Filling in gaps in Clostridium thermocellum metabolism through co-culturing with hydrogenotroph, Methanothermobacter marburgensis

Abstract

Clostridium thermocellum is one of the most promising candidates for consolidated bioprocessing of lignocellulosic biomass to biofuels and chemicals but its low product yield and the presence of unexpected minor fermentation products, such as amino acids, has restricted industrial scale production. Few reports have compared the physiological effects of end-product accumulation and the behavior of C. thermocellum in a natural, consortium setting. This may provide a more detailed understanding of the complex metabolism of C. thermocellum, aiding future engineering efforts to increase product yield. In this study, we grew C. thermocellum in co-culture with Methanothermobacter marburgensis, a hydrogenotrophic methanogen, on cellulose in batch culture at 60 oC to investigate the physiological effects of continuous H2 removal on C. thermocellum. When grown in co-culture, minimal H2 concentration and continuous CH4 production was detected, indicating continuous H2 and CO2 consumption by M. marburgensis. C. thermocellum growth rate did not change significantly, but a faster rate of cellulose hydrolysis was observed. Major end-product profile of C. thermocellum changed with the most notable difference being an increase in acetate, and a decrease in formate and ethanol. Changes in alternate end-products, such as amino acids and TCA cycle intermediates, were also observed. The metabolic shift to H2 and acetate in response to continuous H2 removal was accompanied by changes in the expression of [FeFe] H2ases of C. thermocellum: (a) 10-fold decrease in Cthe_0430, (b) 2-fold decrease in Cthe_0342, (c) 1.5-fold decrease in Cthe_3003. We demonstrated that growth with a naturally occurring hydrogenotrophic partner, such as methanogens, alter the metabolic patterns and H2ase gene expression in C. thermocellum. Low H2 partial pressure required to overcome thermodynamic barriers for the oxidation of reducing equivalents was achieved by continuous H2 removal via methanogen consumption.