432 JOURNAL OF BIOSCIENCE AND BIOENGINEERING © 2007, The Society for Biotechnology, Japan Vol. 104, No. 5, 432–434. 2007 DOI: 10.1263/jbb.104.432 Effect of Adding Cellulolytic Bacterium on Stable Cellulose-Degrading Microbial Community Naoki Narisawa, 1 Shin Haruta, 1 * Zong Jun Cui, 2 Masaharu Ishii, 1 and Yasuo Igarashi 1 Department of Biotechnology, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 1 and College of Agronomy and Biotechnology, China Agricultural University, Haidian District, Beijing 100094, China 2 Received 3 April 2007/Accepted 20 August 2007 The introduction of an exogenous cellulolytic bacterium into a microbial community that was degrading rice straw effectively was evaluated. A stable coexistence of the indigenous and exoge- nous cellulolytic bacteria was achieved by adjusting the cultivation conditions. The obtained com- munity required several subcultures to reach the highest degradation efficiency. [Key words: cellulose degradation, microbial community, Clostridium thermocellum] The introduction of exogenous microorganisms into a mi- crobial community is an attractive means of strengthening and changing the biological activity of the community. Although some experiments on introducing exogenous strains have been reported to be successful, many studies have shown that the exogenous bacteria had only a minor effect on total commu- nity functions (1, 2). The reasons for failure have been con- sidered to be the competitive interactions related to nutrients and space among microorganisms. However, the validation of this presumption is difficult, because these experiments lack sufficient ecological data on the indigenous microbial communities (3). A structurally and functionally well-defined community is essential for investigating the effect of adding on exogenous bacterium. In our laboratory, a bacterial community capable of effec- tively degrading various cellulosic materials (e.g., rice straw) under aerobic static conditions had been developed (4). The major members of the community and their relationships have been well characterized (5, 6). Clostridium straminisolvens CSK1 was the sole cellulose degrading bacterium, which provided metabolites to the other bacterial members. To in- vestigate the effect of introducing a competitor into this mi- crobial community, we selected Clostridium thermocellum as an exogenous bacterium, which is the closest relative to C. straminisolvens CSK1 and has high carboxymethyl cel- lulase activity (8). In this study, we determined whether the coexistence of similarly functional bacteria within our cel- lulose degrading microbial community is possible. The cellulose-degrading microbial community, designated the original community, was maintained on PCS medium containing rice straw (1% w/v) with a loose cap at 50°C (4). C. thermocellum IAM13660T, obtained from the Institute of Molecular and Cellular Biosciences (Tokyo), was grown using DSM122 medium (5, 9) at 60°C under anaerobic con- ditions. Aliquots (100 μl) of each culture, namely, the origi- nal community (8.0 × 10 9 cells/ml) and C. thermocellum (3.0 × 10 7 cells/ml), were coinoculated into 100 ml of fresh PCS medium, and the resulting community was cultivated under static aerobic conditions at 50°C or 60 °C (1st genera- tion). The constructed community was designated the intro- duced community. After 8 d of cultivation, an aliquot (100 μl) of the introduced community was inoculated into 100 ml of fresh PCS medium (2nd generation). This subculture proce- dure was repeated 17 times. A quantification of residual rice straw present at the end of each generation was carried out using a method described by Tailliez et al. (10). DNA was extracted using the method of Zhu et al. (11). To quantify each cellulolytic bacterium, we adapted the following spe- cific primers: CSK198f and CSK439r for strain CSK1 (5), and CTC51f 5′-TCGAGCGGGGATATACGG-3 ′ (positions 51–70 in the Escherichia coli gene) and CTC464r 5 ′-TCTT TGGGTACCGTCACTTC-3 ′ (positions 464–444 in the E. coli gene) for the 16S rRNA gene of C. thermocellum. For standard curves, DNA extracted from each cellulolytic bac- terium was used as templates. Quantitative PCR analysis was performed as described previously (5). Two trials were con- ducted to analyze each sample. PCR-mediated denaturing gradient gel electrophoresis (PCR-DGGE) was performed to characterize the bacterial communities as described pre- viously (4). The sequences of the DGGE bands are available in the DDBJ nucleotide sequence database under accession nos. AB353296 and AB353297. The introduction of the exogenous bacterium cultivated at 50°C did not affect the rice straw degradation or commu- nity structure throughout 18th generations (Figs. 1 and 3). Furthermore, the quantitative PCR analysis at the end of each generation revealed that C. thermocellum was hardly colo- nized within the community (DNA mass lower than 0.01 ng/ml), however, the DNA mass of strain CSK1 was higher than 1.5 ng/ml (Table 1). The noncolonization of the exoge- nous bacterium could have been caused by the competitive disadvantage of the bacterium with respect to the indige- nous bacteria. * Corresponding author. e-mail: aharuta@mail.ecc.u-tokyo.ac.jp phone: +81-(0)3-5841-5144 fax: +81-(0)3-5841-5272