Impact of soil physicochemical factors and heavy metals on co-occurrence pattern of bacterial in rural simple garbage dumping site.

Rural waste accumulation leads to heavy metal soil pollution, impacting microbial communities. However, knowledge gaps exist regarding the distribution and occurrence patterns of bacterial communities in multi-metal contaminated soil profiles. In this study, high-throughput 16 S rRNA gene sequencing technology was used to explore the response of soil bacterial communities to various heavy metal pollution in rural simple waste dumps in karst areas of Southwest China. The study selected three habitats in the center, edge, and uncontaminated areas of the waste dump to evaluate the main factors driving the change in bacterial community composition. Pollution indices reveal severe contamination across all elements, except for moderately polluted lead (Pb); contamination severity ranks as follows: Mn > Cd > Zn > Cr > Sb > V > Cu > As > Pb. Proteobacteria, Actinobacteria, Chloroflexi, and Acidobacteriota predominate, collectively constituting over 60% of the relative abundance. Analysis of Chao and Shannon indices demonstrated that the waste dump center boasted the greatest bacterial richness and diversity. Correlation data indicated a predominant synergistic interaction among the landfill's bacterial community, with a higher number of positive associations (76.4%) compared to negative ones (26.3%). Network complexity was minimal at the dump's edge. RDA analysis showed that Pb(explained:46%) and Mn(explained:21%) were the key factors causing the difference in bacterial community composition in the edge area of the waste dump, and AK(explained:42.1%) and Cd(explained:35.2%) were the key factors in the center of the waste dump. This study provides important information for understanding the distribution patterns, co-occurrence networks, and environmental response mechanisms of bacterial communities in landfill soils under heavy metal stress, which helps guide the formulation of rural waste treatment and soil remediation strategies.

Response of Isovalerate-Degrading Methanogenic Microbial Community to Inhibitors.

Isovalerate is one of the key intermediates during anaerobic digestion treating protein-containing waste/wastewater. Investigating the effect of different kinds of inhibitors on isovalerate-degrading microbial community is necessary to develop measures for improving the effectiveness of the treatment plants. In the present study, dynamic changes in the isovalerate-degrading microbial community in presence of inhibitors (ammonium, sulfide, mixed ammonium and sulfide, and chlortetracycline (CTC)) were investigated using high-throughput sequencing of 16S rRNA gene. Our observations showed that the isovalerate-degrading microbial community responded differently to different inhibitors and that the isovalerate degradation and gas production were strongly repressed by each inhibitor. We found that sulfide inhibited both isovalerate oxidation followed by methanogenesis, while ammonium, mixed ammonium and sulfide, and CTC mainly inhibited isovalerate oxidation. Genera classified into Proteobacteria and Chloroflexi were less sensitive to inhibitors. The two dominant genera, which are potential syntrophic isovalerate oxidizers, exhibited different responses to inhibitors that the unclassified_Peptococcaceae_3 was more sensitive to inhibitors than the unclassified_Syntrophaceae. Upon comparison to acetoclastic methanogen Methanosaeta, hydrogenotrophic methanogens Methanoculleus and Methanobacterium were less sensitive to inhibitors.

Antibiotic resistance and microbiota in the gut of Chinese four major freshwater carp from retail markets.

Fish-associated antibiotic resistance genes (ARGs) have attracted increasing attention due to their potential risks to human beings via the food chain. However, data are scarce regarding the antibiotic resistance in fish themselves. Herein, the antibiotic resistance genes (ARGs) were assessed in the gut of four major Chinese freshwater carp (i.e., silver carp, grass carp, bighead carp, and crucian carp) from food retail markets. Results show that the abundances of target ARGs (e.g., tetA, tetO, tetQ, tetW, sulI, sulII, and blaTEM-1) and class 1 integrase (intI1) were in the range 9.4 × 10-6 - 1.6 × 10-1 and 6.7 × 10-5 - 5.2 × 10-2 gene copies per 16S rRNA gene, respectively. The sulI, sulII, and tetQ strongly correlated with silver and mercury resistance genes (e.g., silE and merR). The microbial taxa of fish gut could be partly separated among retail markets based on the PCA analysis. About 15.0% of the OTUs in fish gut were shared and 74.5% of the shared OTUs were identified as Acidobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Firmicutes, and Proteobacteria. These phyla may constitute the core microbiota in the guts of the four Chinese freshwater carp. The possible ARG hosts were revealed based on the network analysis, and the presence of pathogen-associated resistant genera in fish gut highlights the need to fully understand their potential human health risks.

Growth of Dehalococcoides mccartyi species in an autotrophic consortium producing limited acetate.

The dechlorinating Dehalococcoides mccartyi species requires acetate as carbon source, but little is known on its growth under acetate limiting conditions. In this study, we observed growth and dechlorination of a D. mccartyi-containing mixed consortium in a fixed-carbon-free medium with trichloroethene in the aqueous phase and H2/CO2 in the headspace. Around 4 mM formate was produced by day 40, while acetate was constantly below 0.05 mM. Microbial community analysis of the consortium revealed dominance by D. mccartyi and Desulfovibrio sp. (57 and 22% 16S rRNA gene copies, respectively). From this consortium, Desulfovibrio sp. strain F1 was isolated and found to produce formate and acetate (1.2 mM and 48 µM, respectively, by day 24) when cultivated alone in the above mentioned medium without trichloroethene. An established co-culture of strain F1 and D. mccartyi strain 195 demonstrated that strain 195 could grow and dechlorinate using acetate produced by strain F1; and that acetate was constantly below 25 µM in the co-culture. To verify that such low level of acetate is utilizable by D. mccartyi, we cultivated strain 195 alone under acetate-limiting conditions and found that strain 195 consumed acetate to below detection (5 µM). Based on the acetate consumption and cell yield of D. mccartyi, we estimated that on average 1.2 × 108 acetate molecules are needed to supply carbon for one D. mccartyi cell. Our study suggests that Desulfovibrio may supply a steady but low amount of fixed carbon to dechlorinating bacteria, exhibiting important implications for natural bio-attenuation when fixed carbon is limited.

Bacterial community changes in response to oil contamination and perennial crop cultivation.

We investigated bacterial community dynamics in response to used motor oil contamination and perennial crop cultivation by 16S rRNA gene amplicon sequencing in a 4-year field study. Actinobacteria, Proteobacteria, Chloroflexi, Acidobacteria, and Gemmatimonadetes were the major bacterial phyla, and Rhodococcus was the most abundant genus. Initially, oil contamination decreased the overall bacterial diversity. Actinobacteria, Betaproteobacteria, and Gammaproteobacteria were sensitive to oil contamination, exhibiting clear succession with time. However, bacterial communities changed over time, regardless of oil contamination and crop cultivation. The abundance difference of most OTUs between oil-contaminated and non-contaminated plots remained the same in later sampling years after the initial abundance difference induced by oil spike. The abundances of three oil-favored actinobacteria (Lysinimonas, Microbacteriaceae, and Marmoricola) and one betaproteobacterium (Aquabacterium) changed in different manner over time in oil-contaminated and non-contaminated soil. We propose that these taxa are potential bio-indicators for monitoring recovery from motor oil contamination in boreal soil. The effect of crop cultivation on bacterial communities became significant only after the crops achieved stable growth, likely associated with plant material decomposition by Bacteroidetes, Armatimonadetes and Fibrobacteres.

Effects of Sulfate Reduction on Trichloroethene Dechlorination by Dehalococcoides-Containing Microbial Communities.

In order to elucidate interactions between sulfate reduction and dechlorination, we systematically evaluated the effects of different concentrations of sulfate and sulfide on reductive dechlorination by isolates, constructed consortia, and enrichments containing Dehalococcoides sp. Sulfate (up to 5 mM) did not inhibit the growth or metabolism of pure cultures of the dechlorinator Dehalococcoides mccartyi 195, the sulfate reducer Desulfovibrio vulgaris Hildenborough, or the syntroph Syntrophomonas wolfei In contrast, sulfide at 5 mM exhibited inhibitory effects on growth of the sulfate reducer and the syntroph, as well as on both dechlorination and growth rates of D. mccartyi Transcriptomic analysis of D. mccartyi 195 revealed that genes encoding ATP synthase, biosynthesis, and Hym hydrogenase were downregulated during sulfide inhibition, whereas genes encoding metal-containing enzymes involved in energy metabolism were upregulated even though the activity of those enzymes (hydrogenases) was inhibited. When the electron acceptor (trichloroethene) was limiting and an electron donor (lactate) was provided in excess to cocultures and enrichments, high sulfate concentrations (5 mM) inhibited reductive dechlorination due to the toxicity of generated sulfide. The initial cell ratio of sulfate reducers to D. mccartyi (1:3, 1:1, or 3:1) did not affect the dechlorination performance in the presence of sulfate (2 and 5 mM). In contrast, under electron donor limitation, dechlorination was not affected by sulfate amendments due to low sulfide production, demonstrating that D. mccartyi can function effectively in anaerobic microbial communities containing moderate sulfate concentrations (5 mM), likely due to its ability to outcompete other hydrogen-consuming bacteria and archaea.IMPORTANCE Sulfate is common in subsurface environments and has been reported as a cocontaminant with chlorinated solvents at various concentrations. Inconsistent results for the effects of sulfate inhibition on the performance of dechlorination enrichment cultures have been reported in the literature. These inconsistent findings make it difficult to understand potential mechanisms of sulfate inhibition and complicate the interpretation of bioremediation field data. In order to elucidate interactions between sulfate reduction and reductive dechlorination, this study systematically evaluated the effects of different concentrations of sulfate and sulfide on reductive dechlorination by isolates, constructed consortia, and enrichments containing Dehalococcoides sp. This study provides a more fundamental understanding of the competition mechanisms between reductive dechlorination by Dehalococcoides mccartyi and sulfate reduction during the bioremediation process. It also provides insights on the significance of sulfate concentrations on reductive dechlorination under electron donor/acceptor-limiting conditions during in situ bioremediation applications. For example, at a trichloroethene-contaminated site with a high sulfate concentration, proper slow-releasing electron donors can be selected to generate an electron donor-limiting environment that favors reductive dechlorination and minimizes the sulfide inhibition effect.

Metagenomic and Metatranscriptomic Analyses Reveal the Structure and Dynamics of a Dechlorinating Community Containing Dehalococcoides mccartyi and Corrinoid-Providing Microorganisms under Cobalamin-Limited Conditions.

The aim of this study is to obtain a systems-level understanding of the interactions between Dehalococcoides and corrinoid-supplying microorganisms by analyzing community structures and functional compositions, activities, and dynamics in trichloroethene (TCE)-dechlorinating enrichments. Metagenomes and metatranscriptomes of the dechlorinating enrichments with and without exogenous cobalamin were compared. Seven putative draft genomes were binned from the metagenomes. At an early stage (2 days), more transcripts of genes in the Veillonellaceae bin-genome were detected in the metatranscriptome of the enrichment without exogenous cobalamin than in the one with the addition of cobalamin. Among these genes, sporulation-related genes exhibited the highest differential expression when cobalamin was not added, suggesting a possible release route of corrinoids from corrinoid producers. Other differentially expressed genes include those involved in energy conservation and nutrient transport (including cobalt transport). The most highly expressed corrinoid de novo biosynthesis pathway was also assigned to the Veillonellaceae bin-genome. Targeted quantitative PCR (qPCR) analyses confirmed higher transcript abundances of those corrinoid biosynthesis genes in the enrichment without exogenous cobalamin than in the enrichment with cobalamin. Furthermore, the corrinoid salvaging and modification pathway of Dehalococcoides was upregulated in response to the cobalamin stress. This study provides important insights into the microbial interactions and roles played by members of dechlorinating communities under cobalamin-limited conditions.IMPORTANCE The key chloroethene-dechlorinating bacterium Dehalococcoides mccartyi is a cobalamin auxotroph, thus acquiring corrinoids from other community members. Therefore, it is important to investigate the microbe-microbe interactions between Dehalococcoides and the corrinoid-providing microorganisms in a community. This study provides systems-level information, i.e., taxonomic and functional compositions and dynamics of the supportive microorganisms in dechlorinating communities under different cobalamin conditions. The findings shed light on the important roles of Veillonellaceae species in the communities compared to other coexisting community members in producing and providing corrinoids for Dehalococcoides species under cobalamin-limited conditions.

Changes in the Bacterial Community Structure of Remediated Anthracene-Contaminated Soils.

Mixing soil or adding earthworms (Eisenia fetida (Savigny, 1826)) accelerated the removal of anthracene, a polycyclic aromatic hydrocarbon, from a pasture and an arable soil, while a non-ionic surfactant (Surfynol® 485) inhibited the removal of the contaminant compared to the untreated soil. It was unclear if the treatments affected the soil bacterial community and consequently the removal of anthracene. Therefore, the bacterial community structure was monitored by means of 454 pyrosequencing of the 16S rRNA gene in the pasture and arable soil mixed weekly, amended with Surfynol® 485, E. fetida or organic material that served as food for the earthworms for 56 days. In both soils, the removal of anthracene was in the order: mixing soil weekly (100%) > earthworms applied (92%) > organic material applied (77%) > untreated soil (57%) > surfactant applied (34%) after 56 days. There was no clear link between removal of anthracene from soil and changes in the bacterial community structure. On the one hand, application of earthworms removed most of the contaminant from the arable soil and had a strong effect on the bacterial community structure, i.e. a decrease in the relative abundance of the Acidobacteria, Chloroflexi and Gemmatimonadetes, and an increase in that of the Proteobacteria compared to the unamended soil. Mixing the soil weekly removed all anthracene from the arable soil, but had little or no effect on the bacterial community structure. On the other hand, application of the surfactant inhibited the removal of anthracene from the arable soil compared to the untreated soil, but had a strong effect on the bacterial community structure, i.e. a decrease in the relative abundance of Cytophagia (Bacteroidetes), Chloroflexi, Gemmatimonadetes and Planctomycetes and an increase in that of the Flavobacteria (Bacteroidetes) and Proteobacteria. Additionally, the removal of anthracene was similar in the different treatments of both the arable and pasture soil, but the effect of application of carrot residue, earthworms or the surfactant on the bacterial community structure was more accentuated in the arable soil than in the pasture soil. It was found that removal of anthracene was not linked to changes in the bacterial community structure.

Control of Microthrix parvicella by aluminium salts addition.

Aluminium and iron chloride were added to a biological nutrient removal pilot plant (1,500 population equivalent) treating urban wastewater to investigate the control of Microthrix parvicella bulking and foaming by metallic salts. Monitoring plant performance over two 6-month periods showed a slight impact on the removal efficiencies. Addition of metallic salts (Me; aluminium or aluminium + iron) at a concentration of 41 mmol Me(kg MLSS·d) (MLSS: mixed liquor suspended solids) over 70 days allowed a stabilization of the diluted sludge volume index (DSVI), whereas higher dosages (94 mmol Me(kg MLSS·d) over 35 days or 137 mmol Me(kg MLSS·d) over 14 days induced a significant improvement of the settling conditions. Microscopic observations showed a compaction of biological aggregates with an embedding of filamentous bacteria into the flocs that is not specific to M. parvicella as bacteria from phylum Chloroflexi are embedded too. The quantitative polymerase chain reaction targeting M. parvicella further indicated a possible growth limitation in addition to the flocculation impact at the high dosages of metallic salts investigated. DSVI appeared to be correlated with the relative abundance of M. parvicella.

Stable Carbon Isotope Fractionation During 1,2-Dichloropropane-to-Propene Transformation by an Enrichment Culture Containing Dehalogenimonas Strains and a dcpA Gene.

A stable enrichment culture derived from Besòs river estuary sediments stoichiometrically dechlorinated 1,2-dichloropropane (1,2-DCP) to propene. Sequential transfers in defined anaerobic medium with the inhibitor bromoethanesulfonate produced a sediment-free culture dechlorinating 1,2-DCP in the absence of methanogenesis. Application of previously published genus-specific primers targeting 16S rRNA gene sequences revealed the presence of a Dehalogenimonas strain, and no amplification was obtained with Dehalococcoides-specific primers. The partial sequence of the 16S rRNA amplicon was 100% identical with Dehalogenimonas alkenigignens strain IP3-3. Also, dcpA, a gene described to encode a corrinoid-containing 1,2-DCP reductive dehalogenase was detected. Resistance of the dehalogenating activity to vancomycin, exclusive conversion of vicinally chlorinated alkanes, and tolerance to short-term oxygen exposure is consistent with the hypothesis that a Dehalogenimonas strain is responsible for 1,2-DCP conversion in the culture. Quantitative PCR showed a positive correlation between the number of Dehalogenimonas 16S rRNA genes copies in the culture and consumption of 1,2-DCP. Compound specific isotope analysis revealed that the Dehalogenimonas-catalyzed carbon isotopic fractionation (εC(bulk)) of the 1,2-DCP-to-propene reaction was -15.0 ± 0.7‰ under both methanogenic and nonmethanogenic conditions. This study demonstrates that carbon isotope fractionation is a valuable approach for monitoring in situ 1,2-DCP reductive dechlorination by Dehalogenimonas strains.

Quantitative and functional dynamics of Dehalococcoides spp. and its tceA and vcrA genes under TCE exposure.

This study aimed at monitoring the dynamics of phylogenetic and catabolic genes of a dechlorinating enrichment culture before, during, and after complete dechlorination of chlorinated compounds. More specifically, the effect of 40 µM trichloroethene (TCE) and 5.6 mM lactate on the gene abundance and activity of an enrichment culture was investigated for 40 days. Although tceA and vcrA gene copy numbers were relatively stable in DNA extracts over time, tceA and vcrA mRNA abundances were upregulated from undetectable levels to 2.96 × and 6.33 × 104 transcripts/mL, respectively, only after exposure to TCE and lactate. While tceA gene transcripts decreased over time with TCE dechlorination, the vcrA gene was expressed steadily even when the concentration of vinyl chloride was at undetectable levels. In addition, ratios between catabolic and phylogenetic genes indicated that tceA and vcrA gene carrying organisms dechlorinated TCE and its produced daughter products, while vcrA gene was mainly responsible for the dechlorination of the lower VC concentrations in a later stage of degradation.

Effects of bioaugmentation on enhanced reductive dechlorination of 1,1,1-trichloroethane in groundwater: a comparison of three sites.

Microcosm studies investigated the effects of bioaugmentation with a mixed Dehalococcoides (Dhc)/Dehalobacter (Dhb) culture on biological enhanced reductive dechlorination for treatment of 1,1,1-trichloroethane (TCA) and chloroethenes in groundwater at three Danish sites. Microcosms were amended with lactate as electron donor and monitored over 600 days. Experimental variables included bioaugmentation, TCA concentration, and presence/absence of chloroethenes. Bioaugmented microcosms received a mixture of the Dhc culture KB-1 and Dhb culture ACT-3. To investigate effects of substrate concentration, microcosms were amended with various concentrations of chloroethanes (TCA or monochloroethane [CA]) and/or chloroethenes (tetrachloroethene [PCE], trichloroethene [TCE], or 1,1-dichloroethene [1,1-DCE]). Results showed that combined electron donor addition and bioaugmentation stimulated dechlorination of TCA and 1,1-dichloroethane (1,1-DCA) to CA, and dechlorination of PCE, TCE, 1,1-DCE and cDCE to ethane. Dechlorination of CA was not observed. Bioaugmentation improved the rate and extent of TCA and 1,1-DCA dechlorination at two sites, but did not accelerate dechlorination at a third site where geochemical conditions were reducing and Dhc and Dhb were indigenous. TCA at initial concentrations of 5 mg/L inhibited (i.e., slowed the rate of) TCA dechlorination, TCE dechlorination, donor fermentation, and methanogenesis. 1 mg/L TCA did not inhibit dechlorination of TCA, TCE or cDCE. Moreover, complete dechlorination of PCE to ethene was observed in the presence of 3.2 mg/L TCA. In contrast to some prior reports, these studies indicate that low part-per million levels of TCA (< 3 mg/L) in aquifer systems do not inhibit dechlorination of PCE or TCE to ethene. In addition, the results show that co-bioaugmentation with Dhc and Dhb cultures can be an effective strategy for accelerating treatment of chloroethane/chloroethene mixtures in groundwater, with the exception that all currently known Dhc and Dhb cultures cannot treat CA.

Comparison of targeted peptide quantification assays for reductive dehalogenases by selective reaction monitoring (SRM) and precursor reaction monitoring (PRM).

Targeted absolute protein quantification yields valuable information about physiological adaptation of organisms and is thereby of high interest. Especially for this purpose, two proteomic mass spectrometry-based techniques namely selective reaction monitoring (SRM) and precursor reaction monitoring (PRM) are commonly applied. The objective of this study was to establish an optimal quantification assay for proteins with the focus on those involved in housekeeping functions and putative reductive dehalogenase proteins from the strictly anaerobic bacterium Dehalococcoides mccartyi strain CBDB1. This microbe is small and slow-growing; hence, it provides little biomass for comprehensive proteomic analysis. We therefore compared SRM and PRM techniques. Eleven peptides were successfully quantified by both methods. In addition, six peptides were solely quantified by SRM and four by PRM, respectively. Peptides were spiked into a background of Escherichia coli lysate and the majority of peptides were quantifiable down to 500 amol absolute on column by both methods. Peptide quantification in CBDB1 lysate resulted in the detection of 15 peptides using SRM and 14 peptides with the PRM assay. Resulting quantification of five dehalogenases revealed copy numbers of <10 to 115 protein molecules per cell indicating clear differences in abundance of RdhA proteins during growth on hexachlorobenzene. Our results indicated that both methods show comparable sensitivity and that the combination of the mass spectrometry assays resulted in higher peptide coverage and thus more reliable protein quantification.

Microbial dechlorination activity during and after chemical oxidant treatment.

Potassium permanganate (PM) and sodium persulfate (PS) are used in soil remediation, however, their compatibility with a coinciding or subsequent biotreatment is poorly understood. In this study, different concentrations of PM (0.005-2g/L) and PS (0.01-4.52 g/L) were applied and their effects on the abundance, activity, and reactivation potential of a dechlorinating enrichment culture were investigated. Expression of the tceA, vcrA and 16S rRNA genes of Dehalococcoides spp. were detected at 0.005-0.01 g/L PM and 0.01-0.02 g/L PS. However, with 0.5-2g/L PM and 1.13-4.52 g/L PS no gene expression was recorded, neither were indicator molecules for total cell activity (Adenosine triphosphate, ATP) detected. Dilution did not promote the reactivation of the microbial cells when the redox potential was above -100 mV. Similarly, inoculated cells did not dechlorinate trichloroethene (TCE) above -100 mV. When the redox potential was decreased to -300 mV and the reactors were bioaugmented for a second time, dechlorination activity recovered, but only in the reactors with 1.13 and 2.26 g/L PS. In conclusion, our results show that chemical oxidants can be combined with a biotreatment at concentrations below 0.5 g/L PM and 1g/L PS.

Triclosan enriches for Dehalococcoides-like Chloroflexi in anaerobic soil at environmentally relevant concentrations.

Triclosan is an antimicrobial agent that is discharged to soils with land-applied wastewater biosolids, is persistent under anaerobic conditions, and yet its impact on anaerobic microbial communities in soils is largely unknown. We hypothesized that triclosan enriches for Dehalococcoides-like Chloroflexi because these bacteria respire organochlorides and are likely less sensitive, relative to other bacteria, to the antimicrobial effects of triclosan. Triplicate anaerobic soil microcosms were seeded with agricultural soil, which was not previously exposed to triclosan, and were amended with 1 mg kg(-1) of triclosan. Triplicate control microcosms did not receive triclosan, and the experiment was run for 618 days. The overall bacterial community (assessed by automated ribosomal intergenic spacer analysis and denaturing gradient gel electrophoresis) was not impacted by triclosan; however, the abundance of Dehalococcoides-like Chloroflexi 16S rRNA genes (determined by qPCR) increased 20-fold with triclosan amendment compared with a fivefold increase without triclosan. This work demonstrates that triclosan impacts anaerobic soil communities at environmentally relevant levels.

Regulation of reductive dehalogenase gene transcription in Dehalococcoides mccartyi.

The remarkable capacity of the genus Dehalococcoides to dechlorinate a multitude of different chlorinated organic compounds reflects the number and diversity of genes in the genomes of Dehalococcoides species encoding reductive dehalogenase homologues (rdh). Most of these genes are located in the vicinity of genes encoding multiple antibiotic resistance regulator (MarR)-type or two-component system regulators. Here, the transcriptional response of rdhA genes (coding for the catalytic subunit) to 2,3- and 1,3-dichlorodibenzo-p-dioxin (DCDD) was studied in Dehalococcoides mccartyi strain CBDB1. Almost all rdhA genes were transcribed in the presence of 2,3-DCDD, albeit at different levels as shown for the transcripts of cbrA, cbdbA1453, cbdbA1624 and cbdbA1588. By contrast, 1,3-DCDD did not induce rdhA transcription. The putative MarR CbdbA1625 was heterologously produced and its ability to bind in vitro to the overlapping promoter regions of the genes cbdbA1624 and cbdbA1625 was demonstrated. To analyse regulation in vivo, single-copy transcriptional promoter-lacZ fusions of different rdhA genes and of cbdbA1625 were constructed and introduced into the heterologous host Escherichia coli, and expression levels of the fusions were measured. The cbdbA1625 gene was cloned into a vector allowing a regulation of expression by arabinose and it was transformed into the strains containing the rdh-promoter-lacZ fusion derivatives. CbdbA1625 was shown to downregulate transcription from its own promoter resulting in a 40-50% reduction in the ß-galactosidase activity, giving the first hint that it acts as a repressor.

Compounded effects of chlorinated ethene inhibition on ecological interactions and population abundance in a Dehalococcoides - Dehalobacter coculture.

The development of rational and effective engineered bioremediation approaches for sites contaminated with chlorinated solvents requires a fundamental understanding of the factors limiting the in situ activity of dehalorespiring bacteria. Frequently, multiple dehalorespiring bacteria are present at contaminated sites, particularly when bioaugmentation is applied. The ecological interactions between different dehalorespiring populations can-along with hydrodynamic and other environmental factors-affect their activity and thus the rates and extent of dehalorespiration. An integrated experimental and modeling approach was used to evaluate the ecological interactions between two hydrogenotrophic, dehalorespiring strains. A dual Monod model of dehalorespiration provided a good fit to the chlorinated ethene concentrations measured in a coculture of Dehalococcoides mccartyi 195 and Dehalobacter restrictus growing on tetrachloroethene (PCE) and excess H(2) in a continuous-flow reactor. Inhibition of dehalorespiration by chlorinated ethenes was previously observed in cultures containing Dehalococcoides or Dehalobacter strains. Therefore, inhibition coefficients were estimated for Dhc. mccartyi 195 and Dhb. restrictus. The inhibition effects of PCE and TCE on VC dechlorination by Dhc. mccartyi 195, and of VC on PCE and TCE dechlorination by Dhb. restrictus, were compounded when these strains were grown in coculture, and dehalorespiring population abundance and survival could be accurately predicted only by incorporating these complex interactions into the dual Monod model.

Effect of chloroethene concentrations and granular activated carbon on reductive dechlorination rates and growth of Dehalococcoides spp.

This study focused on the investigation of (i) the tetrachloroethene (PCE) toxicity threshold of a reductively dechlorinating mixed culture containing Dehalococcoides spp., (ii) the adsorption of PCE on different types of granular activated carbon (GAC), and (iii) the bioavailability and reductive dechlorination in the presence of GAC. The abundance of Dehalococcoides spp. detected by quantitative real-time polymerase chain reaction (qPCR) was found to increase by 2-4 orders of magnitude during degradation of PCE. No degradation occurred at dissolved concentrations beyond 420 µM (70 mg/L). Different adsorption isotherms were determined for thermally and chemically activated carbons. The addition of GAC to biological assays reduced the dissolved PCE concentration below the toxicity threshold. The combination of microbial reductive dechlorination with GAC adsorption proved to be a promising method for remediation of groundwater contaminated by high concentrations of chloroethenes.

Effect of coplanar PCB concentration on dechlorinating microbial communities and dechlorination in estuarine sediments.

The effect of concentration of coplanar PCB on the dechlorinating microbial community and dechlorination were investigated in anoxic estuarine sediment collected from Er-Jen River and enriched with 10 and 50 mg L⁻¹ of 3,4,4',5-tetrachlorobiphenyl, 3,3',4,4',5-pentachlorobiphenyl, and 3,3',4,4',5,5'-hexachlorobipheny. Dechlorination rates were similar in the cultures enriched with 10 and 50 mg L⁻¹ of 3,4,4',5-tetrachlorobiphenyl, whereas significantly higher dechlorination rates were observed in cultures enriched with 10 mg L⁻¹ of 3,3',4,4',5-pentachlorobiphenyl. No dechlorination was observed in sediment slurries enriched with 3,3',4,4',5,5'-hexachlorobipheny. Para dechlorination occurred prior to meta dechlorination during reductive dechlorination of 3,4,4',5-tetrachlorobiphenyl and 3,3',4,4',5-pentachlorobiphenyl. GC-MS and denaturing gradient gel electrophoresis (DGGE) were used to detect dechlorination products and dechlorinating microorganisms in the enriched sediment cultures during the process of degradation. Two Chloroflexi phylotypes observed in DGGE were responsible for para and meta dechlorination respectively. Phylotype Cp-1 has 98% similarity to uncultured bacterium N5-12. Phylotype Cm-1 has 99% similarity to uncultured dechlorinating bacterium m1 or SF1 belonging to the ο-17/DF-1 group of PCB-dechlorinating bacteria.

Trichloroethene and cis-1,2-dichloroethene concentration-dependent toxicity model simulates anaerobic dechlorination at high concentrations. II: continuous flow and attached growth reactors.

A model that was used to describe toxicity from high concentrations of chlorinated aliphatic hydrocarbons (CAHs) on reductively dechlorinating cultures in batch reactors (Sabalowsky and Semprini (in press)) was extended here to simulate observations in continuous flow suspended and attached growth reactors. The reductively dechlorinating anaerobic Evanite subculture (EV-cDCE) was fed trichloroethene (TCE) and excess electron donor to accumulate cis-1,2-dichloroethene (cDCE) in a continuous flow stirred tank reactor (CFSTR); and an attached growth recirculating packed column (RPC). A concentration-dependent toxicity model used to simulate the results of batch reactors in part I (Sabalowsky and Semprini (in press) Biotechnol Bioeng) also simulated well the observations for the CFSTR and RPC growth modes. The toxicity model incorporates cDCE and TCE toxicity coefficients that directly increase the cell decay coefficient in proportion with cDCE and TCE concentrations. Simulated estimates of the cDCE and TCE toxicity coefficients indicate reductively dechlorinating cells are most sensitive to high concentrations of cDCE and TCE in batch-fed growth, followed by CFSTR, with attached growth being least sensitive. The greater toxicity of TCE than cDCE, and ratio of the modeled toxicity coefficients, agrees with previously proposed models relating toxicity to partitioning in the cell wall (K(M/B)), proportional to octanol-water partitioning (K(OW)) coefficients.