Date of Award

Fall 2021

Publication Type


Degree Name



Civil and Environmental Engineering

First Advisor

J.A. Lalman

Second Advisor

L. Porter

Third Advisor

N. Biswas


Hydrogen production, Dark fermentation, Glycerol



Creative Commons License

Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.


The focus of this thesis was on the production of H2 from glycerol by mixed anaerobic cultures through dark fermentation. Laboratory scale experiments were conducted to demonstrate H2 production from glycerol. The impact of various factors was evaluated using different analytical and statistical methods. Three pH levels (5.5, 6.5, and 7.5) were examined to determine the effects of the initial pH on H2 production from glycerol. A hydrogen yield of 0.33 ± 0.03 mol H2 mol–1 glycerol was observed in cultures with the initial pH set at 5.5.

Further experiments were focused on increasing the H2 yield using long chain fatty acids (LCFAs) as inhibitors together with glycerol in mixed anaerobic cultures with an initial pH of 5.5. Six LCFAs including lauric acid (LUA), myristic acid (MA), palmitic acid (PA), stearic acid (SA), oleic (OA), and linoleic acid (LA) were examined in this study. Higher H2 yields were observed in cultures fed PA, OA, or LA when compared to cultures fed with only glycerol. The H2 yield for the OA and LA treated cultures were 0.42 ± 0.01 and 0.46 ± 0.03 mol H2 mol–1 glycerol, respectively. In the LA and glycerol fed cultures, the H2 yield was 29% larger when compared to the glycerol control. Based on the electron balance, ethanol (EtOH) (approximately 23.1% of the total electron equivalents) and 1,3-propanediol (1,3 PDO) (approximately 50.0% of the total electron equivalents) were the major metabolites in the LA treated cultures, while approximately 6.5% and 7.9% electron equivalents were directed to H2 and acetate (Ac-) formation, respectively.

A three-factor and three-level BBD model was conducted to maximize the H2 yield in cultures fed glycerol and LA. The initial pH levels (5.5, 6.5, 7.5), glycerol concentrations (1,300, 2,600, 5,110 mg L–1), and operational temperatures (22, 37, 52 °C) were three factors selected in this study. The highest H2 yield was 0.86 ± 0.02 mol H2 mol–1 glycerol at 55 °C, a pH of 5.5, and a glycerol concentration of 2,600 mg L–1. The predicted result was 0.84 mol H2 mol–1 glycerol at 55 °C, a pH of 5.5, and a glycerol concentration of 2,710 mg L–1 using the D-optimality analysis. Based on the designed BBD model, the optimum levels of three factors were significant when predicting the highest H2 yield by the D-optimality analysis.