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AuthorJugdave, Abhita Gyanendra
SubjectMicrobiol-, Biochemical and Food Biotechnology
AbstractThe mining industry has provided researchers access to the deep subsurface. The deep subsurface is known to harbor a treasure of novel genes and proteins that can be exploited in the biotechnology industry. It has been established that microorganisms in the deep subsurface are potentially novel and are able to endure high temperatures and extreme pH with limited nutrients for survival. Unfortunately most of these organisms are unculturable. Due to the lack of nutrients these microorganisms utilize reduced metals and minerals from the environment as a source of survival in a process known as biogeochemical cycling. Two fissure water samples were collected from two borehole sites at the Northam platinum mine and analyzed through molecular approaches. Microbial biodiversity was determined for borehole NO24FW030908 fissure water sample. The microbial biodiversity was based on the 16S rRNA and 18S rRNA gene clone libraries determined through phylogenetic clustering analyses using ARB software and a comparative analysis was done using DGGE profiling. The prokaryote and eukaryote diversity revealed low diversity at the species level but a high intraspecies diversity probably associated with the novelty of the biome. Unique isolates were cultured from borehole NO212FW050508 fissure water sample. Five isolates showed novelty at the species level and one isolate showed novelty at the genus level. Two isolates, Geobacillus sp. A8 and Geobacillus sp. A12 were sent for characterization at the DSMZ, Germany. Both isolates exhibited similarities at the genus level but significant differences at the species level based on the type strain to warrant different taxonomical positions. These isolates along with Thermus scotoductus SA-01 were analyzed for platinum reduction and the possible formation of metallic platinum. All isolates showed the ability to reduce platinum (IV) to platinum (0). Geobacillus sp. A8 was selected for further characterizations of platinum nanoparticle formation. Platinum nanoparticles were characterized with various tools to show the size and shape using TEM and SEM, to show the composition using XRD and EDS, and to show the particle size distribution using the NanoTrac and NiComp 380 ZLS systems. It has been proposed that a classical hydrogenase activated by a cytochrome c3 is responsible for the two-step reduction of platinum. Therefore, hydrogenase inhibition tests and the TTC test for the presence of an active hydrogenase were done to confirm the presence of a hydrogenase in Geobacillus sp. A8. It was then selected for the construction of the metabolic pathway genome database to study the metabolism of the microorganism in order to identify alternate or novel pathway(s) associated with the genome. Platinum reduction activity tests based on subcellular fractionation revealed platinum reduction and deposition that occurred in the periplasm; therefore, the putative protein involved in platinum reduction was probably periplasmic. The periplasmic fraction was separated into three fraction sizes, greater than 30 kDa, between 10 and 30 kDa and less that 10 kDa. The 10 รข 30 kDa fraction revealed positive platinum reduction. The active fraction was analyzed on a SDS-PAGE and revealed three bands that were digested by trypsin and the peptides were analyzed by protein mass spectrometry. Two proteins were identified, an oxidoreductase commonly known as the old yellow enzyme previously shown to reduce chromate (VI), and a hypothetical YajQ protein. Both proteins were expressed and purified and both proteins showed the ability to reduce platinum. Further work would be to elucidate the in situ mechanism involved in the reduction of platinum with hydrogen as the electron donor.
PublisherUniversity of the Free State