CN113181682A - Application of chitin nanofiber-chitosan composite gel microspheres in removal of furan aldehyde stress factors - Google Patents

Application of chitin nanofiber-chitosan composite gel microspheres in removal of furan aldehyde stress factors Download PDF

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CN113181682A
CN113181682A CN202110664234.6A CN202110664234A CN113181682A CN 113181682 A CN113181682 A CN 113181682A CN 202110664234 A CN202110664234 A CN 202110664234A CN 113181682 A CN113181682 A CN 113181682A
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composite gel
gel microspheres
chitosan
chitin
hydrolysate
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郑兆娟
孙慧敏
欧阳嘉
范一民
刘亮
刘青
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Nanjing Forestry University
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    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
    • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof

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Abstract

The invention discloses an application of chitin nanofiber-chitosan composite gel microspheres in removal of furan aldehyde stress factors. Wherein the furan aldehyde stress factor is furfural and/or 5-hydroxymethylfurfural. The chitin nano-fiber and chitosan composite gel microspheres are simple to prepare, special or expensive equipment and reagents are not needed, meanwhile, the composite gel microspheres have high efficiency of adsorbing furan aldehyde stress factors, and the microbial metabolism performance is obviously enhanced when hydrolysate treated by the composite gel microspheres is used for microbial growth or fermentation.

Description

Application of chitin nanofiber-chitosan composite gel microspheres in removal of furan aldehyde stress factors
Technical Field
The invention relates to the technical field of bioengineering, in particular to application of chitin nanofiber-chitosan composite gel microspheres in removal of furan aldehyde stress factors.
Background
Lignocellulose is the most abundant renewable resource on earth and is the only renewable organic carbon resource. The high-efficiency utilization of the lignocellulose has important significance for relieving global energy crisis, reducing greenhouse effect and environmental pollution and realizing green sustainable development of human society. Lignocellulose is generally composed of cellulose, hemicellulose and lignin, which are bound to one another by covalent or non-covalent bonds. But due to the complexity of the structure, the microorganism can be effectively utilized only through a series of pretreatment processes. The pretreatment process can remove lignin in the cellulose, hydrolyze hemicellulose, and simultaneously reduce the crystallinity of the cellulose to loosen the structure of the raw material, thereby improving the enzymolysis efficiency of the cellulase. Meanwhile, pretreatment can cause a series of complex physicochemical reactions of the wood fiber raw material, inevitably generates various stress factors such as furan aldehyde and the like, and seriously hinders the growth of microorganisms and the subsequent fermentation process.
In order to remove stress factors from lignocellulosic hydrolysates, researchers have developed a variety of physical, chemical and biological methods. The physical method is to directly remove toxic substances from the hydrolysate, and the chemical and biological methods are to convert toxic substances into non-toxic substances. The step of removing the stress factor inevitably increases the biorefinery cost, makes the process more complicated, and simultaneously causes the loss of part of the fermentable sugar, so the step has the advantages of simple process, low cost, capability of specifically removing the target stress factor and little influence on the content of the fermentable sugar. Therefore, the invention provides the application of the chitin nano-fiber-chitosan composite gel microspheres in removing furan aldehyde stress factors.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of the prior art and provides an application of chitin nanofiber (PD-NCh)/Chitosan (CS) composite gel microspheres in removal of furan aldehyde stress factors.
The invention idea is as follows: chitin is a natural polysaccharide derived from waste biomasses such as crab shells, shrimp shells, diatoms, squid parietal bones, tubificas and the like, deacetylation products of the chitin are called chitosan, the chitosan is different from the chitosan in functional groups at the C2 position, and the chitin and the chitosan are respectively replaced by an acetamido group and an amino group at the C2 position. The invention uses the waste crab shells rich in chitin with low price as raw materials, prepares the chitin nano-fiber and chitosan composite gel microspheres by a simple process, and applies the chitin nano-fiber and chitosan composite gel microspheres to remove furan aldehyde stress factors in lignocellulose hydrolysate, thereby enhancing the growth and metabolic performance of subsequent microorganisms in the pretreated hydrolysate.
In order to solve the technical problems, the invention discloses application of chitin nanofiber-chitosan composite gel microspheres in removal of furan aldehyde stress factors in lignocellulose hydrolysate.
Wherein the furan aldehyde stress factor is furfural and/or 5-hydroxymethylfurfural.
Preferably, the chitin nano-fiber-chitosan composite gel microspheres are used for removing furan aldehyde stress factors and acetic acid from lignocellulose hydrolysate.
Specifically, the chitin nano-fiber-chitosan composite gel microspheres are added into lignocellulose hydrolysate for incubation; preferably, the incubation temperature is 15-60 ℃, preferably 25-45 ℃; preferably, the incubation is performed at a speed of 0-300rpm, preferably 50-200 rpm; preferably, the incubation time is 0-12 h.
Wherein the lignocellulose hydrolysate comprises but is not limited to bagasse hydrolysate.
Wherein, the lignocellulose hydrolysate is treated by chitin nano-fiber-chitosan composite gel microspheres, and then microorganisms are added for culture; preferably, the lignocellulose hydrolysate is treated by chitin nano-fiber-chitosan composite gel microspheres, and then microorganisms and nutrient substances except carbon sources required by the growth of the microorganisms are added for culture.
Wherein the microorganism is pseudomonas putida.
Wherein, the microorganism is inoculated to the lignocellulose hydrolysate treated by the chitin nano-fiber-chitosan composite gel microspheres according to the volume ratio of 0.5-3.5%; preferably, the microorganisms are inoculated to the lignocellulose hydrolysate treated by the chitin nano-fiber-chitosan composite gel microspheres according to the volume ratio of 2%.
Wherein the nutrients include, but are not limited to, yeast extract and tryptone; preferably, the final concentration of the yeast extract is 1-9g/L, preferably 4-6g/L, and more preferably 5 g/L; preferably, the final concentration of the tryptone is 5-15g/L, preferably 8-12g/L, and more preferably 10 g/L.
Wherein the temperature of the culture is 25-35 ℃, preferably 30 ℃.
Wherein the rotation speed of the culture is 100-400 rpm.
Wherein the culture time is 1-36 h.
Wherein the deacetylation degree of the chitin nano-fibers is 25-35%, and preferably 32.31%.
Wherein the deacetylation degree of the chitosan is 55% or more, preferably 85%.
The chitin nanofiber and the chitosan can be directly purchased in the market, or can be prepared from waste crab shells according to the following method.
The method for preparing chitosan from waste crab shells comprises the following steps: cutting waste crab shell, soaking in 1M NaOH solution for 12-24 hr, washing with distilled water to neutrality, soaking in 1M HCI solution for 12-24 hr, repeating the above acid-base treatment for 3-5 times to remove minerals and proteins, and washing with distilled water to neutrality. Soaking the washed materials in 5-10g/L NaClO2Heating and decolorizing at 70-80 deg.C for 2-3 hr, washing with distilled water to neutrality, and pulverizing with juicer to obtain chitin material. Soaking chitin material in 35-55% (w/w) NaOH solution, water bathing at 90-120 deg.C and 600-800rpm for 3-6 hr, centrifuging to collect precipitate, washing with distilled water to neutrality, and vacuum drying to deacetylate the materialWhen the content is more than 55 percent, the chitosan is obtained.
The method for preparing the chitin nano-fibers from the waste crab shells comprises the following steps: soaking the prepared chitin raw material in 30-35% (w/w) NaOH solution, performing water bath at 90 deg.C and 600rpm for 2-3h, centrifuging, collecting precipitate, and washing with distilled water to neutrality to obtain partially deacetylated chitin (DEChN) with deacetylation degree of 25-35%. Uniformly dispersing DEChN in 0.6-1% (v/v) acetic acid solution under magnetic stirring, adjusting pH to about 3.0 with acetic acid after the solution is uniformly dispersed, placing into a juicer, pulverizing to homogenate state, and preparing into chitin nanofiber with a high-pressure homogenizer.
In the composite gel microsphere, the mass ratio of the chitin nano-fibers to the chitosan is (0.5: 9.5) - (4: 6), and preferably (1: 9) - (3: 7).
Wherein, the preparation method of the chitin nano-fiber-chitosan composite gel microspheres is as follows: liu Liang, Lu Chan He, Jiang Jie, etc. preparation and characterization of chitin nanofiber/chitosan composite aerogel microspheres [ J ]. Nanjing university of industry, school newspaper (Nature science edition), 2016 (2016. 02):51-55.
Preferably, the preparation method of the chitin nanofiber-chitosan composite gel microspheres comprises the following steps:
(1) preparing mixed solution of chitin nano fiber and chitosan with the total volume fraction of 5-20 g/L;
(2) and (2) uniformly dripping the mixed solution prepared in the step (1) into 15-30g/L sodium tripolyphosphate solution, standing, and preparing the composite gel microspheres.
In the step (2), the volume ratio of the mixed solution to the sodium tripolyphosphate solution is 1: (3-7); preferably, the volume ratio of the mixed solution to the sodium tripolyphosphate solution is 1: 5.
in the step (2), standing for 0.5-2 h.
In the step (2), 0.1-0.2M K is used after the composite gel microspheres are prepared2HPO4The solution is washed 2-5 times and stored in distilled water and kept in a refrigerator at 4 ℃ for standby.
Wherein the mass ratio of the total mass of the chitin nanofibers and the chitosan to the furan aldehyde stress factors in the composite gel microspheres is 1: (0.5-1.7), preferably 1: (0.7-1.5).
Has the advantages that: compared with the prior art, the invention has the following advantages,
(1) the waste crab shells rich in chitin in nature have wide sources and low price.
(2) The chitin nano-fiber and chitosan composite gel microspheres are simple to prepare, and special or expensive equipment and reagents are not needed.
(3) The chitosan nanofiber and chitosan composite gel microspheres have high efficiency of adsorbing furan aldehyde stress factors.
(4) The chitin nano-fiber and chitosan composite gel microspheres are easy to separate from the hydrolysate, and secondary pollution is avoided.
(5) When the hydrolysate treated by the chitin nano-fibers and the chitosan composite gel microspheres is used for microbial growth or fermentation, the microbial metabolic performance is obviously enhanced.
(6) The invention has the advantages of low cost of raw materials, simple process operation and remarkable adsorption effect.
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The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
FIG. 1 shows the difference in growth of Pseudomonas putida when the Pseudomonas putida is cultured with the treated simulated hydrolysate and the untreated simulated hydrolysate of composite gel microspheres prepared from waste crab shells.
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
In the following examples, the solvent is deionized water unless otherwise specified.
The detection method of the contents of glucose, xylose, furfural and 5-Hydroxymethylfurfural (HMF) in the following examples is as follows: by Agilent 1260 type liquid chromatograph, Bio-Rad Aminex HPX-87H column (7.8 mm. times.300 mm); the mobile phase was 5mM H2SO4(ii) a The flow rate is 0.6 mL/min; the temperature of the chromatographic column is 55 ℃; the sample volume is 10 mu L; the detector is a differential refractive detector.
The Pseudomonas putida described in the examples below was Pseudomonas putida ATCC 47054.
Example 1: preparation of chitosan
Cutting waste crab shell, soaking in 1M NaOH solution for 12 hr, washing with distilled water to neutrality, soaking in 1M HCI solution for 12 hr, repeating the above steps for 3 times to remove minerals and proteins, and washing with distilled water to neutrality. Soaking the washed material in 5g/L NaClO2And (3) performing heating decolorization treatment for 2h at 70 ℃ in the solution, washing the solution to be neutral by using distilled water after the treatment, and crushing the solution by using a juicer to obtain the chitin raw material.
Soaking chitin raw material in 45% (w/w) NaOH solution, water-bathing at 100 deg.C and 650rpm for 5h, centrifuging, collecting precipitate, washing with distilled water to neutrality, and vacuum drying until the deacetylation degree of raw material reaches 85% to obtain chitosan.
Example 2: preparation of chitin nanofibers (PD-NCh)
The chitin material obtained in example 1 was soaked in 35% (w/w) NaOH solution, water-washed at 90 ℃ and 600rpm for 3h, and then the precipitate was collected by centrifugation and washed with distilled water to neutrality, at which point the deacetylation degree of the material reached 32.31% and the water content was 78.53%, which was partially deacetylated chitin (DEChN).
Under magnetic stirring, adding 2.5g of DEChN in dry weight into 500mL of 1% (v/v) acetic acid solution, adjusting pH to 3.0 with acetic acid, placing into a juicer after the solution is uniformly dispersed, pulverizing to homogenate state, and preparing into chitin nanofiber with concentration of 5g/L by using a high-pressure homogenizer. Concentrating 5g/L chitin nanofiber at 60 deg.C and 60rpm with rotary evaporator, and measuring the concentration of the concentrated chitin nanofiber to 8.5g/L by constant weight method.
Example 3: preparing chitin nano-fiber and chitosan (PD-NCh/CS) composite gel microspheres
Based on example 1 and example 2, 100mL of a total volume fraction of 20g/L and a mass ratio PD-NCh/CS of 1: 9, and compounding the gel microspheres with the compound liquid. The method comprises the following steps: weighing PD-NCh with the dry weight of 0.2g, adding 1% (v/v) acetic acid under magnetic stirring to fix the volume to 100mL, adding 1.8g of chitosan into a water bath at 50 ℃ while stirring for dissolving after the nano-fibers are uniformly dispersed, placing the mixture on a magnetic stirring table after the chitosan is fully dissolved, stirring for 2h until the composite liquid is in a uniform state, and finally placing the mixture under an ultraviolet lamp for irradiating for 12 h.
Before preparing the microspheres, centrifuging the composite solution at 4000rpm to remove bubbles, pouring the mixed solution into a syringe, slowly dripping 5mL of the composite solution into 25mL of 20g/L sodium tripolyphosphate solution by using the action of gravity, keeping a constant liquid level distance, standing for 1h in an ice bath after the solution is completely dripped into the sodium tripolyphosphate, and precipitating the microspheres from a transparent flat shape to the bottom of the bottle to form milky spheres in the process. The sodium tripolyphosphate solution was filtered off with gauze and washed with 0.15M K2HPO4Washing the immobilized microspheres twice with the solution, and finally filtering off K2HPO4The solution was stored in distilled water and placed in a refrigerator at 4 ℃ for subsequent experiments.
Example 4: preparing chitin nano-fiber and chitosan (PD-NCh/CS) composite gel microspheres
On the basis of example 1 and example 2, 100mL of a total volume fraction of 20g/L and a mass ratio PD-NCh/CS of 2: 8, the composite gel microsphere composite liquid. The method comprises the following steps: weighing PD-NCh with the dry weight of 0.4g, adding 1% (v/v) acetic acid under magnetic stirring to fix the volume to 100mL, adding 1.6g chitosan into a water bath at 50 ℃ while stirring for dissolving after the nano-fiber is uniformly dispersed, placing the mixture on a magnetic stirring table after the chitosan is fully dissolved, stirring for 2h until the composite liquid is in a uniform state, and finally placing the mixture under an ultraviolet lamp for irradiating for 12 h.
Before preparing the microspheres, the composite solution is centrifuged at 4000rpm to remove bubbles, the mixed solution is poured into a syringe, and 5mL of the composite solution is slowly dropped into 25mL of 20 g/L-concentration tris (hydroxymethyl) phosphonium chloride under the action of gravityAnd (3) keeping a constant liquid level distance in the sodium polyphosphate solution, after the solution is completely dripped into sodium tripolyphosphate, standing for 1h in an ice bath, and in the process, depositing the microspheres from a transparent flat shape to the bottom of the bottle to form milky spheres. The sodium tripolyphosphate solution was filtered off with gauze and washed with 0.15M K2HPO4Washing the immobilized microspheres twice with the solution, and finally filtering off K2HPO4The solution was stored in distilled water and placed in a refrigerator at 4 ℃ for subsequent experiments.
Example 5: preparing chitin nano-fiber and chitosan (PD-NCh/CS) composite gel microspheres
Based on example 1 and example 2, 100mL of a total volume fraction of 20g/L and a mass ratio PD-NCh/CS of 3: 7, the composite gel microsphere composite liquid. The method comprises the following steps: weighing PD-NCh with the dry weight of 0.6g, adding 1% (v/v) acetic acid under magnetic stirring to fix the volume to 100mL, adding 1.4g chitosan into a water bath at 50 ℃ while stirring for dissolving after the nano-fiber is uniformly dispersed, placing the mixture on a magnetic stirring table after the chitosan is fully dissolved, stirring for 2h until the composite liquid is in a uniform state, and finally placing the mixture under an ultraviolet lamp for irradiating for 12 h.
Before preparing the microspheres, centrifuging the composite solution at 4000rpm to remove bubbles, pouring the mixed solution into a syringe, slowly dripping 5mL of the composite solution into 25mL of 20g/L sodium tripolyphosphate solution by using the action of gravity, keeping a constant liquid level distance, standing for 1h in an ice bath after the solution is completely dripped into the sodium tripolyphosphate, and precipitating the microspheres from a transparent flat shape to the bottom of the bottle to form milky spheres in the process. The sodium tripolyphosphate solution was filtered off with gauze and washed with 0.15M K2HPO4Washing the immobilized microspheres twice with the solution, and finally filtering off K2HPO4The solution was stored in distilled water and placed in a refrigerator at 4 ℃ for subsequent experiments.
Example 6: adsorption simulation of furan aldehyde in hydrolysate by chitin nano-fiber and chitosan composite gel microspheres
Simulated hydrolysate composition (L): 20g of xylose; 5g of glucose; 0.4g of furfural; HMF 1 g; m9 solution (Na)2HPO43.77g；KH2PO4 1.5g；NH4Cl 0.5g；NaCl 0.25g；MgSO40.24 g; 2.5mL of A9 microelement solution, which comprises the following formula: HBO3 0.3g/L；ZnCl2 0.05g/L；MnCl·4H2O 0.03g/L；CoCl2 0.2g/L；CuCl2 0.01g/L；NiCl·6H2O 0.02g/L；NaMoO4·2H2O0.03 g/L). And (3) adding the composite gel microspheres prepared in the example 3 into 15mL of simulated hydrolysate, and measuring the residual amounts of furfural and HMF in the simulated hydrolysate after reacting for 2 hours at the temperature of 30 ℃ and at the rpm of 200. The removal rate of the final composite gel microspheres to furfural and HMF is more than 98%.
Example 7: adsorption simulation of furan aldehyde in hydrolysate by chitin nano-fiber and chitosan composite gel microspheres
Simulated hydrolysate composition (L): 20g of xylose; 5g of glucose; 0.4g of furfural; HMF 1 g; m9 solution (Na)2HPO43.77g；KH2PO4 1.5g；NH4Cl 0.5g；NaCl 0.25g；MgSO40.24 g; 2.5mL of A9 microelement solution, which comprises the following formula: HBO3 0.3g/L；ZnCl2 0.05g/L；MnCl·4H2O 0.03g/L；CoCl2 0.2g/L；CuCl2 0.01g/L；NiCl·6H2O 0.02g/L；NaMoO4·2H2O0.03 g/L). And (3) adding the composite gel microspheres prepared in the example 5 into 15mL of simulated hydrolysate, and measuring the residual amounts of furfural and HMF in the simulated hydrolysate after reacting for 2 hours at the temperature of 30 ℃ and at the rpm of 200. The removal rate of the final composite gel microspheres to furfural and HMF is 90% and 80%, respectively.
Example 8: adsorption simulation of furan aldehyde in hydrolysate by chitin nano-fiber and chitosan composite gel microspheres
Simulated hydrolysate composition (L): 20g of xylose; 5g of glucose; 0.8g of furfural; HMF 1.5 g; m9 solution (Na)2HPO43.77g；KH2PO4 1.5g；NH4Cl 0.5g；NaCl 0.25g；MgSO40.24 g; 2.5mL of A9 microelement solution, which comprises the following formula: HBO3 0.3g/L；ZnCl2 0.05g/L；MnCl·4H2O 0.03g/L；CoCl2 0.2g/L；CuCl2 0.01g/L；NiCl·6H2O 0.02g/L；NaMoO4·2H2O0.03 g/L). And (3) adding the composite gel microspheres prepared in the example 3 into 15mL of simulated hydrolysate, and measuring the residual amounts of furfural and HMF in the simulated hydrolysate after reacting for 2 hours at the temperature of 30 ℃ and at the rpm of 200. The removal rate of the final composite gel microspheres to furfural and HMF is 90% and 85% respectively.
Example 9: adsorbing furan aldehyde simulation hydrolysate component (L) in the simulation liquid by the chitin nano-fiber and chitosan composite gel microspheres: 20g of xylose; 5g of glucose; 0.8g of furfural; HMF 1.5 g; m9 solution (Na)2HPO4 3.77g；KH2PO41.5g；NH4Cl 0.5g；NaCl 0.25g；MgSO40.24 g; 2.5mL of A9 microelement solution, which comprises the following formula: HBO30.3g/L；ZnCl2 0.05g/L；MnCl·4H2O 0.03g/L；CoCl2 0.2g/L；CuCl2 0.01g/L；NiCl·6H2O 0.02g/L；NaMoO4·2H2O0.03 g/L). And (3) adding the composite gel microspheres prepared in the example 5 into 15mL of simulated hydrolysate, and measuring the residual amounts of furfural and HMF in the simulated hydrolysate after reacting for 2 hours at the temperature of 30 ℃ and at the rpm of 200. The removal rate of the final composite gel microspheres to furfural and HMF is 80% and 74% respectively.
Example 10: furanal in simulated liquid adsorbed by chitin nanofiber and chitosan composite gel microspheres
Simulated hydrolysate composition (L): 20g of xylose; 5g of glucose; 1g of furfural; HMF 2 g; m9 solution (Na)2HPO4 3.77g；KH2PO4 1.5g；NH4Cl 0.5g；NaCl 0.25g；MgSO40.24 g; 2.5mL of A9 microelement solution, which comprises the following formula: HBO3 0.3g/L；ZnCl2 0.05g/L；MnCl·4H2O 0.03g/L；CoCl2 0.2g/L；CuCl2 0.01g/L；NiCl·6H2O 0.02g/L；NaMoO4·2H2O0.03 g/L). And (3) adding the composite gel microspheres prepared in the example 5 into 15mL of simulated hydrolysate, and measuring the residual amounts of furfural and HMF in the simulated hydrolysate after reacting for 2 hours at the temperature of 30 ℃ and at the rpm of 200. Removal of furfural and HMF by final composite gel microspheresThe removal rates were 70% and 68%, respectively.
Example 11: pseudomonas putida was cultured using the simulated hydrolysate treated with the composite gel microspheres and the untreated simulated hydrolysate in example 7, respectively
Activating Pseudomonas putida in LB culture medium (yeast extract 5 g/L; tryptone 10 g/L; NaCl 10g/L) at 30 deg.C and 200rpm for 12h, inoculating to the gel microsphere treated simulated solution and untreated simulated solution at transfer amount of 2% (v/v), culturing at 30 deg.C and 200rpm for 12h, sampling every 2h, and determining thallus OD600The value is obtained. As shown in figure 1, the growth of the pseudomonas putida in the simulated hydrolysate treated by the gel microspheres is obviously faster than that in the untreated simulated hydrolysate, the lag phase is only 2 hours, and the thallus OD is 12 hours later600The value reached 2.0. The amount of pseudomonas putida is not obviously increased after being cultured in untreated simulated hydrolysate for 12 hours. The experimental results show that the chitin nano-fiber and chitosan composite gel microspheres prepared based on the waste crab shells can eliminate the growth stress of furan aldehyde compounds in simulated hydrolysate on strains, and the elimination effect is very obvious.
Example 12: culturing pseudomonas putida by using bagasse hydrolysate treated by composite gel microspheres and untreated bagasse hydrolysate respectively
Preparing bagasse hydrolysate: bagasse is dried in the sun, ground and sieved, 2g of bagasse powder is weighed, mixed with 20mL of dilute sulfuric acid with the mass fraction of 0.9 percent, and treated in an oil bath at 150 ℃ for 1 h. And (3) after the oil bath is finished, performing centrifugal filtration, collecting supernatant, adjusting the pH value of the hydrolysate to 7.0 by using a 5M NaOH solution, standing in a refrigerator at 4 ℃ for 24 hours, and then centrifuging again to remove insoluble impurities, wherein the obtained supernatant is the bagasse hydrolysate. The HPLC detection shows that the composition is (g/L): 6.00 parts of glucose; 19.03 parts of xylose; 6.08 parts of acetic acid; HMF 0.28; and 1.23 of furfural.
The composite gel microspheres prepared in example 5 were added into 15mL of bagasse hydrolysate, and the residual amounts of furfural and HMF in the bagasse hydrolysate were measured after 2 hours of reaction at 30 ℃ and 200 rpm. The removal rates of the final composite gel microspheres to furfural and HMF are 80% and 83%, respectively, and 33% of acetic acid can be removed simultaneously.
Pseudomonas putida is activated in LB culture medium (yeast extract 5 g/L; tryptone 10 g/L; NaCl 10g/L) at 30 ℃ and 200rpm for 12h, and then inoculated into bagasse hydrolysate treated by gel microspheres and untreated bagasse hydrolysate in an transfer amount of 2% (v/v), and yeast extract 5g/L and tryptone 10g/L are additionally added into the hydrolysate. Culturing at 30 deg.C and 200rpm for 24h, sampling every 3h to determine the OD of the cells600The value is obtained. The growth of the pseudomonas putida in bagasse hydrolysate treated by the gel microspheres is obviously faster than that in untreated bagasse hydrolysate, and the thallus OD is cultured for 24h600The value reached 8.0. Pseudomonas putida had no sign of growth in the untreated bagasse hydrolysate. The experimental results show that the chitin nanofiber and chitosan composite gel microspheres prepared based on the waste crab shells can eliminate the growth stress of furfural compounds in bagasse hydrolysate on strains, and the elimination effect is very obvious.
The invention provides a thought and a method for application of chitin nanofiber-chitosan composite gel microspheres in removal of furan aldehyde stress factors, and a method and a way for realizing the technical scheme are numerous, the above description is only a preferred embodiment of the invention, and it should be noted that for a person skilled in the art, on the premise of not departing from the principle of the invention, a plurality of improvements and decorations can be made, and the improvements and decorations are also regarded as the protection scope of the invention. All the components not specified in the present embodiment can be realized by the prior art.

Claims (10)

1. An application of chitin nano-fiber-chitosan composite gel microspheres in removal of furan aldehyde stress factors in lignocellulose hydrolysate is characterized in that the furan aldehyde stress factors are furfural and/or 5-hydroxymethylfurfural.
2. The use of claim 1, wherein the chitin nano-fiber-chitosan composite gel microspheres are used for removing furan aldehyde stress factors and acetic acid from lignocellulose hydrolysate.
3. The use of claim 1 or 2, wherein the lignocellulose hydrolysate is treated by chitin nano-fiber-chitosan composite gel microspheres, and then cultured by adding microorganisms.
4. Use according to claim 3, wherein the microorganism is Pseudomonas putida.
5. The use of claim 3, wherein the microorganism is inoculated to the lignocellulose hydrolysate treated by the chitin nano-fiber-chitosan composite gel microspheres according to the volume ratio of 0.5-3.5%.
6. The use according to claim 3, wherein the temperature of the cultivation is 25-35 ℃.
7. The use of claim 1, wherein the chitin nanofibers have a degree of deacetylation of 25% to 35%.
8. Use according to claim 1, wherein the chitosan has a degree of deacetylation of 55% or more.
9. The use of claim 1, wherein the mass ratio of the chitin nanofibers to the chitosan in the composite gel microspheres is (0.5: 9.5) - (4: 6).
10. The use of claim 1, wherein the mass ratio of the total mass of the chitin nanofibers and chitosan to the furan aldehyde stress factors in the composite gel microspheres is 1: (0.5-1.7).
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