Physiology of Wood-Rotting BasidiomycetesII. Nutritive Composition of Mycelium Grown in Submerged Culture"'

MARSHALL W. JENNISON, CARL G. RICHBERG, AND ARTHUR E. KRIKSZENS

Department of Bacteriology and Botany, Syracuise University, Syracuse. New York

Received for publication September 28, 1956

As regards nutritional value, the chemical composi-tion and vitamin content of relatively few kinds offungi have been reported (see review by Thatcher,1954). Strains of yeast have been studied most exten-sively (Dunn, 1952), bacteria and higher fungi to alesser extent. -Most analyses of filamentous fungi, more-over, have been carried out on nonuniform surfacemats, whereas uniform cellular composition and physi-ologic homogeneity of mycelium can be attained onlyin nonstationary cultures, and spores rarely are formedin submerged aerated culture (Foster, 1949).The wood-rotting Basidiomycetes, as well as certain

related forms (mushrooms), may be propagated readilyin submerged culture, forming pellets of mycelium(Humfeld, 1948; Jennison, 1948; Humfeld and Sugihara,1952; Sugihara and Humfeld, 1954; Jennison et al.,1955). Although the two types of wood-rot fungi-browi-n rots and white rots are unlike in their action onthe constituents of wood (Campbell, 1952), and maybe differentiated by their reactions on special culturemedia (Davidson et al., 1938; Preston and McLennan,1948), there appear to be no significant differences innutritional requirements in synthetic media betweenthe two groups (Jennison et al., 1955). Apparentlythere are no reports in the literature, other than threepreliminary papers from this laboratory (Jennison et al.,1953; Fagan and Jennison, 1955; Richberg and Jenni-son, 1956), on the nutritive value of the mycelium ofthe wood-rotting fungi.The present paper is a study of the nutritive com-

position (chemical characteristics and vitamin content)of the mycelium of 17 representative species (10genera) of wood-rotting Basidiomycetes, grown in sub-merged, aerated culture in two types of media syn-thetic and nonsynthetic. Both brown-rot and white-rotforms are included.

1 These studies were aided b- a contract betw-een the Officeof Naval Research, Department of the Navy, and SyracuseUniversity (N-onr-669(06)). The studies were under the di-rection of M. W. .Jennison. This report constitutes a technicalreport under the albove contract. Reproduction in whole or inpart is permitted for any purpose of the United States Govern-ment.

2 Portions of this investigation constituted part of a thesissubmitted by Carl Richberg to the Graduate School of SyracuseUniversitv in partial fulfillment of the requirements for thePh.D. in Microbiology.

EXPERIMENTAL METHODSCulture MIethods

Most of the 17 organisms used (see table 1) werethose for which we had previously reported detailedgrowth data (Jennison et al., 1955). Stock cultures weremaintained on potato glucose agar (Difco, dehydrated).Shake cultures were prepared from the stock slants;standardized, blended pellets of growth from the shakecultures were used to inoculate large submerged-culturevessels for quantity production of each organism forsubsequent analysis. All incubation was at 28 C.Throughout, rigorous sterile precautions were ob-served, and controls for contamination were run rou-tinely.The standard inoculum wAas prepared as follows.

Small bits of mycelium were transferred from a stockculture into 70 ml of 1.0 per cent malt extract (Difco,dehydrated) solution in 250-ml Erlenmeyer flasks,which were incubated for 7 days on a reciprocatingshaking machine. The whole contents of a flask werethen blended for 30 sec in a sterile Waring Blendor, thematerial in the resulting homogeneous suspensionwashed with sterile distilled water three times bycentrifuging at 2000 rpm for 2 min, and resuspended in30 ml of distilled water. This 30 ml of washed suspensionconstituted the standard inoculum for the large fer-mentors.Mass cultivation of the organisms in submerged cul-

ture was carried out under forced aeration in 21-galair-lift fermentors (Lundgren and Russell, 1956) con-taining 6 L of culture medium and 2 ml of Antifoam A.3Sterile air was metered at the rate of 1.5 L per minuteper L of medium. The compressed air was sterilized bypassage through glass wool in a Kelly infusion bottle,then humidified by bubbling through a Selas bacterio-logic filter candle (porosity no. 10; size 1 in by 8 in)immersed in distilled water. Cultures were grown for7 days before harvesting; the approximate maximumamount of mycelium (5 to 10 g, dry weight, per L ofmedium) was attained in this time. The mycelium washarvested by draining and washing rapidly but thor-oughly on a stainless steel sieve, followed by resuspend-ing in water and centrifuging. This procedure, ratherthan centrifugation alone, was essential in some cases;3Dow Corning Corp., 1lidland, Michigan.

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TABLE 1. Chemical and vitamin composition (dry basis) of mycelium of wood-rot fungi grown under forced aeration for 7 days in 1.0 percent malt extract solution

Chemical Composition, Per Cent Vitamin Composition, pg per gOrganism, Culture Number, Brown Rot (B), or

White Rot (W) Protein Ribo Panto- Fo *c(N X Fat Fiber Ash N-re Thiamin Rb-Niacin' Biotin thenic Foc Pyi6.25) extract flavin acid acid doxine

Daedalea quercina FP57076-S (B).... 24.9 8.0 11.5 2.23 53.3 13.8 29.4 420 0.67 5.6 0.49 2.0Fomes annosus FP90898-R (W) ...... 37.0 10.0 11.6 4.52 36.8 18.0 32.4 466 1.23 13.6 6.60 4.7Fomes geotropus FP55521-S (W) . . ........36.67.5 14.6 3.72 37.6 5.8 24.0 178 0.76 52.0 2.20 5.7Fomes subroseus Snell20 (B) ......... 40.4 6.2 7.8 4.08 41.5 4.8 44.0 280 1.40 20.0 1.90 3.5Hydnum putlcherrimum FP81027-It

(B) ...... 23.2 10.0 6.1 5.14 55.6 1.0 25.8 192 0.32 7.2 8.08 6.0Lentinus lepideuts Mad. 534 (B) ...... 36.0 5.7 8.4 1.84 48.1 25.4 38.0 170 1.36 12.8 1.70 3.1Lentinus tigrinusMad. 466 (W) ...... 21.7 9.3 7.4 3.66 57.9 8.3 22.0 182 0.37 30.0 9.10 0.6Lenzites trabeaMad. 539 (B) ......... 34.2 5.5 9.1 3.69 47.5 2.6 10.0 100 1.20 26.4 0.34 3.6Peniophora gigantea FP56475-S (W) 25.5 12.9 10.3 2.21 49.1 16.2 22.0 164 0.64 18.0 4.26 7.7Poria monticola Mad. 575 (B) ....... 38.9 5.5 11.5 2.69 41.4 10.8 46.0 305 2.32 10.8 5.52 4.3Poria subacida FP71955 (W). 22.8 6.6 10.0 8.10 52.5 1.0 20.0 260 0.50 9.8 3.36 3.6Poria xantha FP192-Sporo (B) 38.6 2.3 12.7 2.00 44.4 56.1 21.8 316 1.28 12.1 7.52 7.5Polyporus anceps FP58526-R (W) 27.4 5.5 14.9 3.64 48.6 8.0 96.0 172 0.47 22.4 6.32 5.0Polyporus palustris FP94152 (B) ..... 22.7 2.8 16.3 2.89 55.3 6.0 24.0 325 1.20 15.0 1.80 7.0Polyporus tulipiferus Mad. 517 (W).. 40.8 2.9 5.9 3.38 47.1 23.1 46.0 350 0.78 19.2 5.58 9.8Ptychogaster rubescensUIFP716 (B).. 28.0 7.2 4.3 3.83 56.7 14.4 41.2 250 0.62 25.1 8.83 5.0Trametes serialis FP11977 (B) ....... 21.9 8.9 12.9 1.38 54.9 4.2 7.4 44 1.30 3.1 0.39 2.6

Avg ..................... 30.6 7.0 10.4 3.47 48.7 12.9 32.3 245 0.96 17.8 4.35 4.8

cultures sometimes contained a precipitate, and certainorganisms produced a slime which could not readily beseparated from the mycelium by centrifugation alone.The washed mycelium was immediately lyophilized andstored over Drierite.

MediaTwo types of culture media-nonsynthetic and syn-

thetic were used to obtain mycelium for comparativeanalysis. These were: (1) 1.0 per cent malt extract(Difco, dehydrated) solution. This solution had a totalnitrogen content of approximately 0.012 per cent,4contained about 0.9 per cent carbohydrate and had apH of 4.9 after sterilization. Seventeen fungi weregrown in this medium. (2) Synthetic media optimalfor the maximal amount of growth of each of 8 of the17 organisms. These chemically defined media all con-tained glucose, mineral salts, L-glutamic acid and thi-amin, but in different proportions (for composition, seeJennison et al., 1955). The optimal nitrogen concentra-tion varied from 0.02 to 0.14 per cent, depending uponthe species of fungus; the optimal pH differed for mostspecies. The glutamic acid, thiamin, glucose, mineralsalts and antifoam were autoclaved separately, thencombined aseptically. With one organism (Polyporuspalustris), used for determining amino acids of themycelial protein, a basal synthetic medium (Jennisonet al., 1955) was used for growth.

Analytical ProceduresChemical and vitamin methods used were those of

the Association of Official Agricultural Chemists4This value was erroneously given as 0.004 per cent in

Jennison et al., 1955.

(A.O.A.C.), or other accepted procedures. In severalcases various methods of analysis were compared, asregards reproducibility, for the components of thefungal mycelium; the methods or modifications finallyused were those which gave the most consistent results.In the preparation of material for analysis, all samplesof previously lyophilized mycelium were ground to passthrough a sieve having 1-mm holes (official method,A.O.A.C., 1955, Sec. 22.2), and kept in a desiccatorover Drierite. Before analysis, samples for chemicaldeterminations were dried to constant weight in avacuum oven at 80 C; samples for vitamin assay wereweighed directly from the desiccator. Analytic data arereported on a moisture-free- basis. All figures are aver-ages of at least duplicate determinations on one myce-lial sample; in many cases replicate mycelial sampleswere averaged. Amino acids in hydrolyzates of mycelialprotein were determined chromatographically, usingstandard methods (Block et al., 1955); the protein washydrolyzed with HCl, or, for tryptophan identification,with Ba(OH)2.

Chemical methods.Crude protein: N X 6.25-Official micro-method

for total nitrogen, A.O.A.C. (1955), Sec. 37.9-37.11.Crude fat: Mycelium extracted with petroleum ether

for 24 hr.Crude fiber: Official method, A.O.A.C. (1955), Sec.

22.31-22.33.Ash: Modification of official method, A.O.A.C.

(1955), Sec. 13.6: sample ashed at 550 C for 5 hr, with1 ml of concentrated HNO3 added after 2 hr as anashing aid (Jacobs, 1951).

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N-free extract (carbohydrates): By difference: 100-(protein + fat + fiber + ash).Amino acids-qualitative: Ascending, two-dimen-

sional paper chromatography (Block et al., 1955).Solvents: phenol and butanol-acetic acid; color-devel-oping reagent: ninhydrin. After development of spotson the paper, specific amino acids in the hydrolyzateswere identified by comparison with the Rf values of22 known standards and from the pattern of spots ofknown mixtures.Amino acids-quantitative: Spot-area method of

Fisher (Block et al., 1955). Chromatograms developedas above, using ninhydrin except for proline (isatinreagent). Methionine was converted to the sulfonewith H202 to eliminate overlapping with valine.

Vitamin assay methods.Thiamin (HCl): Official fluorometric method,

A.O.A.C. (1955), Sec. 38.22-38.28.Riboflavin: Official fluorometric method, A.O.A.C.

(1955), Sec. 38.37-38.40.Niacin: Microbiologic method of Snell and Wright

(1941), using Lactobacillus arabinosus 17-5 (A.T.C.Cno. 8014).

Biotin: Modified (Johnson, 1948) microbiologicmethod of Wright and Skeggs (1944), using L. arabi-nosus) 17-5 (A.T.C.C. no. 8014).

Pantothenic acid: Modified (Johnson, 1948) micro-biologic method of Hoag et al., (1945), using L. arabi-nosus 17-5 (A.T.C.C. no. 8014).

Folic acid: Official (microbiologic) method, A.O.A.C.(1955), Sec. 38.48-38.51, using Streptococcus faecalis(A.T.C.C. no. 8043).Pyridoxine (HCl) (B6 complex): Microbiologic method

of Atkin et al., (1943), using Saccharomyces carlsbergen-sis (A.T.C.C. no. 4228).

EXPERIMENTAL RESULTS

Table 1 summarizes the chemical and vitamin com-

position of 17 wood-rot fungi grown in air-lift fer-

mentors in malt extract medium. Table 2 shows thecomposition of the mycelium of eight of the abovespecies grown in optimal synthetic media; the averages

for these same eight organisms from malt extract alsoare given (computed from table 1). In table 3, valuescomputed from tables 1 and 2 allow comparison of theaverage composition of the brown rots and the whiterots harvested from different media. In connectionwith table 3, it should be noted that the total numberof species of brown rots (10) and of white rots (7) grownin the malt extract solution (column a) probably isadequate for computing reliable averages for the my-

celial components of these two groups in this medium;fewer organisms of each group (5 brown rots, 3 whiterots) were cultured in synthetic media and the averages

for these media (column b), as well as the mean valuesin column c, are therefore less reliable.

Chemical Composition of MyceliumCrude protein. It is recognized that the conventional

conversion factor, N X 6.25, may not give a very ac-

curate estimate of the protein content of mold my-

celium, but data are not available for precise evaluationof the protein and nonprotein nitrogen. For protein andother cellular constituents, however, we are interestedprimarily in comparable data rather than in absolutevalues.As regards individual organisms, both species and

media differences are evident in the amounts of proteinsynthesized; the latter differences are in general lessmarked. Among species, the highest protein content wasabout twice the lowest, in both types of culture media(tables 1, 2). The average per cent of protein was vir-tually the same for the 17 species grown in malt ex-

tract, for the eight organisms in synthetic media, andfor the same eight organisms in malt extract.

Eight species from the malt extract contained be-tween 34 and 40 per cent of protein; in the chemicallydefined media, four organisms fell within this range.

TABLE 2. Chemical and vitamin composition (dry basis) of the mycelium of certain wood-rot fungi grown underforced aeration for 7 daysin synthetic media optimal for each organism

Chemical Composition, Per Cent Vitamin Composition, Ag per g

OrganismPrti (rtenX Fa Fie As N-free Th Ribo- Panto- Foi Pyi6 25) Fat iber Ash extract iamin flavin Niacin Biotin thenic Foidc Pyri-6.25) ~~~~~~~~~~~~~~~~~acidacd oxn

Daedalea quercina . ................. 21.6 7.8 9.6 4.82 56.2 194.8 20.4 134 0.68 8.9 1.20 5.0Fomes geotropus .. ................. 38.2 8.6 6.8 8.97 37.4 21.6 35.6 181 0.80 36.0 9.58 5.7Fomes subroseus ................... 29.3 1.9 8.3 6.59 53.9 8.3 21.4 148 0.56 3.2 1.00 1.5Lentinus tigrinus................... 31.6 4.5 7.6 5.35 51.0 20.4 21.0 332 1.06 80.0 3.30 8.0Lenzites trabea .. ................... 36.4 4.3 11.8 3.54 44.0 159.0 22.2 32 0.58 8.0 1.40 3.5Polyporus palustris . ............... 26.4 3.4 6.8 4.53 61.4 10.4 26.2 150 1.68 8.5 6.78 8.0Polyporus tulipiferus............... 40.9 1.8 6.5 4.34 46.5 94.0 56.2 300 1.13 11.4 5.02 7.5Trametes serialis. 34.4 5.8 5.8 3.63 50.4 32.4 28.2 168 0.80 2.8 1.70 3.0

Avg ............................ 32.4 4.8 7.8 5.22 50.1 67.6 28.9 181 0.91 19.9 3.75 5.3

Avg. for same species in malt ex-tract medium (table 1) ....... 30.4 6.3 10.7 3.13 49.4 8.6 25.6 235 0.96 21.4 2.72 4.4

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TABLE 3. Average comiposition (dry basis) of mycelium of brownrots and of white rots, harvested from malt extract and front

optimal synthetic mediaComputed from tables 1 and 2

Ca b From Malt Extract

From Malt Extract From Synthetic Medium (table 1);Medium (table 1) Media (table 2) Same Organisms as

Analyses in Column b

Brown White Brown White Brown Whiterots rots rots rots rots rots(10 (7 (5 (3 (5 (3

species) species) species) species) species) species)

Average composition, per cent

ChemicalProtein 30.9 30.3 29.6 36.9 28.8 33.0Fat 6.2 7.8 4.6 6.5 6.3 6.6Fiber 10.1 10.7 8.5 7.0 11.5 9.3Ash 3.00 4.18 4.62 6.22 2.85 3.59N-free 49.9 47.1 53.2 45.0 50.5 47.5

extract

Average composition, jig per g

VitaminThiamin 13.9 11.5 81.0 45.3 6.3 12.4Riboflavin 28.8 37.5 23.7 37.6 23.0 30.7Niacin 240 253 126 271 234 237Biotin 1.17 0.68 0.86 1.00 1.15 0.64Pantothenic 13.8 23.6 6.3 42.5 14.0 33.7

acidFolic acid 3.7 5.4 2.4 6.0 0.98 5.63Pyridoxine 4.5 5.3 4.2 7.1 3.7 5.4

Two organisms (both white rots)-Fomes geotropus andPolyporus tulipiferus synthesized about 40 per cent ofprotein in both types of media. In table 3, it is seen

that in mycelium from malt extract there was little(column c) or no (column a) difference in the average

protein content of brown rots and white rots; in syn-

thetic media (column b) the white rots averaged some-

what more protein than did the brown rots.Crude fat. The fat content of mycelium varied both

among species (5-fold) and between types of media;the average for organisms in synthetic media was defi-nitely less than in malt extract. In synthetic media,only two organisms contained as much as 8 per cent offat; in malt extract, six species attained or exceededthis figure. Three high-protein organisms-Fomes an-

nosus from malt extract, Trametes serialis from a syn-thetic medium, and F. geotropus from both types ofmedia-showed fat contents greater than the averagesfor the respective media. White-rot species synthesizedsomewhat more fat than did brown-rot forms, in bothtypes of media (table 3).

In developing the fat extractions for the mycelialmaterial, it was noted that the apparent fat contentvaried with the antifoam used in the fermentation.With Vegifat Y5 as an antifoam, the crude fat of the

5 Nopco Chemical Company, Harrison, New Jersey.

mycelium averaged 2 to 3 times that obtained usingAntifoam A. Vegifat Y, according to the manufacturer,is "composed of long and short chain fatty acids," andis soluble in petroleum ether. Apparently this antifoamwas adsorbed or metabolized by the mycelium duringthe fermentation, and then was extracted in the fatdetermination, giving spurious values for mycelial fat.Antifoam A, a silicone product, is not soluble in ether.However, it was adsorbed by the mycelium in culture,and removed physically during the fat extraction, ap-pearing as a scum in the extraction flasks. Care had tobe taken not to remove this scum when the ether wasdecanted prior to evaporation. Also, in a few samples ofmycelium which were prehydrolyzed (refluxed with 2per cent HCl for 2 hr) before extraction of the fat, thevalues for fat were about twice those of the samemycelium without prehydrolysis. Although the datain the tables are for the standard extraction, apparentlyonly a portion of the fat was removed by this procedure.

Crude fiber. This analysis was carried out on theextracted residues from the fat determinations. Thegreatest range of variation in crude fiber among specieswas about 4-fold (in malt extract); the average amountof fiber in organisms from synthetic media was sig-nificantly less than in those grown in malt extract. Thebrown rots, on the average, contained (table 3) aboutthe same (column a) or larger (columns b and c)amounts of fiber than did the white rots.

Ash. The higher average ash of organisms from thesynthetic media may reflect qualitative or quantitativedifferences in mineral content between the malt ex-tract medium and the chemically defined substrates.Variations in ash content among species was greaterin mycelium from malt extract (6-fold) than fromsynthetic media (2.5-fold). The average ash contentof the white rots was considerably higher than thatof the brown rots, in all media.A few determinations were made of the calcium and

the phosphorus content of the ash. Phosphorus (as P),averaged about 1.5 per cent of the dry mycelial weight;calcium (as Ca) was about 0.2 per cent.

Nitrogen-free extract. "Carbohydrates," calculated bydifference, varied less than -other cellular constituents;the averages were virtually the same for all media.The brown rots showed higher average values thanthe white-rot species, regardless of the nutrient sub-stratum.

Vitamin Content of MyceliumThiamin. We have previously shown (Jennison et

al., 1955) that, for the organisms used in the presentstudy, thiamin is the only vitamin which must beadded to synthetic media. Although the malt extractpresumably contained a variety of vitamins, only thia-min was present in the solutions of known composi-tion. Except for thiamin, therefore, vitamins present

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in fungal mycelium from the chemically defined solu-tions represent a total synthesis from components ofthe media. In mycelium from malt extract, the thia-min probably results both from synthesis and absorp-tion from the medium.A wide range in thiamin content was evident among

species-56-fold in mycelium from malt extract andsome 24-fold in material from synthetic media. Theaverage thiamin content of the 17 organisms frommalt extract was only one-fifth as large as the averagefor the eight species from synthetic media; for thesame organisms in the two types of media, the averagefor those in the synthetic substrates was eight timesthat for species from malt extract. Daedalea quercinaand Lenzites trabea showed especially high thiaminvalues in synthetic media. In malt extract, only Poriaxantha was relatively high in thiamin, but this amountwas only one-quarter of that found in D. quercina froma medium of known composition. The high thiaminvalues for mycelium from synthetic media probablyreflect the addition of this vitamin to the media andabsorption by the organisms. As between the brownand white rots (table 3), there was no consistencyrelative to which group was better in the synthesisand/or absorption of this vitamin (cf., columns band c).

Riboflavin. The variation in amounts of this vitaminamong species was 13-fold in malt extract and 3-foldin chemically defined media. The average riboflavincontent of the eight species in synthetic media wascomparable to the average both for the same organismsand for all organisms in malt extract. Polyporus an-ceps had the highest content of riboflavin in maltextract, and P. tulipiferus in a synthetic medium. Thewhite-rot fungi averaged significantly larger amountsof riboflavin than did the brown rots.

Niacin. The range of variation in niacin amongspecies was about 10-fold in both types of media.There was a significantly higher average niacin con-tent in mycelium of organisms from malt extract thanfrom synthetic media. In synthetic media, Lentinustigrinus and P. tulipiferus had the highest niacin val-ues; D. quercina and F. annosus were especially out-standing in malt extract. The white-rot species, espe-cially in synthetic media, had a higher average niacincontent than the brown-rot forms.

Biotin. Although the differences in biotin contentamong species was greater in malt extract (7-fold) thanin synthetic media (3-fold), the average values werenearly identical for the two types of media. The high-est biotin content was found in Poria monticola frommalt extract medium. In the case of this vitamin,mycelium of the brown rots from malt extract (table3, columns a and c) assayed higher than that of thewhite rots, although the reverse was true for the re-spective groups grown in synthetic media (column b).

Pantothenic acid. Large variations in pantothenicacid are seen among species-17-fold in mycelium frommalt extract and 29-fold in cultures from syntheticmedia. Average values for the two types of media arenearly the same, however, whether all organisms frommalt extract are considered or only those also grownin the synthetic nutrient solutions. Outstandingly highamounts of pantothenic acid were found in F. geotropusfrom malt extract and in L. tigrinus from a syntheticmedium. The white-rot fungi contained markedlylarger amounts of pantotheniic acid than did the brown-rot types; the synthesis of this vitamin by the whiterots in chemically defined media was especially striking(table 3, column b).

Folic acid. The differences in folic acid among specieswas greater in organisms from malt extract (27-fold)than from synthetic media (10-fold). Although the av-erage folic acid content was somewhat greater for the17 species from the first medium compared with the 8forms from the second, this relationship was reversedwhen data for the same organisms in the two types ofmedia are compared (table 2). F. geotropus, in a chemi-cally defined medium, synthesized the largest amountof folic acid; two organisms from malt extract ap-proached this value. As was the case with pantothenicacid, the white-rot organisms assayed markedly higheramounts of folic acid than the brown rots, in all culturemedia.

Pyridoxine. The assay method used for vitamin B6did not differentiate among the three forms in thiscomplex-pyridoxine, pyridoxal and pyridoxamine-hence all are included as "pyridoxine."

Variation in pyridoxine among species from maltextract was 5-fold and from the optimal media was16-fold. The average amount of this vitamin was some-what greater in organisms from the synthetic mediathan in those from malt extract. P. tulipiferus had thehighest pyridoxine content of any organism from maltextract; this organism also was one of the three bestin synthetic media. Pyridoxine was found in signifi-cantly greater amounts in mycelium of white-rot fungithan of brown-rot species, regardless of the nutrientsubstrate.

Amino Acids of Polyporus palustrisTable 4 shows the amino acids found in the mycelial

protein of P. palustris; the 7-day-old growth was froma basal synthetic medium (Jennison et al., 1955) con-taining glutamic acid as a nitrogen source. The sameamino acids, and similar quantities, also were found inthe protein of this organism grown in malt extract andin the basal synthetic medium with ammonium nitrateas a nitrogen source. It is apparent that most of thecommon amino acids were present in the protein of thisbrown-rot fungus. The following were not detected:asparagine, cystine, cysteine, hydroxyproline, and orni-

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TABLE 4. Amino acids in hydrolyzates of Polyporus palustrismycelium grown in a basal synthetic medium for 7 days under

forced aeration

Amino Acid Per Cent of Dry Per Cent of TotalMycelium Amino Acids

a-Alanine ................. 3.4 10.5Arginine .................. 2.6 8.0Aspartic acid ............. 2.5 7.7Glutamic acid ............. 2.7 8.3Glycine ................... 1.6 4.9Histidine ................. 1.6 4.9Leucine + isoleucine 2.9 9.0Lysine .................... 2.1 6.5Methionine sulfone 1 .3 4.0Phenylalanine ............. 1 .8 5.6Proline ................... 2. 1 6.5Serine .................... 2 .4 7.4Threonine ................. 1.8 5.5Tryptophan............... 0.8 2.5Tyrosine ...............0. .8 2.5Valine .................... 2.0 6.2

Totals .................. 32.4 100.0

thine. The protein content of P. palustris myceliumfrom the basal synthetic medium was virtually thesame (32.4 per cent) whether calculated from the aminoacid data (table 4) or obtained from a determinationof total nitrogen (33.1 per cent).

Animal Feeding Experiments

Preliminary feeding trials were carried out withyoung white mice (weight 9 to 15 g) and guinea pigs(260 to 340 g weight) fed fresh, drained, uncookedmycelium of each of the organisms in table 1, harvestedfrom the malt extract medium. One feeding, ad libitum,of such mycelium to animals which had been starvedfor 24 hr showed no toxic effect in any case. In a 1-month mouse-feeding experiment, using a diet of 50per cent P. palustris mycelium and 50 per cent labora-tory rations, the mice gained weight and had no ap-

parent symptoms of toxicity or of dietary deficiency.Mice that were fed a diet of 100 per cent P. palustrismycelium lost weight and died on the eighth day,exhibiting symptoms of dietary deficiency. It may

tentatively be concluded that the mycelia of the 17wood-rot fungi used contained no toxic principle per se.

The mycelium of P. palustris did not by itself constitutean adequate diet over a period of time.

DIscussIoN

Among the factors which are known to influencecellular constituents of fungi are the strain of the or-

ganism, aeration, age of culture, and composition ofthe medium (see Foster, 1949). None of the abovefactors was investigated as such in the present study.The present research is essentially a "screening" ofrepresentative wood-rot fungi to determine their chemi-

cal and vitamin composition when grown under certainspecified conditions. The synthetic media, quantita-tively different in composition for each organism, were"optimal" only for producing the maximal amount ofgrowth, not for any specific mycelial constituent. Noattempt was made experimentally to increase any my-celial component; further research is in progress forthis purpose.

Except for three brief reports from this laboratory(Fagan and Jennison, 1955; Jennison et al., 1953;Richberg and Jennison, 1956), the last two of whichstudies are a part of the present paper, there are vir-tually no data available on the chemical and vitamincomposition of the basidiomycetous wood-rotting fungi.The results obtained in the present study can thereforeonly be compared with analyses of related kinds offungi.Our average values for the chemical and vitamin

composition of wood-rot mycelia (except for the highthiamin in organisms from synthetic media) are similarto the data given by Humfeld and Sugihara (1949) forsubmerged-culture mycelia of two strains of a Ba-sidiomycete related to the wood-rot fungi, the com-mercial mushroom, Agaricus campestris. The nutritivecomposition of the spore structure of this organism(Anderson and Fellers, 1942) closely resembles that ofthe mycelium. The vitamin content of the wood rotsalso was similar (except for thiamin) to that of sub-merged-culture mycelium of Agaricus blazei (Block etal., 1953); no data, except kinds of amino acids, weregiven for the chemical co'mposition of this organism.Our very high thiamin values in the organisms fromsynthetic media probably reflect the addition of thisvitamin to the media; in the papers cited above, non-synthetic media without added vitamins were used.The latter authors detected only 14 amino acids inthe protein of A. blazei, in contrast to the 17 which wefound in P. palustris. The 17 amino acids in P. palustrismycelium included those now considered essential inhuman nutrition and those known to be required bycertain animals (Albritton, 1954, table 12). We havedemonstrated the same 17 amino acids to be presentin the protein of P. palustris and of D. quercina grownin several synthetic and nonsynthetic media (Faganand Jennison, 1955). It also is of interest that theprotein of P. palustris contained good amounts oflysine and threonine (table 4), amino acids that oftenare low in plant protein, for example, wheat gluten(Albritton, 1954, table 73). Submerged-culture myce-lium of another mushroom-the edible morel, Mor-chella hortensis (an Ascomycete)-has been shown inour laboratory (Koda, 1951) to contain the sameamino acids as does P. palustris, although in differentproportions. References to other papers on variouschemical constituents of higher fungi, including wood-rotting Basidiomycetes, are given by Ralph (1949); the

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PHYSIOLOGY OF WOOD-ROTTING BASIDIOMYCETES. II

distribution of vitamins in microorganiisms is reviewedby Van Lanen and Tanier (1948) and by Thatcher(1954).There was great variation among individual species

in the amounts of different mycelial constituents syn-thesized by the wood-rot fungi. To some extent thisvariation results from qualitative and (uantitative dif-ferences in composition of the culture media, and insome degree reflects differences in synthetic abilityamong various organisms in the same medium. No onespecies of wood-rot was the "best" in all-around nutri-tive composition under the conditions used in the pres-ent study. The high-protein organisms often were lowin fat content, and vice versa, although F. annosus inmalt extract, T. serialis in a synthetic medium andF. geotropus in both media synthesized greater thanaverage amounts of these two mycelial components.Despite the marked differences in composition of thetwo types of culture media, the average amount ofprotein was the same in organisms grown in each type;the fat content was lower in mycelium from syntheticmedia. As regards vitamins in the mycelium, certainorganisms grown in malt extract contained well-above-average amounts of, usually, one growth factor. A fewspecies from this medium, for example, P. xantha andP. tulipiferus, were above average in several differentvitamins. L. trigrinus and P. tulipiferus from syntheticmedia are seen to contain relatively large quantities ofeach of three vitamins; certain other species were highin onily one. The three high-protein, high-fat organismsmentioned above F. annosus, F. geotropus and T.serialis-were not outstanding in vitamin content; F.annosus, from malt extract, was perhaps the best ofthe three. As regards the relation of the type of culturemedium to the average vitamin content of mycelia,there was little difference between the synthetic andthe inonsynthetic substrates except in the special caseof thiamin. The markedly higher average value forthiamin in mycelia from synthetic media (tables 1 and2) probably results, in part at least, from the additionof this vitamin to the chemically defined substrates.The high thiamin content in this case, therefore, doesniot necessarily reflect cellular synthesis only. In myce-lia from chemically defined solutions, all vitaminsother than thiamin were synthesized from the constit-uents of the media. For organisms harvested frommalt extract, the amounts of at least certain vitaminsrepresent both absorption from the medium and meta-bolic activity of the organiism.

There were fairly marked quantitative differencesbetween the white-rot fungi and the brown rots inmost of the important mycelial constituents. The for-mer species averaged usually in both types of culturemedia-more protein, fat, ash, riboflavin, niacin, pan-tothenic acid, folic acid, pyridoxine and, in syntheticmedia, more biotin. On the basis of the above data, the

white rots appear to offer more promise than the brownrots for further investigation of the synthesis of myce-lial constituents. Also, because white-rot fungi attacklignin in addition to cellulose, whereas the brown rotsact primarily on cellulose (Campbell, 1952), the formertypes may advantageously be grown in a greater vari-ety of complex nutrients, including fermentable wastes.The nutritive quality of the wood-rot fungi, as deter-

mined by animal feeding experiments, needs furtherevaluation. Our preliminary results indicated that, whilethese fungi probably did not contain a toxic principle,the mycelium was not nutritionally adequate as a solesource of food. Similar results have been observed withother kinds of fungi which have been assessed for nu-tritional value (see Thatcher, 1954).

Reasons for the current interest in the synthesis offood by microorganisms, as well as a review of thetechnical literature, are given by Thatcher (1954).'Many factors, biologic and economic, enter into thequestion of what kinds of microbes may be of practicaluse. The production of yeast as a supplement for hu-man food and for animal and poultry feeds has attainedcommercial proportions (Wiley et al., 1950; Dunn,1952); the practicability of large-scale, submerged cul-ture of commercial mushroom mycelium in certainwaste materials has been demonstrated (Humfeld,1952, 1954). There is increasing recognition of the po-tentialities of the fermentation processes of yeast andother fungi as efficient methods-for the utilization ofcarbohydrate wastes of agriculture and industry(Holderby and Wiley, 1950).The wood-rot fungi have one fermentative ad-

vantage not shared by most other microbes: theirunique ability to split the cellulose-lignin complex ofwood. The cellulolytic activity of these organisms (forexample, Campbell, 1952; Siu and Reese, 1953; Kunzet al., 1954), and ability to utilize a variety of carboncompounds (Neewcomb and Jennison, 1954) and differ-ent forms of nitrogen (Jennison et al., 1955), attest totheir adaptability to a diversity of substrates. Thewood-rot fungi produce good growth in many cheapsubstrates and plant wastes, such as potato, bran andcorn steep liquor (Jenniison, 1948), molasses fermenta-tion residues and spent brewers' grains (Fagan andJennison, 1955), corn cobs, wood wastes, coffee grounds,cannery wastes, and spent sulfite liquor (unpublishedobservations). The products of fermentation and syn-thesis of the wood-rot fungi have been studied rela-tively little (see Ralph, 1949; Walter, 1954); the forma-tion of polysaccharide, of pigments, and of oxalic andother acids has been observed in our laboratory witha number of species in submerged culture. The poten-tialities of the wood-rotting fungi for the disposal offermentable wastes, for the production by fermentationor synthesis of useful substances, and as sources of food,warrant further investigation.

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M. W. JENNISON, C. G. RICHBERG AND A. E. KRIKSZENS [VO.

ACKNOWLEDGMENTSThe technical assistance of John Seo, Francis Mi-

lazzo, and Chester Koda was greatly appreciated.

SUMMARYA study was made of the chemical and vitamin

composition of the mycelium of 17 representativespecies of wood-rotting Basidiomycetes, both brownrots and white rots. The organisms were grown for 7days in submerged culture under forced aeration. Eachof the 17 species was cultured in a malt extract me-

dium, and 8 of these also were grown in syntheticmedia. The media were chosen only because theysupported good growth, not because they enhancedthe synthesis of any particular constituent.

Quantitatively, the composition of mycelium variedmarkedly with the species of organism and with themedium. For chemical constituents (expressed as per

cent of dry mycelium), over-all averages for the differ-ent species from both types of media were as follows:protein, 31.5; fat, 5.9; fiber, 9.1; ash, 4.35; carbohy-drates, 49.2. For vitamins (expressed as microgramsper gram of dry mycelium), over-all averages were:

thiamin, 40.3; riboflavin, 30.6; niacin, 213; biotin, 1.32;pantothenic acid, 18.8; folic acid, 4.05; pyridoxine,5.1. The mycelial protein of one organism was shownto contain 17 amino acids; the amount of each was

determined.No one species of wood rot was "best" in all-around

nutritive composition. Three high-protein, high-fat or-

ganisms were not outstanding in vitamin content.Certain other species contained relatively large amountsof one or more vitamins, but showed low values forprotein and fat. There was, in general, little differencebetween synthetic and nonsynthetic media as regardstheir effect on the average amounts of mycelial con-

stituents produced. Mycelia grown in synthetic mediaaveraged somewhat less fat, but more niacin and thia-min (a special case), than when harvested from maltextract. As between the brown rots and the white rots,the latter averaged larger amounts of most of the nu-

tritionally important mycelial components.Preliminary experiments in feeding the uncooked

mycelia of wood rots to mice and guinea pigs indicatedthat none of the 17 species caused any acute toxicity.Mycelium of Polyporus palustris the only species in-vestigated in more detail-was not adequate over a

period of time as a sole source of food for these ani-mals.Some general aspects of the synthesis of nutritionally

valuable substances by microorganisms, and the po-

tentialities of wood-rot fungi in this regard, are men-

tioned briefly.

REFERENCES

ALBRITTON, E. C., editor. 1954 Standard values in nutrition

ANDERSON, E. E. AND FELLERS, C. R. 1942 The food value ofmushrooms (Agaricus campestris). Proc. Am. Soc.Hort. Sci., 41, 301-304.

Association of Official Agricultural Chemists. 1955 Officialmethods of analysis of the association of official agriculturalChemists, 8th ed. Washington.

ATKIN, L., SCHULTZ, A. S., WILLIAMS, W. L., AND FREY, C. N.1943 Yeast microbiological methods for determination ofvitamins. Pvridoxine. Ind. Eng. Chem. (Anal. Ed.)15, 141-144.

BLOCK, R. J., DURRUM, E. L., AND ZWEIG, G. 1955 A manualof paper chromatography and paper electrophoresis. Aca-demic Press, New York.

BLOCK, S. S., STERNS, T. W., STEPHENS, R. L., AND MCCAND-LESS, R. F. J. 1953 Mushroom mycelium. Experimentswith submerged culture. J. Agr. Food Chem., 1, 890-893.

CAMPBELL, W. G. 1952 The biological decomposition ofwood. In Wood chemistry, 2nd ed., edited by L. E. WISEAND E. C. JAHN, Vol. 2, pp. 1061-1116. Reinhold Publish-ing Corp., New York.

DAVIDSON, R. W., CAMPBELL, W. A., AND BLAISDELL, D. J.1938 Differentiation of wood-decaying fungi by theirreactions on gallic or tannic acid medium. J. Agr. Re-search, 57, 683-95.

DUNN, C. G. 1952 Food yeast. Wallerstein LaboratoriesCommunications, 15, 61-79.

FAGAN, AI. ANI) JENNISON, M. W. 1955 Nutritive compo-sition of wood-rotting fungi. (Abstract). Bacteriol.IProc., 1955, 28.

FOSTER, J. W. 1949 Chemical activities of futngi. AcademicPress, New York.

HOAG, E. H., SARRETT, H. P., AND CHELDELIN, V. H. 1945Use of Lactobacillus arabinosuis 17-5 for microassay ofpantothenic acid. Ind. Eng. Chem. (Anal. Ed.), 17,60-62.

HOLDERBY, J. AI4. AND WILEY, A. J. 1950 Biological treat-ment of spent liquor from sulfite pulping process. Sewageand Ind. Wastes, 22, 61-70.

HUMFELD, H. 1948 The production of mushroom mycelium(Agaricuis (ampestris) in submerged culture. Science,107, 373.

HUtMFELD, H. 1952 Production of mushroom mycelitum.U. S. Patent 2,618,900.

HUTMFELD, H. 1954 Production of mushroom mycelium bysubinerged culture in a liquid medium. U. S. Patent2,693,665.

HUMFELD, H. AND SUGIHARA, T. F. 1949 Mushroom my-celium production by submerged propagation. FoodTechnol., 3, 355-356.

HUMFELD, H. AND SUGIHARA, T. F. 1952 The nutrient re-quirements of Agaricus campestris grown in submergedculture. Mycologia, 44, 605-620.

JACOBS, M. B. 1951 Chemical analysis of food and food prod-ucts, -2nd ed. D. Van Nostrand Co., Inc., New York.

JENNISON, M. W. 1948 The growth of wood-destroying fungiin aerated liquid culture. (Abstract). Proc. Soc. Am.Bacteriologists, 1, 48.

JENNISON, M. W., KODA, C., AND FAGAN, M. 1953 Aminoacids of the mycelium of a wood-rotting fungus. (Abstract).Bacteriol. Proc. 1953, 16.

JENNISON, M. W., NEWCOMB, M. D., AND HENDERSON, R.1955 Physiology of the wood-rotting Basidiomycetes.I. Growth and nutrition in submerged culture in syntheticmedia. Mycologia, 47, 275-304.

JOHNSON, B. C. 1948 Methods for vitamin determination.Burgess Publishing Co., Minneapolis.

KODA, C. F. 1951 Fundamental nutritional studies andamino acid composition of Morchella hortensis, Boud.

94 [VOL. 5

and metabolism. W. B. Saunders Company, Philadelphia.

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ACTINOMYCIN FORMATION BY STREPTOMYCES CULTURES

Ph.D. dissertation, Department of Plant Sciences, Syra-cuse University.

KUNZ, E. C., WESTON, W. H., JENNISON, MN. W., SWEET, H.R., AND KITCHENS, G. C. 1954 Process for forming alignin concentrate. U. S. Patent 2,671,751.

LUNDGREN, D. G. ANI) RUSSELL, R. T. 1956 An air-liftlaboratory fermentor. Appl. Microbiol., 4, 31-33.

NEWCOMB, H. R. AND JENNISON, M. W. 1954 Oxidativerespiration of the wood-destroying fungus, Polyporuspaltustris. (Abstract). Bacteriol. Proc., 1964, 114.

PRESTON, A. AND MCLENNAN, E. I. 1948 The use of dyes inculture media for distinguishing brown and white wood-rotting fungi. Ann. Botany, 12, 53-64.

RALPH, B. J. F. 1949 The chemistry of wood-rotting fungi.Ph.D. thesis, University of Liverpool, England.

RICHBERG, C. G. AND JENNISON, M. W. 1956 Vitamin con-tent of the mycelium of wood-rotting Basidiomycetes.(Abstract). Bacteriol. Proc., 1956, 29.

Siu, R. G. H. AND REESE, E. T. 1953 Decomposition ofcellulose by microorganisms. Botan. Rev., 19, 377-416.

SNELL, E. E. AND WRIGHT, L. D. 1941 A microbiological

method for the determination of nicotinic acid. J. Biol.Chem., 139, 675-686.

SUGIHARA, T. F. AND HUMFELD, H. 1954 Submerged cultureof the mycelium of various species of mushrooms. Appl.Microbiol., 2, 170-172.

THATCHER, F. S. 1954 Foods and feeds from fungi. Ann.Rev. Microbiol., 8, 449-472.

VAN LANEN, J. M. AND TANNER, F. W., JR. 1948 Vitaminsin microorganisms-distribution and quantitative syn-thesis. In Vitamins and hormones, edited by R. S. HARRISand K. V. THIMANN, Vol. 6, pp. 163-224. Academic Press,New York.

WALTER, G. R., JR. 1954 Organic acid production by somewood-rotting Basidiomycetes. Ph.D. dissertation, De-partment of Plant Sciences, Syracuse University.

WILEY, A. J., DUBEY, C. A., LUECK, B. F., AND HUGHES, L.F. 1950 Torula yeast grown on spent sulfite liquor.Ind. Eng. Chem., 42, 1830-1833.

WRIGHT, L. D. AND SKEGGS, H. R. 1944 Determination ofbiotin with Lactobacillus arabinosus. Proc. Soc. Exptl.Biol. Med., 56, 95-98.

Actinomycin Formation by Streptomyces Cultures'

WILLIAM A. GOSS AND EDWARD KATZ

Institute of Microbiology, Rutgers, The State University, New Brunswick, New Jersey

Received for publication October 1, 1956

Actinomycin was first isolated from a culture ofStreptomyces antibioticus by Waksman and Woodruff(1940). Since then, this antibiotic has been obtainedfrom the culture medium of a number of Streptomycesspecies by various investigators and has been readilyisolated in crystalline form. The various crystallineproducts have been designated A, B, C, D, I, J, and X(Waksman, 1954). It has been shown, however, thatthese actinomycins are not homogeneous substancesbut consist of a number of closely related biologicallyactive components (Brockmann and Gr6ne, 1954;Roussos and Vining, 1956; Pugh et al., 1956). Recentevidence suggests that the A, B, D, and X complexesmay consist of the identical components but differ inthe relative amount of each component present (Rous-sos and Vining, 1956).

Structural studies have revealed that the actinomy-cin molecule is composed of two polypeptides linkedto a chromophoric quinonoid moiety which is probablythe same for all components (Brockmann and Voh-winkel, 1954; Brockmann and Muxfeldt, 1955; Angyalei al., 1955; Brockmann et al., 1956). The peptides of a

given actinomycin component, however, may or maynot be identical. Differences in the components of an

actinomycin complex or of different complexes are

'This investigation was supported bv a research grant fromthe United States Public Health Service and by funds suppliedby Rutgers Research and Educational Foundation.

probably due solely to variations in the number, ar-rangement, and kinds of amino acids present in thepeptides (Brockmann et al., 1956). For example, cer-tain components of actinomycin C, actinomycin C2and C3, can be distinguished from those of actinomy-cins A, B, D, I, and X by the presence of D-alloiso-leucine in the peptide part of the molecule (Dalglieshet al., 1950; Brockmann et al., 1953; Waksman, 1954).All actinomycin complexes thus far analyzed possessthe amino acids sarcosine, D-valine, L-proline, L-threo-nine, and N-methyl valine.

In a previous investigation, Goss et al. (1956)2 ob-served that a significant change in the composition ofan actinomycin complex occurred during growth of aStreptomyces culture on either a complex organic orchemically defined medium. During the early stages ofgrowth only the actinomycin B type complex was pro-duced; on continued incubation of the culture only theactinomycin A type complex was recoverable from themedium. To determine whether this process was ofgeneral occurrence, a study of actinomycin formationby a number of Streptomyces cultures was undertaken

2 Goss et al. (1956) reported that during the growth ofStreptomyces strain 3720 a shift from the B- to the I-typeactinomycin complex occurred. On the bases of the investi-gations of Roussos and Vining (1956), the actinomycin complexproduced in the late stages of growth has been positivelyidentified by us as the A type.

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