Indirect Evidence Link PCB Dehalogenation with Geobacteraceae in Anaerobic Sediment-Free Microcosms

Although polychlorinated biphenyls (PCBs) production was brought to a halt 30 years ago, recalcitrance to degradation makes them a major environmental pollutant at a global scale. Previous studies confirmed that organohalide-respiring bacteria (OHRB) were capable of utilizing chlorinated congeners as electron acceptor. OHRB belonging to the Phyla Chloroflexi and Firmicutes are nowadays considered as the main PCB-dechlorinating organisms. In this study, we aimed at exploring the involvement of other taxa in PCB dechlorination using sediment-free microcosms (SFMs) and the Delor PCB mixture. High rates of congener dehalogenation (up to 96%) were attained in long-term incubations of up to 692 days. Bacterial communities were dominated by Chloroflexi, Proteobacteria, and Firmicutes, among strictly simplified community structures composed of 12 major phyla only. In a first batch of SFMs, Dehalococcoides mccartyi closely affiliated with strains CG4 and CBDB1 was considered as the main actor associated with congener dehalogenation. Addition of 2-bromoethanesulfonate (BES), a known inhibitor ofmethanogenic activity in a second batch of SFMs had an adverse effect on the abundance of Dehalococcoides sp. Only two sequences affiliated to this Genus could be detected in two (out of six) BES-treated SFMs, contributing to a mere 0.04% of the communities. BES-treated SFMs showed very different community structures, especially in the contributions of organisms involved in fermentation and syntrophic activities. Indirect evidence provided by both statistical and phylogenetic analysis validated the implication of a new cluster of actors, distantly affiliated with the Family Geobacteraceae (Phylum d-Proteobacteria), in the dehalogenation of low chlorinated PCB congeners. Members of this Family are known already for their dehalogenation capacity of chlorinated solvents. As a result, the present study widens the knowledge for the phylogenetic reservoir of indigenous PCB dechlorinating taxa

Dynamics of an Oligotrophic Bacterial Aquifer Community during Contact with a Groundwater Plume Contaminated with Benzene, Toluene, Ethylbenzene, and Xylenes: an In Situ Mesocosm Study{dagger}

An in situ mesocosm system was designed to monitor the in situ dynamics of the microbial community in polluted aquifers. The mesocosm system consists of a permeable membrane pocket filled with aquifer material and placed within a polypropylene holder, which is inserted below groundwater level in a monitoring well. After a specific time period, the microcosm is recovered from the well and its bacterial community is analyzed. Using this system, we examined the effect of benzene, toluene, ethylbenzene, and xylene (BTEX) contamination on the response of an aquifer bacterial community by denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA genes and PCR detection of BTEX degradation genes. Mesocosms were filled with nonsterile or sterile aquifer material derived from an uncontaminated area and positioned in a well located in either the uncontaminated area or a nearby contaminated area. In the contaminated area, the bacterial community in the microcosms rapidly evolved into a stable community identical to that in the adjacent aquifer but different from that in the uncontaminated area. At the contaminated location, bacteria with tmoA- and xylM/xylE1-like BTEX catabolic genotypes colonized the aquifer, while at the uncontaminated location only tmoA-like genotypes were detected. The communities in the mesocosms and in the aquifer adjacent to the wells in the contaminated area consisted mainly of Proteobacteria. At the uncontaminated location, Actinobacteria and Proteobacteria were found. Our results indicate that communities with long-term stability in their structures follow the contamination plume and rapidly colonize downstream areas upon contaminatio

EIF2S3 Mutations Associated with Severe X-Linked Intellectual Disability Syndrome MEHMO

Impairment of translation initiation and its regulation within the integrated stress response (ISR) and related unfolded-protein response has been identified as a cause of several multisystemic syndromes. Here, we link MEHMO syndrome, whose genetic etiology was unknown, to this group of disorders. MEHMO is a rare X-linked syndrome characterized by profound intellectual disability, epilepsy, hypogonadism and hypogenitalism, microcephaly, and obesity. We have identified a C-terminal frameshift mutation (Ile465Serfs) in the EIF2S3 gene in three families with MEHMO syndrome and a novel maternally inherited missense EIF2S3 variant (c.324T>A; p.Ser108Arg) in another male patient with less severe clinical symptoms. The EIF2S3 gene encodes the gamma subunit of eukaryotic translation initiation factor 2 (eIF2), crucial for initiation of protein synthesis and regulation of the ISR. Studies in patient fibroblasts confirm increased ISR activation due to the Ile465Serfs mutation and functional assays in yeast demonstrate that the Ile465Serfs mutation impairs eIF2gamma function to a greater extent than tested missense mutations, consistent with the more severe clinical phenotype of the Ile465Serfs male mutation carriers. Thus, we propose that more severe EIF2S3 mutations cause the full MEHMO phenotype, while less deleterious mutations cause a milder form of the syndrome with only a subset of the symptoms

The great screen anomaly—a new frontier in product discovery through functional metagenomics

Functional metagenomics, the study of the collective genome of a microbial community by expressing it in a foreign host, is an emerging field in biotechnology. Over the past years, the possibility of novel product discovery through metagenomics has developed rapidly. Thus, metagenomics has been heralded as a promising mining strategy of resources for the biotechnological and pharmaceutical industry. However, in spite of innovative work in the field of functional genomics in recent years, yields from function-based metagenomics studies still fall short of producing significant amounts of new products that are valuable for biotechnological processes. Thus, a new set of strategies is required with respect to fostering gene expression in comparison to the traditional work. These new strategies should address a major issue, that is, how to successfully express a set of unknown genes of unknown origin in a foreign host in high throughput. This article is an opinionating review of functional metagenomic screening of natural microbial communities, with a focus on the optimization of new product discovery. It first summarizes current major bottlenecks in functional metagenomics and then provides an overview of the general metagenomic assessment strategies, with a focus on the challenges that are met in the screening for, and selection of, target genes in metagenomic libraries. To identify possible screening limitations, strategies to achieve optimal gene expression are reviewed, examining the molecular events all the way from the transcription level through to the secretion of the target gene product

Acidophilic bacteria in Deep Groundwaters of the Bohemian Cretaceous Basin after Extensive Uranium Leaching

The Stráž pod Ralskem region is a part of the Bohemian Cretaceous Basin. The geological profile of the site consists of Quaternary river sediments, Middle Turonian sandstone, Lower Turonian siltstone and Cenomanian collector formed by friable sandstone containing uranium bearing minerals and upper fucoid sandstone forming an overlying back of the ore. The uranium deposit Stráž was exploited by underground acidic leaching between 1968 and 1996. More than 14 000 tons of uranium were produced during this period. Minimum 4 million tons of H2SO4, 300 thousand tons of HNO3, 120 thousand tons of NH3 and other chemicals were injected into the Cenomanian sandstones. The chemical mining of uranium resulted in a large contamination of the groundwater collectors in Stráž block of North Bohemian cretaceous table and caused serious ecological problems. The lower Cenomanian aquifer containing the U deposit was mainly afflicted. Total salinity of the Cenomanian solutions reaches up to 80 g/l. Overflow of the highly contaminated Cenomanian groundwater to the Turonian collector endangers the sources of drinking water in Northern Bohemia. Our study was focused on analyzing the microbial communities and their nitrate and sulfate reduction potential regarding developing of appropriate bioremediation strategies in the region. Numerical ecology tools were applied for correlating of 16S rRNA gene denatured gradient gel electrophoresis phylogenetic profiles with environmental variables and for selecting of water habitats for further sequencing analysis of 16S rRNA genes, and functional genes for sulfate reduction – aprA, and denitrification – nirS. Significant differences were found between the microbial compositions of the two groundwater collectors. Marker genes for dissimilative reduction of nitrates were detected in waters with pH > 7. Cenomanian waters were characterized by higher diversity and distinct phylogenetic composition of denitrifying bacteria. Regardless the acidic conditions and low bacterial density - 2.7x102 to 5.3x103 cells.ml-1, three aprA gene polymorphs for the -subunit of adenosine-5’-phosphosulfate reductase were identified in the Cenomanian aquatic samples representing the highly polluted groundwaters of Bohemian Cretaceous basin. The prevalent gene variants found in the most contaminated sample C29 (41.9 g/l sulfuric acid, pH 1.68) were affiliated with the obligate chemolithoautotrophic and relevant to in situ bioremediation Thiobacillus and Desulfovibrio spp. The biocatalytic potential of the present extremophile bacteria will be used in development of pilot sulfate-reducing passive bioreactors based on the experience from acid mine drainage treatment

Multidisciplinary approach for evaluation of the complete removal of chloroethenes in contaminated aquifer

Chlorinated ethenes (CEs) pose a long-lasting risk for humans and the environment. Nowadays, they are the major groundwater contaminant worldwide and are of particular concern since they form a dense non-aqueous phase liquid (DNAPL). Reductive dehalogenation of the CEs to the non-toxic end-products ethane and ethene is used for nearly twenty years as the most efficient and used remediation technique called enhanced reductive dehalogenation (ERD). However, in some cases the reduction of CEs is halted to 1,2-dichloroethene (1,2-DCE) or to the carcinogenic vinyl chloride (VC). The dechlorination is known to be a complex process and many factors were identified to play an important role in quantitative and qualitative reduction of the CEs in-situ. This study was designed to show new methodological approach and demonstrated the importance of both, the biological and environmental variables, for the performance of ERD. The combination of analytical data, molecular biological analysis and statistical methods could be used as a tool for finding of a proper efficient remediation procedure for the removal of chlorinated ethenes

An ecological approach for understanding the reductive dechlorination process of chlorinated ethenes in contaminated aquifers

Chlorinated ethenes, frequently encountered contaminants in groundwater, are often found to be incompletely dechlorinated to dichloroethene and vinyl chloride, more toxic products that accumulate in the aquifer. Some of the concerns in designing remediation approaches based on biological dechlorination are i) the presence of bacteria capable of completely reducing chlorinated ethenes, and ii) the competition events for available electron donors between classical biogeochemical processes and the reductive dechlorination process. For this purpose, an assessment methodology was developed to explore the existing relations between autochthonous bacterial communities, degradation state of chlorinated ethenes and redox processes, including reductive dechlorination. Three steps are involved with first the acquisition of abiotic environmental data, followed by assessing the biochemical potential for complete dechlorination and an analysis of bacterial community structures, and finally a statistical Multiple Factor Analysis combining datasets from geochemistry and microbiology. This approach was applied for diagnosing a Swiss aquifer for its in situ potential of complete reductive dechlorination, and for monitoring a Czech aquifer under bioremediation by successive cheese whey injections. The Swiss site contained the microbiological potential for complete dechlorination but the global redox conditions were not enough reducing and dichloroethene reduction was in competition locally with manganese reduction. The follow-up of the Czech site has shown that during the remediation process, complete reductive dechlorination was favored under sulfate-reducing conditions, but was in competition with other anaerobic bacterial processes leading to a transient accumulation of dichloroethene and vinyl chloride. In both cases, antagonist biogeochemical processes were identified as the reasons restricting the reductive dechlorination at the level of toxic intermediates. This approach is now formalized in a user-oriented manual, and could be integrated as tool for eligibility investigation of aquifers for a bioremediation strategy through natural attenuation

Assessment of bioremediation potential and monitoring of biological reductive dechlorination in sites contaminated with chlorinated ethenes

Chlorinated ethenes (CEs), such as perchloroethene (PCE) and trichloroethene, are one of the most common classes of groundwater contaminants. In this project, the contaminant biodegradation capacities of two aquifers, presenting both dichloroethene (DCE) and vinyl chloride (VC) accumulation, was carried out. Aquifers are considered nowadays as dynamic ecosystems, showing multiple interactions between the physical, chemical and biotic components. In this sense, an integrative methodology using multivariate statistics and combining together bacterial community structures, detection of dechlorinating bacteria and genes and water geochemical data were used to investigate these aquifers. Results from multifactorial analysis of data collected from a PCE-contaminated site in Switzerland (25 groundwater samples) showed that manganese reduction (MR) was a key terminal electron accepting process, suggesting a potential competition between MR and DCE degradation to VC. Dehalococcoides sp. and VC reductive dehalogenase genes were detected but ethene concentration was below 0.007mg/L. Potential for a complete natural biodegradation of PCE was present in this aquifer. However, DCE reduction will be strongly inhibited under local conditions as long as oxidized manganese resources are present. The second site located in Czech Republic (Velamos) and sampled at 7 different dates (35 groundwater samples) was under active biostimulation process. Multifactorial analysis showed that successive cheese whey injections modified the aquifer habitat that became favourable not only for a complete dechlorination, but also for sulfate reduction (SR) and methanogenesis. DCE and VC accumulated along with the production of ethene, methane and hydrogen sulphide, indicating a competition between CEs dechlorination and SR and methanogenesis. This possibly explained the transitional slower reaction of CEs dechlorination observed during the remediation process. In conclusion, the used methodology allows evaluation of the bioremediation potential present in contaminated aquifers and monitoring biostimulation processes. This study was funded by grant No. TA02020534 - TECHTOOL of the Technology Agency of the CR, and the Swiss Federal Office for the Environment FOEN

Validation of an Integrative Methodology to Assess and Monitor Reductive Dechlorination of Chlorinated Ethenes in Contaminated Aquifers

Bioremediation of tetra-and trichloroethene-contaminated aquifers is frequently hampered due to incomplete dechlorination to the more toxic dichloroethene (DCE) and vinyl chloride (VC), indicating insufficient knowledge about the biological mechanisms related to aquifer functioning. A methodology based on the joint analysis of geochemical and microbiological datasets was developed to assess the presence of the biochemical potential for complete reductive dechlorination to harmless ethene and to explain the reasons for which degradation often stalls at the more toxic intermediates. This methodology is composed of three successive steps, with i) the acquisition of geochemical data including chlorinated ethenes, ii) the detailed analysis of the bacterial community structures as well as the biochemical potential for complete dechlorination using microcosms and molecular detection of organohalide-respiring bacteria and key reductive dehalogenases, and iii) a statistical Multiple Factor Analysis combining the above mentioned abiotic and biotic variables in a functional modelling of the contaminated aquifer. The methodology was validated by analyzing two chlorinated ethenes-contaminated sites. Results from the first site showed that the full biochemical potential for ethene production was present in situ. However, redox potential was overall too high and locally manganese reduction out-competed chlorinated ethenes reduction, explaining the reasons for the local accumulation of DCE and VC to a lesser extent. The second contaminated aquifer was under bioremediation by successive cheese whey injections. Analysis demonstrated that cheese whey additions led to increasingly reduced redox conditions and that hampered reductive dechlorination was not due to competition with other anaerobic respiration processes. Complete reductive dechlorination to ethene was preferentially occurring under methanogenic conditions. DCE and VC accumulation was probably induced first by low pH resulting from whey fermentation and at a later stage by phosphate limitation. In conclusions, the proposed methodology successfully allowed the identification of biogeochemical processes limiting or supporting complete dechlorination in both aquifers. The integrative approach provided fundamental information about the functional heterogeneity of the contaminated aquifers in time and space, and can be used as a reliable tool to support corrective decision-making in the development of remediation strategies based on natural attenuation of chlorinated ethenes