Physiology and application of sulfur-reducing microorganisms from acidic environments

Sulfur cycle is one of the main geochemical cycles on Earth. Oxidation and reduction reactions of sulfur are mostly biotic and performed by microorganisms. In anaerobic conditions – marine and some freshwater systems, dissimilatory sulfur- and sulfate-reducing bacteria and archaea are key players in the decomposition of organic carbon releasing sulfide as the product of their metabolism. Sulfide can then be used as terminal reductant by anoxygenic photosynthetic microorganisms or it can be used as electron donor for aerobic or nitrate-reducing bacteria, etc. One particular case of the sulfur cycle is the naturally occurring oxidation of metallic sulfide-ores, which produce sulfur-rich waters with low pH and high heavy metals content. Extremophilic sulfur-reducing microorganisms are of scientific and technological interest. They are abundant in natural conditions in extreme environments, so they are environmentally relevant. Although hydrogen sulfide is corrosive and odorous, its production can be beneficial for industrial activities such as the precipitation and recovery of heavy metals. Therefore, sulfur reducers have also potential for extending the range of operating conditions of metal precipitation. This thesis describes the isolation and characterization of acidotolerant sulfur-reducing bacteria, providing a first understanding on their metabolism of sulfur compounds and insights on the beneficial microbial interactions for biotechnological purposes. In <strong>Chapter 2</strong>, the ecology and physiology of sulfur-reducing prokaryotes is investigated. The ability of sulfur reduction is wide-spread phylogenetically over the microbial tree of life, found in more than 70 genera. Elemental sulfur reduction can occur via direct cell attachment to the solid substrate or with polysulfide as an intermediate. At least four different enzymes are described to be involved in sulfur reduction pathways, and these enzymes were also detected in several microorganisms that are potential sulfur reducers, but were not reported as such in literature so far. The ecological distribution of sulfur respiration seems to be more widespread at high temperatures with neutral pH values. However, some sulfur reducers can grow at pH as low as 1 and the strategies adopted by microorganisms to face high proton concentrations in the environment were commented in this chapter. The sulfide produced from sulfur reduction can be used to selectively precipitate metals by varying the pH values from 2 to 7, depending on the target metal. Economic calculations were presented to show that sulfur reduction is more advantageous then sulfate reduction due to the cost savings of the electron donor needed. Therefore, acidophilic sulfur reducers are of particular interest for application in selective precipitation and recovery of heavy metals from metalliferous waste streams and the suitable technologies for that purpose are also discussed. Enrichments for sulfur reducers with various electron donors at low pH and mesophilic conditions were performed from sediments of the acidic Tinto river (Spain). A solid-media with colloidal sulfur was developed to facilitate the isolation of true elemental sulfur reducers at low pH. This strategy resulted in the isolation of a sulfur-reducing bacterium, strain TR1, belonging to the <em>Desulfurella</em> genus. The enrichment and isolation procedure were described in <strong>Chapter 3</strong>. The growth and activity of the isolate was tested at different pH values, temperature conditions, utilization of electron donors, and growth in the presence of heavy metals in solution. The isolate showed tolerance to metals, and growth in a broad temperature and pH, revealing its feasibility to precipitate and recover heavy metals from acidic wastewater and mining water, without the need to neutralize the water before treatment. In <strong>Chapter 4</strong>, the morphological, biochemical and physiological characterization of the isolate is provided, for which the name <em>Desulfurella amilsii</em> TR1 sp. nov. was proposed. <em>D. amilsii</em> is affiliated to the <em>Deltaproteobacteria</em> class showing 97% of 16S rRNA gene identity to the four species described in the <em>Desulfurella</em> genus. In the presence of elemental sulfur, <em>D. amilsii</em> utilized acetate, formate, lactate, pyruvate, stearate, arginine and H2/CO2 as substrates, completely oxidizing them to H2S and CO2. Besides elemental sulfur, thiosulfate was used as an electron acceptor and the isolate also grew in the absence of external electron donor, by disproportionation of elemental sulfur into sulfide and sulfate. The draft genome sequence of <em>Desulfurella amilsii</em> TR1 and a comparative genomic analysis with the members of <em>Desulfurellaceae </em>family are reported in <strong>Chapter 5</strong>. Based on average nucleotide identity and <em>in silico </em>DNA hybridization values<em>, D. multipotens</em> and <em>D. acetivorans</em> were revealed to belong to the same species. Reclassification was therefore suggested. Regarding sulfur metabolism, the analysed genomes encode different sulfur-reducing enzymes per genus. <em>Hippea</em> species encode polysulfide reductase and a sulfide dehydrogenase. The analysed genomes of <em>Desulfurella</em> especies do not possess the polysulfide reductase but possess the sulfide dehydrogenase<em>. D. amilsii </em>is the only member of the family encoding sulfur reductase. Since <em>D. amilsii</em> is able to grow at the lowest pH value, this enzyme was suggested to play a role in sulfur reduction when the microorganism grows in acidic conditions. Genes encoding resistance to acidic conditions were reported for all <em>Desulfurellaceae </em>members, countering physiological tests that showed ability to grow at low pH only for <em>D. amilsii</em> and <em>D. acetivorans</em>. Sulfur respiration by <em>D. amilsii</em> was studied in more detail in <strong>Chapter</strong> <strong>6</strong>, in which the requirement for cell-sulfur interaction at acidic (pH 3.5) and circumneutral (pH 6.5) conditions was evaluated. <em>D. amilsii</em> was shown to benefit from contact with the insoluble substrate, as activity and number of cells decreased when sulfur was sequestered from the medium in dialysis bags of 6-8 kDa pore size. Besides, the abundance of enzymes possibly involved in sulfur respiration, acid resistance and chemolithotrophic growth were investigated by proteomics. Sulfur reductases were not detected in the dataset, but the limitations of the method might leave membrane-bound proteins underrepresented in the study. Different rhodanese-like proteins were detected in high abundance at low and neutral pH, while sulfide dehydrogenase seems to function as a ferredoxin:NADP oxidoreductase. We suggest that the sulfurtransferases might play a key role in sulfur/polysulfide reduction in <em>D. amilsii</em>. Proteomic data also showed that genes involved in acid resistance are constitutively expressed in this microorganism. Some proteins were exclusively detected at low pH, but with very few overlapping with proteins reported to be involved in acid resistance. Moreover, analysis of the proteome revealed the involvement of the hydrogenase HydABC for oxidation of hydrogen during chemolitotrophic growth, as well as the complete pathway for CO2 fixation via the reductive TCA cycle. More aspects of the sulfur metabolism by <em>D. amilsii</em> were investigated in<strong> Chapter 7</strong>. Cultures grown on acetate with sulfur or thiosulfate as electron acceptor and cultures grown by disproportionation of elemental sulfur, all at pH 6.5, had their proteomes compared. Rhodanese-like sulfurtransferases were abundant in all the analyzed conditions, with specific differences in the sequences. In sulfur respiration and disproportionation, sulfurtransferases were the only sulfur enzymes detected and so, they are likely to play a central role in the process. The respiration of thiosulfate is likely to happen via a thiosulfate reductase and a dissimilatory sulfite reductase, highly abundant in this specific condition. Analysis on the heterotrophic cultures revealed the ability of <em>D. amilsii</em> to activate acetate to acetyl-CoA via the acetyl-CoA synthetase enzyme and its oxidation via the TCA cycle being this the first report of acetate activation happening via acetyl-CoA synthetase in sulfur-reducing bacteria. The isolation and characterization of another acidotolerant sulfur respirer, <em>Lucifera butyrica</em> strain ALE, and its growth in co-culture with <em>D. amilsii</em> were described in <strong>Chapter 8</strong>. <em>L. butyrica</em> was shown to use a wide range of substrate, such as glucose, lactose, ethanol, glycerol glycogen, peptone, etc. When growing on glycerol, a cheap substrate, by fermentation or by respiration of elemental sulfur, <em>L. butyrica</em> produced acetate, ethanol and 1,3-propanediol as major products. Elemental sulfur reduction by this bacterium, however, was not efficient and led to the production of maximum 2.5 mM of sulfide. When <em>L. butyrica </em>grew in a co-culture with <em>D. amilsii, </em>the acetate produced by the first was consumed by the latter and the production of sulfide was boosted in the culture. As <em>D. amilsii</em> is not able to degrade glycerol, the co-culture represents a strategy to broaden the applicability of sulfur reduction at low pH with different sources of electron donors.