全 文 ：现代食品科技 Modern Food Science and Technology 2009, Vol.25, No.5
474

Effect of Metal Ions on Biomass and Total Fatty Acids
Content of Chlorella vulgaris

HUANG Guan-hua, CHEN Feng
（College of Light Industry and Food sciences, South China University of Technology, Guangzhou 510640, China）
Abstract: Eight metal ions were studied for their contributions on biomass and the total fatty acid content (TFAC) of C. vulgaris
cultivated heterotrophically in the dark. Cu2+, Zn2+ were limited nutritional factors for the alga growth and the biomass can be improved from
1.24 g/l to 3.60 g/l by adding 0.08 mg/l CuSO4·5H2O. Similarly, the biomass can be improved from 1.71 to 3.96 g/l by adding 0.2 mg/l
ZnSO4·7H2O. Under the conditions without adding Mg2+, the highest TFAC can be obtained, but the biomass was decreased. Mo6+, Mn2+ and
Fe3+ showed little effect on biomass and the TFAC, indicating that they had no special contribution to the growth of C. vulgaris. Increasing Fe2+
concentration enhanced the TFAC of C. vulgaris. As for Ca2+, the maximal biomass and TFAC can be obtained in the medium containing 50
mg/l and 100 mg/l CaCl2·2H2O respectively.
Key words: Chlorella vulgaris , Metal ion, Biomass, Total Fatty Acid Content (TFAC)
CLC number: Q949; Document code: 1673-9078(2009)05-0474-05
金属离子对小球藻 Chlorella vulgaris 生物量和总
脂肪酸含量的影响的研究
黄冠华，陈峰
（华南理工大学轻工与食品学院，广东 广州 510640）
摘要：本文研究了无光照异养条件下八种金属离子对小球藻 Chlorella vulgaris 的生物量和总脂肪酸含量（TFAC）的影响。Cu2+，
Zn2+是小球藻生长的限制性营养因子，通过添加 0.08 mg/L CuSO4·5H2O 可使小球藻生物量从 1.24 g/L 提高到 3.60 g/L；添加 0.2 mg/L
ZnSO4·7H2O，可使小球藻生物量从 1.71 g/L 提高到 3.96 g/L。Mg2+缺失条件可得到最高总脂肪酸含量，但是生物量有所减少。Mo6+, Mn2+
和 Fe3+对小球藻的生物量和油脂含量无明显的影响，说明这些金属离子对小球藻的生长没有特殊的贡献。提高 Fe2+的浓度有利于总脂
肪酸的提高。添加 50 mg/L 和 100 mg/L 的 CaCl2·2H2O 可分别得到最大生物量和最高总脂肪酸含量。
关键词：小球藻；金属离子；生物量；总脂肪酸含量（TFAC）

A small quantity of metal ions is required for
microalgae growth, which can greatly affect the
metabolic growth of microalgae. Metal ions play different
physiological functions on metabolic growth process of
microalgae, so it is necessary to add various metal ions in
cultures to maintain the normal growth of microalgae. At
present, only a few research papers were available
( Knauss and Porter, 1953; Nielsen et al., 1969; Al-Qun-
aibit et al., 2005; Ting et al., 1989) to discuss the effect of
metal ions on biomass and intracellular fatty acid of
收稿日期：2008-11-24，改回日期:2008-12-20
基金项目：国家自然科学基金-广东省自然科学基金联合基金重点项目“微
藻异养转化积累油脂与生物柴油制备”（项目编号U0633009）
作者简介：黄冠华（1981-），女，博士生，主要从事微藻生理和应用的研究
microalgae. Jiang et al. (2005) studied that the alga
growth and the fatty acid composition changed with the
concentration of Fe3+. Liu et al. (2007) also had done
some research about the effect of Fe3+ on cellular lipid
accumulation of Chlorella vulgaris and found that
cellular density could be enhanced when chelated Fe3+
was applied in the late logarithmic growth phase, but
intracellular lipid accumulation can not be induced
markedly. However, the lipid content can be improved to
56.6 % of dry weight when Fe3+ concentration was
improved to 1.2×10-5 mol/l. Lam et al. (1999)
investigated the Ca2+ and Cu2+ for their individual and
associated effect on alga growth and the results showed
that both of the metal ions can restrain the alga growth. In
DOI:10.13982/j.mfst.1673-9078.2009.05.001
现代食品科技 Modern Food Science and Technology 2009, Vol.25, No.5
475
this research, we investigated the effect of eight metal
ions on biomass and the total fatty acid content (TFAC)
of C. vulgaris to offer some useful information to
improve the fatty acids yield (TFAY) from microalgae,
which may help us to know more information about the
physiological characteristics of lipid accumulation in
Chlorella affected by mental ions and further explore
microalgae resources for biodiesel production.
Material and Methods
Microalgae cultures
Alga seed solution preparation
The freshwater microalga Chlorella vulgaris
obtained from CSIRO Marine laboratory (Hobart,
Australia) were grown and maintained on sterilized
modified watanabe medium without adding glucose. The
experimental process were monitored at 20±1 ℃ with the
light intensities at 4000 Lx and shaken at 150 r/min on an
orbital for one week.
Heterotrophic growth in watanabe medium
1 ml fresh alga seed solution with about 106/ml cell
concentration was transferred to a 250 ml flask with
150ml sterilized watanabe medium (Oh-hamat and
Miyachis, 1988) which contained 10 g/l glucose, 2.0 g/l
potassium nitrate, 0.01 mol/l phosphate buffer solution
(The molar ratio of KH2PO4 to K2HPO4 in the phosphate
buffer solution is 5.12), 1.25 g/l MgSO4·7H2O, 20 mg/l
FeSO4·7H2O and 1ml A5 solution (H2BO3: 2.86 g/l,
Na2MoO4·2H2O: 0.039 g/l, ZnSO4·7H2O: 0.222 g/l,
MnCl2·4H2O: 1.81 g/l, CuSO4·5H2O: 0.074 g/l). The pH
value of the medium was adjusted to 6.0 before
sterilization (sterilization Temperature: 116 ℃; Time: 20
min). Alga cells were grown at 23±1 ℃ in the dark and
shaken at 150 r/min on an orbital shaker.
Heterotrophic growth in mediums different concen-
tration of metal ions
Basic nutrition components in these culture
conditions were the same as watanabe medium, but the
concentration of different metal ions were changed
according to the experiment design (see Table 1). It is
supposed that the concentration of metal ion components
were the same as that in watanabe medium under
common condition if no special explanation can be given
in this experiment. The concentration of glucose and
potassium nitrate and other cultivation conditions were
unchanged in the whole cultivation process.
Analytical methods
Growth analysis
Cell numbers were counted under a microscope
(OPTIKA, Italy) with a haemacytometer.
Glucose and nitrate analysis
Residual glucose in the medium was assayed with
the method of 3,5-disalicylic acid (Miller, 1959).
Different concentrations of glucose standard solutions x
are linearly correlated with the absorption values y which
can be expressed as follows: y = 2.5416 x, R2 = 0.9973.
The residual nitrate in the medium was assayed according
to Catalado et al. (Catalado et al., 1975) The residual
concentration of nitrate (g/l) x in the medium can be
calculated according to the absorption values y, and
expressed as follows: y = 0.9878 x, R2 = 0.9976. Glucose
and nitrate concentrations in the cultivation system can be
quantified by using spectrophotometer (UV 2300).
Samples analysis: 1 ml culture solution was
centrifuged at 13000 rpm for 3 min, and the supernatant
was removed to test tubes for the assay of the content of
residual glucose and residual nitrate. The whole operation
process was performed according to above assay methods.
The assay values were calculated by above calibration
equations.
Dry weight analysis
At the late logarithmic phase of alga cultivation, 8
ml alga solution was removed into 10 ml weighted empty
test tube and centrifuged for 5 min (Centrifugation
parameter: speed: 8000 rpm, temperature: 10 ). Later, ℃
the algae cells were washed by distilled water for three
times. The cells were harvested and dried in freeze dryer
(Modul YOD-230) for later biomass calculation and lipid
analysis.
Analysis of fatty acid
About 20 mg dry alga powder and 2 ml
CH3OH-KOH (0.5 mol/l) were added to a test tube which
was put in a water bath at 75 ℃ for 15 min and later
trans-methylation with 2 ml BF3-methanol (1:2, v/v)
regents in a water bath at 75 ℃ immediately after
homogenization for 15 min. The internal standard C19:0
fatty acid (Nonadecanoic acid) was added before
tran-methylation. 1ml saturated salt solution and 2 ml
现代食品科技 Modern Food Science and Technology 2009, Vol.25, No.5
476
hexane were added and mixed equably until the system
was cooled down at room temperature. Hexane layer was
extracted when the reaction system was rested on for 1
min. Hexane layer was analyzed with a FPD Plus gas
chromatograph-mass spectrometer (Agilent Technologies
6890N/5975I) on a (30 m × 0.25 mm ID) DB-23 column.
Helium was used as carrier gas. Initial column
temperature was set at 130 ℃ which was later raised to
200 ℃ at 5 ℃ min-1 and once again raised to 230 ℃ at 40
℃ min-1 for 3 min. The injector and detector were kept at
230 ℃ with an injection volume of 3 μL. Fatty acid
methyl esters were identified and quantitatively analyzed
by GC-MS using C19:0 fatty acid as the internal standard.
Results

Fig.1 Growth curve of C. vulgaris by heterotrophic cultivation
in the darkness

Fig.2 Changes of glucose and potassium nitrate in the medium
by heterotrophic cultivation in the darkness
We can see from Fig.1 that the cell numbers per
milliliter reached the maximum for 11 days. Further
prolonging the cultivation time for 1day decreased the
cell numbers per millilitre which, however, maintained in
the following days of 13-15. The alga cells were
harvested in these days. Fig.2 showed that the
concentration of glucose and potassium nitrate decreased
Table 1 The effects of eight metal ions on Biomass, TFAC and
Total FA Yield of C. vulgaris
Metal Ion
Ion
Concentration
(mg/L)
Biomass
(g/l)
Total FA
Content
(%)
Total FA
Yield
(g/l)
0 2.40±0.21 46.9±0.03 1.14±0.13
500 3.70±0.31 25.2±0.02 0.93±0.08
1000 3.92±0.50 19.8±0.02 0.77±0.10
1500 3.82±0.34 18.6±0.01 0.71±0.06
Mg2+ (MgSO4
7H2O)
2000 4.06±0.24 18.9±0.02 0.76±0.05
0 3.85±0.23 19.4±0.02 0.75±0.05
20 4.43±0.25 19.4±0.01 0.86±0.05
40 3.90±0.15 19.6±0.04 0.76±0.04
60 3.84±0.33 23.7±0.03 0.91±0.09
Fe2+ (FeSO4
⋅7H2O)
80 3.92±0.25 23.6±0.02 0.92±0.06
0 1.71±0.13 25.9±0.02 0.44±0.03
0.2 3.96±0.12 19.1±0.01 0.75±0.02
0.4 4.03±0.20 20.4±0.02 0.82±0.04
0.6 4.06±0.23 21.5±0.04 0.87±0.03
Zn2+
(ZnSO4⋅7H2O)
1.0 4.01±0.25 22.3±0.03 0.89±0.06
0 4.12±0.24 22.3±0.02 0.91±0.06
0.5 4.38±0.20 22.4±0.04 0.98±0.02
1.0 4.07±0.14 22.5±0.05 0.91±0.04
2.0 4.00±0.21 22.1±0.02 0.88±0.04
Mn2+ (MnCl2
⋅2H2O)
3.0 4.14±0.15 20.8±0.02 0.86±0.04
0 1.24±0.05 22.1±0.96 0.27±0.02
0.08 3.60±0.13 20.0±0.17 0.72±0.03
0.12 3.45±0.12 21.3±0.06 0.74±0.04
0.2 3.51±0.21 21.6±0.60 0.83±0.18
Cu2+ (CuSO4
⋅5H2O)
0.3 3.54±0.23 24.2±0.98 0.86±0.09
0 4.03±0.23 21.1±0.30 0.85±0.06
0.02 4.18±0.32 21.5±0.20 0.90±0.08
0.04 4.24±0.30 19.6±0.40 0.83±0.07
0.08 4.32±0.25 21.0±0.87 0.91±0.09
MoO4-2
(Na2MoO4⋅2H2
O)
0.1 4.52±0.22 19.5±0.66 0.88±0.07
0 3.81±0.10 20.3±0.70 0.85±0.18
50 4.27±0.07 20.4±0.50 0.87±0.04
100 4.12±0.01 25.6±0.26 1.05±0.01
150 4.11±0.02 20.8±0.10 0.85±0.01
Ca2+
(CaCl2⋅2H2O)
200 4.09±0.05 19.5±0.15 0.80±0.01
0 3.75±0.02 21.8±0.96 0.81±0.04
3.0 3.87±0.06 21.3±0.65 0.82±0.04
5.0 3.74±0.01 22.6±0.72 0.84±0.02
10.0 3.75±0.06 19.1±0.35 0.71±0.02
Fe3+
(FeCl3⋅6H2O)
15.0 3.78±0.05 20.9±0.17 0.79±0.02
sharply after 7 days. Glucose was exhausted in the day of
现代食品科技 Modern Food Science and Technology 2009, Vol.25, No.5
477
11, but potassium nitrate was not run out in two weeks.
Considering growth characteristics of C. vulgaris in 17
days, we concluded that the best harvesting time of algal
cells is within the days of 13-15 under this cultivation
condition. Mg2+, Fe2+, Zn2+, Mn2+, Cu2+, Mo6+, Ca2+, and
Fe3+ metal ions were studied respectively for their effects
on biomass, TFAC and TFAY. Fig.1 showed that the
highest biomass (4.06 g/l) can be obtained by adding
2000 mg/l MgSO4·7H2O, but the highest TFAC was
obtained using the medium without adding MgSO4·7H2O
(46.9 %). Fig.2 showed that 20 mg/l FeSO4·7H2O in the
medium lead to the highest biomass (4.43 g/l) and the
TFAC can be enhanced from 19.4 g/l to 23.6 g/l by
increasing Fe2+ concentration. As can be seen from Table
1, absence of Zn2+ showed little effect on the increase of
the TFAC but greatly decreased the biomass (1.71 g/l).
Besides, the biomass and the TFAC had little changes in
the mediums with 0.2 ~ 1.0 mg/l ZnSO4·7H2O. Using 0.5
mg/l MnCl2·2H2O improved biomass of alga cells to the
highest value (4.38 g/l), but had little effect on TFAC.
Cu2+ was an important limited element for alga growth,
without which the biomass is only 1.24 g/l.
Na2MoO4·2H2O also benefited the alga growth and the
biomass greatly increased with the increase of the MoO42-
concentration. But it had week effect on fatty acids
accumulation. Ca2+ had obvious effect on both biomass
and TFAC. The highest biomass (4.27 g/l) were achieved
in the mediums with 50 mg/l CaCl2·2H2O and 5 % TFAC
was increased by adding another 50 mg/l CaCl2·2H2O. It
was found that FeCl3·6H2O had little effect on the both of
biomass and TFAC, which indicated that Fe3+ had no
special contribution on algae growth.
Discussion
Macro-element Mg2+ and Fe2+ had different effect on
intracellular fatty acid production. Mg2+ played an
important role in holding back intracellular fatty acids
synthesis and its deficiency can induces a lots of fatty
acids accumulation in C. vulgaris cells, and TFAC can
reached 46.9 % of dry weight. Contrarily, Fe2+ had
notable effect on cellular fatty acids accumulation. The
TFAC increased with adding higher concentration of Fe2+.
Micro-element Mn2+, Mo6+ and Fe3+ had no obvious
effect on biomass and TFAC of C. vulgaris in this
experiment. It was inferred that these ions were important
contributor in photosynthesis growth process,
participating in oxygen yield metabolic activities by
photosynthetic electric transfer chain (Wydrzynski, 1982).
So, they had little effect on heterotrophic growth of C.
vulgaris in the dark. Moreover, the effect of Fe3+ on
biomass and fatty acids in previous conclusion (Nielsen
et al., 1969) had some inconsistent with the previous
research that Fe3+ had no special effect on biomass and
the TFAC production, these conclusion may be explained
from above photosynthesis theory. Zn2+ deficiency was
beneficial for producing fatty acids but was
disadvantageous for C. vulgaris growth. 0.3 mg/l Cu2+
was suitable for both biomass and TFAC production.
Ca2+ was useful for improving C. vulgaris growth and
TFAC when the concentration was ranged from 50 ~ 100
mg/l. This research provided references for optimal
cultivation technologies.
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