Hydrogen sulfide (H₂S) in environments with temperatures below 100 °C is generally assumed to be of microbial origin, while abiotic H₂S production is typically restricted to higher temperatures (T). In this study, we report an abiotic process for sulfidogenesis through the reduction of elemental sulfur (S⁰) by hydrogen (H₂), mediated by pyrite (FeS₂). The process was investigated in detail at pH 4 and 80 °C, but experimental conditions ranged between 40 and 80 °C and pH 4–6. The experiments were conducted with H₂ as reducing molecule, and µm-sized spherical (but not framboidal) pyrite particles that formed in situ from the H₂S, S⁰ and Fe²⁺ present in the experiments. Fe monosulfides, likely mackinawite, were identified as potential pyrite precursors. The absence of H₂ production in controls, combined with geochemical modelling, suggests that pyrite formation occurred through the polysulfide pathway, which is unexpected under acidic conditions. Most spherical aggregates of authigenic pyrite were composed of nanometric, acicular crystals oriented in diverse directions, displaying varying degrees of organization. Although it was initially hypothesized that the catalytic properties were related to the surface structure, commercially sourced, milled pyrite particles (< 50 μm) mediated H₂S production at comparable rates. This suggests that the catalytic properties of pyrite depend on particle size rather than surface structure, requiring pyrite surfaces to act as electron shuttles between S⁰ and H₂.

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Recently, we isolated two marine strains, F1^T and F21^T, which together with Kiritimatiella glycovorans L21-Fru-AB^T are the only pure cultures of the class Kiritimatiellae within the phylum Verrucomicrobiota. Here, we present an in-depth genome-guided characterization of both isolates with emphasis on their exopolysaccharide synthesis. The strains only grew fermentatively on simple carbohydrates and sulfated polysaccharides. Strains F1^T, F21^T and K. glycovorans reduced elemental sulfur, ferric citrate and anthraquinone-2,6-disulfonate during anaerobic growth on sugars. Both strains produced exopolysaccharides during stationary phase, probably with intracellularly stored glycogen as energy and carbon source. Exopolysaccharides included N-sulfated polysaccharides probably containing hexosamines and thus resembling glycosaminoglycans. This implies that the isolates can both degrade and produce sulfated polysaccharides. Both strains encoded an unprecedently high number of glycoside hydrolase genes (422 and 388, respectively), including prevalent alpha-L-fucosidase genes, which may be necessary for degrading complex sulfated polysaccharides such as fucoidan. Strain F21^T encoded three putative glycosaminoglycan sulfotransferases and a putative sulfate glycosaminoglycan biosynthesis gene cluster. Based on phylogenetic and chemotaxonomic analyses, we propose the taxa Pontiella desulfatans F1^T gen. nov., sp. nov. and Pontiella sulfatireligans F21^T sp. nov. as representatives of the Pontiellaceae fam. nov. within the class Kiritimatiellae.

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Methanol is generally metabolized through a pathway initiated by a cobalamine-containing methanol methyltransferase by anaerobic methylotrophs (such as methanogens and acetogens), or through oxidation to formaldehyde using a methanol dehydrogenase by aerobes. Methanol is an important substrate in deep-subsurface environments, where thermophilic sulfate-reducing bacteria of the genus Desulfotomaculum have key roles. Here, we study the methanol metabolism of Desulfotomaculum kuznetsovii strain 17^T, isolated from a 3000-m deep geothermal water reservoir. We use proteomics to analyze cells grown with methanol and sulfate in the presence and absence of cobalt and vitamin B12. The results indicate the presence of two methanol-degrading pathways in D. kuznetsovii, a cobalt-dependent methanol methyltransferase and a cobalt-independent methanol dehydrogenase, which is further confirmed by stable isotope fractionation. This is the first report of a microorganism utilizing two distinct methanol conversion pathways. We hypothesize that this gives D. kuznetsovii a competitive advantage in its natural environment.

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Anaerobic syntrophic acetate oxidation (SAO) is a thermodynamically unfavorable process involving a syntrophic acetate oxidizing bacterium (SAOB) that forms interspecies electron carriers (IECs). These IECs are consumed by syntrophic partners, typically hydrogenotrophic methanogenic archaea or sulfate reducing bacteria. In this work, the metabolism and occurrence of SAOB at extremely haloalkaline conditions were investigated, using highly enriched methanogenic (MSAO) and sulfate-reducing (S-SAO) cultures from south-western Siberian hypersaline soda lakes. Activity tests with the M-SAO and S-SAO cultures and thermodynamic calculations indicated that H₂ and formate are important IECs in both SAO cultures. Metagenomic analysis of the M-SAO cultures showed that the dominant SAOB was 'Candidatus Syntrophonatronum acetioxidans,' and a near-complete draft genome of this SAOB was reconstructed. 'Ca. S. acetioxidans' has all genes necessary for operating the Wood-Ljungdahl pathway, which is likely employed for acetate oxidation. It also encodes several genes essential to thrive at haloalkaline conditions; including a Na⁺dependent ATP synthase and marker genes for 'salt-out'strategies for osmotic homeostasis at high soda conditions. Membrane lipid analysis of the M-SAO culture showed the presence of unusual bacterial diether membrane lipids which are presumably beneficial at extreme haloalkaline conditions. To determine the importance of SAO in haloalkaline environments, previously obtained 16S rRNA gene sequencing data and metagenomic data of five different hypersaline soda lake sediment samples were investigated, including the soda lakes where the enrichment cultures originated from. The draft genome of 'Ca. S. acetioxidans' showed highest identity with two metagenome-assembled genomes (MAGs) of putative SAOBs that belonged to the highly abundant and diverse Syntrophomonadaceae family present in the soda lake sediments. The 16S rRNA gene amplicon datasets of the soda lake sediments showed a high similarity of reads to 'Ca. S. acetioxidans' with abundance as high as 1.3% of all reads, whereas aceticlastic methanogens and acetate oxidizing sulfate-reducers were not abundant (≤0.1%) or could not be detected. These combined results indicate that SAO is the primary anaerobic acetate oxidizing pathway at extreme haloalkaline conditions performed by haloalkaliphilic syntrophic consortia.

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For the anaerobic biological treatment of saline wastewater, Anaerobic Digestion (AD) is currently a possibility, even though elevated salt concentrations can be a major obstacle. Anaerobic consortia and especially methanogenic archaea are very sensitive to fluctuations in salinity. When working with Upflow Sludge Blanket Reactor (UASB) technology, in which the microorganisms are aggregated and retained in the system as a granular biofilm, high sodium concentration negatively affects aggregation and consequently process performances. In this research, we analysed the structure of the biofilm and granules formed during the anaerobic treatment of high salinity (at 10 and 20 g/L of sodium) synthetic wastewater at lab scale. The acclimated inoculum was able to accomplish high rates of organics removal at all the salinity levels tested. 16S rRNA gene clonal analysis and Fluorescence In Situ Hybridization (FISH) analyses identified the acetoclastic Methanosaeta harundinacea as the key player involved acetate degradation and microbial attachment/granulation. When additional calcium (1 g/L) was added to overcome the negative effect of sodium on microbial aggregation, during the biofilm formation process microbial attachment and acetate degradation decreased. The same result was observed on granules formation: while calcium had a positive effect on granules strength when added to UASB reactors, Methanosaeta filaments were not present and the degradation of the partially acidified substrate was negatively influenced. This research demonstrated the possibility to get granulation at high salinity, bringing to the forefront the importance of a selection towards Methanosaeta cells growing in filamentous form to obtain strong and healthy granules.

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The impact of humic acid (HA) on methanogenic activity was investigated. Methanogenic crushed granular sludge and pure cultures of mesophilic methanogens were incubated in batch cultures with HA. Initial methane production rates and substrate consumption rates were quantified. In the presence of 1 kg m⁻³ HA, the methane production rate of all hydrogenotrophic methanogens was inhibited by more than 75%, except Methanospirillum hungatei that was not inhibited up to 5 kg m⁻³ HA. The acetoclastic Methanosarcina barkeri was completely inhibited by HA ≥1 kg m⁻³. However, Methanosaeta concilii was only slightly affected by HA up to 3 kg m⁻³. When methanogenic granular sludge was incubated with HA, the specific methanogenic activity (SMA) tests showed less inhibition, when compared to the pure cultures of methanogens. The SMA test with H₂/CO₂, formate and acetate showed reduced initial methane production rate of 42%, 23% and 40%, respectively. Differences in HA susceptibility were explained by differences in cell wall structure.

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Inhibition effect of humic acid (HA) on anaerobic digestion of cellulose and xylan and the mitigation potential of the inhibition were evaluated in controlled fed batch reactors at 30 °C and a hydraulic retention time (HRT) of 20 days. Reactor performances were evaluated by biogas production and metabolite measurements for 220 days. Microbial population dynamics of the reactors were monitored with next-generation 16S rRNA gene sequencing at nine different sampling times. Our results showed that increasing levels of HA inhibited the hydrolysis efficiency of the digestion by 40% and concomitantly reduced the methane yield. Addition of hydrolytic enzymes helped to reverse the negative effects of HA, whereas calcium addition did not reverse HA inhibition. Microbiological analyses showed that the relative abundance of hydrolytic/fermentative bacterial groups such as Clostridiales, Bacteroidales and Anaerolineales was significantly lowered by the presence of HA. HA also affected the archaeal populations. Mostly hydrogenotrophic methanogens were negatively affected by HA. The relative abundance of Methanobacteriaceae, Methanomicrobiales-WCHA208 and Unassigned Thermoplasmata WCHA1-57 were negatively affected by the presence of HA, whereas Methanosaetacea was not affected.

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Anaerobic oxidation of methane (AOM) is catalyzed by anaerobic methane-oxidizing archaea (ANME) via a reverse and modified methanogenesis pathway. Methanogens can also reverse the methanogenesis pathway to oxidize methane, but only during net methane production (i.e., "trace methane oxidation"). In turn, ANME can produce methane, but only during net methane oxidation (i.e., enzymatic back flux). Net AOM is exergonic when coupled to an external electron acceptor such as sulfate (ANME-1, ANME-2abc, and ANME-3), nitrate (ANME-2d), or metal (oxides). In this review, the reversibility of the methanogenesis pathway and essential differences between ANME and methanogens are described by combining published information with domain based (meta)genome comparison of archaeal methanotrophs and selected archaea. These differences include abundances and special structure of methyl coenzyme M reductase and of multiheme cytochromes and the presence of menaquinones or methanophenazines. ANME-2a and ANME-2d can use electron acceptors other than sulfate or nitrate for AOM, respectively. Environmental studies suggest that ANME-2d are also involved in sulfate-dependent AOM. ANME-1 seem to use a different mechanism for disposal of electrons and possibly are less versatile in electron acceptors use than ANME-2. Future research will shed light on the molecular basis of reversal of the methanogenic pathway and electron transfer in different ANME types.

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Haloalkaliphilic microorganisms that grow optimally at high-pH and high-salinity conditions can be found in natural environments such as soda lakes. These globally spread lakes harbour interesting anaerobic microorganisms that have the potential of being applied in existing technologies or create new opportunities. In this review, we discuss the potential application of haloalkaliphilic anaerobic microbial communities in the fermentation of lignocellulosic feedstocks material subjected to an alkaline pre-treatment, methane production and sulfur removal technology. Also, the general advantages of operation at haloalkaline conditions, such as low volatile fatty acid and sulfide toxicity, are addressed. Finally, an outlook into the main challenges like ammonia toxicity and lack of aggregation is provided.

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A sulfate-reducing bacterium, strain WW1, was isolated from a thermophilic bioreactor operated at 65°C with methanol as sole energy source in the presence of sulfate. Growth of strain WW1 on methanol or acetate was inhibited at a sulfide concentration of 200 mg l-1, while on H 2/CO2, no apparent inhibition occurred up to a concentration of 500 mg l-1. When strain WW1 was co-cultured under the same conditions with the methanol-utilizing, non-sulfate-reducing bacteria, Thermotoga lettingae and Moorella mulderi, both originating from the same bioreactor, growth and sulfide formation were observed up to 430 mg l -1. These results indicated that in the co-cultures, a major part of the electron flow was directed from methanol via H2/CO2 to the reduction of sulfate to sulfide. Besides methanol, acetate, and hydrogen, strain WW1 was also able to use formate, malate, fumarate, propionate, succinate, butyrate, ethanol, propanol, butanol, isobutanol, with concomitant reduction of sulfate to sulfide. In the absence of sulfate, strain WW1 grew only on pyruvate and lactate. On the basis of 16S rRNA analysis, strain WW1 was most closely related to Desulfotomaculum thermocisternum and Desulfotomaculum australicum. However, physiological properties of strain WW1 differed in some aspects from those of the two related bacteria.@en

Two formate dehydrogenases (CO2-reductases) (FDH-1 and FDH-2) were isolated from the syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans. Both enzymes were produced in axenic fumarate-grown cells as well as in cells which were grown syntrophically on propionate with Methanospirillum hungatei as the H2 and formate scavenger. The purified enzymes exhibited extremely high formate-oxidation and CO2-reduction rates, and low Km values for formate. For the enzyme designated FDH-1, a specific formate oxidation rate of 700 U·mg-1 and a Km for formate of 0.04 mM were measured when benzyl viologen was used as an artificial electron acceptor. The enzyme designated FDH-2 oxidized formate with a specific activity of 2700 U·mg-1 and a Km of 0.01 mM for formate with benzyl viologen as electron acceptor. The specific CO2-reduction (to formate) rates measured for FDH-1 and FDH-2, using dithionite-reduced methyl viologen as the electron donor, were 900 U·mg-1 and 89 U·mg-1, respectively. From gel filtration and polyacrylamide gel electrophoresis it was concluded that FDH-1 is composed of three subunits (89 ± 3, 56 ± 2 and 19 ± 1 kDa) and has a native molecular mass of approximately 350 kDa. FDH-2 appeared to be a heterodimer composed of a 92 ± 3 kDa and a 33 ± 2 kDa subunit. Both enzymes contained tungsten and selenium, while molybdenum was not detected. EPR spectroscopy suggested that FDH-1 contains at least four [2Fe-2S] clusters per molecule and additionally paramagnetically coupled [4Fe-4S] clusters. FDH-2 contains at least two [4Fe-4S] clusters per molecule. As both enzymes are produced under all growth conditions tested, but with differences in levels, expression may depend on unknown parameters.@en