Biomass and lipid productivity of scenedesmus deserticola under heterotrophic cultivation

AGU International Journal of Sciences – 2019, Vol. 7 (4), 39 – 48<br /> <br /> <br /> <br /> <br /> BIOMASS AND LIPID PRODUCTIVITY OF SCENEDESMUS DESERTICOLA UNDER<br /> HETEROTROPHIC CULTIVATION<br /> <br /> Pham Duy Thanh1<br /> 1<br /> Ho Chi Minh City University of Food<br /> <br /> Information: ABSTRACT<br /> Received: 17/10/2018<br /> Biodiesel production from oleaginous microalgae shows great potential as a<br /> Accepted: 13/08/2019<br /> promising alternative to conventional fossil fuels. In this experiment,<br /> Published: 11/2019<br /> biomass and lipid productivities of S. deserticola under different growth<br /> Keywords: conditions were investigated. The heterotrophic experiments were grown in<br /> Microalgae, heterotrophic complete darkness to prevent the algae from photosynthesizing. Three<br /> growth, heterotrophic carbon sources such as glucose, acetate or glycerol were utilized under<br /> cultivation, fatty acid, heterotrophic cultivation.<br /> biodiesel, Scenedesmus<br /> deserticola The strain S. deserticola can grow well heterotrophically without light and<br /> maximum algal biomass was 11,22 g/l after cultivation of 14 days. Highest<br /> lipid content under heterotrophic growth was 52,58%, which was about 2,4<br /> times that in photoautotrophic cultivation.<br /> Fatty acid profile was analyzed by gas chromatography. Algal oil was<br /> dominated by palmitic acid (C16:0), oleic acid (C18:1), linoleic acid<br /> (C18:2) and linolenic acid (C18:3) which were desirable feedstocks for<br /> biofuel production. The results in this study suggested that S. deserticola is<br /> potential candidate for biodiesel production and heterotrophic cultivation is<br /> more promising than conventional autotrophic cultivation.<br /> <br /> <br /> <br /> 1. INTRODUCTION source in photosynthesis (Zheng, 2013). During<br /> Biodiesel from algae oil drew the attention of growth and development, some microalgae<br /> many scientists over the past decade and is species have heterotrophic growth mechanisms,<br /> considered a biodegradable fuel (Zheng, 2013; meaning that no light is required and the CO2<br /> Xu et al, 2006). A number of microalgae fixation under autotrophic conditions is<br /> species have been selected for oil separating replaced by the dissolved carbon source in the<br /> and biodiesel production trials thanks to their environment. The heterotrophic culturing<br /> high lipid content, short growth cycle and less environment is the same as in the autotrophic<br /> need of cultivated land compared to some one except the addition of carbon to the<br /> plants used as raw materials for biodiesel culturing environment.<br /> production. (Chisti, 2007). Traditional microalgae culturing methods are<br /> Currently, most microalgae culturing methods difficult to achieve high biomass density<br /> are autotrophic, in which cells use natural or because the penetration of light in the water<br /> artificial light energy and use CO2 as a carbon environment decreases when the biomass<br /> <br /> <br /> <br /> <br /> 39<br /> AGU International Journal of Sciences – 2019, Vol. 7 (4), 39 – 48<br /> <br /> density is high. Moreover, the low biomass were added to the culture medium. Cell<br /> density contributes to the cost of biomass biomass increase, lipid yield and algal fatty acid<br /> collection. Compared to the autotrophic composition were analyzed in this study.<br /> method, the heterotrophic model allows 2. MATERIALS AND METHODS<br /> increasing the growth of microalgae, helping<br /> 2.1 Material<br /> cells to accumulate biomass and lipids in a<br /> short time without the need for light and easy to Microalgae: In this study, the green algae<br /> control operation parameters when rearing Scenedesmus deserticola was used to conduct<br /> (Khan et al., 2016) the experiments. This microalgae is kept at the<br /> Laboratory of Biotechnology - Environment,<br /> In order to reduce the cost of the culturing<br /> Ho Chi Minh City University of Food Industry.<br /> environment, there have been a number of<br /> studies testing different sources of carbon for Culture and growth condition: Microalgae was<br /> culturing microalgae with heterotrophic cultured in Bold’s Basal Medium (BBM).<br /> methods. El - Sheekh et al., (2012) Medium components included the following<br /> experimented to raise Scenedesmus obliquus substances (for 1 liter): 0.25g NaNO3; 0.175g<br /> and Chlorella vulgaris with a carbon source of K2HPO4; 0.1g KH2PO4 ,; 0.075g MgSO4.7H2O;<br /> wheat bran sugarified by the fungus Pleurotus 0.025g CaCl2.2H2O; 0.025g NaCl; 0.031g<br /> ostreatus and Trichoderma viride. The results KOH; 0.05 g Na2EDTA; 4.98 mg FeSO4.7H2O;<br /> showed that the highest value of microalgae 11.42mg of H3BO3; 8.82mg ZnSO4.7H2O;<br /> biomass was 4.99 g / l and 3.37 g / l, 1.44mg MnCl2.7H2O; 0.71mg MoO3; 1.57mg<br /> respectively. Xu et al. (2006) used the enzyme CuSO4.5H2O; 0.49mg Co (NO3) 2.6H2O. The<br /> alpha amylase and glucoamylase to hydrolyze mediums were sterilized in autoclaves at 121oC,<br /> cornstarch and the products were used as a 1.5 atm and 20 minutes. Microalgae were kept<br /> carbon source for feeding Chlorella in a 250 ml conical flask containing 100 ml of<br /> prothecoides. The maximum biomass achieved the medium, at a temperature of 25° C, shaken<br /> was 3.92 g / l. Molasses was also used as a 120 rpm, the light intensity is 2500 lux, the<br /> carbon source to cultivate heterotrophic lighting period is 12: 12.<br /> microalgae Chlorella sp. and algae biomass 2.2 Method<br /> peaked at day 5 of culture, with a value of 7.18<br /> 2.2.1 Culturing method<br /> g / l (Leesing and Kookkhunthod, 2011). Salim<br /> (2013) used the product from the Adaptation culture: Microalgae were cultured<br /> saccharification of wheat starch as a carbon in BBM medium with glucose 1g / l. D-Glucose<br /> source for culturing Ankistrodesmus sp used in this study is manufactured by Xilong<br /> heterotrophically. Chen and Walker (2011) Company, China. 250 ml conical flask<br /> made use of crude glycerol, a byproduct of containing 100 ml of microalgae medium, was<br /> biodiesel production, as a carbon supply for kept at 25 ° C, shaken 120 rpm. The flasks were<br /> heterotrophic growth of Chlorella covered with waterproof cotton plugs and<br /> protothecoides and biomass reaching a value of covered with foil to prevent light that allowed<br /> 45.1 g / l. the microalgae to carry out photosynthesis.<br /> After a week, the cells adapted to the<br /> In this study, Scenedesmus deserticola<br /> heterotrophic environment and were used to<br /> microalgae was tested to grow in lightless<br /> grow experiments with different carbon sources<br /> conditions. Three sources of carbon substrate,<br /> (Doucha & Livansky, 2011; Chen & Walker,<br /> such as glucose, glycerol and sodium acetate<br /> 2011).<br /> <br /> <br /> <br /> <br /> 40<br /> AGU International Journal of Sciences – 2019, Vol. 7 (4), 39 – 48<br /> <br /> <br /> <br /> <br /> a. Microalgae in petri b. Microalgae in c. Microalgae in<br /> culturing flask with culturing flask<br /> light without light<br /> Figure 1. Microalgae adaptation culture<br /> Heterotrophic method: In this study, three In this study, algae biomass parameters were<br /> carbon sources were used separately in three used to evaluate the growth of S. deserticola.<br /> different culture media, respectively, glucose, Cell biomass was determined by weight<br /> sodium acetate and glycerol. Specifically, when method, the results were expressed in grams of<br /> tested with glucose as carbon source, 10g of dry biomass per liter of medium. The procedure<br /> glucose was added to 1 liter of BBM medium was as follows: take 50 ml of microalgae<br /> then Scenedesmus deserticola microalgae was containing centrifuged microalgae for 10<br /> added with 10% (v / v) content (Leesing & minutes at 2000 rpm; then wash twice with<br /> Kookkhunthod, 2011; Chen & Walker, 2011). distilled water, dry biomass at 70 ° C to<br /> Similarly, when tested with acetate or glycerol constant weight to calculate microalgae<br /> as carbon sources, 1 liter of BBM medium were biomass (Kong et al, 2011). In this study, cells’<br /> also added with 10 grams of sodium acetate or forms and colors were also observed, described<br /> glycerol (Ren et al., 1013; Xu et al., 2006). by optical microscopy at 1600-time<br /> Initial biomass concentrations in the three magnification.<br /> treatments with carbon sources glucose, 2.2.3 Method of lipid determination<br /> glycerol and acetate, were respectively 1.48 g /<br /> In this study, lipids in total was determined by<br /> l; 1.53 g / l and 1.53 g / l.<br /> the Folch method. A chloroform-methanol (2: 1<br /> In each treatment, microalgae cells were grown v / v) solvent mixture is used for lipid<br /> in a 1000 ml flask containing 700 ml of extraction. The ratio of algae biomass and<br /> medium. The flask was covered with aluminum solvent mixture is 1: 20 (grams of algae / ml<br /> foil to prevent light, placed in a shaker of 125 solvent). 0.5% NaCl solution was added to the<br /> rpm and at a temperature of 25 ° C. Duration of solvent mixture to obtain a chloroform /<br /> each batch is 14 days. Microalgae culture will methanol / water ratio 8: 4: 3. The lower phase<br /> be taken periodically every 48 hours to analyze consists of chloroform, lipid and biomass; Filter<br /> the growth and forms of microalgae . The to remove biomass then evaporate chloroform<br /> analytical sampling process was carried out at 55 ° C in a fume cupboard. Lipid content was<br /> under aseptic conditions and each treatment measured by weight method. The steps to<br /> was repeated 3 times. Treatments were analyze microalgae lipid was described in<br /> conducted in the same batch. detail in the previous study of the authors<br /> 2.2.2 Methods of measuring microalgae (Pham Duy Thanh et al., 2017). After the<br /> growth<br /> <br /> <br /> 41<br /> AGU International Journal of Sciences – 2019, Vol. 7 (4), 39 – 48<br /> <br /> experiment, algae biomass obtained will be 3.1 Microalgae growth<br /> used to determine lipid content. Experimental results showed an increase in the<br /> 2.2.4 Lipid yield biomass of microalgae in all three culture media<br /> Lipid yield was calculated using the formula: in the first 10 days and then gradually<br /> Plipid = (Clipid x X)/t (g/l .day) decreased. Table 1 presents the findings of<br /> biomass growth of S. deserticola in BBM<br /> in which Clipid was the lipid content of the cell; t<br /> medium supplemented with respectively<br /> is the incubation time and X is the biomass<br /> glucose, acetate or glycerol as the carbon<br /> concentration in the log phase.<br /> source.<br /> 2.2.5 Analysis of fatty acid composition in<br /> In the absence of light, with glucose as the<br /> algae oil<br /> carbon source, the value of microalgae biomass<br /> Algae oil will be converted to biodiesel in gradually increased, reaching the highest value<br /> accordance with ISO 5009: 94 method. The of 11.22 g / l on day 10; after 14 days the<br /> sample will then be used for gas biomass value was 7.60 g / l. In the case that<br /> chromatographic analysis to determine fatty carbon sources in the BBM were glycerol and<br /> acid composition. The sample was analyzed at sodium acetate, there was also an increase in<br /> the laboratory analysis service center of Ho Chi biomass which reached the highest average<br /> Minh City, Department of Science and values, respectively, 8.37 g / l and 6.88 g / l on<br /> Technology of Ho Chi Minh City. day 10 of all the treatments. The growth rates of<br /> 2.2.6 Data processing methods particularly S. deserticola in the log phase<br /> corresponding to carbon sources glucose,<br /> Data were processed using Microsoft Excel<br /> glycerol, acetate, reached 0.20 (ngày-1), 0.17<br /> Office 2010 and Statgraphics XV, Version<br /> (ngày-1) and 0.15 (ngày-1) respectively.<br /> 15.1.02. ANOVA and Multiple Range Tests<br /> Statistical analysis results indicated that there<br /> analysis method with 95% confidence were<br /> was a difference in the specific growth rate of<br /> used to determine the significant differences<br /> S.deserticola when culturing algae with<br /> between the sample mean values.<br /> different carbon sources (Pvalue< 0.05).<br /> 3. RESULTS AND DISCUSSION<br /> Table 1. Microalgae biomass in culture media<br /> <br /> Carbon Experiment timetable<br /> sources Beginning Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 Day 14<br /> 8,65 10,73<br /> 1,48 3,22 5,20 11,22 10,43 7,60<br /> Glucose ± ±<br /> ± 0,03(a) ± 0,10(b) ± 0,17(b) ± 1,10(e) ± 0,58(e) ± 0,18(e)<br /> 0,48(c) 1,04(d)<br /> 6,42 7,85 8,37 6,15<br /> 1,53 3,00 4,70 8,20<br /> Glycerol ± ± 0,05 ± ±<br /> ± 0,06(a) ± 0,67(b) ±0,13(b) ± 0,59(d)<br /> 0,20(c) (c)<br /> 0,15(d) 0,13(d)<br /> 6,88 4,18<br /> 1,53 3,28 4,73 5,83 ± 6,23 4,88<br /> Acetate ± ±<br /> ± 0,06(a) ± 0,29(b) ± 0,23(b) 0,06(bc) ±0,13(cd) ± 0,30(e)<br /> 0,23(d) 0,43(f)<br /> (*) Different characters in the same row indicate significant differences at the 95% confidence<br /> <br /> <br /> <br /> 42<br /> AGU International Journal of Sciences – 2019, Vol. 7 (4), 39 – 48<br /> <br /> Table 1 shows that microalgae grew and In this study, optical microscopy results showed<br /> reached their highest biomass when the carbon that there were differences in forms of cells<br /> source was glucose, followed by glycerol and when microalgae heterotrophically grew. Under<br /> acetate. This result was not different from autotrophic conditions, cells in the unicellular<br /> previous studies. Ren et al (2013) used 7 form consisted of 2, 4 or 8 cells but usually 4<br /> different carbon sources to grow Scenedesmus adjoining cells. The cells stick together in the<br /> sp using heterotrophic culture method. Carbon middle of the cell and line up in the same row.<br /> substrates include glucose, acetate, fructose, The cell was rhomboid; the cell tip was spiked;<br /> maltose, propionate, sucrose and butyrate. Test sometimes the cell was slightly spiked and bend<br /> results showed that glucose was the best carbon inwards. Cell size 2 ÷ 4 x 6 ÷ 12 µm (Pham<br /> source. Gami et al., (2014) when experimented Duy Thanh et al, 2017). In this study, when the<br /> on Chlorella protothecoides raised in glucose- microalgae culture tank was placed in a shaker,<br /> enriched medium, the maximum microalgae the cells tended to separate, sometimes in pairs,<br /> biomass was 17.18 g / l; in another case using in groups of 3 or individually. Cell size was<br /> cornstarch as the carbon source, biomass was also larger, ranging from 4 ÷ 6 x 8 ÷ 12<br /> 17.14 g / l after 11 days of rearing. Research micrometers, often bulging horizontally. Some<br /> results of El - Sheekh et al. (2012) also cells had a different shape than the original one<br /> indicated that glucose was the best carbon and had a large size (Figure 2). Cells had a<br /> source for microalgae growth and lipid yellowish color if bred in the dark while they<br /> production. were usually green if cultured under the light.<br /> Compared to studies conducted on the same In terms of endoplasm, heterotrophic cells had<br /> Scenedesmus breed with heterotrophic method many particles whose diameters varied from 0.8<br /> using glucose carbon source, Scenedesmus ÷ 1.2 micrometres. Changes in the size, color,<br /> deserticola in this study had a biomass of 11.22 shape of microalgae cells were similar to the<br /> g / l, 2.8 times as high as that of Scenedesmus descriptions in the study of Ditattamart et al.<br /> obliquus (Ren et al, 2013) and 3.24 times as (2014); Kim and Hur (2013); Xu et al. (2006).<br /> high as that of Scenedesmus sp. (Dittamart et According to these authors, the particles in the<br /> al., 2014). aforementioned microalgae are lipid-containing<br /> particles in the cell.<br /> 3.2 Forms of cells<br /> <br /> <br /> <br /> <br /> a. Cells in culture medium b. Particles in cells<br /> Figure 2. Forms of cells cultured heterotrophically<br /> <br /> <br /> <br /> <br /> 43<br /> AGU International Journal of Sciences – 2019, Vol. 7 (4), 39 – 48<br /> <br /> 3.4 Microalgae lipid content when the carbon source was acetate (52.58%)<br /> After 14 days of culture, microalgae biomass or glycerol (50.82%). However, the lipid<br /> was collected by centrifugal method. The production value of S. deserticola still had the<br /> biomass was then washed with distilled water highest results when the carbon source was<br /> twice to remove dissolved salts and used for glucose (480 mg / liter per day). The statistical<br /> lipid content analysis. The analysis results show analysis showed that there was a difference<br /> that, in the basic environment supplemented between the content of microalgae lipid when<br /> with glucose as carbon source, the lipid content the carbon source was glucose and the other<br /> is low (42.72%). While this value was high two carbon sources were glycerol and acetate.<br /> <br /> <br /> <br /> <br /> a. Microalgae lipid content and output b. Microalgae oil<br /> Figure 3. Lipid content and lipid yield of microalgae<br /> According to Ren et al (2013), the lipid content yields of 636 mg / day and 602 mg / day,<br /> and production of microalgae of Scenedesmus respectively.<br /> sp were valued 43.4% and 250 mg / l per day, When cultured in the traditional autotrophic<br /> respectively, when reared in heterotrophically method, Scenedesmus deserticola had a lipid<br /> with carbon source of glucose for 6 days. . In content of 18.05% (Pham Duy Thanh et al,<br /> this study, those values obtained on 2017), whereas if raised in heterotrophic<br /> Scenedesmus deserticola were 42.72% and 480 environment with glucose as carbon source, the<br /> mg / l per day, respectively. Gami et al., (2014) lipid level 2.4 times increased. The study of Xu<br /> tested the culture of Chlorella protothecoides. et al. (2006) indicated that the content of<br /> The results showed that when the carbon source Chlorella protothecoides lipid when growing<br /> from corn starch, the microalgae had higher heterotrophically was 3.16 times. Table 2<br /> lipid content (40.82%) than the case with summarizes the maximum biomass values, lipid<br /> glucose as carbon source (38.57%), and lipid content of some microalgae heterotrophically<br /> growing with different carbon sources.<br /> <br /> <br /> <br /> <br /> 44<br /> AGU International Journal of Sciences – 2019, Vol. 7 (4), 39 – 48<br /> Table 2. Biomass and lipid content of Microalgae with different carbon sources<br /> <br /> Substrate Lipid<br /> Lipid<br /> Carbon amount Biomass Yield<br /> Microalgae content References<br /> Sources (g/l) (g/l) (mg/l per<br /> (%)<br /> day)<br /> Scenedesmus sp Glucose 10 3,46 43,4 250,27<br /> Chlorella sp Glucose 10 3,76 15,6 58,8<br /> C. saccharophila Glucose 40 1,1 37 58,5<br /> Ren et al., 2013<br /> C. sorokiniana Glucose 40 3,2 56 256<br /> Nanochloropsis sp Glucose 10 3,83 19,3 74<br /> Monoraphidium sp. Glucose 10 3,39 37,56 148,74<br /> C. prothecoides Glucose 12 17,18 38,57 602<br /> Glucose Gami et al.,<br /> C. prothecoides from corn 12 17,14 40,82 636 2014<br /> starch<br /> C. protothecoides<br /> Glucose 30 46 53 3040<br /> (*)<br /> C. protothecoides<br /> Glycerol 30 43,3 53 2800<br /> (*)<br /> Crude<br /> Glycerol<br /> C. protothecoides<br /> from 30 45,1 54 2990<br /> (*) Chen & Walker,<br /> biodiesel<br /> 2011<br /> production<br /> C. protothecoides Glucose 15 4,2 13 130<br /> C. protothecoides Glucose 10 3,7 55 -<br /> C. protothecoides Glucose 30 9,1 53 -<br /> C. protothecoides Glucose 15 10,4 - -<br /> C. protothecoides Glucose 40 14,2 - 510<br /> Glucose<br /> from<br /> Ankistrodermus sp 5 1,26 30 -<br /> wheat<br /> starch Salim, 2013<br /> Glucose<br /> from<br /> Ankistrodermus sp 10 1,12 28 -<br /> wheat<br /> starch<br /> <br /> <br /> <br /> 45<br /> AGU International Journal of Sciences – 2019, Vol. 7 (4), 39 – 48<br /> <br /> Substrate Lipid<br /> Lipid<br /> Carbon amount Biomass Yield<br /> Microalgae content References<br /> Sources (g/l) (g/l) (mg/l per<br /> (%)<br /> day)<br /> Scenedesmus sp<br /> Glucose 10 2,78 8,43 -<br /> (**)<br /> Scenedesmus sp Dittamart et al.,<br /> Glycerol 4,6 0,38 14,52 -<br /> (**) 2014<br /> Scenedesmus sp Acetate<br /> 4,1 0,30 12,08 -<br /> (**) natri<br /> S. deserticola Glucose 10 11,22 42,38 480<br /> Acetate<br /> S. deserticola 10 8,37 53,91 420 This study<br /> natri<br /> S. deserticola Glycerol 10 6,88 51,49 360<br /> (*): Fed-batch; (**): Mixotrophic culture<br /> 3.5 Composition of fatty acids in algae oil palmitic acid (16.94%), oleic acid (14.80%),<br /> Composition and content of fatty acids in raw linoleic acid (14.53%) and linolenic acid<br /> materials will affect the characteristics of (13.01%). The fatty acid composition was<br /> biodiesel. Therefore, the analysis of algae oil similar to the microalgae oil Chlorella<br /> fatty acid composition is very necessary. The sorokiniana, raised in the lightless condition.<br /> study also conducted the analysis and its results The content of palmitic acid, oleic acid, linoleic<br /> were presented in Table 3. acid were 13.7%; 14.4% and 14.1%<br /> respectively and these were the suitable fatty<br /> The analysis results showed that the fatty acids<br /> acids for biodiesel production (Zheng, 2013).<br /> accounted for high proportion are respectively<br /> Table 3. Fatty acid composition in microalgae oil of S. deserticola<br /> <br /> Content<br /> No Names of acid Molecular formula<br /> (% chromatograph)<br /> <br /> 1 Octanoic acid (C8:0) C8H16O2 0,09<br /> 2 Lauric acid (C12:0) C12H24O2 0,28<br /> 3 Myristic acid (C14:0) C14H28O2 0,63<br /> 4 Pentadecanoic acid (C15:0) C15H30O2 0,14<br /> 5 Palmitic acid (C16:0) C16H32O2 16,94<br /> 6 Palmitoleic acid (C16:1) C16H30O2 1,15<br /> 7 Margaric acid (C17:0) C17H34O2 0,31<br /> 8 Stearic acid (C18:0) C18 H36O2 4,10<br /> 9 Oleic acid (C18:1) C18 H34O2 14,80<br /> <br /> <br /> <br /> 46<br /> AGU International Journal of Sciences – 2019, Vol. 7 (4), 39 – 48<br /> <br /> Content<br /> No Names of acid Molecular formula<br /> (% chromatograph)<br /> <br /> 10 Linoleic acid (C18:2) C18 H32O2 14,53<br /> 11 Linolenic Acid (C18:3) C18 H30O2 13,01<br /> 12 Arachidic acid (C20:0) C20H40O2 0,21<br /> 13 Gadoleic acid (C20:1) C20H38O2 0,31<br /> 14 Arachidonic acid (C20:4) C20H32O2 1,19<br /> 15 Lignoceric Acid (C24:0) C24H48O2 0,21<br /> 16 Behenic acid (C22:0) C22H44O2 0,27<br /> 17 Erucic acid(C22:1) C22H42O2 0,07<br /> <br /> <br /> 4. CONCLUSION Dittamart, D., Pumas, C., Pekkhoh, J.,<br /> Scenedesmus deserticola species has Peerapornpisal, Y. (2014), Effects of<br /> heterotrophic growth mechanisms. It can grow organic carbon source and light – dark<br /> and develop in BBM medium with the addition period on growth and lipid accumulation of<br /> of soluble carbon sources such as glucose, Scenedesmus sp., J. Sci. Techno. 8 (02), 198<br /> glycerol or sodium acetate at a concentration of – 206.<br /> 10 g / l. S. deserticola grows fast and has the El – Sheekh M. M., Bedaiwy . M., Osman E.<br /> highest lipid yield when the carbon substrate is M., Ismail M. M. (2012), Mixotrophic and<br /> glucose. Heterotrophic culture has higher heterotrophic growth of some microalgae<br /> biomass content and lipid yield than traditional using extract of fungal – treated wheat bran,<br /> culture. Algae oil is a suitable raw material for International Journal of Recycling of<br /> biodiesel production. 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