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Zoogloea oleivorans, capable of using toluene as a sole source of carbon and energy, was earlier found to be an active degrader under microaerobic conditions in aquifer samples. To uncover the genetic background of the ability of microaerobic toluene degradation in Z. oleivorans, the whole-genome sequence of the type strain Buc T was revealed. Metatranscriptomic sequence reads, originated from a previous SIP study on microaerobic toluene degradation, were mapped on the genome. The genome (5.68 Mb) had a mean G + C content of 62.5%, 5005 protein coding gene sequences and 80 RNA genes. Annotation predicted that 66 genes were involved in the metabolism of aromatic compounds. Genome analysis revealed the presence of a cluster with genes coding for a multicomponent phenol-hydroxylase system and a complete catechol meta-cleavage pathway. Another cluster flanked by mobile-element protein coding genes coded a partial catechol meta-cleavage pathway including a subfamily I.2.C-type extradiol dioxygenase. Analysis of metatranscriptomic data of a microaerobic toluene-degrading enrichment, containing Z .  oleivorans as an active-toluene degrader revealed that a toluene dioxygenase-like enzyme was responsible for the ring-hydroxylation, while enzymes of the partial catechol meta-cleavage pathway coding cluster were responsible for further degradation of the aromatic ring under microaerobic conditions. This further advances our understanding of aromatic hydrocarbon degradation between fully oxic and strictly anoxic conditions.

The availability of oxygen is often a limiting factor for the degradation of aromatic hydrocarbons in subsurface environments. However, while both aerobic and anaerobic degraders have been intensively studied, degradation betwixt, under micro- or hypoxic conditions has rarely been addressed. It is speculated that in environments with limited, but sustained oxygen supply, such as in the vicinity of groundwater monitoring wells, hypoxic degradation may take place. A large diversity of subfamily I.2.C extradiol dioxygenase genes has been previously detected in a BTEX-contaminated aquifer in Hungary. Older literature suggests that such catabolic potentials could be associated to hypoxic degradation. Bacterial communities dominated by members of the Rhodocyclaceae were found, but the majority of the detected C23O genotypes could not be affiliated to any known bacterial degrader lineages. To address this, a stable isotope probing (SIP) incubation of site sediments with 13C7-toluene was performed under microoxic conditions. A combination of 16S rRNA gene amplicon sequencing and T-RFLP fingerprinting of C23O genes from SIP gradient fractions revealed the central role of degraders within the Rhodocyclaceae in hypoxic toluene degradation. The main assimilators of 13C were identified as members of the genera Quatrionicoccus and Zoogloea, and a yet uncultured group of the Rhodocyclaceae.

Microbial communities are the driving force behind the degradation of contaminants like aromatic hydrocarbons in groundwater ecosystems. However, little is known about the response of native microbial communities to contamination in pristine environments as well as their potential to recover from a contamination event. Here, we used an indoor aquifer mesocosm filled with sandy quaternary calciferous sediment that was continuously fed with pristine groundwater to study the response, resistance and resilience of microbial communities to toluene contamination over a period of almost two years, comprising 132days of toluene exposure followed by nearly 600days of recovery. We observed an unexpectedly high intrinsic potential for toluene degradation, starting within the first two weeks after the first exposure. The contamination led to a shift from oxic to anoxic, primarily nitrate-reducing conditions as well as marked cell growth inside the contaminant plume. Depth-resolved community fingerprinting revealed a low resistance of the native microbial community to the perturbation induced by the exposure to toluene. Distinct populations that were dominated by a small number of operational taxonomic units (OTUs) rapidly emerged inside the plume and at the plume fringes, partially replacing the original community. During the recovery period physico-chemical conditions were restored to the pristine state within about 35days, whereas the recovery of the biological parameters was much slower and the community composition inside the former plume area had not recovered to the original state by the end of the experiment. These results demonstrate the low resilience of sediment-associated groundwater microbial communities to organic pollution and underline that recovery of groundwater ecosystems cannot be assessed solely by physico-chemical parameters., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Hydrocarbon contaminants in groundwater can be degraded by microbes under different redox settings, forming hot spots of degradation especially at the fringes of contaminant plumes. At a tar-oil-contaminated aquifer in Germany, it was previously shown that the distribution of anaerobic toluene degraders as traced via catabolic and ribosomal marker genes is highly correlated to zones of increased anaerobic degradation at the lower fringe of the plume. Here, we trace the respective distribution of aerobic toluene degraders over a fine-scale depth transect of sediments taken at the upper fringe of the plume and below, based on the analysis of 16S rRNA genes as well as catabolic markers in intervals of 3-10 cm. Well-defined small-scale distribution maxima of typical aerobic degrader lineages within the Pseudomonadaceae, Comamonadaceae and Burkholderiaceae are revealed over the redox gradient. An unexpected maximal abundance of 9.2 × 10⁶ toluene monooxygenase (tmoA) genes per g of sediment was detected in the strongly reduced plume core, and gene counts did not increase towards the more oxidized upper plume fringe. This may point towards unusual ecological controls of these yet unidentified aerobic degraders, and indicates that competitive niche partitioning between aerobic and anaerobic hydrocarbon degraders in the field is not yet fully understood. These findings demonstrate the potential of catabolic marker gene assays in elaborating the ecology of contaminant plumes, which is a prerequisite for developing integrated monitoring strategies for natural attenuation., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Bioavailability of electron acceptors is probably the most limiting factor in the restoration of anoxic, contaminated environments. The oxidation of contaminants such as aromatic hydrocarbons, particularly in aquifers, often depends on the reduction of ferric iron or sulphate. We have previously detected a highly active fringe zone beneath a toluene plume at a tar-oil-contaminated aquifer in Germany, where a specialized community of contaminant degraders codominated by Desulfobulbaceae and Geobacteraceae had established. Although on-site geochemistry links degradation to sulphidogenic processes, dominating catabolic (benzylsuccinate synthase α-subunit, bssA) genes detected in situ appeared to be more related to those of Geobacter spp. Therefore, a stable isotope probing (SIP) incubation of sediment samples with (13)C(7)-toluene and comparative electron acceptor amendment was performed. We introduce pyrosequencing of templates from SIP microcosms as a powerful new strategy in SIP gradient interpretation (Pyro-SIP). Our results reveal the central role of Desulfobulbaceae in sulphidogenic toluene degradation in situ, and affiliate the detected bssA genes to this lineage. This and the absence of (13)C-labelled DNA of Geobacter spp. in SIP gradients preclude their relevance as toluene degraders in situ. In contrast, Betaproteobacteria related to Georgfuchsia spp. became labelled under iron-reducing conditions. Furthermore, secondary toluene degraders belonging to the Peptococcaceae detected in both treatments suggest the possibility of functional redundancy among anaerobic toluene degraders on site., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Global groundwater resources are constantly challenged by a multitude of contaminants such as aromatic hydrocarbons. Especially in anaerobic habitats, a large diversity of unrecognized microbial populations may be responsible for their degradation. Still, our present understanding of the respective microbiota and their ecophysiology is almost exclusively based on a small number of cultured organisms, mostly within the Proteobacteria. Here, by DNA-based stable isotope probing (SIP), we directly identified the most active sulfate-reducing toluene degraders in a diverse sedimentary microbial community originating from a tar-oil-contaminated aquifer at a former coal gasification plant. On incubation of fresh sediments with (13)C(7)-toluene, the production of both sulfide and (13)CO(2) was clearly coupled to the (13)C-labeling of DNA of microbes related to Desulfosporosinus spp. within the Peptococcaceae (Clostridia). The screening of labeled DNA fractions also suggested a novel benzylsuccinate synthase alpha-subunit (bssA) sequence type previously only detected in the environment to be tentatively affiliated with these degraders. However, carbon flow from the contaminant into degrader DNA was only ∼50%, pointing toward high ratios of heterotrophic CO(2)-fixation during assimilation of acetyl-CoA originating from the contaminant by these degraders. These findings demonstrate that the importance of non-proteobacterial populations in anaerobic aromatics degradation, as well as their specific ecophysiology in the subsurface may still be largely ungrasped.

A toluene-degrading microbial consortium was enriched directly in a BTEX-contaminated aquifer under sulfate-reducing conditions using in situ microcosms consisting of toluene-loaded activated carbon pellets. Degradation of toluene and concomitant sulfide production by the consortium was subsequently demonstrated in laboratory microcosms. The consortium was physiologically and phylogenetically characterized by isotope tracer experiments using nonlabeled toluene, [(13)C]-alpha-toluene or [(13)C(7)]-toluene as growth substrates. Cells incubated with [(13)C]-alpha-toluene or [(13)C(7)]-toluene incorporated 8-15 at.%(13)C and 51-57 at.%(13)C into total lipid fatty acids, respectively, indicating a lower specific incorporation of (13)C from [(13)C(7)]-toluene. In order to identify the toluene-assimilating bacteria, the incorporation of carbon from both [(13)C]-alpha-toluene and [(13)C(7)]-toluene into rRNA was analyzed by stable isotope probing. Time and buoyant density-resolved 16S rRNA gene-based terminal restriction fragment length polymorphism profiles, combined with cloning and sequencing, revealed that an uncultured bacterium (99% sequence similarity) related to the genus Desulfocapsa was the main toluene-degrading organism in the consortium. The ratio of the respective terminal restriction fragments changed over time, indicating trophic interactions within this consortium.

Microbial degradation is the only sustainable component of natural attenuation in contaminated groundwater environments, yet its controls, especially in anaerobic aquifers, are still poorly understood. Hence, putative spatial correlations between specific populations of key microbial players and the occurrence of respective degradation processes remain to be unraveled. We therefore characterized microbial community distribution across a high-resolution depth profile of a tar oil-impacted aquifer where benzene, toluene, ethylbenzene, and xylene (BTEX) degradation depends mainly on sulfate reduction. We conducted depth-resolved terminal restriction fragment length polymorphism fingerprinting and quantitative PCR of bacterial 16S rRNA and benzylsuccinate synthase genes (bssA) to quantify the distribution of total microbiota and specific anaerobic toluene degraders. We show that a highly specialized degrader community of microbes related to known deltaproteobacterial iron and sulfate reducers (Geobacter and Desulfocapsa spp.), as well as clostridial fermenters (Sedimentibacter spp.), resides within the biogeochemical gradient zone underneath the highly contaminated plume core. This zone, where BTEX compounds and sulfate--an important electron acceptor--meet, also harbors a surprisingly high abundance of the yet-unidentified anaerobic toluene degraders carrying the previously detected F1-cluster bssA genes (C. Winderl, S. Schaefer, and T. Lueders, Environ. Microbiol. 9:1035-1046, 2007). Our data suggest that this biogeochemical gradient zone is a hot spot of anaerobic toluene degradation. These findings show that the distribution of specific aquifer microbiota and degradation processes in contaminated aquifers are tightly coupled, which may be of value for the assessment and prediction of natural attenuation based on intrinsic aquifer microbiota.

Benzylsuccinate synthase (Bss) is the key enzyme of anaerobic toluene degradation and has been found in all anaerobic toluene degrading bacterial isolates tested. However, only a few pure cultures capable of anaerobic toluene oxidation are available to date, and it is important to understand the relevance of these model organisms for in situ bioremediation of hydrocarbon-contaminated aquifers. Due to their phylogenetic dispersal, it is not possible to specifically target anaerobic toluene degraders using marker rRNA genes. We therefore established an assay targeting a approximately 794 bp fragment within the Bss alpha-subunit (bssA) gene, which allows for the specific detection and affiliation of both known and unknown anaerobic degraders. Three distinct tar-oil-contaminated aquifer sites were screened for intrinsic bssA gene pools in order to identify and compare the diversity of hydrocarbon degraders present at these selected sites. We were able to show that local diversity patterns of degraders were entirely distinct, apparently highly specialized and well-adapted to local biogeochemical settings. Discovered at one of the sites were bssA genes closely related to that of Geobacter spp., which provides evidence for an importance of iron reduction for toluene degradation in these sediments. Retrieved from the other two sites, dominated by sulfate reduction, were previously unidentified bssA genes and also deeply branching putative bssA homologues. We provide evidence for a previously unrecognized diversity of anaerobic toluene degraders and also of other hydrocarbon degraders using fumarate-adding key reactions in contaminated aquifers. These findings enhance our current understanding of intrinsic hydrocarbon-degrading microbial communities in perturbed aquifers and may have potential for the future assessment and prediction of natural attenuation based on degradation genes.

Microorganisms are essential in the degradation of environmental pollutants. Aromatic hydrocarbons, e.g., benzene, toluene, ethylbenzene, and xylene (BTEX), are common aquifer contaminants, whose degradation in situ is often limited by the availability of electron acceptors. It is clear that different electron acceptors such as nitrate, iron, or sulfate support the activity of distinct degraders. However, this has not been demonstrated for the availability of nitrate vs. nitrite, both of which can be respired in reductive nitrogen cycling. Here via DNA-stable isotope probing, we report that nitrate and nitrite provided as electron acceptors in different concentrations and ratios not only modulated the microbial communities responsible for toluene degradation but also influenced how nitrate reduction proceeded. Zoogloeaceae members, mainly Azoarcus spp., were the key toluene degraders with nitrate-only, or both nitrate and nitrite as electron acceptors. In addition, a shift within Azoarcus degrader populations was observed on the amplicon sequence variant (ASV) level depending on electron acceptor ratios. In contrast, members of the Sphingomonadales were likely the most active toluene degraders when only nitrite was provided. Nitrate reduction did not proceed beyond nitrite in the nitrate-only treatment, while it continued when nitrite was initially also present in the microcosms. Likely, this was attributed to the fact that different microbial communities were stimulated and active in different microcosms. Together, these findings demonstrate that the availability of nitrate and nitrite can define degrader community selection and N-reduction outcomes. It also implies that nitrate usage efficiency in bioremediation could possibly be enhanced by an initial co-supply of nitrite, via modulating the active degrader communities. [ABSTRACT FROM AUTHOR]

The degradation of benzene, toluene, ethylbenzene and xylene (BTEX) contaminants in groundwater relies largely on anaerobic processes. While the physiology and biochemistry of selected relevant microbes have been intensively studied, research has now started to take the generated knowledge back to the field, in order to trace the populations truly responsible for the anaerobic degradation of BTEX hydrocarbons in situ and to unravel their ecology in contaminated aquifers. Here, recent advances in our knowledge of the identity, diversity and ecology of microbes involved in these important ecosystem services are discussed. At several sites, distinct lineages within the Desulfobulbaceae, the Rhodocyclaceae and the Gram-positive Peptococcaceae have been shown to dominate the degradation of different BTEX hydrocarbons. Especially for the functional guild of anaerobic toluene degraders, specific molecular detection systems have been developed, allowing researchers to trace their diversity and distribution in contaminated aquifers. Their populations appear enriched in hot spots of biodegradation in situ. 13C-labelling experiments have revealed unexpected pathways of carbon sharing and obligate syntrophic interactions to be relevant in degradation. Together with feedback mechanisms between abiotic and biotic habitat components, this promotes an enhanced ecological perspective of the anaerobic degradation of BTEX hydrocarbons, as well as its incorporation into updated concepts for site monitoring and bioremediation. [ABSTRACT FROM AUTHOR]