Lignocellulose is a generic term used to describe the cell wall of plants which constitutes of cellulose (38–50%), hemicellulose (17–32%), and lignin (15–30%), where cellulose and hemicellulose consist of pentose and hexose sugar monomers, whereas lignin consists of polyphenol aromatics (Jönsson and Martín, 2016; Elumalai et al., 2018; Garlapati et al., 2020). The structural functionality of lignocellulose can be elucidated as following, where cellulose forms the structure of the cell walls, while hemicellulose assists in the cross-linking between the non-cellulosic and cellulosic polymers via covalent bonding (Figure 1). On the other hand, lignin which resembles phenol formaldehyde resin encloses the polysaccharide polymers and functions as “glue” to hold the lignocellulose structure together, thus providing additional mechanical strength to the cell walls for resistance from insects or during wet conditions. Besides, lignin functions as the hydrophobic barrier that enables transport of water and many essential nutrients through the cell wall. Several studies have reported that these polymeric substances are more prominent in larger concentrations within the primary and secondary cell walls of the plant, although the fractional composition may vary significantly based on their species, genetics, or environmental factors (Sun and Cheng, 2002).

In recent times, research on lignocellulosic material and its conversion into bioenergy has been growing at a relentless pace owing to the dwindling fossil fuel reserves and the environmental pollution associated with the exploitation of these resources. In an attempt to gradually transit toward a bio-based economy, various renewable energy sources have been explored to date, such as solar, wind, hydrothermal, and biomass. However, biomass-derived energy has remained the only promising renewable energy source that can potentially supersede petroleum-based fuel for transportation or to produce chemicals. The high chemical energy content of lignocellulosic biomass owing to the 75% carbohydrates from cellulose and hemicellulose has been ideal for bioenergy use (Chen, 2014; Sanusi et al., 2021). Besides, furfural, 5-hydroxymethylfurfural, and levulinic acid obtained from lignocellulose during biorefining are very favorable building blocks for the generation of energy fuels by chemical or biological synthesis processes (Elumalai et al., 2018).

Advantageously, lignocellulosic biomass is available abundantly from agriculture and forestry with an estimated annual production of 1.7-2 × 10¹¹ tonnes per year (Elumalai et al., 2018). Besides, in 2011, 38 million tonnes of biomass were used for biofuels in the EU, out of 1.2 billion tonnes of biomass generated from various crops (Scarlat et al., 2015). In addition, it is reported that by 2030, the supply of lignocellulose biomasses in the United States alone would be 450 million dry tonnes per year, which can generate 67 million gallons of ethanol per year (Valdivia et al., 2016).

Despite these advantages, biomasses are recalcitrant in nature owing to their cellulose crystallinity and non-reactive lignin. As such, pretreatment techniques are required to allow the cellulose to be amenable to the enzymatic hydrolysis process that subsequently assists in the extraction of fermentable sugars prior to the biofuel generation processes. Besides, technological limitations such as high cost and inadequacy in the existing infrastructure have posed major challenges in attaining high quality and yield of bioenergy from lignocellulosic material. In view of these limitations, studies have reported various optimization strategies in pretreatment stages as well as enzymes and fermentation stages to enhance bioenergy production in an energy efficient and cost-effective manner. Recently, the growing interest in nanomaterials and their exceptional properties have been vastly exploited to enhance bioenergy generation. As such, in this review, first, the fundamental principles of nanomaterials and their significance in bioenergy generation are discussed thoroughly. Section 2 will detail the application of nanotechnology for biomass conversion particularly in lignocellulosic pretreatment and for enzyme immobilization. Next, the utilization of a nanobiocatalyst in bioenergy production will be reviewed. Last, the challenges and perspectives for future progress in bioenergy generation from lignocellulosic biomasses are highlighted.

The International Standardization Organization (ISO) has defined nanomaterials as any material having external, internal, or surface structure dimensions in the nanoscale range of 1–100 nm (ISO/TS 27687:2008 Nanotechnologies, 2008). Nanoparticles (NPs) can be broadly classified into four groups: carbonic, organic, inorganic, and composites. The characteristics of NPs are dependent on their chemical structure which influences their functionality in various environments. Besides, it is interesting to note that the physical and chemical properties of NPs vary significantly from their macro counterparts which can be exploited for use in various applications such as bioenergy, electronics, medicine, ionic liquids, and consumer products (Li et al., 2005; Shuttleworth et al., 2014; He et al., 2020; Production et al., 2020). Moreover, NPs have a faster reaction rate than larger particles while being essential in the bioenergy generation process. The faster reaction rate is associated with the extremely fine particle sizes of NPs that provide a large surface area to volume ratio which in turn increases the number of active sites for various reactions or processes to occur (Misson et al., 2015).

From the perspective of bioenergy production, nanomaterials can be envisioned in many aspects to meet the rapidly growing global energy requirements owing to their exceptional properties such as large surface area, high catalytic activity, crystallinity, durability, stability, adsorption capability, and efficient storage (Zhang et al., 2011; Hamawand et al., 2020). All the aforementioned advantages of NPs can enhance the productivity, hydrolysis, and stability of cellulase enzymes for various bioenergy productions. Generally, NPs in bioenergy production can be advantageous in cellulase production, for cellulase thermal stability, in pretreatment of biomass, waste management, or sugar and biohydrogen production. For instance, in a study by Srivastava et al., it was reported that an efficient cellulase process is essential for enzymatic hydrolysis, and the use of NPs such as Fe₃O₄ and Fe₃O₄/alginate showed higher hydrolysis efficiency and enhanced cellulase production by 35 and 40% respectively (Srivastava et al., 2015). A similar group of authors also investigated the efficacy of nickel cobaltite (NiCo₂O₄) NPs and reported 40% more cellulase production with an enhanced thermal stability at 80°C for 7 h (Srivastava et al., 2014). The thermal and pH stability of crude cellulase have also been investigated using zinc oxide (ZnO)-based NPs, and it was shown to exhibit an enhanced thermal stability at 65°C for 10 h with a maximum pH alkaline value of 10.5; all of which are highly desirable in the field of bioethanol and biohydrogen production (Srivastava et al., 2016). In another study by Dutta et al., calcium hydroxyapatite-based NPs increased the thermal stability and enhanced the production of d-xylose by 35% (Dutta et al., 2014). The authors reported that the uses of both nanoparticle-activated xylanase and nanoparticle-activated cellulase systems are exclusive for demonstrating high activity at elevated temperatures up to 80°C. It was further added that utilization of cofactor calcium in an NP form conferred the increased thermostability and activity to the cellulase and xylanase enzymes.

Magnetic nanoparticles (MNPs) have been most commonly used in comparison with other nanoparticles in bioenergy production since the magnetic properties allow easy recoverability, and have the ability to bind to multiple target compounds, excellent biodegradability, low cytotoxicity to biomass cells, and the ease of synthesis (Pena et al., 2012). The large surface area to volume ratios of MNPs have also been utilized as carriers for the immobilization of enzymes, which leads to increased stability for use in biodiesel or bioethanol production. For instance, Xie and Ma reported that immobilization on the support with magnetic Fe₃O₄ NPs showed excellent pH tolerance and thermostability with conversion to biodiesel fuels up to 90%, while the immobilized lipase was reusable four more times without compromising its activity or performance (Xie and Ma, 2009). Other MNPs that were used for immobilization of enzymes include TiO₂ for the hydrolysis of lignocellulosic material for bioethanol production (Ahmad and Sardar, 2014), a mix of TiO₂–ZnO catalyst for biodiesel production (Madhuvilakku and Piraman, 2013), and manganese dioxide (MnO₂) for bioethanol production. Moreover, the usage of nanoparticles or their microemulsion was shown to have a hydrolytic effect that is identical to that observed with chemical pretreatment in lignocellulosic biomass processing. This was seen in the study carried out by Pena and a colleague whereby when wheat straw was treated with perfluoroalkylsulfonic (PFS) and alkylsulfonic (AS) acid-functionalized magnetic nanoparticles, it produced 46% more sugar than the control (35%). The addition of nanoparticles to PFS and AS acid may have led to the stabilization and allotted hemicellulose hydrolysis which has comparable acid strength of sulfuric acid solutions (Wang et al., 2012). The authors highlighted the main advantage of using magnetic nanoparticles which lies in their notable function in pretreating lignocellulosic biomass with little quantities while still being recyclable for future usage. In another study of pretreatment, the combined effect of alkaline pretreatment with magnetite (Fe₃O₄) nanoparticles on rice straw for biogas production was explored (Khalid et al., 2019). The application of MNPs demonstrated a major advancement in biogas production that increases biogas and methane yield by 100 and 129%. Nanoparticles have also been used to improve sugar production at the pretreatment level. Yellow poplar sawdust was processed to 96 percent (w/w) hemicellulose at 175°C using 0.8 percent (w/v) sulfuric acid. The application of sulfuric acid with PFS and AS nanomaterials converted hemicelluloses by 66 and 61%, respectively, at 50- and 400-fold acid levels (Arora et al., 2020). Some other studies have reported that the high coercivity and excellent paramagnetic properties of MNPs are highly desirable for biogas and methane production. For instance, Abdelsalam et al. reported that Fe₃O₄ MNPs with a concentration up to 20 mg/L reduced the time required to achieve highest biogas and methane production (Abdelsalam et al., 2017). Besides, the Fe₃O₄ MNPs assisted in the effective distribution of the iron ions in the solution which ensured a sustainable supply of iron ions in the bioreactor; all of these bio-stimulated the anaerobic digestion process, thus increasing the production of biogas and methane. Table 1 summarizes some of the most recent utilization of nanoparticles to generate bioenergy such as biodiesel, bioethanol, and biogas from biomasses.

Pretreatment of LB is the first step in any bioenergy conversion which breaks down the complex structure of LB to release polymers. The pretreatment of LB is an important stage, and choosing the best pretreatment procedure is critical. However, suitable pretreatment procedures must fulfill the specific criteria, including the following: 1) improving the generation of sugars from LB or having the ability to generate sugars eventually via hydrolysis, 2) high resisting ability of the degradation or depletion of sugars, 3) they must limit the creation of by-products that obstruct succeeding hydrolysis and fermentation processes, and 4) the technique must be cost-efficient (Ingle et al., 2019).

The nanotechnology method is widely used in numerous industries, including biofuel production (Rai et al., 2019). In the nanotechnology-based approaches, nanomaterials are used, and due to their smaller size, they can easily penetrate lignocellulosic biomass cell walls and interact with lignocellulosic components (cellulose and hemicellulose) to release monomeric and oligomeric sugars (Rai et al., 2017). Nanomaterials employed for pretreatment are capable of improving the chemistry of biomass at a molecular basis. Magnetic nanoparticles are preferred because they aid in their retrieval out from reaction media for reusing, as a consequence, making the entire procedure more economical (Pena et al., 2012). The usage of nanoparticles or their microemulsion has been shown to have a hydrolytic effect that is identical to that seen with chemical pretreatment for LB processing. This was seen in the study carried out by Pena and a colleague whereby when wheat straw was treated with perfluoroalkylsulfonic (PFS) and alkylsulfonic (AS) acid-functionalized magnetic nanoparticles, it produced 46% that is more sugar than the control (35%). The addition of nanoparticles to PFS and AS acid may have caused the stabilization and allotted hemicellulose hydrolysis which has comparable acid strength of sulfuric acid solutions (Wang et al., 2012). The main advantage of using nanoparticles is their notable function in pretreating LB substances with little quantities while still being recyclable for future usage. In another study of pretreatment, the combined effect of alkaline pretreatment with magnetite (Fe₃O₄) nanoparticles on rice straw for biogas production was explored. The application of magnetite nanoparticles (MNPs) demonstrated a major advancement in biogas production that increases biogas and methane yield by 100 and 129%, respectively (Khalid et al., 2019). Nanoparticles have been used to improve sugar production at the pretreatment level. Yellow poplar sawdust was processed to 96 percent (w/w) hemicellulose at 175°C using 0.8 percent (w/v) sulfuric acid. The application of sulfuric acid with PFS and AS nanomaterials converted hemicelluloses by 66 and 61%, respectively, at 50- and 400-fold acid levels (Arora et al., 2020).

Nano-shear hybrid alkaline (NSHA) pretreatment is another emerging approach of nano-based pretreatment of LB. This technique involves a combination of high-speed shear and chemical reagent synergy, as well as a gentler thermal action. This reaction commonly is performed in a specific reactor known as nanomixing. The high shearing work axis is transported to the biomass nanostructure, allowing efficient lignin removal with cellulose and hemicellulose exposure in a short time. Wang developed a corn stover pretreatment method using the NSHA technique (Wang et al., 2013). In the study of using the NSHA method, findings indicated that hemicellulose and lignin were removed from the original content, leaving with up to 82% cellulose content (Arora et al., 2020). The synergetic action of cellulase enzymes degraded biomass recalcitrance and created nanoscale polysaccharide agglomeration that can be readily converted to simple sugars by NSHA pretreatment. Indeed, utilizing immobilized cellulase, the NSHA pretreatment technique exhibited a four-fold and five-fold increase in enzymatic degradation of cellulose and hemicellulose into sugars. Owing to the high shear rate of synergic effects, this mechanism indicates benefits in adding high shearing into current pretreatment procedures and many other chemical products (de Oliveira et al., 2017).

In an alternative study of the NSHA pretreatment of corn stover, a positively charged polyelectrolyte composing poly (diallyldimethylammonium chloride) (PDAC) is employed as an additive to alter the cellulosic surfaces and stabilize lignin (Duque Leidy Eugenia Peña, 2013). The morphology and chemical components of pretreated biomass were studied using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Lignin develops a globular aggregate with PDAC polyelectrolyte, altering the cell wall’s shape. The procedure using polyelectrolyte offers a benefit in the reduction of chemical consumption. Despite the potential advantages, reports on biomass pretreatment utilizing nanoparticles are scarce at the moment, as the application of nanotechnology in biomass refineries is still in its infancy.

Hydrolysis of lignocellulosic biomass utilizing nanoparticles or nanomaterials can be accomplished in two approaches: physical adsorption or covalent bonding of different enzymes on nanomaterials (Elumalai et al., 2018), and employing functionalized nanoparticles (Garlapati et al., 2020). In the biofuel production process, enzymatic hydrolysis is considered a critical stage; however, the stability of the enzymes employed in the process is a significant concern. Nanotechnology is applied in this step as it can help to stabilize these biocatalysts. Nanotechnology has recently advanced to the point that it can be utilized to immobilize enzymes. Immobilizing enzymes on nanostructures is a novel method of improving enzyme catalytic efficiency (Song et al., 2018). The term “nanobiocatalyst” refers to enzymes immobilized on NPs, and it is evidence of nanobiotechnology’s constantly developing and pioneering discoveries (Misson et al., 2015). Enzyme immobilization provides benefits in regards to simple enzyme reutilization, proving itself as a key tool for the development of second-generation biorefineries (Zanuso et al., 2021). Because of the spacer, enzyme immobilization on nanomaterials by cross-linking causes it to be more specific for substrates and more versatile (Ingle et al., 2017).

Adsorption, covalent bonding, entrapment/encapsulation, and cross-linking are the four basic types of immobilization strategies, based on how the enzymes engage with the support or with one another as shown in Figure 3. Each has its pros and cons; for example, the adsorption technique is regarded as a low-cost and straightforward approach. The weakening of the interactions and the possibility of enzyme leakage when medium conditions such as pH, temperature, or polarity are altered are the major disadvantages of this approach (Nguyen and Kim, 2017). Because covalent bonding enhances enzyme stabilization and the generated bonds are strong enough to resist enzyme outflow, it is commonly utilized for enzyme immobilization (Zanuso et al., 2021). Owing to the presence of inhibitors in lignocellulose hydrolysates, the encapsulation technique provides an advantage by its ability to generate a microenvironment that protects the enzymes from hostile environments. Cross-linking is considered a different kind of immobilization approach where it involves the creation of enzyme aggregation, also known as cross-linking enzyme aggregates (CLEAs).

The matrix utilized to immobilize enzymes differs, and it comprises nanofibers, nanoparticles (NPs), and organic metal frames, all of which serve the same purpose (Khoshnevisan et al., 2017). MNPs, nickel nanoparticles (NiNPs), iron nanoparticles (FeNPs), and other metal oxide nanoparticles have all been utilized as transporters for enzyme immobilization. The table below displays nanomaterials used for enzyme immobilization in treating LB.

Carbon nanotubes are a potential material for immobilizing enzymes. In a study by Tang et al., they used the adsorption technique to immobilize glucose oxidase (GOD) on a carbon nanotube (CNT) electrode coated with platinum nanoparticles (Tang et al., 2004). The use of nanotubes enhances the immobilization process. In another study, CD spectra investigation found that lipase bound to carbon nanotubes retained 62 percent of the native lipase’s a-helix composition (Ji et al., 2010). In general, enzyme immobilization on nanoparticles or other relevant support elements was used to improve the enzyme’s characteristics, allowing it to be used in LB pretreatment operations at a lower cost. Immobilization has been shown to counteract several inhibitory effects by preventing inhibitors from binding to the enzyme’s active site. Inhibitor tolerance may have improved as a result of this (Qi et al., 2018). The thermal stability of cellulase enzymes is essential for large-scale industrial lignocellulosic operations because high temperatures enhance biomass degradation. Immobilized cellulases have higher thermal stability, which is one of their greatest benefits for large-scale production (Ahmad and Khare, 2018). One main limitation of cellulase immobilization is that the majority of research on cellulase immobilization employs artificial cellulose as the substrate, for instance, microcrystalline cellulose or carboxymethyl cellulose. The structure of microcrystalline seems to have a consistent diameter of approximately 20–200 mm, which makes for easier mass transport as contrasted to actual LCM substrates (Zanuso et al., 2021). Mo et al. proposed the construction of scaffolds with a larger specific area and open porous structure to give more locations for enzyme immobilization to address these impacts on enzyme immobilization. In addition to offering more surface area for the enzyme to uptake, an open porosity shape makes it easier for the substrate to reach the enzyme (Mo and Qiu, 2020).