Krumholzibacteriota and Deltaproteobacteria contain rare genetic potential to liberate carbon from monoaromatic compounds in subsurface coal seams.

Biogenic methane in subsurface coal seam environments is produced by diverse consortia of microbes. Although this methane is useful for global energy security, it remains unclear which microbes can liberate carbon from the coal. Most of this carbon is relatively resistant to biodegradation, as it is contained within aromatic rings. Thus, to explore for coal-degrading taxa in the subsurface, this study reconstructed relevant metagenome-assembled genomes (MAGs) from coal seams by using a key genomic marker for the anaerobic degradation of monoaromatic compounds as a guide: the benzoyl-CoA reductase gene (bcrABCD). Three MAGs were identified with this genetic potential. The first represented a novel taxon from the Krumholzibacteriota phylum, which this study is the first to describe. This Krumholzibacteriota MAG contained a full set of genes for benzoyl-CoA dearomatization, in addition to other genes for anaerobic catabolism of monoaromatics. Analysis of Krumholzibacteriota MAGs from other environments revealed that this genetic potential may be common, and thus, Krumholzibacteriota may be important organisms for the liberation of recalcitrant carbon in a broad range of environments. Moreover, the assembly and characterization of two Syntrophorhabdus aromaticivorans MAGs from different continents and a Syntrophaceae sp. MAG implicate the Deltaproteobacteria class in coal seam monoaromatic degradation. Each of these taxa are potential rate-limiting organisms for subsurface coal-to-methane biodegradation. Their description here provides some understanding of their function within the coal seam microbiome and will help inform future efforts in coal bed methane stimulation, anoxic bioremediation of organic pollutants, and assessments of anoxic, subsurface carbon cycling and emissions.IMPORTANCESubsurface coal seams are highly anoxic, oligotrophic environments, where the main source of carbon is "locked away" within aromatic rings. Despite these challenges, many coal seams accumulate biogenic methane, implying that the coal seam microbiome is "unlocking" this carbon source in situ. For over two decades, researchers have endeavored to understand which organisms perform these processes. This study provides the first descriptions of organisms with this genetic potential from the coal seam environment. Here, we report metagenomic insights into carbon liberation from aromatic molecules and the degradation pathways involved and describe a Krumholzibacteriota, two Syntrophorhabdus aromaticivorans, and a Syntrophaceae MAG that contain this genetic potential. This is also the first time that the Krumholzibacteriota phylum has been implicated in anaerobic dearomatization of aromatic hydrocarbons. This potential is identified here in numerous MAGs from other terrestrial and marine subsurface habitats, implicating the Krumholzibacteriota in carbon-cycling processes across a broad range of environments.

Methanogenic archaea in subsurface coal seams are biogeographically distinct: an analysis of metagenomically-derived mcrA sequences.

The production of methane as an end-product of organic matter degradation in the absence of other terminal electron acceptors is common, and has often been studied in environments such as animal guts, soils and wetlands due to its potency as a greenhouse gas. To date, however, the study of the biogeographic distribution of methanogens across coal seam environments has been minimal. Here, we show that coal seams are host to a diverse range of methanogens, which are distinctive to each geological basin. Based on comparisons to close relatives from other methanogenic environments, the dominant methanogenic pathway in these basins is hydrogenotrophic, with acetoclastic being a second major pathway in the Surat Basin. Finally, mcrA and 16S rRNA gene primer biases were predominantly seen to affect the detection of Methanocellales, Methanomicrobiales and Methanosarcinales taxa in this study. Subsurface coal methanogenic community distributions and pathways presented here provide insights into important metabolites and bacterial partners for in situ coal biodegradation.

Microbial Community Shifts on Organic Rocks of Different Maturities Reveal potential Catabolisers of Organic Matter in Coal.

The global trend of transiting to more renewable energy sources requires transition fuels, such as coal seam gas, to supplement and secure energy needs. In order to optimise strategies and technologies for enhancing gas production, an understanding of the fundamental microbial processes and interactions would be advantageous. Models have recently begun mapping the microbial roles and interactions in coal seam environments, from direct coal degradation to methanogenesis. This study seeks to expand those models by observing community compositional shifts in the presence of differing organic matter by conducting 16S rRNA microbial surveys using formation water from the Surat and Sydney Basins grown on varying types of organic matter (black and brown coal, oil shale, humic acid, and lignin). A total of 135 microbes were observed to become enriched in the presence of added organic matter in comparison to carbon-free treatments. These surveys allowed detailed analysis of microbial compositions in order to extrapolate which taxa favour growth in the presence of differing organic matter. This study has experimentally demonstrated shifts in the microbial community composition due to differing carbon sources and, for the first time, generated a conceptual model to map putative degradation pathways regarding subsurface microbial consortia.

The characterisation and provenance of crude oils stranded on the South Australian coastline. Part II: Potential parent petroleum systems.

In 2014-2016 more than 600 specimens of semi-solid crude oil were recovered from 30 ocean beaches along the coastline of South Australia, as part of the recently completed Great Australian Bight Research Program. All are believed to be products of submarine oil seepage. Their source-specific biomarker signatures provide the basis for their assignment to sixteen oil families, some previously unrecognised. Two of these families (asphaltite and asphaltic tar) likely originated from Cretaceous marine source rocks in the offshore Bight Basin. The others comprise waxy oils of lacustrine, fluvio-deltaic and marine source affinity. Their biomarker characteristics do not match those of any Australian crude oil. However, they are strikingly similar to those of oils found in Cenozoic and Mesozoic basins throughout the Indonesian Archipelago and elsewhere in Southeast Asia.

Aromatic compound-degrading taxa in an anoxic coal seam microbiome from the Surat Basin, Australia.

Methane is an important energy resource internationally, and a large proportion of this methane is produced by microbial communities living in coal seams. Despite the value of this resource for human energy security, our understanding of the metabolic roles played by specific taxa during the biodegradation of coal to methane in situ is quite limited. In order to develop a greater understanding of microbial catabolism on coal, a community from a coal seam in the Surat Basin, Australia, was incubated on 10 different aromatic organic compounds: coronene, benzo[a]pyrene, pyrene, phenanthrene, naphthalene, ethylbenzene, phenol, benzoate, vanillate and syringate. Each of these aromatic compounds either occurs in coal or is a possible product of the coal biodegradation process. 16S rRNA sequencing revealed substantial changes to each community in response to each aromatic carbon substrate provided. Abundant taxa from these substrate-specific communities were identified and their probable catabolic roles proposed based on literature searches of related taxa. This study is the first to link specific coal seam taxa to aromatic substrates available in coal seam environments. Two conceptual models of the putative degradation pathways and key taxa responsible are proposed.

The characterisation and provenance of crude oils stranded on the South Australian coastline. Part I: Oil types and their weathering.

Semi-solid crude oil has been known to wash ashore along the South Australian coastline for over 120 years. The early reports pre-date offshore petroleum exploration and tanker shipping activities in Australian waters, suggesting that this stranded oil originates from natural offshore seepage. Three physically distinct varieties are represented: waxy bitumen, asphaltite and tar. In order to distinguish this natural "background" contamination of the coastline from any potential anthropogenic sources of petroleum, such as oil spills, whole-oil GC-MS analysis was employed to identify at least seven geochemically different types of stranded oil, based on a suite of 633 specimens collected from the coastline during three annual surveys of 30 ocean beaches between 2014 and 2016. The waxy bitumens, which in terms of their biomarker alkanes display an atypical pattern of alteration due to weathering in the marine environment, are more severely altered than similar specimens collected 25 years ago.

The natural hydrocarbon loading of the South Australian coastline.

Crude oil released from natural offshore seeps may strand in coastal environments. Understanding the different types of oil which accumulate on a given coastline, in addition to their spatial distribution and abundance, may be used to establish an environmental baseline for natural "background" petroleum contamination. Here we summarise the hydrocarbon loading of thirty beaches on Australia's southern margin based on three annual surveys in 2014-2016. Comparison with the results of surveys conducted in 1990 and 1991 reveals a marked reduction in hydrocarbon loading. Furthermore, modern samples of the most commonly encountered oil, attributed to a lacustrine petroleum system in the Indonesian Archipelago, are significantly more degraded than those of prior studies. We attribute this reduction in hydrocarbon loading to prolonged oil production in Southeast Asia, which in turn results in reduced reservoir pressures and the eventual cessation of formerly active offshore seepage.

Who eats what? Unravelling microbial conversion of coal to methane.

Microbial communities in subsurface coal seams are responsible for the conversion of coal organic matter to methane. This process has important implications for both energy production and our understanding of global carbon cycling. Despite the environmental and economic importance of this process, little is known about which components of the heterogeneous coal organic matter are biodegradable under methanogenic conditions. Similarly, little is known about which taxa in coal seams carry out the initial stages of coal organics degradation. To identify the biodegradable components of coal and the microorganisms responsible for their breakdown, a subbituminous coal was fractionated into a number of chemical compound classes which were used as the sole carbon source for growth by a coal seam microbial community. This study identifies 65 microbial taxa able to proliferate on specific coal fractions and demonstrates a surprising level of substrate specificity among members of this coal-degrading microbial consortia. Additionally, coal kerogen, the solvent-insoluble organic component of coal often considered recalcitrant to microbial degradation, appeared to be readily converted to methane by microbial degradation. These findings challenge our understanding of coal organic matter catabolism and provide insights into the catabolic roles of individual coal seam bacteria.

Remotely constraining the temporal evolution of offshore oil systems.

An understanding of the temporal evolution of a petroleum system is fundamental to interpreting where hydrocarbons may be trapped in the subsurface. However, traditional exploration methods provide few absolute constraints on the timing of petroleum generation. Here we show that 187Re/187Os geochronology may be applied to natural crude oil seepage to determine when petroleum generation occurred in offshore sedimentary basins. Using asphaltites collected from the South Australian coastline, our determined Re-Os age (68 ± 15 million years ago) is consistent with their derivation from a Late Cretaceous source rock in the nearby Bight Basin, an interpretation similarly favoured by source-specific biomarker constraints. Furthermore, the calculated initial 187Os/188Os composition of the asphaltites, a value inherited from the source rock at the time of oil generation, suggests that the source rock represents the later stage of Oceanic Anoxic Event 2. Our results demonstrate a new approach to identifying the origin of crude oils encountered in coastal environments by providing direct constraints on the timing of petroleum generation and potential source rock intervals in poorly characterised offshore sedimentary basins prior to exploratory drilling.

Succession Patterns and Physical Niche Partitioning in Microbial Communities from Subsurface Coal Seams.

The subsurface represents a largely unexplored frontier in microbiology. Here, coal seams present something of an oasis for microbial life, providing moisture, warmth, and abundant fossilized organic material. Microbes in coal seams are thought to syntrophically mobilize fossilized carbon from the geosphere to the biosphere. Despite the environmental and economic importance of this process, little is known about the microbial ecology of coal seams. In the current study, ecological succession and spatial niche partitioning are explored in three coal seam microbial communities. Scanning electron microscopic visualization and 16S rRNA sequencing track changes in microbial communities over time, revealing distinct attached and planktonic communities displaying patterns of ecological succession. Attachment to the coal surface is biofilm mediated on Surat coal, whereas microbes on Sydney and Gunnedah coal show different attachment processes. This study demonstrates that coal seam microbial communities undergo spatial niche partitioning during periods of succession as microbes colonize coal environments.