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 SH
Trang 1CAN 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
Trang 2CAN 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
Trang 3EFFECTS 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
Trang 41 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 26oC until reaching the highest density of 5.106-6.106 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
Trang 5Microbiological 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 NO2, NH4/NH3 and CaCO3 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
Trang 6Statistical 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)
1
2
3
4
5
6
7
12±3.30a 28±4.65b 31±1.23bc 37±2.46c
34±7.11 a 52±1.17 b 64±1.72 bc 66±2.50 c
66±3.90 a 73±1.38 ab 75±3.66 bc 80±3.84c
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
Trang 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)
Total bacteria (CFU/mL)
10 6575±913a 5087±643a 3930±519c
Vibrio bacteria (CFU/mL)
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
Preservation time (day)
Trang 83.2 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 27.17oC to 30.38oC (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 28oC and 31oC In this temperature range, larvae shrimp obtained high survival rate and developmental stage
Figure 2 Variation of temperature (oC) during shrimp larvae rearing
The environmental parameter was almost the same among feeding treatments Average pH and alkalinityvaried in the ranges 8.3-8.4, and 135-143mg CaCO3/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 NO2 varied from 0.33 to 0.39 mg/L in which the chitosan 100 mg/L had higher content of TAN and
NO2 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 NO2 range for larvae shrimp was smaller than 1 mg/L (Pham Van Tinh, 2004) Water quality in the
25
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27
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30
31
o C)
Culture day Morning Afternoon
Trang 9above 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
Control Chitosan 40 Chitosan 70 Chitosan 100
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
NO2- (mg/L) 0.33±0.13 a
0.36±0.13 a 0.36±0.18 a 0.39±0.18 a Alkalinity (mg
CaCO3/L) 143.20±12.66
a
139.22±7.89 a 143.20±8.95 a 135.24±13.0a
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
Developmental stage of shrimp
Control Chitosan 40 Chitosan 70 Chitosan 100
Trang 10The 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
2.10±0.17 a 4.04±0.15 b 5.02±0.21 b Chitosan 40 0.84±0.05a
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 25oC to 39.3oC and temperature fluctuated during rearing period and was not significant difference among treatments Nguyen Van Man (2012) reported that mean temperature maintained at
28oC giving the best growth rate of blood cockle compared to 32 or 34oC
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
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25
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30
o C)
Culture day
Morning Afternoon