ARTICLE IN PRESS

Water Research 37 (2003) 4561–4572

Mesophilic and thermophilic anaerobic digestion

of primary and secondary sludge. Effect of
pre-treatment at elevated temperature
Hariklia N. Gavala*, Umur Yenal, Ioannis V. Skiadas,
Peter Westermann, Birgitte K. Ahring
The Environmental Microbiology and Biotechnology Group (EMB), BIOCENTRUM-DTU, Bldg 227,
Technical University of Denmark, Lyngby 2800, Denmark
Received 26 June 2002; received in revised form 5 June 2003; accepted 19 June 2003

Abstract

Anaerobic digestion is an appropriate technique for the treatment of sludge before final disposal and it is employed
worldwide as the oldest and most important process for sludge stabilization. In general, mesophilic anaerobic digestion
of sewage sludge is more widely used compared to thermophilic digestion. Furthermore, thermal pre-treatment is
suitable for the improvement of stabilization, enhancement of dewatering of the sludge, reduction of the numbers of
pathogens and could be realized at relatively low cost especially at low temperatures. The present study investigates (a)
the differences between mesophilic and thermophilic anaerobic digestion of sludge and (b) the effect of the pre-
treatment at 70
C on mesophilic and thermophilic anaerobic digestion of primary and secondary sludge. The pre-
treatment step showed very positive effect on the methane potential and production rate upon subsequent thermophilic
digestion of primary sludge. The methane production rate was mostly influenced by the pre-treatment of secondary
sludge followed by mesophilic and thermophilic digestion whereas the methane potential only was positively influenced
when mesophilic digestion followed. Our results suggest that the selection of the pre-treatment duration as well as the
temperature of the subsequent anaerobic step for sludge stabilization should depend on the ratio of primary to
secondary sludge.
r 2003 Elsevier Ltd. All rights reserved.

Keywords: Activity; Kinetics; Mesophilic digestion; Pre-treatment; Primary sludge; Secondary sludge; Thermophilic digestion

1. Introduction sludge is more widely used compared to thermophilic

The residue generated during the primary, the
secondary and the tertiary wastewater treatment is often
called ‘‘sludge’’. Anaerobic digestion is an appropriate
technique for the treatment of sludge before final
disposal and it is employed worldwide as the oldest
and most important process for sludge stabilization [1].
In general, mesophilic anaerobic digestion of sewage
*Corresponding author. Fax: +45-45883276.
E-mail address: hng@biocentrum.dtu.dk (H.N. Gavala).
digestion, mainly because of the lower energy require-
ments and higher stability of the process. However, the
thermophilic anaerobic digestion process is usually
characterized by accelerated biochemical reactions,
higher growth rate of microorganisms and accelerated
interspecies hydrogen transfer resulting in an increased
methanogenic potential at lower hydraulic retention
times [2]. Also, the enhanced hygienization effect of the
thermophilic process [3,4] complies with the EU policy
for elimination of pathogens, originating mainly
from humans and animals; it has been reported that

0043-1354/$ - see front matter r 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0043-1354(03)00401-9
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4562 H.N. Gavala et al. / Water Research 37 (2003) 4561–4572
thermophilic anaerobic digestion of sewage sludge can
lead to EPAs class A biosolids, which are suitable for
subsequent land application [4].
The microbiology of anaerobic digestion is compli-
cated involving several bacterial groups forming a
complex interdependent food web. However, four major
steps can be distinguished. In the first hydrolysis step,
both solubilization of insoluble particulate matter and
biological decomposition of organic polymers to mono-
mers or dimers take place. Acidogenesis and acetogen-
esis follow in the second and third step while in the
fourth and final step methane is produced by methano-
genic archaea [5]. The methanogenesis is, in most of the
cases, the rate-limiting step of the overall process;
however, hydrolysis could also be the rate-limiting step
during the anaerobic digestion of wastewater rich in
organic solids [6], such as activated sludge [7]. A number
of pre-treatment processes have been developed and
investigated in order to improve and enhance the
disintegration and solubilization of sludge solids and
are reviewed by Muller. [8]. Thermal pre-treatment is
suitable for the improvement of stabilization, enhance-
ment of dewatering of the sludge, reduction of the
numbers of pathogens and could be realized at relatively
low costs [8].
The effect of thermal pre-treatment on the anaerobic
biodegradability and toxicity of activated (secondary)
sludge was investigated in the study of Stuckey and
McCarty [9]. The temperature range studied was
between 150
C and 275
C. They observed an optimum
in methane production after pre-treatment at 175
C
whereas at more elevated temperatures, a decline in
methane production and sludge biodegradability was
observed which was attributed to the formation of toxic,
refractory compounds. Another experiment involving
pre-treatment of primary and secondary sludge for 1 h at
temperatures between 120
C and 220
C was described
by Pinnekamp [10]. A decrease in gas production below
that of the nonpre-treated sludge was observed for
temperatures higher than 180
C; however, the differ-
ences in gas yield increase at pre-treatment temperatures
between 120
C and 180
C were not considerable. Li and
Noike [7] focussed on the thermal pre-treatment of
secondary sludge and they reported 170
C and 60 min as
the most favourable pre-treatment temperature and
duration regarding COD removal and gas production
during mesophilic (37
C) anaerobic digestion yielding
an increase of approximately 100% compared to the
nonpre-treated sludge. However, higher temperature
pre-treatment has high energy requirements and is
difficult to operate. Thus, thermal pre-treatment at a
lower temperature, i.e. below 100
C becomes more and
more attractive. In the study of Wang et al. [11]
the effectiveness of lower temperature pre-treatment
(60–100
C) on mesophilic (37
C) anaerobic digestion of
activated sludge was investigated. It was concluded that
thermal pre-treatment resulted in a significant increase
(30–52%) in methane yield; however, no significant
differences were observed between pre-treatments at
60
C, 80
C and 100
C. The rate of the methane
generation was higher after the pre-treatment at 60
C
compared with 80
C and 100
C. One can see that there
are already numerous studies investigating the effect of
the pre-treatment temperature on the anaerobic diges-
tion of sludge. However, most studies focus on the
investigation of the temperature selection and pre-
treatment duration using one type of sludge while in
most municipal treatment plants primary and secondary
sludge streams are combined prior to anaerobic diges-
tion. Furthermore, no study exists focusing on the
different characteristics and response of different kind of
sludge to a thermal pre-treatment process. Therefore,
the investigation of the pre-treatment duration in
relation with the different type of sludge treated is of
great interest and it will significantly contribute to the
progress of the specific scientific field.
The scope of the present study was to investigate (a)
the differences between mesophilic and thermophilic
anaerobic digestion of sludge concerning the digester’s
performance characteristics and the relative microbial
activities expressed as the different substrate consump-
tion activities and (b) the effect of the pre-treatment at
70
C on mesophilic and thermophilic anaerobic diges-
tion of primary and secondary sludge. For this purpose,
two semi-continuous digesters were started-up and
operated at 20 days retention time at 37
C and 55
C,
respectively. Also, two kinds of batch experiments were
carried out: (a) experiments in order to measure the
activity of the mesophilic and thermophilic digester,
respectively, regarding butyrate, propionate, acetate,
formate and hydrogen consumption and (b) experiments
aiming at the examination of the effect of the pre-
treatment at 70
C on the methane potential and the
methane production rate during the mesophilic and
thermophilic digestion of primary and secondary sludge.
The results obtained confirmed the initial hypothesis and
suggest that the optimal pre-treatment duration as well
as the temperature of the subsequent anaerobic step for
sludge stabilization depends on the ratio of primary to
secondary sludge.
2. Materials and methods
2.1. Analytical and computational methods
Determinations of the total (TS) and volatile (VS)
solids were carried out according to Standard Methods
[12]. For the quantification of volatile fatty acids,
acidified samples with 17% H3PO4 were analysed on a
gas chromatograph (Hewlett Packard 5890 series II)
with a flame ionization detector and a capillary column
 ARTICLE IN PRESS
H.N. Gavala et al. / Water Research 37 (2003) 4561–4572
(Hewlett Packard FFAP 30 m, inner diameter 0.53 mm,
film 1 mm). Biogas composition in methane was quanti-
fied with a gas chromatograph (Shimadzu GC-8A)
equipped with a flame ionization detector and a packed
column (Porapak Q, 80/100-mesh). Hydrogen partial
pressure was measured with a Trace Analytical RGA-3
Reduction Gas Analyser. The medium (BA medium)
used in batch experiments was prepared from the
following stock solutions (chemicals in g l1 of distilled
water): (A) NH4Cl, 100; NaCl, 10; MgCl2  6H2O, 10;
CaCl2  2H2O, 5; (B) K2HPO4  3H2O, 200; (C) resazurin,
0.5; (D) trace metals and selenite solution: FeCl2  4H2O,
2; H3BO3, 0.05; ZnCl2, 0.05; CuCl2  2H2O, 0.038;
MnCl2  4H2O, 0.05; (NH4)6Mo7O24  4H2O, 0.05; AlCl3,
0.05; CoCl2  6H2O, 0.05; NiCl2  6H2O, 0.092; ethylene-
di-amine-tetra-acetate, 0.5; Na2SeO3  5H2O, 0.1; HCl
37%, 1 ml; (E) vitamin solution according to Wolin et al.
[13]. The following volumes of stock solutions were
added to 916 ml of distilled water: (A) 10 ml, (B) 2 ml,
(C) 1 ml, (D) 1 ml, (E) 10 ml. Fifty millitres of a 52 g l1
NaHCO3 solution were added as well. The medium was
gassed with 80% N2–20% CO2, dispensed and auto-
claved. Before inoculation the medium was reduced with
a 25 g l1 Na2S  9H2O solution to a ratio of 0.1 ml/10 ml
of medium.
Simulation of the experimental data and estimation of
the individual kinetic parameters along with their
associated statistics was made using the Levenberg–
Marquardt method for nonlinear regression analysis
[14,15].
Fig. 1. The set-up of the digesters used for the mesophilic and thermophilic digestion of the mixed sludge.
4563
2.2. Draw and fill experiments
Two 1 l (useful volume) digesters inoculated with
anaerobic sludge were fed with mixed (dewatered
mixture of primary and secondary) sludge from the
Lundofte municipal wastewater treatment plant located
at Lyngby in Denmark. The anaerobic sludge used as
inoculum was acclimated to primary sludge, household
wastes and cow manure. The digesters were fed daily in a
draw and fill manner at a hydraulic retention time of 20
days and were kept in two incubators at 37
C and 55
C,
respectively. In Fig. 1 the set-up of the digesters is
shown. Their performance was followed by measuring
the pH, biogas production and composition, volatile
fatty acids, total and volatile solids concentration.
Biogas production corresponded to the volume of the
liquid displaced to the third bottle (Fig. 1).
2.3. Batch kinetic experiments
Batch experiments were carried out in triplicates at
35
C and 55
C in 58 ml serum vials sealed with butyl
rubber stoppers and aluminium crimps. The serum
bottles contained 15 ml of BA medium and acetate or
propionate or butyrate or formate as sodium salts at a
final concentration of approximately 20 mM. The serum
bottles were inoculated with 5 ml of anaerobic mixed
liquor from the mesophilic or the thermophilic digester.
When hydrogen was used as the substrate, the vials
were pressurized to 2 atm with a gas mixture (20/80) of
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4564 H.N. Gavala et al. / Water Research 37 (2003) 4561–4572
CO2/H2. Triplicate controls were also prepared contain-
ing only 15 ml of BA medium and 5 ml of anaerobic
mixed liquor (no substrate added). VFA concentration
and methane production were followed throughout the
experiments. The initial activities of the anaerobic
cultures regarding butyrate, propionate and acetate
consumption as well as the kinetic parameters of the
utilization of the different substrates were calculated. In
case of experiments with hydrogen and formic acid as
substrates only the initial activities based on the
methane production were calculated.
2.4. Experiments with pre-treated sludge at 70
C
In total, four sets of batch experiments were carried
out aiming at the investigation of the effect that pre-
treatment at 70
C had on: (a) mesophilic digestion of
primary sludge, (b) mesophilic digestion of secondary
sludge, (c) thermophilic digestion of primary sludge and
finally (d) thermophilic digestion of secondary sludge.
Batch experiments were carried out in triplicate in
117 ml serum vials. For each experimental set, 5
triplicates were prepared containing 30 ml of sludge
pre-treated at 70
C for 0, 1, 2, 4 and 7 days, respectively,
and inoculated with 10 ml of anaerobic liquor. Two
controls duplicates were prepared for each triplicate; one
containing 30 ml of sludge and the other containing
10 ml of anaerobic liquor. Anaerobic mixed liquor from
the two digesters was used as inoculum for studies of the
effect of the pre-treatment at 70
C. VFA concentration,
hydrogen gas concentration and methane production
and production rate were followed throughout the
experiments.
2.5. Kinetic expressions and calculations
The initial activities (Ac ) were calculated using the
following expression for butyrate, propionate and
acetate consumption [16,17]:
 
1 dS
Ac ¼  ; ð1Þ
x0 dt
where x0 is the initial VS concentration and S is the
substrate concentration.
The following equation was used for the calculation of
initial activities based on the methane production in
batch experiments with hydrogen and formate as
substrates.
 
1 dP
Ac ¼ ; ð2Þ
X0 dt
where X0 is the initial VS mass and P is the amount of
methane produced.
Acetate, propionate and butyrate consumption was
assumed to follow Monod kinetics as it is directly
related to microbial growth:
dS umax S
 ¼ x0 ; ð3Þ
dt KS þ S
where umax is the maximum specific substrate utilization
rate, KS is the half saturation constant and x0 is the
initial VS concentration. It should be noted that umax
includes the total VS percentage, f ; which is responsible
for the specific bioconversion of the substrate S; the
maximum specific growth rate, mmax and the biomass
yield factor, Yx=S [18]:
mmax f
umax ¼ : ð4Þ
Yx=S
Consequently, umax reflects the ability of the anaerobic
sludge to degrade specific substrates; the higher the umax
value the faster is the biodegradation of the substrate, S:
This can be due to either domination of different species
of microorganisms characterized by increased mmax and/
or decreased Yx=S or higher percentage (f ) of micro-
organisms responsible for the consumption of the
specific substrate or to both of them. The biomass was
considered to be constant during the activity experi-
ments, since the theoretical increase of VS was too low
compared to the initial VS concentration in the different
experiments. Therefore, it can be assumed that it had no
significant effect on the degradation rate [16]. The data
from the activity experiments were used to estimate the
values of the kinetic parameters, umax and KS ; according
to Eq. (3), for each triplicate of batch experiments
(a triplicate for each substrate) after subtracting the
corresponding acid concentration in the control tripli-
cate. Applying the Levenberg–Marquardt method for
nonlinear regression analysis did the fitting of the
theoretical equation to the experimental data.
3. Results and discussion
3.1. Draw and fill experiments
Two batches of mixed sludge were stored at 4
C and
used for the feeding of the two draw and fill digesters.
The TS/VS content of the two batches of mixed sludge
was 37.319/24.971 and 48.470/32.332 g/l, respectively.
The digesters were operated for approximately 7 months
and their performance was stabilized after the first 2
months. The mean values of the characteristics of the
mesophilic (A) and thermophilic (B) draw and fill
digesters during the last 5-months operation period are
shown in Table 1. It is evident that the characteristics of
the mesophilic and the thermophilic anaerobic digester
are similar under the specific operating conditions. Both
show the same efficiency of organic solids removal and
methane production. However, the concentration of the
VFA in the thermophilic digester was higher than in the
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H.N. Gavala et al. / Water Research 37 (2003) 4561–4572 4565

Table 1
Characteristics of the mesophilic and thermophilic draw and fill digesters during the operation period

Characteristics Mesophilic (37
C) Digester A Thermophilic (55
C) Digester B

pH 7.670.3 7.870.2
TS (g/l) 24.0174.28 24.2573.97
VS (g/l) 13.2371.86 13.4971.48
COD (g/l) 21.2673.22 22.3672.63
Biogas production (ml/d) 406768 426756
Methane content in biogas (%) 61.674.9 60.275.7
Acetate (mM) 0.5170.55 4.4371.97
Propionate (mM) — 1.0670.98

25 25

20 20
Acetate (mmol/l)

Acetate (mmol/l)
15 15

10 10

5 5

0 0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140
(a) Time (h) (b) Time (h)

Fig. 2. Experimental and theoretical acetate concentration profiles for acetate fed (a) mesophilic and (b) thermophilic anaerobic
cultures. Points represent measurements from the experiments in triplicate and lines represent theoretical predictions.

mesophilic digester. The latter is in accordance with
Zinder [19] who reported that high effluent VFA
concentrations are often found in thermophilic anaero-
bic digesters due to the relatively high sensitivity of
thermophilic anaerobic microorganisms to intermediate
compounds. Also Lettinga [20] reported that thermo-
philic digestion suffers from a higher degree of product
and/or substrate inhibition.
3.2. Batch kinetic experiments
Batch experiments were performed in order to
compare the microbial activities of the mesophilic and
thermophilic digester regarding butyrate, propionate,
acetate, formate and hydrogen consumption. Experi-
mental data as well as theoretical predictions based on
Eq. (2) for mesophilic and thermophilic anaerobic
sludge fed with acetate are shown in Figs. 2a and b,
respectively. Propionate and butyrate consumption
profiles are shown in Figs. 3 and 4, respectively and
the calculated initial activities and kinetic parameters
yielded by the fitting process for acetic, propionic and
butyric acids are presented in Table 2. The initial
methanogenic activities for hydrogen and formic acid
are shown in Table 2 as well. It is evident that the
thermophilic anaerobic inoculum was characterized by a
higher ability to utilize the methanogenic substrates
(acetate, formate and hydrogen) as well as propionate
compared to the mesophilic inoculum as shown by the
calculated initial activities and kinetic constants. This is
in accordance with the study of Kiyohara et al. [21]
where the activities of acidogenic and methanogenic
bacteria fed with raw sludge were about 1.8 and 1.6
times higher under thermophilic conditions than under
mesophilic conditions at a retention time of 20 days.
One can observe that there is a difference between the
calculated KS values under thermophilic and mesophilic
conditions, especially for acetate and butyrate (Table 2).
These values represent just a tendency since the standard
deviation of the KS values is high due to the limited
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4566 H.N. Gavala et al. / Water Research 37 (2003) 4561–4572

30 30

Propionate (mmol/l) 25 25

Propionate (mmol/l)
20 20

15 15

10 10

5 5

0 0
0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120 140 160
(a) Time (h) (b) Time (h)
Fig. 3. Experimental and theoretical propionate concentration profiles for propionate fed (a) mesophilic and (b) thermophilic
anaerobic cultures. Points represent measurements from the experiments in triplicate and lines represent theoretical predictions.

25 25

20 20
Butyrate (mmol/l)
Butyrate (mmol/l)

15 15

10 10

5 5

0 0
0 20 40 60 80 0 20 40 60 80
(a) Time (h) (b) Time (h)
Fig. 4. Experimental and theoretical butyrate concentration profiles for butyrate fed (a) mesophilic and (b) thermophilic anaerobic
cultures. Points represent measurements from the experiments in triplicate and lines represent theoretical predictions.

number of experimental points at the KS values area.
Nevertheless, this is consistent with other values found
in the literature, i.e. Siegrist et al. [22], reported a KS
value of 40 mg COD l1 (0.63 mmol l1) for acetate
degradation during the mesophilic digestion of primary
sludge whereas the corresponding value under thermo-
philic conditions was 300 mg COD l1 (4.69 mmol l1).
Oppositely, KS values of 280 mg COD l1 (1.75 mmol l1)
and 30 mg COD l1 (0.19 mmol l1) have been reported
for butyrate consumption under mesophilic [23] and
thermophilic [24] conditions, respectively. This suggests
that microorganisms with different affinities to acetate
and butyrate dominate under mesophilic and thermo-
philic conditions.
3.3. Experiments with pre-treated sludge at 70
C
3.3.1. Effect of the pre-treatment on anaerobic mesophilic
digestion of primary sludge
After inoculation of the pre-treated primary sludge at
70
C with mesophilic anaerobic mixed liquor from
digester A, the production of methane and volatile fatty
acids concentration were followed until they reached a
stationary level. Then the total amount of methane as
well as the mean methane production rate was
calculated. In Table 3 the net production of methane
per gram of volatile solids added (after subtracting the
methane production in control vials with anaerobic
inoculum) as well as the mean methane production rate
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H.N. Gavala et al. / Water Research 37 (2003) 4561–4572 4567

Table 2
Calculated initial activities and kinetic parameters concerning acetate, propionate and butyrate consumption by the mesophilic and
thermophilic anaerobic sludge

Substrate Initial activity umax (mmol g-VS1 h1) KS (mmol l1)

mmol g-VS1 h1 mmol CH4 g-VS1 h1

Mesophilic anaerobic sludge

Acetate 6.14  102 5.82  10275.98  103 2.73  10274.65  101
Propionate 4.54  102 5.01  10279.53  103 0.8471.93
Butyrate 12.58  102 15.56  10272.72  102 2.8272.25
Formate — 4.66  102 — —
Hydrogen — 6.77  102 — —

Thermophilic anaerobic sludge

Acetate 9.66  102 15.59  10275.27  102 8.5276.34
Propionate 7.22  102 8.73  10271.74  102 0.9972.18
Butyrate 12.36  102 12.64  10277.28  101 0.2770.07
Formate — 6.70  102 — —
Hydrogen — 6.94  102 — —

The values of the kinetic parameters are presented along with their standard deviation.

Table 3
The net production of methane and the mean methane production rate per gram of volatile solids added during mesophilic and
thermophilic digestion of pre-treated primary and secondary sludge

Pre-treatment duration (days) Net methane production Mean methane production

(mmol CH4/gr VS) rate (mmol CH4/d/gr VS)

Experimental set (a)—mesophilic digestion of primary sludge

0 21.22570.592 2.67470.075
1 19.78070.267 2.92170.039
2 19.75870.940 2.80670.134
4 24.66070.217 2.25970.020
7 22.85170.347 2.56370.039

Experimental set (b)—mesophilic digestion of secondary sludge

0 8.29870.243 1.06270.031
1 9.94070.295 1.46470.043
2 10.20870.360 1.71970.061
4 10.28370.422 2.59870.051
7 10.44970.478 1.99070.091

Experimental set (c)—thermophilic digestion of primary sludge

0 13.70970.350 1.56770.040
1 19.98870.623 2.56570.080
2 18.90470.654 2.76670.096
4 24.71170.468 2.09670.040
7 25.48570.557 2.37570.052

Experimental set (d)—thermophilic digestion of secondary sludge

0 10.90370.185 1.10470.019
1 10.20470.340 1.15070.038
2 10.46770.276 1.31570.035
4 10.94470.197 1.52770.028
7 10.95470.280 1.74770.045
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Table 4
The production of methane per gram of volatile solids in control vials with primary/secondary sludge and the methane production in
control vials with anaerobic sludge

Pre-treatment duration (days) Methane production in Methane production in

control vials with sludge control vials with anaerobic
(mmol CH4/gr VS) sludge (mmol CH4)

Experimental set (a)—mesophilic digestion of primary sludge

0 4.61871.079 0.17170.009
1 0.61870.875 0.23370.014
2 0 0.26670.006
4 0 0.22870.007
7 0 0.16470.003

Experimental set (b)—mesophilic digestion of secondary sludge

0 4.03270.023 0.18970.006
1 0.05870.005 0.25970.014
2 0.24870.144 0.25770.013
4 0.25270.171 0.30770.010
7 0 0.19370.007

Experimental set (c)—thermophilic digestion of primary sludge

0 0 0.22670.020
1 0 0.36970.069
2 0 0.62570.103
4 0 0.60970.013
7 0 0.30570.019

Experimental set (d)—thermophilic digestion of secondary sludge

0 0 0.28470.007
1 0 0.46270.040
2 0 0.48870.016
4 0 0.45370.0004
7 0 0.25470.048

are shown. Pre-treatment of 4 and 7 days at 70
C had a
slightly positive effect on the total methane production
resulting in an increase of 16.2% and 7.7%, respectively,
while the 1 and 2 days pre-treatment had no marked
effect. On the other hand, 1 and 2 days pre-treatment
resulted in an increased mean methane production rate
(9.2% and 4.9%, respectively). The mean methane
production rate in the vials containing 4 and 7 days
pre-treated primary sludge was comparable or lower
compared to the untreated raw primary sludge. No
methane production was observed during the pre-
treatment of the primary sludge at 70
C. In control
vials containing only primary sludge, methane produc-
tion was observed during incubation at 37
C in vials
with no and 1 day pre-treatment (Table 4). This implies
that there are indigenous microorganisms in the primary
sludge capable of methane production; however, in-
cubation at 70
C for a prolonged period (more than 1
day) resulted in their inactivation. Incubation of
primary sludge at 70
C led to increased H2 levels
(Table 5). However, H2 was stabilized at lower levels
similar to that of anaerobic sludge in all experimental
vials after approximately 3 days of anaerobic digestion
at 37
C (Table 5).
3.3.2. Effect of the pre-treatment on anaerobic mesophilic
digestion of secondary sludge
The net methane production as well as the mean
methane production rate was followed in the secondary
sludge pre-treated at 70
C and inoculated with meso-
philic anaerobic mixed liquor from digester A (Table 3).
A gradual increase in methane production as a function
of the pre-treatment duration was observed, starting
with an increase of 19.8% for 1 day up to 25.9% for 7
days pre-treatment, respectively. Furthermore, a 144.6%
increase in the mean methane production rate was
observed for 4 days pre-treatment compared to the
untreated secondary sludge. The methane potential of
the primary sludge was found to be almost 2.5 times
higher than the potential of the secondary sludge.
It can be concluded that pre-treatment at 70
C
is more efficient for the secondary sludge with respect
to methane potential and mean methane production
rate compared to the primary sludge. No methane
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H.N. Gavala et al. / Water Research 37 (2003) 4561–4572 4569

Table 5
Hydrogen levels after pre-treatment of primary/secondary sludge and during mesophilic/thermophilic anaerobic digestion

Pre-treatment duration (days) H2 level after the pre-treatment at 70
C H2 level in the triplicate vials during
(% H2) anaerobic digestion (% H2)

Experimental set (a)—mesophilic digestion

of primary sludge
0 2.19  10572.37  104
1 Not measured 3.39  10573.69  104
2 7.60  10475.90 104 3.22  10574.67  104
4 3.81  10371.27  104 2.73  10573.11  104
7 1.25  10278.18  104 5.85  10575.71  104

Experimental set (b)—mesophilic digestion

of secondary sludge
0 8.97  10574.24  104
1 1.14  10 78.48  10
2 5
9.37  10574.46  104
2 5.93  10371.35  104 1.34  10476.75  104
4 1.20  10271.64  105 1.59  10474.21  104
7 1.13  10276.80  104 1.20  10476.77  104

Experimental set (c)—thermophilic

digestion of primary sludge
0 1.34  10372.76  103
1 2.76  10 77.17  10
6 7
9.93  10473.68  103
2 8.70  10576.68  105 8.95  10472.65  103
4 3.11  10471.19  104 8.76  10473.02  103
7 7.20  10573.88  105 9.92  10472.63  103

Experimental set (d)—thermophilic

digestion of secondary sludge
0 7.41  10471.20  103
1 2.61  10 71.54  10
3 3
7.25  10471.16  103
2 4.48  10374.79  104 6.89  10471.09  103
4 5.16  10373.20  104 5.89  10471.22  103
7 4.88  10373.95  104 6.13  10471.29  103

production took place during the pre-treatment of
neither primary nor secondary sludge. However, in
control vials containing only secondary sludge, methane
production was observed during the period of incuba-
tion at 37
C in vials with no, 1, 2 and 4 days pre-
treatment (Table 4). Probably the indigenous methano-
gens in secondary sludge are more resistant to high
temperatures than those present in primary sludge.
Incubation of secondary sludge at 70
C led to even
higher H2 levels compared to the primary sludge.
However, H2 was stabilized at lower levels similar to
that of anaerobic sludge in all triplicate vials after
approximately 1 day of incubation at 37
C (Table 5). H2
levels were in the same range during mesophilic
digestion of both primary and secondary sludge.
3.3.3. Effect of the pre-treatment on anaerobic
thermophilic digestion of primary sludge
The inoculum for these experiments was thermophilic
anaerobic mixed liquor from digester B. The net
production of methane as well as the mean methane
production rate during thermophilic digestion of non-
and pre-treated at 70
C primary sludge are shown in
Table 3. Both parameters were greatly affected: the
methane production increased with the pre-treatment
duration (37.9–85.9%) as in the mesophilic secondary
sludge digestion while the pre-treatment of 2 days
yielded the most efficient mean methane production
rate (76.5% higher compared to the raw sludge).
Thermophilic anaerobic digestion of primary sludge
yielded lower methane potential and mean methane
production rate compared to the mesophilic anaerobic
digestion under the specific experimental conditions.
This may be due to the presence of inhibitory
components to the more sensitive thermophilic metha-
nogenic microorganisms or to the increased levels of H2
(Table 5). It is noticeable that H2 levels after the pre-
treatment at 70
C were lower than these measured
during the thermophilic anaerobic digestion of raw and
pre-treated primary sludge (Table 5). No methane
production was observed either during the pre-treatment
of primary sludge or in the control vials containing only
 ARTICLE IN PRESS
4570 H.N. Gavala et al. / Water Research 37 (2003) 4561–4572

primary sludge during the incubation at 55
C (Table 4).

This means that there were not indigenous thermophilic
methanogens in the primary sludge.

3.3.4. Effect of the pre-treatment on anaerobic

thermophilic digestion of secondary sludge
During this set of batch experiments, the pre-treated
secondary sludge at 70
C was inoculated with thermo-
philic anaerobic mixed liquor from digester B. The net
methane production as well as the mean methane
production rate are shown in Table 3. The methane
potential of the pre-treated secondary sludge remained
almost the same as for the untreated raw sludge. The
mean methane production rate increased gradually with
the pre-treatment duration from 4.2% up to 36.8%.
Thermophilic digestion of secondary sludge was ap-
proximately 31% more efficient than the mesophilic
digestion regarding the methane potential (Table 3).
However, pre-treatment at 70
C did not seem to
influence the methane potential. Like in the previous
batch experiments, no methane production was ob-
served during the pre-treatment of the secondary sludge
at 70
C. Also, no methane was produced in the control
vials containing only secondary sludge during their
incubation at 55
C. This is probably an indication that
no indigenous thermophilic methanogens are present in
the secondary sludge. Similar to the experimental set in
Section 3.3.3, incubation of secondary sludge at 70
C
led to increased H2 levels. However, after 1 day of
incubation at 55
C, H2 levels were stabilized at a slightly
higher level than observed for mesophilic digestion of
secondary sludge.
In Figs. 5a and b the percentage increase of the
methane potential and production rate after pre-treat-
ment during mesophilic and thermophilic anaerobic
digestion of primary and secondary sludge is shown. It is
obvious that the effect of the pre-treatment at 70
C on
the mesophilic digestion of primary sludge is minimal
with respect to both methane potential and production
rate. Pre-treatment had, however, very positive effects
on thermophilic digestion of primary sludge, where the
methane potential and production rate showed the
highest increase after 4, 7 (80–86%) and 1, 2 (64–77%)
days of pre-treatment, respectively. The methane pro-
duction rate from secondary sludge showed the highest Fig. 5. (a) Percentage increase of the methane potential and
increase after the pre-treatment at 70
C during production rate with the pre-treatment duration during the
both mesophilic (43–145% increase) and thermophilic mesophilic (M) and thermophilic (T) anaerobic digestion of
(4–58% increase) digestion. The methane potential of primary sludge. (b) Percentage increase of the methane
secondary sludge is only positively influenced when potential and production rate with the pre-treatment duration
mesophilic digestion follows (20–26%). The higher during the mesophilic (M) and thermophilic (T) anaerobic
methane potential of primary sludge compared to digestion of secondary sludge.
secondary sludge (Table 3) and the different effects of
the pre-treatment in each case might be explained by the
different composition of the two types of sludges. constitute 18% and 10%, respectively [25]. Secondary
Primary sludge is solid organic material composed sludge consists mainly of bacterial cells, characterized by
mainly of carbohydrates (55%) while proteins and lipids higher protein content (36%) and much lower amounts
 ARTICLE IN PRESS
H.N. Gavala et al. / Water Research 37 (2003) 4561–4572
of carbohydrates (20%) than primary sludge [26].
Furthermore, the integrity of bacterial cells hampers
the biological decomposition of secondary sludge. It has
been suggested that thermal pre-treatment destroys the
cell walls and makes the proteins accessible for
biological degradation [8]. Conclusively, our experimen-
tal results suggest that the selection of the pre-treatment
duration as well as the temperature of the subsequent
anaerobic step for sludge stabilization should depend on
the composition of the sludge, i.e. whether it is primary,
secondary, a mixture of primary and secondary and
their ratio.
4. Conclusions
The maximum specific utilization rates of acetate and
propionate as well as the initial activity of methane
production from hydrogen and formic acid were higher
when the thermophilic inoculum was used. Conse-
quently, thermophilic anaerobic digestion is more
efficient in terms of organic matter removal and
methane production than the mesophilic process. The
fact that no significant differences were observed in the
characteristics of the thermophilic digester compared to
the mesophilic under the specific experimental condi-
tions and relatively high retention time suggests that the
benefits of the thermophilic anaerobic digestion become
evident at lower retention times.
In the study of the effect of the pre-treatment on the
methane potential and production rate during the
mesophilic and thermophilic digestion of primary and
secondary sludge, nonmethanogenic biological activity
was observed during the pre-treatment step as increased
hydrogen percentage. The pre-treatment step showed
very positive effect on the methane potential and
production rate upon subsequent thermophilic digestion
of primary sludge. The methane production rate was
mostly influenced by the pre-treatment of secondary
sludge followed by mesophilic and thermophilic diges-
tion whereas the methane potential only was positively
influenced when mesophilic digestion followed. The
presented results suggest that the selection of the pre-
treatment duration as well as the temperature of the
subsequent anaerobic step for sludge stabilization
should depend on the ratio of primary to secondary
sludge.
Acknowledgements
The authors wish to thank the Commission of the
European Communities for the financial support of this
work under Marie Curie grant No HPMF-CT-1999-
00371.
4571
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