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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 similar to 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.

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.

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.

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).

openaire: EC/H2020/730480/EU//ITERAMS The changes in water quality owing to recirculation of water in mineral processing plants can compromise the plant performance as well as maintenance needs. Therefore, mining process water quality assessment is becoming critical. Nevertheless, very few studies have investigated the suitability of the current analysis methodology practiced in certified laboratories for evaluating mining process water quality. This article presents two case studies to highlight the major issues encountered when performing sampling for physicochemical and chemical parameters in process water at two European mine sites using procedures from two certified laboratories. In addition, microorganisms were shown to be abundant in process waters and likely affect the mining water chemistries. However, the protocols used for microbial studies are not optimal for mining process samples, and need to be improved. The results showed difficulties in providing satisfactory results when analyzing control samples. Additionally, the analysis results presented a strong imbalance in TDS and sulfur compounds. Several potential causes associated with the poor quality of the analysis results have been outlined with a specific focus on the preservation methods. A literature review on the degradation of thiosalts suggested that the current preservation procedures are not suitable for preserving sulfur compounds. Moreover, the results indicated that the water matrix strongly influenced the validity of the chosen analysis method. In conclusion, the analysis methods should be customized for the different mining water matrix types in order to ensure the accuracy and reproducibility of the results.

Microorganisms enter the flotation process mainly from intake water and ore material. The diversity and number of microorganisms can vary significantly from mine to mine. In flotation, the conditions including oxygen levels, temperature, and nutrients from ore, intake water, and reagents are often favorable for the microbial growth. The mining industry aims to close the water loops, which is expected to result in the accumulation of microorganisms in the process waters with potential effects on flotation performance. Bioflotation, bioleaching, and bio-oxidation have been studied for decades as tools for concentrating and dissolving minerals. In contrast, there is limited scientific literature or industrial knowledge about microorganisms that naturally inhabit and prevail in minerals processing applications over a wide pH range. Microorganisms affect minerals when they selectively attach to the surfaces, produce extracellular polymeric substances (EPS) and polysaccharides, oxidize or reduce the minerals, change the pH and Eh of the process solution, and degrade organic flotation chemicals. Microorganisms contain different structural components that affect their surface chemistry, charge, and behavior in flotation, but these properties may also change via adaptation and solution conditions. Almost all studies on flotation have focused on chemical and physical parameters, and the role of naturally occurring microorganisms has remained underexplored. Advances in genomics and proteomics offer possibilities to describe not only which microorganisms are present, but also which physiological functions are being exercised. This article reviews the current knowledge of microorganisms in various mineral processes, identifies potential microbe–mineral interactions in flotation, describes the gaps in current knowledge, and concludes with the potential effects of microorganisms on flotation, especially in closed water loops.

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.

We studied the uptake of Ni2+ and Ag+ by bacterial strains of Paenibacillus, Pseudomonas, Burkholderia and Rhodococcus isolated from an acidic nutrient-poor boreal bog. The tests were run in two different growth media at two temperatures; +4 °C and +20 °C. All bacterial strains removed Ni2+ and Ag+ from the solution with highest efficiencies shown by one of the Pseudomonas sp. and one of the Paenibacillus sp. strains. Highest Ni2+ uptake was found in 1% Tryptone solution, whereas the highest removal of Ag+ was obtained using 1% Yeast extract. Temperature affected the uptake of Ni2+ and Ag+, but statistically significant difference was found only for Ni2+. Based on tests carried out for the bacteria in nutrient broths and for fresh samples taken from varying depth up to seven meters from the ombrotrophic bog, from which the bacteria were isolated, we estimated that in in situ conditions of the bog the uptake of Ni2+ by bacteria accounts for approximately 0.02% of the total sorption in the uppermost moss layer, 0.01% in the peat layer, 0.02% in the gyttja layer and 0.1% in the bottom clay layer of the bog. For Ag+ the corresponding values were 2.3% in the moss layer, 0.04% in the peat layer, 0.2% in the gyttja and 0.03% in the clay layer.

Copper is used in various applications in environments favoring and enabling formation of biofilms by naturally occurring microbes. Copper is also the chosen corrosion barrier for nuclear waste in Finland. The copper canisters should have lifetimes of 100,000 years. Copper is commonly considered to be resistant to corrosion in oxygen-free water. This is an important argument for using copper as a corrosion protection in the planned canisters for spent nuclear-fuel encapsulation. However, microbial biofilm formation on metal surfaces can increase corrosion in various conditions and provide conditions where corrosion would not otherwise occur. Microbes can alter pH and redox potential, excrete corrosion-inducing metabolites, directly or indirectly reduce or oxidize the corrosion products, and form biofilms that create corrosive microenvironments. Microbial metabolites are known to initiate, facilitate, or accelerate general or localized corrosion, galvanic corrosion, and intergranular corrosion, as well as enable stress-corrosion cracking. Sulfate-reducing bacteria (SRB) are present in the repository environment. Sulfide is known to be a corrosive agent for copper. Here we show results from corrosion of copper in anoxic simulated ground water in the presence of SRB enriched from the planned disposal site.

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.

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.

The microbial consortia in sulphate-reducing bioreactors with different operating conditions were studied and compared to the sulphate reduction efficiencies. The results showed vast differences in microbial communities among the reactors. The fraction of sulphate-reducing bacteria correlated with the bioreactor performance. This study sheds new light to the biological sulphate-reducing process applied in bioreactors, which is traditionally seen as a black box.

Microbial communities in deep subsurface environments comprise a large portion of Earth's biomass, but the metabolic activities in these habitats are largely unknown. Here the effect of CO2 and carbonate on the microbial community of an isolated groundwater fracture zone at 180 m depth of the Outokumpu Deep Scientific Drill Hole (Finland) was tested. Outokumpu groundwater at 180 m depth contains approximately 0.45 L L-1 dissolved gas of which methane contributes 76%. CO2, on the other hand, is scarce. The number of microbial cells with intracellular activity in the groundwater was low when examined with redox staining. Fluorescence Assisted Cell Sorting (FACS) analyses indicated that only 1% of the microbial community stained active with the redox sensing dye in the untreated groundwater after 4 weeks of starvation. However, carbon substrate and sulfate addition increased the abundance of fluorescent cells up to 7%. CO2 and CO2 + sulfate activated the greatest number of microbes, especially increasing the abundance of Pseudomonas sp., which otherwise was present at only low abundance in Outokumpu. Over longer exposure time (2 months) up to 50% of the bacterial cells in the groundwater were shown to incorporate inorganic carbon from carbonate into biomass. Carbon recapture is an important feature in this ecosystem since it may decrease the rate of carbon loss in form of CO2 released from cellular processes.

Selenite (Se(IV), SeO32−) uptake and the effect of selenite supplement on protein synthesis was investigated in two Pseudomonas sp. strains isolated from a boreal bog. These aerobic bacteria efficiently reduced Se(IV) with intracellular reduced Se0 observed in the cytoplasm under dark aerobic conditions. The proteome analysis of Se(IV) supplement and temperature responses by SDS-PAGE gel electrophoresis showed variations in the protein expression on the 40–60 kDa regions following these stress factors, probably through enzymes associated to oxidative stress or temperature adaptation. NO3−/NO2−/SO42− addition enhanced Se(IV) uptake in both bacteria, but Se(IV) uptake sustained also under sulphur and nitrogen starvation. Our findings suggest two different transport mechanisms for Se(IV) uptake in these Pseudomonas sp. strains; a low affinity transport system up-regulated by NO3−/NO2−/SO42− and a distinct Se(IV)O32− regulated transport system. Following transport, Se(IV) is reduced in the cytoplasm, forming Se0 granules, visible in TEM and verified using EDX.

Selenite (Se(IV), SeO32-) uptake and the effect of selenite supplement on protein synthesis was investigated in two Pseudomonas sp. strains isolated from a boreal bog. These aerobic bacteria efficiently reduced Se(IV) with intracellular reduced Se 0 observed in the cytoplasm under dark aerobic conditions. The proteome analysis of Se(IV) supplement and temperature responses by SDS-PAGE gel electrophoresis showed variations in the protein expression on the 40-60 kDa regions following these stress factors, probably through enzymes associated to oxidative stress or temperature adaptation. NO3-/NO2-/SO42- addition enhanced Se(IV) uptake in both bacteria, but Se(IV) uptake sustained also under sulphur and nitrogen starvation. Our findings suggest two different transport mechanisms for Se(IV) uptake in these Pseudomonas sp. strains; a low affinity transport system up-regulated by NO3-/NO2-/SO42- and a distinct Se(IV)O32- regulated transport system. Following transport, Se(IV) is reduced in the cytoplasm, forming Se 0 granules, visible in TEM and verified using EDX.