Journal of Hazardous Materials 164 (2009) 15681572

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Journal of Hazardous Materials
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Short communication
Ex-situ bioremediation of polycyclic aromatic hydrocarbons in sewage sludge
Sille Bendix Larsen
a,1
, Dimitar Karakashev
a
, Irini Angelidaki
a
, Jens Ejbye Schmidt
a,b,
a
Department of Environmental Engineering, Technical University of Denmark, DK-2800 Lyngby, Denmark
b
NRG - Biosystems Department, Ris National Laboratory for Sustainable Energy, Technical University of Denmark, P.O. 49, DK-4000 Roskilde, Denmark
a r t i c l e i n f o
Article history:
Received 8 November 2007
Received in revised form 20 August 2008
Accepted 21 August 2008
Available online 27 August 2008
Keywords:
Bioremediation
PAH
Sewage sludge
a b s t r a c t
Polycyclic aromatic hydrocarbons (PAH) are regarded as environmental pollutants. A promising approach
to reduce PAH pollution is based on the implementation of the natural potential of some microorgan-
isms to utilize hydrocarbons. In this study Proteiniphilum acetatigenes was used for bioaugmentation of
sewage sludge to improve the PAH removal. Bioaugmentation experiments were performed in parallel
semi-continuously fed reactors started up with digested primary and secondary sludge. Three bioaug-
mentation approaches were investigated: A1, addition of bacteria once during starting up; A2, addition of
bacteria at the beginning and then every 2nd day and A3, addition of encapsulated bacteria once during
starting up. Removal of PAH was found to be both biotic and abiotic. All three approaches had a positive
effect of the biological removal of PAH. Highest biological removal of individual PAH (up to 80%) was
observed using continuous addition (approach A2) of the bacteria to the reactors. In general, the effect of
bioaugmentation was higher in the reactors fed with primary sludge compared to the reactors fed with
mixedsludge. Bioaugmentationresultedinbiological removal of lowmolecular weight PAHinthe reactors
fed with primary sludge using all three approaches while clear biological removal of the medium- and
high molecular weight PAH only was observed if the bacteria were added continuously (approach A2).
2008 Elsevier B.V. All rights reserved.
1. Introduction
Polycyclic aromatic hydrocarbons (PAH) are regarded as envi-
ronmental pollutants by environmental and health agencies
because they have toxic, mutagenic and carcinogenic effects on the
living organisms [1].
PAH are formed during the combustion of different organic
materials e.g. coal, cigarettes, fossil fuels and in relation to
domestic heating (Polycyclic aromatic hydrocarbons (PAHs) [2]).
Through the air, the PAH can deposit to water bodies and
soil [3]. PAH also deposits on surfaces of roads by, which
they enter the sewage system through runoff after rainy events
[4]. Through the common sewer system they end up at the
wastewater treatment plants (WWTP). Due to the hydrophobic-
ity of PAH, they absorb to the particulate matter in wastewater
sludge, hence they end up in the sewage sludge fraction. Sewage
sludge have been disposed through several ways, among these
are agricultural land application, ocean dumping and incin-
eration, where land application is the most frequently used.
However, disposal of sewage sludge on land posses a potential

Corresponding author. Tel.: +45 46774195.

E-mail address: jens.ejbye.schmidt@risoe.dk (J.E. Schmidt).
1
Present address: EnviDan, Fuglebkvej 1B, DK-2770 Kastrup, Denmark.
risk to the environment especially with respect to PAH in the
sludge.
A promising approach to reduce PAH pollution is the utilization
of the natural potential of microorganisms to utilize hydrocarbons
since the bioremediation techniques are cheaper than the other
alternatives (soil washing, solidication and stabilization, inciner-
ation, thermal treatment or advanced oxidationprocesses) used for
cleaning up of contaminated sites [5].
Bioaugmentation have been previously used as a tool for biore-
mediation of wastewaters, especially in relation to activated sludge
[6,7]. Anaerobic bioremediation of PAH contaminated anaerobic
environments such as aquifers and marine sediments has been
widely studied [8]. Anaerobic PAH biodegradation in synthetic
medium was also previously studied with a mixed microbial con-
sortium[9] and with a pure culture [10] isolated froma wastewater
treatment plant. However, anaerobic PAHbiodegradationinsewage
sludges is often limited and other approaches have to be imple-
mented to remove PAH in the sludge. To our knowledge, addition
of anaerobic microorganisms known to degrade PAH in order
to enhance the biodegradation has not been successfully imple-
mented until now. An important limitation of bioaugmentation
is deactivation or loss of microorganisms responsible for PAH
degradation shortly after the addition due to abiotic (nutrients
availability, toxic levels of pollutants etc.) and biotic stress (com-
petitive and antagonistic interactions with respect to indigenous
0304-3894/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jhazmat.2008.08.067
S.B. Larsen et al. / Journal of Hazardous Materials 164 (2009) 15681572 1569
Table 1
Characteristics of the different sludges collected
Parameter Unit Primary sludge
(meanS.D.
a
)
Mixed sludge
(meanS.D.)
Total COD gL
1
11.1 1.50 25.7 1.10
VS gL
1
10.8 0.8 19.2 1.10
Naphthalene gL
1
6.3 0.22 6.9 0.24
Acenaphtene gL
1
4.6 0.15 3.0 0.11
Anthracene gL
1
9.6 0.39 7.1 0.25
Phenanthrene gL
1
1 0.04 1.2 0.04
Fluoranthene gL
1
12.5 0.42 13.7 0.05
Pyrene gL
1
9 0.37 11.6 0.42
Benzo(k)uoranthene gL
1
2.5 0.10 2.8 0.12
Benzo(a)pyrene gL
1
1 0.04 1.2 0.05
a
Standard deviations (S.D.) were based on triplicate analysis.
microora) [11,12]. Therefore, development of bioaugmentation
methods and technologies is very important.
In our study three different bioaugmentation approaches were
tested with respect to obtain maximum removal of PAH in pri-
mary and mixed sewage sludge. An isolate of Proteiniphilum
acetatigenes, able to degrade PAH in synthetic media under
anaerobic methanogenic conditions, was used for bioaugmenta-
tion.
2. Materials and methods
2.1. Sludge collection and characterization
Primary, mixed (primary:secondary sludge =1:3) and anaero-
bically digested sewage sludge were collected from a wastewater
treatment plant (Lundtofte, Denmark) and analysed for total
chemical oxygendemand(COD), volatilesolids (VS) andPAH(naph-
thalene, acenaphtene, anthracene, uoranthene, phenanthrene,
pyrene, benzo(k)uoranthene, benzo(a)pyrene) content (Table 1).
In order to have homogenous substrate for the bioaugmentation,
the primary and mixed sludge were degraded with anaerobically
digestedsewage sludge as inoculum(50%) under mesophilic (37

C)
anaerobic conditions and hydraulic retention time of 20 days in
continuously stirred tank reactors (CSTR) with working volume
2L. These digested sludges were used for the bioaugmentations
experiments.
2.2. Bioaugmentation semi-continuous experiments
120ml anaerobically digested sludge (primary or mixture of
primary and secondary sewage sludge) were dispensed in 300ml
glass bottles and closed with Teon coated rubber stoppers and
aluminium caps. Bottles headspace was ushed with nitrogen to
ensure anaerobic conditions. The retention time was 20 days. Every
second day 12ml of sludge was removed and a newdigested sludge
was added to the bottles.
Three different bioaugmentation approaches (named as A1, A2
and A3) were studied: rst (A1), with addition of bacteria in the
beginning of the experiment; second(A2), withadditionof bacteria
in the beginning and every 2nd day and third (A3), with addition
of encapsulated bacteria in the beginning (Fig. 1). 12ml bacterial
culture (OD
600
=0.9) of P. acetatigenes (OD) was used for bioaug-
mentation at the corresponding day (approach 1 and approach 2).
62g of alginate beads carrying 12ml bacterial culture of P. acetati-
genes (OD
600
=0.9) was used for bioaugmentation in approach 3.
An abiotic control (only batch) to evaluate the amount of PAH
removed by abiotic processes (adsorption, volatilisation) was set
up for all experiments. The abiotic control was prepared by auto-
claving 120ml sludge for 30min at 140

C in order to ensure no
biological activity. Also a biotic control to evaluate the amount of
PAH degraded without bioaugmented bacteria was included in all
experiments. The biotic control consisted of 120ml sludge which
was not bioaugmented. All bioaugmented reactors and controls
were incubated in dark at 37

C for approximately 40 days until

no changes in PAH removal rate, volatile fatty acids and methane
productionwas registered. Everyseconddayliquidandgas samples
were withdrawn for evaluation of batch performance. Liquid sam-
ples were containing sludge were used for determination of PAH
and volatile fatty acids content respectively. Gas samples from the
headspace were used for analysis of methane content.
2.3. Bacterial culture used for bioaugmentation
A microbial culture of P. acetatigenes, utilizing PAH at strict
anaerobic methanogenic conditions, was previously isolated
(manuscript in preparation) from activated sludge received from a
municipal wastewater treatment plant (Lundtofte, Denmark). The
stock culture was maintained at 4

C on agar slants containing

0.5% nutrient broth (MPB) through regular subculturing. A loopfull
from an agar slant of this culture was used to prepare an inocu-
Fig. 1. Overview of the bioaugmentation approaches studied.
1570 S.B. Larsen et al. / Journal of Hazardous Materials 164 (2009) 15681572
lum in basic anaerobic (BA) medium [13] autoclaved at 115

C for
20min and supplemented aseptically with naphthalene and lter
(0.45m) sterilized yeast extract to nal concentration of 0.2gL
1
and 2gL
1
, respectively. Prior to inoculation, the mediumwas dis-
pensed under anaerobic conditions by ushing the headspace of
the vessels with sterile gas mixture of N
2
:CO
2
(80:20, v/v). After
3 days incubation at 37

C cell suspensions were centrifuged and

washed three times in sterile BA mediumin order to remove nutri-
ents. Washed cells diluted in BA medium to OD
600
=0.9 were used
as inoculum in the bioaugmentation experiments.
2.4. Encapsulation in alginate beads
Alginate beads for bacterial immobilisation were prepared by
adding 0.8g of alginic acid sodium salt (Fluka 71238) to 80ml
of exponentially grown cells of P. acetatigenes. The mixture was
dropped into a sterile 0.2M CaCl
2
solution as previously described
[14]. The resultedbeads (diameter 2mm) were collectedaseptically
and used for bioaugmentation experiments.
2.5. Analyses
Total chemical oxygen demand (COD) and volatile solids (VS)
of the sludges were measured according to standard methods
[15]. Methane and volatile fatty acids (VFA) content were analysed
by gas chromatography equipped with FID detector as previously
described [16]. Concentration of the individual PAH (naphthalene,
acenaphtene, anthracene, uoranthene, phenanthrene, pyrene,
benzo(k)uoranthene, benzo(a)pyrene was monitored by using
GCMS with ame ionization detection [17].
2.6. Calculation of PAH removal
PAHremoval was made on the base of mass balances taking into
account the mass and volume of the efuents and concentration of
individual PAH in those efuents.
3. Results and discussion
3.1. Initial anaerobic digestion of the sludges collected
Initial methanogenic anaerobic digestion of the primary and
mixed sewage sludge was done to produce a homogeneous inocu-
lum for the bioaugmentation experiments. During this process
total COD and VS was reduced with 382.5% and 332.0%
for primary sludge and with 413.5% and 49%3.8 for mixed
sludge respectively. The VFA production was stable with acetate
and propionate as the main intermediates for methane produc-
tion.
No signicant total PAH removal (less than 10% from the ini-
tial concentration) was observed during initial anaerobic digestion
of the sludges collected (data not shown). The small decrease in
PAH concentration can be explained with abiotic processes, such
as adsorption to rubber stoppers and internal glass surface of
the bottles [8,11]. The concentration variations were not due to
biodegradation as the level of easiest degradable PAH with low
molecular weight such as naphthalene, uoranthene and phenan-
threne was quite stable over time.
The COD and VS content of the primary sludge was relative
low compared to the mixed sludge (Table 1). It was also observed
that during the collection of the sludges at the WWTP the primary
sludge was very diluted with low TS content, around 13gL
1
. The
WWTP had also problems with the biological removal of N and P
due to lowCOD loads to the plants in the period where the sludges
were collected.
3.2. Batch performance of bioaugmentation approach A1:
addition of bacteria in the beginning
Bioaugmentation approach A1 aimed to determine whether or
not the bioaugmented bacteria after single addition, could main-
tain sufcient growth and PAHbiodedgradation in sewage sludges,
primary and mixed.
Inall bioaugmentedreactors, bothwithprimary andwithmixed
sludge, methane production and VFA concentration were from
1.5 to 2 times higher compared to methane production in the
biotic control (data not shown). This indicated that bioaugmented
bacteria facilitated the biodegradation of organic material to inter-
mediates (organic acids) converted to methane by methanogenic
archae naturally presented in the indigenous microbial consortium
[18]. All abiotic controls had a very low methane and VFA content
indicated absence of any microbial activity.
An overview of the PAH concentration changes in both bioaug-
mented sludges compared to biotic and abiotic controls is given in
Table 2. The concentrations of PAHwas from2 (for benzo(a)pyrene)
to 30 (for naphthalene) times lower compared to the PAH concen-
trations in the sludges (Table 1) fed to the reactors, indicating that
the PAH was removed either biological or abiotic.
For the primary sludge, low molecular weight PAH such as
naphtalene, acenaphtene and anthracene were found in up to 80%
lower concentrations while Phenanthrene was found in around
30% higher concentration in the bioaugmentated reactors com-
pared to the abiotic control. Results obtained indicate that the
enhanced removal of low molecular weight PAH was mainly due
Table 2
PAH degradation with primary and mixed sludge and bioaugmented bacteria added in the beginning (A1)
PAH Primary sludge Mixed sludge
Average
concentration of
PAH (gL
1
)
*
(meanS.D.)
a
Concentration change
in the bioaugmented
batch compared to
biotic control
Concentration change
in the bioaugmented
batch compared to
abiotic control
Average
concentration of
PAH (gL
1
)
*
(meanS.D.)
a
Concentration change
in the bioaugmented
batch compared to
biotic control
Concentration change
in the bioaugmented
batch compared to
abiotic control
Naphthalene 0.21 0.02 (50%) (5080%) 0.86 0.08 (50%) (5080%)
Acenaphtene 1.00 0.08 (50%) (5080%) 2.03 0.02 (0) (50%)
Anthracene 2.03 0.2 (50%) (5080%) 0.52 0.05 (0) (0/)
Phenanthrene 0.58 0.04 (50%) +(30%) 1.77 0.16 (0) (50%)
Fluoranthene 1.46 0.13 (5080%) (0) 2.41 0.20 (0) (0)
Pyrene 1.51 0.15 (5080%) (0) 2.06 0.20 (0) +(30%)
Benzo(k)uoranthene 0.43 0.03 (5080%) (0) 0.86 0.07 (0) (50%)
Benzo(a)pyrene 0.49 0.04 (50%) (0) 0.76 0.06 (0) (50%)
*
From day 3140.
a
Standard deviations (S.D.) were based on triplicate analysis.
S.B. Larsen et al. / Journal of Hazardous Materials 164 (2009) 15681572 1571
Table 3
PAH degradation with primary and mixed sludge and bioaugmented bacteria added continuously (A2)
PAH Primary sludge Mixed sludge
Average
concentration of
PAH (gL
1
)
*
(meanS.D.)
a
Concentration change
in the bioaugmented
batch compared to
biotic control
Concentration change
in the bioaugmented
batch compared to
abiotic control
Average
concentration of
PAH (gL
1
)
*
(meanS.D.)
a
Concentration change
in the bioaugmented
batch compared to
biotic control
Concentration change
in the bioaugmented
batch compared to
abiotic control
Naphthalene 6.53 0.55 (50%) (5080%) 12.29 1.11 (50%) (5080%)
Acenaphtene 0.85 0.08 (50%) (5080%) 1.34 0.12 (0) (0)
Anthracene 1.14 0.11 (50%) (5080%) 1.48 0.13 +(30%) +(3050%)
Phenanthrene 1.48 0.13 (50%) (0) 2.77 0.25 (50%) (5080%)
Fluoranthene 2.38 0.23 (50%) (5080%) 4.48 0.41 +(3050%) +(3050%)
Pyrene 2.77 0.26 (5080%) (5080%) 4.39 0.42 +(3050%) +(3050%)
Benzo(k)uoranthene 0.41 0.40 (50%) (5080%) 0.80 0.08 +(30%) +(30%)
Benzo(a)pyrene 0.62 0.06 (50%) (5080%) 1.00 0.09 +(30%) +(30%)
*
From day 3140.
a
Standard deviations (S.D.) were based on triplicate analysis.
to biodegradation. No biological removal of medium and high
molecular weight PAH in primary sludge was registered with this
bioaugmentation approach. Abiotic sorption was the main reason
of the removal of uoranthene, pyrene, benzo(k)uoranthene and
benzo(a)pyrene.
For the mixed sludge, no signicant changes were observed
in the PAH concentrations except for naphtalene that was found
in up to 80% lower concentrations compared to abiotic control.
The results indicated that the bioaugmentation only enhanced the
removal of naphthalene, when mixed sludge was treated in the
reactors.
Bioaugmented reactors fed with primary sludge showed higher
removal of PAH compared to bioaugmented reactors with mixed
sludge (Table 2). This nding can be attributed to higher organic
matter content, respectively COD of the mixed sewage sludge
(Table 1) resulting in more rm binding of PAH into the organic
matrix, thereby decreasing the PAH bioavailability. It was previ-
ously estimated that between 1020% of PAH content is complexly
bound in the particulate organic matter, which would limit its
bioavailability and thereby its biodegradation [12,19].
3.3. Batch performance of bioaugmentation approach A2:
continuous addition of bacteria
Bioaugmentation approach A2 aimed to clarify if the wash out
of the added bacteria could be the limiting factor of anaerobic
PAH biodegradation in sewage sludge. Possibility of adjusting the
sludges indigenous microbial consortiumto the exogenous bioaug-
mented bacteria by adding themcontinuously was studied through
this approach.
Trendlines for methane and VFA production were similar as in
the previous bioaugmentation approach A1 indicating a facilitated
anaerobic biodegradation of the sludge during bioaugmentation.
An overviewof the PAHconcentrations changes in both bioaug-
mented sludges compared to biotic and abiotic controls is given in
Table 3. The concentrations of PAH were up to 8 times lower com-
pared to the PAH concentrations in the sludge fed to the reactors
(Table 1), indicating that the PAH was removed either biological or
abiotic.
Results obtained with primary sludge (Table 3) revealed that
there was up to 80% biological removal of low- (except for
phenanthrene), medium- and high molecular weight PAH in the
bioaugmented reactors.
Bioaugmented primary sludge showed higher removal of PAH
compared to bioaugmented mixed sludge (Table 3) which can be
explained again with limited bioavailability of PAH due to high
organic matter content of the mixed sludge. The removal of PAH
in bioaugmented mixed sludge was only registered for naphtha-
lene and phenanthrene while for the other PAHthe concentrations
were higher or similar in the bioaugmented reactors compared
to the biotic and abiotic control. Most reasonable explanation for
the increased PAH concentration in the bioaugmented batches, is
that some evaporation might have taken place during opening the
bottles for the continuous addition of bacteria which resulted in
up-concentration of the PAH content. Thus, higher concentrations
were measured.
Table 4
PAH degradation with primary and mixed sludge and bioaugmented encapsulated bacteria added in the beginning (A3)
PAH Primary sludge Mixed sludge
Average
concentration of
PAH (gL
1
)
*
(meanS.D.)
a
Concentration change
in the bioaugmented
batch compared to
biotic control
Concentration change
in the bioaugmented
batch compared to
abiotic control
Average
concentration of
PAH (gL
1
)
*
(meanS.D.)
a
Concentration change
in the bioaugmented
batch compared to
biotic control
Concentration change
in the bioaugmented
batch compared to
abiotic control
Naphthalene 8.60 0.79 (0) (50%) 37.86 2.5 +(3050%) (5080%)
Acenaphtene 0.30 0.02 (0) (50%) 0.28 0.02 +(30%) (0)
Anthracene 0.19 0.01 (0) (50%) 0.28 0.01 +(30%) (50%)
Phenanthrene 0.60 0.05 (0) (0) 0.72 0.05 (0) (50%)
Fluoranthene 0.37 0.03 (0) (50%) 1.04 0.09 (0) (0)
Pyrene 0.38 0.03 (0) (50%) 0.88 0.07 (50%) (0)
Benzo(k)uoranthene 0.10 0.01 +(30%) +(30%) 0.19 0.01 (0) (0)
Benzo(a)pyrene 0.11 0.01 (0) (0) 0.19 0.01 (50%) (0)
*
From day 3140.
a
Standard deviations (S.D.) were based on triplicate analysis.
1572 S.B. Larsen et al. / Journal of Hazardous Materials 164 (2009) 15681572
Results obtained indicate that the enhanced removal of naph-
thalene and Phenanthrene in mixed sludge was mainly due to
biological activities.
3.4. Batch performance of bioaugmentation approach A3:
addition of encapsulated bacteria in the beginning
Bioaugmentation approach A3 aimed to clarify if the encapsula-
tion in alginate beads will have protect the bacteria from protozoa
or other predators insewage sludge, thereby enhance the biodegra-
dation of PAH compared to other approaches.
Results obtained showed that methane production in the
bioaugmented reactors with the encapsulated cells was strongly
inhibited due to tannins production during alginate lysis (data not
shown). VFA levels were in all cases very low, approximately below
1mM (data not shown). It has been previously shown that tannins
inhibit methanogens resulting in inhibition of anaerobic biodegra-
dation [20].
An overview of the PAH concentration changes in both bioaug-
mented sludges compared to biotic and abiotic controls is given
in Table 4. The concentrations of PAH were from around 2 (for
phenanthrene) to 50 (for anthracene) times lower compared to the
concentration of PAH in the sludge fed to the reactors (Table 1)
except for naphthalene, indicating that the PAHwas removedeither
biological or abiotic.
Results obtained (Table 4) showed that there was up to
50% biological PAH removal due to bioaugmentation in the pri-
mary sludge except for phenanthrene, benzo(k)uoranthene and
benzo(a)pyrene. Comparing with the biotic and abiotic controls
indicates that the removal was mainly biotic for naphthalene, ace-
naphtene, anthracene, uoranthene and pyrene.
The effect of bioaugmentation in the mixed sludge reactors
was limited, only naphthalene, anthracene and phenanthrene were
found to be biologically removed. Clear preference for naphthalene
removal was also registered. This could be due to PAH diffusion
limitations through the alginate beads. Among the studied PAH,
naphthalene has the lowest molecular weight resulting in easi-
est diffusion through the alginate beads towards bacterial cells.
Furthermore, the bioaugmented bacteria have been grown on
naphthalene for inoculum preparation, hence it could be expected
that they have a larger ability for degradation of naphthalene.
4. Conclusions
In our study three different bioaugmentation approaches
employing P. acetatigenes were investigated with respect to anaer-
obic biodegradation of PAH in primary and mixed sewage sludge
under mesophilic semi-continuous conditions. Results obtained
showed that primary sewage sludge having lower strength than
mixed sludge showed higher potential for anaerobic PAHbiodegra-
dation through bioaugmentation compared to mixed sludge.
Bioaugmentation resulted in biological removal of low molecular
weight PAH in the reactors fed with primary sludge using all three
approaches. Singleadditionof bacteria(nonimmobilized, approach
A1 and encapsulated, approach A3) was found to be not efcient
for biodegradation of medium and high molecular weight PAH.
Bioaugmentation with continuous addition of bacteria (approach
A2) seems most promising approach allowing highest (up to 80%)
biological removal of low, mediumand high molecular weight PAH.
Acknowledgements
The nancial support of BIOWASTE project (QLK5-CT-2002-
01138) and XENOMIC project (MEIF-CT-2003-500956) is gratefully
acknowledged.
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