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Aerated swine-wastewater treatment with K-carrageenan-immobilized Spirulina maxima
Bioresource Technology 47 (1994) 89-91
Short Communication
Aerated Swine-Wastewater Treatment With K-Carrageenan-Immobilized Spirulina maxima
the organisms. Immobilized living cells have some advantages over suspended cells: they simplify the treatment of liquids, the final separation of cells is unnecessary and cell metabolic activity remains constant for longer periods (Lukavsky, 1986). The aim of this work was to study the depuration efficiency of S. maxima immobilized in K-carrageenan for the tertiary treatment of swine wastes.
Abstract The immobilization of Spirulina maxima for tertiary treatment of diluted aeration-stabilized swine waste was investigated. The technique employed for the immobilization was active entrapment in K-carrageenan. The entrapped cells were used for repeated batch-culture nutrient-removal. The immobilized algae were submitted to a number of repeated cycles for the effluent, seven or 10 days each, depending on the initial nutrient concentration of the swine waste. The ammonium nitrogen and total-phosphorus removal efficiencies were the same at 25% dilution of the swine waste and over 90%. For 50% dilution the removal efficiency for ammonium was over 80% and 90% for total-phosphorus.
METHODS Organism The culture of Spirulina maxima was supplied by Sosa Texcoco Co., Mexico. Inoculum preparation Spirufina cells were cultivated in Fernbach flasks, with 2500 ml of Bourrely's medium (Bourrely, 1948). The culture was sparged with sterile air at room temperature (25 + 3°C), under continuous artificial white light (2000 lux) given by fluorescent lamps. After cell growth, a sample was taken to carry out the immobilization experiments.
Key words: Swine waste, immobilized cells, tertiary treatment, cyanobacteria, K-carrageenan.
Preparation of the swine waste (lfiiguez, 1983) One kilogram of hog manure from a single pig, fed on a balanced commercial diet (Prepartina, Purina, Co., containing protein, amino acids and alfalfa) was suspended in 20 liters of aerated tap water. The mix was mechanically stirred for 20 min. After this time, it was sieved through a 30 mesh Tylor net and settled for 3 h at room temperature. Then it was decanted and the supernatant was fed to a 15 liters reactor magnetically stirred to carry out the stabilization of the swine waste by means of vigorous aeration ( 15 liters air min- 1) for 24-96 h, depending on the initial chemical oxygen demand (COD) of the swine waste. Once no change in COD values was registered over a period of 12 h the stabilization of the effluent was considered as completed (total time 48 h). The treated swine waste was refrigerated at 4°C for 72 h until settled. After this time, the supernatant was ready to be used as a culture medium for the growth experiments. For nutrient-removal experiments, the final effluent was diluted with distilled water to 25 and 50% (v/v) (25 ml swine waste plus 75 ml water for 25% dilution, and so on). As the pH of the swine waste was 7"5, to attain a good growth of Spirulina sodium bicarbonate was
INTRODUCTION The utilization of microalgae for tertiary treatment of different wastes has been described by many authors (Shelef et al., 1969; Benemann et al., 1980; Lukavsky, 1986; de la Noiie & de Pauw, 1988; de la Noiie & Proulx, 1988; Oswald, 1988). The utilization of microalgae for the treatment of swine wastes, in particular, was described by Goh (1986) and for other animal wastes by Lincoln et al. (1977) and Lincoln and Hill (1980). Tsai (1980) showed that aerobically digested swine manure could be utilized as a nutrient medium for Spirulina platensis. Chiu et al. (1980) carried oul studies with S. platensis cultivated on aerobically digested swine manure. Other studies were done by de la Noiie and Proulx (1988) and de la Noiie and Bassbres (1989) utilizing the cyanobacterium Phormidium bohneri immobilized in chitosan and they reported removals of over 90% for organic nitrogen as well as orthophosphates. It was found in all cases that the ammonium nitrogen concentration in the medium was very important for Bioresource Technology 0960-8524/94/S07.00 © 1994 Elsevier Science Limited, England. Printed in Great Britain 89
90
Aerated swine-wastewater treatment
added to the swine waste until a pH of 8.0-9.0 was reached.
Table 1. Aeration-stabilized swine manure characterization at different concentrations
Parameter
Immobilization of cells
The algae were used while in their logarithmic growth phase (72 h). Samples of 2 liters of culture were vacuum filtered (Millipore membrane HAWP 0"45 /~m); then the cells were washed twice with 5% KCI solution. The resulting filtered cells (2 g cell, wet basis) were resuspended in 50 ml of deionized water and mixed with 150 ml of 1"5% (w/v) K-carrageenan (Type III, Sigma Chemical Co., St Louis, MS) at 38°C. The gel had been previously dissolved in hot distilled water and 0-25% (w/v) of karaya gum (Food-grade) added. The cells-gel mixture obtained was added drop by drop via a syringe needle and a peristaltic pump (Cole Parmer Mod. 701400) to a magnetically stirred 0"3 M KCI solution, at room temperature (25+3°C). The resulting carrageenan-gel beads (mean diameter 3 mm) were soaked in the KCI solution for 24 h, to increase stability. They were then washed with distilled water and stored under refrigeration in Bourrely's medium until used. Beads showed enough strength and good mechanical properties to allow their incubation in a continuously air-agitated system. The optimal cell concentration for immobilization was 2 g of filtered cells (wet basis) per 200 ml total volume. Cell concentrations up to 3 g of filtered cells caused problems for bead formation. Bioreactor
A glass airlift reactor, with two external loops and a total volume of 250 ml operating with 100 ml liquid, was used. An airflow (0"5 vvm) was pumped into the reactor via the bottom to keep the beads suspended. The reactor was packed with 80 ml of beads, and different dilutions of the swine waste (25 and 50%) were added. Batch cultures were conducted by submitting the immobilized algae successively to a number of renewal cycles for the effluent of 7 days each for 25% dilution and 10 days each for 50% dilution and sampling every 24 h for ammonium and phosphorus analysis. Maximum nutrient removal was used as a criterion to set the lengths of the cycles. When this was attained, the effluent from the reactor was drained off and more untreated effluent added, starting in this way a new treatment cycle for the effluent, but using the same immobilized cells. Analysis
Ammonium (N--NH4) was quantified by Nessler (APHA, 1985) and total phosphorus (total-P) by Fiske-Subbarow (1980) methods. The presence of algae in the supernatant was estimated by chlorophyll determination (APHA, 1985). Results
The composition and characteristics of the swine waste are shown in Table 1. At 25% dilution of the swine waste, the maximum N--NH~- and total-P removal was
Total phosphorus (mg/liter) Ammonia nitrogen (mg/liter) COD (mg/liter) BOD (mg/liter) pH a
Swine waste concentration 100%
50%
25%
80 120 400 520 9
40 60 215 260 9
23 28 110 130 9
"Adjusted.
reached in seven days, when N - - N H 4 concentration had been reduced to 2 mg/liter (92% removal) and total-P to 2 mg/liter (91% removal). At 25% dilution, for both nutrients the immobilized Spirulina accomplished 13 renewal cycles for the effluent (over 91 days), with a steadily increasing loss of removal efficiency for ammonium (from 92% for the first cycle to 75% for the last). For total-phosphorus removal, efficiency fell little over the first nine cycles, but then decreased rapidly (from 91% for the first cycle to 87% for the ninth and 80% for the last). Maximum nutrient-removal percentages at 50% dilution was attained in 10 days. When working at 50% dilution, there were 10 renewal cycles for the effluent. For the first cycle, the maximum ammonium removal was 83% and after 10 cycles (over 100 days) it was 70%, a 13% loss in removal efficiency for the immobilized system. For total-P the loss in efficiency was 41% (93% for the first renewal cycle to 52% for the last). In each case efficiency declined fairly steadily. The carrageenan beads maintained their natural physiological characteristics in both sets of experiments. Carrageenan-immobilized cells of Spirulina maxima could be used for the tertiary treatment of aerated swine wastes, although these might have to be diluted. ACKNOWLEDGEMENT The authors wish to express their gratitude to C O N A C Y T of Mexico for their support in this work included in the Collaborating Project between Mexico and Cuba, 1990. REFERENCES
APHA (1985). Standard Methods for the Examination of Waters and Wastewaters (16th edn). American Public Health Association, Washington, DC. Benemann, J. R., Koopman, B. L., Weissman, J. C., Eisenher, D. M. & Oswald, W. J. (1980). Cultivation on sewage of microalgae harvestable by microstrainers. Progress Report San. Engn. Res. Lab. Univ. California, Berkeley, CA. Bourrely, P. (1948). L'Algotheque du Laboratoire de Cryptogamie de Musrum National d'Histoire Naturelle, Paris, 14 pp. In Arch. Hydrobiol. Suppl. 39, Algal Studies, 2/3 (1970) 86-126.
Aerated swine- wastewater treatment Chiu, R. J., Liu, H. I., Chen, C. Ci., Chi, Y. C., Shao, H., Soong, P. & Has, P. (1980). The cultivation of Spirulina platensis on fermented swine manure. In Proc. Int. Symp. on Biogas, Microalgae and Livestock Wastes, Sept. 15-19, Taipei, Taiwan, pp. 435-6. de la Noiie, J. & de Pauw, N. (1988). The potential of microalgal biotechnology: A review of production and uses of microalgae. Biotechnol Adv., 6, 725-70. de la Noiie, J. & Proulx, D. (1988). Biological tertiary treatment of urban waste water with chitosan-immobilized Phormidium. Appl. Microbiol. Biotechnol., 29, 292-7. de la Noiie, J. & Basstres, A. (1989). Biotreatment of anaerobically digested swine manure with microalgae. Biol. Wastes, 29, 17-31. Fiske-Subbarow. (1980). Determinaci6n de f6sforo total. In
Manual de Procedimientos de Bioquirnica Clinica ENCB1PN, Mtxico D. F. Instituto Polittcnico Nacional. Escuela Nacional de Ciencias Bi61ogicas del Instituto Polittonico Nacional. pp. 38-90. Goh, A. (1986). Production of microalgae using pig waste as a substrate. In Algal Biomass Technologies. An Interdisciplinary Perspective, ed. W. R. Barclay & R. P. Mcintosh. J. Cramer, Berlin, pp. 235-44. Ifiiguez, C. G. (1983). Manejo y aprovechamiento integral del estitrcol de cerdo. Congreso Nacional AMVEC-83, Puerto Vallarta, Julio 1983, Mtxico. Lincoln, E. P., Hill. D. T. & Nordstedt, R. A. (1977). Microalgae as means of recycling animal wastes. J. Am. Soc. Agr. Eng., 5026-31. Lincoln, E. P. & Hill, D. T. (1980). An integrated microalgae system. In Algae Biomass, ed. G. Shelef & C. J. Soeder. Elsevier, Amsterdam, pp. 229-44.
91
Lukavsky, J. (1986). Methabolic activity and cell structure of immobilized algae cells. Arch. Hydrobiol. Suppl. 73, 2, Algological Studies, 43, 261-79. Oswald, W. J. (1988). Microalgae and waste water treatment. In Microalgal Biotechnology, ed. A. Borowitzka & L. Borowitzka. Cambridge University Press, New York, pp. 306-28. Shelef, G., Oswald, W. J. & Golueke, C. G. (1969). The continuous culture of algal biomass on wastes. In Continuous Cultivation of Microorganisms, ed. I. Malek. Academy, Prague, pp. 601-29. Tsai Pi-Hsin. (1980). Mass culture and utilization of Spirulina platensis grown on fermented hog manure. In Proc.
Int. Symp. on Biogas, Microalgae and Livestock Wastes, Sept. 15-19, Taipei,Taiwan. pp. 399-414.
R. O. Cafiizares, L. Rivas, C. Montes, A. R. Dominguez Centro de Investigaci6n y de Estudios Avanzados del Instituto Politecnico Nacional (CINVESTAV-IPN), PO Box 14-740, 7300 Mexico D. E
L. Travieso & F. Benitez Centro Nacional de Investigaciones Cientificas (CNIC), PO Box 6990, L a Habana, Cuba (Received 17 D e c e m b e r 1992; revised version received 8 May 1993; accepted 12 May 1993)
Short Communication
Aerated Swine-Wastewater Treatment With K-Carrageenan-Immobilized Spirulina maxima
the organisms. Immobilized living cells have some advantages over suspended cells: they simplify the treatment of liquids, the final separation of cells is unnecessary and cell metabolic activity remains constant for longer periods (Lukavsky, 1986). The aim of this work was to study the depuration efficiency of S. maxima immobilized in K-carrageenan for the tertiary treatment of swine wastes.
Abstract The immobilization of Spirulina maxima for tertiary treatment of diluted aeration-stabilized swine waste was investigated. The technique employed for the immobilization was active entrapment in K-carrageenan. The entrapped cells were used for repeated batch-culture nutrient-removal. The immobilized algae were submitted to a number of repeated cycles for the effluent, seven or 10 days each, depending on the initial nutrient concentration of the swine waste. The ammonium nitrogen and total-phosphorus removal efficiencies were the same at 25% dilution of the swine waste and over 90%. For 50% dilution the removal efficiency for ammonium was over 80% and 90% for total-phosphorus.
METHODS Organism The culture of Spirulina maxima was supplied by Sosa Texcoco Co., Mexico. Inoculum preparation Spirufina cells were cultivated in Fernbach flasks, with 2500 ml of Bourrely's medium (Bourrely, 1948). The culture was sparged with sterile air at room temperature (25 + 3°C), under continuous artificial white light (2000 lux) given by fluorescent lamps. After cell growth, a sample was taken to carry out the immobilization experiments.
Key words: Swine waste, immobilized cells, tertiary treatment, cyanobacteria, K-carrageenan.
Preparation of the swine waste (lfiiguez, 1983) One kilogram of hog manure from a single pig, fed on a balanced commercial diet (Prepartina, Purina, Co., containing protein, amino acids and alfalfa) was suspended in 20 liters of aerated tap water. The mix was mechanically stirred for 20 min. After this time, it was sieved through a 30 mesh Tylor net and settled for 3 h at room temperature. Then it was decanted and the supernatant was fed to a 15 liters reactor magnetically stirred to carry out the stabilization of the swine waste by means of vigorous aeration ( 15 liters air min- 1) for 24-96 h, depending on the initial chemical oxygen demand (COD) of the swine waste. Once no change in COD values was registered over a period of 12 h the stabilization of the effluent was considered as completed (total time 48 h). The treated swine waste was refrigerated at 4°C for 72 h until settled. After this time, the supernatant was ready to be used as a culture medium for the growth experiments. For nutrient-removal experiments, the final effluent was diluted with distilled water to 25 and 50% (v/v) (25 ml swine waste plus 75 ml water for 25% dilution, and so on). As the pH of the swine waste was 7"5, to attain a good growth of Spirulina sodium bicarbonate was
INTRODUCTION The utilization of microalgae for tertiary treatment of different wastes has been described by many authors (Shelef et al., 1969; Benemann et al., 1980; Lukavsky, 1986; de la Noiie & de Pauw, 1988; de la Noiie & Proulx, 1988; Oswald, 1988). The utilization of microalgae for the treatment of swine wastes, in particular, was described by Goh (1986) and for other animal wastes by Lincoln et al. (1977) and Lincoln and Hill (1980). Tsai (1980) showed that aerobically digested swine manure could be utilized as a nutrient medium for Spirulina platensis. Chiu et al. (1980) carried oul studies with S. platensis cultivated on aerobically digested swine manure. Other studies were done by de la Noiie and Proulx (1988) and de la Noiie and Bassbres (1989) utilizing the cyanobacterium Phormidium bohneri immobilized in chitosan and they reported removals of over 90% for organic nitrogen as well as orthophosphates. It was found in all cases that the ammonium nitrogen concentration in the medium was very important for Bioresource Technology 0960-8524/94/S07.00 © 1994 Elsevier Science Limited, England. Printed in Great Britain 89
90
Aerated swine-wastewater treatment
added to the swine waste until a pH of 8.0-9.0 was reached.
Table 1. Aeration-stabilized swine manure characterization at different concentrations
Parameter
Immobilization of cells
The algae were used while in their logarithmic growth phase (72 h). Samples of 2 liters of culture were vacuum filtered (Millipore membrane HAWP 0"45 /~m); then the cells were washed twice with 5% KCI solution. The resulting filtered cells (2 g cell, wet basis) were resuspended in 50 ml of deionized water and mixed with 150 ml of 1"5% (w/v) K-carrageenan (Type III, Sigma Chemical Co., St Louis, MS) at 38°C. The gel had been previously dissolved in hot distilled water and 0-25% (w/v) of karaya gum (Food-grade) added. The cells-gel mixture obtained was added drop by drop via a syringe needle and a peristaltic pump (Cole Parmer Mod. 701400) to a magnetically stirred 0"3 M KCI solution, at room temperature (25+3°C). The resulting carrageenan-gel beads (mean diameter 3 mm) were soaked in the KCI solution for 24 h, to increase stability. They were then washed with distilled water and stored under refrigeration in Bourrely's medium until used. Beads showed enough strength and good mechanical properties to allow their incubation in a continuously air-agitated system. The optimal cell concentration for immobilization was 2 g of filtered cells (wet basis) per 200 ml total volume. Cell concentrations up to 3 g of filtered cells caused problems for bead formation. Bioreactor
A glass airlift reactor, with two external loops and a total volume of 250 ml operating with 100 ml liquid, was used. An airflow (0"5 vvm) was pumped into the reactor via the bottom to keep the beads suspended. The reactor was packed with 80 ml of beads, and different dilutions of the swine waste (25 and 50%) were added. Batch cultures were conducted by submitting the immobilized algae successively to a number of renewal cycles for the effluent of 7 days each for 25% dilution and 10 days each for 50% dilution and sampling every 24 h for ammonium and phosphorus analysis. Maximum nutrient removal was used as a criterion to set the lengths of the cycles. When this was attained, the effluent from the reactor was drained off and more untreated effluent added, starting in this way a new treatment cycle for the effluent, but using the same immobilized cells. Analysis
Ammonium (N--NH4) was quantified by Nessler (APHA, 1985) and total phosphorus (total-P) by Fiske-Subbarow (1980) methods. The presence of algae in the supernatant was estimated by chlorophyll determination (APHA, 1985). Results
The composition and characteristics of the swine waste are shown in Table 1. At 25% dilution of the swine waste, the maximum N--NH~- and total-P removal was
Total phosphorus (mg/liter) Ammonia nitrogen (mg/liter) COD (mg/liter) BOD (mg/liter) pH a
Swine waste concentration 100%
50%
25%
80 120 400 520 9
40 60 215 260 9
23 28 110 130 9
"Adjusted.
reached in seven days, when N - - N H 4 concentration had been reduced to 2 mg/liter (92% removal) and total-P to 2 mg/liter (91% removal). At 25% dilution, for both nutrients the immobilized Spirulina accomplished 13 renewal cycles for the effluent (over 91 days), with a steadily increasing loss of removal efficiency for ammonium (from 92% for the first cycle to 75% for the last). For total-phosphorus removal, efficiency fell little over the first nine cycles, but then decreased rapidly (from 91% for the first cycle to 87% for the ninth and 80% for the last). Maximum nutrient-removal percentages at 50% dilution was attained in 10 days. When working at 50% dilution, there were 10 renewal cycles for the effluent. For the first cycle, the maximum ammonium removal was 83% and after 10 cycles (over 100 days) it was 70%, a 13% loss in removal efficiency for the immobilized system. For total-P the loss in efficiency was 41% (93% for the first renewal cycle to 52% for the last). In each case efficiency declined fairly steadily. The carrageenan beads maintained their natural physiological characteristics in both sets of experiments. Carrageenan-immobilized cells of Spirulina maxima could be used for the tertiary treatment of aerated swine wastes, although these might have to be diluted. ACKNOWLEDGEMENT The authors wish to express their gratitude to C O N A C Y T of Mexico for their support in this work included in the Collaborating Project between Mexico and Cuba, 1990. REFERENCES
APHA (1985). Standard Methods for the Examination of Waters and Wastewaters (16th edn). American Public Health Association, Washington, DC. Benemann, J. R., Koopman, B. L., Weissman, J. C., Eisenher, D. M. & Oswald, W. J. (1980). Cultivation on sewage of microalgae harvestable by microstrainers. Progress Report San. Engn. Res. Lab. Univ. California, Berkeley, CA. Bourrely, P. (1948). L'Algotheque du Laboratoire de Cryptogamie de Musrum National d'Histoire Naturelle, Paris, 14 pp. In Arch. Hydrobiol. Suppl. 39, Algal Studies, 2/3 (1970) 86-126.
Aerated swine- wastewater treatment Chiu, R. J., Liu, H. I., Chen, C. Ci., Chi, Y. C., Shao, H., Soong, P. & Has, P. (1980). The cultivation of Spirulina platensis on fermented swine manure. In Proc. Int. Symp. on Biogas, Microalgae and Livestock Wastes, Sept. 15-19, Taipei, Taiwan, pp. 435-6. de la Noiie, J. & de Pauw, N. (1988). The potential of microalgal biotechnology: A review of production and uses of microalgae. Biotechnol Adv., 6, 725-70. de la Noiie, J. & Proulx, D. (1988). Biological tertiary treatment of urban waste water with chitosan-immobilized Phormidium. Appl. Microbiol. Biotechnol., 29, 292-7. de la Noiie, J. & Basstres, A. (1989). Biotreatment of anaerobically digested swine manure with microalgae. Biol. Wastes, 29, 17-31. Fiske-Subbarow. (1980). Determinaci6n de f6sforo total. In
Manual de Procedimientos de Bioquirnica Clinica ENCB1PN, Mtxico D. F. Instituto Polittcnico Nacional. Escuela Nacional de Ciencias Bi61ogicas del Instituto Polittonico Nacional. pp. 38-90. Goh, A. (1986). Production of microalgae using pig waste as a substrate. In Algal Biomass Technologies. An Interdisciplinary Perspective, ed. W. R. Barclay & R. P. Mcintosh. J. Cramer, Berlin, pp. 235-44. Ifiiguez, C. G. (1983). Manejo y aprovechamiento integral del estitrcol de cerdo. Congreso Nacional AMVEC-83, Puerto Vallarta, Julio 1983, Mtxico. Lincoln, E. P., Hill. D. T. & Nordstedt, R. A. (1977). Microalgae as means of recycling animal wastes. J. Am. Soc. Agr. Eng., 5026-31. Lincoln, E. P. & Hill, D. T. (1980). An integrated microalgae system. In Algae Biomass, ed. G. Shelef & C. J. Soeder. Elsevier, Amsterdam, pp. 229-44.
91
Lukavsky, J. (1986). Methabolic activity and cell structure of immobilized algae cells. Arch. Hydrobiol. Suppl. 73, 2, Algological Studies, 43, 261-79. Oswald, W. J. (1988). Microalgae and waste water treatment. In Microalgal Biotechnology, ed. A. Borowitzka & L. Borowitzka. Cambridge University Press, New York, pp. 306-28. Shelef, G., Oswald, W. J. & Golueke, C. G. (1969). The continuous culture of algal biomass on wastes. In Continuous Cultivation of Microorganisms, ed. I. Malek. Academy, Prague, pp. 601-29. Tsai Pi-Hsin. (1980). Mass culture and utilization of Spirulina platensis grown on fermented hog manure. In Proc.
Int. Symp. on Biogas, Microalgae and Livestock Wastes, Sept. 15-19, Taipei,Taiwan. pp. 399-414.
R. O. Cafiizares, L. Rivas, C. Montes, A. R. Dominguez Centro de Investigaci6n y de Estudios Avanzados del Instituto Politecnico Nacional (CINVESTAV-IPN), PO Box 14-740, 7300 Mexico D. E
L. Travieso & F. Benitez Centro Nacional de Investigaciones Cientificas (CNIC), PO Box 6990, L a Habana, Cuba (Received 17 D e c e m b e r 1992; revised version received 8 May 1993; accepted 12 May 1993)