JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 87, No. 5, 642-646. 1999

Investigation of the Utility of Pineapple Juice and Pineapple Waste Material as Low-Cost Substrate for Ethanol Fermentation

by Zymomonas mobilis KENJI TANAKA, ZAKPAA D. HILARY, AND AYAAKI ISHIZAKI*

Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, 6-10-I Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

Received 19 October 19981Accepted 15 February 1999

The utility of the juice of rotten or discarded pineapples and the waste material of the production of pineapple juice as low-cost substrates for ethanol production by Zymomonas mobilis was investigated. Z. mobilis ATCC 10988 produced 59.0gX1 ethanol in undiluted pineapple juice without nutritional supplementation and without the regulation of the pH while 42.5 g-Z-’ ethanol was obtained using a 125 g.Z-l sucrose medium supplemented with 10 g-Z-l yeast extract and mineral salts. Ethanol fermentation using uuhydrolyzed and en- zymatically hydrolyzed pineapple waste material was also investigated under various culture conditions. When a 15% (v/v) dilution of unhydrolyzed waste material without nutritional supplementation was used, more than 3.5 g-l-l ethanol was produced. When the media containing 15,30, and 40% (v/v) of the hydrolyzate consisting of a 60% (v/v) suspension of pineapple waste material were used, final concentrations of ethanol were 5.0 g- Z-l, 7.6 gel-l, and 9.3 g-l-l, respectively. These results suggest that pineapple juice and the waste material can be useful low-cost substrates for ethanol production by Z. mobilis without supplementation with expensive organic nitrogen complexes such as yeast extract and without the regulation of the pH during cultivation, leading to the reduction in the production costs.

[Key words: ethanol fermentation, Zymomonas mobilis, pineapple juice, pineapple waste material]

Some of the most important issues for the production of ethanol as a biofuel on a commercial scale are the production costs, the availability of a carbon source and the availability of other nutrients required for the growth of microorganisms used. The ethanol-producing bacterium, Zymomonas mobilis, utilizes glucose, fruc- tose or sucrose as an energy source but the growth of this anaerobic bacterium is restricted in simple synthetic culture media. Z. mobilis requires complex organic nitrogen sources and/or growth factors for satisfactory cell growth (1). The first study of the nutritional require- ments of Z. mobilis was carried out by Belaich and Senez (2). Cell growth rate and biomass yield in synthetic cul- ture media including amino acids or NH&l were only half of the values obtained in a natural medium includ- ing yeast extract. Attempts to restore the cell growth rate of Z. mobilis by supplementing the synthetic medium with various kinds of nutritional compounds were unsuc- cessful. Therefore, the development of an economical fermentation process using cheap carbon sources and organic nitrogen compounds is essential for the produc- tion of biofuel ethanol on a commercial scale. Tripet- chkul et al. investigated the feasibility of using the natural rubber serum powder (NRSP), the spray-dried waste material obtained from the refining process of latex rubber, and soybean protein hydrolyzate (Mieki) as or- ganic nitrogen sources in ethanol fermentation (3). They showed that NRSP and Mieki could be a substitute for very expensive organic nitrogen complexes such as yeast extract. On the other hand, sago palm starch can be used as a carbon source for economical ethanol production (4).

The number of pineapple (Ananas comosus) produc-

* Corresponding author.

tion is increasing in tropical regions and 12.8 x 106 tons of pineapples were produced worldwide in 1997. Most pineapples are consumed as either fresh products or pro- cessed fruit (mainly canned) but only very high-quality fruit is selected for processing and shipment. Low-quality fruit is therefore left to rot on the farms due to the lack of a market. A large proportion of pineapples are processed into juice, leaving a large amount of unusable pulp as waste material. This pulpy waste material still contains a substantial amount of sucrose in addition to starch and hemicellulose. Therefore, it is anticipated that the juice from discarded fruit as well as the waste material can be utilized in an ethanol fermentation proc- ess.

The aim of this study is to investigate the feasibility of using the juice from discharged pineapples and waste material as low-cost substrates for ethanol fermentation by Z. mobilis ATCC 10988.

MATERIALS AND METHODS

Microorganism Z. mobilis ATCC 10988 was used throughout this study. Stock cultures were incubated in YM liquid medium (Difco Laboratories, Michigan, USA) for 18 h at 30°C and stored for up to 3 weeks in YM liquid medium at 4°C.

Sample preparation Samples of Del Monte pineap- ples were purchased from a supermarket and left to fully ripen at room temperature for 10 d. The pineapples were peeled and blended in a household juicer for 3 min. The juice was then extracted by filtering it through a double- fold of cotton gauze cloth, and particulate matter was removed by centrifuging at 10,000~g for 10min at 4°C. The undiluted juice and the pineapple waste material obtained from the extraction of juice were stored sepa-

642

VOL. 87, 1999 PRODUCTION FROM PINEAPPLE JUICE AND WASTE 643

rately at -20°C until use. The undiluted juice, which was used in our study, contained approximately 125.Og.I-’ sucrose and trace amounts of glucose and fructose. Fruc- tose and glucose concentrations of less than 5.0 g. I-’ were occasionally detected in the pineapple juice but these sugars were not detected in the pineapple juice that we used in the fermentation process by Z. mobilis. The undiluted pineapple juice also contained Mg, 120 rng.l-I; Ca, 160mg.I-1; P, 9mg.l-l; Fe, 3mg.l-‘; Na, l.Omg. 1-l; and K, 150mg.l-1.

Enzymatic hydrolysis of pineapple waste Meicel- lase, a commercial cellulase provided by Meiji Seika CO. Ltd. (Tokyo), was used for the batch-wise enzymatic hydrolysis of the pineapple waste material. A 60% (v/v) suspension of pineapple waste material was first pre- pared and the pH was adjusted to 4.8 with 5.0 M NaOH. Enzymatic hydrolysis of the waste was carried out at 50°C for 24 h. The enzyme was used at a protein concentra- tion of 0.3 mg/ml with a specific activity of 1.82 (units/ mg) in a filter-paper assay. The reaction was terminated by heating the waste suspension in boiling water for 10min.

termined by gas chromatography ((X-8 APE; Shimadzu Co. Ltd., Kyoto) equipped with a PEG column (80/100 mesh). The temperatures in the column oven and the injection room were set to 70°C and 9O”C, respectively. Glucose concentration was determined using a glucose analyzer (Model 23 A, Yellow Spring Instrument Co. Ltd., OH, USA). Sucrose concentration was determined using /?-fructosidase (Boehringer Mannheim, Germany). The supernatant of the culture broth was diluted with 2.0 M acetate buffer (pH 4.6) containing meicellase and the solution was then incubated at 56°C for 90min. The concentration of liberated glucose was determined by the glucose analyzer.

RESULTS

Preparation of culture media When unhydrolyzed pineapple waste material was used for the fermentation process, the waste material was appropriately diluted with distilled water before use. The pH was adjusted to 5.5 with 5.0 M NaOH and then the diluted waste material was autoclaved at 121 “C for 20 min. When enzymatically hydrolyzed waste material was used, the hydrolyzate was diluted with distilled water to give 40x, 30% and 15% (v/v) aliquots. Two hundred milliliters of each diluted hydrolyzate was transferred into 300-ml flasks with cot- ton stoppers. Three sets of flasks were prepared for each solution. The first set of flasks was not supplemented with any organic nutrients or mineral salts. The second set of flasks was supplemented with 5.Og.l-1 yeast ex- tract. The third set of flasks was supplemented with 0.5 g1 1-l MgS04. 7Hz0. Control cultures were also car- ried out using natural culture media containing sucrose or glucose as a carbon source. The natural media was composed of 10 g .I-’ yeast extract; 1 gel-’ KH2P04; 1 g.l-1 (NH& SO4 and 0.5 g. 1-l MgS04.7H20, and was autoclaved at 121°C for 20min before use. The pHs of all culture media were adjusted to 5.5 with 5 M NaOH, then autoclaved at 121 “C for 20 min.

Culture conditions A starter culture of Z. mobilis was prepared using 0.5 ml of the stock culture and to inoculate 1Oml of YM liquid medium in a test tube and then incubating at 30°C for 18 h without shaking. Ten milliliters of the broth was transferred to 40ml of YM medium and incubated for 18 h at 30°C. Cells were har- vested by centrifugation at 10,000 x g for 10 min at 4”C, then rinsed with 50ml of autoclaved distilled water and recentrifuged. The cells were suspended in 50 ml of autoclaved distilled water and used for flask culture and batch culture with an inoculum size of 10% (v/v). Kinet- ic studies on fermentation using pineapple juice were carried out by batch culture using a glass-jar fermentor with a total volume of 11. The working volume was 500 ml and the agitation speed was 200 rpm. The temper- ature was maintained at 30°C. In some cases, the pH was automatically maintained at 5.5 by the addition of 2.0 M NaOH with a pH controller (PHC-2201, Biott Co. Ltd., Tokyo). Investigations using pineapple waste were carried out using 300-ml conical flasks with cotton stop- pers at 30°C for 24 h.

Ethanol production from undiluted pineapple juice by Z. mobilis lo988 Ethanol production by Z. mobilis ATCC 10988 using undiluted pineapple juice was first investigated by batch culture. A control culture was also carried out using natural culture medium containing 125 g. 1-l as a carbon source. Figure 1 shows the time course of ethanol production. The pH was not con- trolled by the addition of alkali during cultivation. The ethanol production rate in the early phase of the culture using pineapple juice was relatively slow but rapidly increased after 12 h. Fermentation using pineapple juice was completed after 21 h. The final concentration of ethanol was 59.Og.l-1 while that using the 125 g.l-1 sucrose medium was 42.5 g. 1-l. Figure 2 shows the change in residual concentrations of glucose and sucrose in each cultivation. Sucrose was hydrolyzed to glucose and fruc- tose and then utilized by Z. mobilis ATCC 10988. In the cultivation using pineapple juice, sucrose in the culture liquid was exhausted by 15 h. However, approximately 3Og.l-’ ethanol was produced after 15 h (Fig. 1). On the other hand, in the cultivation using sucrose medium, sucrose was still present in the culture liquid at a concen- tration of 6.4 g+l-’ even after 27 h and approximately lOgal-’ ethanol was produced after sucrose consump- tion by the cells (15 h). The inconsistency between the time period required for ethanol production and that required for sucrose consumption in these two cultures is discussed later. The productivity of ethanol in the culti- vations using pineapple juice and sucrose medium was

Cultivation time (h)

Analytical methods Ethanol concentration was de-

FIG. 1. Time course of ethanol production in batch cultures of 2. mobilis ATCC 10988 using undiluted pineapple juice and natural culture medium without the regulation of pH. Symbols: A, undiluted pineapple juice; 0, natural medium containing 125 g .I-’ sucrose.

644 TANAKA ET AL. J. BIOSCI. BIOENG.,

o 3 6 9 12 15 18 21 24 27

Cultivation time (h)

FIG. 2. The changes in residual sugar concentration in batch culture of Z. mobilis ATCC 10988 using undiluted pineapple juice and natural culture medium without the regulation of pH. Symbols: A, undiluted pineapple juice; 0, natural medium containing 125 g.lPi sucrose. Solid lines indicate glucose concentration and broken lines indicate sucrose concentration.

2.81 and 1.57 g .1-l. h-l, respectively. In ethanol fermen- tation using sucrose as the sole carbon source, the theo- retical value for ethanol yield is 0.51 (g-ethanol/g- sucrose). The yield of ethanol in the batch culture using pineapple juice in our study was 0.47 (g-ethanol/g- sucrose), which corresponds to 92.4% of the theoretical ethanol yield. On the other hand, in the cultivation using 125 g . I- l sucrose medium, the ethanol yield was only 0.36 (g-ethanol/g-sucrose), which corresponds to 70.5% of the theoretical ethanol yield. In this study, the cell growth of 2. mobilis was not monitored because mea- surement of the optical absorbance of the culture broth was not possible due to the turbidity of the pineapple juice and the waste material. However, the fermentation results obtained in our study suggest that pineapple juice contains adequate amounts of organic nitrogen com- pounds which are essential for satisfactory cell growth of 2. mobilis. According to the report by Lozano-de- Gonzalez et al., filtered pineapple juice contains 17.1 g. 1-l protein, 2.6g.l-’ citric acid, 0.7 g.l-l ascorbic acid, and 0.6 g-l-’ phenolics (6). Therefore, it was expected that Z. mobilis can ferment the juice extracted from

0 3 6 9 12 15 16 21 24 27

Cultivation time (h) -0 3 6 21 24 27

FIG. 3. The changes in pH in batch cultures of Z. mobilis FIG. 4. Ethanol production in batch cultures of Z. mobilis ATCC ATCC10988 using undiluted pineapple juice and natural culture 10988 using undiluted pineapple juice with the regulation of pH by medium without regulation of pH. Symbols: A, undiluted pineapple automatic addition of 2 M NaOH. Symbols: n , ethanol concentra- juice; 0, natural medium containing 125 g. IPi sucrose. tion; A, sucrose concentration; A, glucose concentration.

TABLE. 1. The results of ethanol fermentation by Zmobilis ATCC 10988 using pineapple juices supplemented with 5.Og/l yeast

extract or 0.5 g/l MgS04.7HrO

Nutrients Fermentation supplemented

Ethanol yield referred time to theoretical yield

to pineapple juice (h) (%I 5.0 g/l yeast extract 21 97.7

0.5 g/l MgS04.7HzO 21 78.3

rotten or discarded pineapples as well as the waste material without supplementation with yeast extract. Figure 3 shows the change in pH during batch culture. In batch culture using the natural medium with 125 g-1-l sucrose, the pH decreased to 3.8 after 12 h of cultivation. In the culture using pineapple juice, the decrease in pH was very slow and the pH was higher than 4.8 throughout fermentation. It is thought that pineapple juice is strong- ly buffered against pH change, and that the organic nitrogen compounds and/or other nutrients contained in pineapple juice cause the increase in productivity and yield of ethanol yield from sucrose compared to those of the cultivation using sucrose medium.

The effects of nutritional supplementation and regula- tion of pH on ethanol production from pineapple juice It is known that magnesium ions are cofactors for a vari- ety of enzymes of the Entner-Doudoroff pathway (7). Furthermore, magnesium ions have the ability to restore the stability of outer membrane permeability of the cell, therefore the addition of magnesium ions is useful for the restoration of ethanol-damaged Z. mobilis cells and for the prevention of the damage due to the leakage of intermediates from cells. This is particularly important when ethanol concentration in the culture system is increased. It has also been reported that neither cell growth nor ethanol production of Z. mobilis was im- proved by the addition of MgS04. 7Hz0 to final concen- tration greater than 0.5 g. I-’ (8). Therefore, the effect of the supplementation of undiluted pineapple juice with 0.5 g. I-l MgS04.7Hz0 on ethanol production by Z. mobilis was investigated by batch culture without con- trolling pH. The effect of the addition of 5.0 g. 1-l yeast extract on ethanol fermentation was also investigated. The fermentation results are shown in Table 1. The use

VOL. 87, 1999 PRODUCTION FROM PINEAPPLE JUICE AND WASTE 645

TABLE 2. The effect of the supplementation of hydrolyzed waste with MgS04 or yeast extract on ethanol production by Z. mobilis

Aliquots of hydrolyzed Glucose concentration Nutritions supplemented to Ethanol concentration pineapple waste (g/O hydrolyzed waste (g/O

15% (v/v) 9.0 Not supplemented 5.0 15% (v/v) 9.0 0.5 g/l MgS04 5.1 15% (v/v) 9.0 5.0 g/l Yeast extract 5.5 30% (v/v) 18.0 Not supplemented 7.6 30% (v/v) 18.0 0.5 g/l MgS04 8.1 30% (v/v) 18.0 5.0 g/l Yeast extract 7.6 40% (v/v) 24.0 Not supplemented 9.3 40% (v/v) 24.0 0.5 g/l MgS04 10.7 40% (v/v) 24.0 5.0 g/l Yeast extract 9.6

Glucose medium 12.1 Natural medium 6.3

of yeast extract slightly increased the ethanol yield from the pineapple juice, however, the use of MgS04.7HzO somewhat decreased the ethanol yield. The effect of controlling pH during cultivation on ethanol production with undiluted pineapple juice was also investigated. The pH was maintained at 5.5 by the addition of 2.0 M NaOH automatically with a pH controller. The fermenta- tion profile of the batch culture using pineapple juice with controlling pH virtually corresponded to that of the batch culture without controlling pH (Fig. 4). This result is supported by the fact that there was no drastic de- crease in pH in cases using undiluted pineapple juice.

Ethanol production from pineapple waste by 2. mobi- lis The bulk of the waste material discarded from the pineapple juice manufacturing process is fruit pulp. This bulky waste is rich in fiber, carbohydrates and unextract- ed juice. Therefore, ethanol fermentation using the pineapple waste was also investigated. The waste is semi- solid, hence, flask cultures were carried out using both unhydrolyzed and enzymatically hydrolyzed waste materi- als. Figure 5 shows the concentrations of ethanol after 24 h of cultivation when various dilutions of unhydro- lyzed pineapple waste material were used. More than 3.5 g.l- l ethanol was produced in the culture using a 15% (v/v) dilution of unhydrolyzed waste material. In the control cultures using 5 g-1-l sucrose and 5 g.l-’ glu- cose media, ethanol production was 1.3 g-Z-l and 2.3 g. IPI, respectively. Ethanol production by Z. mobilis with hydrolyzed waste material was also investigated and the results are summarized in Table 2. When the media con- taining 15, 30, and 40% (v/v) of the hydrolyzate consist- ing of 60% (v/v) suspension of hydrolyzed pineapple waste material were used, the amounts of ethanol pro- duced were 5.Og.l-l, 7.6g.I-l, and 9.3g.lP1, respectively. The addition of 5.0 g .I-’ yeast extract to the hydrolyzed

5 g/1 Sucrose medium

5 g/l Glucose medium

2.5 %(v/v) Unhydrolyze pineapple waste 5.0 %(v/v) Unhydrolyze pineapple waste

7.5 %(v/v) Unhydrolyzed pineapple waste

0 1 2 ii Ethanol concentration (g/l)

FIG. 5. Ethanol production in flask cultures of Z. mobilis ATCC 10988 using various dilutions of unhydrolyzed pineapple waste material.

waste material did not promote ethanol production by Z. mobilis. On the other hand, the addition of MgS04. 7Hz0 to a final concentration of 0.5 g. I-’ into the hydro- lyzed waste solutions slightly increased ethanol produc- tion. The material balance for ethanol production in fer- mentation with hydrolyzed waste was not completely de- termined in this study. The percentage of pulpy waste in the pineapples used in this study was about 50% (w/w) and the waste material contained 80% (w/w) water and 10% (w/w) saccharides. The glucose concentration of the undiluted hydrolyzed waste material was 60.1 g .1-l. When a 60% (v/v) suspension of pineapple waste mate- rial was hydrolyzed and the medium containing 30% (v/v) of the hydrolyzate was used for fermentation, 7.6 g. 1-l ethanol was produced. Therefore, the ethanol yield in the case using the medium containing 30% (v/v) of the hydrolyzate was estimated to be 0.42 (g-ethanol/g-glu- case), which corresponds to 82.6% of the theoretical ethanol yield.

DISCUSSION

According to the calculation by Claude ef al., the quantities of ethanol and biofuel that can be produced from the pineapple crop per l-ha soil corresponds to 482 I/month and 386 m3/month, respectively (9). The quantity of ethanol produced is estimated to be marked- ly less than that produced by sugar cane (710 I/month) (10). However, when the juice of discarded rotten fruit and the waste material are used as the substrate for ethanol production, the production costs are considera- bly reduced. Alain et al. reported on the production of ethanol from pineapple juice by the most-productive yeast strains (11). When Saccharomyces cerevisiae var. sake was used, the maximum ethanol yield (89% of the theoretical yield) was obtained but the productivity was 1.44g.IP1.h-‘. When S. cerevisiae CM1 was used, the maximum ethanol productivity (2.22 g. f-l. h-l) was obtained but the ethanol yield was 77% of the theoretical yield. In our study using Z. mobilis ATCC 10988, the ethanol yield from pineapple juice was 92.4% of the the- oretical yield and the productivity was 2.81 g.l-I. h-1. Furthermore, it was shown that pineapple juice and the waste material contain a large amount of organic nitro- gen compounds which are essential for the growth of this microorganism, and that the controlling of pH by the addition of alkali need not to be performed. These results suggest that the juice from discarded pineapples and the waste material can be used as low-cost substrates for the production of biofuel ethanol by Z. mobilis.

The inconsistency between the time period required for ethanol production and that required for sucrose

646 TANAKA ET AL. J. BIOSCI. BIOENG.,

consumption, which was observed in cultivation using undiluted pineapple juice without the regulation of the pH (shown in Figs. 1 and 2), cannot be explained at present although one possibility is that fructose, which was liber- ated by the hydrolysis of sucrose, was still present in the culture liquid at a high concentration and Z. mobilis cells produced ethanol from the remaining fructose after 15 h. In actual that in cultivation using glucose as a sole carbon source, the time period of ethanol production was consistent with that of glucose consumption (data not shown). On the other hand, in cultivation using sucrose medium, about 10 g. 1-l ethanol was produced after the cells had almost exhausted the sucrose supply (after 15 h). In this case, one possibility may be that a large portion of remaining fructose was converted to prod- ucts other than ethanol. It is known that in ethanol fer- mentation by Z. mobilis, the ethanol yield from fructose is lower than that from glucose and that polysaccharides such as levans are produced from sucrose. However, we could not prove the evidence for the above speculation regarding the increase in ethanol concentration after the exhaustion of sucrose because the concentrations of fruc- tose, ethanol and other metabolites during fermentation were not monitored in our experiments.

The addition of magnesium sulfate to the hydrolyzed waste material slightly increased ethanol production by Z. mobilis. However, the reason why the ethanol yield decreased in the culture using pineapple juice which was supplemented with MgS04 to a final concentration of 0.5 g.I-’ is not clear at present. Our research into the use of pineapple waste in ethanol production continues.

REFERENCES

1. Swings, J. and de Ley, J.: The biology of Zymotnonas. Bac- teriol. Rev., 41, l-46 (1977).

2. Belaich, I. P. and Senez, J. C.: Influence of aeration and pan- tothenate on growth yields of Zymomonus. J. Bacterial., 89, 119.5-1200 (1965).

3. Tripetchkul, S., Tonokawa, M., and Ishizaki, A.: Ethanol production by Zymomonas mobilis using natural rubber waste as a nutritional source. J. Ferment. Bioeng., 74, 384-388 (1992).

4. Ishizaki, A.: Diversity utilization of sago palm starch as tro- pical bioresource, p. 251-252. In Yoshida, T. (ed.), Annual reports of ICBiotech, ~01.18. Faculty of Engineering, Osaka University, Osaka (1995).

5. Bartolome, A. P., Ruperez, P., and Fruster: Freezing rate and frozen storage effects on color and sensory characteristics of pineapple fruit slices. J. Food Sci., 61, 154-156 (1996).

6. Lozano-de-Gonzalez, P. G., Barrett, D. M., Wrolstad, R. E., and Durst, R. W.: Enzymatic browning inhibited in fresh and dried apple rings by pineapple juice. J. Food Sci., 58, 399-404 (1993).

7. Osman, Y. A. and Ingram, L. 0.: Mechanism of ethanol inhibi- tion of fermentation in Zymomonus mobilis CP4. J. Bacterial., 164, 173-180 (1985).

8. Park, S. C. and Baratti, J.: Batch fermentation kinetics of sugar beet molasses by Zymomonar; mobilk. Biotechnol. Bioeng., 38, 304-313 (1991).

9. Claude, P. Y., Lacoeuilhe, J. J., and Claude, T.: The pineap- ple, cultivation and uses, p. 474-477. In Coste, R. (ed.), Tech- niques agricoles et productions tropicales, vol. 37. G.-P. Maisonneuve & Larose, Paris (1985).

10. Carvalho, V. A., Clemente, P.R., Chitarra, M. I. F., and Carvalho, J. G.: Estudo dos components quicos de fruitos e parte vegetative do abacaxixeiro (A, comosw) visando approveita- mento industial. VI. Congress0 Brasileiro de Fruti cultura. Anais, 1, 156-162 (1981).

11. Alain, K., Georges, A. N., and Aka, Y.: Ethanol production from pineapple juice in Cote d’Ivoire with preselected yeast strains. J. Ferment. Technol., 65, 475-481 (1987).