高效纤维素降解菌的筛选及其特性研究
|
摘要
纤维素是自然界中存在最广泛的一类碳水化合物,同时它也是地球上数量最大的再生资源。据不完全统计,全球每年通过光合作用产生的植物物质高达1.55×109t,其中有89%尚未被利用或未被合理利用(如直接焚烧)。目前全世界被开发利用的农林纤维副产物不足2%。因此将这部分可再生资源转变成可利用的能源,不仅可以减轻农业废弃物对环境的污染,更对能源危机的缓解具有重要意义。氢气作为清洁的、高品位二次能资源,越来越受到全世界的关注。在多种制氢方法中,利用微生物厌氧发酵生物质(有机废水、秸秆类农林业废弃物、有机垃圾等)制氢,具备治污、环保和产能等多重优越性,开发前景广阔,已成为各国关注的研究课题。
本文从牛粪堆肥中筛选到1株纤维素降解菌G3与2株同步降解纤维素产氢菌(G2、G1),通过对G3进行16S rDNA鉴定,发现与G3最相近的菌株为痢疾杆菌Shigalle flexneri strain FBD002,同源性为99%,对G1和G2的16S rDNA鉴定发现,G1、G2与鹑鸡肠球菌Enterococcus gallinarum strain LMG 13129最相近,同源性也达99%。
通过研究发现G3为一株纤维素糖化菌株,以PH-101Avicel为底物发酵40h,G3纤维素糖化率达到48.2%。G3在温度30-40oC和pH6.0-7.0的范围内都可以有效降解纤维素,最佳纤维素降解温度为37oC左右,最佳pH为6.5左右。G3的最适底物浓度5g/l,酵母粉最加添加量为1.5g/l。G1和G2利用纤维素产氢的单位体积产氢量(YH2)分别为97.5ml/L和82.0ml/L。纤维素降解率达到42.6%和36%,G1、G2在温度35-45oC、pH6.5-7.0的范围内都可以有效降解纤维素产氢,最佳纤维素降解温度为40oC左右,最佳pH为6.5左右。G3的最适底物浓度5g/l,氮源(Yesat extract)最适添加量为1.5g/l。
实验还用倍比稀释法从瘤胃液中分离得到一组最小的降解纤维素功能菌群(RCB),以5g/l PH-101Avicel为底物,温度40℃,初始pH6.2条件下,RCB纤维素降解率为40%,利用纤维素产氢的单位体积产氢量(YH2)达到70 ml/L。
利用G1、G3二株纤维素降解菌和己糖产氢菌B49进行复配产氢实验,发现菌种复配时的纤维素降解和产氢能力大大高于各单菌株能力。尤其是G1+G3+B49二步法复配,静态发酵100h,以PH-101Avicel为底物时,纤维素降率和YH2分别达到60.5%和208.0ml/L。与G1单独发酵时相比,菌种复配的纤维素降解能力提高了20.6%,产氢量提高了53.1%。产氢能力达到4.0 mmolH2/g·纤维素。所以G1+G3+B49是纤维素降解菌与己糖产氢菌的复配方式。
将瘤胃中分离得到的降解纤维素最小功能菌群(RCB)与B49二步法复配,结果表明当RCB与B49的接种比例为5:8时,体系有最佳的纤维素降解率与产氢量,分别为55.1%和165.7ml/L,分别较RCB体系的纤维素降解率与产氢量提高了27.2%和51.5%。
Cellulose is the most extensive carbohydrates in the nature, and it is also the world's largest number of renewable resources. According to incomplete statistics, every year around the world the plant material through photosynthesis up to 1.55×109t, of which 89% has not been used or unreasonable use (such as direct burning). At present, the utilization of fiber-product in the world is less than 2%. So how to effectively use these renewable resources into the available energy can not only reduce agricultural waste on the environment pollution, ease the energy crisis of great significance. As advanced renewable energy, hydrogen is attracted more and more attention. In most methods of producing hydrogen, using microbe to ferment biomass such as organic waste water,agriculture solid wastes and organic rubbish in anaerobic condition to produce hydrogen is becoming a noticeable problem all over the world, because of the advantage of getting rid of wastes,protecting environment and producing energy. And using low-cost agriculture organic wastes to produce hydrogen is a hot problem. While the correlative researchs are very few and stay in the stage of library, because of the problem of low utility efficiency and the gain and culture of hydrogen producing bacterium. Basing on the standpoint of interaction in biohydrogen production, the research screened out strains that could hydrolysis cellulose effectively, and mixed them with hexose-hydrogen producing bacterium, hoping to achieve highly cellulose degradation and hydrogen producing capability.
This research isolated one cellulose degradation strain G3 and two strains G1, G2 that can degrade cellulose to procuce hydrogen, through the test of 16S rDNA, the most similar bacterium with G3 is Shigalle flexneri strain FBD002, and the comparability is about 99%, the most similar bacterium with G1 and G2 is Enterococcus gallinarum strain LMG 13129, and the comparability is also about 99%.
Through the test we found that G3 is a saccharolytic strain. The cellulose saccharity rate of bacteria G3 with PH-101 Avicel as substrate is 48.2% in 40h. G3 can hydrolysis cellulose under the condition of T 30-40oC、pH 6.0-7.0, and the best result was achieved with T 37oC and pH 6.5 with 5g/l substrate and 1.5g/l nitrogen source (Yeast extract). The YH2 of bacteria G2 and G3 are 97.5ml/L and 82.0ml/L, respectively as PH-101 Avicel substrate. Under the condition of T 35-45oC, pH 6.5-7.0, G1, G2 could degradation cellulose to hydrogen production, and the best result was achieved with T 40oC and pH 6.5. The optimal substrate yeast extract concentration is similar to the strain G3.
The research on isolating the minimum functional consortia (RCB) were also conducted by multiple proportion diluted method. Cellulose hydrolysis rate and hydrogen production were 40% and 80ml/L were gained under the condition of T 40oC, the initial pH 6.2, making 5g/l PH-101Avicel as substrate. Under the condition of different mixed culture with G1, G3 and B49, we found the cellulose hydrolysis and hydrogen producing capability in mixed culture is much higher than that in pure culture.Especially the mixed manner of G1+G3+B49 with two-step inoculation, the best result achieved were, cellulose hydrolysis rate of 60.5%, and hydrogen producing capability YH2 208.3ml/L with 5g/l PH-101 Avicel substrate.So G1+G3+B49 with two-step inoculation was the best co-culture method.
The result of the co-culture system of RCB & B49 presented that the optimal cellulose hydrolysis ratio and hydrogen production would obtained when the proportion is 5(RCB):8(B49), which was 27.2% higher cellulose hydrolysis ratio and 51.5% higher quantity of H2 produced than that of the RCB, the cellulose hydrolysis ratio and hydrogen production were 55% and 165 ml/L, respectively.
本文从牛粪堆肥中筛选到1株纤维素降解菌G3与2株同步降解纤维素产氢菌(G2、G1),通过对G3进行16S rDNA鉴定,发现与G3最相近的菌株为痢疾杆菌Shigalle flexneri strain FBD002,同源性为99%,对G1和G2的16S rDNA鉴定发现,G1、G2与鹑鸡肠球菌Enterococcus gallinarum strain LMG 13129最相近,同源性也达99%。
通过研究发现G3为一株纤维素糖化菌株,以PH-101Avicel为底物发酵40h,G3纤维素糖化率达到48.2%。G3在温度30-40oC和pH6.0-7.0的范围内都可以有效降解纤维素,最佳纤维素降解温度为37oC左右,最佳pH为6.5左右。G3的最适底物浓度5g/l,酵母粉最加添加量为1.5g/l。G1和G2利用纤维素产氢的单位体积产氢量(YH2)分别为97.5ml/L和82.0ml/L。纤维素降解率达到42.6%和36%,G1、G2在温度35-45oC、pH6.5-7.0的范围内都可以有效降解纤维素产氢,最佳纤维素降解温度为40oC左右,最佳pH为6.5左右。G3的最适底物浓度5g/l,氮源(Yesat extract)最适添加量为1.5g/l。
实验还用倍比稀释法从瘤胃液中分离得到一组最小的降解纤维素功能菌群(RCB),以5g/l PH-101Avicel为底物,温度40℃,初始pH6.2条件下,RCB纤维素降解率为40%,利用纤维素产氢的单位体积产氢量(YH2)达到70 ml/L。
利用G1、G3二株纤维素降解菌和己糖产氢菌B49进行复配产氢实验,发现菌种复配时的纤维素降解和产氢能力大大高于各单菌株能力。尤其是G1+G3+B49二步法复配,静态发酵100h,以PH-101Avicel为底物时,纤维素降率和YH2分别达到60.5%和208.0ml/L。与G1单独发酵时相比,菌种复配的纤维素降解能力提高了20.6%,产氢量提高了53.1%。产氢能力达到4.0 mmolH2/g·纤维素。所以G1+G3+B49是纤维素降解菌与己糖产氢菌的复配方式。
将瘤胃中分离得到的降解纤维素最小功能菌群(RCB)与B49二步法复配,结果表明当RCB与B49的接种比例为5:8时,体系有最佳的纤维素降解率与产氢量,分别为55.1%和165.7ml/L,分别较RCB体系的纤维素降解率与产氢量提高了27.2%和51.5%。
Cellulose is the most extensive carbohydrates in the nature, and it is also the world's largest number of renewable resources. According to incomplete statistics, every year around the world the plant material through photosynthesis up to 1.55×109t, of which 89% has not been used or unreasonable use (such as direct burning). At present, the utilization of fiber-product in the world is less than 2%. So how to effectively use these renewable resources into the available energy can not only reduce agricultural waste on the environment pollution, ease the energy crisis of great significance. As advanced renewable energy, hydrogen is attracted more and more attention. In most methods of producing hydrogen, using microbe to ferment biomass such as organic waste water,agriculture solid wastes and organic rubbish in anaerobic condition to produce hydrogen is becoming a noticeable problem all over the world, because of the advantage of getting rid of wastes,protecting environment and producing energy. And using low-cost agriculture organic wastes to produce hydrogen is a hot problem. While the correlative researchs are very few and stay in the stage of library, because of the problem of low utility efficiency and the gain and culture of hydrogen producing bacterium. Basing on the standpoint of interaction in biohydrogen production, the research screened out strains that could hydrolysis cellulose effectively, and mixed them with hexose-hydrogen producing bacterium, hoping to achieve highly cellulose degradation and hydrogen producing capability.
This research isolated one cellulose degradation strain G3 and two strains G1, G2 that can degrade cellulose to procuce hydrogen, through the test of 16S rDNA, the most similar bacterium with G3 is Shigalle flexneri strain FBD002, and the comparability is about 99%, the most similar bacterium with G1 and G2 is Enterococcus gallinarum strain LMG 13129, and the comparability is also about 99%.
Through the test we found that G3 is a saccharolytic strain. The cellulose saccharity rate of bacteria G3 with PH-101 Avicel as substrate is 48.2% in 40h. G3 can hydrolysis cellulose under the condition of T 30-40oC、pH 6.0-7.0, and the best result was achieved with T 37oC and pH 6.5 with 5g/l substrate and 1.5g/l nitrogen source (Yeast extract). The YH2 of bacteria G2 and G3 are 97.5ml/L and 82.0ml/L, respectively as PH-101 Avicel substrate. Under the condition of T 35-45oC, pH 6.5-7.0, G1, G2 could degradation cellulose to hydrogen production, and the best result was achieved with T 40oC and pH 6.5. The optimal substrate yeast extract concentration is similar to the strain G3.
The research on isolating the minimum functional consortia (RCB) were also conducted by multiple proportion diluted method. Cellulose hydrolysis rate and hydrogen production were 40% and 80ml/L were gained under the condition of T 40oC, the initial pH 6.2, making 5g/l PH-101Avicel as substrate. Under the condition of different mixed culture with G1, G3 and B49, we found the cellulose hydrolysis and hydrogen producing capability in mixed culture is much higher than that in pure culture.Especially the mixed manner of G1+G3+B49 with two-step inoculation, the best result achieved were, cellulose hydrolysis rate of 60.5%, and hydrogen producing capability YH2 208.3ml/L with 5g/l PH-101 Avicel substrate.So G1+G3+B49 with two-step inoculation was the best co-culture method.
The result of the co-culture system of RCB & B49 presented that the optimal cellulose hydrolysis ratio and hydrogen production would obtained when the proportion is 5(RCB):8(B49), which was 27.2% higher cellulose hydrolysis ratio and 51.5% higher quantity of H2 produced than that of the RCB, the cellulose hydrolysis ratio and hydrogen production were 55% and 165 ml/L, respectively.
引文
1宋颖琦,杨谦.纤维素降解菌的筛选及其降解特征的研究.哈尔滨工业大学学报.2002,34(2):197～199
2刘倩,宋金柱等.影响玉米秸秆微生物降解制肥的因素浅析.环境卫生工程. 2000, 10(3):103～106
3陈光,张真妮.淀粉发酵生产微生物多糖的研究现状.吉林农业大学学报. 2001, 23(1):111～113
4刘翔,何国庆.利用木素纤维素生产燃料乙醇的微生物代谢工程.粮油加工与食品机械.2003,(8):67～69
5 MGlancer.Process biochemistry [J].1989,24(3):109～113
6陈庆森,刘剑虹等.利用多菌种共发酵技术转化玉米秸杆的研究.食品与发酵工业.1999,25(5):1～4
7张继泉,王瑞明.利用木质纤维素生产燃料酒精的研究进展.酿酒科技. 2003(1): 39～42
8李建法,宋湛谦.纤维素制备高吸水材料研究进展.林产化学与工业.2002,22 (2):81～85
9 Yan B X. Domain Structure and Conformation of a Cellobiohydrolase from Trichoderma Pseudokonjingiis-38[J].Journal of Protein Chemistry .1997(16):59
10高培基,曲音波,汪天虹等.微生物降解纤维素机制的分子生物学研究进展.纤维素科学与技术.1995,3(2):1～19
11李素芬,霍贵成.纤维素酶的分子结构组成及其功能.中国饲料.1997(13):12～14
12阎伯旭,高培基.纤维素酶分子结构与功能研究进展.生命科学. 1995,7(5):22～25
13阎伯旭,齐飞,张颖舒等.纤维素酶分子结构和功能研究进展.生物化学与生物物理进展.1999,26(3):233～235
14王春卉,汪天虹,高培基.纤维素酶分子的纤维素吸附区的研究进展.纤维素科学与技术.1997,5(4):1～10
15 Eriksson K-E L, Blanchette R A, Ander P. Michobil and Enzymatic Degradation of Wood and Wood Compents Springer-verlag [C].Berlin Heidelberg,1990,89
16 Marx Figioni M. The Control of Molecular Weight and Molecular WeightDistribution in the Biogenesis of Cellulose [A]. Jrced Cellulose and other Natural Polymer Systems Biogenesis. Structure and Degradation. Plenum [Z]. Newyork, 1982.243～268.
17 Amano Yoshihiko, Kanda Takahisa. New Insights into Cellulose Degradation by Celluloses and Related Enzymes. Trends in Glycoscience and Glyco Take care, Echnology 2002,14:27～34.
18 Tomme P, Warren R A, Gilkes N R. Cellulose Hydrolysis by Bacteria and Fungi [J]. Advanced Microbiology and Physiology,1995,37(1):1～81.
19阎伯旭,孙迎庆,高培基.有限酶切拟康氏木霉纤维素酶分子研究其结构域的结构与功能.纤维素科学与技术.1998,6(3):1～9
20高培基,刘洁.山东大学.天然纤维素在生物降解过程中超分子结构的变化.自然科学进展.1998,8(4):391～397
21 Woodward J.M.Lima, M.Lee. Biochem.[J].1988,255:898～899
22张树政.酶制剂工业.科学出版社.北京,1984
23 M.P.Coughlan著,吴克谦摘译,细菌和真菌降解纤维素的机理,国外畜牧科学.1991,18(6):28～31
24袁勤生.现代酶学.第一版,上海,华东理工大学出版社.2001
25刘家建.纤维素酶的研究及应用综述.林产化工通讯.1995,1:6～10
26张树政.酶制剂工业.北京,科学出版社.1998,595～615
27 Mandels M, Weber J. The production of cellulases [J] .Reese ET Cellulases and theirs application,1996(95):391～413.
28 Robson L M, Chambliss G H. Cellulases of Bacteria Origin [J]. Enzyme Microb Technol, 1989,(11):626～644.
29王兰芬.纤维素酶的作用机理及开发应用,酿酒科技.1997,84(6):16～17
30齐飞.生孢噬纤维细菌的分离纯化及鉴定.山东大学学报,.1999,34(4): 484～487
31李日强.纤维素类废弃物的综合利用.中国环境科学.2002,22(1):24～27
32刘敏雄.反刍动物消化生理学[M].北京:北京农业大学出版社,1991.
33霍鲜鲜,侯先志,李翠英.瘤胃微生物对纤维素物质的粘附作用[J].内蒙古畜牧科学,2001,22(3);:19~21
34刘敏雄,反刍动物消化生理学[M],北京:北京农业大学出版社,1991.
35张辉.瘤胃内真菌降解粗纤维的研究进展[J].黄牛杂志,1997,3(2):49~51
36杨文博译.微生物学[M].北京:科学出版社,1991
37任南琪,王爱杰.哈尔滨工业大学.厌氧生物技术原理与应用.化学工业出版社.2004
38 Ueno Y., Sato, S., MorimotoM. Hydrogen Production from Industrial Waste Water by Anaerobic Microflora in Chemostat Culture [J]. Ferment.Bioeng. 1996, 82(2):194～197
39 LayJ.J. Biohydrogen Generation by MesopHilic Anaerobic Fermentation of Microcrystalline Cellulose [J]. Biotech.Bioeng. 2001,74(4):280～287
40 Evvyernie D, etal. Conversion of Chitinous Wastes to Hydrogen Gas by Clostridium Paraputrificum M-21 [J]. Biosci.Bioeng. 2001, 91(4):339～343
41卢怡,张无敌,宋洪川等.稻草发酵产氢潜力的研究.能源工程.2003,2:26～28
42樊耀亭,侯红卫,张高生.一种由农作物秸秆及酒糟生产氢气的有效方法.中国发明专利公告.CN:1522805A.2004
43 Yokoi H, Saitsu, A.Uchida, etal. Microbial Hydrogen Production from Sweet Potato Starch Residue [J].Biosci.Bioeng. 2001,91(1):58～63
44王之中.台湾大学.以梭状芽孢杆菌进行废弃活性污泥厌氧消化产氢.[硕士学位论文],2002
45 Tong Zhang, Hong Liu, Herbert H.P, etal. University of Hong Kong Biohydrogen Production from Starch in Wastewater under Thermophilic Condition.Journal of Environmental Management. 2003,69:149～156
46林明,任南琪,王爱杰等.哈尔滨工业大学.混合菌种在发酵法生物产氢中的协同作用.环境科学.2003,24(2):54～59
47林明,任南琪.哈尔滨工业大学.混合菌种非固定化技术制氢反应器产氢效能.哈尔滨工业大学学报.2002,34(6):815～819
48樊耀亭,李晨林,侯红卫.郑州大学.天然厌氧微生物氢发酵生产生物氢气的研究.中国环境科学.2002,22(4):370～374
49樊耀亭,廖新成.郑州大学.有机废弃物氢发酵制备生物氢气的研究.环境科学.2003,24(3):132～135
50闫有旺.聊城大学.世纪绿色可再生资源—生物质.贵州化工.2003,28(5):1～3
51魏桃员,张素琴,邵林广,游映玖.一株纤维素降解细菌的分离及特性研究.环境科学与技.2004,27(5)
52 Kocherginskaya S A, Aminov R I, White B A. Analysis of The Rumen Bacterial Diversity Under Two Different Diet Conditions Using Denaturing Gradient Gel Electrophoresis, Random Sequencing and Statistical Ecology Approaches[J].
37任南琪,王爱杰.哈尔滨工业大学.厌氧生物技术原理与应用.化学工业出版社.2004
38 Ueno Y., Sato, S., MorimotoM. Hydrogen Production from Industrial Waste Water by Anaerobic Microflora in Chemostat Culture [J]. Ferment.Bioeng. 1996, 82(2):194～197
39 LayJ.J. Biohydrogen Generation by MesopHilic Anaerobic Fermentation of Microcrystalline Cellulose [J]. Biotech.Bioeng. 2001,74(4):280～287
40 Evvyernie D, etal. Conversion of Chitinous Wastes to Hydrogen Gas by Clostridium Paraputrificum M-21 [J]. Biosci.Bioeng. 2001, 91(4):339～343
41卢怡,张无敌,宋洪川等.稻草发酵产氢潜力的研究.能源工程.2003,2:26～28
42樊耀亭,侯红卫,张高生.一种由农作物秸秆及酒糟生产氢气的有效方法.中国发明专利公告.CN:1522805A.2004
43 Yokoi H, Saitsu, A.Uchida, etal. Microbial Hydrogen Production from Sweet Potato Starch Residue [J].Biosci.Bioeng. 2001,91(1):58～63
44王之中.台湾大学.以梭状芽孢杆菌进行废弃活性污泥厌氧消化产氢.[硕士学位论文],2002
45 Tong Zhang, Hong Liu, Herbert H.P, etal. University of Hong Kong Biohydrogen Production from Starch in Wastewater under Thermophilic Condition.Journal of Environmental Management. 2003,69:149～156
46林明,任南琪,王爱杰等.哈尔滨工业大学.混合菌种在发酵法生物产氢中的协同作用.环境科学.2003,24(2):54～59
47林明,任南琪.哈尔滨工业大学.混合菌种非固定化技术制氢反应器产氢效能.哈尔滨工业大学学报.2002,34(6):815～819
48樊耀亭,李晨林,侯红卫.郑州大学.天然厌氧微生物氢发酵生产生物氢气的研究.中国环境科学.2002,22(4):370～374
49樊耀亭,廖新成.郑州大学.有机废弃物氢发酵制备生物氢气的研究.环境科学.2003,24(3):132～135
50闫有旺.聊城大学.世纪绿色可再生资源—生物质.贵州化工.2003,28(5):1～3
51魏桃员,张素琴,邵林广,游映玖.一株纤维素降解细菌的分离及特性研究.环境科学与技.2004,27(5)
52 Kocherginskaya S A, Aminov R I, White B A. Analysis of The Rumen Bacterial Diversity Under Two Different Diet Conditions Using Denaturing Gradient Gel Electrophoresis, Random Sequencing and Statistical Ecology Approaches[J].
2刘倩,宋金柱等.影响玉米秸秆微生物降解制肥的因素浅析.环境卫生工程. 2000, 10(3):103～106
3陈光,张真妮.淀粉发酵生产微生物多糖的研究现状.吉林农业大学学报. 2001, 23(1):111～113
4刘翔,何国庆.利用木素纤维素生产燃料乙醇的微生物代谢工程.粮油加工与食品机械.2003,(8):67～69
5 MGlancer.Process biochemistry [J].1989,24(3):109～113
6陈庆森,刘剑虹等.利用多菌种共发酵技术转化玉米秸杆的研究.食品与发酵工业.1999,25(5):1～4
7张继泉,王瑞明.利用木质纤维素生产燃料酒精的研究进展.酿酒科技. 2003(1): 39～42
8李建法,宋湛谦.纤维素制备高吸水材料研究进展.林产化学与工业.2002,22 (2):81～85
9 Yan B X. Domain Structure and Conformation of a Cellobiohydrolase from Trichoderma Pseudokonjingiis-38[J].Journal of Protein Chemistry .1997(16):59
10高培基,曲音波,汪天虹等.微生物降解纤维素机制的分子生物学研究进展.纤维素科学与技术.1995,3(2):1～19
11李素芬,霍贵成.纤维素酶的分子结构组成及其功能.中国饲料.1997(13):12～14
12阎伯旭,高培基.纤维素酶分子结构与功能研究进展.生命科学. 1995,7(5):22～25
13阎伯旭,齐飞,张颖舒等.纤维素酶分子结构和功能研究进展.生物化学与生物物理进展.1999,26(3):233～235
14王春卉,汪天虹,高培基.纤维素酶分子的纤维素吸附区的研究进展.纤维素科学与技术.1997,5(4):1～10
15 Eriksson K-E L, Blanchette R A, Ander P. Michobil and Enzymatic Degradation of Wood and Wood Compents Springer-verlag [C].Berlin Heidelberg,1990,89
16 Marx Figioni M. The Control of Molecular Weight and Molecular WeightDistribution in the Biogenesis of Cellulose [A]. Jrced Cellulose and other Natural Polymer Systems Biogenesis. Structure and Degradation. Plenum [Z]. Newyork, 1982.243～268.
17 Amano Yoshihiko, Kanda Takahisa. New Insights into Cellulose Degradation by Celluloses and Related Enzymes. Trends in Glycoscience and Glyco Take care, Echnology 2002,14:27～34.
18 Tomme P, Warren R A, Gilkes N R. Cellulose Hydrolysis by Bacteria and Fungi [J]. Advanced Microbiology and Physiology,1995,37(1):1～81.
19阎伯旭,孙迎庆,高培基.有限酶切拟康氏木霉纤维素酶分子研究其结构域的结构与功能.纤维素科学与技术.1998,6(3):1～9
20高培基,刘洁.山东大学.天然纤维素在生物降解过程中超分子结构的变化.自然科学进展.1998,8(4):391～397
21 Woodward J.M.Lima, M.Lee. Biochem.[J].1988,255:898～899
22张树政.酶制剂工业.科学出版社.北京,1984
23 M.P.Coughlan著,吴克谦摘译,细菌和真菌降解纤维素的机理,国外畜牧科学.1991,18(6):28～31
24袁勤生.现代酶学.第一版,上海,华东理工大学出版社.2001
25刘家建.纤维素酶的研究及应用综述.林产化工通讯.1995,1:6～10
26张树政.酶制剂工业.北京,科学出版社.1998,595～615
27 Mandels M, Weber J. The production of cellulases [J] .Reese ET Cellulases and theirs application,1996(95):391～413.
28 Robson L M, Chambliss G H. Cellulases of Bacteria Origin [J]. Enzyme Microb Technol, 1989,(11):626～644.
29王兰芬.纤维素酶的作用机理及开发应用,酿酒科技.1997,84(6):16～17
30齐飞.生孢噬纤维细菌的分离纯化及鉴定.山东大学学报,.1999,34(4): 484～487
31李日强.纤维素类废弃物的综合利用.中国环境科学.2002,22(1):24～27
32刘敏雄.反刍动物消化生理学[M].北京:北京农业大学出版社,1991.
33霍鲜鲜,侯先志,李翠英.瘤胃微生物对纤维素物质的粘附作用[J].内蒙古畜牧科学,2001,22(3);:19~21
34刘敏雄,反刍动物消化生理学[M],北京:北京农业大学出版社,1991.
35张辉.瘤胃内真菌降解粗纤维的研究进展[J].黄牛杂志,1997,3(2):49~51
36杨文博译.微生物学[M].北京:科学出版社,1991
37任南琪,王爱杰.哈尔滨工业大学.厌氧生物技术原理与应用.化学工业出版社.2004
38 Ueno Y., Sato, S., MorimotoM. Hydrogen Production from Industrial Waste Water by Anaerobic Microflora in Chemostat Culture [J]. Ferment.Bioeng. 1996, 82(2):194～197
39 LayJ.J. Biohydrogen Generation by MesopHilic Anaerobic Fermentation of Microcrystalline Cellulose [J]. Biotech.Bioeng. 2001,74(4):280～287
40 Evvyernie D, etal. Conversion of Chitinous Wastes to Hydrogen Gas by Clostridium Paraputrificum M-21 [J]. Biosci.Bioeng. 2001, 91(4):339～343
41卢怡,张无敌,宋洪川等.稻草发酵产氢潜力的研究.能源工程.2003,2:26～28
42樊耀亭,侯红卫,张高生.一种由农作物秸秆及酒糟生产氢气的有效方法.中国发明专利公告.CN:1522805A.2004
43 Yokoi H, Saitsu, A.Uchida, etal. Microbial Hydrogen Production from Sweet Potato Starch Residue [J].Biosci.Bioeng. 2001,91(1):58～63
44王之中.台湾大学.以梭状芽孢杆菌进行废弃活性污泥厌氧消化产氢.[硕士学位论文],2002
45 Tong Zhang, Hong Liu, Herbert H.P, etal. University of Hong Kong Biohydrogen Production from Starch in Wastewater under Thermophilic Condition.Journal of Environmental Management. 2003,69:149～156
46林明,任南琪,王爱杰等.哈尔滨工业大学.混合菌种在发酵法生物产氢中的协同作用.环境科学.2003,24(2):54～59
47林明,任南琪.哈尔滨工业大学.混合菌种非固定化技术制氢反应器产氢效能.哈尔滨工业大学学报.2002,34(6):815～819
48樊耀亭,李晨林,侯红卫.郑州大学.天然厌氧微生物氢发酵生产生物氢气的研究.中国环境科学.2002,22(4):370～374
49樊耀亭,廖新成.郑州大学.有机废弃物氢发酵制备生物氢气的研究.环境科学.2003,24(3):132～135
50闫有旺.聊城大学.世纪绿色可再生资源—生物质.贵州化工.2003,28(5):1～3
51魏桃员,张素琴,邵林广,游映玖.一株纤维素降解细菌的分离及特性研究.环境科学与技.2004,27(5)
52 Kocherginskaya S A, Aminov R I, White B A. Analysis of The Rumen Bacterial Diversity Under Two Different Diet Conditions Using Denaturing Gradient Gel Electrophoresis, Random Sequencing and Statistical Ecology Approaches[J].
37任南琪,王爱杰.哈尔滨工业大学.厌氧生物技术原理与应用.化学工业出版社.2004
38 Ueno Y., Sato, S., MorimotoM. Hydrogen Production from Industrial Waste Water by Anaerobic Microflora in Chemostat Culture [J]. Ferment.Bioeng. 1996, 82(2):194～197
39 LayJ.J. Biohydrogen Generation by MesopHilic Anaerobic Fermentation of Microcrystalline Cellulose [J]. Biotech.Bioeng. 2001,74(4):280～287
40 Evvyernie D, etal. Conversion of Chitinous Wastes to Hydrogen Gas by Clostridium Paraputrificum M-21 [J]. Biosci.Bioeng. 2001, 91(4):339～343
41卢怡,张无敌,宋洪川等.稻草发酵产氢潜力的研究.能源工程.2003,2:26～28
42樊耀亭,侯红卫,张高生.一种由农作物秸秆及酒糟生产氢气的有效方法.中国发明专利公告.CN:1522805A.2004
43 Yokoi H, Saitsu, A.Uchida, etal. Microbial Hydrogen Production from Sweet Potato Starch Residue [J].Biosci.Bioeng. 2001,91(1):58～63
44王之中.台湾大学.以梭状芽孢杆菌进行废弃活性污泥厌氧消化产氢.[硕士学位论文],2002
45 Tong Zhang, Hong Liu, Herbert H.P, etal. University of Hong Kong Biohydrogen Production from Starch in Wastewater under Thermophilic Condition.Journal of Environmental Management. 2003,69:149～156
46林明,任南琪,王爱杰等.哈尔滨工业大学.混合菌种在发酵法生物产氢中的协同作用.环境科学.2003,24(2):54～59
47林明,任南琪.哈尔滨工业大学.混合菌种非固定化技术制氢反应器产氢效能.哈尔滨工业大学学报.2002,34(6):815～819
48樊耀亭,李晨林,侯红卫.郑州大学.天然厌氧微生物氢发酵生产生物氢气的研究.中国环境科学.2002,22(4):370～374
49樊耀亭,廖新成.郑州大学.有机废弃物氢发酵制备生物氢气的研究.环境科学.2003,24(3):132～135
50闫有旺.聊城大学.世纪绿色可再生资源—生物质.贵州化工.2003,28(5):1～3
51魏桃员,张素琴,邵林广,游映玖.一株纤维素降解细菌的分离及特性研究.环境科学与技.2004,27(5)
52 Kocherginskaya S A, Aminov R I, White B A. Analysis of The Rumen Bacterial Diversity Under Two Different Diet Conditions Using Denaturing Gradient Gel Electrophoresis, Random Sequencing and Statistical Ecology Approaches[J].