(796e) Use of Oxygen Transport Limitations to Mediate a Fuel Producing Symbiosis

We have demonstrated an oxygen transport mediated symbiosis between the cellulolytic anaerobe, Clostridium phytofermentans, and the ethanol producing yeast Saccharomyces cerevisiae cdt-1 or Candida molischiana for ethanol production from cellulose. The consortium produce over two times more ethanol from α-cellulose when grown under simultaneous saccharificaiton and fermentation conditions. Oxygen transport induces a symbiotic cooperation between the two organisms which is both stable and scalable. These results demonstrate the power of microbial consortia for lignocellulosic biofuel production processes and the use of environmentally mediate symbioses presents a fresh look on a natural paradigm for regulating consortium function.

The process of consolidated bioprocessing (i.e., simultaneous biological hydrolysis and fermentation) of lignocellulosic is an increasingly feasible solution for the production of sustainable, liquid transportation fuels. However, the lack of a suitable organism capable of efficient hydrolysis and fermentation has limited the implementation of consolidated bioprocessing. As an alternative to single organism bioprocessing, mixed cultures represent a potential solution to achieve the appropriate combination of hydrolytic and fermentative capabilities. The stability of mixed consortia and lack of simplistic bioprocessing are often cited as challenges to this approach.  This work presents the development and characterization of a semi-synthetic consortium of the cellulolytic mesophile, Clostridium phytofermentans, and a cellodextrin fermenting yeast, Candida molischiana or Saccharomyces cerevisiae cdt-1, for direct ethanol production from cellulose. The symbiotic cooperation is induced by the diffusion of oxygen into the culture medium. Oxygen acts to inhibit the growth of the obligate anaerobe, C. phytofermentans but when provided a soluble carbon source from C. phytofermentans hydrolysis, the yeast metabolizes oxygen relieving the inhibitory effect. When fermenting cellobiose, a mutually accessible carbon source, under semi-aerobic conditions C. phytofermentans is “rescued” from oxygen inhibition by yeast growth. This paradigm was extended to cellulose hydrolysis in static, aerobic filter paper degradation which was only possible using co-cultures. We then applied this symbiotic consortium to cellulose fermentation and found that the rate of oxygen transfer had a profound impact on population control as well as maintenance of the desired fuel product, ethanol. Using oxygen diffusion through submerged neoprene tubing we achieved efficient consortia-mediated cellulose fermentation and found that populations were stable up to 2 months while the system appeared to be hydrolysis rate limited. In order to overcome this rate limitation, we simulated improved hydrolysis capability by adding exogenous endoglucanase from Trichoderma viride at a moderate level of 400 mg/L. Using this simultaneous saccharification and fermentation approach the consortium produced greater than two times more ethanol than either mono-culture. The ethanol production level was greater than the sum of the mono-culture titers suggesting a synergism in the system. This work represents a viable demonstration of the utility of environmentally controlled microbial consortia in the production of liquid transportation fuels from cellulosic biomass. Our future work will focus on characterizing the physical interactions in the consortium system as well as developing mathematical models to predict dynamics to improve productivity. Ultimately, we aim to expand the paradigm of symbiotic biofuels production to more advanced biofuel products and true lignocellulosic substrates. In this presentation we will present our previous work in light of new advancements in understanding the consortium bioprocess.