Comprehensive utilization of Aspergillus niger mycelium waste from citric acid fermentation on the basis of steam explosion and ionic liquid pretreatment


 Background Aspergillus niger mycelium waste, a by-product from citric acid fermentation is considered a good raw material to produce fungal chitosan and glucosamine (GlcN). However, its successful application has always been a challenge.Results In this work, the effects of combined pretreatment involving steam explosion and ionic liquid (IL) on mycelium waste were investigated. Results show that pretreatment of mycelium waste at 2.5 MPa for 1 min improved the GlcN hydrochloride yield by 65.49%, the release of soluble sugar reached 0.46 g/g of sample, and the deacelation degree of fungal chitosan by hydrolysis of chitin deacetylase from Rhodococcus equi CGMCC14861 (ReCDA) was from scratch and a 1.3-fold increase by combined with IL pretreatment. In addition, a recycling strategy for by-products, which promoted the fermentation of ReCDA used for fungal chitosan, was efficiently developed.Conclusion This is the first report of comprehensive utilization of Aspergillus niger mycelium waste from citric acid fermentation on the basis of steam explosion and ionic liquid pretreatment. This study demonstrates a green-like technology for the comprehensive utilization of A. niger mycelium waste from citric acid fermentation.

5 many materials, such as hemicellulose [15], lignocellulose, lignin [16,17], chitin [18], and corncobs (Teng et al., 2010). The pretreated material becomes porous and accessible to subsequent enzymatic hydrolysis. In addition, the use of ionic liquids (ILs) for cellulose and chitin pretreatment has also been increasingly recognized as a novel, efficient, and environment-friendly approach for biomass deconstruction [19,20]. Some ILs can influence the activity and stability of enzymes [21][22][23]. IL pretreatment can reduce the crystallinity of biopolymers, improving the surface area, porosity, and accessible binding sites during enzymatic catalysis [24].
To date, no attempt has been made in utilizing residual fibers, proteins, and mycelia in the mycelium waste from citric acid fermentation by SE combined with ionic liquid treatment, which needs to be discussed and solved especially in the current situation of environmental protection. At present, corn is used as a raw material for industrial production of citric acid [25]. Thus, there are a large amount of corn husks exist in mycelium waste, including 382 g of cellulose, 445 g of hemicellulose, 66 g of lignin, 19 g of protein, and 28 g of ash per kg of dry matter [26]. Hence, macromolecules, including chitin and cellulose, should be focused to comprehensively recycle mycelium waste. And, the structure composition of chitin is very similar to cellulose, which is reasonable to treat them at the same conditions and the treatment also can reduce resource utilization cost. In this study, SE and IL treatment was used in pretreating mycelium waste for the first time. First, the optimum conditions of SE toward mycelium waste were investigated. Then, SE and IL were used in the pretreatment of mycelium waste. The effect of combined pretreatment on enzymatic hydrolysis by cellulase and CDA was evaluated. This study demonstrates a possible approach for utilizing the soluble components and mycelia of waste from citric acid fermentation industry. 6 Results and discussion Understanding the structural and chemical changes of mycelium waste following SE pretreatment Initially, optimization experiments of SE pretreatment were performed. Then, the pretreated waste was divided into two parts: solid content containing mycelia and cellulose and soluble components released by A. niger containing sugar and proteins. Both components were analyzed to evaluate the effect of SE on the mycelium waste.

SE
Changes of various pretreated mycelium waste were analyzed after SE pretreatment. Fig. 1 shows the changes of color, shape, and particle size of materials pretreated by SE. With the increase of SE pressure from 1.0 MPa to 2.5 MPa, the color of materials gradually deepened ( Fig. 1(A)). Meanwhile, the proportion of small particles increased remarkably above 1.5 MPa of SE, (Fig. 1(B)), and the differential pore increased remarkably at 2-10 nm ( Fig. 1(C)). These changes indicate that SE showed a positive effect on the expansion and crushing of citric acid fermentation residues, which is consistent with a previous report [27].
But, it is difficult to recognize A. niger mycelia in figure 1(B), which may be due to the industrialization process of fermentation residue of citric acid including high temperature sterilization, plate frame filtration and full washing.
X-ray diffraction (XRD) analysis showed that the diffraction peaks at 19°-20° were obviously stretched, indicating the perceptible changes of the crystal structure of materials during SE treatment (Fig. 2(A)) [28]. Fig. 2(C) shows the comparative analysis of A 1560 /A 1070 from Fourier-transform infrared (FT-IR) data ( Fig. 2(B)). The deacetylation degree (DD) of chitin could be estimated by FT-IR spectra using two peaks associated with the C-O stretching band of the acetamide group (1070 cm -1 ) and the amide II band of amino and acetamide groups (1560 cm -1 ) [29]. The A 1560 /A 1070 value of samples treated under 2.0 and 2.5 MPa decreased from 0.69 of the control to 0.61 and 0.60, respectively, indicating that partial deacetylation of chitin from mycelium waste of citric acid fermentation occurred above 2.0 MPa of SE, which is consistent with other reports [29,30].
To further characterize the effect of SE, the solid content produced after pretreatment was set as starting materials for the extraction of GlcN by hydrochloric acid (30%). As shown in Table 1 3 and Table 2), including total soluble sugar, total soluble protein, and total soluble solids. The content of all components increased continuously with the increase of pressure. Compared with the control, the content of total soluble sugar, total soluble protein, and total soluble solids increased to varying degrees (Fig. 3). The content of total soluble solids increased from 7.40 wt % to 28.60 wt % with a growth rate of 21.20%. Higher SE pressure leads to greater degree of cell fragmentation and release of intracellular substances. This study is the first to apply SE to degrade the cell wall of mycelium waste.  Table 2 shows the sugar released from solid content by cellulase degradation with mycelium waste under different pretreatment conditions. Compared with the control, SE and IL pretreatment showed positive effect on the hydrolysis of cellulase toward mycelium waste. The concentrations of total sugar and single sugar, including glucose, arabinose, and cellobiose, increased obviously. When further pretreated by IL after SE, the concentration of total sugars increased to 64.49 g/L (0.46 g/g of sample) with a growth rate of 26.97% compared with the control. The maximum sugar yield was higher than that from distillers' dried grains with solubles (0.38 g/g) [34].

Application of by-products for ReCDA fermentation
As stated above, the soluble substances released from SE pretreatment under 2.5 MPa were rich in soluble sugar and proteins. Meanwhile, the solid content from SE could be further processed by IL pretreatment and enzymatic hydrolysis to yield fermentable sugars. On this basis, we used their mixtures as nutritional ingredients and added them into ReCDA media for ReCDA fermentation. After the preparation of ReCDA using the same experimental conditions, when 0.2g residues was added into 100mL media, the production of ReCDA exceeded the control after 30 h of fermentation, and the final product was approximately 1.5-fold higher than that of the control (Fig. 4). This finding indicates that the addition of soluble components produced by SE pretreatment was beneficial for R. equi fermentation. One possible reason may be that the degradation products, especially the degradation products of chitin, released from A. Niger fermentation residue treated by SE and cellulase hydrolysis is conducive to the expression of ReCDA. As reported results, substances such as furfural, which can inhibit microbial fermentation, are produced during the SE treatment of cellulose [15,35]. As Fig. 4, ReCDA fermentation was seriously inhibited with the addition of 0.4% residues, which might indicate that there are much inhibitors in the residues.
However, the content of sugar produced by enzymatic hydrolysis was not high. Two main reasons can explain this phenomenon. First, the raw material was sprayed for liquefaction at 0.1 MPa before citric acid fermentation. Afterward, the material was treated with SE at low pressure, and further SE treatment did not exert much effect on corn fiber decomposition. Second, the amylase and glycosylase used in the production of citric acid had cellulase activity, and the corn fiber was hydrolyzed by cellulase to some extent.

ReCDA
To investigate the effect of combined pretreatment on chitin recycling from mycelium waste, ReCDA was used to hydrolyze the solid content after sequential pretreatment. chitin and promote the cracking and dissolution of chitin chain, which enhanced the deacetylation reaction due to more reactive sites exposed [28,37].

Overall understanding and implications
To our knowledge, no study has comprehensively utilized mycelium waste from citric acid fermentation. This study was therefore carried out through investigating the pretreatment methods and cascaded enzymatic hydrolysis on the waste. One part of pretreated products could be hydrolyzed for soluble sugars by cellulase, which could be used as nutrients for microorganisms. The other part, mycelia, could be used to produce chitin derivates, which is promoted by SE and IL pretreatment (Fig. 5).
Although a comprehensive utilization strategy of mycelium waste was proposed, the production cost and efficiency, especially the efficiency of ReCDA, are key issues that need to be considered before application. As shown in Table 3, ReCDA provided an acetic acid yield of 554.48 mg/L after 6 h, which proved that pretreatment improved the efficiency of ReCDA. However, this value is too low to be considered for industrial application. Nevertheless, it is still worth studying because chitosan and GlcN from A. niger mycelia are important products with various advantages, such as continuous supply, free of heavy metals, and suitability for individuals with restrictive diets [39]. Thus, future large-scale studies should focus on the decrystallization of chitin by pretreatment and improvement of enzymatic hydrolysis involving chitinase and CDA [40][41][42]. 13

Conclusions
In this study, SE was first employed for the pretreatment of mycelium waste from citric acid fermentation. The optimal pretreatment condition of SE was 2.

Materials
The mycelium waste from citric acid fermentation used in this study was obtained

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 0.02 0 at a scan rate of 4 0 /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  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].  Comparison of soluble components of various pretreatments of fermentation residue.

Abbreviations
28 Figure 4 CDA fermentation in different media containing residue pretreated by SE and cellulolytic hyd