Biotechnology Letters 24: 1173–1176, 2002.

© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

1173

Denitrification kinetics of simulated fish processing wastewater at

different ratios of nitrate to biomass

O. Soto, O. Sánchez, E. Aspé & M. Roeckel∗

Departamento de Ingenierı́a Quı́mica, Facultad de Ingenierı́a, Casilla 160 C, Correo 3, Universidad de Con-
cepción, Concepción, Chile
∗ Author for correspondence (Fax: (56) (41) 243750; E-mail: mroeckel@diq.udec.cl)

Received 4 February 2002; Revisions requested 15 February 2002; Revisions received 7 May 2002; Accepted 7 May 2002

Key words: catabolism/anabolism uncoupling, denitrification, fishing effluents, saline effluents

Abstract
Denitrification was studied in anoxic batch cultures of a simulated fish processing wastewater at 37˚C and pH 7.5,
using a denitrifying enrichment culture from fishery wastewater. Different initial nitrate to biomass ratios (So/Xo)
were used: nitrate and biomass varied from 7.5 to 94.7 mg NO3 -N l−1 , and from 20 to 4300 mg volatile suspended
solids l−1 , respectively. The specific maximum denitrification rate (rm ) and the cell yield (YX/S ) depended on the
So/Xo ratio under anoxic conditions: rm increased from 1.2 to 1584 mg NO3 -N g−1 VSS h−1 and YX/S decreased
from 42 to 0.03 mg VSS mg−1 NO3 -N when So/Xo varied from 5.5 · 10−3 to 9.3 mg NO3 -N/mg VSS.

Nomenclature
CNO3−N nitrate concentration, mg NO3 -N l−1
KS saturation constant, mg NO3 -N l−1
rm specific maximum denitrification rate, mg NO3 -N g−1 VSS h−1
So initial substrate concentration, mg l−1
t time, h
TOC total organic carbon
VSS volatile suspended solids
x biomass concentration, g VSS l−1
Xo initial biomass concentration, g VSS l−1
YX/S substrate to biomass cell yield, mg VSS/mg N
Greek symbols:
µm maximum specific growth rate of the anoxic microbial population, 1 h−1

Introduction from experimental work in anaerobic and anaerobic

The organic substrate to biomass initial ratio (So/Xo)
has a strong influence on kinetic parameter estimation
in aerobic biodegradation of organic matter (Chudoba
et al. 1992). Liu (1996) observed higher values for
the cell yield (YX/S ) at small So/Xo ratios; this ef-
fect has been explained by a physiological uncoupling
of catabolism and anabolism (Dauner et al. 2001).
A model for YX/S has been proposed and verified
treatment processes (Liu 1996, Liu et al. 1998).
Anoxic batch experiments have been used for the
determination of parameters in the design and oper-
ation of denitrifying processes (Quevedo et al. 1996).
Little attention has been given to the nitrate to biomass
ratio effect. Therefore, the main goal of this work was
to study the influence of So/Xo on the denitrifying
process. While in the previously reported processes
1174
So signifies the initial electron donor concentration, in
this work So signifies acts as the initial electron accep-
tor concentration. A saline-simulated, fish processing
waste was used since denitrification is part of a whole
biological treatment for fish industries effluents (Vidal
et al. 1997).
Materials and methods
Cultivation of a denitrifying culture for batch
experiments
A denitrifying enrichment culture was grown in a 10 l
laboratory sequencing batch reactor (SBR). The SBR
inoculum was obtained from an anaerobic pilot plant
treating fishery wastewater. To adapt the inoculum, a
synthetic medium was used (Akunna et al. 1994). This
medium was modified by the addition of 24 g NaCl l−1
to mimic a fish processing wastewater. Acetic acid
was added as an electron donor and KNO3 as an
electron acceptor. The mass C/N ratio was 4.5:1 mg
TOC mg−1 NO3 -N to ensure an excess of electron
donor source. The composition of the medium was
as follows: KH2 PO4 : 3 g l−1 ; K2 HPO4 : 3 g l−1 ;
NaHCO3 : 0.4 g l−1 ; MgSO4 · 7H2 O: 0.005 g l−1 ;
NaCl: 24 g l−1 ; yeast extract: 0.05 g l−1 ; peptone:
0.12 g l−1 ; glacial acetic acid: 1.22 ml l−1 ; trace ele-
ments: 1 ml l−1 (FeSO4 · 7H2 O: 1.096 g l−1 ; CaCl2 :
0.5 g l−1 ; KCl: 0.5 g l−1 ; CoCl2 : 0.1 g l−1 ); KNO3 :
721 mg l−1 (100 mg NO3 -N l−1 ). The pH value and
temperature of the medium was adjusted to 7.5 and
37 ◦ C, respectively. A solid retention time (SRT) of 60
days was maintained. The reactor was operated at four
daily cycles of 6 h and mean value of biomass con-
centration was 400 mg VSS l−1 . Reactor performance
was monitored by periodically measuring the effluent
VSS and influent and effluent NO3 -N and NO2 -N con-
centrations. Upon attainment of steady state after eight
months, mixed liquor was withdrawn from the SBR
and used in all batch tests.
Determination of maximum specific growth rate
Three anoxic batch assays were performed in order
to determine the maximum specific growth rate (µm ).
Anoxic assays were carried out in 300 ml Erlenmeyer
flasks filled with the same concentration of organic
matter plus different amounts of nitrate so as to reach
different So/Xo ratios of 2, 7 and 10 mg N/mg VSS.
Samples, 1 ml, were collected hourly. Samples were
fixed in formalin and the total cell counts were de-
termined by a fluorescent-microscopy method with
acridine orange as described previously (Hobbie et al.
1977).
Batch anoxic assays conditions
Denitrification assays were carried out in 300 ml Er-
lenmeyer flasks, each containing 250 ml of reaction
mixture of an anoxic medium previously described us-
ing 24 g NaCl l−1 and acetic acid in order to mimic
fishing effluents. The medium was kept at pH 7.5. The
flasks were incubated in an orbital agitator at 200 rpm
and 37 ◦ C. Anoxic conditions were maintained ini-
tially gassing with N2 for 5 min, and sealing each
flask with a butyl stopper. Acetic acid, measured as
TOC was in excess for all cases (C/N > 1.07 mg
TOC/mg NO3 -N). Given the nature of the carbon
source complete denitrification was assured without
ammonification (Akunna et al. 1993).
Estimations of kinetic parameters
Mass balance for nitrate in a batch reactor is given by:
dCNO3 −N CNO3 −N
= rm · x. (1)
dt KS + CNO3 −N
The integration of Equation (1) was carried out
using the fixed path 4–4 Runge Kutta method. A
search for the values of model parameters (rm and
KS ) that result in the least-square error between the
data and the model solution was carried out using the
Levenberg–Marquard method.
The algorithm was programmed using a source
written in MATLAB v. 4.2 software. All the exper-
iments for the parameters estimation have not been
run for more than 6 h; therefore it was assumed that
the x remained constant for purposes of the numerical
fitting.
Chemical analysis
Nitrite (NO2 -N) and nitrate (NO3 -N) concentrations
were measured using a flow injection analysis (FIA)
equipment (Foss Tecator, Sweden). In this equip-
ment, nitrite is measured at 540 nm after reaction with
sulfanilamide diazotized with N-ethylenediamine di-
hydrochloride; and nitrate is reduced to nitrite in a
Cd+2 column and measured as described for nitrite
(APHA 1992). TOC was measured as the difference
between total carbon and inorganic carbon in a Shi-
mazdu Analyzer (TOC 5000A). VSS were measured
by standard methods (APHA 1992).
 1175
Table 1. Maximum specific denitrification rates at different So/Xo ratios in anoxic batch cultures at pH 7.5 and 37 ◦ C.

So/Xo ratio, Biomass initial Substrate initial C/N ratio, Maximum specific denitrification rate,
mg NO3 -N/ mg VSS concentration, mg VSS l−1 concentration, mg NO3 -N l−1 mg TOC/mg NO3 -N mg NO3 -N/g VSS h−1

5.5×10−3 1733 10 47 1
7.3×10−3 1029 8 60 1
1.5×10−2 1029 15 30 2
3.4×10−1 111 38 12 76
3.7×10−1 53 20 23 70
6.6×10−1 53 35 13 120
7.2×10−1 27 19 24 168
1.2 41 49 9 239
1.2 77 93 5 223
1.9 14 27 17 509
1.9 20 39 12 430
2.1 27 58 8 556
2.3 41 95 5 370
4 14 58 8 1025
4.3 7 31 15 1148
4.6 12 57 8 1093
5.5 11 57 8 1397
7.6 8 64 7 1514
7.8 7 58 8 1150
8 6 50 9 1788
9.3 6 58 8 1584

Results and discussion

Table 1 shows results from batch data evaluation using

different initial substrate and biomass concentrations.
As shown, rm increases three orders of magnitude
when So/Xo varies from 5.5 × 10−3 to 9.3 mg NO3 -
N/mg VSS, demonstrating that rm can change with
experimental conditions. Previous reported kinetic
studies proposed a constant value for this parameter
(Akunna et al. 1994, Delanghe et al. 1994, Murray
et al. 1989) but little attention has been paid to the
uniqueness of parameters in wastewater process mod-
eling. As shown in Figure 1, a clear dependence is
observed when rm is plotted against the So/Xo ratio.
Fig. 1. The effect of So/Xo on the denitrification rate in anoxic batch
In Table 1 it appears that rm depends not only on the cultures of a simulated fishing effluent at pH 7.5 and 37 ◦ C.
So/Xo ratio, but on the biomass initial concentration;
nevertheless, different rm were obtained for same VSS
values. So/Xo ratios. Therefore, the variation in rm for dif-
When the data was analyzed, it was determined ferent So/Xo values can be only addressed due to the
that the affinity constant (KS ) from Monod’s model variation of YX/S . The following relationship between
was not affected by So/Xo (data not shown), and its rm and µm can be obtained:
mean value was 0.46 ± 0.17 mg NO3 -N l−1 . µm
In this work, µm was determined and a constant rm = (2)
YX/S
value of 0.045 ± 0.003 1 h−1 was obtained for all
1176
Fig. 2. The effect of So/Xo on the cell yield using nitrate as substrate
in anoxic batch cultures of a saline media at pH 7.5 and 37 ◦ C.
Figure 2 shows the relationship between So/Xo and
YX/S . As shown, an increase in So/Xo ratio diminishes
YX/S . This pattern has been only shown for aerobic
heterorotrophic mixed culture when So represents the
initial electron donor concentration and an uncoupling
of the anabolism and the catabolism have been pro-
posed to explain this phenomena (Liu 1996, Dauner
et al. 2001).
According to the results of this work, the decrease
in energetic efficiency observed for processes where
So is the initial electron donor concentration can be ex-
tended to those cases where So acts as initial electron
acceptor concentration.
Nitrogen concentrations in fishery wastewater may
reach 1.9 g N l−1 (Aspé et al. 1997). In high salinity
wastewater treatments the biomass concentration may
vary from 0.2 to 38 g VSS l−1 , for activated sludge
systems and SBR, respectively (Eilersen et al. 1995,
Glass & Silverstein 1999).Then, for treating fishery
wastewater So/Xo may vary from 0.05 to 9.5 g NO3 -
N/g VSS. As seen from Figure 2, a four orders of
magnitude variation in rm can be obtained when So/Xo
is increased from 0.05 to 9.5. Therefore, laboratory
concentration ranges should be careful established in
kinetic parameters determination assays.
Conclusions
The denitrification kinetics is affected by So/Xo
when So is the initial electron acceptor concentra-
tion, validating this behavior formerly reported for
heterotrophic cultures where So is the initial electron
donor concentration. Experimental results showed a
relationship between So/Xo and YX/S ; therefore ki-
netic general models for substrate consumption rate
should be completed.
Acknowledgement
This work was possible through the project from the
Chilean Council FONDECYT 2000129.
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