Production of Oxalic Acid by a Wood-Rotting FungusGEORGE TSU-NING TSAO'

Divisiont of Chemnical Development, Tennessee Valley Authority, JVilson Dam, Alabanma

Received for publicatioln 11 Janiuary 1963

ABSTRACT

TSAO, CGEORGE Tsu-NING (Tenniiessee Valley Authority,Wilson Dam, Ala). Productioni of oxalic acid by a wood-rottinig fungus. Appl. Microbiol. 11:249-254. 1963. Thewood-rotting fungus Pleurotus ostreatus NRRL 2366 wasgrowin successfully in submerged shaker cultures in whichit produced oxalic acid from simple carbohydrates asefficiently as did Aspergillus niger. P. ostreatus also pro-duced oxalic acid from mixtures of sawdust and CaCO3,and from the solid residue from the acid hydrolysis of woodwhen the culture was supplemented with inorganic nu-trients. A. niger produced oxalic acid from the liquid hy-drolysate.

Large quanitities of nonllumiber hardwoods are availablein the Tennessee Valley, and processes to utilize this niatu-ral resource would add considerably to the economy of theregion. Oxalic acid is desirable as a starting material for thepreparationi of oxamide, which has possibilities as a nitro-gen fertilizer, and a search was made for organisms thatproduce oxalic acid fromi wood, preferably without pre-liminary chemical treatment of the wood.The presence of oxalates in higher and fleshy funigi is

well knowni. De Bary (1887) founid calcium oxalate crystalson mycelia of P'salliota camtpestris. Zellner (1907) fouid freeoxalic acid in Boletus sulfureus and Calvaria flora, andoxalate was founid in the cultures of many other higherfungi including Coniophora cerebella (Birkinshaw, Findlay,and Webb, 1940; Nord and Vitucci, 1947a), Polyporusanceps (Perlman, 1949), Merulius niveus, Mll. tre,nellosus,MU. confluens, Foitues annosus (Nord and Vitucci, 1947b, c),Tramitetes cinnabarina (DeStevenis, De Baun, and Nord,1951), anld Botrytis cinerea (Gentile, 1954). Shimazono(1955) studied the formationi and decomposition of oxalicacid in cultures of 19 species of wood-rotting funigi.Although the occurrence of oxalate in higher fungi has

been knowni for 50 years, no extensive study of the pro-ductioni of oxalic acid by wood-rotting fungi is reported inthe literature. The production of oxalic acid and otherorganic chemnicals by the common mold Aspergillus nigerhas been studied, but A. niger canniot utilize wood directlyat any appreciable rate. Oxalic acid produced by A. nigerfrom simple carbohydrates, such as glucose, by usual fer-menitation techniques is more expensivre than that pro-

' Present address: Unioin Starch and Refining Co., GraniteCity, Ill.

duced chemically, as by alkali fusion of cheap cellulosematerials or by heating sodium formate in the presence ofNaOH or Na2CO3 (Foster, 1949).

Furthermore, both surface and submerged cultures ofA. niger, as in the fermentation of simple carbohydrates,are vulnerable to contamination by wild organisms, andprevention of contaminiation considerably increases thecost of the fermentation process. Wood-rotting fungigrowing directly on solid wood wastes would be much lesssusceptible to contamination by other organisms, and amaterial intended for use in the manufacture of a fertilizerneed not meet the standards of purity required for pharma-ceuticals, foodstuffs, and most organic chemicals.

In a search for organisms that would produce oxalicacid directly from wood, four organisms were cultured oncellulosic media. The organisms were obtained from theU.S. Department of Agriculture Culture Collection inPeoria, Ill. Of these, two (A. niger NRRL 364 andPleurotus ostreatus NRRL 2366) grew satisfactorily; theother two (B. cinerea NRRL 1649 and Collybia velutipesNRRL 2367) showed too little growth to warrant furtherstudy. P. ostreatus produced relatively large amounts ofoxalic acid, both in submerged cultures from glucose andin solid cultures from wood. In addition, P. ostreatus pro-duces an edible, palatable mushroom which has possi-bilities as a valuable by-product.

P. ostreatus is one of the white-rot fungi which attackboth cellulose and the lignin fraction of wood. Its fruitingbodies are known as oyster mushrooms because the formof the plant sometimes suggests the outline of an oystershell. The organism grows naturally on dead tree trunks andbranches. It is widely distributed in America and Europe,and it has been reported in Africa, Australia, and Japan.P. ostreatus is one of the mushroom species successfullycultivated by Lohwag (1951) in Austria by the method ofBadcock (1941, 1943). Tsao (1956) cultivated the organismon sawdust and discussed the possibility of using it com-mercially as a food mushroom; this study is being con-tinued (Han, 1957; Block, Tsao, and Han, 1958, 1959).

P. ostreatus, like the basidiomycetes in general, has notbeen studied thoroughly, and the literature contains littleinformation on its physiology and metabolism. The presentpaper presents the results of a study of the growth of P.ostreatus on simple carbohydrates, unitreated sawdust, andproducts of the acid hydrolysis of wood.

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MATERIALS AND AIETHODSTests with simple carbohydrates. The stock culture of

P. ostreatus was maintained on agar slants in test tubes.Petri-dish and flask cultures were inoculated with myceliafrom 10- to 20-day-old agar slants.

Inorganic nutrients for the cultures usually were sup-plied by a solution containing (g per liter): NaNO3, 2;KH2PO4, 1.5; and MgSO4 7H20, 1. The agar medium forthe stock culture also contained (g per liter): glucose, 20;yeast extract, 2; and agar, 20. Most of the media weresterilized at 121 C for 15 min. In all experiments, the incu-bation temperature was 28 to 30 C.

In submerged-culture experiments, 50 ml of culture solu-tion were placed in a 250-ml Erlenmeyer flask which wasinoculated, plugged with cotton, and incubated on agyratory shaker operated at 240 rev/min. At the end of theincubation period, the weight of the wet mycelia was takenas a measure of the growth. In some experiments, solidssuch as CaCO3 were added to the cultures, and the myceliathen could not be weighed accurately; in these, growthwas estimated visually as none, poor, fair, or good.

Oxalate was determined by a modification of the methodof Perlman (1948), in which the sample was acidified withH2SO4 and filtered. Oxalate was then precipitated at pH5.5 to 7.0 as the calcium salt, which was filtered andwashed, dissolved in dilute H2SO4, and titrated with per-manganate. Glucose and other reducing sugars were de-termined by the modified Shaffer and Somogyi method(Neish, 1952) in which Cu2O was precipitated and thendissolvted in an excess of KI solution. The solution wasacidified and the iodine titrated with thiosulfate. In eachdetermination, a blank and a standard glucose solutionwere titrated also.

In the petri-dish cultures, 10 ml of agar medium wereplaced in each dish and, when set, inoculated in the center.At the end of the incubation period, the diameter of thecolony was taken as a measure of the growth. In some ofthese experiments, however, the diameters of two or morecolonies were the same but the amounts of aerial myceliawere different; the amounts of mycelia then were estimatedvisually and rated as poor, fair, or good.

Tests with sawdust and the products of acid hydrolysis ofwood. Sawdust, obtained from a carpenter shop whichuses mostly shortleaf pine, was screened to separate the6- to 12-mesh fraction for use in the fermentation studies.A few runis were made with oak sawdust, but no compari-son was made of sawdusts from different species. Tsao(1956) made a brief comparison of oak and gum sawdusts,but observed little difference between them as culturemedia for P. ostreatus.Most of the runs were made in 250- and 500-ml Erlen-

meyer flasks and in modified tall-form 9-liter bottles; a fewruns were made in no. 10 cans, polyethylene bags, and 30-gal tanks. The usual charge of dry sawdust was 20 g ina 250-ml flask, 40 g in a 500-ml flask, or 600 to 700 g in a9-liter bottle.

Not all the culture media were sterilized; some weresterilized with steam at 121 C (250-ml flasks, 15 min; 9-literbottles, 90 min).

Charges in 250-ml flasks were inoculated with 10- to 20-day-old slant-agar cultures; larger charges were inoculatedwith cultures grown on sterilized sawdust. The inoculumwas about 10 %o of the fresh medium; the charge in a 500-mlflask was inoculated with one-fourth of the culture in a 250-ml flask, and that in a 9-liter bottle with the cultures intwo 500-ml flasks. The incubation temperature was usually28 to 30 C.The extent of growth of P. ostreatus on sawdust

depended upon the thoroughness of mixing of the inoculumwith the fresh medium. To ensure maximal growth, largelumps of the inoculum were broken up before addition tothe fresh medium, and the inoculated mixtures were shakenthoroughly by hand.

Oxalic acid in the sawdust cultures was determined byanalysis of aqueous sulfuric acid extracts of the cultures.The calcium oxalate was precipitated at pH -5.5; at higherpH levels, the precipitate was colored, apparently by or-ganic matter.

RESULTS

Tests with simple carbohydrates. To investigate the pro-duction of oxalate from glucose by P. ostreatus, submergedshaker cultures were made in which additional organicmatter was added as oatmeal, yeast extract, or peptonie;CaCO3, CaHPO4, or (NH4)2HP04 was added to neutr-alizeor "trap" the acid. The cultures were sterilized with thetrapping agents in the flasks. With (NH4)2HP04, however,the cultures became dark brown during sterilization, andthis trapping agent was sterilized separately and addedto the sterile culture.The results, summarized in Table 1, show that no

oxalate accumulated in the absence of a trapping agent.Of the trapping agents tested, only CaCO3 was effective.In the presence of CaCO3, P. ostreatus produced about 60g of (COOH)2 per 100 g of glucose consumed. It was sur-prising that very little oxalate was obtained in the presenceof peptone, for A. niger was reported to produce oxalicacid from peptone alone (Foster, 1949).

In a study of the effect of the glucose concentration,submerged cultures were made with CaCO3 and with 30,90, 180, and 360 g of glucose per liter. Growth was goodand oxalic acid was produced at the lowest concentrationof glucose, but growth was only fair and little oxalic acidwas produced when the culture solution contained 90 g ofglucose per liter, and growth was poor at higher concentra-tions of glucose.

In tests of the effect of the oxalate concentration on thegrowth of P. ostreatus, culture solutions were prepared with30 g of glucose and 2 g of yeast extract per liter and withdifferent amounts of ammonium oxalate. The cultures wereinoculated and incubated for 40 days in submerged culture.The growth of the organism, as measured by the weight of

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wet mycelia produced per liter, decreased almost linearlywith the increase in oxalate from 484 g of mycelia with nooxalate to 20 g of mycelia with 100 g of (COONH4)2 H20per liter.To investigate sources of nitrogen for P. ostreatus, the

NaNO3 in the culture solution was replaced by its nitrogenequivalent of (NH4)2HP04, (NH4)2SO4, or peptone (16%N). To the solutions were added 30 g of glucose and 20 g ofagar per liter and potassium phosphate to give pH 5.5; theagar cultures were inoculated with P. ostreatus and incu-bated for 15 days. NaNO3 and the two ammonium saltsproduced colonies about 4.5 cm in diameter, but the growthin each was rated poor from the anmounts of mycelia pro-duced. Peptone produced colonies about 4 cm in diameter,but the growth was good.The standard culture solution contained 0.34 g per liter

of inorganic N as NaNO3. When the concentration of thisN was increased to 2.4 g per liter, there was some retarda-tion of the growth of P. ostreatus in both pour-plate andshaker cultures.

Tests were made of the effect of pH on the growth ofP. ostreatus in both shaker and pour-plate cultures. TheKH2PO4 in the solution was replaced by its phosphateequivalent of H3PO4, and the pH was adjusted to the de-sired value with KOH. To the solutions were added 30 gof glucose and 1 g of yeast extract per liter. The pour-platecultures were incubated for 15 days and the shaker cul-tures for 21 days. The results, summarized in Table 2,show that growth was best at pH 5.5. When P. ostreatuswas cultivated on a solid mixture of sawdust and CaCO3,

TABLE 1. Production of oxalic acid fronm glucose by Pleurotusostreatuts in subbmerged culture*

Charge In 4 weeks In 6 weeks

08 E < F el' o o E n 8 0

50 None None Poor 0 16 - 0

50 Oatmieal 10 Noise Good 0 14 - 8CaCO3 Fair 16 20 24 11CaHP(4 Fair 0 5 1 0(NH4)2HP04 None - - - -

40 Yeast 2 None GTood 0 9 - 0extr-act

CaCO3 Good 9 19 21 5(NH4)2HP04 Poor 0 37 - -

40 Peptone 2 None Good 0 30 - 24CaCO3 Good 1 19 -

(NH4)2HP(4 Poor 0 37 -

* Basis: 1 liter of basal solution containing inorganic nutrients;trapping agent supplied at rate of 0.84 mole per liter; 50 ml ofsolution used in each 250-ml Erlenmeyer flask.

the initial pH was 7.5, but oni inicubation the pH droppedto 5.5.

In a study of the effect of phosphorus on P. ostreatus,the KH2PO4 in the culture solution was replaced by themixture of KH2PO4 and K2HPO4 that produced pH 5.5 andin amounts ranging from 0.33 to 3.3 g of P per liter (thestandard culture solution contained 0.66 g of P per liter).Change in the amount of phosphorus made no significantdifference in the growth of P. ostreatus in either pour-

plate or shaker culture.Vitamin B1 is known to promote the growth of a number

of basidiomycetes, and its stimulating effect on P. ostreatuswas noted by Block et al. (1959). To confirm this effect,pour-plate cultures were made with culture solutions con-

taining 30 g of glucose per liter and differing amounts ofthe vitamin. After incubation for 16 days, colonies on cul-tures containing no thiamine hydrochloride were 3.1 cm indiameter; additions of 10-11, 10-1', and 10-9 g of thiaminehydrochloride per liter increased the diameters of the colo-nies to 3.5, 4.7, and 5.4 cm, respectively.

In one series of tests of different carbohydrates as sources

of energy for P. ostreatus, shaker cultures were made withsolutions to which had been added 50 g of CaCO3 and 30 g

of carbohydrate per liter. After incubation for 21 days, theorganism had produced (from the 30 g of carbohydrate)5.2 g of oxalic acid from glucose, 4.4 g from starch, 3.2 g

from sucrose, 2.0 g from lactose, and 1.8 g from maltose.In another series in which the cultures were incubated for60 days, the organism produced 28.2 g of oxalic acid fromglucose, 14.2 g from fructose, 13.6 g from xylose, 13.0 g

from arabinose, and none from galactose. The ability of P.ostreatus to make oxalic acid from the pentoses xylose andarabinose indicates that the organism should be able toproduce oxalic acid directly from the hemicellulose ofwood.To determine the ability of P. ostreatus to make oxalate

from other organic anions, shaker cultures were made withsolutions to which had been added 50 g per liter of CaCO3and organic acids to supply 12 g of C per liter. After incu-bation for 6 weeks, growth was fair in the solution con-

taining pyruvate, and 4.6 g of oxalic acid per liter was

produced. Growth was poor with gluconate, and no oxalic

TABLE 2. Effect of pH on grouth of Pleurotus ostreatus

pH Pour plate* Shakert

cm g/liter

3.6 0.0 254.0 0.4 524.5 2.8 2344.9 3.5 3565.5 6.3 5725.8 5.0 5566.1 5.5 5487.8 2.7 376

* Results expressed as diameter size of colony.t Results expressed as wet weight of mycelia.

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acid was produced. There was nio growth with formate,acetate, glycolate, lactate, malate, succinate, fumarate, ortartrate.

Tests with sawdust and the pr-oducts of acid hydrolysis ofwood. On moist sawdust alone, both A. niger and P.ostreatus grew poorly, but oln moist mixtures of sawdustwith 10% of rice hulls or 87% of oatmeal, both organismsgrew well. When the sawdust was mixed with 25 % of in-organic compounds, growth of P. ostreatus was none withCa(OH)2, (NH4)2HP04, or (NH4)2SO4; poor with Ca(N03)2,NaNO3, or KNO3; fair with calcium acetate; and goodwith CaCO3 or CaHPO4. The initial growth with CaHPO4was less than that with CaCO3, but in 6 weeks growth wasthe same on both mixtures.Growth of P. ostreatus oni sawdust mixed with only

CaCO3 was encouraging, because contamination with wildorganisms on such a mediunm should not be serious. In testsin no. 10 cans, plastic bags, and 9-liter bottles, 5 volumes ofunsterilized mixtures of sawdust and CaCO3 were mixedwith 1 volume of 3- to 4-week-old sawdust cultures andincubated at 28 to 30 C. Contaminating organisms ap-peared in some of the mixtures and dominated in the earlystages. In 2 or 3 weeks, however, the contaminants ceasedto grow, presumably because of exhaustion of the simplecarbohydrates in the sawdust, and P. ostreatus took overand eventually spread throughout the mixtures, althoughin some batches the P. ostr-eatus had been completely in-hibited by vigorous growth of the wild organisms. Heavyinoculation and thorough aeration of the culture assistedin promoting growth of P. ostreatus.On incubation at room temperature (24 to 26 C), P.

ostreatus grew poorly oni mixtures of sawdust and CaCO3alone. Addition of organic matter such as yeast extract orrice hulls gave good growth, but these mixtures had to besterilized and protected froimi contamination by wild or-ganisms which would have ruined the cultures.

10

(8

0

cn

0a

N.1

N4

I00

( 2

00 20 40 60

TIME, DAYS80 100 120

FIG. 1. Production of oxalic acid from sawduist by Pleur)otuisostreatus.

Charges in no. 10 cans were about 3 in. deep; P. ostreatusstarted to grow on the top surface and spread downward.Charges in 9-liter bottles were about 7 in. deep, and forcedaeration was required for growth of the organism. Eachbottle, with its bottom cut out, was inverted, and the saw-dust was placed on a perforated porcelain plate that restedon the shoulder of the bottle. The bottle was closed withstoppers, and filtered air was blown through the culture.In some runs, liquor was recirculated through the culturewith an air lift in an attempt to extract oxalate for pre-cipitation with CaCO3 in a separate vessel. The mycelia ofP. ostreatus formed a gelatinous mat, however, much likethat formed in cultures of Acetobacter .rylinuin, that en-trained a considerable amount of liquor, and recirculationof the liquor was discontinued.

In liquid culture, the mycelia of P. ostreatus formed gelat-inous balls. Wheni the gelatinous material formed in 14days of incubationi in a submerged shaker culture waspressed to free the entrained liquid, the liquid was foundto contain 26.8 g per liter of glucose, whereas the liquor ofthe culture contained only 14.3 g per liter of glucose. Se-questration of the glucose by the gelatinous mass ofmycelia may provide an improved environment for thegrowth of the organism, but no study was made of thephenomenon.Study of the production of oxalic acid from mixtures of

sawdust and CaCO3 was made largely in 500-ml flasks.Each flask was charged with a mixture of 40 g of sawdustand 10 g of CaCO3. In onie series, 100 ml of tap water wereadded, and the mixture was sterilized, inoculated with P.ostreatus, and incubated at 28 to 30 C. In a second series,the tap water was replaced by 100 ml of a solution con-taining (g per liter): NaNO3, 2; KH2PO4, 1.5; andMgS04* 7H20, 1. The results, summarized in Fig. 1, showthat the iinorganic nutrients markedly increased the pro-duction of oxalic acid.

In the acid hydrolysis of wood (Gilbert, Hobbs, andLevine, 1952), a solution of wood sugar and a residue of thelignin and the acid-resistant fraction of the cellulose areproduced. To study the action of wood-rotting fungi on the

TABLE 3. Acid hydrlolysis of sawduist for 90 7sbin at 21 psigage and 135 C

Hydrolysis

ChargeSawdust, g........................

Acid\'olume, ml......................

Concn, X, H2504...ProductResidue, g

Wet .............................

D)ry .............................

HydrolysateN'olume, ml......................

Stigar (as glucose), g per liter.

Batch

1 2

300 200

3,1005

1,0000.5

500 480- 134

2,500 1,0609.2 16.5

WATER SUPPLIED AS /

A - TAP WATER /

0 - INORG. NUTRIENTSOLUTION O

/ -/_ 0 AD0

0~~~~~~~00

0/

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hydrolysis products, two batches of sawdust werehydrolyzed with dilute sulfuric acid, with the results shownin Table 3. Because the autoclave in which the hydrolyseswere carried out had a maximal operating pressure of 21psi gage, instead of the 100 to 150 psi used in previouswork, the yields of sugar were relatively low.Growth of P. ostreatus was poor in a mixture of either

hydrolysate and an excess of CaCO3 over that required toneutralize the acidity. Addition of a small amount of thesolid residue to the mixture did not improve the growth.The organism would not grow on either untreated solidresidue or on a mixture of the residue from batch 1 witheither CaCO5 or yeast extract. Growth was good on theresidue from batch 1, however, when the residue was neu-tralized with CaCO3 and mixed with yeast extract. Growthof P. ostreatus on the residue from batch 2 was good whenthe residue was mixed with yeast extract alone or withCaCO3 and rice hulls, but only fair when the residue wasmixed with CaCO3 and yeast extract. The results show thatP. ostreatus grows best in a slightly acid medium, but thatgrowth is inhibited by excess acid. Failure of the organismto grow on the residues without addition of yeast extractor rice hulls indicated that some essential material hadbeen removed or destroyed by the acid hydrolysis.When a mixture of 25 g of the wet residue (7 g dry) from

batch 2, 5 g of CaCO3, and 10 ml of the inorganic nutrientsolution was incubated with P. ostreatus for 35 days at 28to 30 C, growth was good, and 10 to 13 g of oxalic acid wereproduced per 100 g of dry residue. Addition of 40 mg ofyeast extract increased the yield of oxalic acid to 18 g per100 g of dry residue. Since, as shown in Table 3, the dryresidue was 67 %O of the initial sawdust, this is a yield of 12g of oxalic acid per 100 g of sawdust, which is about 2.5times that (4.4 g per 100 g of sawdust) obtained in thesame length of time from cultures of P. ostreatus onmixtures of untreated sawdust, CaCO3, and inorganic nu-trients.

Although P. ostreatus did not grow well in the liquidhydrolysates, A. niger produced about 65 g of oxalic acidper 100 g of total wood sugar (calculated as glucose) insubmerged shaker cultures of either hydrolysate, to whichhad been added excess CaCO3 and some of the wet residue.Hence, both A. niget and P. ostreatus might be used in theproduction of oxalic acid from wood.

DIscussIoNThe production of oxalic acid by A. niger was studied by

Wehmer (Foster, 1949), who found that free oxalic acidwas decomposed almost as fast as it was formed and thatonly traces of the acid remained in the cultures. When analkali was added to neutralize the acid, however, oxalateaccumulated in the cultures. Shimazono (1955) studied theproduction and decomposition of oxalic acid by 19 speciesof wood-rotting fungi, and he concluded that a soluble en-zyme, oxalic acid decarboxylase, decomposed the acid. Freeoxalic acid accumulated in cultures of "brown-rot" fungi,

but not in cultures of "white-rot" fungi, the group that in-cludes P. ostreatus. Shimazono did not investigate theeffect of trapping agents on the accumulation of oxalatesin the cultures. As shown by the data in Table 1, oxalateaccumulates in cultures of P. ostreatus that contain CaCO3.It appears that calcium oxalate, which is quite insolublein water, is not attacked by oxalic acid decarboxylase.

Results of the tests of the effects of concentration ofglucose, oxalate, nitrate, and phosphate showed that P.ostreatus cannot tolerate high concentrations of solutes.Within the ranges of concentrations ordinarily used inculture media, however, P. ostreatus suffered no retarda-tion of growth.

P. ostreatus produced oxalic acid from all the simplecarbohydrates tested except galactose. Higher yields ofoxalate were obtained from glucose than from xylose,arabinose, or other carbohydrates. In the tests with wood,higher yields of oxalate were obtained from the solid resi-due of the acid hydrolysis than from the untreated saw-dust, from which it was concluded that the hydrolysisresulted in considerable rupture of the molecules of thewood, thus making them more suitable as sources of energyfor P. ostreatus. Dilute acid hydrolyzes most of the hemi-cellulose and some of the cellulose of wood, so that theresidue contains less hemicellulose than the untreatedsawdust. Hydrolysis of the hemicellulose, which producesxylose and arabinose, thus removes carbohydrates that arerelatively poor sources of oxalate, and it is not surprisingthat higher yields of oxalate are obtained from the residuefrom which the undesirable carbohydrates were removed.The mechanism by which CaCO3 promotes the growth

of P. ostreatus on sawdust is unknown, but its effect on thepH may be a contributing factor. The initial moist mixtureof sawdust and CaCO3 was dark brown and had a pH of6.8 to 7.2. As P. ostreatus grew in the mixture, the partsreached by mycelia became light brown and had a pH ofabout 5.5; the change in color was a convenient indicatorof the extent of the growth of the organism. When the mix-ture was acidified with H2SO4 and warmed, the hydrolysatewas light brown; addition of NaOH turned the solutiondark brown.The oxalate in the solid cultures was determined by ex-

tracting the mixture with dilute sulfuric acid and analyzingthe filtered extract. This treatment not only kills P.ostreatus, preventing further utilization of the culture forthe production of mushrooms, but also would be expensivein a commercial process. The calcium oxalate and unre-acted CaCO3 probably could be separated from the residualwood by blunging with water, but this step was not in-vestigated.

LITERATURE CITED

BADCOCK, E. C. 1941 New methods for the cultivation of wood-rotting fungi. Trans. Brit. Mycol. Soc. 25:200-205.

BADCOCK, E. C. 1943. Method for obtaining fructification of wood-rotting fungi in culture. Trans. Brit. Mycol. Soc. 26:127-132.

BIRKINSHAW, J. H., W. P. K. FINDLAY, AND R. A. WEBB. 1940.

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Biochemistry of the wood-rotting fungi. II. The acids pro-duced by Coniophora cerebella Pers. Biochem. J. 34:906-916.

BLOCK, S. S., G. TSAO, AND L. H. HAN. 1958. Production of mush-rooms from sawdust. J. Agr. Food Chem. 6:923-927.

BLOCK, S. S., G. TSAO, AND L. H. HAN. 1959. Experiments on thecultivation of Pleurotus ostreatus. Mushroom Sci. 4:309-325.

DE BARY, A. 1887. Comparative morphology and biology of fungi,mycetozoa, and bacteria. Clarendon Press, Oxford.

DESTEVENS, G., R. M. DEBAUN, AND F. F. NORD. 1951. Themechanism of enzyme action. XLV. The role of certain di-carboxylic acids in the formation of oxalic acid by wood-destroying molds. Arch. Biochem. Biophys. 33:304-313.

FOSTER, J. W. 1949. Chemical activities of fungi, p. 326-350.Academic Press, Inc., New York.

GENTILE, A. C. 1954. Carbohydrate metabolism and oxalic acidsynthesis by Botrytis cinerea. Plant Physiol. 29:257-261.

GILBERT, N., I. A. HOBBS, AND J. D. LEVINE. 1952. Hydrolysis ofwood using dilute sulfuric acid. Ind. Eng. Chem. 44:1712-1720.

HAN, L. H. 1957. Examination of some of the unit processes inmushroom production and evaluation of methods for increas-ing efficiency. M.S. Thesis, University of Florida, Gainesville.

LOHWAG, K. 1951. Crops from sawdust. Mushroom Growers'Assoc. Bull. 22:20-21.

NEISH, A. C. 1952. Analytical methods for bacterial fermentations,p. 35-36. Natl. Res. Council Can., Saskatoon, Rept. 46-8-3.

NORD, F. F., AND J. C. VITUCCI. 1947a. Enzymic hydrolysis ofcellulose by Coniophora cerebella. Nature 160:261.

NORD, F. F., AND J. C. VITUCCI. 1947b. Enzyme studies on themechanism of wood decay. Nature 160:224-225.

NORD, F. F., AND J. C. VITUCCI. 1947c. The mechanism of enzymeaction. XXIX. The acetate metabolism of certain wood-destroying molds and the mechanism of wood decay. Arch.Biochem. 14:229-241.

PERLMAN, D. 1948. The nutrition of Sclerotium delphinii. Am. J.Botany 35:360-363.

PERLMAN, D. 1949. Studies on the growth and metabolism ofPolyporus anceps in submerged culture. Am. J. Botany 36:180-184.

SHIMAZONO, H. 1955. Oxalic acid decarboxylase, a new enzymefrom the mycelium of wood-destroying fungi. J. Biochem.(Japan) 42:321-340.

TSAO, G. T. 1956. Investigations on a new method for mushroomproduction. M.S. Thesis, University of Florida, Gainesville.

ZELLNER, J. 1907. Chemie der hoheren Pilze. Leipzig Verlag Wil-helm Engelmann. (Cited in Foster, 1949).

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