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Utilization of beet molasses and whey for fat biosynthesis by a yeast
Agricultural Wastes 18 (1986) 19-26
Utilization of Beet Molasses and Whey for Fat Biosynthesis by a Yeast W. Bednarski, J. L e m a n & J. T o m a s i k Institute of Food Engineeringand Biotechnology, University of Agriculture and Technology, 10-957 Olsztyn-Kortowo,B1. 43, Poland
ABSTRACT From a number of yeast strains capable of fat biosynthesis on a medium with beet molasses, Candida curvata D was selected as being most efficient. The optimum conditions for fat biosynthesis by C. curvata D were pH, 5.4; temperature, 30°C; sucrose content of the medium, 25g litre- 1 Cultivation of C. curvata D on a medium with beet molasses and whey gave good fat and biomass yields. The biomass, preserved separately or together with the spent medium, could be used as a fat-protein component of feeds for poultry, pigs or cattle.
INTRODUCTION One of the possibilities of processing by-products from the food industry is to utilize them for the cultivation of biomass. The yeasts from the genera Torula, Candida and Rhodotorula are capable of fat biosynthesis (Rattray, 1975). The efficiency of fat biosynthesis and its composition depend on genetic properties of the yeast strains, culturing conditions and the kind of medium. Studies in America showed that the yeast, Candida curvata, cultivated on whey medium, synthesized 15-8g of fat from 1 litre of the medium and that the chemical composition of this fat was close to that of cocoa butter (Hammond et al., 1981). In this work an attempt was made to biosynthesize fat by cultivating selected yeast strains on beet molasses or on molasses-whey media. The 19 Agricultural Wastes 0141-4607/86/$03-50 © Elsevier Applied Science Publishers Ltd, England, 1986. Printed in Great Britain
Fat synthesis by a yeast
21
Efficiency of fat biosynthesis was calculated per unit of the medium volume or per 100 g of sugars utilized by the yeasts. Dry matter and the crude protein in the yeast biomass were also determined by A O A C (1970b) methods.
RESULTS AND DISCUSSION Efficiency of fat biosynthesis in the yeasts was 7.3 g litre- 1 in the biomass of Trichosporon cutaneum 24, 7.6 g litre- 1 in the biomass of Trichosporon cutaneum 40, 7-9 g litre- 1 in the biomass of Candida curvata R and 8.5 g litre- 1 in the biomass of Candida D; the latter being selected for further studies. The effect of pH on growth of C. curvata D was studied. The sucrose content of the medium was 55 g litre- 1. The yield of the biosynthesis, calculated per 100 g of the sucrose utilized, changed from 7.95 g at pH 5.0 to 10.75g at pH 5.4. Reductions in COD were 75.3% and 86.8%, respectively (Fig. 1). The yeast was then cultured for 84 h at pH 5-4 and a sucrose content of 55 g litre- 1. The most effective yield of fat (10-75 g per 100g of sucrose utilized) was obtained at 30°C (Fig. 2). Analysis of the results suggests that the efficiency of fat biosynthesis would not, Yield offot//1OOg sucrose
g fo 1//lOOg 1 % COD % fat./D~
o---o COD reduction
sucrose
10.5,
rO0~
60-
10-
9o!
5O-
9.5.
e--..e For contents % of Dhl.
.< ,~o .
I,zj Q:
30,
8.5-
60-
8-
10.
?
~o
~.~
518
Fig. 1. Effect of pH on the efficiency of fat biosynthesis and COD reduction by C. curvata D cultured on molasses medium. Culturing conditions: sucrose content, 50g litre-1; temperature, 30°C; time of cultivation, 84h.
22
I4z. Bednarski, J. L e m a n , J. T o m a s i k
!
gr=l 7oogI
% COD % f a
Yield of fat I lOOg sucrose
OHI
sucrose
o=~o COD reduction 100.
13-
Fat confenfs% of DM
60-
~ 5o-
90.
~ ~o80"
ta.
~ 30-
11Q
t~ 70"
20-
10.
9"
60
tO r
""
~5
io
J5
*c
Temperature
Fig. 2. Effectof temperature on the efficiencyof fat biosynthesis and COD reduction. Culturing conditions: sucrose content, 50 g dm- 3, pH, 5-4; time of cultivation, 84 h.
in practice, be affected by the temperature. Reduction in C O D was within the range from 79.8% after yeast culturing at 25°C to 86.8% after culturing at 30°C (Fig. 2). Studying the effect o f the sucrose content o f the medium at pH 5-4, cultivation was carried out for 84 h at 30°C. Efficiency o f fat biosynthesis depended on the sucrose level in the medium, which ranged from 10"75 g per 100g of the sucrose utilized at a sucrose concentration of 5 5 g litre -1 to 16.25g per 100g o f sucrose in the m e d i u m containing 25 g o f sucrose per litre (Fig. 3). These results showed that the efficiency o f fat biosynthesis by C. curvata D cultured on beet molasses depended on the sucrose levels in the medium. Satisfactory results were obtained at low sucrose levels. On the other hand, differences in the sucrose contents in the medium were less important with regard to the a m o u n t utilized and C O D reduction, which ranged from 81.2% to 85.1% (Fig. 3). Most probably, in the case of yeast cultured in the media with the higher sucrose levels, part o f the energy was transferred to non-fatty components o f the biomass.
23
Fat synthes~ by a yeast
Yield of f o t / lOO g s u c r o s e
g fot/7oo~
%COD %fot/DNll
o~o
sucrose
17-
:
60 i
tO0-
COD reduction -- Fat contents % of OAf.
16-
5090-
15
u.
Q
P--.
4oQ
~ 30-
Q
~ 12-
Q ~
Q Q
~, 7o-
~. 2o-
60"
10-
\
1110-
~5
~'5 Sucrose
;0
;5 g/dm5
contents.
Fig. 3. Effect of sucrose concentration on the efficiency of fat biosynthesis and COD reduction. Culturing conditions: temperature, 30°C; pH, 5.4; time of cultivation, 84h.
Dependence of fat biosynthesis efficiency on the sucrose contents, i.e. on the degree of molasses dilution with water, suggested that other substances present in the molasses might also affect the process. In order to test this suggestion, the results of fat biosynthesis on beet molasses medium at a sucrose level of 50 g litre-1 were compared with the efficiency of fat biosynthesis on Czapek's medium in which the sucrose level was also 50 g litre-1. Efficiency of fat biosynthesis was higher on the synthetic medium, at 14.88 g per 100 g of sucrose utilized, compared with only 11.99g per 100g of sucrose on the medium with diluted molasses. This lower efficiency of fat biosynthesis was probably determined by some mineral salts present in the beet molasses. Studies were also made on the possibility of using beet molasseswhey as the medium, the proportions of the two components being 4: 96. The cultures were performed in the same conditions as on molasses medium. Fat biosynthesis by C . c u r v a t a D was much more effective on the molasses-whey medium. Fat content in the yeast biomass cultivated on this medium was 40.19% compared with 30.5% on the beet-molasses medium. Also, the highest fat biosynthesis of 22.56 g per 100 g of sucrose
W. Bednarski, J. Leman, J. Tomasik
24
+ lactose (calculated as sucrose equivalent) was obtained using the molasses-whey medium. Differences were observed in the dynamics of lactose and sucrose utilization by yeast during the cultivation. Lactose was utilized predominantly. The final degrees of sucrose and lactose utilization were 63.0% and 92%, respectively (Table 1). Sixty per cent of the total biomass synthesis occurred in the first 48 h of the culture. This was reflected in the yield of dry matter--20.54g litre -1 after 48 h and 29.45g litre-1 after 84h (Table 1), Comparison of the efficiency of dry matter biosynthesis after 84h, 60h and 48h suggests that it is possible to shorten the time of cultivation provided that the biomass is used as a whole, together with the medium. The prolonged culturing is necessary in order to utilize as much as possible of the medium components and to reduce the COD of the effluent which is discarded after separation of the yeast biomass. The utilization of the molasses-whey medium improved the efficiency of fat biosynthesis and molasses-whey may be used in areas where by-products are easily available. The fats synthesized by C. curvata D are characterized by high contents of unsaturated fatty acids. Some differences, mostly in the contents of oleic and linoleic acids, were observed in the compositions of fats synthesized on different media (Table 2). The differences in
TABLE 1 Characteristics of Candida curvata D Cultivation o n W h e y - B e e t Molasses M e d i u m
Time of cultivation
Yeast Sucrose utilization Lactose utilization Reduction o f COD (%)~ (%? (%) (g litre- t)
(h) 0
12 24 36 48 60 72 84
.
6-72 16-47 18.56 20.54 23.18 23.88 29.45
.
.
8-5 18.2 24.3 45-1 60.0 62.0 63.0
a Sucrose c o n t e n t was 22.20 g l i t r e - t . b Lactose c o n t e n t was 41.76g litre-1.
.
31-0 52.9 60.5 77.3 80.1 85.0 92.0
17.1 28.1 35.2 49.3 58.4 67.8 78.5
25
Fat synthesis by a yeast TABLE 2
Composition of Fatty Acids of Triglyceridesof Candida curvata D Grown in Different Media Fatty acids
Medium
(%)
C16:o C16:1 C1s:o C18:1 Cls:2 Ct8:3
Beet molasses ~
Molasses + wheyb 4:96
25"90 1'21 7'37 56'02 6"90 0.46
30-4 0"8 11"4 51'0 6.2 0-4
° Medium with: 5.5% sucrose; pH, 5.4; 30°C. bMedium with: 6.4% carbohydrates (sucrose + lactose); pH, 5.4; 30°C. fatty acid composition suggest, however, that the medium is o f little importance with regard to fat quality. At present, studies are being carried out in order to obtain a fat similar to cocoa butter from Candida curvata. It seems that this might be achieved by genetic improvement of yeasts as shown by Glatz et al. (1984). In summary, it can be stated that our results confirm the possibility of fat biosynthesis by yeasts cultivated on beet molasses or molasseswhey media. Besides the valuable fats, the biomass of C. curvata D also contains 2 3 % - 3 5 % protein. If the biomass, together with the spent medium, is preserved, the fat-protein preparation so obtained could be used in feed production. Initial experiments have shown that these preparations can be well used in poultry, pig or cattle feeding. REFERENCES AOAC (1970a). Official methods o f analysis (1 lth edn), Association of Official Analytical Chemists, Washington, DC. AOAC (1970b). Official and tentative methods of analysis (3rd edn), American Oil Chemists Society, Champaign, I11, USA. Glatz, B. A., Hammond, E. G., Hszi, K. H., Behman, L., Bati, N., Bednarski, W., Brown, D. & Floeteumeyer, M. (1984). Production and modification of fats and oils by yeast fermentation. Iowa State University Reports. Paper No. 1-10774, 163-76.
26
W. Bednarski, J. Leman, J. Tomasik
Hammond, E. G., Glatz, B. A., Choi, Z. & Teasdale, M. (1981). Oilproduction by Candida curvata D and extraction, composition and properties. New sources of fats and oils. AOCS Monograph No. 9, Champaign II1., 17187. Rattray, J. B. M. (1975). Lipids of yeast. Bacteriol. Rev., 39, (3), 19-21. Zadernowski & Sosulski, F. (1978). Composition of total lipids in rapeseed. J. Am. Oil Chem. Soc., 55, 870-2.
Utilization of Beet Molasses and Whey for Fat Biosynthesis by a Yeast W. Bednarski, J. L e m a n & J. T o m a s i k Institute of Food Engineeringand Biotechnology, University of Agriculture and Technology, 10-957 Olsztyn-Kortowo,B1. 43, Poland
ABSTRACT From a number of yeast strains capable of fat biosynthesis on a medium with beet molasses, Candida curvata D was selected as being most efficient. The optimum conditions for fat biosynthesis by C. curvata D were pH, 5.4; temperature, 30°C; sucrose content of the medium, 25g litre- 1 Cultivation of C. curvata D on a medium with beet molasses and whey gave good fat and biomass yields. The biomass, preserved separately or together with the spent medium, could be used as a fat-protein component of feeds for poultry, pigs or cattle.
INTRODUCTION One of the possibilities of processing by-products from the food industry is to utilize them for the cultivation of biomass. The yeasts from the genera Torula, Candida and Rhodotorula are capable of fat biosynthesis (Rattray, 1975). The efficiency of fat biosynthesis and its composition depend on genetic properties of the yeast strains, culturing conditions and the kind of medium. Studies in America showed that the yeast, Candida curvata, cultivated on whey medium, synthesized 15-8g of fat from 1 litre of the medium and that the chemical composition of this fat was close to that of cocoa butter (Hammond et al., 1981). In this work an attempt was made to biosynthesize fat by cultivating selected yeast strains on beet molasses or on molasses-whey media. The 19 Agricultural Wastes 0141-4607/86/$03-50 © Elsevier Applied Science Publishers Ltd, England, 1986. Printed in Great Britain
Fat synthesis by a yeast
21
Efficiency of fat biosynthesis was calculated per unit of the medium volume or per 100 g of sugars utilized by the yeasts. Dry matter and the crude protein in the yeast biomass were also determined by A O A C (1970b) methods.
RESULTS AND DISCUSSION Efficiency of fat biosynthesis in the yeasts was 7.3 g litre- 1 in the biomass of Trichosporon cutaneum 24, 7.6 g litre- 1 in the biomass of Trichosporon cutaneum 40, 7-9 g litre- 1 in the biomass of Candida curvata R and 8.5 g litre- 1 in the biomass of Candida D; the latter being selected for further studies. The effect of pH on growth of C. curvata D was studied. The sucrose content of the medium was 55 g litre- 1. The yield of the biosynthesis, calculated per 100 g of the sucrose utilized, changed from 7.95 g at pH 5.0 to 10.75g at pH 5.4. Reductions in COD were 75.3% and 86.8%, respectively (Fig. 1). The yeast was then cultured for 84 h at pH 5-4 and a sucrose content of 55 g litre- 1. The most effective yield of fat (10-75 g per 100g of sucrose utilized) was obtained at 30°C (Fig. 2). Analysis of the results suggests that the efficiency of fat biosynthesis would not, Yield offot//1OOg sucrose
g fo 1//lOOg 1 % COD % fat./D~
o---o COD reduction
sucrose
10.5,
rO0~
60-
10-
9o!
5O-
9.5.
e--..e For contents % of Dhl.
.< ,~o .
I,zj Q:
30,
8.5-
60-
8-
10.
?
~o
~.~
518
Fig. 1. Effect of pH on the efficiency of fat biosynthesis and COD reduction by C. curvata D cultured on molasses medium. Culturing conditions: sucrose content, 50g litre-1; temperature, 30°C; time of cultivation, 84h.
22
I4z. Bednarski, J. L e m a n , J. T o m a s i k
!
gr=l 7oogI
% COD % f a
Yield of fat I lOOg sucrose
OHI
sucrose
o=~o COD reduction 100.
13-
Fat confenfs% of DM
60-
~ 5o-
90.
~ ~o80"
ta.
~ 30-
11Q
t~ 70"
20-
10.
9"
60
tO r
""
~5
io
J5
*c
Temperature
Fig. 2. Effectof temperature on the efficiencyof fat biosynthesis and COD reduction. Culturing conditions: sucrose content, 50 g dm- 3, pH, 5-4; time of cultivation, 84 h.
in practice, be affected by the temperature. Reduction in C O D was within the range from 79.8% after yeast culturing at 25°C to 86.8% after culturing at 30°C (Fig. 2). Studying the effect o f the sucrose content o f the medium at pH 5-4, cultivation was carried out for 84 h at 30°C. Efficiency o f fat biosynthesis depended on the sucrose level in the medium, which ranged from 10"75 g per 100g of the sucrose utilized at a sucrose concentration of 5 5 g litre -1 to 16.25g per 100g o f sucrose in the m e d i u m containing 25 g o f sucrose per litre (Fig. 3). These results showed that the efficiency o f fat biosynthesis by C. curvata D cultured on beet molasses depended on the sucrose levels in the medium. Satisfactory results were obtained at low sucrose levels. On the other hand, differences in the sucrose contents in the medium were less important with regard to the a m o u n t utilized and C O D reduction, which ranged from 81.2% to 85.1% (Fig. 3). Most probably, in the case of yeast cultured in the media with the higher sucrose levels, part o f the energy was transferred to non-fatty components o f the biomass.
23
Fat synthes~ by a yeast
Yield of f o t / lOO g s u c r o s e
g fot/7oo~
%COD %fot/DNll
o~o
sucrose
17-
:
60 i
tO0-
COD reduction -- Fat contents % of OAf.
16-
5090-
15
u.
Q
P--.
4oQ
~ 30-
Q
~ 12-
Q ~
Q Q
~, 7o-
~. 2o-
60"
10-
\
1110-
~5
~'5 Sucrose
;0
;5 g/dm5
contents.
Fig. 3. Effect of sucrose concentration on the efficiency of fat biosynthesis and COD reduction. Culturing conditions: temperature, 30°C; pH, 5.4; time of cultivation, 84h.
Dependence of fat biosynthesis efficiency on the sucrose contents, i.e. on the degree of molasses dilution with water, suggested that other substances present in the molasses might also affect the process. In order to test this suggestion, the results of fat biosynthesis on beet molasses medium at a sucrose level of 50 g litre-1 were compared with the efficiency of fat biosynthesis on Czapek's medium in which the sucrose level was also 50 g litre-1. Efficiency of fat biosynthesis was higher on the synthetic medium, at 14.88 g per 100 g of sucrose utilized, compared with only 11.99g per 100g of sucrose on the medium with diluted molasses. This lower efficiency of fat biosynthesis was probably determined by some mineral salts present in the beet molasses. Studies were also made on the possibility of using beet molasseswhey as the medium, the proportions of the two components being 4: 96. The cultures were performed in the same conditions as on molasses medium. Fat biosynthesis by C . c u r v a t a D was much more effective on the molasses-whey medium. Fat content in the yeast biomass cultivated on this medium was 40.19% compared with 30.5% on the beet-molasses medium. Also, the highest fat biosynthesis of 22.56 g per 100 g of sucrose
W. Bednarski, J. Leman, J. Tomasik
24
+ lactose (calculated as sucrose equivalent) was obtained using the molasses-whey medium. Differences were observed in the dynamics of lactose and sucrose utilization by yeast during the cultivation. Lactose was utilized predominantly. The final degrees of sucrose and lactose utilization were 63.0% and 92%, respectively (Table 1). Sixty per cent of the total biomass synthesis occurred in the first 48 h of the culture. This was reflected in the yield of dry matter--20.54g litre -1 after 48 h and 29.45g litre-1 after 84h (Table 1), Comparison of the efficiency of dry matter biosynthesis after 84h, 60h and 48h suggests that it is possible to shorten the time of cultivation provided that the biomass is used as a whole, together with the medium. The prolonged culturing is necessary in order to utilize as much as possible of the medium components and to reduce the COD of the effluent which is discarded after separation of the yeast biomass. The utilization of the molasses-whey medium improved the efficiency of fat biosynthesis and molasses-whey may be used in areas where by-products are easily available. The fats synthesized by C. curvata D are characterized by high contents of unsaturated fatty acids. Some differences, mostly in the contents of oleic and linoleic acids, were observed in the compositions of fats synthesized on different media (Table 2). The differences in
TABLE 1 Characteristics of Candida curvata D Cultivation o n W h e y - B e e t Molasses M e d i u m
Time of cultivation
Yeast Sucrose utilization Lactose utilization Reduction o f COD (%)~ (%? (%) (g litre- t)
(h) 0
12 24 36 48 60 72 84
.
6-72 16-47 18.56 20.54 23.18 23.88 29.45
.
.
8-5 18.2 24.3 45-1 60.0 62.0 63.0
a Sucrose c o n t e n t was 22.20 g l i t r e - t . b Lactose c o n t e n t was 41.76g litre-1.
.
31-0 52.9 60.5 77.3 80.1 85.0 92.0
17.1 28.1 35.2 49.3 58.4 67.8 78.5
25
Fat synthesis by a yeast TABLE 2
Composition of Fatty Acids of Triglyceridesof Candida curvata D Grown in Different Media Fatty acids
Medium
(%)
C16:o C16:1 C1s:o C18:1 Cls:2 Ct8:3
Beet molasses ~
Molasses + wheyb 4:96
25"90 1'21 7'37 56'02 6"90 0.46
30-4 0"8 11"4 51'0 6.2 0-4
° Medium with: 5.5% sucrose; pH, 5.4; 30°C. bMedium with: 6.4% carbohydrates (sucrose + lactose); pH, 5.4; 30°C. fatty acid composition suggest, however, that the medium is o f little importance with regard to fat quality. At present, studies are being carried out in order to obtain a fat similar to cocoa butter from Candida curvata. It seems that this might be achieved by genetic improvement of yeasts as shown by Glatz et al. (1984). In summary, it can be stated that our results confirm the possibility of fat biosynthesis by yeasts cultivated on beet molasses or molasseswhey media. Besides the valuable fats, the biomass of C. curvata D also contains 2 3 % - 3 5 % protein. If the biomass, together with the spent medium, is preserved, the fat-protein preparation so obtained could be used in feed production. Initial experiments have shown that these preparations can be well used in poultry, pig or cattle feeding. REFERENCES AOAC (1970a). Official methods o f analysis (1 lth edn), Association of Official Analytical Chemists, Washington, DC. AOAC (1970b). Official and tentative methods of analysis (3rd edn), American Oil Chemists Society, Champaign, I11, USA. Glatz, B. A., Hammond, E. G., Hszi, K. H., Behman, L., Bati, N., Bednarski, W., Brown, D. & Floeteumeyer, M. (1984). Production and modification of fats and oils by yeast fermentation. Iowa State University Reports. Paper No. 1-10774, 163-76.
26
W. Bednarski, J. Leman, J. Tomasik
Hammond, E. G., Glatz, B. A., Choi, Z. & Teasdale, M. (1981). Oilproduction by Candida curvata D and extraction, composition and properties. New sources of fats and oils. AOCS Monograph No. 9, Champaign II1., 17187. Rattray, J. B. M. (1975). Lipids of yeast. Bacteriol. Rev., 39, (3), 19-21. Zadernowski & Sosulski, F. (1978). Composition of total lipids in rapeseed. J. Am. Oil Chem. Soc., 55, 870-2.