Fermentative Production of Fumaric Acid by Candida hydrocarbofumarica n. sp.

Transcription

1 [Agr. Biol. Chem., Vol. 34, No. 5, p , 1970] Studies on the Utilization of Hydrocarbons by Microorganisms Part XVIII. Fermentative Production of Fumaric Acid by Candida hydrocarbofumarica n. sp. By Koichi YAMADA, Toshiro FURUKAWA and Tadaatsu NAKAHARA Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo Received September 8, 1969 Screening was carried out to obtain microorganisms which produce organic acids from n-paraffins. Three strains of yeast like microorganisms showed strong ability to produce fumaric acid. One of them turned out to be a new species and was named Candida hydro carbofumarica n. sp. Shaking culture of this microorganism was carried out in a medium containing 8% (v/v) n-paraffins, mineral salts (NH4Cl 0.3%, KH2PO4 0.05%, K2HPO4 0.05%, MgSO4. 7H2O 0.05%, FeSO4.7H2O 0.001%, MnSO4.4H2O %, ZnSO4.7H2O %, CaC %) 0.02% yeast extract and 4% CaCO3. We found that the yield of fumaric acid reached as high as 65% of n-paraffin added after 7 days cultivation. In these days, the development of petro chemical industries have provided the pos sibility of obtaining abundant quantities of n-paraffins in low cost. Concerning with the organic acids, researches on the fermentative production of ƒ -ketoglu taric acid from hydrocarbons using microorganisms were reported by Tsugawa et al.1 `3) and Tanaka et al.,4) and that of citric acid was presented by Abe et al.5) Screening was carried out to obtain from soil microorganisms which produce organic 1) R. Tsugawa, T. Nakase, T. Kobayashi, K. Yamashita and S. Okumura, Agr. Biol. Chem., 33, 158 (1969). 2) R. Tsugawa and S. Okumura, ibid., 33, 676 (1969). 3) R. Tsugawa, T. Nakase, T. Kobayashi, K. Yamashita and S. Okumura, ibid., 33, 929 (1969). 4) K. Tanaka, K. Kimura, T. Suzuki, K. Yama guchi and S. Kinoshita, J. Ferment. Technol., 47, 291 (1969). 5) U. Tanaka, Y. Tahara, T. Tabuchi and M. Abe, Annual meeting of the Agricultural Chemical Society of Japan, Tokyo, April, acids or other useful metabolites from n-para fiins. Three strains of microorganisms which produce large quantities of fumaric acid (ab breveated to FuA) were isolated. One of them turned out to be a new species and was named Candida hydrocarbofumarica n. sp. Although it has been known that some yeasts have ability to produce ƒ -ketoglutaric acid and/or citric acid from n-parafiins, the pro duction of FuA has not been reported. The present paper describes the methods and the results of the taxonomical studies of the microorganism (Et-15-2) and the determina tion of optimal conditions for production of FuA by this strain. MATERIALS AND METHODS 1) n-paraffin. Commercial product of n-paraffin mixture was used. The carbon composition of n- paraffrn was as follows; C10, 0.5%; C11, 1.6%; C12, 14.6%; C13, 42.0%; C14, 39.7% and C15, 1.6%. The carbon numbers of individual n-alkanes used were from 10 to 18, and purity of each n-alkane was

2 Studies on the Utilization of Hydrocarbons by Microorganisms. Part XVIII 671 over 98%. TABLE I. COMPOSITION OF THE ISOLATION MEDIA 2) Microorganism. A microorganism (Et-15-2) was isolated from soil. 3) Inoculum. Cells grown in liquid medium for two or three days at 30 C were used as an inoculum. 4) Methods of analysis 1) Growth of microorganisms. Growth of microorga nisms was estimated by measuring the optical density at 660mƒÊ of the culture broth diluted fold with dil. HCl by Nakahara's solvent.6) 2) Qualitative analysis of organic acids. Organic acids were determined qualitatively by paper chromato graphy using bromcresol green as a detective agent. FuA has UV absorption by it's double bond and de tected by UV ray. Toyo filter paper No. 51A was used. The composition of developing solvents were as follows; (A) n-butanol, 4; acetic acid, 1; water, 1, and (B) phenol, 4; water, 1. 3) The assay method of FuA. FuA was assayed by the method of oxidation with 0.1 N KMnO4 soln.7) RESULTS AND DISCUSSION 1) Isolation media and isolation method The media used for the isolation are shown in Table I. The isolation of hydrocarbon ph was adjusted to 6.5 by NaOH. utilizing microorganism from soil was carried out by the method of Yamada et al.8) The enrichment culture and plate culture were alternately repeated for the isolation of those microorganisms as shown in Fig. 1. 2) Identification of the acid The following experiments were carried out for the identification of the acid. The Rf values of the product were 0.85 and 0.62 with the solvent system A and B, and those of authentic FuA were 0.86 and 0.62 respectively. IR spectrum of the precipitates from culture broth agreed with that of authentic FuA as shown in Fig. 2. Elementary analysis of the product. Found: C, 41.90%; H, 3.38%. Cald. for C4- H4O4; C, 41.39%; H, 3.47%. 6) T. Nakahara, K. Hisatsuka and K. Yamada, J. Fermentation Association, Japan, 26, 413 (1968). 7) "Jikken Kagaku Koza," Vol. 25, Maruzen Co., L td., Japan, p ) K. Yamada, J. Takahashi, K. Kobayashi and Y. Imada, Agr. Biol. Chem., 27, 390 (1963). 3) Identification of the microorganism The characteristics of the microorganism (Et-15-2) are shown in Table II. From the experimental facts, our strain was determined to belong to genus Candida, however, there is no species (in Lodder and Kreger-van Rij's Manual) corresponding to our strain. This yeast resembles to Candida steatolytica, a new

3 672 K. YAMADA, T. FURUKAWA and T. NAKAHARA FIG. 2. Infrared Absorption Spectra of the Product and Authentic FuA in Nujol. TABLE II. DESCRIPTION OF Et-15-2: Candida hydrocarbofumarica n. sp. Growth in YM broth: After 3 days at 25 C, cells are round, short oval or oval, (2 `5)x(2 `7)p, and occur singly, in pairs or in chains. A thin, smooth pellicle and sediment are formed. Growth on YM agar: After 20 days at 25 C, the streak culture is smooth or slightly wrinkled, dull, soft, and has a ciliate margin. Sporulation: Absent. Dalmau plate culture on potato dextrose agar: Pseudo- and true mycelia develop abundantly. Blasto spores are round to oval, in chains, in verticils or in clusters. Potassium nitrate is not utilized as the sole nitrogen source. Arbutin is weakly splitted. Urease is not detected on Christensen's medium. Gelatin Vitamin is not liquefied. is not required. a) latent and weak b) latent TABLE III. COMPARISON OF C. steatolytica AND C. hydrocarbofumarica n. sp. yeast species reported by D. Yarrow,9) but as shown in Table III some properties are dif ferent between them. Authors propose a name Candida hydrocarbo- 9) D. Yarrow, Antonie van Leeuwenhoek, 35, 24 (1969).

4 Studies on the Utilization of Hydrocarbons by Microorganisms. Part XVIII 673 fumarica n. sp. for this strain. 4) Determination of the optimal conditions of FuA production by Candida hydrocarbofumarica n. sp. Shaking flasks (capacity about 500 ml) were employed for the experiment. Concentration of the n-paraffins added to the medium was varied from 4% (v/v) to 8% (v/v). Sterilization of the media was done at 115 C for 10 min. Detailed composition of the medium is described in each Table. Cell suspension cultivated for two or three days at 30 C were inoculated into shaking flask. Incubation was carried out usually at 30 C for 5 to 7 days. (1) Effects of various inorganic nitrogen sources. In Table IV, effects of various inorganic nit- TABLE IV. EFFECT OF INORGANIC NITROGEN SOURCES ON GROWTH AND FuA PRODUCTION TABLE V. EFFECT OF NH4Cl CONCENTRATION ON GROWTH AND FuA PRODUCTION Basal medium M-2; n-paraffin 8 ml/dl, yeast ex tract 0.02g/dl, CaCO3 4g/dl. Fermentation was carried out at 30 C for 6 days using shaking flasks. but the maximal FuA production was obtain ed at the ammonium chloride concentration of the 0.3 g/dl. (3) Incubation temperature. Table VI show ed that the optimal temperature for growth and FuA production was about 30 C. TABLE VI. EFFECT OF INCUBATION TEMPERA- TURE ON GROWTH AND FuA PRODUCTION Basal medium M-2; n-paraffin 6 ml/dl, yeast ex tract 0.02 g/dl, CaCO3 3 g/dl. Fermentation was carried out at 30 C for 5 days using shaking flasks. Togen sources were shown. The yeast was able to utilize all of the ammonium salts tested but nitrate was not assimilated. Among them, ammonium phosphate and ammonium chloride showed good results. (2) Concentration of ammonium chloride. As shown in Table V, the growth of the yeast increased with the increasing concentration of ammonium chloride in the culture medium, Basal medium M-2; n-paraffin 6 ml/dl, NH4C1 0.4 g/dl, YE 0.02 g/dl (for 5 days). (4) Inorganic salts. Results shown in Table VII indicated that the optimal concentration of mono-potassium phosphate for the FuA pro duction was 0.05g/dl, and that of magnesium sulfate 7 hydrate was 0.05 g/dl. (5) Heavy metal ions. As shown in Table VIII, ferrous, zinc and manganese ions are essential for FuA production. This medium contained many kinds of trace elements de riving from yeast extract, so details will be reported in the next paper.

5 674 K YAMADA, T. FURUKAWA and T. NAKAHARA TABLE VII. EFFECT OF INORGANIC SALTS TABLE X. FuA PRODUCTION FROM C10 TO C18 ALKANES a) K2HPO4 0.05g/dl, MgSO4.7H2O 0.1g/dl. b) K2HPO4 0.05g/dl, KH2PO4 0.05g/dl. Basal medium M-2; n-paraffin 8m1/dl (30 Ž for 7 days). Basal medium M-2; n-alkane 4ml/dl. (NH4)2SO4 0.2g/dl, CaCO3 2g/dl (30 Ž for 5 days). TABLE VIII. EFFECT OF HEAVY METAL IONS Basal medium M-2; n-paraffin 8m1/dl, NH4Cl 0.3g/dl, YE 0.04g/dl, CaCO3 4g/dl, (30 Ž for 7 days). TABLE IX. EFFECT OF YEAST EXTRACT AND CORN STEEP LIQUOR ON GROWTH AND FuA PRODUCTION TABLE XI. EFFECT OF INITIAL ph ON GROWTH AND FuA PRODUCTION Basal medium M-2; n-paraffin 8ml/dl. 0.3g/dl, CaCO3 4g/dl (30 Ž for 7 days). NH4Cl Basal medium M-2; n-paraffin 8ml/dl. 0.3g/dl, CaCO3 4g/dl (30 Ž for 7 days). NH4Cl (6) Effect of yeast extract and corn steep liquor. In order to promote the growth and the yield of FuA, effects of yeast extract and corn steep

6 Studies on the Utilization of Hydrocarbons by Microorganisms. Part XVIII 675 liquor (CSL) were examined. As shown in Table IX, the growth and the FuA production of the yeast increased with the increasing concentration of CSL in the culture medium, and the maximal growth and FuA production were obtained by the addition of more than 0.1g/dl of CSL. On the other hand, yeast extract showed the same effect within the concentrations used in this experi ment. (7) Productivity of FuA from C10 to C18 n- alkanes. Fermentative production of FuA from various individual n-alkanes of carbon number from 10 to 18 was examined as given in Table X. Higher yield was obtained from alkanes of C12 to C14. On the other hand, n- octadecane was given the best growth. (8) Effect of initial ph. Studies for obtaining optimal initial ph were carried out, and the results are shown in Table XI. The initial ph of 7 to 7.5 was optimal for FuA production. Acknowledgement. Authors wish to express their sincere thanks to Drs. H. Mitsuki and T. Nakase of the Central Res. Inst. of Ajinomoto Co. for their kind help in identification of the newly isolated yeast.