of the conserved pentapeptide GXSXG essential for lipase activity. Alignment with known sequences of proteins showed no more than 52% identity with different lipases, confirming the discovery of a novel gene sequence. The lipase was cloned and expressed in Streptomyces lividans and further purified by a combination of hydrophobic interaction and size-exclusion chromatography. Spectrophotometric assays with different p-nitrophenyl esters demonstrated a preference for long-length acyl chains, especially p-nitrophenylmyristate (C14). Moreover, the enzyme presented an optimal activity at 60 degrees C and at alkaline pH of 10.5.

Fate of chlortetracycline- and tylosin-resistant bacteria in an aerobic thermophilic sequencing batch reactor treating swine waste.

Antibiotics have been added to animal feed for decades. Consequently, food animals and their wastes constitute a reservoir of antibiotic-resistant bacteria. The objective of this work was to characterize the impact of an aerobic thermophilic biotreatment on aerobic, antibiotic-resistant bacteria in swine waste. The proportion of tylosin- and chlortetracycline-resistant bacteria grown at 25 degrees C, 37 degrees C, and 60 degrees C decreased after treatment, but they were still abundant (10(2) to 10(8) most probable number ml(-1)) in the treated swine waste. The presence of 14 genes conferring resistance to tylosin and chlortetracycline was assessed by polymerase chain reaction in bacterial populations grown at 25 degrees C, 37 degrees C, and 60 degrees C, with or without antibiotics. In 22 cases, genes were detected before but not after treatment. The overall gene diversity was wider before [tet(BLMOSY), erm(AB)] than after [tet(LMOS), erm(B)] treatment. Analysis by denaturing gradient gel electrophoresis of amplified 16S ribosomal DNA (rDNA) fragments generally showed a reduction of the bacterial diversity, except for total populations grown at 60 degrees C and for tylosin-resistant populations grown at 37 degrees C. The latter were further investigated by cloning and sequencing their 16S rDNA. Phylotypes found before treatment were all closely related to Enterococcus hirae, whereas six different phylotypes, related to Pseudomonas, Alcaligenes, and Pusillimonas, were found after treatment. This work demonstrated that the aerobic thermophilic biotreatment cannot be considered as a means for preventing the dissemination of aerobic antibiotic-resistant bacteria and their resistance genes to the environment. However, since pathogens do not survive the biotreatment, the effluent does not represent an immediate threat to animal or human health.

Bacterial diversity of a consortium degrading high-molecular-weight polycyclic aromatic hydrocarbons in a two-liquid phase biosystem.

High-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs) are pollutants that persist in the environment due to their low solubility in water and their sequestration by soil and sediments. Although several PAH-degrading bacterial species have been isolated, it is not expected that a single isolate would exhibit the ability to degrade completely all PAHs. A consortium composed of different microorganisms can better achieve this. Two-liquid phase (TLP) culture systems have been developed to increase the bioavailability of poorly soluble substrates for uptake and biodegradation by microorganisms. By combining a silicone oil-water TLP system with a microbial consortium capable of degrading HMW PAHs, we previously developed a highly efficient PAH-degrading system. In this report, we characterized the bacterial diversity of the consortium with a combination of culture-dependent and culture-independent methods. Polymerase chain reaction (PCR) of part of the 16S ribosomal RNA gene (rDNA) sequences combined with denaturing gradient gel electrophoresis was used to monitor the bacterial population changes during PAH degradation of the consortium when pyrene, chrysene, and benzo[a]pyrene were provided together or separately in the TLP cultures. No substantial changes in bacterial profiles occurred during biodegradation of pyrene and chrysene in these cultures. However, the addition of the low-molecular-weight PAHs phenanthrene or naphthalene in the system favored one bacterial species related to Sphingobium yanoikuyae. Eleven bacterial strains were isolated from the consortium but, interestingly, only one-IAFILS9 affiliated to Novosphingobium pentaromativorans-was capable of growing on pyrene and chrysene as sole source of carbon. A 16S rDNA library was derived from the consortium to identify noncultured bacteria. Among 86 clones screened, 20 were affiliated to different bacterial species-genera. Only three strains were represented in the screened clones. Eighty-five percent of clones and strains were affiliated to Alphaproteobacteria and Betaproteobacteria; among them, several were affiliated to bacterial species known for their PAH degradation activities such as those belonging to the Sphingomonadaceae. Finally, three genes involved in the degradation of aromatic molecules were detected in the consortium and two in IAFILS9. This study provides information on the bacterial composition of a HWM PAH-degrading consortium and its dynamics in a TLP biosystem during PAH degradation.

Microeukaryote diversity in a marine methanol-fed fluidized denitrification system.

The marine methanol-fed fluidized denitrification system operated by the Montreal Biodome includes carriers on which a denitrifying biofilm has developed. Previous observations showed a high abundance of microeukaryotes living in and around the biofilm. These eukaryotes may influence the system's denitrification efficiency. The composition of the microeukaryote population was determined. Microscopic observations showed at least 20 different morphologies that included large numbers of ciliates. Molecular analyses of an 18S ribosomal RNA (rDNA) gene library revealed 31 different phylotypes. Alveolobiontes were the most abundant phylotypes and made up 75% of the 159 screened clones. Other eukaryotic groups, including Stramenopiles, Fungi, Amoebozoa, and nematodes, were also present. From 18S rDNA specific sequences, one of the Amoebozoa-affiliated phylotypes was visualized by fluorescence in situ hybridization. It had a rod-like irregular shape and measured less than 5 mum in length. We determined the impact of protozoans on the denitrifying activity. In a laboratory-scale batch culture assays, the denitrifying biofilm was treated with cycloheximide and nystatin that eliminated the protozoans. No difference in the denitrification rate was found. However, planktonic bacteria were more abundant in the treated culture medium.

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 (14)C methanol in the presence of nitrate, suggesting their involvement in denitrification. Despite their abundance, Methylophaga spp. did not assimilate methanol under those conditions.

Long-term storage conditions for carriers with denitrifying biomass of the fluidized, methanol-fed denitrification reactor of the Montreal Biodome, and the impact on denitrifying activity and bacterial population.

The Montreal Biodome has a denitrification system to stabilize the nitrate concentration in its 3 million liter seawater aquarium. However, this microbial process has failed periodically due to various technical problems. The system can take several weeks to recover its full denitrification capacity. In order to provide the denitrification system with a backup of active biomass, different freezing conditions for the denitrifying biomass were tested. The biomass was conserved for 1 week-17 months at -20 degrees C with and without glycerol or at -80 degrees C with and without glycerol, and the denitrifying activity was tested in batch culture for 140 h periods at various intervals. Our results showed that glycerol was required for fast recovery of the microbial community's denitrifying activity. The -20 degrees C and -80 degrees C conservation temperatures with glycerol gave similar results although there was a short period of nitrite accumulation in the -20 degrees C sample. There were no substantial changes in the microbial community of any of the frozen samples after 17 months of conservation as monitored by denaturing gradient gel electrophoresis. This is the first report on the long-term conservation of a complex denitrifying population by freezing.

Occurrence and expression of crdA and cprA5 encoding chloroaromatic reductive dehalogenases in Desulfitobacterium strains.

Desulfitobacterium hafniense PCP-1 (formerly frappieri PCP-1) has two reductive dehalogenases (RDases) that have been characterized. One is a membrane-associated 2,4,6-trichlorophenol RDase, which is encoded by crdA, and the other is a 3,5-dichlorophenol RDase encoded by cprA5. In this report, we determined the occurrence of these two RDase genes in seven other Desulfitobacterium strains. The presence or absence of these two RDases may explain the differences in the spectrum of halogenated compounds by these Desulfitobacterium strains. crdA gene sequences were found in all of the tested strains. It was expressed in strain PCP-1 regardless of the absence or presence of chlorophenols in the culture medium. crdA was also expressed in D. hafniense strains DCB-2 and TCE-1. cprA5 was detected only in D. hafniense strains PCP-1, TCP-A, and DCB-2. In these strains, cprA5 transcripts were detected only in the presence of chlorophenols. We also examined the expression of putative cprA RDases (cprA2, cprA3, and cprA4) that were shown to exist in the D. hafniense DCB-2 genome. RT-PCR experiments showed that cprA2, cprA3, and cprA4 were expressed in D. hafniense strains PCP-1, DCB-2, and TCP-A in the presence of chlorophenols. However, contrary to cprA5, these three genes were also expressed in the absence of halogenated compounds in the culture medium.

Seawater denitrification in a closed mesocosm by a submerged moving bed biofilm reactor.

The performance of a submerged moving bed biofilm reactor (MBBR) for the denitrification of seawater in a 3.25 million closed circuit mesocosm was investigated at pilot scale, using methanol as a carbon source at various C/N ratios. Nitrate accumulation in closed systems where water changes are expensive and problematic may cause toxicity problems to marine life. Seawater was pretreated in a recirculated fixed bed to remove oxygen prior to the denitrification step. The 110l MBBR was partly filled (25%) with spherical positively buoyant polyethylene carriers with an effective surface area of approximately 100 m2 m(-3), which represents 35% of the total surface area. Carriers were maintained submerged by a conical grid and circulated by the downflow jet of an eductor. The MBBR mixing system was designed to prevent dead mixing zones and carrier fouling to avoid sulfate reduction while treating seawater containing as high as 2150 mg SO4-Sl(-1). NO3-N reduction from 53 to as low as 1.7+/-0.7 mg l(-1) and a maximum denitrification rate of 17.7+/-1.4 g Nm(-2) d(-1) were achieved at 4.2-4.3 applied COD/N (w/w) ratio. Methanol consumption corresponded to denitrification stoichiometric values, indicating the absence of sulfate reduction. Denitrification rates and effluent residual dissolved organic carbon were proportional to the C/N ratio. Such reactors could be scaled up in closed systems where water changes must be minimized.

Cryptanaerobacter phenolicus gen. nov., sp. nov., an anaerobe that transforms phenol into benzoate via 4-hydroxybenzoate.

An anaerobic bacterium that transforms phenol and 4-hydroxybenzoate (4-OHB) into benzoate, strain LR7.2T, was isolated from a culture originating from a mixture of swamp water, sewage sludge, swine waste and soil. Cells of strain LR7.2T are Gram-positive short rods (1 x 2 microm) that are electron-dense when observed by electron microscopy. The optimum pH and temperature for growth and transformation activity of 4-OHB are 7.5-8.0 and 30-37 degrees C, respectively. The bacterium does not use sulphate, thiosulphate, nitrate, nitrite, FeCl3, fumarate or arsenate as an electron acceptor. It does not normally use sulphite, although stimulation of growth and 4-OHB transformation activity at a low concentration (up to 2 mM) has been reported previously under different culture conditions. The presence of 4-OHB or phenol is essential for growth; transformation of 4-OHB or phenol into benzoate is used to produce energy for growth. Using [6D]-phenol, 4-OHB was shown to be an intermediate in the transformation of phenol into benzoate. No spore was observed. The bacterium has a DNA G+C content of 51 mol% and its major membrane fatty acid is anteiso-C(15 : 0). The 16S rRNA gene sequence of strain LR7.2T shows only 90 % similarity to its closest relative (Pelotomaculum thermopropionicum). From these results, a new taxon is proposed: Cryptanaerobacter phenolicus gen. nov., sp. nov. The type strain is LR7.2T (=ATCC BAA-820T=DSM 15808T).

Purification, cloning, and sequencing of a 3,5-dichlorophenol reductive dehalogenase from Desulfitobacterium frappieri PCP-1.

A membrane-associated 3,5-dichlorophenol reductive dehalogenase was isolated from Desulfitobacterium frappieri PCP-1. The highest dehalogenase activity was observed with the biomass cultured at 22 degrees C, compared to 30 and 37 degrees C, where the cell suspensions were 2.2 and 9.6 times less active, respectively. The reductive dehalogenase was purified 12.7-fold to apparent homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 57 kDa. Its dechlorinating activity was not inhibited by sulfate and nitrate but was completely inhibited by 2.5 mM sulfite and 10 mM KCN. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activities, suggesting the involvement of a corrinoid cofactor. Several polychlorophenols were dechlorinated at the meta and para positions. The apparent K(m) for 3,5-dicholorophenol was 49.3 +/- 3.1 microM at a methyl viologen concentration of 2 mM. Six internal tryptic peptides were sequenced by mass spectrometry. One open reading frame (ORF) was found in the Desulfitobacterium hafniense genome containing these peptide sequences. This ORF corresponds to a gene coding for a CprA-type reductive dehalogenase. The corresponding ORF (named cprA5) in D. frappieri PCP-1 was cloned and sequenced. The cprA5 gene codes for a 548-amino-acid protein that contains a twin-arginine-type signal for secretion. The gene product has a cobalamin binding site motif and two iron-sulfur binding motifs and shows 66% identity (76 to 77% similarity) with some tetrachloroethene reductive dehalogenases. This is the first CprA-type reductive dehalogenase that can dechlorinate chlorophenols at the meta and para positions.

Swine waste treatment by self-heating aerobic thermophilic bioreactors.

Pig manure represents a very high-strength wastewater that is well suited for a self-heating aerobic thermophilic treatment. Here we report the use of 59-L Aerobic Thermophilic Sequencing Batch Reactors (AT-SBR) to study the treatment of pig manure with a HRT of 6 days. Temperatures up to 75 degrees C were reached without external heating by using Venturi-type aerators but these conditions were detrimental for the respiratory activity of the microflora. For COD removal, better performances were achieved when the temperature was limited to 50 degrees C. However, higher temperatures increased the rate of phosphorus crystallisation and the volatilisation of ammonia. A temperature of 50 degrees C was enough to eliminate faecal coliforms and Campylobacter spp., but 60 degrees C was needed for the efficient destruction of Clostridium perfringens. Consequently, an operating temperature of 60 degrees C appears to be a good compromise. Under these conditions, the BOD(5) decreases from 50.5 to 1.0 g L(-1), yielding a 98% removal.

Improving the biotreatment of hydrocarbons-contaminated soils by addition of activated sludge taken from the wastewater treatment facilities of an oil refinery.

Addition of activated sludge taken from the wastewater treatment facilities of an oil refinery to a soil contaminated with oily sludge stimulated hydrocarbon biodegradation in microcosms, bioreactors and biopile. Microcosms containing 50 g of soil to which 0.07% (w/w) of activated sludge was added presented a higher degradation of alkanes (80% vs 24%) and polycyclic aromatic hydrocarbons (PAHs) (77% vs 49%) as compared to the one receiving only water, after 30 days of incubation at room temperature. Addition of ammonium nitrate or sterile sludge filtrate instead of activated sludge resulted in a similar removal of PAHs but not of alkanes suggesting that the nitrogen contained in the activated sludge plays a major role in the degradation of PAHs while microorganisms of the sludge are active against alkanes. Addition of sludge also stimulated hydrocarbon biodegradation in 10-kg bioreactors operated during 60 days and in a 50-m3 biopile operated during 126 days. This biopile treatment allowed the use of the soil for industrial purpose based on provincial regulation ("C" criteria). In contrast, the soil of the control biopile that received only water still exceeded C criteria for C10-C50 hydrocarbons, total PAHs, chrysene and benzo[a]anthracene. The stimulation effect of sludge was stronger on the 4-rings than on 2-rings PAHs. The soil of the biopile that received sludge was 4-5 times less toxic than the control. These results suggest that this particular type of activated sludge could be used to increase the efficiency of the treatment of hydrocarbon-contaminated soils in a biopile.

Purification, cloning and sequencing of an enzyme mediating the reductive dechlorination of 2,4,6-trichlorophenol from Desulfitobacterium frappieri PCP-1.

A new membrane-associated 2,4,6-trichlorophenol reductive dehalogenase from Desulfitobacterium frappieri PCP-1 was isolated. Initial characterization of the crude preparation showed that the dechlorinating activity was sensitive to oxygen, and its optimum pH was 7.0. Its dechlorinating activity was not inhibited by sulphate, was completely inhibited by 1 mM sulphite, and partially inhibited by 5 mM sodium azide and by more than 5 mM nitrate. Several polychlorophenols were dechlorinated in the ortho position with respect to the hydroxy group. A dehalogenase was purified to apparent homogeneity. SDS gel electrophoresis revealed a single protein band with a molecular mass of 37 kDa. However, after two-dimensional gel electrophoresis, this band was composed of three isoforms. MS analyses showed that the three isoforms were from the same protein and the molecular mass of the most abundant isoform is 33800 Da. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activity, suggesting the involvement of a corrinoid cofactor. The apparent K(m) value for 2,4,6-trichlorophenol and pentachlorophenol were 18.3+/-2.8 microM and 26.8+/-2.9 microM respectively, at a methyl viologen concentration of 2 mM. The N-terminal amino acid sequence and an internal tryptic peptide sequence were determined. One open reading frame (ORF) was found in the Desulfitobacterium hafniense genome containing these peptides sequences. The corresponding ORF in D. frappieri PCP-1 was cloned and sequenced. This ORF, that we designated crdA, showed no homology with any known dehalogenase, suggesting a distinct reductive dehalogenase.