【作者】 张建峰；

【导师】 王丕武；

【作者基本信息】 吉林农业大学 ， 作物生物技术， 2012， 博士

【摘要】 我国年产秸秆总量已经超过了7亿吨,居世界第一位,由于缺乏有效的处理手段大量的秸秆被露天焚烧,造成了环境污染,浪费了宝贵的生物资源。而在众多秸秆利用的方法中,微生物菌剂处理秸秆有着较为独特的优势。微生物发酵秸秆可以提供高品质的饲料,微生物菌剂降解秸秆可以提高秸秆利用率,微生物菌剂堆肥秸秆可以提供高品质的有机肥料。而微生物处理的关键是需要优良的菌种。我国东北地区秸秆资源占全国的前三位,由于气温低等因素使其更加难于有效的利用,也更加缺乏优良的菌种。为了提高东北地区的秸秆利用率,本试验进行了东北地区秸秆降解工程菌的选育方面的研究,并制备了秸秆速腐菌剂。取得了以下主要成果：1、利用多种微生物育种方法获得以下秸秆降解菌从树林中腐烂的树叶、田间地头腐烂的秸秆、放牛场中腐败的牛粪、天然堆沤秸秆肥料中采集样品,利用CMC-Na平板初筛分离纯化培养后,筛选出了40株纤维素降解能力较强的菌株。利用刚果红透明圈法复筛获得了10株菌(H1-H10)。通过CMC酶活和滤纸酶活测定,结果显示酶活情况都很好。选取其中综合性能都好的6株菌,进行形态学和分子生物学鉴定,结果表明H2为假单孢杆菌；H1为缺陷短波单孢菌；H3为荧光单胞菌；H4为解淀粉芽孢杆菌；H7为铜绿假单胞菌；H6为解淀粉芽孢杆菌。利用基因工程手段,克隆黑曲霉木聚糖酶基因xynB,并成功插入穿梭质粒,获得重组质粒pGAPz-xynB。PCR、双酶切及测序结果显示克隆的目的基因xynB正确,构建的重组质粒成功。将pGAPz-xynB利用电击法转入毕赤酵母X-33,使用博来霉素筛选得到阳性克隆,经PCR鉴定和SDS-PAGE鉴定,表明转入成功。通过对表达的木聚糖酶酶活测定显示,工程菌成功构建,胞外酶活性可达到8.33IU/mL。重组工程菌命名为PP-XYNB。利用原生质体融合手段,对绿色木霉AS3.3711和康宁木霉AS3.2774进行了融合。首先研究了2种木霉菌原生质体的制备条件。试验结果表明,原生质体制备条件为：菌龄为22h,酶浓度为13mg/mL(0.5%溶壁酶+0.5%蜗牛酶+0.3%纤维素酶),酶解时间2h,渗透压稳定剂为STC,酶解温度31℃。在此条件下,原生质体制备率70%,再生率为62%。选取酶活力较高突变株,制备原生质体,再进行细胞融合,通过刚果红初筛和发酵产酶复筛,得到一株酶活力高,遗传稳定的突变株T-VK10。其CMC酶活达到119IU/ml。利用分子生物学手段,以pMD18-T-35S.EGI.NOS为模板,将纤维素酶EGI基因、35S启动子、NOS终止子作为一个整体克隆出来,并将其插入到粟酒裂殖酵母表达系统的pESP-2的多克隆位点处,结果证明pESP-2-35S.EGI.NOS表达载体构建成功,将构建成功的载体导入到粟酒裂殖酵母菌体中,并使外源基因得到表达。最后获得了酶活性达16U/mL的表达纤维素酶粟酒裂殖酵母菌体,并命名为SP-EGI。2、进行菌株的复合配比试验,并成功制备复合菌剂利用平板培养法进行拮抗实验,结果证明实验室筛选的6株纤维素分解菌株,实验室保存的绿色木霉(TV)、康宁木霉(TK)、平菇菌丝(PO)、枯草芽孢杆菌(BS)、酿酒酵母(SC)、啤酒酵母(BY),工程改造的毕赤酵母(PP-XYNB)、粟酒裂殖酵母(SP-EGI)、原生质体融合菌T-VK10共15株菌之间没有明显的拮抗作用,均可以进行复合菌剂的复配实验。将备选的15株菌进行正交实验,通过观察各种组合对液体培养基中滤纸条溃烂的情况,确定高效降解秸秆的最优菌种复合系为：假单孢杆菌(H2)+荧光单胞菌(H3)+解淀粉芽孢杆菌(H6)+枯草芽孢杆菌(BS)+毕赤酵母(PP-XYNB)+粟酒裂殖酵母(SP-EGI)+原生质体融合菌(T-VK10)。制备复合菌剂干粉,通过单因素试验筛选了20种常见的干燥保护剂,其中效果突出的是海藻糖、吐温80、司班60,效果最好的是海藻糖。通过正交试验设计,得出5%海藻糖、2%吐温80、2%司班60干燥剂组合保护效果最好,CMAP活菌率可以达到83.39%；另外2%司班60、2%吐温80的干燥剂组合效果也不错,CMAP活菌率达到77.43%,完全可以进行工业化生产,关键是其造价低廉。最后确定了CMAP生产条件：培养到适当菌龄的菌液按比例混合,加入2%吐温80、2%司班60为干燥剂,利用板框压滤机压榨到含水率低于65%,挤出造粒Φ1mm×2mm,干燥温度90℃,最终含水率7%左右。3、通过功效验证,自制的复合菌剂综合性能优良通过堆肥试验验证复合菌剂的功效,结果显示：添加复合菌剂的实验组堆温升高为最快,在第4天进入了高温发酵期,并于第7天达到了最高温度64℃。比CK2先进入高温期5天左右,而且高温期的温度也比CK2高出约13℃,缩短了腐熟时间,并且有利于有害病菌的死亡。堆肥pH值测定结果：实验组堆肥的pH值在7天以后就处于7.4-8.0之间,优于CK2和CK1,这样的偏碱环境下非常有利于微生物降解秸秆,利于堆肥品质的提高。通过平板计数法测定各堆肥处理在不同堆肥时间各种相关微生物变化,结果表明：堆肥过程中以好气细菌、兼气细菌数量的增长最为迅速。其次以产纤维素酶的菌株、放线菌生长最快,有利于玉米秸秆的降解。实验组较空白对照而言,堆体中微生物数量明显较多,实验组和CK2微生物数量比CK1高出一个数量级,而实验组又比CK2高出30%以上,大量的微生物繁殖使堆温上升较快且温度高,高温期持续时间较长,能明显促进堆肥腐熟。接种外源菌剂对细菌、真菌、放线菌的数量变化与自然堆肥相比有显著的影响。通过对各处理不同时段采样测定有效元素,实验结果表明：实验组有效氮的含量先增后减,表明了微生物大量繁殖和作用的过程,总的来讲,有效氮含量是增加的。从总体来看,有效钾在堆肥过程中的变化不大,实验组和CK2有效钾的含量高于CK1,实验组比CK2略好。堆肥过程中速效磷的变化较大,三个处理堆肥后都比堆肥前提高较多,以实验组增加最为明显,实验组高出对照组69.41%。更多还原

【Abstract】 The total annual production of straw in our country is above7hundred million tons, ranking first in the world, but due to lack of effective management, a large quantity of straw is burnt in open field, which pollutes the environment and wastes the valuable biological resource. Microbial agent has more unique advantages comparing with other methods of straw utilization. Microbial fermentation of straw can provide high-quality feed, microbial agent degradation of straw can boost utilization ratio, and microbial compost of straw can offer high-quality organic fertilizer. The key of microbial measure is having excellent strains. Straw resources of northeast account for the top three in China, but it is hard to get making full use of them as a result of such factors as low temperatures and lack of excellent strains. In order to improve the utilization of straw of northeastern region, this paper researches breeding of the engineered bacteria used on the Northeast straw degradation and preparing the straw-speed rot agents. The main results as the following:1. we obtained the following straw degrading bacteria by various microbial breeding methods.Collecting samples from decaying leaves in the woods, rotting straw in the fields, putrid cow dung in the cattle field and natural composting straw fertilizer, the auther screened out40well ability strains of cellulose degradation after initial screening separation and the pure culture by CMC-Na tablet. After secondary screening,10strains H1～H10were obtained by means of Congo red transparent circle. The results showed that enzyme activity is good to get through enzyme activity determination of the CMC and filter paper. Selecting six strains that have good comprehensive performance to make morphological and molecular biological identification, the results show that H2is Pseudomonas mendocina; H1is Brevundimonas diminuta; H3is Pseudomonas fluorescens; H4is Bacillus amyloliquefaciens; H7is Pseudomonas aeruginosa; H6is Bacillus amyloliquefaciens.Through genetic engineering means, cloning xynB which is xylanase gene of Aspergillus niger, and inserting into the shuttle plasmid successfully, the recombinant plasmid pGAPz-xynB can be obtain. PCR, double enzyme digestion and sequencing results showed that the cloned target gene xynB is correct and the recombinant plasmid constructed is successful. Transferring pGAPz-xynB to Pichia pastoris X-33with electric impact method and screening positive clones by bleomycin, it indicates that the transferred is successful by PCR and SDS-PAGE. Determination of the expression of xylanase activity showed that the engineered bacteria was successfully constructed, and extracellular enzyme activity can be achieved8.331U/mL. Recombinant engineering bacteria is named PP-XYNB.Trichoderma viride3.3711and Trichoderma koningii were fused by means of protoplast fusion. Firstly, we researched preparation conditions of the two kinds of Trichoderma protoplast. The results indicate that the protoplast preparation conditions are as follows:strain age is22h, the concentration of enzyme is13mg/mL (0.5%lywallzyme+0.5%snail enzyme+0.3%of cellulase), hydrolysis time is2h, the osmotic stabilizer is STC, hydrolysis temperature is31℃. Under these conditions, the rate of protoplast formation is70%and regeneration rate is62%. Selecting mutants having higher enzyme activity to prepare protoplast and make cells fusion, the mutant T-VK10which has high enzyme activity and genetic stability can be obtained by means of screening with Congo red and rescreening with enzyme production. Its CMC activity reaches1191U/ml.Using molecular biology methods and as pMD18-T-35S.EGI.NOS as a template, cellulases EGI gene,35S promoter and NOS terminator were cloned as a whole, then inserted into the multiple cloning sites pESP-2of Schizosaccharomyces pombe expression system, the results demonstrate pESP-2-35S.EGI.NOS expression vector was successfully constructed, which was imported into the Schizosaccharomyces pombe yeast body and made the foreign gene had been expressed. We get Schizosaccharomyces pombe cell that expresses cellulase enzyme, activity reaching16U/mL, and named SP-EGI.2. We prepared the composite microbial agent successfully through test concentration configuration of strains.The results of Plate culture antagonistic test show that the15strains are no obvious antagonism each other, they included the six cellulose-decomposing strains from laboratory screening, Trichoderma viride (TV), Trichoderma koningii (TK), P. ostreatus (PO), Bacillus subtilis(BS), Saccharomyces cerevisiae (SC), beer yeast (BY) from laboratory saved, and Pichia pastoris (PP-XYNB), Schizosaccharomyces pombe (SP-EGI), protoplast fusion of bacteria (T-VK10) from engineering renovation. So its are appropriate for formulation experiment of composite microbial agent.Doing the orthogonal experiment with alternative15strains, by observing the situation that the various combinations fester filter strips in the liquid medium, it can be confirmed that the optimal composite microbial agent of efficient degradation of straw is Pseudomonas mendocina (H2)+Pseudomonas fluorescens (H3)+bacillus amyloliquefaciens (H6)+Bacillus subtilis (BS)+Pichia pastoris (PP-XYNB)+Schizosaccharomyces pombe (SP-EGI)+protoplast fusion strain (T-VK10).In the tests of preparation for CMAP(composite microbial agent powder), Screening20kinds of common drying protective agents through single-factor tests, it had prominent effect that Tween-80, span60and trehalose, while the trehalose had the best effect. By orthogonal experimental design, it can be summarized that the desiccant combination including5%trehalose,2%Tween-80and2%Span60had the best protection effect, and viable rate of CMAP can achieve83.39%. In addition, the effect of the combination including2%Span60and2%Tween-80is also good, viable rate of CMAP can achieve77.43%. It can be used for industrial production completely, and the key is its low cost. The production conditions of CMAP are confirmed that the bacterial liquid at proper cell age is mixed in proportion, and added2%Tween-80and2%Span60as drying protective agents, then pressed moisture content to below65%with frame filter, extrusion granulation is Φ1mm×2mm, drying temperature is90℃and final moisture content is about7%.3. Our composite microbial agent had good performance, through the functional validation.The results of efficacy of the composite microbial agent verified through composting test showed that the experimental group added composite microbial agent had the fastest rise in temperatures, which was into the high-temperature fermentation period on the fourth day, reached the maximum temperature of64℃on the seventh day,5days prior to CK2into the high temperature period, and it’s temperature of high temperature period is about13℃higher than CK2, which greatly shortening the maturity time and is conducive to the death of harmful bacteria. The compost pH value indicates that the experimental group was in7.4-8.0after7days better than CK2and of CK1, whose slightly alkaline environment is very conducive to microbial degradation of straw and can improve the quality of compost.Determining the changes of variety of microorganisms in the different composting time by the plate count method, the results showed that the number of aerobic bacteria and facultative bacteria has most rapid growth in the composting process. Followed fastest growth strains are the strains produced cellulase and actinomycetes, which make for the degradation of corn stover. The experimental group had more microorganisms in the pile than blank control group significantly, and the number of microorganisms of the experimental group and CK2was an order of magnitude higher than that of CK1, while the experimental group was30%higher than CK2. The large number of microorganisms breeding made temperature rise faster and resulted in long duration of high temperature, which can promote the composting significantly. It had remarkable influence to inoculate exogenous strains for bacteria, fungi and actinomycetes by comparison natural compost in the number change.Determining effective elements of each treatment group at different times, the experimental results showed that the effective nitrogen content of the experimental group increases first and then decreases, indicating the process of reproduction and impact of microorganisms. In general, the effective nitrogen content is increased. Overall, effective potassium had only little changes in the composting process, the effective potassium content of experimental group and CK2was higher than that of CK1, the experimental group is slightly better than CK2. Quick-acting phosphorus content had a greater change in the composting process, after compost improve more than before in three treatments, and the most obvious increase is the experimental group that was69.41%higher than the control group.更多还原