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Methods for increasing the biogas potential from the

Water Science and Technology Vol 41 No 3 pp 189194 2000 IWA Publishing and the authors
recalcitrant organic matter contained in manure
I. Angelidaki1 and B.K. Ahring1,2
1Department of Biotechnology, Building 227, The Technical University of Denmark, 2800 Lyngby, Denmark

(E-mail: ria@ibt.dtu.dk)
2School of Engineering and Applied Science, Department of Civil and Environmental Engineering,

The Technical University of Denmark, 2800 Lyngby, Denmark

Abstract The biogas potential of manure could be significantly increased by treatment of the recalcitrant
organic matter (biofibers) contained in the manure. Several treatment methods were tested. Mechanical
maceration resulted in an average increase of the biogas potential of approximately 17% as shown by the
continuous stirred reactor experiment. In general the smaller the fibers the higher the biogas potential was.
The best results showed an approximately 20% increase of the biogas potential with fibers smaller than 0.35
mm as measured by batch experiments. The increase was approximately 16% with fibers of size 2 mm.
Chemical treatment of the fibers with bases such as NaOH, NH4OH or a combination of bases also resulted
in an increased methane potential. However, combination of maceration and chemical treatment did not
result in a further increase of the methane potential. There was not any significant difference of the biogas
potential from fibers in the range 520 mm. Treatment of the fibers with hemicellulolytic or cellulolytic
enzymes did not result in any significant increase of the methane potential. However, biological treatment of
the fibers of the manure with the hemicellulose degrading bacterium B4 resulted in a significant increase of
the biogas potential of manure. An increase of approximately 30 % in methane potential was achieved
compared to controls.
Keywords Biofibers; biogas; manure; chemical treatment; biological treatment; maceration

Introduction
Anaerobic degradation of organic waste is an environmentally attractive way of treating
organic waste. Anaerobic treatment of organic wastes in large scale biogas plants has been
supported the last decade in many European countries and substitutes for the disposal of
organic wastes in landfills, a solution that in modern times is considered undesirable. Many
centralized full-scale biogas plants have been built in Denmark operating with a mixture of
mainly animal manure supplemented with industrial organic wastes. Economic evaluation
of these plants reveals that the plants can be economically viable, only if special taxation
measures economically favor energy from biogas, compared to energy from conventional
fossil fuels. Additionally the economy of biogas plants can be improved by using high bio-
gas potential substrates in addition to manure. However, as more biogas plants have
emerged and many more are planned the supply of industrial waste becomes scarce. It is
therefore necessary to find ways to increase the biogas potential of manure and thus be able
to keep biogas plants economically viable based only on manure.
A large part (4050%) of the total solids in manure and other wastes consists of biofibers.
Biofibers can only partly be degraded during the biogas process. Increase of the biodegrad-
ability of biofibers will be an important factor for the economic feasibility of manure based
full scale biogas plants.
Biofibers consist of lignocellulosic matter that forms the support structure of most
plants. Biofibers consist of a core of carbohydrates, holocellulose (cellulose and hemicellu-
lose) which makes up 6378% of the fiber lignocellulose structure (Crawford, 1981).
Holocellulose is packed and supported by lignin, a natural and very stable binder which
makes up 1538% of the lignocellulose structure (Sarkanen and Heregeret, 1971). 189
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Microbial hydrolysis of natural lignocellulose is a slow and difficult process, due to the
chemical and physical construction of lignocellulose. The lignin is tightly associated to
hemicellulose, which covers the cellulose and creates a physical barrier for the hydrolytic
enzymes. Furthermore, the cellulose arranges itself in crystalline structures, which are also
difficult to degrade. Finally, there is a relatively small surface area where the micro-
organisms can attack the fibers and break down their structure.
I. Angelidaki and B.K. Ahring

Holocellulose is degradable organic matter, while the lignin part of fibers is almost total-
ly recalcitrant to microbial degradation (Sarkanen and Heregeret, 1971). Therefore, effi-
cient utilization of lignocellulosic material requires that the holocellulose part is made
accessible for the hydrolytic enzymes produced by bacteria that are able to degrade cellu-
lose and hemicellulose. To improve the biodegradability of lignocellulosic materials, sev-
eral treatment methods have been investigated, having as a primary goal to decrease the
association of lignin with the degradable part of biofibers and thereby increase the substrate
accessibility to the bacteria. Other less important goals are to break down the crystalinity of
cellulose to increase the pore size of cellulose and to increase the specific surface area
(Sarkanen and Heregeret, 1971).
However, most other investigations have been centred on increasing the biodegradabili-
ty of straw or other dry lignocellulosic material for the purpose of monosaccharide produc-
tion for ethanol production.
In this study we have investigated mechanical, physical, chemical, microbiological and
enzymatic treatment methods for increasing the biogas potential of manure. The treated
manure was then tested in anaerobic batch and continuous reactor experiments to ascertain
any possible effect of the treatment.

Materials and methods

Substrates
As substrates for the experiments, cattle manure or fibers separated from cattle manure
from a full-scale biogas plant have been used. The manure was kept frozen until use.
The treatment methods applied are divided into three groups: physical, chemical and
biological treatment.

Treatments methods tested

Physical treatment: As physical treatment maceration was tested. Fresh manure was macer-
ated with a blender resulting in manure with different sizes of fibers. Also separated fibers
from digested manure were blended and were sieved in order to achieve different sizes of
fibers. In addition to maceration mechanical destruction of the fibers was achieved by pres-
surizing manure to 100 atm or more and then forcing the pressurized manure through a hole
of 1.2 mm to atmospheric pressure. This decompression results in an explosion of the fibers
and thus affects the lignocellulosic structure of the fibers.

Chemical treatment: The separated fibers were treated with NaOH, or NH4OH or a combi-
nation of bases (2:2:1) NaOH:KOH:Ca(OH)2 for a period of 24 to 48 hours.

Biological treatment: The separated fibers were treated with the hemicellulose degrading
bacterium B4. The bacterium was cultivated with xylan as substrate. When the culture was
growing in the exponential phase, the bacteria were concentrated by centrifugation and 1ml
of the bacteria concentrate was added to the fibers. The fibers were incubated 7 days at 70C,
which is the optimal growth temperature of this bacterium. As controls, vials with fibers and
190 the following combinations were included: with autoclaved B4, with B4 but without
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incubation before testing, and without B4. Furthermore, controls where fibers were incu-
bated at 70C for 7 days without addition of B4 were included.

Evaluation of the treatment methods

Batch and CSTR experiments were performed for evaluation of the treatment methods.

I. Angelidaki and B.K. Ahring

Batch experiments: The batch experiments were performed in 117 ml vials. The vials con-
tained: fibers (1 g volatile solids (VS) of fibers) to be tested, 15 ml of BA-medium
(Angelidaki et al., 1990) and 15 ml of inoculum. As inoculum digested manure from a ther-
mophilic (55C) lab scale reactor was used. The mixture was gassed with N2:CO2 (4:1) and
a further 0.3 ml of Na2S 8H2O and 0.3 ml of vitamin solution of DSM (1989) were added to
assure optimum anaerobic conditions. The vials were closed with rubber stoppers and alu-
minium crimps. As controls vials without fibers only with growth medium and inoculum
were used. The methane produced in the headspace of the vials was measured every two to
three days for a period of 45 to 60 days. All experiments were run in triplicate.

CSTR reactor experiments: For this experiments 4.5 l, automated lab-scale reactors with a
liquid working volume of 3 l were used. The reactors had a slowly moving stirring blade,
which was activated for 1 min every 3 min. Gas production was measured automatically.
The reactor temperature was kept at 55C. The reactors were started up with 100% inocu-
lum (digested manure from a thermophilic lab scale reactor) and then fed for approximately
1 months, until steady state was assumed, with untreated or macerated cattle manure.
Thereafter the experiments were initiated.
Two experiments were performed with above reactors. In the first experiment the effect
of maceration was tested. In one reactor (R1) untreated manure was fed, while the other
reactor was fed with macerated manure (R2). Data are shown in Figure 2 starting when the
reactors had run under these conditions for approximately 15 days, corresponding to one
hydraulic retention time (HRT). The two reactors were further run for approximately two
HRT with different feed (macerated/untreated for R1 and R2 respectively), whereafter both
reactors were fed with macerated manure in order to verify that the measured effect was due
to maceration only. In this experiment both reactors were fed manually twice a day, as auto-
matic pumping was difficult due to the large fiber size of the untreated manure resulting in
clogging of the tubing. For the feeding a syringe attached through a valve to the reactor was
used. After the syringe was attached to the reactor the valve was opened, thus no contact
with air was permitted minimizing the risk for oxygen contamination of the reactors.
In the second experiment, the combined effect of maceration and NaOH treatment of
fibers was tested. In the beginning of this experiment both reactors were fed with macerated
cattle manure. At day 9 (Figure 3) the influent macerated manure was treated as follows:
Macerated manure was centrifuged in order to separate the manure into a fiber fraction and
a liquid fraction. Then the fiber fraction was mixed with NaOH and kept for one day before
it was mixed back with the liquid fraction of the manure. The two reactors were fed auto-
matically with pumps four times a day. One reactor was fed with manure where the fibers
were treated with 40 g/kg VS NaOH (R40-NaOH), while the other was fed with manure
where fibers were treated with 80 g/kg VS NaOH (R80-NaOH).

Analytical methods
Methane and volatile fatty acids (VFA) were measured by gas chromatography as
previously described Srensen et al. (1991). 191
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Table I Effect of the different treatments on the methane potential

achieved from cattle manure

CH4 potential
Treatment increase (%)

Maceration < 0.35 mm 20

Maceration 2 mm 16
I. Angelidaki and B.K. Ahring

Decompression explosion 17
NaOH 20 g/kg VS 13
NaOH 40 g/kg VS 23
NH4OH <20 g/kg VS 0
NH4OH 40 g/kg VS 23
NaOH:KOH:Ca(OH)2 40 g/kg VS 20
Hemicellulose degrading bacterium B4 30

Results
Batch experiments
The results from the batch experiments are summarized in Table 1.
The fiber size had a significant effect on the degradability of the fibers. The smaller the
fibers were the higher the methane potential of the macerated manure compared to untreat-
ed manure was. When manure was macerated to a size smaller than 0.35 mm the methane
potential was 20% higher than for untreated manure. When the size was between 12 mm
the increase was 16%. However, decrease of the size from 20 to 5 mm did not significantly
increase the methane potential of manure. Similar results were reported by other investiga-
tions. Sharma et al. (1988) reported that macerating straw in different sizes results in a high-
er potential. However, particle sizes smaller than 6 mm did not result in much methane
potential improvement as compared to particle reduction to between 6 and 30 mm.
Decompression explosion showed similar results as when manure was macerated to
12 mm. Other forms of explosion, such as ammonia-freeze-explosion have also been
reported to increase the availability of fibers to microbial degradation (Dale and Moreira,
1989).
The observed effect of maceration and decompression explosion can be explained by the
physical destruction of the lignocellulosic structure and increase of the relative surface area
of the lignocellulosic particles. By investigation of the microflora in the stomach of cows it
has been observed that bacteria attack fibers at the free ends and through destroyed surfaces

192 Figure 1 Biological treatment of fibers with hemicellulose degrading bacterium B4

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I. Angelidaki and B.K. Ahring

Figure 2 Effect of maceration on the biogas potential of fibers from cattle manure in CSTR experiment

(Hobson and Wheatley, 1993). Furthermore physical treatment has probably reduced the
crystallinity of lignocellulose.
Chemical treatment with NaOH, NH4OH, or the base combination
NaOH:KOH:Ca(OH)2 (2:2:1) (Table 1) showed a significant effect on the methane poten-
tial of manure. The bases had to be added at a concentration of at least 20 g base/kg VS.
However, 40 g base/kg VS was required before any effect was measured for NH4OH. The
effect observed in this study with chemical treatment is in agreement with the known effect
of chemical destruction of lignocellulosic structure. Chemical treatment of lignocellulosic
material is used to a wide extent in the paper industry, which in many years have used a pulp
process with base treatment in order to de-lignify the lignocellulose, which gives paper with
higher strength (Fan et al., 1982; Hobson and Wheatley, 1993). Furthermore, chemical
treatment has been used for years for improving straws quality as animal feed.
Biological treatment of fibers by addition of hemicellulose degrading bacterium B4
exhibited a significant increase of the biogas potential (Figure1). Considering losses due to
biomass synthesis and assuming lignin as inert the theoretical maximum yield from degra-
dation of the manure used has been estimated to be in the range 300 to 350 ml methane/g
VS, (Figure 1). The actual methane production obtained for treated manure reached

Figure 3 Effect of combined treatment by maceration and NaOH treatment, on the biogas potential of cattle
manure, in CSTR experiment 193
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approximately 300 ml/g VS, which is close to the estimated maximum methane potential.
All the controls showed a biogas production of approximately 230 ml/g VS (Figure1),
showing that treatment of the fibers in manure with hemiclullose degrading bacterium B4
resulted in an approximately 30% increase of the methane potential (Table 1).

CSTR reactor experiments

I. Angelidaki and B.K. Ahring

Reactor R2 receiving macerated manure produced approximately 17% more biogas than
reactor R1 receiving untreated manure.
In addition the process was more balanced as indicated by the slightly higher volatile
fatty acids (VFA) concentration in the reactor receiving untreated manure.
However, the difference in VFA could only explain approximately 20% of the difference
in biogas production in the two reactors, indicating that the hydrolysis of organic macro-
molecules to intermediates such as VFA was also lower in R1 than in R2.
At day 28 (Figure 2) when both reactors were fed with macerated manure, the biogas
production in both reactors became similar, indicating that the difference measured for the
first period of the experiment was indeed due to the differences in the fiber size of the feed.
The results from the CSTR reactor experiments are in agreement with the results from the
batch experiments, further supporting maceration as one promising method of increasing
the biogas potential from livestock wastes.

Conclusion
The investigations performed have revealed several possible ways/methods to improve the
biogas yield of manure.
Maceration of manure seems to be a promising and straightforward option to implement
on full-scale plants. Also the biological treatment with specific hydrolytic could be very
interesting, especially if the bacterium could be cheaply cultivated on the plant.
Chemical methods showed promising results, however the feasibility depends on the
dosing/price relation and equipment cost involved. Further evaluation and optimization is
required.

Acknowledgement
This program was supported by grants from the research program of The Danish Energy
Council, no, 1383/95-002.

References
Angelidaki, I., Petersen, S.P. and Ahring, B.K. (1990). Effects of lipids on thermophilic anaerobic digestion
and reduction of lipid inhibition upon addition of bentonite. Appl. Microbiol. Biotechnol., 33, 469472.
Crawford, D.L. (1981). Microbial conversion of lignin to useful chemicals using a lignin-degrading strepto-
myces. Biotechnol. Bioeng. Symp., 11, 275291.
Cunningham, R.L., Carr, M.E. and Bagby, M.O. (1985). Improved hemicellulose recovery from wheat straw:
Biotechnol. Bioeng. Symp., 15, 1726.
Dale, B.E. and Moreira, M.J. (1989). A freeze technique for increasing cellulose hydrolysis: Biotechnol.
Bioeng., 33, 3143.
Fan, L.T., Lee, Y.-H. and Gharpuray, M.M. (1982). The nature of lignocellulosics and their pretreatments for
enzymatic hydrolysis. Adv. Biochem. Eng., 23, 157187.
Hobson, P.N., Wheatley, A.D. (1993). In: Anaerobic digestion: Modern theory and practice, 149174,
Elsevier Applied Science, London and New York.
Sarkanen, K.V. and Heregeret, H.L. (1971). Lignins Occurrence, formation, structure and reactions, p. 43,
Wiley-Interscience, New York.
Sharma, S.K., Mishra, I.M., Sharma, M.P., and Saini, J.S. (1988). Effect of particle size on biogas generation
from biomass residues. Biomass, 17, 251263.
Srensen, A.H., Winther-Nielsen, M., and Ahring, B.K. (1991). Kinetics of lactate, acetate and propionate in
unadapted and lactate-adapted thermophilic, anaerobic sewage sludge: the influence of sludge adaptation
194 for start-up of thermophilic UASB- reactors. Appl. Microbiol. Biotechnol., 34, 823827.