Process for production of yeast cells from butanols
United States Patent 1 Matsumoto et al.  
 PROCESS FOR PRODUCTION OF YEAST CELLS FROM BUTANOL S Inventors: Shun-ichi&#39;Matsumoto, Kamakura;  
 Eiji Sato, Zushi. both of Japnn Assignee: Filed: Jan. 31&#39;. 1974 Appl. N0.: 438,390  
 Foreign Application PriorityData Toray Industries. Inc.. Tokym jupin 1 11 3,879,261 1451 Apr. 22, 1975 1581. Field ofSe arch 195/49. 82. 28 R; 51/75 References Cited FOREIGN PATENTS OR APPLICATIONS 1.210.770 10/1970, United Kingdom 195/49 Primary E.\&#39;uminer&#39;-Lionel M. Shapiro Assistant Examiner- R. B. Penland ABSTRACT Method for producing yeast cells by growing a yeast strain in a culture medium containing a normal and/or secondary butanol. I 1  
 ll Claims, No Drawings PROCESS FOR PRODUCTION OF YEAST CELLS FROM BUTANOLS DESCRIPTION OF THE PRIOR ART Commercial production of yeast cells from normal paraffin has been realized in some countries, and utilization of the products as feed has been developing. Various strains of yeast have been reported which can assimilate methanol. ethanol. acetic acid and the like as their main carbon source. and some of these strains are expected to be used for production of yeast cells.  
  S. Omata et al. investigated the capability of various yeast strains for assimilating lower alcohols. They reported in Journal of the Fermentation Association. Japan. vol. 26, page 313 to 3 l6 I968). that none of the yeast strains tested by them actually utilized secondary butanol while only a few utilized normal butanol.  
  We now have found new species of yeast strains which have the capability of growing by assimilating normal and/or secondary butanol as their main carbon source. with a possibility of producing yeast cells from In accordance with this invention. yeast cells can be products of the hydrocarbon-cracking process for preparing ethylene.  
 The yeast strains which may be employed according to the present invention are strains of the generaCam dida or Tricl&#39;losporon. Typical examples of the strains belonging to these genera and having ability to assimilate nand/or secbutanol are as follows: Yeast species assimilating both normal and secondary butanols Candida bulanolpliila nov. sp. FERM-P I882 Candida Imlunolyrim nov. sp. PERM-P 1883 Candida alcopliila nov. sp. FERM-P 1884 I Tric-lwx nn&#39;on hulano p/iilum nov. sp. FERM-P I885 Triclms nn&#39;un altoplu&#39;lum nov. sp. FERM-P 1886 Yeast species assimilating only normal butanol Candida .spllerica nov. sp. FERM-P I91 I Candida ellipsis nov. sp. FERM -P 1908 Candida splilica nov. sp. FERM-P I909 Tric/mspuron opalum nov. sp. FERM-P I910 These species were classified with respect to their genera. and were confirmed to be novel species on the basis of their taxonomic characteristics mentioned below: Taxonomic Characteristics at. The growing properties of the vegetative cells in various media are reported in Table l which appears hereinafter. b. Production of ascospore: I  
 Absent for all species on Gorodkwa agar and gyp sum block (at C. for 7 days). c. Production of ballistospore:  
  Absent for all species on Malt-Yeast extract agar. d. Physiological properties are reported in Table II which appears hereinafter.  
 e. Assimilation of various carbon sources. This is reported in Table III which appears hereinafter.  
 f. Fermentation of sugars. This is reported in Table IV which appears hereinafter.  
  Observation of FE RM-P 1882, FERM-P 1883, FERM-P 1884 FE RM-P 1911, FERM-P s, FERM-P 1909 FE RM-P 1 Medium and condition growth a. Butanophila C.Butanol1 tica c. Alcophila c. Spherica 0. Em a 0. Splitica T. emm ri i izin i &#39;i z iii aitfi q u n 1. Shape Oval or long ovaL. Cylindrical-. Sh0rt-0val.. Splicrical..... 0val... Long oval.-....... Oval Ova1 Oval.  
 6 x (58 (5-10 4) (2-5 x (37 (2-5 x (515u) (ti-12 x (IO-20 (7-14 x (1020 (2-5 x (3-8 Budd1ng--..... Budding-- Budding--...- Budding.- Budding. Bud ding. F y and thick Rlng formation&#34; Heavy and thick Thin and smooth Thin pelliclc, Pelliclc formapellicle pcllicle. pellicle. pellicle, crcepcreeping ring tion. ing ring forformation. mation.  
 2. Size.-. (3-8 x (8-14 (24m) x (715u) (2 3. Vegetative rc- Budding- Budding. Budding production. Thin andsmooth Thin and smooth I Thin and smooth Heav 4. Pelllclc pellicle, creeppelliele, creeping ring formg ring [ormation. mation. 5. Production of Abscnt.. Absent.... Abscnt....... Absent............ Absent..... Absent. Absent Absent...&#34; Absent.  
 Malt-yeast extract medium-Peptone, 5 g.; Yeast extract, 3 3.; Malt extract, 3 g.; 1)- glucose, 10 g.; Dist. water, 1,000 ml. (30 0., 2-7 days).  
 gas. 6. Turbidity of Slight. Moderate...... M0derate... do........ M0derate..... Moderate..... Slight&#34;. do. Moderate,  
 medium. 7. Sediment for- Compact sedi- Compact and Compact and Compact scdi- Block sediment. Flaky and com- Block sediment. Block sediment- Block sediment. block sediment. block sediment. ment. pact sediment. No change No change No change. No change No change&#34; No change. No change.. No change No change.  
 ment.  
 dium  
 Table 11 Physiological Properties FERM Number FERM-P FERM-P FERM-P FERM-P FERM-P FERM-P FERM-P FERM-P FERM-P 1882 1883 1884 1911 1908 1909 1885 1886 1910 Name of Species C. buruno- Cbuumo- (.alcophila C. spherica C .ellipsis C .splitica T. butanu- &#39;I.alco- Tiopalum phiIa lytica philum philum Optimum pH 3.0-7.0 3.0-7.0 3.0-7.0 3.0-7.0 3.0-7.0 3.0-7.0 3.0-7.0 3.0-7.0 3.0-7.0  
 growing conditemp. 20-35 C 20-35 C 20-35 C 20-36 C 2036 C 20-36 C 20-35&#34; C 20-35 C 20-36 C tion Limit pH scanty scanty scanty scanty scanty scanty scanty scanty scanty growth at growth at growth at growth at growth at growth at growth at growth at growth at of pH 7.8 pH 7.8 pH 7.8 pH 7.8 pH 7.8 pH 7.8 -pH 7.8 pH 7.8 pH 7.8  
  temp. scanty scanty scanty scanty scanty scanty scanty scanty scanty growth growth at growth at growth at growth at growth at growth at growth at growth at growth at 40C 40C 40C 42C 42C 42C 40C 40C 42C Assimilation of absent absent absent absent absent absent absent absent absent potassium nitrate Splitting of absent absent absent observed absent observed absent absent absent arbutin Ethanol as sole abundant abundant abundant abundant abundant abundant abundant abundant abundant source of carbon growth growth growth growth growth growth growth growth growth Action in litmus no change no change no change yellow and no change yellow and no change no change no change milk solidificasolidification tion Urease test I Production of absent absent absent absent absent absent absent absent absent carotenoid pigment Production of absent absent absent observed absent observed absent absent absent starch-like compounds Vitamin t t i i i i requirements Production of absent absent absent absent absent absent absent absent absent organic acid Limit condition 10% 10% 10% 10% 10% 10% 10% 10% 10% of growth in NaCl solution Butanol as sole (sec-) (sec-) (sec-) (n-) (n-) (n-) (sec-) (sec-) (n-) source of carbon abundant abundant abundant abundant abundant abundant abundant abundant abundant growth growth growth growth growth growth growth growth growth Gelatin liqueabsent absent absent observed observed observed absent absent observed faction Table III Assimilation of various carbon sources I Yeast FERM-P FERM-P FERM-P FERM-P FERM-P FERM-P FERM-P FERM-P I FERM-P species 1882 1883 1884 1911 1908 1909 1885 1886 1910 Carbon C .burano- C. bu lana- C .alcaphila C .spherica C. ellipsis C .splitica T. butano- T.alco- T.opaIum sources phila lylica philum philum D-glucose D-galactose 1 Sucrose Maltose Lactose Raffinose Esculin lnulin Dextrin Melibiose D-arabinose Soluble starch Trehalose D-Mannose a-Methyl- D-glucose D-Xylose Table IV Fermentation of Sugars FERM-P FERM-P FERM-P FERM-P FERM-P FERM-P FERM-P FERM-P FERM-P Yeast 1882 1883 1884 1911 1908 1909 1885 1886 1910 species C .bumno- C .bu mm C. alcaphila C .rpherica C .ellipsis C .splilica T.bumn0- T.alc0- T.opalum Sugars phila Iylica philum philum D-glucose D-galactose Sucrose i Maltose Lactose Raffinose Six species of the yeasts cited above (FERM-P 1882. 1883.1884. 1911,1909 and 1910) do not form ascospores. ballistospores or arthrospores. Their vegetative cells are oval or cylindrical with some variations in size. They reproduce by budding and form true or pseudomycelia. Carotinoid pigment is not produced.  
  Thus it is appropriate to consider these species as Candida on the basis of the description in The Yeasts. a Taxonomic Study&#34; by .I. Lodder and N. W. J. Kreger Van Rij (1952).  
  On comparison of morphological and physiological characteristics of FERM-P 1882, 1883 and 1884 (C. bulunophila, C. butanolytica and C. alcophila) with those of the described species of Candida. they are similar to C. rugosa in the properties of fermentation and assimilation of sugars. However. they differ considerably from C. rugosa in terms of ethanol assimilation, action on litmus milk and the absence of long cylindrical cells.  
 FERM-P 191 l (C. spherica) is similar to C. solani. C.  
 guilliermoudii and C. molibiosi in the properties of fermentation and assimilation of sugars. But FERM-P 1911 differs from these described species in assimilation of lactose. action in litmus milk. and shape of vegetative cell.  
  FERM-P 1908 (C. ellipsis) is similar to C. rugosa in the properties of fermentation and assimilation of sugars. However. FERM-P 1908 differs from this described species in assimilation of ethanol. action on litmus milk and shape of vegetative cell.  
  FERM-P 1909 (C. splilit&#39;a) is similar to C. humicolu and C. (lll&#39;llllll in terms of no fermentation of sugars, and assimilation of D-glucose, D-galactose, saccharose and maltose. But PERM-P 1909 differs from (1. luunicula in assimilation of ethanol, and morphology of pseudomycelium. and C. curvaza in shape of vegetative cell and splitting of arbutin.  
  Three species cited above. FERM-P 1885. 1886 and 1910 identified as the genus Trichosporon on the following grounds. These species neither form ascospores nor ballistospores. They form arthrospores. The vegetative cells are oval and reproduce by budding. Carotinoid pigment is not formed.  
  FERM-P 1885 (T. butunop/rilum) resembles T. sericeum and T. capimmm in terms of sugars fermentation and assimilations of glucose and galactose. But FERM- P 1885 is distinguished from T. sericeum by nitrate assimilation and true mycelium formation on potato agar. T. capitatum is not identical with it in respect of ethanol assimilation and some morphological characteristics.  
  FERM P 1886 (T. ulcophilum) is similar to T. cumneum in terms of fermentation and assimilation of sugars. But they are not identical in assimilation of galactose and ethanol and in the shape of vegetative cells.  
  FERM-P 1910 (T. upulum) resembles T. sericeum and T. t-upimlum in fermentation of sugars and assimilation of D-glucose and D-galactose. But FERM-P 1910 is distinguished from the described yeasts in morphology of true mycelium.  
  From these grounds, we have concluded that these yeasts should be titled as new species and have named them as mentioned above.  
  In the production of yeast cells, one of the yeast strains may be grown in a medium containing normal and/or secondary butanol as the main carbon source. nitrogen compounds. nutrient salts and growthpromoting materials. Regarding the composition of the culture medium. one of either synthetic or natural origin can be used so long as it contains the above mentioned materials.  
 The concentration of normal and/or secondary butanol in the medium should be carefully determined. 1f the concentration is too high, inhibition of growth of the yeast would result. Furthermore loss of butanols into exhaust gas becomes greater. For this reason. butanols are preferably added to the culture medium stepwise or continuously during the time of fermentation. so as to maintain an appropriate concentration.  
  ln addition to normal and/or secondary butanols, utilizable carbon sources such as sugars or alcohols can be added to the culture medium in order to promote the growth of yeast strains.  
  Nitrogen source of the medium can be selected deliberately from nitrogen containing materials utilizable by the yeast. Usually, good results are obtained when ammonia. urea or ammonium salts such as ammonium chloride. ammonium sulfate or ammonium phosphate are used as a nitrogen source. in a concentration between about 0.1 4%.  
  Regarding the matter of ingredients other than carbon and nitrogen sources. nutrient salts and growthpromoting materials which include all or some of such compounds as potassium phosphate. magnesium sulphate, compounds containing iron. manganese, zinc and other minerals, vitamins. amino acids and nucleotides, can be added.  
  The yeasts are grown under submerged aerobic con ditions for about 1 3 days at a temperature from about 20 to 30 C. The pH of the growth medium is preferably maintained between about 3 and 7. Owing to production of organic acids from butanols and/or consumption of ammonium ions in the culture medium, decrease of pH may occur. To keep the pH of the medium within a desirable range, it is necessary either to add calcium carbonate or a buffer solution, or to titrate it during the course of culture with an ammonia or alkali solution.  
  When the culture is terminated. the yeast cells can be harvested from the culture medium either by filtration or centrifugation. The cells are washed and dried to produce the final crop.  
  The invention will be described in further detail with reference to the following examples.  
 EXAMPLE 1 A medium consisting of KH PO 0.02% (w/v). K- .HPO 0.12% (w/v). MgSO .7H O 0.05% (w/v). (NH SO 0.3% (w/v) and yeast extract (Difco Laboratories) 0.3% was dispensed. in a 50 ml amount. into three Sakaguchi culture flasks (500 ml) and sterilized by autoclaving at 120 C for minutes. After the medium was cooled. secondary butanol was added to each flask to give a final concentration of secondary butanol of 0.5% (w/v). The pH of the medium was 6.0 before and after sterilization.  
  FERM-P 1882 (C. butanophila). FERM-P 1883 (C. butanolytica) and FERM-P 1884 (C. alcophila) were individually inoculated into separate flasks and grown with shaking at 30C.  
  The concentration of secondary butanol in the culture medium was determined by gas chromatography. Within 18 hours of culture. secondary butanol in the culture medium was consumed nearly completely. Then. sec-butanol was added to 0.5% (v/v). This process was repeated five times to obtain a high density of the cells. The pH of the medium during culture was maintained between 4 and 6 by titrating with 10% ammonia. The yeast cells were harvested by centrifuga tion. washed with water and dried in vacuo. The yields of dry cells were 14.0 g/l-broth for FERM-P 1882 (C. butanophila). 10.0 g/l-broth for FERM-P 1883 (C. butanolytica) and 12 g/l-broth for FERM-P 1884 (C. alcophila). The rate of propagation of these yeasts in the media were 2.5. 2.9 and 3.2 hours of doubling time respectively. Data regarding elemental analysis of dried cells of these yeasts are shown in Table 4.  
 1 EXAMPLE 2 FERM-P 1885 (T. bulanophilum and FERM-P 1886 (T. uIcup/rilum) were individually inoculated into separate flasks containing the same medium as used in Example l and culturing was carried out similarly. The consumption of secondary butanol in the medium was complete within 24 hours. Secondary butanol was added 6 times to give 0.5% (v/v) of butanol concentra: tion at each addition. The cells were harvested. washed and dried. The yield of dried cells (g/l-broth) was 10.0 for FERM-P 1885 (T. Imtanop/tilum) and 8.0 for FERM-P 1886 (T. aIc/mp/rilum The rates of propagation of these yeasts were 2.8 and 3.2 hours of doubling time. respectively. Data regarding elemental analysis of the dried cells are shown in Table 4.  
 EXAMPLE 3 To the medium prepared in Example 1 was added normal butanol to give final concentration of 0.5% (weight by volume).  
 Six species of Candida and three species of Trichosporon cited in Table 5 were individually inoculated into separate flasks. and cultured with shaking at 30 C for 24 hours.  
  Yeast cells were harvested and treated in the same manner as Example 1. The yield of cells are shown in Table 5.  
 The following is claimed:  
  1. A process for preparing yeast cells which comprises growing at least one yeast strain belonging to a genus selected from the group consisting of genera Candida and Trichosporon in a culture medium containing at least one butanol selected from the group consisting of normal and secondary butanol. and harvesting the yeast cells.  
  2. The process according to claim 1 wherein the yeast strain is FERM-P 1882 (Candida hutanophila).  
  3. The process according to claim 1 wherein the yeast strain is FERM-P 1883 (Candida lmtanolytr&#39;ca).  
  4. The process according to claim 1 wherein the yeast strain is FERM-P 1884 (Candida butanophila).  
  5. The process according to claim 1 wherein the yeast strain is FERM-P 1885 (Trichosporon butanap/rilum 6. The process according to claim 1 wherein the yeast strain is FERM-P 1886 (Trichosporon alcupliilum).  
  7. The process according to claim 1 wherein the butanol is normal butanol and the yeast strain is FERM-P 191 l (Candida splrerica).  
  8. The process according to claim 1 wherein the butanol is normal butanol-and the yeast strain is FERM-P 1908 (Candida ellipsis).  
  9. The process according to claim 1 wherein the hutanol is normal butanol and the yeast strain is F ERM-P 1909 (Candida splitica).  
  10. The process according to claim 1 wherein the butanol is normal butanol and the yeast strain is FERM-P l9 l0 Trichosporon apalum 11. The process according to claim 1 wherein the butanol is prepared by hydrating C olefins obtained from the products of hydrocarbon cracking.