CN114561317A - Application method of degrading bacteria for degrading phenols and lignin - Google Patents
Application method of degrading bacteria for degrading phenols and lignin Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 34
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
- D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
- D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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Abstract
Embodiments of the present disclosure disclose degrading bacteria application methods for degrading phenols and lignin. One embodiment of the method comprises: inoculating a strain frozen in a glycerin pipe at the temperature of-80 ℃ into a solid culture medium for culturing for 14 to 16 hours in a streaking manner under the environment of the temperature of 30 ℃ to obtain first-class seeds, wherein the strain is used for degrading phenols and lignin; picking a single colony from the first-stage seed, transferring the single colony into a liquid culture medium, and culturing the single colony for 14 to 16 hours at the temperature of 30 ℃ under the environment of 150 revolutions per minute of a shaking table; adjusting the pH value of the liquid culture medium inoculated with the first-stage seeds to 7 to obtain second-stage seeds; inoculating the second-level seeds into the pretreated raw material in an inoculation amount of 3-20%, and degrading for 1-6 days in an environment with the temperature of 30-40 ℃ to obtain a target product. The mode can realize the degradation of phenols and lignin at the same time, and in addition, the degradation degree is high, the generation of solid waste is less, and the influence on the environment is small.
Description
Technical Field
The embodiment of the disclosure relates to the field of biochemical engineering, in particular to an application method of a degrading bacterium for degrading phenols and lignin.
Background
The lignocellulosic feedstock is composed mainly of cellulose, hemicellulose and lignin. Lignin is second only to cellulose in nature and is considered to be an extremely important aromatic resource in biomass energy. During the utilization of plants, lignin often needs to be removed, for example, in the process of paper pulping, in order to retain cellulose, the lignin is usually removed by a method of strong acid and strong base.
Hemicellulose of the wood fiber raw material is easy to hydrolyze into monosaccharide, and hemicellulose hydrolysate rich in pentose can be obtained after being treated by dilute acid under the environment of the temperature of 90-140 ℃ generally. While cellulose components in the wood fiber raw material are difficult to hydrolyze, and the common method is dilute acid high-temperature hydrolysis, concentrated acid hydrolysis or enzyme hydrolysis. Among them, enzymatic hydrolysis can efficiently hydrolyze cellulose to produce monosaccharides. However, the raw material needs to be pretreated before the enzymatic hydrolysis, wherein the main purposes of the pretreatment are to reduce the crystallinity of the cellulose, improve the porosity of the matrix, increase the contact area of the enzyme and the cellulose and further improve the enzymolysis efficiency. During pretreatment, the hemicellulose hydrolysate produces a number of fermentation-inhibiting byproducts, such as furfural from xylose degradation, acetic acid from hemicellulose deacetylation, and phenolics from lignin degradation. These substances will have some inhibitory effect on the growth of microorganisms, and therefore, the hydrolysate is usually detoxified before fermentation. In the detoxification of hydrolysate, various detoxification methods such as lime milk neutralization, organic solvent extraction, activated carbon adsorption, vacuum concentration and ion exchange are tried to remove fermentation inhibitors, and certain achievements are achieved in the aspects of improving the fermentation performance of hydrolysate and increasing the yield.
Biological detoxification is a detoxification method for selectively degrading inhibitors in bagasse hemicellulose diluted acid hydrolysate by using microorganisms with specific activity and reserving main carbohydrate substances in the dilute acid hydrolysate, and has attracted general attention due to the characteristics of simplicity and environmental protection. The published literature has many studies on the biodegradation of aldehydes, but reports on the simultaneous degradation of phenols and lignin in complex environments are few. Therefore, the important significance is achieved in seeking strains for degrading phenols and lignin and expanding the application scenes of the strains.
In summary, the problems of the prior art are as follows:
firstly, the simultaneous degradation of phenols and lignin in complex environments cannot be achieved by using microorganisms.
Secondly, non-biological detoxification is adopted, sugar loss is large, solid waste is large, and the influence on the environment is large.
Disclosure of Invention
This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
Some embodiments of the present disclosure propose methods of using degrading bacteria for degrading phenols and lignins to address one or more of the technical problems mentioned in the background section above. The specific method comprises the following steps: inoculating strains frozen in a glycerin pipe at the temperature of-80 ℃ into a solid culture medium for culturing for 14 to 16 hours in a streaking manner at the temperature of 30 ℃ to obtain first-grade seeds, wherein the strains are used for degrading phenols and lignin; picking a single colony from the first-stage seeds, transferring the single colony into a liquid culture medium, and culturing for 14 to 16 hours at the temperature of 30 ℃ under the environment of 150 revolutions per minute of a shaking table; adjusting the pH value of the liquid culture medium inoculated with the primary seeds to 7 to obtain secondary seeds; inoculating the secondary seeds into a pretreatment raw material in an inoculation amount of 3-20%, and degrading for 1-6 days in an environment with the temperature of 30-40 ℃ to obtain a target product, wherein the pretreatment raw material comprises phenols and/or lignin.
The present disclosure has the following beneficial effects:
firstly, phenols and lignin in complex environment are simultaneously degraded by microorganisms.
Secondly, the microbial detoxification is adopted, the degradation degree is high, the generation of solid waste is less, and the influence on the environment is small.
Drawings
Fig. 1 is a flow diagram of a degrading bacteria application method for degrading phenols and lignin according to some embodiments of the present disclosure.
Detailed Description
The word "embodiment" as used herein, is not necessarily to be construed as preferred or advantageous over other embodiments, including any embodiment illustrated as "exemplary". Performance index tests in the examples of this application, unless otherwise indicated, were performed using routine experimentation in the art. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; other test methods and techniques not specifically mentioned in the present application are those commonly employed by those of ordinary skill in the art.
The terms "substantially" and "about" are used throughout this disclosure to describe small fluctuations. For example, they may mean less than or equal to ± 5%, such as less than or equal to ± 2%, such as less than or equal to ± 1%, such as less than or equal to ± 0.5%, such as less than or equal to ± 0.2%, such as less than or equal to ± 0.1%, such as less than or equal to ± 0.05%. Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. Such range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of "1 to 5%" should be interpreted to include not only the explicitly recited values of 1% to 5%, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values, such as 2%, 3.5%, and 4%, and sub-ranges, such as 1% to 3%, 2% to 4%, and 3% to 5%, etc. This principle applies equally to ranges reciting only one numerical value. Moreover, such an interpretation applies regardless of the breadth of the range or the characteristics being described.
In this disclosure, including the claims, all conjunctions such as "comprising," including, "" carrying, "" having, "" containing, "" involving, "" containing, "and the like are to be understood as being open-ended, i.e., to mean" including but not limited to. Only the conjunctions "consisting of … …" and "consisting of … …" are closed conjunctions.
In the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present disclosure. It will be understood by those skilled in the art that the present application may be practiced without some of these specific details. In the examples, some methods, means, instruments, apparatuses, etc. known to those skilled in the art are not described in detail in order to highlight the subject matter of the present application. On the premise of no conflict, the technical features disclosed in the embodiments of the present application may be combined arbitrarily, and the obtained technical solution belongs to the content disclosed in the embodiments of the present application.
The present invention will be described in detail below with reference to the accompanying drawings.
Fig. 1 is a flow diagram 100 of a degrading bacteria application method for degrading phenols and lignins according to some embodiments of the present disclosure, wherein the method comprises the steps of:
101, in the environment of 30 ℃, streaking and inoculating the strain frozen in a glycerin pipe with the temperature of-80 ℃ into a solid culture medium for culturing for 14 to 16 hours to obtain first-grade seeds.
In some embodiments, the seed culture frozen in a-80 ℃ glycerol tube is streaked into a solid medium for 14 to 16 hours at 30 ℃ to obtain first-grade seeds. Wherein, the strain can be used for degrading phenols and lignin.
Alternatively, the above-mentioned bacterial species may be Pseudomonas putida (Pseudomonas putida). Wherein the Pseudomonas putida has a number of CCTCC (China Center for Type Culture Collection) M2020800. The code of the preservation unit of the pseudomonas putida is as follows: CCTCC (China center for type communication); the address is as follows: china, wuhan university. Whether survival is carried out: is that; the preservation date is as follows: year 2020, 11, 30; the preservation number is: m2020800; and (3) classification and naming: pseudomonas putida.
Alternatively, the components of the solid medium may include: 10 g/l of sodium chloride, 5 g/l of yeast extract powder, 10 g/l of peptone and 15 g/l of agar.
In some embodiments, single colonies from the primary seeds are transferred to liquid medium and incubated at 30 ℃ for 14 to 16 hours on a shaker at 150 rpm.
Alternatively, the components of the liquid medium may include: 10 g/l of sodium chloride, 5 g/l of yeast extract powder and 10 g/l of peptone.
And 103, adjusting the pH value of the liquid culture medium inoculated with the primary seeds to 7 to obtain secondary seeds.
In some embodiments, the ph of the liquid medium inoculated with the primary seed is adjusted to 7 to obtain the secondary seed. Wherein, the pH value of the liquid culture medium can be adjusted by sodium hydroxide solution.
And 104, inoculating the second-level seeds into the pretreated raw material in an inoculation amount of 3-20%, and degrading for 1-6 days in an environment with the temperature of 30-40 ℃ to obtain a target product.
In some embodiments, the secondary seeds are inoculated into the pretreated feedstock at an inoculum size of 3% to 20% and degraded at a temperature of 30 to 40 degrees celsius for 1 to 6 days to yield the target product. Wherein the inoculation amount may be a ratio of a volume of the secondary seed to a volume of the pretreated raw material after inoculation.
In some optional implementations of some embodiments, the inoculating the second-stage seed into the pretreated feedstock in an inoculation amount of 3% to 20%, and degrading in an environment at a temperature of 30 to 40 ℃ for 1 to 6 days to obtain the target product may include the following steps:
the first step, inoculating the secondary seeds into the pretreated raw materials for degradation treatment for 1 to 5 days at the temperature of 30 to 40 ℃.
Wherein the second seed may be inoculated in an amount of 3% to 20%.
And secondly, carrying out enzymolysis saccharification on the degraded pretreated raw material with the solid content of 15-30% for 48 hours by using cellulase to generate the target product.
Wherein the cellulase has a unit of 5 to 30 filter paper enzyme activities per gram of dry matter.
Alternatively, the pretreated feedstock may be produced by:
in a first step, a lignocellulosic feedstock is comminuted to between 1 and 25 millimeters to produce a comminuted lignocellulosic feedstock.
And secondly, carrying out impregnation treatment on the crushed lignocellulose raw material by using a dilute sulfuric acid solution with the mass fraction of 1-5% according to a solid-to-liquid ratio of 1: 1-1: 3 to obtain a first initial raw material.
And thirdly, standing the first initial raw material for 12 hours, and reacting for 30 to 60 minutes in an environment of 120 to 160 ℃ to obtain the acid pretreatment raw material.
And fourthly, adjusting the pH value of the acid pretreatment raw material to 5-7 by using a sodium hydroxide solution to obtain the pretreatment raw material.
Wherein the sodium hydroxide solution may be present in an amount of 10% by mass. The pretreated raw material may contain phenols and lignin, and the phenols are produced by degradation of the lignin.
In some optional implementations of some embodiments, the inoculating the second-stage seed into the pretreated feedstock in an inoculation amount of 3% to 20%, and degrading the pretreated feedstock in an environment with a temperature of 30 to 40 ℃ for 1 to 6 days to obtain the target product, may further include the following steps: inoculating the secondary seeds into the pretreated raw material at the temperature of 30-40 ℃ for degradation treatment for 1-3 days to generate the target product.
Wherein the second seed may be inoculated in an amount of 3% to 20%.
Optionally, the pretreated feedstock can also be produced by:
in a first step, a lignocellulosic feedstock is comminuted to between 1 and 25 millimeters to produce a comminuted lignocellulosic feedstock.
And secondly, impregnating the crushed lignocellulose raw material by using a sodium hydroxide solution with the mass fraction of 1-2% according to a solid-to-liquid ratio of 1: 8-1: 10 to obtain a second initial raw material.
And thirdly, standing the second initial raw material for 12 hours, and reacting for 60 to 90 minutes in an environment of 70 to 90 ℃ to obtain the alkali pretreatment raw material.
And step four, adjusting the pH value of the alkali pretreatment raw material to 7 by using a dilute sulfuric acid solution with the mass fraction of 10% to obtain the pretreatment raw material.
In some optional implementations of some embodiments, the inoculating the second-stage seed into the pretreated feedstock in an inoculation amount of 3% to 20%, and degrading the pretreated feedstock in an environment with a temperature of 30 to 40 ℃ for 1 to 6 days to obtain the target product, may further include the following steps: responding to the pretreatment raw material containing phenols, inoculating the secondary seeds into the pretreatment raw material to carry out degradation treatment for 3 to 6 days at the temperature of 30 to 40 ℃ so as to generate the target product.
Wherein the inoculation amount of the secondary seeds is 3-20%.
Alternatively, the concentration of phenols in the above pretreated raw material may be 100 to 400 mg/l.
The technical details are further illustrated in the following examples.
Example 1
The application method of the degrading bacteria for degrading phenols and lignin disclosed in example 1 may comprise the following steps:
first, the Pseudomonas putida frozen in a glycerin pipe at-80 ℃ is streaked and inoculated in a solid culture medium by using an inoculating loop, and cultured in an incubator at 30 ℃ for 16 hours (h) to obtain first-grade seeds.
And secondly, selecting a single colony from the primary seeds, inoculating the single colony in a liquid culture medium, culturing for 16h under the conditions of 30 ℃ and 140 revolutions per minute (rpm) of a shaking table, and adding a proper amount of sodium hydroxide (NaOH) solution to adjust the pH value (pH) to 7 so as to obtain secondary seeds.
Wherein the liquid medium is contained in a 250 milliliter (ml) triangular flask in an amount of 50 milliliters (ml).
Thirdly, crushing bagasse into 1-25 millimeters (mm) by a crusher, and storing the crushed bagasse raw material in a dry environment for later use.
And fourthly, pre-soaking the crushed bagasse raw materials with a dilute sulfuric acid solution with the mass fraction (w/w) of 5% according to the solid-to-liquid ratio of 1:1, standing for 12 hours, reacting for 30 minutes (min) in an environment with the temperature of 120 ℃ to obtain lignocellulose raw materials, and placing the lignocellulose raw materials in a refrigerator with the temperature of 4 ℃.
Fifthly, adjusting the pH of the lignocellulose raw material to 5 by using a 10% (w/w) sodium hydroxide solution, and inoculating the secondary seeds into the lignocellulose raw material.
Wherein the inoculation amount is 20% (based on the liquid amount), the temperature is 30 ℃, and the time is 1 day.
And sixthly, performing enzymolysis saccharification on the degraded lignocellulose raw material with the solid content of 30% for 48 hours by using cellulase to obtain a target product.
Wherein the unit of the cellulase is 5 filter paper enzyme activities per gram of dry matter raw materials. And analyzing a target product, wherein the cellulose enzymolysis rate is 63 percent, and the lignin degradation rate is 15 percent. Under the same conditions, the hydrolysis rate of untreated cellulose is 32%, and the lignin degradation rate is 0%.
Example 2
The application method of the degrading bacteria for degrading phenols and lignin disclosed in example 2 may comprise the following steps:
firstly, inoculating the pseudomonas putida frozen in a glycerin pipe with the temperature of minus 80 ℃ into a solid culture medium by a loop for streak inoculation, and culturing for 14h in a constant temperature incubator with the temperature of 30 ℃ to obtain first-grade seeds.
And secondly, selecting a single colony from the primary seed, inoculating the single colony in a liquid culture medium, culturing for 14 hours at the temperature of 30 ℃ and the speed of 140rpm of a shaking table, and adding a proper amount of NaOH solution to adjust the pH to 7 so as to obtain a secondary seed.
Thirdly, crushing bagasse into 1-25mm by a crusher, and storing the crushed bagasse raw material in a dry environment for later use.
And fourthly, pre-soaking the crushed bagasse raw materials with 1% (w/w) dilute sulfuric acid solution according to a solid-to-liquid ratio of 1:3, standing for 12 hours, reacting for 60min at 160 ℃ to obtain lignocellulose raw materials, and storing the lignocellulose raw materials in a refrigerator at 4 ℃.
Fifthly, adjusting the pH of the lignocellulose raw material to 7 by using a 10% (w/w) sodium hydroxide solution, and inoculating the secondary seeds into the lignocellulose raw material.
Wherein the inoculation amount is 3 percent (based on the liquid amount), the temperature is 30 ℃, and the time is 5 days.
And sixthly, performing enzymolysis saccharification on the degraded lignocellulose raw material with the solid content of 15% by using cellulase for 48 hours to obtain a target product.
Wherein the unit of the cellulase is 30 filter paper enzyme activities per gram of dry matter raw materials. And analyzing a target product, wherein the cellulose enzymolysis rate is 88 percent, and the lignin degradation rate is 24 percent. Under the same conditions, the hydrolysis rate of untreated cellulose is 80%, and the lignin degradation is 0%.
Example 3
The application method of the degrading bacteria for degrading phenols and lignin disclosed in example 3 may comprise the following steps:
firstly, inoculating the pseudomonas putida frozen in a glycerin pipe with the temperature of minus 80 ℃ into a solid culture medium by a loop for streak inoculation, and culturing for 16h in a constant temperature incubator with the temperature of 30 ℃ to obtain first-grade seeds.
And secondly, selecting a single colony from the primary seed, inoculating the single colony in a liquid culture medium, culturing for 14 hours at the temperature of 30 ℃ and the speed of 140rpm of a shaking table, and adding a proper amount of NaOH solution to adjust the pH to 7 so as to obtain a secondary seed.
Thirdly, crushing the corncobs to 1-25mm by a crusher, and storing the crushed corncob raw materials in a dry environment for later use.
And fourthly, pre-soaking the crushed corncob raw material with 1% (w/w) dilute sulfuric acid solution according to the solid-to-liquid ratio of 1:3, standing for 12 hours, reacting for 60min at the temperature of 160 ℃ to obtain a lignocellulose raw material, and storing the lignocellulose raw material in a refrigerator at the temperature of 4 ℃.
Fifthly, adjusting the pH of the lignocellulose raw material to 7 by using a 10% (w/w) sodium hydroxide solution, and inoculating the secondary seeds into the lignocellulose raw material.
Wherein the inoculation amount is 3 percent (based on the liquid amount), the temperature is 30 ℃, and the time is 5 days.
And sixthly, performing enzymolysis saccharification on the degraded lignocellulose raw material with the solid content of 30% for 48 hours by using cellulase to obtain a target product.
Wherein the unit of the cellulase is 30 filter paper enzyme activities per gram of dry matter raw materials. And analyzing the target product, wherein the cellulose enzymolysis rate is 84%, and the lignin degradation rate is 19%. Under the same conditions, the hydrolysis rate of untreated cellulose is 60%, and the lignin degradation is 0%.
Example 4
The application method of the degrading bacteria for degrading phenols and lignin disclosed in example 4 can comprise the following steps:
firstly, inoculating the pseudomonas putida frozen in a glycerin pipe with the temperature of minus 80 ℃ into a solid culture medium by a loop for streak inoculation, and culturing for 16h in a constant temperature incubator with the temperature of 30 ℃ to obtain first-grade seeds.
And secondly, selecting a single colony from the primary seed, inoculating the single colony in a liquid culture medium, culturing for 14 hours at the temperature of 30 ℃ and the speed of 140rpm of a shaking table, and adding a proper amount of NaOH solution to adjust the pH to 7 so as to obtain a secondary seed.
Thirdly, crushing the corncobs to 1-25mm by a crusher, and storing the crushed corncob raw materials in a dry environment for later use.
And fourthly, pre-soaking the ground corn cob raw material with 1% (w/w) NaOH solution according to the solid-to-liquid ratio of 1:10, standing for 12 hours, and reacting for 90min at the temperature of 90 ℃ to obtain the lignocellulose raw material.
And fifthly, carrying out vacuum filtration on the lignocellulose raw material through a vacuum filtration device, and then diluting by 10 times to obtain a pretreatment solution.
Sixthly, adjusting the pH of the pretreatment solution to 7 by using 10% (w/w) dilute sulfuric acid, and inoculating the secondary seeds into the pretreatment solution to obtain a target product.
Wherein the inoculation amount is 20% (based on the liquid amount), the temperature is 40 ℃, and the time is 3 days. And analyzing the target product, wherein the degradation of free phenol is 96% and the degradation of lignin is 46%.
Example 5
The application method of the degrading bacteria for degrading phenols and lignin disclosed in example 5 may comprise the following steps:
firstly, inoculating the pseudomonas putida frozen in a glycerin pipe with the temperature of minus 80 ℃ into a solid culture medium by a loop for streak inoculation, and culturing for 14h in a constant temperature incubator with the temperature of 30 ℃ to obtain first-grade seeds.
And secondly, selecting a single colony from the primary seed, inoculating the single colony in a liquid culture medium, culturing for 14 hours at the temperature of 30 ℃ and the speed of 140rpm of a shaking table, and adding a proper amount of NaOH solution to adjust the pH to 7 so as to obtain a secondary seed.
Thirdly, crushing bagasse into 1-25mm by using a crusher, and storing the crushed bagasse raw material in a dry environment for later use.
Fourthly, pre-soaking the crushed bagasse raw material with 2 percent (w/w) NaOH solution according to the solid-to-liquid ratio of 1:8, standing for 12 hours, and reacting for 60min at the temperature of 70 ℃ to obtain the lignocellulose raw material.
And fifthly, performing vacuum filtration on the lignocellulose raw material through a vacuum filtration device, and then diluting by 10 times to obtain a pretreatment solution.
Sixthly, adjusting the pH of the pretreatment solution to 7 by using 10% (w/w) dilute sulfuric acid, and inoculating the secondary seeds into the pretreatment solution to obtain a target product.
Wherein the inoculation amount is 3% (based on liquid amount), the temperature is 30 ℃, and the time is 1 day. The target product was analyzed, free phenol degraded by 63% and lignin degraded by 13%.
Example 6
The application method of the degrading bacteria for degrading phenols and lignin disclosed in example 6 may comprise the following steps:
firstly, inoculating the pseudomonas putida frozen in a glycerin pipe with the temperature of minus 80 ℃ into a solid culture medium by a loop for streak inoculation, and culturing for 16h in a constant temperature incubator with the temperature of 30 ℃ to obtain first-grade seeds.
And secondly, selecting a single colony from the primary seed, inoculating the single colony in a liquid culture medium, culturing for 14 hours at the temperature of 30 ℃ and the speed of 140rpm of a shaking table, and adding a proper amount of NaOH solution to adjust the pH to 7 so as to obtain a secondary seed.
And thirdly, inoculating the secondary seeds into wastewater with the phenol concentration of 100 milligrams per liter (mg/L) to obtain a target product.
Wherein the inoculation amount is 20%, the temperature is 30 ℃, and the time is 6 days. The target product was analyzed and the degree of phenol degradation was 92%.
Example 7
The application method of the degrading bacteria for degrading phenols and lignin disclosed in example 7 may comprise the following steps:
firstly, inoculating the pseudomonas putida frozen in a glycerin pipe with the temperature of minus 80 ℃ into a solid culture medium by a loop for streak inoculation, and culturing for 16h in a constant temperature incubator with the temperature of 30 ℃ to obtain first-grade seeds.
And secondly, selecting a single colony from the primary seed, inoculating the single colony in a liquid culture medium, culturing for 14 hours at the temperature of 30 ℃ and the speed of 140rpm of a shaking table, and adding a proper amount of NaOH solution to adjust the pH to 7 so as to obtain a secondary seed.
And thirdly, inoculating the secondary seeds into wastewater with the phenol concentration of 400mg/L to obtain a target product.
Wherein the inoculation amount is 3%, the temperature is 40 ℃, and the time is 3 days. The target product was analyzed and the degree of phenol degradation was 53%.
The foregoing description is only exemplary of the preferred embodiments of the disclosure and is illustrative of the principles of the technology employed. It will be appreciated by those skilled in the art that the scope of the invention in the embodiments of the present disclosure is not limited to the specific combination of the above-mentioned features, but also encompasses other embodiments in which any combination of the above-mentioned features or their equivalents is made without departing from the inventive concept as defined above. For example, the above features and (but not limited to) technical features with similar functions disclosed in the embodiments of the present disclosure are mutually replaced to form the technical solution.
Claims (10)
1. A method of using a degrading bacterium for degrading phenols and lignins, comprising:
streaking and inoculating a strain frozen in a glycerin pipe at the temperature of-80 ℃ into a solid culture medium at the temperature of 30 ℃ to culture for 14-16 hours to obtain a first-class seed, wherein the strain is used for degrading phenols and lignin;
picking a single colony from the primary seed, transferring the single colony into a liquid culture medium, and culturing for 14 to 16 hours at the temperature of 30 ℃ under the environment of 150 revolutions per minute of a shaking table;
adjusting the pH value of the liquid culture medium inoculated with the primary seeds to 7 to obtain secondary seeds;
inoculating the secondary seeds into a pretreatment raw material in an inoculation amount of 3-20%, and degrading for 1-6 days in an environment with the temperature of 30-40 ℃ to obtain a target product, wherein the pretreatment raw material comprises phenols and/or lignin.
2. The method of claim 1, wherein the bacterial species is pseudomonas putida.
3. The method of claim 2, wherein the composition of the solid medium comprises: 10 g/l of sodium chloride, 5 g/l of yeast extract powder, 10 g/l of peptone and 15 g/l of agar.
4. The method of claim 3, wherein the composition of the liquid medium comprises: 10 g/l of sodium chloride, 5 g/l of yeast extract powder and 10 g/l of peptone.
5. The method of claim 4, wherein the pretreated feedstock is produced by:
comminuting a lignocellulosic feedstock to between 1 and 25 millimeters to produce a comminuted lignocellulosic feedstock;
impregnating the crushed lignocellulose raw material by using a dilute sulfuric acid solution with the mass fraction of 1-5% according to a solid-to-liquid ratio of 1: 1-1: 3 to obtain a first initial raw material;
placing the first initial raw material for 12 hours, and reacting for 30-60 minutes in an environment of 120-160 ℃ to obtain an acid pretreated raw material;
adjusting the pH value of the acid pretreatment raw material to 5-7 by using a sodium hydroxide solution to obtain the pretreatment raw material, wherein the sodium hydroxide solution accounts for 10% by mass, and the pretreatment raw material comprises phenols and lignin.
6. The method of claim 5, wherein the inoculating of the secondary seed into the pretreated feedstock at an inoculum size of 3% to 20% and degradation at a temperature of 30 to 40 ℃ for 1 to 6 days to obtain the target product comprises:
inoculating the secondary seeds into the pretreatment raw material at the temperature of 30-40 ℃ for degradation treatment for 1-5 days, wherein the inoculation amount of the secondary seeds is 3-20%;
and (3) carrying out enzymatic saccharification on the degraded pretreated raw material with the solid content of 15-30% by using cellulase for 48 hours to generate the target product, wherein the unit of the cellulase is 5-30 filter paper enzyme activities per gram of dry matter.
7. The method of claim 4, wherein the pretreated feedstock is produced by:
comminuting a lignocellulosic feedstock to between 1 and 25 millimeters to produce a comminuted lignocellulosic feedstock;
impregnating the crushed lignocellulose raw material by using a sodium hydroxide solution with the mass fraction of 1-2% according to a solid-to-liquid ratio of 1: 8-1: 10 to obtain a second initial raw material;
standing the second initial raw material for 12 hours, and reacting for 60 to 90 minutes at 70 to 90 ℃ to obtain an alkali pretreatment raw material;
and adjusting the pH value of the alkali pretreatment raw material to 7 by using a dilute sulfuric acid solution with the mass fraction of 10% to obtain the pretreatment raw material.
8. The method of claim 7, wherein the inoculating of the secondary seed into the pretreated feedstock at an inoculum size of 3% to 20% and degradation at a temperature of 30 to 40 ℃ for 1 to 6 days to obtain the target product comprises:
inoculating the secondary seeds into the pretreatment raw material for degradation treatment for 1 to 3 days under the condition of 30 to 40 ℃ to generate the target product, wherein the inoculation amount of the secondary seeds is 3 to 20 percent.
9. The method of claim 4, wherein the inoculating of the secondary seed into the pretreated feedstock at an inoculum size of 3% to 20% and degradation at a temperature of 30 to 40 ℃ for 1 to 6 days to obtain the target product comprises:
in response to the pretreatment raw material comprising phenols, inoculating the secondary seeds into the pretreatment raw material for degradation treatment for 3 to 6 days at a temperature of between 30 and 40 ℃ to generate the target product, wherein the inoculation amount of the secondary seeds is between 3 and 20 percent.
10. The process of claim 9, wherein the concentration of phenols in the pretreated feedstock is from 100 to 400 mg/l.
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