Stable coexistence of five bacterial strains as a cellulose-degrading community

Abstract

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, no change 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.

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%.

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.

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.