Author ORCID Identifier

Date Available


Year of Publication


Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation




Biosystems and Agricultural Engineering

First Advisor

Dr. Jian Shi


Anaerobic digestion (AD) is a widely used biowaste conversion method. Recent studies have explored arrested methanogenesis as an alternative approach to leverage existing AD facilities and produce volatile fatty acids (VFAs) instead of biogas. VFAs have been recognized as precursors for a range of value-added chemicals and bioproducts. Arrested methanogenesis involves inducing acidic fermentation by halting the AD process before methanogenesis. It has been found that higher organic loading and thermophilic conditions enhance VFA accumulation and stabilize the acidic fermentation (AF) system.

Brewer's spent grain (BSG), a significant by-product of the brewing industry, was employed as the feedstock for VFA production. However, previous studies utilizing raw BSG reported relatively low yields of VFAs. To address this issue, a synergistic ball-milling and enzymatic hydrolysis pretreatment was employed to release fermentable sugars from raw BSG. The approach allowed for a thermophilic AF system with a high substrate-to-inoculum ratio and VFA yield, without the addition of any methanogenesis inhibitors. Several strategies were then investigated to further boost VFA production by timely removal of the produced VFAs to alleviate potential product inhibition.

Direct replacement of the acid-rich supernatant, however, did not significantly increase the overall VFA production. This is likely caused by the concurrent removal of other compounds from the supernatant that weakened the alkalinity of the AF system. To enable the continuous removal of VFAs while preserving alkalinity, a membrane extraction system was developed using an omniphobic membrane and hydrophobic deep eutectic solvent (DES). The VFA extractability by different hydrophobic DESs under varying temperatures and pH conditions were investigated using synthetic VFA solutions and real fermentate effluent. Molecular dynamic simulations were employed to elucidate interactions between DES and VFA components.

In situ conversion of VFAs into esters is another potential strategy for VFA removal. However, VFA esterification in aqueous medium is challenging due to the abundant water driving the reaction toward hydrolysis. To overcome this problem, two commercial lipases from Aspergillus oryzae (AoL) and Candida rugosa (CrL) were immobilized on lignin nanoparticles and embedded into hydrogel beads, providing a confined hydrophobic space for the esterification reaction in aqueous solution. The immobilization process was optimized, and the hydrogel biocatalyst beads were compared to free enzymes and commercial catalysts in terms of their hydrolysis and esterification activities.

To gain a better understanding of lipase catalyzed VFA esterification in DES, the compatibility of various hydrophilic and hydrophobic DESs with two commercial lipases were studied using an optimized hydrolysis activity assay. Generally, lipase CrL exhibited higher activity and tolerance compared to AoL while the pH of the system played a crucial role in the lipase performance. Docking simulations were employed to examine the interactions between lipases and the various components of DESs, aiming to establish a rational approach for predicting the impact of different DES species on lipase activity.

In conclusion, this study developed and implemented a process-intensified approach for fermentative VFA production from BSG, along with methods to enhance VFA yield and recovery through novel VFA extraction and esterification techniques enabled by green solvents and enzyme immobilization. These findings provide a potential solution to the sustainable utilization of BSG waste from the brewing industry.

Digital Object Identifier (DOI)