Acceleration of perchloroethylene dechlorination by extracellular secretions from Microbacterium in a mixed culture containing Desulfitobacterium.

The study was conducted to demonstrate the influence of extracellular secretions from Microbacterium on the reductive dechlorination of tetrachloroethene (PCE). A series of mixed cultures were established from a paddy soil sample. In the mixed cultures amended with extracellular secretions from Microbacterium, PCE was rapidly and completely converted into cis-1,2-dichloroethene (cis-DCE) and trans-1,2-dichloroethene (trans-DCE) within 40 days. The unamended mixed cultures showed weak signs of dechlorination after a pronounced lag phase, and trichloroethene (TCE) was accumulated as a major end product. This result means that amendment with extracellular secretions from Microbacterium shortened the lag phase, increased the dechlorination velocity and promoted the production of less-chlorinated chloroethene. The results were corroborated by defined subculture experiments, which proved that microorganisms from unamended mixed cultures could also be stimulated by extracellular secretions from Microbacterium. Desulfitobacterium was identified as the main dechlorinating population in all mixed cultures by direct PCR. Additionally, the 16S rRNA gene copies of Desulfitobacterium increased by one or two orders of magnitude with PCE dechlorination, which provided corroborative evidence for the identification result. The volatile fatty acids were monitored, and most interestingly, a close association between propionate oxidation and dechlorination was found, which has rarely been mentioned before. It was assumed that the oxidation of propionate provided hydrogen for dechlorination, while dechlorination facilitated the shift of the reaction toward propionate oxidation by reducing the partial pressure of hydrogen.

Overview of organohalide-respiring bacteria and a proposal for a classification system for reductive dehalogenases.

Organohalide respiration is an anaerobic bacterial respiratory process that uses halogenated hydrocarbons as terminal electron acceptors during electron transport-based energy conservation. This dechlorination process has triggered considerable interest for detoxification of anthropogenic groundwater contaminants. Organohalide-respiring bacteria have been identified from multiple bacterial phyla, and can be categorized as obligate and non-obligate organohalide respirers. The majority of the currently known organohalide-respiring bacteria carry multiple reductive dehalogenase genes. Analysis of a curated set of reductive dehalogenases reveals that sequence similarity and substrate specificity are generally not correlated, making functional prediction from sequence information difficult. In this article, an orthologue-based classification system for the reductive dehalogenases is proposed to aid integration of new sequencing data and to unify terminology.

The transcriptional regulator CprK detects chlorination by combining direct and indirect readout mechanisms.

The transcriptional regulator CprK controls the expression of the reductive dehalogenase CprA in organohalide-respiring bacteria. Desulfitobacterium hafniense CprA catalyses the reductive dechlorination of the terminal electron acceptor o-chlorophenol acetic acid, generating the phenol acetic acid product. It has been shown that CprK has ability to distinguish between the chlorinated CprA substrate and the de-halogenated end product, with an estimated an estimated 10(4)-fold difference in affinity. Using a green fluorescent protein GFPUV-based transcriptional reporter system, we establish that CprK can sense o-chlorophenol acetic acid at the nanomolar level, whereas phenol acetic acid leads to transcriptional activation only when approaching micromolar levels. A structure-activity relationship study, using a range of o-chlorophenol acetic-acid-related compounds and key CprK mutants, combined with pKa calculations on the effector binding site, suggests that the sensitive detection of chlorination is achieved through a combination of direct and indirect readout mechanisms. Both the physical presence of the bulky chloride substituent as well as the accompanying electronic effects lowering the inherent phenol pKa are required for high affinity. Indeed, transcriptional activation by CprK appears strictly dependent on establishing a phenolate-K133 salt bridge interaction, rather than on the presence of a halogen atom per se. As K133 is strictly conserved within the CprK family, our data suggest that physiological function and future applications in biosensing are probably restricted to phenolic compounds.

Dechlorination and organohalide-respiring bacteria dynamics in sediment samples of the Yangtze Three Gorges Reservoir.

Several groups of bacteria such as Dehalococcoides spp., Dehalobacter spp., Desulfomonile spp., Desulfuromonas spp., or Desulfitobacterium spp. are able to dehalogenate chlorinated pollutants such as chloroethenes, chlorobenzenes, or polychlorinated biphenyls under anaerobic conditions. In order to assess the dechlorination potential in Yangtze sediment samples, the presence and activity of the reductively dechlorinating bacteria were studied in anaerobic batch tests. Eighteen sediment samples were taken in the Three Gorges Reservoir catchment area of the Yangtze River, including the tributaries Jialing River, Daning River, and Xiangxi River. Polymerase chain reaction analysis indicated the presence of dechlorinating bacteria in most samples, with varying dechlorinating microbial community compositions at different sampling locations. Subsequently, anaerobic reductive dechlorination of tetrachloroethene (PCE) was tested after the addition of electron donors. Most cultures dechlorinated PCE completely to ethene via cis-dichloroethene (cis-DCE) or trans-dichloroethene. Dehalogenating activity corresponded to increasing numbers of Dehalobacter spp., Desulfomonile spp., Desulfitobacterium spp., or Dehalococcoides spp. If no bacteria of the genus Dehalococcoides spp. were present in the sediment, reductive dechlorination stopped at cis-DCE. Our results demonstrate the presence of viable dechlorinating bacteria in Yangtze samples, indicating their relevance for pollutant turnover.

Physiological adaptation of Desulfitobacterium hafniense strain TCE1 to tetrachloroethene respiration.

Desulfitobacterium spp. are ubiquitous organisms with a broad metabolic versatility, and some isolates have the ability to use tetrachloroethene (PCE) as terminal electron acceptor. In order to identify proteins involved in this organohalide respiration process, a comparative proteomic analysis was performed. Soluble and membrane-associated proteins obtained from cells of Desulfitobacterium hafniense strain TCE1 that were growing on different combinations of the electron donors lactate and hydrogen and the electron acceptors PCE and fumarate were analyzed. Among proteins increasingly expressed in the presence of PCE compared to fumarate as electron acceptor, a total of 57 proteins were identified by mass spectrometry analysis, revealing proteins involved in stress response and associated regulation pathways, such as PspA, GroEL, and CodY, and also proteins potentially participating in carbon and energy metabolism, such as proteins of the Wood-Ljungdahl pathway and electron transfer flavoproteins. These proteomic results suggest that D. hafniense strain TCE1 adapts its physiology to face the relative unfavorable growth conditions during an apparent opportunistic organohalide respiration.

Divergent roles of CprK paralogues from Desulfitobacterium hafniense in activating gene expression.

Gene duplication and horizontal gene transfer play an important role in the evolution of prokaryotic genomes. We have investigated the role of three CprK paralogues from the cAMP receptor protein-fumarate and nitrate reduction regulator (CRP-FNR) family of transcriptional regulators that are encoded in the genome of Desulfitobacterium hafniense DCB-2 and possibly regulate expression of genes involved in the energy-conserving terminal reduction of organohalides (halorespiration). The results from in vivo and in vitro promoter probe assays show that two regulators (CprK1 and CprK2) have an at least partially overlapping effector specificity, with preference for ortho-chlorophenols, while meta-chlorophenols proved to be effectors for CprK4. The presence of a potential transposase-encoding gene in the vicinity of the cprK genes indicates that their redundancy is probably caused by mobile genetic elements. The CprK paralogues activated transcription from promoters containing a 14 bp inverted repeat (dehalobox) that closely resembles the FNR-box. We found a strong negative correlation between the rate of transcriptional activation and the number of nucleotide changes from the optimal dehalobox sequence (TTAAT-N4-ATTAA). Transcription was initiated by CprK4 from a promoter that is situated upstream of a gene encoding a methyl-accepting chemotaxis protein. This might be the first indication of taxis of an anaerobic bacterium to halogenated aromatic compounds.

Sustained generation of electricity by the spore-forming, Gram-positive, Desulfitobacterium hafniense strain DCB2.

Desulfitobacterium hafniense strain DCB2 generates electricity in microbial fuel cells (MFCs) when humic acids or the humate analog anthraquinone-2,6-disulfonate (AQDS) is added as an electron-carrying mediator. When utilizing formate as fuel, the Gram-positive, spore-forming bacterium generated up to 400 mW/m2 of cathode surface area in a single-chamber MFC with a platinum-containing air-fed cathode. Hydrogen, lactate, pyruvate, and ethanol supported electricity generation, but acetate, propionate, and butyrate did not. Scanning electron microscopy indicated that strain DCB2 colonized the surface of a current-generating anode but not of an unconnected electrode. The electricity was recovered fully within minutes after the exchange of the medium in the anode chamber and within a week after an exposure of a colonized anode to 90 degrees C for 20 min. Of the six strains of Desulfitobacteria tested, all of which would reduce AQDS, only D. hafniense strain DCB2 continued to reduce AQDS and generate electricity for more than 24 h, indicating that reduction of the humate analog alone is insufficient to sustain electrode reduction.