Farai Maphosa

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.

Impact of long-term diesel contamination on soil microbial community structure.

ABSTRACT Microbial community composition and diversity at a diesel-contaminated railway site were investigated by pyrosequencing of bacterial and archaeal 16S rRNA gene fragments to understand the interrelationships among microbial community composition, pollution level, and soil geochemical and physical properties. To this end, 26 soil samples from four matrix types with various geochemical characteristics and contaminant concentrations were investigated. The presence of diesel contamination significantly impacted microbial community composition and diversity, regardless of the soil matrix type. Clean samples showed higher diversity than contaminated samples (P < 0.001). Bacterial phyla with high relative abundances in all samples included Proteobacteria, Firmicutes, Actinobacteria, Acidobacteria, and Chloroflexi. High relative abundances of Archaea, specifically of the phylum Euryarchaeota, were observed in contaminated samples. Redundancy analysis indicated that increased relative abundances of the phyla Chloroflexi, Firmicutes, and Euryarchaeota correlated with the presence of contamination. Shifts in the chemical composition of diesel constituents across the site and the abundance of specific operational taxonomic units (OTUs; defined using a 97% sequence identity threshold) in contaminated samples together suggest that natural attenuation of contamination has occurred. OTUs with sequence similarity to strictly anaerobic Anaerolineae within the Chloroflexi, as well as to Methanosaeta of the phylum Euryarchaeota, were detected. Anaerolineae and Methanosaeta are known to be associated with anaerobic degradation of oil-related compounds; therefore, their presence suggests that natural attenuation has occurred under anoxic conditions. This research underscores the usefulness of next-generation sequencing techniques both to understand the ecological impact of contamination and to identify potential molecular proxies for detection of natural attenuation.

Metagenome analysis reveals yet unexplored reductive dechlorinating potential of Dehalobacter sp. E1 growing in co‐culture with Sedimentibacter sp.

The importance of Dehalobacter species in bioremediation as dedicated degraders of chlorinated organics has been well recognized. However, still little is known about Dehalobacters full genomic repertoires, including the genes involved in dehalogenation. Here we report the first insights into the genome sequence of Dehalobacter sp. E1 that grows in strict co-culture with Sedimentibacter sp. B4. Based on the co-culture metagenome and the genome of strain B4 (4.2 Mbp) we estimate the genome sequence of strain E1 to be 2.6 Mbp. Ten putative reductive dehalogenase homologue (Rdh)-encoding gene clusters were identified. One cluster has a putative tetrachloroethene Rdh-encoding gene cluster, similar to the pceABCT operon previously identified in Dehalobacter restrictus. Metagenome analysis indicated that the inability of strain E1 to synthesize cobalamin, an essential cofactor of reductive dehalogenases, is complemented by Sedimentibacter. The metagenomic exploration described here maps the extensive dechlorinating potential of Dehalobacter, and paves way for elucidation of the interactions with its co-cultured Sedimentibacter.

Ecogenomics of microbial communities in bioremediation of chlorinated contaminated sites

Organohalide compounds such as chloroethenes, chloroethanes, and polychlorinated benzenes are among the most significant pollutants in the world. These compounds are often found in contamination plumes with other pollutants such as solvents, pesticides, and petroleum derivatives. Microbial bioremediation of contaminated sites, has become commonplace whereby key processes involved in bioremediation include anaerobic degradation and transformation of these organohalides by organohalide respiring bacteria and also via hydrolytic, oxygenic, and reductive mechanisms by aerobic bacteria. Microbial ecogenomics has enabled us to not only study the microbiology involved in these complex processes but also develop tools to better monitor and assess these sites during bioremediation. Microbial ecogenomics have capitalized on recent advances in high-throughput and -output genomics technologies in combination with microbial physiology studies to address these complex bioremediation problems at a system level. Advances in environmental metagenomics, transcriptomics, and proteomics have provided insights into key genes and their regulation in the environment. They have also given us clues into microbial community structures, dynamics, and functions at contaminated sites. These techniques have not only aided us in understanding the lifestyles of common organohalide respirers, for example Dehalococcoides, Dehalobacter, and Desulfitobacterium, but also provided insights into novel and yet uncultured microorganisms found in organohalide respiring consortia. In this paper, we look at how ecogenomic studies have aided us to understand the microbial structures and functions in response to environmental stimuli such as the presence of chlorinated pollutants.

Small-scale oxygen distribution determines the vinyl chloride biodegradation pathway in surficial sediments of riverbed hyporheic zones

Surficial riverbed sediments are often characterized by sharp redox gradients between the aerobic benthic sediment and underlying anoxic sediment, potentially representing an ideal niche for aerobic and anaerobic vinyl chloride (VC) degraders. To test this, the fate of VC in aerobic and anaerobic microcosms containing surficial sediment of a riverbed hyporheic zone receiving VC-contaminated groundwater was explored. Quantitative PCR showed that Dehalococcoides 16S rRNA gene and VC reductive dehalogenase-encoding genes (vcrA, bvcA) were highly enriched in anaerobic microcosms, with stoichiometric conversion of VC to ethene. In aerobic microcosms, etnC and etnE involved in aerobic ethene/VC oxidation were enriched with concomitant low or no accumulation of ethene. However, Dehalococcoides 16S rRNA gene, vcrA and bvcA copy numbers were also enriched in oxygen-exposed microcosms containing sediment with high organic carbon and small grain size, whereas they were reduced in oxygen-exposed sediment with low organic carbon and larger grain size in line with extensive oxygen penetration into the sediment. These results suggest the coexistence and coactivity of anaerobic and aerobic VC degraders in the same small volume of surficial sediment and that oxygen distribution, as determined by sediment grain size and organic matter content, affects the local VC-degrading bacterial community and VC biodegradation pathway.

Genomic, Proteomic, and Biochemical Analysis of the Organohalide Respiratory Pathway in Desulfitobacterium dehalogenans

Desulfitobacterium dehalogenans is able to grow by organohalide respiration using 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) as an electron acceptor. We used a combination of genome sequencing, biochemical analysis of redox active components, and shotgun proteomics to study elements of the organohalide respiratory electron transport chain. The genome of Desulfitobacterium dehalogenans JW/IU-DC1(T) consists of a single circular chromosome of 4,321,753 bp with a GC content of 44.97%. The genome contains 4,252 genes, including six rRNA operons and six predicted reductive dehalogenases. One of the reductive dehalogenases, CprA, is encoded by a well-characterized cprTKZEBACD gene cluster. Redox active components were identified in concentrated suspensions of cells grown on formate and Cl-OHPA or formate and fumarate, using electron paramagnetic resonance (EPR), visible spectroscopy, and high-performance liquid chromatography (HPLC) analysis of membrane extracts. In cell suspensions, these components were reduced upon addition of formate and oxidized after addition of Cl-OHPA, indicating involvement in organohalide respiration. Genome analysis revealed genes that likely encode the identified components of the electron transport chain from formate to fumarate or Cl-OHPA. Data presented here suggest that the first part of the electron transport chain from formate to fumarate or Cl-OHPA is shared. Electrons are channeled from an outward-facing formate dehydrogenase via menaquinones to a fumarate reductase located at the cytoplasmic face of the membrane. When Cl-OHPA is the terminal electron acceptor, electrons are transferred from menaquinones to outward-facing CprA, via an as-yet-unidentified membrane complex, and potentially an extracellular flavoprotein acting as an electron shuttle between the quinol dehydrogenase membrane complex and CprA.

Impact of a wastewater treatment plant on microbial community composition and function in a hyporheic zone of a eutrophic river

The impact of the installation of a technologically advanced wastewater treatment plant (WWTP) on the benthic microbial community of a vinyl chloride (VC) impacted eutrophic river was examined two years before, and three and four years after installation of the WWTP. Reduced dissolved organic carbon and increased dissolved oxygen concentrations in surface water and reduced total organic carbon and total nitrogen content in the sediment were recorded in the post-WWTP samples. Pyrosequencing of bacterial 16S rRNA gene fragments in sediment cores showed reduced relative abundance of heterotrophs and fermenters such as Chloroflexi and Firmicutes in more oxic and nutrient poor post-WWTP sediments. Similarly, quantitative PCR analysis showed 1–3 orders of magnitude reduction in phylogenetic and functional genes of sulphate reducers, denitrifiers, ammonium oxidizers, methanogens and VC-respiring Dehalococcoides mccartyi. In contrast, members of Proteobacteria adapted to nutrient-poor conditions were enriched in post-WWTP samples. This transition in the trophic state of the hyporheic sediments reduced but did not abolish the VC respiration potential in the post-WWTP sediments as an important hyporheic sediment function. Our results highlight effective nutrient load reduction and parallel microbial ecological state restoration of a human-stressed urban river as a result of installation of a WWTP.

Microbial Community- And Metabolite Dynamics of an Anoxic Dechlorinating Bioreactor

Monitoring and quantification of organohalide respiring bacteria is essential for optimization of on-site bioremediation of anoxic subsurface sites contaminated with chloroethenes. Molecular monitoring and model simulations were applied to determine degradation performance of an in situ dechlorinating bioreactor and its influence on the contamination plume. Dehalococcoides was the dominant dechlorinating microorganism as revealed by qPCR targeting 16S rRNA- and chloroethene reductive dehalogenase-encoding genes (tceA, vcrA, bvcA). The presence of all three reductive dehalogenases genes indicated coexistence of several distinct organohalide respiring bacterial populations in the bioreactor and groundwater. Mass balancing revealed that main dechlorinating activities were reduction of cis-dichloroethene and vinyl chloride. Analysis of growth kinetics showed that when performance of the bioreactor improved due to especially the addition of molasses, dechlorinating microorganisms were growing close to their maximum growth rate. Once near-complete dehalogenation was achieved, Dehalococcoides only grew slowly and population density did not further increase. The bioreactor influenced dechlorinating populations in the plume with subsequent decrease in chlorinated compound concentrations over time. In the present study, a combination of molecular diagnostics with mass-balancing and kinetic modeling improved insight into organohalide respiring bacteria and metabolite dynamics in an in situ dechlorinating bioreactor and showed its utility in monitoring bioremediation.

Kinetics of dechlorination by Dehalococcoides mccartyi using different carbon sources

Stimulated anaerobic dechlorination is generally considered a valuable step for the remediation of aquifers polluted with chlorinated ethenes (CEs). Correct simulation and prediction of this process in situ, however, require good knowledge of the associated biological reactions. The aim of this study was to evaluate the dechlorination reaction in an aquifer contaminated with trichloroethene (TCE) and its daughter products, discharging into the Zenne River. Different carbon sources were used in batch cultures and these were related to the dechlorination reaction, together with the monitored biomarkers. Appropriate kinetic formulations were assessed. Reductive dechlorination of TCE took place only when external carbon sources were added to microcosms, and occurred concomitant with a pronounced increase in the Dehalococcoides mccartyi cell count as determined by 16S rRNA gene-targeted qPCR. This indicates that native dechlorinating bacteria are present in the aquifer of the Zenne site and that the oligotrophic nature of the aquifer prevents a complete degradation to ethene. The type of carbon source, the cell number of D. mccartyi or the reductive dehalogenase genes, however, did not unequivocally explain the observed differences in degradation rates or the extent of dechlorination. Neither first-order, Michaelis-Menten nor Monod kinetics could perfectly simulate the dechlorination reactions in TCE spiked microcosms. A sensitivity analysis indicated that the inclusion of donor limitation would not significantly enhance the simulations without a clear process understanding. Results point to the role of the supporting microbial community but it remains to be verified how the complexity of the microbial (inter)actions should be represented in a model framework.

Meta-omics approaches to understand and improve wastewater treatment systems

Biological treatment of wastewaters depends on microbial processes, usually carried out by mixed microbial communities. Environmental and operational factors can affect microorganisms and/or impact microbial community function, and this has repercussion in bioreactor performance. Novel high-throughput molecular methods (metagenomics, metatranscriptomics, metaproteomics, metabolomics) are providing detailed knowledge on the microorganisms governing wastewater treatment systems and on their metabolic capabilities. The genomes of uncultured microbes with key roles in wastewater treatment plants (WWTP), such as the polyphosphate-accumulating microorganism “Candidatus Accumulibacter phosphatis”, the nitrite oxidizer “Candidatus Nitrospira defluvii” or the anammox bacterium “Candidatus Kuenenia stuttgartiensis” are now available through metagenomic studies. Metagenomics allows to genetically characterize full-scale WWTP and provides information on the lifestyles and physiology of key microorganisms for wastewater treatment. Integrating metagenomic data of microorganisms with metatranscriptomic, metaproteomic and metabolomic information provides a better understanding of the microbial responses to perturbations or environmental variations. Data integration may allow the creation of predictive behavior models of wastewater ecosystems, which could help in an improved exploitation of microbial processes. This review discusses the impact of meta-omic approaches on the understanding of wastewater treatment processes, and the implications of these methods for the optimization and design of wastewater treatment bioreactors.