Microstructure of Anaerobic Granules Bioaugmented with Desulfitobacterium frappieri PCP-1

Oligonucleotide probes were used to study the structure of anaerobic granular biofilm originating from a pentachlorophenol-fed upflow anaerobic sludge bed reactor augmented with Desulfitobacterium frappieri PCP-1. Fluorescence in situ hybridization demonstrated successful colonization of anaerobic granules by strain PCP-1. Scattered microcolonies of strain PCP-1 were detected on the biofilm surface after 3 weeks of reactor operation, and a dense outer layer of strain PCP-1 was observed after 9 weeks. Hybridization with probes specific for Eubacteria and Archaea probes showed that Eubacteria predominantly colonized the outer layer, while Archaea were observed in the granule interior. Mathematical simulations showed a distribution similar to that observed experimentally when using a specific growth rate of 2.2 day⁻¹ and a low bacterial diffusion of 10⁻⁷ dm² day⁻¹. Also, the simulations showed that strain PCP-1 proliferation in the outer biofilm layer provided excellent protection of the biofilm from pentachlorophenol toxicity.     

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.     

Biodegradation of pentachlorophenol in a continuous anaerobic reactor augmented with Desulfitobacterium frappieri PCP-1.

In this work, a strain of anaerobic pentachlorophenol (PCP) degrader, Desulfitobacterium frappieri PCP-1, was used to augment a mixed bacterial community of an anaerobic upflow sludge bed reactor degrading PCP. To estimate the efficiency of augmentation, the population of PCP-1 in the reactor was enumerated by a competitive PCR technique. The PCP-1 strain appeared to compete well with other microorganisms of the mixed bacterial community, with its population increasing from 10(6) to 10(10) cells/g of volatile suspended solids within a period of 70 days. Proliferation of strain PCP-1 allowed for a substantial increase of the volumetric PCP load from 5 to 80 mg/liter of reaction volume/day. A PCP removal efficiency of 99% and a dechlorination efficiency of not less than 90.5% were observed throughout the experiment, with 3-Cl-phenol and phenol being observable dechlorination intermediates.     

Biodegradation of Pentachlorophenol in a Continuous Anaerobic Reactor Augmented with Desulfitobacterium frappieri PCP-1

In this work, a strain of anaerobic pentachlorophenol (PCP) degrader, Desulfitobacterium frappieri PCP-1, was used to augment a mixed bacterial community of an anaerobic upflow sludge bed reactor degrading PCP. To estimate the efficiency of augmentation, the population of PCP-1 in the reactor was enumerated by a competitive PCR technique. The PCP-1 strain appeared to compete well with other microorganisms of the mixed bacterial community, with its population increasing from 10⁶ to 10¹⁰ cells/g of volatile suspended solids within a period of 70 days. Proliferation of strain PCP-1 allowed for a substantial increase of the volumetric PCP load from 5 to 80 mg/liter of reaction volume/day. A PCP removal efficiency of 99% and a dechlorination efficiency of not less than 90.5% were observed throughout the experiment, with 3-Cl-phenol and phenol being observable dechlorination intermediates.     

Rapid method for detecting Desulfitobacterium frappieri strain PCP-1 in soil by the polymerase chain reaction

A rapid method was developed for detecting in soil Desulfitobacterium frappieri strain PCP-1, an anaerobic gram-positive bacterium, isolated from a methanogenic consortium degrading pentachlorophenol. The method involved the establishment of a protocol for extracting total DNA from soil with the least contamination, and the use of the polymerase chain reaction (PCR) to detect strain PCP-1 with primers targeted with PCP-1 16S rRNA. To optimize the DNA extraction conditions, a glass mill homogenizer and a low-salt buffer containing polyvinylpolypyrrolidone were used on a black soil rich in organic matter. Recovered DNA was further purified with phenol/chloroform extractions, ammonium acetate precipitation and a G200 Sephadex gel-filtration column. DNA was extracted from soil supplemented with different concentrations of PCP-1 cells. Detection of PCP-1 was by PCR. The limit of detection was 800 added PCP-1 cells/g dry soil. This level of detection was achieved when the T4 gene-32 protein and 1 μg soil DNA were added to the PCR mixture followed by a nested PCR. This method is quick, sensitive, and can process several samples at the same time. Received: 22 October 1996 / Received revision: 24 January 1997 / Accepted: 10 February 1997     

Monitoring of Desulfitobacterium frappieri PCP-1 in pentachlorophenol-degrading anaerobic soil slurry reactors

Anaerobic biodegradation of pentachlorophenol (PCP) was studied in rotative bioreactors containing 200 g of PCP-contaminated soil and 250 ml of liquid medium. Reactors were bioaugmented with cells of Desulfitobacterium frappieri strain PCP-1, a bacterium able to dehalogenate PCP to 3-chlorophenol. Cells of strain PCP-1 were detected by quantitative PCR for at least 21 days in reactors containing 500 mg of PCP per kg of soil but disappeared after 21 days in reactors with 750 mg of PCP per kg of soil. Generally, PCP was completely removed in less than 9 days in soils contaminated with 189 mg of PCP per kg of soil. Sorption of PCP to soil organic matter reduced its toxicity and enhanced the survival of strain PCP-1. In some non-inoculated reactors, the indigenous microorganisms of some soils were also able to degrade PCP. These results suggest that anaerobic dechlorination of PCP in soils by indigenous PCP-degrading bacteria, or after augmentation with D. frappieri PCP-1, should be possible in situ and ex situ when the conditions are favourable for the survival of the degrading microorganisms.     

Microstructure of anaerobic granules bioaugmented with Desulfitobacterium frappieri PCP-1

Oligonucleotide probes were used to study the structure of anaerobic granular biofilm originating from a pentachlorophenol-fed upflow anaerobic sludge bed reactor augmented with Desulfitobacterium frappieri PCP-1. Fluorescence in situ hybridization demonstrated successful colonization of anaerobic granules by strain PCP-1. Scattered microcolonies of strain PCP-1 were detected on the biofilm surface after 3 weeks of reactor operation, and a dense outer layer of strain PCP-1 was observed after 9 weeks. Hybridization with probes specific for Eubacteria and Archaea probes showed that Eubacteria predominantly colonized the outer layer, while Archaea were observed in the granule interior. Mathematical simulations showed a distribution similar to that observed experimentally when using a specific growth rate of 2.2 day(-1) and a low bacterial diffusion of 10(-7) dm(2) day(-1). Also, the simulations showed that strain PCP-1 proliferation in the outer biofilm layer provided excellent protection of the biofilm from pentachlorophenol toxicity.     

Spectrum of the Reductive Dehalogenation Activity of Desulfitobacterium frappieri PCP-1

Desulfitobacterium frappieri PCP-1 was induced for ortho- and para-dechlorinating activities by different chlorophenols. Dehalogenation rates ranging from 25 to 1,158 nmol/min/mg of cell protein were observed according to the chlorophenol tested and the position of the chlorine removed. D. frappieri shows a broad substrate specificity; in addition to tetrachloroethylene and pentachloropyridine, strain PCP-1 can dehalogenate at ortho, meta, and para positions a large variety of aromatic molecules with substituted hydroxyl or amino groups. Reactions of O demethylation and reduction of nitro to amino substituents on aromatic molecules were also observed.     

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°C, compared to 30 and 37°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 μM 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.     

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.     

Riboflavin- and cobalamin-mediated biodegradation of chloroform in a methanogenic consortium

Chloroform (CF) is an important priority pollutant contaminating groundwater. Reductive dechlorination by anaerobic microorganisms is a promising strategy towards the remediation of CF. The objective of this study was to evaluate the use of redox active vitamins as electron shuttles to enhance the anaerobic biodegradation of CF in an unadapted methanogenic consortium not previously exposed to chlorinated compounds. Only negligible degradation of CF was observed in control cultures lacking redox active vitamins. The addition of riboflavin (RF), cyanocobalamin (CNB12), and hydroxycobalamin (HOB12) enabled biodegradation of CF. The reactions were predominantly catalyzed biologically as evidenced by the lack of any CF conversion in heat-killed controls amended with the cobalamins or minor conversion with RF. In live cultures, significant increases in the rate of CF conversion was observed at substoichiometric molar ratios as low as 0.1 to 0.01 vitamin:CF for RF and CNB12, respectively. At the highest molar vitamin:CF ratios tested of 0.2, the first-order rate constant of CF degradation was 5.3- and 91-fold higher in RF and CNB12 amended cultures, respectively, compared to the unamended control culture. The distribution of biotransformation products was highly impacted by the type of redox active vitamin utilized. Cultures supplemented with RF provided high yields of dichloromethane (DCM). On the other hand, cobalamins promoted the near complete mineralization of organochlorine in CF to inorganic chloride and lowered the yield of DCM. In cultures where no or little CF bioconversion occurred, prolonged exposure to CF resulted in cell lysis, as evidenced by the release of intracellular chloride. The results taken as a whole suggest that the anaerobic bioremediation of CF-contaminated sites can greatly be improved with strategies aimed at increasing the concentration of redox active vitamins.     

五氯酚在污染沉积物泥浆固液两相中厌氧生物降解

研究了污染沉积物泥浆液、固两相五氯酚(PCP)厌氧生物降解.结果表明,投加10g·kg^-1厌氧颗粒污泥,经31d处理泥浆液、固两相PCP降解率达98.9%,平均降解速率达到80mg·kg^-1·d^-1,对照处理平均降解速率仅为4.4mg·kg^-1·d^-1,颗粒污泥生物强化作用明显.作为泥浆修复过程的调控因子,有机溶剂、共基质和表面活性剂对PCP降解效应不同,投加乙醇,可提高PCP解吸和降解速率,4d内两相PCP降解速率达到54.3mg·kg^-1·d^-1;而投加共基质和非离子表面活性剂乙二醇丁醚后,液、固两相PCP降解均出现迟滞,两者均不同程度地抑制PCP降解.     

Anaerobic biodegradation of pentachlorophenol in a liquor obtained after extraction of contaminated chips and wood powder

Recovery of 97.5% of the pentachlorophenol (PCP) in contaminated wood powder was obtained after extraction with 0.1% KOH solution at 60 degrees C for 75 min. Extraction with NaOH and Na2CO3 was less effective than KOH. The neutralized extract was treated using a methanogenic consortium in an upflow anaerobic fixed-film reactor. The reactor was operated at 29 degrees C for over 600 d. The best performance of the reactor was observed when the PCP liquor was supplemented with glucose and formate. Complete dechlorination of PCP and phenol removal was obtained for a PCP loading rate of 13.3-18.0 mg l(-1) of reactor volume d(-1) with recirculation of the effluent and a hydraulic retention time (HRT) of 0.5-0.6 d.     

Anaerobic biodegradation of polychlorinated biphenyls by a microbial consortium originated from uncontaminated paddy soil

Polychlorinated biphenyls (PCBs) in Kanechlor-300 and -400 mixtures dissipated significantly compared with a sterilized control under anaerobic conditions in three Japanese paddy soils with no history of PCB contamination, demonstrating the anaerobic microbial degradation of PCBs. The PCB-degrading activity was maintained successfully in a static flooded soil medium for more than 3 years by serial transfer at intervals of 56 days (13 transfers). Ortho-, meta-, and para-substituted PCBs, 15.2 ± 9.9 mol% in total, were significantly degraded after 56 days of incubation. Analysis of menaquinones-6 and -7 and cloning of 16S rRNA gene fragments from a polymerase chain reaction denaturing gradient gel electrophoresis (DGGE) profile indicated the predominance of Firmicutes in the consortium. A PCR-based identification of the gene fragments showed the frequent presence of Desulfitobacterium sp., but not Dehalobacter sp. or Dehalococcoides sp., in the consortium. It is proposed that Japanese paddy soils with no history of PCB contamination contain an anaerobic microbial consortium consisting predominantly of Firmicutes that have the potential for anaerobic degradation of PCB.     

Anaerobic degradation of toluene and o-xylene by a methanogenic consortium.

Toluene and o-xylene were completely mineralized to stoichiometric amounts of carbon dioxide, methane, and biomass by aquifer-derived microorganisms under strictly anaerobic conditions. The source of the inoculum was creosote-contaminated sediment from Pensacola, Fla. The adaptation periods before the onset of degradation were long (100 to 120 days for toluene degradation and 200 to 255 days for o-xylene). Successive transfers of the toluene- and o-xylene-degrading cultures remained active. Cell density in the cultures progressively increased over 2 to 3 years to stabilize at approximately 10(9) cells per ml. Degradation of toluene and o-xylene in stable mixed methanogenic cultures followed Monod kinetics, with inhibition noted at substrate concentrations above about 700 microM for o-xylene and 1,800 microM for toluene. The cultures degraded toluene or o-xylene but did not degrade m-xylene, p-xylene, benzene, ethylbenzene, or naphthalene. The degradative activity was retained after pasteurization or after starvation for 1 year. Degradation of toluene and o-xylene was inhibited by the alternate electron acceptors oxygen, nitrate, and sulfate. Degradation was also inhibited by the addition of preferred substrates such as acetate, H2, propionate, methanol, acetone, glucose, amino acids, fatty acids, peptone, and yeast extract. These data suggest that the presence of natural organic substrates or contaminants may inhibit anaerobic degradation of pollutants such as toluene and o-xylene at contaminated sites.     

Analysis of a 2,4,6-trichlorophenol-dehalogenating enrichment culture and isolation of the dehalogenating member Desulfitobacterium frappieri strain TCP-A

An anaerobic, 2,4,6-trichlorophenol ortho-dehalogenating mixed culture was enriched from sediment of the river Saale (Germany). Two isolated dechlorinating colonies (MK1 and MK2) consisted of rods of different lengths and thicknesses, indicating heterogeneity. Following subcultivation with thiosulfate as alternative electron acceptor and cocultivation with Clostridium celerecrescensT, the 2,4,6-trichlorophenol-dehalogenating bacterium Desulfitobacterium frappieri strain TCP-A was isolated and characterized regarding its taxonomic properties and the spectrum of chlorophenols that it dehalogenated. Four other bacterial strains were coenriched and identified as organisms with closest phylogenetic relatedness to the Clostridium type strains C. indolis, C. glycolicum, C. hydroxybenzoicum and C. sporosphaeroides (16S rDNA sequence identities of 99.5, 99.2, 94.4, and 93.5%, respectively). Amplified ribosomal DNA restriction analysis of the original dehalogenating cultures MK1 and MK2 (when not exposed to thiosulfate) confirmed the microbial heterogeneity and revealed the presence of two additional species related to the type strains of C. celerecrescens and Clostridium propionicum. Only one copy of the 16S rRNA genes of Desulfitobacterium frappieri in each of the clone libraries of MK1 and MK2 (containing 136 and 56 clones, respectively) was found by dot-blot hybridization, suggesting a relatively low number of the dehalogenating bacterium within the enrichment culture.     

Microbial consortium bioaugmentation of a polycyclic aromatic hydrocarbons contaminated soil

In this study we evaluated the capacity of a defined microbial consortium (five bacteria: Mycobacterium fortuitum, Bacillus cereus, Microbacterium sp., Gordonia polyisoprenivorans, Microbacteriaceae bacterium, Naphthalene-utilizing bacterium; and a fungus identified as Fusarium oxysporum) isolated from a PAHs contaminated landfarm site to degrade and mineralize different concentrations (0, 250, 500 and 1000 mg kg(-1)) of anthracene, phenanthrene and pyrene in soil. PAHs degradation and mineralization was evaluated by gas chromatography and respirometry, respectively. The microbial consortium degraded on average, 99%, 99% and 96% of the different concentrations of anthracene, phenanthrene and pyrene in the soil, in 70 days, respectively. This consortium mineralized 78%, on average, of the different concentrations of the 3 PAHs in soil after 70 days. Contrarily, the autochthonous soil microbial population showed no substantial mineralization of the PAHs. Bacterial and fungal isolates from the consortium, when inoculated separately to the soil, were less effective in anthracene mineralization compared to the consortium. This signifies synergistic promotion of PAHs mineralization by mixtures of the monoculture isolates (the microbial consortium).     

Anaerobic biofilm enriched with an ammonia tolerant methanogenic consortium to improve wastewater treatment in the fishing industry

Biotechnology Letters - The digestion efficiency of liquid industrial wastes increases when using bioreactors colonized by microbial biofilms. High concentrations of proteins derived from the fish...