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.

Eubacterium maltosivorans sp. nov., a novel human intestinal acetogenic and butyrogenic bacterium with a versatile metabolism.

A novel anaerobic, non-spore-forming bacterium was isolated from a faecal sample of a healthy adult. The isolate, designated strain YIT, was cultured in a basal liquid medium under a gas phase of H2/CO2 supplemented with yeast extract (0.1 g l-1). Cells of strain YIT were short rods (0.4-0.7×2.0-2.5 µm), appearing singly or in pairs, and stained Gram-positive. Catalase activity and gelatin hydrolysis were positive while oxidase activity, indole formation, urease activity and aesculin hydrolysis were negative. Growth was observed within a temperature range of 20-45 °C (optimum, 35-37 °C), and a pH range of 5.0-8.0 (optimum pH 7.0-7.5). Doubling time was 2.3 h when grown with glucose at pH 7.2 and 37 °C. Besides acetogenic growth, the isolate was able to ferment a large range of monomeric sugars with acetate and butyrate as the main end products. Strain YIT did not show respiratory growth with sulfate, sulfite, thiosulfate or nitrate as electron acceptors. The major cellular fatty acids of the isolate were C16 : 0 and C18 : 0. The genomic DNA G+C content was 47.8 mol%. Strain YIT is affiliated to the genus Eubacterium, sharing highest levels of 16S rRNA gene similarity with Eubacterium limosum ATCC 8486T (97.3 %), Eubacterium callanderi DSM 3662T (97.5 %), Eubacterium aggregans DSM 12183T (94.4 %) and Eubacterium barkeri DSM 1223T (94.8 %). Considering its physiological and phylogenetic characteristics, strain YIT represents a novel species within the genus Eubacterium, for which the name Eubacterium maltosivorans sp. nov. is proposed. The type strain is YIT (=DSM 105863T=JCM 32297T).

Desulfurella amilsii sp. nov., a novel acidotolerant sulfur-respiring bacterium isolated from acidic river sediments.

A novel acidotolerant and moderately thermophilic sulfur-reducing bacterium was isolated from sediments of the Tinto River (Spain), an extremely acidic environment. Strain TR1T stained Gram-negative, and was obligately anaerobic, non-spore-forming and motile. Cells were short rods (1.5-2 × 0.5-0.7 µm), appearing singly or in pairs. Strain TR1T was catalase-negative and slightly oxidase-positive. Urease activity and indole formation were absent, but gelatin hydrolysis was present. Growth was observed at 20-52 °C with an optimum close to 50 °C, and a pH range of 3-7 with optimum between pH 6 and 6.5. Yeast extract was essential for growth, but extra vitamins were not required. In the presence of sulfur, strain TR1T grew with acetate, formate, lactate, pyruvate, stearate, arginine and H2/CO2. All substrates were completely oxidized and H2S and CO2 were the only metabolic products detected. Besides elemental sulfur, thiosulfate was used as an electron acceptor. The isolate also grew by disproportionation of elemental sulfur. The predominant cellular fatty acids were saturated components: C16 : 0, anteiso-C17 : 0 and C18 : 0. The only quinone component detected was menaquinone MK-7(H2). The G+C content of the genomic DNA was 34 mol%. The isolate is affiliated to the genus Desulfurella of the class Deltaproteobacteria, sharing 97 % 16S rRNA gene sequence similarity with the four species described in the genus Desulfurella. Considering the distinct physiological and phylogenetic characteristics, strain TR1T represents a novel species within the genus Desulfurella, for which the name Desulfurella amilsii sp. nov. is proposed. The type strain is TR1T ( = DSM 29984T = JCM 30680T).

Description of Trichococcus ilyis sp. nov. by combined physiological and in silico genome hybridization analyses.

Species of the genus Trichococcus share high similarity of their 16S rRNA gene sequences (>99 %). Digital DNA-DNA hybridization values (dDDH) among type strains of all described species of the genus Trichococcus (T. flocculiformis DSM 2094T, T. pasteurii DSM 2381T, T. collinsii DSM 14526T, T. palustris DSM 9172T, and T. patagoniensisDSM 18806T) indicated that Trichococcus sp. strain R210T represents a novel species of the genus Trichococcus. The dDDH values showed a low DNA relatedness between strain R210T and all other species of the genus Trichococcus (23-32%). Cells of strain R210T were motile, slightly curved rods, 0.63-1.40×0.48-0.90 µm and stained Gram-positive. Growth was optimal at pH 7.8 and at temperature of 30 °C. Strain R210T could utilize several carbohydrates, and the main products from glucose fermentation were lactate, acetate, formate and ethanol. The genomic DNA G+C content of strain R210T was 47.9 mol%. Based on morphological, physiological and biochemical characteristics along with measured dDDH values for all species of the genus Trichococcus, it is suggested that strain R210T represents a novel species within the genus Trichococcus, for which the name Trichococcus ilyis sp. nov. is proposed. The type strain is R210T (=DSM 22150T=JCM 31247T).

Desulfosporosinus acididurans sp. nov.: an acidophilic sulfate-reducing bacterium isolated from acidic sediments.

Three strains of sulfate-reducing bacteria (M1(T), D, and E) were isolated from acidic sediments (White river and Tinto river) and characterized phylogenetically and physiologically. All three strains were obligately anaerobic, mesophilic, spore-forming straight rods, stained Gram-negative and displayed variable motility during active growth. The pH range for growth was 3.8-7.0, with an optimum at pH 5.5. The temperature range for growth was 15-40 °C, with an optimum at 30 °C. Strains M1(T), D, and E used a wide range of electron donors and acceptors, with certain variability within the different strains. The nominated type strain (M1(T)) used ferric iron, nitrate, sulfate, elemental sulfur, and thiosulfate (but not arsenate, sulfite, or fumarate) as electron acceptors, and organic acids (formate, lactate, butyrate, fumarate, malate, and pyruvate), alcohols (glycerol, methanol, and ethanol), yeast extract, and sugars (xylose, glucose, and fructose) as electron donors. It also fermented some substrates such as pyruvate and formate. Strain M1(T) tolerated up to 50 mM ferrous iron and 10 mM aluminum, but was inhibited by 1 mM copper. On the basis of phenotypic, phylogenetic, and genetic characteristics, strains M1(T), D, and E represent a novel species within the genus Desulfosporosinus, for which the name Desulfosporosinus acididurans sp. nov. is proposed. The type strain is M1(T) (=DSM 27692(T) = JCM 19471(T)). Strain M1(T) was the first acidophilic SRB isolated, and it is the third described species of acidophilic SRB besides Desulfosporosinus acidiphilus and Thermodesulfobium narugense.

Sulfur Reduction in Acid Rock Drainage Environments.

Microbiological suitability of acidophilic sulfur reduction for metal recovery was explored by enriching sulfur reducers from acidic sediments at low pH (from 2 to 5) with hydrogen, glycerol, methanol and acetate as electron donors at 30 °C. The highest levels of sulfide in the enrichments were detected at pH 3 with hydrogen and pH 4 with acetate. Cloning and sequencing of the 16S rRNA gene showed dominance of the deltaproteobacterial sulfur-reducing genus Desulfurella in all the enrichments and subsequently an acidophilic strain (TR1) was isolated. Strain TR1 grew at a broad range of pH (3-7) and temperature (20-50 °C) and showed good metal tolerance (Pb(2+), Zn(2+), Cu(2+), Ni(2+)), especially for Ni(2+) and Pb(2+), with maximal tolerated concentrations of 0.09 and 0.03 mM, respectively. Different sources of sulfur were tested in the enrichments, from which biosulfur showed fastest growth (doubling time of 1.9 days), followed by colloidal, chemical and sublimated sulfur (doubling times of 2.2, 2.5, and 3.6 days, respectively). Strain TR1's physiological traits make it a good candidate to cope with low pH and high metal concentration in biotechnological processes for treatment of metal-laden acidic streams at low and moderately high temperature.

Microbacter margulisiae gen. nov., sp. nov., a propionigenic bacterium isolated from sediments of an acid rock drainage pond.

A novel anaerobic propionigenic bacterium, strain ADRI(T), was isolated from sediment of an acid rock drainage environment (Tinto River, Spain). Cells were small (0.4-0.6×1-1.7 µm), non-motile and non-spore-forming rods. Cells possessed a Gram-negative cell-wall structure and were vancomycin-resistant. Strain ADRI(T) utilized yeast extract and various sugars as substrates and formed propionate, lactate and acetate as major fermentation products. The optimum growth temperature was 30 °C and the optimum pH for growth was pH 6.5, but strain ADRI(T) was able to grow at a pH as low as 3.0. Oxidase, indole formation, and urease and catalase activities were negative. Aesculin and gelatin were hydrolysed. The predominant cellular fatty acids of strain ADRI(T) were anteiso-C15 : 0 (30.3 %), iso-C15 : 0 (29.2 %) and iso-C17 : 0 3-OH (14.9 %). Major menaquinones were MK-8 (52 %) and MK-9 (48 %). The genomic DNA G+C content was 39.9 mol%. Phylogenetically, strain ADRI(T) was affiliated to the family Porphyromonadaceae of the phylum Bacteroidetes. The most closely related cultured species were Paludibacter propionicigenes with 16S rRNA gene sequence similarity of 87.5 % and several species of the genus Dysgonomonas (similarities of 83.5-85.4 % to the type strains). Based on the distinctive ecological, phenotypic and phylogenetic characteristics of strain ADRI(T), a novel genus and species, Microbacter margulisiae gen. nov., sp. nov., is proposed. The type strain is ADRI(T) ( = JCM 19374(T) = DSM 27471(T)).

Ercella succinigenes gen. nov., sp. nov., an anaerobic succinate-producing bacterium.

A novel anaerobic succinate-producing bacterium, strain ZWB(T), was isolated from sludge collected from a biogas desulfurization bioreactor (Eerbeek, the Netherlands). Cells were non-spore-forming, motile, slightly curved rods (0.4-0.5 µm in diameter and 2-3 µm in length), and stained Gram-negative. The temperature range for growth was 25-40 °C, with an optimum at 37 °C. The pH range for growth was 7.0-9.0, with an optimum at pH 7.5. Strain ZWB(T) was able to ferment glycerol and several carbohydrates mainly to H2, succinate and acetate. Sulfur and fumarate could be used as electron acceptors by strain ZWB(T). The G+C content of the genomic DNA was 37.6 mol%. The most abundant fatty acids were iso-C14 : 0 and iso-C16 : 0 DMA. On the basis of 16S rRNA gene sequence similarity, strain ZWB(T) belongs to the family Ruminococcaceae and it is distantly related to Saccharofermentans acetigenes JCM 14006(T) (92.1%). Based on the physiological features and phylogenetic analysis, strain ZWB(T) represents a novel species of a new genus, for which the name Ercella succinigenes gen. nov., sp. nov. is proposed. The type strain of Ercella succinigenes is ZWB(T) ( = DSM 27333(T) = JCM 19283(T)).

Harnessing the potential of the microbial sulfur cycle for environmental biotechnology.

The sulfur cycle is a complex biogeochemical cycle characterized by the high variability in the oxidation states of sulfur. While sulfur is essential for life processes, certain sulfur compounds, such as hydrogen sulfide, are toxic to all life forms. Micro-organisms facilitate the sulfur cycle, playing a prominent role even in extreme environments, such as soda lakes, acid mine drainage sites, hot springs, and other harsh habitats. The activity of these micro-organisms presents unique opportunities for mitigating sulfur-based pollution and enhancing the recovery of sulfur and metals. This review highlights the application of sulfur-oxidizing and -reducing micro-organisms in environmental biotechnology through three illustrative examples. Additionally, it discusses the challenges, recent trends, and prospects associated with these applications.

A novel mechanism for dissimilatory nitrate reduction to ammonium in Acididesulfobacillus acetoxydans.

The biological route of nitrate reduction has important implications for the bioavailability of nitrogen within ecosystems. Nitrate reduction via nitrite, either to ammonium (ammonification) or to nitrous oxide or dinitrogen (denitrification), determines whether nitrogen is retained within the system or lost as a gas. The acidophilic sulfate-reducing bacterium (aSRB) Acididesulfobacillus acetoxydans can perform dissimilatory nitrate reduction to ammonium (DNRA). While encoding a Nar-type nitrate reductase, A. acetoxydans lacks recognized nitrite reductase genes. In this study, A. acetoxydans was cultivated under conditions conducive to DNRA. During cultivations, we monitored the production of potential nitrogen intermediates (nitrate, nitrite, nitric oxide, hydroxylamine, and ammonium). Resting cell experiments were performed with nitrate, nitrite, and hydroxylamine to confirm their reduction to ammonium, and formed intermediates were tracked. To identify the enzymes involved in DNRA, comparative transcriptomics and proteomics were performed with A. acetoxydans growing under nitrate- and sulfate-reducing conditions. Nitrite is likely reduced to ammonia by the previously undescribed nitrite reductase activity of the NADH-linked sulfite reductase AsrABC, or by a putatively ferredoxin-dependent homolog of the nitrite reductase NirA (DEACI_1836), or both. We identified enzymes and intermediates not previously associated with DNRA and nitrosative stress in aSRB. This increases our knowledge about the metabolism of this type of bacteria and helps the interpretation of (meta)genome data from various ecosystems on their DNRA potential and the nitrogen cycle.IMPORTANCENitrogen is crucial to any ecosystem, and its bioavailability depends on microbial nitrogen-transforming reactions. Over the recent years, various new nitrogen-transforming reactions and pathways have been identified, expanding our view on the nitrogen cycle and metabolic versatility. In this study, we elucidate a novel mechanism employed by Acididesulfobacillus acetoxydans, an acidophilic sulfate-reducing bacterium, to reduce nitrate to ammonium. This finding underscores the diverse physiological nature of dissimilatory reduction to ammonium (DNRA). A. acetoxydans was isolated from acid mine drainage, an extremely acidic environment where nitrogen metabolism is poorly studied. Our findings will contribute to understanding DNRA potential and variations in extremely acidic environments.

Biological S0 reduction at neutral and acidic conditions: Performance and microbial community shifts in a H2/CO2-fed bioreactor.

Sulfidogenesis is a promising technology for the selective recovery of chalcophile bulk metals (e.g. Cu, Zn, and Co) from metal-contaminated waters such as acid mine drainage (AMD) and metallurgy waste streams. The use of elemental sulfur (S0) instead of sulfate (SO42-) as electron acceptor reduces electron donor requirements four-fold, lowering process costs, and expanding the range of operating conditions to a more acidic pH. We previously reported autotrophic S0 reduction using an industrial mesophilic granular sludge as inoculum under thermoacidophilic conditions. Here, we examined the effect of pH on the S0 reduction performance of the same inoculum, in a gas-lift reactor run at 30°C under neutral (pH 6.9) and acidic (pH 3.8) conditions, continuously fed with mineral media and H2 and CO2. Steady-state volumetric sulfide production rates (VSPR) dropped 2.5-fold upon transition to acidic pH, from 1.79 ± 0.18 g S2-·L-1·d-1 to 0.71 ± 0.07 g S2-·L-1·d-1. Microbial community composition was analyzed using 16S rRNA gene amplicon sequencing. At neutral pH (6.9), the high relative abundance of the S0-reducing genus Sulfurospirillum, previously known only for heterotrophic members, combined with the presence of Acetobacterium and detection of acetate, suggests an important role for heterotrophic S0 reduction facilitated by acetogenesis. Conversely, at acidic pH (3.9), S0 reduction appeared autotrophic, as indicated by the high relative abundance of Desulfurella.