2005--Stable Coexistence of Five Bacterial Strains as a Cellulose-Degrading Community

A cellulose-degrading defined mixed culture (designated SF356) consisting of five bacterial strains (Clostridium straminisolvens CSK1, Clostridium sp. strain FG4, Pseudoxanthomonas sp. strain M1-3, Brevibacillus sp.strain M1-5, and Bordetella sp. strain M1-6) exhibited both functional and structural stability; namely, nochange in cellulose-degrading efficiency was observed, and all members stably coexisted through 20 subcultures. In order to investigate the mechanisms responsible for the observed stability, “knockout communities” in which one of the members was eliminated from SF356 were constructed. The dynamics of the community structure and the cellulose degradation profiles of these mixed cultures were determined in order to evaluate the roles played by each eliminated member in situ and its impact on the other members of the community. Integration of each result gave the following estimates of the bacterial relationships. Synergistic relationships between an anaerobic cellulolytic bacterium (C. straminisolvens CSK1) and two strains of aerobic bacteria(Pseudoxanthomonas sp. strain M1-3 and Brevibacillus sp. strain M1-5) were observed; the aerobes introduced anaerobic conditions, and C. straminisolvens CSK1 supplied metabolites (acetate and glucose). In addition, there were negative relationships, such as the inhibition of cellulose degradation by producing excess amounts of acetic acid by Clostridium sp. strain FG4, and growth suppression of Bordetella sp. strain M1-6 by Brevibacillus sp. strain M1-5. The balance of the various types of relationships (both positive and negative) is thus considered to be essential for the stable coexistence of the members of this mixed culture.

2005——降解纤维素菌群中的五株细菌稳定共存

一种降解纤维素的混合培养物（命名为SF356），由五株细菌（梭状芽孢杆菌CSK1、梭状芽孢杆菌FG4、假黄单胞菌M1-3、短杆菌M1-5和波氏杆菌M1-6）组成，具有功能和结构稳定性；即，通过20次继代培养，观察到纤维素降解效率无变化，所有成员稳定共存。为了研究观察到的稳定性的机制，我们构建了一个从SF356中剔除一个成员的“淘汰社区”。测定了这些混合培养物的群落结构和纤维素降解曲线的动态，以评估每一个原位消除的成员所起的作用及其对群落其他成员的影响。观察到一株厌氧纤维素分解菌（C.straminisolvens CSK1）与两株需氧菌（假黄单胞菌M1-3和短杆菌M1-5）之间的协同关系；需氧菌引入厌氧条件，C.straminisolvens CSK1提供代谢物（乙酸盐和葡萄糖）。此外，有抑制作用，梭状芽孢杆菌FG4通过产生过来的乙酸对纤维素的降解有抑制作用，菌株短杆菌M1-5对波氏杆菌M1-6的生长有抑制作用。不同类型的关系（积极和消极）的平衡被认为是这种混合文化成员稳定共存的必要条件。

图1。SF356中各细菌的相对丰度及第2代和第22代第6天的敲除群落。数据显示了从两个独立的亚文化群落（A和B）获得的结果。这些值表示为两次实时PCR实验的平均值。同一DNA样本的实时PCR实验之间的差异小于20%。

FIG. 1. Relative abundance of each bacterium in SF356 and the knockout communities at day 6 of the 2nd and 22nd generations. The data show the results obtained from two independent subcultured communities (designated A and B). The values are expressed as the means of two replications of real-time PCR experiments. The variation between the real-time PCR experiments for the same DNA sample was less than 20%.

图2。SF356与敲除菌群的滤纸降解过程比较。（A） 滤纸在4天（开条）或8天（灰条）周期内降解；（B）pH值；（C）低聚糖积累；（D）乙酸盐积累；（E）乙醇积累。代谢物的累积表示为在8天的培养期（毫克）内，每降解8天（克）的滤纸中代谢物的增加。值表示为两个培养液样本的平均值。误差线表示标准偏差。星号与SF356有显著性差异（P 0.05）。N、 A.不适用，因为滤纸未发生降解。

FIG. 2. Comparisons of the filter paper degradation processes of SF356 and the knockout communities. (A) Filter paper degradation over a period of 4 days (open bars) or 8 days (gray bars); (B) pH value; (C) accumulation of oligosaccharides; (D) accumulation of acetate; (E) accumulation of ethanol. Accumulation of the metabolites is represented as an increase in the metabolites during an 8-day period of cultivation(milligrams) per amount of filter paper degraded over a period of 8 days (grams). Values are expressed as the means of two culture solution samples. The error bars indicate standard deviations. Asterisks represent a significant difference (P  0.05) from SF356. N.A., not applicable, because filter paper degradation did not occur.

TABLE 1. Pure culture of noncellulolytic isolates in PCS basal medium and in the cell-free culture filtrate of C. straminisolvens CSK1

表1 非纤维素降解菌株在PCS基础培养基和梭状芽孢杆菌CSK1无细胞培养滤液中的纯培养

图3。SF356物质流网络模型及各个物种的假定作用。实线表示物质流。每条线的厚度表示SF356中特定路径的相对贡献。虚线表示与纤维素降解效率有关的促进或抑制因子。物质的名称用方框表示。这些菌株的名称以卵形图显示。

FIG. 3. Network model of substrate flow and the putative roles of each member in SF356. The solid lines indicate the flow of the substrates. The thickness of each line represents the relative contribution of a particular pathway in SF356. The dotted lines indicate promoting or inhibiting factors with respect to the efficiency of cellulose degradation. The names of the substrates are presented in boxes. The designations of the bacterial strains are presented in ovals.