| Uranium, as a source of nuclear fuel, with rapid development of nuclear industry (eg. nuclear electricity, ship industry), the increasing application has gradually led to the exhaustion of high-grade ore reserves and accumulation of large quantities of low-grade ores/tailings. Bioleaching is a method by which the low-grade ores can be processed economically and eco-friendly, however, there are some problem in the application of uranium bioleaching, such as operational parameters regulation, microbial tolerance and so on. Therefore, it is of utmost valuable to explore how to well regulate the operational parameters and optimize microbial community structure, and it is theoretically meaningful for their rational isolation and adaptation to investigate the fluoride tolerant mechanism of bioleaching microorganisms at functional genomics level.In this study, effects of multiple factors on bioleaching of low-grade uranium, and fluoride tolerant mechanism for bioleaching microorganisms were investigated. On one hand, effects of multiple parameters on uranium dissolution efficiency in bioleaching of low-grade ores were analyzed, and it revealed the microbial community succession in the process. On the other hand, the fluoride tolerant mechanism of pure culture and mix culture was compared and investigated by using gene microarray technology. There are six aspects for detail contents and results in this study.1Effects of multiple parameters on column bioleaching of low-grade uranium ores were explored.The performance of a mesophilic microbial enrichment in column bioleaching of low-grade uranium ores was investigated, and the microbial enrichment showed strong performance. A uranium recovery of96.82%was achieved in97days column leaching process including33days acid pre-leaching stage and64days bioleaching stage. At the bioleaching stage,74.5%uranium recovery was achieved, which was3/4of the grass quantity, while the acid consumption was less than3/8in the process. It was reflected that indirect leaching mechanism took precedence over direct. It was revealed appropriate regulating the microbial community structure and iron or other ions in the leaching solution could improve the redox potential, and then indirectly improve the uranium dissolution.Moreover, the correlation of operational parameters and uranium dissolution efficiency was analyzed by Canoco for Windows (version4.5). The results showed, at the initial stage (day1-27), uranium dissolution rate was at lag phase, and the pH, spraying intensity and acid consumption played leading roles; at the middle stage (day39-87), uranium dissolution rate reached a peak phase, and the Eh, Fe3+/Fe2+and liquid/solid ratio were the predominant factors; at the later stage (day88-97), the dissolution rate was at a decline phase, and the liquid/solid ratio played a dominant role.2Microbial community successions in the column bioleaching of uranium were analyzed.Microbial community structure in the feed solution and leach liquor and on the mineral surface was analyzed by using Amplified Ribosomal DNA Restriction Analysis. The results A. ferrooxidans and L. ferriphilum were were predominant no matter where in the solution or on the residual surface. The microorganisms on the residual surface were more diverse than that in the solution. A. ferrooxidans was the dominant species in the solution and L. ferriphilum on the residual surface.3Fluoride tolerances of five representive bioleaching microorganisms were compared.The growth state and iron/sulfur oxidation activities of five representive bioleaching microorganisms(A. ferrooxidans ATCC23270, L. ferriphilum YSK, S. thermosulfidooxidans ST, A. thiooxidans A01, A. caldus S1) upon different fluoride stress were compared. It showed, the fluoride tolerance of A. ferrooxidans was best, secondly A. thiooxidans, thirdly L. ferriphilum and A. caldus, worst S. thermosulfidooxidans. Additionally, moderate thermophiles were inhibited more than mesophiles.4Gene regulation mechanism of A. ferrooxidans upon fluoride stress was studied by the whole-genome array.The gene expression profile of A. ferrooxidans ATCC23270upon4.8mM fluoride stress was investigated using the whole-genome array. The results showed,1354genes were differentially expressed upon fluoride stress for240min (2fold as threshold, P<0.05), was42.08%of microarray probes. In addition, the genes associated with fluoride stress were involved with cell membrane, energy metabolism, transport and binding proteins, DNA metabolism, cellular processes, proteins synthesis and fate, biosynthesis of cofactors, and so on, it provided some insight for quickly isolation of bioleaching strain with highly fluoride tolerance.Further analysis revealed, A. ferrooxidans could regulate the composition or ratio or structure or conformation of murein sacculus, peptidoglycan, polysaccharides, lipopolysaccharides and lipoprotein of cell membrane upon fluoride stress, to maintain membrane bioactivities such as osmotic balance and mobility and etc. The resistance proteins, ion transporter protein and transportation regulator involved with detoxification were induced, meantime, some ion channel was closed and turned to the pathway favorable for fluoride resistance. Secondly, it enhanced the phosphate metabolism to synthetize phospholipid, and further to maintain the membrane integrity and mobility. Additionally, at the short time fluoride stress, the bacteria could improve the carbon metabolism to provide the energy for resisting the severe environment. Lastly, the cells could enhance the phosphate metabolism to relieve or repair the protein and nuclear acid damage by fluoride stress. Interestingly, fluoride stress inhibited the growth of A. ferrooxidans, but improved the iron oxidation rate of single cell.5Fluoride tolerance of the co-culture of five bioleaching bacteria and the community dynamics upon the fluoride stress was studied.The growth and iron/sulfur oxidation rate of the co-culture of the five bioleaching bacteria was analyzed. The results showed, the growth of the co-culture was inhibited upon fluoride stress, and kept the stable cell density at high fluoride stress. As for iron oxidation activity, the co-culture was worse than the pure iron-oxidating bacteria. As for sulfur oxidation activity, the co-culture was better. The sulfur activity of the pure sulfur-oxidation bacteria was almost disappeared, while the co-culture's was not affected by fluoride stress.Microbial community dynamics of the co-culture was analyzed by real-time PCR. It showed that L. ferriphilum YSK and A. caldus S1was always the dominant species at fluoride stress or non-stress, while the minor was A. ferrooxidans ATCC23270, S. thermosulfidooxidans ST, A. thiooxidans A01. Besides, S. thermosulfidooxidans was inhibited most obviously in the co-culture, secondly A. caldus, A. thiooxidans, A. ferrooxidans, while the L. ferriphilum kept a very stable growth.6Gene regulation mechanism of the co-culture upon fluoride stress was studied by the functional gene array.The gene expression profile of the co-culture upon4.8mM fluoride stress was investigated using the functional gene array. The results showed, the genes associated with fluoride stress were involved with sulfur metabolism, cell membrane, electron transport, detoxification, carbon fixation, nitrogen metabolism, and so on. Additionally, majority of genes was induced upon the short time (<60min) fluoride stress, while repressed upon the long time (>120min) fluoride stress.The effects of fluoride stress on different microbial population in the co-culture were different. The results revealed that the dominant species in the co-culture played the crucial role for resisting fluoride stress, while the minor species to a large extent assisted the oxidation ability preservation and growth of the dominant species.
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