Aquacult Int (2008) 16:85–90 DOI 10.1007/s10499-007-9125-y
Experimental evaluation of a composted seaweed extract as microalgal culture media Daniela Alvarado Æ Esperanza Buitrago Æ Marı´a Sole´ Æ Khenia Frontado
Received: 7 February 2007 / Accepted: 20 June 2007 / Published online: 2 August 2007 Springer Science+Business Media B.V. 2007
Abstract An organic culture media based on a composted liquid seaweed extract was developed and tested for growth of Chaetoceros muelleri. The extract was evaluated at two concentrations and then compared with two commercial media: Walne and agricultural fertilizer (AF). The concentrations of nitrogen and phosphorous differed between the two commercial media and the seaweed extract; however, growth of Chaetoceros muelleri was similar. The successful culture of Chaetoceros muelleri in composted liquid seaweed extract suggests the feasibility of and potential use for this natural, organic fertilizer in aquaculture. The seaweed extract is simple to obtain and inexpensive. Keywords
Chaetoceros muelleri Culture media Marine diatom Seaweed extract
Introduction Beach-cast macroalgae and seagrasses in tropical, subtropical and temperate regions as consequence of hydrodynamic activity are well documented (Lemus and Balza 1995; Ortiz and Alvarez 1998). In Venezuela, beach-cast material is frequently found on the northeastern shores of Margarita Island. This is a consequence of the local coastal geomorphology and the prevailing northeasterly winds. The use of this accumulated material to create a seaweed extract represents an innovative application for large quantities of beachcast seaweed. The seaweed extract could be used in culture media of microalgae which are utilized in aquaculture as live feed for molluscs, larval stages of crustaceans and some fish. Mass production of microalgae is frequently carried out by utilizing commercial inorganic fertilizers (De Pauw et al. 1983), which generally are laboratory prepared culture media of
D. Alvarado (&) E. Buitrago M. Sole´ K. Frontado Estacio´n de Investigaciones Marinas, Apdo. 144, Pta de Piedras, Isla de Margarita 6301, Venezuela e-mail:
[email protected]
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relatively high economic cost (Soeder 1979). However, trials using low-cost alternative organic fertilizers have been carried out. Panigua and Buckle (1985) used liquid extract to culture Monochrysis lutheri and Skeletonema costatum; their results indicated that the nutrients contained in the seaweed extract were sufficient to support a growth up to 79% when compared to control media. Also, Valenzuela et al. (2002) concluded that there is no difference between proximate composition and growth of the microalgae Isochrysis aff. galbana obtained with agricultural fertilizer and f/2 media. The aim of this study is to evaluate the application of seaweed extract as a nutrient source for culturing for microalgae. Chaetoceros muelleri was chosen as the test organism due to its frequent use in aquaculture. The development of a low cost method for largescale culture of C. muelleri would be of great benefit to the marine aquaculture industry worldwide.
Materials and methods Preparation of the liquid seaweed extract Beach-cast seaweed was composed of 99% brown macroalgae, including Sargassum filipendula var. filipendula C. Agardh, S. filipendula var. montagni (Bailey) Grunow, S. furcatum Ku¨tzing, S. rigidulum Ku¨tzing and S. vulgare C. Agardh. In addition there was (1%) green and red macroalgae as well as the marine seagrass Thalassia testudinum Banks & Solander ex Ku¨tzing. Beach-cast material was washed twice with fresh water to eliminate excess salt and sand. The material was then cut in small pieces, around 1 cm · 1 cm, to facilitate the decomposition process. The pieces were mixed and covered with a plastic, non-transparent cover to maintain compost humidity. The compost was aerated and swirled every 2 days for 12 weeks. Subsequently, 1 kg of the compost was filtered by using a funnel, an Advantec Mfs1 Inc. grade No. 1 filter paper, and 200 ml of distilled water. The liquid filtered was newly added to the mass compost until an intense brown color liquid was observed. The extract was analyzed for total nitrogen (Carrillo et al. 1998), total phosphorus (AOAC 1997), total potassium (COVENIN 1979) calcium, magnesium and microelements (COVENIN 1981).
Algal culture Chaetoceros muelleri was obtained from the phytoplankton laboratory of the Margarita Marine Research Station (EDIMAR) (Buitrago et al. 1999). Cultures were grown in 800 ml of culture medium in 1 l glass bottles at an irradiance of 45 lmol 2 s 1 under white fluorescent light. The microalgae was maintained at 22.7C (standard deviation = 1.20) a salinity of 40% in constantly aerated microfiltered sea water. The culture media used were (1) Walne (Conway Medium) (Walne 1966); (2) agricultural fertilizer (AF) (SEAFDEC 1984); and (3) the macroalgae compost extract at two different nitrogen concentrations 243 g l 1 of nitrogen (Compost Media CM-10) and 486 g l 1 of nitrogen (CM-20). In addition, to sustain the formation of the siliceous frustules, di-sodium silicate was added to all media. The media were filtered through both fibreglass GF/C and membrane filters (0.45 lm). The culture media was inoculated with 100,000 cells ml 1 C. muelleri which was obtained from a stock culture pre-conditioned in the respective experimental culture media. Cell density of four replicates for each culture media was
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measured daily. Biochemical composition of the microalgae was calculated after 4 days of culture. Lipids were extracted following Marsh and Weinsten (1966), using tripalmitin as a standard. Carbohydrates were calculatedusing glucose as standard, according to Dubois et al. (1956), and protein was analyzedusing albumin as standard, following Lowry et al. (1951).
Results and discussion Macroalgal compost The seaweed compost was analyzed to determine macro and micronutrient contents and pH (Table 1). The concentration of nitrogen and phosphorus in the compost extract were 162% higher and 44% lower than in the AF media, respectively. The microelement manganese and iron had concentrations similar to the AF media. These chemical analyses of the seaweed compost extract compost indicate that the media contain many of the important nutrients required by microalgae to grow in culture. If 20 ml of the compost (CM-20) is used, the culture media offers suitable chemical contents for C. muelleri growth. The concentration of nitrogen and phosphorus in the media are different among the two commercial media and the compost at 20 ml, though growth parameters of the algae are similar, suggesting that growth of the algae could be affected by nutrient source instead of concentration.
Algal growth and biochemical composition The cultured algae grow similarly using either the Walne, AF or CM-20 culture media during the exponential phase between the first and third day. For all treatments, there were no lag phases (Fig. 1). Maximum cell density was 3.94, 3.99, 3.09 and 1.06 · 106 cells/ml for Walne, AF, CM-20 and CM-10, respectively. Chaetoceros muelleri growth in the experimental media and Walne media and AF is shown in Table 2. Using Walne, we found that AF and CM-20, the microalgae remained in the exponential growth phase for 2 days, whereas with CM-10 exponential growth is maintained for only 1 day. The biochemical content of Chaetoceros muelleri was not significantly different (P < 0.02) following cultivation in AF and CM-20 media (Fig. 2). A higher percentage of carbohydrate was obtained following culture with CM-20 than with the other media. The highest C. muelleri protein content was obtained following culture with Walne media, and the lowest with CM-10.
Table 1 Chemical analysis (%) of the macroalgal compost Tot. N
Tot. P
Tot. P2O5
Tot. K2O
Ca
Mg
Fe
Zn
Mn
Hum
pH
2.43
0.42
1.08
10.93
3.67
1.22
0.11
0.02
0.07
4.00
8.13
Tot.: Total Hum: Humidity pH: Hydrogen potential
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Log cells/ml
Fig. 1 Growth of cultures of Chaetoceros muelleri in different media, Walne, agricultural fertilizer (AF), compost macroalgae 486 g l 1 of nitrogen (CM-20) and compost macroalgae 243 g l 1 of nitrogen (CM-10)
1000000
100000 0
1
2
3
4
5
6
Days walne
F.A.
cm10
cm20
Table 2 Growth parameters of C. muelleri cultured with Walne, agricultural fertilizer and seaweed liquid compost at two different concentrations (SD) Growth parameters
P
Walne
AF
Compost. M 20
Compost. M 10
Maxim cellular density (cells/ml 106)
1.06 (0.82)
K (div day 1)
1.53 (0.29)
TD (days div 1)
1.00 (0.00)
<
2.00 (2.00)
Algal growth and biochemical composition of the microalgae cultured in CM-20 were similar to the two commercial media (Walne and AF). Biochemical composition of the microalgae depends mainly on the concentration of nutrients in the media, and the physiological state of the algae. (Emdadi and Berland 1989; Herrero et al. 1991; Abalde et al. 1997). In conclusion, Chaetoceros muelleri was successfully cultured using the macroalgae compost at a concentration of 20 ml of the compost in 1 l of sea water (CM-20). Our results show the feasibility of the use of this seaweed extract for culture media of microalgae Chaetoceros muelleri, suggesting its potentiality as an organic fertilizer alternative. Further studies to test the success of the algae compost for other important microalgae for aquaculture are recommended. The seaweed compost extract compost represents a cheaper organic culture media than other commercial inorganic media.
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Percentage
Lipid
Protein
Carbohydrate
50 40 30 20 10 0 W
CM-10
CM-20
FA
Fig. 2 Lipids, proteins and carbohydrates of Chaetoceros muelleri cultured in Walne (w), agricultural fertilizer (AF), compost at 10 ml (CM-10) and compost at 20 ml (CM-20)
Acknowledgements The chemical analysis of seaweed extract compost was funded by Ceporejun-Margarita. Thanks to Cooperative Algas Marinas (El Tirano) for help in the elaboration of compost.
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