Nitrogen Sources and the Growth Response of Pleurotus ostreatus Mushroom Mycelium

Can. Inst. Food Sel. Teehnol. J. Vol.

21,

No.

2,

pp.

194-199, 1988

RESEARCH

Nitrogen Sources and the Growth Response of Pleurotus ostreatus Mushroom Mycelium W. Manu-Tawiah and A.M. MartinI Food Science Program, Department of Biochemistry, Memorial University of Newfoundland. St. John's, Newfoundland Canada AlB 3X9

ducted in propagating edible mushroom mycelium in liquid-phase fermentations (Litchfield, 1967a; Martin, 1986). The objectives of these studies were the production of mycelial biomass to be used as a food, food additive or food supplement. One of the mushroom species most often studied in submerged culture is Pleurotus ostreatus (Zadrazil, 1975, 1978; Dijkstra, 1976; Labeneiah et al., 1977; Manu-Tawiah and Martin, 1987a). Several substrate sources have been tested in those studies. Peat (ManuTawiah and Martin, 1986) and acid-extracts from peat (Manu-Tawiah and Martin, 1987a) have been recently used in the growth of both P. ostreatus fruiting bodies and mushroom mycelium. Peat extracts have been employed as substrate for several fermentation processes (Quierzy et al., 1979; Boa and LeDuy, 1982; Martin, 1983a,b; Martin and White, 1985). Peat could provide a complex mixture of nutrients, mainly carbohydrates, and growth promoters to the culture medium. However, the amounts of some specific components present in peat could be insufficient to support active microbial growth. One of these components is nitrogen. The need for a certain nutrient to be present in a culture medium at specific concentrations is a function of the demand for that nutrient from the species being cultivated. Few studies have been conducted concerning the P. ostreatus mycelial growth demand for nitrogen (Sugimori et al., 1971; Bukhalo and Solomko, 1978). Manu-Tawiah and Martin (l987b) fpund that the P. ostreatus mycelial growth was poor in a peat extract culture medium without nitrogen supplementation. This work presents a study of the effect of different nitrogen sources on the growth of P. ostreatus mycelium grown in a peat extract medium and in a synthetic medium. Because it has been found that the mycelial biomass growth is related to the carbonto-nitrogen (C:N) ratio in the culture media (Litchfield, 1967a), the relationships between the dry biomass concentrations of the fungus and the C:N ratio, in both the peat extract and the synthetic media, have been investigated.

Abstract The effect of different sources of nitrogen on the mycelial growth of the edible mushroom Pleura/us as/rea/us has been studied. The organic nitrogen sources tested were yeast extract, peptone, and urea, and the inorganic nitrogen sources were ammonia solution, ammonium phosphate dibasic, ammonium citrate, ammonium nitrate and potassium nitrate. The concentrations of the different sources of nitrogen were calculated to provide 0.5 g/L of nitrogen to both a synthetic and a peat extract medium. The ammonia solution was used to provide nitrogen and also to adjust the pH of the peat extract medium. Different concentrations of yeast extract (1.0, 3.0, 5.0, 7.5 and 10.0 g/L), and (NH4)2HP04 (0, 2.5, 5.0 and 7.5 g/L) (which were the best nitrogen sources added to the peat extract medium) were studied for their effects on growth and on crude protein production. Ammonium citrate was the best nitrogen source added to the synthetic medium, producing 14.7 g/L of dry mycelium, which was the highest biomass concentration obtained in this work. The effect of the carbon: nitrogen ratios in the culture media on the mycelial growth is presented.

Resume L'effet de differentes sources d'azote sur la croissance du mycelium a ete etudie chez le champignon Pleura/us astreatus. Les sources d'azote organique qui ont ete etudiees furent I'extrait de levure, la peptone, l'uree, et les sources d'azote inorganique furent l'ammoniaque, le phosphate dibasique d'ammonium, le citrate d'ammonium, le nitrate d'ammonium et le nitrate de potasse. Les concentrations des differentes sources d'azote furent calculees pour apporter 0.5g/L d'azote a un milieu synthetique et a un milieu d'extrait de tourbe. L'ammoniaque fut utilisee pour apporter de I'azote et aussi pour ajuster le pH du milieu a l'extrait de tourbe. Differentes concentrations d'extrait de levure (1.0,3.0,5.0,7.5 et 10.0 g/L) et du (NH 4)2 HP0 4 (0, 2.5, 5.0, et 7.5 g/L) (qui furent les meilleures sources d'azote ajoutee au milieu d'extrait de tourbe) furent etudiees pour les effets de croissance et sur la production de proteine brute. Le citrate d'ammonium fut la meilleure source d'azote ajoutee au milieu synthetique produisant 14.76 OIL de 4 mycelium sec, qui fut la plus forte concentration de biomasse obtenue dans cette etude. On y presente I'effet des ratios carbone: azote dans le milieu de culture sur la croissance du mycelium.

Introduction Since 1950, when it was first reported that mushroom mycelium could be cultivated in submerged culture (Humfeld, 1948), several studies have been conITo whom all correspondence should be addressed.

Copyright

©

1988 Canadian Institute of Food Science and Technology

194

Materials and Methods

Preparation of peat extract· The peat utilized in this work was a high-moor Sphagnum peat moss of low degree of decomposition from bogs near the city of St. John's, Newfoundland. The humification value corresponded to H2 in the von Post scale (Fuchsman, 1980). The peat, as received (with a moisture content of approximately 80070), was air-dried (to a moisture content of 20%), and the peat extract was prepared according to the method of Martin and Bailey (1983). It involved mixing peat with 1.5% H 2S04 in a ratio of 20 g of dry peat to 100 g of acid solution, and autoclaving it at 15 psi (121 ± 1°C) for 2 h. The extract was separated by pressing the autoclaved product in a Carver Laboratory Press (Model C, F.S. Carver Inc., WI), followed by filtration through Whatman no. 1 filter paper. The peat extract was stored in plastic containers at 4°C until required for use. When employed, the extract was diluted to a 1: 1 ratio with distilled water.

Preparation of the synthetic medium The basal medium was prepared, according to the method of Srivastava and Bano (1970), by dissolving the following chemicals in a liter of deionized water: KH 2P04, 1 g; MgS04·7H20, 0.5 g; MnS04·H20, 3 mg; ZnS0 4,7H 20, 3 mg; FeS04,7H 20, 3 mg; Na2Mo0 4, 3 mg. Glucose was added to a concentration of 45 g/L. To this solution was added a mixture of vitamins containing thiamine, niacin, riboflavin, pantothenic acid and p-aminobenzoic acid, each at the rate of 100 j.tg/L, and cyanocobalamin, biotin, pyridoxine, and folic acid, each at the rate of 50 j.tg/L.

Nitrogen supplementation of the culture media The effect of different sources of nitrogen (ammonium citrate, ammonium phosphate dibasic, ammonium sulfate, ammonium nitrate, potassium nitrate and urea) were studied in the synthetic medium. The same sources of nitrogen, plus ammonia solution, yeast extract and peptone were also studied to determine the possibility of supplementing the low nitrogen content in the peat extract. The ammonia solution was used to provide nitrogen and also to adjust the pH of the peat extract medium. Different concentrations of yeast extract (1.0, 3.0, 5.0, 7.5 and 10.0 g/L) and (NH4)2HP04 (0, 2.5, 5.0 and 7.5 g/L), which were the best nitrogen sources, were added to the peat extract medium to study their effects on growth and on crude protein production. The concentrations of the different sources of nitrogen used were calculated to provide 0.5 g/L of nitrogen to the culture media. This was the most suitable concentration found in previous experiments on the cultivation of mushroom mycelium in peat extract media (Martin and Bailey, 1985). The effect of different concentrations of ammonium citrate (which was found to be the best nitrogen source in the synthetic medium) on the growth of the P. ostreatus mycelium, was also studied. Con. Inst. Food Sri. Technol. J. Vol. 21, No. 2, 1988

Culture conditions The culture of P. ostreatus No. 152 was obtained from the Dept. of Plant Sciences, University of Western Ontario, London, Canada. The inoculum was prepared as reported by Manu-Tawiah and Martin (1987a). The growth and biomass production of the P. ostreatus mycelium in the peat extract and in the synthetic medium were studied in shake flask fermentations. The pH of the growth medium was adjusted to the required values by the addition of either lactic acid (to the synthetic medium) or 10 M NaOH (to the peat extract medium) before sterilization at 121 ± 1°C for 30 min. The following optimal growth conditions were employed: a temperature of 28°C, an initial pH of 5, an inoculum ratio of 5.0% (v/v), an agitation speed of 150 rpm and a fermentation time of 192 h.

Analytical methods For the determination of the dry biomass weight, the total biomass in the culture medium after fermentation was filtered through oven-dried (at 105°C to constant weight) Whatman no. 1 filter paper. The filter paper with the mycelium was washed twice with distilled water to remove the fermentation broth and oven dried at 60-65°C to a constant weight. The initial inoculum dry weight was subtracted from the total biomass dry weight to obtain the dry weight of the mycelium produced. The biomass yield was calculated as grams of the mycelium dry weight produced per gram of the total carbohydrate consumed, and the efficiency was calculated as grams of the mycelium dry weight produced per gram of the total carbohydrate supplied. The total carbohydrate concentrations of the growth medium before and after fermentations were determined by the anthrone reagent method (Morris, 1948; Neish, 1952). The total nitrogen contents of the peat extract and the mycelium of P. ostreatus were determined by a modified micro-Kjeldahl method (AOAC. 47.021; 1980). The crude protein content in the sample was calculated from the % nitrogen content using the conversion factor (N x 6.25).

Experimental design and statistical analysis The design of the research consisted of series of randomized experiments with the two basic substrates: peat extract and the synthetic medium. The process variable selected for investigation in the first series was the nitrogen source, and in the second series it was the concentration of the nitrogen sources which had produced the best responses (dry biomass concentration, yield) in the first series. Protein content and efficiency were incorporated as measured responses in the second series of experiments. All experiments were conducted in triplicate. Since yields and efficiencies data were binomial and covered a range of values, each percentage was transformed Manu-Tawiah and Martin / 195

Table I. Effect of different sources of nitrogen on the growth of P. ostreatus mycelium in the peat extract. Dry Final Nitrogen biomass Yield pH of source conc. (0.5 g/L N) (g/L) (070) medium 1.06 ± 0.02a I 35.35 ± 1.92a None Ammonia 1.35 ± 0.02b 35.05 ± 1.07a 4.43 solution (29.8%) Ammonium citrate 1.01 ± 0.02a 29.55 ± 1.76b 5.17 Ammonium phosphate (dibasic) 37.30 ± I.l4a 4.95 3.40 ± 0.04c Ammonium sulfate 4.31 1.20 ± 0.03d 28.20 ± 1.92bc Ammonium 4.35 nitrate 0.91 ± 0.07a 23.20 ± 1.48d Potassium 4.68 nitrate 1.32 ± 0.02b 14.10 ± 0.85e Yeast 60.02 ± 1.I4f 6.47 extract 4.98 ± 0.05e 31.00 ± 1.90b 7.39 Peptone 2.89 ± 0.07f Urea 2.46 ± 0.02g 26.20 ± 1.I4c 5.98 I All data are means (standard deviation) of results obtained with three replicates. Percent yields were subjected to angular transformation (Snedecor and Cochran, 1980). Means in the vertical columns not followed by the same letter are significantly different (P :5 0.05) according to Duncan's Multiple Range Test. 2Means of three replicates. No statistical analysis was conducted for the pH values.

to the Arcsine of its square root in order to stabilize the variance (Snedecor and Cochran, 1980) and the means were analyzed using the Duncan's Multiple Range Test (Montgomery, 1984).

Results and Discussion Preliminary experiments in the growth of P. ostreatus in a non-nitrogen supplemented peat extract produced very little mycelial growth. Therefore, various organic and inorganic sources of nitrogen were individually used to supplement the peat extract (Manu-Tawiah and Martin, 1987b). Studies were also conducted using a synthetic medium with various ammonium salts as sources of nitrogen. Nitrogen supplementation, in most cases, produced higher mycelial concentrations than a non-supplemented peat extract (Table 1). The effect of the nitrogen supplementation

Table 2. Effect of different nitrogen sources on the growth of P. ostreatus mycelium in the synthetic medium. Dry Final biomass Nitrogen pH of Yield conc. source (%) medium (g/L) (0.5 g/L N) • • None trace Ammonium 14.68 ± 0.69a l 64.17 ± 1.62a citrate Ammonium phosphate 3.11 4.78 ± 0.36b 31.91 ± 1.02b (dibasic) Ammonium 3.58 5.20 ± 0.19c 38.88 ± 1.I3c sulfate Ammonium 4.05 nitrate 1.76 ± 0.19d 21.46 ± 0.75d Potassium nitrate trace • 4.95 5.96 Urea 9.06 ± 0.82e 52.93 ± 1.73e 1All data are means (standard deviation) of results obtained with three replicates. Percent yields were subjected to angular transformation (Snedecor and Cochran, 1980). Means in the vertical columns not followed by the same letter are significantly different (P :5 0.05) according to Duncan's Multiple Range Test. 2Means of three repligates. No statistical analysis was conducted for the pH values. • = Not determined.

on the growth of the P. ostreatus mycelium depended on the nitrogen source. Maximal growth values were obtained in the peat extract supplemented with yeast extract as the nitrogen source, which confirms previous findings concerning the beneficial effect of yeast extract on the growth of mushroom mycelium (Martin and Bailey, 1985; Manu-Tawiah and Martin, 1987b). In the synthetic medium, ammonium citrate produced the best growth (Table 2). Ammonium phosphate produced the second best growth in the peat extract medium. However, in the synthetic medium, urea and ammonium sulfate were more effective than ammonium phosphate in enhancing the mycelial growth.

Ability of the mushroom mycelium to utilize ammonium salts as nitrogen sources in submerged culture Fungi differ in their ability to utilize various ammonium salts as nitrogen sources for their growth (Hawker, 1968). In this work, P. ostreatus grew very

Table 3. Effect of different concentrations of ammonium phosphate on the growth and the crude protein content of the P. ostreatus mycelium in the peat extract. ~

~&

Ammonium phosphate conc. (g/L)

biomass protein (% of dry conc. Yield Efficiency (%) (%) mycelium) (g/L) o 1.06 ± 0.08a 1 25.35 ± 1.92a 7.55 ± 0.65a 9.81 37.30 ± 1.14b 22.65 ± 1.21b 13.75 2.5 3.40 ± O.04b 32.90 ± 1.50c 18.25 ± 1.29c 22.19 5.0 1.10 ± 0.05a • • 7.5 trace • 1All data are means (standard deviation) of results obtained with three replicates. Percent yields and efficiencies were subjected to angular transformation (Snedecor and Cochran, 1980). Means in the vertical columns not followed by the same letter are significantly different (P :5 0.05) according to Duncan's Multiple Range Test. 2Means of three replicates. No statistical analysis was conducted for the crude protein contents. • = Not determined.

196 / Manu-Tawiah and Martin

J. Inst. Can. Sci. Technol. Aliment. Vol. 21. No. 2, 1988

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well in the synthetic medium with ammonium citrate as a nitrogen source, but Brock (1951) found that ammonium citrate was toxic to Morchella esculenta. Ghosh and Sengupta (1978) observed very good gr0'Yt~s of Termitomyces clypeatus and Pana/olus papllllOnaceus when the medium was supplemented with ammonium phosphate, while the reverse was o?served for Gymnopilus chrysimyces. The apparent differences in the utilization of the various ammonium ~ompounds m~y b~ due to increases in the hydrogen Ion concentration III the media. Hawker (1968) has reported that when a fungus was grown with an ammonium salt of an inorganic acid as a source of nitrogen, the medium became more acidic. That acidity resulted from the rapid utilization of the ammonium ion. Srivastava and Bano (1970) have reported that when ammonium salts of inorganic acids were used as sole sources of nitrogen for the growth of Pleurotus flabellatus, the final pH of the medium dropped !o 3.0. Th~refore, the low pH values which developed III the medIUm when some ammonium salts were used as supplements could have been responsible for the poor growth observed in the peat extract medium (Hashimoto and Takahashi, 1974). The good growth obtained when ammonium phosphate was used, however, was due to the strong buffering capacity of the phosphate ions. Thus, they were able to maintain the pH values of the medium over a r~nge. suitable for growth of the mushroom mycelium (Lltchfleld, 1967a). However, this strong buffering Can. Inst. Food Sci. Technol. J. Vo!. 21, No. 2, 1988

AMMONIUM CITRATE CONC. (gIll

Fig. 2. Effect of different concentrations of ammonium citrate on the growth and the crude protein content of P. astreatus mycelium grown in a synthetic medium. Dry biomass concentration (.); yield (_); efficiency (A); crude protein content (0); final pH (.0,).

capacity was lost at higher concentrations of the ammonium salt, and acid reactions began to increase in the medium (Reusser et al., 1958a; Shannon and Stevenson, 1975). In this work, it was observed that the growth of P. ostreatus decreased when the concentration of ammonium phosphate was increased from 2.5 to 5.0 g/L. (Table 3). Ammonium citrate did not enhance the growth of !he P. ostreatus mycelium in the peat extract, although It was the best nitrogen source for the fungus in the synthetic medium. It could be that the ability to utilize a particular nitrogen source might depend, among other factors, on the nature of the carbon source used in the medium (Hawker, 1968). P. ostreatus grew poorly, in both the synthetic medium and the peat extract, when potassium nitrate or ammonium nitrate was used as the nitrogen source. A similar observation was made by Srivastava and Bano (1970) with P. flabellatus, and by Hashida et al. (1967) with Collybia velutipes. Hacskaylo et al. (1954) have reported that basidiomycetes utilize nitrate nitrogen very slowly or not at all.

Effect of increasing concentrations of nitrogen on the mycelial growth and the crude protein concentration Increased concentrations of nitrogen had a positive Manu-Tawiah and Martin / 197

influence on the growth and the crude protein concentration of the P. ostreatus mycelium. There was a smooth increase in the growth and in the crude protein concentration when the concentration of yeast extract in the peat extract medium (Figure 1), and the concentration of ammonium citrate in the synthetic medium (Figure 2) were increased. Maximal growth values were obtained at 7.5 g/L yeast extract in the peat extract medium, and at 3.5 g/L ammonium citrate in the synthetic medium. Higher concentrations produced a less favorable effect on growth. There was no significant difference (P > 0.05) between the corresponding values of the growth parameters at 5.0 and 7.5 g/L yeast extract in the peat extract or at 3.0 g/L and 4.0 g/L ammonium citrate in the synthetic medium. However, higher concentrations of the yeast extract and of the ammonium citrate produced an increase in the crude protein content in the mycelium. With the addition of ammonium phosphate to the peat extract, the growth followed a similar pattern. Table 3 shows that concentrations of ammonium phosphate higher than 2.5 g/L had a detrimental effect on the growth, but it produced an increase in the protein content of the mycelium. This corresponds with the observations made by other investigators, that the mycelial protein concentration of a variety of mushrooms could be increased markedly by increasing the amount of nitrogen in the medium (Humfeld and Sugihara, 1952; Reusser et al., 1958a; Falanghe, 1962; Litchfield, 1963, 1967b; Srivastava and Bano, 1970). Increased concentrations of yeast extract or ammonium phosphate also produced increased pH values in the media during the growth of the fungal mycelium in the peat extract. However, while the pH of the synthetic medium increased during the mycelial growth for ammonium citrate concentrations up to 3.5 g/L, higher concentrations produced a decrease in the pH.

Effect of the C:N ratio on the mushroom mycelial growth Several factors may influence the growth of the mushroom mycelium in submerged culture. Among these factors are the adequate amounts and the kinds of carbon and/or nitrogen sources, and a favorable quantitative balance (C:N ratio) of these nutrients. Moustafa (1960) and Falanghe et al. (1964) have reported that an optimal C:N ratio was needed for the effective reduction of the lag period, a maximum sugar consumption, and a good yield. Figure 3 shows the dry biomass concentration of the P. ostreatus mycelium as a function of the C:N ratios in the synthetic medium and in the peat extract. The optimal C:N ratio for P. ostreatus was about 40: 1 in both media. This compares very well with the optimal C:N ratio for the growth of P. flabellatus (Srivastava and Bano, 1970). The optimal C:N values for mushroom biomass production vary widely depending upon the species, the growth medium, and such operating conditions as temperature, pH, agitation and aeration. Reusser et al. (1958b) reported that the highest yields of Agaricus campestris NRRL 2335 mycelium grown 198/ Manu-Tawiah and Martin

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in a glucose/ammonium tartrate synthetic medium were obtained in the C:N range of 20: 1 to 25: 1. However, with another strain of this organism (NRRL 2334), Moustafa (1960) observed the best growth in a malt sprout extract/glucose medium in the C:N range of 8:1 to 12:1. Shannon and Stevenson (1975) obtained the best growth for Calvatia gigantea mycelium grown in brewery waste/ammonium sulfate medium, in the C:N range of 30: 1 to 40: 1. The approximately similar optimal C:N ratio found for the growth of the P. ostreatus mycelium in the synthetic medium and in the peat extract could mean that the C:N ratio which affords the maximum growth of mushroom mycelium in submerged culture is independent of the carbon and the nitrogen sources. Similar observations have been made by other investigators. For example, Morchella hortensis grown in a glucose/ammonium phosphate synthetic medium alone, and in a medium supplemented with corn steep liquors, produced the highest growth at a C:N range of 5:1 to 10: 1 (Litchfield, 1963). Litchfield and Overbeck (1965) obtained optimal yields of three species of morel mushrooms at a C:N ratio of 8: 1 in pumpkin waste and in cheese whey media.

Conclusions In this work, the response of the growth parameters dry biomass concentration, yield and efficiency of P. ostreatus mushroom mycelium to nitrogen supplementation of the culture medium depended on the nitrogen source. Also, the effects of the nitrogen sources utilized were affected by the kind of substrate, whether peat extract or synthetic medium, to which they were added. However, the C:N ratio composition in the culture media for optimal growth of the fungus was found to be independent of the substrate type employed.

Acknowledgment This work was supported in part by an Operating Grant from the Natural Sciences and Engineering J. Inst. Can. Sci. Technol. Aliment. Vo!. 21. No. 2, 1988

Research Council of Canada. The authors would like to thank Ms. Susan Legeza, Department of Plant Sciences, University of Western Ontario, for supplying the culture of P. ostreatus. The assistance of Mr. Paul Bemister, Biochemistry Department, Memorial University of Newfoundland, is appreciated.

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Can. Inst. Food Sri. Technol. J. Vo!. 21. No. 2, 1988

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Submitted August 3, 1987 Revised December 3, 1987 Accepted December 5, 1987

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