Optimizing Cellulases Production by Trichoderma atroviride AD-130 under Submerged Fermentation using Low-Cost Substrates

A newly isolated strain of Trichoderma atroviride AD-130 was used to produce cellulases under submerged fermentation (SmF) using low-cost lignocellulosic substrates, water hyacinth, and municipal solid wastes. Although, the synthesis of β-glucosidase enzyme remains deficient in most of the Trichoderma spp. but the same was found considerably higher in the newly isolated fungus. Untreated and dried lignocellulosic biomasses of water hyacinth ( Eichhornia crassipes ) and municipal solid wastes were used as abundant and cost-effective substrates. Trichoderma atroviride AD-130 showed the highest cellulase activity on the fourth day of incubation at pH 7.0, temperature 30°C, 2% substrate concentration, and agitation speed of 180 rpm under submerged fermentation. The cellulase production decreased sharply when any of the optimized factors, whether pH, temperature, substrate concentration, or agitation speed was either increased or decreased beyond the optimized ones. Also, the poised proportion of FPase and β-glucosidase secreted by the cited fungus could have helped in completing the hydrolytic procedure efficiently.


Introduction
Over the long-time, cellulolytic enzymes have found their widespread applications in various industrial sectors (Kuhad et al., 2011) such as agriculture, textile, food, biofuels, etc. Concurrently, researchers have shown keen interest in these enzymes because of their role in the bioconversion of lignocellulosic biomasses (LB) into bioethanol as it decreases our dependency on petroleum fuel, enhances the environmental quality and reduces the burden of waste management practices (Devi et al., 2022).Studies have shown that low-cost lignocellulosic (LB) materials, such as wheat straw, rice straw, corn cob, and sugar cane straw, offer both environmental and socio-economic benefits (Lin, 2022).The conversion of lignocellulosic biomass (LB) to bioethanol is a three-step process: pretreatment, enzymatic hydrolysis, and fermentation.The first step is pretreatment which helps to breakdown the ligninhemicellulose-cellulose complex of any biomass and makes it more susceptible to enzymatic hydrolysis during which cellulase and xylanase enzymes act on the cellulosic and hemicellulosic components of the LB, converting them into their respective monomeric sugars (Romero-Martínez et al., 2021).These glucose units can then undergo fermentation to produce bioethanol.
Cellulose is a naturally occurring polysaccharide that is the main component of plant cell walls.It is a linear, unbranched polymer of glucose subunits.Glucose subunits can be obtained by depolymerizing plant cellulose into its smaller basic building blocks with the help of cellulases, a group of enzymes that includes β-glucosidases, cellobiohydrolases, and endoglucanases (Singhania et al., 2010).Bioethanol, which is produced by fermenting glucose subunits, has become increasingly important in recent years as a potential alternative to petroleum-based transportation fuels (Iqbal et al., 2022).One of the major factors owing to which this conversion is still not commercialized is the high price of commercial cellulases available in the market (Chen et al., 2022).
Albeit many studies recognized the synthesis of high of cellulases from the isolation and screening of various groups of microorganisms, dwelling in a broad range of environmental niches, including cellulolytic bacteria and fungi (Balla et al., 2022).However, higher levels of the enzymes are found to be produced by the fungi (mainly by filamentous fungi: Penicillium, Trichoderma, and Aspergillus species) than those collected from yeast and bacteria (Sulyman et al., 2020).Filamentous fungi, which are Generally Regarded as Safe (GRAS) strains, are the most commercially important microbes for cellulases production because they produce high levels of extracellular enzymes that can be easily recovered from the fermenting medium (Canassa & Baldin, 2022).Therefore, researchers are focusing on isolating and screening efficient lignocellulolytic enzymes that can produce high titers of cellulases (Duraimurugan et al., 2022).
Along with the isolated fungus, the choices of substrate and fermentation technology are another two important concerns that determine the success of cellulase production (Sahebzadeh et al., 2021).Moreover, to make the production of cellulase enzymes more economical, the exploitation of low-cost substrate as well as process optimization is of great consideration (Taiwo et al., 2022).Traditionally, submerged fermentation (SmF) is used for the large-scale production of cellulases because it is easier to handle and allows for better control over parameters such as pH, temperature, and substrate concentration (Sirohi et al., 2019).Therefore, envisaging the above facts, this study aimed to explore cellulases production using cost-effective feedstocks of Eichhornia crassipes (water hyacinth) and municipal solid waste (MSW) as substrates using newly isolated fungus Trichoderma atroviride AD-130 under submerged fermentation (SmF) conditions.Water hyacinth, an aquatic noxious weed capable to grow fast and having adverse impacts on aquatic life, can be seen as a potential feedstock for the production of valuable chemicals and fuels (Sindhu et al., 2017).Its biomass composition ranges from 20-30% cellulose, 40-48% hemicellulose, 13.5-16% lignin, and low lignin content makes it more favorable for its suitability as the feedstock for bioethanol production by its commercial cultivation (Rathod et al., 2018).Similarly, MSW was also used as a substrate because it is inexpensive, does not compete with food crops, and offers a waste management practice (Martau et al., 2021).

Collection and Preparation of Substrates
Lignocellulosic biomass (LB), viz., Eichhornia crassipes (water hyacinth) was collected from the pond in Kharawar village (28.8400°N and 76.6860°E) on NH 10, Rohtak and Municipal Solid Waste (MSW) from the dumping sites for garbage in Rohtak city (28.8909°N and 76.5796°E).The biomass was collected and subsequently subjected to a thorough washing with distilled water.Following the washing process, it was carefully dried in an oven at a temperature of 70°C.Both feedstocks underwent grinding to ensure they could pass through a sieve with a mesh size of 1.0 mm.The resulting material was then stored in separate sealed plastic bags, maintaining ambient room temperature, for future use in upcoming experiments.

Inoculum Preparation
To prepare the inoculum, 20-40 mL of sterilized normal saline solution containing 0.1% (v/v) Tween-80 was added to Trichoderma atroviride AD-130 slants that had been cultured on potato dextrose agar (PDA) media for 5 days.After harvesting the spores with a sterile wire loop, they were vigorously shaken and a homogenous microbial suspension containing 1x10 7 spores per one milliliter of inoculum was created.
The 'one variable at one-time' approach was utilized to optimize the culture parameters, including incubation period, pH, temperature, inducer substrate concentration, and agitation speed, to enhance the yield of cellulolytic enzymes.Specifically, for the incubation period variable, the fermentation was conducted for a total of 6 days.A series of flasks were harvested every 24 hours, beginning from the 48 th hour, and the resulting culture filtrate was employed for enzyme assays.The investigation of the remaining variables (pH, temperature, substrate concentration, and agitation speed) was solely conducted on the 4 th day, as it was determined to be the day of maximum enzyme production during the examination of the incubation period's effect.

Enzyme Assays
The method of Ghose (1987) was followed to examine the filter paperase (FPase) and carboxymethylcellulase (CMCase) activities.The activity of FPase was determined using the principle that 0.37 Filter Paper Units (FPU) of the enzyme would release 2.0 mg of glucose under standardized assay conditions.Similarly, the activity of CMCase (U/mL) was quantified based on the principle that 0.185 units of the enzyme would liberate 0.5 mg of glucose under the assay conditions.The assay for βglucosidase followed the method outlined by Ghose and Bisaria (1987), where its activity (U/mL) was defined as the amount of enzyme that releases one micromole of p-nitrophenol per minute under standardized assay conditions.

Estimation of Reducing Sugars
The amount of reducing sugars or equivalent release due to the activity of enzymes in various assays was determined with the help of the DNSA (3,5-dinitrosalicylic acid) method adopted by Miller (1959).

Statistical Analysis
All the experiments were performed in triplicate and the data was presented as an average mean ± standard error calculated in MS Excel.

Impact of Incubation Period on the Production of Cellulases
The incubation period is a critical factor that significantly impacts the production of cellulolytic enzymes and other metabolites.To analyze this effect, Figure 1 showcases the production of FPase, CMCase, and BGL at regular intervals using E. crassipes and Municipal Solid Waste (MSW).These experiments were conducted under submerged fermentation (SmF) conditions, maintaining a pH of 5.0, a temperature of 30°C, and an agitation speed of 180 rpm.
Figure 1 demonstrates the varying activity levels (U/mL) of FPase, CMCase, and BGL on E. crassipes, ranging from 0.24 to 0.68, 0.17 to 0.72, and 0.09 to 0.24, respectively.Notably, the highest activity levels for FPase, CMCase, and BGL (0.68, 0.72, and 0.24U/mL, respectively) on E. crassipes were observed on the 4 th day of the fermentation period.
Similarly, on the 4th day of the fermentation period, the highest values (U/mL) for FPase (0.92), CMCase (1.56), and BGL (0.69) on MSW were attained, as demonstrated in Figure 1, which showcases the production of these enzymes by T. atroviride AD-130.The production ranges for FPase, CMCase, and BGL on MSW were measured between 0.27 to 0.92U/mL, 0.31 to 1.56U/mL, and 0.17 to 0.69U/mL, respectively.
The activity levels of BGL, FPase, and CMCase showed an upward trend with the increase in the incubation period, reaching their maximum on the 4th day after inoculation for both E. crassipes and MSW substrates.Subsequently, there was a gradual decline in enzyme activity.Interestingly, when comparing the two substrates, MSW exhibited higher suitability for cellulase production during submerged fermentation, as evidenced by the higher levels of all cellulolytic enzymes produced.The observation of decreased cellulase production by T. atroviride AD-130 after the fourth day of incubation is not unique to this study.Similar findings have also been reported by Ogel et al. (2001) and Shruthi et al. (2020).Several factors may contribute to this decline, such as nutrient depletion in the medium (Bedade et al., 2017), the accumulation of cellobiose resulting during the breakdown of cellulosic material (Ojumu et al., 2003), or the release of phenolic compounds from residual lignin during the enzymatic hydrolysis process (Leu and Zhu, 2012).

Impact of pH on the Production of Cellulases
In this study, the influence of the initial pH of the culture medium on cellulases production by T. atroviride AD-130 was investigated in the context of submerged fermentation using E. crassipes and MSW as substrates.The pH of the culture medium was adjusted within the range of 4 to 8 using 1N HCl/1N NaOH, and the results are presented in Figure 2.
Figure 2 demonstrates that the highest (U/mL) of FPase, CMCase, and BGL (0.79, 1.19, and 0.67 respectively) were attained on the fourth day of incubation at 30°C, 180 rpm, using 1.0% (w/v) E. crassipes biomass as the substrate, with an initial pH of 7.0.Notably, a sharp decline in cellulase production was observed at pH 8.0.Similarly, when 1.0% (w/v) MSW was used as the substrate, Figure 2 also shows that the maximum activities (U/mL) of FPase, CMCase, and BGL were 1.14, 3.84, and 1.55 respectively, on the fourth day of incubation at pH 7.0, 30°C, and 180 rpm.
The results of the study revealed that MSW served as a more favorable carbon source for cellulase production compared to E. crassipes.This conclusion was based on the superior release of cellulases observed when MSW was utilized as a substrate.The results presented in this study demonstrate that the highest levels of FPase, CMCase, and BGL were achieved when T. atroviride AD-130 was cultivated in a medium with an initial pH of 7.0, using either E. crassipes or MSW residues as the substrate.
The pH of the culture medium could have influenced the microorganisms' morphology, thereby affecting the extracellular secretion of enzymes (Papagianni, 2004).Additionally, the neutral pH of 7.0 may have positively influenced the stability of the microbes.Similar findings have been reported by other researchers, such as Sherief et al., (2010) and Karmakar and Ray (2011), who also observed maximum cellulase production within the pH range of 5.0-7.0 in different Trichoderma spp.
Although the optimal pH for fungal cellulases varies among species, these enzymes generally exhibit high activity over a broad pH range, typically from 3.0 to 6.0 (Grigorevski-Lima et al., 2013).Different studies have reported varying pH ranges for achieving maximum cellulase production by different cellulolytic fungi.For instance, Deshpande et al. (2009) observed significant cellulases production by T. reesei within the pH range of 6.0 to 8.0, while Li et al. (2013) found that T. reesei exhibited maximum FPase, CMCase, and BGL production at pH 5.0, 4.5, and 5.5, respectively.
Given the wide variation in pH requirements among cellulolytic fungal species, a pH of 7.0 was selected as the optimized standard for analyzing successive factors, such as temperature, substrate concentration, and speed of agitation.

Impact of Temperature on the Production of Cellulases
The optimal temperature for cellulase production by T. atroviride AD-130 was determined by conducting experiments at different temperatures (ranging from 24°C to 37°C) using E. crassipes and MSW as substrates, under pH 7.0 and 180 rpm conditions.The results, depicted in Figure 3, demonstrate that the activity levels of FPase, CMCase, and BGL varied within the ranges of 0.23-0.79,0.11-1.19,and 0.16-0.67U/mL,respectively, on E. crassipes while on MSW, the activity levels ranged from 0.34-1.29U/mLfor FPase, 0.46-3.84U/mLfor CMCase, and 0.29-1.55U/mLfor BGL.
The cellulase production exhibited a gradual increase as the incubation temperature was raised from 24°C to 30°C.However, elevating the temperature further to 33°C or higher led to a significant decline in cellulase production.On the fourth day of submerged fermentation at 30°C, utilizing 1.0% (w/v) E. crassipes as the substrate, the highest levels of activity (U/mL) were observed for FPase (0.79), CMCase (1.19), and BGL (0.67).
Similarly, when 1.0% (w/v) MSW was used as the substrate, the maximum activity levels (U/mL) for FPase (1.29), CMCase (3.84), and BGL (1.55) were achieved on the 4 th day at 30°C.Furthermore, the choice of microorganism strain plays a crucial role in achieving efficient enzyme production.Various studies have highlighted different strains that demonstrate maximum cellulase production at 28°C.For instance, Trichoderma viride (Zhou et al., 2008), Trichoderma harzianum (Ahmed et al., 2009), Trichoderma asperullum (Bara et al., 2003), and Aspergillus niger (Kang et al., 2004) have all shown optimal cellulases production at the temperature of 28°C.Thus, based on various reports by researchers, it can be concluded that the optimal production of cellulases requires consideration of both the microorganism strain and the temperature variations of the culture conditions.Although most cellulolytic fungi are best suited for mesophilic conditions, some are also stable under thermophilic conditions.Unlike the present fungal isolate T. atroviride AD-130, which grows best at 30°C, other fungi may exhibit better growth under thermophilic conditions.

Impact of Substrate Concentration on the Production of Cellulases
In this study, the production of cellulases using MSW and E. crassipes as carbon sources investigated under submerged fermentation conditions, employing T. atroviride AD-130 with a substrate ratio of 1:4% (w/v).The culture flasks were incubated at 30°C and 180 rpm, with the initial pH adjusted to 7.0.The highest activity levels (U/mL) of FPase (1.48), CMCase (4.56), and BGL (1.67) were achieved using 2% MSW (w/v) as the substrate (Figure 4), while FPase (1.01), CMCase (1.28), and BGL (0.80) exhibited their highest activities using E. crassipes at a substrate concentration of 2% (w/v) (Figure 4).The production of all cellulases decreased at substrate concentrations higher than 2% (w/v).Corroboratively, Deshpande et al. (2009) also observed a marked reduction in cellulase yield with increasing substrate concentration.In various studies, researchers have investigated the production of cellulases by different fungi using various substrates, for example, Ibrahim et al. (2013) found that T. asperellum UPM1 and A. fumigatus had the maximum activity at 1% substrate concentration of pretreated oil palm empty fruit branch, while Irfan et al. (2011) observed higher cellulases production at substrate concentrations of 1.0% for rice straw and 1.5% for wheat straw.In the study conducted by Razak et al. (2012), T. asperellum UPM1 and A. fumigatus UPM2 were utilized for crude cellulases production using 4% untreated palm decanter cake as a substrate.This choice was based on the lower content of cellulose and hemicellulose present in palm decanter cake.Similarly, Linggang et al. (2012) achieved optimal cellulase production using 1% untreated pith of the sago plant.Another study by Pothiraj et al. (2016) demonstrated that, after 72 hours of incubation, T. viride yielded maximum cellulases activity of 68.3 IU whereas A. niger yielded 46.3 IU at 5% substrate concentration (w/v).
The studies show that an increase in substrate concentration can lead to reduced cellulase yield due to enzyme inactivation, which is influenced by induction and repression mechanisms (Jorgersen et al., 2003).Cellobiose has been identified as the key inducer in β-glucosidase production, but it can also act as an inhibitor at higher concentrations (Sukumaran et al., 2005).Additionally, it has been observed that increasing the substrate proportion leads to a decrease in medium porosity, which in turn affects the rate of oxygen transfer (Gomathi et al., 2012).Therefore, considering all these factors, it can be predicted that T. atroviride AD-130, which yields higher cellulases production on MSW than on E. crassipes, can induce cellulases in a balanced proportion at a substrate concentration of 2%.This is mainly because the chemical nature of both substrates varies due to differences in their cellulose and hemicellulose composition.

Impact of Agitation on the Production of Cellulases
In this study, the impact of agitation on cellulases production was examined in the flasks containing a substrate concentration of 2% (w/v) which were cultivated at an initial pH of 7.0 and various rotational speeds (60, 120, 180, 240, and 300 rpm) in an orbital shaker at 30°C.The results demonstrated that, on the fourth day of fermentation at 180 rpm, utilizing E. crassipes biomass as the carbon source resulted in a significant increase in FPase activity (1.01U/mL), CMCase activity (1.28U/mL), and BGL activity (0.80U/mL) (Figure 5), while employing MSW as the substrate led to higher levels of FPase (1.48U/mL), CMCase (4.56U/mL), and BGL (1.69U/mL) production (Figure 5).Agitation plays a dual role in enzyme production, as it promotes uniform nutrient distribution in the culture medium and provides aeration, which enhances fungal cell growth and enzyme yield (Matkar et al., 2013).Corroboratively, cellulase production was found to decrease significantly at much higher or lower agitation speeds.It is noteworthy that although the production of the enzyme β-glucosidase is deficient in most Trichoderma spp., the cited fungus produced sufficient amounts of this enzyme (Table 1).

Conclusion
In the present study, it was observed that the highest levels of FPase (1.01U/mL), CMCase (1.28U/mL), and BGL (0.80U/mL) on E. crassipes biomass, and FPase (1.48U/mL), CMCase (4.56U/mL), and BGL (1.69U/mL) on MSW were achieved at an agitation speed of 180 rpm.These results were obtained under submerged fermentation conditions with a substrate concentration of 2%, pH 7.0, and a temperature of 30°C after a four-day incubation period.Therefore, it can be concluded that T. atroviride AD-130 produced balanced amounts of all the cellulolytic extracellular enzymes, including FPase, CMCase, and BGL, enabling efficient hydrolysis of both biomasses of MSW and Eichhornia crassipes.Notably, although BGL production is usually deficient in most Trichoderma spp., the cited fungus produced sufficient amounts of all the cellulase enzymes.The proportion of FPase and BGL secreted by T. atroviride AD-130 may have facilitated the complete conversion of cellulose to glucose, thereby eliminating cellobiose inhibition.Therefore, , T. atroviride AD-130 could be a suitable candidate for the commercial production of cellulases because the results indicate its potential for saccharifying lignocellulosic biomass into reducing sugars, which can then be utilized for the large-scale production of bioethanol.However, further optimization of the inoculum age, inoculum size, use of inducers (such as lactose or cellobiose), substrate particle size, and culture medium may improve cellulases production by T. atroviride AD-130.

Figure 1 .
Figure 1.Effect of incubation time on cellulases production by T. atroviride AD-130 under SmF using E. crassipes (EC: 1% w/v) and MSW (1%) residues as substrate Several studies, including those conducted by Gomathi et al. (2012) and Dedavid et al. (2009), have reported varying findings regarding the optimal incubation times for achieving maximum cellulases production in different fungi.Singh et al. (2009) observed that the highest activity levels of FPase and CMCase (3.2 IU/mL and 83 IU/mL, respectively) were achieved on the 5 th day of incubation at a temperature of 30°C.In a separate study conducted by Sarkar and Aikat (2014), notable levels (IU/mL) of CMCase (2.31), FPase (0.261), and xylanase (48.33) were achieved after a 5-day incubation period.However, as the incubation time increased beyond 5 days, there was a significant decrease in enzyme activity.Likewise, Naher et al. (2021) observed the highest production of cellulases using T. reesei and A. awamori through submerged fermentation of rice straw on the 5 th and 3 rd days of incubation, respectively.

Figure 3 .
Figure 3.Effect of temperature on cellulases production by T. atroviride AD-130 under SmF using E. crassipes (EC: 1% w/v) and MSW (1% w/v) residues as substrate Various studies, including Kang et al. (2004) and Jiang et al. (2011), have testified optimized temperature ranges of 25-30°C for generating cellulases using Aspergillus and Trichoderma spp.Furthermore, the choice of microorganism strain plays a crucial role in achieving efficient enzyme production.Various studies have highlighted different strains that demonstrate maximum cellulase production at 28°C.For instance, Trichoderma viride(Zhou et al., 2008), Trichoderma harzianum(Ahmed et al., 2009), Trichoderma asperullum(Bara et al., 2003), and Aspergillus niger(Kang et al., 2004) have all shown optimal cellulases production at the temperature of 28°C.

Table 1 .
Cellulases Production by T. atroviride AD-130 Using MSW and E. crassipes Biomasses as Carbon Source at 2% (w/v) Concentration: Submerged Fermentation Study at pH 7.0, 30°C, 180 rpm on the 4th Day of Incubation