全 文 ：小头端菱形藻生长的最适光照
强度及氮磷比
晏妮 1，胡晓红 2，陈椽 1* （1.贵州师范大学生
命科学学院，贵州贵阳 550001；2.贵州师范大
学地理与环境科学学院，贵州贵阳 55001）
摘 要 ［目的］ 为进一步探索小头端菱形藻
(Nitzschia capitellata Hust.) 作为优质饵料、生
物能源的潜能，在室内条件下对小头端菱形藻
生长的最适光照强度、氮磷比等培养条件进行
研究。 ［方法］设定不同光照度梯度，在相同营
养、温度 (25±1)℃以及光周期 (12 h/12 d)下进
行培养，筛选得出最适光照强度，其次将氮磷
比设置为 5∶1、6∶1、7∶1 和 8∶1，置于相同条件进
行培养。 ［结果］ 在 3 000 lx 的光照度下，小
头端菱形藻的藻比增殖率和现存量最高，分别
为 0.51 d-1 和 7.97×104 cells/ml。 在氮磷比为
6∶1 条件下，小头端菱形藻生长最佳。［结论］小
头端菱形藻的最适光照度为 3 000 lx，最适氮
磷比为 6∶1。
关键词 小头端菱形藻；光照强度；比增殖率；
现存量；氮磷比
基金项目 贵州省科学技术基金（黔科合 J 字
（2010）2067 号）。
作者简介 晏妮 （1981- ），女，贵州贵阳人，
副教授，从事植物学、水域生态学方面的研究，
E-mail：yandy600@sohu.com。 * 通讯作者 ，教
授， 从事植物学、 水域生态学方面的研究，E-
mail：cchuan66@163.com。
收稿日期 2014-02-10
修回日期 2014-03-14
Optimal Light Intensity and Nitrogen-to-
Phosphorus Ratio for Growth of Nitzschia
capitellata Hust.
Ni YAN1, Xiaohong HU2, Chuan CHEN1*
1. Institute of Life Science, Guizhou Normal University, Guiyang 550001, China;
2. Institute of Geography and Environmental Science, Guizhou Normal University, Guiyang 550001, China
Supported by Guizhou Provincial Science&Technology Fund ((2010)2067).
*Corresponding author. E-mail: yandy600@sohu.com
Received: February 10, 2014 Accepted: March 14, 2014A
Agricultural Science & Technology, 2014, 15(3): 453-456
Copyright訫 2014, Information Institute of HAAS. All rights reserved Animal Science and Feeds
I t is well known that light is key forplant growth and development.Besides as source of photosyn-
thesis, light has a control on plant de-
velopment, including algae, namely
photomorphogenesis [1]. The develop-
ment of algae is frequently affected by
light, involving nutrient and reproduc-
tive structure. Among multiple influen-
tial factors, nitrogen and phosphorus
are widely accepted as important fac-
tors influencing alga growth, besides
sunlight. On the other hand, nitrogen
interplays with phosphorus and the
demands on nitrogen and phosphorus
are diversified upon alga species.
Currently, many researches are avail-
able on unicellular algae, such as
chlorella, and dinoflagellate, but less
attention is paid to diatom in fresh
water and ocean[2]. Planktonic diatoms
are a major group of algae easily be
digested, with abundant nutrients and
a major natural bait for filter feeders [3],
especially for Nitzschia which contains
lots of excellent and highly-valued al-
gae[4-5], whilst Bacillariophyta contains
higher fats and are believed one of al-
gae with promising potential for indus-
trialization[6].
Currently, Nitzschia has totaling
350 species in the world and over 76
species can be found in China. Never-
theless, researches available only
concentrate on few species in our
country, including Nitzschia alexan-
dria, Nitzchiaceae closterium, and
Nitzschia plea, et al. [7 -11]. Nitzschia
capitellata is a unicellular alga in fresh
water, in Bacillariophyta. It is re-
searched that total lipids of Nitzschia
capitellata reach 9.25% and fatty acid
consists of myristic acid (C14∶0), palmitic
acid (C16∶0), palmitoleic acid (C16∶1), oleic
acid (C18 ∶1), linoleic acid (C18 ∶2) and
therapic acid (C18 ∶4). The unsaturated
fatty acid takes up over 63% [12]. Al-
though lower compared with diatom
average (22.7%) in ocean[13], total lipid
content in Nitzschia capitellata is high-
er than measured values of diatoms in
the test conducted by Lin et al. [14].
Abstract [Objective] The aim was to explore the potential of Nitzschia capitellata as
excellent baits and bioenergy, and the optimal light intensity and nitrogen-to-phos-
phorus (N/P) ratio suitable for its growth were researched as well. [Method] With
light intensity gradient set, Nitzschia capitellata was cultured with the same nutrients
at (25 ±1) ℃ and light cycle at 12 h/d to select optimal light intensity. Then,
Nitzschia capitellata was cultured with the same condition, and nitrogen-to-phospho-
rus ratios at 5∶1, 6∶1, 7∶1 and 8∶1. [Result] With light intensity at 3 000 lx, specific growth
rate and standing stock achieved the highest at 0.51 d-1 and 7.97×104 cells/ml. The
growth condition with nitrogen-to-phosphorus ratio at 6 ∶1 was the most suitable for
Nitzschia capitellata growth. [Conclusion] The optimal light intensity and nitrogen-to-
phosphorus ratio were 3 000 lx and 6∶1, respectively, for Nitzschia capitellata.
Key words Nitzschia capitellata Hust.; Light intensity; Specific growth rate; Standing
stock; Nitrogen-to-phosphorus ratio
DOI:10.16175/j.cnki.1009-4229.2014.03.029
Agricultural Science & Technology 2014
What’s more, the life circle of
Nitzschia capitellata is short and
biomass is high. Therefore, it is of
great significance for fish farming de-
velopment in fresh water with energy
shortage to research of cultivation con-
ditions for Nitzschia capitellata in order
to further explore highly-qualified baits
and development of application value
of biological energy source.
Materials and Methods
Cultivation of Nitzschia capitellata
Culture medium used in the test
was D1 medium, and glass wares
were all strictly washed and sterile with
a high pressure sterilization pot at 1
kg/cm at 120 ℃ for 30 min. Light was
provided by fluorescent lamp; test
bottle was a conical flask (250 ml)
where 100 culture solutions were
added.
Design of light gradient
According to documents avail-
able, the suitable light range of diatom
is in 1 000-5 000 lx[15] and the light gra-
dient , therefore , was set at 1 000,
2 000, 2 500, 3 000, 4 000 and 5 000
lx, respectively. The same glass pane
was lighted from two sides and light
intensity was adjusted with a modula-
tor tube. Among different treatments, a
black paper was made into conical-
flask shape room in case of influence
of nearby lights. Practical light intensity
was measured in every test bottle with
an illuminometer provided from JIB IA
Shanghai Jiading Xuelian Equipment
Industry. The initial alga density was at
103 cells/ml for inoculation. Every illu-
mination series included three repeti-
tions with cultivation temperature at
(25±1) ℃ , and light-shading circle at
12 h/12 h. The test bottle sites were-
moved and the bottle shaken for every
3 h.
Design of N/P ratio
Parent solution in the test was
mixed well and added to culture solu-
tion as per N/P ratios at 5 ∶1, 6 ∶1, 7 ∶1,
and 8∶1, according to Survey Standard
of Fishery and Natural resources in
Inland Water Area. Meanwhile, a con-
trol group was prepared with N/P ratio
at 0∶0. The culture solution was at 100
ml and every treatment was set with
three repetitions.
Measurement methods
Population increase was mea-
sured with Cell Counting Method. For
example, since the following day of
inoculation, materials were sampled at
10:00 in the morning and algal solution
in a conical flask was well shaken.
Then, the culture solution was suck up
at 1 ml with a sterile pipette and added
into a test tube. After shaking, 0.1 ml of
solution was suck into a counting
pane. It is notable that the whole sam-
pling process was conducted in a ster-
ile condition. Cells were counted with a
binocular microscope in multiple times
to get averages, and the diagram of
algal cell growth was drawn.
It can be concluded that the in-
crease rate of algal cell is as follows:
μ=ln(Xn /Xn-1)/(tn-tn-1)
where Xn represents the number
of cell on that very day; Xn-1 represents
the number of cell in the day before; tn
refers to culture time (d) corresponding
to Xn ; tn-1 refers to the culture time (d)
corresponding to Xn-1. The average of
specific growth rate of algae was then
computed (d-1)[16] to be an estimate of
algae in a specific condition.
In addition, standing stock was as
follows:
X=(A/Ac)/V×(n/m)[17-19]
where X is the number of phyto-
plankton or cells per milliliter in cul-
ture solution; A refers to the area of
counting frame (mm2); Ac is a view-
ing area (mm2); V represents volume
of counting frame; n refers to phy-
toplankton or cell numbers; m repre-
sents viewing frequency. The aver-
age of maximal standing stock would
be calculated and data in every
group were analyzed.
Results and Analysis
Specific growth rate of Nitzschia
capitellata with different light inten-
sities
As shown in Fig.1 and Fig.2,
light has effects on growth of
Nitzschia capitellata. Within light inten-
sity in 1 000-3 000 lx, specific growth
rate of Nitzschia capitellata was in-
creasing upon light intensity. With light
intensity at 3 000 lx, in particular,
Nitzschia capitellata grew well and
rapidly. When the value of μgrew to
maximum at 0.51 d -1, cell density
achieved peak at 7.97 ×104 cells/ml,
which was significantly higher com-
pared with other treatments. When
light intensity exceeded 3 000 lx, how-
ever, the value of μ was declining.
In culture of Nitzschia capitellata,
both of cell concentration and matrix
concentration tended to be volatile and
cell growth experienced lag phase, log
phase, stationary phase and death
phase. With lower light intensity (1 000
lx), Nitzschia capitellata entered log
Fig.1 Growth curve of Nitzschia capitellata with different light intensities
Fig.2 Specific growth rate of Nitzschia capitellata with different light intensities
454
Agricultural Science & Technology2014
Table 1 Changes of Nitzschia capitellata cell concentrations with different N/P ratios
Day∥d
N/P
0∶0 5∶1 6∶1 7∶1 8∶1
1 0.10 0.23 0.40 0.40 0.40
2 0.40 0.44 0.50 0.60 0.70
3 0.90 0.95 1.10 0.70 1.20
4 1.70 1.82 2.00 2.30 2.60
5 2.20 3.20 6.20 4.50 5.10
6 3.40 6.80 8.80 7.20 8.00
7 5.10 9.40 13.40 11.60 11.90
8 6.70 11.50 16.70 14.50 13.60
9 8.80 13.70 18.80 16.30 15.50
10 10.20 14.20 21.10 18.10 16.30
11 8.10 12.30 20.10 16.10 14.60
12 6.60 9.90 17.90 15.50 13.20
phase through 2 d adaptation, be-
cause of light shortage, and in the
phase, the alga grew slowly and log
phase last long. As a result, algal cell
density was only 3.75×104 cells/ml af-
ter 4 d cultivation. When light intensity
was adjusted at 3 000 lx, log phase re-
duced and Nitzschia capitellata en-
tered stationary phase when algal
density was 2.1 times of that with light
density at 1 000 lx and growth rate was
3 times. Generally speaking, in the
range of 1 000-3 000 lx, the absorbed
light by unit cells increased upon light
intensity, and both of photosynthesis
and cell division sped up. When light
intensity was adjusted in 3 000-6 000
lx, in contrast, the harvested quantity
of algae were declining upon light in-
tensity. It is clear that there is a light
saturation point for algae. Within the
range, alga growth speeds up with the
increase of light intensity. When light
intensity exceeds the point, however,
photosynthetic rate would be weaker
or even stop. Yan et al. [20] researched
that Chlorella sp. and Scenedesmus
obliquus grew the fastest with light in-
tensity at 5 000 lx when light cycle was
at 12 h ∶12 h, with light intensity gradi-
ent set at 500, 1 000, 3 000, 5 000,
and 7 000 lx. Liu et al.[21] pointed out that
the optimal light intensity of chlorella,
Isochrysis zhanjiangensis, Platymonas
helgolandica and Dunaliella viridis was
5 000 lx at 25 ℃. The test also demon-
strated that there is a light saturation
point for algae, and alga growth would
be inhibited if light intensity breaks the
point. Nitzschia capitellata grew slowly
with light intensity at 1 000 lx, but the
phenomena, such as decoloring or
death, were not found, indicating that
light intensity at 1 000 lx is not the low-
er limit of light intensity required by
algae. With light intensity at 5 000 lx,
photosynthesis of Nitzschia capitellata
dropped and growth rate declined, but
still growing, which indicated that light
intensity at 5 000 lx is not the upper
limit of Nitzschia capitellata, either, and
light intensity from 1 000 to 5 000 lx is
just a middle interval of corresponding
light intensity range. Hence, Nitzschia
capitellata is of high adaptability to light
intensity, but 3 000 lx constitutes an
optimal light intensity.
Optimal N/P ratio of Nitzschia
capitellata with light intensity at
3 000 lx
Some researches suggested that
N/P ratio in plant body is about 7∶1 and
even exceeds 8∶1 in natural water with
high productivity. Still, based on ab-
sorption characteristics of diatom, the
ratio of N to P should be controlled
at (6-7)∶1[22]. Hence, the test set tem-
perature at (25±1)℃, light cycle at 12
h∶12 h, light intensity at 3 000 lx, inoc-
ulation density at 1×103 cells/ml, and
N/Pratios at 5∶1, 6∶1, 7∶1 and 8∶1.
It can be concluded from Table 1
that Nitzschia capitellata grew in cul-
ture solutions with N/P ratio gradients
from 5 ∶1 to 8 ∶1 and the growth per-
formed better compared with control
group, showing significant differences
with control group (P<0.01). Specifi-
cally, 3 d before cultivation, algae in
different groups kept stable growth
and entered log phase from the 4th d.
In the end of log phase, cell number
achieved peak with N/P at 6 ∶1 at 2.9×
105 cells/ml and the number was the
lowest at 1.42×105 cells/ml with N/P at
5 ∶1. The number tended to be in the
range between the maximum and
minimum with N/P at 7 ∶1 and 8 ∶1. Ac-
cording to paired sample t-test (N/P at
7∶1 and 8∶1), the value of t was 1.308,
of df was 11 and of P was 0.217, sug-
gesting that none of significant differ-
ences existed in the two groups (P>
0.05). In terms of the whole growth
term, however, algal cell density was
higher with N/P at 7 ∶1, compared with
the condition with N/P at 8 ∶1. The re-
search results are in consistent with
researches available, in terms of N/P
ratio from 6 ∶1 to 7 ∶1. On the other
hand, based on comparisons of treat-
ment with N/P at 6 ∶1, with other treat-
ments, the values of t, df, and P were
4.003, 11 and 0.001 in the treatment
with N/P at 5∶1, 4.102, 11 and 0.002 in
the treatment with N/P at 7 ∶1, and
4.297, 11 and 0.001 in the treatment
with N/P at 8∶1 by paired sample t-test.
The results incorporated that signifi-
cant differences were found of the
treatment with N/P at 6 ∶1 with other
treatments (P<0.01), and the ratio at
6 ∶1 is the most suitable for Nitzschia
capitellata, when the use efficiency of
N and P achieves the peak.
Conclusions
Light is a crucial ecological factor
for algae growth and development, as
well as major energy source. With the
value of pH and temperature fixed,
light intensity and duration play a key
role in algal growth rate[23-24]. Still, light
effects differ on alga growth upon algal
species. In a proper range, photosyn-
thesis would be sped upby increase of
light intensity, but inhibited if light in-
tensity exceeds a saturation point [25]
causing slow growth and even death
of algae. The research also demon-
strated that the most suitable light in-
tensity for Nitzschia capitellata growth
is 3 000 lx with which growth rate is the
highest and the number of the alga
achieves the peak, laying foundation
for large-scale cultivation of the alga
and obtaining baits or bioenergy.
Nitrogen and phosphorus are
both nutritional factors limiting primary
productivity and N/P ratio has effects
455
Agricultural Science & Technology 2014
Responsible editor: Xiaoxue WANG Responsible proofreader: Xiaoyan WU
on alga growth. The research proved
that the most suitable N/P ratio for
Nitzschia capitellata is 6 ∶1 with light
intensity at 3 000 lx, which would
control alga growth in a rational and
effective way to promote fishing in-
dustry development.
References
[1] KENDRICK, RE, KRONENBERG GHM
(eds), 1994. Photomorphogenesis in
Plants, 2nd ed Dordrecht: Kluwer Aca-
demic, Netherlands.
[2] WANG W (王伟), LIN JM (林均民), JIN
DX (金德祥 ). Photocontrol of Develop-
metn in Algae(藻类的光控发育)[J].Chi-
nese Bulletin of Botany (植物学通报 ),
1998, 15(5): 31-39.
[3] SU JG(苏建国), ZHANG Y(张玥), XU Y
(许英), YANG GS(杨公社). The Optimal
Demands of Diatom in Fresh Water on
Nitrogen and Phosphorus (淡水浮游硅
藻对氮磷的最适需求量 ) [J].Journal of
Northwest A&F University(Natural Sci-
ence Edition)(西北农林科技大学学报
(自然科学版)), 2002, 30(1): 99-102.
[4] ROBERTS RD, CAROLINE L. Effect of
starvation on the growth and survival of
post-larval abalone [J]. Aquaculuture.
2001.200: 323-338.
[5] ROBERTS RD, CAROLINE L. Effect of
delayed metamorphosis on larval com-
petence, the post-larval survival and
growth, in the abalone Haliotis iris Gem-
lin[J]. J.Exp.Mar.Biol.Ecol., 2001. 258:
1-13.
[6] XU F(徐芳), CAI ZL(蔡昭铃), CONG W
(丛威) et al. Research Progress of Cul-
tivating and Developing PUFA from Mi-
cro-algae(微藻培养生产多不饱和脂肪酸
的研究进展 )[J] . Journal of Chongqing
Institute of Technology: Natural Sci-
ence Edition(重庆工学院学报: 自然科学
版), 2005, 19(1): 96-100.
[7] HUANG HZ(黄鸿洲), Tang YY(康燕玉),
LIANG JR (梁君荣 ) et al. Analysis of
Fatty Acid Composition in Five Strains
of Diatoms Used as the Food of A-
balone (五种底栖硅藻(鲍鱼饵料)的脂肪
酸组成分析)[J], Plant Physiology Com-
munications (植物生理学通讯 ), 2007,
43（2）: 349-354.
[8] ZHANG YJ(张英俊), YAN RM(颜日明),
ZHANG ZB (张志斌 ) et al. Effects of
Different Factors on Growth of Nitzschia
Palea(不同环境因子对谷皮菱形藻生长
的影响 ) [J]. Food Science 食品科学 ,
2007, 28（10）: 369-372.
[9] Effect of Cd (Ⅱ)and Zn (Ⅱ) on growth
and biochemical composition of alga
Nitzschia closterium(Cd(Ⅱ)、Zn(Ⅱ)对新
月菱形藻生长及生化成分的影响 ) [J],
Journal Of Dalian Fisheries University
(大连水产学院学报), 2010, 25(1): 178-
182.
[10] YANG RJ(杨茹君), TANG HJ(唐洪杰),
WANG XL (王修林 ). Size Effect on
Nitzschia closterium When Exposed to
Nutrients of Different Concentrations
(不同营养盐浓度下新月菱形藻生长的
粒径效应)[J], Periodical Of Ocean U-
niversity Of China(中国海洋大学学报),
2009, 39(1): 119-124.
[11] Effects of CO2 Enrichment on Growth
and Photosynthesis of Nitzschiaceae
closterium (补充 CO2对光生物反应器
培养新月菱形藻的影响)[J], Journal Of
Hydroecology（水生态学杂志), 2008, 1
(2): 35-38.
[12] YAN N (晏妮), XU XH (徐兴华), NING
AL(宁爱丽), PAN J(潘静), CHEN C(陈
椽 ).Content and component analysis
of lipid in three freshwater diatoms[J].
贵州农业科学, 2011, 39(7): 239-242.
[13] HU Q, ZHANG CW, SOMMERFELD
M. Biodiesel from Algae: Lessons
Learned Over the Past 60 Years and
Future Perpspectives. Juneau, Alaska:
Annual Meeting of the Phycological
Society of America [C]. 2006, July7-
12, pp.40-41(Abstract).
[14] LINXZ(林学政),LIGY(李光友). Biomass,
Total Liqids and Fatty Acid EPA/DHA
Composition of 11 Species of Microal-
gae(11种微藻脂类和 EPA ／DHA组成的
研究)[J]. Journal of Oceanography of
Huanghai & Bohai Seas (黄渤海海洋),
2000, 18(2): 36-40.
[15] LIN FH(林繁会). Research of Purifica-
tion and Cultivation of Unicellular Di-
atom (单细胞硅藻纯化培养实验研究
初报)[J]. Journal Of Mudanjing Normal
University(Natural Sciences Edition(牡
丹江师范学院学报(自然科学版)), 2008
(4).
[16] SHI XL (史小丽). Effect of disturbance
on the movement of exogenous phos-
phorus in simulative aquatic eco-sys-
tems(扰动对外源性磷在模拟水生态系
统中迁移的影响 ) [J]. China Environ-
mental Science(中国环境科学), 2002,
22(6): 537- 541.
[17] ZHANG ZS(章宗涉), HUANG XF(黄祥
飞 ). Limnoplankton Research Method
(淡水浮游生物研究方法 )[M]. Beijing:
Science Press (北京 : 科学出版社 ),
1991.
[18] SHEN YF (沈韫芬), ZHANG ZS (章宗
涉), GONG XJ (龚循矩) et al. New
Monitoring Technology of Microorgan-
ism(微型生物监测新技术)[M]. Beijing:
China Architecture & Building Press(北
京: 中国建筑工业出版社), 1990.
[19] JIN XC (金相灿 ), TU QY (屠清瑛 ),
ZHANG ZS (章 宗 涉 ). Research of
Lake Eutrophication (湖泊富营养化调
查规范 (第二版))[M]. Beijing: Chinese
Research Academy OF Environment
Sciences Press(北京: 中国环境科学出
版社), 1990.
[20] YAN MJ(严美娇), WANG YD(王银东),
HU XJ (胡贤江). Impact of Illumination
on Chlorella vulgaris and Scenedes-
mus Obliquusz Growth Rate and
Chlorophyll Contents(光照对小球藻斜
生栅藻生长速率及叶绿素含量的影响)
[J]. Anhui Agricultural Science Bulletin
(安徽农学通报), 2007, 13( 23): 27-31.
[21] LIU Q (刘青), ZHANG XF (张晓芳), LI
TW (李太武) et al. Effects of Light on
Growth Rate, Chlorophyll Level and
Cell Cycyle(光照对 4 种单胞藻生长速
率、 叶绿素含量及细胞周期的影响)[J],
Journal of Dalian Fishery University(大
连水产学院学报), 2006, 21(1): 24-30.
[22] SU JG(苏建国), ZHANG WY(张王月),
XU Y (许英). The Optimal Nitrogen-to-
Phosphorus Proportion in Cultivation
of Planktonic Diatom(培养淡水浮游硅
藻所需的最适氮磷比 ) [J], Journal of
Animal Science and Veterinary
Medicine(畜牧兽医杂志), 2002, 21(1):
11-13.
[23 ]SHEN YJ(沈英嘉), CHEN DH(陈德辉).
Effects of Light Cycles on the Cell
Proliferations of Microcystis aerugi-
nosa and Green Microalgae(不同光照
周期对铜绿微囊藻和绿色微藻生长的
影响 ) [J]. Lake Science (湖泊科学 ),
2004(3): 21-25.
[24] SUN RY(孙儒泳), JI B(季博), ZHU GY
(诸葛阳) et al. Ecology (普通生态学 )
[M]. Beijing Higher Education Press
(北京: 高等教育出版社), 1993.
[25] LI WQ(李文权), HUANG XM(黄贤芒).
Determination of Niobium in Poly-
metallic Nodules by 5-Br-PADAP
Spectrophotometry(4 种海洋单胞藻生
化组成的环境因子效应研究 )[J]. Acta
Oceanologica Sinica(海洋学报), 1999,
21(3): 59-65.
456