Metabolic mechanisms and regulation of mixed culture fermentation

Abstract

Fermentation of waste streams is an emerging method for production of biofuels and commodity chemicals, and mixed-culture fermentation allows for robustness and flexibility in the range of waste streams that can be processed, and in varying product mix from the fermenter. However, mechanistic models of mixed culture fermentation are limited, with mixed culture fermentation generally resulting in acetate, butyrate, and ethanol, with even these production modes are not clearly understood. Results from pure-culture fermentation, as well as those from methanogenic reactors indicate the possibility to produce high-value lactate and propionate if the cellular mechanisms which control the expression of these production modes can be harnessed and understood. Lactate and propionate are key chemicals in production of bioplastics, pesticides, preservatives, and food additives, among many other uses. Their production by mixed cultures has been observed at high substrate concentrations (shock loads), and inconsistently at high pH. They are produced via metabolic pathways which branch off of the primary energy-generation pathways. In general, they are thought to be produced by microbes as a way to redirect excess carbon flow in transient states, and are also thought to be produced as an electron sink. In this work, both of these possible production influences are explored for steady state production of lactate and propionate. By increasing substrate from limiting to non-limiting quantities, and thereby increasing the carbon load on a fermentation system, lactate production was successfully achieved and maintained during steady state only when substrate was in excess. Substrate consumption at this point was 14.1±0.8 gCOD·L-1, and lactate accounted for 25±1% of the total product COD. Upon return to substrate-limiting conditions, the process returned to butyrate production. This demonstrates the reversibility of this production mode. Taxonomic analysis via Illumina pyrosequencing of the 16S rRNA gene revealed that the microbial community experienced a shift from Megasphaera-dominant during butyrate production to Ethanoligenens-dominant during lactate production. Taxonomy was also regained upon return to substrate-limiting conditions. However, taxonomy alone could not explain the product shift because Ethanoligenens produces little to no lactate in cultured references. A mechanistic description of the product shift therefore required molecular analysis of the metabolic mechanisms, and this was achieved through metaproteomics via SWATH-MS. The evidence suggests that at high carbon loads, toxic methylglyoxal is formed in many microbe groups, including Megasphaera, due to limited processing capacity of the dihydroxyacetone-P intermediate of glycolysis. The methylglyoxal is converted into lactate for disposal. This detoxification need is abated upon return to substrate-limited conditions. The impact of redox stress was examined using cathodic electro-fermentation with soluble mediator chemicals ranging from slightly negative (-119 mV vs SHE) to near that of the intracellular NAD(P) and Fd pools (-394 mV). Propionate production was successfully achieved and maintained during steady state at 17±3% of total product COD (5.68±0.02 gCOD·L-1 substrate consumed) using methyl viologen as a mediator. However, minimal cathodic current was measured in the system. Taxonomic analysis revealed a gradual change from Megasphaera to Pectinatus with mediators of increasingly negative redox potential. This corresponded with a shift from butyrate production to propionate production. Pectinatus is a known propionate producer, and so this describes the product spectrum, but not the switching mechanism. Metaproteomic analysis revealed increased expression of both oxidative stress enzymes and electron mediation in mediated systems compared to non-mediated systems. Oxidative stress response in particular appears to be the causative factor for the change in product spectrum, and this could explain why others have also reported very low current compared to the reduction of the product spectrum. Additionally, expression of oxidative stress response enzymes was inversely related to expression of propionate pathway enzymes (p < 0.01). This mechanism could explain the occasional observation of propionate formation at high pH, which is also inversely related to oxidative stress response. It is hypothesized that in general, propionate formation is repressed by oxidative stress. However, in this system, propionate formation was due to the inhibition of bacteria other than Pectinatus. In general, it is proposed that mixed culture lactate production is a response to high carbon loads. This includes transient states, such as exponential growth and inter-steady-state periods, where the substrate supply is greater than the biomass processing capacity. By contrast, propionate production is an electron mediation response at low oxidative stress. Transiently increased substrate loads may also induce propionate formation via the same mechanism due to increased NAD reduction from increased substrate processing. However, at steady state this effect appears to diminish in favor of lactate production. This may occur due to electron rebalancing to other reduced products, such as ethanol. These conclusions are not substantively linked, and not related to the simpler (and more intuitive) models previously proposed. In this thesis, while there are still limitations in database information, the metaproteomic approach was critical to identify functional mechanisms in comparison with the more traditional taxonomic approaches.