Ammonia stripping by duckweed and its feasibility in circulating aquaculture

Aquatic Botany, 13 (1982) 125--131 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

125

A M M O N I A S T R I P P I N G B Y D U C K W E E D A N D ITS F E A S I B I L I T Y IN CIRCULATING AQUACULTURE

DAN PORATH and JOAN POLLOCK

Jacob Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, Sede Boqer Campus (Israel) (Accepted for publication 19 October 1981)

ABSTRACT Porath, D. and Pollock, J., 1982. Ammonia stripping by duckweed and its feasibility in circulating aquaculture. Aquat. Bot., 13: 125--131. The duckweed Lemna gibba L. was evaluated for its potential as a biological ammonia stripper which thus improves protein production in a combined circulating aquaculture of fish. Ammonia uptake was compared with respect to varying conditions of circulating water, temperature, pH, and nitrate concentrations. Results indicate that uptake is an active process with preference for ammonia over nitrate. In an axenic culture of 0.1--0.3% duckweed biomass, Lemna gibba stripped 50% of the ammonia present at levels 10 -4 M NH3~-NH+ in 5 h, while the nitrate level (10 -: M NO~) remained constant. Circulation of fish effluent under a duckweed mat promoted an uptake of 80% ammonia in less than 48 h. The role of duckweed in direct conversion of ammonia into plant protein as an abridgement of the nitrogen cycle is discussed.

INTRODUCTION The i n t e n s i f i c a t i o n o f circulating a q u a c u l t u r e is limited p r e d o m i n a n t l y b y the a p p e a r a n c e o f a m m o n i a as a toxic substance. A m m o n i a seems t o be t h e m a j o r n i t r o g e n o u s waste p r o d u c t o f aquatic animals (Alabaster a n d L l o y d , 1980). A t t e m p t s t o eliminate the t o x i c e f f e c t o f NH3 t o fish in circulating s y s t e m s have thus far been m a d e using microbial d e c o m p o sition (Meske, 1 9 7 6 ; Naegel et al., 1 9 7 6 ; Naegel, 1977). R e c e n t l y it was s h o w n t h a t d u c k w e e d can be useful in the removal o f n u t r i e n t s f r o m d i f f e r e n t animal effluents ( S u t t o n and Ornes, 1 9 7 5 ; Stanley, 1977). It was also claimed b y F e r g u s o n and Bollard ( 1 9 6 9 ) t h a t the d u c k w e e d Spirodela oligorrhiza (Kurz) Hegelm. is capable o f preferential a m m o n i a u p t a k e in the presence o f a similar nitrate c o n c e n t r a t i o n . In t h e p r e s e n t , s t u d y , a t t e n t i o n was given to the q u a n t i t a t i v e u p t a k e o f a m m o n i a b y the d u c k weed L e m n a gibba L. in the presence o f nitrate. Testing was c o n d u c t e d in aseptic c o n d i t i o n s as well as circulating a n d n o n - c i r c u l a t i n g s y s t e m s o f fish effluent. The Hurfeish c l o n e was utilized because o f its efficient g r o w t h rate and p o t e n t i a l as an aquatic c r o p ( P o r a t h et al., 1981). The u n i q u e 0304-3770/82/0000--0000/$02.75 © 1982 Elsevier Scientific Publishing Company

126 nitrogen metabolism of duckweed as compared to other higher plants (Hansen, 1979) is discussed with respect to its feasibility in circulating aquaculture. MATERIALS AND METHODS

Plant materials For both axenic culture and circulating batches, Lemna gibba Hurfeish clone (Porath et al., 1980) was used, which had been propagated in sterile conditions as published previously (Porath and Ben Shaul, 1971). Experiments with circulating fish effluents were carried out in 200-1 asbestos tanks (150 X 40 X 30 cm) in partial shade, to minimize algal growth.

Chemical analysis of water Fish effluent was taken from a 500-1 tank containing ca. 1.0 kg fish

(Tilapia sp.) which had been fed daily on 50 g drum-dried algae (Richmond and Preiss, 1980). The effluent was filter-sterilized (Seitz filter S-1 size 6, Milldale, CT), and transferred aseptically to 250-ml Erlenmeyer flasks. Axenic duckweed cultures were maintained on the effluent in standard conditions (Porath and Ben Shaul, 1971). Such an effluent contained ammonia and nitrate in similar molar concentrations (approx. 8 x 10-s M). Other nutrients were determined. Ammonia assessment, in most cases, was made colorimetrically using a Nessler reagent according to the procedure outlined by Rand et al. (1975). NA-K tartrate was used as the stabilizer reagent. Colorimetric readings were taken in a Klett--Summerson apparatus using filter no. 42 (blue). In axenic cultures where standard growth medium was used, the Nessler m e t h o d proved unsatisfactory due to precipitate formation, Instead, Ammonia Electrode no. 95-10 (Orion Research Incorporated, Cambridge, MA 02139) was used. The electrode was attached to a digital pH meter (Radiometer, Copenhagen). Nitrate assessment was also done colorimetrically (filter no. 54, green) according to a standard procedure Szechrome NAS reagent (diphenylamine sulfonic acid chromogen). The methods and reagents were developed by the Research and Development Authority of the Ben-Gurion University of the Negev, Beer Sheva, Israel. Temperature and dissolved oxygen were measured, the latter by using a portable oxygen meter (WTW D 812 Weilheim). RESULTS In axenic culture, the addition of ca. 2.0 mg 1-~ ammonia was rapidly t a k e n up by L. gibba even in the presence of high concentrations of nitrate.

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Fig. 1. A preferential a m m o n i a uptake by axenic d u c k w e e d in standard conditions of m e d i u m and growth (about 150 mg fresh weight in 100 ml). An uptake of 50% of the added a m m o n i a (as NH4C1 ) can be observed after 5 h. A m m o n i a level was measured with a specific electrode (Orion no. 95-10) attached to a pH meter (Radiometer Copenhagen), at pH 6.0. Fig. 2. A preferential a m m o n i a uptake from fish effluents by an axenic culture of d u c k w e e d (about 100 mg fresh weight in 100 ml). Slight changes in pH were effected by adding concentrated phosphoric acid. ( A m m o n i a level was measured colorimetrically.)

Within 5 h, 50% of the ammonia was stripped, and within ca. 30 h the molar ratio of ammonia to nitrate had dropped from an initial 1 : 1 0 0 to 1 : 1 0 0 0 (Fig. 1). Again in axenic culture, when filter-sterilized fish effluent was used as the sole source of nutrients, and ammonia to nitrate molar ratios were ca. 1:1, duckweed still exhibited a preferential uptake of ammonia. Fifty percent of the ammonia was stripped within the first 8 h (Fig. 2). In o u t d o o r experiments, the ammonia level of stagnant fish effluent showed a rise in both a plant-free asbestos tank and one covered by a mat of duckweed during the first 20 h. Circulation of the effluent resulted in a rapid decline in the ammonia level (50% within 24 h after initiation of circulation, 90% within 48 h) in the tank containing duckweed. In contrast the ammonia level remained constant in the plant-free tank during the first 48 h of circulation, showing a gradual decrease thereafter (Fig. 3) due to aquatic bacterial activity. The mechanism of preferential ammonia uptake is still under investiga-

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129 DISCUSSION In a proposed combined circulating aquaculture of fish and duckweed (Porath et al., 1981), the primary protein product originates from separate intensive algaculture (Richmond and Preiss, 1980). The advantages of duckweed in such a system are as follows. (a) A rather efficient preferential ammonia uptake in the presence of nitrate can be observed (Figs. 1--3). This biological active uptake (Table I) seems to be different, however, from the mechanism shown to take place in some algae (Shilo and Shilo, 1961). In duckweed, ammonia uptake is temperature-sensitive, occurs only in living plants, and lies quite consistently in the pH range of 6--8, as shown by the different experiments. (b) When a combined circulating aquaculture of fish and duckweed is operated, the nitrogen is transformed directly from its reduced form of NH$~NH3 to plant protein, rather than having to first undergo oxidation to nitrates (Fig. 4). In addition, utilization of nitrogen by duckweed in the form of ammonia is an anabolic, sunlight-driven process, as compared to the catabolic, energy-consuming nitrification performed by the activated sludge (Meske, 1976; Naegel et al., 1976; Naegel, 1977). (c) The duckweed biomass formed in the combined system can be utilized as an additional protein product (Porath et al., 1980; R u s o f f et al., 1980), thereby improving the overall net protein production of the system.

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130 A basic question arises as to w het her the efficient am m oni a stripping by duckweed resulting in a solution having an a m m o n i a / n i t r a t e molar ratio o f up to l:100O (Fig. 1) is a specific mechanism found only in Lemnaceae. In terrestrial higher plants, such as wheat seedlings (Minotti et al., 1969), as well as in the duckw e e d Spirodela oligorrhiza, high ammonia levels repress nitrate uptake and reduction (Ferguson and Bollard, 1969). This fact m a y account, at least in part for enhanced ammonia uptake. However, Hansen (1979) showed that terrestrial plants like Lolium multiflorum Lamk., cultivated in growth solution, preferred nitrate nutrition to ammonia. Miflin and Lea (1977) suggested that the high glutamine synthesase activity in a relatively small organism like Lemna (Stewart and Rhodes, 1977) m ay result in a rather unusually high rate of ammonia uptake. Most of the ammonia available for terrestrial plants is rapidly nitrified in the soil; t her e f or e an accurate measurement of its direct uptake also presents difficulties (Beevers, 1976). However, it was shown recently that when t o m a t o e s were cultivated in a growth solution which contained a similar molar c o n c e n t r a t i o n of NH3 and NO~, nitrate uptake proceeded continuously for 21 days (Ben Asher and Ben Yaakov, 1981). With respect to ammonia metabolism on a biotic level, one may observe three differents shunts in the nitrogen cycle (Fig. 4). A microbial-driven shunt includes nitrogen fixation, nitrification and denitrification. A mixed shunt driven by plants and bacteria results in reduction to organic nitrogenou compounds. The third shunt is pe r f or m e d by microorganisms, plants and animals, while nitrogen appears only in its reduced form. In the proposed combined circulating aquaculture (Porath et al., 1981), microbial decomposition can be minimized, allowing enhanced net protein p r o d u c t i o n due to the ammonia stripping mechanism of the duckweed which completes an abridged and reduced nitrogen cycle. ACKNOWLEDGEMENTS This study was supported by the German Israel Fund for International Research and Development -- GIFRID. The manuscript was prepared with the aid o f Helen Strumpf.

REFERENCES Alabaster, J.S. and Lloyd, R., 1980. Water Quality Criteria for Freshwater Fish. Butterworth, London, pp. 85--102. Beevers, L., 1976. Nitrogen Metabolism in Plants. Elsevier, New York, pp. 1--3. Ben Asher, J. and Ben Yaakov, S., 1981. An approach for monitoring of NO~ and K+ level in solution by four-electrode conductivity sensor. Soil Sci. Am. J., submitted. Ferguson, A.R. and Bollard, E.G., 1969. Nitrogen metabolism of Spirodela oligorrhiza. I. Utilization of ammonium nitrate and nitrite. Planta, 88: 344--352. Hansen, G.K., 1979. Influence of nitrogen form and absence on utilization of assimilates for growth and maintenance in tops and roots of Lolium multiflorum. Physiol. Plant, 46: 165--168.

131 Meske, Ch., 1976. Fish culture in a recirculating system with water turnover by activated sludge. In: FAO Technical Conference on Aquaculture, Kyoto, 1976, E 62, pp. 1--7 Miflin, B.J. and Lea, P.J., 1977. Amino acid metabolism. Ann. Rev. Plant Physiol., 28: 299--329. Minotti, P.L., Williams, D.C. and Jackson, W.A., 1969. The influence of ammonium on nitrate reduction in wheat seedlings. Planta, 86: 267--279. Naegel, L.C.A., 1977. Combined production of fish and plants in recirculating water. Aquaculture, 10: 17--24. Naegel, L., Meske, C. and Mudrack, K., 1976. Untersuchungen zur Intensivhaltung yon Fischen in Warmwasserkreislauf. Arch. Fisch. Wiss., 22: 9--23. Oron, G., Porath, D., Pollock, J. and Richmond, A., 1981. Ammonia stripping of fish influent by duckweed in a circulating system. In: Reuse of Water, Washington, DC, in press. Porath, D. and Ben Shaul, Y., 197]. Structural and physiological changes during "heat bleaching" in Spirodela oligorrhiza. Isr. J. Bot., 20: 152--168. Porath, D., Efrat, Y. and Arzee, T., 1980. Morphological patterns and heterogeneity in populations of duckweed. Aquat. Bot., 9: 159--168. Rand, M.C., Greenberg, A.E. and Tarns, M.J. (Eds.), 1975. Standard Methods for the Examination of Water and Wastewater. 14th edn., Am. Publ. Health Assoc., pp. 412--415. Richmond, A. and Preiss, R., 1980. The biotechnology of algaculture. Interdiscip. Sci. Rev., 5: 60--70. Rusoff, L.L., Blakeney, E.W., Jr. and Culley, D.D., Jr., 1980. Duckweed (Lemnaceae Family): A potential source of protein and amino acids. J. Agric. Food Chem., 28: 848--850. Shilo, M. and Shilo, M., 1961. Osmotic lysis of Prymnesium parvum by weak electrolytes. Verh. Internat. Vet. theor, angew. Limnol., 14: 905--911. Stanley, R.A., 1977. Methods of biological recycling of nutrients from livestock wastes; A literature review and system analysis. Y-80, Tennessee Valley Authority, Muscle Shoals, Alabama, 45 pp. Stewart, G.R. and Rhodes, D., 1977. Control of enzyme levels in regulation of nitrogen assimilation. In: H. Smith (Ed.), Regulation of Enzyme Synthesis and Activity, London, Academic Press, pp. 1--22. Sutton, D.L. and Ornes, W.H., 1975. Phosphorus removal from static sewage effluents using duckweed. J. Environ. Qual., 4: 362--370.