Proceedings of the Twentieth (2010) International Offshore and Polar Engineering Conference

Beijing, China, June 2025, 2010

Copyright 2010 by The International Society of Offshore and Polar Engineers (ISOPE)
ISBN 978-1-880653-77-7 (Set); ISSN 1098-6189 (Set); www.isope.org

Salinity Acclimation of Chlorella pynenoidosa and Its Application in Mariculture Wastewater Treatment
Haiyan Hu, Yong Liu, Yibing Deng, Weihong Jin
M arine Science College, Zhejiang Ocean University
Zhoushan, Zhejiang, China

Chlorella pynenoidosa has been regarded as a nice nurture for people

recently and has been made into all kinds of food and medicine
products. Chlorella pynenoidosa has also been applied in ammonia
removal in fresh water and showed an excellent result. And it has also
been reported that Chlorella pynenoidosa can be grown in seawater.

ABSTRA CT
We attempted to evaluate the possibility of using Chlorella
pynenoidosa to remove ammonia and COD from mariculture effluent
through a series of experiments. The results showed that Chlorella
pynenoidosa can depend on ammonia as necessary nutrient for growth
after the process of domestication culture. High concentration ammonia
has a toxic effect on Chlorella pynenoidosa. The higher the
concentration, the stronger the effect is. The appropriate ammonia
range is below 50mg/L. The best working condition of Chlorella
pynenoidosa for ammonia removal is pH 7-9temperature 25-30,
algae delivery amount 8 105cell/mL. Further experiment results
showed that the acclimated algae can reduce the ammonia
concentration below 0.02mg/l in 7 days.

In this study, we attempted to evaluate the possibility of using

Chlorella pynenoidosa to remove ammonia and COD from mariculture
effluent through a series of experiments. And the manifold Chlorella
pynenoidosa can make an economical effect.

Experi mental
Algae and Culture
The Chlorella pynenoidosa used in this work was supplied by Ocean
University of China, which was originally found in freshwater.
Chlorella pynenoidosa grows fast and produces new biomass
efficiently in the solution displayed in Table 1(A5 and B6 are illustrated
in Table 2). In this study, we attempted to acclimate the algae through 2
ways. The first method is to culture the algae in seawater-diluted
solution directly. And the other way is to increase the seawater percent
in the solution by and by according certain program (the last two rows
of Table 1).
Table 1 Composition of acclimating solution
Composition
Quantity
NaCl
1.0 g
CaCl2
0.4 g
NaNO 3
2.5 g
FeSO 4
7H2O
0.01 g
EDTA
0.08 g
K 2SO4
1.0 g
M gSO 4
0.2 g
K 2HPO 4
0.5 g
NaHCO 3
16.8 g
A5
1l
B6
1l
Disinfected seawater*
200/400/600/800/1000 mL
Deionized water*
800/600/400/200/0 mL
* Disinfected seawater referred in this paper is natural seawater filtered
through 0.45m film and boiled at 121 for 20min. Deionized water
was also been boiled at 121 for 20min for disinfection.

KEY WORDS: Ammonia; Chlorella pynenoidosa; mariculture;

salinity acclimation.

INTRODUCTION

Researches on the application of biological nitrogen removal

techniques to mariculture have recently attracted much attention due to
the increase in the numbers of seafood farms and the pollution of the
coastal area(Seo, 2001). Traditional nitrogen removal process depends
on aerobic nitrification and anaerobic denitrification to eliminate
nitrogen from the water system(Abeysinghe, 1996). The nitrification
process transforms ammonia to more oxidized nitrogen compounds
such as nitrite or nitrate, which are converted to nitrogen gas in the
following denitrification process(Chang, 2005; Anthony, 1998).
Biofiltration by plants, such as algae, is assimilative, and therefore adds
to the assimilative capacity of the environment for nutrients(Cheng,
2003; Lin, 2005). With solar energy and the excess nutrients, plants
photosynthesize new biomass. Algae, and in particular seaweeds, are
most suitable for biofiltration because they probably have the highest
productivity of all plants and can be economically cultured(Zimmo,
2003). Algae use sunlight to build their biomass, while assimilating
dissolved inorganic nutrients removed from the water. If properly
cultured, the organisms of both extractive groups can turn pollutant
nutrients into commercial crops and loaded effluents into clean
water( David, 2002).

998

Table2 Composition of trace element solutions

A5
Composition
Concentration (g/L)
ZnSO 4
7H 2O
0.222
CuSO 4
5H 2O
0.079
M oO 3
0.015
H3BO 3
2.86
M nCl2
4H 2O
1.81
B6
Composition
Concentration (g/L)
NH4VO3
229.610-4
K2Cr2(SO4)4
24H 2O
96010-4
NiSO4
7H 2O
478.510-4
Na2WO 4
2H 2O
179.410-4
Co(NO 3)2
6H 2O
439.810-4
Ti2(SO 4)3
400.010-4

3.6

2.8

Algae Biomass (10 cell)

3.2

2.4
2.0
1.6
1.2
0.8
0.4
0.0
0

Timed

100

Ammonia Removal Rate(%)

90

Instrumentation and Experimentation

Take certain amount of well grown Chlorella pynenoidosa as the
studying object, ensuring that it can adapt to seawater. Change the
condition parameters such as pH, temperature, ammonia concentration,
organics concentration (described as COD value) and algae delivery
one by one. Examine ammonia and Chlorella pynenoidosa biomass to
see about environment change impacts. When one of the parameters is
changed, all the others are fixed. The original condition is set as
biomass 104cell/mLammonia10mg/L.
M ix well grown Chlorella pynenoidosa and mariculture dischar ged
wastewater, making sure that condition parameters are optimized
according to the results of the former experiments. Examine ammonia
and Chlorella pynenoidosa biomass every day to make clear the
decontaminating ability of Chlorella pynenoidosa for mariculture
wastewater.
Salinity, pH and dissolved oxygen (DO) were measured by HA CH
Port able M ult ip aramet er M et er
(HA CH
s ens IO N tm 156
Comp any, U SA ) . AmmoniaCOD Mn were determined according to
the methods described in Chinese Seawater Quality Standard. Biomass
was determined by microscope and counting meter.

80
70
60
50
40
30
20
10
0

Time (d)

()Using seawater directly ()Improving seawater proportion gradually

(a) Algae growth (b) Ammonia removal rate
Fig.1The acclimation results of two different culture modes
Optimization of growing and decontamination conditions
Fig.2-6 show the result of Chlorella pynenoidosas growing,
reproduction and its removal rate under a series parameter changes.
Ammonia could be absorbed by Chlorella pynenoidosa as a kind of
nutrient. But high concentration of ammonia has a toxic effect on
Chlorella pynenoidosa. The higher the concentration, the stronger the
effect is. Experiments were carried out to determine the safe
concentration range for Chlorella pynenoidosa growth. From the results
shown in figure 2 we can see that Chlorella pynenoidosa can grow
normally when ammonia is controlled under 50mg/L and ammonia
concentration must be maintained below 20mg/L to get good harvest
and water quality. Or else the algae will grow slowly and cannot
remove the ammonia effetely and it may die under worse conditions.

Results and discussion

Acclimation of Chlorella pynenoidosa
Fig.1 shows the results of salinity acclimation of Chlorella pynenoidosa
through the two ways. From the data we can see that salinity increase
can reduce its ability to grow. As the time goes on, Chlorella
pynenoidosa can get used to the environment change. The algae
reproduces faster and faster and ammonia concentration accordingly
decreases. By comparing the 2 curves in the figure we also can
conclude that increasing the salinity gradually can get a better result.
Chlorella pynenoidosa has a good capability to acclimate itself to the
changed environment. It has been found in several places such as field,
pond, swamp, streams and so on. It has been recently reported that
Chlorella pynenoidosa is rich in proteins, vitamins, minerals, fibrins,
nucleic acids and chlorophyll, which are necessary for p eoples health.
It is now widely accepted that Chlorella pynenoidosa is a new resource
for healthy food products. New ways are seeking to culture Chlorella
pynenoidosa for lower cost and better harvest.

4.0

3.2
80

7-day Algae Biomass (10 cell)

7-day Ammonia Removal Rate(%)

100

3.6

2.8

7-day Algae Biomass

7-day Ammonia Remove Rate

2.4

60

2.0
1.6

40

1.2
20

0.8
0.4

0
0.0
0

10

15

20

25

30

35

40

45

50

55

60

65

70

Original Ammonia Concentrationmg/L

Fig.2 Ammonia removal rate and algae biomass at different ammonia

concentration
From figure 3 we can see that Chlorella pynenoidosa grows well and
ammonia is effectively removed when pH is set from 6-11. Bad results
come out when it is out of this pH range. When pH is below 4 or
above12, Chlorella pynenoidosa will suffer biomass loss and can not
develop or reproduce normally. The best pH range for growing is 7-9.
The decontaminating effect is closely interrelated to algae growth.

999

4.0

110

7-day Algae Biomass

7-day Ammonia Remove Rate

3.6

80
2.8

60

2.0
1.6

40

1.2
20

0.8

7-day Ammonia Removal Rate(%)

3.2

2.4

7-day Algae Biomass (10 cell)

100

7-day Algae Biomass (10 cell)

3.6

105
3.2
100

2.8

2.4

95

7-day Algae Biomass

7-day Ammonia Remove Rate

2.0
90
1.6

1.2
0

0.4

100

0
1

10

11

12

13

200

300

400

500

85
600

Organisms Concentration(mg/L)

0.0
0

7-day Ammonia Removal Rate(%)

4.0

14

Fig.5 Ammonia removal rate and algae biomass at different organic

matters concentration

pH

Fig.3 Ammonia removal rate and algae biomass at different pH value

We can demonstrate from figure 5 that delivery of organic matters
could accelerate the growth and reproduction of Chlorella pynenoidosa.
The positive correlation between biomass and organic matter delivery
(represented by the data of COD) was indicated from the figure, too.
But we must notice that the correlation is not linear. When original
COD exceeds 100mg/L, more delivery does not has an obvious effect
on growth acceleration. This phenomenon may be explained as the
result of competition between algae cells. So, in order to get a good
profit and graceful decontamination rate, we should make a delivery of
organic matters at the concentration of 50-100 mg/L.

Temperature is very important for algae metabolism, especially

infecting the course of degradation and photosynthesis. The key
reaction of photosynthesis mainly relies on catalysis devoted by
enzymes, and the activity of enzymes is very sensitive to temperature.
Figure 4 shows how the algae reproduce by absorbing ammonia at
different temperature. Comparing the results we can conclude that
Chlorella pynenoidosa can grow normally between hte temperature
from 15 to 40, and the best temperature range is 25 to 30, in
which Chlorella pynenoidosa grow and reproduce fairly well and can
remove ammonia from water at a rate more than 90%. It may be
because that at low temperature photosynthesis is restricted and at high
temperature chloroplasts will be destroyed. M ost of the algaes are
reported to be adapted to the temperature range from 28 to 35.

The degradation of ammonia in this study is actually through t he

absorption ability of Chlorella pynenoidosa. So, in theory, the more
biomass, the more ammonia is removed from the water. But in fact it is
not the case. Beside, cost and economic factors must be considered in
practical operation.

4.0

4.0

7-day Ammonia Removal Rate(%)

100

2.0
60

7-day Algae Biomass

7-day Ammonia Remove Rate

1.6
1.2
0.8
10

2.8

100

2.4
2.0

95

1.6

7-day Algae Biomass

7-day Ammonia Remove Rate

1.2

90

0.8

85

40
15

20

25

30

35

40

7-day Ammonia Removal Rate(%)

80
2.4

105

3.2

2.8

110

3.6

7-day Algae Biomass (10 cell)

3.2

7-day Algae Biomass (10 cell)

3.6

45

0.4

Temperature ()

0.0

80
0

Fig.4 A mmonia removal rate and algae biomass at different

temperature

10

12

Algae Dilivery(10 cell/mL)

Fig.6 Ammonia removal rate and algae biomass at different algae

delivery

Appropriate pH, temperature and illumination are elementary points for

algae growing. Well, nutrients are also crucial in most circumstances.
Ammonia in the mariculture can be utilized as the necessary nitrogen
source, and at the same time organic matters must be available to make
the nutrient structure balanced and reasonable. Certain organic matters
can help improving the growing velocity of Chlorella pynenoidosa.

Further analysis of figure 6 can make it clear how much seed biomass
should be delivered for good results. Addition of algae delivery does
not lead to higher production and better decontamination. The probable
reason is that high density of algae may prevent the sunlight partly so
the average growing velocity is slowing down. Considering the result in
figure 6 and the integrated cost, we regard the best algae delivery as

1000

8105cell/mL.

Because in this study ammonia is being absorbed instead of being

transferred, so there are no by-products from nitrogen degradation. We
can judge the water quality only be ammonia concentration and COD
value. The data in the table indicates it is fairly reasonable to use
Chlorella pynenoidosa to treat mariculture wastewater. Common
mariculture wastewater could satisfy the requirement of EPA and
Chinese Seawater Quality Standard after treatment with pertinence.

Treatment of real mariculture wastewater

Though all the paramters of condition had been confirmed, further
experiments must be carried out to test whether it is feasible to use
Chlorella pynenoidosa as a mariculture wastewater cleaner. Table 3
shows the treatment of 3 maricluture wastewaters. It can be concluded
that waste removal rate has a reverse correlation with the level of
contamination. Ammonia is far more difficult to remove than COD,
which can be totally taken off within 7 days.

Table3 Aquaculture wastewater treatment by Chlorella pynenoidosa

Original wastewater quality
Decontamination effect by Chlorella pynenoidos
Ammonia
Parameters

pH

Ammonia

COD

COD
Concentration

Remove Rate

Concentration

Remove Rate

Unit

mg/L

mg/L

mg/L

mg/L

8.2

6.7

4.5

0.018

99.7

100

7.9

55.3

21.4

1.493

97.3

100

7.8

152.8

49.6

8.159

94.7

100

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CONCLUSIONS
The best working condition of Chlorella pynenoidosa for mariculture
decontamination is pH 7-9temperature 25-30, organic matters 50100 mg/L, algae delivery 8105cell/mL. Further experimental results
showed that the acclimated algae could reduce the ammonia
concentration below 0.02mg/l in 7 days for fairly contaminated
wastewater, in which ammonia concentration is below 50 mg/L. And
the best result came out when ammonia concentration was below
20mg/L. The effluent water quality could satisfy the requirement of
EPA and Chinese Seawater Quality Standard. But the researches of
technology and craft for Chlorella pynenoidosa harvest are to be
continuously carried out in the future.

ACKNOW LEDGEM ENTS

This work was supported by 2008s Study on Spirulina platensis
culturing technologies by using mariculture wastewater No.
Y200804859 from Project of Zhejiang Education Bureau.

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