CN110283334B - Pretreatment method for improving biodegradation effect of locust wood - Google Patents
Pretreatment method for improving biodegradation effect of locust wood Download PDFInfo
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Abstract
A pretreatment method for improving the biodegradation effect of guaiacyl-syringyl lignin agricultural and forestry wastes relates to a locust wood pretreatment method. Aims to solve the problem that the guaiacyl-syringyl lignin in the locust tree is difficult to biodegrade due to high content. The method comprises the following steps: firstly, grinding the locust tree, and sieving the ground locust tree through a 20-mesh sieve to obtain locust tree fragments; secondly, adding inorganic salt into the locust tree fragments for treatment; and thirdly, filtering out inorganic salt solution, washing with distilled water, filtering, adjusting the pH value, and drying to constant weight to obtain the product. According to the invention, by a chemical pretreatment method and inorganic salt pretreatment, the smooth and compact structure of the surface of the natural lignocellulose becomes rough and irregular, the surface fiber bundle is damaged, and the raw material becomes loose and porous, so that the subsequent biological treatment can enter the interior more easily, the contact area of the material and the enzyme, the contact site of the enzyme and the enzyme load are increased, and the biodegradation effect is further improved. The invention is used in the field of natural lignocellulose degradation.
Description
Technical Field
The invention relates to a method for pretreating locust trees.
Background
By the end of the 20 th century, renewable energy sources have produced energy that accounts for 13% of the world's total energy consumption, a large portion of which is based on energy provided by biomass materials. The application of lignin biomass in all countries of the world is already in use on a certain scale, and in Canada with abundant wood resources, the energy generated by biomass materials accounts for 5% of the total energy of residents and 17% of the total industrial consumption. Lignocellulose is cheap and abundant renewable resources, and the full utilization of the lignocellulose is beneficial to improving the problems of resource shortage, environmental pollution and the like at present, and has important significance for realizing sustainable development of society.
The pagoda tree is a common tree species, is widely distributed in China, is particularly common in northern China, and is also widely planted in Guangdong, Taiwan, Gansu, Sichuan, Yunnan and other places. The tree is used as street tree for wind prevention, sand fixation, material and economic forest. Because the wood is straight, the processing performance is good, the wood is not easy to age, and the wood is antiseptic and insect-proof, has good stability, and is suitable for being made into furniture, floors and the like. The sophorae is a typical broad-leaf natural lignocellulose (the lignin of the sophorae is guaiacyl-syringyl lignin which is formed by dehydropolymers of coniferyl alcohol and sinapyl alcohol), the degradation difficulty of the sophorae under natural conditions is far greater than that of straws and common laboratory materials such as arbors, the materials are subjected to microbial degradation, and the generated degradation enzyme system also has lower enzyme activity. The existing research shows that the chemical pretreatment can improve the related enzyme activity in the process of degrading natural lignocellulose by microorganisms, but most of the chemical pretreatment is strong acid and strong alkali, so that the problems of high treatment cost, generation of a large amount of byproducts, no secondary pollution to the environment and the like exist. Therefore, the efficient and economic degradation mode of the sophorae can obviously affect the harmless treatment of the guaiacyl-syringyl lignin agricultural and forestry wastes.
Disclosure of Invention
The invention aims to solve the problems that the guaiacyl-syringyl lignin in the sophorae is high in content and difficult to biodegrade, and provides a pretreatment method for improving the biodegradation effect of the sophorae.
The pretreatment method for improving the biodegradation effect of the locust wood comprises the following steps:
firstly, grinding treatment:
removing impurities in the locust tree, physically grinding the locust tree, and sieving to obtain 20-mesh locust tree fragments;
secondly, inorganic salt pretreatment:
adding an inorganic salt solution into the locust tree chips treated in the first step, and treating for 46-50 h at 55-65 ℃; the inorganic salt is FeCl with the mass concentration of 9-13%2A solution; wherein the solid-to-liquid ratio of the locust tree fragments to the inorganic salt solution is 1 (9-11) g/mL;
and thirdly, filtering out inorganic salt solution, washing the residue with distilled water until the pH value is not changed, filtering, adjusting the pH value to 6-7, and drying at 55-65 ℃ to constant weight to complete the process.
And step three, regulating the pH value by using a citric acid-sodium citrate buffer solution, wherein the concentration of the citric acid-sodium citrate buffer solution is 0.1mol/L, and the pH value is 3.
The drying method in the third step comprises the following specific steps: drying in a forced air drying oven.
The invention has the beneficial effects that:
according to the invention, by a chemical pretreatment method and inorganic salt pretreatment, the smooth and compact structure of the surface of the natural lignocellulose becomes rough and irregular, the surface fiber bundle is damaged, and the raw material becomes loose and porous, so that the subsequent biological treatment can enter the interior more easily, the contact area of the material and the enzyme, the contact site of the enzyme and the enzyme load are increased, and the biodegradation effect is further improved.
The inorganic salt pretreatment for removing lignin and hemicellulose changes the structure of the lignocellulose matrix and promotes the improvement of the enzyme yield. During the pretreatment of the inorganic salt, key ester bonds such as ferulic acid, p-hydroxybenzoic acid, p-hydroxycinnamic acid and the like between the connecting polysaccharide and the lignin are broken, hydrogen bonds between hemicellulose and cellulose are weakened, and the change of the functional groups obviously promotes the dissolution of the lignin, so that more effective contact of ligninase is caused. The material treated by the inorganic salt increases the specific surface area of the fiber and has more porosity, so that the substrate treated by the inorganic salt is more suitable for the growth of filamentous fungi.
By researching that the optimal inorganic salt species for efficiently degrading the locust tree by the aspergillus fumigatus is FeCl2The concentration is 11%, the activity of the strain ligninogenic enzyme is highest, wherein the maximum value of the activity of the manganese peroxidase is 2244U/L, and is improved by 3 times compared with a control group; the maximum value of the lignin peroxidase activity is 292.3U/L, which is improved by 2.1 times compared with the control group, and the maximum value of the lignin peroxidase activity appears 3d ahead of time.
Drawings
FIG. 1 shows the effect of various pretreatment modes on the activity of Aspergillus fumigatus G-13 to produce ligninolytic enzymes.
FIG. 2 shows FeCl in different concentrations2The enzyme activity of the manganese peroxidase is changed after the locust tree is pretreated.
FIG. 3 shows FeCl in different concentrations2Enzymatic activity change of lignin peroxidase after pretreatment of locust wood。
FIG. 4 is a scanning electron microscope image of Japanese pagodatree that has not been pretreated and biodegraded.
FIG. 5 is a scanning electron microscope image showing the best pretreatment effect without biodegradation.
FIG. 6 is a scanning electron microscope image of biodegradation without pretreatment.
FIG. 7 is a scanning electron microscope image of biodegradation performed with optimal pretreatment.
FIG. 8 is a Fourier infrared spectrum of Sophora japonica before and after different pretreatments and biodegrades.
FIG. 9 is a XRD crystallinity spectrum of Japanese pagodatree before and after different pretreatments and biodegradation.
Detailed Description
The first embodiment is as follows: the pretreatment method for improving the biodegradation effect of the locust wood in the embodiment comprises the following steps:
firstly, grinding treatment:
removing impurities in the locust tree, physically grinding the locust tree, and sieving to obtain 20-mesh locust tree fragments;
secondly, inorganic salt pretreatment:
adding an inorganic salt solution into the locust tree chips treated in the first step, and treating for 46-50 h at 55-65 ℃; the inorganic salt is FeCl with the mass concentration of 9-13%2A solution; wherein the solid-to-liquid ratio of the locust tree fragments to the inorganic salt solution is 1 (9-11) g/mL;
and thirdly, filtering out inorganic salt solution, washing the residue with distilled water until the pH value is not changed, filtering, adjusting the pH value to 6-7, and drying at 55-65 ℃ to constant weight to complete the process.
Physically grinding the locust tree to 20 meshes, and then treating the locust tree by adopting a chemical pretreatment method. The purpose of the grinding treatment is to increase the contact area with the lignin-degrading enzyme by reducing its particle size or destroying its structural regularity. After the natural lignin is crushed, the action of some chemical reagents on the lignin is improved, and the lignin degradation by ligninase is promoted.
The second embodiment is as follows: the first difference between the present embodiment and the specific embodiment is: and in the second step, the treatment is carried out for 47-49 hours at 60 ℃. The rest is the same as the first embodiment.
The third concrete implementation mode: the present embodiment differs from the first or second embodiment in that: in the second step, the inorganic salt solution is FeCl with the mass concentration of 9-13%2And (3) solution. The other is the same as in the first or second embodiment.
The fourth concrete implementation mode: the present embodiment differs from the first or second embodiment in that: in the second step, the inorganic salt solution is FeCl with the mass concentration of 11 percent2And (3) solution. The other is the same as in the first or second embodiment.
The fifth concrete implementation mode: the difference between this embodiment and one of the first to fourth embodiments is: and in the second step, the solid-to-liquid ratio of the locust tree fragments to the inorganic salt solution is 1:11 g/mL. The other is the same as one of the first to fourth embodiments.
The sixth specific implementation mode: the difference between this embodiment and one of the first to fifth embodiments is: in the third step, citric acid-sodium citrate buffer solution is used for adjusting the pH value. The other is the same as one of the first to fifth embodiments.
The seventh embodiment: the sixth embodiment is different from the sixth embodiment in that: the concentration of the citric acid-sodium citrate buffer solution is 0.1mol/L, and the pH value is 3. The rest is the same as the sixth embodiment.
The specific implementation mode is eight: the present embodiment differs from one of the first to seventh embodiments in that: the drying method in the third step comprises the following specific steps: drying in a forced air drying oven. The other is the same as one of the first to seventh embodiments.
The specific implementation method nine: the present embodiment differs from the first to eighth embodiments in that: in the third step, the pH value is adjusted to 6.5. The rest is the same as the first to eighth embodiments.
The detailed implementation mode is ten: the present embodiment differs from one of the first to ninth embodiments in that: drying at 60 deg.C to constant weight. The other is the same as one of the first to ninth embodiments.
The following examples are given to illustrate the present invention, and the following examples are carried out on the premise of the technical solution of the present invention, and give detailed embodiments and specific procedures, but the scope of the present invention is not limited to the following examples.
Example 1:
the pretreatment method for improving the biodegradation effect of the locust wood comprises the following steps:
firstly, grinding treatment:
removing impurities in the locust tree, physically grinding the locust tree, and sieving to obtain 20-mesh locust tree fragments;
secondly, inorganic salt pretreatment:
adding an inorganic salt solution into the locust tree chips treated in the first step, and treating for 46-50 h at 55-65 ℃; the inorganic salt is FeCl with the mass concentration of 9-13%2A solution; wherein the solid-to-liquid ratio of the locust tree fragments to the inorganic salt solution is 1 (9-11) g/mL;
and thirdly, filtering out inorganic salt solution, washing the residue with distilled water until the pH value is not changed, filtering, adjusting the pH value to 6-7, and drying at 55-65 ℃ to constant weight to complete the process.
The concentration of the citric acid-sodium citrate buffer solution is 0.1mol/L, and the pH value is 3.
The following experiments were carried out on the locust tree obtained in the above manner:
culturing in a solid culture mode, adding 3g of pretreated locust tree sample into a 150mL conical flask, adding macroelement nutrient salt solution according to the solid-to-liquid ratio of 1:1.5 of the locust tree to the macroelement nutrient salt solution, adding a microelement nutrient salt solution with the volume about 0.1% of the macroelement nutrient salt solution, and sterilizing at 120 ℃ for 20min under high pressure. Then inoculating 3.5mL of spore suspension, culturing at 30 ℃, and sampling at 3 rd, 6 th, 9 th, 12 th, 15 th, 18 th and 21 th days of culture to perform fermentation liquor ligninase activity determination. Each set of 3 replicates.
Preparation of spore suspension: inoculating Aspergillus fumigatus G-13 with inoculating loop, inoculating into sterile water to obtain a solution with concentration of 106Spore suspension of each/mL is preserved in a refrigerator at 4 ℃ for later use; said Aspergillus fumigatus G-13 has been already mentionedIs published in article "establishment of self-immobilized cell system of lignin-degrading fungal mycelium pellets".
(II) preparing a crude enzyme solution: taking the solid matrix after fermentation into a centrifuge tube, adding 5mL200mmol/L acetic acid-sodium acetate (pH value 4.5) buffer solution according to 1g substrate, and leaching for 40min at 30 ℃ under 100r/min shaking. Centrifuging at 6000r/min for 10min at room temperature to obtain supernatant as crude enzyme solution.
And (III) measuring the activity of the ligninase:
determining the activity of the manganese peroxidase: adding 3.4mL of 200mmol/L acetic acid-sodium acetate buffer solution with the pH value of 4.5 and 0.1mL of 1.6mmol/L MnSO into the reaction system4The solution and 0.4mL of the crude enzyme solution were added, and 0.1mL of 1.6mmol/L H was added2O2The reaction was started with the solution, and the reaction was carried out at 30 ℃ for 3min, and the change in absorbance was measured at 240 nm. 1 enzyme activity unit (U/L) is defined as: within 3min before the reaction, 1 mu mol Mn is oxidized every min2+To Mn3+Average enzyme requirement of (2). Each sample was averaged 3 times in parallel.
And (3) measuring the activity of the lignin peroxidase: adding 1.5mL of 250mmol/L buffer solution with pH value of 3 tartaric acid-sodium tartrate, 1mL of 15mmol/L veratrum solution and 0.4mL of crude enzyme solution into the reaction system, and finally adding 0.1mL of 20mmol/L H2O2The reaction was started with the solution, and the reaction was carried out at 30 ℃ for 3min, and the change in absorbance was measured at 310 nm. 1 enzyme activity unit (U/L) is defined as: within 3min before the reaction, oxidizing 1 mu mol of veratryl alcohol per min to obtain the average enzyme demand of veratraldehyde. Each sample was averaged 3 times in parallel.
(IV) fermentation substrate treatment: washing the centrifugal residue with distilled water, vacuum filtering until the color of the filtrate is unchanged, adding water into the filter residue, centrifuging at 5000r/min for 10min, washing for 3 times, and drying the precipitate at 60 deg.C to constant weight for subsequent characterization of scanning electron microscope, infrared spectrum and X-ray diffraction.
The results are shown in FIG. 1. FIG. 1 shows the effect of various pretreatment modes on the ligninolytic enzyme production activity of Aspergillus fumigatus (A. Fumigatus) G-13.
FIG. 1 shows the data for the most active ligninase produced in 21d for various pre-treated fermentation substratesHigh values, where Mnp and Lip enzyme activity 15d reaches a maximum, with 11% FeCl2And 11% FeCl3The Lip enzyme activity of the pretreated locust tree reaches the maximum at 12 d. From FIG. 2, it can be seen that FeCl is present at a concentration of 11%2，FeSO4And ZnCl2The solution has obvious improvement on enzyme activity, and Mnp is respectively improved by 3 times, 2.7 times and 2.1 times compared with a control group; the Lip is increased by 2.1 times, 2.4 times and 2.3 times compared with the control group respectively. Although FeCl2The pretreated Lip enzyme has higher activity than FeSO4And ZnCl2Treating low, but FeCl2The highest Lip enzyme activity occurred 3d earlier in the pretreatment. The best pretreatment inorganic salt for A.Fumigatus G-13 fermented locust wood is FeCl2。
FIGS. 2 and 3 show FeCl in different concentrations2And (3) a Mnp and Lip enzyme activity change trend chart after the locust tree is pretreated. Changes in the activities of lignin peroxidase and manganese peroxidase were studied at different times, using a solid culture medium of locust tree, and continuously tested at 30 ℃ for 21 days. The results show that the activity of 2 lignin degrading enzymes shows a trend of increasing firstly and then decreasing, the maximum value of the enzyme activity is reached near 15d, wherein the FeCl with the concentration of 11 percent2The solution is pretreated to compare 2 percent FeCl and 8 percent FeCl2The activity of the treatment enzyme is obviously improved, and the activity of Mnp is obviously improved compared with that of Lip. Mnp and Lip of Japanese pagodatree reach maximum values at 15d and 12d, respectively, and the maximum values are 2244U/L and 292.3U/L, respectively. The results show that 11% FeCl concentration2The solution pretreatment of the locust tree can promote enzyme production and increase the enzyme production speed.
(V) scanning Electron microscopy analysis
FIG. 4 is a scanning electron microscope image of Japanese pagodatree that has not been pretreated and biodegraded; FIG. 5 is a scanning electron microscope image without biodegradation with optimal pretreatment; FIG. 6 is a scanning electron microscope image of biodegradation without pretreatment; FIG. 7 is a scanning electron microscope image of biodegradation performed with optimal pretreatment. As shown in fig. 4-7, the sheet-like substances or the covers on the surface of the locust tree which is not pretreated have compact and uniform physical and chemical structures and are arranged closely and orderly; via FeCl2The dense structure of the surface of the locust wood pretreated by the solution and biologically degraded by the aspergillus fumigatus strain is destroyed, and the surface structure becomes rough and irregularThen, the loosening is obvious, the fiber bundles on the surface of the locust tree are peeled off and even broken, and a series of irregular micropores and cracks are shown. 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 the enzyme loading. Improves the accessibility of the ligninase to the materials, destroys the natural barrier of the lignocellulose, thereby improving the enzymolysis efficiency.
(VI) Infrared analysis
FIG. 8 is a Fourier infrared spectrum of Japanese pagodatree before and after pretreatment and biodegradation, and the measured wave number range is 4000-400cm-1. In fig. 8, curve a is an infrared spectrum of the locust tree which is not pretreated and is not biodegraded, curve B is an infrared spectrum of the locust tree which is not pretreated and is not biodegraded with the best pretreatment effect, curve C is an infrared spectrum of the locust tree which is not pretreated and is biodegraded, and curve D is an infrared spectrum of the locust tree which is biodegraded with the best pretreatment effect.
As can be seen from FIG. 8, there are many similarities between the 4 spectra, indicating that there is no significant change in the major groups in the structure of the treated locust tree. But the relative intensity of the transmittance of each group is changed, which shows that the components of the locust tree are obviously changed in amount. 1736cm-1The absorption peak of ester bond (C ═ O) is more obvious in untreated locust wood, and the intensity of the peak is weakened after the optimal pretreatment, which shows that the key ester bonds such as ferulic acid, p-hydroxybenzoic acid, p-hydroxycinnamic acid and the like connecting between the polysaccharide and the lignin are broken, and the further biological treatment of the pretreated sample can obviously weaken the intensity of the peak, which shows that the combined treatment can break more key ester bonds between the lignin and the polysaccharide. The length of the original locust tree is 1618cm-1And 1507cm-1A strong characteristic absorption peak is near, the peak is the stretching vibration of a benzene ring framework and represents the extension of a lignin component, FeCl2The pre-treated locust wood fermented by a. funigatus was not evident in this absorption peak, indicating that most of the lignin was removed. And 1618cm-1，1507cm-1And 1319cm-1The infrared absorption peak of lignin is typically close to the infrared absorption peak, and the comparison of A and B, C, D shows that the peak intensity is obviously weakened along with the application of the treatment means, which indicates that the pretreatment promotes the bacterial strain to produce ligninase,inducing the strain to further degrade the locust tree.
(VII) X-ray diffraction analysis
Cellulose crystallinity reflects the relative proportion of crystalline regions in cellulose, rather than the absolute proportion. Studies have shown that the reduction of amorphous lignin and hemicellulose also affects the proportion of crystalline regions of cellulose in the sample. As shown in fig. 9, a curve a shows that the locust tree is not pretreated and is not biodegraded, a curve B shows that the locust tree is not biodegraded after the optimal pretreatment, a curve C shows that the locust tree is not biodegraded before the optimal pretreatment, and a curve D shows that the locust tree is biodegraded after the optimal pretreatment.
And (4) obtaining the crystallinity of the locust tree according to XRD analysis and related calculation. After the locust tree is pretreated, the crystallinity (CrI) of the cellulose is reduced, which indicates that FeCl2The crystal structure is destroyed; the crystallinity of the locust tree fermented directly with aspergillus fumigatus without pretreatment was rather higher than the CrI of the untreated locust tree, probably because ligninase produced during the aspergillus fumigatus fermentation degraded amorphous lignin, but crystalline cellulose remained in the locust tree structure, resulting in an increase in CrI of the directly fermented locust tree. The crystallinity of the pre-treated Aspergillus fumigatus fermented locust wood is reduced due to FeCl2The pretreatment can break the connection of key ester bonds and ether bonds between the connecting polysaccharide and the lignin, weaken the hydrogen bond bonding between the hemicellulose and the cellulose, and degrade and dissolve out the pretreated lignin, the hemicellulose and other amorphous substances; subsequently, ligninase produced by fermentation of aspergillus fumigatus can effectively degrade lignin, so that an amorphous region of cellulose is exposed, cellulose in the locust tree is remarkably swelled, the crystalline index of a lignocellulose structure is reduced, and a cellulose crystalline region is damaged, so that the crystallinity is reduced.
Claims (8)
1. A pretreatment method for improving the biodegradation effect of locust trees is characterized by comprising the following steps:
firstly, grinding treatment:
removing impurities in the locust tree, physically grinding the locust tree, and sieving to obtain 20-mesh locust tree fragments;
secondly, inorganic salt pretreatment:
adding inorganic salt into the locust tree chips treated in the first step, and treating for 46-50 h at 55-65 ℃; wherein the solid-to-liquid ratio of the locust tree fragments to the inorganic salt solution is 1 (9-11) g/mL;
thirdly, filtering inorganic salt solution, washing residues with distilled water until the pH value is not changed, adjusting the pH value to 6-7 after suction filtration, and drying at 55-65 ℃ to constant weight to complete the process; the biological degradation is to degrade the locust tree by using aspergillus fumigatus;
in the second step, the inorganic salt is FeCl with the mass concentration of 11 percent2And (3) solution.
2. The pretreatment method for improving the biodegradation effect of locust wood according to claim 1, wherein the second step is carried out at 60 ℃ for 46-50 h.
3. The pretreatment method for improving the biodegradation effect of locust wood according to claim 1, wherein the solid-to-liquid ratio of the locust wood chips to the inorganic salt solution in the second step is 1:11 g/mL.
4. The pretreatment method for improving the biodegradation effect of locust tree according to claim 1, wherein the pH value is adjusted by using citric acid-sodium citrate buffer solution in the third step.
5. The pretreatment method for improving the biodegradation effect of locust wood according to claim 4, wherein said citric acid-sodium citrate buffer solution has a concentration of 0.1mol/L and a pH of 3.
6. The pretreatment method for improving the biodegradation effect of the locust trees according to claim 1, wherein the drying in the third step is specifically as follows: drying in a forced air drying oven.
7. The pretreatment method according to claim 1, wherein the pH is adjusted to 6.5 in the third step.
8. The pretreatment method according to claim 1, wherein the pretreatment is carried out at 60 ℃ to achieve constant weight in the third step.
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