生 物 质 化 学 工 程
Biomass Chemical Engineering
第 45 卷第 5 期
2011 年 9 月
Vol． 45 No． 5
Sep． 2011
·研究报告———生物质能源·
Effect of Nutrient Elements on Growth and Lipid
Accumulation of Phaeodactylum tricornutum
*
CHEN Yu1 ，QIU Yu-jing2 ，ZHANG Wei1 ，L Su-juan2 ，LIU Tian-zhong1
（ 1． Key laboratory of Biofuel，Qingdao Institute of Bioenergy and Bioprocess Technology，Chinese Academy of Sciences，
Qingdao 266101，China； 2． School of Food Science and Engineering，
China Ocean University，Qingdao 266003，China）
Abstract： Effects of nutrient elements，such as nitnogen，phosphor，silicon and iron，on growth and lipid accumulation of
Phaeodactylum tricornutum were investigated by Nile-red fluorescence method． The results showed that higher nitrogen content
would enhance the growth of algal cell and the optimal concentration is 1． 76 mmol / L． Nitrogen deficiencies （ 0． 22－0． 44 mmol / L）
induced the lipid synthesis remarkably． Phosphor deficiencies inhibited the growth of cells，but greatly induced the lipid synthesis．
Silicon deficiencies caused a slow down on cell growth，but little effect on lipid accumulation． Either iron deficiency or iron
excess （ 0． 048 mmol / L） would enhance the lipid accumulation in P． tricornutum cells． In two-stage culture method，the first
step is to grow cell in the nutrients-abundant medium and then transfer it to nitrogen / phosphor-deficienct medium for lipid
induction in the second stage． The fat content of algal cell can be improved significantly by two-step culture method． Seawater is
feasible to be used as a good lipid induction medium for P． tricornutum．
Key words： Phaeodactylum tricornutum； nutrient of culture medium； lipid accumulation； Nile-red fluorescent method
CLC number： TQ517； TQ91
Document code： A
Article ID： 1673 － 5854（ 2011） 05 － 0001 － 06
营养元素对三角褐指藻生长和脂类积累的影响
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陈 昱 ，邱昱晶 ，张 维 ，吕素娟 ，刘天中
（ 1． 中国科学院 青岛生物能源与过程研究所生物燃料重点实验室，山东 青岛 266101；
2． 中国海洋大学 食品科学与工程学院，山东 青岛 266003）
摘
要：研究了不同营养元素（ 氮、磷、硅、铁） 在不同培养浓度条件下，对三角褐指藻生长以及细胞脂类积累的影响作
用。结果显示： 藻细胞生长随氮含量的增加而增加，但超过一定浓度后作用不明显，最适合三角褐指藻生长的氮含量为
1． 76 mmol / L。缺氮和低氮（ 0． 22～0． 44 mmol / L） 培养能够明显提高藻细胞脂类含量。缺磷培养能有效促进藻株脂类积
累，但是藻细胞生长受到明显抑制。缺硅导致藻细胞的生长减缓，但是对藻细胞的油脂积累影响不显著。在缺铁和高铁
（ 0． 048 mmol / L） 条件下，藻的总脂含量均高于正常的培养条件。实验证明通过两步培养的方法，先在营养充足的培养基
中进行藻细胞生长，然后将藻细胞转移到海水中培养，可以明显提高藻细胞的油脂含量。最终本实验确定海水为诱导三
角褐指藻脂类积累的最佳诱导培养基。
关键词：三角褐指藻； 营养元素； 脂类积累； 荧光光谱测定法
Microalgae can be used to produce numerous high-value bio-actives like carbohydrates，proteins，lipids
（ unsaturated fatty acids） ，and pigments （ chlorophylls and carotenoids） ［1－2］． In recent decades，microalgae
are widely recognized as one of the most promising feedstock for biofuels［3－5］． Phaeodactylum tricornutum is
收稿日期：2011 － 03 － 14
基金项目：山东省科技攻关项目资助（ 2008GG20007002） ； 中国科学院太阳能计划资助（ KGCX2 － YW － 374 － 4）
作者简介：陈 昱（ 1983 － ） ，男，四川崇州人，硕士，助理研究员，主要从事微藻生物能源的研究
* 通讯作者：刘天中，研究员，博士生导师，从 事 能 源 微 藻培养与 加 工 利 用、生 物 反 应 器 及 其 CFD 模 拟 等 研 究； 联 系 电 话：0532 －
80662735； E-mail： Liutz@ qibebt． ac． cn。
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one of the most oleaginous algal species，which is recognized as an important potential source of both neutral
lipid and eicosapentaenoic acid［6－8］． But the employment of this alga in aquaculture is very controversial
because the culture conditions can cause very important variations in the biochemical composition of the cells，
and especially the nutrients composition in culture medium is thought to be important factors for cell growth
and lipid content［9－11］．
The object of this work is to detect the relationship among the composition of culture medium，growth
rate，and lipid induction behavior during P． tricornutum culturing． These factors included contents of
nitrogen，phosphor，silicon and iron in culture medium． In order to monitor the lipid synthesis process，
modified Nile-red fluorescent method was introduced in this work． On the other hand，the cultivation of
P． tricornutum in sterilized seawater was also carried out to investigate the lipid induction effect of seawater on
P． tricornutum．
1
Materials and Method
1． 1
Algal strains and preparation of microalgal inoculum
P． tricornutum was kindly gifted by Dr． Zhu B． Ocean University of China． Its basic culture medium is
f /2［12］． Seawater was taken from Huiquan Bay of Qingdao，China．
A stock culture of P． tricornutum was cultured in an Erlenmeyer flask with 500 mL working volume of
modified f /2 medium under （ 25 ± 1 ） ℃ and 150 μmol / （ m2·s） ． After 6 days，the microalgal cells were
obtained by centrifugation at 4 000 g for 5 min，washed and centrifuged twice with sterilized sea water and
then re-suspended in 100 mL fresh medium with different nutrients for further culturing．
1． 2
Microalgal cultures，medium and chemicals
To investigate the effect of different factors （ nitrogen，phosphor，silicon and iron） ，P． tricornutum cells
were grown in different f /2 medium as follows： 1） using NaNO3 as nitrogen resource to prepare six-gradient
nitrogen，i． e． ，0，0． 22，0． 44，0． 88，1． 76 and 3． 52 mmol / L； 2 ） using NaH2 PO4·2H2 O as phosphor
resource to prepare gradient phosphor，i． e． ，normal phosphor concentration of 0． 032 mmol / L and nonphosphor media； 3） using Na2 SiO3·9H2 O as silicon resource to prepare gradient silicon，i． e． ，normal silicon
concentration of 0． 25 mmol / L and non-silicon media； 4） using FeCl3·6H2 O as iron resource to prepare fourgradient iron，i． e． ，0，0． 012，0． 024 and 0． 048 mmol / L． All the chemicals are from Sigma-aldarich．
1． 3
Analysis methods
1． 3． 1
Biomass
OD values at 540 nm［10］ （ WFJ 7200） was meas-
ured to represent the growth of algal cells． The relationship between
OD540 and cell number per millilitre by hemocytometer was plotted in
Fig． 1． The fitted formula is：
N = 1． 05 × 10 6 × OD540 ，R2 = 0． 95
1． 3． 2
Lipid monitoring
Modified Nile-red fluorescent method，
［13］
originally introduced by Lee
，Elsey［14］ and Huang［15］，was used to
monitor the lipid accumulation during culture process of P． tricornutum． The protocol is as follows： 100 μL culture media with OD540 （ A）
was sampled in 1 mL centrifuge tube，10 μL Nile-red acetone solution
Fig． 1
The relationship between cell
density and OD540
（ 0． 1 g / L） and 890 μL distilled water were added． After ice-bathing for 90 s，the fluorescence intensity （ F）
was measured using Hitachi F-2500 fluorescence spectrophotometer with an excitation wavelength of 530 nm
and emission wavelength of 575 nm． The relative fluorescence intensity （ F / A） which is defined as fluorescence
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intensity （ F） divided by optical density OD540 （ A） was used to present the lipid content in microalgal cells．
2
2． 1
Results and Disscussions
Effects of nitrogen in culture media on growth and lipid accumulation
Fig． 2 shows the changes of cell density under five nitrogen gradients in media during culturing．
Obviously，both the growth rate and final cell density of P． tricornutum increased with the increase of nitrogen
concentration in media． The cell would not proliferate anymore without nitrogen in media． However，as
nitrogen increased to about 1． 76 mmol / L，higher nitrogen would lead to higher cell density，which could
reach to 11． 06 × 10 6 / mL． It indicates that under this condition of static culture in flask without aeration，
nitrogen is not the limit factor．
Conversely，low nitrogen concentration is helpful to the lipid induction of P． tricornutum （ Fig． 2） ． After
two days，the relative fluorescence intensity （ F / A） increased quickly in 0． 22 and 0． 44 mmol / L nitrogen
media． It indicates fast lipid accumulation． The relative fluorescence intensity （ F / A） has little increase in the
whole culture process while under higher initial nitrogen concentration． This indicates that no lipid could be
induced．
The effect of nitrogen depletion deletion on cell growth and lipid induction can be further validated by
other experiments shown in Fig． 3． Cultures with a little higher inoculums in normal f /2 medium （ a） and f /2
medium without nitrogen （ b and c） were carried out． It could be seen that，in the first six days，f /2 medium
（ a） resulted in higher cell growth rate but no lipid accumulation （ F / A） ，and f /2 medium without nitrogen （ b
and c） resulted in lower cell growth rates but obvious lipid increase （ F / A） ． However at the sixth day，when
additional nitrogen was added in one of f /2 medium without nitrogen medium （ c） ，cell growth was obviously
but its lipid （ F / A） dropped dramatically to the lipid level as that of in f /2 medium （ a） ．
speeded up，
Fig． 2
2． 2
Effects of initial nitrate concentrations on cell
Fig． 3
Effect of nitrogen depletion on cell
growth and relative fluorescence intensity of
growth and relative fluorescence
P． tricornutum
intensity of P． tricornutum
Effects of phosphor in culture media on growth and lipid accumulation
Apart from nitrogen，phosphor in culture media was also taken as a stress factor on microalgae cell growth
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and lipid induction［16］． As shown in Fig． 4，f /2 medium without phosphor almost inhibited the growth of
P． tricornutum，but greatly induced the lipid synthesis．
2． 3
Effects of silicon in culture media on growth and lipid accumulation
Silicon is thought as the important cell wall component of P． tricornutum． Fig． 5 gave the results of cell
density and relative fluorescence intensity （ F / A） （ lipid） in normal f /2 and silicon-depletion media during
culture． Silicon-depletion caused a slow down on cell growth，but little difference on lipid accumulation，
which does not conform to reference results． Paulanne chelf confirmed that silicon depletion can result in
increased lipid accumulation in two diatom species［17］． One possible reason is that，in the experiments，both
normal and silicon-depletion culture media were prepared with seawater，the silicon content in seawater is
higher than the stress-point of silicon．
Fig． 4
2． 4
Effect of phosphor depletion on cell
Fig． 5
Effect of silicon depletion on cell
growth and relative fluorescence
growth and relative fluorescence
intensity of P． tricornutum
intensity of P． tricornutum
Effects of ferrous in culture media on growth and lipid accumulation
Fig． 6 gave the changes of cell growth and relative fluorescence intensity （ F / A） under different iron
concentration in culture media． It can be found that both excess and depletion of iron would inhibit the growth
of P． tricornutum cells，but had little effect on the relative fluorescence intensity （ F / A） ．
2． 5
Seawater induction effects on P． tricornutum
Lipid accumulation in P． tricornutum is the results of nutrients stress such as nitrogen，phosphor etc．
Thus from the view of lipid induction，transfer of microalgae cells harvested from nutrient-rich medium to
nutrient-depletion medium would promote the accumulation of lipid in cells，which was conventionally taken as
two-stage method． As seawater is an abundant nutrient-depletion medium，investigating the induction of
seawater for lipid accumulation of P． tricornutum in two-stage method is interesting． Fig． 7 show the changes of
cell density and relative fluorescence intensity （ F / A） when microalgae cells were transferred in normal f /2
medium and seawater． In normal f /2 medium，the final cell density could reach 16． 2 × 10 6 / mL，twice as
much as that in seawater． The final relative fluorescence intensity （ F / A） in seawater is almost twice as much
as that in normal f /2 medium，which indicated both have almost the same lipid productivity． As seen in Fig．
8，through the induction in seawater，the drop of lipid oil by Nile-red fluorescent staining （ Fig． 8，c，d） is
more concentrated and bigger than that in normal f /2 medium （ Fig． 8，a，b） ． This indicated that cheap
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seawater would be a good lipid induction medium for P． tricornutum when two-stage process is used to produce
microalgae lipid．
3
3． 1
Conclusions
P． tricornutum is one of the most oleaginous algal species． The composition of culture is demonstrated to
be the important factor on cell growth and lipid accumulation． It should be pointed out that the nutrientabundant mediums would increase the cell density and growth rate，but decrease the lipid content． On the
other hand，nutrients limitation such as nitrogen and phosphor deficiency results in a significant increase in the
lipid content． Furthermore，it's observed that silicon deficiency has caused a slow-down on cell growth and no
significant change on lipid accumulation．
3． 2
Either iron deficiency or iron excess would enhance the lipid accumulation，but reduce the growth rate．
Seawater could be used as the induction medium for lipid accumulation of P． tricornutum． Co-effect investigation of these nutrient factors on the lipid accumulation is the aim of our subsequent work．
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