Bioresource Technology 102 (2011) 567–575

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Biodegradation kinetics of peptone and 2,6-dihydroxybenzoic acid

by acclimated dual microbial culture
Emine Ubay Cokgor a,1, Guclu Insel a,1, Tugce Katipoglu a,1, Derin Orhon a,b,*,2
a
Environmental Engineering Department, Faculty of Civil Engineering, Istanbul Technical University, 34469 Maslak, Istanbul, Turkey
b
Turkish Academy of Sciences, Piyade Sokak No. 27, 06550 Çankaya, Ankara, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: This study evaluated the kinetics of simultaneous biodegradation of peptone mixture and 2,6-dihydroxy-
Received 9 June 2010 benzoic acid (2,6-DHBA) by an acclimated dual microbial culture under aerobic conditions. A laboratory-
Received in revised form 27 July 2010 scale sequencing batch reactor was sustained at steady-state with peptone mixture feeding. During the
Accepted 28 July 2010
study period, peptone mixture feeding was continuously supplemented with 2,6-DHBA. Related experi-
Available online 1 August 2010
mental data were derived from three sets of parallel batch reactors, the first fed with the peptone mix-
ture, the second with 2,6-DHBA and the third one with the two substrates, after acclimation of microbial
Keywords:
culture and simultaneous biodegradation of both organics. A mechanistic model was developed for this
2,6-Dihydroxybenzoic acid
Biodegradation, process kinetics
purpose including the necessary model components and process kinetics for the model calibration of
Acclimated culture, dual biomass relevant experimental data. Model evaluation provided all biodegradation characteristics and kinetics
for both peptone mixture and 2,6-DHBA. It also supported the development of a dual microbial commu-
nity through acclimation, with the selective growth of a second group of microorganisms specifically
capable of metabolizing 2,6-DHBA as an organic carbon source.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction tion of the tested microbial community to the particular xenobiotic

Xenobiotics are now essential ingredients for a wide spectrum of
industrial activities. Significant portion of these chemicals end up in
wastewater streams. As they are mostly synthetically produced,
they are alien to microbial communities and therefore resistant to
biodegradation within biological wastewater treatment systems.
The same also applies to 2,6-dihydroxybenzoic acid (2,6-DHBA) se-
lected for this study; this chemical can be considered as a xenobi-
otic when it enters a wastewater treatment plant, although, it is a
naturally occurring organic compound imparted to olive mill
wastewaters. Impact of xenobiotics is often assessed in terms of
short-term biodegradation tests which generally reflect significant
inhibition and toxic effects upon the microbial community exposed
to the selected chemical the first time during the test (Ricco et al.,
2004). These tests, although indicative, are not too meaningful,
mainly because they do not consider and evaluate possible acclima-
* Corresponding author at: Environmental Engineering Department, Faculty of
Civil Engineering, Istanbul Technical University, 34469 Maslak, Istanbul, Turkey.
Tel.: +90 212 2856587; fax: +90 212 2856576.
E-mail address: orhon@itu.edu.tr (D. Orhon).
1
Tel.: +90 212 2856587; fax: +90 212 2856576.
2
Tel./fax: +90 212 285 3793.
at continuous exposure, eventually utilizing it as an additional
organic carbon source. This aspect, while quite meaningful for
many xenobiotics and especially for 2,6-DHBA (2,6-dihydrobenzoic
acid) which is discharged seasonally and intermittently to treat-
ment plants primarily designed for domestic sewage, has not been
studied sufficiently and still remains a challenging issue of major
practical importance.
Among many chemicals taking part in industrial processes, bio-
degradation studies focused extensively on phenolic compounds
(Di Gioia et al., 2001; Lee and Chen, 2009). Phenol derivatives and
mostly polyphenols occur in natural systems and they were identi-
fied and studied as significant constituents of olive oil wastewater
(Garcia et al., 2000; Dogruel et al., 2009). Similarly, 2,6-DHBA is a
naturally existing phyto-toxin exerting a defense mechanism
against the natural (microbial) deterioration of olives. However, it
is alien to the microbial community of the domestic treatment
systems where olive wastewaters are usually discharged. Its
biodegradation characteristics, although important for the biologi-
cal treatment of olive oil wastewaters, have not been fully explored
aside from a number of pure culture studies (Yoshida et al., 2007;
Lee and Chen, 2009).
Modeling is now regarded as a useful instrument for under-
standing and interpreting biodegradation and related mecha-
nisms of substrate utilization (Rezouga et al., 2009; Damayanti

0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.07.112
568 E.U. Cokgor et al. / Bioresource Technology 102 (2011) 567–575
et al., 2010). Recent models structured for this purpose operate
with two major conceptual developments: (i) they use chemical
oxygen demand (COD) and the organic carbon parameter and in-
clude COD fractions with different biodegradation characteristics
based on the pioneering works of Dold et al. (1980) and Ekama
et al. (1986); (ii) they incorporate dissolved oxygen concentration
(S0) as a significant model component which allow to calibrate
and evaluate oxygen uptake rate profiles (Spanjers and Vanrolleg-
hem, 1995). The mechanistic model, commonly known as ASM1
and proposed by Henze et al. (1987), should be considered as a
milestone in the modeling approach, involving for the first time
the above mentioned concepts with the assumption that sub-
strate is solely utilized for microbial growth. Later, recognition
of the formation of internal biopolymers in systems operated
with feeding/starvation (feast/famine) transients established the
basis for another model (ASM3) which postulated that all sub-
strate was first stored as internal storage products then utilized
for microbial growth (Krishna and van Loosdrecht, 1999). Obvi-
ously these two models represented extremes for substrate utili-
zation and they were primarily conceived for domestic sewage.
Therefore, for real-case situations involving treatment systems
with cyclic feeding as in the case of sequencing batch reactors,
models designed for simultaneous substrate storage and micro-
bial growth were developed and successfully used for the kinetic
interpretation of related experimental data (Karahan-Gul et al.,
2003). Similarly, new models were developed with additional
COD fractions as model components and corresponding biochem-
ical processes to analyze the biodegradation kinetics of specific
chemicals (Cokgor et al., 2007) and mixtures of complex indus-
trial effluents (Lubello et al., 2009). These approaches are impor-
tant because they set the basis for the modeling exercise adopted
for this study.
The study reported in this paper is part of a comprehensive
investigation involving the fate of a synthetic substrate and
2,6-dihydroxybenzoic acid in an enrichment culture under aero-
bic conditions. The synthetic substrate was a mixture of meat
extract and peptone – defined as peptone mixture in the text
for simplicity. It was selected mainly because it is prescribed
as the standard substrate for the ISO inhibition test (ISO 8192,
1995); the peptone mixture does not have the same particle size
distribution without the particulate COD fractions, but it is very
similar to domestic sewage in terms of biodegradation charac-
teristics, as it contains a similar balance between readily biode-
gradable COD and slowly biodegradable COD fraction. This
property is well documented in the literature and sets the basis
for the selection of the peptone mixture in experimental biodeg-
radation studies (Insel et al., 2006). Furthermore, it can be easily
adjusted to sustain the same characteristics throughout the
experiments. Different phases of the study covered the follow-
ing: (i) biodegradation kinetics of peptone mixture (Orhon
et al., 2009a); (ii) the adverse impact of continuous 2,6-DHBA
feeding on peptone mixture utilization and acclimation of the
mixed culture to 2,6-DHBA (Orhon et al., 2009b) and the effect
of sludge age on the impact of 2,6-DHBA (Katipoglu et al., 2010).
The objective of the study was to assess the biodegradation
kinetics of the peptone/2,6-DHBA mixture by an acclimated
microbial culture. A suitable mechanistic model was developed
and experimentally tested for this purpose. Experimental data
including profiles of oxygen uptake rate, COD and polyhydrox-
yalkanoates (PHA) were used for the calibration of the selected
model to derive the corresponding biodegradation kinetics for
the synthetic substrate (peptone mixture), 2,6-DHBA and the
substrate mixture. Special emphasis in the modeling was placed
upon testing the development of a group of microorganisms
upon acclimation, specifically capable of metabolizing 2,6-DHBA
as a organic carbon source.
2. Methods
2.1. Evaluation approach
The evaluation approach was primarily designed to assess the
biodegradation kinetics of peptone mixture and 2,6-DHBA by the
acclimated culture. In essence, an SBR (sequencing batch reactor)
system, fed with both peptone and 2,6-DHBA, was run for acclima-
tion. After the acclimation period, three different batch tests
seeded with the acclimated biomass taken from the SBR system,
were carried out to evaluate the response of the biomass to differ-
ent substrates, i.e. the peptone mixture, 2,6-DHBA alone and a mix-
ture of peptone and 2,6-DHBA. A model developed for this purpose
was calibrated with the experimental data collected after the accli-
mation period to generate appropriate values for kinetic and stoi-
chiometric coefficients of the model.
A laboratory-scale SBR system fed with the synthetic peptone
mixture substrate and operated at steady-state at a sludge age of
10 days was the primary source of the experimental data. The
SBR was seeded with activated sludge from a domestic wastewater
treatment plant and had a net volume of 5 L. Its operation was ad-
justed to one cycle a day with pulse feeding at the beginning of
each cycle. Pulse feeding was provided an initial peptone mixture
concentration of around 500 mg COD/L in the reactor to represent
real domestic wastewater. The system was operated with an initial
volatile suspended solids (VSS) concentration of 1865 mg/L and
more than 95% removal efficiency was achieved for peptone mix-
ture at steady-state. The resulting food to microorganisms ratio
(S0/X0) was 0.3 mg COD/mg VSS. The synthetic substrate contained
peptone and meat extract as the organic carbon source which is
rich in amino acids, peptides, organic acids, minerals and vitamins;
it is referred to as peptone mixture throughout the text for simplic-
ity. The stock substrate solution was prepared to contain 16 g pep-
tone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl22H2O, 0.2 g
MgSO47H2O and 2.8 g K2HPO4 in 1 L of distilled water as described
in ISO 8192 (1995).
An acclimation procedure was applied to acclimate mixed
microbial culture to 2,6-DHBA and peptone mixture feed. Since
the olive mill wastewater generation is seasonal and often comes
as transient discharge pulses into the treatment system, the 2,6-
DHBA concentration may well vary in the range of 100–1000 mg
COD/L. Therefore, both peptone mixture and 2,6-DHBA concentra-
tions selected for the study represented meaningful average values
for the practical application (Aktas et al., 2001; Drouiche et al.,
2004). At the start of the acclimation period, VSS and total sus-
pended solids (TSS) concentration of the SBR system were around
2000 and 2500, respectively. The peptone mixture removal perfor-
mance of the reactor remained the same throughout the acclimation
period. Complete removal of 2,6-DHBA was achieved after 15 days
of acclimation. After 30 days of acclimation, VSS and TSS concentra-
tion increased to around 2300 and 3000 mg/L, respectively.
The experimental period was set as 32 days of SBR operation
after establishment of steady-state conditions during which the
reactor received on a continuous basis pulse feeding of 2,6-DHBA
at the beginning of each daily cycle. Consequently, each daily cycle
was started with around 500 mg COD/L of peptone mixture and
500 mg COD/L of 2,6-DHBA. SBR was operated at a temperature
of 20 ± 1 °C and the air supply to the reactor was adjusted to secure
a dissolved oxygen concentration of around 3.0 mg/L. pH was
maintained around 7.5 in the reactor volume.
The experimental evaluation was conducted using the experi-
mental data generated by means of three different sets of batch
reactors run during the last 2 days of observation (Run 1 – day
31; Runs 2 and 3 – day 32), a stage where the SBR operation ex-
ceeded a three times of sludge age period (SRT) and indicated that
the biomass was fully acclimated to utilize both peptone mixture
 E.U. Cokgor et al. / Bioresource Technology 102 (2011) 567–575
Table 1
Outline and basic information for batch experiments.
Runs Initial COD
Peptone mixture COD
(mg COD/L)
Run 1 – 31 day – 620
Run 2 – 32 day 545
Run 3 – 32 day 285
and 2,6-DHBA with more than 95% removal. The volume of each
batch reactor was set as 4.0 L; the reactors were fed and operated
at exactly the same conditions as the continuous SBR. Basic infor-
mation related to these batch experiments is outlined in Table 1.
An initially prepared single set was simply split into two reactors
to create identical conditions: The first one was used for respiro-
metric tests and the other for different substrate measurements,
namely the COD, 2,6-DHBA and polyhydroxyalkanoate (PHA) com-
ponents. The major PHA components monitored as internal storage
compounds were polyhydroxybutyrate (PHB), polyhydroxyvaler-
ate (PHV) and 3-hydroxy-2-methylvalerate (3H2MV). The batch
reactors were started with biomass taken from the SBR at the
endogenous respiration stage and continued with the addition of
the selected substrate. Respirometric analyses consisted of mea-
suring and recording the oxygen uptake rate (OUR) profile in the
reactor after substrate addition. OUR measurements were per-
formed with a Ra-Combo-1000 (Applitek Co., Nazareth, Belgium)
continuous respirometer with a sampling interval of 1 min. A nitri-
fication inhibitor (Formula 2533TM, Hach Company) was used to
prevent any possible interference induced by nitrification.
2.2. Analytical procedures
Assessment of total suspended solids (TSS) and volatile sus-
pended solids (VSS) within the SBR system and the batch sets were
performed according to Standard Methods (2005). COD and 2,6-
DHBA samples were filtered through 0.45 lm Millipore membrane
filters. The COD samples were preserved with H2SO4 and analyzed
as described in ISO 6060 method (ISO 6060, 1986). 2,6-DHBA anal-
yses were conducted by using a Lambda 25 UV/Vis spectrometer at
306.17 nm as proposed by Davey et al. (2001). COD equivalent of
2,6-DHBA results were subtracted from total COD results to deter-
mine COD associated with the peptone mixture.
PHA samples taken from mixed liquor were preserved with form-
aldehyde in order to prevent biological activity. The liquid phase
was removed by centrifugation and biomass was washed with K-P
buffer. The samples were exposed to freeze-drying. Hydrochloric
acid, 1-propanol, and dichloroethane mixture was used for extrac-
tion, hydrolysis and esterification of samples at 100 °C as
described by Beun et al. (2000). After the removal of free acids by
water extraction, the organic phase was analyzed by an Agilent
6890N gas chromatograph. The PHB, PHV, and 3H2MV fractions of
PHA were determined with benzoic acid being used as the internal
standard. Duplicate samples were taken for the analysis of each
parameter. Standard deviation of COD, 2,6-DHBA, and PHA measure-
ments were reported to be lower than 3%, 1% and 3%, respectively.
2.3. Modeling and parameter identifiability
Modeling consisted of analyzing and calibrating the OUR and PHA
profiles using the mechanistic model developed for this purpose.
Kinetic and stoichiometric parameters of the adopted model were
estimated using the approach defined by Insel et al. (2003) by using
SIMPLEX algorithm. In identifiability study, the OUR profile and PHA
measurements were taken into consideration. The AQUASIM simula-
tion program was used in modeling studies (Reichert et al., 1998).
569
VSS (mg VSS/L) Initial COD/VSS ratio
(mg COD/mg VSS)
2,6-DHBA COD
(mg COD/L)
1950 0.32
– 2050 0.27
285 2150 0.27
The UNCSIM module (Brun et al., 2001) was used for the calculation
of collinearity indices, cK in order to illustrate the best identifiable
parameter subsets in modeling. In summary, the method of Brun
et al. (2001) combines the sensitivity measures and determines the
linear dependency of the parameters described with the collinearity
index (cK) was applied. This method provides a systematic approach
and compares the collinearity indices of parameter subsets and
checks the degree of identifiability of parameter subsets. Accord-
ingly, subset K out of n model parameters is first decided using the
dimension-free sensitivity function, si,j and their sensitivity mea-
sures of dmsqr
j as expressed in Eqs. (1) and (2) by ranking in ascending
order. Hence, the constitution of a parameter subset is carried out via
grouping the parameters from the top of the ranking.
Dhj @yi
si;j ¼ ð1Þ
sci @hj
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
u n
u1 X 1  
dmsqr
j ¼t s2 ¼ pffiffiffi si;j  ð2Þ
n i¼1 i;j n
where, si,j is scaled sensitivity matrix, sci is scaling factor for mea-
surements, @yi/@hj is sensitivity function of parameter, hj to model
output, y. In Eqs. (1) and 2, n is number of observations (i) and m
is number of model parameters (j).
The collinearity index, cK (see Eq. (3)) is the measure of approx-
imate linear dependency of selected parameter subsets and it also
has a close relationship to the condition number. In Eq. (3), ~ SK is a
sub-matrix of ~ S that corresponds to the parameters in K; b is the
vector of coefficients and kk is the smallest Eigen value of ~ STK ~
SK .
1 1
cK ¼   pffiffiffiffiffi
~  ¼ kk ð3Þ
minkbk¼1 SK b
A collinearity index of 20 means that the effect of an estimated
parameter on the measurement can be compensated by the change
of 5% in other parameters (Brun et al., 2001). Practically, it is sug-
gested that the condition number should fulfill the criteria of cK < 20.
3. Results and discussion
Experimental data considered for model calibration and evalu-
ation covered results of three consecutive batch tests conducted
with the acclimated biomass after 30 days of continuous 2,6-DHBA
feeding together with peptone mixture. The results included OUR,
PHA and COD profiles; they were derived from batch reactors
where (i) 2,6-DHBA; (ii) peptone mixture, and (iii) peptone/2,6-
DHBA mixture sequentially served as the organic carbon source
for the corresponding biodegradation experiments.
For each experimental set, the model structured for this pur-
pose was calibrated simultaneously using the OUR, COD, 2,6-DHBA
and PHA profiles associated with each experimental run. The cali-
bration defined the appropriate values of the kinetic and stoichi-
ometric coefficients which provided consistently best fits with
the experimental data; it also yielded the corresponding values
of the state variables, namely the concentration of active biomass
and relevant biodegradable COD fractions.
570 E.U. Cokgor et al. / Bioresource Technology 102 (2011) 567–575
3.1. Model structure
The experimental design was organized in three consecutive
steps for collecting the data using an acclimated biomass. Basically,
the data collection steps involved the data sets of (a) peptone mix-
ture degradation only; (b) 2,6-DHBA degradation only and (c) pep-
tone and 2,6-DHBA mixture degradation. Initially, the parameter
estimation using peptone mixture degradation data enabled the
estimation of maximum growth rate (l ^ H ) together with active het-
erotrophic biomass (Insel et al., 2003).
A number of modeling scenarios were sequentially tested and
the model structure adopted for the study was the end product
of the extensive testing exercise which proved most appropriate
in terms of both consistency and identifiability. Furthermore, state
variables including active biomass fractions (XH, XHB) and the set of
model parameters were not derived from a single OUR experiment,
but from three interrelated OUR experiments conducted on syn-
thetic substrate, 2,6-DHBA and the synthetic substrate/2,6-DHBA
mixture so that each model calibration step yielded its own values,
which were verified with the next calibration step.
The initial OUR levels in all batch experiments could be charac-
terized with the OUR response of the sum of two biomass by set-
ting the same endogenous decay rate level in model. On the
other hand, the OUR profile could be simulated by separating the
heterotrophic biomass into two fractions of XH and XHB responsible
for (a) peptone mixture degradation and (b) 2,6-DHBA degradation,
respectively. This assumption is compatible with the literature that
two different organic matters having different molecular structure
could be degraded via different metabolic pathways such as acetyl-
CoA, catechol (Fritsche and Hofrichter, 2005). As a result, two types
of different microbial communities (dual biomass) are expected to
play role in co-metabolic degradation of peptone mixture and 2,6-
DHBA by adjusting their internal/external enzymatic systems
depending on the availability of external substrate. The term ‘‘dual
biomass” is specifically associated with modeling where the con-
sortium of different species is conveniently grouped into the het-
erotrophic biomass, XH. In this sense it defines a particular
metabolic activity towards utilization of the substrate. In our
study, since 2,6-DHBA could only be biodegraded by another
microbial community developed after acclimation, the correspond-
ing biochemical model had to include two biomass components
hence a ‘‘dual biomass” system.
Furthermore, calibration did not only involve OUR profiles but
also all the substrate utilization data (i.e. COD, PHA, 2,6-DHBA),
therefore not allowing arbitrary adjustments but yielding parame-
ter values as the model outputs. Finally, the validity of model cal-
ibration was controlled and justified by detailed identifiability
analysis.
The experimental data provided in the following sections sup-
ported considerable amount of substrate storage that has to be
considered in model building. So, the storage process was added
Table 2
Matrix representation of the adopted model structure.
Process SO2 SS SSB SH1
Growth of XH  1Y H  Y1H
YH
Growth of XHB  1Y H  Y1H
YH
Hydrolysis of SH1 1 1
Hydrolysis of SH2 1 1
Storage of PHA by XH (1  YSTO) 1
Growth on XSTO by XH  1Y H
YH
Disintegration 1 1
Decay of XH (1  fP)
Decay of XHB (1  fP)
to the model matrix by incorporating an additional state variable
of XSTO. This state variable is the sum of all storage materials mea-
sured as PHB, PHV, and 3H2MV.
The part of the model related to peptone mixture biodegrada-
tion included the basic elements – model components; processes
– for direct microbial growth with simultaneous substrate storage,
successfully utilized in similar studies (Karahan-Gul et al., 2003).
This model was also used in an earlier study, which explored the
validity of using Monod kinetics with constant coefficients, for
the biodegradation of peptone mixture at two different sludge ages
(Orhon et al., 2009a). The model defined peptone mixture by
means of three COD fractions, namely the readily biodegradable
COD, SS and two slowly biodegradable COD fractions, SH1 and SH2.
The PHA concentration reflecting the overall internal storage is in-
cluded as a model parameter. Active heterotrophic biomass, XH,
and dissolved oxygen are the other essential components of the
model. Accordingly, two microbial growth processes, namely,
growth on SS and growth on internal storage products, XSTO; two
hydrolysis steps involving breakdown of SH1 and SH2 and decay of
XH by means of endogenous respiration are selected and defined
as the necessary biochemical processes.
The second part of the model dealing with the biodegradation of
2,6-DHBA was again tailored to a great extent through interpreta-
tion of the experimental results and specifically the corresponding
OUR profile which indicated three major points (i) the existence of
an initial rate limiting step. This step is well interpreted in the lit-
erature by means of cleavage of the aromatic ring, a common path-
way suggested for phenolic compounds (Fritsche and Hofrichter,
2005). In the adopted model, a similar overall disintegration step
was formulated which breaks the 2,6-DHBA concentration, CB
down to readily biodegradable COD, SSB (ii) the development of a
selective group of biomass (XHB) through acclimation, which can
selectively utilize 2,6-DHBA as the organic carbon source (Orhon
et al., 2009b). This observation was accounted for in the model
by including a specific group of active biomass, XHB capable of uti-
lizing SSB for microbial growth, the same way as SS. (iii) Generation
of internal storage products were negligible during 2,6-DHBA bio-
degradation; consequently the model did not include any provision
for this process. A parallel approach for the endogenous decay of
XHB was considered. The modeling of respirometric data for 2,6-
DHBA degradation revealed that a lag phase should have been
added in order to pick up the gradual increase in the OUR profile.
This transient response was delineated by the disintegration process
which summarizes the enzymatic breakdown of complex organic
matter (2,6-DHBA) into intermediate biodegradable substrate
(SSB). Endogenous decay was also defined to generate non biode-
gradable microbial products; XP as commonly proposed in similar
modeling studies (Lindblom et al., 2009).
In accordance with similar models, microbial growth was
defined by the Monod expression for both biomass components;
hydrolysis rates were expressed as saturation-type surface
SH2 CB XH XHB XSTO XP Rate equation
1 l^ H K SSþS
S
XH
S
1 l^ HB K SBSþS
SB
X HB
SB
kh1 K XSþS
H1 =X H
XH
H1 =X H
kh2 K XXSH2 =X H
þSH2 =X H X H
YSTO kSTO K STOSSþSS X H
1  Y1H rGXSTO
kdis K DCþC
B
B
X HB
1 fP bHXH
1 fP bHXHB
 E.U. Cokgor et al. / Bioresource Technology 102 (2011) 567–575 571

was defined by means of an hyperbolic-type enzyme reaction sim-

ilar to Monod kinetics. The integrated model structure is given in a
matrix format in Table 2 (Henze et al., 1987), also indicating the
basic stoichiometry of model state variables.
The final model structure used in kinetic evaluation of experi-
mental data was developed based upon an iterative approach.
Within the framework of arguments given above, a progressive
model building was performed by evaluating two objectives of
(a) obtaining the best fit of simulation trajectories on multi-re-
sponse data and (b) the selection of optimal model complexity,
in turn maximum number of parameter (subsets) within an
acceptable identifiability limit.

3.2. Biodegradation kinetics of 2,6-DHBA only

The first batch experiment evaluated the biodegradation kinet-

Fig. 1. Modeling of (a) COD and (b) OUR profile associated with 2,6-DHBA
biodegradation (Run – 1, day 31).
reaction kinetics. Endogenous decay was formulated by the com-
monly adopted first order reaction with respect to corresponding
biomass concentration, XH or XHB. Disintegration of 2,6-DHBA
ics of 2,6-DHBA after an acclimation period of 30 days (Run – 1, day
31). In the earlier phases of SBR operation with continuous 2,6-
DHBA feeding, this chemical was not removed and totally re-
mained in solution; its biodegradation took place at a slow rate
only after 15 days (Orhon et al., 2009b). As clearly indicated in
Fig. 1a, biomass became fully acclimated and achieved full removal
of 2,6-DHBA after 30 days.
The test was started with an initial 2,6-DHBA concentration cor-
responding to 650 mg/L. The resulting OUR curve and substrate re-
moval data (Fig. 1a and b) confirmed the compatibility of the
selected model including an initial rate limiting step of disintegra-
tion generating cleavage products which then could be utilized for
microbial growth (Fritsche and Hofrichter, 2005). Model calibra-
tion of the 2,6-DHBA data and simulation of the corresponding
SSB profile given in the Fig. 1b showed that SSB remained around
25 mg/L during the course of the biodegradation experiment,
therefore with a minor effect (5%) on COD measurements.
The exclusion of PHA and related storage/utilization mecha-
nisms were also justified as the experimental results indicated no
generation of internal storage products. The specific shape of the

Table 3
Results of model calibration for the biodegradation of 2,6-DHBA, peptone mixture and peptone/2,6-DHBA mixture with the acclimated culture.

Model parameter and statea Unit Peptone mixture 2,6-DHBA Peptone mixture Peptone/2,6-DHBA mixture
0 days 31 days 32 days 32 days
Maximum growth rate for XH l^ H 1/day 4.1 – 5.6 5.6
Half saturation constant for growth of XH KS mg COD/L 4.0 – 10 10
Maximum growth rate for XHB l^ HB 1/day – 5.6 – 5.6
Half saturation constant for growth of XHB KSB mg COD/L – 20 – 20
Maximum hydrolysis rate for SH1 kh1 1/day 4.34 – 4.34 4.34
Hydrolysis half saturation constant for SH1 KX g COD/g COD 0.03 – 0.08 0.08
Maximum hydrolysis rate for SH2 kh2 1/day 1.94 – 2.60 2.60
Hydrolysis half saturation constant for SH2 KXX g COD/g COD 0.08 – 0.08 0.08
Disintegration rate for CB kdis 1/day – 5.4 – 5.4
Half saturation constant for disintegration of CB KD mg COD/L – 9 – 9
Maximum storage rate of PHA by XH kSTO 1/day 0.90 – 0.90 2
Half saturation constant for storage of PHA by XH KSTO mg COD/L 0.41 – 0.41 0.41
Maximum growth rate on PHA for XH rG 1/day 1.25 – 1.25 3.5
Endogenous decay rate for XH and XHB bH 1/day 0.20 0.20 0.20 0.20
Yield coefficient for XH and XHB YH g COD/g COD 0.60 0.60 0.60 0.60
Storage yield of PHA YSTO g COD/g COD 0.80 – 0.80 0.80
State variables
Initial active heterotrophic biomass XH1 mg COD/L 900 900 1050 1100
Initial active 2,6-DHBA degraders XHB1 mg COD/L – 720 450 450
Initial biodegradable peptone mixture COD CS1 mg COD/L 565 0 545 285
Initial biodegradable 2,6-DHBA COD CB1 mg COD/L – 650 – 285
Initial readily biodegradable COD generated from CB1 SSB1 mg COD/L – 0 – 0
Initial readily biodegradable peptone mixture COD SS1 mg COD/L 51 (9%) – 49 (9%) 26 (9%)
Initial readily hydrolysable peptone mixture COD SH1 mg COD/L 247 (44%) – 240 (44%) 125 (44%)
Initial slowly hydrolysable peptone mixture COD SH2 mg COD/L 267 (47%) – 256 (47%) 134 (47%)
Initial PHA of peptone mixture fed biomass XSTO1 mg COD/L 70 40 50 50
a
fP is assumed as 0.2.
572 E.U. Cokgor et al. / Bioresource Technology 102 (2011) 567–575

(a) 160 OUR curve with no initial peak at the beginning and a gradual in-
140 OUR_data crease afterwards to around 100 mg O2/L h was well simulated
OUR_model_total with the model. The slight discrepancy between model simulation
120
OUR_model_XH and the OUR curve after the sharp decrease was possibly due to the
OUR (mg O2/L.h)

100 OUR_model_XHB hydrolysis of a small portion of the intermediate cleavage products

80 before utilization, a process although not significant, not accounted
60 for in the model.
As summarized in Table 3, the model was calibrated with a dis-
40
integration rate coefficient, kdis of 5.4/day and a maximum specific
20 growth rate, l ^ HB of 5.6/day for the portion of the biomass, XHB
0 which developed the enzymatic capability for cleavage and utiliza-
-100 0 100 200 300 400 500 tion of 2,6-DHBA as the organic carbon source for energy and bio-
Time (min) synthesis. The initial XHB concentration was similarly determined
as 720 mg cell COD/L. The calibration was performed with a yield
(b) 140 coefficient YH of 0.60 mg cell COD/mg COD, totally compatible with
PHA_data
120 PHB_data the oxygen utilization of around 248 mg O2/L computed as the area
PHV_data under the OUR curve, excluding the endogenous decay portion. The
100 3H2MV_data calibration of the initial endogenous respiration part of the OUR
COD (mg/L)

PHA_model
80 curve also yielded an endogenous decay coefficient, bH of 0.20/
day, in agreement with the commonly reported values for this pro-
60
cess (Orhon et al., 2009c).
40 The interesting observation of the calibration exercise was
related to the observed OUR level due to endogenous respiration
20
before substrate addition to the batch reactor. It indicated aside a
0 XHB level of 720 mg cell COD/L, a second group of active biomass
0 100 200 300 400 500 600 700 800 concentration, XH of 900 mg cell COD/L, interpreted to be
Time (min) associated with peptone mixture biodegradation in the main SBR
which supplied the biomass seed for the batch experiment. In this
Fig. 2. Modeling of peptone mixture biodegradation with the acclimated culture
experiment, the sole function of the identified XH fraction was
(Run – 2, day 32) (a) OUR profile and (b) PHA profile.

(a) 160 (c) 220

OUR_data OUR_data
200
140 OUR_model_specific biomass OUR_model_total biomass
180
OUR (mg O2/L.h)

120 160
OUR (mg O2/L.h)

100 140
120
80
100
60 80
40 60
40
20
20
0 0
-100 0 100 200 300 400 500 -100 0 100 200 300 400 500
Time (min) Time (min)

(b) 120 (d) 200

OUR_data OUR_data
180 OUR_model_total biomass
OUR_model_specific biomass
100 160
OUR (mg O2/L.h)
OUR (mg O2/L.h)

140
80
120
60 100
80
40
60

20 40
20
0 0
-100 0 100 200 300 400 500 -100 0 100 200 300 400 500
Time (min) Time (min)

Fig. 3. Simulation of the OUR profile: with specific biomass fraction for (a) biodegradation of peptone mixture, (b) 2,6-DHBA; with total biomass for (c) biodegradation of
peptone mixture and (d) 2,6-DHBA.
 E.U. Cokgor et al. / Bioresource Technology 102 (2011) 567–575 573

endogenous respiration in the absence of peptone mixture. This
observation will be elaborated in the evaluation of the following
experimental results.
3.3. Biodegradation kinetics of peptone mixture only
The second of a series of batch experiments was carried out to
provide a model evaluation for peptone mixture biodegradation
alone, after the acclimation period (Run – 2, day 32). The batch test
was started with peptone mixture as the sole organic carbon
source supplied at an initial concentration of 545 mg COD/L. A typ-
ical OUR profile was obtained, as illustrated in Fig. 2, reflecting the
specific fingerprint defining peptone mixture utilization (Insel
et al., 2006) with an early peak reaching above 150 mg O2/L h, with
a sequential decline afterwards indicating hydrolysis of two COD
fractions at different rates. The OUR curve yielded an oxygen con-
sumption value of 186 mg O2/L approximating and confirming the
YH value of 0.60 mg cell COD/mg COD adopted for model calibra-
tion. As summarized in Table 3, the calibration exercise defined
5.6/day for the maximum specific growth rate, l ^ H for XH as the
appropriate best fit value. The hydrolysis rates were markedly dif-
ferent for the two slowly biodegradable COD fractions, SH1 and SH2
with a kh1 of 4.34/day and a much lower kh2 of 2.60/day. The cali-
bration was best performed with an active biomass concentration,
XH of 1050 mg COD/L and the same value of 0.2/day for the endog-
enous decay coefficient, bH as in the previous batch experiment
according to Ekama et al. (1986). It also defined the magnitude of
whereas if it is started with the total biomass (XH + XHB) the OUR
profile would be overestimated, as shown in Fig. 3c and d.
3.4. Biodegradation kinetics of peptone/2,6-DHBA mixture
The batch test was carried out with initial concentrations of
both peptone mixture and 2,6-DHBA adjusted to 285 mg COD/L
in order to approximate the initial COD level in the other
experiments (Run 3 – day 32). The resulting OUR profile is given
in Fig. 4; it closely reflects the superposition of the OUR curves ob-
tained in the experiments conducted separately on each substrate,
with necessary concentration adjustments, basically indicating
simultaneous utilization of both substrates. The PHA breakdown
was given in Fig. 4c to indicate the relative magnitude of each frac-
tion on the overall PHA profile. The observations indicated that the
relative contributions have not changed before and after acclima-
tion of the culture to 2,6-DHBA. As previously mentioned, 2,6-
DHBA biodegradation did not involve PHA storage. The adopted
model calibrated individually for the two substrates provides, as
shown in Fig. 4a, a close simulation of the experimental OUR pro-
file with the kinetic and stoichiometric coefficients given in Table
3: basically the same model coefficients ascertained for each sub-
(a) 180
OUR_data
160
OUR_model_total
140
OUR (mg O2/L.h)

OUR_model_XH
the three COD fractions involved, subdividing the total initial pep- 120
OUR_model_XHB
tone mixture COD into SS (9%), SH1 (44%) and SH2 (47%). 100
The results summarized above may be compared with a similar 80
model evaluation for peptone mixture conducted at the beginning 60
of the experiment (i.e. day 0) before the first addition of 2,6-DHBA 40
to the SBR systems, in other words, before the exposure of the 20
microbial culture to 2,6-DHBA (Orhon et al., 2009a). The data re- 0
lated to this experiment, also summarized in Table 3, provide a -100 0 100 200 300 400 500
clear indication that the recovery of the biomass was complete Time (min)
after the strong adverse effect of 2,6-DHBA feeding with almost
the same biodegradation characteristics, except for (i) a slight dif- (b) 600
ference in the growth kinetics, and (ii) a similar variation in the Total COD_data
500 Peptone COD_data
magnitude of generation and subsequent utilization of internal
2,6-DHBA COD_data
storage products. These results were expected in view of the fact
COD (mg/L)

400 Total COD_model

that previous studies provided evidence for a variable Monod
300 Peptone COD_model
kinetics and substrate utilization pattern – shifts between growth 2,6-DHBA COD_model
and storage – depending on the characteristics of the microbial cul- 200
ture, quite reasonable for a recovered and newly adapted culture in
a competing dual biomass system. 100
As in the previous batch experiment, model evaluation supplied 0
conclusive support for the existence of the establishment of a dual 0 100 200 300 400 500
biomass system after the acclimation phase, a group utilizing pep- Time (min)
tone mixture alone and the other group with the acquired ability to
utilize 2,6-DHBA. In fact, calibration of the part of the OUR profile (c) 140
prior to substrate addition indicated the existence of XHB so that PHA_data
120 PHB_data
the total active biomass concentration was 1500 mg cell COD/L
with two components of XH (1050 mg cell COD/L) and XHB 100 PHV_data
COD (mg/L)

450 mg cell COD/L). 80 3H2MV_data

The validity of the identified biomass fractions were also tested PHA_model
60
with the following model simulations: each OUR profile has two
segments (i) endogenous respiration phase before substrate addi- 40
tion, and (ii) the following substrate utilization phase. It is imper- 20
ative that the endogenous phase involves both biomass fractions
0
and the substrate utilization phase is functionally related solely
to the specific biomass fraction. 0 100 200 300 400 500
Model simulations conducted on peptone mixture and 2,6- Time (min)
DHBA experiments indicated, as illustrated in Fig. 3a and b that if Fig. 4. Modeling the biodegradation of peptone/2,6-DHBA mixture with the
the endogenous phase is started with the specific biomass fraction acclimated culture (Run – 3, day 32) (a) OUR profile (b) COD profile and (c) PHA
(XH or XHB) the whole OUR profile would be underestimated, profile.
574 E.U. Cokgor et al. / Bioresource Technology 102 (2011) 567–575

XHB and the resulting XH/XHB ratio remained nearly the same. But
280 OUR_data
these values differed in the first experiment on 2,6-DHBA, with less
240 OUR_model_total XH and more XHB. This result is quite explainable in view of the fact
OUR (mgO2 /L.h)

200 OUR_model_Peptone degradation that the model evaluates in essence metabolic functions and not
OUR_model_2,6-DHBA degradation strictly microbial communities as in a microbiological and/or ge-
160
netic test. In fact, the result showed that a fraction of the heterotro-
120 phic biomass, XH could utilize 2,6-DHBA as substrate in the
80 absence of peptone mixture. This observation, although interesting
40 is not practically important because absence of the main carbon
source will never be an issue in treatment systems.
0
-100 0 100 200 300 400 500
Time (min) 3.5. Identifiability analysis

Fig. 5. Simulation of the OUR and PHA profiles for the biodegradation of peptone/
The identifiability analyses were conducted for the simulations
2,6-DHBA mixture with a single biomass.
runs of (i) 2,6-DHBA degradation (Run – 1) and (ii) mixture of pep-
tone/2,6-DHBA degradation (Run – 3) by using dual biomass mod-
el. In the identifiability analysis of 2,6-DHBA degradation, only the
strate remained valid for the biodegradation of the mixture, with
OUR profile was taken into consideration since no storage com-
the exception of a relatively higher maximum storage rate of
pound was experimentally determined in this run. On the other
PHA, kSTO and maximum growth rate for growth on PHA, rG. A mod-
hand, the model identifiability study for the mixture of peptone/
el simulation exercise conducted with storage parameters previ-
2,6-DHBA covers both the OUR profile and XSTO measurements.
ously estimated for peptone mixture and 2,6-DHBA experiments
The sensitivity rankings of the model parameters were first cal-
indicated discrepancy between experimental data and model sim-
culated in order to select the best identifiable parameter subsets
ulation. The discrepancy was quite significant for the PHA profile
for evaluating the changes in model parameter values according
justifying the change in the corresponding kinetic coefficients.
to Brun et al. (2001) (results not shown). Then, respective condi-
Fig. 4 also shows the simulated profiles for each substrate, com-
tion number, cK for the selected parameter subsets was calculated
bined to fit the experimental OUR data. They were derived using
as shown in Table 4. The identifiable limit (cK) for the condition
two different components of the active biomass as defined in the
number was targeted as 20. Hence, the number of parameter was
model and confirmed with the previous experiments. The model
increased by keeping the identifiability criteria of cK < 20.
also calibrated well with the COD and PHA data (Fig. 4b and c).
As shown in Table 4, 5 out of 25 model parameters can be esti-
The modeling exercise performed with a single biomass approach
mated using the OUR profile for the model run of 2,6-DHBA degra-
provides, as shown in Fig. 5, a similar support for the existence
dation (Run – 1). The maximum growth rate for XHB1 (l ^ HB ) can be
of two active components of the biomass separately responsible
estimated together with the active biomass fraction. The identifi-
for peptone mixture and 2,6-DHBA utilization when both sub-
ability study conducted for peptone/2,6-DHBA mixture (Run – 3),
strates are simultaneously fed to the reactor. The model parame-
biodegradable COD fractions (SS1, SH1), active biomass fractions
ters (maximum growth rate and half saturation constant)
(XH1, XHB1), maximum hydrolysis rates (kh1, kh2) together with
characterizing the growth process is known to be dependent on
the parameters of storage process can be accepted as identifiable
the sludge history (Ferenci, 1999; Insel et al., 2007). By using a sin-
when in parallel OUR and XSTO measurements are included in the
gle biomass in model building, it is expected to have at least closer
identifiability study. In this case, 14 out of 25 model parameters
maximum growth rates for all experimental setups all operated at
can be extracted from the experimental data.
10 days of SRT. However, the use of model with a single biomass
for peptone mixture and 2,6-DHBA data showed that half of the
maximum growth rates were required to get an reasonable fit. In 4. Conclusions
addition, the modeling effort carried out using single biomass
(XH) also necessitated many switching functions to mimic the dif- Model analysis separately identified kinetic and stoichiometric
ferent response of biomass during different time segments of OUR model parameters for the microbial community fractions responsi-
experiment. ble for the utilization of peptone mixture and 2,6-DHBA after accli-
In the last two experiments conducted with peptone mixture mation. Results provided conclusive support for the existence of a
and peptone/2,6-DHBA mixture respectively – that is, when pep- dual microbial community and development of another microbial
tone mixture was present – (Runs 2 and 3) the levels of XH and group specifically capable of metabolizing 2,6-DHBA under com-

Table 4
Collinearity index (cK) calculation for parameter subsets of dual biomass model.

Run Parameter subset cK

1 2 3 4 5 6 7 8 9 10 11 12 13 14
2,6-DHBA KD l^ HB XH1 XHB1 YH 5.6
kdis KSB l^ HB XH1 YH 10.8
KSB l^ HB XH1 XHB1 YH 11.4
KD kdis KSB l^ HB YH 19.3
KD kdis KSB l^ HB XH1 19.3
Peptone/2,6-DHBA mixture SS1 SH1 XH1 XHB1 kh1 kh2 l^ H l^ HB kSTO KSTO rG KS YH YSTO 18.2
SS1 SH1 XH1 XHB1 kh1 kh2 l^ H l^ HB kSTO KD KSTO rG KS YH 18.3
SS1 SH1 XH1 XHB1 kh1 kh2 l^ H l^ HB kSTO rG KS KSB YH YSTO 18.4
SS1 SH1 XH1 XHB1 kh1 kh2 l^ H l^ HB kSTO KSTO rG KS KSB YH 18.6
SS1 SH1 XH1 XHB1 kh1 kh2 l^ H l^ HB kSTO KSTO KS KSB YH YSTO 18.7
 E.U. Cokgor et al. / Bioresource Technology 102 (2011) 567–575
petitive growth conditions. Metabolic adaptation of a new micro-
bial community to new growth conditions with two substrates
was identified and kinetically defined.
The adopted approach offers new perspectives for the evalua-
tion of biodegradation of xenobiotics with two components – bio-
mass models in real systems. These studies should also be
supported by molecular/genetic techniques.
References
Aktas, E.S., Imre, S., Ersoy, L., 2001. Characterization and lime treatment of olive mill
wastewater. Water Res. 35, 2336–2340.
Beun, J.J., Paletta, F., van Loosdrecht, M.C.M., Heijnen, J.J., 2000. Stoichiometry and
kinetics of poly-b-hydroxybutyrate metabolism in aerobic, slow growing
activated sludge cultures. Biotechnol. Bioeng. 67, 379–389.
Brun, R., Reichert, P., Künsch, R.K., 2001. Practical identifiability analysis of large
environmental simulation models. Water Resour. Res. 37, 1015–1030.
Cokgor, E.U., Arslan-Alaton, I., Erdinc, E., Insel, G., Orhon, D., 2007. Effect of
photochemical pre-treatment on COD fractionation of a non-ionic textile
surfactant. Water Sci. Technol. 55, 155–163.
Damayanti, A., Ujang, Z., Salim, M.R., Olsson, G., Sulaiman, A.Z., 2010. Respirometric
analysis of activated sludge models from palm oil mill effluent. Bioresour.
Technol. 10, 144–149.
Davey, R.J., Blagden, N., Righini, S., Alison, H., Quayle, M.J., Fuller, S., 2001. Crystal
polymorphism as a probe for molecular self-assembly during nucleation from
solutions: the case of 2,6-dihydroxybenzoic acid. Cryst. Growth Des. 1, 59–
65.
Di Gioia, D., Fava, F., Bertin, L., Marchetti, L., 2001. Biodegradation of synthetic and
naturally occurring mixtures of mono-cyclic aromatic compounds present in
olive mill wastewaters by two aerobic bacteria. Appl. Microbiol. Biotechnol. 55,
619–626.
Dogruel, S., Olmez-Hanci, T., Kartal, Z., Arslan-Alaton, I., Orhon, D., 2009. Effect of
Fenton’s oxidation on the particle size distribution of organic carbon in olive
mill wastewater. Water Res. 43, 3974–3983.
Dold, P.L., Ekama, G.A., Marais, G.V.R., 1980. A general model for the activated
sludge process. Prog. Water Technol. 12, 47–77.
Drouiche, M., Le Mignot, V., Lounici, H., Belhocine, D., Grib, H., Pauss, A., Mameri, N.,
2004. A compact process for the treatment of olive mill wastewater by
combining UF and UV/H2O2 techniques. Desalination 169, 81–88.
Ekama, G.A., Dold, P.L., Marais, G.V.R., 1986. Procedures for determining influent
COD fractions and the maximum specific growth rate of heterotrophs in
activated sludge. Water Sci. Technol. 18, 91–114.
Ferenci, T., 1999. Growth of bacterial cultures, 50 years on: towards an uncertainty
principle instead of constants in bacterial growth kinetics. Res. Microbiol. 150,
431–438.
Fritsche, W., Hofrichter, M., 2005. Aerobic degradation of recalcitrant organic
compounds by microorganisms. In: Jördening, H.J., Winter, J. (Eds.),
Environmental Biotechnology: Concepts and Applications. Wiley-VCH Verlag,
Weinheim, pp. 203–226.
Garcia, I.G., Pena, P.R.J., Venceslada, J.L.B., Martin, A.M., Santos, M.A.M., Gomez, E.R.,
2000. Removal of phenol compounds from olive mill wastewater using
Phanerochaete chrysosporium, Aspergillus niger, Aspergillus terreus and
Geotrichum candidum. Process. Biochem. 35, 751–758.
Henze, M., Grady Jr., C.P.L., Gujer, W., Marais, Gv.R., Matsuo, T., 1987. Activated
Sludge Model No. 1. IAWPRC Scientific and Technical Report No. 1. IAWPRC,
London, UK.
575
Insel, G., Orhon, D., Vanrolleghem, P.A., 2003. Identification and modelling of
aerobic hydrolysis mechanism-application of optimal experimental design. J.
Chem. Technol. Biotechnol. 78, 437–445.
Insel, G., Karahan, O., Ozdemir, S., Pala, I., Katipoglu, T., Cokgor, E.U., Orhon, D., 2006.
Unified basis for the respirometric evaluation of inhibition for activated sludge.
J. Environ. Sci. Health A 41, 1763–1780.
Insel, G., Celikyilmaz, G., Ucisik Akkaya, E., Yesiladali, K., Cakar, P., Tamerler, C.,
Orhon, D., 2007. Respirometric evaluation and modeling of glucose utilization
by Escherichia coli under aerobic and mesophilic cultivation conditions.
Biotechnol. Bioeng. 96, 94–105.
ISO 6060, 1986. Water Quality-determination of the Chemical Oxygen Demand.
International Standards Organization, Switzerland.
ISO 8192, 1995. Water Quality-test for Inhibition of Oxygen Consumption by
Activated Sludge. International Standards Organization, Switzerland.
Karahan-Gul, O., Orhon, D., van Loosdrecht, M.C.M., 2003. Modification of activated
sludge model no. 3 considering direct growth on primary substrate. Water Sci.
Technol. 47, 219–225.
Katipoglu, T., Cokgor, E.U., Insel, G., Orhon, D., 2010. Response of mixed microbial
culture to 2,6-dihydroxybenzoic acid and peptone mixture at low sludge age –
effect of culture history. J. Environ. Sci. Health A 45, 1–9.
Krishna, C., van Loosdrecht, M.C.M., 1999. Substrate flux into storage and growth in
relation to activated sludge modeling. Water Res. 33, 3149–3161.
Lee, P.Y., Chen, C.Y., 2009. Toxicity and quantitative structure–activity relationships
of benzoic acids to Pseudokirchneriella subcapitata. J. Hazard. Mater. 165,
156–161.
Lindblom, E., Press-Kristensen, K., Vanrolleghem, P.A., Mikkelsen, P.S., Henze, M.,
2009. Dynamic experiments with high bisphenol-A concentrations modelled
with an ASM model extended to include a separate XOC degrading
microorganism. Water Res. 43, 3169–3176.
Lubello, C., Caffaz, S., Gori, R., Munz, G., 2009. A modified activated sludge model to
estimate solids production at low and high solids retention time. Water Res. 43,
4539–4548.
Orhon, D., Cokgor, E.U., Insel, G., Karahan, O., Katipoglu, T., 2009a. Validity of Monod
kinetics at different sludge ages – peptone biodegradation under aerobic
conditions. Bioresour. Technol. 100, 5678–5686.
Orhon, D., Cokgor, E.U., Katipoglu, T., Insel, G., Karahan, O., 2009b. Fate of
2,6-dihydroxybenzoic acid and its inhibitory. Impact on the biodegradation of
peptone under aerobic conditions. Bioresour. Technol. 101, 2665–2671.
Orhon, D., Germirli Babuna, F., Karahan, O., 2009c. Industrial Wastewater Treatment
by Activated Sludge. IWA Publishing, London.
Reichert, P., Ruchti, J., Simon, W., 1998. AQUASIM 2.0. Swiss Federal Institute for
Environmental Science and Technology (EAWAG), CH-8600 Duebendorf,
Switzerland.
Ricco, G., Tomei, M.C., Ramadori, R., Laera, G., 2004. Toxicity assessment of common
xenobiotic compounds on municipal activated sludge: comparison between
respirometry and Microtox (R). Water Res. 38, 2103–2110.
Rezouga, F., Hamdi, M., Sperandio, M., 2009. Variability of kinetic parameters due to
biomass acclimation: case of para-nitrophenol biodegradation. Bioresour.
Technol. 100, 5021–5029.
Spanjers, H., Vanrolleghem, P.A., 1995. Respirometry as a tool for rapid
characterization of wastewater and activated sludge. Water Sci. Technol. 31,
105–114.
Standard Methods, 2005. Standard Methods for the Examination of Water and
Wastewater, 21st ed. American Public Health Association/American Water
Works Association/Water Environment Federation, Washington, DC.
Yoshida, M., Oikawa, T., Obata, H., Abe, K., Mihara, H., Esaki, N., 2007. Biochemical
and genetic analysis of the c-resorcylate (2,6-dihydroxybenzoate) catabolic
pathway in Rhizobium sp. Strain MTP-10005: identification and functional
analysis of its gene cluster. J. Bacteriol. 189, 1573–1581.