PCB-degrading Bacteria Research Articles

Isolation and assessment of Pseudomonas aeruginosa and Bacillus cereus as PCB-degrading bacteria in environmental bioremediation

Classical methods of treatment of polychlorinated biphenyls (PCBs) in environmental matrices such as soil encapsulation, high-temperature heating, thermal desorption, and chemical decontamination, have been costly, and they leave behind problems of environmental footprints. They must therefore give way to bioremediation techniques which are less costly and result in eco-friendly products. The aim of the study was to assess the suitability of PCB degrading bacteria (using published data) from their environmental matrix for use in environmental bioremediation. Initial isolation of the bacteria was obtained by culturing ash samples taken from the combustion chamber of an MP400 Incinerator of infectious waste at Komfo Anokye Teaching hospital (KATH) in Kumasi, Ghana, and composite soil samples taken from a transformer-servicing site at an electricity substation in Sunyani, Ghana. Test for thermotolerance was carried out by the culturing of bacteria from the soil samples and exposing them to higher temperatures (55 °C and 65 °C) to assess their resilience in an incubator for 48 h. Growing cultures were sub-cultured, after which a cocktail of bacteria isolate characterization assays was carried out on the growing isolates. These included biochemical tests on the bacterial suspensions using the analytical profile index (API 20E) for examining Enterobacteriaceae and other non-fastidious bacteria. This was followed by MALDI-ToF-MS analyses of the bacterial strains to profile and image their proteins. A molecular step was needed which was the conventional polymerase chain reaction (PCR) method, using specific primer pairs, to amplify and accentuate sections of the bacterial DNA extracts. Results of the study showed a confirmed sample site in Ghana for harvesting bacteria capable of degrading polychlorinated biphenyls (PCBs). MALDI-ToF-MS analyses revealed the possible species as Pseudomonas aeruginosa and Bacillus cereus. The conventional PCR and gel electrophoresis steps confirmed the presence of bph genes encoding the 2,3-dioxygenase enzymes in bacteria with PCB-degrading ability as documented in published data. The serial dilution of the bacterial suspensions was modified from 10−7 to 10−5 which was enough for distinct colonies to be counted, and it was a novel contribution to environmental microbiology. The confirmation of Pseudomonas and Bacillus species isolated from a transformer oil-impacted soil in this study shows promise in growing these strains in various environmental media such as in a gaseous medium to mineralize gas-phase PCBs from combustion smoke or in bottom ash residues to degrade solid-phase PCBs. It can also be applied in bioremediation of waste liquid systems containing PCBs.

Aerobic polychlorinated biphenyl-degrading bacteria isolated from the Tohoku region of Japan are not regionally endemic.

In the Tohoku region of Japan, 72% of the land comprises mountain forest zones. During winter, severe climatic conditions include heavy snowfall. In such an environment, which is considered high in biodiversity, we assumed that aerobic bacteria would be diverse and would possess the ability to degrade polychlorinated biphenyls (PCBs). In this study, 78 environmental samples were collected from the Tohoku region and 56 aerobic PCB-degrading bacterial strains were isolated. They belonged to the genera Achromobacter, Rhodococcus, Pseudomonas, Stenotrophomonas, Comamonas, Pigmentiphaga, Xenophilus, Acinetobacter, and Pandoraea. Previously reported aerobic PCB-degrading bacterial strains isolated in Japan belonged to the same genera, except that the genera Acidovorax and Bacillus were not identified in the present study. In particular, the isolated Comamonas testosteroni strains YAZ2 and YU14-111 had high PCB-degrading abilities. Analysis of the sequences of the YAZ2 and YU14-111 strains showed that the gene structures of the bph operon, which encode enzymes associated with PCB degradation, were the same as those of the Acidovorax sp. KKS102 strain. Moreover, 2,3-biphenyl dioxygenase activity was responsible for the degradation characteristics of all the isolated strains. Overall, this study suggests that aerobic PCB-degrading bacteria are not specifically endemic to the Tohoku region but distributed across Japan.

Augmentation of an Engineered Bacterial Strain Potentially Improves the Cleanup of PCB Water Pollution.

ABSTRACTPolychlorinated biphenyls (PCBs) are recalcitrant organohalide pollutants, consisting of 209 congeners. PCB cleanup in natural landscapes is expected to be achieved by the metabolic activity of microorganisms, but aerobic PCB-degrading bacteria that inhabit sites polluted by PCBs cannot degrade all PCB congeners due to the specificity of their enzymes. In this study, we investigated the degradability of PCBs when a genetically modified PCB-degrading bacterium was compounded with wild-type PCB-degrading bacteria. We used two bacterial strains, Comamonas testosteroni YAZ2 isolated from a PCB-uncontaminated natural landscape and Escherichia coli BL21(DE3) transformed with a biphenyl dioxygenase (BphA) gene from a well-known PCB degrader, Burkholderia xenovorans LB400. The enzymatic specificities of BphA were 2,3-dioxygenation in the YAZ2 and 2,3- and 3,4-dioxygenations in the recombinant E. coli. For the PCB-degrading experiment, a dedicated bioreactor capable of generating oxygen microbubbles was prototyped and used. The combined cells of the recombinant and the wild-type strains with an appropriate composite ratio degraded 40 mg/L of Kaneclor KC-300 to 0.3 ± 0.1 mg/L within 24 h. All of the health-toxic coplanar PCB congeners in KC-300 were degraded. This study suggested that the augmentation of an engineered bacterial strain could improve the cleanup of PCB water pollution. It also revealed the importance of the ratio of the strains with different PCB-degrading profiles to efficient degradation and that the application of oxygen microbubbles could rapidly accelerate the cleanup.IMPORTANCE PCB cleanup technique in a natural environment relies on the use of enzymes from microorganisms, primarily biphenyl dioxygenase and dehalogenase. Herein, we focused on biphenyl dioxygenase and created a recombinant PCB-degrading E. coli strain. Despite the development of environments for the field use of transgenic microbial strains around the world, verification of the applicability of transgenic microbial strains for PCB cleanup in the field has not yet been reported. We tentatively verified the extent to which degradability could be obtained by an augmentation model of a transgenic strain, the enzyme expression of which is easily regulated in rivers and lakes with PCB pollution. Our experiments used a dedicated bioreactor to model the natural landscape and produced results superior to those of bioremediation or biostimulation methods. The application of micro-nano bubbles, which has recently been discussed, to the cleanup of environmental pollution was also found to be useful in this study.

Biodegradation of PCB congeners by Paraburkholderia xenovorans LB400 in presence and absence of sediment during lab bioreactor experiments.

Experiments were conducted to measure biodegradation of polychlorinated biphenyl (PCB) congeners contained in mixture Aroclor 1248 and congeners present in wastewater lagoon sediment contaminated decades earlier at Altavista, Virginia. A well-characterized strain of aerobic PCB-degrading bacteria, Paraburkholderia xenovorans LB400 was incubated in laboratory bioreactors with PCB-contaminated sediment collected at the site. The experiments evaluated strain LB400's ability to degrade PCBs in absence of sediment and in PCB-contaminated sediment slurry. In absence of sediment, LB400 transformed 76% of Aroclor 1248 within seven days, spanning all homolog groups present in the mixture. In sediment slurry, only mono- and di-chlorinated PCB congeners were transformed. These results show that LB400 is capable of rapidly biodegrading most PCB congeners when they are freely dissolved in liquid but cannot degrade PCB congeners having three or more chlorine substituents in sediment slurry. Finally, using GC/MS-MS triple quadrupole spectrometry, this work distinguishes between physical (sorption to cells) and biological removal mechanisms, illuminates the process by which microorganisms with LB400-type congener specificity can selectively transform lower-chlorinated congeners over time, and makes direct comparisons to other studies where individual congener data is reported.

Exploring bacterial community structure and function associated with polychlorinated biphenyl biodegradation in two hydrogen-amended soils

Hydrogen (H2) is a universal energy source supplying survival energy for numerous microbial functions. Diffusive fluxes of H2 released by rhizobacterial symbiont nodules in which H2 is an obligate by-product of dinitrogen fixation may act as an additional energy input shaping microbial community structure and function in soils. However, the effects of H2 at the soil-nodule interface on soil contaminant degradation processes are poorly understood. Here, we mimicked the hydrogen conditions present at the soil-nodule interface (10,000 ppmv) to test the impact of elevated H2 concentrations on soil microbial removal of 3, 3′, 4, 4′-tetrachlorobiphenyl (PCB77) and examined the associated bacterial communities and their functions by conducting a microcosm experiment using two different soil types at three PCB contamination levels (0.5, 1.0 and 5.0 mg kg−1). After incubation for 84 days the PCB77 removal rates in the elevated H2 treatments in the Paddy soil were significantly promoted (by 4.88 to 6.41%) compared with the control (0.5 ppmv H2) but no significant effect was observed in a Fluvo-aquic soil. This is consistent with changes in the abundance of functional genes for PCB-degraders as shown by quantitative real-time PCR (Q-PCR) and phylogenetic investigation of bacterial communities by reconstruction of unobserved states (PICRUSt). 16S amplicon sequencing was conducted to explore bacterial community structure and correlate the genera to potential PCB degradation. The abundance of a total of four potentially PCB-degrading bacterial genera (Bacillus, Streptomyces, Ramlibacter and Paenibacillus) increased with increasing H2 level. In addition, the abundance of hydrogenase in the elevated H2 treatments was higher than in the control across different contamination levels in both soil types. Thus, elevated H2 stimulated soil PCB degradation with direct effects (aerobic PCB-degrading bacteria directly utilized H2 as an energy source for growth and thus enhanced PCB degradation efficiency) and indirect effects (aerobic PCB-degrading bacteria acted synergistically with other hydrogenotrophs to enhance PCB degradation efficiency by exchange of substances and energy). These results help to further understand the role of elevated hydrogen amendment in the PCB biodegradation process and provide evidence that H2 supports metabolic and energetic flexibility in microorganisms supplying a range of ecosystem services.

Supplementing resuscitation-promoting factor (Rpf) enhanced biodegradation of polychlorinated biphenyls (PCBs) by Rhodococcus biphenylivorans strain TG9T

The biodegradation of polychlorinated biphenyls (PCBs) occurs slowly when the degrading bacteria enter a low activity state, such as a viable but nonculturable (VBNC) state, under unfavorable environmental conditions. The introduction of resuscitation-promoting factor (Rpf) can re-activate VBNC bacteria. This study tested the feasibility of enhancing PCB biodegradation via supplementing Rpf in liquid culture and soil microcosms inoculated with Rhodococcus biphenylivorans strain TG9T. Exogenous Rpf resuscitated TG9T cells that had previously entered the VBNC state after 90 d of nutrient starvation, resulting in the significantly enhanced degradation of PCB by 24.3% over 60 h in liquid medium that originally contained 50 mg L−1 Aroclor 1242. In soil microcosms containing 50 mg kg−1 Aroclor 1242 and inoculated with VBNC TG9T cells, after 49 d of supplementation with Rpf, degradation efficiency of PCB reached 34.2%, which was significantly higher than the control. Our results confirmed that exogenous Rpf resuscitated VBNC TG9T cells by stimulating endogenous expression of rpf gene orthologs. The enhanced PCB-degrading capability was likely due to the increased cell numbers and the strong expression of PCB catabolic genes. This study demonstrated the role of Rpf in enhancing PCB degradation via resuscitating PCB-degrading bacteria, indicating a promising approach for the remediation of PCB contamination.

Polychlorinated Biphenyl (PCB)-Degrading Potential of Microbes Present in a Cryoconite of Jamtalferner Glacier

Aiming to comprehensively survey the potential pollution of an alpine cryoconite (Jamtalferner glacier, Austria), and its bacterial community structure along with its biodegrading potential, first chemical analyses of persistent organic pollutants, explicitly polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) as well as polycyclic aromatic hydrocarbons (PAHs), revealed a significant contamination. In total, 18 PCB congeners were detected by high resolution gas chromatography/mass spectrometry with a mean concentration of 0.8 ng/g dry weight; 16 PAHs with an average concentration of 1,400 ng/g; and 26 out of 29 OCPs with a mean concentration of 2.4 ng/g. Second, the microbial composition was studied using 16S amplicon sequencing. The analysis revealed high abundances of Proteobacteria (66%), the majority representing α-Proteobacteria (87%); as well as Cyanobacteria (32%), however high diversity was due to 11 low abundant phyla comprising 75 genera. Biodegrading potential of cryoconite bacteria was further analyzed using enrichment cultures (microcosms) with PCB mixture Aroclor 1242. 16S rDNA analysis taxonomically classified 37 different biofilm-forming and PCB-degrading bacteria, represented by Pseudomonas, Shigella, Subtercola, Chitinophaga, and Janthinobacterium species. Overall, the combination of culture-dependent and culture-independent methods identified degrading bacteria that can be potential candidates to develop novel bioremediation strategies.

Advances and perspective in bioremediation of polychlorinated biphenyl-contaminated soils.

In recent years, microbial degradation and bioremediation approaches of polychlorinated biphenyls (PCBs) have been studied extensively considering their toxicity, carcinogenicity and persistency potential in the environment. In this direction, different catabolic enzymes have been identified and reported for biodegradation of different PCB congeners along with optimization of biological processes. A genome analysis of PCB-degrading bacteria has led in an improved understanding of their metabolic potential and adaptation to stressful conditions. However, many stones in this area are left unturned. For example, the role and diversity of uncultivable microbes in PCB degradation are still not fully understood. Improved knowledge and understanding on this front will open up new avenues for improved bioremediation technologies which will bring economic, environmental and societal benefits. This article highlights on recent advances in bioremediation of PCBs in soil. It is demonstrated that bioremediation is the most effective and innovative technology which includes biostimulation, bioaugmentation, phytoremediation and rhizoremediation and acts as a model solution for pollution abatement. More recently, transgenic plants and genetically modified microorganisms have proved to be revolutionary in the bioremediation of PCBs. Additionally, other important aspects such as pretreatment using chemical/physical agents for enhanced biodegradation are also addressed. Efforts have been made to identify challenges, research gaps and necessary approaches which in future, can be harnessed for successful use of bioremediation under field conditions. Emphases have been given on the quality/efficiency of bioremediation technology and its related cost which determines its ultimate acceptability.

Regional analysis of potential polychlorinated biphenyl degrading bacterial strains from China.

Polychlorinated biphenyls (PCBs), the chlorinated derivatives of biphenyl, are one of the most prevalent, highly toxic and persistent groups of contaminants in the environment. The objective of this study was to investigate the biodegradation of PCBs in northeastern (Heilongjiang Province), northern (Shanxi Province) and eastern China (Shanghai municipality). From these areas, nine soil samples were screened for PCB-degrading bacteria using a functional complementarity method. The genomic 16S rDNA locus was amplified and the products were sequenced to identify the bacterial genera. Seven Pseudomonas strains were selected to compare the capacity of bacteria from different regions to degrade biphenyl by HPLC. Compared to the biphenyl content in controls of 100%, the biphenyl content went down to 3.7% for strain P9-324, 36.3% for P2-11, and 20.0% for the other five strains. These results indicate that a longer processing time led to more degradation of biphenyl. PCB-degrading bacterial strains are distributed differently in different regions of China.

Isolation of the PCB-degrading bacteria Mesorhizobium sp. ZY1 and its combined remediation with Astragalus sinicus L. for contaminated soil

ABSTRACTA bacterial strain ZY1 capable of utilizing PCBs as its carbon source was isolated from the root nodules of Chinese milk vetch (Astragalus sinicus L.). The strain was identified as Mesorhizobium sp. according to its physiological-biochemical properties and the analysis of its 16S rRNA gene sequence. When the initial OD600 was 0.15, 62.7% of 15 mg L−1 3,3′,4,4′-TCB in a liquid culture was degraded by Mesorhizobium sp. ZY1 within 10 days. Mesorhizobium sp. ZY1 also greatly increased the biotransformation of soil PCBs. Pot experiments indicated that the soil PCB concentrations of a single incubation of strain ZY1 (R) and a single planting of A. sinicus (P) decreased by 20.5% and 23.0%, respectively, and the concentration of PCBs in soil treated with A. sinicus and strain ZY1 decreased by 53.1%. We also observed that A. sinicus-Mesorhizobium sp. ZY1 treatment (PR) improved plant biomass and the concentration of PCBs in plants compared with a single A. sinicus planting treatment (P). The results suggest that the synergistic association between A. sinicus and PCBs-degrading Mesorhizobium sp. ZY1 can stimulate the phytoextraction of PCBs and the rhizosphere microflora to degrade PCBs, and might be a promising bioremediation strategy for PCB-contaminated soil.

Transformation of hydroxylated derivatives of 2,5-dichlorobiphenyl and 2,4,6-trichlorobiphenyl by Burkholderia xenovorans LB400

The polychlorinated biphenyl (PCB)-degrading bacterium, Burkholderia xenovorans LB400, was capable of transforming three hydroxylated derivatives of 2,5-dichlorobiphenyl (2,5-DCB) (2'-hydroxy- (2'-OH-), 3'-OH-, and 4'-OH-2,5-DCB) when biphenyl was used as the carbon source (i.e., biphenyl pathway-inducing condition), although only 2'-OH-2,5-DCB was transformed when the bacterium was growing on succinate (i.e., condition non-inductive of the biphenyl pathway). On the contrary, hydroyxlated derivatives of 2,4,6-trichlorobiphenyl (2,4,6-TCB) (2'-OH-, 3'-OH-, and 4'-OH-2,4,6-TCB) were not significantly transformed by B. xenovorans LB400, regardless of the carbon source used. Gene expression analyses showed a clear correlation between the transformation of OH-2,5-DCBs and expression of genes of the biphenyl pathway. The PCB metabolite, 2,5-dichlorobenzoic acid (2,5-DCBA), was produced following the transformation of OH-2,5-DCBs. 2,5-DCBA was not further transformed by B. xenovorans LB400. The present study is significant because it provides evidence that PCB-degrading bacteria are capable of transforming hydroxylated derivatives of PCBs, which are increasingly considered as a new class of environmental contaminants.

Isolation and identification of aerobic polychlorinated biphenyls degrading bacteria

Aims: The purpose of this study was to isolate and identify aerobic polychlorinated biphenyls (PCBs) degrading bacteria. Materials and Methods: This study was performed in lab scale aerobic sequencing batch biofilm reactor. Polyurethane foams were used as bio-carrier and synthetic wastewater was prepared with PCBs in transformer oil as the main substrate (20-700 μg/l) and acetone as a solvent for PCBs as well as microelements. After achieving to adequate microbial population and acclimation of microorganisms to PCB compounds with high efficiency of PCB removal, identification of degrading microbial species was performed by 16s rRNA gene sequencing of isolated bacteria. Results: Gene sequencing results of the isolated bacteria showed that Rhodococcus spp., Pseudomonas spp., Pseudoxanthomonas spp., Agromyces spp., and Brevibacillus spp. were dominant PCB-degrading bacteria. Conclusion: PCB compounds can be degraded by some microorganisms under aerobic or anaerobic conditions or at least be reduced to low chlorinated congeners, despite their chemical stability and toxicity. Based on the results of the study, five bacterial species capable of degrading PCBs in transformer oil have been identified.

Biochemical Studies and Ligand-bound Structures of Biphenyl Dehydrogenase from Pandoraea pnomenusa Strain B-356 Reveal a Basis for Broad Specificity of the Enzyme

Biphenyl dehydrogenase, a member of short-chain dehydrogenase/reductase enzymes, catalyzes the second step of the biphenyl/polychlorinated biphenyls catabolic pathway in bacteria. To understand the molecular basis for the broad substrate specificity of Pandoraea pnomenusa strain B-356 biphenyl dehydrogenase (BphB(B-356)), the crystal structures of the apo-enzyme, the binary complex with NAD(+), and the ternary complexes with NAD(+)-2,3-dihydroxybiphenyl and NAD(+)-4,4'-dihydroxybiphenyl were determined at 2.2-, 2.5-, 2.4-, and 2.1-Å resolutions, respectively. A crystal structure representing an intermediate state of the enzyme was also obtained in which the substrate binding loop was ordered as compared with the apo and binary forms but it was displaced significantly with respect to the ternary structures. These five structures reveal that the substrate binding loop is highly mobile and that its conformation changes during ligand binding, starting from a disorganized loop in the apo state to a well organized loop structure in the ligand-bound form. Conformational changes are induced during ligand binding; forming a well defined cavity to accommodate a wide variety of substrates. This explains the biochemical data that shows BphB(B-356) converts the dihydrodiol metabolites of 3,3'-dichlorobiphenyl, 2,4,4'-trichlorobiphenyl, and 2,6-dichlorobiphenyl to their respective dihydroxy metabolites. For the first time, a combination of structural, biochemical, and molecular docking studies of BphB(B-356) elucidate the unique ability of the enzyme to transform the cis-dihydrodiols of double meta-, para-, and ortho-substituted chlorobiphenyls.

BACTERIAL DEGRADATION AND BIOREMEDIATION OF CHLORINATED HERBICIDES AND BIPHENYLS

Chlorinated herbicides (e.g. s-triazines) and polychlorobiphenyls (PCBs) are persistent organic pollutants (POPs) that are widely distributed in the environment. s-Triazine herbicides are used in agriculture and forestry in diverse regions of the world. PCBs were produced worldwide for industrial applications, and an important amount of these compounds have been released into the environment. PCBs and s-triazines are toxic compounds that could act as endocrine disrupters and cause cancer. Therefore, environmental pollution with s-triazines and PCBs is of increasing concern. Bioremediation is an attractive technology for the decontamination of polluted sites. Microorganisms play a main role in the removal of POPs from the environment. Diverse bacteria able to degrade s-triazines and PCBs have been characterized. Bacterial degradation of s-triazine herbicides involves hydrolytic reactions catalyzed by amidohydrolases encoded by the atz genes. Anaerobic and aerobic bacteria are capable of biotransforming PCBs. Higher chlorinated PCBs are subjected to reductive dehalogenation by anaerobic microorganisms. Lower chlorinated biphenyls are oxidized by aerobic bacteria. Genome analyses of PCB-degrading bacteria have increased the knowledge of their metabolic capabilities and their adaptation to stressful conditions. For the removal of s-triazines and PCBs from the environment, efficient bioremediation processes have to be established. In this report, bacterial degradation of s-triazines and PCBs is described and novel strategies to improve bioremediation of these POPs are discussed.

Identification and Analysis of Polychlorinated Biphenyls (PCBs)-Biodegrading Bacterial Strains in Shanghai

As one of China's great metropolises, Shanghai is vulnerable to various forms of industrial and agricultural contamination associated with its development. Polychlorinated biphenyls (PCBs) are man-made chemicals that never existed in nature until the 1900s when they started to be released into the environment. PCBs are hazardous environmental contaminants that bind strongly to soil. In this study, four soil samples were screened for the presence of PCB-degrading bacteria. The 16 S rDNAs were amplified from those genomes and the products (~1.5 kb) were purified and sequenced for the isolation and identification of bacterial species. Four Pseudomonas strains (strain 1-212 from sample 1; strain 2-241 from sample 2; strain 3-318 from sample 3; and strain 4-150 from sample 4) were selected for analysis by HPLC. Setting the content of the biphenyl in CK as 100%, the biphenyl contents was 2.32% in 1-212, 73.11% in 2-241, 69.83% in 3-318, and 86.16% in 4-150. The results of this study suggest directions for future research, including genetic screening, cloning and restructuring, and provide guidance for the cultivation of PCBs-degrading bacteria.

The effects of individual PCB congeners on the soil bacterial community structure and the abundance of biphenyl dioxygenase genes

Polychlorinated biphenyls (PCBs) are toxic environmental contaminants that represent a class of 209 congeners characterized by different degrees of chlorination and substitution patterns. Most of experimental studies about microbial degradation of PCBs have been conducted on PCB mixtures, even though evidence accumulated in bacteria and other organisms shows that exposure to different congeners may have different biological effects. Microcosm experiments were conducted using aerobic agitated soil slurries individually exposed to PCB congeners with different degrees of chlorination: PCB-3, 15, 28, and 77, and the commercial mixture Aroclor 1242. After four weeks of incubation, PCBs were analyzed by gas chromatography/mass spectrometry (GC/MS) showing different transformation extents: With the exception of PCB-15 that was not significantly transformed (7%), biodegradation rates decreased with the degree of chlorination, from 75% for PCB-3 to 22% for PCB-77 and Aroclor 1242. The bacterial abundance, as measured by colony counting and 16S rDNA quantification by real-time PCR, was lower (of about 40%) in soil microcosms exposed to the higher-chlorinated congeners, PCB-28, PCB-77, and Aroclor 1242, as compared to non-exposed soils and soils exposed to the lower-chlorinated congeners, PCB-3 and PCB-15. The relative abundance of different taxonomic groups, as determined by real-time PCR, revealed an increase of β-Proteobacteria and Actinobacteria in all microcosms exposed to PCBs, as compared with non-exposed soil. In addition, exposure to PCB-77 and Aroclor 1242 resulted in a higher abundance of α-Proteobacteria and Acidobacteria. Globally, these results suggest that exposure to PCBs (and especially to higher-chlorinated congeners and Aroclor 1242) selected bacterial groups involving most known PCB degraders, i.e., β-Proteobacteria and Acidobacteria. The quantification of biphenyl dioxygenase (BPH) genes — involved in the aerobic degradation of PCBs — using real-time PCR showed that exposure to all PCB congeners and Aroclor 1242 resulted in a marked increase of two out of the four BPH genes tested, similarly suggesting the selection of PCB-degrading bacteria. This paper showed that exposure to different PCB congeners leads to different structures of the soil bacterial community and BPH genes expression patterns.

Biodegradative potential and characterization of psychrotolerant polychlorinated biphenyl-degrading marine bacteria isolated from a coastal station in the Terra Nova Bay (Ross Sea, Antarctica)

Antarctic marine bacteria were screened for their ability to degrade polychlorinated biphenyls (PCB) as the sole carbon and energy source at both 4 °C and 15 °C. PCB-degrading isolates (7.1%) were identified by sequencing their 16S rDNA as Pseudoalteromonas, Psychrobacter and Arthrobacter members. One representative isolate per genera was selected for evaluating the biodegradative potential under laboratory scale and phenotypically characterized. Removal of individual PCB congeners was between 35.6% and 79.8% at 4 °C and between 0.4% and 82.8% at 15 °C. Differences in the removal patterns of PCB congeners were observed in relation to the phylogenetic affiliation: Arthrobacter isolate showed similar biodegradation efficiencies when growing at 4 °C and 15 °C, while Pseudoalteromonas better degraded PCBs at 15 °C. No biodegradation was detected for Psychrobacter isolate at 4 °C. Results obtained highlight the occurrence of PCB-degrading bacteria in Antarctic seawater and suggest the potential exploitation of autochthonous bacteria for PCB bioremediation in cold marine environments.

Polychlorinated biphenyl (PCB)-degrading bacteria associated with trees in a PCB-contaminated site.

The abundance, identities, and degradation abilities of indigenous polychlorinated biphenyl (PCB)-degrading bacteria associated with five species of mature trees growing naturally in a contaminated site were investigated to identify plants that enhance the microbial PCB degradation potential in soil. Culturable PCB degraders were associated with every plant species examined in both the rhizosphere and root zone, which was defined as the bulk soil in which the plant was rooted. Significantly higher numbers of PCB degraders (2.7- to 56.7-fold-higher means) were detected in the root zones of Austrian pine (Pinus nigra) and goat willow (Salix caprea) than in the root zones of other plants or non-root-containing soil in certain seasons and at certain soil depths. The majority of culturable PCB degraders throughout the site and the majority of culturable PCB degraders associated with plants were identified as members of the genus Rhodococcus by 16S rRNA gene sequence analysis. Other taxa of PCB-degrading bacteria included members of the genera Luteibacter and Williamsia, which have not previously been shown to include PCB degraders. PCB degradation assays revealed that some isolates from the site have broad congener specificities; these isolates included one Rhodococcus strain that exhibited degradation abilities similar to those of Burkholderia xenovorans LB400. Isolates with broad congener specificity were widespread at the site, including in the biostimulated root zone of willow. The apparent association of certain plant species with increased abundance of indigenous PCB degraders, including organisms with outstanding degradation abilities, throughout the root zone supports the notion that biostimulation through rhizoremediation is a promising strategy for enhancing PCB degradation in situ.

Crystal structure of 4-chlorobenzoate:CoA ligase/synthetase in the unliganded and aryl substrate-bound states.

4-Chlorobenzoate:CoA ligase (CBAL) is a member of a family of adenylate-forming enzymes that catalyze two-step adenylation and thioester-forming reactions. In previous studies, we have provided structural evidence that members of this enzyme family (exemplified by acetyl-CoA synthetase) use a large domain rotation to catalyze the respective partial reactions [A. M. Gulick, V. J. Starai, A. R. Horswill, K. M. Homick, and J. C. Escalante-Semerena, (2003) Biochemistry 42, 2866-2873]. CBAL catalyzes the synthesis of 4-chlorobenzoyl-CoA, the first step in the 4-chlorobenzoate degredation pathway in PCB-degrading bacteria. We have solved the 2.0 A crystal structure of the CBAL enzyme from Alcaligenes sp. AL3007 using multiwavelength anomalous dispersion. The results demonstrate that in the absence of any ligands, or bound to the aryl substrate 4-chlorobenzoate, the enzyme adopts the conformation poised for catalysis of the adenylate-forming half-reaction. We hypothesize that coenzyme A binding is required for stabilization of the alternate conformation, which catalyzes the 4-CBA-CoA thioester-forming reaction. We have also determined the structure of the enzyme bound to the aryl substrate 4-chlorobenzoate. The aryl binding pocket is composed of Phe184, His207, Val208, Val209, Phe249, Ala280, Ile303, Gly305, Met310, and Asn311. The structure of the 4-chlorobenzoate binding site is discussed in the context of the binding sites of other family members to gain insight into substrate specificity and evolution of new function.

Growth of polychlorinated-biphenyl-degrading bacteria in the presence of biphenyl and chlorobiphenyls generates oxidative stress and massive accumulation of inorganic polyphosphate.

Inorganic polyphosphate (polyP) plays a significant role in increasing bacterial cell resistance to unfavorable environmental conditions and in regulating different biochemical processes. Using transmission electron microscopy of the polychlorinated biphenyl (PCB)-degrading bacterium Pseudomonas sp. strain B4 grown in defined medium with biphenyl as the sole carbon source, we observed large and abundant electron-dense granules at all stages of growth and following a shift from glucose to biphenyl or chlorobiphenyls. Using energy dispersive X-ray analysis and electron energy loss spectroscopy with an integrated energy-filtered transmission electron microscope, we demonstrated that these granules were mainly composed of phosphate. Using sensitive enzymatic methods to quantify cellular polyP, we confirmed that this polymer accumulates in PCB-degrading bacteria when they grow in the presence of biphenyl and chlorobiphenyls. Concomitant increases in the levels of the general stress protein GroEl and reactive oxygen species were also observed in chlorobiphenyl-grown cells, indicating that these bacteria adjust their physiology with a stress response when they are confronted with compounds that serve as carbon and energy sources and at the same time are chemical stressors.