ARTICLE IN PRESS

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Microbial processes associated to the decontamination anddetoxification of a polluted activated sludge during itsanaerobic stabilization

Lorenzo Bertina, Serena Capodicasaa, Fabio Occultia, Stefano Girottib,Leonardo Marchettia, Fabio Favaa,�

aDICASM, Faculty of Engineering, University of Bologna, viale Risorgimento 2, I-40136 Bologna, ItalybSMETEC, University of Bologna, via S. Donato 15, I-40127 Bologna, Italy

a r t i c l e i n f o

Article history:

Received 2 August 2006

Received in revised form

8 February 2007

Accepted 26 February 2007

Available online 16 April 2007

Keywords:

Contaminated activated sludge

Anaerobic digestion

Ecotoxicological tests

Polychlorinated biphenyls

Polycyclic aromatic hydrocarbons

Methanogenesis

nt matter & 2007 Elsevie.2007.02.046

thor. Tel.: +39 051 2093212fabio.fava@unibo.it (F. Fav

a b s t r a c t

Xenobiotic compounds accumulate in activated sludge resulting from wastewater

treatment plants serving both civil and industrial areas. The opportunity to use anaerobic

digestion for the decontamination and beneficial disposal of a contaminated activated

sludge was investigated in mesophilic and thermophilic microcosms monitored through an

integrated chemical, microbiological and ecotoxicological procedure. The 10 months

anaerobic sludge incubation at 35 1C resulted in an extensive production of a methane-

rich biogas, a marked reduction of pathogenic cultivable bacteria and, importantly, a

marked biodegradation of the sludge-carried organic pollutants, including some poly-

chlorinated biphenyls and polycyclic aromatic hydrocarbons, along with a relevant sludge

detoxification. The sludge decontamination seemed to occur mostly under methanogenic

conditions and was not significantly affected by the addition of yeast extract or molasses.

Lower bioremediation and biomethanization yields were observed under thermophilic

conditions.

& 2007 Elsevier Ltd. All rights reserved.

1. Introduction

Wastewater treatment plants produce high amounts of

sewage sludge (about 8 million tons per year in the sole

Europe; Trably et al., 2003). They are generally recycled/

disposed through landfill operations and applied as fertilizers

(EC, 1986). However, the increasing domestic use of chemical

compounds along with the growing tendency to send

industrial wastewater to civil wastewater treatment plants

resulted in the increasing occurrence of organic xenobiotics

in the sludge collected downstream to conventional waste-

water treatment plants (Blanchard et al., 2004; Katsoyiannis

and Samara, 2004; Abad et al., 2005; Busetti et al., 2006). This

evidence has induced the European Commission to propose

r Ltd. All rights reserved.

; fax: +39 051 2093220.a).

measures specifically addressed to classify a sludge on the

basis of its pollutant content and toxicity and to restrict its

use in agriculture (CEC, 2000; Benabdallah El-Hadj et al., 2006).

According to this proposal, sludge containing polycyclic

aromatic hydrocarbons (PAHs) or polychlorobiphenyls (PCBs)

at concentrations higher than 6 and 0.8 mg/kgdry sludge,

respectively, should be managed through incineration or

disposal in specific dump sites (CEC, 2000). These procedures

are expensive and unable to offer any type of beneficial reuse

of sludge. The opportunity to manage contaminated sludge

through anaerobic digestion/stabilization has been recently

proposed (Trably et al., 2003; Christensen et al., 2004; Patureau

and Trably, 2006; Benabdallah El-Hadj et al., 2006). Indeed, this

strategy might offer the opportunity to markedly reduce the

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WAT E R R E S E A R C H 4 1 ( 2 0 0 7 ) 2 4 0 7 – 2 4 1 62408

sludge volume and content of pathogenic bacteria by also

permitting its valorization through biogas production and

decontamination via microbial degradation of pollutants (De

Leon and Jenkins, 2002; Dahab and Surampalli, 2002;

Onyeche, 2004). On the other hand, the biological degradation

of several aliphatic and aromatic (halogenated) hydrocarbons

has been observed in a variety of anaerobic contaminated

matrices, including some anaerobically digested activated

sludge (Ye et al., 1992; Phelps et al., 1996; Chang et al., 2002;

Fava et al., 2003). However, the majority of data available on

the pollutant biodegradation in sludge have been obtained on

sludge spiked with specific mixtures of pollutants and

incubated under well defined conditions (Chang et al., 1999;

Jianlong et al., 2000; Gerecke et al., 2006), whereas little is

known about the occurrence of the same processes in actual-

site contaminated sludge (Trably et al., 2003; Patureau and

Trably, 2006) and in particular on microorganisms mainly

responsible for them (Christensen et al., 2004) and on the

impact of the treatment on the final sludge toxicity.

The present study was undertaken to investigate the effects

of a prolonged anaerobic digestion on the main chemical and

biological features of an activated sludge obtained from the

wastewater treatment plant of Fusina (Venice, Italy) con-

taminated by several organic priority pollutants, including

PCBs and PAHs. To such an aim, a series of slurry-phase

anaerobic microcosms consisting of activated sludge sus-

pended in its own water were developed, incubated under

mesophilic and thermophilic conditions and monitored along

a 10 months period through an integrated chemical, micro-

biological and ecotoxicological procedure. Further, the possi-

bility of enhancing the processes through yeast extract or

molasses addition and the role of indigenous sludge microbes

in the biodegradation of the main pollutants were also

studied by performing parallel experiments in microcosms

amended with specific microbial inhibitors. To the very best

of our knowledge, this is the first report in which such an

integrated approach has been applied to investigate and

characterize the actual potential of anaerobic digestion in the

beneficial disposal of actual-site contaminated activated

sludge.

2. Materials and methods

2.1. Chemicals and activated sludge employed

The analytical standards employed in the characterization of

PCBs and PAHs occurring in the activated sludge were

purchased by Ultra Scientific (North Kingstown, RI, USA).

Dichloromethane as well as all chemicals and antibiotics

employed in the microcosm preparation were provided by

Sigma Aldrich (Steinheim, Germany). The solvents employed

in the chromatographic analyses were obtained from Baker

Italia (Milan, Italy). Cultural media for pathogenic bacteria

and yeast extract were from Biolife (Milano, Italy). Molasses

were kindly purchased by the COPROB sugar beet-refinery

(Bologna, Italy). The activated sludge employed in the study

was collected from the aeration basin of the wastewater

treatment plant of Fusina (Venice, Italy), which serves both

urban and industrial areas of the city of Venice (Busetti et al.,

2006). The sludge was analysed for its pH, density, COD, total

suspended solids, pathogenic indicator microorganisms and

for its content of organic pollutants, heavy metals and anions

(i.e., CH3COO�, Cl�, NO3� and SO4

2�).

2.2. Anaerobic digestion of the activated sludge:approach, microcosms preparation and management

The anaerobic digestion of the Fusina activated sludge was

studied by developing several sets of 120-ml anaerobic

microcosms. Different experimental conditions were applied

in order to investigate (a) the influence of the temperature on

the process, (b) the possibility of biostimulating the process

through exogenous substrate addition and (c) the indigenous

bacteria potentially involved in the pollutants transforma-

tion. In particular, yeast extract or molasses (each at the

concentration of 1.77 mg/l) were added as exogenous sources

of substrates, while bromoethanesulfonate (BES, at the

concentration of 380 mg/l), sodium molybdate (290.3 mg/l)

and two antibiotics (D-cycloserine and penicillin G, at the

concentrations of 3.1 and 62 mg/l, respectively) were em-

ployed to inhibit methanogenic, sulphate-reducing and

fermentative/acidogenic bacteria, respectively (Fava et al.,

2003; Ye et al., 1999). As methanogens and sulfate-reducing

archaeabacteria may be adversely affected by eubacteria-

inhibiting antibiotics, depending on fermentative eubacteria

for substrates (Ye et al., 1999; Ward and Wrinfey, 1985),

methanol, formate and acetate (each at the concentration of

7 g/l) were also added, as substrates for archaeabacteria (Ward

and Wrinfey, 1985). To determine the contribution of in-

digenous spore-forming species on the process, an additional

set of microcosms was subjected to pasteurization (15 min-

heat treatment in a water batch at 90 1C ) according to Ye et al.

(1992, 1999). Abiotic controls were prepared by autoclave

sterilization (three cycles at 121 1C for 1 h, separated by 48 h of

incubation at 30 1C). The different experimental conditions

were developed according to Table 1, where the initial COD

related to each of them is as well reported. Two bottles per

each set of microcosms were incubated statically at 3571 1C

in the dark, while other two bottles per set were incubated

statically at 5571 1C in the dark.

Microcosms were developed according to the following

procedure: the sludge was purged with filter-sterilized O2-free

N2:CO2 (70:30) until redox potential reached the value of

�254 mV; then, 50 ml aliquots of it were transferred into 120-

ml serum bottles, each equipped with a magnetic bar and

subjected to purging with filter-sterilized O2-free N2:CO2

(70:30), and amended with 1.67 ml of filter sterilized water or

a filter sterilized aqueous solution according to Table 1. Each

microcosm was sealed with Teflon-coated butyl stopper and

aluminium crimp sealer.

During the 10 months of incubation, all microcosms were

periodically monitored for the concentration of the pollutants

and their main possible metabolites, nitrate, sulphate,

chloride ions and acetate ions as well as for the quantity

and composition of produced biogas. The microcosm-sam-

pling procedure (applied at the 7th day of the experiment,

then monthly during the subsequent 24 weeks of incubation

and finally at the end of the study), consisted in the following

steps: the head-space gas was quantified by aseptically

ARTICLE IN PRESS

Table 1 – List of the developed experimental conditions expressed by agents added to the sludge and by microcosm pre-treatments.

Amendments/operations

Objective Initial COD(g/l)

microcosms incubatedat 35 1C

microcosms incubatedat 55 1C

NoneDecontamination under actual

conditions17.1170.02 2 2

+Yeast extractBiostimulation with electron

donors17.5570.02 2 2

+MolassesBiostimulation with electron

donors19.0370.03 2 2

+BES+molybdateRole of fermentative/acidogenic

bacteria19.8770.01 2 2

+BES+antibioticsRole of sulphate-reducing

bacteria49.2970.00 2 2

+Molybdate+antibiotics Role of methanogenic bacteria 47.4570.01 2 2

Pasteurization Role of spore-forming bacteria 17.3370.42 2 2

Sterilization Abiotic control 17.3470.31 2 2

For each condition the research target, the initial COD and the number of developed microcosms are indicated.

WAT E R R E S E A R C H 41 (2007) 2407– 2416 2409

connecting the microcosm head-spaces to a Mariotte system

(Bertin et al., 2004), then 0.5 ml sample of the head-space gas

were taken with a 1 ml syringe from each serum bottle and

injected into the gas-chromatograph. Finally, each sealed

microcosm was mixed vigorously via magnetic stirring for

5 min, the stopper of the sampled microcosm was removed

and, while the microcosm content was being mixed magne-

tically and flushed with filter-sterilized O2-free N2:CO2 (70:30),

duplicate 0.3 ml samples were collected from each micro-

cosm. The head space was then flushed with filter-sterilized

O2-free N2:CO2 (70:30) and the microcosm recapped with

sterile Teflon-coated, butyl stoppers. Original activated sludge

and sludge resulting from the treatment performed in the

absence of chemical inhibitors were also analysed for

pathogenic bacteria and ecotoxicity.

2.3. Pollutant extraction procedures and analyticalmethods

A 0.25 ml of activated sludge samples collected from micro-

cosms was subjected to extraction with anhydrous diethyl

ether according to Fava et al. (2003); the obtained organic

phases were then analysed for organic pollutants by using a

GC-ECD chromatograph and analytical procedure reported by

the same authors (Fava et al., 2003). The extraction procedure

was tested by using 2,3,4,5,6-pentachlorobiphenyl (Fava et al.,

2003) as a standard and it provided a recovery yield of 8674%.

The amount of sludge occurring in the samples subjected to

extractions was periodically measured and it was generally of

about 0.015 g of dry weight. Qualitative analyses of the sludge-

carried PCBs and PAHs were performed by comparing the

retention time (relative to octachloronaphtalene) of each GC

peaks obtained from the analysis of the sludge organic

extracts with those of pure PCBs and PCBs of standard

Aroclor1242 and Aroclor1254 and of pure PAHs analysed

under identical conditions. Only some of the GC peaks could

be ascribed to specific compounds. The fate of compounds

responsible for the non-characterized peaks was studied by

using the peak area. The weighted average removal of sludge

organic pollutants was calculated as a sum of the single

compounds weighted removals, calculated by multiplying the

area of each peak at the end of the incubation for the ratio

between the initial peak area and the sum of the areas of all

the monitored peaks at the initial time.

Other 0.05 ml of each 0.30 ml samples originally collected

from microcosms were diluted with milliQ water up to 1 ml;

the samples obtained were then subjected to centrifugation

followed by ion-chromatography analyses (Fava et al., 2003).

The collected head space gas was analysed for CH4, CO2, N2

and O2 with a Varian TCD 3300 gas chromatograph equipped

with a Carbosieve S-II stainless steel column (3 m by 18 internal

diameter) (Supelco, Inc. Bellefonte, PA) and thermal conduc-

tivity detector (TCD) (Varian Instruments, Texas, USA) as

reported by Fava et al. (2003). pH and the oxidation–reduction

potential of the slurry-phase microcosms were measured at

the beginning (in additional microcosms identical to those

used in the experiment) and at the end of the experiment

(directly in the microcosms used in the experiment) by using

selective Orion electrodes (81-04 model and 97-78/SC model,

respectively) (Orion Research Inc., Beverly, MA, USA). COD

was spectrophotometrically measured following the Hach

Mn(III) method (Miller et al., 2001). The occurrence of heavy

metals in the activated sludge was determined at the

beginning of the experiment according to the methods EPA

7062/94 (Arsenic), EPA 7471A/94 (Mercury) and EPA 6010B/96

(other metals).

The concentration of cultivable Coliform and Streptococcus

bacteria occurring in the activated sludge before and after the

anaerobic incubation was determined through plate-counting

techniques on selective agar-media according to the proce-

dure reported by APAT-IRSA (2004).

Ecotoxicity assays were performed by using an ecotoxico-

logical method developed by Bolelli et al. (2006) for high-COD

contaminated matrices. In brief, the sludge samples were

boiled for 30 min and then centrifuged. The recovered super-

natants were amended with NaOH (to adjust their pH to 7)

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WAT E R R E S E A R C H 4 1 ( 2 0 0 7 ) 2 4 0 7 – 2 4 1 62410

and with NaCl (to a final concentration of 3% w/v). In order to

determine their long-term (18 h) toxic effects, three test

strains of marine luminescent bacteria belonging to Vibrio

genus were applied, i.e., strain ‘‘UCIBO’’, which was isolated

from the Mediterranean seawater, strain ‘‘Russian’’, which

was purchased from Biophysics Institute of the Russian

Academy of Science (RAS), Siberian Branch, Novosibirsk,

Russia, and the Vibrio fisheri strain employed in the official

assay European standard EN ISO 11348, which was purchased

by the Institut Pasteur Laboratories, Paris, France. The

procedure was applied on the original activated sludge and

on those resulting from the treatment in the absence of

inhibitors.

3. Results

3.1. Characterization of the activated sludge employed

The main physical, chemical and biological properties of the

Fusina plant activated sludge are reported in Table 2. The

sludge was contaminated by a complex mixture of organic

pollutants, such as PCBs (total concentration: 1.243 mg/kgdry

sludge), PAHs (327.1 mg/kgdry sludge), dichloromethane

(117.5 mg/kgdry sludge) and a number of organic unidentified

compounds (Table 3). Several heavy metals, such as arsenic

(15.0 mg/kgdry sludge), chromium (143.5 mg/kgdry sludge), mer-

cury (4.2 mg/kgdry sludge), nickel (44.4 mg/kgdry sludge), lead

(137.7 mg/kgdry sludge), copper (318.6 mg/kgdry sludge), iron

Table 2 – Initial physical and chemical sludge parameters

Density (g/l) Total suspended

solids (g/l)

pH CH3COO� (mg/

kgdry sludge)

983.0372.48 59.0070.57 6.80 354073.55

Table 3 – Initial sludge pollution characterization: relative reteoctachloronaphtalene) and areas of the 14 revealed GC-peakscompounds

Peak RRT (min/min) Peak area (mV�min) Comp

0.034 251183.378891.0 Dic

0.051 171267.276752.1

0.078 133728.9742127.6

0.104 37759.973617.0

0.180 18271.875120.4 Ac

0.185 74242.6726690.5 2-Me

0.205 133495.1711221.5

0.208 112704.0733532.3

0.257 93770.3725285.1

0.486 147891.9719177.4 2,4

0.696 105472.2714505.8 2,3,4,4

0.738 88636.1716923.2 3,

0.908 68088.477744.1

0.921 52319.075442.5

n.i., not identified.

(14906.0 mg/kgdry sludge), zinc (970.6 mg/kgdry sludge) and alu-

minium (28966.0 mg/kgdry sludge) were detected in the sludge

collected from the plant. It also exhibited a neutral pH, a high

content of total suspended solids (Table 2) and of pathogenic

cultivable bacteria (Table 4).

3.2. Anaerobic digestion of activated sludge:biodegradation of pollutants and associated microbialprocesses

The majority of compounds detected in the activated sludge

underwent microbial transformation in the biologically active

microcosms throughout the 10 months of incubation at 35

and 55 1C. In general, some of the higher hydrophobic/

molecular weight compounds, including 2,3,4,40/2,3,30,40-tet-

rachlorobiphenyl (2,3,4,40/2,3,30,40-tetraCB) and 3,4,40,5-tetra-

chlorobiphenyl (3,4,40,5-tetraCB) were depleted whereas some

lighter/more polar compounds, probably products of the

microbial transformation of the former pollutants, accumu-

lated in such microcosms (Figs. 1 and 2). Most of the pollutant

depletions occurred between the 1st and the 9th week of

incubation. Only limited pollutant depletions were observed

in the sterilized microcosms during the first 5 weeks of

incubation (weighted average pollutant removals were 9.65%

and 9.29% at 35 and 55 1C, respectively); importantly, biogas

and methane productions occurred in those microcosms after

the 5th week of incubation, so that they were not under

sterile conditions any more. Under mesophilic conditions, the

Cl� (mg/kgdry

sludge)

NO�3 (mg/kgdry

sludge)SO2�

4 (mg/kgdry

sludge)

1280715.50 8.8172.03 24.91711.60

ntion times (with respect to the retention time ofalong with initial concentrations of the identified

ound Concentration (mg/kgsludge dry weight)

lhoromethane 11774.10

n.i.1

n.i.2

n.i.3

enaphthylene 28.678.01

thylnaphtalene 2987107

n.i.4

n.i.5

n.i.60,5/2,4,40-triCB 0.15770.0200/2,3,30,40-tetraCB 0.29770.041

4,40,5-tetraCB 0.78970.151

n.i.7

n.i.8

ARTICLE IN PRESS

Table 4 – Pathogenic indicator microorganism concentrations in the initial sludge and in sludge samples collected at theend of the experiment from microcosms without amendments, with molasses or with yeast extract, both for mesophilic(35 1C) or thermophilic (55 1C) experimental conditions

Escherichia coli (CFU/ml) Coliform bacteria (CFU/ml) Fecal streptococcus (CFU/ml)

Sludge before treatment 1.33E0470.46E04 1.83E0470.45E04 1.40E0470.00E04Experimental conditions

Sludge 35 1C 4.50E0370.03E03 n.d. 2.27E0370.06E03

55 1C n.d. n.d. n.d.Sludge plus molasses 35 1C 6.40E0270.40E02 2.50E0170.50E01 n.d.

55 1C n.d. n.d. n.d.Sludge plus yeast extract 35 1C 2.80E0270.05E02 2.20E0171.20E01 n.d.

55 1C n.d. n.d. n.d.

n.d., not detected.

0.00E+00

2.00E+05

4.00E+05

6.00E+05

8.00E+05

1.00E+06

1.20E+06

1.40E+06

Are

a

0.00E+00

2.00E+05

4.00E+05

6.00E+05

8.00E+05

1.00E+06

1.20E+06

1.40E+06

dichlo

rometh

ane

n.i.1

n.i.2

n.i.3

acen

aphth

ylene

2-meth

yl-na

phtha

lene

n.i.4

n.i.5

n.i.6

2,4',5

/2,4,4

'-triC

B

2,3,4,

4'/2,3

,3',4'

-tetra

CB

3,4,4'

,5-tet

raCB

n.i.7

n.i.8

Are

a

A

B

Fig. 1 – GC-peak areas related to sludge samples collected at the beginning of the experiment (’) and at its end from

microcosms without amendments (&), with molasses ( ) or with yeast extract ( ) incubated at 35 1C (A) or 55 1C (B). Peaks are

reported in order of growing GC retention time. Each value represents the average of the values related to measurements

performed within samples collected from the two parallel microcosms. Standard deviations are reported as error bars.

WAT E R R E S E A R C H 41 (2007) 2407– 2416 2411

weighted average removal of pollutants observed in the non-

amended microcosms at the end of 10 months incubation

was 38%. However, 2,3,4,40/2,3,30,40-tetraCB and 3,4,40,5-tet-

raCB were completely removed, while 2,40,5/2,4,40-trichloro-

biphenyl (2,40,5/2,4,40-triCB) was depleted by 68%.

Dichloromethane was removed by 21%, whereas acenaphthy-

lene and 2-methyl-naphtalene were biodegraded by 18% and

58%, respectively (Fig. 1A). The addition of yeast extract or

molasses had only small effects on the pollutant removal

extent and pattern (Fig. 1A): while the biodegradation of

chlorinated compounds appeared to be slightly inhibited by

the two additives, that of acenaphthylene and 2-methyl-

naphtalene was slightly enhanced, in particular when yeast

extract was used (Fig. 1A).

Under the same mesophilic conditions, significant deple-

tions of high retention time compounds along with a marked

ARTICLE IN PRESS

0.00E+00

2.00E+05

4.00E+05

6.00E+05

8.00E+05

1.00E+06

1.20E+06

1.40E+06

Are

a

0.00E+00

2.00E+05

4.00E+05

6.00E+05

8.00E+05

1.00E+06

1.20E+06

1.40E+06

dichlo

rometh

ane

n.i.1

n.i.2

n.i.3

acen

aphth

ylene

2-meth

yl-na

phtha

lene

n.i.4

n.i.5

n.i.6

2,4',5

/2,4,4

'-triC

B

2,3,4,

4'/2,3

,3',4'

-tetra

CB

3,4,4'

,5-tet

raCB

n.i.7

n.i.8

Are

a

A

B

Fig. 2 – GC-peak areas related to sludge samples collected at the beginning of the experiment (’) and at its end from

microcosms with antibiotic and BES (&), with antibiotic and molybdate ( ) with BES and molybdate ( ) or pasteurised ( )

incubated at 35 1C (A) or 55 1C (B). Peaks are reported in order of growing GC retention time. Each value represents the average

of the values related to measurements performed within samples collected from the two parallel microcosms. Standard

deviations are reported as error bars.

WAT E R R E S E A R C H 4 1 ( 2 0 0 7 ) 2 4 0 7 – 2 4 1 62412

accumulation of low retention time pollutants were observed

in the microcosms amended with antibiotics and molybdate,

i.e., under methanogenic conditions (Fig. 2A), where a

weighted average removal of pollutants of 31% was observed.

The removal of PCBs was the highest recorded in the study. In

particular, 2,40,5/2,4,40-triCB was almost totally degraded in

these microcosms (Fig. 2A) where, in addition, the transient

accumulation of 3,30,4-trichlorobiphenyl, 2,40/2,3-dichlorobi-

phenyl and 2-chlorobiphenyl was observed from the 4th and

the 6th month of incubation (data not shown). On the

contrary, a lower depletion of the same pollutants and PAHs

were observed in the presence of antibiotics and BES, i.e.,

under sulphidogenic conditions, and in the presence of

molybdate and BES, i.e., where fermentative/acidogenic

bacteria were probably the only active population in the

microcosms (Fig. 2A). A detectable pollutant biotransforma-

tion was also observed in the parallel pasteurized micro-

cosms, where weighted average pollutant depletion

percentages of 16% along with pollutant biotransformation

patterns similar to those observed in the non-amended

microcosms were observed (Fig. 2A). Notably, the identified

PAHs were totally depleted in these microcosms.

Under thermophilic conditions, the pollutants were gen-

erally depleted through patterns often similar to those

observed in the parallel mesophilic microcosms (Figs. 1B

and 2B). However, lower pollutant biodegradation yields along

with a more extensive accumulation of intermediates were

very often observed in these microcosms with respect to

those incubated at 35 1C (Figs. 1B and 2B).

A significant accumulation of Cl� (ranging from 540 to

1350 mg/kgdry sludge) was detected in several of the developed

microcosms since the 5th week of incubation. The process

was more extensive in the non-amended microcosms and in

those supplemented with antibiotics and molybdate incu-

bated under mesophilic conditions than in all the other

microcosms (data not shown).

Ecotoxicity test data shown in Fig. 3 indicate that a marked

depletion of the original activated sludge toxicity took place

during its anaerobic incubation at 35 1C in the absence of

amendments (Fig. 3). The presence of yeast extract or

molasses did not affect significantly the sludge detoxification.

Pasteurized activated sludge subjected to the same treatment

also exhibited a marked detoxification. No significant detox-

ification was observed under thermophilic conditions (Fig. 3).

ARTICLE IN PRESS

0

10000

20000

30000

40000

50000

60000

70000

80000

initia

l slud

ge

sludg

e 35°C

sludg

e 55°C

patso

rized

slud

ge 35

°C

pasto

rized

slud

ge 55

°C

sludg

e plus

mola

sse 35

°C

sludg

e plus

mola

sse 55

°C

sludg

e plus

yeas

t extr

act 3

5°C

sludg

e plus

yeas

t extr

act 5

5°C

RL

U

Experimental conditions

Fig. 3 – Relative light unit (RLU) related to bioluminescent assays applied with Ucibo ( ), Vibrio fisheri ( ) or Russian ( ) test

microorganism on the initial sludge along with sludge samples collect at the end of the experiment from microcosms

without amendments, pasteurised, with molasses or with yeast extract, incubated at 35 1C or 55 1C. Each value represents the

average of three replicates test performed within a mixture of sludge samples collected from the two parallel microcosms

(1:1). Standard deviations are reported as error bars.

050

100150200

250300350400

sludg

e 35°C

sludg

e 55°C

sludg

e plus

mola

sses 3

5°C

sludg

e plus

mola

sses 5

5°C

sludg

e plus

yeas

t extr

act 3

5°C

sludg

e plus

yeas

t extr

act 5

5°C

sludg

e plus

antib

iotics

and B

ES 35°C

sludg

e plus

antib

iotics

and B

ES 55°C

sludg

e plus

antib

iotics

and m

olybd

ate 35

°C

sludg

e plus

antib

iotics

and m

olybd

ate 55

°C

sludg

e plus

BES an

d moly

bdate

35°C

sludg

e plus

BES an

d moly

bdate

55°C

paste

urize

d slud

ge 35

°C

paste

urize

d slud

ge 55

°C

Vol

ume

(ml)

/init

ial C

OD

(g)

Experimental conditions

Fig. 4 – Total biogas (&) and methane (’) produced during the whole experiment in all the experimental conditions. Each

value, expressed as ml of biogas or methane per COD supplied, represents the average of the values related to measurements

performed in the two parallel microcosm. Standard deviations are reported as error bars.

WAT E R R E S E A R C H 41 (2007) 2407– 2416 2413

A marked (over 50%) and rapid depletion (within the

first 5 weeks) of nitrate and sulphate ions originally occurring

in the activated sludge was observed in all microcosms under

both mesophilic and thermophilic conditions, with the

exception of the ones amended with molybdate, where

sulphate ions persisted until the end of the treatment (data

not shown).

Biogas production occurred in all the biologically active

microcosms (Fig. 4) since the beginning of the experiment.

The highest biogas production was generally measured after 5

weeks of incubation (38–48% of the biogas produced all along

the 10 months experiment), with the exception of the

mesophilic microcosms amended with yeast extract and

molasses, where the highest biogas productions were

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WAT E R R E S E A R C H 4 1 ( 2 0 0 7 ) 2 4 0 7 – 2 4 1 62414

measured after 9 weeks (54% and 47% of the biogas produced

all along the 10 months experiment, respectively).

Under mesophilic conditions, biogas with an average

methane content of 55% (v/v) was extensively accumulated

in the non-amended microcosms. The occurrence of yeast

extract or molasses did not significantly affect the biogas

production yield (Fig. 4). Larger volumetric productions of

biogas and methane were observed in the microcosms

amended with antibiotics and molybdate under mesophilic

conditions, whereas only a little biogas without methane was

produced at the same temperature in the antibiotic and BES

amended microcosms and in the BES and molybdate

amended ones (Fig. 4). Methane produced in pasteurized

microcosms was the same as the one revealed in the non-

amended microcosms. A slightly higher evolution of biogas

poorer of methane (47–49% v/v) was generally observed under

thermophilic conditions in the non-inhibited microcosms

(Fig. 4).

An extensive depletion of cultivable pathogenic target

microorganisms was revealed in the non-amended micro-

cosms incubated under mesophilic conditions, where the

complete disappearance of coliform bacteria was observed at

the end of treatment. The addition of yeast extract or

molasses boosted the disinfection of activated sludge by

inducing the complete disappearance of fecal Streptococcus sp.

microflora and reducing of Escherichia coli counts by 2–3 logs

(Table 4). A complete depletion of pathogenic bacteria was

achieved in all the parallel microcosms incubated at 55 1C

(Table 4).

4. Discussion

This study was undertaken to primarily determine and

characterize the fate of organic pollutants occurring in an

activated sludge of a wastewater treatment plant when

subjected to anaerobic digestion under mesophilic and

thermophilic conditions. The purpose was to verify the actual

intrinsic potential of the selected sludge to undergo microbial

decontamination and to determine the different indigenous

populations potentially responsible for it. Thus, no exogenous

acclimated inocula were employed in the study.

A high microbial activity was generally recorded in all

biologically active sludge microcosms, where an initial

extensive nitrate and sulphate-reduction followed by a

marked production of a CH4-rich biogas were observed. In

particular, under mesophilic conditions methane represented

the 55% (v/v) of the produced biogas in the non-amended

microcosms (Fig. 4). Similar production yields have been

reported in previous studies (Shang et al., 2005; Bouskova et

al., 2005; Song et al., 2004) and these findings confirm the

actual role that anaerobic mesophilic digestion of activated

sludge might have in the bioenergy production.

Larger production of biogas was obtained under thermo-

philic conditions. However, the content of methane was lower

than under mesophilic conditions (Fig. 4). Similar findings

have already been reported in the literature (Bouskova et al.,

2005; Song et al., 2004) and ascribed to the higher specific

growth rate of biogas-generating thermophilic microorgan-

isms with respect to the mesophilic ones (Mladenovska and

Ahring, 2000). These data, taken together, suggest that

fermentative bacteria of the Fusina activated sludge were

favoured by thermophilic conditions whereas methanogen-

esis prevailed under the mesophilic ones. This conclusion is

supported by the evidence that methanogenesis, which was

the main microbial activity occurring in the microcosms

supplemented with antibiotics and molybdate, decreased

markedly by moving from the microcosms incubated at

35 1C to those incubated at 55 1C and by the finding that

biogas evolution in the parallel microcosms amended with

BES and molybdate, where fermentative bacteria prevailed on

the other microbes, increased by moving in the same

direction (Fig. 4).

No stimulation effects on the biogas and methane produc-

tion were observed upon addition of yeast extract and

molasses (Fig. 4). However, on the basis of available data, it

cannot be excluded that the two agents enhanced the activity

of other microorganisms occurring in the microcosms. The

latter finding is consistent with that recently published by

Dionisi et al. (2006) and Gerecke et al. (2006), but in contrast

with the results obtained by Moeller-Chavez and Gonzales-

Martinez (2002), who reported a doubling of methane

production upon yeast extract addition. These discrepancies

might be ascribed to the relevant differences in chemical,

physical and microbial properties often occurring among

sludges applied in different studies. However, the mechanism

of yeast extract stimulatory effect has not been explained yet

and may be different for different microorganisms (Gonzalez-

Gil et al., 2003). Concerning molasses, to the best of our

knowledge, this is the first report where such an agent was

applied as possible enhancer in the anaerobic digestion of an

activated sludge.

A complete removal of pathogenic microorganisms was

observed under thermophilic conditions, where a larger

production of acids probably accounted for this phenomenon

(Table 4). However, a marked disinfection of the activated

sludge was also achieved under mesophilic conditions (Table

4). The reduction of target pathogens observed in this study

was higher with respect to those observed in other studies

(Song et al., 2004).

Relevant removal percentages of activated sludge-carried

pollutants were observed in all biologically active microcosms

at the end of 10 months treatment (Figs. 1 and 2). Several

pollutants were extensively removed within the first 9 weeks

of incubation under both mesophilic and thermophilic

conditions. Unfortunately, the occurrence of some biological

activities (methane production) in the sterilized microcosms

after such an experimental period did not allow us to evaluate

the contribution of abiotic losses on the pollutant depletions

observed at the end of treatment. However, abiotic losses

were below the 10% up to the 5th week of incubation, i.e., in

the experimental period during which most of the abiotic

transformations probably occurred, and this allow to spec-

ulate that the detected depletions were mostly due to

pollutant biodegradation. The highest activities (in terms of

both number of pollutants removed and removal percentages

achieved) were observed in the non-amended microcosms

under mesophilic conditions (Fig. 1A). The highest produc-

tions of methane were also observed in these microcosms

(Fig. 4), thus suggesting that methanogenic bacteria were

ARTICLE IN PRESS

WAT E R R E S E A R C H 41 (2007) 2407– 2416 2415

responsible for the pollutant biodegradation. This hypothesis

is supported by the evidence that a quite extensive removal of

pollutants was also observed in the parallel mesophilic

microcosms amended with antibiotics and molybdate

(Fig. 2A) where marked productions of methane were

observed (Fig. 4). In particular, under these conditions the

highest PCB removals achieved in the study were observed.

On the other hand, the biotransformation of a number of

chlorinated aromatic and aliphatic chlorinated organic com-

pounds as well as PAHs under methanogenic mesophilic

conditions has been already reported in the literature (Freed-

man and Gosset, 1989; Bedard and Quensen, 1995; Chang

et al., 2006). Moreover, high amounts of chloride ions, which

are the final product of chlorinated pollutants mineralization,

accumulated in the microcosms with antibiotics and molyb-

date and in the non-amended ones under mesophilic condi-

tions. This indicates that PCBs occurring in the sludge

underwent extensive reductive dehalogenation. The occur-

rence of such processes has been observed in a large variety

of anaerobic contaminated habitats and matrices and very

often under methanogenic conditions (Bedard and Quensen,

1995; Wiegel and Wu, 2000). However, this is the second study

in which the occurrence of these processes in an actual-site

contaminated sludge subjected to anaerobic digestion is

documented (Patureau and Trably, 2006).

A significant activated sludge detoxification was observed

in the non-amended microcosms incubated at 35 1C (Fig. 3),

condition under which the highest pollutant removal was

achieved. This finding, combined with the fact that no GC

detectable compounds accumulated and that the amounts of

chloride ions increased in such microcosms, allow to spec-

ulate that pollutants were significantly mineralized under

such mentioned condition.

Lower activated sludge decontamination yields were gen-

erally achieved in the mesophilic microcosms amended with

BES or subjected to pasteurization (Fig. 2A) and in the parallel

microcosms incubated under thermophilic conditions prob-

ably for the lower methanogenic activity occurring in all of

them. This could be also ascribed to the fact that a sludge

collected from a mesophilic treatment plant was employed in

the study.

5. Conclusions

In conclusion, the prolonged anaerobic incubation of the

actual site contaminated activated sludge of the Fusina’s

wastewater treatment plant resulted in an extensive produc-

tion of methane-rich biogas, a marked reduction of patho-

genic cultivable bacteria and, importantly, a marked

biodegradation of the sludge-carried PCBs, PAHs and organic

pollutants with a relevant sludge detoxification. The sludge

decontamination seemed to be strictly correlated to metha-

nogesis and both bioremediation and biomethanization of the

activated sludge were maxima under mesophilic conditions.

Similar observations have been already reported in the

literature (Benabdallah El-Hadj et al., 2006; Chang et al.,

1999; Christensen et al., 2004; Dionisi et al., 2006; Gerecke

et al., 2006; Jianlong et al., 2000; Katsoyiannis and Samara,

2004; Patureau and Trably, 2006; Trably et al., 2003) but the

original aspects of this work are that the findings reported

above were obtained on an actual-site contaminated acti-

vated sludge, that the chemical analysis of pollutants was

flanked by ecotoxicity monitoring and that a special attention

was paid to correlate pollutant and ecotoxicity depletions to

microbial populations involved in the sludge digestion. The

results of this study clearly indicate that anaerobic stabiliza-

tion merits to be reconsider as possible strategy for an

effective and environmental and economical sustainable

disposal of activated sludge resulting from treatment plants

fed with industrial wastewaters. Indeed, under mesophilic

conditions, it might offer the opportunity to conjugate an

effective mineralization and disinfection of activated sludge

with other key features poorly exploited so far, such as the

decontamination and detoxification of activated sludge and

its valorization through the production of large amounts of

methane-rich biogas.

Acknowledgements

The Authors sincerely thank Prof. F. Cecchi and Dr. D.

Bolzonella (Scientific and Technological Department, Univer-

sity of Verona, Italy) and the COPROB sugar-refinery (Bologna,

Italy) for providing the activated sludge and the molasses,

respectively, employed in the study. The authors also thank

Dr. L. Bolelli (SMETEC, University of Bologna, Italy) for his help

in the bioluminescent assays. The project was funded by the

Italian MIUR (COFIN/PRIN 2003).

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