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Feasibility study of the anaerobic digestion of dewatered pig slurry
by means of polyacrylamide
E. Campos a, M. Almirall a, J. Mtnez-Almela b, J. Palatsi c, X. Flotats
a
c,*
Laboratory of Environmental Engineering, Centre UdL-IRTA, Rovira Roure 191, E-25198 Lleida, Spain
b
SELCO MC SL, Pza. Tetuán 16, E-12001 Castellón, Spain
c
GIRO Technological Centre, Rambla Pompeu Fabra 1, E-08100 Mollet del Vallès, Barcelona, Spain
Received 15 January 2004; received in revised form 20 September 2006; accepted 6 December 2006
Abstract
Liquid livestock waste can be managed by separating liquid and solid fractions then treating each separately by applying best available technology, such as anaerobic digestion for the solid fraction. There is an increasing use of polyacrylamide (PAM) as a flocculant
agent to improve solid–liquid separation. In the present work, the anaerobic toxicity of PAM residues and the optimal range of total
solids concentration for maximum methane production were studied as a function of PAM dosage. Results showed that dry matter
and its volatile solids content increased significantly with increasing PAM dosage. Batch anaerobic tests showed that methane yield
decreased linearly with increasing total solids, while the methane production per unit of raw substrate reached a maximum at 16.4% total
solids. No PAM toxicity was measured for PAM concentrations below 415 g/kg total solids, but some indirect inhibitory phenomena
were observed, such as a limited hydrolysis rate due to particle aggregation, and inhibition of methanogenesis by high ammonia
concentration.
2007 Elsevier Ltd. All rights reserved.
Keywords: Anaerobic digestion; Pig slurry; PAM; Polyacrylamide; Solid–liquid phase separation
1. Introduction
Modern pig production, which has a very intensive and
concentrated character, generates a large pig slurry surplus
that often cannot be used as an agricultural fertiliser in the
same geographical area, thus making its transport a limiting factor. One management strategy consists of separating
the solid and liquid fractions, then treating the liquid fraction prior to using it for irrigation on nearby land, while
treating the solid fraction in order to stabilise it and to
reduce volume before transporting it to areas with nutrient
and/or organic matter demand. Stabilisation of the solid
fraction, prior to land application, can be achieved by aerobic composting and/or anaerobic digestion. The second
option provides a better energy balance and can be comple*
Corresponding author. Fax: +34 935796785.
E-mail address: xavier.fl[email protected] (X. Flotats).
mented by further aerobic composting in order to produce
a higher quality end product. The efficiency of anaerobic
digestion of this solid fraction can be negatively affected
by high total solids concentration (Itodo and Awulu,
1999; Bujoczek et al., 2000).
The main fraction of organic matter found in pig slurry
takes the form of small suspended particles, mainly in colloidal form, which are not easily separated by applying a
simple mechanical system (Hill and Tollner, 1980). The efficiency of suspended solids separation using filters and
presses is limited, and for colloids agglutination a chemical
coagulation process is required (Sievers et al., 1994). Treatment with polyacrylamide (PAM) polymers, prior to
mechanical removal or gravity settling, has the potential
to enhance solid–liquid separation, thus concentrating
nitrogen, phosphorous and organic carbon (Vanotti and
Hunt, 1999). Since most suspended particles in wastewaters
and aqueous solutions, such as livestock and poultry
0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2006.12.008
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manure, have a negative charge at pH values higher than 4,
the addition of cationic coagulants to these wastewaters
would be more effective than the addition of anionic ones
(Sievers et al., 1994).
Polyacrylamide (PAM) is widely used in sewage sludge
treatment to enhance dewatering. The polyelectrolyte concentration in mechanically dewatered cakes is relatively
high and typically in the range 2.5–5.0 g/kg dry matter
(TS), and it can be degraded by abiotic processes in cultivated soils without toxicity problems (ICON, 2001). Therefore, it has been suggested that it presents low toxicity, with
LD50 value greater than 5 g/kg TS, but this potential toxicity has not been studied yet for anaerobic digestion.
PAM can degrade to acrylamide monomer, which is
highly toxic (IPCS, 1985), followed by a rapid degradation
to ammonia and acrylic acid, which is not toxic and in turn
degrades to CO2 and water (ICON, 2001). El-Mamouni
et al. (2002) demonstrated that PAM is highly recalcitrant
to aerobic or anaerobic microbial degradation, suggesting
that this recalcitrance is linked to the high molecular
weight, thus making it inaccessible to microbial attack.
These authors found that PAM is very susceptible to UV
photolysis, enhancing a further microbial degradation process, but without intermediate production of acrylamide.
Studies on PAM degradation in cultivated soils (Kay-Shoemake et al., 1998) demonstrated that indigenous bacteria
could use PAM as a source of nitrogen, biotransforming
the polymer to long chain polyacrylate, which may be further degraded by biological processes without toxicity
problems. In an extended review, Caulfield et al. (2002)
concluded that there is no evidence to suggest that PAM
can undergo biodegradation to form free acrylamide
monomer units. Caulfield et al. (2002) also concluded that
PAM can act as a carbon source for microbial growth only
when some other physical or chemical process lowers the
molecular weight of the polymer beforehand. These results
suggest that toxicity by acrylamide is not probable during
anaerobic digestion processes without previous physical
or chemical pre-treatment to enhance PAM degradation.
Chu et al. (2003) studied the anaerobic digestion of
PAM flocculated activated sludge, comparing the effect of
cationic, non-ionic and anionic polyacrylamide. They
found that anionic and non-ionic PAM had no effect on
methane yield at doses below 15 g/kg TS. For cationic
PAM, methane yield decreased with increasing dosage,
showing a small variation in relation to the control assay
for 1 and 5 g/kg TS, and a significant decrease for 15 and
40 g/kg TS. Since dosed polymers had no apparent toxicity
to the inoculum, although an anaerobic toxicity test was
not performed, Chu et al. (2003) suggested that the much
greater floc size obtained with cationic PAM increased
the mass transfer resistance. No methane production from
PAM degradation was measured in these experiments.
Chang et al. (2001) found that the methane production
obtained from anaerobic batch digestion of a commercial
PAM, consisting of a copolymer of acrylamide and acryloyloxyethyltrimethylammonium chloride, was consistent
with a complete degradation of the second monomer, but
not with the degradation of PAM or acrylamide. No inhibition phenomena were reported in this study.
The optimum cationic PAM dose varies with the type of
manure and the amount of total suspended solids (TSS) in
the liquid manure, the dose increasing with TSS concentration (Vanotti and Hunt, 1999; Chastain et al., 2001). In a
study of the separation of different types of pig slurry,
Walker and Kelley (2003) found that optimal PAM dosage
was in the range 0.9–1.8 g/kg TS for efficient separation of
SS, TSS and COD, and in the range 4.2–10.9 g/kg TS for
efficient separation of nitrogen (N) and phosphorous (P).
In general, the TSS removal efficiencies achieved range
from 76% to 99%. These values contrast with the efficiencies of the screening process alone (without using PAM),
which range from 5% to 14%.
The objectives of the present work were to study the
anaerobic digestion of the solid fraction of pig slurry separated using PAM at different dosages, to characterize this
solid fraction and to study the anaerobic biodegradability
and toxicity of PAM, in order to determine whether the
polymer or its possible degradation products can affect
anaerobic microorganisms during the pig slurry digestion
process.
2. Methods
2.1. Materials
The pig slurry and its associated solid fraction came
from a treatment plant in Modena, Italy. This plant used
the SELCO-EcopurinTM solid/liquid separation system
(Martı́nez-Almela and Barrera, 2005), using cationic
PAM as the coagulant agent. The four different materials
identified in Table 1 were used: raw pig slurry (PS), solid
fraction of pig slurry using a PAM dose of 120 mg/l
(SFPS), which is the usual dose in the plant, solid fraction
of pig slurry using a PAM dose of 140 mg/l (SFPS-1) and
solid fraction of pig slurry without using PAM (SFPS-0).
Anaerobically digested sewage sludge from a mesophilic
digester was used as inoculum for batch tests.
2.2. Analytical methods
Analytical methods for the determination of total and
volatile solids (TS and VS), total and volatile suspended
solids (TSS and VSS), total and soluble chemical oxygen
demand (CODt and CODs), total Kjeldahl nitrogen
(NTK), ammonia nitrogen ðNHþ
4 –NÞ and pH were adapted
from Standard Methods for the Examination of Water and
Wastewater (APHA, 1995). Total and partial alkalinity
(TA, PA) were analysed according to the method proposed
by Hill and Jenkins (1989).
Methane and carbon dioxide concentration in the biogas
were measured with a GC 8000 Top Series gas chromatograph (CE Instruments, Italy), fitted with PORAPAK-N
(80/100 mesh) packed column (2 m · 2 mm) and a Thermal
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3
Table 1
Identification and basic characterisation, average of three replicates, of inoculum and substrates used (g/kg of substrate or inoculum)
PAMa dose (mg/l)
SFPS-1
SFPS
SFPS-0
PS
Inoculum
a
mg/l
g/kg TS
140
120
0
–
–
14.27
12.23
0
–
–
TS (g/kg)
VS (g/kg)
COD (g/kg)
NTK (g/kg)
N–NHþ
4 (g/kg)
313.60 ± 41.41
136.12 ± 6.06
13.57 ± 0.24
9.81 ± 0.36
39.45 ± 0.16
233.58 ± 29.98
100.36 ± 3.79
7.38 ± 0.51
5.53 ± 0.70
22.65 ± 0.93
271.99 ± 36.17
96.30 ± 24.37
17.07 ± 6.15
6.17 ± 3.78
28.22 ± 4.24
18.81 ± 0.78
7.79 ± 3.45
2.03 ± 0.20
0.90 ± 0.58
–
3.06 ± 0.00
1.89 ± 0.43
1.51 ± 0.07
0.83 ± 0.00
–
PAM dose applied to raw pig slurry to obtain the corresponding solid fraction used as substrate.
Conductivity Detector (TCD). Helium (He) was used as a
carrier gas (20 ml/min), and temperatures of the injector
port (J70), column and TCD were 130, 30 and 120 C
respectively.
Volatile fatty acids (VFA) – acetate (Ac), propionate
(Pro), iso-butyrate (Iso-But), n-butyrate (n-But), iso-valerate (Iso-Val) and n-valerate (n-Val) – were determined from
samples after centrifugation (2790g for 20 min), filtration
(0.45 lm) and acidification/extraction (with HCl and
diethyl-ether 1/1) with a Trace 2000 gas chromatograph
(Thermo Instruments, Italy), fitted with a FFAP capillary
column (30 m · 0.250 mm · 0.25 lm), with flame ionization detector (FID) and equipped with auto sampler (Autosampler AS2000, Italy). The FID was supplied with H2 and
synthetic air, while He was used as make-up gas with a flow
rate of 30 ml/min. Samples of 1 ll were injected in splitsplitless mode, with a constant carrier gas flow rate of
1 ml/min, a split ratio of 20/1 and a septum purge ratio
of 5/1. The initial oven temperature was 90 C for 4 min,
after which it was increased to 155 C at 6 C/min then
to 255 C at 12.5 C/min, with a final isotherm of 2 min.
The injector and detector temperatures were set constant
at 240 C.
NH3–N concentration was calculated by using Eq. (1),
1
þ
½NH3 –N ¼ ½NH4 –Nt
;
ð1Þ
10ðpKpHÞ þ 1
35 C in a closed and dark incubator for 21 days and the
amount of gas accumulated in the headspace was measured
twice a week.
Six treatments were carried out (Table 2), with five different PAM concentrations between 0 and 126 mg/l and
one additional treatment with a much higher concentration, about 2775 mg/l. These concentrations correspond
to 0–18.7 g PAM/kg TS of sludge, and the sixth concentrated treatment corresponds to 415 g/kg TS, which is an
unusual and extremely high value.
An anaerobic biodegradability test, following Soto et al.
(1993), was also carried out in order to study the biodegradability of PAM in an anaerobic environment. The
120-ml vials were filled with 50 ml of medium, containing
macronutrient and micronutrient solutions, alkalinity solution and anaerobically digested sewage sludge (222 g/l),
giving a solids concentration of 6.7 g TS/l and 5.1 g VS/l.
The initial concentration of PAM was 259.8 ± 4.4 mg/l in
the culture liquid, corresponding to 38.5 ± 0.6 g/kg TS.
pH was adjusted to neutrality. After displacement of air
from the headspace with N2/CO2 gas (80/20 v/v), the vials
were tightly closed with rubber stoppers. Finally, a reducing solution (0.1 ml of 50 g Na2S/l) was injected into every
vial to achieve a reduced medium. The vials were incubated
at 35 C in a closed and dark incubator for 33 days. The
accumulated methane production was determined by periodic headspace analysis.
with a pK value of 8.938 at 35 C (Bonmatı́ and Flotats,
2003).
2.4. Batch anaerobic tests
2.3. Polyacrylamide toxicity and biodegradability test
The PAM toxicity test was carried out according to Soto
et al. (1993). The culture medium consisted of 223 g/l of
digested sewage sludge as inoculum, macro and micronutrient solutions, a mixture of volatile fatty acids (2.95 g
acetate/l, 0.59 g propionate/l and 0.25 g butyrate/l) as substrate (Soto et al., 1993) and the corresponding PAM concentration (from 0 – control – to 2775 mg/kg sludge TS).
Batch reactors were 120 ml glass vials filled with 50 ml of
culture medium. After displacement of air from the headspace with N2/CO2 gas (80/20 v/v) for 3 min, the vials were
tightly closed with rubber stoppers. Finally, a reducing
solution (0.1 ml of 50 g Na2S/l) was injected into every vial
to achieve a reduced medium. The vials were incubated at
Four substrate mixtures (Table 1) were prepared and
mixed in various ratios to produce five different combinations of TS and PAM concentrations, corresponding to five
different treatments for batch anaerobic tests (Table 3). An
additional ‘‘blank’’ treatment – water plus inoculum – was
also prepared to evaluate the methane production from
inoculum. The methodology of batch tests was adapted
from Campos et al. (2000): 120 ml glass vials were filled
with 30 g of mixture (90% substrate and 10% inoculum,
digested sewage sludge). After displacement of air from
the headspace with N2/CO2 gas (80/20 v/v) for 3 min the
vials were tightly closed with rubber stoppers. Finally, a
reducing solution (0.1 ml of 50 g Na2S/l) was injected into
every vial to achieve a reduced medium. The vials were
incubated at 35 C in a closed and dark incubator, and
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Table 2
Results from the PAM toxicity test
Treatment
PAM dose (mg/l)
PAM dose (g/kg TS)
ACm, 7 daya (g COD/g VS day)
Final methane yield (ml CH4/vial)
T1
T2
T3
T4
T5
T6
0.00
33.29
66.89
99.27
125.55
2775.19
0.00
4.96
10.07
14.99
18.67
414.80
0.118 ± 0.025
0.114 ± 0.025
0.116 ± 0.018
0.117 ± 0.007
0.111 ± 0.012
0.107 ± 0.026
76.30 ± 12.53 a
75.24 ± 9.06 a
76.94 ± 3.51 a
75.59 ± 6.42 a
78.42 ± 5.87 a
86.96 ± 6.55 b
A
A
A
A
A
A
Letters: results of Duncan test at 5% significance; different letters indicate statistically significant differences.
a
ACm, maximum methanogenic activity, observed at seventh day.
Table 3
Characterisation of treatments used in the anaerobic digestion test (three replications per treatment)
Treatment
T1
T2
T3
T4
T5
T6 (blank)
PAM dose
Substrate composition (% w/w)
Substrate characterisation (g/kg)
g/kg TS
mg/l
SFPS-1
SFPS
SFPS-0
TS
VS
COD
14.27
13.25
12.23
10.40
0
0
140
ffi130
120
ffi100
0
–
100
50
0
0
0
0
0
50
100
85
0
0
0
0
0
15
100
0
313.60 ± 41.41
210.04 ± 17.34
136.12 ± 6.06
98.74 ± 3.03
13.56 ± 0.24
–
233.58 ± 29.98
155.85 ± 15.47
100.36 ± 3.79
71.41 ± 1.83
7.52 ± 0.51
–
271.99 ± 36.17
200.65 ± 4.97
96.30 ± 24.37
85.00 ± 7.34
14.84 ± 6.15
–
monitored for 82 days. Vials were shaken by hand once a
day. A complete analytical characterisation was performed
at both the beginning and the end of the experiment: TS
and VS, TSS and VSS, CODt and CODs, NTK, NHþ
4 –N,
pH, TA, PA and VFA. The accumulated methane production was determined by periodic headspace analysis.
2.5. Calculations
2.5.1. Separation efficiency
The separation efficiency (Et) is defined as the total mass
recovery of nutrients in the solid fraction as a proportion
(%) of the total input of solids or nutrients (Møller et al.,
2002),
Et ¼
U Mc
100;
Q Sc
ð2Þ
where U (kg) is the quantity of solid fraction, Mc (g/kg) is
the concentration of TS or NTK in the solid fraction; Q (kg)
is the amount of manure treated; and Sc (g/kg) is the concentration of TS or NTK in the manure.
2.5.2. Methanogenic activity
The methanogenic activity, ACm (g COD/g VS Æ day), in
the toxicity test was calculated as methane production during the maximum growth period, by using the following
expression adapted from Soto et al. (1993):
AC m ¼
R
f V ½SSV
ð3Þ
where R is the methane production rate (ml CH4/day), f is
a factor to transform methane volume to grams of COD
(350 ml of CH4/g COD for Normal Conditions), and
[SSV] is the concentration of SSV in the culture medium
with a volume V.
2.6. Statistical methods
The confidence limits of average values of experimental
data were calculated using Eq. (4),
pffiffiffi
x tðs= nÞ;
ð4Þ
where x is the average value, s is the standard deviation, n is
the number of replicates and t is the corresponding t-statistical distribution value, depending on the number of samples and on the degree of confidence (95% in the present
study).
Mean separation tests were performed using statistical
analysis software (SAS Institute, 1989) and by applying a
Duncan test with a significance level of 5%. Significant differences have been indicated with different letters. Regression analyses were done using the Levemberg–Marquardt
algorithm.
3. Results and discussion
3.1. Effect of polyacrylamide on substrate characteristics
The basic characterisation of the different original materials – shown in Table 1 – was carried out prior to the anaerobic tests. The solid fraction of pig slurry obtained from the
usual dose of PAM (120 mg/kg) – SFPS – showed a TS
concentration higher than 13% (136.1 g TS/kg). A slight
increase in the PAM dose, from 120 to 140 mg/kg (SFPS-
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1), led to a very significant increase in the total solids concentration of the solid fraction, which surpassed 30% of
the total weight (313.6 g TS/kg). The solid fraction of pig
slurry obtained when PAM was not used (SFPS-0) showed
a very low TS and NTK content and it was slightly higher
than raw pig slurry (PS). This fact shows that the separation
process was not effective without PAM dosage, which is
consistent with results from Vanotti and Hunt (1999).
The separation efficiency for 120 and 140 mg PAM/kg
raw slurry was higher than the usual values reported for
other mechanical separation methods (Møller et al., 2000,
2002), as shown in Table 4. The differences were especially
important for the separation efficiency of total nitrogen,
with values obtained exceeding 50%. Similar or higher
removal efficiencies have been obtained by other authors
using PAM as an additive in the separation process (Vanotti and Hunt, 1999; Walker and Kelley, 2003). As can be
observed in Table 1, the organic fraction (VS) of total
solids, the COD/NTK ratio and the N TK =N–NHþ
4 ratio
increased with PAM dose, indicating an increasing separation efficiency for organic materials.
5
Evolution of accumulated methane production (ml) per
vial is shown in Fig. 1. Treatments T1–T5 showed a very
similar evolution, without statistically significant differences between treatments. However, treatment T6 (corresponding to 415 g PAM/kg TS) showed higher and
statistically significant accumulated methane production
at the end of the experiment. The difference in methane
production for this treatment was 112.8 ± 38.0 ml CH4/g
PAM added, an average 23% of the maximum theoretical
methane yield predicted based on molecular composition.
Chang et al. (2001) found that low methane production
from PAM was due to the degradation of acryloyloxyethyltrimethylammonium chloride, a second monomer contained in the PAM used for their work. However, PAM
used in the work described here did not contain this monomer. Therefore, findings from Chang et al. (2001) cannot
explain the measured methane production. Taking into
account the high dose used in treatment T6, this low but
significant production could have been caused by the presence of impurities and additives.
Maximum methanogenic activity for the six treatments
was found on the seventh day of digestion, and showed
no statistically significant differences when applying the
Duncan test (Table 2 and Fig. 2). These results indicate
that the polymer compound used cannot be considered
toxic for anaerobic microorganisms at the concentrations
studied. If PAM is degraded in some way, products are also
non-inhibitors even at the high concentration used in T6.
No statistically significant differences were found in the
biodegradability test (Fig. 3) between the treatment with
PAM as substrate and the control treatment. This fact indicates that the polymer is not significantly biodegradable by
anaerobic microorganisms. The low and non-statistically
3.2. Polyacrylamide toxicity and biodegradability study
The maximum concentration of PAM that could be
found in the solid fraction of pig slurry, assuming that all
PAM was associated with the separated solids, was
14.27 g PAM/kg TS for 140 mg PAM/l dose (Table 1).
With treatments T2–T5 (Table 2) the toxicity study covered
usual PAM concentrations. The study was contrasted both
with an extremely high PAM concentration (T6) and with a
control assay without PAM dosage (T1). Table 2 shows the
results from the toxicity test.
Table 4
Separation efficiencies obtained compared with literature values
Technology
Substrate
Reference
U/Q (%)
PAM 120 mg/l
PAM 140 mg/l
Centrifuge
Centrifuge
Centrifuge
Centrifuge
Centrifuge
Centrifuge
Centrifuge
Centrifuge
Screw press
Screw press
Screw press
Tilted plane screen
Pressing screw
Pressing screw
Two-stage separator
Belt press separator
PS
PS
PS
PS
PS
PS
ADPS
ADPS
ADPS
ADPS
PS
ADPS
ADPS
PS
PS
PS
PS
PS
Present study
Present study
Møller et al. (2002)
Møller et al. (2002)
Møller et al. (2002)
Møller et al. (2002)
Møller et al. (2002)
Møller et al. (2002)
Møller et al. (2002)
Møller et al. (2002)
Møller et al. (2002)
Møller et al. (2002)
Møller et al. (2002)
Møller et al. (2000)
Møller et al. (2000)
Møller et al. (2000)
Møller et al. (2000)
Møller et al. (2000)
6.09
2.59
13.10
8.28
5.67
4.69
13.72
14.11
8.82
9.91
5.23
3.85
2.88
30.00
5.00
7.30
24.00
17.50
PS
SFPS
Mc/Sca
Et
TS
NTK
TS
NTK
TS (%)
NTK (%)
TS
NTK
9.81
9.81
53.20
47.90
17.10
25.50
56.20
65.30
35.50
37.40
53.20
56.20
37.40
56.60
56.60
56.60
56.60
56.60
0.90
0.90
4.20
4.40
2.20
3.90
4.20
5.00
3.80
3.30
4.20
4.20
3.30
4.10
4.10
4.10
4.10
4.10
136.12
313.60
245.60
279.30
187.30
178.20
280.80
252.70
299.90
201.90
364.70
268.40
298.40
117.00
317.00
219.00
167.00
192.00
7.79
18.81
9.4
9.88
7.79
10.91
7.41
10.99
10.89
7.89
6.61
6.31
6.89
4.6
4.8
4
5.3
6.4
84.6
82.9
60.5
48.3
62.1
32.8
68.6
54.6
74.5
53.5
35.9
18.4
23.0
62.0
28.0
28.2
70.8
59.4
53.0
54.4
29.3
18.6
20.1
13.1
24.2
31.0
25.3
23.7
8.2
5.8
6.0
33.7
5.9
7.1
31.0
27.3
13.9
32.0
4.6
5.8
11.0
7.0
5.0
3.9
8.4
5.4
6.9
4.8
8.0
2.1
5.6
3.9
3.0
3.4
8.7
21.0
2.2
2.2
3.5
2.8
1.8
2.2
2.9
2.4
1.6
1.5
2.1
1.1
1.2
1.0
1.3
1.6
PS, pig slurry; SFPS, separated solid fraction of pig slurry; ADPS, anaerobically digested pig slurry.
a
Concentration ratio Mc/Sc (see Eq. (2)).
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Accumulated CH4 (mL)
100
80
T1-0 g/kgTS
T2-5 g/kgTS
60
T3-10 g/kgTS
40
T4-15 g/kgTS
T5-19 g/kgTS
20
T6- 415 g/kgTS
0
0
2
4
6
8 10 12 14 16 18 20 22 24
Time (days)
Fig. 1. Accumulated methane production in toxicity test. Confidence
intervals calculated for 95% confidence level.
release ranging from 17% to 69% of PAM organic N (data
not shown), with high deviation within and between treatments and without a defined tendency. Since no anaerobic
biodegradability was measured and no polymer chain
break took place, this ammonia release suggests that deamination of PAM occurred to some extent, as described by
Kay-Shoemake et al. (1998) and Caulfield et al. (2002).
It can be concluded from the toxicity and biodegradability tests that the polymer or its degradation products did
not produce toxicity to anaerobic digestion, suggesting that
acrylamide was not produced, in agreement with KayShoemake et al. (1998); Caulfield et al. (2002) and ElMamouni et al. (2002).
3.3. Study of the initial total solid concentration effect to
anaerobic batch tests
Methanogenic activity 7d
(g COD/g VS·d)
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0
2
4
6
8
10
12
14
PAM concentration (g/kg TS)
16
18
20
Fig. 2. Maximum methanogenic activity index, found at day 7, as a
function of PAM dose expressed as g of PAM/kg of total dried solids.
Accumulated CH4 (ml)
35
30
25
PAM-250 mg/l
20
Control
15
10
5
0
0
5
10
15
20
25
Days of incubation
30
35
Fig. 3. Accumulated methane generation in the anaerobic biodegradability test.
significant difference in methane production has the same
order of magnitude as that obtained for the T6 treatment
in the toxicity test, low enough to be disregarded at usual
PAM dosages.
The NTK and NH4–N measurements in the biodegradability and toxicity experiments showed an ammonia
Methane production results from the batch anaerobic
tests are shown in Table 5 and Figs. 4–6. Methane production related to substrate weight (M), Table 5 and Fig. 4,
increased with TS concentration or PAM dose until treatment T3, decreasing with higher PAM doses with a minimum methane production value for treatment T1 (14.27 g
PAM/kg TS). Treatments T2 and T3 showed no statistically significant differences, but treatment T2 showed an
increasing methane production during the last days of the
incubation period (Fig. 4), suggesting that methane values
were not the maximum obtainable in spite of the extended
incubation period.
Different behaviour was detected when methane yield
(B) was measured. Yield is expressed as ml CH4 produced
related to initial added VS or related to initial added COD
(Table 5). When related to initial VS concentration, accumulated methane showed statistically significant differences
for the five treatments of the test, with methane yield
increasing with decreasing TS concentration or PAM dose
and a maximum value for the control treatment (T5).
When related to initial COD concentrations, the global
response was very similar but without statistically significant differences between treatments T3 and T4. Treatment
T2 showed a clear increasing yield value at the end of the
experiment (Fig. 5), suggesting that the final value could
have been higher if incubation time were longer. Although
with a lower slope, treatment T1 also showed a slight
increase at the end of the experiment (Figs. 4 and 5), suggesting that the digestion process rate was decreased significantly by the increase in TS or PAM dose, but not
stopped.
Studying the relationship between methane yield (B) and
initial TS (Fig. 6), a clear linear decrease associated with
the increase in TS content of the substrate can be observed.
Other authors have pointed out this tendency; Bujoczek
et al. (2000) found that for total solids concentrations
above 4%, maximum methane production rate decreased
with total solids content, following a linear tendency; Itodo
and Awulu (1999) observed that methane yield from different types of animal waste tended to decrease when total
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7
Table 5
Accumulated production of methane in the anaerobic batch test
PAM dose (g/kg TS)
CH4 (M) (ml/g sub)
CH4 yield (B) (ml CH4/g VSinitial)
CH4 yield (B) (ml CH4/g CODinitial)
T1
T2
T3
T4
T5
3.85 ± 1.13
20.41 ± 1.61
20.73 ± 0.73
18.27 ± 0.63
4.04 ± 0.14
16.46 ± 4.83 A
130.99 ± 10.33 B
206.58 ± 7.24 C
255.81 ± 8.87 D
538.00 ± 18.92 E
14.14 ± 4.15
101.74 ± 8.02
215.29 ± 7.55
214.89 ± 7.45
272.56 ± 9.59
14.27
13.25
12.23
10.40
0
A
C
C
B
A
A
B
C
C
D
Different letters indicate statistically significant differences among means by columns, with a significance level of 5%.
M (ml CH4/g subs)
M (ml CH4/g subs)
25
T1-14.3 g/kgTS
15
T2-13.3 g/kgTS
T3-12.2 g/kgTS
10
T4-10.4 g/kgTS
T5-0 g/kgTS
5
R2 = 0.9989
250
200
150
y = -897TS + 302.23
50
0
5
10
0
20
30 40 50 60
Days of incubation
70
80
100
R2 = 0.9599
0
10
350
300
0
0
B (ml CH4/g CODini)
B
y = -787.23TS + 257.67TS + 0.5842
22
20
18
16
14
12
10
8
6
4
2
0
20
M
15
20
10.40 12.23
25
30
13.25
35
TS (%)
14.27g PAM/kg TS
90
Fig. 6. Methane production (M) and yield (B) as functions of the total
solids of the substrate, obtained with the indicated PAM dose.
Fig. 4. Accumulated methane production (M) per gram of substrate
obtained in the batch anaerobic test, for different PAM doses.
B (ml CH4/g CODini)
300
T1-14.3 g/kg TS
250
T2-13.3 g/kg TS
T3-12.2 g/kg TS
200
T4-10.4 g/kg TS
T5-0 g/kg TS
150
100
The rate of hydrolysis can be measured by the removal
of particulate matter during the batch anaerobic digestion, expressed as the reduction of organic nitrogen
ðN TK –N–NHþ
4 Þ, of TSS, of VSS or of particulate COD
(CODp = CODt CODs). The corresponding removals
are shown in Fig. 6. It should be noted that the general
trend was a decreasing removal rate of particulate matter
as TS or PAM dose was increased (see Fig. 7).
Mass transfer limitation due to high solids concentration could produce a local high accumulation of VFA. Its
accumulation in the biowaste bed over inhibitory levels
(40–50 g COD VFA/l) can inhibit the hydrolysis process,
50
0
10
20
30 40 50 60
Days of incubation
70
80
90
Fig. 5. Accumulated methane yield (B) per gram of initial COD in the
batch anaerobic test for different PAM doses.
solids content increased. In the case of pig slurry, this
decrease only took place for TS values above 10%.
Since PAM toxicity was not demonstrated in the present
study, the explanation for the lower methane production in
the most concentrated treatments could be: (a) inhibition
of enzymatic hydrolysis due to the colloidal aggregation,
decreasing effective particle surface and increasing internal
mass transfer resistance due to the increase in floc size, as
suggested by Chu et al. (2003), or (b) specific inhibition
of another process step.
% reduction of particulate matter
90
0
80
N org
70
TSS
60
VSS
CODp
50
40
30
20
10
0
0
5
10
15
20
25
30
35
% TS
Fig. 7. Average reduction of particulate matter, for different initial total
solids concentration values, expressed as organic N, TSS, VSS or
particulate COD.
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E. Campos et al. / Bioresource Technology xxx (2007) xxx–xxx
as has been reported previously (Veeken and Hamelers,
2000). The volatile fatty acid concentration at the end of
the experiment for T1 (highest solid concentration treatment) was very high, close to 40 g COD VFA/kg, a level
at which hydrolysis is completely inhibited by VFA (Veeken
and Hamelers, 2000). Validated models describing VFA
inhibition of hydrolysis of particulate matter have been
developed with satisfactory results (Angelidaki et al.,
1999; Vavilin and Angelidaki, 2005).
The high concentration of acetate at the end of the process in T1 (Table 7) together with the ammonia concentration (Table 6), above 6 g N–NHþ
4 =l, could also explain an
inhibition of acetoclastic methanogenic microorganisms
by free ammonia. Other VFA were also accumulated, but
at lower levels than acetate. Accumulation of acetate could
also have caused an accumulation of other longer chain
acids, since a high concentration of acetate can inhibit
the acetogenic process (Ahring and Westermann, 1988).
In most of the treatments, ammonia nitrogen concentration increased towards the end of the process (Table 6). In
treatments T1–T4, ammonia nitrogen concentration practically doubled and reached extremely high levels, above 6 g
N/kg. This increase in ammonia nitrogen and of pH
throughout the process caused a significant increase in free
ammonia at the end of the process (Table 6). The measured
values were higher than the values described as inhibitory
for a methanogenic population by some authors (Hashim-
oto, 1986; Gallert et al., 1998), although below the inhibition threshold value for adapted acetate-utilizing bacteria
(Angelidaki and Ahring, 1993; Hansen et al., 1998).
The high concentration of propionate and the high value
of the propionate/acetate (P/A) ratio (Table 7) at the end
of treatment T2 (corresponding to 130 mg/kg PAM dose)
showed that the process had been strongly inhibited. This
also explains the shape of the accumulated methane curve,
with a longer lag phase than in the most diluted treatments
(Figs. 3 and 4), probably indicating an overloading of the
methanogenic population due to the high ratio of organic
matter/inoculum.
The other treatments (T3–T6) showed much lower levels
of VFA at the end of the experiment (Table 7), and in many
cases only acetate was detectable, showing that the
methanogenic phase was not inhibited, as can also be
deduced from the shape of accumulated methane production curves.
Summing up, the decrease in solids removal rate and
methane yield of the solid fraction of pig slurry separated
by PAM as PAM dose increased, with consequent
increased TS concentration, can be explained by a combination of two phenomena: resistance to enzymatic hydrolysis due to organic matter aggregation and transport
limitations, and inhibition of the methanogenic step due
to high NH4–N concentration, which increased in the solid
fraction separated as PAM dose increased.
Table 6
Nitrogen measurements at the beginning and at the end of the anaerobic batch tests (mixture of substrate and inoculum)
PAM dose (mg/kg TS)
NTK (g N/kg)
NHþ
4 –N ðg N=kgÞ
Initial
Final
Initial
Final
Initial
Final
T1
T2
T3
T4
T5
T6
15.68 ± 0.67
11.73 ± 0.59
7.87 ± 0.66
6.45 ± 0.25
2.01 ± 0.09
0.35 ± 0.06
3.07 ± 0.28
2.19 ± 0.12
2.46 ± 0.09
1.78 ± 0.10
1.37 ± 0.08
0.25 ± 0.21
6.47 ± 1.10
4.96 ± 0.12
3.57 ± 0.27
3.29 ± 0.27
1.62 ± 0.08
0.20 ± 0.05
7.42
7.29
7.99
7.26
7.58
7.00
7.60
7.87
7.96
7.98
8.19
7.65
90.52
48.10
248.91
36.23
57.68
2.86
286.23
387.89
337.70
328.97
246.31
9.93
14.27
13.25
12.23
10.40
0
–
pH
NH3 (mg N/kg)
Average of three replicates.
Table 7
Volatile fatty acid concentrations at the beginning and at the end of batch experiments (average of three replicates)
PAM (mg/kg TS)
Individual volatile fatty acid concentration (mM)
Ac
Initial
T1
T2
T3
T4
T5
T6
14.27
13.25
12.23
10.40
0
–
75.48
59.33
50.23
48.00
42.34
0.52
Final
T1
T2
T3
T4
T5
T6
14.27
13.25
12.23
10.40
0
–
206.23
8.14
1.44
1.03
0.13
0.13
Pro
Iso-But
n-But
5.84
10.51
22.90
19.64
9.56
0.33
6.99
6.92
2.63
2.30
1.38
0.08
54.71
77.59
0.10
0.00
0.00
0.00
27.00
12.55
0.06
0.01
0.00
0.00
Total VFA (mM)
Total VFA (g COD/kg)
Iso-Val
n-Val
3.44
5.90
6.27
5.60
4.42
0.01
10.96
10.19
3.99
3.51
1.88
0.10
0.35
0.84
1.57
1.37
1.04
0.02
103.05 ± 13.73
93.69 ± 35.72
87.60 ± 2.60
80.43 ± 4.74
60.61 ± 4.55
1.06 ± 1.09
9.50 ± 1.14
9.32 ± 3.25
8.35 ± 0.37
7.55 ± 0.43
5.31 ± 0.48
0.07 ± 0.07
39.53
0.16
0.00
0.00
0.00
0.00
39.49
18.98
0.08
0.33
0.00
0.00
2.11
0.19
0.00
0.00
0.00
0.00
369.08 ± 31.34
117.61 ± 13.26
1.68 ± 1.03
1.37 ± 0.98
0.13 ± 0.21
0.13 ± 0.12
38.61 ± 4.87
15.21 ± 1.21
0.13 ± 0.08
0.14 ± 0.24
0.01 ± 0.01
0.01 ± 0.01
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The maximum of curve M (Fig. 6) provides an estimate
of the total solids concentration value (16.4%) which would
have produced maximum methane volume per unit of raw
substrate. This maximum was obtained with a low methane
yield per unit of COD (155.39 ml CH4/g CODinitial), and it
poses the question of the elucidation of the optimal level of
total solids for the anaerobic digestion of the solid fraction
of pig slurry separated by PAM. An approximation of the
level of total solids for optimal methane production could
be made by assuming that this must provide the maximum
of the product M Æ B. Adjusting M experimental values to a
second degree polynomial model and B to a linear model
(Fig. 6), the total solids level that maximises the M Æ B
product is 10.9%. It is interesting to note here that the correlations used have no physical or biological meaning, but
they are useful in obtaining a value which could be considered as indicative evidence for a first estimation in a further
design stage.
4. Conclusions
The solid–liquid phase separation process applied to pig
slurry is very sensitive to the dose of PAM used as coagulant agent. An increase from 12 to 14 g PAM/kg TS is
capable of almost tripling the total solids content of the
solid fraction, reaching a total solids concentration as high
as 31% w/w.
The use of a PAM concentration higher than 12 g/kg TS
is not recommended for further anaerobic treatment, since
symptoms of inhibition of the hydrolysis step, probably
due to the strong colloidal aggregation, and of inhibition
of the methanogenic step by free ammonia nitrogen, were
observed in the most concentrated treatments.
Anaerobic toxicity by PAM, or by its degradation products, was not observed for concentrations lower than 415 g
PAM/kg TS.
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