Algae – its role, species and production requirements Live food aquaculture training course www.aquatrain.org

Role of algae in aquaculture First link in the chain of live food manufacture and nutrition. Culture diet for rotifers Enrichment diet for rotifers Green water technique Provides a direct source of nutrition for larvae Provides background rotifer feeding Other zootechnical benefits Historically algae has provided for marine fin fish culture the fundamental building block required for the mass production and enrichment of the rotifer Branchionus plicatilis. The importance of marine unicellular algae as the first link in the chain of live food production and nutritional enhancement was most evident in the development of larval rearing techniques for Pagrus major and other marine fin fish species in Japan in the late 60 and early 70’s. Enrichment of rotifers with algae provides a variety of essential fatty acids and other nutritional factors for the first feeding fish larvae. The use of algae in the larval rearing tanks as “ green water technique” provides essential background feeding for the remaining uneaten rotifers helping to maintain their health status and contribute to the maintenance of their nutritional value. Other zootechnical characteristics essential for the successful larval rearing of several marine species are contributed to by the employment of green water techniques. Live food aquaculture training course www.aquatrain.org

Role of algae in the green water larval rearing technique An anti-bacterial agent In situ biological filter and producer of oxygen Light filter Promoter in the location of prey organisms Stimulation of enzymatic synthesis and onset of feeding in young larvae Algae usage in the green water technique is not limited to the purely nutritional side of larval rearing. Algae has been reported to act in the following ways although it is true to say that in some cases the mechanism are not clearly understood. As an antibacterial agent – Extracts from Tetraselmis suecia have been found to inhibit bacterial activity within 15 minutes of addition to fish tanks and for periods up to 4 hours. In addition specific polysaccharides in the algae cell wall are thought to stimulate a non-specific immune response in young larvae. Algae has been reported to act as an In situ biological filter removing potentially harmful metabolites from the water by stripping off nitrogenous substances and algae also produces oxygen through photosynthesis Algae acts as a light filter and diffuser facilitating an even distribution of live food and larvae within the tank system. It acts as a promoter and background for the location of prey organisms hence playing a particularly important role in the critical first feeding stage of larvae. Algae has been shown to stimulate the enzymatic synthesis and onset of feeding in young larvae. So as you can see despite the development of excellent culture and enrichment diets for rotifers marine unicellular algae used in the green water concept are still considered essential for the production of many marine fin fish species. Larval survival and quality can be related to the availability and quality of the algal species produced in commercial fin fish hatcheries. Live food aquaculture training course www.aquatrain.org

Practical usage of algae in the hatchery Maintenance of master/stock rotifer cultures Feeding or co-feeding of rotifer mass cultures Enrichment diet for rotifers prior to feeding the larvae Used in the larval rearing tanks as “Green water techniques” Live food aquaculture training course www.aquatrain.org

Species commonly used in aquaculture (a) Chlorophyceae – green algae Chlorella salina (8 μm) Chlorella sp. Dunaliella sp. Nannochloris atomus (4 μm) Prasinophyceae – greenish coloured algae Tetraselmis chui (14 μm) Tetraselmis suecica ( 12μm) Eustigmatophyceae – greenish yellow algae Nannochloropsis oculata ( 3 μm) Nannochloropsis sp. (4 μm) Nannochloropsis gaditana (4 μm) Most of the successful algal species commonly used in aquaculture belong to 1 of the 6 algal classes which are shown here. All the strains listed are commonly used as food organisms in aquaculture either for larval shellfish or for feeding invertebrates rotifers. They are generally easy to maintain and are of a suitable cell size. Live food aquaculture training course www.aquatrain.org

Species commonly used in aquaculture (b) Prymnesiophyceae - golden brown flagellates Isochrysis galbana (7 μm) Isochrysis sp. (Tahitian) (9 μm) Monochrysis Pavlova lutheri (7 μm) Cryptophyceae – naked flagellates Rhodomonas sp. (17 μm) Chroomonas salina (17 μm) Bacillariophyceae – diatoms Chaetoceros calcitrans (5 μm) Skeletonema costatum (9 μm) Thalassiosira pseudonana (9 μm) I have also included the diatoms here, which are primarily used in shellfish culture and their relevance to fin fish culture is limited. In reality and particularly in the Mediterranean two species dominate namely Nannochloropsis sp. and Isochrysis sp. Tahitian. These have been particularly successful with the culture of sea bream and are commonly used in combination with each other. Isochrysis Tahitian or TISO CCAP 927/14 as it also designated is preferred as it can tolerate higher temperatures that Isochrysis galbana CCAP 927/1. In practice many farms find it more difficult to culture Isochrysis species and during production algal culture often becomes a monoculture of Nannochloropsis. Given the availability of commercial enrichment diets which are both complete and nutritionally customized this failure has in fact little affect on the over all success of production. Live food aquaculture training course www.aquatrain.org

Various algal species (CCAP-M, Oban) Chaetoceros sp. Nannochloropsis salina CCAP 849/2 Dunaliella Here we can see some examples of the algal species. These photographs are not to scale or relative to one and other in terms of cell size The non motile Nannochloropsis is 4micron and Rhodomonas 17 microns We can see clearly the flagella of Dunaliella. Rhinomonas reticulata var reticulata (‘Rhodomonas’) CCAP 995/2 Live food aquaculture training course www.aquatrain.org

Live food aquaculture training course Sources of algae CCAP Culture collection of algae and protozoa, Oban, UK. www.ife.ac.uk/ccap/ Algobank Microalgae Strain Bank, Universite de Caen, France. www.unicaen.fr SAG Sammlung von Algenkulturen, Gottingen, Germany www.gwdg.de/~botanik/phykolgia CCMP Provasoli-Guillard National Centre for culture of marine phytoplankton, Westboothbay harbour ME, USA. http://ccmp.bigelow.org UTEX Culture collection of algae at the univesity of Texas at Austin www.bio.utexas.edu/research/utex From a practical point of view I have listed some of the algal culture collections centres where axenic master cultures can be obtained for a charge. This list of sources of algae is not exhaustive and several university and research centres also have collections of the more commercially used species. Several commercial companies also can supply. ( Florida sea Farms on agar). It should always be borne in mind however that algal cultures have the potential to contain harmful pathogens and care should be taken to ensure that master and production cultures are kept as clean as possible and that if samples are taken from or exchanged with other fish producers this risk is understood. Good husbandry practices dictate that regular renewal from axenic stocks is preferable to last minute help from a neighbouring farm. As previously discussed many of the commonly used algal species were developed within the framework of warm water fin fish culture. It is perhaps worthy to mention that recent cold water species development in cod and Halibut could benefit from the isolation and identification of dominant local species commonly associated within the areas of wild stock breeding grounds. The suitability of these species for commercial production is a separate issue and would have to be studied. Live food aquaculture training course www.aquatrain.org

Calculating algal requirements Hatchery - Algae requirements   Sea bream General assumptions 1) Av. daily algal consumption/m3 larval volume (L) 20 2) Av daily requirement per 100M rotifer production (L) 100 3) Algal production volume/m3 daily production (m3) 6,7 Annual production target (2g juveniles) Initial larval rearing vol (m3) 62 123 185 308 Daily larval algae requirement (m3) 1,23 2,46 3,69 6,15 Daily rotifer production (M) 615 1231 1846 3077 Daily rotifer algae requirement (m3) 0,62 1,85 3,08 Total daily algal production (m3) 1,8 3,7 5,5 9,2 Algal production volume needed (m3) 12 25 37 61 No of 0.5m3 bags needed 28 56 54 50 No of 4m3 tanks required 4 Requirements 2.000.000 4.000.000 6.000.000 10.000.000 Total algal production volume (m3) 14 43 73 Live food aquaculture training course www.aquatrain.org

Live food aquaculture training course Calculating the volume of algae required for rotifer production. (6 million juveniles at 20 x 106 cells/ml Nannochloropsis) Stock cultures Approx. 150 - 300 litres per day Mass cultures Using algae and yeast there is an average daily requirement of 100 litres of algae for every 100 million rotifers produced. A 6 million hatchery would use 2 x 109 rotifers per day => 2m3 of algae per day. (3 production runs of 2 mill) Rotifer enrichment. If all the rotifers were enriched at 2million/ml need 1m3 of tank space and at least 1m3 of algae. Live food aquaculture training course www.aquatrain.org

Live food aquaculture training course Calculating the volume of algae required for green water larval rearing (6 million juveniles at 20 x 106 cells/ml Nannochloropsis) 20 L of algae per m3 of larval rearing per day at 20 x 106 cells/ml A 6 million production hatchery will have approximately 185 m3 of larval rearing. Daily larval rearing requirement of 3.7 m3 Live food aquaculture training course www.aquatrain.org

Daily algal requirement Rotifer stock cultures 0.3 m3 Rotifer mass cultures 2.0 m3 Rotifer enrichment 1.0 m3 Green water larval rearing 3.7 m3 Total requirement 7.0 m3 /per day Live food aquaculture training course www.aquatrain.org

Typical cell densities achieved The relative cells densities achievable in different culture volumes are shown above:- Generally speaking volumes up to 20 litres achieve greater cell densities than the sack cultures of 200-500 littres and this is due to light availability. Note that in indoor situations high density cultures only reach half of their capability when compared to outdoor cultures using ambient light. In Northern climates such as these the efficiency and method of lighting will play an important role in the success of algal culture facilities and the cost of producing algae. Live food aquaculture training course www.aquatrain.org

Algal culture techniques The culture conditions of the different culture volumes are summarized here Diffuse – 1000 lux max Continuous 3000-4000 lux Sacks >4000 lux Salinity 28-37 ppt Live food aquaculture training course www.aquatrain.org

Fluorescent shelf and overhead metal halide lighting Here we can see a mixture of fluorescent lighting and overhead metal halide lights. This photograph shows well the arrangement of fluorescent tubes on the far wall for the culture volumes ranging from 500 ml through 2 litre and up to 20 litres. The distances between the shelves should be designed to meet the scaling up procedure from post master to sack inoculation and match the choice of culture vessels to be used. Note also the white walls and the carefully sloping epoxy painted floors which facilitate cleanliness and avoid water retention an build up of contaminants - important points to be considered when designing commercial production units Live food aquaculture training course www.aquatrain.org

Transparent walls and lighting Here we can see another unit with central fluorescent lighting for sack culture and an insulated transparent wall for additional ambient light. Live food aquaculture training course www.aquatrain.org

Live food aquaculture training course Shelf lighting Here you can see a classic set up with 2l, 10L and 20l cultures. A variety of culture vessels are available ranging from pyrex glass to polycarbonate carboys shown here and in fact many units use cheap disposable plastic tubular sacking in a variety of sizes. It should be noted that large glass vessels are not recommended as they can slip easily out of ones hands and have been known to cause serious injury to hatchery personnel. Live food aquaculture training course www.aquatrain.org

Under shelf and side lighting Here we can see under and over shelf lighting using heavy duty glass as the shelving material. This is not ideally suitable for the reason mentioned previously. Live food aquaculture training course www.aquatrain.org

Fluorescent sack lighting Sack cultures of 300 and 500Litres can be grown indoors in parallel rows with central fluorescent lighting. However leaking sacks can lead to short circuiting as even spray proof fittings are liable to fail. Again the safety aspects of all electrical appliances in a sea water environment need to be well respected and equipment and circuitry should comply to IP 16 regulations with trip switches. Fluorescent lights have heat emitting ballasts which can in some production units require addition temperature control. The intensity and duration of illumination are both important. It is difficult to measure the total light or the photosynthetically active light received by a culture and confusing to describe it when measured because of the different kinds of units used. As the most common and least expensive type of measuring device is the hand held light meter it is hardly surprising that the unit of lighting intensity the lux ( lumens/m2) is used for the purpose of plant lighting rather than the energy units. For example the cool white fluorescent bulbs have an energy rating of 1000μw/cm2 microwatts per cm2 this is approximately the equivalent of 3020 lux The intensity of the fluorescent tubes should be checked regularly as they deteriorate with time. Generally these lights will have to be replaced once a year. For most hatcheries 24 hour illumination is used as it is much more difficult to saturate a dense carboy or sack culture than a stock culture because of the shading effect. Live food aquaculture training course www.aquatrain.org

Natural light greenhouse structures Of course in the right environment, natural lighting is the cheapest and most efficient source of light energy for algal culture. Here we can see a greenhouse structure with a central walkway arranged with a drip fed continuous culture system in Spain and a tank system installed recently in Singapore. The tank system does have the ability to have overhead night time lighting. Live food aquaculture training course www.aquatrain.org

Live food aquaculture training course Indoor bag cultures The mass sack culture provides the bulk of the algae for for purposes although 1L and 10L cultures can also be used. The sacks can either be cultured in a batch manner or a continuous way. The initial stages for both methods are the same. The bags are half filled with sterilized sea water, nutrients added and the culture medium vigorously aerated. It is inoculated with about 9 litres of culture and left for 3 days to become established. For batch cultures after 3 days the culture is topped up completely with sterile sea water and nutrients and left until ready for harvesting. If the culture is healthy a small volume about 20-30% is left and the cycle restarted. If found to be contaminated or unsuitable the bag is discarded. If continuous culture is to be used a small flow is added to the culture from a header tank supplied with sterile sea water and nutrients dosed into the system by peristaltic pump. The flow rate is adjusted to equal the culture volume over the period of normal growth. If the culture volume is 300litres and the growing phase 6 days the the flow rate will be 35ml per min. This usually works out about 15-20% of the total culture volume per day. When the culture volume reaches its maximum level it is overflowed through a small tube into a collecting pipe which then passes to a collection tank. The algae in this collection tank can be harvested as required. Many medium sized marine hatcheries will require at least 3 m3 of algae per day which would require the establishment of a total culture volume of 20m3 or 67 x 300L sacks. The collected algae is then pumped by centrifugal pump to the larval rearing and rotifer production facilities. If set up properly such a bag can continue for several weeks before needing replacement. When the bag is replaced it is simply disconnected from the system and a new one placed in its position Live food aquaculture training course www.aquatrain.org

High density culture systems In recent years photobioreactor systems have provided an efficient alternative to sack culture systems. They are both labour saving, automated and cost effective. They have the advantage that the cell concentration obtained is up to 10 times greater than that achievable within the sack cultures which of course makes moving large volumes of algae unnecessary. This 600 L unit produces 200L of algae per day at a density of nearly 300 x 10 ^ 6 cells/ml. This is the same number of cells that would be obtained from 2 m3 of sack culture production. pH is controlled by the addition of carbon dioxide and sterilized sea water and nutrients are added automatically. Harvesting being achieved by overflow collection from the header tank. Undoubtedly the success of such systems is dependent upon light availability and Mediterranean climates are particularly suitable in the Autumn to Spring periods. Live food aquaculture training course www.aquatrain.org

Photo-bioreactor algal culture Live food aquaculture training course www.aquatrain.org