Microalgae: Its Application And Potential

July | August Feature heading: Microalgae Feature title: Microalgae: its application and potential International Aquafeed is published five times a year by Perendale Publishers Ltd of the United Kingdom. All data is published in good faith, based on information received, and while every care is taken to prevent inaccuracies, the publishers accept no liability for any errors or omissions or for the consequences of action taken on the basis of information published. ©Copyright 2009 Perendale Publishers Ltd. All rights reserved. No part of this publication may be reproduced in any form or by any means without prior permission of the copyright owner. Printed by Perendale Publishers Ltd. ISSN: 1464-0058

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Microalgae

Microalgae

Microalgae: its application and potential

of these components, particularly so in some species rich in ω-3 and ω-6 fatty acids. Today, commercial microalgae production is used to produce algal biomass for direct use as feed additives in the food and feed industry or for the extraction of high-valued components such as vitamins (C & D2), ω- fatty acids, pigments and antioxidants (B carotene, astaxanthin, lutein) and stable isotope bio-chemicals.

Commercial production

- a valuable resource for the 21st Century by Dr Elizabeth Sweetman, Ecomarine Ltd, Livadi, 28200 Lixouri, Cephalonia, Greece Email: [email protected]

M

icroalgae have been studied since the late 19th Century and approximately 40,000 freshwater and marine algal species have been identified. Cultured algal species account for barely 10 percent of these.

discharged as a waste product according to the formula: CO2 + H2O ------ Cn(H2O)n + O2

Photosynthesis takes place during the light phase and pigments capture light to generate ATP and NADPH2. These energy rich components are then used in the Calvin cycle to convert carbon dioxide into organic molecules (C3 sugars) catalysed by Recently considerable effort has been enzymes which combine to form molecules made to understand more fully and exploit of glucose (see Figure 1). Glucose can then the unique characteristics of certain species be converted to polysaccharides or fatty for a variety of applications in the food, acids which are important building blocks feed, cosmetic, nutriceutical, chemical and in many cell processes. biodiesel industries. Some microalgae species can use a variMicroalgae are typically unicellular and ety of organic carbon sources either as a like their plant relatives grow photosynsupplement to photosynthesis (mixotrophs) thetically using light, carbon dioxide and or replace it completely in heterotrophic other nutrients but in an aerated liquid production which takes place in the dark. culture medium. In heterotrophic growth microalgae assimiMicroalgae normally grow photosynlate organic substances, usually glucose, acetate thetically using light energy to fix carbon or glycerol to cover their carbon and energy dioxide into hydrocarbons with oxygen requirements. The organic substances Figure 1: Simple overview of the photosynthetic process are respired in mitochondria with oxygen acting as an electron receptor similarly to respiration in animal cells.

Where is algae used? Microalgae are rich sources of protein, carbohydrates and lipids. 28 | InternatIonal AquAFeed | July-august 09

Dr Elizabeth Sweetman Dr Elizabeth Sweetman is an oceanographic chemist with 25 years of experience in marine larval rearing and has been responsible for the establishment of numerous commercial microalgae and live feed production units for various aquaculture species. Her consultancy company Ecomarine Ltd can be contacted at [email protected] or Tel: +30 6944554498. The amino acid profile of almost all algae compares favourably to other food protein sources, the carbohydrate components are highly digestible, however, it is the lipid content that is especially interesting because of the functionality

In order to produce a commercial product from microalgae, either as whole cells or an extract, algal biomass must first be produced in a cost effective manner, be consistent in batch quality and produced a biosecure, contaminant free product. Production is either based on high volumes of low quality product or low volumes of high quality product. Almost all commercial scale production currently takes place in open outdoor circulating raceways or ponds that typically range in size from 5m3 to 1000m3. Open ponds are generally restricted to tropical and subtropical zones with high levels of natural sunlight, low rainfall and low cloud cover. The major disadvantage of open outdoor systems is that they are susceptible to contamination by other microbes, zooplankton and other indigenous algal species and it is for this reason that commercial production has been limited to a relatively small number of species due to their niche environmental growth conditions. The 1960s saw the development of continuous large scale methods in Japan for the mass production of Chlorella producing today approximately 2000 tonnes of dry product per year for whole-cell dietary supplements. Arthrospira autotrophic cultivation (formerly known as Spirulina) takes place in open raceway ponds in USA, China and India and produces some 3000 tonnes of product per year again used directly for food supplements. Approximate 1200 tonnes per year are produced of another green alga Dunaliella salina from which β-carotene is extracted in Australia, USA, Japan, China and India. β-carotene can constitute up to 14 percent of its dry weight. About 500 tonnes per year of the green alga Haematococcus pluvialis, containing up to six percent astaxanthin is produced autotrophically or mixotrophically in raceways, ponds and photobio-

reactors in USA, Israel and India primarily for use in the salmon feed industry. Smaller scale commercial production takes place of a number of other species: Porphyridium sp. (Arachidonic acid), Phaeodactylum tricornutum (Eicosapentaenoic acid) and Scenedesmus obliquus (Carotenoids). Many of these systems have been in operation for more than 20 years and their success has been dependant on developing a good understanding of the physiology, biochemistry and ecology of the algae being cultured and the application of appropriate engineering principles to the design system. The same period has seen Picture1: Photobioreactors at Kefalonia significant advances made Fisheries, Greece - a bass and bream hatchery in cultivation methods to improve productivity. The introduction of highly controlled closed or nearly closed photobioreactors (PBR), where energy is supplied either by direct sunlight or by artificial lighting, or fermentors where energy is supplied from organic carbon sources. D e s i g n aspects of these systems have been reviewed by Ugwu et al, 2008 for photobioreactors and by Chen & Chen, 2006 for heterotrophic microalgal production. (see The premier meeting point for the feed and food industry in 2010. Addressing common concerns and identifying opportunities. picture 1) Closed or Join us in Cancun, Mexico!. For more information visit: nearly closed production systems allow greater Hosted jointly by in co-operation control over steFAO & IFIF with Conafab rility and culture

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Microalgae

Microalgae

conditions (Light, carbon dioxide, nutrient levels and temperature). Under optimal culture conditions higher cell densities can be achieved than in open pond culture systems corresponding to 5g dry weight of phototrophic biomass and 60g dry weight of heterotrophic biomass being produced per litre (Muellar –Feuga, 2004). Behrens 2005 reports that when just the energy costs of producing a kilo of dry algal biomass are considered heterotrophic production of algae will generally be more economic than phototrophic growth using artificial lighting (US$2.01 versus US$11.22 respectively).

A variety of bag and tank culture facilities have been used in either natural or artificial light environments. In house production enabled the hatcheries to control own product, its quality and biosecurity. These units were essential design components, vital to the operation of the hatcheries either for feeding shellfish larvae or growing and enriching rotifers for feeding in the early fish larval stages or for using in the larval rearing tanks for the “green water techniquea”. (see picture 2.) Significant advances were made in the commercialisation of photobioreactor technology in the mid 1990s and the introduction of such systems to the Mediterranean sea bass and sea bream industry resulted in a dramatic improvement in algal productivity with concentrations of microalgae of up to 10 times that achieved with the traditional systems and this reduced the algal production cost to the hatcheries. During the same period Nannochloropsis sp was also being produced commercially in the USA in open ponds for the production of aquaculture pastes. The last 12 years has seen a rise in the number of companies producing concentrated algal pastes and an increase in the variety of species available for use in the aquaculture industry. These algal pastes, depending on the species cultured, range from preserved to live paste and from fresh to freeze dried products. However, the early concerns of the industry was the biosecurity of Photo 2: Green water larval rearing such products but now processing technologies have in many cases eliminated these concerns. The cost and Several species of microalgae Tetraselmis, availability of safe paste products has Nannochloropsis, Isochrysis, Pavlova, Chlorella, resulted in some recently constructed Chaetoceros, Skelotonema, Thassiosira, and hatcheries, particularly in the emergNitzschia are grown commercially for use ing cod industry, to rely entirely on in the marine fish, crustacean and shellfish purchased paste products and therefore aquaculture industry and approximately avoid the capital and operational costs of one-fifth of the algal biomass produced is live algal production. reported to be used in aquaculture hatcherIn Japan freshwater Chlorella destined ies (Mueller-Fuega 2004). for the aquaculture industry is produced Traditionally most fish and shellfish heterotrophically as a valued added prodhatcheries have had their own algal prouct and manipulated to improve its fatty duction facilities producing a variety of acid profile and mineral content using species suitable for the animals being proprietary osmotic technologies. cultured. 30 | InternatIonal AquAFeed | July-august 09

Table 1: Oil yields of various crops

Crop

Oil Yield litre/ha/ year Corn 168 Cotton 327 Soybean 440 Mustard sees 570 Sunflower 950 Rapeseed / Canola 1600 Jatropha 1800 Oil Palm 6000 Safflower 780 Castor 1400 Jojoba 1800 Coconut 2700

Algae (20mg/m2/day at 11,200 15% TAG) Algae (50g/m2/day at 93,000 50% TAG)

This provides an algal cell as a bioparticle rich in a number of components not naturally available and at concentrations that enable efficient culture and enrichment of zooplankton feeds, in order to better match the nutritional requirements of the marine larvae being cultured.

Heterotrophic production Large scale conventional fermentation methods have been used to grow heterotrophically the dinophycean Crypthecodinium cohnii (>100m3) and thraustochytrids Schizochytrium and Ulkenia sp. (80m3). These species are rich in docosahexaenoic acid (DHA) a long chain polyunsaturated fatty acid and can be considered a sustainable source of this essential fatty acid. DHA productivity is reported to reach 3gDHA / litre /day with Schitzochytrium. The dried product has been shown to be effective in enrichment diets for rotifers and Artemia (Harel et al, 2002, Yamasaki et al., 2007) and early larval weaning diets due to their high yields of DHA. They are also used to provide DHA- rich oils for human nutrition (Kyle et al 1992). Interest has developed further in the production of these and other closely related species as they offer a source of long-chain PUFA’s that have the potential to become an alternative sustainable

source and therefore help to meet the increasing demand for fish oil that is a critical limiting factor for the future expansion of aquaculture activities. Oil extracted from the algae can successfully replace marine fish oil in the diet for Atlantic salmon parr without affecting their growth (Miller et al., 2007). Dried algae of Schizochytrium sp. has been shown to be a good source of DHA for sea bream larvae (Ganuza et al., 2008).

Future work The cultivation of microalgae offers much potential and many new opportunities. There is strong interest in microalgae production for products of pharmaceutical interest and particularly in biofuels. Recent advances have been made in identifying suitable species, engineering photosynthesis and enhancing and manipulation of crucial metabolic pathways. This uses environmental factors to redirect cellular function towards the synthesis of preferred products and can even expand the processing capabilities of the microalgae. Chlorella protothecoides cultured heterotrophically has been shown to accumulate as much as 55 percent of its dry weight as oil compared to 14 percent in cells grown photoautotropically (Wu and Miao 2006). In heterotrophic production systems, a b i o - av a i l a b l e nitrogen deficiency has been shown to result in a higher lipid synthesis and therefore higher oil accumulation in starved cells (Sheehan et al 1998) and a higher accumulation of July-august 09 | InternatIonal AquAFeed | 31

astaxanthin in cells of Haematococcus pluvialis (Boussiba 2000). The cost price of production is important and many new companies and research groups are developing activities in these areas. For example, Zaslavskaia et al, (2001) transformed Phaeodactylum tricornutum from an obligate phototroph to a heterotroph thriving on glucose in the absence of light using the Glut 1 gene from human erythrocytes. This offers the potential to produce an algal species in either manner.

Biofuels The technologies exist today for the commercial production of biofuels from food crops such as grains, sugar beet and oil seeds, the first generation biofuels and these are well developed and understood in a mature market. However, many questions have been raised about their sustainability and their ability to substitute for oil products as the food crops required for biofuel produc-

Microalgae tion appears to create undue competition for land and water resources used for food production therefore driving up food prices. Second generation biofuels utilise more abundant non-food biomass such as agricultural and forestry residues (cereal straw, bagasse and vegetative grasses). However, the technologies used have not yet reached large scale commercial production but offer potential for increased production efficiency and cost reductions. In the EU biofuel production has focussed on biodiesel due to the higher fuel economy of diesel engines and the agricultural practises in Europe. Biodiesel is produced from the triglycerides, a major component of vegetable oil, which are broken down or transesterified with alcohols such as methanol or ethanol into smaller straight chained alkyl esters similar to the standard diesel hydrocarbons.

fossil derived diesel fuel). The production of microalgae as an alternative feedstock for biodiesel production has several key advantages. • It is environmentally sustainable • It does not compete with agriculture as it does not require soil for growth • Microalgae are highly bio-degradeable, containing no sulphur therefore there are zero sulphur dioxide emissions. • Microalgae can be cultivated in a variety of systems and depending on the choice of this can be used in a variety of locations with limited water requirements. • If grown using photosynthetic methods microalgae production can be considered carbon neutral.

Conclusion Microalgae possess many unique and interesting biochemical properties that are playing an increasingly important role in many aspects of our daily lives from nutrition through to energy production. Aquaculture already successfully uses many species as an essential component of the live food chain in the production of a number of high valued species. The potential that microalgae has, to act as a partial replacement of fish oils, offers the opportunity for the continued expansion of global aquaculture production while at the same time maintaining the well publicised nutritional benefits that aquaculture products offer to human nutrition and health. Microalgae species research and the development of cost effective and industrial scale production processes are ongoing and their successful application will enable microalgae to develop their full potential as a new and exciting resource of valuable organisms in the 21st century.

"Microalgae possess many unique and interesting

biochemical properties that are playing an increasingly

important role in many aspects

of our daily lives from nutrition through to energy production" Microalgae can be far more productive than other feedstuffs with the oil yield per hectare of microalgae significantly exceeding other common oil sources such as soya and rapeseed. (see Table 1). Microalgae like higher plants produce storage lipids in the form of triglycerols (TAGs) which can be used to synthesize fatty acid methyl esters (a substitute for

32 | InternatIonal AquAFeed | July-august 09

References Behrens P.W. (2005). Photobioreactors and Fermentors: the light and dark sides of growing algae. In: Algal Culturing Techniques. Edited by R.A. Andersen. Elsevier Academic Press, 189204. Boussiba S. (2000). Carotenogenesis in the green algal Haematococcus pluvialis: cellular physiology and stress response. Physiol Plant 108, 111-117 Chen F., Chen G.Q. (2006). Growing phototrophic cells without light. Biotechnol Lett 28, 607-616. Ganuza E., Benitez-Santana T., Atalah E., VegaOrellana O., Ganga, R., Izquierdo M.S. (2008). Crypthecodinium cohnii and Schizochytrium sp. as potential substitutes to fisheries-derived oils from seabream (Sparus aurata) microdiets. Aquaculture 277, 109–116. Harel M., Koven W., Lein I., Bar, Y., Behrens P., Stubblefield J., Zohar Y., Place A.R. (2002). Advanced DHA, EPA and ArA enrichment materials for marine aquaculture using single cell heterotrophs. Aquaculture 213, 347–362. Li M.H., Robinson E.H., Tucker C.S., Manning B.B., Khoo L. (2009). Effects of dried algae Schizochytrium sp., a rich source of docosahexaenoic acid, on growth, fatty acid composition, and sensory quality of channel catfish Ictalurus punctatus, Aquaculture (2009), doi:10.1016/j.aquaculture.2009.04.033 Mueller-Fuega A. (2004). Microalgae for aquaculture: the current global situation and future trends. In: Handbook of Microalgal Culture. Edited by Richmond A., Blackwell Science, pp 352-364 Sheehan J., Dunahay T., Benemann J., Roessier P. (1998). A look back at the U.S. Department of Energy’s Aquatic Species program : Biodiesel from Algae. Golden, Colorado: TP-580-24190 National Renewable Energy Laboratory. Ugwu C.U., Aoyagi H., Uchiyama H. (2008) Photobioreactors for mass cultivation of algae. Bioresour Technol 99, 4021-4028. Wu Q., Miao X. (2006). Biodiesel production from heterotrophic microalgal oil. Bioresour Technol 97, 841-846.

Dry with 20-50% less energy

Yamasaki T., Aki T., Mori Y., Yamamoto T., Shinozaki M., Kawamoto S., Ono, K. (2007). Nutritional enrichment of larval fish feed with thraustochytrid producing polyunsaturated fatty acids and xanthophylls. J Biosci Bioeng 104, 200–206. Zaslavskaia L.A., Lippmeier J.C., Shih C., Ehrhardt D., Grossman A.R., Apt K.E. (2001). Trophic conversion of an obligate photoautotrophic organism through metabolic engineering. Science 292, 2073-2075.

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