Asian Journal of Advances in Agricultural Research

Volume 25, Issue 7, Page 38-58, 2025; Article [Link].138952

ISSN: 2456-8864

Recent Advances in Pre-treatment

Strategies for Anaerobic Digestion of
Lignocellulosic Biomass: A Review
Nitin Karwasra a, Yadvika a and Kanishk Verma a*
a Department of Renewable and Bio-Energy Engineering, COAE&T, CCSHAU, Hisar, India.

Authors’ contributions

This work was carried out in collaboration among all authors. All authors read and approved the final
manuscript.

Article Information

DOI: [Link]

Open Peer Review History:

This journal follows the Advanced Open Peer Review policy. Identity of the Reviewers, Editor(s) and additional Reviewers,
peer review comments, different versions of the manuscript, comments of the editors, etc are available here:
[Link]

Received: 24/04/2025
Review Article Accepted: 26/06/2025
Published: 02/07/2025

ABSTRACT

This review critically examines recent advancements in the anaerobic digestion (AD) of
lignocellulosic biomass for biogas production, highlighting its promise as a sustainable alternative to
fossil fuels. Lignocellulosic residues from agriculture and forestry, generated at an estimated 180–
200 billion tonnes annually, represent a vast and renewable energy resource. Under optimal
conditions, these substrates can yield 0.35–0.40 m³ of methane per kilogram of volatile solids.
However, their complex matrix of cellulose, hemicellulose, and lignin poses significant barriers to
microbial degradation. The review categorizes and evaluates a wide array of pretreatment
strategies—physical, chemical, hydrothermal, biological, and hybrid—based on their mechanisms,
effectiveness, energy demands, and impact on methane yield. Additionally, the synergistic role of
co-digestion, feedstock variability, and microbial dynamics is explored to identify process
optimization opportunities. Despite technological progress, key challenges persist, including
feedstock heterogeneity, process inefficiencies, and economic constraints. The review concludes by
_____________________________________________________________________________________________________

*Corresponding author: Email: kanishk57@[Link];

Cite as: Karwasra, Nitin, Yadvika, and Kanishk Verma. 2025. “Recent Advances in Pre-Treatment Strategies for Anaerobic
Digestion of Lignocellulosic Biomass: A Review”. Asian Journal of Advances in Agricultural Research 25 (7):38-58.
[Link]
 Karwasra et al.; Asian J. Adv. Agric. Res., vol. 25, no. 7, pp. 38-58, 2025; Article [Link].138952

identifying critical research gaps, such as the need for integrated pretreatment systems and robust
microbial consortia, to enhance the scalability and economic feasibility of lignocellulosic biogas
systems.

Keywords: Lignocellulosic biomass; anaerobic digestion; biogas production; pretreatment strategies;

renewable energy.

1. INTRODUCTION uptake (Zheng et al.,2017; Hagman et al., 2018).

Recent progress, including novel reactor
1.1 Energy Context designs and enzyme cocktails, has begun to
narrow this gap (Dahmen et al., 2018).
Global energy consumption is projected to rise Third-generation biofuels explore advanced
from 580 EJ in 2018 to nearly 700 EJ by 2050, systems such as algae-based lipids and
driven by population growth and expanding microbial engineering to produce drop-in
industrial activities (Nations, 2022). Despite rapid fuels; while promising high yields and
growth in wind and solar installations, fossil- reduced land footprint, these approaches
based sources—coal, oil, and natural gas— remain at pilot or demonstration scale due to
remain the backbone of global energy systems, high capital and operational costs (Shrestha et
accounting for over 75% of primary energy al., 2021).
supply and contributing significantly to
greenhouse gas emissions (Olabi and 1.3 Why Lignocellulose for Anaerobic
Abdelkareem, 2022). This continued reliance on Digestion
carbon-intensive fuels exacerbates climate
change and undermines sustainability objectives Among alternative biofuel pathways, anaerobic
outlined by the Paris Agreement. Moreover, the digestion (AD) of lignocellulosic biomass holds
production and disposal of agricultural and particular promise by simultaneously generating
forestry residues present environmental renewable methane and valorising waste
challenges that demand sustainable streams. Globally, an estimated 180–200 billion
valorisation pathways (Islam et al., 2020). tonnes of lignocellulosic residues are produced
Addressing these interlinked issues requires annually from agriculture and forestry (Gatto and
integrated approaches that couple renewable Drago, 2021), yet only a fraction undergoes
energy generation with waste management, biological conversion. Under optimal AD
thereby fostering circular economy practices conditions, these residues can yield up to 0.35–
and reducing dependency on imported 0.40 m³ of methane per kilogram of volatile
hydrocarbons. solids, offering energy returns comparable to
liquid biofuels (Nigam and Singh, 2011).
1.2 Generations of Biofuels Furthermore, AD produces nutrient-rich digestate
that can be recycled as biofertilizer, closing
Biofuel technologies have undergone three nutrient loops and displacing synthetic fertilizers
distinct developmental stages, each driven by (Kamaraj et al., 2019). Despite these
evolving feedstock availability and advantages, the rigid structure of lignocellulose—
environmental imperatives. First-generation composed of cellulose, hemicellulose, and
biofuels utilize edible crops such as maize and lignin—impedes microbial accessibility,
sugarcane to produce ethanol or biodiesel; their necessitating pretreatment strategies to
deployment proved the technical viability of enhance biodegradability (Shahid et al., 2021;
bioenergy but sparked concerns about food Islam et al., 2020). This review synthesizes
security and land-use change (Khan et al., 2022). existing physical, chemical, biological, and
The second generation, emerging in the early hybrid pretreatment methods, evaluating their
2000s, shifted focus to lignocellulosic impacts on methane yield, process economics,
feedstocks—agricultural residues, forestry and environmental footprint. By consolidating
byproducts, and dedicated energy crops—to recent techno-economic analyses alongside
avoid direct competition with food resources. laboratory performance data, we aim to provide a
However, the inherent recalcitrance of roadmap for selecting appropriate pretreatment
lignocellulose posed challenges in pretreatment, techniques to advance sustainable biogas
hydrolysis, and fermentation, limiting commercial production.

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2. ANAEROBIC DIGESTION OF 2.3 The Role of Pre-treatment in Biogas

LIGNOCELLULOSIC BIOMASS Production

2.1 Importance of Biogas in Renewable While anaerobic digestion represents a

Energy Systems promising renewable energy solution, the
effective conversion of lignocellulosic biomass
Biogas plays a significant role in meeting remains hindered by the structural complexity of
renewable energy goals and has potential its components—primarily cellulose,
applications in both energy production and hemicellulose, and lignin. The overall conversion
transportation, especially with further process typically includes three key stages: pre-
technological advancements. Considerable treatment, enzymatic hydrolysis, and anaerobic
research has been dedicated to various aspects digestion. Among these, pre-treatment is
of biogas systems, including its generation, recognized as the most energy-intensive phase
production enhancement (Prabhu et al., due to its role in disrupting the rigid
2021), and the optimization of process lignocellulosic matrix, solubilizing hemicellulose,
configurations (Mirmohamadsadeghi et al., and partially removing or altering lignin content to
2019). One particularly efficient method of enhance microbial accessibility (Poddar et al.,
utilizing renewable biomass resources is the 2021). Integrating pre-treatment into the AD
production of biogas from lignocellulosic workflow helps address inherent limitations in
materials such as forest residues, agricultural substrate digestibility and leads to improved
waste, energy crops, organic fractions of biogas yields (Yu et al., 2019).
municipal solid waste, and various
industrial wastes including wood, paper, and A wide array of pre-treatment strategies has
pulp. been explored to enhance the suitability of
lignocellulosic biomass for AD. These include
2.2 Anaerobic Co-Digestion and physical, thermal, chemical, biological,
Challenges with Lignocellulosic combined, and emerging pre-treatment
technologies. Ongoing research has also
Feedstocks
extended to non-conventional and green pre-
treatment methods suitable for biorefinery
In recent years, anaerobic co-digestion has
integration, as well as the application of
gained momentum as a strategy to enhance
nanotechnology, biological agents, and
methane yield and nutrient recovery while
enzymatic enhancements to boost biomethane
maintaining overall digester stability (Yang et al.,
production. Each of these approaches aims to
2021). Among the most abundant feedstocks for
improve process efficiency while maintaining
biogas production are agricultural crop residues,
environmental and economic sustainability.
which when subjected to anaerobic digestion
(AD), not only contribute to renewable energy 2.4 Microbial Dynamics and Process
generation but also aid in mitigating the
Optimization in AD
environmental impact of agricultural waste.
While considerable attention has been directed Roopnarain and Adeleke (2017) have clearly
toward the digestion of manure, sludge, outlined the sequential stages involved in the
and food waste (Kumar et al., 2021), the anaerobic digestion process: hydrolysis,
anaerobic conversion of crop residues remains acidogenesis, acetogenesis, and
a challenge. This is primarily due to the methanogenesis (Fig. 1). These biological
complex physicochemical characteristics of transformations rely on complex microbial
lignocellulosic materials, which include high lignin consortia, each adapted to a specific phase of
and cellulose content, structural rigidity, low bulk degradation. The pivotal role of microorganisms
density, poor fluid dynamics, a high carbon-to- in the breakdown of organic matter and the
nitrogen (C/N) ratio, and a deficiency in trace production of biogas is well recognized, as
elements. These features, along with variable originally detailed by Chaiprasert (2011).
particle size and morphology, necessitate Although biogas typically contains a lower
the development and implementation of methane concentration (50–60%) compared to
tailored technologies to improve their liquefied petroleum gas (LPG) or compressed
biodegradability and conversion efficiency (Sun natural gas (CNG), global efforts continue to
et al., 2021). support its commercialization as a renewable,

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Fig. 1. Schematic representation of the anaerobic digestion process (Roopnarain, A., & Adeleke, R. (2017))

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cost-effective, and environmentally preferable (Salminen and Rintala, 2002). By enabling the
alternative to conventional fossil-based fuels simultaneous digestion of two or more
(Garcia et al., 2019). substrates, co-digestion offers a strategic
advantage over mono-digestion in terms of
To maximize the efficiency of anaerobic digestion methane yield and economic feasibility (El-
systems, it is imperative to apply scientific and Mashad and Zhang, 2010; Li et al., 2010;
engineering approaches that optimize methane Siddique and Wahid, 2018). This method allows
recovery across a variety of feedstocks. better balancing of carbon-to-nitrogen (C/N)
Parameters such as substrate composition, ratios, enhanced buffering capacity, and dilution
operating conditions, and digester configuration of potential toxic compounds.
play critical roles in determining overall system
performance (Kumar et al., 2015). The natural For instance, a study conducted by Yong et al.
degradation of organic matter highlights the (2015) in China examined the co-digestion of
ubiquity of microbial agents capable of breaking food waste and straw. Utilizing a 1-liter enclosed
down diverse compounds, and a deeper reactor maintained at 35°C with a total organic
understanding of these microbial dynamics, load of 5 g VS/L, they assessed various food
coupled with comprehensive feedstock waste–straw mixtures. The optimal ratio of food
characterization, offers an avenue for developing waste to straw (5:1) achieved a peak methane
precise and sustainable solutions to current and production yield (MPY) of 0.392 m³/kg-VS,
future energy challenges. In this context, the representing increases of 39.5% and 149.7%
present review examines anaerobic digestion compared to individual digestion of food waste
strategies aimed at enhancing biogas production and straw, respectively. Gas production (GP) and
from lignocellulosic feedstocks. methane content reached 0.58 m³/kg-VS and
67.62%, respectively. The study further analysed
2.5 Co-Digestion Strategies for Enhanced particle size effects and recommended a straw
Biogas Production size of 0.3–1 mm as optimal for maximizing
energy efficiency and cost-effectiveness
The global energy crisis necessitates the (Almomani and Bhosale, 2020).
development of environmentally sustainable and
scalable solutions. To achieve meaningful In a separate investigation, Almomani and
progress in commercialization and industry Bhosale (2020) explored the anaerobic co-
integration, innovation rooted in scientific digestion of agricultural solid wastes (ASWs) and
research is essential. Traditionally, anaerobic cow dung (CD), as well as the impact of sodium
digestion systems have focused on mono- bicarbonate (NaHCO₃) alkalinity treatment. Their
substrate digestion; however, their limited results demonstrated that a 60:40 ASW to CD
efficiency across varying substrate types has ratio yielded the highest cumulative methane
prompted a shift toward co-digestion strategies. production (CMP) of 297.99 NL/kg-VS. Notably,
The co-digestion of multiple organic substrates the addition of NaHCO₃ at a dose of 1.0 g/gVS
has gained increasing attention as a viable enhanced the CMP to 386.3 NL/kg-VS,
approach to improve methane yields and process highlighting the potential of alkalinity regulation in
stability. Emerging evidence suggests that co- improving process efficiency.
digestion of complementary biodegradable
wastes results in significantly higher methane 3. FEEDSTOCK AND BIOMASS
production compared to single-substrate COMPOSITION
digestion systems. Organic substrates inherently
contain a range of macro- and micronutrients 3.1 Feedstock for Biogas Production
necessary for the growth and function of both
aerobic and anaerobic microbial communities. The exploration and utilization of organic wastes
Importantly, these nutrient profiles can vary have gained considerable momentum due to
based on species, cultivation conditions, and advances in sustainable development,
material maturity (Hagos et al., 2017). particularly in the domains of renewable energy
production and waste management. While
Anaerobic co-digestion has become a widely anaerobic digestion systems were originally
adopted method for improving biogas output in employed for the treatment of animal and
digesters. Numerous studies have focused on vegetable wastes, their application has now
combining livestock manure with other organic broadened significantly. Today, biogas can be
materials to boost methane production rates generated from a diverse array of feedstocks,

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including agricultural, industrial, and municipal efficiency (Brown and Li, 2013; Almomani et al.,
waste streams. The quality and stability of biogas 2019). These advancements are crucial for
production depend heavily on the properties of maximizing energy recovery from a broad range
the feedstock. Parameters such as moisture of organic waste resources.
content, volatile solids concentration, nutrient
availability, and particle size play a significant 3.2 Composition of Lignocellulosic
role in influencing the efficiency and stability of Biomass
the anaerobic digestion process (Dobre et al.,
2014). Lignocellulosic biomass is composed primarily of
three major biopolymers: cellulose (typically 80–
To enhance the effectiveness of feedstock 90%), hemicellulose (10–50%), and lignin (5–
utilization, advanced processing strategies have 35%), along with minor components such as
been introduced. Technological innovations such proteins, ash, and pectin (Xu and Li, 2017). The
as optimized feedstock selection, impurity relative proportions of these components vary
removal systems, co-digestion trials, and pre- significantly depending on the biomass source,
treatment technologies have shown great as illustrated in Fig. 2.
potential in improving biogas yield and process

Fig. 2. Structure of a biomass in general

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Cellulose, the most abundant polymer, is a linear sinapyl alcohols. These precursors generate the
polysaccharide made up of D-glucose subunits structural phenylpropanoid units known as p-
linked through β-(1,4)-glycosidic bonds. This hydroxyphenyl (H), guaiacyl (G), and syringyl (S)
polymer forms a crucial part of the structural groups, respectively (Sluiter et al., 2008). Lignin
framework in plant cell walls (Kumar et al., 2017). contributes mechanical strength and rigidity to
Though cellulose is generally insoluble in water plant tissues and constitutes 15–40% of the dry
under neutral conditions, it can be dissolved in weight of plant biomass such as wood and straw.
specific solvents such as ionic liquids (ILs) and It is significantly more resistant to microbial and
N-methylmorpholine-N-oxide (NMMO), which aid enzymatic degradation compared to cellulose
in its processing and application (Kumar et al., and hemicellulose. As one of the most abundant
2020). Cellulose is valued for its biocompatibility, organic polymers in nature, lignin plays essential
hydrophilicity, stereoregularity, and potential roles in plant structure and carbon cycling. It also
applications in fuel production and the finds numerous applications in agriculture and
development of bio-based chemicals (Jedvert industry. For instance, in the paper industry,
and Heinze, 2017). lignin must be removed during the pulping
process to produce high-quality paper (Echresh
Hemicellulose, the second principal component, et al., 2019).
comprises a branched heteropolymer consisting
of several sugar monomers, including xylan, 3.3 Biomass Recalcitrance and Pre-
galactomannan, glucuronoxylan, glucomannan, treatment
arabinoxylan, and xyloglucan. These are linked
by glycosidic bonds in a more amorphous and One of the primary challenges in converting
less polymerized structure compared to lignocellulosic biomass to bioenergy is its
cellulose. Due to its relatively low crystallinity, inherent recalcitrance, which refers to the
hemicellulose is more readily hydrolyzed and is structural resistance to chemical and biological
widely utilized in industrial applications such as degradation. Effective utilization of lignocellulosic
hydrogels and drug delivery systems (Liu et al., feedstocks requires overcoming obstacles such
2019). as the crystallinity of cellulose, the high lignin
content, and the complex, heterogeneous
Lignin is a highly complex aromatic polymer architecture of plant cell walls. Pretreatment
derived from the radical coupling of three main processes play a critical role in disrupting this
monolignols: p-hydroxycinnamyl, coniferyl, and rigid structure. Through mechanisms such as

Fig. 3. Different lignocellulosic biomass pretreatment techniques

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lignin depolymerization, hemicellulose polymerization, and decreases cellulose

solubilization, and reduction in cellulose crystallinity, thereby weakening the rigid plant
crystallinity, pretreatment enhances substrate matrix (Dahunsi, 2019). Often performed as an
accessibility and digestibility (Zoghlami and initial step in pretreatment, milling accelerates
Paës, 2019). fermentation by allowing faster enzymatic or
microbial degradation of substrates. Moreover,
Over the past few decades, numerous finely milled particles exhibit enhanced buffering
pretreatment strategies have been developed to capacity, which can help resist acidification
address these challenges. As depicted in Fig. 3, during digestion (Mayer-Laigle et al., 2018).
these methods are broadly categorized into
physical, chemical, biological, and combined The advantages of milling include its chemical-
approaches. The selection of an appropriate free operation and room-temperature processing,
pretreatment technique is closely tied to the which eliminates costs associated with chemical
feedstock composition, particularly the relative procurement and post-treatment washing or
abundance of cellulose, hemicellulose, and lignin filtration. However, the method is energy-
(Dadhah et al., 2017). Each method targets intensive and requires high-capacity mechanical
specific structural barriers, and their equipment, making it expensive and less feasible
effectiveness can vary depending on the at scale unless integrated with other techniques
biomass source. In subsequent sections, various (Baruah et al., 2018; Mankar et al., 2021).
pretreatment techniques will be discussed in
detail, examining their impacts on the separation Multiple milling technologies are employed in
and conversion of lignocellulosic components lignocellulosic preprocessing, such as two-roll
(Baruah et al., 2018). milling, rod milling, ball milling, wet disk milling,
hammer milling, centrifugal milling, and colloid
4. PRETREATMENT milling. The appropriate milling method depends
on factors including biomass type, moisture
Pretreatment is a crucial step in the anaerobic content, target particle size, and the intended
digestion of lignocellulosic biomass due to the downstream application (Sitotaw et al., 2021;
natural recalcitrance of its structural components. Zhang et al., 2019).
The primary goal of pretreatment is to break
down the complex architecture of lignin, Among these, wet disk milling is particularly
cellulose, and hemicellulose, thereby enhancing noted for its lower energy consumption (Baruah
microbial accessibility and improving biogas et al., 2018). This method utilizes two grooved,
yields. Pretreatment methods are generally counter-rotating discs—or a combination of a
categorized as physical, chemical, biological, or stationary and a rotating disc moving in opposite
a combination of these approaches (Table 1). directions—to create shear forces that break
down biomass in a continuous flow. In scenarios
4.1 Physical Pretreatment where chemical agents are applied concurrently,
the system must be built with corrosion-resistant
Physical pretreatment involves the application of materials. Although wet disk milling is efficient, it
mechanical processes such as chipping, has minimal effect on the crystallinity and
grinding, milling, extrusion, and cavitation to alter polymerization degree of lignocellulose
the physical characteristics of lignocellulosic (Rajendran et al., 2018). Additionally, it requires
biomass. The primary aim is to disrupt the plant a large volume of water to maintain low solid
cell wall’s waxy surface and lignin encrustation, loading, which can be a limitation.
thus increasing the substrate's surface area. This
facilitates greater microbial accessibility and Ball milling is considered one of the most robust
accelerates the breakdown of cellulose and and effective methods for mechanical
hemicellulose during anaerobic digestion (Akhtar pretreatment. It significantly reduces particle size
et al., 2016). while decreasing both the crystallinity and
polymerization degree of cellulose. However, its
4.1.1 Milling high energy consumption, costly equipment, and
lack of lignin removal capabilities make it
Milling employs various mechanical forces— economically impractical as a standalone
impact, compression, friction, and shear—to technique. Consequently, ball milling is often
induce both physical and chemical alterations in paired with other pretreatment methods to
the biomass structure. This process effectively achieve more comprehensive biomass
reduces particle size, lowers the degree of breakdown (Rizal et al., 2018).

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Table 1. Different lignocellulosic biomass pretreatment methods, their effects and drawbacks

Pretreatment Method Process Effect on biomass Drawback Reference

Physical Milling Increases the surface area and One drawback is the potential Dahunsi, 2019; Mayer-Laigle et al.,
accessibility of lignocellulosic for increased energy 2018; Baruah et al., 2018; Mankar
biomass for subsequent enzymatic consumption during the milling et al., 2021; Sitotaw et al., 2021;
hydrolysis process. Zhang et al., 2019

Extrusion Disrupts lignocellulosic biomass One drawback is the potential Hjorth et al., 2011; Konan et al.,
structure, improving enzymatic for high energy consumption 2022; Duque et al., 2017; Victorin
accessibility for bioconversion and equipment costs. et al., 2020

Microwave Irradiation Enhances lignocellulosic biomass One drawback is uneven Aguilar-Reynosa et al., 2017;
enzymatic digestibility by breaking heating leading to localized hot Calcio Gaudino et al., 2019; Li et
down structural barriers spots in the biomass. al., 2016

Ultrasonication Increases lignocellulosic biomass high equipment cost associated Pilli et al., 2011; Dhandayutha- pani
enzymatic digestibility by disrupting with ultrasonication devices et al., 2021
its structure at a molecular level
Hydrothermal Steam explosion Enhances lignocellulosic biomass One drawback is the potential Zheng et al., 2021; Sun and Cheng,
enzymatic digestibility by breaking for formation of inhibitors like 2002; Lam, 2011; Menardo et al.,
down hemicellulose and lignin furfural and acetic acid, which 2013; Datar et al., 2007
barriers can hinder downstream
processes and high energy
consumption associated with
the explosion process.
Freezing Disrupts lignocellulosic biomass One drawback is the potential Rooni et al., 2017; Guan et al.,
structure, facilitating enzymatic for cell wall damage due to ice 2021; Smichi et al., 2016; Cano et
hydrolysis crystal formation, affecting al., 2015; Yang et al., 2021; Jan et
biomass integrity al., 2008
Chemical Alkali Delignifies lignocellulosic biomass, One drawback is the potential Badiei et al., 2014; Rabelo et al.,
improving enzymatic hydrolysis for lignin redistribution and 2009; Park and Kim, 2012.
efficiency formation of alkali-resistant
compounds, limiting enzymatic
digestibility

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Pretreatment Method Process Effect on biomass Drawback Reference

Acid Solubilizes hemicellulose and generation of inhibitors like Guan et al., 2021; Monlau et al.,
enhances enzymatic digestibility of furfural and 2013; Syaichurrozi et al., 2019;
lignocellulosic biomass hydroxymethylfurfural, which Taherdanak et al., 2016; Tsai et al.,
can inhibit downstream 2021.
fermentation processes
Biological Fungal Enzymatically degrades requirement for specific fungal Zheng et al., 2014; Taherzadeh and
lignocellulosic biomass, increasing strains, which may limit its Karimi, 2008
its digestibility for bioconversion applicability and efficiency.
processes
Microbes Enhances lignocellulosic biomass One drawback is the potential Zhang et al., 2019; Badiei et al.,
degradation and enzymatic for longer processing times and 2014, Taherzadeh and Karimi,
accessibility for bioconversion the need for strict control of 2008
microbial conditions

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4.1.2 Extrusion which result in dielectric polarization and

subsequent molecular collisions. These
Extrusion is a physical pretreatment technique interactions rapidly and uniformly heat the
that integrates multiple mechanical actions into a material, disrupt its structural integrity, and
single continuous process. In this method, enhance its susceptibility to microbial
lignocellulosic biomass is fed into one end of the degradation (Aguilar-Reynosa et al., 2017; Calcio
extruder and transported along a barrel by a Gaudino et al., 2019).
rotating screw. As the material advances, it
experiences friction against the screw and barrel, Microwave irradiation may be applied under
which generates heat and promotes mixing and atmospheric or high-pressure conditions. High-
shearing. The barrel typically contains distinct pressure microwave pretreatment is conducted in
zones, including a compression zone at the closed reactors and typically operates at
midpoint and an expansion or wear zone near temperatures ranging from 150°C to 250°C (Li et
the outlet. Upon release of pressure, significant al., 2016). In a comparative study, Huang et al.
abrasion occurs, weakening the structural (2016) evaluated microwave pyrolysis across
integrity of the biomass (Hjorth et al., 2011; seven types of biomass, including corn stover,
Konan et al., 2022). This abrasion facilitates the rice straw, rice husk, sugarcane bagasse,
depolymerization of macromolecules, improving sugarcane peel, coffee grounds, and bamboo.
biodegradability through both external and The results demonstrated that microwave
internal friction (Duque et al., 2017). pyrolysis effectively converts biomass into
bioenergy and green materials. Both temperature
The effectiveness of extrusion as a pretreatment and heating rate were found to correlate linearly
has been demonstrated in multiple studies. For with microwave power levels up to 250 W.
example, extrusion of rice straw resulted in a Compared to conventional thermal methods,
32.5% and 72.2% increase in methane microwave pretreatment is notably faster,
production when compared to milled and consumes less energy, and results in greater
untreated rice straw, respectively (Tsapekos et weight loss due to more aggressive thermal
al., 2015). Similarly, Kozlowski et al. (2019) decomposition. It also exhibits distinct kinetic
reported an increase in biogas and methane parameters and reaction dynamics.
production by 7.50% and 8.51%, respectively,
following extrusion pretreatment. Despite these However, a key limitation of microwave
improvements in yield, the application of irradiation lies in the inherently low dielectric loss
extrusion in full-scale biogas plants has faced factor of dry biomass, which limits its ability to
economic challenges. In particular, studies in absorb microwave energy effectively. This
Poland have shown that the energy demand of challenge persists until sufficient char is
the extrusion process renders it economically generated to improve microwave coupling
unjustifiable in certain operational contexts. efficiency (Salema et al., 2017).

Although extrusion can be conducted under 4.1.4 Ultrasonication

relatively mild conditions—such as low pH,
moderate temperatures, and short retention Ultrasonication is a modern and highly effective
times—it remains constrained by its high energy mechanical pretreatment technique that has
consumption and the significant capital cost of demonstrated considerable potential in
the required machinery (Victorin et al., 2020). enhancing the biodegradability of biomass,
particularly sludge. This method involves the
4.1.3 Microwave irradiation application of high-frequency ultrasonic waves
that disrupt the physical structure, alter chemical
Microwave irradiation is another physical bonds, and influence the biological accessibility
pretreatment method that uses non-ionizing of organic matter. The effectiveness of
electromagnetic radiation, typically in the ultrasonication depends on both the
frequency range of 300 to 300,000 MHz with physicochemical properties of the biomass and
wavelengths between 1 mm and 1 m, to deliver key operational parameters such as frequency,
energy to the biomass (Huang et al., 2016). This amplitude, and duration of sonication. In full-
form of non-conventional heating has been scale applications, ultrasonication has resulted in
extensively applied to the treatment of up to a 50% increase in biogas production.
lignocellulosic materials. The mechanism Energy balance analyses have further indicated
involves dipole rotation and ionic conduction, a favourable energy return, with an average net

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energy gain to electricity use ratio of 2.5 (Pilli et chemicals for hydrolysate conditioning. By
al., 2011). increasing the accessible surface area of
cellulose, HTP improves its enzymatic
In addition to sludge treatment, ultrasonication digestibility (Sato et al., 2021).
has shown promising results with microalgal
biomass. Dhandayuthapani et al. (2021) In a comparative study, Chandra et al. (2012)
conducted a study using defatted green evaluated the performance of untreated and
microalgae biomass derived from Chlorella pretreated rice straw substrates using NaOH and
sorokiniana NITTS3 as a substrate for ethanol hydrothermal pretreatments. Their findings
fermentation by Saccharomyces cerevisiae showed that NaOH-pretreated biomass achieved
NITTS1. Ultrasonic pretreatment of the defatted the highest biogas and methane yields, though
biomass led to a significant increase in ethanol HTP-treated straw also outperformed untreated
production, with yields rising by 25.83 g/L samples. Similarly, Hashemi et al. (2019)
compared to untreated biomass. Analysis of the demonstrated that hydrothermal pretreatment
hydrolysate obtained after sonication revealed improved biogas production from safflower straw,
elevated concentrations of simple sugars, with the best methane yields observed under the
notably glucose and xylose, which are readily mildest pretreatment conditions. These findings
fermentable by yeast. Optimal fermentation emphasize the efficiency of HTP in enhancing
conditions—30°C, pH 4, and agitation at 200 substrate digestibility and methane production,
rpm—resulted in an ethanol yield of 52.10 ± 0.12 especially under optimized process conditions.
g/L, corresponding to 86.70 ± 0.52 mg of
bioethanol per gram of dry moldy bran. 4.2.1 Steam explosion

These findings underscore the versatility of Steam explosion, initially developed by Mason in
ultrasonication as a pretreatment approach not 1926, is a widely adopted hydrothermal
only for improving anaerobic digestion but also pretreatment technique that utilizes high-
for enhancing fermentative biofuel production by pressure saturated steam to disrupt biomass
increasing substrate accessibility and sugar structure. The process typically involves
release. exposing biomass particles to temperatures
between 160°C and 260°C for a short duration,
4.2 Hydrothermal Pretreatment followed by rapid decompression. This sudden
pressure release causes the biomass to undergo
Hydrothermal pretreatment (HTP) is recognized explosive decompression, which mechanically
as one of the most environmentally sustainable breaks down the cell walls, hydrolyses
methods for biomass processing due to its hemicellulose, and alters the structure of lignin
reliance solely on water and lignocellulosic (Zheng et al., 2021; Sun and Cheng, 2002).
substrate, without the need for chemical
additives. Key parameters influencing the During steam explosion, organic acids—such as
efficiency of HTP include temperature and acetic acid—are often released from the biomass
residence time, both of which play critical roles in itself and can aid in hemicellulose hydrolysis. In
disrupting the rigid lignocellulosic structure (Guan addition, water at elevated temperatures
et al., 2021). During HTP, biomass—such as rice contributes to hydrolysis due to its mildly acidic
straw—is subjected to high-pressure, high- behaviour. The combined physical and chemical
temperature water, which facilitates cellulose action of steam explosion significantly improves
hydration and enables the partial removal of the degradability and bioconversion potential of
hemicellulose and lignin. Importantly, this lignocellulosic materials.
process avoids the need for corrosive chemicals
or specialized hydrolysis reactors made from Several studies have investigated the fuel
corrosion-resistant materials. characteristics and biogas potential of steam-
exploded biomass. Lam (2011) reported that
Unlike several other pretreatment techniques, steam-exploded wood displayed favourable fuel
HTP can be performed without prior size properties, such as high heating value, low
reduction of biomass on a commercial scale, moisture uptake, and excellent pelletizing
leading to substantial energy savings in qualities. Pellets produced from steam-exploded
preprocessing (Kumari and Singh, 2018). biomass exhibited higher density and structural
Furthermore, the process generates fewer integrity compared to untreated wood. Similarly,
residues requiring neutralization and uses fewer Biswas et al. (2011) found that steam-exploded

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salix biomass exhibited lower alkali metal content types. For example, Li et al. (2010) reported
and improved physical properties, including cellulose yields between 75% and 77% from
enhanced density, impact resistance, and bamboo subjected to this pretreatment. Similarly,
abrasion resistance. Although char reactivity Smichi et al. (2016) demonstrated that freezing–
showed a slight decline at higher temperatures thawing was the most effective method among
and longer residence times, ash fusion behaviour several tested for achieving the highest glucose
remained largely unaffected. concentration from Juncus maritimus. These
improvements are attributed to enhanced
Steam explosion can be implemented in both substrate permeability and structural loosening.
continuous and batch reactor configurations.
Continuous systems are typically preferred for Beyond plant biomass, freezing–thawing
industrial-scale applications, while batch reactors pretreatment has also been investigated for its
are commonly used in research settings. The effects on sludge biodegradability and
technique has been successfully applied to a subsequent anaerobic digestion. Studies by
wide range of biomass types, including wheat Cano et al. (2015) and Yang et al. (2021), have
straw, corn stalks, Miscanthus, hardwoods, shown promising results, particularly in
softwoods, sugarcane bagasse, and food enhancing sludge solubilization and organic
processing residues such as citrus pulp and matter availability. In one notable study, Rooni et
potato waste, as well as aquatic biomass like al. (2017) examined the freezing pretreatment of
seaweed. barley straw for bioethanol production. Their
laboratory-scale experiments revealed that
Several studies have reported significant applying four freeze–thaw cycles resulted in a
improvements in methane yields following steam maximum hydrolysis efficiency of 19.42%,
explosion pretreatment. For example, methane highlighting the potential of this low-cost and
production from wheat straw has been shown to chemical-free method.
increase by 20–30% compared to untreated
biomass (Menardo et al., 2013; Datar et al., While additional work, such as that by Li et al.
2007). These enhancements affirm steam (2019), may further clarify the scope and
explosion as a robust and scalable pretreatment limitations of freezing–thawing pretreatment, the
strategy for improving the anaerobic digestibility existing evidence supports its utility as an
of lignocellulosic feedstocks. environmentally benign and operationally simple
approach to enhance biomass conversion
4.2.2 Freezing efficiency.

Freezing is a simple yet effective hydrothermal 4.3 Chemical Pretreatment

pretreatment method that utilizes repeated
freeze–thaw cycles to disrupt the lignocellulosic Chemical pretreatment techniques involve the
matrix and improve biomass digestibility. As application of acids, alkalis, oxidants, or solvents
described by Rooni et al. (2017), the process to disrupt the structural integrity of lignocellulosic
involves mixing milled lignocellulosic biomass biomass. These methods aim to improve the
with water and subjecting it to sub-zero digestibility of cellulose and hemicellulose by
temperatures, typically as low as –18°C, followed altering or removing lignin, decrystallizing
by thawing at ambient temperature cellulose, and solubilizing hemicellulose.
(approximately 22°C). This freeze–thaw process Chemical pretreatments are particularly effective
is usually repeated across multiple cycles to when integrated with physical or thermal
maximize structural damage. The mechanical strategies and can significantly enhance
stress caused by the expansion of water during enzymatic hydrolysis and microbial degradation
freezing increases the internal water content of in downstream processes.
plant stalks and results in the expansion and
rupture of cell walls. Consequently, the 4.3.1 Alkali pretreatment
lignocellulosic structure becomes more porous,
enhancing the accessible surface area for Alkali pretreatment involves the immersion of
enzymatic or microbial action (Guan et al., 2021). lignocellulosic biomass in alkaline solutions such
as sodium hydroxide (NaOH), potassium
The effectiveness of freezing–thawing hydroxide (KOH), ammonium hydroxide
pretreatment in isolating cellulose-rich fractions (NH₄OH), or lime (Ca(OH)₂). This process
has been confirmed across a range of biomass modifies the structural components of the

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biomass by disrupting ester and ether linkages in mineral and organic acids such as sulfuric acid
lignin and between lignin and carbohydrates. It (H₂SO₄), hydrochloric acid (HCl), acetic acid
also results in partial decrystallization of cellulose (CH₃COOH), and phosphoric acid (H₃PO₄) to
and facilitates the solvation and removal of disrupt the biomass matrix. These acids
hemicellulose. A subsequent neutralization step effectively hydrolyse hemicellulose and, to a
helps to separate lignin from inhibitory lesser extent, cellulose, thereby increasing the
compounds, reducing the formation of accessibility of carbohydrates for enzymatic or
fermentation inhibitors and improving overall microbial digestion (Guan et al., 2021). Acid
biodegradability. Compared to other chemical pretreatment is applicable using either
pretreatment methods, alkali pretreatment concentrated or dilute acid solutions. While
typically operates under milder temperature and concentrated acids demonstrate high efficacy in
pressure conditions (Badiei et al., 2014). depolymerizing structural carbohydrates, their
corrosiveness, toxicity, and associated recovery
This method enhances microbial accessibility to costs limit their practical application at scale.
lignin by disrupting its cross linkages within the
plant cell wall matrix, thereby facilitating more The effectiveness of acid pretreatment has been
efficient enzymatic or microbial degradation. demonstrated in multiple studies. Monlau et al.
Alkali pretreatment has demonstrated broad (2013) optimized methane production from
efficacy across various biomass types, including sunflower oil cake by applying the highest
maize stover, switchgrass, bagasse, wheat effective concentration of H₂SO₄ at 17°C,
straw, rice straw, hardwoods, and softwoods. resulting in enhanced methane yields. Similarly,
Syaichurrozi et al. (2019) examined the effects of
One specific variant, lime pretreatment, uses sulfuric acid pretreatment on Salvinia molesta
CO₂ for in situ neutralization, which eliminates biomass. Their results indicated that increasing
the need for a solid-liquid separation step. It is the acid concentration from 2% to 6% (v/v) over
considered cost-effective and energy-efficient a two-day batch process at ambient temperature
compared to other alkaline techniques. and pressure improved biogas yield. This was
Additionally, CaCO₃ can be recovered via CO₂ attributed to lignin solubilization and an increase
precipitation (Rabelo et al., 2009). While alkali in nitrogen-free extract content. Taherdanak et
pretreatment generally incurs high operational al. (2016) further demonstrated that dilute sulfuric
costs—mainly due to the use of chemical acid pretreatment of wheat plants significantly
catalysts—it generates fewer inhibitory enhanced methane yield, lignin removal, and
compounds and can be combined with other xylan degradation during anaerobic digestion.
methods for high-solids biomass treatment. The
approach has shown significant potential for Despite its efficacy, acid pretreatment presents
improving sugar yields from various several challenges. The low pH of acid-treated
lignocellulosic sources. biomass requires neutralization prior to
anaerobic digestion, typically achieved through
Park and Kim (2012) investigated the alkali addition or extensive water washing.
effectiveness of alkali pretreatment on Moreover, under high-temperature conditions,
eucalyptus residue for ethanol production. Their acid pretreatment can generate toxic degradation
study revealed that enzymatic digestibility of byproducts such as 5-hydroxymethylfurfural
pretreated biomass was five times greater than (HMF), furfural, and phenolic compounds. These
that of untreated eucalyptus. Moreover, a compounds are known to inhibit microbial activity
percolation-based pretreatment process resulted and reduce biogas production efficiency.
in more than double the degree of delignification Consequently, additional steps such as
and a twelvefold increase in enzymatic detoxification or thorough washing are often
digestibility compared to soaking-based necessary to remove these inhibitors and ensure
methods, demonstrating the efficiency of alkali the safety and effectiveness of downstream
pretreatment in facilitating biomass conversion anaerobic digestion processes (Tsai et al., 2021).
for biofuel applications.
4.4 Biological Pretreatment
4.3.2 Acid pretreatment
Biological pretreatment utilizes naturally
Acid pretreatment is another widely used occurring microorganisms—primarily fungi and
chemical method for enhancing the digestibility of bacterial consortia—to break down complex
lignocellulosic biomass. This approach employs lignocellulosic structures under mild operational

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conditions. This approach is environmentally after a 7-day pretreatment at 35.2°C. However,

friendly and energy-efficient, offering a not all microbial combinations are effective. For
sustainable alternative to chemical and thermal example, mixing garden waste compost and
methods. By targeting structural components fungus from maize silage and applying the
such as lignin and hemicellulose, biological mixture to manure biofibres did not result in
pretreatment enhances the accessibility of significant improvements in methane production.
cellulose, thereby improving the efficiency of
subsequent anaerobic digestion and biogas Biological pretreatment strategies also extend to
production. the use of formulated microbial agents, such as
freeze-dried powders that combine pure cultures
4.4.1 Fungal pretreatment of yeast and cellulolytic bacteria. In one study,
maize straw inoculated with this microbial
Fungal pretreatment aims to exploit specific fungi formulation and incubated for 15 days at ambient
capable of selectively degrading lignin and temperature exhibited a 75% increase in
hemicellulose while minimizing cellulose loss. methane yield and a 34.6% reduction in digestion
Since cellulose is more resistant to fungal time. These results highlight the importance of
degradation, its preservation is crucial for microbial synergy and environmental adaptation
downstream biogas production. The degradation in successful pretreatment design.
of lignin and hemicellulose increases the porosity
of biomass, thereby improving enzymatic and Ongoing research also focuses on using
microbial access to cellulose and enhancing microbes and enzymes to facilitate targeted
digestibility during anaerobic digestion (Zheng et degradation pathways. In addition to lignin
al., 2014). Among the various fungi studied, removal, biological agents can be tailored to
white-rot fungi have shown the highest reduce specific inhibitory or antimicrobial
effectiveness due to their ligninolytic enzyme compounds present in biomass. Fungal species
systems. These fungi possess the ability to such as Sporotrichum, Aspergillus, Fusarium,
modify lignin structure through the secretion of and Penicillium have been employed in solid-
oxidative enzymes such as lignin peroxidase, state fermentation processes to increase
manganese peroxidase, and laccase. Fungal feedstock availability and reduce antimicrobial
pretreatment is generally conducted under content, thereby further enhancing biogas yield
sterilized or semi-sterilized conditions to control (Taherzadeh and Karimi, 2008).
microbial competition and ensure consistent
degradation performance (Taherzadeh and 5. CONCLUSION
Karimi, 2008).
Anaerobic bioconversion of lignocellulosic
4.4.2 Microbial pretreatment wastes represents a promising and sustainable
approach to addressing global energy demands
In contrast to isolated fungal treatments, and mitigating environmental degradation caused
microbial consortia pretreatment employs diverse by fossil fuel dependence. With a rapidly growing
microbial communities—often sourced from population and intensifying ecological
natural habitats such as decaying biomass or challenges, advancements in bioenergy
compost piles—to degrade lignocellulosic technologies offer a viable pathway to reduce
materials. These consortia are typically more greenhouse gas emissions and transition toward
versatile and capable of degrading cellulose and a circular, low-carbon economy. Anaerobic
hemicellulose in addition to lignin. Zhang et al. digestion processes, when optimized for
(2019) developed a thermophilic microbial lignocellulosic biomass, can effectively convert
consortium by enriching microbes from decaying agricultural residues and organic waste into
straw and landfill environments. When applied to renewable biogas, contributing to both energy
cassava residues at 55°C in the presence of security and environmental conservation.
distillery effluent, this consortium resulted in a
96% increase in methane yield over untreated A wide range of pretreatment techniques has
residues after 12 hours of pretreatment. been developed to enhance the efficiency of
lignocellulosic bioconversion. These methods
Badiei et al. (2014) introduced a microbial focus on improving substrate accessibility by
enrichment group (MEG) system through multi- altering factors such as cellulose crystallinity,
generation selection, achieving a 25% surface area, and the protective effects of lignin
improvement in biogas yield from cotton stalks and hemicellulose. Targeted disruption of these

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structural barriers enhances enzymatic Akhtar, N., Gupta, K., Goyal, D., & Goyal, A.
hydrolysis, thereby improving biogas yields. (2016). Recent advances in pretreatment
Additionally, maximizing the utilization of technologies for efficient hydrolysis of
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co-digestion of yard waste and food waste
Author(s) hereby declare that NO generative AI for biogas production. Bioresource
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COMPETING INTERESTS 21(6), 1202–1235.
Cano, R., Pérez-Elvira, S., & Fdz-Polanco, F.
Authors have declared that no competing (2015). Energy feasibility study of sludge
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