Characterization of cell growth, substrate utilization, end-product synthesis and gene expression patterns in cellulose degrading co-cultures of Clostridium termitidis CT1112 and Clostridium intestinale URNW

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Thinesh, Peranantham
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Co-cultures of selected fermentative bacteria have been shown to improve rates of substrate conversion and yields of some fermentation end-products. We have tested this hypothesis using a co-culture of the mesophilic, cellulolytic bacterium Clostridium termitidis CT1112 and the mesophilic, saccharophylic bacterium, Clostridium intestinale URNW. C. termitidis can utilize cellulose as a sole carbon source. It releases glycosyl hydrolases that hydrolyze cellulose, cellobiose and glucose. C. intestinale URNW, which was isolated as a contaminant from a cellobiose stock bottle of C. termitidis (Ramachandran et al., 2011), is not capable of hydrolyzing cellulose, but can utilize the cellobiose and glucose released by cellulose hydrolysis to grow in co-culture with C. termitidis. Based on the faster doubling-time of C. intestinale URNW on cellobiose, it was expected that the “soluble sugar free” environment will stimulate C. termitidis to hydrolyse cellulose at faster rate and which in-turn will result in increased substrate utilization and end-product synthesis compared to the monoculture of C. termitidis. The designed co-culture was characterized in depth with the use of microbial quantification studies (multiplex quantitative Real Time Polymerase Chain Reaction – qPCR) and ‘Omics techniques to understand the population dynamics and gene product expression in each species in the co-culture versus their respective monocultures at the molecular level. Inoculation of co-culture with diffferent initial ratios of the C. termitidis and C. intestinale resulted a fixed ratio of approximately 13:1 (C. termitidis : C. intestinale) at 168 hour post-inoculation (h pi). A statistical difference in substrate utilization and total cell mass production, but not end-product cocentrations, was observed at 168 h pi in cultures with an initial C. termitidis : C. intestinale ratio of 1:1 and 1:0.2). No statisical differences in substrate utilization, biomass accumulation, or end-product synthesis concentrations were observed for all other initial C. termitidis : C. intestinale ratios, or for co-cultures in where the C. termitidis : C. intestinale ratio was 1:25. Thus, the hypothesis that synergistic interactions between species in co-cultures can stimulate substrate consumption and end-product synthesis was supported under very limited conditions in this co-culutre system. Unlike other co-cultures reported in literature, the co-culture of C. termitidis and C. intestinale did not show large increases in substrate utilization or end-product concentrations synthesized when cultured on 2 g/L α-cellulose. This may be due to the slower cellulose degradation ability of C. termitidis in the co-culture, (compared to the cellulose degraders in other co-cultures such as C. thermocellum), which is the main contributor to the growth of C. intestinale in the co-culture and the competition for the same substrate (cellobiose) by both the species in the co-culture.
cellulose degraders