新型聚乙烯醇降解菌的筛选及降解特性的研究
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摘要
聚乙烯醇(PVA)是一种通过水解聚乙酸酯而得到的水溶性高分子聚合物,被广泛运用于工业产品中,例如在黏合剂和纺织浆料之中。除此之外,聚乙烯醇在固定化微生物细胞技术中也有应用。近年来,聚乙烯醇因为具有可以被微生物降解的含有糖类或脂肪酸支链的聚合物的基本链式结构而受到了广泛的关注。根据有效统计,聚乙烯醇的产量比其他任何水溶性的人工合成聚合物都要大,全球范围内的聚乙烯醇年产量可以达到650000吨,大量废弃的聚乙烯醇日渐成为重要的环境问题。聚乙烯醇是很难被天然降解的物质,因此在排放之前应当做适当处理。由于国内外仅有少量的从事聚乙烯醇生物降解的研究,从而使得本课题具有一定的研究价值。本文从活性污泥中筛选出了具有聚乙烯醇降解能力的单一菌种(P7)和它的共生菌种(B2)。着重对具有聚乙烯醇降解能力的菌种(P7)和共生菌群(P7和B2)的降解能力进行了研究,并对单一菌种的降解曲线也进行了讨论。
基于16S rRNA基因序列的细菌分类学分析表明P7属于新鞘氨醇杆菌属(Novosphingobium sp),与Novosphingobium sp.的基因序列有98.2%的相似度。B2的分析结果表明其属于黄色(无芽胞)杆菌属(Xanthobacterflavus)并与Xanthobacterflavus的16S rRNA基因序列有100%的相似度。以前学者的研究结果表明,PVA的分解离不开细胞外酶和细胞内酶共同作用,前人发现的PVA降解菌需添加酶或辅酶来辅助降解,而本研究表明,P7只要在有氮源存在的环境中就能降解聚乙烯醇,降解率可达95%,这是以前的研究中所未发现的菌种。我们发现P7不仅能产生出细胞外酶分解PVA,还能产生细胞内酶进一步分解PVA初步水解后形成的中间产物。而在P7与不同种类氮源共同培养的实验当中,我们发现有机氮比无机氮更适合细菌的生长和降解聚乙烯醇。
为了在活性污泥中找到P7菌的共生菌,我们把活性污泥中分离出来的其他菌种与P7菌进行共同培养。通过把其他菌种接种到含P7菌的聚乙烯醇基础培养液中(不含氮源),经过一定时间的培养后,仅发现含P7菌和B2菌的溶液中的聚乙烯醇几乎完全降解,降解率可达94.4%。而在另外的试验中,我们把B2菌放在含有氮源的聚乙烯醇培养液中培养一周后,发现B2菌即使在氮源存在的环境中也并没有聚乙烯醇的降解能力。由于P7菌具有降解聚乙烯醇的能力而B2菌不具备,与此同时,P7菌在不存在氮源的情况下几乎不具备聚乙烯醇降解能力,而在P7菌与B2菌的共同存在情况下,即使没有氮源存在情况下也能降解聚乙烯醇。因此我们可以推断B2可以产生能充当氮源的有机物质,在P7和B2之间存在着共生现象。
Poly(vinyl alcohol)(PVA),a water soluble polymer prepared by the hydrolysis of poly(vinyl acetate),is widely used in industrial applications,such as adhesives and sizing agent in textiles.It has also been used for cell immobilization.Recently,PVA has attracted attention as the biodegradable backbone in sugar and fatty acid branched polymers.PVA is produced in greater quantities than any other water-soluble synthetic polymer.The world production of PVA is about 650000 tons per year and the large amount of used and discarded PVA has become a significant waste problem.PVA is difficult to be degraded in natural environment and must be removed from the waste water before discharge.The little information available regarding microbial degradation of PVA,despite the large amounts consumed,has made study of its degradation necessary.In the paper,single microorganism(P7) and symbiotic culture(P7&B2) capable of degrading PVA have been identified from activated sludge,we present the study on the degradation behavior of PVA by an acclimated PVA-degrading microorganism(P7),and the symbiotic microbial culture (P7&B2) involved.Degradation patterns of PVA by the isolated strains are also discussed.
Taxonomic studies and the results of analysis based on 16S rRNA gene sequences revealed that strain P7 belongs to Novosphingobium sp.with 98.2%similarity.While strain B2 was classified as Xanthobacter flavus and showed 100%identities with the 16S rRNA gene of Xanthobacter flavus.As previously reported,extracellular enzyme should coactted with intracellular enzyme in PVA degradation process,and the identified microorganisms needed the addition of enzymes or coenzyme to degrade PVA.However,our experiments displayed that strain P7 could degrade PVA only with the addition of nitrogen source.The PVA degradation rate was 95%,this was an unprecedented find.We also found that strain P7 not only produce extracellular PVA- degrading enzymes to degrade PVA,but also produce intracellular PVA-degrading enzymes for the further utilization of PVA- degrading intermediates.Strain P7 was cultivated in different nitrogen sources,in which organic nitrogen source was more suitable for the microorganism to grow and degrade PVA than inorganic nitrogen source.
To identify the symbiotic bacteria with P7 in activated sludge,PVA degradation by the co-culture was examined.Several strains isolated from sludge samples were inoculated into the modified PVA medium(without nitrogen source) with strain P7.In the mixed culture of strain B2 and P7,PVA was degraded completely,the degradation rate was 94.4%.The experiment that PVA- degradability of strain B2 with nitrogen source has also been carried out for a week,and the result showed that strain B2 has no PVA- degrading activity.Strain P7 exhibited a capability of PVA degradation while strain B2 could not utilize PVA as a carbon source.PVA was degraded slowly by a pure culture of strain P7 without the addition of nitrogen source.However,the degradation was markedly enhanced by a mixed culture of P7 and B2.This suggests that the reaction proceeds in the degradation of PVA due to secretion of the nitrogen source by the symbiotic strain.
基于16S rRNA基因序列的细菌分类学分析表明P7属于新鞘氨醇杆菌属(Novosphingobium sp),与Novosphingobium sp.的基因序列有98.2%的相似度。B2的分析结果表明其属于黄色(无芽胞)杆菌属(Xanthobacterflavus)并与Xanthobacterflavus的16S rRNA基因序列有100%的相似度。以前学者的研究结果表明,PVA的分解离不开细胞外酶和细胞内酶共同作用,前人发现的PVA降解菌需添加酶或辅酶来辅助降解,而本研究表明,P7只要在有氮源存在的环境中就能降解聚乙烯醇,降解率可达95%,这是以前的研究中所未发现的菌种。我们发现P7不仅能产生出细胞外酶分解PVA,还能产生细胞内酶进一步分解PVA初步水解后形成的中间产物。而在P7与不同种类氮源共同培养的实验当中,我们发现有机氮比无机氮更适合细菌的生长和降解聚乙烯醇。
为了在活性污泥中找到P7菌的共生菌,我们把活性污泥中分离出来的其他菌种与P7菌进行共同培养。通过把其他菌种接种到含P7菌的聚乙烯醇基础培养液中(不含氮源),经过一定时间的培养后,仅发现含P7菌和B2菌的溶液中的聚乙烯醇几乎完全降解,降解率可达94.4%。而在另外的试验中,我们把B2菌放在含有氮源的聚乙烯醇培养液中培养一周后,发现B2菌即使在氮源存在的环境中也并没有聚乙烯醇的降解能力。由于P7菌具有降解聚乙烯醇的能力而B2菌不具备,与此同时,P7菌在不存在氮源的情况下几乎不具备聚乙烯醇降解能力,而在P7菌与B2菌的共同存在情况下,即使没有氮源存在情况下也能降解聚乙烯醇。因此我们可以推断B2可以产生能充当氮源的有机物质,在P7和B2之间存在着共生现象。
Poly(vinyl alcohol)(PVA),a water soluble polymer prepared by the hydrolysis of poly(vinyl acetate),is widely used in industrial applications,such as adhesives and sizing agent in textiles.It has also been used for cell immobilization.Recently,PVA has attracted attention as the biodegradable backbone in sugar and fatty acid branched polymers.PVA is produced in greater quantities than any other water-soluble synthetic polymer.The world production of PVA is about 650000 tons per year and the large amount of used and discarded PVA has become a significant waste problem.PVA is difficult to be degraded in natural environment and must be removed from the waste water before discharge.The little information available regarding microbial degradation of PVA,despite the large amounts consumed,has made study of its degradation necessary.In the paper,single microorganism(P7) and symbiotic culture(P7&B2) capable of degrading PVA have been identified from activated sludge,we present the study on the degradation behavior of PVA by an acclimated PVA-degrading microorganism(P7),and the symbiotic microbial culture (P7&B2) involved.Degradation patterns of PVA by the isolated strains are also discussed.
Taxonomic studies and the results of analysis based on 16S rRNA gene sequences revealed that strain P7 belongs to Novosphingobium sp.with 98.2%similarity.While strain B2 was classified as Xanthobacter flavus and showed 100%identities with the 16S rRNA gene of Xanthobacter flavus.As previously reported,extracellular enzyme should coactted with intracellular enzyme in PVA degradation process,and the identified microorganisms needed the addition of enzymes or coenzyme to degrade PVA.However,our experiments displayed that strain P7 could degrade PVA only with the addition of nitrogen source.The PVA degradation rate was 95%,this was an unprecedented find.We also found that strain P7 not only produce extracellular PVA- degrading enzymes to degrade PVA,but also produce intracellular PVA-degrading enzymes for the further utilization of PVA- degrading intermediates.Strain P7 was cultivated in different nitrogen sources,in which organic nitrogen source was more suitable for the microorganism to grow and degrade PVA than inorganic nitrogen source.
To identify the symbiotic bacteria with P7 in activated sludge,PVA degradation by the co-culture was examined.Several strains isolated from sludge samples were inoculated into the modified PVA medium(without nitrogen source) with strain P7.In the mixed culture of strain B2 and P7,PVA was degraded completely,the degradation rate was 94.4%.The experiment that PVA- degradability of strain B2 with nitrogen source has also been carried out for a week,and the result showed that strain B2 has no PVA- degrading activity.Strain P7 exhibited a capability of PVA degradation while strain B2 could not utilize PVA as a carbon source.PVA was degraded slowly by a pure culture of strain P7 without the addition of nitrogen source.However,the degradation was markedly enhanced by a mixed culture of P7 and B2.This suggests that the reaction proceeds in the degradation of PVA due to secretion of the nitrogen source by the symbiotic strain.
引文
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