【作者】 李乾；

【导师】 刘学端；

【作者基本信息】 中南大学 ， 生物工程， 2012， 博士

【摘要】 铀作为核燃料的一种能源,随着核工业的日益发展,高品位铀矿逐步耗竭,造成了低品位矿/尾矿的大量累积。生物冶金技术由于经济、环保等优势适合处理这些低品位矿/尾矿,但生物浸铀技术在工业应用过程中仍面临着工艺因素的合理调控与菌种的耐受性等难题,因此,探讨如何合理调控生物氧化浸铀工艺与优化微生物群落结构有很有价值的意义,以及从功能基因组学角度研究浸矿微生物的耐氟机理对高耐氟菌种的合理选育和驯化具有很强的理论指导作用。本文针对以上难题开展了低品位铀矿微生物浸出过程中的多因素影响规律以及浸矿菌种耐氟机理两方面的研究。一方面,针对生物浸铀工艺调控的合理性,探讨了低品位铀矿生物浸出体系中的多种工艺因素对铀浸出效率的影响规律,并且分析了浸出过程中的微生物群落结构；另一方面,针对浸矿菌种对氟的耐受性问题,从功能基因组学角度研究了单一菌与混合菌的耐氟机理。具体研究内容与结果主要包括以下六个方面。1、探讨了低品位铀矿生物柱浸过程的工艺因素影响规律低品位铀矿微生物柱浸研究表明,常温微生物富集培养物对低品位铀矿展现出了良好的浸矿性能,97天柱浸过程中(包括33天酸预浸和64天微生物浸出)铀的浸出率达到了96.8%。其中微生物作用阶段铀的浸出率有74.5%,占总浸出量的约3/4,而耗酸量不到总量的3/8。分析表明,可以通过适当控制溶浸液中的微生物群落结构,以及铁和其他离子的含量,来间接调节氧化还原电位,从而促进铀的快速浸出。并且,采用相关性分析软件Canoco for Windows (version4.5)对工艺因素与铀浸出效率的相关性(CCA)进行分析,结果表明,浸出初期(1-27天),铀的浸出速率处于延缓阶段,pH、喷淋强度及耗酸量对铀浸出效率起到了主导作用；浸出中期(39-87天),铀的浸出速率处于较为快速的阶段,Eh、Fe3+/Fe2+比及液固比是对铀浸出效率的关键因素；在浸出后期(88-97天),铀的浸出速率已经非常缓慢,受液固比的影响比较大。2、研究了低品位铀矿生物柱浸过程中微生物群落演替规律运用分子生物学技术(ARDRA)检测了低品位铀矿柱浸体系中游离微生物(溶浸液与浸出液)及吸附微生物(矿物表面)的群落结构。微生物群落分析结果表明,A. ferrooxidans和L. ferriphilum一直都是铀矿浸出体系中的主要群落,不论在溶液中还是矿物表面；而且吸附在矿物表面的微生物多样性比游离在溶液中的微生物更丰富；溶浸液和浸出液中的优势种群是A. ferrooxidans,而吸附在矿物表面的优势种群是L. ferriphilum。3、对比研究了五种典型浸矿微生物的耐氟性状通过比较五株浸矿微生物(A. ferrooxidans ATCC23270, L. ferriphilum YSK, S. thermosulfidooxidans ST, A. thiooxidans A01, A. caldus S1)在不同浓度氟胁迫下的生长状态与铁硫氧化活性,结果表明,A. ferrooxidans的氟耐受性是最高的,其次是A. thiooxidans,再次是L. ferriphilum与A. caldus,最差的是S. thermosulfidooxidans,并表明,中度嗜热浸矿菌比常温浸矿菌更容易被氟离子所抑制。4、应用全基因组芯片研究了嗜酸氧化亚铁硫杆菌在氟胁迫下的基因调控机理通过全基因组芯片对A. ferrooxidans在4.8mM氟胁迫下的基因表达谱研究,结果显示,在氟胁迫的240min内,A. ferrooxidans ATCC23270总共有1354个基因受到氟胁迫后表达发生了差异表达(均以2倍变化为临界值,T检验,P<0.05),约占全基因组芯片所检测基因总数(3217)的42.08%。分析表明,与氟胁迫密切相关的基因信息主要涉及到细胞膜、能量代谢、转运与结合蛋白、DNA代谢、细胞处理、蛋白质的合成与命运、生物因子的合成等多个方面功能的代谢途径,这些基因信息为芯片快速筛选高耐氟菌株提供了重要的依据。通过生物信息学相关分析,初步阐述了A. ferrooxidans在氟胁迫下的基因调控机理：A. ferrooxidans在应对氟胁迫时,通过调节细胞膜上胞壁质、肽聚糖、多聚糖和脂蛋白的组成以及非饱和脂肪酸的比例乃至结构或构象,来维持细胞膜的渗透平衡与流动性等生物活性；以及高效表达解毒系统的相关抗性蛋白、离子运输通道蛋白、及转运调节子等,同时关闭某些离子通道进而转向对氟耐受性更有利的途径；其次,还能通过加强磷代谢合成磷脂分子来维持细胞膜的完整性和流动性。另外,在短期氟胁迫下,通过加强中间碳代谢来为细菌抵抗不良环境提供能量。最后,细胞能够通过加强氮磷代谢相关途径来减少或修复氟胁迫对蛋白质、核酸造成的损伤。有意思的是,氟胁迫虽然抑制了A. ferrooxidans的生长繁殖,却能提高单个细胞的铁氧化速率。5、研究了五种浸矿微生物共培养体系的耐氟特性及其在氟胁迫下的种群动态通过分析五种浸矿细菌的共培养体系在不同浓度的氟胁迫下的生长状态和铁硫氧化活性,表明氟胁迫在一定程度上抑制了该共培养体系的生长繁殖,对于高浓度的氟胁迫共培养体系保持较为平稳的生长延滞状态；单就铁氧化活性来说,共培养体系不如单一的铁氧化细菌；就硫氧化活性来说,单一的硫氧化细菌在氟胁迫时硫氧化活性基本消失,而共培养体系硫氧化活性的保持能力得到了明显的增强。通过Real-time PCR技术进行种群动态分析,结果表明,L.ferriphilum YSK和A. caldus S1为该共培养体系的优势种群；而A.ferrooxidans ATCC23270、S. thermosulfidooxidans ST、A. thiooxidansA01为劣势种群。共培养体系中受氟胁迫影响最大的是S.thermosulfidooxidans ST;其次(?)(?)A. caldus S1、A. thiooxidans A01、A. ferrooxidans ATCC23270;而L. ferriphilum YSK在氟胁迫下保持非常稳定的生长。6、应用功能基因芯片研究了浸矿微生物共培养体系在氟胁迫下的基因调控机制通过功能基因芯片(FGA-Ⅱ)对该共培养体系在4.8mM氟胁迫下的基因表达谱研究,探明了该共培养体系中与氟胁迫相关的基因主要涉及到硫代谢、细胞膜、电子传递、解毒、碳固定、氮代谢等多个方面功能的代谢途径,而且各个途径在短时间(<60min)氟胁迫倾向于高效表达,而长时间(>120min)氟胁迫各个途径更倾向于低效表达。芯片图谱分析表明,氟胁迫下共培养体系中起主导调节作用的是其中的优势种群,但是劣势种群在氟胁迫时很大程度上辅助了优势种群的生长及其氧化活性的保持。更多还原

【Abstract】 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.更多还原