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This work was supported by the Earth and Natural Sciences (ENS) Doctoral Studies Programme, funded by the Higher Education Authority (HEA) of Ireland through the Programme for Research at Third Level Institutions, Cycle 5 (PRTLI-5), co-funded by the European Regional Development Fund (ERDF). As the human population grows on the planet so does the generation of waste and particularly that of food waste. In order to tackle the world sustainability crisis, efforts to recover products from waste are critical. Here, we anaerobically recovered volatile fatty acids (VFAs) from food waste and analysed the microbial populations underpinning the process. An increased contribution of fungi relative to bacteria was observed throughout the reactor operation, with both kingdoms implicated into the main three steps of anaerobic digestion occurring within our systems: hydrolysis, acidogenesis and acetogenesis. Overall, Ascomycota, Proteobacteria and Firmicutes were found to drive the anaerobic digestion of food waste, with butyrate as the most abundant VFA likely produced by Clostridium using lactate as a precursor. Taken together we demonstrate that the generation of products of added-value from food waste results from cross-kingdoms microbial activities implicating fungi and bacteria. Publisher PDF

The soil microbiome is inherently complex with high biological diversity, and spatial heterogeneity typically occurring on the submillimetre scale. To study the microbial ecology of soils, and other microbiomes, biomolecules, that is, nucleic acids and proteins, must be efficiently and reliably co-recovered from the same biological samples. Commercial kits are currently available for the co-extraction of DNA, RNA and proteins but none has been developed for soil samples. We present a new protocol drawing on existing phenol-chloroform-based methods for nucleic acids co-extraction but incorporating targeted precipitation of proteins from the phenol phase. The protocol is cost-effective and robust, and easily implemented using reagents commonly available in laboratories. The method is estimated to be eight times cheaper than using disparate commercial kits for the isolation of DNA and/or RNA, and proteins, from soil. The method is effective, providing good quality biomolecules from a diverse range of soil types, with clay contents varying from 9.5% to 35.1%, which we successfully used for downstream, high-throughput gene sequencing and metaproteomics. Additionally, we demonstrate that the protocol can also be easily implemented for biomolecule co-extraction from other complex microbiome samples, including cattle slurry and microbial communities recovered from anaerobic bioreactors, as well as from Gram-positive and Gram-negative pure cultures.

Summary The overarching aim of microbiome analysis is to uncover the links between microbial phylogeny and function in order to access ecosystem functioning. This can be done using several experimental strategies targeting different biomolecules, including DNA (metagenomics), RNA (metatranscriptomics) and proteins (metaproteomics). Despite the importance of linking microbial function to phylogeny there are currently no visualization tools that effectively integrate this information. Chordomics is a Shiny-based application for linked -omics data analysis, allowing users to visualize microbial function and phylogeny on a single plot and compare datasets across time and environments. Availability and implementation Chordomics is available on GitHub: https://github.com/kevinmcdonnell6/chordomics; software is coded in R and JavaScript and a demonstration version is available at https://kmcd.shinyapps.io/ChordomicsDemo/. Supplementary information Supplementary data are available at Bioinformatics online.

Aims: The objective of this work was to provide functional evidence of key metabolic pathways important for anaerobic digestion processes through the identification of highly expressed proteins in a mixed anaerobic microbial consortium. Methods and Results: The microbial communities from an anaerobic industrial-like wastewater treatment bioreactor were characterized using phylogenetic analyses and metaproteomics. Clone libraries indicated that the bacterial community in the bioreactor was diverse while the archaeal population was mainly composed of Methanocorpusculum-like (76%) micro-organisms. Three hundred and eighty-eight reproducible protein spots were obtained on 2-D gels, of which 70 were excised and 33 were identified. The putative functions of the proteins detected in the anaerobic bioreactor were related to cellular processes, including methanogenesis from CO2 and acetate, glycolysis and the pentose phosphate pathway. Metaproteomics also indicated, by protein assignment, the presence of specific micro-organisms in the bioreactor. However, only a limited overlap was observed between the phylogenetic and metaproteomic analyses. Conclusions: This study provides some direct evidence of the microbial activities taking place during anaerobic digestion. Significance and Impact of Study: This study demonstrates metaproteomics as a useful tool to uncover key biochemical pathways underpinning specific anaerobic bioprocesses.

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This research was financially supported by Science Foundation Ireland (Grants RFP 276 08/RFP/EOB1343 and 06/CP/E006). The Welcome Trust, (grant number GR06281AIA), which funded the purchase of the ABSciex QStar XL mass spectrometer at the University of St. Andrews, is also gratefully acknowledged. Anaerobic digestion (AD) is an attractive wastewater treatment technology, leading to the generation of recoverable biofuel (methane). Most industrial AD applications, carry excessive heating costs however, as AD reactors are commonly operated at mesophilic temperatures while handling waste streams discharged at ambient or cold temperatures. Consequently, low-temperature AD represents a cost effective strategy for wastewater treatment. The comparative investigation of key microbial groups underpinning laboratory-scale AD bioreactors operated at 37°C, 15°C and 7°C was carried out. Community structure was monitored using 16S rRNA clone libraries, while abundance of the most prominent methanogens was investigated using qPCR. In addition, metaproteomics was employed to access the microbial functions carried out in situ. While δ-Proteobacteria were prevalent at 37°C, their abundance decreased dramatically at lower temperatures with inverse trends observed for Bacteroidetes and Firmicutes. Methanobacteriales and Methanosaeta were predominant at all temperatures investigated while Methanomicrobiales abundance increased at 15°C compared to 37°C and 7°C. Changes in operating temperature resulted in the differential expression of proteins involved in methanogenesis, which was found to occur in all bioreactors, as corroborated by bioreactors’ performance. This study demonstrated the value of employing a polyphasic approach to address microbial community dynamics and highlighted the functional redundancy of AD microbiomes. Postprint

Some of the most transformative discoveries promising to enable the resolution of this century's grand societal challenges will most likely arise from environmental science and particularly environmental microbiology and biotechnology. Understanding how microbes interact in situ, and how microbial communities respond to environmental changes remains an enormous challenge for science. Systems biology offers a powerful experimental strategy to tackle the exciting task of deciphering microbial interactions. In this framework, entire microbial communities are considered as metaorganisms and each level of biological information (DNA, RNA, proteins and metabolites) is investigated along with in situ environmental characteristics. In this way, systems biology can help unravel the interactions between the different parts of an ecosystem ultimately responsible for its emergent properties. Indeed each level of biological information provides a different level of characterisation of the microbial communities. Metagenomics, metatranscriptomics, metaproteomics, metabolomics and SIP-omics can be employed to investigate collectively microbial community structure, potential, function, activity and interactions. Omics approaches are enabled by high-throughput 21st century technologies and this review will discuss how their implementation has revolutionised our understanding of microbial communities. peer-reviewed

System approaches to elucidate ecosystem functioning constitute an emerging area of research within microbial ecology. Such approaches aim at investigating all levels of biological information (DNA, RNA, proteins and metabolites) to capture the functional interactions occurring in a given ecosystem and track down characteristics that could not be accessed by the study of isolated components. In this context, the study of the proteins collectively expressed by all the microorganisms present within an ecosystem (metaproteomics) is not only crucial but can also provide insights into microbial functionality. Overall, the success of metaproteomics is closely linked to metagenomics, and with the exponential increase in the availability of metagenome sequences, this field of research is starting to experience generation of an overwhelming amount of data, which requires systematic analysis. Metaproteomics has been employed in very diverse environments, and this review discusses the recent advances achieved in the context of human biology, soil, marine and freshwater environments as well as natural and bioengineered systems.

Granular biomass from a laboratory-scale anaerobic bioreactor trial was analysed to identify changes in microbial community structure and function in response to temperature and trichloroethylene (TCE). Two bioreactors were operated at 37 degrees C, while two were operated at 15 degrees C. At the time of sampling, one of each temperature pair of bioreactors was exposed to process failure-inducing concentrations of TCE (60 mg L-1) while the other served as a TCE-free control. Bacterial community structure was investigated using denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library analysis. Temperature was identified as an important factor for bacterial community composition, while minor differences were associated with trichloroethylene supplementation. Proteobacteria was the dominant phylum in all bioreactors, while clone library analysis revealed a higher proportion of Bacteroidetes-, Chloroflexi-, and Firmicutes-like clones at 15 degrees C than at 37 degrees C. Comparative metaproteomics in the presence and absence of TCE was carried out by two-dimensional gel electrophoresis (2-DGE), and 28 protein spots were identified, with putative functions related to cellular processes, including methanogenesis, glycolysis, the glyoxylate cycle, and the methyl malonyl pathway. A good agreement between metaproteomic species assignment and phylogenetic information was observed, with 10 of the identified proteins associated with members of the phylum Proteobacteria. This work was supported by Enterprise Ireland, The Irish Environmental Protection Agency, and Science Foundation Ireland. peer-reviewed

The feasibility of metaproteomic analysis of the microbial communities underpinning anaerobic wastewater treatment relies on efficient protein extraction and separation techniques. The microorganisms involved in anaerobic digestion (AD) of wastewater typically aggregate to form tightly organised, spherical granules, from which protein extraction is challenging. Here, we compare two methods of protein extraction, one using a French press previously used successfully to analyse the proteome of an activated sludge [Wilmes, P., Bond, P. L., Environ. Microbiol. 2004, 6, 911-920.] and one using sonication developed in the context of pure culture [Abram, F., Wan-Lin, S., Wiedmann, M., Boor, K. J., Coote, P., Botting, C., Karatzas, K. A. G., O'Byrne, C. P., Appl. Environ. Microbiol. 2008, 74, 594-604.]. We used 2-DE to carry out this comparison. The protein extraction using the sonication method resulted in a significant increase in the number of protein spots and higher quality 2-D gels.