Renewable Energy 171 (2021) 958e970

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Renewable Energy
journal homepage: www.elsevier.com/locate/renene

Granular activated carbon supplementation enhances anaerobic

digestion of lipid-rich wastewaters
Lea Chua Tan a, c, *, Richen Lin b, c, Jerry D. Murphy b, c, Piet N.L. Lens a, c
a
Microbiology Department, School of Natural Sciences, National University of Ireland, Galway, University Road, Galway, Ireland
b
Environmental Research Institute, School of Engineering, University College Cork, Cork, Ireland
c
SFI MaREI Centre for Climate, Energy and Marine, Ireland

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

Article history: This study investigated the application of a conductive material, granular activated carbon (GAC), as an
Received 5 July 2020 approach to improve anaerobic lipid degradation and methane production. Anaerobic biomethane po-
Received in revised form tential (BMP) assays were performed in 120 ml batch anaerobic digestion (AD) vials using 5 gVS/L
15 January 2021
anaerobic sludge as inoculum. Different BMP assays were carried out testing the impact of increasing
Accepted 15 February 2021
Available online 19 February 2021
GAC concentrations (0e33 g/L), use of different sludge types (granular vs. crushed), different substrates
(oleate C18:1, butter and dairy wastewaters) and different temperatures (15, 37 and 55
C). Experimental
results and model fitting showed that addition of GAC supports faster methane production, i.e. the lag-
Keywords:
Activated carbon
phase decreased by 2e1000% depending on the GAC concentration and AD temperature. GAC addition
Long-chain fatty acid also showed faster consumption of both volatile fatty acid and long-chain fatty acid, particularly
Lipid wastewater palmitate (C16:0). Thermodynamic modelling suggested that GAC-induced direct interspecies electron
Direct interspecies electron transfer transfer is kinetically superior to conventional indirect hydrogen transfer during AD of oleate. However,
Methane potential assay when the GAC concentration exceeded 8.0 g/L, there was a 20e50% decrease in the maximum methane
production compared to the control. Overall, GAC supplementation has a significant potential to improve
the digestion of lipid-rich wastewater which benefits design of modern bioenergy systems.
© 2021 Published by Elsevier Ltd.

1. Introduction equaled to 8 billion litres of liquid milk, 251,000 tonnes of butter

To move towards a climate neutral energy future, production of
bioenergy (such as methane) from organic solid waste and waste-
water plays a significant role in providing renewable energy [1].
Thus, fugitive methane emissions from wastes, particularly animal
manures, are mitigated whilst simultaneously producing renew-
able gaseous fuel and biofertilisers [2]. The International Energy
Agency (IEA) has estimated that full utilisation of the biogas and
methane potential may cover 20% of today’s gas demand world-
wide [1]. The availability of sustainable feedstock and its conver-
sion technologies are key parameters that need to be considered to
achieve such an ambitious goal. In the European context, dairy
farming and product processing is one of the largest industries [2].
For example in Ireland alone, there are over 18,000 dairy farmers
producing a range of dairy products where production during 2019
* Corresponding author. Microbiology Department, School of Natural Sciences,
National University of Ireland, Galway, University Road, Galway, Ireland.
E-mail address: lea.tan@nuigalway.ie (L.C. Tan).
and 142,500 tonnes of skin milk powder [3]. As a result, huge
amounts of lipid-rich wastewaters are generated, with a fluctuating
lipid concentration range of 20 mg/L to 25 g/L, which require
treatment in a wastewater facility [4].
Anaerobic digestion (AD) provides a green alternative in treating
waste streams by simultaneously degrading organic waste and
generating energy through biogas production [4]. However, lipids
deteriorate the AD operation, by sludge washout or inhibition of
biological activity. Therefore, most AD plants separate lipids from
the waste streams via dissolved air flotation (DAF) processes
upfront of the anaerobic reactor. DAF processes separate lipids by
injecting fine air bubbles with addition of coagulant/flocculant
chemicals resulting in floatation of lipids, which can then be
skimmed off from the wastewater surface for disposal. This skim-
med fat-rich substance is called DAF sludge. Disposing of DAF
sludge in landfills or by land spreading is a linear economy
approach and represents a loss of resources, i.e. the energy content
of the lipids [5,6]. The recent European Union (EU) action plan
promotes development of a circular bioeconomy, setting clear tar-
gets for sustainable waste reduction and management [7].

https://doi.org/10.1016/j.renene.2021.02.087
0960-1481/© 2021 Published by Elsevier Ltd.
L.C. Tan, R. Lin, J.D. Murphy et al. Renewable Energy 171 (2021) 958e970

Nomenclature DG0f Standard free energy of formation for substrates or

products
LCFA Long-fatty acids
Abbreviations n Mole electron per reaction
AC Activated carbon N Mole electrons carried per hydrogen molecule
[Acetate] Acetate concentration [Ole] Oleate concentration
AD Anaerobic digestion pCH4 Methane partial pressure
ANOVA Analysis of variance pCO2 Carbon dioxide partial pressure
BMP Biomethane potential Pm Maximum methane potential yield
CM Conductive material p-value Probability value
COD Chemical oxygen demand GAC Granular activated carbon
d Distance between microbial cells R Ideal gas constant
DAF Dissolve air flotation Rm Peak methane production rate
Df Diffusion constant of hydrogen in water RSME Root mean square error
DIET Direct interspecies electron transfer Scell Surface area of the cells
Eq Equation Sconduit Cross sectional area of the electron conduit
EU European Union SMA Specific methanogenic activity
F Faraday’s constant T Temperature
GAC Granular activated carbon Tm Peak time of methane production
[H2] Hydrogen concentration TS Total solid
i_DIET Maximum electron transfer flux VFA Volatile fatty acids
i_IHT Maximum indirect hydrogen transfer flux VS Volatile solid
IHT Indirect hydrogen transfer
IEA International Energy Agency Greek letters
GC Gas chromatograph L Lag-phase time
DG00 Standard Gibbs free energy changes S Electrical conductivity of granular activated carbon

When compared to carbohydrates and proteins, lipids can be
considered ideal substrates for the AD process since more biogas
can theoretically be produced from lipid degradation: 0.95 L CH4/g
volatile solid (VS) as compared to 0.57 L CH4/gVS for protein and
0.35 L CH4/gVS for carbohydrates at standard conditions [8]. Indeed,
a higher net energy production was reported in many AD plants
when lipid fractions are combined in the waste stream [4]. How-
ever, without proper strategy, the intermediate long-chain fatty
acids (LCFA) formed during lipid degradation can accumulate over
time resulting in process operational failure [9]. The LCFA inhibition
mechanism on AD processes has been mainly linked to the LCFA
layer coating the microbial cell surface which can lead to limiting
substrate to product contact, to biogas entrapment within the
anaerobic sludge, inhibition of acetoclastic and hydrogenotrophic
methanogens as well as sludge flotation and washout [10].
Different strategies to tackle LCFA degradation in AD have been
investigated, including feeding modes [6,11,12], pre-treatment
methods [13] and adjusted reactor configurations [2,14,15]. These
methods either require very low and/or intermittent organic
loading rates, high chemical and energy costs as well as modifica-
tion of existing AD plants to accommodate changes in reactor
configuration. Addition of conductive materials (CM) to the AD of
lipid-rich wastewaters has thus far received little attention [16e18].
Previous studies have demonstrated that CM are capable of
improving biogas production in AD through the promotion of direct
interspecies electron transfer (DIET), which is a more efficient way
to exchange electrons among microorganisms than indirect
hydrogen transfer (IHT) [19,20]. However, most studies have been
limited to using pure cultures and/or focusing on single soluble
substrates that are typically rich in carbohydrates and easily
degraded as compared to lipids [19]. The latter are semi-soluble
with low solubility in water and are thus harder to degrade. This
study, therefore, conducted a series of biomethane potential (BMP)
assays to investigate the use of granular activated carbon (GAC) for
the degradation of lipid-rich wastewaters under different
959
experimental conditions. BMP assays performed ranged from
testing the impact of GAC dosage, changing the sludge type, using
real lipid-rich wastewater and incubating at different temperature.
Theoretical electron transfer flux calculations along with kinetic
modeling of methane yields and the adsorption capacity of GAC
were also assessed to provide supporting information to elucidate
the mechanism of enhanced AD of lipids by GAC supplementation.
2. Materials and methods
2.1. Conductive material, inoculum and substrates
The GAC (granular activated carbon, Alfa Aesar™ Carbon, Norit
ROW) was purchased from Fisher Scientific (Dublin, Ireland). The
GAC was rod shaped with an average diameter and length of
0.8 mm and 3 mm, respectively. Prior to use, GAC was soaked in
demi water overnight to remove any particulate carbon attached to
the GAC and subsequently air dried.
The inoculum used for this study was anaerobic granular sludge
collected from a full-scale AD plant treating dairy wastewater at
ambient/psychrophilic temperature (Kilconnell, Galway, Ireland).
Granular sludge has been previously reported to be effective for
LCFA degradation in non-CM supplemented systems [15,21].
However, in order to allow for better contact between the GAC and
the sludge, the granular sludge was crushed using a mortar and
pestle. Crushed sludge was used for all experimental conditions,
except for the sludge configuration experiment. The inoculum had
a total solid (TS) content of 62.5 gTS/kg wet granular sludge. The VS/
TS ratio was 89%, yielding a concentration of 55.6 gVS/kg wet
granular sludge. The inoculum for the BMP was provided at a
concentration of 5 gVS/L.
Both synthetic and real lipid-rich wastewaters were used as the
substrates. The synthetic wastewater was composed of oleate (so-
dium oleate, 82% purity, Sigma-Aldrich), mineral medium and a
trace element solution. Mineral media and trace element solution
L.C. Tan, R. Lin, J.D. Murphy et al.
were prepared according to the composition reported by Stams
et al. [22]. In order to be within the range of a real industrial
wastewater in terms of chemical oxygen demand (COD) concen-
tration [23], oleate was provided at 1.0 g/L, which corresponds to
approximately 3.3 (±0.2) gCOD/L. Butter and dairy wastewater was
used as the real lipid-rich wastewaters. Butter wastewater was
taken from the butter processing facility of a dairy manufacturing
plant (Mitchelstown, Cork, Ireland), while the dairy wastewater
was taken from the same location as the inoculum (Kilconnell,
Galway, Ireland). The COD concentration of the butter wastewater
was 4.5 (±0.1) g/L with a TS concentration of 3.5 (±0.2) g/L and a
total lipid content of 2.4 (±0.8) g/L. The COD concentration of the
dairy wastewater was 5.0 (±0.7) g/L with a TS concentration of 4.3
(±0.1) g/L and a total lipid content of 4.1 (±0.8) g/L. When not in use,
inoculum and real wastewaters were kept at 4
C. Images of the
sludge and GAC used are shown in supplementary information
Figure S1.
2.2. Biomethane potential (BMP) assays
Biomethane potential (BMP) assays for different experimental
conditions were evaluated with and without GAC supplementation
as shown in Table 1. The sampling of the BMP assay was carried out
in two ways: non-sacrificial and sacrificial. In the non-sacrificial
assays, liquid and gas samples were taken at different sampling
periods. In the sacrificial assays, an entire bottle was taken down at
a certain sampling point to analyze both insoluble and soluble
components. The sacrificial assays were employed to quantify all of
the LCFA (C10 to C18:1) content since not all LCFA are entirely
soluble during the digestion, i.e. palmitate is insoluble in water
Initial COD in solution  Final COD in solution
Adsorption percentage ¼
Initial COD in solution
with a low solubility of 7.2 mg/L [24]. Each condition was carried
out in triplicate. The negative control used was inoculum alone; the
accumulated methane was subtracted from the tested conditions.
Non-sacrificial BMP assays were performed in 120 ml bottles
with a working volume of 70 ml. Pressure reading was done before
gas and liquid samples were taken. Gas samples were taken using a
10 ml syringe with a valve to prevent leakage and were analyzed for
methane content the same day. Liquid samples (1 ml) were taken
each time for volatile fatty acid (VFA) analysis. After each sampling,
bottles were vented to allow for atmospheric pressure in the
bottles.
Sacrificial BMP assays were performed using 60 ml bottles with
a working volume of 30 ml. At each sampling interval, 3 bottles for
each condition were taken down and the entire content was
analyzed for LCFA, VFA, biogas volume and composition. Pressure
Table 1
Experimental conditions tested in this study.
BMP assay Experiment GAC concentration
(g/L)
(A) GAC dosagea 0.0/0.5/2.0/4.0/8.0/16.0/25.0/33.0
(B) Sludge type 0.0 and 2.0
(C) Real wastewater 0.0 and 2.0
(D) Incubation temperature 0.0 and 2.0
a
Concentration of GAC applied was based on previous studies where a reported concentration used were in the ranged of 3.3e50.0 g/L [19].
Renewable Energy 171 (2021) 958e970
reading was recorded first before the gas sample was taken for
methane content analysis. Only 1 ml of liquid sample was taken for
VFA analysis and the rest was freeze dried to collect the solids for
LCFA extraction and quantification.
BMP assays at different temperatures were incubated at
different temperatures: psychrophilic AD (15
C), mesophilic AD
(37
C) and thermophilic AD (55
C). Bottles were purged with
nitrogen gas to ensure anaerobic conditions. Initial pH of the so-
lution was not further adjusted and was 7.2 (±0.2). Initial COD
values were measured for each bottle and methane production
were converted to COD equivalent (1.0 g CH4 ¼ 4.0 g CH4-
COD ¼ 3.0, 3.2, and 3.4 L CH4-COD at 15, 37 and 55
C, respectively).
For 1 g CH4-COD, the theoretical methane yield is 350 mL at stan-
dard conditions (0
C and 1 atm).
2.3. Adsorption capacity test
Apart from being conductive, GAC is an adsorbent [25], thus the
adsorption capacity of GAC at different concentrations, substrates
and temperatures was determined. The adsorption test evaluated
the decreased amount of COD in the liquid fraction after 7 days of
incubation. Adsorption tests were done in 120 ml bottles with a
working volume of 70 ml. Bottles were made anaerobic by flushing
with nitrogen gas for 5 min, incubated at 37
C (except for the
temperature test) and mixed at 120 rpm. The adsorption test that
contained only the GAC plus the substrate under different tem-
peratures was conducted to determine the potential amount of COD
that the GAC can adsorb at certain substrate loading and temper-
ature conditions. The adsorption percentage was calculated as
shown in Eq. (1).
(1)
2.4. Analytical methods
COD, TS and VS of substrate and inoculum and total lipid content
were measured following standard methods [26]. Biogas pressure
was measured using a pressure transducer (0e4 bar range abs
Mano LEO1, Keller Druckmesstechnik, Sweden) and the methane
volume was calculated using the ideal gas formula taking note of
the headspace of the bottles at each sampling point. The methane
content was measured using a gas chromatograph (CP-3800 GC,
Varian) with a flame ionization detector [23]. Nitrogen was used as
gas carrier at a flow rate of 25 ml/min.
Liquid samples for VFA analysis (C2 to C7) were prepared by
adding a known amount and concentration of internal standard
(ethyl butyric acid in 10% phosphoric acid) and filtering with a
0.20 mm filter syringe (Chromafil Xtra Syringe Filters, PET-20/25)
Sludge type Substrate Temperature Sampling method Feed to biomass ratio
(
C) (F:M)
Crushed Oleate 37 Non-sacrificial 0.66
Crushed & granular Oleate 37 Non- sacrificial 0.66
Crushed Butter & dairy 37 Non- sacrificial 0.90 & 1.0
Crushed Oleate 15, 37 & 55 Sacrificial 0.66
960
L.C. Tan, R. Lin, J.D. Murphy et al.
[27]. For LCFA analysis (C10 to C18), samples were prepared by
lyophilizing at 56
C and 0.050 mbar for 1 week using a freeze
dryer L-200 basic with Edwards nXDS6iC dryscroll vacuum pump
(Buchi, Mason Technology). After lyophilization, LCFA was extracted
and esterified using methanol and hexane following the procedure
described by Guihe
neuf et al. [28] with modification. The detailed
process for the LCFA extraction is described in the supplementary
information.
VFA and LCFA were quantified using a Varian Saturn 2000 GC
equipped with an automatic sampler (CombiPal) and a flame
ionization detector. The GC column used for VFA and LCFA analysis
was BP21 and BPX70, respectively. Both GC columns were FFAP
capillary with 30 m length, 0.25 mm internal diameter and 0.25 mm
filter (SGE analytical science). The carrier gas was helium at a flow
rate of 1 ml/min. The volume injected was set at 1 ml with the
injector temperature set at 250
C and detector set at 300
C. The
VFA GC oven method was set to the following sequence: 1) 10 s hold
at 60
C and ramp up at a rate of 30
C/min to 110
C and hold for
20 s; 2) ramp up from 110
C to 200
C at a rate of 10
C/min and
hold for 2 min [27]. The LCFA GC oven method was set starting at
80
C, hold for 5 min before ramping up to 200
C at a rate of 20
C/
min and hold for 8 min.
2.5. Calculations
2.5.1. Modified gompertz kinetic model
Averaged methane values were fitted using the modified
Gompertz model [29] to simulate the methane production pattern
(Eq. (2)) using the kinetic parameters of Pm (maximum methane
potential yield in mg CH4-COD/L), Rm (peak methane production
rate in mg CH4-COD/L$d), l (lag-phase time in d) and the peak time
of methane production Tm in d (Eq. (3)). The modified Gompertz
model is the most common kinetic model used when the AD pro-
cess has a lag-phase period [29]. Kinetic fitting was done using
OriginPro 2018 (OriginLab Corporation, US) software.

 
Rm *2:7183
P@t ¼ Pm *exp  exp * ðl  tÞ þ 1 (2)
Rm
Pm
Tm ¼ þl (3)
Rm *2:7183
2.5.2. Statistical analysis
Statistical analysis of variance using one-way analysis of vari-
ance (ANOVA) with Tukey and Fisher comparison were performed
in Minitab17 Statistical (Minitab Inc., US) software to evaluate
whether estimated kinetic parameters for each condition are
significantly different from each other. A value of p < 0.05 was
considered statistically significant. An extended report on the sta-
tistical grouping analysis for each experimental condition is shown
in supplementary information Table S1.
2.5.3. Theoretical thermodynamics and interspecies electron
transfer flux
The standard Gibbs free energy changes (DG00 ) of the possible
reactions for oleate degradation were determined based on the
relationship as expressed in Eq. (4):
0
X X
DG0 ¼ DGf 0 ðproductsÞe DGf 0 ðsubstratesÞ (4)
in which DG0f refers to the standard free energy of formation for
substrates or products. Standard free energy of formation of oleic
acid was estimated using the Joback method [30], while standard
961
Renewable Energy 171 (2021) 958e970
free energy of formation of other compounds (including CO2, CH4,
acetate, Hþ, OH and H2O) in the reactions were adopted from
Madigan et al. [31].
Gibbs free energy changes at non-standard conditions were
determined based on Eq. (5).
!
0 00 ½Cc ½Dd
G ¼ G þ 2:3  RT  log (5)
½Aa ½Bb
where DG’ refers to Gibbs free energy change (25
C, pH 7) deter-
mined at non-standard conditions, DG00 is the Gibbs free energy
change determined at standard conditions (solute concentrations
of 1 M, gas partial pressure of 105 Pa and temperature of 25
C) and
pH 7; [A] and [B] are substrate concentration (mol/l); [C] and [D] are
product concentration (mol/l); a and b are stoichiometric coeffi-
cient of substrates; c and d are stoichiometric coefficients of
products; R is ideal gas constant and T is temperature (K).
The calculations of theoretical interspecies electron transfer via
DIET or IHT are based on previously proposed models [7,32]. The
maximum electron transfer via the GAC based DIET was calculated
using the Nernst equation and Ohm’s law as described in Eq. (6):
Sconduit DG0
i DIET ¼ s (6)
d nF
in which i_DIET is the maximum electron transfer flux, s is the
electrical conductivity of the conductive material, Sconduit is the
cross sectional area of electron conduit, d is the distance between
microbial cells, n is mole electron per reaction and F is the Faraday’s
constant, DG0 can be calculated based on the conversion of oleate
oxidation and CO2 reduction.
The maximum electron transfer via IHT was calculated based on
Fick’s diffusion law as shown in Eq. (7):
Scell  
i IHT ¼ Df ½H2 highest  ½H2 lowest NF (7)
d
where i_IHT is the maximum electron transfer flux, Df is the
diffusion constant of hydrogen in water, Scell is the surface area of
the cells, d is the distance between cells, N is mole electrons carried
by hydrogen molecule, F is the Faraday’s constant, [H2]highest is the
highest hydrogen concentration produced by acetogens, and
[H2]lowest is the lowest hydrogen concentration reached by
methanogens. The highest and lowest hydrogen concentration was
calculated according to, respectively, the electron-donating and
electron-consuming reaction.
The following parameters were used to determine the ther-
modynamic values: oleate concentration ([Ole]) of 1.77 mM (or
0.5 g/L), acetate concentration ([Acetate]) of 15.93 mM, CH4 partial
pressure (pCH4) of 0.6 atm, and CO2 partial pressure (pCO2) of
0.4 atm. An interbacterial distance (d) of 0.5 mm was assumed with
cells having a cylindrical shape (assuming diameter ¼ 0.5 mm, and
length ¼ 2.5 mm). The electrical conductivity of GAC (s) was
assumed as 3000 mS/cm [33], and the shape of the GAC conduit was
assumed as a cuboid shape. The diffusion constant of H2 in water at
35
C was used as 5.9  105 cm2/s.
3. Results
3.1. Effect of GAC concentration on oleate methanation
Fig. 1 shows the methane production and rate from oleate as the
substrate at different GAC concentrations. Table 2 gives the corre-
sponding estimated parameters from the modified Gompertz
model.
L.C. Tan, R. Lin, J.D. Murphy et al.
Fig. 1. Methane (a) production and (b) production rate profile using oleate as model substrate with different GAC concentrations (g/L) incubated at 37
C, 120 rpm and for 38 days.
Initial oleate concentration provided was approximately 3276 (±266) mg COD/L. At standard pressure (1 atm) and temperature (0
C), 1 g CH4-COD equals to 350 mL CH4.
Increasing amounts of added GAC had a major impact on
decreasing the lag-phase for methane production. Except for the
GAC concentration at 0.5 and 1.0 g/L, all other conditions showed a
significant decrease (p < 0.05) in estimated lag-phase time shown
in Table 2 from 16.6 d (control) to 9e13 d (2.0e8.0 g/L) and further
down to 2 d (16 g/L to 33.0 g/L). Fig. 2c shows that the relation of
lag-phase time with GAC concentration is decreasing linearly and
becoming steady at a GAC concentration higher than 8.0 g/L.
Similarly, a faster peak of methane production was observed at
different GAC concentrations, achieving the peak about 2e10 days
faster than without GAC (Fig. 1b). The peak methane production
rate, Rm, was significantly different (p < 0.05) with different GAC
concentrations. A linear increasing relationship (Fig. 2b) was
observed from 0.0 to 4.0 g/L GAC resulting in an increase in Rm
values from 138.2 to 410.5 mg CH4-COD/L$d (Table 2, please note
that at standard pressure and temperature 1 g CH4-COD/L equals to
350 mL CH4/L). This is followed by a rapid decrease to 164.9 mg
CH4-COD/L$d at 8.0 g/L GAC and 79.1 mg CH4-COD/L$d at 33.0 g/L
GAC (Table 2).
In contrast to the lag-phase and peak methane production rate
and time, the maximum yield in methane did not show huge dif-
ferences with various GAC concentrations (Table 2). A GAC con-
centration starting at 0.5 g/L to 1.0 g/L and 8.0e16 g/L showed no
significant difference (p > 0.05) from the control in terms of
maximum methane yield, producing approximately 2200 mg CH4-
COD/L or 65% of COD conversion to methane. A GAC concentration
of 2.0 and 4.0 g/L resulted in a 20% higher maximum methane yield
at 2500e2900 mg CH4-COD/L, approximately 85% conversion of
COD to methane. Interestingly, GAC concentrations of 25.0 and
33.0 g/L reduced the maximum methane production by 50%
compared to the control, resulting in 1300e1400 mg CH4-COD/L or
45% COD conversion. The Pm estimated from the kinetic model was
962
Renewable Energy 171 (2021) 958e970
close to the measured values with, on average, 3.2% difference
(Table 1).
3.2. Effect of sludge type (crushed vs. granular)
Following the dosage concentration experiment, a GAC con-
centration of 2.0 g/L was chosen to be used for all other experi-
ments. To test whether the sludge configuration, crushed or
granules, has an effect in the incubation with GAC addition, BMP
assays were carried out. Results for BMP assay results (methane
production, rate and total VFA profile) and kinetic parameters are
shown in Fig. 3 a1 to 3 a3 and Table 2, respectively.
When compared to the control, both sludge types showed that
addition of GAC achieved faster methane generation (Fig. 3 a1) with
a peak production reached 3 days faster (Fig. 3 a2). However, when
comparing between the methane production profiles of crushed
and intact granules with GAC addition, no significant difference was
observed (p > 0.05). The peak methane production rate, Rm, was not
significantly different for all conditions tested, ranging between
185 e 200 and 270e300 mg CH4-COD/L$d for the control and GAC
supplementation, respectively. Peak production was achieved at an
estimated peak time, Tm, of 50e60 d and 43 d for control and GAC
amended incubations, respectively (Table 2). The methane yield
was approximately 2700 mg CH4-COD/L or 75% COD conversion
without GAC for both sludge types. On the other hand, addition of
GAC for both sludge configurations led to a methane yield of
approximately 2950 mg CH4-COD/L or 90% COD conversion. The
calculated maximum methane production, Pm, was < 17% different
with measured data. The lag-phase time for granular and crushed
sludge was estimated at 17.1 and 18.3 d, which is 1.3-fold higher
with GAC (Table 2). Apart from a faster methane yield and
decreased lag-phase time, VFA consumption was also faster in the
L.C. Tan, R. Lin, J.D. Murphy et al. Renewable Energy 171 (2021) 958e970

Table 2
Estimated parameters of methane production from the modified Gompertz model and GAC adsorption capacity (after 7 days of incubation) from different experimental
conditions. Pm, maximum methane potential; Rm, peak methane production rate; l, lag-phase time of methane production; RSME, root-mean-square prediction error; and Tm,
peak time. At standard pressure and temperature, 1 g CH4-COD equals to 350 mL CH4.

Conditions COD conversion to Pmeasured Pm Difference Rm l R2 RSME Tm Adsorption ratio

CH4 (t ¼ 7 d)

% (mg CH4-COD/ (mg CH4-COD/ (%) (mg CH4-COD/L- (d) (mg CH4-COD/ (d) (%)
L) L) d) L)

(a) Different GAC concentration (using oleate)

0.0 g/L GAC 66.3 (±2.0) 2277.3 2446.5 9.3 138.2 16.6 0.9815 94.4 64.7 e
(±176.5)
0.5 g/L GAC 62.7 (±8.9) 2327.7 2364.1 11.2 176.9 16.3 0.9849 90.7 53.1 25.7 (±6.0)
(±351.3)
1.0 g/L GAC 65.9 (±6.4) 2212.4 2177.4 6.1 192.0 15.5 0.9966 42.3 46.4 40.2 (±3.9)
(±232.7)
2.0 g/L GAC 89.6 (±6.1) 2980.7 2894.7 7.8 310.8 13.2 0.9958 65.5 40.5 39.3 (±1.8)
(±185.5)
4.0 g/L GAC 82.2 (±11.0) 2508.4 2469.1 7.5 410.5 12.0 0.9881 97.7 31.7 65.1 (±5.0)
(±278.2)
8.0 g/L GAC 60.9 (±8.7) 2217.4 2166.7 4.4 164.9 9.5 0.9975 37.1 45.3 80.7 (±1.9)
(±273.4)
16.0 g/L GAC 68.6 (±4.8) 2160.0 2167.5 5.6 114.9 2.6 0.9925 59.1 54.0 92.4 (±2.0)
(±153.9)
25.0 g/L GAC 44.1 (±1.4) 1360.2 (±46.7) 1314.2 4.9 72.7 1.6 0.9767 63.8 51.3 90.1 (±2.8)
33.0 g/L GAC 45.3 (±2.3) 1453.0 (±85.2) 1389.6 5.1 79.1 1.6 0.9739 71.8 50.0 92.0 (±2.7)

(b) Sludge configuration (using oleate)

Crushed, 0.0 g/L GAC 75.0 (±12.3) 2712.2 2555.6 8.5 201.0 17.1 0.9759 124.9 52.4 e
(±400.0)
Crushed, 2.0 g/L GAC 91.1 (±1.9) 2898.4 2872.9 5.2 271.0 14.2 0.9953 67.6 43.2 39.3 (±1.8)
(±144.1)
Granules, 0.0 g/L 74.3 (±6.3) 2730.5 3028.9 16.9 184.6 18.3 0.9883 90.5 63.0 e
GAC (±253.6)
Granules, 2.0 g/L 88.0 (±11.4) 3053.8 2991.1 2.4 301.1 15.8 0.9923 88.9 43.0 42.1 (±2.6)
GAC (±359.1)

(c) Different wastewater

Oleate, 0.0 g/L GAC 72.2 (±9.9) 2420.3 2279.1 18.4 186.8 16.4 0.9734 120.5 50.3 e
(±393.9)
Oleate, 2.0 g/L GAC 83.8 (±13.2) 2699.0 2606.6 9.9 204.8 12.6 0.9977 42.1 47.3 39.3 (±1.8)
(±401.3)
Butter, 0.0 g/L GAC 50.5 (±2.4) 2280.7 2639.1 19.4 130.9 17.0 0.9999 9.1 73.5 e
(±105.5)
Butter, 2.0 g/L GAC 62.0 (±3.0) 2799.2 3339.1 15.9 160.6 16.6 0.9989 22.9 71.4 47.5 (±0.4)
(±134.3)
Dairy, 0.0 g/L GAC 40.0 (±6.4) 1992.3 2195.6 11.9 103.0 12.2 0.9962 37.8 72.6 e
(±373.1)
Dairy, 2.0 g/L GAC 51.5 (±8.7) 2589.0 2899.1 15.1 120.6 6.5 0.9623 161.0 70.2 70.9 (±1.8)
(±475.7)

(d) Different temperature (using oleate)

15
C, 0.0 g/L GAC 2.4 (±0.1) 78.6 (±3.7) 14994.4 19046.9 25.7 94.9 0.9956 1.9 670.3 e
15
C, 2.0 g/L GAC 13.8 (±0.3) 249.6 (±10.1) 576.3 131.3 80.1 30.0 0.9956 8.6 90.9 64.7 (±1.4)
37
C, 0.0 g/L GAC 66.5 (±8.1) 2171.4 2119.0 7.0 102.2 12.9 0.9856 101.9 69.6 e
(±230.5)
37
C, 2.0 g/L GAC 67.9 (±9.7) 2217.8 2061.2 10.1 155.2 10.8 0.9877 83.0 47.2 39.3 (±1.8)
(±267.7)
55
C, 0.0 g/L GAC 4.1 (±0.4) 134.1 (±11.5) 814.4 512.3 4.3 25.3 0.8731 11.8 533.7 e
55
C, 2.0 g/L GAC 65.1 (±10.0) 2130.4 1898.2 12.0 124.9 16.4 0.9686 106.5 58.4 86.1 (±0.6)
(±280.6)

presence of GAC (Fig. 3 a3). Total VFA was mainly composed of
acetate, comprising 60e80% of the fraction at the peak concentra-
tion (supplementary information, Figure S2) for all conditions
tested.
3.3. Use of real lipid-rich wastewaters
Compared to butter wastewater, dairy wastewater showed sig-
nificant improvement in the BMP when GAC was added (Fig. 3 b1).
Dairy wastewater with GAC addition showed no lag-phase with an
initial linear increase in the observed sampling time interval (Fig. 3
b1). When fitted to the modified Gompertz kinetic model, an
963
estimated lag-phase time of 6.5 d was calculated, which was
approximately 50% shorter than without GAC addition (Table 2). It
should be noted that the R2 value for the modified Gompertz fitting
was low for dairy wastewater with GAC, which suggests that the
actual lag-phase was shorter or even absent.
Another small decrease in lag-phase time from 17.0 to 16.6 d was
observed for butter wastewater upon GAC addition. However, GAC
addition resulted in a higher maximum methane production of
2799.2 (±134.3) mg CH4-COD/L when compared to the control at
2280.7 (±105.5) mg CH4-COD/L (p < 0.05). A higher COD conversion
to methane was also obtained with GAC addition at 62.0 (±3.0)%
compared to 50.5 (±2.4)% without GAC. Opposite this, the peak
L.C. Tan, R. Lin, J.D. Murphy et al. Renewable Energy 171 (2021) 958e970

Fig. 2. Correlation of GAC dosage concentration with different estimated parameters calculated for (a) Pm: methane yield potential, (b) Rm: peak methane production rate, (c) l:
lag-phase time and (d) Tm: peak time from modified Gompertz kinetic model as well as the (e) COD adsorption percentage. At standard pressure (1 atm) and temperature (0
C), 1 g
CH4-COD equals to 350 mL CH4.

methane production rate and peak time were not significantly
different from each other for both butter and dairy wastewater
(Table 2). The total VFA profiles showed low VFA concentrations in
the bottles with no major differences between wastewater and
with or without GAC addition (Fig. 3 b3).
3.4. Degradation profiles with and without GAC at different
incubation temperatures
Addition of GAC had a positive impact not only on the methane
production (37 and 55
C) but also degradation of fatty acids (all
temperatures investigated). A very low methane production of 78.6
(±3.7) mg CH4-COD/L was observed at 15
C, which increased to
249.6 (±10.1) mg CH4-COD/L when GAC was present in the medium
(Table 2). The palmitate concentration increased over time in the
presence of GAC, while a slower oleate degradation was observed
without GAC. Degradation of oleate was also faster with GAC,
reducing to < 20% of the initial oleate concentration by day 33
(Fig. 4b) compared to the control at day 50 (Fig. 4a). By day 50, a
higher VFA concentration was observed with GAC with low LCFA
concentrations. The lag-phase time of the 15
C incubations was
estimated to be 3-fold faster with GAC compared to without
(Table 2).
When incubated at 37
C, methane production started at day 13

964
L.C. Tan, R. Lin, J.D. Murphy et al. Renewable Energy 171 (2021) 958e970

Fig. 3. Methane production (a1 and b1), methane production rate (a2 and b2) and total VFA production (a3 and b3) profile for experiments comparing different sludge configuration
(a e crushed “cGAC”; granular “gGAC”) and using different wastewater (b e butter wastewater “Butter”; dairy wastewater “Dairy”). Closed shapes represent control conditions,
without GAC addition, while open shapes represent conditions with GAC. Initial COD concentration was provided at approximately 3359 (±309), 4516 (±144) and 5046 (±708) mg
COD/L for oleate, butter and dairy wastewater, respectively. GAC was provided at 2.0 g/L. At standard pressure (1 atm) and temperature (0
C), 1 g CH4-COD equals to 350 mL CH4.

with GAC compared to the control which started at day 17 (Fig. 4c
and d). The lower accumulation and faster generation of VFA also
occurred with GAC amended to the 37
C incubations. The oleate
concentration dropped to < 40% and 20% for control and with GAC,
respectively, by day 3. The palmitate fraction increased to 57.2
(±1.1)% by day 5 and quickly decreased to 13.3 (±3.8)% after 12 days
with GAC supplementation. Opposite to this, control bottles took 10
days longer to produce palmitate and another 10 days to convert to
VFA. About 67% COD conversion was achieved for both conditions at
the end of the 37
C incubation period (Table 2).
When oleate degradation was tested at 55
C, fast oleate
degradation and high VFA accumulation were observed in the
control without GAC (Fig. 4e). The VFA concentration without GAC
was stable at 0.4e0.5 gCOD/gCODinitial starting from day 5 until day
50, with a very low methane yield at 134.1 (±11.5) mg-CH4-COD/L.
Opposite to this, incubations in the presence of GAC showed
already VFA consumption at day 25 (Fig. 4f) and resulted in a final
maximum methane production of 2130.4 (±280.6) mg CH4-COD/L
which is 65.1 (±10.0)% COD conversion to methane. The palmitate
965
fraction was maintained at approximately 0.15 gCOD/gCODinitial for
the entire incubation with GAC (Fig. 4f). A doubling (0.15e0.30
gCOD/gCODinitial) was observed at day 27 before decreasing
(0.30e0.15 gCOD/gCODinitial) at day 43 for the 55
C incubations
without GAC (Fig. 4e). The lag-phase time of 55
C incubations with
GAC addition was estimated to be 16.4 d, 54% faster compared to
the control at 25.3 d (Table 2).
3.5. Adsorption percentage of GAC
The adsorption percentage of total COD (initial COD range of
3e5 g COD/L) from synthetic oleate and real wastewater at GAC
concentrations of 0.5e33.0 g/L was evaluated. The total COD
adsorption percentages ranged from 25.7% to 92.0% (Table 2), with
increasing adsorption percentages for increasing the GAC concen-
trations. Total COD adsorption percentage was > 90% at GAC con-
centration higher than 8 g/L. When added to the wastewaters at
2.0 g/L GAC, the total COD adsorption percentage was 47.5% and
70.9% for butter and dairy, respectively. More adsorption occurred
L.C. Tan, R. Lin, J.D. Murphy et al. Renewable Energy 171 (2021) 958e970

Fig. 4. Degradation by-production profile (g COD product per g COD initial fed) for different temperature incubations at 15
C (a and b), 37
C (c and d) and 55
C (e and f) with and
without GAC addition for 50 days at 120 rpm. Batch experiments were done using the sacrificial assay to monitor the products over time. Dashed line represents 1.0 g COD/g
CODinitial. GAC and initial oleate concentration were, respectively, 3259 (±117) mg COD/L and 2 g/L. At standard pressure (1 atm) and temperature (0
C), 1 g CH4-COD equals to
350 mL CH4. Total VFA are composed of C2 to C6 while LCFAothers were composed of C10 to C14.

Table 3
Reactions involved in oleate biodegradation to methane via direct interspecies electron transfer (DIET) and indirect hydrogen transfer (IHT).

Process Reaction DG00 (kJ/reaction)

 þ
(A) Electron-producing reaction 1. IHT: C18H34O2 þ 16H2O / 9CH3COO þ 9H þ15H2 196.4
2. DIET: C18H34O2 þ 16H2O / 9CH3COO þ 39Hþ þ30 e- 998.5
(B) Electron-consuming reaction 3. IHT: 15H2 þ 15/4 CO2 / 15/4 CH4 þ 15/2 H2O 490.1
4. DIET: 30Hþ þ 30 e þ 15/4 CO2 / 15/4 CH4 þ 15/2 H2O 704.8
(C) Acetate-consuming reaction 5. 9CH3COO þ 9Hþ / 9CH4 þ 9CO2 322.7
Overall 6. C18H34O2 þ 17/2H2O / 51/4CH4 þ 21/4CO2 616.4

Note: Values are calculated under standard conditions (25
C, 1 atm, and neutral pH). Negative value indicates the reaction is thermodynamically favorable and proceeds
spontaneously.

at both 15
C and 55
C by more than 2 to 3 times compared to 37
C
using synthetic wastewater at 2.0 g/L GAC.
3.6. Theoretical thermodynamic reaction for oleate degradation
Theoretical calculations of DIET or IHT process reactions
involved for oleate degradation are shown in Table 3. Theoretical
electron transfer flux calculations show that DIET exhibits a more
negative Gibbs free energy change than IHT in terms of electron-
producing reaction (998.5 vs. 196.4 kJ/reaction), suggesting that
the degradation of oleate to acetic acid is more efficient via DIET
(Table 3). On the other hand, the reduction of CO2 (electron-
consuming reaction) via DIET is less advantageous than that via IHT
(704.8 vs. 490.1 kJ/mol), due to the energy conservation of the
overall reaction (C18H34O2 þ 17/2H2O / 51/4CH4 þ 21/4CO2,
DG00 ¼ 616.4 kJ/reaction). Based on the thermodynamic models
exemplified with mesophilic digestion of oleate, DIET shows a
maximum electron transfer of 2.4  103 mA as compared to
7.1  106 mA for IHT.
4. Discussion
4.1. AD of lipid-rich wastewaters with GAC supplementation
This study showed that addition of GAC improved both
acetogenesis (LCFA degradation to acetate) (Fig. 4) and methano-
genesis (acetate conversion to methane) (Figs. 3 and 4). There was a
clear increase in oleate/palmitate degradation and VFA consump-
tion for all GAC-supplemented assays of more than 30% (Fig. 4).
Only a few studies have reported the application of CM in the AD of
lipid-rich wastewater. Improved AD from oleate (0.1e4.0 g COD/L)
was observed by Mostafa et al. where systems provided with
magnetite (4.1 g/L) or carbon nanotubes (1.0 g/L) led to a 10e300%
increase in maximum methane yield [16]. However, no decrease in
the lag-phase was observed by Mostafa et al. [16], which is in
contrast to this study where lag-phase time was drastically
decreased (Table 1). This indicates that different types of CM could
have a different effect on the AD of lipid-rich wastewater. Similarly,
Zhang et al. tested the impact of 12.0 g/L granular activated carbon
(GAC) on degrading typical waste lipid-rapeseed oil and showed
that bottles amended with GAC showed a 20% higher methane yield
[18], similar to the butter wastewater tested in this study with a 12%
higher methane yield (Table 2). However, both studies did not
quantify the VFA and LCFA degradation profiles and it is unknown if
the addition of the CM also improved the acetogenesis step in those
studies.
In an AD process, lipids are readily hydrolysed forming LCFA and
glycerol [34]. The subsequent acetogenesis phase, where the
degradation of LCFA to acetate occurs via the b-oxidation pathway,
is the rate-limiting step. In particular, palmitate degradation is the

966
L.C. Tan, R. Lin, J.D. Murphy et al.
bottleneck in treating lipid-rich wastewaters since it is the main
degradation by-product from oleate: Oleate þ 2 H2O /
Palmitate þ Acetate þ H2 þ Hþ [18,35]. Approximately 90% of the
COD is conserved as LCFA after lipid hydrolysis and as such, efforts
in improving LCFA degradation have been the focus in research on
AD of lipid-rich wastewaters [4]. Shin et al. highlighted that un-
saturated LCFA, i.e. oleate, are especially toxic to acetogens leading
to high VFA accumulation [36]. As such, faster fatty acid con-
sumption attained with GAC addition (Fig. 4) clearly prevented the
toxic impact of LCFA that can lead to process failure caused by
palmitate or VFA accumulation. In addition, faster palmitate
degradation was clearly observed under GAC-supplemented assays,
33% faster as compared to the control (Fig. 4c vs. 4d), indicating a
reduction in the bottleneck reaction and improvement in LCFA
degradation.
Application of GAC to real wastewaters showed different effects
(Fig. 3 b1). Using dairy wastewater, both lag-phase time and
maximum methane production was improved with GAC supple-
mentation (Table 2). Opposite to this, butter wastewater showed
only a higher maximum methane production but no change in lag-
phase time (Table 2). Lu et al. similarly reported no decreased in
lag-phase time but instead 32.5% higher maximum methane yield
from an oil substrate at 35
C using granular pine-based biochar
(0.5e1.0 mm) at a concentration of 10 g/L [17]. Differences in
methane production could be due to the different lipid wastewater
matrix. Lipid-rich wastewater can be assessed as either fat or oil
based, where fat, i.e. dairy wastewater, contains saturated LCFA
while oils, i.e. butter wastewater, typically contain unsaturated
fatty acids [4,24]. Saturated LCFA degradation, i.e. palmitate, occurs
via the b-oxidation pathway [4,35]. However, for unsaturated LCFA,
i.e. oleate, it is unknown whether fatty acid saturation is required
before b-oxidation can be activated [10,35]. Degradation of unsat-
urated LCFA requires two steps, first hydrogenation, cleaving off the
double bond from oleate (C18:1) to form stearate (C18:0) followed by
b-oxidation [24]. However, it is uncertain if this step can be per-
formed by the same microorganism or would require another
microorganism in conjunction [10]. As such, the type of LCFA pre-
sent, saturated (i.e. dairy wastewater) or unsaturated (i.e. butter
wastewater), in wastewater (Fig. 3 b1) thus impacts the degrada-
tion mechanism and, therefore, the effect of GAC supplementation
on the AD process.
Activated carbon (AC) has a cost involved in purchasing com-
mercial AC depending on the quality and particle size. An alterna-
tive can be to use biochar which can be manufactured from waste
materials and has a low production cost [37] in comparison to
commercial AC, and can also improve methane generation by up to
25% from oil substrate [17]. Different types of low-cost carbon-
based CM can thus be explored as optimal materials to enhance
methane formation at minimal cost. Moreover, different surface
areas have different effects on the enhancement of the AD process
with studies reporting smaller particle sizes resulting in better
methane generation [17,38]. However, using smaller particle size
carbon-based CM may prove difficult to retain within the
biodigester.
Overall, implementation of GAC in an existing AD plant requires
some modification for the reactor to prevent GAC washout and
repeated GAC addition in a long-term operation. A novel reactor
configuration such as the hybrid upflow anaerobic sludge blanket
(bottom section) and anaerobic filter (upper section) used in the
pilot-scale AD by Paulo et al. [23] would be a reactor option to be
used for the GAC-supplemented system. With the hybrid configu-
ration, this allows for sludge to have contact with the GAC via a
recirculation flow, while also preventing GAC washout. Designing
the reactor dimensions including for the volume ratio of filter to
sludge bed can be modelled with a recommended maximum GAC
967
Renewable Energy 171 (2021) 958e970
concentration observed in this study of 8 g/L GAC (Fig. 1). However,
long-term application of the AD process with addition of GAC
should also be investigated to test the durability of repeated LCFA
exposure on their degradation and methane generation. Zhang
et al. tested repetitive exposure of lipid-rapeseed oil in a batch
operated GAC-amended microbial system [18]. The authors
observed that the system with GAC allowed for a robust biological
process without process failure, which resulted in steady state
production of methane and lower VFA accumulation as compared
to the system without GAC. Thus, future research should look into
comparing the long-term process stability and performance be-
tween a reactor with and without GAC in a continuous or fed-batch
system.
4.2. Role of GAC in AD of lipids
4.2.1. Thermodynamic advantages of DIET with GAC
supplementation
LCFA degradation is accomplished through syntrophic cooper-
ation between methanogenic and anaerobic microbial commu-
nities [4] and as such, improving the relationship between these
syntrophic communities allows for better biodegradation of LCFA to
methane [4,39]. GAC can thus act as an electron conduit to help in
mass and electron transfer between substrate-microbes and
bacteria-archaea, respectively [20,40]. Theoretical calculations of
the maximum electron transfer flux gave further insights in
quantifying the kinetic difference between DIET and IHT (Table 3).
The significantly higher electron transfer via DIET (3 orders of
magnitude higher than that via IHT) in the theoretical calculation
indicates that the potential shift of IHT to the DIET pathway induced
by conductive GAC may lead to a more effective conversion of
oleate to methane. Mei et al. determined that, using ethanol as the
substrate, only half of the electrons generated are transferred via
DIET, even though 30 kJ/mol more energy is available compared to
complete hydrogen production [41]. Similarly, Lin et al. demon-
strated that DIET is kinetically more advantageous than IHT in AD of
different substrates including ethanol and glycine [7,32]. It is worth
mentioning that no drastic difference, in the methane production
profile was observed, between granular and crushed sludge
(Fig. 3a). Providing a higher biomass surface was hypothesized to
lessen the impact of the LCFA coating as well as offering more
exposure and contact surface between the microorganisms and the
CM. Since no difference was observed when using granular or
crushed sludge (Fig. 3 a1), this could indicate that DIET was not the
main cause for improved methane production for this study but
could still contribute to the overall improved AD process.
Another drawback in AD of lipids is that fatty-acid degrading
bacteria are generally low in abundance with slow growth rates
[12]. Rinzema et al. modelled the toxic effect of LCFA on AD and
concluded that obligate hydrogen producing acetogenic bacteria
and hydrogenotrophic methanogens recover first followed by
acetotrophic methanogens [42]. Remarkably, the SMA assays con-
ducted in this study also indicate that the hydrogenotrophic activity
showed better performance compared to acetoclastic activity with
GAC addition (supplementary information, Figure S3). This could
indicate that GAC potentially allowed for faster recovery of hydro-
genotrophic methanogens leading to faster methane generation.
Change in microbial community profile with and without GAC-
supplemented system should be considered in the future work.
This is to identify which microorganisms are growing in abundance
when fed with GAC as well as quantification of the carbon and
electron flow via labeled isotopes coupled with microscopic im-
aging, i.e. fluorescence in situ hybridization of acetogens and
methanogens [2,43].
L.C. Tan, R. Lin, J.D. Murphy et al.
4.2.2. Role of GAC as an adsorbent
The long lag-phase time in both LCFA breakdown to VFA and
finally to methane was shortened considerably with GAC addition
(Fig. 4), potentially because GAC acts as an adsorbent relieving
substrate inhibition by allowing LCFA to coat the GAC instead of the
biomass. GAC showed high adsorption percentages of COD, up to
90% (Table 1), which could potentially indicate that the incubations
with GAC were able to circumvent the toxicity of LCFA or their in-
termediates. AD of lipids causes for LCFA biosorption onto the
sludge leading to inhibition of the biological activity. Oleate and
palmitate substrate inhibition can occur at low concentrations, and
their IC50 (half maximal inhibitory concentration) amounts to
50e75 mg/L and 1100 mg/L, respectively [44]. LCFA inhibition by
adsorption to biomass has been reported to be reversible over a
long period of time where the methane production rate can sharply
increase once the adsorbed LCFA are metabolized [18,45]. However,
a decrease in the maximum methane production (Fig. 1) was
observed corresponding with to the increase in COD% adsorbed
onto the GAC, reaching 90% COD adsorbed after 7 days of incubation
with the GAC concentrations of 16.0e33.0 g/L (Table 2). Florentino
et al. similarly observed that at a higher GAC concentration of
33.0 g/L, methane yield drastically decreased by 3 orders of
magnitude when treating blackwater when comparing to condition
without GAC [46]. From these observations, it can be concluded that
the GAC addition concentration added in wastewater treatment is a
critical factor and should be optimized for each type of wastewater
and CM.
The adsorption tests carried in this study do not distinguish
whether adsorbed COD onto the GAC is inaccessible by the organ-
isms and therefore reducing the methane potential yield of the
system or that the adsorbed COD onto GAC was still able to be
further utilized. Bioregeneration of activated carbon has been
investigated in previous studies treating inorganic and organic
compounds and showed that organisms are indeed able to utilize
adsorbed compounds to regenerate the activated carbon given the
approximate operation conditions and adsorption-desorption
contact time are provided [25,47]. Islam et al. demonstrated that
simultaneous adsorption and degradation of oil sands process-
affected water in GAC is possible provided sufficient biofilm
growth has occurred on the GAC surface [48]. Further adsorption
studies should be conducted to test whether adsorbed COD in GAC
is accessible to microorganisms or whether adsorption saturation
of GAC does not lead to improved methane generation in the AD of
lipid-rich wastewater.
4.3. Effect of GAC on AD of lipids at different temperatures
Degradation tests at different temperatures showed that GAC
supplementation improved the AD by decreasing fatty acid accu-
mulation at all three temperatures investigated (Fig. 4). Previous
studies have tested the degradation of LCFA at high or low tem-
perature, but so far none has explored the effect of adding GAC.
Operating at a higher temperature (55
C) has been reported to
provide faster recovery from LCFA inhibition [44]. At low temper-
atures (15
C), degradation of LCFA is difficult; partly as LCFA turn
insoluble due to their low solubility in water [21]. Without addition
of GAC, degradation of LCFA took longer at 15
C conditions, while
VFA accumulated in the 55
C incubations. However, GAC-amended
conditions for all incubation temperatures alleviated accumulation
of fatty acids, leading to faster methane production and LCFA/VFA
consumption. Lu et al. concluded that providing powdered biochar
and operating at 55
C allowed for faster acclimatization of mi-
crobial communities leading to increased methane production by
13.3% [17]. The authors noted that the biochar amended conditions
also alleviated acetate and propionate accumulation compared to
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Renewable Energy 171 (2021) 958e970
the assessment without biochar. The degradation tests incubated at
55
C similarly showed accumulation of VFA without GAC addition
(Fig. 4e).
Lin et al. highlighted through thermodynamic calculations that
the electron transfer of graphene-based DIET during incubation at
37
C and 55
C were at similar levels, thus suggesting that tem-
perature might not be a significant factor affecting DIET thermo-
dynamics [49]. However, the authors observed that the abundance
of bacteria and archaea was temperature-dependent in 37
C and
55
C. Lin et al. offered a different perspective in DIET process in
correlation with the performance of anaerobic bioreactors operated
at different temperature (20, 37 and 55
C) with carbon cloth as the
CM and using ethanol and/or propionate as the substrate [50]. They
observed that the DIET process was temperature-dependent but
with distinct correlation between biomass conductivities, pilA
gene, and heme-binding proteins leading to various DIET routes. As
such, it is important to operate the AD at an appropriate or optimal
temperature when supplementing CM as it can impact the degra-
dation by-products and breakdown profile of lipid-rich wastewa-
ters (Fig. 4).
5. Conclusion
To address the research gap in developing a cost-effective
approach to enhance AD of lipid-rich wastewaters, application of
GAC to AD can be considered as an alternative strategy for dealing
with LCFA degradation with minimal modification to existing AD
plants. The lag-phase time can be decreased by a minimum of 1.5-
fold (2.0 g/L GAC) to a maximum of 10-fold (33.0 g/L GAC)
compared to digestion without GAC addition. Both VFA and LCFA
were consumed faster with the addition of GAC, indicating that
apart from methanogenesis, also acetogenesis was enhanced.
Enhancing the AD process through GAC addition was similarly
confirmed at 15 and 55
C as well as when applied to real lipid-rich
wastewater streams. It should be noted that providing more than
8.0 g/L GAC lowers the maximum methane production in the range
of 20e50%, potentially due to the higher amount of COD adsorbed,
thus reducing available substrates for methane conversion. Future
work is needed to assess the effect of GAC supplementation on lipid
AD in continuous operation and at demonstration sale.
CRediT authorship contribution statement
Lea Chua Tan: conception and design, Investigation, data
acquisition, analysis and interpretation, writing e original and
revised manuscript (drafting, submitting and revising). Richen Lin:
theoretical data conception, Writing e original draft, preparation,
Writing e review & editing, Writing e review & editing, Funding
acquisition. Jerry D. Murphy: Writing e review & editing, Funding
acquisition.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgement
The authors would also like to acknowledge the assistance given
by Dr. Corine Nzeteu (NUIG, Galway, Ireland) through providing the
expertise and training for the SMA and BMP assay. The study was
funded through the Science Foundation Ireland (SFI) Research
Professorship Programme Innovative Energy Technologies for Bio-
fuels, Bioenergy and a Sustainable Irish Bioeconomy (IETSBIO3;
L.C. Tan, R. Lin, J.D. Murphy et al.
award no. 15/RP/2763) and the Research Infrastructure research
grant Platform for Biofuel Analysis (grant no. 16/RI/3401). The au-
thors would also like to acknowledge the support from SFI through
the MaREI Centre for Energy, Climate and Marine under grants no.
12/RC/2302 P2 and 16/SP/3829. This study was also supported by
the European Union’s Horizon 2020 research and innovation pro-
gramme under the Marie Skłodowska-Curie grant (No. 797259) and
Ireland Environmental Protection Agency (EPA) Research Pro-
gramme 2014e2020 (No. 2018-RE-MS-13).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.renene.2021.02.087.
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