高效纤维素酶菌种的筛选及菌群的构建

发布时间：2018-07-04 15:41

  本文选题：纤维素复合菌群 + 纤维素酶活　； 参考：《石河子大学》2015年硕士论文

【摘要】：为了实现高效降解秸秆的目的,依据降解秸秆所需酶系的不同进行微生物间协调组配增加菌种多样性的理论基础,进行了纤维素降解菌的筛选及菌群构建,获得了高效降解纤维素的混合菌群,以期解决秸秆类资源充分利用的问题。本试验首先采用刚果红透明圈的初筛方法,从腐烂棉秆、牛瘤胃液、森林朽木等样品中初步分离筛选出10株能够降解纤维素的菌株,并对降解作用较好的菌株采用3,5-二硝基水杨酸比色定糖法(DNS)进行菌株CMC酶活测定,得到高效降解纤维素的菌株N05、N13及N21,将3株高效菌株进行复合菌群的构建;通对优化菌群的培养条件从而使菌群降解能力达到最大;采用失重法和扫描电镜观察研究菌群的降解能力;最后对N05、N13及N21这3株菌进行形态及分子鉴定。通过上述试验得到以下结果:(1)用刚果红纤维素培养基对采集的样品进行初筛依据透明圈直径/菌落直径的比值(D/d),得到10株具有较强分解纤维素能力的菌株。然后将这些菌株接种到液体产酶培养基中测定其CMC酶活性进行复筛,最终筛选出N05、N13、N21 3株高效纤维素降解菌株。将这3株菌株进行组合培养,通过对酶活性的测定比较,得到一个效果最好组合SDP(N05+N13+N21)。(2)在液体产酶培养条件下研究温度、培养时间、初始pH和氮源对菌群SDP纤维素酶活性的影响。结果显示,菌群降解秸秆发酵培养条件的优化组合为14(A3 B1 C5 D3),即培养温度为30℃,培养时间周期为5 d,氮源为NH4NO3,初始pH值为6.0。(3)菌群SDP对玉米秸秆的降解能力较强,在被降解之前含有纤维素36.58%、半纤维素25.60%和木质素16.35%。到第8 d培养结束时纤维素降低了25.37%,半纤维素降低了27.32%,木质素降低了15.29%。通过对比玉米秸秆的被微生物降解前后的扫描电镜图可知,经过微生物的降解作用,木质纤维素的结构遭到破坏,变得松散,结晶度下降,混合菌群对玉米秸秆的降解效果要比单一菌株好。(4)对菌株N05、N13和N21进行形态学观察和16S r DNA及18S r DNA基因测序和序列分析,经鉴定N05、N13和N21分别为真菌属的塔宾曲霉(Aspergillus tubingensis),巨大芽孢杆菌(Bacillus megaterium)和枯草芽孢杆菌(Bacillus sp.LX-102)。在国内外已有的研究报道中,纤维素降解菌主要集中在纤维单胞菌(Cellu-lomonas)、假单胞菌(Pseudomonas)、梭菌(Clostridium)和芽孢杆菌(Bacillus),而关于塔宾曲霉降解纤维素的研究,还几乎未见报道。混合菌群纤维素降解效果高于单一菌株,本试验为微生物混合培养降解纤维素提供了理论基础。
[Abstract]:In order to degrade straw efficiently, the selection and construction of cellulosic degrading bacteria were carried out on the basis of the different enzyme systems needed to increase the diversity of microbial species. In order to solve the problem of making full use of straw resources, a mixture of cellulose-degrading bacteria was obtained. In this experiment, 10 strains which can degrade cellulose were isolated from rotting cotton stalk, bovine rumen fluid and forest wood by the method of Congo red transparent circle. The enzyme activity of CMC was determined by DNS-DNSS, and the strains N05N _ (13) and N _ (21) were obtained, and the three high-efficient strains were used to construct the complex bacterial community of N05N _ (13) and N _ (21). Tongduan optimized the culture conditions of the microflora so as to maximize the biodegradation ability of the microflora; observed the degradation ability of the flora by weightlessness method and scanning electron microscope; finally, carried out morphological and molecular identification of the three strains N05N13 and N21. The results were as follows: (1) according to the ratio of transparent circle diameter to colony diameter (D / d), 10 strains with strong cellulolysis ability were obtained by using Congo red cellulose medium. Then these strains were inoculated into liquid enzyme producing medium to determine their CMC enzyme activity and screened again. Finally, N05N 13 N 21 3 strains of high efficiency cellulose degradation were screened. The three strains were cultured in combination. The best combination, SDP (N05 N13 N21). (2), was obtained by measuring and comparing the enzyme activity, and the temperature and time of culture were studied under the condition of liquid enzyme production, and the results showed that SDP (N05 N13 N21). (2) was the best combination of the three strains. Effects of initial pH and nitrogen sources on cellulase activity of SDP. The results showed that the optimum culture conditions were 14 (A3B1C5D3), that is, the culture temperature was 30 鈩，

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