Optimization of Production and Characterization of Cellulase Enzymes from <i>Nigrospora oryzae </i>(Berk and Br.) Petch for Biomass Saccharification and Ethanol Production

Dukuzimana Olivier; George Omwenga Isanda; Mathew Piero Ngugi

doi:10.20944/preprints202504.1594.v1

Submitted:

17 April 2025

Posted:

21 April 2025

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Abstract

This study aimed to isolate and optimize cellulase production by Nigrospora oryzae from decaying wood. N.oryzae was screened for cellulase enzymes production using carboxyl methyl cellulase congo red. Cellulase enzymes production was carried out using maize cobs and sugarcane bagasse as substrates and optimize using one variable at a time procedure. Maximum total cellulase activity (Fpase) were 11.3±0.94 IU/ml and 8.9±0.47IU/ml on maize cobs and sugarcane bagasse respectively after 9 days of incubation. Highest endoglucanase activity were 19.7±1.74 IU/ml and 15.5±0.76 IU/ml of maize cobs and sugarcane bagasse respectively after 12days of incubation. Maximum exoglucanase activity were 3.46 ±0.25IU/ml and 2.06±0.11IU/ml on maize cobs and sugarcane bagasse after 9 and 15 days of incubation respectively. Effect of pH on cellulase production varied on the substrates after 3 and 6 days of incubation. Inorganic nitrogen sources (NH4)2SO4 and NH4N03 organic peptone and urea significantly increased cellulase production. Several soluble carbon sources such as fructose, mannitol, galactose, sucrose and glucose also enhance cellulase enzyme production. A mixture substrate ratio of 1:2.5to 1:3.0 resulted in maximum cellulase production. Enzyme laoding of 5% and substrate loading of 15% for maize cobs and 10% sugarcane bagasse resulted. In maximum reducing sugars production Maximum ethanol was obtained with substrate concentration 5%, 10% and 15% by simultaneous saccharification and fermentation with saccharomyces cerevisaie and 10FPu enzymes.

Keywords:

maize cobs

;

sugarcane bagasse

;

Nigrospora oryzae

;

fermentation

;

enzymes production

;

pH

;

incubation time moisture

;

nitrogen and carbon supplementation

;

surfactants

;

saccharification

;

ethanol production

Subject:

Biology and Life Sciences - Biology and Biotechnology

1. Introduction

Energy and a clean ecosystem are the key ingredient of human life in the world. Currently there is a worldwide energy insufficient problematic , extremely in developing countries (Sulyman et al.,2020) brought about by global fossil fuel demand-supply problems, such as excessive exploitation and utilization, brutality and economic and government disasters around the supply regionals, increasing severe pollution, and climate change make renewable energy responses more necessary now than before. Applying renewable energy source to replace fossil fuels reducing world warming and ecosystem degradation, since about 85% of the annual greenhouse gas emission due to fossil energy use. On the opposite, proper use of biomass e.g lignocellulose biomass does not result in ecosystem degradation, making biomass strengthen energy source with great accessibility (Amaefule et al., 2023).

Lignocellulose plant biomass is one of the most abundant, economic and renewable resource that can be using in production value added products such as biofuels and as substrates for the enzymes production of such as cellulases (Kim, et al., 2014; Visser, et al., 2015). Lignocellulose is the main compound of the plant cell wall and is essentially major unit of cellulose (35-50%), hemicellulose (25-30%) and lignin (25-30%) (Mostafa et al, 2024). Lignocellulose is efficiently hydrolyzed and recycled in nature by microorganism that produce 3 classes of enzymes-hydrolases, that can involve cellulases and hemicellulases; lyases and oxidoreductases, that can be involved the lignin degrading enzymes (Christopher, et al., 2023). Cellulose is a polysaccharide of glucose monomers linked by β-1,4-glycosidic bonds. Cellulose can be hydrolyzed to glucose by acid treatment or by cellulase enzymes. Cellulases are a complex of three types of enzymes namely endoglucanse, cellobiohydrolase (exoglucanase) and β-glycosidase it works cooperatively to hydrolyze cellulose to glucose (Escuder-Rodríguez et al., 2018). Endoglucanases randomly cleave the β-1,4-D-glycosidic affinity to generate new reducing and non-reducing ends of cellulose molecules. Exoglucanases further break down cellulose chains into cellobiose units at their ends. Finally, β-glucosidases hydrolyze glucose dimers and oligomers into individual glucose molecules completing the hydrolysis process (Deswal, et al., 2011).

Production of cellulase has been produced from a broad different microorganism including bacteria and fungi. Therefore, filamentous fungi are more suitable commercial enzyme production, due to their amount of the enzymes that can be produced by these cultivation great than those found from yeast and bacteria. Industrially fundamental cellulase enzymes traditionally have been found in submerged fermentation (SmF) due to ease of manipulation , high control of environmental factors such as temperature and pH, using cellulose of different purity as substrates, which leads to high costs of the product because of low concentration of the enzyme and additional cost of downstream purification processes (Yoo, et al., 2014). Therefore, solid state fermentation (SSF) method ameliorated production and reducing the cost of enzyme produced. Filamentous fungi are the most common utilized microorganisms in SSF due to easy possibility to grow on solid materials with low water content. However a number of records describing utilizing agro industrial wastes in production of cellulase enzyme including wheat straw, wheat bran and rice straw as substrates. Filamentous fungi have been shown to grow well on these solid materials which resembles the conditions of their natural habitat (Mostafa, et al., 2024) and secrete the enzymes extracellularly (Behera and Ray, 2016). The other benefits of SSF include high productivity, simple method to use; little cost investment, little energy required and less water output, great product recovery and absence of foam build up and is recorded to be preferable proper procedure for cellulase enzyme production in developing countries (MaMrudula, 2011). Currently there is a great demand for cellulases in many industries such as food, paper and pulp, textiles, pharmaceuticals, alcoholic beverages, starch processing, and biofuel production (Boondaeng, 2024 ).

Cellulases is the 3rd largest industrial enzymes and its demand is projected to grow strongly in the future due to commercial biofuel production ( Szijártó, et al., 2013; Rodrigues and Odaneth, 2021). However, this will require a reduction in the cost of enzyme production to make the process economically feasible (Bhardwaj et al., 2021). Cellulase enzymes production is an inducible process affected by biochemical and physical parameters namely, microbial species, carbon source, incubation period, temperature, pH,etc., (Kuhad et al., 2011 ; Sharada et al., 2013 ; Patel et al., 2014). Different cellulase producing microorganisms require optimization of nutritional parameters such as carbon, nitrogen and mineral sources and physical parameters include pH and temperature (Sethi et al., 2013) for enhanced enzyme production. When the enzyme is produced, the cellulase enzyme can be practiced for different purposes such as saccharification of lignocellulose in production of glucose that can be fermented to bioethanol (Srivastava et al., 2023). In this study, Nigrospora oryzae was isolated and identified from decaying wood, and used to produce cellulases under SSF using locally sourced maize cobs and sugarcane bagasse as substrates. Various nutritional and physical chemical parameters were evaluated the maximum cellulase production using one variable at time (OVAT) approach. Factors contributing to high cellulase enzyme production were applied in enzyme production and whole fermented broth obtained was used for saccharification of maize cobs and sugarcane bagasse for production of ethanol utilized simultaneous saccharification and fermentation with Saccharomyces cerevisiae.

2. Materials and Methods

2.1. Collection of Samples

Decaying wood specimens were collected from Kenyatta university environments. Samples were taken to the Laboratory of Microbial Biotechnology, Kenyatta University and stored at appropriate required temperature.

2.2. Isolation of Fungi

The decaying wood samples were sectioned into 1cm×2cm pieces. Each specimen were sterilized using 1% sodium hypochlorite for 1 min and rinsed five times with sterile distilled water and then both dried with sterilize filter paper. Samples were directly plated on 1% carboxymethycellulose (CMC)-congo red agar plates supplemented with 200 mg/l gentamycin. The plates were incubated at accepted temperature and monitored daily for fungal growth. Emerging calories were sub-cultured into CMC agar media for isolation of cellulase decaying fungi.

2.3. Subculturing Cellulolytic Fungi

Samples growing on CMC- Congo red agar were subculture on potato dextrose agar (PDA) medium prepared from an infusion of 200 g of red potato, 40 g of dextrose and 15 g of agar and sterilized at 121⁰C,15 Psi for 15 minutes. The medium was supplemented with 200 mg/l gentamycin to inhibit bacterial growth.

2.4. Molecular Identification of Nigrospora oryzae

Total genome DNA was isolated as described by Xin and chen j (2012). The ITS sequence of Nigrospora aryzae was amplified by PCR utilizing ITS4R (5-TCCTCCGCTTATTGATATGC-3’) and ITS86F(5-GTGAATCATCGAATCTTTGAA-3’) primers (Op deBeeck et al, 2014). Total volume of 22 ul PCR reaction mixture was used consisting of 10.6 ul ad H₂0 ,2 ul forward, primers, 2 ul reverse primers ,4 ul myTaq buffer ,04 units 0.4 ul) of myTaq polymerase (bioline USA) and 3 ng template DNA. PCR conditions were as follow cycle at 95⁰C initial denaturation for 3 minutes; 35 cycles of 95⁰c denaturation for 3 seconds ,55⁰C annealing for 30 seconds and extension for 1 minute at 72⁰C. A final extension at 72⁰C for 10 minutes and holding stage at 5⁰C. The PCR products were resolved on 1.2% agarose gel. The PCR products were cleared and sent for sequencing using sanger dideoxy sequencing by Microgen Inc (Netherlands).

2.5. Solid State Fermentation (SSF) for Cellulase Enzymes Production

2.5.1. Cellulosic Substrates

Sugarcane bagasse and maize cobs were locally sourced and evaluated as substrates production of cellulase enzymes. The cellulase substrate were ground using electric mill and sieved through a 2 mm sieve.

2.5.2. Solid State Fermentation (SSF) for Cellulase Enzymes Production

SSF was used for cellulase enzymes production in 350ml bottles. Five (5) g of substrate (sugarcane bagasse and maize cobs) were placed in the bottles and moistened with 10 ml of Bushnell-Hans solution [(w/v) 0.02 g CaCl₂ ,0.2 MgSO₄•7H₂O g, 1g KH₂PO₄ ,0.1g FeCl₃, 1g NH₄NO₃, 1g peptone, pH5.3]. The contain within the bottles were autoclaved at 121⁰C, 15 psi for 15 minutes. The bottles were inoculated with inoculum after cooling to room temperature.

2.5.3. Preparation of Inoculum

Seven (7) day old cultures of N. oryzae cultured on PDA were used to prepare inoculum. The fungal mycelium was scrapped from the plates and mixed with sterile physiological saline solution and then blended for 30 seconds. The mycelia mixture was used as SSF inoculum.

2.5.4. Enzyme Extraction

Crude cellulase enzyme was extracted by adding 50 ml of 50 mM citrate buffer pH 4.8 to each bottle. The bottles were vigorously shaken and left standing at the room temperature for 2 hours to extract the enzyme. The crude enzymes extract was filtered through a muslin cloth and a centrifuged at 10,000rpm for 10 minutes. The enzyme extract was stored at -20⁰C until used for bioassays.

2.6. Cellulase Enzyme Assays

The crude enzyme extracts were utilized to determine Total cellulase activity (Fpase), Endoglucanase and exoglycanase activity as described by Kamande, et al., (2024). One unit of Fpase, endoglucanase and exoglucanase was described as the amount of enzymes releasing 1umol of reducing sugar per minute, under assay conditions.

2.7. Optimization of Culture Conditions for Enzyme Production

Production of cellulase enzyme was optimized by one variable at a time (OVAT) approach. Effect of time of incubation on cellulase enzyme production was determined by taking samples at 3 days intervals up 18 days and determining enzyme activity. Effect of pH was determined by adjusting initial pH of Bushenell-Haas, solution to pH (2-8) before dispensing to bottles with substrates. Effect of moisture was determined by adding Bushnell-Haas solution at the following ratios (w/v): 1:1, 1:1.5, 1:2.0, 1:2.5, 1:3.0, 1:3.5, 1:4.0 and 1:4.5 to substrate containing bottles. Effect of Nitrogen were determined by supplementing Bushnell-Hass solution with peptone, urea, asparagine, yeast extract, NH₄N0_3, (NH₄)₂SO₄, and NaNO₃, and dispensing 10 ml to substrate containing bottles at final concentration of 20 mM and 80 mM. Concentration of yeast extract and peptone were determined using their percentage nitrogen content. Effect of supplemental carbon was done by adding glucose, galactose, fructose, sucrose, mannitol and maltose at 20 mM, and 80mM. Effect of surfactants was determined by incorporating 0.4% and 1.2% Tween-20, Triton X-100 and Sodium dodecyl sulfate (SDS). For effect of nitrogen, carbon and surfactants, bottles without supplementation were used as control and samples were taken on the 3rd and 6th days for enzyme bioassays.

2.8. Production of Cellulase Enzymes for Saccharification and Ethanol Production

The time of incubation and various physiochemical and nutritional parameters giving maximum enzyme activity were used to produce cellulases for saccharification and ethanol production using maize cobs and sugarcane bagasse. The produced cellulases were concentrated five-fold by freeze drying.

2.9. Effect of Enzymes Loading on Reducing Sugar Production Utilizing Maize Cobs and Sugarcane Bagasse Substrates and Nigrospora oryzae Cellulase Enzyme Extract

The effect of cellulase enzyme loading on reducing sugar production from maize cobs and sugarcane bagasse was determined using 5% maize cobs and sugarcane bagasse and 1%,2%,3%,4% and 5% enzymes loading over 72 hour incubation period. The total saccharification mixture of 25 ml contained substrate, enzyme, 100 mg/l gentamycin and fluconazole. Samples were taken at 12 hour intervals and the amount of reducing sugars produced were determined by the DNS method (Kamande, et al., 2024).

2.10. Effect of Substrate Loading on Reducing Sugar Production by Nigrospora oryzae Cellulase Enzymes

The effect of different substrate loading on reducing sugar production from maize cobs and sugarcane bagasse using Nigrospora oryzae cellulase enzymes, were determined by using 1%, 5%, 10% and 15% substrate loading over a 72-hour incubation period. Samples were taken at 12hour intervals and reducing sugars produced determined by the DNS method (Kamande, et al., 2024).

2.11. Simultaneous Saccharification and Fermentation of Maize Cobs and Sugarcane Bagasse Using Nigrospora oryzae Cellulase and Saccharomyces cerevisiae

Simultaneous saccharification fermentation was used to produce ethanol from maize cobs and sugarcane bagasse using Nigrospora oryzae cellulase and Saccharomyces cerevisiae with over a 72-hour period. The fermentation mixture contained, the substrate, the optimized cellulase enzyme titer, 1x10⁶ ml yeast cells, and 100mg/l gentamycin in a total volume of 25 ml using Falcon tubes. Five (5) FPU of commercial cellulase was used as positive control. Samples were taken at 12 hours’ interval and ethanol produced determined by the Potassium dichromate method (Caputi, et al., 1968) and quantified using an ethanol standard curve.

2.12. Statistical Analysis

Enzyme activity absorbance data was transformed into international units (IU/ml) utilizing glucose standard curves as detailed Rahnama et al.2024. Ethanol produced found in triplicates was typed in Excel spreadsheet and transformed into mg/ml of ethanol. The data was imported in R software version 3.5.1 (2018) and evaluated for normality utilizing Shapiro-Wilk and equality of variance utilizing Levene's test. Data in triplicates were statistically analyzed utilizing one-way ANOVA at P ≤ 0.05 significant level. Any significant varied in the factors affecting enzyme and ethanol product were determined by Tukey's HSD Post Hoc test. Molecular analysis sequences were compared with the sequences in the nucleotide database (NCBI) utilized BLAST technique. The MEGA software version X program was utilized for phylogenetic analysis and multiple alignments utilized CLUSTAL (Horiike, T ;2016). Tables and figures were utilized to present the data.

3. Results

3.1. Isolation of Nigrospora Oryzae

The ability of the fungus designated as KU_05 to grow on 1% carboxymethyl cellulose and congo red (Figure 1A) was used to determine its cellulolytic activity (Figure 1B)

Molecular Identification of Nigrospora oryzae

Nigrospora oryzae was identified based on the internal transcribed spacer region (ITS) sequence located between the 18S rRNA and 5.8S rRNA coding genes. This regional is widely utilizing for analyzing fungal diversity (Rittenour, et al., 2014). The alignment of the ITS fragment (574bp) in the NCBI database by the BLASTN program reported that it was Nigrospora oryzae (97.5% identity). The result of the nucleotide sequence of 5.8S rRNA were deposited in the GeneBank database under the accession number PQ896652.

The evolutionary history of Nigrospora oryzae was inferred using the Neighbor-Joining technique (Saitou et al., 1987). The percentage of replicate trees that can be associated with taxa clustered cooperation in the bootstrap test (1000 replicates) are indicated above the branches (Felsenstein; 1985). The evolutionary distances were computed utilizing the Kimura 2-parameter method (Kimura M.; 1980) and are in the units of the number of base substitutions per site. The rate of variation between sites was modeled with a gamma distribution (shape parameter = 1). Evolutionary analyses were carry out inMEGA11 (Tamura et al., 2021) (Figure 2)

3.2. Effect of Incubation in Production of Celluloses Enzymes by Nigrospora oryzae

Incubation time take a key fundamentals of metabolic activity of microorganisms since it is directly associated to the production of enzymes and various metabolites. Fpase production increased from 3rd day of incubation and reached a maximum on the 9th day at 11.3 ±0.94 IU/ml for maize cobs and decreasing 2.6 fold by the 18th day of cultivation. On sugarcane bagasse, the highest Fpase was reported on the 9th day which however was not significantly higher than that reported on the 3rd and 6th days of cultivation. Fpase production on the two substrate did not differ significantly except on the 15th day of incubation (Table 1). Endoglucanase production on maize cobs reached a maximum at 19.7±1.74IU/ml on the 12th of cultivation which did not differ significantly from that reported on the 3rd and 15th days of incubation. On sugarcane bagasse, the highest endoglucanase activity was reported of 15.5 ±0.76 IU/ml on the 12th day of cultivation which was only significantly higher than that reported on the 6th and 18th day of incubation (Table 1). Endoglucanase activities between the two substrates were significantly different only the 18th day of incubation. Exoglucanase production on maize cobs peaked at 3.46±0.25IU/ml on the 9th day of cultivation but was not significantly higher than that reported on the 3rd and 6th days of incubation. On sugarcane bagasse, the highest exoglucanase activity was 1.96 ±0.16 IU/ml which was only significantly higher than that reported on the 18th day of cultivation. Significantly higher exoglucanase activities were reported on maize cobs compared to sugarcane bagasse over the incubation period except on the 12th day of cultivation at which they did not significantly differ (Table 1).

3.3. Effect of pH on Production of Cellulase Enzymes by Nigrospora oryzae

pH is fundamentals key element in production of cellulase enzymes. The control of pH during the growth cellulase producing microorganisms is important as it affects the morphology of the microorganisms and their ability to secrete cellulase enzymes. The highest Fpase activities were recorded on the 3rd day of incubation and were 19.0±1.33 IU/ml for maize cobs and 19.1±1.0 IU/ml for sugarcane bagasse recorded at pH 7 and 3 respectively (Table 2). These Fpase activities did not differ significantly from those recorded with the other pH values. On the 6th day of cultivation, the highest Fpase activity on maize cobs was 18.6±1.62 IU/ml recorded with pH 5.0 which was only significantly higher that reported at pH 2, 3 and 8. On sugarcane bagasse, the highest Fpase recorded on the pH 2 on the 6th day. Nigrospora oryzae produced endoglucanase activities that did not differ significantly over the pH values on the 3rd day of incubation when cultivated on maize cobs (Table 2). On sugarcane bagasse, the highest endoglucanase activity of 42.6±0.86 IU/ml was produced at pH 4 and was significantly higher compared to the other pH values. On the 6th day of cultivation the highest endoglucanase activity on maize cobs was 33.4±1.27 IU/ml recorded on pH 2 and was only significantly higher than that reported at pH 3. On sugarcane bagasse, the highest endoglucanase activity on the 6th day of cultivation was 33.0±1.160IU/ml reported on pH 2 and was only significantly higher than that recorded at pH 7. Exoglucanase activities on maize cobs and sugarcane bagasse showed interesting trends at the different pHs tested. On maize cobs the highest exoglucanase activity was 6.02 ±0.03 IU/ml on the 3rd day of cultivation which however did not differ significantly from those reported on the other pH values. For sugarcane bagasse the highest exoglucanase activity of 7.85 ±0.31 IU/ml was reported at pH 8 on the 3rd day of cultivation and was only significantly higher than that reported on the pH 7. On the 6th day of cultivation the highest exoglucanase activity of 7.58 ±0.47 IU/ml reported on maize cobs at pH 2.0 was not significantly varied from those reported on the other pH values. On sugarcane bagasse highest exoglucanase of 9.13 ±0.68 IU/ml at pH 2.0 was not significantly higher than that reported with pH 6 after 6 days of incubation.

3.4. Effect of Nitrogen Supplementation on Production of Cellulase Enzymes by Nigrospora oryzae

The type of and amount of nitrogen are fundamentals factors in the synthesis and secretion of enzymes and other microbial proteins. Therefore, the best nitrogen source is the one that initiate enzymes synthesis and secretion. In this study seven different kinds of nitrogen sources were investigated for the production of cellulase by Nigrospora oryzae at two concentrations (20 mM and 80 mM) at two incubation periods (3 and 6). The results are summarized in Table 3a–f The highest Fpase activity on maize cobs supplemented with 20mM was 24.413 ±3.26 IU/ml using (NH₄)₂SO₄ which was not significantly different to with Fpase recorded with asparagine, peptone and urea after 3 days of incubation. Increasing the concentration to 80mM resulted in the highest Fpase activity of 25.4 ±0.95 IU/ml with urea supplementation which was not significantly higher than supplementation with asparagine, NH₄N0_3, peptone and yeast extract. On sugar cane bagasse the highest Fpase activity was 19.6±1.66 IU/ml with 20 mM supplementation with (NH₄)₂SO₄ which was only significantly higher than supplementation with asparagine and NaNO₃. Increasing the concentration of nitrogen to 80 mM resulted in highest Fpase activity of 23.0±1.42 IU/ml with (NH₄)₂SO₄ which was statistically comparable to that produced by peptone and yeast extract on maize cobs. On the 6th day of incubation supplementation with 20 mM and 80 mM of (NH₄)₂SO₄ resulted in the highest Fpase activities of 23.3±1.64 IU/ml and 24.5±1.46 IU/ml on maize cobs respectively. On sugarcane bagasse the highest Fpase activities of 18.9 ±0.78 IU/ml and 20.8±1.15 IU/ml were reported with 20mM (NH₄)₂SO₄ and 80mM urea respectively. Endoglucanase activity produced by Nigrospora oryzae varied with the type of nitrogen and substrate used. The highest endoglucanase activity recorded on the 3rd day of incubation was 45.3 ±1.47 IU/ml recorded with 20 mM NH₄N0₃ which was significantly higher compared to those produced by the other nitrogen sources and control. Similarly increasing the nitrogen concentration to 80 mM resulted in the highest endoglucanase activity of 38.4±1.160 IU/ml with NH₄N0₃ which was only significantly higher than that recorded with urea supplementation and in the control. On sugarcane bagasse the highest endoglucanase activities of 31.2 ±0.30 IU/ml and 42.1±0.81 IU/ml were reported with 20mM peptone and 80 mM (NH₄)₂SO₄ respectively. These endoglucanase activities were significantly higher compared with that recorded in control and with the other nitrogen sources. On the 6th day of incubation 20 mM urea and 80 mM peptone produced the highest endoglucanase activity of 42.7 ±1.52 IU/ml and 40.1±1.33 IU/ml accordingly on maize cobs. On sugarcane bagasse 20 mM NH₄N0₃ and 80 mM NaN0₃ supplementation resulted in the highest endoglucanase activities of 33.5±0.95 IU/ml and 37.3±0.56 IU/ml respectively. The highest exoglucanase activities on maize cobs on 3rd day of incubation were 9.66±1.12 IU/ml and 8.8±0.57 IU/ml recorded with 20 mM and 80 mM supplementation with NH₄N0_3. On sugarcane bagasse 20 mM peptone and 80 mM (NH₄)₂SO₄ supplementation resulted in exoglucanase activities of 8.70±0.65 IU/ml and 8.62±0.32 IU/ml respectively. On the 6th day of incubation the highest exoglucanase activity on maize cobs was 8.96 ±0.78 IU/ml with 20 mM yeast extract which was only significantly higher than that recorded with 20 mM asparagine, NaN0₃ and in control_. Increasing the concentration to 80 mM resulted in the highest exoglucanase activity of 7.92±0.46 IU/ml in control which was only significantly higher that supplementation with 80mM (NH₄)₂SO_4. On sugarcane bagasse the highest exoglucanase activity was achieved on the control which was only significantly higher compared to supplemented with 20 mM urea, peptone and yeast extract. Increasing the concentration of nitrogen supplementation to 80 mM resulted in the highest exoglucanase of 7.18±0.22 IU/ml with NaN0₃ which was only significantly higher compared with supplementation with NH₄N0_3.

Table 3a. Filter paper activity after Nitrogen supplementation at 3rd day of incubation.

Substate	Nitrogen	Mean ± sem	Mean ± sem
		20 mM	80 mM
	Control	16.3 ± 1.08a	11.2 ± 0.78c
	(NH₄)₂SO₄ Asparagine	23.6 ± 3.26a 20.2 ± 1.33a	11.8 ± 0.45c 20.4 ± 0.97ab
Maize cobs	NaNO₃ NH₄NO₃ Peptone	17.6 ± 1.26a 17.4 ± 1.67a 18.3 ± 0.76a	19.8 ± 1.42b 24.1 ± 1.80ab 21.2 ± 1.08ab
	Urea	20.1 ± 1.49a	25.4 ± 0.95a
	Yeast Extract	17.8 ± 1.29a	25.1 ± 1.18ab
	Control	16.3 ± 0.80ab	10.7 ± 0.25d
	(NH₄)₂SO₄ Asparagine	19.6 ± 1.66a 11.8 ± 1.20b	23.0 ± 1.42a 15.4 ± 0.90c
Sugarcane bagasse	NaNO₃ NH₄NO₃ Peptone	13.6 ± 1.24b 19.6 ± 1.43a 16.2 ± 0.90ab	16.4 ± 0.49c 18.5 ± 0.74bc 19.4 ± 0.96abc
	Urea	16.6 ± 0.97ab	17.8 ± 0.49c
	Yeast Extract	13.7 ± 0.80ab	22.3 ± 1.06ab

Note: Treatments with means of varied letter in the similar columns for substrate are significantly varied at p≤0.05.

Table 3b. Filter paper activity after Nitrogen supplementation at 6th day of incubation.

Substate	Nitrogen	Mean ± sem	Mean ± sem
		20 mM	80 mM
	Control	14.8 ± 0.83b	16.2 ± 1.21bc
	(NH₄)₂SO₄ Asparagine	18.1 ± 1.61ab 16.7 ± 1.06b	18.5 ± 0.65b 16.8 ± 0.88bc
Maize cobs	NaNO₃ NH₄NO₃ Peptone	17.4 ± 1.19ab 23.3 ± 1.64a 20.3 ± 0.85ab	19.2 ± 0.84b 24.5 ± 1.46a 20.7 ± 1.22ab
	Urea	16.5 ± 1.12b	13.6 ± 0.92cd
	Yeast Extract	19.2 ± 1.18ab	10.4 ± 0.40d
	Control	16.7 ± 1.39ab	12.0 ± 0.47d
	(NH₄)₂SO₄ Asparagine	16.1 ± 0.14ab 10.2 ± 0.46d	16.4 ± 0.15bc 17.1 ± 0.94bc
Sugarcane bagasse	NaNO₃ NH₄NO₃ Peptone	13.8 ± 0.70bc 18.9 ± 0.78a 15.8 ± 0.46ab	12.8 ± 0.52d 19.8 ± 0.80ab 14.4 ± 0.70cd
	Urea	13.6 ± 0.35bcd	20.8 ± 1.15a
	Yeast Extract	12.0 ± 0.58cdb	17.9 ± 0.98abc

Note: Treatments with means of varied letter in the similar columns for substrate are significantly varied at p≤0.05.

Table 3c. Endoglucanase activity after Nitrogen supplementation at 3rd day of incubation.

Substate	Nitrogen	Mean ± sem	Mean ± sem
		20 mM	80 mM
	Control	29.3 ± 1.38cd	21.0 ± 0.27c
	(NH₄)₂SO₄ Asparagine	24.7 ± 1.39de 35.7 ± 1.78b	38.2 ± 3.45a 33.4 ± 1.93ab
Maize cobs	NaNO₃ NH₄NO₃ Peptone	33.1 ± 1.19bc 45.3 ± 1.47a 35.0 ± 0.57bc	31.4 ± 0.59ab 38.4 ± 1.60a 32.8 ± 1.05ab
	Urea	33.0 ± 0.94bc	27.2 ± 0.84bc
	Yeast Extract	20.4 ± 0.50e	32.2 ± 1.43ab
	Control	23.8 ± 0.66b	25.5 ± 1.60cd
	(NH₄)₂SO₄ Asparagine	20.8 ± 0.98bc 15.7 ± 0.24de	27.5 ± 0.94bc 29.8 ± 0.40b
Sugarcane bagasse	NaNO₃ NH₄NO₃ Peptone	15.0 ± 0.58e 18.1 ± 0.43cde 31.2 ± 0.3a	19.6 ± 0.31e 42.1 ± 0.81a 21.6 ± 0.73de
	Urea	18.5 ± 0.92cd	15.4 ± 0.20f
	Yeast Extract	16.5 ± 0.80de	25.4 ± 0.88cd

Note: Treatments with means of varied letter in the similar columns for substrate are significantly varied at p≤0.05.

Table 3d. Endoglucanase activity after Nitrogen supplementation at 6th day of incubation.

Substate	Nitrogen	Mean ± sem	Mean ± sem
		20 mM	80 mM
	Control	28.7 ± 1.15bc	26.5 ± 0.74bc
	(NH₄)₂SO₄ Asparagine	27.2 ± 1.71c 33.8 ± 1.65b	35.5 ± 4.82ab 25.7 ± 0.63bc
Maize cobs	NaNO₃ NH₄NO₃ Peptone	27.3 ± 0.34bc 27.5 ± 0.89bc 28.2 ± 0.58bc	28.6 ± 1.35abc 27.3 ± 1.00bc 40.1 ± 1.23a
	Urea	42.7 ± 1.52a	30.7 ± 0.93ab
	Yeast Extract	26.5 ± 1.39c	17.9 ± 0.48c
	Control	24.8 ± 0.97c	18.7 ± 1.18d
	(NH₄)₂SO₄ Asparagine	30.9 ± 1.43ab 17.0 ± 0.68d	24.0 ± 0.98c 29.7 ± 0.86b
Sugarcane bagasse	NaNO₃ NH₄NO₃ Peptone	31.3 ± 1.28ab 33.5 ± 0.95a 22.2 ± 0.54cd	37.3 ± 0.56a 18.9 ± 0.77d 28.4 ± 1.01bc
	Urea	27.3 ± 1.12bc	35.8 ± 1.25a
	Yeast Extract	30.6 ± 1.44ab	33.1 ± 1.19ab

Note: Treatments with means of varied letter in the similar columns for substrate are significantly varied at p≤0.05.

Table 3e. Exoglucanase activity after Nitrogen supplementation at 3rd day of incubation.

Substate	Nitrogen	Mean ± sem	Mean ± sem
		20 mM	80 mM
	Control	7.33 ± 0.29ab	5.48 ± 0.09bc
	(NH₄)₂SO₄ Asparagine	6.61 ± 0.48b 6.92 ± 0.04b	5.11 ± 0.43c 7.15 ± 0.61ab
Maize cobs	NaNO₃ NH₄NO₃ Peptone	9.66 ± 1.12a 5.14 ± 0.65b 6.96 ± 0.35b	8.80 ± 0.57a 6.91 ± 0.28abc 7.77 ± 0.36a
	Urea	7.58 ± 0.48ab	7.34 ± 0.21ab
	Yeast Extract	5.34 ± 0.13b	8.01 ± 0.27a
	Control	6.22 ± 0.23b	5.36 ± 0.18bc
	(NH₄)₂SO₄ Asparagine	6.33 ± 0.27b 6.07 ± 0.35b	8.62 ± 0.32a 6.79 ± 0.30b
Sugarcane bagasse	NaNO₃ NH₄NO₃ Peptone	5.72 ± 0.50b 5.17 ± 0.10b 8.70 ± 0.65a	5.42 ± 0.30bc 6.02 ± 0.42bc 6.80 ± 0.34b
	Urea	4.52 ± 0.26b	5.28 ± 0.38bc
	Yeast Extract	5.62 ± 0.31b	4.50 ± 0.24c

Note: Treatments with means of varied letter in the similar columns for substrate are significantly varied at p≤0.05.

Table 3f. Exoglucanase activity after Nitrogen supplementation at 6th day of incubation.

Substate	Nitrogen	Mean ± sem	Mean ± sem
		20 mM	80 mM
	Control	5.16 ± 0.40b	7.92 ± 0.46a
	(NH₄)₂SO₄ Asparagine	6.97 ± 1.32ab 4.97 ± 0.06b	4.50 ± 0.25b 7.38 ± 0.63a
Maize cobs	NaNO₃ NH₄NO₃ Peptone	4.45 ± 0.86b 7.10 ± 0.77ab 7.71 ± 0.35ab	7.36 ± 0.47a 7.38 ± 0.61a 7.32 ± 0.67a
	Urea	6.34 ± 0.37ab	5.72 ± 0.27ab
	Yeast Extract	8.96 ± 0.78a	7.34 ± 0.05a
	Control	8.21 ± 0.71a	5.92 ± 0.47ab
	(NH₄)₂SO₄ Asparagine	6.89 ± 0.45ab 5.75 ± 0.41ab	7.15 ± 0.20a 6.70 ± 0.53ab
Sugarcane bagasse	NaNO₃ NH₄NO₃ Peptone	6.80 ± 0.53ab 7.47 ± 0.37ab 7.01 ± 0.28b	7.18 ± 0.22a 4.90 ± 0.48b 5.96 ± 0.38ab
	Urea	5.78 ± 0.25b	5.53 ± 0.48ab
	Yeast Extract	5.65 ± 0.30b	5.29 ± 0.30ab

Note: Treatments with means of varied letter in the similar columns for substrate are significantly varied at p≤0.05.

3.5. Effect of Supplementation of Carbon on Cellulase Enzyme Production by Nigrospora oryzae

The carbon elements supplied to microorganisms influence production and suppression of cellulase enzymes production at the gene level. Table 4a–f shows the outcomes of supplement of soluble carbon sources of cellulase enzymes produced by Nigrospora oryzae. On maize cobs the highest Fpase activities were 17.1 ±0.51 IU/ml and 18.0±1.33 IU/ml recorded with 20mM mannitol and 80 mM maltose respectively on the 3rd day of incubation. On the 6th day of incubation 20 mM sucrose and 80 mM fructose produced the highest Fpase activities of 23.7±1.03 IU/ml and 25.1±1.59 IU/ml respectively. On sugarcane bagasse the highest Fpase of 24.0±1.61 IU/ml and 16.7±1.43 IU/ml were recorded with supplementation of 20 mM and 80 mM mannitol accordingly. Supplementation with the different soluble carbon sources produced varied endoglucanase activities on both maize cobs and sugarcane bagasse on the 3rd and 6th days of incubations. For maize cobs supplementation with 20 mM fructose and 80 mM mannitol resulted in the highest endoglucanase activities of 28.3±1.00 IU/ml and 42.6±2.06 IU/ml respectively on the 3rd day of incubation, while on the 6th day of culturing 20 mM sucrose and 80 mM glucose resulted in the highest endoglucanase activities of 35±1.34 IU/ml and 32.8±0.98 IU/ml accordingly. Supplementation of sugarcane bagasse with 20 mM galactose and 80 mM sucrose resulted in the highest endoglucanase activity of 31.6±1.17 IU/ml and 34.6±2.11 IU/ml respectively on the 3rd days of incubation, while 20 mM glucose and 80 mM mannitol resulted in the highest endoglucanase activities of 41.9±0.62 IU/ml and 36.3±2.08 IU/ml on the 6th day of incubation. Influence of supplementation of maize cobs and sugarcane bagasse with soluble carbon source on exoglucanase activity varied depending on the concentration and types of carbon source and incubation period. On maize cobs 20 mM mannitol and 80mM fructose resulted in the highest exoglucanase activities of 8.95±0.55 IU/ml and 8.39±0.28 IU/ml on the 3rd day of incubation. On sugarcane bagasse the highest exoglucanase activities was 6.87 ±0.40 IU/ml recorded with 20 mM galactose supplementation which however was not significantly higher compared to that produced with 20 mM fructose, glucose, mannitol and control. There was no influence on exoglucanase production when concentration of the sugars were increased to 80 mM on sugarcane bagasse on the 3rd day of incubation. On the 6th day of incubation supplementation of maize cobs with 20 mM mannitol resulted in the highest exoglucanase activities of 6.53±0.26 IU/ml which however was not significantly higher than supplementation with 20 mM fructose, glucose, maltose, sucrose and control. Increasing the concentration of the sugar to 80 mM resulted in the highest exoglucanase activities of 10.40±0.67 IU/ml with addition of sucrose which however was not significantly higher than that produced with 80 mM mannitol. On sugarcane bagasse addition of 20 mM glucose resulted in the highest exoglucanase activity of 7.47±0.37 IU/ml which was only higher than that recorded with 20 mM galactose. The highest exoglucanase activity of 8.55±0.45 IU/ml was recorded with 80 mM fructose on sugarcane bagasse on 6th day of incubation.

Table 4a. Filter Paper activity after carbon supplementation at 3rd day of incubation.

Substate	Carbon	Mean ± sem	Mean ± sem
		20 mM	80 mM
	Control	16.6 ± 0.90ab	11.0 ± 0.88c
	Fructose	10.2 ± 0.68c	12.9 ± 0.69bc
	Galactose	15.6 ± 1.15ab	16.3 ± 1.45ab
Maize cobs	Glucose	12.4 ± 0.87bc	10.9 ± 0.47c
	Maltose	14.1 ± 0.78abc	18.0 ± 1.33a
	Mannitol	17.1 ± 0.51a	17.0 ± 0.51ab
	Sucrose	15.8 ± 1.32ab	13.3 ± 1.18abc
	Control	16.3 ± 0.80a	10.3 ± 0.55bc
	Fructose	13.4 ± 0.08ab	11.4 ± 0.71abc
	Galactose	15.2 ± 0.47a	12.7 ± 1.03abc
Sugarcane bagasse	Glucose	12.7 ± 1.28b	9.18 ± 0.72c
	Maltose	13.0 ± 0.79a	11.4 ± 0.89abc
	Mannitol	6.13 ± 0.40c	14.8 ± 0.44a
	Sucrose	6.93 ± 0.22a	14.3 ± 1.33ab

Note: Treatments with means varied letter in the similar columns for substrate are significantly varied at p≤0.05.

Table 4b. Filter Paper activity after carbon supplementation at 6^the day of incubation.

Substate	Carbon	Mean ± sem	Mean ± sem
		20 mM	80 mM
	Control	15.2 ± 0.55b	16.8 ± 0.77bc
	Fructose	14.2 ± 0.92bc	25.1 ± 1.59a
	Galactose	13.3 ± 0.58bc	13.9 ± 0.71cd
Maize cobs	Glucose	9.5 ± 0.18d	13.6 ± 0.82cd
	Maltose	10.9 ± 0.86cd	14.0 ± 0.95cd
	Mannitol	20.3 ± 0.92a	19.1 ± 1.03b
	Sucrose	23.7 ± 1.03a	9.4 ± 0.53d
	Control	15.2 ± 0.55b	11.5 ± 0.95bcd
	Fructose	14.2 ± 0.92bc	7.25 ± 0.58d
	Galactose	13.3 ± 0.58bc	15.8 ± 1.06ab
Sugarcane bagasse	Glucose	9.5 ± 0.18d	15.1 ± 0.31abc
	Maltose	10.9 ± 0.86cd	10.9 ± 1.05cd
	Mannitol	20.3 ± 0.92a	16.7 ± 1.43a
	Sucrose	23.7 ± 1.03a	10.2 ± 0.65d

Note: Treatments with means of varied letter in the similar columns for substrate are significantly varied at p≤0.05.

Table 4c. Endoglucanase activity after carbon supplementation at 3rd day of incubation.

Substate	Carbon	Mean ± sem	Mean ± sem
		20 mM	80 mM
	Control	27.1 ± 0.79a	21.0 ± 0.27de
	Fructose	28.3 ± 1.00a	25.8 ± 1.34cd
	Galactose	18.4 ± 0.89bc	29.5 ± 1.51bc
Maize cobs	Glucose	15.2 ± 0.72c	17.1 ± 0.68e
	Maltose	20.3 ± 1.30b	32.8 ± 0.74b
	Mannitol	27.3 ± 1.22a	42.6 ± 2.06a
	Sucrose	21.1 ± 0.78b	22.4 ± 1.69de
	Control	23.8 ± 0.66bc	25.5 ± 1.6bc
	Fructose	28.1 ± 1.24ab	23.7 ± 1.94bc
	Galactose	31.6 ± 1.17a	18.3 ± 0.55bc
Sugarcane bagasse	Glucose	22.8 ± 1.16c	21.8 ± 0.99c
	Maltose	26.3 ± 1.00bc	25.3 ± 2.15bc
	Mannitol	15.9 ± 1.02d	30.2 ± 0.93ab
	Sucrose	23.9 ± 1.15bc	34.6 ± 2.11a

Note: Treatments with means of varied letter in the similar columns for substrate are significantly varied at p≤0.05.

Table 4d. Endoglucanase activity after carbon supplementation at 6th day of incubation.

Substate	Carbon	Mean ± sem	Mean ± sem
		20 mM	80 mM
	Control	25.0 ± 0.70b	26.0 ± 1.12abc
	Fructose	21.1 ± 1.07b	31.2 ± 2.19a
	Galactose	25.3 ± 1.29b	21.9 ± 1.48bc
Maize cobs	Glucose	13.6 ± 0.48c	32.8 ± 0.98a
	Maltose	32.9 ± 1.43a	28.5 ± 1.13ab
	Mannitol	33.1 ± 1.51a	31.7 ± 1.84a
	Sucrose	35.5 ± 1.34a	21.2 ± 1.00c
	Control	24.1 ± 1.48cd	19.6 ± 0.76c
	Fructose	18.7 ± 0.65d	14.0 ± 0.83d
	Galactose	12.1 ± 1.15e	31.3 ± 0.85ab
Sugarcane bagasse	Glucose	41.9 ± 0.62a	22.8 ± 1.25c
	Maltose	28.1 ± 1.36bc	29.6 ± 1.02b
	Mannitol	33.0 ± 1.77b	36.3 ± 2.08a
	Sucrose	26.5 ± 1.39c	18.4 ± 0.05cd

Note: Treatments with means of varied letter in the similar columns for substrate are significantly varied at p≤0.05.

Table 4e. Exoglucanase activity after carbon supplementation at 3rd day of incubation.

Substate	Carbon	Mean ± sem	Mean ± sem
		20 mM	80 mM
	Control	7.36 ± 0.26b	5.11 ± 0.16c
	Fructose	4.99 ± 0.19c	8.39 ± 0.28a
	Galactose	4.22 ± 0.14c	4.95 ± 0.39c
Maize cobs	Glucose	7.65 ± 0.41abc	6.93 ± 0.56ab
	Maltose	7.13 ± 0.12b	6.60 ± 0.36abc
	Mannitol	8.95 ± 0.55a	5.76 ± 0.50bc
	Sucrose	6.51 ± 0.27b	6.20 ± 0.17bc
	Control	6.37 ± 0.16a	4.77 ± 0.46a
	Fructose	5.71 ± 0.46ab	5.31 ± 0.27a
	Galactose	6.87 ± 0.40a	4.40 ± 0.09a
Sugarcane bagasse	Glucose	5.43 ± 0.39abc	5.18 ± 0.22a
	Maltose	3.91 ± 0.12c	4.93 ± 0.35a
	Mannitol	5.42 ± 0.34abc	4.02 ± 0.31a
	Sucrose	4.57 ± 0.26bc	4.16 ± 0.05a

Note: Means expressed of varied letter in the similar columns for substrate are significantly varied at p≤0.05.

Table 4f. Exoglucanase activity after carbon supplementation at 6th day of incubation.

Substate	Carbon	Mean ± sem	Mean ± sem
		20 mM	80 mM
	Control	5.27 ± 0.34ab	7.70 ± 0.57bc
	Fructose	5.56 ±0.14a	5.18 ± 0.12d
	Galactose	3.97 ±0.09b	5.62 ± 0.23cd
Maize cobs	Glucose	5.60 ±0.44a	4.54 ± 0.25d
	Maltose	5.25 ±0.45ab	3.77 ± 0.18d
	Mannitol	6.53 ±0.26a	8.79 ± 0.67ab
	Sucrose	6.41 ±0.32a	10.40 ± 0.89a
	Control	7.23 ± 0.74ab	6.17 ± 0.31bc
	Fructose	5.83 ± 0.17a	8.55 ± 0.45a
	Galactose	5.09 ± 0.06b	4.43 ± 0.23c
Sugarcane bagasse	Glucose	7.47 ± 0.37a	6.40 ± 0.26b
	Maltose	7.24 ± 0.39ab	5.40 ± 0.37bc
	Mannitol	6.16 ± 0.58a	5.10 ± 0.41bc
	Sucrose	6.93 ± 0.22a	5.53 ± 0.45bc

Note: Treatments with means of varied letter in the similar columns for substrate are significantly varied at p≤0.05.

3.6. Effect of Moisture Levels on Production of Cellulase Enzymes by Nigrospora oryzae

Moisture content is very interested key element in SSF process since it affect the ability of growing of microorganisms, biosynthesis and secretion of cellulase enzymes. In this regard the effect of moisture on cellulase enzymes production was investigate by adjusting the substrate to moisture of ratios between 1:1 to 1:4.5 on the 3rd and 6th days of incubation and the outcomes are summarized in Table 5). The highest Fpase activities on maize cobs on the 3rd and 6th days of incubation were 17.4±0.30 IU/ml and 18.6±0.83 IU/ml respectively with substrate: moisture ratios of 1:30 which however were not significantly different from those reported with a substrate moisture ratio of 1:2 and 1:2.5 on both days. On sugarcane bagasse, the highest Fpase activities were 16.8±0.44I U/ml and 20.0±0.72 IU/ml on the 3rd and 6th days of respectively with a substrate moisture ratio of 1:2.5. The highest Fpase activity reported on the 6th of incubation was not significantly varied compared to that reported with substrate to moisture ratios of 1:3.0. The highest endoglucanase activities on maize cobs were 28.2 ±0.36 IU/ml and 28.3±0.35 IU/ml with substrate to moisture ratios of 1:2.5 and 1:30 for 3rd and 6th days of incubation accordingly. Endoglucanase activity produced on maize cobs with substrate to moisture ratios 1:2.0, 1:2.5 and 1:3.0 on the 3rd day of incubation and 1:1.5, 1:2.0, 1.2.5 and 1:3.0 on the 6th day of incubation were not significantly varied. On sugarcane bagasse, the highest endoglucanase activities were obtained with. substrate to moisture ratio of 1:3.0 of 26.2±0.54 IU/ml on the 3rd day of incubation which was not significantly different from endoglucanase obtained with ratio 1:2.5 and 31.8±0.5 IU/ml on the 6th of incubation which was not significantly different from those obtained with 1:2.0 and 1.25 ratios. The substrate moisture ratio of 1:3.0 on maize cobs resulted in the highest exoglucanase activities of 8.8±0.12 IU/ml and 8.47±0.03 IU/ml on the 3rd and 6th days of incubation respectively. Exoglucanase activities produced on maize cobs with substrate to moisture ratios of 1:2.5 and 1:3.0 on the 3rd day and 1:2.0, 1:2.5 and 1:3.0 on the 6th day of incubation were not significantly varied. On sugarcane bagasse, the highest exoglucanase activities were 9.64±0.21 IU/ml and 9.57±0.07 IU/ml reported with substrate to moisture ratios of 1:3.0 and 1.2.5 on the 3rd and 6th days of incubation respectively. Exoglucanse activities recorded with substrate to moisture ratios of 1:2.5 and 1:3.0 on the 6th day of incubation were not significantly varied.

3.7. Effect of Surfactant Levels on Production of Cellulase Enzymes by Nigrospora oryzae

Surfactants are amphiphilic molecules with the capacity to reduce surface tension and modify the composition of plant biomass. In this study the results of surfactants on cellulase enzymes producing by Nigrospora oryzaie cultivated on maize cobs and sugarcane bagasse was evaluated by incorporating Tween-20, Triton X-100 and SDS at 0.4% and 1.2%. The results are summarized in Table 6a–c). The highest Fpase activities on maize cobs and sugarcane bagasse were 22.4±0.76 IU/ml and 19.4±0.46 IU/ml with 0.4% Tween-20 and Triton X-100 accordingly after 3 days of incubation. However, the trend was reversed when the surfactants concentration was increased to 1.2% in which the highest Fpase activities were 15.4±0.94 IU/ml and 20.0±0.99 IU/ml with Triton X-100 and Tween-20 for maize cobs and sugarcane bagasse respectively. On the 6th day of incubation, the highest Fpase activities for maize cobs were 15.4 ±0.29 IU/ml and 18.3±0.22 IU/ml with 0.4% Tween-20 and 1.2% Triton X-100. The trend was reversed on sugarcane bagasse with highest Fpase activities of 22.3±1.84 IU/ml and 11.8±0.97 IU/ml with 0.4% Triton X-100 and 1.2% Tween 20 respectively. The results of surfactants on endoglucanase producing varied with the highest activity on maize cobs of 25.5±1.16 IU/ml and 29.9±1.62 IU/ml recorded with 0.4% Tritonx-100 and Tween-20 respectively on the 3rd day of incubation. For sugarcane bagasse the highest endoglucanase activities were 19.66 ±0.66 IU/ml and 24.5±1.00 IU/ml with 0.4% Tween-20 and SDS on 3rd day of incubation. On the 6th day of incubation, the highest endoglucanase activities on maize cobs were 28.6±0.33 IU/ml and 45.5±0.94 IU/ml with 0.4 SDS and 1.2% Tween-20 respectively. For sugarcane bagasse 0.4% and 1.2% Tween-20 resulted in production of highest endoglucanase activities of 22.0±1.32Iu/ml and 26.3±0.7 IU/ml respectively. The highest exoglucanase activities with both maize cobs and sugarcane bagasse were recorded on control compared with addition of 0.4% surfactants while increasing surfactants concentration to 1.2% resulted in exoglucanase activities of 7.21±0.32 IU/ml and 6.0±0.72 IU/ml with Triton X-100 and SDS on maize cobs and sugarcane bagasse respectively on the 3rd day of incubation. On the 6th day of incubation, the highest exoglucanase activities on maize cobs were 7.40±0.58 IU/ml and 9.0±0.51 IU/ml with 0.4% Tween-20 and 1.2% SDS respectively. For sugarcane bagasse the highest exoglucanase activities were 6.12±0.35 IU/ml and 7.19±0.3 IU/ml in the control and 1.2% SDS.

Table 6a. Effect of Surfactants on Fpase Activity by Nigrospora oryzaie cultured on Maize cobs and Sugarcane bagasse.

Note: Treatments with means of varied letter in the similar columns for substrate are significantly varied at p≤0.05.

Table 6b. Effect of Surfactants on Endoglucanse Activity by Nigrospora oryzaie cultured on Maize cobs and Sugarcane bagasse.

Note: Treatments with means of varied letter in the similar columns for substrate are significantly varied at p≤0.05.

Table 6c. Effect of Surfactants on Exoglucanase Activity by Nigrospora oryzae cultured on Maize cobs and Sugarcane bagasse.

Note: Treatments with means varied letter in the similar columns for substrate are significantly varied at p≤0.05.

3.8. Effect of Enzymes Loading on Reducing Sugar Production Using Maize cobs and Sugarcane Bagasse Substrates and Nigrospora oryzae Cellulase Enzyme Extract

Methodical enzymatic hydrolysis is determined by the optimal ratio between enzyme and substrate. Therefore, the impact of the different quantity of the enzymes on reducing sugar producing was evaluated in this study on different concentrations 1% ,2%,3%,4% and 5% enzymes loading over 72 hours’ incubation period. The results are summarized in Table 7, the highest reducing sugar produced from both maize cobs and sugarcane bagasse occurred with 5% enzyme concentration after 72 hours of incubation. There was increase in the quantity of reducing sugars produced from 12 hours to 72 hours for both maize cobs and sugarcane bagasse for all the enzymes concentration tested with highest recorded after 72 hours of incubation.

3.9. Effect of Substrate Loading on Reducing Sugar Production by Nigrospora oryzae Cellulase Enzymes

The impact of the different quantity of the substrate on reducing sugar producing was evaluated in this study by utilizing different quantity 1%, 5%, 10% and 15% over a 72-hour incubation period. For maize cobs the highest amounts of reducing sugars were produced with 15% substrate loading over the 72-hour incubation which were significantly higher compared to 1%, 5%, 10% and control except at 24 and 72 hours at which the reducing sugars from 10% substrate loading were not significantly different compared to 15% substrate loading. The lowest reducing sugars were produced with 1% substrate loading (Table 8). For sugarcane bagasse, the highest reducing sugars were produced using 10% substrate loading except at 36 and 48 hours of incubation and 72 hours at which reducing sugars produced with 5%, 10%, 15 % and control and 10% and control were not significantly different. The lowest reducing sugars were produced with 1% substrate loading. (Table 8).

3.10. Simultaneous Saccharification and Fermentation of Maize Cobs and Sugarcane Bagasse Using Nigrospora oryzae Cellulase and Saccharomyces cerviceaie

The fermentability of the saccharification results were further investigated by utilizing S. cerevisiae as the fermenting specimen over a period of 72 hours. S. cerevisiae was selected as it is an efficient ethanolic fermenter, able fermenting glucose from the breakdown of cellulose (Shukla et al.,2023). Ethanol from simultaneous saccharification and fermenting of maize cobs and sugarcane bagasse increased from 12 hours of incubation and peaked at 72 hours for the different substrate and enzyme concentrations, similar simultaneous saccharification and fermentation of sugarcane bagasse with 5 FPU resulted in significantly comparable ethanol production between 60 and 72 hours for 10% and 15% substrate concentration. Simultaneous saccharification and fermenting of maize cobs and sugarcane bagasse with 10 FPU resulted in significantly higher ethanol production using 10% substrate concentration after 72 hours of incubations (Table 9).

4. Discussions

4.1. Effect of Incubation Period on Enzyme Production

Incubation period plays a significantly positive influence in the metabolic activity of microorganisms and cellulase enzymes production. Incubation time is directly related to the producing of enzymes and other metabolites. Enzymes being the primary metabolites are synthesized during the logarithm phase of fungal growth cycle (Munyasi, et al., 2024). The producing of the component of cellulase by Nigrospora oryzae varied depending on the substrate used. Maximum Fpase production for both maize cobs and sugarcane bagasse occurred on the 9th day of incubation with no significant difference in FPase reported on the 6th day for maize cobs and 3rd and 6th days for on sugarcane bagasse. These results agrees with observations by many workers that maximum Fpase occurs between 5-7 days of incubation. For instance (Bharti,,et al, (2018) cultured Talamyces stipitatus on Parthenium hysterophorus substrate and obtained maximum Fpase of 2.85 FPU gds on the 5th day of cultivation. Similarly, by Boondaeng, et al. (2024) recorded maximum Fpase of 0.25±0.02 U/g on soybean residue on the 5th day of cultivation using Aspergillus sp Ezecho et al. (20219) cultured Trichoderma asperellium on raw oil palm ford leaves and recorded the highest Fpase of 15. ±0.02 U/g on the 3rd day of incubation. The producing of endoglucanase by Nigrospora oryzae on maize cobs and sugarcane bagasse did not significantly differ on the 3rd, 12th and 15th days of incubation with maximum production occurring on the 12 day incubation. Narra, et al. (2014) cultured Aspergillus terreus for 11 days and obtained maximum endoglucanase of 11.18±0.625IU/ml on microcellulose on day 5, 9.97±0.169IU/ml on corn stalk on day 5 and 8.15±0.13IU/ml and 3.96±0.72 IU/ml on wheat straw and rice straw respectively on day 8. Exoglucanase production by Nigrospora oryzae remained relatively constantly between the 3 and 9th day and 3rd and 15 days of incubation on maize cobs and sugarcane bagasse respectively. Exoglucanase continuously catalyzes the hydrolysis of cellulose from reducing and non-reducing leads to release cellobiose (Munyasi, et al., 2024), describing its relatively constant production by Nigrospora oryzae observed in our study. Other workers have observed maximum exoglucanase at different time periods of incubations. Sharada, et al. (2013) observed maximum exoglucanase production by Emericella niveus cultured on rice straw and wheat bran under SSF occurred on the 3rd day of cultivation. Seratale, et al. (2014) obtained the highest exoglucanase activity of 24.22 U/g on the 5th day of cultivation when Phanerochaete chrysosporium was cultivated on grass powder. The decrease on Fpase, endoglucanase and exoglucanase production with increase in incubation time reported in our study could be attributed to heterogenicity of the substrate, change in pH during fermentation, production of inhibitory by-products and diminishment of nutrient in the medium (Narra, et al.,2014).

4.2. Effect of pH on Cellulase Enzyme Production

Among physical/chemical element the pH of the growing medium plays a significant positive impact role by inducing morphologically change in microorganisms and enzymes production (Gupta, et al; 2016). The optimum pH for cellulase enzymes producing varies among microorganism and substrate used. Cellulase enzyme production using maize cobs and sugarcane bagasse was evaluated by adjusting the pH of the moistening agent between 2-8 and culturing at for 3 and 6 days. FPase production by N. oryzaie was not influenced by pH on the 3rd day of incubation on both maize cobs and sugarcane bagasse. On the 6th day of cultivation maximum Fpase was recorded at pH 4,6 and 7, while culture of N. oryzae on sugarcane bagasse produced comparable amounts of FPase over the tested pHs. These observations are similar to Tai,et al. (2018) who noted that initial pH ranges of 4.5 to 8.5 had no significant effect on FPase production when Aspergillus niger DWAB was cultured on oil palm for between 1 and 8 days. These observations are contrary to several studies which identifies specific pH at which maximum FPase is produced. A study by Boondaeng et al. (2024) recorded the highest FPase of 0.24 ±0.00IU/g at pH 7.0 when Asperigillus sp IN5 was cultured on soybean residue after 7 days of incubation. Aggawal et al (2024) cultured Penicillium citrinum on Parthenium hysterophorus and obtained highest FPase at pH 6 after 7 days of incubation. Similarly, Munyasi, et al. (2024) reported highest FPase at pH 5.0 by Chaetomium globosum cultured on maize cobs after 7 days of incubation. Nigrospora oryzae produced both endoglucanase and exoglucanase over a broad range of pH when cultured on maize cobs. These observations are contrary to observations made by several workers in which maximum endoglucanase and exoglucanase are attained at specific pHs. For instance, Munyasi et al. (2024) recorded maximum endoglucanase of 12.20±0.13IU/ml and 10.8±0.55IU/ml at pH 5 and 9 respectively when Chaetomium globosum was cultured on maize cobs for 7 days. Endoglucanase and exoglucanase production by Nigrospora oryzae on sugarcane bagasse was generally favored by acidic pH. Similar observation have been noted by Singla, et al. (2024) when Scytalidium thermophilum was cultured on agro wastes resulting in the highest endoglucanase at pH 5-6 and Li et al. (2013) who observed maximum exoglucanase at pH 5 for Fusarum oxysporium cultured on maize cobs.

4.3. Effect of Nitrogen Sources on Cellulase Enzymes Production

In solid state fermentation, the solid substrate is preferable factor in cellulase enzymes producing as it provides all the necessary nutrients in addition to providing on anchorage to the growing microorganisms. Therefore, there a number of the nutrients may be found in sub-optimal concentration or not even present in the solid substrate (Saratale et al., 2014). Therefore, maize cobs and sugarcane bagasse were supplemented with various nitrogen sources and their effect on cellulase enzymes evaluated. Previous studies have shown the nature and the amount of nitrogen sources are powerful key element that enhance cellulase producing by wood rotting basidiomycetes (Kachlishvili, et al., 2006). With varieties nitrogen sources significant increase in FPase production were recorded with addition of inorganic (NH₄)₂SO₄ and NH₄NO₃ or organic peptone and Urea were added to maize cobs or sugarcane bagasse. Endoglucanase production by Nigrospora oryzae was enhanced by addition of inorganic (NH₄)₂SO₄ or NH₄NO₃ or organic peptone or yeast extract. Supplementation of NaNo₃ or peptone to maize cobs and (NH₄)₂SO₄ or peptone to sugarcane bagasse lead to an increase in exoglucanase producing. These observations are in agreement with Saratale et al. (2016), Fernando et al. (2015) and Kalsoon et al. (2019) who observed that both organic and inorganic nitrogen sources observed significant results on cellulase enzyme producing from various microorganisms.

4.4. Effect of Carbon Supplementation on Cellulase Enzyme Production

Microorganisms cultured under SSF for cellulase enzymes production derive their nutrients by hydrolysis of organic compounds such as cellulose are within the solid substrate. With the complex and recalcitrant nature of these lignocellulose substrates usually offer limited nutrients to induce microbial growth (Tai et al., 2019). Therefore, the supplementation of the lignocellulose substrates with soluble carbohydrates may have an inductive result on cellulase enzyme production. In our study addition of 20 mM maltose, 80mm mannitol, 20 mM and 80 mM mannitol to maize cobs and sugarcane bagasse also increased FPase production by Nigrospora oryzae. Endoglucanase production was enhanced by addition of fructose, mannitol, galactose, sucrose and glucose mannitol. These observations suggested enhancement of cellulase production on the two substrate by the soluble carbon sources occurred in a substrate dependents manner. In addition, some soluble carbon sources seemed to have had a repressive effect on cellulase enzyme production in some substrate. Similar observations have been recorded by Saini and Aggarwal (2019) who noted the addition of fructose, lactose and starch to Parthenium hysterophorus enhanced cellulase production by Steptomyces sp. NAA2 but glucose and galactose strongly repressed cellulase production whereas cellobiose, sucrose and mannitol had an inhibitory effect. Tai et al. (2019) reported that none of the soluble carbon sources tested in their study induced higher cellulase production by Aspergillus niger DWA8 cultured on oil palm food contrary to the observation in the current study. Domingues et al. (2000) indicated that lactose acted as an inducer in cellulase producing by Trichoderma reesii RUTC-30 and Aspergillus nidulans.

4.5. Effect of Moisture on Cellulase Enzymes Production

Moisture component is one of interested key elements in SSF procedure because it affect the ability lead growing of microorganisms and the biosynthesis and secretion of enzymes (Munyasi, et al., 2024). In addition moisture enables microorganisms to uptake nutrients from the solid substrate. In our study we determined the effect of different substrate to moisture ratios on FPase, endoglucanase and exoglucanase production by N. oryzae. The optimum substrate to moisture ratio varied among the substrates. Optimum substrate to moisture ratio for FPase and endoglucanase ranged from 1:2.0 to 1:3.0 for maize cobs and 1:2.5 for FPase and 1:2.5 to 1:3.0 for endoglucanse on sugarcane bagasse. Optimum substrate to moisture ratio for maximum exoglucanase production were 1:1.25 to 1:3.0 for maize cobs and sugarcane bagasse. Our observations are in agreement with Kalsoomet et al. (2019) who reported 75% (1:3.0 substrate to moisture ratio) moisture content as optimum for cellulase production by Trichoderma sp. cultured on Kallargrass, cotton stalk and rice husks. Tai et al. (2019) and Li et al. (2013) recorded the optimum moisture of 75% for the production cellulase by Aspergillus niger DWA8 Aspergillus niger USM AI1 respectively. Our observation differ from those of Munyasi, et al. (2024) who reported a substrate to moisture ratios of 1:2.0 as optimum for cellulase production by Chretomium globosum cultured on maize cobs and sugarcane bagasse. Aita et al. (2019) reported a 40% moisture levels as being optimum for endoglucanase and exoglucanase production by Metarhizium anisopliae IBCB 348 cultured on white rice, malt bagasse and sugarcane bagasse. Therefore, fungi require appropriate moist environment facilitated easily growing enhance cellulase enzymes production. Low moisture content in SSF may cause reduction in solubility of nutrients, cause low degree swelling and high water tension of the solid substrate. High moisture content leads to decrease in porosity, changes particles structures of the substrate, promotes the developments of stickness all of which lowers oxygen transfer (Saratali et al., 2014) resulting in a decrease in enzyme production due to depletion of contact surface of substrate particles (Munyasi, et al., 2024).

4.6. Effect of Surfactant on Cellulase Enzymes Production

Surfactants have been shown to have a beneficial impact facilitating enzymes activities particularly in SSF by increasing the surface area (Nanjundaswamy, et al., 2020). Several surfactants such as Tween-80, Tween-20, Triton X-100 and many others have been evaluated for their effect on cellulase enzymes production by fungi under SSF. In our study, Tween-20, TritonX-100 and SDS were investigated for their effect on cellulase enzymes production by Nigrospora oryzae using maize cobs and sugarcane bagasse substrate at 0.4% and 1.2%, Tween-20 and TritonX-100 enhanced FPase production when incorporated into maize cobs and sugarcane bagasse at 0.4% respectively. Increasing the concentration to 1.2% revised the effect with Triton X-100 enhancing FPase production in maize cobs and Tween-20 in sugarcane bagasse on the 3rd day of incubation. The same trend was reported on the 6th day of culturing. Endoglucanase production by Nigrospora oryzae was highest with incorporate of Tween-20, Though high activity was also recorded with Tritnx-100 and SDS on sugarcane bagasse at 1.2% on 3rd day and 0.4% on 6th day. Effect of the surfactant on exoglucanase production did not follow any specific pattern a through the 3 surfactants lead to maximum production at different concentration on different days of incubation and on differently on the two substrates. These observations mirror observations by other researchers. For instance, Saratale et al., (2024) evaluated addition of PEG-1500-6000, Tween-20, Tween-80 and Triton X-100 and noted that increase in molecular weight of PEG lead to a gradual increase in FPase as the molecular weight increased, while the other surfactants showed a slight positive effect on enzyme production. Kshirsagar, et al., (2017) tested PEG -4000 Tween-20, Tween-80, Triton X-100 and SDS at 1% and noted that Tween -80 resulted in the production of 2.28±0.04 IU/ml FPase, 21.56±0.21 IU/ml exoglucanase and 78±0.55IU/ml endoglucanase but Triton X-100 showed lower yield of cellulase enzyme activities due to inhibition of Nocardiopsis sp KNUC. Triton X-100 has been shown to inhibit growth of microorganisms and thereby cellulase production (Pardo;1996). Kalsoon et al; (2019) tested the effect of Tween -20 and Tween-80 on cellulase enzyme production by Trichoderma sp cultured on Kallargrass, cotton Stalk and rice husk and noticed that Tween-20 at 0.4% resulted in optimum cellulase production of 1.073 IU/ml. The enhancement of cellulase production by surfactants has been attributed to their ability to reduce pellet formation increases cellulase production since the fungus becomes more active in degradation of cellulase (Kalsoon,et al., 2019). In addition, surfactants have been shown to increase permeability of the cell membrane allowing more rapid secretion of enzymes and release of cell bound enzymes (Kshirsagar, et al., 2017). Surfactants may also increasing enzyme production during SSF by increased penetration of water into solid substrate matrix and increase the surface regional for microbial growth (Chugh, et al., 2016).

4.7. Effect of Enzymes Loading on Reducing Sugar Production Using Maize Cobs and Sugarcane Bagasse Substrates and Nigrospora oryzae Cellulase Enzyme Extract

The cost of saccharification of lignocellulose biomass using cellulases is proportional to the cost of cellulases. Therefore, enzymes loading plays an important role in enzymes hydrolysis lignocellulose. There it is suggested to use minimal enzymes loading in the saccharification process (Zhang et al., 2018). On this account we investigated the effect of enzymes loading on production of reducing sugars from maize cobs and sugarcane bagasse at different concentration 1%,2%,3%,4% and 5% over a 72 hour incubation period. The study recorded an increasing in the quantity of reducing sugars resulted from 12 hours to 72 hours for both maize cobs and sugarcane bagasse. The highest reducing sugars were produced using 5% enzymes loading over the 72 hours incubations period. Similarly, Munyasi et al. (2024) recorded an increasing in the production of reducing sugars from maize cobs and sugarcane bagasse with increase in enzymes loading using crude cellulases produced by Chaetomium globosum. Interesting between the 60 and 72 hours of incubations cellulases of Nigrospora oryzae at 4%, 5% and control did not lead to significant increase in reducing sugar production. Similarity at 72 hours of incubation, enzymes loading of 3%;4%;5% and control resulted in production of reducing sugars that did not differ significantly. This observation could be due to limited substrate availability for hydrolysis with increase in enzymes concentration (Deng, et al., 2018). The observations are also in agreement with those of Alabdalall, et al. (2021) who used cellulases produced by Aspergillus niger under SSF and noted an increase in reducing sugar producing with an increasing in enzymes loading.

4.8. Effect of Substrate Loading on Reducing Sugar Production by Nigrospora oryzae Cellulase Enzymes

Saccharification to produce reducing sugars is faced with challenges due to insolubility and heterogeinity of lignocellulose materials (Robak, et al., 2018). Therefore there is a need to determine concentration of biomass that will support optimal and efficient conversion of biomass to produce high content of ethanol or chemicals per volume (Wang, et al., 2019). The study therefore sought to determine the substrate concentration which could produce the highest reducing sugars from maize cobs and sugarcane bagasse using 1%, 5%,10% and 15%. The substrate loading for production of high amount of reducing sugars differed between the two lignocellulose biomass substrate loading of 15% and 10% resulted in highest production of reducing sugars from maize cobs and sugarcane bagasse respectively. These difference in substrate loading for production of highest amount of reducing sugars could be due to technical challenges such as rheological challenges due to unable in mixing, lack of free water for enzymatic reactions resulting in increasing inhibitors concentrations and insufficient heat and mass transfer (Robert, et al., 2011). The lack of free water could also be caused by the absorption capacity of different lignocellulose biomass which could result in the sugar monomers and oligomers produced during hydrolysis to be in close proximity with enzymes causing feedback inhibition (Agrawal et al., 2021).

4.9. Simultaneous Saccharification and Fermentation of Maize Cobs and Sugarcane Bagasse Using Nigrospora oryzae Cellulases and Saccharomyces cerevisiae

Maize cobs and sugarcane bagasse at substrate loading of 1%,5%,10% and 15% were saccharifized by 5 FPU and 10 FPU of Nigrospora oryzae cellulase enzymes and the hydrolysates fermented to ethanol by simultaneous saccharifications and fermentations using Saccharomyces cerevisiae. In simultaneous saccharifications and fermentations, the substrate is hydrolyzed and the resulting glucose is simultaneous fermented to ethanol by the yeast (Dearman, et al., 2022). The amount of ethanol produced increased with increased substrate loading and enzymes loading as the glucose generated is utilized by yeast and converted to ethanol simultaneously thus removing feedback inhibition due to accumulation of glucose on β-glucosidase (Singhania, et al., 2011). However at 72 hours of fermentations ethanol production using 5%; 10% and 15% substrate loading were not significantly. Similar observation have been made by Abada et al. (2018) who observed an increased in ethanol production with fermentation time of sesame seed residue saccharifized by cellulase enzymes produced by Bacillus cereus. The reduction in ethanol production at high solid loading as fermentation time increases has also be reported by Zhu et al., (2012) who noted that simultaneous saccharification and fermenting of cassava palp above 16% substrate loading lead to a decrease in fermentation efficiency which could have been due to difficult of heat and mass transfer at high solid loading record and inhibition of enzymes by increasing concentrations of glucose and/or ethanol (Nguyen et al., 2017). The increase in ethanol production observed with high solid leading also requires the use of yeast that are resistant to ethanol concentration (Singhania et al., 2011). Therefore decrease in ethanol production as substrate loading increased could have been caused by the use of yeast not resistant to increase in ethanol concentration.

5. Conclusions

In this study, Nigrospora oryzaie successfully isolate from decaying wood, identified using molecular techniques and evaluated for its cellulase enzyme production using maize cobs and sugarcane bagasse as substrate. Cellulase enzyme production was conducted and utilizing one variable at a time (OVAT) by considering effect of incubation time, pH, supplementation carbon and moisture content, nitrogen and surfactant. The optimized parameters were used to produce cellulase enzymes that were used for saccharification of maize cobs and sugarcane bagasse for ethanol production by simultaneous saccharification and fermentation using Saccharomyces cerevisiae.

Author Contributions

DUKUZIMANA OLIVIER and George Isanda Omwenga study designing and revising manuscript; DUKUZIMANA OLIVIER samples collection and drafting research data, Mathew Piero Ngugi revised the entire document. All authors revised, understood and confirmed the entire document before publishing.

Funding

This study did not receive any funding.

Ethical approval

This study did not involve human subjects. All data used in this are present in this paper.

Acknowledgments

The authors thanks every one, I am very grateful for the help from Department of Biochemistry, Microbiology and biotechnology, school of pure and applied sciences Kenyatta university Kenya and the National commission for science and technology granted permission to conducted this study in KENYA. Special thanks go to Dr. George I. Omwenga and Prof Mathew P. Ngugi for invaluable support during my research. Special thanks also go to my parent BUTUTIRE and MUNDANIKURE for invaluable moral support and encouragement, special thanks go to NTAKIRUTIMANA FRANCOIS SAVIER for their support during my research. I can’t forget my colleagues and friends from Kenyatta University for their help. Finally GOD IS GOOD ALL THE TIME lead me from the start up to end.

Conflicts of Interest

The author has no conflicts of interest in this paper's research, authorship, or publishing.

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Figure 1. Growth of Nigrospora Oryzae on Carboxymethyl cellulose (A) and Potato dextrose agar (B).

Figure 2. Phylogenetic analysis of 16S rRNA sequence of Nigrospora oryzae KU-05.

Table 1. Time of Incubation on FPase, Endoglucanase and Exoglucanase Activities.

substrate	Day	Fpase	Endoglucanase	Exoglucanase
	3	8.3 ± 0.64b	13.7 ± 1.79abc	3.31 ± 0.25ab
	6	10.3 ± 0.87ab	12.3 ± 1.01bc	3.11 ± 0.16abc
	9	11.3 ± 0.94a	12.5 ± 1.73bc	3.46 ± 0.25a
Maize cobs	12	2.8 ± 0.36c	19.7 ± 1.74a	2.19 ± 0.15d
	15	4.0 ± 0.11c	18.9 ± 1.44ab	2.53 ± 0.08bcd
	18	4.4 ± 0.18c	7.0 ± 0.16c	2.42 ± 0.15cd
	3	8.7 ± 0.26a	14.6 ± 0.97ab	1.77 ± 0.11ab
	6	8.8 ± 0.36a	10.4 ± 0.72b	1.80 ± 0.10ab
	9	8.9 ± 0.47a	15.2 ± 0.74ab	1.72 ± 0.10ab
Sugarcane bagasse	12	2.8 ± 0.11c	15.5 ± 0.76a	1.96 ± 0.16a
	15	2.5 ± 0.05c	14.1 ± 1.90ab	2.06 ± 0.11a
	18	4.6 ± 0.04b	11.1 ± 0.37b	1.40 ± 0.06b

Note: Treatments with means of varied letter in the same columns for substrate are significantly varied at p≤0.05.

Table 2. FPase, Endoglucanase and Exoglucanase Activities Under different pH.

substrate	pH	Fpase	Fpase	Endoglucanase	Endoglucanase	Exoglucanase	Exoglucanase
		Day 3	Day 6	Day 3	Day 6	Day 3	Day 6
	2	17.1 ± 1.62a	13.0 ± 1.67b	33.5 ± 1.76a	33.4 ± 1.27a	6.02 ± 0.03a	7.58 ± 0.47a
	3	16.3 ± 1.26a	11.3 ± 1.05b	31.9 ± 1.33a	24.5 ± 0.83b	5.24 ± 0.31a	5.79 ± 0.44a
	4	16.7 ± 0.96a	14.6 ± 0.92ab	28.5 ± 1.09a	32.0 ± 1.69a	4.84 ± 0.41a	6.05 ± 0.35a
Maize cobs	5	16.2 ± 1.37a	18.6 ± 1.62a	34.1 ± 1.05a	30.0 ± 1.92ab	5.72 ± 0.06a	7.22 ± 0.78a
	6	15.9 ± 0.84a	14.6 ± 0.68ab	29.4 ± 3.09a	29.2 ± 0.17ab	4.95 ± 0.43a	6.85 ± 0.21a
	7	19.0 ± 1.33a	15.4 ± 0.45ab	28.8 ± 0.49a	30.8 ± 1.21ab	5.75 ± 0.59a	5.98 ± 0.16a
	8	17.2 ± 0.65a	12.7 ± 0.51b	32.9 ± 1.18a	32.8 ± 1.49a	5.58 ± 0.36a	7.04 ± 0.48a
	2	17.9 ± 0.24a	17.6 ± 1.17a	29.3 ± 0.45cd	33.0 ± 1.60a	5.94 ± 0.36ab	9.13 ± 0.68a
	3	19.1 ± 1.01a	16.2 ± 1.18a	25.0 ± 1.32d	31.1 ± 1.49a	6.75 ± 0.53ab	6.71 ± 0.24bc
	4	18.7 ± 1.58a	14.7 ± 0.66a	42.6 ± 0.86a	32.0 ± 1.64a	5.99 ± 0.27ab	6.65 ± 0.46bc
Sugarcane bagasse	5	18.0 ± 0.46a	15.5 ± 0.76a	35.7 ± 1.50bc	27.9 ± 1.00ab	8.16 ± 0.87a	5.98 ± 0.47c
	6	17.7 ± 0.55a	14.9 ± 0.91a	36.0 ± 1.77b	28.1 ± 0.96ab	6.57 ± 0.40ab	8.23 ± 0.54ab
	7	18.9 ± 1.21a	15.8 ± 1.17a	31.2 ± 1.27bcd	23.2 ± 1.61b	4.53 ± 0.21b	6.53 ± 0.41bc
	8	18.6 ± 1.31a	16.3 ± 1.71a	31.9 ± 1.57bc	32.1 ± 1.70a	7.85 ± 0.31a	6.14 ± 0.34bc

Note: Treatments with means of varied letter in the same columns for substrate are significantly varied at p≤0.05.

Table 5. FPase, endoglucanse and exoglucanase activities of Nigrospora oryzae cellulases at different substrate to moisture levels.

substrate	Moisture	Fpase	Fpase	Endoglucanase	Endoglucanase	Exoglucanase	Exoglucanase
	Ratio	Day3	Day6	Day3	Day6	Day3	Day6
	1:1.0	10.8 ± 0.36de	10.7 ± 0.90b	11.7 ± 0.11de	13.2 ± 0.86b	4.77 ± 0.18cde	5.43 ± 0.13b
	1:1.5	14.1 ± 0.55bc	12.3 ± 0.79b	23.8 ± 0.06bc	25.8 ± 0.47a	5.90 ± 0.30bc	6.25 ± 0.13b
	1:2.0	16.2 ± 0.29ab	16.2 ± 0.13a	24.3 ± 0.71ab	25.7 ± 0.73a	6.63 ± 0.31b	8.23 ± 0.06a
Maize cobs	1:2.5	16.7 ± 0.06ab	16.7 ± 0.44a	28.2 ± 0.36ab	28.1 ± 0.34a	8.08 ± 0.09a	8.22 ± 0.03a
	1:3.0	17.4 ± 0.30a	18.6 ± 0.83a	28.0 ± 0.27a	28.3 ± 0.35a	8.81 ± 0.12a	8.47 ± 0.33a
	1:3.5	13.2 ± 0.16cd	12.7 ± 0.35b	17.1 ± 0.36cd	13.9 ± 0.49b	4.50 ± 0.39e	5.79 ± 0.17b
	1:4.0	11.3 ± 1.24cde	12.3 ± 0.59b	15.0 ± 0.33cde	14.1 ± 0.89b	5.78 ± 0.28bcd	5.59 ± 0.37b
	1:4.5	9.6 ± 0.65e	10.2 ± 0.96b	17.6 ± 0.70e	27.0 ± 0.55ab	4.56 ± 0.28de	3.85 ± 0.18c
	1:1.0	9.33 ± 0.57d	11.6 ± 0.28d	11.8 ± 0.45d	15.1 ± 0.39f	3.29 ± 0.26e	4.10 ± 0.26e
	1:1.5	13.7 ± 0.23bc	15.3 ± 0.47c	16.6 ± 0.10c	20.5 ± 0.09de	4.81 ± 0.13d	5.27 ± 0.05d
	1:2.0	14.1 ± 0.73b	17.5 ± 0.31b	22.1 ± 1.02b	30.1 ± 0.86ab	6.74 ± 0.18c	7.99 ± 0.26b
Sugarcane bagasse	1:2.5	16.8 ± 0.44a	20.0 ± 0.72a	22.5 ± 0.29ab	28.0 ± 0.82b	8.09 ± 0.17b	9.57 ± 0.07a
	1:3.0	12.7 ± 0.25bc	19.6 ± 0.49a	26.2 ± 0.54a	31.8 ± 0.57a	9.64 ± 0.21a	9.03 ± 0.14a
	1:3.5	14.3 ± 0.37b	15.5 ± 0.21bc	20.1 ± 1.54bc	23.8 ± 0.77c	6.07 ± 0.15c	6.97 ± 0.14c
	1:4.0	11.8 ± 0.67c	13.8 ± 0.19c	16.8 ± 0.80c	17.7 ± 0.54ef	4.62 ± 0.26d	5.67 ± 0.31d
	1:4.5	13.1 ± 0.12bc	13.9 ± 0.43c	18.8 ± 0.42bc	21.1 ± 0.77c	4.49 ± 0.30d	5.18 ± 0.14d

Note: Treatments with means of varied letter in the similar columns for substrate are significantly varied at p≤0.05.

Table 7. Effect of Enzymes of loading using maize cobs and Sugarcane Bagasse and Nigrospora oryzae in production of reducing sugar.

Enzymes concentration

Substrate

Time(h)

1%

2%

3%

4%

5%

Control

Mc

12

8.39±0.62dE

26.1±0.42eD

30.0±0.41dC

34.5±0.17cdA

33.7±1.24cAB

8.39±0.62bBC

Mc

24

21.6±0.56cD

27.5±0.32deC

34.3±0.61cB

39.9±1.31bA

39.4±0.5cA

21.6±0.56bAB

Mc

36

24.8±1.02cD

29.9±0.11dC

31.2±0.2cdC

33.9±1.2dBC

45.4±0.44abA

24.8±1.02bB

Mc

48

32.1±0.74bE

35.3±0.24cD

39.8±0.34bC

39.7±0.59bcC

48.0±0.41aA

32.1±0.74aB

Mc

60

38.1±0.86aC

38.4±1.27bC

41.0±1.06bBC

43.9±1.43abABC

50.5±2.56aA

38.1±0.86aAB

Mc

72

38.6±0.66aC

41.3±0.27aBC

45.1±1.21aABC

49.1±1.37aA

52.9±3.11aA

38.6±0.66aAB

Note: Treatments with means of varied letter in the similar columns for substrate are significantly varied at p≤0.05.

Enzymes

concentration

Substrate

Time

1

2

3

4

5

Control

Sb

12

16.4±0.91cAB

17.5±0.34dA

15.1±1.51dAB

5.89±0.7dB

9.90±0.59cAB

8.3±0.47bAB

Sb

24

21.8±0.27cB

34.3±2.24cA

35.0±2.52cA

42.3±2.62cA

38.0±0.19bA

21.2±3.23bB

Sb

36

35.0±3.39bB

48.9±2.13bB

46.3±1.39bB

48.1±1.23bcAB

62.7±0.56aA

42.3±2.83aB

Sb

48

37.2±1.17abC

53.0±0.64abB

49.3±1.91abB

49.8±0.32bB

62.0±1.34aA

44.2±0.10ab B

Sb

60

43.2±0.3aD

55.0±0.39abB

53.5±0.87abBC

52.2±0.61abBC

62.7±1.06aA

48.7±1.97aC

Sb

72

44.0±0.41aC

58.2±1.23aAB

55.3±1.65aB

58.0±1.19AB

63.8±0.44aA

49.0±1.95aC

Note. Note: Treatments with means of varied letter in the similar columns for substrate are significantly varied at p≤0.05.

Table 8. Effect of substrate loading using Maize Cobs and Nigrospora oryzae in production of reducing sugar.

Substrate concentration

Substrate

Time

1

5

10

15

Control

Mc

12

3.35±0.8bD

7.69±0.37cC

11.6±0.29cB

13.8±0.48dA

6.84±0.18dC

Mc

24

3.33±0.39bC

9.24±0.35bcB

13.6±0.2bA

15.0±0.11cA

8.06±0.44cdB

Mc

36

3.97±0.12bE

10.2±0.19bC

14.3±0.16bB

15.6±0.2bcA

8.84±0.37cD

Mc

48

4.29±0.18bD

10.4±0.06C

15.2±0.25abB

16.3±0.16abA

10.9±0.21bC

Mc

60

4.45±0.2abD

10.8±0.06abC

15.30.05abB

16.6±0.2abA

11.6±0.42bC

Mc

72

5.70±0.33aC

11.9±0.57aB

17.0±0.79aA

17.2±0.06aA

13.9±0.6aB

Note: Treatments with means of varied letter in the similar columns for substrate are significantly varied at p≤0.05.

Substrate concentration

substrate

Time

1

5

10

15

Control

Sb

12

3.41±0.04dD

7.99±0.75dAB

9.15±0.39eA

6.19±0.21cBC

5.93±0.42eC

Sb

24

4.26±0.09cC

10.1±83cdAB

11.2±0.6dA

8.27±0.3bcB

9.23±0.4dAB

Sb

36

5.04±0.006bB

12.2±0.14bcA

12.9±0.2cA

10.0±1.4bA

11.4±0.42cdA

Sb

48

5.45±0.23bC

12.3±0.38bcB

14.4±0.26bcA

13.9±0.56aAB

13.0±0.52BCAB

Sb

60

6.53±0.17aC

14.6±0.26abAB

15.4±0.28abA

14.3±0.1aB

14.0±0.21bB

Sb

72

7.29±0.3aC

16.6±0.25aA

16.7±0.23aA

15.2±0.25aB

17.0±0.4aA

Note: Treatments of similar lower case letter in similar column are not significantly varied. Treatments with the similar upper case letter across each row are not significantly varied at p≤0.05.

Table 9. simultaneous saccharification fermentation of maize cobs and sugarcane bagasse using Nigrospora oryzae cellulase and Saccharomyces cervisaie.

Substrate concentration 5 FPU

Substrate

Incubation Time

1

5

10

15

Control

Mc

12

1.85±0.03c

6.32±0.47d

6.64±0.4d

7.61±0.6b

4.37±0.34e

Mc

24

2.16±0.009c

7.01±0.6d

8.03±0.14c

7.77±0.62b

4.42±0.26e

Mc

36

5.14±0.26b

8.46±0.16c

8.16±0.56bc

8.40±0.94b

6.39±0.21d

Mc

48

5.42±0.29b

8.85±0.14bc

9.06±0.43abc

8.98±0.42ab

7.97±0.47c

Mc

60

7.22±0.28a

9.71±0.26ab

9.4±0.74ab

10.2±0.52a

9.64±0.39b

Mc

72

7.21±0.24

9.88±0.06a

10.1±0.46a

10.2±0.13a

11.4±0.62a

Substrate concentration 10 FPU

Substrate

Incubation Time

1

5

10

15

Control

Mc

12

2.05±0.03e

6.92±0.7c

6.32±0.46c

4.87±0.17d

Mc

24

2.21±0.16e

7.50±1.14

7.16±0.89c

5.95±0.47c

Mc

36

5.23±0.18d

9.34±0.6ab

9.11±0.25b

9.01±0.42b

Mc

48

6.2±0.14c

10.6±0.47a

9.34±0.09b

9.38±0.4ab

Mc

60

7.69±0.44b

10.5±0.89a

9.99±0.69ab

9.59±0.53ab

Mc

72

9.19±0.16a

10.9±0.87a

11.0±0.29a

10.1±0.23a

Substrate concentration 5 FPU

Substrate

Incubation Time

1

5

10

15

Control

Sb

12

3.30±0.39c

7.29±0.22b

6.60±0.33c

4.38±0.36d

4.37±0.34e

Sb

24

3.38±0.22c

7.59±0.35ab

7.98±0.51b

7.90±0.86c

4.42±0.26e

Sb

36

4.96±0.79b

7.87±0.58ab

8.74±0.3b

8.21±0.27c

6.39±0.21e

Sb

48

5.07±0.17b

9.92±0.72ab

8.97±o.4b

9.06±0.21bc

7.97±0.47d

Sb

60

5.97±0.3ab

8.36±0.44ab

8.99±.42b

10.0±0.2ab

9.64±0.39b

Sb

72

6.99±0.65a

8.78±0.13a

11.7±0.64a

11.1±1.26a

11.4±0.62a

Substrate concentration 10FPU

Substrate

Incubation Time

1

5

10

15

Control

Sb

12

4.88±0.39c

6.01±0.41d

6.93±0.3d

6.72±0.29b

Sb

24

4.96±0.24c

8.17±0.16c

6.27±0.49d

6.74±0.67b

Sb

36

6.62±0.58b

9.02±0.43bc

7.85±0.43c

6.76±0.79b

Sb

48

8.65±0.29a

9.47±0.35b

9.17±0.5b

8.9±0.84a

Sb

60

9.18±0.39a

10.9±0.33a

9.0±0.48bc

9.58±0.61a

Sb

72

9.37±0.28a

11.0±0.48a

10.8±0.27a

9.89±0.44a

Incubation Time 5FPU

Substrate

Substrate
Conc

12

24

36

48

60

72

Sb

1

3.30±0.39c

3.38±0.22b

4.96±0.79b

5.0±0.17b

5.97±0.3c

6.99±0.65c

Sb

5

7.29±0.22a

7.59±0.35a

7.87±0.58a

7.92±0.72a

8.36±0.44b

8.78±0.13bc

Sb

10

6.60±0.33a

7.98±0.51a

8.74±0.3a

8.97±0.4a

8.99±0.42ab

11.7±0.64a

Sb

15

4.38±0.36b

7.90±0.86a

8.21±0.27a

9.06±0.21a

10.0±0.2a

11.1±1.26a

Sb

control

5.18±0.15b

7.68±0.99a

8.05±1.47a

8.52±0.68a

9.88±0.61a

10.3±0.46ab

Incubation Time 10FPU

Substrate

Substrate
Conc

12

24

36

48

60

72

Sb

4.88±0.39c

4.96±0.24c

6.62±0.58b

8.65±0.29a

9.18±0.39b

9.37±0.28c

Sb

5

7.80±0.8a

8.66±0.45a

8.63±0.93a

9.73±0.37a

9.57±0.49b

10.3±0.11ab

Sb

10

6.01±0.41bc

8.17±0.16a

9.0±0.43a

9.47±0.35a

10.9±0.33a

11.0±0.48a

Sb

15

5.93±0.3bc

6.27±0.49b

7.85±0.43ab

9.17±0.5a

9.0±0.48b

10.8±0.27ab

Sb

control

6.72±0.29ab

6.74±0.67b

6.76±0.79b

8.9±0.84a

9.58±0.61b

9.89±0.44bc

Incubation Time 5FPU

Substrate

Substrate
Conc

12

24

36

48

60

72

Mc

1

1.85±0.03d

2.16±0.09c

5.14±0.26b

5.42±0.29c

7.22±0.28b

7.21±0.24c

Mc

5

6.32±0.47b

7.01±0.6a

8.46±0.16a

8.85±0.14ab

9.71±0.26a

9.88±0.06b

Mc

10

6.64±0.4ab

8.03±0.14a

8.16±0.56a

9.06±0.43a

9.4±0.74a

10.1±0.46b

Mc

15

7.61±0.6a

7.77±0.62a

8.40±0.94a

8.98±0.42a

10.2±0.52a

10.2±0.13b

Mc

control

4.37±0.34c

4.42±0.26b

6.39±0.21b

7.97±0.47a

9.64±0.39a

11.4±0.62a

Incubation Time 10FPU

Substrate

Substrate
Conc

12

24

36

48

60

72

Mc

1

2.05±0.03c

2.21±0.16b

5.23±0.18c

6.2±0.14d

7.69±0.44b

9.19±0.16b

Mc

5

6.53±0.38a

7.38±0.28a

7.26±0.1b

7.36±0.24c

7.92±0.31b

9.06±0.47b

Mc

10

6.92±0.7a

7.50±1.14a

9.34±0.6a

10.6±0.47a

10.5±0.89a

10.9±0.87a

Mc

15

6.32±0.46a

7.16±0.89a

9.11±0.25a

9.34±0.09b

9.99±0.69a

11.0±0.29a

Mc

control

4.87±0.17b

5.95±0.47a

9.01±0.42a

9.38±0.4b

9.59±0.53a

10.1±0.23ab

Note: Treatments with similar lower case letter in the similar column are not significantly varied. Treatments with the similar upper case letter across each raw are not significantly varied.

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Optimization of Production and Characterization of Cellulase Enzymes from Nigrospora oryzae (Berk and Br.) Petch for Biomass Saccharification and Ethanol Production

Abstract

Keywords:

Subject:

1. Introduction

2. Materials and Methods

2.1. Collection of Samples

2.2. Isolation of Fungi

2.3. Subculturing Cellulolytic Fungi

2.4. Molecular Identification of Nigrospora oryzae

2.5. Solid State Fermentation (SSF) for Cellulase Enzymes Production

2.5.1. Cellulosic Substrates

2.5.2. Solid State Fermentation (SSF) for Cellulase Enzymes Production

2.5.3. Preparation of Inoculum

2.5.4. Enzyme Extraction

2.6. Cellulase Enzyme Assays

2.7. Optimization of Culture Conditions for Enzyme Production

2.8. Production of Cellulase Enzymes for Saccharification and Ethanol Production

2.9. Effect of Enzymes Loading on Reducing Sugar Production Utilizing Maize Cobs and Sugarcane Bagasse Substrates and Nigrospora oryzae Cellulase Enzyme Extract

2.10. Effect of Substrate Loading on Reducing Sugar Production by Nigrospora oryzae Cellulase Enzymes

2.11. Simultaneous Saccharification and Fermentation of Maize Cobs and Sugarcane Bagasse Using Nigrospora oryzae Cellulase and Saccharomyces cerevisiae

2.12. Statistical Analysis

3. Results

3.1. Isolation of Nigrospora Oryzae

Molecular Identification of Nigrospora oryzae

3.2. Effect of Incubation in Production of Celluloses Enzymes by Nigrospora oryzae

3.3. Effect of pH on Production of Cellulase Enzymes by Nigrospora oryzae

3.4. Effect of Nitrogen Supplementation on Production of Cellulase Enzymes by Nigrospora oryzae

3.5. Effect of Supplementation of Carbon on Cellulase Enzyme Production by Nigrospora oryzae

3.6. Effect of Moisture Levels on Production of Cellulase Enzymes by Nigrospora oryzae

3.7. Effect of Surfactant Levels on Production of Cellulase Enzymes by Nigrospora oryzae

3.8. Effect of Enzymes Loading on Reducing Sugar Production Using Maize cobs and Sugarcane Bagasse Substrates and Nigrospora oryzae Cellulase Enzyme Extract

3.9. Effect of Substrate Loading on Reducing Sugar Production by Nigrospora oryzae Cellulase Enzymes

3.10. Simultaneous Saccharification and Fermentation of Maize Cobs and Sugarcane Bagasse Using Nigrospora oryzae Cellulase and Saccharomyces cerviceaie

4. Discussions

4.1. Effect of Incubation Period on Enzyme Production

4.2. Effect of pH on Cellulase Enzyme Production

4.3. Effect of Nitrogen Sources on Cellulase Enzymes Production

4.4. Effect of Carbon Supplementation on Cellulase Enzyme Production

4.5. Effect of Moisture on Cellulase Enzymes Production

4.6. Effect of Surfactant on Cellulase Enzymes Production

4.7. Effect of Enzymes Loading on Reducing Sugar Production Using Maize Cobs and Sugarcane Bagasse Substrates and Nigrospora oryzae Cellulase Enzyme Extract

4.8. Effect of Substrate Loading on Reducing Sugar Production by Nigrospora oryzae Cellulase Enzymes

4.9. Simultaneous Saccharification and Fermentation of Maize Cobs and Sugarcane Bagasse Using Nigrospora oryzae Cellulases and Saccharomyces cerevisiae

5. Conclusions

Author Contributions

Funding

Ethical approval

Acknowledgments

Conflicts of Interest

References

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