Bioresource Technology 102 (2011) 7845–7849

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Optimization of biogas production from cattle manure by pre-treatment

with ultrasound and co-digestion with crude glycerin
L. Castrillón ⇑, Y. Fernández-Nava, P. Ormaechea, E. Marañón
Chemical Engineering and Environmental Technology Department, University Technology Institute of Asturias (IUTA), University of Oviedo, Campus of Gijón, 33203 Gijón, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Biogas production by co-digestion of cattle manure with crude glycerin obtained from biodiesel produc-
Received 23 February 2011 tion was studied after pre-treatment of the cattle manure or mixtures of cattle manure with different
Received in revised form 18 May 2011 amounts of added glycerin with ultrasound. Batch experiments with 1750 mL of medium containing
Accepted 20 May 2011
1760 g of screened cattle manure or mixtures of cattle manure (screened or ground) and 70–140 mL or
Available online 26 May 2011
crude glycerin were incubated under mesophilic and thermophilic condition in stirred tank reactors.
Under mesophilic conditions, the addition of 4% glycerin to screened manure increased biogas production
Keywords:
by up to 400%. Application of sonication (20 kHz, 0.1 kW, and 4 min) to a mixture of manure + 4% glycerin
Cattle manure
Crude glycerin
increased production of biogas by up to 800% compared to untreated manure. The best results were
Biogas obtained under thermophilic conditions using sonicated mixtures of ground cattle manure with 6% added
Ultrasound glycerin (348 L methane/kg COD removed were obtained).
Renewable energy Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction improve the degradability of the biorefractory materials are

In areas with high dairy cattle populations such as Asturias in
northern Spain, disposal of manure can be challenging as more
manure is produced than can be safely applied as fertilizers and
therefore alternative methods for manure utilization are required.
Laboratory-scale studies with upflow anaerobic sludge blanket
(UASB) reactors have demonstrated that anaerobic treatment of
manure at mesophilic and thermophilic resulted in high organic
matter removal rates, but low biogas production (Marañón et al.,
2001, Castrillón et al., 2002). At a HRT of 16 days (Organic Loading
Rate of 3.7 kg COD/m3 day), COD removal was 69.7% and the pro-
duction of biogas was 0.20 m3/kg COD removed, with a methane
percentage of 73.7%.
The low biogas production levels could be due to the poor bio-
degradability of fibrous materials which can amount to 40–50% of
the total solids (TS) in cattle manure (Boe and Angelidaki, 2009). In
an anaerobic digestion process, with a typical hydraulic retention
time (HRT) of 15–30 days, only part of the fibers were degraded,
resulting in an average methane yield of 0.20–0.25 m3/kg volatile
solids (VS), which is about half of the theretical yield of
0.40–0.45 m3/kg VS added for cow manure (Hartmann et al., 2000).
For this reason, it is difficult to run biogas plants with finan-
cially profitable results if the process is based solely on cattle man-
ure. Co-digestion strategies and pre-treatments of the substrate to
⇑ Corresponding author. Tel.: +34 985182382; fax: +34 985182337.
E-mail address: cleonor@uniovi.es (L. Castrillón).
therefore required to increase biogas yields. Macias-Corral et al.
(2008) studied the anaerobic co-digestion of dairy cow manure,
the organic fraction of municipal waste and cotton gin waste.
Maximum biogas production was obtained with cow manure and
cotton gin waste (87 m3 methane/ton of dry waste). Callaghan
et al. (2002), studying the co-digestion of cattle slurries with fruit
and vegetable wastes (FVW) and with chicken manure, found that
increasing the proportion of FVW from 20% to 50% improved the
methane yield from 0.23 to 0.45 m3 CH4/kg VS added. Capela
et al. (2008) evaluated the technical feasibility of anaerobic co-
digestion with three types of organic solid waste under mesophilic
conditions: the organic fraction of municipal solid waste (OFMSW),
industrial sludge and cattle manure. Increasing the OFMSW in the
mixture generally resulted in higher methane production and vol-
atile solids reduction. The treatment of solid slaughterhouse waste,
fruit-vegetable waste and manure in a co-digestion process re-
sulted in methane yields in the range of 0.27–0.35 m3/kg VS added
and VS reductions of between 50% and 67% (Alvarez and Liden,
2008). Large-scale biogas plants co-digesting manure and other
biomass report the production of as much as 97.5 m3 biogas/t of
waste (Seadi, 2000).
Increases in methane production of 13% and 35% were
measured as a result of alkaline (13% NaOH) or thermal (170 °C
and 7 bar) pre-treatments of swine waste (Gonzalez-Fernández
et al., 2008). Low intensity ultrasound has been shown to decrease
chemical oxygen demand (COD) of anaerobic sludge by 30% (Xie
et al., 2009).

0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.05.047
7846 L. Castrillón et al. / Bioresource Technology 102 (2011) 7845–7849
The current study aimed to determine if crude glycerin, a by-
product of biodiesel production, could be used for co-digestion of
cattle manure. Currently the production of crude glycerin exceeds
commercial demand for purified glycerin (Siles López et al., 2009),
and alternative uses of crude glycerin are limited due to the pres-
ence of methanol, water, inorganic salts (residue from the catalyst),
unreacted mono-, di- and triglycerides and methyl esters. These
impurities potentially inhibit microorganisms in biological pro-
cesses; however, a positive impact of glycerin on methane produc-
tion was demonstrated by Amon et al. (2006) who showed that the
addition of 6% crude glycerin to a mixture of maize silage, pig man-
ure and rapeseed meal at 38–40 °C increased CH4 production from
0.57 to 0.68 m3 CH4/kg VS. To test the utility of crude glycerin and
ultrasound pre-treatment of increasing methane yields from cattle
manure, batch trials were carried out under mesophilic and ther-
mophilic conditions. The ultimate goal of the studies is to deter-
mine optimum conditions for biogas production in a continuous
process.
2. Methods
2.1. Materials
Crude glycerin was collected from a plant located in Asturias,
Spain that produces biodiesel from used vegetable oil. Cattle man-
ure was collected in 20-L plastic bottles from the cesspit of a dairy
farm after agitation of the cesspit contents and stored at 4 °C.
2.2. Equipments employed
Ultrasonic treatments were carried out with a Bandelin Sonoplus
HD 3200 homogenizer equipped with a 13-mm diameter UW 3200
sonotrode, operating at 20 kHz. Digestions of 1750 mL of waste were
done in 2-L jacketed stirred batch reactors made of glass and pro-
vided with automatic temperature and pH control. Biogas was col-
lected in TEDLAR bags and samples from the digested waste
(digestate) were taken from an outlet located near the bottom of
the reactors. The biogas inside the Tedlar bags was aspired by means
of a vacuum pump coupled to a previously calibrated ball flow me-
ter. The volume of biogas inside the bags was calculated after mea-
suring the time needed to empty the bag.
2.3. Analytical methods
Samples from the reactors (digestate) and of the produced bio-
gas were taken twice a week, and COD was determined following
Method 5220 D (closed reflux, colorimetric method) of the Stan-
dard Methods for the Examination of Water and Wastewater
(APHA, 1998) using a Perkin Elmer Lambda 35 Visible–UV system.
Ammonium nitrogen was determined by titration with boric acid
after distillation using a FOSS TECATOR Kjeltec 2200 Auto Distilla-
tion System. Total nitrogen and total phosphorus were determined
by ion chromatography (861 Advanced Compact IC 2.861.0010)
after transformation into nitrates and phosphates, respectively,
by digestion under pressure with H2O2 and HNO3 in a microwave
oven (Milestone Ethos 1 Advanced Microwave Digestion
Labstation). The methane and carbon dioxide content of the biogas
was measured on an Agilent gas chromatograph using a TCD detec-
tor and a Porapack N packed column plus a molecular sieve,
employing the following temperature ramp: starting temperature
35 °C (1.5 min) increasing up to 55 °C at a rate of 1.5 °C/minute.
Ca, K and Na in the crude glycerin were determined using a
Perkin–Elmer 310 Atomic Absorption Spectrophotometer after acid
microwave digestion with HNO3 and H2O2 in a proportion of 1:3.
Methanol, glycerin, soap, ester, monoglycerides, diglycerides, and
triglycerides were determined at an external laboratory (Cemitec,
Fundacion Cetena, Navarra, Spain).
2.4. Methods
A series of experiments using CSTR containing cattle manure
(screened) or mixtures of cattle manure (screened or ground) with
different proportions of crude glycerin were conducted at 35 °C
and 55 °C. All the experiments were carried out in duplicate. In
one case, the cattle manure was strained through a 2-mm sieve
to remove straw so as to simulate the treatment applied on some
farms that use a press screw separator. In the other case, the man-
ure was ground using a triturator. A jug-type household blender
was used (500 W) over a period of 2 min to obtain a maximum par-
ticle size after trituration of 5 mm.
To study the influence of ultrasound, both screened cattle man-
ure and mixtures of screened cattle manure and crude glycerin
were treated with the ultrasonic equipment for different periods
of time (from 1 to 8 min) to determine the most suitable sonication
time to achieve maximum COD solubilization. Each sample was
mixed for 3–5 min before ultrasound treatment so as to obtain a
homogeneous mixture, using a volume of 250 mL in all cases.
The amplitude was set at 30% (48 lm), 50% (80 lm) and 70%
(112 lm). Temperature and COD values (total and soluble) were
measured for the analysis of the results.
To describe the energy consumption in the pre-treatment of
screened or ground cattle manure with or without added glycerin,
the specific energy (Es) was used as an indicator for comparison.
Thus, Es was calculated for each sample on the basis of microwave
irradiation dose exposure and the solids content of the sample:
Es = (P
t)/(VTSo) (kJ/kg TS) where P is the microwave irradiation
power (kW), in our case 0.1 kW; t is the time(s) of exposure of
the sample to microwave irradiation; V is the volume (mL) of the
sample treated; and TSo is the initial Total Solids.
Experiments were carried out using the following as substrates:
screened cattle manure, screened cattle manure + 4% glycerin,
screened cattle manure (sonicated) + 4% glycerin, screened cattle
manure + 4% glycerin (mixture sonicated), screened cattle manure
(sonicated) + 6% glycerin, screened cattle manure + 6% glycerin
(mixture sonicated), ground cattle manure + 4% glycerin (mixture
sonicated) and ground cattle manure + 6% glycerin (mixture soni-
cated). All the quantities are expressed in wt%. No inoculum was
needed to start up the reactors, as previous results obtained in
our laboratory (not published) had shown similar behavior in the
reactors regardless of whether an inoculum was added or not.
3. Results and discussion
3.1. Characterization of the cattle manure and crude glycerin
Table 1 shows the characteristics of the screened (SM) and
ground manure (GM). The screened cattle manure had a total
Table 1
Physico-chemical characteristics of screened and ground cattle manure.
Parameter Screened cattle manure Ground cattle manure
pH 7.31 ± 0.16 7.61 ± 0.06
Water content (%) 94.92 ± 1.12 92.5 ± 0.36
Total solids (g/L) 52.51 ± 11.35 67.97 ± 12.53
Volatile solids (g/L) 35.15 ± 14.08 32.93 ± 2.89
Total COD (g/L) 45.39 ± 5.77 45.74 ± 2.69
Soluble COD (g/L) 34.58 ± 1.48 30.99 ± 3.92
Total nitrogen (g/L) 2.71 ± 0.50 2.23 ± 0.40
Ammonium nitrogen (g/L) 1.47 ± 0.09 1.21 ± 0.04
Total phosphorus (g/L) 1.2 ± 0.3 1.45 ± 0.16
 L. Castrillón et al. / Bioresource Technology 102 (2011) 7845–7849 7847

solids content of around 52 g/L (volatile solids representing around 50 40

67%) and a total COD (CODt) of around 45 g/L. Nitrogen was found
in both organic and ammonium forms at average values of 2.7 and 40

Temperature (ºC)
35
1.5 g/L, respectively. Total phosphorous values were around 1.2

COD (g/L)
30 SM (Total COD)
g/L. The ground cattle manure had a total solid content of 68 g/L 30
SM (Soluble COD)
(volatile solids representing around 49%) and a CODt of around 20
Temperature
45.7 g/L. Crude glycerin (Table 2) had a high salt and methanol,
25
was dark brown, and had a high organic content with a COD of 10
840 g/L (825 g/kg and density 1018 g/L). This COD was thus lower
than that reported by Siles López et al. (2009) of 1010 g/kg. 0 20
0 2 4 6 8
Nitrogen was not detected. 80 40

3.2. Pre-treatment by sonication 70

35

Temperature (ºC)
COD (g/L)
3.2.1. Sonication time 60
Fig. 1 shows the influence of ultrasound treatment on the total 30
and soluble COD of SM and for a mixture of SM plus 4% and 6% 50
Total COD (SM + 4% glycerin)
glycerin using an amplitude of 50%. In all cases (SM, SM + 4% glyc-
Soluble COD (SM + 4% glycerin) 25
erin and SM + 6% glycerin), maximum COD solubilization was ob- 40
Temperature
tained using an amplitude of 50%, although the difference was
not very high in the studied range (10% differences were found). 30 20
0 2 4 6 8
100 60
3.2.2. Energy consumption evaluation
55
Tang et al. (2010) stated that the CODS/CODt ratio is used as a 90

Temperature (ºC)
50
general indication of the extent of solubilization in most reports
COD (g/L)
on sludge. In our case, we represent the ratio CODS/CODS0 (Soluble 80 45
COD at time t/Soluble COD at time 0) as an indication of the extent 40
of solubilization. Fig. 2 presents the variation in the ratio CODS/ 70 Total COD (SM + 6% glycerin) 35
CODS0 versus specific energy (Es) applied to SM and GM. The low- Soluble COD (SM + 6% glycerin)
30
60
est specific energy that produces the highest CODS/CODS0 ratio was Temperature
25
2857 kJ/kg ST for SM and mixtures of SM + 4% glycerin and 2791 kJ/ 50 20
kg ST for mixtures of SM + 6% glycerin. In the case of GM, the low- 0 2 4 6 8
est specific energy was 1685 kJ/kg ST for GM, 1644 kJ/kg ST for Sonication time (min)
GM + 4% glycerin and 1345 kJ/kg ST for GM + 6% glycerin.
Fig. 1. Influence of ultrasound on total and soluble COD and variation in
temperature in cattle manure and mixtures of cattle manure and 4% or 6% crude
3.3. Methane production glycerin.

3.3.1. Production of biogas (screened manure) gas production increased over 400% (5.88 m3 biogas/ton of wet
Fig. 3 shows the production of biogas over time in the reactors mixture). Pre-treatment of the mixture (manure + 4% glycerin) by
operating at 35 °C with the different substrates. The values repre- ultrasound increased biogas production to 11.6 m3 biogas/ton of
sented are the mean values of the two replicas, the difference wet sonicated mixture. This represents an increase of 800% with
resulting always lower than 5%. When adding only screened cattle respect to the untreated manure without the addition of glycerin.
manure to the reactor, 1.4 m3 biogas/ton of wet manure, with a Crude glycerin from biodiesel can produce a very low cost biogas,
methane content of around 60% was obtained. When the screened mainly because of its high biodegradable organic matter content
cattle manure was pre-treated by ultrasound, biogas production (Schievano et al., 2009). Moreover, at temperatures above 30 °C,
increased by 121% (3.1 m3 biogas/ton of wet sonicated manure), glycerin remains as a fluid and can be easily pumped into digesters.
and the methane content of the gas was similar to that of untreated Furthermore, owing to its solubility in water, glycerin is readily
manure. When adding 4% glycerin (in volume) as co-substrate, bio- available to microorganisms. Table 3 shows the initial COD and
VS for each mixture, as well as the specific biogas production,
Table 2
Physico-chemical characteristics of crude glycerin.
per COD removed or per initial VS. The best results at 35 °C were
obtained when adding 4% glycerin as co-substrate. Robra et al.
Parameter Mean values (2010) studied the influence of adding crude glycerin (5%, 10%
pH 7.6 ± 0.12 and 15% w/w) on biogas production in CSTR digesters at mesophilic
Water content (%) 0.3 ± 0.02 conditions. They found the highest biogas production when adding
Total COD (g/kg) 837 ± 59.3
5% and 10% crude glycerin (similar values for both percentages,
Ash (%) 4.8 ± 0.3
Methanol (% w/w) 5.6 ± 0.2 825 mL biogas/g VS). The results obtained at 55 °C were better than
Glycerin (% w/w) 49.4 ± 0.3 at 35 °C, and a maximum production of biogas with mixtures of 6%
Soaps (% w/w sodium oleate) 27.4 ± 0.0 glycerin of 476.4 L biogas/kg COD removed with a methane con-
Glycerol ester (% w/w) 9.6 ± 0.1
tent of 59.8% (v/v), was achieved (Fig. 4 and Table 3). The methane
Monoglycerides (% w/w) 0.28 ± 0.08
Diglycerides (% w/w) 0.02 ± 0.01
content of the biogas was similar at both temperatures, ranging
Triglycerides (% w/w) n.d. between 56.3% (8% glycerin) and 59.8% (6% glycerin).
Ca (mg/kg) 1.7 ± 0.1
K (mg/kg) 19.5 ± 0.7
3.3.2. Production of biogas (ground manure)
Na (mg/kg) 16759.96 ± 56.9
Fig. 5 shows the results obtained regarding the production of
n.d.: not detected. biogas in the mesophilic anaerobic digestion of ground manure.
7848 L. Castrillón et al. / Bioresource Technology 102 (2011) 7845–7849

1.20 Table 3
Biogas production and composition in batch mesophilic and thermophilic anaerobic
co-digestion of screened cattle manure and crude glycerin.
CODs /CODs (initial)

1.15
SM Mixture Initial COD Initial VS Lbiogas/ Lbiogas/ CH4
(g/kg) (g/kg) kgCODrem kg VS (%)
SM + 4%glycerin 35 °C 35 °C 35 °C
1.10
SM+6%glycerin 55 °C 55 °C 55 °C
(SM + 4% 73.98 56.38 365.4 235.1 57.8
1.05 glycerin)sonic. 228.3 140.5 58.7
(SM + 6% 89.58 72.55 335.1 168.9 58.7
glycerin)sonic. 476.4 298.7 59.8
1.00 (SM + 8% 100.53 69.91 192.3 96.7 58.4
0 500 1000 1500 2000 2500 3000 3500 glycerin)sonic. 409.9 261.4 56.3
Specific energy (kJ/kg TS)

1.25
40000

1.20
CODs /CODs initial

GM

Biogas volume, mL
30000
1.15 (SM + 4% glycerin) sonic.
GM + 4% glycerin
(SM + 6% glycerin) sonic.
GM + 6% glycerin (SM + 8% glycerin) sonic.
1.10 20000

1.05
10000
1.00
0 500 1000 1500 2000 2500 3000 3500
Specific Energy (kJ/kg TS) 0
0 10 20 30 40
Fig. 2. Influence of the specific energy of ultrasound on COD solubilization in
Time (days)
screened and ground cattle manure with and without crude glycerin.
Fig. 4. Biogas production in batch thermophilic anaerobic co-digestion of previ-
ously sonicated mixtures of screened cattle manure and crude glycerin.

25000
SM
SM sonic.
25000
Biogas volume, mL

20000 SM + 4% glycerin (GM + 4% glycerin)sonic.

SM sonic. + 4% glycerin (GM + 6% glycerin) sonic.
Biogas volume, mL

(SM + 6% glycerin) sonic. 20000 (GM + 8% glycerin) sonic.

15000
SM sonic. + 6% glycerin
(SM + 4% glycerin) sonic. 15000
10000
10000
5000
5000
0
0 5 10 15 20 25 30 35 40 0
Time (days) 0 10 20 30 40
Time (days)
Fig. 3. Effect of sonication and of the addition of crude glycerin on biogas
production in batch mesophilic anaerobic digestion of screened cattle manure. Fig. 5. Biogas production in batch mesophilic anaerobic co-digestion of previously
sonicated mixtures of ground cattle manure and crude glycerin.

As with screened manure, the best results were obtained, when

adding 4% glycerin as co-substrate, although biogas production Table 4
Biogas production and composition in batch mesophilic and thermophilic anaerobic
was higher when using screened manure (Table 4).
co-digestion of ground cattle manure and crude glycerin.
As with screened manure, the highest biogas production was
obtained in the thermophilic range, although the biogas amounts Mixture Initial COD Initial VS Lbiogas/ Lbiogas/ CH4
(g/kg) (g/kg) kgCODrem kg VS (%)
obtained using ground manure were lower than those obtained
35 °C 35 °C 35 °C
using screened manure, with the exception of the mixture contain- 55 °C 55 °C 55 °C
ing 6% glycerin. In this case, 581.6 L biogas/kg COD removed were
(GM + 4% 72.14 53.00 245.5 116.6 58.7
obtained, with a methane content of 59.8 (Fig. 6 and Table 4). As glycerin)sonic. 141.9 86.0 57.4
with the screened manure, the best results were obtained using (GM + 6% 89.02 71.18 136.1 72.7 57.7
6% glycerin. glycerin)sonic. 581.6 291.6 59.7
In agreement with results from other studies (Yang et al., 2008; (GM + 8% 103.24 73.34 147.4 70.2 58.3
glycerin)sonic. 284.3 190.9 52.5
Fountoulakis et al., 2010; Robra et al., 2010), glycerol as co-
substrate for anaerobic digestion was shown to increasing biogas
production; however, the studies differed in the optimum amount
of glycerol required. For example, Fountoulakis et al. (2010) found in the feed could not exceed 1% for a stable digestion process.
that adding 1% v/v crude glycerol to sewage sludge doubled CH4 Robra et al. (2010) found that the optimal proportion of glycerol
production in the reactor (at 35 °C), but that the amount of glycerol in the anaerobic digestion of cattle slurry at 35 °C ranged between
 L. Castrillón et al. / Bioresource Technology 102 (2011) 7845–7849
40000
35000
Biogas volume, mL
30000
25000
20000 (GM + 4% glycerin) sonic.
(GM + 6% glycerin) sonic.
15000
(GM + 8% glycerin) sonic.
10000
5000
0 production from manure. Water Research 43 (1), 166–172.
0 10 20 30
Time (days)
Fig. 6. Biogas production in batch thermophilic anaerobic co-digestion of previ-
ously sonicated mixtures of ground cattle manure and crude glycerin.
5% and 10%. Since the composition of crude glycerin is not given in
these researches, it is not possible to compare results. Besides,
some studies are carried out at 35 °C and others at 55 °C. Yang
et al. (2008) found better performance in a reactor with crude glyc-
erin and synthetic wastes at 55 °C than at 35 °C, in agreement with
our results. The different results suggest that the optimum amount
of glycerol depends on various factors, such as nature of substrates,
digestion temperature and composition of the crude glycerin.
4. Conclusions
Applying ultrasound as a pre-treatment increased the produc-
tion of biogas in the anaerobic digestion of screened cattle manure
or mixtures of screened cattle manure as did the addition of crude
glycerol as co-substrate. The optimum amount of glycerol as sup-
plement to cattle manure in anaerobic digestion ranged between
4% when operating at 35 °C and 6% when operating at 55 °C. The
best result (348 L methane/kg COD removed) was obtained in the
thermophilic anaerobic digestion of a previously sonicated mixture
of ground manure with 6% glycerin. Continuous experiments need
to be performed in order to evaluate the economic feasibility of the
process.
Acknowledgement
The authors gratefully acknowledge the funding received from
the Spanish Ministry of Education (PROBIOGAS) (Ref. PSE
120000-2007-16).
7849
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