城市生活垃圾堆肥复合菌剂的前期开发研究
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摘要
目前如何提高城市生活垃圾堆肥处理的技术水平、提高处理效率、改善产品质量，一直是相关科研人员所重点关注的。开发高效堆肥复合菌剂，从根本上优化堆肥过程更是大家研究的热点。由于堆肥中难降解的木质纤维素类物质一直是制约堆肥快速腐熟的关键因素，所以在已有的关于堆肥复合菌剂的报道中，都不约而同地使用了纤维素和木质素降解菌。但是在已有的这些报道中，对复合菌剂中纤维素和木质素降解菌的选择都具有比较明显的主观性，缺乏详细的菌株之间关系的研究，所得到的结果有一定的偶然性，适用性有限。本文的思路是以从堆肥中分离的纤维素和木质素降解菌为基础，在对其产酶过程和条件有了深入了解后，进行两类菌的初步混合培养，寻找高效的纤维素和木质素降解菌组合。为以后在此基础上复合菌剂的开发提供基础。
首先从堆肥本身出发，进行了堆肥物料中纤维素降解菌的分离和筛选，通过纤维素琼脂平板的分离，刚果红琼脂平板和实际降解效果实验的筛选，最终得到了四株效果较好的纤维素降解菌，其中尤其以X菌株在纤维素酶的产生和对稻草的降解上效果最优。
其次，选择这株菌进行了培养条件埘其产酶过程的研究。研究结果表明液料比在整个发酵过程中对产酶影响最大，温度在发酵初期影响较大，初始pH和接种量的影响均不显著。总体看来，培养温度、初始pH值、液料比和接种量分别为30℃、4、7和5％是比较合适的。通过对不同碳源对产酶影响的考察，发现添加适量的麦芽糖和纤维二糖对产酶是有利的，纤维二糖主要是可以促进CMC酶的分泌。通过对不同氮源对产酶影响的考察，发现该菌株对氮源有很大的选择性，适量的酵母膏和(NH_4)_2SO_4作为氮源是合适的。在物料中添加0.1％的吐温80也被证明是对产酶有利的。
最后，在上述研究的基础上，结合购买的菌株，挑选了纤维素降解菌6株和木质素降解菌8株进行了初步的混合培养实验。在葡萄糖牛肉膏蛋白胨的基础琼脂上对其两两混合培养并观察它们的菌落生长形态，最后得到的在一起能较好共存的菌株对有以下9对：S+10、AF93251+2、AF93251+10、X+3、X+10、AF93252+2、AF93252+5、P+5、P+10。
选取其中的三对(AF93251+2、AF93252+5、X+10)进行了固态发酵试验，通过测定发酵过程中的半纤维素酶、纤维素酶、漆酶和木素过氧化物酶活考察了它们在混合培养时的产酶情况。实验结论表明AF93252+5这个组合通过混合培养在纤维素酶和半纤维素酶的产生上有
城市生活垃圾堆肥复合菌剂的前期开发研究
较明显的改善，虽然在木质素降解酶系上无明显优势，但仍是一个可取
的，并能进一步进行研究的菌株组合。而其他两个组合的混合培养并没
有体现出优势。
通过上述试验发现，纤维素降解菌和木质素降解菌虽然大部分都是真
菌，但是它们中并不是任何两株就可以通过简单混合来获得较好的降解
和产酶效果的，菌株与菌株之间仍然存在着很多我们还不是很清楚的相
互制约、竞争和促进的关系。在复合菌剂的制备中，要想获得最好的效
果，就必须对’所用菌株之间的各种可能关系进行深入研究。
In recent years, developing new technology, increasing efficiency and improving the quality of product were the key points of the research on municipal solid waste composting. Developing the high efficient complex microbial community was the hot point in this area especially. In the recent reports about efficient complex microbial community, the cellulose decomposing microbe and lignin decomposing microbe were used widely. It was often objective that selecting the cellulose and lignin decomposing microbe was, being lack of studying on the relationship between these microbes. So it was often accidentally to get proper results, and the applicability of there results were limited. In this paper, the propose was to separate and screen cellulose decomposing microbe from the composting process, study the process of cellulase production in solid state fermentation, carry out the primary study on mixed culture in order to find proper combination of these special microbes to decompose the cellulose or lignin.
First, four high efficient cellulose decomposing microbes were screened from the composting process by the separating by cellulose agar and the screening by cellulose-congo red agar and solid state fermentation. The microbe named X was the best one in cellulase production and cellulose decomposing.
Then, in order to see the process of cellulase production and the culture conditions, the effects of different conditions on cellulase activity in cellulase production by the microbe named X were studied by solid state fermentation. The liquid-solid ratio is the most greatly influencing factor during the whole process of the solid state fermentation. In the initial stage of the fermentation, the temperature can affect the cellulase production relatively greatly because it can affect the growth of the microorganisms. The effects of initial pH and the number of inoculum were not significant. Considering the whole process of the fermentation, the temperature, initial pH, liquid-solid ratio and the number of inoculum
were suggested to be 30C, 4, 7 and 5%, respectively. By investigation on the effects of different carbon sources on the cellulase production, adding the proper amount of maltose and cellobiose was proved to be useful for cellulase production. The primary function of cellobiose is stimulating the endoglucanase production. The differences of effects of defferent nitrogen sources are great. Some nitrogen sources are hard to be utilized by the microorganism. The yeast extract and (NH4)2SO4 are proper nitrogen sources for this microorganism. Adding the 0.1% of surfactant of Tween 80 was proved to be be useful for cellulase production.
At last, basing above results, the mixed culture of six cellulose decomposing microbes and eight lignin decomposing microbes were studied. Nine couples of microbes were selected by their modalities on the basic agar, named S + 10, AF93251+2, AF 93251 + 10 , X+3 , X+10, AF93252+2, AF93252+5, P+5, P+10.
The cellulase, xylanase, laccase and lignin peroxidase activities of three couples named AF93251+2, AF93252+5, X+10 were studied by mixed culture in solid state fermentation. The results, indicated that the couple named AF93252 + 5 was the best one in cellulase and xylanase production by mixed culture. This couple could be taken further study although it wasn't the best in laccase and lignin peroxidase production.
From above results, it was obvious that the better effects on decomposing and enzyme production could not be acquired by mixing cellulose and lignin decomposing microbes randomly, although these microbes were all fungi. Many interactions such as competition and symbiosis between these microbes were unknown to us. So, it was necessary to study these interactions to make high efficient complex microbial community for municipal solid waste composting.
首先从堆肥本身出发，进行了堆肥物料中纤维素降解菌的分离和筛选，通过纤维素琼脂平板的分离，刚果红琼脂平板和实际降解效果实验的筛选，最终得到了四株效果较好的纤维素降解菌，其中尤其以X菌株在纤维素酶的产生和对稻草的降解上效果最优。
其次，选择这株菌进行了培养条件埘其产酶过程的研究。研究结果表明液料比在整个发酵过程中对产酶影响最大，温度在发酵初期影响较大，初始pH和接种量的影响均不显著。总体看来，培养温度、初始pH值、液料比和接种量分别为30℃、4、7和5％是比较合适的。通过对不同碳源对产酶影响的考察，发现添加适量的麦芽糖和纤维二糖对产酶是有利的，纤维二糖主要是可以促进CMC酶的分泌。通过对不同氮源对产酶影响的考察，发现该菌株对氮源有很大的选择性，适量的酵母膏和(NH_4)_2SO_4作为氮源是合适的。在物料中添加0.1％的吐温80也被证明是对产酶有利的。
最后，在上述研究的基础上，结合购买的菌株，挑选了纤维素降解菌6株和木质素降解菌8株进行了初步的混合培养实验。在葡萄糖牛肉膏蛋白胨的基础琼脂上对其两两混合培养并观察它们的菌落生长形态，最后得到的在一起能较好共存的菌株对有以下9对：S+10、AF93251+2、AF93251+10、X+3、X+10、AF93252+2、AF93252+5、P+5、P+10。
选取其中的三对(AF93251+2、AF93252+5、X+10)进行了固态发酵试验，通过测定发酵过程中的半纤维素酶、纤维素酶、漆酶和木素过氧化物酶活考察了它们在混合培养时的产酶情况。实验结论表明AF93252+5这个组合通过混合培养在纤维素酶和半纤维素酶的产生上有
城市生活垃圾堆肥复合菌剂的前期开发研究
较明显的改善，虽然在木质素降解酶系上无明显优势，但仍是一个可取
的，并能进一步进行研究的菌株组合。而其他两个组合的混合培养并没
有体现出优势。
通过上述试验发现，纤维素降解菌和木质素降解菌虽然大部分都是真
菌，但是它们中并不是任何两株就可以通过简单混合来获得较好的降解
和产酶效果的，菌株与菌株之间仍然存在着很多我们还不是很清楚的相
互制约、竞争和促进的关系。在复合菌剂的制备中，要想获得最好的效
果，就必须对’所用菌株之间的各种可能关系进行深入研究。
In recent years, developing new technology, increasing efficiency and improving the quality of product were the key points of the research on municipal solid waste composting. Developing the high efficient complex microbial community was the hot point in this area especially. In the recent reports about efficient complex microbial community, the cellulose decomposing microbe and lignin decomposing microbe were used widely. It was often objective that selecting the cellulose and lignin decomposing microbe was, being lack of studying on the relationship between these microbes. So it was often accidentally to get proper results, and the applicability of there results were limited. In this paper, the propose was to separate and screen cellulose decomposing microbe from the composting process, study the process of cellulase production in solid state fermentation, carry out the primary study on mixed culture in order to find proper combination of these special microbes to decompose the cellulose or lignin.
First, four high efficient cellulose decomposing microbes were screened from the composting process by the separating by cellulose agar and the screening by cellulose-congo red agar and solid state fermentation. The microbe named X was the best one in cellulase production and cellulose decomposing.
Then, in order to see the process of cellulase production and the culture conditions, the effects of different conditions on cellulase activity in cellulase production by the microbe named X were studied by solid state fermentation. The liquid-solid ratio is the most greatly influencing factor during the whole process of the solid state fermentation. In the initial stage of the fermentation, the temperature can affect the cellulase production relatively greatly because it can affect the growth of the microorganisms. The effects of initial pH and the number of inoculum were not significant. Considering the whole process of the fermentation, the temperature, initial pH, liquid-solid ratio and the number of inoculum
were suggested to be 30C, 4, 7 and 5%, respectively. By investigation on the effects of different carbon sources on the cellulase production, adding the proper amount of maltose and cellobiose was proved to be useful for cellulase production. The primary function of cellobiose is stimulating the endoglucanase production. The differences of effects of defferent nitrogen sources are great. Some nitrogen sources are hard to be utilized by the microorganism. The yeast extract and (NH4)2SO4 are proper nitrogen sources for this microorganism. Adding the 0.1% of surfactant of Tween 80 was proved to be be useful for cellulase production.
At last, basing above results, the mixed culture of six cellulose decomposing microbes and eight lignin decomposing microbes were studied. Nine couples of microbes were selected by their modalities on the basic agar, named S + 10, AF93251+2, AF 93251 + 10 , X+3 , X+10, AF93252+2, AF93252+5, P+5, P+10.
The cellulase, xylanase, laccase and lignin peroxidase activities of three couples named AF93251+2, AF93252+5, X+10 were studied by mixed culture in solid state fermentation. The results, indicated that the couple named AF93252 + 5 was the best one in cellulase and xylanase production by mixed culture. This couple could be taken further study although it wasn't the best in laccase and lignin peroxidase production.
From above results, it was obvious that the better effects on decomposing and enzyme production could not be acquired by mixing cellulose and lignin decomposing microbes randomly, although these microbes were all fungi. Many interactions such as competition and symbiosis between these microbes were unknown to us. So, it was necessary to study these interactions to make high efficient complex microbial community for municipal solid waste composting.
引文
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