摘 要

微生物产生的多糖等代谢物，对土壤团聚体的形成有重要影响。研究采集了南京紫金山地区野芝麻根际和非根际的土壤，并对其中的产多糖细菌进行分离筛选及生物学特性的分析，初步了解了产多糖细菌的生理生化特性。研究表明，采集地区根际土壤中的产多糖细菌的数量约为4.8x10⁹株/kg，而非根际土壤中的产多糖细菌数约为2.5x10⁹株/kg，远小于根际土壤。从根际和非根际土壤中各筛选1株多糖高产菌，产多糖量分别达961.671mg/L和283.702 mg/L。对这两株细菌的生物学特性进行研究，结果表明，2株菌的菌落形态相似，适应环境能力都较强，在高低温、高酸碱性、高盐度、以及不同抗生素环境下几乎都能正常生长。此外，对这2株细菌16S rDNA序列进行了测定，并对其系统发育关系进行了分析，发现二者可能分属于Rhizobium（根瘤菌）属和Pantoea（泛菌）属。本研究为进一步研究产多糖细菌对土壤团聚体形成的作用奠定了一定的基础。

关键词：产多糖细菌；分离筛选；生理生化特征；适应性；系统发育学分析

ISOLATION AND SCREENING OF POLYSACCHARIDE-PRODUCING BACTERIA AND THE RESEARCH ON THEIR BIOLOGICAL CHARACTERS

ABSTRACT

The cohesion of metabolites of microbes, including polysaccharide, can have a great impact on the formation of soil aggregates. We collected rhizosphere soil and non-rhizosphere soil from Lamium barbatum Sieb.etZucc. in Purple Mountain in Nanjing, and preliminary learned the physiological and biochemical characteristics of polysaccharide-producing bacteria, through the isolation and screening of them and the analysis of their biological characteristics. It showed that, the amount of the polysaccharide-producing bacteria in rhizosphere soil is about 4.8x10⁹, much higher than that in non-rhizosphere soil which is about 2.5x10⁹. We chose one that had the highest amount of producing polysaccharide from the rhizosphere soil and the other from the non-rhizosphere soil. And the amounts were up to 961.671mg/L and 283.702 mg/L.The study of the two bacteria displayed that they had the similar colony morphology and all had a strong adaptation in different environments. They could be alive in almost all tough conditions such as high and low temperature, pH and different antibiotics. Furthermore, by testing the 16S rDNA sequence and analyzing the phylogenic relation of the two bacteria, we found they were possibly identified as Rhizobium and Pantoea. It laid a good foundation on the further study on how the polysaccharide-producing bacteria work on the formation of soil aggregates.

Key words: polysaccharide-producing bacteria；isolation and screening；physiological and biochemical characteristics；adaptation ；phylogenetic analysis

目 录

1文献综述……………………………………………………………………………………………… 1

1.1产多糖细菌生物学特性研究的国内外现状 …………………………………………… 1

1.1.1微生物胞外多糖的概述…………………………………………………………………… 1

1.1.2 土壤团聚体的概述………………………………………………………………………1

1.1.3细菌胞外多糖在土壤团聚体形成中的作用…………………………………………1

1.1.4国内外研究现状…………………………………………………………………………2

1.2 研究目的和意义……………………………………………………………………3

2产多糖细菌的分离筛选及系统发育学分析…………………………………………………4

2.1 材料…………………………………………………………………………………………4

2.1.1样品来源……………………………………………………………………………………4

2.1.2培养基………………………………………………………………………………4

2.1.3试剂……………………………………………………………………4

2.2方法………………………………………………………………………………………4

2.2.1产多糖细菌的分离纯化……………………………………………………………………4

2.2.2细菌产多糖量的测定…………………………………………………………………5

2.2.3产多糖细菌系统发育学分析………………………………………………………………5

2.3结果与分析………………………………………………………………………………6

2.3.1产多糖细菌的分离纯化…………………………………………………………………… 6

2.3.2细菌产多糖量的测定…………………………………………………………………7

2.3.3产多糖细菌系统发育学分析………………………………………………………………7

2.4 讨论 ………………………………………………………………………………………………9

2.5本章小结……………………………………………………………………………10

3 产多糖细菌的生物学特性………………………………………………………………………………11

3.1 材料………………………………………………………………………………………………11

3.1.1 供试菌株………………………………………………………………………………… 11

3.1.2 培养基…………………………………………………………………………………… 11

3.1.3 试剂……………………………………………………………………………………… 11

3.2 方法………………………………………………………………………………………………11

3.2.1 供试菌株菌落形态学特征……………………………………………………………… 11

3.2.2 供试菌株的环境适应性………………………………………………………………… 11

3.3 结果与分析…………………………………………………………………………………… 12

3.3.1 供试菌株菌落形态学特征………………………………………………………………12

3.3.2 供试菌株的环境适应性…………………………………………………………………13

3.4 讨论………………………………………………………………………………………………15

3.5 本章小结…………………………………………………………………………………………15

结论…………………………………………………………………………………………………………16

致谢…………………………………………………………………………………………………………17

参考文献……………………………………………………………………………………………………18

1文献综述

1.1 产多糖细菌生物学特性研究的国内外现状

1.1.1 微生物胞外多糖的概述

微生物胞外多糖（Exopolysaccharide，EPs）是一种被分泌到细胞外的多糖,它或者以荚膜的形式附着在细胞表面,或者以粘液的形式分散在胞外环境中,分别被称为附着胞外多糖和游离胞外多糖(或荚膜多糖和粘液多糖)。EPS广泛产生于细菌和海藻中,以及少数酵母和真菌中,在自然环境中,EPS通常起着保护微生物细胞的作用,它可以避免细胞干燥脱水、遭噬菌体侵染、受抗生素或有毒物质(如有毒金属离子、二氧化碳、乙酸)损害、原生动物伤害,还可稳定渗透压、支持细胞结构、参与细胞信息传递和细胞构成等。一般认为多数产游离EPS的细菌都没有代谢自身EPS的能力。在病原菌中,如Streptococcus pneummiae和Streptococcus ogalactae,均可以通过吸附胞外多糖从而引起机体免疫反应。

1.1.2 土壤团聚体的概述