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Tree decline is a global concern and the primary cause is often unknown. Complex interactions between fluctuations in nitrogen (N) and acidifying compounds have been proposed as factors causing nutrient imbalances and decreasing stress tolerance of oak trees. Microorganisms are crucial in regulating soil N available to plants, yet little is known about the relationships between soil N-cycling and tree health. Here, we combined high-throughput sequencing and qPCR analysis of key nitrification and denitrification genes with soil chemical analyses to characterise ammonia-oxidising bacteria (AOB), archaea (AOA) and denitrifying communities in soils associated with symptomatic (declining) and asymptomatic (apparently healthy) oak trees (Quercus robur and Q. petraea) in the United Kingdom. Asymptomatic trees were associated with a higher abundance of AOB that is driven positively by soil pH. No relationship was found between AOA abundance and tree health. However, AOA abundance was driven by lower concentrations of NH4+, further supporting the idea of AOA favouring lower soil NH4+ concentrations. Denitrifier abundance was influenced primarily by soil C:N ratio, and correlations with AOB regardless of tree health. These findings indicate that amelioration of soil acidification by balancing C:N may affect AOB abundance driving N transformations, reducing stress on declining oak trees.

Naphthenic acids (NAs) are carboxylic acids with the formula (C n H 2n + Z O2) and are among the most toxic, persistent constituents of oil sands process‐affected waters (OSPW), produced during oil sands extraction. Currently, the proteins and mechanisms involved in NA biodegradation are unknown. Using LC‐MS/MS shotgun proteomics, we identified proteins overexpressed during the growth of Pseudomonas fluorescens Pf‐5 on a model NA (4′‐n‐butylphenyl)‐4‐butanoic acid (n‐BPBA) and commercial NA mixture (Acros). By day 11, >95% of n‐BPBA was degraded. With Acros, a 17% reduction in intensity occurred with 10–18 carbon compounds of the Z family −2 to −14 (major NA species in this mixture). A total of 554 proteins (n‐BPBA) and 631 proteins (Acros) were overexpressed during growth on NAs, including several transporters (e.g., ABC transporters), suggesting a cellular protective response from NA toxicity. Several proteins associated with fatty acid, lipid, and amino acid metabolism were also overexpressed, including acyl‐CoA dehydrogenase and acyl‐CoA thioesterase II, which catalyze part of the fatty acid beta‐oxidation pathway. Indeed, multiple enzymes involved in the fatty acid oxidation pathway were upregulated. Given the presumed structural similarity between alkyl‐carboxylic acid side chains and fatty acids, we postulate that P. fluorescens Pf‐5 was using existing fatty acid catabolic pathways (among others) during NA degradation., Oil sands mining operations produce vast amounts of oil sands process‐affected waters (OSPW) that contain recalcitrant and toxic naphthenic acids (NAs), yet little is known about the mechanisms of NA biodegradation and the enzymes involved. Here, we identified several proteins overexpressed in Pseudomonas fluorescens Pf‐5 during growth on NAs and included multiple transporters (e.g. ABC transporters), suggesting a cellular protective response from NA toxicity, alongside proteins associated with the fatty acid, lipid, and amino acid metabolism. We postulate that P. fluorescens Pf‐5 is using existing fatty acid catabolic pathways (among others) during NA degradation and these protein targets could be exploited in future biotechnological efforts for OSPW reclamation.

Bioaerosols (or biogenic aerosols) have largely been overlooked by molecular ecologists. However, this is rapidly changing as bioaerosols play key roles in public health, environmental chemistry and the dispersal ecology of microbes. Due to the low environmental concentrations of bioaerosols, collecting sufficient biomass for molecular methods is challenging. Currently, no standardized methods for bioaerosol collection for molecular ecology research exist. Each study requires a process of optimization, which greatly slows the advance of bioaerosol science. Here, we evaluated air filtration and liquid impingement for bioaerosol sampling across a range of environmental conditions. We also investigated the effect of sampling matrices, sample concentration strategies and sampling duration on DNA yield. Air filtration using polycarbonate filters gave the highest recovery, but due to the faster sampling rates possible with impingement, we recommend this method for fine ‐scale temporal/spatial ecological studies. To prevent bias for the recovery of Gram‐positive bacteria, we found that the matrix for impingement should be phosphate‐buffered saline. The optimal method for bioaerosol concentration from the liquid matrix was centrifugation. However, we also present a method using syringe filters for rapid in‐field recovery of bioaerosols from impingement samples, without compromising microbial diversity for high ‐throughput sequencing approaches. Finally, we provide a resource that enables molecular ecologists to select the most appropriate sampling strategy for their specific research question.

Supplementary Table S1

Naphthenic acids (NAs) are carboxylic acids with the formula (CnH2n+ZO2) and are the toxic, persistent constituents of oil sands process-affected waters (OSPW), produced during oil sands extraction. Currently, the proteins and mechanisms involved in NA biodegradation are unknown. Using LC-MS/MS shotgun proteomics, we identified proteins overexpressed during the growth of Pseudomonas fluorescens Pf5 on a model NA (4-n-butylphenyl)-4-butanoic acid (n-BPBA) and commercial NA mixture (Acros). By day 11, >95% of n-BPBA was degraded. With Acros, a 17% reduction in intensity occurred with 10-18 carbon compounds of the Z family -2 to -14 (major NA species in this mixture). A total of 554 proteins (n-BPBA) and 631 proteins (Acros) were overexpressed during growth on NAs; including several transporters (e.g. ABC transporters), suggesting a cellular protective response from NA toxicity. Several proteins associated with fatty acid, lipid and amino acid metabolism were also overexpressed; including acyl-CoA dehydrogenase and acyl-CoA thioesterase II, which catalyze part of the fatty acid beta-oxidation pathway. Indeed, multiple enzymes involved in the fatty acid oxidation pathway were upregulated. Given the presumed structural similarity between alkyl-carboxylic acid side chains and fatty acids, we postulate that P. fluorescens Pf-5 was using existing fatty acid catabolic pathways (among others) during NA degradation.

Particle size is a significant factor in determining the dispersal and inhalation risk from bioaerosols. Green-waste composting is a significant source of bioaerosols (including pathogens), but little is known about the distribution of specific taxa across size fractions. To characterise size fractionated bioaerosol emissions from a compost facility, we used a Spectral Intensity Bioaerosol Sensor (SIBS) to quantify total bioaerosols and qPCR and metabarcoding to quantify microbial bioaerosols. Overall, sub-micron bioaerosols predominated, but molecular analysis showed that most (>75%) of the airborne microorganisms were associated with the larger size fractions (>3.3 µm da). The microbial taxa varied significantly by size, with Bacilli dominating the larger, and Actinobacteria the smaller, size fractions. The human pathogen Aspergillus fumigatus dominated the intermediate size fractions (>50% da 1.1–4.7 µm), indicating that it has the potential to disperse widely and once inhaled may penetrate deep into the respiratory system. The abundance of Actinobacteria (>60% at da

In grasslands, N mineralization and nitrification are important processes and are controlled by several factors, including the in situ microbial community composition. Nitrification involves ammonia oxidising archaea (AOA) and bacteria (AOB) and although AOA and AOB co-exist in soils, they respond differently to environmental characteristics and there is evidence of AOA/AOB niche differentiation. Here, we investigated temporal variation in N mineralization and nitrification rates, together with bacterial, archaeal and ammonia-oxidiser communities in grassland soils, on different geologies: clay, Greensand and Chalk. Across geologies, N mineralization and nitrification rates were slower in the autumn than the rest of the year. Turnover times for soil ammonium pools were

Airborne microorganisms are very difficult to assess accurately under field conditions owing to differences in the sample collection efficiency of the selected sampler and variations in DNA extraction efficiencies. Consequently, bioaerosol abundance and biodiversity can be underestimated, making it more difficult to link specific bioaerosol components to diseases and human health risk. Owing to the low biomass in air samples, it remains a challenge to obtain a representative microbiological sample to recover sufficient DNA for downstream analyses. Improved sampling methods are particularly crucial, especially for investigating viral communities, owing to the extremely low biomass of viral particles in the air compared with other environments. Without detailed information about sampling, characterization and enumeration techniques, interpretation of exposure level is very difficult. Despite this, bioaerosol research has been enhanced by molecular tools, especially next-generation sequencing approaches that have allowed faster and more detailed characterization of air samples.

Silver nanoparticles (AgNPs) enter estuaries via wastewater treatment effluents, where they can inhibit microorganisms, because of their antimicrobial properties. Ammonia-oxidising bacteria (AOB) and archaea (AOA) are involved in the first step of nitrification and are important to ecosystem function, especially where effluent discharge results in high nitrogen inputs. Here, we investigated the effect of a pulse addition of AgNPs on AOB and AOA ammonia monooxygenase (amoA) gene abundances and benthic nitrification potential rates (NPR) in low-salinity and mesohaline estuarine sediments. Whilst exposure to 0.5 mg L-1 AgNPs had no significant effect on amoA gene abundances or NPR, 50 mg L-1 AgNPs significantly decreased AOB amoA gene abundance (up to 76% over 14 days), and significantly decreased NPR by 20-fold in low-salinity sediments and by twofold in mesohaline sediments, after one day. AgNP behaviour differed between sites, whereby greater aggregation occurred in mesohaline waters (possibly due to higher salinity), which may have reduced toxicity. In conclusion, AgNPs have the potential to reduce ammonia oxidation in estuarine sediments, particularly where AgNPs accumulate over time and reach high concentrations. This could lead to long-term risks to nitrification, especially in polyhaline estuaries where ammonia-oxidation is largely driven by AOB.

High-molecular-weight polycyclic aromatic hydrocarbons (HMW-PAHs) are natural components of fossil fuels that are carcinogenic and persistent in the environment, particularly in oil sands process-affected water (OSPW). Their hydrophobicity and tendency to adsorb to organic matter result in low bioavailability and high recalcitrance to degradation. Despite the importance of microbes for environmental remediation, little is known about those involved in HMW-PAH transformations. Here, we investigated the transformation of HMW-PAHs using samples of OSPW and compared the bacterial and fungal community compositions attached to hydrophobic filters and in suspension. It was anticipated that the hydrophobic filters with sorbed HMW-PAHs would select for microbes that specialize in adhesion. Over 33 days, more pyrene was removed (75% ± 11.7%) than the five-ring PAHs benzo[ a ]pyrene (44% ± 13.6%) and benzo[ b ]fluoranthene (41% ± 12.6%). For both bacteria and fungi, the addition of PAHs led to a shift in community composition, but thereafter the major factor determining the fungal community composition was whether it was in the planktonic phase or attached to filters. In contrast, the major determinant of the bacterial community composition was the nature of the PAH serving as the carbon source. The main bacteria enriched by HMW-PAHs were Pseudomonas , Bacillus , and Microbacterium species. This report demonstrates that OSPW harbors microbial communities with the capacity to transform HMW-PAHs. Furthermore, the provision of suitable surfaces that encourage PAH sorption and microbial adhesion select for different fungal and bacterial species with the potential for HMW-PAH degradation.

Anammox, an important N2 loss pathway in marine waters, is not well understood in rivers. In situ measurements of N2 production in UK rivers reveal that anammox can be the dominant N2 loss pathway in permeable but not in impermeable riverbeds. Rivers are an important global sink for excess bioavailable nitrogen: they convert approximately 40% of terrestrial N runoff per year (∼47 Tg) to biologically unavailable N2 gas and return it to the atmosphere1. At present, riverine N2 production is conceptualized and modelled as denitrification2,3,4. Anaerobic ammonium oxidation, known as anammox, is an alternative pathway of N2 production important in marine environments, but its contribution to riverine N2 production is not well understood5,6. Here we use in situ and laboratory measurements of anammox activity using 15N tracers and molecular analyses of microbial communities to evaluate anammox in clay-, sand- and chalk-dominated river beds in the Hampshire Avon catchment, UK during summer 2013. Abundance of the hzo gene, which encodes an enzyme central to anammox metabolism, varied across the contrasting geologies. Anammox rates were similar across geologies but contributed different proportions of N2 production because of variation in denitrification rates. In spite of requiring anoxic conditions, anammox, most likely coupled to partial nitrification, contributed up to 58% of in situ N2 production in oxic, permeable riverbeds. In contrast, denitrification dominated in low-permeability clay-bed rivers, where anammox contributes roughly 7% to the production of N2 gas. We conclude that anammox can represent an important nitrogen loss pathway in permeable river sediments.

Both polycyclic aromatic hydrocarbons (PAHs) and naphthenic acids (NAs) are natural components of fossil fuels, but they are also widespread toxic and environmentally persistent pollutants. They are the major cause of environmental toxicity in oil-sands-process waters (OSPW). This study aimed to investigate the anaerobic biodegradation of the PAHs pyrene and 2-methylnaphthalene, and the NAs adamantane-1-carboxylic acid and a “natural” NA mixture (i.e., acid-extractable NAs from OSPW) under sulfate-reducing and methanogenic conditions by a microbial community derived from an oil sands tailings pond. Using gas-chromatography mass spectrometry (GC–MS), the rate of biodegradation was measured in relation to changes in bacterial community composition. Only 2-methylnaphthalene was significantly degraded after 260 days, with significantly more degradation under sulfate-reducing (40%) than methanogenic conditions (25%). During 2-methylnaphthalene biodegradation, a major metabolite was produced and tentatively identified as 2-naphthoic acid. Denaturing gradient gel electrophoresis (DGGE) demonstrated an increase in intensity of bands during the anaerobic biodegradation of 2-methylnaphalene, which derived from species of the genera Fusibacter, Alkaliphilus, Desulfobacterium, Variovorax, Thaurea, and Hydrogenophaga. Despite the biodegradation of 2-methylnaphthalene, this study demonstrates that, under anaerobic conditions, NAs and high-molecular-weight PAHs are the predominant molecules likely to persist in OSPW. Therefore alternative remediation strategies are required.

Naphthenic acids (NAs) are among the most toxic organic pollutants present in oil sands process waters (OSPW) and enter marine and freshwater environments through natural and anthropogenic sources. We investigated the effects of the acid extractable organic (AEO) fraction of OSPW and individual surrogate NAs, on maximum photosynthetic efficiency of photosystem II (PSII) (FV/FM) and cell growth in Emiliania huxleyi and Chlorella vulgaris as representative marine and freshwater phytoplankton. Whilst FV/FM in E. huxleyi and C. vulgaris was not inhibited by AEO, exposure to two surrogate NAs: (4′-n-butylphenyl)-4-butanoic acid (n-BPBA) and (4′-tert-butylphenyl)-4-butanoic acid (tert-BPBA), caused complete inhibition of FV/FM in E. huxleyi (≥10 mg L−1 n-BPBA; ≥50 mg L−1 tert-BPBA) but not in C. vulgaris. Growth rates and cell abundances in E. huxleyi were also reduced when exposed to ≥10 mg L−1 n- and tert-BPBA; however, higher concentrations of n- and tert-BPBA (100 mg L−1) were required to reduce cell growth in C. vulgaris. AEO at ≥10 mg L−1 stimulated E. huxleyi growth rate (p ≤ 0.002), yet had no apparent effect on C. vulgaris. In conclusion, E. huxleyi was generally more sensitive to NAs than C. vulgaris. This report provides a better understanding of the physiological responses of phytoplankton to NAs which will enable improved monitoring of NA pollution in aquatic ecosystems in the future.

To date, few studies have examined the aerosol microbial content in Metro transportation systems. Here we characterised the aerosol microbial abundance, diversity and composition in the Athens underground railway system. PM10 filter samples were collected from the naturally ventilated Athens Metro Line 3 station “Nomismatokopio”. Quantitative PCR of the 16S rRNA gene and high throughput amplicon sequencing of the 16S rRNA gene and internal transcribed spacer (ITS) region was performed on DNA extracted from PM10 samples. Results showed that, despite the bacterial abundance (mean = 2.82 × 105 16S rRNA genes/m3 of air) being, on average, higher during day-time and weekdays, compared to night-time and weekends, respectively, the differences were not statistically significant. The average PM10 mass concentration on the platform was 107 μg/m3. However, there was no significant correlation between 16S rRNA gene abundance and overall PM10 levels. The Athens Metro air microbiome was mostly dominated by bacterial and fungal taxa of environmental origin (e.g. Paracoccus, Sphingomonas, Cladosporium, Mycosphaerella, Antrodia) with a lower contribution of human commensal bacteria (e.g. Corynebacterium, Staphylococcus). This study highlights the importance of both outdoor air and commuters as sources in shaping aerosol microbial communities. To our knowledge, this is the first study to characterise the mycobiome diversity in the air of a Metro environment based on amplicon sequencing of the ITS region. In conclusion, this study presents the first microbial characterisation of PM10 in the Athens Metro, contributing to the growing body of microbiome exploration within urban transit networks. Moreover, this study shows the vulnerability of public transport to airborne disease transmission.

Graphene oxide (GO) materials loaded with silver nanoparticles (AgNPs) have drawn considerable attention due to their capacity to efficiently inactivate bacteria though a multifaceted mechanism of action, as well as for presenting a synergetic effect against bacteria when compared to the activity of AgNPs and GO alone. In this investigation, we present an inexpensive and environmentally-friendly method for synthesizing reduced GO sheets coated with silver nanoparticles (AgNPs/r-GO) using a coffee extract solution as a green reducing agent. The physical and chemical properties of the produced materials were extensively characterized by scanning electron microscopy (SEM), field-emission gun transmission electron microscopy (FEG-TEM), ultraviolet and visible absorption (UV–Vis), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), inductively coupled plasma-optical emission spectroscopy (ICP-OES) and ion release determination. The results demonstrated that AgNPs/r-GO composites were successfully produced, revealing the formation of micrometer-sized r-GO sheets decorated by AgNPs of approximately 70 nm diameter. Finally, bactericidal and photobactericidal effects of the AgNPs/r-GO composites were tested against Staphylococcus aureus, in which the results showed that the composites presented antimicrobial and photoantimicrobial activities. Moreover, our results demonstrated for the first time, to our knowledge, that an efficient process of bacterial inactivation can be achieved by using AgNPs/r-GO composites under blue light irradiation as a result of three different bacterial killing processes: (i) chemical effect promoted by Ag+ ion release from AgNPs; (ii) photocatalytic activity induced by AgNPs/r-GO composites, enhancing the bacterial photoinactivation due to the excited-Plasmons of the AgNPs when anchored on r-GO; and (iii) photodynamic effect produced by bacterial endogenous photosensitizers under blue-light irradiation. In summary, the present findings demonstrated that AgNPs/r-GO can be obtained by a non-toxic procedure with great potential for biomedical-related applications.

Ammonia oxidising microorganisms (AOM) play an important role in ammonia removal in wastewater treatment works (WWTW) including rotating biological contactors (RBCs). Environmental factors within RBCs are known to impact the performance of key AOM, such that only some operational RBCs have shown ability for elevated ammonia removal. In this work, long-term treatment performance of seven full-scale RBC systems along with the structure and abundance of the ammonia oxidising bacteria (AOB) and archaea (AOA) communities within microbial biofilms were examined. Long term data showed the dominance of AOB in most RBCs, although two RBCs had demonstrable shift toward an AOA dominated AOM community. Next Generation Sequencing of the 16S rRNA gene revealed diverse evolutionary ancestry of AOB from RBC biofilms while nitrite-oxidising bacteria (NOBs) were similar to reference databases. AOA were more abundant in the biofilms subject to lower organic loading and higher oxygen concentration found at the distal end of RBC systems. Modelling revealed a distinct nitrogen cycling community present within high performing RBCs, linked to efficient control of RBC process variables (retention time, organic loading and oxygen concentration). We present a novel template for enhancing the resilience of RBC systems through microbial community analysis which can guide future strategies for more effective ammonia removal. To best of the author’s knowledge, this is the first comparative study reporting the use of next generation sequencing data on microbial biofilms from RBCs to inform effluent quality of small WWTW. Keywords: Biofilm, Rotating biological contactor, Nitrification, Wastewater treatment, Ammonia oxidising bacteria, Ammonia oxidising archaea

International audience; Air samples from four contrasting outdoor environments including a park, an arable farm, a waste water treatment plant and a composting facility were analysed during the summer and winter months. The aim of the research was to study the feasibility of differentiating microbial communities from urban, rural and industrial areas between seasons with chemical and molecular markers such as microbial volatile organic compounds (MVOCs) and phospholipid fatty acids (PLFAs). Air samples (3 l) were collected every 2 h for a total of 6 h in order to assess the temporal variations of MVOCs and PLFAs along the day. MVOCs and VOCs concentrations varied over the day, especially in the composting facility which was the site where more human activities were carried out. At this site, total VOC concentration varied between 80 and 170 μg m−3 in summer and 20–250 μg m−3 in winter. The composition of MVOCs varied between sites due to the different biological substrates including crops, waste water, green waste or grass. MVOCs composition also differed between seasons as in summer they are more likely to get modified by oxidation processes in the atmosphere and in winter by reduction processes. The composition of microbial communities identified by the analysis of PLFAs also varied among the different locations and between seasons. The location with higher concentrations of PLFAs in summer was the farm (7297 ng m−3) and in winter the park (11,724 ng m−3). A specific set of MVOCs and PLFAs that most represent each one of the locations was identified by principal component analyses (PCA) and canonical analyses. Further to this, concentrations of both total VOCs and PLFAs were at least three times higher in winter than in summer. The difference in concentrations between summer and winter suggest that seasonal variations should be considered when assessing the risk of exposure to these compounds.

Silver nanoparticles (AgNPs) enter estuaries via wastewater treatment effluents, where they can inhibit microorganisms, because of their antimicrobial properties. Ammonia-oxidising bacteria (AOB) and archaea (AOA) are involved in the first step of nitrification and are important to ecosystem function, especially where effluent discharge results in high nitrogen inputs. Here, we investigated the effect of a pulse addition of AgNPs on AOB and AOA ammonia monooxygenase (amoA) gene abundances and benthic nitrification potential rates (NPR) in low-salinity and mesohaline estuarine sediments. Whilst exposure to 0.5 mg L

Research spanning over 40 years has examined many aspects of the microbial ecology of the Colne estuary (Essex, United Kingdom) and it is arguably the most comprehensively understood temperate estuary in the Northern hemisphere. The hypernutrified Colne estuary exhibits strong gradients of nutrient concentrations (nitrate, ammonium, phosphate, dissolved organic nitrogen) from river and treated sewage inputs at the top of the estuary, with concentrations decreasing towards the estuary mouth. These strong concentration gradients facilitate the study of the biogeochemical process rates and the microbial communities responsible. Planktonic primary production is at an oligotrophic level because of light limitation in the turbid water, but a mixed water column maintains planktonic photosynthesis despite low light. Dense microphytobenthic biofilms occur throughout the estuary, with high rates of annual primary production typical of NW European nutrient-rich estuaries, but benthic primary production accounts for only a small proportion of the N load to the estuary. Organic matter degradation is focussed in the estuarine sediments, with greatest organic content in the upper estuary, decreasing towards the mouth. Benthic biogeochemical processes, leading to organic mineralisation and element recycling, including O2 uptake, sulphate reduction, methanogenesis, nitrate reduction, denitrification and anammox have all been quantified along the estuary, together with chemolithotrophic nitrification. Benthic denitrification removes a significant proportion of the N load to the estuary. Molecular techniques, including profiling, identification and quantification of 16S rRNA and key functional genes, have provided an understanding of the microbial communities in relation to position and biogeochemical activity. The concentrations and emissions of biological volatile compounds (nitrous oxide, methane, hydrogen sulphide and isoprene) have also been measured, and their ecological significance elucidated. Tidal exchange between the saltmarshes and the main estuary channel shows seasonal variation. When nitrate in tidal water is high in spring, the marsh sediments remove it, but tidally export ammonium and small particles of organic nitrogen to the estuary in summer when nitrogen in coastal water is low. Overall, the saltmarshes show a balanced nitrogen cycle suggestive of a stable climax community, but capable of responding to and removing increased nitrate concentrations in estuarine waters. These data, originating from the long-term study of a single system, are unique and this paper highlights how the Colne estuary microbial ecology observatory has contributed to our understanding of estuarine microbial ecology and biogeochemistry.