Bioresource Technology 101 (2010) 26572664

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Bioresource Technology
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Anaerobic co-digestion of meat-processing by-products and sewage sludge

Effect of hygienization and organic loading rate
Sami Luste a,*, Sari Luostarinen b
a
University of Kuopio, Department of Environmental Science, Yliopistonranta I E, FI-70211 Kuopio, Finland
b
MTT Agrifood Research Finland, Lnnrotinkatu 5, FI-50100 Mikkeli, Finland

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

Article history: Anaerobic co-digestion of a mixture of animal by-products (ABP) from meat-processing industry and of
Received 10 July 2009 sewage sludge was studied at 35 C for co-digesting such by-products in digesters at wastewater treat-
Received in revised form 19 October 2009 ment plants. The three reactors were fed with ABP mixture and sewage sludge (1) in a ratio of 1:7 (v/v),
Accepted 26 October 2009
(2) in the same ratio but with hygienization (70 C, 60 min) and (3) in a ratio of 1:3 (v/v). Hydraulic reten-
tion time (HRT) was decreased from 25 to 20 days and nally to 14 days, while organic loading rates (OLR)
ranged from 1.8 to 4.0 kg VS/m3 day. The highest specic methane yields were achieved with 20-days-HRT
Keywords:
(1) 400 30, (2) 430 40, (3) 410 30 m3 CH4/t VS. Hygienization improved methane production to a
Anaerobic co-digestion
Hygienization
level above the highest OLR applied (feed ratio 1:3 (3)), while the quality of the digestate remained similar
Meat-processing by-products to the other reactors.
Organic loading rate 2009 Elsevier Ltd. All rights reserved.
Sewage sludge

1. Introduction 2003; Climent et al., 2007; Lu et al., 2008). Another option is to

Efcient waste management is increasingly required due to sev-
eral environmental and economical concerns such as climate
change, eutrophication, and the diminishing resources for fossil
energy and raw materials. Anaerobic digestion is considered a sus-
tainable option for management of organic wastes and by-products
as it produces renewable energy in the form of biogas and enables
recycling of materials, especially nutrients. Simultaneously, it en-
ables controlled stabilization and thus decreased emissions from
the treated waste materials. By replacing non-renewable energy
and materials with the use of biogas and digestate, the greenhouse
gas and other emissions from energy production and consumption
and from petrochemical and fertilizer industries can be decreased.
Anaerobic digestion of sewage sludge is a usual process in many
municipal wastewater treatment plants. Mass reduction, stabiliza-
tion, methane production and improved dewatering properties are
the main features of the process (Mata-Alvarez et al., 2000). How-
ever, slow degradation (>20 days) and the relatively low volatile
solids (VS) removal (3040%) are often the disadvantages of the
process as the digesters are rarely optimized for biogas production
and are operated with too low C/N ratio and organic loading rate
(OLR; Murto et al., 2004; Climent et al., 2007). To intensify the pro-
cess, several pre-treatments (chemical, thermal, biological,
mechanical) have been studied (Gavala et al., 2003; Kim et al.,
* Corresponding author. Tel.: +358 405143694; fax: +358 15226578.
E-mail address: sami.luste@uku. (S. Luste).
co-digest the sludge with other, easily degradable materials, which
may improve also the degradation of the sludge and thus the sta-
bility of the digestate (Mata-Alvarez et al., 2000).
Animal by-products (ABP) from meat-processing industry con-
tain several different materials. Meat and fatty tissue containing
materials have high energy potential because of typically high
grease and protein content. On the contrary, e.g. digestive tract con-
tent consists mainly of partly digested fodder. Anaerobic digestion
of both types of ABPs is challenging either due to high possibility for
ammonia and/or fatty acid inhibition (Salminen and Rintala, 2002)
or the recalcitrance of cellulose and lignin compounds (Rosenwin-
kel and Meyer, 1999; Buenda et al., 2008; Luste et al., 2009). Lignin
compounds act as glue between polysaccharide laments and bres
thus slowing down their degradation, while 12% of cellulose is esti-
mated to remain in the otation layer of and biogas reactor and the
C/N ratio may be suboptimal (Rosenwinkel and Meyer, 1999).
Co-digestion of different ABPs and sewage sludge could be ben-
ecial due to dilution of inhibitive substrates, improved nutrient
content and synergistic effects between the treated materials
resulting in better degradation of both (Mata-Alvarez et al.,
2000). Moreover, the addition of different ABPs into a sewage
sludge digester increases the OLR of the digester, thus resulting
in higher methane production. Co-digestion of sewage sludge with
grease trap sludge from a meat-processing plant is already re-
ported to be very effective (Luostarinen et al., 2009). As the co-
digestion also increases the nutrient content (ammonium nitrogen,
potassium, phosphorous, calcium, magnesium) of the digestate as

0960-8524/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2009.10.071
2658 S. Luste, S. Luostarinen / Bioresource Technology 101 (2010) 26572664

compared to digesting sewage sludge alone (Field et al., 1985), the
digestate may also have better prospects for end-uses in e.g. gar-
dening and agriculture, provided the hygienic quality and low con-
tent of harmful substances, such as heavy metals, have been
ensured.
Anaerobic digestion eliminates pathogens from the treated
materials due to the temperature ranges and the relatively long
HRT used. Especially thermophilic digestion (55 C) is reported to
reduce the pathogen content sufciently (Huyard et al., 2000; Lu
et al., 2008), while mesophilic process alone may not be adequate
(Iranpour et al., 2004) depending on the feed materials. In order to
ensure hygienization of the treated materials, a separate hygieni-
zation treatment can be added either before or after the digester.
Hygienization treatment (70 C, 60 min, particle size <12 mm) rec-
ommended or demanded for ABPs (1774/2002/EC) and for sewage
sludge before the mesophilic digestion (ENV.E.3/LM, 2000) is re-
ported to reduce the pathogen content adequately for fertilizer
use of the digestate (Bendixen, 1999).
When hygienization is performed before the digester, it also
serves as a thermal pre-treatment possibly increasing the degrada-
bility of the treated materials. Thermal pre-treatments differenti-
ate liquid organic material from solids, loosen the structure of
the remaining solid particles via pressure changes and concentrate
the treated material due to evaporation of water (Bougrier et al.,
2005). In previous studies, pre-treatment temperatures below
100 C have been found more effective in increasing biogas produc-
tion than higher temperatures (Gavala et al., 2003; Climent et al.,
2007), thus indicating the pre-treatment potential of hygieni-
zation. In this study, hygienization was studied from the perspec-
tive of possible enhanced hydrolysis and pathogen elimination
was not veried.
In this study, the anaerobic co-digestion of sewage sludge and a
mixture of ABPs from meat-processing industry was studied in a
ratio of real middle-sized companies in Finland and in an optimal
ratio described in the literature. The aim was to evaluate the pos-
sibility to co-digest such by-products in existing digesters at
wastewater treatment plants. The effect of different OLRs and
hygienization was studied in three semi-continuous reactors at
35 C.
2. Methods
2.1. Studied materials
The studied materials (Table 1) were chosen according to their
annual production rates in middle-sized Finnish wastewater treat-
ment plant and meat-processing industry as well as their availabil-
ity for treatment in Finland. The ABP materials were received from
a slaughterhouse (Lappeenranta, Finland) and a meat-processing
plant (Mikkeli, Finland) handling cows and pigs. At the time of
sampling, approximately 5300 tons of digestive tract content,
drumsieve waste and dissolved air otation (DAF) sludge were pro-
duced annually in the slaughterhouse and 75100 tons of grease
sludge in the meat-processing plant. Digestive tract content and
drumsieve waste were mixed (82:18 v/v) in the slaughterhouse
according to produced amounts. It was mixed with the other stud-
ied materials, DAF sludge and grease trap sludge, according to their
produced wet weight (w.w.) ratios (53:34:13, respectively). The
resulting ABP mixture was frozen at 18 C prior to melting for
feeding.
Sewage sludge and inoculum (digested sewage sludge) were
collected from a municipal wastewater treatment plant (Mikkeli,
Finland; Table 1). The plant treats wastewaters not only from res-
idential areas, but also from small and medium-sized industries
and produces approximately 36,400 m3 sewage sludge per year.
The sludge was collected at the plant once a month and kept at
4 C prior to feeding.
2.2. Reactor experiment
The reactor experiment was conducted in three ve liter glass
reactors with a liquid volume of 4 l at 35 C. The reactors were con-
stantly mixed using magnetic stirrers (300 rpm; Heidolph MR
3001, Germany). Feeding of reactors was performed once a day,
5 days per week using a 100 ml syringe. The volume of withdrawal
was 5% smaller than the feed in order to maintain constant liquid
volume in the reactors. The produced biogas was collected into alu-
minum gas bags (Tesseraux Spezialverpackungen GmbH, Ger-
many) through lead-ins on the reactor top. HRT was reduced
from 25 (days 043) to 20 days (days 44126) and nally to
14 days (127175) with OLR increasing accordingly. Some varia-
tion in OLR was also due to changes in the characteristics of the
sewage sludge.
The feed ratio of ABP mixture and sewage sludge for reactor 1
(R1) and reactor 2 (R2) was 1:7 (v/v), respectively, and for reactor
3 (R3) 1:3 (v/v; Table 1). The feed for R1 and R2 represented the
annual production ratio of the materials, while the feed for R3 rep-
resented the reported optimum co-digestion ratio from the litera-
ture (sewage sludge with industrial food waste or slaughterhouse
waste and/or municipal food waste; Rosenwinkel and Meyer,
1999; Murto et al., 2004; Sosnowski et al., 2008). The feed for R2
was also hygienized (70 C, 60 min) by rstly heating it to 70 C
using the heater in a magnetic stirrer (Heidolph MR 3001, Ger-
many) and then keeping it in an incubator (Termaks TS 8056, Nor-
way) at 70 C for one hour. Before the feeding it was cooled to
35 C.
2.3. Analyses
Biogas volume was measured with water displacement and
methane content with gas chromatography (Agilent 6890N: Per-
kinElmer Elite-Alumina column 30 m  0.53 mm, ame ioniza-
tion detector 225 C, oven 100 C, inlet 225 C, carrier gas helium

Table 1
Characteristics of the feeds of R1, R2 and R3 and their separate feed materials as determined in previous studies (dtc = digestive tract content; dw = drumsieve waste; Ds = DAF
sludge; gts = grease trap sludge; Luste et al., 2009; sewage sludge = ss; Luostarinen et al., 2009).

Reactor/feed TS (%) VS (%) CODsol CODsol/VS VFAtot LCFA NH4-Nsol (g/l) CODsol/NH4- pH CH4 CH4
material (g/l) (g/l) (mg/l) Nsol (m3 CH4/t VS) (m3 CH4/t w.w.)
R1 6.3 0.7 4.6 0.3 6.6 2 1.4 4.7 1 3.042 0.3 0.1 19 2 6.26.6 - -
R2 7.3 0.8 5.4 0.5 9.2 3 1.7 5.8 1 2.727 0.4 0.1 24 2 5.96.5 - -
R3 7.2 0.6 5.6 0.3 7.8 2 1.4 5.3 1 3.022 0.4 0.1 22 2 6.16.5 - -
dtc 13 1 12 1 4.0 0.1 0.4 7.2 400 42
dw 14 2 14 2 0.9 0.1 0.1 6.6 230 30
Ds 7.8 0.6 6.8 0.6 5.6 0.2 1.6 6.8 340 12
gts 15.9 0.9 15.8 0.8 6.6 0.1 0.6 5.6 900 99
ss 4.5 0.8 3.0 0.6 7.2 300 7.8
 S. Luste, S. Luostarinen / Bioresource Technology 101 (2010) 26572664 2659

10 ml/min, split ratio 35:1, injection volume 100 ll). TS and VS
were analyzed according to standard methods (APHA, 1998). COD-
sol was determined after ltration through Whatman GF/A glass
microbre-lters (Scheicher & Schuell, Germany) according to
the Finnish standard method SFS 5504. pH was measured with
WTW 340i pH-meter and electrode (Germany) and alkalinity was
measured according to the European standard ISO 9963-1. Soluble
ammonium nitrogen (NH 4 -Nsol) was analyzed photometrically
(HACH LANGE DR 2800 VIS photometer, Germany) from ltered
samples (Whatman GF/A) using cuvette tests (HACH LANGE
LCK302, 47130 mg/l, Germany). Particle size distribution (PSD)
was analyzed using high-end dispersion analyzer LUMiSizer
(L.U.M. GmbH, Germany), measuring average particle sizes as
arithmetic average volume diameters (nm). To give an exact diam-
eter of particles, the density of the particles should be known.
However, as the studied materials were heterogeneous compos-
ites, particle density was considered constant (1.0) and the results
were reported with percentual comparison of particle sizes (R1 vs.
R2).
Volatile fatty acids (VFA; acetic, propionic, isobutyric, butyric,
isovaleric, valeric, caproic acid) and long chain fatty acids (LCFA;
palmitic C16, oleic acid C18:1) were measured using gas chroma-
tography with ame ionization detector (Agilent 6890N GC-FID,
column Agilent HP-FFAP 30 m  0.32 mm  0.25 lm). The param-
eters for VFA were: oven 100160 C 25 C/min, injector 225 C,
detector 230 C, carrier gas helium 2.6 ml/min, split ratio 2.3:1,
injection volume 1 ll, and for LCFA: oven 50230 C, 10 C/min,
injector and detector 230 C, carrier gas helium 3.3 ml/min, split
ratio 2.3:1 and injection volume 1 ll. For VFA, the samples were l-
tered through 0.45 lm syringe lters (VWR International Ltd.).
LCFA (palmitic 16:0 and oleic acids 18:1) analysis was made from
2 ml of ltered samples (Whatman GF/A glass microbre-lters,
1.6 lm), extracted with tert-metylbutylether (TMBE, Merck; ISO
5508). Oleic and palmitic acids were chosen for analysis as they
are the most common LCFAs in animal fats (68%) and sewage
sludge (65%; Miron et al., 2000; Fernndez et al., 2005).
2.4. Calculations
HRTs and OLRs were calculated for 5 feeding days per week (25,
20, 14 days; used in discussion) and taking into account the week-
ends with no feed (actual HRTs 35, 28, 20 days, respectively). COD-
sol/VS ratio was used as an indicator of hydrolysis during
hygienization to ignore the change of VS via possible evaporation
of water.
Energy input (Ei) of hygienization describes the energy needed
to increase the temperature of daily feed: (qQc(T1  T2) and to
maintain the wanted temperature ((qQc)(T1  T3))t70 Cacc (Lu
et al., 2008)
Ei kJ=day qQ cT 1  T 2 qQ cT 1  T 3 t 70  C acc; 1
where q = specic density of sludge (1.2 g/ml) calculated from the
specic volume of the weighted daily feed; Q = daily feed to the
reactor (0.16, 0.2, 0.28 l/day); c = specic heat capacity of water is
used as a specic heat of the feed containing ABP and sewage sludge
(0.00419 kJ/g C); T1 = terminal temperature of thermal treatment
(70 C); t70 C = duration of treatment (60 min), T2 = digestion tem-
perature (35 C); T3 = selected detection temperature of cooling
material (69 C); acc = measured average cooling constant of ther-
mally treated daily feed (0.2 l/day), when heating power of oven
is switched off and temperature starts to decrease
(0.125 0.025 C/min) toward to T3. acc was measured during
hygienization treatment with ten repetitions during reactor opera-
tion at the HRT of 20 days.
Energy output (Eo) describes the differences in methane pro-
duction of R1 and R2
Eo kJ=day HCH4 V R2 nR2 Q R2  V R1 nR1 Q R1 ; 2
where HCH4 = caloric value of methane (802 kJ/mol); n = feed VS;
Q = daily feed; V = methane potential of digesters (Lu et al., 2008).
Theoretical methane potential was calculated for the studied
HRT of 20 days due to the steadiest daily methane production
and the highest specic methane production achieved
V mol CH4 g1 VS1  SMPp=RT 2 ; 3
where SMP = specic methane production, p = air pressure
(1.013 bar), R = gas constant (0.08314 bar dm3/mol K).
3. Results
Anaerobic co-digestion of ABP mixture and sewage sludge was
studied in semi-continuous experiments at 35 C. R1 and R2 were
fed with the respective substrates in the ratio on 1:7 and R3 in the
ratio of 1:3. The feed for R2 was hygienized. The HRT was de-
creased from 25 to 20 days and further to 14 days with simulta-
neous increase in OLR (Table 2). OLR of R1 ranged from
1.8 kg VS/m3 day to 3.3 kg VS/m3 day, that of R2 from 2.1 kg VS/
m3 day to 3.7 kg VS/m3 day, and that of R3 from 2.2 kg VS/m3 day
to 4.0 kg VS/m3 day.
3.1. Experimental phase with HRT 25 days
The rst applied HRT, i.e. 25 days (days 043), served as an
start-up for the reactors, as dened by uctuations in all reactor
parameters, especially increasing specic methane production
and high CODsol and total VFA especially during the rst 23 days
(Fig. 1). On days 2443, the specic methane production from R1
was 340 30 m3 CH4/t VS, from R2 370 40 m3 CH4/t VS and from
R3 340 30 m3 CH4/t VS (Table 3). The daily methane production

Table 2
Hydraulic retention time, organic loading rate and characteristics of co-digested efuents of R1, R2 and R3. The results are presented from days 24 to 43 for HRT of 25 days, from
days 80 to 126 for HRT of 20 days and from days 127 to 175 to represent the steadiest study phases for each (as determined by little changes in the measured parameters).

HRT OLR TS (%) VS (%) CODsol CODsol/VS VFAtot LCFA NH4-Nsol Alkalinity pH
(days) (g VS/day l) (g/l) (g/l) (mg/l) (g/l) (g CaCO3/l)
R1 25 1.8 0.1 2.9 0.6 1.7 0.3 0.7 0.2 0.4 0.15 0.03 1.4 0.4 1.0 0.1 4.5 0.6 7.47.5
20 2.4 0.1 5.1 0.2 3.0 0.1 1.0 0.1 0.3 0.2 0.1 0.8 0.3 1.30 0.1 6.0 0.4 7.47.5
14 3.3 0.3 4.6 0.5 2.7 0.3 1.1 0.1 0.4 0.09 0.01 21 1.4 0.1 5.9 0.9 7.47.5
R2 25 2.1 0.1 3.9 0.9 2.4 0.6 1.7 0.6 0.7 0.9 0.4 11 1.1 0.2 5.3 0.6 7.4 7.5
20 2.8 0.2 5.8 0.1 3.4 0.1 1.0 0.2 0.2 0.10 0.02 0.60 0.09 1.5 0.1 6.8 0.9 7.57.6
14 3.7 0.4 5.6 0.5 3.4 0.3 1.2 0.2 0.4 0.08 0.01 1.6 0.7 1.6 0.1 6.4 0.5 7.47.5
R3 25 2.2 0.1 4.0 0.9 1.9 0.4 1.2 0.8 0.5 0.5 0.2 1.6 0.5 1.1 0.1 5.0 0.1 7.47.5
20 2.9 0.2 5.7 0.3 3.7 0.2 0.9 0.2 0.3 0.10 0.02 1.0 0.3 1.4 0.1 6.5 0.9 7.47.6
14 4.0 0.2 5.3 0.6 3.5 0.4 1.1 0.1 0.3 0.08 0.02 44 1.4 0.1 6.1 0.6 7.47.5
2660 S. Luste, S. Luostarinen / Bioresource Technology 101 (2010) 26572664

25 HRT & OLR 5 500 CH4 production 10

20 4 400 8

m3 CH4 / t VS
kg VS/m3 d

l CH4 / d
15 3 300 6
d

10 2 200 4

5 1 100 2

0 0 0 0
0 25 50 75 100 125 150 175 0 25 50 75 100 125 150 175

1.2 VFAtot
3 CODsol

2.25
0.8
g/ CODsol/l

g VFAtot/l
1.5
0.4
0.75

0 0
0 25 50 75 100 125 150 175 0 25 50 75 100 125 150 175

80 VS removal NH4-Nsol & pH 8

1.6
60 7.8

g NH4-Nsol/l
1.2

pH
40 7.6
%

0.8

20 0.4 7.4

0 0 7.2
0 25 50 75 100 125 150 175 0 25 50 75 100 125 150 175
Days Days

Fig. 1. HRT (), OLR, specic methane production (SMP), daily methane production (DMP), VS removal of R1 (D), R2 () and R3 (h) and the NH
4 -Nsol, CODsol, VFAtot and pH
in the digestates.

Table 3
Daily biogas and methane productions and specic methane productions during co-digestion of sewage sludge and ABP from meat-processing industry. The results are presented
from days 24 to 43 for HRT of 25 days, from days 80 to 126 for HRT of 20 days and from days 127 to 175 to represent the steadiest study phases for each (as determined by little
changes in the measured parameters).

Reactor HRT (days) Biogas (ml/day) CH4 (ml/day) SMP (m3 CH4/kg VS) SMP (m3 CH4/t w.w.) CH4 (%)
R1 25 3600 500 2000 300 340 30 15 1 56 3
20 5200 500 3300 300 400 30 20 2 63 1
14 6400 700 4200 600 380 20 18 1 65 4
R2 25 3600 400 2200 400 370 40 17 2 59 4
20 6300 500 4100 400 430 40 24 2 65 1
14 7400 800 4800 700 400 20 20 2 64 4
R3 25 3800 600 2400 500 340 30 18 2 62 2
20 6200 500 4000 300 410 30 24 1 65 1
14 7600 800 5100 600 390 30 22 1 67 4

Table 4
Measured VFAs in the efuents of co-digestion of sewage sludge and ABP from meat-processing industry with different HRTs studied. The results are presented from days 24 to 43
for HRT of 25 days, from days 80 to 126 for HRT of 20 days and from days 127 to 175 to represent the steadiest study phases for each (as determined by little changes in the
measured parameters).

Reactor HRT Acetic acid Propionic acid Isobutyric acid Butyric acid Isovaleric acid Valeric acid Caproic acid
(days) (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) (mg/l)
R1 25 70 50 19 3 BDL BDL 09* 012* 053*
20 200 100 24 9 013* 012* 92 014* 026*
14 40 10 18 1 013* 11 1 07* BDL 043*
R2 25 800 400 29 6 13 4 15 2 18 3 BDL 016*
20 50 8 22 4 012* 018* 014* 014* 050*
14 40 10 18 2 010* 10 1 82 BDL 016*
R3 25 400 200 29 2 11 1 12 1 12 3 010* 018*
20 50 6 21 8 018* 013* 011* 012* 045*
14 40 10 18 3 017* 11 1 08* BDL 015*

BDL = below determination limit (<5 mg/l).

*
One or more of the values are below the detection limit and average values or standard deviation cannot be calculated.
 S. Luste, S. Luostarinen / Bioresource Technology 101 (2010) 26572664
of R2 was 10% higher than that of R1. R3 with the highest OLR
(2.2 g VS/day l) produced 20% more methane daily than R1 and
9% more than R2 (Fig. 1, Table 3).
Average VS removal of R1 was 61%, while R2 removed 60% and
R3 65% from the feed VS (Fig. 1). Total VFA comprised of 75% of
CODsol in the feed of R1 (4.3 0.3 g VFAtot/l), 66% of R2
(5.2 1 g VFAtot/l) and 68% of R3 (4.7 0.5 g VFAtot/l; Table 1,
Fig. 1). In the digestates, VFAtot varied between 0.20.9 g/l in all
reactors (Table 2), and of CODsol it comprised on 21%, 50% and
42% in R1, R2 and R3, respectively. The VFA in the digestates was
mostly acetic acid (Table 4) with the content of 90 5% of VFAtot
in R2 and R3 (Fig. 1) and 50% in R1. Of the two measured LCFA,
oleic acid was completely removed from R2 and R3 and only
1 mg 0.2 of palmitic acid was detected (data not shown). More-
over, NH 4 -Nsol concentration increased on average by 220 40%
in all three reactors (Tables 1 and 2).
3.2. Experimental phase with HRT 20 days
During the HRT of 20 days, the highest methane productions
were achieved during days 73126 (Table 3, total study period
shown in Fig. 1). The average specic methane production was
400 30 m3 CH4/t VS in R1, 430 40 m3 CH4/t VS in R2 and
410 30 m3 CH4/t VS in R3. The daily methane production of the
hygienized R2 was 24% higher than that of R1, while R3 produced
21% more methane than R1.
The average VS removal of all the reactors was 38 1% with the
HRT of 20 days (Fig. 1). VFAtot increased or remained high during
the rst 23 weeks after lowering the HRT in all reactors (Table 2,
Fig. 1), but on days 73126 for R2 and R3, VFAtot was 60.1 g/l in
the digestates. Simultaneously, acetic acid content of VFAtot de-
creased from 90 5% to 50 3%. VFAtot content of CODsol in the
feeds were 69% in R1 (4.8 1.6 g VFAtot/l), 57% in R2 (5.8 1.6 g
VFAtot/l) and 73% in R3 (5.8 1.1 g VFAtot/l) and 23%, 10% and
12%, respectively, in the digestates (Table 3). Average LCFA content
of the digestates was 0.8 0.2 mg/l (Table 2) with complete re-
moval of oleic acid in R2 and R3. NH 4 -Nsol concentration increased
by 250 20% in all reactors and its content varied between 1.2 and
1.6 g/l (Table 2).
3.3. Experimental phase with HRT 14 days
When the HRT was decreased to 14 days (day 126), specic
methane production of the reactors decreased shortly by
14 0.7% (days 131140; Fig. 1). After this transition state, specic
methane production was restored but remained lower than with
HRT of 20 days (R1: 380 20 m3 CH4/t VS, R2: 400 20 m3 CH4/
t VS, R3: 390 30 m3 CH4/t VS). Daily methane production was
15.2% higher in R2 and 24% higher in R3 than with R1 (Table 3).
VS removal of R1 remained at 40%, when the removals from R2
and R3 decreased to 33% and 35% from the original VS content
(Fig. 1). VFAtot remained 60.09 g/l with acetic acid content of
48 2% in all reactors (Tables 2 and 4, Fig. 1). VFAtot content of
CODsol in the feeds were R1: 69% (4.4 2.1 g VFAtot/l), R2: 54%
(5.2 2.5 g VFAtot/l) and R3: 64% (5.0 2.2 g VFAtot/l), and in the
digestates 8.8%, 6.7% and 7.6%, respectively (Table 1). LCFA in-
creased in the feeds in the beginning of the HRT of 14 days due
to new grease trap sludge, being 20 17 mg LCFA/l with R1,
14 10 with R2 and 12 7.8 with R3 (with the earlier HRTs: R1;
7 1, R2; 6 2, R3; 5 2 mg LCFA/l, Table 1). LCFA in the digestates
increased to 1.6 0.8 mg LCFA/l (Table 2) and oleic acid (00.005 g
oleic acid/l) was found in all the reactors (data not shown). NH 4-
Nsol content increased by 260 20% in all reactors, being in the
range of 1.31.7 g /l (Table 2).
2661
3.4. Effect of hygienization treatment to the feed of R2
Hygienization increased the VS content of the feed to R2 (1:7 v/
v) on average by 17 4%, CODsol by 40 10% (CODsol/VS by 23%),
VFAtot by 23 2% (VFAtot/VS by 8%), NH
4 -Nsol by 25 7% (NH4 -
Nsol/VS by 8 4%) and CODsol/NH4 -Nsol by 13 2%, when com-
pared to the untreated feed of R1 (Table 1). With the HRTs of 25
and 20 days, the hygienization did not affect the LCFA content,
but with the HRT of 14 days, 30% of LCFA was removed during
hygienization. Hygienization also decreased the feed pH from
6.26.6 to 5.96.5, while alkalinity was 12 2% higher than in R1.
Hygienization decreased the average particle size inside the PSD
by 10 3%, when compared to the untreated particles. This could
also be seen directly in the appearance of the feed, which was more
homogenous than the feed for R1 with little of the original partic-
ulate structure of the ABPs to be seen. Moreover, the particles in
the digestate of R2 (HRT 20 days) were averagely by 4.5 1.5%
smaller than in R1.
Theoretical methane potential (V) of R1 was 0.0157 mol/g VS,
0.0178 mol/g VS for R2 and 0.165 mol/g VS for R3 during the HRT
of 20 days. Simultaneously, the energy intensity (Ei) for the
hygienized daily feed of R2 was 40 kJ/day of which heating the feed
(0.2 l/day) to 70 C consumed 35 kJ/day and the 1 h of maintaining
this temperature 4.4 kJ/day. R2 produced on average 800 ml/day
more methane than R1 during the steadiest phase (days 80127)
and thus the theoretic energy output (Eo) of the extra methane
produced due to hygienization was 47 kJ/day. Thus, the net surplus
energy production due to the hygienization was 7.2 kJ/day. Eo for
R1 was 120 kJ/day, for R2 170 kJ/day and for R3 160 kJ/day.
4. Discussion
Anaerobic co-digestion of ABP mixture and sewage sludge was
studied in two different feed ratios (1:7 and 1:3 v/v) and with
and without hygienization pre-treatment (ratio 1:7 v/v) at 35 C.
The three laboratory-reactors produced 380430 m3 CH4/t VS
(HRT of 20 and 14 days) which is comparable or higher to the
yields reported with digestive tract content/otation tailings and
sewage sludge (feed ratio 1:3; methane yield 280480 m3 CH4/
t VS; Rosenwinkel and Meyer, 1999) and to those achieved when
co-digesting slaughterhouse wastes with manure and fruit and
vegetable waste (270350 m3 CH4/t VS; Alvarez and Liden, 2008).
Co-digestion of ABP mixture and sewage sludge produced signi-
cantly more methane than the sewage sludge from the same
wastewater treatment plant alone (260 m3 CH4/t VS; Luostarinen
et al., 2009). Still, co-digestion did not improve VS removal which
was approximately 3565%, both in the present study and the
study by Luostarinen et al. (2009).
4.1. The different HRT phases studied
During the rst experimental period of 25 days HRT, the meth-
ane production and the characteristics of the digestates uctuated
due to the inoculum adapting to the new feed. R1 sported the high-
est specic methane production, the lowest VFAtot content in
efuent, the highest VS removal and the highest efuent NH 4 -Nsol
most likely due to the lowest OLR of the three reactors. Even
though the methane production was the lowest of the whole study,
VS removal was the most effective (approx. 60%) due to longest
HRT. Similar ndings were made by Rosenwinkel and Meyer
(1999), who reached the VS removal of 5261% when co-digesting
stomach content/otation tailings and sewage sludge (1:3) at an
HRT of 25 days. Further, a maximum VS removal in a single pass
mode reactor digesting ABPs alone has been reported to be 50%
(HRT: 30, 20, 15 days; Banks and Wang, 1999).
2662 S. Luste, S. Luostarinen / Bioresource Technology 101 (2010) 26572664
During the 25 days HRT, digestates from R2 and R3 contained
relatively high concentrations of acetic acid and the highest ob-
served concentrations of branched VFA (isovalerate, isobutyrate)
indicating slow growth of the acetate-utilizing methanogenic mi-
cro-organisms (Kalle and Menon, 1984; Wang et al., 1999). Also,
butyric acid concent of R1 digestate was below the detection limit,
when those of R2 and R3 were 15 2 and 12 1 mg/l, respectively.
Because butyric acid is the precursor for acetate (Kalle and Menon,
1984; Wang et al., 1999), this indicates that R1 was already pro-
ducing mostly acetate and consuming it to methane, while R2
and R3 were still adapting to the new feed. LCFA content was the
highest in the feed of R1 (1:7), when the feed of R3 (1:3) contained
LCFA the least. This was most likely due to the higher sewage
sludge addition to R1 as sewage sludge has a typical LCFA content
of 40% (Miron et al., 2000). Also, the higher VFA contents of R2 and
R3 may have promoted the degradation of unsaturated oleic acid to
saturated stearic acid (Miron et al., 2000) as oleic acid was not de-
tected in the digestates of R2 and R3 during the HRTs of 25 and
20 days.
When HRT was lowered to 20 days, methane production main-
tained a linear increase due to the increased OLR. During days 73
126, specic methane production of all reactors was at their height
(400430 m3 CH4/t VS) and the increased methane production due
to the hygienization pre-treatment the most notable. Despite the
lower OLR and addition of ABP mixture, R2 produced approxi-
mately as much or slightly more methane than R3 with its opti-
mized feed ratio of one part ABP mixture and three parts sewage
sludge (Rosenwinkel and Meyer, 1999; Murto et al., 2004; Sosnow-
ski et al., 2008). The reasons for the differences in methane produc-
tion are discussed in more detail in Section 4.2 of the discussion.
During the HRT of 20 days, the VS removals of all the processes
were on average 38 1%. When also low CODsol and VFAtot were
observed in efuents, it is presumable that the reactors found
operational stability during the HRT of 20 days (Rosenwinkel and
Meyer, 1999). Still, the VS removal was lower as compared to the
60% achieved at the HRT of 25 days and the 4767% (HRT 10
50 days) reported by Rosenwinkel and Meyer (1999) and Alvarez
and Liden (2008). This might have been due to the differences on
the production sites and the relative proportions of fats, proteins
and carbohydrates. In the present study, most of the ABPs con-
tained more slowly degraded plant materials from the digestive
tract content and drum sieve waste consisting mostly of partly di-
gested fodder and its high concentration of cellulose-related com-
pounds (Rosenwinkel and Meyer, 1999; Fernandes et al., 2009).
At the same time, VFAtot in the digestates of R2 and R3 de-
creased and settled to 60.1 g/l and most of the time only acetic
and propionic acid was detected (Table 4). However, in R1, the
VFAtot content increased and uctuated between 0.13 and
0.32 g/l for signicantly longer. The increased acetic acid content
of VFAtot (69%) indicates that acetate utilizing methanogens of
R1 were not as viable with the shorter HRT as the methanogens
in R2 and R3. Consumption of the produced acetic acid increased
notably for approximately days 4455 in R2 and days 4470 in
R3. After day 55, butyric and isobutyric acid contents of R2 were
23% lower when compared to those in R1 and R3, indicating the in-
creased production and utilization of acetate acid by acetoclastic
methanogens (Kalle and Menon, 1984; Wang et al., 1999). Lu
et al. (2008) have reported higher VFA consumption in anaerobic
processes with hygienization pre-treatment at 70 C. Hence,
hygienization treatment seemed also presently to accelerate the
microbial adaptation to increased OLR and to increase the alkalin-
ity of R2. The alkalinity was similarly higher in R3 (+12.5 0.5%)
than R1, indicating higher buffering capacity and resistance toward
possible organic overload due to higher ion content (Alvarez and
Liden, 2008). The higher buffering capacity and higher ion content
are contributed to the higher OLRs in R2 and R3. After reaching
operational stability, however, the reactors did not have to con-
sume ions to regulate against the pH changes.
When HRT was decreased to 14 days, specic methane produc-
tion of all the three reactors decreased and steadied to 380
400 m3 CH4/t VS. Though the specic methane production was
lower than during HRT of 20 days, less VFA was measured from
the digestates. VFAtot/CODsol ratio of all the reactor efuents de-
creased to 7.5 0.5%, having been at 30 11% at the longer HRTs.
This indicates that the processes were not inhibited (Angelidagi
and Ahring, 1993), at least not do to high concentrations of VFA
(Cirne et al., 2007). As the VS removal of R1 remained at 40% and
those of R2 and R3 decreased to 34 1%, it may be argued that
hydrolysis was weaker and less VFA was formed than with the
longer HRTs studied. This is supported by the moderately increased
LCFA content in efuents, though this was partly due to a new
batch of grease trap sludge. Though the concentrations were well
below reported levels for inhibition (Alves et al., 2001; Lalman
and Bagley, 2001) and higher LCFA content is reported to acceler-
ate the consumption rate of VFA/LCFA (Palenzuela-Rollon, 1999),
high LCFA is also said to slow down the degradation rate of lipids
(Cirne et al., 2007). Additionally, high LCFA enhances protein
hydrolysis (Palenzuela-Rollon, 1999), which may explain the pres-
ent slightly increased NH 4 -Nsol in the efuents at this HRT. In-
creased, but not yet inhibitory LCFA content is also reported to
increase the methanogenic activity (Palenzuela-Rollon, 1999;
Miron et al., 2000), which may support the presently noticed in-
creased methane content in biogas.
All-in-all, the results for LCFA and VFA do not give clear indica-
tion to why methane production was decreased at the HRT of
14 days. The increased OLR (3.34.0 kg VS/m3 day) was probably
too high to ensure efcient hydrolysis and to thus increase the spe-
cic methane production with the decreased HRT. A reported high-
est achievable OLR for reactors co-digesting meat-processing
wastes is 1.32.9 kg VS/m3 day for the non-pre-treated (Alvarez
and Liden, 2008; Rosenwinkel and Meyer, 1999) and 3.9
4.2 kg VS/m3 day for the mechanically pre-treated material (Murto
et al., 2004). After these threshold values the biogas production de-
creases. It is also possible that a longer study period would have al-
lowed for more adaptation and restoration of higher methane
production.
4.2. Effect of hygienization
Hygienization caused a clear morphological difference between
the feed of R1 and the feed of R2. The feed of R2 was more amor-
phous than the other feeds in which ABP and sewage sludge were
more as distinct phases despite mixing. Hygienization also de-
creased the average particle sizes inside the PSD by 10 3% indi-
cating degradation of solid material. Feed pH was also decreased
as a sign of acid formation, while simultaneously every hydrolysis
parameter (except LCFA) increased as such and in relation to the
slightly increased VS content. Decreasing particle size, increase of
soluble compounds (VFA, ammonium, hydrogen) and increase of
methane production are reported to be the indicators of the suc-
cessful thermal pre-treatment (Gavala et al., 2003; Kim et al.,
2003; Climent et al., 2007; Fernandes et al., 2009). Accordingly,
the hygienization applied worked as an efcient pre-treatment,
ruptured the original structure of ABP and sewage sludge and re-
leased material to the soluble phase.
Hygienization also facilitated higher degradation rate during
digestion and apparently worked in favour of protein degradation
as shown with the highest NH4-Nsol content in the digestate
(+14%). Hygienization decreased LCFA concentration slightly prob-
ably as the hydrophobicity of lipids prevented direct solubilization
to liquid phase and the already existing LCFA was degraded to
shorter chained fatty acids (Mendes et al., 2006). The latter is sup-
 S. Luste, S. Luostarinen / Bioresource Technology 101 (2010) 26572664
ported by the fact that the content of the longest measured VFA,
caproic acid, increased by 73% during the hygienization, while
the shorter VFA increased on average by 18 2%.
R2 with the pre-hygienization sported the highest specic
methane production throughout the study period. Its feed had
the highest CODsol and VFA contents, but the lowest VFA/CODsol
ratio. This is most likely due to higher production of other CODsol
than VFA, though increased degradation and/or volatilization of
VFAs during the hygienization or methane production by indige-
nous micro-organisms in the primary sludge are also possible
(Gavala et al., 2003). In any case, the present VFAtot increase
(23 2%) via hygienization (70 C, 1 h) was lower than reported
previously with a mixture of sludge and abattoir waste (43
175%; 924 h; Climent et al., 2007; Wang and Banks, 2003). The
longer treatment time applied can be the decisive factor explaining
the difference, though also the original VFA was presently higher
(4.75.8 g/l) than the 0.53.7 g/l reported by Rosenwinkel and
Meyer (1999) and Wang and Banks (2003).
Despite of the highest feed concentrations, the efuent of R2
contained the least VFAtot and CODsol as compared the efuents
of R1 and R3. Thus, also the methane production was the highest
with R2. Hygienization apparently caused the feed of R2 to be ini-
tially hydrolyzed and more easily degraded than those of R1 and
R3. Higher content of soluble material allows the bacteria to ex-
ploit the substrate more efciently and to achieve a faster diges-
tion rate and subsequently higher biogas production, which also
has a positive effect on further hydrolysis in the reactor (Palenzu-
ela-Rollon, 1999; Miron et al., 2000). Also, the composition of R2
feed was more homogenous than those of R1 and R3 increasing
the surface area for further hydrolysis and allowing better contact
between the bacteria and the substrate with less mixing.
4.3. Energy calculations
If the reported indicative calculations about the energy balance
of hygienization (surplus net energy production due to hygieni-
zation 7.2 kJ/day as compared to digestion without hygienization)
are extended to cover yearly production rates of ABPs and sewage
sludge in the example middle-sized facilities (41,800 t/a; ratio 1:7),
the Ei to achieve the hygienization temperature would be
2.04 GWh/a (formula (1)). Eo from the methane obtained due to
the hygienization varies from 2.6 GWh/a (1 m3 CH4 = 10.4 kWh;
Davidsson et al., 2007) to 2.7 GWh/a (calculated with formula
(2)). The surplus net energy production would therefore be 0.56
0.66 GWh/a. As oil, the surplus amounts to 5666 m3 of oil (1 l of
oil approximately equals to 10 kWh).
These calculations presuppose that the material going through
hygienization would be pre-warmed close to the mesophilic tem-
perature (35 C) with heat recovery from the process. However,
Ei exhausted to hygienization should then be notably lower, result-
ing in a lower specic heat capacity of the feed containing sludge
and ABPs than that of the water used in the calculations of the
present study. Thus, the surplus net energy with the use of hygien-
ization may be higher in real biogas plants than presently
calculated.
The wastewater treatment plant of the present study produces
approximately 2.5 GWh (1 m3 CH4 = 10.4 kWh; Davidsson et al.,
2007) of energy per year from digestion of sewage sludge alone
(Luostarinen et al., 2009). If it would co-digest the sludge with
the ABPs studied presently, the energy production would grossly
be signicantly higher, about 8.3 GWh/a without hygienization
and 10.8 GWh/a with hygienization. With the non-pre-treated
higher ABP feed (R3; 1:3), but the same total amount of sludge,
about 10.5 GWh/a of energy would be produced. It is very likely
that with the hygienization pre-treatment this could still be in-
creased. Thus, co-digestion of by-products from meat-processing
2663
industry with sewage sludge in municipal wastewater treatment
plants may offer an efcient way to increase the effectiveness of
the existing biogas plants and nding a sound treatment for the
ABPs.
5. Conclusions
Co-digestion of by-products from meat-processing industry and
sewage sludge (respective feed ratios 1:7 and 1:3), gave the highest
methane yield and the steadiest digestate quality at 20-days-HRT.
At 14-days-HRT, methane production decreased indicating too
high OLR. Hygienization pre-treatment of the feed ratio 1:7 was
found efcient at improving degradation and thus increasing
methane production, the latter being higher than with the digester
of feed ratio 1:3. Hygienization caused an estimated 0.55
0.66 GWh/a more energy despite the energy consumed by the
pre-treatment itself.
Acknowledgements
We are grateful for Maj and Tor Nessling Foundation, the Euro-
pean Union and the City of Mikkeli for nancing the study. We also
gratefully acknowledge Jrvi-Suomen Portti Ltd. for providing the
materials. Laboratory of Applied Environmental Chemistry at Uni-
versity of Kuopio is appreciated for providing the facilities for the
study.
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