本文選題：海洋細菌　切入點：原位富集　出處：《集美大學》2015年碩士論文

【摘要】：海洋環(huán)境中含有豐富的微生物資源,基于技術(shù)等原因,只有少部分的海洋微生物被分離、研究及應(yīng)用。由于生活環(huán)境不同于陸地,多數(shù)海洋微生物具有獨特的生理生化及代謝功能,具有耐鹽、嗜熱、嗜冷等特征,可以說海洋環(huán)境比陸地蘊含更多極端微生物。深入研究海洋微生物,能夠為人類獲得更多的生物資源。甘蔗渣是甘蔗制糖后的殘留物,是一種生物質(zhì)資源,主要成分為纖維素、半纖維素和木質(zhì)素,本研究采用原位富集方法,于近海及深海環(huán)境中富集甘蔗渣降解菌。首先利用分子生態(tài)學方法研究了兩個富集位點下未培養(yǎng)甘蔗渣降解菌的菌群組成情況。結(jié)果顯示兩個文庫均有各自較為明顯的優(yōu)勢菌群:深海文庫優(yōu)勢細菌類群為變形菌門,其中γ-變形菌綱在深海文庫中的豐度最大;近海文庫優(yōu)勢細菌類群為擬桿菌門。同時,從兩個富集樣品分離得到29株細菌,主要屬于變形菌門與厚壁菌門兩個類群,與未培養(yǎng)得到的優(yōu)勢菌群一致。對從深海環(huán)境中分離到的兩株芽孢桿菌GZ03、GZ04降解甘蔗渣的情況進行了分析,結(jié)果發(fā)現(xiàn)兩株菌均能在甘蔗渣降解上具有應(yīng)用價值:兩個菌株能夠降解甘蔗渣產(chǎn)較高濃度還原糖,離子色譜結(jié)果顯示降解產(chǎn)物為葡萄糖、果糖混合液;通過GZ03菌株產(chǎn)還原糖單因素條件優(yōu)化實驗發(fā)現(xiàn),菌株產(chǎn)還原糖最適培養(yǎng)基為2%甘蔗渣(60目)、3%Na Cl、蒸餾水配置,產(chǎn)還原糖最適條件為:4%接種量、35℃搖床培養(yǎng)60 h;GZ04菌株采用固體發(fā)酵方法可降解甘蔗渣半纖維素,于28℃恒溫發(fā)酵5 d,半纖維素質(zhì)量減少20%。本研究也針對這兩株菌一些參與甘蔗渣降解的酶類進行了研究。對菌株GZ03進行全基因組測序,分析測序結(jié)果,對其中編碼果聚糖蔗糖酶基因GH5進行克隆表達。果聚糖蔗糖酶的最適水解溫度為30℃,最適p H為6.0,最適蔗糖濃度為400 m M。果聚糖最適合成條件為:30℃、p H7.8、蔗糖濃度400 m M,并且在36 h合成量最大。對GZ04菌株產(chǎn)的木聚糖酶進行酶學性質(zhì)研究,發(fā)現(xiàn)該酶的最適溫度為40℃,最適p H值為5.0,在40℃環(huán)境下具有良好的穩(wěn)定性,并且在不同p H環(huán)境下均能維持較高酶活力。合理使用甘蔗渣,減少生產(chǎn)廢料,實現(xiàn)蔗渣資源的可循環(huán)、可發(fā)展利用日益受到關(guān)注,而開發(fā)海洋來源的細菌降解甘蔗渣制糖是當前高效利用甘蔗渣的新途徑。綜上所述,本研究從海洋環(huán)境中分離得到兩株芽孢桿菌均有潛在的應(yīng)用價值,能夠為合理使用甘蔗渣提供新思路,在利用海洋微生物降解甘蔗渣制糖應(yīng)用上提供了理論依據(jù),發(fā)掘了海洋微生物功能基因,為木質(zhì)纖維的合理利用提供了更為廣闊的研究及應(yīng)用前景。
[Abstract]:The marine environment is rich in microbial resources. For reasons such as technology, only a small number of marine microbes are separated, studied and applied. Most marine microbes have unique physiological, biochemical and metabolic functions, and have the characteristics of salt tolerance, thermophilic, cold, etc. It can be said that the marine environment contains more extreme microbes than on land. Bagasse is a kind of biomass resource, which is mainly composed of cellulose, hemicellulose and lignin. Bagasse degrading bacteria were enriched in offshore and deep sea environment. Firstly, the microflora composition of uncultured bagasse degrading bacteria at two enrichment sites was studied by molecular ecology method. The results showed that the two libraries had their own characteristics. Obvious dominant flora: the dominant bacteria group in deep sea library is Proteus. Among them, 緯 -Proteus is the most abundant in the deep sea library, and the dominant bacteria group is Bacteroid phylum in the offshore library. At the same time, 29 strains of bacteria are isolated from two enriched samples, which mainly belong to two groups: Proteus and phylum thuringiensis. The degradation of bagasse by two strains of Bacillus sp. GZ03GZ04 isolated from deep-sea environment was analyzed. The results showed that the two strains could be used in the degradation of bagasse. Two strains could degrade the high concentration of reducing sugar from bagasse. Ion chromatography showed that the degradation product was glucose and fructose mixture. By optimizing the single factor condition of reducing sugar production of GZ03 strain, it was found that the optimum medium for reducing sugar production of the strain was 2% bagasse, 60 mesh, 3% NaCl3, and distilled water, and the optimum medium for reducing sugar production was 2% bagasse. The optimum condition for reducing sugar production was that the strain GZ04 could degrade hemicellulose from bagasse by solid fermentation at 35 鈩，

本文編號：1659413