Enzymes are biological catalysts and are able to catalyse, i.e. speed up biological reactions by means of lowering the activation energy of the reactions that it part takes in. Enzymes have a role in all biological systems and are an imperative part constituting the living world. In the later parts of the 19^th and earlier parts of the 20^th centuries, notable progress was achieved in the isolation, characterization, and commercial application of various enzymes. However, it wasn’t until the 1920s that the crystallization of enzymes unveiled their association with protein molecules, establishing the proteinaceous nature of catalytic activity. A paradigm shift occurred during the late 20^th century, in the 1980s when the discovery was made that certain ribonucleic acid (RNA) molecules, termed ribozymes, possessed catalytic capabilities. These ribozymes were identified as key contributors to gene expression processes. Concurrently, within the same epoch, biochemists developed methodologies for generating antibodies endowed with catalytic properties, colloquially referred to as ‘abzymes’. The emergence of abzymes presented substantial prospects for innovative applications both as catalysts in industrial processes and as therapeutic agents. This transformative period in enzymology expanded the conceptual framework beyond the exclusive protein-centric view, acknowledging the catalytic potential inherent in RNA molecules and engineered antibodies. Consequently, a diversification of enzymatic entities occurred, with ribozymes and abzymes contributing significantly to the understanding and application of catalytic processes.¹ There has been a recent shift to use bio-catalysts in order to promote “green chemistry”. This approach has led to many research and development studies that have focused on cost-effective enzyme production. One such enzyme is Cellulase, that degrade cellulose. Cellulose, as studies have suggested is the most abundant biological polymer that is a homopolymer in nature comprising D-glucose units which are connected by β-1, 4 bonds, stands as the pre-eminent organic polymer. Functioning as a crucial structural constituent within the primary cell wall of plants which are green in colour comprising of 50% of its total biomass in dry weight.² Cellulose holds promise as a renewable natural resource and has wide applications in various industries. Consequently, heightened economic interest surrounds the establishment of systems geared towards way to efficiently manage and utilize cellulose-containing industrial wastes, and presenting it as cost-effective carbon sources.³

Cellulose rich materials have been in use for the production of cellulases, especially using agro-residues such as tea and coffee waste,⁴ fruit waste, rice bran wastes, etc. as has been reported.⁵ The utilization of such economical and low-cost substrates poses to be a good alternative for enzyme production. Cellulases, have distinct and diverse activities. The degradation of cellulose rich lignocellulosic biomass. The general division of cellulase is done into exoglucanases (EC 3.2.1.74), endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91), and b-glucosidases (EC 3.2.1.21). Exoglucanases exhibit enzymatic activity on either reducing or nonreducing ends of cellulose chains, liberating glucose units. Conversely, endoglucanases catalyze the random hydrolysis of b-1,4-glycosidic bonds within cellulose chains, yielding dextran chains of variable lengths. Cellobiohydrolase specifically cleave glycosidic bonds at nonreducing ends, releasing cellobiose units. These enzymes play a crucial role in the hydrolysis of crystalline cellulose, owing to their processivity. b-glucosidases contribute to the hydrolysis of cellobiose units, thereby converting them into monomeric glucose. The complete hydrolysis of cellulose involves a synergistic effect among these enzymes, manifesting as synergy between endoglucanases and exoglucanases (endo-exo synergy), exoglucanases acting on both reducing and nonreducing ends (exo-exo synergy), synergy between cellobiohydrolases and b-glucosidases, as well as between catalytic and carbohydrate binding domains. Various technologies and substrates have been in use for the production of cellulase. This review seeks to elucidate the microbes used, the current technologies that are being employed and the limitations and future prospects, all pertaining to cellulase production. Figure 1 demonstrates the action of cellulase on cellulose containing materials.

A synergy of three enzymes constituting cellulase complex degrade the lignocellulosic biomass into its constituent monomeric units.

Microbial cellulases
Cellulolytic enzymes, characterized by significant enzymatic activity and stability variations, are predominantly derived from microbial sources. Notably, variations in physical parameters such as pH and temperature contribute to the observed diversity in enzymatic properties. A plethora of microorganisms, including bacteria, protozoans, fungi, etc. have been documented for their capacity to produce cellulases. There is a need for enumeration of such microbes, namely, bacteria, fungi, actinobacteria and yeasts for the potential in production of cellulolytic enzymes. Bacteria and fungi, exhibit proficiency as producers of cellulolytic enzymes, with fungi proving particularly suitable producers of enzymes owing to their extracellular properties. Therefore, the ongoing exploration for novel microorganisms, is imperative to align with industrial requirements. At an industrial scale, various fungal and bacterial strains have been harnessed for cellulase production. Notably, the use of commercial substrates such as cellulose or carboxymethyl cellulose (CMC) has been prevalent, albeit associated with high costs. Microbial cellulases have drawn significant academic interest because they are more economically viable than conventional methods. In contrast, alternative techniques like chemical hydrolysis, physical treatments, thermochemical processes, and solvent-based methods often involve higher costs due to their energy-intensive nature, the use of costly reagents, complex machinery, and negative environmental impacts. As a result, microbial cellulases are seen as a more sustainable and cost-effective option in various industrial applications. Consequently, scientific endeavours have been directed towards substituting these costly substrates with naturally abundant cellulose-rich biomass derived from agricultural and forest residues.^6,7

Fungal cellulases
Fungi, formerly regarded as antagonistic microorganisms, have now become an indispensable industrial raw material across diverse applications. The dynamic cellulose decomposers inherent in fungi may be implicated in the global decomposition processes. Additionally, the preferential framework for cellulase synthesis through fungal cellulosomes has been identified as more resilient to environmental variabilities.⁸ In comparative analyses involving Aspergillus and Humicola species, Trichoderma species have been identified as particularly suitable for cellulase synthesis and industrial utilization.⁹ The most extensively studied cellulolytic microorganisms encompass white-rot fungi, such as Phanerochaete chrysosporium, and soft-rot fungi, including Fusarium solani, Penicillium funiculosum, Talaromyces emersonii, Trichoderma koningii, and Trichoderma reesei. Prominent among commercially employed microorganisms exhibiting significant cellulolytic capabilities are Aspergillus fumigatus, Aspergillus nidulans, Aspergillus aculeatus, Aspergillus niger, Aspergillus oryzae, and Aspergillus niger;¹⁰ Within the Indo-Myanamar Biodiversity Hotspot, three cellulolytic fungi, namely Talaromyces verruculosus SGMNPf3, Trichoderma gamsii SGSPf7, and Trichoderma atroviride SGBMf4, were meticulously identified.¹¹

Bacterial cellulases
Bacterial cellulases have been a part of many industrial applications and there have been many species which have been used for the production of cellulase. Seven bacterial isolates originating from the genera Ochrobactrum, Acinetobacter, Pseudoxanthomonas, Paenibacillus, Stenotrophomonas, and Comamonas were identified in the composting leachate derived from wheat straw.¹² The screening of the cellulolytic activity of 398 bacteria isolated from various ecosystems in Algeria (cave, ruins, chott, thermal station, and rhizosphere of arid and semi-arid region). Studies have established that five isolates that belonged to the genus Bacillus. viz, B. velezensis, B. subtilis, B. mojavensis and

B. cereus were able to flourish after being subjected to various stress conditions.¹³ Specific enzymes produced by microorganisms; however, cellulases of bacterial origin attract more interest due to their natural diversity and ability to inhabit a variety of niches, allowing the selection of cellulolytic strains resistant to environmental stresses. Several reported isolates also belonged to the aforementioned genus with the species being, B. flexus, B. licheniformis and B. paralicheniformis.¹⁴ The existence of cellulolytic microorganisms in a variety of habitats is highlighted by the isolation of cellulase-producing bacterial isolates from different settings. These results suggest that favourable environmental factors may aid in initiating the formation of cellulase. Pseudomonas, Paenibacillus, Acidothermus and Micromonospora represent distinctive taxa within cellulolytic species, that make them promising candidates for the exploration of novel enzymes involved in the degradation of polysaccharides, particularly cellulose. These microorganisms highlight the ecosystem as a favourable platform for investigating enzymatic mechanisms associated with cellulose degradation.¹⁵

Yeast cellulases
Essentially, only a small number of yeasts exhibit the capacity to break down cellulose. A few of them have been elucidated as of now, with the recent research even forging towards the untouched arena of marine yeast. The first ever report on cellulase producing yeast was by Dennis in 1972,¹⁶ where they isolated various strains from the Rhubarb plant out of which nine (out of 12 strains) of Trichosporon cutaneum and 12 out of 13 strains of T. pullulans showed cellulolytic activity. Since then, several new species have been identified that have the potential to degrade cellulose via the production of cellulase such as yeasts obtained from rotten dahlias for the degradation of coconut oil cake belonging to the genus Meyerozyma, named M. guillermondii CBS 2030 which has a relatively high cellulase activity of 102.96 U/mL.² Naturally, relatively few yeasts have the capacity to break down cellulose. Rhodotorula glutinis, a psychrophile by nature, was reported that purported to be the first of its type. Aureobasidium sp. is the major cellulolytic yeast identified in the habitat of Forcipomyia taiwana, also called the biting midge, according to research on the subject. It has been observed that crude lignocellulosic substrates may be hydrolyzed using yeast strains such as Dendrobium spathilingue, Moesziomyces sp., Candida easanensis, and Saccharomyces species.¹⁷ 51 species of yeast were recovered from rotting wood; these species were classified into the following genera: Pichia, Candida, Filobasidium, Kluyveromyces, and Aureobasidium. When cultivated on carboxymethyl cellulose or wood chips, only Aureobasidium microstictum and Trichosporon pullulans showed cellulase activity.¹⁸

Actinobacterial cellulases
Actinobacteria constitute a taxonomically diverse group of Gram-positive bacteria that predominantly inhabit the soil. They also exhibit a notable capacity for prolific mycelial growth and the formation of chains of aerial conidia similar to that of imperfect fungi, Actinobacteria are characterized by their saprophytic lifestyle. Their presence in various ecological niches makes them one of the most diverse bacterial groups in the natural environment. The physiological traits of Actinobacteria span a wide spectrum, encompassing anaerobic unicellular organisms as well as aerobic, filamentous, and spore-forming lineages. This inherent variability in metabolic and morphological features contributes to the adaptability and ecological success of Actinobacteria across a wide array of habitats. Within the wide sphere of cellulase-producing actinomycetes, considerable attention has been devoted to the investigation of Cellulomonas fimi, Microbispora bispora, and Thermobifida fusca. In particular, Thermobifida fusca emerged as a thermophilic, spore-forming actinomycete species. The genomic constitution of T. fusca comprises 3.6 billion base pairs organized in a singular circular chromosome, hosting 3117 coding sequences. Notably, the genomic composition manifests a G+C content of 67.5%, thereby imparting stability to DNA under conditions of extreme temperature.¹⁹ Several studies have proposed the isolation of actinomycetes from marine sediments as a promising strategy for obtaining novel actinomycetal strains capable of yielding potentially valuable new products. Concurrently, actinomycetes are recognized for their proficiency in producing diverse extracellular enzymes, cellulase being one. The proficiency of Streptomyces strains, namely Streptomyces sp. LCJ10A, Streptomyces sp. LCJ11A, Streptomyces sp. LCJ13A, Streptomyces sp. LCJ14A, and Streptomyces sp. LCJ16A, isolated from the Pichavaram mangroves has also been documented.²⁰ These strains were found to exhibit high efficiency in the production of economically significant enzymes, including lipase, cellulase, and asparaginase. The identified enzymes hold substantial potential as valuable assets for innovative biotechnological processes. The overview of the same has been depicted in Table 1.

Table (1):
Bacterial, fungal, actinobacterial and yeast cellulases

+ : Production reported. , – :  Not reported.

Current technologies in microbial cellulase production
Predominantly, two fermentation techniques are utilized for the production of cellulase, viz. submerged fermentation (SmF) and solid-state fermentation (SSF). In SmF, microorganisms, predominantly fungi are cultivated in a liquid medium containing cellulose or other cellulosic substrates as the primary carbon source. This method allows meticulous control over crucial parameters including pH, temperature, and nutrient availability, facilitates the obtainment of elevated yields of cellulase enzymes. In contrast, SSF involves the utilization of a solid substrate, typically agro-residues, in place of a liquid medium.

In SSF, solid substrates such as bagasse, bran, paper pulp, and rice straw are typically employed, facilitating the gradual metabolism of the substrate by microorganisms.^51,52 This technique is particularly advantageous for fungi (with the exception of yeasts) and other microorganisms that necessitate diminished moisture conditions.⁵³ Nonetheless, SSF encounters several challenges including the precise regulation of environmental parameters, scalability, heat dissipation, and mass transfer efficiency. Despite these obstacles, SSF presents benefits such as enhanced yield, cost-effectiveness, and a more streamlined operational process. Conversely, SmF entails the cultivation of microorganisms on either soluble or insoluble substrates, which are either dissolved or submerged within liquid media.^54,55 Examples of liquid media comprise domestic wastewater and hydrolysate derived from grape pomace⁵⁴. SmF facilitates uniform substrate metabolism and affords manageable control over external conditions. This approach is more apt for bacteria, yeasts, and other microorganisms that require elevated moisture levels. While SmF simplifies handling processes and improves heat transfer dynamics, it also poses challenges such as the viscosity of the culture broth, which can result in foaming and incomplete saccharification of lignocellulosic biomass. Ultimately, the enhanced ease of handling and the regulated external conditions render SmF a more efficient and manageable fermentation process.

Active research endeavours are directed towards the refinement and optimization of both fermentation methodologies with the aim of enhancing cellulase production efficiency and mitigating associated costs, thereby enhancing accessibility for diverse industrial application.⁵⁵ A brief comparative overview of the fermentation technologies has been highlighted in Table 2.

Table (2):
Comparative overview of fermentation technologies employed for cellulase production

Solid state fermentation
Solid-state fermentation involves the utilization of solid substrates such as bagasse, bran, and paper pulp. A notable benefit of employing these substrates lies in the facile recycling of nutrient-rich waste materials. In this fermentation methodology, the substrates are metabolized gradually, enabling prolonged utilization of the same substrate throughout extended fermentation periods. Consequently, this technique facilitates the controlled release of specific nutrients. Solid-state fermentation is particularly well-suited for processes involving fungi and microorganisms that thrive under low moisture conditions. However, its applicability is constrained in fermentation processes that encompass a diverse range of organisms.⁵⁶ Solid-state fermentation surpasses submerged fermentation in cellulase yield, and effective cost reduction is achievable through appropriate technology, enhanced bioreactor design, and a streamlined cellulase production process. The constituents of the medium play a pivotal role in influencing enzyme synthesis by various bacteria⁵⁷ β–fructans and α-galactans, which are polymers of glucose, fructose and galactose, respectively. The synthesis of these compounds is catalyzed by glycosyltransferases (glycansucrases). The challenges associated with scaling up solid-state fermentation for industrial applications include difficulties in maintaining optimal temperature, pH, and moisture levels. Additional issues encompass the absence of homogeneous mass transfer, uneven fungal growth within the SSF media, and a lack of methodologies to accurately estimate fungal biomass concentrations.⁵⁸ Once the production is complete, the recovery of the crude enzyme is done which are made to undergo certain downstream processes, viz., Solid-liquid extraction in which the enzyme is typically extracted by washing the solid substrate with a suitable buffer solution; Filtration or Pressing where the liquid (containing the enzyme) is filtered using filter presses, mesh filters or vacuum filters; Clarification is then done to remove fine particles and debris from the extracted liquid by processes such as centrifugation or by microfiltration; post this step, the enzyme is concentrated by ultrafiltration and precipitation with agents like ammonium sulfate to precipitate the enzyme. The ultimate step of obtaining the pure enzyme is purification in which the purity of cellulase is enhanced by removing other proteins and contaminants by means of processes like chromatography, dialysis, etc. The enzyme is then refined and stabilized using stabilizers such as glycerol for making it competent for storage.⁵⁹ The overview of the process of SSF has been depicted in Figure 2.

Submerged fermentation
It is operationally characterized as the fermentation process conducted in an environment where water is present in excess up to 95%.⁶⁰ The prevailing practice in numerous large-scale enzyme production facilities involves the utilization of SmF owing to its established technological efficacy, facilitating enhanced monitoring capabilities and simplified handling procedures.⁶¹ One of the most imperative reasons for the prevalence of submerged fermentation is due to the fact that the environmental factors such as pH and temperature can be controlled thereby reducing errors and cost.⁶² Enzymes for commercial applications are presently synthesized through submerged fermentation as a result of its cost-effectiveness and reduced enzyme consumption. Numerous potential applications of these enzymes have been successfully industrialized. Submerged fermentation exhibits advantages such as lower labor costs, reduced space requirements, enhanced control over environmental conditions, and less complexity in scaling up, when compared to solid-state fermentation.⁵²

Within the context of submerged fermentation, extracellular endoglucanase activity was identified, and four strains exhibited similar patterns of enzyme secretion. The Klebsiella sp. (B2) strain displayed the lowest extracellular activity, although this disparity did not achieve statistical significance when contrasted with the activity of the other strains. The findings on petri dishes consistently mirrored CMC growth patterns and enzymatic hydrolysis profiles.⁵⁸ It has been documented that during the implementation of submerged fermentation for cellulase production, several challenges are observed which generally reoccur.⁸ One of these challenges include an increase in the culture broth’s viscosity induced by cellulase production. Attempts to mitigate this viscosity increase through heightened agitation may result in unmanageable foaming. Additionally, the presence of secreted cellulase in the culture broth may lead to partial saccharification of lignocellulosic biomass and concentration of sugars (specifically glucose and xylose). Furthermore, a decline in dissolved oxygen percentage (DO%) in the broth is noted due to the heightened oxygen uptake by the proliferating fungus. Despite the multi-fold advantages presented by solid-state fermentation process-based bioreactors as compared to submerged fermentation bioreactors, the stirred-tank reactor with an automatic process control system prevails and emerges as the preferred choice for large-scale production of cellulase enzyme. This preference is attributed to its ease of handling, operation, and superior heat transfer characteristics.⁶³ The overview of the process has been elucidated in Figure 3 which showcases the various steps of submerged fermentation.

Applications of cellulase enzyme
Cellulase exhibits significant potential across various industrial sectors. In the domain of industrial waste management, this enzyme is imperative for the degradation of cellulosic materials present in both domestic and industrial refuse, thereby enhancing the overall effectiveness of waste management protocols. Its contribution to bioethanol production is equally paramount, as it facilitates the conversion of cellulose into fermentable sugars, consequently improving the efficiency of biofuel production from cellulosic feedstocks. Moreover, in the paper and pulp industry, cellulase enzymes augment product quality by refining cellulose fibers and optimizing manufacturing processes.

Within the textile sector, cellulase enzymes are essential for environmentally sustainable processing by substituting conventional chemical techniques. They are extensively employed in denim washing to achieve a soft, aged appearance without compromising fabric integrity, as well as in fabric modification to enhance texture and quality. These enzymes also assume a critical function in dye removal and biofinishing applications, contributing to fabric softness, durability, and superior dye absorption. By supplanting traditional chemical methodologies in biodesizing and bioscouring, cellulase enzymes not only enhance operational efficiency but also diminish water usage and mitigate environmental repercussions, thereby endorsing sustainable practices.

In the agricultural sector, cellulase enzymes improve soil health and promote plant growth by facilitating the breakdown of crop residues and recycling vital nutrients back into the soil. They also assist in the decomposition of cellulosic substances within animal feed, enhancing nutrient assimilation and livestock productivity. In composting processes, these enzymes expedite the decomposition of organic matter, yielding nutrient-rich compost for soil enrichment. The applications extend further into bioremediation, where cellulase enzymes facilitate the degradation of cellulose-based pollutants, thereby aiding environmental remediation efforts. Bioethanol represents a sustainable energy source. Notably, second-generation bioethanol production is gaining traction due to the availability of economical raw materials. Lignocellulosic biomass, the primary feedstock, encompasses agricultural residues, herbaceous and woody crops, forestry waste, and various municipal and industrial by-products. Lignocellulose constitutes the most prevalent renewable biomass. Global lignocellulose yield is estimated at around 200 billion metric tons annually. Utilizing these feedstocks for bioethanol production offers numerous benefits. It effectively facilitates the disposal of non-edible plant materials. Its lower pollutant emissions render it environmentally advantageous. Crucially, this process does not compromise food security as it avoids the use of food crops pre-harvest. Moreover, it provides a year-round supply of raw materials.

Conversely, a significant limitation of this production method lies in the structural composition of lignocellulosic biomass. This biomass is predominantly comprised of lignin, cellulose, and hemicellulose, forming a highly stable matrix. To liberate fermentable sugars from this substrate, a pretreatment is necessary to disrupt the stable structure, wherein cellulase enzymes play a crucial role.⁷¹

Additionally, they find extensive application in the food and beverage industry for juice extraction, flavor enhancement in wine production, and product clarification, while also playing an indispensable role in biofuel generation and pharmaceutical applications.⁶

Strain improvement
The enhancement of cellulase production can be achieved through strain improvement techniques such as involving the genetic modification of microbial strains, employing advanced methodologies, that can be used to attain significant results. Genetic modifications and random mutagenesis of parental strains play a pivotal role in amplifying the commercial production of cellulases. Additionally, the molecular cloning of cellulase genes and the augmentation of gene copy numbers exert a substantial influence on the degradation of cellulosic materials and the production of high-quality value-added products.

Strain improvement emerges as a promising strategy for elevating cellulase production Wild-type or normal strains may possess distinct characteristics suitable for industrial applications. To fully exploit the potential of wild-type microorganisms, strain improvement is imperative, allowing for the modification or elimination of specific traits. Recombinant DNA technology, site-directed mutagenesis, recombination protoplast fusion, and random mutagenesis, contribute significantly to enhancing commercial production. Immobilizing cellulase on a solid support represents another method to improve the catalytic efficiency of enzymes for industrial applications.⁷² Introducing a novel strategy for enhancing catalytic activity involves the application of substrate-induced gene-expression screening (SIGEX) coupled with fluorescence-activated cell sorting (FACS). Additionally, bacterial mutagenesis has been employed to elevate cellulase activity in thermophilic cellulase-producing organisms. For an extended duration, the limited efficacy in gene targeting has posed a significant challenge in achieving a substantial yield of transformants through homologous integration or expression cassette deletion. These obstacles stem from the difficulties in executing such processes. Strains capable of thriving on more cost-effective substrates have emerged as promising reservoirs for metabolites of industrial interest. Extensive research endeavours and practical applications over the years have contributed to a comprehensive understanding of fungal genetics.⁷³ Using fluorescence-activated cell sorting (FACS) in conjunction with substrate-induced gene-expression screening (SIGEX) is one way to improve catalytic activity. In thermophiles that produce cellulases, there was an increase in cellulase activity by bacterial mutagenesis. For instance, changed clones were obtained when NTG (N-methyl-N′-nitro- N-nitrosoguanidine) was applied as a mutagen to Bacillus sp. strain C1.⁷⁴ Analysis of the properties of bacterial cellulose generated by Komagataeibacter sp. nov. CGMCC 17276 under static and agitated conditions revealed the presence of strongly cross-linked cellulose nanomaterial.^69,75 Studies⁷⁶ based on phylogenetic position and phenotypic characteristics, the high-yield bacteria cellulase-producing strain Komagataeibacter sp. nov. CGMCC 17,276 was assigned as a novel species in the Komagataeibacter genus with good properties of rapid cell growth and high bacterial cellulase production. Sequential mutagenesis employing three agents-ultraviolet irradiation (UV), N-methyl-N′-nitro-N-nitrosoguanidine (NTG), and ethyl methanesulfonate-resulted in enhanced activity of Streptomyces durhamensis EMS.^77-79 Following mutagenesis, the cellulase activity of mutant Streptomyces durhamensis GC23 exhibited a 1.86-fold improvement compared to the wild-type strain (vs15). Subsequent optimization of the cellulase activity in mutant Streptomyces durhamensis GC23 led to a twofold increase relative to the wild type, as documented by Ega et al.⁷⁸

The various strategies to enhance the production of cellulases include molecular docking, by employing computational methodologies and strategies to forecast enzyme-substrate interactions, facilitating enhanced enzyme engineering. Molecular docking insights on the cellulase production of bacterium Bacillus amyloliquefaciens indicate that the cellulase enzyme derived from the Bacillus amyloliquefaciens strain elh1 exhibits a robust and specific affinity A critical factor for its cellulolytic activity.⁸⁰ These findings facilitate subsequent optimization and augmentation of cellulase performance; Inclusion of multifunctional cellulase genes along with the combination of various cellulase genes can help provide a means to lower enzyme costs by reducing the complexity of enzyme cocktails while aiding to increase the efficiency of enzyme hydrolysis. Use of effective promoters can upregulate the transcription of the enzyme leading to heightened enzyme production. This was elucidated in a study,⁸¹ where overexpression of the transcription factors  Xyr1 and Ace3 of Trichoderma orientalis EU7-22 was done which led to the increased production of cellulase and hemicellulase in the recombinant strain dxyA-8 illustrating the efficacy of the chosen promoters in attaining elevated enzyme activity. The overview of the strategies is depicted in Figure 4.

Challenges in cellulase production
Lignocellulosic biomass represents a promising reservoir for the generation of biofuels, biofertilizers, animal feed, and chemicals, in addition to serving as a primary constituent for the paper industry. The harnessing of this renewable resource necessitates either chemical or biological treatment methods, with cellulases emerging as prominently favoured agents in the latter approach over the preceding decades. Investigative endeavours have elucidated the intricacies underlying microbial cellulase production mechanisms, leading to the evolution of technologies dedicated to the production and application of cellulose-degrading enzymes; namely cellulase. Despite these advancements, a challenge persists in the absence of a singular, cost-effective, and efficient process for converting natural lignocellulosic materials into valuable metabolites or biofuel. The utilization of existing commercial cellulase preparations for the bioconversion of lignocellulosic waste is presently deemed economically impractical as there is still a debate as to which method is deemed suitable for the production of cellulase.⁷⁴ Some practical drawbacks include enzyme synergy, improperly treated lignocellulosic biomass, excess solid concentrations, sub-optimal ratio of critical factors, etc.⁸²

The following points delve into the challenges faced in cellulase production

• Fermentation Techniques: Various fermentation methodologies, including submerged and solid-state fermentation, present distinct challenges in the optimization of cellulase yield. Each methodology possesses its own array of operational difficulties that can significantly influence the efficiency of enzyme production.⁸³
• Scale-Up Issues: The transition from laboratory-scale to pilot-scale production frequently encounters bottlenecks, complicating the maintenance of efficiency and cost-effectiveness.⁷⁴

Cost and Economic Feasibility

• Production Costs: The substantial expenses associated with cellulase production continue to pose a significant barrier, prompting the necessity for the investigation of more economical substrates and enhanced microbial strains.⁸⁴
• Monitoring and Control: The effective monitoring of the fermentation process is paramount for cost reduction; however, the intricate nature of the bioprocess presents challenges in the formulation of precise models.⁸⁵

Optimization of Microbial Strains

• Genetic Modification: Although the genetic modification of microbial strains has the potential to augment cellulase production, the initial yield of isolated strains is frequently suboptimal, thereby necessitating further refinement.⁸⁴

Challenges faced during isolation, screening and growth on various substrates

• Cultivation Recalcitrance: Numerous cellulolytic microbes exhibit recalcitrance to laboratory cultivation, restricting access to a plethora of potential cellulase-producing organisms found in natural ecosystems.
• Diversity of Microbial Communities: The extensive diversity within microbial communities that synthesize cellulases complicates the isolation of specific strains, thereby hindering the identification and characterization of the most effective cellulolytic microbes.
• Variability in Fermentation Conditions: Variations in fermentation parameters, including media composition and raw materials, influence cellulase production across different microbial strains, thereby complicating comparative analyses of cellulase yields and activities.
• Assay Method Discrepancies: The variability in methodologies employed to assay cellulase activity across studies creates challenges in result comparison and in determining the most effective cellulolytic strains.
• Technological Limitations: Conventional methods for isolating cellulolytic microbes may fail to encompass the complete spectrum of potential cellulase producers, particularly those that are unculturable, necessitating advanced methodologies such as metagenomics to access these elusive microbial resources.
• Environmental Factors: The extreme conditions prevalent in the natural habitats of cellulolytic microbes (e.g., high temperature, salinity) complicate isolation efforts, making it imperative to comprehend their specific environmental requirements for successful cultivation.⁸⁶

In spite of these obstacles, ongoing research endeavours strive to enhance cellulase production through innovative biotechnological strategies and a more profound comprehension of enzymatic mechanisms, which could ultimately facilitate the development of more sustainable biofuel production methodologies.

Future prospects in cellulase production
Cellulases are imperative enzymes as they are employed in degradation of lignocellulosic biomass for their subsequent conversion to biofuels. Use of micro-organisms not only makes the process eco-friendly and sustainable but also adds to the cost-effectiveness of the process.

The following points elucidate the future prospects that can aid in the effective and enhanced production of cellulase enzyme. The methods by which effective cellulase production can be achieved by,

• Bacteria mediated transformation; chemical and/or natural.⁷⁵
• Genetic engineering for multifunctional cellulase genes: Creating genetic modification to cellulase genes to enhanced production capabilities and efficiency,
• Combining different cellulase genes: Strategically combining genes from various sources,
• Identification of genes from cellulose-degrading insects: Studying insects like termites to discover novel and potentially powerful cellulase genes,
• Use of effective promoters: Use of genetic promoters to improve the expression and production of cellulase enzymes,
• Molecular docking: Utilizing computational tools and techniques to predict enzyme-substrate interactions, enabling improved enzyme design.
• Consolidated Bioprocessing (CBP): Creating one-pot procedures that integrate the conversion of biomass and the synthesis of cellulase into a single stage. This simplifies the process and lowers manufacturing costs by eliminating the requirement for distinct enzyme purification processes.