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 costs, 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 FeSO4, Fe2(SO4)3, FeCl2, FeCl3, NaCl, CaCl2, ZnCl2 and MgCl2 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 15 d. It can be seen from the table that the pretreatment of FeCl2 and FeSO4 solution improved the enzyme activity significantly. After pretreatment with this 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, FeCl2 and FeSO4 were selected as the best pretreatment inorganic salts of A. Fumigatus G-13 fermented Robinia.
Table 1. Effect of different types of inorganic salt pretreatment on ligninase activities produced by A. Fumigatus G-13 on Robinia
3.3 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 FeCl2 and FeSO4 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 were 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 FeCl2 solution and FeSO4 solution. As can be observed in Fig. 3 (a) and (b) and Fig. 4 (a) 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 FeCl2 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 U/L, respectively. When the ratio of solid to liquid was 1:16 and 1:21, the pretreatment effect was about the same as that of Mnp and Lip produced by 1:11 (Mnp was 1655.98 U/L, Lip was 302.4 U/L). 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 FeCl2 solution.
When used FeSO4 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 FeSO4 solution.
3.4 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 (FeCl2 solution) 1:16 (FeSO4 solution) and 8%, respectively. The effects of FeCl2 and FeSO4 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 FeCl2 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 FeSO4 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 FeCl2 and FeSO4 solution was 48 h.
3.5 Effects of pretreatment with different inorganic salts concentrations on ligninase activity produced by A. fumigatus G-13 fermented Robinia
Figure 7 and Fig. 8 were the trend diagrams of enzyme activities after pretreatment of Robinia with different concentrations of FeCl2 and FeSO4 solutions. Maintain pretreatment temperature, time and solid-liquid ratio of 60°C, 48 h, 1:11 (FeCl2 solution) 1:16 (FeSO4 solution), respectively. Under different concentrations, the changes of lignin peroxidase and manganese peroxidase activities were examined. The results showed that the activities of the two lignin-degrading enzymes were all increased first and then decreased. The highest value of ligninase activity in the fermented substrate pretreated by FeCl2 and FeSO4 solution appeared near 15 d. As can be seen from the figure below, the pretreatment with 11% concentration of two inorganic salts was better than 2% and 8% concentration, and the Lip activity of the Robinia pretreated with 11% concentration of FeCl2 solution reached the maximum at 12 d.
After pretreatment of Robinia with 11% FeCl2 solution, Mnp and Lip activities reached maximum at 15 d (2244 U/L) and 12 d (292.3 U/L), respectively. Compared with the control group (no inorganic salt pretreatment), Mnp and Lip enzyme activities were increased by 3 times and 2.1 times, respectively. For 11% FeSO4 solution, the Mnp and Lip enzyme activities reached a maximum at 18 d (2094 U/L) and 15 d (313.6 U/L), respectively. 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% FeCl2 solution pretreatment, Mnp activity was higher than FeSO4 pretreatment, although Lip enzyme activity was lower than FeSO4 treatment, but after FeCl2 solution pretreatment, Lip enzyme activity peak appeared 3 days earlier than FeSO4 pretreatment. Therefore, the higher concentration of FeCl2 (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 FeCl2 with a concentration of 11%.
3.6 Effect of optimal condition pretreatment and A. fumigatus G-13 fermentation on surface structure of lignocellulosic materials
Scanning electron microscopy has become an important technical means to observe the surface structure characteristics of lignocellulosic substrates. In order to further understand the reasons of FeCl2 solution pretreatment, A. Fumigatus fermentation treatment and FeCl2 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).
Figure 9 (a) 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 9 (b) 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% FeCl2 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. 9 (a) and (c)), it can be found that the Robinia treated by A. Fumigatus G-13 was obviously loose and the surface becomes rough, and presents a series of irregular micro-holes and cracks. Figure 9 (d) was the SEM image of Robinia that was pretreated with FeCl2 solution and then treated with A. Fumigatus fermented. Observing and comparing Fig. 9 (d) 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 FeCl2 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.
3.7 Effect of optimal condition pretreatment and A. fumigatus G-13 fermentation on chemical structure of lignocellulosic materials
3.7.1 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.
Table 2. Attribution of key chemical bonds or group characteristics in lignocellulose matrix
Wavenumber /cm-1
|
Attribution
|
Lignocellulose Structurally Related
|
3455-3410
|
O-H Stretching vibration
|
Internal hydrogen bond cleavage of cellulose molecules
|
2920-2850
|
C-H Stretching vibration
|
Waxy aliphatic related
|
2920
|
C-H Stretching vibration
|
Cellulose methyl/methylene break
|
1745
|
C=O Ester bond
|
Related to lignin side chain removal
|
1720
|
Carboxylic acid and Ester bond
|
Related to hemicellulose removal
|
1595-1605
|
Aromatic benzene ring stretching
|
Related to lignin removal
|
1510
|
β-Glycosidic bond vibration and Benzene ring stretching
|
Carbohydrate and lignin removal characteristic peaks
|
1426
|
CH2 and CH3 Bending vibration
|
—
|
1329
|
C-C and C-O Skeleton vibration
(Syringyl ring C-O)
|
Related to lilac-based condensation lignin
|
1260
|
Ester absorption
(Guaiacyl ring C-O)
|
Uronic acid removal related
|
1218-1245
|
C-O absorption in hemicellulose and lignin
|
Derived from acetyl group removal
|
1059
|
C=O stretching
|
Related to cellulose/ hemicellulose content
|
900
|
Sugar C1 group or sugar ring frequency, β- glucoside bond
|
Non-crystalline cellulose
|
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 key 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 FeCl2 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 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 FeCl2 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 FeCl2 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.
3.7.2 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.
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 FeCl2 solution, indicated that FeCl2 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 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 FeCl2 solution decreased from 44.84–43.11%. This was due to the pretreatment of FeCl2 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).