Towards increasing lignocellulose to biofuel conversion by Clostridium thermocellum: co-culturing for increased hydrolysis and characterization of pyruvate phosphate dikinase for understanding atypical metabolism
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Date
2021-01
Authors
Froese, Alan
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Abstract
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 end-products, 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 pyruvate phosphate dikinase (PPDK), which may act to increase net ATP yields from glycolysis. The PPDK was cloned into E. coli, expressed, purified, and characterized, confirming its function as a PPDK for glycolysis and revealing strong activation by ammonium. The kinetic characterization of the PPDK will help inform future studies that measure and model levels of important intracellular metabolites, such as pyrophosphate and ammonium, to better understand the metabolism of C. thermocellum and allow further metabolic engineering to increase ethanol yields.
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Keywords
Clostridium thermocellum, biofuel, co-culturing, pyruvate phosphate dikinase
Citation
Froese A, Schellenberg J, Sparling R (2019) Enhanced depolymerization and utilization of raw lignocellulosic material by co-cultures of Ruminiclostridium thermocellum with hemicellulose-utilizing partners. Can J Microbiol 65:296–307. https://doi.org/10.1139/cjm-2018-0535
Froese AG, Nguyen T-N, Ayele BT, Sparling R (2020) Digestibility of Wheat and Cattail Biomass Using a Co-culture of Thermophilic Anaerobes for Consolidated Bioprocessing. BioEnergy Res 13:325–333. https://doi.org/10.1007/s12155-020-10103-0
Froese AG, Nguyen T-N, Ayele BT, Sparling R (2020) Digestibility of Wheat and Cattail Biomass Using a Co-culture of Thermophilic Anaerobes for Consolidated Bioprocessing. BioEnergy Res 13:325–333. https://doi.org/10.1007/s12155-020-10103-0