Chalcopyrite bioleaching efficacy by extremely thermoacidophilic archaea leverages balanced iron and sulfur biooxidation.

Factors that contribute to optimal chalcopyrite bioleaching by extremely thermoacidophilic archaea were examined for ten species belonging to the order Sulfolobales from the genera Acidianus (A. brierleyi), Metallosphaera (M. hakonensis, M. sedula, M. prunae), Sulfuracidifex (S. metallicus, S. tepriarius), Sulfolobus (S. acidocaldarius), Saccharlobus (S. solfataricus) and Sulfurisphaera (S. ohwakuensis, S. tokodaii). Only A. brierleyi, M. sedula, S. metallicus, S. tepriarius, S. ohwakuensis, and S. tokodai exhibited significant amounts of bioleaching and were investigated further. At 70-75 °C, Chalcopyrite loadings of 10 g/l were leached for 21 days during which pH, redox potential, planktonic cell density, iron concentrations and sulfate levels were monitored, in addition to copper mobilization. S. ohwakuensis proved to be the most prolific bioleacher. This was attributed to balanced iron and sulfur oxidation, thereby reducing by-product (e.g., jarosites) formation and minimizing surface passivation. Comparative genomics suggest markers for bioleaching potential, but the results here point to the need for experimental verification.

Phenotype-driven assessment of the ancestral trajectory of sulfur biooxidation in the thermoacidophilic archaea Sulfolobaceae.

Certain members of the family Sulfolobaceae represent the only archaea known to oxidize elemental sulfur, and their evolutionary history provides a framework to understand the development of chemolithotrophic growth by sulfur oxidation. Here, we evaluate the sulfur oxidation phenotype of Sulfolobaceae species and leverage comparative genomic and transcriptomic analysis to identify the key genes linked to sulfur oxidation. Metabolic engineering of the obligate heterotroph Sulfolobus acidocaldarius revealed that the known cytoplasmic components of sulfur oxidation alone are not sufficient to drive prolific sulfur oxidation. Imaging analysis showed that Sulfolobaceae species maintain proximity to the sulfur surface but do not necessarily contact the substrate directly. This indicates that a soluble form of sulfur must be transported to initiate cytoplasmic sulfur oxidation. Conservation patterns and transcriptomic response implicate an extracellular tetrathionate hydrolase and putative thiosulfate transporter in a newly proposed mechanism of sulfur acquisition in the Sulfolobaceae.IMPORTANCESulfur is one of the most abundant elements on earth (2.9% by mass), so it makes sense that the earliest biology found a way to use sulfur to create and sustain life. However, beyond evolutionary significance, sulfur and the molecules it comprises have important technological significance, not only in chemicals such as sulfuric acid and in pyritic ores containing critical metals but also as a waste product from oil and gas production. The thermoacidophilic Sulfolobaceae are unique among the archaea as sulfur oxidizers. The trajectory for how sulfur biooxidation arose and evolved can be traced using experimental and bioinformatic analyses of the available genomic data set. Such analysis can also inform the process by which extracellular sulfur is acquired and transported by thermoacidophilic archaea, a phenomenon that is critical to these microorganisms but has yet to be elucidated.

Complete genome sequence for the thermoacidophilic archaeon Metallosphaera sedula (DSM:5348).

The complete genome sequence of the thermoacidophilic archaeon Metallosphaera sedula (DSM 5348) is reported here. M. sedula, originally isolated from a volcanic field in Italy, is a prolific iron-oxidizing archaeon with applications in bioleaching of sulfide minerals.

Complete genome sequence for the extremely thermophilic bacterium Anaerocellum danielii (DSM:8977).

The complete genome sequence of the extremely thermophilic bacterium Anaerocellum (f. Caldicellulosiruptor) danielii (DSM:8977) is reported here. A. danielii is a fermentative anaerobe and capable of lignocellulose degradation with potential applications in biomass degradation and production of chemicals and fuels from renewable feedstocks.

Metabolic engineering of Caldicellulosiruptor bescii for 2,3-butanediol production from unpretreated lignocellulosic biomass and metabolic strategies for improving yields and titers.

The platform chemical 2,3-butanediol (2,3-BDO) is used to derive products, such as 1,3-butadiene and methyl ethyl ketone, for the chemical and fuel production industries. Efficient microbial 2,3-BDO production at industrial scales has not been achieved yet for various reasons, including product inhibition to host organisms, mixed stereospecificity in product formation, and dependence on expensive substrates (i.e., glucose). In this study, we explore engineering of a 2,3-BDO pathway in Caldicellulosiruptor bescii, an extremely thermophilic (optimal growth temperature = 78°C) and anaerobic bacterium that can break down crystalline cellulose and hemicellulose into fermentable C5 and C6 sugars. In addition, C. bescii grows on unpretreated plant biomass, such as switchgrass. Biosynthesis of 2,3-BDO involves three steps: two molecules of pyruvate are condensed into acetolactate; acetolactate is decarboxylated to acetoin, and finally, acetoin is reduced to 2,3-BDO. C. bescii natively produces acetoin; therefore, in order to complete the 2,3-BDO biosynthetic pathway, C. bescii was engineered to produce a secondary alcohol dehydrogenase (sADH) to catalyze the final step. Two previously characterized, thermostable sADH enzymes with high affinity for acetoin, one from a bacterium and one from an archaeon, were tested independently. When either sADH was present in C. bescii, the recombinant strains were able to produce up to 2.5-mM 2,3-BDO from crystalline cellulose and xylan and 0.2-mM 2,3-BDO directly from unpretreated switchgrass. This serves as the basis for higher yields and productivities, and to this end, limiting factors and potential genetic targets for further optimization were assessed using the genome-scale metabolic model of C. bescii.IMPORTANCELignocellulosic plant biomass as the substrate for microbial synthesis of 2,3-butanediol is one of the major keys toward cost-effective bio-based production of this chemical at an industrial scale. However, deconstruction of biomass to release the sugars for microbial growth currently requires expensive thermochemical and enzymatic pretreatments. In this study, the thermo-cellulolytic bacterium Caldicellulosiruptor bescii was successfully engineered to produce 2,3-butanediol from cellulose, xylan, and directly from unpretreated switchgrass. Genome-scale metabolic modeling of C. bescii was applied to adjust carbon and redox fluxes to maximize productivity of 2,3-butanediol, thereby revealing bottlenecks that require genetic modifications.