Pyrite formation in modern stromatolite at hot spring in Fukiagesawa, Osaki City, Miyagi, Japan

Geothermal surface alteration of basalts, Krýsuvík Iceland—Alteration mineralogy, water chemistry and the effects of acid supply on the alteration process

The microbial sulfur cycle plays an important role in acidic environments such as acid mine drainage (AMD) and acidic volcanic hot pools. The reduction of inorganic sulfur compounds such as sulfate (SO42-) and elemental sulfur (S80) results in the production of sulfide (H2S) (biosulfidogenesis). H2S reacts with dissolved metals to form metal sulfides, which precipitate out of solution and thereby lower metal concentrations in AMD environments. In addition, at low pH biosulfidogenesis from SO42- is proton-consuming, mitigating the acidity. (Bio)sulfidogenesis furthermore is an attractive technology for selective recovery of metals as metal sulfides from mining and metallurgy process waters. In industry this has been applied predominantly with neutrophilic sulfate-reducing bacteria (SRB). Process economics can be improved, however, by using S80 instead of SO42- as electron acceptor, as this enables a fourfold decrease in electron donor requirements. The use of S80 could also enable operation at more acidic pH and higher temperatures, as so far no extremely acidophilic (pHopt < 3.0) SRB are known, while multiple (thermo)acidophilic S80-reducing bacteria and archaea have been described. Biosulfidogenesis at (thermo)acidophilic conditions can further reduce process costs by enabling the combination of sulfidogenesis and metal precipitation from AMD or hot acidic metallurgy process waters in one reactor. In this thesis we investigated sulfidogenesis from S80 and SO42- at acidic pH and low to high temperature, focusing on laboratory-scale processes for application (chapter 2 and 3), and bioremediation of AMD-impacted environments (chapter 4 and 5)—acidic mine pit lakes and AMD sediments.In chapter 2 we describe a novel abiotic process for sulfidogenesis from S80 and H2, mediated by catalytic pyrite. The catalytic properties of pyrite likely are related to their size, since both pyrite formed in situ from the S80, H2S and Fe2+ present in the medium, and externally sourced, milled pyrite mediated sulfidogenesis from H2 and S80. The process was investigated in detail at pH 4, 80°C, but also occurred at lower temperatures (40 °C) and higher pH (pH 6). A combination of mineralogical techniques showed spherical pyrite particles composed of acicular pyrite nanocrystals. The identification of pyrrhotite nanocrystals suggested this was an intermediate in pyrite formation in these incubations. Yeast extract (YE) lengthened the lag phase preceding sulfidogenesis, likely through limiting pyrite formation, and appeared to result in less organized FeS2 nanocrystals on the surface of the pyrite spheroids. Spherical pyrite particles formed both in the presence and absence of YE, countering the hypothesis that spherical pyrite is a proxy for the presence of organic matter. Based on the absence of H2 production during pyrite formation, the presence of excess S80 and geochemical modeling, we propose that pyrite formation in our incubations occurs via the polysulfide pathway, which was unexpected at acidic pH.In chapter 3 we investigated S80 reduction at lower temperatures (30°C) at neutral (6.9) and acidic (3.8) pH in a continuous laboratory-scale process, using H2/CO2 as electron donor and carbon source, and industrial mesophilic granular sludge as inoculum (Emmtec, The Netherlands). We assessed the effect of pH on VSPR and the microbial community composition. The steady-state VSPR dropped 2.3-fold upon transition to acidic pH, from 1.79 ± 0.18 to 0.71 ± 0.07 g S2-·L-1·d-1. Our results indicate that VSPR from S80 at acidic pH are limited by S80 bioavailability rather than system limitations. With regards to temperature, VSPR achieved here at acidic pH were higher than those obtained with a similar system configuration at higher temperatures and acidic pH (pH 3.5, 60 °C), suggesting that the inoculum used in our study is not suitable for high temperature processes. Microbial community analysis of the reactor at neutral and acidic pH via 16S rRNA gene amplicon sequencing showed that at pH 6.9, Sulfurospirillum, Sulfurovum and Desulfurella were the most abundant S80-reducers, while at pH 3.9, Desulfurella dominated the sequenced reads. The detection of acetic acid and the abundance of Acetobacterium at pH 6.9 pointed towards acetogenesis, explaining the dominance of the heterotrophic genus Sulfurospirillum in this H2 and CO2–fed bioreactor.In chapter 4 we investigated biosulfidogenesis in the water column of two acidic pit lakes (APL) in the Iberian Pyrite Belt (IPB), Filón Centro (Tharsis) (FC) and La Zarza (LZ). In both APL a natural attenuation of acidity and toxic metal concentrations occurred towards the lake bottom, which was more pronounced in FC. The detection of Cu and Zn sulfides in the monimolimnion of FC indicated the presence of biogenic sulfide. This was supported by the detection of sulfidogenic taxa in the 16S rRNA gene amplicon sequence reads from the monimolimnion of both APL. Metal sulfide precipitation was likely mediated by biosulfidogenesis from S80 reduction and disproportionation in the younger APL (LZ), as indicated by the abundance of S80-reducing Acidianus and -disproportionating Desulfocapsa in the sequenced reads. In the older APL (FC), SO42- reduction became dominant, indicated by the abundance of reads assigned to the putative SRB Desulfomonile (58 %). The water columns of these APL provide fewer opportunities for the formation of protective microenvironments than AMD sediments, and the species detected in these APL are therefore likely more resistant to the poly-extreme conditions, making them of great interest for application in bioremediation technologies.In chapter 5 we describe a novel species from a novel genus of acidotolerant SRB, Acididesulfobacillus acetoxydans, previously isolated from AMD sediment. We investigated the dominant acid stress resistance mechanisms enabling this species to grow at low pH. Of great interest was its capacity to completely oxidize organic acids to CO2. This is uncommon among the moderately acidophilic SRB described so far, and likely provides an important mechanism to cope with acid stress. Comparative proteogenomic and membrane lipid analysis further indicated that the presence of saturated ether-bound lipids in the membrane, and their relative increase at lower pH, could be a protection mechanism against acid stress, while a canonical acid stress resistance mechanisms such as a Donnan potential and increased active charge transport did not appear to be active.

We investigated bacterial and archaeal communities along an ice-fed surficial hot spring at Kverkfjöll volcano-a partially ice-covered basaltic volcano at Vatnajökull glacier, Iceland, using biomolecular (16S rRNA, apsA, mcrA, amoA, nifH genes) and stable isotope techniques. The hot spring environment is characterized by high temperatures and low dissolved oxygen concentrations at the source (68°C and <1mg/L (±0.1%)) changing to lower temperatures and higher dissolved oxygen downstream (34.7°C and 5.9mg/L), with sulfate the dominant anion (225mg/L at the source). Sediments are comprised of detrital basalt, low-temperature alteration phases and pyrite, with <0.4 wt. % total organic carbon (TOC). 16S rRNA gene profiles reveal that organisms affiliated with Hydrogenobaculum (54%-87% bacterial population) and Thermoproteales (35%-63% archaeal population) dominate the micro-oxic hot spring source, while sulfur-oxidizing archaea (Sulfolobales, 57%-82%), and putative sulfur-oxidizing and heterotrophic bacterial groups dominate oxic downstream environments. The δ13 Corg (‰ V-PDB) values for sediment TOC and microbial biomass range from -9.4‰ at the spring's source decreasing to -12.6‰ downstream. A reverse effect isotope fractionation of ~3‰ between sediment sulfide (δ34 S ~0‰) and dissolved water sulfate (δ34 S +3.2‰), and δ18 O values of ~ -5.3‰ suggest pyrite forms abiogenically from volcanic sulfide, followed by abiogenic and microbial oxidation. These environments represent an unexplored surficial geothermal environment analogous to transient volcanogenic habitats during putative "snowball Earth" scenarios and volcano-ice geothermal environments on Mars.

A comprehensive research of water and cyanobacterial mats in Mogoysky and Shurindinsky thermal springs (Baikal rift zone) was carried out by hydrochemical, chemical, microbiological, and mineralogical methods. Detailed descriptions of the springs location and their characteristics were given. According to their chemical composition, the springs were classified as fluoride-bicarbonate (Mogoysky) and bicarbonatesulfate (Shurindinsky) types with a high concentration of fluorine. This is explained by the interaction of infiltration waters with embedding rocks. A wide diversity of cyanobacteria (14 species of 7 genera) was revealed in the investigated springs. The development of cyanobacteria in microbial mats was observed at water outflows at the temperatures of 37.8 to 76.6°C. Chlorophyll a was the predominant pigment in microbial mats of the studied springs, indicating predominance of cyanobacteria in the mat. Deposition of various pyrite forms, celestite (SrSO4), fluorite (CaF2), calcium carbonate, elemental sulfur, barite, and amorphous silica was found in microbial mats.

The Vatukoula caldera is semi-elliptical in shape with the long axis trending north-easterly and occupies about 14 square miles of an undulating topographical basin located near the central north coast of Viti Levu, the largest island of the Fiji Group. The caldera formed when Tertiary basalts collapsed after prolonged explosion from a central vent area. The ensuing subsidence, which appears to have been cyclic, was accompanied by the deposition of andesitic volcanic material to form 5,000 to 7,000 feet of rhythmic tuffs, breccias and agglomerates partly under lacustrine conditions. The peripheral basalts were shattered during the stages of collapse forming a ring fault zone around the caldera. The depositional and subsidence stages were followed by an intrusive augite andesitic one from which extensive cone sheets formed in the caldera rocks. Radial and tangential dykes formed around the caldera in the peripheral basalts. After a time interval, the comparatively shallow central depression of the caldera received biotite andesitic pyroclastics and flows. Biotite andesite dykes followed a similar structural pattern to the augite andesitic ones. Finally, plug like bodies of porphyrite and monzonite intruded into the highly fractured zones, particularly the ring fault zone in the peripheral basalts. An important younger structural development with economic significance was the formation of a north-westerly shear system across the caldera. Flatly dipping structures formed in the peripheral basalts from the resettling of major blocks around the caldera. After the monzonite intrusions, epithermal mineralisers were liberated with economic amounts of gold in the form of telluride and auriferous pyrite. The mineralisers favoured the north-westerly shear system and, in the peripheral basalts, the accompanying flatly dipping structures. Thermal spring activity appears to mark the last phase of volcanicity.