Bacteria for decomposing oil

Abstract

The invention discloses a bacterial agent for decomposing grease, which is prepared by the following method: inoculating a composite bacterial species in a seed culture medium to obtain a seed liquid, and then inoculating the seed liquid into an expansion culture medium for expansion culture. The bacterial agent for decomposing grease disclosed by the invention has good grease decomposition ability.

Description

Technical Field

[0001] The invention relates to the technical field of grease-decomposing bacteria, and in particular to a bacterial agent for decomposing grease. Background Art

[0002] In recent years, the catering industry has risen and grown rapidly with the improvement of residents' living standards, but the discharge of catering wastewater has also increased. Catering wastewater contains high concentrations of grease and a large amount of suspended solids, which is one of the serious pollution sources in some places. At present, the treatment process for grease wastewater mainly includes ecological treatment, biological treatment and other means. When using bioreactors to treat grease wastewater, grease will be wrapped on the surface of sludge, hindering mass transfer and greatly reducing the conversion rate of substrates. In addition, filamentous bacteria multiply in large numbers in the reactor, and grease adheres to the surface of sludge, which can easily cause sludge sedimentation difficulties and cause a large amount of active sludge loss. The reactor is also prone to problems such as blockage and the spread of foul odors. Therefore, in the process of catering wastewater treatment, microorganisms with high-efficiency grease degradation ability need to be used to solve difficult-to-degrade substances such as grease and protein. It is of great significance to separate and screen microorganisms with high-efficiency grease degradation ability, develop high-efficiency and low-cost microbial agents and use them for grease wastewater treatment.

[0003] Polylactic acid is a new type of environmentally friendly fiber that was industrialized in the 1990s. It has the advantages of abundant sources, biodegradability, excellent physical and mechanical properties and processing performance. However, polylactic acid fiber belongs to aliphatic polyester fiber, and there are a large number of ester bonds in its molecular structure, which has poor hydrophilicity, affects compatibility with other substances, and the degradation cycle is difficult to control. These problems have prompted people to conduct in-depth research on modified polylactic acid. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a bacterial agent for decomposing oil.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A bacterial agent for decomposing grease is prepared by the following method: inoculating a composite bacterial species into a seed culture medium to obtain a seed solution, and then inoculating the seed solution into an expansion culture medium to obtain the bacterial agent for decomposing grease.

[0007] The composite bacterial strain is one or a mixture of more than one of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides.

[0008] Preferably, the composite bacterial species is a mixture of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides in a mass ratio of (1-10):(1-10):(1-10):(1-10):(1-10).

[0009] Most preferably, the composite bacterial species is a mixture of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides in a mass ratio of 5:3:3:2:3.

[0010] The components of the seed culture medium are: sodium chloride, ammonium acetate, magnesium nitrate, calcium nitrate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, yeast extract, chitosan and distilled water.

[0011] Preferably, the components of the seed culture medium are: 1-2 parts by weight of sodium chloride, 4-5 parts by weight of ammonium acetate, 0.1-0.4 parts by weight of magnesium nitrate, 0.1-0.5 parts by weight of calcium nitrate, 0.1-1.5 parts by weight of dipotassium hydrogen phosphate, 0.1-2 parts by weight of potassium dihydrogen phosphate, 8-12 parts by weight of yeast extract, 0.1-1.2 parts by weight of chitosan, and 900-1100 parts by weight of distilled water.

[0012] Most preferably, the components of the seed culture medium are: 1.5 parts by weight of sodium chloride, 4.5 parts by weight of ammonium acetate, 0.2 parts by weight of magnesium nitrate, 0.2 parts by weight of calcium nitrate, 1.2 parts by weight of dipotassium hydrogen phosphate, 0.5 parts by weight of potassium dihydrogen phosphate, 10 parts by weight of yeast extract, 1 part by weight of chitosan, and 1000 parts by weight of distilled water.

[0013] The culture conditions of the seed solution are: the seed solution is obtained after culturing under light.

[0014] Preferably, the culture conditions of the seed solution are: culture at 27-38°C, 60-180rpm, 800-1200lux light until the bacterial concentration reaches 10 8 -10 10 CFU / mL to obtain the seed solution; the pH of the seed culture medium is 6.8-7.2.

[0015] Most preferably, the culture conditions of the seed solution are: culture at 30°C, 120rpm, 1000lux light until the bacterial concentration reaches 10 9 CFU / mL to obtain the seed solution; the pH of the seed culture medium is 7.0.

[0016] The components of the expansion culture medium are: beef extract, peptone, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, distilled water, and hydrophilic porous polylactic acid particles.

[0017] Preferably, the components of the expansion medium are: 8-12 parts by weight of beef extract, 7-15 parts by weight of peptone, 0.1-5.5 parts by weight of potassium dihydrogen phosphate, 0.1-3 parts by weight of dipotassium hydrogen phosphate, 900-1100 parts by weight of distilled water, and 550-650 parts by weight of hydrophilic porous polylactic acid particles.

[0018] Most preferably, the components of the expansion culture medium are: 10 parts by weight of beef extract, 10 parts by weight of peptone, 5 parts by weight of potassium dihydrogen phosphate, 2 parts by weight of dipotassium hydrogen phosphate, 1000 parts by weight of distilled water, and 600 parts by weight of hydrophilic porous polylactic acid particles.

[0019] The culture conditions for the expanded culture are: adding the seed liquid to the expanded culture medium and culturing under light; centrifuging and preserving the precipitate to obtain the bacterial agent for decomposing oil.

[0020] Preferably, the culture conditions for the expanded culture are: adding 8-20 mL of the seed solution to 1 L of the expanded culture medium, culturing at 27-38°C, 120-240 rpm, and 800-1200 lux; the pH of the expanded culture medium is 6.8-7.2; when the bacterial cell concentration reaches 10 14 -10 16 CFU / mL, centrifuge, and store the precipitate in a lightless environment with a relative humidity of (70-85)% and an oxygen content of (5-20)% to obtain the bacterial agent for decomposing grease.

[0021] Most preferably, the culture conditions for the expanded culture are: adding 10 mL of the seed solution to 1 L of the expanded culture medium, culturing at 30°C, 180 rpm, and 1000 lux; the pH of the expanded culture medium is 7.0; when the bacterial cell concentration reaches 10 15 CFU / mL, centrifuge, and store the precipitate in a lightless environment with a relative humidity of 80% and an oxygen content of 15% to obtain the bacterial agent for decomposing grease.

[0022] The preparation method of the hydrophilic porous polylactic acid particles is:

[0023] ① Mix and stir porcine pancreatic lipase, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and distilled water at 43-48°C to obtain an enzyme treatment solution;

[0024] ② The porous polylactic acid particles and the enzyme treatment solution obtained in step ① are mixed, stirred, filtered and then dried to obtain the hydrophilic porous polylactic acid particles.

[0025] Preferably, the preparation method of the hydrophilic porous polylactic acid particles is:

[0026] ① Mix porcine pancreatic lipase, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and distilled water at 43-48°C and stir at 240-360 rpm for 20-40 min to obtain an enzyme treatment solution with a pH of 8.3-8.6 and a porcine pancreatic lipase concentration of 0.4-0.7 g / L;

[0027] ② The porous polylactic acid particles and the enzyme treatment solution obtained in step ① are mixed in a mass ratio of (0.8-1.2): (2-4), stirred at 40-45°C and 240-360rpm for 10-16h, filtered and dried at 45-55°C for 4-6h to obtain the hydrophilic porous polylactic acid particles.

[0028] Most preferably, the preparation method of the hydrophilic porous polylactic acid particles is:

[0029] ① Mix porcine pancreatic lipase, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and distilled water at 45°C and stir at 300 rpm for 30 min to obtain an enzyme treatment solution with a pH of 8.5 and a porcine pancreatic lipase concentration of 0.5 g / L;

[0030] ② The porous polylactic acid particles and the enzyme treatment solution obtained in step ① were mixed in a mass ratio of 1:2.5, stirred at 43°C and 300 rpm for 12 hours, filtered and dried at 50°C for 5 hours to obtain the hydrophilic porous polylactic acid particles.

[0031] The preparation method of the porous polylactic acid particles is:

[0032] (1) Mixing polylactic acid, cellulose acetate and n-butyl propionate and stirring to obtain a two-phase solution;

[0033] (2) mixing n-pentanol and a precipitation aid and stirring to obtain a precipitating agent;

[0034] ⑶ Cooling and stirring the two-phase solution obtained in step ⑴, and adding the precipitant obtained in step ⑵ to obtain the porous polylactic acid particles.

[0035] Preferably, the preparation method of the porous polylactic acid particles is:

[0036] (1) At 90-96° C., polylactic acid, cellulose acetate and n-butyl propionate were mixed in a mass ratio of (1-1.5):(0.8-1.2):(6-9) and stirred at 540-660 rpm for 2-3.5 h to obtain a biphasic solution;

[0037] (2) At 28-35° C., n-pentanol and a precipitation aid were mixed in a mass ratio of (90-110):(0.25-0.3) and stirred at 540-660 rpm for 2-5 h to obtain a precipitant;

[0038] ⑶ Cool the two-phase solution obtained in step ⑴ to 45-55°C and stir at 300-420rpm, and add the precipitant obtained in step ⑵ and cooled to 1-5°C to obtain the porous polylactic acid particles; the mass ratio of the two-phase solution and the precipitant is (0.8-1.5):(4-6).

[0039] Most preferably, the preparation method of the porous polylactic acid particles is:

[0040] (1) At 95°C, polylactic acid, cellulose acetate and n-butyl propionate were mixed in a mass ratio of 1.2:1:7 and stirred at 600 rpm for 2.5 h to obtain a biphasic solution;

[0041] (2) At 30° C., n-pentanol and a precipitation aid were mixed in a mass ratio of 100:0.28 and stirred at 600 rpm for 3 h to obtain a precipitant;

[0042] ⑶ The two-phase solution obtained in step ⑴ is cooled to 50°C and stirred at 360rpm, and the precipitant obtained in step ⑵ and cooled to 4°C is added to obtain the porous polylactic acid particles; the mass ratio of the two-phase solution to the precipitant is 1:5.

[0043] The precipitation aid is one of xanthan gum and carbomer or a mixture of the two. Preferably, the precipitation aid is a mixture of xanthan gum and carbomer in a mass ratio of (0.1-5):(0.1-5). Most preferably, the precipitation aid is a mixture of xanthan gum and carbomer in a mass ratio of 1:1.

[0044] The porous carrier can be used to store the bacteria in the porous structure of the carrier, thereby enhancing the stability of the obtained bacterial preparation and the ease of storage and transportation. When in use, the polylactic acid particles prepared by the present invention have a suitable surface tension range due to their unique surface structure, and therefore have good hydrophilicity and lipophilicity and spontaneously diffuse between the water and oil phases. At the same time, the bacteria are quickly sucked out of the porous carrier due to the polarity of water and the surface tension, and fully diffuse at the water-oil interface, and then carry out the physiological activity of decomposing oil.

[0045] Xanthan gum and carbomer have different molecular structures, and the ratio of polar groups to non-polar groups in their respective molecules is different, so that the two have different surface tensions in a two-phase solution obtained by stirring polylactic acid, cellulose acetate and n-butyl propionate. The difference in surface tension between the two makes the porous polylactic acid precipitated when n-pentanol is added have different surface morphologies; when xanthan gum and carbomer are compounded and used as precipitation aids, the average particle size distribution and wetting angle range of the porous polylactic acid obtained after proportion control are more conducive to the final bacterial agent for decomposing grease being evenly distributed at the oil-water interface of grease wastewater, which is convenient for the bacteria in the preparation to quickly diffuse out and reproduce and decompose grease, thereby improving the ability of the bacterial agent to decompose grease.

[0046] Polyfiber belongs to aliphatic polyester, and there are a large number of ester bonds in the molecular structure, which has poor hydrophilicity and will lead to a long degradation cycle; the use of porcine pancreatic lipase to modify polylactic acid can convert the ester bonds exposed in the outer layer of polylactic acid particles into polar hydroxyl and carboxyl groups, so that the obtained polylactic acid particles can be better distributed at the interface between the water phase and the oil phase, so that the bacteria in the particles can be better diffused at the oil-water interface, which is conducive to the rapid reproduction and increase of the bacteria; and when the decomposition activity occurs at the oil-water interface, the glycerol and low-level fatty acids in the decomposition products can quickly diffuse into the water to avoid the accumulation of decomposition products. The oil decomposition effect of the microbial agent for decomposing oils and fats obtained by hydrophilic modification of porous polylactic acid particles with porcine pancreatic lipase is better than that of the fluorescent Pseudomonas lipase, because porcine pancreatic lipase can achieve higher activity under milder and lower cost conditions due to its unique spatial three-dimensional molecular structure, and the ratio of polar groups to ester groups on the surface of the obtained polylactic acid particles is more moderate, which is more conducive to the uniform distribution of the microbial agent at the oil-water interface, and improves the ability of the microbial agent to decompose oils and fats.

[0047] Carboxymethyl cellulose has more polar groups. The presence of these polar groups will result in particles with poor surface uniformity when porous polylactic acid particles are obtained by precipitation with n-pentanol. These particles with poor surface uniformity will rub and collide with each other and break and shatter during subsequent transportation and storage, that is, they will become ineffective before they are officially put into use. The proportion of polar groups in cellulose acetate is lower, and polylactic acid particles with better surface uniformity and easy transportation and storage can be precipitated, thereby improving the ability of the bacterial agent to decompose oil.

[0048] The beneficial effects of the present invention are as follows: a bacterial agent for decomposing grease and a preparation method thereof are provided, and a bacterial agent for decomposing grease having good grease decomposition ability is obtained. DETAILED DESCRIPTION

[0049] The above content of the present invention is further described in detail below in conjunction with specific implementation modes, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments.

[0050] Introduction of some raw materials in this application:

[0051] Bacillus subtilis: Bacillus subtilis, CGMCC: 1.9083, purchased from China General Microbiological Culture Collection Center.

[0052] Bacillus licheniformis, CGMCC: 1.6510, was purchased from China General Microbiological Culture Collection Center.

[0053] Pseudomonas chengduensis, CGMCC: 1.15626, was purchased from China General Microbiological Culture Collection Center.

[0054] Pseudomonas xanthomarina: Pseudomonas xanthomarina, CGMCC: 1.10904, purchased from China General Microbiological Culture Collection Center.

[0055] Rhodobacter sphaeroides, CGMCC: 1.2569, was purchased from China General Microbiological Culture Collection Center.

[0056] Porcine pancreatic lipase, CAS: 9001-62-1, purchased from Shanghai Aladdin Co., Ltd., Enzyme commission (EC) Number: 3.1.1.3, purity: 15-35 units / mg, RTECS: TO9776500, item number: L111237-100g.

[0057] Polylactic acid, CAS: 26100-51-6, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number: P169115-5g, molecular weight: 60000.

[0058] Cellulose acetate, CAS: 9012-09-3, was purchased from J&K Technology Co., Ltd., product number: C17782.

[0059] Xanthan gum, CAS: 11138-66-2, purchased from Sane Chemical Technology (Shanghai) Co., Ltd., brand: Sigma-Aldrich, number: 42663, specification and purity: USP grade.

[0060] Carbomer, CAS: 54182-57-9, was purchased from Hubei Xinrunde Chemical Co., Ltd., product number: Carbomer 934.

[0061] Yeast extract, CAS: 8013-01-2, was purchased from West Asia Chemical Technology (Shandong) Co., Ltd., order number: A48126-500g, specification: BR.

[0062] Chitosan, CAS: 9012-76-4, was purchased from West Asia Chemical Technology (Shandong) Co., Ltd., order number: A14854-100g, specification: BR.

[0063] Beef extract, CAS: 68990-09-0, purchased from West Asia Chemical Technology (Shandong) Co., Ltd., order number: A21486-500g, specification: BR.

[0064] Peptone, CAS: 73049-73-7, purchased from West Asia Chemical Technology (Shandong) Co., Ltd., order number: A12846-500g, specification: BR.

[0065] Pseudomonas fluorescens lipase, CAS: 9001-62-1, purchased from Shanghai Aladdin Co., Ltd., Enzyme commission (EC) Number: 232-619-9, purity ≥ 20,000 U / g, RTECS: TO9776500, item number: L299014-50g.

[0066] Carboxymethyl cellulose, CAS: 9004-32-4, was purchased from West Asia Chemical Technology (Shandong) Co., Ltd., order number: A11895-250g, specification: BR.

[0067] Example 1

[0068] A bacterial agent for decomposing grease is prepared by the following method: a composite bacterial species is inoculated into a seed culture medium to obtain a seed solution, and then the seed solution is inoculated into an expansion culture medium to obtain the bacterial agent for decomposing grease.

[0069] The composite bacterial strain is a mixture of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides in a mass ratio of 5:3:3:2:3.

[0070] The seed culture medium comprises: 1.5 parts by weight of sodium chloride, 4.5 parts by weight of ammonium acetate, 0.2 parts by weight of magnesium nitrate, 0.2 parts by weight of calcium nitrate, 1.2 parts by weight of dipotassium hydrogen phosphate, 0.5 parts by weight of potassium dihydrogen phosphate, 10 parts by weight of yeast extract, 1 part by weight of chitosan, and 1000 parts by weight of distilled water.

[0071] The culture conditions of the seed solution are: culture at 30°C, 120rpm, 1000lux light until the bacterial concentration reaches 10 9CFU / mL to obtain the seed solution; the pH of the seed culture medium is 7.0.

[0072] The components of the expansion culture medium are: 10 parts by weight of beef extract, 10 parts by weight of peptone, 5 parts by weight of potassium dihydrogen phosphate, 2 parts by weight of dipotassium hydrogen phosphate, 1000 parts by weight of distilled water, and 600 parts by weight of hydrophilic porous polylactic acid particles.

[0073] The culture conditions of the expansion culture are as follows: 10 mL of seed solution is added to 1 L of expansion culture medium, and cultured at 30°C, 180 rpm, and 1000 lux; the pH of the expansion culture medium is 7.0; when the bacterial cell concentration reaches 10 15 CFU / mL, centrifuge, and store the precipitate in a lightless environment with a relative humidity of 80% and an oxygen content of 15% to obtain the bacterial agent for decomposing grease.

[0074] The preparation method of the hydrophilic porous polylactic acid particles is:

[0075] ① Mix porcine pancreatic lipase, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and distilled water at 45°C and stir at 300 rpm for 30 min to obtain an enzyme treatment solution with a pH of 8.5 and a porcine pancreatic lipase concentration of 0.5 g / L;

[0076] ② The porous polylactic acid particles and the enzyme treatment solution obtained in step ① were mixed in a mass ratio of 1:2.5, stirred at 43°C and 300 rpm for 12 hours, filtered and dried at 50°C for 5 hours to obtain the hydrophilic porous polylactic acid particles.

[0077] The preparation method of the porous polylactic acid particles is:

[0078] (1) At 95°C, polylactic acid, cellulose acetate and n-butyl propionate were mixed in a mass ratio of 1.2:1:7 and stirred at 600 rpm for 2.5 h to obtain a biphasic solution;

[0079] (2) At 30° C., n-pentanol and a precipitation aid were mixed in a mass ratio of 100:0.28 and stirred at 600 rpm for 3 h to obtain a precipitant;

[0080] ⑶ The two-phase solution obtained in step ⑴ is cooled to 50°C and stirred at 360rpm, and the precipitant obtained in step ⑵ and cooled to 4°C is added to obtain the porous polylactic acid particles; the mass ratio of the two-phase solution to the precipitant is 1:5.

[0081] The precipitation aid is a mixture of xanthan gum and carbomer in a mass ratio of 1:1.

[0082] Example 2

[0083] A bacterial agent for decomposing grease is prepared by the following method: a composite bacterial species is inoculated into a seed culture medium to obtain a seed solution, and then the seed solution is inoculated into an expansion culture medium to obtain the bacterial agent for decomposing grease.

[0084] The composite bacterial strain is a mixture of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides in a mass ratio of 5:3:3:2:3.

[0085] The seed culture medium comprises: 1.5 parts by weight of sodium chloride, 4.5 parts by weight of ammonium acetate, 0.2 parts by weight of magnesium nitrate, 0.2 parts by weight of calcium nitrate, 1.2 parts by weight of dipotassium hydrogen phosphate, 0.5 parts by weight of potassium dihydrogen phosphate, 10 parts by weight of yeast extract, 1 part by weight of chitosan, and 1000 parts by weight of distilled water.

[0086] The culture conditions of the seed solution are: culture at 30°C, 120rpm, 1000lux light until the bacterial concentration reaches 10 9 CFU / mL to obtain the seed solution; the pH of the seed culture medium is 7.0.

[0087] The components of the expansion culture medium are: 10 parts by weight of beef extract, 10 parts by weight of peptone, 5 parts by weight of potassium dihydrogen phosphate, 2 parts by weight of dipotassium hydrogen phosphate, 1000 parts by weight of distilled water, and 600 parts by weight of hydrophilic porous polylactic acid particles.

[0088] The culture conditions of the expansion culture are as follows: 10 mL of seed solution is added to 1 L of expansion culture medium, and cultured at 30°C, 180 rpm, and 1000 lux; the pH of the expansion culture medium is 7.0; when the bacterial cell concentration reaches 10 15 CFU / mL, centrifuge, and store the precipitate in a lightless environment with a relative humidity of 80% and an oxygen content of 15% to obtain the bacterial agent for decomposing grease.

[0089] The preparation method of the hydrophilic porous polylactic acid particles is:

[0090] ① Mix porcine pancreatic lipase, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and distilled water at 45°C and stir at 300 rpm for 30 min to obtain an enzyme treatment solution with a pH of 8.5 and a porcine pancreatic lipase concentration of 0.5 g / L;

[0091] ② The porous polylactic acid particles and the enzyme treatment solution obtained in step ① were mixed in a mass ratio of 1:2.5, stirred at 43°C and 300 rpm for 12 hours, filtered and dried at 50°C for 5 hours to obtain the hydrophilic porous polylactic acid particles.

[0092] The preparation method of the porous polylactic acid particles is:

[0093] (1) At 95°C, polylactic acid, cellulose acetate and n-butyl propionate were mixed in a mass ratio of 1.2:1:7 and stirred at 600 rpm for 2.5 h to obtain a biphasic solution;

[0094] (2) At 30° C., n-pentanol and a precipitation aid were mixed in a mass ratio of 100:0.28 and stirred at 600 rpm for 3 h to obtain a precipitant;

[0095] ⑶ The two-phase solution obtained in step ⑴ is cooled to 50°C and stirred at 360rpm, and the precipitant obtained in step ⑵ and cooled to 4°C is added to obtain the porous polylactic acid particles; the mass ratio of the two-phase solution to the precipitant is 1:5.

[0096] The precipitation aid is xanthan gum.

[0097] Example 3

[0098] A bacterial agent for decomposing grease is prepared by the following method: a composite bacterial species is inoculated into a seed culture medium to obtain a seed solution, and then the seed solution is inoculated into an expansion culture medium to obtain the bacterial agent for decomposing grease.

[0099] The composite bacterial strain is a mixture of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides in a mass ratio of 5:3:3:2:3.

[0100] The seed culture medium comprises: 1.5 parts by weight of sodium chloride, 4.5 parts by weight of ammonium acetate, 0.2 parts by weight of magnesium nitrate, 0.2 parts by weight of calcium nitrate, 1.2 parts by weight of dipotassium hydrogen phosphate, 0.5 parts by weight of potassium dihydrogen phosphate, 10 parts by weight of yeast extract, 1 part by weight of chitosan, and 1000 parts by weight of distilled water.

[0101] The culture conditions of the seed solution are: culture at 30°C, 120rpm, 1000lux light until the bacterial concentration reaches 10 9 CFU / mL to obtain the seed solution; the pH of the seed culture medium is 7.0.

[0102] The components of the expansion culture medium are: 10 parts by weight of beef extract, 10 parts by weight of peptone, 5 parts by weight of potassium dihydrogen phosphate, 2 parts by weight of dipotassium hydrogen phosphate, 1000 parts by weight of distilled water, and 600 parts by weight of hydrophilic porous polylactic acid particles.

[0103] The culture conditions of the expansion culture are as follows: 10 mL of seed solution is added to 1 L of expansion culture medium, and cultured at 30°C, 180 rpm, and 1000 lux; the pH of the expansion culture medium is 7.0; when the bacterial cell concentration reaches 10 15CFU / mL, centrifuge, and store the precipitate in a lightless environment with a relative humidity of 80% and an oxygen content of 15% to obtain the bacterial agent for decomposing grease.

[0104] The preparation method of the hydrophilic porous polylactic acid particles is:

[0105] ① Mix porcine pancreatic lipase, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and distilled water at 45°C and stir at 300 rpm for 30 min to obtain an enzyme treatment solution with a pH of 8.5 and a porcine pancreatic lipase concentration of 0.5 g / L;

[0106] ② The porous polylactic acid particles and the enzyme treatment solution obtained in step ① were mixed in a mass ratio of 1:2.5, stirred at 43°C and 300 rpm for 12 hours, filtered and dried at 50°C for 5 hours to obtain the hydrophilic porous polylactic acid particles.

[0107] The preparation method of the porous polylactic acid particles is:

[0108] (1) At 95°C, polylactic acid, cellulose acetate and n-butyl propionate were mixed in a mass ratio of 1.2:1:7 and stirred at 600 rpm for 2.5 h to obtain a biphasic solution;

[0109] (2) At 30° C., n-pentanol and a precipitation aid were mixed in a mass ratio of 100:0.28 and stirred at 600 rpm for 3 h to obtain a precipitant;

[0110] ⑶ The two-phase solution obtained in step ⑴ is cooled to 50°C and stirred at 360rpm, and the precipitant obtained in step ⑵ and cooled to 4°C is added to obtain the porous polylactic acid particles; the mass ratio of the two-phase solution to the precipitant is 1:5.

[0111] The precipitation aid is carbomer.

[0112] Comparative Example 1

[0113] A bacterial agent for decomposing grease is prepared by the following method: a composite bacterial species is inoculated into a seed culture medium to obtain a seed solution, and then the seed solution is inoculated into an expansion culture medium to obtain the bacterial agent for decomposing grease.

[0114] The composite bacterial strain is a mixture of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides in a mass ratio of 5:3:3:2:3.

[0115] The seed culture medium comprises: 1.5 parts by weight of sodium chloride, 4.5 parts by weight of ammonium acetate, 0.2 parts by weight of magnesium nitrate, 0.2 parts by weight of calcium nitrate, 1.2 parts by weight of dipotassium hydrogen phosphate, 0.5 parts by weight of potassium dihydrogen phosphate, 10 parts by weight of yeast extract, 1 part by weight of chitosan, and 1000 parts by weight of distilled water.

[0116] The culture conditions of the seed solution are: culture at 30°C, 120rpm, 1000lux light until the bacterial concentration reaches 10 9 CFU / mL to obtain the seed solution; the pH of the seed culture medium is 7.0.

[0117] The components of the expansion culture medium are: 10 parts by weight of beef extract, 10 parts by weight of peptone, 5 parts by weight of potassium dihydrogen phosphate, 2 parts by weight of dipotassium hydrogen phosphate, 1000 parts by weight of distilled water, and 600 parts by weight of hydrophilic porous polylactic acid particles.

[0118] The culture conditions of the expansion culture are as follows: 10 mL of seed solution is added to 1 L of expansion culture medium, and cultured at 30°C, 180 rpm, and 1000 lux; the pH of the expansion culture medium is 7.0; when the bacterial cell concentration reaches 10 15 CFU / mL, centrifuge, and store the precipitate in a lightless environment with a relative humidity of 80% and an oxygen content of 15% to obtain the bacterial agent for decomposing grease.

[0119] The preparation method of the hydrophilic porous polylactic acid particles is:

[0120] ① Mix porcine pancreatic lipase, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and distilled water at 45°C and stir at 300 rpm for 30 min to obtain an enzyme treatment solution with a pH of 8.5 and a porcine pancreatic lipase concentration of 0.5 g / L;

[0121] ② The porous polylactic acid particles and the enzyme treatment solution obtained in step ① were mixed in a mass ratio of 1:2.5, stirred at 43°C and 300 rpm for 12 hours, filtered and dried at 50°C for 5 hours to obtain the hydrophilic porous polylactic acid particles.

[0122] The preparation method of the porous polylactic acid particles is:

[0123] (1) At 95°C, polylactic acid, cellulose acetate and n-butyl propionate were mixed in a mass ratio of 1.2:1:7 and stirred at 600 rpm for 2.5 h to obtain a biphasic solution;

[0124] (2) Stir n-pentanol at 600 rpm for 3 h at 30°C to obtain a precipitant;

[0125] ⑶ The two-phase solution obtained in step ⑴ is cooled to 50°C and stirred at 360rpm, and the precipitant obtained in step ⑵ and cooled to 4°C is added to obtain the porous polylactic acid particles; the mass ratio of the two-phase solution to the precipitant is 1:5.

[0126] Comparative Example 2

[0127] A bacterial agent for decomposing grease is prepared by the following method: a composite bacterial species is inoculated into a seed culture medium to obtain a seed solution, and then the seed solution is inoculated into an expansion culture medium to obtain the bacterial agent for decomposing grease.

[0128] The composite bacterial strain is a mixture of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides in a mass ratio of 5:3:3:2:3.

[0129] The seed culture medium comprises: 1.5 parts by weight of sodium chloride, 4.5 parts by weight of ammonium acetate, 0.2 parts by weight of magnesium nitrate, 0.2 parts by weight of calcium nitrate, 1.2 parts by weight of dipotassium hydrogen phosphate, 0.5 parts by weight of potassium dihydrogen phosphate, 10 parts by weight of yeast extract, 1 part by weight of chitosan, and 1000 parts by weight of distilled water.

[0130] The culture conditions of the seed solution are: culture at 30°C, 120rpm, 1000lux light until the bacterial concentration reaches 10 9 CFU / mL to obtain the seed solution; the pH of the seed culture medium is 7.0.

[0131] The components of the expansion culture medium are: 10 parts by weight of beef extract, 10 parts by weight of peptone, 5 parts by weight of potassium dihydrogen phosphate, 2 parts by weight of dipotassium hydrogen phosphate, 1000 parts by weight of distilled water, and 600 parts by weight of porous polylactic acid particles.

[0132] The culture conditions of the expansion culture are as follows: 10 mL of seed solution is added to 1 L of expansion culture medium, and cultured at 30°C, 180 rpm, and 1000 lux; the pH of the expansion culture medium is 7.0; when the bacterial cell concentration reaches 10 15 CFU / mL, centrifuge, and store the precipitate in a lightless environment with a relative humidity of 80% and an oxygen content of 15% to obtain the bacterial agent for decomposing grease.

[0133] The preparation method of the porous polylactic acid particles is:

[0134] (1) At 95°C, polylactic acid, cellulose acetate and n-butyl propionate were mixed in a mass ratio of 1.2:1:7 and stirred at 600 rpm for 2.5 h to obtain a biphasic solution;

[0135] (2) At 30° C., n-pentanol and a precipitation aid were mixed in a mass ratio of 100:0.28 and stirred at 600 rpm for 3 h to obtain a precipitant;

[0136] ⑶ The two-phase solution obtained in step ⑴ is cooled to 50°C and stirred at 360rpm, and the precipitant obtained in step ⑵ and cooled to 4°C is added to obtain the porous polylactic acid particles; the mass ratio of the two-phase solution to the precipitant is 1:5.

[0137] The precipitation aid is a mixture of xanthan gum and carbomer in a mass ratio of 1:1.

[0138] Comparative Example 3

[0139] A bacterial agent for decomposing grease is prepared by the following method: a composite bacterial species is inoculated into a seed culture medium to obtain a seed solution, and then the seed solution is inoculated into an expansion culture medium to obtain the bacterial agent for decomposing grease.

[0140] The composite bacterial strain is a mixture of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides in a mass ratio of 5:3:3:2:3.

[0141] The seed culture medium comprises: 1.5 parts by weight of sodium chloride, 4.5 parts by weight of ammonium acetate, 0.2 parts by weight of magnesium nitrate, 0.2 parts by weight of calcium nitrate, 1.2 parts by weight of dipotassium hydrogen phosphate, 0.5 parts by weight of potassium dihydrogen phosphate, 10 parts by weight of yeast extract, 1 part by weight of chitosan, and 1000 parts by weight of distilled water.

[0142] The culture conditions of the seed solution are: culture at 30°C, 120rpm, 1000lux light until the bacterial concentration reaches 10 9 CFU / mL to obtain the seed solution; the pH of the seed culture medium is 7.0.

[0143] The components of the expansion culture medium are: 10 parts by weight of beef extract, 10 parts by weight of peptone, 5 parts by weight of potassium dihydrogen phosphate, 2 parts by weight of dipotassium hydrogen phosphate, 1000 parts by weight of distilled water, and 600 parts by weight of hydrophilic porous polylactic acid particles.

[0144] The culture conditions of the expansion culture are as follows: 10 mL of seed solution is added to 1 L of expansion culture medium, and cultured at 30°C, 180 rpm, and 1000 lux; the pH of the expansion culture medium is 7.0; when the bacterial cell concentration reaches 10 15 CFU / mL, centrifuge, and store the precipitate in a lightless environment with a relative humidity of 80% and an oxygen content of 15% to obtain the bacterial agent for decomposing grease.

[0145] The preparation method of the hydrophilic porous polylactic acid particles is:

[0146] ① Mix fluorescein Pseudomonas lipase, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and distilled water at 45°C and stir at 300 rpm for 30 min to obtain an enzyme treatment solution with a pH of 8.0 and a fluorescein Pseudomonas lipase concentration of 0.5 g / L;

[0147] ② The porous polylactic acid particles and the enzyme treatment solution obtained in step ① were mixed in a mass ratio of 1:2.5, stirred at 43°C and 300 rpm for 12 hours, filtered and dried at 50°C for 5 hours to obtain the hydrophilic porous polylactic acid particles.

[0148] The preparation method of the porous polylactic acid particles is:

[0149] (1) At 95°C, polylactic acid, cellulose acetate and n-butyl propionate were mixed in a mass ratio of 1.2:1:7 and stirred at 600 rpm for 2.5 h to obtain a biphasic solution;

[0150] (2) At 30° C., n-pentanol and a precipitation aid were mixed in a mass ratio of 100:0.28 and stirred at 600 rpm for 3 h to obtain a precipitant;

[0151] ⑶ The two-phase solution obtained in step ⑴ is cooled to 50°C and stirred at 360rpm, and the precipitant obtained in step ⑵ and cooled to 4°C is added to obtain the porous polylactic acid particles; the mass ratio of the two-phase solution to the precipitant is 1:5.

[0152] The precipitation aid is a mixture of xanthan gum and carbomer in a mass ratio of 1:1.

[0153] Comparative Example 4

[0154] A bacterial agent for decomposing grease is prepared by the following method: a composite bacterial species is inoculated into a seed culture medium to obtain a seed solution, and then the seed solution is inoculated into an expansion culture medium to obtain the bacterial agent for decomposing grease.

[0155] The composite bacterial strain is a mixture of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides in a mass ratio of 5:3:3:2:3.

[0156] The seed culture medium comprises: 1.5 parts by weight of sodium chloride, 4.5 parts by weight of ammonium acetate, 0.2 parts by weight of magnesium nitrate, 0.2 parts by weight of calcium nitrate, 1.2 parts by weight of dipotassium hydrogen phosphate, 0.5 parts by weight of potassium dihydrogen phosphate, 10 parts by weight of yeast extract, 1 part by weight of chitosan, and 1000 parts by weight of distilled water.

[0157] The culture conditions of the seed solution are: culture at 30°C, 120rpm, 1000lux light until the bacterial concentration reaches 10 9 CFU / mL to obtain the seed solution; the pH of the seed culture medium is 7.0.

[0158] The components of the expansion culture medium are: 10 parts by weight of beef extract, 10 parts by weight of peptone, 5 parts by weight of potassium dihydrogen phosphate, 2 parts by weight of dipotassium hydrogen phosphate, 1000 parts by weight of distilled water, and 600 parts by weight of hydrophilic porous polylactic acid particles.

[0159] The culture conditions of the expansion culture are as follows: 10 mL of seed solution is added to 1 L of expansion culture medium, and cultured at 30°C, 180 rpm, and 1000 lux; the pH of the expansion culture medium is 7.0; when the bacterial cell concentration reaches 10 15 CFU / mL, centrifuge, and store the precipitate in a lightless environment with a relative humidity of 80% and an oxygen content of 15% to obtain the bacterial agent for decomposing grease.

[0160] The preparation method of the hydrophilic porous polylactic acid particles is:

[0161] ① Mix porcine pancreatic lipase, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and distilled water at 45°C and stir at 300 rpm for 30 min to obtain an enzyme treatment solution with a pH of 8.5 and a porcine pancreatic lipase concentration of 0.5 g / L;

[0162] ② The porous polylactic acid particles and the enzyme treatment solution obtained in step ① were mixed in a mass ratio of 1:2.5, stirred at 43°C and 300 rpm for 12 hours, filtered and dried at 50°C for 5 hours to obtain the hydrophilic porous polylactic acid particles.

[0163] The preparation method of the porous polylactic acid particles is:

[0164] (1) At 95°C, polylactic acid, carboxymethyl cellulose and n-butyl propionate were mixed in a mass ratio of 1.2:1:7 and stirred at 600 rpm for 2.5 h to obtain a biphasic solution;

[0165] (2) At 30° C., n-pentanol and a precipitation aid were mixed in a mass ratio of 100:0.28 and stirred at 600 rpm for 3 h to obtain a precipitant;

[0166] ⑶ The two-phase solution obtained in step ⑴ is cooled to 50°C and stirred at 360rpm, and the precipitant obtained in step ⑵ and cooled to 4°C is added to obtain the porous polylactic acid particles; the mass ratio of the two-phase solution to the precipitant is 1:5.

[0167] The precipitation aid is a mixture of xanthan gum and carbomer in a mass ratio of 1:1.

[0168] Test Example 1

[0169] Test of the ability to decompose grease: According to GB / T 22500-2008 "Standard for the determination of ultraviolet absorbance of animal and vegetable oils", the ultraviolet spectrophotometry of animal and vegetable oils in the grease wastewater before and after treatment with the bacterial agents for decomposing grease obtained in the above embodiments and comparative examples is measured, and the grease concentration before and after treatment is calculated according to the absorbance value standard curve. Among them, the initial grease concentration is 30 mg / L. The grease wastewater is treated with the bacterial agents for decomposing grease obtained in the above embodiments and comparative examples at 30°C, 1000 lux light, and 300 rpm. The treatment time is 48 hours, and the mass ratio of the bacterial agents for decomposing grease to the grease wastewater is 1:50. Grease decomposition rate = (initial grease concentration - grease concentration after treatment) / initial grease concentration × 100%. The test results are shown in Table 1.

[0170] Table 1 Grease decomposition rate of the bacterial agent used to decompose grease of the present invention

[0171] Oil decomposition rate (%) Example 1 94.2 Example 2 92.8 Example 3 91.5 Comparative Example 1 84.1 Comparative Example 2 74.0 Comparative Example 3 87.6 Comparative Example 4 87.4

[0172] Test Example 2

[0173] Test of the distribution of bacterial agent carrier particles in water-oil mixture: animal and plant mixed oil and distilled water were mixed at a mass ratio of 1:1 at 65°C and stirred at 600rpm for 1h to obtain a water-oil mixture; the animal and plant mixed oil was composed of lard and peanut oil at a mass ratio of 1:1. When the water-oil mixture was naturally cooled to 25°C, the bacterial agents for decomposing oil obtained in the above-mentioned embodiments and comparative examples were added respectively and stirred at 600rpm for 30min; the mass ratio of the bacterial agent to the water-oil mixture was 1:10. Then, after standing at 25°C for 2h, the carrier particles distributed in the oil phase, water phase, and water-oil interface were counted respectively, and the proportion of carrier particles in the three states was calculated, and the proportion = (the number of carrier particles in the oil phase or water phase or water-oil interface) / the total number of carrier particles × 100%.

[0174] Table 2 Proportion of carrier particles of the bacterial agent for decomposing grease of the present invention distributed in the oil phase, water phase, and the interface between water and oil phases

[0175]

[0176] The oil degradation rate of Example 1 (using a mixture of xanthan gum and carbomer in a mass ratio of 1:1 as the precipitation aid) = 94.2%, and the water-oil two-phase ratio = 88.4% are significantly better than those of Example 2-3 (using one of xanthan gum and carbomer as the precipitation aid) = (91.5-92.8)%, and the water-oil two-phase ratio = (80.5-83.3)%. This is because: the molecular structures of xanthan gum and carbomer are different, and the ratios of polar groups and non-polar groups contained in their respective molecules are different, so that the two are in the polylactic acid, ethyl acetate, etc. The two-phase solution obtained by stirring cellulose ether and n-butyl propionate has different surface tensions. The difference in surface tension between the two makes the porous polylactic acid precipitated when n-pentanol is added have different surface morphologies. When xanthan gum and carbomer are compounded as precipitation aids, the average particle size distribution and wetting angle range of the porous polylactic acid obtained after proportional regulation are more conducive to the final bacterial agent for decomposing grease to be evenly distributed at the oil-water interface of grease wastewater, which is convenient for the bacteria in the preparation to quickly diffuse out and reproduce and decompose grease, thereby improving the ability of the bacterial agent to decompose grease.

[0177] The oil degradation rate of Example 1 (hydrophilic porous polylactic acid particles prepared by a specific method) = 94.2%, the water-oil two-phase ratio = 88.4% is significantly better than that of Comparative Example 2 (polylactic acid particles are not hydrophilically modified) and Comparative Example 3 (hydrophilic porous polylactic acid particles are prepared using Pseudomonas fluorescens lipase) = (74.0-87.6)%, the water-oil two-phase ratio = (25.4-74.9)%, this is because: polylactic acid fibers belong to aliphatic polyesters, there are a large number of ester bonds in the molecular structure, and the hydrophilicity is poor. , and will lead to a longer degradation cycle; the use of porcine pancreatic lipase to modify polylactic acid can convert the ester bonds exposed in the outer layer of polylactic acid particles into polar hydroxyl and carboxyl groups, so that the obtained polylactic acid particles can be better distributed at the interface between the water phase and the oil phase, so that the bacteria in the particles can be better diffused at the oil-water interface, which is conducive to the rapid reproduction and increase of the bacteria; and when the decomposition activity occurs at the oil-water interface, the glycerol and low-level fatty acids in the decomposition products can quickly diffuse into the water to avoid the accumulation of decomposition products. The oil decomposition effect of the microbial agent for decomposing oils obtained by hydrophilic modification of porous polylactic acid particles using porcine pancreatic lipase is better than that of using fluorescent Pseudomonas lipase, because porcine pancreatic lipase can achieve higher activity under milder and lower cost conditions due to its unique spatial molecular structure, and the ratio of polar groups to ester groups on the surface of the obtained polylactic acid particles is more moderate, which is more conducive to the uniform distribution of the microbial agent in the oil-water interface, and improves the ability of the microbial agent to decompose oils.

[0178] The oil degradation rate of Example 1 (preparing porous polylactic acid particles using cellulose acetate) = 94.2%, the water-oil two-phase ratio = 88.4% is significantly better than the oil degradation rate of Comparative Example 1 (not using the precipitation aid) and Comparative Example 4 (using carboxymethyl cellulose) = (84.1-87.4)%, the water-oil two-phase ratio = (72.5-73.3)%, this is because: carboxymethyl cellulose has more polar groups, the presence of these polar groups will result in the use of n-pentanol precipitation to obtain porous polylactic acid particles. The particles with poor surface uniformity will rub and collide with each other during subsequent transportation and storage, and break and crush, that is, they have already failed before they are officially put into use; while the proportion of polar groups in cellulose acetate is low, and polylactic acid particles with better surface uniformity and convenient transportation and storage can be precipitated, thereby improving the ability of the bacterial agent to decompose oil.

[0179] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, a person skilled in the art should understand that the technical solutions of the invention may be modified or replaced by equivalents without departing from the scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. A bacterial agent for decomposing oil, characterized in that: The method is prepared by the following method: the composite bacteria are inoculated into a seed culture medium to obtain a seed solution, and then the seed solution is inoculated into an expansion culture medium for expansion culture to obtain a bacterial agent for decomposing oil; The components of the expansion culture medium are: beef extract, peptone, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, distilled water, and hydrophilic porous polylactic acid particles; The preparation method of the hydrophilic porous polylactic acid particles is: ① Mix and stir porcine pancreatic lipase, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and distilled water at 43-48°C to obtain an enzyme treatment solution; ② Mixing the porous polylactic acid particles and the enzyme treatment solution obtained in step ①, stirring, filtering and drying the precipitate to obtain the hydrophilic porous polylactic acid particles; The preparation method of the porous polylactic acid particles is: ⑴ polylactic acid, cellulose acetate and n-butyl propionate are mixed and stirred to obtain a two-phase solution; ⑵ n-pentanol and a precipitation aid are mixed and stirred to obtain a precipitant; ⑶ Cooling and stirring the two-phase solution obtained in step ⑴, and adding the precipitant obtained in step ⑵ to obtain the porous polylactic acid particles.

2. The bacterial agent for decomposing fats and oils as claimed in claim 1, characterized in that: The composite bacterial species is a mixture of two or more of Bacillus subtilis, Bacillus licheniformis, Pseudomonas chengduensis, Pseudomonas aureus, and Rhodobacter sphaeroides.

3. The bacterial agent for decomposing fats and oils according to claim 1, characterized in that: The components of the seed culture medium are: sodium chloride, ammonium acetate, magnesium nitrate, calcium nitrate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, yeast extract, chitosan and distilled water.

4. The bacterial agent for decomposing fats and oils according to claim 1, characterized in that: The culture conditions of the seed solution are: the seed solution is obtained after culturing under light.

5. The bacterial agent for decomposing fats and oils as claimed in claim 1, characterized in that: The culture conditions of the expansion culture are: adding the seed solution to the expansion culture medium and culturing under light; Centrifuge and save the precipitate to obtain a bacterial agent for decomposing oil.

6. The bacterial agent for decomposing fats and oils as claimed in claim 1, characterized in that: The precipitation aid is one of xanthan gum and carbomer or a mixture of the two.

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