LIBRARY Michigan State University This is to certify that the thesis entitled Methanol Utilization by Yeasts presented by Ibrahim Saad Al-Mohizea has been accepted towards fulfillment of the requirements for M.S. Food Science degree in {KM Major professor Date 11/6/78 0-7 639 METHANOL UTILIZATION BY YEASTS By Ibrahim Saad Alélbhizea A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of EASTER OF SCIENCE Department of Food Science and Human Nutrition 1978 6 537;; a (2" L; 69’ ABSTRACT METHANOL UTILIZATION BY YEASTS By Ibrahim Saad Al-Mohizea ‘ The present study was carried out to investigate the characteristics of some methanol-grown yeasts. Six yeasts grown on methanol were isolated by means of a batch enrichment technique. One isolate, identified as a strain of Candida boidinii, was selected for further study; the isolate had temperature and pH growth optima of 28° C and 4 to 6, respectively. Although biotin enhanced growth, the growth proceeded slowly in vitamin-free medium. The yeast grew in media containing up to 10%»meth- anol. The duration of the lag phase was prolonged when methanol rather than glucose was used as the carbon source. The growth rate was retarded by high concentra- tions of methanol. The maximum cell dry weight (8.6 g/l) was obtained with 5% (v/v) methanol. Cells grown on from I to 496 methanol contained 41% protein and 4.8% nucleic acid (NA). "IN THE NAME OF GOD, HOST GRACIOUS, MOST MEBCIFUL" ii ACKNOWLEDGMENTS The author would like to express his deep appreci- ation to his major professor, Dr. K. E. Stevenson, for his guidance, his very detailed and helpful reviews and for his constructive criticisms during the course of the labo- ratory work and during the preparation of this manuscript. Appreciations also go to the other members of the author's committee: Dr. E. S. Beneke, Dr. L. G. Harmon, and Dr. P. Markakis for their guidance and their careful proofreading of this manuscript and to his colleagues for their valuable assistance. Lastly, the author would like to pay tribute to his mother, his brother Mohammed, and his wife whose influence and encouragement were a source of much needed moral sup- port. iii LIST OF LIST OF INTRODU TABLE OF CONTENTS TABLES . . . . . FIGURES . . . . . . CTION O O O 0 O O O O LITERA’I‘TJRE REVIEI’J e e e e 0 Historical Aspects . Methanol as a Novel Production . . . . Substrate for Yeast as the Microbe of Choice in Production of SCP from Methanol Methanol Utilization by Yeasts . Dissimilation of Methanol . . . . MATERIALS AND METHODS . . . Microorganisms . . . EES'LTS Isolation of Yeasts . Identification of the Screening Test . . . Growth Studies . . Analytical Techniques Experimental Design . Isolates . iv Nutritional Value of Methanol-Grown 11 14 14 14 16 l6 17 18 22 Taxonomic Studies . . . . . . . . . . . Morphology . . . . . . . . . . . Reproduction . . . . . . . . . . Physiological Characteristics . Growth Characteristics . . . . . . . . Effect of Temperature . . . . . Effect of pH . . . . . . . . . . Vitamin Requirements . . . . . . Effect of Methanol Concentration Macromolecular Composition . . . . . . Protein Content . . . . . . . . Nucleic Acid Content . . . . . . DISCUSSION . . . . . . . . . . . . . . . . . . SUMMARY AND CONCLUSIONS . . . . . . . . . . . BIBLIOGRAPHY . . . . . . . . . . . . . . . Page 22 22 22 23 24 24 24 24 28 32 32 53 34 46 48 LIST OF TABLES Table Page I. Yeasts Identified as Methanol Utilizers . . . 8 2. Essential Amino Acids in Some Microorganisms Grown on Methanol . . . . . . . . . . . . . l2 3. The Growth Parameters of Candida boidinii ST in YNB + Methanol Broth Containing Various Methanol Concentrations . . . . . . . . . . 29 4. Comparison of Growth Parameters of Candida boidinii ST Grown on Glucose or MethanoI . 52 5. Protein and Nucleic Acid Content of Cells of Candida boidinii ST Grown on Methanol and Glucose I O I I O O O O O O O 0 O O O O O O 32 vi LIST OF FIGURES l. A schematic diagram of the main experi- ments 0 O O O O O O O O O O O O O O O O 2. Effect of temperature on the growth of andida boidinii ST in a medium contain- ing 1% (v7v5 methanol at pH 5.5 . . . . 5. Effect of pH on growth of Candida boidinii ST at 28° C in a medium containing 1% (v/v) methanol . . . . . . . . . . . . . 4. Effect of biotin on growth of Candida boidinii ST in a vitamin—free broth at 23° C and PH 5.5 e e e e e e e e e e e e 5. Standard curve showing the relationship between cell dry weight and optical density (OD) at 600 nm . . . . . . . . . 6. Effect of initial concentration of methanol on growth of Candida boidinii ST at pH 5.5 and a cultivation temperature of 28° C. O O O O O O O O O O O O O O O O O O 0 vii Page 21 25 26 27 30 31 INTRODUCTION Because of overpOpulation and the scarcity of ara- ble land, a shortage of food, particularly food high in protein, is anticipated, even in the so-called advanced nations (Han 93 §;., 1971). This fact stimulated many scientists from all over the world to put forth a large ef- fort exploring novel sources for food, especially protein- rich food. As a result of this interest, many approaches to the problem have been suggested, such as: leaf protein concentrate (Kohler and Knuckles, 1977), fish protein con- centrate (Pigott, 1976) and single cell protein (SCP) (Mateles and Tannenbaum, 1968; Lipinsky and Litchfield, 1970, 1974; Tannenbaum and Wang, 1975; Litchfield, 1977). Of these products, SCP seems to be the more practical ap— proach and the most promising source, at least in the near future (Lipinsky and Litchfield, 1974). Such sources of protein must fulfill a set of economic, nutritional, health and social criteria. The protein source has to be avail- able and at a low cost, nutritionally valuable and free of any toxic compounds, and acceptable when used as food or as a food additive. Many studies on SCP have been concerned with the l criteria cited above. Since raw material cost represents a major part of the total cost of SCP production (Wang, 1968), this aspect has been studied extensively. Investi- gations have shown the economic feasibility of utilizing many raw materials as possible substrates for SCP produc- tion (Kihlberg, 1972). One of these substrates, methanol has many advantages--enough to make it the substrate of choice for SCP production (Cooney and Levine, 1972). Methanol could be produced cheaply from natural gas by catalytic oxidation of methane, which would otherwise be flared, particularly in the Middle East. In a country like Saudi Arabia, which utilizes less than 20% of its daily 6 x 109 cubic feet production of natural gas with a methane content of 62.2% (Collins, 1976; Huval, 1976; Hatch and Matar, 1977), SCP production from methanol could potentially be produced in the near future. It is for these reasons that I have been attracted to the study of methanol utilization by microorganisms. Yeasts have been selected as the microorganisms of choice in this study for their many advantages which will be dis- cussed later. The objective of this study was to review some work which has been done in this vital area, as well as to focus on the results of investigations which I have completed con- cerning growth of yeasts on methanol. LITERATURE REVIEW Historical Aspects Yeasts were used by the ancient Egyptians more than 4,000 years ago (Phaff 33 al., 1968). The yeast was used for leavening as much as 50 kinds of breads, by fermenting fruit juices, and for making foods palatable and nutri- tious (Hulse, 1974). The possibility of growing yeast as a direct human food was first explored in'Berlin by Delbruk gfi 3;. in 1910 (Scrimshaw, 1975). Thousands of tons of the yeast Candida utilis, formerly Torula utilis, were consumed in Germany during World War I and II as a meat substitute for different sectors of the pepulation (Scrimshaw, 1975). Also, food yeast was incorporated into rations by the Russians and the Japanese during World war II (Scrimshaw, 1975). After that time and until the mid-60's, SCP did not appear as a likely source of food. The reason for that was the relatively rapid developments in improved methods of field crap production and pest control. In my Opinion, these two were the major factors which contributed to the delay of development of SCP as a potential supplement to conventional protein sources. The situation has changed as 4 prices have risen due to the relative scarcity of protein sources and the growing demand which appeared as an in- evitable result of the world population increase over a limited arable land. According to Senez (1972), the annual rate of population increase is about 2.5%rin the so-called Third World and 1.2%»in the so-called industrialized coun- tries. With these rates of pOpulation increase, it is expected that world population will increase to seven bil— lion in 2,000 A.D. It is for these reasons that many in- vestigators over the world began to attach great signifi- cance to the production of SCP. Methanol as a Novel Substrate for SCP Productiog Since the raw material cost represents a major part of the SCP production, methanol has attracted many investi- gators as a substrate for SCP production. Many substrates have the potential to serve as substrates for the produc- tion of SCP, e.g. molasses (Bunker, 1965), agricultural wastes (Dunlap, 1975), cellulose (Han 33 al., 1971), sauerkraut wastes (Hang 33 al., 1972), food processing wastes (Church g§,§;., 1975), petroleum (Champagnat, 1965), ethanol (Gate 23 al., 1975) and methanol (Cooney and Levine, 1972). Of these substrates, methanol seems to be the most promising due to the following features: 1. Methanol is completely miscible with water in all portions. This avoids three-phase problems and 6. the residual methanol is readily washed from cells (Cooney and Levine, 1972 and 1975; Gow 93; $1., 1975). It is readily available and can be produced at a low cost from a wide variety of hydrocarbon sources, ranging from methane to naphtha (Gow 33 gal” 1975). It is highly purified by distillation. Usually it is sold in a form which is 99.85%1pure so the carryover of polycyclic aromatic compounds is minimized (Mehta and Pan, 1971). It requires less oxygen for its metabolism by microorganisms and a lower cooling load than methane (Abbott and Clamen, 1975). It is less explosive than.methane-oxygen mix- tures (Gow 22 31., 1975). It is easy to handle and store (Cooney and Levine, 1972, 1975). Its use by microorganisms is restricted which helps maintain a pure culture during fermenta- tion (Cooney and Levine, 1975). Yeast as the Microbe of Choice in the Production of SCP from Methanol Yeasts have been selected for production of SCP for the following reasons: 1. Their ease of cultivation; they are not nutri- tionally fastidious. Yeasts are not affected appreciably by changes in pH. Nearly all species can grow within a wide range of pH values (Phaff 3E 31., 1968). This criterion helps yeasts to grow in different media and to overgrow bacteria, particularly at low pH. Yeasts grow relatively rapidly. They provide a high quality and quantity protein. The average crude protein content of yeast cells is usually from 45—50% of the dry weight. They form a rich source of many of the B vitamins (Phaff 33 31., 1968; Bressani, 1968). Recovery of yeast by either centrifugation or fil- tration is significantly easier and cheaper than recovery of bacteria due to the larger cell diam- eter of yeasts (Levine and Cooney, 1975). Yeasts have been used for a long time as a food additive so they would be psychologically more acceptable than other microbes for human con- sumption. Production of yeast is independent of climate, requires a small area and uses less water than most food processing plants (Kihlberg, 1972). Methanol Utilization by Yeasts Assimilation of methanol by bacteria is well known. In 1906, Sohngen reported for the first time that methanol was utilized by bacteria (Cooney and Levine, 1972). On the other hand, methanol was thought to be among the sub- strates which were not utilized by any yeast, particularly since Wickerham and Burton (1948) reported none of the strains they tested could utilize methanol. As a result, methanol has been neglected as a criterion in yeast classi- fication. Japanese investigators (Ogata 33 31., 1969, 1970 a, b) reported for the first time that a strain of yeast, Kloeckera 32. No. 2201, could grow on methanol as the sole carbon and energy source. Some microbiologists insist that this yeast is a strain of Candida boidinii (Fukui 33 31., 1975b). There have been relatively rapid increases in the amount of work done on methanol utilization by yeasts. This is clear from the increase in the number of yeasts re- ported to utilize methanol (Table 1) and also from research which has been done on the mechanism of methanol dissimila- tion. Dissimilation of Metha331 Since the discovery of methanol utilization by yeasts, much interest has been devoted to the dissimilatory Table l. Yeasts Identified as Methanol Utilizers. Yeast Species Specific code Reference Candida parapsilosis IFO 0585 Okumura 33 31. (1970) N - l6 Fujii and Tono- N - 17 mura (1972) methanolica Oki 33 31. (1972) boidinii CBS 2428 Hazeu et 31. (19727 boidinii Sahm and Wagner, (1972) alcomigas Uragami 33 31. (1973) Hansenula wickerhamii NRRL-YE-4745 Okumura 33 31. wickerhamii CBS 4705 (1970) capsulata CBS 1995 glucozyma CBS 5766 henricii CBS 5765 Hazeu et 31. EYEEEE" CBS 1708 (19727 nonfermentans CBS 5764 philodendra CBS 6075 01 or ha CBS 4752 pon3orpha NRRL-Y-756O Levine and Cooney (1973) Kloeckera 33. No. 2201 Ogata et 31. Pi hi h l hi1 CBS 2028 0k§19697 t 1 c a a op a ura e a . pastoris CBS 704 (1970).. -‘ pastoris Kato et 31. 1 CBS 744 H (197E; 1 p nus azeu e a . trehalophila CBS 5561 (19727 “' Rhodotorula Asano et 31. S h H 1 F {$27fijd T acc MMfi __-___ 11,31 an 0110-- mura (1972) metha-nonfoams Uragami 33 31. (1973) Torulopsis methanolovescens Oki et al. (19727- glabrata Asthana 33 31. (1971) nemodendra nitratophila Hazeu et 31. inus (19727 methanofloat enukii Uragami 33 31. methanoPhila (1975) Table 1.--Continued. Yeast Species Specific code Reference methanosorbosa kaote et 31. methanodomercqii (1974)- M-I Fujii and Tono- mura (1972) pathway of methanol. The pathway for dissimilation of methanol by bacteria has been studied extensively and pro- ceeds as follows (Kaneda and Roxburgh, 1959; Large and Quayle, 1965; Lawrence 33 31., 1970; Quayle, 1972): NAD NADH; NAD NADHZ NAD NADHg angers—55:41» HCHO >411» HCOOH—¥+ 002 methanol formaldehyde formate dehydrogenase dehydrogenase dehydrogenase Tani 33 31. (1972 a, b) reported for the first time the isolation of a flavin-dependent alcohol oxidase from yeasts grown on methanol. They found no evidence for the presence of the hydrogen acceptor NAD. The enzyme was re- sponsible for catalyzing the transformation of methanol to formaldehyde and peroxide. Fujii and Tonomura (1972) studied the overall oxida- tion of methanol to carbon dioxide by yeast. In their work with Candida N313, they have also been able to isolate an alcohol oxidase. They examined the enzyme activity in cells grown on glucose and methanol and found the enzyme was inducibly formed in response to the presence of methan- 01. Nevertheless, the enzyme had a broad specificity for lO alcohols such as methanol, ethanol, n-propanol, n-butanol and n-amyl-alcohol. The same authors also found that when a pure form of alcohol oxidase was used to catalyze the oxidation of methanol, one mole of oxygen was consumed as one mole of formaldehyde was produced. In experiments using cell extracts, one mole of 02 was consumed as two moles of formaldehyde were produced. This suggested the involvement of catalase in the oxidation of methanol to formaldehyde. Apparently, catalase reacted with hydrogen peroxide which was produced by the alcohol oxidase. Later, Roggenkamp 33 31. (1975) confirmed this assumption when.thqy found that both enzymes were present in the same location, the peroxisome. Similar results were obtained by Fukui g: 21. (1975 a. b). A formaldehyde dehydrogenase has been found to mediate the conversion of formaldehyde to formate, while a formate dehydrogenase was found to be responsible for the oxidation of formate to C02. Both enzymes require NAD (Fujii and Tonomura, 1972; Fukui, 33 31., 1975 a, b; Sahm 33 31. , 1975). Until recently, nothing was known about the location of assimilatory and dissimilatory enzyme systems involved in the methanol metabolism in yeast cells. However, van Dijken 33 31. (1975 a, b), Fukui 33 31. (1975 a, b), and Sahm 33 31. (1975) found, independently, that yeast cells grown on methanol, when examined by electron microscopy, 11 contained microbodies, while cells grown on glucose did not have or had very small microbodies. Roggenkamp 33 31. (1975) found that the microbodies of methanol-grown yeasts, characterized by their enzyme complement, were peroxisomes. Alcohol oxidase and catalase have been con- firmed to be located in these structures (Roggenkamp 33 31. 1975). 3 On the other hand, formaldehyde dehydrogenase and formate dehydrogenase were found in the cytoplasmic frac- tion (Sahm 33 31., 1975; Fukui 33 31., 1975 a, b). Nutritional Value of Meth3301 Grown Yeasts Since SCP is intended to compete economically with protein from conventional resources, it has to have com- patible characteristics. According to Cooney and Levine (1972), high protein, low nucleic acid, low carbohydrate, low lipid content and balanced content of amino acids are desired in sources of SCP. Despite the increase in the number of yeasts which can grow on methanol, little data are available on the composition of methanol-grown yeasts. Data on the protein content of yeasts grown on methanol are within the range of protein content of yeast cells grown on conventional substrates. Protein quality usually is determined in terms of the amino acid profile. In general, the amino acid profiles of yeast cells grown on methanol (Table 2) compare favorably with the standards set 12 Table 2. Essential Amino Acids in Some Microorganisms Grown on Methanol. a b c d e f Amino Acid FAO Kloeckera Candida Hansenula TM 123 . ref. 3 N. boIdInIi pm 20 aeruginosa 01 g of amino acid per g of protein Isoleucine 4.2 5.1 5.78 5.1 5.7 5.57 Leucine 4.8 7.1 5.57 5.54 6.7 5.62 Lysine 4.2 7.5 6.01 8.1 5.5 4.88 Methionine 2.2 0.7 0.86 1.45 1.81 2.00 Phenyla- lanine 2.8 4.0 5.59 4.76 4.18 2.85 Threonine 2.8 5.1 4.42 5.17 4.52 5.81 Trypto- phane 1.4 -- -- -- -- 0.74 Valine 4.2 5.5 4.59 6.21 5.85 4.54 aJones (1974). b Ogata 33 31. (1970). cSahm and Wagner (1972). dLevine and Cooney (1975). eHaggstrom (1969). f60w 22 21. (1975). by the Food and Agricultural Organization of the United Nations (PAC) except for deficiencies in methionine and tryptophane (Jones, 1974). Single-cell protein is commonly deficient in methionine (Cooney 33 31., 1975). However, bacteria have relatively balanced amino acid composition 13 (Haggstrom, 1969; Rosenzweig and Ushio, 1974; Dostalek and Molin, 1975). Methanol-grown yeasts contain a fairly low content of nucleic acid (5-7%) compared with yeasts grown on other substrates. This is ascribed to the slow growth rate exhibited by yeast during growth on methanol. It is well known that the nucleic acid content of yeasts is propor- tional to the growth rate (Kihlberg, 1972). MATERIALS AND METHODS Microorggnisms Methanol-utilizing yeasts were isolated from natu- ral sources, using enrichment techniques. In addition, sixty-seven yeast strains from the Food Microbiology Labo- ratory at Michigan State University were tested for their ability to assimilate methanol as the sole source of car- bon and energy. 1301ation of Yeasts Samples were obtained from various fruits (pine- apples, apples, oranges, strawberries, cactus and pears), vegetables (tomatoes, lettuce and cabbage), and soils (fruit garden soil, vegetable garden soil and greenhouse soil). A suspension was prepared from each sample by blend- ing with distilled, deionized water. Enrichment techniques were used to isolate methanol- assimilating yeasts from these samples. The enrichment experiments were carried out in 500-ml Erlenmeyer flasks containing 100 ml of the isolation medium. The medium (YNB- methanol broth) contained 0.67% yeast nitrogen base (YNB) l4 15 (Difco Laboratories, Detroit, Michigan), 1% (v/v) methanol, and 0.5% KH2P04. Yeast nitrogen base medium was prepared according to Difco manual (1955). Methanol was aseptical- 1y, added to the medium. The pH was, initially, adjusted to pH 4 by adding 1N HCl; pH readings were taken by a pH meter (Model 7, Corning Scientific Instruments, Medfield, Mass.). A 2-ml quantity of each sample suspension was added to each flask containing YNB-methanol broth; the flasks were incubated for seven days in a gyrotary shaker (Model G-25, New Brunswick Scientific Co., New Brunswick, New Jersey) deve10ping 200 rpm in the dark at 25° C. One ml of this culture was added to 100 m1 of the same medium contain- ing 0.1% glucose, and the flask was incubated for four days; one m1 of this subculture was transferred to 100 ml of YNB- methanol broth and the culture incubated for three days. Two successive subcultures were made in YNB-methanol broth if visible growth was present. All the cultures were obser- ved periodically using a light microscope. A culture which showed growth in the last subculture was presumed to be a methanol-assimilating yeast; this presumptive result was confirmed by streaking on a plate containing YNB-methanol plus 2%1agar. Cultures which grew were subcultured on YNB- methanol agar plates in order to obtain pure cultures. Methanol assimilation was further confirmed by running con- trol tests, using either the same isolate and the same 16 conditions except that methanol was not added, or using Candida steatolytica and Q. utilis, which do not utilize methanol and adding methanol as the sole carbon source. The methanol-utilizing isolates were maintained on YMPG agar slants (yeast extract, 0.3%6 malt extract, 0.5%; peptone, 0.5%; glucose, 1.0%; agar, 2.0%; pH 5.5), and subjected to further studies. Identification of the Isolates The taxonomic studies were conducted in accordance with the methods described by Lodder (1970). Screening Test The yeasts were maintained either on Gorodkowa agar or YMPG-agar slants. Sixty-two yeast cultures were iso- lates from various natural sources, and they have not been classified. The remainder were: Candida steatolytic3 Y—51 Candida utilis Y-59 Hansenula anomala YA6O Pichia _3. Y-5 Rhodotorula glutinis Screening test was made by growing the yeasts either on YNB-methanol agar plates or in YNB—methanol broth tubes. The tubes were incubated on a rotary drum (Model G-6, New 1? Brunswick Scientific Co., New Brunswick, New Jersey), operating at 6 rpm. Both plates and test tubes were incu- bated at room temperature (25° C) for 1-2 weeks. Growth Studies Yeasts were precultured in YMPG broth for 18 hours at 25° C. Then, cells were harvested by centrifugation and washed twice with sterile, distilled, deionized water. The cells were suspended in sterile, distilled, deionized water and used as inocula for growth experiments. In all growth studies, except those to determine vitamin requirements, the medium was the same for isolation except that the methanol concentration and pH were varied. Vitamin-free yeast base (Difco) was used to study vitamin requirements and was prepared according to the manufactur- er's instructions except that initial pH was adjusted to 5.5 instead of 4.5. All growth experiments were carried out in 500-m1 Erlenmeyer flasks containing 100 m1 of media. The flasks were inoculated with a cell suspension and incubated at the appropriate temperatures in a New Brunswick gyrotary shaker, developing 200 rpm. The initial pH was adjusted in all growth experiments by adding 1N NaOH or 1N HCl. Cell concentrations were measured with a spectro- photometer (Spectronic 20, Bauch and Lomb, Rochester, New York) at a wave length of 600 nm. A standard curve was 18 prepared by plotting Optical density (0.D.) vs. cell dry weight (g/l) . Analytical Techniques To determine dry weight, cells were harvested by centrifugation at 9000 x g for 10 min. and washed twice with deionized water. The washed cells were dried in pre- dried, tared aluminum weighing dishes for 16 hr. at 100° C. The weight of the dried pellets was used to determine the dry cell weight in g/l (AOAC, 1975; Levine and Cooney, 1973). Protein was determined by two methods. A modified biuret method (Herbert 33 31., 1971) was used: 2-ml ali- quots of washed cell suspension containing 1-5 mg of cell dry weight per ml broth were transferred to 5-m1 centrifu- gation tubes; 1 ml of 5N NaOH was added to each tube; the tubes were placed in a boiling water bath for 5 min. and then cooled; 1 m1 of 2.5% CuSOa was added to each tube, the tube was shaken thoroughly and allowed to stand at room temperature for 5 min. and centrifuged. A set of standard protein solutions (2, 4, 6 and 8 mg standard protein/tube) were prepared and treated in a similar manner. The Optical densities of the supernatant from the cell sus- pensions and protein standards were read against the reagent blank in a spectroPhotometer (Spectronic 20, Bauch and Lomb) using a wave length of 555 nm. A standard curve was drawn 19 by plotting 0.D. vs protein concentration. For the micro-Kjeldahl method, the following modifi- cations of the AOAC (1975) procedure described by Shannon and Stevenson (1975a) was used: 4-m1 aliquots of a di- gestion mixture containing 5g CuSO4 5H20, 5g SeOg and 500 m1 cone. H2S04 was added to a micro-Kjeldahl flask contain- ing approximately 0.1 g of dried cells. When the samples cleared during heating, the sides of the flask were rinsed with deionized water, 1 ml of 50%»H202 was added and heat- ing was continued for another hour. The total nucleic acids content of the cells was estimated by the method described by Levine and Cooney (1975). According to this method, a 2-ml volume of culture broth, containing 1 g of cell dry weight/l was centrifuged, washed with 5 m1 of cold, distilled water and centrifuged. The supernatant was saved and the pellets were extracted with 5 m1 of 0.5 N perchloric acid (PCA) at 0° C for 50 min., centrifuged, and the supernatant was saved. The pel- lets were extracted with 0.5 N PCA at 70° C for 20 min., centrifuged, and the supernatant was saved. The absorbance at 260 nm of the wash and two PCA extracts were measured on a Beckman DB-G spectrophotometer (Beckman Inst., Fullerton, California). The total nucleic acid content was calculated by assuming an absorptivity of 52 ml/mg-cm for the nucleic acids as determined by Ohta 33 31., (1971) in a study with Candida utilis. 20 Experimental Design Six strains of yeast were isolated which utilized methanol. These yeasts were subjected to preliminary in- vestigations concerning growth rate, dry cell weight and average cell size when grown on a methanol-containing medium. Based on these preliminary investigations (data not shown), one strain, isolated from pineapple, was se- lected for further studies. Figure 1 outlines the main ex- periments which were conducted to characterize this isolate. None of the sixty-seven yeast strains from the Food Microbiology Laboratory culture collection were able to utilize methanol. Therefore, further experiments were not conducted with these strains. 21 Isolate ST Identified according to Lodder (1970) Growth studies Vitamin Effect of - Effelt of Effect of require. methanol conc. temperature pH VFB YNB-methanol YNB-methanol YNB-methanol 28° C, pH 5.5 28°CL pH 5.5 pH 5.5 28° C biotin and/or range l-10% range temp. range pH 2-8 thiamin 20-55° 0 Optimum conditions for growth Media (1) YNB + methanol (2) YNB + glucose I Analysis Nucleic Protein Dry weight acid content Micro-Ejeldahl Biuret Figure l. A schematic diagram of the main experiments. RESULTS Taxonomic Studies Morphology Growth at 25° C in YMPG broth: After incubation for three days, cells were oval to long oval (5-6) x (6-10)“, and occurred singly or in pairs; a sediment was formed. A pellicle was formed after one day and became creeping after 2-5 days. Growth at 25° C on YMPG agar: After 5-7 days, the streak culture was raised slightly, slightly wrinkled (umbilicate after 15 days), shiny, butyrous and had com- plete margins. The margins were fringed after 10 days. Dalmau plate and slide culture on YMPG: A well de- veloped pseudomycelium was formed after incubation for 2-5 days at 25° C. Blastospores were formed after 4-5 days. True mycelia were not observed. ggproductigp Vegetative reproduction: Proceeds by multilateral budding. Sporulation: Absent on both Gorodkowa and V-8 media after 1, 2, 5, 4 and 9 weeks. 22 23 Physiological Characteristics Fermentation: Only glucose was fermented. Assimilation of carbon source: Glucose + Galactose + (very weak) Sorbose - Sucrose - Maltose - Cellibiose - Trehallose - Lactose - Melibiose - Raffinose - Inulin - Soluble starch - Xylose - Arabinose - Rhamnose - Ethanol + Mannitol + Salicin - Inositol - Methanol + Assimilation of NaNO;: + Vitamin requirements: None. However, growth was stimulated by biotin. Sodium chloride tolerance: Up to 10% Gelatin liquification: -- Optimum temperature: 28° 0 Maximum temperature: 55° 0 Optimum pH: 4-6 Source: Pineapple According to the scheme of Lodder (1970), the charac- teristics of this yeast strain were in a good agreement with those of Q. 33131311, thus it has been identified as Q. boidinii ST. 24 Growth Characteristics Effect of Temperature A temperature of 28° C was optimum for growth (the highest specific growth rate) of g. boidinii ST (Figure 2). Using 1%:methanol as the carbon source at pH 5.5 and 28° C, the specific growth rate was 0.165 hr"l which corresponds to a generation time of 4.2 hours. The maximum temperature for growth (after which the growth was no longer detected) was 55° C. The lag phase was rather prolonged while the maximum cell concentration was unaffected when the isolate was grown at 25° C, whereas at 50° C a prolonged lag phase and lower maximum cell con- centration were observed as shown in Figure 2. Effect ofggg The organism grew well over a wide range Of pH (from 5.0 to 7.0) with an optimum pH for specific growth rate in the range from 4 to 6 (Figure 5). Slow growth was detected at both pH 2.5 and 7.5. Vitamin Requirements Biotin was found to enhance growth. A concentration of 20 jig/1 was required to give the maximum growth-enhancing effect. However, growth proceeded slowly in vitamin-free broth (Figure 4). Optical Density (CD) at 600 nm 25 17 1.0 ——EL—- 28° C —0— 30° 0 0.1 + 25° 0 OIXfi 40 50 60 Incubation Time (hrs) Figure 2. Effect of temperature on the growth of Candida boidinii ST in a medium containing 1% (v v methanoI at pH 5.5. Optical Density (CD) at 600 nm 26 F- ,, E _ : gK", .. v j ‘ — e i a i “ i l v | 100 '— : . l a . g : //4 i . i ~ I I 0.10__ Pd 5 & 6 F pH 4 _ pH 3 I pH 7 " -—49-——- pH 2.5 0.05‘ l 1 1 1 0 10 20 30 40 50 Incubation Time (hrs) Figure 5. Effect of pH on growth of Candida boidinii ST at 28° C in a medium containing 1% (v/v) methanol. Optical Density (OD) at 600 nm 27 1.0— " —e—— 20 jag/1 Biotin ' —-A— mfg/1 Biotin '4?!" 5pg/1 Biotin 0'1 r --O—- 1rg/l Biotin : —-7(—- 0 Vitamin 0. I ' ' ‘ ‘ ‘ 5 8 10 2O 30 40 SO Incubation Time (hrs) Figure 4. Effect of biotin on growth of Candida boidinii ST in a vitamin-free broth at 28° C and pH 5.5. 28 Effect of Methanol Concentration A prolonged lag phase was associated with using methanol as the sole source of carbon; the extent of the lag phase was variable and, generally, increased with an increase in the concentration of methanol (Table 5). The specific growth rate was appreciably affected by methanol concentration. The growth rate decreased as the concentration of methanol was increased above 0.5%. At 40.5% methanol, the highest specific growth rate was obtained (Table 5). Cell concentration was expressed in terms of opti- cal density. The Optical density was found to have a linear relationship with cell dry weight (Figure 5). The final cell concentration increased as the initial methanol concentration increased, reaching its maximum value at a methanol concentration of approximately 5%t Increases in methanol concentrations above 5%, up to 10%, resulted in decreases in the final cell concentration. At methanol concentrations above 10%, growth was no longer detected. The growth yield (g of cell dry weight/g of sub- strate) followed a different pattern. With initial methanol concentrations of 40.7%, the growth yield increased as the methanol concentration increased. At methanol concentra- tions above 0.7%” growth yield decreased as the initial methanol increased. The maximum growth yield of 0.59 was obtained with a methanol concentration of 0.7%'(Table 5). 29 Table 5. The Growth Parameters of Candida boidinii ST in YNB + Methanol Broth Containing Various Methanol Concentrations. cone. of duration tion growth weight (g D.C. wt./ methanol (hrs) time rate (g/l) g sub.) (ml/1) (hrs) (hr-1) 5 6-8 5.6 0.195 .8 .338 8-10 5.6 ' 0.195 1.4 .55 7 8-10 5.6 0.195 2.1 0.58 10 8-10 4.2 0.165 2.7 0.54 20 11 4.8 0.144 4.1 0.26 50 15 6.5 0.110 5.8 0.24 40 19 7.8 0.090 7.1 0.22 50 3 3 3 8.6 0.21 60 3 3 3 8.1 0.17 70 3 3 3 7.5 0.13 aIn calculating growth yield, methanol lost by evap- oration was ignored. bThe growth curves were so complicated to allow cal- culating these parameters. When glucose was substituted for methanol, there was a relatively short lag phase. In addition, the generation time was as short as one hour, and the final cell concen- tration was slightly higher than when methanol was used as the sole carbon source (Figure 6 and Table 4). 50 —v 10 - Cell Dry Weight (g/l) , l I I I I I I I l 0 2 ,4 6 8 10 12 14 16 .18 20 Optical density (GB) at 600 nm Figure 5. Standard curve showing the relationship between 6011 dry weight and Optical density (CD) at 0 nm. Optical Density (0D) at 600 nm 51 ‘y llllII \\ T P]: v 1.0 I.— a L- l.- __.o_— 0.8% (w/v) (glu) ___*3__ .5% (v/v) methanol '- . 1% n u U“ 2% n H —-k')*‘"' 3% n n 1.0 _ I : 4?6 H H I: 0.02 l l l L l 0 20 40 60 80 103 120 Incubation Time (hrs) Figure 6. Effect of initial concentration of methanol on growth of 93ndida boidinii ST at pH 5.5 and a cultivation témperature of 28° C. 52 Table 4. Comparison of Growth Parameters of Candida boidinii ST Grown on Glucose or MethanoI. Initial Lag Genera- Specific Dry Growth conc. phase tion growth cell yield of glu duration time rate weight (g D.C. wt/ 8/1 (hrs) (hrs) (hr-1) s/1 8 Sub.) Glucose 8 4 l .69 5.2 .40 Macromolecular Composition. Protein Content The true protein content of Q. boidinii ST grown on methanol or glucose as the sole sources of carbon was 41%, while the crude protein was 47-48%I(Table 5). There was no significant difference in the protein contents of cells grown on methanol or glucose. Table 5. Protein and Nucleic Acid Content of Cells of Candida boidinii ST Grown on Methanol and Glu- cose. Carbon Crude True % Nucleic 96 NA Source Protein Protein Acid (NA) Cr. Protein Methanol 47 41 4.8 ' 10.2 Glucose 48 41 5.2 10.8 53 Nucleic33cid Content The total nucleic acid content was approximately 4.8% of dry cell weight when cells were grown on methanol. When the cells were grown on glucose, a slightly higher nucleic acid content (5.2%>Of dry cell weight) was ob- tained (Table 5). DISCUSSION Enrichment techniques with minimal media are widely used for isolating microorganisms from different natural sources. For this investigation, Difco yeast nitrogen base (YNB) was used in the minimal medium since it con- tained all the necessary growth factors which are required by the most fastidious yeasts, and in addition it contained ammonium sulfate which is utilized by all yeasts as a ni- trogen source. Methanol was the only carbon source added to the initial culture while glucose (0.1%) was added to the first subculture to stimulate the growth of the methanol-utilizing yeasts recovered during the initial en- richment procedure. Methanol was the only carbon source in subsequent subcultures. By this mechanism, six strains of methanol-utilizing yeasts were isolated. The assimilation of methanol by the isolates was confirmed by streaking on YNB-methanol agar and by running control experiments using nonmethanOl-utilizing yeasts. According to Lodder (1970), methanol-utilizing yeasts are ubiquitous; they were isolated from soil, fruit, vegetables, flowers, insects, tree barks . . . etc. Fur- thermore, Hazeu 33 31. (1972) related the high incidences of isolation of methanol-utilizing yeasts from environments 54 35 rich in compounds such as lignin and pectin to the pre- sence of methoxy compounds which might have enhancing ef- fects on methanol-utilizing yeasts. In our study, our isolates came mostly from immature fruits and vegetables since these were the primary sources used. The results Of this investigation support the above hypothesis by Hazeu 33 31. (1972) since the isolates came from samples that were rich in methoxy-containing substances. The isolate ST was selected for further investiga- tions since preliminary experiments indicated that this isolate could grow efficiently on a wide range of methanol concentration.' The fermentation and assimilation patterns, and the morphological and physiological characteristics of this isolate were in good agreement with those of Candida boidinii. However, there were slight differences in the assimilation of galactose. vanUden and Buckley (1970) described 9. boidinii as unable to utilize galactose and by having biotin as a growth-enhancing vitamin. Therefore, this slight difference was deemed insufficient to classify this isolate as a new species Of Candida resembling Q. boidinii, and instead it was identified as a new strain of 3. boidinii. Ramerez in 1955 and Santamaria in 1958, how- ever, reported the weak assimilation of galactose by some strains of Q. boidinii (Lodder, 1970). Sahm and wagner (1972) isolated a methanol-utilizing yeast which was identi- fied as a strain of 3. boidinii which required biotin for 56 growth. These slight differences among the strains within the species could be ascribed to the effect of natural habitat. One of the purposes of this study was to establish the Optimum conditions for growth and investigate the effect of methanol concentration on growth of our isolate 3. boidinii ST. Temperature is an important parameter which affects lthe growth of any microorganism. In general, any micro- organism has three important temperatures; the minimum, the optimum and the maximum for growth. The last two tem- peratures are very important in SCP production. Candida boidinii had an optimum temperature (28° 0) similar to that found for another strain of Candida boidinii by Sahm and wagner (1972). A rather prolonged lag phase and a lower growth rate were observed when cells were grown at 25° C or 50° C. These results were expected since the two temperatures were below and above the optimum temperature of growth, respectively (Brock, 1974). The reason for the low final concentration of cells when grown at 50° C may be explained by the fact that methanol is a toxic substance. Hence, at a temperature higher than the optimum, the toxi- city becomes more apparent (Chalfan and Mateles, 1971), or it might be ascribed to the increased cell maintenance (Cooney and Levine, 1975). Recently, several investigators have described other methanol-utilizing yeasts. Oki 33 31. (1972) isolated a new 37 yeast, 9. methanolica, which resembled Q. boidinii; the Optimum temperature of this yeast was in the range of 25° to 55° C. Tozuka (1975) also isolated a new methanol- utilizing yeast which was identified as closely resembling g. boidinii, and the optimum temperature of this yeast was 28.5° C and the maximum was 40° 0. 0f the yeasts, which can utilize methanol, only a few can grow well at elevated temperatures. In this regard, Levine and Cooney (1975) isolated a strain of 3. polyporpha which can grow well at 57° C. The significance of this criterion becomes impor- tant when SCP production is carried out in tropical and subtropical areas (Mateles, 1968) where the cost of cooling comprises a major part of the total cost of production. Temperature of cultivation also may play an impor- tant role in determining the nucleic acid content of the yeast. In a study conducted by Alroy and Tannenbaum (1975) on the influence of the cultivation temperature on the macromolecular composition of 3. utilis, they have found that by increasing the cultivation temperature from 15 to 57.5° C, while other conditions were constant, the nucleic acid content decreased markedly (from 7.94% to 4.56%). Yeasts grow readily over a wide range of pH. The isolate 3. boidinii ST grew from pH 2.5 to 7.5. Although there was no significant difference in growth rate within the optimum pH (4-6) range, pH 5.5 was chosen arbitrarily for subsequent experiments in this investigation. Sahm and Wagner (1972) found that Q. boidinii could grow over a wide fl 'Pfifl‘me‘” '11-‘11 .‘ar' 58 range of pH (2-9). The difference between the pH ranges for these two strains are slight and may be due to the ef- fect of the natural habitat from which they were isolated or due to the differences in the cultural conditions. The optimum pH range of the isolate used in this investigation provides a mechanism for inhibition of many bacteria, especially when coupled with relatively high methanol con- centrations. This inhibitory effect might be helpful in the production of SCP since these operations are usually conducted under nonaseptic conditions. The pH of the medi- um plays an important role in the protein and the amino acid content of the cells. According to Yamada 33 31. (1968) in a study with Candida tropicalis grown on hydro- carbons, the protein content declined with increases in pH of the cultivation medium from 4 to 8, while the content of essential amino acids reached a maximum at pH 6; the yield of dried cells reached a maximum at pH 7. However, the ability of methanol-grown yeasts to grow over a wide range of pH could be utilized efficiently to manipulate SCP pro- duction, i.e., production at a low pH could be utilized to obtain a high content of crude protein and/or to inhibit bacteria. Yeasts vary widely in their vitamin requirements, i.e., some yeasts grow in vitamin-free media whereas other yeasts have an absolute requirement for certain vitamins. According to Phaff 33 31. (1968), biotin is the vitamin most commonly required by yeasts. In the present study, the 59 isolate ST required biotin for rapid growth, while growth proceeded slowly when grown in a vitamin-free medium. This result may be ascribed to the fact that some yeasts have the ability to synthesize slowly their vitamin require- ments (Phaff 33_31., 1968). Under Optimum conditions, pH 5.5 and 28° C, the strain of Q. boidinii used in this investigation grew over a wide range of initial methanol concentrations (up to 10%0 in comparison to several other methanol-utilizing yeasts. Sahm and Wagner (1972) reported that another strain of Q. boidinii was inhibited by 5% (v/v) methanol. Asthana 33 31. (1971) isolated a strain of the yeast 1. glabrata which was inhibited by methanol concentrations higher than 5%I(v/v). YOkote 33 31. (1974) isolated a yeast which was classified as 1. methanosorbosa. This strain could grow in up to 5%I(v/v) methanol and it grew well at 1%Imethanol. The value of the relatively high tolerance of yeasts to methanol is related to the fact that high concentrations of methanol could be used to produce SCP. This method can ef- ficiently inhibit many extraneous organisms since methanol is toxic to many organisms, especially when present at relatively high concentrations. The initial methanol concentration had an effect on lag phase, growth rate, growth yield and cell concentration. The lag phase was prolonged, even if the inocula were taken from cultures in the exponential phase. The extent to which the lag phase was prolonged was related to the 40 initial methanol concentration. The lag phase may be regarded as a period of adapta- tion where specific enzymes (adaptive enzymes) are formed under the influence of the substrate (Monod, 1949). The reasons for the relatively long lag phase which was asso- ciated with methanol utilization could be ascribed to the fact that methanol is slightly inhibitory to the micro- organism. This assumption was supported by the extension Of lag phase in relation to the initial methanol concentra- tion. The specific growth rate (ft) was markedly affected by the initial methanol concentration. Growth was retarded by relatively low-methanol concentrations. This result came out as a deviation of the Monod model of growth: C ”'fims'r'o' wherefi andf" max are the specific, and maximum specific growth rate, respectively. 0 is the concentration of an essential nutrient. S is the concentration of this nu- trient at which the rate is one-half the maximum. Accord- ing to this equation, the specific growth rate is linearly prOportional to the concentration of an essential nutrient at relatively low concentrations. After a certain concentra- tion, the specific growth rate reaches the maximum, i.e., .j" a J“ max and is no longer affected by the concentration of the nutrient. In this study the specific growth rate decreased with higher concentrations of methanol as shown 41 in Figure 6. Similar results were reported in other studies. Levine and CoOney (1975) reported the inhibition of 3. polymorpha DL-l in shake flask experiments by methanol concentrations greater than 1% and in chemostat experiments by methanol concentrations greater than 0.5%. Asthana 33 31. (1971) in a study on the effect of methanol concentration on growth of 1. glabrata found that growth was inhibited by high concentrations of methanol. However, in a study on the effects of methanol and other substrates on the growth of Q. methanolica and 1. methanolovescens, Goto 33 31. (1976) found that as long as growth occurred the specific growth rate remained unaffected by the me- thanol concentrations, i.e., the specific growth rate was independent of methanol concentration. Cell concentration and the growth yield were also affected by the initial methanol concentration. The re- sults indicate that cell concentration increased as the initial methanol concentration increased to 5% (v/v) where the maximum cell concentration was reached. Higher me- thanol concentrations resulted in a decline in cell concen- tration and, subsequently, in cell dry weight. This de- cline was probably due to the inhibitory effect of high methanol concentrations. The growth yield (g cell dry weight/g substrate) is an important parameter, particularly where the commercial production of SCP is desired since it measures the 42 efficiency of converting the substrate to cells. The growth yield which was obtained for 3. boidinii ST (0.59) was within the acceptable range for yeast. In comparison to other methanol-grown yeasts, this isolate had a higher growth yield than that obtained by Sahm and Wagner (1972) for another strain of Candida boidinii (0.29), and it is similar to the growth yield (0.57) obtained by Levine and Cooney (1975) for 3. p0 yporpha. However, higher growth yields were reported by Asthana 33 31. (1971) for 1. glabrata (0.40 - 0.57) and by Gate 33 31. (1976) for Q. methanolica (0.545) and 1. methanolovescence (0.512). The wide discrepancy in the reported growth yields may be due to various factors related to the organism itself or to the techniques applied in the cultivation, i.e., whether continuous or batch cultures were used, whether pH was con- trolled or not, and whether the culture was aerated or not. Under normal conditions of cultivation, protein forms the major component of yeast cell. Yeast, however, have been reported to contain crude protein in the range of 40 - 60%>of cell dry weight (Stewart, 1975). The protein content of our isolate (41% andl47%, true and crude pro- tein, respectively) when grown on methanol falls within the acceptable range for yeasts grown on conventional substrataa There was no significant difference in the protein content between methanol-grown cells and glucose-grown cells when grown under the same conditions. In comparison to another methanol-grown yeast, Sahm and Wagner (1972) obtained only 45 about 55% crude protein for Q. boidinii. Higher protein contents for methanol-grown yeasts were reported by other workers. YOkote 33 31. (1974) reported a crude protein content of 1. methanosorbosa in the range of 47.4 - 51.1% while Levine and Cooney (1975) Obtained 46% (biuret pro- tein) for 3. 01 or ha, and Ogata 33 31. (1969 and 1970b) reported 45% crude protein for Kloecker3 33. no. 2201. So, regard to the protein content, 3. boidinii ST compares well with other methanol-grown yeasts. 0n the other hand, work- ing with other substrates, Reiser (1954) obtained up to 55% crude protein for Q. utilis grown on potato starch wastes. However, Vananuvat and Kinsella (1975) reported 42%Icrude protein for §. fragilis when grown on crude lactose, and Shannon and Stevenson (1975b) obtained 44.5%.crude protein for 3. steatolytica when grown on brewery wastes. The wide variation in protein contents of yeasts was influenced by the strain, the cultivation conditions, substrate on which the yeasts were cultivated and finally by the analytical techniques. The high nucleic acid content of single-cell protein (SCP) is one of the major problems which limits its use. In general, yeasts have a high nucleic acid content, vary- ing from 8 to 25% of the crude protein content (Sinskey and Tannenbaum, 1975). The phenomenon of high nucleic acid con- tent seems to be normal for organisms which have a rela- tively rapid growth (Scrimshaw, 1975). The nucleic acid content of Q. boidinii ST was found to be in the lower 44 limit of the range reported for yeasts. This result is similar to those reported by other researchers working with methanol-grown yeasts. Yokote 33 31. (1974) reported total nucleic acids were 2.82 - 5.56%»of cell dry weight for 1. methanosorbosa, and Levine and Cooney (1975) found nucleic acids were 5 - 7% of cell dry weight of 3. polyporpha. Ogata 33 31. (1969 and 1970b) reported a total nucleic acid content 5 - 7% for Kloeckera _p. no. 2201. On the other hand, yeasts grown on other substrates generally were reported to have higher nucleic acid contents. Vananuvat and Kinsella (1975) reported the total nucleic acid content of crude lactose-grown §. fragilis was 12% of the cell dry weight. Castro 33 31. (1971) reported total nucleic acids to be in the range of 7.5 - 9.0%Iof cell dry weight for Q. utilis grown on glucose. The relatively low nucleic acid content of methanol- grown yeasts probably is related to the fact that nucleic acid content is usually prOportional to the growth rate. Knowing this and the fact that high concentrations of methanol retard the growth rate as shown previously and in other studies (Asthana 33 31., 1971; Cooney and Levine, 1972; Levine and Cooney, 1975), the low nucleic acid con- tent can be ascribed to the relatively low growth rate of methanol-grown yeast. However, more research needs to be done on the effect of methanol concentration on NA content. The nucleic acid content of SCP can be reduced to the extent that a relatively large quantity could be 45 ingested by humans without undue hazard. Several methods have been suggested for the reduction of the nucleic acid content of yeasts. These methods include: controlling the cell growth rate (Kihlberg, 1972), chemical extraction of the cell RNA (Kihlberg, 1972), disruption of cells (Dunell and Lilly, 1975), degradation of cell RNA by exogenous enzymes (Castro 33 313.1971), degradation of cell RNA by endogenous enzymes (Maul 33 31., 1970) and, finally, at- tempts to produce mutants with low RNA contents (Sinskey and Tannenbaum, 1975). SUMMARY AND CONCLUSIONS In 1969, it was first reported that yeasts can utilize methanol as the sole carbon source. Since that time, a considerable amount of research has been done on this subject. The present study was undertaken to investi- gate the characteristics of methanol-grown yeasts which serve as a possible source of protein. Sixty-seven strains of yeast were tested for their ability to utilize methanol as the sole source of carbon. As a matter of fact, none of these strains could utilize methanol. However, six methanol-utilizing yeasts were isolated from natural sources by means of a batch enrichment technique. One iso- late, identified as a strain of Candida boidinii, was se- lected for further study. The isolate had temperature and pH growth optima of 28° C and 4 to 6, respectively. Al- though biotin was found to enhance growth, the growth pro- ceeded slowly on vitamin-free methanol. The yeast grew in media containing up to 10%Imethanol. The duration of the lag phase was prolonged when methanol, rather than glucose, was used as the sole carbon source. The growth rate was retarded by high concentration of methanol. The maximum cell dry weight (8.6 g/l) was obtained with 5% (v/v) methanol. The protein content of the isolate was 41% and 46 47 the total nucleic acid (NA) content was 4.8% of cell dry weight. When glucose was substituted for methanol, a short lag phase and generation time were obtained. While there was no significant difference between methanol-grown cells and glucose-grown cells in the protein content, the glucose-grown cells had higher nucleic acid contents. In conclusion, methanol-grown cells of Q. boidinii ST exhibited relatively long lag phase, low growth rate and low nucleic acid content. The isolate appeared to be suit- able for SCP production from methanol. Taking into account the cost of raw materials, methanol compares favorably with the other substrates which could be utilized for SCP pro- duction. However, before establishing SCP production from methanol, using the isolate Q. boidinii ST, a continuous culture study would be necessary. BIBLIOGRAPHY 4s BIBLIOGRAPHY Abbott, B. and Clamen, A. 1975. The relationship of sub- strate, growth rate and maintenance coefficient to single cell protein production. Biotechnol. Bio- eng. 15:117. Alroy, Y. and Tannenbaum, S. R. 1975. The influence of environmental conditions on the macromolecular composition of Candida utilis. Biotechnol. Bioeng. 15:259. AOAC. 1975. Official methods of analysis. 12th edition. ’ Association of Official Agricultural Chemists. Washington, D. C. Asano, H., Watnabe, T. and Tokuyama, T. 1972. As cited by Ogata, K., Tani, Y. and Kato, N. 1975. Oxidation of methanol b yeasts. In "Microbial growth on 01- compounds." Free. Int. Symp. on Cl-Compounds. PP. 99—119. Soc. Ferment. Technol., Tokyo, Japan. Asthana, H., Humphrey, A. E. and Moritz, V. 1971. Growth of yeast on methanol as the sole carbon substrate. Biotechnol. Bioeng. 15:925. Bressani, R. 1968. The use of yeast in human foods. In "Single cell protein 1." Eds. Mateles, R. I. and Tannenbaum, S. R. PP. 90-121. MIT Press. Cam- bridge, Mass. Brock, T. D. 1974. Biology of microorganisms. PP. 285-522 Prentice-Hall, Inc., Englewood Cliffs, New Jersey. Bunker, H. J. 1965. Microbial Food. In "Biochemistry of industrial microorganisms." Eds. Rainbow, 0. and Rose, A. H. PP. 54-55. Academic Press, New York. Castro, A. C., Sinskey, A. J. and Tannenbaum, S. R. 1971. Reduction of nucleic acid content in yeast cells by bovine pancreatic ribonuclease treatments. Appl. Microbiol. 22:422. Chalfan, Y. and Mateles, R. I. 1972. New pseudomunad util- izing methanol for growth. Appl. Microbiol. 25:155. 49 50 Champagnat, A. 1965. Protein from petroleum. Scient. Amer. 215:15. Church, B. D., Erickson, E. E. and Wimer, C. M. 1975. Fungi digestion of food processing wastes. Food Technol. 27(2):56. Collins, B. 1976. Middle east plans are geared to big budgets and high prices. Oil Gas J. 24:71. Cooney, C. L. and Levine, D. W. 1972. Microbial utiliza- tion of methanol. Adv. Appl. Microbiol. 15:557. Cooney, C. L. and Levine, D. W. 1975. Single cell pro- tein production from methanol by yeast. In "Single cell protein 11." Eds. Tannenbaum, S. R. and Wang, D. I. C. PP. 402-425. MIT Press, Cambridge, Mass. Cooney, C. L., Levine, D. W. and Shedecor, B. 1975. Pro- duction of sin 1e cell protein from methanol. Food Technol. 29(12 :52. ~ Difco Laboratories. 1955. Difco manual. 9th ed. Detroit, Michigan. vanDijken, J. P., Veenhuis, M., Kreger-van Rig, N. J. W. and Harder, W. 1975a. Microbodies in methanol- assimilating yeasts. Arch. Mikrobiol. 102:41. vanDijken, J. P., Veenhuis, M. Vermeulen, C. A. and Harden fl. 1975b. Cytochemical location of catalase activ- ity in methanol-grown Hansenu13 polymorpha. Arch. Mikrobiol. 105:261. Dostalik, M. and Molin, N. 1975. Studies of biomass pro- duction of methanol-oxidizing bacteria. In "Single cell protein 11." Eds. Tannenbaum, S. R. and Wang, D. I. 0. PP. 586-401. MIT Press. Cambridge, Mass. Dunlap, C. E. 1975. Economics of producing nutrients from cellulose. Food Technol. 29(12):62. Dunell, P. and Lilly, M. D. 1975. Protein extraction and recovery from microbial cells. In "Single cell protein 11." Eds. Tannenbaum, S. R. and Wang, D. I. C. PP. 158-178. MIT Press. Cambridge, Mass. Fujii, T. and Tonomura K. 1972. Oxidation of methanol, formaldehyde and formate by a Candida pp. Agr. Biol. Chem. 56:2297. Fukui, S., Tanaka, A., Kawamato, S., Yasuhara, S. Terannxu, Y. and Osumi, M. 1975a. Ultrastructure of methanol- utilizing yeast cells: appearance of microbodies 51 in relation to high catalase activity. J. Bac- teriol. 125(1):5l7. Fukui, S., Kawamoto, S., Yasuhara, S., Tanaka, A., Osumi, A. and Imaizumi, F. 1975b. Microbody of methanol- grown yeasts: location of catalase and flavin- dependent alcohol oxidase in the isolated micro- body. Eur. J. Biochem. 59:561. Goto, S., Kitai, A. and Ozaki, A. 1975. Continuous yeast cell production from ethanol with a multi- stage tower fermenter. J. Ferment. Technol. 51:582. Goto, S. Okamoto, R., Kumajima, T. and Takamatsu, A. 1976. Growth characteristics Of methanol-assimilat- ing yeasts on various substrates. J. Ferment. Technol. 54:215. Gow, J. S. Littlehailes, J. D., Smith, S. R. L. and Walter, R. B. 1975. Single cell protein production from ethanol bacteria. In "Single cell protein 11." Eds. Tannenbaum, S. R. and Tang, D. I. 0. PP. 570- 584. MIT Press. Cambridge, Mass. Haggstrom, L. 1969. Studies on methanol oxidizing bac- teria. Biotechnol. Bioeng. 11:1045. Han, Y. H., Dunla p, C. E. and Callihan, C. D. 1971. Single cel protein from cellulosic wastes. Food Technol. 25:150. Hang, Y. D. and Splittstoesser, D. F. 1972. Sauerkraut wastes: a favorable medium for cultivating yeasts. Appl. Microbiol. 24:1007. Hatch, L. F. and Matar, S. 1977. From h drocarbons to petrochemicals. Hyd. Process. 56( ):l92. Hazeu, W., deBruyn, J. C. and Bos, P. 1972. Methanol assimilation by yeasts. Arch. Mikrobiol. 87:185. Herbert, D., Phipps, P. J. and Stange, R. E. 1971. Chemi- cal analysis of microbial cells. In "Methods in microbiology VB.” Eds. Norris, J. R. and Ribbons, g. 3. PP. 209-544. Academic Press. New York, New or . Hulse, J. H. 1974. The protein enrichment of bread and baked products. In "New protein foods, IA." Ed. Altschul, A. M. PP. 155-250. Academic Press. New York, New York. Huval, M. 1976. Industrial base aim of huge Saudi gas project. Oil Gas J. 74:86. 52 Jones, A. 1974. World protein resources. PP. 229-247. Halsted Press. New York, New Yark. Kaneda, T. and Roxburgh, J. M. 1959. A methanol- utilizing bacterium, II. Studies on the pathway of methanol assimilation. Can. J. Microbial. 5:187. Kata, N., Tani, Y. and Ogata, K. 1974. Enzyme system for methanol oxidation in yeasts. Agr. Biol. Chem. 58:675. Kihlberg, R. 1972. The microbe as a source of food. Ann. Rev. Microbial. 26:427. Kohler, G. 0. and Knuckles, B. E. 1977. Edible protein from leaves. Food Technol. 51(5):191. Large, P. J. and Quayle, J. R. 1965. Microbial growth on Cl-compaunds. Biochem. J. 87:586. Lawrence, A. J., Kemp, M. B. and Quayle, J. R. 1970. Al- ternative carbon assimilation pathways in methanol- utilizing bacteria. J. Gen. Microbiol. 65:571. Lipinsky, E. S. and Litchfield J. H. 1970. Algae, bac- teria and yeasts as food or feed. CRC Crit. Rev. Food Technol. 1:581. Lipinsky, E. S. and Litchfield, J. H. 1974. Single cell protein in perspective. Food Technol. 28(5):16. Levine, D. W. and Cooney, C. L. 1975. Isolation and characterization of a thermotolerant methanol- utilizing yeast. Appl. Microbial. 26:982. Litchfield, J. H. 1977. Single cell protein. Food Tech- nal. 51(5):175. Ladder, J. Ed. 1970. The yeasts, a taxonomic study. 2nd ed. North-Haaland Pub. Co. Amsterdam, Netherlands. Mateles R. I. 1968. Application of continuous culture. In "Single cell protein I." Eds. Mateles, R. I. and Tannenbaum, S. R. PP. 208-216. MIT Press. Cam- bridge, Mass. - Mateles, R. I. and Tannenbaum, S. R. Eds. 1968. Single cell protein I. MIT Press. Cambridge, Mass. Maul, S. G., Sinskey, A. J. and Tannenbaum, S. R. 1970. New process for reducing the nucleic acid content. Nature. 228:181. 53 Mehta, D. D. and Pau, W. W. 1971. Purify methanol this way. Hyd. Process. 50(2):115. Monode, J. 1949. The growth of bacterial cultures. Ann. Rev. Microbial. 5:571. Ogata, K., Nishikawa, H. and Ohsugi, M. 1969. A yeast capable of utilizing methanol. Agr. Biol. Chem. 33:1519. Ogata, K., Nishikawa, H., Ohsugi, M. and Tochikura, T. 1970a. Studies on the production of yeast. 1. A yeast utilizing methano as a sole carbon source. J} Ferment. Technol. 48:584. Ogata, K., Nishikawa, H., Ohsugi, M. and Tochikura, T. 1970b. Studies on the production of yeast. II. The cultural conditions of methanol-assimilating yeast, Klaeckera 33 Na. 2201. J. Ferment. Technol. 48:470. Ogata, K., Tani, Y. and Kata, N. 1975. Oxidation of methanol by yeasts. In "Microbial growth on C1- campounds." Prac. Int. Symp. on Microbial growth on Cl-campounds. PP. 99-119. Sac. Ferment. Technol. Takya, Japan. Ohta, S., Maul, S., Sinskey, A. J. and Tannenbaum, S. R. 1971. Characterization of a heat-shack process for reduction of the nucleic acid content of Candida utilis. Appl. Microbial. 22:415. Oki, T., Kauna, K., Kitai, A. and Ozaki, A. 1972. New yeast capable of assimilating methanol. J. Gen. Appl. Microbial. 18:295. Okumura, S. 1970. English Patent No. 1, 210, 770 as cited by Cooney, C. L. and Levine, D. W. 1975. Single cell protein production from methanol by yeasts. In "Single cell protein II." Eds. Tannenbaum, S. R. and Wang, D. I. C. PP. 402-425. MIT Press. Cam- bridge, Mass. Phaff, H. J., Miller, M. W. and Mrak, E. M. 1968. The fiife of yeasts. 2nd ed. MIT Press. Cambridge, ass. Pigott, G. H. 1976. New approaches to marketing fish. In "New protein foods I ." Ed. Altschulg, A. M. PP. 1-57. Academic Press. New York, New York. Quayle, J. R. 1972. The metabolism of one carbon compound by microorganisms. Adv. Microbial. Physiol. 7:119. 54 Reiser, C. 0. 1954. Tarula yeast from potato starch wastes. J. Agr. and Food Chem. 2:70. Ruggenkamp, R., Sahm, H., Hinkelmann, W. and Wagner, F. 1975. Alcohol oxidase and catalase in peroxisomes of methanol-grown Candida boidinii. Eur. J. Bia- chem. 59:251. Rosenzweig, M. and Ushio, S. 1974. Protein from Methanol. Chem. Eng. 81(1):62. Sahm, H. and Wagner, F. 1972. Mikrobielle verwentung van methanol: Isolietung und charakterisierung der hefe Candida boidinii. Arch. Mikrobiol. 24:155. Scrimshaw, N. S. 1975. Single cell protein for human can- sumptian. In "Single cell protein II." Eds. Tannenbaum, S. R. and Wang, D. I. C. PP. 24-45. MIT Press. Cambridge, Mass. Senez, J. 1972. Single cell proteins. The present and potential role of yeasts grown on alkanes. In "Proteins from hydrocarbons." Ed. Gaunelle de Pantanel, H. PP. 5-26. Academic Press. New York, New York. Shannan L. J. and Stevenson, K. E. 1975a. Growth of fungi and BOD reduction in selected brewery wastes. J. Food Sci. 40:826. Shannan L. J. and Stevenson, K. E. 1975b. Growth of Calvatia i antea and Candida steatal tics in brewery wasées for microEiaI protein and BOD re- duction. J. Food Sci. 40:850. Sinskey, A. J. and Tannenbaum, S. R. 1975. Removal of nucleic acids in sifigle cell protein. In "Single cell protein II." 5., Tannenbaum, S. R. and gang, D. I. 0. PP. 158-178. MIT Press. Cambridge, ass. Stewart, P. R. 1975. Analytical methods for yeasts. In "Methods in cell bialo XII.” Ed. Prescott, D. M. PP. 111-147. Academic see. New York, New York. Tani, Y., Miya, T., Nishikawa, H. and Ogata, K. 1972a. The microbial metabolism of methanol. I. Forma- tion and crystallization of methanol-oxidizing en- zyme in a methanol-utilizing yeast, Kloeckera 33. No. 2201. Agr. Biol. Chem. 56:68. Tani, Y., Miya, T. and Ogata, K. 1972b. The microbial metabolism of methanol. II. Properties of crystal- line alcohal oxidase from Kloeckera 3p, No. 2201. Agr. Biol. Chem. 56:76. 55 . Tannenbaum, S. R. and Wang, D. I. C. Eds. 1975. Single cell protein II. MIT Press. Cambridge, Mass. Tozuka, H., Nakahara, T., Minada, Y. and Yamada, K. 1975. Production of yeast cells from methanol. Agr. Biol. Chem. 39(1):285. vanUden, N. and Buckley, H. 1970. Candida Berkhout. In "The Yeasts." Ed. Ladder, J. PP. 895-1087. North Holland Pub. 00. Amsterdam, Netherlands. Uragami, T. and Domi, R. 1975. As cited by Ogata, K., Tani, Y. and Kata, N. 1975. Oxidation of methanol by yeasts. In "Microbial growth on Cl-campaunds." Proc. Int. Symp. on Microbial Growth an C1-compounda PP. 99-119. Sac. Ferment. Technol. Tokyo, Japan. Vananuvat, P. and Kinsella, J. E. 1975. Production of yeast protein from crude lactose. J. Food Sci. 40:556. Wang, D. I. C. 1968. Proteins from petroleum. Chem. Eng. 26:99. Wickerham, L. J. and Burton, K. A. 1948. Carbon assimila- tion tests for the classification of yeasts. J. Bacterial. 56:565. Yamada, K., Takahashi J., Kawabata, Y., Okada, T. and Onihara, T. 1968. Single cell protein from yeast and bacteria grown on hydrocarbons. In "Single cell protein .” Eds. Mateles, R. I. and Tannen- fiaum, S. R. PP. 192-207. MIT Press. Cambridge, ass. Yakote, Y., Sugimoto, M. and Abe, S. 1974. 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