Growth of Fungi in Sea Water Medium'

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1 Growth of Fungi in Sea Water Medium' WILLIAM D. GRAY, PATRICK V. C. PINTO, AND S. G. PATHAK Department of Botany and Plant Pathology, The Ohio State University, Columbus, Ohio Received for publication 27 June 1963 ABSTRACT GRAY, WILLIAM D. (The Ohio State University, Columbus), PATRICK V. C. PINTO, AND S. G. PATHAK. Growth of fungi in sea water medium. Appl. MNicrobiol. 11: The effect of sea water on protein synthesis and growth of some species of Fungi Imperfecti was investigated. The dry weight of mycelia was greater from sea water medium than from distilled water medium in most instances; however, in a few there was a marked reduction in growth. The beneficial effect could not be ascribed to sodium choride or phosphate ion but may be due to the magnesium ion in sea water. This is indicated by the increase in mycelial yield and protein synthesis, even above that obtained with sea water, when magnesium ion was added to the medium. The reduction in yield and protein synthesis in some instances may be attributed to metal interactions; this possibility is being investigated further. At the initiation of an extensive program designed to explore the potential of fast-growing form species of Fungi Imperfecti as synthesizers of edible protein (Gray, 1962a), it was necessary to devise a single medium in which many such organisms would grow. The medium finally selected was a variation of one which had been used for other culture work in this laboratory and was compounded as follows: dextrose, 20.0 g; KH2PO4, 5.0 g; NH4NO3 or NH4Cl, 0.6 or 1.0 g; corn steeping liquor, 2.0 ml; trace elements solutions, 1.0 ml; FeCl3 solution (1.92 g per liter), 1.0 ml; ZnSO4.7H20 solution (44 g per liter), 1.0 ml; vitamin solution, 1.0 ml. The ph was adjusted to 6.0 with 0.1 N NaOH, and distilled water was added to a final volume of 1 liter. The stock vitamin solution and the trace elements solution were prepared as follows. The vitamin solution contained: p-aminobenzoic acid, 50.3 mg; thiamine hydrochloride, 99.6 mg; riboflavine, 50.0 mg; niacinamide, mg; inositol, mg; calcium pantothenate, mg; pyridoxine, 50.2 mg; and distilled water to a volume of 1 liter. The solution was refrigerated until use. The trace element solution contained: H3BO3, g; (NH4) 6Mio7 024*4H20, g; CuSO4*7H20, g; M\InCl2 *4H20, g; ZnSO4 7H2O, g; and distilled water to a volume of 1 liter. 1 Paper no. 659 from the Department of Botany and Plant Pathology, The Ohio State University. 501 The choice of whether NH4NO3 or NH4Cl was used as the nitrogen source for a particular organism was based on results from screening experiments in which it was determined which nitrogen salt permitted greatest yields of dry mycelium during a 4-day growth period in medium adjusted to an initial ph of 6.0. With the better nitrogen salt (for each individual test fungus) as the source of nitrogen, a wide variety of imperfect fungi were examined; as might be expected, their total dry weight yields as well as their protein content varied considerably. Some organisms were so inefficient in the conversion of substrate carbon to tissue carbon that they received no further attention; others were quite efficient in this respect and hence were judged to have greater immediate potential. Consequently, details of optimal temperature, ph, concentration of various medium ingredients, etc. were investigated further. Among the more promising organisms were Heterocephalum aurantiacum and Epicoccum sp., which in large-bottle, aerated cultures yielded 55.2 and 81.7 g, respectively, of dried mycelia from 100 g of glucose. Crude protein content varied from 9.11 to 35.0% (dry weight basis) depending on species and environmental conditions. With results such as those mentioned, it became apparent that, among the Fungi Imperfecti, forms exist which might well be of considerable value in the conversion of cheap carbohydrate and inorganic salts of nitrogen to high-protein fungus mycelium. In view of the carbohydrate excesses and protein deficiencies existing in many areas of the world, such a conversion process could make tremendous contributions to the world protein pool (Gray, 1962b). However, one major criticism of such a process might be that, to attain production of a magnitude that could add significantly to man's protein supply, tremendous volumes of water would be required. In many areas of the world, fresh water simply is not available to the extent that would be required; hence, experiments were initiated to determine whether sea water could be substituted, at least in part, for fresh water in the preparation of culture medium: The isolation of new fungal species from the oceans (Johnson, 1956; van Uden and Castelo- Branco, 1961) and the reports (Carlucci and Pramer, 1960a, b; MIacLeod, Onofrey, and Norris, 1954; MIacLeod and Onofrey, 1956; Vishniac, 1955) of the beneficial effect of sea water on growth or product biosynthesis are other factors that prompted investigation of the effect of sea

2 502 GRAY, PINTO, AND PATHAKA APPL. MICROBIOL. water on protein synthesis and growth of the Fungi Imperfecti. EXPERIMENTAL PROCEDURE AND RESULTS Sea water was obtained by filling 1-gal bottles with water dipped directly from the Gulf of -Mexico or the Atlantic Ocean along the Florida coast. In the event that sand was in the bottle, the water was filtered prior to use; otherwise, it was not treated in any special fashion and was substituted directly for distilled water in the preparation of the medium described above. The first experiment was conducted with H. aurantiacum. Two lots of media were prepared, one with sea water and the other with distilled water. Both were adjusted to ph 6.0 prior to making to final volume, and 50-ml portions were then dispensed in 250-ml Erlenmeyer flasks which were cotton-stoppered and autoclaved for 20 min at 15 psi of steam pressure. Six flasks containing sea water medium and six containing distilled water medium were then inoculated by adding 0.1 ml of spore suspension (prepared by suspending the spores of a fresh slant culture in 10 ml of sterile distilled water) to each flask. The flasks were then incubated at 25 C with continuous agitation in a constant-temperature water bath shaking apparatus. After 4 days, the mycelia were recovered from each flask (by filtration on weighed filter papers), dried, and weighed; mycelial yields per flask were calculated. The average weight of mycelium recovered from each flask containing distilled water medium was 207 mg, and the average from flasks containing sea water medium was 271 mg an increase in yield of 30.9 %. In view of the results obtained with H. aurantiacum, similar experiments were conducted with 19 other imperfect fungi, one Basidiomycete (Collybia velutipes, OSU no. 313), one Ascomycete (Morchella cr-assipes, OSU no. 146), and one Phycomycete (Rhizopus nigr icans, OSU no. 211). Since the Basidiomycete and Ascomycete did not sporulate in culture, inocula of each of these fungi were prepared by macerating a 10-mm mycelial pellet in 50 ml of sterile medium for 7 sec in a sterile Waring Blendor and then using 1 ml of the resultant suspension of mycelial fragments to inoculate each 50-ml portion of medium. The results obtained in these experiments are listed in Table 1. The data of Table 2 were calculated from the results in Table 1 and are presented here to illustrate the potential of each of the test organisms in the conversion of substrate carbon to tissue carbon. From the results presented in Table 1, it is apparent that, in all except two instances, dry weight yields of mycelium were greater from sea water medium than from distilled water medium. The two exceptions were 1-99 and 1-92 (Sepedoniumn sp. and Hormiactella sp.), the growth of these two fungi being decreased rather than increased by sea water. Additional experiments were conducted with both of these fungi, and the results obtained verified the original findings. The greatest percentage of increase in TABLE 1. Comparison of mycelial yields (dry weight) obtained in medium prepared with sea water and medium prepared with distilled water Yield (mg) Per cent Nitrogen change OSU no. Fungus source Sea Distilled disetillsd water water water) I-14 Phoma NH4NO I-36 Cephalothecium NH4NO I-101 Cylindrocephalum NH4NO I-114 Geomyces NH4NO I-100 Linderina NH4C Sepedonium NH4NO I-134 Spicaria NH4C I-104 Tritirachium NH4NO I-41 Bispora NH4Cl I-81 Brachysporium NH4NO I-83 Cladosporium NH4C Curvularia NH4C Hormiactella NH4NO I-80 Pullularia NH4C I-21 Stemphylium NH4NO I-111 Verticillicladium NH4C I-157 Epicoccum NH4NO I-11 Myrothecium NH4NO I-19-8 Spegazzinia NH4NO I-9 Heterocephalum NH4NO Rhizopus NH4NO Collybia NH4NO Morchella NH4NO TABLE 2. Comparison of the efficiencies of conversion of substrate carbon to tissue carbon by fungi cultured in sea water and distilled water media* Glucose required for production OSU no. Fungus of 1 mg of mycelium Sea water Distilled water nlg mrg I-4 Curvularia I-21 Stemphylium Phoma Geomyces I-lll Verticillicladium I-83 Cladosporium Pullularia I-81 Brachysporium Cephalothecium Rhizopus Spicaria Epicoccum Hormiactella I-ll Myrothecium I-19-8 Spegazzinia Linderina Cylindrocephalum Heterocephalum I-104 Tritirachium I-41 Bispora Collybia I-99 Sepedonium Morchella * Fungi are listed in the order of decreasing efficiency in sea water, and calculations are based on quantity of glucose supplied.

3 VOL. 11 X 1963 GROWTH OF FUNGI IN SEA WATER MIEDIUM 503 mycelial yield was obtained with I-104 (Tritirachium sp.) and the smallest percentage of increase was obtained with I-ll (Myrothecium sp.). The average percentage of increase for the 21 fungi whose yields were increased when they were cultured in sea water medium was 51.7 %. The generally beneficial effect of sea water upon efficiency of conversion of substrate carbon to tissue carbon is illustrated in Table 2. From this table, it may be noted that 12 of the 23 test organisms required less than 2 mg of sugar for the production of 1 mg of mycelium in sea water medium, whereas only four grew so efficiently in distilled water medium. The exceptionally high efficiencies of the first three fungi listed in Table 2 are somewhat unusual for fungi; the values listed in connection with these forms should not be accepted as absolute, since, in addition to sugar, carbon-containing compounds were also added in the form of corn steeping liquor. In medium prepared with distilled water, a small amount of precipitate usually appeared during autoclaving; however, in medium prepared with sea water, rather copious quantities of white precipitate appeared during autoclaving. After such media were inoculated and mycelial growth had become obvious, the precipitate slowly disappeared, and usually by the end of 3 days the media were clear. It was thought initially that the beneficial effect of sea water might be due to the fact that greater quantities of phosphate were precipitated out of this medium and that, as the remaining phosphate in solution was utilized by the growing fungus mycelium, precipitated phosphates slowly dissolved and in turn were utilized by the mycelium. To test this possibility, sea water medium was prepared as usual, adjusted to ph 6.0, and made to volume. It was then dispensed in 50-ml portions in 12 Erlenmeyer flasks (250-ml). All 12 flasks were autoclaved and, after cooling, the precipitate was removed from 6 flasks by filtration. All flasks were then autoclaved a second time and inoculated with spores of H. aurantiacum. After 4 days of incubation, the average yield per flask in unfiltered sea water medium was 380 mg, and that in filtered medium was 363 mg. Thus, the removal of precipitate effected a slight reduction in yield of mycelium, but this reduction was so slight (4.5 %) that phosphates being precipitated from the solution could scarcely be accepted as a plausible explanation of the beneficial effect of sea water on fungus growth. Although there are many salts in sea water, one of the principal ones is NaCl; therefore, two experiments were conducted to determine whether the observed effects could be due simply to the presence of the added NaCl. A medium was compounded in the usual manner except that 0.9 % NaCl solution was used instead of distilled water or sea water. Yields obtained in this type of medium were compared with yields obtained in flasks containing distilled water medium which were incubated at the same time. The average yield per flask of I-9 (Heterocephalum) was 342 mg in distilled water medium compared with only 178 mg in medium containing 0.9 % NaCl. Similar results were obtained with I-100 (Linderina), although the inhibitory effect of NaCl was much less pronounced with this latter fungus; the average yield was 324 mg in NaCl medium compared with 335 mg in distilled water medium. Obviously, the observed beneficial effects of sea water cannot be ascribed to an effect of greater NaCl concentration. Since artificial sea water compositions (M\acLeod et al., 1954; MacLeod and Onofrey, 1956) indicated a high magnesium content, 12 organisms were selected and cultured in distilled water medium containing in addition 0.1 % magnesium sulfate heptahydrate under the same conditions employed for the earlier work. MIedia with distilled water and sea water served as dual controls. The data obtained (Table 3) indicate that the mycelial yields, per cent nitrogen, and total nitrogen content with magnesium sulfate were equal to or better than the corresponding values for distilled water or sea water in most instances. The only exceptions are Bispora, Hormiactella, and Sepedoniumn (I-41, 1-92, 1-99) when mycelial yields or total nitrogen are compared, and Cladosporiumn and TABLE 3. Comparison of mycelial weight, per cent nitrogen, and total nitrogen content of 12 form species of Fungi Imperfecti when cultured on distilled water mediurm, sea water medium, and distilled water medium with added magnesium Distilled water Sea water Distilled water + Mg++ Organism Mycelial Per cent Total N Mycelial Per cent Total N Mycelial Per cent Total N wt N (m/lakvt N (mg/flask) wt N (mg/dask) (mg/flask) (mg/flask) (mg/aask) Heterocephalum (I-9) Phoma (1-14) Spegazzinia (I-19) Bispora (I-41) Cladosporium (I-75) Brachysporium (1-81) Cladosporium (I-83) Hormiactella (1-92) Sepedonium (I-99) Linderina (1-100) Verticillicladium (I-ill) Spicaria (I-134)

4 5)04 GRAY, PINTO, AND PATHAKA APPL. MICROBIOL. C TABLE 4. Comparison of ntycelial weight, per cent nitrogen, and total nitrogen content of five form species of Fungi Imperfecti when cultured on distilled water mediumi, sea water mediuim, distilled water and calcium, distilled water and magnesium, and distilled water plus magnesium and calcium Distilled water Sea water Distilled water + Mg Distilled water + Ca Distilled water + Ca + MIg Organism Mlycelial Per Total N Mycelial Per Total N Mycelial Per Total N Mycelial Per Total N Mycelial Per Total N wt cent (mg/ wt cent (m/ wt cent (mg/ wt cent (mg/ wt~, cent (mg/ N N flask) Nt flask) flask) N flask) flask) flask) flask) flask) Heterocephalumt (1-9) Bispora (1-41) Brachysporiuim (1-81) Sepedonium (1-99) Linderina (1-100) Horiniactella (1-83 and 1-92) when per cent nitrogen is compared. Reports on calcium-magnesium interaction (Vishniac, 1955; Cochrane, 1958), as well as other metal interactions that suggested a critical function of the relative concentrations of metals (Darby and MIardy, 1956; Fottergill and Rainie, 1954; Fottergill and Ashcroft, 1955; Fottergill and Yeoman, 1957; Vishniac, 1955), led to the study of the effect of calcium chloride (0.04%), singly and in combination with magnesium sulfate, on the mycelial yields and protein synthesis of a few of these organisms. The experiment was conducted in a manner similar to that of the magnesium sulfate experiment (Table 4). Calcium singly was inhibitory to Heterocephalum but had no effect on Brachysporium and Linderina, was very slightly stimulatory to Bispora (less than magnesium), and was equally stimulatory in the case of Sepedoniunti. In combination with magnesium, the toxicity of calcium to Heterocephalumt was overcome. In the case of Bispora, there was an increase as compared with either employed singly, but the increase was not as great as that obtained with sea water. With Brachysporium, calcium was not toxic but it reversed the effect of magnesium; with Linderina, there was not only reversal but an inhibition that resulted in yields below those obtained with distilled water. No antagonism was observed with Sepedoniui.i_ DISCUSSION From the results presented above, it is apparent that all of the test fungi not only are capable of growing in media prepared with untreated sea water but that growth of the majority was actually enhanced. The bulk of the experiments reported were conducted with various imperfect fungi, and, if the sampling from this group of fungi is at all representative, it may be anticipated that sea water will have a beneficial effect on the growth of many more such fungi. Thus, from the purely practical standpoint the re(luirement for vast (luantities of water for the mass production of fungus protein need not be a handicap in areas where there is a paucity of fresh water provided there is access to ocean water. The underlying cause of the beneficial-effect of sea water in the present investigation appears to be the magnesium ion, present in the sea water. The antagonistic effect of calcium and the reversal of the calcium or magnesium effect by the other ion are other indications that it is the magnesium ion that is involved. In some instances, calcium may substitute for magnesium, although in quite a few one may expect antagonism. The data do not indicate the reasons for the exceptions observed with addition of magnesium. It may be the concentration of magnesium or the relative concentrations of magnesium and calcium that are important (e.g., Bispora), or there may be metal interaction involving other metals (Hormiactella). An investigation of other metals like Fe, i\in, Zn, and KxHyPO4, as well as the effect of varying concentrations of sea water Carlucci and Pramer, 1960a, b; Jann, Walch, and Salle, 1953; Painter, 1954), with other organisms would help in better understanding the role of metal ion concentrations on growth and protein synthesis. The report of Sverdrup and Fleming (1941) on the variability of sea water composition indicates that further study with different sea water samples is necessary to ascertain more fully the effect of sea water. Finally, the commonly employed definition of marine organisms (those that develop better on media prepared with sea water than on media prepared with fresh or distilled water) should be re-examined. MIacLeod and Onofrey (1956) suggested the requirement for sodium as the critical factor for classifying an organism as marine, but the difficulty of obtaining sodium-free media and the possibility of other ions (like K, Rb, or Cs) replacing sodium in some or all environments make the evaluation of this suggestion very difficult. ACKNOWLEDGMENTS The authors are indebted to the I\Iershon Committee for Education in National Security (The Ohio State University) and the Rockefeller Foundation for financial assistance which made this investigation possible. LITERA\TURE CITED CARLUCCI, A. F., AND D. PRAMER. 1960a. An evaluation of factors affecting the survival of Escherichia coli in sea water. I. Experinieital procedure. Appl. Microbiol. 8:

5 VOL. 11, 1963 GROWTH OF FUNGI IN SEA WATER MEDIUM 505 CARLUCCI, A. F., AND D. PRAMER. 1960b. An evaluation of factors affecting the survival of Escherichia. coli in sea water. II. Salinity, ph, and nutrients. Appl. Microbiol. 8: COCHRANE, V. M Physiology of fungi. John Wiley & Sons, Inc., New York. DARBY, R. T., AND G. R. MARDY Inorganic nutrition of Myrothecium verrucaria. Mycologia 46: FOTTERGILL, P. G., AND R. ASHCROFT The nutritional requirements of V. inequalis. J. Gen. Microbiol. 12: FOTTERGILL, P. G., AND L. C. D. P. RAINE The mineral requirements of Mucor hiemalis. J. Gen. Microbiol. 10: FOTTERGILL, P. G., AND M. M. YOEMAN Mineral nutrition of Mucor stolonifer. J. Gen. Microbiol. 17: GRAY, W. D. 1962a. Microbial protein for the space age. Develop. Ind. Microbiol. 3: GRAY, W. D. 1962b. Fungi as a nutrient source. Biologistics for space systems symposium. Technical Documentary Report No. AMRL-TDR , p th Aerospace Medical Research Laboratories, Wright-Patterson Air Force Base, Ohio. JANN, G. J., H. A. WALCH, JR., AND A. J. SALLE Enhancement of antibiotic production by the use of sea water media. Appl. Microbiol. 1: JOHNSON, T. W., JR Marine fungi. I. Leptosphaeria and Pleospora. Mycologia 48: MAcLEOD, R. A., AND E. ONOFREY Nutrition and metabolism of marine bacteria. II. Observations on the relation of sea water to the growth of marine bacteria. J. Bacteriol. 71: MACLEOD, R. A., E. ONOFREY, AND M. E. NORRIS Nutrition and metabolism of marine bacteria. I. Survey of nutritional requirements. J. Bacteriol. 68: PAINTER, H. A Factors affecting the growth of some fungi associated with sewage purification. J. Gen. Microbiol. 10: SVERDRUP, H. U., AND R. H. FLEMING The waters off the coast of southern California, Mar.-Jul Scripps Institute of Oceanography. Calif. Univ. Tech. Ser. 4: VAN UDEN, N., AND R. CASTELO-BRANCO Metschnikowiella zobelli sp nov. and M. krissi sp nov.: 2 yeasts from the Pacific Ocean, pathogens for Daphnia magna. J. Gen. Microbiol. 26: VISHNIAC, H. S Nutritional requirements of isolates of Labyrinthula spp. J. Gen. Microbiol. 12: Downloaded from on January 15, 2019 by guest