Date of Award
5-2025
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Environmental Engineering
Committee Chair/Advisor
Sudeep Popat
Committee Member
David Freedman
Committee Member
David Ladner
Committee Member
Cindy Lee
Abstract
Anaerobic co-digestion with fats, oils, and grease (FOG) waste can increase energy production due to the high methane potential of lipids. However, adding FOG can lead to operational issues caused by accumulating saturated long-chain fatty acids (LCFA). My research explored why saturated LCFAs are often the primary intermediate that accumulates and the microbial sensitives of the populations responsible for their degradation. In each batch or semi-continuous experiment, all intermediates of FOG degradation, including LCFAs, volatile fatty acids (VFAs), Hydrogen gas (H2), and endpoint methane production, were paired with temporal quantitative polymerase chain reaction (qPCR) of the Syntrophomonas genus, a known LCFA b-oxidizing microorganism, or 16S ribosomal ribonucleic acid (rRNA) sequencing. Monitoring all potential intermediates, including H2 partial pressure, was a novel addition to FOG anaerobic co-digestion studies. The prevailing explanation in literature of why palmitic acid accumulates revolves around increased H2 partial pressure leading to non-spontaneous b-oxidation.
The research analyzed the pH sensitivity of anaerobic co-digestion of FOG. Buffering the pH as a strategy to mitigate the inhibitory effects of FOG was explored with a semi-continuous experiment. Also, a batch study investigated the pH sensitivity of species belonging to the Syntrophomonas genus and palmitic acid degradation, the primary analyte observed in inhibited anaerobic co-digesters. Apparent first-order growth rate constants for palmitic acid and growth rate constants for Syntrophomonas sp. were calculated. Results indicated that microbes involved in lipid anaerobic digestion, specifically the genus Syntrophomonas, are highly sensitive to even small changes in pH and that buffering can help overcome the inhibitory effects associated with LCFA accumulation by promoting the growth of Syntrophomonas sp. during co-digester start-up or LCFA overloaded conditions.
The research also explored the biochemical pathways of five common saturated and unsaturated LCFAs observed in wastewater. All intermediates produced from myristic, palmitic, stearic, oleic, and linoleic acid were monitored in batch study to identify differences in the degradation pathways of unsaturated vs saturated LCFA degradation and differences in the microbial communities involved. Specifically, the batch study allowed exploration of whether unsaturated LCFA degradation requires hydrogenation and whether species belonging to Syntrophomonas genus are involved in that step. Unsaturated LCFA degradation most likely involves an initial hydrogenation step, as the dominant b-oxidizing genus present, Syntrophomonas, experienced a lag in growth while unsaturated LCFAs were converted to saturated LCFAs. My results indicated that unsaturated LCFAs do not directly enter the b-oxidation cycle and the Syntrophomonas genus were not involved in unsaturated degradation to saturated LCFAs. The research also supported the conclusion that saturated LCFA accumulation was not due to H2 partial pressure, as concentrations never reached high enough levels to cause a positive Gibbs free energy.
Saturated LCFAs, primarily palmitic acid, are the primary analyte observed in stalled anaerobic co-digesters despite FOG waste containing predominantly unsaturated fatty acid tails. Saturated LCFAs degrade rapidly under ideal conditions when fed as the substrate; however, they accumulate when produced from unsaturated LCFAs. My research explored if the presence of unsaturated LCFAs inhibited the degradation of palmitic acid and the growth of Syntrophomonas sp. through a batch study. Assays were spiked with palmitic acid and oleic, linoleic, or stearic acid. Palmitic and stearic acid are saturated LCFAs, while oleic and linoleic acid are unsaturated LCFAs. The study revealed significantly smaller first-order palmitic acid degradation rate constants in the presence of oleic and linoleic acid compared to stearic acid, indicating that unsaturated LCFAs inhibit b-oxidation. Syntrophomonas sp. concentrations also initially decreased during hydrogenation within the first two days of the experiment. These results indicated potentially different inhibition mechanisms between unsaturated and saturated LCFAs. Unsaturated LCFAs could promote permanent toxicity due to lysing and the ultimate decay of cells. Saturated LCFAs could promote temporary inhibition due to adsorption to cells, limiting substrate transport and nutrient uptake. H2 partial pressure never reached concentrations high enough to create a positive Gibbs free energy for b-oxidation. However, the presence of unsaturated LCFA in digester feed causing decay of b-oxidizers like Syntrophomonas sp. could be a potential explanation of why saturated LCFAs are the primary bottleneck.
Finally, VFAs are an alternative resource to methane generated from lipid-rich waste streams like FOG. Adding acetic acid, a VFA, can enhance biological nutrient removal at water resource reclamation facilities (WRRF). Excess methane not used to heat digesters or provide onsite electricity is often flared, resulting in CO2 emissions. VFAs from onsite FOG fermentation would reduce the chemical costs and greenhouse gas emissions (GHG). VFA production from FOG requires manipulating the lipid anaerobic food web to enhance b-oxidation and inhibit methane production. VFA production of lipids under slightly acidic, pH 6.7, and basic conditions, pH 8.2, was explored as a potential means of promoting the growth of b-oxidizers such as Syntrophomonas sp. while inhibiting methanogens. Apparent first-order growth rate constants of Syntrophomonas sp. and apparent first-order degradation rate constants of palmitic acid at pH 6.7 and 8.2 were calculated in a batch study. Both apparent first-order degradation rate constants of palmitic acid and growth rate constants of Syntrophomonas sp. were greater at pH 8.2 compared to pH 6.7. Another batch study spiked with FOG was used to determine if efficient b-oxidation of LCFAs produced from lipid hydrolysis is possible at pH 8.2 or 6.7. My study showed acetate as the primary VFA produced from the FOG and that at pH 8.2, there was a point at which acetate concentrations reached 80-100% of the influent COD added. A semi-continuous study further analyzed VFAs produced from FOG fermentation, pH control for methane inhibition, and solids residence time (SRT) selection. Caproate was the dominant VFA produced in both pH 6.7 and pH 8.2 FOG fermentation reactors, most likely due to the biological conversion of glycerol into caproic acid via chain elongation. VFA production efficiency was similar between pH 6.7 and 8.2. Semi-continuous buffering was not effective in controlling the pH in either fermentation reactor. A continuous experimental design would be necessary to identify the best pH and SRT selection, as continuous buffering and pH adjustment would allow for better stabilization at the desired reactor conditions.
Recommended Citation
Funk, Julia Ann, "Understanding Conversion of Long Chain Fatty Acids in Anaerobic Digestion Systems" (2025). All Dissertations. 3957.
https://open.clemson.edu/all_dissertations/3957