Agler, Matthew2012-06-282017-06-012012-01-31bibid: 7745319https://hdl.handle.net/1813/29427IMPROVING PERFORMANCE AND PRODUCT SPECIFICITY OF THE CARBOXYLATE PLATFORM FOR PRODUCTION OF BIOCHEMICALS Matthew T. Agler, Ph.D. Cornell University, January 2012 The carboxylate platform converts organic feedstocks to short- or medium-chain carboxylates with reactions that are catalyzed by undefined mixed cultures in anaerobic systems. The undefined mixed cultures first convert substrate biomass to primary fermentation products (e.g., ethanol, acetate, propionate, lactate, n-butyrate, hydrogen, and carbon dioxide). Next, secondary fermentation reactions (e.g., carboxylate oxidation, methanogenesis, or carboxylate chain elongation) couple oxidation and reduction of primary products to achieve a final product spectrum. The most successful application of the carboxylate platform, thus far, has been anaerobic digestion, because most substrate is efficiently and almost exclusively converted to methane and carbon dioxide. Additionally, problems are relatively easy to address because of years of experience with anaerobic digesters and a good understanding of the underlying microbial processes. The carboxylate platform can also be applied to production of liquid bioproducts, such as carboxylates. Until now, this application has suffered from low product specificity and poor yields due to unclear links between operating conditions and performance and a poor understanding of the underlying microbial communities. In this dissertation, we improve carboxylate production with undefined mixed cultures by using molecular biology tools to guide engineering of carboxylate-producing systems. First, we studied the efficiencies of thermophilic bioreactors producing n-butyrate for 421 days to determine how bioreactor operating conditions (substrate pretreatment and undissociated carboxylic acid toxicity reductions) affected the undefined mixed cultures. We used high-throughput 16S rRNA gene analysis, combined with ordination strategies, to observe the connection between development and performance of the bacterial community structure. Also, machine learning enabled us to probe the taxonomic structure to understand why performance did not increase with decreased carboxylic acid toxicity at pH 5.8. Finally, we implicated lactate in decreased n-butyrate specificity because of competing n-caproate production (i.e., lactate + n-butyrate  n-caproate), indicating that secondary fermentation reactions are important factors in determining the efficiency of carboxylate-producing systems. We concluded that in-situ product-specific carboxylate removal and secondary reaction control would be necessary to further decrease toxicity and increase performance of carboxylate-producing bioreactors. Because n-butyrate is relatively difficult to recover, next, we sought to study production of n-caproate, which, with a more hydrophobic nature is easier to remove from solution. Thus, we performed a functional metagenomics study of bioreactors operated at three temperatures (55oC, 40oC, and 30oC) to produce n-caproate and ncaprylate. We combined an electron pushing strategy (i.e., ethanol supplementation to promote coupling of ethanol oxidation and short-chain carboxylate elongation) and insitu product specific extraction to optimize n-caproate and n-caprylate formation. Functional metagenomics could map the dynamics between operating conditions and performance, indicating that better extraction efficiency could further improve performance. In the current setup, we achieved a maximum 52% n-caproate/n- caprylate specificity (i.e., the ratio of n-caproate and n-caprylate COD to the COD of ii all other fermentation products) and maximum rates that were six times those without extraction. Further, we challenged traditional knowledge by suggesting that hydrogenotrophic methanogenesis and carboxylate production can occur simultaneously, and that the community function may have depended on management of the hydrogen partial pressure by methanogens. Overall, we showed that many principles driving product specificity in anaerobic digestion (product removal and directed secondary fermentations) also apply to efficient production of liquid carboxylates. iiien-USCarboxylatebiochemical16S rRNA geneundefined mixed culturebutyratecaproatecaprylatemetagenomicsdissertation or thesis