Seasonal variation in metabolic profiles and microbial communities in a subarctic ore processing plant.

The mining industry strives to reduce its water footprint by recycling water in ore processing. This leads to build-up of ions, flotation chemicals and microbial biomass, which may affect the process. The Boliden Kevitsa mine in Northern Finland is exposed to seasonal change and recycles up to 90% of the process water. We studied the variation in size, composition and putative functions of microbial communities in summer and winter in the ore processing plant. The raw water, Cu and Ni thickener overflow waters had statistically significantly higher bacterial numbers in winter compared to summer, and specific summer and winter communities were identified. Metagenomic analysis indicated that Cu and Hg resistance genes, sulphate/thiosulphate, molybdate, iron(III) and zinc ABC transporters, nitrate reduction, denitrification, thiosulphate oxidation and methylotrophy were more common in winter than in summer. Raw water drawn from the nearby river did not affect the microbial communities in the process samples, indicating that the microbial communities and metabolic capacities develop within the process over time in response to the conditions in the processing plant, water chemistry, used chemicals, ore properties and seasonal variation. We propose that the microbial community structures are unique to the Boliden Kevitsa mine and processing plant.

Altering environmental conditions induce shifts in simulated deep terrestrial subsurface bacterial communities-Secretion of primary and secondary metabolites.

The deep terrestrial subsurface (DTS) harbours a striking diversity of microorganisms. However, systematic research on microbial metabolism, and how varying groundwater composition affects the bacterial communities and metabolites in these environments is lacking. In this study, DTS groundwater bacterial consortia from two Fennoscandian Shield sites were enriched and studied. We found that the enriched communities from the two sites consisted of distinct bacterial taxa, and alterations in the growth medium composition induced changes in cell counts. The lack of an exogenous organic carbon source (ECS) caused a notable increase in lipid metabolism in one community, while in the other, carbon starvation resulted in low overall metabolism, suggesting a dormant state. ECS supplementation increased CO2 production and SO4 2- utilisation, suggesting activation of a dissimilatory sulphate reduction pathway and sulphate-reducer-dominated total metabolism. However, both communities shared common universal metabolic features, most probably involving pathways needed for the maintenance of cell homeostasis (e.g., mevalonic acid pathway). Collectively, our findings indicate that the most important metabolites related to microbial reactions under varying growth conditions in enriched DTS communities include, but are not limited to, those linked to cell homeostasis, osmoregulation, lipid biosynthesis and degradation, dissimilatory sulphate reduction and isoprenoid production.

Anionic nanocellulose as competing agent in microbial DNA extraction from mine process samples.

Microorganisms in flotation and minerals processing may significantly affect the grade and yield of metal concentrates. However, studying the phenomena requires working techniques to detach microorganisms and their DNA from mineral particles to which they strongly adhere. We developed a new method utilizing the competitive properties of anionic nanocellulose to block sorption of DNA to and detach microbial cells from mineral particles from ore processing. In general, up to one ng DNA mL-1 sample was obtained with the custom anionic nanocellulose method (CM) compared to DNA amounts below the Qubit assay's detection limit for extractions with a commercial kit (KIT). Similarly, 0.5-4 orders of magnitude more bacterial 16S and fungal 5.8S rRNA gene copies were detected by qPCR from CM treated samples compared to KIT extractions. A clear difference in the detected microbial community structure between CM and KIT extracted samples was also observed. Commercial kits optimized for mineral soils are easy to use and time efficient but may miss a considerable part of the microbial communities. A competing agent such as anionic nanocellulose may decrease the interaction between microorganisms or their DNA and minerals and provide a comprehensive view into the microbial communities in mineral processing environments.

Implications of a short carbon pulse on biofilm formation on mica schist in microcosms with deep crystalline bedrock groundwater.

Microbial life in the deep subsurface occupies rock surfaces as attached communities and biofilms. Previously, epilithic Fennoscandian deep subsurface bacterial communities were shown to host genetic potential, especially for heterotrophy and sulfur cycling. Acetate, methane, and methanol link multiple biogeochemical pathways and thus represent an important carbon and energy source for microorganisms in the deep subsurface. In this study, we examined further how a short pulse of low-molecular-weight carbon compounds impacts the formation and structure of sessile microbial communities on mica schist surfaces over an incubation period of ∼3.5 years in microcosms containing deep subsurface groundwater from the depth of 500 m, from Outokumpu, Finland. The marker gene copy counts in the water and rock phases were estimated with qPCR, which showed that bacteria dominated the mica schist communities with a relatively high proportion of epilithic sulfate-reducing bacteria in all microcosms. The dominant bacterial phyla in the microcosms were Proteobacteria, Firmicutes, and Actinobacteria, whereas most fungal genera belonged to Ascomycota and Basidiomycota. Dissimilarities between planktic and sessile rock surface microbial communities were observed, and the supplied carbon substrates led to variations in the bacterial community composition.

Sulfate-reducing bioreactors subjected to high sulfate loading rate or acidity: variations in microbial consortia.

Sulfate-reducing bioreactors are used in e.g. the mining industry to remove sulfate and harmful metals from process waters. These bioreactors are expected to be run for extended periods of time and may experience variations in the influent quality, such as increasing sulfate loading rate and decrease in pH, while being expected to function optimally. In this study we followed the sulfate removal rate and variation in microbial communities over a period of up to 333 days in three different up-flow anaerobic sludge blanket (UASB) bioreactors being submitted to increasing sulfate loading rate or decreasing pH. Sodium lactate was used as the sole carbon source and electron donor. All three bioreactors contained highly diverse microbial communities containing archaea, fungi and bacteria. Sulfurospirillum and Desulfovibrio were the most prominent bacterial genera detected in the bioreactors receiving the highest sulfate loading rates, and the greatest relative abundance of methanogenic archaea and the fungal genus Cadophora coincided with the highest sulfate reduction rates. In contrast, Sulfuricurvum was dominant in the bioreactor receiving influent with alternating pH, but its relative abundance receded in response to low pH of the influent. All bioreactors showed excellent sulfate removal even under extreme conditions in addition to unique responses in the microbial communities under changing operational conditions. This shows that a high diversity in the microbial consortia in the bioreactors could make the sulfate removal process less sensitive to changing operational conditions, such as variations in influent sulfate loading rate and pH.

Epilithic Microbial Community Functionality in Deep Oligotrophic Continental Bedrock.

The deep terrestrial biosphere hosts vast sessile rock surface communities and biofilms, but thus far, mostly planktic communities have been studied. We enriched deep subsurface microbial communities on mica schist in microcosms containing bedrock groundwater from the depth of 500 m from Outokumpu, Finland. The biofilms were visualized using scanning electron microscopy, revealing numerous different microbial cell morphologies and attachment strategies on the mica schist surface, e.g., bacteria with outer membrane vesicle-like structures, hair-like extracellular extensions, and long tubular cell structures expanding over hundreds of micrometers over mica schist surfaces. Bacterial communities were analyzed with amplicon sequencing showing that Pseudomonas, Desulfosporosinus, Hydrogenophaga, and Brevundimonas genera dominated communities after 8-40 months of incubation. A total of 21 metagenome assembled genomes from sessile rock surface metagenomes identified genes involved in biofilm formation, as well as a wide variety of metabolic traits indicating a high degree of environmental adaptivity to oligotrophic environment and potential for shifting between multiple energy or carbon sources. In addition, we detected ubiquitous organic carbon oxidation and capacity for arsenate and selenate reduction within our rocky MAGs. Our results agree with the previously suggested interaction between the deep subsurface microbial communities and the rock surfaces, and that this interaction could be crucial for sustaining life in the harsh anoxic and oligotrophic deep subsurface of crystalline bedrock environment.

First insights to the microbial communities in the plant process water of the multi-metal Kevitsa mine.

Metallurgical processes demand large quantities of water. However, in many locations, water is becoming scarce and process water recycling is needed. Closing water loops can be challenging due to build-up of flotation chemicals, metal ions and microorganisms in the recycled water affecting the flotation performance. Here, we have characterized the microbial communities over a 2-month period in different locations of the multi-metal Kevitsa mine in Northern Finland, by microbiome sequencing, enumeration of bacteria, archaea and fungi by qPCR, and cultivation. The microbial communities showed high diversity, but were dominated by Alpha- and Gammaproteobacteria. In addition, various fungal taxa were detected, whereas the archaeal taxa were only sparsely detected from the sequence data. The number of bacterial 16S rRNA gene copies in Process water and Ni thickener overflow varied between 0.5-3.3 × 105 mL-1, whereas the Flotation tailings showed two orders of magnitude lower amounts. Fungi were present at 3.0 × 102-8.1 × 104 5.8S rRNA gene copies mL-1 in all samples, while the number of archaea fluctuated between 8.8 × 101-3.2 × 105 16S rRNA gene copies mL-1. The number of all microbial groups were generally lower in September than in August. When tested on 8 different cultivation media, the microorganisms generally responded positively to organic carbon, and were also shown to oxidize thiosulfate, which may indicate that build-up of organic flotation chemicals and sulfur species from the ore may cause the microbial numbers to increase. This study is part of the H2020 ITERAMS project (Grant agreement# 730480), which strives to improve the recycling of water and minimize the environmental impact of mines.

Ultradeep Microbial Communities at 4.4 km within Crystalline Bedrock: Implications for Habitability in a Planetary Context.

The deep bedrock surroundings are an analog for extraterrestrial habitats for life. In this study, we investigated microbial life within anoxic ultradeep boreholes in Precambrian bedrock, including the adaptation to environmental conditions and lifestyle of these organisms. Samples were collected from Pyhäsalmi mine environment in central Finland and from geothermal drilling wells in Otaniemi, Espoo, in southern Finland. Microbial communities inhabiting the up to 4.4 km deep bedrock were characterized with phylogenetic marker gene (16S rRNA genes and fungal ITS region) amplicon and DNA and cDNA metagenomic sequencing. Functional marker genes (dsrB, mcrA, narG) were quantified with qPCR. Results showed that although crystalline bedrock provides very limited substrates for life, the microbial communities are diverse. Gammaproteobacterial phylotypes were most dominant in both studied sites. Alkanindiges -affiliating OTU was dominating in Pyhäsalmi fluids, while different depths of Otaniemi samples were dominated by Pseudomonas. One of the most common OTUs detected from Otaniemi could only be classified to phylum level, highlighting the uncharacterized nature of the deep biosphere in bedrock. Chemoheterotrophy, fermentation and nitrogen cycling are potentially significant metabolisms in these ultradeep environments. To conclude, this study provides information on microbial ecology of low biomass, carbon-depleted and energy-deprived deep subsurface environment. This information is useful in the prospect of finding life in other planetary bodies.

Data on the optimization of an archaea-specific probe-based qPCR assay.

Estimation of archaeal numbers by use of fluorescent DNA binding dyes is challenging, because primers targeting the archaeal 16SrRNA genes readily also bind to bacterial 16S rRNA gene sequences, especially when the relative abundance of bacteria is greater than that of archaea. In order to increase specificity, we optimized a fluorescent probe-based assay using previously published archaeal primers and probe. The assay was tested on genomic DNA of pure bacterial and archaeal cultures and optimized using PCR amplicons of the archaeal pure cultures. The used bacterial strains showed slight amplification using the fluorescent dye assay, whereas all archaeal strains could be amplified with the archaea primers used. Due to differences in genome size and number of 16S rRNA gene copies between the tested archaeal strains, the amplification level varied greatly between the strains. Therefore, we also tested the amplification using PCR amplified fragments of the archaeal 16S rRNA genes. The tests with the archaeal 16S rRNA gene amplicons showed good amplification, although the amplification efficiency still varied between archaeal strains. The qPCR assay was used to estimate the archaeal numbers in process water of a multi-metal mine's metallurgical plant [1] and will be used in similar future microbiological analysis included in the H2020 ITERAMS project (Grant agreement# 730480).

Rock Surface Fungi in Deep Continental Biosphere-Exploration of Microbial Community Formation with Subsurface In Situ Biofilm Trap.

Fungi have an important role in nutrient cycling in most ecosystems on Earth, yet their ecology and functionality in deep continental subsurface remain unknown. Here, we report the first observations of active fungal colonization of mica schist in the deep continental biosphere and the ability of deep subsurface fungi to attach to rock surfaces under in situ conditions in groundwater at 500 and 967 m depth in Precambrian bedrock. We present an in situ subsurface biofilm trap, designed to reveal sessile microbial communities on rock surface in deep continental groundwater, using Outokumpu Deep Drill Hole, in eastern Finland, as a test site. The observed fungal phyla in Outokumpu subsurface were Basidiomycota, Ascomycota, and Mortierellomycota. In addition, significant proportion of the community represented unclassified Fungi. Sessile fungal communities on mica schist surfaces differed from the planktic fungal communities. The main bacterial phyla were Firmicutes, Proteobacteria, and Actinobacteriota. Biofilm formation on rock surfaces is a slow process and our results indicate that fungal and bacterial communities dominate the early surface attachment process, when pristine mineral surfaces are exposed to deep subsurface ecosystems. Various fungi showed statistically significant cross-kingdom correlation with both thiosulfate and sulfate reducing bacteria, e.g., SRB2 with fungi Debaryomyces hansenii.

Ni(II) Interactions in Boreal Paenibacillus sp., Methylobacterium sp., Paraburkholderia sp., and Pseudomonas sp. Strains Isolated From an Acidic, Ombrotrophic Bog.

The uptake of nickel [Ni(II)] by Paenibacillus sp., Methylobacterium sp., Paraburkholderia sp., and Pseudomonas sp. strains isolated from a boreal bog was studied using batch experiments. All strains removed Ni(II) from the solution and the uptake efficiency was affected by the nutrient source, incubation temperature, time, and pH. As highest Ni uptake (with a maximum Kd of 1890 L/kg DW) was recorded for the Pseudomonas sp. strains, these bacteria were used in the following protein expression (SDS-PAGE and MALDI-TOFF), transmission electron microscopy (TEM) and EDS experiments. In addition, Freundlich and Langmuir sorption isotherms were determined. In the Ni(II) treated cells, dense crystalline intra-cellular accumulations were observed in TEM examinations, which were identified as Ni accumulations using EDS. SDS-PAGE and MALDI-TOFF spectra of Ni(II) treated cells showed several changes in the protein profiles, which can indicate active accumulation of Ni in these bacteria. Concurrently, we observed Ni(II) uptake to follow Freundlich and Langmuir isotherms, suggesting straight cellular biosorption in addition to the intra-cellular accumulation. The role of cellular (cell membrane and cell wall) functional groups involved in Ni(II) binding were therefore studied using Fourier transformation infrared spectroscopy. These analyses supported the potential role of the alcoholic hydroxyl, carboxyl and amine groups in Ni(II) binding in these bacteria, therefore suggesting two different Ni(II) uptake mechanisms; (i) intra-cellular accumulation [possibly connected to detoxification of Ni(II)], and (ii) straight biosorption on cell membrane/wall functional groups.

The reduction of selenium(IV) by boreal Pseudomonas sp. strain T5-6-I - Effects on selenium(IV) uptake in Brassica oleracea.

Selenium (Se) is an essential micronutrient but toxic when taken in excessive amounts. Therefore, understanding the metabolic processes related to selenium uptake and bacteria-plant interactions coupled with selenium metabolism are of high importance. We cultivated Brassica oleracea with the previously isolated heterotrophic aerobic Se(IV)-reducing Pseudomonas sp. T5-6-I strain to better understand the phenomena of bacteria-mediated Se(IV) reduction on selenium availability to the plants. B. oleracea grown on Murashige and Skoog medium (MS-salt agar) with and without of Pseudomonas sp. were amended with Se(IV)/75Se(IV), and selenium transfer into plants was studied using autoradiography and gamma spectroscopy. XANES was in addition used to study the speciation of selenium in the B. oleracea plants. In addition, the effects of Se(IV) on the protein expression in B. oleracea was studied using HPLC-SEC. TEM and confocal microscopy were used to follow the bacterial/Se-aggregate accumulation in plants and the effects of bacterial inoculation on root-hair growth. In the tests using 75Se(IV) on average 130% more selenium was translocated to the B. oleracea plants grown with Pseudomonas sp. compared to the plants grown with selenium, but without Pseudomonas sp.. In addition, these bacteria notably increased root hair density. Changes in the protein expression of B. oleracea were observed on the ∼30-58 kDa regions in the Se(IV) treated samples, probably connected e.g. to the oxidative stress induced by Se(IV) or expression of proteins connected to the Se(IV) metabolism. Based on the XANES measurements, selenium appears to accumulate in B. oleracea mainly in organic C-Se-H and C-Se-C bonds with and without bacteria inoculation. We conclude that the Pseudomonas sp. T5-6-I strain seems to contribute positively to the selenium accumulation in plants, establishing the high potential of Se0-producing bacteria in the use of phytoremediation and biofortification of selenium.

Highly Diverse Aquatic Microbial Communities Separated by Permafrost in Greenland Show Distinct Features According to Environmental Niches.

The Greenland Analog Project (GAP) study area in the vicinity of Kangarlussuaq, Western Greenland, was sampled for surface water and deep groundwater in order to determine the composition and estimate the metabolic features of the microbial communities in water bodies separated by permafrost. The sampling sites comprised a freshwater pond, talik lake, deep anoxic groundwater, glacier ice and supraglacial river, meltwater river and melting permafrost active layer. The microbial communities were characterized by amplicon sequencing of the bacterial and archaeal 16S rRNA genes and fungal ITS1 spacer. In addition, bacterial, archaeal and fungal numbers were determined by qPCR and plate counts, and the utilization pattern of carbon and nitrogen substrates was determined with Biolog AN plates and metabolic functions were predicted with FAPROTAX. Different sample types were clearly distinguishable from each other based on community composition, microbial numbers, and substrate utilization patterns, forming four groups, (1) pond/lake, (2) deep groundwater, (3) glacial ice, and (4) meltwater. Bacteria were the most abundant microbial domain, ranging from 0.2-1.4 × 107 16S rRNA gene copies mL-1 in pond/lake and meltwater, 0.1-7.8 × 106 copies mL-1 in groundwater and less than 104 copies mL-1 in ice. The number of archaeal 16S and fungal 5.8S rRNA genes was generally less than 6.0 × 103 and 1.5 × 103, respectively. N2-fixing and methane-oxidizing Actinomycetes, Bacteroidetes and Verrucomicrobia were the dominant microorganisms in the pond/lake samples, whereas iron reducing Desulfosporosinus sp. dominated the deep anaerobic groundwater. The glacial ice was inhabited by Cyanobacteria, which were mostly Chloroplast-like. The meltwater contained methano- and methylotrophic Proteobacteria, but had also high relative abundances of the nano-sized Parcubacteria. The archaea composed approximately 1% of the 16S rRNA gene pool in the pond/lake samples with nano-sized Woesearchaeota as the dominating taxon, while in the other sample types archaea were almost negligent. Fungi were also most common in the pond/lake communities, were zoospore-forming Chytridiomycetes dominated. Our results show highly diverse microbial communities inhabiting the different cold Greenlandic aqueous environments and show clear segregation of the microbial communities according to habitat, with distinctive dominating metabolic features specifically inhabiting defined environmental niches and a high relative abundance of putatively parasitic or symbiotic nano-sized taxa.

Oil degradation potential of microbial communities in water and sediment of Baltic Sea coastal area.

Two long-term potentially oil exposed Baltic Sea coastal sites near old oil refineries and harbours were compared to nearby less exposed sites in terms of bacterial, archaeal and fungal microbiomes and oil degradation potential. The bacterial, archaeal and fungal diversities were similar in oil exposed and less exposed sampling sites based on bacterial and archaeal 16S rRNA gene and fungal 5.8S rRNA gene amplicon sequencing from both DNA and RNA fractions. The number of genes participating in alkane degradation (alkB) or PAH-ring hydroxylation (PAH-RHDα) were detected by qPCR in all water and sediment samples. These numbers correlated with the number of bacterial 16S rRNA gene copies in sediment samples but not with the concentration of petroleum hydrocarbons or PAHs. This indicates that both the clean and the more polluted sites at the Baltic Sea coastal areas have a potential for petroleum hydrocarbon degradation. The active community (based on RNA) of the coastal Baltic Sea water differed largely from the total community (based on DNA). The most noticeable difference was seen in the bacterial community in the water samples were the active community was dominated by Cyanobacteria and Proteobacteria whereas in total bacterial community Actinobacteria was the most abundant phylum. The abundance, richness and diversity of Fungi present in water and sediment samples was in general lower than that of Bacteria and Archaea. Furthermore, the sampling location influenced the fungal community composition, whereas the bacterial and archaeal communities were not influenced. This may indicate that the fungal species that are adapted to the Baltic Sea environments are few and that Fungi are potentially more vulnerable to or affected by the Baltic Sea conditions than Bacteria and Archaea.

Microbial communities in a former pilot-scale uranium mine in Eastern Finland - Association with radium immobilization.

The bacterial, fungal and archaeal communities were characterized in 17 top soil organic and mineral layer samples and in top sediment samples of the Paukkajanvaara area, a former pilot-scale uranium mine, located in Eno, Eastern Finland, using amplicon sequencing and qPCR. Soil and sediment samples were in addition analyzed for radium (226Ra), sulfate (SO42-), nitrate (NO3-) and phosphate (PO43-) concentrations. New bacterial strains, representing Pseudomonas spp., were isolated from the mine and reference area and used in laboratory experiments on uptake and leaching of radium (Ra). The effect of these strains on the sulfate leaching from the soil samples was also tested in vitro. Between 6 × 106 and 5 × 108 copies g-1 DW (dry weight) of bacterial 16S rRNA genes, 5 × 105-1 × 108 copies g-1 DW archaeal 16S rRNA genes and 1 × 105-1 × 108 copies g-1 DW fungal 5.8S rRNA genes were detected in the samples. A total of 814, 54 and 167 bacterial, archaeal and fungal genera, respectively, were identified. Proteobacteria, Euryarchaeota and Mortiriella were the dominant bacterial, archaeal and fungal phyla, respectively. All tested Pseudomonas spp. strains isolates from Paukkajanvaara removed Ra from the solution, but the amount of removed Ra depended on incubation conditions (temperature, time and nutrient broth). The highest removal of Ra (5320 L/kg DW) was observed by the Pseudomonas sp. strain T5-6-I at 37 °C. All Pseudomonas spp. strains decreased the release of Ra from soil with an average of 23% while simultaneously increasing the concentration of SO42- in the solution by 11%. As Pseudomonas spp. were frequent in both the sequence data and the cultures, these bacteria may play an important role in the immobilization of Ra in the Paukkajanvaara mine area.

Diversity and functionality of archaeal, bacterial and fungal communities in deep Archaean bedrock groundwater.

The diversity and metabolic functions of deep subsurface ecosystems remain relatively unexplored. Microbial communities in previously studied deep subsurface sites of the Fennoscandian Shield are distinctive to each site. Thus, we hypothesized that the microbial communities of the deep Archaean bedrock fracture aquifer in Romuvaara, northern Finland, differ both in community composition and metabolic functionality from the other sites in the Fennoscandian Shield. We characterized the composition, functionality and substrate preferences of the microbial communities at different depths in a 600 m deep borehole. In contrast to other Fennoscandian deep biosphere communities studied to date, iron-oxidizing Gallionella dominated the bacterial communities, while methanogenic and ammonia-oxidizing archaea were the most prominent archaea, and a diverse fungal community was also detected. Potential for methane cycling and sulfate and nitrate reduction was confirmed by detection of the functional genes of these metabolic pathways. Organotrophs were less abundant, although carbohydrates were the most preferred of the tested substrates. The microbial communities shared features with those detected from other deep groundwaters with similar geochemistry, but the majority of taxa distinctive to Romuvaara are different from the taxa previously detected in saline deep groundwater in the Fennoscandian Shield, most likely because of the differences in water chemistry.

Microbial Community Structure and Functions in Ethanol-Fed Sulfate Removal Bioreactors for Treatment of Mine Water.

Sulfate-rich mine water must be treated before it is released into natural water bodies. We tested ethanol as substrate in bioreactors designed for biological sulfate removal from mine water containing up to 9 g L-1 sulfate, using granular sludge from an industrial waste water treatment plant as inoculum. The pH, redox potential, and sulfate and sulfide concentrations were measured twice a week over a maximum of 171 days. The microbial communities in the bioreactors were characterized by qPCR and high throughput amplicon sequencing. The pH in the bioreactors fluctuated between 5.0 and 7.7 with the highest amount of up to 50% sulfate removed measured around pH 6. Dissimilatory sulfate reducing bacteria (SRB) constituted only between 1% and 15% of the bacterial communities. Predicted bacterial metagenomes indicated a high prevalence of assimilatory sulfate reduction proceeding to formation of l-cystein and acetate, assimilatory and dissimilatory nitrate reduction, denitrification, and oxidation of ethanol to acetaldehyde with further conversion to ethanolamine, but not to acetate. Despite efforts to maintain optimal conditions for biological sulfate reduction in the bioreactors, only a small part of the microorganisms were SRB. The microbial communities were highly diverse, containing bacteria, archaea, and fungi, all of which affected the overall microbial processes in the bioreactors. While it is important to monitor specific physicochemical parameters in bioreactors, molecular assessment of the microbial communities may serve as a tool to identify biological factors affecting bioreactor functions and to optimize physicochemical attributes for ideal bioreactor performance.

Reactivation of Deep Subsurface Microbial Community in Response to Methane or Methanol Amendment.

Microbial communities in deep subsurface environments comprise a large portion of Earth's biomass, but the microbial activity in these habitats is largely unknown. Here, we studied how microorganisms from two isolated groundwater fractures at 180 and 500 m depths of the Outokumpu Deep Drillhole (Finland) responded to methane or methanol amendment, in the presence or absence of sulfate as an additional electron acceptor. Methane is a plausible intermediate in the deep subsurface carbon cycle, and electron acceptors such as sulfate are critical components for oxidation processes. In fact, the majority of the available carbon in the Outokumpu deep biosphere is present as methane. Methanol is an intermediate of methane oxidation, but may also be produced through degradation of organic matter. The fracture fluid samples were incubated in vitro with methane or methanol in the presence or absence of sulfate as electron acceptor. The metabolic response of microbial communities was measured by staining the microbial cells with fluorescent redox sensitive dye combined with flow cytometry, and DNA or cDNA-derived amplicon sequencing. The microbial community of the fracture zone at the 180 m depth was originally considerably more respiratory active and 10-fold more numerous (105 cells ml-1 at 180 m depth and 104 cells ml-1 at 500 m depth) than the community of the fracture zone at the 500 m. However, the dormant microbial community at the 500 m depth rapidly reactivated their transcription and respiration systems in the presence of methane or methanol, whereas in the shallower fracture zone only a small sub-population was able to utilize the newly available carbon source. In addition, the composition of substrate activated microbial communities differed at both depths from original microbial communities. The results demonstrate that OTUs representing minor groups of the total microbial communities play an important role when microbial communities face changes in environmental conditions.