Biological properties and the nutrition value of an Isochrysis strain as a live food for geo-duck larvae

Biological properties and the nutrition value of an Isochrysis strain as a live food for geo-duck larvae Le Thi Phuong Hoa1, Nguyen Thi Hoai Ha2, Pham Thi Bich Dao2, Lưu Thị Thùy Giang2

Biological properties and the nutrition value of an Isochrysis strain as a

live food for geo-duck larvae

Le Thi Phuong Hoa1, Nguyen Thi Hoai Ha2, Pham Thi Bich Dao2, Lưu Thị Thùy Giang2

, Luong Thanh Hao3

1

Faculty of Biology, Hanoi National University of Education

2

Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi

3

Faculty of Biology, College of Natural Sciences

Abstract

The addition of Isochrysis galbana H5 ensures a balanced supply of the omega3 and

omega6 fatty acids for the survival and healthy of geoduck larvae This study has shown that the critical factor in this diet for geoduck larvae is the content of polyunsaturated fatty acids (PUFAs), accounted for more than 40 % of total fatty acids, and docosahexaenoic acid (DHA-5.53 mg per mg fresh weight), which play an important role in the membrane lipids Medium f/2

was selected as best medium for microalgae culture Isochrysis galbana H5 is recommended as a

live food for growing larvae and juvenile of fish, bivalves and crustaceans in aquaculture

Keyword: fatty acid, marine microalgae, geoduck

1 Introduction

Marine microalgae provide the food base which supports the entire animal population of the open sea They contain a wide range of fatty acids especially polyunsaturated fatty acids (PUFAs), which cannot be produced in sufficent quantities for metabolic functioning in most marine animals [3,5,12] Many microalgae are believed to improve the nutritional conditions of the larvae as well as their growth and survival [7,12] They are widely used in aquatic cultivation

as food for bivalves, crustaceans and fish especially in their larval stage [12] The

prymnesiophyte Isochrysis is the one of the most commonly used in mariculture, which is rich in

essential fatty acids, omega6-linoleic acid (C18:2n-6) and omega3-linolenic acid (C18:3n-3), and the highly polyunsaturated omega3 fatty acids, octadecatetraenoic acid (C18:4n-3), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) [2,10,12]

Geo-duck is a bivalve species, which inhabits commonly in Quang Ninh and is served as

a specialty with high nutrition [6] In this study, Isochrysis strains isolated, in a way to find

suitable food for sustainable development and conservation of geo-ducks in their larval stage

2 Materials and Methods

Isochrysis strains were isolated based on their morphological properties using

micropipettes [9] and jellylike desert dish according to Shirai et al [8] They were grown at room temperature and illuminated under neon light (Philips daylight tubes) on 10: 14 h light: dark cycles Media f/2, ASW and ESM [4] were used to select culture medium for the growth and development Cells were cultured in 250 ml conical flask and harvested every two days Cell density was determined with a Neubauer haemocytometer in three replicates

Total DNA was extracted and 18S rDNA-coding region were amplified and sequenced according to An et al [1] The 18S rDNA sequences were analyzed using BLAST tool

Biomass for fatty acid analysis was obtained from 1-4 liter aerated culture suspensions in plastic containers Cells were harvested at the log phase to get healthy cells Fatty acid composition was determined according to Krienitz et al [5] by gas chromatography (Finnigan Trace GC) using an ultra-column BPX70 Fatty acids were identified by comparing retention times with those of a calibration standard solution

3 Results and discussion 3.1 Isolation, and classification of Isochrysis strains

Isochrysis strains were isolated based on their morphological properties One of them is

signified as H5 (Fig 1)

Figure 1 Microscopic morphology of

Isochrysis H5

Figure 2 Agarose gel electrophoresis of 18S

rDNA SSU rDNA sequence comparison has proved to be a powerful alternative to morphology for inferring phylogenetic relationships at all taxonomic levels The result on analysis and

alignment of 18S rDNA sequences confirms Isochrysis H5 identification as Isochrysis galbana

Figure 2 shows the18S rDNA amplification products

The phylogenetic tree constructed from neighbour – joining analysis of the sequences of

18SrDNA depiciting relationships of species (Figure 3)

3.2 Selection of culture medium

Optimization of culture conditions for the selected strains is essential for algal mass culture Furthermore, growth characteristics have been shown to have a significant impact on the lipid and fatty acid profiles [1,7] In this study, medium f/2, ASW and ESM were used The cells were collected every two days and counted (Table 1)

H5 Isochrysis galbana

Gephyrocapsa oceanica Emiliania huxleyi

Coccoid haptophyte

88

100

100

Phaeocystis antarctica

96

Prymnesium nemamethecum Prymnesium patelliferum Chrysochromulinda polylepis

100

Chrysochromulina hirta

100

Coccolithus pelagicus Coccolithus braarudii Cruciplaccolithus neohelis

100

Pleurochrysis carterae Pleurochrysis carterae

100

100

87

73

0.005

100

Figure 3 The phylogenetic tree constructed from neighbour – joining analysis of the sequences of

18SrDNA

Table 1 Cell density of Isochrysis galbana grown on different media

Time (days)

Cell density (x 106/ml)

Isochrysis galbana H5 grew the better in f/2 than in the ESM media and ASW media

The growth of Isochrysis galbana H5 reached the highest peak after 10 days culture (7.15 x 106 cell/ml) It is suggested that f/2 is the most suitable for culturing Isochrysis galbana H5 and can

be applied for biomass production in aquaculture

3.3 Fatty acid composition

Lipid has its own specificity in each species of living organisms Fatty acids are mostly located in the cell membrane, which is much enough to apply to taxonomic characterization

[3,12] Fatty acid components of Isochrysis galbana H5 were analysed (Table 2)

Table 2 Composition of fatty acids in Isochrysis galbana H5

weight)

The total fatty acids in Isochrysis galbana H5 are 74.37 g/mg fresh weight Isochrysis

galbana H5 has a great variety of fatty acids ranging from 14C to 22C, among C16:0, C18:4n-3

and C22:0 are the most abundant fatty acids The compossition is different from previous report

[10] but major variations in the fatty acid compositions of Isochrysis galbana have been reported [12] More than 40 % of total fatty acids (30.9 g/mgfresh weight) in Isochrysis galbana H5 is polyunsaturated fatty acids, suggesting the high quality of Isochrysis galbana H5 and the

potential to be applied in aquatic food webs such as for feeding geo-ducks In addition, DHA had

remarkably high content (5.53 g) while EPA was at low abudance, smilar to Isochrysis sp Clone

T.ISO in previous report [12] DHA and EPA play an important role in the membrane lipids

3.4 Application of Isochrysis galbana H5 for feeding geo-duck at Vandon, Quangninh

Isochrysis galbana H5 was cultured in F/2 medium, grown at room temperature and

illuminated under neon light (Philips daylight tubes) 4000 - 5000 Lux as the following scheme with illustrated pictures

Microalgae volume was used depending on geo-duck larvae density.Geoduck larvae with 2.5x105 -16.6x105 individuals per m3 were fed in 120 – 180 liters microalage with cell density 7.7 - 8.2

106 cells/ml per day

Conclusions

The Isochrysis strain were successfully isolated from Vandon, Quangninh and identified

as Isochrysis galbana The suitable medium for strain was f/2

The Isochrysis galbana strain showed a huge range of fatty acids among, contained

remarkable amount of PUFA and considerate level of EPA and DHA which play an essential role

in cell membrane physiology and hormone metabolism, suggesting high value as food for amimals, specially for feeding geo-duck larvae

Stock culture

Cell density

1.25-2.1 105 cells/ml

2-6 litre flask

After 5-7 days, cell density

4.2 106 cells/ml

30–60 litre plastic bag

Cell density 5.7- 7.15 106

cells/ml after 4-6 days

160 litre tank

After 4-6 days, cell density 7.7

- 8.2 106 cells/ml

10 litre container

After 2-3 days, cell density

5.3-6.4 106 cells/ml

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