Biodegradation of Phenolic Mixtures
Subject Area 5.1: Microbial Waste Disposal
Research Article
Biodegradation of Phenolic Mixtures in a Sequencing Batch Reactor
A kinetic study
Maria Concetta Tomei1* and Maria Cristina Annesini2
1 Water Research Institute, C.N.R., Via Reno 1, 00198 Rome, Italy
2 Chemical Engineering Department, La Sapienza University of Rome, Via Eudossiana 18, 00184 Rome, Italy
* Corresponding author (tomei@irsa.cnr.it)
DOI: http://dx.doi.org/10.1065/espr2007.12.470
Please cite this paper as: Tomei MC, Annesini MC (2008):
Biodegradation of Phenolic Mixtures in a Sequencing Batch
Reactor. A kinetic study. Env Sci Pollut Res 15 (3) 188–195
Abstract
Goal, Scope and Background. In this study, attention was fo-
cused on substituted phenols because of their widespread pres-
ence in industrial effluents originating from many different
sources: they are major constituents of wastewater from coal
conversion processes, coke ovens, petroleum refineries and pet-
rochemical industries, resin and fibreglass manufacturing and
herbicide production. Moreover, for their characteristics of toxic-
ity to humans and aquatic life (1 mgl–1 is enough to detect the
effects), they are included in the USEPA list of priority pollutants.
Toxicity is higher in substituted phenols and is dependent on the
nature and numbers of substituent groups. Objective of the present
paper is to give a contribution to the modelling of phenolic mix-
ture biodegradation by kinetic studies in which the different com-
pounds are followed separately: this can be easily attained with
an experimental apparatus such as the Sequencing Batch Reac-
tor (SBR). Two substituted phenols, 4-nitrophenol (4NP) and
3,4-dimethylphenol (3,4DMP), were utilized as substrates and
their degradation kinetics were investigated to evaluate the pro-
cess parameters both in single compound and in mixture tests.
Methods. Single compound and mixture kinetic tests have been
carried out during the reaction phase of the working cycle of
the SBR reactor. The single substrates and their mixture were
utilized as sole carbon and energy sources. Moreover, in order
to verify data reproducibility, all kinetic tests have been car-
ried out in at least two replicates under the same operating
conditions.
Results and Discussion. Kinetic data showed the presence of sub-
strate inhibition, to model this experimental evidence the Haldane
equation, that is usually employed for substrate inhibited kinet-
ics, was rearranged in a different form with parameters which
have a precise meaning in relation to the process kinetics and, at
the same time, make the integration procedure easier. The deriva-
tion of the equation is shown in an Appendix at the end of the
paper. Kinetic parameters obtained are suitable for application. It
was observed that the 4-nitrophenol removal rate in single com-
pound tests is significantly higher than the 3,4-dimethylphenol
removal rate in the whole range of investigated concentrations
(up to 80 mg COD l–1). A faster 4-nitrophenol biodegradation
188
Subject Area 5.
was also observed in mixture tests. Moreover, it is worth noting
that the two compounds were simultaneously degraded and no
diauxic growth was observed. The comparison between single
compound and mixture degradation kinetics showed that the 4-
nitrophenol degradation rate was comparable in the two cases
while a significantly beneficial effect (by increase by about 80%
of the maximum removal rate) was detected for 3,4-dimethyl-
phenol degradation in the mixture.
Conclusions. Results of this study showed that the biodegrada-
tion kinetics of substituted phenols in mixture can be signifi-
cantly different from that observed in single compound tests: in
fact, the presence of a faster degradable compound (the 4NP)
seems to exert a positive effect on the removal of a slower de-
gradable compound (the 3,4DMP). The higher removal rate
detected for 4NP, both in single compound and mixture tests,
confirmed the key role of the biomass acclimatization in deter-
mining the biodegradation kinetics of xenobiotic compounds.
The experimental approach and the original method applied
for data analysis are of general validity and can be extended to
the investigation of different classes of compounds.
Recommendations and Perspectives. A relevant aspect related
to the process applicability is the demonstrated possibility of
easily adapting an enriched culture grown on a specific xenobiotic
(in our case the 4NP) for the removal of similar single com-
pounds or in mixtures. When biological process are considered
for xenobiotic removal, this suggests a possible strategy of de-
veloping enriched cultures on target compounds that can be ef-
ficiently utilized on more complex matrices with reduced start
up and acclimatization periods.
Keywords: 3,4-dimethylphenol; 4-nitrophenol; biodegradation
kinetics; Haldane equation; phenolic mixtures; SBR (sequenc-
ing batch reactor)
Introduction
In the scientific literature referring to wastewater contain-
ing phenol compounds, the word 'mixture' is utilized for
defining both 'real' mixtures of phenol and its derivatives,
and aqueous matrices containing only phenol which is con-
sidered to be a target compound (Veeresh et al. 2005). This
last approach is frequently adopted in the field of xenobiotic
biodegradation, where there are intense difficulties in find-
ing the formulation of the synthetic mixtures, which are
Env Sci Pollut Res 15 (3) 188 – 195 (2008)
© Springer-Verlag 2008
Subject Area 5.1
representative of real wastewater, and due to the complex-
ityof the analysis required in following the different com-
pounds during the biodegradation process. The single tar-
get compound approach can be utilized to have an approxi-
mate estimation of the process kinetics, but no information
is obtainable on the mutual effects of different compounds
for the kinetic mechanisms involved in the global degrada-
tion process of the mixture. Indeed, this kind of data is nec-
essary in order to set up reliable process models for predict-
ing mixture biodegradation.
In this study, attention was focused on phenols because of
their widespread presence in industrial effluents originated
from many different sources: they are major constituents of
wastewater from coal conversion processes, coke ovens,
petroleum refineries and petrochemical industries, resin and
fibreglass manufacturing and herbicide production. Concen-
tration values detected in these effluents are quite high rang-
ing from 10 to 17 103 mg l–1 while the related COD fraction
varies from 40 to 80% of the total COD (Suidan et al. 1983,
Tsai & Folsom 1982, Luthy et al. 1983). This 'rough data'
is sufficient to give an idea of the enormous impact of this
class of compounds on the water pollution. Moreover, sub-
stituted phenols are quite stable in the environment and
hardly biodegradable, but can penetrate easily through the
soil into surface and groundwater, and diffuse in the air: the
result is their progressive accumulation in water, air and soil
(Kahru et al. 2002, Morville et al. 2006). Due to their char-
acteristics of toxicity for humans and aquatic life (1mg l–1 is
enough to detect the effects), they are included in the USEPA
list of priority pollutants. Toxicity is higher in substituted
phenols and is depending on the nature and numbers of sub-
stituent groups (Walker 1989).
Removal of phenols can be attained by physical-chemical
(i.e. solvent extraction, activated carbon adsorption, Ad-
vanced Oxidation Processes (AOPs) (Ugurlu & Kula 2007))
and biological processes. In the last years more attention was
paid to the biological treatment because, unlike the most com-
mon physical-chemical processes, that is solvent extraction
and adsorption, which 'concentrate' the compound in a solid
or liquid phase, a complete mineralization is potentially achiev-
able with the biological processes. Moreover, biological pro-
cesses are less expensive if compared to AOPs that can also
give the complete mineralization of the compound.
Due to their relevant presence in industrial wastewater, the
biological removal of phenolic compounds was extensively
investigated in the last decades utilizing both aerobic and
anaerobic processes. Pure culture studies on their aerobic
biodegradability were performed with microorganisms iso-
lated from activated sludge plants (Buitron & Gonzalez 1996,
Buitron et al. 1998, Viggor et al. 2002) and a number of
bench and pilot scale investigations were carried out with
aerobic biofim reactors (Ray et al. 1999, Xing et al. 1999,
Bhatti et al. 2002). Since 1990, anaerobic process applica-
tion received considerable attention with consequent pro-
duction of bench scale studies and also full scale plants mainly
realized with the UASB (Upflow Anaerobic Sludge Blanket)
configuration (Razo-Flores et al 2003, Veeresh et al. 2005).
Env Sci Pollut Res 15 (3) 2008
Biodegradation of Phenolic Mixtures
A promising alternative technology is the employment of
Sequential Batch Reactors (SBRs) that, in the last years, were
proposed and successfully experimented for xenobiotic bio-
degradation. In fact, the high variability of the operating
conditions in these systems could induce in the biomass en-
zymes the metabolic pathways required for biodegrading
these compounds (Ellis et al. 1986). Authors of the present
paper carried out an experimental programme on 4-nitro-
phenol (4NP) biodegradation realized in a lab-scale SBR
reactor operated with aerobic–anoxic cycles. Results were
quite satisfactory: the reactor was operated with suspended
biomass, 4NP was the sole carbon and energy source and
kinetics of both aerobic and anoxic reactions are of the same
order of magnitude of that commonly adopted in full scale
wastewater treatment plants (Tomei et al. 2004, Tomei &
Annesini 2005).
Objective of the present paper is to give a contribution to
the modeling of phenolic mixture biodegradation by kinetic
studies in which the different compounds are separately fol-
lowed: this can easily be attained with an experimental ap-
paratus such as the SBR. Two substituted phenols (4-
nitrophenol and 3,4-dimethylphenol (3,4DMP)) were utilized
as substrates and their degradation kinetics were investigated
to evaluate the process parameters both in single compound
and mixture tests.
1 Material and Methods
1.1 Chemicals
4-Nitrophenol and 3,4-dimethylphenol were in granular
form (purity >98%) and supplied by Fluka (Italy) and Merck
(Germany), respectively. All other chemicals were commer-
cial grade and supplied by Carlo Erba (Italy).
1.2 Bacterial culture
A mixed culture previously acclimatized to 4NP as the sole
carbon source was used in the experiments. The original
biomass inoculum was a mixed liquor sample from a full
scale urban wastewater treatment plant; the details of the
acclimatization procedure are reported in previous papers
(Tomei et al. 2003, Tomei et al. 2004).
In order to progressively acclimatise the biomass to the
3,4DMP, the culture was fed with solutions of the compound
in the range of concentration 30–60 mg l–1, then, when the
complete removal was attained, two aliquots of biomass
degrading 4NP and 3,4DMP were mixed and utilized as in-
oculum in the SBR reactor employed for the mixture bio-
degradation.
To ensure the required contribution of nutrients and micro-
elements, in all cases the feed consisted of a pure compound
solution or mixture with the addition of the mineral me-
dium MSV (Williams et al. 1989). The composition of the
MSV solution, prepared in deionized water, was as follows,
in mg l –1 units: (NH 4 )SO 4 500, MgSO 4 ·7H 2 O 100,
CaCl2·2H2O 50, K2HPO4 110, KH2PO4 85, FeCl3·6H2O 2,
NaEDTA 2. The C:N:P ratio in the influent was 100:5:1
with respect to the 4NP and 3,4DMP carbon.
189
Biodegradation of Phenolic Mixtures Subject Area 5.1

1.3 Sequencing batch reactors Table 1: Operating parameters of the SBR reactor

SBR reactors are glass vessels of 5 litres volume with a Parameter Value Unit
thermostated water jacket to control the operating tempera- Hydraulic retention time 0.67 d
ture. Dissolved oxygen and pH are on-line monitored and Sludge retention time (SRT) 16 d
controlled by WTW instruments (CellOx 325 and Ino Lab Biomass concentration 400–800 mg l–1 VSS
pH level 2, respectively). Feeding, sludge wasting, effluent Influent 4NP concentration 40–60 b mg l–1
discharge and acid/base addition for pH control were per- Influent 3,4DMP concentration 30–60 a 20–40 b mg l–1
formed by peristaltic pumps (Cellai, Perinox SF3) through Temperature 20±0.5 °C
openings located in the reactor cover. Mixing was ensured Dissolved Oxygen 3–4 mg l–1
by a magnetic stirrer. Air was supplied by variable flow com- pH 7–8
pressors through a glass diffuser. Working Volume 4.2 l
Time operational sequence and control strategies have been Residual Volume 2.1 I
automatically operated by a personal computer interfaced a
in single compound tests
b
to the reactor. Specialised software has been developed un- in mixture
der Labview-Windows 3.1 environment to manage time
definition of the working cycle phases, dissolved oxygen adopted acclimatization procedure and the time intervals be-
(DO) and pH monitoring and control by on-off strategies tween one test and the subsequent are the same (≈3 Sludge
and driving of stirrer, compressors and pumps. Retention Time) for single compound and mixture tests. De-
tails of the operating procedures for kinetic tests carried out
1.4 Analytical methods with single compounds and mixtures are given below.
Volatile and Total Suspended Solids concentrations have been Single compound. Kinetic tests were carried out during the
determined according to Standard Methods (APHA 1998). reaction phase of the working cycle at 3,4DMP feed con-
centrations (CDMP) of 30 and 60 mg l–1. 3,4DMP concentra-
COD Cell Tests (MERCK) based on potassium dichromate tion was measured at time intervals varying from 5 to 10
oxidation and spectrophotometric determination (Spectro- min, while COD and VSS concentrations have been followed
quant Nova30) have been employed. at longer time intervals (30-40 min). Furthermore, DO was
4-Nitrophenol analysis was performed on samples filtred on continuously measured during the test. Table 2 shows the
syringe nylon membrane filtres (0.45 µm pore-size) acidified test plan for 3,4DMP kinetic tests.
in order to stop the 4NP biodegradation by the residual biom- Moreover, in order to verify data reproducibility, all kinetic
ass not retained in the filtre. They were then analysed by mea- tests have been carried out in at least two replicates under
suring the UV absorbance at 320 nm using a spectrophotom- the same operating conditions.
eter Varian (model Cary 1). Interference of other compounds
in the aqueous matrix was excluded by preliminary tests. Mixture. In the second series of kinetic tests the reactor was
fed with a mixture of 4NP and 3,4DMP. The two compounds
3,4-dimethylphenol concentration was measured by a gas- as well as DO, COD and VSS have been measured during
chromatograph HP 5890A equipped with a capillary col- the feed and reaction phases at the same time intervals of
umn Mega SI52 and a FID detector. The splitless injector the previous series. Also in mixture tests, data reproducibil-
was operated with a ratio1:60, the transport gas was He- ity was verified by performing two replicates of each test
lium, head column pressure was 15 psi, temperature 225°C under the same operating conditions.
and a volume of 4ml was employed for the measure.
Table 3 shows the test plan for the mixture biodegradation
1.5 Reactor operation
experiments.
A typical SBR work cycle lasted 8 hours distributed as fol- Control experiments. Abiotic control experiments have been
lows: FILL 30 min, REACTION 340 min, WASTAGE 3 min, carried out in order to exclude the effects of stripping phe-
SETTLE 92 min, DRAW 15 min. Fill phase was mixed and nomena on the removal. Tests were performed in the same
aerated. Table 1 shows the main operating parameters of the mixing and aeration conditions of kinetic tests without the
reactor. Two series of kinetic tests were carried out under the biomass and with the same substrate concentrations uti-
operating conditions indicated in Table 1 by varying the in- lized in the kinetic tests. Substrates were detected at regu-
fluent concentration as described in detail in the following. lar time intervals for a total time equal to the maximum
duration of a kinetic test. Control experiments excluded
After any change of operating conditions, the SBR was op-
erated for the appropriate time (at least two weeks) required Table 2: Kinetic test plan for 3,4DMP
to obtain stable reactor performance before carrying out the Test Influent 3,4DMP Biomass concentration X a
kinetic tests. concentration (mg l–1 VSS)
(mg l–1)
1.6 Kinetic tests S1.a 30 810
S1.b 30 707
The main goal of the experiments was to investigate the
S2.a 60 786
mutual effect of the two compounds on the process kinetics so
S2.b 60 626
the experimental programme was designed to minimize the a
mean value during the run
effects of the acclimatization on the biomass performance. The

190 Env Sci Pollut Res 15 (3) 2008

Subject Area 5.1 Biodegradation of Phenolic Mixtures

Table 3: Kinetic test plan the mixture biodegradation experiments

Test Influent 4NP concentration Influent 3,4DMP concentration Biomass concentration X a
(mg l–1) (mg l–1) (mg l–1 VSS)
M1.a 40 20 483
M1.b 40 20 463
M2.a 50 30 340
M2.b 50 30 330
M3.a 60 40 345
M3.b 60 40 340
a
mean value during the run

abiotic removal due to stripping phenomena for both single
compounds and mixtures, no variation in concentration
were detected for the investigated times corresponding to
the duration of kinetic tests.
Moreover, in order to verify and quantify the presence of
adsorption phenomena, control experiments were carried
out with biomass deactivated with HgCl under the same
operating conditions and substrate/biomass ratios utilized
in the kinetic tests. Also in this case, only minor changes
(≤5%) in substrate concentration (not relevant in terms of
removal) were observed.
Finally, to verify the complete biodegradation of the com-
pounds, at the end of the kinetic tests (both pure compound
and mixture) the COD residual concentration was regularly
measured in the effluent. Quite low residual COD values,
always in the range of 10–30 mg l–1, were detected that are
plausibly attributable to biomass lysis products.
Oxygen Uptake Rate (OUR). The amount of oxygen trans-
ferred from the reactor headspace was evaluated with a con-
trol experiment performed with tap water, previously de-
aerated by fluxing a nitrogen stream, in the SBR reactor
filled up to the work volume. The oxygen concentration in
the liquid vs. time has been continuously detected keeping
the air compressor off and the stirrer on (as in the kinetic
experiments) and with atmospheric air in the head space.
Results of the control test showed that the oxygen trans-
ferred from the reactor headspace is negligible if compared
to the oxygen supplied with the compressor.
Oxygen Uptake Rate (OUR) was obtained from continu-
ously measured and recorded DO data when, as a conse-
quence of the on-off control strategy, the oxygen is not sup-
plied to the system:
concentration remains practically constant throughout each
run; therefore, in order to have a direct evaluation of the
3,4DMP removal kinetics, the concentration data are reported
vs. the product X·t: in this way the slope of the curve in each
point gives the specific removal rate at the corresponding con-
centration. Fig. 1 shows an overview of the results obtained
for tests S1 and S2 (in the figure the time scale is arbitrarily
shifted in order to overlap the initial substrate concentration
in the different runs so allowing an immediate comparison of
the concentration profiles that is of the removal rates).
The observed overlap of the experimental concentration
profiles demonstrates that the process kinetics does not sig-
nificantly differ in the two tests so all the data were analysed
together. Furthermore, data analysis of the two runs shows
a substrate inhibition effect in the investigated range of con-
centration. To model this inhibitory effect, the experimental
data were correlated by the Haldane equation that is usu-
ally employed for substrate inhibited kinetics:
C C
rS = v = k * ⋅X
C
2
C2
C + Ks + C + Ks + (2)
KI KI
where
rS is the substrate consumption rate,
X and C are the biomass and 3,4DMP concentration, re-
spectively.

OUR = (DO2–DO1)/(t2–t1) (1)

where OUR is the oxygen consumption rate [M L–3 T–1] and

DO1 and DO2 are the dissolved oxygen concentrations at
time t1 and t2, respectively. According to the control test
results, the oxygen transferred from the headspace was not
considered in Eq. (1).

2 Results and Discussion

2.1 3,4DMP removal
Objectives of this first experimental part were to obtain a cul- Fig. 1: Experimental and calculated 3,4DMP concentration profiles in single
ture acclimatized to 3,4DMP and to evaluate the removal ki- compound tests S1 and S2. The arrow indicates the starting point of the
netics of this compound. It was observed that the biomass experiments S1a/b

Env Sci Pollut Res 15 (3) 2008 191

Biodegradation of Phenolic Mixtures
In this model, three fitting parameters, the rate constant k*
[MDMPMVSS–1T–1] and the saturation and inhibition constants,
Ks and KI [ML–3], are included. In the classical form of the
Haldane equation (Eq. (2)), it is worth noting that the param-
eters k*, Ks and KI do not have a precise meaning in terms of
process kinetics, in fact no direct information is given on the
maximum consumption rate and on the critical substrate con-
centration. In order to have an equation with more represen-
tative parameters in relation to the process kinetics, the
Haldane equation was rearranged in a different form:
C/C *
rS = k max ⋅ X(2 + β)
1 + β(C/C *) + (C/C *)
2 (3)
where the parameters are redefined as follows:
C* = K s ⋅ K I is the substrate concentration where the
maximum removal rate occurs,
kmax is the maximum specific removal rate observed at C=C*
β = K I K s is a parameter that accounts for the extent of
the inhibitory effects (the smaller β the larger the removal
rate reduction at high substrate concentration).
A limit curve corresponding to the maximum inhibitory ef-
fect predicted by this model is obtained for β=0. The effect
of β and C* on the kinetics is represented in Fig. 2 where the
ratio rS/(X·kmax) is reported vs. the substrate concentration
S: the two plots (a) and (b) display the effects of the param-
eters C* and β, respectively.
In our opinion, having a direct indication from the kinetic
parameters of the possible maximum rate or of the critical
substrate concentration value, is quite useful for evaluating
the process applicability. Furthermore, in our experience,
working with β and C* in the data fitting is easier and reduces
some numerical problems that are often found with Ks and
KI in applying the classical form of Haldane equation.
The procedure applied to derive Eq. (3) from Eq. (2) is re-
ported in the Appendix at the end of the paper.
Fig. 2: Effect of β and C* on substrate removal kinetics: a) fixed C*=20, increasing β; b) fixed β=0, increasing C*
192
Subject Area 5.1
In the data analysis of 3,4DMP removal, Eq. (3) was uti-
lized with the further assumption that, in agreement with
the experimental results, the biomass concentration remains
practically constant throughout each run. The values of kmax,
β and C* were evaluated by applying the standard, least-
square, best-fitting procedure. It results, kmax=0.0296 mg
COD mgVSS–h–1, C*=14.0 mgCOD l–1 and β=0. The stan-
dard errors (S.E.) of the estimated parameters are 0.0003
for kmax, 0.2 for C* and 0.006 for β. In Fig. 1, the curve
obtained with the Haldane model is compared with the ex-
perimental data: the figure clearly shows that the agreement
is quite good, as it is also confirmed by the high value of the
correlation coefficient (0.99).
The oxygen uptake rate (OUR) data continuously evaluated
during the kinetic tests were utilized to calculate the growth
rate yield coefficient according to the procedure reported in
Tomei et al. (2004). A Y value of 0.59±0.15 was estimated
on COD base. The yield coefficient is in the range of values
reported in literature for heterotrophic bacteria degrading
more easily biodegradable organics i.e. the carbonaceous
substrate present in sewage (Kappeler and Gujer 1992).
In order to evaluate the feasibility of the process in real sys-
tems an useful parameter is the maximum growth rate
µmax=kmax·Y, for the 3,4DMP removal process µmax= 0.28 d–1
was obtained. This value is of the same order of magnitude
of that assumed for nitrifying bacteria (0.36–0.5 d–1) oper-
ating in nitrogen removal full scale plants (Henze et al. 1997).
2.2 Comparison between 4NP and 3,4DMP removal
Kinetics of 4NP has been extensively investigated with the
same apparatus by the authors of the present paper and the
results published in previous papers (Tomei et al. 2003, Tomei
et al. 2004, Tomei & Annesini 2005). The experimental data
clearly showed that 4NP removal kinetics can be described
by the Haldane model so that the kinetic parameters
k*=0.688 mgCOD mgVSS–1 h–1, Ks=83.0 mgCOD l–1 and
KI = 22.65 mgCOD l–1 (corresponding to kmax=0.142 mg
COD mgVSS–1 h–1, C*=43.36 mgCOD l–1 and β≅0.52) were
obtained (Tomei & Annesini 2005).
Env Sci Pollut Res 15 (3) 2008
Subject Area 5.1 Biodegradation of Phenolic Mixtures

0.16 40
M3a
0.14 M3b
M2a
M2b
0.12
rsp (mgCOD mgVSS h )

30
-1 -1

M1a
M1b

4NP (mgCOD l-1)

0.10 4NP pure cal
4NP mix cal
0.08 20

0.06

3,4 DMP
0.04 4NP 10

0.02

0 20 40 60 80 100 0 200 400 600 800 1000 1200

-1 . -1 .
mgCOD l X t (mg l VSS) h

Fig. 3: Specific reaction rate vs. substrate concentration. Simulation curves Fig. 5: 4NP concentration profiles: experimental data in mixture tests,
are obtained with the best fitting parameters of the experimental data for simulated curves for pure compound and mixture
3,4DMP (this study) and 4NP (Tomei & Annesini 2005)

A comparison of the Haldane parameters for the biodegra-
dation process of the two considered substrates (3,4DMP
and 4NP) shows a faster and less inhibited 4NP removal rate
(higher kmax and β values); furthermore, the higher C* value
found for 4NP indicates that the substrate inhibitory effect on
4NP kinetics is detected at higher concentration with respect
to 3,4DMP. Fig. 3 shows the specific substrate removal rate
(rsp = rS/X) vs. substrate concentration for 3,4DMP and 4NP.
Curves are obtained by simulation performed with the best
fitting parameters of the experimental data.
The higher 4NP removal rate, in the whole range of investi-
gated concentrations, can be reasonably attributed to the
long acclimatization period that produced an enriched cul-
ture highly specialized for 4NP biodegradation. 3,4DMP is
a similar compound in terms of structure formula so that
the biomass is able to degrade it, even if at lower kinetics.
2.3 Mixture biodegradation
Fig. 4 shows the typical pattern for 4NP and 3,4DMP con-
centration profiles observed for mixture kinetic tests. A first
data analysis shows a faster biodegradation of 4NP, thus
confirming the results of single compound tests. It is also to
note that the two compounds are simultaneously degraded
and no diauxic growth was observed. Moreover, the observed
capability of the biomass of degrading the 3,4DMP with
kinetics high enough to be proposed for application can rea-
sonably exclude cometabolic mechanisms in the mixture
biodegradation.
Data analysis for mixture kinetic tests was performed sepa-
rately for 4NP and 3,4DMP in order to compare the behav-
iour of each compound in the mixture with that observed in
single compound tests (when it was the sole carbon and en-
ergy source) and to evaluate the mutual effect of the two
substrates in mixture biodegradation.
In order to have an overview of the experimental results, all
the experimental concentration profiles for each compound
were reported (the plotting procedure was the same as in
Fig. 1) vs. (X·t): in this way, it is possible to have the com-
parison in terms of specific kinetics by taking into account
the biomass concentration effect.
Fig. 4 Figs. 5 and 6 show the
4NP and 3,4DMP data respectively.

50 50
M1a
M1b
M2a
40 40
3,4DMP exp M2b
4NP exp M3a
3,4DMP (mgCOD l-1)

3,4DMP calc M3b

4NP calc 3,4 DMP pure cal
30
mgCOD l-1

30 3,4 DMP mix cal

20 20

10 10

0 50 100 150 200 0 200 400 600 800 1000 1200 1400 1600 1800

time (min) X.t (mg l-1VSS).h

Fig. 4: The typical pattern for 4NP and 3,4DMP concentration profiles Fig. 6: 3,4DMP concentration profiles: experimental data in mixture tests,
(test M3b) simulated curves for pure compound and mixture

Env Sci Pollut Res 15 (3) 2008 193

Biodegradation of Phenolic Mixtures Subject Area 5.1

Regarding 4NP biodegradation, a first preliminary qualita-
tive analysis shows that there are not significant differences
among removal rates in the different runs; moreover, the ki-
netics in the mixtures is close to that observed in the single
compound tests. According to this experimental evidence, all
the data were fitted with the Haldane model, assuming
C*=43.36 mgCOD l–1 and β≅52as obtained from single com-
pound tests. The fitted value kmax= 0.154 mgCOD mgVSS–1 h–1
(S.E. =0.0022; correlation coefficient = 0.99) is very close to that
found for the pure compound (0.142 mgCOD mgVSS–1 h–1),
thus confirming that the 4NP removal rate is practically
comparable in mixture and pure compound tests.
The behaviour of 3,4DMP, reported in Fig. 6, is quite differ-
ent. A qualitative analysis of the experimental results shows
that the 3,4DMP removal rate of the first replicate tests M1a/b
is significantly lower than that observed in the successive
tests M2a/b and M3a/b.
This behaviour could be attributable to a still too short accli-
matization time of the biomass to the mixture: in fact, M1a
and M1b were the first tests carried out with the mixture and,
probably, this more complex substrate requires a longer con-
tact period for the biomass to fully express its potentialities.
For 4NP, it is plausible, due to the long previous contact pe-
riod (about four years), that the culture quickly reached the
maximum removal efficiency even in the presence of another
compound while the 3,4DMP is a quite new substrate for the
biomass and the best removal performance for this compound
in this new condition could require a longer acclimatization.
Moreover, in M1 experiments, the 3,4DMP concentration
range explored is quite low and only a few data are available
to be analysed, so that this run seems to be less significant
than the other ones and only tests M2a/b and M3a/b were
considered in order to evaluate the achievable 3,4DMP re-
moval efficiency of the culture fed with the mixture. From the
fitting of the experimental data of these runs, with
C*=14.0 mgCOD l–1 and β=0(as determined from single sub-
strate tests) a value of kmax= 0.0535 mgCOD mgVSS–1 h–1 (S.E.=
0.0007; correlation coefficient = 0.99) was obtained with an
increase of about 80% respect to the pure compound value.
The very good removal kinetics observed in mixture tests
suggest, for practical application, the direct acclimatization
of the biomass to the mixture. This procedure could enhance
the degradation efficiency and reduce the time required for
adapting the culture.
Finally, the comparison between experimental and calcu-
lated concentration profiles of 4NP and 3,4DMP vs. time,
reported in Fig. 4 for test M3b, shows a very good agree-
ment between experimental and predicted values.
3 Conclusions
• Results of this study showed that the biodegradation ki-
netics of substituted phenol in mixture can be signifi-
cantly different from that observed in single compound
tests: in fact, the presence of a faster degradable com-
pound (the 4NP) seems to exert a positive effect on the
removal of a slower degradable compound (the 3,4DMP).
• The higher removal rate detected for 4NP, both in single
compound and mixture tests, and the improvement of
3,4DMP biodegradation kinetics in the subsequent mix-
ture tests confirmed the key role of the biomass accli-
matization in determining the biodegradation kinetics of
xenobiotic compounds.
• The experimental approach and the original method ap-
plied for data analysis are of general validity and can be
extended to the investigation of different classes of com-
pounds.
• An interesting aspect related to the process applicability
is the possibility of easily adapting an enriched culture
grown on a specific xenobiotic (in our case the 4NP) to
the removal of similar compounds found either singulary
or in mixtures. This suggests a possible strategy when
biological processes are considered for xenobiotic re-
moval: to develop enriched cultures on target compounds
that can be efficiently utilized on more complex matri-
ces with reduced start up and acclimatization periods. It
is worth noting that the strategy efficiency is strongly de-
pendent on the degree of similarity between compounds
and on the history of the biomass.

Appendix: A new form for the Haldane equation

Starting from the Haldane equation in the classical form: C C

C C rS = k * ⋅X C* = β ⋅ k * ⋅X C*
rS = v = k * ⋅X C KS C2 C  C 
2
(A4)
C
2
C2
+ + β⋅ +1+  
C + KS + C + KS + (A1) C * C * C * ⋅K I C*  C*
KI KI
known that the maximum removal rate vmax, observed at
C=C*, is given by:
and assuming:

k * ⋅X k * ⋅X k * ⋅X ⋅ β
v max = = =
C* = K S K I (A2) KS (1 + )
2 2+β
(1 + 2 ) (A5)
KI β

KI v max
β= (A3) Finally, if we define k max = , we obtain:
KS X
C/C *
rS = k max ⋅ X(2 + β)
1 + β(C/C *) + (C/C *) (A6)
2
the following equation is obtained:

194 Env Sci Pollut Res 15 (3) 2008

Subject Area 5.1
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* Corresponding author (anne@kbfi.ee)
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Received: June 21st, 2007
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OnlineFirst: December 18th, 2007
of oil product sand 35 mg of PAHs per g dwt) was analyzed chemi-
cally (HPLC) and toxicologically (Flash-Assay using Vibrio fischeri)
for the leaching of phenols during shaking of soil-water slurriesfor
24 h. Only 5.8% of the total concentration of phenols was water-
extractable, whereas about 50% of the leached amount was biode-
graded by the soil microorganisms. Phenol and cresols were biode-
graded by 80%, but the concentration of dimethylphenols practically
did not change. The pollutants (measured as total water-extractable
toxicity) were desorbed from the soil particles by the 8th h of extrac-
tion, whereas the toxicity of the aqueous phase continued to increase,
probably due to the formationof toxic metabolites. The concentration
of water-extractable phenols was too low to explain the toxicity of
the extract. Also the impact of PAHs and oil products was excluded.
Thus, the relatively low concentration of phenols in the oil-shale re-
gion soils is most probably the reflection of both natural attenuation
and pollution aging. Therefore, the impact of phenolic compounds to
the net bioavailable hazard is probably not so remarkable as it has
been considered. The actual pollutants causing the toxicity of the soils
from the oil-shale region, however, need to be elucidated.
Keywords: Bioavailability; biodegradability; cresols; dimethylphenols;
phenol; photobacteria; polluted soils, solid phase flash assay; sorp-
tion; toxicity; water extractable toxic hazard
195