Declining carbohydrate solubilization with increasing solids loading during fermentation of cellulosic feedstocks by Clostridium thermocellum: documentation and diagnostic tests

Matthew R Kubis; Evert K Holwerda; Lee R Lynd

doi:10.1186/s13068-022-02110-4

Declining carbohydrate solubilization with increasing solids loading during fermentation of cellulosic feedstocks by Clostridium thermocellum: documentation and diagnostic tests

Biotechnol Biofuels Bioprod. 2022 Feb 5;15(1):12. doi: 10.1186/s13068-022-02110-4.

Authors

Matthew R Kubis^{1

2}, Evert K Holwerda^{3

4}, Lee R Lynd^{1

2}

Affiliations

¹ Thayer School of Engineering, Dartmouth College, 14 Engineering Drive, Hanover, NH, 03755, USA.
² The Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, 37831, USA.
³ Thayer School of Engineering, Dartmouth College, 14 Engineering Drive, Hanover, NH, 03755, USA. evert.k.holwerda@dartmouth.edu.
⁴ The Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, 37831, USA. evert.k.holwerda@dartmouth.edu.

Abstract

Background: For economically viable 2nd generation biofuels, processing of high solid lignocellulosic substrate concentrations is a necessity. The cellulolytic thermophilic anaerobe Clostridium thermocellum is one of the most effective biocatalysts for solubilization of carbohydrate harbored in lignocellulose. This study aims to document the solubilization performance of Clostridium thermocellum at increasing solids concentrations for two lignocellulosic feedstocks, corn stover and switchgrass, and explore potential effectors of solubilization performance.

Results: Monocultures of Clostridium thermocellum demonstrated high levels of carbohydrate solubilization for both unpretreated corn stover and switchgrass. However, fractional carbohydrate solubilization decreases with increasing solid loadings. Fermentation of model insoluble substrate (cellulose) in the presence of high solids lignocellulosic spent broth is temporarily affected but not model soluble substrate (cellobiose) fermentations. Mid-fermentation addition of cells (C. thermocellum) or model substrates did not significantly enhance overall corn stover solubilization loaded at 80 g/L, however cultures utilized the model substrates in the presence of high concentrations of corn stover. An increase in corn stover solubilization was observed when water was added, effectively diluting the solids concentration mid-fermentation. Introduction of a hemicellulose-utilizing coculture partner, Thermoanaerobacterium thermosaccharolyticum, increased the fractional carbohydrate solubilization at both high and low solid loadings. Residual solubilized carbohydrates diminished significantly in the presence of T. thermosaccharolyticum compared to monocultures of C. thermocellum, yet a small fraction of solubilized oligosaccharides of both C₅ and C₆ sugars remained unutilized.

Conclusion: Diminishing fractional carbohydrate solubilization with increasing substrate loading was observed for C. thermocellum-mediated solubilization and fermentation of unpretreated lignocellulose feedstocks. Results of experiments involving spent broth addition do not support a major role for inhibitors present in the liquid phase. Mid-fermentation addition experiments confirm that C. thermocellum and its enzymes remain capable of converting model substrates during the middle of high solids lignocellulose fermentation. An increase in fractional carbohydrate solubilization was made possible by (1) mid-fermentation solid loading dilutions and (2) coculturing C. thermocellum with T. thermosaccharolyticum, which ferments solubilized hemicellulose. Incomplete utilization of solubilized carbohydrates suggests that a small fraction of the carbohydrates is unaffected by the extracellular carbohydrate-active enzymes present in the culture.

Keywords: Biofuels; Biomass deconstruction; Cellulose; Clostridium thermocellum; Coculture; Corn stover; Hemicellulose; High solid loading; Lignocellulose; Switchgrass; Thermoanaerobacterium thermosaccharolyticum.