复合菌系降解木质纤维素特性及其菌群动态
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摘要
化石燃料的储量有限性以及过度利用化石燃料所造成严重的温室效应,迫使人类寻求化石燃料的替代品。生物质能源由于兼具能源和环境效益受到了高度认可。木质纤维素是生物质的主要组成成分,同时也是生物质利用的瓶颈。如何高效的实现木质纤维素的水解酸化一直是困扰生物法利用生物质资源的难题,为了提高木质纤维素的降解效率,不同领域的研究学者已经做了大量的科研工作,近年来随着环境生物技术的发展,微生物的协同作用逐渐受到重视,利用复合菌系进行木质纤维素降解成为研究的热点。本论文的主要研究内容和研究结果如下:
系统地研究了一组木质纤维素降解复合菌系的形态特征、生长特性及生态条件。通过显微观察发现,复合菌系主要由杆菌组成,普遍周生鞭毛,胞外分泌粘液,表面形貌多样。在降解过程中复合菌系对氧的需求量较低,反应体系的DO值的波动范围很小,始终维持在0.07~0.25 mg/L范围内。体系的pH值呈现出先降低后上升的趋势,表明复合菌系对发酵产物的反馈抑制有较强的缓冲能力。
对复合菌系的发酵特性进行了较为详细的探讨,其对木质纤维素的降解表现出较强的能力,即在216 h内稻草降解了75 %,纤维素降解了81 %,半纤维素降解了92 %,木质素降解了46 %,并且前60 h为高效降解阶段,期间对稻草、纤维素、半纤维素和木质素的降解量分别占总降解量的59.0 %、48.5 %、86.8 %、44.0 %。而从复合菌系的发酵类型分析,其属于典型的乙酸型发酵,90%以上的代谢产物是乙酸,其次是少量的乙醇、丁酸和丙酸。发酵108 h后,乙酸浓度达到最大值2.58 g/L,此时总挥发酸也达到最大值2.90 g/L,该发酵类型为下游两相厌氧消化的产甲烷相的稳定、高效运行提供重要物质基础。发酵体系的COD值呈现出先增加后降低的总体趋势,说明了底物由固态的木质纤维素转变为液态的挥发酸,再转变为气态的CO2和H2的过程。总凯氏氮和氨态氮的变化趋势表明,复合菌系的活性在降解后期降低的原因不是氨态氮的累积而是营养的耗尽。
复合菌系的生长规律表明,其整个生长发酵过程划分为三个时期,即0~108 h的对数生长期、108~144 h的稳定期、144~216 h的衰亡期。并利用变形梯度凝胶电泳(即DGGE)对复合菌系的菌群组成及动态进行了探讨,结果表明,该复合菌系主要由14个微生物种群组成;随着发酵降解的逐渐进行,复合菌系的菌群出现了一定的波动。DGGE指纹图谱的聚类分析显示,整个降解过程可以划分为三个阶段:0~12 h为降解初期,24~120 h为降解中期,144~216 h为降解末期,与生长曲线大致吻合。
在降解初期微生物菌群之间竞争激烈,其中,Proteolyticum ethanoligenes strain GW、Uncultured Acidobacteria bacterium clone RDM3-B02、Uncultured Bacillus sp. clone 126、Aneurinibacillus danicus gene等种群数量相对较多,由于这些好氧菌的生长速率相对较快,引起反应体系的DO值和pH的迅速下降,从而为复合体系内微好氧菌群的生长发酵提供了适宜的条件。随着发酵的进行,这些种群的活性逐渐地受到抑制,从而转入休眠状态。降解中期是稻草水解的主要阶段,数量较多的微生物种群主要有Clostridium sp. PML14、Clostridium islandicum strain AK1、Brevibacillus borstelensis clone US12、Ureibacillus sp. A3.03、Uncultured bacterium tbr1-10、Bacillus sp. R-30914、Brevibacillus borstelensis strain U404。这些微生物都属于内生芽孢的革兰氏阳性菌,对温度和pH值变化的适应性强,可以产生胞外水解酶。其中Uncultured bacterium tbr1-10、Bacillus sp. R-30914、Brevibacillus borstelensis strain U404、Clostridium islandicum strain AK1是功能微生物,多种微生物的协同作用可以解除木质素降解过程中产生的有毒物质对降解的抑制,这是复合菌系高效降解木质纤维素的重要原因之一。
进入降解末期后,随着底物营养的逐渐耗尽,内源性呼吸作用加强,从而导致部分功能菌群数量减少,如Clostridium islandicum strain AK1、Brevibacillus borstelensis clone US12、Ureibacillus sp. A3.03、Uncultured bacterium tbr1-10、Bacillus sp. R-30914;部分菌群数量出现了相反的趋势,即在降解后期数量表现明显增加的迹象,逐渐成为优势菌,如:Bacillales bacterium 01QDX、Uncultured Bacilli bacterium clone SHBZ1189和Aneurinibacillus danicus;而一些菌群在整个发酵过程中始终保持相对较高的数量,如:Clostridium sp. PML14、Brevibacillus borstelensis strain U404。
As reserves of fossil fuels are limited and greenhouse effect caused by excessive use of fossil fuels is getting serious, the human are forced to seek alternatives to fossil fuels. Biomass energy is highly recognized as a result of both energy and environmental benefits. Lignocellulose is the main component of biomass and also the bottleneck for the usage of biomass. Thus, how to achieve efficient hydrolysis and acidification of lignocellulose has been a difficult problem for the usage of biomass resources. With the development of environmental biotechnology, microbial synergy gradually is taken seriously, and the application of microbial community in lignocellulose degradation has become a hot research. The main contributions to the current understanding of this topic are described below:
Growth characteristics, morphological characteristics and ecological conditions of the microbial community were studied respectively. Microscopic observation reveals that the microbial community is mainly composed of rod-shape bacteria. Those bacteria are mostly peritrichous flagellation with extracellular mucus layer, and their surface morphologies are diverse. The microbial community demands low oxygen, and DO was kept in 0.07~0.25 mg/L during degradation. The pH shows a trend of increase after initial decrease, which demonstrates the strong buffer capacity of the microbial community for the feedback inhibition of fermentation product.
Fermentation characteristics of microbial community were explored in detail and showed stronger capacity of lignocellulose degradation. The degradation rate of rice straw, cellulose, hemi cellulose and lignin may reach 75 %, 81 %, 92 %, and 46 % respectively when the fermentation terminated. The initial 60 h play an important role in degradating process, in which the degradation of rice straw, cellulose, hemi cellulose and lignin reached 59.0 %, 48.5 %, 86.8 % and 44.0 % of total degradation amount respectively. The fermentation is typically acetic acid type owing to 90% metabolite is acetic acid, and there are also a little ethanol, butyric acid and propionic acid in fermentative products. At 108 h, acetic acid and total volatile acids concentration reached maximum, they were 2.58 g/L and 2.90 g/L, respectively. The COD value of reactive system shows a trend that it increased at initial stage and decreased graduately. This demonstrates changes of substrate from solide lignocellulose to liquid volitail fatty acid, then to gaseous CO2 and H2. Furthermore, the change of Total Kjeltic Nitrogen (TKN) and ammonia nitrogen show that degradation capacity of microbial community declined owing to mainly depletion of the substrates, but not ammonia accumulation in 144-216 h.
Growth curve of the microbial community includes typical three periods, they are logarithmic phase (0~108 h), stationary phase (108~144 h) and decline phase (144~216 h) respectively. Community composition and development were investigated by the means of DGGE, and results showed that microbial community was constituted of 14 kinds of bacteria and presented fluctuation in some degree with fermention. Furthmore, result of cluster analysis of DGGE profile was accordant with growth curve, and the degradation process was divided into three stages: initial stage (0~12 h), intermediate stage (24~144 h) and end stage (144~216 h).
In initial stage, bateria competed fiercely, some bateria amount were dominant, such as Proteolyticum ethanoligenes strain GW,Uncultured Acidobacteria bacterium clone RDM3-B02,Uncultured Bacillus sp. clone 126 and Aneurinibacillus danicus, DO and pH of reactive system decreased rapidly owing to these aerobic bacteria’s relative faster grow rate, this will provide fitting conditions for grow and fermentation of facultative bacteria. In intermediate stage, these species were gradually being inhibited and transferred to a dormant state.
Straw degradation occurred mainly in intermediate stage. In this stage Clostridium sp. PML14,Clostridium islandicum strain AK1,Brevibacillus borstelensis clone US12,Ureibacillus sp. A3.03,Uncultured bacterium tbr1-10,Bacillus sp. R-30914,Brevibacillus borstelensis strain U404 existed in a large amount. These microorganisms are gram-positive bacteria with endogenous spore, which can adapt to changes in temperature and pH value and produce extracellular hydrolase. Uncultured bacterium tbr1-10、Bacillus sp. R-30914、Brevibacillus borstelensis strain U404、Clostridium islandicum strain AK1 performed as functional species group. Synergy of microbial community can also lift inhibition produced by toxic substances of the lignin degradation. This is one of the important reasons for the effective degradation of lignocellulose.
After entering end stage, with gradual depletion of the nutrients in substrate, part of functional species group declined as a result of endogenous respiration, such as Clostridium islandicum strain AK1、Brevibacillus borstelensis clone US12、Ureibacillus sp. A3.03、Uncultured bacterium tbr1-10、Bacillus sp. R-30914;Part of species shows the contary trend,and gradually develop into dominant species group, such as Bacillales bacterium 01QDX、Uncultured Bacilli bacterium clone SHBZ1189 and Aneurinibacillus danicus;While some species have always been to maintain a relatively high number, such as Clostridium sp. PML14 and Brevibacillus borstelensis strain U404。
系统地研究了一组木质纤维素降解复合菌系的形态特征、生长特性及生态条件。通过显微观察发现,复合菌系主要由杆菌组成,普遍周生鞭毛,胞外分泌粘液,表面形貌多样。在降解过程中复合菌系对氧的需求量较低,反应体系的DO值的波动范围很小,始终维持在0.07~0.25 mg/L范围内。体系的pH值呈现出先降低后上升的趋势,表明复合菌系对发酵产物的反馈抑制有较强的缓冲能力。
对复合菌系的发酵特性进行了较为详细的探讨,其对木质纤维素的降解表现出较强的能力,即在216 h内稻草降解了75 %,纤维素降解了81 %,半纤维素降解了92 %,木质素降解了46 %,并且前60 h为高效降解阶段,期间对稻草、纤维素、半纤维素和木质素的降解量分别占总降解量的59.0 %、48.5 %、86.8 %、44.0 %。而从复合菌系的发酵类型分析,其属于典型的乙酸型发酵,90%以上的代谢产物是乙酸,其次是少量的乙醇、丁酸和丙酸。发酵108 h后,乙酸浓度达到最大值2.58 g/L,此时总挥发酸也达到最大值2.90 g/L,该发酵类型为下游两相厌氧消化的产甲烷相的稳定、高效运行提供重要物质基础。发酵体系的COD值呈现出先增加后降低的总体趋势,说明了底物由固态的木质纤维素转变为液态的挥发酸,再转变为气态的CO2和H2的过程。总凯氏氮和氨态氮的变化趋势表明,复合菌系的活性在降解后期降低的原因不是氨态氮的累积而是营养的耗尽。
复合菌系的生长规律表明,其整个生长发酵过程划分为三个时期,即0~108 h的对数生长期、108~144 h的稳定期、144~216 h的衰亡期。并利用变形梯度凝胶电泳(即DGGE)对复合菌系的菌群组成及动态进行了探讨,结果表明,该复合菌系主要由14个微生物种群组成;随着发酵降解的逐渐进行,复合菌系的菌群出现了一定的波动。DGGE指纹图谱的聚类分析显示,整个降解过程可以划分为三个阶段:0~12 h为降解初期,24~120 h为降解中期,144~216 h为降解末期,与生长曲线大致吻合。
在降解初期微生物菌群之间竞争激烈,其中,Proteolyticum ethanoligenes strain GW、Uncultured Acidobacteria bacterium clone RDM3-B02、Uncultured Bacillus sp. clone 126、Aneurinibacillus danicus gene等种群数量相对较多,由于这些好氧菌的生长速率相对较快,引起反应体系的DO值和pH的迅速下降,从而为复合体系内微好氧菌群的生长发酵提供了适宜的条件。随着发酵的进行,这些种群的活性逐渐地受到抑制,从而转入休眠状态。降解中期是稻草水解的主要阶段,数量较多的微生物种群主要有Clostridium sp. PML14、Clostridium islandicum strain AK1、Brevibacillus borstelensis clone US12、Ureibacillus sp. A3.03、Uncultured bacterium tbr1-10、Bacillus sp. R-30914、Brevibacillus borstelensis strain U404。这些微生物都属于内生芽孢的革兰氏阳性菌,对温度和pH值变化的适应性强,可以产生胞外水解酶。其中Uncultured bacterium tbr1-10、Bacillus sp. R-30914、Brevibacillus borstelensis strain U404、Clostridium islandicum strain AK1是功能微生物,多种微生物的协同作用可以解除木质素降解过程中产生的有毒物质对降解的抑制,这是复合菌系高效降解木质纤维素的重要原因之一。
进入降解末期后,随着底物营养的逐渐耗尽,内源性呼吸作用加强,从而导致部分功能菌群数量减少,如Clostridium islandicum strain AK1、Brevibacillus borstelensis clone US12、Ureibacillus sp. A3.03、Uncultured bacterium tbr1-10、Bacillus sp. R-30914;部分菌群数量出现了相反的趋势,即在降解后期数量表现明显增加的迹象,逐渐成为优势菌,如:Bacillales bacterium 01QDX、Uncultured Bacilli bacterium clone SHBZ1189和Aneurinibacillus danicus;而一些菌群在整个发酵过程中始终保持相对较高的数量,如:Clostridium sp. PML14、Brevibacillus borstelensis strain U404。
As reserves of fossil fuels are limited and greenhouse effect caused by excessive use of fossil fuels is getting serious, the human are forced to seek alternatives to fossil fuels. Biomass energy is highly recognized as a result of both energy and environmental benefits. Lignocellulose is the main component of biomass and also the bottleneck for the usage of biomass. Thus, how to achieve efficient hydrolysis and acidification of lignocellulose has been a difficult problem for the usage of biomass resources. With the development of environmental biotechnology, microbial synergy gradually is taken seriously, and the application of microbial community in lignocellulose degradation has become a hot research. The main contributions to the current understanding of this topic are described below:
Growth characteristics, morphological characteristics and ecological conditions of the microbial community were studied respectively. Microscopic observation reveals that the microbial community is mainly composed of rod-shape bacteria. Those bacteria are mostly peritrichous flagellation with extracellular mucus layer, and their surface morphologies are diverse. The microbial community demands low oxygen, and DO was kept in 0.07~0.25 mg/L during degradation. The pH shows a trend of increase after initial decrease, which demonstrates the strong buffer capacity of the microbial community for the feedback inhibition of fermentation product.
Fermentation characteristics of microbial community were explored in detail and showed stronger capacity of lignocellulose degradation. The degradation rate of rice straw, cellulose, hemi cellulose and lignin may reach 75 %, 81 %, 92 %, and 46 % respectively when the fermentation terminated. The initial 60 h play an important role in degradating process, in which the degradation of rice straw, cellulose, hemi cellulose and lignin reached 59.0 %, 48.5 %, 86.8 % and 44.0 % of total degradation amount respectively. The fermentation is typically acetic acid type owing to 90% metabolite is acetic acid, and there are also a little ethanol, butyric acid and propionic acid in fermentative products. At 108 h, acetic acid and total volatile acids concentration reached maximum, they were 2.58 g/L and 2.90 g/L, respectively. The COD value of reactive system shows a trend that it increased at initial stage and decreased graduately. This demonstrates changes of substrate from solide lignocellulose to liquid volitail fatty acid, then to gaseous CO2 and H2. Furthermore, the change of Total Kjeltic Nitrogen (TKN) and ammonia nitrogen show that degradation capacity of microbial community declined owing to mainly depletion of the substrates, but not ammonia accumulation in 144-216 h.
Growth curve of the microbial community includes typical three periods, they are logarithmic phase (0~108 h), stationary phase (108~144 h) and decline phase (144~216 h) respectively. Community composition and development were investigated by the means of DGGE, and results showed that microbial community was constituted of 14 kinds of bacteria and presented fluctuation in some degree with fermention. Furthmore, result of cluster analysis of DGGE profile was accordant with growth curve, and the degradation process was divided into three stages: initial stage (0~12 h), intermediate stage (24~144 h) and end stage (144~216 h).
In initial stage, bateria competed fiercely, some bateria amount were dominant, such as Proteolyticum ethanoligenes strain GW,Uncultured Acidobacteria bacterium clone RDM3-B02,Uncultured Bacillus sp. clone 126 and Aneurinibacillus danicus, DO and pH of reactive system decreased rapidly owing to these aerobic bacteria’s relative faster grow rate, this will provide fitting conditions for grow and fermentation of facultative bacteria. In intermediate stage, these species were gradually being inhibited and transferred to a dormant state.
Straw degradation occurred mainly in intermediate stage. In this stage Clostridium sp. PML14,Clostridium islandicum strain AK1,Brevibacillus borstelensis clone US12,Ureibacillus sp. A3.03,Uncultured bacterium tbr1-10,Bacillus sp. R-30914,Brevibacillus borstelensis strain U404 existed in a large amount. These microorganisms are gram-positive bacteria with endogenous spore, which can adapt to changes in temperature and pH value and produce extracellular hydrolase. Uncultured bacterium tbr1-10、Bacillus sp. R-30914、Brevibacillus borstelensis strain U404、Clostridium islandicum strain AK1 performed as functional species group. Synergy of microbial community can also lift inhibition produced by toxic substances of the lignin degradation. This is one of the important reasons for the effective degradation of lignocellulose.
After entering end stage, with gradual depletion of the nutrients in substrate, part of functional species group declined as a result of endogenous respiration, such as Clostridium islandicum strain AK1、Brevibacillus borstelensis clone US12、Ureibacillus sp. A3.03、Uncultured bacterium tbr1-10、Bacillus sp. R-30914;Part of species shows the contary trend,and gradually develop into dominant species group, such as Bacillales bacterium 01QDX、Uncultured Bacilli bacterium clone SHBZ1189 and Aneurinibacillus danicus;While some species have always been to maintain a relatively high number, such as Clostridium sp. PML14 and Brevibacillus borstelensis strain U404。
引文
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Bayer E A, Lamed R, Himmel M E. 2007. The potential of cellulases and cellulosomes for cellulosic waste management [J]. Current Opinion in Biotechnology. 18:237~245
Baldrian P. 2004. Increase of laccase activity during interspecific interactions of white rot fungi [J]. FEMS Microbiology ecology. 50(3): 245~253
Du C Y, Sze K C L, Koutinas A et al. 2008. A wheat biorefining strategy based on solid-state fermentation for fermentative production of succinic acid [J]. Bioresource Technology 99: 8310~8315
Fischer S G, Lerman L S. 1979. Length-independent separation of DNA restriction fragments in two dimensional gel electrophoresis [J]. Cell. 16: 191~200
Goto K, Fujita R, Kato Y et al. 2004 Reclassification of Brevibacillus brevis strains NCIMB 13288 and DSM 6472 (=NRRL NRS-887) as Aneurinibacillus danicus sp. nov. and Brevibacillus limnophilus sp. nov [J]. International journal of systematic and evolutionary microbiology. 54: 419~427
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Ishii K, Fukui M, Takii S. 2000. Microbial succession during a composting process as evaluated by denaturing gradient gel electrophoresis analysis [J]. Journal of applied microbiology. 89(5): 768~777
Kato S, Haruta S, Cui Z J. 2005. Stable coexistence of five bacterial strains as a cellulose-degrading community [J]. Applied and environmental microbiology. 71(11): 7099~7106
Kiyohiko N, Le T, Yoshito I et al. 2009. Comparison of organic matter degradation and microbial community during thermophilic composting of two different types of anaerobic sludge [J]. Bioresource technology. 100: 676~682
Michio K. 2003. Oraging adaptation and the relationship between food-web complexity and stability [J]. Science. 299: 1388~1391
Muyzer G, Waal E C, Uitterlinden A G. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes encoding for 16S rRNA [J]. Applied and Environmental Microbiology. 59: 695~700
MacNaughton S J, Stephen J R, Venosa A D et al. 1999.Microbial population changes during bioremediation of an experimental oil spill[J]. Applied and Environmental Microbiology. 65: 3566~3574
Niu L L, Song L, Dong X Z et al, 2008 Proteiniborus ethanoligenes gen. nov., sp. nov., an anaerobic protein-utilizing bacterium[J]. International journal of systematic and evolutionary microbiology. 58:1~6
Nuebel U, Engelen B, Felske A. 1996. Sequence heterogeneities of genes encoding 16S rRNAs in paenibacillus polymyxa detected by temperature gradient gel electrophoresis [J]. Journal of bacteriology. 178: 5636~5643
Okuda N, Soneura M, Ninomiya K, et al. 2008 Biological detoxification of waste house wood hydrolysate using Ureibacillus thermosphaericus for bioethanol production[J]. Journal of bioscience and bioengineering. 106(2):128~133
O-Thong S, Prasertsan P, Intrasungkha N, et al. 2008 Microbial community analysis of a thermophilic mixed culture sludge for biohydrogen production [J]. Journal of applied microbiology. accepted in revise
Sakae H, Yoh S, Yasunori N et al. 2008. Profiling of a microbial community under confined conditions in a fed-batch garbage decomposer by denaturing gradient gel electrophoresis [J]. Bioresource technology. 99: 3084~3093
Shi, J, Chinn M S, Sharma R R et al. 2008. Microbial pretreatment of cotton stalks by solid state cultivation of Phanerochaete chrysosporium [J]. Bioresource Technology. 99: 6556~6564
Stepanova E V, Koroleva O V, Vasilehenko L G et al. 2003. Fungal decomposition of oat straw during liquid and solid-state fermentation [J]. Applied biochemistry and microbiology. 39(l): 65~74
Tang J C, Atsushi S, Zhou Q X et al. 2007. Effect of temperature on reaction rate and microbial community in composting of cattle manure with rice straw [J]. Journal of bioscience and bioengineering. 104(4): 321~328
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