Title: Unlocking the Potential of Carbonaceous Resource Recovery from the Arrested Anaerobic Digestion of Food Waste
Description: Organic waste-fueled carbonaceous resource recovery using approaches such as arrested anaerobic digestion generates economically attractive products such as volatile fatty acid (VFA). The production of VFA expands the applications of anaerobic biotechnologies beyond the traditionally produced biogas. Compared to biogas, VFA is produced and recovered in a concentrated form in the aqueous phase, which is more conducive to direct utilization in downstream bioplastic, biodiesel production, and nitrogen/phosphorus removal in water resource-recovery facilities. However, this application is limited by the variability in VFA yield and composition as obtained from different complex solids streams. Additionally, the lack of understanding of the nexus between the performance-structure-function of the microbial community within the arrested anaerobic digestion process leads to the massive gap between the optimized engineering regulations and the high-throughput VFA production. Consequently, this dissertation aimed to unlock the potential of VFA production with maximized yield and regulated composition through the manipulation of the operational parameter (hydraulic retention time (HRT)) and the feedstock condition (thermal hydrolysis pretreatment (THP)).

In response, meta-omics-derived approaches were applied to elucidate the dynamic changes of microbial structure, potential, and extant functionality in terms of the two processes (hydrolysis and acidification) within arrested anaerobic digestion of food waste. Specifically, the objectives were (1) Performance: Evaluate the hydrolysis and acidification performance changes including hydrolysis yield, VFA yield, VFA composition, methane yield, etc. under different HRTs and feeding THP or non-THP food waste. (2) Microbial structure: characterize and compare the significance of HRT and feedstock condition in shaping microbial structures. (3) Functional analysis: Interpret the community-level dynamic changes of potential and extant functions within the (3.1) customized acidification metabolic networks and the (3.2) carbohydrate hydrolysis niches. The highlighted findings are as follows: (1) Performance of the arrested anaerobic digestion (including hydrolysis and acidification processes): Neither the hydrolysis yield nor the VFA yield was improved by the extended HRT from 4 to 8 days (P > .05).

The inclusion of THP on feedstock didn’t improve the hydrolysis yield (P > .05) while the VFA yield was significantly decreased (P = .003). Among all conditions, the methane production was less than 5% of the chemical oxygen demand (COD) and a propionic acid-dominant type product was robustly formed. (2) Microbial structures in the arrested anaerobic digestors (including core hydrolyzers and acidification microbial communities): Both HRT and the inclusion of THP on feedstock shaped distinct microbial structures in the arrested anaerobic digestors (P = .02 and .01). Although the extension of HRT didn’t change the Shannon diversity Index (P > .05), it was significantly decreased after feeding with THP food waste (P = .03), which might stem from the reduced indigenous microbes in the initial food waste feedstock. Prevotella was always the most abundant genus under all conditions, which might contribute to the dominantly produced propionic acid among all conditions. The successfully suppressed growth of methanogenic archaea was reflected in terms of the low relative abundance (<1.5%) among all conditions. (3.1) Functional analysis of the customized acidification metabolic networks: Under the two selected HRTs, the potential and extant functions of acidification were unchanged between the two reactors (P > .05), which indicated a community-level redundancy in convergent potential and extant acidification functions even under a completely shifted microbial structure.

However, the inclusion of THP diminished the potential and extant functions of acidification, in the meantime, shifting the main producer of butyric acid from Bacteroides to Prevotella through the expression of gene buk2. Among all conditions, the highest potential and extant functions in propionic acid production corresponded to the propionic acid-dominant acid profile in all reactors. The prevalently enriched Prevotella contributed to the stable propionic acid-dominant production via the acryloyl-CoA to propionyl phosphate to the propionic acid pathway. (3.2) Functional analysis of the carbohydrate hydrolysis niches: The extension of HRT from 4 days to 8 days didn’t impact the potential and extant functions of carbohydrate-activated enzymes (CAZys) and the hydrolysis of polysaccharides. Only two intermediate steps (gene malQ and lplD) during the hydrolysis of starch and pectin were enhanced with higher absolute transcriptional activities (mRNA/DNA RPKM) under HRT 8 days. The abundance ratio of the two main hydrolysis phyla Firmicutes: Bacteroidetes was unchanged between the two HRTs.

When feeding with THP feedstock, the potential and extant functions of CAZys were both enhanced. All steps within the hydrolysis of cellulose (polysaccharides) exhibited increased absolute transcriptional activities (mRNA/DNA RPKM). The abundance ratio of Firmicutes: Bacteroidetes was decreased after the inclusion of THP on feedstock, which corresponded to the increased hydrolysis of polysaccharides- cellulose. Although the carbohydrate hydrolysis functions were improved after feeding with THP food waste, the total hydrolysis yield was not enhanced. The hydrolysis of other compounds such as proteins and lipids could also contribute to the total hydrolysis yield. The taxonomic analysis revealed that in all four conditions, the genus Prevotella presented with the highest potential functions in CAZys, while the genus Pararhodospirillum exhibited the highest extant functions in CAZys. This indicated that distinct bacteria were endowed with different functional potentials of CAZys and mobilized these functions differently.

Overall, this research provides practical suggestions for engineering designs to maximize the VFA production profits from arrested anaerobic digestion of food waste: (1) A properly controlled HRT enables a long-term high-throughput production of VFA with stable yield and the unchanged dominant acid type (2) The inclusion of THP to the feedstock was not suggested to be applied to maximize the VFA yield even the dominant acid type may not change. (3) The dominantly produced propionic acid could be targeted by enriching the Prevotella genus to produce the propionic acid through the acryloyl-CoA to propionyl phosphate to the propionic acid pathway. Besides the engineering aspect, this research also specifically elucidates the long-time lumped and simplified acidification and carbohydrate hydrolysis processes with the extended metabolic databases including each reaction, key intermediates, enzymes, and corresponding genes. This expanded database served as an essential upstream process, which could be integrated into the current anaerobic digestion model.

Additional applications could be extended to the human digestion systems' microbiome and be exploited commercially for other mixed-culture biosynthesis processes such as bioplastic and biodiesel production. Finally, the application of meta-omics-derived methodology revealed the functional redundancy and the potential discrepancy between the most abundant group and the most actively functional group underlying the formed black box of VFA production performance. This discussion of the nexus of performance-structure-function suggested the importance of applying meta-omics approaches in engineering practice, especially when feeding the mixed-culture community with real complex solid streams. The targeted VFA profiles cannot be reached without identifying the actual functional bacteria under selected engineering conditions.