4.1 Geochemical characteristics of the mine water
Studies on dammed or collected water from technical processes such as the flooded mine water often show a diverse and relevant microbial community, which could have a deep impact on the overall biogeochemical cycles of the elements in mine water and on its quality. In general, flooded mine water quality is determined by the solubility of the minerals from the mine and by chemical changes due to oxidation of the exposed ore and host rock (Bernhard et al. 1996). In addition, the methodology used during mineral extraction may also influence mine-water quality (e.g., acidification of host rock for ore extraction) (Arnold et al. 2011). Water from both the mines (Schlema-Alberoda and Pöhla) showed a circumneutral pH. The Schlema-Alberoda water is characterized by a much higher SO42− concentration (335 mg/L) compared to that of the Pöhla sample (0.5 mg/L). However, an acidification is not to be expected since the supply of SO42− is limited and high amounts of carbonates support the neutralization of the water as was previously described by Hiller and Schuppan (2008). The higher concentrations of Mg, Na, K and Ca observed in the Schlema-Alberoda samples may originate from the chemical or microbiological alteration of granite (containing feldspar and plagioclase minerals) or dolomite (calcium magnesium carbonate), poly- and monomineralic minerals as described by Naumov et al. (2017) in the Schlema-Alberoda mine. Differences in EH values may be due to the architecture of the Pöhla mine, being probably more "hermetic" than the Schlema-Alberoda mine, where infiltration of rainwater and O2 are possible. The considerably high As concentrations in both mines are probably caused by oxidation of the arsenic minerals in the ore veins, according to Paul et al. (2013).
The chemical behaviour of the uranyl ion in natural waters may be partly influenced by pH, EH and dissolved ions (Bernhard et al. 1998). Thermodynamic calculations predict a calcium uranyl carbonate complex [Ca2UO2(CO3)3(aq)] such as the dominant species in the Schlema-Alberoda mine water. By cryo-TRLFS measurements of the Schlema-Alberoda mine water combined with PARAFAC, two species were detected. We observed an analogy in the positions for Ca2UO2(CO3)3(aq) complexes. A slight shift of the fluorescence bands to the left for the second one matches with a uranyl carbonate complex [UO2(CO3)34−] (Wang et al. 2004). Furthermore, our results are consistent compared to those of Bernhard et al. (1996, 1998, 2001), where calcium uranyl carbonate complex was reported as the major species in the Schlema-Alberoda water. Since the U concentration in the Pöhla samples is extremely low, no detectable U signal was obtained by cryo-TRLFS measurements. On the other hand, by thermodynamic calculation of the predominance fields of U species, U is mostly found in the form of uraninite mineral (UO2).
4.2 Impact of microbial populations on mine-water biogeochemical processes
Many countries have implemented different management and remediation strategies to reduce the negative environmental impact of U mine water, generated by the U mining and milling activities. These remediation technologies should meet the set water quality regulatory standard for beneficial reuse of the U mine water for different purposes (e.g., irrigation, especially in water-stressed regions in the world) within the concept of circular economy (Annandale et al. 2017). Exploring the links between the geochemistry and microbial diversity of U mine water will provide insights into how microbial communities survive and thrive in such extreme contaminated environments and help the design of efficient bioremediation strategies.
It is well known that the structure and composition and activity of U mine water microbial communities are shaped by physicochemical factors such as pH, total organic carbon (TOC), dissolved oxygen (DO), concentrations of anions (e.g., NO3−, SO42−), cations (e.g., Fe, Mn) and toxic heavy metals/metalloids (e.g., U, Pb, As). Schippers et al. (1995), Fields et al. (2005) and Shuaib et al. (2021) showed that heavy metal and radionuclide contamination reduce the microbial diversity of the environment. In this study, similar richness and diversity were found in the bacterial community of water from both mines despite the fact that the concentration of U(VI) is a hundred times higher in the Schlema-Alberoda mine than in the Pöhla mine. However, the diversity of the fungal community was higher in the Pöhla mine water compared to the Schlema-Alberoda samples.
4.2.1 In situ bacterial community composition and structure
The bacterial community composition of water from both U mines displayed similarities at phylum level with that of other U-contaminated environments previously reported (Rastogi et al. 2010a, b; Lusa et al. 2019), but they exhibited differences regarding the proportions of some taxa. The phyla Campilobacterota, Proteobacteria, Patescibacteria, Verrucomicrobiota and Nitrospirota showed a higher relative abundance. The representative of these bacterial phyla has evolved mechanisms of resistance and tolerance to environmental toxicity of heavy metals and radionuclides. In addition, they play a major role in the biogeochemical cycles of elements such as S, N, Fe which subsequently effect that of U.
Bacteria involved in N/S redox cycling (nitrate reducers and sulfur oxidizers) from the phyla Campilobacterota and Proteobacteria were identified in water from both U mines. Abundant distribution of nitrate reducers and sulphur oxidizers from the genera Sulfuricurvum, Sulfurovum and Sulfurimonas of the phylum Campilobacterota in water from both U mine has been observed. They were reported to be distributed in heavy metals and radionuclide impacted environments (Chang et al. 2005; Shen et al. 2013; Zeng et al. 2019; Povedano-Priego et al. 2022) and to play a key role in the maintenance of reduced U species stability through coupling nitrate reduction to S-compound oxidation, and subsequently promote the growth of metal-reducing micro-organisms (e.g., Proteobacteria as SRB) (Chang et al. 2005; Huang et al. 2021). Nitrate might negatively influence the microbial reduction of U(VI). Nitrate, ferric ion and sulphate serve as thermodynamically more favourable final electron acceptors than U, and subsequently they would be reduced earlier than this radionuclide (Finneran et al. 2002). Therefore, anaerobic micro-organisms usually prefer nitrate as the first electron acceptor, followed by ferric iron and sulphate (Jroundi et al. 2020). In our study, the concentration of nitrate and nitrite remained below 0.07 mg/L, suggesting an adequate correlation of the microbial activity of these nitrate/nitrite reducers with the biogeochemical cycle of nitrogen.
Proteobacterial nitrate reducers and sulphur oxidizers including Sulfuritalea, Thiovirga and an unidentified genus of the family Hydrogenophilaceae also constitute a considerable microbial population in water from both U mines. They are well known for surviving in oligotrophic environments, and previously reported for their ability to reduce and tolerate metals (You et al. 2021; Bärenstrauch et al. 2022). Sulfuritalea can reduce nitrate to molecular nitrogen under anoxic conditions and oxidize thiosulfate, elemental sulphur and hydrogen (Kojima and Fukui, 2011). Furthermore, Thiovirga is a sulphur oxidizer (Ito et al. 2005). Peng et al. (2020) reported the role of the Hydrogenophilaceae family as beneficial and important in the sulphur cycle. Hydrogenophilaceae is able to oxidize sulphide compounds (e.g., S2−, HS− and H2S) to SO42−, which could be used by SRB (Peng et al. 2020). Its role in the reduction of nitrate to nitrite in microaerophilic members has also been reported (Orlygsson and Kristjansson 2014). Highly increased sulphate concentrations were observed in the Schlema-Alberoda mine water compared to the Pöhla mine water. The high sulphate concentration could be correlated with the role of sulphur oxidizing bacteria (SOB). The increased SO42− concentration could support the proliferation of SRB. For example, the phylum Desulfobacterota which contains several anaerobic genera of SRB including the genus Desulfurivibrio. Desulfurivibrio was mainly identified in the Schlema-Alberoda mine water where the sulphate concentration was higher (Jroundi et al. 2020). This is consistent with the assumption that SRB proliferated in the presence of higher sulphate concentrations. Moreover, an unidentified genus of the Thermodesulfovibrionia family (Nitrospirota) which couples H2 oxidation to sulphate reduction was also identified in the water samples (Rempfert et al. 2017; Nothaft et al. 2021; Umezawa et al. 2021). The reduced products of sulphate as hydrogen sulphides are able to, chemically reduce U(VI) as the FeRBs do (North et al. 2004).
In addition to Fe oxidizing bacteria, U mine water also harbour an unidentified genus of the family Rhodocyclaceae which include Fe-reducing bacteria (FeRB) with the ability to reduce U(VI) (Cummings et al. 1999; Porsch et al. 2009). Fe(III) reduction products have been reported to be able to chemically reduce U(VI) as well (Lovley et al. 1993; North et al. 2004; Wilkins et al. 2006). The abundant distribution of FeOB and FeRB in Schlema-Alberoda is correlated with its high Fe concentration. Furthermore, members of the Rhodocyclaceae family were reported to be able to grow lithotrophically by respiring U(VI) together with H2 oxidation and to be responsible for U(VI) bioreduction coupled with organic electron donors (Zhou et al. 2014).
In addition to phyla involved in the biogeochemical cycle of S, Fe, N and U, microbial diversity analysis has unveiled the presence of bacterial communities described for their adaptation to extreme environments including U contaminated sites. Amongst them, the phylum reported as Patescibacteria has an ultra-small cell size, highly simplified membrane structures and a greatly reduced metabolism highly adapted to U-contaminated environments by so far unknown mechanisms (Tian et al. 2020; Povedano-Priego et al. 2022). Nayak et al. 2021 identified sequences of unclassified Candidatus Moranbacteria (Parcubacteria), in radon- and heavy-metal contaminated water. Candidatus Omnitrophus (Verrucomicrobiota) is a chemolithoautotrophic bacterial genus that thrives in anoxic environments. This genus and its phylum have been previously reported by other authors in different contaminated environments (Underwood et al. 2022). The role they play is unknown, but they have generally been associated with environments contaminated by low concentrations of U, and could become a possible indicator for monitoring these contaminations as reported by Mumtaz et al. 2018.
4.2.2 In situ fungal community composition and structure
Bacteria have been the heroes during the last decade in the biogeochemical cycles of elements in environments contaminated by heavy metals and radionuclides (You et al. 2021; Povedano-Priego et al. 2022). Fungi have also been identified in such contaminated environments and contribute to many biogeochemical transformations (Gadd and Fomina 2011; Passarini et al. 2022). It is well documented that fungi can interact with U, mainly by biomineralization and biosorption processes (Schaefer et al. 2021). In addition, fungi are good metal chelators forming metal-organic complexes through the secretion of products from their secondary metabolism such as low-molecular-weight carboxylic acids (oxalic, succinic, malic and formic acids) (Gadd and Fomina 2011).
The fungal diversity of water from both U mines s was dominated by Ascomycota (phylum with the highest number of fungal genera) and Basidiomycota. Likewise, but with a low proportion, the phylum Rozellomycota was mainly identified in the Pöhla water mass. These results are in agreement with those reported by Zirnstein et al. (2012) and Harpke et al. (2022) where these phyla were described in environments contaminated by U. Furthermore, Ascomycota, Rozellomycota and Basidiomycota, have been reported in previous studies as phyla that could potentially play a key role in the decomposition and degradation of lignocellulosic biomass (Young et al. 2018; Liu et al. 2022). At the genus level, the water samples from the two U mines were characterized by the distribution of genera that have been previously reported in heavy metal and radionuclide contaminated habitats. These include Cadophora, Lecanicillium, Exophiala, and unidentified genera of different taxa (e.g., order Helotiales (Ascomycota) and Cystobasidium (Basidiomycota), class Sordariomycetes (Ascomycota) and family Fomitopsidaceae (Basidiomycota) (Dos Santos Utmazian et al. 2007; Dirginčiute-Volodkiene and Pečiulyte 2011; Jasrotia et al. 2014; Văcar et al. 2021; Passarini et al. 2022). Fomitopsis annosa (Fomitopsidaceae) was reported to accumulate U (Nakajima and Sakaguchi 1993). To the best of our knowledge, this is the first study to describe the identification of genera such as Neodevriesia and Cyphellophora (Ascomycota) in these types of extreme environments. They are able to produce melanin, a substance that protects the cell and participates in the immobilization of metals and radionuclides such as U (Fogarty and Tobin 1996; Turick et al. 2008; Oh et al. 2021). The most representative genera based on their role in the removal and biomineralization of U phosphates were Penicillium and Aspergillus (Ascomycota) (Schaefer et al. 2021; Zhang et al. 2022). But despite its high adaptive potential Penicillium was poorly reported in the Schlema-Alberoda samples and Aspergillus in the Pöhla samples.
4.3 Changes in mine-water geochemistry by microbial biostimulation
The inner walls of Schlema-Alberoda mine are covered with spruce and pine boards to prevent the collapse of the floors. Although no data are available on the type of wood in Pöhla mine, an abundance of conifers left by mining activities can be assumed for both mines. During mining, wood degradation through microbial activity (mainly fungi) was observed. With beginning of the flooding process, the mine water comes into contact with the wood, causing the further degradation processes. The natural and fungal-mediated decomposition of the wood releases cellulose and lignin as well as low molecular weight molecules (carbohydrates, saccharic acids, vanillin, vanillic acid and gluconic acid, amongst others) that may act as electron donors for U-reducing bacteria (Ander et al. 1980; Hedges et al. 1988; Baraniak et al. 2002; Mansour et al. 2020). Glycerol has previously been reported by other authors as an electron donor, in addition to lactate, acetate, methanol, and others (Madden et al. 2007; Newsome et al. 2015). These electron donors might stimulate the growth of SRB of the phylum Desulfobacterota (e.g., Desulfurivibrio), distributed in minor proportions in the Schlema-Alberoda mine, that may play an important role in U(VI) reduction (Chang et al. 2001; Geissler and Selenska-Pobell 2005; Moon et al. 2010). Biostimulation is a simple and effective bioremediation strategy that has previously been reported in situ and at laboratory scale by other authors (Yabusaki et al. 2007; Williams et al. 2013; Xu et al. 2017). In order to study the potential of the natural microbial community in the reduction of U(VI) in Schlema-Alberoda mine water, we amended a set of anoxic mine water microcosms with three different electron donors (glycerol, gluconic acid and vanillic acid). At the end of the experiment, remarkable changes were observed in the microcosm doped with glycerol where U(VI) concentration was reduced by ~ 99%. The concentration of total Fe (~ 95% reduction), SO42− (~ 58% reduction) and EH were affected as well, mainly by glycerol. It suggests that biostimulation with glycerol could promote the growth of SRB and FeRB which may be involved in U(VI) reduction. Madden et al. (2007) and Newsome et al. (2015) reported similar U reduction rates using glycerol phosphate and glycerol.