Sequential Solid-State and Slurry Fermentation for Direct Succinic Acid Production from Non-Hydrolyzed Lignocellulosic Biomass using Mixed Fungal Cultures

Date of Award


Degree Name

Doctor of Philosophy


Chemical and Paper Engineering

First Advisor

Dr. Andro Mondala

Second Advisor

Dr. James R. Springstead

Third Advisor

Dr. Brian Young

Fourth Advisor

Dr. John Miller


The current industrial process of producing bio-based succinic acid is thru bacterial fermentation wherein sugars from energy crops are commonly used as the substrate. The competition for biofuels and costly nutritional requirements are the drawbacks of producing succinic acid by bacterial fermentation. To address these concerns, this study investigated an alternative route of producing succinic acid from direct bioconversion of lignocellulosic biomass in a two-step fermentation process using mixed fungal culture. The two-step fermentation process involved a solid-state fermentation pre-culturing phase followed by a slurry fermentation phase. In the solid-state fermentation pre-culturing phase, the cellulolytic and acidogenic fungus Aspergillus niger and cellulolytic fungus Trichoderma reesei were co-cultured on milled soybean hull substrate to induce cellulolytic and hemicellulolytic enzyme production. The white-rot ligninolytic fungus Phanerochaete chrysosporium was grown separately on milled birchwood chips to produce ligninolytic enzymes. During the slurry fermentation phase, the seven-day-old fermented biomass substrates were combined and submerged in an acetate buffer for five days to induce simultaneous saccharification and succinic acid production.

This research investigated succinic acid production from non-hydrolyzed lignocellulosic biomass using a two-step fermentation process employing a lignocellulolytic-acidogenic mixed fungal culture. The first study addressing Research Objective 1 examined the solid-state fermentation pre-culturing phase for lignocellulosic enzyme production. The enzyme activities and the consequential biomass degradation, saccharification, and organic acid production were discussed. The second study, which addressed Research Objectives 2 focused on statistical optimization and the effects of batch slurry fermentation variables on succinic acid yield. There have been several studies carried out on optimizing solid-state fermentation variables for cellulolytic enzyme production. Batch slurry fermentation variables such as acetic acid concentration, initial pH, and solids loading were optimized for the maximum succinic acid yield, and the effects of these variables on succinic acid yield were determined. The interaction between acetic acid concentration and initial pH could have affected the acetate and succinate anion permeability, resulting in the accumulation of succinic acid in the broth. The third study addressing Research Objectives 3 tested kinetic models to understand the kinetic behavior of fungal succinic acid production during the slurry fermentation phase under optimum conditions. The experimental data were fitted to the proposed kinetic models, and the kinetic parameters were calculated using the fourth order Runge-Kutta method and method of least squares. The logistic, Luedeking-Piret, and carbon balance rate equations were used to describe the mixed fungal biomass growth, substrate consumption, and succinic acid production, respectively. In bacterial fermentation, carbon dioxide is externally supplied to shift the metabolic pathway towards succinate production route. In this study, the probable internal sources of carbon dioxide are from carbon dioxide evolved from mixed fungal metabolism, and as an end-product of lignin degradation.

The results of this study could be useful for future process improvement and scale-up of the two-step fermentation process. This fermentation technology uses relatively inexpensive substrates and nutritional requirements, which could potentially reduce the cost of fungal succinic acid, making it potentially more cost-competitive than those generated using existing bacterial fermentation process or obtained from petroleum feedstock.

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