Enhancing the lipid content of the diatom Nitzschia sp. by 60Co-γ irradiation mutation and high-salinity domestication

Energy 78 (2014) 9e15

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Enhancing the lipid content of the diatom Nitzschia sp. by irradiation mutation and high-salinity domestication

60

Co-g

Jun Cheng*, Jia Feng, Jing Sun, Yun Huang, Junhu Zhou, Kefa Cen State Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou 310027, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 October 2013 Received in revised form 2 June 2014 Accepted 4 June 2014 Available online 25 June 2014

To improve the lipid accumulation of diatom cells, Nitzschia sp. was mutated by 60Co-g-ray irradiation and cultivated with high salinity. The biomass and lipid yields of Nitzschia sp. mutated by 60Co-g-ray irradiation at 900 Gy were increased by 53.8% and 28.1%, respectively. The lipid content of Nitzschia sp. mutant cells cultivated with gradually increased salinity of up to 30‰ was increased from 11.9% to 27.2%. This was because that the increased demand for nitrogen to keep cells healthy growth under high salinity conditions resulted in lipid accumulation, which was beneficial to prevent cell membranes from being destroyed by changing osmotic pressure. When the Nitzschia-ZJU1 strain obtained from Nitzschia sp. by irradiation mutation and salinity domestication was cultured in the growth media with no nitrogen and no silicon, the lipid content of cells was dramatically increased to 51.2%. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Biomass Diatom Irradiation mutation Lipid Salinity domestication

1. Introduction Microalgae are used to absorb solar energy to be used as raw materials to produce biodiesel and other clean renewable fuels. Microalgae plays an important role in solving the crisis of petroleum depletion [1,2]. Microalgae rapidly grows and has strong physiological metabolism and high photosynthetic efficiency. Lipids can be enriched by green and clean biochemical processes in microalgae cells through the use of seawater resources and the absorption of large amount of CO2 [3e5]. The heat value of biodiesel produced by lipids extracted from microalgae cells can reach up to 41 MJ kg
1 [6]. Therefore, microalgae biodiesel can replace fossil diesels for transportation applications and offer the advantage of not releasing SO2 [7]. The annual potential yield of biodiesel derived from microalgae is 58700e136900 L ha
1, which is 130e300 times higher than that from soybean (446 L ha
1) and 10e23 times higher than that from oil palm (5950 L ha
1) [8,9]. Previous studies related to the screening of microalgae for biodiesel production have indicated that the original LCs (lipid contents) and growth rates of diatoms have great advantages [8,10,11]. However, an inverse relationship exists between growth rate and lipid enrichment, which means that most microalgae species able to reach optimized growth rates also exhibit low LCs [8,12].

* Corresponding author. Tel.: þ86 571 87952889; fax: þ86 571 87951616. E-mail address: [email protected] (J. Cheng). http://dx.doi.org/10.1016/j.energy.2014.06.009 0360-5442/© 2014 Elsevier Ltd. All rights reserved.

However, for bio-oil production, microalgae should have both characteristics of rapid growth rate and high LC, and do not compete directly with food crops for land [13,14]. The following four main methods of isolating microalgae with high lipid yields have been reported. (1) Natural strain selection. The United States Department of Energy has confirmed that the LCs of diatom algae are very high [8]. Under natural conditions, the LC of diatom cells can reach up to 10%e30%. (2) Irradiation mutagenesis. Bougaran et al. [15] developed a mutation-selection method aimed at increasing neutral lipid productivity of the microalgae Isochrysis affinis galbana by a two-step method based on UV irradiation. In the process, neutral lipid productivity increased by 80% from 262 ± 21 mg$TFA$(gC)
1 to 409 ± 64 mg$TFA$(gC)
1 after the incorporation of a second mutationeselection step. Using a similar procedure, Alonso et al. [16] successfully increased the Eicosapntemacnioc Acid (EPA) content of the diatom Phaeodactylum tricornutum by 37%. (3) Highesalinity screening: Ben-Amotz et al. [17] confirmed that under high-salinity conditions, some microalgae species contain very high LCs; Cohen et al. [18] also found that under high-salinity conditions, Porphyridium cruentum algae cells contain a higher proportion of linoleic acid (LA) and arachidonic acid (AA), which comprise the main fatty acids of triglycerides, together with palmitic acid. (4) Macroscopic growth factor regulation and control. The U.S. National Renewable Energy Laboratory, University of California, Berkeley and other major research institutes developed various kinds of diatoms and green algae rich in lipids [10,19]. The LC of microalgae cells can reach up to 40%e66%,

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J. Cheng et al. / Energy 78 (2014) 9e15

Nomenclature AA AC AL ACCase DGAT EPA LA LC LCA MB ML OD PA PG TG

arachidonic acid mean glomerular cell area mean glomerular lipid area of the cells acetyl-CoA carboxylase diglyceride acyltransferase Eicosapntemacnioc Acid linoleic acid lipid content lipid content calculated by area biomass dry weight dry weight (g) of lipids extracted from algae cells optical density palmitic acid phosphatidylglycerol triacylglycerol

which is 3e12 times greater than natural yields when algae cells are cultivated under nitrogen- and silicon-deficient conditions. Wu and Hsieh [20] investigated the effects of salinity, nitrogen concentration, and light intensity on lipid productivity. They recorded up to 76% increase in the production of lipids for specific growth conditions compared with more typical growth processes. The natural method of isolating high lipid productivity microalgae strains is preferred, and mutagenesis by nuclear irradiation is seldom studied, especially for Nitzschia sp [21]. Genome sequencing for Nitzschia sp. and other microalgae has not been completed, so microalgae mutants with fast growth rates and high lipid productivities might be obtained by nuclear irradiation of the original strain. In the present study, Nitzschia sp. was mutated by 60 Co-g-ray irradiation and domesticated under high-salinity conditions. The mutant Nitzschia-ZJU1 whose cell LC increased from the original value 11.9%e51.2% was obtained by controlling growth and metabolism factors. 2. Materials and methods 2.1. Microalgae strains and culture media The diatom strains derived from Luojia Mountain of Wuchang were obtained from the Freshwater Algae Culture Collection of Hydrobiology, Chinese Academy of Sciences, China. The diatom strain was identified as Nitzschia sp. according to Phylogenetic tree of 18S rDNA gene sequences which were aligned with Clustal X. D1 solution (containing 0.12 g of NaNO3, 0.07 g of MgSO4$7H2O, 0.02 g of CaCl2$2H2O, 0.04 g of KH2PO4, 0.08 g of K2HPO4, 0.1 g of Na2SiO3, 0.0002 g of MnSO4, 0.005 g of ferric citrate, 20 mL of soil extract, 1 mL of A5 solution and 979 mL of distilled water) was used as cultivation medium. The main components of A5 solution were 100 mL of distilled water with 286 mg of H3BO3, 186 mg of MnC12$4H2O, 22 mg of ZnSO4$4H2O, 39 mg of Na2MoO4$2H2O, 8 mg of CuSO4$5H2O and 5 mg of Co(NO3)2$6H2O. The soil extract was derived from the supernatant of a boiled soil solution. All strains were maintained at 18  C under a 12 h/12 h light/dark cycle illumination of 1000 Lux. 2.2. Mutation of Nitzschia sp. by nuclear irradiation Nitzschia sp. cells in 50 mL aliquots [optical density at 680 nm (OD680) ¼ 0.45] were irradiated with 100, 500, 700, 900, 1000, 1500, 2000 and 4000 Gy of 60Co-g-rays at a dose rate of 15 Gy min
1. The 60 Co-g-rays were generated from 55,000 Ci of g cells at the Institute

of Nuclear-Agricultural Sciences, Zhejiang University, China. The mutants were cultured by the methods of Cheng et al. [22] recovery and pre-cultivation. Cells from 40 mL of pre-cultivated samples were inoculated into D1 medium in a 1 L triangular flask (800 mL of medium) at 25  C with air bubbling under 12 h/12 h light/dark cycle for 15 days. Light absorbance and biomass dry weight were measured to compare microalgae growth after mutagenesis [22]. Chloroform and methanol were used to extract lipids from cells. The LC of microalgae cells were calculated using the following equation [23,24]:

LC ¼

ML  100% MB

(1)

where ML is the dry weight (g) of lipids extracted from algae cells, and MB (g) is the biomass dry weight. 2.3. Domestication of Nitzschia mutants under high-salinity conditions To increase the tolerance of algae cells to high-salinity conditions, the mutants were domesticated by gradually increasing salinity from 0‰ (m/m) to 30‰ (m/m) using a mixture of f medium and artificial seawater. The f medium contained the following components：0.15 g of NaHCO3, 0.02 g of KH2PO4, 0.027 g of VB1, 1.5  10
6 g of VB12, 0.1 g of Na2SiO3$9H2O, 1.0 g of NaNO3, 0.0005 g of biotin, 1 mL of microelement solution, and 980 mL of artificial seawater. The following were the main ingredients of the microelement solution: 1000 mL of distilled water with 4.35 g of Na2EDTA, 7.3 mg of Na2MoO4$2H2O, 12 mg of CoCl2$6H2O, 3.9 g of FeC6H5O7$5H2O, 10 mg of CuSO4$5H2O, 23 mg of ZnSO4, 178 mg of MnCl2$4H2O, and 600 mg of H3BO3. The following was the recipe for preparing the artificial seawater (30‰): 1000 mL of distilled water with 21.2157 g of NaCl, 3.407 g of Na2SO4, 0.3577 g of KCl, 9.3042 g of MgCl2$6H2O, 1.3044 g of CaCl2, 0.0862 g of KBr, 0.2760 g of NaF, and 0.0219 g of SrCl2 $6H2O. First, a 30 mL culture of airgrown mutants cultured with f freshwater medium was inoculated into 270 mL of f medium (salinity 3 ± 1‰ using artificial seawater) bubbled with air at a rate of 80 mL min
1. The initial pH of the culture was adjusted to 8.5. After 10 days, microalgae growth reached a steady state, after which, 30 mL of the culture was collected and inoculated into another 270 mL of f medium with salinity of 6 ± 1‰ in another bioreactor for fresh cultivation under the same conditions. Similarly, after 10 days, 30 mL of this culture was again inoculated to 270 mL f medium with a salinity of 9 ± 1‰ for 10 days. By analogy algae cells were inoculated in the medium with a salinity of 30 ± 1‰. Finally, the cells were recultured with a salinity of 30 ± 1‰ for four generations. Afterwards, a strain was obtained and named Nitzschia-ZJU1. All domestication experiments were performed at 27  C under the same conditions described by Cheng et al. [22]. Each culture was sampled daily, and the microalgae dry weight was measured after drying the microalgae sludge dewatered by centrifugation. LC was measured after harvesting the dry biomass. 2.4. Medium macroscopic condition optimization of nuclear mutant Nitzschia-ZJU1 cells were cultured in a column bioreactor containing 300 mL of f medium (salinity of 30 ± 1‰) with various concentrations of nitrogen and silicon and continuously aerated with air at a rate of 80 ml min
1. The different nitrogen contents used were 0.00, 2.35, 4.70, 7.05, 9.40, and 11.75 mmol/L. The different silicon contents used were 0.00, 0.07, 0.14, 0.21, 0.28 and 0.35 mmol/L. The initial pH was adjusted to 8.5 using 0.1 M HCl and 0.1 M NaOH. 6000 Lux of illumination was supplied onto the

J. Cheng et al. / Energy 78 (2014) 9e15

bioreactor surface under a 12 h/12 h of light/dark cycle and other conditions were the same as above mentioned. 2.5. Comparison of cellular constituents Four different microalgae strains were used, including nonmutagenic Nitzschia sp., Nitzschia sp. mutated with 900 Gy, mutagenic algae Nitzschia-ZJU1, and nitrogen- and silicon-deficient algae Nitzschia-ZJU1. The biochemical compositions of the four strains were determined in terms of total protein, total lipids and carbohydrate content. The lipids were extracted according to methods described in the literature [23]. The proteins were determined by the Kjeldahl technique and carbohydrates were determined according to the Dubois method [25]. The strains were then dyed using Nile Red (9-diethylamino-5H-benzo[a]phenoxazine-5-one; Sigma). The fluorescence of lipid droplets was measured using an inverted microscope (TI-S, Nikon) equipped with a B-2A light filter (EX450-490 nm) and an analysis software (NIS-ELEMENTS D) [26]. All measurements were repeated in triplicate, and the mean values were reported. 3. Results and discussion 3.1. Screening of Nitzschia strains mutated by irradiation

Co-g-ray

60

The moisture content of microalgae medium is very high (99.9%) [22]. The biological effects of g-rays were determined by affecting the atoms and molecules in cells, especially the interaction between water molecules. Compared with the limited penetration effect of UV-B, g-rays can generate free radicals, which change the composition of cells [27]. Thus, 60Co-g-rays were chosen for irradiation because of their strong penetration capability. The growth curve of the surviving Nitzschia strains is shown in Fig. 1. The growth rate of mutants increased with increasing irradiation dose below 1000 Gy. The biomass yield increased from 0.13 g L
1 to 0.20 g L
1 (by 53.8%) at the optimum irradiation dose of 900 Gy. Algae cells irradiated at higher doses (1000 Gy) slowly decayed to death and decomposed after irradiation in 1e4 week. These results showed that lower doses of g-ray irradiation improved microalgae cell growth. However, when the algae cells were irradiated at very high doses, the cells broke down or disintegrated if they failed to recover completely or lose their self-repair ability. Given that different strains have different irradiation sensitivities to nuclear

Biomass dry weight (g/L)

0.1 120

160

200

20

27

18

0.24

24

16

0.21

21

0.18

18

0.15

15

0.12

12

0.09

9

6

0.06

6

4

0.03

3

2

0.00

0

0

240

280

320

360

400

0

14 12 10 8

100 200 300 400 500 600 700 800 900 Irradiation dose (Gy)

Growth time (h) Fig. 1. Growth curves of microalgae irradiated by different doses of

60

Co-g-ray.

Lipid dry weight (mg/L)

0.2

80

30

0.27

0.3

40

22

Lipid dry weight Biomass dry weight Lipid content

0.30

0.4

0

33

0.33

Nitzschia sp. irradiated at 900Gy Nitzschia sp. irradiated at 700Gy Nitzschia sp. irradiated at 500Gy Nitzschia sp. irradiated at 100Gy Nitzschia sp.

0.5

irradiation [27], the microalgae cells were still consistently slightly damaged under low doses of g-ray irradiation, and the cells recovered to normal levels within a short time [28]. Protein synthesis in cells was observed to decrease with increased dose of gray irradiation, which can probably lead to enhanced photosynthetic photoinhibition [29]. To provide a continuous supply of proteins involved in the electron-transfer chain, cell photosynthesis should be significantly enhanced. Thus, the growth of cells should be stimulated. By contrast, higher irradiation doses seriously damage cell metabolism regulation system. Cell growth stops if cells lose their self-repair ability during damage recovery [29]. Fig. 2 shows the biomass dry weights and cell LCs of mutant strains under different irradiation doses and the total lipid dry weights in culture medium. The cell LC was found to decrease with increased irradiation dose. At zero irradiation, the cell LC was 11.9%, which decreased to 9.9% upon irradiation at 900 Gy (16.8% decrease). Compared with the 53.8% increase in growth rate, the decrease in cell LC was very small. The following reasons may explain the above results. First, given the increase in growth rate, the activity of the activating enzyme of the Calvin cycle increased [29]. Consequently, the catalytic carboxylation reaction of 3-phosphoglyceric acid was promoted. Cell LC was decreased because lipid synthesis during cell metabolism recovery was inhibited. Second, higher irradiation doses generated large amounts of free radicals in the cells. Free radicals attacked the polyunsaturated fatty acids of the phosphatide of biological membrane, thereby leading to lipid peroxidation to damage the metabolic processes in cells [22,29,30]. Phosphatide also influenced with the metabolism of DGAT (diglyceride acyltransferase) which is encoded by a gene homologous to phosphatide. DGAT can utilize phosphatide as the acyl donor and diacylglycerol as the acyl acceptor to synthesize TG (triacylglycerol) and lysophosphatide [31]. To repair the damage to cell metabolism caused by phosphatide, which was ultimately caused by irradiation, the cells tended to accelerate phosphatide synthesis to restore the lipids. This phenomenon explained the smaller magnitude of reduced LC of the cells. Methods of screening high-lipid-yield algae mutants should consider both cell growth rate and LC [8]. Figs. 1 and 2 show that the mutant strains produced the highest lipid yields at the irradiation dose of 900 Gy. The lipid dry weight of the mutant was 19.76 mg L
1, which was 28.1% higher than the yield of 15.43 mg L
1 of the original strain. Thus, the Nitzschia mutants irradiated at 900 Gy were chosen as strains for lipid production.

Lipid content (%)

Optical density (absorbance OD680)

0.6

11

Fig. 2. Biomass and lipid yields of microalgae irradiated by different doses of ray.

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Co-g-

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J. Cheng et al. / Energy 78 (2014) 9e15

3.2. Gradient domestication of Nitzschia mutants under highsalinity conditions

54

0.6

36 Biomass dry weight Lipid content

30

0.4

24

0.3

18

0.2

12

0.1

6

Lipid content (%)

Biomass dry weight (g/L)

0.5

0

0.0 0

3

6

9

12

15

18

21

24

27

30

Salinity ( ) Fig. 3. Biomass dry weight and lipid content of microalgae mutant cultured with different salinity.

45

Lipid content (%)

The main factors affecting microalgae growth and chemical composition include light, nutrients, temperature, and pH. However, other factors can also crucially affect some microalgae species, such as salinity. The total lipid yield of the diatom Chaetoceros cf. wighamii cultured without CO2 at 20  C slightly increased with the increasing salinity from 25‰ to 35‰. But the total lipid yield of this diatom specie cultured with CO2 bubbling at higher temperatures (25  C and 30  C) slightly decreased with the increasing salinity [25]. However, different microalgae species have various degrees of tolerance to salinity [32]. To further optimize the LC of the Nitzschia mutants, the growth status and LC as affected by gradient changes in salinity were studied. A comparison of mutant strains cultured under different salinity conditions (as described in Section 2.3) with the original strain revealed that the biomass yield and LC increased with increased salinity (Fig. 3). Results showed that the LC of the mutant strain Nitzschia-ZJU1 was 27.2% for the strain cultivated with 30‰ salinity, which was 1.9 times higher than the value of 9.9% in undomesticated cells cultivated at 0‰ salinity. The biomass dry weight of Nitzschia-ZJU1 increased by 26.8% from 0.41 g L
1 to 0.52 g L
1. The following four reasons may explain the above results: First, when cells cultured from the 0‰ salinity medium were directly inoculated into the 30‰ salinity medium, a substantial difference in cell osmotic pressure occurred, which led to decreased growth rate [25]. The cells were unable to rapidly adjust to the change in osmotic conditions, thereby affecting the nutrient absorption and the metabolism of cells. However, with increased salinity in smaller increments, only small changes in osmotic pressure occurred and increased the probability of cell survival. Under this condition, cells accumulated large amounts of free amino acids, especially proline [33-35]. This phenomenon was due to osmolytes stressed by the increase in salt concentration to relieve the limitation of the change in osmotic pressure, thereby counteracting the decrease in growth. Second, increasing the salinity of the medium may induce the cells to produce more lipids, which counteracted the damaging effects of drastic changes in osmotic pressure to preserve cell membrane integrity [36,37]. A substantial increase in PG (phosphatidylglycerol) was observed in salt-stressed cells, which was actually due to an increase of the LA (linoleic acid) levels [37,38].

36 27 18 9 0 0

0.00 0.07 0.14 0.21 0.28

2.35 4.70 7.05 9.40 11.75

N=0 Si = 0

Si content (mmol/L) N= 11.75 mmol/L

N content (mmol/L) Si = 0.35 mmol/L

Fig. 4. Lipid content of diatom Nitzschia-ZJU1 cultured with different nitrogen and silicon concentrations.

Cohen et al. [18] discovered that salt-stressed microalgae cells produce a higher proportion of LA and AA (arachidonic acid), which are the major fatty acids esterified to storage TG together with PA (palmitic acid). The above mentioned aliphatic acids improved the ability of cells to adjust to changes in osmotic pressure under salt stress and simultaneously increased the LC. Meanwhile, LA and PG are the main components of the photosynthetic membrane, and can restore normal cell photosynthesis. A higher aliphatic acid content may interfere with the activity of solvent molecules around the cell membrane in the culture medium [36]. Polyunsaturated fatty acid may result in a higher fluidity of the cell membrane, thereby preventing water loss from cells. Interactions among the aforementioned factors ensured normal growth of microalgae cells. Third, the photosynthetic efficiency of microalgae cells is affected when placed in a high-salinity medium. Protein synthesis is also affected in some species [39]. To ensure normal cell growth, the expression of nitrate reductase and nitrite reductase should be enhanced by nitrogen metabolism, which means that the demand for nitrogen in cells should be increased [40], as previously demonstrated in Chlamydomonas reinhardtii. The lipid productivity of microalgae is known to increase in nitrogen-deprived cultures. Thus, the LC of Nitzschia mutants increased because of the increased demand for nitrogen in cells and the relatively lower nitrogen concentration of the medium. Fourth, the mutants were not derived from a single cell, which is advantageous for screening cells with high LC resulting from selfadaption to high-salinity conditions. Mutant cells at a competitive disadvantage decrease in number and eventually die out under salt stress. Selection enables mutant cells to adapt to high-salinity conditions and survive, which can explain the observed increase in LCs and growth rates of the Nitzschia sp. in the culture medium from the salinity concentration of 0‰, to 15‰ and to 30‰. In summary, the Nitzschia-ZJU1 strain was isolated by salinity gradient screening, which can be a better choice of LC optimization in macroeconomic control experiments. 3.3. Optimizing the LC of mutant Nitzschia-ZJU1 Fig. 4 shows the LCs of mutant Nitzschia-ZJU1 cells cultured using different kinds of f media. LC was found to monotonously increase with a decrease in silicon content and fluctuate in an increase tendency with a decrease in nitrogen content. LC reached up to 43.1% when the cells were cultured without silicon in the

J. Cheng et al. / Energy 78 (2014) 9e15

3.4. Organic compositions of microalgae mutants domesticated under high-salinity, nitrogen deprivation and silicon deprivation conditions The following results were obtained by comparing the compositions of the original Nitzschia strain (Nitzschia-0Gy), the Nitzschia mutant strain irradiated at 900 Gy (Nitzschia-900Gy), the NitzschiaZJU1 strain, and the Nitzschia-ZJU1 strain (Nitzschia-ZJU1 with no N&Si) cultured under nitrogen and silicon deprivation (Fig. 5): First, protein and LCs in cells decreased upon nuclear irradiation, but carbohydrate content increased. This finding proved that nuclear irradiation damaged protein and lipid in cells because of the outbreak of reactive oxygen [30,43]. To repair the damage, carbohydrate synthesis was enhanced and protein metabolism increased to protect the activity of enzymes and maintain the normal cell growth. Second, high salinity increased the LC of Nitzschia and decreased the protein content, consistent with the results of Ben-Amotz et al. [17] and Fabregas et al. [39]. Salinity had little effect on carbohydrate synthesis, which may be due to the increase in osmotic pressure in the salt-pressed medium, consistent with previous results [25,44]. Third, nitrogen and silicon deprivation increased LC and decreased carbohydrate, and protein contents. The microalgae were unable to completely utilize the mineral when cultured in a 30‰ salinity medium, which impaired cellular metabolism but enhanced lipid synthesis [25]. The four types of Nitzschia strains were diluted to twice the original volume. Afterwards 100 mL of the resulting solution was

70

Carbohydrates Protein Lipid Other constituents

60

Composition content (% DW)

medium (N ¼ 11.75 mmol/L), 40.3% when the cells were cultured in a nitrogen-deficient medium (N ¼ 4.7 mmol/L, Si ¼ 0.35 mmol/L), and 51.2% when the medium did not contain silicon and nitrogen. The increase in LC was 0.9 times higher than that of the original strain (27.2%). The study of Roessler showed that the LC of the diatom Cyclotella cryptic rapidly increased in the medium without silicon and the carbon absorbed to synthesize carbohydrate decreased by 50% [41]. At the same time, non-lipid substances were converted to lipids, which led to increased LC. On the other hand, silicon deficiency increased the LC because the activity of ACCase (acetyl-CoA carboxylase) in cells was enhanced [32]. As in animal cells, lipid synthesis was initiated from the catalytic carboxylation reaction of ACCase, followed by several rounds of chain elongation and desaturation. ACCase activity increased in silicon-deficient medium, which improved lipid synthesis. Most neutral lipids (mainly TG) in cells increased because of the nitrogen deficiency in the medium [20]. By contrast, a few microalgae species such as Dunaliella tertiolecta, Biddulpa aurita and Synedra ulna did not accumulate more lipids under nitrogendeficient conditions, suggesting differences in metabolic processes in various microalgae species [42]. In the present study for high silicon content in the medium, LC fluctuated with decreased nitrogen concentration in the medium, but LC increased with decreased silicon concentration for high nitrogen concentration of 11.75 mmol/L. This phenomenon was due to the absence of nitrogen in the depot lipids and the fact that most membrane lipids can be synthesized under nitrogen-deficient condition. However, other molecules such as protein and nucleic acids cannot be synthesized in large amounts. When protein synthesis sharply decreased, the synthesis of other molecules such as carbohydrates and lipids was relatively enhanced. Lipid synthesis was enhanced by both interactions when Nitzschia-ZJU1 cells were cultured in a medium without silicon and nitrogen. The catalysis of ACCase was enhanced, so the lipid amount was enhanced and cell LC increased to 51.2% with lipid yield of 133.2 mg L
1.

13

50 40 30 20 10 0 Nitzschia-0Gy

Nitzschia-900Gy

Nitzschia-ZJU1

Nitzschia-ZJU1 with no N&Si

Fig. 5. Organic compositions of microalgae mutants domesticated with high salinity and cultured with nitrogen and silicon deprivation.

mixed with Nile Red at a final concentration of 1 mg mL
1. Fluorescence images (400 times) were obtained under a microscope after 6 min (Fig. 6). The morphology of Nitzschia excited by blue light (480 nm) was observed under the fluorescence microscope. When stained with Nile Red, the intracellular lipid droplets showed characteristic yellow fluorescence, whereas other parts of cells appeared red. The following notable differences were observed among the four strains. First, the mean fluorescence of Nitzschia-ZJU1 with no N&Si was higher than those of the other strains, whereas the mean fluorescence of the Nitzschia-0Gy strain was the lowest. The built-in software of the microscope was used to calculate LC using the following equation:

LCA ¼

AL  100% AC

(2)

where AC is the mean glomerular cell area, and AL is the mean glomerular lipid area of the cells. The LCA of Nitzschia-0Gy, Nitzschia-900Gy, Nitzschia-ZJU1 and Nitzschia-ZJU1 with no N&Si were 10.6%, 9.6%, 28.5%, and 50.4%, respectively, which were pretty much similar to with the actual LC values. Second, the cell size was changed by nuclear irradiation. The lengths of Nitzschia-0Gy cells ranged from 11 mm to 13 mm, but the lengths of Nitzschia-900Gy cells were smaller and ranged from 7 mm to 10 mm. The observed differences in morphology were similar to those found by Wang and Zhao [45]. Moreover, the length of cells decreased, the width increased, and LC of cells increased. The lengths and widths of Nitzschia-ZJU1 with no N&Si cells ranged from 7 mm to 9 mm and 4 mm to 6 mm, respectively, whereas those of Nitzschia-ZJU1 cells were 8 mme11 mm and 3 mme5 mm, respectively. Third, the different strains showed variations in biomass yield. The amount of Nitzschia-0Gy cells was 0.5  106 cells mL
1, and the amount of Nitzschia-ZJU1 cells was 2.0  106 cells mL
1, which were the same as the biomass dry weight of the strains. 4. Conclusions It was effective to enhance biomass yield and lipid content of diatom Nitzschia sp. by nuclear irradiation mutation and high salinity domestication. The lipid content of Nitzschia sp. mutated by 60 Co-g-ray irradiation at 900 Gy and domesticated with high salinity of 30‰ was increased to 27.2% with biomass productivity of 0.52 g L
1. The lipid content in cells was further increased to 51.2%

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Fig. 6. Fluorescence microscope images of microalgae mutants dyed with Nile Red.

with lipid yield of 133.2 mg L
1 when Nitzschia-ZJU1 strain was cultured with nitrogen and silicon deprivation. It is necessary to further explore the transformed functional genes and regulated metabolic network, which are related to lipid accumulation in Nitzschia sp. cells mutated by nuclear irradiation and domesticated with high salinity. Acknowledgement This work was supported by the National Natural Science Foundation of China (51176163), National High Technology R&D Program of China (2012AA050101), International Sci. & Tech. Cooperation Program of China (2012DFG61770), Zhejiang Provincial Natural Science Foundation of China (LR14E060002), Program for New Century Excellent Talents in University (NCET-11-0446), Specialized Research Fund for the Doctoral Program of Higher Education (20110101110021). References ^ Malcata FX. Microalgae: an alternative as sustainable [1] Amaro HM, Macedo AC, source of biofuels? Energy 2012;44(1):158e66. [2] Maity JP, Bundschuh J, Chen C-Y, Bhattacharya P. Microalgae for third generation biofuel production, mitigation of greenhouse gas emissions and wastewater treatment: present and future perspectives e a mini review. Energy 2014;78:104e13. [3] Dismukes GC, Carrieri D, Bennette N, Ananyev GM, Posewitz MC. Aquatic phototrophs: efficient alternatives to land-based crops for biofuels. Curr Opin Biotechnol 2008;19(3):235e40. [4] Schenk PM, Thomas-Hall SR, Stephens E, Marx UC, Mussgnug JH, Posten C, et al. Second generation biofuels: high-efficiency microalgae for biodiesel production. Bioenergy Res 2008;1(1):20e43. [5] Scott SA, Davey MP, Dennis JS, Horst I, Howe CJ, Lea-Smith DJ, et al. Biodiesel from algae: challenges and prospects. Curr Opin Biotechnol 2010;21(3): 277e86. [6] Vijayaraghavan K, Hemanathan K. Biodiesel production from freshwater algae. Energy Fuel 2009;23(11):5448e53. [7] Sun A, Davis R, Starbuck M, Ben-Amotz A, Pate R, Pienkos PT. Comparative cost analysis of algal oil production for biofuels. Energy 2011;36(8):5169e79.

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