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. Müller, G. Haider
Uni6ersität Stuttgart, Institut für Siedlungswasserbau, Wassergüte- und Abfallwirtschaft,
Arbeitsbereich Biologie, Bandtäle 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 denitrifica-
tion. 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 poly-

mers; Solid substrates

1. Introduction

In aquaculture systems nitrate removal is a problem which has not always found
satisfactory solutions in practice. Modern technology of water treatment in recircu-
lating 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 0 1 4 4 - 8 6 0 9 ( 0 0 ) 0 0 0 3 3 - 9
76 A. Boley et al. / Aquacultural Engineering 22 (2000) 75–85

Denitrification is defined as the biological nitrate reduction sequence NO− 3 “

NO− 2 “ 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; Stöver and Roennefahrt, 1990).
The disadvantage of this treatment process is the need of a close, rather sophisti-
cated 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 (Müller et
al., 1992; Wurmthaler, 1995).
The scheme in Fig. 2 elucidates the difference between conventional denitrifica-
tion 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− 3 , soluble organic substrate and trace elements.
End-products are N2, H2O, CO2 and biomass. The new system with biodegradable

Fig. 1. Scheme of a modern recirculated aquaculture system.

Fig. 2. 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+ −
4 and HCO3 , which are excreted via gills (Eq. (1);
Forster and Goldstein, 1969):
NH2CH2COOH + 1.5 O2 “NH+ −
4 + 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+ −
4 +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− 3 .
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+ −
4 +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.

2. Materials and methods

The examination of solid substrates in form of biodegradable polymers for

denitrification purposes in aquaculture has been carried out in simple laboratory-
scale 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+ bi-
ofilter) 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. 3. Aquarium system for testing denitrification. Period 1: Carbon removal + nitrification only; period
2: Carbon removal +nitrification + denitrification.

Table 1
Material characterization

Short name PHB PCL Bionolle

Trade name, type BIOPOL D400 GN TONE P 787 Bionolle c 6010

Chemical formula [C4H6O2]n [C6H10O2]n [C6H8O4]n
Mass (g) per unit 265 264 294
Total surface (m2) 0.52 0.39 0.46
Manufacturer Monsanto Union Carbide 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 2 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−2 ). 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

temperature, pH, oxygen, conductivity, NH+ − −
4 , NO2 , NO3 , PO4
3−
were conducted.
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−2 was not included into the model.
The NO− 3 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 = 0 is deter-
mined by the relation:
cA0 =mNO3/QD (6)
where cA is the cA0NO− − −
3 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− 3 ); 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− 3 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 );
3
and VD is the denireactor volume (l).

3. Results

Due to a preconditioning of the biofilters, ammonium and nitrite concentrations

were low during the whole test-periods 1 and 2. NH+ 4 did not exceed 0.1 mg/l
(N-NH+ 4 ), NO −
2 was below 0.05 mg/l N-NO −
2 after the first day. Temperature was
stable in a range of 25.1 – 26.1°C. DOC values increased slowly during the tests,
beginning with 3 – 4 mg/l they did not exceed 5–7 mg/l at the end of the tests.
In period 1 NO− 3 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− 3 .
Denitrification with PHB started 8 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. 4 and Table 2). For Bionolle
two phases of activity could be observed, an explanation cannot yet be given.
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A. Boley et al. / Aquacultural Engineering 22 (2000) 75–85
Table 2
Estimated maximal denitrification velocities of tested materials

Solid substrate Specific surface Temp. (°C) Flowrate (l/h) Concentration range 5–40 mg/l N-NO−
3
(m2/l)

Volumetric rates (mg N-NO−

3 / Surface related rates (mg N-NO− 2
3 /(mh))
(lh))

PHB 1.49 20–25 0.4–0.6 7–41 5–28

PCL 0.87 20–25 0.2–0.3 21–166 20–160
-Bionolle, period 1 1.22 20–25 0.3–0.6 1.5–10; 12–77 1.3–9; 10.5–67
and 2
 A. Boley et al. / Aquacultural Engineering 22 (2000) 75–85 81

As Fig. 4 shows the theoretical concentration limits (about 5 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− 3 ). 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− 3 . 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 2.
As the acid neutralizing capacity of the tap-water was low (ANC= 1 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. 4. Nitrate concentrations in testsystems. Temperature: 25 – 26°C.

Fig. 5. 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 3
Denitrification velocities in fixed bed reactors with different substrates

Carrier-material Spec. surface Substrate Temp. (°C) Volumetric rate (mg Surface related rate (mg N- Type of water and in-

A. Boley et al. / Aquacultural Engineering 22 (2000) 75–85

(m2/l) N-NO−3 /(lh)) NO− 2
3 /(mh)) stallation

Sand 1.5 Methanol 12 145 97 Wastewater, labora-

tory-scalea
Burned clay 1.3 Acetic Acid 12 14–34 10–26 Drinking water plantb
Burned clay 0.9 Ethanol 12–13 49–59 54–66 Drinking water plantc
PHB 1.55 PHB 10 16 11 Tap water, labora-
tory-scaled
PHB 1.6 PHB 15 22 14 Tap water, labora-
tory-scalee
PCL 1.2 PCL 15 13 10 Tap water, labora-
tory-scalee
Sand (d = 0.3–0.9 mm) Dissolved organic 22.5–27 36 Fluidized bed, aqua-
substrates culture systemf
Brick granules 2.2 Ethanol 20 100 Marine closed aqua-
culture systemg

a
Hawkins et al., 1978.
b
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.
 A. Boley et al. / Aquacultural Engineering 22 (2000) 75–85 83

Table 4
Estimated costs of substrates for nitrate removal

Substrate Price of substrate Consumption of substrate (kg Costs of denitrification

(€/kg substrate) substrate/kg N-NO−
3 ) (€/kg N-NO− 3 )

Methanol: 1.00 2.08–3.98 2.0–4.0

CH3OH
Ethanol: 1.20 2.0 2.4
C2H5OH
Acetic acid: 2.40 3.5 8.0
CH3COOH
PCL (C6H10O2)n 5.00 1.33–1.77 6.6–8.9
PHB (C4H6O2)n 10.00 2.1–2.7 21.0–37.2
Bionolle c 6010 Commercially not available
(C6H4O2)n

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.

4. 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 conven-
tional denitrification processes is shown in Table 3. 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 4. 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. Denitrifi-
cation 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

5. 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 require-
ment, 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. 1. 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 denitrifica-
tion) Stuttg. Ber. Siedl. Wass. Wrtsch., Bd. 135, R. Oldenbourg, München.
Jestin, J.-M., Philipot, J.-M., Berdou, C., Moulinot, J.-P., 1986. Maitrise d’un processus biologique: la
dénitrification à Eragny. T.S.M.-L’Eau, pp. 359 – 362.
Müller, W.-R., Wurmthaler, J., Heinemann, A., 1992. Biologische nitratelimination in kleinen wasserw-
erken (Biological nitrate elimination in small drinking water treatment plants). WAP 5, 231 – 235.
Partos, J., Richard, Y., 1984. Dénitrification d’eau potable par cultures fixées 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, Liège, 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 kontinuier-
lichen Festbettverfahren. (Denitrification of drinking water in a fixed bed reactor with biodegradable
polymers) Diploma Thesis, Lehrstuhl für Wasserchemie, Universität Karlsruhe, Germany.
 A. Boley et al. / Aquacultural Engineering 22 (2000) 75–85 85

Stöver, T., Roennefahrt, K.W., 1990. Biologische denitrifikation in der trinkwasseraufbereitung. (Biolog-
ical 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 Trinkwasseraufbere-
itung. (Biological nitrate elimination in drinking water treatment with a solid substrate). Stuttg. Ber.
Siedl. Wass. Wrtsch. Bd. 132, R. Oldenbourg, München.

.
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