sustainability

Review
Impacts of Anaerobic Co-Digestion on Different Influencing
Parameters: A Critical Review
Mohammed Kelif Ibro 1 , Venkata Ramayya Ancha 1 and Dejene Beyene Lemma 2, *

1 Faculty of Mechanical Engineering, Jimma Institute of Technology, Jimma University,

Jimma P.O. Box 378, Ethiopia; mohamedkelifa@obu.edu.et (M.K.I.); dra.venkata@ju.edu.et (V.B.A.)
2 Faculty of Civil and Environmental Engineering, Jimma Institute of Technology, Jimma University,
Jimma P.O. Box 378, Ethiopia
* Correspondence: dejene.beyene@ju.edu.et

Abstract: Lignocellulosic feedstocks are year-round, available bio-residues that are the right candi-
dates for counteracting the energy crises and global warming facing the world today. However, lignin
leads to a slow hydrolysis rate and is a major bottleneck for biogas production via anaerobic digestion.
Anaerobic co-digestion (AcoD) is an economical method available, which overcomes the limitation of
a single feedstock’s properties in an anaerobic digestion process. This paper critically reviews the
impacts of co-digestion on lignocellulosic biomass degradation, process stability, various working
parameters, and microbial activities that improve methane yields. A combination of compatible
substrates is chosen to improve the biomethane yield and conversion rate of organic matter. AcoD is a
promising method in the delignification of lignocellulosic biomass as an acid pretreatment. Ultimate
practices to control the impact of co-digestion on system performances include co-feed selection,
in terms of both carbon-to-nitrogen (C/N) and mixing ratios, and other operating conditions. A
detailed analysis is performed using data reported in the recent past to assess the sensitivity of
influencing parameters on the resultant biogas yield. For the investigators motivated by the basic
principles of AcoD technology, this review paper generates baseline data for further research work
around co-digestion.
Citation: Ibro, M.K.; Ancha, V.R.;
Lemma, D.B. Impacts of Anaerobic
Keywords: biomethane potential (BMP); synergistic effect; biodegradability; anaerobic co-digestion;
Co-Digestion on Different
lignocellulosic biomass; operating parameters
Influencing Parameters: A Critical
Review. Sustainability 2022, 14, 9387.
https://doi.org/10.3390/su14159387

Academic Editor: Alessio Siciliano 1. Introduction

Received: 13 June 2022 Bioenergy is a key component in biorefinery, with bio-residues from other green
Accepted: 12 July 2022 economy sectors being used as raw material in bioenergy conversion processes. In this
Published: 31 July 2022 respect, biomass is one of the most promising renewable sources of energy generation.
It offers an opportunity to switch to renewable energy, making bioenergy (biogas) cost
Publisher’s Note: MDPI stays neutral
competitive. Biogas plants are the most competitive technology for energy generation
with regard to jurisdictional claims in
through organic waste management and an expected, respectable applicant for the future
published maps and institutional affil-
iations.
of renewable energy supply [1] under the umbrella of circular biorefinery. Anaerobic
digestion (AD) is the microbial degradation of complex organic matter in oxygen-limited
conditions [2], as shown in Figure 1. Several studies were conducted on the AD process
using sole feedstocks for biogas production [2–4]. However, feeding a single substrate to a
Copyright: © 2022 by the authors. biodigester does not produce sufficient methane yields due to several problems such as the
Licensee MDPI, Basel, Switzerland. stubborn nature of lignocellulosic material and lack of diversified microbes [4], the low and
This article is an open access article imbalanced C/N ratio [5], and the effect of operating conditions.
distributed under the terms and The concept of co-digestion to improve the methane yield using different substrate
conditions of the Creative Commons combinations is called anaerobic co-digestion (AcoD); it may create the best synergisms in
Attribution (CC BY) license (https:// the biodigester. There have been various pieces of research performed on the AcoD of lig-
creativecommons.org/licenses/by/ nocellulosic feedstocks with other different organic biomasses, such as animal manure [6,7],
4.0/).

Sustainability 2022, 14, 9387. https://doi.org/10.3390/su14159387 https://www.mdpi.com/journal/sustainability

Sustainability 2022, 14, 9387 2 of 19

food waste,
Sustainability 2022, 14, x FOR PEER REVIEW aquatic plants, and algal biomass, to improve the methane yield [8–10], as
2 of 20
shown in Figure 2.

Figure 1.
Figure 1. Microbiology
Microbiologyofof
organic material
organic in the
material in anaerobic co-digestion
the anaerobic systemsystem
co-digestion (adapted from
(adapted from [1]).
[1]).
AcoD may deliver significant advantages, including improved stability of the sys-
The concept of co-digestion to improve the methane yield using different substrate
tem [11], neutralization of toxic compounds [12], encouragement of a multiple microbe soci-
combinations is called anaerobic co-digestion (AcoD); it may create the best synergisms
ety, and better nutrient balance (appropriate C/N ratio and delivery of trace elements) [13],
in the biodigester. There have been various pieces of research performed on the AcoD of
providing the needed
lignocellulosic feedstocksamount of moisture
with other differentfor substrates
organic [14], increasing
biomasses, the organic
such as animal manure loading
rates
[6,7], (OLRs) [15],
food waste, and improving
aquatic the rate
plants, and algal of organic
biomass, degradation
to improve the methanein the digester
yield [8–10], [9]. As
per one review,
as shown in Figurethe
2. beneficial aspect of an enriched biogas yield of 400% was reported
for co-digestion compared
AcoD may deliver to digestion
significant of aincluding
advantages, sole feedstock
improved [1]. In addition,
stability regardless of
of the system
the
[11],substrate to inoculum
neutralization ratio, the methane
of toxic compounds yield increased
[12], encouragement by 120%
of a multiple with society,
microbe co-digestion of
and better
vicuñas nutrient
(VM) and balance
amaranth (appropriate C/N ratio
(AS) compared to and deliverydigestion
anaerobic of trace elements)
of a single[13],
substrate,
providing
which wasthe needed
higher amount of
compared to moisture for substrates
other studies [14], increasing
on co-digestion the organic
of animal load- with an
byproducts
ing rates (OLRs)
increment [15], and
in volatile improving
solid the rate ofoforganic
(VS) conversion about degradation
75% [16]. Ininthisthe regard,
digester this
[9]. review
As per one review, the beneficial aspect of an enriched biogas yield of 400%
paper details the impacts of a co-substrate on system performance, various operational was reported
for co-digestion compared to digestion of a sole feedstock [1]. In addition, regardless of
parameters, microbe status, and recent achievements. In addition, the status of the AcoD
the substrate to inoculum ratio, the methane yield increased by 120% with co-digestion of
process, current progress, and the future trend for further enhancements are discussed.
vicuñas (VM) and amaranth (AS) compared to anaerobic digestion of a single substrate,
which was higher compared to other studies on co-digestion of animal byproducts with
2. Microbiological Pathways in Anaerobic Co-Digestion Condition
an increment in volatile solid (VS) conversion of about 75% [16]. In this regard, this review
paperThe science
details behindofthe
the impacts AcoD process
a co-substrate is complex
on system as the biological
performance, degradation of
various operational
organic matter is performed by groups of anaerobic microorganisms through
parameters, microbe status, and recent achievements. In addition, the status of the AcoD a multi-step
process
process,in an oxygen-limited
current environment.
progress, and the future trendAn
for anaerobic condition involves
further enhancements hydrolysis, aci-
are discussed.
dogenesis, acetogenesis, and methanogenesis as the methane-producing last step (Figure 1).
2. Microbiological
The Pathways in critical
first step is a deliberately Anaerobic Co-Digestion
rate-limiting stageCondition
due to the barrier of lignin at the
Thethe
start of science behind the
AD process [1].AcoD process
The AcoD is complex
system makesas the
the biological
first step degradation of or-
stable by combining sub-
ganic matter
strates is performed
with high and slowbyhydrolysis
groups of anaerobic microorganisms
rates [2]. The second stage through a multi-step
is called the fermentation
process in
process, in an oxygen-limited
which all products environment.
of hydrolysisAnare
anaerobic
convertedcondition involves hydrolysis,
into alcohols, volatile fatty acids
acidogenesis, acetogenesis, and methanogenesis as the methane-producing
(VFAs), H2 , acetates, and CO2 with byproducts such as H2 S and NH last[17].
step (Fig-
In the third
3
ure 1). The first step is a deliberately critical rate-limiting stage due to the barrier of lignin
stage, acetogenesis, microorganisms convert organic acid into acetates, H2 , and CO2 , which
at the start of the AD process [1]. The AcoD system makes the first step stable by combin-
are utilized by methane-producing groups of microbes [18]. In the last stage, methanogen
ing substrates with high and slow hydrolysis rates [2]. The second stage is called the fer-
archaea
mentation use acetates,
process, H2, and
in which CO2 to form
all products methane
of hydrolysis are[19].
converted into alcohols, volatile
To sustain the activities of the acidifying and methane-producing
fatty acids (VFAs), H2, acetates, and CO2 with byproducts such as H2S and bacteria,
NH3 [17].theInmethano-
genesis
the third stage, acetogenesis, microorganisms convert organic acid into acetates, H2,and
stage should be carried out at a pH above 6.6, ideally between 6.8 and 7.2 [20].
Caruso et al. [21] identified some conditions that may block microbial activity, including a
shortage of nutrients and the existence of barrier chemicals, such as sulfide, which cause a
Sustainability 2022, 14, 9387 3 of 19

drop in pH and excess VFA accumulation [21]. Thus, co-digestion provides the micro- and
macronutrients for microbial growth [22]. Generally, the AcoD process is a promising strat-
egy for keeping the optimum pH constant and permitting digester stability by buffering
the extreme acidification/alkalinity conditions for groups of archaea [23].

3. Principal Parameters and Factors Affecting Microbial Activity during AcoD

3.1. Temperature
Based on the set-up condition, the optimum temperature of the AcoD route is classified
as thermophilic (55 ◦ C) [24], psychrophilic (20 ◦ C) [25], or mesophilic (35 ◦ C) [26]. In ther-
mophilic conditions, the anaerobic process improves pathogen reduction, achieves greater
conversion rates, and achieves a shorter digester hydraulic retention time [27]. However,
thermophilic conditions are harder to control and demand more energy to heat to keep the
AD at ideal condition [28]. Thermophilic groups are more sensitive to changes in operating
conditions and environmental fluctuations than mesophilic conditions [27]. In addition,
temperature influences digester performance when it is less than optimum and reduces the
substrate consumption in the digester (i.e., causes substrate turnover), microbial growth
rates, microbial diversity [29], and methane production rate, while higher temperatures
increase the degradation, VS removal rate, and process stability [27]. Uma et al. [30] com-
bined food waste (FW) and switchgrass (SG) at different mixing ratios, focusing on the
effect of temperature on the process performances. They achieved maximum methane
yields of 267 mL/g and 234 mL/g (VS) from FW:SG (1:1) under 35 ◦ C and 55 ◦ C, respec-
tively. They revealed that the mesophilic temperature (35 ◦ C) showed the highest methane
yield with better performance.

3.2. Total Solid (TS) and Volatile Solid (VS)

The volatile solid (VS) and total solids (TS) content are useful indicators of biogas
conversion rates [31]. The TS of a feed indicate how much moisture is available in the
substrates. The higher the TS content, the more volatile fatty acids (VFAs) are accumu-
lated [32]. Similarly, reactors containing lower TS content showed higher methane yield in
mono-digestion of dairy manure [25]. Combining two or more substrates may improve
the degradability of TS in organic waste due to moisture increments in the digester [32].
VS content allows evaluation of the AD performance, biogas potential approximation, and
biomass decomposition rate. For instance, Elsayed et al. [33] investigated the conversion
efficiency of organic matter at different mixing ratios of primary sludge (PS), fruit, and
vegetable wastes (FVW) through batch mesophilic conditions. The highest VS removal rates
of 73% and 70% were observed at mixing ratios of 70:30 and 50:50, respectively. However,
the maximum cumulative methane yield was produced at PS:FVW 50:50. Klarosk et al. [34]
defined VS as the amount of organic matter that may be converted to biogas. The greater
the VS amount, the better the anaerobic digestion process (higher biogas production). VS
removal efficiency indicates the synergistic effect of co-digestion due to a blend of supple-
ment substrates [35]. Considering this benefit, Hamrouni [36] digested food waste with
sewage sludge (SS) at different mixing ratios. They revealed that a co-substrate improved
the VS removal efficiency for SS.

3.3. Carbon to Nitrogen Ratio

The archaea are highly sensitive to the C/N proportion that indicates deficient nutrient
levels of feedstocks. Assessing C/N balance is the method for minimizing or avoiding the
issue of ammonia in the anaerobic digestion process [37]. When the C/N ratio is greater
than the ideal (Tables 1 and 2), it means deficient nitrogen (or underuse of carbon), which is
consumed rapidly by methanogens and leads to low biogas production [38]. Nitrogenous
substrates, such as animal manure and chicken droppings, might be required to boost
biogas generation in this situation. A low C/N ratio indicates insufficient carbon content
or nitrogen underuse, resulting in ammonia buildup, low pH, and the presence of phenolic
chemicals [39]. Carbon-rich substrates, such as kitchen waste and energy crops, might
Sustainability 2022, 14, 9387 4 of 19

be required to improve biogas production [38]. As a result, the digestion of energy crops
alone may result in significantly low CH4 yield (i.e., CO2 -rich biogas with poor CH4 ) if an
optimum C/N ratio (20–30:1) is not achieved [40].
The AcoD process is more suitable for optimizing the value of the C/N ratio than
single substrate-based mono-digestion [1]. Substrates with the best carbon to nitrogen
ratio may ensure the desired nutrition for the microbes’ activities. In a previous study, a
C/N ratio of 25 resulted in the best biogas yields when rice straw and Hydrilla verticillata
substrates were co-digested [41]. In other experiments, biogas yield was boosted by 100.2%
over the control with a C/N ratio of 25:1 over the value of 9 [42]. When the ratio of
food waste to rice straw was tuned at C/N 30, pH 7.32, and F/M 1.87, the CH4 yield
increased by 94.41% over rice straw mono-digestion [10]. Contrastingly, the oily biological
sludge’s C/N ratio is lower than the ideal 20–30 ratio required by anaerobic digestion
technology for desired biogas yield [37]. The C/N proportions of various single feedstocks
are shown in Table 1. However, determining the appropriate C/N ratio for co-substrates is
difficult because many parameters, such as substrate type, trace element content, chemical
components, and biodegradability, can all influence the best value. When the C/N ratio
deviates from the ideal, the system becomes unstable, and biogas output suffers.

Table 1. Different substrates characterized by C/N ratios lower and higher than the optimum value.

Comparatively Lower C/N Value Materials Comparatively Higher C/N Value Materials
Lower C/N
Substrates References Substrates Higher C/N Value > 24 References
Value < 23
Chicken manure 9.27 [43] Corn stover 42.92 [43]
Vicuñas (VM) 15.40 [16] Olive mill solid waste (OMSW) 31.4 [44]
Rugulopteryx okamurae 15.2 [44] R. Okamurae—OMSW 27.4 [44]
CCF 13 [8] Raw llama dung 26.8 [45]
Pig manure 11.70 [46] Buckwheat hull 43.8 [47]
Raw dromedary dung 22.2 [45] Cardboard (CB) 163 [48]
Palm oil mill effluent 9.7 [49] Corn stover 40.8 [46]
Slaughterhouse waste 13.7 [47] Brewery trub 33 [47]
Cucumber residues 14.76 [46] Fruit wastes 44.7 [47]
Sewage sludge 8.5–12 [2,47] Sophora flavescens residues 65.64 [2]
Dairy manure 22.5 [47] Coffee husk 86 [20]
Water hyacinth 19.5 [50] Cactus 27.9 [51]
MSW 18.4 [50] Decanter cake 49.54 [49]
Llama manure (LM) 17.40 [16] Food waste 24.6 [2]
Microalgae 15.3 [52]
Empty fruit bunch 12.86 [49]

3.4. Retention Time

The average time it takes slurry to transfer between the digester’s entrance and exit
is hydraulic retention time (HRT). Another retention time is solid retention time (SRT),
described as the total length of time bacteria spend in the digester [25,53]. Microorganisms
require sufficient retention time to convert organic substrates into desired products [54].
Long HRT improves the effluent quality [55], but, with longer residence time, the reaction
rate decreases. If HRT is less than the optimal value, there is an accumulation of VFAs that
inhibit bacterial activity, resulting in low biogas yields [56]. The proliferation of acetic acid
is the main cause of the process disturbance observed at short HRTs [24]. In addition, low
C increases the risk of biomass removal from the bioreactor, which may negatively affect
the constancy of the entire system with VFA accumulation and increasing alkalinity [25].
Furthermore, the time it takes anaerobic digestion to degrade any waste is determined
by the feedstock and ambient factors such as temperature. Anaerobic mono-digestion of
lignocellulosic material is difficult due to low moisture content and slow mass transfer
within the blend of matter in the reactor and requires a long retention time to degrade. To
compensate for the drawbacks of AD of lignocellulosic biomass, Wickramarachchi et al. [57]
conducted three serial experiments on co-digestion of rice straw (RS) and cow dung (CD),
recycling slurry from the first to the second and from the second to the third reactors as
Sustainability 2022, 14, 9387 5 of 19

inoculum under batch mesophilic conditions. In the second experiment, the lag phase
dropped from 14 days to zero, with increments of methane yields of 104%. The optimum
HRT for different temperatures is shown in Table 2.

Table 2. Operating parameters and optimum ranges for the anaerobic digestion process.

Operational Parameter Optimum Reference

pH overall 6–8.5 (ideal 6.8–7.2)
Methanogenesis 6.8–8.0 [20,58]
Alkalinity 1000–5000 mg/L as CaCO3
C/N 20–30:1 [37]
70–80 days at a psychrophilic
temperature, 12–40 days at mesophilic
HRT [53,59]
temperature, and 15–20 days at
thermophilic temperature
OLR 0.5–4.7 kg VS/m3 d [60]
Particle size Less than 10 mm is recommended [60]
Semi-dry, wet, dry 10–20%, ≤10%, ≥20%, respectively [14]

3.5. Ammonia
Biodegradation of nitrogenous materials produces ammonia as a byproduct. Above
threshold concentrations, free ammonia nitrogen (FAN), dependent on pH and tempera-
ture [61], is a significant inhibitor species of total ammonia in an anaerobic digester [62].
Thus, the excess accumulation of ammonia in the digester increases pH and finally causes
the failure of a process [63]. Due to the synergistic effect, the AcoD process recovers the
excess ammonia inhibition by combining nitrogen-rich and carbon-rich substrates. Pig
manure (PM) is rich in ammonia that might affect anaerobic microbial activity. For example,
pig manure was added to carbon-rich organic matter as a neutralizing agent to assist in
optimizing the AcoD process via avoiding acidification through VFA neutralization in
the digestion of olive mill waste that was poor in nitrogen [39]. In another experiment,
Fadairo et al. [64] mixed poultry litter and cow dung with water hyacinth (2:2:1) and
achieved the highest biogas yield of 3.073 L/kg VS over sole digestion of water hyacinth
without addition of any improving agent.

4. Effects of Anaerobic Co-Digestion on Different Parameters

4.1. As Chemical Pretreatment
To improve the hydrolysis rates of lignocellulosic biomasses, different pretreatment
methods have been tried; however, pretreatment is always related to cost and high-energy
requirements. In addition, some pretreatment leads to the formation of compounds toxic to
microorganisms, resulting in low biogas production [65,66]. The anaerobic co-digestion
system overcomes the problem of chemical pretreatment by simultaneously introducing
two compatible substrates in a sole bioreactor (Figure 3). As acid pretreatment, food waste
(FW) that may be able to acidify during the digestion of a corncob was explored for eight
days under mesophilic conditions [67]. Specifically, the effects of FW as pretreatment on
different parameters such as VFAs, pH, lignocellulosic organic matter, and strength were
considered. The result demonstrated that the addition of food waste increased the amount
of VFA in the digester and enhanced the activities of microbial enzymes. A hydrolysis
efficiency improvement of 28% over the control substrate, reduction of crystallinity by 6.7%,
and an increment of cellulose digestibility of 13.2% were achieved. In the methanogenesis
step, the biodigester offered the best stability with better efficiency in hydrolysis with pH
recovery in the optimum range (pH 6.3–7.2). In addition, the result of kinetics analysis
revealed that the pretreatment of FW might assist cellulose digestibility [67].
 of VFA in the digester and enhanced the activities of microbial enzymes. A hydrolysi
efficiency improvement of 28% over the control substrate, reduction of crystallinity b
6.7%, and an increment of cellulose digestibility of 13.2% were achieved. In the methano
genesis step, the biodigester offered the best stability with better efficiency in hydrolysi
Sustainability 2022, 14, 9387 6 of 19
with pH recovery in the optimum range (pH 6.3–7.2). In addition, the result of kinetic
analysis revealed that the pretreatment of FW might assist cellulose digestibility [67].

75% YW+25% FW

Pecentages of co-substrates
100%YW
100%CB
75%DSCG + 25%STW
FW:SFR(7:3)
CS:CM(1:2)
CS(1:0)
GM (1:0)
GM:CG (0:1)
0 100 200 300 400 500 600 700

Comparison of theoretical and experimental methane yields

Experimental CH4 (mL/gvs) Theoretical maximum CH4 (mL/gvs)

Figure
Figure 2. 2.
The The
bestbest theoretical
theoretical and experimental
and experimental methanemethane yieldsfrom
yields obtained obtained from lignocellulosic
lignocellulosic biomass bio
mass mono/co-digestion with food waste and animal manure versus mixing
mono/co-digestion with food waste and animal manure versus mixing ratios (Table 3). Defatted ratios (Table 3). Defa
ted spent
spent coffee grounds
coffee grounds (DSCG); macroalgae,
(DSCG); macroalgae, MCsp.);
MC (Cladophora (Cladophora sp);
glycerin (G); glycerin
spent (G); spent coffe
coffee grounds
grounds
(SCG); spent (SCG); spent
tea waste tea chicken
(STG); waste (STG);
manurechicken manure
(CM); cotton (CM);(CG);
gin trash cotton
goatgin trash(GM);
manure (CG);corn
goat manur
(GM);and
stover; corn stover;
Sophora and Sophora
flavescens flavescens
residues (SFR). residues (SFR).

700
The maximum theoretical and

600
experimental methane

500
400
300
200
100
0

C/N ratios

Theoretical maximum CH4 (mL/gvs) Experimental CH4 (mL/gvs)

Figure3.3.Comparison
Figure Comparisonof of
thethe maximum
maximum theoretical
theoretical and and experimental
experimental methane
methane of lignocellulosic b
of lignocellulosic
omass mono/co-digestion
biomass withfood
mono/co-digestion with foodwastes
wastes and
and animal
animal manure
manure versus
versus carbon-to-nitrogen
carbon-to-nitrogen ratio ratio (Ta
ble 3).
(Table 3).

InInthe classical
the anaerobic
classical system,
anaerobic chemical
system, buffering
chemical methods
buffering using chemicals
methods such
using chemicals suc
as sodium bicarbonate, calcium oxide [68], HCl, and NaOH, were reported as able to
as sodium bicarbonate, calcium oxide [68], HCl, and NaOH, were reported as able t
maintain the ideal pH setting [43,69]. Nevertheless, these agents are said to have a cost
maintain the ideal pH setting [43,69]. Nevertheless, these agents are said to have a cos
and may unfavorably hinder bioacid- and methane-forming microbes [68]. As a result, an
and may
organic unfavorably
co-substrate hinder bioacid-
that primarily and
operates as amethane-forming
stabilizing agent tomicrobes
maintain [68]. As a result, a
pH during
organic co-substrate
single-stage AD of FW is that primarily
urgently operates
needed. as of
In light a stabilizing agent toanaerobic
this, the mesophilic maintainco- pH durin
single-stage AD of FW is urgently needed. In light of this, the mesophilic
digestion of FW and grass clippings (GC) feedstock was examined as an environmentally anaerobic co
digestion
beneficial of FW andagent.
neutralizing grassThe
clippings (GC) feedstock
results showed that using was
GC toexamined
prevent pH asdecline
an environmentall
in the
biodigester
beneficialhelped to alleviate
neutralizing the The
agent. redoxresults
environment
showed andthat
increase
usingtheGCchosen bioproducts,
to prevent pH decline i
making the process more economical [68]. The acetic-acid-rich food waste assisted the
derivation of lignin and depolymerization of cardboard without the help of the pretreatment
step. In addition to focusing on expanding the potential of furfural wastewater as a low-cost
acid pretreatment agent by substituting conventional acid pretreatment, Wang et al. [70]
combined crop stalk and furfural wastewater at 20, 35, and 50 ◦ C for 3, 6, and 9 days and
conducted batch experiments for 25 days at 35 ◦ C. The result showed that the maximum
Sustainability 2022, 14, 9387 7 of 19

total biogas product (196.68 mL/g VS) was produced by the treatment at 35 ◦ C for six
days, which was 59.28% greater than that produced by crop stalk without treatment. They
revealed furfural wastewater as a feasible pretreatment agent for improving biogas in
anaerobic co-digestion.

4.2. Synergistic Effect

4.2.1. Theoretical Biomethane Potentials
In the AcoD process, the biomethane potential (BMP) test may give the parameters
to predict the theoretical BMPs of two or more co-substrates. Recently, several studies
were performed on the effect of co-substrates on theoretical biomethane potentials (Table 3).
For example, Kaur et al. [71] studied the effect of goat manure on the theoretical methane
potential of lignocellulosic under different mixing ratios. The result indicated that the
co-substrates did not enhance the theoretical methane of cotton gin trash in all mixing
proportions. The maximum theoretical methane yield was obtained from mono-digestion
of cotton gin trash with low biodegradability at a C/N ratio of 36. In addition, the co-
digestion that obtained the maximum experimental methane yield did not show a similar
trend for theoretical methane estimates [71]. In contrast, the maximum theoretical and
experimental methane yield from mixtures of 25% yard waste (YW) + 75% FW [72] and
combinations of 80% food waste and 20% cardboard [48] were attained (Table 3). Similarly,
the better theoretical and experimental methane yield was obtained at the same mixing
ratio [73].

Table 3. Theoretical and experimental methane yields of lignocellulosic biomass mono/co-digestion

with food wastes and animal manure.

Theoretical
Experimental CH4
Mono/Co-Substrates C/N Mode Conditions Maximum CH4 References
(mL/g VS)
(mL/g VS)
Cotton gin trash (0:1) 36 BMP test, 36 ◦ C ± 1 451.0 169.6 [71]
Goat manure:cotton gin trash (0.1:0.9) 32.2 BMP test, 36 ◦ C ± 1 428.5 189.0 [71]
Goat manure (1:0) 15 BMP test, 36 ◦ C ± 1 290 274.1 [71]
Goat manure:cotton gin trash (0.9:0.1) 17.7 BMP test, 36 ◦ C ± 1 313.0 261.4 [71]
Corn stover (1:0) 42.9 Batch scale, 37 ◦ C 555.81 240 [43]
Chicken manure (0:1) 9.27 batch scale, 37 ◦ C 401.32 298.21 [43]
Corn stover:chicken manure (1:2) 21 Batch scale, 37 ◦ C 452.82 280 [43]
FW (10:0) 24.61 Batch, 37 ◦ C 513 nd [2]
FW:Sophora flavescens residues (7:3) 25.8 Batch, 37 ◦ C 503 nd [2]
100% DSCG 24 Batch, 37 ◦ C 483 336 [74]
75% DSCG:25% STW 24.3 Batch, 37 ◦ C 481 231 ± 12 [74]
25% DSCG:75% MC 24.2 Batch, 37 ◦ C 333.7 260 [74]
CB 160 Batch, 37 ◦ C 450 [48]
80% FW and 20% CB 77.9 Batch, 37 ◦ C 610 240 [48]
YW 74 BMP test, 37 ◦ C 497.9 49 [72]
25% YW + 75% FW 29 BMP test, 37 ◦ C 637.4 360 [72]
75% YW + 25% FW 59 BMP test, 37 ◦ C 509 165 [72]

Comparatively, the maximum theoretical and experimental methane yields were

achieved from co-digestion of yard waste and food waste (Figure 2). However, for most
studies, the trend of theoretical and experimental methane yields was the opposite in the
combination of two or more substrates. When theoretical methane yields decreased, the
experimental methane yields increased for the different mixing ratios in the co-digestion
of other substrates (Table 3 and Figure 2). In Figure 3, the effects of the carbon to nitrogen
ratio on the production of theoretical and experimental methane yield are presented. This
graph does not specify the effect of the C/N ratio on theoretical methane yield. The better
theoretical methane yields were produced at the low or high values of C/N ratios. However,
the maximum methane yields were produced at the optimum values of the C/N ratio (24
and 29) (Table 3 and Figure 3).
Sustainability 2022, 14, 9387 8 of 19

4.2.2. Biogas Yield

Lignocellulosic biomass is stubborn, it is difficult to degrade it without the aid of
pretreatment, and microorganisms consume resources quickly, resulting in an imbalance
between acidogenesis and methanogenesis during the first phase of AD. This fluctuation
is owing to the excess formation of volatile fatty acids (VFAs) [19,75]. In the past, in
AD, different wastes, such as municipal solid waste, food waste, kitchen waste, and
animal manure, were used for biogas generation [50,76–78]. However, recently, attention
has shifted to the co-digestion of lignocellulose material with kitchen waste, food waste,
and animal manure to generate biogas due to various advantages [14,47,79]. The major
drawback of food waste digestion is the fast hydrolysis rate and the resulting drop in pH
(<5.5) because of the accumulation of volatile fatty acids [80]. Therefore, co-digestion of
food waste with stubborn feedstocks can help to retard the hydrolysis rate and reduce
the VFAs [81]. El et al. [81] co-digested agricultural waste, such as wheat straw and cow
manure, with food waste in semi-continuous mesophilic AD and considered the effect of
mixing ratio, C/N ratio, and variation of organic loading rates from 2 to 3.6 kg VS/m3 d on
the digester performance. The result demonstrated that the highest biogas yield increased
by 119.97% for the organic load of 3.6 kg VS/m3 d fed with the optimum mixing ratio of
FW:CM (75:25) and a C/N ratio of 20.03.
As discussed before, the essential need for co-digestion is to achieve synergy between
feedstocks in terms of yields of biogas and methane. This means that biogas obtained
from digesting two feedstocks at once is higher than the amount produced by substrate
mono-digestion (AC > A + C) [80,82]. The synergistic index (α) is used to assess the
interaction between two or more feedstocks fed into a biodigester at the same time as
co-substrates. If α > 1, the synergistic effect occurs; if α < 1, an antagonistic effect occurs.
The antagonistic effect is caused when incompatible feedstocks are co-digested [72], and,
if α = 1, the interaction between co-substrates is unclear [43]. This parameter is used to
indicate the performance of the process; however, it is not used to indicate the maximum
methane yield, which means that the maximum synergistic index does not promise the
highest methane yield [83,84]. Accordingly, both specific methane yield and the synergistic
index can be measured to decide the optimum mixing ratio in AcoD. The best synergistic
index, mixing ratios, and methane yields from several studies are shown in Figure 4
and Table 4. Furthermore, the approaches to improve the biogas yields of lignocellulosic
biomasses based on co-digestion are shown in Figure 5.
Other important advantages attained with anaerobic co-digestion are the neutral-
ization of inhibitory compounds, cost reduction through digestion of two or more sub-
strates [85–87] and subsequent greenhouse emissions reduction [20]. Moreover, it decreases
the hydrolysis rate [88]. Cow manure contributes to maintaining the digesters’ optimal
pH values, although its strong neutralizing power is unrelated to the C:N ratio. In another
study, meadow grass and wheat straw digested with cattle manure in thermophilic (53 ◦ C)
conditions exhibited enhancement of biomethane by 20–24% over straw digestion alone.
Furthermore, they achieved optimum co-digestion with a maximum yield of methane at
25%, the smallest lag of 6–7 days after 75% of organic matter was initiated from cattle
manure, and the overall biodegradability, as compared to single feedstock digestion, was
enriched and boosted methane yield [89].
 Sustainability
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2022,
2022,
2022, 14,9387
x FOR PEER
x FOR REVIEW
PEER REVIEW 9 of2019
9 of9 20
of

Synergistic effect
Synergistic effect
1.4 1.4
co-digestion interaction performances
co-digestion interaction performances

1.2 1.2

1 1

0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2

0 0

Co-substrates
Co-substrates

Figure 4. The
Figure
Figure 4. co-digestion
4. The
The interaction
co-digestion
co-digestion index
interaction
interaction (α)(α)
index
index of various
(α) of co-feedstocks
of various
various (Table
co-feedstocks
co-feedstocks 4). 4).
(Table
(Table 4).

Figure 5. Innovative
Figure
Figure 5. approaches
5. Innovative
Innovative based
approaches on on
based co-digestion to improve
co-digestion
co-digestion to lignocellulosic
to improve
improve biomass
lignocellulosic
lignocellulosic viavia
biomass
biomass AD.
viaAD.
AD.

Table 4. Operating
Other
Otherimportantparameters
important for attained
advantages
advantages AcoD andwith
attainedtheir respective
anaerobic
with achievements
co-digestion
anaerobic in are
are
co-digestion terms
thetheofneutraliza-
biomethane
neutraliza-
yields
tiontion ofwith bettercompounds,
of inhibitory
inhibitorybiodegradability.
compounds,cost reduction
cost reductionthrough digestion
through of two
digestion or more
of two or moresubstrates
substrates
[85–87]
[85–87] andand
subsequent
subsequent greenhouse
greenhouse emissions
emissions reduction
reduction [20]. Moreover,
[20]. Moreover, it decreases
it decreases thethe
Mode and Synergistic
Co-Substrate hydrolysis
hydrolysis C/N
raterate
[88]. BD
Cow
[88]. Cow
th (%)
manure
manure contributes
contributes to to
maintaining
maintaining Methane
thethe Yields
digesters’
digesters’ optimalReferences
optimal pHpH
Condition Effect
values,
values,
Cabbage cauliflower and FW (0.36:0.64) although
although
45 its its
strong
98 neutralizing
strong BMP test atpower
neutralizing 37 ◦power
C is unrelated
is unrelated
0.9 to 475
themL
to C:N
the ratio.
STPC:N
CH VSIn In
ratio.
4 /g another
another
[8]
study,
study,
Cabbage and cauliflower FW (0.14:0.86) meadow 56 grass
meadow and
grass 85andwheat
wheatstraw
BMP test,digested
straw 37 ◦digested
C withwithcattle
0.85 manure
cattle manure
433 inCH
mLSTP thermophilic
in thermophilic
4 /g VS
(53(53
[8]
°C)°C)
Corn stover:chicken waste (1:2) conditions 21 exhibited
conditions exhibited enhancement
70.60 Labscale,of
enhancement biomethane
37 ◦of
C biomethane 1 by by20–24%
20–24% over
319.70 straw
over
mL/g VSstrawdigestion
digestion
[43]
alone.
Corn stover:chicken waste (1:1) alone.Furthermore,
Furthermore,
26 they
theyachieved
60.02 achieved optimum 37 ◦ C co-digestion
optimum
Labscale, co-digestion
1 with
witha 287.28
maximummL/g VSyield
a maximum of me-
yield of
[43]me-
FW:CB (0.8:0.2) thane
thane at 25%,
at 25%,
60the smallest
the smallest
39 lag of
lag 6–7
of days
6–7 days after
Pilot scale, 37 C◦ 75%
after of
75% 0.7organic
of organic matter
matterwaswas
240 mL/g VS initiated from
initiated from
[48]
FW:SFR (7:3) cattle
cattlemanure,
manure,
25.8 and andthe overall
the
58.83 biodegradability,
overall biodegradability,
Batch, 37 C ◦ as compared
as compared
1.19 to single
to feedstock
single
640 mL/g VS feedstockdiges-
diges-
[2]
tion,
Food waste:Sophora flavescens residues was
tion,
(5:5) enriched
was enriched
27.3 andandboosted
boosted
58.11 methane
Batch, 37yield
methane [89].
◦ C yield [89].1.21 629 mL/g VS [2]
Food waste:sewage sludge (3:1) 28 40 Batch, 37 ◦ C 0.88 452 mL/g VS [36]
4.2.3. Microbes
4.2.3.
Defatted spent coffee grounds:spent tea
Delivery
Microbes Delivery
24.2 66.4 BMP, 37 ◦ C 1.06 318 mL/g VS [74]
grounds (0.5:0.5)
Different
Different substrates
substratescontain microorganisms
contain microorganisms important
important forfor
biodegradation,
biodegradation, simulta-
simulta-
Defatted spent coffee grounds:macroalgae
neously sustaining various
neously sustaining
24.2 77.9 archaea
various archaeathrough
BMP, 37 C co-digestion
through
◦ 1.01 and
co-digestion limiting
and limitingthethe
260 mL/g riskrisk
VS of microbe
of microbe
[74]
(0.25:0.75)
wash wash
Defatted spent coffee grounds:spent coffee
away.
away.Co-digestion
Co-digestion has the
has potential
the potential to ensure
to a
ensure microbial
a population
microbial population with
witha more
a more
24.8 BMP, 37 ◦ C
grounds (0.75:0.25) complex
complex diversity than
diversity a64.3
than sole substrate
a sole substrateas varied
as varied 0.9
microbes
microbes areare 306 mL/g VS
constantly
constantlyhosted
hostedfrom [74]
fromco-co-
substrates
Meadow grass:wheat straw:cattle manure [90].
substrates 34
Thus,
[90]. anaerobic
Thus, anaerobic
83
co-digestion
co-digestion
Batch, 53 ◦ C
of compatible
of compatible
1.18
substrates
substratesimproves
improves
351 mL/g VS
thethe
stabil-
stabil-
[89]
(0.75:0.75:0.25)
Sustainability 2022, 14, 9387 10 of 19

4.2.3. Microbes Delivery

Different substrates contain microorganisms important for biodegradation, simulta-
neously sustaining various archaea through co-digestion and limiting the risk of microbe
wash away. Co-digestion has the potential to ensure a microbial population with a more
complex diversity than a sole substrate as varied microbes are constantly hosted from
co-substrates [90]. Thus, anaerobic co-digestion of compatible substrates improves the
stability of the bacteria population. In classical AD, it is mostly groups of phyla, such as Fir-
micutes, Bacteroidetes, Fibrobacteres, Proteobacteria, and Actinobacteria, that are dominant [91].
However, microorganism structures depend on the types of feed in a digester. For example,
a sewage-sludge-fed AD system sustained Microtrichaceae, the mono-digestion of cellulose
had a bacterial population dominated by Ruminococcaceae (79.20%), and, for hemicellulose,
Clostridiaceae dominated by 84.57%. In the same study, the co-digestion of sewage sludge
and lignocellulosic biomass was dominated by Actinobacteria (40%), Proteobacteria (14.38%),
and Chloroflexi (23.89%), which showed improved bacterial population diversity over a sole
substrate [91].
In another study, Zhang et al. [92] co-digested FW, cattle manure (CM), and corn
straw (CS) in a mesophilic batch experiment focusing on the relation between synergy and
microbes. The result showed that 65% FW + 35% CM digestion maintained the principal
growth of hydrogenotrophic methanogens (68.9%) with maximum synergy because of
dual-effect neutralizing inhibitory compounds. Different mixing fractions of feedstocks and
working conditions also affect the microbe structure. For example, Shi et al. [29] studied
the effects of working conditions and co-feed ratio on microbe structure and the stability of
the system using the co-digestion of wheat straw and food waste under batch thermophilic
and mesophilic conditions. The result indicated that, for a FW sole substrate, both digesters
were disturbed. Further, an excess concentration of VFA failed in a mesophilic digester,
and a thermophilic digester showed better stability than the mesophilic one. In addition,
raising the fraction of FW supported the bacteria group of the phylum Thermotoga with
the largest numbers in the thermophilic digester, whereas the phylum Bacteroidetes was
the largest in the mesophilic digester [29]. It is worth noting that microbial diversity is not
the only thing to examine when assessing the efficacy of AD processes; the community’s
functional status and resilience are also crucial. It is well acknowledged that co-feedstocks
improve stability and overall digestibility by facilitating enzymatic activities through a
better balance of micronutrients and trace minerals [90,93,94].

4.3. Biodegradability (BD)

Biodegradation involves microbiological degradation of biowaste by microbes under
oxygen-deficient conditions for anaerobic digestion or oxygen-sufficient conditions for
aerobic. Thus, it is used to realize the theoretical biomethane potential of biomasses. The
realization of the theoretical potential is equal to the actual yields divided by theoretical
potentials. Mathematically can be defined as

Actual yields
%BDth = ∗ 100 (1)
Theoratical poteantials

There are various physiochemical and natural aspects that affect the biodegradabil-
ity of an organic substance, including bioaccessibility, temperature, pH, and moisture
content [1]. There are several pretreatment methods, such as physical, chemical [95], bi-
ological [42], and physiochemical methods, and their combination, which are applied to
improve the digestibility of lignocellulosic materials before the AD process to enhance the
volume of biogas of sufficient quality by facilitating lignin removal and the destruction of
the complex structure of organic biomass [96,97]. For example, as per reported studies on
pretreatment approaches for biogas production, a 1200% boost in the yield of biogas was
achieved with ionic liquid pretreatment of lignocellulose [98]. In addition, the pretreatment
must be effective and economical and ideally meet the following requirements: expose
lignin to enzymatic destruction, have a lesser effect on hemicellulose and cellulose destruc-
Sustainability 2022, 14, 9387 11 of 19

tion, minimize the production of an inhibitory compound for enzymes and fermenting
microbes, and minimize cost and the energy requirement [97,99]. Each of these methods
has individual negative and positive effects. As a result, one technique cannot apply to all
kinds of lignocellulosic material. Thus, a single pretreatment technique that achieves all
criteria for various types of feedstocks is still not available.
In the AcoD process, the biochemical methane potential (BMP), as in [2], may help to
anaerobically evaluate the biodegradability and the quantum of organic matter in various
feedstocks that change to biomethane during anaerobic digestion [100]. In addition, the
BMP may be helpful for determining the organic content (VS) in the substrates that change
to methane (biogas) in a given amount of time and remains for further management. It
can also help researchers to figure out the best mixing ratios for the co-digestion pro-
cess [47,95,101]. Hamrouni [36] co-digested Mediterranean FW and sewage sludge (SS) in
both batch and semi-continuous experiments to predict the degradation using the BMP
test. The report showed that FW had a higher biodegradability than SS. In line with this
observation, Hamrouni [36] also found that, when mixed at SS:FW 1:3, the feed resulted in
Sustainability 2022, 14, x FOR PEER REVIEW 12 of 20
better biogas production and minimum biodegradability, which dropped the BD of FW to
40% (Figure 6).

100

80
biodegradability %

60

40

20

0
C/N ratios

45 56 21 26 60 25.8 27.3 28 24.2 24.2 24.8 34

Figure 6.
Figure 6. Realization
Realization of
of theoretical
theoreticalmethane
methanepotential
potentialofoforganic
organicwaste
wasteco-digestion versus
co-digestion C/N
versus ra-
C/N
tios (Table 4).
ratios (Table 4).

Cardboard
Cardboard and and acidified
acidified foodfood waste
waste were
were co-digested
co-digested to to improve
improve digestibility
digestibility under
under
mesophilic conditions.
mesophilic conditions. The highest
highest methane yield of 0.24 L/kg and biodegradability of
methane yield of 0.24 L/kg and biodegradability of
39%
39% was obtained, with better stability
was obtained, with better stability without
without any any pretreatment
pretreatment method.
method. However,
However, due due
to
to low
low moisture
moisture content
content and
and slow
slow mass
mass transfer
transfer within
within thethe reactor’s
reactor’s blend of matter,
matter, solid
solid
anaerobic
anaerobic co-digestion
co-digestion of of lignocellulosic
lignocellulosic material
material had had limitations
limitations [48].
[48]. For
For instance,
instance, based
based
on
on the
the benefits
benefits ofof AcoD,
AcoD, the
the balance
balance ofof pH
pH for
for acidogenesis
acidogenesis and and methane-forming
methane-forming archaea archaea
was studied under mesophilic conditions to improve system stability
was studied under mesophilic conditions to improve system stability and yield. The find- and yield. The
findings demonstrated that adding 20% of food waste to garden
ings demonstrated that adding 20% of food waste to garden waste enhanced methane and waste enhanced methane
and organic
organic material
material elimination
elimination by 83%
by 83% (VS)(VS)
[34].[34].
BMP
BMP tests may indicate the parameters totopredict
tests may indicate the parameters predictthethedegradation
degradation of of organic
organic mat-
matter.
ter.
In theIn co-digestion
the co-digestionrole,role, combining
combining different
different feedstocks
feedstocks mightmight at least,
at least, improve improve the
the biodi-
biodigestibility of one substrate as the balance of the BD of both.
gestibility of one substrate as the balance of the BD of both. With this baseline, Atelge et With this baseline,
Atelge
al. [74]etstudied
al. [74] digestion
studied digestion of defatted
of defatted spent grounds
spent coffee coffee grounds
(DSCG) (DSCG)
without without
other other
feed-
feedstocks and co-digestion with other feedstocks, such
stocks and co-digestion with other feedstocks, such as macroalgae (MC), spentas macroalgae (MC), spent tea
tea
grounds
grounds(STG),(STG),and
andspent
spent coffee
coffeegrounds
grounds (SCG),
(SCG), at batch mesophilic
at batch conditions
mesophilic for 49for
conditions days49
to improve
days the performance
to improve the performanceof theofsystem. The result
the system. demonstrated
The result that, for
demonstrated a sole
that, for afeed,
sole
in the case of VS conversion, the better removal efficiency of 35.48% reflected that the
feed, in the case of VS conversion, the better removal efficiency of 35.48% reflected that
biodegradability of organic biowaste improved with maximum methane yield from oil-
the biodegradability of organic biowaste improved with maximum methane yield from
extracted spent coffee grounds because of the organic composition of proteins, sugars,
oil-extracted spent coffee grounds because of the organic composition of proteins, sugars,
and lipids. At the same time, oil extraction assisted as a pretreatment agent. For co-feed,
they achieved the maximum yield with the best biodegradability from DSCG and STG
(50%:50%); however, biodegradability was not the highest (Figure 7) [74]. However,
Awais et al. [89] observed a better BD (83%) and maximum methane yield with mixed
Sustainability 2022, 14, 9387 12 of 19

and lipids. At the same time, oil extraction assisted as a pretreatment agent. For co-feed,
they achieved the maximum yield with the best biodegradability from DSCG and STG
(50%:50%); however, biodegradability was not the highest (Figure 7) [74]. However, Awais
et al. [89] observed a better BD (83%) and maximum methane yield with mixed meadow
Sustainability 2022, 14,
grass, x FOR straw,
wheat PEER REVIEW
and cattle manure at a ratio of 0.75:0.75:0.25, with better performance of 13 of 20

the system.

C/N %BD

100
biodegradability and C/N ratios

80

60

40

20

Mixing ratios of co-substrates

Figure 7. ComparingFigure 7. Comparing the biodegradability and C/N ratios to mixing ratios of different organic wastes
the biodegradability and C/N ratios to mixing ratios of different organic wastes
in mono- and co-digestion (data from [74]). Defatted spent coffee ground (DSCG); macroalgae, MC
in mono- and co-digestion (data from [74]). Defatted
(Cladophora sp.); glycerin (G); spent
spent coffee coffee(SCG);
grounds ground (DSCG);
spent macroalgae,
tea waste (STG). MC
(Cladophora sp.); glycerin (G); spent coffee grounds (SCG); spent tea waste (STG).
4.4. Moisture Contents
4.4. Moisture Contents
Moisture content determines the kind of biodigester based on the total solid compo-
Moisture content
sitiondetermines
in the organicthe kindadded.
matter of biodigester
Based on based on thecontents,
the moisture total solid composi- can be
the biodigester
tion in the organicclassified
matter added. Based
as a solid, on the
semi-solid, ormoisture contents,
liquid anaerobic the [14].
digester biodigester canthis
In line with bebenefit,
anaerobic
classified as a solid, co-feeding
semi-solid, is theanaerobic
or liquid best potential pretreatment
digester [14]. Inmethod
line withthatthis
combines high and
benefit,
anaerobic co-feeding is the best potential pretreatment method that combines high and and
low moisture feedstocks into a bioreactor at the same time, ensuring proper growth
free mobility of microbes in the bioreactor, and elevating yields of the bioreactor. Animal
low moisture feedstocks into a bioreactor at the same time, ensuring proper growth and
manures contain high moisture content, which might be responsible for sufficient mois-
free mobility of microbes in the bioreactor, and elevating yields of the bioreactor. Animal
ture for the formation of archaeal function [14] and create benefits in anaerobic co-diges-
manures contain high moisture
tion. Concerning content, which might
this significance, they be responsible
achieved the bestfor sufficient
yield of biogasmoisture
(307-cm3 CH4/g
for the formation (VS))
of archaeal
from the digestion of pig manure, water hyacinth, and poultryco-digestion.
function [14] and create benefits in anaerobic droppings via a mix-
Concerning this significance, they achieved the bestconditions
yield of biogas 3
ing ratio of 15:40:45 under mesophilic [102]. (307-cm CH4 /g (VS))
from the digestion of pig manure, water hyacinth, and poultry droppings via a mixing ratio
4.5. Stability conditions [102].
of 15:40:45 under mesophilic
4.5.1. pH
4.5. Stability The pH value is a prominent operational parameter that strongly affects microbial
4.5.1. pH activities and biomethane yields. Most microorganisms prefer a neutral pH in the biogas
The pH valuegeneration process. operational
is a prominent Although a different
parametergroupthat
of microbes
stronglyplays a great
affects role that needs
microbial
varied optimum pH settings for their metabolism and anaerobic digestion, keeping a pH
activities and biomethane yields. Most microorganisms prefer a neutral pH in the biogas
range of 6.3–7.2 is important to achieve a high biogas yield [23,39,67]. Methanogens are
generation process. Although
extremely a different
sensitive group of
to pH changes andmicrobes
prefer a pHplays
of 7.0a [20],
great roleacidogenic
while that needs microbes
varied optimum pH settings
require a pHfor their
range of metabolism and anaerobic
4–8.5. The co-substrate digestion,
may permit keeping
operational a pH of the
constancy
range of 6.3–7.2 isdigester
important to achieve
by buffering the aextreme
high biogas yield [23,39,67].
acidification/alkalinity Methanogens
condition. As per aare
reviewed
extremely sensitive to pH
study, in changes andit prefer
co-digestion, a pH
is simple and of 7.0to[20],
easy keepwhile
ideal pH acidogenic microbes
settings constant during the
require a pH range period of theThe
of 4–8.5. digestion process compared
co-substrate may permit to during the digestion
operational of a substrate
constancy of the alone
[1,23].
digester by buffering the extreme acidification/alkalinity condition. As per a reviewed
study, in co-digestion, it is simple and easy to keep ideal pH settings constant during the
4.5.2. Organic Removal Efficiency
period of the digestion process compared to during the digestion of a substrate alone [1,23].
As the significance of AcoD rises, it is essential to find a method to assess the biodeg-
radability of substrates and the biogas production performance. In light of this, the BMP
Sustainability 2022, 14, 9387 13 of 19

Sustainability 2022, 14, x FOR PEER REVIEW 14 of 20

4.5.2. Organic Removal Efficiency
As the significance of AcoD rises, it is essential to find a method to assess the biodegrad-
ability of substrates and the biogas production performance. In light of this, the BMP test
test may
may givegive useful
useful information
information to predict
to predict the the overall
overall performance
performance of the
of the biogas
biogas digester.
digester. In
In the AcoD system, VS/COD removal is very crucial since biogas
the AcoD system, VS/COD removal is very crucial since biogas yield, in terms of VS/COD yield, in terms of
VS/COD removal, can provide an overview of the efficiency of the anaerobic
removal, can provide an overview of the efficiency of the anaerobic process [103]. From this process [103].
From this perspective,
perspective, a product aofproduct
biogas isofinformed
biogas is informed
in terms ofinVS/COD
terms of VS/COD
removal.removal.
COD balance COD
balance indicates
indicates the synergistic
the synergistic effect ofeffect of co-digestion
co-digestion due to due to a of
a blend blend of supplement
supplement feed-
feedstocks.
stocks. Food waste was confirmed as the best way to speed up AcoD
Food waste was confirmed as the best way to speed up AcoD for surprising biogas produc-for surprising biogas
production
tion due to thedueformation
to the formation of highin
of high acidity acidity in the
the initial initial
stage stage [36,104].
[36,104]. In addition,In addition,
Elsayed
Elsayed et al. [35] mixed cow manure, linen (Ln), and wheat straw,
et al. [35] mixed cow manure, linen (Ln), and wheat straw, focusing on the effects focusing on the effects
of the
of the mixing
mixing ratio onratio
processon performance
process performance in a mesophilic
in a mesophilic digester
digester using using(sludge)
inoculum inoculum in
a(sludge) in a BMP experiment.
BMP experiment. They demonstrated
They demonstrated that the methane
that the maximum maximumyield methane
of 351yieldmL/g of
351was
VS mL/g VS wasatobtained
obtained a mixingatratio
a mixing ratio with
of 50:25:25 of 50:25:25 with VS
VS removal removal
efficiency of efficiency
72.87% [35]. of
72.87% [35].
Further, Further,
the better the better
strategies strategies
to improve to improve
biogas biogas
yields and yields and
VS removal VS removal
efficiency effi-
is shown
ciency
in is shown
Figure 8. in Figure 8.

Figure 8. Relations
Figure 8. Relations among
among some factors affecting the performance
performance of AcoD [103]
[103] and
and strategies
strategies to
to
improve
improve biogas
biogas yields.
yields.

4.5.3.
4.5.3. Organic
Organic Loading
Loading Rates
Rates (OLRs)
(OLRs)
The organic loading rates are one of the major parameters that indicates the capacity
The organic loading rates are one of the major parameters that indicates the capacity
of the bioreactor. The real feeding rate of organic matter into the reactor depends on the
of the bioreactor. The real feeding rate of organic matter into the reactor depends on the
types of waste. Increasing organic loading rates boosts microbe activities, which results in
types of waste. Increasing organic loading rates boosts microbe activities, which results in
enhanced biogas production to some extent [105,106]. However, introducing a substrate into
enhanced biogas production to some extent [105,106]. However, introducing a substrate
the digester without considering the optimum rate reduces the biogas volume. Overloading
into the digester without considering the optimum rate reduces the biogas volume. Over-
the digester may block the mobile microorganisms, cause the over-concentration of VFA,
loading the digester may block the mobile microorganisms, cause the over-concentration
which mainly influences methane-producing bacteria, and result in low biogas yield [107].
of VFA, which mainly influences methane-producing bacteria, and result in low biogas
Underload (introducing a small amount of organic substance) leads to the formation of
yield [107]. Underload (introducing a small amount of organic substance) leads to the for-
alkalinity in the biodigester, resulting in low biogas yields [14]. In addition, increasing OLR
mation of alkalinity in the biodigester, resulting in low biogas yields [14]. In addition,
exhibited a decrease in biogas yield by 168% even for the co-digestion process [15] and
increasing OLR exhibited a decrease in biogas yield by 168% even for the co-digestion
formed instability in the biodigester by influencing alkalinity [108].
process
The[15] and formed
advantage instability
of AcoD in the biodigester
over mono-digestion byAcoD
is that influencing alkalinity
has a higher [108].
organic loading
and significant substrate composition variation [9,15,44,109,110]. In addition, methane load-
The advantage of AcoD over mono-digestion is that AcoD has a higher organic yield
ing
is and significant
boosted with the substrate
optimumcomposition
AcoD process variation
through[9,15,44,109,110].
tolerable organicInloading
addition,
andmethane
a solid
yield is boosted
retention period, with
whichthe optimum
may improve AcoD process
organic matterthrough
removal tolerable
and VFA organic loading
conversion. and a
Further,
solid retention period, which may improve organic matter removal and VFA
it leads to a reduction of biosolids odorous releases [13]. Kesharwani et al. [15] explored conversion.
Further,
the effectitofleads
OLRs to on
a reduction
biogas at of biosolids
a pilot scaleodorous releases conditions.
under ambient [13]. Kesharwani
They et al. [15]
achieved
explored the effect of OLRs on biogas at a pilot scale under ambient
the highest biogas yield from co-digestion of food waste with cow dung compared conditions. Theyto
achieved the highest biogas yield from co-digestion of food waste
mono-digestion of cow dung (Table 4) without disturbance of inhibitory compounds.with cow dung com-
In
pared
the to mono-digestion
laboratory of cow production
study, the methane dung (Tablevia4) mono-digestion
without disturbanceof cowofdung
inhibitory com-
was higher
pounds. In the laboratory study, the methane production via mono-digestion of cow dung
was higher at lower OLRs, whereas the trend was the opposite in the co-digestion of cow
dung and grass silage [25].
Sustainability 2022, 14, 9387 14 of 19

at lower OLRs, whereas the trend was the opposite in the co-digestion of cow dung and
grass silage [25].

4.5.4. VFA, TA, and VFA/TA Ratio

VFA is a key indicator of the stability of an anaerobic digester. The excess formation
of VFA can drop the pH in biodigesters, affecting active, methane-producing archaea and
causing a disturbance of digester stability. The only balance for VFAs that leads to process
stability is their removal, i.e., biogas conversion. High VFAs (28.88 g/L) cause long-term
yield and methanogenic microbe suppression [105]. Therefore, anaerobic co-digestion is
a promising method that ensures the achievement of higher yields in a short retention
time with the addition of greater OLRs when compared with anaerobic mono-digestion of
organic substrates.
Alkalinity (TA) is the acid-neutralizing ability of an aqueous solution. It is a more
reliable indicator of digester imbalance than direct pH testing. The archaea produce
alkalinity in the form of CO2 and bicarbonate that offsets the pH drop. Pig manure (PM)
contains a lot of ammonia that might repress the process anaerobically. PM coupled to
assist the AcoD process as a buffering agent achieved a synergetic effect by avoiding
acidification during co-digestion through VFA neutralization in olive mill wastewater,
which is a low-nitrogen, low-pH substrate. As a result, a high biogas yield and stability
were attained [39].
VFA/TA ratio is another parameter that measures the performance of the anaerobic
digestion process that is more reliable than measuring the pH directly. In anaerobic condi-
tions, depression in alkalinity or over-rising of VFA causes a quick drop in pH [30]. Thus, it
is considered “the early warning indicator of process failure” within an optimum range
of 0.2–0.4 [15]. It is a good parameter in the buffering of anaerobic process analysis [108].
VFA/TA ratios are used to assessing the effects of increasing the OLRs on the digester
performance. For example, Mu et al. [72] experimented on SS, FW, and YW co-digestion
under a BMP test and semi-continuous mesophilic reactor for 60 days. They achieved
enhancement of the stability process and archaea/total microbe ratio by 17.1% from SS
(25% vs. basis) in FW, which could be due to the delivery of trace metals by SS which
buffered the inhibitory matter that affects microbe competition.

5. Conclusions and Recommendations for Future Studies

AcoD is a cutting-edge technology that is commercially and environmentally viable
with novel approaches to increasing biomethane generation. The handling of AcoD tech-
nology requires a good understanding of the effect of different operating conditions. The
effects of co-substrates on the performance of the AcoD process have been mentioned and
detailed. Additionally, some recommendations for future work are worthwhile: The effects
of co-digestion on theoretical biomethane production, volatile solid conversion rates, and
the relation between theoretical biomethane, biodegradability, and volatile conversion rates
need further investigation. Consideration of the effect of co-substrates on the start of the
anaerobic digestion process and hydrolysis rate may also be required in future works. In
addition, further studies may be necessary on the accompanying co-feed types with the
groups of microbes and their activities.

Author Contributions: All authors contributed their share from the inception of the critical review.
All authors have read and agreed to the published version of the manuscript.
Funding: This review received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors are very grateful to the ExiST project: Excellence in Science and
Technology-Ethiopia funded by KfW, Germany, through Jimma Institute of Technology, Center of
Sustainability 2022, 14, 9387 15 of 19

Excellence (KfW Project No. 51235, BMZ No. 2011 66 305, JiT CoE-CRJE RESOURCE CART FUNDS)
for providing financial assistance to conduct and publish this study.
Conflicts of Interest: The authors declare no conflict of interest.

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