The identity of the fungal isolate was determined based on the cultural and morphological characteristics (Figure 1). The black colony color, conidial head biseriate and small conidia 2.7-3.5µm exhibited by this isolate are typical of Aspergillus niger as reported previously by Klich (2002) and Samson et al. (2007). The phylogenetic analysis using rDNA ITS sequences and the BLAST search against the GenBank reference strains revealed that the isolate was a close relative of Aspergillus species with the closest sister being the Aspergillus niger (MT620753). Li et al. (2021) had reported the applications of A. niger (MT620753) in rice wine saccharification. However, despite being 99% identical to the GenBank Aspergillus niger strains, our isolate separated distinctively from her closely related GenBank strains (Figure 2) suggesting that the isolate could probably have underwent unique evolutionary events in the recent past.
The physicochemical characteristics of the isolate were evaluated on media with different pH values, NaCl concentrations and temperature levels. The fungal isolate showed good growth in pH range of 4-12, temperature 25-30oC and 0-12% (w/v) NaCl concentrations. The wide ambient pH and temperatures exhibited by the isolate indicated that it was a strong haloalkalitolerant strain of A. niger; it could as well be that the strain was under transition in alkaline soil since it was also able to grow well in neutral media. The alkaliphiles have been reported to have the ability to survive under salt concentrations ≥5% w/v and high pH ≥9 (Grum-Grzhimaylo et al. 2016).
Pineapple peels are produced in high quantities across many fruit processing factories worldwide and their disposal still remain a great challenge. Their utilization as raw materials for the production of value added products such as enzymes may be a welcome move by the bioprocessing companies. In the present study, the fermentation of pineapple peels proved to be a viable cost-effective strategy of producing enzymes with widespread industrial applications. Crude enzymatic extract displayed strong cellulolytic and xylanolytic activities of 20.73U/mL, 34.57U/mL and 118.03U/mL for CMCase, Fpase and xylanase respectively. The cellulolytic and xylanolytic activity of crude extract from this isolate was higher than most filamentous fungi reported in the literature (Jampala et al. 2017). Similarly, xylanase and CMCase activities of 91.9 U/mL and 5.61 U/mL respectively were reported in fermentation broth of barley straw. In this study, the pineapple peels substrates must have influenced the high enzyme activities displayed in the fermentation broth. Elisashvili et al (2008) had previously reported that the growth substrate have great influence on the production enzyme production and activities.
The response surface methodology was used to develop the experimental design for evaluating the optimum conditions and the interaction effects of the process parameters on the response. The probability (p) ˃F<0.05, the F-value = 7.39 and a low p-value (p=0.000) suggested that the model terms were significant. The coefficient of determination (R2 =0.87) indicated that the experimental and predicted values were in good agreement, and that the model can well be used to predict process performance and optimization. The lack-of-fit (F-value of 1.1) for regression of Eq. 2 was not significant (p-value=0.475). Non-significant lack-of-fit is a good proof that the model equation is adequate to predict the response under any combination of values of the variables. Non-interactive effect of the variables (p > 0.05) on mushroom hydrolysis (Table 3) implies that these variables had additive effects on mushroom hydrolysis. Similar results had been reported by de Almeida et al. (2016) where non-interactive effect of reaction variables resulted in additive effects on the enzymatic saccharification of pineapple peels.
The effect of independent variables and their interaction on mushroom saccharification were visualized using three dimensional response surface plots (Figure 3). The interaction effect of temperature, incubation time, pH and enzyme loading had influence on glucose yield. In a similar study, Sattler et al. (1989) reported 31% increase in glucose yield from bioconversion of cellulose from pretreated poplar wood with increase in the enzyme loading and temperature. Pan et al. (2005), Manonmani and Sreekantiah (1987) and Kaur et al. (1998) also noted that the increase in temperature and enzyme loading up to the optimum levels favored the enzymatic hydrolysis of cellulose from softwood, sugarcane bagasse and rice straw respectively. However, the temperature and enzyme load required to achieve a complete conversion of cellulose into glucose vary with raw material used. Increasing enzyme loading beyond optimum level resulted in minimal or no increase in glucose yield because all the glycosidic bonds available for hydrolysis may have been exhausted. Saini et al. (2013) reported that the hydrolysis of the enzyme-susceptible cellulose linkages occur simultaneously when enzyme is absorbed into the suspended substrate particles. Increasing temperature beyond 50oC resulted in a decrease in glucose yield. This is in agreement with the study by Daniel and Danson (2013) that reported enzyme inactivation at elevated temperature due to thermal inactivation. Variation in pH of a reaction mixture beyond optimal pH value negatively affected mushroom hydrolysis and glucose yield. Reactions at pH and temperatures beyond optimum levels of pH 5.5 and temperatures of 50oC respectively affect enzyme activity and conformation (Althuri and Banerjee 2016). This corresponded well with the present study where temperature of 50oC and pH 6.5 displayed maximum glucose yield.
A response optimizer (Figure 4) was used to create optimum conditions of temperature 50oC, incubation time of 12h, pH 6.5 and enzyme loading of 5% (v/v) with predicted yield of 1.494 mg mL−1. A validation experiment under the optimal model conditions produced 1.639 ±0.04 mg mL−1 of glucose after saccharification, which is 1.1 folds higher than the predicted value; suggesting that the predicted and experimental response values are in good agreement. This further validates the RSM model, showing that the model was adequate for the optimization of enzymatic saccharification of mushroom experiments. The reported glucose yield (1.639 ±0.04 mg mL−1) in the present study is above glucose yields obtained from Pleurotus species previously reported (Sławińska et al. 2020, Zhou et al. 2912). This could have resulted from the efficient hydrolysis of mushroom biomass and the presence of adequate balance between different enzymes in the crude extract. Van Dyk and Pletschke (2012) noted that biomass degradation is a function of a balanced enzyme proportions that act in synergy to breakdown the complex structure of the lignocellulose. The accessory enzymes that are important in mushroom cell-wall degradation include glucanases, chitinases and proteases (Harman et al. 2004, Kubicek et al. 2014). These enzymes are also important during mushroom pathogenesis by the pathogenic fungi such as Trichoderma harziunum (Wang et al. 2016). The extract contains antifungal properties that may be employed in the management of many fungal related complications.
Enzymes have been used in mushroom processing to recover high value products. Banjongsinsiri et al. (2016) used commercial bromelain and papain enzymes to enhance recovery of protein from mushroom biomass. Similarly, Poojary et al (2017) digested mushroom biomass with commercial enzymes to recover amino acids responsible for umami taste. However, for a long time, the cost of commercial enzymes has remained a major bottleneck in industrial bioprocesses. Increasing research into the applications of the crude enzyme extracts in the bioconversion processes is being driven majorly by the need to make enzyme-based processing more competitive. Mahamud and Gomes (2012) applied crude enzymes in saccharification of sugarcane bagasse for the production of bioethanol production; the crude enzyme extract of Trichoderma sp. displayed CMCase, Fpase and xylanase activities of 0.977, 0.110 and 9.280 U/ml which had overall degree of saccharification 45.71%. Similarly, Kumar and Sharma (2012) used crude enzyme extract in juice clarification. In the same study, the crude enzymatic extract was more competitive compared to commercial enzymes and the combination of crude and commercial enzymes produced even better results. However, despite potential of crude enzymes as a viable approach to lowering the cost of bioprocessing, limited or probably no study is available on its application in mushroom processing. This study therefore provides baseline information necessary for the application of crude enzymatic extracts in mushroom processing.