Salinity Acclimation of Chlorella pynenoidosa and Its Application in Mariculture Wastewater Treatment

Haiyan Hu, Yong Liu, Yibing Deng, Weihong Jin Marine Science College, Zhejiang Ocean University

Zhoushan, Zhejiang, China

ABSTRACT

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.

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).

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.

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.

Experimental 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

NaNO3 2.5 g FeSO47H2O 0.01 g

EDTA 0.08 g

K2SO4 1.0 g

MgSO4 0.2 g

K2HPO4 0.5 g NaHCO3 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.

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

998

Table2 Composition of trace element solutions

A5

Composition Concentration (g/L)

ZnSO47H2O 0.222

CuSO45H2O 0.079

MoO3 0.015

H3BO3 2.86

MnCl24H2O 1.81

B6

Composition Concentration (g/L)

NH4VO3 229.610-4

K2Cr2(SO4)424H2O 96010-4

NiSO47H2O 478.510-4

Na2WO42H2O 179.410-4

Co(NO3)26H2O 439.810-4

Ti2(SO4)3 400.010-4

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. Mix well grown Chlorella pynenoidosa and mariculture discharged

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 HACH

sensION tm156 Port able Mult ip aramet er Met er (HACH

Comp any, USA). AmmoniaCODMn were determined according to the methods described in Chinese Seawater Quality Standard. Biomass

was determined by microscope and counting meter.

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 peoples 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.

0 2 4 6 80.0

0.4

0.8

1.2

1.6

2.0

2.4

2.8

3.2

3.6

Alg

ae

Bio

ma

ss (

10

4ce

ll)

Timed

a

0 2 4 6 810

20

30

40

50

60

70

80

90

100

Am

mo

nia

Re

mo

va

l R

ate

(%)

b

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.

0 5 10 15 20 25 30 35 40 45 50 55 60 65 700.0

0.4

0.8

1.2

1.6

2.0

2.4

2.8

3.2

3.6

4.0

7-d

ay A

mm

on

ia R

em

ova

l R

ate

(%)

7-day Algae Biomass

7-day Ammonia Remove Rate

Original Ammonia Concentrationmg/L

7-d

ay A

lga

e B

iom

ass (

10

6ce

ll)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70

0

20

40

60

80

100

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

0 1 2 3 4 5 6 7 8 9 10 11 12 13 140.0

0.4

0.8

1.2

1.6

2.0

2.4

2.8

3.2

3.6

4.0

7-d

ay A

mm

on

ia R

em

ova

l R

ate

(%)

7-day Algae Biomass

7-day Ammonia Remove Rate

pH

7-d

ay A

lga

e B

iom

ass (

10

6ce

ll)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

0

20

40

60

80

100

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

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. Most of the algaes are

reported to be adapted to the temperature range from 28 to 35.

10 15 20 25 30 35 40 450.8

1.2

1.6

2.0

2.4

2.8

3.2

3.6

4.0

7-d

ay A

mm

on

ia R

em

ova

l R

ate

(%)

7-day Algae Biomass

7-day Ammonia Remove Rate

7-d

ay A

lga

e B

iom

ass (

10

6ce

ll)

10 15 20 25 30 35 40 4540

60

80

100

Fig.4 Ammonia removal rate and algae biomass at different

temperature

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.

0 100 200 300 400 500 6001.2

1.6

2.0

2.4

2.8

3.2

3.6

4.0

7-d

ay A

mm

on

ia R

em

ova

l R

ate

(%)

7-day Algae Biomass

7-day Ammonia Remove Rate

Organisms Concentration(mg/L)

7-d

ay A

lga

e B

iom

ass (

10

6ce

ll)

0 100 200 300 400 500 60085

90

95

100

105

110

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

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.

The degradation of ammonia in this study is actually through the 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.

0 2 4 6 8 10 120.0

0.4

0.8

1.2

1.6

2.0

2.4

2.8

3.2

3.6

4.0

7-d

ay A

mm

on

ia R

em

ova

l R

ate

(%)

7-day Algae Biomass

7-day Ammonia Remove Rate

Algae Dilivery(105cell/mL)

7-d

ay A

lga

e B

iom

ass (

10

6ce

ll)

0 2 4 6 8 10 1280

85

90

95

100

105

110

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

delivery

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

Temperature ()

1000

8105cell/mL.

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.

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.

Table3 Aquaculture wastewater treatment by Chlorella pynenoidosa

Original wastewater quality Decontamination effect by Chlorella pynenoidos

Parameters pH Ammonia COD

Ammonia COD

Concentration Remove Rate Concentration Remove Rate

Unit mg/L mg/L mg/L % mg/L %

1 8.2 6.7 4.5 0.018 99.7 0 100

2 7.9 55.3 21.4 1.493 97.3 0 100

3 7.8 152.8 49.6 8.159 94.7 0 100

CONCLUSIONS

The best working condition of Chlorella pynenoidosa for mariculture

decontamination is pH 7-9temperature 25-30, organic matters 50-100 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 LEDGEMENTS

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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