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Growth rates, phycocolloid yield and quality of the red seaweeds, Gracilaria sp., Pterocladia capillacea, Hypnea musciformis, and Hypnea cornuta, in field studies in Israel
Aquaculture, 40 (1984) 57-66 Elsevier Science Publishers B.V., Amsterdam
57 - Printed
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GROWTH RATES, PHYCOCOLLOID YIELD AND QUALITY OF THE RED SEAWEEDS, GRACILARIA SP., PTEROCLADIA CAPILLACEA, HYPNEA MUSCIFORMIS, AND HYPNEA CORNUTA, IN FIELD STUDIES IN ISRAEL
M. FRIEDLANDER
and N. ZELIKOVITCH
Department of Botany, Tel Aviv (Israel) (Accepted
28 October
The George S. Wise Faculty of Life Sciences,
Tel Aviv University,
1983)
ABSTRACT Friedlander, M. and Zelikovitch, N., 1984. Growth rates, phycocolloid yield and quality of the red seaweeds, Gracilaria sp., Pterocladia capillacea, Hypnea musciformis, and Hypnea cornuta, in field studies in Israel. Aquaculture, 40: 57-66. The agarophytes Gracilaria sp. and Pterocladia capillacea, and the carrageenophytes Hypnea musciformis and H. cornuta were cultured under field conditions. Specific growth rates appeared to be related to temperature and light intensity. Attached seaweeds had higher annual yields per mz than free-floating samples. The specific growth rate of all four seaweeds was positively related to phycocolloid content in the main growing season. In agar extracts high gel strength coincided with high 3,6 anhydrogalactose content, with high gelling and melting temperature, and with low sulfate content. In the carrageenan extracts low gel strength coincided with low 3,6 anhydrogalactose content, with low gelling and melting temperatures and with high sulfate content. Chemical composition and physical properties of cultured and natural seaweeds were almost the same.
INTRODUCTION
The possibility of tropical seaweed mariculture as a source of phycocolloids (Doty, 1979) has been suggested for the eastern Mediterranean (Friedlander and Lipkin, 1982). In previous studies, seaweed growth rates in the field have been shown to be highly variable due to fluctuations of natural factors (Rama Rao, 1970; Dawes et al., 1974; Durako and Dawes, 1980; Guist et al., 1982). Furthermore, phycocolloid extract such as agar and carrageenan are known to be affected by seasonal changes (Dawes et al., 1974; Hoyle, 1978; Mshigeni, 1976; Simpson and Shachlock, 1979). Chemical composition as well as physical properties of phycocolloids also change with environmental conditions (Asare, 1980; Whyte et al., 1981; Bird et al., 1981). The dominant factors affecting seaweed growth in tanks appear to be: light intensity, temperature, salinity (Bird et al., 1979; Hanisak, 1979;
0044-8486/84/$03.00
o 1984 Elsevier
Science
Publishers
B.V.
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Lapointe, 1981), nitrogen enrichment (DeBoer et al., 1978; Lapointe and Ryther, 1979; Ryther et al., 1981; Bird et al., 1981, 1982), water motion (Parker, 1982) and plant density (Lapointe and Ryther, 1978). Little information is available regarding effects of fluctuating field conditions on growth. The present study was undertaken to determine growth rates and phycocolloid yield and quality under changing field conditions, and to compare these relationships with the variations found in previous studies. MATERIALS
AND METHODS
Gracilariu sp. (not yet identified, voucher specimen at the Tel Aviv University herbarium), Pterocladiu cupillacea (Gmelin) Born. et Thur., Hypnea musciformis (Wulfen) Lamouroux and H. cornuta (Lamouroux) J. Agardh, were collected at Mikhmoret on the eastern Mediterranean coast during April 1981 to April 1982. The main experimental site was a sea water pond of 0.5 ha area, near the coast of Ma’agan Michael. A second pond of 400 m* received warm sea water from a power station in the winter, with a turnover of less than one volume per day. The ponds were 1 m deep with sandy bottoms. The seaweeds were separated into 50 pieces of ca. 2 g each, and tied to ropes on a 1 X 1 m polypropylene tube frame. The frame was placed in an anchored net basket of the same surface area, 25 cm under water. New plants were established bimonthly because of epiphytization. In a second experiment samples of 100 g were placed in baskets of the same size without tying. All experiments were conducted without additional water motion or nutrient enrichment. The sea water had a stable salinity (33%). Environmental factors were measured weekly at noon time. Water temperature and salinity were determined with a S.C.T. meter (Y.S.I. model 33); light intensity was measured with a LI-170 quantum meter (LI-COR). Damp dried seaweeds were weighed biweekly and then returned to their initial weight. Specific growth rate (S.G.R.) was calculated as M= doublings per day [p = l/t X log (N/N,) X 3.32, where t is the number of days between primary fresh weight (No ) and final fresh weight (iV)] . Seaweed samples were collected every l-3 months for phycocolloid analysis from the main experimental site and from natural populations. Dry weight was measured after air drying and oven drying at 60°C for 24 h. Agar and carrageenan were extracted as described by Young et al. (1971). Gel strength, melting and dynamic gelling temperatures were measured as described by Friedlander and Lipkin (1982). D-galactose content was measured by a modification of the phenolsulfuric acid test (Dubois et al., 1956), 3,6 anhydrogalactose was measured by a modification of the resorcinol test (Friedlander et al., 1981). Sulfate content was measured by a micromethod of Jackson and McCandless (1978).
59
RESULTS
The specific growth rate (S.G.R.) of the attached seaweeds and its relationship with temperature and light intensity is shown in Fig. 1 and Table I. Gracilaria sp. reached a maximum of 0.07 doublings per day in August, with a positive correlation with temperature (P <0.05). Pterocladiu capitlacea reached a maximum of 0.08 doublings per day in April, and similar but noncontinuous rates in the following summer, with a positive correlation with temperature and light intensity (P <0.05). Hypnea musciformis reached two maxima of 0.12 and 0.09 doublings per day in July and November, respectively. Hypnea cornutu reached a maximum of 0.19 doublings per day in July, which was the highest S.G.R. observed. Positive correlations of S.G.R. with temperature and light intensity (P
S.G.R. versus temperature
S.G.R. versus light intensity
Gracilaria sp. Pterocladia capillacea Hypnea musciformis Hypnea cornuta
0.617* 0.692* 0.805** 0.720**
0.420 0.539* 0.728** 0.737**
* Significant at the level of P <0.05.
** Significant at the level of P
Carrageenan content (11-55s) was higher than agar content (5-32s) in the respective seaweeds, and carrageenan content of H. cornu tu was generally higher than that of H. musciformis (Table III). All four species had a decrease in phycocolloid content from early summer to fall and showed a positive relationship between S.G.R. and phycocolloid content during that period. The 3,6 anhydrogalactose content in the extracts of agar was 30-43s (the higher values were in P. capillacea) and increased from summer to fall, with a final decrease towards winter. The 3,6 anhydrogalactose content of the extracts of carrageenan was lower than that of agar and fluctuated in a range of 18-
60
25%. Sulfate content of extracts of agar fluctuated between 1 and 2%, that of carrageenan showed a considerably higher range of values with fluctuations (4-27%). High 3,6 anhydrogalactose content coincided low sulfate content in the agar extracts, while low 3,6 anhydrogalactose
AMJ 1981
while wider with con-
JASONOJFMA 1982
Fig. 1. Annual temperature and light intensity profile, and S.G.R. variations (doublings per day f S.E.) of four cultured seaweeds in: (A) Regular sea water pond (n = 50); (B) Heated sea water pond (n = 10). Gracilario sp. (G); P. capillaceo (P); H. musciformis (Hm); H. cotnutu (Hc).
61 TABLE II Annual yield of attached and free floating Gracilaria sp., Pterocladia musciformis and H. cornuta in culture. Fifty single pieces of 2 g each and 100 g of seaweeds formed the free-floating inoculum in two 1 x density per ml). Yields were calculated as g fresh weight m-* year-* * Species
Attached
Free-floating
Gracilaria sp. Pterocladia capillacea H. musciformis H. cornuta
872+ 70 1272 f 140 2444 r 220 2828 f 339
506 414 1314 1758
capillacea, Hypnea were tied to ropes, 1 m baskets (same 1 S.D.
* 116 f 87 f 236 t 264
TABLE III Comparative seasonal variations of chemical constituants and physicd properties of four regularly cultured seaweeds: Gracilaria sp. (G), Pterocladia capillacea (P), Hypnea musciformis (Hm) and H. cornu to (Hc) Species
Month Jun. Jul.
Aug.
Oct.
Nov.
Dec.
13.0 11.0 24.0 42.0
-
7.5 5.5 15.0 38.0
10.0 10.0 6.5 20.0 25.0
5.5 34.0 28.0
33.7 42.7 24.8 20.9
36.2 33.7 24.7 20.7
33.5 29.8 21.9 18.6
1.2 8.0 8.0
0.8 1.6 6.0 27.0
1.4 2.5 6.0 5.0
1.6 2.1 4.0 6.0
110 420 -
G
-
P Hm Hc
22.0 43.0 33.0
32.0 25.0 55.0
3,6 anhydrogalactose (% of phycocolloid)
G P Hm Hc
-
37.2 20.7 23.3
29.6 34.0 20.8 23.2
-
35.0 22.3 24.4
Sulfate content (W of phycocolloid)
G P Hm Hc
1.3 9.0 14 .o
2.0 1.7 15.0 15.0
-
2.0 15.0 7.0
Gel strength (g cm-‘)
G
-
-
-
P Hm Hc
340 <14 14
950 15 26
100 250 -
440 -
28
14
G
-
-
Agar or Carrageenan (4’0of dry weight)
Melting temperature (“C)
Gelling temperature (“C)
P Hm Hc G P Hm Hc
93 27 26
92
23 25
-
-
30 7 8
37.1 24.0
97 30 32
-
28 25 6
-
-
78 99
25 32
-
120 150 -
81 99
27 10 12
125 200 -
-
-
26
27 10 8
75 95
10.5 11.0 15.0
Jan.
-
-
-
85 92
28 29
-
-
28 21
13.2 f 10.1 23.5 f 6.1
27.7 + 12.7 34.9 t 7.1
33.7 * 12.9 38.4 f 1.1
Cultured Natural
Cultured Natural
Cultured Natural
Pterocladia capillacea
Hypnea musciformis
Hypnea cornu ta
* Significant at the level of P <0.05.
22.1 f 2.5 23.8 f 2.3
23.3 f 2.3 22.1 f 0.1
34.7 f 4.6 42.5 t 2.5
32.7 f 2.8 35.3 +_0.9
3,6 anhydrogaiactose (%)
10.86 + 5.93 9.44 f 0.25
8.62 f 4.21 14.22 + 3.87*
1.77 f 0.46 1.86 f 0.13
1.45 f 0.50 1.75 f 0.49
Sulfate (%)
** Significant at the level of P < 0.01
2.2 2.9**
10.1 + 18.3 f
Cultured Natural
Gracilaria sp.
Agar or carrageenan (%)
Source
Species
19.4 t 16.4 f
12.5 +_ 32.1 f
5.9 2.9
3.5 2.5**
392.8 + 268.6 526.0 f 107.8
113.7 +_ 11.1 122.2 f 49.1
Gel strength (g cm-*)
26.0 f 4.5 28.7 f 8.1
27.0 t 1.4 27.0 f. 3.5
95.3 f; 3.2 90.0 t 3.0
79.7 f 4.2 78.3 f 8.0
8.6 + 2.7 7.7 f 2.4
9.0 f 1.7 9.1 f 1.9
27.4 + 3.6 27.0 f 1.3
27.3 + 1.5 25.5 f 3.9
Gelling temp. (“C)
of four seaweeds:
Melting temp. (“C)
Comparison of chemical constituants and physical properties of cultured and natural populations Gracilaria sp., Pterocladia capillacea, Hypnea musciformis and H. cornuta (average + S.D.)
TABLE IV
63
tent coincided with high sulfate content in the carrageenan extracts. Gel strength of Gracilaria sp. agar fluctuated around 100 g cmm2, while that of P. capillacea agar was considerably higher and fluctuated much more, with a maximum of 950 g cm-‘. Carrageenan from Hypnea species had low gel strengths ranging between 10 and 28 g cmm2. Melting and gelling temperatures were considerably higher in the extracts of agar, with minor seasonal fluctuations in all four seaweeds. The agar of P. capillacea showed higher melting temperatures than the agar of Gracilaria sp. which coincided with the higher gel strength of the agar of P. capilhcea. High melting temperatures in the agar extracts coincided with high gel strength, high 3,6 anhydrogalactose and low sulfate content. In the carrageenan extracts low gelling and melting temperatures coincided with low gel strength, low 3,6 anhydrogalactose and high sulfate content. Chemical compositions and physical properties of cultured and natural populations of the four species were similar, except for three differences: agar content of Gracilaria sp. (P
Light intensity was determined at noon once a week. The data are similar to previous years and show the seasonal changes in available light over the year. Growth rates of Gracilaria sp. had a positive relationship to changes in temperature, but not to light intensity (Table I), suggesting light saturation. A positive relationship between growth and light intensity was shown in field studies of G. uerrucosa (Hudson) Papenfuss (Whyte et al., 1981), and in tank cultures of G. tikuahiae McLachlan (as G. foliifera var. angustissima) (Lapointe, 1981) and G. tikvakiae (changed name for G. foliifera; Lapointe and Ryther, 1978). These contradictions to our data may be caused by a cold climate ecotype in the first case, and by a much higher growth rate in tanks in the second case. However, the S.G.R. of P. capillacea did positively relate to both temperatures and light intensity (Fig. 1, Table I). Both agarophytes showed almost the same maximum S.G.R. of 0.07-0.08 doublings per day, but P. capilhcea reached a 50% higher annual yield (Table II) because of an extended time of high level S.G.R. The effective culturing season in the field would be April-September and April-November for Gracilaria sp. and P. capillacea, respectively. A g-month growing season (April-December) of more than 0.05 doublings per day, was positively related in both Hypnea species to temperature and to light intensity (Fig. 1, Table I). These relationships confirm the results of studies with H. musciformis in tank cultures by Lapointe et al. (1976). Both Hypnea species had 2-3 times higher annual yields than the agarophytes (Table II), and Hypnea cornuta reached the highest specific growth rate of 0.19 doublings per day. The relatively low S.G.R. of the sea-
64
weeds in this experiment did not express their maximal growth potential, because no water flow, nutrient enrichment and other means of enhancement were used in the cultures. In a preliminary experiment in which the density of H. cornutu was doubled, maximum annual yield was increased by 60% (data not shown). A warm sea water supply (22°C) can extend the growing season to more than 10 months a year, as shown by reinitiation of growth 3 months earlier, when compared to the regular pond (Fig. 1 A,B). The higher correlation of S.G.R. with temperature in the carrageenophytes (Table I) may be caused by the rapid increase in growth in January and February, when compared to the agarophytes in the same experiment (Fig. 1B). The attached plants showed higher annual yield than the free-floating ones (Table II), perhaps due to shading effects by the detached plants gathered in a small section of the basket (Friedlander and Lipkin, 1982). Seasonal variations in S.G.R., chemical composition and physical properties, which were observed in the seaweed cultures (Table III), confirm similar changes in G. uerrucosa (Whyte et al., 1981), G. tikuahiae and Neoagardhiella baileyi (Harvey ex Kiitzing) Wynne and Taylor (Asare, 1980), H. musciformis (Durako and Dawes, 1980), Eucheuma species (Dawes, 1977), and G. bursapastoris (Gmelin) Silva and G. coronopifolia J. Agardh (Hoyle, 1978). The major relationship found during the main growing season in this study was that S.G.R. was positively related to phycocolloid content (Fig. 1, Table III). This relationship differs from the inverse correlation between growth rate and phycocolloid accumulation found in nitrogen-enriched H. musciformis (Guist et al., 1982), and G. tikuahiae (Bird et al., 1981) and between biomass and agar content in vegetative and tetrasporic plants of G. uerrucosa (Whyte et al., 1981). However, these correlations refer either to higher levels of growth rate obtained under nutrient enrichment which decreased phycocolloid accumulation or to distinctly different environmental systems. The positive relationship of 3,6 anhydrogalactose content with gel strength, and gelling and melting temperatures, and the inverse relationship with sulfate content, in these polysaccharides (Table III), confirm the model described by Yaphe and Duckworth (1972), in which high gel strength was due to lower sulfate and higher 3,6 anhydrogalactose contents. The considerably higher gel strength of P. capillacea as compared to Gracilaria sp. was not reflected by a comparable difference in sulfate content, but was certainly affected by higher content of 3,6 anhydrogalactose (Table III). It might also be affected by longer polymer molecules (Selby and Wynne, 1973), as suggested by a higher melting temperature. The average chemical composition and physical properties in both cultured and natural populations of seaweeds were similar (Table IV).
65 ACKNOWLEDGEMENTS
The authors wish to thank Prof. C.J. Dawes (Department of Biology of the University of South Florida) for reading the manuscript and making constructive comments, and Prof. J. Ryther (Harbor Branch Institution, Florida) for reviewing the manuscript. Thanks are also given to Mr. E. Cohen (Department of Botany, Tel Aviv University) for assistance in the field experiments. This research was supported by “Ramot” (The Cooperative Fund of Tel Aviv University and the Ministry of Industry and Commerce), and by The National Council for Research and Development of Israel.
REFERENCES Asare, S.D., 1980. Seasonal changes in rmlphate and 3,6 anhydrogalactose content in phycocolloids from two red algae. Bot. Mar., 23: 595-598. Bird, N.L., Chen, L.C.M. and McLachlan, J., 1979. Effects of temperature, light and salinity on growth in culture of Chondrus crispus, Furcellaria lumbricalis, Gracilaria tikvahiae and Fucus serratus. Bot. Mar., 22: 521-527. Bird, K.T., Hanisak, M.D. and Ryther, J., 1981. Chemical quality and production of different nitrogen enrichment agars extracted from Gracilaria tikvahiae grown in conditions. Bot. Mar., 24: 441-444. Bird, K.T., Habig, C. and DeBusk, T., 1982. Nitrogen allocation and storage patterns in Gracilaria tikvahiae. J. Phycol., 18: 344-348. Dawes, C.J., 1977. Seasonal reproductive aspects of plant chemistry and icarrageenan from Floridian Eucheuma. Bot. Mar., 20: 137-147. Dawes, C.J., Lawrence, J.M., Cheney, D.P. and Mathieson, A.C., 1974. Ecological studies of Floridian Eucheuma. 3: Seasonal variations of carrageenan, total carbohydrate, protein and lipid. Bull. Mar. Sci., 24: 286-299. DeBoer, J.A., Guigly, H.J., Israel, T.L. and D’Elia, CF., 1978. Nutritional studies of red algae. I. Growth rate as a function of nitrogen source and concentration. J. Phycol., 14: 261-266. Doty, M.S., 1979. Status of marine agronomy with special reference to the tropics. Proc. Int. Seaweed Symp., 9: 35-38. Dubois, M.K., Gilles, A., Hamilton, J.K., Reber, P.A. and Smith, F., 1956. Calorimetric methods for the determination of sugars and related substances. Anal. Chem., 28: 350-356. Durako, M.J. and Dawes, C.J., 1980. A comparative seasonal study of two populations of Hypnea musciformis from the east and west coasts of Florida. 1. Growth and chemistry. Mar. Biol., 59: 151-156. Friedlander, M. and Lipkin, Y., 1982. Rearing agarophytes and carrageenophytes under field conditions in the east Mediterranean. Bot. Mar., 25: 101-105. Friedlander, M., Lipkin, Y. and Yaphe, W., 1981. Composition of agars from Gracilaria cf. verrucosa and Pterocladia capillacea. Bot. Mar., 24: 595-598. Guist, G.G. Jr., Dawes, C.J. and Castle, J.R., 1982. Mariculture of the red seaweed Hypnea musciformis. Aquaculture, 28: 375-384. Hanisak, M.D., 1979. Growth pattern of Codium fragile ssp. tomentosoides in response to temperature, irradiance, salinity and nitrogen source. Mar. Biol., 50: 319-332. Hoyle, M.D., 1978. Agar studies in two Gracilaria spp. from Hawaii. 2: Seasonal aspects. Bot. Mar., 21: 347-352.
Jackson, S.G. and McCandless, E.L., 1978. Simple, rapid, turbidometric determination of inorganic sulfate and/or protein. Anal. Biochem., 90: 802-808. Lapointe, B.E., 1981. The effect of light and nitrogen on growth, pigment content and biochemical composition of Gracilaria foliifera v. angustissima. J. Phycol., 17: 90-95. Lapointe, B.E. and Ryther, J.H., 1978. Some aspects of the growth and yield of Gracilaria tikvahiae in culture. Aquaculture, 15: 185-193. Lapointe, B.E. and Ryther, J.H., 1979. The effect of nitrogen and seawater flow rate on the growth and biochemical composition of Gracilaria foliifera v. angustissima in mass outdoor cultures. Bot. Mar., 22: 529-537. Lapointe, B.E., Williams, L.D., Goldman, J.C. and Ryther, J.H., 1976. The mass outdoor culture of macroscopic algae. Aquaculture, 8: 9-20. Mshigeni, K.E., 1976. Field cultivation of Hypnea spores for carrageenan: prospects and problems. Bot. Mar., 19: 227-230. Parker, H.S., 1982. Effect of simulated current on the growth rate and nitrogen metabolism of Gracilaria tikuahiae. Mar. Biol., 69: 137-149. Rama Rao, K., 1970. Studies in growth cycle and phycocolloid content in Hypnea musciformis. Bot. Mar., 163-165. Ryther, J.H., Corwin, N., DeBusk, T.A. and Williams, L.D., 1981. Nitrogen uptake and storage by the red algae Gracilaria tikvahiae. Aquaculture, 26: 107-115. Selby, H.H. and Wynne, W.H., 1973. Agar. In: R.L. Whistler (Editor), Industrial Gums, Polysaccharides and their Derivatives. Academic Press, New York, pp. 29-48. Simpson, F.J. and Shachlock, P.E., 1979. The cultivation of Chondrus crispus. Effect of temperature on growth and carrageenan production. Bot. Mar., 22: 295-298. Whyte, J.N.C., Englar, J.R., Saunders, R.G. and Lindsay, J.C., 1981. Seasonal variations in the biomass, quantity and quality of agar from the reproductive and vegetative stages of Gracilaria (uerrucosa type). Bot. Mar., 24: 493-501. Yaphe, W. and Duckworth, M., 1972. The relationship between structures and biological properties of agars. Proc. Int. Seaweed Symp., 7 : 16-22. Young, K., Duckworth, M. and Yaphe, W., 1971. The structure of agar. 3: Pyruvic acid, a common feature of agars from different agarophytes. Carbohydr. Res., 16: 446-448.
57 - Printed
in The Netherlands
GROWTH RATES, PHYCOCOLLOID YIELD AND QUALITY OF THE RED SEAWEEDS, GRACILARIA SP., PTEROCLADIA CAPILLACEA, HYPNEA MUSCIFORMIS, AND HYPNEA CORNUTA, IN FIELD STUDIES IN ISRAEL
M. FRIEDLANDER
and N. ZELIKOVITCH
Department of Botany, Tel Aviv (Israel) (Accepted
28 October
The George S. Wise Faculty of Life Sciences,
Tel Aviv University,
1983)
ABSTRACT Friedlander, M. and Zelikovitch, N., 1984. Growth rates, phycocolloid yield and quality of the red seaweeds, Gracilaria sp., Pterocladia capillacea, Hypnea musciformis, and Hypnea cornuta, in field studies in Israel. Aquaculture, 40: 57-66. The agarophytes Gracilaria sp. and Pterocladia capillacea, and the carrageenophytes Hypnea musciformis and H. cornuta were cultured under field conditions. Specific growth rates appeared to be related to temperature and light intensity. Attached seaweeds had higher annual yields per mz than free-floating samples. The specific growth rate of all four seaweeds was positively related to phycocolloid content in the main growing season. In agar extracts high gel strength coincided with high 3,6 anhydrogalactose content, with high gelling and melting temperature, and with low sulfate content. In the carrageenan extracts low gel strength coincided with low 3,6 anhydrogalactose content, with low gelling and melting temperatures and with high sulfate content. Chemical composition and physical properties of cultured and natural seaweeds were almost the same.
INTRODUCTION
The possibility of tropical seaweed mariculture as a source of phycocolloids (Doty, 1979) has been suggested for the eastern Mediterranean (Friedlander and Lipkin, 1982). In previous studies, seaweed growth rates in the field have been shown to be highly variable due to fluctuations of natural factors (Rama Rao, 1970; Dawes et al., 1974; Durako and Dawes, 1980; Guist et al., 1982). Furthermore, phycocolloid extract such as agar and carrageenan are known to be affected by seasonal changes (Dawes et al., 1974; Hoyle, 1978; Mshigeni, 1976; Simpson and Shachlock, 1979). Chemical composition as well as physical properties of phycocolloids also change with environmental conditions (Asare, 1980; Whyte et al., 1981; Bird et al., 1981). The dominant factors affecting seaweed growth in tanks appear to be: light intensity, temperature, salinity (Bird et al., 1979; Hanisak, 1979;
0044-8486/84/$03.00
o 1984 Elsevier
Science
Publishers
B.V.
58
Lapointe, 1981), nitrogen enrichment (DeBoer et al., 1978; Lapointe and Ryther, 1979; Ryther et al., 1981; Bird et al., 1981, 1982), water motion (Parker, 1982) and plant density (Lapointe and Ryther, 1978). Little information is available regarding effects of fluctuating field conditions on growth. The present study was undertaken to determine growth rates and phycocolloid yield and quality under changing field conditions, and to compare these relationships with the variations found in previous studies. MATERIALS
AND METHODS
Gracilariu sp. (not yet identified, voucher specimen at the Tel Aviv University herbarium), Pterocladiu cupillacea (Gmelin) Born. et Thur., Hypnea musciformis (Wulfen) Lamouroux and H. cornuta (Lamouroux) J. Agardh, were collected at Mikhmoret on the eastern Mediterranean coast during April 1981 to April 1982. The main experimental site was a sea water pond of 0.5 ha area, near the coast of Ma’agan Michael. A second pond of 400 m* received warm sea water from a power station in the winter, with a turnover of less than one volume per day. The ponds were 1 m deep with sandy bottoms. The seaweeds were separated into 50 pieces of ca. 2 g each, and tied to ropes on a 1 X 1 m polypropylene tube frame. The frame was placed in an anchored net basket of the same surface area, 25 cm under water. New plants were established bimonthly because of epiphytization. In a second experiment samples of 100 g were placed in baskets of the same size without tying. All experiments were conducted without additional water motion or nutrient enrichment. The sea water had a stable salinity (33%). Environmental factors were measured weekly at noon time. Water temperature and salinity were determined with a S.C.T. meter (Y.S.I. model 33); light intensity was measured with a LI-170 quantum meter (LI-COR). Damp dried seaweeds were weighed biweekly and then returned to their initial weight. Specific growth rate (S.G.R.) was calculated as M= doublings per day [p = l/t X log (N/N,) X 3.32, where t is the number of days between primary fresh weight (No ) and final fresh weight (iV)] . Seaweed samples were collected every l-3 months for phycocolloid analysis from the main experimental site and from natural populations. Dry weight was measured after air drying and oven drying at 60°C for 24 h. Agar and carrageenan were extracted as described by Young et al. (1971). Gel strength, melting and dynamic gelling temperatures were measured as described by Friedlander and Lipkin (1982). D-galactose content was measured by a modification of the phenolsulfuric acid test (Dubois et al., 1956), 3,6 anhydrogalactose was measured by a modification of the resorcinol test (Friedlander et al., 1981). Sulfate content was measured by a micromethod of Jackson and McCandless (1978).
59
RESULTS
The specific growth rate (S.G.R.) of the attached seaweeds and its relationship with temperature and light intensity is shown in Fig. 1 and Table I. Gracilaria sp. reached a maximum of 0.07 doublings per day in August, with a positive correlation with temperature (P <0.05). Pterocladiu capitlacea reached a maximum of 0.08 doublings per day in April, and similar but noncontinuous rates in the following summer, with a positive correlation with temperature and light intensity (P <0.05). Hypnea musciformis reached two maxima of 0.12 and 0.09 doublings per day in July and November, respectively. Hypnea cornutu reached a maximum of 0.19 doublings per day in July, which was the highest S.G.R. observed. Positive correlations of S.G.R. with temperature and light intensity (P
S.G.R. versus temperature
S.G.R. versus light intensity
Gracilaria sp. Pterocladia capillacea Hypnea musciformis Hypnea cornuta
0.617* 0.692* 0.805** 0.720**
0.420 0.539* 0.728** 0.737**
* Significant at the level of P <0.05.
** Significant at the level of P
Carrageenan content (11-55s) was higher than agar content (5-32s) in the respective seaweeds, and carrageenan content of H. cornu tu was generally higher than that of H. musciformis (Table III). All four species had a decrease in phycocolloid content from early summer to fall and showed a positive relationship between S.G.R. and phycocolloid content during that period. The 3,6 anhydrogalactose content in the extracts of agar was 30-43s (the higher values were in P. capillacea) and increased from summer to fall, with a final decrease towards winter. The 3,6 anhydrogalactose content of the extracts of carrageenan was lower than that of agar and fluctuated in a range of 18-
60
25%. Sulfate content of extracts of agar fluctuated between 1 and 2%, that of carrageenan showed a considerably higher range of values with fluctuations (4-27%). High 3,6 anhydrogalactose content coincided low sulfate content in the agar extracts, while low 3,6 anhydrogalactose
AMJ 1981
while wider with con-
JASONOJFMA 1982
Fig. 1. Annual temperature and light intensity profile, and S.G.R. variations (doublings per day f S.E.) of four cultured seaweeds in: (A) Regular sea water pond (n = 50); (B) Heated sea water pond (n = 10). Gracilario sp. (G); P. capillaceo (P); H. musciformis (Hm); H. cotnutu (Hc).
61 TABLE II Annual yield of attached and free floating Gracilaria sp., Pterocladia musciformis and H. cornuta in culture. Fifty single pieces of 2 g each and 100 g of seaweeds formed the free-floating inoculum in two 1 x density per ml). Yields were calculated as g fresh weight m-* year-* * Species
Attached
Free-floating
Gracilaria sp. Pterocladia capillacea H. musciformis H. cornuta
872+ 70 1272 f 140 2444 r 220 2828 f 339
506 414 1314 1758
capillacea, Hypnea were tied to ropes, 1 m baskets (same 1 S.D.
* 116 f 87 f 236 t 264
TABLE III Comparative seasonal variations of chemical constituants and physicd properties of four regularly cultured seaweeds: Gracilaria sp. (G), Pterocladia capillacea (P), Hypnea musciformis (Hm) and H. cornu to (Hc) Species
Month Jun. Jul.
Aug.
Oct.
Nov.
Dec.
13.0 11.0 24.0 42.0
-
7.5 5.5 15.0 38.0
10.0 10.0 6.5 20.0 25.0
5.5 34.0 28.0
33.7 42.7 24.8 20.9
36.2 33.7 24.7 20.7
33.5 29.8 21.9 18.6
1.2 8.0 8.0
0.8 1.6 6.0 27.0
1.4 2.5 6.0 5.0
1.6 2.1 4.0 6.0
110 420 -
G
-
P Hm Hc
22.0 43.0 33.0
32.0 25.0 55.0
3,6 anhydrogalactose (% of phycocolloid)
G P Hm Hc
-
37.2 20.7 23.3
29.6 34.0 20.8 23.2
-
35.0 22.3 24.4
Sulfate content (W of phycocolloid)
G P Hm Hc
1.3 9.0 14 .o
2.0 1.7 15.0 15.0
-
2.0 15.0 7.0
Gel strength (g cm-‘)
G
-
-
-
P Hm Hc
340 <14 14
950 15 26
100 250 -
440 -
28
14
G
-
-
Agar or Carrageenan (4’0of dry weight)
Melting temperature (“C)
Gelling temperature (“C)
P Hm Hc G P Hm Hc
93 27 26
92
23 25
-
-
30 7 8
37.1 24.0
97 30 32
-
28 25 6
-
-
78 99
25 32
-
120 150 -
81 99
27 10 12
125 200 -
-
-
26
27 10 8
75 95
10.5 11.0 15.0
Jan.
-
-
-
85 92
28 29
-
-
28 21
13.2 f 10.1 23.5 f 6.1
27.7 + 12.7 34.9 t 7.1
33.7 * 12.9 38.4 f 1.1
Cultured Natural
Cultured Natural
Cultured Natural
Pterocladia capillacea
Hypnea musciformis
Hypnea cornu ta
* Significant at the level of P <0.05.
22.1 f 2.5 23.8 f 2.3
23.3 f 2.3 22.1 f 0.1
34.7 f 4.6 42.5 t 2.5
32.7 f 2.8 35.3 +_0.9
3,6 anhydrogaiactose (%)
10.86 + 5.93 9.44 f 0.25
8.62 f 4.21 14.22 + 3.87*
1.77 f 0.46 1.86 f 0.13
1.45 f 0.50 1.75 f 0.49
Sulfate (%)
** Significant at the level of P < 0.01
2.2 2.9**
10.1 + 18.3 f
Cultured Natural
Gracilaria sp.
Agar or carrageenan (%)
Source
Species
19.4 t 16.4 f
12.5 +_ 32.1 f
5.9 2.9
3.5 2.5**
392.8 + 268.6 526.0 f 107.8
113.7 +_ 11.1 122.2 f 49.1
Gel strength (g cm-*)
26.0 f 4.5 28.7 f 8.1
27.0 t 1.4 27.0 f. 3.5
95.3 f; 3.2 90.0 t 3.0
79.7 f 4.2 78.3 f 8.0
8.6 + 2.7 7.7 f 2.4
9.0 f 1.7 9.1 f 1.9
27.4 + 3.6 27.0 f 1.3
27.3 + 1.5 25.5 f 3.9
Gelling temp. (“C)
of four seaweeds:
Melting temp. (“C)
Comparison of chemical constituants and physical properties of cultured and natural populations Gracilaria sp., Pterocladia capillacea, Hypnea musciformis and H. cornuta (average + S.D.)
TABLE IV
63
tent coincided with high sulfate content in the carrageenan extracts. Gel strength of Gracilaria sp. agar fluctuated around 100 g cmm2, while that of P. capillacea agar was considerably higher and fluctuated much more, with a maximum of 950 g cm-‘. Carrageenan from Hypnea species had low gel strengths ranging between 10 and 28 g cmm2. Melting and gelling temperatures were considerably higher in the extracts of agar, with minor seasonal fluctuations in all four seaweeds. The agar of P. capillacea showed higher melting temperatures than the agar of Gracilaria sp. which coincided with the higher gel strength of the agar of P. capilhcea. High melting temperatures in the agar extracts coincided with high gel strength, high 3,6 anhydrogalactose and low sulfate content. In the carrageenan extracts low gelling and melting temperatures coincided with low gel strength, low 3,6 anhydrogalactose and high sulfate content. Chemical compositions and physical properties of cultured and natural populations of the four species were similar, except for three differences: agar content of Gracilaria sp. (P
Light intensity was determined at noon once a week. The data are similar to previous years and show the seasonal changes in available light over the year. Growth rates of Gracilaria sp. had a positive relationship to changes in temperature, but not to light intensity (Table I), suggesting light saturation. A positive relationship between growth and light intensity was shown in field studies of G. uerrucosa (Hudson) Papenfuss (Whyte et al., 1981), and in tank cultures of G. tikuahiae McLachlan (as G. foliifera var. angustissima) (Lapointe, 1981) and G. tikvakiae (changed name for G. foliifera; Lapointe and Ryther, 1978). These contradictions to our data may be caused by a cold climate ecotype in the first case, and by a much higher growth rate in tanks in the second case. However, the S.G.R. of P. capillacea did positively relate to both temperatures and light intensity (Fig. 1, Table I). Both agarophytes showed almost the same maximum S.G.R. of 0.07-0.08 doublings per day, but P. capilhcea reached a 50% higher annual yield (Table II) because of an extended time of high level S.G.R. The effective culturing season in the field would be April-September and April-November for Gracilaria sp. and P. capillacea, respectively. A g-month growing season (April-December) of more than 0.05 doublings per day, was positively related in both Hypnea species to temperature and to light intensity (Fig. 1, Table I). These relationships confirm the results of studies with H. musciformis in tank cultures by Lapointe et al. (1976). Both Hypnea species had 2-3 times higher annual yields than the agarophytes (Table II), and Hypnea cornuta reached the highest specific growth rate of 0.19 doublings per day. The relatively low S.G.R. of the sea-
64
weeds in this experiment did not express their maximal growth potential, because no water flow, nutrient enrichment and other means of enhancement were used in the cultures. In a preliminary experiment in which the density of H. cornutu was doubled, maximum annual yield was increased by 60% (data not shown). A warm sea water supply (22°C) can extend the growing season to more than 10 months a year, as shown by reinitiation of growth 3 months earlier, when compared to the regular pond (Fig. 1 A,B). The higher correlation of S.G.R. with temperature in the carrageenophytes (Table I) may be caused by the rapid increase in growth in January and February, when compared to the agarophytes in the same experiment (Fig. 1B). The attached plants showed higher annual yield than the free-floating ones (Table II), perhaps due to shading effects by the detached plants gathered in a small section of the basket (Friedlander and Lipkin, 1982). Seasonal variations in S.G.R., chemical composition and physical properties, which were observed in the seaweed cultures (Table III), confirm similar changes in G. uerrucosa (Whyte et al., 1981), G. tikuahiae and Neoagardhiella baileyi (Harvey ex Kiitzing) Wynne and Taylor (Asare, 1980), H. musciformis (Durako and Dawes, 1980), Eucheuma species (Dawes, 1977), and G. bursapastoris (Gmelin) Silva and G. coronopifolia J. Agardh (Hoyle, 1978). The major relationship found during the main growing season in this study was that S.G.R. was positively related to phycocolloid content (Fig. 1, Table III). This relationship differs from the inverse correlation between growth rate and phycocolloid accumulation found in nitrogen-enriched H. musciformis (Guist et al., 1982), and G. tikuahiae (Bird et al., 1981) and between biomass and agar content in vegetative and tetrasporic plants of G. uerrucosa (Whyte et al., 1981). However, these correlations refer either to higher levels of growth rate obtained under nutrient enrichment which decreased phycocolloid accumulation or to distinctly different environmental systems. The positive relationship of 3,6 anhydrogalactose content with gel strength, and gelling and melting temperatures, and the inverse relationship with sulfate content, in these polysaccharides (Table III), confirm the model described by Yaphe and Duckworth (1972), in which high gel strength was due to lower sulfate and higher 3,6 anhydrogalactose contents. The considerably higher gel strength of P. capillacea as compared to Gracilaria sp. was not reflected by a comparable difference in sulfate content, but was certainly affected by higher content of 3,6 anhydrogalactose (Table III). It might also be affected by longer polymer molecules (Selby and Wynne, 1973), as suggested by a higher melting temperature. The average chemical composition and physical properties in both cultured and natural populations of seaweeds were similar (Table IV).
65 ACKNOWLEDGEMENTS
The authors wish to thank Prof. C.J. Dawes (Department of Biology of the University of South Florida) for reading the manuscript and making constructive comments, and Prof. J. Ryther (Harbor Branch Institution, Florida) for reviewing the manuscript. Thanks are also given to Mr. E. Cohen (Department of Botany, Tel Aviv University) for assistance in the field experiments. This research was supported by “Ramot” (The Cooperative Fund of Tel Aviv University and the Ministry of Industry and Commerce), and by The National Council for Research and Development of Israel.
REFERENCES Asare, S.D., 1980. Seasonal changes in rmlphate and 3,6 anhydrogalactose content in phycocolloids from two red algae. Bot. Mar., 23: 595-598. Bird, N.L., Chen, L.C.M. and McLachlan, J., 1979. Effects of temperature, light and salinity on growth in culture of Chondrus crispus, Furcellaria lumbricalis, Gracilaria tikvahiae and Fucus serratus. Bot. Mar., 22: 521-527. Bird, K.T., Hanisak, M.D. and Ryther, J., 1981. Chemical quality and production of different nitrogen enrichment agars extracted from Gracilaria tikvahiae grown in conditions. Bot. Mar., 24: 441-444. Bird, K.T., Habig, C. and DeBusk, T., 1982. Nitrogen allocation and storage patterns in Gracilaria tikvahiae. J. Phycol., 18: 344-348. Dawes, C.J., 1977. Seasonal reproductive aspects of plant chemistry and icarrageenan from Floridian Eucheuma. Bot. Mar., 20: 137-147. Dawes, C.J., Lawrence, J.M., Cheney, D.P. and Mathieson, A.C., 1974. Ecological studies of Floridian Eucheuma. 3: Seasonal variations of carrageenan, total carbohydrate, protein and lipid. Bull. Mar. Sci., 24: 286-299. DeBoer, J.A., Guigly, H.J., Israel, T.L. and D’Elia, CF., 1978. Nutritional studies of red algae. I. Growth rate as a function of nitrogen source and concentration. J. Phycol., 14: 261-266. Doty, M.S., 1979. Status of marine agronomy with special reference to the tropics. Proc. Int. Seaweed Symp., 9: 35-38. Dubois, M.K., Gilles, A., Hamilton, J.K., Reber, P.A. and Smith, F., 1956. Calorimetric methods for the determination of sugars and related substances. Anal. Chem., 28: 350-356. Durako, M.J. and Dawes, C.J., 1980. A comparative seasonal study of two populations of Hypnea musciformis from the east and west coasts of Florida. 1. Growth and chemistry. Mar. Biol., 59: 151-156. Friedlander, M. and Lipkin, Y., 1982. Rearing agarophytes and carrageenophytes under field conditions in the east Mediterranean. Bot. Mar., 25: 101-105. Friedlander, M., Lipkin, Y. and Yaphe, W., 1981. Composition of agars from Gracilaria cf. verrucosa and Pterocladia capillacea. Bot. Mar., 24: 595-598. Guist, G.G. Jr., Dawes, C.J. and Castle, J.R., 1982. Mariculture of the red seaweed Hypnea musciformis. Aquaculture, 28: 375-384. Hanisak, M.D., 1979. Growth pattern of Codium fragile ssp. tomentosoides in response to temperature, irradiance, salinity and nitrogen source. Mar. Biol., 50: 319-332. Hoyle, M.D., 1978. Agar studies in two Gracilaria spp. from Hawaii. 2: Seasonal aspects. Bot. Mar., 21: 347-352.
Jackson, S.G. and McCandless, E.L., 1978. Simple, rapid, turbidometric determination of inorganic sulfate and/or protein. Anal. Biochem., 90: 802-808. Lapointe, B.E., 1981. The effect of light and nitrogen on growth, pigment content and biochemical composition of Gracilaria foliifera v. angustissima. J. Phycol., 17: 90-95. Lapointe, B.E. and Ryther, J.H., 1978. Some aspects of the growth and yield of Gracilaria tikvahiae in culture. Aquaculture, 15: 185-193. Lapointe, B.E. and Ryther, J.H., 1979. The effect of nitrogen and seawater flow rate on the growth and biochemical composition of Gracilaria foliifera v. angustissima in mass outdoor cultures. Bot. Mar., 22: 529-537. Lapointe, B.E., Williams, L.D., Goldman, J.C. and Ryther, J.H., 1976. The mass outdoor culture of macroscopic algae. Aquaculture, 8: 9-20. Mshigeni, K.E., 1976. Field cultivation of Hypnea spores for carrageenan: prospects and problems. Bot. Mar., 19: 227-230. Parker, H.S., 1982. Effect of simulated current on the growth rate and nitrogen metabolism of Gracilaria tikuahiae. Mar. Biol., 69: 137-149. Rama Rao, K., 1970. Studies in growth cycle and phycocolloid content in Hypnea musciformis. Bot. Mar., 163-165. Ryther, J.H., Corwin, N., DeBusk, T.A. and Williams, L.D., 1981. Nitrogen uptake and storage by the red algae Gracilaria tikvahiae. Aquaculture, 26: 107-115. Selby, H.H. and Wynne, W.H., 1973. Agar. In: R.L. Whistler (Editor), Industrial Gums, Polysaccharides and their Derivatives. Academic Press, New York, pp. 29-48. Simpson, F.J. and Shachlock, P.E., 1979. The cultivation of Chondrus crispus. Effect of temperature on growth and carrageenan production. Bot. Mar., 22: 295-298. Whyte, J.N.C., Englar, J.R., Saunders, R.G. and Lindsay, J.C., 1981. Seasonal variations in the biomass, quantity and quality of agar from the reproductive and vegetative stages of Gracilaria (uerrucosa type). Bot. Mar., 24: 493-501. Yaphe, W. and Duckworth, M., 1972. The relationship between structures and biological properties of agars. Proc. Int. Seaweed Symp., 7 : 16-22. Young, K., Duckworth, M. and Yaphe, W., 1971. The structure of agar. 3: Pyruvic acid, a common feature of agars from different agarophytes. Carbohydr. Res., 16: 446-448.