Nova Biotechnol Chim (2022) 21(2): e1226 DOI: 10.36547/nbc.1226 1 Nova Biotechnologica et Chimica Optimization of procedural factors for advanced xylanase synthesis by Lysinibacillus fusiformis using Kolanut husk as substrate Suliat Olatidayo Omisore, Daniel Juwon Arotupin, Michael Tosin Bayode Department of Microbiology, Federal University of Technology, P.M.B. 704, Akure, Nigeria  Corresponding author: omisore34@gmail.com Article info Article history: Received: 13th October 2021 Accepted: 1st December 2022 Keywords: Kolanut husk Lysinibacillus fusiformis Optimization Substrate Xylan Xylanase Abstract Xylan is a complex hetero-polysaccharide consisting of different monosaccharides held together by glycosidic and ester bonds. Extracellular xylanase fashioned by numerous microbes principally from bacterial species such as Bacillus species are responsible for cleaving the glycosidic linkages. Microbial xylanases exhibit different substrate specificities and biochemical peculiarities. This study was carried out for optimization of cultivation conditions for xylanase production using the bacterium Lysinibacillus fusiformis and Kolanut husk as a component of cultivation medium. The bacterium was isolated from Kolanut plantation waste soil and screened for the production of xylanase qualitatively on xylan nutrient agar and quantitatively under submerged fermentation. The different conditions optimized included substrate concentration, additional sugars, incubation period, temperature, initial pH, nitrogen supplementation and inoculum mass through one factor at a time approach. Maximum xylanase production was obtained at substrate concentration of (1 % xylan and 1.5 % Kolanut husk), nitrogen source (yeast extract plus peptone), carbon source (sucrose), incubation period (24 h), pH (5.0), temperature (35 oC) and inoculum size (1 %). Lysinibacillus fusiformis has been proven to be a promising bacterium for xylanase production using Kolanut husk as substrate. The use of Kolanut husk as foremost carbon source is predominantly precious as being an agricultural waste, affordable, and locally available compared to expensive commercially sold xylan. Introduction Hemicellulose serves as the second most ample source of polysaccharides in nature, exhibiting 25 % 30 % of the dry weight of lignocellulose biomass (Coman et al. 2011; Walia et al. 2015). It is the major amount of lignocellulose, comprising of varied 5- and 6-carbon sugars such as arabinose, galactose, glucose, mannose, and xylose (Girisha 2018). Xylan is a polymer of xylose molecules which plays the pivotal role in holding the plant cell walls together (Irfan et al. 2016; Girisha 2018). Considerable xylanolytic enzymes comprising α-arabino-furanosidase, act collectively for total disintegration of xylan. The most important endo β- 1, 4-xylanase plays an imperative part to catalyze the breakdown of xylan into diminutive oligosaccharides (Irfan et al. 2012; Khusro et al. 2016). Udey et al. (2016) reported the latent applications of xylanase in numerous fields such as saccharification of lignocellulosic biomass for ethanol and xylo-oligosaccharides generation in the biofuel industry. Its use as a fading agent and mailto:omisore34@gmail.com Nova Biotechnol Chim (2022) 21(2): e1226 2 lighting up of pulp to reduce chlorine in paper and pulp production operations was also reported (Raghukumar et al. 2004; Sridevi et al. 2016; Patel and Dudhagara 2020a). It helps in the smooth relocation of the water core in bread dough ensuring a softer dough formation (Beg et al. 2001; Garg et al. 2010). Furthermore, xylanolytic enzymes have been reported by Garg et al. (2010) for its application in the animal industry for the conversion of lignocellulose into feedstocks and fuels. Xylanases are produced by a diverse number of microbes such as bacteria, fungi and actinomycetes (Battan et al. 2006; Chakdar et al. 2016; Ramanjaneyulu et al. 2017). However, xylanases from bacteria are the most-preferred for industrial applications due to thermo-stability and ability to withstand alkali-substrates (Nagar et al. 2013; Aarti et al. 2015; Mandal et al. 2015). Xylanolytic bacteria are the generally striking producers of high-level extracellular xylanase as compared to fungi and actinomycetes, which have time- consuming generation time alongside the production of extremely glutinous polymers and deprived oxygen transport. There is limited exploitation of xylanase commercially due to its high cost of production. In this view, the search for cost-effective means of production, such as the use of low-cost agro agricultural residues has captivated the attention of scientists (Patel and Dudhagara 2020b). Gautério et al. (2020) and Ratnadewi et al. (2020) also buttressed the utility of agriculture waste derivatives like wheat bran, corn cobs, rice straw for exploitation as a low- grade substrate for xylanase production. Kolanut husk alongside its testa has been proven as a budding substrate to produce the microbial enzyme particularly xylanase (Fabunmi et al. 2018). Therefore, the study was carried out to determine the optimal process parameters needed to improve the yield of xylanase produced from simple inexpensive Kolanut husk using Lysinibacillus fusiformis. In biotechnological process, the best results are achieved when the individual and combined effects of parameters involved are considered (Tandon et al. 2016). The current study aims at optimizing the cultural conditions such as carbon source, nitrogen supplementation, temperature, incubation time and inoculum sizes for xylanase production by Lysinibacillus fusiformis, an isolate of Kolanut plantation waste soil using a medium with Kolanut husk as substrate in other to know the best conditions for highest xylanase. Experimental Kolanut plantation soil and Kolanut husk collection Kolanut husks and Kolanut plantation soil from the Kolanut husk dumpsite were obtained from a Kolanut farm at Ile Ife City, Osun State, Nigeria (7.4905° N, 4.5521° E). Sample transported to the laboratory of Microbiology Department, Federal University of Technology Akure, Ondo State in a ziploc bag. The soil samples were preserved at -20 ºC before use. Isolation and identification of bacteria isolates One gram of the soil sample was suspended in 100 mL of distilled water then incubated in a control incubator shaker (Gallenkamp, Cambridge, UK) at 200 rpm for 30 min. Mixtures were allowed to settle, and serial dilutions of up to 104 diluents were prepared using sterile distilled water. Bacterial isolation was conducted by aseptically plating 100 µL from the diluent on xylan nutrient agar (nutrient agar supplemented with 1 % xylan) and incubated aerobically for 37 ºC for 24 h. Purification of bacteria was done by repeated streaking and sub-culturing for purity and preserved at 4 ºC until use (Kartik et al. 2016). Authentication of the xylanolytic ability of isolates by well plate diffusion assay The organism was however authenticated qualitatively for positive xylanase production by growing isolates on nutrient agar medium containing 1 % birchwood xylan. The media was sterilized, poured, and allowed to solidify. Upon solidification, wells were made with the aid of sterile cork-borer of 10 mm diameter. To each well bored in the solidified media, 100 µL of the overnight pure bacterial culture was poured and incubated for 24 – 36 h. The plate was flooded with Nova Biotechnol Chim (2022) 21(2): e1226 3 0.25 % Congo red solution for 15 min after which it was decolorized with 0.5 % acetic acid for 15 min and finally washed twice by 1 M NaCl for de-staining as described (Chan et al. 2016). Clear zones around the well further authenticated xylanolytic activity. Submerged fermentation for xylanase production Submerged fermentation was conducted using Kolanut husk as the substrate in addition to minimal salt medium containing (g.L-1): xylan 10.0, K2HPO4 0.1, CaCl2 0.5, MgSO4.7 H2O 0.5, peptone 2.0, FeSO4·7 H2O in trace amount, pH 6.8, sterilized at 121 ºC for 15 min. 50 mL of the sterile minimal salt medium in 250 mL flasks were inoculated with 1 mL of bacterial culture in broth and incubated at 37 oC for 24 h in a rotary shaker (HunterLab Digital Colorimeter, Reston, USA) with agitation speed of 150 rpm. After the incubation period, the enzyme production medium was subjected to cold centrifugation at 10,000 rpm for 10 min to eliminate the bacterial cells and superfluous elements. The supernatant considered as source of the crude enzyme was gently decanted into sterile vials (Sari et al. 2016). Xylanase assay Xylanase activity in the supernatant was assayed by determining the concentration of reducing sugar released by the activity of the crude enzyme on its substrate xylan by 3,5-dinitrosalicylic acid (DNSA) reagent (Miller 1959). Reaction mixture containing 0.5 mL of culture enzyme with 0.5 mL 1 % of Birchwood xylan solution primed in phosphate buffer pH 6.8 was incubated for 20 min at 45 oC. The control experiment was set up to authenticate the results from the actual testing; with the control tube comprising a similar volume of a substrate with enzyme solution being replaced with sterile distilled water. After incubation, the reaction was terminated by adding 1 mL of DNSA reagent as described by Miller (1959). The tubes enclosing the resultant blend were excited in the boiling water bath for 10 min, observed for brick red colour development and cooled swiftly in a water bath at room temperature. The optical density of the cooled mixture was measured with a spectrophotometer (Lab-Tech Digital Colorimeter) at 540 nm against a blank and articulated as xylose equivalent. One unit of endo-1,4-β-xylanase was defined as the amount of enzyme requisite to discharge 1 µmol of xylose per minute in model assay conditions as depicted by Sharma and Bhajaj (2005); Bhalla et al. (2015). Protein estimation The amount of protein released in the supernatant was estimated as described Bradford (1976) using Bovine Serum Albumin (BSA) (Sigma-Aldrich, Darmstadt, Germany) as standard. 1mL of the enzyme extract was taken in a glass tube and added to it 3 mL of Bradford’s reagent, the mixture was incubated at room temperature for 7 – 10 min. The absorbance was measured at 595 nm with a spectrophotometer (HunterLab Digital Colorimeter, Virginia, USA) and compared with the Bovine Serum Albumin standard curve. Optimization of cultural and nutritional conditions The optimization of xylanase production by Lysinibacillus fusiformis was carried out using “one factor at a time technique” (varying one factor while keeping the rest constant) in order to estimate xylanase activity for each varied parameter. Parameters such as the effect of Kolanut husk as carbon source at concentration (0.5 – 2.5 %), Kolanut husk induction with xylan (0.5 – 1.5 %), additional carbon source at 0.5 % (glucose, fructose, sucrose, galactose and lactose), nitrogen supplementation at 0.25 % (yeast extract, urea, gelatin, tryptone, casein, ammonium chloride, ammonium nitrate, ammonium sulfate, potassium nitrate), incubation period (0 – 42 h), pH (2 – 10), temperature (25 – 45 oC) and inoculum size (0.5 – 2.5 %) were optimized for enhanced xylanase production by Lysinibacillus fusiformis. The fermentation broth was separated from the bacterial cells by centrifugation, the supernatant served as a source of a crude enzyme tested for xylanase activity. The entire experimentations were done in triplicates with each mean shown in the result section. Each experiment had its control with the control tube not having the tested process parameters. Nova Biotechnol Chim (2022) 21(2): e1226 2 Statistical analysis The data obtained after the optimization studies were statistically evaluated using the one-way analysis of variance (ANOVA) at significance level of P < 0.05 using the statistical package IBM SPSS Statistics v. 23.0 (Chicago, USA). Results Screening and authentication of obtained xylanolytic bacteria Isolate appeared cream-coloured, regularly shaped, round edges with 1 – 2 mm diameter on nutrient agar plate. It was Gram-positive and rod-shaped and conforms to its earlier identification by Fabunmi et al. (2018) as L. fusiformis. Organisms showed zones of hydrolysis on xylan agar plates after incubation, indicating its ability to degrade xylan in the media alongside its growth during the incubation period with an average of 29 mm diameter zone of hydrolysis on xylan agar medium. Optimization of culture and nutritional conditions The effect of different substrate (Kolanut husk) concentration (0.5 – 2.5 %) as sole source of xylan in submerged fermentation by L. fusiformis is in Table 1. Result indicated that the highest enzyme activity was at Kolanut husk concentration of 2.0 % with value of 419.28 µmol.mL-1.min-1 while the least at 0.5 % with a value of 201.52 µmol.mL-1.min-1. Table 1. Upshot of substrate concentration (Kolanut husk) on xylanase production by L. fusiformis under SmF. Values are obtainable as Mean ± S.E (n = 3). Values with different superscripts are statistically significantly different (P < 0.05). SmF – Submerged fermentation. Effect of xylan supplementation with Kolanut husk on xylan production by L. fusiformis The effect of different concentrations of a husk (0.5, 1.0, 1.5, 2.0, and 2.5 %) in combination with a commercial birchwood xylan was supplemented to the fermentation medium for enhanced production of xylanase as shown in Table 2. Xylan supplementation as inducer showed maximum activity at 1.5 % Kolanut husk supplemented with 1 % commercial xylan with xylanase activity of 422.63 µmol.mL-1.min-1 and lowest at 1 % Kolanut husk with 0.5 % xylan with activity of 303.06 µmol.mL-1.min-1. Effect of carbon source on xylanase production by Lysinibacillus fusiformis Effect of sugar optimization revealed sucrose to having the highest activity effect amongst others with enzyme activity of 549.75 µmol.mL-1.min-1 exceeding other sugars as shown in Table 3. The monosaccharide galactose gave the least activity of 494.5 µmol.mL-1.min-1, the remaining sugars gave moderate improvement on enzyme production but not comparable to sucrose. Kolanut husk [%] Substrate optimization Absorbance Enzyme activity [µmol.min-1.mL-1] Protein concentration Specific enzyme activity [µmol.min-1.mg-1] 0.5 0.54 ± 0.00a 206.52 ± 2.02a 1.96 ± 0.06a 100.18 ± 0.16b 1.0 0.70 ± 0.00b 266.83 ± 2.04b 2.17 ± 0.01b 124.28 ± 0.11e 1.5 0.92 ± 0.00c 341.43 ± 0.29c 3.89 ± 0.02d 88.34 ± 0.08a 2.0 1.11 ± 0.00e 419.28 ± 0.36e 3.91 ± 0.01d 106.83 ± 0.12c 2.5 1.01 ± 0.00d 381.36 ± 0.31d 3.11 ± 0.06c 121.65 ± 0.03d 4 Nova Biotechnol Chim (2022) 21(2): e1226 3 Table 2. Effect of Xylan (X) supplementation as inducer with varied Kolanut husk concentration on xylanase production by L. fusiformis under SmF. Values are obtainable as Mean ± S.E (n = 3). Values with different superscript are statistically significantly different (P < 0.05). SmF – Submerged fermentation. Table 3. Effect of sugar supplementation (carbon source) on xylanase production by Lysinibacillus fusiformis under SmF. Carbon sources [%] Carbon sources optimization Absorbance Enzyme activity [µmol.min-1.mL-1] Protein concentration Specific enzyme activity [µmol.min-1.mg-1] Control (peptone water) 0.86± 0.02a 320.66 ± 0.88a 1.79 ± 0.05d 177.61 ± 0.87a Glucose 1.39 ± 0.01d 521.74 ± 0.80e 2.06 ± 0.02e 250.47 ± 0.86b Fructose 1.38 ± 0.01d 518.02 ± 1.13d 1.21 ± 0.04c 452.10 ± 0.58d Sucrose 1.45 ± 0.02e 550.58 ± 0.71f 1.08 ± 0.02b 500.57 ± 0.86e Galactose 1.32 ± 0.01c 495.16 ± 0.44c 0.88 ± 0.01a 566.38 ± 2.34f Lactose 1.25 ± 0.02b 476.16 ± 1.48b 1.76 ± 0.02d 264.52 ± 0.86c Values are obtainable as Mean ± S.E (n = 3). Values with different superscript are statistically significantly different (P < 0.05). SmF – Submerged fermentation. Effect of organic nitrogen on xylanase production by Lysinibacillus fusiformis Nitrogen sources revealed yeast extract giving the highest xylanase titre (437.75 µmol.mL-1.min-1) and gelatin (339.38 µmol.mL-1.min-1) displayed the lowest enzyme titre (Table 4). Table 4. Effect of organic nitrogen source on xylanase production by Lysinibacillus fusiformis under SmF. Inoculum [%] Organic sources optimization Absorbance Enzyme activity [µmol.min-1.mL-1] Protein concentration Specific enzyme [µmol.min-1.mg-1] Control (peptone water) 1.05 ± 0.00b 401.06 ± 0.23b 2.76 ± 0.07b 149.06 ± 0.38b Gelatin 0.86 ± 0.01a 340.12 ± 0.47a 2.79 ± 0.06b 116.61 ± 0.45a Casein 1.12 ± 0.00c 426.90 ± 0.31c 2.72 ± 0.01b 155.44 ± 0.29c Urea 1.13 ± 0.00c 430.72 ± 0.14d 2.39 ± 0.01a 181.06 ± 0.52e Tryptone 1.15 ± 0.00d 437.98 ± 0.89e 2.36 ± 0.01a 184.73 ± 0.63f Yeast extract 1.15 ± 0.00d 438.55 ± 0.47e 2.70 ± 0.01b 162.89 ± 0.58d Values are obtainable as Mean ± S.E (n = 3). Values with different superscript are statistically significantly different (P < 0.05). SmF – Submerged fermentation. Effect of inorganic nitrogen on xylanase production by Lysinibacillus fusiformis Table 5 showed the effect of inorganic nitrogen with KNO3 resulting in the highest xylanase activity (464.25 µmol.mL-1.min-1) and NH4CO3 with the least (325.70 µmol.mL-1.min-1). Kolanut husk + Xylan [%] Substrate optimization Absorbance Enzyme activity [µmol.min-1.mL-1] Protein concentration Specific enzyme activity [µmol.min-1.mg-1] K1.0 + X0.5 0.80 ± 0.00a 303.06 ± 0.54a 1.87 ± 0.01a 162.84 ± 0.07d KI.0+ X1.0 1.01 ± 0.00d 384.13 ± 0.59e 2.09 ± 0.05b 190.69 ± 0.85f K0.5+X1.0 0.97 ± 0.01c 367.12 ± 1.16c 2.38 ± 0.01c 153.37 ± 0.49b K1.5+X0.5 0.79 ± 0.00a 374.65 ± 0.87d 2.38 ± 0.07c 166.72 ± 0.43d K1.5+X1.0 1.12 ± 0.00e 422.54 ± 1.44f 2.39 ± 0.01c 178.55 ± 0.23e 2.0K.H 0.92 ± 0.00b 348.36 ± 0.31b 2.36 ± 0.02c 147.21 ± 1.17a 5 Nova Biotechnol Chim (2022) 21(2): e1226 2 Table 5. Outcome of inorganic nitrogen source on xylanase production by Lysinibacillus fusiformis under SmF. Inoculum [%] Inorganic sources optimization Absorbance Enzyme activity [µmol.min-1.mL-1] Protein concentration Specific enzyme [µmol.min-1.mg-1] Control (peptone water) 1.05 ± 0.00b 400.56 ± 0.86b 2.68 ± 0.02a 148.96 ± 1.15e KNO3 1.22 ± 0.01d 463.74 ± 0.90d 3.83 ± 0.01c 121.27 ± 0.36d (NH4)2SO4 1.05 ± 0.00c 398.01 ± 0.57b 3.52 ± 0.02b 113.49 ± 0.86c NaNO3 1.07 ± 0.01c 405.86 ± 0.59c 4.30 ± 0.05d 96.44 ± 0.28b NH4CO3 0.85 ± 0.00 325.70 ± 1.19a 4.50 ± 0.01e 92.45 ± 0.27a Values are obtainable as Mean ± S.E (n = 3). Values with different superscript are statistically significantly different (P < 0.05). SmF – Submerged fermentation. Effect of pH on xylanase synthesis by Lysinibacillus fusiformis Media pH is an extra significant dynamic considered in Table 6, enzyme activity was highest value of 537.79 µmol.mL-1.min-1 at pH of 5.0 and the lowest enzyme activity at pH of 2.0 (368.20 µmol.mL-1.min-1). A supplementary boost in pH above 5.0 resulted in a significant drop-off in the activity recorded. Table 6. Upshot of pH on xylanase synthesis by Lysinibacillus fusiformis under SmF. pH pH optimization Absorbance Enzyme activity [µmol.min-1.mL-1] Protein concentration Specific enzyme [µmol.min-1.mg-1] 2 0.88 ± 0.08a 368.20 ± 0.90a 3.55 ± 0.02d 102.69 ± 0.11a 3 1.11 ± 0.01bc 424.08 ± 0.46b 3.52 ± 0.03cd 121.53 ± 0.03b 4 1.20 ± 0.00cd 456.88 ± 1.55c 3.05 ± 0.02b 148.83 ± 0.06d 5 1.41 ± 0.00g 537.79 ± 1.10h 3.73 ± 0.12e 141.29 ± 0.12c 6 1.39 ± 0.00fg 528.31 ± 0.84g 3.35 ± 0.07c 161.22 ± 0.14g 7 1.31 ± 0.00ef 497.30 ± 0.84f 3.14 ± 0.03b 159.14 ± 0.13f 8 1.28 ± 0.00de 486.26 ± 1.86d 3.10 ± 0.05b 161.25 ± 0.14g 9 1.20 ± 0.00cd 458.18 ± 0.92cc 2.99 ± 0.05b 152.71 ± 0.11e 10 1.09 ± 0.00b 490.56 ± 0.72e 2.75 ± 0.02a 141.44 ± 0.02c Values are obtainable as Mean ± S.E (n = 3). Values with different superscript are statistically significantly different (P < 0.05). SmF – Submerged fermentation. Effect of temperature on xylanase production by Lysinibacillus fusiformis Xylanase production was at its peak at 35 oC (572.83 µmol.mL-1.min-1) and least at 45 oC (347.33 µmol.mL-1.min-1) (Table 7). Table 7. Effect of temperature on xylanase synthesis by Lysinibacillus fusiformis under SmF. Temp [ºC] Temperature optimization Absorbance Enzyme activity [µmol.min-1.mL-1] Protein concentration Specific enzyme [µmol.min-1.mg-1] 25 1.20 ± 0.00c 457.64 ± 1.30c 3.07 ± 0.06a 151.82 ± 0.03c 30 1.39 ± 0.00d 531.41 ± 0.33d 3.11 ± 0.11a 169.65 ± 0.17d 35 1.51 ± 0.00e 572.77 ± 1.29e 3.50 ± 0.05b 167.62 ± 1.44d 40 1.10 ± 0.00b 419.86 ± 0.32b 3.39 ± 0.05b 124.51 ± 0.86b 45 0.91 ± 0.00a 346.11 ± 0.67a 2.94 ± 0.02a 119.35 ± 0.87a Data are presented as Mean ± S.E (n = 3). Values with different superscript are statistically significantly different (P < 0.05). SmF – Submerged fermentation. 6 Nova Biotechnol Chim (2022) 21(2): e1226 3 Effect of incubation time on xylanase production by Lysinibacillus fusiformis Incubation period optimization results showed that 24-h cultivation (624.29 µmol.mL-1.min-1) gave the best production as seen in Fig. 1 and further increase revealed a corresponding reduction in enzyme activity. Fig.1. Effect of time on xylanase production by Lysinibacillus fusiformis under submerged fermentation. Effect of inoculum size on xylanase production by Lysinibacillus fusiformis Inoculum size is a vital dynamic in enzyme synthesis and maximum enzyme activity was recorded at 1.5 % with a value of 647.63 µmol.mL- 1.min-1 and the least observed at 2.5 % with 616.07 µmol.mL-1.min-1 (Table 8). Table 8. Effect of inoculum size on xylanase production by Lysinibacillus fusiformis under SmF. Inoculum [%] Inoculum optimization Absorbance Enzyme activity [µmol.min-1.mL-1] Protein concentration Specific enzyme [µmol.min-1.mg-1] 0.5 1.62 ± 0.00a 615.27 ± 0.36a 0.89 ± 0.01b 697.68 ± 0.71d 1.0 1.67 ± 0.00b 624.07 ± 0.51b 0.96 ± 0.01c 653.53 ± 0.74b 1.5 1.70 ± 0.00d 647.63 ± 0.32d 1.25 ± 0.01d 518.78 ± 0.32a 2.0 1.69 ± 0.00c 643.35 ± 0.86c 0.97 ± 0.01c 671.07 ± 0.52c 2.5 1.62 ± 0.00a 616.07 ± 0.51a 0.84 ± 0.00a 732.31 ± 0.56e Data are presented as Mean ± S.E (n = 3). Values with different superscript are statistically significantly different (P < 0.05). SmF – Submerged fermentation. 7 Nova Biotechnol Chim (2022) 21(2): e1226 2 Discussion Kolanut husk in nature is composed of hemicellulose (40.41 %), lignin (21.29 %), cellulose (38.72 %) as reported by Adeyi (2010) and Patel and Dudhagara (2020a), making it an appropriate substrate for xylanase production. They are mostly disposed of as agricultural waste at production site of Kolanut forming a landfill and environmental problems. Nevertheless, as a substrate for enzyme production could help to solve environmental pollution problems as well as reduction in production cost of xylanase. Kolanut husk as an enzyme source was optimized as a component of the microbial fermentation medium. Among the concentration of Kolanut husk tested 2.0 % gave the maximum xylanase production. Concentration above 2.0 % demonstrated a declined xylanase production but not as low as when lower concentrations of 0.5 % and 1.0 % was used (Table 1). This indicated that adding further substrate does not show any noteworthy change in xylanase synthesis. A general boost in substrate absorption could lead to swell in enzyme output velocity at certain point till it reaches substrate saturation where no enzyme activity could be recorded (Heskia et al. 2018). Furthermore, the fermentation medium was induced with commercial birchwood xylan alongside Kolanut husk. This was intended to boost xylanase production in the medium. Optimum xylanase production was attained at a lesser substrate concentration in contrast to medium with solely Kolanut husk. Different xylan concentrations have also been reported to give maximum xylanase activity of 0.5 % (Annamalai et al. 2009; Heskia et al. 2018). Thus, indicating that xylanase stimulation is an intricate trend in which the intensity of reaction varies with altered concentration combinations. Decreases in enzyme activity at higher substrate concentration might be owed to the formation of thick suspension in the presence of higher substrate concentration which could have resulted in improper mixing of the substrate or non-uniform circulation of a nutrient under agitation condition (Karim et al. 2015). Karim et al. (2015) has revealed that substrate concentration used is a decisive element for utmost xylanase optimization and that there are different concentrations for various ligno-cellulose substrates. Supplementation of medium with additional carbon sources involving both monosaccharide and disaccharide improved the enzyme activity produced. Sucrose had the highest enzyme activity and closely followed by other sugars (Table 2 and Table 3). The results obtained agree with Singh et al. (2011) who stated that amylase synthesis is favored by galactose, linking to the fact growth and enzyme productions of any organism are greatly influenced by both environmental conditions as well as the nutrient present in the growth medium. However, these findings contradict reports that wholesome sugars only have good growth but poor xylanase synthesis (Shah and Madamwar 2005) and that of Seyis and Aksoz (2005) that sucrose and glucose gave inhibitory effect on enzyme activity. Their reports were attributed to catabolite repression of xylanase genetic material which is guarded at two levels as reported by De Graaff et al. (1994), repressed gene transcription and ultimately by repressed transcriptional activator. In addition to carbon sources, nitrogen sources have a significant end product on the synthesis of xylanolytic enzymes produced by bacteria. Overall, crude sources gave a higher xylanase production than inorganic sources (Table 4 and Table 5). This agrees with the report of Bajaj et al. (2010), who stated that peptone combined with yeast extract as the largely reasonable and accustomed nitrogen sources in terms of elevated output of xylanase from diverse bacteria. Nagar et al. (2010) reported maximum xylanase production by B. pumilus SV- 85S using peptone followed by yeast extract as nitrogen source. However, it is contrary with the report of Sepahy et al. (2011) where tryptone in combination with yeast extract gave the maximum xylanase titre. Peptone and yeast extract are multifaceted nitrogen sources rich in diverse growth factors such as minerals and vitamins and hence improve the growth of bacteria and enzyme synthesis (Bibi et al. 2014) as few among the inorganic KNO3 resulted in higher titre among others. Sepahy et al. (2011) stated the pH of the medium has a strong influence on enzymatic processes as it affects the growth and metabolic activities of 8 Nova Biotechnol Chim (2022) 21(2): e1226 3 individual microorganisms. Influences of pH ranging from acidic to alkaline were tested for xylanase production as B. megaterium had the highest production at pH 5.0, indicating that acidic favours this particular organism when acting on Kolanut husk as substrate. The alkalinity or acidity of the medium affect bacteria growth coupled with other enzymatic reactions through transport of ions, metabolites, and enzyme structure (Liang et al. 2010). Our results revealed that as the pH of the medium increases from extremely acidic (pH 2.0) to slightly acidic (pH 5.0), so does the enzyme activity increased but tending towards neutral pH, there was a drop in the value and the least value recorded at alkaline pH (Table 6). This report is in contrary with the observations of Sepahy et al. (2011); Irfan et al. (2016) who reported the highest xylanase synthesis at pH 8.0 using B. mojanvensis and B. subtilis under submerged fermentation. This could possibly be attributed to the alkaline medium possessing an inhibitory influence on Lysinibacillus fusiformis and moreover each microorganism has its pH for optimum growth and enzyme production. Temperature is another key dynamic that influences the success of optimization system. The best medium temperature was 35 oC which is close to the optimal temperature for utmost xylanase titre in submerged fermentation (SmF) (Table 7) from other Bacillus species reported as 37 °C (Qureshi et al. 2002; Battan et al. 2007; Geetha and Gunasekaran 2010; Nagar et al. 2012) but contradicts the report of 50 °C temperature (Sa- Pereira et al. 2002; Azeri et al. 2010; Sepahy et al. 2011), 50 – 55 °C (Anuradha et al. 2007; Sharma et al. 2011) and 55 °C (Annamalai et al. 2009). This indicates that productions of xylanase are favored at ambient temperature as compared to extremophilic temperature. This could be due to the fact that mesophilic temperature favors the growth of organisms and hence their ability to produce the desired end product. Though, thermophilic temperature is best preferred as it diminishes the risk of mesophilic microbial defects (Yeoman et al. 2010). Generally, Bacillus sp. shows ability to grow and produce enzymes at 37 oC under suitable fermentation conditions (Sanghi et al. 2008; Nagar et al. 2010). The finest temperature for microbial enzyme synthesis may fluctuate as it is linked to their proliferation. The organism used in this study showed relatively high xylanase production within a short period of incubation which could be attractive for large-scale xylanase production. Xylanase activity was found to decrease with prolonged incubation which could be linked to depletion in the available nutrient as time progresses and accumulation of end-products (Fig. 1). Production of toxic metabolites inhibits enzyme synthesis also (Irfan et al. 2016). Apparently, the increased incubation period affected any xylanase yield but reduced enzyme activity. This appears to be similar to Simphiwe et al. (2011) who reported 24-h as an utmost incubation period using digested bran as substrate. In another report (Anuradha et al. 2007), xylanase synthesis by Bacillus species was growth-related with highest production at 24-h incubation period. However, Prakash et al. 2011 reported contrarily with maximum production after 48-h of fermentation with B. habdurans. The decline in xylanase production during the later stage of fermentation could be attributed to the liberation of a limited volume of protease from the mature cells penetrating into autolysis or shortage of impenetrable xylan particles in the medium. Limited fermentation time presented better and gainful production which is a favourable condition in large-scale industrial production. Optimal inoculum size is crucial for preserving balance between growth sizes and obtainable nutrients in order to obtain the utmost yield. The outcome of this study as illustrated in Table 8 is parallel to Irfan et al. (2012) where 1.5 % inoculum size as peak of yield using B. megaterium on corn cob but in variance with that of Nagar et al. (2010) who tested higher inoculum size (1.0 – 5.0 %) for xylanase production. The reduction might be related to the depletion of available nutrients from the fermentation medium as inoculum level increases which could have resulted to decline in enzyme synthesis. Conclusion The search for agricultural waste such as Kolanut husk as potential source of enzyme production rather than environmental nuisance is desirable as it reduces cost of production and increases bioconversion of waste to value-added compounds 9 Nova Biotechnol Chim (2022) 21(2): e1226 2 by economically feasible techniques. The results obtained therein showed that L. fusiformis can produce xylanase via Kolanut husk as a substrate under submerged fermentation. Results indicated that cultural and nutritional parameters are paramount to enzyme production. 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