Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Metaproteomic Insights into Mechanisms Enabling Anaerobic, Thermophilic Microbiomes to Achieve Undiminished Fractional Carbohydrate Solubilization at High Solids Loading


Evert K. Holwerda1,2* (, Payal Chirania1,3, Matthew R. Kubis1,2, Richard J. Giannone1,3, Yannick J. Bomble1,4, Robert L. Hettich1,3, Lee R. Lynd1,2, and Gerald A. Tuskan1,3


1Center for Bioenergy Innovation, Oak Ridge National Laboratory; 2Dartmouth College; 3Oak Ridge National Laboratory; and 4National Renewable Energy Laboratory



The Center for Bioenergy Innovation (CBI) vision is to accelerate domestication of bioenergy-relevant, non-model plants and microbes to enable high-impact innovations along the bioenergy and bioproduct supply chain while focusing on sustainable aviation fuels (SAF). CBI has four overarching innovation targets: (1) Develop sustainable, process-advantaged biomass feedstocks, (2) Refine consolidated bioprocessing with cotreatment to create fermentation intermediates, (3) Advance lignin valorization for biobased products and aviation fuel feedstocks, and (4) Improve catalytic upgrading for SAF blendstocks certification.


Economically viable production of cellulosic biofuels requires (1) operation at high solids loadings of lignocellulosic feedstocks—on the order of 150 g/L, and (2) complete utilization of solubilized carbohydrates. Around two-thirds of the mass content of lignocellulose is carbohydrate. An efficient sugar-to-liquid-biofuel microbial metabolism can achieve an end-product yield of 0.5 g ethanol/g sugar. Not considering product titer restrictions and solids handling issues, 150 g/L solids loading would result in a maximum biofuel titer for ethanol of ~50 g/L. However, the recalcitrant character of lignocellulosic feedstocks impedes biological conversion and represents a major cost barrier. To this end, the team characterized Nature’s ability to deconstruct and utilize lignocellulosic feedstocks at increasing solid loadings using defined cultures of anaerobic bacteria as well as anaerobic methanogenic microbiomes.

While the microbial community exhibits undiminished fractional carbohydrate solubilization near 0.7 at loadings ranging from 30g/L to 150g/L (Chirania et al. 2022), the defined culture shows decreasing solubilization at increasing solids loadings up to 80 g/L (Kubis et al. 2022). Note that fractional carbohydrate solubilization is defined as the portion of carbohydrate removed or solubilized per the original total amount of carbohydrate in a sample of biomass. The defined cultures reach high levels of solubilization they also leave behind small but distinct amounts of solubilized yet unutilized oligosaccharides. An ideal lignocellulose-to-biofuel process would employ characteristics of both these biocatalyst systems: the ability to (1) make a single liquid biofuel or intermediate as the metabolic end-product and (2) maintain high solubilization and utilization at high solids loadings.

To gain insight into the differences between solubilization and utilization of these two biological systems team members characterized microbiomes using metaproteomics, particularly focused on Carbohydrate Active enzymes (CAZymes) and diagnosed the defined cultures via fermentation- centered studies.

An anaerobic, thermophilic, semi-continuously fed, methanogenic microbial enrichment cultivated over an extended period (550 days), referred to as the lignocellulose-fermenting microbiome, was sampled at various solids loadings at steady state. The samples were fractionated to identify key microbes and enzymes. Researchers documented changes in the abundance of CAZymes across fractions and the details of the methanogenesis pathways. Significant enrichment of auxiliary activity family 6 enzymes at higher solids suggests a role for Fenton chemistry. Stress-response proteins accompanying these reactions are similarly upregulated at higher solids, as are β-glucosidases, xylosidases, carbohydrate-debranching, and pectin-acting enzymes—all of which indicate that removal of deconstruction inhibitors is important for observed undiminished solubilization.

The defined cultures reached solubilization levels as high as 75% on unpretreated feedstock in batch fermentations of corn stover and switchgrass. Cocultures consisting of cellulolytic and saccharolytic organisms showed increased solubilization and utilization over monocultures of cellulolytic bacteria. However, solubilization diminished at increasing solid loadings and this was not recovered via diagnostic experiments: the addition of a concentrated cell pellet to a running high solids fermentation did not increase solubilization compared to an equal volumetric addition of water-only. The culture was also able to ferment additional pulses of model soluble (cellobiose) and insoluble (cellulose) substrates without issue, but this did not result in adverse or beneficial effects on the solubilization of the corn stover. Dilution with no-carbon source medium additions did partly recover the high solubilization characteristics, as did supplementation of the defined cultures with microbiome isolates or co-inocula.

Our work provides insights into the mechanisms by which natural microbiomes effectively deconstruct and utilize lignocellulose at high solids loadings and sets us on a path for engineering bacterial strains in the development of defined cultures for efficient bioconversion.


Chirania, P., et al. 2022. “Metaproteomics Reveals Enzymatic Strategies Deployed by Anaerobic Microbiomes to Maintain Lignocellulose Deconstruction at High Solids,” Nat Commun 13, 3870. DOI:10.1038/s41467-022-31433-x.

Kubis, M. R., et al. 2022. “Declining Carbohydrate Solubilization with Increasing Solids Loading During Fermentation of Cellulosic Feedstocks by Clostridium thermocellum: Documentation and Diagnostic Tests,” Biotechnol Biofuels 15, 12. DOI:10.1186/s13068-022-02110-4.

Funding Information

Funding was provided by the Center for Bioenergy Innovation (CBI) led by Oak Ridge National Laboratory. CBI is funded as a U.S. Department of Energy Bioenergy Research Centers supported by the Office of Biological and Environmental Research in the DOE Office of Science under FWP ERKP886. Oak Ridge National Laboratory is managed by UT- Battelle, LLC for the U.S. Department of Energy under contract no. DE-AC05-00OR22725.