The constant depletion of fossil fuels and the air pollution resulting from burning fossil fuels exacerbate the current world. It is envisioned that the consumption of the globally most abundant source of biomass is lignocellulose, while the eco-friendly hydrolysis by cellulases makes these enzymes highly desirable in bioenergy (biofuels, viz. methane and ethanol) research and industry. Biofuels generated using lignocellulosic biomass are second-generation biofuels, which are superior to the food crop-dependent first-generation biofuels [17]. Conversely, the complexity of the lignocellulosic biomass due to cellulose, lignin, and hemicellulose is a challenge that manufacturers of second-generation biofuels face. The multistep transformation of the lignocellulosic biomass into usable and value-added commodities is not as easy as it sounds. The biggest challenge is to break down the raw biomass into simple sugars, followed by the bioconversion of these components into biofuels and value-added commodities [18]. Environmentally hazardous chemical hydrolysis is fully replaceable by eco-friendly enzymatic hydrolysis [19, 20]. One eighty billion tons of raw lignocellulosic biomass on our planet makes it the most desirable inexpensive raw material, and the cellulases, in turn, have a plethora of applications in industry [21]. However, the high production prices of the cellulases are a bottleneck to making this commercially viable. It is envisaged that using lignocellulosic biomass for ethanol production in the transportation sector would make cellulases the most in-demand industrial enzyme. The greatest potential of fungal cellulolytic enzymes lies in ethanol production from biomass through the enzymatic hydrolysis of cellulose; however, low thermostability and low titer cellulase production results in high enzymatic costs [21, 22]. Nonetheless, merchantable production of ethanol from lignocellulosic biomass depends on the production of cellulases, whose finances require upgrading.

The importance of microbial cellulases in prospective applications to the lignocellulosic biomass started gaining attention in the early 1950s. The widespread need for cellulases is because the cellulose polysaccharide constitutes 50% of the globally most abundant source of biomass, i.e., lignocellulose, thereby making it an industrially desired raw material for manufacturing products that add value to the bioenergy sector. Annually, 100 billion tons of lignocellulosic biomass is accumulated from various agricultural and waste resources. Efficient and sustainable utilization of lignocellulosic biomass can be achieved by cellulolytic hydrolysis by cellulases. Indeed, Anselme Payen first achieved the discovery and isolation of cellulolytic enzymes from plants [23]. Marine algae viz. Ulva and numerous prokaryotes, i.e., bacteria from the genus Glucanobacter and Agrobacterium, also contribute to the hydrolysis of lignocellulosic biomass [24, 25]. However, fungi were concluded to be the dominant and efficient cellulase producers globally.

Cellulases have played a significant role as biocatalysts for many decades, which has enlightened their possible manufacturing applications. Value-added products from lignocellulosic biomass by employing cellulases are a recent concept. Industrial demand for fungal cellulases has risen significantly over the last few decades, especially for strains capable of producing stable enzymes under harsh industrial conditions. The possibility of actively converting cellulose into simpler components for conversion to transportation fuel can make cellulases the most desirable industrial class of enzymes [26].

Widespread applications of cellulases in food are diverse, viz. cereal grains, polishing, feed supplements, and flavor enhancements. Moreover, cellulases are involved in improving the digestibility of animal feeds [27]. Flavonoid extraction from the seeds and flowers is also carried out by cellulases. The food sector utilizes cellulases for bakery, juice, and wine processing, as cellulases provide enhanced wine filtration, improved pulp hydrolysis in the juice industry, and enriched texture and quality of bakery products [28]. Cellulases are efficient and preferred in extraction because they ensure less heat damage and higher yields. The major cellulase contributors to the food industry originate from Aspergillus and Trichoderma [29]. Additionally, cellulases are commercially produced by companies such as Novozymes, Badische Anilin- und Sodafabrik (BASF), DuPont Danisco, and Dutch State Mines (DSM) [30].

Biofuel production using lignocellulosic biomass hydrolyzed by cellulases has the potential to be the world’s largest industry, yet requires these enzymes. Further, the pulp and paper industry has great market demand for stable cellulases. Cellulase cocktails are very effective in sustainable, environment-friendly detergents, and the utilization of cellulases in the textile, paper, and detergent industries is massive. Hence, demand has increased [31, 32]. In the textile industry, pumice stone washing of the jeans reduced the capacity of the jean load, while a fifty percent higher jean load can be achieved by replacing stone washing with cellulases [33]. Additionally, acidic T. reesei endoglucanase II is employed in nontoxic fabric biopolishing [34].

Cellulases comprise approximately 3/4 of the total demand for industrial enzymes in many industries mentioned above, making them a frontrunner in global enzyme production and consumption [35]. The most recent global market value of cellulases was estimated at 1.621 billion dollars in 2022, which is expected to increase at a compounded annual growth rate (CAGR) of 6.94% from 2022 to 2032 [36]. In comparison, global biofuel enzymes are projected to increase from 905.2 million dollars (2020) to 1.3 billion dollars by 2026 with a CAGR of 7.3% [36], while the total enzyme market will grow from 10 billion dollars in 2019 to 14.7 billion in 2024 [37]; the CAGR of industrial enzymes is placed at 6.5% in terms of value [38].

The extensive research on cellulases by different manufacturers in the past two decades has led to the production of diverse blends of cellulases. The current market is dominated by Novozymes (Denmark) and Genencor, which have merged with DuPont (Netherlands), DSM (Netherlands), Solvay enzymes (Germany), and Dyadic (USA) [38]. In fact, 75% of the entire industrial enzyme market, not just cellulases, is concentrated within the first three hosts listed [39]. The indices used to indicate enzymatic activity are diverse; thus, the market value of cellulases is not standardized based on the quality of the product [40]. The inconsistencies between commercial indices used in the market present another major hurdle: inappropriate production improvement and economic analyses of biofuel production from cellulases.

Owing to the diverse composition of the lignocellulosic biomass obtained from various sources, a specific cocktail of enzymes cannot provide a widespread solution. This allows us to identify crucial limitations in producing the ideal enzyme or ideal blend of enzymes that combine for a specific application. The Carbohydrate-Active EnZymes (CAZy) database provides substantial information and plays a fundamental role in the improvisations of industrially needed enzyme preparations. The three approved approaches for preparing the desired mix of enzymes are (i) crude enzyme preparations from microbial consortia, (ii) defined cellulolytic enzyme preparation obtained from synergistic microbial interactions by fungal decomposers, and (iii) single component enzymes obtained from simple synergistic hydrolyzing systems for specific applications [41]. Next-generation biological modification tools (-omics centered and high-throughput selection) are the driving force in characterizing these enzymes and promoting the progress in improved production of cellulases from diverse fungal strains along with studies on their synergism, the ideal cellulase cocktail, and relevant applications in the desired industry.

Due to the inducible expression of the cellulases, the chief expensive and demanding feature of industrial fungal cellulase production is delivering a suitable inducer. Additionally, bacterial cellulases are less desirable compared to fungal cellulases since bacterial cellulases mostly lack either of the three cellulolytic activities, particularly exoglycanase activity. From an economical and ease of process point of view, fungal cellulases are preferred due to simpler downstream processing. Fungal cellulases also have higher activities than their bacterial counterparts.

There are many economic challenges associated with producing cellulases, thereby making their applications expensive. Therefore, enzyme industries aim to reduce production costs. Hence, sustainable, economically reasonable, and readily procurable sources of nutrients for cellulase-producing microbes can significantly lower enzyme costs [42]. However, the economic repercussions of producing cellulases from renewable biomass are many-fold and encompass multistep evaluations of the production process, from raw material selection to the market dynamics affecting the final product price.

Owing to its abundance and diversity, lignocellulosic biomass is an inexpensive and attractive substrate for cellulase production [43]. However, sole dependence on conventional lignocellulosic biomass, such as forest residues, municipal waste, and agro-industrial resources, may eventually raise questions about land management and affordable availability due to their abundant applications [44]. Additionally, lignocellulosic pretreatment is an energy-demanding and expensive affair. Therefore, it is also advisable to explore unconventional resources, such as naturally growing unwanted weeds. For instance, Parthenium hysterophorus is among seven globally abundant and unwanted weeds growing in Australia, Asia, America, and Africa [45]. Microbial fermentation of such renewable biomass would also allow environmental protection from these weeds and economic cellulase production.

Finally, Life Cycle Analysis (LCA) should be included to evaluate the environmental cost of utilizing various renewable resources. Life cycle assessment of cellulase production from solid-state fermentation of coffee husks has been reported [46]. Similarly, a techno-economic evaluation of cellulase production by submerged fermentation was reported to compare the batch versus fed-batch process feasibility, whereby the fed-batch was concluded to be a more economically viable process [47]. These studies imply that the feasibility of using renewable resources depends on overall process energy costs, fermentation time, and additional nutrient components. Cellulases are inducible enzymes, and the addition of lactose has been widely explored to induce their production, which also increases the final costs. There is no doubt that renewable resources and biomass management align with sustainability goals, yet the long-term availability and stability should also be considered.

To manufacture value-added products via fermentation, the most common and simplest feedstocks are readily fermentable sugars, such as glucose, fructose, sucrose, galactose, alongside many other mono, di, or oligosaccharides [81, 82, 83, 84]. However, decades of research have shown that each product can be produced in higher amounts using certain types of feedstocks compared to others [81, 83, 85]. For example, in the case of citric acid, simple sugars such as glucose and fructose have been shown to be ideal feedstocks compared to others [85]. The definition of a microbial feedstock is also ambiguous as it sometimes contains all the nutrient requirements of the microbial species, whereas sometimes, additional macro or micronutrients are required in a prepared feedstock. As mentioned earlier, fungal cellulase production depends on the presence of cellulose in the media [5]. However, cellulose is not soluble in the medium, creating an accessibility problem for the fungal cells under submerged fermentation. Conversely, simple sugars such as glucose are also not feasible for fungal cellulase production because they do not induce cellulase production in fungal cells. Therefore, media containing soluble cellulose fibrils can induce enzyme production. Indeed, Iram et al. [9] recently proved the theoretical basis of this concept.

Several low-cost feedstocks containing cellulose in the media are ideal to meet the above-mentioned conditions. Some famous examples include sugarcane bagasse [71, 72, 73], wheat or rice straw [66, 69], corn cobs or husks [86], and many other agricultural waste products, as mentioned in Table 2. The main problem with all high cellulose feedstocks is that they need harsh pretreatments to release simple cellulose fibrils for enzyme induction and simple fermentable sugars for the initial growth of the fungal species. In addition, all such feedstocks also lack the essential nitrogen sources required to produce microbial proteins efficiently. Distillers’ Dried Grains with Soluble (DDGS) are a byproduct of bioethanol production, which shows high potential to produce fungal cellulases with the help of optimization strategies mentioned below [14]. DDGS contains 26–33% proteins that help produce fungal enzymes, although it also has fibers (33–40%) that induce cellulase production [14]. The enzyme production (0.66 IU/mL) was highest in mildly acid-treated DDGS, compared to crystalline cellulose (0.4 IU/mL) or glucose (0.3 IU/mL) [9]. Conclusively, DDGS is an ideal feedstock for fungal enzyme production under SmF.

Nutrient optimization means making a microbial medium recipe that is best for a particular microbial outcome or product. Thus, for fungal cellulase production, cellulase activity, yield, and stability are some of the crucial factors. Alternatively, growth is not a desired factor, as dense mycelial clumps in the media will hinder the effective distribution of nutrients and dissolved oxygen in the media [5, 12]. Any nutrient element such as nitrogen source, minerals, or vitamins can positively or negatively affect the desired outcome. Therefore, it is crucial to know the effect (positive or negative) of each element and the ideal concentration in the media.

Many statistical techniques, such as the Plackett–Burman design (PBD), can be used to screen the effectiveness of each nutrient element. In addition, a range of concentrations can be optimized for various media elements using statistical optimization techniques, such as the response surface methodology (RSM). RSM is a method used to set pre-determined variables in such combinations that the desired level (maximum, minimum, or a specific value) can be obtained throughout a low number of experiments. The method is a fractional factorial design, which also provides response values at corresponding values of the dependent variables [87]. After an initial screening via PBD or other methods, RSM can be applied to optimize three or more variables for one or more response variables. This technique has been extensively used in the past few decades to optimize nutrient formulations to obtain maximum levels of specific fermentation products, as described in an example below.

In a study by the authors, statistical optimization of various minerals and nitrogen sources was evaluated using DDGS hydrolysate as the main feedstock. It was determined that additional minerals do not significantly positively affect fungal cellulase production under submerged fermentation [10]. This could be because DDGS, a byproduct of a microbial fermentation process (bioethanol production), already has some minerals. In addition, the pretreatment and subsequent neutralization step can further enhance the salt concentrations in DDGS hydrolysates. On the other hand, RSM helped produce a 262% increase in cellulase production [10].

Many other studies show the positive effect of nitrogen source amendment and optimization on fungal enzyme production. For example, in a study by Acharya et al. [88], peptone and ammonium sulfate were optimized to maximize cellulase production. The maximum cellulase activity was obtained at 0.125% peptone and 0.14% ammonium sulfate. On the other hand, using only 0.03% urea produced 0.15 IU/mL of cellulase activity. The main feedstock in this study was sawdust, and the concentration of sawdust was also optimized. Similar results were also obtained by Ghori et al. [89], where urea showed significant enzyme production. All such studies show the efficacy of using inexpensive feedstocks to analyze and optimize nitrogen sources for maximum enzyme production.

Culture parameters are pH, temperature, aeration, agitation, and inoculum size. All such parameters directly influence growth rate, enzyme production, and the production of undesirable products. There are many studies where such parameters have been evaluated and optimized using RSM for maximum fungal cellulase production under submerged fermentation. The main difference between these studies is the choice of feedstock and fermentation scale (shake-flask or benchtop bioreactors or pretreatment method before the fermentation stage). All such parameters, however, show a direct influence on cellulase production. Using DDGS as the main feedstock, Iram et al. [13] optimized the effect of inoculum size (1–10%), aeration (0.5–2 vvm), and agitation (100–500 rpm) in benchtop bioreactors (1.5 L) for fungal cellulase production under the submerged condition [13]. Cellulase production increased by 37% following fermentation parameter optimization. The optimum conditions were 6.5% inoculum size with 1.4 vvm aeration and 310 rpm agitation.

Several other studies have shown the effect of fermentation parameter optimization on cellulase production [5, 88]. In the study by Acharya et al. [88], pH, inoculum size, temperature, and agitation were evaluated for maximum enzyme production. An optimal pH of 4, a temperature of 28 °C, and an inoculum size of 10 discs were reported.

The effect of agitation is crucial for fungal fermentation under submerged conditions for several reasons. Agitation directly affects the distribution of nutrients and dissolved oxygen in the media [90]. However, it also affects negatively by imposing shear stress on the fungal mycelial structures. Therefore, optimizing this culture parameter is crucial [12]. In addition to the agitation rate, the selection and design of the impeller are also important as it can influence the shear stress on microbial cells [91]. The impeller helps in the dispersal of oxygen bubbles in the media. There are several types of impellers, yet the two main types are Rushton (radial flow) impellers and axial flow impellers. Flat-blade or Rushton impellers have shown high shear stress to mycelial mass in submerged fermentation [92]. Therefore, innovative impeller designs specifically for fungal fermentation have been reported to reduce the shear stress on fungal morphology [92]. Fig. 1 (Ref. [92]) shows one of the innovative impellers designed specifically for submerged fungal fermentation.

Fig. 1.

Impeller design for fungal submerged fermentation. (a) Conventional impeller. (b) Novel impeller. Reproduced with permission from [92].

Among various techniques to increase the productivity of the enzymes for a particular industrial product, enzyme kinetics can help better interpret results and optimize the production process. Enzyme kinetics involves modeling the rate of enzymes with respect to substrate conversion. Furthermore, quantifying enzyme–substrate interactions can contribute to a better understanding of hydrolysis, yield, and any reasons that may reduce the reaction rate. Due to the heterogeneous nature of the lignocellulosic biomass, the study of cellulase kinetics has always been simplified with different assumptions [93]. Different sets of assumptions result in varying degrees of model accuracy, yet all ensure different strategies to improve the degree of hydrolysis. For example, in a study by Paulraj et al. [94] enzyme kinetics were used to assess the effect of inorganic salts on cellulase activity.

In fermentation process improvement, growth kinetics also play an important role in defining the quality of the desired product. Predominantly, enzyme kinetics (e.g., substrate consumption) are closely correlated with the growth of the microorganism. In a study by Sasikumar and Viruthagiri [95] growth kinetics were used to predict the constants in the polysaccharide fermentation process. Similarly, in a study by these authors, substrate conversion was correlated to cellulase production trends, and it was concluded that higher enzyme activity results from low substrate concentration in the fungal media [12]. Therefore, it can be supposed that modeling enzyme and growth kinetics can provide useful insights into the control of the cell mass and enzyme activity in fungal fermentation systems.

Cellulase is a set of several different enzymes that act in synergy for the effective hydrolysis of cellulose. However, not all fungal species or their specific strains can produce all these enzymes, with most strains only producing a subset [6]. Therefore, for effective adaptation at industrial scales, microbial strains that can produce many different types of cellulases are preferred due to the production of an efficient enzyme cocktail. For example, Trichoderma reesei RUT-C30, which is a top producer of cellulase enzymes, produces higher amounts of exoglucanases [6]. Therefore, screening the best fungal strains for the desired cellulase is extremely important. With DDGS as the main fermentation feedstock, several A. niger and T. reesei strains showed promise for the production of many different types of fungal cellulases [6].

In addition to the ideal strains, a mixture or a co-culture of several fungal strains has also been explored for maximum cellulase production under submerged fermentation [80, 96]. While the efficacy of using multiple microbial strains for a specific outcome has been proven many times for maximum product formation, undesirable microbial metabolic interactions can also hinder optimal production. Therefore, co-cultures should be designed by their synergetic effect on each other to produce the maximum output.

Biofilm reactors are a special type of bioreactors, whereby the microbial species are facilitated with solid or semi-solid surfaces where they can form biofilms for the growth and production of desired products [97]. Several microbial species can form biofilms following mutual interactions to create the desired product formation. However, in the case of filamentous fungal species, the main incentive is to give fungus support for mycelial growth while tuning its environment via fermentation parameter optimization for maximum productivity. A recent example of this is shown in the study by Xiros and Studer [79], where they used multiple fungal species for cellulase activity. However, the concept is still in its infancy and should be explored further to assess the full potential of this technique for fungal submerged cellulase production.

Recently, a newer technique where the microbial medium is supplemented with inert microparticles, such as aluminum oxide, magnesium silicate, or titanium oxide, to control the morphology of filamentous fungi under submerged fermentation has been explored [15]. Several studies have been shown to increase microbial product formation, while decreasing the mycelial clumps in liquid media. Some examples include the studies conducted by Coban et al. [98] on phytase production and Coban and Demirci [99] on lactic acid production. Iram et al. [100] assessed the effect of microparticles (e.g., aluminum oxide and magnesium silicate) on fungal cellulase production using a shake-flask and 1.5 L benchtop bioreactor scales. The addition of microparticles increased cellulase production by several folds while decreasing the size of mycelial clumps in the media, resulting in a better distribution of nutrients and dissolved oxygen. These results show the positive effect of microparticles on fungal cellulase production under submerged fermentation.

Wild-type strains of fungi have been studied tremendously for cellulase production. The research studies underline the major drawbacks of wild-type strains, such as the unavailability of hyper-producing efficient strains, incapacity of industrial-scale production, and limited tolerance to extreme conditions in industrial settings. Improvement in the catalytic properties of cellulases can withstand industrial-scale requirements. The high demand for improved production and desired properties of cellulases underlines the importance of tailored genetic manipulations to construct novel fungal strains that can be exploited industrially. Time-consuming and tedious conventional genetic manipulations are also not viable solutions.

Innovative sequencing methods and molecular-level tools in the present times reinforce the efficacy of genetic manipulations of filamentous fungi for improved properties and industrial-scale production. Moreover, next-generation sequencing has enabled access to the genome sequences of several industrially appropriate filamentous fungal strains to design relevant studies. The most rigorously studied and the major cellulase-producer filamentous fungi include Trichoderma reesei [101], Aspergillus niger [22, 102], Neurospora crassa [103], and Penicillium oxalicum [104]. Natural processes are slow owing to the scarcity of bioavailable nutrient components; therefore, industrial scale-up is required to speed up the processes and cell factory manipulations to enhance their enzyme production speeds and capacities.

Implementation of metabolic engineering for efficient production and improved catalytic properties of cellulolytic enzymes by fungal strains is linked to understanding the mode of action of cellulases and their transcription level expression. As opposed to traditional genome editing tools, CRISPR/CAS has been exploited to test multiple target sites (Table 3, Ref. [102, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119]). Clustered, regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR-CAS) technology has emerged for developing competent genetically modified cellulolytic fungi strains. The preferred level of cellulase production and the futuristic approaches require a better understanding of the mode of action of cellulases and transcription level regulation of enzyme expression, which has progressed by utilizing technology such as CRISPR-CAS. Genetic manipulations are also employed to improve the enzymes’ properties, namely stereospecificity, enantioselectivity, substrate specificity, enzyme activity, enzyme stability, and tolerance to environmental factors.