| 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. |
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