Materials
The mycelium waste from citric acid fermentation used in this study was obtained from Weifang Ensign Industry Co., Ltd, China. The moisture content of a sample was adjusted to 30% before SE treatment. IL, [Emim][Cl], was purchased from Shanghai Macklin Biochemical Co., Ltd., China. Standard sample of GlcN hydrochloride was obtained from Nantong Foreign Trade Medicine and Health Products Co., Ltd. CDA-producing strain, Rhodococcus equi CGMCC14861 (ReCDA), was stored by the China General Microbiological Culture Collection Center.
SE pretreatment of mycelium waste from citric acid fermentation
SE pretreatment was conducted in an explosion device composed of a 5 L reaction chamber, a steam generator, and a sample collector. During the SE pretreatment process, only high-pressure steam was pushed into the reaction chamber to control the reaction pressure. After 1 min treatment, the samples were suddenly discharged into the collector. Samples were collected for further analysis and characterization.
Sequential pretreatment of SE and IL toward mycelium waste from citric acid fermentation
When combined pretreatment was performed, 0.5 g of different pretreated materials by SE was added into 10 g of [Emim][Cl], and the mixture was heated at 100 °C for 6 h [43]. Then, samples were thoroughly washed with distilled water, and the solid residues were recovered by filtration and freeze drying. The supernatant containing IL and water was kept for recycling [44].
Changes in structure and morphological properties of mycelium waste after pretreatment
Residues obtained after various pretreatments were characterized using SEM, FT-IR spectroscopy, and XRD. Materials treated with SE were covered with gold before being observed using a SU1510 FE-SEM (Hitachi, Japan). SEM was conducted in accordance with a previously reported method with slight modifications [45]. For FT-IR spectroscopy, samples were scanned with an IS50 spectrometer (Nicolet, USA) at a range of 4000 cm-1 to 400 cm-1 and resolution of 4 cm-1 [46]. XRD was performed in accordance with a previously reported method with slight modifications [47]. Samples were analyzed using a D8 Advance diffractometer (Brucker, Germany), with incident radiation CuKα (λ = 1.54) at voltage of 36 kV and current of 20 mA. The range of investigation corresponded to 5° < 2θ < 60° with a step scan of 0.020 at a scan rate of 40/min. The pore size distribution of samples was analyzed using a specific surface and aperture analyzer (3H-2000PS1/2, Beishide Instrument-S&T. (Beijing) Co., Ltd.).
Composition analysis of mycelium waste after pretreatment
The contents of total soluble sugars and total soluble proteins were determined in accordance with the protocols described by Dubois et al. [48] and Bradford [49], respectively. Total soluble solids were determined by gravimetric method. Samples were washed with deionized water until the total soluble sugar was not detected. Then, they were dried at 65 °C for 10-12h to constant weight, weighed, and used to calculate the content of total soluble solids. Analysis of glucose, arabinose, and cellobiose was conducted by high-performance liquid chromatography (HPLC) following a previously reported method [34].
Chemical preparation of GlcN hydrochloride from mycelium waste
In accordance with a previously reported method [39], 10 g of samples treated at different pressures and dried at 65 °C for 10-12h to constant weight was added to 25 mL of hydrochloric acid (30%), incubated at 90 °C for 2 h, and filtered [50]. The filtrate was collected, dried to constant weight, and weighed for calculating the yield of crude GlcN hydrochloride.
Enzymatic hydrolysis by cellulase toward pretreated mycelium waste
Mycelium waste pretreated after combined pretreatment was hydrolyzed by cellulase in accordance with a previously reported method with slight modification (Pielhop et al. 2016). Samples (140 g/L) were incubated in a shaker at 50 °C and 210 rpm for 24 h and then analyzed for sugars in the supernatant.
Enzymatic preparation of chitosan by CDA from pretreated mycelium waste
CDA produced by ReCDA was used for the deacetylation of mycelium waste. To prepare ReCDA, the producing strain was cultivated on Luria Bertani medium at 37 °C and 200 rpm for 24 h. The bacterial cells were washed and diluted with 0.2 M phosphate buffer (pH 7.0) and then homogenized by grinding with liquid nitrogen in a mortar. The enzyme activity assay of crude CDA was mainly performed as described previously [51]. One unit of CDA activity was defined as the amount of enzyme that catalyzes the release of 4-nitroaniline per hour from 4-nitroacetanilide.
Approximately 0.1 g of untreated and pretreated samples was added into 5 mL of crude ReCDA, stirred evenly, and subjected to vibration reaction under 37 °C for 6 h. Samples were immediately boiled for 5 min and filtered. Then, the production of acetic acid in the liquid was analyzed by HPLC [52, 53]. The solids were freeze-dried and detected by FT-IR spectroscopy to analyze the deacetylation degree (DD) [29].