Recent advances in bioethanol production from lignocelluloses: a comprehensive review with a focus on enzyme engineering and designer biocatalysts

Department of Biotechnology, Punjabi University, Patiala-147002, Punjab, India. Faculty of Applied Medical Sciences, Lovely Professional University, Phagwara-144411, Punjab, India. School of Water, Energy and Environment, Cranfield University, Cranfield MK43 0AL, UK. Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut, India. Biochemical Conversion Division, Sardar Swaran Singh National Institute of Bio-Energy, Kapurthala-144601, India. Department of Biotechnology, Engineering School of Lorena (EEL), University of São Paulo, Lorena-SP12606452, Brazil.


Introduction
The ever-increasing demands for energy due to rapid increase of global population, industrialization, and geopolitical factors have called for the search for alternative and carbon neutral sources of energy (Souza et al., 2017;Chandel et al., 2020). For many years, the primary sources of energy have been non-renewable fossil fuels, oil, natural gas, and coal. However, these energy sources are inadequate to fulfil today`s most significant requirements of the societies in particular from the environmental and public health perspectives. More specifically, the widespread application of conventional energy resources has contributed to serious challenges, including global warming and climate change by releasing greenhouse gases (GHGs) like carbon dioxide (CO2), methane (CH4), nitrous oxide (N2O), and chlorofluorocarbons (Kiran et al., 2014). In fact, these adverse impacts have overshadowed the previous justifications used, including burgeoning petroleum prices, finite nature of fossil fuels, and have encourage the government and non-government agencies to find environmentally friendly, renewable, and sustainable energy resources for transportation, heating, and electricity generation (Nikolić et al., 2016).
Among these alternative energy carriers, ethanol has attracted a great deal of attention. It should be noted that ethanol is also widely used in a number of other industries and sectors among which the healthcare sector is largely highlighted due to the current COVID-19 pandemic and the recommendations by the World Health Organization (WHO) on the use of disinfectants containing alcohols like ethanol and isopropanol for adequate inactivation of coronavirus (Kratzel et al., 2020). This has for sure intensified the global demands for this valuable commodity and hence, a larger magnitude of production is required which in turns imposes more pressure on the already limited feedstocks available, i.e., first-generation feedstocks such as sugars and starch.
Given the above, lignocellulosic biomass (LCB) used as economic, widelyavailable non-edible feedstock for second-generation (2G) biofuels are highlighted more than ever (Kuila et  Three major steps are involved in LCB conversion into fuel ethanol, viz., pretreatment, saccharification, fermentation and distillation. Lignocelluloses are composed of complex polysaccharides, which are highly resistant to degradation by chemical and enzymatic methods, due to closed packing within recalcitrant lignin structure (Haldar and Purkait, 2020). Hence, in spite of their high availability and cost-effectiveness, the production of fuel ethanol and other high value-added products with high yield and productivity is a challenge (Kumar et al., 2008). The pretreatment process is performed to remove or redistribute the lignin, to reduce the cellulose crystallinity, and to increase the porosity significantly (McMillan, 1994). Subsequent saccharification or hydrolysis is done by acids or enzymes to hydrolyze the polymeric cellulose and hemicellulose into fermentable monomeric sugars (hexoses and pentoses). Enzymatic hydrolysis is preferred over acid hydrolysis due to lower energy requirements and reduced by-products formation. Nevertheless, enzymatic hydrolysis is influenced by several factors such as accessible surface area, cellulose crystallinity and degree of polymerization, lignin content, and enzyme synergy and effectiveness ( The selection of microorganisms for industrial bioethanol production depends upon their ability to utilize a wide range of substrates, being resistance against various inhibitory products, and tolerance to high sugar and ethanol concentrations . The yield and productivity of ethanol is much less with wild microbial strains, hence, developing genetically-modified microbial strains capable of meeting these requirements at industrial scale has been a primary focus. In light of these, the aim of the present article is to review and critically discuss the advanced approaches used for the pretreatment of LCB, enzymatic saccharification, development of modified microbial strains to improve bioethanol yield, and different action mechanisms for bioethanol production using wild and genetically-modified strains. It also provides a summary of various integration approaches used for fermentative production of bioethanol. The review articles published in last five years in this domain are tabulated in Table 1.

Pretreatment
As mentioned earlier, pretreatment is a necessary step to unwind the complex structure of LCB composing mainly of cellulose, hemicellulose, and lignin (Kassaye et al., 2017). A suitable pretreatment method is key in breaking down/redistribute the recalcitrant lignin structure leading to the accessibility of polysaccharides towards hydrolytic enzymes for their conversion into monosaccharides. In fact, an efficient pretreatment method largely facilitates the hydrolysis process leading to improved yields of monomeric sugars, reduced degradation of carbohydrates, and reduced formation of inhibitory by-products (Procentese et al., 2017). Therefore, finding an effective biomass pretreatment which is at the same time convenient to perform, environment friendly, and economically feasible, is highly critical (Ravindran et al., 2018). A variety of pretreatment methods have been developed for LCB conversion over the past few decades; however, there is no single strategy available so far that could be suitable for all types of feedstocks.

Conventional pretreatment approaches for lignocellulosic biomass
The most commonly used pretreatment technologies for LCB conversion include physical, (thermo)chemical, physicochemical, and biological methods (Behera et al., 2014;Kumar and Sharma, 2017;Baruah et al., 2018;Gabhane et al., 2020;Hans et al., 2020). These are extensively studied methods but are associated with a variety of limitations such as low yield, high processing cost, and negative environmental impacts, and therefore, more efficient green technologies are being explored continuously to overcome these challenges (Capolupo and Faraco, 2016). Figure 1 shows the different pretreatment approaches along with their pros and cons.

Green pretreatment approaches
Recently, the "Green Chemistry" concept has gained attention with a possible solution to the challenges of negative environmental impacts associated with the conventionally used pretreatment methods for LCB, involving the use of hazardous chemicals. Ionic liquids (ILs)-and deep eutectic solvents (DES)-based pretreatments are among the most promising alternative methods owing to their ability to pretreat and selectively dissolve the constituents of biomass in a non-hazardous manner. 18 Review on pretreatment methods and ethanol production from cellulosic water hyacinth Composition of LCB; cell wall composition of water hyacinth (WH); pretreatment methods along with their advantages and disadvantages; sugar production from WH; fermentation and ethanol production considering the latest studies on ethanol production from WH Rezania et al. 19 Harnessing the potential of bio-ethanol production from lignocellulosic biomass in Nigeria-A review Potential LCB feedstocks in Nigeria; bioethanol production from sugarcane bagasse, corn cobs, mango peels, sorghum straw, and rice husks; cellulosic biomass capacity for bioethanol production in Nigeria; pathways to bioethanol production; challenges of LCB conversion to bioethanol; commercialization of biomass to bioethanol processes  23 Second generation bioethanol production: On the use of pulp and paper industry wastes as feedstock LCB composition; production of 2G bioethanol through pretreatment, hydrolysis/ saccharification, fermentation, recovery, and dehydration; bioethanol production from kraft pulp, spent sulfite liquor, and paper and pulp sludge; conversion of paper and pulp mills into biorefineries Branco et al.

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Bioethanol production from renewable raw materials and its separation and purification: A review Biorefinery and bioethanol production; raw materials and their pretreatment for bioethanol production; bioethanol production from raw materials containing sugar, starch, and LCB; bioethanol separation and purification Lignocellulosic biomass for bioethanol: An overview on pretreatment, hydrolysis and fermentation processes Sources and composition of LCB; bioethanol production steps including pretreatment, hydrolysis, and fermentation; integrated processes in bioethanol production Overview on different steps for production of first, second, third, and fourth generation biofuels; details on production of fourth generation biofuels including cultivation and accumulation of carbohydrates, harvesting, recycling of water, and nutrients after cultivation; saccharification and fermentation; distillation, concentration, transportation and use of bioethanol; comparison of bioethanol productivities from plants and microalgae Silva and Bertucco (2019) 35 An overview on bioethanol production from lignocellulosic feedstocks Physico-chemical properties of bioethanol; steps involved in bioethanol production; feedstocks for bioethanol production; fermentation process and mechanism; types of fermentation in bioethanol production (SHF, SSF, BF, FBF, CF, SoSF, NSSF, SSCF, SSFF, CBP); advantages of CBP

Ionic liquid pretreatment
ILs are known as "green solvents" due to their higher thermal and chemical stability, low vapor pressure, and non-flammable nature (Wu et al., 2014;Wahlström and Suurnäkki, 2015). ILs are liquids composed of ions with strong electrostatic bonding, making them less volatile, and electrochemicallystable (Socha et al., 2014). Moreover, ILs` characteristic could be designed by altering the combination of cations and anions. The cations contained in ILs are organic, while the anions could be either inorganic or organic (Brandt et al., 2011). Existing processes of lignocellulosic bioethanol production; conventional fermentation technology to produce bioethanol; conventional separation processes and their limitations; emerging membrane-based processes for lignocellulosic bioethanol production; advanced membrane-based enzymatic saccharification and fermentation for bioethanol production; advanced SSFF strategy for lignocellulosic bioethanol production; advanced membrane separation processes for recovery of bioethanol; status of lignocellulosic bioethanol production at international and national levels; economic aspects of lignocellulosic bioethanol production Although as reported in the studies, ILs pretreatment approaches are environment friendly but the high cost of these strategies may limit their use in large scale biorefineries (Hou et al., 2013).  Table 3.

Deep eutectic solvent pretreatment
Several studies have reported DES as a reasonable reaction media for enzymatic reactions as compared to conventional organic media (Gill and Vulfson, 1994;Erbeldinger et al., 1998). DESs are also promising solvents for dissolving a considerable proportion of the lignin contained in LCB Another report showed similar results for DES-pretreated food wastes (pretreatment conditions: 150°C for 16 h), such as apple residues, potato peels, and brewer's spent grains (Procentese et al., 2018). In this study, ChCl-U pretreatment lowered the energy requirement by about 28% as compared to NaOH pretreatment.
The effect of three different DESs, namely, ChCl-LA, ChCl-U, and choline chloride-glycerol (ChCl-G), during the pretreatment of OPEFB at 120°C for 3 h with the solid-to-liquid ratio of 1:10 (w/v) was studied and compared to acid and alkaline solvents (Thi and Lee, 2019). ChCl-LA (1:2) showed the highest reducing sugars yield (20.7%) and was found more effective than acid and alkaline solvents in preventing sugars loss and exposing the cellulose fraction to enzymatic saccharification.

Enzymatic saccharification of lignocellulosic biomass
Conversion of LCB into pentose/hexose sugars with industrially desired yields is one of the major bottlenecks in the production of 2G biofuels since there are several challenges associated with achieving high process efficacies. Acid hydrolysis is the most convenient and widely employed method for hydrolysis of polysaccharides into monomers via breakdown of hydrogen bonds between cellulose chains and converting its crystalline form into entirely amorphous state. However, its corrosive nature, degradation of released sugars, difficulties in sugar and acid recovery from the mixture, high energy requirements, formation of fermentation inhibitors, and several environment-related issues increase the process cost, and hence, limit its use

Carbohydrate esterases (CEs)
Carbohydrate esterases formulate a distinct class of hydrolytic enzymes that are involved in the removal of ester flags from carbohydrates (Cantarel et al., 2009;Nakamura et al., 2017). These enzymes have been classified into 16 CE families ranging from CE1 to CE16; however, CE family 10 has been abolished since most of the members corresponding to this family were found to be active against non-carbohydrate substrates (Nakamura et al., 2017). There are diverse biotechnological applications assigned to CE proteins where the majority of the enzymes catalyze the elimination of ester-based alterations form mono, oligo, and polysaccharides. Therefore

Auxiliary activity (AA) proteins
The conventional hydrolytic model for degradation of lignocelluloses has been updated with the discovery of a novel class of oxidative enzymes. These enzymes are capable of triggering the cleavage of glycosidic bonds within the glucose polymers through oxidative route and are referred to as AA proteins (Ezeilo et  . Therefore, these enzymes can be used for boosting the efficiency of lignocellulolytic GHs.

Strain improvement strategies for hyper-producing deregulated lignocellulolytic strains
The cost of lignocellulolytic enzymes is one of the limiting factors in biorefineries; therefore, continuous efforts are being made to (i) minimize the cost of enzymes and (ii) increase overall yields of the enzymes with desired productivity. For making the concept of biorefineries economically feasible, several approaches such as random mutagenesis, site-directed mutagenesis, heterologous expression of proteins, clustered regularly interspaced short palindromic repeats (CRISPR-Cas) system, and genome and metabolic engineering have been used in recent years to improve the enzymatic expression by microbial strains (Fujii et  Site-directed mutagenesis is also a highly accepted technique that induces specific alterations in the known DNA sequences. Error-prone PCR (EP-PCR) applied in tandem with site-directed mutagenesis has been reported to increase EG activity (by up to 7.93 folds) in Bacillus amyloliquefaciens DL-3, and enhance alkaline tolerance of EGIII in T. reesei (Wang et al., 2005;Vu and Kim, 2012). A study has suggested that the product of bgl2 gene catalyzes conversion of cellobiose into glucose, which in turn inhibits the expression of cellulases through feedback inhibition of cre1 gene. Also, cellobiose acts as an inducer for transcription factor clrB, which activates the expression of cellulases. Therefore Genetic engineering is also regarded as a powerful tool for putting together multiple traits of interest in a single organism. Therefore, this technique could be very useful in (i) increasing enzyme titer, (ii) reducing the production cost of enzymes, and (iii) developing process-specific enzymes ( The three cellulases PaCel6A, PaCelB, and PaCel6C (Podospora anserine) functionally expressed in Pichia pastoris were reported to hydrolyze amorphous and crystalline celluloses but were found to be inactive against hydroxyethyl cellulose, mannan, galactomannan, xyloglucan, arabinoxylan, arabinan, xylan, and pectin (Poidevin et al., 2013). A study has reported the expression and production of CBH II from T. reesei into P. pastoris, and its application has also been proved in the hydrolysis of corn stover and rice straw (Fang and Xia, 2015). The cloning and expression of GH11 xylanase gene from A. fumigatus MKU1 has been reported where two exons of the gene were amplified separately and fused using overlap extension PCR. The fused product was then cloned in pPICZB, and expressed in P. pastoris under the control of AOX1 promoter  Gong et al. (2013) reported cloning of aufaeA, a gene encoding for type-A feruloyl esterase, in A. usamii E001. The gene was expressed in a heterologous host P. pastoris GS115. One of the transformants, P. pastoris GSFaeA4-8 showed high expression of the recombinant auFae A with an enzyme activity of 10.76 U/ml. A gene encoding CDH was cloned from Neurospora crassa strain FGSC 2489 and successfully expressed in a heterologous host P. pastoris under the control of AOX1 methanol inducible promoter (Zhang et al., 2011). A novel laccase gene pclac2 was cloned from Phytophthora capsici using pPIC9K expression vector and expressed in P. pastoris host system (Feng and Li, 2014). A thermo-alkali stable laccase was cloned from B. licheniformis and expressed in P. pastoris. The expressed protein showed remarkable stability at 70˚C with a half-life (t1/2) of 6.9 h (Lu  et al., 2013). The cloning of a novel LPMO (PMO9A_MALCI) from thermophilic fungus Malbranchea cinnamomea and its expression in P. pastoris has been recently reported where the expressed AA9 protein was capable of hydrolysis of both cellulose and pure xylan (Basotra et al., 2019).
In case of bacteria, more reports concern introduction of cellulase and hemicellulase genes into strains of Escherichia coli. There is a report on the cloning of first functional gene from Paecilomyces thermophila where a 681bp xylanase gene (Pt xynA) was expressed in E. coli BL21, and the recombinant protein was purified using nickel-nitrilotriacetic acid (Ni-NTA) and Sephadex G50 columns. The characterization of the recombinant xylanase indicated that the enzyme is thermostable and has a great potential in various industries

Fermentation
Fermentation is a critical step for the production of industrially important fuels and chemicals where monomeric sugars released by hydrolysis of feedstock are converted into these products by the microbial action. The wildtypes of microorganisms tested in the ethanol fermentation are Calonectria brassicae, Candida (Scheffersomyces) shehatae, E. coli, S. cerevisiae, Mucorindicus, Pachysolen tannophilus, Pichia (Scheffersomyces) stipitis, and Zymomonas mobilis (Sanchez and Cardona, 2008). There are many factors affecting fermentation process for bioethanol production such as temperature, pH, aeration rate, salt concentration, carbohydrate concentration, and ethanol concentration (Arora et al., 2017; Selim et al., 2018; Ding et al., 2020). There are three major modes of fermentation for ethanol production: batch, fed-batch, and continuous fermentation, and each process has its own advantages and limitations. Batch fermentation is the most traditional type of fermentation where high concentration of initial substrate is converted into high concentration of product, and a fresh batch is run after the end of each batch (Olsson and Hahn-Hägerdal, 1996). A modification of batch fermentation is repeated batch fermentation in which immobilized microbial cells are used instead of free ones to make the system more efficient (Jain and Chaurasia, 2014). Fed-batch method is another type of fermentation, which is a combination of batch and continuous mode, with intermittent additions of fresh substrates without removing products. This type of fermentation is more economical compared to batch type method due to shorter fermentation time, higher ethanol productivity, higher dissolved oxygen in media, and less toxicity of media components (Cheng et al., 2009). There is a constant addition of substrate and nutrients with continuous removal of products from bioreactor in the continuous type of fermentation. Continuous fermentation is the most common type of fermentation, which has been used for industrial bioethanol production due to easy process control, elimination of unproductive time required for cleaning, less investment cost, and less labor-intensive process (Sanchez and Cardona, 2008;Kumar et al., 2015).
Techno-economic analysis of a pilot-scale production of bioethanol with high yields using Z. mobilis, revealed that ethanol production using this bacterium could save the cellulosic ethanol production facility by $2 million/yr (Kremer et al., 2015). Another study on pilot-scale production of bioethanol from dilute sulphuric acid-pretreated wheat straw by using recombinant E. coli FBR5 in a simultaneous saccharification and fermentation (SSF) system achieved an improved ethanol yield (0.29 g/g), and productivity (0.43 g/L/h) (Saha et al., 2015). The consolidated alcohol dehydration and oligomerization (CADO) approach, a one-step conversion process, is estimated to reduce the operating plus annual capital costs from $2.00/GJ to $1.44/GJ, i.e., 28% reduction in the conversion of wet ethanol to fungible blend-stocks. This approach has enhanced the liquid hydrocarbon yield (36% of theoretical), decreased ethanol conversion cost (12-fold), and scaled up the process by 300-fold (Hannon et al., 2020). A recent study proved an enhanced bioethanol production of 20.6 g/L with volumetric productivity of 1.0 g/L/h from food waste in a SSF system using the mixed culture of F. oxysporum F3 and S. cerevisiae. The supplementation of glucoamylase into the mixed culture resulted in further enhancement of ethanol production and productivity by 30.3 g/L and 1.4 g/L/h, respectively, and hence, proved the feasibility of on-site production of multienzyme system and bioethanol production from food waste (Prasoulas et al., 2020). Similarly, a pilot-scale continuous tubular reactor (PCTR) technology is expected to achieve a high ethanol yield of 11.0 to 11.3 kg of ethanol per 100 Kg of untreated biomass by overcoming the challenges related to biomass recalcitrance (Pérez-Pimienta et al., 2020).
To achieve maximum yield and productivity in bioethanol production, the selected microbial strain should have some unique features such as a broad range of substrate utilization, ability to withstand high concentrations of sugar, ethanol, and by-products produced during pretreatment step, and minimum by-products formation (Lugani and Sooch, 2018). However, most of the naturally-occurring microbial strains employed for the alcoholic fermentation possess the ability to ferment hexose sugars only with very low ethanol yields and productivities. The wild pentose sugars fermenting microbial strains such as P. stipitis, P. tannophilus, and C. shehatae are sensitive to low pH, high concentration of ethanol, and inhibitors . Therefore, it is very difficult for wild microbial strains to fulfil the features, which are required for their selection as industrially important, and hence, over the last few years, the focus has been placed on the development of genetically modified microorganisms to ensure their use in industrial applications.

Strain development for improved bioethanol fermentation
There are various previous studies, which have been done using adaptive evolution to create mutant strains, which are resistant to high temperatures, salt concentrations, acetic acid concentrations, freeze-thawing, pentose sugars, and various stress inducers (Wati et  The ethanol-resistant strains are produced by global transcription machinery engineering (gTME), which is a powerful tool for selection of mutant library (Yang et al., 2011). Many in-silico tools like dynamic flux balance model and dynamic simulations are used for analysis of bioethanol production by genetically modified microorganisms in co-culture fermentation (Parambil and Sarkar, 2015). The main focus of developing genetically modified microbial strains is on accelerating the rate of reaction, shifting the existing metabolic pathway towards production of useful products, enhancing substrate specificity, and altering enzyme activity for producing novel structures (Doğan et al., 2014). Many previous studies have already been reported on the production of enhanced bioethanol using recombinant microbial strains  (Dien et al., 2003). Previously, it has been observed that S. cerevisiae mutant with disrupted ura7 or gal6 showed increased resistance to different kinds of stressors including ethanol. The mutant yeast strain also showed enhanced glucose consumption at low temperatures compared to wild strains (Yazawa et al., 2007). Both ethanol tolerance and fermentation capacity of sake yeast strains were enhanced by overexpression of msn2 (Watanabe et al., 2009). The enhanced effective and rapid ethanol production (with 90% of maximum theoretical yield) was achieved with Geobacillus thermoglucosidasius by up-regulating the expression of pyruvate dehydrogenase, and disruption of pyruvate formate lyase and lactate dehydrogenase genes (Cripps et al., 2009). The overexpression of sugar transporter (Hxt) in Fusarium oxysporum resulted in enhanced glucose and xylose transport capacity with 39% increase in ethanol yield (Ali et al., 2013).
Cell surface engineering is an innovative tool in molecular breeding for displaying functional proteins on the surface of microorganisms used in consolidated bioprocessing (CBP) system. Cell display system is very useful for ethanol production from starch in CBP because various amylases are displayed on the yeast cell surface, which can utilize starch as the sole carbon source for ethanol production (Sakuragi et al., 2011). The recombinant thermophilic strain of Kluyveromyces marxianus has been developed using cell surface engineering, and the recombinant strain displayed both β-glucosidase and endoglucanase on cell surface. The recombinant strain was used for bioethanol production in a CBP system using β-glucan as substrate, and the improved bioethanol production of 0.47 g/g of carbohydrate consumed, was observed at the end of fermentation (Hasunuma and Kondo, 2012). E. coli is considered as one of the important industrial bacteria, and is commonly used in most of the recombinant studies. Xylose metabolic pathway was introduced into Z. mobilis from E. coli for producing recombinant strain having GRAS (generally recognized as safe) status. The recombinant strain showed minimum nutrient requirements, and could tolerate high temperatures and low pH values (McEwen and Atsumi, 2012). Similarly, constitutive promoter substitution and xylose metabolic integration was done in S. cerevisiae for producing an engineered strain EBY101-X5CC, and the engineered yeast strain had the ability for co-fermentation of cellulose and either sucrose or xylose. The recombinant strain produced 4.3 g/L ethanol from 10 g/L carboxymethyl cellulose (CMC) in a CBP system (Li et al., 2017). The engineering of both feedstock as well as microorganism has reportedly resulted in an enhanced bioethanol production. This strategy also provided the feasibility of ethanol production at commercial scale using lignocellulosic waste materials (Ko et al., 2018).
Safe and stable expression of cellulase gene (sestc) and glyceraldehyde-3phosphate dehydrogenase gene (gpd) promoter was achieved using the CRISPR -Cas9 approach in S. cerevisiae chromosome using gRNA expression vector from Agaricus biporus. The recombinant yeast strain showed increased expression of endo-1,4-β-glucanase and exo-1,4-β-glucanase, and 37.7-fold improved ethanol production compared to its native strain . In a recent study, industrially engineered S. cerevisiae MF01-PHO4 was produced by protoplast formation and pho4 gene replacement, and the mutant strain was observed to be stable for up to 30 generations. An enhanced ethanol yield of 114.71 g/L was achieved with the genetically engineered strain, accounting for 5.30% increase in ethanol yield and 12.5% decrease in fermentation time (Wu et al., 2020). There is no clear evidence on side effects of genetically modified microorganisms on environment; thus, there is still a need to take preventive measures to ensure environmental safety. Federal government renewable fuel standards (FGRFS) should be adapted before introducing genetically modified microorganisms into large scale bioethanol production.

Strain development for co-fermentation of glucose and xylose
The conversion of LCB into ethanol is associated with challenges such as co-utilization of pentose and hexose sugars, and presence of fermentative inhibitory compounds such as phenolic derivatives, acetic acid, and furfurans. However, only a few strains such as S. shehatae, S. stipitis, and K. marxianus have the ability to assimilate pentose sugars but the production of ethanol is not up to the industrial standards, and hence the cofermentation of pentose and hexose sugars is a major obstacle in efficient conversion of LCB to ethanol (Kim et  For instance, among the organisms capable of converting sugars into ethanol, S. cerevisiae is the most widely used at the industrial scale due to its versatile characteristics such as high tolerance to ethanol, ability to withstand low pH values, ability to ferment under anaerobic condition, tolerance to high osmotic pressures, and less prone to bacteriophage infections (Robak and Balcerek, 2018). However, the yeast S. cerevisiae exhibits weak expression of pentose pathway gene, and have poor/no xylose uptake ability.
Over the last few years, the considerable developments in genetic engineering has changed the metabolic engineering paradigm. Specialized tool boxes are currently available for pathway manipulation of microbial strains by overexpression and knock-out of genes targeting metabolic pathways, molecular transport capability, cellular tolerance, and catabolite sensing (Selim et al., 2018).

Xylose metabolism
The pentose sugar xylose is metabolized by microorganisms during xylose metabolism through two different pathways. In filamentous yeasts, the oxidoreductase pathway having two-step reaction is involved. In the initial step, xylose is reduced to xylitol through NAD(P)H-and/or NADHdependent xylose reductase (XR) (EC 1.1.1.30) encoded by xyl1, xyl1p, and in the second step, xylitol is oxidized to xylulose by NAD + -dependent xylitol dehydrogenase (XDH) (EC 1.1.1.9) encoded by xyl2, xyl2p. In case of bacteria, xylose is directly converted into 5-xylulose using xylose isomerase (XI) (EC 5.3.1.5) encoded by xylA without any co-factor usage. The 5-xylulose is phosphorylated by xylulokinase (XK) (EC 2.7.1.17) to xylulose-5-phosphate, which is an intermediate for the phosphoketolase and non-oxidative pentose pathways. The pentose pathway can be further classified into two distinct pathways, namely non-oxidative and oxidative pentose phosphate pathways. Most of the yeasts use the non-oxidative pathway to metabolize xylulose-5-phosphate as a precursor for nucleic acid and amino acids production and also convert it to three, four, five, six, and seven atom carbon sources, which serves as intermediate to glycolysis. While the oxidative pathway is used as a defensive mechanism against oxidative stress and to generate NADPH, which is a major precursor for biomass formation, and is also a driving element of XR Kwak and Jin, 2017).

Utilization of xylose by engineering oxidoreductase pathway
Even though S. cerevisiae encodes for putative pentose pathway genes, the expression level of these genes is weak, and as a result, the microorganism is unable to assimilate xylose as sole carbon source. Hence, there is a requirement of heterologous complementation and significant metabolic engineering. Among the pentose-utilizing strains, S. stipitis is the most studied organism as its xylose pathway of converting pentose sugars to ethanol is well curated (Harner et al., 2015). Most of the heterologous expression genes related to the pentose pathway were used from S. stipitis as compared to other eukaryotic organisms. Kötter et al. (1990) isolated xyl1 and xyl2 genes encoding XR and XDH from S. stipitis genomic DNA, respectively, cloned them in S. cerevisiae for the first time, and showed oxidative utilization of xylose. Later, more work on expressing xyl1 and xyl2 genes from S. stipitis in S. cerevisiae was carried out (Ho et  overexpressed with XI gene Li et al., 2016). The XR-XDH is an oxidoreductases-based enzyme, and requires a balance for complete assimilation of xylose. Under anaerobic condition, the NADH cannot be reoxidized to NAD + using oxygen as terminal electron acceptor; hence, there is an excess accumulation of NADPH and reduced NAD + availability. Further the XR in P. stipitis has a higher affinity for NADPH (Km= 3.2 µmol/L) as compared to NADH (Km=40 µmol/L) and XDH completely relies on NAD + , which causes severe disparity in the redox balance, leading to excess xylitol and reduced ethanol production (Jeffries and Jin, 2004).
To overcome the xylitol accumulation due to the redox imbalance, several strategies were employed including addition of external electron acceptor such as acetoin, acetaldehyde, furfural, and 5-hydroxymethylfurfural (HMF). Addition of acetoin and furfural showed reduced xylitol accumulation and increased ethanol production from 0.62 mol ethanol/mol xylose to 1.35 mol ethanol/mol xylose by decreasing the flux by oxidative pentose pathway as the reduction of acetoin and furfural required NADH (Wahlbom and Hahn-Hägerdal, 2002). Almeida et al. (2009) reported that the overexpression of the gene encoding furaldehyde reductase as a co-factor used in HMF reduction, significantly influenced ethanol production. They reported that the NADHdependent reductase exhibited carbon conservation by reducing glycerol formation and enhancing NAD + availability for XDH, which eased xylose uptake and reduced xylitol accumulation.
In S. cerevisiae, ammonium assimilation involves glutamate dehydrogenase. Basically, glutamate dehydrogenase catalyzes the synthesis of glutamate from ammonium and 2-ketoglutarate. Two glutamate dehydrogenases namely NADPH-dependent glutamate dehydrogenase and NADH dependent glutamate dehydrogenase are responsible for ammonium assimilation in S. cerevisiae. Cofactor imbalance in the recombinant S. cerevisiae can be reduced by modifying ammonium assimilation through the deletion of gdh1 encoding NADPHdependent glutamate dehydrogenase (EC 1.4.1.4) and the overexpression of gdh2 encoding NADH-dependent glutamate dehydrogenase (EC 1.4.1.2). This strategy was reported to improve ethanol yield from 0.43 to 0.51 cmol/cmol while reducing xylitol accumulation by 44% (Roca et al., 2003). The increase in ethanol production and reduced xylitol production was due to the increase in the NAD + availability for XDH, which directed xylose towards product and biomass formation (Roca et al., 2003). The re-oxidation of NADH can be achieved by channelizing the carbon flux through recombinant phosphoketolase pathway. Overexpression of phosphotransacetylase and acetaldehyde dehydrogenase in combination with the native phosphoketolase in xylose-fermenting S. cerevisiae strain TMB3001c showed reduced glycerol and xylitol accumulation, while ethanol yield was increased by 25% (Sonderegger et al., 2004).
The accumulation of xylitol was reduced when glucose was used as a cosubstrate; however, this costs in reduced xylose assimilation due to the competition among the sugar transporters (Hallborn et al., 1994). A recombinant S. cerevisiae strain with an XR to XDH ratio of 0.06 showed no xylitol and acetic acid formation, and depicted a good ethanol yield as compared to the strain with a higher XR:XDH ratio (Walfridsson et al., 1997). Multicopy integration of xyl2 gene encoding XDH in the recombinant S. cerevisiae strain elevated xylulose accumulation and reduced xylitol formation reflecting that the activity of XK inhibits the assimilation and utilization of xylose on such cells (Jeffries and Jin, 2004). Further inefficient XK activity will tend to accumulate excess xylulose, and reduce intracellular levels of ATP and the ATP/ADP ratio with the subsequent overexpression of XR, XDH, and XK substantially enhancing the production of ethanol in S. cerevisiae (Richard et al., 2000). The engineered S. cerevisiae strain consisting of XR isozyme for wild type, and mutant showed an ethanol yield of 0.47 g/g emphasizing on the role of XR in increasing ethanol yield (Jo et al., 2017).

Utilization of xylose by engineering isomerase pathway
The XI pathway is evident in most of the bacterial species as compared to yeast and in contrast to oxidoreductase pathway, it does not require co-factor and convert xylose directly to xylulose. Most of the heterologous expression of XI in S. cerevisiae strain shows lower functionality; probably due to suboptimal internal pH, absence of specific metal ion, post-translational modification and protein misfolding. Heterologous expression of XI from Piromyces sp. E2 (pirXI), an anaerobic fungus, increased the flux of xylose towards ethanol production; however, misfolding of the protein was evident which restrained the enzyme activity (Lee et al., 2017). Co-expression of cytoplasmic chaperonin complex Gro EL-Gro ES complex from E. coli in the recombinant S. cerevisiae cloned with XI from the bacterium Propionibacterium acidipropionici displayed proper folding of XI and efficiently converted xylose to ethanol with a yield of 0.44 g ethanol/g xylose (Temer et al., 2017). Walfridsson et al. (1996) expressed xylA gene encoding XI from Thermus thermophiles, which showed a high specific activity (1.0 U/ mg of protein) at 80°C but had a poor performance (with a specific activity of 0.04 U/ mg of protein) at 30°C, and accumulated xylitol and acetate as byproduct. On the similar lines, Lönn et al. (2003) performed a study by overexpressing multicopy xylA gene from T. thermophiles and found xylitol formation by activity of non-specific endogenous aldose reductase (GRE3) which reduced the activity of non-oxidative pentose pathway and XK. The deletion of gre3 and over-expression of an extra copy of XK in the recombinant strain improved ethanol productivity and reduced xylitol production. The bottleneck related to xylitol accumulation and increasing xylose flux was addressed by over-expressing enzymes such as xylulokinase ( (Kuyper et al., 2005).
The affinity of XI from Ruminococcus flavefaciens towards xylose could be improved by adapting modifications to the 5'-end of the gene, sitedirected mutagenesis, and codon optimization. The modified enzyme showed 4.8-fold higher activity as compared to the native enzyme with a Km= 66.7 mM and specific activity of 1.41 µmol/min/mg. The recombinant S. cerevisiae harboring the modified enzyme along with cellobiose phosphorylase, cellobiose transporters, the endogenous genes gal2 (encoding transporter gene) and xk and disruption of the native pho13 (encoding p-nitrophenylphosphatase) and gre3 genes resulted in four-fold higher xylose consumption even in the presence of lignocellulosic inhibitors and showed higher ethanol concentration (Aeling et al., 2012). Likewise, the xylose consumption could be increased by overexpressing the heterologous sugar transporter (PsSUT1) and xk in the engineered strain containing xylA from the fungus Orpinomyces, showing an ethanol yield of 0.48 g/g and low xylitol yield of 0.04 g/g when grown in a complex medium supplemented with 0.01M borate (Madhavan, et al., 2009). Brat et al. (2009) compared the performance of XI isolated from anaerobic bacterium Clostridium phytofermentans and Piromyces sp., and reported low inhibition of xylitol in the strain cloned with C. phytofermentans XI with an ethanol yield of 0.43 g/g and xylitol production of 0.18 g/g. Cloning and expression of XI gene (xylA) of Burkholderia cenocepacia in S. cerevisiae showed better co-consumption of glucose and xylose under anaerobic condition and also resulted in a higher ethanol yield of 0.45 g/g without xylitol accumulation (Peng et al., 2015). Ota et al. (2013) showed that the cell surface display of xylA from C. cellulovorans with the over-expression of xk resulted in 0.5 g/g ethanol yield under anaerobic condition.
To overcome the challenges related to redox imbalance, some studies have been focused on altering NADH/NADPH ratio for efficient performance of the XR and XDH. Since NADPH co-factor is majorly generated through the oxidative pentose phosphate pathway, the deletion of zwf1 (glucose-6-phosphate dehydrogenase, EC 1.1.1.49) and gnd1 (6phosphogluconate dehydrogenase, EC 1.1.1.44) genes reduced the xylitol production with a low XR/XDH ratio. However, the mutant having Δzwf1 and Δgnd1 also showed reduced growth rate due to a significant drop in NADPH levels. To subsidize the negative effect caused by deletion of zwf1, over-expression of gpd 1 encoding NADP + -dependent glyceraldehyde-3phosphate dehydrogenase (Kluyveromyces lactis GPD 1, EC 1.2.1.13) was performed which resulted in 52% alleviation in ethanol yield and 48% lower xylitol accumulation (Verho et al., 2003). The overexpression of water-forming NADH oxidase (EC 1.6.99.3) gene noxE from Lactococcus lactis in recombinant S. cerevisiae led to a significant decrease in glycerol and xylitol production, and hence, increased final ethanol production during xylose metabolism. The ethanol yields of 0.294 g/g and 0.211 g/g, respectively, were observed with recombinant and control strains of S. cerevisiae, which clearly revealed the effect of co-factor imbalance on the production of by-products, i.e., ethanol and xylitol (Zhang et Table 4.

Engineering of transporters for xylose uptake
Yeast shows an efficient transport system for the endogenous metabolism of hexose sugars but a limited exogenous xylose metabolism and a low affinity for xylose as it is dependent on the hexose transport system. Considerable efforts have been made to engineer the xylose transporters to improve the simultaneous uptake of hexose and pentose sugars (Sharma et al., 2018a). The strength of the xylose transporter can be improved by targeting and engineering the existing sugar transporters or searching for novel heterologous xylose affinity/glucose repressor-based transporters (Kwak and Jin, 2017). S. cerevisiae has 18 hexose transporters among which Hxt 1-17 and Gal2 are responsible for glucose permeation across the cell membrane, while Hxt 1-7 acts as glucose facilitator. Several hexose transporters such as Hxt1, Hxt2, Hxt4, Hxt5, Hxt7, and Gal2 facilitate xylose uptake in S. cerevisiae; however, these transporters have low affinities towards xylose in the presence of glucose and are inefficient in xylose transportation at lower concentrations (Hamacher et al., 2002;Saloheimo et al., 2007). Among the hexose transporters, Hxt7 and Gal2 show higher affinities for xylose, but in the presence of glucose, these transporters are repressed and xylose uptake rate is reduced.
To enhance the uptake of xylose in S. cerevisiae, Leandro and co-workers expressed C. intermedia PYCC 4715 transporter proteins (glucose/xylose symporter -Gxs1 and glucose/xylose facilitator -Gxf1) in S. cerevisiae. The recombinant strain exhibited a higher growth rate in a xylose-containing medium with Km=0.2 mM, but in the presence of glucose in the medium, the affinity towards xylose was significantly reduced. These results concluded that the activity of transporter proteins Gxs1 and Gxf1 is directly proportional to the glucose concentration (Leandro et al., 2006). Young et al. (2011) expressed C. intermedia Gxs1 and Gxf1 along with S. stipitis Xut1 and Xut2 in a hexose null mutant which barely showed any improvements on a xylose-containing medium. When Gxf1 was expressed in S. cerevisiae, the recombinant strain showed a higher xylose uptake at lower concentrations of xylose, but it was unchanged even at higher concentrations of xylose in aerobic condition. The strain also exhibited a higher ethanol production and xylose uptake under anaerobic condition (Runquist et al., 2009). Young et al. (2011) created a mutant of transporter protein C. intermedia Gxs1 and S. stipitis Xut1 through the directed evolution method and expressed these mutant genes in a hexosenull S. cerevisiae mutant. The recombinant strain showed a substantial growth and uptake of xylose in a glucose/xylose medium. Further improvement in Vmax and Km was observed by point mutating amino acid, Phe40 in Gxs1 and Glu538 in Xut1. Similarly, the single nucleotide polymorphism was created by point mutating Phe79Ser in HXT7, which showed a co-utilization of glucose and xylose sugars with a higher xylose uptake ability with Vmax=186.4 nmol/mL/min as compared to the wild type with Vmax=101.6 nmol/mL/min (Apel et al., 2016). Based on the sequence similarity of Gxs1 with other xylose transporters, a conserved motif sequence G-G/F-XXX-G has been identified and successive mutation in the amino acids Phe38, Ile39, and Met40 showed a two-fold improvement in the xylose uptake rate (Young et al., 2014).
The presence of three sugar transporters Sut1, Sut2, and Sut3 in P. stipitis leads to a higher affinity towards glucose than xylose; however, Sut1 has a higher Vmax for xylose as compared to the other transporters. Overexpression of P. stipitis Sut1 in a recombinant S. cerevisiae harboring XR-XDH-XK genes showed a higher uptake of xylose in glucose/xylose fermentation with an ethanol yield of 0.44 g/g sugar (Katahira et al., 2008). Goncalves et al. (2014) over-expressed Hxt1, Hxt2, Hxt5, and Hxt7 permeases in a hexose-null mutant strain (hxt1Δ-hxt7Δ and gal2Δ) harboring xyl1, xyl2, and xk genes. The results revealed that Hxt7 had a higher xylose consumption ability compared to the other transporters; however, the substrate affinity was 200 folds higher for glucose as compared to xylose in the medium containing glucose/xylose mixture making xylose the second choice even in the presence of low concentrations of glucose. While Hxt1 showed higher sugar uptake and ethanol productivity in co-fermentation of glucose and xylose but severely repressed xylose uptake in the presence of glucose showing diauxic growth profile. To overcome the barrier related to transporter repression, Farwick et al. (2014) conducted homology modelling for xylose transporters to transport D-xylose without any inhibition by D-glucose. This study showed that glucose-insensitive xylose transporters could be obtained by mutations in Gal2 and Hxt7 transporters, and hence it contributed to the understanding of sugar-transport mechanisms. More specifically, single point mutation in N376-F region of Gal2 and N370-S region of Hxt7 led to higher affinity towards xylose and loss of ability to transport hexose sugars. Nijland et al. (2017) adapted an evolutionary engineering strategy to develop a chimeric HXT36 by the fusion of functional hexose transporter Hxt3-Hxt6. An amino acid substitution at N367A of Hxt36 enabled the coconsumption of glucose and xylose. The genome sequence analysis showed that co-repressors such as CYC8 and SSN6 were responsible for phenotypic characteristics of the non-evolved strain. Inactivation of CYC8 showed a higher activity of Hxt, which in turn increased the xylose transport and led to less sensitivity to D-glucose repression (Nijland et al., 2017). Wei et al. (2018) found 11 transcriptional factors in glycolysis and pentose pathway of yeast that varied with the concentration of xylose and glucose/xylose in the medium. Knockout of THI2 promoted ribosome synthesis, enhanced xylose uptake rate and ethanol production by 26.8% and 32.4%, respectively. Also, the over-expression of cell cycle related transcriptional factor Nrm1 further improved the xylose utilization rate by 30% and ethanol production by 76.6% in a glucose and xylose containing medium. An overview of metabolic engineering in yeast for simultaneous uptake of glucose and xylose is depicted in Figure 4.

Elimination of by-products for efficient production of ethanol
In microbial fermentation, the production of by-products is inevitable, which in turn diverts the carbon flux from the main product, thereby reducing the desired product titers (Arora et al., 2019). In order to overcome this bottleneck, appropriate rewiring of metabolic pathway is indispensable. In S. cerevisiae, glycerol is one of the major by-products, which accounts for 2-3% of sugar bioconversion. Even though glycerol is one of the platform chemicals, its separation during ethanol fermentation is not economically viable (Prior and Hohmann, 1997). In S. cerevisiae, glycerol formation is a twostep process. In the initial step, the NADH-dependent glycerol-3-phosphate dehydrogenases (GPD) catalyze the conversion of dihydroxyacetone phosphate to glycerol-3-phosphate followed by dephosphorylation of glycerol-3-phosphate to glycerol (Gancedo et al., 1968;Påhlman et al., 2001). Glycerol is usually accumulated in the cell during osmotic stress condition and acts as osmolytes (Luyten, 1995). Jain et al. (2011) eliminated the gdp1 (osmotically induced) and gdp2 (anaerobically induced) but the growth of the strain was hindered under anaerobic conditions due to the excess accumulation of NADH. The redox imbalance was mitigated by introduction of oxido-reductase gene (which converts NADH to NAD + by production of sorbitol and propane-1,2diol) and ethanol yield was maintained at 0.48 g/g glucose. On the similar lines, Papapetridis et al. (2017), deleted gdp2 and aldehyde dehydrogenase (ALD6) genes and replaced it with native gdp of an archaeal NADP + -preferring enzyme in an acetate reducing S. cerevisiae strain. The mutant strain was able to grow under anaerobic conditions with a high osmolarity and through the consumption of acetic acid without producing glycerol. Acetic acid is another product usually observed in S. cerevisiae fermentation. It is also one of the major inhibitors present in lignocellulosic hydrolysate. Wei et al. (2013) proposed co-utilization of xylose and acetic acid for the production of ethanol by combining the NADH-producing xylose utilization pathway and NADH-consuming acetate reduction pathway. For this, they deleted gdp1 and gdp2 in order to reduce glycerol formation and introduced XR-XDH from P. stipitis, and adhE and mphF (proteins that are part of a bifunctional aldolase-dehydrogenase complex involved in 4-hydroxy-2-ketovalerate catabolism) from E. coli. The adhE and mphF genes aided in reduction of acetate to ethanol by generating 2 NAD + molecules. In xylose assimilation pathway, 1 mole of NADH is generated by the oxidation of xylitol to D-xylulose. The co-factor is exchanged between these two pathways showing improved ethanol production.

Integration approaches
The most extensively used method of ethanol production using LCB is separate hydrolysis and fermentation (SHF), which involves two consecutive steps of enzymatic hydrolysis and fermentation in separate reactors. In this process, each process is optimized separately to achieve better enzymatic hydrolysis and microbial fermentation. However, some of the major limitations associated with this method are high production cost, less product yield, and high chances of contamination.
The fermentation of hexoses and pentoses are performed in different reactors during SHF, which further increases the processing time (Chandel et al., 2007;Offei et al., 2018;Tandon and Sharma, 2019). Based on the limitations associated with SHF, various integration processes such as simultaneous saccharification and fermentation/co-fermentation (SSF/SSCF) and CBP approaches have been adapted for commercial production of bioethanol (Fig.  5) (Arora et al., 2015a). CBP encompasses two strategies, i.e., engineering of wild microbial strains to improve product-related properties (titre and yield), and expression of heterologous cellulase system for cellulose utilization by high product-yielding non-cellulolytic microbes (Lynd et al., 2005).

Simultaneous saccharification and fermentation/co-fermentation (SSF/SSCF)
In SSF and SSCF processes, both enzymatic hydrolysis and fermentation processes are carried out simultaneously in the same reactor to maintain a low concentration of glucose as the accumulation of glucose inhibits the enzymatic activity. The fermentation of hexoses is performed by hexose-fermenting microorganisms in SSF whereas, both pentoses and hexoses are fermented in the same reactor in SSCF. In this process, the saccharification of cellulose and fermentation/co-fermentation of pentose and hexose sugars do not occur simultaneously, but in a sequential manner .
These integrated approaches are less susceptible to contamination due to immediate conversion of sugars into ethanol in the same reaction vessel, which also leads to higher ethanol yields due to avoidance of feedback inhibition to enzyme. Moreover, these approaches offer easy process design and short reaction time, and are easy to operate with reduced process cost. However, there are also some limitations associated with these processes. One of the major challenges is the optimization of reaction conditions to make the system more efficient because separate optimal conditions are required for enzymatic hydrolysis and microbial fermentation.
SSF and SSCF processes are operated normally at 30-35 o C to accommodate both microbial growth and ethanol fermentation (Canilha et al., 2012;Nikolić et al., 2016;Azhar et al., 2017). Moreover, the fermentation media used for bioethanol production is very viscous in nature due to presence of lignin content of LCB, and it is very difficult to separate lignin from the cellulosic part before fermentation. This results in difficulty in heat and mass transfer, and homogenous mixing of culture and media components. Hence, the energy consumption is high for distillation of fermentation broth and treatment of distillate .
The promising microorganisms for bioethanol fermentation in SSF system are S. cerevisiae and Z. mobilis (Nigam and Singh, 1995). The other yeast strains, which have been reported for bioethanol production in SSF system are K. marxianus, K. fragilis, P. pastoris, and Hansenula polymorpha (Mejía-Barajas et al., 2016). It has been found that microwaveassisted liquefaction (80 W for 5 min) of cornmeal (cornmeal to water ratio Please cite this article as: Lugani Y., Rai R., Prabhu A.A., Maan P., Hans M., Kumar V., Kumar S., Chandel A.K., Sengar R.S. Recent advances in bioethanol production from lignocelluloses: a comprehensive review with a focus on enzyme engineering and designer biocatalysts. Biofuel Research Journal 28 (2020) 1267-1295. DOI: 10.18331/BRJ2020.7.4.5 of 1:3) increased bioethanol production by 13.4% using S. cerevisiae var. ellipsoideus in SSF process (Nikolić et al., 2016). In another study, SSF system was used for bioethanol production from recycled paper sludge using P. stipitis CBS 5773. Celluclast®1.5 L supplemented with Novozym®188 was used for enzymatic hydrolysis, which resulted in 100% saccharification. The ethanol concentration of 19.6 g/L was achieved after 179 h of fermentation (Marques et al., 2008). Similarly, bioethanol production was reported using pretreated municipal solid waste via SSF using S. cerevisiae in a fed-batch mode with 25% (w/w) substrate loading and achieved an ethanol concentration of 30 g/L (Ballesteros et al., 2010).
In a study, the thermotolerant yeast strain S. cerevisiae KNU5377 was used for ethanol production from pretreated waste newspaper (250 g/L, solid loading) in a SSF system, and the ethanol production of 8.4% (v/v) was obtained at 50 o C after 72 h in a 5 L fermenter (Park et al., 2010). In another study, SSF was conducted using S. cerevisiae under shaking conditions (60 rpm) using 1% (v/v) inoculum under semi-anaerobic conditions for ethanol production from dates juice, and 88% of the substrate was converted into bioethanol at the end of fermentation with a product yield of 0.51 g/g sugar (Taouda et al., 2017).
In a more recent study, bioethanol production of 82.1 g/L was reported using sulphite-pretreated momentary pine slurry (25%, w/w) in a SSF system. Prehydrolysis was done at 50℃ for 24 h and 200 rpm followed by fermentation at 28℃ or 35℃ using 5 g/L dry inoculum of S. cerevisiae (Dong et al., 2018). Many previous studies have also been reported on bioethanol production using SSCF fermentation (Erdei et

Consolidated bioprocessing (CBP)
The concept of CBP strategy was evolved from direct microbial conversion, but wild microbial strains are not available for commercial bioethanol production using this approach . In CBP, all the steps of bioethanol production, i.e., enzymes production, cellulose hydrolysis, and fermentation, are conducted in a single vessel, and single microbial community is used for both production of cellulases and fermentation, which makes the process cost-effective. Lynd  . Filamentous fungi, Fusarium oxysporum, possesses the potential of bioethanol production from lignocelluloses in the CBP system . In the past few years, thermophilic anaerobic cellulolytic bacteria such as T. ethanolicus, C. thermohydrosulfuricum, T. mathranii, Thermoanaerobium brockii, and C. thermosaccharolyticum have been explored for bioethanol production using the CBP approach due to their ability for direct conversion of cheaper biomass feedstocks into bioethanol at extreme temperatures. However, these extremophiles are sensitive to ethanol concentration, which is a major hurdle for their use (Lynd et al., 2005;Vazirzadeh and Robati, 2013). The economic commercial production of bioethanol (66 million gallons at a breakeven price of $1.31 per gallons) from pure sugarcane bagasse feed using the CBP platform has been reported by Raftery and Karim (2017). In another study, pine needle biomass was pretreated using IL followed by fermentation using S. cerevisiae and P. stipitis in a CBP system. The ethanol yield of 0.148 g/g was obtained after 72 h, and the fermentation efficiency of system was found at 41.39% (Vaid et al., 2018). Recently, recombinant S. cerevisiae ER T12 and M2n T1 strains (harboring integrated temA and temG Opt gene cassettes) simultaneously expressing αamylase and glucoamylase, produced 89.35 g/L and 98.13 g/L ethanol from starchy biomass in a single step CBP system at 30℃ after 192 h with carbon conversion of 87% and 94%, respectively (Cripwell et al., 2019). Beri et al. (2020) proved the consumption of 85% recalcitrant glucuronoarabinoxylan (GAX) contained in from corn fiber by the isolated Herbinix spp. strain LL1355, and reported that six enzymes were involved in the hydrolysis of GAX linkages. They argued that the successful expression of up to four genes in Thermoanaerobacterium thermosaccharolyticum increased the GAX consumption and ethanol yield by 78% and 28%, respectively.
Recently, the cell-free extract reaction (CFER) system was developed in Clostridium thermocellum to identify potential metabolic limitations and to offer potential metabolic engineering interventions to enhance ethanol titers (Cui et al., 2020). Although CBP method is much improved for ethanol production compared to other existing methods due to less production cost of enzymes, yet there are some gaps concerning the commercial use of CBP systems. Future studies should be directed towards understanding the metabolic pathways of microorganisms, synergistic action between microbes and their enzymes for simultaneous pretreatment, hydrolysis and fermentation, and developing recombinant strains and bio-design strategies for enhanced ethanol production with improved yields.

Concluding remarks and future prospects
Lignocellulosic or 2G ethanol is being considered as one of the longterm sustainable alternative to the environmentally-degrading crude oil reserves. However, there are several technical and economic challenges associated with bioethanol refineries. Low-cost pretreatment to overcome biomass inherent recalcitrance in an eco-friendly manner is the first major hindrance that needs to be addressed. The choice of pretreatment method relies on the type of biomass selected for 2G sugars production at competitive prices. Thermo-mechanical extrusion method is considered one of the most efficient pretreatment methods which can be used in combination with other technologies such as particle-size reduction and green solvent pretreatment for efficient ethanol production. Pretreatment cost and chemical waste generation can also be reduced by altering lignin structure of LCB and expressing novel microbial enzymes in plants, which results in decreased molecular weight of lignin without compromising the biomass yield.
High cost associated with the commercially available cellulase/hemicellulase enzymes is another bottleneck that should be addressed by formulating indigenous tailor-made enzyme cocktails that are highly efficient against a wide range of agro-residues even at low protein loadings. On-site production of enzymes could be an effective strategy to reduce the production cost of bioethanol. The innovative technologies like protein engineering and computational protein design can be used for generation of cost-effective and industrially important novel biocatalysts. The future research should also target designing integrated approaches for simultaneous pretreatment and saccharification of biomass, and fermentation of the released sugars.
The third major challenge in 2G ethanol processing is the limited uptake of xylose by fermenting yeasts in the presence of glucose. The ethanol production efficiency can also be improved by using genetically modified microbes, which possess the ability to ferment pentose and hexose sugars simultaneously in the presence of fermentation inhibitors by eliminating the detoxification step. Therefore, the future research should be focused on the development of robust engineered yeast having suitable transporters for simultaneous uptake of glucose and xylose with equal assimilation rates. Metabolic engineering, cell surface engineering, and synthetic biology are other promising approaches being used for the synthesis of engineered host fermentation system to improve the production of bioethanol. CRISPR-Cas9 is a simple but powerful gene-editing tool for safe and stable gene expression, which can be used for synthesis of engineered microbial strains. Among the various production platforms, CBP seems more efficient for economic bioethanol production because all the steps are performed in a single reactor by a single microorganism capable of producing hydrolytic enzymes and fermentation.
Finally, development of biorefineries seems critical for economical utilization of LCB. Focusing on a single product or bioethanol may not be an economically viable option. A biorefinery may be designed in such a fashion to valorize each and every component of lignocelluloses into biofuels and biochemicals for sustainable development of circular bioeconomy . Therefore, a major focus should be placed on the development of such kinds of systems to reduce production cost and improve production efficiency.
Addressing the above-mentioned challenges could help to provide solutions for escalating global energy demands while mitigating the climate-related challenges as well.