Proteogenomic analysis of Georgfuchsia toluolica revealed unexpected concurrent aerobic and anaerobic toluene degradation

Denitrifying Betaproteobacteria play a key role in the anaerobic degradation of monoaromatic hydrocarbons. We performed a multi-omics study to better understand the metabolism of the representative organism Georgfuchsia toluolica strain G5G6 known as a strict anaerobe coupling toluene oxidation with dissimilatory nitrate and Fe(III) reduction. Despite the genomic potential for degradation of different carbon sources, we did not find sugar or organic acid transporters, in line with the inability of strain G5G6 to use these substrates. Using a proteomics analysis, we detected proteins of fumarate-dependent toluene activation, membrane-bound nitrate reductase, and key components of the metal-reducing (Mtr) pathway under both nitrate- and Fe(III)-reducing conditions. High abundance of the multiheme cytochrome MtrC implied that a porincytochrome complex was used for respiratory Fe(III) reduction. Remarkably, strain G5G6 contains a full set of genes for aerobic toluene degradation, and we detected enzymes of aerobic toluene degradation under both nitrate- and Fe(III)-reducing conditions. We further detected an ATP-dependent benzoyl-CoA reductase, reactive oxygen species detoxification proteins, and cytochrome c oxidase indicating a facultative anaerobic lifestyle of strain G5G6. Correspondingly, we found diffusion through the septa a substantial source of oxygen in the cultures enabling concurrent aerobic and anaerobic toluene degradation by strain G5G6.This work was supported by Wageningen University & Research through its investment theme Resilience, the Technology Foundation (STW), the Applied Science Division of the Dutch Research Council (NWO; project 08053), NWO grant 016.Vidi.189.050, and a Gravitation grant of the Netherlands Ministry of Education, Culture and Science and NWO (project 024.002.002 SIAM). B.K. was supported by the Villum foundation, Denmark (VYI Grant 25491).info:eu-repo/semantics/publishedVersio

Organohalide-respiring Desulfoluna species isolated from marine environments

The online version of this article (https://doi.org/10.1038/s41396-019-0573-y) contains supplementary material, which is available to authorized usersThe genus Desulfoluna comprises two anaerobic sulfate-reducing strains, D. spongiiphila AA1T and D. butyratoxydans MSL71T, of which only the former was shown to perform organohalide respiration (OHR). Here we isolated a third strain, designated D. spongiiphila strain DBB, from marine intertidal sediment using 1,4-dibromobenzene and sulfate as the electron acceptors and lactate as the electron donor. Each strain harbors three reductive dehalogenase gene clusters (rdhABC) and corrinoid biosynthesis genes in their genomes, and dehalogenated brominated but not chlorinated organohalogens. The Desulfoluna strains maintained OHR in the presence of 20?mM sulfate or 20?mM sulfide, which often negatively affect other organohalide-respiring bacteria. Strain DBB sustained OHR with 2\% oxygen in the gas phase, in line with its genetic potential for reactive oxygen species detoxification. Reverse transcription-quantitative PCR revealed differential induction of rdhA genes in strain DBB in response to 1,4-dibromobenzene or 2,6-dibromophenol. Proteomic analysis confirmed expression of rdhA1 with 1,4-dibromobenzene, and revealed a partially shared electron transport chain from lactate to 1,4-dibromobenzene and sulfate, which may explain accelerated OHR during concurrent sulfate reduction. Versatility in using electron donors, de novo corrinoid biosynthesis, resistance to sulfate, sulfide and oxygen, and concurrent sulfate reduction and OHR may confer an advantage to marine Desulfoluna strains.We thank Johanna Gutleben and Maryam Chaib de Mares for sediment sampling, W. Irene C. Rijpstra for fatty acid analysis, and Andreas Marquardt (Proteomics Centre of the University of Konstanz) for proteomic analyses. We acknowledge the China Scholarship Council (CSC) for the support to PP and YL. The authors thank BE-BASIC funds (grants F07.001.05 and F08.004.01) from the Dutch Ministry of Economic Affairs, ERC grant (project 323009), the Gravitation grant (project 024.002.002) and the UNLOCK project (NRGWI.obrug.2018.005) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO), and National Natural Science Foundation of China (project No.51709100) for funding.info:eu-repo/semantics/publishedVersio

A benzene-degrading nitrate-reducing microbial consortium displays aerobic and anaerobic benzene degradation pathways

All sequence data from this study were deposited at the European Bioinformatics Institute under the accession numbers ERS1670018 to ERS1670023. Further, all assigned genes, taxonomy, function, sequences of contigs, genes and proteins can be found in Table S3.In this study, we report transcription of genes involved in aerobic and anaerobic benzene degradation pathways in a benzene-degrading denitrifying continuous culture. Transcripts associated with the family Peptococcaceae dominated all samples (2136% relative abundance) indicating their key role in the community. We found a highly transcribed gene cluster encoding a presumed anaerobic benzene carboxylase (AbcA and AbcD) and a benzoate-coenzyme A ligase (BzlA). Predicted gene products showed >96% amino acid identity and similar gene order to the corresponding benzene degradation gene cluster described previously, providing further evidence for anaerobic benzene activation via carboxylation. For subsequent benzoyl-CoA dearomatization, bam-like genes analogous to the ones found in other strict anaerobes were transcribed, whereas gene transcripts involved in downstream benzoyl-CoA degradation were mostly analogous to the ones described in facultative anaerobes. The concurrent transcription of genes encoding enzymes involved in oxygenase-mediated aerobic benzene degradation suggested oxygen presence in the culture, possibly formed via a recently identified nitric oxide dismutase (Nod). Although we were unable to detect transcription of Nod-encoding genes, addition of nitrite and formate to the continuous culture showed indication for oxygen production. Such an oxygen production would enable aerobic microbes to thrive in oxygen-depleted and nitrate-containing subsurface environments contaminated with hydrocarbons.This study was supported by a grant of BE-Basic-FES funds from the Dutch Ministry of Economic Affairs. The research of A.J.M. Stams is supported by an ERC grant (project 323009) and the gravitation grant “Microbes for Health and Environment” (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science. F. Hugenholtz was supported by the same gravitation grant (project 024.002.002). B. Hornung is supported by Wageningen University and the Wageningen Institute for Environment and Climate Research (WIMEK) through the IP/OP program Systems Biology (project KB-17-003.02-023).info:eu-repo/semantics/publishedVersio

Effect of carbonate precipitating bacteria on strength and hydraulic characteristics of loess soil

Microbial-induced calcite precipitation (MICP) is one of the environmentally friendly techniques that has recently become popular amongst geotechnical engineers. Two bacterial species of Bacillus family, i.e., B. pasteurii and B. megaterium have been used to improve the loess soil properties. A set of unconfined compressive, permeability, ultrasonic, and collapse potential tests have been applied to assess the characteristics of natural soil compared to those of MICP-treated ones. The effects of curing time (1, 3, and 7 days), bacterial optical density (OD = 0.5, 1, and 1.5), and soil density (13, 14, and 15 kNm3) have been investigated. Results indicate that biological enhancement has improved the engineering properties of the loess soil. MICP-treated soil using B. megaterium provides higher strength improvement ratios (1.15�4.4 times) rather than B. pasteurii-treated samples (1.05�3.4 times). Correspondingly, specimens containing B. megaterium have greater permeability reduction ratios (3.9�93.7) compared with those of B. pasteurii ones (2�95). Moreover, scanning electron microscope (SEM) analysis has been employed to confirm the findings. It is worth noting that various bacteria concentrations, curing periods, and soil densities can affect the stress-strain curve considerably. The results indicated that MICP reduced the collapse potential between 24 and 54.8 and increased the longitudinal wave velocity between 1.1 and 2.4 times more than the untreated soil. © 2020, Springer-Verlag GmbH Germany, part of Springer Nature

Primers That Target Functional Genes of Organohalide-Respiring Bacteria

Halogenated organic hydrocarbons are problematic environmental pollutants that can be reductively dehalogenated by organohalide-respiring bacteria (OHRB) in anoxic environments. This energy-conserving process is mediated by reductive dehalogenases (RDases). To amplify the diversity of reductive dehalogenase-encoding genes, degenerate primers have been designed, most of which target the conserved regions of the encoded protein sequences of the catalytic subunit, RdhA. In addition, specific primer sets have been developed and widely used to quantify and characterise OHRB and the reductive dehalogenase homologous (rdh) genes in the environment. The specific primers have been applied to multiple molecular techniques including regular and quantitative PCR (qPCR), Southern blot hybridisation, terminal restriction fragment length polymorphism (T-RFLP) and reverse transcriptase PCR (RT-PCR). The hunt for novel rdhA genes has benefited greatly from next-generation sequencing techniques, including primer-dependent amplicon sequencing and primer-independent metagenomic analyses. This chapter provides an overview of most primers targeting RDase-encoding genes described to date and their applications, and it discusses the developing trend of leveraging primer-(in)dependent techniques for better understanding of OHRB and their RDase gene pool

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 pathwa

Microbial community response of an organohalide respiring enrichment culture to permanganate oxidation

While in situ chemical oxidation (ISCO) is often used to remediate tetrachloroethene (PCE) contaminated locations, very little is known about the influence of oxidation on organohalide respiration (OHR) activity and especially microbial community structure. Here, we investigate the impact of oxidation with permanganate on OHR rates, the abundance of organohalide respiring bacteria (OHRB) and reductive dehalogenase (rdh) genes using quantitative PCR, and microbial community composition and dynamics based on sequencing of partial 16S rRNA gene fragments. A PCE degrading enrichment culture was treated with multiple rounds of low (25 µmol), medium (50 µmol), or high (100 µmol) permanganate doses, or no oxidant treatment (biotic control). Results indicate that under mild permanganate treatments (25 µmol or 50 µmol), chemical oxidation stimulated biodegradation leading to higher OHR rates and enrichment of a number of OHRB and rdh genes, as compared to the biotic control. Improved degradation rates can be attributed to enrichment of (1) OHRB able to also utilize Mn oxides as a terminal electron acceptor and (2) non-dechlorinating community members of the order Clostridiales and Deltaproteobacteria who provide essential co-factors to OHRB. In contrast, 100 µmol permanganate treatment disrupted biodegradation activity beyond cis-DCE and caused at least a 2-4 orders of magnitude reduction in the abundance of all measured OHRB and rdh genes, as compared to the biotic control. High permanganate treatments yielded the development of a notably divergent microbial community, with increased abundances of organisms capable of dissimilatory Mn reduction associated with Campylobacterales and Oceanospirillales and a decrease in the abundance of known supporters of OHRB . Although OTUs classified as syntrophic members of the order Clostridiales and OHRB increased in abundance over the course of 213 days of incubation following the final 100 µmol permanganate treatment, only limited regeneration of PCE biodegradation was observed in one of three microcosms, suggesting that strong chemical oxidation treatments can irreversibly disrupt OHR. Overall, this detailed investigation into microbial community structure changes due to permanganate treatment provides insight into the mechanisms of OHR stimulation or disruption upon chemical oxidation

Microbial dynamics during and after in situ chemical oxidation of chlorinated solvents

In situ chemical oxidation (ISCO) followed by a bioremediation step is increasingly being considered as an effective biphasic technology. Information on the impact of chemical oxidants on organohalide respiring bacteria (OHRB), however, is largely lacking. Therefore, we used quantitative PCR (qPCR) to monitor the abundance of OHRB (Dehalococcoides mccartyi, Dehalobacter, Geobacter, and Desulfitobacterium) and reductive dehalogenase genes (rdh; tceA, vcrA, and bvcA) at a field location contaminated with chlorinated solvents prior to and following treatment with sodium persulfate. Natural attenuation of the contaminants tetrachloroethene (PCE) and trichloroethene (TCE) observed prior to ISCO was confirmed by the distribution of OHRB and rdh genes. In wells impacted by persulfate treatment, a 1 to 3 order of magnitude reduction in the abundances of OHRB and complete absence of rdh genes was observed 21 days after ISCO. Groundwater acidification (pH500 mV) due to persulfate treatment were significant and contributed to disruption of the microbial community. In wells only mildly impacted by persulfate, a slight stimulation of the microbial community was observed, with more than 1 order of magnitude increase in the abundance of Geobacter and Desulfitobacterium 36 days after ISCO. After six months, regeneration of the OHRB community occurred, however, neither D. mccartyi nor any rdh genes were observed, indicating extended disruption of biological natural attenuation (NA) capacity following persulfate treatment. For full restoration of biological NA activity, additional time may prove sufficient; otherwise addition electron donor amendment or bioaugmentation may be required