Appl Microbiol Biotechnol (2019) 103:8267–8279
All the statistical analyses were performed using the SAS
package procedures (Littell et al. 1996). Fitting of the
Gompertz model to measurements was performed using the
PROC NLIN. The parameter values were estimated according
to the Gauss-Newton method.
The specific methane yield was equal to Mmax g−1 VS in the
reactor. The theoretical Mmax of the WCO + PS treatment was
calculated as the sum of the Mmax of the individual substrates
(WCO + HM and PS). A t test was used to compare the
theoretical and actual Mmax of the WCO + PS treatment.
Results
Initial conditions
The pig slurry used in this experiment had a low concentration
of volatile solids (Table 1), due to the pig housing and manure
management system (without straw litter, and with high wash-
ing water volumes) and to the effluent storage practices (ani-
mal effluents in open tanks are diluted by rainwater), which
are common in the farms of our region. For this reason, the pig
slurry contribution to the volatile solid content, in the reactors
including pig slurry, was limited (Table 2).
The C/N value of the treatments was heavily affected by
the inoculum, which lowered the ratio with its N contribution.
Otherwise, it would have been much higher, because the N
content of fats is very low. The C/N value of WCO + PS was
lower than that of WCO + HM, due to the contribution of
nitrogen by pig slurry.
In the WCO + HM reactors, the organic nitrogen (0.012 g)
was supplied only by the inoculum, the hydration medium
contributing exclusively to the NH4-N content (Table 1). In
the WCO + PS reactors, pig slurry increased by 27% the NH4-
N amount and nearly doubled the amount of organic nitrogen,
in comparison with WCO + HM. The pig slurry organic ni-
trogen content is represented almost exclusively by undigest-
ed proteins, endogenous enzymes and mucus, and free
aminoacids (Huang et al. 2018; Jensen and Sommer 2013).
The organic nitrogen in the inoculum is mainly cellular (such
as enzymes, structural proteins).
The pig slurry addition increased by 20% (Table 2) the
alkalinity of the reaction mixture, in comparison with WCO
Table 2 Selected nutrient contents in the reactors. WCO waste cooking oil, HM hydration medium, PS pig slurry
Recipe Substrate VS, g Organic C, g TKN, g C/N
WCO + HM 0.500 0.496 0.023 21.0
WCO + PS 0.656 0.576 0.035 16.6
PS 0.156 0.194 0.035 5.6
1
Lipids other than waste cooking oil are those supplied by inoculum and/or pig slurry
8271
+ HM. In WCO + HM the alkalinity was due to the inoculum
contribution and, to a far lesser extent, to the hydration medi-
um. As the inoculum contribution was the same in all treat-
ments and ash content differences between pig slurry and
hydration medium were limited, in the end all the reactors
contained the same amount of mineral salts (Table 2).
Both inoculum and pig slurry contain lipids (Table 1). The
lipid contribution of pig slurry (12.7 mg per reactor, calculated
from data in Table 1), was nearly twice that of the inoculum
(6.6 mg per reactor). Pig slurry lipids are undigested dietary
lipids (Jorgensen et al. 2000); those of the inoculum are cell
components, as the inoculum feedstock had been completely
exhausted in the preliminary degassing phase.
Lipids in waste cooking oil were mainly triglycerides (Fig.
1a). Conversely, lipids in pig slurry included phospholipids,
di- and monoglycerides and free sterols, free fatty acids and
hydrocarbons, whereas triglycerides were almost absent. The
lipid composition of the inoculum, deriving from pig slurry
digestion, was similar to that of pig slurry, differing only for a
lower free fatty acids content. The pig slurry and inoculum
fatty acids differed, in percentage of presence and in compo-
sition, from those prevailing in waste cooking oil, and a large
percentage of them could not be identified (Fig. 2a).
After addition of waste cooking oil to the aqueous phase,
three layers were recognizable, in agreement with Suto et al.
(2006): a top floatable layer (primarily waste cooking oil), a
middle aqueous layer and a bottom layer formed by the settled
inoculum. However, the addition of waste cooking oil to pig
slurry involved the formation of a very stable emulsion. In
visual tests of emulsion stability, in the WCO + HM cylinders
the hydration medium was already separated from the fat
3 min after vortexing, although it became opaque. On the
contrary, in the WCO + PS cylinders, an intermediate zone
with a glomerular appearance formed between the oil and the
aqueous phase after vortexing. The oil took on a milky ap-
pearance. This situation was still stable after 2 h.
Biogas production
The models of CH4 accumulation were different, depend-
ing on the formulation (Fig. 3a). The CH4 accumulation
curve of WCO + HM was of sinusoidal type. The CH4
NH4-N, g Organic N, g Alkalinity Mineral salts Lipids other
(CaCO3, mg L−1) (ashes), g than WCO1, mg
0.011 0.012 79 0.222 6.6
0.014 0.021 95 0.212 19.3
0.014 0.021 95 0.213 19.3
8272 Appl Microbiol Biotechnol (2019) 103:8267–8279

Fig. 1 Feedstock fat composition

(a) and temporal evolution of fat
composition (b) at 9, 14, 21, 28
and 35 days after inoculum for
waste cooking oil alone (−) or in
co-digestion with pig slurry (+).
WCO, waste cooking oil; PS, pig
slurry; Ino, inoculum. PL, phos-
pholipids; DG, MG, FSt: diglyc-
erides, monoglycerides and free
sterols; FFA, free fatty acids; TG,
triglycerides; HY: hydrocarbons

accumulation curve of WCO + PS was almost linear until
the plateau was reached.
In the WCO + PS reactors, the maximum CH4 accumula-
tion rate (8.8 mL day−1, Table 3) was lower than in the WCO +
HM reactors (13.9 mL day−1), and this entailed a longer time
to reach the plateau (68.4 instead of 53.1 days). However, the
lag phase was much shorter (8.43 days) with than without pig
slurry (20.2 days). The highest value of Mmax, that is the max-
imum CH4 yield per reactor, was observed for WCO + PS. In
co-digestion the volatile solids of pig slurry contributed to the
increase of total CH4 production (+ 15.5%), for the same re-
action volume.
Fat degradation
At the start of the incubation period, the oily phase was clearly
recognizable and floating on the surface of the aqueous phase.
A week after the start of incubation the oil was no more de-
tectable. It was replaced by floating irregular whitish granules.
Their appearance was slightly different, depending on the
presence or absence of pig slurry. In the pig slurry-
containing reactors, the granules were smaller, with a tenden-
cy to sediment together with the suspended hydro-soluble
material in the washing phase of granule recovery. On the
contrary, in the absence of pig slurry, they remained floating
during the various washing phases.
The floating fat extract at the time of measurement repre-
sents the residual amount of fat still to be digested. This
amount decreased much more quickly in WCO + PS than in
WCO + HM (Fig. 4). Indeed, it decreased in geometric pro-
gression in WCO + HM, whereas the most suitable model for
WCO + PS was that of exponential decay.
Nine days after the start of incubation, the floating fat was
64% of the initial, in the WCO + HM reactors, whereas it was
only 24% of the initial, in the WCO + PS reactors. When
comparing fat degradation (Fig. 4) and CH4 accumulation
curves (Fig. 3a), the amount of fat recovered at the last granule
collection (35 days after the start of incubation) was close to 0,
whereas CH4 production was 47% of the maximum, in the
WCO + PS reactors, and 35% of the maximum in the WCO

Fig. 2 Percentage composition of

long chain fatty acids in the raw a) b)
materials (a) and in the floating 100% 100%
Others
fat of the reactors (b) with WCO, 90% 90%
with (WCO + PS) or without pig C24:0
slurry (WCO + HM), 35 days 80% C22:080%
after the start of the incubation 70% C20:070%
LCFA Concentration

60% C18:360%
50% C18:250%
40% C18:140%

30% C18:030%

20% C16:1
20%
C16:0
10% 10%
C14:0
0% 0%
Inoculum Pig slurry WCO WCO + HM WCO + PS
Appl Microbiol Biotechnol (2019) 103:8267–8279 8273

Fig. 3 Curves of CH4 production 600

during incubation of WCO + HM a)
(white dots, dashed line) and
WCO + PS (black dots, 500

Cumulative CH4(STP), mL
continuous line). a Cumulative
CH4 production, measured (dots) 400
and estimated (lines) values. b
Mean measured daily CH4 pro-
duction rate. Vertical bars are the 300
standard deviations of the
measurements
200

100

0
0 15 30 45 60 75 90
Days of incubation

35
b)
30
CH4(STP), mL d-1

25

20

15

10

0
0 15 30 45 60 75 90
Days of incubation

+ HM reactors. The maximum daily CH4 production for both Composition changes accompanying the decrease in fat
treatments was only detected 42 days after the start of the amount differed depending on the treatment (Fig. 1b). In
incubation (Fig. 3b), that is when all the floating fat had the WCO + PS reactors triglycerides in the floating fat had
completely disappeared. already disappeared at the first measurement date, a week

Table 3 Parameters of the CH4 accumulation curves and model evaluation. WCO waste cooking oil, HM hydration medium, PS pig slurry

Treatment Curve parameter estimates Specific CH4 yield1 Final pH1

Lambda Rmax Mmax Time to Mmax F value Number of

observations
days mL CH4(STP) day−1 mL CH4(STP) days mL CH4(STP) g−1 VS

WCO + HM 20.2 13.9 457 53.1 1162*** 39 922 (17.9) 7.12 (0.13)
WCO + PS 8.43 8.80 528 68.4 4136*** 42 811 (26.5) 7.25 (0.05)
PS 0.61 6.18 52.0 9.0 2187*** 18 333 (12.5) 7.30 (0.03)
1
In parentheses, standard deviation
***Highly significant (P < 0.001)
8274 Appl Microbiol Biotechnol (2019) 103:8267–8279

0.6 days after the start of the incubation, when there were very few
0.5
granules left to degrade, the residual fat contained mainly
palmitic acid (C16:0; Fig. 2b). The percentage of palmitic acid
Extracted fat, g

0.4 was 68% higher in the WCO + PS treatment than in WCO +

0.3
HM. In the WCO + PS reactors, the oleic acid percentage
y1 = 0.0004x2 - 0.0275x + 0.509
R² = 0.9955
decreased over time (Fig. 5a) and the palmitic increased
0.2 (Fig. 5b) faster than in those containing WCO + HM.
y2 = 0.5e-0.139x
0.1 R² = 0.9463

0
0 10 20 30
Days of incubation
Fig. 4 Amount of floating fat during the first 35 days of incubation of
WCO + HM (white dots, dashed line) and WCO + PS (black dots,
continuous line). Dots are the observed points, lines are the predicted
points. y1: amount of fat recovered from the WCO + HM reactors; y2:
amount of fat recovered from the WCO + PS reactors. Vertical bars are the
standard deviations of the measurements
after the start of incubation, leaving a place to free fatty
acids. On the contrary, in the WCO + HM reactors, triglyc-
erides were still detectable in the floating fat granules col-
lected 3 weeks after the start of the incubation. They were
no longer detectable after 4 weeks. Triglyceride degrada-
tion was accompanied in both treatments by an increase in
the amounts of phospholipids, diglycerides, monoglycer-
ides and free sterols, and hydrocarbons, already present
in the pig slurry feedstock and in the inoculum.
Fatty acid composition
Fatty acids determined by gas chromatography in the floating fat
include both those already free in the reaction mixture and those
released by methylation of the triglycerides still not degraded at
the time of measurement. It should be kept in mind that these are
percentages of presence, and not absolute values. In fact, the
total amount of fat decreased over time (Fig. 4).
Oleic acid (C20:1) and, to a lesser extent, linoleic acid
(C20:2), had the highest percentage of presence in the feed-
stock waste cooking oil (Fig. 2a). At the last sampling time, 35
40 Discussion
Anaerobic digestion
A few data are available in the literature concerning the anaer-
obic digestion of fats alone. Meng et al. (2015) tested the CH4
production potential of floatable oil skimmed from food waste,
in stirred conditions, and they obtained up to 900 mL CH4 mL−1
of fat. In their experiment, the start of the digestion was greatly
delayed and the CH4 production rate much lower than those
estimated for food waste in mono-digestion. A long lag phase
and a slow CH4 production rate characterized the anaerobic
digestion of waste cooking oil also in our experiment.
However, co-digestion of fat and pig slurry reduced the initial
adaptation phase, as already observed for swine manure in co-
digestion with waste cooking oil sludge (Fierro et al. 2014).
Even though the positive difference in Mmax (+ 71 mL
CH4) observed for oil in co-digestion with pig slurry in com-
parison with oil in hydration medium was higher than the
Mmax value of pig slurry alone (52 mL CH4), in the end, the
overall methane production in co-digestion (Mmax = 528 mL
CH4(STP)) was not significantly different from the theoretical
(Mmax = 509 mL CH4(STP)), calculated as the sum of the indi-
vidual contributions of the recipe ingredients.
The specific methane yield for pig slurry (333 mL CH4(STP)
g−1 VS) was expected for this type of material (Møller et al.
2004; Burton and Turner 2003). The actual specific methane
yield for WCO + HM (922 CH4(STP) g−1 VS) was close to that
calculated using the Buswell formula based on elemental
composition (Morales-Polo et al. 2018). The specific methane

Fig. 5 Change in the percentage 100 100

of long-chain fatty acids over time a) b)
Palmitic acid, %

in the floating fat removed from 80 80

Oleic acid, %

the reactors containing waste

cooking oil with (dark bars) or 60 60
without (bright bars) pig slurry. a
Oleic acid percentage. b Palmitic 40 40
acid percentage. Values at time 0
are for the waste cooking oil 20 20
feedstock. Vertical bars are the
standard deviations 0 0
0 11 14 20 26 34 0 11 14 20 26 34
Days of incubation Days of incubation
Appl Microbiol Biotechnol (2019) 103:8267–8279
yield in co-digestion (WCO + PS) was 811 mL CH4(STP) g−1
VS. Sunada et al. (2018) obtained the maximum yield of
291.4 L biogas kg−1 VS, by adding 64 g of waste cooking
oil per kilogramme of swine manure volatile solids. Fierro
et al. (2014) studied the anaerobic co-digestion of swine ma-
nure with the sludge from a plant using waste cooking oil as
feedstock for biodiesel production. This sludge contained a
39% aqueous phase, and its fat content was only 18.1% of
the total. Conversely, swine manure had a higher total solids
content than the pig slurry used in our experiment. They ob-
tained 540 mL CH4 g−1 VS for the anaerobic digestion of used
cooking oil sludge alone, and 225–325 mL CH4 g−1 VS, de-
pending on the amount of added waste oily sludge (from 1 to
10%), in co-digestion with pig manure. Maximum CH4 pro-
ductions between 339 and 681 mL CH4 g−1 VS were reported
for co-digestion of fat, oil and grease with sewage sludge
(Long et al. 2012). In co-digestion experiments, the specific
methane yield of waste oil digested with pig slurry depends on
the quantities in co-digestion. It is expected to be lower than
that of oil without pig slurry, because the high-yielding vola-
tile solids of fat are “diluted” with those of pig slurry, which
are less energy-yielding. In general, differences in specific
methane yield among recipes can be traced back to differences
in organic carbon amount and quality per gramme of volatile
solids, as methane yields are influenced by both carbon avail-
ability and suitability to anaerobic digestion, as well as by the
possible presence of inhibiting factors.
Waste cooking oil contains very little amounts of nitro-
gen, which can be found mainly in sphingolipids (Salas
et al. 2011). A lowering of the C to N ratio was expected,
due to pig slurry addition. However, differences between
treatments were not so large because also the inoculum
lowered the C/N, in both treatments. The resulting values
were close to those reported as suitable for methanogens
(25–30; Paritosh et al. 2017). It remains to be ascertained
what could happen in case of different pig slurry to fat
volume ratios, with consequent changes in the C to N
ratio and ammonium amounts.
The ammonium content in the pig slurry-containing treat-
ments was of the same level as that in the hydration medium,
that is suitable for methanogenic activity. However, an overall
higher nitrogen availability could have contributed to the im-
provement of the digestion performances, as it will be further
discussed below.
The buffering effect of pig slurry is linked to the presence
of CO2 in equilibrium with carbonic acid in solution (alkalin-
ity), total ammonium nitrogen and VFA (Japenga and
Harmsen 1990). In our experiment, the presence of pig slurry
increased the total alkalinity of the medium (Table 2). The
final pH was however neutral in both treatments, with and
without pig slurry, indicating the absence of pH unbalances.
Therefore, the reasons for the positive effect of pig slurry must
be sought elsewhere.
8275
Static conditions and high oil to water ratios were adopted
for our biphasic system, based on previous experiences
(Fiume et al. 2017). These process conditions may have con-
tributed to the results, on the one hand, by lengthening the
digestion time, due to a lower probability of contact between
the settling microorganisms and the floating substrate, and, on
the other hand, by improving the overall digestion perfor-
mances. Indeed, good specific methane yields were obtained,
and it has been shown that the reduction in mixing intensity
improves the performances of anaerobic digestion (Lindmark
et al. 2014). However, mixing is needed in the digesters to
disrupt the crusts. Our process conditions are therefore not
transferable to full-scale applications, due both to the exces-
sively long times required for the completion of the process,
and because the static conditions lead to the formation of
surface crusts, which complicate the management of the di-
gester (Lienen et al. 2013; Long et al. 2012). They are how-
ever useful to better understand the system behaviour in a
simplified context.
Fat degradation
The time taken by the floating granules to disappear was an
indicator of the fat degradation level, because they were de-
tectable shortly after the inoculation, and their disappearing
was accompanied by that of triglycerides (Fig. 1). The earlier
and temporary formation of floating granules has already been
observed in the anaerobic digestion of oil-in-water biphasic
systems. Rinzema et al. (1993) detected their formation in a
study for the development of continuous high-rate anaerobic
systems suitable for the digestion of lipids. Pitk et al. (2014),
studying the anaerobic digestion of dairy manure and slaugh-
terhouse waste, which usually contains high percentages of
fats, identified these granules as agglomerates of long-chain
fatty acid calcium salts.
In our experiment the fat composition changed over
time, as consequence of fat degradation, and the anaerobic
digestion performances were clearly related to these chang-
es. Faster degradation of triglycerides was observed in co-
digestion with than without pig slurry. In the presence of
pig slurry, methanogenesis started about a week after the
start of incubation (precisely after 8.43 days; Table 3),
whereas in the reactors with WCO + HM, it started nearly
3 weeks after the start of the incubation (20.2 days;
Table 3). In both conditions, methanogenesis started only
when all the triglycerides had been hydrolyzed (Fig. 1b). In
other words the lag phase duration was affected more by
the time needed for triglycerides to be hydrolyzed than by
long-chain fatty acid accumulation, the latter being the re-
sult of triglycerides hydrolysis. Consequently, triglyceride
hydrolysis appears to be the first bottleneck for fat anaer-
obic digestion. Affes et al. (2017) suggested the addition of
8276
lipase to overcome this drawback. A microbial adaptation
is also needed, as pointed out by Amha et al. (2017).
Whereas the lag phase duration was controlled by the rate
of triglycerides hydrolysis, the methane production rate was
influenced by the hydrolysis products. The maximum meth-
ane production rate was lower (lower Rmax values, Table 3) in
the WCO + PS treatment, possibly due to the negative effect
of earlier accumulation of free fatty acids (Fig. 1b), especially
of palmitic acid (C16:0), the most represented fatty acid in the
granules before their complete disappearance (Fig. 4b). The
accumulation of palmitic acid as intermediate in fat digestion
has been reported also by other authors, who observed its
reversible inhibiting effect on microorganisms (Dasa et al.
2016; Fernández et al. 2005; Pereira et al. 2002; Soni 2018).
This effect was reversible also in our experiment, as shown by
the fact that palmitic acid completely disappeared, and the
specific methane yield in WCO + PS was not significantly
different from the theoretical. The negative effect of palmitic
acid can be either physical or biological. It has been demon-
strated that palmitic acid forms a physical barrier around mi-
crobial aggregates, which hinders the transfer of substrates
and products (Pereira et al. 2005). Moreover, syntrophic
acetogens that pave the way for methanogens are sensitive
to high long-chain fatty acid concentrations more than
methanogens (Ma et al. 2015). Therefore, palmitic acid ap-
pears as the second bottleneck in waste cooking oil digestion,
because it tends to accumulate and to be digested after the
other fatty acids. The accumulation of long-chain fatty acids
can become a problem when their amounts are too high to be
endured by microorganisms (Cirne et al. 2007; Hanaki et al.
1981; Hwu et al. 1996).
Influence of pig slurry composition on fat
degradation
In our experiment, an oil-in-water emulsion formed when pig
slurry was added to waste cooking oil. Emulsions are mixtures
of two or more liquids that are normally immiscible. Emulsion
phases tend to separate unless emulsifiers are added, which
can reduce droplet size and increase emulsion stability. Finer
and more stable emulsions increase the surface-to-volume ra-
tio of the oil droplet, and the probability for microorganisms in
the aqueous phase to meet the oily substrate. Several emulsi-
fiers are available (EFEMA 2015), a most important category
being that of soaps, which are Na or K salts of long-chain fatty
acids. Saponification has been proposed to improve anaerobic
digestion of fat-containing substrates (Affes et al. 2013). Soap
may have formed in our experiment due to the alkalinity of pig
slurry, able to increase the emulsion stability in the biphasic
system. Alkalinity was found to positively influence anaerobic
digestion in model systems containing waste cooking oil
(Fiume et al. 2017).
Appl Microbiol Biotechnol (2019) 103:8267–8279
Proteins also have emulsifying properties (Cabra et al.
2008; Hoffmann and Reger 2014). In the reactors containing
pig slurry the organic N, representative of the protein-N con-
tent, was nearly twice that in the reactors without pig slurry
(Table 2), and this suggests an effect on the emulsion stability.
Moreover, Shon et al. (2002) reported that triglycerides deg-
radation by lipolytic bacteria is faster when proteins are added
to the medium, whereas ammonium nitrogen does not exert
the same effect. Inoculum proteins, besides being equally
present in all the reactors, were not expected to have any
effect, due to their different nature (cellular instead of
extracellular).
Phospholipids and mono- and diglycerides of fatty acids
are also well-known emulsifiers (Faia 2012), and they are
commonly used in food and cosmetic formulations to favour
the mixing of the fat with the other hydrophilic components.
In our reactors the main contribution to the presence of phos-
pholipids and of diglycerides, monoglycerides and free sterols
was from pig slurry and inoculum, whereas these lipid cate-
gories were present in waste cooking oil in very small
amounts (Fig. 1a). As the amount of fat deposited in each spot
of the TLC was the same, it is evident that pig slurry phos-
pholipids were present in a percentage close to that of the
inoculum. However, the amount of fats supplied by pig slurry
in the WCO + PS reactors was twice that of the inoculum. It
follows that the amounts of phospholipids and of diglycerides,
monoglycerides and free sterols in the pig slurry-containing
reactors was three-fold that in the reactors of the treatment
without pig slurry (Table 2).
Lipases are known to accelerate the anaerobic digestion
of lipid-based substrates (Cirne et al. 2007). Lipolytic en-
zymes were detected in the intestinal tract of pigs (Corring
1982), and a probability exists for their passage to excreta
(Jensen and Sommer 2013). Strains of Clostridium and
Lactobacillus have been identified in the pig slurry micro-
biota (Peu et al. 2006), which can survive during pig slurry
storage and are also common representatives of the anaer-
obic biogas-producing consortia. These genera include li-
polytic strains (Cirne et al. 2006; Katz et al. 2002; Willis
1960). It is known that co-digestion affects bacterial diver-
sity in the reactors (Mata-Alvarez et al. 2014), and this is
particularly true in case of pig slurry as feedstock, which
can contribute with its own rich microflora.
In conclusion, in our laboratory experiment, pig slurry was
particularly suitable as an ingredient in co-digestion with
waste cooking oil, and its composition and physico-chemical
effects on the oil-in-water system seem to play a role. Several
pig slurry components: alkalinity, proteins, phospholipids,
have possibly favoured the emulsion of fats in the aqueous
phase, favouring the access of microorganisms to the sub-
strate. Pig slurry could also make available supplemental mi-
croorganisms and/or lipases for triglyceride hydrolysis, in ad-
dition to those supplied by the inoculum.
Appl Microbiol Biotechnol (2019) 103:8267–8279
In this experiment, a single combination of fat and pig
slurry volumes was tested. Changes in the amount of both
could imply changes in the C to N ratio and in nitrogen con-
tribution, possibly affecting the co-digestion performances.
Equally, higher contents of volatile solids in pig slurry could
affect the results. This issue clearly deserves further research.
Changes in microbial composition during anaerobic diges-
tion were not monitored, and we cannot say if the observed
effects were directly related either to the type and physico-
chemical composition of the feedstock or to the microorgan-
isms involved. This aspect also deserves further investigation,
as well as the understanding of why pig slurry accelerates
oleic acid degradation and how to remove the inhibiting effect
of palmitic acid.
From a practical point of view, the anaerobic co-digestion
of waste cooking oil with pig slurry is currently subject to law
restraints in Italy because these two ingredients belong to dif-
ferent waste sectors, the former being classified as civil and
the latter as agri-food waste. The use in anaerobic digestion of
civil waste faces more legal restrictions than that of agro-
industrial waste. Despite the possible difficulties in practical
application of co-digestion, due to both regulatory and logistic
constraints, nevertheless, the understanding of the reasons for
better performances in co-digestion can allow an improve-
ment in biogas production from waste cooking oil.
Acknowledgements We gratefully acknowledge the contribution of
Anna Orsi to laboratory activities.
Funding information Funding for this research was provided by
Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria
(CREA), within the framework of a collaboration between CREA and
DICAM, of the University of Bologna.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
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