Acetic acid stress response of the acidophilic sulfate reducer Acididesulfobacillus acetoxydans.

Acid mine drainage (AMD) waters are a severe environmental threat, due to their high metal content and low pH (pH <3). Current technologies treating AMD utilize neutrophilic sulfate-reducing microorganisms (SRMs), but acidophilic SRM could offer advantages. As AMDs are low in organics these processes require electron donor addition, which is often incompletely oxidized into organic acids (e.g., acetic acid). At low pH, acetic acid is undissociated and toxic to microorganisms. We investigated the stress response of the acetotrophic Acididesulfobacillus acetoxydans to acetic acid. A. acetoxydans was cultivated in bioreactors at pH 5.0 (optimum). For stress experiments, triplicate reactors were spiked until 7.5 mM of acetic acid and compared with (non-spiked) triplicate reactors for physiological, transcriptomic, and membrane lipid changes. After acetic acid spiking, the optical density initially dropped, followed by an adaptation phase during which growth resumed at a lower growth rate. Transcriptome analysis revealed a downregulation of genes involved in glutamate and aspartate synthesis following spiking. Membrane lipid analysis revealed a decrease in iso and anteiso fatty acid relative abundance; and an increase of acetyl-CoA as a fatty acid precursor. These adaptations allow A. acetoxydans to detoxify acetic acid, creating milder conditions for other microorganisms in AMD environments.

Alcohol dehydrogenase system acts as the sole pathway for methanol oxidation in Desulfofundulus kuznetsovii strain TPOSR.

Desulfofundulus kuznetsovii is a thermophilic, spore-forming sulphate-reducing bacterium in the family Peptococcaceae. In this study, we describe a newly isolated strain of D. kuznetsovii, strain TPOSR, and compare its metabolism to the type strain D. kuznetsovii 17T. Both strains grow on a large variety of alcohols, such as methanol, ethanol and propane-diols, coupled to the reduction of sulphate. Strain 17T metabolizes methanol via two routes, one involving a cobalt-dependent methyl transferase and the other using a cobalt-independent alcohol dehydrogenase. However, strain TPOSR, which shares 97% average nucleotide identity with D. kuznetsovii strain 17T, lacks several genes from the methyl transferase operon found in strain 17T. The gene encoding the catalytically active methyl transferase subunit B is missing, indicating that strain TPOSR utilizes the alcohol dehydrogenase pathway exclusively. Both strains grew with methanol during cobalt starvation, but growth was impaired. Strain 17T was more sensitive to cobalt deficiency, due to the repression of its methyl transferase system. Our findings shed light on the metabolic diversity of D. kuznetsovii and their metabolic differences of encoding one or two routes for the conversion of methanol.

Coupled C, H, N, S and Fe biogeochemical cycles operating in the continental deep subsurface of the Iberian Pyrite Belt.

Microbial activity is a major contributor to the biogeochemical cycles that make up the life support system of planet Earth. A 613 m deep geomicrobiological perforation and a systematic multi-analytical characterization revealed an unexpected diversity associated with the rock matrix microbiome that operates in the subsurface of the Iberian Pyrite Belt (IPB). Members of 1 class and 16 genera were deemed the most representative microorganisms of the IPB deep subsurface and selected for a deeper analysis. The use of fluorescence in situ hybridization allowed not only the identification of microorganisms but also the detection of novel activities in the subsurface such as anaerobic ammonium oxidation (ANAMMOX) and anaerobic methane oxidation, the co-occurrence of microorganisms able to maintain complementary metabolic activities and the existence of biofilms. The use of enrichment cultures sensed the presence of five different complementary metabolic activities along the length of the borehole and isolated 29 bacterial species. Genomic analysis of nine isolates identified the genes involved in the complete operation of the light-independent coupled C, H, N, S and Fe biogeochemical cycles. This study revealed the importance of nitrate reduction microorganisms in the oxidation of iron in the anoxic conditions existing in the subsurface of the IPB.

Comparative genomics and proteomics of Eubacterium maltosivorans: functional identification of trimethylamine methyltransferases and bacterial microcompartments in a human intestinal bacterium with a versatile lifestyle.

Eubacterium maltosivorans YIT is a human intestinal isolate capable of acetogenic, propionogenic and butyrogenic growth. Its 4.3-Mb genome sequence contains coding sequences for 4227 proteins, including 41 different methyltransferases. Comparative proteomics of strain YIT showed the Wood-Ljungdahl pathway proteins to be actively produced during homoacetogenic growth on H2 and CO2 while butyrogenic growth on a mixture of lactate and acetate significantly upregulated the production of proteins encoded by the recently identified lctABCDEF cluster and accessory proteins. Growth on H2 and CO2 unexpectedly induced the production of two related trimethylamine methyltransferases. Moreover, a set of 16 different trimethylamine methyltransferases together with proteins for bacterial microcompartments were produced during growth and deamination of the quaternary amines, betaine, carnitine and choline. Growth of strain YIT on 1,2-propanediol generated propionate with propanol and induced the formation of bacterial microcompartments that were also prominently visible in betaine-grown cells. The present study demonstrates that E. maltosivorans is highly versatile in converting low-energy fermentation end-products in the human gut into butyrate and propionate whilst being capable of preventing the formation of the undesired trimethylamine by converting betaine and other quaternary amines in bacterial microcompartments into acetate and butyrate.

Anaerobic microbial methanol conversion in marine sediments.

Methanol is an ubiquitous compound that plays a role in microbial processes as a carbon and energy source, intermediate in metabolic processes or as end product in fermentation. In anoxic environments, methanol can act as the sole carbon and energy source for several guilds of microorganisms: sulfate-reducing microorganisms, nitrate-reducing microorganisms, acetogens and methanogens. In marine sediments, these guilds compete for methanol as their common substrate, employing different biochemical pathways. In this review, we will give an overview of current knowledge of the various ways in which methanol reaches marine sediments, the ecology of microorganisms capable of utilizing methanol and their metabolism. Furthermore, through a metagenomic analysis, we shed light on the unknown diversity of methanol utilizers in marine sediments which is yet to be explored.

The bacterial sulfur cycle in expanding dysoxic and euxinic marine waters.

Dysoxic marine waters (DMW, < 1 µM oxygen) are currently expanding in volume in the oceans, which has biogeochemical, ecological and societal consequences on a global scale. In these environments, distinct bacteria drive an active sulfur cycle, which has only recently been recognized for open-ocean DMW. This review summarizes the current knowledge on these sulfur-cycling bacteria. Critical bottlenecks and questions for future research are specifically addressed. Sulfate-reducing bacteria (SRB) are core members of DMW. However, their roles are not entirely clear, and they remain largely uncultured. We found support for their remarkable diversity and taxonomic novelty by mining metagenome-assembled genomes from the Black Sea as model ecosystem. We highlight recent insights into the metabolism of key sulfur-oxidizing SUP05 and Sulfurimonas bacteria, and discuss the probable involvement of uncultivated SAR324 and BS-GSO2 bacteria in sulfur oxidation. Uncultivated Marinimicrobia bacteria with a presumed organoheterotrophic metabolism are abundant in DMW. Like SRB, they may use specific molybdoenzymes to conserve energy from the oxidation, reduction or disproportionation of sulfur cycle intermediates such as S0 and thiosulfate, produced from the oxidation of sulfide. We expect that tailored sampling methods and a renewed focus on cultivation will yield deeper insight into sulfur-cycling bacteria in DMW.

Acetotrophic sulfate-reducing consortia develop active biofilms on zeolite and glass beads in batch cultures at initial pH 3.

Sulfate-reducing microbial communities remain a suitable option for the remediation of acid mine drainage using several types of carrier materials and appropriate reactor configurations. However, acetate prevails as a product derived from the incomplete oxidation of most organic substrates by sulfate reducers, limiting the efficiency of the whole process. An established sulfate-reducing consortium, able to degrade acetate at initial acidic pH (3.0), was used to develop biofilms over granular activated carbon (GAC), glass beads, and zeolite as carrier materials. In batch assays using glycerol, biofilms successfully formed on zeolite, glass beads, and GAC with sulfide production rates of 0.32, 0.26, and 0.14 mmol H2S/L·d, respectively, but only with glass beads and zeolite, acetate was degraded completely. The planktonic and biofilm communities were determined by the 16S rRNA gene analysis to evaluate the microbial selectivity of the carrier materials. In total, 46 OTUs (family level) composed the microbial communities. Ruminococcaceae and Clostridiaceae families were present in zeolite and glass beads, whereas Peptococcaceae was mostly enriched on zeolite and Desulfovibrionaceae on glass beads. The most abundant sulfate reducer in the biofilm of zeolite was Desulfotomaculum sp., while Desulfatirhabdium sp. abounded in the planktonic community. With glass beads, Desulfovibrio sp. dominated the biofilm and the planktonic communities. Our results indicate that both materials (glass beads and zeolite) selected different key sulfate-reducing microorganisms able to oxidize glycerol completely at initial acidic pH, which is relevant for a future application of the consortium in continuous bioreactors to treat acidic streams. KEY POINTS: • Complete consumption of glycerol and acetate at acidic pH by sulfate reduction. • Glass beads and zeolite are suitable materials to form sulfate-reducing biofilms. • Acetotrophic sulfate-reducing bacteria attached to zeolite preferably.

Sulfur Reduction at Hyperthermoacidophilic Conditions with Mesophilic Anaerobic Sludge as the Inoculum.

Sulfur reduction at hyperthermoacidophilic conditions represents a promising opportunity for metal sulfide precipitation from hot acidic metallurgical streams, avoiding costly cooling down. The suitability of mesophilic anaerobic sludges as the inoculum for sulfur-reducing bioreactors operated at high temperature and low pH was explored. We examined sludges from full-scale anaerobic reactors for sulfur-reducing activity at pH 2.0-3.5 and 70 or 80 °C, with H2 as an electron donor. At pH 3.5 in batch experiments, sulfidogenesis started within 4 days, reaching up to 100-200 mg·L-1 of dissolved sulfide produced after 19-24 days, depending on the origin of the sludge. Sulfidogenesis resumed after removing H2S by flushing with nitrogen gas, indicating that sulfide was limiting the conversion. The best performing sludge was used to inoculate a 4 L gas-lift reactor fed with H2 as the electron donor, CO2 as the carbon source, and elemental sulfur as the electron acceptor. The reactor was operated in semibatch mode at a pH 3.5 and 80 °C, and stable sulfide production rates of 60-80 mg·L-1·d-1 were achieved for a period of 24 days, without formation of methane or acetate. Our results reveal the potential of mesophilic anaerobic sludges as seed material for sulfur-reducing bioprocesses operated at hyperthermoacidophilic conditions. The process needs further optimization of the volumetric sulfide production rate to gain relevance for practice.

Insight into the sulfur metabolism of Desulfurella amilsii by differential proteomics.

Many questions regarding proteins involved in microbial sulfur metabolism remain unsolved. For sulfur respiration at low pH, the terminal electron acceptor is still unclear. Desulfurella amilsii is a sulfur-reducing bacterium that respires elemental sulfur (S0 ) or thiosulfate, and grows by S0 disproportionation. Due to its versatility, comparative studies on D. amilsii may shed light on microbial sulfur metabolism. Requirement of physical contact between cells and S0 was analyzed. Sulfide production decreased by around 50% when S0 was trapped in dialysis membranes, suggesting that contact between cells and S0 is beneficial, but not strictly needed. Proteome analysis was performed under the aforementioned conditions. A Mo-oxidoreductase suggested from genome analysis to act as sulfur reductase was not detected in any growth condition. Thiosulfate and sulfite reductases showed increased abundance in thiosulfate-reducing cultures, while rhodanese-like sulfurtransferases were highly abundant in all conditions. DsrE and DsrL were abundantly detected during thiosulfate reduction, suggesting a modified mechanism of sulfite reduction. Proteogenomics suggest a different disproportionation pathway from what has been reported. This work points to an important role of rhodaneses in sulfur processes and these proteins should be considered in searches for sulfur metabolism in broader fields like meta-omics.

Novel haloalkaliphilic methanotrophic bacteria: An attempt for enhancing methane bio-refinery.

Methane bioconversion into products with a high market value, such as ectoine or hydroxyectoine, can be optimized via isolation of more efficient novel methanotrophic bacteria. The research here presented focused on the enrichment of methanotrophic consortia able to co-produce different ectoines during CH4 metabolism. Four different enrichments (Cow3, Slu3, Cow6 and Slu6) were carried out in basal media supplemented with 3 and 6% NaCl, and using methane as the sole carbon and energy source. The highest ectoine accumulation (∼20 mg ectoine g biomass-1) was recorded in the two consortia enriched at 6% NaCl (Cow6 and Slu6). Moreover, hydroxyectoine was detected for the first time using methane as a feedstock in Cow6 and Slu6 (∼5 mg g biomass-1). The majority of the haloalkaliphilic bacteria identified by 16S rRNA community profiling in both consortia have not been previously described as methanotrophs. From these enrichments, two novel strains (representing novel species) capable of using methane as the sole carbon and energy source were isolated: Alishewanella sp. strain RM1 and Halomonas sp. strain PGE1. Halomonas sp. strain PGE1 showed higher ectoine yields (70-92 mg ectoine g biomass-1) than those previously described for other methanotrophs under continuous cultivation mode (∼37-70 mg ectoine g biomass-1). The results here obtained highlight the potential of isolating novel methanotrophs in order to boost the competitiveness of industrial CH4-based ectoine production.