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Biochemical Engineering Journal 91 (2014) 283289

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Regular Article

Acute impact of tetracycline and erythromycin on the storage

mechanism of polyhydroxyalkanoates
Tugce Katipoglu-Yazan a, , Ilke Pala-Ozkok a , Emine Ubay-Cokgor a , Derin Orhon a,b
Faculty of Civil Engineering, Environmental Engineering Department, Istanbul Technical University, 34469 Maslak, Istanbul, Turkey
The Science Academy, 34353 Besiktas, Istanbul, Turkey

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

Article history: The study investigated acute impact of tetracycline and erythromycin on substrate storage under aerobic
Received 21 March 2014 conditions. A ll and draw reactor fed with peptone mixture was maintained at steady-state at a sludge
Received in revised form 29 July 2014 age of 10 days; the acclimated biomass was used in a series of batch runs. The rst run served as control
Accepted 1 September 2014
reactor with organic substrate alone and the others were started with antibiotic doses of 50 mg/L and
Available online 6 September 2014
200 mg/L for assessing intracellular storage. Parallel batch reactors were also conducted for recording
oxygen uptake rate proles. Both antibiotics enhanced substrate storage, leading to higher levels of
polyhydroxyalkanoates incorporated into biomass, but they impaired its internal utilization for microbial
Substrate storage
growth. The observed decrease in oxygen consumption under the acute effect of antibiotics could partially
Kinetic parameters be related to substrate storage except for 50 mg/L of erythromycin dosing suggesting an additional
Dissolved oxygen substrate binding mechanism by antibiotics, leading to residual biodegradable substrate.
Peptone 2014 Elsevier B.V. All rights reserved.

1. Introduction converted to storage products under pulse feeding [5,6]. They also
indicated that the magnitude of storage was likely to exhibit signif-
Storage of intracellular biopolymers is now recognized as a sig- icant variations depending on the nature of substrate, the feeding
nicant auxiliary process during substrate utilization by microbial regime and culture history i.e. sludge age of the microbial culture
cultures. It was mainly observed under transient feeding condi- [714]. Expected storage would be substantially lower in com-
tions, where sequential feast and famine phases trigger substrate plex substrate mixtures such as domestic sewage where only a
storage [1]. In essence, storage results from an imbalance between small percent of the organic matter consist of readily biodegrad-
removal of available substrate and microbial growth potential; able compounds favoring formation of intracellular biopolymers.
while substrate may be removed, limitations on the metabolic reac- In fact, in a study using a peptone-meat extract mixture as the
tions leading to growth may prevent consumption of all energy organic carbon source with biodegradation characteristics similar
and divert a fraction of the substrate for generating intracellu- to domestic sewage, storage of only 30 mg COD/L of intracellular
lar biopolymers [2]. Batch reactors are often selected as the most biopolymers was observed, corresponding to 60% of the available
suitable experimental tool for investigating different aspects of readily biodegradable COD at a sludge age of 8 days [15].
substrate biodegradation, mainly because they offer accurate eval- The storage mechanism is also important when studying inhi-
uation of transient responses and resulting concentration proles bition of substrate biodegradation under the impact of adverse
of major parameters [3,4]. Dynamic conditions sustained in batch chemicals and xenobiotics. Traditionally, inhibition was inter-
reactors approximate pulse feeding and induce a physiological preted by means of enzyme analogy affecting only microbial
adaptation for the microbial community, often leading to substrate growth [16,17], simply because corresponding substrate utiliza-
storage. tion was only characterized by a single overall parameter such
The storage mechanism is often studied with simple, readily as BOD or COD. After recognition and experimental assessment
biodegradable substrates like acetate or glucose, generating poly- of COD fractions with different biodegradation characteristics in
hydroxybutyrate (PHB) and glycogen as storage products. Reported domestic sewage and industrial wastewaters [15], interpretation of
results suggested that up to 70% of the simple substrate could be biodegradation involved all these fractions and related biochemi-
cal processes [18]. While multi-component models incorporated
all these processes, they initially disregarded substrate storage
Corresponding author. Tel.: +90 2122856542; fax: +90 2122853781. [19,20]. Later, these models were modied to account for simulta-
E-mail address: (T. Katipoglu-Yazan). neous substrate utilization for growth and storage [21,22]. Thanks
1369-703X/ 2014 Elsevier B.V. All rights reserved.
284 T. Katipoglu-Yazan et al. / Biochemical Engineering Journal 91 (2014) 283289

Table 1
Nomenclature Operating characteristics of batch experiments [33].

Runs Antibiotic Biomass (mg VSS/L) S0 /X0 (mg T ( C)

Cs initial biodegradable substrate (mg COD/L) COD/mg VSS)
CSG fraction of substrate directly utilized for microbial
Run 1 920 0.55 21
growth (mg COD/L) Run 2.1 50 mg TET/L 1005 0.50 21
CSGP fraction of the initial substrate potentially available Run 2.2 200 mg TET/L 995 0.51 24
for microbial growth (mg COD/L) Run 3.1 50 mg ERY/L 995 0.51 25
CSR residual fraction of the initially available biodegrad- Run 3.2 200 mg ERY/L 1035 0.49 25

able substrate (mg COD/L) Peptone mixture having 506 mg COD/L was added in all runs.
CST fraction of substrate diverted to storage (mg COD/L)
CSTR substrate COD equivalent of residual PHA entrapped
in biomass (mg COD/L)
under aerobic conditions. The rst part involved running a ll and
CSTU fraction of substrate stored and internally utilized
draw reactor with biomass taken from a domestic wastewater
(mg COD/L)
treatment plant. The system was maintained at steady-state at a
kSTO maximum storage rate (1/day)
sludge age of 10 days, a value compatible with the level selected for
KSTO half saturation coefcient for storage and internal
biological treatment systems targeting combined organic carbon
growth (mg COD/L)
removal and nitrication [30].
OUR oxygen uptake rate (mg O2 /L h)
The acclimated biomass from the ll and draw reactor was used
PHA polyhydroxyalkanoates
for starting a series of batch runs, each run including two parallel
PHB polyhydroxybutyrate
batch reactors, one for the assessment of intracellular biopolymers
PHV polyhydroxyvalerate
and the other, for the respirometric analysis of oxygen uptake rate
SO oxygen consumed (mg/L)
(OUR) proles. The initial COD concentration of the peptone mix-
SS readily biodegradable substrate (mg COD/L)
ture was adjusted to around 500 mg/L. The rst run served as the
SOT theoretical oxygen demand (mg/L)
control reactor; the other two runs included in a feed having ini-
XSTO stored PHA (mg COD/L)
tial dose of 50 mg/L and 200 mg/L of the antibiotic for its acute
XSTR residual PHA entrapped in biomass (mg COD/L)
impact, primarily on substrate storage. The highest antibiotic level
YH yield coefcient (mg COD/mg COD)
was selected mainly to approximate efuent discharges of phar-
YSTO storage yield (mg COD/mg COD)
maceutical industries and hospitals which were reported to reach
3H2MV 3-hydroxy-2-methylvalerate
up to 100500 mg/L [23,31]. As an example, tylosin antibiotic was
detected in the range of 20200 mg/L in an antibiotic wastewater
[32]. The lowest concentration was selected based on the respiro-
to these modeling tools, interpretation of inhibitory substances metric studies previously conducted on three different antibiotics
was no longer limited to substrate utilization for microbial growth, indicating oxygen uptake rate reduction by more than 50% for the
but it could also cover all related biochemical mechanisms such same substrate [17]. Batch experiments were conducted separately
as hydrolysis, endogenous respiration and substrate storage [14]. for tetracycline and erythromycin.
In this context, the storage mechanism is particularly important,
mainly because it is most likely to be affected under stress con-
ditions, i.e. diversion of substrate from growth to storage under 2.2. Batch experiments
adverse impact and when disregarded it may signicantly distort
the kinetics of parallel biochemical processes, leading to misinter- Batch experiments were conducted in aerated 2 L cylindrical
pretation of the inhibitory impact. benchtop reactors having 30 cm diameter. The dissolved oxygen
The previous works investigated the impact of selected antibi- was supplied air supplying diffusers connected to airline. The oxy-
otics on either heterotrophic or autotrophic bacteria in terms of gen level in the reactors was maintained above 3.0 mg/L (33%
general process kinetics. The main objective of this part of the study oxygen saturation) by mixing with mechanical impellers to pre-
was to evaluate the acute impact of two selected inhibitors on the vent any oxygen limitations on biochemical reactions. In order to
mechanism of substrate storage in a nitrifying microbial commu- determine the effects of selected antibiotics on substrate storage
nity. Two major antibiotics, namely tetracycline and erythromycin mechanism, related parameters such as COD and storage com-
were selected as the inhibitor compounds, due to the fact that they pounds were monitored. Batch reactors were supplied with 50
take place in our daily life, discharged into the wastewaters and to and 200 mg/L of TET or ERY solutions in addition to peptone mix-
the environment [23] and well studied for their inhibitory effects ture. Initial food to microorganisms ratio (S0 /X0 ) was adjusted
[2426]. The acute impact was evaluated on the biodegradation to 0.490.55 mg COD/mg VSS where macro (320 g/L K2 HPO4 , and
of peptone-meat extract mixture called thereafter peptone mix- 160 g/L KH2 PO4 ) and micro nutrients (15 g/L MgSO4 7H2 O, 0.5 g/L
ture for simplicity under aerobic conditions. It was selected as FeSO4 7H2 O, 0.5 g/L, ZnSO4 7H2 O, 0.41 g/L MnSO4 H2 O, 2.65 g/L,
the complex organic carbon source because it is prescribed as the CaCl2 2H2 O g/L) were also fed to support microbial activity. The sys-
standard substrate by ISO 8192 [27] in similar inhibition studies tem was fed once on a daily basis and continuously aerated to have
[28] and it approximates the COD fractionation and biodegradation a hydraulic retention time of 1 day. Activated sludge was settled
characteristics of domestic sewage [29]. for 1 h and efuent was decanted to 2 L. COD removal efciencies
and internal storage biopolymers, polyhydroxyalkanoates (PHAs)
2. Materials and methods including polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV)
and 3-hydroxy-2-methylvalerate (3H2MV) were monitored during
2.1. Experimental rationale the experiments for the determination of biological activity [33].
The tests lasted for 24 h and pH remained at neutral levels for this
The experiments were conducted as part of a comprehensive time period. Information related to batch experiments was given in
study investigating the impact of antibiotics on the heterotrophic Table 1. Repeated experiments yielded the same proles of related
and autotrophic communities in single sludge systems operated parameters.
T. Katipoglu-Yazan et al. / Biochemical Engineering Journal 91 (2014) 283289 285

2.3. Measurements of storage products

Samples taken from mixed liquor were ltered through AP40

glass bers for suspended solids (SS) and volatile suspended solids
(VSS) analyses as described in Standard Methods [34]. For COD anal-
ysis, 0.45 m Millipore membrane lters were used. The ltrate
was preserved with H2 SO4 and analyzed according to ISO 6060 [35].
pH was monitored by Orion 520 A pH meter. The pH meter was con-
nected with an online dosing system for a bicarbonate-carbonate
buffer solution that will keep the pH at neutral ranges.
Mixed liquor was directly preserved with formaldehyde and
biological activity was prevented in PHAs samples. K-P buffer solu-
tion was used for washing and samples were freeze dried. Boiling
at 100 C resulted in extraction, hydrolysation, and esterication
of the samples [5]. The free acids were water extracted and the
remaining organic phase was measured by gas chromatography
Fig. 1. Storage proles in the control reactor for PHA and individual storage com-
(Agilent 6890 N). Benzoic acid was used as internal standard. ponents PHB, PHV and 3H2MV [33].
Samples were analyzed in duplicate for each parameter. Standard
deviations of COD were 3%. feast and famine conditions abundance and absence of substrate
favoring substrate storage [1].
The peptone mixture, selected as the source of organic matter in
2.4. Respirometric measurements the study is a complex substrate with a COD fractionation quite sim-
ilar to domestic sewage and most industrial wastewaters: previous
Previous works involving respirometric analysis indicated that studies indicated that a small fraction less than 10% of its COD
evaluation of OUR proles enabled determination of the effects of content was of readily biodegradable nature and the rest included
inhibitors on substrate biodegradation kinetics in terms of model two fractions of slowly biodegradable components with different
coefcients [28]. These model coefcients exert variable sensitivity hydrolysis rates [15,40]. Therefore, the substrate fraction diverted
at different regions of OUR prole that are used to identify the most to the generation of intracellular biopolymers was generally lower
sensitive parameter [10,15,36]. as compared with simple/readily biodegradable compounds like
Modeling exercise involved analyzing and calibrating the OUR acetate or glucose [9,41].
and PHA proles. Kinetic and stoichiometric parameters were Substrate storage observed for the control reactor oper-
estimated by using SIMPLEX algorithm and AQUASIM simulation ated without antibiotic dosage was illustrated in Fig. 1:
program [37]. Parameter identiability procedure was also imple- It essentially involved polyhydroxyalkanoates (PHA), including
mented to determine best identiable parameter subsets. The polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV) and 3-
UNCSIM module proposed by Brun et al. [38] was used for the cal- hydroxy-2-methylvalerate (3H2MV) with no detectable glycogen
culation of sensitivity rankings of model parameters as previously often associated with carbohydrates. As shown in this gure, the
reported [29]. observed PHA prole exhibited an initial peak, reecting PHA accu-
In this study computer controlled oxygen uptake rate (OUR) mulation due to diversion of a fraction of available substrate to
measurements, with well proven reliability, were conducted with storage; then, it was characterized by a descending curve result-
TET and ERY having identical conditions with batch experiments. ing from biochemical consumption of the stored PHA as internal
Biomass seeding alone to obtain the initial endogenous OUR level substrate for microbial growth.
was supplied with peptone mixture and antibiotic solutions to The prole started with a slight initial PHA pool of 35 mg COD/L
have desired S0 /X0 ratios. A continuous lab-scale online respirom- incorporated into the biomass seed during the continuous opera-
eter (Ra-Combo) was used for OUR measurements (Applitek Co., tion of the ll and draw reactor. As shown in Fig. 1, PHA gradually
Nazareth, Belgium). Oxygen depletion was measured with highly increased and reached to a plateau with the rst 2.5 h. A maximum
developed dissolved oxygen probes located in closed vessels of the of 22 mg COD/L PHA was stored. Stored PHA was consumed as inter-
respirometer [39]. Biological activity was monitored with addi- nal substrate and decreased down to the initial pool level within
tional COD sampling. 69 mg/L of dissolved oxygen level was the next 20 h. Although PHA data is not available after 6 h, the PHA
maintained in the reactor and pH was recorded during the exper- plateau indicated a clearly denable gradual decrease within next
iments. Respirometric data was analyzed considering pH and 18 h. PHA storage mainly consisted of PHB (18 mg COD/L) and a
temperature corrections. slight PHV fraction (4 mg COD/L). The initial PHA pool consisted of
30 mg COD/L 3H2MV (Fig. 1). It is interesting to note that while
3. Results and discussion the initial pool essentially composed of 3H2MV (30 mg COD/L), no
detectable accumulation of 3H2MV was observed during the test.
The nature and magnitude of substrate storage mainly depends The results indicated that the amount of substrate stored was rel-
on substrate characteristics, the feeding regime and the culture his- atively minimal i.e. less than 5% with respect to the initial level
tory i.e. the sludge age of the microbial culture. As in this study, of 506 mg COD/L selected as the initial level of peptone mixture.
utilization/biodegradation of organic substrate is now evaluated
by means of respirometric procedures in waste/wastewater [15]; 3.1. Impact of tetracycline
they require the use of batch reactors for assessing the evolution of
the oxygen uptake rate (OUR) prole associated with the selected Tetracycline was observed to inict a signicant change on the
experimental conditions. Accordingly, all auxiliary measurements storage mechanism. As shown in Fig. 2, the modied shape of
supporting OUR proles are also derived from parallel batch reac- the PHA prole signaled a different storage mechanism under the
tors, which conveniently yield concentration transients of relevant inuence of the antibiotic. At 50 mg/L dose, the PHA level rapidly
parameters. Batch reactors start with an initial substrate concen- increased to 60 mg COD/L and the increase was continued at a
tration, acting as a pulse feeding, which then leads to a sequence of slower pace to reach a nal plateau of around 90 mg COD/L within
286 T. Katipoglu-Yazan et al. / Biochemical Engineering Journal 91 (2014) 283289

Fig. 2. Storage proles at tetracycline dosing of (a) 50 mg/L and (b) 200 mg/L for Fig. 3. Storage proles at erythromycin dosing of (a) 50 mg/L and (b) 200 mg/L [33]
PHA and individual storage components PHB, PHV and 3H2MV. for PHA and individual storage components PHB, PHV and 3H2MV.

the rst 4 h. Afterwards, the PHA prole exhibited a slight decrease 3.2. Impact of erythromycin
of only 26 mg COD/L at the end of the monitoring period of 18 h,
indicating that its utilization as an internal substrate was partially The impact of erythromycin on the storage mechanism, while
blocked due to inhibitory action of tetracycline. The same trend was equally signicant, was different as compared with tetracycline
also observed for the two major components, PHB and 3H2MV; no in the sense that a much larger portion of the substrate ini-
appreciable generation could be measured for PHV (Fig. 2a). This tially available for microbial growth was diverted to storage: At
way, 50 mg COD/L of PHA was generated and incorporated into 50 mg/L dose, the PHA prole exhibited the same rapid increase
biomass. Based on storage yield, YSTO of 0.80 mg PHA COD/mg COD, to around 50 mg COD/L; however, the increase continued to
it could be estimated that 64 mg COD/L, corresponding to 13% of the reach 119 mg COD/L after 5 h of monitoring. At this level PHA
peptone mixture was diverted to intracellular storage rather direct was composed of 46 mg COD/L of PHB, 33 mg COD/L of 3H2MV
utilization for microbial growth. This level is almost three times and 40 mg COD/L of PHV (Fig. 3a). Calculations yielded an over-
higher than the one associated with the control reactor. Further- all PHA accumulation of around 80 mg COD/L, indicating that
more, at the end of the observation period, 25 mg COD/L of PHA, 100 mg COD/L of the peptone mixture was diverted to stor-
equivalent to 32 mg COD/L of peptone mixture initially available age under the effect of erythromycin. Similar to the observed
for biodegradation, was not utilized and remained entrapped in effect of tetracycline, erythromycin exerted a retardation effect of
the biomass. internal utilization of storage products; the slower consumption
Increase of the tetracycline dose to 200 mg/L reduced the total resulted in a residual PHA of 46 mg COD/L after 19 h of monitor-
PHA accumulation, which remained around 30 mg COD/L, only ing, corresponding to 58 mg/L of substrate initially converted to
slightly higher than the level in the control reactor. However, it fur- PHA.
ther decreased the internal utilization of the stored PHA down to When the erythromycin dose was increased to 200 mg/L, PHA
only 5 mg COD/L; at the end of the observation period, 25 mg COD/L accumulation basically remained the same as 83 mg COD/L within
of PHA remained unutilized and still entrapped within the biomass, the rst 6.7 h. However, the higher dose further decreased the rate
as in the case of the lower TET dose. The higher TET dose also of internal PHA utilization, which was limited to 21 mg COD/L of the
appeared to affect the metabolism of individual storage compo- generated PHAs (Fig. 3b). The slower internal utilization increased
nents: As plotted in Fig. 2b, 3H2MV was still the major component the residual PHA to 62 mg COD/L, i.e. 78 mg/L of initially available
around 30 mg COD/L and a slight generation of PHV was observed substrate converted to PHA remained incorporated into biomass at
at the expense of a lower PHB formation. the end of the experiment.
T. Katipoglu-Yazan et al. / Biochemical Engineering Journal 91 (2014) 283289 287

200 Table 2

180 OUR Model coefcients associated with storage assessed by model calibration [33].

160 O2=O2_growth+O2_storage+O2_growth on PHA Experimental runs kSTO (1/day) 

STO (1/day)
OUR (mg O2/L.h)

140 Control 1.30 0.33

TET 50 2.20 0.20
120 TET 200 4.00 0.08
100 Endogenous respiration level
ERY 50 3.16 0.22
ERY 200 3.16 0.16
40 all processes related to organic carbon removal. Detailed analysis
O2 of the modeling exercise is reported elsewhere [33]. Values of the
model coefcients conrm and provide numerical support for the
-1 1 3 5 7 9 11 13 15 17 observed impact of the antibiotics on the storage mechanism.
Oxygen uptake rate (OUR) proles were also obtained from
Time (h)
O2_endogenous respiration parallel batch experiments. It is now well known that the OUR
proles provide two signicant information related to the course
Fig. 4. Evaluation of OUR prole for microbial growth, substrate storage, and of biochemical reactions taking place as well as to the stoichio-
endogenous respiration.
metric balance between utilization of biodegradable substrate and
consumption of oxygen as the nal electron acceptor [40]: (i) bio-
3.3. Theoretical concepts chemical reactions related to substrate utilization are terminated
when the OUR level drops down to endogenous respiration level;
These results may better be evaluated when supported by mod- (ii) the area of the OUR curve above the endogenous respiration
eling and the respirometric data collected together with substrate level yields oxygen consumed as related to substrate actually uti-
storage tests. Respirometry is an extensively used method for the lized in Eq. (3):
experimental assessment of biodegradation characteristics of sub-
strates. Obtained oxygen uptake (OUR) prole is interpreted by O2 = So = (1 YH )CS (3)
modeling to estimate biodegradable substrate COD fractions and
where So is the concentration of oxygen consumed; CS , the con-
biodegradation kinetics. The area under OUR curve, above endoge-
centration of biodegradable substrate and YH , the yield coefcient.
nous decay level reects the amount of oxygen consumed for
When there is simultaneous storage, Eq. (3) has to be modied,
available substrate [39].
with the assumption that growth on internal substrate proceeds
In this study, mass balance on PHA storage and microbial
with the same yield coefcient:
growth were based on overall oxygen utilization, O2 , exclud-
ing endogenous respiration. This term refers to the net amount of O2 = SO = (1 YSTO YH )CST + (1 YH )CSG (4)
oxygen utilized for growth and storage processes after subtracting
oxygen requirement for endogenous respiration (cells mainte- where CST is substrate diverted to storage, CSG , substrate utilized
nance/respiration processes) as clearly indicated in Fig. 4. for microbial growth and YSTO , the storage yield. Eq. (4) basi-
Some studies considered similar material balances of biomass cally applies to the respirometric results of the control reactor.
precursors like NADH2, ATP, AcCoA, substrate, and nutrients as Since the tested antibiotics inhibited internal utilization of stored
basis for modeling. However, they focused on production of storage biopolymers, leaving a residual PHA concentration at the end of the
compounds mainly in pure cultures [42,43]. In this study however, observation period, related mass balance equation should account
evaluation of oxygen uptake rate enabled estimation of oxygen con- for the COD fractions, namely COD stored and internally utilized,
sumption for substrate storage and external substrate utilization in CSTU , substrate COD equivalent to the residual PHA (XSTR ) entrapped
mixed cultures. in the biomass, CSTR , and the fraction directly utilized for microbial
Modeling of substrate storage involves two biochemical reac- growth, CSG (Eq. (5)):
tions, namely the storage mechanism and internal use of storage O2 = SO = (1 YSTO YH )CSTU + (1 YSTO )CSTR + (1 YH )CSG (5)
products for microbial growth. Generation of intracellular storage
products, YSTO is generally dened in terms of the following surface- Observation of the OUR proles shows, as illustrated in Fig. 5, a
saturation type of a rate expression as given in Eq. (1) where SS is signicant decrease in the amount of oxygen consumed between
readily biodegradable substrate [20]: the control reactor and those operated with antibiotic dosing,
despite the fact that they were all started with the same substrate
= YSTO kSTO XH (1) concentration which should theoretically be available for microbial
dt SS + KSTO
uptake. For the control reactor, the mass balance dened by means
where kSTO is the maximum storage rate; KSTO half saturation coef- of Eq. (4) is satised by a YH of 0.63 mg cell COD/mg COD, a value
cient for storage, and XH active heterotrophic biomass; a typical quite in agreement with the default value of 0.64 mg cell COD/mg
Monod expression is usually adopted for the growth process on COD suggested in ASMs [20] and 0.60 mg cell COD/mg COD adopted
stored products Eq. (2): for model calibration of the biodegradation of the same peptone
mixture [26,33,44]. This balance also conrms, as expected, com-
STO XH (2) plete utilization of the available substrate as plotted in Fig. 5a, also
including the observed substrate storage.
where YH is the heterotrophic yield coefcient,  STO , the maximum The same balance is also calculated for every experiment con-
internal growth rate and KSTO , half saturation coefcient for inter- ducted with antibiotic dosing, based on Eq. (5); This evaluation
nal growth. Calibration of the kinetic expressions dened above, outlined in Table 3 gives a clear indication that the impact of antibi-
yielded the set of model coefcients outlined in Table 2, which otics disturbs the basic stoichiometry for substrate utilization and
provided best ts with the experimental data on substrate stor- identies a residual fraction of the initially available biodegradable
age, as illustrated in Figs. 13. It should be noted that the model substrate, CSR which remains unutilized, despite the fact the corre-
evaluation also covered oxygen uptake rate proles and included sponding OUR proles suggest completion of related biochemical
288 T. Katipoglu-Yazan et al. / Biochemical Engineering Journal 91 (2014) 283289

Table 3
Stoichiometry and mass balance between oxygen consumed, stored PHA and substrate utilized for microbial growth.

Experimental runs Antibiotic Stored PHA, XSTO Residual PHA, XSTR Oxygen consumed, Theoretical oxygen Substrate utilized Residual
concentration (mg COD/L) (mg COD/L) SO (mg COD/L) demand, SOT (mg/L) for growth, CSG (mg biodegradable
(mg/L) COD/L) substrate, CSR (mg

Control 22 185 185 463

TET-50 50 50 25 153 175 354 59
TET-200 200 30 25 58 182 132 336
ERY-50 50 81 46 180 182 397 7
ERY-200 200 83 62 150 178 327 75

In all runs initial substrate concentration, CS , was 506 mg.

reactions. In the traditional inhibition concept, the observed COD available for microbial growth, CSGP , may also be calculated the
difference is explained in terms of uncompetitive inhibition, where a same way from mass balance. Following this explanatory note, cal-
fraction of the available substrate remains bound with the inhibitor culations for the TET-200 experiment for example, would give that
[17]. It should be noted that substrate binding should now be con- a total amount of 38 mg/L of COD was diverted to storage and only
sidered as a simplistic explanation covering any possible metabolic 6 mg/L could be internally utilized (CSTU = 6 mg/L; CSTR = 32 mg/L);
impact impairing substrate utilization for growth. The results pre- as the initial COD level of the peptone mixture in the experiment
sented above also show that enhanced substrate storage is likely to was 506 mg/L, that leaves a CSGP of 468 mg/L that may be poten-
be one of the important factors inducing lower oxygen consump- tially utilized for microbial growth. Substituting this information
tion. in Eq. (5) yields three signicant results, also listed in Table 3: (1)
A clear explanation on the structure of Table 3 should rst be the theoretical oxygen consumption, SOT based on CSTU , CSTR and
provided, for properly interpreting the implications of related mass CSGP i.e. the expected oxygen consumption if all available substrate
balance: The rst part of the table reports observed experimental were to be utilized for growth, aside from storage; for TET-200, SOT
results, namely stored PHA, XSTO , the residual/unutilized fraction was calculated as 182 mg/L, much higher than the observed value
of the PHA at the end of the experiment, XSTR and the observed of only 58 mg/L, showing that all available substrate was indeed not
oxygen consumption SO calculated from the OUR proles in par- utilized. (2) Substrate actually utilized for growth, CSG , calculated
allel respirometric experiments. The rst two parameters enable by using the observed oxygen consumption in Eq. (5); as shown in
to calculate COD equivalents of stored PHA fractions, CSTU and CSTR the table, the CSG level corresponding to 58 mg/L of O2 consump-
using the previously adopted value of 0.80 mg PHA COD/mg COD tion was 132 mg/L for the TET-200 run. (3) Residual biodegradable
for the storage yield, YSTO ; the fraction of the initial COD potentially substrate, CSR , calculated from mass balance (CSGP CSG ), again cal-
culated as 336 mg/L for the TET-200 experiment.
In this context, the stoichiometric evaluation presented in
200 Table 3 indicated that antibiotic dosing signicantly reduced the
theoretical oxygen consumption corresponding to full utilization
180 OUR_Total_Data
of the substrate fraction available for microbial growth, paral-
lel to storage. The level of residual biodegradable substrate was
OUR (mg O2/L.h)

140 much higher for tetracycline compared to erythromycin. In fact,

120 for 50 mg/L dose of erythromycin, the slight change in the level
100 of oxygen consumption could be explained solely on the basis of
80 enhanced substrate storage within limits of analytical precision
60 associated with oxygen and COD measurements.
0 4. Conclusions
-1 1 3 5 7 9 11 13 15 17
Time (h) The major outcome of the experimental results outlined above
was (i) a noticeable increase in the magnitude of the biodegradable
(b) substrate diverted to intracellular storage, and (ii) inhibition and
retardation of the utilization of the stored biopolymers for internal
120 growth so that a residual PHA was always observed at the end of the
experiment. The impact of erythromycin in diverting a higher frac-
OUR (mg O2/L.h)

tion of the available substrate toward internal storage was much
80 more pronounced as compared to tetracycline.
Tetracycline and erythromycin signicantly affected substrate
60 storage. While the magnitude of the PHA incorporated into biomass
40 was increased, its internal utilization for microbial growth was
severely impaired. Results suggested that OUR is an essential tool
20 for understanding the impact of antibiotics. Moreover, kinetic and
stoichiometric evaluations on inhibitory impacts should not be
limited to microbial growth alone, but they should cover all bio-
-1 1 3 5 7 9 11 13 15 17
chemical processes including substrate storage, where relevant.
Time (h)
The observed decrease in the level of oxygen consumed under the
Fig. 5. OUR proles obtained (a) in the control reactor (b) in the reactor started with acute effect of antibiotics could not be solely attributed to enhanced
200 mg/L tetracycline dose [45]. storage except in the experiment started with 50 mg/L of
T. Katipoglu-Yazan et al. / Biochemical Engineering Journal 91 (2014) 283289 289

erythromycin suggesting an additional substrate binding mech- [21] O. Karahan-Gul, N. Artan, D. Orhon, M. Henze, M.C.M. van Loosdrecht, Experi-
anism by antibiotics. mental assessment of bacterial storage yield, J. Environ. Eng.-ASCE 128 (2002)
[22] C. Krishna, M.C.M. van Loosdrecht, Effect of temperature on storage polymers
Acknowledgments and settleability of activated sludge, Water Res. 33 (1999) 23742383.
[23] K. Kmmerer, Drugs in the environment: emission of drugs, diagnostic aids and
disinfectants into wastewater by hospitals in relation to other sources, review,
This study was conducted as a part of the project Evaluation of Chemosphere 45 (2001) 957969.
the Biodegradation Characteristics and Toxicity/Inhibition Effects [24] B. Halling-Srensen, G. Sengelv, J. Tjrnelund, Toxicity of tetracyclines
of Selected Antibiotics on Nitrication Systems and supported by and tetracycline degradation products to environmentally relevant bacteria,
including selected tetracycline-resistant bacteria, Arch. Environ. Contam. Tox-
joint doctoral degree program of Turkish Academy of Sciences and icol. 42 (2002) 263271.
Scientic Research Fund of Istanbul Technical University. [25] J.N. Louvet, H. Heluin, G. Attik, D. Dumas, O. Potier, M.N. Pons, Assessment of
erythromycin toxicity on activated sludge via batch experiments and micro-
scopic techniques (epiuorescence and CLSM), Process Biochem. 45 (2010)
References 17871794.
[26] I. Pala-Ozkok, E.U. Cokgor, Z.P. Cakar, D. Orhon, Acute impact of erythromy-
[1] M.C.M. van Loosdrecht, M.A. Pot, J.J. Heijnen, Importance of bacterial storage cin on substrate utilization by activated sludge: effect of sludge age, J. Chem.
polymers in bioprocesses, Water Sci. Technol. 35 (1997) 4147. Technol. Biotechnol. (2013),
[2] G.T. Daigger, C.P.L. Grady Jr., The dynamic of microbial growth on soluble sub- [27] ISO 8192, Water Quality-Test for Inhibition of Oxygen Consumption by Acti-
strates: a unifying theory, Water Res. 16 (1982) 368382, Review paper. vated Sludge, International Standards Organization, Switzerland, 2007.
[3] C.B. Youssef, A. Zepeda, J. Gomez, Modelling the combined inhibition and time- [28] G. Insel, O. Karahan, S. Ozdemir, I. Pala, T. Katipoglu, E. Ubay-Cokgor, D. Orhon,
dependent inactivation effects of toluene on ammonia and nitrite oxidation Unied basis for the respirometric evaluation of inhibition for activated sludge,
using a nitrifying sludge, Biochem. Eng. J. 80 (2013) 3744. J. Environ. Sci. Health A 41 (2006) 17631780.
[4] A. Chiavola, G. Farabegoli, F. Antonetti, Biological treatment of olive mill [29] E.U. Cokgor, G. Insel, T. Katipoglu, D. Orhon, Biodegradation kinetics of peptone
wastewater in a sequencing batch reactor, Biochem. Eng. J. 85 (2014) 7178. and 2,6-dihydroxybenzoic acid by acclimated dual microbial culture, Bioresour.
[5] J.J. Beun, F. Paletta, M.C.M. van Loosdrecht, J.J. Heijnen, Stoichiometry and kinet- Technol. 102 (2011) 567575.
ics of poly--hydroxybutyrate metabolism in aerobic, slow growing, activated [30] D. Orhon, S. Sozen, E. Ubay, Assessment of nitrication-denitrication potential
sludge cultures, Biotechnol. Bioeng. 67 (2000) 379389. of Istanbul domestic wastewaters, Water Sci. Technol. 30 (1994) 2130.
[6] K. Dircks, J.J. Beun, M. van Loosdrecht, J.J. Heijnen, M. Henze, Glycogen [31] D.G. Larsson, C. de Pedro, N. Paxeus, Efuent from drug manufactures con-
metabolism in aerobic mixed cultures, Biotechnol. Bioeng. 73 (2001) 8594. tains extremely high levels of pharmaceuticals, J. Hazard. Mater. 148 (2007)
[7] F. Carta, J.J. Beun, M.C.M. Van Loosdrecht, J.J. Heijnen, Simultaneous storage and 751755.
degradation of PHB and glycogen in activated sludge cultures, Water Res. 35 [32] S. Chelliapan, T. Wilby, P. Sallis, Treatment of pharmaceutical wastewater con-
(2001) 26932701. taining tylosin in an anaerobicaerobic reactor system, Water Pract. Technol.
[8] A.S. Ciggin, S. Rossetti, M. Majone, D. Orhon, Effect of feeding and sludge age 5 (2010) 116,
on acclimated bacterial community and fate of slowly biodegradable substrate, [33] T. Katipoglu-Yazan, I. Pala-Ozkok, E.U. Cokgor, D. Orhon, Acute impact of eryth-
Bioresour. Technol. 102 (2011) 77947801. romycin and tetracycline on the kinetics of nitrication and organic carbon
[9] A.S. Ciggin, G. Insel, M. Majone, D. Orhon, Respirometric evaluation and mod- removal in mixed microbial culture, Bioresour. Technol. 144 (2013) 410419.
elling of acetate utilization in sequencing batch reactor under pulse and [34] A.P.H.A., Standard Methods for the Examination of Water and Wastewater, 21st
continuous feeding, Bioresour. Technol. 107 (2012) 6169. ed., 2005, Washington, DC.
[10] A.S. Ciggin, D. Orhon, D. Capitani, A. Miccheli, C. Puccetti, M. Majone, Aerobic [35] ISO 6060, Water Quality-Determination of the Chemical Oxygen Demand,
metabolism of mixed carbon sources in sequencing batch reactor under pulse International Standards Organization, Switzerland, 1986.
and continuous feeding, Bioresour. Technol. 129 (2013) 118126. [36] G. Insel, D. Orhon, P.A. Vanrolleghem, Identication and modelling of aerobic
[11] D. Dionisi, M. Majone, V. Papa, M. Beccari, Biodegradable polymers from organic hydrolysis mechanism-application of optimal experimental design, J. Chem.
acids by using activated sludge enriched by aerobic periodic feeding, Bio- Technol. Biotechnol. 78 (2003) 437445.
technol. Bioeng. 85 (2004) 569579. [37] P. Reichert, J. Ruchti, W. Simon, AQUASIM 2.0, in: Swiss Federal Institute
[12] T. Katipoglu, E.U. Cokgor, G. Insel, D. Orhon, Biodegradation kinetics of 2,6- for Environmental Science and Technology (EAWAG), 1998, Duebendorf,
dihydroxybenzoic acid and peptone mixture by acclimated microbial culture Switzerland.
at low sludge age, J. Environ. Sci. Health. A 45 (2010) 18851891. [38] R. Brun, P. Reichert, R.K. Knsch, Practical identiability analysis of large envi-
[13] I. Pala-Ozkok, D. Orhon, Chronic effect of erythromycin on substrate biodegra- ronmental simulation models, Water Resour. Res. 37 (2001) 10151030.
dation kinetics of activated sludge, Biochem. Eng. J. 81 (2013) 2939. [39] D. Orhon, F. Germirli Babuna, O. Karahan, Industrial Wastewater Treatment by
[14] I. Pala-Ozkok, G. Kor-Bicakci, A. Ural, T. Katipoglu-Yazan, N. Yagc, E. Ubay- Activated Sludge, IWA Publishing, London, 2009.
Cokgor, D. Orhon, Acute impact of sulfamethoxazole on the utilization of simple [40] I. Pala-Ozkok, A. Rehman, N. Yagci, E. Ubay-Cokgor, D. Jonas, D. Orhon, Charac-
and complex substrates by fast growing microbial culture, J. Chem. Technol. teristics of mixed microbial culture at different sludge ages: effect on variable
Biotechnol. (2013), kinetics for substrate utilization, Bioresour. Technol. 126 (2012) 274282.
[15] D. Orhon, E.U. Cokgor, G. Insel, O. Karahan, T. Katipoglu, Validity of Monod kinet- [41] G. Insel, G. Celikyilmaz, E. Ucisik Akkaya, K. Yesiladali, P. Cakar, C. Tamer-
ics at different sludge ages peptone biodegradation under aerobic conditions, ler, D. Orhon, Respirometric evaluation and modeling of glucose utilization
Bioresour. Technol. 100 (2009) 56785686. by Escherichia coli under aerobic and mesophilic cultivation conditions, Bio-
[16] A. Mulchandani, J.H.T. Luong, Microbial inhibition kinetics revisited, Enzyme technol. Bioeng. 96 (2007) 94105.
Microb. Technol. 11 (1989) 6673. [42] C. Chatzidoukas, G. Penloglou, C. Kiparissides, Development of a structured
[17] I. Pala-Ozkok, T. Katipoglu-Yazan, E.U. Cokgor, G. Insel, I. Talinli, D. Orhon, dynamic model for the production of polyhydroxybutyrate (PHB) in Azohy-
Respirometric assessment of substrate binding by antibiotics in peptone dromonas lata cultures, Biochem. Eng. J. 71 (2013) 7280.
biodegradation, J. Environ. Sci. Health A 43 (2011) 15881597. [43] J.M.L. Dias, L.S. Seram, P.C. Lemos, M.A.M. Reis, R. Oliveira, Mathematical
[18] H. Spanjers, P. Vanrolleghem, Respirometry as a tool for rapid characterization modelling of a mixed culture cultivation process for the production of poly-
of wastewater and activated sludge, Water Sci. Technol. 31 (1995) 105114. hydroxybutyrate, Biotechnol. Bioeng. 92 (2005) 209222.
[19] F.G. Babuna, D. Orhon, E.U. Cokgor, G. Insel, B. Yaprakl, Modelling of activated [44] T. Katipoglu-Yazan, E.U. Cokgor, G. Insel, D. Orhon, Is ammonication the rate
sludge for textile wastewaters, Water Sci. Technol. 38 (1998) 917. limiting step for nitrication kinetics? Bioresour. Technol. 114 (2012) 117125.
[20] M. Henze, W. Gujer, T. Mino, M. van Loosdrecht, Activated sludge models ASM1, [45] T. Katipoglu-Yazan, Evaluation of the Biodegradation Characteristics and Tox-
ASM2, ASM2d and ASM3, in: Scientic and Technical Report 9, IWA Publishing, icity/Inhibition Effects of Selected Antibiotics on Nitrication Systems (PhD
London, 2000. thesis), Istanbul Technical University, Turkey, 2013.