【作者】 林惠荣；

【导师】 郑平； 陈英旭；

【作者基本信息】 浙江大学 ， 环境工程， 2010， 博士

【摘要】 稻田土壤不同区域中氧化还原电位变化影响功能微生物硫氧化菌和硫还原菌的种群分布,导致土壤硫形态转化,并影响重金属迁移转化。功能微生物作用下的金属硫化物的形成和氧化对于控制重金属的迁移转化有重要意义。随着环境分子科学及分子生物学的发展和研究方法的突破,从分子水平深入认识重金属在环境中的反应机制成为环境科学新的研究热点。本论文在传统分析方法的基础上,通过引入同步辐射x射线吸收光谱技术(XAS)、X射线荧光(XRF)微区XAFS以及PCR-DGGE、FISH和分子克隆等技术,研究水稻土壤重金属和硫分子形态转化的规律及其相互作用,考察硫氧化还原菌种群结构,探索其对金属形态转化的作用机制。主要研究结论为：明确了土壤-水稻系统中重金属Cu和硫的形态转化及相互关系。水稻根际土及非根际土中Cu的螯合官能团差异较大。根际土中Cu主要以Cu(Ⅱ)为主,而非根际土中有部分Cu(Ⅰ)存在。根际土中氧化态硫的含量比非根际土中高。稻田土壤不同区域的氧化还原电位变化导致硫的形态发生转化,并影响Cu的形态。Cu的解毒机理可能与铜和硫基团的高结合性有关。Cu-组氨酸和Cu-柠檬酸类似基团是水稻根中铜的主要分子形态,此外还有少量的Cu-谷胱甘肽类似基团,这些基团与重金属Cu的络合可能参与了水稻体内Cu的解毒。揭示了硫底物对重金属Cu、Zn、Pb的生物有效性及土壤微生物群落结构的影响。硫氧化菌在氧化条件下氧化低价态硫,降低土壤pH值,导致Cu、Zn、有效性显著提高,而Pb的有效性没明显变化。PCR-DGGE结果表明铜处理水稻根际土的微生物群落结构发生显著变化,而铅处理土壤微生物多样性的变化并不显著。硫底物作用下有特异性条带产生,经鉴定为硫杆菌。硫的形态转化对土壤重金属的有效性产生重要影响。明确了水稻根际硫氧化还原菌的种群结构及其与重金属Pb的相互作用关系。FISH结果表明水稻根际土中硫氧化菌的数量比硫还原菌多,硫氧化菌和硫还原菌共同作用促进硫的形态转化,导致重金属沉淀或释放,控制重金属迁移转化。根际土中硫的形态主要为氧化态,还有少量的负二价硫存在。水稻根际有少量耐氧的SRB子群促进硫还原反应进行。随着铅浓度的增加,根际土Eh值下降,pH值上升,SRB活性增强。水稻根际存在高效的SRB子群,能够存在于较高浓度Pb污染的土壤环境中,这对于较高Pb污染土壤中Pb污染控制具有重要意义。从水稻根际土中筛选出一株硫氧化菌HT1,证明该菌株属于硫氧化菌中的盐硫杆菌属Halothiobacillus。该菌株可利用硫代硫酸盐,连四硫酸盐、单质硫和亚硫酸盐等硫底物当电子供体,且耐盐(0-3 MNaCl)和耐重金属。菌株HT1不含soxB基因,其代谢途径是通过形成连四硫酸盐作为中间产物并被氧化的硫代硫酸盐,营该代谢途径的细菌会在体内聚集硫球,硫的K边XANES分析表明菌株HT1体内硫球形态主要为S8。初步探讨了硫氧化菌HT1与重金属的相互作用机制。吸附Cu2+,Pb2+和Zn2+后菌株HT1体内多数硫转化成半胱氨酸以络合重金属离子,起到提高耐性和解毒的作用。与金属硫化物作用后菌株HT1氧化硫化物中的负二价硫,形成高价硫。还原态硫也转化半胱氨酸,参与重金属的解毒。HT1生物膜上层、内层与培养基交界层是活性区域,氧含量对细胞膜中细菌的活性有重要影响。在LB培养基上与金属硫化物作用后,HT1生物膜上分别吸附了一定量的Pb、Cu,但基本不吸附Zn。XFR研究结果表明在CuS表面生长的HT1生物膜Cu主要分布在内层培养基界面层,其次是上层,而中间层铜含量较低。而在PbS上HT1生物膜Pb主要分布在上层。HT1对不同金属有选择性吸收或吸附,Zn对HT1毒性不大,因此ZnS上HT1的活细菌比CuS和PbS上的多。μ-XAFS研究结果表明生物膜不同水平微区与铜结合的基团有所不同,而Cu-半胱氨酸类似基团是生物膜中铜结合的重要配体,参与Cu的解毒。更多还原

【Abstract】 In paddy soils, changes of redox conditions in different zones led to microbially mediated sulfur transformation, thus affecting heavy metal behavior. Microbe-mineral interactions play an important role in affecting geochemical transformations of heavy metals in the soil environment. With the developing of molecular environmental science and the evolution of molecular biology technologies in recent years, a late hotspot in the environmental science is how to understand the reaction mechanisms of heavy metals in environment medium at molecular scale. Based on the traditional analysis methods, the synchrotron radiation X-ray absorption spectroscopy (XAS), the synchrotron radiation X-ray fluorescence (XRF), as well as PCR-DGGE, FISH and molecular cloning were used in present study. The aim of our study was to clarify heavy metals and sulfur transformation and the relations between these processes under different conditions, and microbial community diversity and changes of biochemistry characters. The main results were as follows:The speciation of sulfur and copper in rice rhizosphere and bulk soil was investigated using integrated approaches including sequential extraction and XANES. Cu speciation exhibited some differences in rhizosphere and bulk soil. Most Cu in the rhizosphere existed as Cu(II), whereas part of Cu transformed to Cu(I) in the bulk soil. Sulfur XANES showed the presence of multiple both oxidized and reduced forms of sulfur, with more oxidized sulfur in the rhizosphere than in the bulk soil. Changes in redox potential and microbial action shifted the sulfur oxidation and reduction reaction and affected the Cu speciation. Combined action of organisms maintained Cu homeostasis through cation binding to bioactive molecules. Cu bond to histidine and citrate groups as well as glutathione in rice could be defenses against toxic copper.The effect of sulfur on the availability of Cu, Zn, Pb and the microbial community composition in rice rhizosphere soil was studied. Under the impact of sulfur, the availibity of Cu and Zn increased dramatically, while Pb did not. Accordingly, PCR-DGGE experiment suggested that sulfur action led to dramactic changes of soil bacteria composition in Cu polluted soil, while there were no obivious differences in Pb polluted soil. Some specific clones found in S addition soils had high similarity to Thiobacillus, which indicated relatively high rates of potential S oxidation.The compositions of SOB and SRB and their relationship with Pb were studied in rice rhizosphere. FISH results showed more SOB than SRB. Combined action of SOB and SRB contribute to transformation of heavy metals. The formation of metal sulfide, which is mediated by SRB through contributing to sulfate reduction is an important pathway for heavy metal stabilization in anoxic soil. The mechanism of SOB and SRB on transformation of sulfur and heavy metals was studied. PCR-DGGE fingerprinting and real-time PCR results showed increasing numbers of SRB with Pb addition, which corresponded with increases in soil pH and reduction in Eh, suggesting the enhancement of sulfur reduction and SRB activity. Sulfur K-edge XANES revealed reduced states of sulfur. The SRB mediated the sulfate reduction and contributed to the formation of reduced sulfur which interacted with Pb, leading to the formation of stable metal sulfide. In return, acclimated SRB populations developed in Pb-polluted conditions. Hence stabilization of reduced sulfur by Pb enhanced the activity of SRB and sulfate reduction in rice rhizosphere.A mesophilic, chemolithoautotrophic, SOB named HT1 was isolated from a rice rhizosphere soil polluted by Pb. The 16S rRNA gene sequence showed that it was a S oxidizing obligate chemolithotroph belonging to Grammproteobacteria, Halothiobacillus utilizing sulfide, elemental sulfur, thiosulfate and sulfite as substrates. Strain HT1 was able to use CO2 as a carbon source and was responsible for the reduction of nitrate to nitrite and represented a halophilic SOB capable of growth within 0 to 3 M NaCl and a heavy-metals-tolerant SOB. The soxB gene could not be detected in strain HT1. Its metabolism pathway was’S4 intermediate’(S4I) pathway. Sulfur globules accumulated in strain HT1 were mainly S8.It was illustrated that cysteine was involved in chelation within strain HT1 when interacted with Cu2+, Pb2+and Zn2+. Strain HT1 could oxidize sulfide in CuS, PbS and ZnS and cysteine was also synthesized for the tolerance against heavy metal. There were two active zones in the biofilm of HT1:air-biofilm interface and medium-biofilm interface. Oxygen played an important role in the distribution of live cells. The biofilm of HT1 presented different spatial distribution when reacted with different metal sulfides. Strain HT1 did not absorb Zn which resulted in more live cells of HT1 on ZnS than on CuS or PbS. XRF results showed that Pb was mainly on the upper layer while Cu was mainly on the under layer of the biofilm. Withμ-XAFS, it was found that ligands bond to Cu were different. Whiel Cu-cysteine was found to be an important ligand both in the middle and edge of the biofilm.更多还原