Impact of Culture Media Composition, Nutrients Stress and Gamma Radiation on Biomass and Lipid of the Green Microalga, Dictyochloropsis splendida as a Potential Feedstock for Biodiesel Production

Biodiesel production from microalgae depends on the biomass and lipid production. Both biomass and lipid accumulation is controlled by several factors. The effect of various culture media (BG11, BBM, and Urea), nutrients stress [nitrogen (N), phosphorous (P), magnesium (Mg) and carbonate (CO3)] and gamma (γ) radiation on the growth and lipid accumulation of Dictyochloropsis splendida were investigated. The highest biomass and lipid yield of D. splendida were achieved on BG11 medium. Cultivation of D. splendida in a medium containing 3000 mg L N, or 160 mg L P, or 113 mg L Mg, or 20 mg L CO3, led to enhanced growth rate. While under the low concentrations of nutrients caused a marked increase in the lipid content. Cultures exposure to 25 Gy of γ-rays, led to an increase in lipid content up to 18.26 ± 0.81 %. Lipid profile showed the maximum presence of saturated fatty acids (SFAs, 63.33%), and unsaturated fatty acids (UFAs, 37.02%). Fatty acids (FAs) recorded the predominance of C16:0, C18:2, C15:0 and C16:1, which strongly proved D. splendida is a promising feedstock for biodiesel production.


Introduction:
Renewable, sustainable, and eco-friendly biofuels are development fields and attractive research that are much needed because of fossil fuels depletion and environmental pollution. Biodiesel has several advantages such as high biodegradable, absence of any aromatic compounds and 90% reduction in air toxicity may conduct to 95% decrease in the applicable cancer cases and have similar properties of fossil diesel 1,2 .
Biodiesel can be classified according to their source into 1) biodiesel produced from edible oil (first generation) such as soybeans, rapeseed, and sunflower seeds 3 . About 7% of global edible vegetable oil supplies were utilized for biodiesel production in 2007. However, vast use of edible oils may cause food supplies versus fuel issue (food crisis) 4, 2 biodiesel produced from waste cooking oil, animal fats and nonedible vegetable oils (second generation) such as jatropha 5 , and 3) third generation biodiesel is produced from microalgae 6 .
The advantages of microalgae over higher plants as a source of biodiesel: 1) synthesize and accumulate large quantities of neutral lipids, 2) Possess a high photosynthetic efficiency and growth rate, 3) Grow on saline/brackish water and nonarable land as well as it can utilize nitrogen (N) and phosphorous (P) of wastewater, 4) Can grow in photobioreactors with higher biomass production. 5) Sequester CO 2 through photosynthesis and so reducing greenhouse gas emission 7 .
Current research into increasing lipid accumulation in microalgal cells mainly focuses on the optimization of culture conditions, screening microalgae species, and the transformation of microalgae by genetic engineering. Limitation of nutrients in culture media is a commonly technique used to increase lipid inside the microalgal cells. N and P starvation besides magnesium (Mg) and carbon supplementation can induce biosynthesis of FAs 8, 9 . Little information is available on the effect of γradiation on the physiological mechanism and biochemical composition of microalgae 10,11 . The objectives of this study were to investigate the effect of culture media composition, nutrients concentration (nitrogen, phosphorous, magnesium, and carbonate) as well as the dosage of γradiation on both algal growth and lipid parameters of microalga, Dictyochloropsis splendida

Materials and Methods: Cultivation of microalgae
The green alga, Dictyochloropsis splendida was provided by the algal culture collection from the Laboratory of Phycology in Botany and Microbiology Department, Faculty of Science, Cairo University, Egypt. The alga was cultivated on BG-11 medium 12 and incubated under a continuous light intensity of 40 µE m -2 s -1 (daylight fluorescent lamps, Philips, TLD18W/54-765) at 25± 1 o C and aeration with constant sterilized bubbling of air (by a 0.22µm filter) for 25 days.

Influence of media composition on growth and lipid production of D. splendida
To evaluate the impact of media composition on the growth and lipid content, D. splendida was cultured in 1L glass flasks in BG11 medium 12 , BBM 13 and urea medium 14 . Flasks were incubated under the same pervious conditions.

Influence of nutrients concentrations on growth and lipid production of D. splendida
In all experiments, D. splendida was grown in BG11 medium 12 under continuous illumination with aeriation rate of 1.25 L/min at 25± 1 o C for 25 days. Nitrogen was used in the form of NaNO 3 in concentrations 0, 380, 750, 1500, and 3000 mg L −1 . Phosphorous (P) was used in the form of K 2 HPO 4 in concentrations of 0, 40, 80, 160, and 320 mg L −1 . Magnesium (Mg) was used as MgSO 4 •7H 2 O in concentrations 19,38,75,113 and 150 mg L −1 . Carbonate (CO 3 ) as Na 2 CO 3 in concentrations of 0, 10, 20 40 and 80 mg L −1 . Growth parameters and lipid content were determined at each experiment.

Influence of γ-radiation on growth and lipid production of D. splendida
Cultures of D. splendida were irradiated by different γ-doses 0, 25, 50, 100, 200,300, 500, 1000 Gy of 60 Co γ-rays. Irradiation was performed by 60 Co γ-rays (Gamma cell 4000-A-India) at National Center for Radiation Research and Technology (NCRRT), Egyptian Atomic Energy Authority (EAEA), Egypt at a dose rate of 1.296 KGy/h. Cultures after irradiation were incubated under previous conditions and growth was determined as optical density at 680 nm. The lipid content was calculated at the end of the experiment.

Cell growth measurements
From 1L incubated algal culture (900 ml BG11 + 100 ml algal inoculum), optical density (OD at 680nm) of the microalgal sample (3ml) was determined at regular interval of 5 days (in triplicates) using spectrophotometer (UV-Vis spectrophotometer, T60, UK). Twenty ml of washed filtered culture were dried at 105°C for 24 hrs., chilled in a desiccator, and the algal dry weight was determined and expressed as g L -1 . The maximum specific growth rate, μ max (d -1 ), was evaluated as: Where X f and X o are the biomass concentrations (g L -1 ) at the final and the start of a batch run, respectively; and t is the time span of the run (day). The biomass productivity (BP) (mg L -1 d -1 ) and biomass yield (BY, g L -1 ) were assessed as follows 15 : Where X f and X o are the biomass concentrations (g L -1 ) at the final and the start of a batch run, respectively; and T 1 and T 2 (day) represent the incubation time of an experiment at the start time day and the final day of incubation, respectively.

Determination of lipid content
Lipids were extracted at the final incubation time by a 1:1:0.9 ratios of chloroform: methanol: deionized water mixture on volumetric basis 16 where 5 ml chloroform, 10 ml methanol, and 4 ml of deionized water were initially added to 0.3 g dried sample (0.3 g dried algal biomass/1L algal culture). Then, the mixture was shaken for 10 min, and then another 5 ml chloroform and 5 ml deionized water were added and shaken for overnight. The algal-solvent mix was refined to eliminate the algal precipitates. The chloroform layer of the filtrate was removed, solvent was volatilized at 40-45°C and the lipid was weighed. Lipid content was determined as percentage of cell dry weight:

Transesterification and Fatty acid analysis
Lipid was transesterified to produce fatty acid methyl ester (FAMEs) using 2% sulphuric acid in methanol 19 . FA analysis was achieved in Central Laboratory, Faculty of Agriculture, El-Azhar University by gas chromatography (Perkin Elmer Auto System XL) using DB5 silica gel capillary column (60 m ×0.32mm i.d.) with flame ionization detector and Helium was applied as the carrier gas (at the flow rate of 1 ml min -1).

Statistical analysis
All the experiments were conducted in 3 replicates. One-way ANOVA with 95% confidence (probability limit of p < 0.05 was utilized to estimate the significant difference in dependent variables, and Tukey's test at a reliability level of (p<0.05) was used to identify differences between each level of treatment. The statistical analyses were achieved using Minitab software (V18, Minitab Inc., State College, PA, USA).

Results and Discussion Influence of media composition on growth and lipid accumulation
The effect of various culture media (BG11, BBM and Urea) composition on the growth of D. splendida were assessed as outlined in Figure1. The highest BY of D. splendida (0.90 ± 0.01 g L -1 ) resulted in culturing on BG11 medium. With this medium, the maximum μ max and BP were 0.097 ± 0.002 d -1 and 32.96 ± 0.54 mg L -1 d -1 , respectively. Also, highest LC, LB and LY were 16.92 ± 0.07 %, 5.58 ± 0.07 mg L -1 d -1 and 0.152 ± 0.001 g L -1 , respectively while urea medium showed the lowest LC (10.43 ± 0.79 %) as illustrated in (Figure 2A,B). The increase in the LY of D. splendida when cultured on the BG11 medium may be back to the high N concentration (1.5 g L -1 ) in the BG11 medium which led to an increasing μ max , where LY is the product of the BY multiplied by the LC 18 . This finding went parallel with Chandra et al 20 who studied the effect of different culture media (BG-11, modified CHU-13 and BBM medium) on the growth and lipid production of Chlorella minutissima. Maximum BY and LY were achieved by modified CHU-13 medium (970 ± 0.21 and 356.63 ± 0.51 mg L −1 , respectively) succeed in descending order by those produced by BG-11 medium (850 ± 0.12 mg L −1 and 243.65 ± 0.30 mg L −1 , respectively) and the minimum values were recorded by BBM medium (730 ± 0.42 mg L −1 and 196.83 ± 0.43 mg L −1 , respectively).
In another study, Chlorella sp. and Scenedesmus sp. were cultivated in media with more or less nutrients. Accumulation of lipid was higher in media deficient of nutrients whereas μ max and LP were reduced 21 . Furthermore, micronutrients such as iron, cobalt, zinc, copper and manganese and nickel are the most essential trace metals required by algae for several metabolic functions 22 . This supports our results, where the highest μ max and LP of D. splendida were recorded on BG11 medium followed in descending order by BBM and urea medium, which may be due to the availability (or not) of nutrients in the media 23 . On other hand, several studies used nitrate in source of N in culture media, whereas urea has been highly applied in large-scale algal cultivation due to its competent low cost compared to the others. Nevertheless, the manipulation of urea concentration through the cultivation is the challenge. Urea can liberate urease or be hydrolyzed to ammonia in basic conditions which lead to the growth of inhibition at high levels 24 .

Impact of nutrients concentrations on growth and lipid formation Nitrogen
The impact of initial concentrations of N on the growth of D. splendida was represented in Figure 3A. Increasing the P and N, was accompanied by an increase in growth. The highest BP and BY of 42.06 ± 2.25 mg L -1 d -1 and 1.15 ± 0.05 g L -1 , respectively were obtained by cultivation with a start N concentration of 3000 mg L -1 ( Table  1). Elevation of the N concentration from 0 to 3000 mg L −1 showed an obvious increment in biomass and growth rate, but a decline in lipid accumulation. The highest LC of 18.09 ± 0.03 % was recorded under N depletion (380 mg L −1 ) as illustrated in Figure 4A. The LY of D. splendida was significantly influenced by the N concentration (P <0.05). The highest LP (5.37 ± 0.12 mg L -1 d -1 ) and LY (0.0152 ± 0.001 g L -1 ) were recorded at N concentration of 1500 mg L -1 .
Nitrogen is the most commonly reported nutrient-limiting factor in the growth and lipid accumulation of microalgae 24 . The obtained results agrees with Ishika et al. 25 who reported that N deficiency results in an increment in lipid and /or carbohydrate accumulation of microalgae and a decline in growth rate, photosynthetic efficacy, and protein amounts. Rehman and Anal 26 noted that the LC of Chlorococcum sp. TISTR 8583 increased by 1.7 folds when cultured on N-deficient medium and optimized light intensity. Similarly, Yodsuwan et al. 27 reported that the maximum LC of P. tricornutum (53.04 ± 3.26% %) was noted under N-deficient condition.

Phosphorous
The growth curve of D. splendida in the growth medium for different initial P concentrations are shown in Figure 3B. Reasonably, the maximum cell density increased with an increase in initial P concentration. From the ANOVA results, we found that P had a remarkable effect (p< 0.05) on biomass production of D. splendida. The maximum μ max, BP and BY of 0.111 ± 0.010 d -1 , 41.01 ± 3.96 mg L -1 d -1 and 1.10 ± 0.11 g L -1 were obtained at 160 mg L -1 , respectively. Increasing the P concentration from 40 mg L -1 to 320 mg L -1 had an insignificant effect (p> 0.05) on BY ( Table 1). The lipid accumulation of D. splendida under different initial P concentrations was given in Figure 4B. While deficiency in P significantly promoted lipid accumulation (p< 0.05). The highest LC (18.39 ± 1.22 %), LP (7.06± 0.82 mg L -1 d-1) and LY (0.189 ± 0.023 g L -1 ) were recorded at 80 mg L -1 P as shown in Table 1and Figure 4B.
Phosphorous is the main player in cellular metabolic processes, which are connected to photosynthesis and energy transfer. The results agreed with those of Guschina and Harwood 28 who mentioned that under P deficiency, the photosynthetic rates decreased, the cell division rates reduced, and this may lead to the accumulation of triacylglycerols. Also, under P limitation, the LC of Tisochrysis lutea 29 and P. tricornutum 30 were increased. In addition, the total FAs content increased over two folds under P depletion, conversely total FAs content was inversely proportional with P concentration over a factor of ten 31 . Figure 3C illustrates Mg, respectively (Table 1). On the contrary, the increasing Mg concentration exhibited a negative impact on the LC. The maximum LC (20.06 ± 0.15 %) was achieved at 19 mg L −1 of Mg ( Figure 4C). Further, the LP and LY of the tested microalga were significantly affected by alteration in the Mg concentration (P<0.05).

Magnesium
Mg plays a key role in the growth of microalgae, whereas it is the central atom of chlorophyll and as a co-factor of some enzymes in the metabolic pathway 32 . There are limited studies on microalgae responses during Mg limitation in terms of biomass growth and lipid accumulation 33 . The lipid yield and growth of microalgae were improved by Mg supplementation, whereas the starvation of Mg ions anticipates the decrease in mitotic division, hinder of chlorophyll formation and, so, the biomass yields 34 .
In harmony with the obtained data, Gorain et al. 35 found a marked increase in the neutral lipid content of Chlorella vulgaris and Scenedesmus obliquus in Mg-and Ca-free medium. Also, Increasing the concentration of Mg exhibited positive effects on BY of C. vulgaris and S. obliquus, and at concentration (150 mg L −1 ) the BY was elevated up to 1.5 g L −1 (36% rise) for S. obliquus and 1.6 g L −1 (33% rise) for C. vulgaris on the 18 th day of incubation. While the LC was increased with maximum up to 27% and 26%, respectively at 100 mg L −1 of Mg. The function of Mg ions in switch on the enzyme Acetyl-CoA carboxylase and catalyzing the first stage of FA production was proved 36 . In addition, the productivity of microalgae is augmented when Mg 2+ concentration is in the range of 2-8 mg/L 24 .

Carbonate
High and low sodium carbonate concentration in the growth medium had significant influence (p< 0.05) on growth ( Figure 3D) and lipid production of D. splendida ( Figure 4D). Table 1 summarizes the biomass and lipid parameters of D. splendida under different concentrations of sodium carbonate. At 20 mg L -1 of CO 3 , the maximum BY (0.90 ± 0.01 g L -1 ) was recorded, whereas, rising the CO 3 concentration showed a significant decrease in the growth parameters (μ max and BP) (p<0.05). The highest LC of 19.46 ± 0.32 % was showed at 40 mg L -1 as presented in Figure 4D. The LP and LY ranged between 1.66-5.37 mg L -1 d -1 and 0.051-0.152 mg L -1 , respectively.
Most investigations that have been done on the effect of inorganic carbon supply and lipid formation in microalgae cultures have converged on the addition of CO 2 37 . In some works, NaH 2 CO 3 has been utilized as a source of carbon on experimenting growth and biochemical composition in various microalgae species 38 and induced the accumulation of triacylglycerol in microalgal species. On the contrary, Zhao et al. 39 recorded that the addition of sodium bicarbonate in the culture medium of Scenedesmus quadricauda had a negative influence on the lipid production and the highest LC was obtained under air. On the other hand, Li et al. 40 found that the maximum LC of 494 mg g −1 and LP of 44.5 mg L −1 d −1 of C. vulgaris were recorded at 160 mM NaHCO 3 and pH 9.5, and 10 mM NaHCO 3 was the optimal concentration for cell growth and elevating NaHCO 3 from 10 to 160 mM prosecute an inhibition to biomass. Figure 3E shows the growth curve of D. splendida under different gamma radiation doses. The data exhibited that high doses of γ-ray had a negative effect on growth. The maximum μ max was decreased with elevating irradiation dose ( Table 2). The BY declined from 0.90 ± 0.01 g L -1 to 0.21 ± 0.02 g L -1 (decreased by 76.67 %) when cultures were displayed to irradiation dosage of 1000 Gy. The LC of D. splendida given in Figure 4E, the highest LC of 18.26 ± 0.81 % was achieved when the alga cell exposed to 25 Gy. While the higher irradiation doses had negative impact on the lipid accumulation. The maximum LP (5.37 ± 0.12 mg L -1 d -1 and 5.24 ± 0.43 mg L -1 d -1 ) was recorded at zero and 25 Gy, respectively.

Gamma radiation
Gamma rays can generate free radicals (ROS), which have the ability to change the composition of cells in comparison with the slight penetration influence of UV-B 41 . Hence, 60 Co-γrays were selected for irradiation due to their powerful penetration ability. In concomitant with the obtained results, Cheng et al. 10 found that the lipid amount of Nitzschia sp. declined with increased irradiation dose (0-900 Gy). Agarwal et al. 42 reported that the high irradiation doses extremely injure cell metabolism regulation complex and growth cease if cells lose their selfrepair potential through injury recuperation. Considering that various strains had diverse irradiation vulnerability to nuclear irradiation, whereas under low dosages of γ-ray irradiation, some microalgal cells were still slightly damaged and recuperate their normal states within a brief period 43 .

Fatty acid composition
The fatty acid composition of D. splendida was given in Table 3. The FAME mainly contains saturated fatty acids (SFAs, 63.33 %) and unsaturated fatty acids (UFAs, 37.02%), also, the carbon chain lengths were from C12 to C24. Among the identified FAs, C16:0 was found to be  FA, fatty acid Regarding the biodiesel formation from D. splendida, the green microalgal lipid usually has a FAs content of mostly C16 and C18 FAs that is alike to that of vegetable oils, and so appropriate for biodiesel formation 44 . The C16-C18 FAs of D. splendida were 80.55%, which can give the best relation between oxidative stability and cold flow properties 45 . MUFAs, which mainly formed of C16:1 and C18:1, are regarded as the most favorable components for forming biodiesel, and they give the best compromise between oxidative stability and cold flow properties 46 .
The tested microalga had a distinctly higher amounts of C16 and C18 which were closer to those of Haematococcus pluvialis (76.6%) 47 . Also, D. splendida demonstrated considerable amount of C18:2 and C18:3, formed in low melting points, and are preferable for the improvement of the low temperature properties of biodiesel 48 .

Conclusion:
The impact of media components, nutrients stress and γradiation on the biomass and lipid production of D. splendida was studied. The highest BY and LY were achieved when alga culturing on BG11 medium. The maximum µ max was obtained at high N, P and Mg as well as low CO 3 . While the highest LC was observed under nutrients limitation. Additionally, high γ-radiation doses expressed a negative influence on both growth and lipid production. The C16-C18 FAs of D. splendida were 80.55% which firmly manifested that D. splendida is a promising source for biodiesel formation.