Anaerobic Digestion of Lignocellulosic Biomass: Substrate Characteristics (Challenge) and Innovation

Abstract

Modern society is characterised by its outstanding capacity to generate waste. Lignocellulosic biomass is most abundant in nature and is biorenewable and contains energy sources formed via biological photosynthesis from the available atmospheric carbon dioxide, water, and sunlight. It is composed of cellulose, hemicellulose, and lignin, constituting a complex polymer. The traditional disposal of these types of waste is associated with several environmental and public health effects; however, they could be harnessed to produce several value-added products and clean energy. Moreover, the increase in population and industrialisation have caused current energy resources to be continuously exploited, resulting in the depletion of global fuel reservoirs. The overexploitation of resources has caused negative environmental effects such as climate change, exacerbating global greenhouse gas emissions. In the quest to meet the world’s future energy needs and adequate management of these types of waste, the anaerobic digestion of lignocellulosic biomass has remained the focus, attracting great interest as a sustainable alternative to fossil carbon resources. However, substrate characteristics offer recalcitrance to the process, which negatively impacts the methane yield. Nevertheless, the biodigestibility of these substrates can be enhanced through chemical, physical, and biological pretreatment methods, leading to improvement in biogas yields. Furthermore, the co-digestion of these substrates with other types and adding specific nutrients as trace elements or inoculum will help to adjust substrate characteristics to a level appropriate for efficient anaerobic digestion and increased biogas yield.

1. Introduction

Biomass resources can be categorised into first-generation biomass (food crops), second-generation biomass (lignocellulosic biomass), and third- and fourth-generation biomass (microalgae) [1]. Lignocellulosic biomass is an abundant and renewable resource originating from plants, and its major composition is polysaccharides (cellulose and hemicellulose) added to lignin (an aromatic polymer) [2]. It is viewed as the only sustainable source of organic carbon on Earth with net-zero carbon emission; thus, it has no adverse environmental effects. The production of lignocellulosic biomass is quick, and it has a lower cost than other types of resource feedstock [3]. It is the non-edible portion of the plants; therefore, it does not interfere with food supplies. These types of waste are biodegradable and biocompatible and could be used in several commercial applications [4].

Clearly, large quantities of forestry, domestic, agricultural, and agro-industrial waste are produced and accumulated each year [5]. Nevertheless, if such wastes cannot be treated rapidly and via harmless approaches, they will become an obvious and striking threat to the health of the population as well as to the development of society on a sustainable basis [6]. The management of these wastes still dominates as the key societal and governance task, especially in urban areas faced with high population growth rates and garbage generation. Agricultural wastes include wastes originating from the growing and processing of agricultural products, e.g., fruits, vegetables, meat, poultry, dairy products, and crops, which can exist in solid, liquid, or slurry forms based on the agricultural product [7,8].

Agricultural wastes are anticipated to increase in the future in less developed countries. In these countries, they are usually managed or disposed of by dumping in landfills or via incineration [9,10]. These methods of disposal lead to environmental pollution (air, water, and soil pollution harming natural life), causing climate change and global warming via the emission of greenhouse gases [11]. They also cause environmental odour and public health hazards owing to the pollution of water with leachate (emanating from the decomposition of wastes and containing diverse toxic chemicals and trace elements and microbial contents) seeping through the soil into environmental water bodies [12], which might lead to infections and diseases in humans [13]. They have also demonstrated hazardous effects on aquatic and animals in the soil. Consequently, there is a quest to improve on or upgrade methods to conveniently managed these wastes, thereby mitigating the deleterious effects associated with the commonly used waste disposal methods [9].

In addition, the strict reliance on fossil fuels for energy production globally results in climate change and global warming, and the effects of greenhouse gas emissions prompt the quest for alternative and complementary sources of energy [14]. More elaborately, technological development along with adverse environmental impacts ensuing from the utilisation of conventional fuels have incited the production of clean energy in great quantities from renewable energy resources on a sustainable basis [15]. The use of biomass presented as a renewable source with the help of diversified technologies is considered a sustainable approach to address the crisis in energy supply and the release of greenhouse gases to the environment [16]. The implementation of biomass offers cost-efficient viability and reduces the quantity of waste discharged into the environment. Energy sources are considered renewable and sustainable if they are produced from sources that are regularly replenished in a human’s lifetime, including wind, hydropower (water), geothermal, biomass, and solar. Amongst these, anaerobic digestion is viewed as the important and popular method for producing renewable energy from biomass, thus becoming the focus of attention [14].

Lignocellulosic biomass is composed of polysaccharides (cellulose and hemicellulose) and lignin, forming a complex three-dimensional structure that hinders hydrolysis by enzymes during anaerobic digestion; therefore, pretreatments are applied on these substrates to render cellulose accessible for hydrolysis, producing monosaccharides. to monosaccharides for hydrolysis [17]. In general, pretreatment methods entail chemical, physical, and biological methods [18]. The selection of substrates for anaerobic digestion is a key parameter that influences the anaerobic digestion process, and finally, the quantities of biogas that are produced as substrates vary in composition in terms of the C:N ratio, trace elements, microbial content, volatile solids, total solids, biochemical oxygen demand (BOD), chemical oxygen demand (COD), as well as the biochemical methane potential (BMP) [19].

Seemingly, the availability of trace elements as micronutrients is critical in the stability and performance of the anaerobic digestion process via microbial growth and enzyme activity [20]. Adequate supplementation with trace elements is essential for microbial reproduction and metabolic processes [21]. This insinuates that the deficiency of trace elements during the anaerobic mono-digestion of lignocellulosic feedstock results in reduced biogas production and process failure [17].

Against this background, the challenges in enzymatic hydrolysis and trace element deficiency during the anaerobic mono-digestion of lignocellulosic biomass can be avoided via pretreatment, inoculation, and co-digestion with different substrates (especially animal manure) in order to achieve and maintain optimum nutrient supply in terms of the C/N ratio as well as the macro- and micronutrients needed for an efficient process.

2. Description of Lignocellulosic Wastes (Agricultural Wastes)

Several types of lignocellulosic biomass do exist, which can be grouped broadly into woody and non-woody biomass solely based on their chemical composition and physical properties [22]. From a bioenergy perspective, lignocellulosic biomass can be categorised based on its source/origin. Biomass can be derived from different sources, including rural (forestry, livestock, agriculture), urban (sewage sludge and municipal solid wastes), and industrial (cellulose and agro-industrial). Lignocellulosic biomasses comprise cellulose (40–60%), hemicellulose (20–35%), and lignin (15–40%) bound into a complex structure. The ratios between these polymers are influenced by the plant′s age, stage of growth, and other conditions. Cellulose comprises an amorphous portion and a crystalline portion owing to the varying orientation of cellulose molecules throughout the structure [23]. Hemicellulose can be hydrolysed to simple sugars, though it appears as a random amorphous structure consisting of heteropolymers (glucomannan, glucuronoxylan, xyloglucan, xylan, and arabinoxylan). The source of the lignocellulose determines the composition of hemicellulose in a biomass material. Accordingly, lignin is hydrophobic in nature and links cellulose to hemicellulose, presenting as a physical barrier against the biological degradation of lignocellulosic biomass [24].

Agricultural wastes are a subgroup of the lignocellulosic biomass and are described as plants or plant-derived wastes that are not used as food or feed and are the most abundant and renewable on the planet Earth [2]. They include manure (grass-fed cattle), corn stover (stalk and leaves), wheat stem, Prairie cordgrass, corncob, unbleached paper, switchgrass, cereal straw, bagasse, rice straw, wheat straw, poplar and wood residues, etc. [25,26]. However, wood residues (e.g., sawdust, wood chips, etc.) have an edge over agricultural residues in that the transportation cost is reduced because of their high bulk density, the possibility of year-round harvest, plus the availability of well-established logistics [27]. Sawdust is an example of plant waste that can be reused to produce a valuable product (i.e., resource recycling) if not discarded in an attempt to prevent the side effects resulting from its disposal [28]. Successful approaches to the management of sawdust waste include the production of particle boards [28], lightweight bricks [29], and wood–plastic composites, as well as mulching, animal bedding, briquette production, household energy generation, and fertilisers [30].

Lignocellulosic wastes are some of the most abundant wastes generated annually, in most developing countries. These types of waste constitute a threat to the environment when not disposed of appropriately, as they pose environmental challenges [31]. Lignocellulosic crop residues, including straw, are usually combusted in an attempt to prepare the land; however, this practice is associated with huge environmental pollution and the loss of nutrients [32]. Owing to the problems associated with waste disposal, an essential way for the application of lignocellulosic wastes is using them for soils and farming. Plant-based materials rich in carbon are effective in producing soil conditioners. The lignocellulose structure in different biomass types varies greatly, which may affect degradability owing to the difference in lignin structure, though having the same lignocellulose concentration. However, straw is described to harbour a high concentration of lignocellulose content relative to maize and grass of low concentration. Straw return is an effective method for the management and disposal of agricultural residues. It has a dual purpose: addressing the problem of excessive straw treatment and preventing the pollution caused by straw burning [33]. Straws can be incorporated (for decomposition) into the soil in preparation for the growth of the subsequent crop. The decomposition of the straw returned into the soil occurs in two phases. The first is a rapid phase, wherein organic materials (cellulose, hemicellulose, protein, etc.) are decomposed, and microbial activities are higher as the soil microorganisms are diverse (bacteria and fungi). In the second phase, which is a slow decomposition phase, the lignin, tannins, waxes, and other substances that were not previously or minimally decomposed are gradually decomposed, lasting for 2–3 years or longer [34]. Wang and colleagues [35] remarked that mixing straw with soil via tillage methods (mixing or ploughing, ditch burying) increases soil microbial activities and soil respiration, which tends to cause higher decomposition of straw, thus improving soil, water, and heat conditions [36]. This will eventually lead to an increase in the content of the soil’s organic matter, as well as nitrogen (long term) and phosphorus and potassium (short term), promoting the growth of the subsequent crop [33].

When introduced into the soil (without tillage), it leads to a reduction in the quantity of plant available nitrogen because the microbial communities involved in the decomposition process require more nitrogen than that found in the plant material; thus, the final product originating from lignocellulosic crop residues tends to be phytotoxic.

Interestingly, Harindintwali and colleagues [37] highlighted that aerobic composting is a rapid, cost-effective, and sustainable alternative process to solve the environmental issues ascribed to the treatment of lignocellulosic crop residues; therefore, it is a better management practice. It is termed an eco-sustainable green approach for the treatment of lignocellulosic crop residues and agricultural development. In this light, although it decomposes slowly, composting with a nitrogen-rich substrate (e.g., manure) results in a nutrient-rich compost. Additionally, Wei and colleagues [38] remarked that the utilisation of different bacteria with both cellulolytic and nitrogen-fixing properties tends to boost the degradation of lignocellulose in straw during composting via the increase in the activities of key enzymes. The achievement of composting and the subsequent application of the compost as a soil conditioner solely depends on the ability of the microflora occurring during the composting process. As a compost, generated from straw, a lignocellulose material, it serves as the main source of precursors for humus production as well as other microbiological biotransformation products. Mature composts constituting plant materials such as carbon improve the physical, biological, and chemical characteristics of the soil when introduced due to their ability to provide better water and air conditions, reduce soil erosion, and enhance soil structure [39]. Therefore, compost can be employed as a soil conditioner, organic fertiliser, and plant growth stimulator, thus avoiding the negative effects associated with chemical fertilisation due to reduced dependence on chemical fertiliser for crop production [37].

Typically, the lignocellulosic content of agricultural plant residues accounts for about 50–90% of total organic matter; therefore, they are often used as bulking agents in composting to produce compost for soil application. Initially, soluble organic compounds are broken down via bacterial activities, but as the process of composting progresses, hemicellulose and cellulose become part of the formation of soluble organic compounds [40], which can be partly converted to a humus compound that is similar in structure to those found in the soil. The degradation of organic matter varies indirectly associated with the initial concentration of lignin in the composted biomass.

3. Traditional Disposal or Management of Lignocellulosic Wastes

The long-established traditional disposal of lignocellulosic biomass includes uncontrolled dumping, combustion, incineration, composting, and landfilling. However, each method is associated with drawbacks.

Table 1. Differences between the three thermal processes alongside composting and landfilling.

Parameters	Thermal Processes			Composting	Landfilling
	Incineration	Gasification	Pyrolysis
Level of oxygen	Excess air	Partial air	Absence of air	5–15%	Little to no oxygen
End products
Liquid	None	None	Pyrolysis oil and water	Water	Leachate
Solid	Slag, ash	Slag, ash	Ash and coke	Compost	Humus
Gas	CO2, H2O, O2, N2	H2, CO, CO2, CH4, H2O, N2	H2, CO, H2O, N2, hydrochloric	CO2	CH4, CO2, NH3, SO2−
Temperature	980–1200 °C	1000 °C (downdraft) 1500 °C (cross-draft)	400–800 °C	32–60 °C	Depends on the height and size, 34–55 °C

shows the differences between the three thermal treatment processes alongside composting and landfilling (adopted from Quina et al. [41] with modifications).

The Economy and Transferability at Real Scale/Applicability of Conventional Methods of Waste Disposal

The organic fraction is a vital portion of lignocellulosic wastes that are generated in different countries, with different states of economy. The implementation of management strategies on a large scale owing to economic growth, rapid urbanisation, rising population growth, and industrialisation faces challenges related to economic feasibility [42]. The degree of implementation and the practical use of the different conventional waste management methods are believed to vary amongst individuals, communities, and countries, based on economic viability. These wastes need to be sustainably managed in a circular economy where, recycling, recovery, and reuse are pertinent to evade environmental and public health effects [43]. Landfilling exists as the oldest form of waste management, which is cost-effective in countries with large open spaces and is associated with fewer fixed or ongoing costs, presenting a very strong competitive advantage [44]. However, the availability of plots of land needed for this purpose as the population increases, as well as the cost involved in the proper management of the site (cost of screening or preselection of the wastes, the disposal fee of the rejected wastes, etc.) in order to mitigate the effects of leachate and greenhouse gas emissions on water, soil, and air, respectively, seem economically challenging [45]. Methane gas is a major contributor to climate change and global warming. In addition, the high price of land cannot be excluded.

Incineration/combustion is considered the most popular means of waste management, and it is usually conducted in sites located far away from where people or communities reside, or at the edge of cities. Therefore, wastes need to be collected and transported to distant sites, causing marked increases in costs. Incineration is the option of choice for large cities but involves large atmospheric pollution and capital costs for the construction of incinerator plants or the upgrade of treatment facilities for co-incineration [44]. In addition, the costs incurred via the use of advanced chemical processes to remove pollutants (sulphur dioxide, hydrogen chloride, nitrogen dioxide, and hydrogen fluoride) from flue gases (composed of dust and gases) after incineration might grossly increase the overall cost of the project [46]. Previous researchers have further mentioned that African countries (South Africa, Egypt, Morocco, Algeria, and Nigeria) have a higher dioxin emission potential via the incineration of solid wastes than the other countries included in their study. This will, however, affect the sustainability and applicability of waste management methods. Moreover, Kim and Jeong [47] mentioned that incineration has played a principal role in waste energy production through heat energy recovery, which is asserted to incur inevitable increases in cost. Jacela and coauthors [48] declared incineration with an energy recovery scheme unfit due to environmental and public health reasons but emphasised that waste-to-energy incineration on a small scale is economically feasible and cost-efficient in treating healthcare wastes based on cost–benefit and cost-effective analysis in the Philippines.

As a result, composting is considered more attractive than landfill and incineration because of the high degradation of organic wastes, yielding a value-added product [45]. Its application leads to fewer environmental effects than landfilling and incineration. It embraces several technological options, which may be used in a small-scale system, such as in an urban apartment, or medium-to-large/full-scale systems, for instance, at the industrial level, and they prevent the loss of organic material due to landfilling [49,50]. Accordingly, the cost of constructing a compost system will vary based on the type and size of the system, the materials employed, and whether an expert was contracted. Economic feasibility affects viability. The cost of operation/implementation includes capital cost and operational cost (utilities, operational personnel, maintenance and repairs, disposal of rejects, etc.), production processes, quality assurance, and compost market. The chain of continuous supply and demand of feedstock may equally vary; therefore, there is no need for a comparison of the cost between plants or countries. For instance, in the case of composting, the operational cost can be affected by the different methods, the organic content of the wastes, as well as the degree of pre-separation. Sabki and colleagues [42] recommended that it is better to improve compost quality so as to set it at a higher price that can generate higher revenue to enhance the long-term economic feasibility of the compost industry. Gillespie and Halog [43] highlighted that community-scale composting is viable when having the right partnerships, financial support, project design, and community awareness. However, the longer duration of the process can be a challenge to its economic feasibility as wastes might accumulate, leading to an environmental burden. Therefore, alternative treatments will be needed, resulting in additional costs for their disposal. Alternatively, storage facilities become crucial, adding to the overall cost, alongside the cost of collection. It is therefore clear that traditional waste management methods cannot be completely abandoned as they can complement each other and even supplement green technologies of waste management.

4. From Waste to Recovery and Energy (Anaerobic Digestion)

Naturally, lignocellulosic waste is restored in great quantities, and gathering it in the environment poses a threat to environmental sustainability. Unsustainable practices employed to manage lignocellulosic wastes are worsened because of rapid urbanisation, as well as financial and institutional limitations that hamper public health interventions and environmental sustainability [7]. In addition, there are financial constraints involved in their disposal; therefore, their potency as renewable and economically viable energy resources needs to be exploited. An effective and more sustainable approach to managing these wastes culminates in alleviating the negative impacts on the environment and public health, conserving resources, and improving the livability of cities [7]. In this light, waste can therefore be transformed or recycled, or reused via physical and biological approaches.

The reuse of wastes as recycled resources from the industrial chain saves costs in terms of raw materials and lessens the dependence on primary resources, in addition to providing an elimination route [4]. Certainly, lignocellulosic biomass can be employed in different types of industries, including paper manufacturing, biorefinery or biomass fuel production, animal feed, or composting tasks for biofertilisers. Lignocellulosic wastes obtained from agriculture are the most dominant, containing diverse sugars that occur as building blocks, and serve as key sources of biofuels and value-added organic products. They can be pretreated and serve as feedstock to produce biofuels (bioethanol), which is an ideal option to meet the increasing demand for energy for transportation and other purposes like heating and industrial processes [51]. In addition, they provide raw and less expensive materials to the chemical industry on a sustainable basis.

Globally, there is a steady rise in energy demand, owing to the rising population, rapid increase in industrialisation, and the quest for better living conditions [52]. This has resulted in permanent damage to the environment known as global warming and climate change because of the release of greenhouse gases from conventional sources of energy (coal, oil, etc.) [53]. In addition, escalating fuel prices, concerns about global warming potential, and a decline in known reserves, together with the growing demand for fossil fuels, have resulted in the uncertainty of petroleum supplies and thus prompted the global shift toward renewable energy [54]. The key sources of renewable energy include wind, solar, hydro, nuclear, and biomass [55]. Considering all renewable energy sources, biomass energy in the form of bioenergy is preferable as it is obtainable in all places across the world. Other energy sources are unable to generate liquid or gaseous fuels for transportation, nor can they be used for combined heat and power generation. Their use is also affected by season and greatly influenced by the weather, atmospheric, or environmental conditions [55]. Some of these renewable energy sources are fraught with environmental and geographical challenges (wind and hydro); photovoltaic, solar thermal, and wind power are only imperative when the available weather conditions are right, and their surplus can only be converted into a storable form with great technical effort. Gangopadhyay et al. [56] concluded that the hybridisation of energy resources (wind, solar, and biomass) will create an avenue for their different individual characteristics to be exploited, compensating for their drawbacks, thereby projecting the maximum utilisation of biomass. Biomass energy provides a reliable and consistent supply of electricity to end users. However, lignocellulosic biomass is considered the third major source of energy production, following petroleum and coal [57].

Although conventional energy sources are associated with adverse environmental and public health effects, the lack of continuous access to plant materials is a severe concern in the long-term sustainability of biogas generation, even as energy crops’ growth has been encouraged in crop rotations in order to increase biomass yields. Biomass can be converted via thermochemical (combustion, gasification, pyrolysis, and liquefaction) and biochemical methods (anaerobic digestion and fermentation) into useful products, depending on their characteristics and the end product [58]. In developing countries, where energy security is a major issue, renewable energy technologies appear to be promising as alternative energy sources to mitigate environmental challenges as well as to alleviate the energy scarcity in these countries. It is clear that crop residues are regarded as a large source of lignocellulosic biomass that is generated after agricultural practices, and they do not have any other use. As a result, they can be left in an open field, where they are burnt, polluting the air, and thus becoming potential sources of greenhouse gas emissions, or they remain unutilised.

The anaerobic digestion process is a technology through which abundant plant wastes can be transformed into valuable end products. It is considered a good way to improve waste management practices involving biodegradable wastes, thus sanitising the environment, a situation very prominent in developing countries [59]. In addition, anaerobic digestion appears to be the most profitable approach in terms of economic viability [59] for waste treatment, curbing environmental and energy concerns. The technology has long been in existence; nevertheless, it remains a very attractive technology for producing energy, especially because it is eco-friendly and replaces fossil-based energy sources [60]. Considering a time period from 1995 to 2023, about 17,200 published articles based on the anaerobic digestion of lignocellulosic wastes were found in the Google Scholar database ranging from studies in laboratories to pilot-scale trials. Also, there are 36,800 articles on the pretreatment of lignocellulosic wastes and 5800 articles on the BMP of lignocellulosic wastes. However, only two patents were found on anaerobic digestion of lignocellulosic wastes.

According to Kougias and Angelidakis [61], anaerobic digestion is valuable as a key alternative for producing energy amongst other renewable sources because it requires less space. It is independent of seasonal variations, the process is less complex, and it can be stably produced. The shift to renewable energy sources presents anaerobic digestion with the greatest advantage as it employs feedstock materials, which are by-products, residues, or waste products of other processes, with no competition for arable land.

4.1. Details of Anaerobic Digestion Process

Anaerobic digestion can also be termed microbial digestion/degradation, occurring without the presence of oxygen. The process involves concerted activities of microorganisms belonging to two major domains: bacteria and archaea. The consortia of microbes belong to four trophic levels, reflecting the four stages of the process, and they perform specific tasks. Accordingly, organisms are described based on the stage of the anaerobic process at which they perform their functions. They include hydrolytic bacteria (hydrolysis), acidogenic bacteria (acidogenesis), acetogenic bacteria (acetogenesis), and lastly, methanogens (methanogenesis) [62]. The variety of these microorganisms, as well as their varying growth requirements and selective ability in performing their task, make anaerobic digestion a complex process [14]. Owing to the sensitivity of microorganisms to nutritional and environmental parameters, it is of utmost importance to create a balance in a range of factors to maximise the possibility of achieving an optimum design and effective implementation [63].

Several studies have highlighted the significant effect of temperature on the growth and metabolism of microorganisms in addition to the interactions between microbial groups [64]. In this light, based on temperature, the anaerobic digestion process can be categorised into psychrophilic, mesophilic, and thermophilic microbial degradation, occurring at different temperature ranges of less than 30 °C, 30–40 °C, and 50–60 °C, respectively [65,66]. Regardless of the temperature at which the biodigester is operated, the feeding mode (continuous or batch), and the feedstock (animal wastes, food wastes, and wastewater sludge), all anaerobic digestion systems adhere to the same underlying principles. Thus, the process is influenced by a set of factors, including the carbon–nitrogen (C/N) ratio, temperature, organic loading rate (OLR), hydraulic retention time (HRT), and pH [19]. Generally, the process produces renewable energy (biogas) and organic-rich by-products (biodigestate products) used in agriculture for improving the growth of crops. In addition, it is used as a waste treatment method, preventing the release of dangerous wastes into the environment that cause pollution as well as diverting organic wastes from landfills.

The key aspects (products) of significance in anaerobic digestion, include the following features:

(a)

Degradation of cellulose and non-degradation of lignin: Cellulose constitutes about 30–50% of the dry weight of lignocellulose, thus presenting as the whole polysaccharide structure of the plant’s cell wall. It contains a linear chain of β (1→4) linked D-glucose units. The cellulose chains are interlinked by hydrogen bonds and Van der Waal’s forces, resulting in high tensile strength microfibrils. Owing to the different orientations of the cellulose molecule throughout the structure, it has varying levels of crystallinity. Moreover, Ahmed et al. [67] mentioned that cellulose microfibrils are connected to one another with the help of pectin and hemicellulose and are then covered with lignin, thus hindering easy degradation. It consists of an amorphous (non-crystalline) and a crystalline domain, with varying degrees of hydrolysis; the amorphous portion hydrolyses first before the crystalline [23]. Cellulose has the propensity to be degraded to simple sugars via the actions of cellulase after pretreatment is applied. Through pretreatment, the inter- and intra-hydrogen bonds are disrupted, making the solid material and macromolecules become hydrolysed and solubilised in the medium. This is achieved through the mechanisms of the extracellular enzymes of acidogenic bacteria, facilitating their utilisation by microorganisms [67]. Lignin may originate from three different building blocks, namely coniferyl alcohol (G), p-coumaryl alcohol (H), and sinapyl alcohol (S). Following cellulose, lignin is referred to as the second most abundant carbon source. It is a non-carbohydrate aromatic heteropolymer, different from cellulose and hemicellulose; therefore, it does not contribute simple monomeric sugars. It is the most complex component of lignocellulose, forming an irregular network with cellulose and hemicellulose. It fills the spaces in the cell wall, which results in the formation of a multistage fibre structure, thus providing lignocellulose with structural stability.

Anaerobic digestion is performed through the concerted activities of different bacterial groups and methanogens; however, the bacterial degradation of lignocellulose is more restricted. More elaborately, during the anaerobic degradation of plant-based materials, hydrolysis (the first microbiological step) becomes rate-limiting. The tight structure between lignin, cellulose, and hemicellulose, termed biomass recalcitrance, limit the hydrolysis step, thereby affecting the overall performance of the anaerobic digestion process. Furthermore, this culminates in poor biogas production [23]. This is because lignin provides strength and hydrophobicity to the plant’s cell wall, as well as shielding polysaccharides from microbial enzymatic degradation [68]. Specifically, recalcitrance is largely conferred by both cellulose (crystallinity and polymerisation) and lignin polymers, presenting physical barriers to the activities of microbial enzymes and therefore resulting in sub-optimal performance [23]. In addition, Serrano et al. [69] highlighted that the anaerobic digestion of lignocellulosic biomass may lead to the collapse of the system owing to the accumulation of volatile fatty acids as these materials are composed of high carbon-to-nitrogen ratio.

Lignin is known to be recalcitrant under anaerobic conditions except when the structure is modified [70]. This shows that lignin needs to be removed, but this is not feasible as the use of the whole biomass for biogas production will improve the economics of the process, in addition to making the process more sustainable [71]. To overcome this limitation in hydrolysis, pretreatment is chosen to weaken the crystallinity of cellulose, reduce the hindering effect of lignin, as well as increase the accessible surface area for enzymatic attack. Khan and Ahring [70] noted that wet oxidation followed by steam explosion pretreatments created noteworthy changes to the lignin structure, allowing for its biodegradation under anaerobic conditions. In addition, supplementing the anaerobic digester with specific microorganisms can help to improve the hydrolytic phase of the anaerobic digestion process involving lignocellulosic biomass. Naturally, bacteria and fungi are known to cause the degradation of lignin, but fungi are classified as the most efficient group responsible for lignin degradation [72]. However, fungal processes occur slowly and are hampered by requirements for specific temperatures (mesophilic range) and pH (acidic range) [73] as well as the slow reaction process. The biological conversion of lignin is majorly limited by the fact that native lignin is less non-degradable in the absence of oxygen; moreover, anaerobic conditions are salient in the production of valuable bioproducts via fermentation [71]. In a previous study, the authors further stated that the wet oxidation of lignin occurring at a temperature of ca 175 °C, while employing oxygen, causes an increase in the degree of methoxylation of lignin and a simultaneous increase in conversion throughout anaerobic digestion. In fact, for the anaerobic degradation of lignin, the first step involves de-methoxylation, followed by the cleavage of the ring and fermentation into methane and carbon dioxide [71].

(b)

Biogas production and valorisation: Biogas is produced via the anaerobic degradation of organic matter via the concerted activities of different microorganisms, occurring at the four distinct phases of the biological process, wherein the metabolites/end products of the previous phase serve as substrates for the subsequent phase [74]. Firstly, complex polymers, including carbohydrates, proteins, and fats, are decomposed into their monomeric units, e.g., sugars, amino acids, and fatty acids by hydrolytic bacteria. The acidogenic or fermentative bacteria utilise monomers, converting them into a blend of short-chain fatty acids. Subsequently, short-chain fatty acids are converted by acetogenic bacteria to produce acetate, carbon dioxide, and hydrogen, which are precursors for methane production. Methane is produced via the process of methanogenesis through either the hydrogenotrophic or acetoclastic pathways of methanogens [75]. However, the quantity of methane produced, which determines its calorific value, varies with the type and source of substrates, as well as the digester’s operating conditions employed in the anaerobic digestion process. Naturally, lignocellulosic materials have very small interior surfaces, especially when dried. The anaerobic co-digestion of lignocellulosic wastes with other substrates exerts synergistic effects on process stability and methane generation, and it enriches the microbial population, thus enabling contact between the substrates and microbes or enzymes as well as increasing the biodegradable components. Anaerobic digestion is the best-suited approach for lignocellulosic waste management and valorisation, producing biogas consisting of a mixture of gases, including methane (55–65%), carbon dioxide (30–35%), hydrogen sulphide, water, and traces of other gases [76].

Biogas can be combusted directly for cooking and lighting or harnessed for power generation [77]. However, carbon dioxide, water, siloxanes, and traces of H₂S exist as impurities in biogas, limiting its utilisation. If not removed, these impurities can lead to varying consequences, including fouling, corrosion, harmful emissions, and a decrease in the energy content of the biogas [78]. Owing to the incombustibility of CO₂, its calorific value is limited, thus affecting its applicability and transportability [79]. Additionally, hydrogen sulphide can corrode appliances, including generators and diesel engines that store, transport, or utilise biogas [78]. To address these limitations, biogas can be upgraded or converted into value-added products, thus increasing the scope of its utilisation as well as reducing greenhouse gas emissions to the environment. The upgrading of biogas can occur via different methods, including absorption, absorption, cryogenic, separation, and in situ techniques into biomethane [77]. According to Kapoor et al. [77], to ensure cost-effectiveness in biogas systems, the individual gases occurring in the biogas need to be valorised. For example, biomethane and bio-carbon dioxide realised during the upgrading of biogas can be employed as fuel for vehicle transportation, the injection of natural gas grids, grain fumigation, and the production of chemicals, respectively. Equally, de Llobet and coauthors [80] highlighted the valorisation of biogas via catalytic decomposition to produce syngas and carbon nanofibers.

(c)

Biodigestate: The residual liquid or solid material discharged from the biodigester after the completion of anaerobic digestion is termed digestate. The successful co-digestion of lignocellulosic wastes with other substrates is a potential economic process, leaving a nutrient-rich residue known as the digestate [81]. During anaerobic digestion, biogas is produced through the degradation of organic matter via bacterial actions. Therefore, the subsequent digestate is a complex matrix, comprising partly degraded organic matter, inorganic compounds, and microbial biomass in volumes, based on the makeup of the biomass feedstock and process parameters (designated temperature and retention time) [82]. This explains the fact that the content of each component in a digestate may differ when recovered from different installations. This is because it is known that most of the nutrients required by plants that occur in raw feedstock are retained during anaerobic digestion; thus, digestate contains all the critical macro- and micronutrients in different portions, reflecting those in the substrates. This indicates that the amount and composition of the digestate synchronises with those of the feedstock [83].

Digestate is a nutrient-rich material; however, its nutrients/physicochemical properties strongly depend on the type of feedstock material(s) (their nature and composition), the type of anaerobic digestion (wet or dry, batch or continuous, or mono- or co-digestion) and operational parameters (retention time) [84]. This might insinuate that the digestate recovered through co-digestion is higher in nutrient content than that from mono-digestion because of the multiplicity of substrates employed. In addition, it is established that the digestate from the anaerobic digestion of food-based feedstock is of higher nutrient value than those obtained from crop- and animal-manure-based materials. Furthermore, the quality and safety of the digestate equally depend on the type of substrates employed for its production [85]. Digestate contains high content of nitrogen in the form of ammonium nitrogen–total nitrogen ratio but a decreased carbon content since the organic matter of the feedstock is metabolised to methane and carbon dioxide. Thus, the digestate is known to have a lower carbon-to-nitrogen ratio than the initial feedstock [86]. It is considered for its valuable application as biofertilisers during crop production, replacing fossil-energy-requiring mineral fertilisers, thus aiding the recycling of nutrients between urban and rural areas [79].

Digestate might harbour biological, physical, or chemical contaminants, and their abundance is based on their initial concentration in the original substrate. Biological contaminants (including pathogens and weeds) are destroyed (inactivation) during anaerobic digestion, based on the temperature at which the process is performed [87]. However, chemical and physical contaminants usually end up in the digestate as they are not altered or affected during anaerobic digestion [84]. This jeopardises its applicability as a fertiliser because it poses a high potential threat to the environment and exerts a negative impact on its quality and sustainability for its use as a fertilisers. Therefore, regardless of the type of anaerobic digestion process through which the digestate is obtained, it needs to be post-treated or processed prior to its application as a biofertiliser for environmental and public health safety.

Digestate processing can be described as partial, targeting a reduction in its volume through relatively simple and cheap techniques. On the other hand, it is complete, meaning the digestate is refined via technologies with varying levels of technical maturity, higher energy consumption, higher investment, and operational costs to yield solid biofertiliser fractions, fertiliser concentrates, and pure water. The separation of the digestate into solid and liquid phases is the first step in its processing, which is inevitable to optimise its characteristics and the benefits associated with its use [83]. Employing digestate in agriculture as a fertiliser is one of the simplest methods of management, providing a solution to lessen or prevent adverse environmental effects and enhance the economic sustainability of biogas production [88]. In addition, returning nitrogen and phosphorus, which are vital nutrients, to the soil helps to offset soil erosion [89]. Unlike undigested organic wastes, digestate is more hygienic and stable microbially, and it is also high in ammonium, making it an excellent alternative to chemical fertilisers, reducing their use. It equally has fertilising properties, enhancing soil respiration [90].

Following the chronicle of information, Lamolinara et al. [83] advised that each digestate should be individually evaluated to know its strengths and weaknesses and its characteristics to determine its most sustainable application. However, Häffner et al. [86] proposed that the differences in the composition between digestates can affect their fertilising properties. The authors further remarked that the advantages of digestate as a soil conditioner are ascribed to the change in the composition of nutrients and organic matter caused by the anaerobic digestion process.

4.2. Characteristics of Substrates Employed in Anaerobic Digestion

A variety of substrates with varying organic fractions are employed in the process of anaerobic degradation, utilised either for waste treatment/management or for renewable energy production [74]. These substrates include wastes produced through activities performed in the agricultural, industrial, domestic, and economic sectors as well as animal farming [74]. Knowing the characteristics of the organic material to be digested will facilitate the detection of the rate-limiting step. Methane yields realised from the anaerobic degradation of organic wastes depend on the anaerobic microbial activity, the composition and characteristics of the crude ingredients (carbohydrates, proteins, lipids, lignin, etc.), and the degree of any inhibitory factors produced during the process. The essential parameters studied in relation to substrate characteristics during anaerobic digestion are as follows:

4.2.1. Biochemical Methane Potential (BMP)

BMP tests are employed to assess the biodegradability of a substrate and its methane potential. In other words, it can be described as a method to evaluate the quantity of organic carbon in a said substrate that can be converted to methane via the anaerobic digestion process. Also, it can be defined as the maximum quantity of methane produced by a substrate per mass of the substrate’s organic matter expressed as volatile solid (VS) or chemical oxygen demand (COD) [91]. It denotes the methane produced per unit of volatile solid (VS) or per unit of chemical oxygen demand (COD) at standard temperature and pressure. Presently, BMP is being widely used to determine the feasibility, installation, operation, and design of the anaerobic digestion process [92].

It is simple, cost-effective, and capable of evaluating the potential of substrates for producing biogas [93]. Therefore, BMP can be termed as a reproducible method that can be employed to relatively differentiate among substrates depending on their anaerobic digestibility and the potential of biogas production [94]. Koch et al. [91] highlighted that performing BMP tests does not require a huge investment of labour, time, and equipment. Janke et al. [95] indicated that BMP tests are amongst the options for determining the effect of pretreatment on the substrate’s degradability. De Vrieze et al. [96] noted that the BMP of a substrate is a vital factor in determining its price when purchased by biogas owners and thus is considered a central factor in estimating the profit margins of anaerobic digestion.

Overall, the BMP procedure consists of batch experiments conducted in a bottle containing an organic substrate and anaerobic inoculum (i.e., inoculum retrieved from an active digester) under anaerobic conditions and specific operational conditions of temperature and pH [94]. Other nutrients are added if needed. Batch tests are highly valued as information generated from these tests can produce reasonable predictions of full-scale behaviour [92,93]. In addition, each BMP test should include a blank (inoculum plus a medium or water but no substrate to exclude methane production from the inoculum), control (the bottle is filled with inoculum, control substrate with known theoretical methane yield, and any needed substrate), and substrate (the bottle is filled with the inoculum and substrate as well as any other substrates). The control is used to evaluate the accuracy of the BMP test. All assays in In every assay, the control, substrate, and blank samples are conducted in triplicates, ensuring the reproducibility of the test and statistical analysis [92].

Initially, BMP could be carried out at two different temperatures, i.e., mesophilic (35 °C) and thermophilic (55 °C), as it is well noted that temperature exerts a profound effect on microbial metabolism. The higher the temperature, the better the microbial activity, and the greater the biogas production [66,97]. Similarly, Hamzah et al. [98] revealed that thermophilic digestion is associated with benefits, including a faster rate of operation, increased reduction of solids, improvement in dewatering, and high-quality digestate, as well as the increased decimation of pathogenic bacteria. On the other hand, the process requires additional energy or power, it is unstable, it has a higher odour potential, and it results in a reduced quality supernatant with large quantities of dissolved solids [19,66]. However, toward the standardisation and elimination of errors in BMP testing, the mesophilic temperature range has been adopted as the bulk of the experimental data were obtained at this temperature unlike a few at the thermophilic temperature.

Based on the consistency of the substrates, BMP assays employ varying volumes of 125–500 mL for homogenous substrates and 500–2000 mL for heterogenous substrates. In general, several authors have explained the different procedures employed for the determination of methane potential. Although all are batch-operated, variations exist in terms of the bottles used, the specific method of measurement of the gas, the ratio of inoculum to the substrate, the mode of mixing, and the retention time [92,94]. According to Filer et al. [92], there is a continuous evolution of methods to determine BMP; thus, there is a lack of standardisation of units and techniques, which in turn affects the comparability and validity of the obtained BMP data. Differences in laboratory-specific experiments, operational conditions (test inoculum, food-to-microorganism ratios, serum bottles, mixing mode, and incubation period), and data presentation (nutrient and methane measurement devices) impede the validity of BMP test results [93].

Despite the variation in the test procedures, there are key factors that must be considered, including the following aspects:

(i)

The substrate-to-inoculum ratio should be above 0.1, and the inoculum must be greatly stabilised to exclude further degradation during the assay [99].

(ii)

The inoculum should be withdrawn from an active digester operating on a complex feed material; thus, it will provide a microbial population that is varied and balanced, e.g., a wastewater treatment plant (WWTP) [93]. The inoculum is responsible for providing the initial microbial population in the anaerobic digestion process. An active inoculum has the tendency to provide extra methane-producing microorganisms and a good source of inoculum is endowed with the potential to positively augment anaerobic biodegradability, shorten the lag phase and thus further stabilise the process. Of high recommendation is the fact that the inoculum should be preincubated for 1 to 5 days at 35 °C to degas and lessen the influence of its methane production [100].

(iii)

The batch process should be carried out using a temperature-controlled system (mesophilic), and the preferred temperature of the bottles must be the same as the temperature of the system from which the inoculum was withdrawn [92].

(iv)

In order to design the test and eradicate the problem of process inhibition, the substrate should be evaluated for total solids, volatile solids, volatile fatty acids, and total Kjeldahl nitrogen, as well as ammonium and alkalinity concentrations [92].

(v)

Most importantly, biogas is the key factor to determine methane potential and the biodegradability of a feedstock; therefore, biogas production has to be monitored very closely so that no significant losses or errors occur during its collection. Correction factors are applied to convert the observed methane potential to that under standard temperature and pressure conditions for standardised results [101].

Theoretical Biochemical Methane Potential (BMP)

BMP gives an indication of the biodegradability of a substrate and its potential to produce methane via anaerobic digestion [102]. It can be obtained via theoretical methods and experimental assays. Theoretical methods are fast and easy to use and very crucial, where access to the laboratory is limited. The BMP obtained via theoretical methods is termed theoretical BMP (TBMP). The TBMP is estimated from the chemical composition, elemental composition, and chemical oxygen demand of a given biomass [103]. The BMP obtained via theoretical methods does not give a true representation of the methane potential of the material as it is often higher than the experimental/measured BMP. According to Yasin and Buyong [104], correction factors are instituted, ranging from 0.96 to 2.55 and from 0.50 to 4.27 when the theoretical BMPs are based on COD composition and elemental composition, respectively. This is because it is assumed that the substrate will be completely degraded, which implies that there is no discrimination between biodegradable and non-degradable portions of the biomass; thus, the lignin fraction is not considered to be recalcitrant during the anaerobic digestion process. Also, the use of substrate is used by microorganisms as an energy source for cell growth, and the synthesis of metabolites and protoplasm is negligible [105]. Notwithstanding, the theoretical BMPs for certain lignocellulosic biomasses are presented in

Table 2. Theoretical BMP of some lignocellulosic biomass.

Lignocellulosic Biomass	Theoretical BMP	References
Napier grass Longkong peel seed Lady finger banana peel	0.46 L CH4/g VS 0.5 L CH4/g VS 0.41 L CH4/g VS	Odedina et al. [106]
Sugarcane bagasse Agave Corn straw Wheat straw	369 mLCH4/g COD 178 mL CH4/ g COD 230 mL CH4/g COD 195 mL CH4/g COD	Buitrόn et al. [107]
Maize straw Wheat straw Corn cob Sugarcane bagasse Almond shell	471.2 mL/g VS 471.5 mL/g VS 436.5 mL/g VS 425.6 mL/g VS 381.2 mL/g VS	Ali et al. [108]
Cooked food waste Uncooked food waste Vegetable waste Fruit waste Garden waste Paper waste Textile waste	487.20 mLCH4/g VS 117.39 mLCH4/g VS 401.17 mLCH4/g VS 362.50 mLCH4/g VS 336.65 mLCH4/g VS 496.84 mLCH4/g VS 743.10 mLCH4/g VS	Yasim and Buyong [104]
Biological sludge Organic fraction municipal solid waste	333.9 mLCH4/g VS 494.3 mLCH4/g VS	Nielfa et al. [109]
Vegetable okra (Abelmoschus esculentus)	444.8 mLCH4/ g VS 342.06 mL CH4/g VS	Ugwu and Enweremadu [110]
Kales Cabbage	449.6350 mLCH4/ g VS 491.6115 mLCH4/g VS	Mbugua et al. [111]

.

Biodegradability Based on Theoretical BMP

Data in BMP allow for a direct assessment of biogas yields obtained via anaerobic digestion. Biodegradability can be explained in terms of elemental composition, describing the extent of biomass degradation, which can be grouped into biodegradability and non-degradability [108]. BMP determination measures substrate biodegradability, insinuating that the greater the BMP value amongst the different substrates, the greater the biodegradability of the said substrate relative to the others [91]. It is affected by the physicochemical characteristics of the feedstock plus the environmental conditions of the anaerobic digestion process [112]. The kinetics of the different steps of the anaerobic digestion process are controlled by the biodegradability characteristics of the substrate together with the inhibitory intermediate products produced and, subsequently, define the shape of the biogas production curve [113].

Proposal of a Real or an Industrial Application of Anaerobic Digestion of Lignocellulosic Biomass, Economics, Limitations, and Advantages

Many localised industries in developing countries produce large quantities of lignocellulosic wastes which are utilised or, if not managed, cause deleterious effects on the environment and humans. Sawdust (forest waste) is described as small discontinuous chips or fine particles of wood, occurring as a by-product, that originates from a few timber manufacturing processes, including sawing, routing, drilling, and furniture manufacturing, as well as joinery. In addition, rice and wheat straws, as well as corn husk and corn stover (cobs, leaves, and stalks), are obtained through harvesting. Traditionally, these wastes can be heaped or piled and burnt, releasing emissions that pollute the atmosphere, thus exacerbating greenhouse gas effects [36]. They may also be added to water bodies through uncontrolled rainfall, having deleterious effects on aquatic organisms [31]. In a circular economy, with the principles of recycling, recovery, and reuse, crop residues and forest waste can serve as feedstock materials for anaerobic degradation, preventing the lignocellulose from becoming waste in the environment. The waste being diverted into an applicable pathway saves costs that would have normally been incurred to purchase the raw material or substrate for the anaerobic digestion process. In a pilot-scale project for waste management in order to make use of lignocellulose wastes, the following processes was are described: From the point of waste generation (saw mill, farms, etc.), the feedstock is collected in clean bags and delivered to the site or an area in proximity, where the biodigester is constructed to perform a batch anaerobic digestion operating at a mesophilic temperature range. The feedstock is preselected to remove physical contaminants (e.g., stones, broken bottles, etc.) and then is air- or oven-dried and mechanically treated (milled) to increase the surface area of accessibility by microorganisms and prepare it for other pretreatments, including chemical and enzymatic methods [16]. At this stage, mechanical treatment might involve the consumption of energy in the form of either manpower or electricity for machine operation; this may further increase the cost of the production process. The use of sodium hydroxide or acids for pretreatment will result in the formation of substances that are inhibitory to the hydrolysis and fermentation processes affecting the quality and yield of the biogas, or they may be toxic, thus needing removal at this stage or during post-treatment when the anaerobic digestion is completed. These procedures will equally incur costs, further raising the overall cost of the process. To overcome the challenges associated with chemical pretreatment, enzyme application appears better since it does not involve chemicals. However, the cost of enzymes is high, posing difficulties to sustain in poor developing countries.

Digested cow manure is retrieved from an existing/ongoing digester, and incubated for 5 days to degas or stabilise the material in order to serve as an inoculum. The physicochemical properties of the feedstock (percentage total solids (TSs), volatile solids (VSs), volatile fatty acids, ammonium and alkalinity concentration, elemental composition, and chemical oxygen demand) are determined. Feedstock and inoculum are mixed in a ratio above 0.1, and the biodigester is charged and batch-operated over a retention time of 30–60 days. Constant monitoring of the operational conditions (pH, temperature, and microbiological population) is needed. The lack of expertise (personnel) in operating the anaerobic digester and monitoring, as well as the maintenance of the anaerobic process, could be challenging. At this juncture, additional cost is also incurred. The operational parameters including pressure, temperature, and gas production can be monitored via the construction of a data acquisition system harbouring all the sensors (which must be bought as well) [114]. On a real scale, for cooking purposes, gas can be channelled through pipes to the cooking stove, but on an industrial scale, a gas-collecting chamber must be provided. It is worth mentioning that, on an industrial scale, the entire process will incur huge costs as every stage carried out in the process will involve huge quantities of feedstock, energy for pretreatment, huge (costly) bioreactors, extensive land for construction, cost of waste treatment generated, etc. In some countries (e.g., Sweden), the government gives subsidies to farmers or company owners operating biodigesters to help cover some of the costs outlined.

4.2.2. Biochemical Oxygen Demand (BOD)

This parameter determines the biodegradable organics in a sludge, and it can be used as a measure to determine the overall effectiveness of the anaerobic digestion process. According to Meegoda et al. [115], BOD measurements give an indication of the microbial metabolism of dissolved oxygen per sample over a period of five days. Therefore, it makes use of aerobic bacteria to oxidise the sludge in the sample. BOD experiments/assays are performed in sealed bottles at a given temperature, in a dark room to avoid any dissolved oxygen production via photosynthesis. Nevertheless, BOD testing is avoided owing to challenges in logistics, specifically in relation to the time taken to complete the test. In addition, obtaining results after five days does not provide accurate insight into the conditions operating in the biodigester at present. This will pose challenges in decision making in order to adjust the operating conditions within the biodigester.

4.2.3. Chemical Oxygen Demand (COD)

This defines the level of oxygen present in a sample of sludge that can be utilised upon reaction with oxidising agents. It measures all the organics occurring in a sample. Performing a COD test occurs within a few hours during which a sludge is refluxed in surplus with a solution comprising two chemicals: potassium dichromate and sulphuric acid. At the end of the reflux process, the amount of excess potassium dichromate can be evaluated based on titration against ferrous ammonium sulphate. The final COD value can be obtained from the quantity of potassium dichromate utilised in the initial reflux. COD measurement can serve as an indicator of the efficiency of the anaerobic digestion process, insinuating that COD reduction mirrors the quantity of the digestion occurring within the precinct of a biodigester as it reflects the consumption of organics. However, the value of COD is usually higher than the BOD because it entails the presence of all the organics in the sample; the ratio of BOD to COD represents the biodegradable portion of a sludge [115].

4.2.4. Total Solids

Regardless of the inorganic or organic nature of a sludge, total solid defines the dry matter content of the substrate. It includes the volatile solids and the ash content; thus, it is considered a valuable parameter in assessing the efficiency of the anaerobic degradation process, since it is the volatile portion that is degraded and transformed into methane. Therefore, it can be obtained by drying a sludge at 105 °C until no further change is observed. In the literature, it is described as either a percentage or a concentration [116].

$$\%\mathrm{Total}\mathrm{solids}=100\times {\mathrm{W}}_{\mathrm{DM}}÷{\mathrm{W}}_{\mathrm{s}}$$

(1)

where W_S = mass of the fresh sample; W_DM = mass of the dried sample.

4.2.5. Volatile Solids

These constitute the biodegradable portion of the substrate. VS reduction during the process is indicative of a reduction in the biodegradable fraction, highlighting the fact that the solids were utilised by microorganisms performing the process, resulting in the production of biogas [14]. VS is also considered a measure of the process efficiency; thus, it can be determined by combusting the remaining solids recovered from the total solid measurement in a furnace at a temperature of 550 °C.

$$\%\mathrm{Volatile}\mathrm{solid}=100\times ({\mathrm{W}}_{\mathrm{DM}}-{\mathrm{W}}_{\mathrm{ash}})÷{\mathrm{W}}_{\mathrm{DM}}$$

(2)

where W_ash = weight of the ash; W_DM = mass of the dried sample.

4.2.6. Carbon–Nitrogen (C:N) Ratio

In relation to the major building block, i.e., lipids (fats), proteins, and carbohydrates, the C/N ratio varies with the types of substrates because each substrate comprises different percentages of carbohydrates, fats, and proteins. Simply, lignocellulosic biomass varies in elemental and chemical composition based on the species. The carbon content is the primary contributor to the total calorific value, while the content of nitrogen and sulphur are relatively low compared with fossil [117]. Therefore, different substrates have different C/N ratios, as shown in

Table 3. Different agricultural wastes and their respective carbon-to-nitrogen ratios.

Agricultural Substrates	Carbon–Nitrogen	References
Cassava leaf silage	18.88:1	Noviadi et al. [119]
Grass clover silage	16:1	Majuga et al. [120]
Sugar beet residues	11:1	Whitmore and Groot [121]
Wheat straw Spruce sawdust	44:1 46:1	Reichel et al. [122]
Bagasse Water hyacinth	58:1 19:1	Hadiyarto et al. [123]
Sugarcane bagasse Sorghum stalk Rice straw Cattle manure Chicken manure Rice husks Maize stover Grass clippings	100:1 73:1 78:1 22:1 5.7:1 87.5:1 68:1 67.9:1	Alabi et al. [124]
Vetiver grass	45.03:1	Srikanlayanukul and Suksabye [125]
Goat dung Rice straw ash	18.04:1 20.83:1	Khalib et al. [126]

. Both C and N are needed at specified concentrations in order to present as a suitable substrate for anaerobic digestion. Carbon is needed for the maintenance of cell structure and nitrogen for the manufacturing of amino acids and proteins. However, a low value of C/N in substrates causes an increase in ammonium nitrogen concentration and the inhibition of methane production, whilst a high value of C/N in substrates indicates a low level of nitrogen for protein synthesis, thus disrupting metabolism and energy transformation in cells [118].

4.2.7. Trace Elements

Elements occurring in very minute quantities in minerals that are not included in the formula of the mineral are termed trace elements. They are known as macro- or micronutrients [127] and include iron (Fe), molybdenum (Mo), nickel (Ni), copper (Cu), cobalt (Co), magnesium (Mg), calcium (Ca), manganese (Mn), selenium (Se), tungsten (W), calcium (Ca), potassium (K), silver (Ag), cadmium (Cd), phosphorus (P), and B12 vitamins. Trace elements are nutrients necessary for cell growth in microbiology as they play a crucial role in organisms, demonstrating functions in enzyme complexes, and can be categorised into essential and non-essential elements [128].

Substances supplied with substrates might inhibit the anaerobic digestion process, as certain compounds, including alkanes, alkenes, alcohols, ketones, and hydrocarbons, are not directly susceptible to hydrolysis except in the presence of extracellular enzymes that harbour trace elements as components [118]. Trace elements can occur as part of enzymes, for instance, as a few nucleic acids, and are relevant to the formation of vitamins. They are also the major elements in the functioning of multiple enzymes within the anaerobic digestion process [67,129]. Microorganisms in the anaerobic digestion process require trace elements as co-factors for enzymes involved in methanogenesis, and the enzymes equally improve the rate of hydrolysis. Examples of substrates deficient in trace elements include manures from chicken, cattle, and pig, whereas food wastes, abattoir wastes, and kitchen wastes are assumed to be endowed with adequate concentrations and quantities of micronutrients [127].

Nevertheless, in terms of the process, the significance of trace elements is in the order of Fe, Ni, Co, Mo, W, and Zn, which need to be controlled in as concentrations above threshold levels for lower biogas production because of the accumulation of organic acids, originating from the inhibition of methanogenic organisms [127]. Supplementing an anaerobic digester with trace elements is one of the avenues observed for improvement in biogas yields through the process of optimisation and scale-up techniques [130]. Therefore, trace elements can be supplemented in isolation or combination with anaerobic digesters [118].

Yao and colleagues [131] reported a better reactor performance in terms of process stability, causing a reduction in volatile fatty acids and diminishing the accumulation of ammonia and the total biogas produced when supplemented with a combination of Co-Ni as opposed to those supplemented with individual elements. The lack of trace elements led to acidification, while the presence of excess trace elements culminated in toxicity [130]. Supplementation with combinations of trace elements can also result in an antagonistic effect [132]. The influence of trace elements in the anaerobic digestion process depends on their content in the digesting material, the temperature operating conditions, the mode of operation of the process, as well as the bioavailability and activity of microbes (the type of methanogens) [118].

In summary, the effect of trace elements in anaerobic digestion can be stimulatory, inhibitory, and toxic. Their negative effects are based on the concentration of trace elements and the pH of the anaerobic digestion process. However, they exert toxic effects because naturally occurring elements are replaced with enzyme prosthetic groups or due to the disruption of enzyme function and restructuring caused by the binding of trace metals with thiols and other groups in protein molecules [127].

4.2.8. Microbial Composition

Hydrolytic bacteria, acidogenic bacteria, acetogens, and methanogens are the key players in the anaerobic digestion process. The interdependence of the microbes in this process is remarkable, and it is the key factor of the process [133]. The quality and type of the substrate determine the type and level of fermentative microorganisms present in the digester. Substrate types determine the differences in microbiomes in anaerobic digesters or the phylogenetic structure of microbial communities in the biodigester [134]. The rate of growth of microorganisms is crucial. This is influenced by processing factors (temperature, organic loading rate, feeding mode, and substrate composition), including the operating parameters. The different classes of microorganisms acting at the various stages/phases respond differently to these parameters [135]. The choice of substrate is paramount owing to the sole dependence of microbial activities on the chemical constituents (macro- and micronutrients, fats, proteins, nucleic acids, and carbohydrates) whose breakdown generate substances that could alter the pH of the medium. Ultimately, it affects the microbial communities as particular organisms have appropriate environmental and nutritional conditions under which they can thrive [134].

Variation in the microbial composition is observed amongst the substrates due to their source of origin, collection, processing (pretreatment), and storage, thus explaining the difference in the microbial population coexisting in a bioengineered reactor [136]. Owing to this variation, co-digestion can be termed as a technique employed for the simultaneous treatment of several organic substrates, wherein better synergism from the diverse co-substrates and microbial activity takes place, resulting in improved methane yield in the process [137]. In addition, Liu et al. [138] stressed that the source of the inoculum during mono-digestion has the capacity to affect the microbial structure involved in the anaerobic digestion process, which in turn influences its effectiveness. Also, Černey et al. [136] alleged that a huge fraction of the microbial community involved in the digestion of crop and manure wastes is of soil and gastrointestinal consortia. Rumen microorganisms are denoted as good facilitators of the hydrolysis of lignocellulosic biomass in anaerobic digestion. Hence, the rumen content can serve a dual purpose: It can be used as an inoculum or as a co-substrate in anaerobic digestion [139]. In addition, Satpathy et al. [140] stated that different substrates and starter inocula determine the microbial structure. Although maize and silage have similarities as substrates, diverse communities dominate the lactate-rich silage, unlike the maize, thus emphasising that the inoculum’s origin has a remarkable role in shaping microbial communities. Furthermore, the feeding mode (either continuous or batch) has been observed to exert an effect on the overall microbial structure [138].

It is worth mentioning that, at the initial stage of the process development, some authors evaluated the microbial level of the substrates in terms of the total viable counts/aerobic/standard plate [141], described as the viable number of bacterial or fungal cells present in the substrate. This is performed via the use of different microbiological media while employing the spread plate technique [142]. In addition, cultivation-based methods are employed to isolate, enumerate, and characterise individual microbes or functional groups [143]. These methods are simple, easy, and culture-based; however, they are biased as they only focus on viable and culturable cells, missing the viable but non-culturable cells (e.g., spore-forming bacteria) [87]. To obtain comprehensive knowledge of the microbial structure/composition of substrates, several molecular-based methods are available, including high-throughput sequencing, cloning, metagenomics, next-generation sequencing, denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP), transcriptomics, metatranscriptomics, metabolomics following DNA extraction, and the amplification of the 16S rRNA (PCR), as the case may be [144]. These microorganisms are described as sensitive to changes or alterations; therefore, in-depth knowledge of their involvement in the process is necessary for the optimisation of the process.

4.2.9. Moisture Content

Even though organic substrates have high moisture content [59], the moisture level is known to vary from one substrate to the next, and it is amongst the factors that can influence the performance of the anaerobic digestion process. Therefore, the moisture content is amongst the physicochemical parameters measured in the tested substrate(s) before charging or feeding, or loading the biodigester [59]. However, the high-water content of manure presents a limitation, hindering high organic loads and volumetric gas production; therefore, blending manure with more energy-dense materials (crop residues) seems to increase the organic loading rate without drastically reducing the hydraulic retention time [145].

According to Cioabla et al. [146], a certain mass of sample is weighed into a dish and dried overnight at 105 °C. Subsequently, the dish and the dried sample are weighed, and mass is noted. The moisture content (in percentage) of the different feedstock materials employed in anaerobic digestion can be determined using the following equation:

$$\%\mathrm{moisture}\mathrm{content}=({\mathrm{m}}_2-{\mathrm{m}}_1)-({\mathrm{m}}_3-{\mathrm{m}}_1)/({\mathrm{m}}_2-{\mathrm{m}}_1)\times 100$$

(3)

where m₁ is the mass (g) of the empty dish, m₂ is the mass (g) of the sample and the empty dish before drying, and m₃ is the mass (g) of the sample and the empty dish after drying.

5. Pretreatment Methods for Lignocellulosic Biomass Involved in Anaerobic Digestion

The presence of lignin in these biomass wastes causes recalcitrance to anaerobic digestion. Equally, the carbohydrate, protein, and lipid contents of these agricultural wastes vary, affecting the stability of the process and, ultimately, biogas yields [9]. The recalcitrance presented by the biomass is termed biomass recalcitrance. Biomass recalcitrance is defined as the anti-degradation properties of native lignocellulose, protecting the cell wall of the plants from attack by pathogens or fermentation by microorganisms and enzymes [75]. The level of recalcitrance varies based on the composition of the biomass, which closely relates to the genotype, environmental conditions, crop management practices, and parts of the plants [147]. In detail, the recalcitrance properties of these materials are due to both chemical and structural factors, including cellulose-specific surface area, the degree of polymerisation, pore size and volume, cellulose crystallinity, hemicelluloses, and acetyl groups, as well as the composition and content of lignin [2]. Hence, the hydrolysis of substrates (occurring during the hydrolysis phase) is considered the most critical of all the phases involved in the entire anaerobic digestion process [16]. This is because the restricted hydrolysis of lignocellulose culminates in the production of insufficient intermediate products that can act as substrates for subsequent metabolic processes across the anaerobic digestion process [148].

The pretreatment step is viewed as a technological hurdle during anaerobic digestion; however, during this process, the lignocellulosic structure is altered, making cellulose accessible for enzyme conversion and thus overcoming its recalcitrance [149]. In precise terms, pretreatment causes changes in both the physical and chemical structure of the lignocellulosic material. The chemical and hydrogen bonds are broken, thereby improving the hydrolysis rates. Accordingly, pretreatment is highly considered an efficient method of releasing nutrients from plant tissues [150]. Similarly, pretreatment causes the modification of the structure of different feedstock materials at all fibre levels, paving the way for hydrolysis, during which lignin and hemicellulose components are broken down to release the cellulose embedded in them. Thus, it facilitates the downstream processes [151]. This creates a wider surface area, allowing for efficient interaction between the microorganisms and substrate particles, and causes a reduction in the crystalline nature of cellulose, thereby improving the digestion operated under anaerobic conditions and, eventually, biogas yields [152]. Equally, the pretreatment process aids in exploiting the recovery of cellulose and reduces the number of enzyme inhibitors that are formed during the process [26].

The selection of the pretreatment method is guided by the type of lignocellulosic biomass, the nature of the subsequent process, and the overall economics of the process [149]. Considering the composition of lignocellulosic materials, it is obvious that cellulose can be isolated to ensure accessibility for degradation. Alternatively, hemicellulose or lignin can be removed or altered to heighten the accessibility to cellulose for degradation. Depending on lignocellulosic materials, pretreatment methods vary in efficiency and can be categorised into physicochemical, chemical, physical, and biological methods [153].

5.1. Physical Treatment

Particle size reduction is of great relevance prior to the employment of other chemical and biological methods [154], as it increases the surface area. This facilitates accessibility to the biomass and increases the susceptibility to microbial and enzymatic attacks, thereby promoting its digestion in the course of the process [153]. This group of pretreatment methods does not involve the use of water and therefore does not produce wastewater. Nevertheless, it requires energy, which is a major hindrance to the implementation of these methods. Remarkably, these pretreatment methods do not produce toxic compounds that will negatively affect the anaerobic digestion process [153].

5.1.1. Mechanical Pretreatment/Comminution

This procedure can be performed via several techniques, including chopping, milling (ball milling, hammer milling, two-roll milling, and colloid milling), and grinding. Usually, grinding and milling are mostly employed to mechanically pretreat lignocellulosic biomass before the actual process of anaerobic digestion [75,153]. According to Olatunji et al. [16], the type of milling, the duration of the operation, and the structure of the feedstock will determine the improvement in a certain surface area, the net polymerisation level, and the final reduction in the crystallinity of cellulose. However, the moisture content of the biomass points towards choosing grinding or milling [155], resulting in particles of varying sizes that can lead to the reduction in the degree of crystallinity of cellulose and the degree of polymerisation of cellulose and hemicellulose, thereby increasing the surface area of cellulose biomass for microbial activity [156]. Therefore, the impact on methane production and hydrolysis kinetics will depend on the pretreatment method, particle size reduction, and the physical structure of the substrate [157]. Certain pretreatment methods require the feedstock to be reduced to a particular size prior to pretreatment. Having different materials with different lignocellulosic compositions of varying particle sizes involved in the process necessitates the precise determination of the particle size [75] because an extreme reduction in particle size may lead to the generation of inhibitory compounds, thus causing a decrease in biogas production. For easily degradable lignocellulosic biomass, grinding/or size reduction can be the sole pretreatment method to reduce the crystallinity of cellulose [16].

5.1.2. Irradiation

Irradiation has the ability to dissociate the glucoside bonds of cellulose, converting cellulose chains into oligosaccharides, brittle fibres, and even cellobiose [75]. Microwave energy is popular and occurs as radiation energy in the electromagnetic spectrum, with wavelengths of 1mm to 1m. The irradiation of the biomass material via microwaving delivers the radiation directly to the biomass, causing rapid heating with a minimal thermal gradient. Because the heating process occurs rapidly, the time and energy consumed are much reduced [153].

The irradiation of lignocellulosic biomass via microwaving as a pretreatment technique employs a catalyst and can be categorised into two distinct groups, namely microwave-assisted solvolysis and microwave-assisted pyrolysis. The former process is performed under mild temperatures below 200 °C, depolymerising the feedstock to release value-added chemicals, whilst the latter involves the pretreatment of lignin at higher temperatures (>400 °C) without oxygen, transforming the feedstock to bio-oil or biogases. Nevertheless, microwave-assisted pyrolysis is preferred and most widespread, owing to the energy shortage and the plan of different countries to ensure sustainability [16]. The effectiveness of microwave pretreatment largely relies on the dielectric properties of the feedstock, defined as the material’s strength to stock electromagnetic energy and convert it to heat [16].

5.2. Chemical Pretreatments

As the name represents, in this type of pretreatment, chemicals are employed to alter the chemical and physical properties of native lignocellulose [75]. Based on the chemicals employed, these methods are grouped into acidic, alkaline, oxidative, and organo-solvent treatments [17]. Ahmad et al. [157] highlighted other chemical pretreatment methods known as advanced chemical pretreatments such as organo-solvents, ionic liquid, and supercritical carbon dioxide pretreatments. Carbohydrates can be hydrolysed via acid treatment, while alkali/oxidative treatment involves the attack on lignin, but the hemicellulose polymer is not fragmented [157]. In detail, strong and diluted acid can be used, and through acidic pretreatments, the lignocellulosic substrate is degraded to their respective monosaccharides, furfural, and volatile compounds, reducing the time for digestion.

Seemingly, Taokaew and Kriangkrai [158] purported that cellulose can be dissolved from the structure of lignocelluloses via an ionic liquid treatment that breaks the hydrogen bonds between the microfibres of celluloses, resulting in increased porosity, reduced crystallinity, and an increase in digestibility. Similarly, lignin and hemicellulose can be removed from the feedstock to enhance access to cellulose. Hemicellulose can be removed by using dilute acid or via thermal treatments. Although the extraction of high quantities of sugars is achieved via acid hydrolysis, the yield and molecular weight of the separated hemicellulose are low because it is easily hydrolysed under acidic conditions [150].

Several dilute bases, namely sodium hydroxide (NaOH), calcium oxide (CaO), potassium hydroxide (KOH), ammonia (NH₃), and calcium hydroxide (Ca(OH)₂), can be employed as alkali substances for pretreatment purposes [156]. Sodium hydroxide (NaOH) is the most typical inorganic alkali solution employed in this process [159]. It causes both the swelling of cellulose and the hydrolysis of hemicellulose, resulting in enhanced dissolution of hemicellulose. This leads to the extraction of high-purity hemicellulose [150]. However, Thomas et al. [159] argued that sodium spreading into agricultural soils should be sidestepped because digestates discharged from agricultural anaerobic digesters are often used as organic fertilisers, and therefore they proposed the use of lime. The authors further outlined other chemicals that could be employed in an alkaline process for the extraction of hemicellulose, including sodium hydroxide in combination with a strong base (ammonium hydroxide, NH₄OH), KOH (potassium hydroxide), H₃BO₃, and hydrogen peroxide (H₂O₂).

Altogether, alkali pretreatments have been considered the most effective in lignin degradation amongst the different types of pretreatments [160], and they are associated with the following pluses: the biomass itself retains some of the alkalis, and the remaining concentration is carried over for the reaction [156]. The alkali helps during acidogenesis to prevent the alteration of the pH, it positively augments (increases) the rate of methanogenesis, and it occurs in the presence of low moisture levels and environmental temperature, which are parameters desirable for other processes. Overall, chemical pretreatment methods occur faster; however, they generate toxic substances and wastewater that would require extra financial expenses for the recycling of chemicals [161].

5.3. Physicochemical Pretreatment

5.3.1. Thermal Pretreatments

These methods involve subjecting the substrates to high temperatures, ensuring hydrolysis while avoiding evaporation. However, the temperature application has a magnitude of effect on the efficacy of the pretreatment process. Thermal treatments incorporate temperatures between 120 °C and 210 °C, but an optimum temperature of 100 °C has been realised to cause an increase in biogas yields [156]. The authors revealed that applying high temperatures of about 160 °C and above leads to the solubilisation of lignin, producing an end product that is reactive and delays the growth of bacteria in the hydrolysis phase.

5.3.2. Hydrothermal Pretreatment

This method involves the use of high pressure and high temperature in the treatment for a short period of time. The temperature, and, to some extent, the duration of operation of this method, have a significant impact on the overall process. This technique involves temperatures from greater than 90 to less than 260 °C [154]. The high pressure in the process is to keep the water in a liquid state at high temperatures of between 200 and 240 °C. The hydrogen ions in water serve as catalysts, providing the efficient medium necessary for hydrolysis. Hydrothermal pretreatment occurring in batch mode addresses the challenges posed by continuous mode (e.g., clogging of the flow valve), but it is faced with the problem of high energy consumption owing to the slow heating of water [156]. Zhou et al. [162] found an increase in biogas production by 21% after the hydrothermal pretreatment of Miscanthus at 170 °C. However, this technique generates inhibitory substances, including 5-hydroxymethylfurfural and furfural, which are reported to affect methanogenesis at 200 °C [154].

5.3.3. Steam Explosion

Steam explosion is another form of pretreatment performed at high temperatures and pressure in which water vapour penetrates the cell wall of lignocellulosic biomass, thereby destroying its structure while separating its components [150]. Taherzadeh and colleagues [163] explained that a steam explosion causes the opening of the lignocellulosic fibre structure, the solubilisation of hemicellulose, and the redistribution of lignin, along with the decrease in the length of the fibre.

These methods are physical methods that do not require the use of chemicals, thus reducing costs and environmental pollution. However, they are associated with drawbacks due to the high temperatures employed in the pretreatment process, resulting in the formation of inhibitors, causing the waste liquors to be further treated before being discharged into the environment [164]. Moreover, the application of high temperature and pressure results in the high involvement of equipment and huge consumption of energy [150].

5.4. Biological Pretreatment

This is an effective method that requires less energy and is environmentally friendly [154]. One major target of biological pretreatment is curtailing the loss of carbohydrates and maximising the removal of lignin. Biological methods involve milder process conditions than chemical methods. The biological treatment utilises microorganisms (fungi), enzymes, or a mixture of microorganisms (microbial consortium) to increase the biological destruction of the biomass. Thus, it can be grouped into three categories according to the tool used in the pretreatment process [75].

5.4.1. Fungal Pretreatment

The biological treatment of feedstock involving fungi is carried out via an oxidative hydrolytic system (attacks solely the phenyl bonds in lignin) and a hydrolytic enzyme system (degrading cellulose and hemicellulose) [75]. According to Banu and colleagues [165], pretreatment focuses on the degrading of lignin, leading to an enhancement in the digestibility of cellulose. White rot fungi, brown rot, and soft rot fungi are well-known fungal species producing ligninolytic enzymes that degrade lignin. The white-rot fungi (Ceriporiopsis subvermispora, Phellinus pini, and Phlebia sp., etc.) produce different ligninolytic extracellular oxidases and are known to be the most effective in their preferential deconstruction of lignin in lignocellulose, releasing cellulose and hemicellulose, after which the wood appears whitish in colour with a fibrous texture [166]. In comparison, brown-rot fungi use the polysaccharides in the lignocellulose, resulting in partial delignification, which causes the wood to shrink and exhibit a brown discolouration of oxidised lignin [166]. On the other hand, bacteria are known as poor degraders of lignin, implying that they have a low capability of degradation. Nevertheless, some bacteria, including members belonging to the alpha and gamma Proteobacteria added to actinomycetes, are known to degrade lignin as they function in the production of ligninolytic enzymes [167].

5.4.2. Microbial Consortium

Contrarily, microbial consortium demonstrates the specific activity of degrading cellulose and hemicellulose as it is endowed with great cellulose and hemicellulose degrading ability [75]. The microorganisms constituting the consortium are screened from the natural environment, harbouring rotten lignocellulosic biomass as substrates [168]. The consortium can be composed of the following microbes: yeast and cellulolytic bacteria, a blend of fungi and composting microbes, Clostridium thermocellum, and heat-treated sludge [169]. Interestingly, Chandel et al. [161] highlighted the implementation of a microbial co-culture system as an ideal strategy for the one-step fermentation of lignocellulosic biomass. The co-culture systems in pretreatment can be grouped into three types, namely fungal, bacterial, and combined bacterial and fungal co-culture systems. In these systems, the burden is not limited to a particular strain since a single bacterium or fungus cannot produce or secrete concurrently all the necessary enzymes needed for a desired effect. In this light, Wu et al. [170] expressed the reduction in the metabolic burden of each strain occurring in a co-culture system as a benefit over the monoculture system. This action helps to improve the overall performance of the system. Zhou et al. [171] described the diversified cellular and conducive environment provided by co-culture systems, allowing for the expression of hydrolases produced by unique pathways and lessening the delay between two working modules.

5.4.3. Bacterial Pretreatment

Bacterial pretreatment involves the use of a wide variety of microorganisms and their hydrolases that occur in nature. Several microorganisms occur in the gut of xylophagous and saprophagous insects, exhibiting the ability to degrade lignocellulosic materials, and therefore serve as prospective options for pretreatment [161]. Owing to the occurrence of lignin, enzymes are used as a biological treatment to enhance the hydrolytic phase, which will eventually result in the optimisation of the process and an increase in the energetic yield. Enzymes implemented in the pretreatment of lignocellulosic biomass include cellulases, laccases, and manganese peroxidases [38]. Owing to the longer pretreatment times (from several weeks to months) associated with biological methods (fungal and microbial consortium) and the cost of the enzymes employed for the treatment of lignocellulosic wastes, biological pretreatment methods are less advantageous. On the other hand, biological pretreatment is preferred to others because of the efficient lignin degradation involved [166] as well as its environmental safety, i.e., it is free from hazardous chemicals [154].

5.4.4. Ensiling

Compared to the above-mentioned physical and chemical pretreatments, ensiling can be considered a mild biological pretreatment strategy [172]. Inadequate or improper storage of biomass can lead to a loss of 70% of biomass that will ultimately exert a negative effect on the methane yield. Energy crops and crop residues with highly endowed biogas potential can be intensively produced as substrates for anaerobic digestion, which will be utilised throughout the year. However, this practice only occurs in the harvesting season; therefore, the storage of the biomass becomes crucial to ensure the annual stable functioning of the biogas plants [173], that is providing constant feeding of the biodigesters. Ensiling, also described as wet storage, is an aerobic–anaerobic process conducted by microorganisms and results in an improvement in biogas yield attributed to the increased biodigestibility of lignocellulosic materials through acid-based hydrolysis and biological degradation [174].

This process maximises the preservation of biomass nutrients and energy, unlike open-air storage or hay, and takes place through four main biochemical and microbiological phases [175] as moist biomass is harvested and transported into silos. Subsequently, they are compacted to ensure maximum removal of air (first, aerobic phase) and later sealed with a cover. Immediately, anaerobic conditions are set (second, anaerobic phase), and the microorganisms involved in ensiling fermentation begin to multiply as follows: The lactic acid-producing bacteria degrade or break down carbohydrates, e.g., sucrose and glucose, within the substrate to lactic acid with or without acetic acid [176]. The authors further mentioned that this action reduces the pH, inhibiting undesirable microorganisms (microbial activity). Kalač [177] indicated that the pH is reduced to a critical pH value (4.1–5.0 based on the moisture content), establishing a successful long-term stable storage (third phase). Finally, silos are opened during the feed-out step for the use of silage, and aerobic degradation can partially occur. Keeping biomass as silage helps to preserve the quality of the biomass and facilitates a continuous supply of the substrate for anaerobic digestion [174]. Sun et al. [174] pointed out several factors that could be adjusted to maximise biogas yields by improving acid-based hydrolysis and biological degradation during ensiling. These parameters include co-storage, buffering capacity, the ratio of carbon to nitrogen, and the inclusion of additives.

Several researchers demonstrated that the co-storage of different substrates can strengthen the initial microbial abundance and the types of fermentation, enhancing the activities of microbes and resulting in more production of organic acids. Consequently, the biodigestibility of lignocellulose is improved via the combined effect of acid-based hydrolysis and biological degradation [178]. In relation to the buffering capacity, which is the ability of biomass to resist pH changes, improving the buffering capacity of lignocellulosic biomass tends to cause a delay in the rapid decline in pH during the early phase of the ensiling process. This is because the buffering capacity determines the quantity of organic acid produced that is needed to attain the critical pH value to ensure stable storage conditions. The improved buffering capacity might cause prolonged microbial activity and biological degradation in terms of duration, which eventually positively augment the biodigestibility of lignocellulose and the production of biomethane [179].

Zhang et al. [180] revealed that the carbon–nitrogen ratio of biomass is usually above the optimum ratio of 25:1; thus, adjusting this ratio can lead to an increase in microbial activity and biological degradation and the ensuing anaerobic fermentation. Lastly, fermentation stimulants (e.g., sugars, molasses, etc.), fermentation inhibitors (acids e.g., formic acid), and fibre enzymes (e.g., hemicellulose and cellulase) or bacteria have been added to regulate the properties of the raw materials to be used in the ensiling process [95,181]. Furthermore, Hillion and colleagues [182] investigated the co-ensiling of sugar beet leaves (agro-industrial waste) and wheat straw (lignocellulosic waste) in vacuum-packed bags for 180 days to effectively store highly fermentable fresh waste evenly with low sugar content. This appeared promising for the constant supply of industrial anaerobic digesters on a long-term basis.

In conclusion, the effectiveness of pretreatment varies with the type of biomass, since the degradability of lignocellulosic substrates can be affected by the percentage of lignin, surface area and solubility, crystallinity, and the grade of polymerisation [183]. No single pretreatment method has been shown to be suitable for all types of lignocellulosic substrates, and any chosen pretreatment method is expected to be economical, simple, eco-friendly, and feasible in addition to the fact that it should not generate inhibitory, toxic substances that will affect the anaerobic digestion process [154]. Therefore, Ahmad et al. [156] stated that pretreatment methods comprising biological, chemical, and physical methods can be used operating as standalone or in combination. Usually, combining two or more of these pretreatment techniques is more economical and can cause a substantial improvement in the effectiveness of the process, resulting in breakthroughs in this area of study as opposed to the use of a single pretreatment method [16].

5.4.5. Bioaugmentation

Alternatively, the biomethane yield of lignocellulosic biomass can be enhanced during the anaerobic digestion process, whereby a pure (specific microbial species) or mixed culture (consortium) is introduced or seeded into the biodigester during anaerobic digestion to improve certain stages of the process owing to their potent lignocellulolytic activities [184]. This procedure is known as bioaugmentation or microbial reinforcement [185]. Bioaugmentation employs different cultures enriched from natural (rumen fluids of goats and cows) or bioengineered cellulolytic environment (e.g., an operating biodigester treating sorghum as a mono-substrate). The complex structure of lignocellulose composed of cellulose, hemicellulose, and lignin offers recalcitrance; thus, diverse groups of enzymes are needed to break down the structure. Moreover, no single strain can produce a range of lignocellulolytic enzymes; therefore, mixed cultures are popular because they harbour synergistic microbial consortia, producing a mixture of enzymes capable to degrade lignocellulose and demonstrating tolerance to stress conditions and environmental changes [38].

The effectiveness of bioaugmentation or the degree of enhancement depends on the type of culture used for enrichment [185]. Researchers demonstrated varied increase levels of 6%, 20%, and 27% in methane yields of bioreactors anaerobically treating wheat straw, enriched with 2% cultures of cow rumen; digested sorghum extracted from an operating biodigester; and cultures of goat rumen, respectively. Henderson et al. [186] clearly expressed that the rumen of ruminants is a unique digestive system. It contains microbiomes that can play a great role in the breakdown of plant polymers and their fermentative conversion to short-chain fatty acids. Thus, the fluid from this organ has been used as inoculum for anaerobic digestion. Roopnarain et al. [187] reported the natural occurrence of biogas-producing microbes attached to water hyacinth, collected from the Hartbeespoort dam in South Africa. Thus, an anaerobically digested water hyacinth can be used as an inoculum.

Čater et al. [188] opined that bioaugmentation is better since it is an eco-friendly technique and does not require any initial pretreatment step, thus curtailing time and costs, and it takes place instantly because it is very simplified. It is vital in the recovery of biodigesters experiencing the accumulation of high concentrations of volatile fatty acids or malfunctioning due to a high charging/loading rate [154]. The authors confirmed that bioaugmentation enhances the startup process and the performance of the entire process, and it also improves the depolymerisation effect of the blend of microbial species.

5.5. Technical and Economic Considerations of the Pretreatment Methods

Firstly, the various pretreatment techniques demonstrate varying effects on the degradation of the components of lignocellulose (lignin, cellulose, and hemicellulose). Secondly, these methods embrace varying levels of energy consumption and rely on the different substrates utilised, and the biogas will equally vary. Thirdly, naturally, no single pretreatment method can be employed for all types of feedstock because the substrates have been shown to respond differently based on their lignocellulose composition as each method exerts specific effects on cellulose, lignin, and hemicellulose [16]. This indicates that only when the feedstock composition appropriately corresponds with the pretreatment technique is the aim of pretreatment fulfilled. Therefore, the selection of the pretreatment method must take into consideration energy balances and economic feasibility [189]. The pretreatment method must be chosen carefully while aiming for optimal biogas yields; this suggests that each method should be evaluated in detail to emphasise their advantages and disadvantages as well as to calculate the net energy balance of the overall process. Some of the methods (physical, physicochemical, and chemical methods) require the purchase and installation of huge equipment and costly chemicals, and they result in the formation of harmful substances that end up in the digestate [23,190]. Therefore, post-treatment is needed for detoxification prior to the release of the digestate into the environment, thus incurring further costs that affect the overall economic viability of the process [16,70]. Moreover, Adewuyi et al. [31] emphasised that the inhibitory substances or chemical wastes produced during pretreatment might negatively influence bacterial activities, which in turn affect the hydrolysis and fermentation processes. In addition, high energy input and cost requirements appear to be the limitations associated with the decomposition of biomass recalcitrance [75]. The existing biological treatments are eco-friendly, precise, and selective, and they require very little energy consumption or chemical additives [190]. However, the huge cost associated with enzymes, making them costly to sustain, renders these methods not economically feasible in developing countries [31] because of the lack of financial capacity to sustain the technology.

6. Anaerobic Co-Digestion of Agricultural Wastes

In general, agricultural by-products are considered one of the promising feedstock materials for anaerobic digestion because, as raw materials, they are endowed with characteristics such as price stability and low cost, and they are potentially inexhaustible fuel sources [191]. These materials have chemical energy stored in bonds in their biomolecules (carbohydrates, proteins, and fats) or polymers, which are formed via photosynthesis, using sunlight. Hence, they serve as a potential source of sustainable and eco-friendly energy relying on renewable resources, which are needed to meet the ever-increasing world’s energy demand, since fossil fuels are faced with many mishaps and deleterious effects [152].

Recently, crop residues (maize, corn straw, sugarcane bagasse, groundnut shell, rice straw, wheat straw, etc.) are known to have lignocellulosic content of substantial quantities and are therefore described to be suitable substrates for biogas production [154]. Maize (Zea mays) is one of the rapidly growing annual energy crops. It is highly efficient in biogas generation and has shown improvements in BMPs when ensiled at the optimum time. Therefore, it is considered the most important energy crop for biogas production [192]. However, the wide-scale use of maize in the anaerobic digestion process suggests competition between energy and food uses. The cultivation of forage maize causes direct destruction to soil quality, soil erosion, and pollution of surface and groundwater [193]. More elaborately, maize cultivation is considered intensive and involves high input of fertilisers in combination with intensive soil cultivation, and it has significant environmental impacts and negative effects on biodiversity due to the use of pesticides during the monoculture of maize [192,194].

Similarly, Miscanthus is observed as the best option or alternative to maize for anaerobic digestion in terms of biogas yield. It is a rhizomatous, C4 grass species recognised as a promising perennial energy crop for biogas production, originating from South East Asia. Whittaker et al. [195] suggested that this crop would lead to a reduction in the erosion of agricultural soils acting as a prolonged soil cover. Moreover, Perrin et al. [196] mentioned that Miscanthus leads to high biomass yields, requires low input (e.g., water and fertilisers), causes reduced soil disturbance and increased soil carbon content, and can be grown on polluted soils. Nevertheless, during the growing season, Miscanthus undergoes great changes based on yield, moisture content, and composition when harvested, and these go a long way to affect the anaerobic digestion of the crop. Sorghum is also regarded as a suitable substrate for anaerobic digestion in agricultural biogas plants [157].

Søndergaard et al. [197] revealed that different agricultural substrates vary in terms of their biochemical methane potential (BMP) (e.g., manure < rapeseed< spring barley, winter wheat, winter barley, ryegrass < meadow grass). The significant variation in biogas yields between the substrates can be based on the season of harvest and the atmosphere of storage [75]. In addition, the ratio of the lignocellulosic contents i.e., cellulose, hemicellulose, and lignin of the substrates tend to differ due to age variation (maturation in both quantity and quality), the culture/growth conditions, and the type of plants and the season of harvest [16,75].

Anaerobic mono-digestion is defined as a process in which one (single) of the substrates is anaerobically digested for the treatment of its organic wastes, producing biogas, a renewable energy source. However, the mono-digestion of agricultural residues or lignocellulosic wastes can lead to inhibition problems, which accelerate fermentation, resulting in reduced methane yields [198]. This could be attributed to nutrient imbalances (high content of carbon and low nitrogen content), imbalanced trace elements, the lack of diversity in microbial communities, and the effect of operating factors [169].

Owing to the high lignocellulose content and nutrient imbalances, the mono-digestion of these wastes is fraught with the challenge of reduced efficiency in degradation. Some of the hurdles against mono-digestion can be mitigated via the use of trace elements or pretreatment methods, inoculation, or co-digestion [155,183]. Evranos and Demirel [199] reported that the anaerobic mono-digestion of maize silage supplemented with trace elements in combination with concentrations of 0.5, 0.5, and 0.25 mg/L of Ni, Co, and Mo respectively, caused improvement in methane yields, with an optimum methane yield of 0.429 L CH4/g V. Similarly, Sambusiti et al. [157] noted an increase in methane yields (by 324:5 0:7 mL CH4/g VS) and kinetic constants (by 0.16 ± 0.00 d⁻¹) during the anaerobic digestion of ensiled sorghum forage ground into different particle sizes and pretreated with an alkali (NaOH).

Co-digestion is a process whereby two or more feedstock substrates are blended and anaerobically treated in a biodigester. These substrates are termed co-substrates, and a co-substrate is selected bearing in mind that it balances the excess metabolites or substances generated by another substrate, demonstrating the inhibitory effect on the methanogens occurring in the process [200]. Therefore, substrates of different nutritional compositions are blended and digested together in a digester to provide process stabilisation, prevent the production of toxic/inhibitory substances, regulate pH and buffer capacity, and facilitate good synergistic actions between microorganisms [201]. Co-digestion is known to improve biogas production because the combination results in the achievement of optimum nutrients that are balanced and required for anaerobic digestion [19]. It can be claimed that co-digestion is a more feasible option through which nutrient supply, buffering capacity, and a balanced C/N are maintained [202], providing a wide range of nutrients needed by the methanogens.

The choice of substrates employed in anaerobic digestion is affected by numerous factors, including availability, the desired application of anaerobic digestion products, environmental conditions, digester technology, and economic benefits [19]. Owing to the ready availability of livestock manure in agricultural farms, it is one of the highly employed substrates in anaerobic digestion; however, it produces lower levels of biogas than other substrates because they are already predigested by the animal’s intestines. Nevertheless, they are usually added as a base substrate for co-digestion with other types of feedstock because of their desirable characteristics [19]. Uzodinma et al. [203] emphasised that animal manure consists of rumen microbes that perform anaerobic digestion at a faster rate than plant microorganisms; nevertheless, an improvement in biogas yield is observed when substrates are blended owing to synergistic effects.

Depending on the agricultural residues (e.g., manure), co-digestion creates avenues for increasing the organic loading rate [138] and allows for the mixing of substrates based on chemical composition, thereby eliminating the differences ascribed to the chemical composition between substrates that emerged from different sources [204]. Thus, energy crops can be co-digested, and although biogas production from energy crops still focuses on maize, combining maize with other energy crops (legumes, ryegrass, flower mixtures, and amaranth) results in a number of advantages. These include limiting the use of artificial trace elements (the presence of high concentrations of artificial trace elements (heavy metals) in the digestate, diminishes its use as a biofertiliser as heavy metals affect the natural element cycle); greater biodiversity in the fields; and enriching the biogas substrate with essential trace elements (Co, Mn, Ni, and Mo) [205]. On the other hand, lignocellulosic substrates can be co-digested, and low trace element conditions can be mitigated by adding animal manure since it contains high levels of trace elements or by supplementing with trace element additives [205]. Clearly, lignocellulosic biomass can be pretreated and co-digested with manure to improve its biochemical methane potential. For example, Thomas et al. [159] demonstrated that Miscanthus pretreated with 5 and 10% CaO and co-digested with cattle manure in dry leach bed reactors resulted in a 14–37% improvement in the BMP of Miscanthus. In addition, Aboudi and colleagues [206] noted increases of 70% and 31% in methane production from the co-digestion of sugar beet by-products with pig and cow manure, respectively, as compared to the mono-digestion of sugar beet by-products.

7. Conclusions

The increase in the use of biogas in society to meet energy demand will curtail the use of fossil-based energy sources and therefore assist in reducing greenhouse gas emissions and allows for the use of a non-renewable energy source while providing sustainable waste management. Anaerobic digestion is considered to be the best option for managing the available lignocellulosic biomass found in the environment, although biomass recalcitrance tends to hinder the process. The innovation via the utilisation of pretreatment techniques, trace elements, inoculum, and co-digestion with other substrates to increase biodigestibility and balance the nutrient requirements improve the feasibility of the anaerobic digestion process as well as biogas yields. Nevertheless, the conventional/traditional methods of waste disposal should not be completely negated, as they can serve as complements to green technologies because it has been shown that composting is quite relevant thus far owing to the value-added product utilised as a soil conditioner for improved agriculture.