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Enhanced Butanol Production Obtained by Reinforcing the Direct Butanol-Forming Route in Clostridium acetobutylicum

ABSTRACT Butanol is an important industrial solvent and advanced biofuel that can be produced by biphasic fermentation by Clostridium acetobutylicum. It has been known that acetate and butyrate first formed during the acidogenic phase are reassimilated to form acetone-butanol-ethanol (cold channel). Butanol can also be formed directly from acetyl-coenzyme A (CoA) through butyryl-CoA (hot channel). However, little is known about the relative contributions of the two butanol-forming pathways. Here we report that the direct butanol-forming pathway is a better channel to optimize for butanol production through metabolic flux and mass balance analyses. Butanol production through the hot channel was maximized by simultaneous disruption of the pta and buk genes, encoding phosphotransacetylase and butyrate kinase, while the adhE1D485G gene, encoding a mutated aldehyde/alcohol dehydrogenase, was overexpressed. The ratio of butanol produced through the hot channel to that produced through the cold channel increased from 2.0 in the wild type to 18.8 in the engineered BEKW(pPthlAAD**) strain. By reinforcing the direct butanol-forming flux in C. acetobutylicum, 18.9 g/liter of butanol was produced, with a yield of 0.71 mol butanol/mol glucose by batch fermentation, levels which are 160% and 245% higher than those obtained with the wild type. By fed-batch culture of this engineered strain with in situ recovery, 585.3 g of butanol was produced from 1,861.9 g of glucose, with the yield of 0.76 mol butanol/mol glucose and productivity of 1.32 g/liter/h. Studies of two butanol-forming routes and their effects on butanol production in C. acetobutylicum described here will serve as a basis for further metabolic engineering of clostridia aimed toward developing a superior butanol producer. IMPORTANCE Renewable biofuel is one of the answers to solving the energy crisis and climate change problems. Butanol produced naturally by clostridia has superior liquid fuel characteristics and thus has the potential to replace gasoline. Due to the lack of efficient genetic manipulation tools, however, strain improvement has been rather slow. Furthermore, complex metabolic characteristics of acidogenesis followed by solventogenesis in this strain have hampered development of engineered clostridia having highly efficient and selective butanol production capability. Here we report for the first time the results of systems metabolic engineering studies of two butanol-forming routes and their relative importances in butanol production. Based on these findings, a metabolically engineered Clostridium acetobutylicum strain capable of producing butanol to a high titer with high yield and selectivity could be developed by reinforcing the direct butanol-forming flux. Renewable biofuel is one of the answers to solving the energy crisis and climate change problems. Butanol produced naturally by clostridia has superior liquid fuel characteristics and thus has the potential to replace gasoline. Due to the lack of efficient genetic manipulation tools, however, strain improvement has been rather slow. Furthermore, complex metabolic characteristics of acidogenesis followed by solventogenesis in this strain have hampered development of engineered clostridia having highly efficient and selective butanol production capability. Here we report for the first time the results of systems metabolic engineering studies of two butanol-forming routes and their relative importances in butanol production. Based on these findings, a metabolically engineered Clostridium acetobutylicum strain capable of producing butanol to a high titer with high yield and selectivity could be developed by reinforcing the direct butanol-forming flux.

Metabolic engineering of Clostridium acetobutylicum for the enhanced production of isopropanol-butanol-ethanol fuel mixture

Butanol is considered as a superior biofuel, which is conventionally produced by clostridial acetone‐butanol‐ethanol (ABE) fermentation. Among ABE, only butanol and ethanol can be used as fuel alternatives. Coproduction of acetone thus causes lower yield of fuel alcohols. Thus, this study aimed at developing an improved Clostridium acetobutylicum strain possessing enhanced fuel alcohol production capability. For this, we previously developed a hyper ABE producing BKM19 strain was further engineered to convert acetone into isopropanol. The BKM19 strain was transformed with the plasmid pIPA100 containing the sadh (primary/secondary alcohol dehydrogenase) and hydG (putative electron transfer protein) genes from the Clostridium beijerinckii NRRL B593 cloned under the control of the thiolase promoter. The resulting BKM19 (pIPA100) strain produced 27.9 g/l isopropanol‐butanol‐ethanol (IBE) as a fuel alcohols with negligible amount of acetone (0.4 g/l) from 97.8 g/l glucose in lab‐scale (2 l) batch fermentation. Thus, this metabolically engineered strain was able to produce 99% of total solvent produced as fuel alcohols. The scalability and stability of BKM19 (pIPA100) were evaluated at 200 l pilot‐scale fermentation, which showed that the fuel alcohol yield could be improved to 0.37 g/g as compared to 0.29 g/g obtained at lab‐scale fermentation, while attaining a similar titer. To the best of our knowledge, this is the highest titer of IBE achieved and the first report on the large scale fermentation of C. acetobutylicum for IBE production.

Inactivating effect of phenolic unit structures on the biodegradation of lignin by lignin peroxidase from Phanerochaete chrysosporium

An imbalance of electron in an intramolecular electron transfer pathway was identified as the central factor causing inefficient degradation of lignin by the lignin peroxidase H8 from Phanerochaete chrysosporium (LiPH8). It was elucidated that dimeric lignins or monolignolic analogs containing free-hydroxyl phenolic groups were not only favorable substrates for the reduction of LiPH8 but also strong inhibitors depressing the enzymatic degradation of lignin. The data collectively demonstrated that disturbing the interaction between the free OH group on the phenolic structure and the surface active sites around Trp171 caused the primary deficiency in electron transport between Trp171 and the heme site, which severely inhibited the efficiency of lignin biodegradation by LiPH8/H2O2.

ABE production from yellow poplar through alkaline pre-hydrolysis, enzymatic saccharification, and fermentation

ABE (acetone-butanol-ethanol) was produced through alkaline pre-hydrolysis, enzymatic saccharification, and fermentation using yellow poplar as a raw material. In alkaline pre-hydrolysis, 51.1% of the biomass remained as a residue. In the main woody components, the degrees of lignin and xylan removal were 94.3 and 62.0%, respectively. A yield of 80.9% for cellulose-to-glucose and 81.2% for xylan-to-xylose were obtained by enzymatic hydrolysis. The sugar composition of enzymatic hydrolysate was 95.1 g/L of glucose and 21.4 g/L of xylose. The enzymatic hydrolysate also contained 0.5 g/L of acetic acid and 0.5 g/L of total phenolics. Furfural and 5-hydroxymethylfurfural (5-HMF) were not detected in this hydrolysate. The yellow poplar hydrolysate (YPH) from enzymatic saccharification was used for the production of ABE using Clostridium acetobutylicum and C. beijerinckii. In YPH fermentation, C. acetobutylicum produced 18.1 g/L total ABE (productivity 0.38 g/L h, and yield 0.42), and C. beijerinckii produced 12.1 g/L (productivity 0.25 g/L h, and yield 0.37). Although the ABE productivity by C. beijerinckii was slightly low, the general performance of ABE fermentation in YPH was similar to or higher than those reported previously. Therefore, alkaline pre-hydrolysis could be a very effective pretreatment step prior to enzymatic hydrolysis.

Kinetic Study of Metabolic Pathways in Clostridium Acetobutylicum

Abstract To develop a commercial fermentation process producing butanol, a kinetic model describing the metabolism in an acetone-butanol-ethanol (ABE)-producing by Clostridium acetobutylicum ATCC 824, was proposed by modifying the previously proposed metabolic pathways. We used an efficient optimization algorithm combining a genetic algorithm and the Levenberg-Marquardt algorithm in order to estimate the kinetic parameters of the model with experimental data obtained from a batch fermentor because of the complexity of the model equations. To evaluate the kinetic parameters estimated with the fermentation experiments using C. acetobutylicum ATCC824, an additional experiment was carried out using the modified clostridium, where pathway deletion was introduced. Thus, this kinetic study will be contributed to designing C. acetobutylicum for enhanced ABE production.

Kinetic Studies on Biobutanol Recovery Process Using Adsorbent Resin

Abstract Adsorption of 1-butanol using adsorbent resin is considered as an energy-efficient method to recover 1-butanol from an acetone-butanol-ethanol (ABE) fermentation broth. To develop the adsorption process, kinetic studies were carried out on adsorption of fermentation products using poly-(styrene- co -divinylbenzene) adsorbent resin. The kinetics for the adsorption of each component were represented in the form of the Langmuir equation and the kinetic parameters were then estimated based on the experimental data. For the parameter estimation of the adsorption model, experiments have been carried out with a batch type slurry adsorption equipment and fermentation model broth, containing acetone, ethanol, 1-butanol, acetic acid, and butyric acid. It was found that the extended Langmuir model derived from single component adsorption can correctly predict the competitive adsorption of multi-component mixture. The developed kinetic models are validated by the experiment using the actual broth. This study is expected to contribute to designing a large-scale biobutanol recovery process.