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Fermentation of Whey Filtrate by Kluyveromyces fragilis: the Effect on Cell Lipids
Can. 2, pp. 117-120, 1984 Can. Inst. Insf. Food Food Sci. Sci. Technol. J. Vol. 17, No. No.2,
RESEARCH NOTE
Fermentation of Whey Filtrate by Kluyveromyces jragilis: fragilis: the Effect on Cell Lipids M.M.A. AI-Shabibi Al-Shabibi and N.A.J. Younis Department of Food Science College of Agriculture University of Baghdad Baghdad, Iraq
fragilis is a lactose utilizing yeast, and Kluveromyces jragi/is fragilis was capable of reducthe latter showed that K. jragi/is ing the chemical oxygen demand (COD), a measure BaD, of cheese whey by at least 60%. of BOD, fragitis which is a non-fat yeast according to Rose K. jragi/is and Harrison (1971) was claimed by Wierzbicki and Kosikowiski (1973) to be capable of producing about 50% 50070 protein measured a total N x 6.25. Furthermore, fragilis Noble and Duitschaever (1973) reported that K. jragi/is produces less than 5% fat while Vrignaud (1976) found that it can produce up to 7-9% fat if grown at 38°C under high areation of the media and pH 3.5. The purpose of this study was to find the best growth conditions for the yeast to reduce the BOD BaD value as estimated by COD and the production of oil through fortification of whey filtrate with salts for nutrient enrichment.
Abstract The utilization of cheese whey filtrate through fermentation was studied. K. fragi/is, jragilis, a lactose fermenting yeast was used. It was found that growing the yeast at 27°C with aeration, agitation and fortification of of whey filtrate with some mineral salts, caused the reduction of of COD by over 90%. This was coupled with an increase in oil content of the yeast (5.6 to 6.8 g oil/liter fermentation liquor). The composition of neutrallipids neutral lipids fraction showed the presence of fatty acids ranging from 14 to 28 carbon atoms, with unsaturated fatty acids amounting amounting to 53 to 770/0. The polar lipids fraction showed the presence of fatty acids ranging from 14 to 32 carbon atoms.
Resume L'utilisation L'utilisation du filtrat de lactoserum de fromage fut etudiee a I'aide jragilis, capable de de la technologie de fermentation. La levure K. fragi/is, Ie lactose, fut I'organisme mis al'essai. 11 II a ete demontre fermenter le fermenter que la demande en oxygene chimique peut etre diminue par plus 90(1,10 par par la culture de cette levure a 27°C avec aeration, agitade 90% et fortification du filtrat avec quelques sels mineraux. Cela fut tion et accompagne d'une augmentation de la teneur en huile de la levure (5.6 a6.8 g d'huile/litre de liqueur fermentee). La composition de la fraction des lipides neutres revela la presence d'acides gras variant 53a 77% etaient insatures. La de 14 a 28 tonnes de carbone dont 53'a fraction des lipides polaires montra la presence d'acides gras variant de 14 a 32 tonnes de carbone. de
Materials and Methods Cultures of K. jragi/is fragilis NCIM 3217 were prepared according to the directions of the supplier. Commercial whey powder (supplied by the New Zealand Dairy Board) was reconstituted to 6.5% total solids, deproteinized by heating to 85°C for 30 min at pH 4.5. The heated reconstituted whey was left undisturbed for 18 h in the cold room (5°C), after which the supernatant was siphoned and passed through a Berkfield porcelain water filter. The pH of the whey filtrate was adjusted to 5.5 using 10% tartaric acid and 50% sodium hydroxide. The whey filtrate was fortified with a number of salts as in Table 1 to determine the best best nutrient enrichment enhancing the production of oil by the yeast. Sterilized whey filtrate (antoclaved at 121°C for 15 min under lKg/cm2 pressure) was used to prepare the yeast inoculum from two-day-old culture by repeated transfer before use and as follows: one loopfull of the yeast culture was transferred to 25 ml medium placed in 100 mL shake flask. The culture was incubated for 48 h, after which the content was transferred to
Introduction One of the major problems that faces the dairy industry is the disposal of whey, a by-product of the cheese industry. The high biological oxygen demand (BOD) of whey makes dumping impossible and cost(BaD) ly sludge treatment necessary. The conversion of carbohydrates to fat and oils by fermentation has been of little interest to workers because of high cost. But the rising disposal costs and environmental regulations necessitate the development of new methods of utilizing whey. Since lactose in whey accounts for about 69070 of the BOD BaD value al., 1974), it becomes necessary to remove (Mickle et aI., this sugar to simplify the disposal of whey. Atkins et al. (1967) and Knight et al. (1972) reported that Copyright Copyright
Cl !Cl
J984 Canadian Canadian Institute Institute of J984 of Food Food Science Science and and Technology Technology
117
Table I.I. Fortification of whey filtrate with salts (WT. %). Treatment I
2 3 4 5 6 7 8 9 10 11 II 12 Control
NH 4H 2P0 4 0.25
NH 40H 0.07
+ + + + + + + + + + + +
+ + + + + + + + + + + +
118/ Al-Shabibi and Younis
MnS04 0.00025
+ + + +
+ + +
+
+
+
+ + +
+ + + +
+
+ +
+
FeSo 4 0.001
+
+
+
+ + + + + +
another shake flask (250 ml) containing 50 mL of the medium. The procedure was repeated three more times in larger flasks containing 100, 150, and 250 mL of the media respectively. The growth media (whey or fortified whey) was also sterilized in an autoclave before use in actual experiments. Two methods were used to study the growth of K. jragilis. The first method was a batch system, using a thermostatically controlled incubator/shaker (100 mL medium in 1 L shaking flasks with agitation speed of 280 rpm) at 27°C to determine the best two treatments enhancing the production of oil. The second method was a semicontinuous system involving a Biolafett fermentor with a 2 L jar used to further study the best two treatments in duplicate. Sterile air was bubbled through the fermentation liquor at a rate of 15 L/min/L liquor. The temperature of the medium was kept constant by circulating water at 27°C through an immersed coil. The medium was continuously mixed by a stirrer with two sets of stirring plates at a rate of 250 rpm. The pH and foam were controlled by a system equipped with three perstaltic pumps for acid (50070 tartaric), base (50070 NaOH) and antifoam (1070 tween 80). These pumps were controlled by a pH and a foam controller. Each treatment was repeated twice. Periodic samples of fixed volume were asceptically drawn from the fermentation liquor. The samples were centrifuged at 9,000xg for 30 min in a Beckman high speed refrigerated centrifuge at 2°C. The supernatant was used for measurement of the chemical oxidation demand (COD) as well as an estimate for the biological (BOD) and according to the procedure oxygen demand (BaD) given by the American Public Health Association (1972). Lactose was determined according to the method of Teles et al. (1978). Total nitrogen was determined according to the official methods of the American Association of Cereal Chemists (1962). The most probable values were determined according to Chou (1969). The precipitated cell paste was washed twice by resuspending in sterile distilled water and recentrifugation as above. The cell yield was determined on freshly drawn samples of the fermentation liquor by drying under vacuum (150 mm Hg) at 45°C for about 20 h or until constant weight. Analysis of the cell for fat and protein content were performed on the harvested, washed yeast cells which
MgS0 4 0.0003
KH 2P0 4 0.5
MgCI 26HP 0.001
were dried under vacuum. The extraction of lipids was performed according to Moon and Hammond (1978) utilizing methanol/benzene. The extract was washed twice with water to remove the insoluble debris. The totallipids total lipids thus obtained were fractionated according to the method of Borgstrom (1952) to neutral lipids and phospholipids (the polar lipid fraction). Fatty acid methyl esters were prepared from both fractions by the methods described by deMan (1964) and Smith and Lowry (1962), respectively. The fatty acid methyl esters were analyzed by gas liquid chromatography using a 571lA equipped with dual Hewlett-Packard model 5711A stainless steel columns (180 x 0.3 cm) packed with 10070 DEGS on 100-120 mesh chromosorb W. DMCS. The instrument was connected to Hewlett-Packard integrator/printer model 3380A.
Results and Discussion Table 2 represents average values of the effect of nutrient enrichment on the growth of K. jragilis in shake flasks. The best two treatments showing the highest production of oil (treatments 5 and 10) were selected. Cellular oil (based on dry weight bases) amounted to 6.8 and 5.6 oil/liter of fermentation liquor. These values are close to the values given by Table 2. Effect of nutrient enrichment on the cell yield, production of oil and protein as well as the consumption of lactose of K. jragilis NCIM 3217 grown in whey filtrate at 27°C. 27°C. Treatment I
2 3 4 5 6 7 8 9 lO 10 II 11 12 Control
Cell Cell Yield Oil (giL) (glL)
26.1 27.3 27.2 33.6 31.0 23.5 25.8 35.2 26.9 28.1 24.9 34.3 22.0
Oil' (giL) (glL) coefficient 7.1 2.9 3.6 8.3 8.3 3.5 3.4 1.3 6.8 15.1 1.8 4.8 4.3 1.6 1.I 3.0 1.1 2.7 6.8 5.6 12.6 2.1 5.4 4.5 1.8 3.4 1.1
Oil
Lactose Protein Consumption
(%)
(%)"
(%)
11.0 13.0 12.8 4.0 22.0 7.6 6.2 3.1 lO.O 10.0 20.0 8.3 5.3 5.0
28.4 26.0 26.7 45.9 21.7 35.0 39.8 49.3 29.8 22.4 32.5 43.3 34.0
81.3 85.2 83.5 77.9 90.4 75.2 75.0 73.0 78.9 88.8 76.8 81.8 64.5
I(g oil produced/liter whey/(IOO)/(g whey/(100)/(g lactose consumed/liter whey) J. J. Inst. Insl. Can. Can. Sci. Sei. Technol. Teehnol. Aliment. Aliment. Vol. Vol. 17, 17, No.2, No. 2,
1984
10 70
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Fermentation time (hours) Fig. I. Consumption of lactose by K. Fragilis NClM NCIM 3217 grown in whey filtrate at 27°C. (_ _) control, (- - -) treatment 5 and (- • - • - .) treatment 10.
jragilis (Noble other workers on oil production of K. fragilis and Duitschaever, 1973; Vrignaud, 1976). Table 2 also shows the ratio of oil to protein was about 1: 1 in these two treatments. It can further be noticed that the best consumption of lactose was achieved in these two treatments. Therefore, it is expected that these two treatments would reduce the BOD value since lactose accounts for about 69% of its value (Mickle et al..
10
SO 50
Fermentation time (hours) Fig. 2. Reduction of chemical oxygen demand (COD) of whey filtrate by K. Fragilis NCIM 3217 when grown at 27°C. (_ _) control, (- - -) treatment 5 and (- •e - •e - .) treatment 10. Table 3. Effect of nutrient enrichment on the component fatty acids of neutral lipid fractions obtained from K. jragilis fragilis NCIM 3217 grown in whey filtrate at 27°C. Treatment Fatty I Acid
Control
5
10
Weight Percent Tr 2 Tr Tr
3
~
20
2
~
"'0
.11 ~
>u
u
10
20
30
40
50
Fermentation time (hours) Fig. 3. Cell Yield of K. Fragilis NCIM 3217 grown in whey filtrate at 27°C. (_ _) control, (- - -) treatment 5 and (- • - • - e) .) treatment 10. Can. InSf. Vol. 17. 17, No. No.2, Inst. Faad Food Sci. Techno/. J. Vo!. 2. 1984
14:0 2.2 1.7 14:1 2.0 5.5 3 16:0 br Tr 18.6 15.5 16:0 14.3 13.7 16:1 7.4 10.0 18:0 br Tr 1.8 2.2 18:0 2.0 9.3 6.7 18:1 27.3 34.3 23.9 18:2 23.5 16.4 13.5 5.4 6.0 18:3 6.1 20:0 Tr Tr 20:1 Tr 22:0 3.2 2.0 Tr 22:1 Tr 23:0 1.2 Tr 24:0 Tr 24:0 Tr 28:0 6.8 1.4 1.4 'Fatty acids are identified by their carbon number; br, represent branched chain fatty acid. 2Tr, represents fatty acids detected in amounts less than I % of the total fatty acids. 3Not detected.
Al-Shabibi and Younis /I 119
Table 4. Effect of nutrient enrichment on the component fatty acids of the polar lipid fractions obtained from K. fragilis NCIM 3217 grown in whey filtrate at 27°C. Treatment Treatment Fatty) Fatty - - - - - - - Fatty! Acid Control 5 10 Acid Control 5 10 Weight Percent Weight Percent 14:0 Tr 2 Tr Tr 22:0 3.3 1.5 2.8 14: 1I Tr Tr 0.3 22: I Tr Tr Tr 16:0 4.5 13.3 1.8 22:2 3.7 11.2 30.0 16: 1 1.2 5.5 0.5 24:0 br Tr Tr Tr 18:0 br Tr Tr 24:0 3.9 1.3 2.0 18:0 1.8 1.2 0.9 26:0 14.9 2.5 18:0 4.7 18: II 5.6 20.9 1.7 27:0 1.4 Tr Tr 18:2 2.2 10.0 0.4 27:1 13.6 1.6 3.1 18:3 1.3 1.2 1.3 28:0 7.7 9.1 20.2 20:0 1.3 0.3 30:0 13.5 12.4 13.1 OJ 20: Tr Tr Tr 32:0 8.5 20: 1I 5.6 16.8 )Fatty !Fatty acids are are identified by their carbon number, br, represent branched chain fatty acid. 2Tr, represent fatty acids detected in amounts less than I% of the total fatty acids.
1974). These two treatments, along the control, were subjected to fermentation for about 45 h at 27°C and pH 5.5 using the semicontinuous system. Figure 1 shows the rate of consumption of lactose during the fermentation period. It can be seen from this figure that at the end of the fermentation period less than 0.750/0 lactose was left in the fermentation liquor. The rate of lactose consumption under the conjragi/is can be utilized ditions used indicated that K. jragilis successfully for the treatment of whey to lower its lactose contents and therefore lower its COD value. The fairly long fermentation period is due to the temperature used (27°C) which is lower than the optimum temperature for K. jragilis jragi/is (38°C) reported by Vrignaud (1976) and 35 ± 2°C reported by Knight et al. (1972).
Figure 2 shows the reduction of COD during the fermentation period. The figure indicates a total COD 92% at the end of the fermentareduction of 93 and 920/0 tion period. This again shows that this yeast is very efficient in reducing the COD of whey. Figure 3 shows the rate of growth of yeast in the fermentation liquor during the fermentation period. Treatment 5 gave the highest yield (2.6% based on dry weight basis). The length of the fermentation period contributed to the increase of yield and also in building up cellular oil (fattening phase). Microscopic examination of the yeast cells in the fattening phase showed the presence of bright yellow colored areas representing oil droplets. The component fatty acids of the cellular lipids of K. jragilis jragi/is are shown in Tables 3 and 4. The neutral K. lipid fractions (Table 3) are made up of fatty acids ranging from 14 to 28 carbon atoms. The effect of nutrient enrichment on the fatty acid composition of neutral lipids is very clear. The total unsaturated fatneutrallipids ty acids in treatment 5 represent 76.9% of the total
120/ AI-Shabibi and and Younis Younis 120 / AI-Shabibi
fatty acids, while those in treatment 10 and the control were 53.4 and 57.2%, respectively. Oleic acid was the predominant acid in all treatment followed by linoleic acid treatment 5. The control is characterized by the highest concentration of saturated fatty acids of 16, 18 and 28 carbon atoms. Table 4 shows the effect of nutrient enrichment on the component fatty acids of polar lipid fractions obtained. These fractions of lipids were found to be made up of fatty acids ranging from 14 to 32 carbon atoms, each but with extreme differences in proportions of each acid.
References American Association of Cereal Chemists Inc. Approved Methods. 1962. Vol. 2, 3340 Pilot Knob Rd. St. Paul, Minnesota, U.S.A. Methods for American Public Health Association, Inc. Standard Methods the Examination of Water and Wastewater. 14th Edition. American Public Health Association, Inc. New York. 1975. Association of Official Analytical Chemist. 1975. Official Methods D.e. Analysis. 12th Edition. A.O.A.C. Washington, D.C. e., Witter, L.D. and Ordal, Z.l. 1967. Continuous proAtkins, c., pagation of Trichosporon cutaneum in cheese whey. 1. App!. Microbol. 15:1339-1344. 15: 1339-1344. Appl. Borgstro, B. 1952. Investigation on lipid separation methods: Separaacids. tion of phospholipids from neutral fat and fatty acids. 25: 101-110. Acta Physiol, Scand. 25:101-110. Chou, Y. 1969. Statistical Analysis. Holt, Rinehart and Winston, New York. deMan, 1. 1.V. V. 1964. Determination of the fatty acid composition of milk fat by dual column temperature programmed gas liquid chromatography. 1. Dairy Sci. 47:546-549. Knight, S., Smith, W. and Mickle, 1.B. (1972) Cheese whey disposal using Saccharomyces fragilis yeast. Cult. Dairy Prod. 1. 17(2): 17-22 Mickle, 1.B., Smith, W., Halter, D. and Knight, S. 1974. Performance and morphology of K/uyveromyces Kluyveromyces fragilis jrogilis and Rhodotoru/a Rhodotorula gracilis grown in cottage cheese whey. 1. Milk Food Technol. 37:481-484. Moon, N.l. and Hammond, E.G. 1978. Oil production by cermentation of lactose and the effect of temperature on the the fatty acid composition. 1. Amer. Oil. Chern. Chem. Soc. 55:684. Noble, A.C. and Duitschaever, C.L. 1973. Fatty acid acid composition of lipids from Saccharomycesfragi/is. Saccharomycesjragi/is. Lipids. 8:655-657. 8:655-657. (Cited from Rattray, 1.B.M., Schibeci, A., A., and Kidby, 197-231). D.K. 1975. Lipids of Yeast. Bacteriol. Rev, 39: 39:197-231). Rose, A.H. and Harrison, 1.S. 1971. The Yeasts. Vol. Vo!. 2 Academic Press Inc., New York. Smith, L.M. and Lowry, R.R. 1962. Fatty acid composition of the milk phospholipids and other lipids in milk. 1. Dairy Sci. 45:481 Teles, F.F.F., Young, C.K. and Stull, Stun, 1.W. 1978. A method ofrapid of rapid determination of lactose. 1. Dairy Sci. 61 :506-508. Vrignaud, Y. 1976. Lactose yeasts - methods of production and their utilization in nutrition. Oesterreichische Milchwirtschaft. Vol. Vo!. 31. No. 21. pp. 405, 408-410. (Cited from Dairy Sci. Abstr. 1977. pp. 2288). Wasserman, A.E. 1960. Whey Utilization IV. Availability of whey nitrogen for the growth of Saccharomyces fragilis. jragi/is. 1. Dairy Sci. 43:1231-1234. Wierzbicki, L.E. and Kosikowski, F.V. 1973. Lactose potential of various microorganisms grown in whey. 1. Dairy Dairy Sci. 56:26-32.
or
Accepted September 13, 1983.
J. J. lnst. Inst. Can. Can. Sci. Sci. Technol. Technol. Aliment. Aliment. Vol. Vol. 17, 17. No.2, No. 2. 1984 1984
RESEARCH NOTE
Fermentation of Whey Filtrate by Kluyveromyces jragilis: fragilis: the Effect on Cell Lipids M.M.A. AI-Shabibi Al-Shabibi and N.A.J. Younis Department of Food Science College of Agriculture University of Baghdad Baghdad, Iraq
fragilis is a lactose utilizing yeast, and Kluveromyces jragi/is fragilis was capable of reducthe latter showed that K. jragi/is ing the chemical oxygen demand (COD), a measure BaD, of cheese whey by at least 60%. of BOD, fragitis which is a non-fat yeast according to Rose K. jragi/is and Harrison (1971) was claimed by Wierzbicki and Kosikowiski (1973) to be capable of producing about 50% 50070 protein measured a total N x 6.25. Furthermore, fragilis Noble and Duitschaever (1973) reported that K. jragi/is produces less than 5% fat while Vrignaud (1976) found that it can produce up to 7-9% fat if grown at 38°C under high areation of the media and pH 3.5. The purpose of this study was to find the best growth conditions for the yeast to reduce the BOD BaD value as estimated by COD and the production of oil through fortification of whey filtrate with salts for nutrient enrichment.
Abstract The utilization of cheese whey filtrate through fermentation was studied. K. fragi/is, jragilis, a lactose fermenting yeast was used. It was found that growing the yeast at 27°C with aeration, agitation and fortification of of whey filtrate with some mineral salts, caused the reduction of of COD by over 90%. This was coupled with an increase in oil content of the yeast (5.6 to 6.8 g oil/liter fermentation liquor). The composition of neutrallipids neutral lipids fraction showed the presence of fatty acids ranging from 14 to 28 carbon atoms, with unsaturated fatty acids amounting amounting to 53 to 770/0. The polar lipids fraction showed the presence of fatty acids ranging from 14 to 32 carbon atoms.
Resume L'utilisation L'utilisation du filtrat de lactoserum de fromage fut etudiee a I'aide jragilis, capable de de la technologie de fermentation. La levure K. fragi/is, Ie lactose, fut I'organisme mis al'essai. 11 II a ete demontre fermenter le fermenter que la demande en oxygene chimique peut etre diminue par plus 90(1,10 par par la culture de cette levure a 27°C avec aeration, agitade 90% et fortification du filtrat avec quelques sels mineraux. Cela fut tion et accompagne d'une augmentation de la teneur en huile de la levure (5.6 a6.8 g d'huile/litre de liqueur fermentee). La composition de la fraction des lipides neutres revela la presence d'acides gras variant 53a 77% etaient insatures. La de 14 a 28 tonnes de carbone dont 53'a fraction des lipides polaires montra la presence d'acides gras variant de 14 a 32 tonnes de carbone. de
Materials and Methods Cultures of K. jragi/is fragilis NCIM 3217 were prepared according to the directions of the supplier. Commercial whey powder (supplied by the New Zealand Dairy Board) was reconstituted to 6.5% total solids, deproteinized by heating to 85°C for 30 min at pH 4.5. The heated reconstituted whey was left undisturbed for 18 h in the cold room (5°C), after which the supernatant was siphoned and passed through a Berkfield porcelain water filter. The pH of the whey filtrate was adjusted to 5.5 using 10% tartaric acid and 50% sodium hydroxide. The whey filtrate was fortified with a number of salts as in Table 1 to determine the best best nutrient enrichment enhancing the production of oil by the yeast. Sterilized whey filtrate (antoclaved at 121°C for 15 min under lKg/cm2 pressure) was used to prepare the yeast inoculum from two-day-old culture by repeated transfer before use and as follows: one loopfull of the yeast culture was transferred to 25 ml medium placed in 100 mL shake flask. The culture was incubated for 48 h, after which the content was transferred to
Introduction One of the major problems that faces the dairy industry is the disposal of whey, a by-product of the cheese industry. The high biological oxygen demand (BOD) of whey makes dumping impossible and cost(BaD) ly sludge treatment necessary. The conversion of carbohydrates to fat and oils by fermentation has been of little interest to workers because of high cost. But the rising disposal costs and environmental regulations necessitate the development of new methods of utilizing whey. Since lactose in whey accounts for about 69070 of the BOD BaD value al., 1974), it becomes necessary to remove (Mickle et aI., this sugar to simplify the disposal of whey. Atkins et al. (1967) and Knight et al. (1972) reported that Copyright Copyright
Cl !Cl
J984 Canadian Canadian Institute Institute of J984 of Food Food Science Science and and Technology Technology
117
Table I.I. Fortification of whey filtrate with salts (WT. %). Treatment I
2 3 4 5 6 7 8 9 10 11 II 12 Control
NH 4H 2P0 4 0.25
NH 40H 0.07
+ + + + + + + + + + + +
+ + + + + + + + + + + +
118/ Al-Shabibi and Younis
MnS04 0.00025
+ + + +
+ + +
+
+
+
+ + +
+ + + +
+
+ +
+
FeSo 4 0.001
+
+
+
+ + + + + +
another shake flask (250 ml) containing 50 mL of the medium. The procedure was repeated three more times in larger flasks containing 100, 150, and 250 mL of the media respectively. The growth media (whey or fortified whey) was also sterilized in an autoclave before use in actual experiments. Two methods were used to study the growth of K. jragilis. The first method was a batch system, using a thermostatically controlled incubator/shaker (100 mL medium in 1 L shaking flasks with agitation speed of 280 rpm) at 27°C to determine the best two treatments enhancing the production of oil. The second method was a semicontinuous system involving a Biolafett fermentor with a 2 L jar used to further study the best two treatments in duplicate. Sterile air was bubbled through the fermentation liquor at a rate of 15 L/min/L liquor. The temperature of the medium was kept constant by circulating water at 27°C through an immersed coil. The medium was continuously mixed by a stirrer with two sets of stirring plates at a rate of 250 rpm. The pH and foam were controlled by a system equipped with three perstaltic pumps for acid (50070 tartaric), base (50070 NaOH) and antifoam (1070 tween 80). These pumps were controlled by a pH and a foam controller. Each treatment was repeated twice. Periodic samples of fixed volume were asceptically drawn from the fermentation liquor. The samples were centrifuged at 9,000xg for 30 min in a Beckman high speed refrigerated centrifuge at 2°C. The supernatant was used for measurement of the chemical oxidation demand (COD) as well as an estimate for the biological (BOD) and according to the procedure oxygen demand (BaD) given by the American Public Health Association (1972). Lactose was determined according to the method of Teles et al. (1978). Total nitrogen was determined according to the official methods of the American Association of Cereal Chemists (1962). The most probable values were determined according to Chou (1969). The precipitated cell paste was washed twice by resuspending in sterile distilled water and recentrifugation as above. The cell yield was determined on freshly drawn samples of the fermentation liquor by drying under vacuum (150 mm Hg) at 45°C for about 20 h or until constant weight. Analysis of the cell for fat and protein content were performed on the harvested, washed yeast cells which
MgS0 4 0.0003
KH 2P0 4 0.5
MgCI 26HP 0.001
were dried under vacuum. The extraction of lipids was performed according to Moon and Hammond (1978) utilizing methanol/benzene. The extract was washed twice with water to remove the insoluble debris. The totallipids total lipids thus obtained were fractionated according to the method of Borgstrom (1952) to neutral lipids and phospholipids (the polar lipid fraction). Fatty acid methyl esters were prepared from both fractions by the methods described by deMan (1964) and Smith and Lowry (1962), respectively. The fatty acid methyl esters were analyzed by gas liquid chromatography using a 571lA equipped with dual Hewlett-Packard model 5711A stainless steel columns (180 x 0.3 cm) packed with 10070 DEGS on 100-120 mesh chromosorb W. DMCS. The instrument was connected to Hewlett-Packard integrator/printer model 3380A.
Results and Discussion Table 2 represents average values of the effect of nutrient enrichment on the growth of K. jragilis in shake flasks. The best two treatments showing the highest production of oil (treatments 5 and 10) were selected. Cellular oil (based on dry weight bases) amounted to 6.8 and 5.6 oil/liter of fermentation liquor. These values are close to the values given by Table 2. Effect of nutrient enrichment on the cell yield, production of oil and protein as well as the consumption of lactose of K. jragilis NCIM 3217 grown in whey filtrate at 27°C. 27°C. Treatment I
2 3 4 5 6 7 8 9 lO 10 II 11 12 Control
Cell Cell Yield Oil (giL) (glL)
26.1 27.3 27.2 33.6 31.0 23.5 25.8 35.2 26.9 28.1 24.9 34.3 22.0
Oil' (giL) (glL) coefficient 7.1 2.9 3.6 8.3 8.3 3.5 3.4 1.3 6.8 15.1 1.8 4.8 4.3 1.6 1.I 3.0 1.1 2.7 6.8 5.6 12.6 2.1 5.4 4.5 1.8 3.4 1.1
Oil
Lactose Protein Consumption
(%)
(%)"
(%)
11.0 13.0 12.8 4.0 22.0 7.6 6.2 3.1 lO.O 10.0 20.0 8.3 5.3 5.0
28.4 26.0 26.7 45.9 21.7 35.0 39.8 49.3 29.8 22.4 32.5 43.3 34.0
81.3 85.2 83.5 77.9 90.4 75.2 75.0 73.0 78.9 88.8 76.8 81.8 64.5
I(g oil produced/liter whey/(IOO)/(g whey/(100)/(g lactose consumed/liter whey) J. J. Inst. Insl. Can. Can. Sci. Sei. Technol. Teehnol. Aliment. Aliment. Vol. Vol. 17, 17, No.2, No. 2,
1984
10 70
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20
30
l.,0 40
§ 20
\
20
\ 10
50
Fermentation time (hours) Fig. I. Consumption of lactose by K. Fragilis NClM NCIM 3217 grown in whey filtrate at 27°C. (_ _) control, (- - -) treatment 5 and (- • - • - .) treatment 10.
jragilis (Noble other workers on oil production of K. fragilis and Duitschaever, 1973; Vrignaud, 1976). Table 2 also shows the ratio of oil to protein was about 1: 1 in these two treatments. It can further be noticed that the best consumption of lactose was achieved in these two treatments. Therefore, it is expected that these two treatments would reduce the BOD value since lactose accounts for about 69% of its value (Mickle et al..
10
SO 50
Fermentation time (hours) Fig. 2. Reduction of chemical oxygen demand (COD) of whey filtrate by K. Fragilis NCIM 3217 when grown at 27°C. (_ _) control, (- - -) treatment 5 and (- •e - •e - .) treatment 10. Table 3. Effect of nutrient enrichment on the component fatty acids of neutral lipid fractions obtained from K. jragilis fragilis NCIM 3217 grown in whey filtrate at 27°C. Treatment Fatty I Acid
Control
5
10
Weight Percent Tr 2 Tr Tr
3
~
20
2
~
"'0
.11 ~
>u
u
10
20
30
40
50
Fermentation time (hours) Fig. 3. Cell Yield of K. Fragilis NCIM 3217 grown in whey filtrate at 27°C. (_ _) control, (- - -) treatment 5 and (- • - • - e) .) treatment 10. Can. InSf. Vol. 17. 17, No. No.2, Inst. Faad Food Sci. Techno/. J. Vo!. 2. 1984
14:0 2.2 1.7 14:1 2.0 5.5 3 16:0 br Tr 18.6 15.5 16:0 14.3 13.7 16:1 7.4 10.0 18:0 br Tr 1.8 2.2 18:0 2.0 9.3 6.7 18:1 27.3 34.3 23.9 18:2 23.5 16.4 13.5 5.4 6.0 18:3 6.1 20:0 Tr Tr 20:1 Tr 22:0 3.2 2.0 Tr 22:1 Tr 23:0 1.2 Tr 24:0 Tr 24:0 Tr 28:0 6.8 1.4 1.4 'Fatty acids are identified by their carbon number; br, represent branched chain fatty acid. 2Tr, represents fatty acids detected in amounts less than I % of the total fatty acids. 3Not detected.
Al-Shabibi and Younis /I 119
Table 4. Effect of nutrient enrichment on the component fatty acids of the polar lipid fractions obtained from K. fragilis NCIM 3217 grown in whey filtrate at 27°C. Treatment Treatment Fatty) Fatty - - - - - - - Fatty! Acid Control 5 10 Acid Control 5 10 Weight Percent Weight Percent 14:0 Tr 2 Tr Tr 22:0 3.3 1.5 2.8 14: 1I Tr Tr 0.3 22: I Tr Tr Tr 16:0 4.5 13.3 1.8 22:2 3.7 11.2 30.0 16: 1 1.2 5.5 0.5 24:0 br Tr Tr Tr 18:0 br Tr Tr 24:0 3.9 1.3 2.0 18:0 1.8 1.2 0.9 26:0 14.9 2.5 18:0 4.7 18: II 5.6 20.9 1.7 27:0 1.4 Tr Tr 18:2 2.2 10.0 0.4 27:1 13.6 1.6 3.1 18:3 1.3 1.2 1.3 28:0 7.7 9.1 20.2 20:0 1.3 0.3 30:0 13.5 12.4 13.1 OJ 20: Tr Tr Tr 32:0 8.5 20: 1I 5.6 16.8 )Fatty !Fatty acids are are identified by their carbon number, br, represent branched chain fatty acid. 2Tr, represent fatty acids detected in amounts less than I% of the total fatty acids.
1974). These two treatments, along the control, were subjected to fermentation for about 45 h at 27°C and pH 5.5 using the semicontinuous system. Figure 1 shows the rate of consumption of lactose during the fermentation period. It can be seen from this figure that at the end of the fermentation period less than 0.750/0 lactose was left in the fermentation liquor. The rate of lactose consumption under the conjragi/is can be utilized ditions used indicated that K. jragilis successfully for the treatment of whey to lower its lactose contents and therefore lower its COD value. The fairly long fermentation period is due to the temperature used (27°C) which is lower than the optimum temperature for K. jragilis jragi/is (38°C) reported by Vrignaud (1976) and 35 ± 2°C reported by Knight et al. (1972).
Figure 2 shows the reduction of COD during the fermentation period. The figure indicates a total COD 92% at the end of the fermentareduction of 93 and 920/0 tion period. This again shows that this yeast is very efficient in reducing the COD of whey. Figure 3 shows the rate of growth of yeast in the fermentation liquor during the fermentation period. Treatment 5 gave the highest yield (2.6% based on dry weight basis). The length of the fermentation period contributed to the increase of yield and also in building up cellular oil (fattening phase). Microscopic examination of the yeast cells in the fattening phase showed the presence of bright yellow colored areas representing oil droplets. The component fatty acids of the cellular lipids of K. jragilis jragi/is are shown in Tables 3 and 4. The neutral K. lipid fractions (Table 3) are made up of fatty acids ranging from 14 to 28 carbon atoms. The effect of nutrient enrichment on the fatty acid composition of neutral lipids is very clear. The total unsaturated fatneutrallipids ty acids in treatment 5 represent 76.9% of the total
120/ AI-Shabibi and and Younis Younis 120 / AI-Shabibi
fatty acids, while those in treatment 10 and the control were 53.4 and 57.2%, respectively. Oleic acid was the predominant acid in all treatment followed by linoleic acid treatment 5. The control is characterized by the highest concentration of saturated fatty acids of 16, 18 and 28 carbon atoms. Table 4 shows the effect of nutrient enrichment on the component fatty acids of polar lipid fractions obtained. These fractions of lipids were found to be made up of fatty acids ranging from 14 to 32 carbon atoms, each but with extreme differences in proportions of each acid.
References American Association of Cereal Chemists Inc. Approved Methods. 1962. Vol. 2, 3340 Pilot Knob Rd. St. Paul, Minnesota, U.S.A. Methods for American Public Health Association, Inc. Standard Methods the Examination of Water and Wastewater. 14th Edition. American Public Health Association, Inc. New York. 1975. Association of Official Analytical Chemist. 1975. Official Methods D.e. Analysis. 12th Edition. A.O.A.C. Washington, D.C. e., Witter, L.D. and Ordal, Z.l. 1967. Continuous proAtkins, c., pagation of Trichosporon cutaneum in cheese whey. 1. App!. Microbol. 15:1339-1344. 15: 1339-1344. Appl. Borgstro, B. 1952. Investigation on lipid separation methods: Separaacids. tion of phospholipids from neutral fat and fatty acids. 25: 101-110. Acta Physiol, Scand. 25:101-110. Chou, Y. 1969. Statistical Analysis. Holt, Rinehart and Winston, New York. deMan, 1. 1.V. V. 1964. Determination of the fatty acid composition of milk fat by dual column temperature programmed gas liquid chromatography. 1. Dairy Sci. 47:546-549. Knight, S., Smith, W. and Mickle, 1.B. (1972) Cheese whey disposal using Saccharomyces fragilis yeast. Cult. Dairy Prod. 1. 17(2): 17-22 Mickle, 1.B., Smith, W., Halter, D. and Knight, S. 1974. Performance and morphology of K/uyveromyces Kluyveromyces fragilis jrogilis and Rhodotoru/a Rhodotorula gracilis grown in cottage cheese whey. 1. Milk Food Technol. 37:481-484. Moon, N.l. and Hammond, E.G. 1978. Oil production by cermentation of lactose and the effect of temperature on the the fatty acid composition. 1. Amer. Oil. Chern. Chem. Soc. 55:684. Noble, A.C. and Duitschaever, C.L. 1973. Fatty acid acid composition of lipids from Saccharomycesfragi/is. Saccharomycesjragi/is. Lipids. 8:655-657. 8:655-657. (Cited from Rattray, 1.B.M., Schibeci, A., A., and Kidby, 197-231). D.K. 1975. Lipids of Yeast. Bacteriol. Rev, 39: 39:197-231). Rose, A.H. and Harrison, 1.S. 1971. The Yeasts. Vol. Vo!. 2 Academic Press Inc., New York. Smith, L.M. and Lowry, R.R. 1962. Fatty acid composition of the milk phospholipids and other lipids in milk. 1. Dairy Sci. 45:481 Teles, F.F.F., Young, C.K. and Stull, Stun, 1.W. 1978. A method ofrapid of rapid determination of lactose. 1. Dairy Sci. 61 :506-508. Vrignaud, Y. 1976. Lactose yeasts - methods of production and their utilization in nutrition. Oesterreichische Milchwirtschaft. Vol. Vo!. 31. No. 21. pp. 405, 408-410. (Cited from Dairy Sci. Abstr. 1977. pp. 2288). Wasserman, A.E. 1960. Whey Utilization IV. Availability of whey nitrogen for the growth of Saccharomyces fragilis. jragi/is. 1. Dairy Sci. 43:1231-1234. Wierzbicki, L.E. and Kosikowski, F.V. 1973. Lactose potential of various microorganisms grown in whey. 1. Dairy Dairy Sci. 56:26-32.
or
Accepted September 13, 1983.
J. J. lnst. Inst. Can. Can. Sci. Sci. Technol. Technol. Aliment. Aliment. Vol. Vol. 17, 17. No.2, No. 2. 1984 1984