Response surface optimization of xylanase diminutive cellulase by a thermo-alkaline tolerant Trichoderma harzanium FC50 under solid state cultivation on Delonix regia pods

Abstract

Xylanase from thermotolerant and alkaline-tolerant fungi plays a vital role in reducing chemical pollution in the bio-pulping process. This study explores the potential of Delonix regia pods for Response Surface Optimization, for the production of xylanase diminutive cellulase. Fungi isolate from hot compost was screened on xylan-agar plate. Enzyme production at 84.10 U/g (xylanase) and 11.17 U/g (cellulase), was significant with Delonix regia pods with Trichoderma harzanium FC50. Using Plackett-Burman design and Central Composite Design, xylanase production was recorded at an optimum temperature of 37°C, pH of 6.0, and 75% moisture content after 120 h of fermentation. The fungus also produced high xylanase over a broad pH range (6–9) and a temperature range (37–49°C). Xylanase increased by 42.8-fold and 1.43-fold with xylan and Delonix regia pods as substrates, respectively. The model was beneficial for predicting optimumal xylanase production and optimizing experimental growth conditions with Delonix regia pods as substrate.

KEYWORDS:

• Delonix regia pods
• thermo- alkaline tolerant
• Trichoderma harzanium
• xylanase
• solid state fermentation
• Response Surface Methodology

1. Introduction

Xylanases are enzymes grouped under the glycoside hydrolase families (O – glycoside hydrolases, EC 3.2.1. X), representing one of the largest groups of commercial enzymes [1]. They catalyze the conversion of xylan to xylose moieties by breaking down the β, 1–4 linkages in the xylan backbone. Xylan is a major structural hemicellulosic polysaccharide in plant cells and the second most abundant polysaccharide in nature after cellulose, measuring up to 1/3 of all renewable organic carbon on earth [2]. The hemicellulose in xylan, is a branched heteropolymer consisting of hexose and pentose sugars, with xylose sugars as the most abundant [3].

Xylanases have found significant applications in different industrial processes of biofuel production, animal feed processing, clarification of juices and wines, improvement of bread quality, processing of fabrics, silage production, waste treatment, etc. They have a good market proportion in the pulp and paper industry and are used to reduce the viscosity and enhance the absorption rate of feeds by degrading the starch polysaccharides in rice fibre and barley-based feeds [4].

Xylanase production by fungi is largely affected by growth factors such as carbon and nitrogen sources, temperature, pH, incubation period, inoculum sizes, and moisture content [5]. To maximize xylanase production, therefore, it is paramount to screen and optimize significant factors that affect its production [6]. In this regard, the classical One-Factor-at-a-Time (OFAT) and optimization study by the Design of Experiment (DOE) is considered suitable for xylanase production optimization studies. Although, the OFAT appears easy to conduct, it is time consuming, and cost-implicative when large factors are involved and does not consider the interactive effects among factors, resulting in unreliable and inaccurate conclusions[7]. Therefore, statistical approaches involving the use of DOE such as Response Surface Methodology (RSM) are employed to combat these limitations associated with OFAT.

Xylanase production from different fungi species by solid-state fermentation has been investigated through RSM. Trichoderma asperellum [8] and Trichoderma reseei QM9414 [9], demonstrate a significant potential for xylanase production and application in the biobleaching of pulp for the separation of lignin from cellulose. However, xylanase from these fungi shows low tolerance to thermophilic and alkaliphilic conditions applied in the biobleaching processes. Xylanase shows optimal activity in pulp biobleaching at temperature range of 50-90°C and alkali pH ranging from pH 8–10 [10]. Thermophilic and alkaliphilic fungi produce enzymes with better characteristics including high optimum activity, and broad pH stability. Several reports have described the thermophilic property of fungi in the production and characterization of thermostable xylanases [11]. However, very few reports are available on the production of thermostable cellulase-free xylanase from thermophilic fungi strains [12]. Therefore, there is a need to source for more thermophilic and alkaliphilic xylanase-producing fungi, particularly those of thermostable cellulase-free xylanase.

Every year, a large amount of lignocellulose is generated through agro-industrial processing, and tree droppings. The utilization of these lignocellulosic wastes for xylanase production is an efficient way to reduce the production cost as well as to utilize them efficiently. Over the years, there has been enormous report describing the use of agro-waste, such as rice husk, sugarcane bagasse, rice straw, and corn cobs as substrate for xylanase production [13–15]. However, there is less attention on the use of tree droppings such as pods as potential substrates. Delonix regia pods are some of the tree droppings produced by the flamboyant tree as underutilized biomass. The pods are oblong, woody, flat, and approximately 55 cm. It is green and turns brown when matured. Recently, D regia pods were utilized as substrates for the production of laccase from Aspergillus carbonarius F5 [16] Sorghum straw biomass generated from Sorghum bicolour, cultivated in Nigeria, contains abundant lignocellulose. The straw consists of stalks and leaves which contain mainly cellulose, hemicellulose, and lignin. The hemicellulose content of sorghum straw is between 19.18 and 28.07% [17,18]. Thus, it is an ideal substrate for the production of xylanase. To our knowledge, no available information on the use of D. regia pods for xylanase production has been documented. Therefore, the D. regia pods and sorghum straw is considered to be an excellent source of carbon to achieve the desired bioprocess parameters for xylanase diminutive cellulase production by the thermotolerant and alkaline tolerant fungi.

In the present study, production of xylanase diminutive cellulase from a thermo-alkaline tolerant fungi isolated from hot compost is described. Consequently, the Central Composite Design (CCD) of Response Surface Methodology (RSM) is utilized to optimize the production and growth factors.

2. Materials and methods

2.1. Raw materials and compost preparation

Sorghum straw was collected from a local farm at Oke-Odo, University Road, Ilorin, Kwara State, Nigeria. The straw was milled to 1–2 cm particle size using a locally fabricated High-Speed Mill (HSM). Dry chicken manure from broiler chicken, fed with TopFeed Finisher Feed was collected from a local poultry farm at Offa-Road, Ilorin, Kwara State, Nigeria. The manure was collected into plastic bag and garbled for the removal of extraneous materials. The Shea-nut cake and effluent were obtained from a local shea-butter factory located at Apa-Ola, Ilorin, Kwara State, Nigeria. The compost was prepared by weighing 16 kg of sorghum straw, 8 kg of chicken manure, and 5.3 kg of shea-nut cake. The mixture was evenly moistened with shea-butter effluent to obtain a 70% moisture content. The resulting heap was properly covered with polyethylene and jute sacs to trap moisture and heat. The pile was turned at a three-day interval and temperature was monitored at 48 h interval using a digital thermometer.

2.2. Sample collection and isolation of thermo-alkaline tolerant fungi

The compost samples were collected at temperatures between 40°C to 65°C by mixing five sub-samples from different parts of the pile. Isolation was performed by suspending 1 g of compost samples into a 50 mL Erlenmeyer flask containing 10 mL sterile distilled water. Following serial dilution, 0.1 mL of sample was spread on a modified xylan-Czapek’s agar [(g/L): xylan, (10); NaNO₃, (1); K₂HPO₄, (1); MgSO₄, (0.5); FeSO₄, (0.01); agar-agar, (20)]. The pH of the media was adjusted to 8.0 using 1 M NaOH. The plates were wrapped with aluminum foil sheets, previously moistened with drops of sterile distilled water (to limit moisture loss), and then incubated at 40°C for five days. The pure cultures of the fungi were maintained on Potato Dextrose Agar (PDA) slants at 4°C for further use.

2.3. Qualitative screening for xylanase production

A culture disk of 5 mm diameter from a 5-day-old culture was placed at the centre of xylan- Czapek’s agar plate and incubated for 7 days at 40°C. After incubation, the plates were flooded with 15 mL of 0.5% (w/v) Congo red staining solution [19], and allowed to stand for 15 mins, then washed with 1 M NaCl to remove the stain. Xylanolytic isolates were selected based on distinct zone of hydrolysis produced around the mycelia. The xylanolytic index was calculated using Equation (1). The isolates with higher xylanolytic index were selected for further quantitative screening for carboxymethyl cellulase (CMCase) and xylanase activities. (1) $\text{XI}=\frac{\text{Diameter of hydrolysis zone}}{\text{Diameter of mycelia}}$(1) where; XI represents the xylanolytic index [13].

2.4. Quantitative screening for xylanase and CMCase production

The secondary screening was done for selected fungal isolates in basal medium [(g/L); MgSO₄.7H₂O (0.05), CaCl₂ (0.005), NaNO₃ (0.005), FeSO₄.7H₂O (0.09), ZnSO₄ (0.002), MnSO₄ (0.012), KCl (0.23), KH₂PO₄ (0.23), yeast extract (7)] containing 1% xylan and 1% carboxymethyl cellulose (CMC) for xylanase and CMCase activities, respectively [7]. The CMCase and xylanase production were done in a 50 mL Erlenmeyer flask containing 15 mL basal medium. Each flask was inoculated with 1 plug of a 5 mm diameter fungi and incubated at 40 °C in an incubator shaker at 120 rpm for 6 days. Samples were taken after 72 h and filtered using Whatman (No. 1) filter paper. The cell-free filtrates were centrifuged at 10,000 × g within 4°C for 15 mins and, the supernatant recovered was used as the crude enzyme.

2.5. Substrate preparation

Mature seed pods of D. regia (authenticated at the Herbarium unit of the Department of Plant Biology, University of Ilorin, Nigeria, with the voucher number UILH/001/990/2021) were collected from a flamboyant tree located within the University of Ilorin campus. The substrate was properly washed with distilled water, dried at 60°C for 24 h, and then milled to 1–2 mm particle size. The sorghum straw was also prepared by the same procedure as described previously.

2.6. Solid state fermentation

Fungi isolates were grown on D. regia pods and sorghum straw under solid-state fermentation according to Ajijolakewu, et al. [7] with little modifications. Four grams of the agro-waste in 250 mL Erlenmeyer’s flask containing 16 mL basal medium [g/L: yeast extract (20), MgSO₄ (0.3), CaCl₂ (0.3), FeSO₄ (0.005), ZnSO₄.7H₂O (0.0016), MnSO₄ (0.002), KH₂PO₄ (2)] adjusted to pH 8.0, was inoculated with 4 mycelial plugs (5 mm diameter each) from a 5-day old fungus and incubated at 40°C for 7 days. The crude enzyme was extracted with 40 mL phosphate buffer (pH 8.0) at 180 rpm for 60 min at 50°C. The fungal biomass was filtered using Whatman (No.1) filter paper and filtrate was further centrifuged at 10,000 × g for 30 mins at 4°C. The supernatant was used for the enzyme assay and fungi showing high xylanase diminutive cellulase activity was selected.

2.7. Proximate and lignocellulose composition analysis of substrate

The proximate analysis (moisture content, crude protein, crude fat, crude fibre, and ash) was carried out using standard procedures [20]. The lignocellulose composition analysis was done using the method described by Jancik, et al. [21].

2.8. Molecular identification of fungal isolate by 18S rDNA polymerase chain reaction (PCR)

The selected fungi isolate was subjected to macroscopic and microscopic examination following the procedure described by Senanayake et al. [22]. The Isolate was identified by amplification of the Internal Transcribed Spacer (ITS) region of the Ribosomal DNA (rDNA) using a forward primer ITS 1(5`-TCCGTAGGTGAACCTGCGG-3`) and reverse primer ITS 4 (5`-TCCTCCGCTTATTGATATGC-3`). The PCR preparation consisted of 10 µL of 5x GoTaq colourless reaction, 3 µL of 25 mM MgCl₂, 1 µL of 10 mM of dNTPs mix, 1 µL of 10 µmol of forward and reverse primer (ITS-1F and ITS-4R) 10.3 units of Taq DNA polymerase (Promega, USA) and 8 µL DNA template. The PCR was carried out in a GeneAmp 9700 PCR System Thermocycler (Applied Biosystem Inc., USA) with the cycling condition as; initial denaturation at 94°C for 5 mins, followed by 35 cycles with melting temperature at 94°C for 30 secs, annealing at 55°C for 1.5 mins, extension at 72°C and a final extension for 7 mins at 72°C. The amplified fragments were sequenced using a sequencer (Genetic Analyzer 3130xl, Applied Biosystems) and Big Dye terminator v3.1 cycle sequencing kit. Multiple sequence alignment was conducted on the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI) server using the Clustal Omega. The similarity search was conducted in-silico using the Nucleotide Basic Local Alignment Search Tool at the National Centre for Biotechnology Information (NCBI) server. The evolutionary distances were computed using the Maximum Composite Likelihood method and are presented in the units of the number of base substitutions per site. The analysis involved 24 nucleotide sequences, and all ambiguous positions with gaps were removed. The phylogenetic and molecular evolutionary analyses were conducted with MEGA version 7 [23] using the neighbour-joining method [24,25]

2.9. Screening of factors affecting xylanase production

Plackett-Burman Design [26] of Mini-Tab 17 software (Mini-Tab LLC, Pennsylvania, USA) was used to determine the most relevant factors among medium components and physical parameters, influencing the production of xylanase. Ten (10) factors (Yeast Extract, CaCl_2, MgSO₄, KH₂PO_4, pH, Tween 80, moisture content, incubation period, inoculum size, and temperature) were screened in 24 runs. The factors were examined at two levels, and the concentration of each factor applied in the experimental design is illustrated in Table 1. The main effect of each factor was calculated as the difference between the means of xylanase production measured using +1 and −1 concentrations. Equation (2), shows the first-order model used for the Plackett-Burman design: (2) $Y=\mathrm{\beta o}+\mathrm{\Sigma \beta iXi}$(2) where: Y = response (xylanase production (U/g), βo =model intercept, βi = linear coefficient for each factor and Xi = concentration of each factor).

Table 1. Variables and levels of screened factors employed in Plackett-Burman design.

Factor	Low level (−1)	High level (+1)
Yeast Extract	2 g/L	20 g/L
CaCl2	0.1 g/L	1 g/L
MgSO4	0.1 g/L	1 g/L
KH2PO4	0.5 g/L	3 g/L
Ph	6.0	9.0
Moisture content	65%	75%
Incubation Period	24 h	120 h
Temperature	37^oC	45 °C
Inoculum Size	3 plugs	5 plugs

2.10. Optimization of significant factors using central composite design (CCD)

Following the Plackett-Burman screening experiments, four factors (temperature, pH, incubation period and moisture content) with significant effect on xylanase production were selected for further optimization using the Central Composite Design (CCD) of Response Surface Methodology (RSM) Design Expert DX10.0 (Start, Ease 2010). A total of 50 experiments were performed in duplicates comprising 4 factors, 32 factorial points, 14 centres and 4 axial points. Optimum temperature, pH, incubation period and moisture content derived from the CCD optimization were used as functions to generate a model for xylanase production (U/g) and analysed based on ANOVA and Regression analysis. The variables and levels used in CCD are illustrated in Table 2. Data obtained from the experimental runs in the CCD design in this study, fitted into Equation (3) (second-order polynomial). (3) $Y={\beta }_0+\sum _{\iota =1}^{\kappa }{\beta }_i{\chi }_i+\sum _{i=1}^{\kappa }{\beta }_{\mathrm{ii}}{{\chi }^2}_i+\sum _{\iota =1,i<\mathrm{jj}=2}^{\kappa -1}{\beta }_{\mathrm{ij}}{\chi }_i{\chi }_j$(3) where, Y = The forecasted response; n = the number of studied factors; ${\chi }_i$ and ${\chi }_j$ are the coded variables; ${\beta }_0=$ the off-set term; ${\beta }_i,{\beta }_{\mathrm{ii}}$ and ${\beta }_{\mathrm{ij}}$ are the second-order, quadratic and interaction effects, respectively; i and j are the index numbers for the factor.

Table 2. Range and values of independent variables for the CCD used in the production of xylanase.

	Variables		Levels
S/No	Parameter code	Parameter	-α	Low (-)	High (+)	+α
1	A	Temperature (°C)	33	37	45	49
2	B	pH	4.5	6	9	10.5
3	C	Incubation period (Hours)	24	24	120	168
4	D	Moisture content (%)	60	65	75	80

2.11. Xylanase and cellulase assay

The xylanase assay was carried out in an assay mixture containing 0.5 mL of 1% (w/v) xylan in phosphate buffer (0.05 M, pH 8.0), 0.5 mL diluted crude enzyme and incubated at 50°C in a water bath for 30 mins. For the CMCase assay, the mixture contained 0.5 mL diluted crude enzyme, 0.5 mL of 1% (w/v) carboxymethyl cellulose (CMC) in phosphate buffer (0.05 M, pH 8.0) and incubated for 30 mins at 50°C. The reducing sugars (xylose and glucose) liberated were measured using the DNS method as described by Miller [27]. The xylanase and CMCase activity was defined as the amount of enzyme required to liberate 1µmoL of xylose and glucose per min under the specified assay conditions and expressed as unit of enzyme activity per mL (U/mL) or activity (U/g).

2.12. Statistical analysis

The experimental data were analysed using Design Expert software (Design Expert DX10.0, Statease Inc., Minneapolis, USA) and the quadratic equation was used to determine the xylanase yield. The fitness of the data into the equation was validated using the co-efficient variation (R²) in the analysis of variance (ANOVA) and statistical significance as measured by the F-test.

3. Results and discussion

3.1. Isolation and screening of thermo-alkali tolerant xylanolytic fungi

A total of seven fungal species were isolated from sorghum straw/shea nut cake-chicken manure compost. Isolates with xylanolytic index greater than 1.20 on Czapek's-xylan agar were screened and selected. Isolate FF50 recorded the highest index (1.34), followed by isolate FC50 (1.26) and isolate FE50 at 1.25 (Table 3). To avoid the ambiguity that could result from the qualitative (primary) screening technique, a secondary screening was done for the fungi isolates. Here, isolate FA50, FC50, FD50, FE50 and FF50 produced considerably high titre of xylanase (Figure 1). Isolate FE50 had higher xylanase production of 3.2 U/mL with a corresponding CMCase production of 1.19 U/mL. Isolate FA50 had higher CMCase production of 1.88 U/mL and a xylanase production of 1.33U/mL. Xylanase/CMCase production by FC50 and FD50 isolates was 2.8/0.6 U/mL and 1.63/1.56 U/mL, respectively. Cellulase production of 1.10 U/mL was observed for isolate FG50, while isolate FB50 produced neither of the two enzymes. Furthermore, isolate FB50 and FG50 which produced a 1.0 xylanolytic index from the qualitative screening did not produce xylanase during the secondary screening. This explains the ambiguity that can result when isolates display activity for one enzyme at the primary screening and the absence of the same activity at the secondary screening. Hence, isolates displaying higher activities for xylanase were selected to be FC50, FE50, and FF50.

Figure 1. Xylanase and CMCase production of fungi isolates grown on xylan substrate.

Table 3. Xylanolytic index during primary screening of fungi isolates.

Fungal isolates	Temperature (°C)	Xylanolytic index (mm)
FA50	40	1.07
FB50	45	1.00
FC50	40	1.26
FD50	50	1.19
FE50	48	1.25
FF50	45	1.34
FG50	43	1.00

3.1. Solid-state fermentation

The fungal isolates were grown on D. regia pods and sorghum straw under SSF fermentation. The results from the study indicated that the three isolates (FC50, FE50 and FF50) produced higher xylanase activities at 84.10, 92.73 and 97.36 U/g, respectively when cultivated on D. regia pods as against 24.88, 34.32 and 22.32 U/g, respectively on sorghum straw substrate (Figure 2). Isolates FC50, FE50 and FF50 had 11.17, 22.68 and 30.04 U/g, of CMCase activities, respectively when grown on D. regia. While CMCase production of 5.71, 10.82 and 17.34 U/g respectively, was observed for sorghum straw substrate. The higher xylanase activities recorded for D. regia could be a result of the higher hemicellulose content as compared to sorghum straw (Table 4). The composition of the lignocellulosic biomass, which is observed to be higher in D. regia pods than sorghum straw substrate is vital in the enzyme production [28]. Substrates with higher xylan composition tend to induce higher production of the enzyme. Similarly, the higher crude protein content from the D. regia pods could also be a contributing growth-promoting factor that influences the metabolism of the fungi. The result suggests that different agro-waste substrate display different rates for xylanase production. Such have also been reported for Cladosporium oxysporium with higher xylanase when grown on wheat bran than corn cob Guan, Zhao [29]. Although higher xylanase activities were observed for isolates FE50 and FF50, their CMCase activities were also observed to be high. Higher xylanase activity is important for the biobleaching process, and the quantity of cellulase needs to be considered to prevent the negative effect associated with cellulase in biobleaching process, such as loss of cellulose, decrease in pulp quality and reduced tear resistance. Therefore, the xylanase must be free from cellulase or produced in very low quantity [12,30,31]. It has however been argued that cellulase and hemicellulase have a positive effect on the modification of bleached softwood fibre [32]. Based on the aforementioned, isolate FC50 was selected and used for further studies due to its lowest CMCase produced.

Figure 2. Xylanase and CMCase production of isolate FC50, FE50 and FF50 grown on sorghum straw and Delonix regia pods.

Table 4. Proximate and lignocellulose composition of Delonix regia pods and Sorghum straw.

Proximate composition	Delonix regia pod	Sorghum straw
Moisture content %	15.17 ± 0.113	13.13 ± 0.11
Ash content %	2.70 ± 0.127	7.50 ± 0.05
Carbohydrate %	65.60 ± 0.106	83.30 ± 0.02
Crude protein %	11.39 ± 0.070	6.40 ± 0.2
Lignocellulose composition
Cellulose (%)	33.01 ± 0.072	35.03 ± 0.067
Hemicelluloses (%)	35.09 ± 0.069	25.05 ± 0.082
Lignin (%)	5.80 ± 0.005	7.33 ± 0.009

Values represented are means of triplicates ± standard deviation.

3.1. Physicochemical composition of sorghum straw and Delonix regia pods

The proximate and lignocellulosic composition of sorghum straw and D. regia pods is represented in Table 4. Pods of D. regia had high carbohydrate content (65.60%), ash content (2.70%) and total protein (11.39%). However, the proximate analysis of sorghum straw revealed carbohydrate content (83.3%), ash content (7.50%) and total protein (6.40%). Delonix regia pods contained 33.01% cellulose, 35.09% hemicellulose and 5.80% lignin, while 35.03% cellulose, 25.05% hemicellulose and 7.33% lignin were recorded for sorghum straw. It can be inferred that the high hemicellulose, protein and lower lignin content of D.regia pods in comparison with sorghum straw makes it a potential substrate for xylanase production. The high protein content in D. regia provides a reduction in the cost of enzyme production by reducing the quantity of organic nitrogen content of the mineral salt medium. Consequently, the protein content in D. regia, could fulfil the nitrogen requirement for xylanase production by the fungi.

3.1. Cultural characterization and molecular identification of isolate FC50

Based on their culturable properties, isolate FC50 has characteristic whitish–greyish colonies with a light brown on the reverse plate, a white margin with a circular form, convex elevation, grey spores and a rough surface. The fungus is fast-growing with a diameter of 36 × 36 mm after five days of incubation. The ITS region of the rRNA gene in the DNA of isolate FC50 was amplified and sequenced. The phylogenetic and molecular evolutionary analyses were conducted using MEGA version 7[23] and the neighbour-joining method [24,25]. Isolate FC50 showed 99.9% similarity with species of Trichoderma harzanium. The phylogenetic tree construction (Figure 3) revealed isolate FC50 to be closely related to strains of Trichoderma harzanium KMISO224 and Trichoderma afroharzanium TM2-4. Strains of Trichoderma harzanium have been widely reported and identified as the most frequently isolated species from forest woods, gardens and, mushroom compost [33]. They are also implicated in the secretion of lignocellulolytic enzymes, among which are cellulases and xylanases [33,34].

Figure 3. Phylogenetic relationship based on homology index for Trichoderma harzanium FC50.

3.1. Xylanase production profile of Trichoderma harzanium FC50 grown on Delonix regia pods

In this study, xylanase production increased steadily with 9.88 U/g after 24 h, reaching its peak with 83.9 U/g after 120 h (Figure 4). This corresponds with the beginning of the stationary phase, before growth ceases, in which cell death and cell growth reaches equilibrium and nutrient begin to deplete. Some fungi also produce secondary metabolites at the stationary phase which may confer negative effect on the activity of the enzyme. However, the FC50 isolate exhibited its full potential for xylanase production with trade-offs to industrial application. Further increase in time of enzyme production, resulted in a decline in xylanase production which could be attributable to the depletion of nutrients by the fungi and/or proteolysis as reported also for Aspergillus awamori and A. niger, respectively [35,36].

Figure 4. Xylanase production profile of Trichoderma harzanium FC50 grown on Delonix regia pods.

3.1. Screening of factors affecting xylanase production using Plackett-Burman design

Wide variation in xylanase production (4.12–133.93 U/g) was observed in the Plackett-Burman experiment as illustrated in Table 5. The individual effects of the factors on xylanase production are identified and presented on the Pareto Chart in Figure 5. The linear correlation model describing the relationship between the factors and the xylanase production is presented in actual units in Equation (4): (4) $\begin{array}{rl}& \text{Xylanase production }(\text{U/g})\\ & =-64.9-2.808\text{Temperature}({}^{\circ }\mathrm{C})-3.861\text{pH}\\ & +2.554\text{Moisture Content}(\mathrm{\%})\\ & +1.2067\text{Incubation Period}(\text{Hours})\\ & -1.76\text{Inoculum size}(\text{mycelial plugs})\\ & +0.993\text{Yeast Extract}\\ & +2.85{\text{K}}_{\text{2}}\text{HP}{\text{O}}_{\text{4}}(\text{g/L})-4.30\text{CaC}{\text{l}}_{\text{2}}(\text{g/L})\\ & +3.17\text{MgS}{\text{O}}_4.7\text{HO}(\text{g/L})\\ & -0.144\text{Tween}80(\text{g/L})\end{array}$(4)

Figure 5. Pareto Chart of the Standardized Effects of Screened factors on xylanase production (U/g) at α = 0.05.

Table 5. Plackett-Burman design layout in coded values and corresponding enzymes activities.

Run	A	B	C	D	E	F	G	H	J	K	Xylanase Activity (U/g)
1	+	+	–	+	–	–	–	+	+	+	86.45 ± 0.001
2	–	+	–	–	–	+	+	+	–	+	4.84 ± 0.005
3	+	–	–	–	+	+	+	–	+	+	6.82 ± 0.003
4	–	–	–	+	+	+	–	+	+	–	95.55 ± 0.000
5	+	+	+	–	+	+	–	+	–	–	7.53 ± 0.005
6	–	–	–	+	+	+	–	+	+	–	95.01 ± 0.000
7	+	+	+	–	+	+	–	+	–	–	7.17 ± 0.004
8	–	–	+	–	+	–	–	–	+	+	8.96 ± 0.006
9	–	–	–	–	–	–	–	–	–	–	6.46 ± 0.006
10	+	+	+	+	–	+	–	–	–	+	100.56 ± 0.006
11	+	+	–	+	+	–	+	–	–	–	82.14 ± 0.012
12	–	–	+	+	–	+	+	–	+	–	132.13 ± 0.014
13	–	–	+	+	–	+	+	–	+	–	133.92 ± 0.008
14	–	–	–	–	–	–	–	–	–	–	6.27 ± 0.009
15	+	+	–	+	–	–	–	+	+	+	85.55 ± 0.005
16	+	+	–	–	+	+	+	–	+	+	5.38 ± 0.002
17	+	+	–	+	+	–	+	–	–	–	82.86 ± 0.007
18	+	+	+	–	–	–	+	+	+	–	6.63 ± 0.017
19	–	–	+	–	+	–	–	–	+	+	9.50 ± 0.014
20	–	–	+	+	+	–	+	+	–	+	115.63 ± 0.005
21	–	–	–	–	–	+	+	+	–	+	4.12 ± 0.018
22	–	–	+	+	+	–	+	+	–	+	117.07 ± 0.008
23	+	+	+	+	–	+	–	–	–	+	102.90 ± 10.008
24	+	+	+	–	–	–	+	+	+	–	5.91 ± 0.016

The factors with p-values < 0.05 were considered to have significant effects on xylanase production. Six of the screened factors (Temperature- 0.000, pH -0.001, moisture content- 0.000, incubation period - 0.000, yeast extract - 0.000 and - KH₂ PO₄ -0.017) were significant for xylanase production while four factors (inoculum size- 0.199, CaCl₂ 0.159, MgSO₄ - 0.291 and tween 80- 0.665) were not (Figure 4). The model was also highly significant with a p-value of 0.000 and R² value of 98.42%. It was observed that runs 12 and 13 gave the highest enzyme production at 132.14 and 133.93 U/g, respectively, while runs 2 and 21 resulted in the lowest xylanase at 4.64 and 4.13 U/g, respectively. Each of these factors were observed to have a significant effect on increased xylanase production at higher concentration level tested. This suggests their optimal concentrations for xylanase production would be closer to higher concentrations used in the experiment than lower concentrations. Similar to the current study, Cui and Zhao [37], observed that out of the several factors screened using the Plackett-Burman design, Tween 80 and inoculum size were not significant for xylanase production by Penicillium sp. Wx-Z1. However, MgSO₄ did not significantly improve xylanase production from a thermotolerant Aspergillus fumigatus var. niveus, [6], as also observed in this study.

3.1. Optimization of significant factors using RSM (Central composite design)

The individual and interactive effects of the incubation period, temperature, pH and moisture content on xylanase production were investigated (Table 6). As shown in the standard order column, the highest xylanase production (143.72 U/g) was recorded in the 50th run, against the lowest (2.87 U/g) in run order 9. This is attributable to the variations in the incubation period, temperature and moisture content. The 50th run showed better combination of temperature (37°C), moisture (75%), pH (6.0) and incubation period of 120 h, therefore, leading to a 70.89% (1.71 folds) increase in xylanase production in contrast to the recorded titer before parametric optimization. Recent studies have reported parametric optimization of xylanase from Penicillium sp. WX-Z1 [37], Thermomyces lanuginosus VAPS-24 [38] and Aspergillus niger [39] using RSM increased by 1.34-folds, 2-folds and 3-folds, respectively.

Table 6. Central composite design layout for xylanase optimization.

	Variables
Run order	A: Temp (°C)	B: pH	C: Incubation Period (Hours)	D: Moisture Content (%)	Xylanase Activity (U/g)
1	0	+α	0	0	8.44 ± 0.004
2	−1	+1	−1	−1	12.67 ± 0.004
3	0	0	+α	0	57.47 ± 0.035
4	−1	+1	+1	+1	96.93 ± 0.006
5	0	-α	0	0	24.87 ± 0.006
6	−1	−1	+1	−1	76.52 ± 0.004
7	−1	+1	+1	−1	59.14 ± 0.003
8	−1	−1	+1	−1	76.92 ± 0.007
9	0	0	-α	0	2.86 ± 0.012
10	+1	+1	−1	+1	12.19 ± 0.007
11	0	0	0	-α	17.69 ± 0.004
12	+1	+1	+1	−1	48.78 ± 0.008
13	+1	−1	−1	+1	16.97 ± 0.011
14	+1	−1	−1	−1	15.78 ± 0.009
15	+1	+1	−1	−1	8.13 ± 0.015
16	−1	−1	−1	+1	20.64 ± 0.006
17	0	+α	0	0	8.68 ± 0.019
18	+1	−1	−1	+1	17.13 ± 0.008
19	−1	−1	−1	−1	17.61 ± 0.024
20	−1	+1	+1	−1	59.46 ± 0.009
21	+1	+1	−1	−1	7.97 ± 0.004
22	0	0	0	0	63.61 ± 0.006
23	0	0	0	-α	17.45 ± 0.008
24	+1	+1	−1	+1	12.19 ± 0.018
25	+α	0	0	0	16.66 ± 0.018
26	−1	+1	−1	+1	12.43 ± 010
27	+1	−1	+1	+1	103.30 ± 009
28	+1	−1	-	−1	15.62 ± 005
29	0	-α	0	0	24.95 ± 0.004
30	0	0	0	0	63.37 ± 0.015
31	+1	−1	+1	−1	56.75 ± 0.011
32	0	0	0	+α	47.58 ± 0.008
33	+1	+1	+1	+1	69.27 ± 0.008
34	0	0	+α	0	57.79 ± 0.007
35	−1	−1	+1	+1	143.48 ± 0.012
36	+1	−1	+1	+1	103.54 ± 0.007
37	-α	0	0	0	42.08 ± 0.019
38	+1	−1	+1	−1	55.79 ± 0.004
39	−1	+1	+1	+1	97.16 ± 0.020
40	−1	+1	−1	+1	12.55 ± 0.063
41	−1	−1	−1	+1	20.80 ± 0.008
42	0	0	-α	0	2.63 ± 0.061
43	−1	-α	−1	−1	17.45 ± 0.043
44	−1	+1	−1	−1	12.91 ± 045
45	+1	+1	+1	+1	69.50 ± 0.019
46	-α	0	0	0	42.32 ± 0.006
47	+α	0	0	0	16.81 ± 0.006
48	+1	+1	+1	−1	48.86 ± 0.026
49	0	0	0	+α	47.50 ± 0.006
50	−1	−1	+1	+1	143.72 ± 0.011

3.1. Regression analysis of the model

A second-order polynomial model was observed to be a better model to predict xylanase production. Processing the data indicated a model with a magnitude of the relationship between the response obtained and the quantum of variables applied in the experimental runs. The following regression equation (in coded terms) between xylanase activity and the model terms was derived based on the RSM-integrated ANOVA. (5) $\begin{array}{rl}& \text{Xylanase activity}\\ & =+63.49-6.68\text{A}-6.82\text{B}+26.99\text{C}\\ & +10.03\text{D}+1.43\text{AB}-5.51\text{AC}-2.14\text{AD}\\ & -5.00\text{BC}-3.53\text{BD}+10.25\text{CD}\\ & -5.01A^2-8.20B^2-4.83C^2-4.24D^2\end{array}$(5)

The level of significance of the model terms is shown in Table 7. The p-value shows the level of significance of each term while the determination coefficient (R²) indicated a perfect coherence between the predicted and experimental values. Higher R² and smaller p-value suggests significant corresponding coefficient. Therefore, as shown in Table 7, where the p-value is less than 0.0001, the probability of having an F value due to noise is 0.0001. The Analysis of variance (ANOVA) of the second-order polynomial regression model established that the computed F-value for the experiment was 13.01 with a p-value of < 0.0001, implying the model is significant. The coefficient of variation (C.V. % = 29.93) indicated a good correlation between experimentally observed and predicted values. The fit of the model can be verified by the determination of co-efficient R², (R² = 0.9088). An R² value of 0.9088 was recorded in the current study (R² values above 0.75 designates fitness of the model). The value of the “Predicted R²” which was 0.7487, has a reasonable agreement with the R² (0.9088) and the adjusted R² (0.8443). Likewise, the adjusted R² value showed a variation of 84.43% in xylanase activity, attributable to the independent values, and only 15.57% of the total variation could not be explained by the model. The model obtained makes it easy to predict the individual and interactive effect of the growth factors on the production of xylanase by the organism.

Table 7. ANOVA table for response surface quadratic model.

Source	Sum of Squares	df	Mean Square	F-Value	Prob > F
Model	51473.98	14	3676.71	13.01	< 0.0001	Significant
A-Temperature	2139.82	1	2139.82	7.57	0.0093	Significant
B-pH	2231.23	1	2231.23	7.89	0.0081	Significant
C-Incubation Period	34970.97	1	34970.97	123.71	< 0.0001	Significant
D-Moisture Content	4826.96	1	4826.96	17.08	0.0002	Significant
AB	65.76	1	65.76	0.23	0.6326
AC	972.88	1	972.88	3.44	0.0720
AD	147.03	1	147.03	0.52	0.4756
BC	799.44	1	799.44	2.83	0.1015
BD	397.85	1	397.85	1.41	0.2435
CD	3359.90	1	3359.90	11.89	0.0015	Significant
A^2	567.36	1	567.36	2.01	0.1654
B^2	1517.08	1	1517.08	5.37	0.0265	Significant
C^2	527.48	1	527.48	1.87	0.1806
D^2	405.99	1	405.99	1.44	0.2388
Std. Dev.	16.81		R-Squared	0.9088
Mean	42.10		Adj R-Squared	0.8443
C.V. %	39.93		Pred R-Squared	0.7487
			Adeq Precision	14.053

3.1. Effect of optimized parameters on xylanase production by Tichoderma harzanium FC50

The ANOVA results in Table 7 suggest that all the optimized linear model terms; A (Temperature), B (pH), C (Incubation Period) and D (Moisture Content) were significant, while only the linear interaction CD (Incubation Period*Moisture Content interaction) and quadratic interaction B² (pH*pH interaction) were significant with p-value less than 0.05. This implies that any change in the values of these factors will significantly affect xylanase production. Ajijolakewu et al [7], reported that pH, moisture content, and inoculum sizes had marked effects on xylanase production. In their study, only the pH showed quadratic interaction, which is also in line with the current study. Among all the factors considered, the incubation period showed the greatest effect (p < 0.0001) on xylanase production, followed by moisture content (p < 0.0002), while the xylanase production was least affected by temperature (p < 0.0093) of the growth medium. It is worth noting that all these factors have been identified to significantly impact on the microbial production of the enzyme. High inoculum often leads to over crowdedness and competition for limited nutrient, thereby extending the lag phase and poor enzyme synthesis. Very high moisture content limit fungal activity by limiting oxygen availability while low moisture increases water stress and reduce accessibility to nutrient [40,41] Lastly, changes in pH affects membrane permeability and stability of the secreted enzymes [42]. In this study, increasing moisture content from 65% to 75% (w/w) gave rise to a gradual increase in xylanase production. The highest moisture content (80%, w/w), reduced xylanase production by the fungus. This may be due to the stickiness of the substrate leading to reduce porosity and oxygen transfer resulting from the change in particle size and texture. [43]. Tai, et al. [44], reported optimum moisture content at 75% for xylanase and cellulase production by an indigenous fungus grown on oil palm fronds [12]. The authors observed 74.73% moisture content for the production of cellulase-free xylanase by Thermomyces dupontii. The initial pH values above or below the optimum may lead to the suppression of ionization in the fermentation medium, thereby slowing down the uptake of nutrients from the substrate and reducing product formation. In this study, the maximum production of xylanase increased maximally at initial pH of 6 (Table 6). A decrease in the pH to 4.5 and an increase to 10.5 greatly reduced the overall enzyme production to 24.95 and 8.69 U/g, respectively. In a previous study, the production of thermostable xylanase by Rhizomucor pusillus was optimum at pH 6.0 [45]. Xylanase production was observed to reach optimum at 37°C. However, being a thermotolerant fungus, it was able to tolerate higher temperatures at 45°C and produce significant titer at 103.55 U/g. Furthermore, xylanase production decreased marginally (16.82 U/g) when the temperature was increased to 49°C. This could result from changes in the physiological properties of fungus at high temperatures, which could lead to the poor synthesis of proteins essential for growth [45]. The fungal strain obtained in this study is one of the very few thermo-alkali tolerant Trichoderma harzanium reported in the literature. Seemakram, et al. [12], reported an optimum temperature of 43.93°C for the production of a cellulase-free thermostable xylanase from Thermomyces sp. Therefore, the optimum temperature for xylanase production can be strain dependent and mainly depends on the source of the isolate.

3.1. Localization of optimum conditions

The response surface plots (Figures 6a–d) depict the interactive effects between optimized factors which were made by selecting the factors with a linear interaction (Incubation period and moisture content) while differing the values of temperature and pH. This implies that for optimal xylanase production by FC50, there must be an interaction between incubation period and moisture content. At optimum interaction between initial moisture content and incubation period (Figure 6a), the three-dimensional response plots showed that the predicted optimum interactive conditions for xylanase production by Trichoderma harzanium FC50 ware 75% and 120 h. It can be inferred that a lower moisture content or lower incubation period is not suitable for xylanase production by the fungus. The xylanase production as predicted by the optimum interaction was 124 U/g. Low moisture content reduces nutrient solubility in the solid substrate and increases water tension. This might be the cause of reduced xylanase production at lower moisture levels. Notwithstanding, altering the values of the other growth parameters at the optimum interaction between the moisture content and incubation period led to corresponding changes in xylanase yield (Figures 6b–d). The cumulative effect between moisture content and incubation period while increasing the pH beyond 6.0 and maintaining temperature at optimum level (37°C) (Figure 6b), caused a slight reduction in the xylanase production from 124 U/g to 116 U/g. Likewise, an increment in temperature beyond 37°C while pH was maintained at optimum level (6.0) caused a decline in the yield of the enzyme (Figure 6c). Similarly, the cumulative effect between moisture content and incubation period while keeping temperature and moisture content above optimum (Figure 6d) led to a slight decrease in the production of xylanase. Hence, an increase in the pH beyond 6.0 and temperature beyond 37°C led to a corresponding decrease in xylanase yield. This suggests that a near neutral-fairly acidic pH range is suitable for xylanase production by Trichoderma harzanium FC50. However, the fungi can withstand and produce considerable amounts of the enzyme at higher pH values (9.0) and temperature of 45°C as shown in the plots. Some thermotolerant fungi have been reported to produce enzymes within the fairly acidic-alkaline pH range of 6.0-8.0 [38]. The suggested optimum conditions for maximum xylanase production were 37 °C, 6.0, 120 h and 75% for temperature, pH, incubation period and moisture content. Several studies showed different variations in the improvement of xylanase production at optimal parameters using RSM (Table 8).

Figure 6. (a-d) Contour plots for thermo-alkali-tolerant xylanase production. (a) interactive effects between C and D at A = 37 and B = 6 (b) interactive effects between C and D at A = 37and B = 6.5 (c) interactive effects between C and D at A = 39 and B = 6 (d) interactive effects between C and D at A = 39 and B = 6.5.

Table 8. Parameters influencing optimal xylanase production by fungi grown on different carbon substrate.

Fungi	Carbon sources	Parameters	Xylanase activity (U/g)	References
Aspergillus niger	Corn-cob	Substrate conc. NaNO3	306.12	Desai et  al. [31]
Rhizopus oryzae UC2	Raw oil palm frond leaves	Temperature, moisture content inoculum size	213.99	Ezeilo et  al. [48]
Rhizopus stolonifer	Corn-cob	Urea, moisture content, ZnSO4 Tween-80	13.90	Zhang et  al. [49]
Aspergillus niger DWA 8	Oil palm frond	Substrate conc. moisture content	5.23	Tai et  al. [44]
Trichoderma harzanium FC50	Delonix regia seed pods	Temperature, moisture content, pH, incubation period	143.72	Current study

Experimental validation of the optimized condition predicted by the RSM was carried out to confirm the accuracy of the model. The xylanase activity was observed to be 120.45 U/g. The experimental result was very close to the predicted value (124 U/g) at optimum growth conditions (Temperature 37°C, pH 6.0, incubation period 120 h, and moisture content of 75%). Maximum production of cellulase free- xylanase from a thermophilic fungus, Thermomyces dupontii KKU-CLD-E2-3 using RSM was achieved after 192 h [12]. Optimization of independent variables using RSM resulted in an increase in xylanase by 1.43-fold compared with unoptimized media (D. regia media) and 42.8-fold compared with xylan media (D. regia replaced with xylan). Similarly, optimization of growth conditions for Fusarium sp. BVKT[46] and Aspergillus candidus [47] resulted in the enhancement of xylanase yield of up to 3.5 and 1.57-folds, respectively.

4. Conclusion

In this study, xylanase-producing fungal strains with the ability to grow on alkali media at elevated temperature were isolated from sorghum straw/shea nut cake-chicken manure hot compost. One potential strain was identified as Trichoderma harzanium FC50 based on the ITS region. This isolate gave higher xylanase and lower CMCase production on D. regia pods substrate. Optimization of growth factors through RSM yielded xylanase with 120.45 U/g in SSF under optimal conditions of 75% moisture content, pH of 6.0, the temperature of 37°C and incubation period at 120 h which was 42.8 -folds higher than media substituted with xylan. The result suggests that the model developed for optimal xylanase by Trichoderma harzanium FC50 is accurate. Thus D. regia pods could be a cheaper substrate for the production of xylanase diminutive cellulase which can be suitably exploited for industrial processes such as pulping. The thermotolerance and alkali pH tolerance, confers a potential robust isolate for the production of thermostable and pH stable xylanase required in industrial biopulping.

Disclosure statement

No potential conflict of interest was reported by the author(s).