Anaerobic biodegradation of pentachlorophenol by a methanogenic consortium

An anaerobic consortium degrading pentachlorophenol (PCP) by methanogenic fermentation was enriched from PCP-contaminated soils. In a semi-continuous reactor, PCP biodegradation was unstable and necessitated periodic additions of unacclimated anaerobic sludge waste to restore the activity. In continuous-flow reactors, PCP degradation activity was more stable when a mixture of glucose and sodium formate was used as secondary carbon source instead of glucose. The analysis of the chlorophenol intermediates suggested that the main pathway of PCP dechlorination was PCP 2,3,5,6-tetrachlorophenol 2,3,5-trichlorophenol 3,5-dichlorophenol 3-chlorophenol phenol. In a laboratory-scale continuous-upflow fixed-film column reactor, a PCP removal of more than 99% was achieved at a PCP loading rate of 60 mol (1 reactor volume)^–1 day^–1 for a hydraulic retention time of 0.7 day. Analysis of culture samples taken at different levels in the reactor have shown that, at this PCP loading rate, only the lower part of the reactor was active. 3-chlorophenol and 3,5- and 3,4-dichlorophenol were detected at the different levels of the reactor. A study of the microorganisms in the biofilm was carried out by scanning electron microscopy and suggested that the microorganisms involved in the consortium were present as a well-structured arrangement. Methanosaeta-like microorganisms were observed mainly at the base of the biofilm whereas, at the surface, a larger diversity of morphotypes was observed in which coccoid or small rod organisms were dominant. This work shows the importance of the design and the control of the operation parameters on the efficiency of the fixed-film reactor.     

Effects of Sulfuroxy Anions on Degradation of Pentachlorophenol by a Methanogenic Enrichment Culture

We studied the degradation of pentachlorophenol (PCP) under methanogenic and sulfate-reducing conditions with an anaerobic mixed culture derived from sewage sludge. The consortium degraded PCP via 2,3,4,5-tetrachlorophenol, 3,4,5-trichlorophenol, and 3,5-dichlorophenol and eventually accumulated 3-chlorophenol. Dechlorination of PCP and metabolites was inhibited in the presence of sulfate, thiosulfate, and sulfite. A decrease in the rate of PCP transformation was noted when the endogenous dissolved H₂ was depleted below 0.11 μM in sulfate-reducing cultures. The effect on dechlorination observed with sulfate could be relieved by addition of molybdate, a competitive inhibitor of sulfate reduction. Addition of H₂ reduced the inhibition observed with sulfuroxy anions. The inhibitory effect of sulfuroxy anions may be due to a competition for H₂ between sulfate reduction and dechlorination. When cultured under methanogenic conditions, the consortium degraded several chlorinated and brominated phenols.     

Influence of a supplemental carbon source on anaerobic dechlorination of pentachlorophenol in granular sludge.

Anaerobic dechlorination of pentachlorophenol (PCP) was studied in two upflow anaerobic sludge blanket reactors. One reactor received glucose (0.9 g liter-1) as an additional carbon source; the other one served as a control. The concentration of PCP in the medium was 4.5 and 3.0 mg liter-1 in the experimental and control reactors, respectively. The reactors were inoculated with granular sludge previously grown on sugar-containing wastewater. After 10 months of continuous operation, the removal of PCP was 99% in the glucose-amended reactor, whereas the removal in the control reactor varied between 32 and 77%. Furthermore, 94% of the PCP was completely dechlorinated in the glucose reactor compared with a maximum of 20% in the control reactor. In the same period, the amount of biomass in the glucose reactor had increased by approximately 150% compared with that in the control reactor, where no growth of the sludge bed occurred. Batch culture activity tests showed that the addition of glucose had a stimulatory effect on the dechlorination rate of PCP per gram of volatile solids. This indicated that the better performance of the glucose-amended reactor was due to a higher concentration of biomass and a direct stimulatory effect of glucose on the dechlorination rate. The pattern of dechlorination of PCP showed that an initial para cleavage was followed by two ortho cleavages.     

Influence of a supplemental carbon source on anaerobic dechlorination of pentachlorophenol in granular sludge.

Anaerobic dechlorination of pentachlorophenol (PCP) was studied in two upflow anaerobic sludge blanket reactors. One reactor received glucose (0.9 g liter-1) as an additional carbon source; the other one served as a control. The concentration of PCP in the medium was 4.5 and 3.0 mg liter-1 in the experimental and control reactors, respectively. The reactors were inoculated with granular sludge previously grown on sugar-containing wastewater. After 10 months of continuous operation, the removal of PCP was 99% in the glucose-amended reactor, whereas the removal in the control reactor varied between 32 and 77%. Furthermore, 94% of the PCP was completely dechlorinated in the glucose reactor compared with a maximum of 20% in the control reactor. In the same period, the amount of biomass in the glucose reactor had increased by approximately 150% compared with that in the control reactor, where no growth of the sludge bed occurred. Batch culture activity tests showed that the addition of glucose had a stimulatory effect on the dechlorination rate of PCP per gram of volatile solids. This indicated that the better performance of the glucose-amended reactor was due to a higher concentration of biomass and a direct stimulatory effect of glucose on the dechlorination rate. The pattern of dechlorination of PCP showed that an initial para cleavage was followed by two ortho cleavages.     

Anaerobic biodegradation of pentachlorophenol in a contaminated soil inoculated with a methanogenic consortium or with Desulfitobacterium frappieri strain PCP-1

Anaerobic biodegradation of pentachlorophenol (PCP) in a contaminated soil from a wood-treating industrial site was studied in soil slurry microcosms inoculated with a PCP-degrading methanogenic consortium. When the microcosms containing 10%–40% (w/v) soil were inoculated with the consortium, more than 90% of the PCP was removed in less than 30 days at 29 °C. Less-chlorinated phenols, mainly 3-chlorophenol were slowly degraded and accumulated in the cultures. Addition of glucose and sodium formate to the microcosms was not necessary, suggesting that the organic compounds in the soil can sustain the dechlorinating activity. Inoculation of Desulfitobacterium frappieri strain PCP-1 along with a 3-chlorophenol-degrading consortium in the microcosms also resulted in the rapid dechlorination of PCP and the slow degradation of 3-chlorophenol. Competitive polymerase chain reaction experiments showed that PCP-1 was present at the same level throughout the 21-day biotreatment. D. frappieri, strain PCP-1, inoculated into the soil microcosms, was able to remove PCP from soil containing up to 200 mg PCP/kg soil. However, reinoculation of the strain was necessary to achieve more than 95% PCP removal with a concentration of 300 mg and 500 mg PCP/kg soil. These results demonstrate that D. frappieri strain PCP-1 can be used effectively to dechlorinate PCP to 3-chlorophenol in contaminated soils. Received: 14 November 1997 / Received revision: 29 January 1998 / Accepted: 24 February 1998     

Evidence for chemiosmotic coupling of reductive dechlorination and ATP synthesis in Desulfomonile tiedjei

Desulfomonile tiedjei (strain DCB-1) was previously shown to conserve energy for growth from reductive dechlorination of 3-chlorobenzoate coupled to formate oxidation. We tested the hypothesis that a chemiosmotic mechanism couples reductive dechlorination and ATP synthesis in D. tiedjei. Dechlorination resulted in an increase in the ATP pool of cells. Uncouplers and ionophores decreased both the dechlorination rate and the ATP pool. However, at low concentrations the inhibitors had relatively greater effects on the ATP pool, and in some cases, even appeared to stimulate dechlorination. Those agents could not completely inhibit ATP synthesis while allowing dechlorination activity. The proton-driven ATPase inhibitor, N,N-dicyclohexylcarbodiimide (DCCD), had similar effects. An imposed pH gradient also resulted in an increase in the ATP pool of cells, and this increase was partially inhibited by DCCD. Addition of 3-chlorobenzoate to cell suspensions caused proton translocation by the cells. Proton translocation was stimulated by the permeant thiocyanate anion and inhibited by uncouplers. A maximum H⁺/3-chlorobenzoate ratio of greater than two was observed. These findings suggest that dechlorination supports formation of a proton-motive force which in turn supports ATP synthesis via a proton-driven ATPase.Abbreviations 3CB 3-chlorobenzoate - CCCP m-chlorophenyl-hydrazone - DCCD N,N-dicyclohexylcarbodiimide - DNP 2,4-dinitrophenol - P proton-motive force - PCP pentachlorophenol     

Changes in the microbial community during repeated anaerobic microbial dechlorination of pentachlorophenol

Pentachlorophenol (PCP) has been widely used as a pesticide in paddy fields and has imposed negative ecological effect on agricultural soil systems, which are in typically anaerobic conditions. In this study, we investigated the effect of repeated additions of PCP to paddy soil on the microbial communities under anoxic conditions. Acetate was added as the carbon source to induce and accelerate cycles of the PCP degradation. A maximum degradation rate occurred at the 11th cycle, which completely transformed 32.3 μM (8.6 mg L^?1) PCP in 5 days. Illumina high throughput sequencing of 16S rRNA gene was used to profile the diversity and abundance of microbial communities at each interval and the results showed that the phyla of Bacteroidates, Firmicutes, Proteobacteria, and Euryarchaeota had a dominant presence in the PCP-dechlorinating cultures. Methanosarcina, Syntrophobotulus, Anaeromusa, Zoogloea, Treponema, W22 (family of Cloacamonaceae), and unclassified Cloacamonales were found to be the dominant genera during PCP dechlorination with acetate. The microbial community structure became relatively stable as cycles increased. Treponema, W22, and unclassified Cloacamonales were firstly observed to be associated with PCP dechlorination in the present study. Methanosarcina that have been isolated or identified in PCP dechlorination cultures previously was apparently enriched in the PCP dechlorination cultures. Additionally, the iron-cycling bacteria Syntrophobotulus, Anaeromusa, and Zoogloea were enriched in the PCP dechlorination cultures indicated they were likely to play an important role in PCP dechlorination. These findings increase our understanding for the microbial and geochemical interactions inherent in the transformation of organic contaminants from iron rich soil, and further extend our knowledge of the PCP-transforming microbial communities in anaerobic soil conditions.     

Introduction of ade novo bioremediation activity into anaerobic granular sludge using the dechlorinating bacterium DCB-2

The strictly anaerobic, pentachlorophenol (PCP) degrading bacterium DCB-2 was immobilized in an Upflow Anaerobic Sludge Blanket (UASB) reactor containing sterile granules. PCP and lactate were fed to the reactor and the concentration of chlorophenols in the effluent were monitored for 641 days. PCP was found to be degraded and transformed into 3.4.5-trichlorophenol in the reactor where DCB-2 was introduced into the granular sludge. PCP was still transformed to 3.4.5-trichlorophenol when the hydraulic retention time was decreased to six hours which was much lower than the generation time of DCB-2 insuring no free living cells in the reactor. This indicated that DCB-2 was immobilized in the granular layer. A control reactor that contained only sterile granules did not dechlorinate PCP indicating that the performance in the inoculated reactor was only due to the introduced bacteria. Immobilization of DCB-2 in the granules was further demonstrated by adding an antibody raised against DCB-2 to sliced granules. Bacteria thus visualized formed a net structure inside the granules. No DCB-2 bacteria could be found in granules from the control reactor. When lactate was omitted from the influent, the reactor still dechlorinated PCP in accordance with our findings that lactate was not used by DCB-2. This suggested that the reducing equivalents for reductive dechlorination were derived from the granules themselves. The reactor performance was 120 mol·l reactor^-1·day^-1, comparable to the best described performance of a UASB-reactor and to aerobic reactors. Our study demonstrates that granules can be constructed which possess specific abilities such as a dechlorinating activity and at the same time be high performing. This result have implications for eco-engineering of granules for anaerobic treatment of contaminated waters.     

Effects of electron donor and microbial concentration on the enhanced dechlorination of carbon tetrachloride by anaerobic consortia

 Reductive dechlorination of carbon tetra-chloride (CCl₄) by anaerobic bacterial communities from anaerobic digester sludge with the amendment of low concentrations of electron donors and microorganisms was undertaken to evaluate the influence of electron donors and microbial concentration on the rate of dechlorination of CCl₄. Humic acid, acetate, and glucose were selected to examine the feasibility of the electron donor with respect to the remediation of a contaminated subsurface. The addition of an electron donor and microorganisms significantly enhanced the dechlorination rate of carbon tetrachloride. The addition of an electron donor increased the cell numbers of anaerobic consortia, thereby increasing the rate of dechlorination. Glucose was a better electron donor than acetate and humic acid under reducing environments. The pseudo-first-order degradation rate constants of CCl₄ ranged from 0.0057 day^-1 to 0.135 day^-1, depending on the conditions of the electron donor and biomass supplemented. Furthermore, the addition of the electron donor in the batches amended with 0.56 mg volatile suspended solids (VSS)/l biomass had a higher enhanced efficiency than those with 1.7 mg VSS/l biomass. These results suggest that there is a potential for stimulating the dechlorinating capability of anaerobic consortia to remedy the chlorinated hydrocarbons in the oligotrophic environment if the conditions of the supplementing electron donor are properly selected. Received: 14 August 1995/Received last revision: 15 March 1996/Accepted: 15 April 1996     

Synchronous anaerobic and aerobic degradation of DDT by an immobilized mixed culture system

Summary For the investigation of a mixed anaerobic and aerobic degradation of xenobiotics the reductive dechlorination of 1,1,1-trichloro-2,2-bis (4-chlorophenyl)ethane (DDT) to 1,1-dichloro-2,2-bis (4-chlorophenyl)ethane (DDD) and the oxidative degradation of the DDT-conversion product 4,4-dichlorodiphenylmethane (DDM) were studied. Enrichments from digested sewage sludge led to the isolation of an Enterobacter cloacae-strain which is able to reductive dechlorination of DDT during the fermentation of lactose. From fresh sewage sludge 11 bacterial strains were isolated in batch-culture and in continuous culture utilizing diphenylmethane, a non chlorinated structural analogon of DDM, as sole source of carbon and energy. One of these isolates, Alcaliaenes sp. cometabolizes DDM during the aerobic growth with diphenylmethane. By coimmobilization of Alcaligenes sp. and Enterobacter cloacae in Ca-alginate a system could be established, in which the reductive dechlorination of DDT and the oxidative degradation of DDM and diphenylmethane proceeds simultaneously in one reactor vessel.     

Isolation and characterization of novel Serratia marcescens (AY927692) for pentachlorophenol degradation from pulp and paper mill waste

Seven aerobic bacterial strains were isolated from pulp paper mill waste and screened for pentachlorophenol (PCP) tolerance on PCP containing mineral salt agar medium (MSM). The organism was characterized by 16S rDNA sequencing which showed 99.7% sequence similarity with Serratia marcescens. PCP degradation was routinely monitored with spectrophotometric analysis and further confirmed by HPLC analysis. Among seven strains, ITRC S₇ was found to degrade up to 90.33% of 1.127 mM (300 mg/l) of PCP and simultaneous release of chloride ion (2.435 mM) emphasized the bacterial dechlorination in the medium in presence of glucose as an additional carbon and energy source under optimized condition within 168 h incubation. In absence of glucose bacterium was unable to utilize PCP indicating the phenomenon of co-metabolism. Bacterium was identified as S. marcescens (AY927692), was a novel and potential aerobic bacterial strain capable of degrading PCP in axenic condition. Further, this strain may be used for bioremediation of PCP containing pulp paper mill waste in the environment.     

Anaerobic dechlorination of pentachlorophenol in fixed-film and upflow anaerobic sludge blanket reactors using different inocula

Longterm performance and stability of two upflow anaerobic sludge blanket (UASB) reactors inoculated with granular sludge and treating a synthetic waste water containing pentachlorophenol (PCP) and phenol were studied. A similar system consisting of two fixed-film reactors inoculated with anaerobic digested sewage sludge were further studied. One reactor in each series received glucose in addition to the phenols. Dechlorination of PCP proceeded via two different dominating pathways in the respective reactor systems, suggesting that two distinct microbial populations were present, probably originating from the different inocula. Dechlorinating activity was maintained for more than 18 months in the UASB reactors and was generally higher than in the fixed-film reactors. In the fixed-film reactors, dechlorination of PCP suddenly decreased after 15.5 months of operation compared to earlier performance. Since no operational parameters had been changed, this indicated that the enriched culture was unstable on a longterm basis. Addition of yeast extract to the medium restored activity. General process stability in both reactor systems was clearly enhanced by the addition of glucose and was superior in the UASB/granular sludge system. The better performance and the higher stability in the UASB/granular sludge reactor highlights the importance of thorough screening of inocular prior to start-up of processes treating waste waters containing xenobiotic compounds.Abbreviations PCP pentachlorophenol - TeCP tetrachlorophenol - TCP trichlorophenol - DCP dichlorophenol - UASB upflow anaerobic sludge blanket - HRT hydraulic retention time     

Dechlorination of pentachlorophenol by membrane bound enzymes of Rhodococcus chlorophenolicus PCP-I

Dechlorination (para-hydroxylation) of pentachlorophenol (PCP) and tetrachloro-para-hydroquinone (TeCH) and O-methylation of TeCH were demonstrated in cell extracts of Rhodococcus chlorophenolicus PCP-I. PCP para-hydroxylating activity was membrane bound, whereas TeCH dechlorinating enzyme was soluble. The PCP para-hydroxylating enzyme was solubilized by Triton X-100 and the requirement for both FAD and NADPH was shown. The dechlorinating activities were inducible in contrast to the constitutive TeCH O-methylating activity. The PCP para-hydroxylation was inhibited by its product TeCH, by anoxic conditions, and by different inhibitors of P₄₅₀. Participation of this cytochrome in the PCP hydroxylation was confirmed by the appearance of a carbon monoxide dependent peak of absorbance at 457 nm in the membrane fraction prepared from PCP degrading cells.     

Dechlorination of three tetrachlorobenzene isomers by contaminated harbor sludge-derived enrichment cultures follows thermodynamically favorable reactions

Dechlorination patterns of three tetrachlorobenzene isomers, 1,2,3,4-, 1,2,3,5-, and 1,2,4,5-TeCB, were studied in anoxic microcosms derived from contaminated harbor sludge. The removal of doubly, singly, and un-flanked chlorine atoms was noted in 1,2,3,4- and 1,2,3,5-TeCB fed microcosms, whereas only singly flanked chlorine was removed in 1,2,4,5-TeCB microcosms. The thermodynamically more favorable reactions were selectively followed by the enriched cultures with di- and/or mono-chlorobenzene as the main end products of the reductive dechlorination of all three isomers. Based on quantitative PCR analysis targeting 16S rRNA genes of known organohalide-respiring bacteria, the growth of Dehalococcoides was found to be associated with the reductive dechlorination of all three isomers, while growth of Dehalobacter, another known TeCB dechlorinator, was only observed in one 1,2,3,5-TeCB enriched microcosm among biological triplicates. Numbers of Desulfitobacterium and Geobacter as facultative dechlorinators were rather stable suggesting that they were not (directly) involved in the observed TeCB dechlorination. Bacterial community profiling suggested bacteria belonging to the phylum Bacteroidetes and the order Clostridiales as well as sulfate-reducing members of the class Deltaproteobacteria as putative stimulating guilds that provide electron donor and/or organic cofactors to fastidious dechlorinators. Our results provide a better understanding of thermodynamically preferred TeCB dechlorinating pathways in harbor environments and microbial guilds enriched and active in anoxic TeCB dechlorinating microcosms.

     

Identification and Characterization of a Novel CprA Reductive Dehalogenase Specific to Highly Chlorinated Phenols from Desulfitobacterium hafniense Strain PCP-1

Desulfitobacterium hafniense strain PCP-1 reductively dechlorinates pentachlorophenol (PCP) to 3-chlorophenol and a variety of halogenated aromatic compounds at the ortho, meta, and para positions. Several reductive dehalogenases (RDases) are thought to be involved in this cascade of dehalogenation. We partially purified a novel RDase involved in the dechlorination of highly chlorinated phenols from strain PCP-1 cultivated in the presence of 2,4,6-trichlorophenol. The RDase was membrane associated, and the activity was sensitive to oxygen, with a half-life of 128 min upon exposure to air. The pH and temperature optima were 7.0 and 55°C, respectively. Several highly chlorinated phenols were dechlorinated at the ortho positions. The highest dechlorinating activity levels were observed with PCP, 2,3,4,5-tetrachlorophenol, and 2,3,4-trichlorophenol. 3-Chloro-4-hydroxyphenylacetate, 3-chloro-4-hydroxybenzoate, dichlorophenols, and monochlorophenols were not dechlorinated. The apparent K_m value for PCP was 46.7 μM at a methyl viologen concentration of 2 mM. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activity, suggesting the involvement of a corrinoid cofactor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partially purified preparation revealed 2 bands with apparent molecular masses of 42 and 47 kDa. Mass spectrometry analysis using Mascot to search the genome sequence of D. hafniense strain DCB-2 identified the 42-kDa band as NADH-quinone oxidoreductase, subunit D, and the 47-kDa band as the putative chlorophenol RDase CprA3. This is the first report of an RDase with high affinity and high dechlorinating activity toward PCP.Halogenated compounds are generally known as toxic environmental pollutants. Hydrogenolytic reductive dehalogenation, a reaction involving the replacement of one halogen atom with one hydrogen atom, is the predominant mechanism for their transformation in anaerobic environments. This process can sustain microbial growth via electron transport-coupled phosphorylation (10, 26, 31). The majority of the known reductive dehalogenases (RDases) belong to the CprA/PceA family. These are single-polypeptide membrane-associated anaerobic enzymes that are synthesized as preproteins with a cleavable twin arginine translocation (TAT) peptide signal. They contain one corrinoid and two iron-sulfur clusters as cofactors.CprA enzymes catalyzing the reductive dechlorination of chloroaromatics have been purified from Desulfitobacterium hafniense strain DCB-2 (6), Desulfitobacterium dehalogenans (30), Desulfitobacterium chlororespirans strain Co23 (12, 14), Desulfitobacterium sp. strain PCE1 (29), and D. hafniense strain PCP-1 (28) and characterized, and PceA enzymes have been purified from Sulfurospirillum multivorans (22, 23), Desulfitobacterium sp. strain PCE-S (18, 19), D. hafniense strain TCE1 (29), Dehalococcoides ethenogenes 195 (15, 16), Desulfitobacterium sp. strain PCE1 (29), Dehalobacter restrictus (17, 25), Desulfitobacterium sp. strain Y51 (27), and Dehalococcoides sp. strain VS (20) and characterized. However, none of these enzymes showed high dechlorinating activity toward highly chlorinated phenols such as pentachlorophenol (PCP).D. hafniense strain PCP-1 is the only known strict anaerobic bacterium which reductively dechlorinates PCP to 3-chlorophenol (3-CP) and a variety of halogenated aromatic compounds at the ortho, meta, and para positions (2, 7). It dechlorinates PCP at the ortho, ortho, para, and meta positions in the following order: PCP → 2,3,5,6-tetrachlorophenol (2,3,5,6-TeCP) → 3,4,5-trichlorophenol (3,4,5-TCP) → 3,5-dichlorophenol (3,5-DCP) → 3-CP (7). Several RDases are thought to operate during this sequence of dechlorinations. Two RDases have already been purified from strain PCP-1. The first one, CrdA, is a membrane-associated enzyme, not related to CprA/PceA-type RDases, that mediates ortho dechlorination of 2,4,6-TCP and several chlorophenols (3). The second enzyme, CprA5, catalyzes the meta and para dechlorination of 3,5-DCP and several chlorophenols (28). Three other putative cprA genes were identified in strain PCP-1 (cprA2, cprA3, and cprA4), which suggests that other RDases with different specificities toward halogenated compounds exist in this strain (8, 31, 32). In this study, we have partially purified and characterized a new CprA-type RDase (CprA3) from strain PCP-1. CprA3 is the first reported RDase with high affinity toward PCP and with high ortho-dechlorinating activity toward PCP and other highly chlorinated phenols.     

Microbial Dechlorination of 2,3,5,6-Tetrachlorobiphenyl under Anaerobic Conditions in the Absence of Soil or Sediment

Bacterial enrichment cultures developed with Baltimore Harbor (BH) sediments were found to reductively dechlorinate 2,3,5,6-tetrachlorobiphenyl (2,3,5,6-CB) when incubated in a minimal estuarine medium containing short-chain fatty acids under anaerobic conditions with and without the addition of sediment. Primary enrichment cultures formed both meta and ortho dechlorination products from 2,3,5,6-CB. The lag time preceding dechlorination decreased from 30 to less than 20 days as the cultures were sequentially transferred into estuarine medium containing dried, sterile BH sediment. In addition, only ortho dechlorination was observed following transfer of the cultures. Sequential transfer into medium without added sediment also resulted in the development of a strict ortho-dechlorinating culture following a lag of more than 100 days. Upon further transfer into the minimal medium without sediment, the lag time decreased to less than 50 days. At this stage all cultures, regardless of the presence of sediment, would produce 2,3,5-CB and 3,5-CB from 2,3,5,6-CB. The strict ortho-dechlorinating activity in the sediment-free cultures has remained stable for more than 1 year through several transfers. These results reveal that the classical microbial enrichment technique using a minimal medium with a single polychlorinated biphenyl (PCB) congener selected for ortho dechlorination of 2,3,5,6-CB. Furthermore, this is the first report of sustained anaerobic PCB dechlorination in the complete absence of soil or sediment.Anaerobic dechlorination of polychlorinated biphenyls (PCBs) has been demonstrated in situ and with laboratory microcosms containing sediment (reviewed in reference 1a). However, sustained PCB dechlorination has never been shown to occur in the absence of soil or sediments. Morris et al. (6) demonstrated a sediment requirement for the stimulation of PCB dechlorination within freshwater sediment slurries. Wu and Wiegel have recently described PCB-dechlorinating enrichments which required soil for the successful transfer of PCB-dechlorinating activity (9). In addition, no anaerobic microorganisms that dechlorinate PCBs have been isolated or characterized, and this may be due in part to the soil or sediment requirement. The inability to isolate dechlorinating organisms or maintain dechlorination without sediment has limited biogeochemical and physiological investigations into the mechanisms of PCB dechlorination.Dechlorination (ortho, meta, and para) of single PCB congeners has been observed following anaerobic incubation of Baltimore Harbor (BH) sediment under estuarine or marine conditions (2). While sediments from several sites within BH are contaminated with PCBs (1, 5), background contamination of sediment is not necessarily a prerequisite for the development of PCB dechlorination in laboratory microcosms. Wu et al. (8) recently demonstrated meta and ortho dechlorination of Aroclor 1260 when it was added to the same BH sediments. These results showed that more than one dechlorinating activity could be developed with these sediments. It has been proposed that discrete microbial populations are responsible for specific PCB dechlorinations (1a). Consistent with this idea, the ortho dechlorination observed with BH sediments may be catalyzed by discrete microbial populations. In addition, these organisms may be able to couple PCB dechlorination with growth. Therefore we have attempted to select for ortho PCB-dechlorinating organisms by enrichment under minimal conditions with high levels of 2,3,5,6-tetrachlorobiphenyl. We also speculated that given the proper conditions, a PCB-dechlorinating population could be maintained in an actively dechlorinating state in the absence of sediment. Here we report that a distinct PCB-dechlorinating activity, namely, ortho dechlorination, was selected for through sequential transfer initiated with sediments from BH and sustained in the absence of soil or sediment. This is the first report of sustained anaerobic PCB-dechlorinating activity in the total absence of sediment.     

Aerobic Mineralization of Hexachlorobenzene by Newly Isolated Pentachloronitrobenzene-Degrading Nocardioides sp. Strain PD653

A novel aerobic pentachloronitrobenzene-degrading bacterium, Nocardioides sp. strain PD653, was isolated from an enrichment culture in a soil-charcoal perfusion system. The bacterium also degraded hexachlorobenzene, a highly recalcitrant environmental pollutant, accompanying the generation of chloride ions. Liberation of ¹⁴CO₂ from [U-ring-¹⁴C]hexachlorobenzene was detected in a culture of the bacterium and indicates that strain PD653 is able to mineralize hexachlorobenzene under aerobic conditions. The metabolic pathway of hexachlorobenzene is initiated by oxidative dechlorination to produce pentachlorophenol. As further intermediate metabolites, tetrachlorohydroquinone and 2,6-dichlorohydroquinone have been detected. Strain PD653 is the first naturally occurring aerobic bacteria capable of mineralizing hexachlorobenzene.Hexachlorobenzene (C₆Cl₆; HCB) is one of the most persistent environmental pollutants. Its average half-life in soil is approximately 9 years (2). When HCB is liberated in environment, it is bioaccumulated in plants, zooplankton, and shellfish. Finally, HCB is accumulated in the human body via the food chain, whereupon its possible toxicity adversely affects human health as a result of long-term exposure and accumulation. Therefore, HCB was listed as one of the 12 persistent organic pollutants in the Stockholm Convention.A number of studies have been attempted to develop cleanup technology for environmental pollutants. Microbial degradation is a promising effective way to remediate environmental pollutants, including persistent organic pollutants. However, heavily chlorinated benzenes, especially HCB, are resistant to microbial degradation. Several studies have been reported on the reductive dechlorination of HCB. Reductive dechlorination of HCB to pentachlorobenzene by cytochrome P-450 was found in rat hepatic microsomes (22). Microbial transformation of HCB to trichlorobenzene and dichlorobenzene by reductive dechlorination was observed in anaerobic sewage sludge and a mixed culture (5, 7). Yeh and Pavlostathis maintained such an HCB-dechlorinating mixed culture for more than 1 year by adding surfactants as carbon sources (30). One of the microorganisms that reductively dechlorinates HCB is “Dehalococcoides” sp. strain CBDB1 (12). Dehalococcoides sp. strain CBDB1 dechlorinated HCB and pentachlorobenzene via dehalorespiration and gave a final end product mixture comprised of 1,3,5-trichlorobenzene, 1,3-dichlorobenzene, and 1,4-dichlorobenzene. These reductive dechlorinating processes take a longer time and leave less-chlorinated compounds such as trichlorobenzene and dichlorobenzene as end products.Strictly aerobic, naturally occurring microorganisms that degrade and completely mineralize HCB have not been found. On the other hand, a microorganism capable of mineralizing pentachlorophenol (PCP), Sphingobium chlorophenolicum strain ATCC 39723, was isolated, and its gene organization involved in PCP metabolism was shown (4). Conversion of HCB to PCP was reported by using the genetically engineered mutant of cytochrome P-450_cam (CYP101) (13). Wild-type CYP101 from Pseudomonas putida had low degrading activity for dichlorobenzene and trichlorobenzene but did not decompose more highly chlorinated benzenes. The F87W/Y96F/V247L mutant showed improved di- and trichlorobenzene-degrading activity, but activity toward highly chlorinated benzenes including HCB was still low. The activity upon highly chlorinated benzenes was further improved in the mutant CYP101, F87W/Y96F/L244A/V247L (6). The rate of HCB degradation was increased 200-fold in the mutant. Yan et al. introduced the mutant CYP101 gene into S. chlorophenolicum strain ATCC 39723 by homologous recombination, to produce a complete HCB degrader (28). This genetically engineered bacterium degraded HCB almost completely within 12 h, together with formation of PCP as an intermediate. However, the application of genetically engineered microorganisms in natural areas is strictly restricted in many countries. HCB-degrading aerobes derived from natural sources are still required for remediation of HCB-contaminated areas.We describe here isolation and identification of a novel aerobic soil bacterial species capable of aerobically mineralizing HCB. The characterization of metabolites caused by oxidative removal of the chlorine groups from HCB is also described.     

Assessment of inhibition kinetics of the growth of strain P5 on pentachlorophenol under steady-state conditions in a nutristat

A bacterium degrading pentachlorophenol (PCP) as the only source of carbon and energy was grown in a nutristat, i.e., a continuous culture with on-line measurement and control of the substrate concentration. We improved the PCP nutristat by incorporation of a personal computer with a proportional integral derivative (PID) algorithm for controlling the medium feed pump. The controlled value deviated from the average (set-point) value by 1% maximally. In the PCP nutristat (30°C), the steadystate dilution rate, and hence, specific growth rate, showed a maximum value of 0.142±0.004 h^-1 at set-point PCP concentrations between 37 and 168 M. At PCP concentrations above 168 M, the steady-state growth rate decreased because of inhibition. The growth yield coefficient was not seriously affected by the PCP concentration, suggesting that uncoupling was not the inhibitory mechanism. It was concluded that the PCP nutristat is very useful for establishing steady-state conditions that maintain growth-inhibitory PCP concentrations and high cell concentrations, conditions for which the chemostat is not suitable.Abbreviations MCA Metabolic control analysis - NTA Nitrilotriacetic acid - PCP Pentachlorophenol - PID Proportional integral derivative     

Isolation and characterization of Dehalospirillum multivorans gen. nov., sp. nov., a tetrachloroethene-utilizing,strictly anaerobic bacterium

A strictly anaerobic bacterium dechlorinating tetrachloroethene (perchloroethylene, PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (DCE) was isolated from activated sludge with pyruvate plus PCE as energy substrates. The organism, called Dehalospirillum multivorans, is a gram-negative spirillum that does not form spores. The G+C content of the DNA was 41.5 mol%. According to 16S rRNA gene sequence analysis, D. multivorans represents a new genus and a new species belonging to the epsilon subdivision of Proteobacteria. Quinones, cytochromes b and c, and corrinoids were extracted from the cells. D. multivorans grew in defined medium with PCE and H₂ as sole energy sources and acetate as carbon source; the growth yield under these conditions was 1.4g of cell protein per mol chloride released. Alternatively to PCE, fumarate and nitrate could serve as electron acceptors; sulfate could not replace fumarate, nitrate, or PCE in this respect. In addition to H₂, the organism utilized a variety of electron donors for dechlorination (pyruvate, lactate, ethanol, formate, glycerol). Upon growth on pyruvate plus PCE, the main fermentation products formed were acetatc, lactate, DCE, and H₂. At optimal pH (7.3–7.6) and temperature (30°C), and in the presence of pyruvate (20mM) and PCE (160M), a dechlorination rate of about 50 nmol min^-1 (mg cell protein)^-1 and a doubling time of about 2.5h were obtained with growing cultures. The ability to reduce PCE to DCE appears to be constitutive under the experimental conditions applied since cultures growing in the absence of PCE for several generations immediately started dechlorination when transferred to a medium containing PCE. The organism may be useful for bioremediation of environments polluted with tetrachloroethene.Abbreviations PCE Perchloroethylene, tetrachloroethene - TCE Trichloroethene - DCE cis-1,2-Dichloroethene - CHC Chlorinated hydrocarbon     

Anaerobic dechlorination and mineralization of pentachlorophenol and 2,4,6-trichlorophenol by methanogenic pentachlorophenol-degrading granules

Anaerobic granules developed for the treatment of pentachlorophenol (PCP) completely minearilized¹⁴C-labeled PCP to¹⁴CH₄ and¹⁴CO₂. Release of chloride ions from PCP was performed by live cells in the granules under anaerobic conditions. No chloride ions were released under aerobic conditions or by autoclaved cells. Addition of sulfate enhanced the initial chloride release rate and accelerated the process of mineralization of¹⁴C-labeled PCP. Addition of molybdate (10 mM) inhibited the chloride release rate and severely inhibited PCP mineralization. This suggests involvement of sulfate-reducing bacteria in PCP dechlorination and mineralization. Addition of 2-bromoethane sulfonate slightly decreased the chloride release rate and completely stopped production of¹⁴CH₄ and¹⁴CO₂ from [¹⁴C]PCP. 2,4,6-trichlorophenol was observed as an intermediate during PCP dechlorination. On the basis of experimental results, dechlorination of 2,4,6-trichlorophanol by the granules was conducted through 2,4-dichlorophenol, 4-chlorophenol or 2-chlorophenol to phenol at pH 7.0–7.2.