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.

For curbing the severe inhibition and toxicity of 1-butanol in a fermentor, which stand as one of the major hurdles on the way to commercialization of biobutanol production processes, an extractive fermentation process that can remove metabolites during the ferementation can be an effective solution. Among various separation techniques, adsorption using poly-(styrene-co-divinylbenzene) adsorbent resin is an effective and energy-efficient technique that holds much promise. In this paper, we have investigated the adsorption-and-desorption characteristics of the fermentation metabolites to aid the design of a new fermentation process equipped with an in situ or ex-situ butanol recovery capability. Specifically, the Langmuir equation and Ideal Adsorption Solution theory (IAST) have been used for developing an adsorption isotherm model, based on which a kinetic model of the adsorption process is developed. For the parameter estimation of the adsorption model, experiments have been carried out with a batch type slurry adsorption process processing a multiple-component mixture containing acetone, ethanol, 1butanol, acetic acid, and butyric acid. It is subsequently confirmed that the adsorption model developed with data from the experiments using the model broth adsorption can accurately predict the adsorption behavior of the actual fermentation broth. To ensure the practical applicability of the adsorption process, desorption experiments of the adsorbent resin have also been performed. It is found that approximately 95% of the adsorbates on the adsorbent can be recovered using 140 °C steam with the steam-to-adsorbent mass ratio of 1. This study on the adsorption-and-desorption characteristics is expected to contribute to designing a large-scale extractive fermentor for biobutanol production.

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.

A dynamic model for a fermentation process equipped with an ex situ butanol recovery (termed “ESBR” hereafter) system is proposed for continuous production of biobutanol. Since the proposed ESBR system integrates a fermenter with a stirred-tank-type adsorption column, the dynamic model includes kinetic models for both the fermentation (the Monod/Luedeking-Piret model) and the adsorption (the extended Langmuir model). Parameters in the kinetic models are initially determined using data from batch and fed-batch fermentation experiments with in situ butanol recovery (ISBR). The initially developed model is then used to find a feasible operating condition for an experimental ESBR system, and its parameter values are further tuned using experimental data from the proposed ESBR system for accurate predictions in the butanol and glucose concentration range seen in the ESBR operation. The approach to improving the model accuracy consists of two steps: (1) identifying the critical parameters by performing a sensitivity analysis and (2) re-estimating the selected parameters using data obtained during cyclic operation of the proposed ESBR system. Accordingly, the developed model based on the kinetics for both fermentation and adsorption can describe and predict the behavior of the proposed ESBR system. Thus, the proposed systematic approach provides a reliable platform for the optimal scale-up design and control studies of the ESBR system.