effects of chitosan on flocculation of chaetoceros algae and ability to apply on blood cockle (anadara granosa) and white leg shrimp (litopenaeus vannamei) rearing

1
CAN THO UNIVERSITY
COLLEGE OF AQUACULTURE AND FISHERIES










EFFECTS OF CHITOSAN ON FLOCCULATION OF
CHAETOCEROS ALGAE AND ABILITY TO APPLY ON
BLOOD COCKLE (Anadara granosa) AND WHITE LEG
SHRIMP (Litopenaeus vannamei) REARING



By



PHAM THI HONG AI

A thesis submitted in partial fulfillment of the requirements for
The degree of Bachelor of Aquaculture Science








Can Tho, December 2014


2
CAN THO UNIVERSITY
COLLEGE OF AQUACULTURE AND FISHERIES








EFFECTS OF CHITOSAN ON FLOCCULATION OF
CHAETOCEROS ALGAE AND ABILITY TO APPLY ON

BLOOD COCKLE (Anadara granosa) AND WHITE LEG
SHRIMP (Litopenaeus vannamei) REARING





By



PHAM THI HONG AI






A thesis submitted in partial fulfillment of the requirements for
The degree of Bachelor of Aquaculture Science




Supervisor

Asc. Prof. Dr. NGO THI THU THAO

Can Tho, December 2014


3

EFFECTS OF CHITOSAN ON FLOCCULATION OF CHAETOCEROS
ALGAE AND ABILITY TO APPLY FOR REARING BLOOD
COCKLE (Anadara granosa) AND WHITE LEG SHRIMP (Litopenaeus
vannamei)

Pham Thi Hong Ai
College of Aquaculture & Fisheries, Cantho University

ABSTRACT

This study was conducted to determine the concentration of chitosan which
was suitably applied to flocculate Chaetoceros algae to feed white leg shrimp
Litopenaeus vannamei and juvenile of blood cockle Andara granosa. There were
three separated experiments, each experiment included 4 treatments and
triplicates per each. In experiment 1, algae were flocculated by difference
concentrations of chitosan of 10, 40, 70, and 100 mg/L. In experiment 2 and 3,
white leg shrimp and blood cockle were fed centrifugal algae, and flocculated
algae with three concentrations of chitosan at 40, 70, 100 mg/L and centrifugal
Chaetoceros was used as control. In experiment 1, algae were flocculated with
chitosan at the concentration of 100mg/L resulting the highest efficiency (92%)
and low bacteria density (1655±302 CFU/mL). The survival of algae cells were
not statistical differences among treatments (P>0.05). In experiment 2, highest
survival rate of shrimp presented (22.5%) in centrifugal algae treatment.
Experiment 3, the highest growth rate of blood cockles was observed in chitosan

flocculated algae at 40 mg/L after 60 days of culture. Survival rate and filtration
rate of blood cockles were not significant difference among treatments (p>0.05).
Results from this study showed that Chaetoceros algae were flocculated by
chitosan at the concentration of 40 mg/L being suitable for feeding blood cockle
juveniles.
Key words: Blood cockle, Anadara granosa, White leg shrimp, Litopenaeus
vannamei, Chaetoceros, Flocculation






4
1 INTRODUCTION
Microalgae, Chaetoceros is a marine diatom widely used in aquaculture
industries, as it is comprised of nutritional value suitable for most marine filter
feeders. However, maintaining fresh microalgal biomass continuously to provide
adequately in seed production of mariculture that is difficult because it depend on
weather, equipment and technical management skills. Produce microalgae biomass
and stored as concentrates help more convenient, initiative to supply feed and
reduce cost in the seed production that need attention. There are many methods to
harvest, but flocculation is preferred for harvesting large cells like microalgae due
to its low costs compared to other methods such as centrifugation and filtration
(Bilanovic et al., 1988). Therefore, the choices of flocculants are important.
Chitosan obtained by deacetylation of chitin, is a cationic polyelectrolyte and is
expected to coagulate negatively charged. The removal of algae by flocculation
and settling using chitosan has limited in the studying. The present study set out to
test the effectiveness of chitosan as a flocculant on flocculation of Chaetoceros
algae and ability to apply on white leg shrimp (Litopenaeus vannamei) and blood

cockle (Andara granosa) rearing.
2 METHODS
2.1 Experiment 1: Evaluating the effects of different concentrations of
chitosan on the flocculation of Chaetoceros
Experiment was designed with 4 treatments, and 3 replicates were run for
each. Algae were flocculated by difference concentrations of chitosan of 10, 40,
70, and 100 mg/L. The experiment included 2 stages as follow:
Stage 1: Flocculation algae with different concentrations of chitosan
Algae were cultured in air-conditioned room at temperature of 26
o
C until
reaching the highest density of 5.10
6
-6.10
6
cell/mL. Chaetoceros algae were
flocculated by four different concentrations of chitosan as mentioned above.
Sample of algae was stopped when flocculation efficiency reach ≥80%.
Flocculation efficiency was evaluated by comparing the remaining cell density in
the clear region with the concentration before treatment.
Stage 2: The quality of flocculated algae were preserved over time
The percentage of viable cells: algae samples were diluted and determined
the density at the beginning and 3 days intervals during the 15-days. The samples
were allowed to stand at room temperature and cells were observed under
microscope. Cell numbers were counted by Improved Neubauer chamber. The
counting process was only determined the algal cells are intact.


5
Microbiological analysis: the density of total bacteria and Vibrio during the

preservation time were determined by colony counting method (Baumann et al.,
1980, cited by Pham Thi Tuyet Ngan, 2012). Algal samples were collected for
bacterial analysis at the beginning and 7 days intervals during preservation in 14
days.
2.2 Experiment 2: Effects of flocculated algae on growth and survival rate of
white leg shrimp
Experiment 2 consisted of four feeding treatments with three replicates per
treatment as follows: 1). Centrifugal algae (control); 2). Flocculated algae with
chitosan at 40 mg/L (chitosan 40); 3). Flocculated algae with chitosan at 70 mg/L
(chitosan 70); 4). Flocculated algae with chitosan at 100 mg/L (chitosan 100).
Larvae shrimp were be reared in 100 liter composite tank at the density of 150
inds./L. Water was maintained at the salinity of 30 ‰. Aeration system was
continuously supplied.
2.3 Experiment 3: Effects of flocculated algae on growth and survival rate of
blood cockle juvenile
This experiment composed four feeding treatments with three replicates per
each treatment and was designed similarly to experiment 2. Blood cockles were
cultured in 100-liter plastic tanks at the density of 50 individuals per tank, water
was supplied to 50 liters/tank at the salinity of 20 ‰. Aeration was continuously
supplied. This experiment was run for 60 days. Blood cockles were fed
Chaetoceros at densities of 10,000 cell/mL with twice a day at 7:30 AM and 2:00
PM.
2.4 Data collection
Daily water temperature was recorded at 7:00 AM and 2:00 PM using a
thermometer. pH was measured weekly by pH meter (YSI 60 Model pH meter,
HANNA instrument, Mauritius). The concentration of NO
2
, NH
4
/NH

3
and CaCO
3

were monitored weekly using test kit (Sera, Germany).
Shrimp survival rate was determined at Zoae 3 (Z3), Mysis 3 (M3), and Post
larvae 3 (PL3). A beaker with volume 1000 ml was used to collect shrimp
randomly at three different places in each rearing tank. Number of shrimp was
counted and calculated the survival rate. At the same time, 10 shrimp in each tank
was randomly collected to determine the length.
Blood cockle samples were conducted every 15 days. All of blood cockles
were taken from each tank to measure shell length and weight. Survival rate of
cockles also were determined every 15 days during experiment. The density of
algae was determined the density 2 times a week to assess filtration rate before and
after feeding.


6
Statistical analysis: data were analyzed for mean value, standard deviation by
using Excel software, and Duncan test (One way ANOVA, SPSS 16.0) to compare
the significant different among treatments at P<0.05.
3 RESULTS AND DISCUSSION
3.1 Effects of different concentrations of chitosan on the flocculation and
quality of Chaetoceros algae
Flocculation efficiency
Flocculation efficiency increased when rising concentration of chitosan and
time. In the first 3 hours, the highest sedimentation efficiency presented in
Chaetoceros flocculated by chitosan at 100 mg/L (66±1.16%) and it was
significant difference from the treatment 1 (P<0.05). After 7 hours, the efficiency
was not significantly difference among treatments (P>0.05), ranging from 88% to

92%. Similar flocculation efficiency (92%) was recorded in chitosan concentration
of 70mg/L and 100mg/L. Harith et al. (2009) assessed the effect of different
flocculants on the flocculation performance of microalgae, Chaetoceros calcitrans.
Authors reported that using sodium hydroxide (NaOH) or potassium hydroxide
(KOH) had harvesting efficiency higher than 90%. Observation on the flocculated
algae with chitosan was high efficiency rate similar to other chemicals. Moreover,
other study confirmed the flocculation was faster when concentrations of chitosan
higher (Divakaran and Pillai, 2002).
Table 1. Flocculation efficiency (%) of Chaetoceros with different chitosan
concentrations
Duration
(hours)
Concentrations of chitosan (mg/L)
10
40
70
100
1
2
3
4
5
6
7
12±3.30
a
28±4.65
b
31±1.23
bc

37±2.46
c
21±8.09
a

35±4.03
b

41±1.76
b
43±2.43
b

34±7.11
a

52±1.17
b

64±1.72
bc

66±2.50
c

55±3.36
a

64±2.79
b

65±1.45
b

68±1.40
b

53±6.65
a

68±1.16
b

67±1.41
b

69±1.14
b

66±3.90
a

73±1.38
ab

75±3.66
bc

80±3.84
c

88±1.90
a

91±1.15
b

92±1.49
b

92±1.14
b

The value in the same row with different letters indicating significant difference (P<0.05)
Quality of Chaetoceros algae during preservation period
The percentage of viable cells had generally downward in 15 days of
preserved time. There was not statistical differences among treatments (P>0.05)
(Figure 1). The highest viable cells of flocculated algae with chitosan at 70mg/L
were 56%. Other study recommended that Chaetoceros algae were deposited by
chitosan had the highest living cell ratio at 52.67% after 14 days of preservation


7
(Tran Thi Ngoc Hanh, 2014). The present study showed that the viable
Chaetoceros cells were similarly among concentrations of chitosan flocculant.
Figure 1. The percentage of viable cells (%) during 15 days of preservation period
In chitosan flocculated algae the density of total bacteria was a slight decline
in preservation period (Table 2). Because chitosan used in food industry as
preservation because of its high antimicrobial activity against various
microorganisms (Dutta et al., 2009). The number of total bacteria in lowest

chitosan concentration (3930±519 CFU/mL) was higher than other treatments. The
significant differences in total bacteria densiy was only dectected in flocculated
algae using the lowest and highest chitosan concentration (P<0.05).
The number of Vibrio bacteria was not significantly different among
treatments (P>0.05) (Table 2). During 15 days of preservation period, the bacteria
densities were fluctuation in all treatments. This study presented that the number
of Vibrio bacteria were similarly among concentrations of chitosan.
Table 2. The density of total bacteria and Vibrio during preservation (CFU/mL)
Chitosan
concentrations (mg/L)
Preservation time (days)
1
7
14
Total bacteria (CFU/mL)
10
6575±913
a

5087±643
a

3930±519
c

40
7175±1673
a

5737±665

a

3231±556
bc

70
8100±1678
a

5475±1036
a

2720±434
ab

100
8487±1941
a

5650±1283
a

1655±302
a

Vibrio bacteria (CFU/mL)
10
25±5.8
a

550±81.9
a

115±30.9
a

40
22±5.8
a

607±153.8
ab

110±24.5
a

70
25±5.0
a

632±130.1
ab

132±35.4
a

100
32±9.6
b

650±162.3
b

140±41.7
a

The value in the same column with different letters indicating significant difference (p<0.05)

0
10
20
30
40
50
60
70
80
90
100
0 3 6 9 12 15
Viable cell %
Preservation time (day)
10 40
70 100


8
3.2 Effects of flocculated algae on growth and survival rate of white leg
shrimp
3.2.1 Water quality parameters

Average values of environmental parameter were illustrated in Table 3. There
was no significant difference of temperature among treatments. Daily mean
temperature ranged from 27.17
o
C to 30.38
o
C (Figure 2). Dao Van Tri (2012)
studied the effects of temperature on the developmental stage and survival rate of
larvae white leg shrimp. The author recommended that the optimal range of
temperature for white leg shrimp was between 28
o
C and 31
o
C. In this temperature
range, larvae shrimp obtained high survival rate and developmental stage.

Figure 2. Variation of temperature (
o
C) during shrimp larvae rearing
The environmental parameter was almost the same among feeding
treatments. Average pH and alkalinity

varied in the ranges 8.3-8.4, and 135-143mg
CaCO
3
/L, respectively. pH is one of the vital environmental characteristics, which
decides the survival and growth of shrimp. For shrimp, pH should be maintained
between 7-8.5 (Nguyen Thanh Phuong et al,. 2003, and Thai Ba Ho et al, 2003). In
addition, Dao Van Tri and Nguyen Thanh Vu (2004) reported that the suitable
range of alkalinity for shrimp was 120-160 mg/L.

The mean concentration of TAN was 0.42-0.75 mg/L and NO
2
varied from
0.33 to 0.39 mg/L in which the chitosan 100 mg/L had higher content of TAN and
NO
2
than other feeding treatments. Whetstone (2002) stated that the toxicity of
ammonia and nitrite for shrimp is greatly dependent on environmental factors such
as pH, dissolved oxygen, salinity, and temperature. Boyd (1990) suggested that the
suitable ammonia value for shrimp was lower than 2 mg/L. Also, Nguyen Thanh
Phuong et al,. (2003) recommended that the content of TAN should be less than
1.5 mg/L for shrimp. Other study found out that the optimal NO
2
range for larvae
shrimp

was smaller than 1 mg/L (Pham Van Tinh, 2004). Water quality in the
25
26
27
28
29
30
31
1 2 3 4 5 6 7 8 9 10 11 12 13
Temperature (
o
C)
Culture day
Morning

Afternoon


9
above study was within acceptable range for white leg shrimp. Therefore, feeding
was the present factor influencing the experiment.
Table 3. Mean values of environmental parameters during shrimp larvae rearing
Criteria
Treatment
Control
Chitosan 40
Chitosan 70
Chitosan 100
pH
8.34±0.18
a
8.37±0.14
a

8.38±0.11
a

8.39±0.13
a

TAN (mg/L)
0.42±0.18
a

0.58±0.25

b

0.64±0.21
bc

0.75±0.22
c

NO
2
-
(mg/L)
0.33±0.13
a

0.36±0.13
a

0.36±0.18
a

0.39±0.18
a

Alkalinity (mg
CaCO
3
/L)
143.20±12.66
a

139.22±7.89
a

143.20±8.95
a

135.24±13.0
a

The value in the same row with different letters indicating significant difference (p<0.05)
3.2.2 Survival and growth rate of larvae shrimp
The shrimp survival rate of all treatments decreased during 13 days ranging
from 13.3% to 22.5% (Figure 3) and there were not statistical difference among
feeding treatments (P>0.05). At P3 stage, the averaged survival rate in control
treatment was the highest (22.5%) and no significant difference (P>0.05) with
chitosan 40 treatment (19.2%). In general, survival rate of white leg shrimp was
lower than the results from study of Nguyen Thanh Mai et al., (2009), in that the
authors used fresh Chaetoceros calcitrans for feeding white shrimps larval from
Nauplius to Mysis 3 with the survial rate from 42% to 76%. Centrifugal and
flocculated algae possibly increased algae clusters, therefore could limit the
filtration of shrimp larvae. The type of food ingested and the feeding mechanisms
are correlated with the characteristics of the different larval stages of shrimp (New,
1979). They found that after 2 and 3 days, the nauplius metamorphosed into zoea
stage and began to feed on phytoplankton of approximately 3-10 μm. In the present
study, survival rate of shrimp was remarkable decrease from Z1 to Z3, and slightly
changed from Z3 and PL3.

Figure 3. Variation of survival rate shrimp during shrimp larvae rearing
0

10
20
30
40
50
60
70
80
90
100
Z1 Z3 M3 P3
Survival rate (%)
Developmental stage of shrimp
Control
Chitosan 40
Chitosan 70
Chitosan 100


10
The results showed that feeding shrimp with chitosan flocculated algae, the
length of shrimp were smaller than the control (Table 4). Final length of
experimental shrimp ranged from 4.88-0.52 mm. Shrimp in chitosan 40 treatment
(4.91±0.24 mm) showed higher length than Chitosan 70 and 100 treatments. Bui
Huu Loc (2013) determined effects of different diets on the maturation and
reproduction of white leg shrimp. The author reported that length of shrimp at Z1
(0.85 to 0.88 mm), Z3 (2.82- 2.87 mm), M3 (4.14 to 4.16 mm), PL1 (4.61 to 4.65
mm). Results from the present study showed that the length of shrimp at different
stages were lower than those from previous study.
Table 4. The length (mm) of shrimp during shrimp larvae rearing

Treatment
The developmental stages
Z1
Z3
M3
P3
Control
0.82±0.06
a

2.10±0.17
a

4.04±0.15
b

5.02±0.21
b

Chitosan 40
0.84±0.05
a
2.04±0.20
a

3.84±0.28
a

4.91±0.24
ab

Chitosan 70
0.83±0.05
a

2.06±0.21
a

3.82±0.28
a

4.90±0.25
ab

Chitosan 100
0.80±0.07
a

2.05±0.18
a

3.78±0.30
a

4.88±0.19
a

The value in the same column with different letters indicating significant difference (p<0.05)
3.3 Effects of flocculated algae on the growth and survival rate juvenile of
blood cockle

3.3.1 Water quality parameters
Average values of environmental parameter were showed in Figure 4 and
Table 5. Daily mean temperature range from 25
o
C to 39.3
o
C and temperature
fluctuated during rearing period and was not significant difference among
treatments. Nguyen Van Man (2012) reported that mean temperature maintained at
28
o
C giving the best growth rate of blood cockle compared to 32 or 34
o
C.


Figure 4. Variation of temperature during rearing period of blood cockles
Environmental parameters were not statistical difference among treatments
(P>0.05) (Table 5). pH value was a leveling off in the ranges 8.35-8.43 during
22
23
24
25
26
27
28
29
30
0 5 10 15 20 25 30 35 40 45 50 55
Temperatute (

o
C)
Culture day
Morning
Afternoon



11
experimental period. The content of TAN and NO
2
-

was 0.41-0.55mg/L and 0.45-
0.63mg/L, respectively. Boyd (1998) claimed that the suitable pH and ammonia
range was 6.5-9 and 0.2-2mg/L, respectively. Alkalinity is the buffering capacity
of the pond and ideal alkalinity range was 75-150 mg CaCO
3
/L (Boyd, 1998).
These factors play an important role in development, growth and survival rate of
aquatic species in aquaculture. Water quality parameters in the present experiment
was within adequate limit for juvenile blood cockle.
Table 5. Mean values of environmental parameters during rearing period of blood
cockles

Treatment

Control
Chitosan 40
Chitosan 70
Chitosan 100
pH
8.41±0.23
a
8.43±0.24
a

8.42±0.24
a

8.35±0.26
a

TAN (mg/L)
0.43±0.19
a

0.41±0.23
a

0.44±0.24
a

0.55±0.18
b

NO

2
-
(mg/L)
0.45±0.23
a

0.51±0.43
ab

0.58±0.45
ab

0.63±0.44
b

Alkalinity (mg/L)
115.75±12.53
a

119.46±12.18
a

116.54±10.14
a

118±11.31
a

The value in the same row with different letters indicating significant difference (p<0.05)
3.3.2 Filtration rate of blood cockle (%)

The filtration rate of blood cockle ranged from 53.17% to 58.71% (Table 6)
and was not significantly different among treatments (P>0.05). The trend generally
had upward for a trial period. In the first day, filtration rate (% /day) in control
treatment was highest (72.87-74.60%/day) significantly different from the other
treatments (P<0.05). During experiment period, filtration rate in all treatments were
slightly fluctuated, probably due to temperature and the preservation time of algae
in experiment. Duong Thi Hoang Oanh et al., (2013) determined the effects of
temperature, density and types of algae on the filtration rate of blood-cockle
(Anadara granosa) and recommended that when temperature and density of algae
increased leading to higher filtration rate.














12
Table 6. Filtration rate of blood cockle (%) during rearing period
Day
Control
Chitosan 40
Chitosan 70

Chitosan100
1
72.87±0.93
b

34.03±0.25
a

34.50±4.09
a

30.67±2.84
a

4
74.60±2.54
b
37.57±0.32
a

40.03±0.81
a

37.77±1.46
a

7
54.83±3.69
b
49.00±0.53

a

48.40±0.50
a

45.93±2.66
a

10
43.53±3.26
a

42.53±0.87
a

42.83±0.95
a

41.50±2.09
a

13
45.67±3.39
a

41.97±2.78
a

42.40±1.65
a

41.33±2.40
a

16
62.83±1.59
a

63.27±0.67
a

61.67±1.66
a

61.93±1.46
a

19
43.93±2.38
a

44.57±1.27
a

43.97±0.81
a

43.87±1.11
a

22
52.30±2.19
a

49.20±2.49
a

51.33±2.01
a

49.67±1.78
a

25
52.83±0.86
a

52.97±1.11
a

52.00±0.79
a

52.30±1.71
a

28
57.23±1.26
a

57.87±0.32
a

58.50±0.53
a

57.80±0.30
a

31
60.90±2.12
a

62.67±1.02
a

62.40±1.99
a

60.10±0.35
a

34
52.83±0.86
a

52.97±1.11
a

52.00±0.79

a

52.30±1.71
a

37
63.77±0.35
a

63.23±2.04
a

63.03±2.37
a

60.87±0.84
a

40
57.23±1.26
a

57.87±0.32
a

58.50±0.53
a

57.80±0.30
a

43
62.83±1.59
a

63.27±0.67
a

61.67±1.66
a

61.93±1.46
a

46
63.27±1.17
a

65.07±3.76
a

63.63±2.57
a

62.30±0.82
a

49
63.77±0.35
a

63.23±2.04
a

63.03±2.37
a

60.87±0.84
a

52
62.10±0.80
a

61.17±1.15
a

61.77±1.91
a

61.37±0.91
a

55
63.77±0.35
a

63.23±2.04
a

63.03±2.37
a

60.87±0.84
a

58
63.27±1.17
a

65.07±3.76
a

63.63±2.57
a

62.30±0.82
a

Mean
58.71±1.60
b

54.55±1.43
a

54.42±1.65
a

53.17±1.33

a

The value in the same row with different letters indicating significant difference (p<0.05)
3.3.3 Survival rate and growth of blood cockle
Survival of blood cockle after 60 days culture was not significantly different
among treatments (P>0.05), ranging from 97% to 99% (Figure 5). Survival of
blood cockle stabilized during experimental period. In the first 15 survival rate
gradually fell, remaining constant between 30 days and 60 days in all treatments.
The highest survival rate of blood cockle was observed in chitosan 70 treatment.
This result indicated that using flocculated algae by differences concentration of
chitosan did not affect the blood cockle survival rate.


13

Figure 5. Variation of survival rate of blood cockle during experimental period
Table 7 showed the growth in shell length and body weight of blood cockle
after 60 rearing days. Initial shell length and body weight of blood cockle were
6.52 mm and 0.06 g, respectively. After 60 days culture, blood cockles in the
control treatment obtained the highest SGR
L
(0.41±0.01%/day), and SGR
W

(1.34±0.03%/day), but significant difference was not observed (P <0.05). Also,
juveniles chitosan 100 obtained lowest growth rate (0.34±0.01%/day and
11.17±0.02%/day for shell length and total weight, respectively). In the first 15
days of rearing, growth rate increased abruptly, so that blood cockles grew faster
than the end of the cultivation period. Moreover, specific growth rate in shell length
and body weight of blood cockle in chitosan 40 and 70 treatment was close to that

control treatment after 45-60 days of cultured period. At the end of experiment, the
higher specific growth rate of shell length and body weight was observed in
chitosan 40 treatment (0.39±0.02 and 1.29±0.07%/day, respectively). The results
in this study were in agreement with the study of Tran Thi Ngoc Hanh (2014), who
found that blood cockles were fed Chaetoceros algae deposited by chitosan
presented high survival rate (100%), growth rate in term of total weight (4,17
%/day) or shell length (1,02 %/day), filtration rate (68,49 %/day) and the results
were higher than those from flocculated algae by other chemical such as NaOH (50
g/m
3
) or PAC (20 g/m
3
). Ly Bich Thuy (2012) evaluated the effects of different
flocculated algae on the growth and survival rate of juvenile hard clam Meretrix
lyrata. Author claimed that after 90 days of culture, the highest survival rate
(15.63%) presented in Chaetoceros flocculated by Al
2
(SO
4
)
3
, significant difference
from the other treatments (P<0.05). However the results from previous study were
lower when compared to using chitosan in present study. Therefore, mineral
coagulants such as alum and ferric chloride might be toxic to animals when
90
91
92
93
94

95
96
97
98
99
100
0 15 30 45 60
survival rate (%)
Culture day
control
Chitosan 40
Chitosan 70
Chitosan 100


14
consumed due to high concentration of residuals in the biomass harvested (Buelna
et al., 1990). Furthermore, the chitosan was a potential flocculant in concentrating
algae due to its low toxicity.
Table 7. The specific growth rate of shell length and body weight (%/day) of blood
cockles during experimental period
Algae
Culture day
15
30
45
60
Specific growth rate of shell length (%/day)
Control
1.48±0.10

b
0.78±0.04
b
0.55±0.03
b
0.41±0.01
b
Chitosan 40
1.06±0.09
a
0.64±0.04
a
0.51±0.03
ab
0.39±0.02
ab
Chitosan 70
1.16±0.19
a
0.69±0.09
ab
0.49±0.06
ab
0.38±0.07
ab
Chitosan 100
1.07±0.04
a
0.60±0.03
a

0.44±0.01
a
0.34±0.01
a
Specific growth rate of total body weight (%/day)
Control
4.43±0.16
b
2.48±0.09
b
1.75±0.09
b
1.34±0.03
c
Chitosan 40
3.13±0.30
a
1.94±0.14
a
1.61±0.06
ab
1.29±0.07
bc
Chitosan 70
3.19±0.25
a
1.92±0.12
a
1.52±0.06
a

1.21±0.07
ab
Chitosan 100
3.43±0.26
a
1.97±0.12
a
1.52±0.07
a
1.17±0.02
a
The value in the same row with different letters indicating significant difference (p<0.05)
4 CONCLUSION AND RECOMMENDATION
4.1 Conclusion
Algae were flocculated with chitosan at the concentration of 70 and 100 mg/L
showing high flocculation efficiency, high viable cell and low bacteria density.
Feeding chitosan flocculated algae did not show the better growth and survival
rates in white leg shrimp larvae.
Juveniles of blood cockle were fed flocculated algae by chitosan at 40 and 70 mg/L
showed high survival and growth rate compared to other treatments.
4.2 Recommendation
Determining the effect of chitosan on flocculation of other algae to feed bivalve.
ACKNOWLEDGE
I would like to express my deep gratitude to my promoter Assoc. Prof. Dr. Ngo Thi
Thu Thao for constant guidance and enthusiastic help during conducting
experiment and patience in correcting thesis.
Special acknowledgements to my teachers of College of Aquaculture and Fisheries,
Can Tho University had taught me the experiences during study.
Finally, I thank to my classmates and my family who have supported and
encouraged me to study and finish my course.

REFFERENCES
Boyd C.E. 1998. Water Quality for Pond Aquaculture. Research and Development
Series No.43. International Center for Aquaculture and Aquatic


15
Environments, Alabama Agricultural Experiment Station, Auburn
University, Alabama.
Bui Huu Loc, 2013. Effects of different diets on the maturation and spawning of
white shrimp (Litopenaeus vannamei). Master thesis. Can Tho University.
Dutta, P. K., Tripathi, S., Mehrotra G. K., Dutta J. (2009). Perspectives for chitosan
based antimicrobial films in food applications. Food Chemistry, Vol. 114,
pp. 1173–1182.
Duong Thi Hoang Oanh, Nguyen Thi Kim Lien and Huynh Truong Giang, 2013.
Effect of temperature, density and type of algae on the filtration rate of
blood-cockle (Anadara granosa) Linne, Scientific Journal 1758 25 (2013):
158-167.
Dao Van Tri, 2011. Research on biological characteristics of artificial breeding and
seed production white leg shrimp (Litopenaeus vannamei, Boone 1931). PhD
thesis in agriculture. Nha Trang University. Abstract.
New, M. B. 1979. The diet of prawn. Working paper based on lectures given to the
2nd Inland Aquaculture course given at National Inland Fisheries Institute,
Bankhem, Thailand (June-August, 1979) 11-19.
Nguyen Van Man, 2012. Effect of different temperature levels on the growth and
survival rate of blood cockles (Anadara grasona). Master thesis. Can Tho
university. Abstract.
Ngo Thi Thu Thao and Truong Quoc Phu, 2012. Molluscan Shellfish Farming. Can
Tho University Publishing House: 136 pages.
Ngo Thi Thu Thao and Ly Thi Bich Thuy, 2013. Effect of different flocculated

algae on the growth and survival rate of juvenile hard clam Meretrix lyrata.
Scientific Jounal of Cantho University 25: 19-26.
Nguyen Thanh Phuong, Tran Ngọc Hai, Tran Thị Thanh Hien và Marcy N. Wilder,
2003. Principles and techniques of freshwater prawn hatchery. Agricultural
Publisher. 127 pages.
Pham Van Tinh, 2004. Technical production of high quality freshwater prawn.
Agricultural publisher. 75 pages.
Tran Thi Ngoc Hanh, 2014. Effect of harvesting methods and preservation on the
quality of Chaetoceros. Master thesis. Can Tho university. Abstract.
Ravi Divakaran and V.N. Sivasankara Pillai, 2001. Flocculation of algae using
chitosan. School of Environmental Studies, Cochin University of Science
and Technology, Foreshore Road, Ernakulam, 682 016, Kerala, India.
Journal of Applied Phycology 14: pp.419–422, 2002.
Whetstone, J,M,, G, D, Treece, C, L, B and Stokes, A, D, 2002, Opporrunities and
Contrains in Marine Shrim Farming, Southern Regional Aquaculture Center
(SRAC) publication No, 2600 USDA,.