Potential feedback mediated by soil microbiome response to warming in a glacier forefield.

Mountain glaciers are retreating at an unprecedented rate due to global warming. Glacier retreat is widely believed to be driven by the physiochemical characteristics of glacier surfaces; however, the current knowledge of such biological drivers remains limited. An estimated 130 Tg of organic carbon (OC) is stored in mountain glaciers globally. As a result of global warming, the accelerated microbial decomposition of OC may further accelerate the melting process of mountain glaciers by heat production with the release of greenhouse gases, such as carbon dioxide (CO2 ) and methane. Here, using short-term aerobic incubation data from the forefield of Urumqi Glacier No. 1, we assessed the potential climate feedback mediated by soil microbiomes at temperatures of 5°C (control), 6.2°C (RCP 2.6), 11°C (RCP 8.5), and 15°C (extreme temperature). We observed enhanced CO2 -C release and heat production under warming conditions, which led to an increase in near-surface (2 m) atmospheric temperatures, ranging from 0.9°C to 3.4°C. Warming significantly changed the structures of the RNA-derived (active) and DNA-derived (total) soil microbiomes, and active microbes were more sensitive to increased temperatures than total microbes. Considering the positive effects of temperature and deglaciation age on the CO2 -C release rate, the alterations in the active microbial community structure had a negative impact on the increased CO2 -C release rate. Our results revealed that glacial melting could potentially be significantly accelerated by heat production from increased microbial decomposition of OC. This risk might be true for other high-altitude glaciers under emerging warming, thus improving the predictions of the effects of potential feedback on global warming.

AsChip: A High-Throughput qPCR Chip for Comprehensive Profiling of Genes Linked to Microbial Cycling of Arsenic.

Arsenic (As) is a ubiquitous toxic element adversely affecting human health. Microbe-mediated cycling of As is largely mediated by detoxification and energy metabolism in microorganisms. We here report the development of a novel high-throughput qPCR (HT-qPCR) chip (AsChip) for comprehensive profiling of genes involved in microbial As cycling (here collectively termed "As genes"). AsChip contained 81 primer sets targeting 19 As genes and the 16S rRNA gene as a reference gene. Gene amplicon sequencing showed high identity (>96%) of newly designed primers corresponding to their targets. AsChip displayed high sensitivity (plasmid template serial dilution test; r = -0.99), with more than 96% of all PCR assays yielding true positive signals. R2 coefficients for standard curves and PCR amplification efficiencies averaged 0.98 and 0.99, respectively. A high correlation between CT values obtained by AsChip and conventional qPCR was obtained ( r = 0.962, P < 0.001). Finally, we successfully applied AsChip on soil samples from a chromium-copper-arsenic-contaminated field site and identified diverse As genes with total abundance average of 0.4 As gene copies per 16S rRNA. Our results indicate that AsChip constitutes a robust tool for comprehensive quantitative profiling of As genes in environmental samples.

Evaluation of pH-Dependent Metal Speciation Artifacts in Whole-Cell Bioreporter Analysis.

Whole-cell bacterial biosensors (bioreporters) are commonly applied for determination of metal toxicity and bioavailability in environmental samples. This is accomplished using a standard procedure whereby the sample is mixed with bioreporter cells suspended in a buffered medium at a fixed pH (set-point pH assay). This experimental approach can alter the sample pH. We therefore hypothesized that metal speciation artifacts compromising our ability to use bioreporters for determination of the "true" metal bioavailability in environmental samples may be introduced. Using the copper-specific bioreporter DF57-Cu15 as a model, we compared the conventional set-point pH assay to a flexible pH assay allowing for bioavailability determination at in situ sample pH. Our results demonstrate that pH-dependent metal speciation bias may occur when using the conventional set-point pH assay, and we recommend performing bioreporter measurements and calibrations at in situ sample pH. Although we only studied copper bioavailability, our results also have implications for bioreporter determination of other analytes displaying pH-dependent speciation such as other metals and some organics. We call for additional bioreporter studies of chemical speciation artifacts as this represents a problem hitherto overlooked in bioreporter literature. We thus conclude that there may be considerable scope for optimization of existing bioreporter assays for assessment of environmental pollutant bioavailability.

Niche differentiation and biogeography of Bathyarchaeia in paddy soil ecosystems: a case study in eastern China.

Bathyarchaeia (formerly Bathyarchaeota) is a group of highly abundant archaeal communities that play important roles in global biogeochemical cycling. Bathyarchaeia is predominantly found in sediments and hot springs. However, their presence in arable soils is relatively limited. In this study, we aimed to investigate the spatial distributions and diversity of Bathyarchaeia in paddy soils across eastern China, which is a major rice production region. The relative abundance of Bathyarchaeia among total archaea ranged from 3 to 68% in paddy soils, and Bathy-6 was the dominant subgroup among the Bathyarchaeia (70-80% of all sequences). Bathyarchaeia showed higher migration ability and wider niche width based on the neutral and null model simulations. Bathy-6 was primarily assembled by deterministic processes. Soil pH and C/N ratio were identified as key factors influencing the Bathyarchaeia composition, whereas C/N ratio and mean annual temperature influenced the relative abundance of Bathyarchaeia. Network analysis showed that specific Bathyarchaeia taxa occupied keystone positions in the archaeal community and co-occurred with some methanogenic archaea, including Methanosarcina and Methanobacteria, and ammonia-oxidizing archaea belonging to Nitrososphaeria. This study provides important insights into the biogeography and niche differentiation of Bathyarchaeia particularly in paddy soil ecosystems.

Impacts of soil type on the temporal dynamics of antibiotic resistance gene profiles following application of composted manure.

Farmland application of composted manure is associated with a risk of dissemination of antibiotic resistance genes (ARGs) in agricultural soils. However, the impact of soil type on the temporal dynamics of ARGs in agricultural soil remains largely unclear. The aims of this study were to study the persistence of composted manure-derived ARGs in six soil types representative for Chinese agriculture and to explore the underlying environmental drivers of soil ARG profiles in a controlled greenhouse experiment. Temporal dynamics of manure-derived ARGs was strongly affected by soil type. High persistence of fertilizer-derived ARGs was evident in red soil, yellow soil and sierozem soil, while a rapid decrease to near pre-fertilization levels (low persistence) was observed in yellow-brown soil, black soil and brown earth soil. The distribution of ARGs was linked to soil properties such as soil texture, pH and concentrations of heavy metals. More complex co-occurrence networks of ARGs and bacteria in red soil, yellow soil, and sierozem soil suggested a higher dissemination potential, which was consistent with the significantly increased abundance of MGEs in these three types of soils. Our findings highlight the necessity for developing tailored fertilization strategies for different soil types to mitigate environmental dissemination of ARGs.

Response of microbial taxonomic and nitrogen functional attributes to elevated nitrate in suburban groundwater.

Anthropogenic nitrogen (N) input has led to elevated levels of nitrate nitrogen (NO3--N) in the groundwater. However, insights into the responses of the microbial community and its N metabolic functionality to elevated NO3--N in suburban groundwater are still limited. Here, we explored the microbial taxonomy, N metabolic attributes, and their responses to NO3--N pollution in groundwaters from Chaobai River catchment (CR) and Huai River catchment (HR) in Beijing, China. Results showed that average NO3--N and NH4+-N concentrations in CR groundwater were 1.7 and 3.0 folds of those in HR. NO3--N was the dominant nitrogen specie both in HR and CR groundwater (over 80 %). Significantly different structures and compositions of the microbial communities and N cycling gene profiles were found between CR groundwater and HR groundwater (p < 0.05), with CR groundwater harboring significantly lower microbial richness and abundance of N metabolic genes. However, denitrification was the dominant microbial N cycling process in both CR and HR groundwater. Strong associations among NO3--N, NH4+-N, microbial taxonomic, and N functional attributes were found (p < 0.05), suggesting denitrifiers and Candidatus_Brocadia might serve as potential featured biomarkers for the elevated NO3--N and NH4+-N concentration in groundwater. Path analysis further revealed the significant effect of NO3--N on the overall microbial N functionality and microbial denitrification (p < 0.05). Collectively, our results provide field evidence that elevated levels of NO3--N and NH4+-N under different hydrogeologic conditions had a significant effect on the microbial taxonomic and N functional attributes in groundwater, with potential implications for improving sustainable N management and risk assessment of groundwater.

Prevalence of antibiotic resistance genes in Pangasianodon hypophthalmus and Oreochromis niloticus aquaculture production systems in Bangladesh.

Antibiotic resistance genes (ARGs) constitute emerging pollutants of significant public health concern. Antibiotics applied in aquaculture may stimulate the proliferation and dissemination of ARGs. This study investigated the prevalence and diversity of ARGs in Pangasianodon hypophthalmus (formerly Pangasius) and Oreochromis niloticus (formerly Tilapia) commercial aquaculture ponds from four economically important divisions (i.e. regions) of Bangladesh using a high-throughput qPCR ARG SmartChip and further aimed to explore effects of aquaculture pond management and water quality on the observed ARG prevalence patterns. A total of 160 ARGs and 10 mobile genetic elements (MGEs) were detected across all samples (n = 33), of which 76 ARGs and MGEs were shared between all regions. Multidrug resistance genes were the most frequently encountered ARGs, followed by ARGs conferring resistance to ß-lactams, aminoglycosides, tetracyclines, and macrolide-lincosamide-streptogramin B (MLSB). Research ponds managed by the Bangladesh Agricultural University had the lowest abundance and diversity of ARGs, suggesting that proper management such as regular water quality monitoring, fortnightly water exchange and use of probiotics instead of antibiotics may mitigate the dissemination of antibiotic resistance from aquaculture ponds. The Adonis test (R2 = 0.35, p < 0.001) and distance decay relationships revealed that the ARGs composition displayed a significant biogeographical pattern (i.e., separation based on geographic origin). However, this effect could possibly be due to feed type as different feed types were used in different regions. In conclusion, our results indicate that there is a vast potential for improving aquaculture pond management practices in Bangladesh to mitigate the environmental dissemination of ARGs and their subsequent transmission to humans.

Evidence for co-selection of antibiotic resistance genes and mobile genetic elements in metal polluted urban soils.

Antibiotic resistance genes (ARGs) constitute emerging environmental pollutants and pose risks to public health. Toxic metals are known to select for metal-resistant bacteria in metal-contaminated soils, but there is growing concern that metal contaminants can also act as co-selective agents thereby causing environmental proliferation of antibiotic resistance. In this study, we quantified ARGs and selected mobile genetic elements (MGEs) known to constitute potential ARG hosts in 50 archived urban and suburban soils from the Belfast metropolitan area using a high-throughput qPCR ARG chip. ARG prevalence was linked to concentrations of individual metals and a soil metal toxicity index calculated based on the relative toxicity of different metals to soil microbial processes. A total of 164 ARGs were detected across the 50 soils analyzed with an average absolute abundance of 3.4 × 107 ARG gene copies per gram of soil. A significant correlation between abundance of ARGs and MGEs was observed, suggesting the importance of horizontal gene transfer for ARG dissemination. Network analysis revealed significant co-occurrence patterns between specific metals (As, Cd, Co, Cr, Cu. Hg, Ni and Zn) and associated ARGs. Path analysis further indicated that the soil metal toxicity index significantly affected the number of detected ARGs (λ = 0.32, P < 0.001) and the abundance of metal co-occurring ARGs (λ = 0.612, P < 0.001) via effects on MGEs. Collectively, our results indicate a role of soil metals in co-selection of ARGs and MGEs in urban and semi-urban soils and suggest a risk for environmental ARG dissemination via horizontal gene transfer.

Feed additives shift gut microbiota and enrich antibiotic resistance in swine gut.

Antibiotic resistance genes (ARGs) are emerging environmental contaminants posing a threat to public health. Antibiotics and metals are widely used as feed additives and could consequently affect ARGs in swine gut. In this study, high-throughput quantitative polymerase chain reaction (HT-qPCR) based ARG chip and next-generation 16S rRNA gene amplicon sequencing data were analyzed using multiple statistical approaches to profile the antibiotic resistome and investigate its linkages to antibiotics and metals used as feed additives and to the microbial community composition in freshly collected swine manure samples from three large-scale Chinese pig farms. A total of 146 ARGs and up to 1.3×1010 total ARG copies per gram of swine feces were detected. ARGs conferring resistance to aminoglycoside, macrolide-lincosamide-streptogramin B (MLSB) and tetracycline were dominant in pig gut. Total abundance of ARGs was positively correlated with in-feed antibiotics, microbial biomass and abundance of mobile genetic elements (MGEs) (P<0.05). A significant correlation between microbial communities and ARG profiles was observed by Procrustes analysis. Network analysis revealed that Bacteroidetes and Firmicutes were the most dominant phyla co-occurring with specific ARGs. Partial redundancy analysis indicated that the variance in ARG profiles could be primarily attributed to antibiotics and metals in feed (31.8%), gut microbial community composition (23.3%) and interaction between feed additives and community composition (16.5%). These results suggest that increased levels of in-feed additives could aggravate the enrichment of ARGs and MGEs in swine gut.

Critical knowledge gaps and research needs related to the environmental dimensions of antibiotic resistance.

There is growing understanding that the environment plays an important role both in the transmission of antibiotic resistant pathogens and in their evolution. Accordingly, researchers and stakeholders world-wide seek to further explore the mechanisms and drivers involved, quantify risks and identify suitable interventions. There is a clear value in establishing research needs and coordinating efforts within and across nations in order to best tackle this global challenge. At an international workshop in late September 2017, scientists from 14 countries with expertise on the environmental dimensions of antibiotic resistance gathered to define critical knowledge gaps. Four key areas were identified where research is urgently needed: 1) the relative contributions of different sources of antibiotics and antibiotic resistant bacteria into the environment; 2) the role of the environment, and particularly anthropogenic inputs, in the evolution of resistance; 3) the overall human and animal health impacts caused by exposure to environmental resistant bacteria; and 4) the efficacy and feasibility of different technological, social, economic and behavioral interventions to mitigate environmental antibiotic resistance.1.

Dissipation of pterosin B in acid soils - Tracking the fate of the bracken fern carcinogen ptaquiloside.

Bracken ferns (Pteridium spp.) are well-known for their carcinogenic properties, which are ascribed to the content of ptaquiloside and ptaquiloside-like substances. Ptaquiloside leach from the ferns and may cause contamination of drinking water. Pterosin B is formed by hydrolysis of ptaquiloside. In soil, Pterosin B is adsorbed more strongly and it is expected to have a slower turnover than ptaquiloside. We thus hypothesized that pterosin B may serve as an indicator for any past presence of ptaquiloside. Pterosin B degradation was studied in acid forest soils from bracken-covered and bracken-free areas. Soil samples were incubated with pterosin B at 3 and 8 µg g-1 for 10 days, whereas sterile (autoclaved) samples were incubated for 23 days. Pterosin B showed unexpected fast degradation in soils with full degradation in topsoils in 2-5 days. Pterosin B dissipation followed the sum of two-first order reactions. The initial fast reaction with half-lives of 0.7-3.5 h contributed 11-59% of the total pterosin B degradation, while the slow reaction was 20-100 times slower than the fast reaction. Total dissipation half-lives were shorter for loamy sand (4 h) than for sandy loam soils (28 h). No degradation of pterosin B took place under sterile conditions assuming observed dissipation during the first 3 h could be attributed to irreversible sorption. Our results demonstrate that pterosin B is microbially degraded and that pterosin B is as unstable as ptaquiloside and hence cannot be used as an indicator for former presence of ptaquiloside in soil.

Activity and population dynamics of heterotrophic and ammonia-oxidizing microorganisms in soil surrounding sludge bands spiked with linear alkylbenzene sulfonate: a field study.

Recent research has documented soil microorganisms to be rather sensitive to linear alkylbenzene sulfonates (LAS), which may enter the soil environment in considerable quantities following sewage sludge disposal. We here report field effects of LAS on selected microbial populations present in a sandy soil surrounding well-defined sludge bands spiked with high but realistic LAS levels (7.1 or 31.3 g/kg). Surprisingly, LAS had no effect on heterotrophic respiration in the sludge compartment per se but stimulated activity and metabolic quotient (microbial activity per unit of biomass) in the surrounding soil. By contrast, autotrophic ammonia oxidation was initially inhibited in the LAS-spiked sludge. This led to dramatic transient increases of NH4+ availability in the sludge and surrounding soil, subsequently stimulating soil ammonia oxidizers. As judged from a Nitrosomonas europaea bioluminescence toxicity assay, however, LAS or other sludge components never accumulated to toxic levels in the soil compartments and the LAS tolerance of the indigenous microbes further remained unchanged following LAS exposure. LAS effects on the investigated microbial populations largely occurred during the first two months and were confined to soil closer than 30 mm from LAS-spiked sludge. Our results strongly suggest that disposal of LAS-contaminated sludge does not pose a major risk to the function of the soil microbial community under field conditions.

Evaluation of the leucine incorporation technique for detection of pollution-induced community tolerance to copper in a long-term agricultural field trial with urban waste fertilizers.

Copper (Cu) is known to accumulate in agricultural soils receiving urban waste products as fertilizers. We here report the use of the leucine incorporation technique to determine pollution-induced community tolerance (Leu-PICT) to Cu in a long-term agricultural field trial. A significantly increased bacterial community tolerance to Cu was observed for soils amended with organic waste fertilizers and was positively correlated with total soil Cu. However, metal speciation and whole-cell bacterial biosensor analysis demonstrated that the observed PICT responses could be explained entirely by Cu speciation and bioavailability artifacts during Leu-PICT detection. Hence, the agricultural application of urban wastes (sewage sludge or composted municipal waste) simulating more than 100 years of use did not result in sufficient accumulation of Cu to select for Cu resistance. Our findings also have implications for previously published PICT field studies and demonstrate that stringent PICT detection criteria are needed for field identification of specific toxicants.

Biomedicine in the environment: cyclotides constitute potent natural toxins in plants and soil bacteria.

Bioactive compounds produced by plants are easily transferred to soil and water and may cause adverse ecosystem effects. Cyclotides are gene-encoded, circular, cystine-rich mini-proteins produced in Violaceae and Rubiaceae in high amounts. Based on their biological activity and stability, cyclotides have promising pharmaceutical and agricultural applications. We report the toxicity of the cyclotides: kalata B1, kalata B2, and cycloviolacin O2 extracted from plants to green algae (Pseudokirchneriella subcapitata), duckweed (Lemna minor L.), lettuce (Lactuca sativa L.), and bacteria extracted from soil measured as [³H]leucine incorporation. Quantification by liquid chromatography-mass spectrometry demonstrated up to 98% loss of cyclotides from aqueous solutions because of sorption to test vials. Sorption was prevented by adding bovine serum albumin (BSA) to the aqueous media. Cyclotides were toxic to all test organisms with EC50 values of 12 through 140 µM (algae), 9 through 40 µM (duckweed), 4 through 54 µM (lettuce), and 7 through 26 µM (bacteria). Cycloviolacin O2 was the most potent cyclotide in all assays examined. This report is the first to document toxic effects of cyclotides in plants and soil bacteria and to demonstrate that cyclotides are as toxic as commonly used herbicides and biocides. Hence, cyclotides may adversely affect soil and aquatic environments, which needs to be taken into account in future risk assessment of cropping systems for production of these highly bioactive compounds.

Comparison of aerobic and anaerobic [3H]leucine incorporation assays for determining pollution-induced bacterial community tolerance in copper-polluted, irrigated soils.

Pollution-induced community tolerance (PICT) constitutes a sensitive and ecologically relevant impact parameter in ecotoxicology. We report the development and application of a novel anaerobic [(3) H]leucine incorporation assay and its comparison with the conventional aerobic [(3) H]leucine incorporation assay for PICT detection in soil bacterial communities. Selection of bacterial communities was performed over 42 d in bulk soil microcosms (no plants) and in rice (Oryza sativa) rhizosphere soil mesocosms. The following experimental treatments were imposed using a full factorial design: two soil types, two soil water regimes, and four Cu application rates (0, 30, 120, or 280 µg g(-1)). Bacterial communities in bulk soil microcosms exhibited similar Cu tolerance patterns when assessed by aerobic and anaerobic PICT assays, whereas aerobic microorganisms tended to be more strongly selected for Cu tolerance than anaerobic microorganisms in rhizosphere soil. Despite similar levels of water-extractable Cu, bacterial Cu tolerance was significantly higher in acid sulfate soil than in alluvial soil. Copper amendment selected for significant PICT development in soils subjected to alternate wetting and drying, but not in continuously flooded soils. Our results demonstrate that soil bacterial communities subjected to alternate wetting and drying may be more affected by Cu than bacterial communities subjected to continuous flooding. We conclude that the parallel use of anaerobic and aerobic [(3) H]leucine PICT assays constitutes a valuable improvement over existing procedures for PICT detection in irrigated soils and other redox gradient environments such as sediments and wetlands.