ELBA BIOFLUX Extreme Life, Biospeology & Astrobiology International Journal of the Bioflux Society Potential application of microbial phytase in aquaculture 1Christopher M. A. Caipang, 2Rande B. Dechavez, and 3Mary Jane A. Amar 1Faculty of Biosciences and Aquaculture, University of Nordland, Bodø 8049, Norway. 2College of Fisheries, Sultan Kudarat State University, Kalamansig, Sultan Kudarat 9808, Philippines. 3National Institute of Molecular Biology and Biotechnology, University of the Philippines Visayas, Miag-ao 5023, Iloilo, Philippines. Corresponding author: C. M. A. Caipang, [email protected] Abstract. Phytases are histidine acid phosphatases that catalyze the hydrolysis of phytic acid to inorganic phosphorus and myo-inositol phosphates. The phosphorus that is being released is used in various metabolic processes. These enzymes are able to promote the growth of animals and plants as well as increase the nutritional value of feedstuffs through dephytinization. Phytases are widespread in nature because they are found in animals, plants and microorganisms. This paper discusses the different microbial phytases that have been characterized and their beneficial roles in aquaculture. In order to be incorporated in the feed, microbial phytases must have the ability to withstand high temperatures during processing, and to be stable in the gastrointestinal tract of the fish, the enzymes must be active at low pH levels. The various effects of microbial phytases on the bioavailability of phosphorus in fish, on growth performance, on the immune responses, on the excretion of phosphorus and on the environment are also reviewed. Key Words: phytase, bacteria, bacterial enzyme, aquaculture. Introduction. The hydrolysis of phytic acid to myo-inositol and inorganic phosphate by phytases (myo-inositol hexakisphosphate phosphohydrolase) is an important reaction in most biological systems. Phytic acid (myo-inositolhexakis phosphate) is an organic form of phosphorus (P) that is abundantly present in plants materials such as legumes, cereals, oilseeds and nuts (Vats & Banerjee 2004; Rao et al 2009). The presence of phytic acid in plant-derived food acts as an anti-nutritional factor because it causes mineral deficiency due to the chelation of metal ions such as Ca2+ Mg2+, Zn2+ and Fe2+ , with proteins, thus affecting their digestion (Harland & Morris 1995). It also inhibits the activities of digestive enzymes such as -amylase, trypsin, acid phosphatase and tyrosinase. The ruminants digest phytic acid through the action of phytases produced by the anaerobic gut fungi and bacteria present in their intestinal microflora. On the other hand, monogastric animals such as pig, chicken and fish utilize phytate phosphorus poorly because they are deficient of phytases in their gastrointestinal tract. Due to the lack of adequate levels of phytases in monogastric animals, phytic acid is excreted in the feces. Therefore, inorganic phosphate is added to their feed to meet the phosphate requirement and to ensure good growth. The antinutritive effect of phytic acid is problematic in the feeding of fish (Richardson et al 1985), due to its short gastrointestinal tract and thus limiting the use of plant-derived protein in fish feed. These problems could be solved by the hydrolysis of phytate through the use of supplemental phytase (Simell et al 1989). Therefore, phytase has become an important industrial enzyme and is the subject of extensive research. By working efficiently on the substrate under optimum conditions, supplemental phytase could diminish the antinutritive effects of phytic acid and reduce the cost of diets by removing or at least reducing the need for supplemental inorganic phosphate. In addition, phytase could be an environmentally friendly product by reducing the amount of phosphorus that enters the environment (Kerovuo 2000). 55 ELBA Bioflux, 2011, Volume 3, Issue 1. http://www.elba.bioflux.com.ro During the last few years, phytases have attracted significant attention in the areas of nutrition, environmental protection, and biotechnology. Increased public concern regarding the environmental impact of high phosphorus levels in animal wastes and uneated feed has resulted in intensive biotechnological development of phytase and its application in animal nutrition. Feeding trials have shown the effectiveness of supplementing microbial phytases for the increased utilization of phytate-P as well as phytate-bound minerals by various animals including fish (Lei & Stahl 2001; Cao et al 2007; Selle & Ravindran 2007; Rao et al 2009). As a result, the phosphorus excretion of these animals can be significantly reduced (Lei et al 1993; Vohra et al 2006). Recent reviews have mainly focused on the production, characteristics and the basic applications of phytases (Greiner & Konietzny 2006; Kaur et al 2007; Fu et al 2008; Rao et al 2009). However, no such reviews have been done on the application of microbial phytases in aquaculture. Hence, this mini-review documents the different studies on microbial phytases and their beneficial effects on fish. Phytic acid, its sources and physiological functions. Phytic acid (myo – inositol hexakis dihydrogen phosphate) is the major storage form of phosphorus in cereals, pollen, legumes and oilseeds. The chemical description of phytic acid is myo-inositol 1, 2, 3, 4, 5, 6-hexakis dihydrogen phosphate (IUPAC-IUB 1977) and the salts of phytic acid are described as phytates. It is considered to be an antinutritional factor since it chelates minerals such as magnesium, zinc and calcium and may also react with proteins, therefore decreasing the bioavailability of protein and nutritionally important minerals. Phytic acid occurs primarily as salts of mono- and divalent cations, e.g., potassium magnesium salt in rice and calcium-magnesium-potassium salt in soybeans. It accumulates in seeds and grains during ripening and is incorporated with other storage substances such as starch and lipids. In cereals and legumes, phytic acid accumulates in the aleurone particles and globoid crystals, respectively (Reddy et al 1989). The endosperm of wheat and rice kernels is almost devoid of phytate, as it concentrates in the germ and aleurone layers of the cells of the kernel. Ferguson & Bollard (1976) found that 99% of the phytate in dry peas was in the cotyledons and 1% in the embryo taxis. The highest amount of phytate among cereals is found in maize, and among legumes in dolique beans (Reddy et al 1989). The physiological roles of phytic acid in plant seeds are the following: 1) acts as storage of phosphorus, 2) source of energy, 3) source of cations, 4) source of myo- inositol, which is a cell wall precursor, and 5) acts as an initiator of dormancy (Reddy et al 1989). Phytic acid also acts as a natural antioxidant in seeds during dormancy (Graf et al 1987). This antioxidant property of phytic acid is based on the assumption that it effectively blocks iron-driven hydroxyl radical formation. Phytic acid has been shown to exert an antineoplastic effect in animal models of both colon and breast carcinomas (Dvorakova 1998). The presence of undigested phytic acid in the colon may protect against the development of colonic carcinoma. Pallauf & Rimbach (1996) have demonstrated that phytic acid showed strong antinutritive effect due to its unusual structure. At complete dissociation, the six phosphate groups of phytic acid carry a total of twelve negative charges. Therefore, phytic acid effectively binds different mono-, di-, and trivalent cations and their mixtures, forming insoluble complexes (Reddy et al 1989). The formation of insoluble phytate mineral complexes in the intestinal tract prevents mineral absorption, thus reducing reducing their bioavailability (Davies 1982). Zinc appears to be the trace element of which its bioavailability is most influenced by phytic acid. Phytic acid interacts with proteins over a wide pH range, forming phytate-protein complexes. At a low acidic pH, phytic acid has a strong negative charge due to total dissociation of the phosphate groups. Under these conditions a negative influence of phytic acid on the solubility of proteins is observed because of the ionic binding between the basic phosphate groups of phytic acid and protonized amino acid (lysyl, histidyl and arginyl) residues (Fretzdorff et al 1995). Under acidic conditions, phytic acid is tightly bound to plant proteins, because the isoelectric point of plant proteins is generally around pH 4.0 - 5.0. In the intermediate pH range (6.0 to 8.0) both phytic acid and plant proteins have a net 56 ELBA Bioflux, 2011, Volume 3, Issue 1. http://www.elba.bioflux.com.ro negative charge. However, under these conditions complex formation occurs between phytic acid and proteins. Possible mechanisms include direct binding of phytic acid to the -NH2 terminal groups groups of lysine residues and multivalent cation-mediated interactions (Cheryan 1980). By binding to plant proteins, phytic acid has lower solubility and digestibility, thereby reducing the nutritive value. In addition to complexing with minerals and proteins, phytic acid interacts with enzymes such as trypsin, pepsin, amylase and galactosidase, resulting in a decreased activity of these important digestive enzymes (Singh & Krikorian 1982; Inagawa et al 1987). Phytase, its sources and biological activities. Phytase is an enzyme known as myo- inositol-hexaphosphate phosphohydrolase (Class 3: hydrolases) and is produced either by microorganisms or present in some plants (Simons et al 1990). It catalyzes the hydrolysis of myo-inositol hexakisphosphate (phytic acid) to inorganic monophosphate and lower myo-inositol phosphates, or in some cases to free myo-inositol. The Enzyme Nomenclature Committee of the International Union of Biochemistry distinguishes two types of phytase, the 3- phytase (EC 3.1.3.8) and 6-phytase (EC 3.1.3.26). 3-Phytase is typical for microorganisms and 6-phytase for plants. Microbial phytase is an enzyme that is used to hydrolyze phytate in plant protein meal-based animal feeds. Table 1 shows the different microbial phytases that have been characterized. Table 1 Optimum culture conditions of different microorganisms for the production and/or activity of phytases Species Optimum Reference pH Tempt Yeasts Arxula adeninivorans 5.5 28 Sano et al (1999) Pichia anomala 6.0 25 Vohra & Satyanarayana (2002) P. rhodanensis 4.5 70 Nakamura et al (2000) P. spartinae 4.5 75 Nakamura et al (2000) Schwanniomyces castellii 4.4 77 Segueilha et al (1992) Bacteria Bacillus subtilis 7.0 37 Kerovuo (2000) B. amyloliquefaciens 6.8 37 Idriss et al (2002) B. pumilus 6.0 35 Dechavez et al (2011) B. megaterium 6.0 35 Dechavez et al (2011) B. coagulans 6.0 35 Dechavez et al (2011) B. licheniformis 6.0 35 Dechavez et al (2011) Escherichia coli 7.0 37 Greiner et al (1993) Klebsiella aerogenes 7.0 30 Tambe et al (1994) Pseudomonas sp. 7.0 15 Lazado et al (2010) Psychrobacter sp. 7.0 15 Lazado et al (2010) Phytases are also broadly categorized into two major classes based on the pH for activity: acid phytases and alkaline phytases. Acidic phytases have been given more attention because of their applicability in animal feeds and broader substrate specificity than those of alkaline phytases. Recently, phytases have also been classified as HAP (Histidine acid phosphatase), BPP (-Propeller phytase), CP (cysteine phosphatase) and PAP (purple acid phosphatase) based on their catalytic properties (Mullaney & Ullah 2003). Phytase is present in grains and oilseeds and in the digestive tract of animals, but its activity is usually not high enough to release phosphorus from phytate to any extent (Bitar & Reinhold 1972; Lall 1991). 57 ELBA Bioflux, 2011, Volume 3, Issue 1. http://www.elba.bioflux.com.ro Monogastric animals cannot produce this enzyme. Presence of phytase in some animals is of microbial origin. In the case of fish, phytase has been detected from gut- associated microbiota (Roy et al 2009; Lazado et al 2010). Microbial phytase either as a dry powder or as a liquid is available commercially. Commercialization requires not only a practical use and delivery of the enzyme but also the ability to produce the enzyme economically. So far, large scale production of cheap phytase for animal feed has been solved through fermentation of genetically modified organisms (Yao & Fan 2000). One key problem that has been solved is instability of the phytase upon heating. Phytase cannot withstand high temperature. Schwarz & Hoppe (1992) found that pelleting a diet with phytase supplementation at 70% reduces the activity by 15 – 25%. Phytases are widely present in the plant kingdom. They have been isolated and characterized from cereals and beans. Phytase activity has also been detected in white mustard, potato, radish, lettuce, spinach, grass and lily pollen (Dvorakova 1998). To a certain extent, traces of of phytase activity has been detected in monogastric animals (Copper & Gowing 1983; Chi et al 1999). Generally, intestinal phytase does not play a significant role in food-derived phytate digestion in monogastric animals (Williams & Taylor 1985). A phytase-like enzyme was also described in the protozoan Paramecium (Freund et al 1992). Microbial phytase activity is most frequently detected in fungi such Aspergillus spp. Shieh & Ware (1968) screened over 2000 microorganisms isolated from soil for phytase production. Most of the positive isolates produced only intracellular phytase, and extracellular phytase activity was observed only in 30 isolates. All extracellular phytase producers are filamentous fungi. Twenty-eight belong to the genus Aspergillus, one to Penicillum and one to Mucor. Of the 28 phytase-producing Aspergillus isolates 21 belong to the A. niger group. Other studies have also demostrated that A. niger strains were the best producers of extracellular phytase (Howson & Davis 1983; Volfova et al 1994). There are various bacteria that have been detected for phytase production, e.g. Aerobacter aerogenes (Greaves et al 1967), Pseudomonas sp. (Irving & Cosgrove 1971; Lazado et al 2010), Bacillus subtilis (Powar & Jagannathan 1982), Klebsiella sp. (Shah & Parekh 1990), Escherichia coli (Greiner et al 1993), Enterobacter sp. (Yoon et al 1996) and Psychrobacter sp. (Lazado et al 2010). The only bacteria producing extracellular phytase are those of the genera Bacillus and Enterobacter. The phytase from E. coli is a periplasmic enzyme. The optimum pH levels of phytases vary from 2.2 to 8. Most microbial phytases, particularly those of fungal origin, have optimum pH values between 4.5 and 5.6. Some bacterial phytases, especially those from Bacillus, optimum pH levels at 6.5 - 7.5. A study done by Dechavez et al (2011) showed that the optimum pH of phytase obtained from the different Bacillus strains ranged from 5.5 to 7. The optimum pH values of plant seed phytases range from 4.0 to 7.5, most having an optimum value between 4.0 and 5.6. Two alkaline plant phytases having an optimum pH value at about 8.0 have been described in legume seeds (Scott 1991) and lily pollen (Hara et al 1985). The optimum temperature of phytases varies from 45 to 77o C. Wyss and co- workers (1998) studied the thermostability of three acid phosphatases of fungal orgin (A. fumigatus and A. niger phytase, and A. niger) by circular dichroism (CD) spectroscopy and fluorescence, and by measuring the enzymatic activity. They concluded that A. niger phytase is not thermostable, neither it has the capacity to refold after heat denaturation. At temperatures between 50 and 55oC it undergoes an irreversible conformational change that result in 70-80% loss of enzyme activity. Compared to two phytases, A. niger had higher intrinsic thermostability. At temperatures up to 80oC, only minor changes in CD spectral characteristics and only slight, but irreversible enzyme inactivation were observed. However, exposure to 90oC resulted in an irreversible conformational change and complete loss of activity. Bacillus sp. strain DS11 phytase (Kim et al 1998) has an optimum temperature at 70o C, which is higher than the optimum temperature values of most of phytases. It is also termostable, i.e., there was 100% residual activity after 10 min incubation at 700 C, in the presence of CaCl . The 2 enzyme stability of the phytase from Bacillus sp. strain DS11 was drastically reduced above 50oC in the absence of CaCl , whereas it was rather stable up to 90oC in the 2 58 ELBA Bioflux, 2011, Volume 3, Issue 1. http://www.elba.bioflux.com.ro presence of CaCl . The phytase of the different Bacillus strains that were studied showed 2 that the enzyme had maximum activity at a 37oC and retained its activity even at 80oC (Dechavez et al 2011). In addition, 50% of the activity was retained at 70oC. As to their activities, phytase from Enterobacter sp is inhibited by Zn2+, Ba2+, Cu2+ and Al3+ (Yoon et al 1996). Similarly, the phytase from B. subtilis strain N-77 was inhibited by metal ions including Zn2+, Cd2+, Ba2+, Cu2+, Fe2+, and Al3+ (Shimizu 1992). Both of these enzymes, as well as two other phytases from Bacillus spp. (Kim et al 1998; Powar & Jagannathan 1982), were inhibited by EDTA, indicating that a metal ion (calcium) is needed for their activity. Wyss et al (1999) reported that Cu2+ considerably reduced the enzyme activities of phytases from E. nidulans and A. terrus, and that several metal ions inhibited A. fumigatus phytase. The activity of A. fumigatus phytase was stimulate by EDTA up to 50%, whereas EDTA had no major effects on the enzymatic activities of the other fungal phytases that were tested such as E. nidulans, A. niger and A. terrus. In addition to the calcium-dependent Bacillus phytases, a phytase obtained from the pollen of Typha latifolia and phytases from some other plants require Ca2+ for optimal activity (Laboure et al 1993; Scott & Loewus 1986). A structural analog of the substrate, myo-inositol hexasulfate, has been shown to be a potent competitive inhibitor of both PhyA and PhyB enzymes from A. ficuum (Ullah & Sethumadhavan (1998). Fluoride is a known inhibitor of different phytases and phosphatases (Nayini & Markakis 1986). The phytase from cotyledons of germinating soybean seeds was strongly inhibited by fluoride, vanadate and inorganic phosphate (Gibson & Ullah 1988). Inorganic phosphate was a competitive inhibitor of soybean seed phytase. Competitive product inhibiton of phytate hydrolysis caused by inorganic phosphate is recognized for bacterial, fungal and oat spelt phytases (Howson & Davis 1983; Greiner et al 1993; Konietzny et al 1995). Molybdate and vanadate are known to inhibit phosphatase enzymes. It has been suggested that these transition metal oxoanions inhibit phosphomonoesterases by forming complexes that resemble the trigonal bipyramidal geometry of the transition state (Zhang et al 1997). Fungal phytase activity has been shown to be inhibited by substrate concentrations exceeding 1 mM (Ullah 1988), whereas maize root and soybean phytases were found to be inhibited at 300 M and 20 mM substrate concentration, respectively (Hubel & Beck 1996; Sutardi & Buckle 1986). Application of microbial phytase in aquaculture. Soybean meal is the most widely used plant-based meal in animal feed formulations. It is used as a primary plant protein source in animal feeds due to its relatively high crude protein level (>40%), high protein availability, good amino acid compound compared to other oilseed meals, and relatively low cost compared to fish meal. Hardy (1995) pointed out that soybean meal is the most promising alternative protein source for fish feeds in terms of future availability. It is widely studied in fish feed research and its use in commercial fish feeds is increasing (Pongmaneerat & Watanabe 1992; Oliva-Teles et al 1994; Vielma et al 2000). One disadvantage of using plant protein meals such as soybean meal in fish feeds is that approximately two-thirds of the total phosphorus in plant protein meals is present as phytate, hence, the bioavailability of phytate to fish is almost absent (NRC 1993). Effects on the bioavailability of phosphorus. Addition of phytase to high phytate striped bass diets improved the absorption and utilization of phosphorus (Hughes & Soares (1998). According to Reddy et al (1982) microbial phytase is effective in improving bioavailability of phytate phosphorus, which could be expected since phytase hydrolyzes to orthophosphate. The treatment of fish feed with phytase resulted in the improvement of protein digestibility and retention in fishes (Storebakken et al 1998; Papatryphon et al 1999; Cheng & Hardy 2002; Usmani & Jafri 2002; Vielma et al 2004; Debnath et al 2005; Baruah et al 2005; Ai et al 2007; Hassan et al 2009). Studies showed that adding microbial phytase to diets have been found to increase phytate hydrolysis and the availability of phosphorus and other minerals that are chelated by phytic acid (Lei et al 1993; Adeola et al 1995). Dietary phytase also improved the 59 ELBA Bioflux, 2011, Volume 3, Issue 1. http://www.elba.bioflux.com.ro nutritive value of canola protein concentrate and decreased phosphorus output in rainbow trout (Forster et al 1999). Several authors reported similar results for different species including rainbow trout (Rodehutscord & Preffer 1995), channel catfish (Li & Robinson 1997), African Catfish (Van Weerd et al 1999), common carp (Schafer et al 1995) and Pangasius pangasius (Debnath 2003). Digestibility of phytate phosphorus increased by as much as 50% in common carp fed a diet containing 500 units of microbial phytase kg-1 (Schafer et al 1995). Microbial phytase is effective in enhancing the bioavailability of phosphorous considerably, thereby reducing the phosphorous output in the feces. Effect on the growth performance of fish. The weight gain rates and specific growth rates of Indian major carp, Labeo rohita significantly decreased when phytic acid was included in diet at levels above 1 percent (Alvi 1994). Similar effects were evident on the growth performance and body composition of Cirrhinus mrigala fry (Usmani & Jafri 2002). It was reported that Chinook salmon, Oncorhynchus tshawytscha, fed semi- purified diets containing various levels of calcium, phosphorous, zinc, and sodium phosphate with a high dietary phytic acid (2.58%) exhibited depressed growth (Richardson et al 1985). In contrast, the growth performance increased when microbial phytase was incorporated in the diets. An increase in weight gain was reported in channel catfish fed phytase supplemented diets containing only plant protein or a combination of plant and animal protein sources (Jackson et al 1996). Weight gain and feed composition increased by 23.52 and 11.59%, respectively, compared to the control group. The same trend was also observed in Pangasius pangasius (Debnath 2003) and African catfish, Clarias gariepinus (Van Weerd et al 1999). The better performance of fish fed phytase- supplemented diets implied that either the phosphorous requirement was met along with other nutrients or that phytase has other positive effect on performance. Phytase derived from Aspergillus niger has been shown to be effective in improving bioavailibity of phytate phosphorous in diets of several animals, including poultry (Simons et al 1990); rainbow trout, Oncorhychus mykiss (Cain & Garling 1995); and common carp, Cyprinus carpio (Schafer et al 1995). Laboratory studies have shown that a concentration of 250 to 500 FTU (phytase unit) microbial phytase kg-1 diet resulted in maximum weight gain and bone phosphorous deposition in channel catfish (Jackson et al 1996; Li & Robinson 1997), and 250 FTU kg-1 can effectively replace the dicalcium phosphate supplement in the diet (Li & Robinson 1997). Results of the study conducted by Jackson et al (1996) in ponds using catfish showed that the level of phytase required for maximum weight gain and bone phosphorus deposition is 500 units phytase kg-1 diet. However, the effective level of microbial phytase according to Jackson et al (1996) was lower than the reported levels (500 to 1,000 units kg-1) of microbial phytase that are effective forr other animals (Simons et al 1990; Rodehutscord & Preffer 1995; Schafer et al 1995). Phytase supplementation increased the apparent absorption of phosphorus, nitrogen (protein), ash, calcium, magnesium, copper, iron, strontium and zinc in low–ash diets containing soybean meal, but had little effect in high–ash diets containing both soybean and fish meal. Effects on the immune system. Supplementation of microbial phytase in the diets of fish to boost the immune system is an area that is least understood and needs intensive research. Lazado et al (2010) extracted crude phytase from Pseudomonas sp. and Psychrobacter sp. Using the shake flask method. When these crude phytases were added to primary cultures of Atlantic cod, Gadus morhua head kidney leukocytes, the enzymes stimulated cell proliferation, induced higher myeloperoxidase and alkaline phosphatase activites. However, extracellular responses such as respiratory burst activities and hydrogen peroxide production were not affected as well as the ability of the cells to inhibit the proliferatio of pathogenic bacteria, Vibrio anguillarum and Aeromonas salmonicida. The preliminary results indicated that the crude microbial phytases when used to incubate immune-related cells such as head kidney leukocytes, resulted in enhanced intracelular immune responses but not the extracellular responses. 60 ELBA Bioflux, 2011, Volume 3, Issue 1. http://www.elba.bioflux.com.ro Effects on fecal phosphorus. Li & Robinson (1997) reported that fecal phosphorus decreased an average of 73% in channel catfish using 250, 500 and 750 units of microbial phytase in comparison to fish fed the basal diet, which did not contain a phosphorus supplement. It appeared supplementation with microbial phytase improved the digestibility of phytate phosphorus. Effects on the environment. The environmental impact assessment of the aquaculture industry is getting increased attention and rigorous restrictions are being set on this industry by governments and environmentalists. Farmers who are involved in freshwater aquaculture and coastal marine operations are facing increasing pressure from various organizations to control farm discharge into the surrounding waterways. This discharge, particularly high in phosphorous content, leads to eutrophication. The phosphorous in the feed ingredients occurs in number of forms, either in the inorganic form or as phosphate complexes of proteins, lipids and carbohydrates. These forms are available to the fish. Phosphorous that is present in most grain and seed by-products is generally unavailable to finfish and monogastric animals. Fish excrete phosphorous and the phosphates have direct Effect on the water quality. The particulate forms accumulate in the sludge and the phosphorous is released slowly to the water. Dissolved reactive phosphorous is usually regarded as the most important factor that affects water quality, because it is the most available nutrient that is needed for the growth of phytoplankton. Microbial phytase supplementation in the diet of fish can overcome this problem. It makes the chelated phosphorous available to fish and hence there is less fecal excretion, thereby reducing environmental pollution (Baruah et al 2004). The use of phytase in feeds also reduces or sometimes eliminates the necessity of mineral supplementation, which would decrease the cost of feeds. Although phytase was first used for environmental reasons, it been recently demonstrated that there are other nutritional and health benefits from using these enzymes (Baruah et al 2004). Conclusions. In summary, microbial phytases have different beneficial roles in aquaculture. When incorporated in the fish diets, these enzymes increased bioavailability of phosphorus, enhanced growth, stimulated the immune response, decreased fecal phosphorus and lessened the risk of environmental degradation. The different microbial phytases that have been characterized to date are physiologically active at wide temperature and pH levels, indicating that they have the potential to be incorporated in the feed during processing and could be estable in the gastrointestinal tract of the fish. Further areas of research in microbial phytases include the development of protocol for their large-scale production and the use of recombinant DNA and protein technologies through cloning of the gene encoding these enzymes and the production of these proteins using various expression systems. Acknowledgements. The authors would like to thank the National Institute of Molecular Biology and Biotechnology (NIMBB), University of the Philippines Visayas and the Aquatic Animal Health Research Group, Faculty of Biosciences and Aquaculture, University of Nordland for the support provided in the preparation of the manuscript. References Ai Q., Mai K., Zhang W., Xu W., Tan B., Zhang C., et al., 2007 Effects of exogenous enzymes (phytase, non-starch polysaccharide enzyme) in diets on growth, feed utilization, nitrogen and phosphorus excretion of japanese seabass, Lateolabrax japonicus. Comp Biochem Physiol A Mol Integr Physiol 147: 502-508. Adeola O., Lawrence B. V., Sutton A. 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