Enhanced lignocellulose utilization, cross-feeding of nutrients, and stability of co-cultures of Ruminiclostridium thermocellum, R. stercorarium, and Thermoanaerobacter thermohydrosulfuricus for consolidated bioprocessing

Ruminiclostridium thermocellum (Rt) is a promising candidate for consolidated bioprocessing, but its low hydrolysis rate on untreated lignocellulosic substrates and its incapability to ferment hemicellulose remain major barriers. Complementing Rt’s cell-wall bound cellulosomes with secreted enzymes from other lignocellulolytic hemicellulose fermenting thermophiles, such as R. stercorarium (Rs) and Thermoanaerobacter thermohydrosulfuricus (Tt), via co-culturing offers a possible solution. Co-culturing may also provide additional benefits such as cross-feeding of nutrients. Rt DSM1237, Rs DSM8532, Tt DSM26960, and various co-cultures of the three species were grown in a minimal, defined medium containing no yeast extract, with 2g/l wheat straw, and passaged repeatedly for 6 weeks. The co-cultures produced up to 80% more total end-products than the mono-culture controls, indicating increased substrate hydrolysis and utilization. The population of each member of the co-cultures maintained stability over the passages. The co-cultures produced similar amounts of end-products as in medium supplemented with yeast extract. To further investigate the seemingly unnecessary role of yeast extract the cultures were grown and passaged 7 times in medium containing no yeast extract, with 2g/l cellobiose. In this minimal medium pure cultures of Rs and Tt grew poorly compared to media supplemented with yeast extract, decreasing over 100-fold in cell numbers, while the co-cultures grew well and contained high levels of Rs and Tt, suggesting that Rt was secreting nutrients required by Rs and Tt for full growth. Growing cells in the presence of extractives released from wheat straw was also found to have a positive effect on cell growth.