【作者】 贾蓉；

【导师】 曲东； 张海；

【作者基本信息】 西北农林科技大学 ， 环境科学， 2012， 硕士

【摘要】 水稻土中的异化Fe(III)还原过程因其具有丰富的碳水化合物作为电子供体以及庞大的微生物物种库而独具特色。土壤脱氢酶活性与微生物代谢活性相关，水稻土在淹水过程中会发生一系列的生物化学变化，其中微生物代谢产氢过程是电子传递链的重要环节。因此可以利用脱氢酶作为土壤微生物的活性指标反映土壤中的碳源被利用的程度。同时，土壤中NO3-、SO42-和Fe(III)在厌氧环境中可作为受氢体，脱氢酶活性也可间接表征土壤厌氧呼吸中反硝化、硫酸盐还原和氧化铁还原等重要的生物学变化过程。在稻田的干湿交替变化中，脱氢酶活性也是表征环境氧化还原状态的一个重要参数。因此，研究水稻土异化铁还原过程中脱氢酶活性的变化、探讨脱氢酶活性与异化铁还原过程的关系对于阐明水稻土微生物Fe (III)还原机理具有重要的意义。该论文以采自我国不同植稻区的4类水稻土为试验材料，对脱氢酶活性测定的影响因素进行研究，探讨了水稻土泥浆脱氢酶的测定方法。采用土壤泥浆培养、土壤浸提液作为微生物接种菌液的混合培养以及典型铁还原菌株的纯培养试验体系，比较了Fe(III)还原与脱氢酶活性变化的相互关系及其添加不同的碳源后对氧化铁还原、体系pH变化和脱氢酶活性的影响，对微生物Fe (III)还原过程和脱氢酶活性的动力学特性进行了表征，为揭示水稻土中Fe(III)还原途径与机理、进一步认识H2-依赖型Fe(III)还原过程对水稻土中微生物铁还原的贡献提供科学依据。研究取得以下主要结果：（1）通过试验确定了水稻土泥浆中脱氢酶活性测定的适宜条件。其TTC浓度为10mg·mL-1，反应时间为15min，乙醇为萃取剂的最优选择，萃取时间选择0.2h，480nm为比色测定的最大吸收峰。（2）在添加不同有机碳源和无定形氧化铁后，葡萄糖和丙酮酸钠能够促进铁还原微生物快速生长代谢，加速Fe(III)还原过程的完成，而无定形氧化铁的加入虽然能够提高铁还原潜势，但是却使铁还原滞后。表明氧化铁的表面吸附作用是研究微生物铁还原过程需要关注的影响因素。（3）添加碳源能够增快脱氢酶的产生，但不同种类的碳源对脱氢酶活性的影响程度存在明显差异。在汉中水稻土中，脱氢酶活性增长快慢表现为葡萄糖和丙酮酸盐处理大于乳酸盐；贵州水稻土脱氢酶活性增长快慢的顺序大致为乳酸钠﹥丙酮酸钠﹥葡萄糖；吉林水稻土的丙酮酸盐处理在培养2天后脱氢酶活性便迅速增加，其次为乳酸盐处理，添加葡萄糖处理的脱氢酶活性增长的最慢；天津水稻土中加入三种碳源的处理中脱氢酶活性相差不大。（4）无定形氧化铁的加入改变了微生物利用碳源的代谢途径，提高微生物发酵底物的脱氢产氢能力，显著提高了脱氢酶的生成速率，最大生成速率对应的时间提前了2-4d。微生物铁还原过程与脱氢酶活性的产生有相关性，当脱氢酶大量产生时，Fe(III)还原开始快速启动，到培养后期，脱氢酶活性有所降低，Fe(III)还原基本完成，SO42－等作为电子受体代替Fe(III)接受[H]。（5）微生物群落在不同淹水时间表现出的差异是导致其对碳源利用程度差异的主要原因，同时也反应出利用不同碳源进行脱氢反应并还原Fe(OH)3的过程有显著差异。在适当的Fe2+浓度范围内，Fe(III)还原过程与微生物的脱氢反应之间存在一定的相关性，且微生物利用葡萄糖和乳酸盐进行脱氢反应时较低的Ks值说明微生物对葡萄糖和乳酸盐的亲和力较强。不同碳源导致的脱氢酶活性出现峰值的时间具有明显的差异，葡萄糖为碳源时脱氢酶出现峰值的时间最早，大约在厌氧培养的5d左右；丙酮酸盐的脱氢酶出现峰值时间居中，一般为10d~15d；利用乳酸盐时的脱氢酶峰值出现的时间最晚，通常在20d~25d之间。表明碳源对产生的脱氢酶种类有明显的影响。（6）4株铁还原菌株对碳源的响应具有显著差异，MR-1、P4、SC-a17、SC-a24均能够以葡萄糖为优势碳源进行脱氢反应和Fe(III)还原，但不能利用乳酸盐为底物。4株铁还原菌代谢葡萄糖的铁还原过程与脱氢酶的变化具有相关性，脱氢酶活性峰值出现时间与Fe(III)还原最大反应速率对应的时间（TVmax）具有显著正相关关系，且脱氢酶活性出现峰值的时间越早Fe(III)还原最大反应速率（Vmax）越大。推测铁还原菌株代谢葡萄糖产生H2从而促进铁还原反应快速发生可能是其主要的铁还原机制。更多还原

【Abstract】 Dissimilatory Fe (III) reduction process of paddy soil was unique because of having richcarbohydrate as electron donor and a lot of microbial species. Soil dehydrogenase activity wasreative to microbial metabolism activity. During the flooding process of paddy soil,therewould be a series of biochemical changes, but hydrogen-producing of microbial metabolismis a important link, so dehydrogenase activity could be considered as a soil microbial activityindex for reflecting the degree of soil carbon source used. At the same time, NO3-、SO42-andFe(III) were main hydrogen acceptors in soil, dehydrogenase activity could indirectlycharacterize the process of denitrification, sulfate reduction and iron reduction in anaerobicrespiration of soil. Furthermore, alternate wetting and drying of paddy soil makes it present aredox alternate, dehydrogenase activity yet is a important index for the status of oxidation andreduction. So researching on the change of dehydrogenase activity in the process ofdissimilatory iron reduction of paddy soil, investigating the relationship betweendehydrogenase activity and dissimilatory iron reduction have vital significance for clarifyingthe mechanism of microbial Fe (III) reduction in paddy soil.In this study four kinds of paddy soil was used as experimental materials which collectedfrom different areas for planting rice in china, got the determine method of dehydrogenaseactivity in paddy soil slurry through researching the influence fators of determining. We tookanaerobic slurry training test, mixed culture test with microbial community vaccination underdifferent flooded time and pure culture test with typical iron reduction strains to compare therelationship between iron reduction and dehydrogenase activity and the effect to ironreduction, pH of incubation system, dehydrogenase activity by adding different carbonsources. Then we characterized dynamic characteristics of microbial iron reduction processand changes of dehydrogenase activity. All above provide the necessary scientific basis forrevealing the pathway and mechanism of iron reduction and get a further understanding ofcontribution to microbial iron reduction of paddy soil made by H2-dependent iron reductionprocess.The mainly conclusions are as follows:(1) The best choice of reaction factors used to determine the dehydrogenase activity of soil slurry as follow: TTC concentration was10mg·mL-1, water reaction time was15min atan even temperature, then using ethanol extracted0.2h, and measuring the amount of TF at480nm wavelength.(2) After adding different organic carbon source and amorphous Fe, glucose andpyruvate could promote the growth metabolism of iron reduction bacteria rapidly andaccelerate Fe(III) reduction process, however, the addition of amorphous Fe could improvethe iron reduction potential, but make the process slow. This shows that when studying theiron reduction process the surface adsorption of iron oxide was a necessary factor need tofocus on when(3) Adding carbon source could speed up dehydrogenase production, but due to thedifferent kinds of carbon sources,there was a significant difference of dehydrogenase activity.When adding glucose and pyruvat the growth rate of dehydrogenase activity in HZ paddy soilwas faster than lactate; the order of growth speed of dehydrogenase activity in GZ paddy soilwas lactate>pyruvate>glucose; in JLpaddy soil, dehydrogenase activity responded quicklywith the addition of pyruvate after incubated2days, secondly was lactate and glucose had theslowest growth.rate; the dehydrogenase activity in TJ paddy soil has no significant differenceamong the three carbon source added.(4) With tne addition of amorphous Fe the microbial metabolic pathway of carbon sourcewas changed, the ability of dehydrogenation and hydrogen production of microorganismfermentation substrates was raised and the generation rate of dehydrogenase was improvedwith the corresponding time to biggest generating rate earlied.2-4d. There was a correlationbetween dehydrogenase activity and microbial iron reduction process, when dehydrogenaseactivity generated largely, Fe(III) reduction began to start quickly, in later period of culture,dehydrogenase activity reduced, Fe (III) reduction completed generally, SO42-accepted [H] aselectron acceptor instead of Fe(III).(5) The difference of microbial community of different flooding time was the mainreason caused the process of using different carbon source for dehydrogenation reaction andreducting Fe(OH)3have significant differences. In an appropriate Fe2+concentration range,there were some correlation between Fe(III) reduction process and microbial dehydrogenationreaction, and the low Ks when microbial use glucose and lactate for dehydrogenation statedthat microbe’s affinity to glucose and lactate was more stronger. The differences of carbonsources lead to obvious differences to the peak time of dehydrgenase activity appeared. Withglucose as a substrate, dehydrogenase activity peak time appeared at about the fifth day efteranaerobic incubation, which was the earliest among the three carbon sources. The peak timeappeared at about the tenth to fifteenth day when pyruvate was as substrates. And the lactate treatment was the latest with dehydrogenase activity peak time appeared at about thetwentieth to twenty-fifth day. All above indicated that carbon sources performace a significanteffect on the dehydrogenase species.(6) The response of4iron reduction strains to carbon source had significant differences,MR-1, P4, SC-a17, SC-a24all could take glucose for superior carbon sources fordehydrogenation reaction and iron reduction, but could’t take lactate as substrate. There wasrelevance between iron reduction process and the change of the dehydrogenase of4ironreduction bacteria for glucose metabolism, the time for dehydrogenase activity peak andcorresponding time to maximum reaction rate of iron reduction (TVmax) had significantpositive correlation, and the earlier the peak value of dehydrogenase activity appeared, thebigger the Fe (III) reduction maximum reaction rate. So we speculate that iron reductionstrains producing H2from glucose metabolism to promote the iron reduction reactions tohappen quickly may be the main iron reduction mechanism.更多还原