Pretreatment of Natural Lignocellulose with Inorganic Salts Improves Ligninase Production Fermented by Aspergillus fumigatus

This work screened out the optimal conditions for pretreatment of natural lignocellulose with inorganic salts and provided a simple, easy-to-operate, low-cost, clean and efficient pretreatment method for the efficient degradation of natural lignocellulose by strains. The results showed that the optimal pretreatment inorganic salt was FeCl2 with a concentration of 11%, pretreatment at 60 °C for 48 h, and the solid–liquid ratio was 1:11 (g/mL). According to the characterization results, after pretreatment of FeCl2 solution, the smooth and dense structure of natural lignocellulose surface became rough and irregular, and surface fiber bundles showed spalling and fracture. Subsequently, the enzymes produced by solid-state fermentation of Aspergillus fumigatus were easier to enter the interior, which increased the contact area between materials and enzymes, and increased the amount of enzymatic loads, thereby improving the biodegradation effect.


Introduction
Lignocellulose is a rich and inexpensive renewable resource. Making ample use of it will help improve the current resource shortage and environmental pollution problems, which also have important implications for the sustainable development of society. Typically, lignocellulosic biomass, widely existing in plants, consists of cellulose, hemicellulose, lignin, as well as small amounts of extract. However, Lignin is a highly heterogeneous polymer derived from a handful of precursor lignols that crosslink in diverse ways. The lignols that crosslink is of three main types, all derived from phenylpropane: coniferyl alcohol (4-hydroxy-3-methoxyphenylpropane) (G, its radical is sometimes called guaiacyl), sinapyl alcohol (3,5-dimethoxy-4-hydroxyphenylpropane) (S, its radical is sometimes called syringyl), and paracoumaryl alcohol (4-hydroxyphenylpropane) (H, its radical is sometimes called 4-hydroxyphenyl) [1]. In the process of degradation and utilization of lignocellulose, lignin and polysaccharide are cross-linked by ester bond and ether bond, resulting in high stability and stubbornness of lignocellulose, poor accessibility of ligninase, resulting in reducing the efficiency of enzymatic hydrolysis. The natural structure of lignocellulose determines its difficulty in resource utilization and harmless treatment, so effective pretreatment is required in its conversion [2]. Pretreatment can change the dense structure of lignocellulose, destroy the physical and chemical connection between the interior, reduce the crystallinity of cellulose, or remove lignin, and increase the porosity of the raw materials. Effectively promote contact between cellulase and cellulose, thereby greatly improving the efficiency of enzymatic hydrolysis [3].
At present, pretreatment methods mainly include physical methods (microwave treatment) [4], chemical methods (acid-base hydrolysis) [5], and biological methods (biological enzymatic method) [6]. Each pretreatment method gets its advantages and disadvantages. Mechanical comminution is a relatively simple physical pretreatment method, which destroys the complex structure of natural lignocellulose to a certain extent by shearing or grinding, and reduces the size of matrix particles, increases the joint point between exposed surface and lignin-degrading enzyme, increases the reaction area. Some research has shown that the lignin enzyme digestion process and degradation efficiency could be improved after the matrix was pulverized [7]. However, the degradation of natural lignocellulose is directly related to the milling time and the degree of comminution. The smaller the particle size, the easier it is to react, but the more energy it needs to provide. Moreover, the amorphous state produced by physical pulverization is precarious and easy to recrystallize, resulting in the limited application. Ultrasonic pretreatment is a pretreatment method reported in recent years [8]. The physicochemical effect is generated by the action of energy, which destroys the intermolecular hydrogen bond and the crystalline structure, changes the size of the fiber crystallization zone, reduces the degree of polymerization, and thereby increases the rate of enzymatic hydrolysis. The Yu research group used the ultrasonic system to pretreat the rice husks and significantly improved the degradation rate of Pleurotus ostreatus on rice hulls. The increase in efficiency was attributed to the structural damage of the rice husks during ultrasonic pretreatment [9]. Even though microwave treatment has the advantages of high efficiency and pollution-free, it is sometimes difficult to obtain industrial applications due to its prohibitive cost. The acid treatment method is applied earlier, among which sulfuric acid is the most widely used. Various diluted inorganic acids, such as HCl, H 3 PO 4 and HNO 3 have also been used to pretreat different lignocellulose materials. Acid pretreatment of lignocellulose can destroy the chemical bonds between lignin and hemicellulose to increase the accessibility of enzymes. At the same time, the average degree of polymerization of cellulose was reduced, which promoted the enzymatic hydrolysis of lignocellulose [10]. The Ali research group pretreated the remaining empty pods of Moringa with dilute H 2 SO 4 . It was found that the degradation of lignin was reduced and the recovery rate of xylan reached 24.7-50.2% [11]. Another team used maleic acid to pretreat lignin in the secondary wall of higher plant cells found that under the condition of low concentration of maleic acid pretreatment, some dense lignin was modified to become more looser, which increased the accessibility of ligninase [12]. Among the alkaline reagents, NaOH and Ca(OH) 2 are the most commonly used, and NaOH works well, but the cost of acid neutralization for subsequent treatment is higher. Currently, Ca(OH) 2 has been used to pretreat various lignocellulosic materials for further enzymatic hydrolysis [13]. Compared with NaOH aqueous solution pretreatment, Ca(OH) 2 pretreatment has lower cost and better environmental benefits because it can be easily recovered by reacting with CO 2 [14].
In recent years, with the development of science and technology, researchers at home and abroad have studied many methods of lignin treatment. Although there are many pretreatment methods that can quickly process lignin, researchers hope to use more environmentally friendly techniques to treat lignin from a cost reduction perspective [15], increase degradation rates, and reduce secondary pollution to the environment during recycling. The biological treatment method has mild conditions, low energy consumption, simple operation and no pollution, and has been proved to be the best way to treat lignocellulose. However, biodegradation has the disadvantages of a long treatment cycle and low degradation efficiency. Referring to previous pretreatment methods, the study used a completely new approach. The method uses an inorganic salt reagent to pretreat natural lignocellulose and then ferment with Aspergillus fumigatus G-13. It is intended to change the dense structure of lignocellulose by inorganic salt pretreatment, and improve the ability of strains to produce ligninase using natural lignocellulosic materials as substrates, thereby improving the degradation efficiency of lignin. For the subsequent release and utilization of cellulose, a method for removing lignin which is relatively clean, efficient, simple in operation and low in cost is provided. In this study, it is expected that different types of inorganic salts will be used to pretreat the Robinia, screen out the inorganic salts of the best pretreated Robinia, and the pretreatment conditions are further optimized. Robinia is a typical broad-leaved natural lignocellulose. Robinia is mainly guaiacyl-syringyl lignin, which is composed of dehydrogenated polymers of coniferyl alcohol and erucyl alcohol.

Strain Sources
Aspergillus fumigatus G-13 (A. fumigatus G-13), which could degrade lignin used in this work was isolated from the samples collected from soil near the sewage draining exit of a paper mill in Harbin.

Natural Lignocellulosic Sample
The Robinia from Songshan Forest Farm, Gongyi City, Henan Province.

Pretreatment Processes
Take the natural lignocellulosic substrate sample Robinia, and the impurities were removed and passed through a 20-mesh sieve. 4 g of the Robinia under the sieve was weighed and placed in a 150 mL Erlenmeyer flask. FeSO 4 , Fe 2 (SO 4 ) 3 , FeCl 2 , FeCl 3 , NaCl, CaCl 2 , ZnCl 2 and MgCl 2 solutions were added to pretreat the samples respectively. After pretreatment, the samples were washed with distilled water until the pH value no longer changed. After filtration, the pH was adjusted with a citric acid-sodium citrate buffer solution (0.1 mol/L, pH 3) to 6. Dry to constant weight at 60 °C.

Preparation of bacterial suspension
The oblique surface of A. Fumigatus PDA medium stored at 4 °C was taken out, and the fungal spores were washed with sterile water to prepare a spore suspension of 10 6 cells/ mL (measured by blood cell counting method). Keep it in a 4 °C refrigerator. The sample was inoculated at 3.5 mL/vial.

Solid-State Fermentation Culture
Used solid-state culture, 3 g of the pretreated natural lignocellulose sample was added to a 150 mL Erlenmeyer flask, and a large amount of elemental nutrient salt solution, and a 0.1% trace element nutrient salt solution were added according to the solid-liquid volume ratio, and autoclaved at 120 °C for 20 min. Afterwards, 3.5 mL of spore suspension was added and cultured at 30 °C under constant temperature. The ligninase activity of the fermentation broth was determined by sampling on the 3rd, 6th, 9th, 12th, 15th, 18th and 21st, respectively, with 3 parallel samples in each group.

Preparation of Crude Enzyme
After fermentation, the solid matrix was added to the centrifuge tube, and then 5 mL of acetic acid sodium acetate solution (pH value 4.5) was added to the 1 g substrate for oscillation extraction at 30℃ and 100 r/min for 40 min. Crude enzyme was collected by centrifugation (20 min, 6000 r/ min) at room temperature.

Manganese Peroxidase Activity Assay
Add 3.4 mL of acetic acid-sodium acetate buffer solution (concentration: 200 mmol/L, pH 4.5) to the reaction system, and add 0.1 mL of 1.6 mmol/L MnSO 4 solution and 0.4 mL of crude enzyme solution. Finally, 0.1 ml of 1.6 mmol/L H 2 O 2 solution was added to start the reaction, and the reaction was carried out at 37 °C for 3 min, and the absorbance at 240 nm was measured. One enzyme unit (U) is defined as the amount of enzyme required to oxidize 1 μmol Mn 2+ per minute to Mn 3+ . Each sample parallels operation three times and then averaged [16].

Lignin Peroxidase Activity Assay
The reaction solution contained 1 mL of 15 mmol/L resveratrol solution, 1.5 mL of sodium tartrate buffer (concentration of 250 mmol/L, pH of 3) and 0.4 mL of crude enzyme solution. Finally, 0.1 mL of 20 mmol/L H 2 O 2 solution was added to initiate the reaction, and the absorbance at 0, 1, 2 and 3 min was measured at 310 nm ultraviolet light. One enzyme unit (U) is defined as the amount of enzyme required to oxidize 1 μmol of resveratrol per minute to become veratraldehyde. Each sample was run in parallel three times and then averaged [17].

Fermentation Substrate Treatment
The centrifugal residue was washed with distilled water and suction filtered until the color of the filtrate did not change. Then, water was added to the residue, and the mixture was centrifuged at 5000 r/min for 10 min, and the precipitate was dried at 60 °C to a constant weight. Subsequent characterization by scanning electron microscopy, infrared spectroscopy and X-ray diffraction.

Scanning Electron Microscope
The scanning electron microscope used a Japanese HITACHI S-3400 M scanning electron microscope with an accelerating voltage of 5.00 kV and a working distance of 13.2 to 18.2 mm. During the observation, a conductive tape was attached to the scanning electron microscope sample stage and a 15 nm thick gold film was plated on the surface of the sample by an E-1010 (HITACHI) type ion sputter coater.

Infrared
Infrared spectroscopy was performed using NEXU type Fourier infrared Raman spectroscopy (FT-IR) from NICO-LET, USA. Using KBr tableting method, the wave number range was 400-4000 cm −1 , and the resolution was higher than 0.09 cm −1 .

X-Ray Powder Diffraction
X-ray powder diffraction (XRD) was performed by a multifunctional X-ray system diffractometer (Philips, Netherlands). The Cu/K α source has a wavelength of 1.5418, and the spectrum was recorded at a current of 40 mA·h and a voltage of 45 kV. The scanning range 2θ was 5°-40°, the step width was 0.03°, and the scanning rate was 10.0 s/step. Cellulose crystallinity (CrI) was calculated on the basis of the diffraction pattern according to the method proposed by Segal [18], See formula 1.

Effects of Different Kinds of Inorganic Salt Pretreatment on Ligninase Activity Produced by A. fumigatus G-13 Fermented Robinia
Acid and alkali pretreatment are currently relatively mature pretreatment methods for the removal of lignin and hemicellulose from lignocellulosic materials. However, residual acid after pretreatment needs to be neutralized, which not only consumes additional reagents, increases cost, but also produces waste and causes pollution. If it is detoxified, the cost will need to be further increased [19]. In this experiment, the effects of pretreatment with eight inorganic salts of FeSO 4 , Fe 2 (SO 4 ) 3 , FeCl 2 , FeCl 3 , NaCl, CaCl 2 , ZnCl 2 and MgCl 2 on the ligninase produced by A. fumigatus fermented Robinia were researched. After pretreatment, the Robinia structure was destroyed, and the destruction of lignocellulosic structure by inorganic salts was used as an indicator of ligninase activity.
Maintain pretreatment temperature, time, solid-liquid ratio and inorganic salt concentration of 50 °C, 48 h, 1:16 (g/mL) and 8%, respectively. The effects of inorganic salt pretreatment on the activity of lignin degradation enzymes of A. fumigatus G-13 fermented Robinia were investigated by changing different kinds of inorganic salts. The results were presented in Table 1, the data in the table were the highest values of ligninase activity produced within 21 days of fermentation of various inorganic salt pretreated fermentation substrates. Among them, Mnp and Lip enzyme activities reached a maximum around day 15. It can be seen from the table that the pretreatment of FeCl 2 and FeSO 4 solution improved the enzyme activity significantly. After pretreatment with these two solutions, Mnp were all increased by 2.2 times compared with the control group, and Lip was increased by 1.8 times and 2.2 times compared with the control group, respectively. The experimental screening, FeCl 2 and FeSO 4 were selected as the best pretreatment inorganic salts of A. Fumigatus G-13 fermented Robinia.

Effects of Pretreatment Temperature on Ligninase Activity Produced by A. fumigatus G-13 Fermented Robinia
Maintain pretreatment time, solid-liquid ratio and inorganic salt concentration of 48 h, 1:16 (g/mL) and 8%, respectively. The effects of FeCl 2 and FeSO 4 solution pretreatment on the activity of lignin degradation enzymes of A. fumigatus G-13 fermented Robinia were investigated by changing different pretreatment temperature. The results were presented in Fig. 1 and Fig. 2. The Mnp and Lip enzyme activities increased with increasing fermentation time and reached a maximum around 15 day. After continued fermentation, the enzyme activities of lignin-degrading enzymes (Mnp and Lip) showed a downward trend. With the increase of pretreatment temperature, the activity of the lignin-degrading enzyme produced by Aspergillus fumigatus fermented Robinia substrates showed an upward trend. When the pretreatment temperature was 60 °C, the enzyme activity of the lignin-degrading enzyme produced by fermentation of two inorganic salts pretreated Robinia substrates reaches a peak. Among them, after pretreatment of FeCl 2 solution, Mnp activity reached 1593.18 U/L, Lip activity was 257.33 U/L, and after FeSO 4 solution pretreatment, Mnp activity reached 1567.23 U/L, and Lip activity was 354.01 U/L. It was indicated that 60 °C was the optimum temperature for pretreatment of Robinia by two inorganic salts.

Effects of Pretreatment Solid-Liquid Ratio on Ligninase Activity Produced by A. fumigatus G-13 Fermented Robinia
Maintain pretreatment temperature, time and inorganic salt concentration of 60 °C, 48 h and 8%, respectively. The effects of FeCl 2 and FeSO 4 solution pretreatment on the activity of lignin degradation enzymes of A. fumigatus G-13 fermented Robinia were investigated by changing different solid-liquid ratio (g/mL). The results are shown in the figure below. Figure 3 and Fig. 4 showed the changes of lignin degradation enzyme activity after pretreatment of Robinia with different solid-liquid ratio FeCl 2 solution and FeSO 4 solution. As can be observed in Figs. 3a and b and 4a and b, when the solid-liquid ratio was 1:6, the Mnp and Lip enzyme activities were lower. This solid-liquid ratio pretreatment cannot fully infiltrate the Robinia, resulting in poor pretreatment effect. For FeCl 2 solution, the volume of the pretreatment solution was increased until the ratio of solid to liquid was 1:16 and 1:21, the highest Mnp activity was reached 1560.9 U/L and 1641.6 U/L, respectively, and the highest Lip activity was reached 269.14 U/L and 265.78 When the solid-liquid ratio was 1:11, the highest enzyme activity appeared 3 days earlier than that of 1:16 and 1:21. Therefore, considering the pretreatment effect and saving raw materials, when the solid-liquid ratio was 1:11, it was the optimum solid-liquid ratio for the pretreatment Robinia with FeCl 2 solution. When used FeSO 4 solution to treat Robinia, it can be seen from 4(a) that when the ratio of solid to liquid was 1:16, the activity of Mnp was most significantly increased, reaching a maximum of 1842.27 U/L. Under this solid-liquid ratio, the Lip activity was 200.46 U/L, which was lower than the 223.77 U/L when the solid-liquid ratio was 1:11, but the enzyme production trend was about the same. Comprehensive consideration, the solid-liquid ratio of 1:16 was chosen as the optimum solid-liquid ratio for the pretreatment Robinia with FeSO 4 solution.

Effects of Pretreatment Time on Ligninase Activity Produced by A. fumigatus G-13 Fermented Robinia
Maintain pretreatment temperature, solid-liquid ratio and inorganic salt concentration of 60 °C, 1:11 (FeCl 2 solution) 1:16 (FeSO 4 solution) and 8%, respectively. The effects of FeCl 2 and FeSO 4 solution pretreatment on the activity of lignin degradation enzymes of A. fumigatus G-13 fermented Robinia were investigated by changing different pretreatment time. In Fig. 5, when the FeCl 2 solution was used, the pretreatment time was less than 48 h, the Mnp and Lip enzyme activities of A. fumigatus fermentation increased with the increase of the pretreatment time. When the pretreatment time was 48 h, the Mnp and Lip enzyme activities reached the maximum on the 15th day, which were 1200.90 U/L and 236.65 U/L respectively. Under other pretreatment time conditions, the trend of enzyme production was regular and stable. If the pretreatment time was extended, the activity of Lip enzyme decreased obviously, and the change of Mnp activity showed that the effect of pretreatment on 60 h and 48 h was equivalent. After prolonging the pretreatment time to 72 h, the enzyme production declined slightly. The possible reason was that under this pretreatment condition, the cellulose content and xylan content in Robinia reduced with the increase of time, resulting in a slight increase in lignin content. Reduced accessibility of enzymes and substrates, resulting in decreased enzyme production efficiency.
As shown in Fig. 6, when the FeSO 4 solution was utilized to pretreat Robinia, the overall trend of enzyme production was approximately the same, reaching the maximum value of A. fumigatus enzyme production in the vicinity of 15 days. When the pretreatment time was 24 h, the Mnp activity achieved the maximum value of 1192.14 U/L, but the overall enzyme production tendency was not as good as the pretreatment for 48 h, the activity of Mnp was always maintained higher and stable. After 21 days of fermentation, when the pretreatment time was 60 h, the Lip enzyme activity reached the maximum value of 255.69 U/L on the 15th day. Compared with pretreatment for 36 h and 48 h, although the highest Lip enzyme activity was not as good as 60 h pretreatment, the highest enzyme activity difference was lower, and the overall enzyme production tendency was better. In summary, the optimum pretreatment time for pretreatment of Robinia with FeCl 2 and FeSO 4 solution was 48 h.  Compared with the control group, Mnp and Lip enzyme activities were increased by 2.6 times and 2.2 times, respectively. To sum up, after 11% FeCl 2 solution pretreatment, Mnp activity was higher than FeSO 4 pretreatment, although Lip enzyme activity was lower than FeSO 4 treatment, but after FeCl 2 solution pretreatment, Lip enzyme activity peak appeared 3 days earlier than FeSO 4 pretreatment. Therefore, the higher concentration of FeCl 2 (11%) solution pretreatment of Robinia can promote the production of enzymes and increase the speed of lignin peroxidase production. The optimal pretreatment inorganic salt of A. Fumigatus G-13 fermented Robinia was FeCl 2 with a concentration of 11%.

Effect of Optimal Condition Pretreatment and A. fumigatus G-13 Fermentation on Surface Structure of Lignocellulosic Materials
Scanning electron microscopy has become a necessary technical means to observe the surface structure characteristics of lignocellulosic substrates. In order to further understand the reasons of FeCl 2 solution pretreatment, A. Fumigatus fermentation treatment and FeCl 2 solution pretreatment after A. Fumigatus fermentation treatment to improve the lignin removal rate, the surface structure of Robinia was observed by scanning electron microscope. As shown in Fig. 9 (all Robinia samples have a particle size of 20 mesh).

Fig. 9
Effects of pretreatment with optimal conditions and A. fumigatus G-13 fermentation. a Not pretreated and not biodegraded. b Not biodegraded after optimal pretreatment. c Not pretreated and biodegraded. d Biodegraded after optimal pretreatment. (Optimal pretreatment inorganic salt was FeCl2 with a concentration of 11%, pretreatment at 60 °C for 48 h, and the solid-liquid ratio was 1:11 (g/mL) Figure 9a was Robinia without any treatment. It can be observed in the figure that the physicochemical structure of the untreated Robinia surface was dense and uniform, and the arrangement was relatively neat. Figure 9b was shown that the dense structure of the original Robinia has been destroyed and the surface structure has become irregular after being treated under optimal pretreatment conditions (11% FeCl 2 solution). And the fiber bundles on the surface of Robinia appeared spalling or even breaking. Comparing the SEM images of Robinia with or without biological treatment (Fig. 9a and c), it can be found that the Robinia treated by A. Fumigatus G-13 was obviously loose and the surface became rough, and presents a series of irregular micro-holes and cracks. Figure 9d was the SEM image of Robinia pretreated with FeCl 2 solution and then treated with A. Fumigatus fermented. Observing and comparing Fig. 9d and a, b, and c, various treatment methods affect the surface structure of Robinia lignocellulosic substrate, but after inorganic salt pretreatment and then the biological fermentation treatment of the Robinia, the structural damage was more thorough. The possible reason was that FeCl 2 solution pretreatment makes the surface of the Robinia rugged, and these structural changes increase the specific surface area of the fiber, thereby increasing the contact area with the enzyme, the contact site of the enzyme and enzyme loading [20]. The change of microstructures enhances the accessibility of ligninase to materials and destroys the natural barrier of lignocellulose, thereby improving the enzymatic efficiency.

Infrared spectra analysis
Infrared spectroscopy is a common means of analyzing the composition and chemical changes of lignocellulose. Table 2 displays the spectra of fundamental chemical bonds and functional groups in the lignocellulose matrix. Figure 10 was an infrared spectrum of Robinia, in which (a), (b), (c)  and (d) respectively represent that Robinia has not been pretreated and has not been biodegraded, not biodegraded after optimal pretreatment, not pretreated and biodegraded, and biodegraded after optimal pretreatment. From the Fourier transform infrared spectroscopy (FTIR) results, it was found that the characteristic peaks of cellulose, hemicellulose, and lignin in Robinia were all significantly changed after pretreatment. The results are presented in Fig. 10. The absorption peak at 3420 cm −1 in the figure was the O-H stretching vibration of the phenolic hydroxyl group and the alcoholic hydroxyl group; the absorption peak at 2920 cm −1 was the stretching vibration of C-H; the absorption peak at 1745-1655 cm −1 represented C=C, C=O (ketones, esters) (Jahan, 2004), and the absorption peak at 1745-1655 cm −1 represented C=C, C=O (ketones, esters). The ester bond (C=O) absorption peak at 1736 cm −1 was more pronounced in untreated Robinia, and the intensity of this peak was weakened after optimal pretreatment. It was indicated that the critical ester bonds between polysaccharide and lignin, such as ferulic acid, p-hydroxybenzoic acid and p-hydroxy cinnamic acid were broken. Further biological treatment of the pretreated sample will significantly reduce the intensity of the peak, indicated that the combined treatment could break more key ester bonds between lignin and polysaccharide [21]. The Robinia has a strong characteristic absorption peak near 1618 cm −1 and 1507 cm −1 . This peak was the stretching vibration of the benzene ring skeleton, which represents the extension of the lignin component. After pretreatment with FeCl 2 solution and then by A. Fumigatus fermentation, the absorption peak of Robinia was not obvious here, indicated that most of the lignin structure was destroyed. And the typical lignin infrared absorption peak was around 1618 cm −1 , 1507 cm −1 and 1319 cm −1 . Compared (a) with (b), (c), and (d), it can be seen that with the application of the treatment means, the peak intensity showed a significant weakening, indicated that the pretreatment promoted the production of ligninase by the strain and induced further degradation of the Robinia.
After the Robinia was treated with optimal pretreatment conditions, the chemical changes in its composition were observed. It was found that Robinia contained two types of lignin, guaiacyl and syringyl, and the characteristic absorption peaks were 1248 cm −1 (guaiac ring C-O) and 1319 cm −1 (syringyl ring C-O). Compared to the infrared spectrum of Robinia, it was found that the characteristic peak of cellulose and hemicellulose (1050 cm −1 ) became wider under the condition of FeCl 2 solution pretreatment of Robinia. It was indicated that the pretreatment can further destroy the structure of cellulose and hemicellulose and change the absorption intensity of the binding bond. In addition, change in strength of 897 cm −1 can reflect the structural changes of crystalline cellulose. It can also be seen from the figure that after pretreatment with FeCl 2 solution and then by A. Fumigatus fermentation, the peak shape became slow and the peak width became large, indicated that the characteristic functional groups of crystalline cellulose were also destroyed, resulted in a decrease in absorption intensity.

XRD Analysis
The crystallinity of the raw materials of Robinia and the crystallinity of the materials obtained after pretreatment by different methods were studied by X-ray powder diffraction. The results were shown in Fig. 11, in which (a), (b), (c) and (d) respectively represent that Robinia has not been pretreated and has not been biodegraded, not biodegraded after optimal pretreatment, not pretreated and biodegraded, and biodegraded after optimal pretreatment (Fig. 12).
Many studies have demonstrated that the reduction of amorphous lignin and hemicellulose affects the proportion of cellulose crystalline regions in the sample [22,23]. Cellulose crystallinity reflects the relative proportion of crystalline regions in cellulose, rather than the absolute proportion. We found through XRD calculation that the crystallinity of Robinia (20 mesh) without any treatment was 44.84%, and the crystallinity decreased to 43.44% after pretreatment with FeCl 2 solution, indicated that FeCl 2 destroyed the crystal structure inside Robinia. The crystallinity of Robinia fermented directly with A. fumigatus G-13 without pretreatment was 46.72%, which was higher than that of Robinia without any treatment. The probable reason was that the ligninase produced during the fermentation of A. fumigatus degraded the amorphous lignin, but the crystalline cellulose Not biodegraded after optimal pretreatment. (c) Not pretreated and biodegraded. (d) Biodegraded after optimal pretreatment. (Optimal pretreatment inorganic salt was FeCl2 with a concentration of 11%, pretreatment at 60 °C for 48 h, and the solid-liquid ratio was 1:11 (g/ mL) remained in the Robinia structure, resulted in an increase in the CrI of the directly fermented Robinia. Compared with the unpretreated Robinia, the crystallinity of Robinia fermented by A. fumigatus after pretreatment with FeCl 2 solution decreased from 44.84 to 43.11%. This was due to the pretreatment of FeCl 2 solution, which could break the linkage between the key ester and ether bonds between the linking polysaccharide and lignin, and weakened the hydrogen bonding between hemicellulose and cellulose. After pretreatment, amorphous substances such as lignin and hemicellulose were degraded and dissolved. Subsequently, the ligninase produced by the fermentation of A. fumigatus was effective in degrading lignin, thereby exposing the amorphous regions of cellulose. The cellulose in the Robinia was significantly swelled, the crystallinity index of the lignocellulosic structure was lowered, and the cellulose crystallization zone was damaged, so that the crystallinity was lowered (Nakashima et al., 2016).

Conclusion
This study used inorganic salt, a simple, low-consumption, non-polluting chemical reagent to pretreat natural lignocellulose (Robinia). It was found that the FeCl 2 solution was an effective pretreatment reagent. The optimal conditions for pretreatment of Robinia by FeCl 2 solution were successfully screened by optimization experiments, and the maximum effectiveness of the pre-processing was achieved. SEM and FTIR analysis showed major structure changed after pretreatment, and it was found that the obstinate structure inside the Robinia was destroyed after pretreatment. Meanwhile, it could effectively promote the solid-state fermentation of A. fumigatus G-13 to produce enzymes, thereby improved the degradation efficiency of Robinia by biological treatment. To the best of our knowledge, there is no study on the pretreatment of natural lignocellulose by mild conditions to promote the production of enzymes by strains. This work provides a basis and reference for further experimental research in the field of biodegradation of natural lignocellulose.

Conflict of interest
The author declares that they have no conflict of interest.

Consent for Publication
All authors agreed to publish.