Manipulating nutrient composition of microalgal growth media to improve biomass yield and lipid content of Micractinium pusillum

1 Department of Environmental Engineering, Yonsei University, Wonju, Gangwon-do 220-710, South Korea. 2 City of Scientific Research and Technology Applications, New Borg El Arab City, Alexandria 21934, Egypt. 3 Geologic Environment Division, KIGAM, Daejeon 305-350, South Korea. 4 Department of Biological Environment, Kangwon National University, Chuncheonsi, Gangwon-do, South Korea. 5 Korea Institute of Energy Research, Daejeon 305-343, South Korea.


INTRODUCTION
Both rapid growth and industrialization of nations have resulted in a steep increase in the production and consumption of fossil fuels. This increase has not only put severe stress on already depleting fossil fuels, but also resulted in an alarming increase in pollution across the globe. The current demand for biofuel as a gasoline substitute is extremely high due to the high cost of petroleum or the potential for a high cost. One such fuel showing great potential is biodiesel that has received much attention recently, as it is made from non-toxic, biodegradable, and renewable resources. Biodiesel also has environmental benefits, because they have fewer *Corresponding author. E-mail: bhjeon@yonsei.ac.kr. Tel: +82 33 760 2446. Fax: +82 33 760 2571.
harmful emissions, such as carbon monoxide and hydrocarbons, and can decrease the greenhouse effect (Gouveia and Oliverira, 2009;Campbell et al., 2011).
Microalgae are emerging as one of the most promising resources of biodiesel with a projected yield of 58,700 to 136,900 L/ha/year (Chisti, 2007). Microalgae have a number of advantages as a potential feedstock to produce biodiesel, including higher photosynthetic efficiency, biomass production, and growth rates than other energy crops (Huang et al., 2010). Many microalgae have the ability to produce substantial amounts (1 to 70% dry cell weight) of triacylglycerols (TAG) as a storage lipid under photo-oxidative stress or other adverse environmental conditions (Richmond, 2004;Cheirsilp and Torpee, 2012). Lipid production from microalgae can be improved by manipulating growth conditions such as nitrogen deprivation (Illman et al., 2000), silicon deficiency (Lynn et al., 2000), phosphate limitation (Reitan et al., 1994), high salinity (Rao et al., 2007), and some heavy metal stress (Guschina and Harwood, 2006).
The nutritional attributes of microalgae depend upon several intrinsic factors, such as their biochemical composition, average size, cell wall digestibility, and lipid accumulation. Dunaliella tertiolecta required inorganic phosphates and trace elements (that is Co, Mo, Fe, and Mn) to be grown optimally . Although several studies of individual nutrients such as carbon (Hosoglu et al., 2012), nitrogen (Shen et al., 2009), and iron (Liu et al., 2008) have been published, the effect of micronutrient concentration on algal growth and lipid production has not been reported.
Therefore, our study evaluated the effects of micronutrients (that is Zn, Mn, Cu, and Co) on biomass production and lipid content of M. pusillum. Furthermore, we investigated the fatty acid composition of this microalgal strain.

Microalgal cultivation and growth analysis
Liquid medium [100 mL at 10% (v/v)] was inoculated with the algal strain in 250 mL Erlenmeyer flasks. The flasks were incubated under continuous white fluorescent light at 40 µmol photon/m 2 /s at 27 ± 2°C for 17 days, while shaking at 150 rpm on a rotary shaker (SH-804, Seyoung Scientific). Algal growth was measured by determining the optical density of the algal cell suspension at 680 nm using a DR/4000 v spectrophotometer (HACH, USA). The OD680 was then converted to dry weight concentration using a linear relationship between OD680 and dry cell weight (g/L) (American Public Health Association, 1998), which was obtained after an extensive data analysis in this study. Experiments were carried out Abou-Shanab et al. 16271 in triplicate, and data are expressed as mean ± standard deviation.

Lipid extraction and fatty acid analyses
The total lipids were extracted from M. pusillum biomass (0.2 g/L) using a slightly modified method of Bligh and Dyer (1959). In brief, cells were harvested and lyophilized. Lipids were extracted with a mixture of chloroform and methanol (1:2, v/v), transferred into a glass tube, and indirectly sonicated for 30 min at a constant frequency of 40 kHz and at a power output of 700 W using a Powersonic 420 bath sonicator, South Korea. The tube was then incubated over night at 27°C with shaking at 100 rpm. An additional aliquot of chloroform (1.25 mL) was added to the tube and the content was sonicated again for 30 min. To separate the chloroform and aqueous methanol layers, 1.25 mL deionized water was added to the tube, which was then centrifuged at 4000 rpm for 10 min. The chloroform layer was collected from the bottom of the tube. A second extraction was performed by adding 2.5 mL chloroform and vortexing. The chloroform layer was gently collected from the bottom of the tube, washed with 5 mL of 5% NaCl solution, and evaporated in a dry oven at 50°C. The percent lipid of total dry biomass was calculated as weight of crude lipids that was used for fatty acid methyl ester analysis. Each experiment was carried out in triplicate and average values were reported. Fatty acids were analyzed using a modification of the method proposed by Lepage and Roy (1984). The crude lipid (~ 10 mg) was dissolved in 2 mL of a freshly prepared chloroform and methanol mixture (2:1, v/v) and transferred to a 10 mL Pyrex tube with a Teflon-sealed screw-cap. 1 mL of chloroform containing an internal standard and transmethylation reagents was added to the tube and mixed for 5 min. The contents were transferred to a 10 mL Pyrex tube, incubated at 100°C for 10 min, cooled to room temperature, and separated into two phases by adding 1 mL deionized water. After 10 min of vigorous mixing and centrifugation at 4000 rpm for another 10 min, the chloroform layer was collected from the bottom of the tube using a hypodermic disposable polypropylene syringe and filtered through 0.2 µm syringe filters. Fatty acid methyl esters (FAMEs) in the extracted liquid were quantified by QP2010 Gas Chromatography-Mass Spectrometry (Shimadzu, Japan) with a flame ionization detector using a HP-5MS capillary column.
The oven temperature was set at 80°C, held for 5 min, raised to 290°C at 4°C/min, and held at 290°C for 5 min, and the temperature for injector and detector were set at 250 and 230°C, respectively. Helium was used as a carrier gas at a flow rate of 1.2 mL/min. The compounds were identified by comparing fragmentation patterns with those in the National Institute of Standards and Technology (NIST) library.

Statistical analysis
All data are represented as mean ± standard deviation of triplicate. Statistical analysis was performed using the SPSS package system version 11.

Effect of media compositions on the growth rate of M. pusillum
Microalgae can grow profusely when supplied with sufficient nutrients under suitable conditions. Algal growth is directly affected by light and nutrient availability, pH and temperature stability, and the initial density of inoculum (Wang et al., 2010a). A certain amount of trace metals (that is Mn, Cu, Zn, and Co) is capable to induce the growth of microalgae, while at the same time higher concentrations of these micronutrients can retard the growth of microalgae (Ilavarasi et al., 2011). Figure 1A shows that depleting individual micronutrients (that is Co, Mn, Zn, and Cu) from the culture media significantly decreased the M. pusillum growth rate compared with the control (paired t-test=3.42, P < 0.01). The average dry biomass concentration of M. pusillum grown in BBM (control) was 0.34 ± 0.01 g/L, while for micronutrientdepleted BBM, the dry biomass ranged from 0.24 ± 0.01 g/L (0X Cu) to 0.28 ± 0.01 g/L (0X Co) after 17 day of cultivation. Micronutrients (Co, Mn, Zn, and Cu) are essential for microalgal growth. These elements play vital roles in the active site of many algal enzymes and are involved in numerous metabolic processes, including photosynthesis and energy storage (Christensen, 1997;Liu et al., 2008;Chen et al., 2011). Thus, depleting micronutrients from the culture medium adversely affected M. pusillum growth. The average dry biomass of M. pusillum increased with the increase of Mn or Cu concentrations (from 2X to 4X) in the growth medium ( Figure 1B ± 0.01 g/L or 0.37 ± 0.02 g/L, respectively, after 17 days of incubation, both of which were significantly higher (paired t-test -2.3, P < 0.05) than the control (0.34 ± 0.01). In contrast, increasing the Zn or Co concentration in the growth media had no noticeable effect on dry weight. Based on these results, further experiments evaluated M. pusillum growth as a function of Mn or Cu concentration in BBM. Increasing the Mn or Cu concentration to 4X, increased the M. pusillum biomass (0.39 ± 0.01 or 0.42 ± 0.01 g/L, respectively) compared to regular BBM (Figures 2 and 3). Interestingly, increasing the Mn or Cu concentration to 5X or higher had no further  Sommerfeld, 1999).

Biomass yield and lipid productivity
We harvested the algal cells after the 17 day incubation and examined lipid content, lipid productivity and biomass yield (Table 1). Depleting Zn, Mn, Co, or Cu from growth medium adversely affected algal biomass and lipid production. M. pusillum grown in BBM with 4X Cu or Mn produced more biomass (1.28 ± 0.04 or 1.25 ± 0.01 g/L) and lipid productivity (0.47 ± 0.05 or 0.45 ± 0.04 g/L) after 17 day of cultivation than the control (Table 1). Increasing the Mn or Cu concentration to 5X or higher had no further effect on the alga dry weight. This finding was consistent with the result of Wang et al. (2010b) who found that the increase of Mn concentrations stimulated the growth of blue green algae, while a further increase in Mn inhibited algal growth. The total lipid contents of M. pusillum in this study ranged from 31 ± 3.5% to 41 ± 1.5% of the dry biomass weight. The highest lipid content (41 ± 1.5%) was presented by the algal strain grown in BBM containing 2X Mn. Cloe¨z et al. (1987) found that lipid synthesis increased by three times after adding manganese, copper, and nickel at 2 mM. Hydrocarbon production was more sensitive to the change in Mn concentration. An increase in hydrocarbon production resulted from the increase of Mn concentrations (Song et al., 2012). Many microalgae species can be induced to accumulate substantial quantities of lipids (Sheehan et al., 1998), resulting in a high oil yield. Lipid contents of 20 to 50% of the dry biomass weight have been reported to be quite common (Spolaore et al., 2006;Li et al., 2008). It has also been reported that lipids accounting for more than 90% of the dry biomass of some microalgae have been reported in some culture conditions (Mata et al., 2010). Table 2 shows the fatty acid composition in M. pusillum harvested from different culture media. Linoleic acid (C18:2n6c) ranged from 49 to 54% of all fatty acids, and was the dominant fraction for all experimental conditions. Linoleic acid was followed by palmitic acid (C16:0) and linolenic acid (C18:3n3) ranging from 24 to 29% and 16 to 22%, respectively. Oleic acid (C18:1n9c) accounted for <5% of all fatty acids. Biodiesel quality depends on the fatty acid composition. Petkov and Garcia (2007) found 14:0, 16:0, 16:1, 18:0, 18:1, 18:2, and α-18:3 fatty acid components from green algae. A large number of double bonds in a fatty acid make it more susceptible to oxidation, thus results in economical loss (Chisti, 2007).

Fatty acid composition
Nutrient composition of the growth medium, cultivation conditions, and growth phase can readily affect the fatty acid composition in algal biomass (Hu et al., 2008). Palmitic acid, oleic acid, and linoleic acid were found as the major fatty acids in both photoautotrophically and heterotrophically grown cultures of Chlorella zofingiensis (Liu et al., 2011). Of all the nutrients evaluated, nitrogen limitation is the single most critical nutrient affecting lipid metabolism in algae. A general trend towards accumulation of lipids, particularly triacylglycerols (TAG), in response to nitrogen deficiency has been observed in numerous species or strains of various algal taxa (Hu et al., 2008).

Conclusion
The present work investigated the effect of culture medium (BBM) supplemented with different concentrations of trace metals on the biomass yield, and lipid production of M. pusillum. The results demonstrate that trace metals play a major role in the algal biomass yield and lipid production. Increasing the Cu or Mn concentration in BBM increased the algal biomass and lipid productivity. BBM amended with 4X concentration of Cu or Mn resulted in 1.6 or 1.5-fold increase in biomass yield and 1.4 or 1.3-fold increase in lipid productivity when compared to control, respectively.
The polyunsaturated fractions ranged from 68 to 73% of the total fatty acids (FA) in microalga cultivated under all experimental variations. The lower percentage of polyunsaturated FA was obtained from alga grown in BBM amended with 4X Mn and 4X Cu. This study underlined the significance of medium development in achieving high-density cultures and lipid contents.