Anaerobic Digestion of Slaughterhouse by-products
Post on 06-Nov-2015
DESCRIPTIONanaerobic digestion of slaughterhouse
Received in revised form
Published online 21 May 2009
by-products from pigs were tested. The methane potential measured by batch assays for
fat, blood, hair, meat, ribs, rawwaste were: 225, 497, 487, 561, 582, 575,
methane potential. Dilution of the by-products had a positive effect on the specificmethane
for food consumption. During the last 60 years, slaughter-
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
(part of infected animals, international catering etc.) and is
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
* Corresponding author. Tel.: +45 45 25 14 29; fax: +45 45 93 28 50.
Avai lab le at www.sc iencedi rect .com
b i om a s s an d b i o e n e r g y 3 3 ( 2 0 0 9 ) 1 0 4 6 1 0 5 4E-mail address: email@example.com (I. Angelidaki).house 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
not allowed to be treated in composting or biogas plants
under any circumstances; category 2, high risk animal by-
products (diseased animals, manure and digestive tractproduction in Denmark. More than 24 million pigs are
slaughtered annually in Denmark . 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
some animal by-products .
According to the legislation slaughterhouse waste must
be treated by different treatments depending on the cate-
gory of the animal byproduct . Three categories of animal
by-products are defined; Category 1 is high risk materialKeywords:
EC regulation No. 1774/2002
Pork production constitutes a s0961-9534/$ see front matter 2009 Publisdoi:10.1016/j.biombioe.2009.03.004yield with the highest dilutions giving the best results. High concentrations of long-chain
fatty acids and ammonia in the by-products were found to inhibit the biogas process at
concentrations higher than 5 g lipids dm3 and 7 gN dm3 respectively. Pretreatment
(pasteurization: 70 C, sterilization: 133 C, and alkali hydrolysis (NaOH) had no effect on
achievedmethaneyields.Mesophilic digestionwasmore stable than thermophilic digestion,
and higher methane yield was noticed at high waste concentrations. The lower yield
at thermophilic temperature and high waste concentration was due to ammonia inhibition.
Co-digestion of 5% pork by-products mixed with pig manure at 37 C showed 40% higher
methane production compared to digestion of manure alone.
2009 Published by Elsevier Ltd.
ant part of meat
human disease CreutzfeldJacob in 2001, has resulted in
increasing awareness of the need for hygiene regulations,
tighter process control, and the prohibition of utilization ofAccepted 17 March 2009 359, 619 dm3 kg1 respectively, corresponding to 50100% of the calculated theoretical11 March 2009 meat- and bone flour,Article history:
Received 13 January 2008
Anaerobic digestion of animal by-productswas investigated in batch and semi-continuously
fed, reactor experiments at 55 C and for some experiments also at 37 C. Separate or mixedAnaerobic digestion of slaugh
Anette Hejnfelt, Irini Angelidaki*
Department of Environmental Engineering, Technical University of D
a r t i c l e i n f o a b s t r a c t
ht tp : / /www.e lsev ierhed by Elsevier Ltd.rhouse by-products
mark, DTU, Building 113, DK-2800 Kgs. Lyngby, Denmark
m/loca te /b iombioe
b i om a s s an d b i o e n e r g y 3 3 ( 2 0 0 9 ) 1 0 4 6 1 0 5 4 1047(catering residues, meat, precooked foods, etc.) approved for
food consumption, must be treated to at least 70 C for 1 h ina closed system .
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 ofmethane, and utilization of
the digestion effluents as fertilizer for application on agricul-
tural fields for nutrient recovery, . However, slaughterhouse
wastes are generally regarded as difficult substrates for anaer-
obic digestion, mainly because of their typically high protein
and lipid content . 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-Nm3 arereported as inhibiting concentrations . 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  requiring gradual adaptation and careful dosing
of lipid rich waste products to avoid LCFA accumulation.
Already at very low concentrations (such as 0.5 kgm3), 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 amore attractive option, co-digestionwith less
concentrated organic waste types, such as manure or waste
water. In co-digestion the concentratedwaste 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 , rumen and cattle blood , blood
and category 3material frompigs . In addition, co-digestion
of manure and rumen  and blood and rumen from cattle
and pigs have been studied in laboratory andpilot scale under
mesophilic conditions (37 C) andwith pre-treatment (heating). Methane yields of 0.520.55 m3 kg1 VS were reportedunder 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 . 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, themethane 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 investi-
gated. The effect of heating (70 C, 1 h) and sterilization (133 C,300 kPa, 20 min) on themethane yield of mixed pig-waste was
tested. Finally co-digestion of animal by-products fromaslaughterhouseandmanurewas tested in semi-continuously
fed reactors at mesophilic and thermophilic temperatures.2. Materials and methods
Five different types of pig slaughterhouse by-products; fat,
blood, raw waste (meat, fat and bones), intermediate product
(pressed rawwaste) andboneflourwere received fromaDanish
animal waste processing company (Daka, Denmark; Lat: N
5525045.3700 Long: E 1147054.7300). All these by-products werepretreated at the factory before delivered for experimental use.
The pretreatment at the factory includedmaceration to particle
size smaller thanapprox. 34 mmof a largeportion, followedby
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 5537050.6100 Long: E 124043.1600). The pretreat-ment of the different waste products are shown in Table 1.
Finally,mixed porkwaste 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
563100.9000 Long: E 818047.7900) was used as inoculum for batchand semi-continuous reactor experiments. For themesophilic
experiments inoculum fromamesophilic biogas plant (Nysted
centralized biogas plant, Denmark; Lat: N 5540039.8100 Long: E1212018.0700) 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.
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 NaOHkg1 VS) treatments and finally autoclaving (133 C and300 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 alltested by-products except for the mixed pork waste for which
the methane yield assay was performed at both 55 C and37 C in order to elucidate the effect of temperature on theanaerobic digestion of mixed pork waste. Four different
concentrations (5%, 20%, 50%, and 100%) of substrate (weight
basis) were prepared by dilution with water. Substrate, basicanaerobic (BA) synthetic media  and inoculum (60% of
active volume) (retrieved 1 or 2 days before the experiments)
with TC detection and chromosorb 108 column (1.1 m 3/16
ncentration % 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 55 C Snertinge biogas plant5, 20, 50, 80 55 C Inoculum from own
reactor experiment5, 20, 50
5 and 10 55 C Snertinge biogas plant
b i om a s s an d b i o e n e r g y 3 3 ( 2 0 0 9 ) 1 0 4 6 1 0 5 41048were added in the bottles under constant flushingwithN2/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 pre-
sented 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. R and
Table 1 Batch experiments, overview.
Waste product Pretreatment Co
Raw wastea Autoclaved in lab. at 300 kPa and
133 C for 20 min. (Jeppesen, 2003)Blooda Autoclaved in lab. At 300 kPa and
133 C for 20 min. (Jeppesen, 2003).Inter-mediate producta Heated to 85 C for 20 min.
Bone floura Sterilized in the process,
133 C for 20 min. (Daka, 2003).Lipida Sterilized in the process at
125 C for one hour (Daka, 2003).Bones Untreated
Heated, 70 C, 1Meat (pure meat) 10 mm
Mixed pork waste Untreated
Heated, 70 C, 1 hSterilized, 133 CNaOH
a Daka products.t,20
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 dailyvariations 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 day
1) respectively. Afterday 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) equippedmolsieve 137) . The biogas produced in CSTRs was
measured through gas meters  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 . Protein
content was calculated from Kjeldahl nitrogen content
multiplying with a conversion factor of Kjeldahl-N, 6.25
20, 50, 80 55 C/37 C Thermo.: Lemvig20, 50, 80 Meso.: Nysted biogas plant
20, 50, 802.6. Calculation on theoretical methane yield
The theoretical methane yield at standard temperature and
pressure (0 C, 100 kPa) was calculated using the followingformula: dm3 kg1X 0.496 + Y 1.014 + Z 0.415
Where: X% proteins of VS, Y% lipids of VS, Z%carbohydrates of VS .
Table 2 Characteristics of different waste productsobtained from a single pig.
Composition of the mixedpork waste product
% Amount inkg
Blood 10.4 2.90
Hair 7.6 2.12
Hair and skin 0.1 0.03
Intestine content Not known 5.00a
Meat fractions not for use, lipids 26.7 7.45
Bones 16.10 4.49
Head Not known 2.78a
Manure 10.7 2.99
Intestine fat 0.5 0.14
Total 100.0 27.90
mixed pork waste while the lowest methane yield were
b i om a s s an d b i o e n e r g y 3 3 ( 2 0 0 9 ) 1 0 4 6 1 0 5 4 1049obtained 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 testedhad high gas production per kg waste. Pork fat had the highest
methane yield per kg waste (562 dm3 kg1 waste) while lowestmethane production per kg waste of 81 dm3 kg1 waste wasobtained from blood.
3.2.1. The effect of pretreatmentResults showed that thermal treatment at 70 C andtreatment by addition of 50 or 100 g NaOH kg1 VS NaOHhad 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 effect3. Results
3.1. Characterization of by-products
Characteristics of the pig by-products are presented in
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 indi-
cates 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
3.2. Batch experiments
The methane production rates and yields for the different
by-products incubated at 37 and 55 C with mesophilicand thermophilic inoculum, respectively, are presented in
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 werecomparable to the theoretical yields of 500750 dm3 kg1 VS.Highest methane yield of 620 dm3 kg1 VS was obtained foron the methane yield, but the methane production per kg
waste was improved, as VS content increased due to
b i om a s s an d b i o e n e r g y 3 3 ( 2 0 0 9 ) 1 0 4 6 1 0 5 41050Daka intermediate product750
Daka raw waste
Swater evaporation. Sterilized waste gave a production of
225 dm3 kg1 waste, while untreated, mixed pork wastewhich gave 125 dm3 kg1 waste.
3.2.2. The effect of temperatureThe 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
Daka bone flour
0 10 20 30 40Time
Fig. 1 Cumulatedmethane production for different pig slaughte
A 5%, 10%,- 20%, :50%, C 80%, B 100% and theoreticalRibs
Meat (50mm pieces)temperature and pH . 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 andthereby the total ammonia load was equal in both the meso-
philic and thermophilic vials. This could explain the severe
inhibition at thermophilic digestion .
3.3. Continuously stirred reactor experiments
3.3.1. The thermophilic reactors (55 C)The process performance of the thermophilic reactors is pre-
sented 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
Mixed pork waste
0 10 20 30 40(Days)
rhouse by-products incubated at 55 8C at different dilutions.
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
VFAwith values reaching up to 120 mM. Propionatewas 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
Thermophilic vs. meso
0 20 40Time
Fig. 2 Cumulated methane production of mixed pork w
b i om a s s an d b i o e n e r g y 3 3 ( 2 0 0 9 ) 1 0 4 6 1 0 5 4 10510
0 20 40 60 80 100 120Time (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.day1 (Fig. 3), corresponding to a specific methane yield ofapprox. 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,20stopped 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).
3.3.2. 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 specificmethane yield of approx. 350 dm3 kg1 VS which is some-what 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 of489 dm3 kg1 VS or approx. 900 dm3 kg1 VS if extra
60 80 100
aste (50% diluted) incubated at C 37 and - 55 8C.philic for 50% of mixed pork wasteproduction is ascribed to pork waste VS only. The yield of
dm3 kg1 VS for pork waste was higher than determinedby 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 to4.5 dm3 day1. The corresponding decrease in yield was
from 489 to 417 dm3 kg1 VS.
The results from the present study showed that slaughter-
house waste in general had a good methane potential
and could produce 225619 dm3 kg1, which corresponds to
Table 4 Results from the continuous reactor experiments. Meastable period and co-digestion with mixed pork waste. The va
Temperature 55 C% Mixed porker waste 20 %
HRT (days) 21
Stable period with manure 6 g VS d1
Methane yield (dm3 kg1) 219.0Methane (%) 69.0
% Of theoretical potential (%) 45.0
Dissolved ammonia (g N dm3) 3.28Free ammonia (g N dm3) 0.99Degradation of VS % 34.0
Total VFA (mM) 111.0
Slaughterhouse waste and manure 12.5 g VS d1
Methane yield (dm3 kg1) 0.0Methane (%) 0.0
Dissolved ammonia (g N dm3) 3.37Free ammonia (g N dm3) 1.33Degradation of VS %
Total VFA (mM) 97.0
b i om a s s an d b i o e n e r g y 3 3 ( 2 0 0 9 ) 1 0 4 6 1 0 5 4105250100% of the theoretical yields. Carpentier et al. ,
investigated thermophilic batch digestion of pig slaughter-
house 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. cor-
responding to the theoretical maximum yield of lipid, indi-
cating that nearly all VS in the tested waste were lipid.
However, it is not possible to verify this assumption, as the
exact composition of thematerial they used was not reported.
Edstrom et al.  carried out mesophilic fed-batch digestionwith mixed, minced animal by-products. The specific
production of biogas was 760 dm3 kg1 VS (490 dm3 kg1 VS)
0 10 20 30 40 50 60 70Time (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.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
sured and calculated parameters for the reactors during thelues are mean values from each period.
55 C 37 C5 % 5 %
6 g VS d1 6 g VS d1
8.3 g VS d1 8.3 g VS d1
128.0 17.0animal 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 corre-
sponding N-concentration (Fig. 5), it can be seen that for total-
N concentrations higher than 7 gN kg1 the process wasseverely inhibited (Fig. 5). The results are in accordance with
previous studies .
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 thermo-
philic 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.  concluded
when he investigated ammonia inhibition in manure at
different temperatures. Angelidaki and Ahring , showed
that mesophilic temperature is a better choice for processes
operating with high N loading because of a slightly lower
ammonia and lipid concentration. The VFA level is lower and
higher amounts of animal by-products could be added
0 5 10 15 20 25
b i om a s s an d b i o e n e r g y 3 3 ( 2 0 0 9 ) 1 0 4 6 1 0 5 4 1053pH 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 methaneyield decreased (Fig. 6).
0 5 10 15 20 25 30Total-N (g kg-1)
Fig. 5 Relation between the total-N (g kgL1), methane
yield (% of theoretical potential) and total VFA
concentration (mM) at 55 8C. Data are from anaerobic batch
assays of different animal by-products.The VFA end concentration increased when the initial
concentration of lipids reached 5 g kg1 (Fig. 6). The inhibi-tion is attributed to accumulation of high concentrations of
accumulated LCFA intermediates [9,10]. A balanced hydro-
lysis 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. Degra-
dation of LCFA has been reported as the rate-limiting step of
lipids . 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 .
Pretreatment had no effect primarily because the by-
products were already easily degradable. This was indicated
by the short lag phase and the high gas yield close to theo-
retical yield for untreated mixed pork waste. This is in
agreement with previously reported in Ref. . Opposite
results were reported by Edstrom et al.,  who found that
pasteurization (70 C, 1 h) of mixed animal by-productincreased the biogas yield from 760 to 1140 dm3 kg1 VS(490740 dm3 kg1 VS). The total solids content was higher inthe 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.In summary this study showed that mesophilic tempera-
tures are preferable for digestion of animal by-products unless
sufficiently diluted by co-digestion with a substrate of lower
Lipids (g kg-1)
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.0
M)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 regula-
tions for category 2 and category 3materials for use in biogas
Weconclude that, animalwaste constitutes a good substrate for
biogas production with a methane potential of mixed animal
waste of 619 dm3 kg1, which is much higher than themethanepotential ofmanures (2030 dm3 kg1).Animalwaste ishowevercontaining high ammonia loads, and due to the susceptibility of
the thermophilic digestion to ammonia, itwouldbe 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.
We acknowledge M. Eiris for her contribution to this work.
Partial fundingwas fromCOOP-CT-2005 (C.N 017641) and from
the EU-FP6 CRAFT project PIGMAN.
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Anaerobic digestion of slaughterhouse by-productsIntroductionMaterials and methodsSubstratePretreatmentMethane yieldContinuously stirred tank reactor experimentsAnalytical methodsCalculation on theoretical methane yield
ResultsCharacterization of by-productsBatch experimentsThe effect of pretreatmentThe effect of temperature
Continuously stirred reactor experimentsThe thermophilic reactors (55degC)The mesophilic reactor (37degC)