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 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.Peer reviewe

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 Pyhasalmi 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 Pyhasalmi 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.Peer reviewe

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

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.Peer reviewe

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

Microbial co-occurrence patterns in deep Precambrian bedrock fracture fluids

The bacterial and archaeal community composition and the possible carbon assimilation processes and energy sources of microbial communities in oligotrophic, deep, crystalline bedrock fractures is yet to be resolved. In this study, intrinsic microbial communities from groundwater of six fracture zones from 180 to 2300aEuro-m depths in Outokumpu bedrock were characterized using high-throughput amplicon sequencing and metagenomic prediction. Comamonadaceae-, Anaerobrancaceae- and Pseudomonadaceae-related operational taxonomic units (OTUs) form the core community in deep crystalline bedrock fractures in Outokumpu. Archaeal communities were mainly composed of Methanobacteriaceae-affiliating OTUs. The predicted bacterial metagenomes showed that pathways involved in fatty acid and amino sugar metabolism were common. In addition, relative abundance of genes coding the enzymes of autotrophic carbon fixation pathways in predicted metagenomes was low. This indicates that heterotrophic carbon assimilation is more important for microbial communities of the fracture zones. Network analysis based on co-occurrence of OTUs revealed possible "keystone" genera of the microbial communities belonging to Burkholderiales and Clostridiales. Bacterial communities in fractures resemble those found in oligotrophic, hydrogen-enriched environments. Serpentinization reactions of ophiolitic rocks in Outokumpu assemblage may provide a source of energy and organic carbon compounds for the microbial communities in the fractures. Sulfate reducers and methanogens form a minority of the total microbial communities, but OTUs forming these minor groups are similar to those found in other deep Precambrian terrestrial bedrock environments.Peer reviewe

Epilithic microbial communities and their functionality in the deep continental bedrock

There is a knowledge gap regarding the microbes attaching to the deep bedrock surfaces and their interactions with minerals. This thesis work characterized bacterial, fungal, and archaeal communities in the mica schist surface biofilms and in the deep groundwater from the depths of 500 and 967 m in the Outokumpu bedrock in Finland. An in-situ biofilm sampler was designed that enabled enrichment of microbes on mica schist surfaces in-situ within the deep subsurface. The attachment process of the deep groundwater microbial communities was followed in similar manner in microcosms. Long-term effects of a low molecular weight carbon compound pulse (methane, methanol, acetate) on evolving microbial communities on the mica schist and in the water phase was assessed. Different microbial morphologies were observed on the mica schist surfaces, such as rods, cocci, spirilli and long microbial filaments. Sessile microbes were shown to produce slimy extracellular polymeric substances (EPS), different attachment related surface appendices as well as tubular structures between cells. Most of the surface attached microbes represented single cells or small colonies, but also more dense biofilm structures were observed. Mineral heterogenicity could lead to uneven distribution of microbial cells on mica schist surface. Epilithic microbial communities on the mica schist generally differed from planktic groundwater communities. Microbial cells attached to both mica schist and glass surfaces, and the communities differed depending on the material. Distinct microbial community developed on mica schist in the in-situ conditions of Outokumpu bedrock in comparison to the mica schist communities developed in the longer enrichments in the laboratory microcosms. After in-situ enrichment in the deep bedrock conditions, mica schist surfaces hosted a diverse fungal community, containing e.g., yeasts adapted to extreme conditions and fungi belonging to the Mortierella-genus. In addition, microcosms also enriched filamentous fungi such as Penicillium and Aspergillus. The fungal community size is generally smaller than that of bacterial communities in the deep groundwaters and mica schist surfaces. Even though deep biosphere is known to host fungi, the actions and role of fungi are not well understood. The fungal communities were versatile and rich, which indicates how well adapted they are to bedrock environment. Genes linked to heterotrophy as well as to the cycling of carbon and sulfur were common in the metagenome assembled genomes (MAGs) of the rock surface attached bacteria. Sulfate reducing bacteria (SRB) attached especially to the mica schist surfaces, which could act as a carbon or sulfur source for the microbes. Mica schist enriched in-situ in the Outokumpu deep drill hole contained various SRB and thiosulfate utilizing bacteria. Genes encoding enzymes needed in the degradation of complex compounds were common across the bacterial groups. This implies the important role of both necromass degradation and recycling in sustaining the deep life.Syvän kallioperän kivipinnoille kiinnittyvistä mikrobeista ja niiden vuorovaikutuksista kivipintojen kanssa tiedetään hyvin vähän. Tässä työssä määritettiin millaisia bakteeri-, sieni- ja arkeoni-yhteisöjä esiintyi pinnoille kiinnittyneenä ja syvässä pohjavedessä 500 ja 967 metrin syvyydellä Outokummun kallioperässä. Tutkimuksessa kehitettiin biofilmikeräin, joka mahdollisti mikrobirikastuksen kivipinnoille in-situ-olosuhteissa syvällä kallioperässä. Syvän pohjaveden mikrobiyhteisöjen kiinnittymistä kivipinnoille tutkittiin myös laboratorio-olosuhteissa mikrokosmeissa. Tutkimuksessa verrattiin lyhytkestoisen hiilipulssin vaikutusta biofilmien muodostukseen pitkällä aikavälillä ja seurattiin pienten hiiliyhdisteiden (metaani, metanoli, asetaatti) vaikutusta kivipintojen ja vesifaasin mikrobiyhteisöjen muodostumiseen. Kivipintojen biofilmeissä todettiin erilaisia mikrobisolutyyppejä kuten sauvoja, kokkeja, spirillejä ja pitkiä mikrobifilamenttejä. Kivipintojen mikrobien todettiin tuottavan solun ulkoista limaa (EPS), erilaisia kiinnittymiseen tarkoitettuja ulokkeita sekä solujen välisiä putkimaisia rakenteita. Suurin osa kivipinnoille kiinnittyneistä mikrobeista esiintyi yksittäisinä soluina tai pieninä soluryppäinä, mutta myös varsinaista biofilmiä havaittiin. Heterogeeninen mineraalirakenne voisi vaikuttaa mikrobien epätasaiseen jakautumiseen kivipinnoille. Kivipintojen mikrobiyhteisöt pääasiassa eroavat syvien pohjavesien mikrobiyhteisöistä. Mikrobit kiinnittyivät sekä kiilleliuske, että lasipinnoille, joiden yhteisöt erosivat toisistaan. Syväreiän in-situ-olosuhteissa kivipinnoille todettiin kehittyneen erilainen mikrobiyhteisö kuin mikrokosmi-rikastuksien jälkeen, mutta pidempi rikastus laboratorio-olosuhteissa on myös voinut edistää biofilmiyhteisön kehittymistä. Tutkimus kartoitti kivipinnoille kiinnittyviä sieniyhteisöjä. Syväreiässä tehdyn rikastuksen jälkeen kiilleliuskeen pinnoilla todettiin monipuolinen sieniyhteisö, jossa kukoistivat erityisesti erilaiset muistakin äärioloista tutut hiivat ja Mortierella-suvun sienet. Mikrokosmeissa rikastuivat myös rihmamaiset homeet kuten Penicillium- ja Aspergillus-suvun sienet. Sieniä esiintyy syvissä pohjavesissä ja kivipinnoilla suhteessa vähemmän kuin bakteereita. Sienet ovat osa syväbiosfääriä, mutta näiden sienien toimintaa eikä täten niiden roolia syväbiosfäärissä vielä kunnolla tunneta. Sieniyhteisöt olivat monipuolisia, mikä osaltaan osoittaisi niiden sopeutuneen syvän kallioperän olosuhteisiin hyvin. Heterotrofiaan, sekä hiilen ja rikin kiertoon liittyvät geenit olivat yleisiä kivipinnoille kiinnittyneiden bakteerien metagenomeista kootuissa genomeissa (MAG). Sulfaatinpelkistäjäbakteerien (SRB) todettiin kiinnittyvän kiilleliuskeelle, joka voisi toimia rikin ja hiilen lähteenä mikrobeille. Outokummun syväreiässä rikastetut kivinäytteet sisälsivät monipuolisen yhteisön erilaisia SRB ja thiosulfaatin kierrättäjämikrobeita. Monimutkaisten yhdisteiden hajottamiseen liittyvien entsyymien koodaamiseen tarvittava geenistö oli runsas eri mikrobiryhmissä, mikä osaltaan voisi viitata kuolleen biomassan hajottamisen merkityksestä syvän kallioperän elämän ylläpidolle ja ravinteiden kierrolle

Epilithic microbial communities and their functionality in the deep continental bedrock

There is a knowledge gap regarding the microbes attaching to the deep bedrock surfaces and their interactions with minerals. This thesis work characterized bacterial, fungal, and archaeal communities in the mica schist surface biofilms and in the deep groundwater from the depths of 500 and 967 m in the Outokumpu bedrock in Finland. An in-situ biofilm sampler was designed that enabled enrichment of microbes on mica schist surfaces in-situ within the deep subsurface. The attachment process of the deep groundwater microbial communities was followed in similar manner in microcosms. Long-term effects of a low molecular weight carbon compound pulse (methane, methanol, acetate) on evolving microbial communities on the mica schist and in the water phase was assessed.Different microbial morphologies were observed on the mica schist surfaces, such as rods, cocci, spirilli and long microbial filaments. Sessile microbes were shown to produce slimy extracellular polymeric substances (EPS), different attachment related surface appendices as well as tubular structures between cells. Most of the surface attached microbes represented single cells or small colonies, but also more dense biofilm structures were observed. Mineral heterogenicity could lead to uneven distribution of microbial cells on mica schist surface.Epilithic microbial communities on the mica schist generally differed from planktic groundwater communities. Microbial cells attached to both mica schist and glass surfaces, and the communities differed depending on the material. Distinct microbial community developed on mica schist in the in-situ conditions of Outokumpu bedrock in comparison to the mica schist communities developed in the longer enrichments in the laboratory microcosms. After in-situ enrichment in the deep bedrock conditions, mica schist surfaces hosted a diverse fungal community, containing e.g., yeasts adapted to extreme conditions and fungi belonging to the Mortierella-genus. In addition, microcosms also enriched filamentous fungi such as Penicillium and Aspergillus. The fungal community size is generally smaller than that of bacterial communities in the deep groundwaters and mica schist surfaces. Even though deep biosphere is known to host fungi, the actions and role of fungi are not well understood. The fungal communities were versatile and rich, which indicates how well adapted they are to bedrock environment.Genes linked to heterotrophy as well as to the cycling of carbon and sulfur were common in the metagenome assembled genomes (MAGs) of the rock surface attached bacteria. Sulfate reducing bacteria (SRB) attached especially to the mica schist surfaces, which could act as a carbon or sulfur source for the microbes. Mica schist enriched in-situ in the Outokumpu deep drill hole contained various SRB and thiosulfate utilizing bacteria. Genes encoding enzymes needed in the degradation of complex compounds were common across the bacterial groups. This implies the important role of both necromass degradation and recycling in sustaining the deep life