绵羊瘤胃主要纤维降解细菌的分离鉴定及不同氮源对其纤维降解能力的影响
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摘要
本研究从内蒙古地区绵羊瘤胃内容物中分离主要纤维素降解细菌并对其进行了系统全面的鉴定。对其中几株典型的、具有代表性的主要纤维素降解细菌的生长特性、纤维降解特性进行了研究。在此基础上研究氮源对瘤胃主要纤维素降解细菌纤维降解能力的影响及初步探讨其可能机理。
从蒙古绵羊瘤胃内容物中分离到23株纤维素降解细菌,通过初步形态学观察和生理生化实验,对所有23株菌进行了初步鉴定。从23株菌中选出12株纤维素降解率较高并具有代表性的菌株进行(G+C) mol%测定,再对其中的11株菌进行16S rDNA测序和系统发育分析研究,结果表明:其中4株溶纤维丁酸弧菌(Butyrivibrio fibrisolvens)、1株白色瘤胃球菌(Rumincoccus albus)、2株黄色瘤胃球菌(R. flavefaciens)、2株产琥珀酸丝状杆菌(Fibrobacter succinogenes)、1株解多糖梭菌(Clostridium polysaccharolyticum)、1株粪肠球菌(Enterococcus faecalis)。从白色瘤胃球菌、黄色瘤胃球菌、产琥珀酸丝状杆菌和溶纤维丁酸弧菌中各选出1株有代表性的菌株进行生长和纤维降解特性研究以及氮源影响研究。
4株菌的生长特性和纤维降解特性研究表明:球菌的生长速度大于杆菌和弧菌。4株菌的纤维素降解率和纤维素酶活力趋势一致,在4株菌中,产琥珀酸丝状杆菌的纤维素降解率和纤维素酶活力均最高。纤维素酶活力测定结果表明:外切-β-1,4-葡聚糖酶是3种纤维素酶中的限速酶;4株菌的纤维素酶大部分是胞内酶或与细胞相连的胞外酶。纤维素与纤维二糖的质量比对纤维素降解率有显著影响,4株菌达到最大纤维素降解率时的纤维素与纤维二糖质量比均在7:3或其附近。
所选4株菌和混合瘤胃微生物既能在氨氮为唯一氮源的合成培养基中生长,又能在含有肽和氨基酸的合成培养基中生长。相对于氨态氮,肽、混合氨基酸和单个氨基酸对所选择的4株纤维素降解细菌的纤维素降解能力具有明显的促进作用。氮源对混合瘤胃微生物、白色瘤胃球菌、黄色瘤胃球菌、产琥珀酸丝状杆菌和溶纤维丁酸弧菌纤维素降解率的影响存在种属差异。在所有氮源中,Phe无论对混合瘤胃微生物还是对4株纤维素降解细菌均具有明显的促进纤维素降解的作用,但作用机制不同。对于4株纤维素降解细菌,酪蛋白酸水解物对纤维素降解率的促进作用大于酪蛋白酶水解物对纤维素降解率的促进作用,而二者对混合瘤胃微生物的影响没有差异。大豆蛋白酸水解物、酪蛋白酸水解物和酪蛋白酶水解物相对于其它氮源对混合瘤胃微生物纤维素降解能力的促进作用较大;酪蛋白酸水解物和大豆蛋白酸水解物对溶纤维丁酸弧菌WH-1的纤维素降解能力具有促进作用;Met、Leu和大豆蛋白酸水解物对白色瘤胃球菌VI3、黄色瘤胃球菌CCQ和产琥珀酸丝状杆菌LBG-1的纤维素降解能力具有促进作用。通过比较固液相菌体蛋白浓度和酶活力发现,4株菌的菌体和酶均有大部分黏附在纤维素颗粒上,而且纤维素降解率与黏附程度基本成正比。从研究结果可见,氮源对本研究所分离的单株纤维素降解细菌的影响与对混合瘤胃微生物的影响存在一定的差异,说明研究氮源对瘤胃主要纤维降解细菌的影响是完全必要的,对我们更好地认识和理解氮源对反刍动物瘤胃纤维素降解的影响机理具有一定指导意义。
Major cellulolytic bacteria were isolated from the rumen of the sheep in Inner Mongolia. The isolates were systemically identified. The characteristics of cell growth and cellulose degradation of the cellulolytic bacteria were studied. The effects of nitrogen sources on cellulolytic activity of the rumen cellulolytic bacteria were discussed and the possible mechanisms for the effects of nitrogen sources on improving cellulolytic activity were suggested.
23 strains of cellulolytic bacteria were isolated from the rumen of the sheep in Inner Mongolia and identified by morphological and physiological characteristics in the first place. 12 strains of 23 strains of cellulolytic bacteria were selected for (G+C) mol% identification. The 16S rDNA gene sequences of 11 strains of the 12 strains of cellulolytic bacteria were sequenced. Based on the 16S rDNA gene sequences, phylogenetic trees were constructed. The result of the identification showed that there were 4 strains of Butyrivibrio fibrisolvens, 1 strain of Rumincoccus albus, 2 strains of R. flavefaciens, 2 strains of Fibrobacter succinogenes, 1 strain of Clostridium polysaccharolyticum and 1 strain of Enterococcus faecalis among the 11 strains of cellulolytic bacteria. One strain of representative bacterium was selected for further research from every type of strain of Rumincoccus albus, Rumincoccus flavefaciens, Fibrobacter succinogenes and Butyrivibrio fibrisolvens respectively.
The following results were concluded by the studies on the characteristics for cell growth and cellulose degradation of the 4 strains. The rumen coccus grows faster than rivibrio and bacillus. The trend in cellulose degradative rate is consistent with the trend in cellulose activity for all the 4 strains. Fibrobacter succinogenes has the highest cellulose degradative rate and highest cellulase activity among the 4 strains. The exo-β-1,4-glucanases is the rate-limiting enzyme of 3 kinds of cellulase out of the 4 strains of cellulolytic bacteria. The cellulase either exists in cells or is attached to cell membrane. The ratio of cellulose to cellobiose affects the degradative rate for cellulose markedly. When the ratio of cellulose to cellobiose is 7:3 or so, the degradative rate for cellulose reaches the maximum for all the 4 strains.
All the 4 strains of cellulolytic bacteria and mixed rumen microbes can grow in the chemically defined medium with ammonia-nitrogen or peptide or amino acid as the sole nitrogen source. Compared with ammonia-nitrogen, peptide and amino acid promote the degradative rate for cellulose of the 4 strains selected significantly. The influence of nitrogen source on the degradative rate for cellulose displays species differences with mixed rumen microbes, Rumincoccus albus, Rumincoccus flavefaciens, Fibrobacter succinogenes and Butyrivibrio fibrisolvens. Phe increases the degradative rate for cellulose of both the mixed rumen microbes and the 4 strains of cellulolytic bacteria, but the effects of Phe on the growth of bacteria are different with strains. Acidic hydrolysates of casein promote the degradation of cellulose of the 4 strains of cellulolytic bacteria, and enzymic hydrolysates of casein don’t. Both acidic hydrolysates of casein and enzymic hydrolysates of casein have the same effect on mixed rumen microbes. Acidic hydrolysates of soybean protein, acidic hydrolysates of casein and enzymic hydrolysates of casein enhance the degradative rate for cellulose of mixed rumen microbes. Acidic hydrolysates of casein and acidic hydrolystes of soybean protein improve the degradative rate for cellulose of Butyrivibrio fibrisolvens WH-1. Met, Leu and acidic hydrolysates of soybean protein increase the degradative rate for cellulose of Rumincoccus albus VI3, Rumincoccus flavefaciens CCQ and Fibrobacter succinogenes LBG-1 respectively. The comparison between BCP concentration and cellulase activity in both the solid and liquid phases indicates that the bacteria and cellulase exist mainly by adhesion to cellulose particles. The degradative rate for cellulose is proportional to the extent of adhension of bacteria and cellulose to cellulose particles. Due to the result that there are differences between the effect of nitrogen sources on mixed rumen microbes and that on the major rumen cellulolytic bacteria, it is necessary to investigate the influence of different kinds of nitrogen sources on the major rumen cellulolytic bacteria. The research contributes to a better understanding of the mechanism of nitrogen sources working on cellulolytic activity in the rumen of ruminants.
从蒙古绵羊瘤胃内容物中分离到23株纤维素降解细菌,通过初步形态学观察和生理生化实验,对所有23株菌进行了初步鉴定。从23株菌中选出12株纤维素降解率较高并具有代表性的菌株进行(G+C) mol%测定,再对其中的11株菌进行16S rDNA测序和系统发育分析研究,结果表明:其中4株溶纤维丁酸弧菌(Butyrivibrio fibrisolvens)、1株白色瘤胃球菌(Rumincoccus albus)、2株黄色瘤胃球菌(R. flavefaciens)、2株产琥珀酸丝状杆菌(Fibrobacter succinogenes)、1株解多糖梭菌(Clostridium polysaccharolyticum)、1株粪肠球菌(Enterococcus faecalis)。从白色瘤胃球菌、黄色瘤胃球菌、产琥珀酸丝状杆菌和溶纤维丁酸弧菌中各选出1株有代表性的菌株进行生长和纤维降解特性研究以及氮源影响研究。
4株菌的生长特性和纤维降解特性研究表明:球菌的生长速度大于杆菌和弧菌。4株菌的纤维素降解率和纤维素酶活力趋势一致,在4株菌中,产琥珀酸丝状杆菌的纤维素降解率和纤维素酶活力均最高。纤维素酶活力测定结果表明:外切-β-1,4-葡聚糖酶是3种纤维素酶中的限速酶;4株菌的纤维素酶大部分是胞内酶或与细胞相连的胞外酶。纤维素与纤维二糖的质量比对纤维素降解率有显著影响,4株菌达到最大纤维素降解率时的纤维素与纤维二糖质量比均在7:3或其附近。
所选4株菌和混合瘤胃微生物既能在氨氮为唯一氮源的合成培养基中生长,又能在含有肽和氨基酸的合成培养基中生长。相对于氨态氮,肽、混合氨基酸和单个氨基酸对所选择的4株纤维素降解细菌的纤维素降解能力具有明显的促进作用。氮源对混合瘤胃微生物、白色瘤胃球菌、黄色瘤胃球菌、产琥珀酸丝状杆菌和溶纤维丁酸弧菌纤维素降解率的影响存在种属差异。在所有氮源中,Phe无论对混合瘤胃微生物还是对4株纤维素降解细菌均具有明显的促进纤维素降解的作用,但作用机制不同。对于4株纤维素降解细菌,酪蛋白酸水解物对纤维素降解率的促进作用大于酪蛋白酶水解物对纤维素降解率的促进作用,而二者对混合瘤胃微生物的影响没有差异。大豆蛋白酸水解物、酪蛋白酸水解物和酪蛋白酶水解物相对于其它氮源对混合瘤胃微生物纤维素降解能力的促进作用较大;酪蛋白酸水解物和大豆蛋白酸水解物对溶纤维丁酸弧菌WH-1的纤维素降解能力具有促进作用;Met、Leu和大豆蛋白酸水解物对白色瘤胃球菌VI3、黄色瘤胃球菌CCQ和产琥珀酸丝状杆菌LBG-1的纤维素降解能力具有促进作用。通过比较固液相菌体蛋白浓度和酶活力发现,4株菌的菌体和酶均有大部分黏附在纤维素颗粒上,而且纤维素降解率与黏附程度基本成正比。从研究结果可见,氮源对本研究所分离的单株纤维素降解细菌的影响与对混合瘤胃微生物的影响存在一定的差异,说明研究氮源对瘤胃主要纤维降解细菌的影响是完全必要的,对我们更好地认识和理解氮源对反刍动物瘤胃纤维素降解的影响机理具有一定指导意义。
Major cellulolytic bacteria were isolated from the rumen of the sheep in Inner Mongolia. The isolates were systemically identified. The characteristics of cell growth and cellulose degradation of the cellulolytic bacteria were studied. The effects of nitrogen sources on cellulolytic activity of the rumen cellulolytic bacteria were discussed and the possible mechanisms for the effects of nitrogen sources on improving cellulolytic activity were suggested.
23 strains of cellulolytic bacteria were isolated from the rumen of the sheep in Inner Mongolia and identified by morphological and physiological characteristics in the first place. 12 strains of 23 strains of cellulolytic bacteria were selected for (G+C) mol% identification. The 16S rDNA gene sequences of 11 strains of the 12 strains of cellulolytic bacteria were sequenced. Based on the 16S rDNA gene sequences, phylogenetic trees were constructed. The result of the identification showed that there were 4 strains of Butyrivibrio fibrisolvens, 1 strain of Rumincoccus albus, 2 strains of R. flavefaciens, 2 strains of Fibrobacter succinogenes, 1 strain of Clostridium polysaccharolyticum and 1 strain of Enterococcus faecalis among the 11 strains of cellulolytic bacteria. One strain of representative bacterium was selected for further research from every type of strain of Rumincoccus albus, Rumincoccus flavefaciens, Fibrobacter succinogenes and Butyrivibrio fibrisolvens respectively.
The following results were concluded by the studies on the characteristics for cell growth and cellulose degradation of the 4 strains. The rumen coccus grows faster than rivibrio and bacillus. The trend in cellulose degradative rate is consistent with the trend in cellulose activity for all the 4 strains. Fibrobacter succinogenes has the highest cellulose degradative rate and highest cellulase activity among the 4 strains. The exo-β-1,4-glucanases is the rate-limiting enzyme of 3 kinds of cellulase out of the 4 strains of cellulolytic bacteria. The cellulase either exists in cells or is attached to cell membrane. The ratio of cellulose to cellobiose affects the degradative rate for cellulose markedly. When the ratio of cellulose to cellobiose is 7:3 or so, the degradative rate for cellulose reaches the maximum for all the 4 strains.
All the 4 strains of cellulolytic bacteria and mixed rumen microbes can grow in the chemically defined medium with ammonia-nitrogen or peptide or amino acid as the sole nitrogen source. Compared with ammonia-nitrogen, peptide and amino acid promote the degradative rate for cellulose of the 4 strains selected significantly. The influence of nitrogen source on the degradative rate for cellulose displays species differences with mixed rumen microbes, Rumincoccus albus, Rumincoccus flavefaciens, Fibrobacter succinogenes and Butyrivibrio fibrisolvens. Phe increases the degradative rate for cellulose of both the mixed rumen microbes and the 4 strains of cellulolytic bacteria, but the effects of Phe on the growth of bacteria are different with strains. Acidic hydrolysates of casein promote the degradation of cellulose of the 4 strains of cellulolytic bacteria, and enzymic hydrolysates of casein don’t. Both acidic hydrolysates of casein and enzymic hydrolysates of casein have the same effect on mixed rumen microbes. Acidic hydrolysates of soybean protein, acidic hydrolysates of casein and enzymic hydrolysates of casein enhance the degradative rate for cellulose of mixed rumen microbes. Acidic hydrolysates of casein and acidic hydrolystes of soybean protein improve the degradative rate for cellulose of Butyrivibrio fibrisolvens WH-1. Met, Leu and acidic hydrolysates of soybean protein increase the degradative rate for cellulose of Rumincoccus albus VI3, Rumincoccus flavefaciens CCQ and Fibrobacter succinogenes LBG-1 respectively. The comparison between BCP concentration and cellulase activity in both the solid and liquid phases indicates that the bacteria and cellulase exist mainly by adhesion to cellulose particles. The degradative rate for cellulose is proportional to the extent of adhension of bacteria and cellulose to cellulose particles. Due to the result that there are differences between the effect of nitrogen sources on mixed rumen microbes and that on the major rumen cellulolytic bacteria, it is necessary to investigate the influence of different kinds of nitrogen sources on the major rumen cellulolytic bacteria. The research contributes to a better understanding of the mechanism of nitrogen sources working on cellulolytic activity in the rumen of ruminants.
引文
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