Isolation and Characterization of a Mixed Culture That Degrades Polychlorinated Biphenyls

A mixed culture composed of two Pseudomonas strains, designated as KKL101 and KKS102, was isolated from soil. This mixed culture had an enhanced ability to degrade various polychlorinated biphenyls (PCBs) which include highly chlorinated components. They did not grow individually on the mineral salts medium supplemented with a highly chlorinated PCB (PCB48, a mixture of mainly tetrachlorobiphenyl) and biphenyl. When the spent medium of KKL101 was added to the washed cell preparation of KKS102, however, the latter grew on these carbon sources, producing yellow compounds which were identified as metabolic intermediates of the carbon sources, biphenyl and PCBs. These results suggest that KKL101 produces a growth factor(s) essential for KKS102 to grow on PCBs and that the growth of KKL101 is supported by the metabolic intermediates produced by KKS102. It appears that these two bacterial strains have a symbiotic relationship. From the analysis of the degradation products of various PCB congeners, it was found that strain KKS102 degrades a wide range of PCBs which have been considered to be refractory to biological degradation.     

Sphingobium fuliginis HC3: A Novel and Robust Isolated Biphenyl- and Polychlorinated Biphenyls-Degrading Bacterium without Dead-End Intermediates Accumulation

Biphenyl and polychlorinated biphenyls (PCBs) are typical environmental pollutants. However, these pollutants are hard to be totally mineralized by environmental microorganisms. One reason for this is the accumulation of dead-end intermediates during biphenyl and PCBs biodegradation, especially benzoate and chlorobenzoates (CBAs). Until now, only a few microorganisms have been reported to have the ability to completely mineralize biphenyl and PCBs. In this research, a novel bacterium HC3, which could degrade biphenyl and PCBs without dead-end intermediates accumulation, was isolated from PCBs-contaminated soil and identified as Sphingobium fuliginis. Benzoate and 3-chlorobenzoate (3-CBA) transformed from biphenyl and 3-chlorobiphenyl (3-CB) could be rapidly degraded by HC3. This strain has strong degradation ability of biphenyl, lower chlorinated (mono-, di- and tri-) PCBs as well as mono-CBAs, and the biphenyl/PCBs catabolic genes of HC3 are cloned on its plasmid. It could degrade 80.7% of 100 mg L ⁻¹ biphenyl within 24 h and its biphenyl degradation ability could be enhanced by adding readily available carbon sources such as tryptone and yeast extract. As far as we know, HC3 is the first reported that can degrade biphenyl and 3-CB without accumulation of benzoate and 3-CBA in the genus Sphingobium, which indicates the bacterium has the potential to totally mineralize biphenyl/PCBs and might be a good candidate for restoring biphenyl/PCBs-polluted environments.     

Isolation and characterization of different plant associated bacteria and their potential to degrade polychlorinated biphenyls

In our experiments the effect of different plants on microbial activities resulting in degradation and PCB removal from long-term contaminated soil was evaluated. Total bacteria and bacteria representing the dominating microflora within rhizosphere of individual plant species – tobacco (Nicotiana tabacum), black nightshade (Solanum nigrum), horseradish (Armoracia rusticana) and goat willow (Salix caprea) planted in PCB contaminated soil as well as from the same, but non-vegetated PCBs soil, were isolated and biochemically characterized. PCB bacterial degraders, stimulated by root exudates of individual plants, were detected after isolation from rhizosphere soil and precultivation on minimal medium with biphenyl as the sole carbon source. Detection of BphA1 gene (first gene of bacterial aerobic PCB degradative pathway) in genomes of rhizosphere microorganisms was performed by nested PCR technique using previously designed specific primers. Rhizosphere of individual plants contained different bacterial species, mostly gram-negative non-fermenting bacteria of Pseudomonas, Agrobacterium, Ochrobactrum and other species. Gene BphA1 was identified in genome of only several of them. From tested species, S. caprea and A. rusticana have shown to be promising candidates for rhizoremediation purposes.     

The effect of polychlorinated biphenyls (PCBs) on the membrane lipids of Pseudomonas stutzeri

In this study we examined the effect of polychlorinated biphenyls (PCBs) on biomass production of a PCB-degrading Pseudomonas stutzeri, and on the fatty acid profile of its major membrane lipids. Growth based on biomass weight was stimulated when PCBs were added at the time of inoculation, but PCB addition three days after inoculation led to a significant decrease in biomass. Simultaneous addition of PCBs plus biphenyl or PCBs plus carvone negatively affected P. stutzeri biomass (addition of biphenyl or carvone at the time of inoculation and PCBs to three-day-old culture). In the presence of PCBs alone the amount of the prevalent fatty acids C16:0 and C17-cyclopropyl fatty acid (C17-CP) of P. stutzeri in total and neutral lipids was significantly reduced. When PCBs were added together with carvone (carvone at the time of inoculation and PCBs after three days) a significant reduction of these fatty acids was obtained, but, in addition, oleic, cis-vaccenic, and cyclononadecanic (C19-CP) acids were increased. When PCBs were combined to biphenyl the prevalent fatty acids were reduced and oleic, cis-vaccenic, and cyclononadecanic acids were increased in total and neutral lipids. Addition of 3-chlorobenzoic acid led to a significant growth inhibition and to the production of oleic and cis-vaccenic acids in the membrane fraction phosphatidylcholine.     

In vitro propagation of Spilanthes mauritiana DC., an endangered medicinal herb, through axillary bud cultures

Summary Spilanthes mauritiana DC., (Compositae), a East African medicinal herb containing pharmaceutically promising secondary metabolites, has successfully been raised in vitro. We have developed a clonal propagation protocol that uses juvenile plants as starting material. The addition of benzylaminopurine (BA) (1.0 μM) and naphthaleneacetic acid (NAA) (0.1 μM) to the culture medium resulted in maximum shooting response with minimal callusing. Shoots rooted best in vitro in MS medium supplemented with indole-3-acetic acid (IAA; 0.2 μM), and plants that had already developed roots showed better growth, with maximum survival rate, in the greenhouse after an initial hardening.     

Characterization of a Defined 2,3,5,6-Tetrachlorobiphenyl-ortho-Dechlorinating Microbial Community by Comparative Sequence Analysis of Genes Coding for 16S rRNA

Defined microbial communities were developed by combining selective enrichment with molecular monitoring of total community genes coding for 16S rRNAs (16S rDNAs) to identify potential polychlorinated biphenyl (PCB)-dechlorinating anaerobes that ortho dechlorinate 2,3,5,6-tetrachlorobiphenyl. In enrichment cultures that contained a defined estuarine medium, three fatty acids, and sterile sediment, a Clostridium sp. was predominant in the absence of added PCB, but undescribed species in the δ subgroup of the class Proteobacteria, the low-G+C gram-positive subgroup, the Thermotogales subgroup, and a single species with sequence similarity to the deeply branching species Dehalococcoides ethenogenes were more predominant during active dechlorination of the PCB. Species with high sequence similarities to Methanomicrobiales and Methanosarcinales archaeal subgroups were predominant in both dechlorinating and nondechlorinating enrichment cultures. Deletion of sediment from PCB-dechlorinating enrichment cultures reduced the rate of dechlorination and the diversity of the community. Substitution of sodium acetate for the mixture of three fatty acids increased the rate of dechlorination, further reduced the community diversity, and caused a shift in the predominant species that included restriction fragment length polymorphism patterns not previously detected. Although PCB-dechlorinating cultures were methanogenic, inhibition of methanogenesis and elimination of the archaeal community by addition of bromoethanesulfonic acid only slightly inhibited dechlorination, indicating that the archaea were not required for ortho dechlorination of the congener. Deletion of Clostridium spp. from the community profile by addition of vancomycin only slightly reduced dechlorination. However, addition of sodium molybdate, an inhibitor of sulfate reduction, inhibited dechlorination and deleted selected species from the community profiles of the class Bacteria. With the exception of one 16S rDNA sequence that had the highest sequence similarity to the obligate perchloroethylene-dechlorinating Dehalococcoides, the 16S rDNA sequences associated with PCB ortho dechlorination had high sequence similarities to the δ, low-G+C gram-positive, and Thermotogales subgroups, which all include sulfur-, sulfate-, and/or iron(III)-respiring bacterial species.The extensive industrial use of polychlorinated biphenyls (PCBs) during the 20th century has resulted in the release of an estimated several million pounds of PCBs into the environment (2). Due to the hydrophobicity and chemical stability of these compounds, PCBs ultimately accumulate in subsurface anaerobic sediments, where reductive dechlorination by anaerobic microorganisms is proposed to be an essential step in PCB degradation and detoxification (6). Although anaerobic reductive dechlorination has been documented in the environment and in the laboratory, attempts to identify and isolate anaerobic PCB-dechlorinating microbes by classical enrichment and isolation techniques have been unsuccessful (for a review, see reference 2). Isolation of anaerobic PCB-dechlorinating microbes has been hindered in part by the inability to maintain and sequentially transfer dechlorinating consortia in defined medium. May et al. (24) were the first to demonstrate that single colonies could be obtained by plating highly enriched PCB-dechlorinating enrichment cultures on agar-solidified media. Although two of the colonies exhibited para dechlorination activity when transferred back to liquid enrichment medium, the colonies contained a mixed community of microorganisms and dechlorination required the addition of sediment to the medium. More recently, highly enriched PCB-ortho-dechlorinating enrichment cultures were developed from Baltimore Harbor sediments in minimal media that contained sediments and a single congener (3) or Aroclor 1260 (37). These were the first confirmed reports of sustained ortho dechlorination of PCBs throughout sequential transfers in medium with estuarine sediments. Finally, Cutter et al. demonstrated that a consortium of PCB-ortho-dechlorinating anaerobes from Baltimore Harbor could be sequentially transferred and maintained in minimal medium without the addition of sterile sediment (9). With the ability to maintain PCB dechlorination in a completely defined medium, highly enriched PCB-dechlorinating consortia could be developed by sequential transfers in medium that contained the minimal growth requirements for dechlorinating species.The current study identifies putative PCB-dechlorinating anaerobes in ortho-dechlorinating enrichment cultures by a comprehensive approach that combines traditional selective enrichment techniques with molecular monitoring (SEMM). Microbial consortia enriched for PCB ortho dechlorination in minimal medium were analyzed by comparative sequence analysis of genes coding for 16S rRNA (16S rDNA) amplified from total community DNAs. Protocols were developed for chromosomal DNA extraction from sediment, 16S rDNA amplification by PCR, cloning of partial 16S rDNA PCR fragments, screening by restriction fragment length polymorphism (RFLP) analysis, and DNA sequencing for comparative sequence analysis. By utilizing these techniques, shifts in the microbial community were monitored as the cultures were further enriched for PCB-dechlorinating anaerobes by elimination of undefined medium components (i.e., sediment), changes in carbon source, and addition of selective physiological inhibitors. The results presented herein demonstrate the applicability of the SEMM approach for the selection and monitoring of highly defined PCB-dechlorinating microbial consortia.     

Molecular assessment of the potential for in situ bioremediation of PCBs from aquatic sediments

Polychlorinated biphenyls (PCBs) are a family of xenobiotic compounds that are ubiquitous and oftentimes persistent environmental pollutants. As such, PCBs are a common target of sediment remediation efforts. Microbial degradation of sediment pollutants such as PCBs offers an environmentally sound and economically favorable alternative to conventional means of remediation such as dredging. This project describes the development of a PCR-based assay to determine the potential for PCB bioremediation by the resident microbial consortium in contaminated sediments. Using PCR and RT-PCR of DNA and RNA, respectively, extracted from aquatic sediments collected from the western basin of Lake Erie and one of its tributaries, we were able to amplify the bphA1 gene that encodes the large subunit of biphenyl dioxygenase. Since other studies have determined that the BphA1 gene product dictates PCB congener specificity, this assay may prove to be a useful screen for endemic catabolic activities for PCB mixtures in aquatic sediments.     

Coping with Polychlorinated Biphenyl (PCB) Toxicity: Physiological and Genome-Wide Responses of Burkholderia xenovorans LB400 to PCB-Mediated Stress

The biodegradation of polychlorinated biphenyls (PCBs) relies on the ability of aerobic microorganisms such as Burkholderia xenovorans sp. LB400 to tolerate two potential modes of toxicity presented by PCB degradation: passive toxicity, as hydrophobic PCBs potentially disrupt membrane and protein function, and degradation-dependent toxicity from intermediates of incomplete degradation. We monitored the physiological characteristics and genome-wide expression patterns of LB400 in response to the presence of Aroclor 1242 (500 ppm) under low expression of the structural biphenyl pathway (succinate and benzoate growth) and under induction by biphenyl. We found no inhibition of growth or change in fatty acid profile due to PCBs under nondegrading conditions. Moreover, we observed no differential gene expression due to PCBs themselves. However, PCBs did have a slight effect on the biosurface area of LB400 cells and caused slight membrane separation. Upon activation of the biphenyl pathway, we found growth inhibition from PCBs beginning after exponential-phase growth suggestive of the accumulation of toxic compounds. Genome-wide expression profiling revealed 47 differentially expressed genes (0.56% of all genes) under these conditions. The biphenyl and catechol pathways were induced as expected, but the quinoprotein methanol metabolic pathway and a putative chloroacetaldehyde dehydrogenase were also highly expressed. As the latter protein is essential to conversion of toxic metabolites in dichloroethane degradation, it may play a similar role in the degradation of chlorinated aliphatic compounds resulting from PCB degradation.     

Anaerobic Degradation of Biphenyl by the Facultative Anaerobic Strain Citrobacter freundii BS2211

Using a synthetic medium supplemented with biphenyl (a polycyclic aromatic hydrocarbon), a new bacterial strain of Citrobacter freundiiwas isolated from enrichment cultures containing soil and industrial wastewater samples of the Serpukhov Condenser Factory. This strain was found to be capable of degrading biphenyl under anaerobic conditions in the course of nitrate reduction. When the initial concentration of biphenyl in the culture medium equaled 150 mg/ml, the culture with a titer of 10⁹ cells/ml degraded up to 26–28% of biphenyl in 3 days (28°C). At 250 mg/ml, the culture with a titer of 10⁷ cells/ml degraded 15% of biphenyl in 21 days. Approximately 10% of the substrate consumed was utilized completely, whereas the remainder underwent transformation.     

Degradation of polychlorinated biphenyl (PCB) by a consortium obtained from a contaminated soil composed of Brevibacterium,Pandoraea and Ochrobactrum

An indigenous polychlorinated biphenyl (PCB)-degrading bacterial consortium was obtained from soils contaminated by transformer oil with a high content of PCBs. The PCB degrader strains were isolated and identified as Brevibacterium antarcticum, Pandoraea pnomenusa, and Ochrobactrum intermedium by 16S rRNA gene sequence phylogenetic analysis. The PCB-degrading ability of the consortium and of individual strains was determined by using GC/MS. The PCB-degrading capacities of the consortium were evaluated for three concentrations of transfomer oil ranging from 55 to 152 μM supplemented with 0.001% biphenyl and 0.1% of Tween 80 surfactant. PCB biodegradation by the consortium was favored in the presence of both additives and the greatest extent of biodegradation (67.5%) was obtained at a PCB concentration of 55 μM. Each bacterial species exhibited a particular pattern of degradation relating to specific PCB congeners. Isolated strains showed a moderate degradation capability towards tetra-, hepta-, and octa-chlorobiphenyls; although no effect on penta-, hexa-, and nona-chlorobiphenyls was observed. Recently, PCB degradation capacity was recognized in a Pandorea member; however, this is the first study that describes the ability of Brevibacterium and Ochrobactrum species to degrade PCBs.     

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.     

The effect of co-substrate activation on indigenous and bioaugmented PCB dechlorinating bacterial communities in sediment microcosms

Microbial reductive dechlorination by members of the phylum Chloroflexi, including the genus Dehalococcoides, may play an important role in natural detoxification of highly chlorinated environmental pollutants, such as polychlorinated biphenyls (PCBs). Previously, we showed the increase of an indigenous bacterial population belonging to the Pinellas subgroup of Dehalococcoides spp. in Anacostia River sediment (Washington DC, USA) microcosms treated with halogenated co-substrates (“haloprimers”), tetrachlorobenzene (TeCB), or pentachloronitrobenzene (PCNB). The PCNB-amended microcosms exhibited enhanced dechlorination of weathered PCBs, while TeCB-amended microcosms did not. We therefore developed and used different phylogenetic approaches to discriminate the effect of the two different haloprimers. We also developed complementary approaches to monitor the effects of haloprimer treatments on 12 putative reductive dehalogenase (rdh) genes common to Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain CBDB1. Our results indicate that 16S rRNA gene-based phylogenetic analyses have a limit in their ability to distinguish the effects of two haloprimer treatments and that two of rdh genes were present in high abundance when microcosms were amended with PCNB, but not TeCB. rdh gene-based phylogenetic analysis supports that these two rdh genes originated from the Pinellas subgroup of Dehalococcoides spp., which corresponds to the 16S rRNA gene-based phylogenetic analysis.     

Potential of autochthonous fungal strains isolated from contaminated soils for degradation of polychlorinated biphenyls

Up to now, most studies on polychlorinated biphenyl (PCB) bioremediation have examined the ability of model fungal strains to biodegrade PCBs. Yet, there is limited information concerning the potential of autochthonous filamentous fungal strains in the biodegradation of PCBs and their possible use in the environmental technologies. In this study, we investigated the capacity of autochthonous fungal strains in the biodegradation of PCBs by isolating 24 taxa from former industrial sites highly contaminated by PCBs. Microscopic and molecular analyses using the internal transcribed spacer (ITS) region revealed that the fungal strains belonged to the phyla Ascomycota (19 strains) and Zygomycota (five strains). The chromatography gas analysis revealed evidence of degradation of seven PCB congeners. With the exception of Circinella muscae which presented no degradation potential, the other fungal strains exhibited a rate of biodegradation ranging from 29 to 85 % after 7 d of incubation in liquid medium. Among these strains, Doratomyces nanus, Doratomyces purpureofuscus, Doratomyces verrucisporus, Myceliophthora thermophila, Phoma eupyrena, and Thermoascus crustaceus showed remarkable degradation ability (>70 %) regardless of the number of chlorine substituents on the biphenyl nucleus and a high tolerance towards PCBs. To our knowledge, this is the first study that demonstrates the ability of PCB degradation by these species and indicates the potential effectiveness of some autochthonous fungal strains in bioremediation systems.