Correlating the succession of microbial communities from Nigerian soils to petroleum biodegradation.

Whilst biodegradation of different hydrocarbon components has been widely demonstrated to occur by specialist oil-degrading bacteria, less is known about the impact on microbial communities as a function of oil composition by comparing the biodegradation of chemically complex fuels to synthetic products. The objectives of this study were (i) to assess the biodegradation capacity and succession of microbial communities isolated from Nigerian soils in media with crude oil or synthetic oil as sole sources of carbon and energy, and (ii) to assess the temporal variability of the microbial community size. Community profiling was done using 16 S rRNA gene amplicon sequencing (Illumina), and oil profiling using gas chromatography. The biodegradation of natural and synthetic oil differed probably due to the content of sulfur that may interfere with the biodegradation of hydrocarbons. Both alkanes and PAHs in the natural oil were biodegraded faster than in the synthetic oil. Variable community responses were observed during the degradation of alkanes and more simple aromatic compounds, but at later phases of growth they became more homogeneous. The degradation capacity and the size of the community from the more-contaminated soil were higher than those from the less-contaminated soil. Six abundant organisms isolated from the cultures were found to biodegrade oil molecules in pure cultures. Ultimately, this knowledge may contribute to a better understanding of how to improve the biodegradation of crude oil by optimizing culturing conditions through inoculation or bioaugmentation of specific bacteria during ex-situ biodegradation such as biodigesters or landfarming.

Methanotrophy by a Mycobacterium species that dominates a cave microbial ecosystem.

So far, only members of the bacterial phyla Proteobacteria and Verrucomicrobia are known to grow methanotrophically under aerobic conditions. Here we report that this metabolic trait is also observed within the Actinobacteria. We enriched and cultivated a methanotrophic Mycobacterium from an extremely acidic biofilm growing on a cave wall at a gaseous chemocline interface between volcanic gases and the Earth's atmosphere. This Mycobacterium, for which we propose the name Candidatus Mycobacterium methanotrophicum, is closely related to well-known obligate pathogens such as M. tuberculosis and M. leprae. Genomic and proteomic analyses revealed that Candidatus M. methanotrophicum expresses a full suite of enzymes required for aerobic growth on methane, including a soluble methane monooxygenase that catalyses the hydroxylation of methane to methanol and enzymes involved in formaldehyde fixation via the ribulose monophosphate pathway. Growth experiments combined with stable isotope probing using 13C-labelled methane confirmed that Candidatus M. methanotrophicum can grow on methane as a sole carbon and energy source. A broader survey based on 16S metabarcoding suggests that species closely related to Candidatus M. methanotrophicum may be abundant in low-pH, high-methane environments.

Response of Mycobacterium smegmatis to the Cytochrome bcc Inhibitor Q203.

For the design of next-generation tuberculosis chemotherapy, insight into bacterial defence against drugs is required. Currently, targeting respiration has attracted strong attention for combatting drug-resistant mycobacteria. Q203 (telacebec), an inhibitor of the cytochrome bcc complex in the mycobacterial respiratory chain, is currently evaluated in phase-2 clinical trials. Q203 has bacteriostatic activity against M. tuberculosis, which can be converted to bactericidal activity by concurrently inhibiting an alternative branch of the mycobacterial respiratory chain, cytochrome bd. In contrast, non-tuberculous mycobacteria, such as Mycobacterium smegmatis, show only very little sensitivity to Q203. In this report, we investigated factors that M. smegmatis employs to adapt to Q203 in the presence or absence of a functional cytochrome bd, especially regarding its terminal oxidases. In the presence of a functional cytochrome bd, M. smegmatis responds to Q203 by increasing the expression of cytochrome bcc as well as of cytochrome bd, whereas a M. smegmatisbd-KO strain adapted to Q203 by increasing the expression of cytochrome bcc. Interestingly, single-cell studies revealed cell-to-cell variability in drug adaptation. We also investigated the role of a putative second cytochrome bd isoform postulated for M. smegmatis. Although this putative isoform showed differential expression in response to Q203 in the M. smegmatisbd-KO strain, it did not display functional features similar to the characterised cytochrome bd variant.

A Functional Carbohydrate Degrading Enzyme Potentially Acquired by Horizontal Gene Transfer in the Genome of the Soil Invertebrate Folsomia candida.

Horizontal gene transfer (HGT) is defined as the acquisition by an organism of hereditary material from a phylogenetically unrelated organism. This process is mostly observed among bacteria and archaea, and considered less likely between microbes and multicellular eukaryotes. However, recent studies provide compelling evidence of the evolutionary importance of HGT in eukaryotes, driving functional innovation. Here, we study an HGT event in Folsomia candida (Collembola, Hexapoda) of a carbohydrate-active enzyme homologous to glycosyl hydrase group 43 (GH43). The gene encodes an N-terminal signal peptide, targeting the product for excretion, which suggests that it contributes to the diversity of digestive capacities of the detritivore host. The predicted α-L-arabinofuranosidase shows high similarity to genes in two other Collembola, an insect and a tardigrade. The gene was cloned and expressed in Escherichia coli using a cell-free protein expression system. The expressed protein showed activity against p-nitrophenyl-α-L-arabinofuranoside. Our work provides evidence for functional activity of an HGT gene in a soil-living detritivore, most likely from a bacterial donor, with genuine eukaryotic properties, such as a signal peptide. Co-evolution of metazoan GH43 genes with the Panarthropoda phylogeny suggests the HGT event took place early in the evolution of this ecdysozoan lineage.

Correlating biodegradation kinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin to the dynamics of microbial communities originating from soil in Vietnam contaminated with herbicides and dioxins.

We studied the succession of bacterial communities during the biodegradation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). The communities originated from a mesocosm with soil from Bien Hoa airbase in Vietnam heavily contaminated with herbicides and dioxins. They were grown in defined media with different carbon and Gibbs energy sources and 2,3,7,8-TCDD. Cultures with dimethyl sulfoxide (DMSO) as the sole carbon and energy source degraded about 95% of 2,3,7,8-TCDD within 60 days of cultivation. Those with an additional 1 mM of vanillin did that in roughly 90 days. Further 16S rRNA gene amplicon sequencing showed that the increase in relative abundance of members belonging to the genera Bordetella, Sphingomonas, Proteiniphilum, and Rhizobium correlated to increased biodegradation of 2,3,7,8-TCDD in these cultures. A higher concentration of vanillin slowed down the biodegradation rate. Addition of alternative carbon and Gibbs energy sources, such as amino acids, sodium lactate and sodium acetate, even stopped the degradation of 2,3,7,8-TCDD completely. Bacteria from the genera Bordetella, Achromobacter, Sphingomonas and Pseudomonas dominated most of the cultures, but the microbial profiles also significantly differed between cultures as judged by non-metric multidimensional scaling (NMDS) analyses. Our study indicates that 2,3,7,8-TCDD degradation may be stimulated by bacterial communities preadapted to a certain degree of starvation with respect to the carbon and energy source. It also reveals the succession and abundance of defined bacterial genera in the degradation process.

Oil Absorbent Polypropylene Particles Stimulate Biodegradation of Crude Oil by Microbial Consortia.

Oil absorbent particles made from surface-modified polypropylene can be used to facilitate the removal of oil from the environment. In this study, we investigated to what extent absorbed oil was biodegraded and how this compared to the biodegradation of oil in water. To do so, we incubated two bacterial communities originating from the Niger Delta, an area subject to frequent oil spills, in the presence and absence of polypropylene particles. One community evolved from untreated soil whereas the second evolved from soil pre-exposed to oil. We observed that the polypropylene particles stimulated the growth of biofilms and enriched species from genera Mycobacterium, Sphingomonas and Parvibaculum. Cultures with polypropylene particles degraded more crude oil than those where the oil was present in suspension regardless of whether they were pre-exposed or not. Moreover, the community pre-exposed to crude oil had a different community structure and degraded more oil than the one from untreated soil. We conclude that the biodegradation rate of crude oil was enhanced by the pre-exposure of the bacterial communities to crude oil and by the use of oil-absorbing polypropylene materials. The data show that bacterial communities in the biofilms growing on the particles have an enhanced degradation capacity for oil.

Degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) by fungi originating from Vietnam.

Three different fungi were tested for their ability to degrade 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid and for the role of laccases and cytochromes P450-type in this process. We studied a white-rot fungus Rigidoporus sp. FMD21, which has a high laccase activity, for its efficiency to degrade these herbicides. A positive correlation was found between its laccase activity and the corresponding herbicide degradation rate. Even more, the doubling of the enzyme activity in this phase corresponded with a doubling of the herbicide degradation rate. It is, therefore, tempting to speculate that laccase is the most dominant enzyme in the degradation of 2,4-D and 2,4,5-T under these conditions. In addition, it was shown that Rigidoporus sp. FMD21 partly relies on cytochromes P450-type for the breakdown of the herbicides as well. Two filamentous fungi were isolated from soil contaminated with herbicides and dioxins located at Bien Hoa airbase. They belong to genera Fusarium and Verticillium of the phylum Ascomycota as judged by their 18S rRNA gene sequences. Both isolated fungi were able to degrade the herbicides but with different rates. Their laccase activity, however, was very low and did not correlate with the rate of breakdown of the herbicides. These data indicate that the white-rot fungus most likely synthesizes laccase and cytochromes P450-type for the breakdown of the herbicides, while the types of enzyme used for the breakdown of the herbicides by the two Ascomycota remain unclear.

Adaptive changes of sediment microbial communities associated with cleanup of oil spills in Nigerian mangrove forests.

The objectives of this study were to assess the influence on microbial communities resulting from i) the physical removal of free oil (pre-treatment or post-treatment), and ii) the level of oiling within a contaminated former mangrove forest. Sediment samples were collected before and after the removal of free oil. Before the process of remediation, a highly biodiverse mangrove microbiome which had adapted to history of recurring oil spills was observed. After removing the surface oil, the microbial diversity of the sediments reduced, with members of the phyla Firmicutes and Proteobacteria becoming dominant. This indicates that while water flushing reduced overall microbial diversity, it stimulated the growth of a more specialized bacterial community reported to be involved in hydrocarbon biodegradation. These results provide new insights on microbial communities and their succession in mangrove forest sediments, that will be useful for monitoring oil cleaning programs using water flushing to remove free oil.

Imaging and modelling of poly(3-hydroxybutyrate) synthesis in Paracoccus denitrificans.

Poly(3-hydroxybutyrate) (PHB) granule formation in Paracoccus denitrificans Pd1222 was investigated by laser scanning confocal microscopy (LSCM) and gas chromatography analysis. Cells that had been starved for 2 days were free of PHB granules but resynthesized them within 30 min of growth in fresh medium with succinate. In most cases, the granules were distributed randomly, although in some cases they appeared in a more organized pattern. The rates of growth and PHB accumulation were analyzed within the frame of a Genome-Scale Metabolic Model (GSMM) containing 781 metabolic genes, 1403 reactions and 1503 metabolites. The model was used to obtain quantitative predictions of biomass yields and PHB synthesis during aerobic growth on succinate as sole carbon and energy sources. The results revealed an initial fast stage of PHB accumulation, during which all of the acetyl-CoA originating from succinate was diverted to PHB production. The next stage was characterized by a tenfold lower PHB production rate and the simultaneous onset of exponential growth, during which acetyl-CoA was predominantly drained into the TCA cycle. Previous research has shown that PHB accumulation correlates with cytosolic acetyl-CoA concentration. It has also been shown that PHB accumulation is not transcriptionally regulated. Our results are consistent with the mentioned findings and suggest that, in absence of cell growth, most of the cellular acetyl-CoA is channeled to PHB synthesis, while during exponential growth, it is drained to the TCA cycle, causing a reduction of the cytosolic acetyl-CoA pool and a concomitant decrease of the synthesis of acetoacetyl-CoA (the precursor of PHB synthesis).

High biodiversity in a benzene-degrading nitrate-reducing culture is sustained by a few primary consumers.

A key question in microbial ecology is what the driving forces behind the persistence of large biodiversity in natural environments are. We studied a microbial community with more than 100 different types of species which evolved in a 15-years old bioreactor with benzene as the main carbon and energy source and nitrate as the electron acceptor. Using genome-centric metagenomics plus metatranscriptomics, we demonstrate that most of the community members likely feed on metabolic left-overs or on necromass while only a few of them, from families Rhodocyclaceae and Peptococcaceae, are candidates to degrade benzene. We verify with an additional succession experiment using metabolomics and metabarcoding that these few community members are the actual drivers of benzene degradation. As such, we hypothesize that high species richness is maintained and the complexity of a natural community is stabilized in a controlled environment by the interdependencies between the few benzene degraders and the rest of the community members, ultimately resulting in a food web with different trophic levels.

Effects of DNA preservation solution and DNA extraction methods on microbial community profiling of soil.

Microbial community profiling using high-throughput sequencing relies in part on the preservation of the DNA and the effectiveness of the DNA extraction method. This study aimed at understanding to what extent these parameters affect the profiling. We obtained samples treated with and without a preservation solution. Also, we compared DNA extraction kits from Qiagen and Zymo-Research. The types of samples were defined strains, both as single species and mixtures, as well as undefined indigenous microbial communities from soil. We show that the use of a preservation solution resulted in substantial changes in the 16S rRNA gene profiles either due to an overrepresentation of Gram-positive bacteria or to an underrepresentation of Gram-negative bacteria. In addition, 16S rRNA gene profiles were substantially different depending on the type of kit that was used for extraction. The kit from Zymo extracted DNA from different types of bacteria in roughly equal amounts. In contrast, the kit from Qiagen preferentially extracted DNA from Gram-negative bacteria while DNA from Gram-positive bacteria was extracted less effectively. These differences in kit performance strongly influenced the interpretation of our microbial ecology studies.

Long-term exposure of activated sludge in chemostats leads to changes in microbial communities composition and enhanced biodegradation of 4-chloroaniline and N-methylpiperazine.

Exposure history and adaptation of the inoculum to chemicals have been shown to influence the outcome of ready biodegradability tests. However, there is a lack of information about the mechanisms involved in microbial adaptation and the implication thereof for the tests. In the present study, we investigated the impact of a long-term exposure to N-methylpiperazine (NMP) and 4-chloroaniline (4CA) of an activated sludge microbial community using chemostat systems. The objective was to characterize the influence of adaptation to the chemicals on an enhanced biodegradation testing, following the OECD 310 guideline. Cultures were used to inoculate the enhanced biodegradability tests, in batch, before and after exposure to each chemical independently in chemostat culture. Composition and diversity of the microbial communities were characterised by 16s rRNA gene amplicon sequencing. Using freshly sampled activated sludge, NMP was not degraded within the 28 d frame of the test while 4CA was completely eliminated. However, after one month of exposure, the community exposed to NMP was adapted and could completely degrade it. This result was in complete contrast with that from the culture exposed for 3 months to 4CA. Long term incubation in the chemostat system led to a progressive loss of the initial biodegradation capacity of the community, as a consequence of the loss of key degrading microorganisms. This study highlights the potential of chemostat systems to induce adaptation to a specific chemical, ultimately resulting in its biodegradation. At the same time, one should be critical of these observations as the dynamics of a microbial community are difficult to maintain in chemostat, as the loss of 4CA biodegradation capacity demonstrates.

Natural Microbial Communities Can Be Manipulated by Artificially Constructed Biofilms.

Biofouling proceeds in successive steps where the primary colonizers affect the phylogenetic and functional structure of a future microbial consortium. Using microbiologically influenced corrosion (MIC) as a study case, a novel approach for material surface protection is described, which does not prevent biofouling, but rather shapes the process of natural biofilm development to exclude MIC-related microorganisms. This approach interferes with the early steps of natural biofilm formation affecting how the community is finally developed. It is based on a multilayer artificial biofilm, composed of electrostatically modified bacterial cells, producing antimicrobial compounds, extracellular antimicrobial polyelectrolyte matrix, and a water-proof rubber elastomer barrier. The artificial biofilm is constructed layer-by-layer (LBL) by manipulating the electrostatic interactions between microbial cells and material surfaces. Field testing on standard steel coupons exposed in the sea for more than 30 days followed by laboratory analyses using molecular-biology tools demonstrate that the preapplied artificial biofilm affects the phylogenetic structure of the developing natural biofilm, reducing phylogenetic diversity and excluding MIC-related bacteria. This sustainable solution for material protection showcases the usefulness of artificially guiding microbial evolutionary processes via the electrostatic modification and controlled delivery of bacterial cells and extracellular matrix to the exposed material surfaces.

Dynamic Metabolic Rewiring Enables Efficient Acetyl Coenzyme A Assimilation in Paracoccus denitrificans.

During growth, microorganisms have to balance metabolic flux between energy and biosynthesis. One of the key intermediates in central carbon metabolism is acetyl coenzyme A (acetyl-CoA), which can be either oxidized in the citric acid cycle or assimilated into biomass through dedicated pathways. Two acetyl-CoA assimilation strategies in bacteria have been described so far, the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). Here, we show that Paracoccus denitrificans uses both strategies for acetyl-CoA assimilation during different growth stages, revealing an unexpected metabolic complexity in the organism's central carbon metabolism. The EMCP is constitutively expressed on various substrates and leads to high biomass yields on substrates requiring acetyl-CoA assimilation, such as acetate, while the GC is specifically induced on these substrates, enabling high growth rates. Even though each acetyl-CoA assimilation strategy alone confers a distinct growth advantage, P. denitrificans recruits both to adapt to changing environmental conditions, such as a switch from succinate to acetate. Time-resolved single-cell experiments show that during this switch, expression of the EMCP and GC is highly coordinated, indicating fine-tuned genetic programming. The dynamic metabolic rewiring of acetyl-CoA assimilation is an evolutionary innovation by P. denitrificans that allows this organism to respond in a highly flexible manner to changes in the nature and availability of the carbon source to meet the physiological needs of the cell, representing a new phenomenon in central carbon metabolism.IMPORTANCE Central carbon metabolism provides organisms with energy and cellular building blocks during growth and is considered the invariable "operating system" of the cell. Here, we describe a new phenomenon in bacterial central carbon metabolism. In contrast to many other bacteria that employ only one pathway for the conversion of the central metabolite acetyl-CoA, Paracoccus denitrificans possesses two different acetyl-CoA assimilation pathways. These two pathways are dynamically recruited during different stages of growth, which allows P. denitrificans to achieve both high biomass yield and high growth rates under changing environmental conditions. Overall, this dynamic rewiring of central carbon metabolism in P. denitrificans represents a new strategy compared to those of other organisms employing only one acetyl-CoA assimilation pathway.

Biodegradation of metformin and its transformation product, guanylurea, by natural and exposed microbial communities.

Metformin (MET) is a pharmaceutical product mostly biotransformed in the environment to a transformation product, guanylurea (GUA). In ready biodegradability tests (RBTs), however, contrasting results have been observed for metformin. The objective of this study was to measure the biodegradation of MET and GUA in RBTs, using activated sludge from the local wastewater treatment plant, either directly or after pre-exposure to MET, in a chemostat. The activated sludge community was cultivated in chemostats, in presence or absence of MET, for a period of nine months, and was used in RBT after one, three and nine months. The results of this study showed that the original activated sludge was able to completely remove MET (15 mg/l) and the newly produced GUA (50% of C0MET) under the test conditions. Inoculation of the chemostat led to a rapid shift in the community composition and abundance. The community exposed to 1.5 mg/l of MET was still able to completely consume MET in the RBTs after one-month exposure, but three- and nine-months exposure resulted in reduced removal of MET in the RBTs. The ability of the activated sludge community to degrade MET and GUA is the result of environmental exposure to these chemicals as well as of conditions that could not be reproduced in the laboratory system. A MET-degrading strain belonging to the genus Aminobacter has been isolated from the chemostat community. This strain was able to completely consume 15 mg/l of MET within three days in the test. However, community analysis revealed that the fluctuation in relative abundance of this genus (<1%) could not be correlated to the fluctuation in biodegradation capacity of the chemostat community.

Circular Spectropolarimetric Sensing of Vegetation in the Field: Possibilities for the Remote Detection of Extraterrestrial Life.

Homochirality is a generic and unique property of all biochemical life, and the fractional circular polarization of light it induces therefore constitutes a potentially unambiguous biosignature. However, while high-quality circular polarimetric spectra can be easily and quickly obtained in the laboratory, accurate measurements in the field are much more challenging due to large changes in illumination and target movement. In this study, we measured various targets in the field, up to distances of a few kilometers, using the dedicated circular spectropolarimeter TreePol. We show how photosynthetic life can readily be distinguished from abiotic matter. We underline the potential of circular polarization signals as a remotely accessible means to characterize and monitor terrestrial vegetation, for example, for agriculture and forestry. In addition, we discuss the potential of circular polarization for the remote detection of extraterrestrial life.

Microbial Communities in Sediments From Four Mildly Acidic Ephemeral Salt Lakes in the Yilgarn Craton (Australia) - Terrestrial Analogs to Ancient Mars.

The Yilgarn Craton in Australia has a large number of naturally occurring shallow ephemeral lakes underlain by a dendritic system of paleodrainage channels. Processes like evaporation, flooding, erosion, as well as inflow of saline, often acidic and ion-rich groundwater contribute to the (dynamic) nature of the lakes and the composition of the sediments. The region has previously been described as an analog environment for early Mars due to its geological and geophysical similarities. Here, we investigated sediment samples of four lake environments aimed at getting a fundamental understanding of the native microbial communities and the mineralogical and (bio)chemical composition of the sediments they are associated with. The dominant mineral phases in the sediments were quartz, feldspars and amphiboles, while halite and gypsum were the only evaporites detected. Element analysis revealed a rich and complex image, in which silicon, iron, and aluminum were the dominant ions, but relative high concentrations of trace elements such as strontium, chromium, zirconium, and barium were also found. The concentrations of organic carbon, nitrogen, and phosphorus were generally low. 16S amplicon sequencing on the Illumina platform showed the presence of diverse microbial communities in all four lake environments. We found that most of the communities were dominated by extremely halophilic Archaea of the Halobacteriaceae family. The dynamic nature of these lakes appears to influence the biological, biochemical, and geological components of the ecosystem to a large effect. Inter- and intra-lake variations in the distributions of microbial communities were significant, and could only to a minor degree be explained by underlying environmental conditions. The communities are likely significantly influenced by small scale local effects caused by variations in geological settings and dynamic interactions caused by aeolian transport and flooding and evaporation events.

Circular spectropolarimetric sensing of higher plant and algal chloroplast structural variations.

Photosynthetic eukaryotes show a remarkable variability in photosynthesis, including large differences in light-harvesting proteins and pigment composition. In vivo circular spectropolarimetry enables us to probe the molecular architecture of photosynthesis in a non-invasive and non-destructive way and, as such, can offer a wealth of physiological and structural information. In the present study, we have measured the circular polarizance of several multicellular green, red, and brown algae and higher plants, which show large variations in circular spectropolarimetric signals with differences in both spectral shape and magnitude. Many of the algae display spectral characteristics not previously reported, indicating a larger variation in molecular organization than previously assumed. As the strengths of these signals vary by three orders of magnitude, these results also have important implications in terms of detectability for the use of circular polarization as a signature of life.

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

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 (21-36% 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.