Manipulating carbon, electron, and nitrogen exchange in syntrophic Clostridium co-cultures for robust, scalable, carbon-negative chemical production

Abstract

The classic acetone-butanol-ethanol (“ABE”) fermentation, which is based on Clostridium acetobutylicum, suffers from low carbon efficiency because 33-50% of carbon from the sugar substrate is lost as CO2 due to decarboxylation reactions in C. acetobutylicum’s central carbon metabolism. Gas fermentation, using acetogenic organisms such as Clostridium autoethanogenum or Clostridium ljungdahlii, can effectively assimilate gaseous carbon in the form of CO, but these organisms grow much more poorly on CO2 compared to CO, and, when grown on CO2, they make primarily acetate instead of more reduced products like alcohols. Our group has recently shown that cocultures between C. acetobutylicum and C. ljungdahlii have the potential to overcome many of the disadvantages associated with using only one of the two organisms in isolation. These C. acetobutylicum-C. ljungdahlii cocultures show improved carbon efficiency, large substrate and product portfolios, and unprecedented prokaryotic interspecies cell fusion events which involve large scale exchange of protein, RNA, and DNA. ☐ Building on and extending this work, this thesis describes an RNAseq study designed to uncover genes in both C. acetobutylicum and C. ljungdahlii which are important to the unique coculture phenotype, especially the interspecies cell fusion events. This study utilized a (to our knowledge) novel approach to test differential gene expression in response to direct microbial interspecies contact by isolating RNA from C. acetobutylicum and C. ljungdahlii grown in a transwell system, separated physically by a permeable membrane, and comparing it with RNA extracted from a normal mixed coculture. Using this methodology, we identified several genes from a putative Type VII secretion system operon in C. acetobutylicum which are upregulated by direct contact with C. ljungdahlii and which may be “fusogen” proteins involved in interspecies cell fusion. The gene expression data also revealed major differential regulation of amino acid metabolism (most especially of arginine, histidine, and tryptophan) in both C. acetobutylicum and C. ljungdahlii in coculture, which, when combined with amino acid secretion kinetics, helped to identify transfer of amino acids from C. acetobutylicum to C. ljungdahlii as a previously unknown layer of syntrophic cross-feeding between the coculture partners. Using this gene expression data, we also reconstructed a (to our knowledge) novel histidine catabolism pathway in C. ljungdahlii which substantially increases the energy efficiency of C. ljungdahlii growth on CO2 and demonstrated that C. ljungdahlii monocultures grown on CO2 with supplemental histidine grow much faster and to higher cell densities than controls grown only on CO2. ☐ Next, this thesis describes how the coculture between C. acetobutylicum and C. ljungdahlii can be repurposed for carbon-negative production of isopropanol from glucose (in which all of the glucose carbon is assimilated to soluble products along with some external CO2). This study presents detailed analysis showing how, due to interspecies electron exchange, the presence of the acetogen, C. ljungdahlii, enables C. acetobutylicum to synthesize much higher yields of acetone (which is then converted to isopropanol by C. ljungdahlii) in the coculture than would be possible in a C. acetobutylicum monoculture. Using high density, small scale pseudo perfusion experiments, we show that higher cell densities (and thus tighter interspecies proximity) strengthen this electron exchange to enable enhanced acetone and isopropanol yields. Finally, we demonstrate how, using a perfusion bioreactor, prolonged high density cocultures of C. acetobutylicum and C. ljungdahlii can produce isopropanol as the sole alcohol product from carbon-negative fermentation for over 100 hours with strong productivity. ☐ Next, this thesis describes how the coculture between C. acetobutylicum and C. ljungdahlii can be converted from obligate commensalism (C. ljungdahlii requires C. acetobutylicum for carbon in the form of CO2, but C. acetobutylicum does not require C. ljungdahlii for growth) to obligate mutualism (C. acetobutylicum requires C. ljungdahlii for nitrogen). This was achieved by designing a minimal medium with nitrate as the sole nitrogen source. C. acetobutylicum cannot use nitrate, but C. ljungdahlii can use nitrate and, when it has more than it needs, converts the excess nitrate to ammonia (a nitrogen source C. acetobutylicum can use) and secretes it into the culture medium. Based on this strategy, we test and demonstrate how varying the nitrogen source ratio in batch cultures and varying the nitrate feed rate in fed-batch cultures can be used to maintain a stable species ratio in the coculture, increase carbon efficiency, and improve yields of isopropanol and butanol. ☐ Finally, this thesis addresses an important open question in the gas fermentation literature: why do acetogens like Clostridium ljungdahlii show such a strong preference for growing on CO compared to CO2? We show that, though the presence of high energy substrates, such as fructose, can produce some form of catabolite repression, the true limitation on CO2 fixation by C. ljungdahlii (and similar acetogens) is the extremely low solubility of H2 (the electron donor for CO2 fixation in C. ljungdahlii) relative to CO2 and CO. By alleviating the H2 mass transfer limitation with increased mixing (via roller bottles) and high H2 partial pressure, we demonstrate (by far) the fastest doubling time ever recorded for C. ljungdahlii (or similar acetogens, to our knowlege) growing on only CO2 and H2, a doubling time equivalent to the fastest doubling time ever recorded by C. ljungdahlii (or similar acetogen) using CO. We discuss the significance of these findings for the future of gas fermentation and describe how coculturing acetogens with solventogenic organisms, such as C. acetobutylicum, can help to overcome H2 insolubility and potentially enable scalable and economically competitive CO2-negative fermentation.