Enhanced degradation of wheat straw by co-cultures of anaerobic clostridia and assessment of population dynamics using cpn60-based quantitative PCR

Consolidated bioprocessing (CBP) simplifies production of second-generation biofuels by combining cellulase production, lignocellulose hydrolysis and fermentation in a single process. However, low rates of hydrolysis in CBP remain a challenge that can potentially be solved using cocultures of micro-organisms with complementary lignocellulose degradation pathways. Few previous co-culture studies have determined dynamics of co-culture sub-populations using updated molecular techniques. Therefore, we combined three clostridial thermophiles whose characteristics indicate potential synergy in cocultures and optimized a qPCR assay based on the universal chaperonin-60 (cpn60) gene to track all three organisms simultaneously. Clostridium thermocellum DSM1237, C. stercorarium DSM8532, and Thermoanaerobacter thermohydrosulfuricus strain WC1 (monocultures), dual cultures comprising all 3 possible pairings and triculture were grown on milled wheat straw at 62C for six days, followed by quantification of gas and liquid end products. Hydrogen production was highest for the triculture at all timepoints, followed by dual cultures. Ethanol production was highest for triculture and C. thermocellum/C. stercorarium coculture. T. thermohydrosulfuricus grew poorly on its own but enhanced end product formation in cocultures. Total end products were approximately twice as high in all cocultures compared to monocultures. Optimized qPCR demonstrates efficiency and specificity of designed cpn60 primer/probe sets to define co-cultures and confirm that C. stercorarium is the most abundant organism in triculture when growing on purified cellulose and xylan. The results of this study provide much-needed insight into population dynamics that may help to explain why co-cultures more effectively degrade and utilize lignocellulosic material for production of biofuels by CBP.