Microbial chain elongation based on methanol

Our society relies heavily on fossil resources to fulfill our energy and commodity demands and this dependence has led to negative economic, environmental and societal consequences. The re-generation rate of fossil resources is much slower than their consumption rate, making these resources a non-renewable feedstock for the supply of energy and goods to our society. Moreover, the rapid consumption of fossil resources releases the carbon sequestrated in the last few million years in a much shorter time span, which contributes to the carbon dioxide (CO2) concentration increase in the atmosphere and potentially global warming. The geographically-uneven distribution of fossil resources also induces social insecurities and political conflicts. An alternative feedstock is necessary for energy and goods supply to our society, and such alternative feedstock should be renewable, economically sustainable, environmentally sound and geographically wide-spread,. Organic waste is an emerging and promising alternative feedstock. The production of organic waste is inevitable, occurs in large quantities and is geographically wide-spread, especially the so-called “mixed organic waste,” e.g. organic fraction of municipal solid waste (OFMSW) and food processing waste. Mixed organic waste contains a large quantity of carbon materials that can be valorised into energy carriers and commodities. However, the extremely heterogeneous composition and the relatively high water content of mixed organic waste make its valorisation via the current waste management methods (e.g. incineration, composting and anaerobic digestion) less efficient and not economically attractive. Given this context, a novel bioprocess based on a mixed culture fermentation, i.e. microbial chain elongation, was developed to promote the valorisation of mixed organic waste. In microbial chain elongation, the diverse, complex organic matter in mixed organic waste are homogenised via hydrolysis and bacterial acidification into basic building blocks; like short chain fatty acids (SCFAs), CO2 and hydrogen (H2). After the homogenisation, energy-rich co-substrates like ethanol are added to these basic building blocks to synthesise medium chain fatty acids (MCFAs) via a mixed culture fermentation. MCFAs are organic compounds with a higher economic value and a higher energy content. Microbial chain elongation can be operated under a non-sterile condition, which makes it applicable to valorise mixed organic waste where diverse microorganisms exist. Caproate is the most dominant product in the microbial chain elongation of mixed organic waste and ethanol, which can be produced at a high rate and selectivity. Caproate has a higher economic value, a lower solubility in water and an interesting market potential. Thus, caproic acid production from mixed organic waste and ethanol via microbial chain elongation is currently undergoing up-scaling and commercialisation. Many studies were done to improve the process of caproate production via microbial chain elongation to make it of industrial interest. The on-going commercialisation of microbial chain elongation also supports the economic feasibility. However, until now, no study addressed the environmental sustainability of microbial chain elongation. Chapter 2 of this thesis took the first attempt in analysing the life-cycle environmental impacts of caproic acid production from organic waste via microbial chain elongation, based on the literature and existing business case. The use of ethanol as a co-substrate (i.e. the electron donor) was shown to be the largest cause the environmental impact. This was found in in all assessed cases and all impact categories studied, and regardless of the feedstocks from which ethanol was produced. An alternative for ethanol as electron donor in microbial chain elongation is, therefore, an effective way to improve the environmental sustainability of microbial chain elongation. In Chapter 3, we investigated the use of methanol as an alternative electron donor in microbial chain elongation, i.e. methanol chain elongation, for butyrate and caproate production. Methanol chain elongation was previously demonstrated using a pure culture, but never with a mixed culture. To employ organic waste as feedstock, the feasibility of applying methanol chain elongation in an open mixed culture condition needs to be investigated. In Chapter 3, it was demonstrated in a batch incubation that methanol chain elongation could occur with a mixed culture, where butyrate was the dominant product (4.2 g/L). Caproate production via methanol chain elongation was also demonstrated, though only in a low concentration (0.1 g/L). In a continuous reactor operation, continuous butyrate production (1.5 g/L.day) was achieved via microbial chain elongation of acetate and methanol. However, caproate was not observed in the continuous methanol chain elongation. Interestingly, microorganisms that can perform methanol chain elongation were likely present in the inoculum taken from a previous ethanol chain elongation reactor without any methanol supplement. In Chapter 4, the use of methanol chain elongation to synthesise a novel product, i.e. isobutyrate, was proposed and investigated. Methanol chain elongation was found to continuously produce butyrate as the main metabolite, the accumulation of which was found to trigger isobutyrate formation in several previous methanogenic anaerobic digestion studies. It was, therefore, hypothesised that by elevating the butyrate concentration in the medium, methanol chain elongation might be able to produce isobutyrate as another metabolite. The result showed that isobutyrate could be produced as the main product, up to 6.2 g/L, when using acidified supermarket food waste and methanol as the substrate. A continuous methanol chain elongation using synthetic medium was also performed, which achieved a production rate of 2.0 g/L.day over five hydraulic retention times. Moreover, the production of isovalerate was also observed. Isobutyrate has a much larger market potential than caproate, though its production relies wholly on fossil-based feedstock. Isobutyrate biosynthesis was demonstrated in previous studies, but was only achieved using metabolically engineered microorganisms as the biocatalyst and glucose as the substrate. Methanol chain elongation, in contrast, could employ derivatives from organic waste as the substrates and a self-regenerating mixed culture biocatalyst for producing isobutyrate. Moreover, methanol chain elongation may be integrated into the current microbial chain elongation production facility without a significant infrastructure retrofit. All these advantages make methanol chain elongation an interesting and promising isobutyrate production process. The relatively large market potential of isobutyrate promotes the application of chain elongation and the use of organic waste for value-added chemical production. In Chapter 5, isobutyrate production was integrated with the caproate production via microbial chain elongation, by concurrently feeding both methanol and ethanol to a mixed culture. The result from Chapter 3 supports the possibility of coexistence of ethanol and methanol chain elongation microorganisms in the same microbiome. In Chapter 4, the possible concurrence of methanol and ethanol chain elongation was also observed. Based on these observations, we hypothesised that methanol and ethanol chain elongation could be integrated to simultaneously produce caproate and isobutyrate. The result showed that such integration was possible when a stable pH was maintained. When pH was controlled between 6.2 – 6.5 and butyrate was supplied in the medium, caproate and isobutyrate could be produced simultaneously. Additionally, increasing the ethanol feeding rate promoted the chain elongation of butyrate to caproate via ethanol chain elongation. The outcome of this chapter demonstrated the possibility of producing two valuable products in a single reactor with a mixed culture which, coupled with further process improvement, may be of industrial interest. In Chapter 6, we reflected on the caproate production performance of methanol chain elongation, in comparison with other electron donors used in microbial chain elongation, i.e. ethanol and lactate. Furthermore, we also reflected on the isobutyrate production via methanol chain elongation, in comparison with other emerging products in microbial chain elongation. These reflections could serve as a benchmark for methanol chain elongation as a waste management strategy. Based on this benchmarking, we proposed that methanol chain elongation is a promising bioprocess for isobutyrate production but not for caproate production. A potential strategy for improving the isobutyrate production via methanol chain elongation was proposed and discussed. The outcomes of this thesis may contribute to future application and assessments of microbial chain elongation in waste management. It may fuel discussion on how to further promote microbial chain elongation for a more sustainable waste management.