Aquacultural Engineering 22 (2000) 75 – 85 www.elsevier.nl/locate/aqua-online Biodegradable polymers as solid substrate and biofilm carrier for denitrification in recirculated aquaculture systems A Boley *, W.-R Muăller, G Haider Uni6ersitaăt Stuttgart, Institut fuăr Siedlungswasserbau, Wasserguăte- und Abfallwirtschaft, Arbeitsbereich Biologie, Bandtaăle 2, D-70569 Stuttgart, Germany Abstract A simple process for nitrate removal is proposed for its application in aquaculture Biodegradable polymer pellets are acting as solid substrate and biofilm carrier for denitrification Laboratory experiments with conventional aquaria and fish were used to examine the feasibility and a first evaluation of the process performance in a recirculated aquaculture system All over the test-period the fish were in a good condition Nitrate concentrations in the aquaria with treatment were low compared to the untreated reference system A further advantage was the stability of the pH in the units with denitrification whereas pH of the untreated water decreased due to nitrification © 2000 Elsevier Science B.V All rights reserved Keywords: Water treatment; Recirculating systems in aquaculture; Denitrification; Biodegradable polymers; Solid substrates Introduction In aquaculture systems nitrate removal is a problem which has not always found satisfactory solutions in practice Modern technology of water treatment in recirculating systems consists of solid waste removal, carbon-removal and nitrification, pH and CO2 control (Fig 1) Consumption of energy and water in those systems can be lowered if the nitrate produced in the aerobic biofilter unit is reduced by a denitrification step This diminishes the fresh water addition and the amount and impact of the wastewater * Corresponding author Tel.: +49-711-6855441; fax: +49-711-6853729 E-mail address: angela.boley@iswa.uni-stuttgart.de (A Boley) 0144-8609/00/$ - see front matter © 2000 Elsevier Science B.V All rights reserved PII: S 4 - ( 0 ) 0 3 - 76 A Boley et al / Aquacultural Engineering 22 (2000) 75–85 Denitrification is defined as the biological nitrate reduction sequence NO− “ NO− “ N2O “N2 We restrict the discussion to the heterotrophic biological process where organisms gain energy and carbon from organic compounds A conventional technique is to add an organic carbon source (e.g ethanol, acetic acid) to a denitrification reactor (Frick and Richard, 1985; Stoăver and Roennefahrt, 1990) The disadvantage of this treatment process is the need of a close, rather sophisticated and costly process control, the risk of overdosing and a deepened knowledge about the operation of this biological system In contrast to conventional treatment units, denitrification with biodegradable polymers presented here is a simple process Microorganisms use the biopolymer in form of pellets as biofilm carrier and simultaneously as water insoluble carbon source for denitrification, which is accessible only by enzymatic attack (Muăller et al., 1992; Wurmthaler, 1995) The scheme in Fig elucidates the difference between conventional denitrification and the new process presented here In conventional denitrification with a fixed bed reactor a biofilm will grow on the inert carrier and denitrification takes place whenever the water contains NO− , soluble organic substrate and trace elements End-products are N2, H2O, CO2 and biomass The new system with biodegradable Fig Scheme of a modern recirculated aquaculture system Fig Denitrification processes with different organic substrates A Boley et al / Aquacultural Engineering 22 (2000) 75–85 77 polymers does not require an external dosing of soluble organic substrate as the polymer itself acts as biofilm carrier and organic carbon source Heterotrophic denitrification positively influences the pH of the water If proteins are metabolized by fish, the end-products of respiration after hydrolysis to amino − acids (e.g glycine) are NH+ and HCO3 , which are excreted via gills (Eq (1); Forster and Goldstein, 1969): − NH2 CH2 COOH + 1.5 O2 “NH+ + HCO3 + CO2 (1) The nitrification equation with biomass formation (Wheaton et al., 1994, Eq (2)) indicates the production of protons (catalyzed by enzymes of, e.g Nitrosomonas and Nitrobacter species): − 1.021 NH+ +1.895 O2 +1.021 HCO3 “0.021 C5H7O2N +NO3− +1.979 H2O+ 0.914 CO2 + H+ (2) Decreasing pH values have to be coped with by adding, e.g NaHCO− The use of a biodegradable polymer as organic carbon substrate, e.g PHB, leads to biomass, carbon dioxide and simultaneous reduction of nitrate to elementary nitrogen With a yield coefficient of 0.45 g biomass/g PHB assumed (Heinemann, 1995), the summarized denitrification equation including biomass formation can be given as: 0.494 C4H6O2 +NO3− “0.130 CO2 +HCO3− +0.415 N2 + 0.169 C5H7O2N+ 0.390 H2O (3) The summary equation (nitrification and denitrification) results in: − 1.021 NH+ +1.021 HCO3 +1.895 O2 + 0.494 C4H6O2 “3.369 H2O +2.044 CO2 +0.415 N2 + 0.190 C5H7O2N (4) CO2 produced can be stripped by aeration If all the nitrate produced is denitrified, the pH remains constant Materials and methods The examination of solid substrates in form of biodegradable polymers for denitrification purposes in aquaculture has been carried out in simple laboratoryscale test systems (Fig 3) We used four commercially available 100-l aquaria operated in parallel Each aquarium was equipped with an aerobic biofilter-unit filled with SIPORAX-packing (Schott, 0.75 l) The total volume (aquarium+ biofilter) was 82.5 l It was filled with tap water and the temperature was adjusted to :25°C The tank was illuminated 10 h/day Each aquarium contained 14 fish (Leuciscus idus) with an initial total biomass of 80 g The feeding rate of one tank was 0.9 g/day with ‘Trouvit pro aqua Brut.00’, (Milkivit–Werke GmbH, protein content 60%, according to manufacturer) 78 A Boley et al / Aquacultural Engineering 22 (2000) 75–85 Fig Aquarium system for testing denitrification Period 1: Carbon removal + nitrification only; period 2: Carbon removal +nitrification + denitrification Table Material characterization Short name PHB PCL Bionolle Trade name, type Chemical formula Mass (g) per unit Total surface (m2) Manufacturer BIOPOL D400 GN [C4H6O2]n 265 0.52 Monsanto TONE P 787 [C6H10O2]n 264 0.39 Union Carbide Bionolle c 6010 [C6H8O4]n 294 0.46 Showa Denko Before starting the experiments the biofilters for carbon removal and nitrification were subjected to a conditioning phase with a medium containing ammonia to secure a good nitrification performance The first period of the experiments was confined to nitrification via biofilter In period the denitrification units were connected They consisted of small fixed bed reactors (‘denireactors’) with a volume of 0.375 l As subsequent aerobic treatment, small aerated fixed bed units (volume 420 ml), with SIPORAX-packing, were installed for polishing to avoid possibly occurring byproducts (e.g NO− ) Different biodegradable polymers pellets (Table 1) were used as packing for the denireactors and, as reference, one was operated with glass beads The polymers to be tested were filled into the closed denireactors without pretreatment and enclosed in the system by a plastic foam bottom and a cover above Water was recirculated from the aquarium to the denireactor and — via the polishing unit — back to the aquarium with flowrates of QD = 0.3–0.5 l/h This low throughput was selected to achieve suitable conditions for denitrification, which depends among others upon sufficiently low oxygen concentrations With an ample residence time, the high oxygen content in the aquarium effluent cA (range 6.8–7.8 mg/l O2) is consumed in the inlet zone of the denireactor by aerobic biodegradation of the polymers This ensures anoxic conditions in the remaining part of the unit A Boley et al / Aquacultural Engineering 22 (2000) 75–85 79 Relevant water-parameters in the tanks were examined Weekly measurements of − − 3− temperature, pH, oxygen, conductivity, NH+ were conducted , NO2 , NO3 , PO4 Occasionally the dissolved organic carbon (DOC) concentration was determined The water volume added for compensation of evaporation was taken into account A simple model for evaluating the performance of the denireactors with different polymer packing has been used after the start-up and beyond the lag-time periods of these units The influence of the oxygen has not yet been taken into account as well as NO− was not included into the model The NO− concentration in the aquarium, considered as complete mixed tank, as function of time can be described as follows: dcA/dt =(QD* (cE −cA) + mNO3)/VA (5) The lowest concentration in the aquarium cA0 to be achieved under steady state conditions, i.e with an effluent concentration of the denireactor cE = is determined by the relation: cA0 =mNO3/QD (6) − − where cA is the cA0NO− conc., aquarium tank (mg/l N-NO3 ); cE is the NO3 conc., − − effluent denireactor (mg/l N-NO3 ); mNO3 is the daily production of NO3 in system (mg/day N-NO− ); QD is the recirculation rate=throughput denireactor (l/h); and VA is the water volume of aquarium tank (l) The overall volumetric denitrification performance rDV in mg/(Lh) N-NO− of a denireactor is given by Eq (7) rDV =QD* (cE −cA)/VD (7) − rDV is the overall volumetric denitrification rate of a denireactor (mg/(lh) N-NO ); and VD is the denireactor volume (l) Results Due to a preconditioning of the biofilters, ammonium and nitrite concentrations were low during the whole test-periods and NH+ did not exceed 0.1 mg/l − − (N-NH+ ), NO was below 0.05 mg/l N-NO after the first day Temperature was 2 stable in a range of 25.1 – 26.1°C DOC values increased slowly during the tests, beginning with – mg/l they did not exceed 5–7 mg/l at the end of the tests In period NO− concentrations increased in all four aquaria in a very similar way (Fig 4) In this period and from the reference aquarium system, the daily production of nitrate could be calculated to 56.1 (9 5) mg/day N-NO− Denitrification with PHB started days after installation of the unit following a lag-time (= period of adaptation of denitrifying microorganisms) The lag-time of PCL and Bionolle was 16 days It was defined as the point when the steepest negative slope of the nitrate concentration versus time occurred This was an indication of the unit to operate at its maximum (Fig and Table 2) For Bionolle two phases of activity could be observed, an explanation cannot yet be given 80 Solid substrate PHB PCL -Bionolle, period and Specific surface (m2/l) 1.49 0.87 1.22 Temp (°C) 20–25 20–25 20–25 Flowrate (l/h) 0.4–0.6 0.2–0.3 0.3–0.6 Concentration range 5–40 mg/l N-NO− Volumetric rates (mg N-NO− / (lh)) Surface related rates (mg N-NO− /(mh)) 7–41 21–166 1.5–10; 12–77 5–28 20–160 1.3–9; 10.5–67 A Boley et al / Aquacultural Engineering 22 (2000) 75–85 Table Estimated maximal denitrification velocities of tested materials A Boley et al / Aquacultural Engineering 22 (2000) 75–85 81 As Fig shows the theoretical concentration limits (about mg/l, Eq (6)) have approximately been achieved with PCL and Bionolle at the end of test Nitrate concentrations in the effluent of these denireactors were below the detection limit (0.23 mg/l N-NO− ) This confirmed our assumptions In contrast to these results the aquarium with the PHB denireactor reached the equilibrium already at a concentration of 18 mg/l N-NO− This decrease of performance (= decrease of denitrification velocity) can probably be explained by clogging and short-circuiting of the denireactor due to excess biomass production, which has been observed after the end of period As the acid neutralizing capacity of the tap-water was low (ANC= mmol/l), pH values decreased with time, due to nitrification (Fig 5) To prevent extensive decrease of pH, it was adjusted twice with NaHCO3, which was added to the reference aquarium (packing with glass beads) at days 71 and 100 For the aquarium with the PHB denireactor NaHCO3 addition was not necessary because at day 71 denitrification had already started The start of denitrification immedi- Fig Nitrate concentrations in testsystems Temperature: 25 – 26°C Fig pH in testsystems Temperature 25–26°C Arrows indicate pH adjustment with NaHCO3 (After 71 days: Reference, PCL, Bionolle; after 100 days: only Reference) 82 Table Denitrification velocities in fixed bed reactors with different substrates Substrate Sand 1.5 Methanol 12 Burned clay Burned clay PHB 1.3 0.9 1.55 Acetic Acid Ethanol PHB 12 12–13 10 14–34 49–59 16 10–26 54–66 11 PHB 1.6 PHB 15 22 14 PCL 1.2 PCL 15 13 10 Sand Brick granules a (d = 0.3–0.9 mm) Dissolved organic substrates 2.2 Ethanol Hawkins et al., 1978 Partos and Richard, 1984 c Jestin et al., 1986 d Wurmthaler, 1995 e Schick, 1998 f Arbiv and Rijn, 1995 g Sautier et al., 1998 b Temp (°C) 22.5–27 20 Volumetric rate (mg N-NO− /(lh)) Surface related rate (mg N2 NO− /(mh)) Type of water and installation 145 97 Wastewater, laboratory-scalea Drinking water plantb Drinking water plantc Tap water, laboratory-scaled Tap water, laboratory-scalee Tap water, laboratory-scalee Fluidized bed, aquaculture systemf Marine closed aquaculture systemg 36 100 A Boley et al / Aquacultural Engineering 22 (2000) 75–85 Carrier-material Spec surface (m2/l) A Boley et al / Aquacultural Engineering 22 (2000) 75–85 83 Table Estimated costs of substrates for nitrate removal Substrate Methanol: CH3OH Ethanol: C2H5OH Acetic acid: CH3COOH PCL (C6H10O2)n PHB (C4H6O2)n Bionolle c 6010 (C6H4O2)n Price of substrate (€/kg substrate) Consumption of substrate (kg substrate/kg N-NO− ) Costs of denitrification (€/kg N-NO− ) 1.00 2.08–3.98 2.0–4.0 1.20 2.0 2.4 2.40 3.5 8.0 5.00 1.33–1.77 10.00 2.1–2.7 Commercially not available 6.6–8.9 21.0–37.2 ately may lead to an increase of pH For the PCL and Bionolle denireactor NaHCO3 was also added at day 71, because denitrification had not yet started Later pH increased too, therefore an adjustment was no more required These results are compatible with Eq (4) After both test-periods the fish were in a good condition and no fish died They almost doubled their initial body weight all together up to 145 g (9 5%) per aquarium Discussion Denitrification systems are not yet common practice in aquaculture and until now they were mostly installed for research purposes The reason is that toxicity of nitrate is low, compared with nitrite and ammonia A comparison of the polymer based denitrification presented here with conventional denitrification processes is shown in Table The volumetric and surface related denitrification rates with PHB and PCL as substrates are lower than the respective rates with methanol and ethanol However the same order of magnitude as with acetic acid as substrate could be observed The costs of the denitrification process depend upon the price of substrates, technical devices and labor costs for operation The costs of different substrates in relation to their denitrification capacity are given in Table Although ethanol and methanol have the best cost-benefit ratio, their use in aquaculture would require additional treatment units to prevent any spill into the recirculated water Denitrification with soluble carbon sources demands a sophisticated process control and continuous monitoring A system based on insoluble solid substrates as carbon source however is an easy to handle process 84 A Boley et al / Aquacultural Engineering 22 (2000) 75–85 Conclusions The denitrification process based on the use of solid substrates (biodegradable polymers) can not yet compete in its performance with the classical treatment units for biological nitrate removal with liquid substrates Preliminary deliberations for this new denitrification process in aquaculture suggest that this is not a low-cost process at present A cost-benefit analysis could not yet be carried out as data close to reality are lacking However when extrapolating these laboratory-scale results and weighing the advantages, which are the user-friendly simplicity and safety of this process in relation to the disadvantages as the high costs of the solid substrates, we remain optimistic A positive expectation is: reduction of clean water requirement, reduction of waste water production, reduction of energy consumption which will contribute to favor an application in the future Acknowledgements This work was supported by Deutsche Bundesstiftung Umwelt and European Communities, INCO-DC References Arbiv, R., Rijn, J., 1995 Performance of a treatment system for inorganic nitrogen removal in intensive aquaculture systems Aquacult Eng 14, 189 – 204 Forster, R.P., Goldstein, L., 1969 Formation of excretory products In: Hoar, W.S., Randall, D.J (Eds.), Fish Physiology, vol Academic Press, New York, pp 313 – 345 Frick, B.R., Richard, Y., 1985 Ergebnisse und erfahrungen mit der biologischen denitrifikation in einem wasserwerk (Experience with biological denitrification in a full scale drinking water treatment plant) Vom Wass 64, 145–154 Hawkins, J.E., Cooper, P.F., Seaman, M.R., 1978 Denitrification of sewage effluent by attached growth technique In: Mattock, G (Ed.), New Processes of Wastewater Treatment and Recovery Ellis Harwood, Chichester, pp 107–124 Heinemann, A., 1995 Denitrifikation mit Co-Immobilisaten aus Bakterien und Poly-b-Hydroxybutyrat (Co-Immobilization of bacteria and Poly-b-Hydroxybutyrate in biopolymer-matrices for denitrification) Stuttg Ber Siedl Wass Wrtsch., Bd 135, R Oldenbourg, Muănchen Jestin, J.-M., Philipot, J.-M., Berdou, C., Moulinot, J.-P., 1986 Maitrise d’un processus biologique: la de´nitrification a` Eragny T.S.M.-L’Eau, pp 359 – 362 Muăller, W.-R., Wurmthaler, J., Heinemann, A., 1992 Biologische nitratelimination in kleinen wasserwerken (Biological nitrate elimination in small drinking water treatment plants) WAP 5, 231 – 235 Partos, J., Richard, Y., 1984 De´nitrification d’eau potable par cultures fixe´es mise en route de la station de Chateau Landon In: Use of Fixed Biomass for Water and Wastewater Treatment, Proceed 37th Internat Conf CEBEDEAU, 23–25 May 1984, Lie`ge, Belgium, pp 163 – 186 Sautier, N., Grasmick, A., Blancheton, J.P., 1998 Biological denitrification applied to a marine closed aquaculture system Water Res 32, 1932–1938 Schick, V., 1998 Denitrifikation von Trinkwasser mit biologisch abbaubaren Polymeren im kontinuierlichen Festbettverfahren (Denitrification of drinking water in a fixed bed reactor with biodegradable polymers) Diploma Thesis, Lehrstuhl fuăr Wasserchemie, Universitaăt Karlsruhe, Germany A Boley et al / Aquacultural Engineering 22 (2000) 7585 85 Stoăver, T., Roennefahrt, K.W., 1990 Biologische denitrifikation in der trinkwasseraufbereitung (Biological denitrification in drinking water treatment) Vom Wass 75, 287 – 305 Wheaton, F.W., Hochheimer, J.N., Kaiser, G.E., Krones, M.J., Libey, G.S., Easter, C.C., 1994 Nitrification filter principles In: Timmons, M.B., Losordo, T.M (Eds.), Aquaculture Water Reuse Systems: Engineering Design and Management Elsevier, Amsterdam, pp 101 – 124 Wurmthaler, J., 1995 Biologische Nitratelimination mit einem Festsubstrat bei der Trinkwasseraufbereitung (Biological nitrate elimination in drinking water treatment with a solid substrate) Stuttg Ber Siedl Wass Wrtsch Bd 132, R Oldenbourg, Muănchen  ... remains constant Materials and methods The examination of solid substrates in form of biodegradable polymers for denitrification purposes in aquaculture has been carried out in simple laboratoryscale... Conclusions The denitrification process based on the use of solid substrates (biodegradable polymers) can not yet compete in its performance with the classical treatment units for biological nitrate... SIPORAX-packing, were installed for polishing to avoid possibly occurring byproducts (e.g NO− ) Different biodegradable polymers pellets (Table 1) were used as packing for the denireactors and, as