Metabolism and cell-to-cell interactions of anaerobic syntrophic Clostridia co-cultures

Date
2019
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Publisher
University of Delaware
Abstract
The greenhouse gas, CO2, in Earth’s atmosphere is a threat to our planet and a cause of global warming. Utilizing CO2 industrial waste gases as fermentation feedstock for biofuel production is a promising technology for the reduction of industrial waste. One of the notable genera of bacteria that can accomplish this is the genus Clostridium. Clostridium spp. are Gram-positive, anaerobic Firmicutes that can metabolize a diverse amount of substrates, including sugars, acids, alcohols, and gases like CO2, H2, and CO. Engineering non-acetogenic clostridia to efficiently consume waste as feedstock has been the subject of many recent studies, but using multiple species of Clostridium in co-culture has been shown to be an achievable and economical option for optimizing fermentation carbon recoveries and production of non-natural products. ☐ Many different co-cultures between Clostridium spp. have been studied, but one pair that has not been investigated previously is between the chain elongating C. kluyveri, and the well-studied solventogen, C. acetobutylicum. Our lab has previously established a co-culture between C. acetobutylicum and the CO2 fixing acetogen, C. ljungdahlii, and this co-culture has been shown to improve carbon efficiency of clostridia fermentation when compared to mono-culture. The addition of a third organism, C. kluyveri, to the co-culture could potentially use the products of syntrophic interactions between C. ljungdahlii and C. acetobutylicum to produce medium-chain fatty acids that can be converted to biofuels. ☐ In this study, we examined the possibilities of C. acetobutylicum and C. kluyveri in co-culture as well as a triple organism co-culture between C. acetobutylicum, C. ljungdahlii, and C. kluyveri. This triple organism co-culture has the potential to use sugars for syntrophic production acids and alcohols, which would be further converted to of medium-chain fatty acids by C. kluyveri. C. acetobutylicum and C. kluyveri co-cultures were shown to be need further optimization due to pH discrepancies between species, but the addition of C. ljungdahlii to the culture was able to produce the medium-chain fatty acid, hexanoate from additional acetate and ethanol in co-culture. In order to further understand syntrophic interactions between Clostridium spp., we designed an anaerobic, highly-fluorescent reporter system using the fluorescence activating protein, FAST, and the fluorogenic ligand, HMBR. Commonly used fluorescent proteins reporters require oxygen for chromophore maturation, and anaerobic fluorescent proteins lack brightness comparable to aerobic fluorescent proteins. FAST does not require oxygen, and fluoresces instantaneously when the fluorogenic ligand, HMBR, is added. In addition to being a successful fluorescent reporter, FAST was also used to successfully tag and view protein localization of the cell division protein, ZapA, in live C. acetobutylicum cells. This fluorescent system opens the door for research on other Clostridium spp. and other anaerobes to study protein interactions with oxygen-independent fluorescence. FAST could be used in the future for further investigation of protein localization, particularly shedding light on localization of proteins that may be involved in syntrophic co-cultures.
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