CN112251373B

CN112251373B - Preparation and application of bacterium-enzyme composite preparation for petroleum hydrocarbon degradation

Info

Publication number: CN112251373B
Application number: CN202011083706.0A
Authority: CN
Inventors: 王卅; 郭书海; 程凤莲; 荆佳维
Original assignee: Institute of Applied Ecology of CAS
Current assignee: Institute of Applied Ecology of CAS
Priority date: 2020-10-12
Filing date: 2020-10-12
Publication date: 2022-05-31
Anticipated expiration: 2040-10-12
Also published as: CN112251373A

Abstract

The invention belongs to the field of soil environment bioremediation, and further discloses a bacterium-enzyme composite preparation for biodegradation of petroleum hydrocarbon pollutants in petroleum hydrocarbon polluted soil, so as to realize efficient and rapid bioremediation of the petroleum hydrocarbon polluted soil. The compound preparation is a microbial inoculum with petroleum hydrocarbon degradation capability and an enzyme agent obtained by crushing the microbial inoculum; the microbial inoculum is culture solution obtained by fermenting and culturing Variovorax sp.JWLC1 of Variovorax to OD value of 1.5-2.5 at 30 +/-1 ℃; wherein the volume ratio of the microbial inoculum to the fermentation culture solution in the enzyme agent is 1:1-1: 3. The bacterium-enzyme composite preparation obviously prolongs the microbial efficient degradation process of petroleum hydrocarbon and improves the bioremediation efficiency of the petroleum hydrocarbon polluted soil.

Description

Preparation and application of bacterium-enzyme composite preparation for petroleum hydrocarbon degradation

Technical Field

The invention belongs to the field of soil environment bioremediation, and further discloses a bacterium-enzyme composite preparation for biodegradation of petroleum hydrocarbon pollutants in petroleum hydrocarbon polluted soil, so as to realize efficient and rapid bioremediation of the petroleum hydrocarbon polluted soil.

Background

The continuous progress of the petroleum industry helps the vigorous development of social economy and brings considerable economic benefits. However, there are accompanying problems of environmental pollution to various degrees. In the processes of crude oil exploitation, transportation, petrochemical product processing and production and the like, the problem of soil petroleum pollution is caused, so that the ecological environment of the soil is destroyed, the function is lost, and repair and treatment are urgently needed. In contrast, many petroleum-contaminated soil remediation technologies are developed, including physical methods such as soil dressing, blocking, landfill, desorption and cleaning, chemical methods such as oxidation, incineration and electrochemical degradation, and biological methods such as phytoremediation, microbial remediation and enzyme preparation degradation. In view of the advantages of economy, effectiveness, simplicity, environmental protection and the like, the microbial remediation technology becomes one of the first-choice technologies for engineering application and treatment in the petroleum-polluted soil industry.

However, the development and application of the microbial remediation technology have the problems that the remediation efficiency is easy to reduce, the organic pollutants are difficult to maintain and degrade efficiently and rapidly, and the like, in the long-term practical application process. In contrast, the development of efficient and durable microbial remediation optimization technology becomes a hot direction in the field of microbial remediation technology research and the like. Accordingly, various combined repair technologies combined with microbial repair technologies, such as chemical oxidation combined microbial repair technology, thermal desorption combined microbial repair technology, and the like, have appeared, however, although the technologies optimize the petroleum hydrocarbon removing capability to a certain extent, the old problems of gradually weakened degradation activity of functional strains, gradually lost metabolic capability of organic pollutants, and the like in the microbial repair stage are still not effectively solved, so that the existing combined repair technology becomes a mechanized segmental effect. In addition, in recent years, in order to break through the bottleneck of low efficiency of microbial remediation and accelerate the biodegradation rate of organic pollutants, the research of enzyme preparations gradually enters the visual field and is vigorously developed towards the engineering application direction. However, although the petroleum hydrocarbon degradation process of the enzyme preparation is rapid, the action is short, and particularly in a soil environment with multidirectional heterogeneity, the disadvantage that biological enzymes are easily inactivated due to the influence of environmental factors such as soil pH, temperature, electrolyte strength and the like is undoubtedly revealed. Although the immobilization technology of the biological enzyme effectively improves the side effect of the environmental factor on the biological enzyme to a certain extent, the application of the enzyme preparation still cannot make up the problems of short efficiency and low efficiency of the microbial remediation technology at present.

Disclosure of Invention

The invention aims to provide a preparation method and application of a bacterium-enzyme composite preparation for petroleum hydrocarbon degradation.

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

a bacterium-enzyme composite preparation for petroleum hydrocarbon degradation is provided, the composite preparation is a bacterium agent with petroleum hydrocarbon degradation capability and an enzyme agent obtained by crushing the bacterium agent; the microbial inoculum is culture solution obtained by fermenting and culturing Variovorax sp.JWLC1 of Variovorax to OD value of 1.5-2.5 at 30 +/-1 ℃; wherein the volume ratio of the fermentation culture solution in the microbial inoculum and the enzyme agent is 1:1-1: 3.

The Variovorax sp JWLC1 is deposited in China center for type culture Collection in 2019, 10.11.8.8.Address, university of Wuhan, China, with the preservation accession number of CCTCC NO: m2019812.

The microbial inoculum is a fermentation culture solution obtained by fermenting Variovorax JWLC 1;

the enzyme agent is a solid-liquid mixture obtained by crushing a fermentation culture solution obtained by fermenting Variovorax JWLC1 and then carrying out immobilization treatment on the crushed solid-liquid mixture.

After the enzyme agent is obtained by fermenting and culturing Variovorax JWLC1, the culture solution is directly subjected to ultrasonic crushing treatment under the conditions of 300-.

And the solid part is carbonized, namely the crushed and separated Variovorax JWLC1 cell residues are dehydrated and air-dried, then carbonized for 1-2h at the temperature of 250-.

The application of a petroleum hydrocarbon degrading bacterium-enzyme composite preparation in degrading petroleum hydrocarbon in petroleum hydrocarbon contaminated soil.

The bacterial-enzyme composite preparation can be applied to petroleum hydrocarbon polluted soil at one time or in multiple times to carry out petroleum hydrocarbon degradation treatment; the application amount of the compound preparation in the polluted soil is 5-10% (mL/g according to the adding proportion of the microbial inoculum); then, the dosage of the enzyme agent is calculated according to the relation of the fermentation culture solution between the microbial inoculum and the enzyme agent.

The application amount of the one-time application composite preparation is 5-10% (mL/g according to the adding proportion of the microbial inoculum), and the condition that the abundance of Variovorax in the composite preparation in soil is not less than 5 multiplied by 10 is ensured⁷CFU·g^-1And (5) drying the soil.

The compound preparation is applied in a grading manner, the microbial inoculum in the compound preparation is applied to the polluted soil according to the adding proportion of 5-10% (mL/g), and when the degradation rate of petroleum hydrocarbon in the soil is reduced to 20-50% of the initial degradation rate, the enzyme agent is applied to the polluted soil (the dosage of the enzyme agent is calculated according to the relation of a fermentation culture solution between the microbial inoculum and the enzyme agent).

The invention has the advantages that:

1) the Variovorax sp.JWLC1 of the Variovorax JWLC1 has a high-efficiency petroleum hydrocarbon degradation function, and the whole enzyme solution extracted from the fermentation culture has the capability of quickly metabolizing petroleum hydrocarbon, so that the speed-limiting step that petroleum hydrocarbon molecules need to cross a membrane to enter cells in the microbial remediation process is broken through, and the petroleum hydrocarbon degradation efficiency is effectively improved;

2) the cell ultrasonication extract (namely holoenzyme solution) of the Variovorax sp.JWLC1 fermentation culture of the Variovorax JWLC1 not only has a Variovorax sp.JWLC1 holoenzyme component, but also has substances such as cell contents (polysaccharide, lipid, protein) and the like, and is an extract in cells of an original strain, so that the strain is easier to metabolize as nutrient substances, and the affinity between a functional strain and a nutrient substrate is enhanced;

3) the Variovorax sp.JWLC1 fermentation culture cell in the invention breaks solid components, and after high-temperature carbonization treatment, the change processes of porous carbon structure, peptidoglycan molecule cracking and the like occur simultaneously, thereby not only providing effective immobilized carbon-based material surface area and enhancing full enzyme adsorption, but also providing good nutrients such as C, N, P for microorganisms in a soil system and having the sustained-release function of nutrients to a certain extent; in addition, as the solid components separated by cell disruption almost completely come from the Variovorax JWLC1, the content ratio of C to N to P serving as nutrient substances in the solid components is basically consistent with the composition of nutrient substrates required by the Variovorax JWLC1, and the nutrient structure requirement of the functional strains is effectively met;

4) in the application process of the microbial inoculum-enzyme agent composite preparation, a mode of firstly adding the microbial inoculum can be selected, and when the degradation rate of the petroleum hydrocarbon is reduced to 30-50% of the initial degradation rate of the petroleum hydrocarbon, the enzyme agent component is added, so that the application mode of the composite preparation has stronger action advantages compared with the conventional biological amplification and biological stimulation method: firstly, the application mode effectively utilizes the domestication process of the real-time pollution substrate composition (including the original substrate and the intermediate product) in the soil to the functional microorganisms in the soil, compared with the additionally applied microorganisms only domesticated by the original petroleum hydrocarbon substrate, the activity of the functional microorganisms aiming at the real-time substrate degradation in a soil system is recovered after the biological stimulation is carried out on the enzyme agent component, so that the application mode has stronger substrate degradation targeting property; and secondly, the application of the enzyme agent comprises original cell contents and nutritional characteristics with similar C: N: P ratio, and has immobilization and slow release capabilities, and the enzyme agent has stronger pertinence compared with the common nutritional auxiliary agent additional supply, so that the enzyme agent and the microbial inoculum form a composite auxiliary agent, and the microbial remediation efficient action period is obviously improved by a delayed supply mode, and the microbial remediation efficiency of the petroleum hydrocarbon polluted soil is greatly improved.

Drawings

Fig. 1 shows the phylogenetic analysis result of the 16S rDNA sequence of the phagocytosis JWLC1(Variovorax sp. JWLC1) according to the embodiment of the present invention.

FIG. 2 is a graph showing the residual rate of petroleum hydrocarbon in soil with treatment time under the conditions of applying the microbial inoculum-enzyme agent complex formulation provided by the embodiment of the invention.

FIG. 3 is a graph showing the degradation rate of petroleum hydrocarbon in soil with treatment time under the applied condition of the microbial inoculum-enzyme agent complex formulation provided by the embodiment of the invention.

FIG. 4 is a graph showing the dynamic change of water-soluble organic carbon content in soil with treatment time under the condition of applying the microbial inoculum-enzyme agent composite preparation provided by the embodiment of the invention.

FIG. 5 is a graph showing the dynamic change of the number of bacterial microorganisms in soil with treatment time under the conditions of applying the microbial inoculum-enzyme agent complex formulation provided by the embodiment of the invention.

Detailed Description

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

Aiming at the defects of long-term low efficiency and the like in the application of microbial remediation technology, the invention provides a preparation capable of effectively improving the problem that the microbial remediation is difficult to maintain the degradation activity for a long time with high efficiency and an application method thereof, and provides a bacterium-enzyme composite preparation. The preparation of the bacterial-enzyme complex preparation and the application thereof to remediation of petroleum hydrocarbon-contaminated soil will be described in detail below with reference to examples.

Example 1 screening and functional characterization of Variovorax sp.JWLC1 Variovorax

The functional bacterial strain is selected and separated from soil polluted by petroleum hydrocarbon in a certain oil field area for a long time, a polluted soil sample is collected and then subjected to enhanced domestication culture by taking the petroleum hydrocarbon as a substrate, and the domestication culture is completed under the condition of shake culture in a shake flask. The initial concentration of the petroleum hydrocarbon is 1g/kg, after shaking culture is carried out for 15 days, the concentration of the petroleum hydrocarbon is increased to 2g/kg, the primary domestication culture is inoculated into a secondary domestication culture bottle, shaking culture is continued for 15 days, the concentration of the petroleum hydrocarbon is increased to 3g/kg, the secondary domestication culture is inoculated into a tertiary domestication culture bottle, shaking culture is carried out again for 15 days, the domestication process is completed, and the total domestication period reaches 45 days. And (3) streaking and separating the domesticated and cultured culture on an inorganic salt solid culture medium containing 1g/L, selecting an independent bacterial colony with good growth vigor for three-stage streaking and purification so as to obtain a single pure bacterial strain, and streaking and storing the purified bacterial strain on a slant culture medium.

In the screening process, the used screening culture medium is an inorganic salt screening liquid culture medium, and the culture medium comprises the following components: 0.5 g.L^-1NaCl，1.0g·L^-1NH₄NO₃，0.2g·L^-1MgSO₄，1.5g·L^-1K₂HPO₄，1.0g·L^-1KH₂PO₄，0.02g·L^-1FeSO₄，0.02g·L^-1CaCl₂The petroleum hydrocarbon content is 1 g.L according to acclimatization level^-1、2g·L^-1And 3 g.L^-1Sterilizing at 121 deg.C for 30min with pH of culture system of 6.5-7.5; the solid culture medium used for purification is prepared by adding 5g/L agar powder into the basic composition of a liquid culture medium, and the content of petroleum hydrocarbon substances on the surface of the solid culture medium is 1 g/L.

Carrying out shake flask culture degradation function verification on the single strain obtained by separation by taking petroleum hydrocarbon as a substrate: inoculating the pure strain to a culture medium containing 5 g.L^-1In a liquid inorganic salt culture medium of petroleum hydrocarbon, shaking culture is carried out for 15 days at the temperature of 30 +/-1 ℃, and the total petroleum residue is measured. The result shows that the separated single bacterial strain has higher activity on the degradation of the petroleum hydrocarbon, and the degradation rate of the petroleum hydrocarbon reaches 43.7 percent after the shaking flask system is cultured for 15 days.

The separated single bacterial strain is JWLC1 bacterial strain, which is preserved in China center for type microorganism preservation in 2019, 10, 11 and has the preservation accession number of CCTCC No: m2019812. The gram-negative bacteria belong to the genus of gram-negative bacteria, the somatic cells of the gram-negative bacteria are slightly bent and rod-shaped, the middle part of the bacterial cells is raised, bacterial colonies are small, the edges of the bacterial cells are neat, the surfaces of the bacterial cells are smooth and wet, no spores are produced, the bacterial cells belong to aerobic bacteria, chemoheterotrophy is realized, the optimal growth temperature is 30 +/-2 ℃, the optimal growth pH is 6.5, fructose and glucose can be utilized, and gelatin can be hydrolyzed.

Performing molecular biological identification on the separated strains: extracting the total genome DNA of the strain JWLC1, carrying out 16S rDNA PCR amplification reaction, adopting a bacterial amplification universal primer: 8 f: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492 r: 5 '-TACGGHTACCTTGTTACGAC TT-3'. The PCR reaction conditions are as follows: 94 ℃ for 5min, 94 ℃ for 1min, 55 ℃ for 1min, 72 ℃ for 3min, 35 cycles, 72 ℃ for 10 min. The sequencing result of the PCR product is known by blast comparison analysis with a Genbank database, the sequence length is 1436bp, and phylogenetic analysis shows that (figure 1) the obtained strain has higher homology with the Variovorax.

>Variovorax sp.

GACCCATGGGCGGCTGCCTTACCATGCAAGTCGAACGGCAGCGCGGGAGCAATCCTGGCGGCGAGTGGCGAACGGGTGAGTAATACATCGGAACGTGCCCAATCGTGGGGGATAACGCAGCGAAAGCTGTGCTAATACCGCATACGATCTACGGAAGCAGGGGATCGCAAGACCTTGCGCGAATGGAGCGGCCGATGGCAGATTAGGTAGTTGGTGAGGTAAAGGCTCACCAAGCCTTCGATCTGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGACCATCGTTTTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGCAGGATGAAGGCCTTCGGGTTGTAAACTGCTTTTGTACGGAACGAAACGGCCTTTTCTAATAAAGAGGGCTAATGACGGTACCGTAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTAATGTAAGACAGTTGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGTGACTGCATTGCTGCATTGCTGGAGTACGGCAGAGGGGGATGGAATTCCGCGTGTAGCAGTGAAATGCGTAGATATGCGGAGGAACACCGATGGCGAAGGCAATCCCCTGGGCCTGTACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATCTGGTAGTCCACGCCCTAAACGATGTCAACTGGTTGTTGGGAATTCACTTTCTCAGTAACGAAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGTTTAACTCGACAACGCGAAAAACCTTACCCACCTTCCCACGGAATTCGCCAGAGATGGCTTAGTGCTCGAAAGAGAACCGTAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCTACATTTAGTTGGGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATAGGTGGGGCTACACACGTCATACAATGGCTGGTACAAAGGGTTGCCAACCCGCGAGGGGGAGCTAATCCCATAAAACCAGTCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGTCACGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGCGGGTTCTGCCAGAAGTAGTTAGCTTAACCGCAAGGAGGGCGATACCACGTCAGGTTCTTTCTA

EXAMPLE 2 preparation of Complex formulation

1) Preparation of enzymatic preparation of Variovorax sp.JWLC1 from Variovorax

The screened Variovorax sp.JWLC1 Variovorax phagemid was inoculated into 1L beef extract peptone medium with pH of 7.0, and cultured at 30 ℃ under 190rpm for 4 days with shaking at constant temperature. Stopping fermentation culture when the OD value of the fermentation culture solution reaches 2.1, carrying out ultrasonic crushing treatment on the fermentation culture under an ice bath condition, wherein the treatment condition is 330W, the total treatment time is 35min, centrifuging the crushed solid-liquid mixture for 10min at 9500rpm, carrying out solid-liquid separation, and collecting supernatant liquid, namely whole enzyme liquid; then collecting solid components such as cell debris and the like, and carrying out anaerobic high-temperature carbonization treatment for 2h at the temperature of 300 ℃ to obtain the solid components in the enzyme preparation.

A Coomassie brilliant blue method is adopted to determine the protein content in the whole enzyme solution, and Coomassie brilliant blue G250 is selected to determine protein staining, and the specific test method comprises the following steps: 100mg of Coomassie brilliant blue G250 dye powder is weighed, dissolved in 50mL of 95% ethanol, added with 120mL of 85% phosphoric acid, diluted with water to 1L, and stored in a brown bottle for later use. Taking a plurality of test tubes, respectively adding a standard protein solution, a dye solution and distilled water into the test tubes, wherein the total volume is 5.1mL, measuring and making a standard curve under OD595 light absorption conditions, the concentration of the standard protein series is 0, 0.02, 0.04, 0.12, 0.16 and 0.2mg/L, and the concentration of the protein in the whole enzyme solution reaches 3.4mg/L through measurement and calculation.

And mixing the whole enzyme solution with the solid component carbonized at high temperature (the volume of the mixed solution is approximately equal to that of a culture solution obtained by fermentation culture of Variovorax JWLC1), wherein the mixing ratio of the total whole enzyme solution (1L) to the carbonized solid component (3.8g) is 263:1(mL/g), and standing the mixture at 4 ℃ for 12 hours to obtain the enzyme preparation. And (3) adopting a TC analyzer to measure the total soluble organic carbon in the enzyme preparation, and obtaining the result that the content of the total soluble organic carbon in the enzyme preparation reaches 1.31 g/L. And concentrating the enzyme preparation to the required volume by using a freeze concentrator for later use.

2) Preparation of microbial inoculum

Inoculating screened Variovorax sp.JWLC1 into 1L beef extract peptone culture medium, wherein the pH of the culture medium is 7.0, and carrying out constant-temperature shaking culture at the temperature of 30 ℃ and the speed of 190rpm for 4 days in the same process as the enzyme preparation fermentation process in the step 1). When the OD value of the fermentation culture solution reaches 2.1, stopping fermentation culture to obtain the microbial inoculum.

3) Preparation of composite preparation

Mixing the obtained microbial inoculum with the obtained enzyme; wherein the volume ratio of the fermentation culture solution in the microbial inoculum (namely the fermentation liquid after fermentation in the step 2) to the fermentation culture solution in the enzyme agent (namely the fermentation liquid obtained before the fermentation in the step 1) is not broken) is 1: 3.

Example 3 Shake flask test functional verification of fungus-enzyme composite preparation

Carrying out a petroleum hydrocarbon degradation test on the composite preparation obtained in the embodiment in a shake flask, and respectively adding a microbial agent and an enzyme agent in the composite preparation:

adopting a screening culture medium formula (namely, recording an inorganic salt screening liquid culture medium in an embodiment), wherein the initial content of petroleum hydrocarbon is 2.5g/L, firstly adding a microbial inoculum (the adding amount is 3% (according to the microbial inoculum adding proportion, mL/g)) in the composite preparation in the embodiment 2, performing shake culture at 30 ℃ and 190rpm for 7 days, and ensuring that the degradation rate of the petroleum hydrocarbon reaches 51.3%; after 7 days of treatment, the enzyme agent in the compound preparation is added (the adding amount of the enzyme agent is calculated by the mixing ratio of the enzyme agent and the compound preparation added in the example 2), and the shaking culture is continued for 5 days, so that the total degradation rate of the petroleum hydrocarbon reaches 92.6 percent.

Meanwhile, directly degrading petroleum hydrocarbon by adopting an enzyme agent, taking a screening culture medium as a substrate, simultaneously adding only the enzyme agent prepared in the example 2 (the addition amount of the enzyme agent is the same as that of the enzyme agent in the compound preparation) and 2.5g/L petroleum hydrocarbon into a culture system, wherein the total treatment volume is 1L, and after 12 hours of standing culture combined with interval mild shaking treatment, the total petroleum hydrocarbon degradation rate is 29.6 percent.

Therefore, the use of the enzyme agent not only directly increases the degradation degree of the petroleum hydrocarbon, but also promotes the microbial inoculum to further degrade the petroleum hydrocarbon.

Example 4 application verification of bacterium-enzyme composite preparation in petroleum hydrocarbon contaminated soil remediation

In the examples, 7 treatments were provided, namely a control group (CK), a single microorganism treatment group (Bio), a single enzyme treatment group (En), a secondary microorganism replenishment treatment group (Bio + Bio), a microbial agent application/inactivation/enzyme replenishment treatment group (Bio + dEn), a microbial agent/enzyme agent common application treatment group (Bio-En), and a microbial agent application/enzyme agent replenishment treatment group (Bio + En), and specific parameters of the test setup are shown in table 1.

The preparation of microbial inoculum was carried out according to the procedure of example 3, culturing 500mL of Variovorax JWLC1 bacterial liquid at 30 deg.C, and adding into 5kg of contaminated soil containing 3.2% petroleum hydrocarbon, wherein OD of the bacterial liquid was 2.2, and the number of bacterial microorganisms in the soil after application was 9.12X 10⁷CFU/g。

Preparing an enzyme agent according to the operation flow of the embodiment 2, culturing 1000mL of a Variovorax JWLC1 bacterial liquid at 30 ℃, performing ultrasonic crushing treatment on a culture solution directly under 350W for 35min after fermentation culture, performing solid-liquid separation on a crushed mixture by centrifugation at 9000rpm, respectively collecting liquid and solid parts, performing carbonization treatment on the solid component at 300 ℃ under an anaerobic condition for 2h, cooling, mixing with all the liquid, standing at 4 ℃ for 10h, and re-suspending to prepare the solid-liquid mixture enzyme agent. The enzyme preparation was concentrated to a total volume of 200mL in a freeze concentrator.

Aiming at a Bio treatment group, 500mL of microbial inoculum is applied to petroleum hydrocarbon polluted soil and then heat preservation and moisture preservation treatment is carried out for 42 d;

aiming at the En treatment group, 200mL of enzyme agent is applied to the petroleum hydrocarbon polluted soil and then heat preservation and moisture preservation treatment is carried out for 42 d;

aiming at a Bio + Bio treatment group, 500mL of microbial inoculum is applied to petroleum hydrocarbon polluted soil and then subjected to heat preservation and moisture preservation treatment for 24d, then microbial inoculum secondary replenishment is carried out, 500mL of microbial inoculum is applied to petroleum hydrocarbon polluted soil after centrifugal concentration, heat preservation and moisture preservation treatment is continued for 18d, and total treatment is carried out for 42 d;

aiming at a Bio + dEn treatment group, 500mL of microbial inoculum is applied to petroleum hydrocarbon polluted soil and then subjected to heat preservation and moisture preservation treatment for 24d, 200mL of enzyme agent subjected to inactivation treatment at 100 ℃ is applied, and heat preservation and moisture preservation treatment is continued for 18d for 42d in total;

aiming at a Bio-En treatment group, centrifuging and concentrating 500mL of microbial inoculum into 300mL, mixing the microbial inoculum with 200mL of enzyme agent, applying the mixture into petroleum hydrocarbon polluted soil together, and carrying out heat preservation and moisture preservation treatment for 42 d;

for the Bio + En treatment group, 500mL of microbial inoculum is applied to petroleum hydrocarbon contaminated soil and then subjected to heat preservation and moisture preservation treatment for 24d, 200mL of enzyme agent is subsequently applied, and heat preservation and moisture preservation treatment is continued for 18d and total treatment is continued for 42 d.

The amount of the enzyme agent in each treatment is equivalent to the volume of the fermentation liquid before crushing in the preparation of the enzyme agent.

The experimental results of the examples show (see fig. 2-5) that the residual rates of the petroleum hydrocarbons treated by Bio-En and Bio + En are 57.38% and 45.10%, respectively, and show that the use of the microbial inoculum-enzyme agent complex preparation prolongs the efficient biodegradation process of the petroleum hydrocarbons and improves the overall degradation efficiency of the petroleum hydrocarbons, and particularly after the enzyme agents in the Bio + En treatment group are applied, the degradation rate of the petroleum hydrocarbons is obviously increased and is almost recovered to the initial level of the degradation treatment. From all soil microThe ecological environment index analysis can know that: the Bio + En treatment group effectively maintains the degradation activity of the microorganisms to a certain extent; from the analysis of the content of the soluble organic carbon in the soil, the addition of the enzyme agent obviously increases the content of the soluble organic carbon in the soil to be more than 3 times of the residual amount after 24 days of treatment, and effectively keeps the easily-utilized carbon in the soil at a higher level, thereby ensuring the number of bacterial microorganisms, wherein the number of the bacterial microorganisms in the Bio + En treatment group reaches 8.51 multiplied by 10⁶The number of the CFU/g is improved by more than 4 times when the microorganism is treated for 42 days compared with the Bio treatment group.

TABLE 1

Sequence listing

<110> Shenyang application ecological research institute of Chinese academy of sciences

<120> preparation and application of bacterium-enzyme composite preparation for petroleum hydrocarbon degradation

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1436

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gacccatggg cggctgcctt accatgcaag tcgaacggca gcgcgggagc aatcctggcg 60

gcgagtggcg aacgggtgag taatacatcg gaacgtgccc aatcgtgggg gataacgcag 120

cgaaagctgt gctaataccg catacgatct acggaagcag gggatcgcaa gaccttgcgc 180

gaatggagcg gccgatggca gattaggtag ttggtgaggt aaaggctcac caagccttcg 240

atctgtagct ggtctgagag gacgaccagc cacactggga ctgagacacg gcccagactc 300

ctacgggagg cagcagtgac catcgttttg gacaatgggc gcaagcctga tccagccatg 360

ccgcgtgcag gatgaaggcc ttcgggttgt aaactgcttt tgtacggaac gaaacggcct 420

tttctaataa agagggctaa tgacggtacc gtaagaataa gcaccggcta actacgtgcc 480

agcagccgcg gtaatacgta gggtgcaagc gttaatcgga attactgggc gtaaagcgtg 540

cgcaggcggt aatgtaagac agttgtgaaa tccccgggct caacctggga actgcatctg 600

tgactgcatt gctgcattgc tggagtacgg cagaggggga tggaattccg cgtgtagcag 660

tgaaatgcgt agatatgcgg aggaacaccg atggcgaagg caatcccctg ggcctgtact 720

gacgctcatg cacgaaagcg tggggagcaa acaggatctg gtagtccacg ccctaaacga 780

tgtcaactgg ttgttgggaa ttcactttct cagtaacgaa gctaacgcgt gaagttgacc 840

gcctggggag tacggccgca aggttgaaac tcaaaggaat tgacggggac ccgcacaagc 900

ggtggatgat gtggtttaac tcgacaacgc gaaaaacctt acccaccttc ccacggaatt 960

cgccagagat ggcttagtgc tcgaaagaga accgtaacac aggtgctgca tggctgtcgt 1020

cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgtcattagt 1080

tgctacattt agttgggcac tctaatgaga ctgccggtga caaaccggag gaaggtgggg 1140

atgacgtcaa gtcctcatgg cccttatagg tggggctaca cacgtcatac aatggctggt 1200

acaaagggtt gccaacccgc gagggggagc taatcccata aaaccagtcg tagtccggat 1260

cgcagtctgc aactcgactg cgtgaagtcg gaatcgctag taatcgtgga tcagaatgtc 1320

acggtgaata cgttcccggg tcttgtacac accgcccgtc acaccatggg agcgggttct 1380

gccagaagta gttagcttaa ccgcaaggag ggcgatacca cgtcaggttc tttcta 1436

Claims

1. A bacterium-enzyme composite preparation for petroleum hydrocarbon degradation is characterized in that: the compound preparation is a microbial inoculum with petroleum hydrocarbon degradation capability and an enzyme agent obtained by crushing the microbial inoculum; the microbial inoculum is culture solution obtained by fermenting and culturing Variovorax sp.JWLC1 of Variovorax to OD value of 1.5-2.5 at 30 +/-1 ℃; the enzyme agent is a solid-liquid mixture obtained by crushing a fermentation culture solution obtained by fermenting Variovorax JWLC1 and then carrying out immobilization treatment; wherein the volume ratio of fermentation culture solution in the microbial inoculum and the enzyme agent is 1:1-1:3, the Variovorax sp.JWLC1 is preserved in the China center for type culture collection 10-11 days 2019, and the preservation accession number is CCTCC NO: m2019812.

2. The bacterial-enzyme complex formulation for petroleum hydrocarbon degradation of claim 1, wherein: the enzyme agent is a solid-liquid mixture prepared by performing fermentation culture on Variovorax JWLC1, performing ultrasonic crushing treatment on a culture solution directly under the condition of 300-350W, performing solid-liquid separation on a crushed mixture by centrifugation at 8000-10000rpm, collecting a liquid part which is a whole enzyme solution, performing carbonization treatment on a solid part, mixing the whole enzyme solution with the solid part at the mixing ratio of 200-300 ml to 1g, standing at 4 ℃ for 8-12h, and re-suspending.

3. The bacterial-enzyme complex formulation for petroleum hydrocarbon degradation of claim 2, wherein: and the solid part is all solids obtained after the crushed and separated Variovorax JWLC1 cell residues are dehydrated and air-dried, carbonized at 250-300 ℃ for 1-2 hours under an anaerobic condition and collected.

4. Use of the petroleum hydrocarbon degrading bacteria-enzyme complex formulation of claim 1 in degrading petroleum hydrocarbons in petroleum hydrocarbon contaminated soil.

5. The use of the petroleum hydrocarbon degrading bacterial-enzyme complex formulation of claim 4, wherein: the bacterium-enzyme composite preparation can be applied to the petroleum hydrocarbon polluted soil for degradation treatment of the petroleum hydrocarbon at one time or in multiple times; the application amount of the composite preparation in the polluted soil is 5-10%, and the condition that the abundance of Variovorax in the composite preparation in the soil is not less than 5 multiplied by 10 is ensured⁷CFU·g^-1And (5) drying the soil.

6. The use of a petroleum hydrocarbon degrading bacterial-enzyme complex formulation as defined in claim 5, wherein: the step of applying is that the microbial inoculum in the compound preparation is applied to the polluted soil according to the adding proportion of 5-10%, and the enzyme agent is applied to the polluted soil when the degradation rate of petroleum hydrocarbon in the soil is reduced to 20-50% of the initial degradation rate.