CN111073874A

CN111073874A - Method for degrading coal by using esterase and rhamnolipid

Info

Publication number: CN111073874A
Application number: CN201911395023.6A
Authority: CN
Inventors: 刘向荣; 石晨; 杨杰; 申文盛
Original assignee: Xian University of Science and Technology
Current assignee: Xian University of Science and Technology
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2020-04-28
Anticipated expiration: 2039-12-30
Also published as: CN111073874B

Abstract

The invention belongs to the technical field of coal biodegradation, and relates to a method for degrading coal by using esterase and rhamnolipid, which comprises the following steps: 1) extracting esterase from pseudomonas japonicus for later use; 2) extracting rhamnolipid from Pseudomonas japonicus for use; 3) preparing a coal sample; 4) respectively adding the esterase obtained in the step 1) and the rhamnolipid obtained in the step 2) into the coal sample obtained in the step 3), dissolving the coal under the combined action, and calculating the coal dissolving rate. The method utilizes the combined action of the rhamnolipid and the esterase to degrade the coal, the rhamnolipid and the esterase have super strong degradation effect on harmful substances in the coal, the dissolution rate of the coal is effectively improved, meanwhile, no harm is caused to the environment, and the clean production of the coal is realized.

Description

Method for degrading coal by using esterase and rhamnolipid

Technical Field

The invention belongs to the technical field of microorganisms, and relates to a method for degrading coal by using esterase and rhamnolipid.

Background

Coal plays a great role in the fields of power generation and combustion, and with the increase of global energy demand, development of coal resources with low rank and huge reserves is necessary. However, due to the limitation of low-rank coal mining technology, carbon dioxide and fine particles generated by coal combustion power generation are released into the atmosphere, and some toxic compounds such as sulfur and nitrogen oxides are released into the atmosphere and the environment to cause the problem of environmental pollution, so that an environment-friendly coal conversion technology and a coal cleaning technology are provided.

Researchers propose that the influence of coal on the environment is reduced by biologically promoting the coal degradation and extracting different valuable materials and chemicals from the coal, but the existing coal biodegradation has the problems of low solvent rate, unsafe degradation process, environmental pollution and the like.

Disclosure of Invention

Aiming at the technical problems of low coal dissolving rate, toxicity, harm and environmental pollution in the conventional coal degradation, the invention provides a method for degrading coal by using esterase and rhamnolipid, which has super-strong degradation effect on harmful substances in coal, effectively improves the coal dissolving rate, does not cause any harm to the environment and realizes clean production of coal.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for degrading coal by using esterase and rhamnolipid comprises the following steps:

1) extracting esterase from pseudomonas japonicus for later use;

2) extracting rhamnolipid from Pseudomonas japonicus for use;

3) preparing a coal sample;

4) and (3) respectively adding the esterase obtained in the step 1) and the rhamnolipid obtained in the step 2) into the coal sample obtained in the step 3), and dissolving the coal under the combined action of the esterase and the rhamnolipid, and calculating the coal dissolving rate.

Further, the extraction process of the esterase in the step 1) is as follows:

1.1) placing the pseudomonas japonicus in a solid culture medium for culturing to obtain a pseudomonas japonicus colony;

1.2) inoculating the pseudomonas japonicus colony obtained in the step 1.1) into a sterilized liquid culture medium by using an inoculating loop, and placing the liquid culture medium in an incubator for culture to obtain a bacterial liquid;

1.3) centrifugally separating the bacterial liquid obtained in the step 1.2), and collecting supernatant to obtain the esterase.

Further, in the step 1.1), the solid culture medium is a beef extract peptone agar culture medium; in the step 1.2), the liquid culture medium is a beef extract peptone culture medium, and the culture conditions in the incubator are as follows: the culture temperature is 20-35 ℃, the oscillation speed is 140-200 r/min, and the culture time is 10 or 15 days until the optical density OD of the supernatant₆₀₀The value is 2.9-3.1.

Further, the extraction process of rhamnolipid in step 2) is as follows:

2.1) adding activated and cultured pseudomonas japonicus into a liquid culture medium, and then placing the culture medium in an incubator for culture to obtain sterile fermentation liquor;

2.2) adjusting the sterile fermentation liquor obtained in the step 2.1) by using 1mol/L HCL solution to ensure that the pH value is 2, standing, adding an extracting agent for extraction, standing for layering, taking an upper methanol layer, and performing rotary evaporation to obtain the rhamnolipid.

Further, the liquid culture medium in the step 2.1) is a solid beef extract peptone culture medium; the conditions for the cultivation in the incubator were: the culture temperature is 20-35 ℃, the oscillation speed is 140-200 r/min, and the culture time is 10 or 15 days until the optical density OD of the supernatant₆₀₀The value is 2.9-3.1.

Further, in the step 3), the preparation process of the coal sample is as follows: crushing and screening the coal sample, selecting the coal sample with the particle size of 0.25-0.5mm for oxidation, soaking the coal sample in nitric acid, washing and filtering until the pH value is 7, sealing the coal sample, and sterilizing for 10-15 minutes to obtain the coal sample.

Further, in the step 4), the mass ratio of rhamnolipid to esterase is 800-1200: 50-10 parts; preferably, the weight ratio of rhamnolipid to esterase is 1100: 20.

An extraction method of esterase comprises the following steps:

1) culturing the pseudomonas japonicus in a solid culture medium to obtain a pseudomonas japonicus colony;

2) inoculating the pseudomonas japonicus colony obtained in the step 1) into a sterilized liquid culture medium by using an inoculating loop, and placing the liquid culture medium in an incubator for culture to obtain a bacterial liquid;

3) and (3) after the bacterial liquid obtained in the step 2) is subjected to centrifugal separation, collecting supernate, and performing salting out, dialysis and chromatography to obtain the purified esterase.

Further, the solid culture medium in the step 1) is a beef extract peptone agar culture medium; the liquid culture medium in the step 2) is a beef extract peptone culture medium.

Further, the conditions for culturing in the incubator in the step 2) are as follows: the culture temperature is 20-35 ℃, the oscillation speed is 140-200 r/min, and the culture time is 10 or 15 days until the optical density OD of the supernatant₆₀₀The value is 2.9-3.1.

The invention has the beneficial effects that:

according to the invention, active substances esterase and rhamnolipid are extracted from the secretion of pseudomonas japonicus, and the extracted rhamnolipid and esterase are used for carrying out microbial degradation on coal, wherein the quality of the rhamnolipid and the quality of the esterase are respectively in the range of 800-1300: 50-10, so that the coal dissolving rate of the inner Mongolia oxidized coal is improved; especially, when the addition amounts of rhamnolipid and esterase are 1100mg and 20mg respectively, the coal dissolving rate can reach 53.8 percent at most, and the coal dissolving rate is increased by 23.8 percent; the rhamnolipid and esterase have super strong degradation effect on harmful substances in coal, and do not cause any harm to the environment.

Drawings

FIG. 1 is an infrared spectrum of a commercial rhamnolipid and extract of the present invention;

FIG. 2 is a graph showing the effect of ammonium sulfate concentration on the precipitated esterase according to the present invention;

FIG. 3 is a polyacrylamide gel electrophoresis pattern of the esterase of the invention;

FIG. 4 is a schematic representation of Pseudomonas degradation of oxidized coal.

Detailed Description

The present invention will now be described in detail with reference to the accompanying drawings and examples.

Example 1 extraction of rhamnolipids

Inoculating the activated strain for 5 times into 1000mL conical flask, wherein the activation is to culture the preserved Pseudomonas japonicus strain in solid culture medium via inoculating loop, and adding the activated Pseudomonas japonicus into 500mL liquid culture medium, culturing at 30 deg.C and 160r/min constant temperature shaking culture box for 10 days until the OD of the supernatant is₆₀₀A value of 3.0; then, adjusting 500mL of sterile fermentation broth with 1mol/L HCl solution to make the pH value of the fermentation broth 2, standing overnight at 4 ℃, and reducing the solubility of rhamnolipid along with the reduction of the pH value to precipitate after the overnight; and adding a chloroform/methanol extractant with the volume ratio of 2:1 for extraction, standing for layering, taking an upper methanol layer, and finally performing rotary evaporation by using a rotary evaporator to obtain a light yellow rhamnolipid solid.

In this example, the liquid medium includes peptone, beef extract, sodium chloride, and distilled water, wherein 5.0g of peptone, 3g of beef extract, 5g of sodium chloride, and 1L of distilled water, and the pH is 7.

In this example, a light yellow rhamnolipid solid extract was characterized by infrared spectroscopy, and the results are shown in fig. 2.

Referring to FIG. 2, it can be seen that at 3445cm^-1An O-H stretching vibration peak of hydroxy group, 2910cm^-1Nearby is the aliphatic chain CH₂And CH₃Symmetrical stretching of the radical, 1460cm^-1The left and right symmetric peaks indicate the presence of an ester carbonyl (C ═ O) in the carboxyl group; 1062cm^-1(1300-1000cm^-1) The C-O telescopic band proves that carbon atoms in a benzene ring structure are connected with hydroxyl and exist; 1640cm^-1The stretching vibration peak of (A) is a C-O-C group, which indicates that a cyclic lactone structure and glycolipid exist in the molecule, and the peaks are characteristic absorption peaks of a biosurfactant rhamnolipid (commercial product), so that the extracted compound is inferred to be the rhamnolipid.

Example 2 extraction of rhamnolipids

In contrast to example 1, the strain (Pseudomonas japonicus) after 4 times activation was inoculated into a conical flask, a liquid medium was added thereto, and the flask was placed in an incubator at 35 ℃ under a shaking speed of 140r/min for 10 days until the OD of the supernatant was reached₆₀₀A value of 3.1; and then adjusting the pH value of the sterile fermentation liquor to 2 by using 1mol/L HCL solution, standing at 4 ℃, adding an extracting agent for extraction, standing for layering, taking an upper methanol layer, and performing rotary evaporation to obtain the light yellow rhamnolipid.

Example 3 extraction of rhamnolipids

In contrast to example 1, 6 times activated strains (Pseudomonas japonicus) were inoculated into Erlenmeyer flask, liquid medium was added thereto, and the flask was placed in an incubator at 20 ℃ under a shaking speed of 200r/min for 15 days until OD of supernatant₆₀₀A value of 2.9; and then adjusting the pH value of the sterile fermentation liquor to 2 by using 1mol/L HCL solution, standing at 3 ℃, adding an extracting agent for extraction, standing for layering, taking an upper methanol layer, and performing rotary evaporation to obtain the light yellow rhamnolipid.

EXAMPLE 4 extraction of esterase

In this embodiment, the method for extracting esterase includes the following steps:

1a) placing the pseudomonas japonicus in a solid culture medium through an inoculating loop for culturing to obtain a pseudomonas japonicus colony;

2a) inoculating the pseudomonas japonicus colony obtained in the step 1a) into a sterilized liquid culture medium by using an inoculating loop, and performing constant-temperature shaking culture to obtain a bacterial liquid;

specifically, a 1000mL conical flask is taken, 400mL of liquid culture medium is added into the conical flask, and the conical flask is sterilized for 15min at 121 ℃; then placing in an incubator, shaking at 30 deg.C and 160r/min, and culturing for 15 days until the optical density OD of the supernatant₆₀₀A value of 3.0;

3a) after the bacterial liquid in the step 2a) is subjected to centrifugal separation, collecting supernatant, and performing salting out, dialysis and chromatography to obtain purified esterase;

specifically, after the culture is finished, a proper amount of bacterial liquid is put into a 50mL centrifuge tube for centrifugal separation, the centrifugal separation is carried out at 10000rpm for 20min, the mixture is kept standing and layered at 4 ℃, and finally supernatant containing esterase is collected.

In the embodiment, the solid medium comprises peptone, beef extract, sodium chloride, agar and distilled water, wherein 5.0g of peptone, 3g of beef extract, 5g of sodium chloride, 15g of agar and 1L of distilled water.

Specifically, in the embodiment, because the coal contains a large amount of lignin-like structures and benzene ring-like substances, Pseudomonas japonicus, namely latin name Pseudomonas japonica, which is a bacterium capable of degrading lignin or aromatic hydrocarbon is selected; the preservation number is CICC 23895, and the preservation time is 2015, 3 months and 31 days; the bacteria are purchased from China center for culture collection and management of industrial microorganisms, and the addresses are as follows: house number 6 of the 24 th city of the zhonglu haoxiangqiao of the Chaoyang area in Beijing.

EXAMPLE 5 extraction of esterase

specifically, a 1000mL conical flask is taken, 400mL of liquid culture medium is added into the conical flask, and the conical flask is sterilized for 15min at 121 ℃; then placing in an incubator, shaking at 20 deg.C at 200r/min for 10 days until the optical density OD of the supernatant₆₀₀A value of 2.9;

specifically, after the culture is finished, a proper amount of bacterial liquid is put into a 50mL centrifuge tube for centrifugal separation, the centrifugal separation is carried out at 10000rpm for 20min, the mixture is kept stand at 3 ℃ for layering, and finally supernatant containing esterase is collected.

EXAMPLE 6 extraction of esterase

specifically, a 1000mL conical flask is taken, 400mL of liquid culture medium is added into the conical flask, and the conical flask is sterilized for 15min at 121 ℃; then placing the mixture in an incubator, and culturing at 35 ℃ and under the shaking speed of 140r/min for 10 days until the optical density OD of the supernatant is reached₆₀₀A value of 3.1;

specifically, after the culture is finished, a proper amount of bacterial liquid is put into a 50mL centrifuge tube for centrifugal separation, the centrifugal separation is carried out at 10000rpm for 20min, the mixture is kept stand at 5 ℃ for layering, and finally supernatant containing esterase is collected.

Further, in order to obtain an esterase with a higher purity, the esterase obtained in example 2 was subjected to a purification test by purifying the collected supernatant containing the esterase by salting out, dialysis, chromatography, and detection by SDS-PAGE polyacrylamide gel electrophoresis.

Purification test

Test groups: example 4 extracted esterase

(1) Preparation of the principal solution

1) Preparation of Coomassie brilliant blue G250 solution

Coomassie brilliant blue G250 is accurately weighed and dissolved in 50mL of 90% (V/V) ethanol solution, then 100mL of 85% (V/V) phosphoric acid is added, and finally distilled water is added to fix the volume to 1L.

2) Preparation of concentration buffer (Stacking buffer)

30.25g of tris (hydroxymethyl) aminomethane and SDS2g were dissolved in an appropriate amount of water and adjusted to pH 6.8 with 1moL of HCl to prepare 500mL of a solution for use.

3) Preparation of 4% concentrated gum

2.5mL of concentration buffer, 0.67mL of 30% acrylamide, 30. mu.L of 10% APS, 12. mu.L of tetraethylethylenediamine and 1.83mL of distilled water.

4)10x SDS protein buffer solution preparation

30.3g of tris (hydroxymethyl) aminomethane, 144.1g of glycine and 10g of Sodium Dodecyl Sulfate (SDS).

5) Preparation of dyeing liquor

1g of Coomassie brilliant blue, 900mL of methanol, 180mL of glacial acetic acid and 900mL of distilled water are mixed to prepare a coloring agent.

6) Preparation of decolouring liquid

400mL of 20% methanol, 100mL of glacial acetic acid and 1500mL of distilled water are mixed to prepare a destaining solution.

(2) Determining the saturation concentration range of ammonium sulfate

Precipitation is a commonly used method of purifying and concentrating proteins when crude proteins are isolated; the solubility of the protein is closely related to the salt concentration, and at low concentrations, the protein concentration increases as the salt concentration increases. However, in a high-concentration salt solution, salt ions compete for binding with water molecules on the surface of the protein, a hydration film on the surface of the protein is damaged, and the solubility is reduced, so that the protein forms a precipitate under the hydrophobic action. The solubility of each protein is different, and therefore different salt concentrations need to be used to precipitate the proteins. In the experiment, ammonium sulfate is added to form different saturated concentrations, so as to judge the optimal saturated ammonium sulfate range. The method comprises the following specific steps:

a: respectively filling the supernatant without the thalli into 9 centrifugal tubes, wherein each centrifugal tube is filled with 40 mL;

b: respectively adding ammonium sulfate into 9 centrifugal tubes to ensure that the saturation degrees of the ammonium sulfate are respectively 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%, slowly adding the ammonium sulfate during operation, and continuously oscillating to ensure that the ammonium sulfate is completely dissolved;

c: standing at 4 deg.C overnight for culture to allow protein to precipitate completely;

d: after centrifugation, the precipitates in the tubes were collected, and the precipitates in 9 centrifuge tubes were dissolved in a small amount of 10 × PBS buffer (pH 7), respectively, and the esterase activity in the supernatant of each tube was measured, and then the test results were compared to find the supernatant with the lowest esterase activity, where the ammonium sulfate concentration was the optimum salting-out concentration, and the results of the precipitation effect of the esterase at different ammonium sulfate concentrations are shown in fig. 2.

As can be seen from FIG. 2, when the saturation concentration of ammonium sulfate was 80%, the esterase activity in the supernatant was the lowest, and the esterase precipitated at that time was the most, so that the optimum saturation concentration of ammonium sulfate was 80%.

(3) Salting out

The solubility of ammonium sulfate is high, and a large amount of NH is formed by dissociation in water₄ ⁺、SO₄ ^2-Ions can be combined with a large number of water molecules, so that the solubility of the protein is reduced and the protein is separated out; in addition, it has a small temperature coefficient and is not easy to denature proteins. As can be seen from fig. 2, the optimum saturation concentration of ammonium sulfate was 80%. Thus, the salting-out process is:

a: taking 300mL of separated supernatant from which the thalli were removed in the example, and slowly adding ammonium sulfate until the saturation degree is 80%;

b: standing overnight at 4 deg.C for culture to allow protein to precipitate completely;

c: centrifuging at 10000rpm for 20 min; the precipitate was collected and dissolved in 2mL of 10 × PBS buffer (pH 7) and stored at 4 ℃.

(4) Dialysis

Preparation of dialysis bags: the dialysis bag was first cut to the appropriate length and placed in a 500mL beaker, followed by 200mL, 5% sodium bicarbonate, and 0.002g EDTA. Boiling for 10min, cooling to room temperature, and storing in distilled water at 4 deg.C.

The dialysis process is:

a: putting the collected mixed protein liquid into a dialysis bag, and clamping by using a clamp;

b: and (3) carrying out dialysis desalting by using a concentrated buffer solution, dialyzing for 24h, and changing the concentrated buffer solution every 2h to obtain a concentrated esterase solution.

(5) Chromatography

Gel filtration chromatography, also known as molecular sieve chromatography and exclusion chromatography, uses the molecular sieve action of a gel having a network structure to separate substances according to their molecular sizes.

Preparation of gel column: selecting dextran G-75 for chromatography, suspending the dry gel particles in 5-10 times of distilled water or eluent for full swelling, and pouring out the superfine particles after swelling for 24 h. Or heating to accelerate swelling, gradually heating the wet gel slurry to near boiling in boiling water bath, and swelling for 1-2 hr.

Filling of gel: the chromatographic column is fixed on a frame vertically to the ground, the lower end outflow port is clamped by a clamp, a larger container with a stirring device can be arranged on the top of the column, the column is filled with eluent, the gel is adjusted into thinner slurry head liquid to be contained in the container on the top of the column, and then the gel is slightly stirred to sink in the column, so that the gel particles rise horizontally until the required height is reached.

The concentrated esterase solution thus obtained was added to a previously prepared Tris-HCl buffer (0.05mol/L, pH ═ 7.0) and equilibrated with a Sephadex G-75 column. Eluting with Tris-HCl buffer solution at flow rate of 1.5mL/min, collecting eluate, measuring enzyme activity of each tube with ultraviolet spectrophotometer, and mixing tubes containing the same enzyme activity.

(6) SDS-PAGE Polyacrylamide gel electrophoresis

And (3) obtaining purified esterase after the ammonium sulfate precipitation, dialysis, chromatography and gel filtration column, and carrying out gel electrophoresis detection on the purified esterase, wherein the specific process comprises the following steps:

a: checking the air tightness of the instrument, installing the instrument, adding distilled water, waiting for 10min, and if the liquid level does not drop, determining that the air tightness is good, otherwise, determining that the air tightness is not good.

B: preparing separation gel, adding to white line, adding water, pouring out water after the separation gel is solidified, adding 4% concentrated gel into instrument, inserting comb, waiting for solidification, wrapping with newspaper, placing into box, wetting with 10xSDS protein buffer, and placing into refrigerator for use.

C: taking down the solidified glass cement plate to convert the direction, then placing the glass cement plate in a glue running device, pouring 1.5L of 10xSDS rounding buffer, taking down a comb, and injecting 3 muL of protein Marker into a first glue hole so as to play a role in judging the size of the induced protein. The second well was filled with 10. mu.L of uninduced protein solution at 30 ℃.

D: and after the sample is added, opening the electrophoresis apparatus, setting the time to be 180min, setting the voltage to be 120V, starting glue running, closing the glue running apparatus when the glue block runs to five sixths, and finishing the glue running.

E: and taking out the rubber block, pouring the rubber block into a dyeing solution for submerging, pouring the dyeing solution after dyeing for 1h, pouring the dyeing solution into a decoloring solution for submerging after washing with water, standing overnight, and observing the rubber running condition on the next day.

The electrophoresis results are shown in FIG. 3, in which: m-protein standard molecular weight; 1-extracellular fluid; 2-crude protein; 3-purification of the protein.

As can be seen from FIG. 3, the electrophoresis test gave a clear band, which was calculated to have a molecular weight of 45kDa, and was a purified esterase.

Example 7

The rhamnolipid extracted in example 1 and the esterase extracted in example 4 were used to biodegrade coal.

1. Preparation of coal samples

In this embodiment, the coal sample comes from a coal mine of the tangjia meeting of inner mongolia, and the specific treatment process is as follows:

1) crushing and screening a coal sample, selecting raw coal with the particle size of 0.25-0.5mm for oxidation, and soaking the raw coal in 6-10 mol/L nitric acid for 24 hours respectively;

2) washing with distilled water after soaking, and filtering until the pH value is about 7;

3) and (3) filling the coal sample into a beaker, sealing the beaker by using a breathable film and newspaper, and sterilizing for 15 minutes for later use.

The raw coal and the coal (8mol/L) subjected to the nitric acid oxidation of the sample were subjected to industrial analysis and elemental analysis, and the results are shown in Table 1.

TABLE 1 coal sample Industrial and elemental analysis

In table 1:M_admoisture, air drying base; a. the_dAsh, dry basis; v_dafVolatilizing, and drying the ashless base; FC_dafFixed carbon, dry ashless base

The comparison in table 1 shows that the content of C, H, S in the internal Mongolia coal pretreated by nitric acid is reduced, the content of O, N in the internal Mongolia coal is increased, oxidation reaction is easy to occur, and the degradation of the coal sample by the strains is facilitated. Researches show that after inorganic matters in coal are oxidized by nitric acid, the ash content is reduced, the volatile content is increased, and the pore diameter is increased. In addition, chemical reactions occur between nitric acid and aromatic ring-containing groups in coal, such as oxidation of aromatic rings and oxidation of aromatic ring side chains to esters, aldehydes, ketones, and the like. The change of the coal structure is beneficial to biological coal dissolution, the dissolution of lignite is increased, the carbon content of raw coal is 61.85 percent, the raw coal belongs to low-rank coal, the sulfur content is 0.12 percent, the sulfur content belongs to low-sulfur coal, and the oxygen content is 32.28 percent, so that the higher the oxidation degree of the coal is, the lower the grade is, and the biological dissolution is easy to realize.

2. Coal degradation test

Through parallel tests, rhamnolipid and esterase with different mass ratios are simultaneously added into a coal sample to degrade the coal. The esterase is the esterase extracted in example 4, and the rhamnolipid is the rhamnolipid extracted in example 1.

Adding 50mL of liquid culture medium into a proper triangular flask, then adding 0.30g of coal sample with granularity of 0.25-0.50 mm and 8mol/L nitric acid oxidation, and adding rhamnolipid and esterase in different proportions, wherein the mass ratio of the rhamnolipid to the esterase is 800mg:50mg respectively; 900mg to 40 mg; 1000mg to 30 mg; 1100mg to 20 mg; 1200mg to 10 mg; finally, 10mL of expanded culture bacterial liquid is added, and the mixture is cultured for 10 days in a full-temperature shaking incubator at 30 ℃ and 160 r/min. Centrifuging at 10000rpm for 20min, wherein the figures of the real object before and after centrifugation are shown in FIG. 4, A is the figure of the degradation liquid phase product after shaking culture is finished, and B is the figure of the degradation liquid phase product after centrifugation.

In this example, the enlarged culture broth was: inoculating the strain obtained after the activation culture into a liquid culture medium, and culturing for 3 days in a constant-temperature shaking incubator at 30 ℃ and 160r/min to obtain the expanded culture solution for dissolving the coal in the microorganism.

After centrifugal separation, the supernatant was diluted 30 times, and absorbance at a450 position was measured using a TU-1900 dual-beam uv-vis spectrophotometer, and the coal-dissolving rate was calculated, and the results are shown in table 2.

TABLE 2 results of the influence of rhamnolipids and esterase on coal dissolution rate

And (4) analyzing results:

1) referring to fig. 4, the dissolution of coal using rhamnolipids and esterase was found to be uniformly black with most of the coal dissolved, and the liquid phase was also black after centrifugation, indicating that a large amount of coal was dissolved.

2) Referring to the table 2, when the added rhamnolipid and esterase have the mass of 800-1200: the coal dissolving rate of the inner Mongolia oxidized coal is between 48.4 and 56.7 percent within the range of 50 to 10, and the coal dissolving rate is the highest when the rhamnolipid and the esterase are respectively 1100mg and 20 mg.

Comparative test for coal degradation property

Further, in order to demonstrate the excellent effect of the coal degradation method provided by the present invention, a control test was performed.

Experimental groups: the results of the degradation of coal by rhamnolipids and esterases in example 7 (table 2).

Control group 1: the rhamnolipid and esterase are not added for coal degradation;

control group 2: only the rhamnolipid extracted in example 1 was added for coal degradation;

control group 3: only the esterase extracted in example 4 is added for coal degradation;

the test process is as follows: in order to make the result of the control experiment more accurate, the control experiment was identical to the other conditions of coal dissolution of example 7 except that the mass ratio of rhamnolipid and esterase was 0: 0; in the control group 2, the mass ratio of rhamnolipid and esterase was 1300 mg: 0 mg; in the control group 3, the mass ratio of rhamnolipid and esterase was 0mg:50 mg.

After the comparative experiment was completed, the coal dissolution rate was calculated, and the results are shown in table 3.

TABLE 3 influence of three control groups on coal dissolution rate

From table 3, it can be seen that:

1) when rhamnolipid and esterase were not added, the coal-dissolving rate of pseudomonas japonicus to the inner Mongolia oxidized coal was 45.8%;

2) when only rhamnolipid is added, the coal dissolving rate of pseudomonas japonicus to the inner Mongolia oxidized coal is 48.9%;

3) when only esterase was added, the coal-dissolving rate of Pseudomonas japonicus to the inner Mongolia oxidized coal was 47.2%.

Comparing the coal degradation results of example 5 (table 2) with the control, it was concluded that:

by adopting the degradation method, when rhamnolipid and esterase are added simultaneously, the coal dissolving rate can be improved by 10.9 percent to the maximum extent;

when rhamnolipid is added, the coal dissolving rate is improved by 7.8% at most by adopting the degradation method provided by the invention;

when esterase is added, the coal dissolving rate is improved by 3.1 percent at most by adopting the degradation method provided by the invention.

Through comparison, the rhamnolipid extracted by the method and the esterase are used for degrading coal under the combined action, so that the dissolution rate of the coal can be improved, the biodegradation of the coal is effectively realized, and the aims of safe degradation and clean production of the coal can be fulfilled.

Claims

1. A method for degrading coal by using esterase and rhamnolipid is characterized in that: the method for degrading coal comprises the following steps:

1) extracting esterase from pseudomonas japonicus for later use;

2) extracting rhamnolipid from Pseudomonas japonicus for use;

3) preparing a coal sample;

2. The method for degrading coal using esterase and rhamnolipid according to claim 1, characterized in that: the extraction process of the esterase in the step 1) comprises the following steps:

3. The method for degrading coal using esterase and rhamnolipid according to claim 2, characterized in that: in the step 1.1), the solid culture medium is a beef extract peptone agar culture medium; in the step 1.2), the liquid culture medium is a beef extract peptone culture medium, and the culture conditions in the incubator are as follows: the culture temperature is 20-35 ℃, the oscillation speed is 140-200 r/min, and the culture time is 10 or 15 days until the optical density OD of the supernatant₆₀₀The value is 2.9-3.1.

4. The method for degrading coal using esterase and rhamnolipid according to claim 1, characterized in that: the extraction process of the rhamnolipid in the step 2) is as follows:

5. The method for degrading coal using esterase and rhamnolipid according to claim 2, characterized in that: the liquid culture medium in the step 2.1) is solidBeef extract peptone medium; the conditions for the cultivation in the incubator were: the culture temperature is 20-35 ℃, the oscillation speed is 140-200 r/min, and the culture time is 10 or 15 days until the optical density OD of the supernatant₆₀₀The value is 2.9-3.1.

6. The method for degrading coal using esterase and rhamnolipid according to claim 1, characterized in that: in the step 3), the preparation process of the coal sample comprises the following steps: crushing and screening the coal sample, selecting the coal sample with the particle size of 0.25-0.5mm for oxidation, soaking the coal sample in nitric acid, washing and filtering until the pH value is 7, sealing the coal sample, and sterilizing for 10-15 minutes to obtain the coal sample.

7. The method for degrading coal using esterase and rhamnolipid according to claim 1, characterized in that: in the step 4), the mass ratio of rhamnolipid to esterase is 800-1200: 50-10 parts; preferably, the weight ratio of rhamnolipid to esterase is 1100: 20.

8. An esterase extraction method, which is characterized in that: the method comprises the following steps:

9. The method for extracting esterase according to claim 8, characterized in that: the solid culture medium in the step 1) is a beef extract peptone agar culture medium; the liquid culture medium in the step 2) is a beef extract peptone culture medium.

10. The method for extracting esterase according to claim 9, characterized in that: culturing in an incubator in the step 2)The conditions of (a) are: the culture temperature is 20-35 ℃, the oscillation speed is 140-200 r/min, and the culture time is 10 or 15 days until the optical density OD of the supernatant₆₀₀The value is 2.9-3.1.