Batch and fed-batch cultivations of Spirulina platensis using ammonium sulphate and urea as nitrogen sources
Introduction
Cultivation of photosynthetic microorganisms can be a profitable process to obtain valuable proteins for both human and animal feeding (Henrikson, 1989). Moreover, it offers the possibility of obtaining fine-chemical products such as pigments, lipids, polyunsaturated fatty acids, polysaccharides, carotenoids, steroids and vitamins (Cohen and Vonshak, 1990, Mahajan and Kamat, 1995, Cohen, 1997, De Philippis and Vincenzini, 1998). For these purposes, research work has been carried out since the early 1950s, especially using the algal genera Chlorella, Scenedesmus and Dunaniella (Vonshak, 1997, Borowitzka, 1999). However, high costs of biomass extraction and drying and low digestibility due to a cellulosic cell wall hindered these processes. None of these problems occurred when using members of Spirulina sp. (Aaronson et al., 1980, Richmond, 1988, Cohen and Vonshak, 1990, Tel-Or et al., 1990, Dillon et al., 1995).
Spirulina sp., a genus of blue-green photoautotrophic and unicellular microalgae, can be easily and cheaply recovered by filtration from the medium, in accordance with its relatively large size. Spirulina platensis has been used as nourishment by inhabitants in Mexico and Africa for a long time: it is in fact easily digestible and has high protein content (up to 70% by dry weight), low nucleic acid content and high-value cell components such as vitamin A, chlorophylls, essential fatty acids, etc. (Ciferri and Tiboni, 1985, Dillon et al., 1995). For these reasons, there is an increasing interest in S. platensis cultivation, and its large-scale production is under consideration worldwide (Becker and Venkataraman, 1984, Chini-Zittelli et al., 1996, Jiménez et al., 2003, Masojídek et al., 2003, Olguín et al., 2003). Nowadays, it is commercialised as pills and bars to be utilized as food integrator, but it also finds application as colorant in the cosmetic, pharmaceutical and food industries, thanks to its high phycocianins and chlorophylls content. Besides, these blue and green pigments are used to substitute artificial dyes (O'Callaghan, 1996), and supplement of S. platensis to food can provide it with green colour and increase its nutritional value (Danesi et al., 2002).
The conventional nitrogen source for S. platensis is nitrate (Zarrouk, 1966, Paoletti et al., 1975, Schlösser, 1982); nevertheless, interesting research work was carried out on using animal wastes (Olguín et al., 2000) and urban effluents (Bustos Aragon et al., 1992) as low-cost nitrogen sources. Spirulina sp. cultivation can then be considered as a promising alternative for nitrogen and phosphorus removal from wastewater (Chuntapa et al., 2003, Olguín et al., 2003). The use of urea (U) as source of nitrogen for S. platensis in batch (Stanca and Popovici, 1996) and fed-batch cultures (Danesi et al., 2002) increased biomass production and ensured equivalent chlorophyll content (Rangel-Yagui et al., 2004). Moreover, it has recently been reported as being a beneficial nutrient for the growth of this cyanobacterium in lagoon water (Costa et al., 2004). Among the possible nitrogen sources—KNO3, NaNO3, urea, NH4NO3, (NH4)2HPO4 and (NH4)2SO4—urea ensured high γ-linolenic acid production (Mahajan and Kamat, 1995) and allowed S. platensis to reach concentrations comparable to those obtained with KNO3 (Danesi et al., 2002).
The general aim of this work was to check the ability of S. platensis to grow in cheaper media where the traditional nitrogen source (nitrate) had been replaced by urea or ammonium sulphate (A). Considering the capability of most microalgae to hydrolyse urea to ammonia, mainly under alkaline conditions (Torre et al., 2003), preliminary batch cultivations were carried out to point out the actual nitrogen requirements of biomass as well as to establish the inhibition threshold of ammonia. Subsequent fed-batch runs, performed according to different feeding protocols, allowed selecting the best conditions for urea supply and demonstrated that the kinetics of growth may be comparable and even better than the ones obtained with the traditional nitrate-based culture media.
Section snippets
Material and methods
The strain S. platensis UTEX 1926, recently classified as Arthrospira (Spirulina) platensis (Nordstedt) Gomont (Silva et al., 1996), was maintained in the medium of Schlösser (1982), containing NaNO3 and NaHCO3/Na2CO3 as nitrogen and carbon sources, respectively.
The experiments were carried out in 5.0-l Erlenmeyer flasks, placed on an orbital shaker at 200 rpm and containing 1.0 l of the above culture medium where NaNO3 had been substituted by ammonium sulphate or urea as nitrogen sources. pH
Batch cultivations
To determine the optimal nitrogen concentration for the growth of S. platensis, preliminary batch experiments were carried out using ammonium sulphate (A) and urea (U) as nitrogen sources. The main results of these tests, performed at initial nitrogen concentrations (No) of 0.56, 1.1 and 1.7 mM and starting biomass level (Xo) of 400 mg l−1, are summarized in Table 1.
The cultivations performed using ammonium sulphate showed that the best No level was in the range of 0.56–1.1 mM, ensuring a
Conclusions
Ammonium sulphate and urea were tested as nitrogen sources for S. platensis cultivations, according to different batch and fed-batch protocols. Comparison of results showed that the use of urea in fed-batch culture led to better growth kinetics. Ammonia accumulation that usually inhibits biomass growth was in fact prevented following an appropriate pulse-feeding pattern. Although the highest productivity during the start-up was obtained with linearly increasing feeding rate, the use of a more
Acknowledgements
The authors acknowledge the support of the Italian MIUR (FIRB prot. RBAU01E83L) for funding this research-work and for the PhD fellowship of Dr. D. Soletto.
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