小麦秸秆降解复合菌群FWD1的构建、降解特性及其微生物群落组成研究

发布时间：2018-05-05 03:15

本文选题：小麦秸秆 + 复合菌群　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：农作物秸秆是一种具有多用途的可再生生物质资源,利用微生物分解这些木质纤维素原料是资源化利用的有效途径。目前,人们已经报道了许多木质纤维素降解菌,但单一菌株存在酶系组成单一、木质纤维素降解效率低等问题。采用限制性培养方法筛选构建的复合菌群,不同菌在群落中各司其职,相互协作,具有较高的木质纤维素降解能力。本研究从秦岭山区采集9个不同林地土壤样品,以秸秆分解率、羧甲基纤维素酶(CMCase)活力及木聚糖酶活力为指标筛选出一份木质纤维素分解能力较强的冷杉林地土壤样品(QLI),以该土壤样品为接种物,小麦秸秆为碳源,连续继代培养,构建了一组稳定高效小麦秸秆分解复合菌群FWD1,并对其分解特性及其微生物群落组成进行研究。主要取得了以下研究成果:1、高效稳定小麦秸秆降解复合菌群FWD1的构建测定所采集的9份土壤样品原始及富集培养4d、8d、12d、16d的CMCase活力、木聚糖酶活力和第16d小麦秸秆失重量,结果表明,冷杉林地土壤样品(QLI)具有最高的CMCase活力和秸秆降解率,以其为接种物,小麦秸秆为碳源,连续继代培养,驯化构建复合菌群。不同代数培养物对小麦秸秆降解能力的变化结果表明,从第9代培养物开始,小麦秸秆失重率均在70%左右,彼此之间没有太大差异;PCR-DGGE结果也表明,从第9代培养物开始,主要细菌组成条带和位置没有太大变化,微生物群落组成稳定。从第9代开始,获得了稳定高效小麦秸秆降解复合菌群FWD1,该菌群对小麦秸秆的降解率达到76.92%。2、小麦秸秆降解复合菌群FWD1的降解特性与实验室筛选的其他3组复合菌群A、B、C相比,复合菌群FWD1具有最高的CMCase酶活(79.64U)、滤纸酶活(75.74U)、木聚糖酶活(1100U)和秸秆降解率(75.22%)。气相色谱法测定FWD1的小麦秸秆降解液组分,结果表明,代谢产物主要为乙酸、丙酸、丁酸和乙醇。乙酸浓度呈现出先升高后降低,再升高的趋势。在发酵l d时,乙酸的含量为140.14 mg·L-1,3 d时就达到了最大值655.54 mg·L-1。而乙醇、丙酸和丁酸分别在1 d、3 d和7 d时达到最大值18.87 mg·L-1、127.17 mg·L-1和27.47 mg·L-1。此外,FWD1还能降解玉米秸秆、滤纸和脱脂棉,7d内降解率分别为52.34%、10%和2.47%。DGGE结果表明,FWD1在降解小麦秸秆、玉米秸秆、滤纸和脱脂棉过程中,各泳道既有一些公共条带,还有一些特殊条带,但公共条带亮度各不相同。此外,随着培养时间的延长,各样品的Shannon指数和丰富度指数整体上都呈先增大后减小的趋势。3、复合菌群FWD1微生物群落组成利用第二代高通量测序技术对复合菌群和菌种来源土壤样品的细菌组成进行分析,结果表明FWD1中主要含有变形菌门、厚壁菌门和拟杆菌门,分别占细菌总量的67.41%、19.25%和13.34%。在种分类水平上,Clostridium sp.BNL1100,uncultured Alcaligenes sp.,uncultured Alcaligenaceae bacterium和Brevundimonas diminuta为优势菌,分别占细菌总量的11.80%,10.30%,6.70%和3.60%。通过连续继代培养,富集了变形菌门和厚壁菌门,更细化分类水平上,富集了以Clostridium sp.BNL1100为主的纤维素降解菌,比例从0.01%上升到11.80%。
[Abstract]:Crop straw is a renewable biomass resource with multipurpose use. The use of microbes to decompose these lignocellulosic materials is an effective way of resource utilization. At present, a number of lignocellulosic degrading bacteria have been reported, but single strains have a single enzyme system and low degradation efficiency of lignocellulose. In this study, 9 different woodland soil samples were collected from the Qinling Mountains mountain area, and a sample of wood was selected from the Qinling Mountains mountain area to select the straw decomposition rate, the carboxymethyl cellulase (CMCase) activity and the xylanase activity as the index. The soil samples of fir woodland (QLI) with strong qualitative cellulose decomposing ability, with the soil sample as inoculant, wheat straw as carbon source and continuous subculture, a group of stable and efficient wheat straw decomposition compound bacteria group FWD1 was constructed, and its decomposition characteristics and microbial community composition were studied. The main achievements are as follows: 1, high The construction and determination of the stable wheat straw degraded compound bacteria group FWD1, 9 samples of soil samples were collected and cultured for 4D, 8D, 12D, 16d, CMCase vitality, xylanase activity and 16d wheat straw weight loss. The results showed that the soil samples (QLI) had the highest CMCase vitality and straw degradation rate, which was the inoculant and wheat. Straw is a carbon source, continuous subculture and domestication to construct complex bacteria groups. The results of the change of wheat straw degradation ability of different algebraic cultures show that the weight loss rate of wheat straw is around 70% from the ninth generation of culture, and the PCR-DGGE results also show that the main bacteria are composed of the main bacteria from the ninth generation of culture. The microbial community composition was stable and the location of the microbial community was stable. From the ninth generation, the stable and efficient wheat straw degradation compound bacteria group FWD1 was obtained. The degradation rate of the wheat straw was 76.92%.2. The degradation characteristics of the wheat straw degraded compound bacteria group FWD1 were compared with the other 3 groups of compound bacteria, B, C, FWD, FWD, and the compound bacterial group FWD. 1 had the highest CMCase enzyme activity (79.64U), filter paper enzyme activity (75.74U), xylanase activity (1100U) and straw degradation rate (75.22%). Gas chromatography was used to determine the component of wheat straw degradation solution in FWD1. The results showed that the metabolites were mainly acetic acid, propionic acid, butyric acid and ethanol. The concentration of acetic acid first increased and then decreased and then increased. In the fermentation of L D When the content of acetic acid is 140.14 mg. L-1,3 D, it reaches the maximum value of 655.54 mg. L-1. and ethanol, propionic acid and butyric acid at 1 D, 3 D and 7 d, respectively, reaching the maximum value 18.87 mg L-1127.17 mg, L-1 and 27.47 vegetables. The degradation rate of corn straw, filter paper and skimmed cotton can be 52.34%, 10% and the result table respectively. In the process of degradation of wheat straw, corn straw, filter paper and skimmed cotton, FWD1 has some public strips and some special strips, but the brightness of the common strip is different. In addition, with the prolongation of the culture time, the Shannon index and the richness index of the samples are increasing first and then decreasing, and the compound flora is.3. The microbial community composition of FWD1 microbial community used second generation high-throughput sequencing technology to analyze the composition of bacteria in the composite bacteria group and the soil samples from the strain source. The results showed that FWD1 mainly contained deformable bacteria gate, thick wall bacteria gate and bacteriobacteria, which accounted for 67.41% of the total bacterial total, 19.25% and 13.34%. at the species classification level, Clostridium sp.BNL1100, Uncu. Ltured Alcaligenes sp., uncultured Alcaligenaceae bacterium and Brevundimonas diminuta were the dominant bacteria, which accounted for 11.80%, 10.30%, 6.70% and 3.60%. of the total bacteria, enriched the deformable bacteria gate and the thicker wall, and enriched the cellulose degrading bacteria based on Clostridium sp.BNL1100 on the more detailed classification level. The proportion rose from 0.01% to 11.80%.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X172;X712

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