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 acetobutylicum 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the fermentation process for acetone- strategy which combines both physical liquid-liquid butanol production was superseded by chemical extraction and biological fermentation processes, and synthetic processes using petroleum-based feed- furthermore to achieve improved production of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 solution 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; medium: 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 employed 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. After 5ml of solvent and 5ml of water containing butanol (13 kg/m3), acetone (5 kg/m3) and ethanol (1 kg/m3) were equilibrated in test tubes by shaking (50 rpm) for 12 h at 37°C, the partition coefficients were determined. The figures in parentheses showthe partition coefficients determined by using cell-free broth containing butanol (5.2 kg/m3), acetone (2.9 kg/m3) and ethanol (0.07 kg/m3) as the aqueous phase.

above 10kg/m3 of butanol. Fine oxocol, C-20 guerbet alcohol, oleic acid, isoste- 2.2 Choice of nontoxic extracting solvents for aric acid, Freon E and octadecafluorodecalin. Table acetone-butanol fermentation 2 shows the partition coefficients of these solvents. Success in extractive fermentation depends ex- The coefficient mwas denned as the ratio of solute clusively on the choice of an extracting solvent suit- concentration in solvent phase to that in aqueous able for the fermentation system of interest. phase at equilibrium at 37°C. Amongthe solvents Generally, the following criteria should be considered tested, Oxocol, C-16 and C-20 guerbet alcohols, oleyl with respect to the solvent to be selected; (1) nontoxic alcohol, Fine oxocol and oleic acid had high partition to microorganism, (2) immiscible with water, (3) high coefficients for butanol. Oxocol, C-16 guerbet al- partition coefficient, (4) low viscosity and large differ- cohol, oleic acid and isostearic acid were too emul- ence in density from water, (5) less emulsible in sible to be separated from aqueous phase. In sub- aqueous phase, (6) sterilizable or autoclavable, and sequent experiments, oleyl alcohol and C-20 guerbet (7) commercially available at relatively low cost. alcohol, which were negligibly emulsible in aqueous As shown in Table 1, toxicities of 29 solvents, phase (Table 2), were chosen on the basis of the including aliphatic alcohols, higher fatty acids and criteria described above. fluorinated alcohols, to the microorganism were in- 2.3 Acetone-butanol fermentation with extracting vestigated using the amount of gas evolved as an solvent indicator. The following were found to be harmless Figure 3 shows time courses of gas evolution, which solvents: Oxocol, C-16 guerbet alcohol, oleyl alcohol, was a convenient indicator of the fermentation proc-

VOL 18 NO. 2 1985 127 Table 3. Reusabilities of extracting solvents Oleyl alcohol C-20 Guerbet alcohol Times t i. , . m*rRelativeL~Jevolution gas[-] mbti-\Relativeevolutiongas[-]

1st 4.1 0.9 3.2 1.0 2nd 3.9 1.0 3.0 1.2 3rd 4.0 1.1 3.1 0.96 4th 4.5 1.0 3.4 0.94 5th 4.5 1.0 3.3 1.1 6th 4.3 1.1 3.2 1.2

The recovered solvents were subjected to simple batch distil- lation for 4 h at 75-80°C under reduced pressure, prior to each use. The relative gas evolution was determined in a similar manner as in Table 1, and mBTwas also determined by using cell-free broth as the aqueous phase as shownin Table 2. Fig. 3. Time course of gas production in extractive fermentation with oleyl alcohol and C-20 guerbet alcohol. The shows the result of long-term fermentation where fermentations were carried out in screw-capped glass bottles containing 100ml of the medium (glucose: 20kg/m3) and oleyl alcohol was used as the extracting solvent. In the 50ml of the extracting solvent. first half of the fermentation, where oleyl alcohol was not added to the medium, the vitality of the microorganism was completely inhibited when butanol concentration reached about 5 kg/m3. This observation coincided with the result shown in Fig. 1. After 320ml ofoleyl alcohol were added to 800ml of the broth, the fermentation wasallowed to take place again, and only butanol and acetone were substantially detect- able in the phase of oleyl alcohol. At 146h, the concentrations of butanol and acetone in the aqueous phase were 4.8 and 6.4kg/m3, respectively, while those in the solvent phase were 20.3 and 2.1 kg/m3, respectively. Throughout the fermentation, 73 kg/m3 of glucose was consumed, and 12.9kg/m3 of butanol and 7.2kg/m3 of acetone were produced. The other products formed were as follows; Cell mass: 3.3kg/m3; gas evolved: 22.7m3/m3-broth; ethanol: 0.35kg/m3; butyric acid: 0.42 kg/m3; and acetic acid: 0.23 kg/m3. Fig. 4. Production of acetone and butanol by extractive fermentation with oleyl alcohol. C. acetobutylicum IAM In this case, the solvent yield was 0.28, which was 19012 was cultivated in a jar fermentor. The broken line about the same as that obtained in the fermentation indicates the time when oleyl alcohol (320ml) was added to without extracting solvent (Fig. 1). Moreover, a the fermentation broth (800ml). The aqueous and solvent similar result was obtained when C-20 guerbet alco- phases were separately agitated (100 rpm) so that they were hol was employed as the extracting solvent (data not completely mixed with each other. not shown). 2.4 Repeated use of extracting solvents ess, using oleyl alcohol and C-20 guerbet alcohol as Reusability of the solvents is important for extracting solvents in acetone-butanol fermentation. economical performance of the extractive fermen- In the absence of the solvent, gas evolution rate tation system. In this study, repeated uses of oleyl significantly decreased after 4 h. On the other hand, in alcohol and C-20 guerbet alcohol were attempted for the fermentation system with the extracting solvent six times, and the partition coefficients for butanol in (oleyl alcohol or C-20 guerbet alcohol), active gas cell-free broth and toxicities to the microorganism, evolution was still observed after 8h. Thus, it is which were evaluated by the amount of gas evolved, evident that the addition of these solvents prevented were investigated each time as shown in Table 3. With metabolic product inhibition. regard to the respective fermentations, no significant Subsequently, resumption of microbial viability changes in the partition coefficients and toxicities wasconfirmed by extractive fermentation. Figure 4 were observed in these runs. Therefore, qualities of

128 JOURNAL OF CHEMICAL ENGINEERING OF JAPAN Table 4. Somephysical properties of oleyl alcohol, C-20 guerbet alcohol and castor oil Oleyl alcohol C-20 Guerbet alcohol Castor oil

Specific gravity 0.85QJ0 0.84218 0.961-0.963j|;| Solidifying or melting point 6-7* _13** -10-18** Boiling point 182-184 152 (under 2.0 x lO2Pa) (under 1.3 x lO2Pa) 4.3 3.5 2.6 0.52 0.27 0.44 0.22 0.17 0.22 0.90 0.88 0.61

* Melting point. ** Solidifying point. The partition coefficients were determined in a similar manner as in Table 2 except that water containing butanol (15 kg/m3) and isopropanol (5 kg/m3) was used as aqueous phase to determine the mIP. The other data were obtained from the references19'20) and the catalogue of KaoCorp. both extracting solvents seemed not to be deteriorated knownthat manyorganic liquids have small partition through repeated use. coefficients in ethanol-water mixture.14) 3. Discussion In the present study, we developed the cultivation system of C. acetobutylicum IAM19012 with in situ The present interest in the acetone-butanol fermen- extraction of butanol, and proposed oleyl alcohol and tation originates from its feasibility of economic C-20 guerbet alcohol, characteristics of which are biomass utilization.1'2'4'8'9'11?13) The microbial proc- summarizedin Table 4, as suitable extracting solvents ess for butanol manufacture may again become a for the acetone-butanol fermentation. Both solvents source of valuable materials, which include: (1) sol- possessed large partition coefficients for butanol be- vent in chemical industry, (2) chemical feedstock, (3) tween 3.5 and 4.3, in contrast to those for acetone and fuel (blending with gasoline or methanol), (4) cosur- ethanol (0.17-0.52); these facts may make it possible factant in micellar flooding (tertiary oil recovery) and to extract specifically butanol out of the aqueous so on. broth. In the acetone-butanol fermentation by C. aceto- Itsumi et al.6) tried to enhance carbohydrate utili- butylicum, generally, nearly twice as much butanol zation by addition of castor oil to a culture broth of as acetone is produced.17) This was also confirmed as C. acetobutylicum. However, castor oil has undesir- shown in Fig. 1. However, it has been shown that able properties as a candidate for extracting solvent in butanol causes damage to cell membranesystems in the acetone-butanol fermentation (Table 4): (1) diffi- C. acetobutylicum, especially to membranetransport culty in separating the oil phase from the aqueous systems and enzymes.12'16) In the present study, it was phase because of its emulsibility and high specific revealed that butanol was more harmful to C. ace- gravity, (2) low partition coefficient for butanol at tobutylicum IAM19012 than acetone (Fig. 2). Thus, 37°C (raBr=2.6), and (3) toxicity to the microor- accumulation of butanol seems to becomea primary ganism of ricinoleic acid (Table 1), which is a main limitation in microbial butanol production. These constituent of castor oil. circumstances closely resemble those of ethanol fer- The present extractive fermentation system using mentation by yeast, Saccharomyces cerevisiae. oleyl alcohol or C-20 guerbet alcohol seems to be Several workers5'10'18) carried out extractive fer- limited by the toxicity of acetone to the microor- mentation for ethanol production, in which ethanol is ganism, because these solvents extracted a smaller continuously removed from fermenting broth as it is amountof acetone in comparison with butanol (Table formed. They reported that aliphatic alcohols and 2). Costa et al.3) reported that 70kg/m3 of acetone paraffins of n=\2 or higher had low toxicities to resulted in the complete growth inhibition of C. Saccharomyces spp. Minier and Goma10)investigated acetobutylicum ATCC 824. It is known that the primary aliphatic alcohols of different chain lengths butanol ratio can be increased at the expense of as ethanol extractor, and preferentially used 1-dode- acetone under some fermentation conditions and that canol (or mixture of 1-decanol and 1-tetradecanol) for a variant of Clostridium sp. preferentially produces a the ethanol fermentation process. At the sametime, greater amount of butanol.17) In such cases, extractive however, they suggested that these solvents had only fermentation will be more useful for butanol small partition coefficients for ethanol below 0.35, production. which is extremely disadvantageous to separation There is an alternative process for microbial bu- efficiency of the solute by liquid-liquid extraction. It is tanol production: isopropanol-butanol fermentation

VOL 18 NO. 2 1985 129 by C. beijerinckii (formerly known as C. butylicum).1) =fermentation time As oleyl alcohol and C-20 guerbet alcohol had fairly high partition coefficients for isopropanol (ca. 0.9) as = specific growth rate shown in Table 4, these solvents would be effectively (Subscripts) applicable to the extractive fermentation for the A = refers to acetone isopropanol-butanol process. Taking account of these BA = refers to ^-butyric acid facts, it maybe possible to convert a greater amount BT = refers to «-butanol of sugar into butanol during one-batch extractive GL = refers to glucose IP = refers to isopropanol (2-propanol) fermentation where the extracting phase is renewed at X = refers to cell mass appropriate intervals. In fact, we could carry out a fed-batch culture of C. acetobutylicum IAM19012 in Literature Cited the extractive fermentation system for a long period. 1) Bahl, H., W. Andersch and G. Gottschalk: Eur. J. Appl. These results will be reported and discussed else- Microbiol. Biotechnol, 15, 201 (1982). where. 2) Clements, L. D., S. R. Beck and C. Heintz: Chem. Eng. Progr., November, 59 (1983). 4. Conclusion 3) Costa, J. M. and A. R. Moreira: "Foundation ofBiochemical Engineering," p. 501, American Chemical Society (1983). 1) Butanol, which is a main product in acetone- 4) Fan, L. T., K. C. Shin, B. Hong and N. H. Choi: Proceedings butanol fermentation, exhibited a greater toxic effect ofPACHEC, Seoul, p. 141 (1983). on C. acetobutylicum IAM19012 than did acetone. 5) Finn, R. K.: /. Ferment. Technol., 44, 305 (1966). 6) Itsumi, F. and M. Tomoeda: Nippon Nogeikagaku Kaishi, 19, 2) An extractive fermentation system was de- 559 (1943). veloped in order to remove inhibiting butanol from 7) Krouwel,P. G.,W. J. Groot,N.W. F. KossenandW. F. M. the fermentation broth. van der Laan: Enzyme Microb. Technol, 5, 46 (1983). 3) As extracting solvents suitable for the acetone- 8) Lenz, T. G. and A. R. Moreira: Ind. Eng. Chem. Process Des. butanol fermentation, oleyl alcohol and C-20 guerbet Dev., 19, 478 (1980). 9) Lin, Y. and H. P. Blaschek: Appl. Environ. Microbiol, 45, 966 alcohol were chosen, based on their nontoxicity to the (1983). microorganism, partition coefficient and so on. 10) Minier, M. and G. Goma: Biotechnol Bioeng., 24, 1565 4) In the fermentation with the solvent, it was (1982). confirmed that the viability of C. acetobutylicum IAM ll) Monot, F. andJ. M. Engasser: Biotechnol Lett., 5, 213 (1983). 19012 was resumed by the liquid-liquid extraction of 12) Moreira, A. R., D. C. Ulmer and J. C. Linden: Biotechnol Bioeng. Symp., No. ll, 567 (1981). butanol from the broth. 13) Nishio, N., H. Biebl and M. Meiners: /. Ferment. Technol, 61, 5) Both solvents could be recovered and reused 101 (1983). without substantial deterioration. 14) Roddy, J. W.: Ind. Eng. Chem. Process Des. Dev., 20, 104 (1981). Acknowledgments 15) Spivey, M. J.: Process Biochem., 13, 2 (1978). The authors are grateful to Drs. H. Muramatsuand T. Ueda, 16) Vollherbst-Schneck, K., J. A. Sands and B. S. Montenecourt: Government Industrial Research Institute, Nagoya, for supplying Appl Environ. Microbiol, 47, 193 (1984). the fluorinated compounds. One author (S.I.) wishes to thank Kao 17) Walton, M. T. and J. L. Martin: "Microbial Technology," Corp. for providing the opportunity to study this topic. This work 2nd ed., Vol. 1, p. 187, Academic Press, Inc. (1979). was supported in part by the Biomass Conversion Project of the 18) Wang, H. Y., F. M. Robinson and S. S. Lee: Biotechnol Ministry of Agriculture, Forestry, and Fisheries (BCP 85-V-l-l). Bioeng. Symp., No. ll, 555 (1981). 19) Weast, R. C. (ed.): "Handbook of Chemistry and Physics," Nomenclature 51st ed., The Chemical Rubber Co. (1970-1971). C = concentration in aqueous broth [kg/m3-broth] 20) Windholz, M., S. Budavari, L. Y. Stroumtsos and M. N. Fertig (ed.): "The Merck Index," 9th ed., Merck & Co., Inc. G = volume of gas evolved per m3 of aqueous broth (1976). [m3/m3 -broth] m = partition coefficient at 37°C [-] n = carbon numberof compound [-] (Presented in part at the 49th Annual Meeting of The Society of P = total amount of product formed per m3 of aqueous Chemical Engineers, Japan, at Nagoya, April, 1984.) broth [kg/m3 -broth]

130 JOURNAL OF CHEMICAL ENGINEERING OF JAPAN