Renewable and Sustainable Energy Reviews 45 (2015) 351–358

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Renewable and Sustainable Energy Reviews

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Effects of lipid inhibition on biogas production of anaerobic digestion

from oily effluents and sludges: An overview
Nazaitulshila Rasit a,b,n, Azni Idris a, Razif Harun a, Wan Azlina Wan Ab Karim Ghani a
a
Department of Chemical and Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan,
Malaysia
b
Department of Engineering Science, Faculty of Science and Technology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu Darul Iman,
Malaysia

art ic l e i nf o a b s t r a c t

Article history: Anaerobic digestion of lipid effluent and sludge is becoming an interest due to its high biodegradability
Received 27 August 2013 in producing biogas. Due to complex polymeric substances, lipid has been investigated as a potential
Received in revised form substrate to produce biogas when digested anaerobically as compared to carbohydrate and protein.
3 September 2014
However, it is known that lipid degradation causes inhibition to biogas production with lag phase
Accepted 25 January 2015
occurrence, sludge floatation and washout. Co-digestion of lipid substrates has shown enhancement to
biogas production of low biodegradability substrates but the experience of slow hydrolysis due to
Keywords: inhibition is still occurred. Long chain fatty acids (LCFAs) produced during lipid hydrolysis is known as
Lipid inhibition primary inhibitor due to its toxicant effects to cell wall adsorption by microorganisms. Currently, the
Anaerobic digestion
study of lipid inhibition and mitigation to induce reduction of lipid toxicity has regained attention. In
Biogas
this overview, a detailed of previous and current literature on lipid biodegradability, inhibition effects,
methods to reduce lipid inhibition in enhancing biogas production are discussed to provide a summary
of available information regarding lipid inhibition and its potential in enhancing biogas production.
& 2015 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
2. Properties of lipid in wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352
3. Lipid degradation in anaerobic digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
4. Lipid biodegradability in anaerobic digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
5. Effects of lipid inhibition on biogas production of anaerobic digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
6. Anaerobic co-digestion of lipid substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
7. Methods to overcome lipid inhibition on anaerobic digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
7.1. Effects of feeding sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
7.2. Effects of adsorbent addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
7.3. Effect of enzymatic hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
7.4. Effect of easily degradable co-substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
7.5. Effect of saponification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
8. Conclusions and directions for future research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

1. Introduction
n
Corresponding author at: Department of Chemical and Environmental
Lipid, the terms used in describing fat, oil and grease (FOG)
Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor Darul Ehsan, Malaysia. Tel.: þ 60 9 6683320; fax: þ 60 9 6694660. contained in wastewater stream (commonly known as oily waste-
E-mail address: nazaitulshila@umt.edu.my (N. Rasit). water) is generated from many sources including municipal such

http://dx.doi.org/10.1016/j.rser.2015.01.066
1364-0321/& 2015 Elsevier Ltd. All rights reserved.
352 N. Rasit et al. / Renewable and Sustainable Energy Reviews 45 (2015) 351–358

as municipal sewage [1,2], industry such as edible oil [3], food
processing [4], slaughterhouse [5] and commercial such as food
trade [6]. Lipids include 25% of organic matter from municipal
sewage concentration that make up this wastewater as oily
wastewater [2]. While, in palm oil mill effluent (POME), lipids
becoming a major pollutant with a concentration ranging from 130
to 18,000 mg/L [3]. Ref. [4] reported the concentration of lipid in
meat and food processing wastewater is relatively high with
average 39,000 mg/L. Lipid is categorized as oil and grease and
listed as conventional pollutant under Clean Water Act. It presents
in wastewater as free oil, grease (oily sludge) or emulsified oil (oily
effluent). The variation on the physical formation of lipid in oily
effluents and sludge greatly depends on their processing and lipid
utilization at source. Lipid accumulation will congeal and reduce
the diameter of sewer pipe in wastewater treatment system [7].
The clogging problem requires regular maintenance on the sewer
pipe to avoid system overflow. A device normally used to retain
lipid is called Grease Trap Waste [8]. It is often used to reduce the
risk mentioned. In wastewater treatment plant, lipid is collected as
scum before being disposed as solid waste. However, improper
lipid management and treatment has caused the oily wastewater
flows into drains and is discharged directly into the watercourses.
Consequently, lipid becoming a part of pollutants that contributes
to water pollution and accounts in blocking of sewer system [1].
Furthermore, oily wastewaters have high content of oil and grease,
chemical oxygen demand (COD) and colour. The lipid accumula-
tion can attract vermin thus lead to pathogen disclosure [1,9,10].
In 2010, Indah Water Konsortium (IWK), a Malaysia’s national
sewerage company received 22,184 inquiries of blockage problems
and unfortunately, the sewage treatment plants are not designed
to handle lipid deposition [11]. In Malaysia, oil and grease
deposition in watercourses are restricted and Table 1 shows the
acceptable limit of oil and grease in related regulations under
Environmental Quality Act 1974 [12].
Considering the stringent standard of the discharge of waste
lipids which has to be strictly complied, removal of this conven-
tional pollutant is becoming a crucial issue for human health and
environmental protection. Unfortunately, most of the collections of
lipid waste then end up at landfill when disposed as solid waste [8].
To reduce the quantity of ultimate disposal into landfill, one of the
options for waste recovery is by implementing anaerobic digestion,
a process in which the biological decomposition of biodegradable
materials occurs in the absence of oxygen [13]. Lipid is known as
one of the potential substrate to produce biogas as shown in Table 2
[14]. Thus, it is known that the utilization of anaerobic digestion of
lipid wastes is becoming a potential source to produce energy.
The positive yield in biogas and methane production by high
lipid wastewater is extensively reported from previous researchers
[3,5,15–21]. Apart from being disposed, the interest towards of
lipid exploitation and its potential of becoming a source of
renewable energy have shown an increase, especially in terms of
waste recovery. Although lipid is degradable through biological
process, the presence of intermediate which is known as long
chain fatty acid (LCFAs) is reported to inhibit and becoming the
main cause of process instability [22–24]. However, considering
the potential of lipid degradation, many studies have exploited
lipid as a co-substrate with low biodegradability substrates such as
waste activated sludge [15–17]. Additionally, an effort to overcome
the drawback of lipid inhibition has been developed. Thus, the aim
of this overview is to perform a detailed descriptive summary of
research regarding lipid inhibition on biogas production and a
strategy to reduce its inhibition. Since the word fat, oil and grease
and lipid are used throughout the literature to describe oily
effluents and sludge, the word lipid waste will be used for clarity
to describe the oily content of the effluents or sludge.
2. Properties of lipid in wastewater
The existence of lipid in aqueous streams is in the form of free
oil and grease or emulsified oil. To identify its characteristics, the
comparison is made based on their droplet size. Having hydro-
phobicity characteristic, free oil and grease are often easier to
identify due to its insolubility in water with droplet size Z150 μm
and appear as a floating layer on the water surface [25]. Physically,
emulsified oil is harder to identify due to its small droplet in size
ranging of 20–150 μm. Free oil and grease and emulsified oil may
disperse on water surface and become hardened, occurring as
solid forms due to metallic salt of fatty acids reaction [26]. In
wastewater treatment plant, emulsified oil may form larger
droplets when accumulating within the wastewater treatment
process and often separates as scum during primary settling. In
the subsequent process, scum containing oil and grease is treated
as sludge in sludge treatment/digestion system. Due to its poor

Table 1
Acceptable condition of oil and grease from sewage and industrial discharges under related regulation of Standard A and B
Environmental Quality Act 1974. (Department of Environment, DOE, Malaysia) [12].

Regulations Unit Standards

A B

Environmental Quality (Sewage) Regulations 2009 mg/L 5.0 10.0

Environmental Quality (Industrial Effluent) Regulations 2009 1.0 10.0

This standard applicable for any discharge into any inland waters within catchment areas listed as A (listed in Third Schedule) and B
(other than listed in Third Schedule of Environmental Quality (Sewage) Regulations 2009).

Table 2
Biogas production potential from lipids, carbohydrates and proteins [14].

Components Lipids Carbohydrates Proteins

Biogas production (L/g) 1.425 0.830 0.921

CH4 percentage (%) 69.5 50.0 68.8
Methanogenic reactions C50H9O6 þ24.5H2O-34.75CH4 þ 15.25CO2 C6H10O5 þH2O-3CH4 þ 3CO2 C16H24O5N4 þ14.5H2O-8.25CH4 þ 3.75CO2 þ 4NH4þ þ 4HCO3
 N. Rasit et al. / Renewable and Sustainable Energy Reviews 45 (2015) 351–358 353

Table 3
Characterization of liquid and semi-solid lipid wastes.

Sources of lipid wastes Parameters

pH TS (g/L) VS (g/L) COD (g/L) AN (g/L) Lipid (g/L) References

Domestic sewage 6.7 0.7 n.d n.d n.d 0.9 [1]

Palm oil mill 4.2 40.0 34.0 51.0 0.04 6.0 [3]
Meat and food processing n.d 36.9 34.4 96.7 0.3 38.8 [4]
Pet food processing 6.7 82.5 51.3 87.5 1.4 83.0 [28]
Olive oil mill 4.81 60.3 51.3 104.9 1.2 17.2 [29]
Dairy processing 4.99 57.0 50.0 65.0 0.03 24.5 [30]

n.d¼ Not detected.

Table 4
Long chain fatty acids found in various effluents. Lipids Proteins Carbohydrate

Sources of Long chain fatty acid (% of total LCFAs) References Hydrolysis

wastewater
C12:0 C14:0 C16:0 C18:0 C18:1 Others
Glycerol Amino Sugar
LCFAs
Alcohol
Grease trap waste 0.5 3.7 62.6 25.0 n.d 8.2 [6]
Slaughterhouse n.d n.d 35 15 50 – [33] β-oxidation
Canned fish n.d 19 260 75 891 – [34] Acidogenesis
processing1
Olive oil mill n.d n.d 10.2 4.1 78.1 7.6 [35] H2 Volatile fatty acids
CO2
C12:0¼ Lauric, C14:0¼ Myristic, C16:0 ¼Palmitic, C18:0¼ Stearic, C18:1 ¼Oleic.
1
Measured as gCOD/kgTS, n.d. ¼not detected. Acetogenesis

solubility in water and low specific gravity, oil and grease accu-
mulates on the top of digester tank then it forms a dense scum
layer before finally being disposed of as a solid waste to landfill.
However, oil and grease are not totally withdrawn during primary
treatment which then resulted in the finely emulsified form in
secondary treatment. As the consequences, the existence of FOG is
becoming an inhibition factor towards biological processes [27].
The characteristics of liquid and semi-solid lipids are listed in
Table 3. The content of lipid can vary greatly depending on types of
the sources of wastes. Listed characteristics as shown in Table 3
proved that the higher content of volatile solid is constituted by
lipid content [21]. Moreover, a lipid constituent is contributes to
high COD content in pet food processing wastewater [28]. High
lipid content in wastewater used for biological processes always
resulted in inhibition effects and delayed in the overall process.
Furthermore, other risks that should be taken into considered is
lower value of pH that may expose to pumps, pipe and tank
corrosion [8].
Long chain fatty acids (LCFAs) is a straight chain aliphatic
carboxylic acids listed as C4 until C22 are considered as a main
component that was induced as an inhibition effects as reported
elsewhere [22,24,31,32]. However, not all types of LCFAs exist in
high percentage in lipid wastewater and it is reported that oleic
acid is the most abundant LCFA in wastewater [33]. However, as
previously stated, the characteristics of LCFAs in effluents are
varying depending on their origin. Table 4 shows the reported of
the most common LCFAs in various effluents.
3. Lipid degradation in anaerobic digestion
There are 4 stages (hydrolysis, acidogenesis, acetogenesis and
methanogenesis) incorporated in lipid degradation of anaerobic
treatment system to produce biogas [13]. As a polymeric sub-
stance, lipid is basically consists of triglycerides and long chain
fatty acids (LCFAs) degraded by hydrolytic bacteria to break the
chemical bond and increase lipid solubility to produce free fatty
acids and glycerol in hydrolysis stage. While carbohydrate
Acetic Acid
Methanogenesis
CH4
CO2
Fig. 1. Complex organic metabolism pathway in anaerobic digestion [37].
hydrolysed to simple sugar and protein to amino acid. Fatty acids,
sugar and amino acid are simple monomer and easier to be
absorbed by bacteria. They are further degraded to carbon dioxide,
hydrogen gas, alcohols and organic acid in acidogenesis stage.
However, LCFAs degradation path is through β-oxidation where
coenzyme A is utilized for LCFAs oxidation for conversion to
acetate and hydrogen [14]. Further degradation in acetogenesis
produced acetic acid, carbon dioxide, hydrogen and ammonia.
Methanogenesis, a final stage of biogas production with acetic acid
as a primary component of volatile organic acids will be used by
methane forming bacteria to produce methane as shown in Fig. 1
[36,37].
4. Lipid biodegradability in anaerobic digestion
Within 3 substrates, lipid is tested to have high biodegradability
and potential to produce more methane compared to protein or
carbohydrates through the catalytic in the process done by acido-
genic bacteria with extracellular lipase as the catalyst [14,22,24].
The activation of lipase enzyme in lipid degradation is occurring via
β-oxidation where fatty acid is activated by lipase and resulted in
the formation of fatty acid chain [38]. The catalytic event thus
induces the biodegradability of lipid substances as compared to
other complex substrates. In theory, based on a standard tempera-
ture and pressure (STP), a comparison were made between 1 g of
oleate and 1 g of glucose showed methane production were 1.01 L
and 0.37 L, respectively [39]. As the consequences, exploitation of
waste containing high lipid content are becoming more attractive
sources in producing biogas from anaerobic treatment system.
354 N. Rasit et al. / Renewable and Sustainable Energy Reviews 45 (2015) 351–358
Table 5
Reported biomethane potential test (BMP) for lipid wastes.
References Lipid samples
[15]  Grease sludge
[16]  Polymer dewatered fat, oil and grease
[17]  Grease trap sludge from meat processing industry
[43]  Fatty residue from urban wastewater treatment plant
[44]  Grease trap waste from dissolved air floatation unit treating sewage
[45]  Grease trap waste from local restaurants
Basically, the method used in determination of lipid biodegrad-
ability is Biomethane Potential Test (BMP), a simple, rapid and
inexpensive method as described by [40] and appropriate protocols
for organic wastes as reported by [41]. In BMP test, an individual
potential substrate to be tested is placed in an anaerobic environ-
ment condition and daily biogas monitoring is done in duration of
50 days or more until ceased. Analysis of BMP is calculated based on
standard temperature and pressure (STP) to ensure comparison
among research findings can be made [41]. Table 5 provides a
summary of BMP test results to determine biodegradability of lipid
contained from various sources based on STP. As expected, the
biodegradability of lipid in 6 studies reported higher potential of
methane yield and some of the finding achieving theoretical value
(1014 N mLCH4/g VS) as experimented by [42].
5. Effects of lipid inhibition on biogas production of anaerobic
digestion
Despite the reported catalytic event that increased the lipid
degradation, it is known that the hydrolysis stage is the accumulation
stage of LCFAs, which are important inhibitors of the anaerobic
microorganisms [46]. When the accumulation of LCFAs occurred,
increasing lag phase in which methane yield may be decreased
initially was detected in methanogen activity as demonstrated by
[24]. Ref. [32] identified the accumulation of LCFAs oleate and stearate
affected the degradation process and become the inhibitory factors to
the delayed hydrolysis of thermophilic digestion process. Shock
loading hampered activation of biogas reactor even though non-
inhibitory concentrations dilutions were made. However, [47] identi-
fied that after the lag phase, anaerobic bacteria are able to digest the
accumulated LCFA and reproduce methane. This is due to the
occurrence of few survival of anaerobic consortium. After the lag
phase, the biogas production process is possible but long retention
times is required.
The inhibition scenario as reported by previous researchers is
rely on several factors and it is suggested that the rate limiting
step occurred during mass transfer that occurs between substrate
and microorganisms and finally into microbial cells. This mass
transfer is related to difficulty in transport and transform from
liquid phase to solid and biological degradation of LCFAs [48]. This
finding is supported by Hwu et al. [33] and Jeganathan et al. [28]
that suggests the limiting in transportation has caused sludge
floatation as a result from LCFAs adsorption into cell walls.
Adsorption onto cell wall is restricted by bacteria coated LCFAs
thus reduce the substrate accessing and yield of biogas [47]. Since
the inhibition mechanism is based on concentration, higher
concentration of LCFAs will exhibit toxicity effects to microbial
cell walls [31,48,49].
6. Anaerobic co-digestion of lipid substrate
As the consequences of low rate of hydrolysis and inhibition
effects, sole degradation of lipid substrate through anaerobic
Methane/biogas biodegradability (N mL CH4/g VS)
 845–928
 993
 918
 570–700
 Methane production of grease trap waste is 50% higher than sewage sludge
 1050
treatment is often not an interest. Thus, a combination of high
lipid substrate with other substrate commonly known as co-
digestion is a subject of interest. According to its high biodegrad-
ability, grease trap waste is considered as a good substrate to be
co-digested with low biodegradability substrate [17]. Co-digestion
of lipid wastewater such as grease trap waste has been extensively
reviewed by Long et al. [8]. Performance of methane yield was
found to rise until 27% at 30% volatile solid added when grease
trap sludge was digested with sewage sludge [15]. Grease trap
sludge from meat processing industry has high methane produc-
tion potential but when tested as a single substrate, it was found to
start slowly and the production of methane starts immediately
when digested with sewage sludge. FOG feed at 45% volatile solid
(VS) added was recorded can attained 66% biogas production
increment [17]. Dewatered FOG when co digested with sewage
sludge was reported to increase 2.95 times of methane production
at 48% VS added as demonstrated by [16]. Apart from that, [19–21]
evaluated the possibility of FOG as co-substrates were successful in
enhancing the methane production. Methane production was also
feasible for plug flow digester treating swine manure and used
cooking grease. It was reported at the concentration of 2.5% used
cooking grease digested, the digester yielded highest biogas with
67% methane. However, higher concentration of used cooking
grease (more than 0.5% on VS basis) did not show any improve-
ment of biogas yield [18]. This experiment supported the inhibi-
tion theory of lipid digestion by [22].
7. Methods to overcome lipid inhibition on anaerobic
digestion
Ref. [8] reported several co-digestion studies of grease trap
waste with sewage sludge to increase the production of biogas of
the system. However, the summary of reported studies still
experienced of deficiency such as low rate of hydrolysis with lag
phase experience. Biodegradability of co-digestion can enhanced
biogas yield but required long time for methane recovery. Thus, to
reduce lagging time and enhance lipid degradation biologically,
various techniques have been employed to focus on recovery
options. Table 6 listed a summary of an approach to reduce
inhibition done by LCFAs. The recovery options in order to improve
the LCFAs toxicity are categorized as feeding sequence, adsorbent
addition, enzymatic hydrolysis, easily degradable substrates addi-
tion and saponification as described hereinafter.
7.1. Effects of feeding sequence
Several researchers reported that the lipid inhibition is inclined
with LCFAs concentration introduced into the system [32,48]. Ref. [32]
identified the accumulation of LCFAs oleate and stearate affected the
degradation process and shock loads hampered activation of biogas
reactor even though non-inhibitory concentrations dilutions were
made. To avert shock load, a pulses feeding sequence have been
investigated where feeding is allowable into the system at intermittent
Table 6
Summary of research trends on approaches to overcome lipid inhibition.

Methods to Operating condition Sources of lipid Methane yield before Methane yield after % Effects on lipid References
overcome inhibition approach inhibition approach Increase inhibition (lag
lipid phase)
inhibition

Feeding  Mesophilic batch experiments Fat-rich dairy wastewater (53% n.s  1st Pulse: 0.45 70.01 g n.s  Diauxic [39]
sequences  Pulse feeding of LCFAs on day 0, 38 and 81 fat) CH4/gCOD behaviour
 Specific pulse load of 1 gCOD/gVSS for each pulse  2nd Pulse: 0.88 7 0.02 g on
CH4/gCOD first pulse
 3rd Pulse: 1.29 7 0.08 g  No lag
CH4/gCOD phase on
three pulses

N. Rasit et al. / Renewable and Sustainable Energy Reviews 45 (2015) 351–358

 Thermophilic continuous stir tank reactor Oleate 253–268 mL CH4/g VS 314–329 mL CH4/g VS 19.0 n.s [50]
 Pulses feeding of sodium oleate on day 104, 111, 133 (max)
 Increasing pulse load of 0.5, 1.0 and 2.0 g/L reactor volume

Adsorbent  Mesophilic batch experiments Oleate 130:5 7 12:2 mgCODCH4 =L day 187:1 7 10:3 mgCODCH4 =L day 43.3 From 6 to 3 days [51]
addition  Inhibition by oleate (0.1 to 1.5 g/L)
 Bentonite was used as synthetic adsorbent to reduce
inhibition effect

 Thermophilic semi-continuous reactor (continue feeding LCFA mixture (sodium oleate, 0.41 L CH4/L day  Bentonite: 1.29 L CH4/ 76% (max) From 4–5 days [5]
after inhibition and recovery action) sodium palmitate and sodium L day to 2–3 days
 Inhibition by LCFAs (4 g/L) stearate)  Fiber: 1.68 L CH4/L day
 Fibers from filtered digested manure and bentonite were
used as adsorbent to reduce inhibition effect
 Addition of fiber and bentonite (5 g VS/L)

 Thermophilic batch and semi-continuous reactor Bentonite bound oil Measured as oil utilization: Measured as oil utilization: 90 (max) n.s [52]
 Inhibition by oil and bentonite bound oil negative oil utilization degree of oil utilization
 Addition of bentonite and calcium carbonate increase 70–90%

Enzymatic  Mesophilic batch reactor in 3 variation temperature Synthetic dairy wastewater 2330 mL 4710 mL after 13 days run at 51 n.s [53]
hydrolysis (30 1C, 37 1C and 45 1C) with enzymatic hydrolysis (1000 mg/L total fat content) 45 1C
(lipase)
 Mesophilic hybrid upflow anaerobic sludge blanket Dairy wastewater (225 g/m3 total n.s 1169 mL at 8.9 kg/m3 day n.s n.s [54]
(UASBh) reactor lipids content)
 Effects of increasing organic loading rates and enzyme
pretreatment (porcine pancreatin)

 Mesophilic batch reactor with variations of oil and Poultry slaughter-house 37 mL 137 mL at 1200 mg/L oil and 73 From 6–7 days [55]
grease contents (150–1200 mg/L) and solid enzymatic wastewater grease, 0.1% enzyme to 4 days
pool concentrations (0.1–1.0% w/v)
 Mesophilic upflow anaerobic sludge blanket (UASB) Synthetic dairy wastewater 61.8 mL 68.5 mL at 600 mg/L oil and 10 n.s [56]
reactor with variations of oil and grease contents (200, grease
600, 1000 mg/L)
 Anaerobic sequencing batch reactor (ASBR) operated at Slaughter-house wastewater n.s 1:09 7 0:65 gCODCH4 =L day n.s n.s [57]
25 1C with enzymatic Pancreatic Lipase 250 hydrolysis containing pork fat particles

Easily  Mesophilic semi-continuous reactor with the addition of Oleate n.s 3.5 L/d n.s n.s [37]
degradable co-substrates, such as glucose and cysteine
substrates
addition

355
356 N. Rasit et al. / Renewable and Sustainable Energy Reviews 45 (2015) 351–358

rate. In this scenario, LCFAs are repeatedly fed as pulse feeding to allow
References

a sufficient time for microorganisms adaptation/tolerance to absorb

LCFAs before being inhibited in subsequent feed. Thus, shock and

[64]

[59]

[66]
excessive loads of LCFAs on cell walls can be minimized [39]. Ref. [50]
studied oleate pulses onto thermophilic anaerobic reactors and
Effects on lipid
inhibition (lag

reported the pulses effects on degradation of volatile fatty acids

Lag phase
(VFA) and also reducing the lag phase during hydrolysis stage. This

duration
reduced.
phase)

agreed with the finding reported by [32] on tolerance of biomass to

n.s

n.s

LCFAs is more achievable by gradually feeding rather than shock

feeding at a time.
50% from

40 (max)
Increase

Drop off

9 (max)
control

7.2. Effects of adsorbent addition

%

The effects of adsorption addition have been studied by [61] with

1324 mL/g VS at thermophilic

the addition of calcium salt to reduce the toxicity and increase the
solubility of LCFAs. LCFAs precipitate with calcium salts to produce
inhibition approach
Methane yield after

approximately 95% fatty acid calcium salt. Due to precipitation,

344 L CH4/kg COD

insoluble salts were performed and simultaneously increased inter-

facial tension. However, according to [24], calcium carbonate may
condition

have enhancement effects on lipid inhibition but the effect is

intermittent. The continuous exposure of LCFAs may precipitate with
n.s

calcium carbonate but inhibition occurrence may exist due to

insolubility of fatty acid calcium salt. Further investigation by
1233 mL/g VS at thermophilic

addition of calcium carbonate showed an enhancement of glyceride

trioleate degradation but slightly lower than the addition of bento-
Methane yield before

nite. Bentonite is types of clay mineral showed similar mechanism

inhibition approach

206 L CH4/kg COD

with calcium carbonate to precipitate LCFAs [52]. However, due to its

characteristic of high porosity and surface area, bentonite provides
greater adsorption capacity towards LCFAs and exhibit higher
condition

adsorption capacity when fitted in Freundlich adsorption isotherm

[51]. In reactor experiments with semi-continuously feed, the lag
n.s

phase as the consequences of LCFAs accumulation has reduced from

4–5 days to 2–3 days with the addition of bentonite and fibers and
(aero-flotation fats and flesh fats

indicate higher LCFAs precipitation [5]. The addition of adsorbent

 Mesophilic and thermophilic batch experiments with waste Fatty slaughter-house waste
 Mesophilic continuously stir tank reactor (CSTR) with waste Slaughter-house fatty waste

addition such as calcium carbonate and bentonite yielded positive

from animal carcasses)

results when adsorbents has the capability to bound LCFAs onto their
surface thus reduce the ability to interpret into microbial cell and
Sources of lipid

finally inducing methane production. However, the existence of

precipitant may have detrimental effects to the microbial community
 Mesophilic two stage reactor with the addition of gallic acid Gallic acid

and lead to sludge accumulation. A further study is needed to

determine the effect of precipitant onto microbial system and to
reduce the sludge accumulation in the anaerobic digester.
saponification pretreatment (70 1C during 60 min) with pulse

7.3. Effect of enzymatic hydrolysis

It is known that the main inhibition is due to difficulties in LCFAs

degradation and enzymatic pre-treatment is becoming of interest to
overcome the problem. The application of enzymatic pre-treatment
may improve LCFAs solubility during hydrolysis stage by increasing
the transportation rate of soluble substrate into biomass to induce
methane conversion. Moreover, the occurrence of lag phase maybe
reduced. Ref. [55] reported the stated scenario with utilization of solid
enzymatic pool retrieved from solid state fermentation of Penicillium
restrictum. Nonetheless, considering the inhibition of LCFAs is con-
centration based, the higher inhibition was recorded in higher LCFAs
Operating condition

content. The positive aspect of enzymatic pretreatment for inhibition

feeding patterns

effects reduction is reported by previous study proved that by using

saponification
and glucose

Pancreatic lipase enzyme in treating slaughterhouse wastewater

containing pork fat particle showed 35% fat have been hydrolysed.
The hydrolysed part of fat particle has become easier to be absorbed
Table 6 (continued )

by microorganism cell walls [57]. Ref. [56] have reported same

Saponification

scenario with 82% fat have been hydrolysed. Using lipase as pre-
n.s: Not stated.
Methods to

inhibition

treated enzyme, an anaerobic digester treating dairy effluents was

overcome

found to increase biogas production approximately 4457

lipid

29 mL as compared to effluents not exposed to enzyme pre-

treatment [62]. Moreover, COD, BOD, lipid content and organic matter
 N. Rasit et al. / Renewable and Sustainable Energy Reviews 45 (2015) 351–358
removal were also observed [53,54,56,62,63]. However, the induction
of enzyme pre-treatment reported were using lab-scaled reactor and
hence, higher cost is required rather than other pre-treatment
especially when commercial enzyme is used. If any large scale reactor
involves applying enzyme pre-treatment, a financial estimation should
be taken into account and further research is needed to estimate the
related cost and applicability on implementing enzyme pre-treatment
on large scale basis.
7.4. Effect of easily degradable co-substrate
It is known that the existence of LCFAs becomes toxicity to the
anaerobic microorganisms and thus performs retardation of gran-
ulation [31]. Ref. [37] suggested inhibition improvement of oleate,
the most abundant LCFAs in wastewater can be achieved together
with enhancement on granular formation with the addition of
easily degradable substrate such as glucose and cysteine. A
methanogenic microorganism activity increases with the increase
of granular formation. This study proved the finding by [58,64]
where glucose and glycine is considered as a growth factor to
adapt to inhibition occurrence by fatty acids. However, the
enhancement on biogas yield caused by this approach is less
reported and further study is needed to figure out the positive
effect of biogas production and inhibition time.
7.5. Effect of saponification
Saponification executes the production of LCFAs salts and
glycerol as resulted from ester of lipid and alkaline reaction. The
conversion of insoluble to soluble LCFA soaps induces the solubili-
sation of insoluble LCFA by improving the transportation through
accessibility in better adsorption between substrate and microor-
ganisms [65]. Biodegradability of saponified fatty wastes exhibits an
improvement on biogas production during hydrolysis stage in batch
tests as compared to unsaponified wastes with biogas yield was
4 times higher. Nevertheless, final volume showed only a slightly
enhancement on biogas production indicated that saponification
enhanced the biodegradation during hydrolysis rate by reducing the
experience of lag phase but not on the subsequent stages [60]. This
situation supported with the finding by [65] that the addition of
alkaline reagent during saponification had influence the pH value in
the biodegradation ecosystem and directed towards toxicity caused
by accumulation of sodium cation concentration. Moreover, the
toxic molecules fostered the inhibition of VFA production and
composition process. Ref. [60] varied the saponification tempera-
ture and further investigation of LCFAs degradation. It is investi-
gated the same scenario occurred as previous study on biogas
production and proved that the low biodegradation of LCFAs
accounted as the cause of the situation [43,59,60]. The selection
of alkaline reagent in saponification processes is also important in
determining the toxic effects towards microorganisms. It is sug-
gested the use of potassium hydroxide (KOH) reduced the toxicity
towards VFA production rather than Sodium Hydroxide (NaOH)
[64]. The effects of saponification on lipid inhibition are proven to
induce bioavailability during hydrolysis stage but unfortunately not
for overall biodegradability when enhancement on biogas produc-
tion is not significant increase. In this configuration, a further study
is needed to focus on LCFAs behaviour on saponification in order to
enhance biogas production.
8. Conclusions and directions for future research
Oily effluents and sludge are considered as potential substrates to
increase biodegradability and bioavailability in biogas production of
anaerobic treatment. The reported inhibition effects such as lag
357
phase during hydrolysis stage, sludge floatation and washout does
not restrain the exploitation of lipid wastes in anaerobic digestion to
produce biogas. The implementation of lipid wastes when co-
digested with low biodegradability substrates showed an improve-
ment in biogas production in terms of biodegradability of the mixed
substrates but not on bioavailability of the lipid wastewaters.
Additional research is needed to determine the dynamics of LCFAs
when co-digestion is implemented. It was found that lipid wastes
characteristics are diverse depending on its origin. Further study is
needed to discover the wide range of lipid wastes characteristics as
it will provide the overview of potential waste recovery rather than
being considered as a waste and discard to landfill. The anaerobic
digestions of these wastes have strong influence on the efficiency of
biogas yield. Improving of lipid inhibition can be achieved by
manipulating the LCFAs degradation through several techniques
and modification methods. Thus, additional research is needed to
determine the effects of modification techniques on lipid degrada-
tion to have better understanding on synergism and antagonism of
LCFAs degradation.
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