Renewable Energy 62 (2014) 73e78

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Renewable Energy
journal homepage: www.elsevier.com/locate/renene

Co-digestion of sewage sludge and crude glycerol from biodiesel

production
E. Athanasoulia*, P. Melidis, A. Aivasidis
Democritus University of Thrace, Dept. of Environmental Engineering, Laboratory of Wastewater Management & Treatment technologies, Vas. Soas 12, 67
100 Xanthi, Greece

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 15 May 2012
Accepted 23 June 2013
Available online 19 July 2013

Biodiesel has become one of the most attractive fuels since it was globally understood the renewability
of its nature. Glycerol is a major byproduct of biodiesel production which is often regarded as a waste
stream which is accompanied by a signicant disposal cost. The effect of glycerol on the performance
of a cascade of two anaerobic continuous stirred tank (CSTR) reactors treating thickened sludge at
mesophilic conditions was investigated. The objective of this study was to evaluate the use of glycerol
as a co-substrate during the anaerobic treatment of sewage sludge. For this purpose, feed mixtures of
sewage sludge supplemented with 0%, 2%, 3% and 4% (v/v) glycerol were tested at hydraulic retention
times between 12.3 d and 19.7 d. By adding 4% of glycerol, the system failed due to overloading. In all
other cases, biodegradability of mixtures estimated to be higher than 88%, while the methane yield
coefcient was 0.8 Lbiogas/g TVSremoved. Moreover, co-digestion improved biogas production by 3.8e4.7
times.
2013 Elsevier Ltd. All rights reserved.

Keywords:
Co-digestion
Sewage sludge
Crude glycerol
Biodiesel
Biogas
Serial digestion

1. Introduction
Anaerobic digestion of sewage sludge is a well-known technology due to the energy gained through biogas production as well
as due to the achieved sludge stabilization and volume reduction.
The heating value (55.5 MJ/kg) of methane, the main component of
biogas, is equivalent to 1.2 kg of diesel or 3.7 kg of wood, therefore
anaerobic digestion constitutes a continuously more promising
technology. Although sewage sludge is digested alone in most
application, the use of co-substrates in anaerobic digestion is a
growing trend for improving methane yields with several associated benets. Co-digestion can enhance the anaerobic digestion
process, because co-substrates can supply nutrients which may be
decient, and at the same time have an overall positive synergistic
effect in the digestion medium, leading to stable digestion and
enhanced gas yields [1].
On the other hand, glycerol is a major byproduct of biodiesel
production. In general, for every 100 kg of biodiesel produced, 10 kg

Abbreviations: CSTR, continuous stirred tank reactor; HRT, hydraulic retention

time; WAS, waste activated sludge; COD, chemical oxygen demand; VFA, volatile
fatty acids; OLR, organic loading rate.
* Corresponding author. Tel.: 30 25410 79390; fax: 30 25410 79376.
E-mail address: eathanas@env.duth.gr (E. Athanasoulia).
0960-1481/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.renene.2013.06.040

of glycerol by-product is approximately generated [2,3]. The signicant increase in biodiesel production has created a glycerol
surplus that has resulted in a dramatic decrease in crude glycerol
prices [4]. Moreover, glycerol is a readily digestible substance,
which can be easily stored over a long period. As a result, glycerol
can be characterized as an ideal co-substrate for the anaerobic
digestion process.
Many researchers have studied the inuence of glycerol as a
co-substrate in anaerobic digestion. During experiments in an
upflow anaerobic sludge blanket (UASB) reactor treating potato
processing wastewater, Ma et al. [5] found that the biogas production increased by 0.74 L biogas per mL glycerol added.
Furthermore, a better biomass yield was observed for the supplemented reactor compared to the control. Additionally, the effects of crude glycerol on the performance of single-stage
anaerobic reactors treating different types of organic waste were
examined by Fountoulakis and Manios [6]. A reactor treating the
organic fraction of municipal solid waste produced 1400 and
2094 mL CH4/d in the absence and presence of glycerol respectively. Further experiments in batch reactors, at mesophilic temperature, using granular and non-granular sludge, were carried
out by Lpez et al. [7]. Results showed a biodegradability of
around 100%, with a methane yield coefcient of 0.306 m3 CH4/kg
when granular sludge-acidied glycerol was used. The organic
loading rate during the above mentioned experimental study was

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E. Athanasoulia et al. / Renewable Energy 62 (2014) 73e78

0.21e0.38 g COD/g VSS d. An inhibition phenomenon was

observed at the highest load. On the contrary, when non-granular
sludge-acidied glycerol was used, the ORL reached values ranging
from 0.12 to 0.26 g COD/g VSS d, methane yield coefcient was
0.28 m3 CH4/kg glycerol and biodegradability estimated nearly
75%. Moreover, Siles et al. [8] studied the anaerobic co-digestion of
glycerol and wastewater derived from biodiesel manufacturing in
batch reactors and mesophilic conditions. Substrate ratios ranged
between 5.02 and 1.48 g VSS/g COD corresponding to organic
loading rates of 0.27e0.36 g COD/g VSS d. Biodegradability was
found to be around 100%, while the methane selectivity coefcient
was 310 mL CH4/g COD removed. However, a 6% supplementation
of glycerine to pig manure and maize silage that was digested
anaerobically, resulted in a signicant increase in CH4 production
from 569 to 679 NL CH4/kg VS [9]. According to Alvarez et al. [10],
the highest biodegradation potential (321 L CH4/kg COD) was
reached in batch experiments when 11% of crude glycerol was
added at a mixture composed of 84% pig manure and 5% sh
waste, while the highest methane production rate (16.4 L CH4/kg
COD d) was obtained by a mixture containing 88% pig manure, 4%
sh waste and 8% biodiesel waste.
However, there is lack of knowledge about pilot scale anaerobic
co-digestion of glycerol, as the preannounced studies were conducted in bench scale reactors. Moreover a necessity to develop a
system of two methanogenic reactors in series was created, based
on its advantages concerning biogas production, VFAs concentrations and efuent quality, over other systems. Conventional onestep CSTR is simple to operate but less efcient in terms of the
efuent quality compared to other reactor congurations such as
upow anaerobic sludge blanket (UASB) reactor or two-phase
reactor system. According to Angelidaki et al. [11], a single CSTR
can lose biogas production due to short-circuit, where a fraction of
the organic material in the feed remains over a shorter duration in
the reactor than the nominal retention time. Concerning the use of
UASB reactors, sludge characteristics (high viscosity and particulate
content) makes UASB reactors unsuitable for sludge treatment. On
the other hand, a two-phase system, where a short acidogenic step
is followed by a long methanogenic step, is sensitive to high organic
load and any separation processes that can be utilized increase costs
[12]. An alternative approach to overcome the above mentioned
problems is to operate two methanogenic reactors connected in
series. Only few studies have been reported about serial digestion of
other substrates as manure [13,14], primary sludge [15] and secondary sludge [16]. Boe [13] demonstrated that serial digestion of
manure, with percent volume distributions of 90/10 or 80/20 between the two methanogenic reactors, improved biogas production
by 11% compared to a traditional one-step CSTR process. According
to Kaparaju et al. [14] serial digestion at 70/30% and 50/50% volume
distribution produced 13.0e17.8% more biogas and methane while
at the same time contained low VFA. The residual methane potential
loss was also lower in the efuent compared to the one-step CSTR
process. In our previous study [16] where anaerobic digestion of
sewage sludge was compared to that of a one stage CSTR system,
results showed that the serial conguration could improve biogas
production by 9.5e40.1%. According to the authors, there are no
other experimental results regarding serial digestion of two methanogenic reactors of a mixture of sewage sludge with glycerol.
In this study, the effect of glycerol on the performance of a pilot
scale cascade of two anaerobic continuous stirred tank (CSTR) reactors treating thickened sludge at mesophilic conditions was
investigated. The objectives were a) to evaluate the use of glycerol
as a co-substrate to improve biogas production and b) to determine
the optimum hydraulic retention time and the most adequate ratios
of different feedstock mixtures that provide an optimized biodegradation rate or methane production.

2. Materials and methods

2.1. Feedstock
The raw material used as co-substrate was a glycerol-containing
waste discharged after the biodiesel manufacturing process at the
ELVI Factory in Kilkis, Greece. In general, this waste contained
glycerol, water, methanol and soaps, as shown in Table 1. Secondary
thickened sludge was obtained from municipal wastewater treatment plant in Komotini, Greece, which operated under extended
aeration conditions.
In order to prepare feed materials, crude glycerol was diluted
ten times and a new glycerol-containing solution of 100 g/L COD
was stocked. By mixing 20% of this solution and 80% of waste
activated sludge (WAS), an inuent mixture of 2% (v/v) crude
glycerol was produced. Similarly, mixtures of 3% and 4% crude
glycerol were prepared.
2.2. Reactor experiments
The mesophilic anaerobic digestion of glycerol and waste activated sludge mixtures was investigated using a cascade of two
continuous stirred tank reactors (CSTR) operated over a range of
hydraulic retention times (12.3 de19.7 d) at organic loading rates
between 1.0 and 1.7 kg COD/m3 day (Fig. 1). The mixture of thickened WAS with glycerol was continuously pumped into the rstdigester, the efuent of which overowed to the second-digester
unit. The working volumes of the digesters were 40 L and 60 L
respectively. Constant sludge volumes inside the reactors were
maintained by a level controller in each digester. Moreover, biogas
production in each reactor was continuously monitored by a
volumetric gas meter. The concentrations of methane and carbon
dioxide in the biogas were continuously measured using an on-line
gas analyzer (BINOS IR, Leybold-Heraeus). The optimal temperature
was controlled at 37  C by circulating hot water inside the water
jacket of the reactors. pH value was maintained constant at 6.8e7.2
by a pH controller.
Three series of experiments were carried out with feed mixtures
of sewage sludge supplemented with 0% (v/v) (control), 2% (v/v), 3%
(v/v) and 4% (v/v) glycerol in order to investigate the limiting
concentration of glycerol in the feed that affect the anaerobic
digestion. For all cases, in order to nd the optimum operation
conditions of the process, hydraulic retention times of 12.3, 14.0,
16.4 and 19.7 d were also examined. According to the available
literature, standard hydraulic retention times in a high-rate
digester without sludge recycle are 15e20 days [17]. However, a
well mixed and heated digester may produce good quality digested
sludge after a 10 day retention time [18,19].
2.3. Analytical methods
Standard Methods for the Examination of Water and Wastewater [20] were used for the estimation of suspended solids (SS),

Table 1
Chemical analysis of glycerol and sewage sludge characteristics.
Parameter

Crude glycerol

Parameter

Sewage sludge

Water
Methanol
Soaps
Glycerin
Catalyst (CH3ONa)
pH
COD

15.7%
7.1%
26.5%
50.6%
0.1%
10.7
1000 g/L

VSS
sCOD
Conductivity
pH

19.6 g/L
0.58 g/L
380 mS/cm
7.12

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E. Athanasoulia et al. / Renewable Energy 62 (2014) 73e78

Biogas

Acid

Biogas

Acid

pHC

pHC
TI

Influent

TI

M
LC

LC

Efluent

Fig. 1. Schematic diagram of the two anaerobic digesters cascade (LC: Level Controller, M: Motor, TI: Temperature Indicator, pHC: pH controller).

volatile suspended solids (VSS), total and dissolved chemical oxygen demand (COD). A PerkinElmer gas chromatograph equipped
with a capillary column and a ame ionization detector was used to
determine the concentrations of volatile fatty acids [21].
3. Results and discussion
Fig. 2 presents biogas production (L/d) for mixtures of 2% and 3%
glycerol, compared to daily biogas production of WAS (0%). The
daily biogas production was found to decrease as the HRT
increased, as expected of course, because of digesters lower
feeding. Moreover, in all cases the addition of glycerol resulted in
higher biogas production. In particular, after 2% addition of glycerol

in sludge, for hydraulic retention time 12.3 d, daily biogas production increased from 30  2.1 L/d to 114  1.7 L/d. For HRT 14 d,
the corresponding increase estimated from 21  0.8 L/d to
100  8.0 L/d. At 16.4 d the increase in biogas production was 3.9
times higher (from 23  2.6 L/d to 90  2.8 L/d). Finally, for HRT
19.7 d, biogas production increased from 20  1.4 L/d to 80  2.6 L/
d. The volumetric biogas production rate at steady state conditions
reached 1.1, 1.0, 0.9 and 0.8 L biogas/L reactor d for 12.3 d, 14 d,
16.4 d and 19.7 d respectively for 2% glycerol mixture, while the
respective of WAS ranged between 0.21 and 0.32 L biogas/L reactor
d. Moreover, Fig. 2 shows that there is a signicant increase in daily
biogas production when 3% of glycerol is used as co-substrate in
anaerobic digestion of sludge. In particular, comparing biogas

Fig. 2. Daily biogas production versus HRT at the rst and the second stage of the cascade and comparison of total biogas production (1st 2nd stage) to the corresponding of WAS.

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E. Athanasoulia et al. / Renewable Energy 62 (2014) 73e78

production of WAS and the corresponding after 3% addition of

glycerol, an increase nearly 360%, 520%, 350% and 335% was estimated as values of 139  7.4 L/d, 130  5.0 L/d, 105  5.5 L/d and
86.5  3.8 L/d estimated for 12.3 d, 14 d, 16.4 d and 19.7 d respectively. The volumetric biogas production rates for this case were 1.4,
1.3, 1.0 and 0.9 L biogas/L reactor d. It is important to note that when
the HRT of the cascade was 12.3 d, the HRT at the rst digester was
4.9 d. This time period was not sufcient for methane formers [12]
and the higher percentage of biogas (59.0  0.4% after 2% and
53.0  1.8% after 3% addition of glycerol) was produced at the
second digester. As the HRT of the system increased to 14 d, the HRT
at the rst digester simultaneously increased to 5.6 days and more
than half of the biogas production of the system took place at the
rst digester (52.0  5.0% after 2% and 52.3  1.9% after 3% addition
of glycerol). Further increase to the cascade HRT to 16.4 and 19.7 d
resulted in the increase of the HRT at the rst stage to 6.6 and 7.9
days respectively and higher biogas production at the rst digester
was observed (60.8  2.6% and 79.2  1.8% respectively for 2%
glycerol in the mixture and 65.9  3.5% and 77.9  3.7% for 3%
glycerol addition respectively).
As shown in Fig. 3, high soluble COD removal efciencies (between 88.0% and 96.4%) were obtained for organic loading rates
(OLRs) between 1.7 and 1.0 kg COD/m3 day. The OLR values were
comparable with those of other researches [7,8], but were not
excessively high when compared to other biodegradable substrates. For food, organic, soft drink and bakery wastes, which their
total COD range from 5000 to 11,000 mg/L, removals of 68e93%
were obtained at organic loadings of 3e27 g COD/L d respectively
[22]. In this work, after 2% glycerol addition in the sludge, the
average value of sCOD that was inserted in the system was
20.7  0.4 g/L, while the efuent sCOD of the system estimated to
be 2.5  0.19, 1.5  0.31, 1.4  0.18 and 0.95  0.21 g/L at HRTs 12.3,
14.0, 16.4 and 19.7 days respectively. Similarly, the sCOD value
when the system fed with 3% glycerol was 28.5  0.9 g/L and the
corresponding values at the efuent of the system were 1.2  0.07,
1.2  0.02, 1.1  0.20 and 1.1  0.11 g/L at HRTs 12.3, 14.0, 16.4 and
19.7 days respectively. It is important that the remaining 1.2  0.2 g
COD/L at the exit of the system comes from sewage sludge, as this
value was measured when only WAS treated anaerobically in the
cascade. As a result, the net glycerol COD degradation is near 100%.
The obtained results are generally consistent with literature data
[7,8]. It is also important to note that as the HRT increases, higher
sCOD removal is taking place in the rst stage of the cascade. In
particular, when the HRT of the cascade was 12.3 days and the
corresponding of the rst digester was 4.9 days, the sCOD reduction
at the rst stage was 35.1% and 52.7% for 2% and 3% glycerol mixture
respectively. In this low HRT, the sCOD removal at the rst stage is
higher at the 3% glycerol mixture compared to the corresponding of

2% because more easily biodegradable compounds are inserted in

the system. By increasing the cascade HRT to 14 days, the HRT at the
rst digester simultaneously increased to 5.6 days and the sCOD
was reduced equally at the rst and the second digester of the
cascade for both mixtures. Further increase to the cascade HRT to
16.4 days (6.6 days at the rst stage) resulted in a corresponding
increase in sCOD reduction at the rst stage of 59.2% and 59.8% for
2% and 3% glycerol mixture respectively. Finally, when the HRT of
the cascade was 19.7 days and the HRT at the rst stage was 7.9
days, 84.1% (for 2% glycerol mixture) and 73.2% (for 3% glycerol
mixture) of sCOD reduction took place at the rst stage of the
cascade.
During the third experimental cycle, the cascade which was fed
with 4% glycerol, at an HRT of 19.7 d, showed almost an immediate
increase of biogas production, however, it failed to reach steady
state conditions. The quick response in gas production (103 L/d),
was likely due to the breakdown of the readily biodegradable soluble COD in the glycerol, however due to the sustained high COD
loading, a build-up of intermediate VFAs occurred (over 10,000 mg/
L), which lead to a substantial pH drop to 5.8, environment which
inhibited methanogenic activity and resulted in an unstable system.
VFA concentrations of acetic, propionic, iso-butyric, butyric and
valeric acid were measured for HRTs 12.3, 14, 16.4 and 19.7 days
and for the 2% and 3% mixtures examined. In both cases, isobutyric, butyric and valeric acid were estimated below the detection limit. VFAs in the inuent mixture present a remarkable
uctuation during all cases that were examined, due to sludge
storage. In the rst digester (D1) of the cascade, VFA concentrations
ranged between 38 and 130 mmol/L when 2% of glycerol was used
as co-substrate, while in the second digester (D2) VFAs concentrations were less than the detection limit because of their total
conversion in biogas (Fig. 4). The corresponding VFA values in the
case of 3% glycerol addition ranged between 64 and 200 mmol/L at
the rst digester, 1.5e2.5 times higher than noticed in 2% mixture,
as it was expected. Moreover, 100% VFAs consumption observed at
the second digester, similar to the previous case. Among the
different tested HRTs tested, the order of magnitude for VFA levels
was highest at the lower HRT and decreased as the HRT increased.
When the HRT of the cascade was 12.3 days, the propionic acid
concentrations were 117 and 161 mmol/L for 2% and 3% glycerol
addition respectively. By increasing the cascade HRT to 14 days, the
propionate decreased to 67 mmol/L after 2% glycerol addition,
while a small increase to 184 mmol/L was observed after 3% glycerol addition. Further rise of the cascade HRT to 16.4 and 19.7 days
increased the HRT at the rst stage, resulting in a corresponding

350

95.9 96.3

96.4

30
25
20
15
10
5
0
12.3

14

16.4

19.7

2%
Influent

12.3

14

16.4

19.7
[d]

3%
1st stage

2nd stage

100
90
80
70
60
50
40
30
20
10
0

removal

Fig. 3. COD removal and COD concentrations versus HRT in the inuent, rst stage and
second stage (efuent) of the cascade, n > 5 (n number of replicates).

VFA's [mmol/l]

93.1

87.9

95.7

removal (%)

concentration (g/L)

35

propionic acid

VFA's

300

COD
93.2 95.3

acetic acid

250
200
150
100
50
0
IN D1 D2 IN D1 D2 IN D1 D2 IN D1 D2 IN D1 D2 IN D1 D2 IN D1 D2 IN D1 D2
12.3

14

16.4

19.7

12.3

14

16.4

19.7

[d]

Fig. 4. VFA concentrations versus HRT in the inuent (IN), rst (D1) and second (D2)
digester, n > 5 (n number of replicates).

77

decrease of propionic acid to 76 and 32 mmol/L for glycerol mixtures 2%, while the respective concentrations were 170 and
54 mmol/L for mixtures of 3% glycerol. It is remarkable that in the
case of 3% glycerol addition in the sludge mixtures, the concentrations of propionate in the rst digester of the cascade are 1.4e2.7
times higher than those in the case of 2% glycerol addition. This
increase in propionic acid concentration which followed the
increased glycerol addition indicates that glycerol is converted to
propionic acid via anaerobic digestion. This observation is in
accordance with the relative ndings reported in the literature
[4,23]. However, due to serial digestion there was total consumption of propionic acid at the second digester and the failure of the
system was avoided. Therefore, when 2% and 3% of glycerol was
added, the concentrations of Volatile Fatty Acids were low, a fact
that proves continuously stable conditions during anaerobic
digestion process. This was also conrmed by Holm-Nielsen et al.
[24], who found that no VFAs were detected under low concentrations (<5 g/L) of glycerol. However, when the glycerol content
was increased (4 g/L), there were clear tendencies of organic
overloading by increasing the VFA concentration during the
fermentation (up to 30 g/L).
Another way of assessing the performance of a digester is to
examine the efciency of the Volatile Suspended Solids (VSS)
reduction. During the digestion process, volatile solids are
degraded to a certain extend and converted into biogas.

Volatile suspended solids reduction %



VSSfeed VSSeffluent
 100

VSfeed
where VSSfeed, VSSefuent are the concentrations of volatile suspended solids in the feed and digested sludge. In terms of VSS
reduction, the values for all cases ranged between 23.4% and 28.7%,
as shown in Fig. 5. The VSS destruction was relatively similar at the
highest and shortest retention time, a fact that is conrmed by
Appels et al. [19] who claims that for SRT values exceeding 12e13
days (at 35  C), changes in increasing volatile solids destruction are
relatively small. According to Babel et al. [25] who co-digested
sewage and brewery sludge, VSS reduction ranged from 14.6% to
33.6%. The optimum VSS reduction should be about 30% after
sludge digestion according to Metcalf and Eddy [26]. Fountoulakis
and Manios [6], found 64.6% VSS average removal efciency, when
a 1:4 mixture of olive mill wastewater and slaughterhouse wastewater was supplemented with 1% v/v crude glycerol. Moreover,
Fig. 5 shows that in all cases, VSS destruction of both mixtures was
lower compared to the corresponding of sludge. This may attribute
to the fact that Fountoulakis et al. [27] and Ma et al. [5] observed,
that the extra organic carbon source, i.e. glycerol addition,
enhanced the growth of active biomass which increased the total
amount of volatile solids.

2%

3%

WAS

45
VSSremoval [%]

40

35
30
25
20
15

10
5
0
12.3

14

[d]

16.4

19.7

Fig. 5. Volatile Suspended Solids degradation for several mixtures and hydraulic
retention times, n > 5 (n number of replicates).

biogas selectivity [L/gTVSr]

E. Athanasoulia et al. / Renewable Energy 62 (2014) 73e78

1.0
0.8
0.6
0.4
0.2

0.0
12.3

14

16.4

19.7

[d]
2%

3%

WAS

Fig. 6. Biogas selectivity (L biogas/g TVSr) at different mixtures and HRTs.

Fig. 6 shows the mean values of the biogas selectivity which

were calculated for 2% and 3% mixtures, with the retention times
being taken into account. For both mixtures that were examined,
the liters of biogas produced were 0.78  0.02 L biogas/g TVS
removed. In bibliography [26,28], the specic gas production lies in
the range of 0.75e1.12 L biogas/g TVS destroyed. Moreover, biogas
selectivity was estimated to be as 0.6 L biogas/g soluble COD
removed (or 0.51 biogas/g total COD removed) or, taking into account that there was 78% methane in the biogas, as 0.47 L CH4/g
soluble COD removed (or 0.39 CH4/g total COD removed). These
values corresponds to the theoretical value of 0.38 L methane/g
COD removed that was proposed by Wheatley [29] and are similar
to the values reported by Ma et al. [5] (0.69 L biogas/g total COD and
0.39 L CH4/g total COD removed), who treated also crude glycerol.
On the contrary, Lpez et al. [7] and Siles et al. [8] measured 0.31 L
CH4/g COD removed.
4. Conclusions
Results show a great ascendancy of 2% and 3% glycerol mixtures
with sludge, as they improve signicantly the biogas production
rate. Specically, the total biogas production, after sludge codigestion with 2% and 3% glycerol, measured to be 3.8e4.7 times
higher. The addition of 3% glycerol at HRT of 12.3 d resulted in the
higher amount of biogas production, while the efuent quality
remained good. However when 4% glycerol added, the system
failed. Overall, digestion systems can operate successfully with the
sewage sludge/crude glycerol waste mixture. The concept of crude
glycerol could be a promising perspective at wastewater treatment
plants as it increases methane production signicantly. However,
the feeding rate should be low in order to prevent overloading
conditions.
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