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Production of Butanol by Clostridium Acetobutyucum in Extractive Fermentation System

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Production of Butanol by Clostridium Acetobutyucum in Extractive Fermentation System PRODUCTION OF BUTANOL BY CLOSTRIDIUM ACETOBUTYUCUM IN EXTRACTIVE FERMENTATION SYSTEM Shigeo ISHII, Masahito TAYA and Takeshi KOBAYASHI Department of Chemical Engineering, Faculty of Engineering, Nagoya University, Nagoya 464 Key Words: Biochemical Engineering, Extractive Fermentation, Butanol Production, Aliphatic Alcohol, Clostridium acetobutylicum Anextractive fermentation system was developed to prevent end-product inhibition of Clostridium aceto- butylicum IAM 19012, which mainly produces butanol and acetone. Butanol exhibited greater toxicity to the microorganism than acetone, and its growth was completely inhibited above 10 kg/m3 of butanol. As an extracting solvent suitable for acetone-butanol fermentation, oleyl alcohol (cw-9-octadecen-l-ol) and C-20 guerbet alcohol (branched-chain alcohol of carbon number, 20) were selected from among29 organic compounds, based on their nontoxicity to the microorganism. These two solvents had high partition coefficients for butanol, and could be reused without deterioration. In fermentation with the solvent (solvent phase : aqueous phase=2 : 5 (v/v)), the viability of the microorganism was resumed by the liquid-liquid extraction of butanol from the broth, and the amount of butanol produced was 2.6 times that in fermentation without extraction. tation by liquid-liquid extraction. To date, a few Intr oduction studies on extractive fermentation have been carried Microbial production of acetone and butanol is a out for ethanol fermentation by yeast.5'10'18) traditional fermentation process. After World War II, The aim of the present study is to develop a new however, the fermentation process for acetone- strategy which combines both physical liquid-liquid butanol production was superseded by chemical extraction and biological fermentation processes, and synthetic processes using petroleum-based feed- furthermore to achieve improved production of bu- stocks, except for a fermentation plant in South Africa.15) tanol and acetone by extractive fermentation. There has recently been renewed interest in 1. Experimental acetone-butanol fermentation as a means of pro- 1.1 Microorganism ducing all or a portion of our future needs of Throughout the experiments, Clostridium aceto- these solvents, because of recent rises in oil prices butylicum IAM19012 (obtained from the collection and the feasibility of biomass utilization.1'2'4'8'9'11'13* of the Institute of Applied Microbiology, University However, the fermentation includes some limitations of Tokyo) was used. This strain was maintained in which delay an economic breakthrough;3'17* the at- a liquid potato medium(see below) and stored at tainable maximumconcentration of acetone and bu- 4°C. tanol is about 20 kg/m3. Above this concentration, the 1.2 Media and culture conditions fermentation completely ceases, owing to inhibitory The basal mediumfor experiments was the follow- effects of the products on the microorganism. As ing composition (per m3 of distilled water); Glucose: product yield is about 30%(w/w), based on substrate, variable; yeast extract: 6kg; tryptone: 10kg; feeds with a maximumsugar content of 60kg/m3 can KH2PO4: 2.5kg; MgSO4-7H2O: 0.25kg; Na2S2O4: be fermented in a batch operation. Therefore, the 3.5x 10~2kg; and resazurin: 1 x 10~3kg. The potato acetone-butanol fermentation requires very large re- mediumcontained (per m3of distilled water): potato actor volume, and high cost is involved in the sepa- extract (obtained from 750kg of potatoes crushed, ration of the products from fermentation broth. A boiled and filtered through cotton cloth); glucose: promising wayto overcomethese problems is extrac- 6kg; CaCO3: 2kg; and NH4C1: 1 kg. The media were tive fermentation in which toxic product(s) can be sterilized by autoclaving for lOmin at 120°C after continuously removed from broth during fermen- boiling under a stream of O2-free N2. Glucose so- lution was separately autoclaved and then added to Received July 19, 1984. Correspondence concerning this article should be addressed to T. Kobayashi. S. Ishii is on leave from WakayamaLaboratory, Kao Corp., Wakayama the sterile media under anaerobic condition. 640. Preparation of inoculum was as follows. After heat VOL 18 NO. 2 1985 125 shocking in boiling water for 1 min, 10 drops of stock culture were transferred to the potato medium(6 ml) in a test tube (12mm0x150mm) with a Pasteur pipette, followed by incubation for a day at 37°C. Inoculum size was 5% (v/v). All cultivations were carried out at 37°C under anaerobiosis, using a butyl- rubber stoppered test tube (15mm</> x 180mm; me- dium: 10ml), a screw-capped glass bottle (300ml; medium: 100ml) and a jar fermentor (Iwashiya Bio- Science, Type MB;medium: 800ml). Shaking (test- tube and glass-bottle cultivations: 50 rpm) and agitat- ing (jar-fermentor cultivation: 100rpm) were em- ployed in extractive fermentations. 1.3 Analyses Cell concentration was determined by measuring optical density at 570 nm with a Shimadzu Bausch & Fig. 1. Time course of acetone-butanol fermentation by C. LombSpectronic 20Acolorimeter, and evaluated on acetobutylicum IAM19012. The fermentation was carried out a dry basis with standard curves of cell weight against in a jar fermentor. optical density. Glucose concentration was measured enzymatically, using a Glucostat reagent kit (Worthington Biochemicals). The analyses of n- butanol, acetone, isopropanol, ethanol, ^-butyric acid and acetic acid were carried out by a ShimadzuGC- 7AGgas chromatograph equipped with a flame ion- ization detector,13) and the concentrations were calcu- lated using isobutanol (2-methyl-l-propanol) as an internal standard. The amounts of gas evolved from broth were determined by collection in a graduated cylinder containing NaCl-saturated solution (test- tube and glass-bottle cultivations) and with a Fig. 2. Effects of butanol and acetone on specific growth rate of C. acetobutylicum IAM19012. The cultivations were Shinagawa WKDi-0.5C wet-type gas meter (jar- carried out on a medium containing 5 kg/m3 of glucose in test fermentor cultivation). Gas volume was corrected and tubes. Butanol or acetone was added independently to the expressed as that at the standard condition mediumat the concentrations indicated. (1.01 x l05Pa and 0°C). 1.4 Chemicals after 22h. The solvent yield (weight of butanol, Dobanol (branched-chain alcohols of n= 12-13), acetone and ethanol produced/weight of glucose con- Oxocol (branched-chain alcohols of n = 14-15), C-16 sumed) was 0.25, which is comparable to those of guerbet alcohol (branched-chain alcohol of n= l6), other acetone-butanol fermentations.3'13'17) The pH Fine oxocol (branched-chain alcohol of n= \8) and profile exhibited a typical time course of acetone- C-20 guerbet alcohol (branched-chain alcohol of butanol fermentation; a sharp drop of pH was ob- n=20) were obtained from Kao Corp., and 1,1- served in the early stage, followed by a rise with dihydroheptafluoro-1-butanol, 1,1-dihydrotrideca- the disappearance of butyric acid. The fermentation fluoro-1-heptanol, Freon E (fluorinated ether of proceeded with smooth cell growth, gas evolution n=%) and octadecafluorodecalin were kindly pro- and glucose consumption up to 15h. Afterwards, vided by Drs. H. Muramatsu and T. Ueda. The however, the fermentation rate gradually slowed other chemicals used were of reagent grade. down and finally became almost zero. This reduction 2. Results of the fermentation rate appeared to result from end-product inhibitions, because it is well known 2.1 Fermentation process and inhibitory effects of that each fermentation product is toxic to C. metabolic products on the microorganism acetobutylicum?A 1 A5) Figure 1 shows the result of a typical batch fermen- To determine the degree of product inhibition, the tation by C. acetobutylicum IAM19012. The strain specific growth rates of the microorganism were consumed 29 kg/m3 of glucose during 22 h, producing examined in the presence of various concentrations of 5kg/m3 ofbutanol, 2kg/m3 of acetone and 1.7kg/m3 acetone or butanol. As shown in Fig. 2, butanol of butyric acid. Acetic acid (0.98 kg/m3) and ethanol exhibited greater toxicity than acetone, and the (0.ll kg/m3) were also detected as minor products growth of this strain was completely inhibited 126 JOURNAL OF CHEMICAL ENGINEERING OF JAPAN Table 1. Effects of various solvents on gas evolution for C. acetobutylicum IAM19012 Solvent Relativeevolution [-] gas Solvent Relativeevolutiongas[-] 1 None 1 C-16 Guerbet alcohol (16) 1 1-Pentanol (5) 0.2 Fine oxocol (18) 1-Hexanol (6) 0.1 Oleyl alcohol (ds-9-octadecen-l-ol) (18) 0.9 1-Octanol (8) 0.2 C-20 Guerbet alcohol (20) 1 1-Decanol (10) 0.1 Ethyl caproate (8) 0.1 1-Dodecanol (12) 0.2 Ethyl salicylate (9) 0.1 1-Tridecanol (1 3) 0.2 0.1 0.1 Dipentene (4-isopropenyl- l-methylcyclohexene) (10) 2-Methyl- l-pentanol (6) Benzyl benzoate (14) 0.2 2-Octanol (8) 0.1 Okie acid (ds-9-octadecenoic acid) (18) 0.9 2-Ethyl-l-hexanol (8) 0.1 Isostearic acid (16-methylheptadecanoic acid) (18) 1 2-Decanol (10) 0.2 Ricinoleic acid (12-hydroxy-cw-9-octadecenoic acid) (1 8) 0.1 2-Tridecanol (1 3) 0.1 1 , 1 -Dihydroheptafluoro- 1 -butanol (4) 0.1 2,4,6,8-Tetramethyl- l-nonanol (1 3) 0.1 1 , 1-Dihydrotridecafluoro- 1-heptanol (7) 0.1 Dobanol (12-13) 0.1 Freon E (8) 1 Oxocol (14-15) 0.6 Octadecafluorodecalin ( 1 0) 1 Thefigures in parentheses showthe carbon numbern of each compound.The cultivations with the test tubes were carried out for 48 h on the medium (glucose: 5 kg/m3), to which each solvent (1 ml) was added, with vigorous agitation every several hours. Table 2. Emulsibilities and partition coefficients (at 37°C) of some solvents Solvent Emulsibility mBT[-] mA[-] Freon E 0.31 0.74 0.20 Octadecafluorodecalin 0.65 0.12 0.74 Oxocol + C-16 Guerbet alcohol 4.7 0.089 0.022 Fine oxocol 4.5 0.44 ND Oleic acid 3.0 Oleyl alcohol 0.14 0.034 Isostearic acid ++ 3.0 0.29 0.047 C-20 Guerbet alcohol 4.3 (4.1) 0.52 (0.45) 0.22 + 2.2 0.15 ND 3.5 (3.2) 0.27 (0.31) 0.17 Key: + +, heavily emulsible; +, moderately emulsible; -, less emulsible; ND, not determined.
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