55AQUACULTURE EXTENSION MANUAL NO. JULY 2013 Culture of marine phytoplankton for aquaculture seed production Milagros R. de la Peña Annie V. Franco Southeast Asian Fisheries Development Center AQUACULTURE DEPARTMENT www.seafdec.org.ph 55AQUACULTURE EXTENSION MANUAL NO. JULY 2013 Culture of marine phytoplankton for aquaculture seed production Milagros R. de la Peña Annie V. Franco Southeast Asian Fisheries Development Center AQUACULTURE DEPARTMENT www.seafdec.org.ph 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 © 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 Live 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 dofiooxrigdaen)iicnptrhoedpurcetsioenncceoomfessufnrloigmhti.nPorhgyatnoipclannuktrtoiennstsp,ewciaetseraraendunCicOe2ll(uclaarrbaonnd 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 μm in length and 9 μm in width. Figure 1. THE DIATOMS AB CD PHOTOS by A Franco A FRANCO PHOTO by D CATEDRAL oSfkSe.letrtoopniecmuma spp. in chains (A); intercalary (B). This alga is fed to shrimp valves nauplii (arrow) (C) and zoea (D) EF PHOTO by D CATEDRAL and A FRNCO M de la PEŃA Chaetoceros calcitrans (E, arrows in inset show setae or spines) is fed to the copepod Acartia (F) GH PHOTO by AV FRANCO PHOTO by D CATEDRAL PHOTOS by M de la PEŃA Thalassiosira sp. (G) and Navicula ramossisima (H, red arrow) being grazed by an abalone veliger (yellow arrow shows its mouth) IJ K PHOTO by M de la PEŃA PHOTO by D CATEDRAL 2 μm N. ramossisima (I, J) is boat-shaped with a raphae (arrow); Amphora sp. (K) under the scanning electron microscope; and the spindle-shaped Nitzchia sp. (L) L 2 μm Chaetoceros spp. They occur as solitary chain of cells (Fig. 1E, inset and F). The most distinguishing feature is the presence of setae. Some cells are solitary. Three species of Chaetoceros are cultured in great quantity as larval feed for shrimps and copepods: C. calcitrans, C. mulleri and C. gracilis. Chaetoceros sp. measures 8 µm in length and 5 µm in width. Thalassiosira spp. They occur as chains or cells embedded as mucilage. Cells in chain are lined by threads extending from the marginal strutted processes. Cells are about ≥ 3-5 µm in diameter and circular (Fig. 1G). This species is also cultured as larval feed for shrimps. The most common cultured species is T. pseudonana. Navicula spp. This species can tolerate a wide range of temperature. Since it is a benthic dweller and can attach to surfaces, this alga is allowed to grow as diatom mats on plastic or corrugated polyvinyl sheets for abalone postlarvae and early juveniles. Navicula can exist as single cells or as ribbon of cells. The valve is boatshaped and each bears a raphe (Fig. 1 H-J). The commonly cultured species is Navicula ramossisima. Navicula sp. measures 8-10 μm in length and 5-8 μm in width. Amphora spp. Amphora sp. (Fig. 1K) is classified as a small periphytic diatom characterized by smooth arched valve on the dorsal side with a weakly convex ventral margin. This species can form flat diatom communities and is highly adhesive, two characteristics considered favorable for abalone larvae. The alga measures 11-13 µm in length and 3-3.5 µm in width. Nitzschia spp. This diatom is cultured in great quantities as food for oyster and abalone. Cells are linear in both valve and girdle view (Fig. 1L). The commonly cultured species are Nitzschia alba and N. closterium. Nitzschia sp. measures 8-10 μm in length and 3 μm in width. Green algae (Chlorophyta) This group of algae has the greatest number of species and is most widely distributed. They are known as chlorophytes because they appear grass green. The green color is due to the presence of chlorophyll a and b in the chloroplasts. The cell wall is composed of cellulose micro-fibril (inner) and pectin (outer). Some species (Platymonas, Tetraselmis, and Chlamydomonas) are motile due to the presence of flagella. The commonly cultured species are: Dunaliella spp (Fig. 2A). The cells are unicellular and the cell walls are indistinct. Cells are elliptical, pyriform or spindle-shaped. The anterior end of the cells has two to eight flagella. It is a halophilic species. Dunaliella-extracted pigments incorporated in shrimp diet showed beneficial effects to shrimp. Two commonly cultured species are D. salina and D. tertiolecta. Dunaliella sp. measures 10-13 μm in length and 10 μm in width. Chlorella spp. These are spherical or ellipsoidal unicells found in fresh and marine waters. Cell size ranges from 2 to 12 µm. Cell shape ranges from globular to wide elliptical (Fig. 2B). C. pyrenoidosa, C. vulgaris, and C. virginica are among the species included in this group. They are commonly used as food for zooplankton (Fig. 2C). Nannochlorum spp. They are small (2-3 μm) coccoid alga that morphologically resembles Chlorella spp. but lack pyrenoid in its cell structure (Fig. 2D). This species are used as food for rotifer and can be easily cultured under tropical conditions. Tetraselmis spp. The local isolate Tetraselmis tetrathele (Fig. 2E) is a fourflagellated prasinophyte characterized by an ovoid body shape and a distinct curved body when viewed sideways. The alga measures 10-16 μm in length, 8-11 μm in width and 4.2-5.0 μm in thickness. Tetraselmis spp. are widely used for crustaceans SEAFDEC Aquaculture Department 3 PHOTO by A FRANCO PHOTOS by M de la PEŃA PHOTO by M de la PEŃA PHOTO by A FRANCO PHOTO by D CATEDRAL A C B Figure 2. THE CHLOROPHYTES: Dunaliella sp (A); Chlorella sp. (B) is fed to Brachionus rotundiformis (C); Nannochlorum sp. (D); motile Tetraselmis tetrathele (E) is fed to the cladoceran Diaphanosoma sp (F) D EF and bivalves. It is also used for mass culturing the cladoceran, Diaphanosoma celebensis (Fig. 2E). Tetraselmis spp. are ideal for culture because they are euryhaline (can tolerate 10-35 ppt) and eurythermal (can tolerate 5-33oC). Golden brown algae (Prymnesiophyta) The golden brown or golden yellow microalgae belong to Prymnesiophyta. They are also called Haptophyta due to the presence of haptonema, a filamentous appendage between the two smooth flagella. The cells are flagellated and have chlorophyll a, c1 and c2 and fucoxanthin as the major carotenoid. The presence of non-oxygenated carotenoid known as phycochrysin is responsible for their coloration. The total amount of pigment contains 75% carotenoid. Cells have periplasts instead of true cell walls. Isochrysis spp. The cells are elliptical or elongate elliptical (Fig. 3A). Cell width ranges 5-6 µm. Isochrysis has been widely used as a mariculture feed due to its high content of long-chain polyunsaturated fatty acids (PUFAs). They are commonly used as food for shellfishes like window-pane oyster or the kapiz shell Placuna placenta and angelwing clam Pholas orientalis. Yellow-green algae (Eustigmatophyta) The class Eustigmatophyceae is a distinct group due to the presence of eyespot (stigma) in the zoospores. They are mostly unicellular and coccoid organisms living in freshwaters and soil. 4 Natural food production PHOTOS by D CATEDRAL A BC PHOTOS by A FRANCO Figure 3. The golden brown microalga Isochrysis galbana (A); the marine green alga Nannochloropsis oculata (B); and the blue-green microalga Spirulina subsalsa (C and inset) Nannochloropsis spp. This is a marine coccoid alga (Fig. 3B). that resembles Chlorella. The cells are small (2-4 µm) and spherical to slightly ovoid in shape. It has a high content of the polyunsaturated fatty acid (eicosapentaenoic acid) and is thus useful to marine animals. N. oculata is the most studied and has been used for rotifer culture. Blue-green algae (Cyanophyta) Blue-green algae are also called cyanobacteria. They are unicellular, colonial and filamentous in form. Some have simple parenchymatous organization with no flagella. The photosynthetic pigments are chlorophyll a, phycocyanin, allophycocyanin (gives the algae their blue color), and the red phycoerythrin pigment. They are nitrogen-fixing so there is no need for external carbon or nitrogen source. Spirulina spp. Arthrospira platensis commonly known as Spirulina is a blue-green alga characterized by helical trichomes. This alga can thrive in high alkaline (pH 8.5-11) and high salinity (>30 ppt) ponds. It is considered as one of the richest natural food. It contains Vitamin B12; has gamma linolenic acid; and is rich in phycocyanin (10-12%), beta carotene, xanthophylls and chlorophyll (1-2%). Spirulina has been cultivated as food supplement due to its high protein content (70%) and high phytochemical content (Fig. 3C). In aquaculture, it is used as feed additive to increase immune resistance of shrimps against white spot syndrome virus. In sandfish, Spirulina powder brushed in polyvinyl corrugated sheet can induce settlement of larvae. NUTRITIONAL VALUE Microalgae offer rich sources of protein and lipids (Table 1). The vitamins and pigments found in microalgae are of better quality and are healthier. Their phycobiliproteins are sources of natural blue pigment dyes used for food and cosmetics, and these mostly come from blue-green algae. SEAFDEC Aquaculture Department 5 Table 1. Protein, lipid and essential fatty acid content of some well-studied algae Species Amphora coffeaformis Crude protein (%) Lipid content (%) Essental fatty acids 20E:P5Aω3 2D2:H6Aω3 20A:R4ωA6 References 29.4 19.7 Renaud et al. 1999 Amphora sp. Chaetoceros sp. 26.481.5 36.7 8.6-17 0.3 0.8 13.9 16.7 4.9 de la Peña 2007; Renaud et al. 1999 3 Renaud et al. 1999; Whyte 1987 Chaetoceros calcitrans Chaetoceros gracilis Chaetoceros muelleri 2.625.1 46 8.916.9 0.2-1.5 2.915.7 1-1.5 11.314.9 12 1.111.8 Fernandez-Reiriz et al. 1989; Millamena et al. 1990; Shamsudin 1992; Thompson et al. 1990; Whyte 1987 Thompson et al. 1990 Hoj et al. 2009 Chlorella sp. Chlorella ellipsoidea Chlorella pyrenoidosa Chlorella vulgaris Dunalliela salina 35.358 57 18.432 13.527.2 13.435.8 11.857.9 6 0.2-0.3 1.127.8 26.6 0.1-3.6 Chisti 2007; James et al. 1989; Millamena et al. 1990 Shifrin & Chisholm 1981 Roessler 1990; Shifrin & Chisholm 1981 2.4 Millamena et al. 1990; Roessler 1990; Spolaore et al. 2006 Spolaore et al. 2006 Dunalliela tertiolecta 17.268.7 1.521.2 0.1 0.1 Shifrin et al. 1981; Thompson et al. 1990; Wilfors et al.1984; Wifkors 1986 Dunalliela sp. 47.4 Millamena et al. 1990 Fragilaria sp. 1 6.8 8.7 Renaud et al. 1999 Isochrysis galbana 13.340 19.936.2 1.7-22 0.1-0.6 Fernandez-Reiriz et al. 1989; Millamena et al. 1990; Shamsudin 1992; Thompson et al. 1990; Whyte 1987; Zhu et al. 1997 Isochrysis sp. 35.638.1 Isochrysis sp. (T-ISO) Nannochloropsis oculata 48 23.444.8 7.136.4 23.530 22.135.8 0.211.4 12-17 0.2-10 0.223.7 0.3-17 0.9-40 Chisti 2007; James et al. 1989; Millamena et al. 1990; Renaud et al. 1991, 1995; Roessler 1990 Brown et al. 1993; Hoj et al. 2009; Whyte 1987 0.9-7.5 Hodgson et al. 1991; Renaud et al. 1991,1999 Nannochloropsis sp. Nannochloris sp. 17.241 17.768 2047.8 0.2-3.8 5-58.4 1.8-7.8 Chisti 2007; James et al. 1989; Lubzens et al. 1995; Maruyama 1986; Sukenik et al. 1993 Chisti 2007; Roessler 1990; Shifrin & Chisholm 1981 6 Natural food production Species Nannochloris atomus Navicula acceptata Navicula sp. Navicula halophila Navicula pelliculosa Nitzschia sp. Nitzschia closterium Nitzschia palea Pavlova sp. Pavlova lutheri Phaeodactylum tricornutum Porphyridium cruentum Skeletonema costatum Skeletonema sp. Spirulina maxima Tetraselmis sp. Tetraselmis chui Tetraselmis suecica Tetraselmis maculata Tetraselmis pseudonana Tetraselmis tetrahele Thalassiosira weissflogii Crude protein (%) Lipid content (%) Essental fatty acids 20E:P5Aω3 2D2:H6Aω3 20A:R4ωA6 References 3.2 1.1 Volkman et al. 1989 19.238.2 16.9 2.1 7.9-9.3 Roessler 1990 Al-Hasan et al. 1994; Lee et al. 2009 31.4 18.4 Kavitha et al. 2006 41.5 22.528.4 16-43 2.617.5 25.1 16-47 0.3 28.637.8 2038.8 0.1-1.3 22.239.5 13.2 3.6-9.7 0.4-1.1 5-8.4 6.528.4 18 16.219.4 28.4 Coombs et al. 1967 6.7-8 Chisti 2007; Renaud et al. 1999 0.2-2.7 Renaud et al. 1995 Roessler 1990; Shifrin & Chisholm 1981 0.4 Patil et al. 2007 Fernandez-Reiriz et al. 1989; Thompson et al. 1990 2.2 Chisti 2007; Fernandez-Reiriz et al. 1989; Patil et al. 2007 22.327.9 28.1 60-71 26.429.9 48.4 40.9 15.638.5 20.449.6 6.1 6 Patil et al. 2007 13.530.3 13.3 1.4-8.5 1.4-1.8 4.9-13 12.9- 13 Millamena et al. 1990; 0.8 Renaud et al. 1999; Shamsudin 1992; Shifrin & Chisholm 1981 0.9-1.4 Renaud et al. 1999 6-7 Spolaore et al. 2006 12.613.8 0.10.2 3.9-4.8 0.2-0.7 Millamena et al. 1990; Renaud et al. 1999; Patil et al. 2007 Millamena et al. 1990 5.924.4 8.320.8 9-26.4 12.819.2 22.224 1.4-3.2 4.3-5.3 7.716.5 0.1-0.3 2.9 Chisti 2007; Fernandez-Reiriz et al. 1989; Volkman et al. 1989; Whyte 1987 Wifkors et al. 1984; Wifkors 1986 Thompson et al. 1990; Whyte 1987 de la Pena & Villegas 2005 Shifrin & Chisholm 1981 SEAFDEC Aquaculture Department 7 The nutritional value of live food organisms plays a significant role in the growth and survival of cultured species. Microalgae are fed to or are prey of larval and postlarval stages of fish, abalones, sandfish, shrimp and other crustaceans. They can be given directly as pure algal cells or in processed form which are incorporated in formulated diets. For fish rearing, high lipid microalgae are used to enrich rotifers to improve growth and survival. The biochemical composition and cellular structure of microalgae determine their nutritional value. These vary among algal species and strains according to genetic and environmental origins, and are affected by culture conditions (nutrient, temperature, pH, illumination, stage of harvest). However, conditions can be optimized to maintain the essential nutrients needed for growth. Microalgae for hatchery use should have a digestible cell wall to make the nutrients available. The cell size, growth rate and stability of culture are also additional factors to be considered in evaluating microalgae as potential food source for aquaculture. It is important to establish the optimum culture methods and conditions of microalgae for the desired cultured species. Besides being an effective source of nutrients, the use of microalgae in the hatchery have many beneficial effects: • Positive effect of beneficial bacteria associated with algal cultures • Presence of active substances that have a positive effect on the digestive or immune system of fish • Provision of visual contrast in the rearing water that have a beneficial effect on the feeding behavior of larvae • Stabilizing effect on the quality of the rearing water • Microalgal secretions can control harmful bacterial population, ie. probiotic effects in the rearing water Protein Protein is a major organic constituent of algae and performs an essential role in the structure and functioning of all living things. The protein content of microalgae ranges 2.6-71 % of dry weight with diatoms having lower values (Table 1). In microalgae, the amino acids are qualitatively similar but are quantitatively different among species. Essential amino acids are provided in the diet because they cannot be synthesized by animals. Lipids The quality and quantity of lipids are of major importance. Several studies have shown that lipids have a significant role in the growth and metamorphosis as well as in the improvement of spawning and egg quality of cultured species. Lipids also play a vital role in the structure of biological membranes. Animals cannot synthesize the essential fatty acids needed for growth and survival. It must be introduced in the diet via the food chain from zooplankton consuming the omega-3 (ω3) long-chain polyunsaturated fatty acids (PUFA) 8 Natural food production synthesizing microalgae. Many studies have shown that the presence of longer chain highly unsaturated fatty acids (HUFAs) in the lipid profile (especially EPA and DHA) in the microalgal diet were associated with high growth rate of bivalve larvae. For instance, Pavlova lutheri with its high PUFA is used in mariculture as food for rotifer; Chaetoceros spp. are valued food for the oyster Ostrea edulis due to the high 22:6ω3 content. The important fatty acids for many marine invertebrates and fish are the following: • Eicosapentaenoic acid (C20:5ω3, EPA) • Docosahexaenoic acid (C22:6ω3, DHA) • Arachidonic acid (C20:4ω6, ARA) The lipid and fatty acid contents vary among microalgal species (Table 1) depending upon growth conditions. The green algae Nannochlorum atomus, Dunaliella tertiolecta and Tetraselmis suecica do not contain high concentration of PUFAs. Freshwater Chlorella are often unsuitable food compared to marine Chlorella which contain high concentration of 20:5 (n-3). The EPA profile of marine Chlorella and Nannochloropsis sp. is species-dependent and varies according to the temperature prevailing in the culture system (Table 1). The diatoms Chaetoceros and Skeletonema have moderate EPA content, while Fragilaria has the highest amino acid content. P. tricornutum contains high EPA, while Isochrysis and Nannochloropsis are rich in DHA. In Cryptomonas and Pavlova, both EPA and DHA are found in moderate concentrations. Fatty acid composition of microalgae can be optimized by adjusting the photon flux density of tropical sunlight in commercial-scale aquaculture facilities. Carbohydrates Carbohydrates are among the most important cell components in microalgae. In green algae Chlorella sp., carbohydrates are accumulated as assimilatory product of photosynthesis. It is an intracellular storage material in the form of starch and several sugars including glucose and polysaccharide. Carbohydrates are used for many cell functions and cellular structures for energy storage. In some algae, carbohydrates are used as intermediary reserves when nitrogen becomes limited in the synthesis of lipids. Benthic diatoms excrete extracellular polysaccharides which serve as food for early larval stages of abalone. In the plant cell wall, carbohydrates are found in the structural components of plant cell wall (cellulose). The carbohydrates found in microalgae range from 4 to 23%. Nannocloropsis oculata and Isochrysis galbana (Tahiti-strain) have lower amounts of carbohydrates when cultured indoors and higher in shaded outdoor culture conditions. SEAFDEC Aquaculture Department 9 CULTURE TECHNIQUES Design of culture facilities Sample layouts of phycology and natural food culture laboratories are shown in Fig. 4. Collection of phytoplankton The source of phytoplankton for mass propagation may come from culture collections or from natural populations. Algal inoculum from culture collections come in pure form (unialgal) though not always bacteria-free. Algal starters coming from natural populations are subjected to isolation and purification to attain a unialgal form. To do this: (1) Collect planktons using 50 µm mesh size (Fig. 5) netting material in open sea or in brackishwater ponds. Keep in sampling bottles. (2) Transport sampling bottles in styrofoam box with ice to maintain 20-250C temperature. (3) Examine the samples under the microscope. (4) Enrich sample with suitable medium at the laboratory. (5) Aerate and expose to appropriate light condition. (6) Isolate the desired species using suitable method/s. Isolation techniques The isolation of a specific phytoplankton from the collected crude sample is a prerequisite for the establishment of unialgal cultures. Unialgal cultures are started from clones propagated from single cell or filament. Several methods are employed in the isolation of single cells depending upon algal size and the characteristics of the desired species for isolation. Combinations of isolation techniques are recommended. Biological isolation. This method of isolation depends on the phototactic response of the organisms to light. This is effective for flagellated organisms (e.g. Tetraselmis, Chlamydomonas, and Isochrysis) which will concentrate towards the light source. The organisms are collected and transferred to sterile seawater using fine pipette. This process may be repeated several times until unialgal cells are attained (Fig. 6). Serial dilution. The collected mixed population is diluted to lower the number and kind of organisms. The crude sample is diluted by means of a series of transfers in a tube containing sterile seawater or enrichment medium (Fig. 7A). The diluted sample is then exposed to ambient temperature and light conditions. Usually the dominant species in the mixed populations are the ones that are successfully isolated. 10 Natural food production Figure 4A. Lay-out of a 6 x 5 m phycology laboratory: main components include (1) isolation room for purifying samples and obtaining new algal strains; (2) stock culture room for maintaining algal stock cultures; (3) working laboratory where culture of algal starters in small culture volumes; (4) hot room for sterilization of glasswares; (5) chemical room for media preparation; and (6) reservoir area for storing filtered clean seawater 4B. A typical larval food / phytoplankton culture area has: (1) wet laboratory or phytoplankton culture area for scaling-up cultures from 10 L to 20 ton tanks. Smaller tanks (10 L) are made of plastic carboys while bigger tanks (500 L 1 ton) are made of fiberglass. Large-scale outdoor tanks (5 – 20 tons) are concrete light banks for 10L culture (carboy) and 30L basins reservoir ozone machine SEAFDEC Aquaculture Department 11 Figure 5. Towing a plankton net (inset) for collecting plankton from the sea rtoopwe bridle hoop bcollotitnhg catcaynlonivntdahgss canvass receiver PHOTO by AJIJO MUYIWA REUBEN Repeated subcultures. The diluted sample is exposed to several media and to different conditions of temperature and light intensities (Fig. 7B). Those species that favor certain culture conditions will grow successfully on each culture vessel. This process can be repeated until unialgal cultures are achieved. Capillary pipette method. This method is the simplest technique for isolating algae. A micropipette is used to separate individual cells through a series of sterile washes under a stereoscope or an inverted microscope (Fig. 7C). Single cells are transferred from the first drop to the next drop until only one single cell is present in a drop of vivid medium. Finally, the single cell is transferred to tubes containing the sterile medium. This technique requires proper training and practice before satisfactory results are obtained. Streak plating. This type of isolation is recommended for small algae with firm cell walls (e.g. Chlorella, Chlamydomonas). This technique uses the agar plates enriched with seawater medium. Crude samples are either streaked or poured on top of solidified agar plates (Fig. 7 D-E). After a few days of incubation, colonies that have grown are picked-up and transferred into sterile medium. Purification techniques Starting from a unialgal culture, one can obtain a pure culture by using some of the isolation techniques with only slight refinements. Suggested methods to obtain pure cultures are as follows: (1) repeated streak plating (2) repeated micropipette washing (3) centrifugation – to remove bacteria, cells are concentrated and transferred repeatedly into a new medium (Fig. 8) (4) antibiotic treatment – involves serial transfer of cells at 1-3 days interval 12 Natural food production PHOTOS by A FRANCO Figure 6. Concentration of algal cells towards light source (above),and collection of microalgae using a pipette at the surface where cells have concentrated (right) original sample 1 2 3 4 5 6 7 8 A tubes with sterile medium B Figure 7. ISOLATION TECHNIQUES: serial dilution (A); repeated cultures (B); capillary pipette method using an inverted microscope (C); and streaking of sample in agar plates (D). Tetraselmis sp. growing in agar slants (E) PHOTO by A FRANCO original sample diluted sample different media D C E PHOTO by M de la PEŃA PHOTO by A FRANCO Figure 8. PURIFICATION TECHNIQUE: Culture tubes are centrifuged at 3500 rpm for 20 minutes to concentrate algae as “pellets” (arrow) through solutions containing penicillin, streptomycin, chloramphenicol or a suitable mixture of the three antibiotics. Preliminary test should be done first to determine the tolerance level for each antibiotic. The minimum effective concentrations are used. Sterilization of culture materials In the culture of phytoplankton, sterilization is mandatory to avoid contamination from other sources. Water treatment. Water which serves as the base for culture medium must be free of any toxic or unwanted sediments. Some of the ways to achieve “clean” water for culture are: (1) physical filtration (Fig. 9) using (a) membrane filters (0.45 µm millipore); (b) cartridge filters (0.22 µm and 3 µm pore sizes); (c) sand filtration to remove silt and debris which is applicable to a large volume of seawater; and filter bag (0.5 µm) to remove fine sediments Figure 9. FILTRATION APPARATUS: 0.45µm air cartridge and 5.0µm filter cartridge (left) installed prior to aerating cultures in light banks and 5 µm filter bag for filtering water in large tanks (right) 14 Natural food production PHOTOS by A FRANCO PHOTOS by A FRANCO Figure 10. STERILIZATION EQUIPMENT: (clockwise from top left) pressure cooker; ozonator; autoclave; and hot air oven (2) chemical treatment where seawater is treated with 10 ppm chlorine for 24 hours then neutralized with equal amount of sodium thiosulfate (3) ultraviolet radiation or ozonation (4) boiling for at least 15 minutes (applicable to small seawater volume) (5) steam sterilization (1210C) Culture vessels. The following methods may be used for sterilizing culture vessels used for culturing algae: (1) hot air ovens for small glasswares; (2) air-drying and exposure to sunlight for bigger culture vessels; (3) soaking or disinfection with dilute HCl solution; and (4) covering glasswares with aluminum foil, cotton or gauze to minimize aerial contamination. Enrichment solution. Prepared media (enriched seawater) are steam-sterilize with the use of autoclave or pressure cooker (15 min. at 15 lb. pressure) (Fig. 10). SEAFDEC Aquaculture Department 15 GROWTH KINETICS OF PHYTOPLANKTON Knowledge of the algal growth phases is necessary to understand the growth of algal population in a limited culture volume. This will help determine the viability of cells to be used as inoculum and to predict time of harvest. Algal growth phase is characterized by a sigmoid growth curve with four distinct phases (Fig. 11). stationary phase cell desity (cells/mL) exponpehnatsieal lag phase death Incubation period (days) Figure 11. Growth kinetics of microalgae Lag phase This phase is characterized by zero growth. The population remains unchanged or decreases. The newly added inoculum adapts to the new culture conditions. Logarithmic or exponential phase The cells in this phase divide fast in constant geometric progression. The cells have high metabolic rate. This is the phase when algae are harvested as inoculum. Stationary phase The population at this phase remains constant or steady. This may be caused by nutrient limitation in medium and aging of cells. At this phase, the culture is light-limited. Usually, the cells in this phase are harvested for feeding cultured invertebrates. Death This is the phase of declining growth. Algal culture collapses and nutrient in the culture medium is exhausted. 16 Natural food production PHOTO by A FRANCO REQUIREMENTS OF AN ACTIVELY GROWING CULTURE Viable inoculum or starter Inoculum or starter is the small volume of algal suspension needed to initiate culture for subsequent mass propagation. The starter must be free of contamination and must come from a reputable institution or reliable source so that the species to be cultured is properly identified. Supply of nutrients and microelements For proper growth and propagation, phytoplankton requires a number of elements classified as macronutrients (nitrogen, N; phosphorus, P; sulfur, S; potassium, K; magnesium, Mg) for cellular building blocks, and micronutrients (iron, Fe; manganese, Mn; copper, Cu; molybdenum, Mo; silica, Si) as catalysts or for other unique functions. These minerals are added in seawater (enriched seawater media) or in distilled water (artificial seawater media) in sufficient amounts. Although artificial media showed consistent results for algal cultures, enriched seawater media is preferably used. It is cheaper and simpler to prepare. The chemical composition of the medium has been derived and modified from basic formulations depending upon the requirement of cultivated algal species. At SEAFDEC/ AQD, three algal media are commonly used for marine microalgae (Appendix I A-C): (1) Gillard and Ryther’s Modified F medium (2) Walne’s Conwy medium (Fig. 12) (3) Liao and Huang’s TMRL medium Figure 12. Walne’s Conwy medium Reagent grade chemicals are used for stock cultures while technical and commercial grade fertilizers are used in large-scale cultures. The use of nonconventional media like the agricultural fertilizers provide cheap alternatives of expensive analytical reagent grade chemicals. Suitable physico-chemical conditions Illumination. Light is a source of energy for photosynthesis. The degree of illumination needed by cultures depends upon the type of algae and the scale of culture. In “controlled rooms”, light comes from cool-white daylight fluorescent tubes in lieu of indirect sunlight. Incandescent bulb or direct sunlight must be avoided because of the heat problem. For outdoor culture, tanks are located in areas that are exposed to morning sunlight. SEAFDEC Aquaculture Department 17 Temperature. In controlled rooms, incubation temperature often ranges from 20 to 250C. The scaling-up of outside culture is usually done in the morning to avoid temperature shock. Aeration. Aeration is provided to keep the algae in suspension, prevent sedimentation, disperse dissolved materials, and avoid adherence of cells to the walls of culture vessels. With continuous bubbling, the carbon needed for plant growth (air contains 0.03% CO2) is supplied. MAINTENANCE AND MASS PROPAGATION Cultures obtained from direct isolation or from culture collection are kept in pure form by establishing a routine aseptic procedure of propagating the stock. Maintenance of the purity of stock is a routine job. Stock cultures are frequently renewed after checking for contamination and viability of cells. Complete fertilization, light manipulation and temperature control are some of the basic requirements for perpetual maintenance of stock cultures (Fig. 13). Figure 13. Stock cultures of microalgae kept in test tubes and flasks are maintained in aseptic and controlled conditions 18 Natural food production PHOTO by A FRANCO Figure 14. MULTI-STEP CULTURE SYSTEM: (a) test tube primary stock culture; (b) flask cultures; (c) 1-3 L cultures; (d) 10 L cultures; (e) 200L tank culture; (f) 1-ton outdoor culture AB CD EF PHOTOS by AV FRANCO Culture vessels and tanks The choice of culture vessels depends upon the system of maintaining stock cultures and of the subsequent food production schedule. For stock cultures, 20 x 20 mm screw-capped culture tubes or a 125 ml Erlenmeyer flask is advisable. For auxiliary stocks (for starting a larger volume of cultures), 125-250 ml Erlenmeyer flasks or dextrose bottles (Baxter type) are preferable. For outdoor culture, a tank size of 1.0-1.5m3 having a depth of 0.5-0.6 m is advisable. Shallow tank is necessary to allow light penetration that is needed for algal growth. The multi-step batch culture system in culturing algae requires a series of containers of increasing volume which is based on inoculum volume (Fig. 14). Stock cultures Stock cultures are kept in agar slants. Those algae that do not grow well in agar are kept in unaerated smaller volumes of culture. Some stock cultures are preserved in the refrigerator for a certain period of time. Stock cultures are exposed to diffused light and are manually shaken (Fig. 15). Stock cultures are carefully handled to avoid contamination (Fig. 16). The presence of bacteria or protozoa has undesirable effects on the culture. SEAFDEC Aquaculture Department 19 Figure 15. Manual shaking of test tubes and flasks for maintenance of stock cultures Figure 16. Sources of contamination in a culture [ILLUSTRATED BY AI de la PEŃA] PHOTO by A FRANCO PHOTOS by A FRANCO Volume of inoculum The volume of inoculum depends upon the type of algal species, the volume of culture vessels and the demand date. For stock culture, a minimal amount of inoculum is used since cultures are allowed to grow for a longer period of time. For larger culture volumes, 10-30% inoculum is needed. Microalgae particularly diatoms which can reach peak growth at a shorter culture period (fast-growing) require less inoculum compared to green algae which are slow-growing and can reach peak growth after 4-5 days. Scaling-up of culture The culture of algae adopts the batch culture system (closed system) where the appropriate medium and viable inoculum are only added once. This system of culture can support cell multiplication for only a limited time since the composition of the medium changes. As cell density increases, light intensity within the culture is also affected. Figure 17. (L-R) First cycle of fully blooming Chaetoceros calcitrans; 50% partial harvest; and second cycle with additional seawater and nutrients to return to original culture volume 20 Natural food production Figure 18. Sample schedule of natural food production in relation to hatchery operations [ILLUSTRATED BY AI de la PEŃA] 3,0005,000L Scaling-up of culture is done according to demand. The usual method of scaling-up adopts the multi-step back-up culture system. Before scaling-up, starter cultures are monitored to check for contamination and to select the quality of seed for the next batch of cultures. During the renewal process, duplicate cultures are necessary to allow the culturist to choose seed or inoculum and to avoid loss due to accident or contamination. It is important to keep the ‘parent’ stock culture during the maintenance process. For large volume of cultures (0.5 to 10 m3), a semi-sustenance culture system is adopted. In this system, a portion of the culture is withdrawn at certain periods and replaced with an equivalent volume of seawater plus nutrients to retain the original culture volume. This procedure is repeated until the system can no longer support algal growth due to contamination or high organic load (Fig. 17). Programming Proper programming of culture is necessary to meet hatchery needs. This is based on the feeding requirement of the cultured species, tank volume and type of algae cultured. Daily renewal of culture is required for fast growing algae (Skeletonema, Chaetoceros) while 3-4 days cycle is needed for the slow growing types (Chlorella, Tetraselmis, Isochrysis, Navicula, Amphora). Figure 18 shows a sample schedule of algal cultures in relation to shrimp and/or fish hatchery operations. SEAFDEC Aquaculture Department 21 REFERENCES Al-Hasan R, Radwan S. 1994. Biomass production of a halophilic Navicula species for use in mariculture. In: Phang et al (eds). Algal Biotechnology in the Asia-Pacific Region. University of Malaya Atlas R, Bartha R. 1998. Microbial Ecology: Fundamentals and Applications, 4th ed. Benjamin Cummings Bai SC, Koo JW, Kim KW, Kim SK. 2001. Effects of Chlorella powder as a feed additive on growth performance in juvenile Korean rockfish, Sebastes schlegeli (Hilgendorf). 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Japan Journal of Phycology 34: 319-325 Millamena OM, Peñaflorida VD, Subosa PF. 1990. The macronutrient composition of natural food organisms mass cultured as larval feed for fish and prawns. The Israeli Journal of Aquaculture-Bamidgeh 42: 77-83 Okauchi M, Fukusho K.1984. Environmental conditions and medium required for mass culture of a minute alga, Tetraselmis tetrahele (Prasinophyceae). Bulletin of the National Institute for Aquaculture 5: 1-11 Parker F, Davidson M, Freeman K, Hair S, Daume S. 2007. Investigation of optimal temperature and light conditions for three benthic diatoms and their suitability to commercial scale nursery culture of abalone (Haliotis laevigata). Journal of Shellfish Research 26 (3): 751-761 Patil V, Kallgvist T, Olsen E, Vogt G, Gislerod H. 2007. Fatty acid composition of 12 microalgae for possible use in aquaculture feed. Aquaculture International 15: 1-9 Renaud SM, Parry DL, Thinh LV, Kuo C, Padovan A, Sammy N. 1991. Effect of light intensity on the proximate biochemical and fatty acid composition of Isochrysis sp. and SEAFDEC Aquaculture Department 23 Nannochloropsis oculata for use in tropical aquaculture. Journal of Applied Phycology 3: 43-53 Renaud SM, Zhou HC, Parry DL, Thinh LV, Woo KC. 1995. Effect of temperature on the growth, total lipid content and fatty acid composition of recently isolated tropical microalgae Isochrysis sp., Nitzchia closterium, Nitzschia paleacea, and commercial species Isochrysis sp. (clone T. ISO). Journal of Applied Phycology 7: 595-602 Renaud SM, Thinh LV, Parry DL. 1999. The gross chemical composition and fatty acid composition of 18 species of tropical Australian microalgae for possible use in mariculture. Aquaculture 170: 147-159 Richmond A. 1986. Handbook of microalgal cultures. CRC Press, USA. 528 p Rodriguez EG, Maestrini SY. 1984. The use of some agricultural fertilizers for the mass production of marine algae. Agriculture 36: 245-256 Roessler PG. 1990. Environmental control of glycerolipid metabolism in microalgae: commercial implications and future research directions. Journal of Phycology 26: 393399 Shamsudin L. 1992. Lipid and fatty acid composition of microalgae used in Malaysian aquaculture as live food for the early stage of penaeid larvae. Journal of Applied Phycology 4: 371-378 Shifrin NS, Chisholm SW. 1981. Phytoplankton lipids: 1981. Interspecific differences and effects of nitrate, silicate and light-dark cycles. Journal of Phycology 17: 374-384 Spolaore P, Joannis-Cassan C, Duran E, Isambert A. 2006. Commercial Applications of microalgae. Journal of Bioscience and Bioengineering 101:87-96 Sukenik A, Zamora O, Carmeli Y. 1993. Biochemical quality of marine unicellular algae with special emphasis on lipid composition. Aquaculture 117: 313-326 Supamattaya K, Kiriratnikom S, Boonyaratpalin M, Borowitzka L. 2005. Effect of Dunaliella extract on growth performance, health condition, immune response and disease resistance in black tiger shrimp (Penaeus monodon). Aquaculture 248: 207-216 Tendencia EA, de la Peña MR. 2003. Investigations of some components of the greenwater system which make it effective in the initial control of luminous bacteria. Aquaculture 218: 115-119 Tendencia EA, de la Peña MR. 2005. Efficiency of Chlorella sp. and Tilapia hornorum in controlling the growth of luminous bacteria in a simulated shrimp culture environment. Aquaculture 249: 55-62 Thompson PA, Harrison PJ, Whyte JNC. 1990. Influence of irradiance on the fatty acid compostion of phytoplankton. Journal of Phycology 26: 278-288 Volkman JK, Jeffrey SW, Nichols PD, Rogers GI, Garland CD. 1989. Fatty acid and lipid composition of 10 species of microalgae used in mariculture. Journal of Experimental Marine Biology and Ecology 128: 219-240 Vonshak A. 2006. Spirulina: growth, physiology and biochemistry. Lecture notes in preconference workshop on measurements of algal growth and photosynthesis; University of Santo Thomas; 9-11 October 2006. 39 p Walne PR. 1974. Culture of Bivalve Molluscs. Whitefriar Press, UK, pp.1-173 Wikfors GH. 1986. Altering growth and gross chemical composition of two microalgal molluscan food species by varying nitrate and phosphate. Aquaculture 59: 1-14 Whyte JNC. 1987. Biochemical composition and energy content of six species of phytoplankton used in mariculture of bivalves. Aquaculture 60: 231-241 Wikfors GH, Twarog JW, Ukeles R. 1984. Influence of chemical composition of algal food sources on growth of juvenile oysters, Crassostrea virginica. Biology Bulletin 167: 251-263 Zhu CJ, Lee YK, Chao TM. 1997. Effects of temperature and growth phase on lipid and biochemical composition of Isochrysis galbana TK1. Journal of Applied Phycology 9: 451-457 24 Natural food production Appendix I. CULTURE MEDIA A. Guillard’s F medium Macronutrients Sodium nitrate Sodium dihydrogen phosphate Ferric chloride Disodium ethylenediaminetetraacetic acid Disodium silicate Micronutrients Copper sulfate Zinc sulfate Cobalt chloride Manganese chloride Sodium molybdate Vitamins Thiamine hydrochloride Cobalamine Biotin Seawater Chemical formula NaNO3 NaH2PO4. H2O FeCl3.H2O Na2EDTA Na2SiO3.9H2O CuSO4.5H2O ZnSO4.7H2O CoCl2.6H2O MnCl2.4H2O NaMoO4.2H2O B1 B12 Amount 84.15 mg 10.00 mg 2.90 mg 10.00 mg 50.00 mg 0.0196 mg 0.0440 mg 0.2000 mg 3.6000 mg 0.0126 mg 0.2 mg 1 μg 1 μg 1 liter SEAFDEC Aquaculture Department 25 B. Walne’s Conwy medium Macronutrients Sodium nitrate Sodium dihydrogen phosphate Boric Acid Disodium ethylenediaminetetraacetic acid Ferric chloride Manganese chloride Micronutrients Zinc chloride Cobalt chloride Ammonium molybdate Copper sulfate Vitamins Thiamine hydrochloride Cobalamine Seawater Chemical formula NaNO3 NaH2PO4.H2O H3BO3 Na2EDTA FeCl3.6H2O MnCl2.4H2O ZnCl2 CoCl2.6H2O (NH4)6Mo7O24.4H2O CuSO4.5H2O B1 B12 Amount 100.000 mg 20.000 mg 33.600 mg 45.000 mg 1.3000 mg 0.360 mg 0.021 mg 0.020 mg 0.009 mg 0.020 mg 0.100 mg 0.005 mg 1 liter C. Liao and Huang’s TMRL medium Macronutrients Potassium nitrate Disodium phosphate Ferric chloride Disodium silicate Seawater Chemical formula KNO3 Na2HPO4.6H2O FeCl3.9H2O Na2SiO3.9H2O Amount 100.00 mg 10.00 mg 3.00 mg 1.00 mg 1 liter 26 Natural food production Appendix II. Multistep culture of phytoplankton at SEAFDEC/AQD CULTURE CONDITION Container Aeration Illumination Temperature Volume of inoculum Grade of reagents Media Water treatment Sterilization of culture vessels Culture period PRIMARY STOCK SECONDARY STOCK Test tube (20-ml agar slant) Test tube (20-ml broth) Flask (50-100 ml) None None (manual shaking) 1 unit 40 watt Fluorescent tube 18-220C 1 drop Analytical reagent Conwy/F Autoclave Steam Autoclave 7-10 days 18-220C 1 drop Analytical reagent Conwy/F 18-220C 5-10 mL Analytical reagent Conwy/F Autoclave Steam Autoclave Steam Autoclave 7-10 days Autoclave 5-7 days STARTER CULTURE Dextrose (1-L) 1-2 units 40 watt Fluorescent tube 22-250C 20-50 mL Analytical reagent Conwy/F Boiled Hot-Air 1-4 days Gallon (3.5-L) Carboy (10-L) Aerated 1-2 units 40 watt Fluorescent tube 22-250C 100-200 mL Analytical reagent Conwy/F Chemical (10 ppm chlorine) Hot-Air 1-4 days 22-300C 1-3 L Technical grade TMRL Chemical (10 ppm chlorine) Chemical (HCl / chlorine) 1-4 days LARGE SCALE CULTURE Fiber glass (200-L) Fiber glass (1000-L) 2-4 units 40 watt Fluorescent tube 27-300C 20-50 L Technical grade TMRL Chemical / sand filtered Chemical / sun-drying  1-4 days Sunlight 27-300C 200-500 L Agricultural fertilizer 16-20-0 + 21-0-0 + 46-0-0 or 14-14-14 Chemical/ sand filtered Chemical / sun-drying 1-4 days Appendix III. Counting of phytoplankton To determine and compute the plankton abundance or density using the haemacytometer, the following materials are needed: • Haemacytometer • Compound microscope • Algae • Lugol’s iodine solution • Test tubes • Cover slips • Pasteur pipettes • Rubber bulbs • Wiping cloth • Wash bottle Counting phytoplankton using a haemacytometer (1) Get to know the haemacytometer: It is a thick rectangular slide with an Hshaped trough forming two counting chambers. Each counting chamber is divided into nine blocks, 1 mm2 in area, giving a total ruled area of 9 mm2. Each of the four corner blocks (A-D) is subdivided into 16 squares while the center block (E) is subdivided into 25 squares 0.04 mm2 in area, each of which is further sub-divided into 16 smaller squares: Chained or large phytoplankton are usually counted using four or five 1 mm2 blocks. Smaller species or very dense samples are usually counted using the smaller 0.04 mm2 counting squares in block E. The number or phytoplankton is tallied in as many squares or portion of squares as is indicated in the desired level of statistical reliability. In all haemacytometers, the fundamental measurement is the average numbers of specimens per 1 mm2 from which the cell density is readily computed. 1 mm 1 mm 1 mm 1 mm 1 mm 1 mm 0.2 mm 0.2 mm With the cover slip in place on the glass support, each area forms a chamber with a depth of 0.1 mm. Therefore, the volume of sample contained in each 1 mm2 blocks is 0.1 mm3 (volume = area x depth), whereas the volume of sample contained in each of the small blocks in block E is 0.04 mm3 (volume, V = 0.04 mm2 x 1 mm). Given that 1 ml = 1,000 mm3, the volume of the bigger counting blocks ABCD is: V = 0.1 mm3 = 0.0001 ml = 1.0 x 10-4 ml. Given that 1 ml = 1,000 mm3, the volume of the smaller counting blocks is: V = 0.004 mm3 = 0.000004 ml = 4.0 x 10-6 ml (2) Prepare the sample: Label test tubes with the name of the test organisms. Put 5-7 ml of the cultured algae in the labeled test tubes. Add one drop of Lugol’s iodine solution to the test tubes. Mix and let tubes stand in rack. (3) Fill in the chamber: Clean the haemacytometer and the cover slip with soap and water or with rubbing alcohol. They must be free from dust, lint or grease Place the cover slip centrally over the ruled areas. Using a clean Pasteur pipette, put a drop of well-mixed algal sample in the entry slit of the chamber, the “V” groove. Fill both chambers. Check for the evenness of cell distribution under low power magnification. If air bubbles are present or if water is overflowing, underfilled and uneven distribution of cells happen, refill the chamber. Allow cells to settle for 3-5 minutes before counting. properly charged counting chamber improperly charged counting chamber good distribution poor distribution SEAFDEC Aquaculture Department 29 (5) Count the microalgae: For cells greater than 6 μm and not too dense cultures, total count of blocks A, B, C, D, and E is done. Start at the top left square and count only those cells which lie within touching the boundary line as shown below. Make a duplicate count in the corresponding blocks in the second chamber. Record the count in individual 1 mm2 blocks. boundary lines left and bottom top and right score board for each small square; total count is 48 For small phytoplankton cells or dense cultures, the sampling is made using the smaller squares in block E. Count only those cells which lie within or touching the boundary line. Record the counts in individual 0.04 mm2 blocks. (4) Calculate the total plankton density (expressed as cells per ml) using the formula: d (cells) = x / v where x = -n--ut-om-t-ab--le-rc--oo-u-f-nb-t-l-o-c-k--s--c-o-u--n-t-e--d-- v = 1.0 x 10-4 ml for the 1 mm2 blocks for ABCD or 4.0 x 10-6 ml for the 0.04 mm2 blocks for block E squares 30 Natural food production GLOSSARY autotrophs: organisms that are capable of synthesizing organic compounds from inorganic substrates using light or chemical energy carotenoid: orange pigments found in algae, e.g. Dunalliela coccoid (=coccal): adjective applied to unicellular, non-flagellate organisms possessing a cell wall, although these often do not have rounded morphology euryhaline: organisms that can live in waters with a wide range of salinity eurythermal: organisms that live in waters with a wide range of temperature fucoxanthin: brown carotenoid pigment occurring in the chloroplasts of brown algae halophilic: able to flourish in a high salinity environment inoculum (or starter): a group of living cells or organisms used to begin a new culture of algae after transfer to a new culture medium lipid: the ester of a fatty acid, insoluble in water parenchymatous: relating to unspecialized plant tissue consisting of simple thin- walled cells with intervening air spaces PUFAs (polyunsaturated fatty acids): fatty acids that have more than one double bond pelagic: organisms inhabiting the upper layers of the open sea periphytic: organisms that attach to underwater surfaces phytoplankton: passively floating or weakly motile photosynthetic aquatic, primarily cyanobacteria and algae plankton: free-floating plants (phytoplankton) and animals (zooplankton) found in both marine and freshwater systems. The phytoplankton function as primary producers in the food chain while the zooplankton serve as the primary herbivores that transfer energy from phytoplankton to secondary consumers prasinophytes: class of unicellular green algae that belongs to the Division Chlorophyta; some are flagellated but some are non-motile phycobiliproteins: collective term for the blue accessory pigment compounds phycocyanin, allophycocyanin, and phycoerythrin giving the blue-green color of the algae pyrenoid: spherical or ellipsoidal structure lying in the chloroplast of chlorophytes. It is the center of carbon dioxide fixation SEAFDEC Aquaculture Department 31 ACKNOWLEDGMENT We thank and recognize the following for their significant contributions in the development of microalgal culture techniques for aquaculture seed production: (1) former SEAFDEC/ AQD researchers Dr. Cesar Villegas, Mr. Roger Gacutan, Ms. Elsie Tech, Ms. Eve Aujero, Mr. Fernando Suñaz, Dr. Susan Baldia, the late Dr. Julia Pantastico; (2) former JICA Expert Dr. Shoichiro Suda who taught us the proper way to maintain the algal culture collection; (3) former AQD technical assistants Ms. Angelita Tillo, Ms. Corazon Espegadera-Co, Mr. Deogracias Reyes Jr, Ms. Florence Escritor-Leonor, Ms. Susan Landoy and Ms. Antonietta Evangelista; and (4) present AQD larval food staff Ms. Ellen Grace Tisuela, Ms. Jilla Alcalde and Mr. Virgilio Futalan Jr. We are also grateful to Dr. Fe Dolores Estepa and Dr. Evelyn Grace Ayson for the valuable comments and encouragement; AQD’s Publications Review Committee for the review [Dr. Relicardo Coloso (chair); Dr. Ma. Rowena Eguia, Dr. EG Ayson, Dr. Luis Ma. Garcia, Dr. Ma. Lourdes Aralar, Dr. Rolando Pakingking Jr, and Dr. Nerissa Salayo (members)]; and Ms. Mila Castaños of the Development Communication Section and her staff for their creativity in simplifying and making this manual possible. 32 Natural food production ABOUT THE AUTHORS Ms. Milagros R. de la Peña has been in SEAFDEC/AQD for 35 years studying microalgal physiology, specifically its biochemical composition. As a researcher working with the live food production team, she has made substantial contributions in the standardization and improvement of microalgal culture techniques and in providing microalgal starters to hatcheries in the Philippines and other countries. She is also instrumental in maintaining and preserving AQD’s algal culture collection. Currently, she is involved in the refinement of abalone culture technology and is doing research in abalone hybridization. She finished her BS Inland Fisheries degree at Mindanao State University, and her M Sc Marine Biology at the University of the Philippines. Ms. Annie V. Franco is a senior technical assistant assigned at AQD’s larval food laboratory. She maintains AQD’s microalgal strains and serves as practical instructor in training courses with live food as component. She has a double bachelor’s degree (BS Chemistry, 1992, Colegio de San Agustin - Bacolod City; and BS Biology 1988, University of St. La Salle - Bacolod). SEAFDEC Aquaculture Department 33 ABOUT SEAFDEC The Southeast Asian Fisheries Development Center (SEAFDEC) is a regional treaty organization established in December 1967 to promote fisheries development in the region. The member countries are Brunei Darussalam, Cambodia, Indonesia, Japan, Lao PDR, Malaysia, Myanmar, Philippines, Singapore, Thailand, and Vietnam. The policy-making body of SEAFDEC is the Council of Directors, made up of representatives of the member countries. SEAFDEC has four departments that focus on different aspects of fisheries development: • The Training Department (TD) in Samut Prakan, Thailand (1967) for training in marine capture fisheries • The Marine Fisheries Research Department (MFRD) in Singapore (1967) for post-harvest technologies • The Aquaculture Department (AQD) in Tigbauan, Iloilo, Philippines (1973) for aquaculture research and development, and • The Marine Fishery Resources Development and Management Department (MFRDMD) in Kuala Terengganu, Malaysia (1992) for the development and management of fishery resources in the exclusive economic zones of SEAFDEC member countries. AQD is mandated to: • Conduct scientific research to generate aquaculture technologies appropriate for Southeast Asia • Develop managerial, technical and skilled manpower for the aquaculture sector • Produce, disseminate and exchange aquaculture information AQD maintains four stations: the Tigbauan Main Station and Dumangas Brackishwater Station in Iloilo province; the Igang Marine Station in Guimaras province; and the Binangonan Freshwater Station in Rizal province. AQD also has a Manila Office in Quezon City. www.seafdec.org.ph