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JOURNAL OF BIOSCIENCE AND BIOENGINEERING © 2006, The Society for Biotechnology, Japan

Vol. 102, No. 4, 323–327. 2006

DOI: 10.1263/jbb.102.323

Utilization of Shochu Distillery Wastewater for Productionof Polyunsaturated Fatty Acids and Xanthophylls

Using Thraustochytrid

Takashi Yamasaki,1 Tsunehiro Aki,2* Masami Shinozaki,2 Masahiro Taguchi,2

Seiji Kawamoto,2 and Kazuhisa Ono2

Department of Chemical and Biological Engineering, Sasebo National College of Technology, 1-1 Okishinmachi,Sasebo, Nagasaki 857-1193, Japan1 and Department of Molecular Biotechnology,

Graduate School of Advanced Sciences of Matter, Hiroshima University,1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan2

Received 19 May 2006/Accepted 14 July 2006

The industrial production of barley shochu, a distilled alcoholic beverage, results in distillerywaste that is currently incinerated or disposed of in landfills, causing environmental pollution.The supernatant of distillery waste contains organic matter such as proteins (~2.5%) and aminoacids (~0.2%). This study demonstrates that the utilization of distillery wastewater as a sole nitro-gen source enables a marine thraustochytrid, Schizochytrium sp. strain KH105, to propagate andaccumulate valuable lipids including docosahexaenoic acid (DHA) and astaxanthin. Under opti-mized culture conditions, the highest DHA and astaxanthin yields were obtained at 3.4 g/l and7.7 mg/l, respectively, after 4 or 5 d of cultivation in a 3-l jar fermentor. The chemical oxygen de-mand of the wastewater was reduced by 35%. About 67% of crude protein content and 85% oftotal free amino acid content also decreased in the culture supernatant. The thraustochytridtherefore serves to upgrade the distillery by-product to one containing value-added lipids forfunctional foods as well as to regulate the environmental contamination.

[Key words: shochu distillery wastewater, docosahexaenoic acid, astaxanthin, thraustochytrid, Schizochytrium]

Shochu is a distilled Japanese alcoholic beverage thatis produced from rice, barley, buckwheat, sweet potato, orsugar cane mainly in Kyushu, Okinawa, and the SouthernIslands of Japan. The production of shochu reached about958,000 kl in 2004 and demand for this beverage is increas-ing (1). During shochu production, shochu kasu (distillerywaste) with a high concentration of organic matter, of whichthe chemical oxygen demand (COD) ranges between 60,000and 90,000 mg/l, is generated. About 15 t of waste is pro-duced from the production of 10 kl of shochu. Because near-ly 90% (w/w) of the waste comprises water, it can be incin-erated resulting in the release of carbon dioxide. A certainportion of the waste is thus incinerated, disposed of in land-fills, or dumped into the ocean, all of which cause environ-mental pollution. Therefore, an effective process for the treat-ment, recycling, or upgrading of shochu distillery waste ishighly desirable.

Because shochu distillery waste is thought to be biologi-cally safe and nutritionally natural, its utilization for the pro-duction of food-related materials has been attempted. For ex-ample, protease (2), chitosan (3), phenolic compounds witha caffeoyl group (4), vinegar with antitumor and some otherphysiological activities (5, 6), and a low-salt seasoning con-

taining γ-aminobutyric acid (7, 8) has been produced bybacterial or fungal fermentation in media containing thewastewater (the supernatant of distillery waste). The waste-water also contains useful bioactive substances such asgrowth-stimulating factors for chick muscle cells (9) andlactic acid bacteria (10), suggesting its possible prebioticeffect.

Here, we aim to utilize shochu distillery wastewater inthe production of polyunsaturated fatty acids and xantho-phylls using a marine eukaryotic protist, that is, a thrausto-chytrid. Thraustochytrids belong to the phylum Labyrin-thulomycota of the kingdom Chromista (11) and are charac-terized by the presence of an ectoplasmic net based at thesagenogenetosome, a multilayered cell wall composed main-ly of L-galactose, and a life cycle consisting of a vegetativecell, a zoosporangium, and a biflagellate heterokont zoo-spore (12, 13). Thraustochytrids contain the genera Th ra us to -chytrium and Schizochytrium, which has been known toproduce lipids rich in polyunsaturated fatty acids such aseicosapentaenoic acid (C20:5n-3), docosapentaenoic acid(C22:5n-6), and docosahexaenoic acid (C22:6n-3; DHA)(14–16). In addition, we previously reported that the DHA-producing Schizochytrium sp. strain KH105 also accumu-lates a significant amount of xanthophylls such as canthax-anthin and astaxanthin (17).

DHA is essential for the functional development and main-* Corresponding author. e-mail: aki@hiroshima-u.ac.jpphone/fax: +81-(0)82-424-7755

YAMASAKI ET AL. J. BIOSCI. BIOENG.,324

tenance of the human brain and retina (18) and shows manypositive effects on human diseases including hypertension,arthritis, atherosclerosis, depression, adult-onset diabetesmellitus, myocardial infarction, thrombosis, asthma, andsome cancers (19). Similarly, astaxanthin exhibits a widevariety of physiological activities such as active oxygenscavenging, photoprotection, and the suppression of lipidperoxidation, inflammation, and carcinogenesis (20). Astax-anthin is also useful for improving meat coloring and nor-mal hatching of cultured fish (21) that require DHA for theirgrowth (22). Thus, DHA, astaxanthin, and the microorgan-isms that produce them have important applications in thenutraceutical, cosmetic, food and feed industries. In thisstudy, the culture conditions of Schizochytrium sp. KH105were evaluated for the production of functional lipids andthe utilization of barley shochu distillery wastewater as amedium component.

MATERIALS AND METHODS

Microorganism and culture media Schizochytrium sp. strainKH105 (17) was maintained on a slant culture with GPY agarmedium, which contained 30 g/l glucose, 10 g/l Polypepton, 5 g/lyeast extract, and 15 g/l agar in 19.5 g/l artificial seawater (50%equivalent of natural seawater; Jamarine laboratory, Osaka). Seedculture medium contained 50 ml of 30 g/l glucose, 20 g/l polypep-tone, and 10 g/l yeast extract in 50% artificial seawater. Distillerywaste generated during shochu production using barley Hordeumvulgare was kindly provided by Iki Shochu Kyogyo Kumiai(Nagasaki). Shochu wastewater was obtained by centrifuging thewaste at 3000 rpm for 10 min, followed by filtrating the superna-tant with a paper filter (no. 1; Advantec, Tokyo). For growth opti-mization, the culture medium was prepared by adding variousamounts of glucose and artificial seasalts in the wastewater followedby the adjustment of pH with sodium hydroxide and autoclaving.Flask cultivation was performed by incubation for 48 h at 25°Cwith reciprocal shaking at 150 rpm.

Jar fermentor culture Fermentation was carried out in a 3-ljar fermentor (model MD-N 3L; B.E. Marubishi, Tokyo) equippedwith two disk turbine propellers with six wings each. The inocu-lum consisted of 200 ml of a 2-day-old culture grown in a flask.Cultivation was performed at 25°C with an agitation speed of500 rpm and an aeration rate of 2 vvm for a working volume of 2 l.Foaming was controlled by adding antifoam (PE-M; Wako PureChemical Industries, Osaka).

Analysis The cells were harvested from the culture broth bycentrifugation at 3000×g and dried at 105°C to measure dry cellweight. Cellular lipids were extracted from wet cells in a chloro-form/methanol mixture (2:1, v/v) and methyl-esterified with 10%hydrochloride in methanol with the addition of eicosanoic acid as aninternal standard (23). Fatty acid methyl esters were extracted withhexane and subjected to gas-liquid chromatography (AutosystemXL; Perkin Elmer Japan, Yokohama) on an apparatus equippedwith a capillary column (TC-70, 0.25 mm×30 m; GL Science,Tokyo) and a flame-ionization detector. Intracellular pigments wereextracted by suspending the harvested wet cells in an acetone/meth-anol mixture (7:3, v/v) and analyzed by thin-layer chromatogra-phy, which was performed on a silica gel plate (Kieselgel 60; Merck,Darmstadt, Germany) using acetone/hexane (3:7, v/v) as a devel-oping agent and authentic astaxanthin as a standard, followed byquantification with a densitometer (17). Glucose concentration inthe medium supernatant was determined using glucose oxidaseaccording to the literature (24). COD, biological oxygen demand(BOD) and the contents of carbon and nitrogen (total and formol

number) in the waste and wastewater were measured by the stan-dard methods (25, 26) and their values are shown in Table 1.

RESULTS

Effect of pH The effect of the initial pH of the culturemedium on the production of lipids was first examined be-cause the low pH of the barley shochu distillery wastewatermight affect microbial growth. In a flask scale cultivation,Schizochytrium sp. KH105 grew well in culture media con-taining 3.0 g/l glucose and 19.5 g/l artificial seasalts in thewastewater adjusted to an initial pH in the range of 6.0–8.0(Fig. 1). Over this effective pH range, the ratios of DHAcontent to biomass were almost constant (~10 mg/g-cell) andDHA accounted for 30–40% of the total amount of fattyacids. COD in the medium with an initial pH of 7.0 (76,000mg/l) decreased by 28% after the cultivation (55,000 mg/l).The maximum biomass and DHA content were observed at

TABLE 1. Composition of shochu distillery wasteand wastewater used in this study

Parameter Total waste Wastewater

pH 4.04 4.03Total nitrogen (%) 0.60 0.40Crude protein (%) 3.73 2.53Formol nitrogen (%) 0.14 0.15Biological oxygen demand (mg/l) 76700 47900Total carbon (mg/l) nd 30000Fatty acid (mg/l)C14:0 9.8 1.0C14:1 30.0 12.2C16:0 2241.6 19.6C16:1 3.1 0.0C18:0 0.0 2.6C18:1n-9 875.8 6.0C18:2n-6 2684.4 19.1

nd, Not determined.

FIG. 1. Effect of initial pH of growth medium on production ofDHA using Schizochytrium sp. KH105. The basal medium contained50% (v/v) shochu distillery wastewater. The initial pH of the mediumwas adjusted to the indicated values with sodium hydroxide prior toautoclaving. Open circles, DHA concentration; closed circles, totalfatty acid concentration; closed triangles, dry cell weight.

UTILIZATION OF SHOCHU DISTILLERY WASTEWATERVOL. 102, 2006 325

an initial pH of 7.5.Effect of glucose concentration Media containing the

shochu wastewater with various concentrations of glucosewere evaluated. As shown in Fig. 2, the strain KH105showed good propagation at an initial glucose concentrationof between 30 and 140 g/l, whereas a poor growth was ob-served in the absence of glucose. This indicated that the or-ganic compounds present in the wastewater (30 g/l; Table 1)were not sufficient as a carbon source to allow a full growthof the strain. Dry cell weight and the contents of total fattyacids and DHA reached the maximum at 80 g/l glucose, atwhich the DHA yield was about 100 mg/l.

Effects of shochu wastewater concentration Theeffect of the wastewater concentration on the lipid produc-tion was examined in the medium containing 80 g/l glucoseat pH 7.5. Figure 3 shows that the growth and DHA pro-

duction of the strain KH105 increased with an increasingamount of wastewater up to 50%, but did not change atwastewater concentrations higher than 50%. Therefore, in thesubsequent experiments, we used medium containing 50%wastewater, in which the working concentration of total ni-trogen (~2.0 g/l) was much higher than that (~0.1 g/l) in me-dium containing 1% polypeptone.

Cultivation in jar fermentor A batch cultivation in a3-l tank fermentor was carried out using a medium com-posed of 80 g/l glucose and 50% shochu distillery waste-water in artificial seawater, pH 7.5, that represented the 50%equivalent of natural seawater. Figure 4 shows a typical fer-mentation profile. The cell growth and total fatty acid con-tent increased until 4 d of cultivation but decreased there-after probably owing to the depletion of carbon sources. Theaccumulation of myristic acid (C14:0), which was a minorcomponent in the wastewater (Table 1), was observed,whereas no significant amount of linoleic acid (C18:2n-6)present in the wastewater at about 30% of the total fatty acidcontent was detected in the cultured cells. Cellular lipid alsocontained oleic acid (C18:1n-9) at about 15%, which wasthe same as the level in the wastewater. The maximum yield

FIG. 2. Effect of initial glucose concentration on DHA productionusing Schizochytrium sp. KH105. The basal medium (pH 7.5) con-tained 50% (v/v) shochu distillery wastewater. Open circles, DHAconcentration; closed circles, total fatty acid concentration; closed tri-angles, dry cell weight.

FIG. 3. Effect of concentration of shochu distillery wastewater onDHA production using Schizochytrium sp. KH105 . The basal medium(pH 7.5) contained 80 g/l glucose and the indicated concentration ofshochu distillery wastewater. Open circles, DHA concentration; closedcircles, total fatty acid concentration; closed triangles, dry cell weight.

FIG. 4. Time course of cultivation of Schizochytrium sp. KH105in 3-l jar fermentor. Culture medium (2 l) contained 80 g/l glucose,50% artificial seawater, and 50% barley shochu distillery waste-water, pH 7.5. The cultivation conditions are described in Materialsand Methods. Closed triangles, Dry cell weight; open circles, glucoseconcentration in culture supernatant; open triangles, COD in culturesupernatant.

YAMASAKI ET AL. J. BIOSCI. BIOENG.,326

of DHA obtained at 4 d of cultivation was 3.4 g/l (25.8% ofthe total fatty acid content; 115 mg/g-cell), which was almostequivalent to the DHA yield in a culture medium containingpolypeptone and yeast extract in a previous study (17).

The composition of the carotenoid pigments in KH105cells harvested at the end of the fermentation was deter-mined to be the following: β-carotene, 4.64 mg/l (24.8% ofthe total carotenoid content); echinenone, 1.13 mg/l (6.1%);canthaxanthin, 0.28 mg/l (1.5%); phoenicoxanthin, 4.95 mg/l(26.4%); astaxanthin, 7.69 mg/l (41.1%). Note that the con-tent ratio of astaxanthin to canthaxanthin was 27.1, which ismuch higher than that (0.2–1.6) obtained when polypeptoneand yeast extract were used as nitrogen and vitamin sourcesin place of the wastewater (17).

The COD of the medium supernatant decreased to 29,500mg/l as a result of fermentation for 5 d, but most of the resi-due was derived from the wastewater because glucose wasconsumed almost completely (Fig. 4). Because about 58%(45,700 mg/l) of COD in the initial medium (79,000 mg/l intotal) was due to the wastewater (data not shown), the de-crease in the COD derived from the wastewater was calcu-lated to be about 35%. About 67% of the crude protein con-tent and 85% of the total free amino acid content in themedium supernatant were reduced (Table 2). Regarding thenitrogen content, the decreases in total nitrogen content by67% and formol-titrated nitrogen content by 77% were ob-served.

DISCUSSION

Barley shochu distillery waste contains significant amountsof proteins and formol-type (free) amino acids (Table 1).Utilizing these nutrients in the propagation of industrial mi-croorganisms enables the application of wastewater to thefermentation of valuable compounds. We described in thisstudy that the barley shochu wastewater contains ingredi-ents utilizable by a marine thraustochytrid, Schizochytriumsp. strain KH105, which produces commercially importantlipids such as DHA and astaxanthin (17). The only mediumcomponents necessary for growth of the strain were glucoseand salts in addition to the distillery wastewater. This meansthat the conventionally used and relatively expensive nitro-gen and vitamin sources such as polypeptone, corn steep li-quor, and yeast extract can be replaced with the wastewater,which is available almost free of charge. Moreover, thewastewater contains water-soluble carbohydrates (Table 1).The lipid production by KH105 in the medium containingno glucose was nearly equivalent to that in the medium con-taining 30 g/l glucose despite the poor growth (Fig. 2). It is

desirable to achieve the optimization of culture conditionsand the breeding of the strain so that no glucose is neces-sary.

The cultivation of KH105 in a 3-l jar fermentor was car-ried out under the semi-optimized conditions and resulted ina maximum DHA production of 3.4 g/l. This value is equiv-alent or superior to those obtained in previous studies usingglucose yeast extract medium (17) and food waste (27, 28).Fan et al. (27) used bread crusts and brewing grain for DHAfermentation using Schizochytrium mangrovei and obtainedthe maximum DHA productions of 82 and 62 mg/l, respec-tively. Although okara powder (soymilk residue) was exam-ined as a substrate for several S. mangrovei strains, only amaximum of 72 mg/l DHA was obtained (28). Strains thatpotentially accumulate higher amounts of lipids and DHAmay be needed to improve the productivity.

An advantage of this study was that a Schizochytriumstrain producing xanthophylls in addition to DHA wasadopted as a biocatalyst. By cultivation of the strain KH105in a jar fermentor, 7.7 mg/l astaxanthin was obtained, whichwas higher than that obtained in our previous study usingthe same strain with medium containing polypeptone andyeast extract (17). Moreover, the ratio of astaxanthin to can-thaxanthin was about 17-fold higher than that in the previ-ous study. Considering the accepted biosynthetic pathwayof astaxanthin in other microorganisms (29) and the caro-tenoid composition in KH105 cells, canthaxanthin could beconverted into astaxanthin by hydroxylase in this strain (17).Some components in the wastewater may positively influ-ence this biosynthetic reaction, resulting in an increase inastaxanthin content. The identification and supplementationof positive factors will be advisable because astaxanthin hasattracted more interest as a functional food material thanother xanthophylls. Astaxanthin can be extracted simply bysuspending the cells with organic solvents in the case ofKH105, unlike other astaxanthin producers, for example,Haematococcus algae (30) and Phaffia yeasts (31), the per-sistent cells of which have to be mechanically disrupted forastaxanthin extraction. The simplification of the manufac-turing process as well as the use of cheap raw materials willbe a significant advantage in terms of lowering the cost ofthe industrial production of astaxanthin.

The other objective of this study is to decrease the dis-posal amounts of the shochu distillery waste-derived carbonand nitrogen compounds, which would be beneficial forthe preservation of the environment. The relative reductionrates of COD and total nitrogen content in the culture super-natant after the jar fermentation were about 35% and 67%,respectively (Fig. 4 and Table 2). The removal of free aminoacids was also significant. However, the COD decrease wassmaller than that in a previous study on the fermentation ofchitosan by the fungus Gongronella butleri using sweetpotato shochu wastewater without nutrient addition (49%)(3). This may be due to the supplementation of glucose thatwould have been preferentially assimilated by the cells. Us-ing a lower concentration of glucose and optimizing cultureconditions for the utilization of the carbon compounds inwastewater will resolve this problem. Through such im-provements in fermentation conditions, an industrial systemcompatible with the economical production of value-added

TABLE 2. Changes in concentrations of nitrogen, protein,and amino acids in shochu wastewater medium during

cultivation of Schizochytrium sp. KH105

ParameterCultivation period (d)

0 2 4

Total nitrogen (%) 0.16 0.11 0.05Crude protein (%) 0.99 0.67 0.32Formol nitrogen (%) 0.11 0.06 0.03Amino acids (mg/100 ml) 451.8 211.8 68.5

UTILIZATION OF SHOCHU DISTILLERY WASTEWATERVOL. 102, 2006 327

lipids and the recycling of shochu distillery waste will beestablished.

REFERENCES

1. Nikkan Keizai Tsushin: The beverage & food statisticsmonthly, p. 133. Nikkan Keizai Tsushin, Tokyo (2005). (inJapanese)

2. Morimura, S., Kida, K., and Sonoda, Y.: Production of pro-tease using wastewater from the manufacture of shochu. J.Ferment. Bioeng., 77, 183–187 (1994).

3. Yokoi, H., Aratake, T., Nishio, S., Hirose, J., Hayashi, S.,and Takasaki, Y.: Chitosan production from shochu distill-ery wastewater by funguses. J. Ferment. Bioeng., 85, 246–249 (1998).

4. Yoshimoto, M., Kurata-Azuma, R., Fujii, M., Hou, D. X.,Ikeda, K., Yoshidome, T., and Osako, M.: Phenolic com-position and radical scavenging activity of sweetpotato-de-rived shochu distillery by-products treated with koji. Biosci.Biotechnol. Biochem., 68, 2477–2483 (2004).

5. Morimura, S., Juan, Y. X., Shigematsu, T., and Kida, K.:Production of vinegar with high physiological activities fromrice-shochu distillery wastewater. Seibutsu-kogaku, 80, 417–423 (2002).

6. Seki, T., Morimura, S., Shigematsu, T., Maeda, H., andKida, K.: Antitumor activity of rice-shochu post-distillationslurry and vinegar produced from the post-distillation slurryvia oral administration in a mouse model. Biofactors, 22,103–105 (2004).

7. Yokoyama, S. and Tarumi, S.: Production and some prop-erties of low-salt seasoning from shochu distillery waste.Seibutsu-kogaku, 79, 211–217 (2001).

8. Yokoyama, S., Hiramatsu, J., and Hayakawa, K.: Pro-duction of γ-aminobutyric acid from alcohol distillery lees byLactobacillus brevis IFO-12005. J. Biosci. Bioeng., 93, 95–97 (2002).

9. Mahfudz, L. D., Nakashima, K., Ohtsuka, A., andHayashi, K.: Growth factors for a primary chick muscle cellculture from shochu distillery by-products. Biosci. Biotech-nol. Biochem., 61, 1844–1847 (1997).

10. Furuta, Y., Takashita, H., Omori, T., Sonomoto, K.,Ishizaki, A., Shimoda, M., and Wada, H.: Growth-stimu-lating effect of shochu wastewater on lactic acid bacteria andbifidobacteria. Ann. NY Acad. Sci., 864, 276–279 (1998).

11. Cavalier-Smith, T., Allsopp, M. T. E. P., and Chao, E. E.:Thraustochytrids are chromists, not fungi: 18S rRNA signa-tures of Heterokonta. Phil. Trans. Royal Soc. London B, 346,387–397 (1994).

12. Olive, L. S.: The Mycetozoans. Academic Press, New York,USA (1975).

13. Moss, S. T.: Biology and phylogeny of the Labyrinthulalesand Thraustochytriales, p. 105–129. In Moss, S. T. (ed.), Thebiology of marine fungi. Cambridge University Press, NewYork, USA (1986).

14. Yongmanitchai, W. and Ward, O. P.: Omega-3 fatty acids:alternative sources of production. Process Biochem., 24, 117–125 (1989).

15. Nakahara, T., Yokochi, T., Higashihara, T., Tanaka, S.,Yaguchi, T., and Honda, D.: Production of docosahexaenoicand docosapentaenoic acids by Schizochytrium sp. isolated

from Yap Islands. J. Am. Oil Chem. Soc., 73, 1421–1426(1996).

16. Huang, J., Aki, T., Yokochi, T., Nakahara, T., Honda, D.,Kawamoto, S., Shigeta, S., Ono, K., and Suzuki, O.:Grouping newly isolated docosahexaenoic acid-producingthraustochytrids based on their polyunsaturated fatty acidprofiles and comparative analysis of 18S rRNA genes. Mar.Biotechnol. (NY), 5, 450–457 (2003).

17. Aki, T., Hachida, K., Yoshinaga, M., Katai, Y., Yamasaki,T., Kawamoto, S., Kakizono, T., Maoka, T., Shigeta, S.,Suzuki, O., and Ono, K.: Thraustochytrid as a potentialsource of carotenoids. J. Am. Oil Chem. Soc., 80, 789–794(2003).

18. Connor, W. E., Neuringer, M., and Reisbick, S.: Essentialfatty acids: the importance of n-3 fatty acids in the retina andbrain. Nutr. Rev., 50, 21–29 (1992).

19. Horrocks, L. A. and Yeo, Y. K.: Health benefits of docosa-hexaenoic acid (DHA). Pharmacol. Res., 40, 211–225 (1999).

20. Guerin, M., Huntley, M. E., and Olaizola, M.: Haemato-coccus astaxanthin: applications for human health and nutri-tion. Trends Biotechnol., 21, 210–216 (2003).

21. Watanabe, T., Itoh, A., Murakami, A., Tsukashima, Y.,Kitajima, C., and Fujita, S.: Bull. Jpn. Soc. Sci. Fish., 50,1023–1028 (1984).

22. Hayashi, M., Matsumoto, R., Yoshimatsu, T., Tanaka, S.,and Shimizu, S.: Isolation of highly DHA-accumulatedLabyrinthulales and their utilization for nutritional enrich-ment of rotifers and Artemia. Nippon Suisan Gakkaishi, 68,674–678 (2002). (in Japanese)

23. Huang, J., Aki, T., Hachida, K., Yokochi, T., Kawamoto,S., Shigeta, S., Ono, K., and Suzuki, O.: Profile of poly-unsaturated fatty acids produced by Thraustochytrium sp.KK17-3. J. Am. Oil Chem. Soc., 78, 605–610 (2001).

24. Okuda, J., Miwa, I., Maeda, K., and Tokui, K.: Rapid andsensitive, colorimetric determination of the anomers of D-glu-cose with D-glucose oxidase, peroxidase, and mutarotase.Carbohydr. Res., 58, 267–270 (1977).

25. Committee, J. I. S.: Testing methods for industrial wastewa-ter, p. 36–38. Japanese Standards Association, Tokyo (1986).

26. Japan Soysauce Research Institute: Methods of soysauceexamination, p. 19–20. Committee for methods of soysauceexamination, Tokyo (1985).

27. Fan, K. W., Chen, F., Jones, E. B. G., and Vrijmoed,L. L. P.: Utilization of food processing waste by thrausto-chytrids, p. 185–194. In Hyde, K. D., Ho, W. H., and Pointing,S. B. (ed.), Aquatic mycology across the millenium, vol. 5.Fungal Diversity Press, Hong Kong, China (2000).

28. Fan, K. W., Chen, F., Jones, E. B., and Vrijmoed, L. L.:Eicosapentaenoic and docosahexaenoic acids production by andokara-utilizing potential of thraustochytrids. J. Ind. Microbiol.Biotechnol., 27, 199–202 (2001).

29. Misawa, N. and Shimada, H.: Metabolic engineering for theproduction of carotenoids in non-carotenogenic bacteria andyeasts. J. Biotechnol., 59, 169–181 (1998).

30. Kobayashi, M., Kakizono, T., and Nagai, S.: Astanxanthinproduction by a green alga, Haematococcus pluvialis, ac-companied with morphological changes in acetate media. J.Ferment. Bioeng., 71, 335–339 (1991).

31. Andrewes, A. G., Phaff, H. J., and Starr, M. P.: Carotenoidsof Phaffia rhodozyma, a red-pigmented fermenting yeast.Phytochemistry, 15, 1003–1007 (1976).