Journal of Hazardous Materials 170 (2009) 263–271

Contents lists available at ScienceDirect

Journal of Hazardous Materials

journal homepage: www.elsevier.com/locate/jhazmat

Mesophilic and thermophilic anaerobic digestion of biologically pretreated

abattoir wastewaters in an upflow anaerobic filter
H. Gannoun a , H. Bouallagui a , A. Okbi a , S. Sayadi b , M. Hamdi a,∗
a
Laboratory of Microbial Ecology and Technology, Department of Biological and Chemical Engineering, National Institute of Applied Sciences and Technology,
B.P. 676, 1080 Tunis, Tunisia
b
Laboratory of bioprocesses, Centre of Biotechnology of Sfax, Sfax, Tunisia

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

Article history: The hydrolysis pretreatment of abattoir wastewaters (AW), rich in organic suspended solids (fats and
Received 7 January 2008 protein) was studied in static and stirred batch reactors without aeration in the presence of natural
Received in revised form 28 April 2009 microbial population acclimated in a storage tank of AW. Microbial analysis showed that the major pop-
Accepted 28 April 2009
ulations which contribute to the pretreatment of AW belong to the genera Bacillus. Contrary to the static
Available online 3 May 2009
pretreatment, the stirred conditions favoured the hydrolysis and solubilization of 80% of suspended mat-
ter into soluble pollution. The pretreated AW, in continuous stirred tank reactor (CSTR) at a hydraulic
Keywords:
retention time (HRT) of 2 days, was fed to an upflow anaerobic filter (UAF) at an HRT of 2 days. The per-
Hydrolysis
Pretreatment
formance of anaerobic digestion of biologically pretreated AW was examined under mesophilic (37 ◦ C)
Anaerobic digestion and thermophilic (55 ◦ C) conditions. The shifting from a mesophilic to a thermophilic environment in the
Abattoir wastewater UAF was carried out with a short start-up of thermophilic condition. The UAF ran at organic loading rates
Mesophilic (OLRs) ranging from 0.9 to 6 g COD/L d in mesophilic conditions and at OLRs from 0.9 to 9 g COD/L d in
Thermophilic thermophilic conditions. COD removal efficiencies of 80–90% were achieved for OLRs up to 4.5 g COD/L d
Disinfection in mesophilic conditions, while the highest OLRs i.e. 9 g COD/L d led to efficiencies of 70–72% in ther-
mophilic conditions. The biogas yield in thermophilic conditions was about 0.32–0.45 L biogas/g of COD
removed for OLRs up to 4.5 g COD/L d. For similar OLR, the UAF in mesophilic conditions showed lower
percentage of methanization. Mesophilic anaerobic digestion has been shown to destroy pathogens par-
tially, whereas the thermophilic process was more efficient in the removal of indicator microorganisms
and pathogenic bacteria at different organic loading rates.
© 2009 Published by Elsevier B.V.

1. Introduction high energy requirements for aeration, large quantities of sludge

production and flotation sludge caused by denitrifying bacteria.
Slaughterhouses and meat processing plants produces a large Anaerobic digestion is becoming the subject of current research
volume of effluents. The wastewaters generated at meat process- of organic waste management for several reasons, it helps to con-
ing industry usually contain high amounts of biodegradable organic vert a large part of degradable organic carbon to biogas to be used
matter, with soluble and insoluble fraction. The insoluble fraction for energy, and it reduces pathogens and minimises odours [3]. The
is formed by colloidal and suspended matter, in forms of fats, pro- advantages of anaerobic processes are biogas production, low gen-
teins and cellulose. In comparison to their treatability with other eration of sludge, no aeration costs and elimination of pathogens
wastewater from many agro-processing industries, the abattoir [4,5].
wastewater has encountered significant problems. The high sus- Anaerobic treatment of abattoir wastewaters is not new and the
pended solid content in the wastewater causes severe problems, use of systems for research, demonstration and full scale applica-
due to their insolubility which slows the rate of degradation, and tion has been reported since the 1950s [6]. However, traditional
its tendency to form scums. anaerobic processes are also limited by low rates of organic matter
Physical–chemical methods and aerobic processes have been removal, long hydraulic retention time, accumulation of excessive
used for the treatment of this type of wastewater [1,2] and they residual organic matter and large reactor volume requirements [7].
are not regarded as suitable treatment options because of odours, The developments of high rate anaerobic biological reactors have
overcome many of these previous objections. Mesophilic digestion
usually requires a long retention time, but is not so efficient in
∗ Corresponding author. Fax: +00216 71 704 329. the reduction of volatiles solids and the deactivation of pathogenic
E-mail address: [email protected] (M. Hamdi). organisms. To overcome these limitations, interest in thermophilic

0304-3894/$ – see front matter © 2009 Published by Elsevier B.V.

doi:10.1016/j.jhazmat.2009.04.111
264 H. Gannoun et al. / Journal of Hazardous Materials 170 (2009) 263–271

digestion, using the higher metabolic rate of thermophilic microor- Table 1

Physico-chemical and microbiological characteristics of raw abattoir wastewater
ganisms has increased. Thermophilic digestion is a little more
(AW) used in this work.
sensitive to operational conditions, such as temperature, and the
organic loading rate, as well as to the characteristics of the influent Parameter Abattoir wastewater
[8]. However, the application of the technology for the treatment pH 6.8–7.4
of wastewaters generated in meat processing plants is still incip- Conductivity (ms/cm) 1.98–2.9
ient, due to the problems with the accumulation of suspended TS (mg/L) 5060–5400
TSS (mg/L) 1500–2500
solids and floating fats in the reactor, which lead to a reduction
TCOD (mg/L) 5800–6100
in the methanogenic activity and biomass washout. The success of SCOD (mg/L) 1800–2500
the technology thus depends on an efficient primary treatment to Total kjeldhal nitrogen (mg/L) 530–810
reduce fats and suspended solids. Many studies have shown that the N–ammoniacal (mg/L) 130–280
Phosphorous (mg/L) 15–50
form of pollutants (suspended, colloidal or soluble) in the influent
Total soluble protein (mg/L) 1950–3600
wastewater greatly affects the performance of high-rate anaerobic Fats (mg/L) 40–410
systems [9]. Removing solids before treatment becomes a com- Total aerobes (CFU/ml) 7 × 107 to 8 × 108
mon practice to remove solids, so that only the soluble part of the Lactic acid bacteria (CFU/ml) 2 × 102 to 6 × 104
wastewater with perhaps small amounts of residual solids (up to Bacillus (CFU/ml) 4 × 105 to 4 × 106
Total coliforms (MPN/ml) 11 × 106 to 20 × 108
500 mg/L) is admitted to the digester without any problems related
Faecal coliforms (MPN/ml) 45 × 103 to 85 × 104
to clogging of solids [10]. Therefore, one way of improving the per- Total Streptococci (MPN/ml) 1.6 × 103 to 2.3 × 103
formance of digesters treating wastewaters with high content of Faecal Streptococci (MPN/ml) 90–300
suspended solids is to promote the hydrolysis of organic matter Pseudomonas* +
Staphylococcus aureus* +
by pretreatment of the substrate. Several pretreatment methods
Salmonella* +
of AW have been reported: physical–chemical methods including,
mechanical [11] or thermo-chemical treatment [12] and biological T: total solids; TSS: total suspended solids; SCOD: soluble COD; TCOD: total COD.
*(+) detected.
methods such as thermophilic bacterial treatment [13] and enzy-
matic hydrolysis pretreatment [14].
In addition, abattoir wastewater carries high levels of pathogenic obtained results, the optimum HRT will be determined to operate
microorganisms that may constitute a serious risk to the human and in continuously anaerobic tank.
animal health. Generally, anaerobic processes can be characterized
from the digestion environments, microorganisms and process con- 2.3. Experimental unit
figuration, and each process has its unique advantages. Although,
better performance at high strength with reduction or deactiva- A schematic representation of the anaerobic digestion systems
tion of pathogenic organisms can be obtained from thermophilic used for the experiments are shown in Fig. 1. A continu-
digestion [15]. ously stirred tank reactor (CSTR) with variable working volume
The aim of this research was the combination and the evalua- (2–15 L) was used to feed the UAF with a biologically pre-
tion of the performance of an efficient biological pretreatment for treated AW. Mixing was assured by the continuous rotation of
solubilization of the suspended solids (proteins and fats) and the the magnetic stirrer. A settler (20 cm in diameter and 37 cm in
anaerobic digestion of biologically pretreated AW under mesophilic height) was used to remove the total suspended solids (TSS).
and thermophilic conditions in an upflow anaerobic filter (UAF). The mesophilic and thermophilic anaerobic digestion of the AW
was carried out in a 5 L continuous upflow anaerobic filter
consisting of glass column of 30 cm in height and 20 cm in diam-
2. Materials and methods eter. The UAF was filled with Flocor (Ф3L3, porosity 95%, specific
surface 230 m2 m−3 ) as a media support entities for the growth of
2.1. AW sampling microorganisms. The anaerobic filter was initially operated during
120 days at the optimal mesophilic temperature range (37 ± 1 ◦ C)
The AW used in this study was collected from an abattoir factory and during 140 days at the optimal thermophilic temperature range
in Tunisia. Abattoir wastewater arises from different steps of the (55 ± 1 ◦ C). The temperature was maintained constant at each con-
process such as washing of animals, bleeding out, skinning, clean- dition by circulating water through the water jacket of the reactor.
ing of animal bodies, cleaning of rooms. The wastewater contains The mesophilic digester was fed initially with an organic loading
blood, particles of skin and meat, excrements and other pollutants. rate of 0.9 g COD/L d and at hydraulic retention time of 5 days. Then,
It also contained varying amounts of wastewater from the washing the organic loading rate (ORL) was increased gradually by vary-
of equipment and premises, which caused a big variation in the ing the HRT, from 2.5 days (ORL = 1.8 g COD/L d) to 18 h (ORL = 6 g
concentration of organic matter. The characteristics of the abat- COD/L d). The start-up of the thermophilic UAF was brought by
toir wastewaters before biological pretreatment are presented in increasing the temperature of the mesophilic UAF from 37 to 55 ◦ C
Table 1. in a single step with a simultaneous decrease of the OLR from 6
to 0.9 g COD/L d. The organic loading rate was increased gradu-
ally by varying the HRT, from 2.5 days (ORL = 1.8 g COD/L d) to 12 h
2.2. Pretreatment procedure (ORL = 9 g COD/L d) at thermophilic condition. The system was fed
by a peristaltic pump connected to a programmable timer.
Pretreatment assay was performed in closed glass flasks with a
total volume of 250 ml containing 50 ml of AW previously accli- 2.4. Analytical methods
mated in a storage tank in the presence of natural microbial
population of the raw AW. Cultivation was conducted at 30 ◦ C The effluent from the anaerobic filter was collected daily, cen-
in a rotary shaker at 100 rpm and in static conditions. Samples trifuged at 7000 rpm for 10 min and analysed for SCOD. The total
were collected for analysis after 1–3 days of growth. Firstly, the and soluble COD were measured spectrophotometrically [16]. Total
effect of stirring on the hydrolysis and solubilization of AW rich in solids (TS), TSS, total nitrogen, nitrogen–ammonium (N–NH4 + ) and
organic suspended solids will be determined, and on the basis of the fats were determined according to the procedure listed in Stan-
 H. Gannoun et al. / Journal of Hazardous Materials 170 (2009) 263–271 265

Fig. 1. Schematic diagram of the laboratory experimental set-up used for abattoir wastewater treatment in two stage process: Biological pretreatment in CSTR and mesophilic
and thermophilic anaerobic digestion in UAF.

dards Methods for the Examination of Water and Wastewater [17]. were calculated as a mean value. The data were analysed using
The total soluble proteins were determined by the method of Brad- ANOVA with statistically significant differences for p < 0.05 [22]. The
ford [18]. The dissociation constant for the ammonium ion (pKa) statistical program used was STATISTICA 6.0. The ANOVA analysis
was calculated based on this equation [19,20] as follows: was performed in order to evaluate the influence of the operating
conditions during the pretreatment (static and stirred conditions),
2729.92
pKa = 0.09018 + ; T is the temperature in ◦ C the anaerobic treatment and the disinfection (OLRs and tempera-
T + 273.25 ture).
The biogas produced was collected daily in plastic bags at room
temperature. The total volume was later determined with a wet
3. Results and discussion
gas meter and time to time the methane content was estimated
using an ORSAT apparatus. In this way the biogas volume pro-
3.1. Microbial pretreatment of abattoir wastewaters
ductions of mesophilic and thermophilic reactors were directly
comparable. Dissolved volatile fatty acids (VFA) in digested efflu-
The main contributors of the TSS forming in the AW are bloods
ents were measured by HPLC (Waters) equipped with a polypore
and colloidal materials and usually during storage, the precipitation
H column (250 mm by 7.8 mm [inside diameter]) connected to a
phenomena and the coagulation process of these components take
detector (RI-401 Waters). The mobile phase was 0.02N H2 SO4 at a
places in the feeding tank. To overcome this problem, the pretreat-
flow rate of 0.6 ml min−1 . It was centrifuged 15 min at 13,000 rpm
ment of abattoir wastewater was conducted in order to standardize
and filtered through 0.22 ␮m filter (Millipore) before use. The vol-
the effluent and to solubilize the TSS into more soluble COD by
ume of injection was 20 ␮l. Bacterial growth was monitored by
the natural bacterial population present in AW without aeration at
direct counting of colony forming units (CFU/ml) determined by
30 ◦ C. The main results obtained with batch pretreatment of AW
plating 0.1 ml of serial dilutions on MRS agar (Man Rogosa and Shap-
under static and stirred conditions in terms of SCOD, TCOD and TSS
man), plate count agar (PCA) and brain heart infusion agar (BHI)
are given in Fig. 2.
(Merck) for the enumeration of the Lactobacillus strains, the total
It was observed that during pretreatment of AW at static condi-
aerobes and the Bacillus strains, respectively. API 50 CHB and API
tions, total COD decreased with the increase of TSS as a result of the
20 E galeries (Biomerieux) were used to test biochemical charac-
precipitation phenomena induced by protein coagulation and bac-
teristics of Bacillus strains. For both mesophilic and thermophilic
terial growth associated with low biodegradation of organic matter
conditions and at each OLRs, the anaerobic treatment was evalu-
(Fig. 2a). Under stirred conditions, the TCOD remained practically
ated for its effectiveness in reducing indicator microorganisms and
constant and the SCOD increased by 64%.
pathogens (total and faecal coliforms, total and faecal Streptococ-
The bacterial populations present in AW are total aerobes, lactic
cus, Pseudomonas, Staphylococcus aureus and Salmonella) using the
acid bacteria (LAB), Bacillus and coliforms (Table 1). The biologi-
most probable number technique (MPN) [21]. Based on dilutions
cal systems are complex, and the natural microflora present in AW
down to nearly 1 remaining bacterium per test tube (three-fold
is competing for nutrients according to the environmental con-
setups repeated two times), the exit concentration can be estimated
ditions. It is likely that rapid growth of a dominant population
statistically.
could restrict the growth of others organisms simply by uptake
of the easily metabilizable nutrients or even by physical occu-
2.5. Statistical analysis pation of available space. In addition, the stirring would provide
the facultative strains with oxygen and as consequence result in
The analyses of the different parameters during the running more favourable growth conditions. Using the morphological and
of anaerobic process were done daily in triplicate at steady-state. biochemical characteristics, the microbial dominant populations
Steady-state conditions were assumed when the coefficient of during the pretreatment under stirred condition were identified as
variation for measured parameters was less than 10%. Average Bacillus species. The different Bacillus species detected were B. circu-
steady-state data and the standard error presented in the paper lans, B. coagulans, B. licheniformis, B. amyloliquifaciens and B. subtilis.
266 H. Gannoun et al. / Journal of Hazardous Materials 170 (2009) 263–271

Table 2
Bacillus spp. and lactic acid bacteria (LAB) count during the pretreatment of AW in static and stirred batch reactors at 30 ◦ C.

CFU/ml Operating conditions p value

Static condition Stirred condition

T=0h T = 24 h T = 48 h T=0h T = 24 h T = 48 h

Bacillus spp. 5 × 105 ± 0.5 6.21 × 105 ± 2 6.7 × 106 ± 1 5.105 ± 0.5 4.37 × 107 ± 2 9 × 108 ± 3 0
Lactic acid bacteria 3 × 103 ± 1 4.62 × 104 ± 1 6.84 × 104 ± 1 3.103 ± 1 1.11 × 103 ± 2 4.2 × 103 ± 1 0

p value 0.001 0.001

p value 0.001 0.001

Results of each experiment are averages of three samples. p-values were determined during the pretreatment and between static and stirred conditions.

In fact, the agitation stimulates Bacillus growth (107 –108 CFU/ml) et al. [35] reported the contribution of proteolytic microbes (Bacil-
and inhibits LAB growth (103 CFU/ml) (Table 2) because of oxygena- lus megaterium) on the pretreatment of cheese whey in order to
tion and shear stress [23]. Statistical analysis of the data indicated improve lactic acid production by Lactobacillus salivarius.
also that the growth of Bacillus and LAB strains was statistically Biological pretreatment of raw AW can also reduce the adverse
significant (p < 0.05) between the static and stirred conditions and impact of the high content of suspended and colloidal components
during the pretreatment (Table 2). on the performance of UAF, leading to clogging the installation
These results support our hypothesis that stirring improved and deterioration of the microbial activity and washout of active
proteins and polymeric carbohydrates degradation efficiency by biomass. Saddoud and Sayadi [36] also reported that the pretreat-
hydrolytic enzymes produced by Bacillus such as proteases and ment of abattoir wastewater in an acidogenic step reduced the
lipases to break down and solubilize the macromolecular structures membrane fouling of the anaerobic membrane bioreactor (AMBR)
into monomers such as amino acids and glycerol and long-chain successive methanogenic step.
fatty acids [24,25]. In fact, bacteria of the genus Bacillus are active According to the obtained results, the pretreatment of raw AW
producers of different enzymes. The strains of Bacillus presenting a using natural microflora for solubilization of suspended solids into
proteolytic activity included B. subtilis, B. licheniformis and B. circu- soluble organic matter could be conducted in 2.5 L stirred tank reac-
lans [25–28]. Under defined and optimized conditions, B. circulans tor in order to feed continuously the UAF. The pretreated AW had
and B. subtilis and B. coagulans were also able to produce lipases an average SCOD, TSS concentration and soluble protein content
[29,31]. of approximately 4.5, 0.4 and 1.044 g/L, respectively, obtained after
The use of microbial population and its hydrolytic enzymes for biological pretreatment and settling (Table 3). From the conditions
the pretreatment of particles-rich wastewater to increase the rate tested, the best results were obtained when the hydrolysis was per-
of solubilization of particulate matter and to improve the anaero- formed for 2 days, reaching important SCOD and stable TSS. The
bic treatment has been demonstrated [32,33]. However, there are enhancement of pretreatment efficiencies was obtained at an HRT
no much works which elucidate the action of Bacillus sp. and its of 2 days. This HRT was applied in continuous system because the
enzymes on the pretreatment of agroindustrial wastewaters. Pre- system was able to maintain above 80% of solubilization of organic
viously, Okuda et al. [34] studied the treatment of lipid-containing matter into soluble COD.
wastewater using Bacillus sp. which assimilates lipids and Vasala

3.2. Mesophilic anaerobic digestion of AW in UAF

Pretreated wastewater was applied initially at an OLR of 0.9 g

COD/L d corresponding to a hydraulic retention time (HRT) of 5
days. The OLR was progressively increased by varying the HRT, from
this value to 6 g COD/L d (HRT = 18 h). The results for the different
loading regimes and hydraulic retention time are presented in Fig. 3.
The UAF showed a stable behaviour up to an OLR of 2.8 g COD/L d
reaching COD removal efficiencies between 90% and 92%. During
this operational period (the first 80 days), the biogas production rate
was increased from 0.24 to 0.95 L/L d by decreasing the HRT from 5
to 1.66 days, respectively. As the digester loading rate was increased
from 2.8 to 4.5 g COD/L d (80–120 days), the COD removal decreased
slightly and ranged between 80% and 85%. Biogas production was
improved by the increase of the OLR until 4.5 g COD/L d; it averaged
from 0.24 (77% of methane) to 1.1 L/L d (68% of methane) (Table 3,
Fig. 3). However, the biogas yield declined from 0.30 to 0.15 L/g COD
removed. Ruiz et al. [37] showed that the decrease of OLR above
3 g COD/L d dropped the COD reduction below 65% in an anaero-
bic mesophilic digestion of slaughterhouse wastewater. However,
Tritt [38] reported that a decrease of COD removal from 80% to 30%
was observed by increasing the OLRs from 2.5 to 18 g TCOD/L d in an
anaerobic filter treating raw slaughterhouse wastewater. Anaerobic
digestion of the same wastewater after 2 h settling period improved
COD reduction by additional 10–15%. These results supported that
the pretreatment step showed a significant improvement in pro-
Fig. 2. TSS (), SCOD () and TCOD (♦) of AW after pretreatment using a natural
bacterial population present in the raw AW in static (a) and stirred (b) batch reactors
cess efficiency as measured by COD removal, and eventually biogas
at 30 ◦ C. conversion.
 H. Gannoun et al. / Journal of Hazardous Materials 170 (2009) 263–271 267

Table 3
pHoutlet , TSSinlet , TSSoutlet , SCODoutlet , methane content, total VFA and N–ammonium obtained with mesophilic and thermophilic anaerobic digestion of AW at different OLRs
and HRTs.

Different organic loading rates; different hydraulic retention time (OLRs: g/L d; HRT:d)

Mesophilic

(0.9; 5) (1.8; 2.5) (2.8; 1.66) (3.6; 1.25) (4.5; 1) (6; 0.75) p value
Runs (d) 1 → 30 31 → 63 64 → 84 85 → 106 107 → 121 122 → 140

pHoutlet 7.49 ± 0.37 7.55 ± 0.41 7.60 ± 0.36 7.62 ± 0.34 7.73 ± 0.28 7.86 ± 0.19 0.03071
TSSinlet (g/L) 0.4 ± 0.05 0.4 ± 0.05 0.45 ± 0.05 0.4 ± 0.02 0.4 ± 0.05 0.4 ± 0.03 nd
TSSoutlet (g/L) 0.012 ± 0.001 0.03 ± 0.005 0.05 ± 0.01 0.068 ± 0.01 0.077 ± 0.01 0.083 ± 0.01 0
SCODoutlet (g/L) 0.39 ± 0.01 0.42 ± 0.02 0.45 ± 0.05 0.65 ± 0.03 0.8 ± 0.02 0.9 ± 0.02 0
CH4 content (%) 77 ± 2.1 75 ± 2 72 ± 2 70 ± 1.4 68 ± 2.3 65 ± 3.1 0
Total VFA (mg/L) 90 ± 5 118 ± 10 124 ± 8 180 ± 14 265 ± 11 390 ± 23 0
N–ammonium (mg/L) 579 ± 28 643 ± 40 752 ± 77 814 ± 71 995 ± 147 1270 ± 180 0.00004

Thermophilic

(0.9; 5) (1.8; 2.5) (2.8; 1.66) (3.6; 1.25) (4.5; 1) (6; 0.75) (7; 0.66) (9; 0.5) p value p value
Runs (d) 1 → 30 31 → 60 61 → 86 87 → 108 109 → 122 123 → 129 130 → 135 126 → 141

pHoutlet 7.58 ± 0.44 7.72 ± 0.64 7.77 ± 0.29 7.97 ± 0.25 8.03 ± 0.16 8.16 ± 0.11 8.26 ± 0.02 8.32 ± 0.35 0.00001 0.00001
TSSinlet (g/L) 0.5 ± 0.01 0.4 ± 0.02 0.45 ± 0.03 0.4 ± 0.05 0.35 ± 0.05 0.45 ± 0.02 0.4 ± 0.05 0.45 ± 0.05 nd nd
TSSoutlet (g/L) 0.023 ± 0.002 0.033 ± 0.001 0.067 ± 0.05 0.07 ± 0.02 0.081 ± 0.05 0.135 ± 0.022 0.142 ± 0.01 0.165 ± 0.01 0 0
SCODoutlet (g/L) 0.30 ± 0.01 0.31 ± 0.04 0.32 ± 0.03 0.32 ± 0.01 0.5 ± 0.1 0.8 ± 0.2 1.08 ± 0.1 1.26 ± 0.5 0.0003 0
CH4 content (%) 78 ± 1.7 76 ± 0.5 75 ± 1.1 74 ± 1.4 74 ± 1.2 70 ± 5 65 ± 2.01 63 ± 5.1 0 0
Total VFA (mg/L) 121 ± 11 153 ± 20 175 ± 14 253 ± 9 320 ± 26 442 ± 17 730 ± 13 965 ± 20 0 0
N–ammonium (mg/L) 627 ± 14 756 ± 21 843 ± 66 917 ± 49 1020 ± 120 1388 ± 70 1570 ± 180 2060 ± 225 0 0

Protein content of the effluent from CSTR (g/L) = 1.044 ± 0.09; SCOD (g/L) = 4.5 ± 0.5; TCOD (g/L) = 6.1 ± 0.2; nd: not determined. Results of each experiment are averages of
three samples. p values were determined for each OLRs in mesophilic and thermophilic conditions and between mesophilic and thermophilic conditions.

At an OLR of 6 g COD/L d, the UAF performance declined to survive at low HRT. It seems that the protein hydrolysis
(Fig. 3). Consequently, a decrease of biogas yield was observed and the amonification rates were higher than the methanization
(0.20–0.15 L biogas/g COD removed) and the COD removal effi- rate, which affects the methanogenic bacteria activity, resulting
ciency ranged between 77% and 80%. The reason of the biogas from a high level of ammonium nitrogen (N–NH4 + ) (995–1270
decrease was due to the inability of mesophilic bacterial biomass mg/L).

Fig. 3. pH variation of the influent (), effluent (), TCODinlet (–), SCODinlet (), the SCODoutlet (䊉), COD removal (), the biogas production rate () and the biogas yield (♦)
at different OLRs () during anaerobic digestion of the mixture AW in UAF at mesophilic condition.
268 H. Gannoun et al. / Journal of Hazardous Materials 170 (2009) 263–271

Fig. 4. pH variation of the influent (), effluent (), TCODinlet (–), SCODinlet (), the SCODoutlet (䊉), COD removal (), the biogas production rate () and the biogas yield (♦)
at different OLRs () during anaerobic digestion of the mixture AW in UAF at thermophilic condition.

During the operational period, the effluent pH remained since stable thermophilic methanogenesis was achieved within a
between 7.5 and 7.9 showing a high buffering capacity in the period of 2 weeks.
digester. The concentration of total volatile fatty acids (VFA) in the Stable removal COD efficiency in the range of 93% was achieved
reactor effluent was between 90 and 390 mg/L. These values were at OLRs ranging from 0.9 to 3.6 g COD/L d The biogas production rate
much lower than the concentrations reported in other studies of increased from 0.35 to 1.4 L/L d as the OLR increased from 0.9 to 4.5 g
poultry slaughtering wastes, in which their accumulation caused COD/L d. The change of temperature leads to enhance the degrada-
inhibition of the anaerobic process [39,40]. tion of organic matter associated with higher biogas production
[45]. As the digester OLR was increased from 3.6 to 4.5 g COD/L d,
3.3. Thermophilic anaerobic digestion of AW in UAF both the COD removal and the biogas yield declined (Fig. 4). From
day 120 to 130, a reduction in removal efficiency to as low as 75%
The start-up of the thermophilic UAF was brought by increas- was observed, this could be related to the combined effects of high
ing the temperature from 37 to 55 ◦ C. When the steady-state was OLR and low HRT.
reached, the OLR was gradually increased from 0.9 to 9 g COD/L d At the end of the 130-day period at OLR of 9 g COD/L d, the bio-
by decreasing the HRT from 5 to 0.5 days. Nevertheless, the opti- gas yield decreased dramatically to achieve 0.15 L biogas/g COD
mal transition of anaerobic digestion systems from mesophilic to removed, indicating the inhibition of methanogenic bacteria. An
thermophilic conditions is not clearly defined. Several researchers important decrease in the methane content from 70% to 63% was
have studied the procedure of the thermophilic start-up in terms observed by increasing the OLR from 6 to 9 g COD/L d (Table 3).
of how to increase the temperature from the mesophilic to ther- Under such conditions, the COD removal efficiency decreased and
mophilic range. Most researchers showed that a one step increasing the VFA was above 965 mg/L. The nitrogen ammonium concentra-
temperature from mesophilic to thermophilic was the best strat- tion of the effluent during different applied OLRs varied between
egy in changing operational temperature in anaerobic digestion 627 and 2060 g/L. The pH of the effluent varied in the range
[41,42]. For the treatment of food waste, Ortega et al. [43] men- of 7.9–8.3 and the highest values were observed at high OLR of
tioned that a fully adapted inoculum was developed by eliminating thermophilic digestion. The increase in the buffer capacity of the
the initial time-consuming acclimatization stage from mesophilic reactor is mainly due to the relatively high concentration of ammo-
to thermophilic conditions. The fast adaptation of the mesophilic nium nitrogen. High concentration of ammonia would decrease the
sludge to the thermophilic conditions indicates the presence of methanogens activity and further accumulation can lead to process
thermophilic microorganisms in the mesophilic inoculum. Mata- failure [46]. Braun et al. [47] working on the anaerobic digestion
Alvarez [44] observed a transition from mesophilic to thermophilic of liquid piggery manure reported that the pH of the effluent was
conditions (35–55 ◦ C in 10 days) requiring significant variations in about 8 and the VFAs accumulated to 316 mg/L. Adjustment of pH
organic loading (from 15% to 40% over 2 days) without permanent to 7.4 led to reutilization of VFAs and lowered VFAs concentrations
effect on the process performance. As expected from the previous to 20 mg/L. It should also be noted that both methanogenic and
reports and also confirming them, the one step increase of temper- acidogenic microorganisms have their optimal pH. Failing to main-
ature coupled to a reduction of OLR was very efficient in our case tain pH within an appropriate range could cause reactor failure
 H. Gannoun et al. / Journal of Hazardous Materials 170 (2009) 263–271 269

Table 4
Removal of indicator microorganisms and pathogenic bacteria at different organic loading rates and hydraulic retention times in mesophilic and thermophilic anaerobic
digestions.

Removal efficiency (as log10 MPN/ml) Operating conditions

Different organic loading rates; different hydraulic retention time (OLRs: g/L d; HRTs:d)

Mesophilic p value

(0.9; 5) (1.8; 2.5) (2.8; 1.66) (3.6; 1.25) (4.5; 1) (6; 0.75)

Total coliforms 3.1 ± 0.1 2.5 ± 0.01 1 ± 0.1 0.8 ± 0.02 0.4 ± 0.3 0.4 ± 0.05 0
Faecal coliforms 1.5 ± 0.35 1.1 ± 0.02 0.8 ± 0.1 0.4 ± 0.1 0.4 ± 0.04 0.2 ± 0.15 0
Total Streptococci 1 ± 0.5 1 ± 0.01 0.8 ± 0.02 0.4 ± 0.2 0.35 ± 0.1 0.3 ± 0.15 0
Faecal Streptococci 1 ± 0.5 0.8 ± 1.1 0.8 ± 0.05 0.4 ± 0.3 0.2 ± 0.2 0.2 ± 0.2 0
Pseudomonas − − − − − +
Staphylococcus aureus − − − − − +
Salmonella − − − − +(*)

Thermophilic

(0.9; 5) (1.8; 2.5) (2.8; 1.66) (3.6; 1.25) (4.5; 1) (6; 0.75) (7; 0.66) (9; 0.5) p value p value

Total coliforms 4 ± 0.5 3 ± 0.03 2.3 ± 0.02 2 ± 0.1 1.9 ± 0.04 1.7 ± 0.1 1 ± 0.5 0.8 ± 0.02 0 0
Faecal coliforms 3 ± 0.2 2.5 ± 0.3 2.5 ± 0.2 2.5 ± 0.05 1.7 ± 0.2 1.8 ± 0.1 1.7 ± 0.15 1 ± 0.02 0 0
Total Streptococci 1.5 ± 0.02 1.3 ± 0.2 1.5 ± 0.3 nd 1 ± 0.1 0.8 ± 0.05 0.4 ± 0.2 0.4 ± 0.15 0.00004 0
Faecal Streptococci − − − − − − − −
Pseudomonas − − − − − − + +
Staphylococcus aureus − − − − − − + +
Salmonella − − − − − − +(*) +(*)

Results of each experiment are averages of three samples and given as log10 MPN/ml. p values were determined for each OLRs in mesophilic and thermophilic conditions and
between mesophilic and thermophilic conditions. (*): presence of S. Arizonae, (−): none detected, (+): detected, nd: not determined.

although ammonia is at a safe level [48]. Zeeman et al. [49] also toxicity value, especially under thermophilic conditions. In fact, the
reported that reducing pH from 7.5 to 7 during thermophilic anaer- dissociation constant for the ammonium ion (pKa) decreased from
obic digestion of cow manure increased the methane production 8.89 to 8.31 by increasing the temperature from 37 to 55 ◦ C. There-
by four times. fore, the concentration of FA increased by increasing the pKa value
[18,19].
3.4. Evolution of performances of the UAF treating pretreated AW In comparison to their treatability with other wastewaters from
from mesophilic to thermophilic conditions many agro-processing industries, the AW has encountered sig-
nificant problems. Recent studies have shown that the anaerobic
The average values of pHoutlet , SCODoutlet , TSSinlet , TSSoutlet , co-digestion of AW with other organic wastewaters has been pro-
methane content, VFA and nitrogen ammonium parameters for posed as a solution to the problems mentioned above. The content
each run are presented in Table 3. At OLRs ranging from 0.9 to of nutrients can thereby be balanced, and the negative effect of
6 g COD/L d, the levels of SCOD in the effluents of mesophilic toxic compounds on the digestion process may be decreased giving
and thermophilic digesters were comparable, 390–900 mg/L and an increased gas yield and energy [56–58].
300–800 mg/L, respectively. Under these conditions, the UAF In fact, the ANOVA analysis showed that pH, TSS, SCOD, VFA,
showed stable operation for both mesophilic and thermophilic N–ammonium of the effluent and the methane content were
digestions. The VFA levels in thermophilic digester increased statistically significant (p < 0.05) between all OLRs in mesophilic
with the increase of OLR from 6 to 9 g COD/L d (442–965 mg/L). and thermophilic conditions. These analyses showed also that
Organic loads used in this work were considerably comparable the different parameters determined were statistically signifi-
with those found in the literature [38,50,51]. Higher COD loadings cant (p < 0.05) between the two tested temperatures 35 and 55 ◦ C
appear to lead to poorer performance. Hence, effective biologi- (Table 3).
cal pretreatment of raw abattoir wastewater to remove suspended The reduction of indicator microorganisms (total and faecal
solids in our study was essential to improve the reactor perfor- coliforms; total and faecal streptococci) and selected pathogens
mance. (Salmonella, Pseudomonas, S. aureus) was examined during the
Many studies on the anaerobic treatment of slaughterhouse mesophilic and thermophilic anaerobic digestions (Table 4). As may
wastewater conducted with anaerobic filter reactors [52], upflow be observed, a satisfactory reduction of total and faecal coliform
anaerobic sludge blanket reactors [53] and anaerobic membrane counts was achieved under mesophilic conditions at OLRs ranging
bioreactors [36] reported the problems of TSS which must be from 0.9 to 3.6 g COD/L d (3.1log10 –0.8log10 and 1.5log10 –0.4log10 ,
removed or solubilized. They suggested that a pretreatment to respectively). The residual numbers ranged from 103 to 104 MPN/ml
hydrolyse a part of the particles could accelerate the anaerobic and from 102 to 103 MPN/ml for total and faecal coliform counts,
treatment of the wastewater. respectively. At an OLR of 4.5 and 6 g COD/L d, the residual num-
The pH of the mesophilic and thermophilic process increased bers present in the digested effluent still high, it was in the range
with the increase of the OLR in both cases. This was a result of of 105 –103 MPN/ml for the total and faecal coliform, respectively.
high conversion level of organic nitrogen to ammonia under high However, only a 1log10 was obtained for total and faecal Streptococ-
OLRs. In fact, high level of free ammonia (FA) would result in cus. The effluent contained less than 102 MNP/ml total and faecal
increased toxicity. Angelidaki and Ahring [54] and Angelidaki et al. Streptococcus. The removal of all tested bacteria decreased then with
[55] showed that the interaction between FA, VFAs and pH may lead the increase of the OLR from 3.6 to 6 g COD/L d and remained low
to an “inhibited steady state”, a condition where the process is run- (<1log10 ). This difference in removal efficiency of the bacteria tested
ning stably but with a lower methane yield. The pH of the reactor in mesophilic conditions may be depending on the decrease of HRT
ranged between 7.5 and 8.3 and probably above critical threshold and to the inefficient mixing, which can present dead zones and
270 H. Gannoun et al. / Journal of Hazardous Materials 170 (2009) 263–271

hydraulic short circuits in the reactor [59]. The ANOVA analysis the Acknowledgements
removal of the indicator and pathogenic bacteria showed significant
differences (p < 0.05) at different OLRs in mesophilic conditions. The authors gratefully acknowledge financial support provided
The thermophilic process was apparently more efficient in by the Tunisian Ministry of Scientific Research, Technology and
the reduction of total (4log10 –1.7log10 ) and faecal coliforms Competences development, Tunis, the International Centre for
(3log10 –1.8log10 ), total Streptococci (1.5log10 –0.8log10 ) at the same Environmental Technologies (Project PRF-eau) and to Mr. Mohamed
OLRs applied in mesophilic conditions (0.9 to 6 g COD/L d). The Kachti (Ellouhoum Society) for cooperation.
residual number of total coliform, faecal coliform and total Strep-
tococci were in the range of 102 –105 , 101 –103 , 102 MPN/ml,
References
respectively. In addition, the populations of faecal Strepto-
cocci, Pseudomonas, S. aureus and Salmonella were undetectable. [1] M.I. Aguilar, J. Sáez, M. Lloréns, A. Soler, J.F. Ortuño, Nutrient removal and sludge
The most resistant microorganisms at relatively high OLRs for production in the coagulation–flocculation process, Water Res. 36 (2002)
2910–2919.
both mesophilic (OLR = 6 g COD/L d) and thermophilic conditions
[2] S. Gariepy, R.D. Tyagi, D. Couillard, F. Tran, Aerobic process for protein recov-
(OLR = 7 and 9 g COD/L d) are faecal Streptococci, Pseudomonas, ery as an alternative to slaughterhouse wastewater treatment, Biol. Wastes 29
S. aureus and Salmonella. Smith et al. [60] demonstrated that (1989) 93–105.
Salmonella spp. is not damaged by mesophilic temperatures, [3] T. Jian, X. Zhang, Bioprocessing of slaughterhouse wastewater and its com-
puterized control and supervising system, Resour. Conserv. Recycl. 27 (1999)
whereas rapid inactivation occurred by thermophilic digestion. The 145–149.
detected Salmonella was identified as S. Arizonae. Although, the Ari- [4] A. Mateu, J. Mata-Alvarez, R. Parés, Enterobacterial and viral decay experimental
zona subgroup may be isolated from a wide variety of nonhuman models for anaerobic digestion of piggery waste, Appl. Microbiol. Biotechnol.
38 (1992) 291–296.
and human sources, the Arizonae are uncommonly recognized as [5] J.C.H. Shih, Ecological benefits of anaerobic digestion, Poultry Sci. 66 (1987)
human pathogens, and surprisingly little is known about their epi- 946–950.
demiology. [6] A.D. Wheatley, Anaerobic digestion: industrial waste treatment, in: P.N. Hob-
son, A.D. Wheatley (Eds.), Anaerobic Digestion: Modern Theory and Practice,
The anaerobic thermophilic digester presents higher efficiency Elsevier, London, 1992, pp. 171–223.
on the removal of pathogens, than the mesophilic digester. The [7] A. Torkian, A. Eqbali, S.J. Hashemian, The effect of organic loading rate on the
important reduction achieved of indicator and pathogens could performance of UASB reactor treating slaughterhouse effluent, Resour. Conserv.
Recycl. 40 (2003) 1–11.
be attributed to different factors which can cause pathogen decay
[8] M. Kim, Y.H. Ahn, R.E. Speece, Comparative process stability and efficiency
during treatment such as temperature, retention time, reactor of anaerobic digestion; mesophilic vs. thermophilic, Water Res. 36 (2002)
configuration, microbial competition, pH value and chemical inter- 4369–4385.
[9] S. Sayed, W. de Zeeuw, The performance of a continuously operated flocculent
actions.
sludge UASB reactor with slaughterhouse wastewater, Biol. Wastes 24 (1988)
The ANOVA analysis of the data indicated that the removal 213–226.
of total and faecal coliforms, total and faecal Streptococci were [10] G.D. Zupančič, M. Stražiščnbar, M. Roš, Treatment of brewery slurry in ther-
statistically significant (p < 0.05) for each OLR in thermophilic con- mophilic anaerobic sequencing batch reactor, Bioresour. Technol. 98 (2007)
2714–2722.
ditions. A comparison of the removal of the bacteria tested between [11] H. Hartmann, I. Angelidaki, B.K. Ahring, Increase of anaerobic degradation of
mesophilic and thermophilic conditions showed also significant particulate organic matter in full scale biogas plants by mechanical maceration,
differences (Table 4). Water Sci. Technol. 41 (2006) 145–153.
[12] V. Patel, M. Desai, D. Madamwar, Thermochemical pretreatment of water
hyacinth for improved biomethanation, Appl. Biochem. Biotechnol. 42 (1993)
67–74.
[13] T. Mori, Treatment of highly concentrated organic wastewaters by thermophilic
aerobic digestion, J. Water Waste 37 (1995) 40–44.
4. Conclusions [14] L. Massé, K.J. Kennedy, S. Chou, Testing of alkaline and enzymatic hydrolysis pre-
treatments for fat particles in slaughterhouse wastewater, Bioresour. Technol.
The microbial pretreatment was based on the hydrolysis and 77 (2001) 145–155.
[15] M. Rojas Oropeza, N. Cabirol, S. Ortega, L.P. Castro Ortiz, A. Noyola, Removal of
solubilization activities of natural microflora present in AW under
(fecal indicator organisms and helminth eggs) from municipal biologic sludge
stirred condition. The dominant bacteria in this wastewater are by anaerobic mesophilic and thermophilic digestion, Water Sci. Technol. 44
identified as Bacillus species. Maximal removal of 80% of TSS (2001) 97–101.
[16] R.J. Knechtel, A more economical method for the determination of chemical
into more soluble COD was obtained under stirred conditions.
oxygen demand, Water Pollut. Control Fed. 50 (1978) 25–29.
The ecological pretreatment could be an attractive solution since [17] Standard Methods for the Examination of Water and Wastewater, 19th edn.,
the TSS considered as a limiting step for the biogas generation American Public Health Association/American Water Works Association/Water
and organic matter removal in the anaerobic digestion pro- Environment Federation, Washington DC, USA, 1995.
[18] M.M. Bradford, A rapid and sensitive method for the quantification of micro-
cess. gram quantities of protein utilizing the principle of protein–dye binding, Anal.
The mesophilic UAF proved to be efficient for the treatment of Biochem. 72 (1976) 241–254.
pretreated AW with an average organic loading rate of 6 g COD/L d. [19] K.H. Hansen, I. Angelidaki, B.K. Ahring, Anaerobic digestion of swine manure:
inhibition by ammonia, Water Res. 38 (1998) 5–12.
Under thermophilic conditions, the anaerobic digestion of pre- [20] B. Calli, B. Mertoglu, B. Inanc, O. Yigun, Effects of high free ammonia concentra-
treated AW showed an improvement in terms of COD removal tions on the performances of anaerobic bioreactors, Process Biochem. 40 (2005)
and biogas yield. These higher performances of thermophilic 1285–1292.
[21] A.L. Koch, Growth measurement, in: P. Gerhardt (Ed. in chief), Manual of Meth-
anaerobic digestion might be mainly attributable to selection of ods For General Bacteriology, ASM Washington, DC, 1981, pp. 197–217, ISBN
the active anaerobic microorganisms, as a result of the sludge 0-914826-30-1.
exchange between mesophilic and thermophilic digesters. How- [22] Statsoft Inc-Statistica for Windows, Computer Program Manual, Tulsa, 1997.
[23] M. Hamdi, S. Hamza, N. Mtimet, N. Hmida, C. Cornelius, S. Zgouli, A.A. Mahjoub,
ever, the increase of the OLR (9 g COD/L d) caused a decrease of
Ph. Thonart, Effect of Corn steep liquor supplementation and scale up on Lac-
the thermophilic UAF performance, especially due to the inhibi- tococcus starter production, Bioprocess Eng. 22 (2000) 23–28.
tion of methanogenic bacteria, resulting from the accumulation of [24] P. Azokpota, D.J. Hounhouigan, M.C. Nago, M. Jakobsen, Esterase and protease
activities of Bacillus spp. from afitin, iru and sonru; three African locust bean
ammonium nitrogen at high OLR. Thermophilic anaerobic diges-
(Parkia biglobosa) condiments from Benin, Afr. J. Biotechnol. 5 (2006) 265–272.
tion of pretreated AW may be considered as an efficient treatment [25] R.S. Prakasham, Ch.S. Rao, R.S. Rao, P.N. Sarma, Alkaline protease production
for organic load reduction and biogas production, and also for by an isolated Bacillus circulans under solid-state fermentation using agro
pathogens removal, being an important contribution for a global industrial waste: process parameters optimization, Biotechnol. Prog. 21 (2005)
1380–1388.
and integrated schema of pollution control and environmental pro- [26] W. Skolpap, S. Nuchprayoon, J.M. Scharer, N. Grisdanurak, P.L. Douglas, M. Moo-
tection. Young, Fed-batch optimization of ␣-amylase and protease-producing Bacillus
 H. Gannoun et al. / Journal of Hazardous Materials 170 (2009) 263–271 271

subtilis using genetic algorithm and particle swarm optimization, Chem. Eng. [44] J. Mata-Alvarez, Biomethanization of the Organic Fraction Municipal Solid
Sci. 63 (2008) 4090–4099. Wastes, IWA Publishing, UK, 2002.
[27] A. Sellami-Kamoun, A. Haddar, N. El-Hadj Ali, B. Ghorbel-Frikha, S. Kanoun, M. [45] J.H. Ahn, C.F. Forster, A comparison of mesophilic and thermophilic anaerobic
Nasri, Stability of thermostable alkaline protease from Bacillus licheniformis RP1 upflow filter, Bioresour. Technol. 73 (2000) 201–205.
in commercial solid laundry detergent formulations, Microbiol. Res. 163 (2008) [46] M.J. Cuetos, X. Gomez, M. Otero, A. Moran, Anaerobic digestión of solid slaugh-
299–306. terhouse waste (SHW) at laboratory scale: influence of co-digestion with the
[28] P. Ravichandra, Ch. Subhakar, J. Annapurna, Alkaline protease production by organic fraction of municipal solid waste (OFMSW), J. Biochemical Eng. 40
submerged fermentation in stirred tank reactor using Bacillus licheniformis (2008) 99–106.
NCIM-2042: effect of aeration and agitation regimes, J. Biochem. Eng. 34 (2007) [47] B. Braun, P. Huber, J. Meyrath, Ammonia toxicity in liquid piggery manure diges-
185–192. tion, Biotechnol. Lett. 3 (1981) 159–164.
[29] S.H. Elwan, M.M. el-Hoseiny, M.S. Ammar, S.A. Mostafa, Lipases production by [48] E.J. Kroeker, D.D. Schulte, A.B. Sparling, H.M. Lapp, Anaerobic treatment process
Bacillus circulans under mesophilic and osmophilic conditions. Factors affecting stability, J. Water Pollut. Control Fed. 51 (1979) 718–727.
lipases production, G. Batteriol. Virol. Immunol. 76 (1983) 187–199. [49] G. Zeeman, W.M. Wiegant, M.E. Koster-Treffers, G. Lettinga, The influence of the
[31] S. Kumar, K. Kikon, A. Upadhyay, S.S. Kanwar, R. Gupta, Production, purifica- total ammonia concentration on the thermophilic digestion of cow manure,
tion, and characterization of lipase from thermophilic and alkaliphilic Bacillus Agric. Wastes 14 (1985) 19–35.
coagulans BTS-3, Protein Expr. Purif. 41 (2005) 38–44. [50] J.R. Campos, E. Foresti, R.P.D. Camacho, Anaerobic wastewater treatment in
[32] A.A. Mendes, E.B. Pereira, H.F. de Castro, Effect of the enzymatic hydrolysis pre- the food processing industry: two cases studies, Water Sci. Technol. 18 (1986)
treatment of lipids-rich wastewater on the anaerobic biodigestion, J. Biochem. 87–97.
Eng. 32 (2006) 185–190. [51] C.E.T. Caixeta, M.C. Cammarota, A.M.F. Xavier, Slaughterhouse wastewater treat-
[33] E. Salminen, J. Einola, J. Rintala, The methane production of poultry slaughtering ment: evaluation of a new three-phase separation system in a USAB reactor,
residues and effects of pre-treatments on the methane production of poultry Bioresour. Technol. 81 (2002) 61–69.
feather, Environ. Technol. 24 (2003) 1079–1086. [52] R. Borja, C.J. Banks, Z. Wang, Performance of a hybrid anaerobic reactor, com-
[34] S.I. Okuda, K. Ito, H. Ozawa, K. Izaki, Treatment of lipid-containing wastewater bining a sludge blanket and a filter, treating slaughterhouse wastewater, Appl.
using bacteria which assimilate lipids, J. Ferment. Bioeng. 71 (1991) 424–429. Microbiol. Biotechnol. 43 (1995) 351–357.
[35] A. Vasala, J. Panula, P. Neubauer, Efficient lactic acid production from high salt [53] S.K.I. Sayed, J. Van der Zanden, R. Wijffels, G. Lettinga, Anaerobic degradation
containing dairy by-products by Lactobacillus salivarius ssp. salicinius with pre- of the various fractions of slaughterhouse wastewater, Biol. Wastes 23 (1988)
treatment by proteolytic microorganisms, J. Biotechnol. 117 (2005) 421–431. 117–142.
[36] A. Saddoud, S. Sayadi, Application of acidogenic fixed-bed reactor prior to [54] I. Angelidaki, B.K. Ahring, Thermophilic digestion of livestock waste: the effect
anaerobic membrane bioreactor for sustainable slaughterhouse wastewater of ammonia, Appl. Microbiol. Biotechnol. 38 (1993) 560–564.
treatment, J. Hazard. Mater. 149 (2007) 700–706. [55] I. Angelidaki, L. Ellegaard, B.K. Ahring, A mathematical model for dynamic sim-
[37] I. Ruiz, M.C. Veiga, P. de Santiago, R. Blázquez, Treatment of slaughterhouse ulation of anaerobic digestion of complex substrates: focusing on ammonia
wastewater in a UASB reactor and an anaerobic filter, Bioresour. Technol. 60 inhibition, Biotechnol. Bioeng. 42 (1993) 159–166.
(1997) 251–258. [56] M. Murto, L. Björnsson, B. Mattiasson, Impact of food industrial waste on anaer-
[38] W.P. Tritt, The anaerobic treatment of slaughterhouse wastewater in fixed-bed obic co-digestion of sewage sludge and pig manure, J. Environ. Manag. 70 (2004)
reactors, Bioresour. Technol. 41 (1992) 201–207. 101–107.
[39] E.A. Salminen, J. Einola, J.A. Rintala, Characterisation and anaerobic batch [57] H. Gannoun, N. Ben Othman, H. Bouallagui, M. Hamdi, Mesophilic and
degradation of materials accumulating in anaerobic digesters treating poultry thermophilic anaerobic co-digestion of olive mill wastewaters and abattoir
slaughterhouse wastes, Environ. Technol. 22 (2001) 577–585. wastewaters in an upflow anaerobic filter, Ind. Eng. Chem. Res. 46 (2007)
[40] E.A. Salminen, J.A. Rintala, Anaerobic digestion of poultry slaughtering wastes, 6737–6743.
Environ. Technol. 20 (1999) 21–28. [58] R. Alvarez, G. Lidén, Semi-continuous co-digestion of solid slaughterhouse
[41] J.B. Van Lier, K.C.F. Grolle, A.J.M. Stams, E.C. de Macario, G. Lettinga, Start-up of waste, manure, and fruit and vegetable waste, Renew. Energy 33 (2008)
a thermophilic UASB reactor with mesophilic granular sludge, Appl. Microbiol. 726–734.
Biotechnol. 37 (1992) 130–135. [59] M.L. Kun, J.B.F. Brunner, E.E. Atal, Destruction of enteric bacteria and
[42] A. Boušková, M. Dohányos, J.E. Schmidt, I. Angelidaki, Strategies for changing viruses during two-phase digestion, J. Water Pollut. Control Fed. 61 (1989)
temperature from mesophilic to thermophilic conditions in anaerobic CSTR 1421–1429.
reactors treating sewage sludge, Water Res. 39 (2005) 1481–1488. [60] S.R. Smith, N.L. Lang, K.H.M. Cheung, K. Spanoudaki, Factors controlling
[43] L. Ortega, S. Barrington, S.R. Guiot, Thermophilic adaptation of a mesophilic pathogen destruction during anaerobic digestion of biowastes, Waste Manage.
anaerobic sludge for food waste treatment, J. Environ. Manag. 88 (2008) 25 (2005) 417–425.
517–525.