Consortia-mediated bioprocessing of cellulose to ethanol with a symbiotic Clostridium phytofermentans/yeast co-culture

Abstract

Background: Lignocellulosic ethanol is a viable alternative to petroleum-based fuels with the added benefit of potentially lower greenhouse gas emissions. Consolidated bioprocessing (simultaneous enzyme production, hydrolysis and fermentation; CBP) is thought to be a low-cost processing scheme for lignocellulosic ethanol production. However, no single organism has been developed which is capable of high productivity, yield and titer ethanol production directly from lignocellulose. Consortia of cellulolytic and ethanologenic organisms could be an attractive alternate to the typical single organism approaches but implementation of consortia has a number of challenges (e.g., control, stability, productivity).

Results: Ethanol is produced from α-cellulose using a consortium of C. phytofermentans and yeast that is maintained by controlled oxygen transport. Both Saccharomyces cerevisiae cdt-1 and Candida molischiana "protect" C. phytofermentans from introduced oxygen in return for soluble sugars released by C. phytofermentans hydrolysis. Only co-cultures were able to degrade filter paper when mono- and co-cultures were incubated at 30°C under semi-aerobic conditions. Using controlled oxygen delivery by diffusion through neoprene tubing at a calculated rate of approximately 8 μmol/L hour, we demonstrate establishment of the symbiotic relationship between C. phytofermentans and S. cerevisiae cdt-1 and maintenance of populations of 105 to 106 CFU/mL for 50 days. Comparable symbiotic population dynamics were observed in scaled up 500 mL bioreactors as those in 50 mL shake cultures. The conversion of α-cellulose to ethanol was shown to improve with additional cellulase indicating a limitation in hydrolysis rate. A co-culture of C. phytofermentans and S. cerevisiae cdt-1 with added endoglucanase produced approximately 22 g/L ethanol from 100 g/L α-cellulose compared to C. phytofermentans and S. cerevisiae cdt-1 mono-cultures which produced approximately 6 and 9 g/L, respectively.

Conclusion: This work represents a significant step toward developing consortia-based bioprocessing systems for lignocellulosic biofuels production which utilize scalable, environmentally-mediated symbiosis mechanisms to provide consortium stability.

Figure 1

Semi-aerobic mono- and co-culture growth on cellobiose.C. phytofermentans mono-culture (A.1 and B.1), C. molischiana mono-culture (A.2),C. phytofermentans/C. molischiana co-culture (A.3), S. cerevisiae cdt-1 mono-culture (B.2) and C. phytofermentans/S. cerevisiae cdt-1 co-culture (B.3) viable cell counts (left) and optical densities (right) in GS2 cellobiose fermentations. Results are representative of at least two independent experiments. Error bars are the standard deviation among drops for colony counting.

Figure 2

Static mono- and co-culture hydrolysis of filter paper. Semi-aerobic cultures in non-degassed GS2 medium with Whatman No. 1 filter paper strips as the carbon source. C. molischiana and C. phytofermentans mono- and co-cultures (A) and S. cerevisiae cdt-1 and C. phytofermentans mono- and co-cultures (B). Photographs taken after 40 days (A) and 15 days (B) static incubation at 30°C with periodic agitation. Image is representative of at least two independent experiments each with two replicates (as shown in the photographs).

Figure 3

Bioreactors designed and utilized for diffusive oxygen transfer. Controlled oxygen transfer reactor with 10 cm neoprene tubing fixed to stainless steel tubing inserted into a sealed 100 mL serum bottle (A). Infors 500 mL reactor containing 100 cm neoprene tubing fixed to stainless steel outlet ports (B). Infors 500 mL reactor in operation with tubing loops submerged in the cellulose-containing medium (C).

Figure 4

Co-culture population dynamics with diffusive oxygen transfer. Population dynamics for 50 mL, 25 g/L α-cellulose co-culture fermentations without added oxygen (A) and with added oxygen (B) and 500 mL, 100 g/L α-cellulose co-culture fermentation (C). Results in A and B are representative of at least 3 independent experiments. Results in C represent a single experiment. Error bars are plus and minus one standard deviation among the drops used for colony counting.

Figure 5

Mono- and co-culture performance under CBP and SSF conditions.C. phytofermentans mono- and co-culture CBP without added enzyme and SSF with 400 mg/L endoglucanase at 30°C. Performance is shown by ethanol, acetate and accumulated reducing sugar concentrations (A) and cellulose conversion (B). Bars represent the average of two independent experiments and error bars are plus and minus one standard deviation among experiments. C. phy indicates C. phytofermentans mono-culture, cdt-1 indicates S. cerevisiae cdt-1 mono-culture and C. phy + cdt-1 indicates C. phytofermentans/S. cerevisiae cdt-1 co-culture. + Endoglucanase indicates an SSF experiment with endoglucanase added as stated in the text. S. cerevisiae cdt-1 mono-cultures and C. phytofermentans/S. cerevisiae cdt-1 culture were grown with oxygen transfer while C. phytofermentans mono-cultures remained completely anaerobic throughout the experiment.

Figure 6

Proposed symbiotic consortium design.C. phytofermentans-yeast symbiotic consortium design based on cellulose hydrolysis to soluble cellodextrins which are utilized by yeast. Both C. phytofermentans and yeast produce ethanol from cellodextrins. Cellodextrins feedback inhibit cellulose hydrolysis, oxygen inhibits anaerobic bacterial growth while yeast oxygen consumption relieves inhibition.