Publications

Recent research has highlighted the importance of dissolved organic matter (DOM) for ecosystem function and because of this paradigm shift, it has become crucial to not only quantify its contribution to river nutrient loads but also to characterise its composition. There has been a significant research effort utilising optical methods, such as fluorescence and UV-Vis spectrophotometry, in order to start exploring DOM character. However, these methods still lack the granularity to understand the chemical composition at the molecular level, which is vital to properly understanding its functional role in freshwater ecosystems. As a direct result, there has been a shift towards including molecular-scale analyses to investigate the in-stream processing of the material. Alongside this, recent methodological advancements, particularly in mass spectrometry are opening new opportunities for probing one of the most complex environmental mixtures. However, in order to fully exploit these opportunities, it is key that the way that samples are collected, processed and stored is considered carefully such that sample integrity is maintained. There are additional challenges when collecting water samples for analysis at molecular scale, for example the ultra-low concentrations of individual compounds within DOM means that the samples are sensitive to contamination. This paper discusses current sample collection, processing and storage protocols for this C, N and P quantification and characterisation in freshwaters, and proposes a new standardised protocol suitable for both nutrient fraction quantification and molecular scale analyses, based on method development and testing undertaken in our UK Natural Environment Research Council large grant programme, characterising the nature, origins and ecological significance of Dissolved Organic Matter IN freshwater Ecosystems (DOMAINE).

AIM This study sought to track the profile of antimicrobial resistance (AMR) genes and pathogens present in hospital settings in Wales, UK, by employing wastewater-based epidemiology (WBE). BACKGROUND Hospitals are recognized as critical hotspots for the proliferation of pathogenic microbes and the rapid emergence and spread of AMR. The effluents discharged from these settings play a significant role in introducing these contaminants into surrounding ecosystems. Tracking the type and load of AMR and pathogens within hospital environments is crucial for informing policy decisions and implementing effective risk mitigation strategies METHODS Twenty-hour composite waste-water samples were collected from hospital outfall pipes using refrigerated autosamplers. These samples were analysed to detect AMR genes and pathogenic microbes via metagenomic sequencing on the Illumina Novaseq 6000 platform. The study also monitored for the presence of antibiotics in the hospital effluents using UPLC-MS/MS. RESULTS AMR genes conferring resistance towards aminoglycosides, beta lactams and macrolides were prevalent. A significant presence of bacterial and fungal pathogens was also noted. Notable spatial and temporal variations were observed in the distribution of these contaminants across the hospital sites. The analysis also showed variations in the relative concentrations of different antimicrobial compounds across the hospitals. CONCLUSIONS The results highlight the role of hospital wastewater as significant reservoirs for AMR genes, antibiotics, and pathogens into the environment. We strongly recommend the pre-treatment of these effluents on-site before their discharge into municipal wastewater treatment systems to mitigate these risks.

Anthropogenic activities have significantly altered global biogeochemical nitrogen (N) cycling leading to major environmental problems such as freshwater eutrophication, biodiversity loss and enhanced greenhouse gas emissions. The soils in the riparian interface between terrestrial and aquatic ecosystems may prevent excess N from entering freshwaters (e.g. via plant uptake, microbial transformations and denitrification). Although these processes are well documented in intensively managed agroecosystems, our understanding of riparian N removal in semi-natural systems remains poor. Our aim was to assess the spatial zoning of soil microbial communities (PLFA), N cycling gene abundance (archaeal and bacterial amoA, nifH, nirK, nirS, nosZ), N processing rates and plant N uptake across an extensively sheep grazed riparian area. As expected, soil properties differed greatly across the riparian transect, with significant decreases in organic matter, NH4 +, carbon (C) and N content closest to the river (<10 m). In addition, different microbial community structures were found along the transect. The abundance of N fixation (nifH) increased with distance from the river (>10 m), while ammonia oxidising archaea (AOA) increased in abundance towards the river. N2O emissions rates were limited by C and to a lesser extent by N with greater emissions close to the river. Plant uptake of urea-derived 15N was high (ca. 55–70% of that added to the soil) but 30–65% of the N was potentially lost by denitrification or leaching. Percentage recovered also suggests that the spatial patterning of plant and microbial N removal processes are different across the riparian zone. Our study provides novel insights into the underlying mechanisms controlling the spatial variability of N cycling in semi-natural riparian ecosystems.

A portion of the nifH (nitrogenase) gene was amplified from twelve samples of boreal forest moss stems (Pleurozium schreberi) from Sweden, using degenerate primers IGK3 and DVV. Sequencing was carried out using the Illumina MiSeq platform, paired-end 300 bp kit. Bioinformatic processing included the RDP FunGene pipeline and a classification and regressions trees (CART) model (Frank et al. 2016, Environ Microbiol Rep 8:905-916). Output from key steps is available here. Raw data are available on NCBI's SRA, accession numbers SRR5366534-SRR5366545.

Soil testing in agriculture is associated with many economic and environmental benefits. However, previous studies have shown that a significant proportion of beef and sheep farmers in the UK do not carry out standard soil index testing (pH, available Mg, P and K); with much fewer again carrying out more extensive soil tests (e.g., organic matter, micronutrients). This study identifies barriers and motivations to soil testing amongst the beef and sheep sector, using a combination of farmer surveys, expert interviews, and a 5-year soil testing dataset from the largest commercial UK soil testing laboratory. Evidence for differences in the adoption of soil tests by beef and sheep farmers compared to the arable and dairy sectors is explained in relation to: (1) the extent of soil pH and nutrient imbalances, linking to the intensity of management in the different sectors; and (2) the extent to which farmers perceive links between their soils and their outputs (profits, yield, livestock health). We show a greater likelihood for farmers to engage with soil testing when the links to declining outputs are clearer. Our results showed that beef and sheep farmers who did engage with soil testing showed greater levels of innovation and were more likely to seek advisory support, most often associated with larger farm sizes. Our data also highlights the importance of an output-driven approach to initiate an interest in soil analyses amongst less engaged farmers. We argue that this avenue offers greater potential for enhancement of farmers’ knowledge of the soil system than a primarily regulatory-driven approach, where soil testing becomes a compulsory action but does not lead to subsequent improvements in farm management.

Infections caused by waterborne Escherichia coli O157 continue to pose a public health risk. An increase in faecal coliform loading of watercourses due to

Prisons are high-risk settings for the transmission of communicable disease. Robust surveillance systems are required to identify and control outbreaks. Wastewater surveillance for SARS-CoV-2 was introduced in four prisons in Wales in March 2022. We investigated its contribution to the COVID-19 surveillance programme.

The current view suggests that in low-temperature acidic environments, archaea are significantly less abundant than bacteria. Thus, this study of the microbiome of Parys Mountain (Anglesey, UK) sheds light on the generality of this current assumption. Parys Mountain is a historically important copper mine and its acid mine drainage (AMD) water streams are characterised by constant moderate temperatures (8–18 °C), extremely low pH (1.7) and high concentrations of soluble iron and other metal cations. Metagenomic and SSU rRNA amplicon sequencing of DNA from Parys Mountain revealed a significant proportion of archaea affiliated with Euryarchaeota, which accounted for ca. 67% of the community. Within this phylum, potentially new clades of Thermoplasmata were overrepresented (58%), with the most predominant group being "E-plasma", alongside low-abundant Cuniculiplasmataceae, 'Ca. Micrarchaeota' and 'Terrestrial Miscellaneous Euryarchaeal Group' (TMEG) archaea, which were phylogenetically close to Methanomassilicoccales and clustered with counterparts from acidic/moderately acidic settings. In the sediment, archaea and Thermoplasmata contributed the highest numbers in V3-V4 amplicon reads, in contrast with the water body community, where Proteobacteria, Nitrospirae, Acidobacteria and Actinobacteria outnumbered archaea. Cultivation efforts revealed the abundance of archaeal sequences closely related to Cuniculiplasma divulgatum in an enrichment culture established from the filterable fraction of the water sample. Enrichment cultures with unfiltered samples showed the presence of Ferrimicrobium acidiphilum, C. divulgatum, 'Ca. Mancarchaeum acidiphilum Mia14', 'Ca. Micrarchaeota'-related and diverse minor (< 2%) bacterial metagenomic reads. Contrary to expectation, our study showed a high abundance of archaea in this extremely acidic mine-impacted environment. Further, archaeal populations were dominated by one particular group, suggesting that they are functionally important. The prevalence of archaea over bacteria in these microbiomes and their spatial distribution patterns represents a novel and important advance in our understanding of acidophile ecology. We also demonstrated a procedure for the specific enrichment of cell wall-deficient members of the archaeal component of this community, although the large fraction of archaeal taxa remained unculturable. Lastly, we identified a separate clustering of globally occurring acidophilic members of TMEG that collectively belong to a distinct order within Thermoplasmata with yet unclear functional roles in the ecosystem.

Urine patches in grazed systems are hotspots for nitrogen (N) cycling and losses to the wider environment. Retention and subsequent recycling of urinary-N is key to minimise losses and increase ecosystem nitrogen use efficiency. Biosynthesis into the microbial organic N pool is an important N pathway but this has not been directly quantified in a urine patch. Herein, we present the results of a time course experiment using soil mesocosms sown with perennial ryegrass (Lolium perenne L.) and treated with 15N-labelled sheep urine to determine partitioning of the applied N between plant, soil biomass pools and leaching losses following simulated rainfall events. 15N-tracing used bulk and compound-specific 15N-stable isotope probing (SIP) to determine the fate of urinary N. Initial high leaching losses (233 kg N ha−1) were comprised of native soil N, ammonium and nitrate derived from urine by urea hydrolysis and nitrification, respectively. Leaching subsequently decreased whilst uptake into plant biomass and microbial biosynthesis increased during periods of low rainfall. Uptake into above and belowground plant biomass was the largest fate of urinary-15N after 94 d (42%), although assimilation into microbial biomass dominated for ca. 1 month after urine deposition (34%). Compound-specific 15N–SIP of amino acids and amino sugars revealed immobilisation of urinary-N following mineralisation was the dominant pathway for biosynthesis, with incorporation into bacterial organic N pools more rapid than into the fungal biomass. There was also intact utilisation of glycine derived from urine. This study provides clear evidence that direct assimilation of urine-derived N into microbial organic N pools is an important process for retaining N in a urine patch, which will subsequently support plant N supply during microbial turnover.

Sulphur (S) is a key macronutrient for all organisms, with similar cellular requirements to that of phosphorus (P). Studies of S cycling have often focused on the inorganic fraction, however, there is strong evidence to suggest that freshwater microorganisms may also access dissolved organic S (DOS) compounds (e.g. S-containing amino acids). The aim of this study was to compare the relative concentration and depletion rates of organic 35 S-labelled amino acids (cysteine, methionine) with inorganic S (Na 2 35 SO 4 ) in oligotrophic versus mesotrophic river waters draining from low nutrient input and moderate nutrient input land uses respectively. Our results showed that inorganic SO 4 2− was present in the water column at much higher concentrations than free amino acids. In contrast to SO 4 2− , however, cysteine and methionine were both rapidly depleted from the mesotrophic and oligotrophic waters with a halving time &lt; 1 h. Only a small proportion of the DOS removed from solution was mineralized and excreted as SO 4 2− (&lt; 16% of the total taken up) suggesting that the DOS could be satisfying a demand for carbon (C) and S. In conclusion, even though inorganic S was abundant in freshwater, it appears that the aquatic communities retained the capacity to take up and assimilate DOS.

Proteins represent the dominant input of organic N into most ecosystems and they also constitute the largest store of N in soil organic matter. The extracellular protease mediated breakdown of proteins to amino acids therefore represents a key step regulating N cycling in soil. In this study we investigated the influence of a range of environmental factors on the rate of protein mineralization in a grazed grassland and fallow agricultural soil. The protein turnover rates were directly compared to the rates of amino acid mineralization under the same conditions. Uniformly 14C-labelled soluble protein and amino acids were added to soil and the rate of 14CO2 evolution determined over 30 d. Our results indicate that the primary phase of protein mineralization was approximately 20 ± 3 fold slower that the rate of amino acid mineralization. The addition of large amounts of inorganic NO3 − and NH4 + to the soil did not repress the rate of protein mineralization suggesting that available N does not directly affect protease activity in the short term. Whilst protein mineralization was strongly temperature sensitive, the presence of plants and the addition of humic and tannic acids had relatively little influence on the rate of soluble protein degradation in this fertile grassland soil. Our results suggests that the extracellular protease mediated cleavage of proteins to amino acids rather than breakdown of amino acids to NH4 + represents the limiting step in soil N cycling.