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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.

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.

Microbial life in the deep subsurface contributes significantly to overall biomass on Earth. Although the microbial communities inhabiting the deep subsurface are abundant, little is known about their diversity, activity, interactions and role in global biogeochemical cycles. The diversity of microbial life in the deep terrestrial subsurface of the Fennoscandian shield was studied with molecular biological methods. The Outokumpu Deep Drill Hole provides access to crystalline bedrock fluids that are estimated to be tens of millions of years old. Characterization of the indigenous bacterial and archaeal communities in addition to microbial communities with important functional properties in bedrock fluids was done from a depth range of 180 m to 2300 m. Microbial community profiling and assessment of possible functional processes was done with molecular fingerprinting, cloning and sequencing methods combined with suitable statistical and bioinformatics analyses. Low cell numbers but high diversity was characteristic to the microbial communities of the Outokumpu deep subsurface. The microbial communities in the fracture zones had in general fewer cells than those in the mixed fluids of the drill hole. Comamonadaceae, Peptococcaceae and Anaerobrancaceae were prevalent bacterial members of the microbial communities in the fracture fluids. Archaea were a minority in microbial communities. Sulfate-reducing bacteria and methanogens were detected at several depths. Microbial communities resembled those detected from other deep Fennoscandian Shield subsurface sites. Furthermore, sulfate reducing communities and archaeal communities resembled those found from the deep subsurface of South Africa. Investigation on carbon assimilation strategies of the microbial communities revealed that mainly heterotrophic Clostridia were responsible for CO2 fixation in this habitat. Representatives of Burkholderiales and Clostridia formed the core microbial community and these were also identified to be the keystone genera. The microbial communities of Outokumpu fractures share similarity with those of serpentinization-driven ecosystems. Energy and carbon substrates formed in serpentinization reactions of ophiolitic rocks in Outokumpu may sustain the microbial communities in this deep subsurface environment. Syvällä maanpinnan alla elävät mikrobit muodostavat merkittävän osan maapallon kokonaisbiomassasta. Vaikka syvällä elää runsaasti mikrobeja, niiden monimuotoisuudesta, aktiivisuudesta, vuorovaikutuksista ja roolista maailmanlaajuisissa biogeokemiallisissa kierroissa tiedetään vielä vähän. Mikrobielämän monimuotoisuutta Fennoskandian kilven kallioperässä tutkittiin käyttäen molekyylibiologisia menetelmiä. Outokummun syvä kairareikä tarjoaa mahdollisuuden tutkia kiteisen kallioperän vesiä, joiden on arvioitu olevan kymmeniä miljoonia vuosia vanhoja. Kallioperän endeemisten bakteeri- ja arkeoniyhteisöjen koostumusta ja merkittäviä toiminnallisia ominaisuuksia luonnehdittiin syvyysvälillä 180 2300 m. Mikrobiyhteisöjen toiminnallisten ominaisuuksien arviointi toteutettiin molekyylibiologisin menetelmin käyttäen sormenjälki-, kloonaus- ja sekvensointimenetelmiä sekä sopivia tilastollisia ja bioinformatiikan analyysejä. Matala solumäärä mutta korkea monimuotoisuusaste leimasivat mikrobiyhteisöjä Outokummun syvässä kallioperässä. Rakovyöhykkeissä oli yleisesti vähemmän soluja kuin sekoittuneessa kairareiän vesipatsaassa. Comamonadaceae-, Peptococcaceae- ja Anaerobrancaceae-sukujen bakteerit olivat vallitsevia rakovyöhykkeiden mikrobiyhteisöissä. Arkeonit olivat vähemmistönä yhteisöissä. Sulfaattia pelkistäviä bakteereita ja metaanintuottajia havaittiin useissa eri syvyyksissä. Mikrobiyhteisöt muistuttivat muista Fennoskandian syvistä ympäristöistä havaittuja mikrobiyhteisöjä. Lisäksi sulfaatinpelkistäjät sekä arkeoniyhteisöt olivat samankaltaisia Etelä-Afrikan syvän kallioperän mikrobiyhteisöjen kanssa. Mikrobien hiiliaineenvaihdunnan tutkimus paljasti heterotrofisten klostridien toimivan pääasiallisina hiilidioksidin sitojina näissä elinympäristöissä. Burkholderiales- ja Clostridia-bakteeriryhmien edustajat muodostivat nk. Outokummun kallioperän ydinmikrobiston, eli näiden ryhmien jäseniä löytyi kaikista syvyyksistä. Lisäksi näiden ryhmien edustajien todettiin olevan yhteisöjen avainlajeja. Outokummun kallioperän rakovyöhykkeiden vesissä elävät mikrobiyhteisöt ovat samankaltaisia kuin ne yhteisöt, joita löytyy ekosysteemeistä joiden energia ja hiili ovat lähtöisin ofioliittisten kivien serpentinisaatiosta. Serpentinisaatioreaktioissa muodostuvat energian- ja hiilenlähteet voivat ylläpitää mikrobiyhteisöjä myös Outokummun syväbiosfäärissä.

Bulletin of the Geological Society of Finland 92(2), 99-110 (2020). doi:10.17741/bgsf/92.2.002, Published by Suomen Geologinen Seura, Helsinki