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AQUACULTURE EXTENSION MANUAL NO. 55

JULY 2013

Culture of marine phytoplankton for aquaculture seed production Milagros R. de la Pe単a Annie V. Franco

Southeast Asian Fisheries Development Center AQUACULTURE DEPARTMENT www.seafdec.org.ph

AQUACULTURE EXTENSION MANUAL NO. 55

Culture of marine phytoplankton for aquaculture seed production Milagros R. de la Pe単a Annie V. Franco

Southeast Asian Fisheries Development Center AQUACULTURE DEPARTMENT www.seafdec.org.ph

JULY 2013

ON THE COVER [CLOCKWISE]: Isochrysis galbana, Navicula ramossisima, Tetraselmis tetrahele, Skeletonema tropicum [PHOTOS by D Catedral]; primary stock cultures in test tubes, 1-10 L jars and 1-ton tank [PHOTOS by AV FRANCO]; use of a haemacytometer in counting microalgae under a microscope [PHOTO by DEVCOM]

Culture of marine phytoplankton for aquaculture seed production [AQUACULTURE EXTENSION MANUAL NO. 55]

JULY 2013

ISSN 0115-5369

Published and printed by: Southeast Asian Fisheries Development Center Aquaculture Department Tigbauan, Iloilo, Philippines Copyright Š 2013 Southeast Asian Fisheries Development Center Aquaculture Department Tigbauan, Iloilo, Philippines

All rights reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher

For comments and inquiries

SEAFDEC Aquaculture Department Tigbauan, Iloilo 5021, Philippines

Tel Fax Email AQD website

(63-33) 511 9172 (63-33) 511 8709 aqdchief@seafdec.org.ph www.seafdec.org.ph

FOREWORD Natural food or plankton production is essential in producing quality seeds for aquaculture. No hatchery for marine species can do without it because live food is crucial to the health and survival of larval and postlarval fishes, shrimps, and mollusks.

The techniques described in this manual are products of hard work by SEAFDEC/ AQD staff. In the early years, microalgae had to be isolated in the laboratory from natural waters, raised in enough volumes to be tested if acceptable to larvae of different cultured species, their mass propagation standardized and improved, and later, starter cultures made available to hatcheries throughout the Philippines and in other countries. While SEAFDEC/AQD maintains and preserves its algal culture collection, it also continues to search for other live food organisms that may be appropriate food during the early larval stages and can be used as substitute for the expensive brine shrimp Artemia.

SEAFDEC/AQD has extended microalgal production techniques to hatchery technicians and other aquaculture stakeholders through hands-on training. And we hope that through this manual, stakeholders can send us feedback on the technology to enable us to refine and improve the culture of live food organisms.

Felix G. Ayson, D.Sc. Chief

Table of contents FOREWORD, v INTRODUCTION, p 1 BIOLOGY OF PHYTOPLANKTON, p 1 Diatoms (Bacillarophyta), p 1 Skeletonema spp., p 1 Chaetoceros spp., p 2 Thalassiosira spp., p 2 Navicula spp., p 3 Amphora spp., p 3 Nitzschia spp., p 3 Green algae (Chlorophyta), p 3 Dunaliella spp., p 3 Chlorella spp., p 3 Nannochlorum spp., p 3 Tetraselmis spp., p 3 Golden brown algae (Prymnesiophyta), p 4 Isochrysis spp., p 4 Yellow-green algae (Eustigmatophyta), p 4 Nannochloropsis spp., p 5 Blue-green algae (Cyanophyta), p 5 Spirulina spp., p 5 NUTRITIONAL VALUE, p 5

Protein, p 8 Lipids, p 8 Carbohydrates, p 9

CULTURE TECHNIQUES, p 10 Design of culture facilities, p 10 Collection of phytoplankton, p 10 Isolation techniques, p 10 Biological isolation, p 10 Serial dilution, p 10 Repeated subcultures, p 12 Capillary pipette method, p 12 Streak plating, p 12 Purification techniques, p 12 Sterilization of culture materials, p 14 Water treatment, p 14 Culture vessels, p 15 Enrichment solution, p 15 GROWTH KINETICS OF PHYTOPLANKTON, p 16 Lag phase, p 16 Exponential or log phase, p 16 Stationary phase, p 16 Death, p 16

REQUIREMENTS OF AN ACTIVELY GROWING CULTURE, p 17 Viable inoculum or starter, p 17 Supply of needed nutrients and microelements, p 17 Suitable physico-chemical conditions, p 17 Illumination, p 17 Temperature, p 18 Aeration, p 18 MAINTENANCE AND MASS PROPAGATION, p 18

Culture vessel and tanks, p 19 Stock cultures, p 19 Volume of inoculum, p 20 Scaling–up of culture, p 20 Programming, p 21 REFERENCES, p 22 APPENDICES, p 25 I. Culture media, p 25 II. Multistep culture of phytoplankton at SEAFDEC/AQD, p 27 III. Counting of phytoplankton, p 28 GLOSSARY, p 31 Acknowledgment, p 32 About the authors, p 33

L

ive food organisms or plankton play a crucial role in all aquaculture systems.

They are organisms that include the plant and animal life grazed upon by young fishes, crustaceans and mollusks. In the marine environment, they serve as the primary (phytoplankton) and secondary (zooplankton) producers of the food web.

Phytoplankton is a group of autotrophic plankton with pigments or chromatophores that are able to produce organic components through photosynthesis. This process of organic production comes from inorganic nutrients, water and CO2 (carbon dioxide) in the presence of sunlight. Phytoplankton species are unicellular and mostly microscopic in size. In the food pyramid, they contribute the highest biomass that serve as the basis of marine biological production.

Zooplankton, on the other hand, is the animal component of plankton. Zooplankton species are often referred to as herbivores or grazers, feeding heavily on phytoplankton. They are considered as secondary producers since small fishes and crustaceans feed on them. They are rich in protein and lipids. They are considered water purifiers since they consume bacteria and detritus.

The culture of live food organisms is considered the heart of the hatchery or the seed production of economically important cultured species. Availability of suitable live food is one of the most critical factors in larval survival and growth. Hatchery operators can not get a continuous supply of plankton from the natural environment because physico-chemical parameters can not be controlled and unwanted species are present. Hence, most hatcheries provide for the culture of live food.

BIOLOGY OF PHYTOPLANKTON There are four major groups of microalgae commonly used in aquaculture seed production:

Diatoms (Bacillarophyta) Diatoms are numerous and the most important aquatic photosynthesizers. They are ubiquitous, occurring in marine and freshwaters, and can be found floating or attached to surfaces. Their most distinguishing feature is the presence of a silica cell wall called the frustule. They are mostly unicellular but can exist as colonies in the shape of filaments. As food producers, they play an important role in the food web.

Diatoms appear in seasons when environmental factors such as temperature and nutrients are most suitable for their growth and reproduction. Due to high carotene and diatoxanthin content, chromatophores appear brown. The commonly cultured diatom species are: Skeletonema spp. This is a widely distributed species that can tolerate a wide range of salinity and temperature. Skeletonema is used as larval feed for shrimps. Cells occur in chains formed by strutted tubular processes arranged in a marginal ring (Fig. 1 A-D). Two commonly cultured species are Skeletonema costatum (temperate strain) and S. tropicum (tropical strain). Skeletonema sp. measures 10 Îźm in length and 9 Îźm in width.