阅读说明：本技术 一种修复海洋溢油污染的固定化菌剂 (Immobilized microbial inoculum for repairing ocean oil spill pollution ) 是由 彭欣 张华伟 刘俊峰 薛峰 于 2020-04-15 设计创作，主要内容包括：本发明提供一种修复海洋溢油污染的固定化菌剂,属于石油及石油产品污染的生物修复技术领域,包括调节物质2-(4-叔丁基-2,6-二甲基苄基)-2-咪唑啉盐酸盐,利用二甲氧基乙烷和3,5-二羟基-3-甲基戊酸进行改性的聚乙烯醇。该固定化菌剂制备时所用聚乙烯醇的量较少,交联反应时间短,在保证较高的机械强度下,能够提高固定化效率和传质性能；能够抑制枯草芽孢杆菌spx基因的转录,提高srfA基因的转录水平,促进表面活性素的生成,提高对原油的乳化能力,进而提高对原油的降解率。(The invention provides an immobilized microbial inoculum for repairing marine oil spill pollution, belonging to the technical field of bioremediation of petroleum and petroleum product pollution, and comprising a regulating substance 2- (4-tert-butyl-2, 6-dimethylbenzyl) -2-imidazoline hydrochloride and polyvinyl alcohol modified by dimethoxyethane and 3, 5-dihydroxy-3-methylvaleric acid. The preparation method of the immobilized microbial inoculum has the advantages that the amount of polyvinyl alcohol used is small, the crosslinking reaction time is short, and the immobilization efficiency and the mass transfer performance can be improved under the condition of ensuring higher mechanical strength; can inhibit the transcription of the spx gene of the bacillus subtilis, improve the transcription level of the srfA gene, promote the generation of surfactant, improve the emulsifying capacity of crude oil and further improve the degradation rate of the crude oil.)

Use of 2- (4-tert-butyl-2, 6-dimethylbenzyl) -2-imidazoline hydrochloride for increasing the biosurfactant yield of bacillus subtilis.

2. The preparation method of the modified polyvinyl alcohol is characterized by comprising the following steps: heating polyvinyl alcohol, stirring to dissolve completely, adding 3, 5-dihydroxy-3-methyl pentanoic acid, adding dimethoxyethane, water bath at 75-80 deg.C, magnetically stirring for 4-6 hr, dialyzing, freeze drying, and vacuum drying at 55-60 deg.C.

3. A preparation method of an immobilized microbial inoculum is characterized by comprising the following steps: the preparation method of the immobilized microbial inoculum comprises the following steps:

s1, sterilizing the modified polyvinyl alcohol, sodium alginate, adjusting substances and activated carbon of claim 2, and dissolving in sterile seawater in a constant-temperature water bath at 74-80 ℃ to obtain embedding material colloid;

s2, cooling to 38-41 ℃, adding the seed bacterial liquid of the petroleum hydrocarbon degrading bacteria with the volume of 1/8-1/10 of the embedding material colloid in the step S1, and uniformly mixing to obtain a raw material mixture;

s3, dripping the raw material mixture obtained in the step S2 into a saturated boric acid solution of calcium chloride with the mass concentration of 2% through a granulating device, stirring, and carrying out cross-linking reaction for 5-8 hours to obtain an immobilized microbial inoculum;

in the step S1, the mass concentration of the modified polyvinyl alcohol in the embedding material colloid is 2-6%, the mass concentration of the sodium alginate is 2-3%, and the mass concentration of the activated carbon is 0.5-0.7%.

4. The production method according to claim 3, characterized in that: the petroleum hydrocarbon degrading bacteria comprise pseudoalteromonas, bacillus subtilis and brevibacillus parabrevis. .

5. The method of claim 4, wherein: the regulating substance in the step S1 comprises 2- (4-tert-butyl-2, 6-dimethylbenzyl) -2-imidazoline hydrochloride.

6. The method of claim 5, wherein: the mass concentration of the 2- (4-tert-butyl-2, 6-dimethylbenzyl) -2-imidazoline hydrochloride in the embedding material colloid in the step S1 is 0.02-0.03%.

7. A petroleum hydrocarbon degrading bacteria immobilized microbial inoculum is characterized in that: the petroleum hydrocarbon degrading bacteria immobilized microbial inoculum is prepared by the preparation method of the immobilized microbial inoculum as set forth in any one of claims 3 to 6.

8. Use of the immobilized microbial agent of claim 7 for remediation of marine oil spill contamination.

The application of 3, 5-dihydroxy-3-methyl pentanoic acid and dimethoxyethane in improving the mass transfer performance of the immobilized microspheres is characterized in that: the immobilized carrier material of the immobilized microspheres comprises polyvinyl alcohol.

Technical Field

The invention belongs to the technical field of bioremediation of petroleum and petroleum product pollution, and particularly relates to an immobilized microbial inoculum for remedying marine oil spill pollution.

Background

In recent years, marine oil spill accidents have been frequent due to various natural or artificial reasons. The oil spill pollution greatly exceeds and seriously damages the self-cleaning capability of the marine ecological environment, thereby directly or indirectly influencing the production and the life of people. The search for an environmentally friendly and low-cost oil spill removal strategy is an urgent problem to be solved by current marine environmental governance. At present, a plurality of methods for alleviating and treating crude oil pollution exist. The traditional physical and chemical method can quickly eliminate oil spill, but in practical application, the traditional physical and chemical method usually only plays a good role in the initial stage of oil spill, has high cost and can cause secondary pollution. The oil spill bioremediation method is to convert crude oil into CO by using the mineralization of petroleum degrading microorganisms₂、H₂Compared with other remediation modes, the method has the advantages of cleanness, high efficiency and low cost, and becomes the most potential treatment strategy for the spilled oil. However, in practical bioremediation applications, many problems need to be overcome, such as maintenance of microbial activity, scouring and dilution in open sea areas, and weak oil spill bioremediation capability of indigenous microorganisms. In view of the above problems, more and more scholars consider that the microorganism immobilization technology is the best solution. Compared with the application of free microorganisms, the microbial immobilization technology has the following advantages: (1) due to high-density aggregation of the degradation microorganisms, the degradation rate is improved; (2) the biodegradation stability and the tolerance to various diluted and predated complex environments are enhanced; (3) stimulating the growth, reproduction and reutilization of microorganisms.

Disclosure of Invention

The invention aims to provide modified polyvinyl alcohol and a preparation method thereof, and when the modified polyvinyl alcohol is used for preparing an immobilized microbial inoculum, the consumption of the polyvinyl alcohol can be reduced, the crosslinking reaction time is shortened, and the immobilization efficiency and the mass transfer performance are improved under the condition of ensuring higher mechanical strength.

The technical scheme adopted by the invention for realizing the purpose is as follows:

provided is a modified polyvinyl alcohol, wherein the preparation method of the modified polyvinyl alcohol comprises the following steps: heating polyvinyl alcohol, stirring to dissolve completely, adding 3, 5-dihydroxy-3-methyl pentanoic acid, adding dimethoxyethane, water bath at 75-80 deg.C, magnetically stirring for 4-6 hr, dialyzing, freeze drying, and vacuum drying at 55-60 deg.C. Preferably, the above polyvinyl alcohol: 3, 5-dihydroxy-3-methylpentanoic acid: dimethoxyethane (m/m) ═ 3-4:2-3: 1-2. In the presence of dimethoxyethane, carboxyl on 3, 5-dihydroxy-3-methyl pentanoic acid can perform esterification reaction with hydroxyl of polyvinyl alcohol, two hydroxyl groups are introduced, when the immobilized microbial agent is prepared by using modified polyvinyl alcohol, intermolecular force of low-concentration PVA in a homogeneous solution is improved by the presence of the two hydroxyl groups, free flow of gel can be resisted, the degree of crosslinking polymerization in the immobilized microsphere is improved, and meanwhile, the immobilized microbial agent can be fully crosslinked in a short crosslinking reaction time, has high mechanical strength and balling property, can reduce damage to bacterial activity, and a porous structure in the immobilized microbial agent is looser by the introduced hydrophobic chain and the modified polyvinyl alcohol with low concentration, so that immobilization efficiency and mass transfer performance are improved, and the degradation rate of crude oil is improved.

The invention also aims to provide an immobilized microbial inoculum for repairing marine oil spill pollution and a preparation method thereof, and the immobilized microbial inoculum can inhibit the transcription of the spx gene of bacillus subtilis, improve the transcription level of the srfA gene, promote the generation of surfactant, improve the emulsifying capacity of crude oil and further improve the degradation rate of the crude oil.

Provides the application of 2- (4-tert-butyl-2, 6-dimethylbenzyl) -2-imidazoline hydrochloride in improving the yield of the biosurfactant of the bacillus subtilis. Surfactin (surfactin) synthesized by bacillus subtilis is the strongest biosurfactant discovered at present, belongs to lipopeptide, can emulsify crude oil into small droplets, increase the solubility of the crude oil in water, increase the effective utilization area of bacteria and the crude oil, and improve the utilization rate of the crude oil. srfA is a synthetic gene involved in the regulation of metabolism of Bacillus subtilis surfactin, and RNA polymerase binds to the-35 region of srfA with the aid of ComA-P during transcription of srfA to initiate complete transcription of the srfA gene. Whereas there is an overlap between ComA-P and the region where the spx protein interacts with the C-terminus of the RNA polymerase alpha subunit, the spx protein inhibits normal transcription of srfA by competitively occupying the domain of the RNA polymerase alpha subunit C-terminus that interacts with ComA-P. The 2- (4-tert-butyl-2, 6-dimethylbenzyl) -2-imidazoline hydrochloride is added to inhibit the transcription of the spx gene of the bacillus subtilis, so that the occupation of a protein domain of the interaction between the C end of the alpha subunit of the RNA polymerase and ComA-P is reduced, the transcription level of the srfA gene is improved, the generation of surfactin is promoted, the emulsifying capacity of crude oil is improved, and the degradation rate of the crude oil is further improved.

Provides a preparation method of an immobilized microbial inoculum, which comprises the following steps:

s1, sterilizing the modified polyvinyl alcohol, sodium alginate, adjusting substances and activated carbon, and dissolving in sterile seawater in a constant-temperature water bath at 74-80 ℃ to obtain embedding material colloid;

s2, cooling to 38-41 ℃, adding 1/8-1/10 volume of petroleum hydrocarbon degrading bacteria seed bacterial liquid of the embedding material colloid, and uniformly mixing to obtain a raw material mixture;

s3, dripping the raw material mixture into a saturated boric acid solution of calcium chloride with the mass concentration of 2% through a granulating device, stirring, and carrying out cross-linking reaction for 5-8h to obtain an immobilized microbial inoculum;

the mass concentration of the modified polyvinyl alcohol in the embedding material colloid is 2-6%, the mass concentration of the sodium alginate is 2-3%, and the mass concentration of the active carbon is 0.5-0.7%.

Preferably, the petroleum hydrocarbon degrading bacteria comprise pseudoalteromonas, bacillus subtilis and brevibacillus parabrevis.

Preferably, the regulating substance in the above step S1 comprises 2- (4-tert-butyl-2, 6-dimethylbenzyl) -2-imidazoline hydrochloride.

Preferably, the mass concentration of the 2- (4-tert-butyl-2, 6-dimethylbenzyl) -2-imidazoline hydrochloride in the embedding material colloid in the step S1 is 0.02-0.03%.

The petroleum hydrocarbon degrading bacteria immobilized microbial inoculum is prepared by the preparation method of the immobilized microbial inoculum.

Provides the application of the immobilized microbial inoculum in repairing marine oil spill pollution.

The application of 3, 5-dihydroxy-3-methyl pentanoic acid and dimethoxyethane in improving the mass transfer performance of the immobilized microspheres is provided, and the immobilized carrier material of the immobilized microspheres comprises polyvinyl alcohol.

The invention has the beneficial effects that:

1) according to the invention, the carboxyl groups on dimethoxyethane and 3, 5-dihydroxy-3-methylvaleric acid are utilized to modify polyvinyl alcohol, when the modified polyvinyl alcohol is used for preparing the immobilized microbial agent, the intermolecular force of low-concentration PVA in a homogeneous solution is improved due to the existence of two hydroxyl groups, the free flow of gel can be resisted, the internal crosslinking polymerization degree of the immobilized microspheres is improved, the crosslinking reaction time is shortened, and the introduced hydrophobic chain and the modified polyvinyl alcohol with lower concentration enable the porous structure in the immobilized microbial body to be loose, so that the immobilization efficiency and the mass transfer performance are improved under the condition of ensuring higher mechanical strength;

2) according to the invention, 2- (4-tert-butyl-2, 6-dimethylbenzyl) -2-imidazoline hydrochloride is added into the immobilized microbial inoculum to inhibit the transcription of the spx gene of the bacillus subtilis, so that the occupation of a protein domain of interaction between the C end of an RNA polymerase alpha subunit and ComA-P is reduced, the transcription level of the srfA gene is improved, the generation of surfactin is promoted, the emulsifying capacity of crude oil is improved, and the degradation rate of the crude oil is further improved.

Drawings

FIG. 1 is a Fourier infrared spectrum of a modified polyvinyl alcohol and an unmodified polyvinyl alcohol in example 1 of the present invention;

FIG. 2 is a graph showing the results of the measurement of the graft ratio in example 2 of the present invention;

FIG. 3 shows the results of the determination of the relative transcription levels of the spx and srfA3 genes and the surfactin content in test example 1;

FIG. 4 shows the results of measurements of the breakage rate, immobilization efficiency and permeability of the immobilized bacteria agent in test example 2 of the present invention;

FIG. 5 is a scanning electron micrograph of an immobilized hollow microsphere in Experimental example 2 of the present invention;

FIG. 6 is a graph showing the results of measuring the surface tension reduction value and the crude oil degradation rate in test example 3 of the present invention.

Detailed Description

The present invention is further described in detail with reference to the following examples: