Production of protein-rich mycelial biomass of a mushroom, Pleurotus sajor-caju, on corn stover

JOURNAl. OF FERMENTATIONAND BIOENGINEERING Vol. 68, No. 5, 334-338. 1989

Production of Protein-Rich Mycelial Biomass of a Mushroom, Pleurotus sajor-caju, on Corn Stover DEVINDER SINGH CHAHAL

University of Quebec, Institut Armand-Frappier, 531 boulevard des Prairies, Laval, Quebec, Canada, H7V 1B7 Received 14 March 1989/Accepted 21 August 1989

Pleurotus sajor-caju was adapted to grow submerged without any loss in the economic yield coefficient on up to 3% wt./vol of corn stover in Mandels and Weber medium without yeast extract or any other growth factors added. The final product (mycelial biomass) contained 40-49% protein (N x 6.25). The amino acid profile of the protein was as good as of any edible fungus except that it was deficient in sulfur amino acids as is characteristic of most fungi. The preliminary feeding trials on rats indicated that about 50% of protein in the diet can be replaced with that of P. sajor-caju without any deleterious effects.

Cultivation of Pleurotus spp. as mushrooms is becoming important throughout the world because of their abilities to grow in a wide range of temperatures (20-35°C) and on various lignocelluloses, and their shorter cultivation time than that of Agaricus spp. Recently it has become popular in Canada (1--4) and in India (5-7). Production of mycelial biomass of various mushrooms in submerged culture is another way to use them as food, food additives, or mushroom-flavor agents. Production o f mycelium of mushrooms, Agaricus spp., in submerged culture, as a substitute of mushrooms for culinary purposes, was started as early as 1949 (8). Later on, mycelia of morel and other mushrooms was produced by other researchers (9-13). Szuecs (Szuecs, J., US Patent 2850841, 1958) got a patent on methods for producing mycelial biomass o f several mushrooms. The mycelium of Pleurotus spp. has been produced in submerged cultures by various researchers (9, 14-18) on various substrates (beer wort, sulfite liquor from wastes of paper manufacturing, hemicellulose fraction, glucose, sugar beet molasses, apple distillery waste, and peat extract). But very little is know about the growth of Pleurotus spp. on lignocelluloses in submerged cultures. This paper reports the production of protein-rich mycelial biomass with P. sajorcaju on pretreated corn stover without its fractionation into cellulose, hemicelluloses, and lignin.

fermentation medium. Fermentation medium Two different media that of Chahal and Gray (22) and that of Mandels and Weber (23), were used in this study. Proteose peptone was replaced with 0.5 g/l of yeast extract (Difco) in Mandels and Weber medium. The nutrients of the fermentation media were added according to the content of total carbohydrates (7 g//) present in the treated corn stover (l°Jo wt./vol) and pH was adjusted to 6.0. The 250-ml Erlenmeyer flasks containing 100ml of medium were sterilized at 121°C for 20min. lnoculum P. sajor-caju was grown at 30°C on Mandels and Weber medium containing 1 ~ glucose as a carbon source in 250-ml Erlenmeyer flasks on a rotary shaker at 200 rpm for 60 h for most of the experiments. Variations in inoculum productions for other experiments are explained at their appropriate places. The inoculum was used at the rate of 1 0 ~ vol/vol. The inoculum was blended in a Waring blender for 1 min under aseptic conditions, to have an homogenous inoculum of well-dispersed mycelial bits. In the experiments on the effects of different media, the inoculum was washed with sterile distilled water twice to eliminate any residual nitrogen or growth factors from yeast extract in the case of Mandels and Weber medium adhering to the mycelium. Dry weight of final product Dry weight (DW) of the final product was measured by filtering 100 ml of sample through a weighed filter paper Whatman no. 4. The residual biomass was washed three times with distilled water, dried overnight at 80°C, then weighed. Total nitrogen in the final product Total nitrogen was measured by the micro-Kjeldhal method of A . O . A . C . (24) and crude protein content was calculated as 6.25 times nitrogen. As it was difficult to separate mycelial biomass from that of unused substrate, the growth rate of P. sajorcaju was calculated on the basis of protein synthesized by its mycelial biomass in the final product.

MATERIALS AND METHODS

The culture of Pleurotus sajor-caju (Ft.) Singer was maintained on yeast malt agar slants (Difco) at 30°C. When fully grown after a week, the cultures were stored at 4°C. Substrate and its pretreatment Corn stover is composed of about 60-70°//00 polysaccharides, 5.5°Jo protein, and 30o/00o f lignin and other materials (19-21). Keeping in view the abundance of corn stover in the world as a renewable carbon source, it was used as a substrate in this study for production of protein-rich mycelial biomass (commonly called single-cell protein-SCP). Corn stover ground to 20 mesh was treated with 1.5°Jo sodium hydroxide (NaOH) wt./vol with a substrate : water ratio of 1 : 10 at 121°C for 60 rain. The substrate was not washed after treatment, and all the solubilized materials (hemicelluloses, lignin, etc.) were retained along with the insoluble cellulose in the Organism

Economic yield coefficient and economic protein productivity The yield coefficient is generally represented as

the yield of the product (g//) divided by the substrate consumed (g//). But in this paper, a term "economic yield coefficient" has been used which is evaluated as the total yield of the product (mycelium or protein) produced (g//) divided by the total substrate (glucose/corn stover) supplied (g//). Because the substrate, corn stover, in this case 334

VoL 68, 1989

MYCELIAL BIOMASS OF A MUSHROOM

335

TABLE 1. Growth of P. sajor-caju with and without nitrogen in various mediaa Total mycelial dry wt. produced (g/l)b

Substrate I. Mandels and Weber medium (complete) II. Mandels and Weber medium (without nitrogen and yeast extract) III. Chahal and Gray medium (complete) (no yeast extract) IV. Chahal and Gray medium (without nitrogen and yeast extract)

Economic yield coefficient (mycelium)

Protein content of mycelium (%)

Total protein synthesized (g/l)c

Economic yield coefficient (protein)

4.9d

0.49

49.0

2.12

0.212

2.3d

0.23

15.2

0.06

0.006

3.8d

0.38

40.7

1.26

0.126

2.0d

0.20

15.0

0.02

0.002

a Both the media contained 10 g of glucose//. b After 57 h of growth in shake flasks (the time when maximum mycelial dry weight was recorded). Initial protein content at 0 h was 0.28 g/l. It was subtracted from the final protein content. d Initial mycelial dry wt. at 0 h was 0.58 g/l. It was subtracted from the final mycelial dry weight. is a mixture of polymers of various sugars (glucose, xylose, galactose, arabinose, sugar acids etc.) mixed with lignin and it was difficult to estimate the residual heterogenous substrate. "Economic protein productivity" is calculated as total protein synthesized (g//) divided by time (h) of fermentation. In fact on industrial bases the "economic yield coefficient" is more meaningful to find how much product is produced from a unit of substrate supplied rather than the substrate consumed. F r o m the economic point of view the high yield coefficient based on substrate consumed has no meaning, if the organism was able to consume only 50% of the substrate supplied under given fermentation conditions.

thetic media unless supplemented with vitamins (28, 29). Hadar and Cohen-Arazi (17) used 0.065% asparagine and 0.050//00 yeast extract; and M a n u - T a w i a h and Martin (18) used 0.5°//00 yeast extract in their culture media containing sugars to grow Pleurotus ostreatus in submerged culture. In our previous experiment P. sajor-caju gave a poor yield of mycelial biomass when grown on Chahal and Gray medium which was devoid of yeast extract and of any other growth factors indicating that the test fungus needs some growth factors for its growth. Use of yeast extract or other growth factors in the culture media for the production of mycelial biomass, to be used as food or food additives, may not be an economical proposition, therefore, P. sajor-caju was adapted to grow on Mandels and Weber

R E S U L T S A N D DISCUSSION Effects of different media P. sajor-caju was grown on Mandels and Weber medium a n d on Chahal and Gray medium with and without any nitrogen source. Glucose was used as a carbon source. It grew very well on Mandels and Weber medium and economic yield coefficients in respect of mycelium and protein were 0.49 and 0.212, respectively (Table 1). The economic yield coefficients on Chahal and Gray medium by P. sajor-caju were lower than that produced o n Mandels and Weber medium. P. sajorcaju produced some mycelium on both the media without any nitrogen source, but there was very little increase in total protein synthesized. This very small increase in protein synthesis could be attributed to the traces of nitrogen f o u n d in other salts used in these media. A small increase in dry weight of mycelium could also be attributed to the synthesis of more mycelial polysaccharides and the possibility of synthesis of some lipids due to lack of nitrogen in these media. It seems that P. sajor-caju failed to fix any atmospheric nitrogen to produce protein-rich mycelial biomass under the tested conditions during its entire period of growth of 57 h. Thus its so-called nitrogen fixation capability reported by some researchers (7, 25, 26) cannot be exploited on a commercial scale for synthesis of single-cell protein from lignocelluloses or other carbon sources. In fact, Shi-li et al. (27) had shown that P. sajor-caju was incapable of fixing atmospheric nitrogen. As Mandels and Weber medium supported better production of mycelial biomass and protein synthesis, it was used in all the following experiments either as such or with modified composition. Elimination of yeast extract from the cultural medium The mycelia of certain mushrooms will not grow in syn-

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FIG. 1. Growth of adapted strain of P. sajor-caju on 1% corn stover in Mandels and Weber medium. Protein produced on medium with yeast extract (e solid lines) and on medium without yeast extract (© dotted lines). Final product dry weight on medium with yeast extract (11 solid lines) and on medium without yeast extract (• dotted lines).

336

J. FERMENT. BIOENG.,

CHAHAL TABLE 2.

Effects of concentration of corn stover on protein production with an adapted strain of P. sajor-caju a,b

conc. of corn stover (g/l)

Time of fermentation (h)

Initial protein conc. (g/l)

Final protein conc. (g/l)

Total protein synthesized (g//)~

Economic protein productivity (g//.h)

Economic yield coefficient (protein)

Growth

10 20 30

54 60 96

0.5 0.9 1.4

2.0 3.6 6.3

1.5 2.7 4.9

0.027 0.045 0.051

O. 15 O. 135 O. 16

0.036 0.031 0.017

rate (h ~)

a Basal medium without yeast extract. Inoculum was produced on corn stover without yeast extract. Conc. of nutrients in the basal medium was correspondingly increased with the increase in conc. of corn stover. c Total protein synthesized (g//)=Final protein concentration (g/l) Initial protein concentration (g/l). 4.0-

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FIG. 3. Growth of adapted strain of P. sajor-caju on 30/00corn stover in Mandels and Weber medium without yeast extract. Symbols: e, protein synthesized; [], final product dry weight.

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Time (h) FIG. 2. Growth of adapted strain of P. sajor-caju on 2%0 corn stover in Mandels and Weber medium without yeast extract. Symbols: e, protein synthesized; [], final product dry weight. m e d i u m w i t h o u t s u p p l e m e n t a t i o n With yeast extract or any o t h e r g r o w t h factors by several c o n t i n u o u s serial subculturings on c o r n stover. W i t h the a d a p t e d strain a l m o s t the s a m e q u a n t i t y o f p r o tein was p r o d u c e d with a n d w i t h o u t yeast extract in 48 and 54 h o f f e r m e n t a t i o n on 1%o c o r n stover (Fig. 1). T h e first i n o c u l u m in this case was p r o d u c e d on M a n d e l s and W e b e r m e d i u m with I °Jo glucose s u p p l e m e n t e d with 0.05 °Joo yeast extract. T h e second i n o c u l u m was p r o d u c e d o n the s a m e m e d i u m w i t h o u t yeast extract o n 1°//oo treated c o r n stover. T h u s , there was no chance o f c a r r y - o v e r o f yeast extract with the i n o c u l u m into the e x p e r i m e n t a l flasks. T h e

p r o t e i n p r o d u c t i v i t i e s in these cases were 0.031 g / l . h with yeast extract and 0.027 g / l . h w i t h o u t yeast extract. T h e g r o w t h rates were 0.033 h - l with yeast extract and 0.036 h -1 w i t h o u t yeast extract in the m e d i u m (Table 2). It indicated that e l i m i n a t i o n o f yeast extract f r o m the m e d i u m did n o t affect the p r o d u c t i v i t y and the g r o w t h rate. T o t a l p r o t e i n p r o d u c t i o n a l m o s t c o r r e s p o n d i n g l y increased with an increase in the c o n c e n t r a t i o n o f c o r n stover in the m e d i u m (Table 2). Similarly the e c o n o m i c protein p r o d u c t i v i t y also increased with the increase in the conc. o f the substrate. T h e e c o n o m i c yield coefficient (protein) was n o t affected with the increase in conc. o f c o r n stover. A slight decrease in g r o w t h rate was noticed with an increase in the c o n c e n t r a t i o n o f c o r n stover to 2%0 in the m e d i u m , but the g r o w t h rate decreased to one h a l f w h e n the conc. o f the substrate was increased to 3%0 (Fig. 2 and 3, T a b l e 2). T h e r e d u c e d g r o w t h rate in the case o f 3%0 c o n c e n t r a t i o n o f substrate in the m e d i u m c o u l d be at-

MYCELIAL BIOMASS OF A MUSHROOM

VoL 68, 1989 TABLE 3.

TABLE 4.

Chemical composition of mycelial biomass of P. sajor-caju P. sajor-caju

Dry matter Crude protein (N × 6.25) Fat Acid detergent fiber Ash Phosphorus Sodium Potassium Magnessium Calcium Iron Zinc Copper Manganese Lead

92.68o/00 40.79%0 7.05%0 8.81% 11.54°Joo 2.02% 0.52% 0.62O/oo 0.240/00 0.84% 124 ppm 48 ppm 3 ppm 29 ppm 12 ppm

5.74 8.84 5.66 2.96 1.83 8.84 2.91 5.08 2.09 3.47 1.14 2.88 4.39 2.04 3.99 0.70

Feeding trial on rats a

Initial weight

Isolated soy protein b P. sajor-caju b,°

61.2 61.8

Body weight in grams Weight Weight after after 1 week 2 weeks 89.3 79.1

115.2 105.4

Weight after 18 d 125.9 117.5

a 10 rats per dietary treatment containing 10% isolated soy protein (1SP). b The diet protein was fortified with methionine. ¢ With 25 %0of required protein from 1SP + 75% of required protein from mycelial biomass of P. sajor-caju.

lignin b e c o m e s part o f the effluent and it can be easily s e p a r a t e d by acidifying the effluent. ACKNOWLEDGMENTS FAO reference

Amino acids as % crude protein Aspartate Glutamate Serine Glycine Histidine Arginine Threonine Alanine Tyrosine Valine Methonine a Isoleucine Leucine Phenylalanine Lysine Cystineb

Diet

337

The author is very grateful to Drs. E.R. Chavez and S.P. Touchburn, Macdonald College of McGill University, Montreal for chemical analysis and preliminary feeding trials of the mycelial biomass of P. sajor-caju. REFERENCES

2.8 2.8 4.2 2.2 4.2 4.8 2.8 4.2 2.0

a AS methionine sulfonate. b AS cysteic acid.

t r i b u t e d to p o o r mass t r a n s f e r in shake flask cultures. Chemical c o m p o s i t i o n o f mycelial biomass The m y c e l i a l b i o m a s s o f P. sajor-caju p r o d u c e d o n glucose was rich in p r o t e i n and m i n e r a l s b u t low in fat (Table 3). T h e a m i n o acid profile i n d i c a t e d that it is deficient in sulfur a m i n o acids as are the mycelial b i o m a s s e s o f o t h e r fungi (30). H o w e v e r , it meets the F A O r e q u i r e m e n t s for the o t h e r essential a m i n o acids. Feeding trial A p r e l i m i n a r y feeding trial o n rats indicated that a b o u t 50%0 p r o t e i n f r o m the diet can be easily r e p l a c e d by the p r o t e i n o f mycelial b i o m a s s o f P. sajorcaju p r o d u c e d o n glucose, because the p e r f o r m a n c e o f rats on diet r e p l a c i n g 7 5 % o f p r o t e i n with mycelial b i o m a s s p r o t e i n was very close to t h o s e on a c o n t r o l diet c o n t a i n i n g the isolated soy p r o t e i n (Table 4). P. sajor-caju c o u l d synthesize 1 . 5 g o f p r o t e i n f r o m 10 g o f treated c o r n stover c o n t a i n i n g 7 g o f polysaccharides (19-21). T h e a m o u n t o f p r o t e i n (1.5 g) synthesized was as high as o f t h a t p r o d u c e d f r o m an e q u i v a l e n t a m o u n t (7 g) o f glucose w h e n calculated f r o m the d a t a in Table 1 (2.12+10x7=l.48g). It is i n f e r r e d f r o m the a b o v e d a t a t h a t there was a l m o s t c o m p l e t e use o f polysaccharides (cellulose and hemicelluloses) present in the treated c o r n stover. T h e m o d i f i c a t i o n s in lignin due to the actions o f v a r i o u s e n z y m e s p r o d u c e d d u r i n g the p e r i o d o f g r o w t h o f P. sajor-caju o n p o l y s a c c h a r i d e s are being studied and will be r e p o r t e d elsewhere. This m o d i f i e d

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