Degree

Doctor of Philosophy (PhD)

Semester of Graduation

Spring 2026

Department

Chemical Engineering

Document Type

Dissertation

Abstract

Methanotrophs are aerobic bacteria that have the ability to convert methane (CH4) into valuable bio-products like lipids while mitigating greenhouse gas (GHG) emissions. Integrating methanotroph-based lipid production process into existing wastewater treatment plants (WWTPs) offers the advantage of utilizing on-site biogas, wastewater effluent, and sludge streams. This approach supports simultaneous CH4 upvaluation and emission reduction as well as resource recovery. However, culture enrichment of robust methanotrophs has several existing challenges that limit the economic feasibility of this system including high cost of bioconversion due to low mass transfer efficiency of CH4 and oxygen demands, low growth rates and metabolic activity, process instability under fluctuating operational conditions, limited amount of methanotroph transformation techniques as well as limited knowledge on how metabolic networks are rearranged on different environments. To overcome these constraints, this study investigated the pivotal role of environmental conditions (mainly under low and high O2 levels) in guiding the source selection of natural methanotrophic consortia since it also governs the production of the target bio-products. Samples from multiple environments were screened to detect methanotrophs which led to the identification of a promising inoculum source containing diverse methanotrophic types. Subsequently, this work systematically evaluated the main and interaction effects of three critical factors, CH4:air volume ratio, initial pH, and culture medium type, with the aim of determining favorable conditions for enhanced methanotrophic growth and activity. The most suitable conditions were found to be synthetic wastewater (SW) medium, initial pH=6.8, and CH4:air volume ratio=1:1 yielding 0.19% (wt. ester/wt. dried biomass) esterifiable lipids. These conditions are operationally favorable as the CH4:air ratio of 1:1 minimizes additional O2 supplementation, near-neutral pH eliminates the need for extensive pH adjustment, and SW medium supports higher lipid production compared to nitrate mineral salts (NMS) medium. These findings served as the basis for subsequent multiple-parameter optimization to determine the threshold of NH4+ as well as its interaction with Cu2+ to boost CH4 consumption, lipid production, and the selective enrichment of a specific type of methanotrophs. With the primary goal of maximizing Types I and X methanotroph abundance, this research established the optimum NH4+ and Cu2+ concentrations to be 361 mg·L-1 and 0.03 mg·L-1, respectively, corresponding to a predicted  increase of 202.51% Types I and X methanotroph relative abundance aligning closely with the experimental findings of 236.02%. This cultivation condition resulted in 22.93% CH4 consumption and 0.88% wt. esterifiable lipid content. To assess the potential of the envisioned methanotroph-based lipid production process, these results were applied and adjusted to aid in the development of the methanotroph cultivation and enrichment process for microbial lipids production via utilizing the by-products of WWTPs: activated sludge as seed source, wastewater effluent (via simulating domestic wastewater) as a culture medium; and CH4 as substrate. In addition, bioaugmentation with methanotroph pure culture under simulated wastewater treatment conditions (in terms of CH4:air replenishments, high NH4+ level, and endogenous Cu2+ concentration) were performed to enhance the process. The introduction of methanotroph pure culture in activated sludge improved CH4 consumption even when biomass concentrations remained in steady conditions. After the 54-day cultivation and enrichment period, apparent CH4 consumption (%vol) reached 59.40% in Cellbag A and 51.92% in Cellbag B. The highest biomass lipid content (%DCW) reached 0.79% and 1.17% in Cellbags A and B, respectively, with corresponding biomass concentrations of 52.13 g·L-1 and 29.06 g·L-1. The 16S rRNA analysis revealed a pronounced community shift toward Types I and X methanotrophs achieving relative abundances of 44.10% (Cellbag A) and 50.39% (Cellbag B), whereas Type II methanotrophs remained below 1%. These findings demonstrate that targeted methanotroph bioaugmentation under simulated wastewater conditions can enhance CH4 utilization, biomass lipid content, and value-added product formation. Finally, this work presented a techno-economic analysis/assessment (TEA) of methanotroph-based lipid production utilizing vented and flared natural gas as feedstock to determine its economic feasibility. At baseline conditions of 839,282 kg/yr and 10% DCW lipid content, the system shows economic viability with a positive net present value (NPV). The break-even price is $19.55/kg and profitability improves at $24.64/kg with higher NPV, return on investment (ROI) and shortened payback period. Sensitivity analysis showed that lipid content strongly impacts process economics. The results imply the need for optimization and process intensification, and focus on high-value applications to attain commercial success. Overall, this approach represents a promising strategy for integrating anaerobic–aerobic bioprocesses in WWTPs that enables simultaneous CH4 upvaluation and mitigation as well as waste-to-resource conversion.

Date

2-6-2026

First Committee Chair

Emmanuel Revellame

First Committee Member

Mark E. Zappi

Second Committee Member

Rafael Hernandez

Third Committee Member

Dhan Lord B. Fortela

Fourth Committee Member

Andrei Y. Chistoserdov

Fifth Committee Member

Remil M. Aguda

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Available for download on Sunday, May 28, 2028

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