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Per-and Polyfluoroalkyl Substances (PFAS), which includes PFOS and PFOA, are recognized as the most important class of emerging contaminants due to their widespread presence in wildlife and humans, their environmental persistence, bioaccumulative potential, and toxicity. Low environmental pH conditions affect the state and physicochemical properties of PFAS, which in turn impacts on their toxicity. Sparse information is available regarding the effect of these compounds on microorganisms, and the possible associated knock-on effect on ecosystem services. The purpose of this study is to investigate the ability of E. coli to tolerate stress in the presence of PFAS compounds. To this end, we carried out phenotypic comparisons of E. coli exposed to a series of environmental conditions in the presence and absence of PFOA and PFOS. Quite remarkably, E. coli growth was not affected by the presence of PFOA and PFOS up to 500 mg/L. The survival of E. coli at pH3, however, decreased by more than three-fold when the medium was supplemented with PFOA. PFOA and PFOS were also found to decrease the growth rates of E. coli in minimal media in the presence of 0.75 and 1M NaCl. We are currently screening further stresses of environmental relevance with the aim to conduct molecular investigations to examine the mechanisms underpinning the effect of PFAS exposure on bacterial stress tolerance. This work will provide some insights into the impact of PFAS on microorganisms, which should shed new light on the assessment of the ecological effects of PFAS.

Proteomics aims to characterise system-wide protein expression and typically relies on mass-spectrometry and peptide fragmentation, followed by a database search for protein identification. It has wide ranging applications from clinical to environmental settings and virtually impacts on every area of biology. In that context

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

All living cells strive to allocate cellular resources in a way that promotes maximal evolutionary fitness. While there are many competing demands for resources the main decision making process centres on whether to proceed with growth and reproduction or to "hunker down" and invest in protection and survival (or to strike an optimal balance between these two processes). The transcriptional programme active at any given time largely determines which of these competing processes is dominant. At the top of the regulatory hierarchy are the sigma factors that commandeer the transcriptional machinery and determine which set of promoters are active at any given time. The regulatory inputs controlling their activity are therefore often highly complex, with multiple layers of regulation, allowing relevant environmental information to produce the most beneficial response. The tension between growth and survival is also evident in the developmental programme necessary to promote biofilm formation, which is typically associated with low growth rates and enhanced long-term survival. Nucleotide second messengers and energy pools (ATP/ADP levels) play critical roles in determining the fate of individual cells. Regulatory small RNAs frequently play important roles in the decision making processes too. In this review we discuss the trade-off that exists between reproduction and persistence in bacteria and discuss some of the recent advances in this fascinating field.

Microbial disease outbreaks related to fresh produce consumption, including leafy green vegetables, have increased in recent years. Where contamination occurs, pathogen persistence may represent a risk for consumers’ health. This study analysed the survival of E. coli and L. innocua on lettuce plants watered with contaminated irrigation water via a single irrigation event and within stored irrigation water. Separate lettuce plants (Lactuca sativa var. capitata) were irrigated with water spiked with Log10 7 cfu/mL of each of the two strains and survival assessed via direct enumeration, enrichment and qPCR. In parallel, individual 20 L water microcosms were spiked with Log10 7 cfu/mL of the individual strains and sampled at similar time points. Both strains were observed to survive on lettuce plants up to 28 days after inoculation. Direct quantification by culture methods showed a Log10 4 decrease in the concentration of E. coli 14 days after inoculation, and a Log10 3 decrease in the concentration of L. innocua 10 days after inoculation. E. coli was detected in water samples up to 7 days after inoculation and L. innocua was detected up to 28 days by direct enumeration. Both strains were recovered from enriched samples up to 28 days after inoculation. These results demonstrate that E. coli and L. innocua strains are able to persist on lettuce after a single contamination event up until the plants reach a harvestable state. Furthermore, the persistence of E. coli and L. innocua in water for up to 28 days after inoculation illustrates the potential for multiple plant contamination events from stored irrigation water, emphasising the importance of ensuring that irrigation water is of a high quality.

Current approaches to interpreting 16S rDNA amplicon data are hampered by several factors. Among these are database inaccuracy or incompleteness, sequencing error, and biased DNA/RNA extraction. Existing 16S rRNA databases source the majority of sequences from deposited amplicon sequences, draft genomes, and complete genomes. Most of the draft genomes available are assembled from short reads. However, repeated ribosomal regions are notoriously difficult to assemble well from short reads, and as a consequence the short-read-assembled 16S rDNA region may be an amalgamation of different loci within the genome. This complicates high-resolution community analysis, as a draft genome’s 16S rDNA sequence may be a chimera of multiple loci; in such cases, the draft-derived sequences in a database may not represent a 16S rRNA sequence as it occurs in biology. We present Focus16, a pipeline for improving 16S rRNA databases by mining NCBI’s Sequence Read Archive for whole-genome sequencing runs that could be reassembled to yield additional 16S rRNA sequences. Using riboSeed (a genome assembly tool for correcting rDNA misassembly), Focus16 provides a way to augment 16S rRNA databases with high-quality re-assembled sequences. In this study, we augmented the widely-used SILVA 16S rRNA database with the novel sequences disclosed by Focus16 and re-processed amplicon sequences from several benchmarking datasets with DADA2. Using this augmented SILVA database increased the number of amplicon sequence variants that could be assigned taxonomic annotations. Further, fine-scale classification was improved by revealing ambiguities. We observed, for example, that amplicon sequence variants (ASVs) may be assigned to a specific genus where Focus16-correction would indicate that the ASV is represented in two or more genera. Thus, we demonstrate that improvements can be made to taxonomic classification by incorporating these carefully re-assembled 16S rRNA sequences, and we invite the community to expand our work to augment existing 16S rRNA reference databases such as SILVA, GreenGenes, and RDP.

All living cells strive to allocate cellular resources in a way that promotes maximal evolutionary fitness. While there are many competing demands for resources the main decision making process centres on whether to proceed with growth and reproduction or to “hunker down” and invest in protection and survival (or to strike an optimal balance between these two processes). The transcriptional programme active at any given time largely determines which of these competing processes is dominant. At the top of the regulatory hierarchy are the sigma factors that commandeer the transcriptional machinery and determine which set of promoters are active at any given time. The regulatory inputs controlling their activity are therefore often highly complex, with multiple layers of regulation, allowing relevant environmental information to produce the most beneficial response. The tension between growth and survival is also evident in the developmental programme necessary to promote biofilm formation, which is typically associated with low growth rates and enhanced long-term survival. Nucleotide second messengers and energy pools (ATP/ADP levels) play critical roles in determining the fate of individual cells. Regulatory small RNAs frequently play important roles in the decision making processes too. In this review we discuss the trade-off that exists between reproduction and persistence in bacteria and discuss some of the recent advances in this fascinating field.

Microbial contamination of fresh produce is a major public health concern, with the number of associated disease outbreaks increasing in recent years. The consumption of sprouted beans and seeds is of particular concern, as these foodstuffs are generally consumed raw, and are produced in conditions favourable for the growth of zoonotic pathogens, if present in seeds prior to sprouting or in irrigation water. This work aimed to evaluate the activity of plasma activated water (PAW) as a disinfecting agent for alfalfa (Medicago sativa) and mung bean (Vigna radiata) seeds, during seed soaking. Each seed type was inoculated with Escherichia coli O157, E. coli O104, Listeria monocytogenes or Salmonella Montevideo, and treated with PAW for different times. A combination of PAW and ultrasound treatment was also evaluated. The germination and growth rate of both seeds were assessed after PAW treatments. PAW was demonstrated to have disinfecting ability on sprouted seeds, with reductions of up to Log10 1.67 cfu/g in alfalfa seeds inoculated with E. coli O104, and a reduction of Log10 1.76 cfu/g for mung bean seeds inoculated with E. coli O157 observed. The germination and growth rate of alfalfa and mung bean sprouts were not affected by the PAW treatments. The combination of a PAW treatment and ultrasound resulted in increased antimicrobial activity, with a reduction of Log10 3.48 cfu/g of S. Montevideo in mung bean seeds observed. These results demonstrate the potential for PAW to be used for the inactivation of pathogenic microorganisms which may be present on sprouted seeds and beans, thereby providing greater assurance of produce safety.

Trichloroethylene (TCE) is a human carcinogen that is commonly found in landfill leachate as a result of anthropogenic activities. Contaminated leachate plumes may be intercepted prior to reaching groundwater and treated in situ using permeable reactive barriers (PRB). This study used a packed column system containing herbal pomace and spruce biochar, previously shown to have TCE adsorptive capabilities, to investigate the feasibility of using pyrolysed waste as a fill material in a PRB. Influent containing raw or autoclaved landfill leachate was used to investigate the potential for environmental micro-organisms to establish a TCE-dechlorinating biofilm on the biochar, in order to prolong the operational life span of the system. TCE removal ≥ 99.7 was observed by both spruce and herbal pomace based biochars. No dichloroethylene (DCE) isomers were present in the column effluents, but cis-1,2 DCE was adsorbed to the biochar treating raw landfill leachate, indicating that dechlorination was occurring biologically in these columns. Known microbial species that are individually capable of complete dechlorination of TCE to ethene were not detected by 16S rRNA gene sequencing, but several species capable of partial TCE dechlorination (Desulfitobacterium spp., Sulfurospirillium spp. and Desulfuromonas spp) were present in the biofilms of the columns treating raw landfill leachate. These data demonstrate that biochar from waste material may be capable of supporting a dechlorinating biofilm to promote bioremediation of TCE. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Actions grant agreement No 748106. The authors would like to acknowledge assistance with column installation from Edward Kilcullen, NUI Galway; biochar contributions from Caspar von Ziegner, NovoCarbo, Germany and Timo Ferdinand, HerbaCarbo, Germany; and supply of landfill leachate from Barry Cormican, East Galway Landfill. peer-reviewed

The Clermont PCR method for phylotyping Escherichia coli remains a useful classification scheme even though genome sequencing is now routine, and higher-resolution sequence typing schemes are now available. Relating present-day whole-genome E. coli classifications to legacy phylotyping is essential for harmonizing the historical literature and understanding of this important organism. Therefore, we present EzClermont – a novel in silico Clermont PCR phylotyping tool to enable ready application of this phylotyping scheme to whole-genome assemblies. We evaluate this tool against phylogenomic classifications, and an alternative software implementation of Clermont typing. EzClermont is available as a web app at www.ezclermont.org, and as a command-line tool at https://nickp60.github.io/EzClermont/.

If pathogens are present in feedstock materials and survive in anaerobic digestion (AD) formulations at 37 &deg, C, they may also survive the AD process to be disseminated in digestate spread on farmland as a fertilizer. The aim of this study was to investigate the prevalence of Salmonella spp., Escherichia coli O157, Listeria monocytogenes, Enterococcus faecalis and Clostridium spp. in AD feed and output materials and survival/growth in four formulations based on food waste, bovine slurry and/or grease-trap waste using International Organization for Standardization (ISO) or equivalent methods. The latter was undertaken in 100 mL Ramboldi tubes, incubated at 37 &deg, C for 10 d with surviving cells enumerated periodically and the T90 values (time to achieve a 1 log reduction) calculated. The prevalence rates for Salmonella spp., Escherichia coli O157, Listeria monocytogenes, Enterococcus faecalis and Clostridium spp. were 3, 0, 5, 11 and 10/13 in food waste, 0, 0, 2, 3 and 2/3 in bovine slurry, 1, 0, 8, 7 and 8/8 in the mixing tank, 5, 1, 17, 18 and 17 /19 in raw digestate and 0, 0, 0, 2 and 2/2 in dried digestate, respectively. Depending on the formulation, T90 values ranged from 1.5 to 2.8 d, 1.6 to 2.8 d, 3.1 to 23.5 d, 2.2 to 6.6 d and 2.4 to 9.1 d for Salmonella Newport, Escherichia coli O157, Listeria monocytogenes, Enterococcus faecalis and Clostridium sporogenes, respectively. It was concluded that AD feed materials may be contaminated with a range of bacterial pathogens and L. monocytogenes may survive for extended periods in the test formulations incubated at 37 &deg, C.

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.

Over three million tonnes of spent mushroom substrate (SMS) are produced in Europe every year as a by-product of the cultivation of Agaricus bisporus. The management of SMS has become an increasing challenge for the mushroom production industry, and finding environmentally and economically sustainable solutions for this organic residue is, therefore, highly desirable. Due to its physical properties and nutrient content, SMS has great potential to be employed in agricultural and horticultural sectors, and further contribute to reduce the use of non-renewable resources, such as peat. However, SMS is often regarded as not being stable and/or mature, which hampers its wide use for crop production. Here, we demonstrate the stabilisation of SMS and its subsequent use as organic fertiliser and partial peat replacement in horticulture. The stabilisation was performed in a laboratory-scale composting system, with controlled temperature and aeration. Physical and chemical parameters were monitored during composting and provided information on the progress of the process. Water soluble carbohydrates (WSC) content was found to be the most reliable parameter to predict SMS stability. In situ oxygen consumption indicated the main composting phases, reflecting major changes in microbial activity. The structure of the bacterial community was also found to be a potential predictor of stability, as the compositional changes followed the composting progress. By contrast, the fungal community did not present clear successional process along the experiment. Maturity and quality of the stabilised SMS were assessed in a horticultural growing trial. When used as the sole fertiliser source, SMS was able to support Lolium multiflorum (Italian ryegrass) growth and significantly improved grass yield with a concentration-dependent response, increasing grass biomass up to 300%, when compared to the untreated control. In summary, the results indicated that the method employed was efficient in generating a stable and mature product, which has a great potential to be applied in horticulture. This study represents a step forward in the management of SMS residue, and also provides an alternative to reduce the use of peat in horticulture, alleviating environmental impacts to peatland ecosystems.

The Clermont PCR method for phylotyping Escherichia coli remains a useful classification scheme even though genome sequencing is now routine, and higher-resolution sequence typing schemes are now available. Relating present-day whole-genome E. coli classifications to legacy phylotyping is essential for harmonizing the historical literature and understanding of this important organism. Therefore, we present EzClermont – a novel in silico Clermont PCR phylotyping tool to enable ready application of this phylotyping scheme to whole-genome assemblies. We evaluate this tool against phylogenomic classifications, and an alternative software implementation of Clermont typing. EzClermont is available as a web app at www.ezclermont.org, and as a command-line tool at https://nickp60.github.io/EzClermont/.

Trichloroethylene (TCE) is an Environmental Protection Agency priority pollutant associated with cancer in humans. With numerous industrial applications and regular landfill disposal, TCE is a common landfill leachate pollutant. In situ treatment barriers use costly fill materials such as granular activated carbon (GAC). Here, we show that while a range of untreated waste materials had little ability to adsorb TCE, waste-derived biochar showed excellent capacity for TCE adsorption. TCE removal efficiencies by spruce and oak-derived biochars were > 99.5 %, outperforming GAC (95 %) and herbal pomace biochar (93 %). A contact time of at least 32 h was required to reach equilibrium for all of these media. Assessment of pollution swapping potential revealed release of phosphate by all biochars. Analysis of media surface characteristics by Fourier Transform Infrared Spectroscopy (FTIR) predicted that GAC should have the highest ability to adsorb TCE, followed by Oak Biochar, Herbal Pomace Biochar 1, and Spruce Biochar 2, which was not in agreement with the experimental adsorption data. These data demonstrate the potential for pyrolysed waste material to be used as an alternative fill material for in situ remediation applications, thereby also addressing the European Circular Economy Strategy. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie Actions grant agreement No 748106. The authors would like to acknowledge laboratory assistance from Tatiana Kelley, NUI Galway and RPS, FTIR analysis by Pól Mac Fhionnghaile, School of Chemistry, NUI Galway, and biochar contributions from: Prof JJ Leahy, University of Limerick, Ireland; Caspar von Ziegner, NovoCarbo, Germany; Timo Ferdinand, HerbaCarbo, Germany, and Thomas Fleming, Brooko GbR, Germany. peer-reviewed 2021-12-17

In agricultural systems based on organic fertilisers, the activity of prokaryotes and fungi is essential for degradation of complex substrates and release of nutrients for plant uptake. Understanding the dynamics of microbial communities in these systems is, therefore, desirable for designing successful management strategies aiming to optimise nutrient availability and to improve plant productivity. Of particular interest is how the microbial inoculum provided by an organic substrate persists in the soil and interacts with soil and plant microbiomes, as these processes may affect the long-term benefits of organic amendments. We aimed to investigate how these dynamics occurred in soil treated with stabilised spent mushroom substrate (SMS), a soil amendment rich in nutrients and complex organic matter. We carried out a 14 weeks soil trial to assess the plant growth promoting properties of the SMS and to monitor the successional processes of the resulting SMS-soil communities compared to a mineral amended control. Bacterial and fungal communities were analysed by high-throughput sequencing at both DNA and RNA (cDNA) levels. Using a combination of computational tools, including SourceTracker and Network analysis, we assessed the persistence of SMS-derived taxa in soil, and the changes in co-occurrence patterns and microbial community structure over time. Prokaryotic and fungal communities presented remarkably distinct trajectories following SMS treatment. The soil prokaryotic communities displayed higher levels of resilience to the changes introduced by SMS treatment and rapidly tended toward a soil-like profile, with low persistence of SMS-derived prokaryotes. In contrast, the SMS fungal community had greater success in soil colonisation during the time monitored. SMS treatment promoted an increase in the participation of fungi in the highly connected fraction of the active community, including fungal taxa of SMS origin. We observed the presence of highly connected microbial guilds, composed by fungal and bacterial taxa with reported capabilities of complex organic matter degradation. Many of these taxa were also significantly correlated with the organic matter content and plant yield, suggesting that these highly connected taxa may play key roles not only in the community structure, but also in the plant-soil system under organic fertilisation.

Inorganic polyphosphate (polyP) is ubiquitous across all forms of life, but the study of its metabolism has been mainly confined to bacteria and yeasts. Few reports detail the presence and accumulation of polyP in Archaea, and little information is available on its functions and regulation. Here, we report that homologs of bacterial polyP metabolism proteins are present across the major taxa in the Archaea, suggesting that archaeal populations may have a greater contribution to global phosphorus cycling than has previously been recognised. We also demonstrate that polyP accumulation can be induced under strictly anaerobic conditions, in response to changes in phosphate (Pi) availability, i.e. Pi starvation, followed by incubation in Pi replete media (overplus), in cells of the methanogenic archaeon Methanosarcina mazei. Pi-starved M. mazei cells increased transcript abundance of the alkaline phosphatase (phoA) gene and of the high-affinity phosphate transport (pstSCAB-phoU) operon: no increase in polyphosphate kinase 1 (ppk1) transcript abundance was observed. Subsequent incubation of Pi-starved M. mazei cells under Pi replete conditions, led to a 237% increase in intracellular polyphosphate content and a > 5.7-fold increase in ppk1 gene transcripts. Ppk1 expression in M. mazei thus appears not to be under classical phosphate starvation control.

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.

Pangenome analysis can help reveal functional and physiological differences between organisms by linking genes present in their genomes with phenotypic characteristics. While this works well for closely-related organisms and large numbers of isolates, analysis of species with a large pangenome may be problematic as optimal sample size increases with intraspecies diversity. Organisms such as E. coli exhibit high intraspecies diversity, resulting in an exceptionally large pangenome, pushing the limits of what constitutes a species. However, the phylogeny of the mobile elements can differ strongly from the phylogeny of the rest of the genome, reflecting how rapidly these elements can be gained or lost. We suggest when studying adaptations on a short genetic time-scale (ie, within species or strain), the mobile regions of the genome may be more informative than the more stable parts of the genome. To this end, we developed a pipeline, Horizontally Acquired Partial Pangenome of Inserted Elements (or ‘happie’), which allows researchers to study the mobile pangenome. Thus, happie could be used as a proxy to detect genes associated with a particular trait amidst the noise of a diverse genetic background. We aim to use happie to compare a panel of known soil-persistent E. coli isolates and a curated panel of non-soil-persistent isolates. This will allow us to determine whether specific mobile elements (or genes carried on those elements) are associated with soil persistence, which would indicate that the cost of harbouring the mobile element was less than the advantage conferred.

The process of anaerobic digestion (AD) is valued as a carbon-neutral energy source, while simultaneously treating organic waste, making it safer for disposal or use as a fertilizer on agricultural land. The AD process in many European nations, such as Germany, has grown from use of small, localized digesters to the operation of large-scale treatment facilities, which contribute significantly to national renewable energy quotas. However, these large AD plants are costly to run and demand intensive farming of energy crops for feedstock. Current policy in Germany has transitioned to support funding for smaller digesters, while also limiting the use of energy crops. AD within Ireland, as a new technology, is affected by ambiguous governmental policies concerning waste and energy. A clear governmental strategy supporting on-site AD processing of agricultural waste will significantly reduce Ireland's carbon footprint, improve the safety and bioavailability of agricultural waste, and provide an indigenous renewable energy source. © 2016 Society of Chemical Industry.

North Atlantic European grassland systems have a low nutrient use efficiency and high rainfall. This grassland is typically amended with unprocessed slurry, which counteracts soil organic matter depletion and provides essential plant micronutrients but can be mobilised during rainfall events thereby contributing to pathogen, nutrient and metal incidental losses. Co-digesting slurry with waste from food processing mitigates agriculture-associated environmental impacts but may alter microbial, nutrient and metal profiles and their transmission to watercourses, and/or soil persistence, grass yield and uptake. The impact of EU and alternative pasteurisation regimes on transmission potential of these various pollutants is not clearly understood, particularly in pasture-based agricultural systems. This study utilized simulated rainfall (Amsterdam drip-type) at a high intensity indicative of a worst-case scenario of ~11 mm hr−1 applied to plots 1, 2, 15 and 30 days after grassland application of slurry, unpasteurised digestate, pasteurised digestate (two conditions) and untreated controls. Runoff and soil samples were collected and analysed for a suite of potential pollutants including bacteria, nutrients and metals following rainfall simulation. Grass samples were collected for three months following application to assess yield as well as nutrient and metal uptake. For each environmental parameter tested: microbial, nutrient and metal runoff losses; accumulation in soil and uptake in grass, digestate from anaerobic co-digestion of slurry with food processing waste resulted in lower pollution potential than traditional landspreading of slurry without treatment. Reduced microbial runoff from digestate was the most prominent advantage of digestate application. Pasteurisation of the digestate further augmented those environmental benefits, without impacting grass output. Anaerobic co-digestion of slurry is therefore a multi-beneficial circular approach to reducing impacts of livestock production on the environment.

In agricultural systems based on organic fertilisers, the activity of prokaryotes and fungi is essential for degradation of complex substrates and release of nutrients for plant uptake. Understanding the dynamics of microbial communities in these systems is, therefore, desirable for designing successful management strategies aiming to optimise nutrient availability and improve plant productivity. Of particular interest is how the microbial inoculum provided by an organic substrate persists in the soil and interacts with soil and plant microbiomes, as these processes may affect the long-term benefits of organic amendments. We aimed to investigate how these dynamics occurred in soil treated with stabilised spent mushroom substrate (SMS), a soil amendment rich in nutrients and complex organic matter. We carried out a 14 week soil trial to assess the plant growth promoting properties of the SMS and to monitor the successional processes of the resulting SMS-soil communities compared to a control treatment containing soil amended with mineral fertiliser. Bacterial and fungal communities were analysed by high-throughput sequencing at both DNA and RNA (cDNA) levels. Using a combination of computational tools, including SourceTracker and Network analysis, we assessed the persistence of SMS-derived taxa in soil, and the changes in co-occurrence patterns and microbial community structure over time. Prokaryotic and fungal communities presented remarkably distinct trajectories following SMS treatment. The soil prokaryotic communities displayed higher levels of resilience to the changes introduced by SMS treatment and rapidly tended toward a soil-like profile, with low persistence of SMS-derived prokaryotes. By contrast, the SMS fungal community had greater success in soil colonisation during the time monitored. SMS treatment promoted an increase in the participation of fungi in the highly connected fraction of the active community, including fungal taxa of SMS origin. We observed the presence of highly connected microbial guilds, composed by fungal and bacterial taxa with reported capabilities of complex organic matter degradation. Many of these taxa were also significantly correlated with soil organic matter content and plant yield, suggesting that these highly connected taxa may play key roles not only in the community structure, but also in the plant-soil system under organic fertilisation.

Low temperature anaerobic digestion remains in its infancy, despite increasing interest for the treatment of complex wastewaters. In this study, the feasibility of low-temperature anaerobic treatment of dairy wastewater was assessed during a 443-day laboratory-scale bioreactor trial. The bioreactors were operated in triplicate at organic loading rates of 7.5–9 kgCODm−3d−1 throughout five operational phases. The structure of the microbial community was analysed using quantitative real-time PCR and amplicon sequencing of 16S rRNA genes from DNA and rRNA. The results indicated that low-temperature treatment of dairy wastewater is feasible at 15 °C, but that reactor configuration remains extremely important. The upflow anaerobic sludge bed (UASB) configuration out-performed the expanded granular sludge bed (EGSB)-based configurations. Decreased temperatures resulted in significant reductions in microbiome diversity. Methanosaeta was identified as a dominant genus throughout the trial, while Lactococcus was identified as an important bacterial genus at low-temperatures. However, the relative abundance of Lactococcus was significantly influenced by reactor configuration.

Promotion of healthier lifestyles has led to an increase in consumption of fresh produce. Such foodstuffs may expose consumers to increased risk of foodborne disease, as often they are not subjected to processing steps to ensure effective removal or inactivation of pathogenic microorganisms before consumption. Consequently, reports of ready-to-eat fruit and vegetable related disease outbreak occurrences have increased substantially in recent years, and information regarding these events is often not readily available. Identifying the nature and source of microbial contamination of these foodstuffs is critical for developing appropriate mitigation measures to be implemented by food producers. This review aimed to identify the foodstuffs most susceptible to microbial contamination and the microorganisms responsible for disease outbreaks from information available in peer-reviewed scientific publications. A total of 571 outbreaks were identified from 1980 to 2016, accounting for 72,855 infections and 173 deaths. Contaminated leafy green vegetables were responsible for 51.7% of reported outbreaks. Contaminated soft fruits caused 27.8% of infections. Pathogenic strains of Escherichia coli and Salmonella, norovirus, and hepatitis A accounted for the majority of cases. Large outbreaks resulted in particular biases such as the observation that contaminated sprouted plants caused 31.8% of deaths. Where known, contamination mainly occurred via contaminated seeds, water, and contaminated food handlers. There is a critical need for standardized datasets regarding all aspects of disease outbreaks, including how foodstuffs are contaminated with pathogenic microorganisms. Providing food business operators with this knowledge will allow them to implement better strategies to improve safety and quality of fresh produce.

Manure and slurry may contain a range of bacterial, viral, and parasitic pathogens and land application of these organic fertilizers typically occurs without prior treatment. In-situ treatment through farm-based anaerobic digestion (AD) of such organic fertilizers co-digested with food-production wastes is multi-beneficial due to energy recovery, increased farm incomes and noxious gas reduction. Before risk assessment can be carried out at field scale an investigation of the fate of relevant target pathogens during the actual AD process must be undertaken, requiring the development of practical test systems for evaluation of pathogen survival. The present study examines miniature (50 mL) and laboratory (10 L) scale AD systems. Treatments included slurry co-digested with fats, oils, and grease (FOG) under typical operating and pasteurization conditions used in farm-based AD, in batch-fed miniature and laboratory mesophilic (37°C) continuously stirred tank reactors. Biogas production, pH, chemical oxygen demand, volatile solids, and ammonia concentration were measured throughout the trial, as were fecal indicator bacteria (FIB) i.e., total coliforms, Escherichia coli, and Enterococcus species. The miniature and laboratory bioreactors performed similarly in terms of physicochemical parameters and FIB die-off. In the absence of pasteurization, after 28 days, enterococci numbers were below the

SummaryThe Clermont PCR method of phylotyping Escherichia coli has remained a useful classification scheme despite the proliferation of higher-resolution sequence typing schemes. We have implemented an in silico Clermont PCR method as both a web app and as a command-line tool to allow researchers to easily apply this phylotyping scheme to genome assemblies easily.Availability and ImplementationEzClermont is available as a web app at http://www.ezclermont.org. For local use, EzClermont can be installed with pip or installed from the source code at https://github.com/nickp60/ezclermont. All analysis was done with version 0.4.0.Contactn.waters4@nuigalway.ie, leighton.pritchard@strath.ac.ukSupplementary informationTable S1: test dataset; S2: validation dataset; S3: results.

Background Nowadays, the vast majority of chemicals are either synthesised from fossil fuels or are extracted from agricultural commodities. However, these production approaches are not environmentally and economically sustainable, as they result in the emission of greenhouse gases and they may also compete with food production. Because of the global agreement to reduce greenhouse gas emissions, there is an urgent interest in developing alternative sustainable sources of chemicals. In recent years, organic waste streams have been investigated as attractive and sustainable feedstock alternatives. In particular, attention has recently focused on the production of caproate from mixed culture fermentation of low-grade organic residues. The current approaches for caproate synthesis from organic waste are not economically attractive, as they involve the use of two-stage anaerobic digestion systems and the supplementation of external electron donors, both of which increase its production costs. This study investigates the feasibility of producing caproate from food waste (FW) without the supplementation of external electron donors using a single-phase reactor system. Results Replicate leach-bed reactors were operated on a semi-continuous mode at organic loading of 80 g VS FW l−1 and at solid retention times of 14 and 7 days. Fermentation, rather than hydrolysis, was the limiting step for caproate production. A higher caproate production yield 21.86 ± 0.57 g COD l−1 was achieved by diluting the inoculating leachate at the beginning of each run and by applying a leachate recirculation regime. The mixed culture batch fermentation of the FW leachate was able to generate 23 g caproate COD l−1 (10 g caproate l−1), at a maximum rate of 3 g caproate l−1 day−1 under high H2 pressure. Lactate served as the electron donor and carbon source for the synthesis of caproate. Microbial community analysis suggested that neither Clostridium kluyveri nor Megasphaera elsdenii, which are well-characterised caproate producers in bioreactors systems, were strongly implicated in the synthesis of caproate, but that rather Clostridium sp. with 99% similarity to Ruminococcaceae bacterium CPB6 and Clostridium sp. MT1 likely played key roles in the synthesis of caproate. This finding indicates that the microbial community capable of caproate synthesis could be diverse and may therefore help in maintaining a stable and robust process. Conclusions These results indicate that future, full-scale, high-rate caproate production from carbohydrate-rich wastes, associated with biogas recovery, could be envisaged. Electronic supplementary material The online version of this article (10.1186/s13068-018-1101-4) contains supplementary material, which is available to authorized users.

The soil is a complex ecosystem where interactions between biotic and abiotic factors determine the survival and fate of microbial inhabitants of the system. Having previously shown that Escherichia coli requires the general stress response regulator, RpoS, to survive long term in soil, it was important to determine what specific conditions in this environment necessitate a functional RpoS. This study investigated the susceptibility of soil-persistent E. coli to predation by the single-celled eukaryotes Acanthamoeba polyphaga and Tetrahymena pyriformis, and the role RpoS plays in resisting this predation. Strain-specific differences were observed in the predation of E. coli strains, with soil-persistent strain COB583 being the most resistant to predation by both protozoans. RpoS and curli, proteinaceous fibres used for attachment to biotic and abiotic surfaces, increased the ability of E. coli to resist predation by A. polyphaga and T. pyriformis. Furthermore, soil moisture content impacted the survival of E. coli BW25113 but wild-type COB583 had similar survival irrespective of soil moisture content. Overall, this study confirmed that RpoS contributes to the resistance of E. coli to protozoan predation and that RpoS is crucial for the increased fitness of soil-persistent E. coli against predation and reduced moisture in soil.

There is increasing interest in methodologies for the simultaneous concentration and detection of multiple targets in individual samples. The aim of this study was to investigate the potential presence of E. coli DNA in beef extract powder used as part of a procedure to concentrate water samples for the simultaneous detection of bacteria, viruses and protozoa. DNA from E. coli was detected in five out of six beef extract lots tested, demonstrating the limitations of its inclusion when being used in assays that will be used for the detection of E. coli in water samples. Further work is required to clarify if this phenomenon also occurs for other microorganisms of interest in water.

This work was supported by Science Foundation Ireland (Charles Parsons Energy Research Award - 06/CP/E006) and the Wellcome Trust (grant number 094476/Z/10/Z) which funded the purchase of the TripleTOF 5600 mass spectrometer at the BSRC Mass Spectrometry and Proteomics Facility, University of St Andrews. The authors thank Matthew A. Fuszard and Sally L. Shirran for their technical assistance with fractionation and mass spectrometry. Geobacter sulfurreducens is a dissimilatory metal-reducing bacterium capable of forming thick electron-conducting biofilms on solid electrodes. Here, we employ for the first time comparative proteomics to identify key physiological changes involved in G. sulfurreducens adaptation from fumarate-respiring planktonic cells to electron-conducting biofilms. Increased levels of proteins involved in outer membrane biogenesis, cell motility and secretion are expressed in biofilms. Of particular importance to the electron-conducting biofilms are proteins associated with secretion systems of Type I, II, V and Type IV pili. Furthermore, enzymes involved in lipopolysaccharide and peptidoglycan biosynthesis show increased levels of expression in electron-conducting biofilms compared to planktonic cells. These observations point to similarities in long-range electron transfer mechanisms between G. sulfurreducens and Shewanella oneidensis, while highlighting the wider significance of secretion systems beyond that of Type IV pili identified to date in the adaptation of G. sulfurreducens to electrode respiration. Postprint

Although Escherichia coli is generally considered to be predominantly a commensal of the gastrointestinal tract, a number of recent studies suggest that it is also capable of long-term survival and growth in environments outside the host. As the extraintestinal physical and chemical conditions are often different from those within the host, it is possible that distinct genetic adaptations may be required to enable this transition. Several studies have shown a trade-off between growth and stress resistance in nutrient-poor environments, with lesions in the rpoS locus, which encodes the stress sigma factor RpoS (σ S ). In this study, we investigated a unique collection of long-term soil-persistent E. coli isolates to determine whether the RpoS-controlled general stress response is altered during adaptation to a nutrient-poor extraintestinal environment. The sequence of the rpoS locus was found to be highly conserved in these isolates, and no nonsense or frameshift mutations were detected. Known RpoS-dependent phenotypes, including glycogen synthesis and γ-aminobutyrate production, were found to be conserved in all strains. All strains expressed the full-length RpoS protein, which was fully functional using the RpoS-dependent promoter reporter fusion P gadX :: gfp . RpoS was shown to be essential for long-term soil survival of E. coli , since mutants lacking rpoS lost viability rapidly in soil survival assays. Thus, despite some phenotypic heterogeneity, the soil-persistent strains all retained a fully functional RpoS-regulated general stress response, which we interpret to indicate that the stresses encountered in soil provide a strong selective pressure for maintaining stress resistance, despite limited nutrient availability. IMPORTANCE Escherichia coli has been, and continues to be, used as an important indicator species reflecting potential fecal contamination events in the environment. However, recent studies have questioned the validity of this, since E. coli has been found to be capable of long-term colonization of soils. This study investigated whether long-term soil-persistent E. coli strains have evolved altered stress resistance characteristics. In particular, the study investigated whether the main regulator of genes involved in stress protection, the sigma factor RpoS, has been altered in the soil-persistent strains. The results show that RpoS stress protection is fully conserved in soil-persistent strains of E. coli . They also show that loss of the rpoS gene dramatically reduces the ability of this organism to survive in a soil environment. Overall, the results indicate that soil represents a stressful environment for E. coli , and their survival in it requires that they deploy a full stress protection response.

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.

The vast majority of bacterial genome sequencing has been performed using Illumina short reads. Because of the inherent difficulty of resolving repeated regions with short reads alone, only ≈10% of sequencing projects have resulted in a closed genome. The most common repeated regions are those coding for ribosomal operons (rDNAs), which occur in a bacterial genome between 1 and 15 times, and are typically used as sequence markers to classify and identify bacteria. Here, we exploit conservation in the genomic context in which rDNAs occur across taxa to improve assembly of these regions relative to de novo sequencing by using the conserved nature of rDNAs across taxa and the uniqueness of their flanking regions within a genome. We describe a method to construct targeted pseudocontigs generated by iteratively assembling reads that map to a reference genome’s rDNAs. These pseudocontigs are then used to more accurately assemble the newly-sequenced chromosome. We show that this method, implemented as riboSeed, correctly bridges across adjacent contigs in bacterial genome assembly and, when used in conjunction with other genome polishing tools, can assist in closure of a genome.

Sigma B (σ B ) is an alternative sigma factor that controls the transcriptional response to stress in Listeria monocytogenes and is also known to play a role in the virulence of this human pathogen. In the present study we investigated the impact of a sigB deletion on the proteome of L. monocytogenes grown in a chemically defined medium both in the presence and in the absence of osmotic stress (0.5 M NaCl). Two new phenotypes associated with the sigB deletion were identified using this medium. (i) Unexpectedly, the strain with the Δ sigB deletion was found to grow faster than the parent strain in the growth medium, but only when 0.5 M NaCl was present. This phenomenon was independent of the carbon source provided in the medium. (ii) The Δ sigB mutant was found to have unusual Gram staining properties compared to the parent, suggesting that σ B contributes to the maintenance of an intact cell wall. A proteomic analysis was performed by two-dimensional gel electrophoresis, using cells growing in the exponential and stationary phases. Overall, 11 proteins were found to be differentially expressed in the wild type and the Δ sigB mutant; 10 of these proteins were expressed at lower levels in the mutant, and 1 was overexpressed in the mutant. All 11 proteins were identified by tandem mass spectrometry, and putative functions were assigned based on homology to proteins from other bacteria. Five proteins had putative functions related to carbon utilization (Lmo0539, Lmo0783, Lmo0913, Lmo1830, and Lmo2696), while three proteins were similar to proteins whose functions are unknown but that are known to be stress inducible (Lmo0796, Lmo2391, and Lmo2748). To gain further insight into the role of σ B in L. monocytogenes , we deleted the genes encoding four of the proteins, lmo0796, lmo0913, lmo2391, and lmo2748. Phenotypic characterization of the mutants revealed that Lmo2748 plays a role in osmotolerance, while Lmo0796, Lmo0913, and Lmo2391 were all implicated in acid stress tolerance to various degrees. Invasion assays performed with Caco-2 cells indicated that none of the four genes was required for mammalian cell invasion. Microscopic analysis suggested that loss of Lmo2748 might contribute to the cell wall defect observed in the Δ sigB mutant. Overall, this study highlighted two new phenotypes associated with the loss of σ B . It also demonstrated clear roles for σ B in both osmotic and low-pH stress tolerance and identified specific components of the σ B regulon that contribute to the responses observed.