Acetate Degradation at Low pH by the Moderately Acidophilic Sulfate Reducer Acididesulfobacillus acetoxydans gen. nov. sp. nov

Abstract

In acid drainage environments, biosulfidogenesis by sulfate-reducing bacteria (SRB) attenuates the extreme conditions by enabling the precipitation of metals as their sulfides, and the neutralization of acidity through proton consumption. So far, only a handful of moderately acidophilic SRB species have been described, most of which are merely acidotolerant. Here, a novel species within a novel genus of moderately acidophilic SRB is described, Acididesulfobacillus acetoxydans gen. nov. sp. nov. strain INE, able to grow at pH 3.8. Bioreactor studies with strain INE at optimum (5.0) and low (3.9) pH for growth showed that strain INE alkalinized its environment, and that this was more pronounced at lower pH. These studies also showed the capacity of strain INE to completely oxidize organic acids to CO₂, which is uncommon among acidophilic SRB. Since organic acids are mainly in their protonated form at low pH, which increases their toxicity, their complete oxidation may be an acid stress resistance mechanism. 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, was a protection mechanism against acid stress. Interestingly, other canonical acid stress resistance mechanisms, such as a Donnan potential and increased active charge transport, did not appear to be active.

Keywords: Acididesulfobacillus; Desulfitobacterium; Desulfosporosinus; acetate oxidation; acid rock/mine drainage; acidophiles; bioremediation; sulfate-reducing bacteria.

Figure 1

Acididesulfobacillus acetoxydans gen. nov. sp. nov. strain INE microscopy images: (A) phase contrast microscopy and (B,C) transmission electron microscopy. Scale bars represent 5 mm, 2 mm, and 200 nm, respectively. Arrows in (B) indicate flagella.

Figure 2

Maximum-likelihood tree showing phylogenetic affiliation of 16S rRNA gene sequences of Acididesulfobacillus acetoxydans (in bold) with related environmental sequences found in acidic environments, and closely related species in the Peptococcaceae family of the Firmicutes phylum. The accession numbers represent sequences retrieved from different acid drainage environments. KJ650710, KJ650737, or KJ650717: sulfidic mine tailings dumps in Botswana; EF042590: an abandoned copper mine in Odiel (Huelva, Spain); HQ730640, JQ433964: Tinto River sediments; EU156143: a thermal spring in Yellowstone National Park (WY, United States); EF464638: acidic enrichment cultures. JF346160 and EF061086: Peptococcaceae bacteria CEB3 and CL4. Scalebar indicates 10% sequence difference.

Figure 3

Glycerol, total acetate (undissociated + dissociated) and sulfate concentrations, and OD₆₀₀ of Acididesulfobacillus acetoxydans strain INE grown in bioreactors at pH 3.9 (A) and pH 5 (B). Concentration profiles of glycerol, sulfate, acetic acid, as well as OD₆₀₀ and HCl addition are shown for one reactor at each pH value for clarity.

Figure 4

Schematic representation of pathways for glycerol oxidation and sulfate reduction as detected in the genome of Acididesulfobacillus acetoxydans. Sat: ATP sulfurylase, Apr: APS reductase, Dsr: dissimilatory sulfite reductase, Qmo: quinone-interacting membrane oxidoreductase complex, Hdr: heterodisulfide reductase, GlpF: glycerol transporter, GlpK: glycerol kinase, Tpi: triose phosphate isomerase, Gap: glyceraldehyde-3-phosphate dehydrogenase, Pgk: phosphoglycerate kinase, Pgm: phosphoglycerate mutase, Eno: phosphoenolpyruvate hydratase, Pyk: pyruvate kinase, Pfor: pyruvate:ferredoxin oxidoreductase, Pta: Phosphate acetyltransferase, Ack: acetate kinase, Acs: acetyl-coA synthase, Codh: carbon monoxide dehydrogenase, Met: methylene-tetrahydrofolate reductase, Fol: methylene-tetrahydrofolate dehydrogenase/methenyl-tetrahydrofolate cyclohydrolase, Fch: 5-formyltetrahydrofolate_cyclo-ligase, Fhs: Formate-tetrahydrofolate ligase/ formyl-H4F synthethase, Fdh: formate dehydrogenase, Nfn: NADH-dependent reduced ferredoxin:NADP⁺ oxidoreductase.