Increase in Chlorella strains calorific values when grown in low nitrogen medium

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1 Enzyme and Microbial Technology 27 (2000) Increase in Chlorella strains calorific values when grown in low nitrogen medium A.M. Illman, A.H. Scragg*, S.W. Shales Department of Environmental Sciences, University of the West of England, Frenchay, Bristol BS16 1QY, UK Received 27 January 2000; received in revised form 19 June 2000; accepted 3 July 2000 Abstract The calorific value of five strains of Chlorella grown in Watanabe and low-nitrogen medium was determined. The algae were grown in small (2L) stirred tank bioreactors and the best growth was obtained with Chlorella vulgaris with a growth rate of 0.99 d 1 and the highest calorific value (29 KJ/g) was obtained with C. emersonii. The cellular components were assayed at the end of the growth period and the calorific value appears to be linked to the lipid content rather than any other component Elsevier Science Inc. All rights reserved. Keywords: Microalgae; Calorific value; Lipid, Biofuel; Bioreactor 1. Introduction * Corresponding author. Tel: ; fax: address: A.Scragg@uwe.ac.uk (A.H. Scragg). Due to the limited stocks of fossil fuels and the production of greenhouse gas carbon dioxide on their combustion alternative sources of energy are being investigated [1]. One renewable energy resource which could be used are biological materials that can be used either directly, converted into ethanol, methanol or methane, or extracted to produce fuel oils such as that extracted from sunflower seeds [2]. One possible source of biological material for fuel production are the microalgae [3,4]. Microalgae are more photosynthetically efficient than higher plants and can be grown in a simple salts medium on a large scale [5]. Microalgae have been proposed as a source of liquid fuel with the production of biodiesel from Botyrycoccus sp [6], and ethanol and methanol after degradation of the algae [4]. Algae can be converted to a gaseous fuel methane [7] and used to produce hydrogen [8]. Microalgae fix carbon dioxide and it has been proposed that the growth of microalgae could be linked to the removal of carbon dioxide from stack and exhaust gasses [9,10]. In all of the uses of microalgae some form of extraction or conversion is required before the algae can be used as a biofuel. The direct use of the microalgal biomass would avoid this processing and should reduce costs. In the original patent Diesel proposed finely powdered coal as one of the fuels for his engine [11] as in compression-ignition engines combustion of small particles is similar to that achieved by liquid fuels [2]. We have run a diesel engine with a high proportion of powdered cellulose (85% cellulose/15% diesel) and dried powdered Chlorella. Chlorella was chosen as it has been cultured extensively, does not aggregate and has a mean diameter of 5 10 m similar to that of powdered coal and cellulose. To act as a fuel the microalgae has to have a high calorific value. It has been known for a considerable time that nitrogen-limiting conditions will increase the lipid content of a number of algae [12 14]. Here we show that the increase in lipid content is mainly responsible for any increase in calorific value rather than changes in any other component. 2. Methods and materials 2.1. Culture and culture conditions Five algal cultures were used. Three Chlorella vulgaris Beijerinck (CCAP 211/11B), Chlorella emersonii Shihira and Kraus (CCAP 211/11N) and Chlorella protothecoides Kruger (CCAP 211/8D) (formerly known as C. pyrenoidosa Chick) were obtained from the Culture Collection of Algae and Protozoa, Ambleside, Cumbria, UK. Chlorella sorokiniana (UTEX 1230) and the marine strain Chlorella minu /00/$ see front matter 2000 Elsevier Science Inc. All rights reserved. PII: S (00) medicinsk robotteknologi

2 632 A.M. Illman et al. / Enzyme and Microbial Technology 27 (2000) Fig. 1. The growth of microalgae in Watanabe medium. (}) C. vulgaris; (*) C. minutissima; ( ) C. emersonii; (x) C. sorokiniana; ( ) C. protothecoides. tissima (UTEX 2341) were obtained from the Culture Collection of Alga at the University of Texas, Austin. The freshwater Chlorella strains were grown and maintained on Watanbe medium [15] consisting of 1.25 g/l KNO 3, 1.25 g/l KH 2 PO 4, 20 mg/l MgSO 4, 20 mg/l FeSO 4, and 1 ml/l A5 solution [16]. The ph was adjusted to 6.0 prior to autoclaving at 120 C for 20 min. The low nitrogen medium contained 203 mg/l (NH 4 ) 2 HPO 4, g/l KCL, g/l MgSO 4, g/l KH 2 PO 4 and 10 mg/l FeSO 4. Cultures were maintained at 25 C in 250 ml flasks containing 100 ml culture under at light intensity of 76 mol m 2 s 1 with 16 hours light, 8 hours dark and shaken at 60 rpm on an orbital shaker. The marine Chlorella was maintained under the same conditions using Guillards Marine medium [17] containing NaNO3 75 mg/l, NaH2PO4 6 mg/l, artificial sea salt 17.5 g/l, trace elements 1 mg/l and vitamins 1 mg/l. In the low nitrogen marine medium the NaNO 3 was reduced to 37.5 mg/l from 75 mg/l. 2.2 Bioreactor A 2 liter stirred bioreactor (LSL Biolafitte) was used to cultivate the Chlorella species. The bioreactor was run at 25 C, agitated at 200 rpm using a 55 mm diameter propeller and illuminated with three Powerglo strip lights giving an illumination of 25 mol m 2 s 1 at the surface of the reactor. Aeration was at 1 l/min with a 5% supplementation with carbon dioxide. Growth was followed by taking sterile samples daily over a 14 day culture period and determining the cell number using a haemocytometer, counting at least 1000 cells per sample. The final biomass was determined by filtering aliquots onto preweighed cellulose nitrate filters (Whatmann 0.2 m), washing with distilled water and dried overnight at 50 C Determination of cell contents Protein determination. 5 ml samples (about 2 mg dry wt.) were centrifuged (5,000 g for 5 min) and the cell pellet resuspended in 0.5 ml 1M NaOH and boiled for 5 min. The protein content was determined using the method of Lowry [18]. Carbohydrate determination 1 ml samples were centrifuged as described above and the pellet resuspended in 1 ml water. The carbohydrate was determined using the phenol sulphuric acid method of Dubois et al. [19] Determination of cell contents Determination of total lipids. This was determined using the method of Bligh & Dyer [20]. Calorific value. The calorific value was determined using an automatic adiabatic bomb calorimeter (Gallenkamp). Samples of algae (100 ml) were filtered onto cellulose nitrate filters (Whatmann) and dried at 50 C for about 4 hours. This sample was combusted in the bomb calorimeter, filters with-

3 A.M. Illman et al. / Enzyme and Microbial Technology 27 (2000) Table 1 Growth of Chlorella strains on Watanabe and low-nitrogen media Growth rate (d 1 ) Doubling time td (d) Dry weight g/l (14 days) Productivity mg dry wt./l/d (9.5)* * biphasic growth out algae and a substance of known calorific value were also used to calibrate the calorimeter. The assays were carried out in triplicate and mean values and standard deviation calculated. 3. Results It was clear that the response of the algal strains to nitrogen limitation was variable both in terms of growth and cell content (Table 1). The growth rate of C. vulgaris was lower on low nitrogen medium but the final cell numbers were a little higher (Figs. 1 and 2). The difference in final dry weights was small but there was a significant increase in lipid content from 18% to 40% and reduction in protein levels from 29% to 7% when C. vulgaris was grown in low nitrogen medium (Table 2). Although the initial growth rate of C. emersonii in Watanabe s medium was rapid growth slows by day 5 whereas in the low nitrogen medium C. emersonii continued to grow producing about three times more cells (Fig. 2). The final dry weights of the two C. emersonii cultures reflected this and there was a considerable increase in the lipid content (29 63%) in the low nitrogen medium. However, the considerable increase in lipid content of C. emersonii only increased the calorific value from 21 to 29% perhaps due to the reduction in carbohydrate content (Table 2). In the cultures of C. protothecoides growth was poor in the medium but this Fig. 2. The growth of microalgae in low-nitrogen medium. (}) C. vulgaris; (*) C. minutissima; ( ) C. emersonii (x) C. sorokiniana ( ) C. protothecoides.

4 634 A.M. Illman et al. / Enzyme and Microbial Technology 27 (2000) Table 2 Cell contents of Chlorella strains grown on Watanabe and low-nitrogen media Protein (%) Carbohydrate (%) Lipid (%) Calorific value KJ/g Calorific yield KJ/l/d improved in the low nitrogen medium. The final biomass was also greater in the low nitrogen medium with an increase in both lipid (11 23%) and calorific value (19 24%). C. sorokiniana cultures grew poorly but growth did improve in the low nitrogen medium. However, there was little increase in either lipid content or calorific value. The marine alga C. minutissima had the same growth rate in the two media but the final number of cells in Watanabe s medium was twice that in the low nitrogen medium. The lipid content of the C. minutissima cells was considerably higher in the low nitrogen medium (57%) but the calorific values remained the same. The highest lipid content was 63% found in C. emersonii grown in low nitrogen medium that also had the highest calorific value of 29 kj/g. C. emersonii grown on low nitrogen medium had the best calorific yield in terms of kj/l/d over the 14 day growth period. The correlation between calorific value and lipid content can be seen in Fig. 3. In all cases except C. sorokiniana the lipid content increased when the cells were cultivated in low nitrogen medium. This increase in lipid was linked to calorific value and was significant at the 5% level. This correlation was not found if carbohydrate or protein were considered. This was not true for all strains as C. minutissima failed to increase its calorific value despite an increase in lipid value that may have been due to a reduction in both protein and carbohydrate content. 4. Discussion Microalgae are a potential source of a number of biofuels [4,6 8] and because of their small size (5 m) and dry powdered microalgae such as C. vulgaris could be used as Fig. 3. The relationship between lipid content and calorific value of the five Chlorella strains.

5 A.M. Illman et al. / Enzyme and Microbial Technology 27 (2000) a fuel supplement in a diesel engine. The growth rates of the five algae varied from (td 0.7d) to 0.19d 1 (td 3.6d) which is similar to values reported in the literature [21]. In general the growth rate in low nitrogen medium was reduced for all strains although in four cases growth continued longer and yielded a higher final cell number. The productivity in terms of mg dry weight per liter per day was maximum at 29 mg/l/d for C. vulgaris with a little reduction in the low nitrogen medium to 28 mg/l/d. C. emersonii had a productivity of 28 mg/l/d which was reduced to 25 mg/l/d in the low nitrogen medium. The maximum productivity found here are low compared with a reported 550 mg/l/d [22] but this was regarded as a very high value. The reduction in nitrogen in the medium increases the lipid content in all the Chlorella strains and in the case of C. emersonii and C. minutissima values of 63 and 56% were obtained. These values are similar to those reported for some other Chlorella strains at 57.9% for C. vulgaris [12], C. luteoviridis 28.8% and C. capsulata 11.4% [13], and C. pyrenoidosa 29.2% [23]. C. vulgaris biomass had a calorific value of 18 kj/g which when grown in low nitrogen medium increased to 23 kj/g. The highest calorific value was for C. emersonii grown in low nitrogen medium at 29 kj/g. The improvement in calorific value is linked to the increase in lipid content rather than any change in other components such as protein and carbohydrates. The calorific value of 29 kj/g is somewhat lower than diesel at 43 kj/g and plantderived oils such as rapeseed oil at 39.5 kj/g. However, Chlorella strains may be suitable for use as diesel replacements and this is under investigation. It may be possible to develop large-scale pond or other growth systems possibly using flue gases for the production of an algal biofuel. References [1] Boyle G. 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