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.

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.

Microbial fouling and corrosion of carbon steel in deep anoxic alkaline groundwater.

Understanding the corrosion of carbon steel materials of low and intermediate level radioactive waste under repository conditions is crucial to ensure the safe storage of radioactive contaminated materials. The waste will be in contact with the concrete of repository silos and storage containers, and eventually with groundwater. In this study, the corrosion of carbon steel under repository conditions as well as the microbial community forming biofilm on the carbon steel samples, consisting of bacteria, archaea, and fungi, was studied over a period of three years in a groundwater environment with and without inserted concrete. The number of biofilm forming bacteria and archaea was 1,000-fold lower, with corrosion rates 620-times lower in the presence of concrete compared to the natural groundwater environment. However, localized corrosion was detected in the concrete-groundwater environment indicating the presence of local microenvironments where the conditions for pitting corrosion were favorable.

Uptake of radioiodide by Paenibacillus sp., Pseudomonas sp., Burkholderia sp. and Rhodococcus sp. isolated from a boreal nutrient-poor bog.

Radionuclides, like radioiodine ((129)I), may escape deep geological nuclear waste repositories and migrate to the surface ecosystems. In surface ecosystems, microorganisms can affect their movement. Iodide uptake of six bacterial strains belonging to the genera Paenibacillus, Pseudomonas, Burkholderia and Rhodococcus isolated from an acidic boreal nutrient-poor bog was tested. The tests were run in four different growth media at three temperatures. All bacterial strains removed iodide from the solution with the highest efficiency shown by one of the Paenibacillus strains with >99% of iodide removed from the solution in one of the used growth media. Pseudomonas, Rhodococcus and one of the two Paenibacillus strains showed highest iodide uptake in 1% yeast extract with maximum values for the distribution coefficient (Kd) ranging from 90 to 270L/kg DW. The Burkholderia strain showed highest uptake in 1% Tryptone (maximum Kd 170L/kg DW). The Paenibacillus strain V0-1-LW showed exceptionally high uptake in 0.5% peptone +0.25% yeast extract broth (maximum Kd>1,000,000L/kg DW). Addition of 0.1% glucose to the 0.5% peptone +0.25% yeast extract broth reduced iodide uptake at 4°C and 20°C and enhanced iodide uptake at 37°C compared to the uptake without glucose. This indicates that the uptake of glucose and iodide may be competing processes in these bacteria. We estimated that in in situ conditions of the bog, the bacterial uptake of iodide accounts for approximately 0.1%-0.3% of the total sorption of iodide in the surface, subsurface peat, gyttja and clay layers.

Heterotrophic communities supplied by ancient organic carbon predominate in deep fennoscandian bedrock fluids.

The deep subsurface hosts diverse life, but the mechanisms that sustain this diversity remain elusive. Here, we studied microbial communities involved in carbon cycling in deep, dark biosphere and identified anaerobic microbial energy production mechanisms from groundwater of Fennoscandian crystalline bedrock sampled from a deep drill hole in Outokumpu, Finland, by using molecular biological analyses. Carbon cycling pathways, such as carbon assimilation, methane production and methane consumption, were studied with cbbM, rbcL, acsB, accC, mcrA and pmoA marker genes, respectively. Energy sources, i.e. the terminal electron accepting processes of sulphate-reducing and nitrate-reducing communities, were assessed with detection of marker genes dsrB and narG, respectively. While organic carbon is scarce in deep subsurface, the main carbon source for microbes has been hypothesized to be inorganic carbon dioxide. However, our results demonstrate that carbon assimilation is performed throughout the Outokumpu deep scientific drill hole water column by mainly heterotrophic microorganisms such as Clostridia. The source of carbon for the heterotrophic microbial metabolism is likely the Outokumpu bedrock, mainly composed of serpentinites and metasediments with black schist interlayers. In addition to organotrophic metabolism, nitrate and sulphate are other possible energy sources. Methanogenic and methanotrophic microorganisms are scarce, but our analyses suggest that the Outokumpu deep biosphere provides niches for these organisms; however, they are not very abundant.

Methanospirillum stamsii sp. nov., a psychrotolerant, hydrogenotrophic, methanogenic archaeon isolated from an anaerobic expanded granular sludge bed bioreactor operated at low temperature.

A psychrotolerant hydrogenotrophic methanogen, strain Pt1, was isolated from a syntrophic propionate-oxidizing methanogenic consortium obtained from granulated biomass of a two-stage low-temperature (3-8 °C) anaerobic expanded granular sludge bed (EGSB) bioreactor, fed with a mixture of volatile fatty acids (VFAs) (acetate, propionate and butyrate). The strain was strictly anaerobic, and cells were curved rods, 0.4-0.5×7.5-25 µm, that sometimes formed wavy filaments from 25 to several hundred micrometres in length. Cells stained Gram-negative and were non-sporulating. They were gently motile by means of tufted flagella. The strain grew at 5-37 °C (optimum at 20-30 °C), at pH 6.0-10 (optimum 7.0-7.5) and with 0-0.3 M NaCl (optimum 0 M NaCl). Growth and methane production was found with H2/CO2 and very weak growth with formate. Acetate and yeast extract stimulated growth, but were not essential. The G+C content of the DNA of strain Pt1 was 40 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Pt1 was a member of the genus Methanospirillum and showed 97.5 % sequence similarity to Methanospirillum hungatei JF1(T) and 94 % sequence similarity to Methanospirillum lacunae Ki8-1(T). DNA-DNA hybridization of strain Pt1 with Methanospirillum hungatei JF1(T) revealed 39 % relatedness. On the basis of its phenotypic characteristics and phylogenetic position, strain Pt1 is a representative of a novel species of the genus Methanospirillum, for which the name Methanospirillum stamsii sp. nov. is proposed. The type strain is Pt1(T) ( = DSM 26304(T) = VKM B-2808(T)).

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.

Application of denaturing high-performance liquid chromatography for monitoring sulfate-reducing bacteria in oil fields.

Sulfate-reducing bacteria (SRB) participate in microbially induced corrosion (MIC) of equipment and H2S-driven reservoir souring in oil field sites. Successful management of industrial processes requires methods that allow robust monitoring of microbial communities. This study investigated the applicability of denaturing high-performance liquid chromatography (DHPLC) targeting the dissimilatory sulfite reductase ß-subunit (dsrB) gene for monitoring SRB communities in oil field samples from the North Sea, the United States, and Brazil. Fifteen of the 28 screened samples gave a positive result in real-time PCR assays, containing 9 × 10(1) to 6 × 10(5) dsrB gene copies ml(-1). DHPLC and denaturing gradient gel electrophoresis (DGGE) community profiles of the PCR-positive samples shared an overall similarity; both methods revealed the same samples to have the lowest and highest diversity. The SRB communities were diverse, and different dsrB compositions were detected at different geographical locations. The identified dsrB gene sequences belonged to several phylogenetic groups, such as Desulfovibrio, Desulfococcus, Desulfomicrobium, Desulfobulbus, Desulfotignum, Desulfonatronovibrio, and Desulfonauticus. DHPLC showed an advantage over DGGE in that the community profiles were very reproducible from run to run, and the resolved gene fragments could be collected using an automated fraction collector and sequenced without a further purification step. DGGE, on the other hand, included casting of gradient gels, and several rounds of rerunning, excising, and reamplification of bands were needed for successful sequencing. In summary, DHPLC proved to be a suitable tool for routine monitoring of the diversity of SRB communities in oil field samples.

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.

Archaeal communities in boreal forest tree rhizospheres respond to changing soil temperatures.

Temperature has generally great effects on both the activity and composition of microbial communities in different soils. We tested the impact of soil temperature and three different boreal forest tree species on the archaeal populations in the bulk soil, rhizosphere, and mycorrhizosphere. Scots pine, silver birch, and Norway spruce seedlings were grown in forest humus microcosms at three different temperatures, 7-11.5°C (night-day temperature), 12-16°C, and 16-22°C, of which 12-16°C represents the typical mid-summer soil temperature in Finnish forests. RNA and DNA were extracted from indigenous ectomycorrhiza, non-mycorrhizal long roots, and boreal forest humus and tested for the presence of archaea by nested PCR of the archaeal 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) profiling and sequencing. Methanogenic Euryarchaeota belonging to Methanolobus sp. and Methanosaeta sp. were detected on the roots and mycorrhiza. The most commonly detected archaeal 16S rRNA gene sequences belonged to group I.1c Crenarchaeota, which are typically found in boreal and alpine forest soils. Interestingly, also one sequence belonging to group I.1b Crenarchaeota was detected from Scots pine mycorrhiza although sequences of this group are usually found in agricultural and forest soils in temperate areas. Tree- and temperature-related shifts in the archaeal population structure were observed. A clear decrease in crenarchaeotal DGGE band number was seen with increasing temperature, and correspondingly, the number of euryarchaeotal DGGE bands, mostly methanogens, increased. The greatest diversity of archaeal DGGE bands was detected in Scots pine roots and mycorrhizas. No archaea were detected from humus samples from microcosms without tree seedling, indicating that the archaea found in the mycorrhizosphere and root systems were dependent on the plant host. The detection of archaeal 16S rRNA gene sequences from both RNA and DNA extractions show that the archaeal populations were living and that they may have significant contribution to the methane cycle in boreal forest soil, especially when soil temperatures rise.

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).

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.

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.

Effect of tree species and mycorrhizal colonization on the archaeal population of boreal forest rhizospheres.

Group 1.1c Crenarchaeota are the predominating archaeal group in acidic boreal forest soils. In this study, we show that the detection frequency of 1.1c crenarchaeotal 16S rRNA genes in the rhizospheres of the boreal forest trees increased following colonization by the ectomycorrhizal fungus Paxillus involutus. This effect was very clear in the fine roots of Pinus sylvestris, Picea abies, and Betula pendula, the most common forest trees in Finland. The nonmycorrhizal fine roots had a clearly different composition of archaeal 16S rRNA genes in comparison to the mycorrhizal fine roots. In the phylogenetic analysis, the 1.1c crenarchaeotal 16S rRNA gene sequences obtained from the fine roots formed a well-defined cluster separate from the mycorrhizal ones. Alnus glutinosa differed from the other trees by having high diversity and detection levels of Crenarchaeota both on fine roots and on mycorrhizas as well as by harboring a distinct archaeal flora. The similarity of the archaeal populations in rhizospheres of the different tree species was increased upon colonization by the ectomycorrhizal fungus. A minority of the sequences obtained from the mycorrhizas belonged to Euryarchaeota (order Halobacteriales).

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.

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.