Licentiate Thesis

Licentiate thesis

Geochemistry

Microbially mediated formation of birnessite-type manganese oxides and subsequent incorporation of rare earth elements, Ytterby mine, Sweden

Susanne Sjöberg

Stockholm 2017

Department of Geological Sciences Stockholm University SE-106 91 Stockholm Abstract

Microbes exert extensive control on redox element cycles. They participate directly or indirectly in the concentration and fractionation of elements by influencing the partitioning between soluble and insoluble species. Putative microbially mediated manganese (Mn) oxides of the birnessite-type, enriched in rare earth elements (REE) + yttrium (Y) were recently found in the Ytterby mine, Sweden. A poorly crystalline birnessite-type phyllomanganate is regarded as the predominant initial phase formed during microbial Mn oxidation. Owing to a higher specific surface area, this biomineral also enhances the known sorption property of Mn oxides with respect to heavy metals (e.g. REE) and therefore has considerable environmental impact.

The concentration of REE + Y (2±0.5% of total mass, excluding oxygen, carbon and silicon) in the Ytterby Mn oxide deposit is among the highest ever observed in secondary precipitates with Mn and/or iron. Sequential extraction provides evidence of a mineral structure where the REE+Y are firmly included, even at pH as low as 1.5. Concentration ratios of Mn oxide precipitates to fracture water indicate a strong preference for the trivalent REE+Y over divalent and monovalent metals. A culture independent molecular phylogenetic approach was adopted as a first step to analyze the processes that microbes mediate in this environment and specifically how the microbial communities interact with the Mn oxides. Plausible players in the formation of the investigated birnessite-type Mn oxides are mainly found within the ferromanganese genera Hyphomicrobium and Pedomicrobium and a newly identified Ytterby Bacteroidetes cluster most closely related to the Terrimonas. Data also indicate that the detected microorganisms are related to the environmental constraints of the site including low constant temperature (8°C), absence of light, high metal content and possibly proximity to the former storage of petroleum products.

Keywords: microbial diversity, manganese oxides, birnessite, rare earth elements, subterranean, Ytterby mine

Sammanfattning

Elementcykler kan drivas av redoxprocesser, vilka sker spontant på Jordens yta så länge de är termodynamiskt fördelaktiga. Mikroorganismer kan påverka och dra nytta av dessa processer så länge den mikrobiella aktiviteten är mer effektiv än den inorganiska processen. Under dessa medlingsprocesser påverkar mikroberna fördelningen mellan lösliga species och olösliga species och vice versa. Genom denna inblandning i produktion och/eller nedbrytning av mineral, har mikroberna stort inflytande över elementcykler och bidrar därmed direkt eller indirekt till koncentration och fraktionering av specifika element. I Ytterby gruva (Sverige) upptäcktes nyligen en ackumulation av en förmodat mikrobiellt bildad variant av manganoxiden birnessit, anrikad med avseende på sällsynta jordartsmetaller (REE) och yttrium (Y). En lågristallin variant av birnessit är den dominerande intiala fasen som produceras vid mikrobiellt bildad manganoxid. Tack vare en större specifik yta har denna mikrobiellt bildade fas egenskaper som ger en förhöjd sorptionskapacitet med avseende på tungmetaller i jämförelse med andra Mn-oxider (som generellt också har mycket hög sorptionskapacitet). Dessa mikrob-medierade Mn-oxider har därför avsevärd påverkan på miljön.

Ytterby-birnessiten innehåller 2±0.5 vikt-% REE+Y (exklusive syre, kol och kisel). Detta är bland de högsta REE+Y-halter som observerats i sekundära utfällningar innehållande REE. Sekventiell lakning av Ytterby-birnessiten antyder en mineralstruktur där REE+Y är fast inkorporerade, även vid pH så lågt som 1.5. Koncentrationsförhållandet mellan de utfällda manganoxiderna och sprickvattnet tyder på en stark preferens för de trivalenta REE+Y över divalenta och monovalenta metaller. Molekylär fylogenetisk analys användes som ett första steg för att förstå processerna som mikrober medlar i denna miljö, och specifikt hur de mikrobiella samhällena interagerar med Mn-oxiderna. Troliga medlare i bildandet av de undersökta Mn-oxiderna av birnessit-typ finns främst bland ferromangansläktena Hyphomicrobium och Pedomicrobium (P. Manganicum) och ett nyupptäckt Bacteroidetes kluster som är närmast släkt med Terrimonas. Data tyder också på att de identifierade mikroorganismerna är relaterade till de faktorer som definierar ackumulationsmiljön: låg konstant temperatur (8°C), avsaknad av ljus, högt metallinnehåll och möjligen också närheten till den tidigare lagringen av petroleumprodukter.

Nyckelord: mikrobiell diversitet, manganoxider, birnessit, sällsynta jordartsmetaller, underjordisk, Ytterby mine List of papers

This thesis is based on the following papers which are referred to in the text by their Roman numerals.

I. Sjöberg S, Allard B, Rattray JE, Callac N, Grawunder A, Ivarsson M, Sjöberg V, Karlsson S, Skelton A and Dupraz C. (2017). Rare earth element enriched birnessite in water-bearing fractures, the Ytterby mine, Sweden. Appl Geochem 78:158-171.

II. Sjöberg S, Callac N, Allard B, Smittenberg R and Dupraz C. (2017). Microbial communities inhabiting a birnessite-type manganese deposit, the Ytterby mine, Sweden. (submitted).

Table of contents

1. Introduction 2. Microbially mediated Mn oxides – formation and trace metal accumulation 2.1 Microbial communities, organic matter and mineral precipitation 2.2 Microbially mediated Mn oxides 2.3 Accumulation of metals by Mn oxides 3. Materials and methods 3.1 Background 3.2 Sampling and characterization of the birnessite-type Mn oxide (paper I) 3.3 Microbial communities inhabiting the birnessite-type Mn oxides (paper II) 4. Results and discussion 4.1 Composition of the birnessite-type Mn oxide (paper I) 4.2 Indicators of microbial involvement in the production of the birnessite-type Mn oxide (paper I) 4.3 Microbial communities inhabiting the YBS (paper II) 4.4 Potential mechanisms for the formation of the birnessite-type Mn oxides (paper II) 4.5 Redistribution of REE and enrichment in the birnessite-type Mn oxide in the Ytterby mine (paper I) 4.6 A metal stressed microbial community? 5. Conclusions 6. Future research areas Acknowledgments References

Appendix A. paper 1 Appendix B. paper 2

1. Introduction

Element cycles can be driven by redox processes, which can spontaneously take place inorganically at the surface of the Earth. Microbes are able to influence these processes and derive advantages from them, provided that their activity is more efficient than the inorganic process (Krauskopf 1957; Pedersen 2005). In these mediations microbes alter the litho- and hydrosphere by transforming soluble species into insoluble precipitates and vice versa (Pedersen 2005). While being involved in production and/or dissolution of minerals, microbes exert extensive control on the cycling of elements and participate, directly or indirectly, in concentration and fractionation of specific elements.

The manganese (Mn) biogeochemical cycle involves cross-linkages between microbial and abiotic processes and multiple element cycles (Hansel et al. 2015). The interaction between Mn and microorganisms in the oxidative Mn cycle may result in the formation of Mn oxides, commonly in the form of poorly crystalline birnessite-type phyllomanganates with hexagonal symmetry (Villalobos et al. 2003; Tebo et al. 2004; Bargar et al. 2005; Villalobos et al. 2006; Santelli et al. 2011; Hansel and Learman 2016). The catalytic function that microbial communities and processes have in the Mn redox cycle is well documented (Hansel and Learman 2016). Nevertheless, the microbial mechanisms driving these processes, and the properties of the mineral product, remain to some extent unknown.

The former quartz and feldspar mine in Ytterby is the type location for scandium, yttrium, tantalum and five of the rare earth elements (REE). Recent investigations have revealed that the mine hosts a black REE+Y enriched Mn deposit, hereafter referred to as the Ytterby black substance (YBS) (Sjöberg 2014). Phase analysis by X-ray diffraction (XRD) and elemental analysis show that the dominating phase is a birnessite-type Mn oxide with traces of organics and low Fe content (Sjöberg 2014). Electron paramagnetic resonance (EPR)-spectroscopy and lipid biomarker analyses indicate that the formation of the Mn deposit is induced by a microbial community (Sjöberg 2014). This site provides a window on the complex interaction between microbes and Mn minerals and the subsequent accumulation of trace elements.

Objectives This thesis aims to increase the understanding of mechanisms involved in the formation and accumulation of the putative microbially mediated REE+Y enriched birnessite-type Mn oxides. Two main questions guide the approaches used in this work: Q1: How are the REE+Y associated with the birnessite-type Mn oxides?

Q2: What microbial communities inhabit the Mn deposit and is it possible within these populations to identify Mn oxidizers and investigate their role in the formation of the birnessite-type Mn oxides?

Preliminary results on composition, structure and origin of the YBS as reported in Sjöberg (2014), are confirmed by repeated elemental, phase, spectroscopic and organic fraction analyses. A sequential extraction procedure is conducted to provide information on the nature of the REE association with the birnessite-type Mn oxides. New analyses of major an- and cations, trace elements (including methods allowing for analysis of the whole series of REE) and DOC for all relevant waters, i.e. groundwater in the mine surroundings and fracture water. Deepened geochemical analyses regarding enrichment of metals in the Mn deposit relative to the fracture water and the YBS preference for trivalent over divalent and monovalent ions (Q1, main topic of paper I). A molecular phylogenetic approach is adopted for characterization of the microbial community associated with and interacting with the YBS and identification of potential candidates for Mn oxidation (Q2, main topic of paper II).

2. Microbially mediated Mn oxides – formation and trace metal accumulation

2.1 Microbial communities, organic matter, and mineral precipitation

Formation of minerals associated with an organic matrix or organic matter can be the result of two main related processes: biomineralization and organomineralization (e.g. Dupraz et al. 2009). Biomineralization involves active pumping of cations and a genetically controlled manner to organize the organic matrix in which the mineralization occurs. The result is the formation of recognizable organism-specific skeletons that, when fossilized, can be used to identify the organism, often at species level. The prerequisite for biomineralization is that the biologically controlled mineralization is recognizable in the end product and that no abiotic processes can produce the same pattern. The mineral product of biomineralization can therefore be interpreted as a proof of life.

Biominerals can be formed within the cells (intracellular) or outside the cell walls (extracellular) (Templeton and Benzerara, 2015). Organomineralization can be (1) active or (2) passive. Active corresponds to biologically induced mineralization which occurs when microbial metabolic activity modifies the microenvironment in a direction favorable for mineral precipitation. Passive organomineralization corresponds to biologically influenced mineralization which occurs when mineralization within a microbial organic matrix is environmentally driven (Dupraz et al. 2009). Organomineralization is simply indicating the nucleation of a mineral in association with organic matter, in contrast to genetically controlled mineralization in which the mineral product provides a direct link to life. This question is particularly sensible when dealing with the study of biosignatures related to the origin of life on Earth or the search for life on other planets. An organomineral is not a direct proof of life because it does not indicate the source of this organic matter, which also can be produced through inorganic processes (e.g. Fischer-Tropsch synthesis, Brasier et al. 2002; Proskurowski et al. 2008). However, organic matter is commonly related to organisms. Identification of organic matter in ancient samples or knowledge of specific minerals that form in association with organic matter is a strong indication that life was in the vicinity to produce the organic matter, i.e. an indirect proof of life.

2.2 Microbially mediated Mn oxides

Precipitation and accumulation of brown to black insoluble Mn oxides is often associated with seepages of anoxic water into oxic environments such as water-bearing rock fractures that crop out in caves or tunnels (Pedersen 1997; Nealson 2006; Frierdich et al. 2011). Mn oxidation is a slow process, which can take years to complete under oxic conditions at circumneutral pH (Krauskopf 1957; Tebo et al. 2004). The liquid-solid and anoxic-oxic interface are known to be favorable conditions for biofilm development and strong microbe- mineral interactions (Pedersen 1997; Donlan 2002). The catalytic function that microbial communities and processes have in the Mn redox cycle is well documented (Hansel and Learman 2016).

Microbial interactions with heavy metals (including Mn)

Microorganisms have a wide array of techniques to adjust to extreme environments, especially to cope with high metal concentrations. Redistribution and mobility of metals in nature is highly controlled by metal bioavailability and subsequent incorporation into biological systems or biogeochemical cycles (Fein 2017). Microorganisms are dealing with the positive (essential micronutrients) and negative (metal toxicity) impacts of metals by uptake into the cell (bio-accumulation) or by extracellular neutralization (Kothe et al. 2005; Haferburg and Kothe 2010; Fein 2017; Lorentz et al. 2012). These processes affect the speciation and, thus, the properties of the given metals, what potentially may lead to the formation of non-toxic compounds through chelation and subsequent sequestration inside the cell, adsorption to the cell wall, binding within extracellular organic matter, and/or organomineralization (Silver and Phung 1996; Kothe et al. 2005; Pedersen 2005; Dupraz et al. 2009; Haferburg and Kothe 2010; Lorentz et al. 2012). In the case of a Mn rich environment, it has been argued that bacterial cells are protecting themselves from the toxicity of heavy metals by extracellularly precipitating Mn species (Tebo et al. 2005; Akob et al. 2014).

Suggested reasons as to why microbes oxidize Mn

Although a range of phylogenetically diverse bacteria and fungi have the ability to oxidize Mn, the reason for possessing this trait remains a subject of discussion (Ghiorse 1984; Tebo et al. 2004; Nealson 2006; Tebo et al. 2010; Hansel and Learman 2016). Suggested reasons as to why microbes oxidize Mn are.  Protection: Use of Mn(II) as an electron donor in superoxide radical reductions, thus eliminating reactive oxygen species (ROS) from the cell (Ghiorse 1984). Sorption of heavy metals to the produced Mn oxides may protect the microbe from potentially toxic metals by neutralizing the metals.  Degradation of recalcitrant carbon: The ability of Mn oxides to degrade complex humic substances into bioavailable low molecular weight carbon (Sunda and Kieber 1994; Hansel and Learman 2016). Thus, by producing Mn oxides, microbes indirectly benefit from it by increasing the amount of carbon available for growth.  Storage: Mn oxides as temporary storage for electron acceptors to be used under anaerobic conditions (e.g. intermediate species such as Mn(III) (Tebo 1983; Tebo et al. 1997; Hansel and Learman 2016).  Energy conservation: Mn(II) oxidation used for energy conservation (ATP synthesis) during lithotrophic metabolisms (Ehrlich 1983; Ehrlich and Salerno 1990; Falamin and Pinevich 2006).  Nutrition: Mn as a trace nutrient for cellular functions (Tebo et al. 2005).  Cofactor: Mn as an essential cofactor for enzymes such as superoxide dismutases (SOD) and in photosystem II.

Mechanisms of microbially mediated Mn oxidation

The ability to catalyze Mn oxidation among microbes is only detected in heterotrophs (Ghiorse 1984; Hansel and Learman 2016) but mediated by phylogenetically diverse bacteria and fungi (Ghiorse 1984; Tebo et al. 2004; Nealson 2006). Microbial catalyses of Mn oxidation can be direct or indirect; possibly involving lithotrophy (Tebo et al. 2004; Nealson 2006; Hansel and Learman 2016) (paper II). Direct refers to an enzymatic pathway in which the microbe utilizes a multicopper oxidase (MCO), a peroxidase or a combination of Mn peroxidase (MnP) and laccase (a specific kind of peroxidase) to directly oxidize Mn. Indirect refers either to microbial modification of the microenvironment in favor of Mn precipitation (e.g. pH and redox conditions) or bacterial production of harmful superoxide radicals (by- products of respiration) and their subsequent reduction by the Mn(II) oxidation to Mn(III, IV) species (Ghiorse 1984) (paper II). In the latter case, Mn(II) serves as antioxidative cell protection. Microbial Mn oxidation is thought to be the onset for further Mn oxide production (Bargar et al. 2005). Soluble Mn(II) ions adsorb onto the surface of the microbially produced mineral, which functions as a solid catalyst and is thereafter oxidized. Mineral products of microbial Mn oxidation Previous research shows that the dominant initial phase formed during microbially mediated Mn oxidation is a poorly crystalline birnessite-type phyllomanganate with hexagonal symmetry (Villalobos et al. 2003; Hansel and Learman 2016). The microbial impact on the properties of these initial precipitates is still not well known but Mn oxides of biological origin and their natural and synthetic counterparts with hexagonal crystal symmetry differ from the more ordered triclinic birnessite variety (Villalobos et al. 2003). The main difference in symmetry is explained by dissimilarities in the cationic positions; whether they are filled by Mn ions (triclinic variety) or whether these positions are vacant (hexagonal variety) (Villalobos et al. 2003). Microbially mediated Mn precipitates are negatively charged because of cation vacancy sites as opposed to the abiotic triclinic variety where the negative charge is due to an exchange of higher valence Mn ions for lower valence Mn ions (Villalobos et al. 2003; Spiro et al. 2008; Kim et al. 2011). These dissimilarities are thought to affect reactivity (e.g. redox potential) and mechanisms for metal sequestration in the birnessite (Villalobos et al. 2003). It is shown that microbial Mn oxides are more reactive than their abiotic counterparts, because they generally display higher specific surface area (Nelson et al. 1999). Mn oxide precipitates are deposited extracellularly, on the cell walls or in the close vicinity of the cell or cellular structures; as a result the microbes commonly become encrusted in the precipitated Mn (Ghiorse 1984; Tebo et al. 2004, 2005; Nealson 2006; Santelli et al. 2011; Hansen and Learman 2016).

The microbial impact on the properties of initial precipitates is still not well known but Mn oxides produced with microbial involvement are commonly referred to as ‘biogenic Mn oxides’ (Tebo et al. 2004; Hansel and Learman 2016). This is a questionable nomenclature since biomineralization refers to a genetically controlled mineralization (Addadi and Weiner 1989). This process generally results in an organism-specific skeleton that, when fossilized, can be used to identify this organism and no abiotic processes could produce the same pattern. Biominerals in the fossil record provide evidence of life. This is not the case for microbially mediated Mn oxides. The term active or passive organomineralization is suggested to avoid ambiguity (e.g, Dupraz et al. 2009). However, nano- and microscale morphology and the emerging growth fabric of early Mn oxides can possibly form recognizable meso- and macroscale morphologies which have the potential to be used as biosignatures in the geologic record.

2.3 Accumulation of metals by Mn oxides

Mn oxides are strong sorbents of heavy metals owing to their high specific surface area and reactivity readiness (Nelson et al. 1999; Kay et al. 2001; Toner et al. 2006; Takahashi et al. 2007; Peña et al. 2010). Oxidation of soluble Mn into insoluble oxides (and concomitant co- precipitation or subsequent sorption of heavy metals) exerts extensive control on the distribution of heavy metals in the environment. Sorption is generally referred to as reaction processes between water and exposed solid phases (formation of complexes or chemical bonding by compounds in solution to an exposed solid surface). Three mechanisms, driven by different factors, are distinguishable: i) physical adsorption (diffuse nonspecific attraction caused by van der Waals forces and always present), ii) electrostatic interaction (ion exchange, species in solution attracted to solid surfaces of opposite surface charge) and iii) chemisorption (formation of surface complexes, chemical bonding to active sites on the solid surface). Two other processes are also often added in this context: iv) precipitation/co- precipitation (formation of a new phase on the surface or inside the solid matrix) and v) substitution (ions in solution exchange places with flexible ions in the solid sorbent) (Allard et al. 1980; Tewari 1981; Torstenfelt et al. 1982 Westall 1987).

Microbially mediated Mn precipitates are negatively charged as a result of cation vacancy sites (Villalobos et al. 2003; Spiro et al. 2008) and therefore attract and adsorb reduced Mn and other available trace elements. It is also shown that metal ions may be incorporated in the Mn mineral structure, either by substitution of Mn(III) or Mn(IV) or by filling an empty layer site in the crystal lattice (Kay et al. 2001; O’Reilly and Hochella 2003; Hansel and Learman 2016). Enrichment of REE and other metals in naturally occurring Mn oxides is a well- established phenomenon (Kolarik 1962; Piper 1974; Elderfield et al. 1981; Ingri and Pontér 1987; Kasten et al. 1998; Leybourne and Johannesson 2008).

Metal redistribution and mobility is largely dependent on complexation with both organic (cells and extracellular organic matter) and inorganic (e.g. cations, mineral phases/surfaces) ligands (Fein 2017). REE partitioning between stationary and suspended solid phases and species in solution is largely governed by the presence of natural organic acids (humics), carbonate levels and pH (Ohta and Kawabe 2001; Davranche et al. 2008; Tanaka et al. 2010; Ohnuki et al. 2015; Kraemer et al. 2017).

3. Materials and methods

3.1 Background

Recent investigations revealed that a tunnel leading to the main shaft of the former Ytterby mine (Sweden) hosts a black REE+Y enriched Mn deposit (Sjöberg 2012, 2014). The tunnel system is 400 m long and situated 29 m below ground surface and 5 m above Baltic Sea mean sea level. It was constructed to convert the former mine into a fuel deposit for the Swedish Armed Forces. The Ytterby black substance (YBS) precipitates from groundwater seeping from the rock wall in the unsaturated zone of the tunnel section and occurs in association with a 2-3 mm thick underlying blanket of CaCO3 (Sjöberg 2014). Water bearing fractures continuously supply water to the fully oxygenated tunnel environment, which holds a year round nearly constant temperature of 8°C. Artificial lighting is used 2-3 hrs/month during mine maintenance in the otherwise completely dark tunnel. The elemental content of the YBS (excluding oxygen, carbon and silicon) was 82% Mn, 13.5% Ca and 2±0.5% REE+Y, with all other metals being less than 2% in total. Phase analysis by X-ray diffraction (XRD) and elemental analyses show that the dominating phase is a birnessite-type Mn oxide with traces of organics and low Fe content. Carbon concentrations were about 1.8 %, one third of which was organic. EPR-spectroscopy and a lipid biomarker analysis indicated a microbial origin for the birnessite-type Mn oxides composing the YBS (Sjöberg 2014).

3.2 Sampling and characterization of the birnessite-type Mn oxide (paper I)

The elemental composition of the birnessite-type Mn oxide was determined by two additional analyses at two different laboratories. Samples were scraped off the deposit surface and stored at 4°C prior to analyses. Existing element data of the water systems were complemented with new analyses of major anions and cations, trace elements (including methods allowing for analysis of the whole series of REE) and DOC for all relevant waters, i.e. groundwater in the mine surroundings and fracture water. Water samples were collected from the fracture water, mine shaft and four groundwater locations surrounding the mine. A sequential extraction procedure was conducted in four steps using acidic leachates: Exchangeable, Reducible, Oxidizable and Residual fractions. In addition a single step extraction using an alkaline leachate was conducted in parallel: Humics (Analytical procedures are described in detail in paper I).

3.3 Microbial communities inhabiting the birnessite-type Mn oxides (paper II)

Characterization of the birnessite-type Mn oxide with respect to associated microbial communities was conducted by EPR-spectroscopy, lipid biomarker analysis and ESEM/cryo- SEM analyses. A molecular phylogenetic approach was adopted for assessment of the microbial communities inhabiting the birnessite-type Mn (extraction and quantification of DNA, cloning of the 16S rRNA gene of archaea and bacteria) and for identification of potential candidates for Mn oxidation. Fracture water and the Mn oxide precipitate YBS were sampled twice, during the winter 2015 and spring 2015, to assess possible seasonal differences in the community. Sterile tubes and spatulas were used and once at the laboratory, all samples were stored at -80°C prior to analyses. Procedures for characterization and analysis are described in detail in paper II.

4. Results and discussion

4.1 Composition of the birnessite-type Mn oxide (paper I)

An empirical formula of the Ytterby birnessite based on total element analyses can be 3+ 4+ assessed as: Mx(Mn ,Mn )2O4•(H2O)n with M = (0.37-0.41) Ca + 0.02 (REE+Y), 0.04 Mg and (0.02-0.03) other metals, and with [Mn3+]/[Mn4+] = 0.86-1.00 to achieve charge balance. This is in agreement with the proposed birnessite phase, as concluded from X-ray diffraction data. Association of REE with birnessite is well documented and usually described as a reversible pH-dependent sorption process (90% adsorption onto Mn oxides at pH above 7-8 and adsorption less than 10-15% at pH below 5-6) (Ohta and Kawabe 2001; Davranche et al. 2008; Pourret and Davranche 2013). The REE in the YBS, however, are firmly included in the birnessite structure even at pH as low as 1.5.

The sequential leaching provided an indication of how the trace elements and particularly the REE+Y are associated with the Mn oxide matrix. Sodium, magnesium, calcium and strontium were to a large extent released during the initial step (A1, exchangeable, pH 4). These elements are loosely attached to the YBS as adsorbates or present as carbonates and/or hydroxides (precipitates). The remaining fraction of these elements together with potassium and barium were released in step two (A2, reducible, pH 5) and three (A3, oxidizable, pH 2), which involve alteration or breakdown of the solid matrix; reducible and/or oxidizable compounds (oxides, sulfides, organic components etc). Aluminum, vanadium, iron, copper and zinc were solubilized during the last step (A4, residual, conc. acid), which involves complete dissolution of all matter but silicates. The REE were mainly released in the last step but also to some extent in A3, possibly in parallel with Mn. Mn was partially released in step A2, A3 and A4, which may indicate the presence of different mineral phases. None of the trace metals (copper, zinc, iron, vanadium) nor REE plus yttrium were merely adsorbed on outer surfaces of the Mn oxide. A single step alkaline leaching showed that none of the metals were present as humic bound species.

4.2 Indicators of microbial involvement in the production of the birnessite-type Mn oxide (paper I)

Electron paramagnetic resonance (EPR)-spectroscopy Results of electron paramagnetic resonance (EPR) spectroscopy, which was used in an attempt to determine whether the Mn oxides in the YBS are microbially mediated or abiotic precipitates, indicate that the YBS contains two or possibly more phases, with one having a microbial signature.

Lipid biomarkers The findings of molecular fragmentation patterns in lipids extracted from the studied Mn oxides revealed the presence of C31 to C35 extended side chain species of hopanoids, which is a distinct sign of bacterial presence.

Micromorphology Cross-sections of a Mn oxide microstructure frequently occuring in the YBS show signs of an iterative growth in the form of alternating light and dark laminae. These laminae mainly express variation in Mn and Ca concentrations but also in the Mn/Ca ratio. ESEM and cryo- SEM images show cell-like structures embedded in these microstructures and suggest that the cell-like shapes occur within specific laminae (Fig. 1c-d). Cryo-SEM imaging which allows for preservation of the 3D relationship in the hydrated sample also shows that the micro- and nanomorphology of the predominant Mn oxide is made of petal-like structures. These structures are similar to morphologies of birnessite-like Mn oxides which are shown to be a product of both bacterial (Nealson 2006) and fungal activity (Santelli et al. 2011; Yu et al. 2012) but also of in vitro laboratory experiments (Yin et al. 2012; Jingping et al. 2013) (Fig. 1a and b). Comparable structures are also observed in other underground environments but it is not known whether they are of biologic origin or purely abiotic (Frierdich et al. 2011). Images also show frequently occuring filaments of varying thickness.

Fig. 1. Scanning Electron Microscopy (SEM) images showing frequently observed microstructures in the YBS. Filaments of varying thickness covering microsperolitic/botryoidal Mn oxides (A). Morphology of the predominant microstructure showing petal-like structures (B). Cross-section of laminated Mn oxides showing cell-like structures embedded in the microsperolitic/botryoidal microstructure. The alternating light and dark layers mainly express variation in Mn and Ca concentrations but also in the Mn/Ca ratio (C and D). Cell-like structures embedded in microsperolitic/botryoidal microstructures suggesting that the cell-like shapes follow the internal laminae (E and F). Figures A, B, E and F are cryo-SEM images. Figures C and D are uncoated thin sections using low pressure ESEM.

4.3 Microbial communities inhabiting the YBS (paper II)

The molecular phylogenetic analysis reveals that extensive and complex microbial communities are associated with the YBS. Bacterial diversity is high and archaeal diversity low with Thaumarchaeota almost exclusively dominating the population. A newly identified Ytterby Nitrospira cluster closely affiliated with clones from ferromanganese environments and detection of a range of Thaumarchaeota sequences suggest a linkage between the Mn and nitrogen cycles. Clone libraries for both bacteria and archaea contain a high percentage of unknown genera that show considerable overlap with studies made in subsurface environments, cold climates, high metal content localities and sites associated with hydrocarbons or calcium carbonates. These data indicate a microbial ability to respond and adapt to the extreme environment in the YBS and thus a controlling role of extrinsic factors on microbial populations. Bacteria are more abundant compared to archaea in all samples. Both bacterial and archaeal abundances are up to four orders of magnitude higher in YBS than in fracture water.

4.4 Potential mechanisms for the formation of the birnessite-type Mn oxides (paper II)

The lack of sunlight forces the microbial community to derive energy from chemical reactions driven by inorganic or organic electron donors. Although chemolithotrophy using reduced Mn as electron donor would be a reasonable process to propose in the Ytterby system, all known Mn oxidizers up to now are heterotrophs that do not oxidize Mn for generation of energy (Ghiorse 1984; Hansel and Learman 2016). Lithotrophy may therefore not be a key microbial metabolic process in the YBS.

The main microbial mechanisms driving Mn oxidation in the Ytterby system likely involve direct (enzymatic) and indirect antioxidative (interactive with Reactive Oxygen Species (ROS)) processes, based on the identified potential Mn oxidizers and previous knowledge of these microbes. Hyphomicrobium and Pedomicrobium belong to the family Hyphomicrobiaceae, a group of hyphal budding bacteria that have shown the ability to oxidize Mn during chemoorganotrophic growth (Santelli et al. 2009). The metabolic moxA gene which encodes a multicopper oxidase (MCO) homolog is known to be responsible for Mn oxidation and laccase-like activity in Pedomicrobium species (Larsen et al. 1999; Ridge et al. 2007). The mechanism of Mn oxidation involves a two-step process in which negatively charged extracellular polymeric substances (EPS) scavenge reduced Mn that is then oxidized by Mn oxidizing bacteria (Ghiorse 1980). This process implies that microbial exudates serve as nucleation sites for the formation of the birnessite-type Mn oxides. The newly identified Ytterby Bacteroidetes cluster most closely related to the Terrimonas, represent other plausible candidates of Mn oxidation. The various factors (intrinsic and extrinsic) that govern the Mn redox cycle in the Ytterby system are illustrated in Fig. 2.

Fig. 2. Schematic of Mn redox cycle in the Ytterby mine

Findings of bacterial clones related to known species capable of Mn oxidation show the potential of microbial mediation in the deposition of the birnessite-type Mn oxides. Potential candidates are mainly found within the ferromanganese genera Hyphomicrobium and Pedomicrobium, and a newly identified Ytterby Bacteroidetes cluster most closely related to the Terrimonas. The newly identified Bacteroidetes cluster is particularly interesting. It is composed of six operational taxonomic units (OTUs, defined on 97% sequence similarity) representing 52 out of a total of 359 clones. The most abundant of these OTUs (also most abundant within the clone library) showed only 94% similarity to the closest cultivated strain Terrimonas ferruginea DSM 30193 (NR_042494, formerly named Flavobacterium ferrugineum (Xie and Yokota 2006). This cluster seems to be specific for the Ytterby mine environment and is likely involved in the Mn oxidation and may also be adapted to or suited for the specific environmental conditions in the tunnels: absence of light, high metal content, low temperature and possibly the presence of hydrocarbons. Hyphomicrobium and Pedomicrobium (notably P.manganicum) are known to be capable of Mn oxidation and observed in a range of Fe- and Mn-rich environments (Tyler and Marshall 1967; Tyler 1970; Ghiorse 1984; Northup et al. 2003; Spilde et al. 2005; Hansel and Learman 2016).

4.5 Redistribution of REE and enrichment in the birnessite-type Mn oxide in the Ytterby mine (paper I) REE patterns in water are largely the result of releases from rocks and should therefore be analyzed in relation to rocks from the site. The fracture water (GW-F) is enriched in HREE compared to MREE and LREE ([Lu/Tb]N = 2.2; [Lu/La]N = 8.0) when compared to a mean rock composition. A depletion of both europium and cerium is visible when fracture water is normalized to mean rock (Eu/Eu* = 0.36; Ce/Ce* = 0.20). The Europium depletion indicates less strong release of europium compared to other REE from the rocks to water. The negative cerium anomaly is likely associated to co-precipitation of cerium by secondary phases such as 3+ 4+ 2+ the YBS-birnessite. Oxidation of Ce to Ce (CeO2 ) by MnO2 during the oxidation of Mn to Mn3+ - Mn4+, often referred to as oxidative scavenging of cerium by Mn oxides, is observed in various environments (e.g., Piper 1974; Ohta and Kawabe, 2001; Neaman et al., 2004; Leybourne and Johannesson, 2008) and leads to an accumulation of cerium in Mn phases.

Explaining the high REE+Y concentrations in the YBS is however more complex than expected at the place known for the discovery of yttrium, scandium and five of the REE. Neither REE+Y nor Mn concentrations in the fracture water from which the YBS precipitate are high but rather consistent with average concentrations in regional groundwater. It is the remarkable enrichment in the Mn precipitates relative to the fracture water that makes the observed process probably unique. Concentrations of Ca, Na and REE in the fracture water are 68, 34 and 0.0027 mg/L, respectively, and Mn 0.003 mg/L. The strong preference for the trivalent REE in comparison with Ca and Na, with similar effective ionic radii (coordination number 6), is indicated by concentration ratios between the solid YBS and fracture water, which is about three and five orders of magnitude higher for REE+Y than for Ca and Na, respectively. Comparable water concentrations are found in other places but do not result in such accumulations. One possibility is that REE+Y and Mn generally exist as colloids and/or larger suspended particles in the fracture water and not as dissolved (≤ 0.22µm) ions. However, as the hypothesis is that the REE+Y are incorporated in the mineral structure rather than adsorbed on the surface of the birnessite-type Mn oxides, this implies that the REE exist as free ions and not complexes in the Ytterby water. It is also possible that the nearby (about 300m distance) previous storage of apolar hydrocarbons, play a role in the accumulation of Mn and REE+Y (in combination with environmental constraints of the mine; dark, cold, oxygenated). Ongoing analyses of metal exchangeability in the birnessite structure point towards exchangeability of the charge balancing elements (REE+Y, Ca, Na and Mn) and a preference for the trivalent REE over divalent and monovalent metals (Allard et al. 2017). The enrichment is clearly lower for the metals partially leached in the initial leaching step than for the other elements.

4.6 A metal stressed microbial community?

The REE+Y are not associated with the organics or biomass in the YBS as shown by leaching experiments, and the microbial inhabitants appear to dispatch metals that are toxic to them into the birnessite-type Mn oxides. The sequestering of metals in the mineral structure may therefore help to reduce the potential bio-availability and thus toxicity of metals (including Mn) to the microbial community (paper II). The production of the birnessite-type Mn oxides is likely microbially induced but the high enrichment of REE in the birnessite lattice may rather reflect a physicochemical reaction. Also, microbial biofilms compared to planktonic cells (fast growing populations of solitary cells) are shown to be less susceptible to metal stress (Harrison et al. 2007). A proposed explanation for this multi-metal resistance or tolerance is the formation of multiple cell types, phenotypic variants, during biofilm growth which is characterized by close proximity, tight metabolic coupling and possible consortia that could help the indirect activation of shared genetic and biochemical pathways used in resistance to toxic metals (Harrison et al. 2007 and references therein). Taken together, these results indicate that the shallow subsurface microbial habitat in the Ytterby mine tunnels is able to respond and adapt to local conditions including stressors such as high metal content.

5. Conclusions

Detailed studies of the precipitate YBS in the Ytterby mine system, as described in paper I and II, lead to the following conclusions and answers to the stated objectives:

 Formation of secondary Mn oxides exceptionally enriched in rare earth elements is established. Concentrations are among the highest observed in secondary ferromanganese precipitates in nature.

 The main component is a birnessite-type Mn oxide, enriched with REE which constitute 1% of the dry mass and 2% of the metal content. The REE are firmly included in the mineral structure even at pH as low as 1.5. An empirical formula based 3+ 4+ on total element analyses can be assessed as, Mx(Mn ,Mn )2O4•(H2O)n with M = (0.37-0.41) Ca + 0.02 (REE+Y), 0.04 Mg and (0.02-0.03) other metals, and with [Mn3+]/[Mn4+] = 0.86-1.00 to achieve charge balance.

 A strong preference for the trivalent REE over divalent and monovalent metals is indicated by Mn oxide precipitate to water concentration ratios.

 The influence of microorganisms in the accumulation of these Mn oxides is demonstrated by EPR-spectroscopy analysis, lipid biomarker analysis and possible cell-like structures shown by ESEM and cryo-SEM images.

 Q-PCR analyses (targeting the bacterial and archaeal 16S rRNA genes in the YBS and the fracture water) indicated that both bacterial and archaeal abundance were up to four orders of magnitude higher in the YBS samples compared to the fracture water samples. Bacteria were dominant with respect to archaea in both the YBS and fracture water, winter and spring samples. The total number of bacteria in the YBS was on the order 1010 cells per g YBS, while the water feeding the fracture was on the order of 106 cells per mL groundwater. The corresponding numbers for archaea were on the order 109 cells per g YBS and 105 cells per mL groundwater. the bacterial and archaeal 16S rRNA genes.

 The detected microorganisms are related to the environmental constraints of the site including the low constant temperature (8ºC), the absence of light, and especially, the high metal content (including REE). This point towards a microbial ability to respond and adapt to this extreme environment.

 Identification of microbial communities inhabiting the Ytterby Mn oxide precipitates and the fracture water revealed the presence of known species capable of Mn oxidation (i.e. Hyphomicrobium spp., Pedomicrobium manganicum, Reyranella soli and Microbacterium pumilum) and a newly identified cluster within the Bacteroidetes likely capable of Mn oxidation and adapted to the environmental constraints in the Ytterby system. 6. Future research areas

Interaction between microbes, EOM and physicochemical parameters in the early formation of the birnessite-type Mn oxides The next step is to conduct further analyses to increase knowledge of the interaction between microbes, extracellular organic matter (EOM) and physicochemical parameters in early precipitation of birnessite-type Mn oxides in the Ytterby mine. Negatively charged EOM has been shown to scavenge and bind reduced Mn, favoring formation of Mn oxides (Ghiorse 1984 and references therein). The EOM binding capacity may thus be an important controlling factor for production of Mn oxides and the location where initial precipitation occurs. The goal is to understand where the initial precipitation takes place and its association with EOM and microbes. Cultivation and isolation experiments are started and will be followed by high resolution microscopy and more molecular work. The intention is to retrieve genomic data for the cultivated bacteria (and possibly fungi) associated with Mn precipitates via single cell genomics (SCG). For the natural samples a technique involving metagenomics binning may be used. By using a method of rapid freezing, Cryo-TEM allows a non- destructive approach preserving the 3D relationship between microbes, EOM and mineral products (birnessite-type Mn oxides) in the highly hydrated biofilm and cultures. Possibly interfering (or diagnostic) autofluorescence of both organic and inorganic particles in the samples have to be investigated.

Organomineralization experiments

Experiments including MnO2, calcium carbonates (CaCO3) and REE will be conducted. The purpose is to look at REE composition as a possible marker in EOM-induced precipitation of Mn oxides and microbial carbonates. Possible morphological imprints on the EOM-induced mineral product will also be investigated (with and without REE). REE have been widely used as tracers in palaeoceanography (e.g. Henderson 1984). However, processes including sorption to inorganic and organic substances may cause REE fractionation (Ingri et al. 2000; Taylor and McLennan 1988). Organic matter plays a critical role in controlling REE speciation in aquatic environments (Davranche et al. 2008). The bacterial role in REE fractionation indicates that bacterial walls have a specific affinity of certain heavy REE (HREE). No study has been done on REE binding by EOM. Acidic bounds in EOM may complex REE thereby producing biosignatures in the nucleating minerals. Various modes of EPS mineralization may also show different REE signatures.

Mn and trace metal speciation in YBS To understand the mechanisms responsible for the sorption of REE onto the birnesssite-type Mn oxides, Mn and REE speciation needs to be further investigated. Possible methods would include X-ray microdiffraction and/or synchrotron based X-ray absorption spectroscopy (XAS) with extended X-ray absorption fine structure (EXAFS). The cations, that balance charge in the birnessite (Ca, Na, as well as REE etc.), are exchangeable, and the proportions of these metals reflect the concentrations in the water phase, and possibly also the speciation in the water phase. Also Mn(III) in the structure may be exchanged by Fe(III) since ionic radii are similar and the charges identical. Effects of exchanges of cations on the birnessite structure should be further demonstrated and quantified.

Acknowledgments

First of all, I would like to thank my supervisor Christophe Dupraz for giving me the chance to continue working in the fascinating Ytterby mine environment and for introducing me into the field of geomicrobiology and microbial geochemistry and Bert Allard for his endless support and for sharing knowledge during invaluable science discussions. I also want to thank my co-supervisor Rienk Smittenberg for keeping me on track during my time as a PhD student. A huge thank you to my dear friends Martin Lundmark (The Swedish Fortifications Agency) and P-O Lindgren (The Swedish Defence) for their generosity in letting me visit the Ytterby mine as often as necessary and for sharing stories and data on this unique locality. I thank Nolwenn Callac for sharing her skills in microbiology and guiding me through the molecular work presented in paper II. This work would not have been possible without her. My other co-authors are thanked for their valuable assistance in analyses and inputs on the papers. I am grateful to Hildred Crill for help to improve my scientific writing, Patrick Crill and Eve Arnold for always having a minute and for solid advice and Barbara Wohlfarth for teaching inspiration and guidance. Dan Zetterberg for his fine work making thin sections, Marianne Ahlbom, Heike Siegmund and Malin Söderman for their assistance with ESEM and isotope analysis, respectively. Joakim Mansfeld for advice and input on rocks in the Ytterby area, Anna Neubeck for advice and support regarding sampling and cultivations, Pär Hjelmquist for advice and assistance in water analyses, Håkan Gustafsson (University of Linköping) for conducting the EPR analysis and for providing valuable knowledge in this field. A big thank you goes to Jonas Fredriksson for helping out in the lab and to Rolf Hallberg who supports and guides me through the challenges involved in the art of cultivating, isolating and visualizing our Mn oxidizers. References

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