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Culture of marine microalgae with natural biodigested resources
Aquaculture, 64 (1987) 249-256 Elsevier Science Publishers B.V., Amsterdam
249
-
Printed
in The Netherlands
Technical Paper
Culture of Marine Microalgae with Natural Biodigested Resources JESUS P.ANIAGUA-MICHEL,
B.C. FARFAN and F. BUCKLE-RAMIREZ
Division of Oceanology, Centro de Investigation Cientifica y de Educacih Ensenada, Espinoza 843, Ensenada, Baja California (Mhico) (Accepted
Superior de
20 October 1986)
ABSTRACT Paniagua..Michel, J., Farfan, B.C. and Buckle-Ramirez, F., 1987. Culture of marine microalgae with natural biodigested resources. Aquaculture, 64: 249-256. The growth response of Pavlova (Monochrysis) lutheri (Droop) Green was assayed in a culture medium enriched with organic extracts aerobically biodigested from cow and chicken manures and from the macroalgae Macrocystis pyrifera (L.) C. Agard. The results indicate that, with a traditional inorganic medium and elutriated, aerobically digested manures, it is possible to attain similar maximum algal densities and equivalent algal growth rates for similar time-periods despite large differences in inorganic nitrogen and phosphorus content. The cause for this is speculated to be the c:onversion of organic nitrogen and phosphorus in the manure medium to inorganic forms and the presence of growth stimulatory substances in the elutriate.
INTRODUCTION
The desire to produce food and energy has led scientists to investigate alternative technologies that use organic wastes. In Ensenada, Baja California (Mexico), cow and chicken manures and the macroalga Macrocystis pyrifera (L. ) C. Agard are abundant organic waste products which are rich in nutrients and trace elements. Recycling these waste products into new plant and animal protein via the cultivation of commercially important shellfish opens interesting perspectives. Shellfish culture requires relatively large quantities of marine microalgae as food. Some of the costs of producing microalgae in shellfish hatcheries could be lowered using organic wastes as a nutrient source. Nutrients from organic wastes can be made available to microalgae by treatments such as aerobic digestion. Several studies have been concerned with the use of swine manure and tertiary and secondary waste-water effluents as nutrient sources for microalgal culture (Dunstan and Menzel, 1971; Dunstan and Tenore, 1972; Goldman and Stanley, 1974; Wong, 1977; De Pauw and De
0044-84816/87,$03.50
0 1987 Elsevier Science Publishers
B.V.
250
Leenheer, 1979; Yang and Huang, 1981) . Recently Granados-Machuca and Buckle-Ramirez (1984) and Paniagua-Michel and Buckle-Ramirez (1985) demonstrated the feasibility of separately utilizing cow and chicken manures and M. p&era aerobically biodigested as the nutrient source for the cultivation of marine microalgae. In this study, we demonstrated that the growth of Pavlova (Monochrysis) lutheri (Droop) Green can be enhanced with a mixture of liquors from the digestion of cow and chicken manures and M. pyrifera. MATERIALS AND METHODS
The organic products were processed separately in laboratory-scale aerobic batch-fed biodigesters with a volume of 11 1 (Fig. 1). Details of the biodigester’s design and optimum loading factors were given by Paniagua-Michel (1984). Treatment temperatures were set at 28 2 1 ‘C. Throughout the 30-day experimental period, liquor samples were withdrawn every 5 days for pH, dissolved oxygen concentration and nutrient analysis; 1200-ml aliquots were also taken and stored frozen for later use in the growth medium elaboration. Dissolved oxygen concentrations were measured with a YSI oxygen meter; pH was determined with a Hach pH meter model DR-EL/4. Using a DR-EL/4 Hach spectrophotometer, the samples were analysed for the following nutrients: ammonia, nitrate, phosphate, silicate, iron, copper and manganese. The analytical procedures were carried out as described in the Hach manual (1980). The liquors of M. pyrifera and cow and chicken manures selected in the growth medium elaboration resulted from 15, 10 and 25 days of treatment respectively. After thawing, aliquots of the liquors were individually autoclaved (1.05 kg/ cm2 and 121 “C) and adjusted with 1 N HCl to a pH of 4-5. To prepare the experimental growth medium, we used the three liquors in the proportions 4 : 2 : 1 ml of M. pyrifera and cow and chicken manure biodigests, respectively, diluted to 100 ml with sea water as recommended by Paniagua-Michel and Granados-Manucha (1981). This solution resulted in the highest concentration of most of the nutrients analysed and the nitrogen/ phosphorus ratio present in conventional phytoplankton culture media (Matthiessen and Toner, 1966; Guillard, 1975). The growth of P. Zutheri was compared in the experimental culture medium and in a control medium (Matthiessen and Toner, 1966). In both cases, unialgal batch cultures were carried out in 2.71-1 acrylic chambers. The culture chambers were stoppered with foam plugs to allow gas exchange. The temperature and continuous illumination conditions, 212 2” C and 2152 lux, were kept constant. Nine replicates of each culture medium were inoculated simultaneously with an equal number of P. lutheri cells, provided by the National Marine Fisheries
251
Fig. 1. Laboratory digester system: 1, gravel bed; 2, bucket support; 3, heater; 4, sampling stopcock; 5, air injection valve; 6, thermometer; 7, scum fractionator; 8, gas venting duct; 9, security spout; 10, sprinkler; 11, perforated bucket; the arrows indicate the liquid flow.
Service at La Jolla, California. During the experimental period, daily estimates of cell density in all of the culture assays were made in samples preserved with Utermijhl solution (Guillard, 1973) by direct counting of cells. For each culture, the logarithmic growth phase was determined by a semilog (base 1.0) plot of algal density versus time. The equation defining this growth phase was determined by least squares linear regression analysis. The slope represents the mean growth rate during the logarithmic phase (Guillard, 1973). Other growth parameters estimated were: daily specific growth rate, defined in the Environmental Protection Agency Algal Assay (1971) as p=log
(N2/Nl)/(t2-t1)
where Nl and N2 are cell densities
at time tl and time t2 respectively;
daily
252
TABLE I Nutrient concentrations in the experimental and the control growth media Nutrients
Control
Experimental
Experimental
(w/ml 1
(w/ml 1
vs. Control (o/o)
NOs PO, SiOz Fe cu Mn NH,
150 20 30 0.01 0.0019 0.36 -
0.796 0.052 1.43 0.0031 0.0031 0.39 0.10
< < < < > > >
99.40 99.40 95.24 69.00 63.00 8.00 100.00
division rate (k) , formulated by Guillard (1973) ; and daily mean generation time ( Tg) , estimated from the equation given by Eppley and Strickland (1968). RESULTS AND DISCUSSION
Although nutrient levels were high in the digest liquors, when mixed with sea water to make up the growth medium (Table 1) , the initial concentrations of most of the nutrients analysed in the experimental medium were 69-99% lower than in the control. Only those of copper and manganese were slightly higher than those in the control, and ammonium was 100% higher since the control medium was lacking in this element. The nitrogen to phosphorus atomic ratio (N : P) was slightly greater than 15. Although it is not necessarily the optimum ratio for culturing all phytoplankton species as reported by Rhee (1978)) it is a value adopted in all conventional chemical culture media (Matthiessen and Toner, 1966; Guillard, 1973). The highest mean cell densities of P. lutheri based on 9 replicate cultures of each experimental and control medium were 27.14 x lo5 and 29.67 x lo5 cells/ml respectively (Table 2). The corresponding growth curves are shown in Fig. 2. The linear fittings of the transformed cell densities against the estimated exponential growth phases of the experimental and control culture media are shown in Figs. 3A and B respectively. The transformed cellular densities during the estimated exponential growth phase in both media and their corresponding replicates were subjected to a covariance analysis, the results of which are given in Table 3 ( Sokal and Rohlf, 1979). The normality hypothesis of residuals was not rejected at a! = 0.05; the Bartlett test indicated homoscedasticity; the explained variability due to the regression model was highly significant ( a = 0.001) . The test detected no significant differences in the adjusted mean cell densities among replicates or between the experimental and control culture media ( CY= 0.001) .
253 TABLE 4: Means* and 95% confidence intervals for the growth parameters of I? lutheri in the experimental (B) and l;he control (A) growth media Time (days)
Cell density (cells/ml X 105)
A Contro I 0 0.12 k 0.00 1 0.27 k 0.03 2 1.75 k 0.38 3 3.53 k 0.42 4 5.01 k 0.73 5 6.39 k 0.83 6 10.45 + 0.81 7 16.68 k 0.46 8 22.56 k 3.88 9 29.68 k 1.49 10 20.33 k 2.29 B Experimental 0 0.12 k 0.00 1 0.26 + 0.02 2 1.81 k 0.18 3 3.50 + 0.53 4 4.64 k 0.63 5 6.02 ?I0.43 6 11.39kO.85 7 15.34 k 0.70 8 27.15 t 0.82 9 26.00 k 0.32 10 20.80 k 0.40
(days )
K (division/day)
P (day)
1.08kO.31 2.26 k 0.96 5.17 k 5.43 1.51 k 0.45 1.52 + 0.29 6.65 k 9.05 3.61 k 2.77
l.01f0.36 0.52 + 0.23 0.35 * 0.19 0.71 k0.24 0.67f0.13 0.44 k 0.29 0.44 f 0.28
0.69 k 0.25 0.35 I!I0.16 0.24 k 0.13 0.49kO.17 0.46 k 0.09 0.29 k 0.18 0.30 k 0.19
1.14 k 0.32 3.59 k 3.15 4.16+ 3.41 1.12 kO.14 2.46 k 0.82 1.22 k 0.13
0.94 + 0.27 0.42 f 0.27 0.37 kO.21 0.84 50.25 0.40f0.17 0.82 f 0.08
0.64kO.19 1.64 k 2.58 0.25 k 0.15 1.05 k 0.66 0.65 k 0.53 0.56 k 0.06
G
“Tg, mean generation time; k, mean division rate; ,u, mean specific growth rate.
The alga was unaffected by the salinity and pH changes of the digest liquors since this species is euryhaline and the volumes of liquors to sea water were low. One factor to be considered in the experimental medium was the slight b.rown colour attained by the liquors. The estimated growth parameters in both culture media were also similar (Table 2 ) . The highest mean division rates (k) in the experimental and control media were 0.93 and 1 division/day respectively, and occurred during the second (day of culture. The comparable growth patterns and kinetics of P. lutheri in both culture media, in spite of the significantly lower concentrations of major inorganic nutrients and most oligoelements in the experimental medium, suggest a microalgal growth partly subsidized by other substances since manures and macroallgae contain all of the major and minor nutrients for microalgal growth and a wide variety of nutrients and trace elements ( Stephenson, 1974; Teusher
254
Fig. 2. Growth curve of P. lutheri in the experimental (- -) and the control (-)
growth media.
Fig. 3. Linear fitness by least squares of P. lutheri densities in the experimental (B) and the control (A) growth media.
and Adler, 1982). In conventional media, the microalgae are restricted to the nutrients supplied while, in the experimental medium, the cells were cultured in a variety of nutrients and other growth substances released during the aerobic digestion of the wastes. During the last two decades, considerable evidence for the ability of microalgae to grow on urea and a variety of purines, amino acids and phosphate organic esters as alternative nutrient sources has been obtained by algal physiologists TABLE 3 Analysis of covariance of the cell concentrations between the experimental and control growth media Source of variation
d.f.
Sum of squares
Mean squares
F
Adjusted means Regression Error Homogeneity Homogeneity error
17 1 116 17 99
0.12 19.34 0.60 0.11 0.49
0.007 19.349 0.005 0.006 0.004
1.3895
1.2999
255
(Kuenzller and Perras, 1965; Wheeler et al., 1974; Antia et al., 1975; Admiral et al., 1984). The enhancement of phytoplankton growth by culture enrichment was first reported by Miquel (1890). Some specific mechanisms for this effect have already been shown and the substances responsible identified. The natural chelating properties of carbohydrates, polyphenols and other exudates of marine macroalgae were recently reviewed by Sueur et al. (1982). The trace e1ement.s contained in the experimental medium may have been chelated by strong o,rganic associations with the compounds from the macroalgae (Milton, 1964) and the humic compounds of the manures (Stumm, 1972). Voropayev and Chechin (1983) have discussed the role in microalgal growth of gibberelins, promoting and regulatory growth hormones isolated from marine macroalgae. There is a possibility that these growth-promoting substances in the experimental culture medium exerted a biostimulatory effect on the utilization of the inorganic nutrients and trace elements. Data presented in this study suggest that phytoplankton may grow better in a medium with a variety of substances and nutrients in low concentrations instead of single nutrients in high concentrations such as those in conventional formulae of culture media. ACKNOWLEDGEMENTS
We are grateful to Dr. Alejandro Chagoya for providing statistical assistance, Dr. W.H:. Thomas of the National Marine Fisheries Center at La Jolla, CA, for the cultures of algae provided and Gregory Hammann, M.Sc., for his constructive critique of this manuscript.
REFERENCES Admiral, W., Laane, R.W. and Peletier, M., 1984. Participation of diatoms in the aminoacid cycle of coastal waters: uptake and excretion in cultures. Mar. Ecol. Prog. Ser., 15: 303-306. Antia, NJ., Berland, B.R., Bonin, D.J. and Maestrini, S.Y., 1975. Comparative evaluation of certain organic and inorganic sources of nitrogen for phototrophic growth of marine microalgae. J. Mar. Biol. Assoc. U.K., 55: 519-539. De la Crux, S.A. and Alfonso, E., 1975. Cultivo masivo de algas planctonicas marinas mediante fertilization. Ciencias, 8: l-25. De Pauw, N. and De Leenheer, L., 1979. Mass culturing of marine and freshwater algae on aerated swine manure. Eur. Maricult. Sot., 4: 441-473. Dunstan, W.M. and Menzel, D.W., 1971. Continuous cultures of natural populations of phytoplankton in dilute treated sewage effluent. Limnol. Ocenaogr., 16 (4): 623-633. Dunstan, W.M. and Tenore, K.R., 1972. Intensive outdoor culture of marinephytoplanktonenriched with treated sewage effluent. Aquaculture, 1: 181-192. Environmental Protection Agency, 1971. Algal Assay Procedure. National Research Eutrophication l?rogram/EPA, pp. l-82. Eppley, R. W. and Strickland, J.D., 1968. Kinetics of marine plankton growth. In: M.R. Droop and
256 E.J. Ferguson (Editors), Advances in Microbiology of the Sea. Academic Press, New York, pp. 23-62. Goldman, J.C. and Stanley, H.I., 1974. Relative growth of different species on marine algae in wastewater-seawater mixtures. Mar. Biol., 28: 17-25. Granados-Machuca, C. and Buckle-Ramirez, L.F., 1984. Cultivo de las microalgas Monochrysis lutheri y Skeletonema costatum con nutrientes producidos por estiercoles digeridos. An. Inst. Biol. Univ. Nat. Auton. Mex. Ser. Cienc. Mar Limnol., 11 (1): 241-256. Guillard, R.R., 1973. Division rates. In: J.R. Stein (Editor), Handbook of Phycological Methods. Cambridge University Press, New York, pp. 289-311. Guillard, R.R., 1975. Culture of phytoplankton for feeding marine invertebrate animals. In: M.L. Smith and M.H. Chanley (Editors), Culture of Marine Invertebrate Animals. Plenum Press, New York, pp. 29-60. Hach Company, 1980. DR-EL/4 methods. In: Hach Manual. Hach Company. Kuenzler, E.J. and Perras, J.P., 1965. Phosphatase of marine algae. Biol. Bull., 128: 271-284. Matthiessen, G.C. and Toner, R.C., 1966. Possible methods of improving the shellfish industry of Martha’s Vineyard, Duke’s County, Massachusetts. Mar. Res. Fund, 138 pp. Milton, R.F., 1964. Liquid seaweeds as fertilizers. Proc. 4th Int. Seaweed Symp., Biarritz, 1961. Pergamon Press, 428 pp. Miquel, P., 1890. De la culture artificielle des diatomees. Diatomiste, pp. 1-18. Paniagua-Michel, J., 1984. Cultivo de fitoplancton marino bajo condiciones controladas en un medio elaborado con productos naturales biodigeridos. Tesis de Maestria CICESE, Ensenada, Mexico, 118 pp. Paniagua-Michel, J. and Buckle-Ramirez, L.F., 1985. Cultivo en condiciones controladas de Manochrysis lutheri y Skeletomena costatum con extractos de macrofitas marinas. An. Inst. Biol. Univ. Nat. Auton. Mex. Ser. Cienc. Mar Limnol., 12 (1) : 59-70. Paniagua-Michel, J. and Granados-Machuca, C., 1981. Obtencion de dos medios economicos para el cultivo de fitoplancton bajo condiciones controladas. Tesis Profesional Escuela Superior de Ciencias Marinas, UABC, Ensenada, Mexico, 81 pp. Provasoli, L., 1968. Media and prospects for the marine algae. In: A. Watanabe and A. Hattori (Editors), Cultures and Collection of Algae. Proc. U.S.-Jpn. Conf., Hakone, September 1966. Japanese Society of Plant Physiology, pp. 63-75. Rhee, Y., 1978. Effect of N : P atomic ratios and nitrate limitation on algal growth, cell composition and nitrate uptake. Limnol. Oceanogr., 23 (1): 10-22. Sokal, R. and Rohlf, F.J., 1979. Biometria. Blume, Spain, 832 pp. Stephenson, W.A., 1974. Seaweed agriculture and horticulture. Conserv. Gard. Farm. Ser., pp. 81-240. Stumm, W., 1972. Die Rolle der Komplexbildung in naturlichen Gewassern und aufallige Beziehungen zur Eutrophierung. Gewasserschutz Wasser Abwasser, 8: 57-88. Sueur, S., Vandenberg, C.M. and Riley, J.P., 1982. Measurement of the metal complexing ability of exudates of marine macroalgae. Limnol. Oceanogr., 27 (3): 536-543. Teusher, B. and Adler, R., 1982. El Suelo y su Fertilidad. CECSA, Mexico, 510 pp. Voropayev, V.M. and Chechin, A.P., 1983. The growth of marine microalgae in cultures in the presence of gibberellins. Hydrobiol. J., 17 (3): 57-59. Wheeler, P.A., North, B.B. and Stephens, G.S., 1974. Aminoacid uptake by marine phytoplankters. Limnol. Oceanogr., 19: 249-259. Wong, M.H., 1977. The comparison of activated and digested sludge extracts in cultivating Chlorella pyrenoidosa and C. salina. Environ. Pollut., 14: 207-211. Yang, P.Y. and Huang, C.J., 1981. Anaerobic and microalgal process kinetics for swine waste. J. Environ. Eng. Div. Am. Sot. Civ. Eng., EE6: 1113-1127.
249
-
Printed
in The Netherlands
Technical Paper
Culture of Marine Microalgae with Natural Biodigested Resources JESUS P.ANIAGUA-MICHEL,
B.C. FARFAN and F. BUCKLE-RAMIREZ
Division of Oceanology, Centro de Investigation Cientifica y de Educacih Ensenada, Espinoza 843, Ensenada, Baja California (Mhico) (Accepted
Superior de
20 October 1986)
ABSTRACT Paniagua..Michel, J., Farfan, B.C. and Buckle-Ramirez, F., 1987. Culture of marine microalgae with natural biodigested resources. Aquaculture, 64: 249-256. The growth response of Pavlova (Monochrysis) lutheri (Droop) Green was assayed in a culture medium enriched with organic extracts aerobically biodigested from cow and chicken manures and from the macroalgae Macrocystis pyrifera (L.) C. Agard. The results indicate that, with a traditional inorganic medium and elutriated, aerobically digested manures, it is possible to attain similar maximum algal densities and equivalent algal growth rates for similar time-periods despite large differences in inorganic nitrogen and phosphorus content. The cause for this is speculated to be the c:onversion of organic nitrogen and phosphorus in the manure medium to inorganic forms and the presence of growth stimulatory substances in the elutriate.
INTRODUCTION
The desire to produce food and energy has led scientists to investigate alternative technologies that use organic wastes. In Ensenada, Baja California (Mexico), cow and chicken manures and the macroalga Macrocystis pyrifera (L. ) C. Agard are abundant organic waste products which are rich in nutrients and trace elements. Recycling these waste products into new plant and animal protein via the cultivation of commercially important shellfish opens interesting perspectives. Shellfish culture requires relatively large quantities of marine microalgae as food. Some of the costs of producing microalgae in shellfish hatcheries could be lowered using organic wastes as a nutrient source. Nutrients from organic wastes can be made available to microalgae by treatments such as aerobic digestion. Several studies have been concerned with the use of swine manure and tertiary and secondary waste-water effluents as nutrient sources for microalgal culture (Dunstan and Menzel, 1971; Dunstan and Tenore, 1972; Goldman and Stanley, 1974; Wong, 1977; De Pauw and De
0044-84816/87,$03.50
0 1987 Elsevier Science Publishers
B.V.
250
Leenheer, 1979; Yang and Huang, 1981) . Recently Granados-Machuca and Buckle-Ramirez (1984) and Paniagua-Michel and Buckle-Ramirez (1985) demonstrated the feasibility of separately utilizing cow and chicken manures and M. p&era aerobically biodigested as the nutrient source for the cultivation of marine microalgae. In this study, we demonstrated that the growth of Pavlova (Monochrysis) lutheri (Droop) Green can be enhanced with a mixture of liquors from the digestion of cow and chicken manures and M. pyrifera. MATERIALS AND METHODS
The organic products were processed separately in laboratory-scale aerobic batch-fed biodigesters with a volume of 11 1 (Fig. 1). Details of the biodigester’s design and optimum loading factors were given by Paniagua-Michel (1984). Treatment temperatures were set at 28 2 1 ‘C. Throughout the 30-day experimental period, liquor samples were withdrawn every 5 days for pH, dissolved oxygen concentration and nutrient analysis; 1200-ml aliquots were also taken and stored frozen for later use in the growth medium elaboration. Dissolved oxygen concentrations were measured with a YSI oxygen meter; pH was determined with a Hach pH meter model DR-EL/4. Using a DR-EL/4 Hach spectrophotometer, the samples were analysed for the following nutrients: ammonia, nitrate, phosphate, silicate, iron, copper and manganese. The analytical procedures were carried out as described in the Hach manual (1980). The liquors of M. pyrifera and cow and chicken manures selected in the growth medium elaboration resulted from 15, 10 and 25 days of treatment respectively. After thawing, aliquots of the liquors were individually autoclaved (1.05 kg/ cm2 and 121 “C) and adjusted with 1 N HCl to a pH of 4-5. To prepare the experimental growth medium, we used the three liquors in the proportions 4 : 2 : 1 ml of M. pyrifera and cow and chicken manure biodigests, respectively, diluted to 100 ml with sea water as recommended by Paniagua-Michel and Granados-Manucha (1981). This solution resulted in the highest concentration of most of the nutrients analysed and the nitrogen/ phosphorus ratio present in conventional phytoplankton culture media (Matthiessen and Toner, 1966; Guillard, 1975). The growth of P. Zutheri was compared in the experimental culture medium and in a control medium (Matthiessen and Toner, 1966). In both cases, unialgal batch cultures were carried out in 2.71-1 acrylic chambers. The culture chambers were stoppered with foam plugs to allow gas exchange. The temperature and continuous illumination conditions, 212 2” C and 2152 lux, were kept constant. Nine replicates of each culture medium were inoculated simultaneously with an equal number of P. lutheri cells, provided by the National Marine Fisheries
251
Fig. 1. Laboratory digester system: 1, gravel bed; 2, bucket support; 3, heater; 4, sampling stopcock; 5, air injection valve; 6, thermometer; 7, scum fractionator; 8, gas venting duct; 9, security spout; 10, sprinkler; 11, perforated bucket; the arrows indicate the liquid flow.
Service at La Jolla, California. During the experimental period, daily estimates of cell density in all of the culture assays were made in samples preserved with Utermijhl solution (Guillard, 1973) by direct counting of cells. For each culture, the logarithmic growth phase was determined by a semilog (base 1.0) plot of algal density versus time. The equation defining this growth phase was determined by least squares linear regression analysis. The slope represents the mean growth rate during the logarithmic phase (Guillard, 1973). Other growth parameters estimated were: daily specific growth rate, defined in the Environmental Protection Agency Algal Assay (1971) as p=log
(N2/Nl)/(t2-t1)
where Nl and N2 are cell densities
at time tl and time t2 respectively;
daily
252
TABLE I Nutrient concentrations in the experimental and the control growth media Nutrients
Control
Experimental
Experimental
(w/ml 1
(w/ml 1
vs. Control (o/o)
NOs PO, SiOz Fe cu Mn NH,
150 20 30 0.01 0.0019 0.36 -
0.796 0.052 1.43 0.0031 0.0031 0.39 0.10
< < < < > > >
99.40 99.40 95.24 69.00 63.00 8.00 100.00
division rate (k) , formulated by Guillard (1973) ; and daily mean generation time ( Tg) , estimated from the equation given by Eppley and Strickland (1968). RESULTS AND DISCUSSION
Although nutrient levels were high in the digest liquors, when mixed with sea water to make up the growth medium (Table 1) , the initial concentrations of most of the nutrients analysed in the experimental medium were 69-99% lower than in the control. Only those of copper and manganese were slightly higher than those in the control, and ammonium was 100% higher since the control medium was lacking in this element. The nitrogen to phosphorus atomic ratio (N : P) was slightly greater than 15. Although it is not necessarily the optimum ratio for culturing all phytoplankton species as reported by Rhee (1978)) it is a value adopted in all conventional chemical culture media (Matthiessen and Toner, 1966; Guillard, 1973). The highest mean cell densities of P. lutheri based on 9 replicate cultures of each experimental and control medium were 27.14 x lo5 and 29.67 x lo5 cells/ml respectively (Table 2). The corresponding growth curves are shown in Fig. 2. The linear fittings of the transformed cell densities against the estimated exponential growth phases of the experimental and control culture media are shown in Figs. 3A and B respectively. The transformed cellular densities during the estimated exponential growth phase in both media and their corresponding replicates were subjected to a covariance analysis, the results of which are given in Table 3 ( Sokal and Rohlf, 1979). The normality hypothesis of residuals was not rejected at a! = 0.05; the Bartlett test indicated homoscedasticity; the explained variability due to the regression model was highly significant ( a = 0.001) . The test detected no significant differences in the adjusted mean cell densities among replicates or between the experimental and control culture media ( CY= 0.001) .
253 TABLE 4: Means* and 95% confidence intervals for the growth parameters of I? lutheri in the experimental (B) and l;he control (A) growth media Time (days)
Cell density (cells/ml X 105)
A Contro I 0 0.12 k 0.00 1 0.27 k 0.03 2 1.75 k 0.38 3 3.53 k 0.42 4 5.01 k 0.73 5 6.39 k 0.83 6 10.45 + 0.81 7 16.68 k 0.46 8 22.56 k 3.88 9 29.68 k 1.49 10 20.33 k 2.29 B Experimental 0 0.12 k 0.00 1 0.26 + 0.02 2 1.81 k 0.18 3 3.50 + 0.53 4 4.64 k 0.63 5 6.02 ?I0.43 6 11.39kO.85 7 15.34 k 0.70 8 27.15 t 0.82 9 26.00 k 0.32 10 20.80 k 0.40
(days )
K (division/day)
P (day)
1.08kO.31 2.26 k 0.96 5.17 k 5.43 1.51 k 0.45 1.52 + 0.29 6.65 k 9.05 3.61 k 2.77
l.01f0.36 0.52 + 0.23 0.35 * 0.19 0.71 k0.24 0.67f0.13 0.44 k 0.29 0.44 f 0.28
0.69 k 0.25 0.35 I!I0.16 0.24 k 0.13 0.49kO.17 0.46 k 0.09 0.29 k 0.18 0.30 k 0.19
1.14 k 0.32 3.59 k 3.15 4.16+ 3.41 1.12 kO.14 2.46 k 0.82 1.22 k 0.13
0.94 + 0.27 0.42 f 0.27 0.37 kO.21 0.84 50.25 0.40f0.17 0.82 f 0.08
0.64kO.19 1.64 k 2.58 0.25 k 0.15 1.05 k 0.66 0.65 k 0.53 0.56 k 0.06
G
“Tg, mean generation time; k, mean division rate; ,u, mean specific growth rate.
The alga was unaffected by the salinity and pH changes of the digest liquors since this species is euryhaline and the volumes of liquors to sea water were low. One factor to be considered in the experimental medium was the slight b.rown colour attained by the liquors. The estimated growth parameters in both culture media were also similar (Table 2 ) . The highest mean division rates (k) in the experimental and control media were 0.93 and 1 division/day respectively, and occurred during the second (day of culture. The comparable growth patterns and kinetics of P. lutheri in both culture media, in spite of the significantly lower concentrations of major inorganic nutrients and most oligoelements in the experimental medium, suggest a microalgal growth partly subsidized by other substances since manures and macroallgae contain all of the major and minor nutrients for microalgal growth and a wide variety of nutrients and trace elements ( Stephenson, 1974; Teusher
254
Fig. 2. Growth curve of P. lutheri in the experimental (- -) and the control (-)
growth media.
Fig. 3. Linear fitness by least squares of P. lutheri densities in the experimental (B) and the control (A) growth media.
and Adler, 1982). In conventional media, the microalgae are restricted to the nutrients supplied while, in the experimental medium, the cells were cultured in a variety of nutrients and other growth substances released during the aerobic digestion of the wastes. During the last two decades, considerable evidence for the ability of microalgae to grow on urea and a variety of purines, amino acids and phosphate organic esters as alternative nutrient sources has been obtained by algal physiologists TABLE 3 Analysis of covariance of the cell concentrations between the experimental and control growth media Source of variation
d.f.
Sum of squares
Mean squares
F
Adjusted means Regression Error Homogeneity Homogeneity error
17 1 116 17 99
0.12 19.34 0.60 0.11 0.49
0.007 19.349 0.005 0.006 0.004
1.3895
1.2999
255
(Kuenzller and Perras, 1965; Wheeler et al., 1974; Antia et al., 1975; Admiral et al., 1984). The enhancement of phytoplankton growth by culture enrichment was first reported by Miquel (1890). Some specific mechanisms for this effect have already been shown and the substances responsible identified. The natural chelating properties of carbohydrates, polyphenols and other exudates of marine macroalgae were recently reviewed by Sueur et al. (1982). The trace e1ement.s contained in the experimental medium may have been chelated by strong o,rganic associations with the compounds from the macroalgae (Milton, 1964) and the humic compounds of the manures (Stumm, 1972). Voropayev and Chechin (1983) have discussed the role in microalgal growth of gibberelins, promoting and regulatory growth hormones isolated from marine macroalgae. There is a possibility that these growth-promoting substances in the experimental culture medium exerted a biostimulatory effect on the utilization of the inorganic nutrients and trace elements. Data presented in this study suggest that phytoplankton may grow better in a medium with a variety of substances and nutrients in low concentrations instead of single nutrients in high concentrations such as those in conventional formulae of culture media. ACKNOWLEDGEMENTS
We are grateful to Dr. Alejandro Chagoya for providing statistical assistance, Dr. W.H:. Thomas of the National Marine Fisheries Center at La Jolla, CA, for the cultures of algae provided and Gregory Hammann, M.Sc., for his constructive critique of this manuscript.
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