Recent advances and sustainable development of biofuels production from lignocellulosic biomass

4.1. Physical method

Physical pre-treatment energy needs are determined by the ultimate particle size and crystallinity reduction of the lignocellulose biomass. Mechanical extrusion, drying, microwave, ultrasound, and pyrolysis are all common physical preparation methods. Grinding, compression and crushing methods comes under this technique. The microstructure of lignocellulosic feedstock can be decreased using the mechanical splintered technique to increase the material's contact surface for further acid or enzyme treatment. Heredia-Olea et al. (2015) investigated the influence of extrusion pretreatment on bioconversion of sweet sorghum biomass into bio-ethanol. Extrusion treatment of sweet sorghum prior to saccharification process yielded around 70% of sugars producing 200 mL of ethanol/kg biomass (Heredia-Olea et al., 2015). A study conducted on testing the impact of ultrasound treatment on sugarcane bagasse’s fermentation and saccharification to produce ethanol using Trichoderma reesei. 90% ethanol production was attained with ultrasound pretreatment of sugarcane bagasse biomass (Velmurugan and Incharoensakdi, 2016).

Thermal pre-treatment is one of the most effective physical pretreatment techniques, where the complex compounds are broke down into sugars, which in turn enhanced the fermentation process and product generation. Thermal pretreatment technique could develop the methanogenic and acidogenic digestibility process of lignocellulosic biomass for the production of biofuels from lignocellulosic biomass (Sarip et al., 2016). In thermal pretreatment, the compounds have been heated above the temperature of 260 °C and pressure 4.5 MPa. Thermal pretreatment progresses the enzyme digestibility of the lignocellulosic biomass and converting the perverse cellulose fraction into glucose for fermentation. Further, the porosity of cellulose rich residue could be increased for greater access of cellulose enzymes. Lignin phase has significant impact on cellulose accessibility which has been disrupted under the thermal pretreatment technique. A research performed by Kong et al. (2018) concluded that there was a slight increase of 12.6% in dextrose sugar production after thermal pre-treatment method which could be further employed for biofuel production (Kong et al., 2018). Microwave pretreatment includes thermal and non-thermal effects and this heating process can significantly reduce pretreatment time and efficiency. In some cases, microwave assisted combined ball milling technique are used. Microwave assisted ball milling technique was effective in reducing the crystallinity index, improving the enzymatic hydrolysis, swelling and fragmentation which enhances the accessibility of the catalysts and substrates and leads the degradation of hemicellulose and lignin in the biomass. In a study involving glucose production using microwave assisted combined ball milling (ball milling for 1 h and microwave for 20 min) could yield higher glucose production compared with ball milling technique at the time period of 6 h (Puligundla et al., 2016). Similarly for biobutanol production from brewer’s spent grain, microwave assisted alkaline pretreatment was carried out. Around 28 kg of butanol per tonne of spent grains was attained with hydrolysis and fermentation by Clostridium sp. following microwave assisted pre-treated samples (Lopez-Linares et al., 2020). Biomass pyrolysis is the most common pre-treatment process employed in most of the researches. It involves the thermal breakdown of organic biomass at a particular range of temperature (300–600 °C) in the absence of free oxygen.

4.2. Chemical method

Chemical pre-treatments have mostly been used to increase cellulose biodegradability by removing lignin and/or hemicellulose, as well as to reduce the degree of polymerization (DP) and cellulose crystallinity to a limited extent. Acid, alkali, ionic liquid and oxidative are the most frequently applied chemical treatment techniques. Acid pre-treatment includes breaking of lignocellulosic material's hard structure with diluted and concentrated acids. The acid pretreatment using concentrated acid suitable for hydrolysis of lignocellulose biomass which cleaves the glycosidic bond in the cellulose structure and the acid stable hemicellulose can decomposes to glucomannan or xylon. It improves the lignocellulose digestibility by solubilisation of lignocellulose, lignin precipitation and rendering better availability of cellulose for further processes. The most significant parameters that affect acid treatment processes are solid to liquid ratio, size of the lignocellulose particle, concentration of the acid, pre-treatment temperatures and duration. Though both dilute and concentrated acids have been used in lignocellulose chemical pretreatment processes, usage of concentrated acid is less appealing due to its eroding, noxious and expensive nature. Gonzales et al. (2017) used dilute acid pre-treatment of pine tree wood pellet – lignocellulosic biomass for the enhanced hydrogen production. Maximal hydrogen production of 1824 mL H₂/L.d was observed following the dilute sulfuric acid treatment procedure. Few researchers conducted a similar study in which they assessed the dilute acid treatment using the combined severity factor (CSF) that considers reaction pH, temperature and time of a certain hydrolysis process. Optimum CSF values were required to attain maximal sugar generation for biohydrogen production. For severity factors of 1.86 and 1.95 optimum rate of H₂ production of 3340 and 2640 mL H₂/L.d were reported for rice husk and empty palm fruit bunch respectively (Gonzales et al., 2016).

Pretreatment of lignocellulosic biomass using bases such as sodium, potassium, calcium, and ammonium hydroxide is known as alkaline pretreatment. The application of an alkali causes ester and glycosidic side chains to degrade, leading to lignin structural changes, partial hemicellulose solvation with crystallization and cellulose dilatation (Bhatia et al., 2020). Mostly, potassium hydroxide (KOH), sodium hydroxide (NaOH) and calcium hydroxide Ca(OH)₂ are used in this pre-treatment procedure which separates lignin, cellulose and hemicellulose. Saponification process majorly helps for hydrolysis process which breaks and modifies the amorphous and crystalline structure of cellulose and cleaves the lignin-carbohydrate linkages. Jiang et al. (2020) examined the effect of NaOH and Ca(OH)₂ pretreatment of Giant reed for photofermentative hydrogen production. This cleavage indirectly affects the hydrogen production by a negative impaction on sugar yield from lignocellulose. NaOH pretreatment of giant reed was found to be more suitable for biofuel production than lime treatment (Jiang et al., 2020). In relative to acid treatment, alkaline pretreatment typically results in less degradation of lignocellulose. Furthermore, this technique demonstrates better efficacy on agriculture based lignocellulose materials (Reilly et al., 2015).

Oxidation treatment method includes the usage of oxidant which aids in the degradation of lignocellulose. Oxidants such as peroxide dissolve the lignocellulose content where only cellulose (crystalline form) remains undissolved and hemicellulose separation from cellulose occur. Photocatalysis, ozonolysis and wet oxidation are some of the prevailing processes in oxidative pre-treatment. Wet oxidation is directly affects the three components of lignocellulosic biomass, lignin is comprehensively undergoes oxidation and cleavage, hemicellulose is broke into sugars and cellulose is degraded into organic acids. Furthermore, wet oxidation improves the cellulose accessibility via the removal of hemicellulose and lignin. dos Santos et al. (2018) analysed the biogas production using oxidative pretreated coffee husks. They determined that the ozonation conditions resulted in better dissolution of hemicellulose, lignin and cellulose. Large quantities of hydroxyl ions might induce ozone molecule to breakdown releasing hydroxyl radicals. Consequently superoxide radicals are generated which act as a potential oxidant for efficient lignocellulose degradation (dos Santos et al., 2018). A study based on improving methane yield from palm fruit bunches by wet oxidation method using hydrogen peroxide resulted in better methane production. Here, following the peroxide treatment, lignocellulose's superstructure is broken apart rather than solubilized, making it easier for enzymes like cellulases to access to lignocellulose materials (Lee et al., 2020). Some of the solvents used in this technique include thiourea/NaOH solutions, N-methyl morpholine N-oxide, tetrafluoroborate, molten salt hydrates (inorganic) and metal complex solutions. The method, also known as cellulose-based treatment, has the ability to dissolve lignocellulosic components such as hemicellulose, cellulose, and lignin selectively. Anti-solvent system is used for the cellulose precipitation when both lignin and cellulose are dispersed in the ionic liquid system (Millati et al., 2020). In a research performed by Mohammadi et al. (2019), 1-H-3-methylmorpholinium chloride was employed as an ionic liquid for the pretreatment of rice straw for enhanced ethanol production. After processing, the rice straws ordered and impenetrable structure was transformed to an accessible structure with the absence of outer covering. Also the particle size and porosity was enhanced resulting in improved ethanol production (Mohammadi et al., 2019).

4.3. Physico-chemical method

Physico-chemical pre-treatment techniques include steam explosion, torrefaction and ammonium fibre explosion. By altering the working conditions (pressure and temperature) in the presence or absence of a chemical, lignin and hemicellulose are eliminated and cellulose is degraded in physico-chemical pretreatment. The torrefaction process us carried out in an inert atmosphere at high temperatures (200 to 300 °C). Torrefied biomass has less moisture content, fibrous nature and larger calorific value relative to raw biomass which results partial depolymerisation of cellulose and lignin and decomposition of hemicellulose. In the course of devolatization and dehydration processes, hydroxyl groups are eliminated from raw lignocellulose biomass making it hydrophobic. Effective breakage of aryl–ether linkages is a vital technique for producing fuels from lignin. A research employing pre-treated rice straw and the torrefaction procedure for ethanol production verified the breakdown of cellulose micro fibrils. It decreased the cellulose crystallinity and improved its availability for further hydrolysis (Sheikh et al., 2013). Li et al. (2014) pretreated the rice straw using torrefaction process and observed better bio-oil yield of around 55%. However at higher torrefaction temperature, there was low conversion of carbohydrate and partial lignocellulose decomposition. Hence the optimum torrefaction temperature should be maintained for better biofuel generation (Li et al., 2014).

In steam explosion pretreatment, the lignocellulosic biomass is primary treated with immersed steam for a specific measure of time. The steam accessibility into the internal structures of biomass is high because of great fume stage dissemination. By doing the condensation steam, the microporous structure of biomass is soaked with hot liquid water which causes release the acids from the hemicellulose and reducing the pH (3.0 to 4.0). The moderate acidic conditions can particularly cleave the lignin ether bonds and hydrolyze the hemicellulose. This separation of lignocellulose structure and the expulsion of hemicellulose enhance the enzymatic digestibility. Steam explosion approach entails the molecular decomposition through shearing and steam caused by the rapid depressurization system. This technique is divided into two phases. For a few seconds, lignocelluloses are subjected to pressurised steam. They are then abruptly depressurised to reach air pressure. It is quite inexpensive in most situations and degrades hemicellulose and lignin compounds. In certain circumstances, severe process conditions resulted in low glucose concentration in wheat straw for ethanol production owing to early cellulose breakdown and glucose loss as residue (Bera et al., 2021). Transformation of lignin with cellulose removal is the principal physico-chemical modifications attributed due to steam explosion pre-treatment technique which aid in improving biomass digestibility to enzymes. The following variables influence steam explosion measurement: moisture levels, temperature, chip size and retention time. Another approach for pretreatment of lignocelluloses is wet oxidation (WO). It has the capacity to fractionate woody materials cellulose rich fraction while forming few inhibitors. Following the WO process, lignocellulose material gets solubilized and decomposition of lignin to carbon dioxide and water occurs. However, optimizing the treatment conditions could increase up the cost of the pre-treatment process. Other treatments like explosion using ammonium fibre and carbon dioxide are rarely used for lignocellulose due to its less efficiency (Shirkavand et al., 2016).

4.4. Biological method

The biological pre-treatment approach uses microorganisms such as bacteria and fungi to modify the lignocellulosic biomass, making it more susceptible to enzymatic digestion. In enzymatic pretreatment, pure enzymes can be used to accelerate the degradation of lignocellulose. Generally, enzymes help to release the fermentable sugars from hemicellulose and cellulose and reduce the recalcitrance of lignocellulose to enhance the biogas generation from the biomass. Enzymatic pretreatment has major impact on the characteristics of recalcitrance for example decreasing crystallinity and polymerization; improving accessible surface area and eliminating lignin content. Removal of lignin and hemicellulose result in improved digestibility of cellulose which is mostly preferred for fermentation process. Most of these bacteria generate lignin degrading enzymes, which results in biomass alteration. Brown rot and white rot fungi are the most commonly studied for this process. The lignolytic enzymes generated by white rot fungus are highly beneficial for biofuel generation by lignin degradation due to their high substrate specificity and high oxidation efficiency. Irpex lacteus (Qin et al., 2018), Polyporus brumalis (Zhou et al., 2017), and Myrothecium verrucaria (Su et al., 2018) were among the fungi used in the pretreatment research. Free radical chain based reaction mechanism occurs in this pretreatment. Initially, formation of high reactive radical species occurs which results in fragments owing to bond breaking during lignin depolymerisation. The surface process is linked to lignin breakdown by white rot fungus. Cellulase enzymes produced by this type of fungi dissolves the waxy coating surface of lignocellulosic substrates paving the way for the fungal hyphae to penetrate into it. Following that, a cascade of enzymes such as hemicellulases, exoglucanases, peroxidases, and endoglycanases are generated, resulting in a more efficient breakdown process. Brown rot fungi are suitable for the pectin decomposition. Fenton’s reaction occurs in brown rot fungi based treatment process and is primarily used for low weight breakdown compounds. The biological pretreatment of wheat straw was investigated using white rot fungi and found that 35% of wheat straw was converted into reducing sugars (Talebnia et al., 2010). However, one drawback of utilising this white rot fungus is the mild alteration of lignin rather than breakdown (Chen et al., 2017, Sindhu et al., 2016, Yadav et al., 2019). Biological pretreatment looks to be a viable technology with several advantages, including low input energy, gentle ambient conditions, an ecologically acceptable operating method and minimal chemical demand. However, its downsides are as obvious as its advantages since biological pretreatment is slow and requires careful management of growing conditions as well as a considerable quantity of area to be performed. Furthermore, most lignolytic organisms solubilize and consume lignin, hemicellulose, and cellulose. As a result, biological pretreatment confronts technological and economic hurdles, making it less appealing commercially.

5.1. Genetic engineering approach

For the efficient conversion of lignocellulose substrate to fuels, both microorganisms and substrate are crucial. There may be less biomass accessibility with native or altered microorganisms. Several genetic engineering techniques have been developed to convert lignocellulose to biofuel. Genetic engineering of lignocellulose biomass is the primary source for enhancing biofuel output. This process was aided by the formation of a molecular matrix between hemicellulose molecules and the resulting complexing with neighbouring cellulose fibrils. Lignin composition varies as a result of genetic modification which improves the digestion of the cell wall polysaccharides (Wang et al., 2015, Madadi et al., 2017). Silencing the gene responsible for lignin production also improves the biomass digestibility. The functional characterization of genes provides potential targets for enlightening saccharification and modifies the lignin content and composition. Genetically engineered approach modifies the lignin pathway which generates transgenic lines with improving enzymatic sugar release proportional to the extent of lignin down regulation. These transgenic approaches used to increase the cell wall traits and characterize the biosynthetic pathway of lignin, reduce the lignin content. Mazarei et al. (2020) studied the improvement in biofuel production from switch grass by silencing Folypolyglutamate synthetase gene. Partial downregulation of this gene slightly altered the lignin composition and increased the ethanol productivity by 18% (Mazarei et al., 2020). Similarly, Lee et al. (2021) investigated lignin modification utilising CRISPR/CAS systems for improved biofuel production using barley. A transgenic plant with a mutant caffeic acid O-methyl transferase 1 mutant was grown and tested for bioethanol production in this work. The mutant barley plant had a 34% greater ethanol concentration and a 14% reduced lignin content (Lee et al., 2021). Lignin biosynthetic genes have also been engineered for easy digestibility of the biomass.

The alteration of strains engaged in the bioconversion process is another option. With the aid of genetic engineering, microbial transformation into requisite cell factories occurs. Genetic modification of organisms offers a greater potential for biomass depolymerisation and conversion of fermented sugar hydrolyzate into free fatty acids in biodiesel synthesis from lignocellulose. In this situation, genetic engineering can be used in one of two ways. First will be engineering of native cellulolytic microbes for improving the product related aspects. Second, non-cellulolytic organisms will be engineered to generate cellulase enzymes. Few studies have focused on using export-related protein fusions in E. coli to extracellularly produce enzymes such xylanases, cellulases, cellobiase, xylobiosidase, and glucosidases. With ionic liquid pre-treated switch grass, this modified strain grew well and enhanced biomass hydrolysis (Lin et al., 2013). In the production of biofuels, high product concentration can sometimes be a hindrance. Butanol, for example, can create a partition in the cytoplasmic membrane, altering its shape. As a result, strains must be modified to improve product tolerance in biofuel-producing microbes. Strains can also be engineered to grow on complicated lignocellulose substrates, increasing biofuel production rate. Lopez-Hidalgo et al. (2021) investigated the effect of a genetically modified E.coli strain on ethanol and hydrogen coproduction from corn stover and wheat straw. Different molecular methods were used to remove genes that produce hydrogenases and reductases in order to improve coproduction in complicated substrates. Ethanol production of 20–30% was observed with this engineered strain (Lopez-Hidalgo et al., 2021, Zheng et al., 2015). Yeast/fungi may also be developed to produce biofuels from lignocellulose substrate, such as ethanol, butanol, methane, and hydrogen. Extracellular enzymes responsible for lignin breakdown are secreted by them. For improved ethanol output, recombinant strains containing genes for intracellular expression of xylitol dehydrogenase, reductase, kinase, and glucosidase from separate species such as Pichia stipitis, Saccharomyces cereviseae, and Aspergillus acleatus have been combined and used in a research (Katahira et al., 2006, Amores et al., 2015). Developing genetically engineered constructs with modified lignolytic enzymes might improve the lignocellulose bioconversion into biofuel.

5.2. Metabolic engineering approach

Development in the generation of chemical by objective metabolic engineering is for the most part performed by (1) upgrading the action of enzymes associated with the product biosynthesis (2) disturbing pathways that enter after carbon substrate or potentially electrons and additionally co-factors. Furthermore, microorganisms should be lenient to high grouping of the substance to permit large scale industrial fermentation. The biosynthetic route for lignocellulose fuel generation is a multi-step operation. The metabolism of microorganisms has thousands of processes that govern the energy process. Recent metabolic engineering approaches have contributed to increased flux for biofuel production. Enzyme modification, scaffold construction, codon optimization, and fermentation conditions are some of the most common tactics used in these engineering procedures. For improved carbon flow in product creation, two major methods have been developed. The first is the push–pull-block approach, in which specific metabolic pathways are inhibited and enzymes are overexpressed to enhance carbon flow. The second strategy is to reduce or block carbon flow in the creation of undesirable by-products, which increases carbon flux in fuel synthesis. Metabolic engineering may provide three main outcomes: high-yielding microorganisms, the capacity to endure harsh environmental conditions, and adaptation to a wide range of lignocellulosic materials. Undesired product formation can also be inhibited by this approach (Hollinshead et al., 2014, Majidian et al., 2018). In case of iso-prenoid derived fuels, mevalonate and deoxy xylulose phosphate pathway can be overexpressed or deregulated by introducing or knocking out the gene in the desired organism to minimize the by-products formation (Choudhary et al., 2020).

Li et al. (2019) investigated the butanol production from lignocellulose substrates using engineered Clostridium tyrobutyricum strain. This bacterial strain with knocked out acetate kinase (ack) gene was designed to overexpress adhE2 gene which encodes alcohol dehydrogenase gene involved in n-butanol synthesis. CtΔack-adhE2, a metabolically modified strain, provides a number of benefits, including absence of acetone generation, increased butanol tolerance in the organism and its production. With this engineered strain, butanol synthesis of high yield 0.3 g/g and titre 15 g/L was achieved (Li et al., 2019). For effective lignocellulose bioconversion, Romani et al. (2015) modified the xylose metabolic pathway in Saccharomyces cerevisiae strains. Hydromycin resistance gene was used in place of URA3 marker gene to develop a superior xylose pathway. Deletion of the GRE gene was also done to improve biofuel production by enhancing the rate of xylose consumption in corn-cob hydrolyzates. Of the estimated theoretical yield, 92% was obtained with this practical study (Romani et al., 2015).

The researchers have lately focused their research on the engineering of plant biosynthetic pathways for fuel synthesis. Lignocellulose substrates have been changed to allow for easy decomposition and the production of fuels with minimum pre-treatment. One of the primary recalcitrance factors, lignin, has been studied in order to decrease its impact on the enzymatic digestion of this biomass. Few researchers have demonstrated in a field study that biomass from Poplar plants with down regulated cinnamoyl-CoA reductase (CCR) gene has the greater potential for bioethanol production. CCR enzyme plays a crucial role in lignin biosynthetic pathway. Down regulation of this enzyme resulted in 161% increase in ethanol yield (Bilal et al., 2018). Designing appropriate microorganisms that synthesis desired biofuels directly from lignin from biomass can increase lignin usage, but this requires a full understanding of engineering approach and lignin consumption mechanisms. Some strategies, such as genome scale modelling, systemic optimization and post translational enzyme modification, have resulted in significant lignocellulose conversion. Native organisms that consume lignocellulose and fuel overproducers can also be used to create hybrid strains for direct fuel synthesis from lignocellulose feedstock (Choi et al., 2020).