象白蚁肠道中一个纤维素降解菌群的分离和特性研究
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摘要
利用滤纸培养基从象白蚁(Nasutitermes sp.)肠道中分离出一个具有纤维素降解能力,能够降解滤纸的混合菌群。在起始pH 6.5,37℃培养条件下培养6d可得到最高的纤维素酶(CMCase和FPase)活性。在优化条件下,混合菌群的滤纸降解率在第15d达到最大值66.3%,显示出较高的滤纸降解效率。酶谱活性染色分析显示,混合菌群在以滤纸为唯一碳源的生长过程中至少表达了8种内切葡聚糖酶和4种木聚糖酶。扫描电镜观察到该混合菌群包含短杆状和球形两种形态的细菌。基于16SrRNA基因的系统发育分析表明,该混合菌群中至少存在两种细菌,分别属于沙雷氏菌属(Serratia)和类芽胞杆菌属(Paenibacillus)。这两种细菌协同降解纤维素的机制值得进一步深入研究。
In this study,a bacterial consortium with the ability of cellulose degradation was isolated from the gut of Nasutitermes sp.using filter paper culture medium.The bacterial consortium was able to degrade the filter paper steadily within 6 days in continuous culture.It showed the maximum cellulase activity(CMCase and FPase)on the 6th day of fermentation at 37℃ with initial pH 6.5.Under the optimum conditions,the degradation rate of filter paper reached 66.3% on the 15th day,indicated a high efficiency of cellulose degradation.Zymogram analysis showed that at least eight endoglucanases and four xylanases were expressed by the bacterial consortium during fermentation.Scanning electron microscopy(SEM)revealed that the bacterial consortium contained bacteria of two morphologies:short rods and spheres.Phylogenetic analysis based on 16 SrRNA genes showed that there were at least two species of bacteria in the bacterial consortium,which belonged to genera Paenibacillus and Serratia.Further studies will be made to investigate the synergistic mechanisms of these two bacteria in the degradation of cellulose.
In this study,a bacterial consortium with the ability of cellulose degradation was isolated from the gut of Nasutitermes sp.using filter paper culture medium.The bacterial consortium was able to degrade the filter paper steadily within 6 days in continuous culture.It showed the maximum cellulase activity(CMCase and FPase)on the 6th day of fermentation at 37℃ with initial pH 6.5.Under the optimum conditions,the degradation rate of filter paper reached 66.3% on the 15th day,indicated a high efficiency of cellulose degradation.Zymogram analysis showed that at least eight endoglucanases and four xylanases were expressed by the bacterial consortium during fermentation.Scanning electron microscopy(SEM)revealed that the bacterial consortium contained bacteria of two morphologies:short rods and spheres.Phylogenetic analysis based on 16 SrRNA genes showed that there were at least two species of bacteria in the bacterial consortium,which belonged to genera Paenibacillus and Serratia.Further studies will be made to investigate the synergistic mechanisms of these two bacteria in the degradation of cellulose.
引文
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[18]Ohkuma M,Yuki M,Oshima K,et al.Draft genome sequence of the alkaliphilic and xylanolytic Paenibacillus sp.strain JCM 10914,isolated from the gut of a soil-feeding termite[J].Genome Announce,2013,2(1):e01144-13.
[2]Ni J F,Tokuda G.Lignocellulose-degrading enzymes from termites and their symbiotic microbiota[J].Biotechnol Adv,2013,31(6):838-850.
[3]Ahmed S,Riaz S,Jamil A.Molecular cloning of fungal xylanases:an overview[J].Appl Microbiol and Biotech,2009,84(1):19-35.
[4]Brune A.Symbiotic digestion of lignocellulose in termite guts[J].Nat Rev Microbiol,2014,12(3):168-180.
[5]Mikaelyan A,Strassert J F H,Tokuda G,et al.The fibre-associated cellulolytic bacterial community in the hindgut of wood-feeding higher termites(Nasutitermes spp.)[J].Environ Microbiol,2014,16(9):2711-2722.
[6]K9hler T,Dietrich C,Scheffrahn R H,et al.Highresolution analysis of gut environment and bacterial microbiota reveals functional compartmentation of the gut in wood-feeding higher termites(Nasutitermes spp.)[J].Appl Environ Microbiol,2012,78(13):4691-701.
[7]Warnecke F,Luginbühl P,Ivanova N,et al.Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite[J].Nature,2007,450(7169):560-565.
[8]Hethener P,Brauman A,Garcia J-L.Clostridium termitidis sp.nov.,a cellulolytic bacterium from the gut of the wood-feeding termite,Nasutitermes lujae[J].System Appl Microbiol,1992,15:52-58.
[9]Tailliez P,Girard H,Millet J,et al.Enhanced cellulose fermentation by an asporogenous and ethanol-tolerant mutant of Clostridium thermocellum[J].Appl Environ Microbiol,1989,55(1):203-206.
[10]Royer J C,Nakas J P.Simple,sensitive zymogram technique for detection of xylanase activity in polyacrylamide gels[J].Appl Environ Microbiol,1990,56(6):1516-1517.
[11]Paul J,Saxena S,Varma A.Ultrastructural studies of the termite(Odontotermes obesus)gut microflora and its cellulolytic properties[J].World J Microbiol and Biotech,1993,9(1):108-112.
[12]Ohkuma M,Shimizu H,Thongaram T,et al.An alkaliphilic and xylanolytic Paenibacillus species isolated from the gut of a soil-feeding termite[J].Microbes and Environ,2003,18(3):145-151.
[13]Dheeran P,Nandhagopal N,Kumar S,et al.A novel thermostable xylanase of Paenibacillus macerans IIPSP3isolated from the termite gut[J].J Ind Microbiol Biotech,2012,39(6):851-860.
[14]Tarayre C,Brognaux A,Bauwens J,et al.Isolation of amylolytic,xylanolytic,and cellulolytic microorganisms extracted from the gut of the termite Reticulitermes santonensis by means of a micro-aerobic atmosphere[J].World J Microbiol Biotech,2014,30(5):1655-1660.
[15]Mathew G M,Mathew D C,Lo S C,et al.Synergistic collaboration of gut symbionts in Odontotermes formosanus,for lignocellulosic degradation and bio-hydrogen production[J].Bioresource Technol,2012,145(10):337-344.
[16]Thayer D W.Facultative wood-digesting bacteria from the hindgut of the termite Reticulitermes hesper[J].J Gen Microbiol,1976,96(2):287-296.
[17]Thayer D W.Carboxymethyl cellulase produced by facultative bacteria from the hindgut of the termite Reticulitermes hesperus[J].J Gen Microbiol,1978,106(1):13-18.
[18]Ohkuma M,Yuki M,Oshima K,et al.Draft genome sequence of the alkaliphilic and xylanolytic Paenibacillus sp.strain JCM 10914,isolated from the gut of a soil-feeding termite[J].Genome Announce,2013,2(1):e01144-13.
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