全 文 ：华 南 理 工 大 学 学 报(自 然 科 学 版 )
第 32卷 第 5 期 Journal of South China University of Technology Vol.32　No.5
2004年 5 月 (Natural Science Edition) May　2004
Article ID:1000-565X(2004)05-0047-04
　
Growth and Biochemical Components of Chlorella Vulgaris
Under Autotrophic , Heterotrophic and Mixotrophic Cultivations＊
Wang Hai-ying　Guo Si-yuan　Zheng Bi-sheng　Li Cun-zhi
(Research Institute of Light Industry and Chemical Engineering , South China Univ.of Tech.,
Guangzhou 510640 , Guangdong , China)
Abstract:Investigated in this paper are the growth and biochemical components , such as soluble protein , soluble
sugar , chlorophyll and fatty acid composition of Chlorella vulgaris , under autotrophic , heterotrophic and mixotrophic
cultivations.The results show that the biomass of Chlorella vulgaris under heterotrophic and mixotrophic cultivations is
much higher than that under autotrophic cultivation , that the specific growth rates are respectively 2.13 and 3 times
that under autotrophic growth , and that the growth of cells under mixotrophic cultivation is affected by illuminence ,
but the effect is not significant at the illuminances of 2.5 and 4.0 klx , especially in the stationary phase.Compared
with autotrophic growth , the content of fat under heterotrophic cultivation obviously increases , while the contents of
soluble protein and saccharide decrease , especially chlorophyll.Heterotrophic cultivation is helpful to the
accumulation of linolenic acid.The adoption of light makes soluble proteins , saccharides and chlorophyll increase
under mixotrophic growth , and the illuminence also has effect on the biochemical component of Chlorella vulgaris.
Key words:Chlorella vulgaris;biochemical component;growth;autotrophic cultivation;heterotrophic cultivation;
mixotrophic cultivation
CLC number:Q 815　　　　Document code:A
　Received date:October 29 , 2003
＊Foundation item:Supported by the Key Research Grant from
Science and Technology Committee of Guangdong Province
(99M01409G)
　Biography:Wang Hai-ying (born in 1974), female , Ph.D.
candidate , mainly researches on microalgae biological technolo-
gy.E-mail:whyzxb@163.com
0　Introduction
Microalgae have found wide practical application in
biological engineering , such as healthy food , agriculture ,
wastewater treatment , aquaculture and food additives.
They are also of great value to the sources of chemicals
and pharmaceuticals[ 1] .
Chlorella vulgaris(C.vulgaris), a kind of unicel-
lular green alga , which contains highly nutritious sub-
stances , is of various biological effects as well as a lot of
vital functions and great commercial value.It is one of
typical microalgae strains that are both photo-autotrophic
and heterotrophic nutrition.In this paper , C.vulgaris
was used as an experimental strain to investigate its growth
and biochemical components under different cultivations ,
such as autotrophic cultivation (AC), heterotrophic culti-
vation(HC)and mixo-trophic cultivation(MC).
1　Materials and Methods
1.1　Strain and Its Cultivation
The strain of C.vulgaris was supplied by the fer-
mentation lab in the College of Food and Biological Engi-
neering , South China Univ.of Tech., with Ba-sal medi-
um used[ 1] .ForAC , the cells were illumina-ted with f lo-
rescent lamps(4.0 klx on the bottle surface).The mass
concentration of glucose as heterotrophic carbon source in
the dark was 10 g/L.For MC , the cells were incubated
in heterotrophic me-dium with illumination.Illuminence
was determined by a ZDS-10 illuminometer and controlled
by varying the number of fluorescent lamps.
1.2　Growth of C .Vulgaris
The cell mass concentration of algae in cultural flu-
ids was determined by the measured absorbance with an
UC-2102 spectrophotometer at 540 nm.The dry cell
weight of C.vulgaris was determined by weighing.
1.3　Specific Growth Rate and Doubl-ing
Generation Time
Specific growth rate (μ)was determined by plot-
ting the natural logarithm of dry cell weight versus time ,
that is , μ=(lnX 2-lnX 1)/ t , where X 1 and X 2 are re-
spectively the initiative and final dry cell weights in the
time period.Doubling generation time DT=ln2/μ[ 2] .
1.4　Preparation of Algae Powder
Cultural solution was collected and centrifugated to
obtain sediment.Algae powder was then obtained by vac-
uum drying and grinding.
1.5 　Deterimination of the Biochemical
Components in C .Vulgaris
Chlorophyll content was measured according to
Arnons method[ 3] .Soluble protein was quantified using
the Folin-phenol reagent[ 4] .The contents of soluble sug-
ars and crude fat were respectively determined by using
sulfuric-phenol reagent[ 4] and Soxhlet method.
1.6　Determination of the Fatty Acids in
C .Vulgaris
The methylation of fatty acids was performed by us-
ing the borontrifluoride (BF3)-methanol method as de-
scribed by Yang et al[ 5] .
The composition of fatty acids in C.vulgaris was
determined by a HP4890D Gas Chromatography.The
chromatograph was fitted with HP-INNOwax (with the
length of 30 m and the inner diameter of 0.32 mm).The
temperature was programmed from 190 ℃ to 220 ℃with
an increasing rate of 2 ℃/min andmaintained at the final
temperature for 13 min.The nitrogen carrier gas was set
with a flowrate of 2 mL/min.The temperature of the in-
jection port was maintained at 250 ℃ and the flame
ionozation detector at 270 ℃.1 μL of the derivatized ex-
tract was injected into the column.
2　Experimertal Results
2.1　Growth of C .Vulgaris in Different
Cultural Conditions
Table 1 and Fig.1 show the growth characteristics
and growth curves of C.vulgaris respectively in different
cultural conditions.It is found that the specific growth
rate and biomass of C.vulgaris under HC and MC are
higher than those under AC , the maximum biomasses are
respectively 4.65 and 5.93 times those under AC , and
the specific growth rates are respectively 2.13 and 3 times
those under AC.Furthermore , the logarithmic phases of
HC and MC extend and rise up.
Table 1　Growth characteristies of C.vulgaris in different cultural
conditions
Cultural
condition
Specific growth
rate/ d-1
Doubling generat ion
time/ d
Maximum biomass
/(g·L -1)
AC 0.15 4.62 0.86
HC 0.32 2.16 4.0
MC 0.45 1.54 5.1
Fig.1　Growth curves of C.vulgaris in different cultural condi-
tions
The absorbance was measured at the wavelength of 540 nm
2.2　Effect of Illuminence on Mixotrophic
Cultivation
Fig.2 and Table 2 show the effect of illuminence
on mixotrophic growth.It can be seen that the growth is
affected by illuminence in mixotrophic culture.With the
increasing of illuminence , the specific growth rate and
maximum biomass increase , and that the effects of the
illuminence at 2.5 and 4.0 klx are not remarkably
48 Journal of South China University of Technology(Natural Science Edition) Vol.32
　
　No.4
　　　Wang Hai-ying et al:Growth and Biochemical Components of Chlorella Vulgaris Under Autotrophic ,
Heterotrophic and Mixotrophic Cultivations
different.The cell mass concentration in stationary phase
at 4.0 klx is almost the same as that at 2.5 klx.
Fig.2　Effect of illuminence on the growth curves of C.vul-
garis under MC
The absorbance was measured at the wavelength of 540 nm
　
Table 2　Effect of illuminence on the growth characteristics of C.
vulgaris under MC
Illuminence
/ klx
Specifi c growth
rate/ d-1
Doubling generation
time/ d
Maximum biomass
/(g·L-1)
1.0 0.35 1.98 4.3
2.5 0.43 1.61 4.9
4.0 0.45 1.54 5.1
2.3 　Biocomponents of C .Vulgaris in
Different Cultural Conditions
Listed in Table 3 are biocomponents of C.vulgaris
in different cultural conditions.As shown in Table 3 , the
biocomponents of C.vulgaris vary with the cultural con-
ditions.The contents of soluble protein and chlorophyll in
autotrophic algae are respectively 1.91 times and 4.18
times those in heterotrophic algae , while the content of
crude fat in heterotrophic algae is 1.71 times that in au-
totrophic algae.The content of soluble sugars in au-
totrophic algae is slightly higher than that in heterotrophic
and mixotrophic algae.MC can improve the contents of
soluble protein and chlorophyll compared with HC.Under
MC , the biocomponents of C.vulgaris , especially the
content of chlorophyll , is affected by the illuminence.
Table 3　Biocomponents of C .vulgaris in different cultural condi-
tions
Cultural
conditon
Mass fract ion
of soluble
protein/ %
Mass fraction
of soluble
sugar/ %
Mass fraction
of crude
fat/ %
Content of
chlorophyll in
dry powder
/(mg·g-1)
AC(4.0 klx) 24.32 14.09 17.68 35.14
HC(0.0 klx) 12.76 11.90 30.23 8.40
MC(1.0 klx) 14.55 12.01 27.89 18.29
MC(2.5 klx) 19.15 12.60 21.56 24.31
MC(4.0 klx) 21.89 12.35 21.25 31.28
2.4　Fatty Acid Compositions of C .Vul-
garis in Different Cultural Condi-
tions
The fatty acid compositions of C.vulgaris under dif-
ferent cultivations are shown in Table 4 , from which it
can be seen that , for AC , the major fatty acids are 16∶0 ,
18∶1 , 18∶2 and 18∶3 , and the mass fractions of linoleic acid
and linolenic acid are respectively 26.24% and 17.
49%.There are significant contents of 16∶0 and 18∶3 un-
der HC and MC.The content of linolenic acid reaches
27.65% in heterotrophic algae.The mass fraction of
linolenic acid under MC is less than that under HC , and
the mass fraction of linoleic acid underMC increases with
the illuminence increasing.
Table 4　Fatty acid compositions of C.vulgaris in different cultural
conditions
Fatty acid
composition
Mass fractions in different cultural conditions/ %
AC
(4.0 klx)
HC
(0.0 klx)
MC
(1.0 klx)
MC
(2.5 klx)
MC
(4.0 klx)
16∶0 17.56 29.41 25.45 26.31 26.5
16∶1 2.05 2.08 2.28 5.72 5.24
16∶2 6.67 14.27 9.47 7.08 8.53
16∶3 4.78 3.66 5.17 4.69 2.83
17∶0 1.09 1.68 5.74 4.83 4.21
18∶0 2.14 2.28 4.51 4.69 6.53
18∶1 21.98 10.09 13.89 11.36 12.24
18∶2 26.24 8.88 11.82 13.74 14.54
18∶3 17.49 27.65 21.67 21.58 19.38
3　Discussion
The growth of C .vulgaris is significantly affected
by the supply of carbon source.The final cell mass con-
centration achieved under AC is very low.Glucose caus-
es a great change in the metabolism system , leading to
49
high cell mass concentration under HC.The specific
growth rate of C .vulgaris under MC is less than the
sum of those under AC and HC.The mixotrophic
growth of C .vulgaris is affected by the illuminence.
At the illuminences of 2.5 klx and 4.0 klx , the growth
of C .vulgaris is almost the same , with a relatively sta-
ble specific growth rate and maximum biomass , espe-
cially in the stationary phase.
Microalgae are usually photoautotrophs , with light
as an energy source and carbon dioxide as a carbon
source.The contents of protein , chlorophyll and carbo-
hydrate achieved under AC are all higher than those un-
der HC and MC.HC is a cost-effective , large-scale at-
tentive method of cultivation that utilizes organic carbon
substances as sole carbon and energy sources.In the ex-
periments , the content of crude fat under HC is much
greater than that in the other two cultural conditions , and
the percentage of linolenic acid is very high.By using
HC , the requirement for light is eliminated.Therefore ,
the cell mass concentration of microalgae will consider-
ably increase , and the commercial production of
linolenic acid becomes possible.The lack of light makes
the accumulation of protein and chlorophyll decrease.
Mixtrophism usually represents the simultaneous use of
light and glucose as energy sources.Mixotrophic
metabolism results in a high energy efficiency with mini-
mal amount of energy dissipated.Compared with those
under HC , the contents of protein and chlorophyll in-
crease significantly under MC.The component of cell is
affected by illuminance , which suggests that light is one
of key factors for chlorella growth.
MC is an efficient way to prepare products of high
cell density and great value simultaneously.Moreover ,
since the penetrating distance of light is inversely propor-
tional to the cell mass concentration , cell growth will be
limited by the availability of light.This may be the rea-
son for the decrease of cell mass concentration at 4.0 klx
in stationary phase.To avoid the trouble , Ogbonna pro-
posed the sequential heterotrophic/autotrophic cultivation
system , which composes of a conventional mini-jar fer-
mentor for the cultivation in heterotrophic phase and a
tubular photobioreactor in autotrophic phase.Products of
great value and high biomass can be obtained by using
this system
[ 6] .
Reference:
[ 1] 　Chen Feng , Jiang Yue.Biotechnology of Microalgae [ M] .Bei-
jing:China Light Industry Press , 1999.61.(in Chinese)
[ 2] 　Wei dong , Zhang Xuechen.Effect of cell growth phase on total
lipid content and fatty acid composition of two marine microal-
gae [ J] .Journal of Ocean University of Qingdao , 2000(3):
503-509.(in Chinese)
[ 3] 　Arnon D J.Copper enzymes in isolated chloroplasts polyphe-
noloxidase in Beta Vulgaris [ J] .Plants Physiol , 1949 , 24:1-
15.
[ 4] 　Li Heshen.Experimental Principle and Technique of Plant
Physiology and Biochemistry [ M] .Beijing:Higher Education
Press , 2000.199-200.(in Chinese)
[ 5] 　Yang Bo , Yao Ruhua , Pan li , et al.Effects of pH control in γ-
linolenic acid fermentation [ J] .China Oils and Fats , 2001
(3):55-57.(in Chinese)
[ 6] 　Ogbonna J C , Masui H , Tanaka H.Sequential heterotrophic/
autotrophic cultivation—An efficient method of producing
Chlorella biomass for health food and animal feed [ J] .Appl
Phycol , 1997(9):359-366. (下转第 55页)
50 Journal of South China University of Technology(Natural Science Edition) Vol.32
Detection of total and hemoly sin-producing Vibrio para-
haemolyticus in shellfish using multiplex PCR amplification of
tlh , tdh and trh [ J] .J of Microbiol Methods , 1999 , 36:215-
225.
Establishment of Colony PCR Method and Its Comparison with
Conventional PCR Method
Xu Li　Cai Jun-peng
(College of Food and Biological Engineering , South China Univ.of Tech., Guangzhou 510640 , Guangdong , China)
Abstract:To establish a quick , sensitive and economical PCRmethod for directly detecting tlh and adh genes-carrying
Vibrio parahaemolyticus in environment and seafood , a colony PCR method was developed and its results were compared
with those of the conventional PCRmethod , thus establishing a colony PCRmethod.By using the Vibrio parahaemolytic-
us strains as positive controls , several marine bacteria strains preserved in the lab were amplified through the colony PCR
method and the conventional PCR method , by which the reliability of colony PCR method for the corresponding gene de-
tection was proved.The comparison shows that the results obtained by colony PCR method are in great agreement with
those obtained by the conventional PCRmethod , and that the tlh and tdh genes in positive controls can be amplified by
these two PCR methods.
Key words:colony PCR;conventional PCR;tdh gene;tlh gene
　(上接第 50页)
自养 、异养和混养下小球藻的生长及生化成分＊
王海英　郭祀远　郑必胜　李存芝
(华南理工大学 轻化工研究所 , 广东 广州 510640)
摘　要:对小球藻在自养 、异养和混养条件下的生长状况及细胞的生化成分(如可溶性蛋白 、可溶性糖 、叶绿
素及脂肪酸组成)进行了研究.结果表明:异养和混养培养的小球藻的生物量远大于自养时的生物量;异养和
混养培养的小球藻的比生长速率分别是自养时的 2.13倍和 3倍;光照对混养条件下的细胞生长有影响 ,但
光照强度为2.5 klx 和 4.0 klx时细胞生长的差别并不明显 ,尤其在稳定生长期;与自养生长相比 ,异养过程
中小球藻的脂肪含量明显增加 ,可溶性蛋白和可溶性糖的含量则有不同程度的降低 ,叶绿素含量大大减少;
异养有利于亚麻酸的积累;在混养条件下 ,光照使可溶性蛋白 、可溶性糖和叶绿素的含量增加 ,其强度对细胞
的生化成分有影响.
关键词:小球藻;生化成分;生长;自养;异养;混养
中图分类号:Q 815　　　　文献标识码:A
文章编号:1000-565X(2004)05-0047-04
责任编辑:傅晓琴
　收稿日期:2003-10-29
＊基金项目:广东省科技攻关项目(99M01409G)
　作者简介:王海英(1974-),女 ,博士生 ,主要从事微藻生物技术研究.E-mail:whyzxb@163.com
55　第 5 期 徐　丽 等:菌落 PCR方法的建立及其与常规 PCR方法的比较