Chemical composition of cyanobacteria grown in diluted, aerated swine wastewater

Bioresource Technology 51 (1995) 111-116 © 1995 Elsevier Science Limited Printed in Great Britain. All rights reserved 0960-8524/95/$9.50 0960-0524(94)00099-9

ELSEVIER

CHEMICAL COMPOSITION OF CYANOBACTERIA GROWN IN DILUTED, AERATED SWINE WASTEWATER R. O. Cafiizares-Villanueva,* A. R. Dolrdnguez, M. S. Cruz & E. R/os-Leal Departamento de Biotecnologia y Bioingenieria, Centro de Investigaci6n y Estudios avanzados del lnstituto Polit~cnico Nacional, Apartado Postal 14-740, M~xico 07000, M&ico (Received 6 August 1994; revised version received 23 September 1994; accepted 27 September 1994)

treated effluents that do not fulfill the regulatory biochemical requirements are allowed to pour into waterbodies. However, swine waste, in particular, can be used as a substrate for microalgae and cyanobacterial growth after a stabilization treatment by aeration or anaerobic digestion has been applied. This makes it possible to obtain a final effluent of an acceptable quality to be used for various purposes. Microalgae grown on swine wastes can be used as dietary supplements for animals (Chung et al., 1978; Lincoln & Hill, 1980; Soong, 1980) in aquaculture for fish, mollusc and crustacean feeding (De la Noiie et al., 1986; De Pauw & Van Vaerenbergh, 1983) as well as for fertilizers, energy sources and fine chemicals production (Benneman et al., 1977). Some studies on the nutritional quality of different species of microalgae, including Spirulina, have demonstrated their usefulness as components in animal and human diets (De la N o i i e & De Pauw, 1988; Pabst, 1978; Gross et al., 1978; Becker, 1980; Ciferri, 1983). Feeding experiments with rats, mice, poultry, pigs, sheep and carp have demonstrated that microalgal meals produced from various strains or species of Chlorella, Scenedesmus and Spirulina are valuable protein sources lacking any acute toxicity (Becker, 1980; 1986). The aim of this work was to determine the chemical compositions of Phormidium sp. and Spirulina maxima grown on aeration-stabilized swine waste (ASSW) and to compare them with those of algae grown on synthetic mineral media (controls). The final purpose was to evaluate the chemical quality of these biomasses for use as dietary supplements for animals.

Abstract The chemical composition of Spirulina maxima and Phormidium sp. biomasses grown on pretreated and diluted swine wastewater was determined. Analyses were carried out on lyophilized samples and compared with data from mineral media (controls). Analyses of Phormidium grown on aeration-stabilized wastewater (ASSW) were: protein ( N x 6"25) 62%, lipids 11%, carbohydrates (calculated by difference) 16%. For Spirulina in the same effluent, data were: protein 36%, lipids 6% and carbohydrates 44%. No crude fiber was found in any of the samples. The fatty acid profiles of both biomasses showed important differences when compared to controls. The biomasses contained all the essential amino acids. The Spirulina biomass had a significantly higher content of pyridoxine, riboflavin and pantothenic and nicotinic acids than Phormidium when grown on ASSW, but in general the vitamin content of both biomasses was practically the same as their respective controls. The results suggest that Phormidium and Spirulina biomasses could be used as dietary supplements in animal feed, but further studies are needed to determine the nutritional value of the product.

Key words: Cyanobacteria, aeration-stabilized swine wastewater, chemical composition, secondary effluent. INTRODUCTION

The waste products of animal husbandry constitute a permanent source of pollution. Manure is not appropriately recycled on farms, and it is released into the environment, provoking serious pollution problems. Manure is not used in Mexico. In Mexico crude livestock wastes are employed on farmlands as inexpensive fertilizer and an irrigation source. Swine waste is rich in inorganic phosphorus and nitrogen, which usually settle in waters, creating nutrient accumulation and generating eutrophic systems. Aerobic digestion is a process for waste treatment in which, as in other systems, excessive volumes of

METHODS Swine wastewater preparation The wastewater was produced according to CafiizaresVillanueva and Dorrdnguez (1993). One kilogram of manure from a single pig was suspended in 201 of aerated tap water. After mixing and settling, the supernatant was stabilized by vigorous aeration. The final liquid was designated ASSW.

*Author to whom correspondence should be addressed. 111

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R. O. Cafiizares-Villanueva, A. R. Dominguez, M. S. Cruz, E. Rios-Leal

Organic medium, called ASSW (Table 1), was used for the culture of both cyanobacteria. Chemical characterization and dilution of the ASSW to 50% in distilled water were performed prior to the growth experiments.

Culture media Zarrouk medium (Zarrouk, 1966) and Dauta medium modified by Lemus (1991) were chosen as control media for the cultivation and maintenance of the strains. Zarrouk medium had the following salts concentration (g/l) NaHCO3, 16.8; K2HPO4, 0"5; NaNO 3, 2.5; K2SO4, 1"0; NaC1, 1"0; MgSOa.7H20, 0"2; CaC12, 0-04; FeSOa.7H20, 0"01; Na2-EDTA.2H20, 0"08; microelements solution 1 ml/l. The microelements solution consisted of the following salts (g/l): H3BO3, 2"86; MnC12.4H20, 1"81; ZnSO4.7H20, 0-222; C u S O 4 . 5H20, 0"08. Dauta medium had the following salts concentration (g/l): NaHCO 3, 25; CaC12 . 2H20, 25; MgSO 4 .7H20, 25; Na2CO3, 5; Na2-EDTA. 2H20, 1.1; KNO3.7H20, 200; K2HPO4, 25; FeSO4.7H20 , 1; microelements solution 1 ml/l. The microelements solution consisted of the following salts (g/l): H3BO3, 2"85; MnC12.4H20, 1.81; ZnSO4.7H20, 0-222; CuSO4.5H20, 0"08; Co(NO3)2.6H20, 0"044; MoO3Na, 0"015. The cyanobacteria were also cultivated in ASSW at 50% dilution in tap water.

Inoeulum preparation Spirulina maxima. The inoculum was prepared under sterile conditions. Suspensions from growth on Zarrouk slants (Zarrouk, 1977) were used to inoculate 250 ml of the same culture medium in 500 ml Erlenmeyer flasks. After incubation at room temperature (25 + 3°C) under aeration (0"5 vvm) and continuous illumination (ca. 1500 lux) over 48 h, aliquots of 200

Table 1. Aerobically-stabilized swine wastewater characterization

Orthophosphates Total-phosphorus Nitrates Ammonium COD (soluble) TS TFS TVS TSS FSS VSS

Range

Mean values

mg/l 2.27-5"7 24.0-49-4 3.25-6.1 42.43 - 125 "0 343.6-840-0 1.48-1"53 0.72-0.74 0.76-0"80 0-084-0"12 0"022-0"03 0"062-0"088

mg/1 3.97 30.18 4.67 83 "4 592-83 1"51 0"73 0-78 0-102 0"26 0'075

COD -- Chemical oxygen demand; TS -- total solids; TFS -total fixed solids; TVS -- total volatile solids; TSS -- total suspended solids; FSS -- fixed suspended solids; VSS -volatile suspended solids.

ml were transferred to Fernbach flasks with 2 1of liquid Zarrouk medium. Phormidium sp. This was cultured under sterile conditions in 500 ml Erlenmeyer flasks with 1.00 ml of mineral Dauta medium containing 1 g/l of FeSO4 (Lemus, 1991). Incubation was carried out at room temperature (25 +3°C) with continuous illumination (2000 lux) over 96 h, with manual agitation once a day. Algal biomass Spirulina maxima. Pyrex carboys of 20 1 capacitywith 15 1 culture medium (Zarrouk or ASSW) were inoculated with 1500 ml Spirulina culture growing exponentially. After incubation as described above, the carboys were placed on a mobile chamber surrounded by fluorescent lamps (20 W each; 20 cm from the carboys) including the bottom of the carboys. At the top of the chamber there was an air connection to bubble filtered air into the carboys. The biomass was collected by vacuum filtration and desiccated by lyophilization. The dry material was used for chemical analysis. Phormidium sp. An oval, open, carrousel-type reactor, made of glass fiber and of 30 1 total capacity containing 28 1 Dauta or ASSW media was used to grow Phormidium (Cafiizares-Villanueva et al., 1994). The reactor was inoculated (10% v/v) and incubated under continuous illumination (3000 lux) by fluorescent lamps placed at the top of the reactor (30 cm from the culture), with a 3-paddle propeller agitation system, at room temperature (25 + 3°C). Bi~nass was collected at the end of the exponential growth phase by sedimentation and dried by lyophilization. Ttie dry material was used for chemical analysis. Analytical determinations Protein, ash and lipids were evaluated according to Official Methods (AOAC, 1984). Amino acids-were determined in a Beckman 118 CL autoanalyzer after acid digestion of the samples.

Fatty acid determination The lipid extract was evaporated to dryness and saponified by boiling for 2 h in 20 ml of 2 M sodium hydroxide solution in 50% ethanol. The unsaponified fraction was extracted with diethyl ether and acidified to pH 2 with hydrochloric acid, and fatty acids were extracted with chloroform. Methyl esters of fatty acids were prepared with a 14% boron trifluoride solution in methanol. Methyl esters of fatty acids were e/ctracted with hexane and were analyzed using a Hewlett Packard 5710-A instrument with ionization flame detector in FFAP 15% column ( 3 m × 2 . 4 m m ID). For-the separation of methyl esters, oven temperature was programmed from 200°C to 230°C at 4°C/rain. Quantification was performed using a Hewlett Packard 3392 integrator and the relative composition was -determined. For identification, the samples were compared with fatty acid methyl ester standards. The analyses were done in triplicate.

Cyanobacteria grown in diluted swine wastewater Water-soluble vitamin determination A 0"5 g sample of dried biomass was placed in a flask containing 20 ml of 1 N H C L and autoclaved at 121°C for 15 min. After cooling to room temperature, the pH of the sample was adjusted to 4.5 by adding 2 N sodium acetate. Following the addition of 5 ml 5% aqueous taka-diastase, the sample was incubated for 3 h at 48°C and-depr0teinized by adding 1 ml 50% trichloroacetic acid and heating in a steam bath for 15 min. The pH was adjusted to 3.5 and the sample was brought to 5 ml with distilled water, filtered through Whatman No. 41 filter paper and finally passed through an activated C~8 Sep-PAK cartridge to remove contaminating substances. The cartridge was subjected to subsequent washes of 2 fnl 0"01 M phosphate buffer pH 7"0 and 2 ml 5% methanol-95% 0"01 M phosphate buffer. Vitamins were eluted from the cartridge with 4 ml of 50% aqueous methanol into 5 ml volumetric flasks, brought to volume with 50% methanol. The sample was analyzed on a Tracor 970 HPLC equipped with a variable wavelength UV detector. Separation of the vitamins was accomplished in a Chromanetics RP18 250 x 4.6 mm, 5 micror/,-isocratically with 30% methanol-70% 0"01 M phosphate buffer pH 4"5 flowing at a rate of 1 ml/min at 28°C. A guard column packed with supelcosil LC~8 was installed in front of the analytical column. Detection was performed at 210 nm for B12 and pantothenic acid, 278 nm for folic acid, 272 nm for riboflavin, 234 nm for thiamin and 218 nm for nicotinic acid. Data were quantified using a Hewlett Packard 3392 integrator. Calibration curves were constructed using standard solu_tions of vitamins. By plotting each peak area against concentration, a linear range was determined. Recover), of vitamins added to the sample before autoclaving was 85.6-96.0%, except for pantothenic acid at 78"6-82-4%. Analyses were performed in triplicate.

Bacteriological methods Plate count, total and fecal coliform bacteria, facultative anaerobes and filamentous fungi and yeasts were evaluated according to Standard Methods for the Examination of Water and Wastewater and to Com-

pendium of Methods for the Microbiological Examination of Foods; both from A P H A ( 1970, 1976). Plate count Triplicate plates were prepared for each biomass sample. Decimal dilution and pour-plate procedure were applied using plate-count agar medium. Inoculated plates were incubated at 35°C for 48 h and colony counts were done at 24 and 48 h.

Total and fecal coliform bacteria The most probable number (MPN) method was used with presumptive test in lactose broth and confirmative test in brilliant green bile broth. For total coliform bacteria, test incubation was done at 35°C for 48 h, while

113

for fecal coliform bacteria the test was at 44"5°C for 48 h.

Facultafive anaerobes Decimal dilution and pour-plate methods were applied using brain heart infusion agar medium. The anaerobic environment was established with the gas pack BBL system. Incubation was done in anaerobic jars at 35°C for 48 h and colony counts were done at 24 and 48 h.

Filamentous fungi and yeasts Decimal dilution and pour-plate methods were applied using potato dextrose agar. Inoculated plates were incubated at 28°C for 72 h and colony counts were done at 24, 48 and 72 h. RESULTS AND DISCUSSION Dilution of the effluent was necessary to reduce the toxic effect of the high ammonium concentration (Pouliot et al., 1984; Konig et al., 1987; De la Noiie & Bassdres, 1989) on the development of Phormidium sp. and to decrease the color of the wastewater which could cause light limitation problems for cyanobacterial growth. The chemical composition of Phormidium and Spirulina grown in ASSW-50% and in their respective synthetic media, as well as the total concentration of the biomass obtained in each case, appear in Table 2. The protein and lipid contents and biomass were highest in Phormidium grown in ASSW-50% and greater, except for biomass, than those of Spirulina grown in the two media studied. Table 3 shows data on the fatty acid content of the two cyanobacteria grown in swine wastewater. The difference in fatty acid content in the two cyanobacterial biomasses was important. The concentration of unsaturated 18-20 carbon fatty acids, particularly palmitoleic and oleic, was highest in the Phormidium biomass grown in ASSW-50%. The same was true for myristic and myristoleic acids, although for Spimlina the fatty acid levels were generally higher in the synthetic medium. T h e pattern of lipid biosynthesis in cyanobacteria is little affected by environmental factors since these microorganisms have intrinsic variability in their fatty acid composition (Materassi et al., 1980). As far as amino acids were concerned (Table 4), their concentration Was higher i n Spirulina than in Phormidium when both were grown in ASSW-50%, except for methionine. However, the concentration of amino acids was still higher in biomasses grown in synthetic media. The vitamin content of the biomass of Phormidium did not vary with the type of culture medium, except in the case of riboflavin, which was higher in algae from synthetic medium than from ASSW-50%. The same was true of Spirulina. However, when the biomasses grown in ASSW-50% were compared, the vitamin content of Spimlina was generally greater than that of

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R. O. Cafiizares-Villanueva, A. R. Dominguez, M. S. Cruz, E. Rios-Leal

Table 2. Chemical composition of Phormidium sp. and Spirulina maxima biomasses grown on swine waste and synthetic media (% dry weight)

Phormidium sp.

Component

Spirulina maxima

Synthetic medium: Dauta

ASSW 50%

Synthetic medium: Zarrouk

ASSW 50%

53.4 9.4 27-5 0.0 9.7 145.0

62.0 11.0 16.0 0"0 11.0 248.0

45.5 2.5 42.5 0"0 9"5 132.0

36-00 6-00 44-00 0-02 14.00 616-0

Protein Lipids Carbohydrates a Crude fiber Ash Biomass (dry basis mg/1) "Calculated by difference. Results are averages of three experiments. ASSW -- Aeration-stabilized swine waste.

Table 3. Fatty acid composition in cyanobacterial biomasses grown on synthetic and swine-waste media

Phormidium sp.

Fatty acid (gO)

Spirulina maxima

Synthetic medium: Dauta

ASSW 50%

Synthetic medium: Zarrouk

ASSW 50%

4.08 2'49 3'55 1.13 28.67 4.27 12"98 5"95 1"10 0.45 2-64

4.25 1"52 7.63 3.76 21.20 11.00 12'76 11'32 1'40 0.54 2.14

3.88 0"52 4.70 1"14 27.10 9.63 5.44 6"12 0.22 0.14 0.62

4.10 0-46 4-02 0-74 27.96 5.29 5"20 3.49 0"10 0-08 0-44

3"31

1.77

2-33

4-12

2.66

2"58

1.57

1.20

Capric acid (C10) Lauric acid (C12) Myristic acid (C14) Myristoleic acid (C14:1 ) Palmitic acid (C16) Palmitoleic acid (C16:1) Stearic acid (C 18) Oleic acid (C18:1) Linoleic acid (C 18 : 2) Linolenic acid (C18:3) Gadoleic acid (C20:1 ) Sat./unsat. ratio Linoleic acid/ linoleic acid ratio Results are averages of three determinations. ASSW -- Aeration-stabilized swine waste.

Table 4. Aminoacid content in cyanobacterial biomasses grown on synthetic and swine waste media

Phormidium sp.

Amino acid (g/100 g protein)

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine lsoleucine Leucine Tyrosine Phenylalanine

Spirulina maxima

Synthetic medium: Dauta

ASSW 50%

Synthetic medium: Zarrouk

ASSW 50%

3.07 1'39 3.22 4.66 4.15 3.67 8"01 2.43 3"98 5.88 3.64 0-74 2.77 5"59 2.63 3"15

1.80 0-85 2.80 4.79 2-62 2.09 5-01 1.14 4.40 3.85 2.14 0.64 2.30 4.12 2.64 2.25

5.10 2-73 7.62 7-04 3-90 4.02 10-93 3"18 3.87 6.08 4.10 0.73 5-20 10-80 3.70 4.60

4.10 2.30 5.50 7.38 4.02 3.97 12"36 3"97 4.05 5.97 4.10 0.53 3"30 6.70 3.20 3.40

Results are averages of two determinations. ASSW -- Aeration-stabilized swine waste.

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Cyanobacteria grown in diluted swine wastewater Table 5. Vitamin content in cyanobacterial biomasses grown on synthetic and swine-waste media

Phormidium sp.

Vitamin (mg/g)

Biotin Pyridoxine (B6) Cyanocobalamin (B 12) Folic acid Panthothenic acid Riboflavin Thiamin (B1) Nicotinic acid

Spirulina maxima

Synthetic medium: Dauta

ASSW 50%

Synthetic medium: Zarrouk

ASSW 50%

0.32 0"18 0.08 0.19 3"14 33" 10 0"51 17.14

0.44 0.20 0.10 0.24 3.84 24.60 0.43 18"16

0.64 10.24 0.16 12.32 45-62 64- 75 0.67 58"90

0.45 9-68 0.10 11.40 42"80 52" 10 0"55 58-60

Results are averages of three determinations. ASSW -- Aeration-stabilized swine waste. Table 6. Essential aminoacid content in cyanobacterial biomasses and nutritional requirements of poultry"

Amino acid (g/100 g sample)

Phormidium sp. ASSW 50%

Spirulina maxima ASSW 50%

Poultry (1) 0-6 Weeks

0"397 2-529 1"395 1.395 1.419 1.091

0' 187 2'39 1.184 1.206 1.433 1.462

0"86 1"6 0"86 1.5 0"8 1"25

Methionine Leucine Isoleucine Phenylalanine Threonine Lysine

"Data from NRC ( 1971 ). Results are averages of two determinations. ASSW -- Aeration-stabilized swine waste. Phormidium, particularly for pyridoxine, riboflavin and folic, nicotinic and pantothenic acids (Table 5). From studies reported in the literature it is known that the chemical composition of Spirulina is 56-62% protein, 2 - 3 % lipid, 0"1-0"9% crude fiber and 6"4-9.0% ash when cultivated in clean water (De la N o i i e & De Pauw, 1988). With regard to the origin of nutrients involved in the cultivation, a distinction can be made between clean water- and wastewater-based production processes (Soeder, 1980). In the former case, strictly defined media are usually employed in which bacteria are of no significant metabolic importance; in the latter case less defined media, such as sewage and manure, are used in which mixed cultures of bacteria and microalgae are active (De la Noiie& De Pauw, 1988). In our study, although protein and lipid contents (36 and 6%, respectively) were inferior to those reported by other authors, the biomass was still appropriate for use as a dietary supplement for animals. The elevated ash content of the biomasses grown in ASSW-50% was largely the result of mineral particles present in the waste. It was not possible to be sure that the washings with distilled water, practised before the biomasses were lyophilized, removed this material, since the stabilized effluent was highly mineralized ( 18-28% ash; Loher, 1974). Since the management of a poultry farm is more appealing from an economic point of view than some

Table 7. Microbiological analysis

Phormidium sp. Spirulina maxima ASSW-50% ASSW-50% Plate count Total coliform bacteria Fecal coliform bacteria Filamentous fungi Yeast Facultative anaerobes

150 × 105/g None None 103 CFU/g 6 × 1 0 3 CFU/g 150 × 104/g

75 x 103/g None None 102 CFU/g < 100 CFU/g 40 x 104/g

other types of animal husbandry, Table 6 compares the content of some essential amino acids in the biomass of the cyanobacteria studied with the nutritional requirements of chickens (0-6 weeks). The algal biomasses can be seen to equal or surpass the standard established concentrations, except in the case of methionine, which would have to be supplemented to complete the amino acid profile. It is noteworthy that the vitamin contents of the biomasses grown in swine wastewater also fulfilled the requirements for chickens (NRC, 1971). Microbiological analysis of cyanobacterial biomasses showed an absence of total- and fecal-coliform bacteria, although the plate counts were 150 × 105/g for the Phormidium biomass and 75 x 103/g for that of Spirulina (Table 7).

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R. O. Cahizares-Villanueva, A. R. Dominguez, M. S. Cruz, E. Rios-Leal

ACKNOWLEDGEMENTS

Sincere appreciation is expressed to Dr Leticia Quintanilla for the English corrections to the manuscript and to: Q. B. P. Joel Alba for performing the microbiological analysis; I. Miguel M~irquez for performing amino acids analysis; I. Cirino Rojas for performing fatty acids analysis.

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