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