Method for enhancing microbial degradation of coal to produce oxygen-containing chemicals by using composite surfactant

Abstract

The invention discloses a method for producing oxygen-containing chemicals by enhancing microbial degradation of coal through a composite surfactant, and relates to the technical field of microbial degradation of coal, and the method specifically comprises the following steps: selecting bacillus XK1, and culturing until OD600 reaches 0.5 to obtain fermentation liquor; adding the fermentation liquor, the sterilized coal sample and sterile surfactants with different concentrations into the liquid culture medium, and centrifuging to obtain supernate and residues after a degradation experiment is finished; filtering the supernate, and measuring the absorbance at 450nm; washing residues and drying to obtain residual coal, and calculating the biodegradation rate of the coal. The added composite surfactant promotes the bacillus XK1 to degrade coal to produce oxygen-containing chemicals, and the oxygen-containing chemicals can be used as a solvent and can also be used as a raw material for synthesizing organic matters; in the presence of the composite surfactant Tr-Rh, the molecular weight of the degraded liquid product is much smaller than that of the coal sample, which indicates that the molecular structure of the coal sample is damaged by the bacillus XK1, and small molecular weight substances are released.

Description

Technical Field

[0001] The invention relates to the technical field of microbial degradation of coal, and in particular to a method for enhancing the microbial degradation of coal to produce oxygen-containing chemicals using a composite surfactant. Background Art

[0002] Low-rank coal is one of China's important coal resources, with abundant reserves and diverse qualities. However, the complex structure of low-rank coal makes its utilization face many challenges. Traditional physical and chemical methods are often characterized by high energy consumption and high pollution when processing coal. As an environmentally friendly technology, coal biodegradation provides a new idea for the efficient utilization of coal. Bacillus XK1 is a microorganism with special metabolic capabilities that can transform coal under certain conditions. However, the interfacial interaction between coal and microbial cells is weak, and the organic components in coal are difficult to be fully obtained and utilized by microorganisms, which limits the biodegradation rate of coal.

[0003] Surfactants, as a class of substances that can significantly change the properties of interfaces, have potential application value in enhancing the interaction between microorganisms and coal. By adding suitable surfactants, the hydrophobicity of the coal surface can be reduced and its dispersibility can be improved, thereby increasing the contact area between microorganisms and coal and promoting the degradation of coal by microorganisms. Due to their synergistic effect, composite surfactants are likely to improve the surface properties of coal more effectively than single surfactants, further increase the biodegradation rate, and promote the production of more oxygen-containing chemicals. Obtaining oxygen-containing chemicals from coal through biodegradation can not only achieve high added value utilization of coal resources, but also reduce dependence on traditional petrochemical raw materials, reduce carbon emissions and environmental pollution in the production process. Summary of the invention

[0004] In order to solve the above technical problems, the present invention discloses a method for enhancing the microbial degradation of coal to produce oxygen-containing chemicals using a composite surfactant. The composite surfactant can help microorganisms better decompose chemical bonds such as CC bonds, CO bonds and CH bonds in coal, generate more types and higher yields of oxygen-containing chemicals, reduce the surface tension of the solution, increase the hydrophilicity of the coal sample, promote the contact between coal and microorganisms, and improve the degradation rate.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The method for producing oxygen-containing chemicals by enhancing microbial degradation of coal by composite surfactants comprises the following specific steps:

[0007] Step 1, selecting Bacillus XK1 on an agar plate, culturing in a liquid culture medium at 30° C. until OD600 reaches 0.5, and obtaining a fermentation broth;

[0008] Step 2, adding fermentation liquid, sterilized coal sample and sterile surfactant of different concentrations into liquid culture medium, degrading under the conditions of 30°C and 160r / min, and culturing for 10 days;

[0009] Step 3: After the degradation experiment is completed, the culture sample is centrifuged at 10,000 rpm for 20 minutes to obtain a supernatant and a residue; Step 4: The supernatant in step 3 is filtered with a 0.22 μm membrane filter, and the absorbance at 450 nm is measured;

[0010] Step 5: Wash the residue in step 3 with 50 mL of distilled water for 5 times, dry at 90° C. for 8 h to obtain residual coal, and calculate the biodegradation rate of the coal.

[0011] Furthermore, in step 2, the surfactant is selected from at least one of Tr or Rh, the final concentration of the two combined surfactants is 400 mg / L, 600 mg / L, 800 mg / L, 1000 mg / L and 1200 mg / L, and the ratios of Tr and Rh are 4:0, 3:1, 2:2, 1:3, 0:4 respectively.

[0012] Furthermore, in step 4, the method for determining the biodegradability of coal includes the following two methods:

[0013] (1) Weighing method

[0014] The residual coal was repeatedly washed with distilled water to sterilize and dried at 90℃ for 8h. The degradation rate of the coal sample was calculated as follows:

[0015] η=(1-w1 / w0)×100%

[0016] Where η is the biodegradation rate of coal, w0 is the mass before biodegradation (g), and w1 is the mass after biodegradation (g);

[0017] (2) A450 value method

[0018] After the biodegradation experiment, the degradation liquid was centrifuged and filtered through a 0.22 μm membrane filter to obtain the supernatant and residual coal. The absorbance of the supernatant at 450 nm was measured by UV-visible spectroscopy, and the absorbance was positively correlated with the degradation rate.

[0019] Furthermore, the effect of the composite surfactant on the degradation activity was as follows: when the concentration of Tr:Rh was 600 mg / L and the ratio of Tr to Rh was 2:2, the degradation activity of Bacillus XK1 was good, the A450 value was 1.15, and the degradation rate was 77.8%.

[0020] Furthermore, the effect of the composite surfactant on bacterial growth characteristics: compared with the individual surfactants Tr and Rh, in the culture medium containing the composite surfactant Tr-Rh, when the culture time was 4d, the OD600 reached a maximum value of 2.891.

[0021] Furthermore, the effect of the composite surfactant on the activity of alkaline protease secreted by Bacillus XK1: in the culture medium containing Tr-Rh, the alkaline protease activity reached 102.33 U / mL, which was increased by 45.22% compared with that without adding surfactant.

[0022] The beneficial effects of the present invention are as follows:

[0023] 1. Improve coal degradation efficiency

[0024] Coal is a complex organic macromolecule with a stable structure. The efficiency of coal degradation by microorganisms alone is low. By using composite surfactants, the physical and chemical properties of the coal surface can be changed. For example, surfactants can reduce the surface tension between coal and water, making it easier for microorganisms to access active sites on the surface of coal particles. Microorganisms can then decompose the organic components in coal more effectively and accelerate the degradation rate, thereby converting more coal into useful oxygen-containing chemicals per unit time.

[0025] 2. Increase the production of oxygen-containing chemicals

[0026] The main goal of this method is to produce oxygen-containing chemicals. Composite surfactants can help microorganisms better decompose chemical bonds such as CC bonds, CO bonds and CH bonds in coal, so that carbon, hydrogen and other elements in coal are released in the form of oxygen-containing functional groups (such as hydroxyl groups, carboxyl groups, etc.), thereby generating more types and higher yields of oxygen-containing chemicals, such as organic acids and alcohols. These oxygen-containing chemicals have wide application value in many fields such as chemical industry and materials.

[0027] 3. Optimize the microbial degradation process

[0028] Composite surfactants can create a more favorable living and metabolic environment for microorganisms. They can reduce the surface tension of the solution, increase the hydrophilicity of coal samples, promote the contact between coal and microorganisms, and increase the degradation rate.

[0029] 4. Explore green and efficient ways to convert coal

[0030] Compared with traditional coal chemical processing methods, the use of microorganisms to degrade coal to produce oxygenated chemicals is a relatively green technology that aims to reduce the high pollution and high energy consumption links in traditional coal processing. By using composite surfactants to enhance the degradation of microorganisms, coal conversion can be achieved under relatively mild reaction conditions, reducing pressure on the environment while improving the utilization efficiency of coal resources, which is in line with the concept of sustainable development. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic diagram of the process of the present invention;

[0032] Figure 2 The figure is the effect of single surfactant Rh and Tr on the degradation activity of Bacillus XK1 in the example;

[0033] Figure 3 The figures are the effects of the composite surfactants of the examples on the degradation activity (a), degradation rate (b) and degradation time (c);

[0034] Figure 4 This is a graph showing the effect of the composite surfactant on the growth of Bacillus XK1;

[0035] Figure 5 This is a graph showing the effect of the composite surfactant on the activity of alkaline protease secreted by Bacillus XK1;

[0036] Figure 6 This is a graph showing the effect of composite surfactant on the contact angle of coal samples;

[0037] Figure 7 This is a graph showing the effect of composite surfactant on the Zeta potential of coal samples;

[0038] Figure 8 The figures are the total ion chromatogram and group component distribution diagram of the liquid product (Tr-Rh) of the example. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] In order to improve the degradation rate of coal, the effects of composite surfactants Tr (octylphenoxy polyethoxy) and Rh (rhamnolipid) on the degradation of coal by Bacillus XK1 were studied. The present invention discloses a method for enhancing the degradation of coal by microorganisms to produce oxygen-containing chemicals using composite surfactants. The flow diagram of the method is shown in FIG. Figure 1 Based on the interaction between low-rank coal, Bacillus XK1 and surfactants, the mechanism of surfactant action in coal biodegradation was explored, providing a theoretical basis for the application of surfactants in coal biodegradation.

[0041] 1. Biodegradation experiment

[0042] The degradation experiment was carried out under aerobic conditions, and the specific process was as follows:

[0043] Bacillus XK1 was selected on an agar plate, cultured in 200 mL of liquid culture medium until OD600 reached 0.5, and a fermentation broth was obtained at 30°C.

[0044] 10 mL of fermentation broth, 0.5 g of sterilized coal sample and sterile surfactant were added to 50 mL of liquid culture medium and cultured at 30°C and 160 r / min for 10 days. The final concentrations of the two combined surfactants were 400 mg / L, 600 mg / L, 800 mg / L, 1000 mg / L and 1200 mg / L.

[0045] After the degradation experiment, the culture sample was centrifuged at 10000 rpm for 20 min to obtain the supernatant and the residue.

[0046] The supernatant was then filtered through a 0.22 μm membrane filter and the absorbance at 450 nm was measured.

[0047] The residue was washed five times with 50 mL of distilled water and dried at 90 °C at constant weight for 8 h.

[0048] The inoculated medium without coal, the uninoculated medium with coal, and the inoculated medium with coal without surfactant were used as controls. Three parallel experiments were conducted in each group. The results are expressed as mean values ​​(p=0.05, n=3±SD).

[0049] In order to further improve the degradation rate, the effect of composite surfactants on the degradation activity was studied, and the composite surfactant Tr-Rh was selected for study. The final concentrations of the two combined surfactants (Tr:Rh) were 400, 600, 800, 1000, and 1200 mg / L, respectively. The ratios of Tr and Rh were 4:0, 3:1, 2:2, 1:3, and 0:4, respectively. A single surfactant was used as a control.

[0050] 2. Determination of biodegradation degree

[0051] The biodegradation degree of coal was evaluated by the following two methods:

[0052] (1) Weighing method

[0053] The residual coal was repeatedly washed with distilled water to sterilize it, and dried at 90 °C for 8 h. The degradation rate of the coal sample was calculated according to formula (1).

[0054] η= (1- w1 / w0)×100% (1)

[0055] Where η is the biodegradation rate of coal, w0 is the mass before biodegradation (g), and w1 is the mass after biodegradation (g).

[0056] (2) A450 value method

[0057] After the biodegradation experiment, the degradation liquid was centrifuged (10000rpm, 20min) and filtered (0.22μm membrane filter) to obtain the supernatant and residual coal. The absorbance of the supernatant at 450nm was determined by UV-visible spectroscopy. The absorbance was positively correlated with the degradation rate.

[0058] 3. Growth curve and alkaline protease activity detection

[0059] Determination of bacterial growth curve: Add activated Bacillus XK1 to liquid culture medium (200mL, 500mL flask) until the OD600 of the liquid culture medium reaches 0.5. Add 1mL of fermentation broth and surfactant to 400mL of liquid culture medium and culture at 30℃ and 160r / min. Collect the culture broth of Bacillus XK1 under sterile conditions every day, and measure the absorbance of the culture broth at 600nm using a UV spectrophotometer, such as Figure 4 shown.

[0060] The activity of alkaline protease was determined by the Folin-phenol method.

[0061] Take 1mL 1% casein solution (pH = 9) and 1mL Bacillus XK1 culture medium and react at 40°C for 10 minutes. After the reaction is completed, immediately add 2ml of 0.4M trichloroacetic acid solution and react at 40°C for 15min. After the reaction is completed, centrifuge the mixture at 2000rpm for 3min to obtain the supernatant. Take 1mL supernatant, 5mL sodium carbonate solution (0.4M) and 1mL Folin reagent, mix, react at 40°C for 20min, and finally measure the absorbance at 680nm.

[0062] The control group did not add Bacillus XK1 culture medium. Under standard conditions, 1 unit of alkaline protease activity is equivalent to releasing 1 μmol / L tyrosine per minute. Figure 6 shown.

[0063] The enzyme activity was calculated according to formula (3):

[0064]

[0065] Wherein, U is the enzyme activity (U / mL), A1 is the absorbance of the experimental group samples at 680nm, A2 is the absorbance of the control group samples at 680nm, K is the tyrosine concentration corresponding to the OD680 value of 1 in the standard curve, and the K value is 98.99.

[0066] 4. Effect of single surfactant on degradation activity

[0067] Figure 2 The results show that in the presence of Rh and Tr, the absorbance value A450 of the supernatant first increased and then decreased. When the concentrations of Rh and Tr were 1000 mg / L and 1000 mg / L, the maximum A450 values ​​were 0.68 and 0.71, respectively.

[0068] When the Tr concentration was lower than 1000 mg / L, the A450 value increased with the increase of Tr. This may be because low concentration of Tr can increase the hydrophilicity of coal samples, promote the adsorption and secretion of Bacillus XK1 on coal, and improve the degradation rate.

[0069] When the Tr concentration was greater than 1000 mg / L, the A450 value decreased. The reason may be that the high concentration of Tr formed a large number of micelles, which prevented the contact between bacteria and their secretions and coal, resulting in a lower A450 value.

[0070] Depend on Figure 2 It can be seen that with the increase of surfactant concentration, the surface tension of the liquid product first decreases and then tends to equilibrium. This is because the surfactant molecules have polar hydrophilic groups and non-polar hydrophobic groups. In the culture medium, the surfactant molecules are arranged on the surface of the solution, changing the contact state between the medium and the air, thereby reducing the surface tension of the solution. Compared with Rh, the surface tension of the supernatant is the lowest when Tr is present, which is conducive to the contact between bacteria and their secretions and coal, and improves the degradation rate.

[0071] 5. Effect of composite surfactants on degradation activity

[0072] The effect of composite surfactant on degradation activity, degradation rate and degradation time, such as Figure 3 When the concentration of Tr:Rh is 600 mg / L and the ratio of Tr to Rh is 2:2, the degradation activity of Bacillus XK1 is the best, the A450 value is 1.15, and the degradation rate is 77.8%. At this time, Tr and Rh have the strongest synergistic effect.

[0073] The degradation rate of the control group was only 52.4%. Compared with the control group (non-surfactant), the degradation rate of the mixed surfactant Tr-Rh increased by 25.4%. The reason may be that when the non-ionic surfactant Tr is combined with the biosurfactant Rh, the non-ionic surfactant molecule Tr is inserted into the micelle of the biosurfactant Rh, thereby increasing the hydrophilicity and degradation rate of the head of the bound surfactant.

[0074] Effects of different surfactants on degradation time Figure 3 (c). When A450 reaches the maximum value, the optimal degradation time under the action of Rh, Tr and Tr-Rh is 9d, 9d and 8d, respectively. Compared with non-surfactants, the presence of single surfactants and composite surfactants shortens the degradation time, and the presence of composite surfactant Tr-Rh shortens the degradation time by 2 days. The reason for this phenomenon is that when the non-ionic surfactant Tr is used together with the biosurfactant Rh, the electrostatic repulsion of the biosurfactant Rh is reduced, the hydrophilicity of the coal sample and the adsorption rate of Bacillus XK1 on the surface of the coal sample increase, and the degradation time is shortened.

[0075] 6. Effect of composite surfactants on bacterial growth characteristics

[0076] Figure 4 The figure shows the effect of composite surfactants on the growth of Bacillus XK1. The three surfactants have similar effects on the growth of Bacillus XK1, and all three surfactants are conducive to the growth of bacteria. In the medium containing surfactants, Bacillus XK1 did not grow significantly in the first two days. This is because Bacillus XK1 needs to adapt to the environment containing surfactants, so it grows slowly. However, when the culture time is 2 to 4 days, Bacillus XK1 increases rapidly in the medium containing surfactants. Compared with the single surfactants Tr and Rh, in the medium containing composite surfactant Tr-Rh, when the culture time is 4 days, OD600 reaches a maximum value of 2.891. This may be because the composite surfactant Tr-Rh can adsorb to the cell surface, change the hydrophobicity of the cell surface, affect the transport between microbial cells and nutrients, and promote cell growth.

[0077] In the biodegradation process, alkaline protease plays a vital role, and its degradation rate is positively correlated with the activity of alkaline protease, that is, as the activity of alkaline protease increases, the degradation rate also increases accordingly. It can be seen that the analysis of alkaline protease activity is of great significance for exploring the effect of surfactants on coal biodegradation.

[0078] Figure 5The figure shows the effect of the composite surfactant on the activity of alkaline protease secreted by Bacillus XK1. The study found that the three surfactants had similar effects on the activity of alkaline protease, and all of them could promote the activity of alkaline protease. It is particularly noteworthy that compared with the use of Rh and Tr alone, the activity of alkaline protease increased in the presence of the mixed surfactant Tr-Rh. When the culture time reached 5 days, the alkaline protease activity reached its maximum value. In the culture medium containing Tr-Rh, the alkaline protease activity could reach 102.33U / mL. Compared with the control group (no surfactant added), the alkaline protease activity increased by 45.22% under the action of Tr-Rh. The reason for this phenomenon may be that the mixed surfactant Tr-Rh participated in the metabolic process of Bacillus XK1, which increased the protein expression and ultimately promoted the growth of Bacillus XK1 and the activity of alkaline protease.

[0079] 7. Effect of composite surfactant on surface properties of coal samples

[0080] Figure 6 The figure shows the effect of composite surfactant on the contact angle of coal samples. Under the condition of culture medium, the contact angle of coal samples is 84.2°, which is due to the large amount of long-chain alkanes and aromatic rings in coal. In the presence of Rh, Tr and Tr-Rh, the contact angles of coal samples are 35.4°, 34.9° and 16.2° respectively. Therefore, in the presence of mixed surfactant Tr-Rh, the contact angle of coal samples is the lowest, which indicates that mixed surfactant can significantly reduce the hydrophobicity of coal samples, improve the wettability of coal, facilitate the contact between coal and microorganisms, and improve the degradation rate.

[0081] Figure 7 The figure shows the effect of composite surfactant on the Zeta potential of coal samples. The surface of coal samples is negatively charged, which is mainly due to the ionization of functional groups such as carboxyl and hydroxyl groups on the surface of coal samples. As the pH value increases, the absolute value of the zeta potential of coal samples increases. The main reason is that the hydrogen ions on the surface of coal samples are freed as the pH value increases. The negative charge of coal samples increases and the hydrophilicity increases. In the solution, Rh will dissociate negatively charged ions, which are adsorbed on the surface of coal samples with their tails facing the surface of coal samples and their heads pointing to the solution, making the surface of coal samples more negatively charged. The non-ionic surfactant Tr has little effect on the zeta potential of coal samples. The reason for this phenomenon is that non-ionic surfactants are not ionized in the culture medium and mainly exist in the form of molecules or micelles. Compared with Rh and Tr, the electronegativity of Tr-Rh increases. This may be because the non-ionic surfactant Tr can shorten the electrostatic distance in the double electron layer arrangement of the anionic surfactant Rh, that is, shorten the Debye length, which leads to an increase in the electronegativity of the coal sample surface, making it easier to adsorb Bacillus XK1, thereby increasing the degradation rate.

[0082] 8. Effect of composite surfactants on degradation products

[0083] Figure 8 The total ion chromatograms and group component distribution diagrams of LP(Tr-Rh) and LP. LP(Tr-Rh) and LP are represented as liquid phase degradation products in the presence of composite surfactants / no surfactants, respectively. It can be seen that a total of 49 different compounds were detected in the extracts, and the main components detected were alkanes, aromatic hydrocarbons, alcohols, esters and amines. The contents of oxygenated organic matter (OOC) in LP(Tr-Rh) and LP were 30.26% and 12.3%, respectively.

[0084] The above results show that the composite surfactant promotes the biodegradation of OOC by Bacillus XK1 in the process of coal production, and OOC can be used as a solvent or as a raw material for the synthesis of organic matter. In addition, in the presence of the composite surfactant Tr-Rh, the molecular weight of the degradation liquid product is much smaller than that of the coal sample, indicating that the molecular structure of the coal sample is destroyed by Bacillus XK1, releasing small molecular weight substances.

[0085] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by technicians in this technical field within the essential scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A method for producing oxygen-containing chemicals by using composite surfactants to enhance microbial degradation of coal, characterized in that: The specific steps are: Step 1, selecting Bacillus XK1 on an agar plate, culturing in a liquid culture medium at 30° C. until OD600 reaches 0.5, and obtaining a fermentation broth; Step 2, adding fermentation liquid, sterilized coal sample and sterile surfactant of different concentrations into liquid culture medium, degrading under the conditions of 30°C and 160r / min, and culturing for 10 days; Step 3: After the degradation experiment is completed, the culture sample is centrifuged at 10,000 rpm for 20 min to obtain the supernatant and the residue; Step 4: The supernatant in step 3 is filtered with a 0.22 μm membrane filter, and the absorbance at 450 nm is measured; Step 5: Wash the residue in step 3 with 50 mL of distilled water for 5 times, dry at 90° C. for 8 h to obtain residual coal, and calculate the biodegradation rate of the coal.

2. The method for producing oxygen-containing chemicals by using composite surfactant to enhance microbial degradation of coal as claimed in claim 1, characterized in that: In step 2, the surfactant is at least one of Tr or Rh, the final concentration of the two combined surfactants is 400 mg / L, 600 mg / L, 800 mg / L, 1000 mg / L and 1200 mg / L, and the ratio of Tr to Rh is 4:0, 3:1, 2:2, 1:3, 0:4 respectively.

3. The method for producing oxygen-containing chemicals by using composite surfactant to enhance microbial degradation of coal as claimed in claim 2, characterized in that: In step 4, the method for determining the biodegradability of coal includes the following two methods: (1) Weighing method The residual coal was repeatedly washed with distilled water to sterilize and dried at 90℃ for 8h. The degradation rate of the coal sample was calculated as follows: η=(1-w1 / w0)×100% Where η is the biodegradation rate of coal; w0 is the mass before biodegradation, g; w1 is the mass after biodegradation, g; (2) A450 value method After the biodegradation experiment, the degradation liquid was centrifuged and filtered through a 0.22 μm membrane filter to obtain the supernatant and residual coal. The absorbance of the supernatant at 450 nm was measured by UV-visible spectroscopy, and the absorbance was positively correlated with the degradation rate.

4. The method for producing oxygen-containing chemicals by using composite surfactant to enhance microbial degradation of coal as claimed in claim 3, characterized in that: Effect of composite surfactant on degradation activity: When the concentration of Tr:Rh was 600 mg / L and the ratio of Tr to Rh was 2:2, the degradation activity of Bacillus XK1 was good, the A450 value was 1.15, and the degradation rate was 77.8%.

5. The method for producing oxygen-containing chemicals by using composite surfactant to enhance microbial degradation of coal as claimed in claim 4, characterized in that: Effect of composite surfactants on bacterial growth characteristics: Compared with the single surfactants Tr and Rh, in the culture medium containing the composite surfactant Tr-Rh, when the culture time was 4d, the OD600 reached a maximum value of 2.

891.

6. The method for producing oxygen-containing chemicals by using composite surfactant to enhance microbial degradation of coal as claimed in claim 5, characterized in that: Effect of composite surfactant on the activity of alkaline protease secreted by Bacillus XK1: In the culture medium containing Tr-Rh, the activity of alkaline protease reached 102.33U / mL, which was increased by 45.22% compared with that without adding surfactant.

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