Sulfur oxidation kinetics of Acidithiobacillus caldus and its inhibition on exposure to thiocyanate present in cyanidation tailings wastewater.

The sulfur oxidation kinetics of an industrial strain of Acidithiobacillus caldus (At. caldus) cultured on elemental sulfur was explored in batch experiments in the absence and presence of thiocyanate (SCN-), a toxin inherent within cyanidation tailings wastewater. The Contois rate expression accurately described At. caldus sulfate generation (R2 > 0.93) and microbial growth (R2 > 0.87). For a culture maintained at 45 °C a maximum specific growth rate (µmax) of 0.105 h-1, sulfate yield from biomass (Ypx) of 4.8 × 10-9 mg SO42-.cell-1, and Contois affinity coefficient (Kx) of 1.56 × 10-8 mg S.cell-1 were established. The presence of SCN- (0 mg/L - 20 mg/L) in the bulk solution inhibited the microbial system competitively. Moreover, SCN- impeded microbial growth differentially; the rate expression was therefore partitioned with respect to SCN- concentration and inhibition constants (Ki) were determined for each region. Adaptation to discrete concentrations of SCN- (1 mg/L and 20 mg/L) improved SCN- tolerance in At. caldus; however, adapted strains exhibited reduced sulfur oxidation potential when cultured under thiocyanate-free conditions relative to the non-adapted control strain. To describe the adapted systems accurately, the Contois affinity coefficient (Kx) was revised to reflect the suspected metabolic decline. The derived Kx values increased in magnitude and affirmed an innate reduction in microbial substrate affinity or substrate adsorption capacity. Inclusion of these updated Kx constants within the rate equation suitably represented the experimental data for both adapted At. caldus strains with R2 > 0.94. Furthermore, the impact of adaptation on the inhibition kinetics and inhibition mechanism associated with SCN- exposure were reviewed. Thiocyanate inhibited sulfur oxidation non-competitively in the adapted strains, and the shift in inhibition mechanism may be attributed to the compromised metabolic state following adaptation.

Sulfur and oxygen isotope constraints on sulfate sources and neutral rock drainage-related processes at a South African colliery.

Understanding the fundamental controls that govern the generation of mine drainage is essential for waste management strategies. Combining the isotopic composition of water (H and O) and dissolved sulfate (S and O) with hydrogeochemical measurements of surface and groundwater, microbial analysis, composition of sediments and precipitates, and geochemical modeling results in this study we discussed the processes that control mine water chemistry and identified the potential source(s) and possible mechanisms governing sulfate formation and transformation around a South African colliery. Compared to various South African water standards, water samples collected from the surroundings of a coal waste disposal facility had elevated Fe2+ (0.9 to 56.9 mg L-1), Ca (33.0 to 527.0 mg L-1), Mg (6.2 to 457.0 mg L-1), Mn (0.1 to 8.6 mg L-1) and SO4 (19.7 to 3440.8 mg L-1) and circumneutral pH. The pH conditions are mainly controlled by the release of H+ from pyrite oxidation and the subsequent dissolution of carbonates and aluminosilicate minerals. The phases predicted to precipitate by equilibrium calculation were green rusts, ferrihydrite, gypsum, ±epsomite. Low concentrations of deleterious metals in solution are due to their low abundance in the local host rocks, and their attenuation through adsorption onto secondary Fe precipitates and co-precipitation at the elevated pH values. The δ34S values of sulfate are enriched (-6.5 ‰ to +5.6 ‰) compared to that of pyrite sampled from the mine (mean -22.5 ‰) and overlap with that of the organic sulfur of coal from the region (-2.5 to +4.9 ‰). The presence of both sulfur reducing and oxidizing bacteria were detected in the collected sediment samples. Combined, the data are consistent with the dissolved sulfate in the sampled waters from the colliery being derived primarily from pyrite probably with the subordinate contribution of organic sulfur, followed by its partial removal through precipitation and microbially-induced reduction.

Characterisation of three novel α-L-arabinofuranosidases from a compost metagenome.

BACKGROUND: The importance of the accessory enzymes such as α-L-arabinofuranosidases (AFases) in synergistic interactions within cellulolytic mixtures has introduced a paradigm shift in the search for hydrolytic enzymes. The aim of this study was to characterize novel AFase genes encoding enzymes with differing temperature optima and thermostabilities for use in hydrolytic cocktails. RESULTS: Three fosmids, pFos-H4, E3 and D3 were selected from the cloned metagenome of high temperature compost, expressed in Escherichia coli and subsequently purified to homogeneity from cell lysate. All the AFases were clustered within the GH51 AFase family and shared a homo-hexameric structure. Both AFase-E3 and H4 showed optimal activity at 60 °C while AFase-D3 had unique properties as it showed optimal activity at 25 °C as well as the ability to maintain substantial activity at temperatures as high as 90 °C. However, AFase-E3 was the most thermostable amongst the three AFases showing full activity even at 70 °C. The maximum activity was observed at a pH profile between pH 4.0-6.0 for all three AFases with optimal activity for AFase H4, D3 and E3 at pH 5.0, 4.5 and 4.0, respectively. All the AFases showed KM range between 0.31 mM and 0.43 mM, Kcat range between 131 s- 1 and 219 s- 1 and the specific activity for AFase-H4, AFases-E3 and was 143, 228 and 175 U/mg, respectively. AFases-E3 and D3 displayed activities against pNP-ß-L-arabinopyranoside and pNP-ß-L-mannopyranoside respectively, and both hydrolysed pNP-ß-D-glucopyranoside. CONCLUSION: All three AFases displayed different biochemical characteristics despite all showing conserved overall structural similarity with typical domains of AFases belonging to GH51 family. The hydrolysis of cellobiose by a GH51 family AFase is demonstrated for the first time in this study.

Liquid Phase Multiplex High-Throughput Screening of Metagenomic Libraries Using p-Nitrophenyl-Linked Substrates for Accessory Lignocellulosic Enzymes.

To access the genetic potential contained in large metagenomic libraries, suitable high-throughput functional screening methods are required. Here we describe a high-throughput screening approach which enables the rapid identification of metagenomic library clones expressing functional accessory lignocellulosic enzymes. The high-throughput nature of this method hinges on the multiplexing of both the E. coli metagenomic library clones and the colorimetric p-nitrophenyl linked substrates which allows for the simultaneous screening for ß-glucosidases, ß-xylosidases, and α-L-arabinofuranosidases. This method is readily automated and compatible with high-throughput robotic screening systems.