CN107119094B - Method for strengthening Fenton reaction pretreatment of waste biomass by using lignin-degrading bacteria - Google Patents
Method for strengthening Fenton reaction pretreatment of waste biomass by using lignin-degrading bacteria Download PDFInfo
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Abstract
The invention discloses a method for strengthening Fenton reaction pretreatment of waste biomass by using lignin-degrading bacteria, and particularly relates to a method for further removing residual lignin in the waste biomass by using the lignin-degrading bacteria (Cupriavidus basilensis B-8 with the preservation number of CGMCC No.4240) and improving the culture condition on the basis of the Fenton reaction pretreatment of the waste biomass, and changing the stable straight rod shape of the waste biomass into loose sheet shape, so that the accessible surface during enzymolysis and saccharification is improved. The method can improve the enzymolysis efficiency of Fenton reaction pretreatment by about 60 percent, can improve the enzymolysis efficiency by nearly 120 percent to the maximum extent, and has the advantages of short treatment time, simple operation, small secondary pollution, low cost and the like.
Description
The technical field is as follows:
the invention belongs to the technical field of biomass new energy, and particularly relates to a method for strengthening Fenton reaction pretreatment of waste biomass by using lignin-degrading bacteria.
Background art:
in recent years, in order to reduce the dependence on fossil fuels, governments and scientific research institutes of various countries have vigorously conducted development and research on renewable energy sources. Lignocellulose in the waste biomass mainly comprises cellulose, hemicellulose and lignin, and a large amount of carbon resources are stored, so that the waste biomass becomes a second-generation biomass energy source, and is the most important carbon source for biological fermentation of green products such as current bioethanol, bioplastic, biodiesel and the like. Hemicellulose and lignin in lignocellulose are closely embedded around the cellulose, and if no treatment is carried out, the saccharification and fermentation efficiency is extremely low. Therefore, the pretreatment becomes the first link for development and utilization of waste biomass, namely loosening or destroying the compact structure of cellulose and the package of lignin and hemicellulose, so that the cellulose, the hemicellulose and the lignin are separated, and the accessibility and the action efficiency of enzyme to the cellulose are improved.
The current pretreatment processes are mainly classified into physical, chemical and biological methods. The Fenton reaction is a non-enzymatic pathway used by part of fungi in the process of attacking the waste biomass, so that the method becomes an effective waste biomass pretreatment method. It is generally believed that the Fenton reaction produces highly reactive hydroxyl radicals that attack the waste biomass causing internal cleavage of the cellulose chain and lignin depolymerization, which increases the hemicellulose content. At present, the deep removal of lignin in waste biomass is realized by adopting a fungus method or fungus-chemical combined pretreatment. However, the time required for the growth of the fungi is too long (10-50 days), which is not beneficial to the process expansion and commercialization. Compared with fungi, the lignin-degrading bacteria have fewer number, but can greatly shorten the inoculation time (<7 days) of biological treatment, so that the pretreatment of waste biomass by using bacteria instead of fungi has important significance. At present, no report related to Fenton reaction pretreatment of lignin-degrading bacteria reinforced waste biomass exists at home and abroad.
Because lignin has natural heterogeneity, variability and extremely strong stability, when the Fenton reaction condition is mild, the lignin is difficult to remove completely, so that the sugar purity is reduced, and subsequent enzymolysis and biotransformation of cellulose are hindered. If high-strength conditions are adopted, excessive damage and loss of cellulose are easily caused. Therefore, weakening the Fenton pretreatment condition, and simultaneously utilizing the microorganisms to deeply remove residual lignin in slag removal is the key for improving the carbon source utilization and resource yield of the waste biomass.
In addition, the waste biomass straws are native lignocellulose and not only comprise lignin, but also comprise cellulose and hemicellulose, and the three are tightly combined and connected; the native lignin is highly polymerized, and the lignin is more complex than pure alkali lignin when the lignin degradation bacteria act on the native lignocellulose, and the action difficulty is higher, so that appropriate culture medium components and conditions are found, and the removal of residual lignin in Fenton reaction pretreatment slag to the greatest extent is also an urgent problem to be solved.
The invention content is as follows:
in order to solve the problems of the existing Fenton reaction pretreatment technology of the waste biomass, the invention provides a method for strengthening Fenton reaction pretreatment of the waste biomass by using lignin-degrading bacteria.
The technical scheme of the invention is as follows:
a method for strengthening Fenton reaction pretreatment of waste biomass by using lignin-degrading bacteria comprises the following steps:
(1) fenton reaction pretreatment: adding FeCl into waste biomass3And H2O2(ii) a Or FeCl2And H2O2Oscillating for reaction, filtering and separating, cleaning the obtained solid until the pH value is neutral, and drying to obtain Fenton reaction pretreated waste biomass;
(2) strengthening pretreatment of lignin degrading bacteria: and (2) inoculating lignin-degrading bacteria Cupriavidus basilensis B-8 with the preservation number of CGMCC No.4240 into a sterile culture medium containing Fenton reaction pretreatment waste biomass, culturing, filtering and separating the obtained solid, cleaning and drying to obtain the waste biomass subjected to bacteria enhanced Fenton reaction pretreatment.
The waste biomass in the step (1) comprises: rice straw, corn stover, wheat straw, sugar cane bagasse, switchgrass, or the like.
Crushing the waste biomass, sieving the crushed waste biomass by a 20-100-mesh sieve, cleaning the crushed waste biomass twice by ultrapure water, and drying the crushed waste biomass at 60 ℃ to constant weight.
Step (1) Fenton reaction pretreatment: the solid-liquid ratio of the waste biomass is 1:5-1:20(g/ml), FeCl with the concentration of 0.01-0.03mol/L is added3And 0.75 to 2.25mol/L of H2O2Or FeCl of 0.01-0.03mol/L2And 0.75 to 2.25mol/L of H2O2And oscillating for 2 hours in a room temperature environment, filtering and separating, cleaning the obtained solid with ultrapure water until the pH value is neutral, and drying to constant weight to obtain the Fenton reaction pretreated waste biomass.
Step (2) lignin degrading bacteria strengthening pretreatment: inoculating lignin-degrading bacteria into a sterile culture medium containing Fenton reaction pretreatment waste biomass, culturing for 1-2 days, filtering and separating the obtained solid, cleaning for 3 times by using ultrapure water, and drying to constant weight to obtain the bacteria-enhanced Fenton reaction pretreatment waste biomass.
The culture condition of the lignin-degrading bacteria in the step (2) is that the inoculation amount is 5-15% (the ratio of the volume of the transferred seed solution to the volume of the culture solution after inoculation), the temperature is 25-40 ℃, the pH is natural, and the culture time is 1-2 days.
The sterile culture medium containing Fenton reaction pretreatment waste biomass in the step (2) comprises the following components: 5-15 g/L (NH) of waste biomass after Fenton reaction treatment4)2SO4 2g/L，K2HPO4 1g/L，KH2PO4 1g/L，MgSO4·7H2O 0.2g/L，CaCl2 0.01g/L，FeSO4·7H2O 0.015g/L，MnSO4·H2O 0.01g/L。
The pretreatment method provided by the invention has the advantages that:
(1) the deep removal of lignin and hemicellulose of waste biomass pretreated by Fenton reaction can be realized by utilizing lignin-degrading bacteria for pretreatment, the structure of the waste biomass is damaged, the structure of the waste biomass becomes loose, and the accessible surface during enzymolysis and saccharification is increased.
(2) When the chemical reaction conditions are mild, lignin is difficult to remove completely, so that the sugar purity is reduced, and subsequent enzymolysis and biotransformation of cellulose are hindered. If the conditions of high strength are changed, the excessive damage and loss of cellulose are easily caused, and the enzymolysis rate is also influenced. Therefore, the method improves the utilization and resource yield of the carbon source in the waste biomass by weakening the chemical pretreatment condition and simultaneously adopting the microorganisms to deeply remove the residual lignin in the slag.
(3) The native lignin in the waste biomass straws is highly polymerized, the action of lignin-degrading bacteria is more complex than that of pure alkali lignin, and the action difficulty is higher.
(4) The enzymolysis saccharification efficiency can be greatly improved, compared with the waste biomass which is not pretreated, the enzymolysis efficiency is improved by more than 3 times, and compared with the traditional Fenton reaction pretreatment, the enzymolysis saccharification efficiency is improved by about 60-120%.
(5) Has the advantages of simple operation, little secondary pollution, short treatment time, low cost and the like.
The lignin-degrading bacteria (Cupriavidus basilensis B-8) used in the invention, the preservation number of which is CGMCC No.4240, are strains which are screened by the applicant and have been subjected to patent preservation and patent application.
Description of the drawings:
FIG. 1: the sugar yield of the waste biomass after pretreatment in the examples was varied;
FIG. 2: in the examples, the components of the pretreated waste biomass were changed;
FIG. 3: in the examples, the waste biomass after pretreatment is analyzed by scanning electron microscopy before and after pretreatment, (a) is untreated rice straw, (B) is rice straw treated by single Fenton reaction, (c) and (d) is rice straw treated by reinforcement of cuprividus basilensis B-8.
Detailed Description
The invention is described in further detail below with reference to the figures and the examples, but without limiting the invention.
Example 1
(1) Crushing rice straws, sieving by a 60-mesh sieve, cleaning twice by ultrapure water, and drying at 60 ℃ to constant weight.
(2) Further placing the waste biomass in a container with proper size, adding FeCl with the concentration of 0.02mol/L according to the solid-to-liquid ratio of 1:10(g/ml)3And 1.5mol/L of H2O2And shaking for 2 hours in a room temperature environment, and filtering and separating to obtain wet residue A.
(3) Further repeatedly washing the wet residue A obtained by filtering and separating with distilled water until the pH value of the washing liquid is neutral, and drying at 60 ℃ to constant weight to obtain dry residue B.
(4) Inoculating the B-8 thallus stored on the LB inclined plane into an LB liquid culture medium, and culturing at the temperature of 30 ℃ for 18h (the optical density at 600nm reaches 0.8-1.0) to obtain a B-8 seed solution; wherein the LB liquid culture medium comprises the following components in percentage by weight: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 1L of distilled water; the LB inclined plane is formed by adding 15g/L agar on the basis of the formula;
(5) centrifuging the B-8 seed solution obtained in the last step for 5 minutes at 12000rpm, removing supernatant, and collecting thalli;
(6) further inoculating the collected B-8 thallus into a dry residue B culture medium according to an inoculation amount of 10% (the ratio of the volume of the transferred seed solution to the volume of the culture solution after inoculation), culturing for 2 days at a temperature of 30 ℃ and natural pH, and filtering and separating to obtain wet residue C; wherein the dry residue B culture medium comprises the following components in percentage by weight: dry slag B10.0g, K2HPO4 1.0g，(NH4)2SO4 2.0g，KH2PO4 1.0g，MgSO4 0.2g，CaCl2 0.01g，FeSO4·7H2O 0.015g，MnSO4·H20.01g of O, and 1L of distilled water;
(7) further repeatedly washing the wet residue C obtained by filtering and separating with distilled water, and drying at 60 ℃ to constant weight to obtain dry residue D;
(8) adding 50mM citric acid buffer solution with pH of 4.8 and cellulase 12PFU/g dry weight of rice straw into dry residue D according to a solid-to-liquid ratio of 1:40(g/ml), and performing enzymolysis for 24h at 50 ℃ to obtain sugar.
Through the implementation of the embodiment, the content of each component of the rice straw is changed remarkably, the Fenton reaction generates high-reactivity hydroxyl radicals to attack the waste biomass, so that the inner part of a cellulose chain is cracked and lignin is depolymerized, the content of hemicellulose is increased, and then the lignin and the hemicellulose are further removed through bacterial strengthening treatment (as shown in figure 2). The lignin and the hemicellulose are removed, so that the structure of the rice straw is easier to be saccharified by enzymolysis; as shown in a scanning electron microscope picture (as shown in figure 3), a straight rod-shaped structure before pretreatment disappears, the surface becomes quite irregular, loose and porous, the subsequent enzymolysis efficiency can be improved, the enzymolysis efficiency after the bacteria reinforced Fenton reaction pretreatment is improved by 5.5 times of that of the untreated bacteria, the reducing sugar content reaches 12.6g/L, and the single Fenton reaction pretreatment under the condition of the invention is improved by nearly one time (as shown in figure 1), and the effect is obvious.
Example 2
(1) Crushing rice straws, sieving by a 60-mesh sieve, cleaning twice by ultrapure water, and drying at 60 ℃ to constant weight.
(2) Further placing the waste biomass in a suitably sized containerFeCl with the concentration of 0.03mol/L is added according to the solid-to-liquid ratio of 1:10(g/ml)3And 2.25mol/L of H2O2And shaking for 2 hours in a room temperature environment, and filtering and separating to obtain wet residue A.
(3) Further repeatedly washing the wet residue A obtained by filtering and separating with distilled water until the pH value of the washing liquid is neutral, and drying at 60 ℃ to constant weight to obtain dry residue B.
(4) Inoculating the B-8 thallus stored on the LB inclined plane into an LB liquid culture medium, and culturing at the temperature of 30 ℃ for 18h (the optical density at 600nm reaches 0.8-1.0) to obtain a B-8 seed solution; wherein the LB liquid culture medium comprises the following components in percentage by weight: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 1L of distilled water; the LB inclined plane is formed by adding 15g/L agar on the basis of the formula;
(5) centrifuging the B-8 seed solution obtained in the last step for 5 minutes at 12000rpm, removing supernatant, and collecting thalli;
(6) further inoculating the collected B-8 thallus into a dry residue B culture medium according to an inoculation amount of 10% (the ratio of the volume of the transferred seed solution to the volume of the culture solution after inoculation), culturing for 2 days at a temperature of 30 ℃ and natural pH, and filtering and separating to obtain wet residue C; wherein the dry residue B culture medium comprises the following components in percentage by weight: dry slag B10.0g, K2HPO4 1.0g，(NH4)2SO4 2.0g，KH2PO41.0g，MgSO4 0.2g，CaCl2 0.01g，FeSO4·7H2O 0.015g，MnSO4·H20.01g of O, and 1L of distilled water;
(7) further repeatedly washing the wet residue C obtained by filtering and separating with distilled water, and drying at 60 ℃ to constant weight to obtain dry residue D;
(8) adding 50mM citric acid buffer solution with pH of 4.8 and cellulase 12PFU/g dry weight of rice straw into dry residue D according to a solid-to-liquid ratio of 1:40(g/ml), and performing enzymolysis for 24h at 50 ℃ to obtain sugar.
The content of reducing sugar in the rice straw enzymatic hydrolysate subjected to the bacteria strengthening pretreatment is improved from untreated 2.3g/L to 9.43g/L, the enzymatic hydrolysis efficiency is improved by 4.1 times (as shown in figure 1), and the enzymatic hydrolysis efficiency is improved by about 45% compared with that of the rice straw enzymatic hydrolysate subjected to the Fenton reaction pretreatment alone under the condition of the invention.
Example 3
(1) Crushing rice straws, sieving by a 60-mesh sieve, cleaning twice by ultrapure water, and drying at 60 ℃ to constant weight.
(2) Further placing the waste biomass in a container with proper size, adding FeCl with the concentration of 0.01mol/L according to the solid-to-liquid ratio of 1:10(g/ml)3And 0.75mol/L of H2O2And shaking for 2 hours in a room temperature environment, and filtering and separating to obtain wet residue A.
(3) Further repeatedly washing the wet residue A obtained by filtering and separating with distilled water until the pH value of the washing liquid is neutral, and drying at 60 ℃ to constant weight to obtain dry residue B.
(4) Inoculating the B-8 thallus stored on the LB inclined plane into an LB liquid culture medium, and culturing at the temperature of 30 ℃ for 18h (the optical density at 600nm reaches 0.8-1.0) to obtain a B-8 seed solution; wherein the LB liquid culture medium comprises the following components in percentage by weight: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 1L of distilled water; the LB inclined plane is formed by adding 15g/L agar on the basis of the formula;
(5) centrifuging the B-8 seed solution obtained in the last step for 5 minutes at 12000rpm, removing supernatant, and collecting thalli;
(6) further inoculating the collected B-8 thallus into a dry residue B culture medium according to an inoculation amount of 10% (the ratio of the volume of the transferred seed solution to the volume of the culture solution after inoculation), culturing for 2 days at a temperature of 30 ℃ and natural pH, and filtering and separating to obtain wet residue C; wherein the dry residue B culture medium comprises the following components in percentage by weight: dry slag B10.0g, K2HPO4 1.0g，(NH4)2SO4 2.0g，KH2PO41.0g，MgSO4 0.2g，CaCl2 0.01g，FeSO4·7H2O 0.015g，MnSO4·H20.01g of O, and 1L of distilled water;
(7) further repeatedly washing the wet residue C obtained by filtering and separating with distilled water, and drying at 60 ℃ to constant weight to obtain dry residue D;
(8) adding 50mM citric acid buffer solution with pH of 4.8 and cellulase 12PFU/g dry weight of rice straw into dry residue D according to a solid-to-liquid ratio of 1:40(g/ml), and performing enzymolysis for 24h at 50 ℃ to obtain sugar.
The content of reducing sugar in the rice straw enzymatic hydrolysate subjected to the bacteria strengthening pretreatment is improved from untreated 2.3g/L to 7.84g/L, the enzymatic hydrolysis efficiency is improved by 2.4 times (as shown in figure 1), and the content is improved by about 58% compared with that of the rice straw enzymatic hydrolysate subjected to the Fenton reaction pretreatment alone under the condition of the invention.
Comparative example 1
The lignocellulose pretreatment method adopted in the comparative example only comprises a high-concentration Fenton reaction pretreatment process, and the specific steps are as follows:
(1) crushing rice straws, sieving by a 60-mesh sieve, cleaning twice by ultrapure water, and drying at 60 ℃ to constant weight.
(2) Further placing the waste biomass in a container with proper size, adding FeCl with the concentration of 0.04mol/L according to the solid-to-liquid ratio of 1:10(g/ml)3And 3mol/L of H2O2And shaking for 2 hours in a room temperature environment, and filtering and separating to obtain wet residue A.
(3) Repeatedly washing the wet residue A obtained by filtering and separating with distilled water until the pH of the washing liquid is neutral, and drying at 60 ℃ to constant weight to obtain dry residue B;
(4) adding 50mM citric acid buffer solution with pH of 4.8 and cellulase 12PFU/g dry weight of rice straw into the dry residue B according to a solid-to-liquid ratio of 1:40(g/ml), and carrying out enzymolysis for 24h at 50 ℃ to obtain the sugar.
FeCl in comparative example 13And H2O2The concentrations of the components are all higher than those of examples 1-3, however, the content of reducing sugar in the rice straw enzymatic hydrolysate pretreated by the comparative example is 7.1g/L, which is lower than that of the examples, and the high-concentration pretreatment effect is not as good as the bacteria strengthening pretreatment under the low-concentration Fenton reaction condition.
Comparative example 2
The lignocellulose pretreatment method adopted in the comparative example only comprises a single Fenton reaction pretreatment process under the conditions of the invention, and the specific steps are as follows:
(1) crushing rice straws, sieving by a 60-mesh sieve, cleaning twice by ultrapure water, and drying at 60 ℃ to constant weight.
(2) Further placing the waste biomass in a container with proper size according to solid-liquid ratioFeCl with a concentration of 0.01mol/L is added in a ratio of 1:10(g/ml)3And 0.75mol/L of H2O2And shaking for 2 hours in a room temperature environment, and filtering and separating to obtain wet residue A.
(3) Repeatedly washing the wet residue A obtained by filtering and separating with distilled water until the pH of the washing liquid is neutral, and drying at 60 ℃ to constant weight to obtain dry residue B;
(4) adding 50mM citric acid buffer solution with pH of 4.8 and cellulase 12PFU/g dry weight of rice straw into the dry residue B according to a solid-to-liquid ratio of 1:40(g/ml), and carrying out enzymolysis for 24h at 50 ℃ to obtain the sugar.
The content of reducing sugar in the rice straw enzymatic hydrolysate pretreated under the mild Fenton chemical conditions in the comparative example is 4.9g/L, which is lower than that in the example, and the content of lignin (15%) after treatment is higher than that in the example (10-13%), so that the sugar purity is reduced, and the subsequent enzymolysis and biotransformation of cellulose are hindered.
Comparative example 3
The lignocellulose pretreatment method adopted in the comparative example only comprises the pretreatment process of the bacterial cupriavidius basilensis B-8, and the specific steps are as follows:
(1) crushing rice straws, sieving by a 60-mesh sieve, cleaning twice by ultrapure water, and drying at 60 ℃ to constant weight.
(2) Inoculating Cupriavidus basilenus B-8 thallus stored on an LB inclined plane into an LB liquid culture medium, and culturing at the temperature of 30 ℃ for 18h (the optical density at 600nm reaches 0.8-1.0) to obtain a seed solution of Cupriavidus basilenus B-8; wherein the LB liquid culture medium comprises the following components in percentage by weight: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 1L of distilled water; the LB inclined plane is formed by adding 15g/L agar on the basis of the formula;
(3) centrifuging the seed solution of Cupriavidus basilensis B-8 obtained in the last step for 5 minutes at 12000rpm, discarding the supernatant, and collecting the thallus;
(4) inoculating the collected Cupriavidus basilensis B-8 thalli into a rice straw culture medium according to the inoculation amount of 20% (the ratio of the volume of the inoculated seed solution to the volume of the culture solution after inoculation), culturing for 3 days at the temperature of 30 ℃ and natural pH, and filtering and separating to obtain wet residues; wherein the rice isThe straw culture medium comprises the following components in percentage by weight: dry slag B10.0g, K2HPO4 1.0g，(NH4)2SO42.0g，KH2PO4 1.0g，MgSO4 0.2g，CaCl2 0.01g，FeSO4·7H2O 0.015g，MnSO4·H20.01g of O, and 1L of distilled water;
(5) repeatedly washing the wet residue obtained by filtering and separating with distilled water, and drying at 60 ℃ to constant weight to obtain dry residue;
(6) adding 50mM citric acid buffer solution with pH of 4.8 and cellulase 12PFU/g dry weight of rice straw into dry residue according to a solid-to-liquid ratio of 1:40(g/ml), and performing enzymolysis at 50 ℃ for 24h to obtain sugar.
The yield of reducing sugar of the rice straw pretreated by the comparative example is about 2.4g/L, which is slightly higher than that of untreated rice straw (2.3g/L), but is far lower than that of the example. Which indicates that the pretreatment by Cupriavidus basilensis B-8 alone can not achieve the ideal effect. On the basis of Fenton pretreatment, the invention produces unexpected effects through the action of Cupriavidus basilensis B-8.
Comparative example 4
The lignocellulose pretreatment method adopted in the comparative example is a pretreatment process of strengthening high-concentration Fenton reaction by using a bacterium Cupriavidus basilensis B-8, and the specific steps are as follows:
(1) crushing rice straws, sieving by a 60-mesh sieve, cleaning twice by ultrapure water, and drying at 60 ℃ to constant weight.
(2) Further placing the waste biomass in a container with proper size, adding FeCl with the concentration of 0.04mol/L according to the solid-to-liquid ratio of 1:10(g/ml)3And 3mol/L of H2O2And shaking for 2 hours in a room temperature environment, and filtering and separating to obtain wet residue A.
(3) Repeatedly washing the wet residue A obtained by filtering and separating with distilled water until the pH of the washing liquid is neutral, and drying at 60 ℃ to constant weight to obtain dry residue B.
(4) Inoculating the Cupriavidus basilenus B-8 thallus stored on an LB inclined plane into an LB liquid culture medium, and culturing at the temperature of 30 ℃ for 18h to obtain a seed solution of the Cupriavidus basilenus B-8; wherein the LB liquid culture medium comprises the following components in percentage by weight: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 1L of distilled water; the LB inclined plane is formed by adding 15g/L agar on the basis of the formula;
(5) centrifuging the seed solution of Cupriavidus basilensis B-8 obtained in the last step for 5 minutes at 12000rpm, discarding the supernatant, and collecting the thallus;
(6) inoculating the collected Cupriavidus basilensis B-8 thalli into a dry residue B culture medium according to the inoculation amount of 10% (the ratio of the volume of the transferred seed solution to the volume of a culture solution after inoculation), culturing for 2 days at the temperature of 30 ℃ and natural pH, and filtering and separating to obtain wet residue C; wherein the rice straw culture medium comprises the following components in percentage by weight: dry slag B10.0g, K2HPO4 1.0g，(NH4)2SO4 2.0g，KH2PO4 1.0g，MgSO4 0.2g，CaCl2 0.01g，FeSO4·7H2O 0.015g，MnSO4·H20.01g of O, and 1L of distilled water;
(7) repeatedly washing, filtering and separating the wet residue C by using distilled water, and drying the wet residue C at the temperature of 60 ℃ to constant weight to obtain dry residue D;
(8) adding 50mM citric acid buffer solution with pH of 4.8 and cellulase 12PFU/g dry weight of rice straw into dry residue D according to a solid-to-liquid ratio of 1:40(g/ml), and performing enzymolysis for 24h at 50 ℃ to obtain sugar.
The content of reducing sugar in the rice straw enzymatic hydrolysate subjected to the bacteria strengthening pretreatment is 7.7g/L, which is still not as high as the yield (8.4-10.3 g/L) of the reducing sugar subjected to the bacteria strengthening pretreatment under the mild and low-concentration conditions, so that the excessive damage and loss of cellulose are easily caused under the high-strength conditions, and the effect of subsequent microbial treatment is influenced.
Comparative example 5
The lignocellulose pretreatment method adopted in the comparative example is the same as the process in the example, only most of the components of the culture medium in the step (6) in the example are changed to be the same as the patent (application No. 201610569477.0), the dry residue B10.0g/L prepared in the example of the invention is used for replacing 1-6g of alkali lignin, and the other components are as follows: (NH)4)2SO4 0.28g，K2HPO4 1g，MgSO40.2g，CaCl20.1g，FeSO40.05g，MnSO40.02g，KH2PO41g, 1000mL of distilled water. As a result, it was found that the bacterial biomass was significantly reduced by using the culture medium conditions of the patent (application No. 201610569477.0), and the enzymatic hydrolysis effect of the rice straw after pretreatment was only about 1/2 in the example. Comparison shows that the culture medium components and the method suitable for the waste biomass pretreatment are obtained through screening of the culture medium components and conditions.
Claims (3)
1. A method for strengthening Fenton reaction pretreatment of waste biomass by using lignin-degrading bacteria is characterized by comprising the following steps:
(1) fenton reaction pretreatment:
the solid-liquid ratio of the waste biomass is 1g to 5-20ml, FeCl with the concentration of 0.01-0.03mol/L is added3And 0.75 to 2.25mol/L of H2O2Oscillating for 2 hours in a room temperature environment, filtering and separating, cleaning the obtained solid with ultrapure water until the pH value is neutral, and drying to constant weight to obtain Fenton reaction pretreated waste biomass; the waste biomass is rice straw,
(2) strengthening pretreatment of lignin degrading bacteria: inoculating lignin-degrading bacteria Cupriavidus basilensis B-8 with the preservation number of CGMCC No.4240 into a sterile culture medium containing Fenton reaction pretreatment waste biomass, filtering and separating the obtained solid after culture, cleaning and drying to obtain the waste biomass subjected to bacteria enhanced Fenton reaction pretreatment;
the sterile culture medium containing Fenton reaction pretreatment waste biomass in the step (2) comprises the following components: 5-15 g/L (NH) of waste biomass after Fenton reaction treatment4)2SO4 2g/L，K2HPO4 1g/L，KH2PO4 1g/L， MgSO4·7H2O 0.2g/L，CaCl2 0.01g/L，FeSO4·7H2O 0.015g/L，MnSO4·H2O 0.01g/L;
The culture conditions of the lignin degrading bacteria in the step (2) are that the inoculation amount is 5-15%, the temperature is 25-40 ℃, the natural pH condition is adopted, and the culture time is 1-2 days.
2. The method for strengthening Fenton reaction pretreatment of waste biomass by using lignin-degrading bacteria according to claim 1, wherein in the step (1), the waste biomass is crushed, sieved by a 20-100 mesh sieve, washed twice by ultrapure water and dried at 60 ℃ to constant weight.
3. The method for strengthening Fenton reaction pretreatment of waste biomass by using lignin-degrading bacteria according to claim 1, wherein the step (2) of strengthening pretreatment of lignin-degrading bacteria comprises: inoculating lignin-degrading bacteria into a sterile culture medium containing Fenton reaction pretreatment waste biomass, culturing for 1-2 days, filtering and separating the obtained solid, cleaning for 3 times by using ultrapure water, and drying to constant weight to obtain the bacteria-enhanced Fenton reaction pretreatment waste biomass.
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