Flying microbes-survival in the extreme conditions of the stratosphere during a stratospheric balloon flight experiment.

Earth's stratosphere is characterized by hypobaric conditions, low temperatures, and high intensities of ultraviolet (UV) and cosmic radiation as well as low water and nutrient availability. While it is not considered a permanent habitat for microorganisms, they can be transported to the stratosphere by storms, volcanic action, or human activity. The impact of those extreme conditions on microorganisms and their survival were tested by sending a sample gondola to the stratosphere. The sample gondola was built to allow exposure of Bacillus subtilis endospores at different angles to the sun. It moreover had holders for three environmental samples to test the effect of stratospheric conditions on complex microbial communities. The gondola attached to a stratospheric balloon was launched near Kiruna, Sweden, ascended to ~25 km, and drifted eastward for ~200 km. Samples were exposed to pressures as low as 2 kPa and temperatures as low as -50°C as well as high UV radiation. Survival rates of B. subtilis were determined by comparing the numbers of colony-forming units (CFUs) for the different exposure angles. Survival was negatively correlated with exposure angle, indicating the significant impact of UV radiation. The effect of stratospheric conditions on environmental samples was assessed by comparing most probable numbers, microbial community composition, and substrate-use profiles to controls that had stayed on the ground. Cultivation was possible from all samples with survival rates of at least 1%, and differences in community composition were observed. Survival of environmental microorganisms might have been supported by the sample matrix, which provided protection from radiation and desiccation. IMPORTANCE: Earth's stratosphere is a hostile environment that has challenged microbial survival. We set out to test the effect of stratosphere exposure on survival of single species (Bacillus subtilis) and complex microbial communities from soils and sediment. B. subtilis survival was strongly impacted by sun exposure, i.e., ultraviolet (UV) radiation, with only 1% survival at full sun exposure. Complex microbial communities had high survival rates, and the soil or sediment matrix may have provided protection against radiation and desiccation, supporting the survival of environmental microorganisms.

Assessment of microbial communities from cold mine environments and subsequent enrichment, isolation and characterization of putative antimony- or copper-metabolizing microorganisms.

Mining activities, even in arctic regions, create waste materials releasing metals and metalloids, which have an impact on the microorganisms inhabiting their surroundings. Some species can persist in these areas through tolerance to meta(loid)s via, e.g., metabolic transformations. Due to the interaction between microorganisms and meta(loid)s, interest in the investigation of microbial communities and their possible applications (like bioremediation or biomining) has increased. The main goal of the present study was to identify, isolate, and characterize microorganisms, from subarctic mine sites, tolerant to the metalloid antimony (Sb) and the metal copper (Cu). During both summer and winter, samples were collected from Finnish mine sites (site A and B, tailings, and site C, a water-treatment peatland) and environmental parameters were assessed. Microorganisms tolerant to Sb and Cu were successfully enriched under low temperatures (4°C), creating conditions that promoted the growth of aerobic and fermenting metal(loid) tolerating or anaerobic metal(loid) respiring organism. Microbial communities from the environment and Sb/Cu-enriched microorganisms were studied via 16S rRNA amplicon sequencing. Site C had the highest number of taxa and for all sites, an expected loss of biodiversity occurred when enriching the samples, with genera like Prauserella, Pseudomonas or Clostridium increasing their relative abundances and others like Corynebacterium or Kocuria reducing in relative abundance. From enrichments, 65 putative Sb- and Cu-metabolizing microorganisms were isolated, showing growth at 0.1 mM to 10 mM concentrations and 0°C to 40°C temperatures. 16S rRNA gene sequencing of the isolates indicated that most of the putative anaerobically Sb-respiring tolerators were related to the genus Clostridium. This study represents the first isolation, to our knowledge, of putative Sb-metabolizing cold-tolerant microorganisms and contributes to the understanding of metal (loid)-tolerant microbial communities in Arctic mine sites.

The influence of coagulant type on the biological treatment of sewage sludge.

Anaerobic digestion (AD) and composting are commonly utilized sludge management methods however, the influence of different coagulant types on these biological processes and their stabilized biomass characteristics have not been fully explained. In this study, the effect of the coagulant used in municipal wastewater treatment on the biological stabilization of sludge was investigated. Fully controlled and monitored small-scale AD and composting bioreactors were utilized. The coagulants tested included an inorganic coagulant (IC), polyaluminium chloride (PAC), and organic coagulants, (OC) polyamine (pAmine) and chitosan (Chit). Overall, the coagulant applied showed a measurable influence on the biological stabilization of sludge. The presence of complex organics compounds from OC in the sludge biomass was found to decrease biomass biodegradability while increasing gas production. During AD, Chit-sludge achieved higher biogas production than pAmine- and PAC-sludges (13 % and 16 %, respectively, in Nm3 CH4 t-1VS). In composting, pAmine-sludge achieved the highest feedstock temperature (34-35 °C) and CO2 gas emissions, followed by Chit- (33 °C) and PAC-sludges (32 °C). Generally, tot-P concentration in PAC-sludge was higher than in pAmine and Chit-sludges both before (20, 17 and 15 g/kg DM, consecutively) and after AD (23, 21 and 20.5 g/kg DM, consecutively), and during the composting (31, 29.5 and 26 g/kg DM, consecutively) process. Tot-N concentrations (g/kg DM) showed a substantial increase after AD (pAmine and PAC ca 50 % and Chit 81 %), while a decrease was observed after composting, specifically in PAC-sludge (PAC 28 %, pAmine and Chit ⁓5 %). The selection of the most suitable coagulant by wastewater treatment facilities depends on the objective of the biological stabilization process. In cases where AD is applied and biogas yield is selected as the target output, the semi-natural OC Chit was found to be the best option among the coagulants tested. Comparably, when the nutrient content of resulting biosolids (AD or composting) is more relevant, it was found that OC-produced sludge contained higher N concentrations, while IC-produced sludge contained slightly higher P concentrations.

Monitoring groundwater quality with real-time data, stable water isotopes, and microbial community analysis: A comparison with conventional methods.

Groundwater provides much of the world's potable water. Nevertheless, groundwater quality monitoring programmes often rely on a sporadic, slow, and narrowly focused combination of periodic manual sampling and laboratory analyses, such that some water quality deficiencies go undetected, or are detected too late to prevent adverse consequences. In an effort to address this shortcoming, we conducted enhanced monitoring of untreated groundwater quality over 12 months (February 2019-February 2020) in four shallow wells supplying potable water in Finland. We supplemented periodic manual sampling and laboratory analyses with (i) real-time online monitoring of physicochemical and hydrological parameters, (ii) analysis of stable water isotopes from groundwater and nearby surface waters, and (iii) microbial community analysis of groundwater via amplicon sequencing of the 16S rRNA gene and 16S rRNA. We also developed an early warning system (EWS) for detecting water quality anomalies by automating real-time online monitoring data collection, transfer, and analysis - using electrical conductivity (EC) and turbidity as indirect water quality indicators. Real-time online monitoring measurements were largely in fair agreement with periodic manual measurements, demonstrating their usefulness for monitoring water quality; and the findings of conventional monitoring, stable water isotopes, and microbial community analysis revealed indications of surface water intrusion and faecal contamination at some of the studied sites. With further advances in technology and affordability expected into the future, the supplementary methods used here could be more widely implemented to enhance groundwater quality monitoring - by contributing new insights and/or corroborating the findings of conventional analyses.

Surface Water Intrusion, Land Use Impacts, and Bacterial Community Composition in Shallow Groundwater Wells Supplying Potable Water in Sparsely Populated Areas of a Boreal Region.

Rural communities often rely on groundwater for potable water supply. In this study, untreated groundwater samples from 28 shallow groundwater wells in Finland (<10 m deep and mostly supplying untreated groundwater to <200 users in rural areas) were assessed for physicochemical water quality, stable water isotopes, microbial water quality indicators, host-specific microbial source tracking (MST) markers, and bacterial community composition, activity, and diversity (using amplicon sequencing of the 16S rRNA gene and 16S rRNA). Indications of surface water intrusion were identified in five wells, and these indications were found to be negatively correlated, overall, with bacterial alpha diversity (based on amplicon sequencing of the 16S rRNA gene). High levels of turbidity, heterotrophs, and iron compromised water quality in two wells, with values up to 2.98 nephelometric turbidity units (NTU), 16,000 CFU/ml, and 2,300 µg/liter, respectively. Coliform bacteria and general fecal indicator Bacteroidales bacteria (GenBac3) were detected in 14 and 10 wells, respectively (albeit mostly at low levels), and correlations were identified between microbial, physicochemical, and environmental parameters, which may indicate impacts from nearby land use (e.g., agriculture, surface water, road salt used for deicing). Our results show that although water quality was generally adequate in most of the studied wells, the continued safe use of these wells should not be taken for granted. IMPORTANCE Standard physicochemical water quality analyses and microbial indicator analyses leave much of the (largely uncultured) complexity of groundwater microbial communities unexplored. This study combined these standard methods with additional analyses of stable water isotopes, bacterial community data, and environmental data about the surrounding areas to investigate the associations between physicochemical and microbial properties of 28 shallow groundwater wells in Finland. We detected impaired groundwater quality in some wells, identified potential land use impacts, and revealed indications of surface water intrusion which were negatively correlated with bacterial alpha diversity. The potential influence of surface water intrusion on groundwater wells and their bacterial communities is of particular interest and warrants further investigation because surface water intrusion has previously been linked to groundwater contamination, which is the primary cause of waterborne outbreaks in the Nordic region and one of the major causes in the United States and Canada.

Assessing the Diversity and Metabolic Potential of Psychrotolerant Arsenic-Metabolizing Microorganisms From a Subarctic Peatland Used for Treatment of Mining-Affected Waters by Culture-Dependent and -Independent Techniques.

Arsenic contamination in water by natural causes or industrial activities is a major environmental concern, and treatment of contaminated waters is needed to protect water resources and minimize the risk for human health. In mining environments, treatment peatlands are used in the polishing phase of water treatment to remove arsenic (among other contaminants), and peat microorganisms play a crucial role in arsenic removal. The present study assessed culture-independent diversity obtained through metagenomic and metatranscriptomic sequencing and culture-dependent diversity obtained by isolating psychrotolerant arsenic-tolerant, arsenite-oxidizing, and arsenate-respiring microorganisms from a peatland treating mine effluent waters of a gold mine in Finnish Lapland using a dilution-to-extinction technique. Low diversity enrichments obtained after several transfers were dominated by the genera Pseudomonas, Polaromonas, Aeromonas, Brevundimonas, Ancylobacter, and Rhodoferax. Even though maximal growth and physiological activity (i.e., arsenite oxidation or arsenate reduction) were observed at temperatures between 20 and 28°C, most enrichments also showed substantial growth/activity at 2-5°C, indicating the successful enrichments of psychrotolerant microorganisms. After additional purification, eight arsenic-tolerant, five arsenite-oxidizing, and three arsenate-respiring strains were obtained in pure culture and identified as Pseudomonas, Rhodococcus, Microbacterium, and Cadophora. Some of the enriched and isolated genera are not known to metabolize arsenic, and valuable insights on arsenic turnover pathways may be gained by their further characterization. Comparison with phylogenetic and functional data from the metagenome indicated that the enriched and isolated strains did not belong to the most abundant genera, indicating that culture-dependent and -independent methods capture different fractions of the microbial community involved in arsenic turnover. Rare biosphere microorganisms that are present in low abundance often play an important role in ecosystem functioning, and the enriched/isolated strains might thus contribute substantially to arsenic turnover in the treatment peatland. Psychrotolerant pure cultures of arsenic-metabolizing microorganisms from peatlands are needed to close the knowledge gaps pertaining to microbial arsenic turnover in peatlands located in cold climate regions, and the isolates and enrichments obtained in this study are a good starting point to establish model systems. Improved understanding of their metabolism could moreover lead to their use in biotechnological applications intended for bioremediation of arsenic-contaminated waters.

Potential of high pH and reduced sulfur for arsenic mobilization - Insights from a Finnish peatland treating mining waste water.

Peatlands, used for purification of mining waste waters, have shown efficient solid-phase sequestration of contaminants such as arsenic (As). However, contaminant re-mobilization may occur related to management changes or chemical alteration of original peatland conditions. For a treatment peatland in Finnish Lapland, we here confirm efficient As retention in near-surface peat layers close to the mining waste water inflow, likely due to binding to FeIII-phases. Seven years into operation of the treatment peatland, there appears to be further retention potential, as large areas downstream still had solid-phase As concentrations at background levels. However, via depth-resolved pore water analysis we observed a hotspot 170 m from the inflow at 10-50 cm depth, where As pore water concentrations exceeded input concentrations by a factor of 20, indicating substantial As re-mobilization. At the same spot, a peak of reduced sulfur (S) species was found. Arsenic species detected were arsenite and up to 26% methylated oxyarsenates, 15% methylated and 7.9% inorganic thioarsenates. We postulate that As mobilization is a result of short-term re-equilibration to a changed inflow chemistry after installation of a process water treatment plant and a long-term consequence of changing pore water pH from acidic to near-neutral, releasing reduced S and As. We infer that the co-occurrence of reduced S and As leads to formation of methylated and/or thiolated As species with known low sorption affinity, thereby further enhancing As mobility. Laboratory incubation studies with two peat cores confirmed a high S-induced As mobilization potential, especially when As-Fe-rich, oxic surface layers were incubated anoxically at near-neutral pH. Highest risk of As re-mobilization from this treatment peatland is expected in a scenario in which mining waste water inflow has stopped but the peatland remains flooded, and near-surface layers transition from oxic to anoxic conditions.

Abundant and diverse arsenic-metabolizing microorganisms in peatlands treating arsenic-contaminated mining wastewaters.

Mining operations produce large quantities of wastewater. At a mine site in Northern Finland, two natural peatlands are used for the treatment of mining-influenced waters with high concentrations of sulphate and potentially toxic arsenic (As). In the present study, As removal and the involved microbial processes in those treatment peatlands (TPs) were assessed. Arsenic-metabolizing microorganisms were abundant in peat soil from both TPs (up to 108 cells gdw -1 ), with arsenate respirers being about 100 times more abundant than arsenite oxidizers. In uninhibited microcosm incubations, supplemented arsenite was oxidized under oxic conditions and supplemented arsenate was reduced under anoxic conditions, while little to no oxidation/reduction was observed in NaN3 -inhibited microcosms, indicating high As-turnover potential of peat microbes. Formation of thioarsenates was observed in anoxic microcosms. Sequencing of the functional genemarkers aioA (arsenite oxidizers), arrA (arsenate respirers) and arsC (detoxifying arsenate reducers) demonstrated high diversity of the As-metabolizing microbial community. The microbial community composition differed between the two TPs, which may have affected As removal efficiencies. In the present situation, arsenate reduction is likely the dominant net process and contributes substantially to As removal. Changes in TP usage (e.g. mine closure) with lowered water tables and heightened oxygen availability in peat might lead to re-oxidation and re-mobilization of bound arsenite.

Design, construction and monitoring of pilot systems to evaluate the effect of freeze-thaw cycles on pollutant retention in wetlands.

Due to the complexity of soil freeze/thaw processes and a variety of factors affecting pollutant removal in treatment wetlands, laboratory pilot systems are powerful tools offering a rare opportunity to observe processes that have a significant impact on year-round purification. This paper describes the design, construction, monitoring and operation of two replicate pilot peat-based wetlands subjected to two simulated freeze-thaw cycles. Undisturbed peat soil and pre-treated gold mine process wastewater were collected from a full-scale treatment wetland operating at a mining site in Northern Finland. The wastewater (pH ~7.8, electric conductivity ~3.6 mS/cm) contained a mix of metals/metalloids (e.g. arsenic 12 µg/L, antimony 19 µg/L) and other contaminants e.g. sulphate (~2 g/L). Fluctuations in removal efficiency of target compounds due to freezing and thawing conditions were observed. Overall, removal of sulphate and arsenic decreased during frost periods, while removal of antimony increased. Monitoring data from the full-scale treatment wetland were used to assess the representativeness of the results obtained. Comparisons of seasonal variations in pollutant concentrations in outflow samples from the full-scale wetland and those measured in the pilot wetlands revealed similar fluctuations in removal efficiency during frost and frost-free periods, suggesting that the pilot wetlands simulated the real system rather well. Carefully designed pilot systems can thus be valuable tools for assessing the effect of harsh winter conditions on wetland processes and operation.

Antimonite Binding to Natural Organic Matter: Spectroscopic Evidence from a Mine Water Impacted Peatland.

Peatlands and other wetlands are sinks for antimony (Sb), and solid natural organic matter (NOM) may play an important role in controlling Sb binding. However, direct evidence of Sb sequestration in natural peat samples is lacking. Here, we analyzed solid phase Sb, iron (Fe), and sulfur (S) as well as aqueous Sb speciation in three profiles up to a depth of 80 cm in a mine water impacted peatland in northern Finland. Linear combination fittings of extended X-ray absorption fine structure spectra showed that Sb binding to Fe phases was of minor importance and observed only in the uppermost layers of the peatland. Instead, the dominant (to almost exclusive) sequestration mechanism was Sb(III) binding to oxygen-containing functional groups, and at greater depths, increasingly Sb(III) binding to thiol groups of NOM. Aqueous Sb speciation was dominated by antimonate, while antimonite concentrations were low, further supporting our findings of much higher reactivity of Sb(III) than Sb(V) toward peat surfaces. Insufficient residence time for efficient reduction of antimonate to antimonite currently hinders higher Sb removal in the studied peatland. Overall, our findings imply that Sb(III) binding to solid NOM acts as an important sequestration mechanism under reducing conditions in peatlands and other high-organic matter environments.

Design parameters for nitrogen removal by constructed wetlands treating mine waters and municipal wastewater under Nordic conditions.

Nitrogen (N) loads from municipal and mine wastewater discharges typically increase N concentrations in recipient water bodies which should get more attention especially in cold-climate regions. This study compared N removal efficiency of six constructed wetlands (CWs) treating mine waters and three CWs polishing municipal wastewater. There were clear impacts of point source N loading to recipient water bodies in all cases studied and >300-fold increase in N was seen in some cases. First-order N removal coefficient was determined for seven of these CWs. All CWs studied were observed to remove N efficiently during the warm growing season but the amount of N released increased significantly during the cold season. Although some year-round purification was achieved by both peat-based and pond-type CWs, removal of nitrate + nitrite-N ((NO3- + NO2-)-N) was low during winter. The first-order N removal coefficient varied from 4.9 ·â€¯10-6 to 1.9 ·â€¯10-3 d-1 and showed that peat-based CWs were slightly more efficient in N removal than pond-type CWs. However, purification efficiency was steadier and higher for pond-type CWs, as lower hydraulic load or longer water residence time compensated for purification performance. Pond-type CWs showed mean removal efficiency of 59% and 46% for ammonium-N (NH4+-N) and (NO3- + NO2-)-N, respectively, whereas peatland-type CWs had lower removal efficiency for NH4+-N (mean of 26%) and in many cases negative removal for (NO3- + NO2-)-N. Correlation analysis revealed no clear, systematic relationship between temperature and N removal. However, in some CWs the highest correlation was between temperature and (NO3- + NO2-)-N, reflecting lower denitrification rate at lower temperature. More than 50% removal was found to require a hydraulic load below 10 mm d-1. In order to achieve 70% of NH4+-N removal, Ntot load lower than 75 g m-2 year-1 and a residence time longer than 80 d are needed in CWs in cold-climate regions.

Arsenic, antimony, and nickel leaching from northern peatlands treating mining influenced water in cold climate.

Increased metal mining in the Arctic region has caused elevated loads of arsenic (As), antimony (Sb), nickel (Ni), and sulfate (SO42-) to recipient surface or groundwater systems. The need for cost-effective active and passive mine water treatment methods has also increased. Natural peatlands are commonly used as a final step for treatment of mining influenced water. However, their permanent retention of harmful substances is affected by influent concentrations and environmental conditions. The effects of dilution, pH, temperature, oxygen availability, and contaminant accumulation on retention and leaching of As, Sb, Ni, and sulfate from mine process water and drainage water obtained from treatment peatlands in Finnish Lapland were studied in batch sorption experiments, and discussed in context of field data and environmental impacts. The results, while demonstrating effectiveness of peat to remove the target contaminants from mine water, revealed the risk of leaching of As, Sb, and SO42- from treatment peatlands when diluted mine water was introduced. Sb was more readily leached compared to As while leaching of both was supported by higher pH of 9. No straightforward effect of temperature and oxygen availability in controlling removal and leaching was evident from the results. The results also showed that contaminant accumulation in treatment peatlands after long-term use can lead to decreased removal and escalated leaching of contaminants, with the effect being more pronounced for As and Ni.

Microbial diversity along a gradient in peatlands treating mining-affected waters.

Peatlands are used for the purification of mining-affected waters in Northern Finland. In Northern climate, microorganisms in treatment peatlands (TPs) are affected by long and cold winters, but studies about those microorganisms are scarce. Thus, the bacterial, archaeal and fungal communities along gradients of mine water influence in two TPs were investigated. The TPs receive waters rich in contaminants, including arsenic (As), sulfate (SO42-) and nitrate (NO3-). Microbial diversity was high in both TPs, and microbial community composition differed between the studied TPs. Bacterial communities were dominated by Proteobacteria, Actinobacteria, Chloroflexi and Acidobacteria, archaeal communities were dominated by Methanomicrobia and the Candidate phylum Bathyarchaeota, and fungal communities were dominated by Ascomycota (Leotiomycetes, Dothideomycetes, Sordariomycetes). The functional potential of the bacterial and archaeal communities in TPs was predicted using PICRUSt. Sampling points affected by high concentrations of As showed higher relative abundance of predicted functions related to As resistance. Functions potentially involved in nitrogen and SO42- turnover in TPs were predicted for both TPs. The results obtained in this study indicate that (i) diverse microbial communities exist in Northern TPs, (ii) the functional potential of the peatland microorganisms is beneficial for contaminant removal in TPs and (iii) microorganisms in TPs are likely well-adapted to high contaminant concentrations as well as to the Northern climate.

Soil Conditions Rather Than Long-Term Exposure to Elevated CO2 Affect Soil Microbial Communities Associated with N-Cycling.

Continuously rising atmospheric CO2 concentrations may lead to an increased transfer of organic C from plants to the soil through rhizodeposition and may affect the interaction between the C- and N-cycle. For instance, fumigation of soils with elevated CO2 (eCO2) concentrations (20% higher compared to current atmospheric concentrations) at the Giessen Free-Air Carbon Dioxide Enrichment (GiFACE) sites resulted in a more than 2-fold increase of long-term N2O emissions and an increase in dissimilatory reduction of nitrate compared to ambient CO2 (aCO2). We hypothesized that the observed differences in soil functioning were based on differences in the abundance and composition of microbial communities in general and especially of those which are responsible for N-transformations in soil. We also expected eCO2 effects on soil parameters, such as on nitrate as previously reported. To explore the impact of long-term eCO2 on soil microbial communities, we applied a molecular approach (qPCR, T-RFLP, and 454 pyrosequencing). Microbial groups were analyzed in soil of three sets of two FACE plots (three replicate samples from each plot), which were fumigated with eCO2 and aCO2, respectively. N-fixers, denitrifiers, archaeal and bacterial ammonia oxidizers, and dissimilatory nitrate reducers producing ammonia were targeted by analysis of functional marker genes, and the overall archaeal community by 16S rRNA genes. Remarkably, soil parameters as well as the abundance and composition of microbial communities in the top soil under eCO2 differed only slightly from soil under aCO2. Wherever differences in microbial community abundance and composition were detected, they were not linked to CO2 level but rather determined by differences in soil parameters (e.g., soil moisture content) due to the localization of the GiFACE sets in the experimental field. We concluded that +20% eCO2 had little to no effect on the overall microbial community involved in N-cycling in the soil but that spatial heterogeneity over extended periods had shaped microbial communities at particular sites in the field. Hence, microbial community composition and abundance alone cannot explain the functional differences leading to higher N2O emissions under eCO2 and future studies should aim at exploring the active members of the soil microbial community.