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Effects of salinity and nutrients on the growth and chlorophyll fluorescence of

Caulerpa lentillifera

Article  in  Chinese Journal of Oceanology and Limnology · March 2014

DOI: 10.1007/s00343-015-4105-y

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 Chinese Journal of Oceanology and Limnology
Vol. 33 No. 2, P. 410-418, 2015
http://dx.doi.org/10.1007/s00343-015-4105-y

Effects of salinity and nutrients on the growth and chlorophyll

fluorescence of Caulerpa lentillifera*

GUO Hui (郭辉)1, 2, YAO Jianting (姚建亭)1, SUN Zhongmin (孙忠民)1,

DUAN Delin (段德麟)1, **
1
Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
2
University of Chinese Academy of Sciences, Beijing 100049, China

Received Apr. 29, 2014; accepted in principle Jul. 22, 2014; accepted for publication Aug. 19, 2014
© Chinese Society for Oceanology and Limnology, Science Press, and Springer-Verlag Berlin Heidelberg 2015

Abstract Caulerpa lentillifera is a green algae that distributes worldwide and is cultivated for food.
We assessed vegetative propagation of C. lentillifera by measuring the specific growth rate (SGR) and
chlorophyll fluorescence of the green algae cultured at different salinities and nutrient levels. The results
indicated that C. lentillifera can survive in salinities ranging from 20 to 50, and can develop at salinities
of 30 to 40. The maximum SGR for C. lentillifera occurred at a salinity of 35. Both chlorophyll content
and the ratio of variable to maximum fluorescence (Fv/Fm) were also at a maximum at a salinity of 35.
Photosynthesis was inhibited in salinities greater than 45 and less than 25. Both the maximum SGR and
maximum chlorophyll content were found in algae treated with a concentration of 0.5 mmol/L of NO3-N
and 0.1 mmol/L of PO4-P. The photosynthetic capacity of photosystem II (PSII) was inhibited in cultures
of C. lentillifera at high nutrient levels. This occurred when NO3-N concentrations were greater than
1.0 mmol/L and when PO4-P concentrations were at 0.4 mmol/L. As there is strong need for large-scale
cultivation of C. lentillifera, these data contribute important information to ensure optimal results.

Keyword: Caulerpa lentillifera; salinity; nutrient level; specific growth rate (SGR); ratio of variable to
maximum fluorescence (Fv/Fm); non-photochemical quenching (NPQ)

1 INTRODUCTION Island (Shi, 2008), little is known about how salinity

and nutrient levels affect growth of C. lentillifera. It is
The siphonous green macroalgae Caulerpa spp. crucial to clarify the effects of these parameters before
distribute across a wide range of tropical and implementing large-scale cultivation.
subtropical seas throughout the world. Caulerpa Salinity is one of the most important abiotic
lentillifera J. Agardh was originally described along environmental factors to influence algal growth and
the Red Sea coast (Agardh, 1837) and was reported to distribution (Lobban and Harrison, 1994). Many
occur at many locations in the tropical Indo-Pacific studies have documented the effects of salinity on
(Hackett, 1977; Taylor, 1977; Menez and Calumpong, several Caulerpa species, including C. paspaloides
1982; Coppejans and Beeckman, 1989; Phillips et al., (O’Neal and Prince, 1988) and C. taxifolia (Theil et
1999; Schils and Coppejans, 2003; Titlyanov et al., al., 2007; West and West, 2007). Deraxbudsarakom et
2012). Morphologically, with terete stolons bearing al. (2003) suggested that a salinity range of 25–30 is
erect fronds of spherical ramuli and filiform rhizoids, required for normal growth of C. lentillifera. In
C. lentillifera is one of the edible algae known as ‘sea addition, Wang (2011) showed that maximum growth
grapes’. It contains high levels of polyunsaturated of C. lentillifera occurred at a salinity of 36. We
fatty acids (PUFA) (Saito et al., 2010) and multiple
essential amino acids and low levels of lipids (Niwano
et al., 2009). Although this alga has been cultivated
* Supported by the Technology Program of Basic Research of Qingdao
widely in the Philippines (Zemke-White and Ohno, (No. 12-1-4-8-(2)-jch)
1999), Okinawa (Kurashima et al., 2003) and Taiwan ** Corresponding author: [email protected]
No.2 GUO et al.: Salinity and nutrients effects on C. lentillifera 411

gives a measure of the efficiency of photosynthesis,

which can be used as an indicator of the health of the
B
plant. Therefore, measuring the ratio of variable to
maximum fluorescence (Fv/Fm) and non-
photochemical quenching (NPQ) can indicate the
physiological status of algae (Maxwell and Johnson,
S 2000). Fluorescence values become stable when algae
grew under favorable conditions. When plants are
exposed to abiotic or biotic stress, decreases in Fv/Fm
are frequently observed (Baker, 2008). For example,
the ratio of variable to maximum fluorescence (Fv/Fm)
R of salinity-stressed Sargassum thunbergii decreased
Fig.1 Diagram of Caulerpa lentillifera, showing a branch
from 0.62 to 0.55 (Liang et al., 2011). Rattan et al.
(B), the stolon (S) and a rhizoid (R) (2012) showed that a depressed Fv/Fm value can
indicate a nutrient deficiency of algae under natural
conditions. NPQ is the release of energy as heat.
studied the C. lentillifera strain from Okinawa, which
Increased levels of NPQ are usually caused by
is likely to differ from strains from Thailand
stressed photosynthetic apparatus (Baker, 2008).
(Deraxbudsarakom et al., 2003) or Vietnam (Wang,
The objective of this study was to determine the
2011). High salinity may influence the photosynthesis
optimal salinity and nutrient levels for the asexual
of macroalgae by inactivating the reaction centers of
reproduction of Caulerpa lentillifera. Understanding
photosystem II (PSII) and inhibiting electron transport
the optimal conditions for maximum growth is
(Xia et al., 2004).
necessary before implementing large-scale
Nitrogen (N) and phosphorus (P) are two essential
commercial propagation of this species.
nutrients for algal growth (Carpenter and Capone,
1983). Insufficient or excess nutrients may impede
2 MATERIAL AND METHOD
growth and affect chlorophyll fluorescence (Yin et al.,
2007; Liang et al., 2008; Qin et al., 2010). However, 2.1 Algal pre-culture
different algal species have different tolerances to
varying concentrations of nutrients. The growth of Fresh Caulerpa lentillifera were transported from
Gracilaria was inhibited at high (60 μmol/L) and low Kumejima, Okinawa of Japan. The algae was washed
(10 μmol/L) concentrations of NO3-N (Huang et al., with sterile seawater several times, then placed in a
2006). Concentrations of over 50 μmol/L of NO3-N flask in an illuminating incubator (GXZ series)
inhibited the growth of Kappaphycus alvarezii (Liu et (Jiangnan, Ningbo, China) and held at 25–26°C. Light
al., 2008). The growth rate of Ulva pertusa was faster was provided using fluorescent tubes with an
at a low concentration (10 mg/L) of nitrate, compared irradiance of 10–20 μmol photons/(m2∙s) and a photo
with a high concentration (30 mg/L) (Qin et al., 2010). period of 12 h:12 h (light:dark). Light irradiance was
The growth rate of Enteromorpha prolifera reached a measured with a LI-250 light meter (LI-COR, USA).
maximum in high N and P concentrations (N: The algal thalli of Caulerpa lentillifera consists of
0.5 mmol/L, P: 0.03 mol/L) (Li et al., 2010). creeping stolons, erect branches and rhizoids (Fig.1).
Deraxbudsarakom et al. (2003) studied C. lentillifera Green, healthy thalli were selected and the erect
in Thailand and concluded that a NO3-N concentration branches were cut into 5 cm sections with a scalpel.
of 0.6 mmol/L and a N:P ratio of 8:1 were optimal for To test the effects of salinity, algae were exposed to
growth. one of nine saline concentrations (15, 20, 25, 30, 35,
Chlorophyll fluorescence parameters are widely 40, 45, 50, and 55). Three replicate algal samples
applied to assess photosynthetic features in algae were tested at each salinity. Saline solutions were
(Lichtenthaler et al., 2005). Energy absorbed by prepared using sea salt (Haidatongyong, Qingdao,
chlorophyll molecules is distributed in one of three China) and distilled water. Salinities were measured
ways, it can be used in photosynthesis using a salinity meter (ATC, China). Algae were
(photochemistry), or re-emitted as heat or as cultured in flasks containing 100 mL of solution.
chlorophyll fluorescence (Lichtenthaler, 1996). Algae were exposed to the temperature 25.0°C and a
Measuring the proportion of energy that is re-emitted light of 40 μmol photons/(m2∙s).
412 CHIN. J. OCEANOL. LIMNOL., 33(2), 2015 Vol.33

3 (Beckman, USA). The concentration of total

2.5 chlorophyll and chlorophyll a was calculated as
follows:
2 Total chlorophyll (mg/L)=8.02A663+20.21A645
SGR (%/d)

1.5 (Arnon, 1949);

Chlorophyll a (mg/L)=11.85A664–1.54A647–0.08A630
1
(Jeffrey and Humphrey, 1975).
0.5 Mass fraction was calculated using to the following
formula:
0
15 20 25 30 35 40 45 50 55 Mass fraction of pigment (mg/g)=(ρV)/m
Salinity (Ma, 2013),
Fig.2 SGR of Caulerpa lentillifera at different salinities in which ρ=mass concentration, V=total volume
The water temperature was 25.0°C and algae were exposed to a light of (10 mL) and m=sample weight.
40 μmol photons/(m2∙s) (n=3). Error bar shows std. deviation.
2.5 Chlorophyll fluorescence parameters
2.2 Preparation of PO4-P and NO3-N solutions
After seven days’ treatment, all the cultured algal
Sodium nitrate (NaNO3) salt was weighed into samples were held in the dark for 10 min. Chlorophyll
batches of 0.043 g, 0.085 g, 0.425 g, 0.850 g, 1.100 g, fluorescence parameters including the ratio of variable
and 3.400 g and then dissolved in distilled water and to maximum fluorescence (Fv/Fm) and NPQ were
made up to a final volume of 10 mL. Monopotassium measured using a fluorescence image system
phosphate (KH2PO4) salt was weighed into batches of (FC1000-H) (PSI, Czech).
0.014 g, 0.136 g, and 0.544 g and dissolved in distilled
2.6 Statistical analysis
water to a final volume of 10 mL. For each treatment,
300 μL of each NaNO3 and KH2PO4 solution was All data were tested with analysis of variance
added into 300 mL of autoclaved seawater (salinity (ANOVA) using the software package PASW 18.0
31). The final concentrations of NaNO3 solutions (SPSS). Tukey’s multiple range tests were applied to
were 0.05, 0.1, 0.5, 1.0, 2.0, and 4.0 mmol/L and the evaluate differences between significant means (Sokal
final concentrations of KH2PO4 were 0.01, 0.1, and and Rohlf, 1969). Analyses had a critical probability
0.4 mmol/L. level of 0.05.
2.3 Specific growth rate measurements 3 RESULT
At the beginning of culture, the fresh weight of 3.1 Effect of salinity on the growth rate of
algae (W0) was measured using an electronic balance C. lentillifera
(Mettler Toledo, Shanghai, China). Seven days later,
algae were observed and the algal fresh weight (W7) Caulerpa lentillifera did not survive at salinities of
was measured. Specific growth rate (SGR) for each 15 and 55, decaying within three days, and gradually
algal sample was calculated as follows: turning white and becoming soft (Fig.3). There was a
SGR=ln(W7/W0)/t×100, where t is time elapsed. significant difference among the SGR of C. lentillifera
at salinities of 20–50 (ANOVA: F=6.084, P<0.05).
2.4 Extraction and measurements of chlorophyll The maximum SGR of 2.038%±0.465% per day
Fresh branches of Caulerpa lentillifera were occurred at a salinity of 35 (Fig.2).
ground in 80% (V/V) acetone using a mortar and At salinities of 20 and 45, only stolons regenerated
pestle, then transferred to a 2-mL centrifuge tube. The from branches. However, new branches with ramuli
tube was placed in the dark for 5 min, and then grew from stolons at salinities of 30–40. The results
centrifuged at 4 000×g for 15 min. The supernatant suggest that the optimum salinity for growth of
was then transferred into a flask and acetone added to C. lentillifera was 35.
make a total volume of 10 mL. The concentration of 3.2 Effect of salinity on photosynthetic
chlorophyll was measured in the solution at characteristics
wavelengths of 630, 645, 647, 663, and 664 nm using
a UV-visible light spectrophotometer (DU650) There was a significant difference in the chlorophyll
No.2 GUO et al.: Salinity and nutrients effects on C. lentillifera 413

15 20 25

30 35 40

45 50 55

Fig.3 Caulerpa lentillifera cultured in different salinities after 7 days

The numbers represent salinity. Scale bar: 1 cm.

0.35 data were generated for algae cultured at a salinity of

Chlorophyll a 50 as regenerated stolons did not form. Chlorophyll a
0.30
Total chlorophyll values were highest in algae cultured at a salinity of
0.25 35. Total chlorophyll increased in algae samples at
Content (mg/g)

30, reaching a maximum at 35 (Fig.4). This suggests

0.20 that a salinity of 35 is optimal for photosynthesis in C.
0.15
lentillifera.
There were significant differences among the
0.10 values for Fv/Fm (ANOVA: F=69.904, P<0.001) and
NPQ (F=14.349, P<0.001) in Caulerpa lentillifera
0.05
cultured at different salinities (Fig.5). Initial Fv/Fm
0
values at different salinities were around 0.85. After
20 25 30 35 40 45 seven days the Fv/Fm value decreased to 0.623 at a
Salinity salinity of 15, and the algae died or became completely
Fig.4 Chlorophyll content of regenerated stolons at white. At a salinity of 55, the Fv/Fm value decreased to
different salinities 0.710. The Fv/Fm values for algae cultured in salinities
Algae were cultured with a water temperature of 25.0°C and light of 25–45 were above 0.80, indicating that
of 40 μmol photons/(m2∙s) (n=3). Error bar shows std. deviation.
C. lentillifera was healthy (Fig.5a). The NPQ value
was highest (0.56) in algae cultured at a salinity of 15.
a content (F=12.198, P<0.05) and total chlorophyll It was also elevated to 0.12 in algae cultured at a
content (F=80.569, P<0.05) of Caulerpa lentillifera salinity of 55, but relatively low (0.10) in algae
cultured at different salinities (Fig.4). No chlorophyll cultured at salinities of 25–45 (Fig.5b).
414 CHIN. J. OCEANOL. LIMNOL., 33(2), 2015 Vol.33

0.90 a 6
PO4-P: 0.01 mmol/L
0.85 PO4-P: 0.1 mmol/L
5
PO4-P: 0.4 mmol/L
0.80
4
0.75

SGR (%/d)
Fv/Fm

0.70 3
0.65
0 day 2
0.60
7 day
0.55 1

0.50
0
15 20 25 30 35 40 45 50 55
0.02 0.1 0.5 1 2 4
Salinity NO3-N (mmol/L)

0.90 b Fig.6 SGR of the erect branches of C. lentillifera cultured at

a range of nitrogen and phosphorus concentrations
0.80 0 day
Error bar shows std. deviation.
0.70 7 day
3.4 Effect of NO3-N on photosynthetic characters
0.60

There was a general trend for chlorophyll content

Fv/Fm

0.50

0.40 (chlorophyll a and total chlorophyll) to increase with

rising NO3-N concentration at low concentrations of
0.30
PO4-P (0.01 mmol/L). In contrast, there was a trend
0.20
for levels of chlorophyll to gradually decrease at
0.10 increasing concentrations of NO3-N, at a PO4-P
0 concentration of 0.4 mmol/L (Fig.7). The maximum
15 20 25 30 35 40 45 50 55 chlorophyll content (0.150 1±0.037 7 mg/g) occurred
Salinity
in algae cultured at a concentration of 0.1 mmol/L of
Fig.5 a. Ratio of variable to maximum fluorescence (Fv/Fm) PO4-P. At a concentration of 0.1 mmol/L of PO4-P,
of the erect branches of C. lentillifera cultured at a
range of salinities (n=3); b. NPQ of the erect branches chlorophyll content decreased at NO3-N concentrations
of C. lentillifera cultured at a range of salinities (n=3) of 1 mmol/L or greater (Fig.7), suggesting that
Measurements were taken at the beginning of the test period and photosynthesis of new branches of C. lentillifera were
again after seven days. Error bar shows std. deviation. inhibited at high NO3-N concentrations.
There were no significant differences among Fv/Fm
values (P>0.05) at PO4-P concentrations of 0.01 and
3.3 Effect of PO4-P on growth 0.1 mmol/L. At a PO4-P concentration of 0.4 mmol/L
the Fv/Fm value decreased rapidly at NO3-N
There were significant differences in SGR
concentrations above 1 mmol/L (Fig.8a). The NPQ
(F=3.374, P<0.05) among algae cultured in different
value increased at high concentrations of NO3-N,
concentrations of NO3-N, when the PO4-P
when the PO4-P concentration was 0.4 mmol/L
concentration was 0.1 mmol/L. However there were
(Fig.8b). These results indicate that high PO4-P and
no significant differences in SGR among algae
NO3-N concentrations have a negative effect on
cultured in different NO3-N concentrations (ANOVA:
photosynthesis in C. lentillifera.
F=2.244, P>0.05) when the PO4-P concentration was
0.01 mmol/L, or 0.4 mmol/L (F=0.782, P>0.05). This 4 DISCUSSION
suggests that the optimal concentration of PO4-P is
0.1 mmol/L. At a PO4-P concentration of 0.1 mmol/L, Our experiments indicate that growth of Caulerpa
the SGR of C. lentillifera increased with NO3-N lentillifera is optimal at a salinity of 35, and although
concentration, from 0.05 to 0.5 mmol/L, then leveled the algae can survive salinities of 20–50, growth only
out (Fig.6). SGR increased linearly with NO3-N occurs in salinities of 20–45. Chlorophyll content and
concentration in the 0.05–0.5 mmol/L range Fv/Fm were also highest at a salinity of 35, indicating
(R2=0.999 3). This implies that growth rate may be optimum photosynthetic capacity was at salinity 35.
inhibited by high NO3-N concentrations. Our results are not consistent with those of
No.2 GUO et al.: Salinity and nutrients effects on C. lentillifera 415

0.2 0.35
a b
0.18
0.3
0.16

Total chlorophyll (mg/g)

Chlorophyll a (mg/g)

0.14 0.25
0.12 0.2
0.1
0.15
0.08
0.06 0.1
0.04
0.05
0.02
0 0
0.01 0.1 0.4 0.01 0.1 0.4
PO4-P (mmol/L) PO4-P (mmol/L)
NO3-N : 0.05 mmol/L NO3-N : 1 mmol/L
NO3-N : 0.1 mmol/L NO3-N : 2 mmol/L
NO3-N : 0.5 mmol/L NO3-N : 4 mmol/L

Fig.7 Ch a content (a) and total chlorophyll content (b) of regenerated stolons of C. lentillifera cultured at a range of
phosphorus and nitrogen concentrations
Water temperature was maintained at 25.0°C and light was 40 μmol photons/(m2∙s) (n=3). Error bar shows std. deviation.

0.90 a Deraxbudsarakom et al. (2003). These authors

0.85 examined the growth of C. lentillifera in Thailand,
and found that salinities of 25–30 were optimal for
0.80
fast growth. Caulerpa letillifera has been recorded to
0.75 occur in the Red Sea at salinities of 38–40 (Friedman
1968), indicating a tolerance to high salinities. In
Fv/Fm

0.70

0.65 PO4-P: 0.01 mmol/L addition, there are some similarities with
PO4-P: 0.1 mmol/L C. paspaloides (O’Neal and Prince, 1988) and
0.60 PO4-P: 0.4 mmol/L C. taxifolia (Theil et al., 2007; West and West, 2007).
0.55 Kirst et al. (1989) suggested that Caulerpa can resist
0.50 osmotic stress using a giant vacuolar system. Here we
0.05 0.1 0.5 1 2 4 deduced that C. lentillifera may possess a complicated
NO3-N (mmol/L) osmotic mechanism, however, further work is
required to examine the vacuolar system of this
1.20
b
PO4-P: 0.01 mmol/L
species.
1.00 PO4-P: 0.1 mmol/L The ratio of variable to maximum fluorescence
PO4-P: 0.4 mmol/L (Fv/Fm) is a sensitive parameter to indicate the degree
0.80 of stress in plants (Chen and Liu, 2007). Values of less
than the optimal value of 0.83 indicate that the plant
NPQ

0.60
has been exposed to stress (Maxwell and Johnson,
0.40 2000). In our study C. lentillifera with (Fv/Fm) values
of less than 0.80 showed relatively slow growth rates,
0.20 and plants with (Fv/Fm) values of less than 0.70 died.
SGR of C. lentillifera were highest at a PO4-P
0
0.05 0.1 0.5 1 2 4
concentration of 0.1 mmol/L and a NO3-N
NO3-N (mmol/L) concentration of 0.5 mmol/L. Deraxbudsarkom et al.
(2003) suggested that a NO3-N concentration of
Fig.8 Ratio of variable to maximum fluorescence (Fv/Fm)
(a) and NPQ (b)of the erect branches of C. lentillifera
0.6 mmol/L and N:P ratio of 8:1 were optimal for the
cultured in a range of phosphorus and nitrogen growth of C. lentillifera, and that nitrate is the
concentrations (n=3) preferable nitrogen source compared with nitrite.
Water temperature was maintained at 25.0°C and light was 40 μmol Huang (2012) found that the wet weight of
photons/(m2∙s). Error bar shows std. deviation. C. lentillifera was the highest when it was cultured in
416 CHIN. J. OCEANOL. LIMNOL., 33(2), 2015 Vol.33

nitrogen concentrations of 15 mg/kg (ca. During our indoor culture experiment, the culture
0.16 mmol/L). Our results indicated that a N:P ratio duration was limited to seven days, and only one
of 5:1 was optimal for growth, differing slightly from nitrogen source was applied. Further experiments
the 8:1 ratio described by Deraxbudsarkom et al. over a longer duration would help to optimize the
(2003). These differences may be due to different parameters for algal growth at a large-scale.
environmental conditions.
The SGR of C. lentillifera was strongly correlated 5 CONCLUSION
with NO3-N concentrations in the range of 0.05 to
In conclusion, optimal conditions for vegetative
0.5 mmol/L (R2=0.999 3), but growth rate was
reproduction of C. lentillifera occurred at a salinity of
relatively stable at NO3-N concentrations of greater
35, with a concentration of 0.1 mmol/L of PO4-P and
than 0.5 mmol/L. These results suggest that a NO3-N
0.5 mmol/L of NO3-N. Further studies are required to
concentration of 0.5 mmol/L is the upper threshold
understand the optimal conditions to culture this algae
for nutritional assimilation and absorption, due to
outdoors and at a large-scale.
complicated enzyme behaviors for nitrate nitrogen
conversion and the reduction of ammonium nitrogen 6 ACKNOWLEDGEMENT
in algae (Hockin et al., 2012). Chlorophyll is a key
compound for restoring nitrogen (Xu et al., 2006), it Thanks to the anonymous reviewers for their
will be degraded and release nitrogen primarily in critical comments and suggestions.
macroalgae when there is lack of nitrogen supply
(Bjomsater and Wheeler, 1990). This may explain References
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