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Some Nutritional Characteristics of Spirulina maxima Algae Grown in Effluents from Biological Treatment Plant
Some Nutritional Characteristics of Spirulina maxima Algae Grown in Effluents from Biologica I Treatment Plant H. T. Nguyen, N. Kosaric and M. A. Bergougnou Chemical and Biochemical Engineering Faculty of Engineering Science The University of Westem Ontario London, Ontario, Canada N6A 3K7
Abstraet
Materials and Methods
Spirulina maxima, a high protein blue-green alga, was grown in effluents from the municipal waste treatment planto The protein content of the algal biomass tended to decrease at the later phase of batch ,culture while the fat and carbohydrate contents increased. The relat:ve protein amino acid composition did not change and was comparable to that of the algae grown in synthetic medium.
Sources of Materials
Résumé· Spirulina maxima, une espece d'algue bleue, est ciltivée dans un milieu de culture composé de la décharge venant du centre municipal de traitement des eaux usées. Discontinue, la teneur en protéines de la biomasse diminue vers la fin de la période de culture tandis que les teneurs en lipides et en glucides augmentent. La proportion relative des acides aminés des protéines est constante et elle est semblable a celle des échantillons cultivés en milieu synthétique.
Introduction Spirulina maxima is a blue-green alga (cyanophyta) belonging to the family of Oscillatoriaceae. It is a large microscopic, multi-cellular alga which grows in the shallow ponds of high alkalinity and high salinity in the tropical countries. Spirulina maxima has been used as food since ancient times in sorne regions 'Of Africa and Mexico. Nutritional studies carried out by the Institut Francais du Petrole (IFP) showed that Spirlllina is one of the richest protein sources ever found; the protein also contains all essential amino acids in a proportion comparable to 'Other conventional protein sources and to the composition recommended by FAO. Tests carried out on rats and chickens showed that Spirlllina maxima has a relatively good nutritive value and digestability (Clement et al., 1967). As a food source Spirulina maxima also supplies a considerable amount of fat, carbohydrate, vitamins and calories (Clement et al., 1967 and Nakamura, 1970) . Considering the nutritional values of Spirulina maxima and the relative simplicity in cultivation and harvesting of this alga because of its large size compared to other microalgae, several pilot plant studies have been performed in France, Algeria and Mexico to produce Spirulina maxima. The algae were cultivated in synthetic media made up mainly of aqueous mineral salt solutions. The cost of chemicals was about 15% of the total cost (Meyer, 1969), therefore a more economical way of Spirulina maxima culture would be beneficial. In this study, Spirulina maxima was grown in effluents from a secondary waste water treatment planto Some nutritional characteristics of the resulting algal biomass were investigated. Can. Inst. Food SeL Technol. J. Vol. 7, No. 2, 1974
The original cultures of Spirulina maxima were received from the Plant Physiology Institute, University of Gottingen, Germany. The waste water effluent samples were obtained from the Greenway Pollution Control Centre which receives and treats municipal sewage in the community of London, Ontario. The synthetic medium describ~d in the IFP patent (Clement et al., 1966) was used ll1 the present study for comparis'On.
Culture System The batch culture experiments were performed in a 5-gallon bottle containing 10 litres of culture medium, which was sterilized in an autoclave at 2 atm and 120°C for 15 minutes and cooled to room temperature. The cultures were illuminated by four "GROLUX" fluorescent tubes of 1,200 Lux. Aeration and mixing were accomplished by air containing 2% COz, which was passed into the culture at the rate of 0.5 ""m and also by a magnetic stirrer. The culture temperature was maintained at 30 ± 2°C in a "walk-in" incubator. pH was controlled in the range of 8.5 to 10 by adjusting the COz content in airo Approximately 0.5 g dry weight of inoculum was added to the bottle at the starting day. The inoculum was taken from a well-developed stock culture grown under the same conditions.
Analytical 111ethods The algal biomass was collected by filtration using a Whatman glass-fiber filter papel' and it was washed several times by distilled water to remove dissolved salts. The samples were then freeze-dried, ground into fine powders and stored at O°C in a refrigerator for chemical analyses. The moisture and ash contents in the freeze-dried sall1ples were determined by the method described by Jacobs (1965). Total carbohydrates were determined colorimetrically using anthrone reagent (Neish, 1952). The fat content was extracted in chlor'Oform and chloroform-methanol mixture, and deterll1ined gravimetrically (Kosaric, 1969). The nitrogen content of the samples was deterll1ined by the COLEMAN model 29A nitrogen analyzer. This method determines total nitrogen that is composed of protein and non-protein fmction. As most of the determined nitrogen is assumed to belong to protein, the non protein nitrogen was neglected. Under this assumption a factor of 6.25 was used to convert the found nitrogen to crude pr'Otein. 114
For amino acid determination, about 200 mg of tlle dry algal biomass was hydrolyzed by refluxing 500 mI of 5.7N Hel in a nitrogen atmosphere for 24 hours. An aliquot equivalent to approximately 2.8 mg of protein was dried undel' vacuum and rediss'Úlved in 2 1111 of a standard buffer solution containing norleucine. [t was then analyzed by an automatic Technicon T8M1 Amino Acid Analyzer.
Results and Discussion The analytical results of the algal biomass growll in Greenway effluent were compared to those grown in synthetic medium under the same growth conditions (Table 1). Table 1.
Chemical composition of Spirulina maxima biomass l . Synthetic Medium 12 days2
"Protein" Carbohydrate Fat Ash Moisture
63.1 16.6 1.0 13.9 5.0
Greenway Effluent 6 days2 50.5 26.5 4.6 14.0 4.8
10 days2
Amino acid spectra were also recorded for the biomass samples gr'Úwn in synthetic medium and Greenway effluent (Figure 1). Table 2 summarizes the results of essential amino acid analyses. Despite the protein content deviation, the composition of essential amino acids in the Spirulina muximu biomass is similar in different samples and comparable to literature values. All essential amino acids are found in the biomass and their levels in the algal "protein" exceed those recommended by F Aü. Methionine is very close to the recommended value as opposed to almost all other single cell proteins that are deficient in this amino acid. According to this finding, good nutritional quality of the algal protein is expected. However, further nutritional studies to establish biological and toxicological values of the "protein" are required.
28.3 43.2 9.2 14.5 4.7
1 % by weight 2 Age of culture
The protein content of Spintlina mQxima grown in synthetic medium was 63.1% (as calculated from total nitrogen) ; it is comparable to the result reported by the IFP (62-68%). However, the algae grown in Greenway effluent showed a difference in characteristics depending on the age of culture. The analysis of biomass after 6 days of batch culture in Greenway effluent showed that the "protein" content decreased from 63.1% to 50.5% while the fat and carbohydrate contents increased. The effect was more significant ufter 10 days 01' at the stationary phuse of growth where the "protein" content of the algal biomass was only 28.3% and the carbohydrate and fat contents increased as high as 43.2% and 9.2% respectively. The ash and moisture contents were relatively unchanged. A possible explanation of the variation in chem'ical composition of the algae is the nutrient deficiency in Greenway effluent during the batch culture. When the limited amount of nutrients (especially nitrogen compounds) in the medium has been used up by the algae during the active growth phase, a decrease in "protein" was observed; as a consequence, higher amounts of carbohydrate and fat were accumulated in the algal cells. Lewin (1962) also mentioned a decrease in nitrogen content from 8 to 10% of the dry weight to 2% and the in crease in fat content as high as 80% in several species of Chlorellu and Scenedesmus algae during the nitrogen deficiency periods. According to Durant and Jolly (1969), by appropriately modifying the culture conditions, the producer could vary the concentration of protein from 7 to 85%, the concentration of fat from 4 to 86%, and the concentration of carbohydrate from ;) to 38% in Chlorella. This response has been considered as an attractive property by Russian scientists since the composition of algae could theoretically be adjusted to suit the nutritional needs of cosmonauts when the algal systems are used in space programs (Lachance, 1968).
115
Fig. 1.
Table 2. Amino Acids
Amino acid spectra of Spimlina maxima in: A: Synthetic medium B: Greenway effluent after 6 days c: Greenway effluent after 10 days Amino acid analyses of Spirulina maxima "protein"l. Synthetic Medium 12 d ays 2
Ile Leu Lys Phe Met Thr Try Val
Greenway Effluents 6 d ays 2
10 days2
5.80 9.31 5.00 4.03 2.28 4.99
5.87 9.60 5.43 4.44 2.12 4.81
5.71 9.26 4.68 4.45 2.10 5.35
6.92
7.55
6.62
Clement et al. FAü (1967) Combination
6.03 8.02 4.59 4.97 1.37 4.56 1.40 6.49
4.2 4.8 4.2 2.8 2.2 2.8
lA 4.2
1 g/16 g of nitrogen 2 Age of culture - not reported J. Inst. Can. SeL Teehnol. Aliment. Vol. 7, No
2, 1974
Acknowledgements This work was supported by The University of vVestern Ontario Research Council. Analytical assistance from Labatt's Breweries Limited is appreciated.
References Clement, o., Rebeller, M., and Zarrouk, C., 1966. Procédé de culture de'algues en mllleu synthétique. Brevet d'invention no. 88, 103, lre addition au no. 1,458,061, France. Clement, O., Riddey, C., and Menzi, R., 1967. Amino acid composltion and nutritive value of the algae Spirulina maxima. J. Scl. Food Agr. 18:497. Durant, N. W., and Jolly, C., 1969. Oreen algae, Chlorella, as a con· tribution to the food supply of mano Fish. Ind. Res. 5:67.
Cs.n. I:lst. Food ScL Technol. J. Vol. 7, No. 2, 1974
Jacobs, M. B., 1965. The Chemical Analysis of Food and Food Products. 3rd editlon, Van Nostrand Co., Inc., 30. Kosaric, N., 1969. Ph.D. Thesls, The Unlverslty of Western Ontario, London, Canada. Lachance, P. A., 1969. Single cell protein in space systems. Single Cell Pl'otein, R. l. Mateles and S. R. Tannenbaum, ed., The M.I.T. Press, Massachusetts. Lewin, R. .A., 1962. Physiology and Biochemlstry of AIgae. Academlc Press, New York. Meyer, C.. 1969. Etude d'une culture d'algues en vue d'une production i\. grande échelle. Industr. Alim. Agr. 86:1445. Nakamura, H., 1970. The mass production of Spirulina: a hellcal blue-green algae as a new food. Report from the Spirulina Development Commlttee of Japan. Nelsh, A. C., 1952. Analytical Methods for Bacterial Fermentatlon. NRC of Canada, Report No. 46-83, 2nd revision, p. 33. Recelved August 1, 1973
116
Abstraet
Materials and Methods
Spirulina maxima, a high protein blue-green alga, was grown in effluents from the municipal waste treatment planto The protein content of the algal biomass tended to decrease at the later phase of batch ,culture while the fat and carbohydrate contents increased. The relat:ve protein amino acid composition did not change and was comparable to that of the algae grown in synthetic medium.
Sources of Materials
Résumé· Spirulina maxima, une espece d'algue bleue, est ciltivée dans un milieu de culture composé de la décharge venant du centre municipal de traitement des eaux usées. Discontinue, la teneur en protéines de la biomasse diminue vers la fin de la période de culture tandis que les teneurs en lipides et en glucides augmentent. La proportion relative des acides aminés des protéines est constante et elle est semblable a celle des échantillons cultivés en milieu synthétique.
Introduction Spirulina maxima is a blue-green alga (cyanophyta) belonging to the family of Oscillatoriaceae. It is a large microscopic, multi-cellular alga which grows in the shallow ponds of high alkalinity and high salinity in the tropical countries. Spirulina maxima has been used as food since ancient times in sorne regions 'Of Africa and Mexico. Nutritional studies carried out by the Institut Francais du Petrole (IFP) showed that Spirlllina is one of the richest protein sources ever found; the protein also contains all essential amino acids in a proportion comparable to 'Other conventional protein sources and to the composition recommended by FAO. Tests carried out on rats and chickens showed that Spirlllina maxima has a relatively good nutritive value and digestability (Clement et al., 1967). As a food source Spirulina maxima also supplies a considerable amount of fat, carbohydrate, vitamins and calories (Clement et al., 1967 and Nakamura, 1970) . Considering the nutritional values of Spirulina maxima and the relative simplicity in cultivation and harvesting of this alga because of its large size compared to other microalgae, several pilot plant studies have been performed in France, Algeria and Mexico to produce Spirulina maxima. The algae were cultivated in synthetic media made up mainly of aqueous mineral salt solutions. The cost of chemicals was about 15% of the total cost (Meyer, 1969), therefore a more economical way of Spirulina maxima culture would be beneficial. In this study, Spirulina maxima was grown in effluents from a secondary waste water treatment planto Some nutritional characteristics of the resulting algal biomass were investigated. Can. Inst. Food SeL Technol. J. Vol. 7, No. 2, 1974
The original cultures of Spirulina maxima were received from the Plant Physiology Institute, University of Gottingen, Germany. The waste water effluent samples were obtained from the Greenway Pollution Control Centre which receives and treats municipal sewage in the community of London, Ontario. The synthetic medium describ~d in the IFP patent (Clement et al., 1966) was used ll1 the present study for comparis'On.
Culture System The batch culture experiments were performed in a 5-gallon bottle containing 10 litres of culture medium, which was sterilized in an autoclave at 2 atm and 120°C for 15 minutes and cooled to room temperature. The cultures were illuminated by four "GROLUX" fluorescent tubes of 1,200 Lux. Aeration and mixing were accomplished by air containing 2% COz, which was passed into the culture at the rate of 0.5 ""m and also by a magnetic stirrer. The culture temperature was maintained at 30 ± 2°C in a "walk-in" incubator. pH was controlled in the range of 8.5 to 10 by adjusting the COz content in airo Approximately 0.5 g dry weight of inoculum was added to the bottle at the starting day. The inoculum was taken from a well-developed stock culture grown under the same conditions.
Analytical 111ethods The algal biomass was collected by filtration using a Whatman glass-fiber filter papel' and it was washed several times by distilled water to remove dissolved salts. The samples were then freeze-dried, ground into fine powders and stored at O°C in a refrigerator for chemical analyses. The moisture and ash contents in the freeze-dried sall1ples were determined by the method described by Jacobs (1965). Total carbohydrates were determined colorimetrically using anthrone reagent (Neish, 1952). The fat content was extracted in chlor'Oform and chloroform-methanol mixture, and deterll1ined gravimetrically (Kosaric, 1969). The nitrogen content of the samples was deterll1ined by the COLEMAN model 29A nitrogen analyzer. This method determines total nitrogen that is composed of protein and non-protein fmction. As most of the determined nitrogen is assumed to belong to protein, the non protein nitrogen was neglected. Under this assumption a factor of 6.25 was used to convert the found nitrogen to crude pr'Otein. 114
For amino acid determination, about 200 mg of tlle dry algal biomass was hydrolyzed by refluxing 500 mI of 5.7N Hel in a nitrogen atmosphere for 24 hours. An aliquot equivalent to approximately 2.8 mg of protein was dried undel' vacuum and rediss'Úlved in 2 1111 of a standard buffer solution containing norleucine. [t was then analyzed by an automatic Technicon T8M1 Amino Acid Analyzer.
Results and Discussion The analytical results of the algal biomass growll in Greenway effluent were compared to those grown in synthetic medium under the same growth conditions (Table 1). Table 1.
Chemical composition of Spirulina maxima biomass l . Synthetic Medium 12 days2
"Protein" Carbohydrate Fat Ash Moisture
63.1 16.6 1.0 13.9 5.0
Greenway Effluent 6 days2 50.5 26.5 4.6 14.0 4.8
10 days2
Amino acid spectra were also recorded for the biomass samples gr'Úwn in synthetic medium and Greenway effluent (Figure 1). Table 2 summarizes the results of essential amino acid analyses. Despite the protein content deviation, the composition of essential amino acids in the Spirulina muximu biomass is similar in different samples and comparable to literature values. All essential amino acids are found in the biomass and their levels in the algal "protein" exceed those recommended by F Aü. Methionine is very close to the recommended value as opposed to almost all other single cell proteins that are deficient in this amino acid. According to this finding, good nutritional quality of the algal protein is expected. However, further nutritional studies to establish biological and toxicological values of the "protein" are required.
28.3 43.2 9.2 14.5 4.7
1 % by weight 2 Age of culture
The protein content of Spintlina mQxima grown in synthetic medium was 63.1% (as calculated from total nitrogen) ; it is comparable to the result reported by the IFP (62-68%). However, the algae grown in Greenway effluent showed a difference in characteristics depending on the age of culture. The analysis of biomass after 6 days of batch culture in Greenway effluent showed that the "protein" content decreased from 63.1% to 50.5% while the fat and carbohydrate contents increased. The effect was more significant ufter 10 days 01' at the stationary phuse of growth where the "protein" content of the algal biomass was only 28.3% and the carbohydrate and fat contents increased as high as 43.2% and 9.2% respectively. The ash and moisture contents were relatively unchanged. A possible explanation of the variation in chem'ical composition of the algae is the nutrient deficiency in Greenway effluent during the batch culture. When the limited amount of nutrients (especially nitrogen compounds) in the medium has been used up by the algae during the active growth phase, a decrease in "protein" was observed; as a consequence, higher amounts of carbohydrate and fat were accumulated in the algal cells. Lewin (1962) also mentioned a decrease in nitrogen content from 8 to 10% of the dry weight to 2% and the in crease in fat content as high as 80% in several species of Chlorellu and Scenedesmus algae during the nitrogen deficiency periods. According to Durant and Jolly (1969), by appropriately modifying the culture conditions, the producer could vary the concentration of protein from 7 to 85%, the concentration of fat from 4 to 86%, and the concentration of carbohydrate from ;) to 38% in Chlorella. This response has been considered as an attractive property by Russian scientists since the composition of algae could theoretically be adjusted to suit the nutritional needs of cosmonauts when the algal systems are used in space programs (Lachance, 1968).
115
Fig. 1.
Table 2. Amino Acids
Amino acid spectra of Spimlina maxima in: A: Synthetic medium B: Greenway effluent after 6 days c: Greenway effluent after 10 days Amino acid analyses of Spirulina maxima "protein"l. Synthetic Medium 12 d ays 2
Ile Leu Lys Phe Met Thr Try Val
Greenway Effluents 6 d ays 2
10 days2
5.80 9.31 5.00 4.03 2.28 4.99
5.87 9.60 5.43 4.44 2.12 4.81
5.71 9.26 4.68 4.45 2.10 5.35
6.92
7.55
6.62
Clement et al. FAü (1967) Combination
6.03 8.02 4.59 4.97 1.37 4.56 1.40 6.49
4.2 4.8 4.2 2.8 2.2 2.8
lA 4.2
1 g/16 g of nitrogen 2 Age of culture - not reported J. Inst. Can. SeL Teehnol. Aliment. Vol. 7, No
2, 1974
Acknowledgements This work was supported by The University of vVestern Ontario Research Council. Analytical assistance from Labatt's Breweries Limited is appreciated.
References Clement, o., Rebeller, M., and Zarrouk, C., 1966. Procédé de culture de'algues en mllleu synthétique. Brevet d'invention no. 88, 103, lre addition au no. 1,458,061, France. Clement, O., Riddey, C., and Menzi, R., 1967. Amino acid composltion and nutritive value of the algae Spirulina maxima. J. Scl. Food Agr. 18:497. Durant, N. W., and Jolly, C., 1969. Oreen algae, Chlorella, as a con· tribution to the food supply of mano Fish. Ind. Res. 5:67.
Cs.n. I:lst. Food ScL Technol. J. Vol. 7, No. 2, 1974
Jacobs, M. B., 1965. The Chemical Analysis of Food and Food Products. 3rd editlon, Van Nostrand Co., Inc., 30. Kosaric, N., 1969. Ph.D. Thesls, The Unlverslty of Western Ontario, London, Canada. Lachance, P. A., 1969. Single cell protein in space systems. Single Cell Pl'otein, R. l. Mateles and S. R. Tannenbaum, ed., The M.I.T. Press, Massachusetts. Lewin, R. .A., 1962. Physiology and Biochemlstry of AIgae. Academlc Press, New York. Meyer, C.. 1969. Etude d'une culture d'algues en vue d'une production i\. grande échelle. Industr. Alim. Agr. 86:1445. Nakamura, H., 1970. The mass production of Spirulina: a hellcal blue-green algae as a new food. Report from the Spirulina Development Commlttee of Japan. Nelsh, A. C., 1952. Analytical Methods for Bacterial Fermentatlon. NRC of Canada, Report No. 46-83, 2nd revision, p. 33. Recelved August 1, 1973
116