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 m
3
consisting of PVC cross-flow medium with a specific surface area of 240 m
2
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 9
10 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.
Fig. 1. Schematic presentation of the experimental treatment system not to scale.
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 aeruginosa and Paracoccus denitrificans. The other, a Pseudomonas isolate,
could not be identified to species level and was deposited as Pseudomonas sp. strain JR12 DSM c 12019 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; KH
2
PO
4
, 0.4 g; NH
4
Cl, 1.0 g; MgSO
4
.7H
2
O, 0.6 g; Na
2
S
2
O
3
5H
2
O, 0.1 g; CaCl
2
2H
2
O, 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 KNO
3
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 oxygentempera-
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 Toward the end of the experimental period between days 200 and 210,
triplicate samples were derived from the fluidized bed reactor 200 g colonized sand, from the trickling filter 36 cm
2
of 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 m
2
; 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 GFC Whatman, UK filtered samples.
Total ammonia NH
3
and NH
4 +
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 temperatureoxy- 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
