Biological phosphate removal in a prototype
recirculating aquaculture treatment system
Yoram Barak, Jaap van Rijn *
The Hebrew Uni6ersity of Jerusalem,Department of Animal Sciences,Faculty of Agricultural,
Food and En6ironmental Quality Sciences,PO Box 12,Reho6ot 76100, Israel
Abstract
Efforts to reduce phosphorus concentrations in aquaculture systems have mainly dealt with improving the bioavailability of phosphorus in fish feed. Once released into the culture water, phosphorus is generally left untreated and discharged with the effluent water. In the present study, results are presented on a prototype recirculating treatment system originally designed for removal of organic matter and inorganic nitrogen. Phosphorus determinations in the various compartments of the treatment system (a digestion basin, a denitrifying fluidized bed reactor and a nitrifying trickling filter) revealed that, after 210 days of operation, more than 90% of the added phosphorus was retained within the organic matter of the trickling filter. By means of batch experiments with bacterial consortia from the reactors and with denitrifying isolates, it was found that denitrifiers were capable of phosphate uptake in excess of their metabolic requirements. The phosphorus content of organic material in the fluidized bed reactor was as high as 11.8% (on a dry-mass basis) while it was much lower in the trickling filter (around 1.9%). Anoxic incubation of the trickling filter material in the presence of an external carbon donor resulted in considerable denitrifi-cation activity and phosphate uptake. This finding served as an additional indidenitrifi-cation for the fact that phosphate removal from the water in the system was mainly mediated by denitrifying organisms. Based on these findings, the feasibility of using denitrification to control phosphate levels in the culture and effluent water of recirculating aquaculture systems is discussed. © 2000 Elsevier Science B.V. All rights reserved.
Keywords:Recirculating systems; Phosphate removal; Denitrification; Effluent treatment
www.elsevier.nl/locate/aqua-online
* Corresponding author. Tel.: +972-8-9481302; fax:+972-8-9465763.
E-mail address:[email protected] (J. van Rijn)
1. Introduction
Water quality deterioration due to excessive nutrient loading is of great concern in intensive, recirculating fish culture systems. Often this concern not only relates to the water quality requirements of the cultured animals but also to the quantity and quality of waste discharge from these systems. The latter concerns arise from more stringent environmental restrictions and concomitant higher levies on waste discharge.
The principal sources of aquaculture wastes are uneaten feed and excreta. The bulk of this waste is in the particulate form and in recirculating systems this is often removed in a concentrated form by gravitational or mechanical methods (Chen et al., 1994). Dissolved organic and inorganic nutrients, making up a smaller fraction of the total waste in these systems, are removed with the effluent water. Among the dissolved inorganic nutrients, nitrate, the end product of nitrification, is usually present at high concentrations in the effluent of recirculating systems. Also phos-phorus effluent concentrations are high due to the fact that much of the phosphos-phorus added with the feed is unutilized by the fish (Rodehutscord and Pfeffer, 1995) and, in addition, due to the lack of appropriate methods for phosphorus removal in these systems.
Enhanced biological phosphorus removal (EBPR) from domestic wastewater in activated sludge plants is accomplished by alternate stages in which the sludge is subjected to anaerobic and aerobic conditions. Under these conditions, phosphorus is released from the bacterial biomass in the anaerobic stage and is assimilated by these bacteria in excess as polyphosphate (poly-P) during the aerobic stage. Phosphorus is subsequently removed from the process stream by harvesting a fraction of the phosphorus-rich bacterial biomass (Toerien et al., 1990). Recently, evidence was provided that some of these organisms are also capable of poly-P accumulation under denitrifying conditions, i.e. with nitrate instead of oxygen serving as the terminal electron acceptor (Barker and Dold, 1996; Mino et al., 1998). As recently reviewed by Mino et al. (1998), studies on poly-P accumulating organisms have revealed the involvement of specific set of metabolic properties under anaerobic, aerobic and anoxic conditions. Under anaerobic conditions, acetate or other low molecular organic compounds are converted to polyhydrox-yalkanoates (PHA), poly-P and glycogen are degraded and phosphate is released. Under aerobic and anoxic conditions, PHA is converted to glycogen, phosphate is taken up and poly-P is intracellularly synthesized. Under the latter conditions, growth and phosphate uptake is regulated by the energy released from the breakdown of PHA.
denitrify-ing bacterial biomass. Differences between bacterial phosphate accumulation by denitrifiers in this system and in EBPR systems are discussed.
2. Materials and methods
2.1.Experimental treatment system
A small prototype treatment system (Fig. 1) was operated at our facilities at the Rehovot campus. The system was comprised of two basins (500 l, each), a denitrifying fluidized bed reactor and a nitrifying trickling filter. One basin served as a digestion basin. From this basin, water was pumped at a rate of 6.0 l min−1
into the fluidized bed reactor (height: 198 cm; diameter: 6.1 cm; volume: 5.8 l) containing 3 l of sand (average diameter: 0.7 mm) as bacterial carrier material. Effluent water from the fluidized bed reactor flowed into the trickling filter basin situated underneath a nitrifying, trickling filter (volume: 1 m3) consisting of PVC
cross-flow medium with a specific surface area of 240 m2 m−3 (Jerushalmi, Israel).
Water from this basin was pumped over the trickling filter at a rate of 42.0 l min−1.
From the trickling filter basin, water was returned by gravity to the digestion basin. The system was operated for a period of 210 days with a weekly water exchange of
910% of the total water volume. Periodically, part of the biofilm developing in the fluidized bed reactor was harvested by removing a portion of the sand, cleaning it of biofilm growth and returning the cleaned sand once more to the fluidized bed reactor. Weekly, four to five daily portions of 400 g feed (30% protein; 1% phosphorus-P) were added to the digestion basin (total number of recorded feeding days: 144).
2.2.Phosphate accumulation and nitrate remo6al by denitrifying isolates
Three denitrifying strains, isolated from a fluidized bed reactor used for nitrate removal in a recirculating fish culture system (van Rijn et al., 1995; Aboutboul et al., 1996), were tested for combined nitrate and phosphate removal. Based on fatty acid profiles and 16S-rDNA, two of these isolates could be identified as Pseu
-domonas aeruginosaandParacoccus denitrificans. The other, aPseudomonasisolate, could not be identified to species level and was deposited asPseudomonassp. strain JR12 (DSM c12019) in the German Collection of Microorganisms and Cell Cultures (van Rijn et al., 1996). The denitrifying organisms were cultured at 30°C in medium containing (per liter): Sodium acetate, 5.6 g; KH2PO4, 0.4 g; NH4Cl, 1.0
g; MgSO4.7H2O, 0.6 g; Na2S2O3 5H2O, 0.1 g; CaCl2 2H2O, 0.07 g, Tris
(Hydrox-ymethyl aminomethane) – hydrochloride, 12 g; and 2 ml of a trace element solution (Visniac and Santer, 1975). The pH of the medium was 7.2. Studies were conducted with cells harvested during the late log phase of growth (after 4 – 5 days). Cells were washed twice and resuspended in the aforementioned synthetic medium with various phosphate and nitrate (as KNO3) levels (Section 3). Determinations of
nitrate and phosphate removal by these isolates under various conditions were conducted in triplicate in a temperature-controlled (30°C) incubation vessel (300 ml), placed on a magnetic stirrer and fitted with nitrate, pH and oxygen/ tempera-ture electrodes. Anaerobic conditions in the vessel were obtained by continuous flushing with prepurified nitrogen gas. Positive pressure within the incubation vessel prevented oxygen penetration, as verified by continuous oxygen monitoring. The experiments were initiated by acetate addition. Periodically, samples were with-drawn, filtered, and analyzed for ammonia, nitrite and phosphorus. Changes in nitrate levels and pH were monitored every 2 – 5 min, whereas protein concentra-tions were determined in aliquots withdrawn at the beginning and end of the experiment. During the various experiments, the bacterial biomass (as measured by protein analysis) did not increase by more than 20%. Ammonia concentrations decreased in correspondence with the increase in bacterial biomass in the medium. An increase in pH (not exceeding 0.6 units) was measured in all experiments.
2.3.Batch studies with bacterial consortia obtained from the laboratory-scale treatment system
Organic matter, making up the biofilms on the PVC and sand carriers in the trickling filter and fluidized bed reactor, respectively, was detached from the carriers by grinding. After washing the detached biofilms in the above described medium, combined nitrate and phosphate removal by the bacterial consortia present in these biofilms was examined by the same experimental protocol used for the bacterial isolates.
2.4.Quantitati6e and qualitati6e phosphorus analyses
triplicate samples were derived from the fluidized bed reactor (200 g colonized sand), from the trickling filter (36 cm2of colonized PVC), from the digestion basin
(3 ml sludge) and from the water in the treatment system (2 l). Dry weight and total phosphorus content of the organic matter present in the samples were determined. Total phosphorus in each of the treatment compartments was calculated based on the following information: total sand dry weight in fluidized bed reactor, 800 g; total surface area of PVC in trickling filter, 240 m2; total sludge dry weight in
digestion basin, 8.8 kg; total water volume in treatment system, 1000 l.
2.5.Analytical procedures
Inorganic nutrients were determined in GF/C (Whatman, UK) filtered samples. Total ammonia (NH3 and NH4+) was determined as described by Scheiner (1976),
nitrite according to Strickland and Parsons (1968) and nitrate was measured with the Szechrome NAS reagent (Ben Gurion University, Applied Research Institute) or, in laboratory batch experiments, with a specific nitrate electrode (Radiometer, Denmark) amplified with a pH meter (Radiometer, model: PHM92). Inorganic orthophosphate (phosphate throughout the text) in filtered samples and total phosphorus (organic, particulate and inorganic orthophosphate) in unfiltered sam-ples was determined with the ascorbic acid method described by Golterman et al. (1978).
Oxygen and temperature were measured with a YSI (model 57) temperature/ oxy-gen probe (Yellow Springs Instruments, USA).
Bacterial dry weight and dry weight of organic matter obtained from the various components of the treatment system were determined after overnight drying of the samples at 105°C. Protein was determined according to Lowry et al. (1951) with bovine serum albumin as standard.
3. Results
3.1.Inorganic nitrogen and phosphorus concentrations in the experimental treatment system
Despite the closed-mode of operation and daily feed supply, inorganic nitrogen and phosphate concentrations in the experimental system (determined in samples obtained from the trickling filter basin), did not accumulate over the 210 days of operation (Fig. 2). Oxygen was at saturation in the trickling filter basin while it was undetectable in the digestion basin and fluidized bed reactor (not shown). The pH of the system fluctuated between 6.9 and 7.6 (not shown).
Fig. 3. Removal (positive values) or production (negative values) of ammonia, nitrite, nitrate and phosphate by the fluidized bed reactor (F.B.R.), trickling filter (T.F.) and digestion basin (D.B.) over the experimental period.
A qualitative analysis revealed that samples derived from the fluidized bed reactor were high in phosphorus content as compared to samples from other treatment compartments (Table 1). Absolute values of total phosphorus were highest in the trickling filter and much lower in the digestion basin, fluidized bed reactor and in the water body of the system (Table 1). The total phosphorus load of the system was 576 g P (144 feeding days×4 g P). We estimated that during the experimental period 16 g P was removed with water exchange and 49 g P with the wasted biomass from the fluidized bed column. Therefore, the total expected phosphorus content of the system over the experimental period was 511 g P. Based on results presented in Table 1, this implies that as much as 9198% of the added phosphorus was retained within the trickling filter, 0.790.1% in the fluidized bed reactor, 4.791.9% in the digestion basin and 2.290.5% in the water of the system.
3.2.Phosphate accumulation by denitrifying and nitrifying consortia
A denitrifying consortium derived from the fluidized bed reactor was incubated under laboratory conditions in the presence or absence of nitrate (Fig. 4). Phos-phate uptake took place in the presence of nitrate whereas after depletion of nitrate from the medium, phosphate was released. In the presence of nitrate, the consor-tium assimilated ammonia and phosphate at a molar N/P ratio of 1.9. Taking into account that the molar N/P ratio of bacterial biomass varies from 5 to 16 (Brock and Madigan, 1991), it can be concluded that in the presence of nitrate, phosphate is assimilated in excess of the metabolic requirements of the bacteria comprising the consortium.
A nitrifying consortium derived from the trickling filter was incubated in the laboratory under aerobic (nitrifying) and anoxic (denitrifying) conditions. Anoxic incubation was conducted in the presence of acetate. Under aerobic conditions (Fig. 5(A)), ammonia was nitrified to nitrate while phosphate concentrations in the medium increased gradually. Trickling filter material was rich in organic matter. Degradation of this organic matter and ammonification of nitrogenous organic compounds to ammonia probably explains the deficit observed between nitrate production and ammonia consumption. Incubation of trickling filter material in the absence of oxygen resulted in a decrease of nitrate and phosphate concentrations in the medium (Fig. 5(B)).
Table 1
Absolute and relative phosphorus content (9S.D.) in different compartments of the experimental treatment system after 210 days of operation
Compartment Total phosphorus (g) Phosphorus in organic matter (mg/g dry weight)
Fluidized bed reac- 3.590.3 118.595.3 tor
Trickling filter 464.7940.0 18.695.2 2.491.1 Digestion basin 24.299.7
11.092.6
Fig. 4. Changes in nitrate ( ) and phosphate () concentrations during batch incubation of a denitrifying consortium derived from the fluidized bed reactor. Arrow indicates time of nitrate addition.
3.3.Phosphate accumulation by denitrifying isolates
All three isolates assimilated phosphate in excess under denitrifying conditions (i.e. in the presence of nitrate) and released phosphate when nitrate became depleted (Fig. 6). Molar N/P ratios of the three different isolates at the end of the denitrifying period were 0.4, 2.1 and 1.3 for P. aeruginosa, P. denitrificans and
Pseudomonas sp. (JR12), respectively.
3.4.Phosphate accumulation and nitrate remo6al in the fluidized bed reactor
The performance of the fluidized bed reactor on selected days throughout the 210 days experimental period are presented in Table 2. It is shown that the denitrifying consortium present in this reactor, assimilated ammonia and phosphate at a molar N/P ratio ranging from 0.5 to 2.4; i.e. phosphate accumulation by this consortium was in excess of the metabolic requirements. With undetectable low inlet concentra-tions of nitrate and nitrite (days 45 and 46), phosphate was released as indicated by the negative phosphate removal values. The latter observation points to the fact that only under denitrifying conditions, i.e. in the presence of nitrate, the denitrify-ing consortium was capable of phosphate uptake in excess of metabolic requirements.
Y
Daily removal rates (per surface area of carrier) of inorganic nitrogen and phosphate by the fluidized bed reactor and calculated ratios between ammonia and phosphate removal at selected sampling daysa
Subsequent reconnection of the water supply was followed by a nitrate increase and a phosphate decrease in the anoxic treatment stage.
4. Discussion
Information on phosphorus dynamics in recirculating fish culture systems is scarce. As determined in more conventional fish culture systems, phosphorus recovery values by fish vary from 10 to 30% of the phosphorus added in the feed (Avnimelech and Lacher, 1979; Boyd, 1985; Schroeder et al., 1991). Of the released phosphorus, roughly 20% is in the soluble form while the remainder is present in
Fig. 7. Changes in nitrate () and phosphate ( ) concentrations in the digestion basin after uncoupling (nitrate off) and reconnecting (nitrate on) the water supply from the trickling filter to the digestion basin.
the organic sludge (Bodvin et al., 1996). Chen et al. (1996) estimated that the total phosphorus in aquaculture sludge is as high as 1.3% of the total solids. Increased environmental concern associated with phosphorus discharge has stimulated re-search on phosphorus reduction in aquaculture systems. Most of the studies in this field have dealt with decreasing phosphorus inputs by increasing the dietary phosphorus availability (Rodehutscord et al., 1994; Rodehutscord and Pfeffer, 1995). Phosphorus released into the culture systems is generally left untreated and discharged with the organic solids and effluent water.
Based on the information available on poly-P organisms in EBPR processes we assume that these bacteria are different from the denitrifying organisms responsible for polyphosphate accumulation in the present study. This assumption is based on the fact that the denitrifying organisms present in the fluidized bed reactor in this study were shown to be capable of polyphosphate synthesis under permanent anoxic conditions. Further evidence for this assumption was obtained from recent studies in our laboratory. Studies on a number of denitrifying strains isolated from the fluidized bed reactor revealed that: (a) polyphosphate synthesis and denitrifica-tion were conducted by organisms incapable of producing PHA; and (b) in organisms capable of producing PHA, polyphosphate synthesis was not coupled to PHA and glycogen degradation (Barak and van Rijn, in press).
A characterization of the phosphate dynamics in the experimental treatment system revealed that, although most of the phosphorus accumulated in the trickling filter, active removal of phosphate was highest in the fluidized bed reactor. The phosphorus content of organic matter attached to the sand particles in the fluidized bed reactor was as much as 11.8% of the dry weight. Similar values were reported for polyphosphate accumulating organisms in wastewater treatment plants (Degre´-mont Ltd., 1991). The high total phosphorus content of the trickling filter sludge can be explained as follows. As our experimental set-up contained no mechanical filtration stage, water led into the trickling filter was rich in organic matter. It may be assumed, therefore, that much of the sloughed denitrifying biomass from the fluidized bed reactor was captured in the trickling filter. This, together with the anoxic areas within the trickling filter resulting from the high organic load, may have resulted in a considerable accumulation of denitrifiers in the trickling filter. Evidence for this assumption was provided by the observed denitrification potential of the trickling filter material upon batch incubation under anoxic conditions (Fig. 5(B)). It is interesting to notice that aerobic incubation of the trickling filter material resulted in a release of phosphorus into the surrounding medium (Fig. 5(A)). A possible explanation for this observation is that phosphorus was released due to carbon limitation of the denitrifying organisms since no external carbon was added. We obtained similar results (phosphorus release under conditions of carbon limitation) in batch experiments with denitrifying isolates (not shown).
5. Conclusions
particulate organic matter to be trapped in this filter. It remains to be examined, therefore, how the addition of a mechanical filtration unit between the fluidized bed reactor and the trickling filter, could serve as a trap for phosphate-rich organic material, and if such phosphate removal will be significant in controlling the inorganic phosphate levels in the culture and effluent water of recirculating systems.
Acknowledgements
This study was supported through grant number 820-0136-98 by the Chief Scientist Office, Ministry of Agriculture and Rural Development, Israel.
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