【作者】 孙丽蓉；

【导师】 曲东；

【作者基本信息】 西北农林科技大学 ， 农业环境保护与食品安全， 2007， 博士

【摘要】 异化Fe(III)还原作用是一种微生物代谢,该过程使有机或无机的电子供体以Fe(III)作为终端电子受体而被氧化,使Fe(III)还原为Fe(II)。在自然界中只要有厌氧环境,几乎都会发生异化Fe(III)还原现象,并发现有异化铁还原微生物存在。然而直到最近10年,异化铁还原的重要性才逐渐为人们所认识。人们推测,异化Fe(III)还原可能是地球上最早的呼吸形式之一,在生命的进化历程中这一遗传烙印在一定范围和一定程度上都会被继承,且势必有更多的微生物种类都具有还原Fe(III)的能力或潜能。因此,加强异化Fe(III)还原研究对于揭示地球早期生命的能量代谢特征和生命进化具有重要的理论意义。水稻土作为研究氧化还原过程的一种模式系统一直受到人们的重视,并且其中铁的氧化还原占有重要地位。渍水土壤中氧化铁的还原作用在很大程度上受微生物的影响。在通常条件下,土壤氧化铁以微生物还原方式为主。所以,研究不同水稻土中Fe(III)微生物还原的特征及影响因素,不仅可深化人们对水稻田微生物生态的认识,而且对于阐明水稻土的形成及稻田生态系统中污染物的转化与修复机理具有重要的意义。水稻土中的异化铁还原过程与电子供体、受体、电子穿梭物质的作用密不可分,受到温度、光照等环境条件的影响。本文采用恒温厌氧培养的方法,较为系统的研究了不同电子供体及浓度、不同电子穿梭物质及浓度、不同电子受体及浓度对异化铁还原过程的影响。针对微生物生长的温度和光照等环境条件,探讨了水稻土中异化铁还原过程的变化。通过富集分离水稻土中的铁还原微生物,采用16S rDNA序列分析对P4菌株进行了系统分类鉴定。本论文的研究内容及获得的主要结果包括以下方面:1.研究了有机酸、糖类物质等电子供体对人工合成无定形氧化铁和水稻土中Fe(III)异化还原的影响。通过接种来源于不同水稻土中的混合微生物群落的混合培养试验发现,在缺乏电子供体的情况下,Fe(OH)3还原过程基本不会发生,表明体系中氧化铁的还原反应是典型的微生物学过程。添加不同电子供体后,不仅可改变铁还原过程中的Fe(II)最大累积量,也将导致Fe(III)还原过程的速率常数改变。利用有机酸盐作为电子供体时Fe(II)最大累积量大小顺序为:乳酸盐﹥丙酮酸盐﹥乙酸盐,铁还原率分别为56.70%(乳酸盐),45.47%(丙酮酸盐),35.10%(乙酸盐),Fe(II)的累积速率常数顺序为乳酸盐(0.816 d-1)﹥乙酸盐(0.520 d-1)﹥丙酮酸盐(0.318 d-1)。有机酸盐作为电子供体对异化铁还原过程的影响作用与其分子结构比如碳链长度、官能团等有关。糖类物质等作为电子供体也可以增加Fe(II)累积量和累积率常数,且在本试验条件下促进效果随浓度增加而增加。Fe(III)还原率分别为94.40%(葡萄糖),93.23%(果糖),97.38%(淀粉)和87.68%(纤维素)。速率常数分别为葡萄糖(0.937 d-1),果糖(1.035 d-1),淀粉(0.218d-1),纤维素(0.183 d-1)。土壤泥浆恒温厌氧培养试验表明,淀粉、纤维素等多糖可以促进水稻土中的异化铁还原,其浓度在0-20 g/L范围内时,Fe(II)最大累积量和速率常数随浓度增加而增大,其对土壤中Fe(III)氧化物异化还原的促进作用与土壤pH、有机质和无定形铁含量有关。对培养过程中pH与异化铁还原动力学数据的比较发现,在微生物正常生长的pH范围内,较低的pH利于Fe(III)的还原。2.研究了Cr(VI)、Cu(II)、As(V)和SO42-作为竞争电子受体时对水稻土中异化铁还原过程的影响。发现Cr(VI)作为竞争电子受体可使Fe(II)生成的时间滞后,且Cr(VI)浓度越大其生成Fe(II)滞后的时间越长。当Cr(VI)完全还原后,Fe(II)的积累才迅速出现。Cr(VI)的影响表现出典型的“生物—非生物联合转化”过程,即Fe(III)通过生物还原产生Fe(II),Fe(II)作为还原剂促使Cr(VI)向Cr(III)的化学转化。Cu(II)作为竞争电子受体时,Fe(II)的累积量和铁还原速率常数降低,且Cu(II)浓度越大对铁还原的影响也越大。当Cu(II)添加量为800 mg/kg时,铁还原被明显抑制。添加不同浓度As(V)后,土壤中Fe(II)的还原也被抑制,且随着浓度的增加抑制越明显。但是,As(V)的还原转化与Cr(VI)的还原过程有所区别。由As(V)和As(III)的形态分布看出,在铁还原同时存在下As(V)不可能完全被还原,反映出Fe(III)在反应过程中具有重要的调节机制。硫酸盐作为竞争电子受体时可增加水稻土中Fe(II)的最大累积量,且随硫酸盐浓度增大而增加,但Fe(II)的累积速率常数却随硫酸盐浓度的增加而持续降低。进一步的研究表明,土壤中异化铁还原微生物可以利用的碳源越多,SO42-对Fe(III)还原的影响越明显。3.采用微生物混合培养和土壤泥浆厌氧培养方法,研究了电子穿梭物质AQDS、黄腐酸(FA)对Fe(OH)3及不同水稻土中铁氧化物异化还原的影响。研究发现,在电子供体充足的情况下,Fe(OH)3的最终还原量并不受AQDS和FA加入量的影响,AQDS和FA对异化铁还原过程的促进作用主要是表现在增加Fe(OH)3还原的反应速率上。分别向浙江、吉林、四川和江西水稻土中添加AQDS后,土壤中Fe(II)的累积量和累积速率常数均有明显增加,表明AQDS对促进土壤中晶体氧化铁的微生物还原具有重要作用。4.采用土壤泥浆厌氧恒温培养和转温培养的方法,研究了温度对水稻土中异化铁还原过程的影响。在10℃-50℃范围内,温度升高不仅可以增加水稻土中Fe(II)的累积量也可以增加Fe(II)的累积速率。水稻土异化铁还原过程中Fe(III)/Fe(II)比值能够反映培养体系中Eh的变化,升高温度可导致Fe(III)/Fe(II)比值迅速降低,还原能力增加。当土壤中有机质含量较高时,有利于微生物铁还原反应的进行,其氧化还原电位(Eh)也相对较低。当温度升高时,加速的有机质的分解,可增加土壤中氧化铁的还原潜势,导致微生物铁还原过程加速,其Eh值也下降迅速。温度升高可促进微生物对于游离铁的利用,从而促进了水稻土中的异化铁还原过程。由转温培养试验看出,温度对铁还原过程的影响可能是加速反应初期有机质的迅速矿化,产生易被铁还原微生物利用的小分子碳源,从而促进土壤中氧化铁的还原。而当培养一段时间后再升温时,易分解的有机质已有所消耗,温度的效应也随之减弱,故Fe(II)的最大累积量也有所降低。5.采用土壤泥浆恒温厌氧培养的方法,模拟自然条件设置光照培养、避光培养、光照转避光培养及避光转光照培养四种模式,研究了光照对水稻土中异化铁还原过程的影响,并采用光学摄影显微镜初步鉴定了光合微生物的特征。研究表明,光照对水稻土异化铁还原过程的影响因土壤种类而异。光照条件对某些酸性土壤中(江西和湖南水稻土)氧化铁的还原过程几乎不产生影响;而在一些石灰性土壤中(四川和天津水稻土),在光照培养初期与避光培养基本相同,均表现为Fe(II)生成量逐渐升高的趋势。随着光照导致的蓝细菌的不断产生,光照处理中Fe(II)浓度开始降低,较之避光培养Fe(II)浓度在2种水稻土中分别降低了54.75%和78.13%。进一步的光照条件转化试验发现,石灰性土壤由避光转为光照培养后,Fe(II)生成量经过一段时间滞后,然后急剧降低,而由光照转为避光培养后,Fe(II)浓度均迅速增大,表明光照可以引起土壤中铁氧化还原平衡状态的变化。对厌氧光照培养条件下出现的绿色微生物菌体进行镜检及叶绿素光谱扫描发现,石灰性土壤在光照培养条件下可产生具有光合放氧能力的蓝细菌,其特征光吸收波长为664nm。验证了光照体系中土壤Fe(II)浓度降低是由于蓝细菌利用光合系统II进行产氧光合作用引起的化学氧化所导致的。根据镜检初步确认,天津水稻土中的蓝细菌主要是念珠蓝细菌属,四川水稻土中主要是鱼腥蓝细菌属。硫酸盐加入后可以显著抑制光合蓝细菌繁殖,延迟Fe(II)的光氧化过程。6.采用富集培养方法,从四川水稻土中分离得到一株兼性厌氧、革兰氏阴性的杆状菌株P4。采用PCR技术获得了1325 bp的铁还原菌株(P4)部分16S rDNA序列。经NCBI的MegaBlast同源性分析和构建铁还原菌株P4与相关菌株16S rDNA的系统进化树,将P4菌株归属为厌氧丁酸梭状芽胞杆菌。不同碳源利用试验表明,铁还原的最大反应速率大小顺序为:葡萄糖>丙酮酸盐>乳酸盐>>琥珀酸盐>丙酸盐>乙酸盐。菌株P4利用葡萄糖的Fe(III)还原率达到63.79%,丙酮酸盐为22.19%,而其他几种碳源的铁还原率均在10%以下,显示出葡萄糖和丙酮酸盐可作为菌株P4的优势碳源。通过本文的研究,对水稻土中电子供体、电子受体及电子穿梭物质等因素与异化铁还原的关系有了更为深入的了解,验证了培养温度及光照等环境条件对土壤泥浆培养过程中异化铁还原反应的影响程度,探讨了水稻土中异化铁还原过程的调控强化措施,加深了对水稻土中异化铁还原过程的认识,并为利用异化铁还原过程处理环境污染及其主要调控措施提供了理论依据,将为土壤环境的自净化潜能研究及污染土壤的生物修复技术建立提供新的思路。更多还原

【Abstract】 Dissimilatory Iron Reduction (DIR), a kind of metabolism of microorganism, and Fe(III) as the terminal electron acceptor was reduced to Fe(II) in this process, coupled with the oxidation of organo/abio electron donors. DIR occurs generally in the natural anaerobic environment, where Dissimilatory Iron Reduction Bacteria (DIRB) exists universally. But, it was not focused on till last decades. Hypothetically, it is the first form of microbial respiration. Since it could be inherited in some degree in the evolution process, there must be much more microbial are capable to dissimilatory reduce Fe(III). Therefore, investigations focused on DIR process could help better understanding the evolution process and the energy metabolism in early stages, theoretically. Redox of iron oxides is prevalent in paddies, the model system in studying the redox process in natural eco-systems. It is influenced by microbial under waterlogging soil system. Study on characteristics of dissimilatory reduction of iron oxides in paddies and its influence factors, might help better understanding the paddies formation, its microbial ecology, also could help to elucidate the remediation and transformation of contamination in paddy soils.DIR is closely related to the electron donors, acceptors and shuttles, affected by environmental conditions such as temperature, light. In the dissertation, effects of electron donors such as organic acids, saccharides, acceptors such as Cr(VI), Cu(II), SO42-, As(V) , and electron shuttles such as AQDS, FA, on the DIR process were investigated, and environmental conditions such as temperature, light were also discussed. 16S rDNA sequencing analyses was employed to genealogical classified the Fe(III)-reducers isolated from paddies using purified amplification culture method. The main objectives and results obtained are listed as follows:1. Effects of electron donors such as organic acids, saccharides on the dissimilatory reduction of amorphous Fe(OH)3 synthesized in the laboratory was studied by Co-culture incubation method. Control experiments showed that there is no obviously accumulation of Fe(II) in the system in the absence of electron donors, which suggested that the DIR is a microbiological process, typically. Addition of electron donors such as organic acids could enhance the accumulation of Fe(II) in the system and the rate constant of the DIR reaction. The dissimatory reduction ratio of Fe(OH)3 is 56.70%, 45.47%, 35.10%, and the rate constant is 0.816, 0.318, 0.520 d-1, in the presence of lactate, pyruvate, and acetate, respectively. The enhancement of organic acids is related to its molecule structure such as carbon chain length and function group. In addition, electron donors such as saccharides could also enhance the accumulation of Fe(II) in the system and the rate constant of the DIR reaction, and the accumulation of Fe(II) and the rate constant could be enhanced by the addition of saccharides concentration increased. The dissimilatory reduction ratio of Fe(OH)3 is 94.40%, 93.23%, 97.38%, 87.68% and the rate constant is 0.937, 1.035, 0.218, 0.183 d-1, in the presence of glucose, fructose, starch, and cellulose, respectively. Effects of amylose such as starch and cellulose on the dissimilatory reduction of iron oxides in the paddy soils were also investigated by soil slurry anaerobic incubation method. The acumulation of ferrous and the rate constant could be enhanced by the addition of starch and cellulose with its concentration increased from 0 to 20 g L-1. The enhancement is correlated with soil characters such as pH, organic matter, and amorphous iron content. Compare with the kinetics parameters of Fe(III) reduction in anaerobic incubation, in the range microbe normal growing pH, the lower pH is more appropriate.2. Cr(VI), Cu(II), As(V) and SO42- could be used as alternative electron acceptor by Dissimilatory Iron Reduction Bacteria. The investigation showed that Fe(III) reduction started only when the reduction of Cr(VI) was accomplished, and the generation of ferrous was delayed by the addition of alternative electron acceptors as Cr(VI), and it was accelerated by the increasing concentration of Cr(VI), suggested that the effect of Cr(VI) on the DIR might be an combined bio-abio transformation, in which Cr(VI) could be reduced by Fe(II) generated from DIR. When Cu(II) was used as alternative electron acceptor, both the accumulation of Fe(II), and the rate constant of DIR were decreased, and accelerated by the increasing concentration. The DIR was inhibited obviously when the concentration of Cu(II) added increased to 800 mg L-1. As(V) could served as an alternative electron acceptor, thus inhibit the DIR with the increasing concentration. Compared with Cr(VI), As(V) could not be reduced entirely, which suggested Fe(III) could regulate dissimilatory reduction of As(V) under iron-reducing conditions. Though the rate constant was decreased with the increasing concentration of sulphate, the cumulation of Fe(II) during the reduction process was increased. And the further investigation showed that the decrease of the rate constant was accelerated with the increasing electron donors could be used by DIRB.3. Co-culture incubation and soil slurry anaerobic incubation method were employed to study the effect of electron shuttles on the dissimilatory reduction of Fe(OH)3 and iron oxides in different paddy soils such as ZJ, JL, SC, and JX. The obtained results showed that the accumulation of Fe(II) caused by dissimilatory reduction of Fe(OH)3 was not affected by the addition of AQDS or FA, but the rate constant was increased when the electron shuttle such as AQDS or FA was added. As contrast, the accumulation of Fe(II) caused by dissimilatory reduction of iron oxides in paddy soils and the rate constant was increased by the addition of AQDS.4. Soil slurry incubated under anaerobic constant and variable temperature to investigate the effect of temperature on DIR process. The result showed that, both Fe(II) accumulation amount and rate constant were increased at higher temperature. The Fe(III)/Fe(II) ratio could has a corelationship with Eh. Decomposition of soil organic matter was improved at higher temperature, thus the reductive capacity of iron oxides in the soils was increased, and the Fe(III)/Fe(II) ratio decreased dramatically with the increase of temperature at the meantime. Therefore, DIR process was improved, and the Eh was decreased at the same time. And the consumption of free iron oxides by DIRB was increased with the increase of temperature, which could also improve the DIR process. The results from soil slurry incubation under variable temperature also showed that, the effect of temperature might closely related to the mineralization of soil organic matter, which could provide a low molecular carbon source-facilitated the consumption by DIRB. Thus, the maximum Fe(II) accumulation amount might decreased under the higher temperature only when soil slurry incubation under constant temperature first. This might attributed to the depletion of soil organic matter which was facilitated to mineralization.5. Soil slurry was incubated under anaerobic constant temperature conditions, treatments including illuminous incubation, dark incubation, illuminous to dark incubation, and dark to illuminous incubation were setup to simulate natural conditions to investigate the effect of illumination on the dissimilatory reduction of iron oxides in paddy soils. Under illuminous conditions, the trend of Fe(II) vs incubation time is different in anaerobic incubated calcareous paddy soils, it increased at the initial stages and then decreased to a very low concentration. To SC and TJ, the accumulation of Fe(II) was decreased in by 54.75% and 78.13%. When illumination was ceased, Fe(II) content increase dramatically. Phototrophic bacteria, identified to be cyanobacteria by Optics Image microscope, can oxidizing Fe(II) to Fe(III) through photosynthesis respiration by photosynthetic system II which producing O2. Added sulphate can inhibit the process of Fe(II) oxidation, and the phototrophs bacteria were poisoned by excess sulphate.6. An anaerobic, gram-negative, rod-shaped organism P4 was isolated from Sichuan paddy soil using purified amplification culture method. Adopting the PCR acquired parts of 16S rDNA sequences of Fe(III)-reducting strain P4 with 1325 bp. Through NCBI MegaBlast homology analysis and 16S rDNA phylogenetic tree construction, strain P4 belonged to Clostridium butyricum. With different carbon sources, the maximal reaction rate of iron reduction was in proper order for: glucose>pyruvate>lactate>>succinate>propionat >acetate. With glucose as carbon source, The dissimatory reduction ratio of Fe(OH)3 is 63.79%, pyruvate is 22.19%, the others are all below 10%, showing glucose and pyruvate be the dominant carbon source for strain P4.Aimed at the dissimilatory reduction of iron oxides in paddy soils, taken the environmental conditions into account, the DIR process was investigated by Co-culture and soil slurry incubation method under anoxic conditions. Effect of intrinsic factor such as electron donors, acceptors, shuttles, and environmental factors such as temperature, light were investigated to disclose mechanism of dissimilatory iron reduction. This work indicated that the DIR process could be regulation and controlled to decontaminate reducible pollutants in natural environments, and will be helpful to understand the DIR process and would also provide basic understanding to the natural attenuation of reducible organic pollutants in the contaminated sediments, subsurface environments, and soil profiles. This investigation provides a feasible remediation technology and might potentially be used in conjunction with the existing remediation strategies.更多还原