Biotechnology Letters 22: 16611665, 2000. 2000 Kluwer Academic Publishers. Printed in the Netherlands.

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Accelerated ethanol fermentation by Saccharomyces cerevisiae with addition of activated carbon

Toru Ikegamai1,2, , Hiroshi Yanagishita1, Dai Kitamoto1 & Kenji Haraya1
1 National 2 Kyushu

Institute of Materials and Chemical Research, 1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan National Industrial Research Institute, Shuku-machi, Tosu, Saga 841-0052, Japan Author for correspondence (Fax: +81-298-61-4674; E-mail: ikegami@home.nimc.go.jp)
Received 11 July 2000; Revisions requested 27 July 2000; Revisions received 25 August 2000; Accepted 1 September 2000

Key words: activated carbon, ethanol, fermentation, glucose consumption rate, yeast

Abstract Fermentation with the addition of activated carbon at 100 g l1 promoted the glucose consumption and ethanol production rates of Saccharomyces cerevisiae by 1.3 and 1.1 times, respectively. With fermentation using spent medium, the consumption rate was maintained at 90% of that in the fresh medium with the addition of activated carbon, while the rate without any addition decreased to about 70%.

Introduction Since the oil crises of the 1970s, the production of ethanol as an alternative fossil fuel energy resource has been a subject of great interest. A strong need therefore exists for efcient ethanol production. Recently, fermentation employing biomass resources as the carbon source has been attracting attention. Strategies for improving the productivity of ethanol are being undertaken based on the concepts of (1) maintaining the active microorganisms at a high density, because productivity is proportional to the biocatalyst concentration; and (2) integrating the effective recovery and concentration processes of ethanol from the fermenter. We have previously reported that the addition of carbon materials, especially activated carbon, is effective in promoting yeast growth in repeated cultivations using spent medium (Ikegami et al. 1998). Moreover, we have also reported that highly concentrated ethanol solutions are obtained by a pervaporation process as a continuous recovery and purication method, using the ethanol-selective silicalite membrane (Ikegami et al. 1997, 1999). Furthermore, it is desired to reuse fermentation broth for long periods of time in order to facilitate the reduction of the cost of raw materials.

The purpose of the present study was to elucidate the effects of the addition of various carbon materials on yeast ethanol fermentation, especially from the viewpoint of medium reuse.

Materials and methods Preparation of fermentation media The following media were used for ethanol fermentation in this study. Medium A: This medium was composed of 300 g glucose l1 , with no other added nutrients. Medium B-1: GP medium (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was used as the basal medium. The GP medium had the following composition (g l1 ): glucose 20, yeast extract 2, polypeptone 5, MgSO4 7H2 O 0.5, and KH2 PO4 1. The modied GP medium with 300 g glucose l1 was referred to as medium B-1. Media B-2 and B-3: After yeast cultivation in the modied GP medium with 100 g glucose l1 , as described a previous report (Ikegami et al. 1998), medium B-2 was prepared by adjusting the glucose concentration to 300 g l1 . Medium B-3 was prepared after carrying out yeast cultivation twice.

1662 Batch fermentation After autoclaving the fermentation media at 121 C for 20 min, 10 g commercially available dry bakers yeast (Oriental Yeast Co., Ltd., Tokyo, Japan) was added to 100 ml of the medium in a 500-ml glass bottle. The viability of cells was about 95%. In these experiments, the cell concentration was prepared at a high level in order to rapidly evaluate the effects of the carbon materials on ethanol fermentation. Batch fermentation was then initiated at 30 C with shaking (4 cm stroke, 130 rpm). Fermentations with the addition of sterilized carbon materials to the medium were performed in a similar manner. Carbon materials The properties of the carbon materials used in the experiments and their sources are listed in Table 1. The surface area of the carbon material was determined by the Brunauer-Emmett-Teller method. All of the carbon materials were sterilized by dry-heating at 170 C before use, as described in a previous report (Ikegami et al. 1998). Analysis The glucose and ethanol concentrations in the medium were determined by high-performance liquid chromatography as described in a previous report (Ikegami et al. 1998). The absorbancy of the fermentation broth from 200 to 400 nm was measured in order to follow the accumulation of nucleotides and proteins originating from inactive cells during the fermentation.
Fig. 1. Effect of the addition of activated carbon-1 to the fermentation medium on glucose consumption and ethanol production over time during fermentation. Fermentation medium: medium A. Weight of added activated carbon-1: 10 g/100 ml.

Results and discussion Effects of addition of carbon materials on ethanol fermentation Using medium A, the effects of the addition of the various carbon materials on the fermentation rate were initially investigated. By the addition of activated carbon-1 (AC-1), the glucose consumption and ethanol production rates were accelerated (Figure 1). The consumption rate was about 1.3 times that of the control. Although glucose molecules are adsorbed on activated carbon, they are fermented to ethanol by yeast cells (Ishibashi et al. 1983). Comparing the

ethanol concentrations when the residual glucose concentrations were equal (ca. 6.5% w/v), the ethanol concentration in the presence of AC-1 (ca. 12% v/v) was lower than that of the control (ca. 13% v/v). The adsorption amount of fermented ethanol by the addition of AC-1 was about 900 mg. Considering that 120 mg ethanol per g AC-1 was adsorbed with an initial ethanol concentration of 12% (v/v) in an ethanol/water mixture, this result was reasonable. The ethanol production rate in the medium increased by about 1.1 times compared to the control. Thus, it is assumed that the ethanol adsorption by the AC-1 contributed to the acceleration of fermentation. The glucose consumption rates are listed for the various carbon materials in Table 2. Although the consumption rate was not accelerated by the addition of coke or charcoal, the activated carbons had promotive actions. Figure 2 shows the relationship between the weight of carbon material added to the medium and the glucose consumption rate. The consumption rate was independent of the added weight of coke. On the other hand, the increase in the consumption rates resulting from the addition of AC-1 became less for the addition of 10 g or more. The addition of 10 g to the medium was enough for fermentation in this case. It has been reviewed that yeast fermentation is inhibited by dissolved CO2 (Jones et al. 1982). How-

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Table 1. Sources and properties of carbon materials. Carbon material Source Form Surface area (m2 g1 ) 0.1> 3.6 757 1080 1210 1480 Volume of mesopores from 5 to 10 nm (ml g1 )

Coke Charcoal Activated Activated Activated Activated

carbon-1 carbon-2 carbon-3 carbon-4

Petroleum Ubame oaka Coal Coal Coconut shell Coal

Granular Fragmentary Granular Granular Granular Granular

0.046 0.040 0.018 0.052

a Quercus phillyraeoides.

Table 2. Effects of the addition of carbon materials to fermentation medium on glucose consumption rate. Carbon material Glucose consumption rate (g l1 h1 ) From 0 to 4 h From 4 to 6 h 42 41 43 52 49 48 52 26 27 24 33 34 33 33

Control (without carbon) Coke Charcoal Activated Activated Activated Activated

carbon-1 carbon-2 carbon-3 carbon-4

Added carbon material: 10 g.

Fig. 2. Relationship between glucose consumption rate and weight of added carbon material.

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Table 3. Effects of the addition of activated carbon-1 to spent medium on glucose consumption rate. Glucose consumption rate (g l1 h1 ) B-1 B-2 04 h 46 h 04 h 46 h Control Addition of 10 g 20 g Pretreatmenta of spent media 42 47 55 27 30 38 39 47 55 45 22 27 27 23

B-3 04 h 30 43 50 42

46 h 21 29 29 28

a Ten g of the activated carbon-1 were immersed in the spent media for 7 h, and the

fermentations were then carried out after the removal of the carbon.

ever, it has also been reported that a decrease of dissolved CO2 caused by the addition of solid materials which act as nucleation sites for CO2 release resulted in the promotion of yeast growth and ethanol fermentation (Takezaki et al. 1993, Fukuda et al. 1996). The larger volume of the mesopores of activated carbon, the higher the consumption rate up to 4 h was on the whole (Table 2). Thus, it is assumed that the mesopores of activated carbon act as nucleation sites. Concerning the relationship between the ethanol fermentation rate and inorganic materials, it has been reported that porous ceramics promote the alcohol dehydrogenase activity of Saccharomyces cerevisiae, which generally reects ethanol prduction (Demuyakor & Ohta 1992). Moreover, Matsuhashi et al. (1995, 1996) have reported that the growth of Bacillus sp. was enhanced by sonic signals resulting from the added carbon materials. It is likely that the carbon materials used have the same action as those inorganic materials. Reuse of spent medium for ethanol fermentation by addition of carbon materials As one of the proposals to reduce the cost of raw materials, the reuse of spent medium was next investigated. Table 3 shows the effect of the addition of AC-1 on the glucose consumption rates in the spent media. In the control fermentation using medium B-3, the consumption rate up to 4 h decreased to about 70% of that in the case of medium B-1. The consumption rate in the presence of AC-1 using the medium B-3 was maintained at 90% of that in the case of medium B-1. Although it was also effective to pretreat the spent media (B-2 and B-3) with the AC-1, the addition of AC-1 was more effective for fermentation than the pretreatment.

Fig. 3. Ultraviolet spectra of spent medium B-3 during fermentation. (a) Fermentation was carried out without activated carbon-1 (AC-1). (b) Fermentation was carried out in the presence of AC-1. (c) AC-1 was added to the spent medium, and fermentation was then carried out after removal of the carbon. 1 The medium was diluted 500 times with distilled water. 2 The medium was diluted 10 times.

1665 The ultraviolet spectra of the spent media during fermentation are compared in Figure 3. A substance(s) having an absorption maximum at approximately 260 nm was accumulated over time during the fermentation (Figures 3a, c). Accumulation was also observed in fermentation using medium A (spectrum not shown). This substance(s) is currently assumed to consist of nucleotides from inactive cells during fermentation. On the other hand, such accumulation was not observed in the presence of AC-1 (Figure 3b). From these results, it is likely that ethanol fermentation was inhibited by the accumulation of the substance(s), and that the accelerated fermentation resulted from the removal of the substance(s) by the AC-1. Therefore, it is possible to reuse spent medium in the presence of AC-1. References
Demuyakor B, Ohta Y (1992) Promotive action of ceramics on yeast ethanol production, and its relationship on pH, glycerol and alcohol dehydrogenase activity. Appl. Microbiol. Biotechnol. 36: 717721. Fukuda T, Hiramatsu M, Sanmoto H, Fukuzaki S (1996) Discharge of dissolved carbon dioxide by the addition of activated carbon particles and its effect on yeast. J. Brew. Soc. Japan 91: 279283 (in Japanese). Ikegami T, Yamada Y, Ando H (1998) Acceleration of yeast growth by addition of carbon to cultivation medium. Biotechnol. Lett. 20: 673677. Ikegami T, Yanagishita H, Kitamoto D, Haraya K, Nakane T, Matsuda H, Koura N, Sano T (1997) Production of highly concentrated ethanol in a coupled fermentation/pervaporation process using silicalite membranes. Biotechnol. Tech. 11: 921924. Ikegami T, Yanagishita H, Kitamoto D, Haraya K, Nakane T, Matsuda H, Koura N, Sano T (1999) Highly concentrated aqueous ethanol solution by pervaporation using silicalite membrane Improvement of ethanol selectivity by addition of sugars to ethanol solution. Biotechnol. Lett. 21: 10371041. Ishibashi T, Saito S, Yamaguchi T, Ishida M, Odawara Y (1984) Alcohol production method from low concentration sugar solution. Japan Patent 1233023 (in Japanese). Jones RP, Greeneld PF (1982) Effect of carbon dioxide on yeast growth and fermentation. Enz. Microbiol. Technol. 4: 210223. Matsuhashi M, Pankrushina A N, Endoh K, Watanabe H, Mano Y, Hyodo M, Fujita T, Kunugita K, Kaneko T, Otani S (1995) Study on carbon material requirements for bacterial proliferation and spore germination under stress conditions: a new mechanism involving transmission of physical signals. J. Bacteriol. 177: 688693. Matsuhashi M, Pankrushina A N, Endoh K, Watanabe H, Ohshima H, Tobi M, Endo S, Mano Y, Hyodo M, Kaneko T, Otani S, Yoshimura S (1996) Bacillus carboniphilus cells respond to growth-promoting physical signals from cells of homologous and heterologous bacteria. J. Gen. Appl. Microbiol. 42: 315323. Takezaki M, Matsuura K, Hirotsune M, Hamachi M (1993) Effect of solids on the growth of yeast. J. Brew. Soc. Japan 88: 319325 (in Japanese).

Conclusions The effects of adding various carbon materials to a medium for ethanol fermentation were investigated in this study. Ethanol fermentation was accelerated by the addition of activated carbon. A signicant promotive effect of the addition of AC-1 on fermentation was observed in the case of reusing the spent media. These suggest the possibility of shortening the fermentation period, and reducing not only the cost of raw materials for fermentation but also the fermentation wastewater.

Acknowledgement The authors wish to sincerely thank Dr Okuyama (Mitsubishi Chemical Corporation Research Center, Yokohama, Japan) for generously providing carbon materials.