asVol. 38

Studies on the Metabolism of Mould Fungi1. PHOSPHORUS METABOLISM IN MOULDS

By T. MANN (Senior Beit Memorial Fellow), Molteno Indstitute, University of Canmridge(Received 22 May 1944)

Phosphorus, together with nitrogen, sulphur, mag-

nesium and organically bound carbon, has been one

of the few elements long recognized as indispensablefor the growth and metabolic activities ofthe mouldfungi (Lafar, 1904). Much later other elements, suchas iron, copper, zinc, m nganese, molybdenum andgallium, were added to the list of essential nutrients(Foster, 1939). It has been repeatedly shown thatmany moulds, including numerous species ofA&per-gillue, PeniciUium and Rhizopu8, are unable to growunless small amounts of phosphate are present inthe nutrient medium..The first systematic study of the phosphorus

metabolism in moulds was carried out by Vorbrodt(1920, 1926, 1928), who demonstrated that growingmycelia ofAspergiUlus niger take up orthophosphatefrom the culture media and store it partly in theinorganic form and partly in the form pf new com-

pounds. More recently, Michel-Durant (1938) ob-served that the uptake of orthophosphate byA. niger is insignificant at the early stage ofmycelialgrowth but that it assumes bigger proportions atthe stage ofthe formation ofthe conidia. Accordingto this author, there are three groups ofphosphoruscompounds in the mycelium, a small 'lipoid fraction'and two ethanol-insoluble fractions, one of which issoluble in trichloroacetic acid ('phosphore organiquesoluble indetermine') and the other acid-insoluble,represented by the non-extractable residue.The paucity of data concerning the chemical

nature-of the phosphorus compounds in moulds andtheir metabolic function is in striking contrast tothe wealth of information available regarding phos-phorus compounds in yeast, bacteria, higher plantsand animal tissues. It seemed, therefore, desirableto investigate the nature and -behaviour of phos-phorus compounds in moulds, and to examine thepossibility of the existence in moulds of a link be-tween the phosphorus metabolism and other pro-

cesses, such as the metabolism of carbohydrate.(Preliminary communication, Mann, 1943.)

EXPERIMENTAL

The stock cultures used for inoculation were grown

on beerwort-agar slopes and were subcultured once

a month. The majority of the experiments was

carried out with A. n4ger v. Tiegh. (Nationhl Collec-tion of Type Cultures (N.C.T.C.) no. 594), but occa-sionally other moulds were used, such as A. nigercitricu Neuberg (N.C.T.C. no. 1692), A. nigerThaysen (N.C.T.C. no. 603), PeniciUium oxaumrCurrie-Thom (N.C.T.C. no. 983) and P. notatumWestipg-Fleming (N.C.T.C. no. 4222). The culturemedia were composed of 10% glucose, 0-5%NaNOs, 0- 1 % MgSO4. 7H,O, with varying amountsof K,HPO4, dissolved in tap water. 50-300 ml.portions of the medium were used arid distributedin crystallizing dishes to a depth not exceeding1-5 cm. The inoculum amounted to-approxiimately1 mg. of spore material (dry wt.) per 100 ml.medium. The mycelia were usually grown at 300under aseptic conditions.

TChe disappearance of glucose from the medinm of thegrowing culture was followed quantitatively by the methodof Benedict. The total amount of organic acids producedand excreted by the mycelium into the medium wa assessedby titration with 0 1 N-NaOH in presence of phenolphthaleinas indicator. Owing to the presence of gluconic acid inlactone form, a rather long time was required to reach theend-point of titration. Besides gluconic acid, the two othermain organic acids produced were citric acid and oxalicacid. Citric acid was determined gravimetrically as penta-bromoacetone and oxalic acid as calcium oxalate.

Before performing any phosphorus analysis of themycelium, the medium was siphoned from beneath themycelium and replaced by the same volume of water.Care was taken not to submerge any part of the surface ofthe mycelium. After two more changes of water at 8 hr.intervals, the mycelium was spread evenly on a glass plateand quickly divided with a sharp knife into several parts.Some of the material was used for the estimation of the dryweight (at 1050) and for the determination of the totalcontent of phosphorus, and the remainder was thoroughlyextracted with cold trichloroacetic acid. This extract, whichcontained the total acid-soluble phosphorus, was used forthe estimation of: (1) the true inorganic phosphorus deter-mined as MgNHIP04; (2) phosphorus determined as phos-phate which reacts directly with ammonium molybdate, PO;(3) the phosphiorus which appears as orthophosphate after7 and 30 min. hydrolysis with u-HCI, P7 and Pao; (4) phos-phorus as the total phosphate after incineration with H2S,0and HN03, Pt (Lohmann & Jendrassik, 1926).

Nitrogen was estimated by the Kjeldahl procedure. Theassays for nicotinic acid, riboflavin and aneurin were verykindly undertaken by Dr E. Kodicek and Dr Y. L. Wangof the Nutritional Laboratory, Cambridge.

T. MANN

Growth and metabolism of Aspergillus niger inpresence of varying concentrations of phosphate

With 0-002% K,HPO4 or less in the culturemedium no growth or only a very poor growth ofA. n4er was observed and conidia appeared verylate, if at all. With 0-02% KIHPO4, thin myceliaappeared 2 days after sowing and developed withinthe next 2 days into stro4g, white, smooth pellicles.U,sually on the 4th day of growth the first blackconidiophores made their appearance. With 0-2-0-5% K2HPO4 the growth was still -quicker, and^within 4 days a very thick mycelium was producedwhich, instead of being white and smooth, showeda creased surface and was of a distinctly yellowishcolour, especially at the surface in contact with themedium. No conidiophores were present at thatstage.'They usually appeared on the 6th or 7th dayof growth.Both the rate at which phosphate was absorbed

from the medium as well as the concentration whichit finally reached in the mycelium were found todepend on the initial amount of K2HPO4 in themedium. With 0-02% K2HPO4 in the medium, therate of disappearance was so rapid that after 3 daysof growth, the medium was completely depleted ofphosphorus; the final concentration of phosphorusreached in the mycelium was about 0-3% P (drywt.). With 0-2-0-5 d/c ]2HPO, in the medium, therate of absorption of phosphate by the growingmycelium was somewhat slower duri4 the firstdays of growth and reached a higher level only at

'944the preconidial stage; the final concentration ofphosphorus in the mycelium was 1-2% P (dry wt.).

Effect of phosphate concentration on metaboli&m.By varying the concentration of phosphate in themedium it was possible to influence markedly boththe carbohydrate and the nitrogen metabolism ofthe mould, as well as its respiration. Table 1 illus-trates the differences between two cultures, Aand B,grown in the presence of 0-02 and 0-2 % K2HPO4,respectively. The cultur6 B grew faster than theculture A; it utilized glucose more rapidly andturned a higher proportion of it into citric acid. Inaddition, it was capable of building up a muchlarger store of nitrogen than the phosphorus-defi-cient culture A. The nitrogen stored in the mycelium,derived from the niitrate of the medium, was foundto be entirely of organic nature; neither nitrate norfree aonia could be detected. Approximatelyone-half of the mycelium-N could be extracted withtrichloroacetic acid or with other deproteinizingreagents. Among the N-containing substances thusextracted were aneurin, riboflavin and nicotinic acid.It is of considerable interest to note that in responseto an increased concentration of inorganic phos-phate in the medium, the formation of all threevitamins was enhanced to such an extent as tocompare favourably with rich vitamin sources suchas liver or yeast. The high riboflavin content isresponsible for the yellow colour of the phosphorus-rich mycelium, and also, it may have some bearingon the fact that such a mycelium, when separatedfrom its medium and transferred to water, continuedto respire for several days much more vigorously

Table 1. Effect of vacrying conentration of orthophosphate on the metabolim of A. niger

Mycelium (5 days old)Appearance

Dry weightQo2 of intact myceliumQo2 of mycelium pulpQo, of pulp + 1% glucoseTotal nitrogen, mg.Total phosphorus, mg.Magnesium, mg.Aneurin, pg.Riboflavin, ,ug.Nicotinic acid, ,ug.

Medium (50 ml.)AppearanceGlucose, %Glucose oxidase activity; 2 ml. medium +10 mg.glucose, pH 7, 30°, take up in 15 min.Total phosphorus, mg.Titratable total acidity, ml. N-acidCitric acid, ml. N-acidOxalic acid, ml. N-acid

Agrown in presence of0-02% K,HPO4

Thin, white, smooth,first conidiophores

460 mg.6-20-4

30-08-11-53-83-2

16-119-4

Colourless3-7

120 1p. 02

0-015-03-80-05

Bgrown in presence of

0-2% K2HPO4Thick, yellowish, noconidiophores

1092 mg.11-41-9

19-023-712-16-8

19-078-7

302-0

Yellow0-130,u. 02

6-316-07.9.0oo

PHOSPHORUS METABOLISM OF MOULDSthan the P-deficient mycelium. Grinding of themycelia with water caused a pronounced fall in the0 uptake, but even then, the pulp obtained fromthe mycelium B had a much higher rate of02 uptakethan that prepared from mycelium A. When, how-ever, glucose was added to the pulp, the 0, uptakeincreased more in A than in B. This was due to a,higher activity ofglucose oxidase in the mycelium A.

The ab8orption of phosphate from the medium andthe formation of phosphorus compounds in themycelium

Absorption of phosphate. The experiments re-corded in Table 2 were carried out with A. nigerv. Tiegh. in crystallizing dishes containing 50 ml.of the medium described, with 0-2% K2HPO4 andgrown at 300. Thin mycelia usually appeared 24 hr.after sowing, and the production of gluconic andcitric acid, at the expense of glucose, soon followed.At the same time, the absorption of phosphate fromthe medium began to be n6ticeable. Occasionallythe rate of the absorption of phosphate by thegrowing mycelium was rather slow during the firstdays of growth and reached a higher velocity onlyat the preconidial stage. In other instances, how-ever, the absorption proceeded rapidly from thebeginning of the growth and was well marked at anearly stage of growth, seveial days before the ap-pearance of the first conidiophores.

Formation ofphosphorus compounds in the mycelia.In order to follow the fate ofthe absorbed phosphate,a study was made ofthe content and the distributionof phosphorus compounds in the mycelia. A large

proportion of the phosphorus which accumulated inthe ihycelia, sometimes as much as 90%, could beextracted by grinding the mycelia thoroughly withtrichloroacetic acid. Only a small proportion of theacid-soluble phosphorus, 10-20% in all, behavedlike ordinary orthophosphate, awyd the value of thephosphorus determined directly (P0) equalled thatin the magnesium-ammonium-phosphate precipi-tate. A large proportion of the total acid-solublephosphorus, as assessed by incineratiQn with H,SO4and HNO3, broke down completely to orthophos-phate after a 7 min. hydrolysis with 1 N-HCI at 1000,and thus behaved like a typical 'easily acid-hydro-lyzable phosphorus compound'. Only a small pro-portion of the phosphorus present in the myceliumwas found to occur in a 'difficultly hydrolyzable'form.The formnation of the phosphorus compounds in

the mycelium usually reached its peak at the timewhen the first conidiophores were appearing. Atthe same time the medium was already depleted of50-90% of the orthophosphate originally present,as well as of glucose, and contained large quantitiesof gluconic acid and citric acid. When the concen-tration of these two acids had reached its maximum,oxalic acid was still absent, and could be found onlylater, after the mycelium had lived for several daysat the expense of the first two organic acids. Assoon as the reserve of gluconic and citric acid wasexhausted, the process of autolysis set in, heraldedby a decrease in the dry weight of the myceliumand accompanied by a rapid decomposition of thephosphorus compounds in the mycelium and thereappearance of orthophosphate in the medium.

Table 2. Phosphorus metabolim in grouing cultures of A. niger

(PO=phosphorus determined as phosphate which reacts directly with ammonium molybdate; P7=the phosphoruswhich appears as orthophosphate after 7 min. hydrolysis with N-HCI; P30 =the phosphorus which appears as orthophos-phate after 30 min. hydrolysis with N-HCI; PtO.=phosphorus as the total phosphate after incineration with H2SO0and HNO,.)

Mycelium Medium (50 ml.)

Age(days) Appearance

0

I 2 Thin, continuous3 White, strong5 First conidia9 Many conidia14 Autolysis

II 2 Thin, continuous4 White, strong6 Few conidia14 Autolysis

III 3 White, strong5 First conidia8 Many conidia12 Onset of autolysis19 Autolysis

I

0l

11

1.

0

1

Trichloroacetic acid extracts TotalDry mg. P Glu- aciditywt. A-_- cose (ml. N-

(g.) PO P7 P,o Ptot. (g.) acid)5X0 0.0

.506 0.4 1-6 3-2.*056 1.5 5.0 5.5 6.5 1.1 15.0*340 1.5 9.0 9.5 11.0 0-1 18-0*728 1.1 12 3 12-9 14-0 0 0 5.51-932 2-6 4*8 0.0 0 3*352 0-2 1-5 15 1-8 1 1 6-51*760 0 7 5.7 6-1 10.0 0 3 12*5.356 2-3 9 0 10-3 12-0 0.0 7.51-816 4-6 6-3 6-3 6.8 0.0 0.00*3200-7921.1500-9200-510

1.50-70-6

5-08-6

10.0 - 120

1.1 13-00-0 15-50.0 8-00.0 2.00.0 0.0

Vol. 38 341

PO(mg.)17-8

10-55.00.08-0

16*0

0.06-0

0-00-311.0

Ptot(mg.)17-8

10.55.00.09*6

16-0

0~06-0

0.00.5

12-0

Citric Oxalicacid acid

(ml. N- (ml. N-acid) acid)

0-6 0.03.9 0Q06.9 0.05.5 0.10.0 0-3

4-0 0.04-6 0-05-0 0.01-0 1-00.0 Trace

T. MANN

Table 3. Formation of an easily acid-ydrolyzable pho&sphoru compound in mwude

SpeciesA. niger v. Tiegh.A. niger citricue NeubergA. niger ThaysenP. oxalicumP. notatum

Dry wt.,(g-)1-1961-0081-2520-6240-960

Mycelium

Trichloroaeotic acidextracts (mg. P)

*p, *P7

2-7 9-40-6 1-35-3 6-60-2 3-01-3 4-7

* See note to Table 2.

r

Glucose(g.)0-00-80-03:3

Trace

Medium (50 mL)

Acidity(ml. x-acid)

9.511-51-50-7510-5

The formation of easily hydrolyzable phosphorusfrom orthophosphate is not restricted to one strainof A. niger but was found also with other moulds(Table 3). However, the amounts of the readilyhydrolyzable phosphorus formed by these mouldswere smaller than with A. niger v. Tiegh.

Effect of oxygen and respiratory inhibitors on phos-phorus metabolism. The absorption of phosphatefrom the medium and the formation of phosphoruscompounds in the mycelium were found to dependstrictly on the presence of oxygen. In this respectthe phosphorus metabolism of moulds differs sub-stantially from the various phosphorylation pro-cesses which occur in yeast and in animal tissues.Cyanide and azide (0-001 N), iodoacetate (0-001 N)and fluoride (0-005N) were found to have a powerfulinhibitory effect on the respiration as well as on thecarbohydrate metabolism of the moulds; at thesame time these inhibitors strongly suppressed thephosphorus metabolism.The effect ofthese inhibitors could best be studied

on mycelia grown directly in the cups of Barcroftdifferential manometers (Table 4).A. niger was sown on 5 ml. medium. During the early

stages of growth the shaking of the manometers had to be

avoided owing to the great fmagility of the fresh mycelium.With a continuous mycelium geiltle shaking could be safelyapplied, but it had little effect on the extent of the oxygenconsumption owing to the fact that the mycelium respiresmainly on the surface. After 4 days of growth at 30° themanometer cups containing the pellicles were attached tothe manometers and the course of the respiration wasfollowed quantitatively. The oxygen uptake of the myceliawas measured at 300, and expressed in terms of

ul. 0, taken up

QOamg. dry wt. x hr.On the addition of inhibitors the absorption of

phosphate from the medium came suddenly to a

standstill. The action of the inhibitors on the respi-ration and the phosphate absorption could be re-

versed provided the mycelium had not been left incontact with the poison for more than a few hours.Otherwise an autolysis set in and the phosphoruscompounds formed in the mycelium broke downrapidly with the liberation of free phosphate.

In order to demonstrate the effect of oxygen on

the phosphate metabolism it was found convenientto growmycelia for several days on media deficient inphosphate (0-01-0-02% instead of 0-2% K,HPO4),but otherwise composed as described above. Ifthesemycelia were then transferred to 0-1 % KH2PO in

Table 4. Effect of respiratory inhibitors on the phosphorus metabolism

Mycelium grown on 5 ml. medium in Barcroft manometer cups; 4 days at 300.

Addition to the mediumf- ~~~~~A

Concentration XSubstance (x)

lodoacetate

Fluoride

Azide

Cyanide

0-0010-0010-0050-0050-010-010-020-020-0010-0010-001

ialyzed ,-after Dry wt.(hr.,, (mg.)

_-- 903 90

24 823

24 80324 80324 603

24 653 90

Mycelium

QO012-7

3-61-6

*Po(mg.)0-070-090-09

5-82-3 0-094-81-9 0-051-3 -

0-5 0-05

2-8 0-071-3 0-09

1-5 0-08* See note to Table 2.

*P7(mg.)0-600-600-40

0-40

0-40

0-400-500-400-55

Medium, A

*Po *P7

(mg.) (mg.)0-08 0-080-09 0-090-30 0-32

0-10

0-20

0-200-100-300-10

0-10

0-25

0-25

0-100-320-10

342 '944

*Po(mg42-5

12-52-5

10-010-0

PHOSPHORUS METABOLISM OF MOULDSthe presence of oxygen, they quickly utilized theinorganic phosphate and built up from it the easilyhydrolyzable phosphorus compound (Fig. 1, curve I).Anaerobically, there was no change in the contentof KH,P04 in the mediuxu (Fig. 1, curve II). When,after a period of anaerobic incubation, oxygen waslet in, the mycelium responded immediately withrenewed absorption of phosphate. However, theabsorption never reached the same level as withmycelia which had not been exposed to anaerobicconditions. The reason is that even a short periodof anaerobiosis has a general adverse effect on the

25,Air

in nitrogen

,20

Jls10

C 1 2 3Time in days

Fig. 1. The absorption of phosphate from the medium inpresence and absence of oxygen

metabolic activities of the mycelium. A prolongedlack of oxygen was found to lead invariably toautolysis.When mycelia were transferred to neutralized

solutions of sodium pyrophosphate or of certain

sugar-phosphoric acid esters instead of to ortho-phosphate, the process ofphosphate absorption wasslower because it was preceded by the breakdownof the phosphoric compounds into orthophosphate(Table 5). Glucose had a slightly delaying effect onthe process of phosphate absorption.

MetaphoophataeIt is essential to use trichloroacetic acid for the

successful extraction ofphosphorus compounds fromthe mycelium. Whelk the myoelium was ground withwater instead of with trichloroacetic acid, the acid-hydrolyzable phosphorus compounds of the pulpbroke down within a short time to orthophosphate.They could be preserved, however, either by heatingthe pulp to 1000 and keeping it at this temperaturefor a few minutes or by rapidly dehydrating thepulp with acetone and converting to a dry powder.No losses in the acid-soluble phosphorus occurredduring the acetone treatment which, however, didremove all acetone-soluble substances includingsome acetone-soluble, difficultly hydrolyzable phos-phorus compound. The distribution of phosphorus,nitrogen and magnesium, between the acetone ex-tract, the acetone powder and the trichloroaceticacid extract from the acetone powder of one my-celium, is illustrated by the scheme shown on p. 344.The assumption that the breakdown of phos-

phorus compounds in the mycelium ground withwater might be due to the presence ofa phosphatasewas strengthened by the observation that if such apulp was brought into contact with a variety ofphosphoric acid derivatives, it decomposed themqtqickly into orthophosphate.The phosphatase contained in the mycelium could

be extracted with water without any loss of activity.The extract (1 ml.; 12 mg. dry wt.), when actingupon 2 ml. substrate (Na salts; 1-2 mg. P) for 3 hr.at pH 5 and 300, converted to orthophosphate70-98% of the following compounds: pyrophos-phate, metaphosphate, hexametaphosphate, mc-phos-phoglycerol, f-phosphoglycerQl, Cori ester (1-phos-phoglucose), Embden ester (6-phospho-hexose),

Table 5. Metabolim of pyrophotphate and tugar-phosphoric acid eaters by the mycdiumnMedia

Mycelium grown in manometer vessel. Incubation , A

On the fifth day medium replaced at 30° QO of P, *P7 Aby 5 ml. of: (hr.) myczlium (mg.) (mg.) (ml

Water 0 8-3 0-0 0016 6-3 0-05 0-06

Sodium pyrophosphate (4 mg. P) 0 9-2 0-0 4-016 10-0 2-5 3-0

K salt of Con ester (6 mg. P) 0 9-4 0-0 6-016 12-8 3-9 4-5

Na salt of Embden ester (6 mg. P) 0 8-6 0-0 0-1le

Lcidity1. N-acid)0-00-250-00-140-00-160-00-08

* See note to Table 2.

Vol. 38 343

11.9 2.0 z D

T. MANNMycelium (3-6 g. dry wt.)

1-08% P, 2-32 0/ N, 0-555% Mg

Acetone extract0-023% P, 0.046% N, 0.01% Mg

Harden-Young ester (1:6-diphospho-fructose), 3-phosphoglycerate, adenosine triphosphate (all threeatoms of phosphorus), muscle adenylate, yeastadenylate, aneurin diphosphate and phytate.The outstanding property of this polyphospha-

tase, and one which sets it apart from other knownphosphatases, is the ease with which it acted on

metaphosphate. This enzyme may therefore bereferred to as a metaphosphatase.The only instance. where metaphosphatase ac-

tivity has been previously described in moulds isthe observation made by Kitasato (1928 a, bJ,'whofound that preparations of 'Taka-phosphatase'from A. oryzae are active towards metaphosphate.He had to use, however, as much as 1 g. of Taka-phosphatase and incubate this preparation for 20 hr.with 250 mg. sodium metaphosphate in order toobtain 10% of the phosphorus in the form of ortho-phosphate. Compared with the metaphosphatase ofA. niger the activity of 'Taka-phosphatase' appears

to be negligible.Metaphosphatose in metabolism fluid. The exami-

nation of the culture media separated from themycelia ofA. niger revealed that they too possessedconsiderable metaphosphatase activity; 2 ml. me-

dium separated from a 4-day-old mycelium andincubated for 5hr. with 2 ml. sodiummetaphosphate(0-6 mg. P) converted it completely to orthophos-plate. Incidentally, the presence ofthe phosphatasein the medium provides an explanation for theobservation that organic phosphorus compoundsadded to the culture media are first dephosphory-lated and then absorbed as orthophosphate.

Purification of metaphosphatase. In order topurify the metaphosphatase and to obtain theenzyme free from glucose oxidase (always present

alike in the mycelium and the medium), the aqueousextracts from the mycelia were dialyzed, the enzymewas precipitated with 3 vol. of cold acetone, re-

dissolved in water and finally purified by adsorptionon tricalcium phosphate gel and alumina C,,. In thismanner it was possible to obtain metaphosphatasepreparations practically free from glucose oxidase.In addition, it was found that 0-01 x-sodium nitratehad a strong inhibitory effect on the activity ofglucose oxidase at pH 5 (but not at pH 7), whereasit did not affect the activity of metaphosphatase atall. The optimum activity of metaphosphatase oc-

Acetone powder1.05% P, 1.87% N, 0550% Mg

Trishloroacetic acid extract (80 ml.)0.85% P (0.024% Po, 0.790% P7)

0.825% N, 0.525% Mg

curred at pH 3-7-4-2. A purified enzyme prepara-tion (601&g.) incubated with a solution of sodiummetaphosphate or pyrophosphate (0-3 mg. P) for2 hr. at pH 5 and 30° converted both compoundsquantitatively to orthophosphate.

DISCUSSIONUnlike the animal tissues and yeast cells, the meta-bolic activities of the mould fungi, especially thoseof the acid-producing species like A8pergiUus andPenicillium, depend to a large extent on the presenceof oxygen. AspergiUus niger, for instance, willoxidize sugar very efficiently in the presence ofoxygen to non-volatile organic acids such as glu-conic, citric and oxalic acid, whereas anaerobicallyit scarcely ferments sugar at all. This phenomenonwas well known to the early investigators, andDuclaux in his Traits de Microbiologie (1898) very

appropriately referred to the process of oxalic acidformation in moulds as 'res iwation oxalique', a

term which is more suitable than the later 'oxalicfermentation' or 'oxidative fermentation'. The ex-

periments described in this paper demonstrate forthe first time that in addition to the carbohydratemetabolism, the phosphorus metabolism of A. nigeris also strictly aerobic in character. In this respectthe moulds differ again from yeast, where the pre-sence or absence of oxygen is said to be of littleimportance so far as the absorption of phosphate isconcerned (Mullins, 1942). The intimate relationshipbetween the phosphorus metabolism and the respi-ration of A. niger is best demonstrated by the factthat various inhibitors ofmould respiration, such as

cyanide, azide, iodoacetate and fluoride, also stopphosphortis metabolism. This fact indicates a con-

nexion between the metabolism of carbohydrateand that of phosphorus compounds in moulds.Moreover, an increase in the content of phosphatein the culture medium enhances both the utilizationof carbohydrate and the formation of organic acids,particularly citric acid. However, the increasedproduction of citric acid may also be due to changesin the nitrogen, metabolism.

Mycelia grown in the presence of high phosphateconcentrations have a much higher nitrogen meta-bolism than those grown on phosphate-deficientmedia and are capable of building up a considerablereserve of various valuable nitrogenous substances.

344 '944

Vol. 38 PHOSPHORUS METABOLISM OF MOULDS 345Another point which emerged is that the mycelia

of various moulds convert a large proportion of theabsorbed orthophosphate into acid-hydrolyzablephosphorus compounds. One of these compoundsbreaks down completely to orthophosphate after a7 min. hydrolysis with NxHCI at 1000, and thusbehaves like pyrophosphate. Another compoundoccurs in the 'difficultly hydrolyzable' fraction.

It is interesting to note that A. niger, which con-tains an abundant store of phosphorus compoundsin the mycelium, also possesses a highly activephosphatase. This enzyme acts optimally under theacid conditions prevailing in the mould culture. Itattacks a variety of derivatives of phosphoric acid,among them metaphosphate, a fact which will becommented upon in the next paper of this series.

SUMMARY1. Mould fungi with a predominantly aerobic

carbohydrate metabolism have also a characteristicaerobic phosphorus metabolism. They absorb ortho-phosphate aerobically and convert it into a nixmberof phosphorus compounds.

2. The rate with which phosphate is absorbedand utilized depends on its initial concentration inthe culture medium. Cultures grown in the presenceof large concentrations of phosphate develop morerapidly than those grown in low concentrations,

have a higher respiratory quotient, utilize glucosemore rapidly, produce more citric acid, have ahigher nitrogen metabolism and exhibit a substan-tially increased content of certain vitamins.

3. Cyanide and azide (0.001 t), iodoacetate(0.001N) and fluoride (0-005N) strongly inhibit therespiration of these moulds, and bring the phos-phorus metabolism to a standstill.

4. A large proportion of the orthophosphate ab-sorbed aerobically is converted to acid-hydrolyzablecompounds. One of these compounds breaks downto orthophosphate after a 7 min. hydrolysis withN-HCI, and thus behaves like a, typical 'easilyhydrolyzable phosphorus compound'. Anothercompound occurs in a 'difficiultly hydrolyzable'form.

5. The formation of phosphorus compounds inthe mycelium reaches its peak at the preconidialstage. During autolysis these compounds are de-composed and excreted into the medium as ortho-phosphate.

6. The occurrence of a metaphosphatase in boththe mycelium and the culture medium ofAspergillusniger is described. The enzyme has been purifiedand separated from glucose oxidase. Whereas theactivity of glucose oxidase is considerably reducedby 0-01 N-NaNO3, the phosphatase activity remainsintact.

REFERENCES

Duclaux, E. (1898). Trait de Microbiologie. Paris: Mason.Foster, J. W. (1939). Bot. Rev. 5, 207.Kitasato, T. (1928a). Biochem. Z. 197, 257.

(1928b). Biochem. Z. 201, 206.Lafar, F. (1904). Handbuch der technologischen Mykooie.

Jena: Fischer.Lohmann, K. & Jendrassik, L. (1926). Biochem. Z. 178, 419.

Mann, T. (1943). Nature. Lon., 151,,619.Miohel-Durant, E. (1938). BuU. Soc. Chim. biol., Paris,

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Studies on the Metabolism of Mould Fungi2. ISOLATION OF PYROPHOSPHATE AND METAPHOSIPHATE

FROM ASPERGILLUS NIGER

BY T. MANN (Senior Beit Memorial Fellow), Moieno In8titute, Univermity of Cambridge

(Received 22 May 1944)

The mycelium of A8pergillu8 niger contains a largeproportion of its phosphorus in the form of com-pounds which can be easily extracted with trichloro-acetic acid and which yield orthophosphoric acidafter acid hydrolysis. In order to obtain moredefinite information with regard to the chemicalnature of these compounds an attempt was made toisolate them from the mycelium.

EXPERIMENTAL

The first step in the purification procedure was theextraction of the mycelia with trichloroacetic acid.This was followed by precipitation with Pb(NO3).,by means of which two distinct fractions were ob-tained which will be referred to as 'Pb fraction I'and 'Pb fraction II'. The first fraction precipitated