Changes in the bacterial community and lin genes diversity during biostimulation of indigenous bacterial community of hexachlorocyclohexane (HCH) dumpsite soil

In this study hexachlorocyclohexane (HCH) contaminated soil (with HCH level 84 g/kg of soil) from HCH dumpsite (Ummari village, Lucknow, India) was used to demonstrate biostimulation approach for HCH bioremediation. Different nutrients (molasses and ammonium phosphate) were used in different pits having contaminated soil to stimulate the indigenous microbial community. There was a substantial reduction in the total HCH content of the soil in 12 months long experiment. Maximum reduction was seen in the pit that received a combination of molasses and ammonium phosphate. A change in the microbial community concomitant with degradation of HCH was observed. Sphingomonads, which are known degraders of HCH, were found to dominate the experimental pits. Moreover changes in linA and linB gene (primary genes involved in HCH degradation) diversity and number were also seen as revealed by T-RFLP and RT-PCR respectively. The study suggests the prospects of biostimulation in decontaminating soils heavily contaminated with HCH.     

Comparative genomic analysis of nine Sphingobium strains: insights into their evolution and hexachlorocyclohexane (HCH) degradation pathways

Background

Sphingobium spp. are efficient degraders of a wide range of chlorinated and aromatic hydrocarbons. In particular, strains which harbour the lin pathway genes mediating the degradation of hexachlorocyclohexane (HCH) isomers are of interest due to the widespread persistence of this contaminant. Here, we examined the evolution and diversification of the lin pathway under the selective pressure of HCH, by comparing the draft genomes of six newly-sequenced Sphingobium spp. (strains LL03, DS20, IP26, HDIPO4, P25 and RL3) isolated from HCH dumpsites, with three existing genomes (S. indicum B90A, S. japonicum UT26S and Sphingobium sp. SYK6).

Results

Efficient HCH degraders phylogenetically clustered in a closely related group comprising of UT26S, B90A, HDIPO4 and IP26, where HDIPO4 and IP26 were classified as subspecies with ANI value >98%. Less than 10% of the total gene content was shared among all nine strains, but among the eight HCH-associated strains, that is all except SYK6, the shared gene content jumped to nearly 25%. Genes associated with nitrogen stress response and two-component systems were found to be enriched. The strains also housed many xenobiotic degradation pathways other than HCH, despite the absence of these xenobiotics from isolation sources. Additionally, these strains, although non-motile, but posses flagellar assembly genes. While strains HDIPO4 and IP26 contained the complete set of lin genes, DS20 was entirely devoid of lin genes (except linKLMN) whereas, LL03, P25 and RL3 were identified as lin deficient strains, as they housed incomplete lin pathways. Further, in HDIPO4, linA was found as a hybrid of two natural variants i.e., linA1 and linA2 known for their different enantioselectivity.

Conclusion

The bacteria isolated from HCH dumpsites provide a natural testing ground to study variations in the lin system and their effects on degradation efficacy. Further, the diversity in the lin gene sequences and copy number, their arrangement with respect to IS6100 and evidence for potential plasmid content elucidate possible evolutionary acquisition mechanisms for this pathway. This study further opens the horizon for selection of bacterial strains for inclusion in an HCH bioremediation consortium and suggests that HDIPO4, IP26 and B90A would be appropriate candidates for inclusion.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1014) contains supplementary material, which is available to authorized users.     

Aerobic degradation of lindane (γ-hexachlorocyclohexane) in bacteria and its biochemical and molecular basis

γ-Hexachlorocyclohexane (γ-HCH, also called γ-BHC and lindane) is a halogenated organic insecticide that causes serious environmental problems. The aerobic degradation pathway of γ-HCH was extensively revealed in bacterial strain Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26. γ-HCH is transformed to 2,5-dichlorohydroquinone through sequential reactions catalyzed by LinA, LinB, and LinC, and then 2,5-dichlorohydroquinone is further metabolized by LinD, LinE, LinF, LinGH, and LinJ to succinyl-CoA and acetyl-CoA, which are metabolized in the citrate/tricarboxylic acid cycle. In addition to these catalytic enzymes, a putative ABC-type transporter system encoded by linKLMN is also essential for the γ-HCH utilization in UT26. Preliminary examination of the complete genome sequence of UT26 clearly demonstrated that lin genes for the γ-HCH utilization are dispersed on three large circular replicons with sizes of 3.5 Mb, 682 kb, and 191 kb. Nearly identical lin genes were also found in other HCH-degrading bacterial strains, and it has been suggested that the distribution of lin genes is mainly mediated by insertion sequence IS6100 and plasmids. Recently, it was revealed that two dehalogenases, LinA and LinB, have variants with small number of amino acid differences, and they showed dramatic functional differences for the degradation of HCH isomers, indicating these enzymes are still evolving at high speed.     

Pseudomonas sp. to Sphingobium indicum: a journey of microbial degradation and bioremediation of Hexachlorocyclohexane

The unusual process of production of hexachlorocyclohexane (HCH) and extensive use of technical HCH and lindane has created a very serious problem of HCH contamination. While the use of technical HCH and lindane has been banned all over the world, India still continues producing lindane. Bacteria, especially Sphingomonads have been isolated that can degrade HCH isomers. Among all the bacterial strains isolated so far, Sphingobium indicum B90A that was isolated from HCH treated rhizosphere soil appears to have a better potential for HCH degradation. This conclusion is based on studies on the organization of lin genes and degradation ability of B90A. This strain perhaps can be used for HCH decontamination through bioaugmentation.     

Rhizosphere microbial community and hexachlorocyclohexane degradative potential in contrasting plant species

The organochlorine 1,2,3,4,5,6 hexachlorocyclohexane (HCH) is a broad-spectrum insecticide that was used on a large-scale worldwide. The soil–plant–microbe system and its influence on HCH biodegradation are evaluated. A greenhouse experiment was designed to evaluate HCH dissipation and several microbial parameters among rhizosphere and bulk soil of two contrasting plants, Cytisus striatus (Hill) Rothm and Holcus lanatus L. Plants were grown for 180 days in three treatments: uncontaminated soil (control), uncontaminated soil inoculated with soil (3% w/w) from a HCH-contaminated site (INOC), and uncontaminated soil inoculated with soil (3% w/w) from the HCH-contaminated site and artificially contaminated to obtain 100 mg HCH kg⁻¹ dry soil (100HCH-INOC). At harvest, plant biomass, soil water-extractable organic C, pH and Cl concentration, rhizosphere microbial densities (total heterotrophs, ammonifiers, amylolytics) and C substrate utilization patterns, and degradation of α-, β-, δ- and γ-HCH isomers were determined in bulk and rhizosphere soils. Soil solution Cl concentration was determined every 30 days throughout the entire growth period. Results demonstrate that both Cytisus striatus and Holcus lanatus can grow in soils with up to 100 mg HCH kg⁻¹. An enhanced degradation of α-HCH, but not β- or δ-HCH, was observed in the rhizosphere. Significant changes in the microbial densities were observed between bulk and rhizosphere soils of Cytisus, and an increase in C source utilization indicated changes in community level physiological profiles (CLPP) in the rhizosphere of this species when grown in contaminated soils. HCH dissipation was also greater in soils planted with this species. In accordance, increases in soil extractable C, Cl concentration and acidity were greater at the rhizosphere of Cytisus. Concentration of Cl in soil solutions also indicates greater HCH dechlorination in soils planted with Cytisus than Holcus. Results suggest that phytostimulation of bacteria present or added to soil is a promising approach to cleaning HCH-contaminated sites, and especially for biodegradation of α-HCH.     

Isolation of hexachlorocyclohexane-degrading Sphingomonas sp. by dehalogenase assay and characterization of genes involved in gamma-HCH degradation

Aim: To screen and identify bacteria from contaminated soil samples which can degrade hexachlorocyclohexane (HCH)‐isomers based on dechlorinase enzyme activity and characterize genes and metabolites. Methods and Results: Dechlorinase activity assays were used to screen bacteria from contaminated soil samples for HCH‐degrading activity. A bacterium able to grow on α‐, β‐, γ‐ and δ‐HCH as the sole carbon and energy source was identified. This bacterium was a novel species belonging to the Sphingomonas and harbour linABCDE genes similar to those found in other HCH degraders. γ‐Pentachlorocyclohexene 1,2,4‐trichlorobenzene and chlorohydroquinone were identified as metabolites. Conclusions: The study demonstrates that HCH‐degrading bacteria can be identified from large environmental sample‐based dehalogenase enzyme assay. This kind of screening is more advantageous compared to selective enrichment as it is specific and rapid and can be performed in a high‐throughput manner to screen bacteria for chlorinated compounds. Significance and Impact of the Study: The chlorinated pesticide HCH is a persistent and toxic environmental pollutant which needs to be remediated. Isolation of diverse bacterial species capable of degrading all the isomers of HCH will help in large‐scale bioremediation in various parts of the world.     

Localization of HCH catabolic genes (lin genes) in Sphingobium indicum B90A

The locations of hexachlorocyclohexane (HCH) catabolic (lin) genes were investigated in HCH degrading sphingomonad, Sphingobium indicum B90A (that was isolated from India). Southern blot analysis revealed the presence of linA1, linC, linDER and linX (linX1 and linX2) on the plasmid DNA in Sphingobium indicum B90A.     

Comparative Metagenomic Analysis of Soil Microbial Communities across Three Hexachlorocyclohexane Contamination Levels

This paper presents the characterization of the microbial community responsible for the in-situ bioremediation of hexachlorocyclohexane (HCH). Microbial community structure and function was analyzed using 16S rRNA amplicon and shotgun metagenomic sequencing methods for three sets of soil samples. The three samples were collected from a HCH-dumpsite (450 mg HCH/g soil) and comprised of a HCH/soil ratio of 0.45, 0.0007, and 0.00003, respectively. Certain bacterial; (Chromohalobacter, Marinimicrobium, Idiomarina, Salinosphaera, Halomonas, Sphingopyxis, Novosphingobium, Sphingomonas and Pseudomonas), archaeal; (Halobacterium, Haloarcula and Halorhabdus) and fungal (Fusarium) genera were found to be more abundant in the soil sample from the HCH-dumpsite. Consistent with the phylogenetic shift, the dumpsite also exhibited a relatively higher abundance of genes coding for chemotaxis/motility, chloroaromatic and HCH degradation (lin genes). Reassembly of a draft pangenome of Chromohalobacter salaxigenes sp. (∼8X coverage) and 3 plasmids (pISP3, pISP4 and pLB1; 13X coverage) containing lin genes/clusters also provides an evidence for the horizontal transfer of HCH catabolism genes.     

Characterization of the novel HCH-degrading strain, Microbacterium sp. ITRC1

A gram-positive Microbacterium sp. strain, ITRC1, that was able to degrade the persistent and toxic hexachlorocyclohexane (HCH) isomers was isolated and characterized. The ITRC1 strain has the capacity to degrade all four major isomers of HCH present in both liquid cultures and aged contaminated soil. DNA fragments corresponding to the two initial genes involved in γ-HCH degradative pathway, encoding enzymes for γ-pentachlorocyclohexene hydrolytic dehalogenase (linB) and a 2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase (linC), were amplified by PCR and sequenced. Their presence in the ITRC1 genomic DNA was also confirmed by Southern hybridization. Sequencing of the amplified DNA fragment revealed that the two genes present in the ITRC1 strain were homologous to those present in Sphingomonas paucimobilis UT26. Both 16S rRNA sequencing and phylogenetic analysis resulted in the identification of the bacteria as a Microbacterium sp. We assume that these HCH-degrading bacteria evolved independently but possessed genes similar to S. paucimobilis UT26. The reported results indicate that catabolic genes for γ-HCH degradation are highly conserved in diverse genera of bacteria, including the gram-positive groups, occurring in various environmental conditions.     

Bioremediation via In Situ Electrotransformation

Bioremediation of polluted sites relies on bacteria to degrade or transform contaminants into less noxious chemicals. To do so, bacteria require genes that encode the degradation enzymes and the capacity to properly express them, which may be lacking in indigenous bacteria. To increase the ability of indigenous bacteria to bioremediate a contaminated site, this research proposes the use of electrotransformation to facilitate bacterial uptake of exogenous degradation genes. As a proof of concept, a lindane degradation gene (linA) located on a broad host-spectrum expression plasmid (pBLN) was introduced into soil bacteria by electroporation both in vitro, in liquid media, and in situ, in soil. In both cases, the electrotransformed bacteria displayed an increase in lindane degradation and an increase in the linA gene copy number. The use of in situ electrotransformation could improve pollutant degradation rates and could provide another tool for bioremediation.     

Reconstructing an ancestral genotype of two hexachlorocyclohexane-degrading Sphingobium species using metagenomic sequence data

Over the last 60 years, the use of hexachlorocyclohexane (HCH) as a pesticide has resulted in the production of >4 million tons of HCH waste, which has been dumped in open sinks across the globe. Here, the combination of the genomes of two genetic subspecies (Sphingobium japonicum UT26 and Sphingobium indicum B90A; isolated from two discrete geographical locations, Japan and India, respectively) capable of degrading HCH, with metagenomic data from an HCH dumpsite (∼450 mg HCH per g soil), enabled the reconstruction and validation of the last-common ancestor (LCA) genotype. Mapping the LCA genotype (3128 genes) to the subspecies genomes demonstrated that >20% of the genes in each subspecies were absent in the LCA. This includes two enzymes from the ‘upper'' HCH degradation pathway, suggesting that the ancestor was unable to degrade HCH isomers, but descendants acquired lin genes by transposon-mediated lateral gene transfer. In addition, anthranilate and homogentisate degradation traits were found to be strain (selectively retained only by UT26) and environment (absent in the LCA and subspecies, but prevalent in the metagenome) specific, respectively. One draft secondary chromosome, two near complete plasmids and eight complete lin transposons were assembled from the metagenomic DNA. Collectively, these results reinforce the elastic nature of the genus Sphingobium, and describe the evolutionary acquisition mechanism of a xenobiotic degradation phenotype in response to environmental pollution. This also demonstrates for the first time the use of metagenomic data in ancestral genotype reconstruction, highlighting its potential to provide significant insight into the development of such phenotypes.     

Genetic manipulations of microorganisms for the degradation of hexachlorocyclohexane

Abstract: Hexachlorocyclohexane (HCH) is an organochlorine insecticide which has been banned in technologically advanced countries. However, it is still in use in tropical countries for mosquito control and thus new areas continue to be contaminated. Anaerobic degradation of HCH isomers have been well documented but until recently there have been only a few reports on aerobic microbial degradation of HCH isomers. The isolation of these microbes made it possible to design experiments for the cloning of the catabolic genes responsible for degradation. We review the microbial degradation of HCH isomers coupled with the genetic manipulations of the catabolic genes. The first part discusses the persistence of residues in the environment and microbial degradation while the second part gives an account of the genetic manipulations of catabolic genes involved in the degradation.     

Metabolic and phylogenetic analysis of microbial communities during phytoremediation of soil contaminated with weathered hydrocarbons and heavy metals

In the current study, the microbial ecology of weathered hydrocarbon and heavy metal contaminated soil undergoing phytoremediation was studied. The relationship of functional diversity, measured as carbon source utilisation in Biolog plates and extracellular enzymatic activities, and genetic diversity of bacteria was evaluated. Denaturing gradient gel electrophoresis was used for community analyses at the species level. Bulk soil and rhizosphere soil from pine and poplar plantations were analysed separately to determine if the plant rhizosphere impacted hydrocarbon degradation. Prevailing microbial communities in the field site were both genetically and metabolically diverse. Furthermore, both tree rhizosphere and fertilisation affected the compositions of these communities and increased activities of extracellular aminopeptidases. In addition, the abundance of alkane hydroxylase and naphthalene dioxygenase genes in the communities was low, but the prevalence of these genes was increased by the addition of bioavailable hydrocarbons. Tree rhizosphere communities had greater hydrocarbon degradation potential than those of bulk soil. Hydrocarbon utilising communities were dominated generally by the species Ralstonia eutropha and bacteria belonging to the genus Burkholderia. Despite the presence of viable hydrocarbon-degrading microbiota, decomposition of hydrocarbons from weathered hydrocarbon contaminated soil over four years, regardless of the presence of vegetation, was low in unfertilised soil. Compost addition enhanced the removal of hydrocarbons.     

Selective loss of <Emphasis Type="Italic">lin</Emphasis> genes from hexachlorocyclohexane-degrading <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> ITRC-5 under different growth conditions

The chlorinated insecticide γ-hexachlorocyclohexane (γ-HCH) is sequentially metabolized by the products of linA, linB, linC, linD, linE, and linF genes to β-ketoadipate, which is subsequently mineralized. Two or more copies of these genes are present in the bacterium Pseudomonas aeruginosa ITRC-5 that was isolated earlier by selective enrichment on technical-HCH. At least one copy of linA, linB, linC, linD, and possibly linE is lost from ITRC-5 upon its growth on γ-HCH. All the lin genes, however, are lost when the bacterium was grown in Luria–Bertani (LB) medium. The loss of lin genes is accompanied with the loss/rearrangement of insertion sequence IS6100 genes. Concomitant to the loss of lin genes, the degradation of HCH-isomers by “γ-HCH grown cells” is slower, when compared with “technical-HCH grown cells”, and is completely lost by “LB-grown cells”. The selective loss of lin genes during different growth conditions has not been reported before and is expected to help in understanding the dynamism of degradative genes.     

Microbial community adaptation to quaternary ammonium biocides as revealed by metagenomics

Quaternary ammonium compounds (QACs) represent widely used cationic biocides that persist in natural environments. Although microbial degradation, sensitivity and resistance to QACs have been extensively documented, a quantitative understanding of how whole communities adapt to QAC exposure remain elusive. To gain insights into these issues, we exposed a microbial community from a contaminated river sediment to varied levels of benzalkonium chlorides (BACs, a family of QACs) for 3 years. Comparative metagenomic analysis showed that the BAC‐fed communities were dramatically decreased in phylogenetic diversity compared with the control (no BAC exposure), resulting presumably from BAC toxicity, and dominated by Pseudomonas species (> 50% of the total). Time‐course metagenomics revealed that community adaptation occurred primarily via selective enrichment of BAC‐degrading Pseudomonas populations, particularly P. nitroreducens, and secondarily via amino acid substitutions and horizontal transfer of a few selected genes in the Pseudomonas populations, including a gene encoding a PAS/PAC sensor protein and ring‐hydroxylating dioxygenase genes. P. nitroreducens isolates were reproducibly recoverable from communities after prolonged periods of no‐BAC exposure, suggesting that they are robust BAC‐degraders. Our study provides new insights into the mechanisms and tempo of microbial community adaptation to QAC exposure and has implications for treating QACs in biological engineered systems.     

Diversity of ‘benzenetriol dioxygenase’ involved in p-nitrophenol degradation in soil bacteria

Ring hydroxylating dioxygenases (RHDOs) are one of the most important classes of enzymes featuring in the microbial metabolism of several xenobiotic aromatic compounds. One such RHDO is benzenetriol dioxygenase (BtD) which constitutes the metabolic machinery of microbial degradation of several mono- phenolic and biphenolic compounds including nitrophenols. Assessment of the natural diversity of benzenetriol dioxygenase (btd) gene sequence is of great significance from basic as well as applied study point of view. In the present study we have evaluated the gene sequence variations amongst the partial btd genes that were retrieved from microorganisms enriched for PNP degradation from pesticide contaminated agriculture soils. The gene sequence analysis was also supplemented with an in silico restriction digestion analysis. Furthermore, a phylogenetic analysis based on the deduced amino acid sequence(s) was performed wherein the evolutionary relatedness of BtD enzyme with similar aromatic dioxygenases was determined. The results obtained in this study indicated that this enzyme has probably undergone evolutionary divergence which largely corroborated with the taxonomic ranks of the host microorganisms.     

Taxonomic and functional diversity of atrazine‐degrading bacterial communities enriched from agrochemical factory soil

Aims: To characterize atrazine‐degrading potential of bacterial communities enriched from agrochemical factory soil by analysing diversity and organization of catabolic genes. Methods and Results: The bacterial communities enriched from three different sites of varying atrazine contamination mineralized 65–80% of ¹⁴C ring‐labelled atrazine. The presence of trzN‐atzBC‐trzD, trzN‐atzABC‐trzD and trzN‐atzABCDEF‐trzD gene combinations was determined by PCR. In all enriched communities, trzN‐atzBC genes were located on a 165‐kb plasmid, while atzBC or atzC genes were located on separated plasmids. Quantitative PCR revealed that catabolic genes were present in up to 4% of the community. Restriction analysis of 16S rDNA clone libraries of the three enrichments revealed marked differences in microbial community structure and diversity. Sequencing of selected clones identified members belonging to Proteobacteria (α‐, β‐ and γ‐subclasses), the Actinobacteria, Bacteroidetes and TM7 division. Several 16S rRNA gene sequences were closely related to atrazine‐degrading community members previously isolated from the same contaminated site. Conclusions: The enriched communities represent a complex and diverse bacterial associations displaying heterogeneity of catabolic genes and their functional redundancies at the first steps of the upper and lower atrazine‐catabolic pathway. The presence of catabolic genes in small proportion suggests that only a subset of the community has the capacity to catabolize atrazine. Significance and Impact of the Study: This study provides insights into the genetic specificity and the repertoire of catabolic genes within bacterial communities originating from soils exposed to long‐term contamination by s‐triazine compounds.     

Diversity of 16S rRNA and dioxygenase genes detected in coal‐tar‐contaminated site undergoing active bioremediation

Aims: In order to develop effective bioremediation strategies for polyaromatic hydrocarbons (PAHs) degradation, the composition and metabolic potential of microbial communities need to be better understood, especially in highly PAH contaminated sites in which little information on the cultivation‐independent communities is available. Methods and Results: Coal‐tar‐contaminated soil was collected, which consisted of 122·5 mg g^?1 total extractable PAH compounds. Biodegradation studies with this soil indicated the presence of microbial community that is capable of degrading the model PAH compounds viz naphthalene, phenanthrene and pyrene at 50 ppm each. PCR clone libraries were established from the DNA of the coal‐tar‐contaminated soil, targeting the 16S rRNA to characterize (i) the microbial communities, (ii) partial gene fragment encoding the Rieske iron sulfur center (α‐subunit) common to all PAH dioxygenase enzymes and (iii) β‐subunit of dioxygenase. Phylotypes related to Proteobacteria (Alpha‐, Epsilon‐ and Gammaproteobacteria), Acidobacteria, Actinobacteria, Firmicutes, Gemmatimonadetes and Deinococci were detected in 16S rRNA derived clone libraries. Many of the gene fragment sequences of α‐subunit and β‐subunit of dioxygenase obtained from the respective clone libraries fell into clades that are distinct from the reference dioxygenase gene sequences. Presence of consensus sequence of the Rieske type [2Fe‐2S] cluster binding site suggested that these gene fragments encode for α‐subunit of dioxygenase gene. Conclusions: Sequencing of the cloned libraries representing α‐subunit gene fragments (Rf1) and β‐subunit of dioxygenase showed the presence of hitherto unidentified dioxygenase in coal‐tar‐contaminated soil. Significance and Impact of the Study: The combination of the Rieske primers and bacterial community profiling represents a powerful tool for both assessing bioremediation potential and the exploration of novel dioxygenase genes in a contaminated environment.     

Bacterial community dynamics during <Emphasis Type="Italic">in-situ</Emphasis> bioremediation of petroleum waste sludge in landfarming sites

In-situ bioremediation of petroleum waste sludge in landfarming sites of Motor Oil Hellas (petroleum refinery) was studied by monitoring the changes of the petroleum composition of the waste sludge, as well as the changes in the structure of the microbial community, for a time period of 14 months. The analyses indicated an enhanced degradation of the petroleum hydrocarbons in the landfarming areas. A depletion of n-alkanes of approximately 75–100% was obtained. Marked changes of the microbial communities of the landfarms occurred concomitantly with the degradation of the petroleum hydrocarbons. The results obtained from terminal restriction fragment length polymorphism (T-RFLP) analysis of polymerase chain reaction (PCR) amplified 16S rRNA genes demonstrated that bacteria originating from the refinery waste sludge and newly selected bacteria dominated the soil bacterial community during the period of the highest degradation activity. However, the diversity of the microbial community was decreased with increased degradation of the petroleum hydrocarbons contained in the landfarms. T-RFLP fingerprints of bacteria of the genera Enterobacter and Ochrobactrum were detected in the landfarmed soil over the entire treatment period of 14 months. In contrast, the genus Alcaligenes appeared in significant numbers only within the 10 month old landfarmed soil. Genes encoding catechol 2,3-dioxygenase (subfamily I.2.A) were detected only in DNA of the untreated refinery waste sludge. However, none of the genes known to encode the enzymes alkane hydroxylase AlkB, catechol 2,3-dioxygenase (subfamily I.2.A) and naphthalene dioxygenase nahAc could be detected in DNA of the landfarmed soils.     

Microbial ecology of chlorinated solvent biodegradation

This study focused on the microbial ecology of tetrachloroethene (PCE) degradation to trichloroethene, cis‐1,2‐dichloroethene and vinyl chloride to evaluate the relationship between the microbial community and the potential accumulation or degradation of these toxic metabolites. Multiple soil microcosms supplied with different organic substrates were artificially contaminated with PCE. A thymidine analogue, bromodeoxyuridine (BrdU), was added to the microcosms and incorporated into the DNA of actively replicating cells. We compared the total and active bacterial communities during the 50‐day incubations by using phylogenic microarrays and 454 pyrosequencing to identify microorganisms and functional genes associated with PCE degradation to ethene. By use of this integrative approach, both the key community members and the ecological functions concomitant with complete PCE degradation could be determined, including the presence and activity of microbial community members responsible for producing hydrogen and acetate, which are critical for Dehalococcoides‐mediated PCE degradation. In addition, by correlation of chemical data and phylogenic microarray data, we identified several bacteria that could potentially oxidize hydrogen. These results demonstrate that PCE degradation is dependent on some microbial community members for production of appropriate metabolites, while other members of the community compete for hydrogen in soil at low redox potentials.