# The Effects of Trace Elements on the Lipid Productivity and Fatty Acid Composition ofNannochloropis oculata

Authors: Xiao Dou; Xiang-Hong Lu; Mei-Zhen Lu; Li-Sheng Yu; Rong Xue; Jian-Bing Ji
Journal: Journal of Renewable Energy
 (2013)

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## Abstract

The effects of trace elements on the lipid productivity and fatty acid composition ofNannochloropis oculata (N. oculata) were studied. The results showed that trace elements had a strong influence on not only the lipid productivity but also the fatty acid composition. The addition of Fe3+, Zn2+, Mn2+, Mo6+, and EDTA and the deletion of Cu2+ and Co2+ can increase the lipid productivity. The optimum concentrations of the trace elements in the culture medium are 6 times of Fe3+ and EDTA, the same concentration of Zn2+, Mn2+, and Mo6+ as the control group, but the optimum medium has no Cu2+ or Co2+. Fe3+, Zn2+, Mn2+, Mo6+, and EDTA are indispensable during the EPA formation of N. oculata. The addition of Fe3+, Zn2+, Mn2+, Mo6+, and EDTA can strongly increase the content of EPA in the lipid of N. oculata, but the concentration of the trace elements had little influence on the level of EPA.


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## Body

## 1. Introduction

In order to reduce the cost of biodiesel from microalgae, we can not only screen out the algal species that both grow well and have high lipid content but also explore the optimal culture condition to improve the lipid productivity of microalgae.N. oculata is a promising source of biodiesel because of its high growth rate and high lipid content, ranging from 31% to 68% [1].Apart from the macroelements such as N, P, and C, the growth rate and lipid content ofN. oculata can also be affected by trace elements. This is because trace elements are indispensable during the growing process of marine algae, and trace elements are indispensable parts of the algal cell itself and the enzyme active center of algal cell [2, 3]. There have been some reports about the effects of trace elements on the growth of microalgae. Liu and Wang [4] reported that high content of Fe3+ was beneficial to increase the lipid content of marine chlorella. Jeffrey and Millton [5] and Alexander and Markus [6] found that Mn2+ was indispensable during the growing process of microalgae and was the catalyst of photosynthesis and the activator of some kind of enzymes in microalgae. Yamochi [7] found that the addition of EDTA which could form a complex with many kinds of metal ions would promote the absorption of metal ions of microalgae and the growth of microalgae, and the complex iron was the inducement of red tide in the nutrition water. Boyer and Brand [8] found that trace elements could cause the abnormal propagation of microalgae, and suitable concentration of Fe3+, Mn2+, and Zn2+ could cause the mass rearing of microalgae. Yamasaki et al. [9] found that Mo6+ could control the growth and material accumulation of Dunaliella salina. Furthermore, too high or too low concentrations of Mo6+ were both harmful to the growth of Dunaliella salina. Their researches showed that trace elements had some effects on the growth density and lipid content of microalgae, but the effects of trace elements on the lipid productivity and fatty acid composition of N. oculata have not been reported yet.In this paper, the effects of trace elements (Fe3+, Cu2+, Zn2+, Co2+, Mn2+, and Mo6+) and EDTA on the lipid productivity and fatty acid composition of N. oculata were studied. The optimum dosage of trace elements that will increase the lipid productivity of N. oculata was determined.


## 2. Materials and Methods

### 2.1. Microalgae

N. oculata was originally obtained from Marine Biological Culture Collection Centre (China) and screened for its potential ability of growth at Zhejiang University of Technology (China). N. oculata cells were maintained in the modified F/2 medium in artificial sea water, which was made by putting 26.726 g NaCl, 2.26 g MgCl2, 3.248 g MgSO4, 1.153 g CaCl2, 0.198 g NaHCO3, 0.721 g KCl, 0.058 g NaBr, 0.058 g H3BO3, 0.0024 g Na2SiO3, 0.0015 g Na2Si4O9, 0.002 g H3PO4, 0.013 g Al2Cl6, 0.002 g NH3, and 0.0013 g LiNO3 in distilled water (per liter), at temperature 20°C, and pH = 8.0 with a light intensity of 3000 μmol·m−2·s−1 in illumination box. The composition of modified F/2 medium (per liter) was shown as follows: 0.225 g NaNO3, 0.045 g NaH2PO4·H2O, 0.02 g Na2SiO3·9H2O, 3.16 mg FeCl3·6H2O, 0.18 mg MnCl2·4H2O, 0.023 mg ZnSO4·7H2O, 4.36 mg Na2 EDTA, 0.01 mg CuSO4·5H2O, 0.012 mg CoCl2·6H2O, 0.07 mg Na2MoO4·2H2O, 1 μg vitamin B1, 1 μg vitamin B12, and 0.5 μg biotin.The mud of algae for inoculation was collected by centrifuging the cells ofN. oculata at the logarithmic phase at 3000 r·min−1 and then washed with distilled water for 3 times to remove the salt in the medium.


### 2.2. Culture System

Growth experiment was done at a different content of trace elements in 1.0 L-Erlenmeyer flasks. The medium and flasks were sterilized at UV irradiation for 30 min in a clean bench in order to prevent any contamination during the early stages of growth.N. oculata cells with the same inoculation density (OD440nm=0.70) grew at room temperature (25±1°C), natural lighting, and pH = 8.0 with bubbling of aseptic air continuously. The concentration of trace elements in different media was listed in Table 1.Table 1
The concentration of trace elements (TEs) in different media.
TE (mg·L−1)
Fe3+
Cu2+
Zn2+
Co2+
Mn2+
Mo6+
EDTA
group number
0
3.16
0.01
0.023
0.012
0.18
0.07
4.36
1
—
0.01
0.023
0.012
0.18
0.07
4.36
2
9.48
0.01
0.023
0.012
0.18
0.07
4.36
3
18.96
0.01
0.023
0.012
0.18
0.07
4.36
4
3.16
0.01
—
0.012
0.18
0.07
4.36
5
3.16
0.01
0.069
0.012
0.18
0.07
4.36
6
3.16
0.01
0.138
0.012
0.18
0.07
4.36
7
3.16
0.01
0.023
0.012
—
0.07
4.36
8
3.16
0.01
0.023
0.012
0.54
0.07
4.36
9
3.16
0.01
0.023
0.012
1.08
0.07
4.36
10
3.16
0.01
0.069
0.012
0.18
—
4.36
11
3.16
0.01
0.138
0.012
0.18
0.21
4.36
12
3.16
0.01
0.023
0.012
0.54
0.42
4.36
13
3.16
0.01
0.023
0.012
1.08
0.07
—
14
3.16
0.01
0.023
0.012
0.18
0.07
8.72
15
3.16
0.01
0.023
0.012
0.18
0.07
17.44
16
3.16
—
0.023
0.012
0.18
0.07
4.36
17
3.16
0.01
0.023
—
0.18
0.07
4.36


### 2.3. Determination of Microalgae Growth Density

The microalgae growth density was determined by measuring the optical density at 440 nm, the highest absorbance ofN. oculata when scanned the wave-length ranging from 220 nm to 700 nm, using a UV-V is spectrophotometer every 24 hours. All measures were carried out in triplicate.


### 2.4. Preparation of Dry Algal Powder

When theN. oculata cells had been at the stationary phase for three days, the pH of the algal culture was adjusted to 10.5 with NaOH which was used for sedimentation, and then the culture stood for 24 hours. The algal mud was obtained by removing the supernatant and washed with distilled water 3 times to remove the salt in the algal mud and then dried for 24 hours at 70°C to get the dry algae powder, and the dry weight was obtained by weighting the algal biomass after being dried for 24 hours at 70°C.


### 2.5. Lipid Extraction

Lipid was extracted using the modified acid method [10]. Firstly, dry algae powder (1 g) was put in a 50 mL centrifuge tube, and then 4 mL of distilled water and 5 mL of HCl were put into the tube. The mixture was heated for 20 min in the water bath at 70°C. Then, 5 mL of ethanol was added into the mixture, and the mixture was cooled naturally. Secondly, 10 mL of diethyl ether was added into the mixture, and the mixture was shaken for 1 min. Next, the mixture was centrifuged for 2 min at 4000 r/min. The ether layer was taken into the round flask. Thereafter, 5 mL of diethyl ether was added into the residue, the above process was repeated, and the ether layer was combined and taken into the round flask. Finally, the lipid was obtained by evaporating the ether under vacuum in a rotary evaporator.


### 2.6. Esterification of Algal Lipid

TheN. oculata lipid was dissolved with chloroform and transferred into the 1.5 mL glass vial. Then, 1 mL of 1 M sulphuric acid-methanol was added into the lipid. The mixture was kept at 100°C for 1 hour under nitrogen protection and was then cooled naturally. 200 μL of distilled water was added into the mixture. The mixture was well mixed by shaking for 1 min and extracted with n-hexane 3 times. The organic phases were combined, stripped with 200 μL of distilled water 3 times, transferred into the 1.5 mL glass vial, and dried by nitrogen blowing. The obtained methyl ester was weighed.


### 2.7. Analysis of Methyl Ester fromN. oculataLipid

The fatty acid composition ofN. oculata lipid was determined by a gas chromatography-mass (Agilent 7890 N), and the content of fatty acid was qualitatively characterized by a gas chromatograph with FID detector (Agilent 7890) using methyl undecanoate as internal standards.Analysis Condition of GC. DB-WAX (30 m × 0.32 mm × 0.50 μm), the temperature program, comprised three phases: the first phase: initially the temperature was ramped from 50°C to 150°C at a rate of 10°C·min−1 and held for 2 min. The second phase: it was then ramped to 200°C from 150°C at a rate of 10°C·min−1 and held for 6 min. The third phase: it was ramped to 230°C from 200°C at a rate of 10°C·min−1 and held for 5 min. Carrier gas (N2) velocity was 3 mL·min−1. The detector was a hydrogen flame detector; the velocity of H2 was 30 mL·min−1, and the velocity of air was 300 mL·min−1. The detector temperature was 300°C. The injector temperature was 280°C.Analysis condition of GC-Mass. DB-5MS (30 m × 250 um × 0.25 um), the temperature program, comprised two phases: the first phase: initially the temperature was ramped from 60°C to 270°C at a rate of 10°C·min−1 and held for 5 min. The second phase: it was then ramped to 300°C from 270°C at a rate of 8°C·min−1 and held for 8 min. The injector temperature was 270°C. The temperature of the connector between GC and mass was 260°C. Carrier gas (He) velocity was 1.0 mL·min−1. The ionization source of mass was EI, 70 eV, and mass range: m/z was 200~550 amu.


## 2.1. Microalgae

N. oculata was originally obtained from Marine Biological Culture Collection Centre (China) and screened for its potential ability of growth at Zhejiang University of Technology (China). N. oculata cells were maintained in the modified F/2 medium in artificial sea water, which was made by putting 26.726 g NaCl, 2.26 g MgCl2, 3.248 g MgSO4, 1.153 g CaCl2, 0.198 g NaHCO3, 0.721 g KCl, 0.058 g NaBr, 0.058 g H3BO3, 0.0024 g Na2SiO3, 0.0015 g Na2Si4O9, 0.002 g H3PO4, 0.013 g Al2Cl6, 0.002 g NH3, and 0.0013 g LiNO3 in distilled water (per liter), at temperature 20°C, and pH = 8.0 with a light intensity of 3000 μmol·m−2·s−1 in illumination box. The composition of modified F/2 medium (per liter) was shown as follows: 0.225 g NaNO3, 0.045 g NaH2PO4·H2O, 0.02 g Na2SiO3·9H2O, 3.16 mg FeCl3·6H2O, 0.18 mg MnCl2·4H2O, 0.023 mg ZnSO4·7H2O, 4.36 mg Na2 EDTA, 0.01 mg CuSO4·5H2O, 0.012 mg CoCl2·6H2O, 0.07 mg Na2MoO4·2H2O, 1 μg vitamin B1, 1 μg vitamin B12, and 0.5 μg biotin.The mud of algae for inoculation was collected by centrifuging the cells ofN. oculata at the logarithmic phase at 3000 r·min−1 and then washed with distilled water for 3 times to remove the salt in the medium.


## 2.2. Culture System

Growth experiment was done at a different content of trace elements in 1.0 L-Erlenmeyer flasks. The medium and flasks were sterilized at UV irradiation for 30 min in a clean bench in order to prevent any contamination during the early stages of growth.N. oculata cells with the same inoculation density (OD440nm=0.70) grew at room temperature (25±1°C), natural lighting, and pH = 8.0 with bubbling of aseptic air continuously. The concentration of trace elements in different media was listed in Table 1.Table 1
The concentration of trace elements (TEs) in different media.
TE (mg·L−1)
Fe3+
Cu2+
Zn2+
Co2+
Mn2+
Mo6+
EDTA
group number
0
3.16
0.01
0.023
0.012
0.18
0.07
4.36
1
—
0.01
0.023
0.012
0.18
0.07
4.36
2
9.48
0.01
0.023
0.012
0.18
0.07
4.36
3
18.96
0.01
0.023
0.012
0.18
0.07
4.36
4
3.16
0.01
—
0.012
0.18
0.07
4.36
5
3.16
0.01
0.069
0.012
0.18
0.07
4.36
6
3.16
0.01
0.138
0.012
0.18
0.07
4.36
7
3.16
0.01
0.023
0.012
—
0.07
4.36
8
3.16
0.01
0.023
0.012
0.54
0.07
4.36
9
3.16
0.01
0.023
0.012
1.08
0.07
4.36
10
3.16
0.01
0.069
0.012
0.18
—
4.36
11
3.16
0.01
0.138
0.012
0.18
0.21
4.36
12
3.16
0.01
0.023
0.012
0.54
0.42
4.36
13
3.16
0.01
0.023
0.012
1.08
0.07
—
14
3.16
0.01
0.023
0.012
0.18
0.07
8.72
15
3.16
0.01
0.023
0.012
0.18
0.07
17.44
16
3.16
—
0.023
0.012
0.18
0.07
4.36
17
3.16
0.01
0.023
—
0.18
0.07
4.36


## 2.3. Determination of Microalgae Growth Density

The microalgae growth density was determined by measuring the optical density at 440 nm, the highest absorbance ofN. oculata when scanned the wave-length ranging from 220 nm to 700 nm, using a UV-V is spectrophotometer every 24 hours. All measures were carried out in triplicate.


## 2.4. Preparation of Dry Algal Powder

When theN. oculata cells had been at the stationary phase for three days, the pH of the algal culture was adjusted to 10.5 with NaOH which was used for sedimentation, and then the culture stood for 24 hours. The algal mud was obtained by removing the supernatant and washed with distilled water 3 times to remove the salt in the algal mud and then dried for 24 hours at 70°C to get the dry algae powder, and the dry weight was obtained by weighting the algal biomass after being dried for 24 hours at 70°C.


## 2.5. Lipid Extraction

Lipid was extracted using the modified acid method [10]. Firstly, dry algae powder (1 g) was put in a 50 mL centrifuge tube, and then 4 mL of distilled water and 5 mL of HCl were put into the tube. The mixture was heated for 20 min in the water bath at 70°C. Then, 5 mL of ethanol was added into the mixture, and the mixture was cooled naturally. Secondly, 10 mL of diethyl ether was added into the mixture, and the mixture was shaken for 1 min. Next, the mixture was centrifuged for 2 min at 4000 r/min. The ether layer was taken into the round flask. Thereafter, 5 mL of diethyl ether was added into the residue, the above process was repeated, and the ether layer was combined and taken into the round flask. Finally, the lipid was obtained by evaporating the ether under vacuum in a rotary evaporator.


## 2.6. Esterification of Algal Lipid

TheN. oculata lipid was dissolved with chloroform and transferred into the 1.5 mL glass vial. Then, 1 mL of 1 M sulphuric acid-methanol was added into the lipid. The mixture was kept at 100°C for 1 hour under nitrogen protection and was then cooled naturally. 200 μL of distilled water was added into the mixture. The mixture was well mixed by shaking for 1 min and extracted with n-hexane 3 times. The organic phases were combined, stripped with 200 μL of distilled water 3 times, transferred into the 1.5 mL glass vial, and dried by nitrogen blowing. The obtained methyl ester was weighed.


## 2.7. Analysis of Methyl Ester fromN. oculataLipid

The fatty acid composition ofN. oculata lipid was determined by a gas chromatography-mass (Agilent 7890 N), and the content of fatty acid was qualitatively characterized by a gas chromatograph with FID detector (Agilent 7890) using methyl undecanoate as internal standards.Analysis Condition of GC. DB-WAX (30 m × 0.32 mm × 0.50 μm), the temperature program, comprised three phases: the first phase: initially the temperature was ramped from 50°C to 150°C at a rate of 10°C·min−1 and held for 2 min. The second phase: it was then ramped to 200°C from 150°C at a rate of 10°C·min−1 and held for 6 min. The third phase: it was ramped to 230°C from 200°C at a rate of 10°C·min−1 and held for 5 min. Carrier gas (N2) velocity was 3 mL·min−1. The detector was a hydrogen flame detector; the velocity of H2 was 30 mL·min−1, and the velocity of air was 300 mL·min−1. The detector temperature was 300°C. The injector temperature was 280°C.Analysis condition of GC-Mass. DB-5MS (30 m × 250 um × 0.25 um), the temperature program, comprised two phases: the first phase: initially the temperature was ramped from 60°C to 270°C at a rate of 10°C·min−1 and held for 5 min. The second phase: it was then ramped to 300°C from 270°C at a rate of 8°C·min−1 and held for 8 min. The injector temperature was 270°C. The temperature of the connector between GC and mass was 260°C. Carrier gas (He) velocity was 1.0 mL·min−1. The ionization source of mass was EI, 70 eV, and mass range: m/z was 200~550 amu.


## 3. Result and Discussion

### 3.1. The Effects of Trace Elements on the Dry Weight ofN. oculata

The effects of trace elements on the dry weight ofN. oculata were presented in Figure 1. As can be seen, there were certain extent effects of trace elements on the dry weight of N. oculata.Figure 1
The effects of trace elements (TEs) on the dry weight ofN. oculata.It was shown that the dry weight ofN. oculata decreased firstly and then increased gradually with the increase of concentration of Fe3+, Zn2+, and EDTA. The dry weight of N. oculata reached the maximum when the concentrations of the trace elements were 6 times of the control group (modified F/2). It was probably because Fe3+ was the assistant factor of many kinds of enzymes, and it was essential for the formation of cytochrome, ferredoxin, and Mo-Fe protein. The photosynthesis of algae depended on Fe3+, and at the same time Fe3+ was an important part of nitrate and nitrite reductase. It could remarkably increase the reduction and transfer efficiency of Nitrate and Nitrite reductase in algae [8]. The carbon source heavily utilized in the algae cell was CO2, but CO2 mainly existed in the form of HCO3- in the cell [8], CO2 should be transferred from HCO3- by the catalysis of carbonic anhydrase before photosynthesis. Zn2+ was a part of carbonic anhydrase [11]. The increase of the concentration of Zn2+ would increase the efficiency of photosynthesis in algae. EDTA was the organic ligand in the medium of algae and could be used as the buffer of metal ions [12], because it could form the stable complex with most of the kinds of metal ions. In our work, the dry weight of N. oculata increased with the increase of the concentration of EDTA, and this might be because EDTA formed a complex with Fe3+. Yang [13] found that the growth rate of algae and the uptake of Fe3+ were higher in complexed iron than that in colloidal iron at the same concentration of Fe3+. The consistent conclusion was drawn in our work.With the increase of concentration of Mn2+, the dry weight of N. oculata decreased firstly, then increased, and then decreased again. When the concentration of Mn2+ was 3 times of the control group, the dry weight of N. oculata reached the maximum. The possible reason was that Mn2+ was the activator of some enzyme in the reaction of glycolysis and tricarboxylic acid cycle and the activator of nitrate reductase. The deletion of Mn2+ would affect the utilization of nitrate [14]. Qin and Zhou [15] and Guo and Yang [16] found that a certain concentration of Mn2+ in water would promote the growth of algae, and the different optimum concentrations of Mn2+ were obtained for the different algae strains. In this experiment, when the concentration of Mn2+ was 3 times of the control group, the dry weight of N. oculata reached the maximum. The addition of Mo6+ restrained the growth of N. oculata. Compared with the control group, the growth density of N. oculata with the deletion of Mo6+ was higher. The dry weight of N. oculata decreased gradually with the increase of the concentration of Mo2+, but when the concentration of Mo2+ became 3 times of the control group, the increase of the concentration of Mo2+ would increase the dry weight of N. oculata, and the results might be because high concentration of Mo2+ impelled the generation of stress protein in N. oculata [9] which would promote algal photosynthesis and increase the g dry weight.


### 3.2. The Effects of Trace Elements on the Lipid Content ofN. oculata

The effects of trace elements on the lipid content ofN. oculata were presented in Figure 2. As can be seen, trace elements had a significant influence on the lipid content of N. oculata. The deletion of Fe3+, Zn2+, Mn2+, Mo6+, and EDTA would greatly cause a decrease of lipid content of N. oculata. In the range of the experimental concentrations, with the increase of Fe3+, the lipid content increased. But with the increase of the concentration of Zn2+, Mn2+, Mo6+, and EDTA, the lipid content of N. oculata increased firstly and then decreased. The lipid content went to the maximum when the concentrations of Mn2+, Mo6+, and EDTA were 6 times of the control group, and the concentration of Zn2+ was the same as the control group. For Zn2+, the lipid content would increase again when the concentration of Zn2+ exceeded 3 times of the control group. The results showed that Fe3+, Zn2+, Mn2+, Mo6+, and EDTA were indispensable during the lipid formation of microalgae, and that appropriate concentration of the trace elements could promote the lipid formation, but too high concentration of the trace elements would produce poisonous effects on microalgae. Furthermore, ultra high concentration of trace elements would impel the generation of stress protein in N. oculata which would make the metabolic pathway turn from producing starch to producing lipid and thus increases the lipid content.Figure 2
The effects of trace elements (TEs) on the lipid content ofN. oculata.However, Cu2+ and Co2+ had negative effects on the lipid formation. Compared with the control group, the deletion of Cu2+ and Co2+ could increase the lipid content in N. oculata. The deletion of Cu2+ and Co2+ had little effects on the growth density as shown in Figure 2. Thus, the optimum culture should have no Cu2+ or Co2+.


### 3.3. The Effects of Trace Elements on the Fatty Acid Composition ofN. oculata

The effects of trace elements on the fatty acid composition ofN. oculata were presented in Table 2.Table 2
The effects of trace elements on the fatty acid composition ofN. oculata.
Trace
Control
Fe3+ (mg/L)
Zn2+ (mg/L)
Mn2+ (mg/L)
elements
0
9.48
18.96
0
0.069
0.138
0
0.54
1.08
C14:0
5.82
4.85
6.02
6.10
4.90
6.45
6.74
0.00
6.01
6.00
C16:0
32.50
34.43
30.42
33.88
33.99
31.30
28.88
25.12
38.70
37.45
C16:1
21.06
39.46
25.75
24.70
38.93
24.15
23.32
40.37
24.40
23.69
C18:0
2.42
0.75
2.43
2.33
0.85
2.43
2.58
0.00
1.89
2.03
C18:1
8.15
12.53
7.62
8.12
9.69
8.04
6.35
22.60
9.51
9.67
C18:2
0.94
0.92
0.64
0.76
1.46
0.59
0.00
11.90
0.88
0.87
C20:4
6.05
1.15
6.71
5.98
1.78
6.50
7.23
0.00
5.10
5.30
C20:5
19.66
5.92
20.42
18.31
8.40
20.56
24.66
0.00
13.52
14.99
Saturated
40.74
40.03
38.87
42.31
39.74
40.18
38.2
25.12
46.6
45.48
Monounsaturated
29.21
51.99
33.37
32.82
48.62
32.19
29.67
62.97
33.91
33.36
Polyunsaturated
26.65
7.99
27.77
25.05
11.64
27.65
31.89
11.9
19.5
21.16
Trace
Mo6+ (mg/L)
EDTA (mg/L)
Cu2+
Co2+
elements
0
0.21
0.42
0
8.72
17.44
0
0
C14:0
3.66
6.07
6.01
4.93
5.88
6.40
6.25
5.82
C16:0
35.62
36.41
34.34
34.98
33.35
34.43
35.60
32.34
C16:1
43.94
25.14
24.59
39.22
23.66
24.02
22.75
22.89
C18:0
0.00
2.07
2.12
0.72
2.30
2.29
2.26
2.40
C18:1
12.14
8.75
8.55
11.26
8.42
8.39
9.03
7.90
C18:2
0.00
0.78
0.82
1.30
0.92
0.00
0.82
0.92
C20:4
0.00
5.45
5.82
1.45
6.11
5.91
5.98
6.58
C20:5
4.64
15.34
17.76
6.14
19.36
18.57
17.32
21.09
Saturated
39.28
44.55
42.47
40.63
41.53
43.12
44.11
40.56
Monounsaturated
56.08
33.89
33.14
50.48
32.08
32.41
31.78
30.79
Polyunsaturated
4.64
21.57
24.4
8.89
26.39
24.48
24.12
28.59As can be seen, palmitic acid, palmitoleic acid, and eicosapentaenoic acid (EPA) were the major fatty acids ofN. oculata, comprising 65% to 85% of total fatty acids. The fatty acid composition shifted in response to the addition of trace elements. The level of saturated fatty acid shifted slightly after the addition of trace elements. Furthermore, the level of monounsaturated and polyunsaturated fatty acids increased sharply after the addition of Fe3+, Zn2+, Mn2+, Mo6+, and EDTA, but the concentration of the trace elements had little influence on the level of monounsaturated and polyunsaturated fatty acids. The deletion of Cu2+ and Co2+ had little effects on all fatty acids in N. oculata.Recently, the utilization of PUFAS, such as eicosapentaenoic acid (EPA) and docosahexaenoic (DHA), for functional food and pharmacological products has received an increased attention [8]. The price of EPA with a purity of 99% in international market is 200 US·g−1. Therefore, it is a good way to increase the economic benefit of biodiesel production by a combined production of EPA from N. oculata.


### 3.4. The Effects of Trace Elements on the Lipid Productivity ofN. oculata

The effects of trace elements on the lipid productivity ofN. oculata was presented in Table 3. The ANOVA of the effects of trace elements on the yield of lipid of N. oculata were presented in Table 4. As can be seen, the P value of the trace elements on the lipid productivity of N. oculata was less than 0.01 which was to say that the trace elements had a large influence on the lipid productivity of N. oculata.Table 3
The effects of trace elements (TEs) on the lipid productivity (mg·L−1·day−1) of  N. oculata.
Concentration(times of the control group)
Fe3+
Zn2+
Mn2+
Mo6+
EDTA
Cu2+
Co2+
None
51.28
28.15
57.6
40.69
55.25
63.48
70.8
One time
62.41
62.41
62.41
62.41
62.41
62.41
62.41
Three times
66.85
42.28
53.11
36.56
70.07
Six times
76.11
61.46
48.77
59.44
82.69Table 4
The ANOVA of the effects of trace elements on the yield of lipid ofN. oculata.
Source of variance
SS
df
F
P value
The critical value ofF
Fe3+
7604.53
7604.53
134.19
2.49
E
-
05
F
0.05 (1.6) = 5.99
Zn2+
4245.81
4245.81
30.50
0.0015
F
0.05 (1.6) = 5.99
Mn2+
5612.17
5612.17
271.21
3.19
E
-
06
F
0.05 (1.6) = 5.99
Mo2+
4469.85
4469.85
50.49
0.00040
F
0.05 (1.6) = 5.99
EDTA
8477.32
8477.32
117.13
3.68
E
-
05
F
0.05 (1.6) = 5.99The order of factors affecting the lipid productivity was Mn2+, EDTA, Fe3+, Mo6+, and Zn2+ according to the magnitude of P value. Based on these results, the proper control of the concentration of trace elements could increase the lipid productivity of N. oculata. The optimum concentrations of the trace elements in the culture medium are 6 times of Fe3+ and EDTA, the same concentration of Zn2+, Mn2+, and Mo6+ as the control group, but the optimum medium has no Cu2+ or Co2+. But when using the optimum concentration of all the trace elements in one medium, we could get the highest lipid productivity; the lipid productivity was 55.53 mg·L−1·day−1 which was lower than the control group. The reason may be that there existed a restrain function among the different concentration of trace elements which prevented the algae from getting the highest lipid productivity.


## 3.1. The Effects of Trace Elements on the Dry Weight ofN. oculata

The effects of trace elements on the dry weight ofN. oculata were presented in Figure 1. As can be seen, there were certain extent effects of trace elements on the dry weight of N. oculata.Figure 1
The effects of trace elements (TEs) on the dry weight ofN. oculata.It was shown that the dry weight ofN. oculata decreased firstly and then increased gradually with the increase of concentration of Fe3+, Zn2+, and EDTA. The dry weight of N. oculata reached the maximum when the concentrations of the trace elements were 6 times of the control group (modified F/2). It was probably because Fe3+ was the assistant factor of many kinds of enzymes, and it was essential for the formation of cytochrome, ferredoxin, and Mo-Fe protein. The photosynthesis of algae depended on Fe3+, and at the same time Fe3+ was an important part of nitrate and nitrite reductase. It could remarkably increase the reduction and transfer efficiency of Nitrate and Nitrite reductase in algae [8]. The carbon source heavily utilized in the algae cell was CO2, but CO2 mainly existed in the form of HCO3- in the cell [8], CO2 should be transferred from HCO3- by the catalysis of carbonic anhydrase before photosynthesis. Zn2+ was a part of carbonic anhydrase [11]. The increase of the concentration of Zn2+ would increase the efficiency of photosynthesis in algae. EDTA was the organic ligand in the medium of algae and could be used as the buffer of metal ions [12], because it could form the stable complex with most of the kinds of metal ions. In our work, the dry weight of N. oculata increased with the increase of the concentration of EDTA, and this might be because EDTA formed a complex with Fe3+. Yang [13] found that the growth rate of algae and the uptake of Fe3+ were higher in complexed iron than that in colloidal iron at the same concentration of Fe3+. The consistent conclusion was drawn in our work.With the increase of concentration of Mn2+, the dry weight of N. oculata decreased firstly, then increased, and then decreased again. When the concentration of Mn2+ was 3 times of the control group, the dry weight of N. oculata reached the maximum. The possible reason was that Mn2+ was the activator of some enzyme in the reaction of glycolysis and tricarboxylic acid cycle and the activator of nitrate reductase. The deletion of Mn2+ would affect the utilization of nitrate [14]. Qin and Zhou [15] and Guo and Yang [16] found that a certain concentration of Mn2+ in water would promote the growth of algae, and the different optimum concentrations of Mn2+ were obtained for the different algae strains. In this experiment, when the concentration of Mn2+ was 3 times of the control group, the dry weight of N. oculata reached the maximum. The addition of Mo6+ restrained the growth of N. oculata. Compared with the control group, the growth density of N. oculata with the deletion of Mo6+ was higher. The dry weight of N. oculata decreased gradually with the increase of the concentration of Mo2+, but when the concentration of Mo2+ became 3 times of the control group, the increase of the concentration of Mo2+ would increase the dry weight of N. oculata, and the results might be because high concentration of Mo2+ impelled the generation of stress protein in N. oculata [9] which would promote algal photosynthesis and increase the g dry weight.


## 3.2. The Effects of Trace Elements on the Lipid Content ofN. oculata

The effects of trace elements on the lipid content ofN. oculata were presented in Figure 2. As can be seen, trace elements had a significant influence on the lipid content of N. oculata. The deletion of Fe3+, Zn2+, Mn2+, Mo6+, and EDTA would greatly cause a decrease of lipid content of N. oculata. In the range of the experimental concentrations, with the increase of Fe3+, the lipid content increased. But with the increase of the concentration of Zn2+, Mn2+, Mo6+, and EDTA, the lipid content of N. oculata increased firstly and then decreased. The lipid content went to the maximum when the concentrations of Mn2+, Mo6+, and EDTA were 6 times of the control group, and the concentration of Zn2+ was the same as the control group. For Zn2+, the lipid content would increase again when the concentration of Zn2+ exceeded 3 times of the control group. The results showed that Fe3+, Zn2+, Mn2+, Mo6+, and EDTA were indispensable during the lipid formation of microalgae, and that appropriate concentration of the trace elements could promote the lipid formation, but too high concentration of the trace elements would produce poisonous effects on microalgae. Furthermore, ultra high concentration of trace elements would impel the generation of stress protein in N. oculata which would make the metabolic pathway turn from producing starch to producing lipid and thus increases the lipid content.Figure 2
The effects of trace elements (TEs) on the lipid content ofN. oculata.However, Cu2+ and Co2+ had negative effects on the lipid formation. Compared with the control group, the deletion of Cu2+ and Co2+ could increase the lipid content in N. oculata. The deletion of Cu2+ and Co2+ had little effects on the growth density as shown in Figure 2. Thus, the optimum culture should have no Cu2+ or Co2+.


## 3.3. The Effects of Trace Elements on the Fatty Acid Composition ofN. oculata

The effects of trace elements on the fatty acid composition ofN. oculata were presented in Table 2.Table 2
The effects of trace elements on the fatty acid composition ofN. oculata.
Trace
Control
Fe3+ (mg/L)
Zn2+ (mg/L)
Mn2+ (mg/L)
elements
0
9.48
18.96
0
0.069
0.138
0
0.54
1.08
C14:0
5.82
4.85
6.02
6.10
4.90
6.45
6.74
0.00
6.01
6.00
C16:0
32.50
34.43
30.42
33.88
33.99
31.30
28.88
25.12
38.70
37.45
C16:1
21.06
39.46
25.75
24.70
38.93
24.15
23.32
40.37
24.40
23.69
C18:0
2.42
0.75
2.43
2.33
0.85
2.43
2.58
0.00
1.89
2.03
C18:1
8.15
12.53
7.62
8.12
9.69
8.04
6.35
22.60
9.51
9.67
C18:2
0.94
0.92
0.64
0.76
1.46
0.59
0.00
11.90
0.88
0.87
C20:4
6.05
1.15
6.71
5.98
1.78
6.50
7.23
0.00
5.10
5.30
C20:5
19.66
5.92
20.42
18.31
8.40
20.56
24.66
0.00
13.52
14.99
Saturated
40.74
40.03
38.87
42.31
39.74
40.18
38.2
25.12
46.6
45.48
Monounsaturated
29.21
51.99
33.37
32.82
48.62
32.19
29.67
62.97
33.91
33.36
Polyunsaturated
26.65
7.99
27.77
25.05
11.64
27.65
31.89
11.9
19.5
21.16
Trace
Mo6+ (mg/L)
EDTA (mg/L)
Cu2+
Co2+
elements
0
0.21
0.42
0
8.72
17.44
0
0
C14:0
3.66
6.07
6.01
4.93
5.88
6.40
6.25
5.82
C16:0
35.62
36.41
34.34
34.98
33.35
34.43
35.60
32.34
C16:1
43.94
25.14
24.59
39.22
23.66
24.02
22.75
22.89
C18:0
0.00
2.07
2.12
0.72
2.30
2.29
2.26
2.40
C18:1
12.14
8.75
8.55
11.26
8.42
8.39
9.03
7.90
C18:2
0.00
0.78
0.82
1.30
0.92
0.00
0.82
0.92
C20:4
0.00
5.45
5.82
1.45
6.11
5.91
5.98
6.58
C20:5
4.64
15.34
17.76
6.14
19.36
18.57
17.32
21.09
Saturated
39.28
44.55
42.47
40.63
41.53
43.12
44.11
40.56
Monounsaturated
56.08
33.89
33.14
50.48
32.08
32.41
31.78
30.79
Polyunsaturated
4.64
21.57
24.4
8.89
26.39
24.48
24.12
28.59As can be seen, palmitic acid, palmitoleic acid, and eicosapentaenoic acid (EPA) were the major fatty acids ofN. oculata, comprising 65% to 85% of total fatty acids. The fatty acid composition shifted in response to the addition of trace elements. The level of saturated fatty acid shifted slightly after the addition of trace elements. Furthermore, the level of monounsaturated and polyunsaturated fatty acids increased sharply after the addition of Fe3+, Zn2+, Mn2+, Mo6+, and EDTA, but the concentration of the trace elements had little influence on the level of monounsaturated and polyunsaturated fatty acids. The deletion of Cu2+ and Co2+ had little effects on all fatty acids in N. oculata.Recently, the utilization of PUFAS, such as eicosapentaenoic acid (EPA) and docosahexaenoic (DHA), for functional food and pharmacological products has received an increased attention [8]. The price of EPA with a purity of 99% in international market is 200 US·g−1. Therefore, it is a good way to increase the economic benefit of biodiesel production by a combined production of EPA from N. oculata.


## 3.4. The Effects of Trace Elements on the Lipid Productivity ofN. oculata

The effects of trace elements on the lipid productivity ofN. oculata was presented in Table 3. The ANOVA of the effects of trace elements on the yield of lipid of N. oculata were presented in Table 4. As can be seen, the P value of the trace elements on the lipid productivity of N. oculata was less than 0.01 which was to say that the trace elements had a large influence on the lipid productivity of N. oculata.Table 3
The effects of trace elements (TEs) on the lipid productivity (mg·L−1·day−1) of  N. oculata.
Concentration(times of the control group)
Fe3+
Zn2+
Mn2+
Mo6+
EDTA
Cu2+
Co2+
None
51.28
28.15
57.6
40.69
55.25
63.48
70.8
One time
62.41
62.41
62.41
62.41
62.41
62.41
62.41
Three times
66.85
42.28
53.11
36.56
70.07
Six times
76.11
61.46
48.77
59.44
82.69Table 4
The ANOVA of the effects of trace elements on the yield of lipid ofN. oculata.
Source of variance
SS
df
F
P value
The critical value ofF
Fe3+
7604.53
7604.53
134.19
2.49
E
-
05
F
0.05 (1.6) = 5.99
Zn2+
4245.81
4245.81
30.50
0.0015
F
0.05 (1.6) = 5.99
Mn2+
5612.17
5612.17
271.21
3.19
E
-
06
F
0.05 (1.6) = 5.99
Mo2+
4469.85
4469.85
50.49
0.00040
F
0.05 (1.6) = 5.99
EDTA
8477.32
8477.32
117.13
3.68
E
-
05
F
0.05 (1.6) = 5.99The order of factors affecting the lipid productivity was Mn2+, EDTA, Fe3+, Mo6+, and Zn2+ according to the magnitude of P value. Based on these results, the proper control of the concentration of trace elements could increase the lipid productivity of N. oculata. The optimum concentrations of the trace elements in the culture medium are 6 times of Fe3+ and EDTA, the same concentration of Zn2+, Mn2+, and Mo6+ as the control group, but the optimum medium has no Cu2+ or Co2+. But when using the optimum concentration of all the trace elements in one medium, we could get the highest lipid productivity; the lipid productivity was 55.53 mg·L−1·day−1 which was lower than the control group. The reason may be that there existed a restrain function among the different concentration of trace elements which prevented the algae from getting the highest lipid productivity.


## 4. Conclusions

N. oculata is a promising source of biodiesel for its high content of lipid and EPA. The trace elements significantly influence the lipid productivity and the fatty acid composition of N. oculata. The results contribute to improving the lipid and EPA production and providing the experimental basis that N. oculata can be used to produce biodiesel.



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*Source: 671545-2013-04-03.xml*