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 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 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 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 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 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. METHODS 2.1 Experiment 1: Evaluating the effects of different concentrations of chitosan on the flocculation of Chaetoceros Experiment was designed with treatments, and replicates were run for each. Algae were flocculated by difference concentrations of chitosan of 10, 40, 70, and 100 mg/L. The experiment included 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 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. 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 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 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 (Z3), Mysis (M3), and Post larvae (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 times a week to assess filtration rate before and after feeding. 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[...]... (p0.05), 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 . 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 103 6 a 2720±434 ab 100 8487±1941 a 5650±1283 a . 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. 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