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J . Sci. Fd Agric. 1973, 24,97-103

A Further Study of the use of Date Extract Dibis forMycological Fat Production in Still and Shaken CulturesKaiser Naguib, I. A . Al-Sohaily and A. S. Al-SultanBiology Department, Collegeof Science, Baghdad University, I raq(Manuscript received 16 August 1972 and acceptedfor publication 18 September 1972)

Fungal growth and fat formation on date extract dibis medium were poor andslow. Additionofcorn-steepl i quor(c.s.1. ) as asource ofnitrogenandothernutritivesubstances edtoaremarkable ncrease n growth and in fat yield. The fat contentin fungal mycelium increased about4 times.Shakencultureswere superior to still cultures with regard to growth and fat yieldonly when a high concentration of c.s.1. was added to dibis medium. Under otherexperimental conditions, still cultures led to heavier mycelium and higher fatcontents than shaken cultures.1. Introduction

Ina previous attempt to study the applicability of date extract known locally as dibisfor mycological fat production, it was found that the addition of nitrogen in the formof asparagine greatly encouraged fungal growth as well as fat formation. In spite ofthis effect, the fat percentage in fungal mycelium did not reach the level previouslyobtained when using synthetic media for fat I n the present study, asource of nitrogen, c.s.1. was found to promote fungal growth and in particular somemetabolic activities such as penicillin5 and streptomycin6 production. C.s.1. contains avariety of amino acids in addition to other nutritive ingredients. It ought to be men-tioned in this connection that when c.s.1. was used to promote fat formation, the resultswere rather different. Murray and WalkerZ ound that c.s.1. improved the performanceof Penicillium soppi , particularly in its ability to produce fat, but they stated that it waseven detrimental to fat formation by F usarium lini. However, in these cases, they usedsynthetic culture media.

The present study also involved the use of shaken cultures, with the aimof promotingfat synthesis through availability of oxygen.

2. Experimental2.1. The organismThe fungus used in the present study wasPenicil lium soppi Zaleski which proved to bean efficient fat-forming fungus when grown on dibis. It was obtained from Centraal-bureau voor Schimmelcultures, Baarn, Holland. It was subcultured every2 weeksonDoxsagar slopes.

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982.2. The substrateDibis is produced on large scale by hot extraction of dates. It is a sweet viscous liquidsimilar to molasses in appearance and contains about 65% soluble sugars, mostlymonosaccharides and traces of nitrogen.2.3. Compositionof media and cultivationThe basal medium was composed of 25% dibis (mass/vol.). Dibis was first dissolved inhot distilled water, pH adjusted to 6.8, other ingredients added if required and themedium was finally made up to volume. The media were distributed in 100-ml conicalflasks each receiving 25 m of medium. The flasks were sterilised by autoclaving at 10Ibfor 15min, cooled and finally inoculated. Inoculation was done by a spore suspensionprepared from1week old slant cultures. The culture flasks were incubated at 25" Candafter specified intervals, triplicate sets were withdrawn for study.2.4. M ethodsof analysisThe fungal mycelia or mats were first separated by filtration. They were then dried andthe fat content determined as previously described by Woodbine, Gregory and Walker.'The filtrate was made up to volume and analysed for its total sugar content by a modifiedSchaffer-Harmann's method after the manner described by Said and Naguib.8

K. Naguib, I .A. A l-Sohaily and A. S.Al-Sultan

3. Results3.1. Fungal growth and fat formation in surface culturesThe fungus was grown on 3 media prepared as follows.

Medium 1:containing 25%dibis.Medium 2: containing25% dibis+50ml/l c.s.1.Medium3: containing 25% dibis+100ml/l c.s.1.

The culture flasks were incubated at 25" Cfor 21 days. After 6,9,12,15,18 and 21 daystriplicate sets were withdrawn for estimation of total sugars in the culture media anddry weight and fat content of fungal mats.3.1.1. Sugar uptakeResults are plotted graphically in Figure 1. It can easily be seen that sugar was slowlyabsorbed from media composed of dibis alone so that about one third of the sugarcontent remained unutilised in spite of the long incubation period. The presence ofc.s.1. greatly enhanced sugar absorption so that the medium was completely depletedwithin 12 days of incubation.3.1.2. Fungal growthMean dry weights of the fungal mats during the incubation period are also shown inFigure 1. It is clear that growth was too slow on dibis alone compared with dibis mediasupplemented with c.s.1. The dry weights of the fungal mats increasedin the presence ofc.s.1. at markedly higher rates than in mediaof dibis alone. The dry weights reached

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I J se of date extract for mycological fat production 99

T i m e ( days )Figure 1. Changes in total sugar content in culture media and the dry weightsof the fungal matsduring the incubation period. - O- o- ,Dibis; -O--O-, dibis+5%c.s.1.; - O- . - O- , ibis+10%c.s.1.;( 0 ) ugar;( 0) ry wt.

maximum by the twelfth day, when the sugar was completely exhausted. The fungalmats on c.s.1. media were almost four times heavier than on dibis alone.3.1.3. Fat content offungal matsThe total fat contents of the fungal mats are graphically presented in Figure 2. Fatformation on media of dibis alone went on quite slowly and ultimately declined whilethe fat content was still low. The presence of c.s.1. accelerated fat formation directlyfrom the beginning to such an extent that a markedly high fat yield was formed within9 days of incubation. It can also be noted that the fat content later dropped rathersteeply in the lower concentration of c.s.1. and relatively slowly in the higher concen-tration denoting that fat has been used up as respiratory substrate by the copious my-celium formed, when the sugar supply has been almost completely consumed.3.2. Fungal growthand fat formation in shaken culturesIn this experiment, three concentrations of c.s.1. were used, namely, 25, 50 and 100ml/l of medium. Four media were prepared as follows.

Medium1:25%dibis.Medium2: 25%dibis+25 ml/l c.s.1.Medium3:25%dibis+50ml/l c.s.1.Medium4 : 25% dibis+100ml/l c.s.1.

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100 K . Naguib, I. A. Al-Sohaily and A. S. Al-Sultan700

Time (days)Figure2. Drift inthe total fat contentof the fungal mats during the incubation period.- 0- 0- , 5%dibis;-0--0-,25%dibis +5 %c.s.l.;-0-.-0-,25%dibis +lO%c.s.l.The culture flasks were prepared as given above. They were divided into two equalsets. One set was fixed to the shaker and the other was left still in the same room ascontrol. A rotatory table shaker operated at 148 rev./min. was employed. The temper-ature was kept at 25 "C i 1. After 9, 12 and 15 days, triplicate sets were selected atrandom for each type of medium from both still and shaken cultures for the deter-mination of dry weight and fat contentof fungal mycelium or mat.

3.2.1. The dry weight o fungal myceliumMean dry weights are given in Table 1. It is clear that fungal growth on dibis alone wasrelatively poor in both still and shaken cultures. Shaking first accelerated growth butlater growth was better in still than in shaken cultures. The presenceof c.s.1. acceleratedfungal growth in both still and shaken cultures with the higher concentrations beingmore effective. The effect of shaking in the presence of c.s.1.was rather peculiar; shakingpromoted growth in the higher concentrations of c.sl.. and in the later period of incu-bation. Otherwise it had a suppressive effect.3.2.2. Fat content o fungal myceliumTable2contains the average values for the total fat content of fungal mycelium in stilland shaken cultures. The results show that the presence of c.s.1. gave rise to markedlyhigher fat yields in both types of cultures. Shaking did not promotefatformation exceptin the highest concentration of c.s.1.Otherwise, still cultures yielded higher fat contents

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Use of date extractfor mycologicalfatproduction 101TABLE. Mean dry weight of fungal mycelium(mg/100ml)

MediumType 25%dibispl usof r \culture 25xdibis 2.5%c.s.l. 5%c.s.l. lO%c.s.l.

After 9daysStill 1228 3276 4192 4768Shake 1332 1696 3140 5384After 12daysStill 1488 3876 5312 4640Shake 796 2088 3678 6484After 15daysStill 1492 4384 4332 4396Shake 880 2332 4736 6560

than shaken cultures. I t can also be noted that inspite of the high fat yields in the presenceof c.s.l., the fat percentage in mycelium did not rise in most cases, denoting that c.s.1.promoted building-up processes including fat synthesis. However, the highest fatyield was obtained in still cultures in the presence of the intermediate concentration of

TABLE. Mean total fat content of fungal mycelium(mg/lOO ml)and fat percentageMedium

25%dibisplus25%dibis ,-Type- .5%c.s.1. 5%C.S.1. 10% .S.1.culture content % content % content % content %of Fat Fat Fat Fat Fat Fat Fat Fat

After 9daysStill 252 20.5 556 17.1 475 11.3 390 8.2Shake 200 15.1 21 7 12.8 361 11.5 581 10.8After 12daysStill 267 17.8 535 13.8 815 15.3 340 7.3Shake 139 17.5 200 9.6 350 9.5 693 10.7

After 15daysStill 213 14.3 478 17.3 502 11.6 220 5.0Shake 108 12.3 127 5.5 256 5.9 514 7.8

c.s.~.,with a fat content of approx. 15%. This percentage is quite near to the highestvalue obtained in cases of low fat yields resulting from handicapped growth. But incases where shaking gave rise to higher fat yields, i.e. in the highest c.s.1. concentration,there wasarise in fat percentage indicating promotion in fat synthesis.

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102 K. Naguib, I . A. Al-Sohailyand A. S.Al-Sultan4. Discussion

Date extract or dibis, though rich in sugar content, did not afford an adequate substratefor mycological fat formation. In a previous attempt (Naguib, A l-Sohaily and Al-Sultan'), it was found that mere addition of asparagine as source of nitrogen promotedboth growth and fat formation. I n the present study, a cheap natural byproduct, thecorn steep liquor, was used as source of nitrogen and other nutritive ingredients.Addition of c.s.1. accelerated rate of sugar consumption sothat the culture media werecompletely depleted within 12 days. When using unsupplemented dibis media, sugarwas slowly utilised so that after 21 days, almost a third of the sugar supply was stillremaining in the culture medium. Prolonged utilization of the sugar led to very slowrate of building up; most of the sugar being used as source ofenergy. Rapid sugar utilis-ation in the presence of c.s.1. led to ahigh rate of growth sothat copious mycelium wasformed. Owing to the presence of high sugar in the culture medium, a good deal of thatsugar was shunted to fat synthesis. It could be shown in this way that the combination oftwo cheap natural sources, one rich in carbohydrates and the other in nitrogen maylead to promising mycological fat formation. The use of c.s.1. by previous workers inprocesses of fat production was not always mentioned as favourable. Thus, Gad andWalker9 using defined media found that addition of c.s.1. enhanced growth but did notraise fat yield in Penicillium javanicum, while in P. spinulosum it resulted in greaterconsumption of sugar and notably enhanced the yield of fat and felt. Using Fusariumlini,M urray and Walker2 mentioned that c.s.1. was detrimental to the production of fat.I t has also been shown that mycological fat formation is favoured by more availabil ityof air or oxygen. This was made possible in simple industrial processes by increasingsurface/volume ratio (L ockwood et a1.l' and Ikeda"). For economy of space, shakingor aeration of cultures was therefore accomplished to avoid large areas of cultivation.In the present study, fungal growth on dibis alone or together with c.s.1. in shakencultures was therefore tried. Results attained here have shown that shaking could notpromote fat synthesis except in the presence of the highest experimental c.s.1. concen-tration. This was not a mere reflection of a growth promoting effect since there was anincrease in fat content and in fat percentage. The use of shaken cultures in mycologicalfat production by several workers led to different conclusions. Thus, N eithammePobserved that more fat is produced in mycelium growing inside the medium, whileFink et al l 3growing Endomyces on molasses, found that agitation and aeration mar-kedly depressed fat yield. Witter and Stotz14 using Fusarium lycopersici found a higherpercentage of fat in stationary cultures than in shaken cultures. Woodbine et aL7havealso shown that cultivation with shaking reduces fat content. It may be said in thisrespect that copious mycelial growth is essential for securing high fat yields and in suchcases shaking would make available more air than do surface cultures. In the case ofthe highest c.s.1. concentration, mycelial growth was too large so that shaking mayhave made possible its better exposure to surrounding atmosphere.References1. Naguib, K.;Al-Sohaily, I. A.; Al-Sultan,A. S.J.Sci. FdAgr i c. 1972,23,845.2. Murray, S.;Walker,T. K. J . Sci. FdAgric. 1956,7,231.3. Naguib, K.; Walker,T. K. J .Exp. Bot. 1958,9,426.

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Useof date extract for mycological fat production 1034. Naguib,K. Can. . Bot. 1959,37, 353.5. Moyer,A . J .; Coghill, R.D. 1946. . Bacteriol. 1946,51, 57.6. Dulaney,E. L . J . Bacteriol. 1948,56, 305.7. Woodbine,M.; Gregory,M.; Walker, T . K. J . Exp. Bot. 1951,2,204,8. Said,H .; Naguib,K. Proc. Egypt. Acad. Sci. 1955,11, 37.9. Gad,A, M.; Walker,T . K . J . Sci. Fd Agric. 1954,5, 339.10. Lockwood, L. B.; Ward, G. E.; May,0.E.; Herrick, H. T .; ONeill, H. T. 11. Bacteriology,1934,90, 411.11. Ikeda,T . J . F erment. Technol.1950,28,69.12. Neithammer,A. F ette Seifen. 1943,50,309.13. Fink, H.; Haenseler,G .; Schmidt,M.Z. Spiritusind. 1937, 60, 74.14. Witter, R. F.; Stotz, E.Arch. Biochem. 1946,9,331.