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A New Dynamic Model For Bioavailability and Cometabolism
A New Dynamic Model For Bioavailability and Cometabolism
A New Dynamic Model For Bioavailability and Cometabolism
Available at www.sciencedirect.com
Article history: Organic micropollutants (OMPs) are present in wastewater and sludge. Their possible
Received 8 February 2011 impact to the environment contributes to their increasing scientific and social interest.
Received in revised form Anaerobic digestion has been shown as a potential biological process for removal of these
25 May 2011 compounds. An accurate description of OMP distribution in the environmental system can
Accepted 31 May 2011 be used to better understand which compartment is used for degradation and to improve
Available online 12 June 2011 their depletion in conventional wastewater treatment technologies. In this work, we
proposed a dynamical model with a four-compartment distribution to describe the Poly-
Keywords: cyclic Aromatic Hydrocarbons (PAHs) fate during anaerobic digestion. The model is cali-
Biodegradation brated and validated using experimental data obtained from two continuous reactors fed
Methanogenic conditions with primary and secondary sludge operated under mesophilic conditions. A non-linear
PAHs least square method was used to optimize the model parameters. The resulted model is
Sorption in accordance with the experimental data. The PAH biodegradation rate is well modeled
Xenobiotic when considering the aqueous fraction (including free and sorbed to dissolved/colloidal
matter PAHs) as the bioavailable compartment. It was also demonstrated in the simula-
tions that the PAHs biodegradation is linked to a mechanism of cometabolism. The model
proposed is potentially useful to better understand the micropollutant distribution, predict
the fate of PAHs under anaerobic condition and help to optimize the operation process for
their depletion.
ª 2011 Elsevier Ltd. All rights reserved.
* Corresponding author.
E-mail addresses: liliana.delgadillo@supagro.inra.fr (L. Delgadillo-Mirquez), lardonl@supagro.inra.fr (L. Lardon), steyer@supagro.inra.fr
(J.-P. Steyer), patureau@supagro.inra.fr (D. Patureau).
0043-1354/$ e see front matter ª 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2011.05.047
4512 w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 4 5 1 1 e4 5 2 1
Table 1 e Primary (PS) and secondary sludge (SS) characteristics (average value and standard deviation from 5
measurements performed at steady state).
Sludge COD DM DCM Proteins Carbohydrates Lipids VFA
Source: Barret et al., 2010d. Chemical oxygen demand (COD), dry matter (DM), dissolved/colloidal matter (DCM) and volatile fatty acids (VFAs).
w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 4 5 1 1 e4 5 2 1 4513
The model describes the physical exchanges of PAHs between Fig. 1 e Representation of the four-compartment model of
compartments in the reactor. Hence, the physical an OMP.
4514 w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 4 5 1 1 e4 5 2 1
pollutant. Indeed, the Table 2 shows the equilibrium gCOD/L) and then soluble substrate (SS, gCOD/L) biodegradation
constants Kp and KDCM calculated for the thirteen PAHs and to biogas.
the two sludge (PS and SS), according to previously developed
khyd
methodology (Barret et al., 2010b). Sp /SS
Ct;liq
Cf ¼ (6) m
SS /X þ CH4 þ CO2
1 þ Kp Sp þ KDCM SS
Table 3 represents the mathematical model with seven
Ct;liq Kp components and nine processes. The metabolism of substrate
cp ¼ (7)
1 þ Kp Sp þ KDCM SS growth is incorporated into the three first processes. Hydro-
lysis is described with a first order kinetics to represent the
Ct;liq KDCM enzymatic degradation of particulate substrate in soluble
cDCM ¼ (8)
1 þ Kp Sp þ KDCM SS substrate. Beside, decay is assumed in the transformation of
Kinetics of sorption and desorption of a pollutant between activated biomass into particulate substrate. Biomass (X, gCOD/
the free dissolved compartment and the particle one and L) growth is linked to soluble substrate uptake and modeled
between the free dissolved compartment and the DCM one are with Monod-type kinetics:
described by Equations (9) and (10):
SS
m ¼ mmax (11)
KS þ SS
cp
part
rsorp=desorp ¼ k1 cp ¼ k1 Kp Cf cp (9)
where mmax (1/d) is the maximum bacterial growth rate and KS
(gCOD/L) is the half-saturation constant associated with the
sorp=desorp ¼ k2 cDCM cDCM ¼ k2 KDCM Cf cDCM
rDCM (10)
soluble substrate SS.
where cp and cDCM are the OMPs particle and DCM concentra- OMP biodegradation may be considered as a classical
tions at equilibrium, respectively. k1 and k2 are the first-order metabolism with a specific OMP-degrader (Fig. 2b) and modeled
kinetic constants of sorption to particle and DCM, with a Monod-type kinetics (Equation (11): mmax,OMP, KS,OMP).
respectively. Nevertheless, the removal of pollutant present only in trace
levels (ng/L or mg/L) could not result in any significant biomass
3.2. Biodegradation growth (Clara et al., 2005). Thus, we assumed that comet-
abolism is the main OMP biodegradation mechanism (Fig. 2a).
A two-steps model has been used to describe the anaerobic Criddle (1993) proposed a cometabolism model between
digestion of sludge: first hydrolysis to particulate matter (Sp, a growing substrate and a non-growing substrate in simple
w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 4 5 1 1 e4 5 2 1 4515
a b
Fig. 2 e Scheme illustrating (a) cometabolism and (b) classic metabolism of OMP.
ecosystem. This equation is based on the assumption that the A sensitivity analysis for OMP total concentration was
cometabolic degradation rate is enhanced by the generation of conducted to identify the most sensitive parameters in the
reductants caused by the degradation of growth substrate (SS) four-compartment model with free dissolved compartment
and, in its absence, the cometabolic transformation is linked (Cf) as the available fraction. In reference to a given set of
to endogenous decay. Moreover, the cometabolic model parameter values, initial condition and characteristics of
includes competitive inhibition between growth and non- pollutants and reactors, eleven parameters were changed over
growth substrate and negative effect of the toxic products. 10%, 20%, 30% and 50% of their based values. In steady
However, we did not include those two last terms in this work state, nine simulations were run at each of these values to
because of the low concentration of OMPs in the system and generate nine concentrations profiles of compartments (Cf, Cp,
a large number of kinetic parameters can complicate the CDCM, Cg) for each parameter. A sensitivity coefficient, sq, of
modeling effort under current conditions. the variable z to the parameter q, defined by Equation (13)
In our case, PAHs are the non-growing substrate and (Bernard et al., 2001; Myint et al., 2007), was calculated to
Criddle’s equation has to be modified. In particular, the quantify the average spread for each parameter.
bioavailability limitation can be accounted for by replacing the
total concentration by the bioavailable one (Cbioav): Ztf
1 zqþDq zq
sq ¼ dt (13)
m C tf zq
bioav 0
rbio ¼ Tc þ kc X (12)
Y KSC þ Cbioav
where tf is the test duration, zq is the variable z associated with
where Tc is the OMPs transformation capacity (mgOMP/gCODSs ) base value of parameter q, and zqþDq is the variable z when the
standing for cometabolic interaction between the soluble parameter q is changed an amount Dq.
substrate metabolism and the OMPs metabolism, kc is the The sensitivity coefficient is presented in Fig. 3. Growth
maximum specific rate of OMPs biodegradation in absence of yield, Y, is the most sensitive parameter. A strong influence of
primary substrate (mgOMP/gCOD-X.d) and KSC is the half satura- parameters linked to soluble substrate uptake (Y, mmax, KS) can
tion constant of OMPs in the Monod formalism (mgOMP/L). The be noted. This is in agreement with cometabolism concept,
kc and KSC parameters are representative of the response of where the micropollutant fate is associated to growth
the OMP metabolic route to the OMP bioavailable fraction substrate degradation. Half saturation constant of OMP KSC
including transporters and enzymes affinity for their and the specific biodegradation rate of OMP kc show a relative
substrate. m is the growth rate (1/d) and Y is the growth yield sensibility. These parameters are representative of the OMP
(gCOD-X/gCODSs ). metabolic route and depend on the type of microbial
Furthermore, the four-compartment distribution may help
us to find which compartment is the real bioavailable fraction
(Cbioav) to be biodegraded. Beside, the four-compartment Y
model can be modified. By this way, the OMPs biodegraded kc
fraction can be assumed to be the free dissolved fraction (Cf e μmax
only process 7) or the aqueous fraction (Cf and CDCM e
KS
processes 7 and 8), or the sum of all fractions (Cf, CDCM and Cp e
Parameter
2 20 40
100 50
1 10 20
0 0 0 0 0
0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60
SS
200 100 3 30 60
2 20 40
100 50
1 10 20
0 0 0 0 0
0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60
Time (d)
Fig. 5 e Fluoranthene behavior in PS and SS reactors: (gray line) influent concentration of PAH, (black circles) experimental
data, (white circles) values estimated from equilibrium constants, (black line) model with cometabolism and (dashed line)
model with classic metabolism.
4518 w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 4 5 1 1 e4 5 2 1
100 40
40 0.5 10
50 20
0 0 0 0 0
0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60
SS
150 80 1 20 60
100 40
40 0.5 10
50 20
0 0 0 0 0
0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60
Time (d)
Fig. 6 e Chrysene behavior in PS and SS reactors: (gray line) influent concentration of PAH, (black circles) experimental data,
(white circles) values estimated from equilibrium constants, (black line) hypothesis 1, (dark-gray line) hypothesis 2 and
(light-gray line) hypothesis 3.
the cell surface (Smith et al., 2009), prior to the diffusion of free saturation constants (KSC) of PAHs estimated in the first case
micropollutant throughout cell membrane. Finally, the (the free dissolved fraction is the bioavailable one) are ten
hypothesis 3 (processes 1e9): All compartments are bioavail- times and hundred times lower than for the two other
able as proposed by Fountoulakis et al. (2006). This is probably hypotheses (aqueous fraction and the sum of all compart-
a mechanism of transport of sorbed micropollutant to vicinity ments, respectively) as shown on Fig. 7.
of microorganism. Consequently, in the hypothesis 1, the degradation rate of
Non-linear least square optimization method has been OMP is faster than that of soluble substrate as shown on
used to estimate PAH parameters (Tc, kc and KSC) for each case, Fig. 7a. This disagrees with the cometabolism studies
thirteen PAHs and two sludge. The residuals value taken up to demonstrating that the cometabolism is relatively slow by
quantify the best fitting do not present a high variation comparison to the metabolism of growth substrate (Chang
between cases, for example the residual values are 0.11, 0.10 et al., 1993; Haws et al., 2006). In addition, half saturation
and 0.12 for free dissolved, aqueous phase and all compart- value estimated in the hypothesis 3 is higher than the OMP
ments as available fraction, respectively, for chrysene in Cf concentration in the free compartment (KSC>>Cf). This
compartment and PS. implies that the free compartment degradation is negligible
Fig. 6 shows the comparison of model predictions (three compared to the particle compartment one and suggests high
hypotheses) for chrysene in PS and SS reactors. Similar affinity for the OMP sorbed to particle. This case is really
behaviors have been obtained for the other PAHs (data not atypical, since it is generally considered that the sorbed
shown). Such results could suggest that the PAH degradation chemicals are unavailable for microorganisms unless
occurs at the same time into free, aqueous and solid fraction, desorption occurs first. Indeed, Feng et al. (2000) have
i.e sorbed fractions into particle and dissolved colloidal matter demonstrated that some bacteria can degrade sorbed chem-
could be bioavailable to degraders. However, the half ical but it is not more important than the OMP aqueous phase
Rate: rSs (gCOD/gCOD.d),
b c
rOMP ( g/gCOD.d)
0.010 0.16
kc/KSC (L/gCOD.d)
0.008
Tc/KSC (L/gCOD)
R2 = 0.50 0.12
R2 = 0.55
0.006
0.08
0.004
2 0.04 2
0.002
R = 0.67 R = 0.49
0 0
150 200 250 300 150 200 250 300
M (g/mol) M (g/mol)
Fig. 8 e Tc/KSC and kc/KSC as a function of molecular masses of PAHs. PS (gray) and SS (black).
degradation. In contrast, hypothesis 2 presents a comet- molecular weight increases. This is in accordance with the
abolism slower than metabolism of soluble substrate (Fig. 7b) idea that high molecular weight PAHs are less efficiently
and the affinity for free compartment and DCM compartment removed (Chang et al., 2003). Moreover, specific biodegrada-
are comparable. Finally, based on the three-compartment tion rate, kc was shown to vary between reactors in a similar
model, Barret et al. (2010c) reported a strong correlation range (PS: 0.70e0.85 and SS: 0.60e0.90 mgOMP/gCOD-X.d).
between PAHs aqueous fraction degradation and the dry Therefore, half saturation constant KSC and kc probably
matter removal and shown that the PAH biodegradation depend on microbial consortium. In this study, it was shown
depended on a combination of bioavailability and comet- that different consortia exhibit different KSC and kc. This
abolism. Thus, the widespread assumption that the aqueous microbial effect could account for biodegradation differences
fraction of PAHs corresponds to their bioavailable compart- reported when bioaugmentation strategy has been developed
ment (Chang et al., 2003; Artola-Garicano et al., 2003; Dionisi (Trably et al., 2003).
et al., 2006; Barret et al., 2010c) was validated by our results.
It is worth noting that PAH volumetric gas fraction does not
exceed 0.05% of micropollutant total concentration for both
5. Conclusion
low and higher molecular weight PAHs (data not shown).
Moreover, hydrophobic character of PAH proves strong PAH
A four-compartment model of the fate of thirteen PAHs during
affinity (higher to 98%) for both particle and DCM while PAH
anaerobic digestion of contaminated sludge was developed
free concentration is hardly detectable (1.5%). However, PAH
and confronted with experimental data. The model includes
sorption to particle in PS reactor (85 5%) was higher than SS
abiotic and biotic processes: volatilization, sorption, biodeg-
(65 5%) one and PAH affinity for DCM was 13 5% and
radation as metabolism or cometabolism. Furthermore, in the
33 5% for PS and SS, respectively. As a consequence in this
case of hydrophobic pollutants, the sorption process can be
study, higher biological removal of individual PAH was
represented by the equilibrium state. The model helps in
observed in SS reactor in contrast with PS one.
elucidating which fraction of the PAHs distribution at equi-
librium state is the real bioavailable compartment. Thus, the
4.3. Kinetic parameters simulation carried out in this study validated the accepted
assumption that the aqueous phase is bioavailable. Indeed,
The results suggest that the three cometabolism parameters biodegradation affinity for OMP free dissolved and OMP sorbed
(Tc, kc and KSC) could explain the different biodegradation rates to DCM are comparable. Furthermore, PAH biodegradation
between PAHs and between bioreactors. This is valid under rate was coupled to cometabolism. The PAH removal was
the assumption that the aqueous fraction (sum of free and linked to soluble substrate uptake in the anaerobic digestion
sorbed to DCM compartments) is the bioavailable compart- of sludge. The modified cometabolism model predicted well
ment. Fig. 8 shows the variation of Tc/KSC and kc/KSC as func- the relation between bioavailability and cometabolism of
tion of molecular weight of PAHs. OMP. As a result of the numerical simulation, the three com-
The transformation capacity values Tc did not present etabolism parameters (Tc, kc and KSC) were shown to be
differences between PAHs and reactors (value close to molecule-dependant. These estimated parameter values
0.05 mgOMP/gCODSs ). As a result, the Tc value could correlate could explain the different biodegradation rates between
with a molecular structure family. Indeed, this term links PAHs and between reactors. Nevertheless, the applied meth-
PAHs degradation to soluble substrate utilization, so it might odology for the parameters identification may converge
play a role in the different fates of PAHs in the reactor fed with toward several values but this study can be considered as
PS and SS. However, previous PS and SS characterizations a starting point, given that the parameter values comparison
presented similar composition and reported slight differences with previously published data was hardly feasible. A limita-
in proteins and lipids content (Table 1). As a result, Tc can tion in this model is that it does not include the OMPs inhi-
present trifling variation between substrates (PS and SS). bition and toxic effect, which could be considered in future
Half saturation constant KSC is likely to vary as a function of work. However, the model proposed is potentially useful to
PAH molecular weight. KSC values indeed increase when PAHs better understand the micropollutant distribution, predict the
4520 w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 4 5 1 1 e4 5 2 1
fate of PAHs under anaerobic condition and help to optimize Dionisi, D., Bertin, L., Bornoroni, L., Capodicasa, S., Petrangeli
the operation process for their removal. Papini, M., Fava, F., 2006. Removal of organic xenobiotics in
activated sludges under aerobic conditions and anaerobic
digestion of the adsorbed species. Journal of Chemical
Technology & Biotechnology 81 (9), 1496e1505.
Acknowledgments Feng, Y., Park, J.H., Voice, T., Boyd, S., 2000. Bioavailability of soil-
sorbed Biphenyl to Bacteria. Environmental Science &
We would like to thank the Ibague University and COLCIEN- Technology 34 (10), 1977e1984.
CIAS (Administrative Department of Science, Technology and Fountoulakis, M.S., Stamatelatou, K., Batstone, D.J., Lyberatos, G.,
2006. Simulation of DEHP biodegradation and sorption during
Innovation) e Colombia for their financial support.
the anaerobic digestion of secondary sludge. Water Science &
Technology 54 (4), 119e128.
Haws, N.W., Ball, W.P., Bouwer, E.J., 2006. Modeling and
references interpreting bioavailability of organic contaminant mixtures
in subsurface environments. Journal of Contaminant
Hydrology 82 (3e4), 255e292.
Alder, A.C., Siegrist, H., Fent, K., Egli, T., Molnar, E., Poiger, T., Joss, A., Zabczynski, S., Göbel, A., Hoffmann, B., Löffler, D.,
Schaffner, C., Giger, W., 1997. The fate of organic pollutants in McArdell, C.S., Ternes, T.A., Thomsen, A., Siegrist, H., 2006.
wastewater and sludge treatment: significant processes and Biological degradation of pharmaceuticals in municipal
impact of compound properties. CHIMIA International Journal wastewater treatment: proposing and classification scheme.
of Chemistry 51 (12), 922e928. Water Research 40 (8), 1686e1696.
Artola-Garicano, E., Borkent, I., Damen, K., Jager, T., Vaes, W.H.J., Joss, A., Andersen, H., Ternes, T., Richle, P.R., Siegrist, H., 2004.
2003. Sorption kinetics and microbial biodegradation activity Removal of estrogens in municipal wastewater treatment
of hydrophobic chemicals in sewage sludge: model and under aerobic and anaerobic condition: consequences for
measurements based on free concentrations. Environmental plant optimization. Environmental Science & Technology 38
Science & Technology 37 (1), 116e122. (11), 3047e3055.
Barret, M., Carrere, H., Latrille, E., Wisniewski, C., Patureau, D., Kordel, W., Hennecke, D., Franke, C., 1997. Determination of the
2010a. Micropollutant and sludge characterization for adsorption-coefficients of organic substances on sewage
modeling sorption equilibria. Environmental Science & sludges. Chemosphere 35 (1e2), 107e119.
Technology 44 (3), 1100e1106. Lindblom, E., Press-Kristensen, K., Vanrolleghem, P.A.,
Barret, M., Patureau, D., Latrille, E., Carrère, H., 2010b. A three- Mikkelsen, P.S., Henze, M., 2009. Dynamic experiments with
compartment model for micropollutants sorption in sludge: high bisphenol-A concentrations modelled with an ASM
methodological approach and insights. Water Research 44 (2), model extended to include a separate XOC degradation
616e624. microorganism. Water Research 43 (13), 3169e3176.
Barret, M., Carrère, H., Delgadillo, L., Patureau, D., 2010c. PAH fate Myint, M., Nirmalakhandan, N., Speece, R.E., 2007. Anaerobic
during anaerobic digestion of contaminated sludge: do fermentation of cattle manure: modeling of hydrolysis and
bioavailability and/or cometabolism limit their acidogenesis. Water Research 41 (2), 323e332.
biodegradation? Water Research 44 (13), 3797e3806. Nikaido, H., 2003. Molecular basis of bacterial outer membrane
Barret, M., Cea Barcia, G., Guillon, A., Carrere, H., Patureau, D., permeability revisited. Microbiology and Molecular Biology
2010d. Influence of feed characteristics on the removal of Reviews 67 (4), 593e656.
micropollutants during the anaerobic digestion of Plósz, B.G., Leknes, H., Thomas, K.V., 2010. Impacts of competitive
contaminated sludge. Journal of Hazardous Materials 181 inhibition, parent compound formation and partitioning
(1e3), 241e247. behavior on the removal of antibiotics in municipal
Bernard, O., Hadj-Sadok, Z., Dochain, D., Genovesi, A., Steyer, J.P., wastewater treatment. Environmental Science & Technology
2001. Dynamical model development and parameter 44 (2), 734e742.
identification for an anaerobic wastewater treatment process. Press-Kristensen, K., Ledin, A., Schmidt, J.E., Henze, M., 2007.
Biotechnology and Bioengineering 75 (4), 424e438. Identifying model pollutants to investigate biodegradation of
Byrns, G., 2001. The fate of xenobiotic organic compounds in hazardous XOCs in WWTPs. Science of the Total Environment
wastewater treatment plants. Water Research 35 (10), 2523e2533. 373 (1), 122e130.
Chang, M.K., Voice, T.C., Criddle, C.S., 1993. Kinetics of Semple, K.T., Doick, K.J., Jones, K.C., Burauel, P., Craven, A.,
competitive inhibition and cometabolism in the Harms, H., 2004. Defining bioavailability and bioaccessibility
biodegradation of Benzene, Toluene, and pXylene by two of contaminated soil and sediment is complicated.
Pseudomonas isolates. Biotechnology and Bioengineering 41 Environmental Science & Technology 38 (12), 228e231.
(11), 1057e1065. Smith, K.E.C., Thullner, M., Wick, L.Y., Harms, H., 2009. Sorption
Chang, B.V., Chang, S.W., Yuan, S.Y., 2003. Anaerobic degradation to humic acids enhances polycyclic aromatic hydrocarbon
of polycyclic aromatic hydrocarbons in sludge. Advances in biodegradation. Environmental Science & Technology 43 (19),
Environmental Research 7 (3), 623e628. 7205e7211.
Clara, M., Kreuzingera, N., Strenna, B., Gansb, O., Kroissa, H., Tiehm, A., Fritzsche, C., 1995. Utilization of solubilized and
2005. The solids retention time-a suitable design parameter to crystalline mixtures of polycyclic aromatic hydrocarbons by
evaluate the capacity of wastewater treatment plants to a mycobacterium sp. Applied Microbiology and Biotechnology
remove micropollutants. Water Research 39 (1), 97e106. 42 (6), 964e968.
Couillard, C.M., Courtenay, S.C., Macdonald, R.W., 2008. Trably, E., Patureau, D., Delgenès, J.P., 2003. Enhancement of
Chemical-environment interactions affecting the risk of polycyclic aromatic hydrocarbons (PAH) removal during
impacts on aquatic organisms: a review with a Canadian anaerobic treatment of urban sludge. Water Science &
perspective - interactions affecting vulnerability. Technology 48 (4), 53e60.
Environmental Reviews 16, 19e44. Trably, E., Delgenes, N., Patureau, D., Delgenes, J.P., 2004.
Criddle, C.S., 1993. The kinetics of cometabolism. Biotechnology Statistical tools for the optimization of a highly reproducible
and Bioengineering 41 (11), 1048e1056. method for the analysis of polycyclic aromatic hydrocarbons
w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 4 5 1 1 e4 5 2 1 4521
in sludge samples. International Journal of Environmental Vavilin, V.A., Fernandez, B., Palatsi, J., Flotats, X., 2008. Hydrolysis
Analytical Chemistry 84, 995e1008. kinetics in anaerobic degradation of particulate organic
Urase, T., Kikuta, T., 2005. Separate estimation of adsorption and material: an overview. Waste Management 28 (6), 939e951.
degradation of pharmaceutical substances and estrogens in Yuan, S.Y., Chang, J.S., Yen, J.H., Chang, B.V., 2001.
the activated sludge process. Water Research 39 (7), Biodegradation of phenanthrene in river sediment.
1289e1300. Chemosphere 43 (3), 273e278.