FORMATION OF BAKER'S YEAST BIOMASS BY ... substituted by date-coat (fleshy part) sugar extrac~ ... In addition to their sugar resources and their rela-.
Bioresource Technology 60 (1997) 67-71 © 1997 Elsevier Science Limited All fights reserved. Printed in Great Britain 0960-8524/97 $17.00 ELSEVIER
PII:SO960-gs24(97)O0004-7
USE OF WASTE DATE PRODUCTS IN THE FERMENTATIVE FORMATION OF BAKER'S YEAST BIOMASS BY SACCHAROMYCES CEREVISIAE Nabil Nancib, a A i c h a N a n c i b a & J o s e p h B o u d r a n t b* aDeparwaent of Microbiology, Insatute of Biology, University of Sen'f, Sen'f, Algeria bLSGC-CNRS-ENSAIA, BP 172, 2, Avenue de la fordt de Haye, 54505 Vandoeuvre-les-Nan~, France
(Received 14 February 1996; revised version received 12 December 1996; accepted 16 December 1996)
and 9-3%), mineral elements (1.9 and 0.9%), fibre (around 3.0%) and water (around 7%). The percentage of total lipid and protein of date-seeds is higher than that of date-coat (Abou-Zeid et al., 1991). Sixteen amino acids are found in both dateseed and date-coat hydrolysate. Arranged in their decreasing content, going from around 400 and 200 to 50 and 10 mg/100 g of dry date coat or seed, respectively, are found glutamic acid, aspartic acid, glycine, serine, lysine, leucine, arginine, tyrosine, alanine, isoleucine, threonine, valine, phenylalanine, histidine, methionine and proline (Booij et al., 1993; Aura et al., 1976; AI-Rawi et al., 1967). The four major mineral constituants are Ca (65 and 25 mg/100g for coat and seed), P (48 and 100 mg/100 g, respectively), K (1000 and 280mg/ 100 g, respectively), and Mg (50 rag/100 g for both parts). The other minerals also arranged in a decreasing order from 5 and 6 rag/100 g, respectively, to zero, are Fe, AI, Na, Cr, Sr, Cu, Zn, Ni, Mn, Pb (Abou-Zeid et al., 1991). In addition to their sugar resources and their relatively long preservation period (Nur et al., 1981), dates offer many technological possibilities. They have been tested as raw material for the production of different metabolites, such as citric acid (AbouZeid & Khoja, 1993; Abou-Zeid & Baghlaf, 1983), oxytetracycline (Abou-Zeid et al., 1991, 1993a, 1993b), ethanol and cattle cake (AI Saady & Benjamin, 1982). Many scientists have studied factors and chemical components of media favouring the fermentation of Saccharomyces cerevisiae. However, the use of date constituents, to our knowledge, except sugars (Khan et al., 1995), has not been investigated. Therefore, the aim of this present work was the use of four date products (date-coat sugar extract, date-seed hydrolysate, date-seed lipid and date-seed ash) as nutrient medium constituents, or additives, for yeast biomass production.
Abstract This work was an approach to waste date products valorization through biomass production with the yeast Saccharomyces cerevisiae. The carbon and nitrogen sources of a semi-synthetic fermentation medifim were substituted by date-coat (fleshy part) sugar extrac~ date-seed hydrolysate, and ammonium nitrate. This modified medium was enriched with date-seed ash and date-seed lipid. Date-coat sugar extract as a carbon source was found to be satisfactory at a concentration of 25 g/l (expressed as its glucose concentration) and date-seed hydrolysate as a nitrogen source was equalty suitable at 25 g/l. The addition to the medium of 1.0 g/l ammonium nitrate increased the efficiency of yeast biomass formation, as did phosphorus, which produced a maximum when the medium was supplemented with about 6.0g/I KH2PO, The presence of I g/l date-seed lipid in the medium also increased the' efficiency of biomass formation. Finally, the addition of date-seed ash (0"6 g/l), as a mineral source, to the fermentation medium could substitute for MgS04 and CaCI2 of the semi-synthetic medium. © 1997 Elsevier Science Ltd.
Key words: Date products, yeast biomass, Saccharomyces cerevisiae. INTRODUCTION In Algeria, a large tonnage of mature dates (60 000 t/ year) is unfortunately lost for various reasons. This date waste comes either directly from the palm grove or from the gap-conditioning station and is unfit for human consumption. Chemical analyses of date-coat and date-seed revealed that they contain a large percentage of sugars (79.2 and 36-3%, respectively), especially glucose, fructose and sucrose (Booij et al., 1993). They also contain protein (2.0 and 3.7%), lipid (0.8
*Author to whom correspondence should be addressed. 67
68
N. Nancib, A. Nancib, J. Boudrant
METHODS Preparation of date extracts
Date fruit is mainly composed of the fleshy part and the seed. Date-seeds were separated from the fleshy parts and the fleshy parts were boiled for 10 rain at 100°C (1 kg dated water). Date juice, obtained after boiling, was centrifuged at 10000g for 10rain to separate cellulosic debris from the sugar-rich supernatant referred to as date-coat sugar extract (DCSE). Generally, the glucose concentration of DCSE is about 100 g/1. The latter was used as a carbon source in the fermentation medium and diluted to reach a given glucose concentration. Air-dry date-seeds (drying, to facilitate grinding, was performed at 50°C for 24 h) were thoroughly ground, and hydrolysed with 400 ml of 1.0 N HC1 in a water bath at 100°C for 12 h, then centrifuged at 10000g for 10rain. The supernatant obtained referred to as date-seed hydrolysate (DSH), was used as the nitrogen source in the fermentations. Date-seeds were ashed (400-500°C for 12 h) and the ash (DSA) was used in the medium as a mineral source. Crude date-seed lipid (CDSL) was extracted by chloroform-methanol (1:1) for 47h from fine seed powder. It was also used in the fermentation medium. Microorganism and maintenance medium
The strain of Saccharomyces cerevisiae used was provided by the industrial plant producing baker's yeast; Bouchegouf (Algeria). This strain was maintained on a medium containing glucose (10.0 g/l, Prolabo); yeast extract (3.0 g/l, Prolabo); Bio-trypticase (5-0 g/l, Difco); agar (20.0 g/l, erolabo); malt extract (3.0 g/l, Difco). Fermentation media and cultivation
Pre-cultures and cultures were carried out in the same semi-synthetic fermentation medium (SSFM) which contained glucose (10.0 g/l, Prolabo); (NH4)2SO4 (10-0 g/l, Prolabo); KH2PO4 (6.0 g/l, Prolabo); MgSO4, 7H20 (3.0 g/l, Merck); CaCI2, H20 (0.1 g/l, Fluka); NaCI (0"1 g/l, Fluka), yeast extract (0"5 g/l, Prolabo). Inoculation rate was 10%. In the first set of experiments, glucose of SSFM was replaced by a solution which contained only datecoat sugar extract (DCSE). In the following experiments, SSFM was then replaced by a DCSE solution supplemented by different constituents; DSH, DSA, CDSL, NH4NO3 and KH2PO4, at increasing concentrations. The initial pH value of these media was adjusted to 4.5. Fermentations were carried out either in 250 ml Erlenmeyer shake flasks (working volume 50 ml), incubated after inoculation, at 30°C on a rotary shaker (200 rpm), or in a 51 LKB fermentor (working volume 2.5 1) fitted with standard equipment: temperature regulation (30°C), agitation (300 rpm), aeration (1 vvm), and dissolved
oxygen regulation (Ingold electrode). The pH was regulated (4.5) with ammonium hydroxide (3 N) or hydrochloric acid (4 N). Analysis
At the end of fermentation periods, the cultivation media were recovered and analysed. Cell concentration was followed by measuring the optical density of the cell suspension, with appropriate dilution, at 660 nm using a spectrophotometer (Spectronic 70) and the optical density was converted to dry cell weight. Due to the fact that during fermentation by Saccharomyces glucose is always utilized first (Rose & Harrison, 1971), only glucose content was determined. For this a Technicon Autoanalyser using the hexokinase method was used. The experiments were performed in triplicate. The indicated results are the mean values of these results, results which have been treated using the ANOVA analysis (Lazar & Lellouch, 1983). This analysis, based on the Fisher test, assumes that the probability for a significant difference between these results is less than 1%. RESULTS AND DISCUSSION
The experiments performed below consisted of the substitution of the carbon and nitrogen sources of a semi-synthetic fermentation medium by date-coat sugar extract and date-seed hydrolysate, and of the enrichment of the natural medium (date-coat sugar extract solution) with NH4NO3, KH2PO4, date-seed ash and date-seed lipid. Effect of date-coat sugar extract (DCSE), as carbon source, on growth rate and yield
These experiments were performed in the fermentor with the SSFM medium containing glucose (control), or DCSE instead of glucose. The results obtained (Table 1) showed that the maximum Table 1. Effect of date-coat sugar extract (DCSE) as carbon source on biomass production by the yeast Saccharomyces cerevisiae. The semi-synthetic medium (SSFM) was used as the control. The composition of SSFM was (g/l): glucose 10.0g/l; (NH4)zSO4 10.0g/l; KH2PO4 6.0 g/I; MgSO4, 7HzO 3.0 g/l; CaCI2, H20 0.1 g/l; NaC! 0.1 g/l, yeast extract 0.5 g/l. DCSE is expressed by its glucose concentration. These data are the mean values of three experiments and the Fisher ratios (see Methods) were 4.54 for the growth data and 4.51 for Yx/~
Medium
SSFM (control) SSFM without glucose
DCSE (g/l) 0 20 25 55 75 95
Maximum specific growth rate (per h) 0.32 0.30 0.38 0.19 0.15 0.11
Yx/o (g/g) 0.22 0.20 0.30 0.11 0.09 0.07
69
Yeast growth on date waste
specific growth rate ( ~ , ~ ) and the biomass yield on glucose (Yxa3) had an optimum value when the fermentation medium (SSFM) contained 25g/I of date-coat sugar extract (expressed in glucose) instead of pure glucose (10 g/l). So this extract, rich in glucose, fructose and saccharose, as reported by Abou-Zeid et al. (1991), seems to be a convenient carbon source for yeast cell biomass production by Saccharomyces cerevisiae. Glucose consumption corresponded here to the prominent growth phase. Measurements of this carbohydrate, generally being the first carbon substrate utilized by this species (Rose & Harrison, 1971), were carried out only on this substrate. Note also that, beside sugars, DCSE contains nitrogen sources, mainly amino acids (Abou-Zeid & Khoja, 1993), and other unknown elements which have a stimulatory effect on the growth of Saccharomyces cerevisiae, probably also explaining the high growth observed at 25 g glucose DCSE/! (Table 1). From these results, we opted for a concentration of 25 g glucose DCSE/I in further experiments. The reason for the decrease of Yocx as a function of DCSE concentration has not yet been identified. Utiifnmtion of date-seed hydrolysate (DSH) as a nitrogen source The following results were obtained in Edenmeyer flasks. The experiments were performed either with the SSFM medium (control), or with a DCSE solution (25 g glucose DCSE/I). The results corresponding to the DCSE solutions show an amount of yeast biomass produced far lower than the amount obtained when SSFM was used (Table 2, lines 1 and 2). This might be explained by a low level of nitrogen in DCSE as reported by Abou-Zeid et al. (1991). To compensate for this, date-seed hydrolysate (DSH) was added at different concentrations to the medium as nitrogen source. An increase in biomass concentration, although low compared to the level seen when using SSFM, was obtained up to 25 g DSH/I (Table 2). This optimum concentration was used in further experiments. Table 2. Effect of date-seed hydrolysate (DSH) as nitrogen source used at different concentrations in a 25 g/! glucose DCSE solution on biomass production by the yeast cer~/s/ae. The semi-synthetic medium
(SSFM) was used as the control. These data are the mean values of three experiments and the Fisher ratio (see Methods) was 6.05 Medium SSFM (control) DCSE (25 g/l)
DSH (g/l)
Dry yeast biomass
0 0 15 25 35
0.450 0.300 0.400 0.420 0.380
(g/l)
at dif-
Table3. ~ of mmeaiem nitrate ( N ~ ) ferent comeenlral~ 1, alwened m its s u h t e (NH4)S04 (10g/l) *'as ~
~ mmmi-sy~mic medium (SSFM) was used as the control ~ data are the mean values of three ~ t s and the Fisher ratio (see Methods) was 4.64
Medium DCSE (25 g/l)+
Ammonium nitrate (g~) 0
Dry yeast biomass (g/l) 0.40
0.5
0.45
(NH4)2SO4
(10 g/l) (control) DCSE (25 g/I)+DSH
(25 g~)
1 2 4 6 10 15
0.48 0.42 0.40 0.35 0.30 0.32
Effect of the addition of ammonium nitrate
(NH4NO3) In fermentation media, nitrogen is generally or frequently introduced under the form of ammonium sulfate. Due to its availability and cost, even if ammonium sulfate gives satisfying results (Table 3), we have used ammonium nitrate (NH4NO3) as the nitrogen source to supplement the natural fermentation medium (25 g glucose DCSE/l+25g/I DSH). The data indicated on Table 3 show that the optimum results were obtained at a concentration of 1 g ammonium nitrated. Growth effect of phosphate (KH2PO4) addition to the natural fermentation medium
Since dates are not rich in phosphorus (50-100 mg/ g, Abou-Zeid et al., 1991), we thought about adding this element to the natural medium containing only DCSE (25 g glucose DCSE/I), DSH (25 g/l) and NH,~O3 (1 g/l). Phosphorus was added in the form of KH2POa at different concentrations. It is worth mentioning at this stage that dates are rich in potasTable 4. Effect of phosphorus, from KH2PO4, at different concentrations, ou biomass production by the yeast Sacchamm--yces cerev/s/ae in medium containing DCSE (25g/i, expressed as its glucose concentration), DSH (25 g/l) and NH4NO3 (1 g/l). The semi-synthetic medium (SSFM) was used as the control. These data are the mean values of three experiments and the Fisher ratio (see Methods) was 5.01
Medium DCSE (25 g/1)+DSn
(25 ga) +nI-hr~o30 g/l)
(g/l) 0
Dry yeast biomass (g/l) 0.330
1 2 4 6
0.345 0.380 0.385 0.385
KH2PO4
70
N. Nancib, A. Nancib, J. Boudrant
sium (Abou-Zeid et al., 1991). Observed growth in the absence of KH2PO4, as shown in Table 4, probably reflects the presence of phosphorus in DCSE and DSH. Nevertheless, the addition of KH2PO4 (6 g/l) showed an increase in biomass production. Therefore it was decided to supplement the natural medium with this concentration of potassium phosphate.
Table 6. Effect of the addition of crude date-seed lipid (CDSL), at different concentrations, to a medium containing DCSE (25 g/I, expressed in its glucose concentration), DSH (25 g/l), NH4NOa (1 g/i), KH2PO 4 (6 g/l) and DSA (0.6 g/l) on biomass production by the yeast Saccharomyces cerevisiae. The semi-synthetic medium (SSFM) was used as the control. These data are the mean values of three experiments and the Fisher ratio (see Methods) was 4.52
Medium Role of date-seed ash (DSA) in yeast biomass production
DCSE (25 g/l) +DSH (25 g/l) + NI--IaNO3 (1 g/l) +KH2PO4 (6 g/I)+DSA (0.6 g/l)
CDSL (g/l)
Dry yeast biomass
(#)
0
0.54
0.25 0.5 1 1.5 2
0.54 0.56 0.60 0.60 0.60
Date-seed ash has been tested as a mineral source. Indeed, DSA contained magnesium, iron, aluminium, calcium, manganese, zinc and nickel (Abou-Zeid et al., 1993a). The results obtained (Table 5) show that DSA influences the formation of yeast biomass. Yeast biomass increases with ash content, reaching a maximum at 0-6 g/l, above which a decrease in yeast biomass was obtained. A complementary experiment was performed in order to compare the efficiency of DSA with regard to the concentrations of MgSO4 (3 g/l) and CaC12 (0.1 g/l) used in the SSFM medium. The result of this experiment, indicated on the last line of Table 5, shows that the addition of DSA (0.6 g/l) could substitute for MgSO4 and CaCI2 of the semi-synthetic medium and gave the maximum production of the yeast biomass. So the concentration of 0.6 g/l of DSA was used in the following experiments.
indicated in Table 6 show that an increase in yeast biomass production was observed when the natural medium (glucose DCSE 25 g/l, DSH 25 g/l, NH4NO3 1 g/l, KH2PO4, DSA 0"6 g/l) was supplemented with CDSL, reaching a maximum at concentrations higher than 1-0 g/l. So this last concentration was used in the last series of experiments.
Use of crude date-seed lipid (CDSL) in yeast biomass production
Yeast biomass production in a natural medium containing date product extracts
A set of experiments was decided in order to add to the medium date-seed lipid (DSL). Lipids are known as being membrane constituents and, therefore, their presence might favour growth. The results Table 5. Effect of the addition of date-seed ash (DSA), at different concentrations, to a medium containing DCSE (25g/!, expressed as its glucose concentration), DSH (25 g/I), NI-][4NO3 (1 g/l) and KH2PO4 (6 g/l) on biomass production by the yeast Saccharomyces cerevisiae. DSA acts as a substituent for MgSO4 (3 g/l) and CaCI2 (0.1 g/ !). The semi-synthetic medium (SSFM) was used as the control. These data are the mean values of three experiments and the Fisher ratio (see Methods) was 4.75
Medium DCSE (25 g/l) +DSH (25 g/l) +NH~O3 (1 g/l) + KH2PO4 (6 g/l)
Control
DSA
(g/l) 0
0.2 0.4 0.6 0.8 1.0 1.5 0
MgSO4 CaCl2 Dry yeast
(g/l) 0
0 0 0 0 0 0 3
(g/l) 0
0 0 0 0 0 0 0.1
As shown in Table 7, the replacement of SSFM by a natural medium based only on date products (glucose DCSE 25 g/l, DSH 25 g/l, DSA 0.6g/l, CDSL 1.0#1) as carbon, nitrogen and mineral sources and two salts (NH,dqO3 1 g/l and KH2PO4 6 g/l) allowed production of a larger biomass concentration (0.620) than the one obtained with SSFM (0.350). This higher yeast biomass output may be attributed to the presence and utilization of miscellaneous components found in DCSE, such as other sugars (fructose and saccharose) and amino acids, in addition to other natural compounds. Further experiments with more analysis (fructose, saccharose, amino acids) will be performed in the future.
biomass (g/l) 0.38
0.38 0.43 0.48 0.42 0.40 0.38 0.44
CONCLUSION
From the results obtained, it is quite clear that date extracts, DCSE and DSH, are good carbon and nitrogen sources suitable for biomass production by this Saccharomyces cerevisiae strain. The natural medium, containing only date extracts (glucose DCSE 25 g/l, DSH 25 g/l, CDSL 1-0 g/l, DSA 0.6 g/l) and two salts (NaNO3 1 g/l and KH2PO4 6 g/l), performed well and gave good and promising results. This natural medium is very suitable economically,
Yeast growth on date waste
71
Table 7. Use of different date preducts in the production of linker's yeast btomass by ~
¢mrev/s/ae. SSFM was used as a ceutroi. DCSE is expressed as its glucose concentration. The semi-synthetic medium (SSFM) was used as the control. These data are the mean values of three experiments and the Fisher ratio (see Metheds) was 4.67
Medium
SSFM DCSE (25 g/I)+DSH (25 g/l)
(g~)
Additives
Dry yeast biomass
(g~)
0 DCSE (25) 0 NH4NO3 (1)+KH2PO, (6)
0.350 0.430 0.475 0.500 0.520
NH4NO3 (1)
0.550
+ KH2PO4 (6) + DSA (0.6) NH4NO3 (1)
0,620
NH4NO3 (1)
+ KH2PO4 (6) +DSA (0.6)+CDSL (1) especially for a major date-producing country such as Algeria. ACKNOWLEDGEMENTS Thanks are due to the industrial plant complex of baker's yeast, Bouchegouf (Algeria), for the yeast strain, and to the industrial complex of ammonia and fertilizer, Annaba (Algeria), for ammonium nitrate, and to Drs M. Benboubatra and M. Ghoul (University of Setif, Algeria) for advice. REFERENCES
Abou-Zeid, A. A., Abdelrahman, N. & Baghlaf, A. O. (1991). The formation of oxytetracycline in date-coat medium. Biores. Techno£, 37, 179-184. Abou-Zeid, A. A., Abdelrahman, N. & Baghlaf, A. O. (1993a). Use of date products in production of oxytetracycline by Streptomyces rimosus. Biosci. Biotechnol. Biochent, S7(6), 987-988. Abou-Zeid, A. A., Baeshin, N. A. & Baghlaf, A. O. (1993b). Utilization of date products in production of oxytetracycline by Streptomyces rimosus. J. Technol. BIOtechnol., 58, 77-79. Abou-Zeid, A. A. & Baghlaf, A. O. (1983). Utilization of date seeds and cheese whey in production of citric acid by Candida lipoO~tica. Agric. Wastes, $, 131-142. Abou-Zeid, A. A. & Khoja, S. M. (1993). Utilization of dates in the fermentative formation of citric acid by Yarrowia h'po!ytica. Zentralbl. Microbiol. , 148, 213-221.
AI-Rawi, N., Marbabis, P. & Bauer, D. H. (1967). Amino acid composition of Iraqi dates. J. Sci. Food Agric., 18, 35-39. AI Saady, G. K & Benjamin, N. (1982). Date technology in Iraq. In Prec. First Symp. on the Date Palm, Saudi Arabia, 23-26 March, King Faical University Arabia, pp. 754-756. Auda, H., AI-Wandawi, H. & AI Adhami, L. (1976). Protein and amino acid composition of the three varieties of Iraqi dates at different stages of development. J. Agric. Food Chem., 24(2), 365-367. Booij, I., Piombo, G., Risterucci, J. M., Thomas, D. & Ferry, M. (1993). Sugar and free amino acid composition of five cultivars of dates from offshoots or Vitroplants in open fields. J. Agric. Food Chem., 41, 1553-557. Khan, J. A., Abulnaja, IC O., Kumosani, T. A. & AbouZaid, A. A. (1995). Utilization of Saudi date sugars in production of baker's yeast. Biores. TechnoL, $3, 63-66. Lazar, P. & Lellouch J. (1983). Statistiques en Experimentation Biologique. Flammarion Edition, Pads, p. 283. Nur, A., Zim, A. M. & Ahmed, A. R. (1981). Physicochemical composition of common Sudanese date cultivars and their suitability for jam making. The Date Palm J., 1, 90-106. Rose, A. H. & Harrison, J. S. (1971). The Yeasts, Physiology and Biochemistry of Yeast, Vol. 2. Academic Press, London and New York, p. 4. Sawaya, W. N., Sail, W. M., Khalil, J. K. & Mashadi, A. S. (1982). Physical measurements, proximate analysis, and nutrient elements content of 25 cultivars grown in Arabia and the Khalal (mature color) and Tamer (ripe) stages. In Prec. First Syrup. on the Date Palm, Saudi Arabia, 23-26 March, King Faical University Arabia, pp. 454-467.
