Improved production of isobutanol in pervaporation-coupled bioreactor using sugarcane bagasse hydrolysate in engineered Enterobacter aerogenes
Graphical abstract
Introduction
Due to the increasing demand for sustainable processes and for reductions of greenhouse gases, bio-based fuels are considered as one of the alternative energy sources in the chemical industry (Balat and Balat, 2009, Mckechnie et al., 2011, Von Blottnitz and Curran, 2007, Sivagurunathan et al., 2017). Among the many organic alcohols, isobutanol is a prominent candidate for gasoline blend stock because of its high energy density, low hygroscopicity, and compatibility with the established infrastructure (Connor and Liao, 2009). Isobutanol is also used to produce jet fuels and is a raw material for synthetic rubber and many chemical additives (Peters and Taylor, 2015, Wang et al., 2012). The microbial metabolic pathways for production of higher alcohols including isobutanol were identified a decade ago (Atsumi et al., 2008). The synthetic isobutanol pathway consists of a valine synthesis pathway with two heterologous enzymes, decarboxylase (KivD) and alcohol dehydrogenase (AdhA). Therefore, various studies on isobutanol production have been carried out harnessing diverse host microorganisms and types of biomass (Lan and Liao, 2013). Most of the studies on isobutanol production have been conducted by means of representative workhorses of bio-processes, e.g., Escherichia coli and Saccharomyces cerevisiae (Atsumi et al., 2008, Baez et al., 2011, Lee et al., 2012, Avalos et al., 2013). A similar strategy has also been implemented in some enteric bacteria like Klebsiella pneumoniae and Enterobacter aerogenes, which have characteristics of natural production of 2,3-butanediol (Jung et al., 2017, Oh et al., 2014). An intermediate in the 2,3-butanediol pathway, acetolactate, can serve as a precursor in the valine synthesis pathway, meaning that those enteric bacteria have a potential to produce isobutanol. They also have attractive physiology because E. aerogenes consumes diverse carbon sources such as glucose, fructose, xylose, arabinose, and glycerol and shows an outstanding growth rate (Jung et al., 2015, Nakashimada et al., 2002).
The toxic effects of the final product prevent high-concentration production of isobutanol. When microorganisms are exposed to this solvent, the structure of the membrane collapses, resulting in an imbalance of intracellular metabolites and cofactors (Aono et al., 1994). It is also known that reactive oxygen species are formed under solvent stress conditions (Shimizu, 2014). Generally, it is difficult to produce over 2% (w/v) of butanol or isobutanol in a simple batch process because of their high toxicity to host microorganisms. Several researchers have developed strains more tolerant to butanol or isobutanol through evolutionary engineering (Winkler and Kao, 2014). In another study, a mutant strain was created via serial adaptation of a bacterial strain, which showed 5-fold improved cellular fitness when exposed to 0.8% (w/v) of isobutanol (Atsumi et al., 2010). Nonetheless, better resistance to a toxic product does not always correspond to higher production of a target product.
Meanwhile, several separation techniques, such as gas stripping, pervaporation and liquid-liquid extraction, can be coupled to a fermentation process for reducing the solvent toxicity to host microorganisms. For example, a solvent extraction process has been integrated with fermentation (Outram et al., 2017). However, an additional separation procedure is necessary to purify the target product from the liquid extractant, resulting in increased process cost (Mohammadi et al., 2005). In case of a gas stripping procedure, a high operational expense is expected to maintain gas flow and condense solvent-holding gases, and a large amount of water is released together with the solvent product (Oudshoorn et al, 2009). A pervaporation process is recognized as superior to other methods due to its low-energy consumption, cost effectiveness, and mild operating conditions (Bowen et al., 2004). Especially, the pervaporation process can save up to 85% of thermal energy compared to the distillation method (Iwamato and Kawamoto, 2009). Therefore, coupling pervaporation to fermentation can minimize energy and cost required for the continuous production of isobutanol from fermentation broth. Several organophilic polymers on a PDMS-based membrane have been demonstrated for pervaporation coupled with acetone–butanol–ethanol (ABE) fermentation (Cai et al., 2017, Li et al., 2014). When applied in ABE fermentation, concentration of the solvent in the bioreactor was maintained below 1% (w/v), which was not lethal for microorganisms and did not disrupt major metabolic pathways. Although there have been examples of ABE fermentation with a pervaporation procedure, fermentative production of isobutanol in this regard has not been reported yet.
In this study, we attempted to engineer the isobutanol producing strain to consume glucose and xylose simultaneously (Fig. 1). Then, pervaporation-coupled fermentation strategy was applied to relieve the toxic effect of isobutanol on the strain. Lab made membrane module (Fig. 1) was utilized and in situ recovery process was effective to increase productivity of isobutanol. The engineered E. aerogenes also made comparable performance using sugarcane bagasse (SCB) hydrolysates in pervaporation-integrated fermentation process.
Section snippets
Strains and plasmids
All the strains and plasmids used in this study are listed in Table 1. The host microorganism, E. aerogenes, was obtained from the Korean Collection for Type Cultures (KCTC, Korea). Mutant strains were constructed previously; their major byproduct pathways were previously eliminated, and they harbor isobutanol pathway enzymes (Jung et al., 2017). The gene related to the PTS, ptsG (NC_015663.1), was removed for coconsumption of lignocellulosic-biomass-derived sugars: glucose and xylose. A gene
Development of the ptsG mutant and flask cultivation
In a previous study, the five heterologous enzymes from K. pneumoniae and Lactococcus lactis were introduced into engineered E. aerogenes (BudB, acetolactate synthase; IlvC, ketol-acid reductoisomerase; IlvD, dihydroxy-acid dehydratase KivD, α-ketoisovalerate decarboxylase; and AdhA, alcohol dehydrogenase; the background: ΔldhA ΔbudA ΔpflB) for isobutanol production with reduced by-products formation (Jung et al., 2017). Approximately 4 g/L isobutanol was produced under semianaerobic condition
Conclusions
The development of lignocellulosic-biomass–utilizing microorganisms is important for the biotechnology industry. A pathway engineered E. aerogenes has advantages, such as in multisugar utilization or in high yield production of pyruvate-driven chemical. The engineered E. aerogenes was used to produce isobutanol from an SCB hydrolysate. To reduce the toxicity of isobutanol to the engineered strain, a pervaporation procedure was coupled to fermentation for in situ recovery of isobutanol. The
Acknowledgement
This study was supported by a grant from the National Research Foundation funded by the Korean Government (2012M1A2A2026560 and 2017R1A2B4008758).
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These two authors made equal contributions to the work described in this paper.