水稻秸秆降解放线菌的分离鉴定及其降解机理研究
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摘要
我国水稻秸秆资源十分丰富,利用微生物使其降解转化为饲料、能源和化工原料对于解决目前面临的环境污染和能源危机具有重大现实意义。
在黑龙江省3个水稻主产区采集27份土壤样品,通过刚果红染色平板初筛;液体摇瓶发酵纤维素酶活测定;水稻秸秆失重率测定;半纤维素和木质素酶活性检测等多重筛选,得到一株高产纤维素酶、对水稻秸秆有较好降解效果的放线菌菌株C-5,该菌株同时具有多种木质纤维素酶活性。通过对该菌株的形态特征、培养特征和生理生化特征观察;16S rDNA序列测定及系统发育分析,将菌株C-5鉴定为灰略红链霉菌(Streptomyces griseorubens)。
测定了灰略红链霉菌C-5对水稻秸秆降解过程中,水稻秸秆主要成分含量的变化和纤维素酶活、半纤维素酶活的连续变化。发酵后,水稻秸秆细胞壁的主要成分得到了有效降解,粗纤维含量明显降低;纤维素、半纤维素酶活均可以在大部分发酵时间内保持在相对较高水平。
采用红外光谱(FTIR)法研究灰略红链霉菌C-5对水稻秸秆的生物降解过程,分析表明,水稻秸秆中含有碳水化合物、木质素和有机硅化物等成分,降解后生成了碳酸盐、硅酸盐和多糖类物质;羧基在发酵过程中以羧酸盐形式存在,一些无机阳离子形成碳酸盐。
对水稻秸秆木质素生物降解过程的红外光谱分析表明,灰略红链霉菌C-5对水稻秸秆木质素的降解主要是氧化作用,发生在木质素单元结构侧链Cα的氧化及Cα-Cβ键的氧化断裂;脂肪醚键发生断裂,苯环侧链的短链脂肪烃被降解;甲氧基含量明显增加,氢醌类物质形成;愈疮木基型木质素的降解程度高于紫丁香基型木质素的降解程度。
利用扫描电镜观察水稻秸秆生物降解过程中的组织结构变化,观察结果表明,在生物降解初期,菌丝通过水稻秸秆端部侵入内部,并在薄壁细胞的腔内繁殖,薄壁细胞开始分解,蜡质-硅化层明显变薄;当菌体大量繁殖后,维管束组织和部分厚壁组织开始降解;降解试验结束时,水稻秸秆的完整结构被严重破坏,薄壁组织被全部降解,蜡质-硅化层大部分也被降解破坏,只剩下部分高度木质化的表皮组织。
研究了不同培养条件对灰略红链霉菌C-5产纤维素酶的影响。结果表明,碳源浓度、氮源浓度、pH和培养温度对CMCase活力的影响均为极显著,碳源浓度是影响CMCase活力的关键性因子;较高浓度的碳源对合成纤维素酶更为有利,外加有机氮源豆饼粉对产纤维素酶的促进作用相对较大,较低浓度的氮源比较高浓度的氮源更能促进纤维素酶的合成;31℃和pH7.5是产酶最佳温度与酸碱度;菌株C-5在最佳产酶条件下的最佳产酶时间为发酵的4~14天。
The rice straw resources are very abundant in China. Its degradation and transformation into feed, energy and chemical raw materials by microorganisms has great realistic meaning of the solution to environmental pollution and energy crisis.
A streptomycete strain C-5 which producing high cellulase activities was selected by screening plate stained with congo red, determination of cellulase activities in liquid culture, determination of rice straw weight loss rate, detection of xylanase and ligninase activity from 27 soil samples which collected from three major rice-producing areas in Hei longjiang province. And it has great potential on the degradation of rice straw. This strain has multi-lignocellulase activities simultaneously. By studying the morphology, cultural characteristics, physiological and biochemical propertices, 16S rDNA and phylogenetic tree, strain C-5 was identified as Streptomyces griseorubens.
Degradation rates of the main components of rice straw and the continuous variation of cellulase activities and xylanase activities were determined during the rice straw fermentation by S. griseorubens C-5. The cell wall composition of rice straw was degradated effectively and the content of crude fiber was obviously reduced after cultivation. The results showed that the cellulase and xylanase activities could still maintain relative higher level in most of the fermentation time.
Fourier transform infrared spectroscopy (FTIR) was used to study the biodegradation process of rice straw. The analysis on the FTIR of rice straw showed that the carbohydrates, lignin and silica were degraded effectively after fermentation. Some of carbonate, silicate and polysaccharide were generated. Carboxyl group in the form of carboxylate in the process of degradation. Some inorganic cations were formed carbonate.
The biodegradation process of rice straw lignin by S. griseorubens C-5 was investigated by FTIR. The results demonstrated that the main function in rice straw lignin biodegradation by S. griseorubens C-5 was oxidation which occurred on side-chain Cαoxidation of lignin monomer and the oxidative cleavage of Cα-Cβbond. Aliphatic ether bond was breaking and some short chain aliphatic hydrocarbons were degraded. The methoxyl content was significantly increased and some hydroquinone substances was formed. The degradation degree of the guaiacyl type lignin was higher than the syringic type lignin by S. riseorubens C-5.
Histological changes of rice straw were observed by scanning electron microscope during fermentation by S. griseorubens C-5. The results showed that in the initial stage of biodegradation, hypha invaded in the rice straw through the end of the rice straw and were already fixed on the parenchyma of rice stem. The parenchyma of rice stem began to degrade and the wax-silicide layer was thinner significantly at the early stage of degradation. The vascular bundles and sclerenchyma began to degrade after the numerous actinomycetes colonized. The rice fragments had lost their intact structures and the parenchyma had completely disappeared and the cuticle wax and silica layer was destroyed partially after fermentation. The remaining tissues in rice stem was the part epidermal tissue which was lignified in high degree.
Different culture conditions on production of cellulase of S. griseorubens C-5 were investigated. The results showed that the carbon concentration, nitrogen concentration, pH and incubation temperature were significant on the activity of CMCase. Carbon concentration was the key factor which affecting CMCase activity. The higher concentrations of carbon sources was more favorable to the synthesis of cellulose. Organic nitrogen soybean could promote the synthesis of cellulase production. The lower concentrations of nitrogen could enhance the cellulose synthesis than the higher concentrations of nitrogen. 31℃and pH 7.5 was the optimal temperature and pH of cellulose synthesis. The optimal time of the cellulase production of S. griseorubens C-5 was between 4~14 fermentation days in the optimal cellulase production condition.
在黑龙江省3个水稻主产区采集27份土壤样品,通过刚果红染色平板初筛;液体摇瓶发酵纤维素酶活测定;水稻秸秆失重率测定;半纤维素和木质素酶活性检测等多重筛选,得到一株高产纤维素酶、对水稻秸秆有较好降解效果的放线菌菌株C-5,该菌株同时具有多种木质纤维素酶活性。通过对该菌株的形态特征、培养特征和生理生化特征观察;16S rDNA序列测定及系统发育分析,将菌株C-5鉴定为灰略红链霉菌(Streptomyces griseorubens)。
测定了灰略红链霉菌C-5对水稻秸秆降解过程中,水稻秸秆主要成分含量的变化和纤维素酶活、半纤维素酶活的连续变化。发酵后,水稻秸秆细胞壁的主要成分得到了有效降解,粗纤维含量明显降低;纤维素、半纤维素酶活均可以在大部分发酵时间内保持在相对较高水平。
采用红外光谱(FTIR)法研究灰略红链霉菌C-5对水稻秸秆的生物降解过程,分析表明,水稻秸秆中含有碳水化合物、木质素和有机硅化物等成分,降解后生成了碳酸盐、硅酸盐和多糖类物质;羧基在发酵过程中以羧酸盐形式存在,一些无机阳离子形成碳酸盐。
对水稻秸秆木质素生物降解过程的红外光谱分析表明,灰略红链霉菌C-5对水稻秸秆木质素的降解主要是氧化作用,发生在木质素单元结构侧链Cα的氧化及Cα-Cβ键的氧化断裂;脂肪醚键发生断裂,苯环侧链的短链脂肪烃被降解;甲氧基含量明显增加,氢醌类物质形成;愈疮木基型木质素的降解程度高于紫丁香基型木质素的降解程度。
利用扫描电镜观察水稻秸秆生物降解过程中的组织结构变化,观察结果表明,在生物降解初期,菌丝通过水稻秸秆端部侵入内部,并在薄壁细胞的腔内繁殖,薄壁细胞开始分解,蜡质-硅化层明显变薄;当菌体大量繁殖后,维管束组织和部分厚壁组织开始降解;降解试验结束时,水稻秸秆的完整结构被严重破坏,薄壁组织被全部降解,蜡质-硅化层大部分也被降解破坏,只剩下部分高度木质化的表皮组织。
研究了不同培养条件对灰略红链霉菌C-5产纤维素酶的影响。结果表明,碳源浓度、氮源浓度、pH和培养温度对CMCase活力的影响均为极显著,碳源浓度是影响CMCase活力的关键性因子;较高浓度的碳源对合成纤维素酶更为有利,外加有机氮源豆饼粉对产纤维素酶的促进作用相对较大,较低浓度的氮源比较高浓度的氮源更能促进纤维素酶的合成;31℃和pH7.5是产酶最佳温度与酸碱度;菌株C-5在最佳产酶条件下的最佳产酶时间为发酵的4~14天。
The rice straw resources are very abundant in China. Its degradation and transformation into feed, energy and chemical raw materials by microorganisms has great realistic meaning of the solution to environmental pollution and energy crisis.
A streptomycete strain C-5 which producing high cellulase activities was selected by screening plate stained with congo red, determination of cellulase activities in liquid culture, determination of rice straw weight loss rate, detection of xylanase and ligninase activity from 27 soil samples which collected from three major rice-producing areas in Hei longjiang province. And it has great potential on the degradation of rice straw. This strain has multi-lignocellulase activities simultaneously. By studying the morphology, cultural characteristics, physiological and biochemical propertices, 16S rDNA and phylogenetic tree, strain C-5 was identified as Streptomyces griseorubens.
Degradation rates of the main components of rice straw and the continuous variation of cellulase activities and xylanase activities were determined during the rice straw fermentation by S. griseorubens C-5. The cell wall composition of rice straw was degradated effectively and the content of crude fiber was obviously reduced after cultivation. The results showed that the cellulase and xylanase activities could still maintain relative higher level in most of the fermentation time.
Fourier transform infrared spectroscopy (FTIR) was used to study the biodegradation process of rice straw. The analysis on the FTIR of rice straw showed that the carbohydrates, lignin and silica were degraded effectively after fermentation. Some of carbonate, silicate and polysaccharide were generated. Carboxyl group in the form of carboxylate in the process of degradation. Some inorganic cations were formed carbonate.
The biodegradation process of rice straw lignin by S. griseorubens C-5 was investigated by FTIR. The results demonstrated that the main function in rice straw lignin biodegradation by S. griseorubens C-5 was oxidation which occurred on side-chain Cαoxidation of lignin monomer and the oxidative cleavage of Cα-Cβbond. Aliphatic ether bond was breaking and some short chain aliphatic hydrocarbons were degraded. The methoxyl content was significantly increased and some hydroquinone substances was formed. The degradation degree of the guaiacyl type lignin was higher than the syringic type lignin by S. riseorubens C-5.
Histological changes of rice straw were observed by scanning electron microscope during fermentation by S. griseorubens C-5. The results showed that in the initial stage of biodegradation, hypha invaded in the rice straw through the end of the rice straw and were already fixed on the parenchyma of rice stem. The parenchyma of rice stem began to degrade and the wax-silicide layer was thinner significantly at the early stage of degradation. The vascular bundles and sclerenchyma began to degrade after the numerous actinomycetes colonized. The rice fragments had lost their intact structures and the parenchyma had completely disappeared and the cuticle wax and silica layer was destroyed partially after fermentation. The remaining tissues in rice stem was the part epidermal tissue which was lignified in high degree.
Different culture conditions on production of cellulase of S. griseorubens C-5 were investigated. The results showed that the carbon concentration, nitrogen concentration, pH and incubation temperature were significant on the activity of CMCase. Carbon concentration was the key factor which affecting CMCase activity. The higher concentrations of carbon sources was more favorable to the synthesis of cellulose. Organic nitrogen soybean could promote the synthesis of cellulase production. The lower concentrations of nitrogen could enhance the cellulose synthesis than the higher concentrations of nitrogen. 31℃and pH 7.5 was the optimal temperature and pH of cellulose synthesis. The optimal time of the cellulase production of S. griseorubens C-5 was between 4~14 fermentation days in the optimal cellulase production condition.
引文
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