Ruminiclostridium thermocellum is one of the most promising candidates for consolidated bioprocessing (CBP) of low-cost lignocellulosic materials to biofuels but it still shows poor performance in its ability to deconstruct untreated lignocellulosic substrates. One promising approach to increase R. thermocellum’s rate of hydrolysis is to co-culture this cellulose-specialist with partners that possess synergistic hydrolysis enzymes and metabolic capabilities. We have created co-cultures of R. thermocellum with two hemicellulose utilizers, Ruminiclostridium stercorarium and Thermoanaerobacter thermohydrosulfuricus, both of which secrete xylanolytic enzymes and utilize the pentose oligo- and monosaccharides that inhibit R. thermocellum’s hydrolysis and metabolism. When grown on milled wheat straw, the co-cultures were able to solubilize up to 58% more of the total polysaccharides than the R. thermocellum mono-culture control. Repeated passaging of the co-cultures on wheat straw yielded stable populations with reduced R. thermocellum cell numbers, indicating competition for cellodextrins released from cellulose hydrolysis, although these stabilized co-cultures were still able to outperform the mono-culture controls. Repeated passaging on Avicel cellulose also yielded stable populations. Overall, the observed synergism suggests that co-culturing R. thermocellum with other members is a viable option for increasing the rate and extent of untreated lignocellulose deconstruction by R. thermocellum for CBP purposes.

Alternative low-carbon transportation fuels, such as biofuels, are needed to replace or supplement fossil fuels in order to lower global greenhouse gas emissions and combat climate change. Lignocellulosic biofuels have relatively low carbon emissions and are created using the non-food parts of crops and other plants, such as the leaves and stems, which are comprised mostly of a tough material called lignocellulose, composed of cellulose, hemicellulose, and lignin. One of the best lignocellulose degraders found in nature that is also capable of fermenting the released sugars to ethanol is Clostridium thermocellum, although improvements in both lignocellulose hydrolysis extent and ethanol yields are needed for commercial viability.C. thermocellum, considered a cellulose-degrading specialist, was co-cultured with two different hemicellulose-specialists, C. stercorarium and Thermoanaerobacter thermohydrosulfuricus. The hypothesis was that the co-cultures might degrade more lignocellulose owing to the additional hydrolytic enzymes supplied by the partners and their ability to uptake the inhibitory hemicellulose sugars. All co-culture combinations were found to solubilize more wheat straw, among other lignocellulose materials, and produce more endproducts, including ethanol, than C. thermocellum alone. These co-cultures were stable over multiple serial passages, on either wheat straw or pure cellulose, although some evidence of carbon competition was observed. The tri-culture was successfully used to screen the digestibility of various lignocellulose materials, revealing substantial difference between cattail harvested in different seasons. Cross-feeding of vital growth factors was observed between the various co-culture members in a defined medium.The metabolism of C. thermocellum is atypical compared to many organisms, including the absence of a pyruvate kinase, and its substitution with both a malate shunt and a putative Thank you to my friends and family for always supporting me along this journey. I would like to thank my supervisor, Dr. Richard Sparling, for giving me this opportunity and for his incredibly valuable guidance and mentorship, the strict scientific rigour helped me develop my critical thinking skills.

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