DISSECTING COMPLEX CELLULAR AND METABOLIC INTERSPECIES INTERACTIONS IN CLOSTRIDIAL CO-CULTURES

Abstract

While microbial fermentation was widely used for the production of commodity chemicals in the early 20th century, petroleum refining has been the dominant approach since the end of World War II. Only ethanol fermentation, using corn sugars as substrate, is practiced commercially in the United States, though this is a subsidized market. The essential issue is the presence of decarboxylation reactions in the central metabolism of most organisms used in fermentative processes, which results in decreased substrate use efficiency and increased CO2 emissions. Fermentation systems that use substrates more efficiently are needed. Among other approaches being pursued (e.g., non-oxidative glycolysis or the CBB cycle), anaerobic, non-photosynthetic mixotrophy (ANP mixotrophy) shows immense promise at the laboratory scale. ANP mixotrophy is possible due to the existence of acetogens. Acetogens are a non-taxonomical class of anaerobic bacteria that employ the Wood-Ljungdahl Pathway (WLP) to fix CO2. When grown on sugar (i.e., grown heterotrophically), the CO2 released by decarboxylation reactions is reincorporated into the metabolism via the WLP. In addition to the CO2 produced by decarboxylation reactions, exogenous CO2 can be fixed simultaneously, which results in carbon-negative fermentations. A promising, recent advance in ANP mixotrophic fermentation technology is the concept of using acetogens in a synthetic consortium to achieve a broader product spectrum, higher productivity, and higher carbon efficiency. The use of more than one organism in a fermentation process introduces additional complexity, particularly in terms of population interactions. Some of these interactions could have been anticipated, but in some cases, interactions with a surprisingly high level of sophistication were observed. Moreover, the tools required to characterize the impact of these interactions (e.g., subpopulation health and dynamics) either have not been adapted for use in the context of synthetic consortia or do not exist. This thesis advances the current understanding of interspecies interactions in acetogenic co-cultures. The first effort is the application of species-specific ribosomal RNA-fluorescent in situ hybridization (rRNA-FISH). This involved the development of novel probes for Clostridium acetobutylicum and Clostridium ljungdahlii, referred to as ClosAcet and ClosLjun, respectively, and an optimization study on the hybridization protocol. It was confirmed that the use of rRNA-FISH does not interfere with other fluorescent labelling techniques, such as dyes and fluorescent proteins. Then, rRNA-FISH was used to determine the growth dynamics and viability of individual subpopulations in acetogenic, clostridial co- and triple-cultures using flow cytometry. The flow cytometry data suggested the existence of a ‘hybrid-cell’ population containing ribosomal material from two species. Microscopy verified the existence of the ‘hybrid-cell’ populations. While these hybrid cells had been shown to propagate in C. acetobutylicum – C. ljungdahlii co-cultures, this approach reported the first evidence of exchange between Clostridium kluyveri and the other two organisms. In addition to ribosomal nucleic acid sharing, the ability of C. acetobutylicum and C. ljungdahlii to share antibiotic-resistant genes (originating from plasmid DNA in C. ljungdahlii) was demonstrated at a frequency similar to known forms of transconjugation. The PacBio sequencing data of the clonal, mutant populations suggested the existence of (at least transient) hybrid-cell populations that contain chromosomal material from the parental species. This thesis also demonstrated the existence of interspecies metabolic cycles formed between C. acetobutylicum and C. ljungdahlii in co-culture. It was found that in the presence of NO3-, C. ljungdahlii activates its alcohol oxidation (e.g., butanol → butyrate) metabolism. The simultaneous reduction of carboxylic acids (e.g., butyrate →butanol) by C. acetobutylicum implies that carbon, in the form of extracellular metabolites, is being cyclically passed between species. Experiments demonstrated that these cycles improve fermentation performance in terms of target product yield. Finally, this thesis describes the creation of the first reported transposon-insertion library in C. ljungdahlii. This library is an essential tool for specific, anticipated studies regarding the exchange of DNA from C. ljundgahlii and for forward genetic studies in this important acetogen, in general. This exploration of nucleic acid sharing, hybrid cell formation, and metabolic cycling highlights the potential of acetogenic co-cultures, particularly the potential arising from newly discovered behaviors.