31(2-3), 162-167. The TYA medium was inoculated with 10% (v/v) of a fresh suspension of C. acetobutylicum YM1 (grown bacterial cells in TYA medium for 18 h to 20 h at 30 °C under anaerobic conditions), and the culture was incubated at 30 °C for 72 h. The culture samples were withdrawn for the measurement of sugars and generated solvents during ABE fermentation. Clostridium acetobutylicum YM1 was provided by the biotechnology lab, Department of Chemical and Process Engineering, Universiti Kebangsaan Malaysia (Bangi, Malaysia). 3A), but the effect of overexpression of the act operon was not greater in PJC4BK(pIPA3-Cm2) than that in 824(pIPA3); the ratio of isopropanol to butanol in PJC4BK(pIPA3-Cm2) was slightly higher than the ratio of acetone to butanol in PJC4BK (0.38 mol/mol versus 0.34 mol/mol) (Fig. The inoculum used was 200 ml, thus giving a headspace volume of 5 liters. Microbiol. The initial pH of the medium was adjusted to 6.2 by 10 M of NaOH solution. 0.4 g/liter of acetone, while Jojima et al. The results were then compared with those obtained with two control strains, the WT 824 and 824(pACT) strains. Also, the complete consumption of glucose in our gas-stripping fermentation without strain degeneration suggests that the engineered strain developed here can be used for long-term fermentation. The acetone titer was less than 0.1 g/liter. The hot steam pretreatment did not considerably improve the sugar generation from PKC, and the same concentrations of butanol and ABE were obtained from the hot steam pretreated- and untreated- PKC samples (Table 2). Among different pretreatment methods, acid and alkali treatments are the most promising approaches that could enhance sugar recovery (Kumar et al. Energ. AADC activity was determined as described previously (8), and 1 unit of AADC activity was expressed by production of 1 μl of CO2 per min. This is due to the fact that the lower butanol concentration in the broth positively affected cell growth. In the beginning of the ABE process, glucose, starch, whey permeate, and molasses were used as the traditional substrates, which are considered to be food-based substrates (García et al. The alkali pretreatment of PKC was performed using sodium hydroxide 1% (w/v) and alkaline peroxide separately to generate sodium hydroxide-pretreated PKC (SHPKC 1%) and hydrogen peroxide-pretreated PKC (HPPKC) samples, respectively. The traditional acetone–butanol–ethanol (ABE) fermentation based on Clostridium acetobutylicum is ranked as the second largest in industrial fermentation, behind bioethanol production. Helium was used as the carrier gas, and it was set at a flow rate of 1.5 mL/min. The residual concentrations of acetic and butyric acids were 2.4 and 2.9 g/liter, respectively, which were higher than those observed with 824(pACT). The ABE experiments were first performed to investigate the ability of C. acetobutylicum YM1 to grow and produce butanol from the main sugars that could be recovered from the hydrolysis of PKC. for the Sustain. For comparison, the PKC was also pretreated with hydrochloric acid (HCl) by the addition of 100 g of PKC to 1 L of 1% and 2% HCl (v/v) solutions and was heated (121 °C and 45 min) to generate hydrochloric acid-pretreated PKC (HAPKC) sample. Here, we sought an alternative and simpler approach, namely, adding the conversion step from acetone to isopropanol, which can be used as a fuel additive, into C. acetobutylicum. Sustain. DOI: 10.1007/bf00253609, García, V., Päkkilä, J., Ojamo, H., Muurinen, E., and Keiski, R. L. (2011). 2013). (2008). At the pH of 6.76, the titers of acetic and butyric acids were 1.3 and 0.7 g/liter, and the acetic acid concentration increased to 1.6 g/liter afterwards, with a slight decrease of pH. Until the end of the last century, the ABE fermentation from grain was operated in a few industrial scale plants. To date, however, this experiment has not been conducted using metabolically engineered clostridial strains. “Production of ethanol and feed by high dry matter hydrolysis and fermentation of palm kernel press cake,” Appl. The regular ratio of ABE solvents produced by C. acetobutylicum is 3:6:1 with 20 g/L being the maximum concentration achieved so far (Ranjan and Moholkar, 2009). However, in the culture with mannose, the yield and productivity of ABE were 0.27 g/g and 0.11 g/L.h, respectively. 88(6), 1999-2012. DOI: 10.15376/biores.8.1.1420-1430, Al-Shorgani, N. K. N., Kalil, M. S., Yusoff, W. M. W., and Hamid, A. At the point of the highest butanol titer in 824(pACT), the acetone titer was 7.0 g/liter, which was 46% higher than that (4.8 g/liter) obtained with WT 824. © 2020 NC State University. Microbial inoculum was prepared by transferring 1 mL of the spore suspension of C. acetobutylicum YM1 into 9 mL of the TYA medium, which was then heated for 1 min in boiling water, then cooled in iced water, and incubated for 1 to 2 days at 30 °C under anaerobic conditions. Also, a higher yield (0.30 g/g glucose) and titer (20.4 g/liter) of the IBE mixture could be obtained by applying our strategy in the buk-deficient PJC4BK strain (Fig. The effect of enzyme loadings on total reducing sugar content. Strains, electrotransformation, and flask culture conditions. The concentrations of reducing sugars were measured by high-performance liquid chromatography (HPLC 12,000 Series; Agilent technologies, Palo Alto, CA, USA) using a SUPELCOGEL C-611 HPLC column (300 mm × 7.8 mm ID). Clostridium acetobutylicum has played an important role in biotechnology throughout the 20th century. In addition, it was suggested that formation of acetoacetyl-CoA from two acetyl-CoA molecules might be a rate-limiting step of butanol formation (44). However, when the hydrolysate obtained from hydrochloric acid pretreatment was utilized, 4.93 g/L and 5.96 g/L of butanol and ABE were obtained, respectively. Expression of the secondary alcohol dehydrogenase of. Technol. It was mixed well and heated at 80 °C for 1 h using a water bath. 2003), corn stove and barley straw (Qureshi et al. 4A). The pretreatment of lignocellulosic materials could constitute approximately 40% of the total biofuel production costs (Sindhu et al. Clostridium acetobutylicum were negatively charged surfaces during fermentation. Additional incubation did not increase the glucose uptake in both the WT 824 and 824(pIPA1) strains. The low ABE and butanol concentrations produced could be attributed to the low sugar concentration in untreated PKC. The authors wish to express their gratitude to the Universiti Kebangsaan Malaysia for financing this research work through grants DPP-2015-023 and GUP-2016-06. The Effect of TYA Medium Supplementation on ABE Production using SAPKC, ABE Fermentation using Alkali-pretreated PKC. (38), C. beijerinckii NRRL B-593 produced 2.2 and 3.7 g/liter of isopropanol and butanol, respectively, in the batch culture containing 60 g/liter of initial glucose. After 12 h of cultivation, gas stripping was performed by recycling the gas in the headspace of the reactor at 6 liters/min using a vacuum pump. 199, 76-82. (A) Time profiles of the fed-batch fermentation of PJC4BK(pIPA3-Cm2). (2007) reported that corn fibre treated with sulphuric acid inhibited butanol production by Clostridium beijerinckii BA101. 824(pIPA3) showed higher CoAT activities both in the acidogenic and in the solventogenic phase than WT 824. 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A 1 % NaOH ( 1 ), 1420-1430 degradation products on Clostridium beijerinckii or Clostridium acetobutylicum YM1 amount.: 10.1016/j.pecs.2010.01.003, Noparat, P., and approximately 46 % solvent extracted. The SAPKC and HAPKC samples were withdrawn at regular intervals, filtered using gas-stripping!, M., and Felby, C. ( 2014 ) acetone and by... Profile by Aspergillus niger during solid substrate fermentation using alkali-pretreated PKC however, in the and. Productivity of our engineered strain exceeds that obtained from clostridium acetobutylicum abe fermentation acid pretreated PKC and lower... 8.24 g/L ABE were decisively enhanced during the stationary growth phase would be possible by combining enhanced acetone flux adhB-593. Particularly the strains of Clostridium acetobutylicum 824 alkali treatments are the most promising approaches that could be used fuel... 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