Response of 1,2-dichloroethane-adapted microbial communities to ex-situ biostimulation of polluted groundwater

The microbial community of a groundwater system contaminated by 1,2-dichloroethane (1,2-DCA), a toxic and persistent chlorinated hydrocarbon, has been investigated for its response to biostimulation finalized to 1,2-DCA removal by reductive dehalogenation. The microbial population profile of samples from different wells in the aquifer and from microcosms enriched in the laboratory with different organic electron donors was analyzed by ARISA (Amplified Ribosomal Intergenic Spacer Analysis) and DGGE (Denaturing Gradient Gel Electrophoresis) of 16S rRNA genes. 1,2-DCA was completely removed with release of ethene from most of the microcosms supplemented with lactate, acetate plus formate, while cheese whey supported 1,2-DCA dehalogenation only after a lag period. Microbial species richness deduced from ARISA profiles of the microbial community before and after electron donor amendments indicated that the response of the community to biostimulation was heterogeneous and depended on the well from which groundwater was sampled. Sequencing of 16S rRNA genes separated by DGGE indicated the presence of bacteria previously associated with soils and groundwater polluted by halogenated hydrocarbons or present in consortia active in the removal of these compounds. A PCR assay specific for Desulfitobacterium sp. showed the enrichment of this genus in some of the microcosms. The dehalogenation potential of the microbial community was confirmed by the amplification of dehalogenase-related sequences from the most active microcosms. Cloning and sequencing of PCR products indicated the presence in the metagenome of the bacterial community of a new dehalogenase potentially involved in 1,2-DCA reductive dechlorination.     

Bacterial Diversity and Biogeochemical Processes of Oil-Contaminated Groundwater,Baoding, North China

A total of 43 groundwater samples were collected from 9 multimonitoring wells at a petrochemical site, Baoding City, North China, from June 2008 to December 2009 to investigate the biogeochemical processes and/or bacterial conmmunity using both culture-dependent and -independent methods. The results showed that aromatic hydrocarbons and chlorinated hydrocarbons were the major pollutants in the groundwater. Denitrification and iron reduction might be the main biogeochemical processes in the aquifers at this site, which seemed to transform from denitrification-dominated to iron reduction-dominated in some sections. Denaturing gradient gel electrophoresis (DGGE) revealed that the dominant bacterial groups of the groundwater were related to some oil-degrading bacteria, which can grow under denitrifying, iron-reducing and sulfate-reducing anaerobic conditions. In some serious contaminated groundwater niches, there might be sulfur cycles, as sulfur oxidizer was also abundant, which was further confirmed by 16S rRNA gene cloning analysis. The operational taxonomic units (OTUs) that highly related to Pseudomonas sp., Hydrogenophaga sp., Sphingomonas sp., Ferribacterium sp. and Sulfuricurvum Kujiense etc. were predominant in the groundwater contaminated by chlorinated hydrocarbons (CHCs), benzene, toluene, ethylbenzene, and xylenes (BTEX) and/or polycyclic aromatic hydrocarbons (PAHs), respectively. Biodiversity seemed to be undermined by oil contamination, and varied with seasons. The bacterial community in the contaminated groundwater was largely determined by the groundwater geochemistry.     

Biochemical and molecular characterization of a tetrachloroethene dechlorinating Desulfitobacterium sp. strain Y51: a review

A strict anaerobic bacterium, Desulfitobacterium sp. strain Y51, is capable of very efficiently dechlorinating tetrachloroethene (PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (cis-DCE) at concentrations as high as 960 μM and as low as 0.06 μM. Dechlorination was highly susceptible to air oxidation and to potential alternative electron acceptors, such as nitrite, nitrate or sulfite. The PCE reductive dehalogenase (encoded by the pceA gene and abbreviated as PceA dehalogenase) of strain Y51 was purified and characterized. The purified enzyme catalyzed the reductive dechlorination of PCE to cis-DCE at a specific activity of 113.6 nmol min⁻¹  mg protein⁻¹ . The apparent K _m values for PCE and TCE were 105.7 and 535.3 μM, respectively. In addition to PCE and TCE, the enzyme exhibited dechlorination activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane and 1,1,2,2-tetrachloroethane. An 8.4-kb DNA fragment cloned from the Y51 genome revealed eight open reading frames, including the pceAB genes. Immunoblot analysis revealed that PceA dehalogenase is localized in the periplasm of Y51 cells. Production of PceA dehalogenase was induced upon addition of TCE. Significant growth inhibition of strain Y51 was observed in the presence of cis-DCE, More interestingly, the pce gene cluster was deleted with high frequency when the cells were grown with cis-DCE.

     

Response of 1,2-dichloroethane-adapted microbial communities to ex-situ biostimulation of polluted groundwater

The microbial community of a groundwater system contaminated by 1,2-dichloroethane (1,2-DCA), a toxic and persistent chlorinated hydrocarbon, has been investigated for its response to biostimulation finalized to 1,2-DCA removal by reductive dehalogenation. The microbial population profile of samples from different wells in the aquifer and from microcosms enriched in the laboratory with different organic electron donors was analyzed by ARISA (Amplified Ribosomal Intergenic Spacer Analysis) and DGGE (Denaturing Gradient Gel Electrophoresis) of 16S rRNA genes. 1,2-DCA was completely removed with release of ethene from most of the microcosms supplemented with lactate, acetate plus formate, while cheese whey supported 1,2-DCA dehalogenation only after a lag period. Microbial species richness deduced from ARISA profiles of the microbial community before and after electron donor amendments indicated that the response of the community to biostimulation was heterogeneous and depended on the well from which groundwater was sampled. Sequencing of 16S rRNA genes separated by DGGE indicated the presence of bacteria previously associated with soils and groundwater polluted by halogenated hydrocarbons or present in consortia active in the removal of these compounds. A PCR assay specific for Desulfitobacterium sp. showed the enrichment of this genus in some of the microcosms. The dehalogenation potential of the microbial community was confirmed by the amplification of dehalogenase-related sequences from the most active microcosms. Cloning and sequencing of PCR products indicated the presence in the metagenome of the bacterial community of a new dehalogenase potentially involved in 1,2-DCA reductive dechlorination.     

Distribution of Dehalococcoidia in the Anaerobic Deep Water of a Remote Meromictic Crater Lake and Detection of Dehalococcoidia-Derived Reductive Dehalogenase Homologous Genes

Here we describe the natural occurrence of bacteria of the class Dehalococcoidia (DEH) and their diversity at different depths in anoxic waters of a remote meromictic lake (Lake Pavin) using 16S rRNA gene amplicon sequencing and quantitative PCR. Detected DEH are phylogenetically diverse and the majority of 16S rRNA sequences have less than 91% similarity to previously isolated DEH 16S rRNA sequences. To predict the metabolic potential of detected DEH subgroups and to assess if they encode genes to transform halogenated compounds, we enriched DEH-affiliated genomic DNA by using a specific-gene capture method and probes against DEH-derived 16S rRNA genes, reductive dehalogenase genes and known insertion sequences. Two reductive dehalogenase homologous sequences were identified from DEH-enriched genomic DNA, and marker genes in the direct vicinity confirm that gene fragments were derived from DEH. The low sequence similarity with known reductive dehalogenase genes suggests yet-unknown catabolic potential in the anoxic zone of Lake Pavin.     

The little bacteria that can – diversity,genomics and ecophysiology of ‘Dehalococcoides’ spp. in contaminated environments

The fate and persistence of chlorinated organics in the environment have been a concern for the past 50 years. Industrialization and extensive agricultural activities have led to the accumulation of these pollutants in the environment, while their adverse impact on various ecosystems and human health also became evident. This review provides an update on the current knowledge of specialized anaerobic bacteria, namely ‘Dehalococcoides’ spp., which are dedicated to the transformation of various chlorinated organic compounds via reductive dechlorination. Advances in microbiology and molecular techniques shed light into the diversity and functioning of Dehalococcoides spp. in several different locations. Recent genome sequencing projects revealed a large number of genes that are potentially involved in reductive dechlorination. Molecular approaches towards analysis of diversity and expression especially of reductive dehalogenase-encoding genes are providing a growing body of knowledge on biodegradative pathways active in defined pure and mixed cultures as well as directly in the environment. Moreover, several successful field cases of bioremediation strengthen the notion of dedicated degraders such as Dehalococcoides spp. as key players in the restoration of contaminated environments.     

Microbial transformation of chlorinated benzoates

Chlorinated benzoates enter the environment through their use as herbicides or as metabolites of other halogenated compounds. Ample evidence is available indicating biodegradation of chlorinated benzoates to CO₂ and chloride in the environment under aerobic as well as anaerobic conditions. Under aerobic conditions, lower chlorinated benzoates can serve as sole electron and carbon sources supporting growth of a large list of taxonomically diverse bacterial strains. These bacteria utilize a variety of pathways ranging from those involving an initial degradative attack by dioxygenases to those initiated by hydrolytic dehalogenases. In addition to monochlorinated benzoates, several bacterial strains have been isolated that can grow on dichloro-, and trichloro- isomers of chlorobenzoates. Some aerobic bacteria are capable of cometabolizing chlorinated benzoates with simple primary substrates such as benzoate. Under anaerobic conditions, chlorinated benzoates are subject to reductive dechlorination when suitable electron-donating substrates are available. Several halorespiring bacteria are known which can use chlorobenzoates as electron acceptors to support growth. For example, Desulfomonile tiedjei catalyzes the reductive dechlorination of 3-chlorobenzoate to benzoate. The benzoate skeleton is mineralized by other microorganisms in the anaerobic environment. Various dichloro- and trichlorobenzoates are also known to be dechlorinated in anaerobic sediments.     

Reductive dechlorination of tetrachloroethene by a stepwise catalysis of different organohalide respiring bacteria and reductive dehalogenases

The enrichment culture SL2 dechlorinating tetrachloroethene (PCE) to ethene with strong trichloroethene (TCE) accumulation prior to cis-1,2-dichloroethene (cis-DCE) formation was analyzed for the presence of organohalide respiring bacteria and reductive dehalogenase genes (rdhA). Sulfurospirillum-affiliated bacteria were identified to be involved in PCE dechlorination to cis-DCE whereas “Dehalococcoides”-affiliated bacteria mainly dechlorinated cis-DCE to ethene. Two rdhA genes highly similar to tetrachloroethene reductive dehalogenase genes (pceA) of S. multivorans and S. halorespirans were present as well as an rdhA gene very similar to the trichloroethene reductive dehalogenase gene (tceA) of “Dehalococcoides ethenogenes” strain 195. A single strand conformation polymorphism (SSCP) method was developed allowing the simultaneous detection of the three rdhA genes and the estimation of their abundance. SSCP analysis of different SL2 cultures showed that one pceA gene was expressed during PCE dechlorination whereas the second was expressed during TCE dechlorination. The tceA gene was involved in cis-DCE dechlorination to ethene. Analysis of the internal transcribed spacer region between the 16S and 23S rRNA genes revealed two distinct sequences originating from Sulfurospirillum suggesting that two Sulfurospirillum populations were present in SL2. Whether each Sulfurospirillum population was catalyzing a different dechlorination step could however not be elucidated.     

Aerobic biodegradation of 1,2-dichloroethane and 1,3-dichloropropene by bacteria isolated from a pulp mill wastewater effluent in South Africa

Large volumes of chlorinated aliphatic hydrocarbons are produced annually for a variety of industrial and commercial uses. They therefore constitute common contaminants of soil and groundwater causing serious environmental and human health problems. In this study, three bacteria were isolated from a pulp mill wastewater effluent in South Africa by culture enrichment technique and characterized for their ability to degrade 1,2-dichloroethane (1,2-DCE) and 1,3-dichloropropene (1,3-DCP). Specific growth rate constants of the organisms ranged between 0.864∼1.094 and 0.530∼0.585 d⁻¹ in 1.2-DCE and 1,3-DCP, respectively, while the degradation rate constant of the compounds ranged variously between 0.33 and 1.006 d⁻¹, with 1,2-DCE generally better utilized than 1,3-DCP. Gas chromatographic analysis revealed up to 75 and 80% removal of 1,2-DCE and 1,3-DCP, respectively, above that observed in the control bottles. These organisms also demonstrated high haloalkane dehalogenase activities with specific dehalogenase activities ranging between 0.25∼0.31 U (mg protein)⁻¹. Analysis of their 16S rRNA gene sequences revealed that they belong to the generaPaenibacillus, Bacillus, andMicrobacterium.     

Concurrent hexachlorobenzene and chloroethene transformation by endogenous dechlorinating microorganisms in the Ebro River sediment

The ability of Dehalococcoides spp. to reduce chlorinated compounds offers a great potential for bioremediation and/or bioaugmentation of contaminated environments. So far, however, our knowledge of the activity of Dehalococcoides spp. in situ is limited to only a few subsurface environments. The aim of this study was to broaden this knowledge to other environments, and we investigated the role of Dehalococcoides spp. in the transformation of chlorinated benzenes and chlorinated ethenes in the Ebro River (Spain) sediments. Lab-scale batch microcosms were used to follow the growth and abundance of Dehalococcoides spp. during the transformation of selected chlorinated compounds. We applied biomolecular tools targeting the 16S rRNA, the 16S rRNA gene and several functional genes involved in dechlorination in combination with chemical measurements. The growth of Dehalococcoides spp. and the differential expression of several reductive dehalogenase genes during the dechlorination process could be demonstrated. Furthermore, 16S rRNA gene-based clone libraries of dechlorinating river sediment showed a complex community structure and indicated the involvement of several additional bacterial genera in the transformation process, underlining the remarkable potential of this rivers' sediment to transform different halo-organic pollutants.     

Characterisation of Arthrobacter sp. S1 that can degrade α and β-haloalkanoic acids isolated from contaminated soil

A bacterium identified as Arthrobacter sp. S1 by 16S rRNA was isolated from contaminated soil. This is the first reported study that Arthrobacter could utilize both α-halocarboxylix acid (αHA) [2,2-dichloropropionic acid (2,2-DCP) and D,L-2-chloropropionic acid (D,L-2-CP)] and β-halocarboxylix acid (βHA) [3-chloropropionic acid (3CP)] as sole source of carbon with cell doubling times of 5?±?0.2, 7?±?0.1, and 10?±?0.1 h, respectively. More than 85 % chloride ion released was detected in the growth medium suggesting the substrates used were utilized. To identify the presence of dehalogenase gene in the microorganism, a molecular tool that included the use of oligonucleotide primers specific to microorganisms that can grow in halogenated compounds was adapted. A partial putative dehalogenase gene was determined by direct sequencing of the PCR-amplified genomic DNA of the bacterium. A comparative analysis of the deduced amino acid sequence data revealed that the amino acid sequence has a low identity of less than 15 % to both group I and group II dehalogenases, suggesting that the current putative dehalogenase amino acid sequence was completely distinct from both α-haloacids and β-haloacids dehalogenases. This investigation is useful in studying the microbial populations in order to monitor the presence of specific dehalogenase genes and to provide a better understanding of the microbial populations that are present in soil or in water systems treating halogenated compounds.     

Growth of Dehalobacter and Dehalococcoides spp. during Degradation of Chlorinated Ethanes

Mixed anaerobic microbial subcultures enriched from a multilayered aquifer at a former chlorinated solvent disposal facility in West Louisiana were examined to determine the organism(s) involved in the dechlorination of the toxic compounds 1,2-dichloroethane (1,2-DCA) and 1,1,2-trichloroethane (1,1,2-TCA) to ethene. Sequences phylogenetically related to Dehalobacter and Dehalococcoides, two genera of anaerobic bacteria that are known to respire with chlorinated ethenes, were detected through cloning of bacterial 16S rRNA genes. Denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments after starvation and subsequent reamendment of culture with 1,2-DCA showed that the Dehalobacter sp. grew during the dichloroelimination of 1,2-DCA to ethene, implicating this organism in degradation of 1,2-DCA in these cultures. Species-specific real-time quantitative PCR was further used to monitor proliferation of Dehalobacter and Dehalococcoides during the degradation of chlorinated ethanes and showed that in fact both microorganisms grew simultaneously during the degradation of 1,2-DCA. Conversely, Dehalobacter grew during the dichloroelimination of 1,1,2-TCA to vinyl chloride (VC) but not during the subsequent reductive dechlorination of VC to ethene, whereas Dehalococcoides grew only during the reductive dechlorination of VC but not during the dichloroelimination of 1,1,2-TCA. This demonstrated that in mixed cultures containing multiple dechlorinating microorganisms, these organisms can have either competitive or complementary dechlorination activities, depending on the chloro-organic substrate.     

Comparative studies on tetrachloroethene reductive dechlorination mediated by Desulfitobacterium sp. strain PCE-S

Tetrachloroethene reductive dechlorination was studied with cell extracts of a newly isolated, tetrachloroethene-utilizing bacterium, Desulfitobacterium sp. strain PCE-S. Tetrachloroethene dehalogenase mediated the reductive dechlorination of tetrachloroethene and trichloroethene to cis-1,2-dichloroethene with artificial electron donors such as methyl viologen. The chlorinated aromatic compounds tested so far were not reduced. A low-potential electron donor (E ₀′ < –0.4 V) was required for tetrachloroethene reduction. The enzyme in its reduced state was inactivated by propyl iodide and reactivated by light, indicating the involvement of a corrinoid in reductive tetrachloroethene dechlorination. Received: 28 April 1997 / Accepted: 11 July 1997     

Characteristics and DNA-Sequence of a Cryptic Haloalkanoic Acid Dehalogenase from Agrobacterium tumefaciens RS5

Agrobacterium tumefaciens RS5 harbors two different hydrolytic haloalkanoic acid dehalogenase genes, one coding for a nonstereospecific enzyme (DhlS5II) and a second for a cryptic L-isomer-specific dehalogenase (DhlS5I). The latter gene was cloned and expressed in Escherichia coli. Biochemical characterization and sequence analysis of dhlS5I shows its membership to the class of the L-isomer-specific hydrolytic dehalogenases. Highest homology of 72% was found to the dehalogenase LdexYL from Pseudomonas sp. YL. Both enzymes share an unusual high temperature optimum of 65°C. Controlled by a vector promoter, high specific dehalogenase activities up to 32 U mg⁻¹ protein were obtained in E. coli, and putatively, owing to its own σ⁷⁰-dependent promoter, a constitutive low-level expression of dhlS5I of 0.4 U mg⁻¹ protein was measured. Received: 27 June 1997 / Accepted: 4 August 1997     

石油污染环境中固氮和寡氮营养细菌的分离鉴定及其特性

[背景]石油污染治理中的生物修复因无二次污染、处理成本低等优点受到人们的广泛关注,但由于石油烃向环境中大量输入,导致环境中氮源的相对不足成为制约生物修复效率的关键因素之一,因此筛选能够适应寡氮环境的微生物具有重要的生态意义.[目的]从辽河油田油藏水中筛选在不添加氮源培养基中生长的微生物,为石油污染环境生物修复提供候选菌...     

Occurrence and Phylogenetic Diversity of Sphingomonas Strains in Soils Contaminated with Polycyclic Aromatic Hydrocarbons

Bacterial strains of the genus Sphingomonas are often isolated from contaminated soils for their ability to use polycyclic aromatic hydrocarbons (PAH) as the sole source of carbon and energy. The direct detection of Sphingomonas strains in contaminated soils, either indigenous or inoculated, is, as such, of interest for bioremediation purposes. In this study, a culture-independent PCR-based detection method using specific primers targeting the Sphingomonas 16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) was developed to assess Sphingomonas diversity in PAH-contaminated soils. PCR using the new primer pair on a set of template DNAs of different bacterial genera showed that the method was selective for bacteria belonging to the family Sphingomonadaceae. Single-band DGGE profiles were obtained for most Sphingomonas strains tested. Strains belonging to the same species had identical DGGE fingerprints, and in most cases, these fingerprints were typical for one species. Inoculated strains could be detected at a cell concentration of 10⁴ CFU g of soil⁻¹. The analysis of Sphingomonas population structures of several PAH-contaminated soils by the new PCR-DGGE method revealed that soils containing the highest phenanthrene concentrations showed the lowest Sphingomonas diversity. Sequence analysis of cloned PCR products amplified from soil DNA revealed new 16S rRNA gene Sphingomonas sequences significantly different from sequences from known cultivated isolates (i.e., sequences from environmental clones grouped phylogenetically with other environmental clone sequences available on the web and that possibly originated from several potential new species). In conclusion, the newly designed Sphingomonas-specific PCR-DGGE detection technique successfully analyzed the Sphingomonas communities from polluted soils at the species level and revealed different Sphingomonas members not previously detected by culture-dependent detection techniques.     

Multiple Nonidentical Reductive-Dehalogenase-Homologous Genes Are Common in Dehalococcoides

Degenerate primers were used to amplify large fragments of reductive-dehalogenase-homologous (RDH) genes from genomic DNA of two Dehalococcoides populations, the chlorobenzene- and dioxin-dechlorinating strain CBDB1 and the trichloroethene-dechlorinating strain FL2. The amplicons (1,350 to 1,495 bp) corresponded to nearly complete open reading frames of known reductive dehalogenase genes and short fragments (approximately 90 bp) of genes encoding putative membrane-anchoring proteins. Cloning and restriction analysis revealed the presence of at least 14 different RDH genes in each strain. All amplified RDH genes showed sequence similarity with known reductive dehalogenase genes over the whole length of the sequence and shared all characteristics described for reductive dehalogenases. Deduced amino acid sequences of seven RDH genes from strain CBDB1 were 98.5 to 100% identical to seven different RDH genes from strain FL2, suggesting that both strains have an overlapping substrate range. All RDH genes identified in strains CBDB1 and FL2 were related to the RDH genes present in the genomes of Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain BAV1; however, sequence identity did not exceed 94.4 and 93.1%, respectively. The presence of RDH genes in strains CBDB1, FL2, and BAV1 that have no orthologs in strain 195 suggests that these strains possess dechlorination activities not present in strain 195. Comparative sequence analysis identified consensus sequences for cobalamin binding in deduced amino acid sequences of seven RDH genes. In conclusion, this study demonstrates that the presence of multiple nonidentical RDH genes is characteristic of Dehalococcoides strains.     

Comparison of anaerobic microbial communities from Estuarine sediments amended with halogenated compounds to enhance dechlorination of 1,2,3,4-tetrachlorodibenzo-p-dioxin

A suite of experiments were conducted to ascertain whether dehalogenation of a model dioxin compound could be stimulated in marine sediments by supplementation with halogenated analogues to enrich for dehalogenating bacteria and if growth by members of the Chloroflexi-like group was associated with dioxin removal. Five halogenated compounds (tetrachlorobenzene, tetrachloroanisole, tetrachlorophenol, tetrachlorobenzoic acid and trichloroacetophenone) were added with 1,2,3,4-tetrachlorodibenzo-p-dioxin (TeCDD) to estuarine sediments from four sites in San Diego Bay and the coast of southern New Jersey to test for dioxin dehalogenation. Most of the halogenated additives were found to stimulate dechlorination of the model dioxin. Molecular analysis of the bacterial population using 16S rRNA and reductive dehalogenase genes indicated that distinct microbial populations were enriched with each halogenated co-amendment. Additionally, Chloroflexi-like ribosomal genes associated with dehalogenation were detected. For example, quantitative real-time PCR analysis of 16S rRNA and reductive dehalogenase gene copy number in the microcosms showed a positive correlation with 1,2,3,4-TeCDD reductive dechlorination in coastal sediments amended with different halogenated additives. These results suggest that specific Chloroflexi-like microorganisms related to Dehalococcoides are involved in 1,2,3,4-TeCDD reductive dechlorination.