biomass and bioenergy 33 (2009) 10461054

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Anaerobic digestion of slaughterhouse by-products

Anette Hejnfelt, Irini Angelidaki*
Department of Environmental Engineering, Technical University of Denmark, DTU, Building 113, DK-2800 Kgs. Lyngby, Denmark

article info

abstract

Article history:

Anaerobic digestion of animal by-products was investigated in batch and semi-continuously

Received 13 January 2008

fed, reactor experiments at 55  C and for some experiments also at 37  C. Separate or mixed

Received in revised form

by-products from pigs were tested. The methane potential measured by batch assays for

11 March 2009

meat- and bone flour, fat, blood, hair, meat, ribs, raw waste were: 225, 497, 487, 561, 582, 575,

Accepted 17 March 2009

359, 619 dm3 kg1 respectively, corresponding to 50100% of the calculated theoretical

Published online 21 May 2009

methane potential. Dilution of the by-products had a positive effect on the specific methane
yield with the highest dilutions giving the best results. High concentrations of long-chain

Keywords:

fatty acids and ammonia in the by-products were found to inhibit the biogas process at

Methane yields

concentrations higher than 5 g lipids dm3 and 7 g N dm3 respectively. Pretreatment

Pasteurization

(pasteurization: 70  C, sterilization: 133  C, and alkali hydrolysis (NaOH) had no effect on

Sterilization

achieved methane yields. Mesophilic digestion was more stable than thermophilic digestion,

Inhibition

and higher methane yield was noticed at high waste concentrations. The lower yield

Pretreatment

at thermophilic temperature and high waste concentration was due to ammonia inhibition.

Temperature

Co-digestion of 5% pork by-products mixed with pig manure at 37  C showed 40% higher

Co-digestion

methane production compared to digestion of manure alone.

EC regulation No. 1774/2002

1.

Introduction

Pork production constitutes a significant part of meat

production in Denmark. More than 24 million pigs are
slaughtered annually in Denmark [1]. Due to higher meat
demand at present compared to the past, the quantity of
organic by-products from slaughterhouses has increased.
Approx. 25% of the total animal weigh slaughtered is not used
for food consumption. During the last 60 years, slaughterhouse waste, rich in proteins and lipids, has been treated and
used for production of animal fodder. However, due to legal
restrictions and environmentally conscious consumers, the
treatment of wastes and by-products has emerged as a major
concern not only in pork industry but also in meat industry in
general. For instance, outbreak of diseases such as bovine
spongiform encephalopathy (BSE) in cattle and the dangerous

* Corresponding author. Tel.: +45 45 25 14 29; fax: +45 45 93 28 50.

E-mail address: ria@env.dtu.dk (I. Angelidaki).
0961-9534/$ see front matter 2009 Published by Elsevier Ltd.
doi:10.1016/j.biombioe.2009.03.004

2009 Published by Elsevier Ltd.

human disease CreutzfeldJacob in 2001, has resulted in

increasing awareness of the need for hygiene regulations,
tighter process control, and the prohibition of utilization of
some animal by-products [2].
According to the legislation slaughterhouse waste must
be treated by different treatments depending on the category of the animal byproduct [3]. Three categories of animal
by-products are defined; Category 1 is high risk material
(part of infected animals, international catering etc.) and is
not allowed to be treated in composting or biogas plants
under any circumstances; category 2, high risk animal byproducts (diseased animals, manure and digestive tract
content) cannot be used as feedstock in composting and
biogas plants, unless they have first been rendered to the
133  C, 300 kPa, 20 min EU pressure-rendering standard
(sterilization); and finally category 3, low risk material

biomass and bioenergy 33 (2009) 10461054

(catering residues, meat, precooked foods, etc.) approved for

food consumption, must be treated to at least 70  C for 1 h in
a closed system [3].
Anaerobic digestion of animal by-products constitutes
a possible method of treating the by-products and at the same
time produce energy in the form of methane, and utilization of
the digestion effluents as fertilizer for application on agricultural fields for nutrient recovery, [4]. However, slaughterhouse
wastes are generally regarded as difficult substrates for anaerobic digestion, mainly because of their typically high protein
and lipid content [5]. Protein degradation releases ammonia,
which at high concentrations is suggested to be inhibitory for
the anaerobic microorganisms [6,7]. It is generally considered
that the unionized form of ammonia is the cause of inhibition
and concentrations ranging from 0.1 to 1.1 kg-N m3 are
reported as inhibiting concentrations [7]. Additionally, lipids
may also cause problems in anaerobic digestion because of
their tendency to promote floating scum and due to possible
accumulation of inhibiting degradation intermediates such as
long-chain fatty acids (LCFAs) [8,6,9]. The breakdown of LCFAs
can be the rate-limiting step in the degradation of complex
substrates [10] requiring gradual adaptation and careful dosing
of lipid rich waste products to avoid LCFA accumulation.
Already at very low concentrations (such as 0.5 kg m3), LCFAs,
especially unsaturated LCFAs, are suggested to be inhibitory to
syntrophic acetogenic and methanogenic bacteria [6,11]. The
relatively high N content, the high total solids (TS) content,
often exclude the possibility of treating animal by-products in
their original undiluted form. Therefore, dilution is typically
necessary or, as a more attractive option, co-digestion with less
concentrated organic waste types, such as manure or waste
water. In co-digestion the concentrated waste types can highly
increase the biogas production compared to dilute wastes,
while dilute wastes can provide process stability and serve as
dilution media while also being treated.
Anaerobic digestion of animal by-products reported in the
literature include studies with by-products from poultry, e.g.
blood, meat and bones [4], rumen and cattle blood [5], blood
and category 3 material from pigs [12]. In addition, co-digestion
of manure and rumen [13] and blood and rumen from cattle
and pigs have been studied in laboratory and pilot scale under
mesophilic conditions (37  C) and with pre-treatment (heating)
[14]. Methane yields of 0.520.55 m3 kg1 VS were reported
under mesophilic conditions for solid slaughterhouse waste
in 2 dm3 continuously stirred tank reactor (CSTR) operated at
0.8 kg VS m3 d1 with 50 days HRT [4]. However, information
about methane yields of different parts of animal by-products
is still lacking. It would be expected that thermophilic
temperature would have better sanitation effect compared to
mesophilic, however, no studies on thermophilic digestion of
animal by-products are yet reported.
In the present study, the methane yields of several types of
by-products from a pig slaughterhouse were determined in
batch assays at different concentrations. Levels for inhibition
under thermophilic and mesophilic conditions were investigated. The effect of heating (70  C, 1 h) and sterilization (133  C,
300 kPa, 20 min) on the methane yield of mixed pig-waste was
tested. Finally co-digestion of animal by-products from
a slaughterhouse and manure was tested in semi-continuously
fed reactors at mesophilic and thermophilic temperatures.

2.

Materials and methods

2.1.

Substrate

1047

Five different types of pig slaughterhouse by-products; fat,

blood, raw waste (meat, fat and bones), intermediate product
(pressed raw waste) and bone flour were received from a Danish
animal waste processing company (Daka, Denmark; Lat: N
55 250 45.3700 Long: E 11 470 54.7300 ). All these by-products were
pretreated at the factory before delivered for experimental use.
The pretreatment at the factory included maceration to particle
size smaller than approx. 34 mm of a large portion, followed by
homogenization in order to take a representative sample. A
smaller portion was thereafter sampled and sent to DTU for
analysis and experiments. In addition, pig hair, fresh meat and
ribs were obtained from a trainee-slaughterhouse (Roskilde,
Denmark; Lat: N 55 370 50.6100 Long: E 12 40 43.1600 ). The pretreatment of the different waste products are shown in Table 1.
Finally, mixed pork waste consisting of all non-commercial
parts of one slaughtered pig, was collected and immediately
delivered for testing from a slaughterhouse. The mixed pork
waste, after removal of bones, was homogenized in a blender
resulting in particle size less than 2 mm (Table 2).
Thermophilically digested manure from a centralized
biogas plant (Lemvig centralized biogas plant, Denmark; Lat: N
56 310 0.9000 Long: E 8 180 47.7900 ) was used as inoculum for batch
and semi-continuous reactor experiments. For the mesophilic
experiments inoculum from a mesophilic biogas plant (Nysted
centralized biogas plant, Denmark; Lat: N 55 400 39.8100 Long: E
12 120 18.0700 ) was used.
Chemical analysis of the above mentioned materials was
performed immediately upon arrival at the laboratory and the
experiments were initiated. The mixed pork waste used for
long term continuous experiments was frozen down in 2 kg
portions and was thawed before use.

2.2.

Pretreatment

The effect of three different pretreatments on characteristics

and methane production potential of the mixed pork waste
was evaluated (Table 1). The pretreatments tested were:
heating (70  C for 1 h); sodium hydroxide (50 or 100 g NaOH
kg1 VS) treatments and finally autoclaving (133  C and
300 kPa for 20 min). As mentioned the Daka products were
pretreated at Daka.

2.3.

Methane yield

Assays for determination of the methane yield of the various

by-products from pigs were carried out in either 0.5 dm3 or
2 dm3 bottles. The assays were carried out at 55  C for all
tested by-products except for the mixed pork waste for which
the methane yield assay was performed at both 55  C and
37  C in order to elucidate the effect of temperature on the
anaerobic digestion of mixed pork waste. Four different
concentrations (5%, 20%, 50%, and 100%) of substrate (weight
basis) were prepared by dilution with water. Substrate, basic
anaerobic (BA) synthetic media [15] and inoculum (60% of
active volume) (retrieved 1 or 2 days before the experiments)

1048

biomass and bioenergy 33 (2009) 10461054

Table 1 Batch experiments, overview.

Waste product
Raw waste

Blooda
Inter-mediate producta
Bone floura
Lipida
Bones
Hair
Meat (pure meat)
Mixed pork waste

Pretreatment

Concentration %

Autoclaved in lab. at 300 kPa and

133  C for 20 min. (Jeppesen, 2003)
Autoclaved in lab. At 300 kPa and
133  C for 20 min. (Jeppesen, 2003).
Heated to 85  C for 20 min.
(Jeppesen, 2003).
Sterilized in the process,
133  C for 20 min. (Daka, 2003).
Sterilized in the process at
125  C for one hour (Daka, 2003).
Untreated
Untreated
Heated, 70  C, 1
10 mm
50 mm
Untreated
Heated, 70  C, 1 h
Sterilized, 133  C
NaOH

Incubation temp.


Inoculum used

5, 20, 50, 100

55 C

Lab.-reactor

5, 20, 50, 100

55  C

Lab.-reactor

5, 20, 50, 100

55  C

Lab.-reactor

5, 20, 50, 100

55  C

Lab.-reactor

5, 20, 50, 100

55  C

Lab.-reactor

5 and 10
5, 20, 50, 80
5, 20, 50
5 and 10

55  C
55  C

Snertinge biogas plant

Inoculum from own
reactor experiment
Snertinge biogas plant

5, 20, 50, 80
5, 20, 50, 80
5, 20, 50, 80
5

55  C/37  C

55  C

Thermo.: Lemvig
Meso.: Nysted biogas plant

a Daka products.

were added in the bottles under constant flushing with N2/CO2

(80%/20%). Finally, the bottles were sealed with butyl rubber
stoppers and aluminium crimps. Bottles containing only
water, BA media and inoculum (60% of active volume) were
used as controls. An overview of all the batch assays is presented in Table 1.

2.4.

Continuously stirred tank reactor experiments

Co-digestion of different mixtures of manure and mixed pork

waste was investigated in three identical semi-continuously
fed CSTRs (referred to as Rt,20, Rt,5 and Rm,5), each with a total
capacity of 5 dm3 and a working volume of 3.2 dm3. Rt,20 and
Rt,5 were operated at 55  C while Rm,5 was operated at 37  C.
Reactors were fed by peristaltic pump at an interval of 8 h
from a continuously stirred feed bottle. On day 0, the reactors
were inoculated. The first 25 days the hydraulic retention time
(HRT) of the thermophilic reactors was 15 days, but because of
high VFA concentrations HRT was increased to 21 days. The
organic loading rate (OLR) operating on pig manure only was
6 g VS day1. Upon reaching stability as indicated by low daily
variations in biogas production and VFA content (lower than
10%), co-digestion of manure with mixed pork waste was
initiated (day 75 for the thermophilic reactors and day 38 for
the mesophilic reactor). 0.15 dm3 (mixed pork waste and
manure) were added per day. Ratios of mixed pork waste to
manure tested were 20% waste in Rt,20 (OLR 12.5 g VS day1),
5% waste in Rt,5 and Rm,5 (8.3 g VS day1) respectively. After
day 63 the composition of the feed in Rm,5 was changed to 2.5%
mixed pork waste in manure, in order to reveal whether
inhibition was present at 5% animal waste additions.

2.5.

Analytical methods

pH was measured with Metrohm 744 pH meter immediately

after each sampling. Methane content in the biogas was
analysed using a Gas Chromatography (GC-14A) equipped

with TC detection and chromosorb 108 column (1.1 m 3/1600

molsieve 137) [6]. The biogas produced in CSTRs was
measured through gas meters [6] Volatile fatty acids (VFAs)
were determined using GC5890-series II equipped with flame
ionisation detection (FID) and HP FFAP column, (0.53 mm/
30 m/1.00 mm). Total Kjeldahl nitrogen, lipids (Soxhlet),
carbohydrates and total solids (TS) and volatile solids (VS)
were determined according to Standard Methods [16]. Protein
content was calculated from Kjeldahl nitrogen content
multiplying with a conversion factor of Kjeldahl-N, 6.25
(for meat).

2.6.

Calculation on theoretical methane yield

The theoretical methane yield at standard temperature and

pressure (0  C, 100 kPa) was calculated using the following
formula: dm3 kg1 X  0.496 + Y  1.014 + Z  0.415
Where: X % proteins of VS, Y % lipids of VS, Z %
carbohydrates of VS [15].

Table 2 Characteristics of different waste products

obtained from a single pig.
Composition of the mixed
pork waste product

Amount in
kg

Blood
Hair
Hair and skin
Intestine content
Meat fractions not for use, lipids
Bones
Head
Manure
Intestine fat

10.4
7.6
0.1
Not known
26.7
16.10
Not known
10.7
0.5

2.90
2.12
0.03
5.00a
7.45
4.49
2.78a
2.99
0.14

Total

100.0

27.90

a Estimated.

4.6
6.4
1.8
93.9
1.0
2.52
4.9
2.4
6.8
31.9
61.3
26.1
28.7
2.6
71.3
0.8
0.4
28.7
28.3
23.6
74.0
2.4
23.2
26.9
3.7
73.1
0.8
0.4
27.8
27.4
23.6
74.0
2.4
41.6
58.9
24.5
41.1

Results

3.1.

Characterization of by-products

a Not measured assumed that 100% of VS is lipid and that bones have the same composition as the meat.

15.6
40.9
25.2
17.2
61.6
21.2
10.6
58.0
47.4
11.1
82.7
6.2

15.6a
40.9a
25.2a
17.2a
61.6a
21.2a

25.6
28.6
3.0
71.4
35.8
39.7
3.9
60.3

99.2
99.4
0.1
0.8
0.7
0.0
0.0
0.0
100.0a
0.0
0.0
16.8
17.9
1.9
82.1
1.0
1.7
27.1
25.4
0.3
94.4
5.3
72.2
95.7
23.6
4.3
0.6
6.4
108.5
102.1
11.5
88.4
0.2
36.6
49.1
12.5
50.9
0.5
3.8
30.8
27.0
16.7
46.1
37.3
37.6
45.8
8.1
54.2
1.1
2.0
24.2
22.3
52.3
37.0
108
%
%
%
%
Kg m3
g N kg1
g N kg1
g N kg1
% of VS
% of VS
% of VS
Volatile solids
Total solids
Ashes
Water content
Density
Dissolved ammonia
Kjeldahl N
Organic N
Lipids
Proteins
Carbohydrates

1049

Characteristics of the pig by-products are presented in

Table 3.
All by-products, except for fat, had higher protein (3794%
of VS) and VS (16.8%99.2%) content than pig manure. In
manure, 32% of the VS was protein and had a VS content of
4.6%. The high protein content of the pig by-products indicates a potential for ammonia inhibition especially under
thermophilic conditions as ammonia-N load was relatively
high already when operated on pig manure alone. Among the
studied by-products, fat had the highest VS content of 99%
followed by bone flour (96%), while blood had the lowest 17%.
Especially the raw waste contained a lot of lipids (52% of the
VS), but also mixed pork waste had high content of lipids (24%
of VS). Pretreatment did not change the characteristic of the
by-products.

33.4
37.9
4.5
62.1
0.9
0.4
34.5
34.1
23.6
74.0
2.4

Heated mix.
pork waste
Untreated mix.
pork waste
Int. product

Bone flour

3.

3.2.

Raw waste

Table 3 Composition of the by-products used in the batch tests.

Blood

Fat

Hair

Meat

Bones

Sterilized mix.
pork waste

Pig manure

biomass and bioenergy 33 (2009) 10461054

Batch experiments

The methane production rates and yields for the different

by-products incubated at 37 and 55  C with mesophilic
and thermophilic inoculum, respectively, are presented in
Fig. 1.
Methane productions in all assays, except for pork fat
which had a lag phase of 20 days, started after an initial lag
phase of 35 days and continued for up to 3035 days. Methane
yields for the studied substrates varied with substrate
concentration, pretreatment and incubation temperature. For
most of the substrates incubation of 5% waste concentration
with 60% inoculum was found optimal with methane yields
ranging between 230 and 620 dm3 kg1 VS. These yields were
comparable to the theoretical yields of 500750 dm3 kg1 VS.
Highest methane yield of 620 dm3 kg1 VS was obtained for
mixed pork waste while the lowest methane yield were
obtained for bone flour when incubated at 5% bone-flour
waste concentration. For meat and bones, 10% waste
concentrations resulted in the highest methane yield which
was 580 dm3 kg1 VS, equal to the respective theoretical
methane yields. Blood had a maximum methane yield of
490 dm3 kg1 VS at 5% concentration. All by-products tested
had high gas production per kg waste. Pork fat had the highest
methane yield per kg waste (562 dm3 kg1 waste) while lowest
methane production per kg waste of 81 dm3 kg1 waste was
obtained from blood.

3.2.1.

The effect of pretreatment

Results showed that thermal treatment at 70  C and

treatment by addition of 50 or 100 g NaOH kg1 VS NaOH
had no significant effect on the biodegradability and
methane yields of mixed pork waste. Untreated mixed
pork waste had a specific methane yield of 600 dm3 kg1
which corresponded to the theoretical yield, thus
pretreatment was not expected to increase the methane
yield. Sterilization of the mixed pork waste had no effect
on the methane yield, but the methane production per kg
waste was improved, as VS content increased due to

1050

biomass and bioenergy 33 (2009) 10461054

dm3 CH4 kg-1 VS

1000

Meat (50mm pieces)

Daka raw waste

750
500
250
0

dm3 CH4 kg-1 VS

1000

Daka intermediate product

Ribs

750
500
250
0

dm3 CH4 kg-1 VS

1000

Daka Blood

Pig hair

Daka bone flour

Mixed pork waste

750
500
250
0

dm3 CH4 kg-1 VS

1000
750
500
250
0

10

20

30

40

10

20

30

40

Time (Days)
Fig. 1 Cumulated methane production for different pig slaughterhouse by-products incubated at 55 8C at different dilutions.
10%,- 20%, :50%, C 80%, B 100% and
theoretical methane yield.
A 5%,

water evaporation. Sterilized waste gave a production of

225 dm3 kg1 waste, while untreated, mixed pork waste
which gave 125 dm3 kg1 waste.

3.2.2.

The effect of temperature

The results showed that there was no difference in methane

yield between thermophilic (55  C) and mesophilic (37  C)
conditions when 5% pork waste was used in batch assays.
Theoretical yields (600 dm3 kg1 VS) were reached under both
conditions. However, when 50% pork waste was used, only
mesophilic digestion was possible (Fig. 2).
The theoretical value was reached, while no methane was
produced under thermophilic conditions. Ionisation of
ammonia is a function of temperature and pH. The free
ammonia concentration is increasing significantly with

temperature and pH [7]. Although the exact free ammonia

concentration was not estimated in the batches, we can
assume that the free ammonia concentration was higher at
55  C compared to 37  C, as the amount of animal waste and
thereby the total ammonia load was equal in both the mesophilic and thermophilic vials. This could explain the severe
inhibition at thermophilic digestion [7].

3.3.

Continuously stirred reactor experiments

3.3.1.

The thermophilic reactors (55  C)

The process performance of the thermophilic reactors is presented in Fig. 3 and Table 4.
The two thermophilic CSTRs were operated equally for 43
days, fed only with pig manure. The biogas production during

1051

biomass and bioenergy 33 (2009) 10461054

1000

dm3 CH4 kg-1 VS

Thermophilic vs. mesophilic for 50% of mixed pork waste

800
600
400
200
0
0

20

40

60

80

100

Time (days)
Fig. 2 Cumulated methane production of mixed pork waste (50% diluted) incubated at C 37 and - 55 8C.

Biogas production (dm3)

the initial phase (days 043), in both Rt,20 and Rt,5, was very
unstable (Fig. 3). During the initial phase very high VFA
concentrations were observed. Acetate was the pre-dominant
VFA with values reaching up to 120 mM. Propionate was found
increasing at the same time (from 10 to 40 mM). The VFA level
in reactor Rt,5 was similar to Rt,20 in this period.
During the next period (days 4375, stable period) the
daily biogas production was approx. 2200 ml per day (69%
methane) with daily variations lower than 0.2 dm3 biogas
6

Rt,20

Rt,5

1
0

120

VFA (mM)

3.3.2.

140

VFA, Rt,5
Ac

100

Prop

80

Iso-bu

60

But

40

Iso-val

20

Val

0
140

VFA (mM)

120

day1 (Fig. 3), corresponding to a specific methane yield of

approx. 220 dm3 kg1 VS, which is rather low for pig
manure. At day 75 addition of mixed pork waste to manure
feed was initiated. Mixed pork waste was applied to the
manure in Rt,20 and Rt,5 in concentrations of 20% and 5%
respectively. After 1 day the gas production in reactor Rt,20
stopped completely. A lot of foam was observed. In reactor
Rt,5 the gas production decreased to 0.73 dm3 day1 after 12
days (Fig. 3). In this period the acetate concentration
decreased, while the propionate concentration increased
slightly to reach 4045 mM (Fig. 3).

VFA, Rt,20

Ac
Prop

100

Iso-but

80

But

60

Iso-val

40

Val
20

The mesophilic reactor (37  C)

The process performance of the mesophilic reactor Rm,5 is

presented in Fig. 4 and Table 4.
Shortly after start up with pig manure, the biogas
production started. After an initial phase of 27 days the
daily
gas
production
was
stable
and
achieved
3.3 dm3 day1 (75% methane), corresponding to a specific
methane yield of approx. 350 dm3 kg1 VS which is somewhat high for pig manure. After 10 days with stable
conditions co-digestion was started. Mixed pork waste was
added to the manure in a concentration of 5%. Within 5
days, gas production increased from 3.3 dm3 day1 to
5.5 dm3 day1, corresponding to an overall specific yield of
489 dm3 kg1 VS or approx. 900 dm3 kg1 VS if extra
production is ascribed to pork waste VS only. The yield of
dm3 kg1 VS for pork waste was higher than determined
by methane potential batch assays, which indicates that
the higher process activity also had positive effect on the
methane production from manure. After 20 days of stable
process conditions the concentration of mixed pork waste
in feed was reduced from 5% to 2.5%. This resulted in
a decrease in biogas production from 5.5 dm3 day1 to
4.5 dm3 day1. The corresponding decrease in yield was
from 489 to 417 dm3 kg1 VS.

0
0

20

40

60

80

100

120

Time (days)
Fig. 3 Thermophilic anaerobic co-digestion of manure
with 5% (reactor Rt,20) and 20% (reactor Rt,5) mixed pork
waste; (a) Biogas production from Rt,5 and Rt,20, (b) VFA
concentrations of Rt,5 and (c) VFA concentrations of Rt,20.

4.

Discussion

The results from the present study showed that slaughterhouse waste in general had a good methane potential
and could produce 225619 dm3 kg1, which corresponds to

1052

biomass and bioenergy 33 (2009) 10461054

Table 4 Results from the continuous reactor experiments. Measured and calculated parameters for the reactors during the
stable period and co-digestion with mixed pork waste. The values are mean values from each period.
Reactor

Rt,5

Rt,20


Temperature
% Mixed porker waste
HRT (days)
Stable period with manure
Methane yield (dm3 kg1)
Methane (%)
% Of theoretical potential (%)
pH
Dissolved ammonia (g N dm3)
Free ammonia (g N dm3)
Degradation of VS %
Total VFA (mM)
Slaughterhouse waste and manure
Methane yield (dm3 kg1)
Methane (%)
pH
Dissolved ammonia (g N dm3)
Free ammonia (g N dm3)
Degradation of VS %
Total VFA (mM)

55 C
20 %
21
6 g VS d1
219.0
69.0
45.0
8.05
3.28
0.99
34.0
111.0
12.5 g VS d1
0.0
0.0
8.23
3.37
1.33

97.0

Biogas production (dm3)

50100% of the theoretical yields. Carpentier et al. [12],

investigated thermophilic batch digestion of pig slaughterhouse waste, category 3. They measured a biogas yield of
1.67 m3kg-1 VS (corresponding to 1085 dm3 kg1 assuming 65%
CH4 in the biogas). This value is very high and is approx. corresponding to the theoretical maximum yield of lipid, indicating that nearly all VS in the tested waste were lipid.
However, it is not possible to verify this assumption, as the
exact composition of the material they used was not reported.
Edstrom et al. [14] carried out mesophilic fed-batch digestion
with mixed, minced animal by-products. The specific
production of biogas was 760 dm3 kg1 VS (490 dm3 kg1 VS)

6
5

4
3
2
1
0
30

VFA (mM)

25

Ac

20

prop

15

Iso-but

10

But
Iso-val

Val

10

20

30

40

50

60

70

Time (days)
Fig. 4 Mesophilic anaerobic co-digestion of manure with
5% (days 3862) and 2.5% (days 6372) mixed pork waste
(Rm,5); (a) Biogas production and (b) VFA concentrations.

55 C
5%
21
6 g VS d1
219.0
69.0
45.0
8.04
3.30
0.98
30.0
117.0
8.3 g VS d1
61.0
71.0
8.10
3.32
1.08
40.0
128.0

Rm,5
37  C
5%
21
6 g VS d1
357.0
75.0
74.0
7.98
3.70
0.40
47.0
7.0
8.3 g VS d1
489.0
74.0
7.97
3.84
0.40
50.0
17.0

for untreated waste, which is lower than the 619 ml dm3 kg1
VS for mixed pork waste in batch assays found in this study.
In most types of by-products the highest specific methane
yields were achieved when the animal by-products were at
the highest dilutions (5%). This indicates that components of
the animal by-products constitute potential inhibitors for the
biogas process. Even 5% of raw waste, bone flour and fat
resulted in methane yields, which were only about 50% of the
theoretical yield. It appears from Table 3 that these substrates
had the highest content of VS, lipids and protein. In addition
animal by-products, due to their high protein and lipid
content, are prone to cause inhibition unless they are diluted.
From the correlation between process quality, as indicated by
the relative methane yield obtained compared to theoretical
yield and VFA levels at end of batch digestion, and the corresponding N-concentration (Fig. 5), it can be seen that for totalN concentrations higher than 7 g N kg1 the process was
severely inhibited (Fig. 5). The results are in accordance with
previous studies [17].
It is generally believed that higher temperatures result in
higher bacterial growth rate and metabolic activities [18,19].
However, in the present study it was shown that thermophilic digestion resulted in lower yields and a more stressed
process. Mainly ammonia were assumed to be the reason
for total process breakdown in the thermophilic batch
assays and reactor (Rt,5 and Rt,20). The VFA level in the
mesophilic CSTR reactor was lower (around 15 mM)
compared to the thermophilic CSTR reactors (around
45 mM) from the beginning of the experiments (Figs. 3 and
4), indicating that the thermophilic reactors were much
more stressed also before mixed pork waste was added to
the manure. This is also what Hansen et al. [7] concluded
when he investigated ammonia inhibition in manure at
different temperatures. Angelidaki and Ahring [17], showed
that mesophilic temperature is a better choice for processes
operating with high N loading because of a slightly lower

1053

% of theoretical CH4 yield

% of theoretical CH4 yield

biomass and bioenergy 33 (2009) 10461054

120
100
80
60
40
20
0

100
80
60
40
20
0
1000

800

Total VFA (mM)

Total VFA (mM)

1000

120

600
400
200

800
600
400
200

0
0

10

15

20

25

30

Total-N (g kg-1)

Fig. 5 Relation between the total-N (g kg ), methane

yield (% of theoretical potential) and total VFA
concentration (mM) at 55 8C. Data are from anaerobic batch
assays of different animal by-products.

pH and lower portion of the total ammonia-N in the free

ammonia (NH3) form.
Concerning inhibition, the same tendency was seen for
lipids as for ammonia in batch assays. When the initial
concentration of lipids reached approx. 4 g kg1 the methane
yield decreased (Fig. 6).
The VFA end concentration increased when the initial
concentration of lipids reached 5 g kg1 (Fig. 6). The inhibition is attributed to accumulation of high concentrations of
accumulated LCFA intermediates [9,10]. A balanced hydrolysis to LCFA and subsequent degradation of LCFA is
required in order to avoid accumulation of LCFA, which may
be obtained in continuous operated systems with careful
introduction/adaptation of lipid rich waste products. Degradation of LCFA has been reported as the rate-limiting step of
lipids [9]. In the batch and CSTR experiments formation of
foam was observed and complicated the biogas process,
especially in the CSTRs. The foam was probably coming from
degradation of LCFA and resulted in reactor or the bottles
overflowing and decreased the bio-accessibility of LCFA and
other particles [4].
Pretreatment had no effect primarily because the byproducts were already easily degradable. This was indicated
by the short lag phase and the high gas yield close to theoretical yield for untreated mixed pork waste. This is in
agreement with previously reported in Ref. [4]. Opposite
results were reported by Edstrom et al., [14] who found that
pasteurization (70  C, 1 h) of mixed animal by-product
increased the biogas yield from 760 to 1140 dm3 kg1 VS
(490740 dm3 kg1 VS). The total solids content was higher in
the pretreated waste probably due to evaporation of water
during heating and sterilization. When the amount of total
solids increased, it was clear that the methane production per
kilo treated waste increased.

10

15

20

25

Lipids (g kg-1)

L1

Fig. 6 Relation between lipids (g kgL1) and methane yield

(% of theoretical potential) and total VFA concentration
(mM) at 55 8C. Data are from anaerobic batch assays of
different animal by-products.

In summary this study showed that mesophilic temperatures are preferable for digestion of animal by-products unless
sufficiently diluted by co-digestion with a substrate of lower
ammonia and lipid concentration. The VFA level is lower and
higher amounts of animal by-products could be added
without inhibition. Although sterilization or pasteurization
did not increase the methane yield of animal waste, it is
necessary in order to comply with the EC Byproduct regulations for category 2 and category 3 materials for use in biogas
reactors.

5.

Conclusions

We conclude that, animal waste constitutes a good substrate for

biogas production with a methane potential of mixed animal
waste of 619 dm3 kg1, which is much higher than the methane
potential of manures (2030 dm3 kg1). Animal waste is however
containing high ammonia loads, and due to the susceptibility of
the thermophilic digestion to ammonia, it would be better to use
mesophilic digestion. Co-digestion is obviously very suitable for
these resources up to a dilution level of 5%.
Pretreatment to satisfy the EU regulations No. 1774/2002
for health and safety categories 2 and 3 was demonstrated to
have no effect on the treatability or methane yield.

Acknowledgement
We acknowledge M. Eiris for her contribution to this work.
Partial funding was from COOP-CT-2005 (C.N 017641) and from
the EU-FP6 CRAFT project PIGMAN.

1054

biomass and bioenergy 33 (2009) 10461054

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