J. Sci. Fd Agric.

1973, 24, 97-103

A Further Study of the use of Date Extract Dibis for

Mycological Fat Production in Still and Shaken Cultures
Kaiser Naguib, I. A. Al-Sohaily and A. S. Al-Sultan
Biology Department, College of Science, Baghdad University, Iraq (Manuscript received 16 August 1972 and accepted for publication 18 September 1972)

Fungal growth and fat formation on date extract dibis medium were poor and slow. Addition ofcorn-steepliquor(c.s.1.) as asource ofnitrogenandothernutritive substances led to a remarkable increase in growth and in fat yield. The fat content in fungal mycelium increased about 4 times. Shakencultureswere superiorto still cultures with regard to growth and fat yield only when a high concentration of c.s.1. was added to dibis medium. Under other experimental conditions, still cultures led to heavier mycelium and higher fat contents than shaken cultures.

1. Introduction

In a previous attempt to study the applicability of date extract known locally as dibis for mycological fat production, it was found that the addition of nitrogen in the form of asparagine greatly encouraged fungal growth as well as fat formation. In spite of this effect, the fat percentage in fungal mycelium did not reach the level previously obtained when using synthetic media for fat In the present study, a source of nitrogen, c.s.1. was found to promote fungal growth and in particular some metabolic activities such as penicillin5 and streptomycin6 production. C.s.1. contains a variety of amino acids in addition to other nutritive ingredients. It ought to be mentioned in this connection that when c.s.1. was used to promote fat formation, the results were rather different. Murray and WalkerZfound that c.s.1. improved the performance of Penicillium soppi,particularly in its ability to produce fat, but they stated that it was even detrimental to fat formation by Fusarium lini. However, in these cases, they used synthetic culture media. The present study also involved the use of shaken cultures, with the aim of promoting fat synthesis through availability of oxygen.
2. Experimental

2.1. The organism

The fungus used in the present study was Penicillium soppi Zaleski which proved to be an efficient fat-forming fungus when grown on dibis. It was obtained from Centraalbureau voor Schimmelcultures, Baarn, Holland. It was subcultured every 2 weeks on Doxs agar slopes.
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K. Naguib, I. A. Al-Sohaily and A. S. Al-Sultan

2.2. The substrate Dibis is produced on large scale by hot extraction of dates. It is a sweet viscous liquid similar to molasses in appearance and contains about 65 % soluble sugars, mostly monosaccharides and traces of nitrogen. 2.3. Composition of media and cultivation The basal medium was composed of 25 % dibis (mass/vol.). Dibis was first dissolved in hot distilled water, pH adjusted to 6.8, other ingredients added if required and the medium was finally made up to volume. The media were distributed in 100-ml conical flasks each receiving 25 ml of medium. The flasks were sterilised by autoclaving at 10 Ib for 15 min, cooled and finally inoculated. Inoculation was done by a spore suspension prepared from 1 week old slant cultures. The culture flasks were incubated at 25 "Cand after specified intervals, triplicate sets were withdrawn for study. 2.4. Methods of analysis The fungal mycelia or mats were first separated by filtration. They were then dried and the 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 modified Schaffer-Harmann's method after the manner described by Said and Naguib.8

3. Results
3.1. Fungal growth and fat formation in surface cultures

The fungus was grown on 3 media prepared as follows. Medium 1 : containing 25 % dibis. Medium 2: containing 25% dibis + 50 ml/l c.s.1. Medium 3: containing 25 % dibis + 100 ml/l c.s.1. The culture flasks were incubated at 25 "Cfor 21 days. After 6,9,12,15,18 and 21 days triplicate sets were withdrawn for estimation of total sugars in the culture media and dry weight and fat content of fungal mats.

3.1.1. Sugar uptake Results are plotted graphically in Figure 1. It can easily be seen that sugar was slowly absorbed from media composed of dibis alone so that about one third of the sugar content remained unutilised in spite of the long incubation period. The presence of c.s.1. greatly enhanced sugar absorption so that the medium was completely depleted within 12 days of incubation. 3.1.2. Fungal growth
Mean dry weights of the fungal mats during the incubation period are also shown in Figure 1. It is clear that growth was too slow on dibis alone compared with dibis media supplemented with c.s.1. The dry weights of the fungal mats increased in the presence of c.s.1. at markedly higher rates than in media of dibis alone. The dry weights reached

IJse of date extract for mycological fat production

99

Time (days)

Figure 1. Changes in total sugar content in culture media and the dry weights of the fungal mats Dibis; -O--O-, dibis 5 % c.s.1.; -O-.-O-, dibis + 10% c.s.1. ; during the incubation period. -O-o-, (0) sugar; (0) dry wt.

maximum by the twelfth day, when the sugar was completely exhausted. The fungal mats on c.s.1. media were almost four times heavier than on dibis alone.

3.1.3. Fat content offungal mats The total fat contents of the fungal mats are graphically presented in Figure 2. Fat formation on media of dibis alone went on quite slowly and ultimately declined while the fat content was still low. The presence of c.s.1. accelerated fat formation directly from the beginning to such an extent that a markedly high fat yield was formed within 9 days of incubation. It can also be noted that the fat content later dropped rather steeply in the lower concentration of c.s.1. and relatively slowly in the higher concentration denoting that fat has been used up as respiratory substrate by the copious mycelium formed, when the sugar supply has been almost completely consumed.
3.2. Fungal growth and fat formation in shaken cultures In this experiment, three concentrations of c.s.1. were used, namely, 25, 50 and 100 ml/l of medium. Four media were prepared as follows.

Medium 1 : 25 % dibis. Medium 2: 25 % dibis + 25 ml/l c.s.1. Medium 3 : 25 % dibis 50 ml/l c.s.1. Medium 4 : 25% dibis 100 ml/l c.s.1.

+ +

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

Time (days)

Figure 2. Drift in the total fat content of the fungal mats during the incubation period. -0-0-, 25 % 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 equal sets. One set was fixed to the shaker and the other was left still in the same room as control. A rotatory table shaker operated at 148 rev./min. was employed. The temperature was kept at 25 "C i 1. After 9, 12 and 15 days, triplicate sets were selected at random for each type of medium from both still and shaken cultures for the determination of dry weight and fat content of fungal mycelium or mat.

3.2.1. The dry weight o f fungal mycelium

Mean dry weights are given in Table 1. It is clear that fungal growth on dibis alone was relatively poor in both still and shaken cultures. Shaking first accelerated growth but later growth was better in still than in shaken cultures. The presence of c.s.1. accelerated fungal growth in both still and shaken cultures with the higher concentrations being more effective. The effect of shaking in the presence of c.s.1. was rather peculiar; shaking promoted growth in the higher concentrations of c.sl.. and in the later period of incubation. Otherwise it had a suppressive effect.

3.2.2. Fat content o f fungal mycelium

Table 2 contains the average values for the total fat content of fungal mycelium in still and shaken cultures. The results show that the presence of c.s.1. gave rise to markedly higher fat yields in both types of cultures. Shaking did not promote fat formation except in the highest concentration of c.s.1.Otherwise, still cultures yielded higher fat contents

Use of date extract for mycological fat production TABLE 1. Mean dry weight of fungal mycelium (mg/100 ml) Medium
Type of culture

101

25 % dibis plus
r
\

25xdibis

2.5%c.s.l. After 9 days 3276 1696 After 12 days 3876 2088 After 15 days 4384 2332

5%c.s.l.

lO%c.s.l.

Still Shake Still Shake Still Shake

1228 1332 1488 796 1492 880

4192 3140 5312 3678 4332 4736

4768 5384 4640 6484 4396 6560

than shaken cultures. It can also be noted that in spite of the high fat yields in the presence of 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 fat yield was obtained in still cultures in the presence of the intermediate concentration of
TABLE 2. Mean total fat content of fungal mycelium (mg/lOO ml) and fat percentage

Type of culture

25 % dibis Fat content Fat

Medium 25 % dibis plus

, -

2.5 % c.s.1. Fat Fat content % After 9 days 17.1 12.8 After 12 days 13.8 9.6 After 15 days 17.3 5.5

5 % C.S.1. Fat Fat content %

10%C.S.1. Fat Fat content %

Still Shake Still Shake Still Shake

252 200 267 139 213 108

20.5 15.1 17.8 17.5 14.3 12.3

556 217 535 200 478 127

475 361 815 350 502 256

11.3
11.5

390 581 340 693 220 514

8.2 10.8 7.3 10.7

5.0

15.3 9.5 11.6 5.9

7.8

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

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4. Discussion

Date extract or dibis, though rich in sugar content, did not afford an adequate substrate for mycological fat formation. In a previous attempt (Naguib, Al-Sohaily and AlSultan'), it was found that mere addition of asparagine as source of nitrogen promoted both growth and fat formation. In the present study, a cheap natural byproduct, the corn steep liquor, was used as source of nitrogen and other nutritive ingredients. Addition of c.s.1. accelerated rate of sugar consumption so that the culture media were completely depleted within 12 days. When using unsupplemented dibis media, sugar was slowly utilised so that after 21 days, almost a third of the sugar supply was still remaining in the culture medium. Prolonged utilization of the sugar led to very slow rate of building up; most of the sugar being used as source ofenergy. Rapid sugar utilisation in the presence of c.s.1. led to a high rate of growth so that copious mycelium was formed. Owing to the presence of high sugar in the culture medium, a good deal of that sugar was shunted to fat synthesis. It could be shown in this way that the combination of two cheap natural sources, one rich in carbohydrates and the other in nitrogen may lead to promising mycological fat formation. The use of c.s.1. by previous workers in processes of fat production was not always mentioned as favourable. Thus, Gad and Walker9 using defined media found that addition of c.s.1. enhanced growth but did not raise fat yield in Penicillium javanicum, while in P. spinulosum it resulted in greater consumption of sugar and notably enhanced the yield of fat and felt. Using Fusarium lini, Murray and Walker2 mentioned that c.s.1. was detrimental to the production of fat. It has also been shown that mycological fat formation is favoured by more availability of air or oxygen. This was made possible in simple industrial processes by increasing surface/volume ratio (Lockwood et a1.l' and Ikeda"). For economy of space, shaking or 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 shaken cultures was therefore tried. Results attained here have shown that shaking could not promote fat synthesis except in the presence of the highest experimental c.s.1. concentration. This was not a mere reflection of a growth promoting effect since there was an increase in fat content and in fat percentage. The use of shaken cultures in mycological fat production by several workers led to different conclusions. Thus, NeithammeP observed that more fat is produced in mycelium growing inside the medium, while Fink et a l l 3 growing Endomyces on molasses, found that agitation and aeration markedly depressed fat yield. Witter and Stotz14using Fusarium lycopersici found a higher percentage of fat in stationary cultures than in shaken cultures. Woodbine et aL7 have also shown that cultivation with shaking reduces fat content. It may be said in this respect that copious mycelial growth is essential for securing high fat yields and in such cases shaking would make available more air than do surface cultures. In the case of the highest c.s.1. concentration, mycelial growth was too large so that shaking may have made possible its better exposure to surrounding atmosphere.
References
1. Naguib, K.; Al-Sohaily, I. A.; Al-Sultan, A. S. J. S c i .Fd Agric. 1972,23, 845. 2. Murray, S.; Walker, T. K. J. S c i .Fd Agric. 1956,7,231. 3. Naguib, K.; Walker, T. K. J. Exp. Bot. 1958,9,426.

Use of date extract for mycological fat production 4. 5. 6. 7. 8. 9. 10.

11. 12. 13. 14.

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Naguib, K. Can. J. Bot. 1959,37, 353. Moyer, A. J.; Coghill, R. D. 1946. J. Bacteriol. 1946,51, 57. Dulaney, E. L. J. Bacteriol. 1948,56, 305. Woodbine, M.; Gregory, M.; Walker, T. K. J . Exp. Bot. 1951,2,204, Said, H.; Naguib, K. Proc. Egypt. Acad. Sci. 1955,11, 37. Gad, A, M.; Walker, T. K. J. Sci. Fd Agric. 1954,5, 339. Lockwood, L. B.; Ward, G. E.; May, 0. E.; Herrick, H. T.; ONeill, H. T. 11. Bacteriology, 1934,90, 41 1 . Ikeda, T. J . Ferment. Technol. 1950,28,69. Neithammer, A. Fette Seifen. 1943,50,309. Fink, H.; Haenseler, G.; Schmidt, M. Z . Spiritusind. 1937, 60, 74. Witter, R. F.; Stotz, E. Arch. Biochem. 1946,9,331.