J Appl Phycol (2009) 21:127–133
DOI 10.1007/s10811-008-9341-5
Growth characteristics of the cyanobacterium Nostoc
flagelliforme in photoautotrophic, mixotrophic
and heterotrophic cultivation
Haifeng Yu & Shiru Jia & Yujie Dai
Received: 2 September 2007 / Revised and Accepted: 7 April 2008 / Published online: 23 May 2008
# Springer Science + Business Media B.V. 2008
Abstract Nostoc flagelliforme is a terrestrial cyanobacteri-
um with high economic value. Dissociated cells separated
from a natural colony of N. flagelliforme were cultivated for
7 days under either phototrophic, mixotrophic or heterotro-
phic culture conditions. The highest biomass, 1.67 g L−1
cell concentration, was obtained under mixotrophic culture,
representing 4.98 and 2.28 times the biomass obtained in
phototrophic and heterotrophic cultures, respectively. The
biomass in mixotrophic culture was not the sum as that in
photoautotrophic and heterotrophic cultures. During the
first 4 days of culture, the cell concentration in mixotrophic
culture was lower than the sum of those in photoautotrophic
and heterotrophic cultures. However, from the 5th day, the
cell concentration in mixotrophic culture surpassed the sum
of those obtained from the other two trophic modes.
Although the inhibitor of photosynthetic electron transport
DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] effi-
ciently inhibited autotrophic growth of N. flagelliforme
cells, under mixotrophic culture they could grow by using
glucose. The addition of glucose changed the response of
N.flagelliforme cells to light. The maximal photosynthetic
rate, dark respiration rate and light compensation point in
mixotrophic culture were higher than those in photoauto-
trophic cultures. These results suggest that photoautotrophic
H. Yu : S. Jia (*) : Y. Dai
Tianjin Key Lab of Industrial Microbiology,
Tianjin University of Science and Technology,
Tianjin 300222, China
e-mail: jiashiru@tust.edu.cn
H. Yu
College of Food and Bioengineering,
Shandong Institute of Light Industry,
Shandong Jinan 250353, China
(photosynthesis) and heterotrophic (oxidative metabolism
of glucose) growth interact in mixotrophic growth of N.
flagelliforme cells.
Keywords Nostoc flagelliforme . Growth characteristics .
Mixotrophic culture . Heterotrophic culture .
Photosynthetic characteristics
Introduction
Nostoc flagelliforme is a terrestrial filamentous cyanobac-
terium that is distributed in some arid and semi-arid areas of
China. It has been used as a kind of food for more than
2,000 years, and its herbal value was recognized more than
400 years ago (Gao 1998). It is named ‘Facai’ (hair-like
vegetable) in China because of its hair-like appearance. In
addition, the pronunciation of ‘Facai’ sounds like another
Chinese word that means to be fortunate and get rich.
Therefore, it is favored by Chinese people, especially in
Guangdong Province and overseas. Hot water extracts of
N. flagelliforme have been reported to have anti-tumor
activity. Moreover, a novel acidic polysaccharide, nosto-
flan, isolated from N. flagelliforme was verified to possess a
remarkable anti-viral effect on a variety of enveloped
viruses, including HSV-1, human cytomegalovirus, and
influenza A virus (Kanekiyo et al. 2005; Jia et al. 2007). N.
flagelliforme grows very slowly in the natural environment
(Dai 1992). Its annual growth rate is less than 6%.
Increased market demands have resulted in excessive
exploitation of N. flagelliforme, which, in turn, has caused
the endangered status of this species and deterioration of
the environment (Gao 1998). Therefore, artificial culture of
N. flagelliforme is needed to meet market demands and to
conserve this endangered species.
128
Some methods of cultivating natural colonial filaments
have been reported both in the laboratory and in the field
(Gao and Yu 2000; Gao and Ye 2003; Su et al. 2005).
Cultivation of free cells isolated from colonial filaments in
liquid medium has also been performed (Su 2006; Liu and
Chen 2003; Li and Hu 2003; Bi and Hu 2004). However,
all these studies have focused on the photoautotrophic
culture of N. flagelliforme. This mode of cultivation has a
number of drawbacks, including low cell concentration and
long cultivation periods. This method cannot be justified
for commercial production of high value-added products
since the cost of product recovery and purification can be
notably high due to low cell densities. Recently, significant
attention has been drawn to several alternative techniques
for mass cultivation of algae, including heterotrophic and
mixotrophic processes (Chen and Zhang 1997). Much work
has been done on heterotrophic and mixotrophic growth of
the green algae Chlorella (Shi et al. 2000, 2006; Xu et al.
2006) and Haematococcus (Kang et al. 2005; Kaewpintong
et al. 2007). These algae can use different organic carbon
sources such as glucose and acetate. Although it has been
reported that some strains of cyanobacteria could grow
heterotrophically and mixotrophically (Stal and Moezelaar
1997), studies on cyanobacteria are rather limited and there
is little information about the utilization of organic carbon
sources for their cultivation.
Some species of cyanobacteria are reported to grow
rapidly and to have a higher cell density under heterotrophic
or mixotrophic conditions than under photoautotrophic
conditions, such as Spirulina (Marquez et al. 1993; Vonshak
et al. 2000; Chen et al. 2006), Anabaena (Yu et al. 2000),
and Synechocystis (Wang et al. 2002; Katarzyna and
Andrzej 2004). The requirement for light is lowered or
eliminated in these modes of growth. In mixotrophic
growth both the photoassimilation of CO2 and oxidative
assimilation of organic carbon sources proceed concomi-
tantly. Therefore, it offers the possibility of greatly increas-
ing the microalgal cell concentration, and hence the
volumetric productivity, in batch systems. This property
may be further improved by using high cell density
techniques, such as fed-batch culture, which have been used
to produce cell densities above 10.24 g L−1 in Spirulina
platensis systems (Chen 1996; Chen and Zhang 1997;
Vonshak et al. 2000). These techniques may be applied to
the production of high economic value, low-volume output
biologics (e.g., pharmaceutical proteins, polysaccharides) in
order to reduce the cost of downstream processing.
Unfortunately, there have been no reports on the mixotro-
phic and heterotrophic culture of N. flagelliforme.
In this article, the potential of mixotrophic and hetero-
trophic cultivation of N. flagelliforme to yield high cell
density was investigated, and growth of N. flagelliforme
with or without addition of glucose was analyzed under
J Appl Phycol (2009) 21:127–133
light or dark conditions. In addition, the photosynthetic
activities of N. flagelliforme cultivated in photoautotrophic
and mixotrophic conditions were also compared.
Materials and methods
The initial cells of N. flagelliforme were isolated from a
field colony that was collected on the eastern side of the
Helan Mountain in Yinchuan, Ningxia, China. Dissociated
(isolated) cells were obtained according to previously
reported methods (Jia et al. 2005; Su et al. 2008). Axenic
cells were screened and cultured in BG-110 (free of
nitrogen) medium.
BG-11 medium without glucose was used for photoau-
totrophic culture, and BG-11 medium with glucose at an
initial concentration 2.53 g L−1 was used for mixotrophic
and heterotrophic cultures. Cells that had been cultivated in
BG-11 medium in a 500 mL shake-flask for 10 days were
used as inoculum (prior to inoculation, the strains were
examined microscopically to make sure that the separate
filament with capsule was the dominant form). The
inoculum was then added to 10% (v/v) (the corresponding
dry mass was 0.075 g L−1) into cultures for autotrophic or
mixotrophic conditions, and grown under conditions of
continuous illumination (10–60 μmol photon m−2 s−1, fluo-
rescent light), whereas for heterotrophic culture the flasks
were wrapped with aluminum foil and incubated in the
dark. Cultivations were conducted at 25°C and the initial
pH was 8.0 with shaking of 140 rpm for 7 days. Each
experiment was conducted with three replicates.
3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was
dissolved in dimethylsulfoxide (DMSO) to form a 50 μM
solution, which was added to the culture as required.
Cell concentration was determined by weighing the dry
cells after filtration through a cellulose nitrate filter
(0.45 μm) and drying at 105°C overnight (the filter was
pre-dried and weighed). Specific growth rate (μ, day−1) was
determined from the linear region of the slope of the growth
curve. μ ¼ ðln x2
ln x1 Þ=ðt2
t1 Þ, where x2 is the cell
concentration at experimental time t2, and x1 the cell
concentration at time t1.
Glucose concentration was determined by the phenol-
sulfuric acid method (Bai et al. 2004).
The rates of photosynthesis and respiration were assayed
by measuring the rate of O2 evolution using an Oxy-lab O2
electrode (Hansatech, Cambridge, UK) in a double jacket
thermoregulated glass vessel. The temperature was kept
constant (25°C), and illumination was provided by a red
slide projector lamp at a photon flux density (PFD) of 0–
500 μmol photon m−2 s−1. Cells in the logarithmic phase of
growth were harvested by centrifugation and resuspended
in fresh BG-11 medium with or without glucose to a final
J Appl Phycol (2009) 21:127–133 129

culture, XM cell
Table 1 Quantitative analysis of Nostoc flagelliforme cells growth in photoautotrophic, mixotrophic
concentration
and heterotrophic
of mixotrophic culture
culture,
conditions.
GM glucose
Data
are expressed as mean
photoautotrophic, mixotrophic
± SEM (n=3). XA Cell concentration
and heterotrophic of photoautotrophic
culture conditions. culture,corresponds
concentration XH cell concentration of heterotrophic
to mixotrophic culture,
cultivation time Xt,M cell
GH
concentration
Data of mixotrophic
are expressed culture,
as mean ± SEM GM glucose
(n=3). XA Cell concentration
concentration of
corresponds
glucosetoconcentration
mixotrophic corresponds
cultivation to
time
heterotrophic
t, GH glucose
cultivation
concentration
time t
photoautotrophic
corresponds to heterotrophic H cell concentration
culture, Xcultivation time t of heterotrophic

Time (days) XA (g L−1) XH (g L−1) XM (g L−1) (XA+XH) /XM GM GH

(g L−1) (g L−1)

1 0.082±0.02 0.079±0.02 0.103±0.05 1.56±0.05 2.52±0.05 2.52±0.05

2 0.143±0.01 0.082±0.01 0.145±0.01 1.55±0.01 2.42±0.06 2.43±0.05
3 0.213±0.05 0.172±0.03 0.304±0.05 1.27±0.05 2.35±0.05 2.20±0.05
4 2.262±0.02 0.374±0.02 0.547±0.02 1.16±0.02 2.06±0.04 1.60±0.05
5 0.288±0.04 0.532±0.03 0.952±0.04 0.86±0.03 1.36±0.03 0.42±0.03
6 0.303±0.02 0.744±0.03 1.524±0.05 0.69±0.05 0.55±0.05 0±0.01
7 0.335±0.06 0.731±0.05 1.670±0.06 0.64±0.06 0±0.01 0±0.01

concentration of 3.8–4.0 mg Chla L−1. The whole process
was controlled by the Oxy-lab procedure, including data
recording and drawing of photosynthetic light-response (P–I)
curves. The net maximum photosynthetic rate (Pm), the net
respiration rate (Rd) and the initial slope (α) at limiting photo
flux densities were determined by fitting a three parameter
mode P ¼ Pm  tanhðα  I=PmÞ þ Rd (Henley 1993),
where P is the net photosynthesis rate and I is the light
level. Chlorophyll-a in the cell was extracted and determined
according to the method reported by Hall and Rao (1994).
Data were analyzed by one-way analysis of variance
(ANOVA), and the significance of the differences (P<0.05)
was estimated by Tukey’s multiple comparison test.
Results
Growth of N. flagelliforme cells in different trophic
conditions
Cell growth under photoautotrophic (without addition of
glucose in the light), mixotrophic (with glucose in the
light), and heterotrophic (with glucose in the dark) cultures
are shown in Table 1. The cells in photoautotrophic culture
showed a linear growth. After 7 days of cultivation, the cell
concentration reached only 0.335 g L−1. The heterotrophic
From the 3rd day, cells in mixotrophic and heterotrophic
cultures exhibited exponential growth and during this
period the glucose concentration decreased rapidly.
In contrast to other reported cyanobacteria (Marquez et al.
1993), the cell concentration in mixotrophic culture was not
equal to the sum of those in photoautotrophic and
heterotrophic cultures. Cell growth rate in the first 4 days
of cultivation in mixotrophic cultures was lower than the
sum of those in photoautotrophic and heterotrophic cul-
tures, but thereafter it was higher than the sum of those in
photoautotrophic and heterotrophic cultures.
The optimal light intensity for phototrophic growth of N.
flagelliforme cells in shake flask cultivation was 40 μmol
photon m−2 s−1(Fig. 1). The effect of light intensity on
mixotrophic growth of N. flagelliforme cells is shown in
Fig. 2. The saturation light intensity for mixotrophic growth of
N. flagelliforme cells was also 40 μmol photon m−2 s−1, i.e.,
the same as that for phototrophic culture. However, when
light intensities were increased from 10 μmol photon m−2 s−1
to 40 μmol photon m−2 s−1, the specific growth rate of
phototrophic cells increased from 0.095 day−1 to 0.12 day−1
1
0.9
0.8
0.7
Dry mass/ g·L-1

growth of N. flagelliforme cells occurred in the dark, with

0.6
glucose as the sole carbon and energy source. Concomitant
with the decrease in glucose, N. flagelliforme cells grew 0.5
quickly and the cell concentration reached 0.744 g L−1 on 0.4
the 7th day, which was 2.22 times that obtained from 0.3
phototrophic culture. As expected, mixotrophic cultures
0.2
grew fastest, and the highest biomass concentration was
achieved. At harvest time, biomass was 1.67 g L−1, which 0.1

was 4.98 and 2.28 times the values obtained in photo- 0

0 4 8 12 16 20
trophic and heterotrophic cultures, respectively. During the
Culture time/d
whole time-course of growth, mixotrophic culture had the Fig. 1 Effect of light intensity (μmol photon m−2 s−1; ● 10, ■ 20, ▲
highest cell concentrations at any given time. In the first 40, □ 60) on growth of Nostoc flagelliforme cells in autotrophic
2 days of the lag phase, a little glucose was consumed. modes. Temperature: 25°C. Error bars SEM (n=3)
130 J Appl Phycol (2009) 21:127–133

2 2
1.8
1.6
1.5
1.4

Dry mass/ g·L-1

Dry mass/ g·L-1

1.2
1
1
0.8
0.6
0.4 0.5

0.2

0
0 1 2 3 4 5
Culture time/d
Fig. 2 Effect of light intensity (μmol photon m−2 s−1;
0, ● 10, ■ 20,
▲ 40, □ 60) on growth of N. flagelliforme cells in mixotrophic modes.
Temperature: 25°C. Error bars SEM (n=3)
accordingly, while the specific growth rate of mixotrophic
cells increased from 0.468 day−1to 0.524 day−1. (Table 2,
P<0.05). Further increase in light intensity did not have
evident effect on the specific growth rate. As evidenced by
the increased specific growth rate, photoautotrophic growth
of N. flagelliforme cells was more sensitive to light. It can be
seen from Table 2 that the ratio of the specific growth rate of
mixotrophic cells to that of photoautotrophic cells (Rmp)
decreased with the light intensity increase under low light
intensity, which indicated that the photosynthesis rate
intensified with light intensity.
6 7 0
0 2 4 6 8
△
Culture time/d
Fig. 3 Effects of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)
supplementation on growth of N. flagelliforme cells under mixotro-
phic and autotrophic cultures. ▲ Autophototrophic culture without
DCMU, △
autophototrophic culture with 50 μM DCMU, □
mixotrophic culture with 50 μM DCMU, ■ mixotrophic culture
without DCMU
cells in autophototrophic culture was inhibited by the
presence of DCMU, whereas the mixtrophic cells were
able to grow because of the presence of glucose. However,
the growth rate was slower than that of mixtrophic cells in
the absence of DCMU. Glucose consumption was also
lower in the presence of DCMU (Fig. 4). This result
indicated that glucose oxidation and photosynthesis interact
in mixotrophic growth of N. flagelliforme cells.

Effect of DCMU Photosynthesis and respiration

DCMU, a photosynthetic inhibitor, affects the reduction Photosynthesis and oxidative glucose metabolism existed
side of photosystem II and inhibits photosynthesis by simultaneously in N. flagelliforme cells grown under
interdicting electron transport on the thylakoid membrane. mixotrophic conditions, and indeed the two processes may
The mechanism involves DCMU competing with QB for the
binding site in photosystem II, inhibiting the reduction of 4
QB, thus interdicting electron transport (Rippka 1972). In 3.5
our previous research (Yu 2007), the real photosynthetic
Glucose concentration/g·L-1

rate of N. flagelliforme cells in phototrophic culture 3

decreased to zero in the presence of 50 μM DCMU. A
2.5
comparison of growth of N. flagelliforme cells in the
presence of DCMU in phototrophic and mixotrophic 2
cultures is shown in Fig. 3. The growth of N. flagelliforme
1.5
Table 2 Specific growth rate of N. flagelliforme cells in different
1
trophic modes

Light intensity (μmol 10 20 40 60 0.5

photon m−2 s−1)
0
−1
0 2 4 6 8
Photoautotrophic (day ) 0.095 0.105 0.120 0.138
Mixotrophic (day−1) 0.468 0.466 0.524 0.539 Culture Time/d
Mixotrophic/ photoautotrophic 4.93 4.44 4.37 3.91 Fig. 4 Time-course of glucose consumption of N. flagelliforme cells
grown in mixotrophic cultures with (▲) or without (■) DCMU
J Appl Phycol (2009) 21:127–133 131

300
in the light saturation point, the light compensation point was
significantly higher in the mixotrophical culture than in the
O2 evolution rate /µmolO2·mgChl-1·h-1

200 photoautotrophic culture (Table 3). The light compensation

point for mixotrophic culture was 50±3.2 μmol m−2 s−1;
100 however, it was only 16±4.5 μmol m−2 s−1 for autotrophic
culture.
0

-100
-200
-300
0 50 100 150 200
Light intensity /µmolphoton·m-2·s-1
Fig. 5 Light response curves of mixotrophic (○ photosynthesis net
rate, △
respiration rate) and photoautotrophic (● photosynthesis net
rate, ▲ respiration rate) cultures of N. flagelliforme cells. Tempera-
ture: 25°C, pH 8.5
affect each other. Comparison of oxygen evolution as a
function of light intensities (P–I curve) in photoautotrophic
and mixotrophic cultures revealed significant differences in
P–I curves (Fig. 5). Some of the photosynthesis parameters
obtained from P–I curves are summarized in Table 3. At a
light intensity of 80 μmol photon m−2 s−1, the photosynthetic
rate of mixotrophic cells was equal to that of autotrophic
cells. At light intensities lower than 80 μmol photon m−2 s−1,
the maximum photosynthetic rate of mixotrophic cells was
lower than that of autotrophic growth cells. However, the
photosynthetic rate of mixotrophic cells was higher than that
of autotrophic cells when the light intensity exceeded
80 μmol photon m−2 s−1. At light saturation point, N.
flagelliforme cells had a comparatively high photosynthetic
rate, with maximum photosynthetic rates of 204.33 μmol O2
(mg Chla h)−1and 162.83 μmol O2 (mg Chla h)−1 for
mixotrophic and autotrophic cultures, respectively. The dark
respiration rate of mixotrophic culture also increased with
light intensity. These results indicated that addition of
glucose not only promotes photosynthesis, but also provides
energy for the growth of N. flagelliforme cells. In addition,
glucose has some effects on the light response of N.
flagelliforme cells. Although there was no obvious change
Discussion
Mixotrophic culture, which usually gives a high biomass, is
very important in high density cultivation of microalgae
(Chen 1996). Cultures with high cell concentration have
several advantages in industrial applications, e.g., they
250 occupy less space and reduce the expense of extraction and
other downstream operations. The prominent feature of
mixotrophic cultures is the presence of two energy sources:
organic carbon sources and light. The former is controlled
by the concentration of organic carbon source, and the latter
is influenced by light intensity. Our investigation found that
N. flagelliforme cells can grow in phototrophic, mixotro-
phic and heterotrophic cultures. A biomass of up to
1.67 g L−1 was achieved with 7 days of mixotrophic
culture, i.e., 4.48 times that obtained from homochronous
phototrophic culture. The cultivation cycle of mixotrophic
culture is 7 days, which was nearly one-third that of the
phototrophic culture reported by Su (2006). For some kinds
of algal strains, photosynthesis and oxidation of organic
substrates proceed independently in an additive manner
under mixotrophic condition so that the growth rate of cells
in mixotrophic condition is equal to the sum of those in
photoautotrophic and heterotrophic cultures. This has been
reported for several strains, such as Chlorella vulgaris
(Ogawa and Aiba 1981; Martinez and Orus 1991), Spirulina
platensis (Marquez et al. 1993) and Haematococcus pluvialis
(Kobayashi et al. 1992). However, Orus et al. (1991)
reported that the mixotrophic growth rate of Chlorella
vulgaris UAM 101 surpassed the sum of photoautotrophic
and heterotrophic growth rates. The results of our experi-
ments indicated that the growth characteristics of N.
flagelliforme cells in mixotrophic culture are different from
those of previously reported algal strains. The cell concen-
tration in mixotrophic culture was not simply the combina-
tion of autotrophic and heterotrophic cell concentrations as

Table 33 Photosynthetic
Photosyntheticparameters
parametersof of N. N.
flagelliforme
flagelliforme
cellscells α initial
in mixotrophic
in and phototrophic
slope [μmol O cultures
2 (mg Chla h)−1time
(culture (μmol
2 days,
photon m−2100
25°C, s−1)mol
−1
],
−2 −1 −1 −2−1 −1
photon m s and
mixotrophic ). Pphototrophic
m Maximum net photosynthesis
cultures rate [μmol
(culture time 2 days,O225°C,
(mgChla h)Ik saturation
], Rd respiration
irradiance
rate [μmol (mgChlamh) s], α),initial
(μmolO2photon Ic compensation
slope [μmol
O2 (mg
100 μmol
Chla h)−1 (μmol
photon m−2 sm −1−2 −1 −1
). sPm) Maximum
], Ik saturation
net photosynthesis
irradiance (μmolratem−2 s−1 ), Ic compensation
irradiance m−2 (μmol
irradiance
(μmol photon s−1). These
m−2 s−1parameters
). These parameters
were averaged
were
−1 −1
[μmol
averaged (mg Chla
O2 from threeh)replicate
], Rd respiration
measurementsrate [μmol O2 (mg Chla h) ], from three replicate measurements

Trophic modes Pm Rd α Ik Ic

Autotrophy 162.83±3.6 −42.26±2.9 1.91±0.06 90±5.9 16±4.5

Mixotrophy 204.33±4.2 −110.08±1.2 2.12±0.63 100±6.7 50±3.2
132
postulated by Marquez et al. (1993). During the first 4 days,
the cell concentration in mixotrophic culture was lower than
the sum of those in photoautotrophic culture and heterotro-
phic culture. However, from the 5th day, the cell concentra-
tion in mixotrophic culture surpassed the sum of those
obtained from other two trophic modes. The reason for this
phenomenon requires further investigation.
To date, there are few reports on the mechanisms by
which cyanobacteria use organic carbon sources. Rippka
(1972) found that cell-free extracts of a Nostoc strain in
heterotrophic growth contain the key enzyme of the
oxidative pentose phosphate cycle for the catabolism of
carbohydrates. We speculate that N. flagelliforme might use
glucose through the phosphopentose pathway, but we are
not sure.
There is a common link between the respiratory and
photosynthetic electrons transport chains in cyanobacteria.
Electrons from the respiratory matrix or photosynthetic
system influence the oxidation–reduction state of the
cytochrome b6/f complex and plastoquinone, which are
the common link components. When the algal cell grows in
different environments, electrons in the common link can
flow to respiratory links as well as to photosystem I. Upon
supplementation of DCMU, autophototrophic growth was
inhibited completely; however, N. flagelliforme cells in
mixotrophic culture could grow by using glucose, although
the growth slowed down. The decrease in the glucose
consumption rate indicated that glucose utilization was
influenced by photosynthetic electron transport. At light
saturation point, N. flagelliforme cells in mixotrophic
culture had a comparatively high photosynthetic rate, which
supports the notion that electrons from glucose metabolism
can enter photosystem I to improve the energy levels of N.
flagelliforme cells. The energy for photosynthesis is
subsequently intensified and the anabolism of N. flagelli-
forme cells can be promoted. These conclusions are in
agreement with results obtained with Synechocystis 6803
under mixotrophic culture (Wang et al. 2000).
According to previous reports, utilization of an external
organic carbon source may affect autophototrophic growth
processes from many aspects, such as photosynthesis and
respiration, though the effects are different for different
algal strains. The addition of glucose made the photosyn-
thetic rate of Chlorella sp. VJ79 somewhat slow and made
the dark respiration rate somewhat fast (Lalucat et al.
1984). However, for Chlorella vulgaris UAM 101 (Orus et al.
1991), both light and dark respiration rates were enhanced by
the addition of glucose, though the net photosynthetic rate
was not influenced. The study of Spirulina platensis showed
that both the photosynthetic and respiration rate were
unchanged upon addition of glucose (Marquez et al. 1993).
The data from our work showed that the addition of glucose
influenced photosynthesis in N. flagelliforme cells. Although
J Appl Phycol (2009) 21:127–133
there was no obvious change in the light saturation point, the
light compensation point was significantly increased. At the
light saturation point, the maximum photosynthetic rate and
dark respiration rate of mixotrophic cells were both increased
significantly. These results suggest that the increased growth
rate of N. flagelliforme cells is due to the synergetic effect of
photosynthesis and glucose oxidation.
Acknowledgements This work was supported by the National
Natural Science Foundation of China (No. 20376061) and by the
Science and Technology Commission of Tianjin municipality (No.
043801611).
References
Bai XJ, Su JY, Zhao SX, Jia SR (2004) Study on the determination
methods of extracellular polysaccharide in culture medium of
Nostoc Flagelliforme cells (in Chinese with English abstract). Sci
Tech Food Ind 25:146–148
Bi YH, Hu ZY (2004) Influence of temperature, nutrients and light
intensity on the growth of Nostoc flagelliforme (in Chinese with
English abstract). Chin J Pro Eng 4:245–249
Chen F (1996) High cell density culture of microalgae in heterotrophic
growth. Trends Biotechnol 14:421–426
Chen F, Zhang YM (1997) High cell density mixotrophic culture of
Spirulina platensis on glucose for phycocyanin production using
a fed-batch system. Enzyme Microb Technol 20:221–224
Chen TF, Zheng WJ, Fang Y, Bai Y, Wang YS (2006) Mixotrophic
culture of high selenium-enriched Spirulina platensis on acetate
and the enhanced production of photosynthetic pigments.
Enzyme Microb Technol 39:103–107
Dai ZJ (1992) Review of Nostoc flagelliforme research (in Chinese
with English abstract). J Ningxia Univ (Nat Sci) 13:71–76
Gao KS (1998) Chinese studies on edible blue-green alga, Nostoc
flagelliforme: a review. J Appl Phycol 10:37–49
Gao KS, Ye CP (2003) Culture of the terrestrial cyanobacterium,
Nostoc flagelliforme (Cyanophyceae), under aquatic conditions. J
Phycol 39:617–623
Gao KS, Yu AJ (2000) Influence of CO2, light and watering on
growth of Nostoc flagelliforme mats. J Appl Phycol 12:185–189
Hall DO, Rao KK (1994) Photosynthesis, 5th edn. The Press
Syndicate of Cambridge, Cambridge, p 43
Henley WJ (1993) Measurement and interpretation of photosynthetic
light-response curves in alga in the context of photoinhibition
and diel changes. J Phycol 29:729–729
Jia SR, Su JY, Qiao CS (2005) Nostoc flagelliforme cells cultivation
and its products. Chinese patent ZL 03119101.0
Jia SR, Yu HF, Lin YX, Dai YJ (2007) Characterization of
extracellular polysaccharides from Nostoc flagelliforme cells in
liquid suspension culture. Biotechnol Bioprocess Eng 12:
271–275
Kaewpintong K, Shotipruk A, Powtongsook S, Pavasant P (2007)
Photoautotrophic high-density cultivation of vegetative cells of
Haematococcus pluvialis in airlift bioreactor. Bioresour Technol
98:288–295
Kang CD, Lee JS, Park TH, Sim SJ (2005) Comparison of
heterotrophic and photoautotrophic induction on astaxanthin
production by Haematococcus pluvialis. Appl Microbiol Bio-
technol 68:237–241
Kanekiyo K, Le JB, Hayashi K, Takenaka H, Hayakawa Y, Endo S,
Hayashi T (2005) Isolation of an antiviral polysaccharide,
J Appl Phycol (2009) 21:127–133
Nostoflan, from a terrestrial cyanobacterium, Nostoc flagelli-
forme. J Nat Prod 68:1037–1041
Katarzyna C, Andrzej N (2004) Evaluation of Spirulina sp. growth in
photoautotrophic, heterotrophic and mixotrophic cultures. En-
zyme Microb Technol 34:461–465
Kobayashi M, Kakizono T, Yamaguchi K, Naomichi N, Nagai S
(1992) Growth and astaxanthin formation of Haematococcus
pluvialis in heterotrophic and mixotrophic conditions. J Ferment
Bioeng 74:17–20
Lalucat J, Imperial J, Pares R (1984) Utilization of light for the
assimilation of organic matter in Chlorella sp. VJ79. Biotechnol
Bioeng 26:677–681
Li YG, Hu ZY (2003) Studies on the cultivation of Nostoc flagelliforme
(in Chinese with English abstract). J Wuh Bot Res 21:411–414
Liu XJ, Chen F (2003) Cell differentiation and colony alteration of an
edible terrestrial cyanobacterium Nostoc flagelliforme, in liquid
suspension cultures. Folia Microbiol 48(5):619–626
Marquez FJ, Sasaki K, Kakizono T, Nishio N, Nagai S (1993) Growth
characteristics of Spirulina platensis in mixotrophic and hetero-
trophic conditions. J Ferment Bioeng 76:408–410
Martinez F, Orus MI (1991) Interactions between glucose and
inorganic carbon metabolism in Chlorella vulgaris strain UAM
101. Plant Physiol 95:1150–1155
Ogawa T, Aiba S (1981) Bioenergetic analysis of mixotrophic growth
in Chlorella vulgaris and Scenedesmus acutus. Biotechnol
Bioeng 23:1121–1132
Orus ML, Marco E, Martinez F (1991) Suitability of Chlorella
vulgaris UAM 101 for heterotrophic biomass production.
Bioresour Technol 38:179–184
Rippka R (1972) Photoheterotrophy and chemoheterotrophy among
unicellular blue-green algae. Arch Microbiol 87:303–322
Shi XM, Zhang XW, Chen F (2000) Heterotrophic production of
biomass and lutein by Chlorella protothecoides on various
nitrogen source. Enzyme Microb Technol 27:312–318
133
Shi XM, Wu ZY, Chen F (2006) Kinetic modeling of lutein
production by heterotrophic Chlorella at various pH and temper-
atures. Mol Nutr Food Res 50:763–768
Stal LJ, Moezelaar R (1997) Fermentation in cyanobacteria. FEMS
Microbiol Rev 21:179–211
Su JY (2006) Study on the photoautotrophic cultivation of Nostoc
flagelliforme cells. Dissertation. Tianjin University of Science
and Technology, Tianjin,China
Su JY, Jia SR, Qiao CS, Jung GK (2005) Culture of Nostoc flagelliforme
on solid medium. Korean J Environ Biol 23:135–140
Su JY, Jia SR, Chen XF, Yu HF (2008) Morphology, cell growth, and
polysaccharide production of Nostoc flagelliforme in liquid
suspension culture at different agitation rates. J Appl Phycol
20:213–217
Vonshak A, Cheung SM, Chen F (2000) Mixotrophic growth modifies
the response of Sprulina (Arthrospira) platensis (cyanobacteria)
cells to light. J Phycol 36:675–679
Wang YH, Ye JY, Mi HL (2000) Relationship between the growth of
Synechocystis sp. PCC6803 on medium with glucose and the
photosynthetic energy transformation. J Integr Plant Biol
42:1122–1125
Wang YH, Li YG, Shi DJ, Shen GM, Ru BG, Zhang SL
(2002) Characteristics of mixotrophic growth of Synechocystis
sp. in an enclosed photobioreactor. Biotechnol Lett 24:1593–
1598
Xu H, Miao XL, Wu QY (2006) High quality biodiesel production
from a microalga Chlorella protothecoides by heterotrophic
growth in fermenters. J Biotechnol 126:499–507
Yu HF (2007) Study on the high cell density culture of Nostoc
flagelliforme cells. Dissertation. Tianjin University of Science
and Technology, Tianjin,China
Yu GC, Xin XF, Cai ZL, Shi DJ, OU YF (2000) Mixotrophic cultures
of Anabaena sp. PCC7120 (in Chinese with English abstract).
Eng Chem Metallurgy 21:53–59