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Article history:
Received 6 March 2009
Received in revised form 1 June 2009
Accepted 6 June 2009
Available online 9 July 2009
Keywords:
Biodiesel manufacturing
Glycerol-containing waste
Anaerobic digestion
Kinetic constants
Biodegradability
a b s t r a c t
The anaerobic digestion of glycerol derived from biodiesel manufacturing, in which COD was found to be
1010 g/kg, was studied in batch laboratory-scale reactors at mesophilic temperature using granular and
non-granular sludge. Due to the high KOH concentration of this by-product, H3PO4 was added to recover
this alkaline catalyst as agricultural fertilizer (potassium phosphates). Although it would not be economically viable, a volume of glycerol was distilled and utilised as reference substrate. The anaerobic
revalorisation of glycerol using granular sludge achieved a biodegradability of around 100%, while the
methane yield coefcient was 0.306 m3 CH4/kg acidied glycerol. Anaerobic digestion could be a good
option for revalorising this available, impure and low priced by-product derived from the surplus of biodiesel companies. The organic loading rate studied was 0.210.38 g COD/g VSS d, although an inhibition
phenomenon was observed at the highest load.
2009 Elsevier Ltd. All rights reserved.
1. Introduction
Concern has recently risen over the use of fossil resources, their
cost, their sustained availability and their impact on global
warming and pollution (Hansen et al., 2005). Since biodiesel fuels
have various advantages such as a complement to petroleumbased fuel, renewable fuel, a favourable energy balance, less harmful emissions and non-toxic fuel, these have drawn much attention
recently (Ito et al., 2005).
During the biodiesel production process, oils/fats (triglycerides)
are mixed with methyl alcohol and alkaline catalysts to produce free
fatty-acid esters, with glycerol as a primary by-product (Chi et al.,
2007). The production of 100 kg of biodiesel yields approximately
10 kg of impure glycerol, with 5590% glycerol (Hazimah et al.,
2003). Glycerol is generated as a by-product not only when biodiesel fuels are produced chemically, but also when they are manufactured enzymatically (Du et al., 2003; Vicente et al., 2004) and during
the production of bioethanol (Yazdani and Gonzalez, 2007). The dramatic growth of the biodiesel industry has created a surplus of glycerol that has resulted in a dramatic 10-fold decrease in crude
glycerol prices over the last few years and generated environmental
concerns associated with contaminated glycerol disposal (Yazdani
and Gonzalez, 2007; Sandun et al., 2007).
At present, due to its properties, pure glycerol has more than
2000 different applications (Elvers et al., 1990). However, the pro* Corresponding author. Tel.: +34 957 218586; fax: +34 957 218625.
E-mail address: a92siloj@uco.es (J.. Siles Lpez).
0960-8524/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2009.06.017
5610
Nomenclature
Alk
alkalinity (mg CaCO3/L)
CODremoved removed chemical oxygen demand (mg/L)
CODsoluble soluble chemical oxygen demand (mg/L)
COD STO added chemical oxygen demand (mg/L)
CODtotal total chemical oxygen demand (mg/L)
G
cumulative methane volume (mL)
cumulative methane volume at innite time (mL)
Gm
experimental maximum methane volume (mL)
GT
specic methane production kinetic constant (L/g VSS h)
KG
apparent kinetic constant (1/h)
K 0G
MS
total mineral solids (mg/L)
MSS
mineral suspended solids (mg/L)
OLR
organic loading rate (g COD/g VSS d; kg COD/m3 d)
Anaerobic digestion is another possible way to revalorise abundant and low-priced glycerol streams. This process may be dened
as the biological conversion of organic material to a variety of end
products including biogas whose main constituents are methane
and carbon dioxide (Gujer and Zehnder, 1983; Olthof and
Oleszkiewick, 1982; Speece, 1983; Wheatley, 1990). The advantages of anaerobic digestion include low levels of biological sludge,
low nutrient requirements, high efciency and the production of
methane, which can be used as an energy source. The stoichiometry of the anaerobic digestion of glycerol can be summarised as follows (Christensen and McCarty, 1975; McCarty, 1975):
r0
specic rate of methane production (mL CH4/g VSS h)
Sremoved removed substrate (g COD)
added substrate (g COD)
STO
t
time (h)
TS
total solids (mg/L)
TSS
total suspended solids (mg/L)
VA
volatile acidity (mg acetic/L)
VS
total volatile solids (mg/L)
VSS
volatile suspended solids (mg/L)
X
biomass concentration (mg VSS/L)
Y CH4 =Sremoved methane yield coefcient (mL CH4/g COD removed)
5611
2.6. Software
Table 1
Composition and features of the acidied, distilled and pure glycerol.
Parameter
Acidied
glycerol
Distilled
glycerol
Pure
glycerol
Density at 20 C (g/mL)
Refraction index at 20 C
COD (g/kg)
Dynamic viscosity at 50 C (mPa s)
Colour
1.044
1.4440
1010
57
Brown
1.260
1.4728
1155
150
Colourless
1.261
1.4746
1217
152
Colourless
Table 2
Composition of the nutrient and trace element solutions added to distilled glycerol.
Nutrient solution
Compound
g/La
Compound
NH4CI
K2HPO43H2O
KH2PO4
MgCl26H2O
Resazurine
FeCl24H2O
NaS9H2O
NaHCO3
0.200
0.100
0.055
0.200
0.001
0.100
0.500
5.000
MnCl24H2O
0.100
CoCl26H2O
0.170
ZnCl2
0.100
CaCl2
0.200
H3BO4
0.019
NiCl26H2O
0.050
Na2MoO4H2O
0.100
Ad 10 mL of trace element solution per litre of diluted
glycerol
g/L
Methane yield coefcient was determined from the experimental maximum methane volume produced (GT) and the nal and initial COD, which were known in all loads. By tting (GT, COD
removed) value pairs to a straight line (Fig. 1), the methane yield
coefcient coincides with the slope of the regression line and
was found to be 292 mL CH4/g COD removed (at 1 atm, 25 C) using
granular sludge-acidied glycerol, 288 mL CH4/g COD removed
using non-granular sludge-acidied glycerol and 356 mL CH4/g
COD removed using granular sludge-distilled glycerol. Similar values have been described in the literature. Gross and Lanting (1988),
for example, studied the anaerobic digestion of wastewater generated during the manufacturing of fuel ethanol from corn, which
contained a highly soluble mixture of weak organic acids such as
acetic, propionic, lactic and butyric acid. The organic strength of
the wastewater typically ranged from 3000 to 5000 mg/L COD soluble. The methane yield coefcient was found to be 330 mL CH4/g
COD removed.
According to Wheatley (1990), and considering the biomass
growth and cell maintenance null, 382 mL of methane are theoretically produced (at 1 atm, 25 C) per gram of COD removed. Experimentally, the effectiveness of the process in each case was: 76%
using granular sludge-acidied glycerol, 75% using non-granular
sludge-acidied glycerol and 93% with granular sludge-distilled
glycerol. When only taking into account these results, granular
sludge-distilled glycerol would be the best option. Studying the removed COD percentage is as important as evaluating the methane
production coefcient in order to determine waste biodegradability. The high biodegradability of glycerol can be demonstrated by
Table 3
pH and volatile acidity/alkalinity ratio values in the efuents of the reactors obtained
for the different substrates and sludge studied.
Substrate and sludge type
pH
VA (eq acetic)/Alk
(eq CaCO3)
7.72 0.24
7.74 0.30
7.81 0.17
0.05 0.02
0.03 0.01
0.05 0.01
5612
3.5
1000
3.0
S removed (g COD)
GT (mL CH4 )
800
600
400
2.5
2.0
1.5
1.0
x = 0.502 g COD
200
0.5
r = 0.9946
3.5
1000
3.0
S removed (g COD)
GT (mL CH4 )
800
600
400
2.5
2.0
1.5
1.0
x = 0.093 g COD
200
0.5
r = 0.9542
3.5
0
1000
3.0
S removed (g COD)
GT (mL CH4 )
800
600
400
2.5
2.0
1.5
1.0
200
x = 0.665 g COD
0.5
r = 0.8925
0.0
0
0
S removed (g COD)
Fig. 1. Variation of the experimental maximum methane volume produced (GT) (at
1 atm, 25 C) with the COD consumed to obtain the methane yield coefcient of the
process using granular sludge-acidied glycerol, non-granular sludge-acidied
glycerol and granular sludge-distilled glycerol.
STO (g COD)
Fig. 2. Plot of the amount of substrate removed against the substrate added for all
the experiments to obtain the biodegradability percentage of the by-product using
granular sludge-acidied glycerol, non-granular sludge-acidied glycerol and
granular sludge-distilled glycerol.
5613
G Gm 1 expK 0G t
1400
1200
1.0 g COD
1.5 g COD
2.0 g COD
3.0 g COD
1.0 g COD
1.5 g COD
2.0 g COD
800
600
200
where Gm is the maximum methane volume accumulated at an innite digestion time; G is zero at t = 0 and the rate of gas production
becomes zero at t equal to innite.
K 0G is an apparent kinetic constant for methane production (h1)
which included the biomass concentration:
0
1400
1200
2
G (mL CH4 )
1000
800
600
400
200
0
1400
1200
1000
G (mL CH4 )
400
K 0G K G X
1.0 g COD
2.0 g COD
3.0 g COD
1000
G (mL CH4 )
800
600
400
200
0
0
10
20
30
40
50
60
Time (h)
Fig. 3. Variation of the methane volume accumulated (G) as a function of time for
the loads of 1.0, 1.5, 2.0 and 3.0 g COD using granular sludge-acidied glycerol, nongranular sludge-acidied glycerol and granular sludge-distilled glycerol.
tration of 25 4 mg PO3
4 =L was obtained. The occurrence of a similar inhibitory phenomenon has been described in the literature
(Ito et al., 2005; Barbirato et al., 1998).
Table 4
Gm, K 0G and KG values obtained for all the loads studied using acidied glycerol-granular sludge, acidied glycerol-non-granular sludge and distilled glycerol-granular sludge.
Substrate and sludge type
Load (g COD)
Gm (mL CH4)
K 0G (h1)
R2
KG (Lg1 h1)
1.0
2.0
3.0
323 5
592 5
857 7
0.2075 0.0117
0.1858 0.0053
0.1316 0.0036
0.9676
0.9909
0.9798
0.0384
0.0338
0.0246
1.0
1.5
2.0
3.0
318 11
451 8
590 15
789 20
0.1655 0.0177
0.1175 0.0151
0.1216 0.0091
0.0749 0.0059
0.9527
0.9960
0.9698
0.9716
0.0203
0.0178
0.0190
0.0128
1.0
1.5
2.0
375 6
578 5
766 15
0.2006 0.0105
0.1372 0.0048
0.0829 0.0046
0.9548
0.9784
0.9697
0.0131
0.0101
0.0068
5614
800
ned as the volume of methane generated per gram of volatile suspended solid and per hour for each set of experiments. As can be
observed in Fig. 5, the r0 values were always higher when using
granular sludge and acidied glycerol at the same time. The lowest
values were reached when using distilled glycerol with a high alkalinity concentration.
600
200
200
400
600
800
1000
Gm(mL CH4 )
Fig. 4. Comparison between the experimental maximum methane production (GT)
values and the theoretical values (Gm) predicted by Eq. (1).
Moreover, the theoretical values of maximum methane production (Gm) were calculated using Eq. (1) and plotted against their
corresponding experimental values (Fig. 4). The deviations obtained were lower than 5% in practically all cases, again suggesting
that the proposed model can be used to accurately predict the
behaviour of the process and that the kinetic parameters obtained
represented the microorganism behaviour affecting the anaerobic
digestion of this by-product.
In order to compare the different substrates and sludge types,
the specic methane production rate, r0 (mL CH4/g VSS h), was de-
The organic loading rate (OLR) for each substrate and sludge
type was determined using the experimental data. The OLR (kg
COD/m3 d) values were not excessively high for any set of experiments (0.922.00 kg COD/m3 d) when compared to other biodegradable substrates. For food, organic chemical, soft drink and
bakery wastes, which range from 5000 to 11,000 mg/L in raw
COD, removals of 6893% were obtained at organic loadings of
327 kg COD/m3 d (Jeris, 1983).
The OLR values, expressed as g COD/g VSS d, take into account
both the biomass concentration and the organic load, thus making
this an interesting variable for comparing the results. The highest
values were obtained using granular sludge-acidied glycerol:
0.210.38 g COD/g VSS d. On the contrary, when non-granular
sludge-acidied glycerol was used, the ORL reached a value ranging from 0.12 to 0.26 g COD/g VSS d and 0.075 to 0.080 g COD/g
VSSd when granular sludge-distilled glycerol was used. Fig. 6
shows the evolution of the specic methane production kinetic
constant, KG (L/g VSSh), with the organic loading rate (g COD/g
VSSd) for each substrate and sludge type studied. A strong decrease in the KG value was observed when the distilled glycerol
OLR was increased. Although the constant also decreased when
using acidied glycerol, the kinetic constant slowdown was much
14
Granular sludge-Acidified glycerol
Non-granular sludge-Acidified glycerol
Granular sludge-Distilled glycerol
12
10
8
1.0 g COD
1.5 g COD
2.0 g COD
3.0 g COD
6
4
2
0
25
20
15
10
0
0
10
20
30
Time (h)
40
50
60
70
10
20
30
40
50
60
70
Time (h)
Fig. 5. Variation of the specic methane production rate, r0 , as a function of time for all the loads using granular and non-granular sludge and acidied and distilled glycerol.
KG (L/g VSSh)
0.05
0.04
0.03
0.02
0.01
0.00
0.0
0.1
0.2
0.3
0.4
0.5
lower. Additionally, this substrate has allowed the reactors to operate in a stable way with higher OLR. Finally, according to economic
criteria, acidied glycerol is the target substrate. Given that the
granular sludge KG shows values that are twice as high as the
non-granular sludge, the glycerol anaerobic treatment could be
carried out using granular sludge in addition to acidied glycerol.
However, one option to improve the OLR could be to mix glycerol with another organic waste such as wastewater generated
during the biodiesel manufacturing process and study its co-digestion. This research would form part of what is known as the Biorenery concept; a relatively new term referring to the conversion
of feedstock into a host of valuable chemicals and energy. Indeed,
this is considered an important strategy for achieving sustainable
development.
4. Conclusions
The results obtained through this research study reveal that
glycerol-containing waste derived from biodiesel production after
pre-treatment has a high level of anaerobic biodegradability and
that a substantial quantity of methane can be obtained this way.
The highest methane yield coefcient was obtained using granular
sludge-distilled glycerol, but a strong inhibition was observed
when the load was increased. The use of granular sludge and acidied glycerol was found to be the best option for revalorising glycerol anaerobically given that biodegradability was found to be
around 100% and the methane yield coefcient was 0.306 m3
CH4/kg acidied glycerol (at 1 atm, 25 C).
Acknowledgements
The authors are very grateful to the BIDA S.A. Factory (Fuentes
de Andaluca, Seville, Spain) and to the Spanish Ministry of Science
and Innovation for funding Jos ngel Siles Lpez through Project
CTM2005-01293 and Grant BES2006-14074. This study was co-nanced by the European Social Fund. We also wish to express
our gratitude to the laboratory technician Inmaculada Bellido
Padillo for her help.
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