一株生孢噬纤维菌的分离纯化、鉴定及降解纤维素滤纸过程的研究
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摘要
本文采用滤纸纤维素平板和纤维素双层平板的方法，从土壤中分离、筛选出一株好氧性纤维素降解细菌。通过对其形态及其生理生化特性的研究，该菌株被鉴定为生孢噬纤维细菌属(Sporocytophaga)的一个新菌株。
生孢噬纤维细菌(Sporocytophaga)是能够在固体表面快速滑动的细菌。它在滤纸和棉花表面上生长时能够将滤纸和棉花完全降解，合成胞外多糖。生孢噬纤维细菌通过与纤维素物质的紧密粘附作用而强烈地降解纤维素；但该菌只能测到极低的胞外CMC酶活，而没有其他的胞外纤维素酶活，因此该菌有特殊的纤维素降解机制。
本文采用扫描电子显微镜观察该菌降解纤维素滤纸的整个过程，分析了该菌株对滤纸纤维素的降解规律和该菌在分解滤纸纤维素这一过程中不同阶段的形态变化。结果表明该菌具有较强的分解纤维素的能力，菌株在降解纤维素滤纸过程中与纤维素物质紧密结合，同时菌体嵌入到滤纸纤维内部。培养48小时，杆状细胞的表面结构发生很大的变化，此时的菌体表面已产生大量的粘性多糖，这些粘性多糖因菌体在纤维素表面滑动而在菌体表面形成突起，即在纤维素被旺盛降解部位的菌体表面产生了大量突起；而产生突起的菌体深入到纤维素分子内部，纤维素表面可以清晰地看到由于菌体嵌入纤维素分子内部而留下的凹陷。在这段时间内测得滤纸纤维素的失重率最大，说明滤纸降解速度最快；因此，我们认为这些粘性多糖突起是该菌旺盛降解纤维素物质的一个重要标志。随着培养时间的延长有小孢囊大量形成，小孢囊可能是通过端生和中间生这两种形式产生。并且这一时期的滤纸降解率开始降低，证明小孢囊并不与纤维素的旺盛降解有关。培养96小时后，滤纸纤维素被降解成碎屑，液体培养基中有大量孢囊存在；培养6—7天后该菌可以将滤纸纤维素完全降解。
A strain of cellulolytic bacterium was isolated from soil by filter paper plate and cellulose fibre double-plate. The properties of morphology and physiology of this strain was studied. According to Bergey's Manual of Determinative Bacteriology, the strain was identified as a new strain of the Sporocytophaga.
Sporocytophaga is a kind of bacteria that can glide on the surface of solid medium .This strain can decompose the cellulose strongly.lt can grow on the surface of cotton and fiter paper, and produce large amount of extracellular polysacchrides during the cellulose degradation. The sporocytophag can only produce a low extracellular carboxymethyl-cellulase(CMC) activity and no other extracellular cellulase activities .So the Sporocytophaga has a special mechanism of cellulose degradation.
In this work, the process that a strain of Sporocytophaga degraded the filter paper fibre was studied by means of scan electronmicroscopy.The filter paper cellulose degraded rule and the morphology change on different phase were analysed. The result showed that this strain have a strong ability to decompose the filter paper cellulose. In the process of degrading fibre ,the strain adhere tightly to the fibre through its bacilliform cell. The bacilliform cell penetrate into interior of the fibre to degrade the cellulose strongly and produced a mass of sticky polysaccharides. After cultured 48 hours ,the bacilliform cell's surface of Sporocytophaga have a great change .At this stage the bacilliform produce a lot of sticky polysaccharides.These sticky polysaccharides associated with the sites where the filter paper was decomposed intensively and form thorns on the surface of the bacillium.At the same time ,the filter-paper weight loss is the greatest and decomposing rate is the fastest ,so we think that the sticky polysaccharides are produced during the cellulose degradation. At the
anaphase of cultured, the bacteria exsist as a cycloidal dormancy body-sporocyst,and the decomposing rate of filter-paper began to decrease.We know the sporocyst is a form that sporocytophaga genus appear in a ill circumstance,so we think the sporocyst didn't have the cellulose decomposing activity.After cultered 96 hours, we can found many sporocysts and filter-paper slices in the fermentable liquid. This strain can decompose the filter paper fibre entirely after cultured 6-7 days.
生孢噬纤维细菌(Sporocytophaga)是能够在固体表面快速滑动的细菌。它在滤纸和棉花表面上生长时能够将滤纸和棉花完全降解，合成胞外多糖。生孢噬纤维细菌通过与纤维素物质的紧密粘附作用而强烈地降解纤维素；但该菌只能测到极低的胞外CMC酶活，而没有其他的胞外纤维素酶活，因此该菌有特殊的纤维素降解机制。
本文采用扫描电子显微镜观察该菌降解纤维素滤纸的整个过程，分析了该菌株对滤纸纤维素的降解规律和该菌在分解滤纸纤维素这一过程中不同阶段的形态变化。结果表明该菌具有较强的分解纤维素的能力，菌株在降解纤维素滤纸过程中与纤维素物质紧密结合，同时菌体嵌入到滤纸纤维内部。培养48小时，杆状细胞的表面结构发生很大的变化，此时的菌体表面已产生大量的粘性多糖，这些粘性多糖因菌体在纤维素表面滑动而在菌体表面形成突起，即在纤维素被旺盛降解部位的菌体表面产生了大量突起；而产生突起的菌体深入到纤维素分子内部，纤维素表面可以清晰地看到由于菌体嵌入纤维素分子内部而留下的凹陷。在这段时间内测得滤纸纤维素的失重率最大，说明滤纸降解速度最快；因此，我们认为这些粘性多糖突起是该菌旺盛降解纤维素物质的一个重要标志。随着培养时间的延长有小孢囊大量形成，小孢囊可能是通过端生和中间生这两种形式产生。并且这一时期的滤纸降解率开始降低，证明小孢囊并不与纤维素的旺盛降解有关。培养96小时后，滤纸纤维素被降解成碎屑，液体培养基中有大量孢囊存在；培养6—7天后该菌可以将滤纸纤维素完全降解。
A strain of cellulolytic bacterium was isolated from soil by filter paper plate and cellulose fibre double-plate. The properties of morphology and physiology of this strain was studied. According to Bergey's Manual of Determinative Bacteriology, the strain was identified as a new strain of the Sporocytophaga.
Sporocytophaga is a kind of bacteria that can glide on the surface of solid medium .This strain can decompose the cellulose strongly.lt can grow on the surface of cotton and fiter paper, and produce large amount of extracellular polysacchrides during the cellulose degradation. The sporocytophag can only produce a low extracellular carboxymethyl-cellulase(CMC) activity and no other extracellular cellulase activities .So the Sporocytophaga has a special mechanism of cellulose degradation.
In this work, the process that a strain of Sporocytophaga degraded the filter paper fibre was studied by means of scan electronmicroscopy.The filter paper cellulose degraded rule and the morphology change on different phase were analysed. The result showed that this strain have a strong ability to decompose the filter paper cellulose. In the process of degrading fibre ,the strain adhere tightly to the fibre through its bacilliform cell. The bacilliform cell penetrate into interior of the fibre to degrade the cellulose strongly and produced a mass of sticky polysaccharides. After cultured 48 hours ,the bacilliform cell's surface of Sporocytophaga have a great change .At this stage the bacilliform produce a lot of sticky polysaccharides.These sticky polysaccharides associated with the sites where the filter paper was decomposed intensively and form thorns on the surface of the bacillium.At the same time ,the filter-paper weight loss is the greatest and decomposing rate is the fastest ,so we think that the sticky polysaccharides are produced during the cellulose degradation. At the
anaphase of cultured, the bacteria exsist as a cycloidal dormancy body-sporocyst,and the decomposing rate of filter-paper began to decrease.We know the sporocyst is a form that sporocytophaga genus appear in a ill circumstance,so we think the sporocyst didn't have the cellulose decomposing activity.After cultered 96 hours, we can found many sporocysts and filter-paper slices in the fermentable liquid. This strain can decompose the filter paper fibre entirely after cultured 6-7 days.
引文
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24．贺延龄等。一个水解纤维素的嗜热厌氧菌新种，微生物学报，1991，31(2)：85-89
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26．张小华、谭培英等。一个分解纤维素的瘤胃梭菌新种，微生物学报，1995，35(6)：397-399
27．韩如旸等。嗜热厌氧纤维素降解细菌的分离、鉴定及其系统发育分析，微生物学报，2002，2(42)：138-144
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2．夏黎明。可再生纤维素资源酶法降解的研究进展，林产化工通报1999，33(1)：23～26
3．张玉忠、陈秀兰等。棉花纤维超微结构的STM观察，生物物理与生物化学学报，2000，32(5)：521-523
4．张玉忠、刘洁等。微晶纤维素超显微结构的STM研究，电子显微学报，1997，16(6)：736-738
5. Y. Z. Zhang, CHEN Xiulan, J. Liu, et al, Pang Size and arrangement of elementary fibrils in crystalline cellulose studied with scanning tunneling microscope, J. Vac. Sxi.Tech. 1997, B15(4), 1502-1505
6．许修宏，肖玉珍。高效纤维素分解菌的分离筛选的研究，东北农业大学学报，1998，29(4)：330-333
7．陈洪章、李佐虎。纤维素原料微生物与生物量的利用，生物技术通报，2002，2：25-30
8．吴元喜。木质纤维素分解菌筛选及木质纤维素降解，华中农业大学学报，1999，16(6)
9．叶汗珍等。优良纤维素分解菌的筛选及产酶研究，南京林业大学学报，1994，(3)：55-58
10．史玉英等。纤维素分解菌的分离和筛选，南京农业大学学报，1996，19(3)：59-62
11．王宜磊、孙迅。五株降解半纤维素细菌的分离鉴定与酶活测定。山东师大学报，1997，12(4)：474-476
12．祝令香，徐建等。一株纤维素降解菌的鉴定，农业生物技术学报，2001，9(3)：255-257
13．周津。绿色木酶A10纤维素酶的分离纯化及理化性质研究，西北大学学报，2001(5)：465～469
14．戴四发。黑曲霉产纤维素酶系各组分特性及酶解条件，华侨大学
学报，2001，22(1)：65～69
15．李宪臻。天然纤维素的微生物降解机理研究进展，《食品发酵工业》，1996，2：74～77
16．赵小蓉、林启美、孙炎鑫等。纤维素分解菌对不同纤维素类物质的分解作用，微生物学杂志，2001，20(3)：12-14
17．张蔚文。混合菌培养在生物降解中的应用，环境科学与技术，1993，(1)：16～20
18．Shetali M．Trivedi, Titendera D. desai，混合培养小柱孢菌和木霉生产纤维酶和β-葡萄糖苷酶，微生物学杂志，1986，4(6)：60-63
19. Highley TL Murmanis L. Micromorphology of degradation in western hemlock and sweetgum by the brown-rot fungus poria placenta, Holzf-orchung 1985, 39 35-40
20. Stockton B C etal., biotechnol. lett, 1991, 13: 57
21. Gao. P. J. etal, progress in natual science, 1998, 8, 117-124
22. Leschine S B. Annu Rev Microbiol, 1995, 49: 399-426
23．谭培英、贺青等。热纤梭菌的分离和鉴定，微生物学报，1991，31(6)：484-487
24．贺延龄等。一个水解纤维素的嗜热厌氧菌新种，微生物学报，1991，31(2)：85-89
25．谭培英等。一株厌氧纤维素分解菌的分离和鉴定，微生物学报，1987，27(3)：211-216
26．张小华、谭培英等。一个分解纤维素的瘤胃梭菌新种，微生物学报，1995，35(6)：397-399
27．韩如旸等。嗜热厌氧纤维素降解细菌的分离、鉴定及其系统发育分析，微生物学报，2002，2(42)：138-144
28．曹月青等。桑粒肩天牛肠道纤维素分解细菌的分离和鉴定，微生物学报，2001，28(1)：9-11
29．官家发等。芽孢杆菌E_2菌株纤维素酶形成条件的研究，微生物学报，1992，32(6)：412-417
30．石晶瑜。分解纤维素菌HT3的筛选及酶活力测定，内蒙古师大学
报，1998，27(1)：66～68
31．刘东波、高培基、王祖农。一株分解纤维素的诺卡氏放线菌的分离、鉴定及降解特性的，微生物学报。1990，30：70-72
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