Manganese (Mn) oxides are ubiquitous in nature and occur as both biological and abiotic minerals, but empirically distinguishing between the two remains a problem. Recently, electron paramagnetic resonance (EPR) spectroscopy has been proposed for this purpose. It has been reported that biogenic Mn oxides display a characteristic narrow linewidth in contrast to their pure abiotic counterparts, which is explained in part by the large number of cation vacancies that form within the layers of biogenic Mn oxides. It was, therefore, proposed that natural samples that display a narrow EPR linewidth, ΔH_pp < 580G, could be assigned to a biogenic origin. However, in poorly crystalline or amorphous solids, both dipolar broadening and exchange narrowing simultaneously determine the linewidth. Considering that the spectral linewidth is governed by several mechanisms, this approach might be questioned. In this study, we report synthetic chemical garden Mn oxide biomorphs that exhibit both morphologically life-like structures and narrow EPR linewidths, suggesting that a narrow EPR line may be unsuitable as reliable evidence in assessment of biogenicity.  

Dissolved inorganic carbon (DIC) levels in freshwaters play a key role in the equilibrium of the carbon cycle between the atmosphere, water and living beings. Standard classical methods for DIC determination generally involve bulky and expensive equipment used in centralized laboratories, resulting in time-consuming processes that do not allow for adequate monitoring in the field. In order to address this challenge, we have developed a distance-based paper analytical device (PAD) for on-site determination of DIC in water. The portable, cost-effective and easy-to-use device was based on the miniaturization and integration of a classical acid-base colorimetric titration on a paper channel, enabling an accurate determination of DIC in less than 20 min. The length of the blue colored line in the detection channel after being filled with the sample was related to the DIC concentration in the sample. The reagent solution used to modify the titration channel was optimized so that DIC concentrations in the range 50–1000 mg L⁻¹ could be measured. The long-term stability of the paper-based device was also evaluated, demonstrating a working stability for more than 70 days after their fabrication, an important characteristic for in-the-field analysis. Finally, the PAD was validated with different water samples, i.e. tap water, commercial bottled drinking water and water samples from a mine, with excellent agreement between the results obtained from the PAD and the standard method. This demonstrates the high potential of the proposed paper analytical device to quantify DIC in situ by minimally-trained personnel without the need for peripheral equipment, which represents an important advance compared to the current limited analysis systems.

A black substance exuding from fractures was observed in 2012 in Ytterby mine, Sweden, and identified in 2017 as birnessite with the composition M_x[Mn(III,IV)]₂O₄∙(H₂O)_n. M is usually calcium and sodium, with x around 0.5. The Ytterby birnessite is unique, with M being calcium, magnesium, and also rare earth elements (REEs) constituting up to 2% of the total metal content. The biogenic origin of the birnessite was established in 2018. Analysis of the microbial processes leading to the birnessite formation and the REE enrichment has continued since then. The process is fast and dynamic, as indicated by the depletion of manganese and of REE and other metals in the fracture water during the passage over the precipitation zone in the mine tunnel. Studies of the exchangeability of metals in the structure are the main objective of the present program. Exposure to solutions of sodium, calcium, lanthanum, and iron led to exchanges and altered distribution of the metals in the birnessite, however, generating phases with almost identical structures after the exchanges, and no new mineral phases were detected. Exchangeability was more efficient for trivalent elements (REE) over divalent (calcium) and monovalent (sodium) elements of a similar size (ionic radii 90–100 pm).

Manganese oxides occur in a wide range of environmental settings either as coatings on rocks, sediment, and soil particles, or as discrete grains. Although the production of biologically mediated Mn oxides is well established, relatively little is known about microbial-specific strategies for utilizing Mn in the environment and how these affect the morphology, structure, and chemistry of associated mineralizations. Defining such strategies and characterizing the associated mineral properties would contribute to a better understanding of their impact on the local environment and possibly facilitate evaluation of biogenicity in recent and past Mn accumulations. Here, we supplement field data from a Mn rock wall deposit in the Ytterby mine, Sweden, with data retrieved from culturing Mn oxidizers isolated from this site. Microscopic and spectroscopic techniques are used to characterize field site products and Mn precipitates generated by four isolated bacteria (Hydrogenophaga sp., Pedobacter sp., Rhizobium sp., and Nevskia sp.) and one fungal-bacterial co-culture (Cladosporium sp.—Hydrogenophaga sp. Rhizobium sp.—Nevskia sp.). Two of the isolates (Pedobacter sp. and Nevskia sp.) are previously unknown Mn oxidizers. At the field site, the onset of Mn oxide mineralization typically occurs in areas associated with globular wad-like particles and microbial traces. The particles serve as building blocks in the majority of the microstructures, either forming the base for further growth into laminated dendrites-botryoids or added as components to an existing structure. The most common nanoscale structures are networks of Mn oxide sheets structurally related to birnessite. The sheets are typically constructed of very few layers and elongated along the octahedral chains. In places, the sheets bend and curl under to give a scroll-like appearance. Culturing experiments show that growth conditions (biofilm or planktonic) affect the ability to oxidize Mn and that taxonomic affiliation influences crystallite size, structure, and average oxidation state as well as the onset location of Mn precipitation.

Microbial mats or biofilms are known to colonize a wide range of substrates in aquatic environments. These dense benthic communities efficiently recycle nutrients and often exhibit high tolerance to environmental stressors, characteristics that enable them to inhabit harsh ecological niches. In some special cases, floating biofilms form at the air-water interface residing on top of a hydrophobic microlayer. Here, we describe biofilms that reside at the air-air interface by forming gas bubbles (bubble biofilms) in the former Ytterby mine, Sweden. The bubbles are built by micrometer thick membrane-like biofilm that holds enough water to sustain microbial activity. Molecular identification shows that the biofilm communities are dominated by the neuston bacterium Nevskia. Gas bubbles contain mostly air with a slightly elevated concentration of carbon dioxide. Biofilm formation and development was monitored in situ using a time-lapse camera over one year, taking one image every second hour. The bubbles were stable over long periods of time (weeks, even months) and gas build-up occurred in pulses as if the bedrock suddenly exhaled. The result was however not a passive inflation of a dying biofilm becoming more fragile with time (as a result of overstretching of the organic material). To the contrary, microbial growth lead to a more robust, hydrophobic bubble biofilm that kept the bubbles inflated for extended periods (several weeks, and in some cases even months).

Microbe-mediated precipitation of Mn-oxides enriched in rare earth elements (REE) and other trace elements was discovered in tunnels leading to the main shaft of the Ytterby mine, Sweden. Defining the spatial distribution of microorganisms and elements in this ecosystem provide a better understanding of specific niches and parameters driving the emergence of these communities and associated mineral precipitates. Along with elemental analyses, high-throughput sequencing of the following four subsystems were conducted: (i) water seeping from a rock fracture into the tunnel, (ii) Mn-oxides and associated biofilm; referred to as the Ytterby Black Substance (YBS) biofilm (iii) biofilm forming bubbles on the Mn-oxides; referred to as the bubble biofilm and (iv) fracture water that has passed through the biofilms. Each subsystem hosts a specific collection of microorganisms. Differentially abundant bacteria in the YBS biofilm were identified within the Rhizobiales (e.g. Pedomicrobium), PLTA13 Gammaproteobacteria, Pirellulaceae, Hyphomonadaceae, Blastocatellia and Nitrospira. These taxa, likely driving the Mn-oxide production, were not detected in the fracture water. This biofilm binds Mn, REE and other trace elements in an efficient, dynamic process, as indicated by substantial depletion of these metals from the fracture water as it passes through the Mn deposit zone. Microbe-mediated oxidation of Mn(II) and formation of Mn(III/IV)-oxides can thus have considerable local environmental impact by removing metals from aquatic environments.

Microorganisms are able to manipulate redox reactions and thus exert extensive control on chemical speciation and element partitioning in nature, affecting the formation and dissolution of certain minerals. One of these redox active elements is manganese (Mn), which in its oxidized states (III/IV), commonly forms Mn oxide-hydroxide minerals. A microbially mediated birnessite-type Mn oxide enriched in yttrium (Y) and rare earth elements (REE) has been studied in our research. The YREE-enriched birnessite was found in a tunnel to the main shaft of the former Ytterby mine in Sweden, well known as the place of discovery of scandium, yttrium, tantalum and five of the REEs. The thesis aims to define preconditions and processes leading to the formation of this Ytterby birnessite, with particular focus on microbial involvement and the potentially promoting role of biofilms. Dynamics and mineral products of the natural system are studied in combination with analyses of Mn phases produced in vitro by microbes isolated from this system. In addition, the nature of the YREE association with the birnessite-type Mn oxides is investigated.

Natural birnessite has the composition M_x(Mn³⁺, Mn⁴⁺)₂O₄•(H₂O)_n  with M ususally being (Na,Ca) and x=0.5. An empirical formula based on element analyses for the Ytterby birnessite has been assessed as M = (Ca_0,37-0,41YREE_0.02Mg_0.04Other metals_0.02-0.03), with [Mn³⁺]/[Mn⁴⁺] = 0.86-1.00 to achieve charge balance. We find that there is a preference for the trivalent YREEs over divalent and monovalent metals. There is also a preferential uptake of light rare earth elements (LREE) relative to heavy rare earth elements (HREE), likely due to mineralogical preferences for charge and ionic radius. The YREEs are strongly bound to the mineral structure and not merely adsorbed on the surface. The Mn deposit subsystems (fracture water, Mn oxide precipitating biofilm and bubble biofilm) are phylogenetically significantly different and the microbial community composition of the feeding water has little impact on the derived biofilms. The signature microbial groups of the Mn oxide producing biofilm Rhizobiales (e.g., Pedomicrobium), PLTA13 Gammaproteobacteria, Pirellulaceae, Blastocatellia and Nitrospira are adapted to the specific characteristics of the biofilm: an emerging extreme environment (low temperature, no light, high metal concentration) which is in part generated by the biofilm components themselves. Known Mn oxidizers are identified among these niched microbial groups and four of the isolated bacterial species (Hydrogenophaga sp., Pedobacter sp., Rhizobium sp. and Nevskia sp.) as well as one fungal species (Cladosporium sp.) are involved in Mn oxide production. Hydrogenophaga sp. and Pedobacter sp. produce Mn oxides independently while results imply a synergistic relationship between Rhizobium sp. and selected species. Members of the Pedobacter and Nevskia genera are previously not known to oxidize Mn. Microstructural characterizaton show that the growth pattern of the birnessite-type Mn oxides is either dendritic/shrublike or spherulitic/botryoidal. Nucleation takes place in close association to the biofilm and initial Mn precipitates are observed at different locations depending on the mediating species. Encrustations of cells and other organic structures by Mn precipitates serve as stable nuclei for further growth. The close relationship appears to decrease in importance as the aggregates of poorly crystalline precipitates grow. In the more developed crystals, a repetitive pattern, Liesegang-type of rings, suggests that abiotic factors take over.

The dominant initial phase formed during microbially mediated manganese oxidation is a poorly crystalline birnessite-type phyllomanganate. The occurrence of manganese deposits containing this mineral is of interest for increased understanding of microbial involvement in the manganese cycle. A culture independent molecular approach is used as a first step to investigate the role of microorganisms in forming rare earth element enriched birnessite-type manganese oxides, associated with water bearing rock fractures in a tunnel of the Ytterby mine, Sweden. 16S rRNA gene results show that the chemotrophic bacterial communities are diverse and include a high percentage of uncultured unclassified bacteria while archaeal diversity is low with Thaumarchaeota almost exclusively dominating the population. Ytterby clones are frequently most similar to clones isolated from subsurface environments, low temperature milieus and/or settings rich in metals. Overall, bacteria are dominant compared to archaea. Both bacterial and archaeal abundances are up to four orders of magnitude higher in manganese samples than in fracture water. Potential players in the manganese cycling are mainly found within the ferromanganese genera Hyphomicrobium and Pedomicrobium, and a group of Bacteroidetes sequences that cluster within an uncultured novel genus most closely related to the Terrimonas. This study strongly suggest that the production of the YBS deposit is microbially mediated.

Microbes exert extensive control on redox element cycles. They participate directly orindirectly in the concentration and fractionation of elements by influencing the partitioningbetween soluble and insoluble species. Putative microbially mediated manganese (Mn) oxidesof the birnessite-type, enriched in rare earth elements (REE) + yttrium (Y) were recentlyfound in the Ytterby mine, Sweden. A poorly crystalline birnessite-type phyllomanganate isregarded as the predominant initial phase formed during microbial Mn oxidation. Owing to ahigher specific surface area, this biomineral also enhances the known sorption property of Mnoxides with respect to heavy metals (e.g. REE) and therefore has considerable environmentalimpact.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 precipitateswith Mn and/or iron. Sequential extraction provides evidence of a mineral structure where theREE+Y are firmly included, even at pH as low as 1.5. Concentration ratios of Mn oxideprecipitates to fracture water indicate a strong preference for the trivalent REE+Y overdivalent and monovalent metals. A culture independent molecular phylogenetic approach wasadopted as a first step to analyze the processes that microbes mediate in this environment andspecifically how the microbial communities interact with the Mn oxides. Plausible players inthe formation of the investigated birnessite-type Mn oxides are mainly found within theferromanganese genera Hyphomicrobium and Pedomicrobium and a newly identified YtterbyBacteroidetes cluster most closely related to the Terrimonas. Data also indicate that thedetected microorganisms are related to the environmental constraints of the site including lowconstant temperature (8°C), absence of light, high metal content and possibly proximity to theformer storage of petroleum products.

Characterization of a black substance exuding from fractured bedrock in a subterranean tunnel revealed a secondary manganese oxide mineralisation exceptionally enriched in rare earth elements (REE). Concentrations are among the highest observed in secondary ferromanganese precipitates in nature. The tunnel is located in the unsaturated zone at shallow depth in the former Ytterby mine, known for the discovery of yttrium, scandium, tantalum and five rare earth elements.

Elemental analysis and X-ray diffraction of the black substance establish that the main component is a manganese oxide of the birnessite type. Minor fractions of calcite, other manganese oxides, feldspars, quartz and about 1% organic matter were also found, but no iron oxides were identified. The Ytterby birnessite contains REE, as well as calcium, magnesium and traces of other metals. The REE, which constitute 1% of the dry mass and 2% of the metal content, are firmly included in the mineral structure and are not released by leaching at pH 1.5 or higher. A strong preference for the trivalent REE over divalent and monovalent metals is indicated by concentration ratios of the substance to fracture water. The REE-enriched birnessite has the general formula Mx(Mn3+,Mn4+)(2)O-4 center dot(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.

The influence of microorganisms on the accumulation of this REE enriched substance is demonstrated by electron paramagnetic resonance spectroscopy. Results show that it is composed of two or more manganese phases, one of which has a biogenic signature. In addition, the occurrence of C-31 to C-35 extended side chain hopanoids among the identified lipid biomarkers combined with the absence of ergosterol, a fungal lipid biomarker, indicate that the in-situ microbial community is bacterial rather than fungal.