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Investigation of Mixotrophic, Heterotrophic and Autotrophic Growth of Chlorella vulgaris Under Agricultural Waste Medium a

a

b

M. A. Mohammad Mirzaie , M. Kalbasi , S. M. Mousavi & B. Ghobadian a

c

Department of Chemical Engineering, Amirkabir University of Technology, Tehran, Iran

b

Biotechnology group, Department of Chemical Engineering, Tarbiat Modares University, Tehran, Iran c

Biosystems engineering department, Tarbiat Modares University, Tehran, Iran Accepted author version posted online: 25 Mar 2015.

Click for updates To cite this article: M. A. Mohammad Mirzaie, M. Kalbasi, S. M. Mousavi & B. Ghobadian (2015): Investigation of Mixotrophic, Heterotrophic and Autotrophic Growth of Chlorella vulgaris Under Agricultural Waste Medium, Preparative Biochemistry and Biotechnology, DOI: 10.1080/10826068.2014.995812 To link to this article: http://dx.doi.org/10.1080/10826068.2014.995812

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Investigation of mixotrophic, heterotrophic and autotrophic growth of Chlorella vulgaris under agricultural waste medium M. A. Mohammad Mirzaie1, M. Kalbasi1, S. M. Mousavi2, B. Ghobadian3 1

Department of Chemical Engineering, Amirkabir University of Technology, Tehran, Iran, 2Biotechnology group, Department of Chemical Engineering, Tarbiat Modares University, Tehran, Iran, 3Biosystems engineering department, Tarbiat Modares University, Tehran, Iran

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Corresponding author to M. Kalbasi: Po. Box: 158754413. E-mail: [email protected]

Abstract Growth of Chlorella vulgaris and its lipid production investigated under autotrophic, heterotrophic and mixotrophic conditions. Cheap agricultural waste molasses and corn steep liquor from industries were used as carbon and nitrogen sources, respectively. Chlorella vulgaris grew remarkably under this agricultural waste medium resulted to a reduction in the final cost of the biodiesel production. Maximum Dry weight of 2.62 g.L-1 obtained in mixotrophic growth with the highest lipid concentration of 0.86 g.L-1. These biomass and lipid concentrations were respectively, 140% and 170% higher than autotrophic growth and 300% and 1200% higher than heterotrophic growth. In mixotrophic growth, independent or simultaneous occurrence of autotrophic and heterotrophic metabolisms was investigated. The growth of the microalgae was observed to take place first heterotrophically to a minimum substrate concentration with a little fraction in growth under autotrophic metabolism and then, the cells grew more autotrophically. It was found that mixotrophic growth was not a simple combination of heterotrophic and autotrophic growth.

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KEYWORDS: Chlorella vulgaris; cultivation medium; lipid content; photosynthesis pigment; agricultural medium; mixotrophic growth

1. INTRODUCTION Biodiesel as a renewable energy sources has a great interest for worldwide energy policy due to its sustainability and reduction in the pollutants which uses fossil fuels. Among the

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conventional raw materials for biodiesel production, such as plant oils and animal fats, microbial oils can play an important role in the world’s future energy supply. Microalgae are considered as a potential biomass feedstock for production of the third different biodiesel generation at large scale. Microalgae have high yields per unit area of land, cultivate in non-arable and unsuitable lands and grow in salt water or even in contaminated water preserved use of fresh water. Moreover, microalgae could grow faster and generate more energy per unit weight compared to other energy sources [1-5].

The production of biodiesel from microalgae in order to replace the conventional diesel has not been economically feasible so far due to the high cost. An increase in biomass productivity and its lipid content and a decrease in cultivation cost using industrial waste materials instead of expensive conventional substrates make microalgae biodiesel more economic. In order to increase the biomass productivity, microalgae growth has been investigated in different trophic modes. In some microalgae species, growth under different trophic conditions of autotrophic, heterotrophic and mixotrophic has been reported to be possible [6, 7] which can be exploited in biotechnological industry [8]. These species of microalgae could change their metabolism according to the changes in

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the environmental conditions [9]. Liang et al. [10] studied biomass and lipid productivity of Chlorella vulgaris (C. vulgaris) under autotrophic, heterotrophic and mixotrophic cultivation and they observed that a higher cellular lipid content was provided in autotrophic cultivation. Expensive substrates of glucose and glycerol were used as organic substrates and glucose showed to have a better effect on biomass productivity. Effect of the substrates of glycerol and glucose was also investigated by Ho Oh et al. [11]

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in the growth of Porphyridium cruentum in different culture conditions. P. cruentum has been grown better in heterotrophic or 12:12 h cyclic mixotrophic growth and glucose has had a better substrate. Liu et al. [12], reported that Chlorella zofingiensis had a higher productivity and better lipid quality in heterotrophic condition. 900% increase in lipid yield has been achieved in heterotrophic cells fed with 30 g.L-1 glucose compared with autotrophic condition. Kim et al. [6] indicated that heterotrophic cultivation had a better potential in higher microalgae dry weight, nitrogen and phosphorus removal rates of Chlorella sorokiniana in waste water treatment system. Chojnacka and Noworyta [13] found a highest specific growth rate for Spirulina sp. in higher level of light intensity (33 W.m2) in mixotrophic cultivation and the lowest rate was reached in lower level of light intensity (17 W.m2) in autotrophic cultivation. In their work, glucose was used as carbon source in heterotrophic and mixotrophic cultivation. Mixotrophic growth in some strains of microalgae demonstrated a higher dry weight or lipid content compare to autotrophic or heterotrophic growths [14-16]. In the mixotrophic growth investigation, it has been strongly reported that microalgae grown mixotrophically, metabolized both heterotrophically and autotrophically [16-19]. But some differences have been observed between the fraction of each metabolism and their occurring times. Some investigators

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reported that mixotrophic growth shown to be a sum of microalgae growth in autotrophic and heterotrophic cultivation (by analyzing dry weight, specific growth rate or lipid content) [16, 18]. Whilst some others revealed that mixotrophic growth was not a simple combination of heterotrophic and autotrophic modes [17, 20]. Understanding how and when these metabolisms happen in mixotrophic growth is essential for scale-up and

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fermentation process design.

In the present research work, C. vulgaris was cultivated in autotrophic, heterotrophic and mixotrophic modes to obtain the best condition for the highest dry weight and lipid content. In the mixotrophic growth, application of existed energy and carbon sources by cells was investigated in heterotrophic and autotrophic metabolisms. Sequential or simultaneous occurrence of these metabolisms and their fractions in mixotrophic growth was studied. Cane molasses and CSL were used as carbon and nitrogen sources, respectively in heterotrophic and mixotrophic conditions. These substrates are agricultural waste materials which reduce the total cost of microalgae growth and lipid production. This observation in the present work may be claimed to be a big challenge in the biodiesel production.

2. MATERIALS AND METHODS 2.1. Microorganism And Cultivation Conditions C. vulgaris (CCAP 211/11B) was obtained from the culture collection of algae and protozoa (CCAP, Scotland). In autotrophic growth, the basic medium for cultivation is Rudic’s culture medium [21] which contains (per liter of distilled water): 300 mg KNO3,

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20 mg KH2PO4, 80 mg K2HPO4, 20 mg NaCl, 47 mg CaCl2, 10 mg MgSO4.7H2O and trace elements consisting of 0.1 mg ZnSO4.7H2O, 1.5 mg MnSO4.H2O, 0.08 mg CuSO4.5H2O, 0.3 mg H3BO3, 0.3 mg (NH4)6Mo7O24.4H2O, 17 mg FeCl3.6H2O, 0.2 mg Co(NO3)2.H2O and 7.5 mg EDTA.

In heterotrophic and mixotrophic cultivations, waste industries of cane molasses (Karaj

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Sugar Company in Iran) and corn steep liquor (CSL) (Gloucosan Corn industry in Iran) were used as cheap substrates of carbon and nitrogen sources, respectively. Composition of these 2 substrates is shown in table 1. For experimental purposes, 20 ml as-received molasses was diluted using 1 L distillated water and after centrifugation in 4500 rpm for 10 min, supernatant solution was used as molasses solution. Also, 10 gr of dried CSL was dissolved in 1 liter distillated water at 50 ºC and centrifuged same as molasses were done. Supernatant solution was used as CSL solution. All experiments were carried out in optimum condition that was determined with statistical methods: light intensity of 65 µmol.m-2.s-1, molasses volume of 180 mL and CSL volume of 150 mL (Details of experiment optimization were reported elsewhere).

2.2. Photobioreactor 3 cylindrical algal photobioreactors were constructed especially for microalgae cultivation. Those for autotrophic and mixotrophic were subjected to a uniform artificial lighting that was produced with six 25 W tungsten lamps with light intensity of 65 µmol.m-2.s-1 in first day. The bioreactor for heterotrophic cultivation was placed in dark. The 5 L reactors with 25 cm height and 17 cm diameter containing 3 L of medium were

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equipped with air sparger and sampling connection. Aeration provides uniform temperature, light and CO2 and other nutrients for all the microalgae and distributes oxygen and other metabolic products in the culture [15]. Sampling was performed daily and growth parameters like microalgae dry weight and lipid content were measured.

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Cell concentration was measured using spectrophotometric results at 550 nm (OD550). According to OD550, microalgae dry weight (Xd) was measured as follow:

X d g .L 1

0.49 OD550 – 0.0215                                    R2

0.9347

(1)

This equation is an acceptable (high R2 value) regression relationship between dry weight and OD550 of microalgae which was derived in 10 separate experiments. In order to obtain the equation (1) for microalgae dry weight measurement, cells were collected and washed twice by centrifuge and then the pellets were dried at 50 ºC for 1 day.

The specific growth rates of microalgae (µ) in each instant were obtained from cell dry weight versus time, using the following equations: μ μ μ

1 dX ; X dt X 1 t X 2 X2 t 2 t1

X2

X1

t 2 t1 X1 X1

X2

X1

1 X2 / 2

(2)

;

where X1 and X2 are the dry weights in times of t1 and t2, respectively. µmax is the maximum specific growth rate obtained from equation (2).

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The lipid weight was measured gravimetrically by chloroform extraction using the modified Bligh and Dyer method. In this method, 15 mL of microalgae collected from the reactor and centrifuged at 4500 rpm for 10 minutes and cell pellets were mixed with 0.8 mL distilled water, 2 mL methanol and 1 mL chloroform to form a single phase solution. After addition of 2 mL distilled water and 2mL chloroform, a double-phase solution was formed but most of the lipid was in the chloroform phase. In first extraction stage,

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heavier phase was pulled over and in the second extraction process, the remaining was filled with 2 mL distilled water and 2 mL chloroform. The heavy chloroform phase from two stages was dried under vacuum and was weighted.

Hydrocarbon content (g.L-1) was measured every day using Phenol sulfuric acid [22]. Briefly, 2 mL of microalgae culture was centrifuged at 4500 rpm for 10 minutes. 1 mL of supernatant solution was mixed with 1 mL of 5% (w/v) phenol solution in a tube and 5 mL of 98% (v/v) sulphuric acid solution was added in less than 10 seconds. The tubes were placed in water bath at 30 ºC for 20 min and then, cooled down. The absorbance of the solution was measured at wavelength of 490 nm using a UV-Vis spectrophotometer. Carbohydrate concentration was determined using a standard absorption curve of glucose (Merck) solution at concentrations up to 0.6 g.L-1.

Chlorophyll-a and chlorophyll-b were measured using methanol solvent extraction method [23]. 2 mL of microalgae culture was centrifuged at 4500 rpm for 10 minutes. The supernatant solution was drawn from the tube and 2 mL methanol

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was added. The tube was then placed in an ultrasonic bath for 20 min and placed in a refrigerator overnight. The tube was centrifuged at 4500 rpm for 5 min. Chlorophyll content was calculated with the following equations according to the measurement of the optical density of the supernatant using spectrophotometer: Ca

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Cb

15.65 OD666 7.340 OD653

(3)

27.05 OD653 11.21 OD666 (4)

where Ca and Cb are the content of chlorophyll-a and chlorophyll-b, respectively.

3. RESULTS AND DISCUSSION 3.1. Dry Weight Of C. Vulgaris Samples of microalgae from autotrophic, heterotrophic and mixotrophic conditions were collected during the experimental procedure. Calculated microalgae dry weights are shown in Fig. 1. In autotrophic cultivation, microalgae grew with a maximum specific rate of 0.085 day-1 and finally 1.08 g.L-1 dry weight was obtained. Microalgae dry weights in heterotrophic condition increased to 0.7 g.L-1 in the first 10 days. After this period of time, microalgae dry weight decreased to a minimum of 0.4 g.L-1. This reduction could be due to a high decrement of carbohydrate concentration in culture medium to 0.015 g.L-1 (section 3.4). In the heterotrophic growth, organic carbon was used as both energy and carbon source and with a high decrement of this source, microalgae was not able to grow and entered the death phase. The best growth rate with a highest dry weight of microalgae was obtained in mixotrophic condition. In this cultivation, maximum specific growth rate of microalgae and its dry weight were 0.12 day-1 and 2.62 g.L-1, respectively. Dry weight in the mixotrophic condition was more than 2.5 and 4

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times higher than in the autotrophic and heterotrophic conditions, respectively and for the autotrophic condition this was 2 times higher than the heterotrophic condition.

3.2. Lipid Content Microalgae produce lipid as energy saving compounds in its cytoplasm. Lipid could be converted to biofuel in esterification process and therefore, it has a great value in

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microalgae fermentation. In this section of the research work, lipid formation in different cultivation modes of C. vulgaris was investigated. The experimental results are shown in Fig. 2. It should be noted that the lipid content is equal to the lipid concentration versus dry weight of microalgae. According to this figure, the highest lipid content was produced in mixotrophic growth; 33% of maximum dry weight of microalgae was lipid (0.86 g.L-1).

According to Fig. 2, in heterotrophic cultivation, there were two phases of (i) synthesis and accumulation of the lipid and (ii) turnover of the storage lipid. After 9 days of initial cultivation, the maximum lipid content of 0.14 g.g-1 was obtained. In this media, substrate was limited (section 3.4) in initial days and energy saved as lipid in the cytoplasm of microalgae. The substrate limitation entered the microalgae to a death phase and the lipid turnover caused degradation of storage lipid to support algal growth [19, 24, 25]. It should be reminded that the lipid is one of the most energy-rich compounds in microalgae. Lipid degradation began very rapidly after the carbon concentration reached to a limit content of 0.1 g.L-1 in the

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medium. Therefore, the saved lipid was consumed to maintain the microorganism to direct to a decrement in lipid content to 0.04 g.g-1.

Microalgae accumulated lipid with an increasing rate after 9 days in mixotrophic growth and 12 days in autotrophic growth.

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Lipid concentration in mixotrophic mode was observed to be higher than the one accumulated in cells in autotrophic mode; even though according to Fig. 2, their lipid content was almost equal. Higher dry weight of microalgae in mixotrophic mode multiplied by lipid content resulted to a higher lipid concentration. Higher dry weight in mixotrophic mode compared to heterotrophic and autotrophic modes was due to the increase in availability of carbon which lead to increased proportion of storage lipids [10]. In mixotrophic condition, both organic carbon (carbon source available in heterotrophic cultivation) and CO2 (carbon source available in autotrophic cultivation) were present.

According to Figs. 1 and 2, C. vulgaris accumulate lipid in its structure simultaneous with the growth. Product formation and its relationship with microalgae may be classified in three categories: (i) product formation is related to microalgae growth; (ii) product formation is partially related to microalgae growth; and (iii) product formation is unrelated to microbial growth [26]. Lipid formation in C. vulgaris under three investigated trophic modes was in class (i) in relation with microorganism growth.

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3.3. Photosynthesis Pigment Content Chlorophylls are greenish pigments. The most important type of chlorophyll is chlorophyll-a which makes photosynthesis possible. Chlorophyll uses as biomass production indicator in cultures with photosynthesis [27]. Another type of chlorophyll is chlorophyll-b that is only in green algae and plants.

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Variation of these pigments has been investigated in autotrophic, heterotrophic and mixotrophic conditions because of its important role in photosynthesis reaction [8]. This variation is demonstrated in Fig. 3. The content of each pigment (mg.g-1) was measured by dividing the pigment concentration (mg.L-1) to microalgae dry weight (g.L-1). Fig. 3a shows the content of chlorophyll-a versus time. In autotrophic growth, chlorophyll-a was increased in initial time of microalgae growth; which is indicative of the production of required pigments by microalgae for photosynthesis which was the only pathway for the metabolism of microorganism. After that a decrement in chlorophyll content was observed due to the needless of more chlorophyll. It should be noted that chlorophyll concentration was found to increase during the time; but the content of chlorophyll decreased.

In mixotrophic growth, as shown in Fig. 3a, chlorophyll-a content was constant in the initial 6 days. Since the chlorophyll concentration is an indicator of photosynthesis, this constant content of chlorophyll demonstrates that photosynthesis happened in a slow rate. Due to a low chlorophyll production, it can be concluded that the microalgae has used preferentially the available organic carbon as energy and carbon sources in heterotrophic

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metabolism than CO2 in autotrophic metabolism [20]. It was after this constant trend that the chlorophyll content was increased. This increment may have been due to the decrement in substrate concentration; which lead to a more production of chlorophyll to change metabolic pathway from heterotrophic to photosynthesis. After 12 days, it was observed that the content of chlorophyll decreased. Chlorophyll decrement in cultivation medium could be due to decreasing of nitrogen [28]. Nitrogen decrement forced cells to

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utilize intracellular nitrogen pool for further cell growth. Photosynthetic pigments like chlorophyll are the most easily consumable intracellular nitrogen pools in depletion of external nitrogen sources [28].

The same results were observed for chlorophyll-b according to Fig. 3b. As shown in experimental results, chlorophyll-a and chlorophyll-b contents in mixotrophic growth increased from 3.07 and 2.78 to 6.40 and 5.80 mg.g-1 in 11th day and then, decreased to 3.49 and 3.82 mg.g-1 in final day, respectively.

A same trend as mixotrophic growth was observed in autotrophic growth. As shown in Fig. 3, the chlorophyll-a and chlorophyll-b content in autotrophic mode increased from 4.19 and 7.36 to 10.43 and 14.01 mg.g-1 in 6th day and decreased to 6.02 and 7.05 mg.g-1 in final cultivation day, respectively.

In heterotrophic cultivation, the chlorophyll content was reduced 10th day which followed by a constant content. As mentioned earlier, intracellular pigments are produced in photosynthesis. But pigment production in heterotrophic growth was negligible and by

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increasing the dry weight, pigment content was decreased.

The color of cultivation media indicates the metabolism pathway as well [20, 29]. As demonstrated in Fig. 4, in heterotrophic growth, the color of cultivation media is yellow due to small amount of chlorophyll; but in mixotrophic and autotrophic growth, chlorophyll produced in cultivation media and the color was green. According to Fig. 4,

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in heterotrophic condition in the absence of light, photosynthesis did not occur and the color of medium was yellow. In autotrophic condition, the color of medium is more greenish than mixotrophic medium due to higher value of chlorophyll and hence, higher photosynthesis.

3.4. Substrate Consumption In heterotrophic and mixotrophic growth, cane molasses and CSL were added as carbon and nitrogen sources, respectively and their carbon sources were presented as carbohydrate concentration. Fig. 5 shows carbohydrate consumption in mixotrophic and heterotrophic cultivation media. In mixotrophic growth, according to Fig. 5, carbohydrate was consumed highly to 10th day and then, its consumption continued with a slow rate. This carbohydrate consumption revealed that the microalgae first selected organic carbon as energy and carbon source. With limitation of carbohydrate, microalgae grew preferentially using photosynthesis. In fact, after 10th day, carbohydrate consumption continued with a slow rate which indicated that microalgae used organic carbon source in heterotrophic metabolism with photosynthetic.

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In heterotrophic growth, carbohydrate was consumed with high rate to 11th day. After that, even though carbohydrate concentration was limited, there was no light for photosynthesis as mixotrophic condition and the microorganism was used remained organic carbon. The consumption rate of substrate in mixotrophic growth was lower than heterotrophic growth. This lower rate revealed that in mixotrophic growth autotrophic

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metabolism took place in addition to the heterotrophic metabolism.

3.5. Heterotrophic And Autotrophic Metabolisms Under Mixotrophic Growth As mentioned in section 3.4, in mixotrophic cultivation medium due to higher carbohydrate consumption in the first 10 days (Fig. 5), the dominant growth pathway was heterotrophic. After limitation of organic carbon in heterotrophic metabolism, microorganism consumed CO2 as another carbon source in photosynthesis in addition to a slow rate of using organic carbon. The use of organic carbon first and the use of CO2 then, may not lead to a conclusion that in mixotrophic cultivation, heterotrophic and autotrophic metabolisms act sequentially. According to the conditions of cultivation in each time, one of these metabolisms has been dominant. In the first 10 days, in addition to heterotrophic growth, photosynthesis was also performed. This is evident by chlorophyll content in Fig. 3. In the first 10 days, the content of chlorophyll in heterotrophic media which consumed mostly organic carbon was observed to be less than the content in mixotrophic media. Higher chlorophyll content revealed that photosynthesis happened in mixotrophic condition in addition to heterotrophic metabolism. Another reason for simultaneous acting of heterotrophic and autotrophic growth in mixotrophic condition was obtained by comparing the dry weight. According

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to Fig. 1, dry weight of microalgae in the first 10 days in heterotrophic condition was less than mixotrophic one and according to Fig. 5, organic substrate was used more in heterotrophic media. Therefore, another carbon source should be used in mixotrophic media which resulted to higher dry weight. As a result, CO2 was consumed in autotrophic metabolism. The CO2 produced from heterotrophic metabolism might also be trapped and

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reused under autotrophic cultivation [18, 30].

In fact, mixotrophic growth of microalgae is not a simple combination of growth under heterotrophic and autotrophic modes [13, 20] unlike that proposed in some previous literatures [16, 31]. In Fig. 1, sum of dry weight of microalgae under heterotrophic and autotrophic modes was demonstrated. According to this curve, dry weight in mixotrophic growth was higher than the sum of dry weight obtained from autotrophic and heterotrophic growth. Heterotrophic and autotrophic modes in mixotrophic growth were not act the same as that happened in heterotrophic or autotrophic growth. In mixotrophic growth, the presence of organic carbon can change autotrophic and heterotrophic modes. In fact, the effect of carbohydrate on photosynthesis and heterotrophic modes can be explained by alteration of the cellular level of photosynthetic enzymes and heterotrophic substrates, respectively [32]. Moreover, the simultaneous occurrence of heterotrophic mode with autotrophic mode, despite their independency, can change the behavior of microalgae in each of these modes. As a result, the summation of dry weight in individual autotrophic and heterotrophic growth was not observed to be equal to the dry weight in mixotrophic condition.

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3.6. Mixotrophic Medium As The Best Growth Condition Of C. Vulgaris Mixotrophic growth of C. vulgaris with molasses and CSL as carbon and nitrogen sources, respectively lead to the highest cell dry weight and lipid content compared to autotrophic and heterotrophic conditions. Microalgae dry weight in mixotrophic growth reached to 2.62 g.L-1 with 33% lipid of 0.864 g.L-1. These values were higher than autotrophic growth with 1.08 g.L-1 cell dry weight and 0.32 g.L-1 lipid and also

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heterotrophic growth with 0.67 g.L-1 cell dry weight and 0.06 g.L-1 lipid. As a result, it can be concluded that the mixotrophic cultivation is the best choice for the growth of used C. vulgaris with the aim of biodiesel production which is the same as that reported previously [6, 10, 13, 15, 16]. Higher productivity reduces the final production cost. It should be noted that the price of required substrates increase the final cost of mixotrophic growth to about 80% of total cultivation medium cost [33]. But in this research work using cheap substrates of molasses and CSL instead of classical carbon sources like glucose, reduces the final cost of cultivation in mixotrophic medium. Moreover, C. vulgaris is one of microalgae species with the ability of the growth in mixotrophic condition; contrary to some strains which cannot use light as energy source in presence of carbohydrates [15]. Therefore, this strain can have advantages of growth in this cultivation medium with a highest biomass productivity and lipid content.

4. CONCLUSIONS In the present investigation, the productivity of C. vulgaris was studied and a maximum cell density of 2.62 g.L-1 was obtained in mixotrophic growth with the highest lipid concentration of 0.864 g.L-1 (33% of dry weight) which is remarkably higher than those

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obtained from autotrophic and heterotrophic growth. As a result, mixotrophic cultivation is the best choice for used C. vulgaris with the aim of biodiesel production. In mixotrophic growth, heterotrophic and autotrophic metabolisms are suggested to function simultaneously. However, the fraction of heterotrophic metabolism was more in first growth days and the fraction of autotrophic metabolism tend to overcome in the last days.

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Table 1. Cane Molasses and corn steep liquor (CSL) composition Molasses

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Major component

CSL % in dry

Mineral

mass

mg per 1 L

Major

molasses

component

% in dry mass

Mineral

mg per 1 L CSL

Ash

14.5

Ca

780

Ash

13

K

6200

Crude protein

2.5

K

2512

Crude Protein

49

Fe

20.6

Crude fiber

0.1

Fe

20

Fat

0.4

Na

200

NDF*

0.8

Mg

100

Lactic Acid

21

Cu

1.6

ADF**

0.5

Na

644

Phytic Acid

6.6

Ni

0.6

Lignin

0.3

Mn

0.8

Nitrogen

7.5

P

0.6

Total sugars

68.2

Zn

1.5

Total Sugars

2.5

Cl

100

Sugar

%

P

180

Glucose

10

Cl

10

Fructose

4

Sucrose

51

22

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Other Sugars 35

* atural detergent fiber

**Acid detergent fiber

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Figure 1. Dry weight of C. vulgaris versus time under different trophic conditions.

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Figure 2. Lipid content (g.g-1) of C. vulgaris versus time in different trophic conditions.

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Figure 3. The content of (a) chlorophyll-a and (b) chlorophyll-b in autotrophic,

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heterotrophic and mixotrophic growth.

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Figure 4. The color of microalgae cells grown under heterotrophic, autotrophic and

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mixotrophic (left to right) conditions.

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Figure 5. Carbohydrate concentration in heterotrophic and mixotrophic growth.

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Investigation of mixotrophic, heterotrophic, and autotrophic growth of Chlorella vulgaris under agricultural waste medium.

Growth of Chlorella vulgaris and its lipid production were investigated under autotrophic, heterotrophic, and mixotrophic conditions. Cheap agricultur...
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