Corn stover and bacterial strain P3
Corn stover was provided by Agriculture and Agri-Food Canada. The air-dried corn stover was chopped and milled to pass through a 50-mesh sieve for this study. The milled sample was stored at ambient temperature in an airtight container until use.
The Bacillus sp. strain P3 (accession No. MF462257) and its enzymatic characteristics have been described previously (Guo et al. 2017a). The strain P3 was stored at -70°C and activated by culturing in Luria-Bertani (LB) broth (10 g/L peptone, 5 g/L yeast extract, 10 g/L NaCl) medium at 37°C and 200 rpm for 12 h as a seed culture of fermentation experiments.
Evaluations of biomass degradation abilities
To assess the biomass degradation ability of the strain P3, different lignocellulosic biomasses including agave, algae, corn stover, Miscanthus, wheat bran, wood dust and pine chip were used as the carbon sources according to the method described by Guo et al. (2017b). Briefly, a 5.0 µl of the overnight grown LB broth culture was dropped or inoculated on the plate containing modified minimal salt (MMS) medium (0.1% NaNO3, 0.1% K2HPO4, 0.1% KCl, 0.05% MgSO4, 0.05% yeast and 0.3% peptone) supplemented with 1.5% agar and 0.5% biomass or CMC or xylan, incubated at 37°C for 48h. The biomass degradation ability was evaluated based on the size of biomass hydrolyzing zone (zone of clearance or halo zone) produced on the plate by the bacterial strain after staining with Gram’s iodine solution.
Optimization of fermentation parameters for cellulolytic enzyme production
To optimize the fermentation conditions for maximum enzyme production, the strain P3 was inoculated (2%, v/v) in the MMS broth medium supplemented with 0.5% (w/v) biomass or CMC, incubated at desired temperature for desired time in a rotary shaker incubator of 200 rpm. To evaluate the influence of different carbon sources on enzyme production, the agave, algae, corn stover, Miscanthus, wheat bran, wood dust, pine chip and CMC were used as the substrates according to the method described previously (Guo et al. 2017b). Following incubation, the broth culture was centrifuged at 12000 g for 3 min to obtain the supernatant, which was used as the crude enzymes for CMCase and xylanase activities analysis. The activities of CMCase and xylanase were determined by measuring the released reducing sugar from substrate. The reducing sugar content was measured by DNS method (Miller 1959). The substrate corn stover (carbon source) was selected to optimize the biomass concentrations for lignocellulosic enzymes production due to its high CMCase and xylanase production ability compared with other biomasses tested herein. For optimization of corn stover concentrations, the MMS broth medium was supplemented with 0.5%, 1.0%, 2.0% and 4.0% corn stover, respectively.
The influences of temperature on CMCase and xylanase productions were investigated by culturing the strain P3 at 30, 37, 45 and 50°C for 24 h, while the effects of different initial pH values of the culture medium on enzyme productions were determined in a wide range of pH from 5.0 to 10.0 at an interval of 0.5. The pH values of medium were adjusted with the addition of hydrochloric acid (HCl) and sodium hydroxide (NaOH).
Effects of temperature and pH on enzyme activities
The crude enzymes harvested under optimal fermentation conditions were taken for evaluating the effects of temperature and pH on CMCase and xylanase activities. However, for determining the effects of incubation temperature and pH of the reaction mixtures with crude enzyme, a wide range of temperatures from 40 to 80°C and pH from 4.0 to 9.5 were set the incubation periods. The 0.05 M citrate and Tris-HCl buffer solutions were used respectively to set the pH of the enzyme reaction mixtures.
Bacterial pretreatment of corn stover
For bacterial pretreatment, dried corn stover (0.5%, w/v) was mixed with MMS broth medium, autoclaved at 121°C for 30 min. An overnight LB broth culture of the strain P3 was inoculated (2%, v/v) in an Erlenmeyer flask containing 50 ml of medium. A control flask of 50 ml medium without bacterial inoculum was also maintained. Bacterial pretreatment was performed at 37°C with 200 rpm for 20d, and all flasks were covered by parafilm to prevent water evaporation in this process (Papavizas et al. 1984). The samples were taken 5–10 d intervals, filtered through a double layered muslin cloth (300 mesh) and the supernatant was collected for the determination of reducing sugar as well as enzymes (CMCase and xylanase) activities. The residue was washed several times with distilled water through a double layered muslin cloth (300 mesh) to remove the bacterial cells, dried at 50°C until constant weight and used to determine the weight loss and cell wall compositions. The pH, enzyme activities and reducing sugar content of supernatant were determined after centrifugation at 1200 g for 3 minutes.
Biomass composition analysis
According to the methods described by Shrestha et al. (2015), the analysis of cellulose and hemicellulose contents was carried out by measuring the contents of glucan and xylan. Anthrone-sulfuric acid and orcinol-hydrochloric acid methods (Leyva et al. 2008; Tomoda 1963) were used to determine the content of hexose and pentose, respectively. The Klason lignin analysis was conducted using the method written by Ibáñez and Bauer (2014).
Enzymatic saccharification of pretreated corn stover
To detect the saccharification effect of the corn stover pretreated by the strain P3, commercial cellulase extracted from Trichoderma reesei ATCC 26921 (Celluclast 1.5 L, Novozymes, Franklinton, NC, USA) was used. The corn stover samples pretreated for 5, 10 and 20 days were saccharified by loading 20 FPU g− 1 of commercial cellulase, the amount of which was set at the maximum to sufficiently hydrolyze the substrate according to the previous study (Singh et al. 2009). Enzymatic saccharification was conducted in 0.05 M citrate buffer (pH 4.8) containing 1% (w/v) pretreated corn stover and 0.005% sodium azide (Ferraz et al. 2017). The reaction mixture was incubated at 50°C with an agitation of 200 rpm for 72h. The non-pretreated corn stover was used as the control group. The reducing sugar was determined using method DNS method (Miller 1959).
All data in our experiments were obtained from the mean of three replicates. To quantify the significant difference between different treatments, statistical analysis was carried out by one-way analysis of variance. Pearson correlation analysis was conducted to explain the main factor resulting in the release of reducing sugar in enzymatic saccharification. Statistical analysis was performed using SPSS (SPSS Inc., USA, version 13.0).