Production and partial purification of extracellular xylanase from Pseudomonas nitroreducens using frugivorous bat (Pteropus giganteus) faeces as ideal substrate and its role in poultry feed digestion

2.1 Faeces collection

Five roosts of P. giganteus were chosen for the collection of faeces from various districts of South India as shown in Table 1 (Dhivahar and Suthakar, 2018). Samples were collected as per the methodology of Akobi et al. (2012).

2.2 Selection of xylanase producing bacteria and estimation of xylanase activity

Basal agar medium constituting 1% birchwood xylan was prepared and autoclaved. The medium was transferred to sterile petriplates aseptically and allowed to cool. Samples were serially diluted, spread on the agar medium, and incubated at 37 °C for 24 h. The potent culture was selected after congo red staining method (Akobi et al., 2012). The xylanase activity of bacterial culture was determined using the method of Khusro et al. (2016). The culture was identified using molecular characterization tools. The 16S rRNA sequence was submitted to GenBank, NCBI.

2.3 Substrate used and solid state fermentation (SSF)

P. giganteus faeces were collected from Srivaikundam (8˚0.62″N:77˚0.91″E) roost colony, Tamil Nadu, South India and dried for few days. The dried sample was ground into powder form for further experiments. The SSF of substrate was carried out as per the methods of Aarti et al. (2017) and supernatant was collected for xylanase activity using the methodology of Khusro et al. (2016) as described earlier.

2.4 Xylanase activity optimization by traditional method

Abiotic and biotic factors such as incubation period (12–96 h), pH (6.0–11.0), temperature (32–60 °C), inoculum (7.31–54.82 log CFU/mL), moisture content (60–120%), carbon sources, and nitrogen sources were optimized by one-factor-at-a-time (OFAT) technique and xylanase production was assessed following the prior method discussed.

2.5 Statistical optimization

Box Behnken design (BBD) of response surface methodology (RSM) was implied for optimizing varied factors towards enhancement of xylanase yield. The BBD contained 17 experiments of three factors (A, B, and C) at three ranges (−1, 0, +1) as shows in Table 2.

The average xylanase activity (Y) was determined using equation as mentioned below:$$Y={\beta }_0+\mathrm{\Sigma }{\beta }_i{X}_i+\mathrm{\Sigma }{\beta }_{\mathit{ii}}{X}_i^2+\mathrm{\Sigma }{\beta }_{\mathit{ij}}{X}_i{X}_j$$

2.6 Partial purification of xylanase

Ammonium sulphate (70% saturation) was added into the collected supernatant of bacterial culture while stirring. After 2 h, the precipitated sample was centrifuged at 8,000 rpm for 20 min at 4 °C, pellet was collected, dissolved in phosphate buffer (pH 7.4), and dialyzed overnight. Sample was purified using ion-exchange chromatographic technique. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine molecular mass of the purified enzyme. Total protein content was determined using Bradford assay (Bradford, 1976).

2.7 Characterization of xylanase

Xylanase stability towards various pH, temperature, metal ions, and solvents was carried out as per the methods of Khusro et al. (2016) and Aarti et al. (2017, 2018).

2.8 Pre-digestion of poultry feed using purified xylanase

Poultry feed was obtained from poultry house, washed thoroughly, dried, and stored in clean bottles. About 15 g of poultry feed were measured in a conical flask and autoclaved using 25–30 mL distilled water. Poultry feed was separately treated with xylanase (5 mL) for 24 h incubation at 40 °C under shaking condition. After incubation, poultry feed was centrifuged at 10,000 rpm for 10 min. Supernatants were incubated with DNS and boiled for 10 min. After water bath treatment, total reducing sugar production was estimated by recording optical density at 540 nm using UV–Vis spectrophotometer (Nagar et al., 2012). Untreated poultry feed was used as control.

2.9 Statistical analysis and software used

Design Expert Version 11.0.0 (Stat-Ease Inc., Minneapolis, MN, USA) statistical software was utilized for optimization. Analysis of variation (ANOVA) was used for validation of various factors.

3.1 Xylanolytic strain identification

Off 18 isolates, 1 bacterial isolate exhibited good xylanase activity and identified as P. nitroreducens strain LLD06 after 16S rRNA sequencing (Accession No: MK530169).

3.2 Solid state fermentation

Strain LLD06 utilized P. giganteus faeces for production of xylanase with maximum activity of 1044.05 ± 303 U/g (Figure not shown).

3.3 OFAT method based optimization

The enzyme activity was optimum at 48 h (1044.05 ± 32.3 U/g) and reduced afterwards (Figure not shown). The xylanase activity was optimum at pH 8 (1144.12 ± 32.3 U/g) and decreased at higher alkaline conditions (Figure not shown). The optimum temperature for the xylanase yield was maximum at 37 °C (1044.05 ± 24.2 U/g) and reduced at high temperature (Figure not shown). The inoculums level showed no significant alteration in xylanase production, being maximum at 18.27 log CFU/mL (Figure not shown). The xylanase productivity was optimum (1323.56 ± 31.3 U/g) with 100% moisture and reduced with lower and higher moisture contents (Figure not shown). The xylanase activity was maximum (1423.14 ± 28.3 U/g) in the presence of birchwood xylan. Similarly, optimum xylanase production (1496.12 ± 31.3 U/g) was estimated in the presence of yeast extract (Figure not shown).

3.4 RSM based optimization

Experimental strategy of parameters (moisture, birchwood xylan, and yeast extract) with 17 runs is demonstrated in Table 3. Xylanase activity (Y) was predicted using model equation as given below:

Y (U/g) = 1150.76 − 74.23A + 379.63B + 348.07C − 140.29AB − 125.82AC − 245.83BC − 282.22A² + 370.88 B² + 195.92C²

Table 4 represents ANOVA for xylanase production. Modelling codes such as BB, CC, BC, A², and B² were significant. The multiple correlation coefficient (R²) was close to 1, indicating an accurate modelling. Parity plot showed the predicted and observed values closeness towards diagonal line (Fig. 1). The interaction effects between parameters are shown in Fig. 2(a–c). The 3D graphs (Fig. 2a–c) represented interaction between two factors.

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Fig. 1

Distribution of experimental/actual and predicated values of xylanase activity by Parity plot.

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Fig. 2

3D plot showing interaction effect between (a) moisture (A:A) and birchwood xylan (B:B), (b) moisture (A:A) and yeast extract (C:C), and (c) birchwood xylan (B:B) and yeast extract (C:C) on xylanase activity (R1).

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3.5 Xylanase purification and characterization

Table 5 shows the purification steps and summary of extracellular enzyme. The SDS-PAGE showed molecular mass of 40 kDa (Figure not shown). Xylanase was stable from slight acidic to alkaline conditions for 4 h (Fig. 3a). Xylanase revealed stability till 65 °C with 25.12% of residual activity (Fig. 3b). Xylanase exhibited stability towards various metal ions, showing activities of 93.32 ± 3.3 to 20.22 ± 1.3% (Fig. 3c). Likewise, xylanase depicted stability towards varied solvents with residual activities of 80.16 ± 2.3 to 28.12 ± 1.3% (Fig. 3d).

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Fig 3

Effect of (a) pH, (b) temperature, (c) various metal ions and (d) various solvents on the stability of xylanase.

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3.6 Pre-digestion of feed

Poultry feed were supplemented with purified xylanase of strain LLD06. The purified xylanase showed increment in the total reducing sugar content (88%) with respect to control (Fig. 4).

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Fig 4

Pre-digestion of poultry feed with purified xylanase on reducing sugar production. Higher reducing sugar was obtained when poultry feed was treated with purified xylanase.

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