全 文 ：化学与生物工程 2008 , Vol.25 No.3　Chemist ry &Bioengineering 35　
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(U0633009),广东省科技攻关计划项目(5006524), 广州市
科技计划项目(2005Z3-E0331)
收稿日期:2007-11-26
作者简介:魏东(1966-),男 , 山东茌平人 , 副教授 ,主要从事工业生物技术研究。 E-mail:few d304@scut.edu.cn。
Optimization of Culture Medium for
Heterotrophic Chlorella Protothecoids Producing Total Fatty Acids
WEI Dong ,LIU Long-jun
(Collegeo f L ight I ndustry and Food Sciences , South China Universi ty o f
Technology , Guangzhou 510640 ,China)
Abstract:The ef fects of g lucose and ni t ra te concentrations in the batch culture medium on the yields of bio-
mass , total lipids and total fatty acids of hetero t rophic Ch lorella protothecoides were inve stig ated.The resul ts
show ed the maximal yields of total lipids(3.83 g ·L-1)and total fat ty acids(1.64 g ·L-1)were achieved in the
cells g rowing in hetero trophic medium containing 40 g · L-1 gluco se and 0.1 mol · L-1 N aNO 3 .Meanwhile ,
C18 fat ty acids w as ove r 69% of to tal fa tty acids as well as a high ratio of total fat ty acids to total lipids
(42.63%)were observed , with a lipid content o f approx imately 24.3%.It indicated that high yield of total fat-
ty acids f rom heterot rophic C.protothecoides could be achieved by opt imizing gluco se and nit rate concentrations
in the medium for further biodie sel production.
Keywords:Chlorella protothecoides;fat ty acid;heterot rophic;lipid
1　Introduction
Biodiesel , known as fatty acid methy l e ste rs de-
riv ed f rom vegetable oils and animal fats , has become
mo re at tractive recently as a renew able , non-toxic
and biodeg radable bio-fuel , which emits less g aseous
po llutants than fo ssil fuels such as pet roleum and
natural gas[ 1 , 2] .However , the high cost fo r raw oil
product ion prevents i ts development and large-scale
use
[ 1 , 3 , 4] .Exploring alternative w ay s to reduce the
co st o f raw oil production is becoming more impo r-
tant in current biodiesel research[ 5] .
Recently , microalgae has been propo sed as an
energy source in a number of w ay s for production of
energy
[ 6] .Some g reen microalgae Chlorel la species
have been suggested as po tential sources o f fuel w i th
high calorific v alue linked to the high lipid con-
tent
[ 7 ～ 9] .Chlorella protothecoides is a unicellular
chlo rophycean micro algae , which show s a high spe-
cific g row th rate , high biomass and high lipid content
in cells unde r hetero t rophic g row th condition[ 10 ～ 12] .
High crude lipid content(46.1%～ 55.3%)was ob-
served w ith the addi tion of glucose and the decrease
of inorganic nit rogen source in the medium , and corn
powder hydroly sate instead of g lucose w as investig a-
ted to increase the biomass and reduce the co st of al-
gae in fermentation.T herefo re , Chlorel la protothe-
coides has been suggested as a candidate for produc-
tion of biodiesel fuel[ 9 , 12] .However , no de tai led data
were provided about the effects of gluco se and ni tro-
gen source and thei r concentra tion variations in the
culture medium on biomass and total lipid produc-
tion.Meanwhile , the fat ty acid composi tion and
y ield , which could determine the co st and quali ty of
biodiesel f rom microalgae , received lit tle attention.
The aim of this study w as to investigate the
ef fects o f medium composition , in particula r , gluco se
and ni t ra te concentration , on the yields of biomass ,
to tal lipids and to tal fat ty acids in the batch culture of
heterot rophic Chlorel la protothecoides.
2　Experimental
2.1　Microalgae and culture conditions
The g reen microalg ae Chlorel la protothecoides
魏　东等:异养小球藻产总脂肪酸的培养基优化/2008 年第 3 期36　
CS-41 w as purchased from the CSIRO M arine Labo-
rato ry (Hobart , Australia).The modified basal me-
dium was adjusted to pH value 6.5 and auto-
claved[ 13] .Erlenmeyer f lasks (250 mL)each contai-
ning 100 mL medium were inoculated wi th 5%inocu-
lum at 28℃ in an o rbital shaker at 160 r ·min-1 un-
der darkness.Fo r investig ating ef fects o f gluco se
concentrations , 10 ～ 50 g ·L-1 g lucose w ith 8.5 g ·
L
-1
NaNO 3 in the medium were used.For investig a-
ting ef fects of nit rogen concentrat ions , 0.05 ～ 0.2
mol·L -1 NaNO 3w ith 40 g ·L-1 gluco se in the medi-
um were used.
2.2　Determination of biomass concentration and spe-
cific growth rate
The dry cell w eight w as determined according to
the method described by Ip & Chen[ 14] .Specific
grow th rate w as de termined by plot ting natural log-
a ri thm of dry w eight against cul ture t ime.
2.3　Determination of glucose and nitrate concentra-
tion
Glucose concentration w as determined by 3 , 5-dini-
tro salicylic acid method[ 15] .Nitrate concentration w as de-
termined by sulphuric acid-salicylic method[ 16] .
2.4　Determination of total lipid and fatty acids
Dried algal cel ls w ere blended wi th 0.5 mL of
distilled w ater and 3 mL of chlo rofo rm/me thano l(2
∶1), and the mix ture w as f ie rcely shaken for 20 min
and centri fuged.T he chlo rofo rm phase w as removed
and the same process w as repeated for five times.
The whole chlo ro fo rm phase w as collected and com-
bined togethe r , then evaporated and dried under vac-
uum.The residue w as immediately weighed to give
the lipid content[ 17] .
Fat ty acids in the algal cells we re ex t racted ac-
cording to the me thod of Cohen & Norman[ 18] .
KOH-CH 3OH (1 mL)was added to 30 mg dried al-
gal cel ls , and the mix ture w as incubated at 75℃ fo r
15 min and coo led to ro om tempe ra ture.
HCl-CH 3OH (1 mL)was added to the mix ture fo r a-
cidification at 75℃ fo r 15 min and cooled to room
temperature.Petroleum benzine (1 mL)and disti lled
w ater (0.3 mL)were added to the mix ture and then
centrifug ed at 3000 r ·min-1 fo r 10 min.The petro-
leum benzine layer w as collected.Fat ty acid esters
were analyzed and ident ified by HP 6890N GC-MS
(Agilent , USA)equipped w ith 5975 inert mass selec-
tive detecto r and G1540N-210 FID detecto r.A high
performance capi llary column (HP-FFAP , 30 m ×
0.25 mm I.D., film thickness 0.25 μm)was used.
Helium was used as carrier gas at a f low rate o f 1 mL
·min-1 .The injecto r temperature w as set at 250℃
and FID detecto r tempe rature w as 300℃.T he pro-
g rammed temperature w as at 150℃ fo r 1 min which
w as subsequently increased to 200℃ at 15℃ ·
min-1 , and then to 212℃ at 1℃·min-1 .Fat ty acid
e ste rs w ere identified by autoscan and direct compari-
son of their mass spect ral pat tern and retention index
(RI)w ith N IS T 05 mass spect ral database.The
quanti ties of individual fat ty acids w ere est imated
f rom the peak areas on the FID chromatog ram using
nonadecanoic acid(C19∶0)as the internal standard.
2.5　Statistical analysis
The reg ression analy sis w as performed in Mi-
crosof t Excel 2003 sof tw are.S tudent t-test analy sis
using Origin 7.0 procedures w as used to est imate the
statistical signif icance betw een coupled mean values.
3　Results and discussion
3.1　Biomass concentration , lipid content and yield of
fatty acids at various glucose concentrations
Fig.1　Biomass concentration and lipid content in
heterotrophic cells of Chlorella protothecoides at
various glucose concentrations and 8.5 g· L-1
NaNO3 in batch culture medium
The results were shown in Fig.1 and further detail
was given in Tab .1.The highest lipid content in the
dried algal cells was approximately 30.12% at 10 g ·L-1
glucose , but the biomass concentration reached the high-
est value (18.4 g · L-1)corresponding to 50 g · L-1
魏　东等:异养小球藻产总脂肪酸的培养基优化/ 2008 年第 3 期 37　
glucose , with a lower lipid content(17.5%).At 40 g ·
L
-1
glucose , a relatively high biomass concentration
(15.8 g · L-1)and lipid content (24.3%)were ob-
tained.The high specific grow th rate(0.66 ～ 0.83 d-1)
was achieved in the medium containing 10 ～ 40 g · L-1
glucose , and 50 g · L-1 glucose inhibited cell growth ,
which w as reflected by a considerably lower specific
grow th rate (0.44 d-1).
Tab.1 Yield and composition of total fatty acids in heterotrophic cells of Chlorella protothecoides at various glucose
concentrations and 8.5 g· L-1 NaNO3 in batch culture medium
Relative con tent of
fat ty acid/ %
Glucose/ g· L -1
10 20 30 40 50
16∶0 23.97±1.02 22.50±1.93 17.40±0.96 17.25±0.85 16.91±1.06
16∶1(Δ9) - 3.76±0.08 5.76±0.36 6.98±0.13 8.11±0.72
16∶2(Δ7 , 10) 9.38±0.71 11.47±0.88 12.32±1.03 14.32±1.32 9.55±0.26
16∶3(Δ7 , 10 , 13) 5.50±0.40 - - - -
17∶0 1.70±0.06 1.53±0.05 0.93±0.03 0.96±0.07 1.19±0.08
18∶0 0.60±0.03 1.10±0.05 0.76±0.03 0.92±0.02 1.21±0.04
18∶1(Δ9) - 21.60±1.63 26.63±1.72 31.76±1.91 43.55±3.58
18∶2(Δ9 , 12) 31.79±2.65 38.10±2.89 36.32±3.01 27.85±1.96 19.53±1.26
18∶3(Δ9 , 12 , 15) 27.06±1.86 - - - -
T FA/DWa 6.09±0.41 6.87±0.51 8.17±0.58 10.36±0.65 9.02±0.62
TFA / T Lb 20.18±1.26 32.85±2.53 37.6±3.03 42.63±3.68 43.70±2.97
Yield of TFA/ g· L-1 0.24±0.01 0.55±0.03 0.92±0.05 1.64±0.08 1.66±0.08
Note:Data are exp ressed as mean S.D. of th ree replicates;“ -” m eans not found
a.TFA/DW:(t otal fat ty acid s/ dry cell w eigh t)×100%
b.TFA/ T L:(t otal fat ty acids/ t otal lipids)×100%
　　Over the w ho le range of gluco se concentrations
tested , C18 fatty acid levels w ere consistent ly high
being over 60% of the total fat ty acids in hete ro-
t rophic cells.C16∶3 and C18∶3 we re detected only
at 10 g ·L-1 glucose.In contrast , C16∶1 and C18∶
1 we re detected at all othe r g lucose concentrations.
The highest cel lular to tal fat ty acid content(10.36%
of dry cell w eight)was obtained at 40 g · L-1 g lu-
cose , but the highest ratio o f to tal fat ty acids to total
lipids(43.70%)and the highest yield of to tal fat ty
acid(1.66 g ·L-1)were obtained at 50 g · L-1 g lu-
cose.However , the yield of total fat ty acid (1.64 g
·L-1)at 40 g · L -1 glucose show ed no stati stical
dif ferential comparing to 1.66 g · L-1 at 50 g · L-1
gluco se.These results suggested that high gluco se
concentration could increase the cellular to tal fat ty
acids content and the proport ion of to tal fat ty acids in
lipids.
Because 40 g ·L-1 g lucose w as the best in terms
of specif ic g row th rate , biomass and cellular fat ty
acids product ion , this glucose concentration w as used
fo r the fur ther investig ation in o rder to obtain higher
product ion yield of to tal lipid and total fat ty acids.
In heterot rophic culture of microalgae , o rganic
carbon source is used as bo th energy and carbon skel-
etons for cell grow th and bio synthesis of prod-
ucts
[ 19] .The present study show ed that gluco se w as
a suitable carbon source for heterot rophic cultiva tion
of C.protothecoides.The low est biomass concentra-
tion w as observed at the low est g lucose concentra tion
(10 g ·L-1), indicat ing that low glucose concentra-
tion w as a limiting factor fo r cel l g row th , although
lipid accumulation w as enhanced.Similar resul ts
were repor ted on o ther Chlorel la species such as C.
saccharophi la and C.sorok iniana [ 17 , 20] .A relatively
high biomass concentration and lipid content w ere ob-
tained at 40 g ·L -1 g lucose.It w as repo rted , howev-
er , the cellular lipid content increased wi th gluco se
concentration in some othe r alg al cultures such as the
diatom N itzschia laev is and the dinoflagel late
魏　东等:异养小球藻产总脂肪酸的培养基优化/2008 年第 3 期38　
Cryp thecodinium cohni i
[ 21 , 22] .Therefore , the ef fect
of gluco se concentration on lipid content in hete ro-
t rophic algal cultures might be species-specific.
3.2　Biomass concentration , lipid content and yield of
fatty acids at various nitrogen concentrations
The results w ere show n in Fig .2 and further de-
tail w as giv en in Tab.2.By using 40 g ·L-1 g luco se ,
the maximal biomass concentration (15.8 g · L-1)
was obtained at 0.1 mol·L -1 NaNO 3 , but the high-
est lipid content (26.61%)was obtained at 0.05 mo l
·L-1 NaNO 3 .Howeve r , the highest yield o f lipids
(3.83 g ·L-1)was achieved at 0.1 mol·L-1 NaNO3
because o f the highest biomass concentration.
Fig.2　Biomass concentration and lipid content in heterotrophic
cells of Chlorella protothecoides at various NaNO3
concentrations and 40 g· L-1 glucose
in batch culture medium
Tab.2 Yield and composition of total fatty acids in
heterotrophic cells of Chlorella protothecoides at
various NaNO3 concentrations and 40 g· L-1
glucose in batch culture medium
Relative content of
fat ty acid s/ %
NaNO 3 concent ration/m ol· L -1
0.05 0.1 0.2
16∶0 15.12±0.63 17.25±0.85 17.92±1.03
16∶1(Δ9) 5.19±0.36 6.98±0.13 5.27±0.42
16∶2(Δ7 , 10) 8.78±0.68 14.32±1.32 12.28±1.07
17∶0 0.64±0.04 0.96±0.07 0.93±0.06
18∶0 8.52±0.23 0.92±0.02 1.98±0.09
18∶1(Δ7) 40.17±3.25 31.76±1.91 32.07±2.38
18∶2(Δ9 , 12) 20.95±1.36 27.85±1.96 29.56±2.63
T FA/DW a 11.98±0.78 10.36±0.65 8.86±0.68
TFA / T Lb 45.2±4.36 42.63±3.68 42.14±2.51
Yield of TFA/ g· L-1 1.62±0.08 1.64±0.08 1.28±0.06
Note:Data are exp ressed as mean S.D.of th ree replicates;
a.TFA/DW:(t otal fat ty acid s/ dry cell w eigh t)×100%;
b.TFA/ T L:(total fat ty acids/ total lipids)×100%
C16∶0 , C16∶2 , C18∶1 and C18∶2 w ere the
main fat ty acids in heterot rophic Ch lorella protothe-
coides at various NaNO 3 concentrations.The amount
of C18 fa tty acids w as near 70% of the to tal fat ty
acids , which w as benef it to prepare high quali ty
biodiesel.The highest content o f total fat ty acids
(11.98% of dry cell w eight)and the highest ratio of
to tal fat ty acids to total lipids (45.2%)we re ob-
tained at 0.05 mol · L-1 NaNO 3 , the maximal yield
of total fat ty acids (1.64 g · L-1)was achieved at
0.1 mo l· L-1 NaNO 3 because of the highest biomass
concentration (15.8 g ·L-1), which show ed no sta-
tistical dif ferential comparing to 1.62 g ·L-1 at 0.05
mol·L -1 NaNO 3 .The results indicated that the de-
cline o f NaNO 3 concentration to 0.05 mol·L-1 could
no t increase the y ield o f total fatty acids because of a
low er biomass concentrat ion(13.5 g ·L-1).
It has been known that ni t ro gen is an impo rtant
nutrient affecting the cell g row th and lipid production
in many microalg ae species.In H.pluv ial is and C.
zof ingiensis , the obvious g row th limitation w as
found in the cell g row ing in the absence o f ni tro-
gen[ 14 , 23] .The significant ly high lipid content w i th
the N-starvat ion in some green algae w as also repo r-
ted[ 7 , 24 , 25] .A t a highe r C/N ratio and excessive car-
bon supplement , synthesis of nit rog en-containing
compounds such as protein and nucleic acids w as cur-
tailed , and cells might begin to accumulate sto rage
lipids[ 26 , 27] .Similar results w ere observed in the
present study.Nitrate as ni t ro gen source w as the
limiting facto r fo r cell g row th , but the limita tion of
nit rate concentration could stimulate the accumula-
tion of total lipids(Fig.2).The possible reason may
invo lve the regulat ion of the metabolic pathw ay relat-
ed to lipid/ fat ty acid biosynthesis , even the carbon/
nit rog en me tabo lic t ransfo rmation in heterot rophic
C.protothecoides cells.
It had been repo rted that diatoms and marine mi-
croalgae Nitzchia laevis , D.bardawil and D.salina
could produce higher contents of polyunsaturated fat-
ty acids under low N-levels[ 24] .Chlorel la protothe-
coides was a f reshwa ter microalgae , and the resul ts of
the present study we re in good ag reement w ith tho se
found in f reshw ate r alg ae Scenedesmus and o ther
魏　东等:异养小球藻产总脂肪酸的培养基优化/ 2008 年第 3 期 39　
Ch lorel la species , in w hich the content of polyunsat-
urated fat ty acids reduced w ith limited N source
[ 28] .
In the previous study , 10 g ·L -1 g lucose as car-
bon source and 0.1 g · L-1 gly cine as nit rogen
source , and the co rn powder hydrolysate instead of
gluco se in the medium were used fo r batch cul ture of
Ch lorel la protothecoides[ 12] . In comparison w i th
tho se results , our highest biomass concentration
(15.8 g · L-1)was 4 times o f the highest v alue
(3.92 g ·L-1), and our highest yield of lipids(3.83
g · L-1)was 76.5% higher than the highest v alue
(2.17 g · L-1)obtained in the previous batch cul-
ture , even if the cellular lipid content w as high up to
55.3%[ 12] .Therefo re , our results indicated that high
gluco se concentration w as preferred to the yield o f bi-
omass and fat ty acids , and NaNO 3 was bet ter than
gly cine as nit rog en source.It also suggested that , fo r
a successfully high yield of fat ty acid production as
biodiesel source , the biomass concentration , fat ty
acid content and composition should be considered
simultaneously , no t the lipid content in biomass on-
ly .
4　Conclusion
The present study indicated that gluco se and ni-
t rate concentrations could remarkably af fect the yield
of biomass , to tal lipid and fat ty acids in heterot rophic
C.protothecoides.High-yield production of total fat-
ty acids as biodiesel could be achieved by optimizing
gluco se and ni trate concentrations in heterot rophic
medium fo r C.protothecoides g row th.Fo r the fur-
ther high yield and low cost production of total fat ty
acids , more cheaper and eff icient carbon and nit rogen
source s should be considered , and optimiza tion of
their concentrations in he terot rophic medium should
be investigated in the future.
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异养小球藻产总脂肪酸的培养基优化
魏　东 ,刘龙军
(华南理工大学轻工与食品学院 ,广东 广州 510640)
　　摘　要:研究了异养小球藻(Chlorella protothecoides)分批培养基中葡萄糖和硝酸盐浓度对生物量 、总脂和总脂肪
酸产量的影响。结果表明 ,当细胞在含有 40 g· L-1葡萄糖和 0.1 mo l· L-1 NaNO 3的异养培养基中生长时 , 总脂和总脂
肪酸的产量可分别达到最高值为 3.83 g· L-1和 1.64 g · L -1 ,同时 C18脂肪酸占总脂肪酸的比例超过 69%, 总脂肪酸
占总脂的比例也高达 42.63%, 总脂含量约为 24.3%。通过优化异养小球藻培养基中的葡萄糖和硝酸盐浓度 , 能够获得
高产总脂肪酸 ,进而生产生物柴油。
关键词:小球藻;脂肪酸;异养;脂类
中图分类号:Q 949.21 +7　　　　　文献标识码:A　　　　　文章编号:1672-5425(2008)03-0035-06
生物法木糖醇新技术通过鉴定
　　化学催化加氢法生产木糖醇 ,不仅工艺复杂 、安全性差 ,而且还必须以纯木糖为原料 ,成本较高 。一直致力于
生物转化法生产木糖醇研究的中科院微生物所江宁研究员领导的课题组 ,与山东威龙工业集团有限责任公司合
作进行工业性试验 ,现已生产出了纯度达 99%以上的木糖醇。日前该项目通过了中科院生命科学与生物技术局
组织的成果鉴定 。
据介绍 ,生物法生产木糖醇的原理是利用细胞内的木糖还原酶 ,将木糖还原为木糖醇。另外 ,生物转化法可使
细胞连续反复使用多次且无需无菌操作 ,不但降低了发酵成本 ,也降低了设备投资和操作费用 ,同时还简化了后处
理过程。该工艺流程在常温常压下进行 ,具有能耗低 、设备简单 、操作安全等特点 。江宁课题组通过选育 ,得到了一
株生产木糖醇的优良菌种 。该菌种的稳定性好 ,可适应较粗放的原料 ,其木糖醇转化率和产率都很高。
据悉 ,微生物所与威龙集团合作建设的年产千吨级生产线 ,以玉米芯水解液以及木糖母液为原料生产木糖醇 ,得到
了纯度 99%以上的结晶木糖醇 ,产品质量不仅明显优于采用化学法以同样原料生产的产品 ,甚至优于化学法以纯木糖
为原料生产的产品。新菌种的获得和新工艺的建立 ,使生物法木糖醇具有明显竞争优势 ,可望成为今后木糖醇生产的
主要方法。生物转化法生产木糖醇新工艺是中国科学院知识创新工程的成果之一 ,拥有自主知识产权。
以中国农业科学院的范云六院士为组长的专家鉴定组认为 ,木糖醇是功能性食品添加剂 ,并有多种其他用
途。该项成果应用生物技术以生物质为原料生产木糖醇 ,对资源的综合利用和发展循环经济有积极的意义。该
项成果在年产千吨级装置上应用成功 ,转化率 、产量和产率等主要生产指标达到较高水平 。专家建议课题组今后
进一步完善该技术 ,结合生物质的高值化综合利用进行工业化生产 ,创造更大的经济和社会效益。 (摘编)