CN107008742B

CN107008742B - Method for accelerating remediation of petroleum-polluted soil

Info

Publication number: CN107008742B
Application number: CN201710417721.6A
Authority: CN
Inventors: 周际海; 樊后保; 濮海燕; 袁颖红; 李威; 廖迎春; 黄荣霞; 唐嘉婕
Original assignee: Nanchang Institute of Technology
Current assignee: Nanchang Institute of Technology
Priority date: 2017-06-06
Filing date: 2017-06-06
Publication date: 2020-11-27
Anticipated expiration: 2037-06-06
Also published as: CN107008742A

Abstract

The invention relates to the technical field of polluted soil remediation, and particularly discloses a method for accelerating remediation of petroleum-polluted soil. The method comprises the following steps: inoculating and culturing the bacteria-eating nematodes on nematode growth medium plates with gradually increased petroleum concentration to obtain domesticated bacteria-eating nematodes; adding organic matters into the petroleum-polluted soil, and uniformly stirring and mixing, wherein the addition amount of the organic matters is 0.5-1% of the mass of the petroleum-polluted soil; inoculating the domesticated bacteria-eating nematodes into petroleum-polluted soil added with organic matters, and repairing the soil by utilizing the bacteria-eating nematodes, the organic matters and microorganisms in the petroleum-polluted soil. The method is simple and convenient to repair the petroleum-polluted soil, is easy to implement, can improve the microbial environment of the soil by adding organic matters, accelerates the reduction of the petroleum concentration of the polluted soil by the combined action of the organic matters and the bacteria-eating nematodes, can effectively play a role in purifying the soil by recycling the method, and realizes the regeneration and the reutilization of the polluted soil.

Description

Method for accelerating remediation of petroleum-polluted soil

Technical Field

The invention belongs to the technical field of polluted soil remediation, and particularly relates to a method for accelerating remediation of petroleum-polluted soil.

Background

The soil nematode is one of the main functional groups of soil animals, is the most abundant metazoan in soil, is widely distributed in the soil of various habitats, and the quantity can reach 7.6 multiplied by 10⁵-9.2×10⁶/m². Soil nematode massage deviceThe nutritional group is mainly divided into 4 categories: the number of the bacteria-eating nematodes in a farmland ecosystem is more than 60 percent, the proportion of the bacteria-eating nematodes in rhizosphere soil is higher and can reach about 90 percent, and the bacteria-eating nematodes have important ecological functions in a soil ecosystem.

At present, the plant repair and microbial repair of the polluted soil developed in China have been researched more, but the research on animal repair of the polluted soil is relatively rare. And related researches show that the ecological functions of the bacteria-eating nematodes not only include self metabolism, but also include stimulation of the number, activity and community structure change of soil microorganisms by taking the feeding microorganisms, and the activity of the microorganisms in the soil can be improved by adding biomass charcoal into the soil, but the functions of the bacteria-eating nematodes in the soil polluted by petroleum and the influence of the interaction between the bacteria-eating nematodes and the microorganisms on the petroleum degradation of the soil are not clear. In order to provide a preliminary scientific basis for a new biological remediation approach of petroleum-contaminated soil, research on remediation of contaminated soil by using animal materials is necessary.

Disclosure of Invention

Aiming at the problem that the research on animal remediation of the contaminated soil is relatively few in the prior art, the method for accelerating the remediation of the petroleum-contaminated soil is provided, the petroleum-contaminated soil is remedied, the method is simple, convenient and easy to implement, organic matters are added to improve the microbial environment of the soil, the organic matters and the bacteria-eating nematodes act together to accelerate the reduction of the petroleum concentration of the contaminated soil, and the method can effectively play a role in purifying the soil and realize the recycling of the contaminated soil.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a method for accelerating the remediation of petroleum-contaminated soil comprises the following steps:

(1) inoculating and culturing the bacteria-eating nematodes on nematode growth medium plates with gradually increased petroleum concentration to obtain domesticated bacteria-eating nematodes;

(2) adding organic matters into the petroleum-polluted soil, and stirring and mixing uniformly; the adding amount of the organic matters is 0.5-1% of the mass of the petroleum-polluted soil;

(3) inoculating the domesticated bacteria-eating nematodes into petroleum-polluted soil added with organic matters, and allowing the bacteria-eating nematodes to enter the petroleum-polluted soil to repair the soil.

And further comprising a determination step (4), namely determining relevant parameters of the repaired soil, and verifying the repair capability of the bacterium-eating nematodes on the petroleum-polluted soil and the repair state of the soil.

Further, the petroleum polluted soil is calculated by dry soil, and 5-20 bactereophagous nematodes are added in each gram of dry soil.

Further, the petroleum polluted soil is calculated by dry soil, and 5 grazing bacterial nematodes are added in each gram of dry soil.

Further, the adding amount of the organic matters is 1 percent of the mass of the petroleum-polluted soil.

Further, the organic matter comprises one of straw, vegetable cake or biochar.

Furthermore, the straw, the vegetable cake and the charcoal are prepared by crushing and sieving with a 2mm sieve.

Further, the domesticated bacteroidal nematode can be used in the oil concentration of 10 g.L^-1Bacteriophagous nematodes growing on the medium.

Further, the bacteria-feeding nematode is a model nematodeCaenorhabditis elegans。

Adopt the produced beneficial effect of above-mentioned technical scheme to lie in: the method for accelerating the remediation of the petroleum-polluted soil is simple, convenient and easy to implement, the organic matter is added to improve the microbial environment of the soil, the organic matter and the bacteria-eating nematodes act together to accelerate the reduction of the petroleum concentration of the polluted soil, and the method can effectively play a role in purifying the soil and realize the recycling of the polluted soil.

Drawings

FIG. 1 is a bar graph showing the number of bacteria-feeding nematodes during the cultivation period in the examples of the present invention;

FIG. 2 is a bar graph showing the effect of organic matter and bacteria-feeding nematodes on the remediation of petroleum-contaminated soil in an embodiment of the present invention;

FIG. 3 is a bar graph of the effect of organic matter and bactereophagous nematodes on the biomass carbon of soil microorganisms in accordance with an embodiment of the invention;

FIG. 4 is a bar graph of the effect of organic matter and bacteria-feeding nematodes on soil base respiration in an embodiment of the invention;

FIG. 5 is a bar graph of the effect of organic matter and bactereophagous nematodes on soil entropy of respiration in an example of the invention;

FIG. 6 is a bar graph of the effect of organic matter and bactereophagous nematodes on soil fluorohydrolase activity in accordance with embodiments of the invention;

FIG. 7 is a bar graph of the effect of organic matter and bactereophagous nematodes on dehydrogenase activity in examples of the invention;

FIG. 8 is a bar graph of the effect of organic matter and bactereophagous nematodes on catalase activity in examples of the invention;

FIG. 9 is a bar graph of the effect of organic matter and bactereophagous nematodes on urease activity in accordance with embodiments of the present invention;

FIG. 10 is a bar graph of the effect of organic matter and bacteria-feeding nematodes on sucrase activity in examples of the invention;

FIG. 11 is a bar graph of the effect of organic matter and bactereophagous nematodes on the total amount of phospholipid fatty acids in soil microorganisms according to an embodiment of the invention;

FIG. 12 is a bar graph of the effect of organic matter and bacteroidal nematodes on the biomass of gram positive, gram negative, bacteria, fungi/bacteria in soil in accordance with an embodiment of the present invention;

FIG. 13 is a bar graph of the effect of organic matter and bacteriophagous nematodes on the biomass of actinomycetes, eukaryotic microorganisms, fungi, arbuscular mycorrhizal fungi in soil in an embodiment of the invention;

in the figure, FSP-high temperature sterilization of petroleum contaminated soil; s-nematode-killing soil; SP-5.0g oil/kg dry oil contaminated soil; oil contaminated soil of SPN-5.0g oil/kg dry soil +5 bacteriophagus nematodes g^-1Drying soil; SPNW-5.0g oil/kg dry soil oil contaminated soil +5 bacteriophagus nematodes g^-1Dry soil + 1% straw; SPNC-5.0g PetroleumOil-contaminated soil per kg of dry soil +5 grazing bacteria nematodes g^-1Dry soil + 1% vegetable cake; oil-contaminated soil of SPNB-5.0g oil/kg dry soil +5 bacteriophagus nematodes g^-1Dry soil + 1% charcoal. Different letters indicate significant differences between treatments.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

The method adopts artificial simulation of petroleum-polluted soil, adds different organic matters into the petroleum-polluted soil, inoculates domesticated and cultured resistant edible bacteria nematodes, and repairs the soil by utilizing the edible bacteria nematodes, the organic matters and microorganisms in the petroleum-polluted soil.

The selected organic matter comprises one of straw, vegetable cake or biochar, wherein the straw is a general term of stem and leaf (ear) parts of mature crops, and generally refers to the rest parts of wheat, rice, corn, potatoes, rape, cotton, sugarcane and other crops (generally coarse grains) after seeds are harvested. More than half of the products of crop photosynthesis exist in the straws, and the straws are rich in nitrogen, phosphorus, potassium, calcium, magnesium, organic matters and the like, and are a multipurpose renewable biological resource; the vegetable cake is prepared by high-temperature treatment, is nontoxic and tasteless, has comprehensive nutrients, proper proportion of nitrogen, phosphorus and potassium and high content of organic matters; the biochar is a solid product generated by high-temperature thermal cracking of a biological organic material (biomass) in an anoxic or anaerobic environment. Can be used as a high-quality energy source and a soil conditioner; the straws, vegetable cakes or biochar are crushed and sieved by a 2mm sieve, and are mixed with the petroleum-polluted soil more fully, so that rich organic matters are provided for soil remediation.

Domestication of bacteria-eating nematodes: inoculating a plurality of bacteria-eating nematodes with sterilized surfaces into a nematode culture medium, placing the nematode culture medium into a constant-temperature incubator at 22 ℃ for amplification culture, selecting a culture dish with the optimal number and the best activity of nematodes after 1 month of culture, inoculating the nematode culture dish onto a nematode growth culture medium plate added with petroleum, and gradually increasing the concentration of the petroleum in the nematode growth culture medium to 2 g.L^-1、4 g·L^-1、8 g·L^-1And 10 g.L^-1. Finally domesticating the petroleum with the concentration of 10 g.L^-1The domesticated bactereophagous nematode has stronger survival ability in the petroleum-polluted soil, so that the capability of repairing the petroleum-polluted soil is obviously improved.

In order to further understand the method for accelerating the remediation of the petroleum-contaminated soil according to the present invention, the following detailed description is given with reference to specific examples.

Example 1

1 experimental part

1.1 Experimental materials

Test soil: the soil is collected from Anhui province and county, large granular substances in the soil are picked out, stones, gravels and plant residues are removed, the soil is sieved by a 2mm sieve, the soil is put into a refrigerator at the temperature of minus 26 ℃ for freezing for three days, then the soil is transferred into a biological incubator at the temperature of 22 ℃ for culturing for one week, and the nematode existing in the soil in advance is killed by repeated freezing and thawing for 5 to 7 times by adopting a repeated freezing and thawing method. The nematodes were isolated by tray method until they were free of nematodes.

Petroleum to be tested: the crude oil used for the test is crude oil which is sourced from China petrochemical Jinnan oil field and has the following basic physicochemical properties: the total content of colloid and asphaltene is 8.4 percent, and the ground relative density is 871.4 kg.m³(25 ℃), the solidifying point is 26.8 ℃, the ground viscosity is 18.7 mPa.s (60 ℃), and the wax content is 24.7%.

Preparing petroleum-polluted soil: artificially simulating contaminated soil, dissolving crude petroleum oil with acetone, slowly adding into soil to be tested, mixing, placing in a fume hood, stirring, volatilizing completely, mixing the contaminated soil with the rest soil to make the petroleum concentration in the contaminated soil 5.0 g/kg^-1。

The bacteriophagous nematodes used in this experiment were model nematodes:Caenorhabditis elegansthe nematode is inoculated to culture medium by single culture method and enriched culture is carried out at 22 deg.C. In order to reduce experimental errors and avoid the pollution of microorganisms carried by the bacteria-eating nematodes to test soil, the nematodes which are subjected to enrichment culture need to be disinfected, and the disinfectant is a mixture of 0.002% of cycloheximide and 0.1% of streptomycin sulfate antibiotics.

1.2 design of the experiment

7 kinds of processing are set:

FSP-high temperature sterilization of petroleum-contaminated soil;

s-nematode-killing soil;

SP-5.0g oil/kg dry oil contaminated soil;

oil contaminated soil of SPN-5.0g oil/kg dry soil +5 bacteriophagus nematodes g^-1Drying soil;

SPNW-5.0g oil/kg dry soil oil contaminated soil +5 bacteriophagus nematodes g^-1Dry soil + 1% straw;

SPNC-5.0g oil/kg dry soil oil contaminated soil +5 bacteriophagus nematode g^-1Dry soil + 1% vegetable cake;

oil-contaminated soil of SPNB-5.0g oil/kg dry soil +5 bacteriophagus nematodes g^-1Dry soil + 1% charcoal.

Wherein, because the water content of different soils is different, the amount of petroleum and bacteria-eating nematodes is calculated after converting the soil into dry soil, for example: petroleum contaminated soil of "SPN-5.0 g petroleum/kg dry soil +5 bacteriophagus nematodes g^-1The term "dry soil" means that the concentration of oil contamination is such that the amount of oil added per kg of dry soil is 5.0g of oil, i.e. 5.0g of oil per kg of dry soil, and then the number of bactereophagous nematodes added is 5 nematodes per g of dry soil, i.e. 5 nematodes g of bactereophagous nematodes^-1Dry soil ".

And (3) the soil is subpackaged into 250 mL triangular bottles, the total number of the triangular bottles is 168, the soil weight in each bottle is equivalent to 120 g of dry soil, and the water content is adjusted to be 50% of the saturated water content. And (3) performing whole-process sterile operation, adding 5 nematodes to each gram of dry soil for treating the SPN, the SPNW, the SPNC and the SPNB, adding 1% of straws, 1% of vegetable cakes and 1% of charcoal respectively for treating the SPNW, the SPNC and the SPNB, and performing constant-temperature culture at 22 ℃. The experiment is destructive sampling, six treatments are randomly selected for sampling in each experiment, and each treatment is repeated for four times. Samples were taken on

days

0, 7, 14, 28, 56, 112, 168 for analysis of nematode numbers, oil residue concentrations, soil microbial biomass carbon, soil basal respiration, soil entropy of respiration, microbial enzyme activity, and soil microbial community structure in soil.

1.3 measurement method

1.3.1 determination of microbial biomass carbon content:

chloroform Fumigation of-K with Vance and Joergensen₂SO₄And (3) an extraction method, wherein microbial biomass carbon in the leaching liquor is measured by a TOC analyzer, and the conversion coefficient of the microbial biomass carbon is 0.38.

1.3.2 determination of the basic respiration content of soil:

the soil basal respiration is determined by gas chromatography.

1.3.3 determination of the Petroleum content in the soil

The residual concentration of the soil petroleum is measured by adopting a micro method of an ultrasonic extraction-enzyme labeling instrument.

1.3.4 determination of soil microbial enzyme Activity

(1) Soil FDA hydrolase activity assay:

determined by an optimized FDA (fluorescein diacetate) hydrolysis method;

(2) determination of soil dehydrogenase Activity:

adopting a TTC reduction method;

(3) determination of soil catalase activity:

a potassium permanganate titration method is adopted;

(4) determination of soil urease activity:

adopting an phenol blue color comparison method;

(5) determination of soil sucrase activity:

milligrams of 1.00 g of soil glucose after 24 hours represent the sucrase activity.

1.3.5 determination of microbial phospholipid fatty acids (PLFA):

the measurement was carried out by GC-MS.

1.3.6 determination of nematode number:

the nematode is separated by a tray method and counted by a stereoscope.

1.4 data calculation and statistical analysis

Data were processed and plotted using sigma plot 13.0 software and for differential significance analysis using SPSS 22.0 statistical software.

2 results and analysis

2.1 Change in the number of bacteria-feeding nematodes in the culture period

As can be seen from fig. 1, there was no significant difference between the treatments at day 0 sampling, and the number of nematodes treated with SPNC was the highest at

day

7, 14 and 28 sampling, and was significantly different from the other treatments; when sampling is carried out on day 28, the number of nematodes of the SPNC is the largest, and the number of nematodes is remarkably different among treatments; at day 56, nematode numbers appeared as: SPNC > SPNW > SPN > SPNB; the number of treated SPNW nematodes was the greatest and the number of SPNB nematodes was the smallest at day 112 and day 168 sampling; the numbers of the treated bactereophagous nematodes added with organic matter were significantly different at the

sampling days

56, 112 and 168.

2.2 Effect of organic matter and bacteriophagous nematode on the remediation Effect of Petroleum contaminated soil

As shown in fig. 2, the overall tendency of the petroleum concentration of the contaminated soil gradually decreased as the culture time increased. At day 0, there was no significant difference between treatments; on day 7, when the samples are sampled, the SPNC is removed, and each treatment has no significant difference with the contrast treatment FSP; at day 14, the soil oil concentration is expressed as: the treatment FSP > SP > SPN > SPNB > SPN > SPNW > SPNC, the treated SPNC has significant difference from other treatments, and the residual concentration of petroleum is the lowest; at the time of sampling on day 56, the concentration of the residual oil in the soil is represented as: FSP > SP > SPN > SPNB > SPNW > SPNC, and it can be obtained that the residual oil quantity in the treatment of adding organic matter is all lower than that in the treatments FSP, SP and SPN, and the residual oil quantity in the treatment of SPNC is the lowest; when sampling is carried out on the 112 th day, the treatment shows significant difference, and the petroleum concentration of the polluted soil in the treatment of adding organic matters and nematodes is lower than that of FSP, SP and SPN; at the time of sampling on day 168, the soil oil concentrations appeared as treatment FSP > SP > SPN > SPNB > SPNW > SPNC. The results show that the addition of organic matter and bacteroidal nematodes can reduce the petroleum concentration of the contaminated soil, and compared with the sampling on day 0, the petroleum concentrations of the treated FSP, SP, SPN, SPNW, SPNC and SPNB are reduced by about 8.26%, 41.15%, 56.79%, 61.89%, 68.66% and 58.91% respectively at the end of the culture; the oil concentrations of treated SP, SPN, SPNW, SPNC and treated SPNB were reduced by about 35.11%, 52.48%, 58.58%, 65.70% and 58.91%, respectively, compared to treatment of FSP; the oil concentrations of the treated SPNW, SPNC and SPNB were reduced by about 12.83%, 27.81% and 4.77%, respectively, compared to the treated SPN.

2.3 influence of organic matter and bacteriophagous nematodes on soil microbial biomass carbon, basal respiration and respiratory entropy

2.3.1 Effect of organic matter and bacteriophagous nematodes on soil microbial Biomass carbon

As shown in fig. 3, the microbial biomass carbon of the soil tended to decrease gradually over the 168 day culture period. The SPNC biomass carbon was highest throughout the culture period. The SPNC treatment is obviously different from other treatments when sampling is carried out on the day 0; the soil microbial biomass carbon content at the 7 th day sampling was shown as SPNC > SPNW > SP > SPN > S > SPNB, with about 8.53%, 89.16%, 422.76% and 4.85% increase in SP, SPNW, SPNC and SPN microbial biomass carbon and about 38.74% decrease in SPNB microbial biomass carbon treatment compared to S, with significant differences between SPNW and SPNC treatment compared to other treatments; at day 14, the soil microbial biomass carbon content was shown as SPNC > SPNW > SP > SPNB > SPN > S, with no significant difference between treatments SP, SPNW, SPNB, and SPN, and significant differences between treatments SPNC and other treatments (P < 0.05); when sampling is carried out on the 28 th day, the treatment of adding the organic matters is obviously different from the treatment S of contrast treatment; at the sampling time of day 56, the differences of the SP, SPNW and SPNC treatments are obvious, the biomass carbon of the microorganism added with organic matters is greater than that of other treatments, at the sampling time of day 112, the SPNW and SPNC are obviously different from other treatments, and the biomass carbon of the soil microorganism of the SPNW is greater than that of the SPNC; at the time of sampling on day 168, the soil microbial biomass carbon can be expressed as SPNW > SPNC > SPNB > SPN > SP > S, and it can be found that the addition of an organic substance increases the microbial biomass carbon in the soil, and the microbial biomass carbon of the SPNW is highest in the late stage of cultivation.

2.3.2 Effect of organic matter and bacteriophagous nematodes on basal respiration of soil

The soil foundation respiration is generally increased and then decreased in the whole culture period, as shown in fig. 4, when sampling is performed on day 0, the respiration of the treated SPNC soil foundation is strongest, and the respiration of the treated soil foundation added with organic matters is greater than that of the control treatment S; at day 7, the basal respiration rates of the SPNP, SPN, SPNW, SPNC and SPNB soils were increased by about 52.39%, 111.78%, 373.15%, 939.14% and 167.53%, respectively, compared to the control treatment S, the basal respiration rates of the SPNW, SPNC and SPNB soils were increased by about 123.41%, 390.67% and 26.32%, respectively, and the basal respiration rates of the SPN soils were increased by about 28.04% compared to the treatment SP. The SPNW processing and the SPNC processing are significantly different from other processing; on the 14 th day of sampling, the total respiration rate of the soil foundation shows a descending trend; when sampling is carried out on the 28 th day, the SPNC soil foundation breathes most strongly; when sampling is carried out on the 56 th day and the 112 th day, the basic respiration of the SPNW is strongest, and the basic respiration of the soil has significant difference among different organic matter treatments; at day 112, there was no significant difference between SP, SPN and SPNB and S; soil basal respiration at day 168 sampling was shown to treat SPNC > SPNW > SPNB > SPN > SP > S, indicating that addition of organic matter can increase basal respiration of soil. Throughout the incubation period, the soil base respiration was stronger for treatment of SPNW and treatment of SPNC than for other treatments.

2.3.3 Effect of organic matter and bacteriophagous nematodes on soil respiration entropy

The microbial respiration entropy of the soil is in a state of increasing and then decreasing and tending to be stable in the culture period of 168 days, as shown in fig. 5, when sampling is carried out on the 0 th day, except for the treatment of SPNC, the respiration entropy of the nematode is far greater than that of SP and S; when sampling is carried out on the 7 th day, the respiratory entropy of the SPNB microorganisms is the highest when the SPNB microorganisms are processed, and the SPNB microorganisms are obviously different from various types of processing; the SPNW respiratory entropy is highest when sampling is carried out on the 14 th day, and the respiratory entropy of each treated microorganism is obviously different from that of the control treatment S; at day 28 sampling, the respiratory entropy is represented as: treating SP > SPNB > SPNW > SPN > SPNC > S, the respiratory entropy of the microorganism decreases sharply; at the sampling time of day 56, the respiratory entropy of microorganisms treated by the SPNW is highest, and no significant difference exists among SPN, SPNC and SPNB treated; at the time of sampling on day 112, the processing SP, SPNW, and SPNC were significantly different from the control processing S; at day 168, the respiratory entropy was expressed as: processing SPNC > SP > S > SPNB > SPNW > SPN. At day 112 and day 168 sampling, except for control treatment S, the respiratory entropy of the treated SPN was lower than that of the other treatments, indicating that the addition of the bacteroidal nematodes had some positive effect on improving the soil environment. During the whole culture period, different organic matters are added to affect the soil environment differently. The SPNC respiratory entropy processing is lower than the SPNW and SPNB processing at the time of sampling on days 0 to 7, the SPNB respiratory entropy processing is lower than the SPNW and SPNC processing at the time of sampling on days 56 to 112, and the SPNW respiratory entropy processing is lower than the SPNC and SPNB processing at the time of sampling on day 168.

2.4 Effect of organic matter and bacteriophagous nematodes on soil enzyme Activity

2.4.1 Effect of organic matter and bacteriophagous nematodes on the Activity of fluorescein hydrolase

As shown in fig. 6, the treated SPNW and the treated SPNC had stronger luciferase activity than the other treatments throughout the entire incubation period. At day 0 sampling, there was no significant difference between the treated SPN and SPNB versus the control treatment S; at the time of sampling on day 7, the fluorohydrolase activity was shown to be SPNW > SPNC > SPNB > SP > SPN > S, the fluorohydrolase activity of SPNW and SPNC being much higher than that of the other treatments; when sampling is carried out on the 14 th day, the SPNW and the SPNC have no significant difference and have significant difference with other processing; at the time of sampling at day 28, the treatment with the added organic matter is significantly different, the fluorescein hydrolase activity of the SPNW is the highest, and the SPNB treatment is not significantly different from S, SP treatment and SPN treatment; at the time of sampling on day 56, the treatments SPNW and SPNC showed significant differences from the other treatments, and except treatment SP, the fluorescein hydrolase activity of each treatment was greater than that of treatment S; no significant difference in the activity of the fluorogenic hydrolase between treatments S, SP, SPN and SPNB at day 112 sampling; the treated SPNW soil has strongest fluorescein hydrolase activity; at the sampling day 168, the FDA hydrolase activity was shown as SPNW > SPNC > SPNB > SPN > S > SP, with no significant difference between treatments other than SPNW and SPNC, with a decrease of about 8.88% for treatment SP compared to control treatment S, an increase of about 2.80%, 233.64% and 123.13% for treatment SPN, SPNW and SPNC compared to control treatment S, respectively, and no change in the luciferase activity for SPNB compared to S, indicating that the addition of organic substances can alter the luciferase activity in contaminated soil.

2.4.2 Effect of organic matter and bacteriophagous nematodes on dehydrogenase Activity

As shown in FIG. 7, the dehydrogenase activity generally showed a tendency of increasing first and then decreasing during the culture period, and the dehydrogenase activity in the petroleum-contaminated treatment was high at the initial stage of the culture. When sampling is carried out from day 0 to day 56, the treatment of adding petroleum is different from the control treatment S in significance, and the treated SPNC dehydrogenase has the highest activity; when sampling is carried out on day 0, the dehydrogenase activity of each treatment is not significantly different except for the SPNB; when sampling is carried out on the 7 th day, the dehydrogenase activity has no significant difference among treatments except the treatment S; at day 14 sampling, the dehydrogenase activity of the treatments SP, SPNW, SPNC, SPNB, and SPN increased by about 200%, 379.70%, 1127.83%, 350.79%, and 394.97%, respectively, compared to the control treatment S; on day 28, the SPN treatment was significantly different from the control treatment S except for the SPN treatment; in the sampling of the 56 th day, except processing SPNC and S, other various processes have no significant difference; on day 168, the dehydrogenase activity was shown as: treatment of SPNB > SPNC > SPNW > SPN > SP > S revealed that addition of organic substances could improve the dehydrogenase activity of the contaminated soil.

2.4.3 Effect of organic matter and bacteriophagous nematodes on Catalase Activity

As shown in FIG. 8, the catalase activity tended to be generally stable over the 168-day culture period. At day 0 sampling, the catalase activity of the SPNW was significantly different from control treatment S, with no significant difference between the other treatments (P > 0.05); at day 7, there was no significant difference between treatment and control treatment S; at the time of sampling at day 14, the SPN treated was not significantly different from S, the other treatments were significantly different from S, and the catalase activities of the SP, SPNW, SPNC, SPNB, and SPN were increased by about 27.37%, 40.06%, 60.54%, 38.10%, and 23.22%, respectively, compared to the treatment S; SPNB had the strongest catalase activity at day 28, day 56, and day 112 samples; at day 168, SP was significantly different from the other treatments and catalase activity was shown as SP > S > SPNB > SPN > SPNW > SPNC.

2.4.4 Effect of organic matter and bacteriophagous nematodes on urease Activity

As shown in FIG. 9, the urease activity generally showed a tendency to increase and decrease again during the culture period. When sampling is carried out on day 0, the treatment of adding organic matters is obviously different from the comparison treatment S; the urease activity is shown to be SPNC > SPNW > SPNB > SPN > SP > S at the sampling time of day 7, and the treatment is significantly different from the control treatment S; at day 14 sampling, the urease activities of treatment SP, SPNW, SPNC, SPNB, and SPN increased by about 102.21%, 670.87%, 162.40%, 130.80%, and 109.24%, respectively, compared to control treatment S; when sampling is carried out on the 28 th day, petroleum is added for treatment, the urease activity is greater than that of the control treatment S, and the treatment of adding the bacteria-eating nematodes is obviously different from the treatment of SP; when sampling is carried out on the 56 th day, the urease activity after organic matter addition treatment is obviously different from that of the urease activity after control treatment S; on the 112 th day of sampling, the other treatments except SPN are all obviously different from S; when the soil is sampled on the 168 th day, the urease activity is represented by SPNW > SPNC > SPN > SPNB > SP > S, and the effect of adding organic matter and bacteria-feeding nematodes on enhancing the urease activity in the soil can be obtained.

2.4.5 Effect of organic matter and bacteriophagous nematodes on sucrase activity

As shown in fig. 10, the sucrase activity was highest in the treatment of SPNC throughout the culture period. At day 0 sampling, the treatments SPNC and SPNB were significantly different from the control treatment S; on day 7, the sucrase activity was shown as SPNC > SPNW > SPN > SPNB > SP > S with no significant difference between treatment SP, SPNB, SPN and control treatment S; at day 28, the sucrase activity was expressed as SPNC > SPNW > S > SPN > SPNB > SP, with sucrase activity increased by about 117.82% and 682.30% for treated SPNC and SPNW, respectively, and sucrase activity decreased by about 23.25%, 10.83%, and 0.72% for treated SP, SPNB, and SPN, respectively, as compared to control treatment S; on the 56 th day of sampling, the SP treatment and the S control treatment are all significantly different; at day 112 sampling, the processing SPNW and SPNC were significantly different from the control processing S; at day 168, the sucrase activity was expressed as SPNC > SPNW > SPNB > S > SPN, with treatment SP and SPN sucrase activities being reduced by about 27.58% and 30.42%, respectively, and treatment SPNW, SPNC, and SPNB sucrase activities being increased by about 80.62%, 313.54%, and 12.40%, respectively, as compared to treatment S. Experiments show that the addition of organic matters, particularly vegetable cakes, is beneficial to the improvement of the activity of sucrase in soil.

2.5 organic matter and bacteriophagous nematode effects on soil microbial community architecture (PLFA)

2.5.1 types of PLFA detected in the soil samples tested:

the microbial PLFAs tested according to the standard nomenclature classification are shown in Table 1.

TABLE 1 PLFA types detected in the soil tested

2.5.2 Effect of organic matter and bacteriophagous nematodes on the Total amount of Phospholipids fatty acids in soil microorganisms

As shown in fig. 11, at the time of sampling on day 0, the treatment SPNC was significantly different from other treatments, and the total amount of microbial phospholipid fatty acids in the soil was much greater than those of other treatments; at day 168, the total PLFA microbial content in the soil was expressed as: SPNC > SPNW > SPNB > SPN > SP > S, indicating that the total amount of phospholipid fatty acids of soil microorganisms in each treatment was greater than that of the control treatment S, and the total amount of PLFA in the treatment with the addition of the bacteroidal nematodes was greater than that of SP and S. The significant difference between the total amount of PLFA treated with SPNC and SPNW compared to the other treatments indicated that the treatment with added organic matter had a higher amount of microbial PLFA compared to the other treatments.

2.5.3 Effect of organic matter and bacteriophagous nematodes on the Biomass of gram-positive, gram-negative, bacterial, fungal/bacterial in soil

As shown in fig. 12, at the time of sampling on day 0, the gram-positive bacteria were the lowest among the treated SPNWs, and the other treatments were not significantly different from the control treatment S except for the treated SPNWs; on day 168, the samples were taken, with petroleum added, and gram-positive bacteria were all greater than control treatment S, with the PLFA levels of gram-positive bacteria appearing as: the treatment of SPNW > SPNB > SPNC > SPN > SP > S indicated that the addition of organic matter can promote the increase in the amount of gram-positive bacteria PLFA in the soil. When sampling is carried out on day 0, the significant difference exists between the gram-negative bacteria for processing the SPNC and each treatment, and the PLFA quantity of the gram-negative bacteria for processing the SPNW is the lowest; at 168 days of sampling, the PLFA amount of gram-negative bacteria is expressed as SPNC > SPNW > SPNB > SPN > SP > S, which indicates that the addition of organic matters can increase the number of gram-negative bacteria in soil, and the addition of vegetable cakes can sharply increase the number of gram-negative bacteria microorganisms in soil during the culture period. When the samples are taken on the 0 th day and the 168 th day, the treatment of the bacteroidal nematodes is added, the number of the bacteroidal PLFA in the soil is large, and the number of the bacteroidal PLFA in the SPNC is the largest, which shows that the addition of the vegetable cake can increase the number of the bacteroidal in the soil. At day 0 and day 168 sampling, both SPNC and SPNB were treated with greater numbers of fungal/bacterial PLFA than the other treatments, and were significantly different from the other treatments. The addition of the straws and the vegetable cakes is beneficial to improving the soil environment and improving the soil quality.

2.5.4 Effect of organic matter and bacteriophagous nematodes on the Biomass of Actinomycetes, eukaryotic microorganisms, fungi, arbuscular mycorrhizal fungi in soil

As shown in fig. 13, the amount of actinomycete PLFA at day 0 sampling was expressed as: SPNB > SPNC > SPN > S > SPNW > SP, the SP and S are significantly different when being processed, and the PLFA quantity of SP actinomycetes is the lowest when being processed; at day 168, the actinomycete PLFA amount was expressed as: SPNW > SPNB > SP > SPNC > SPN > S, the amount of actinomycetes PLFA in each treatment was larger than that in the control treatment S, and was significantly different from that in the control treatment S. When sampling is carried out on day 0, the quantity of eukaryotic microorganisms added with organic matters is greater than that of other eukaryotic microorganisms, and the PLFA for processing the SPNC eukaryotic microorganisms is the highest; at day 168, the eukaryotic microbial PLFA amount appeared as: SPNW > SPNC > SPNB > SP > SPN > S, and the treatments were not significantly different from the control treatment S except for the treatment of SPNW and SPNC, and the results showed that the PLFA amount of the eukaryotic microorganism in the soil increased at the end of the culture period, and the PLFA amount of the eukaryotic microorganism in the treated SPNW was the highest. When sampling is carried out on day 0, the PLFA amount of each treatment fungus is obviously different from that of the control treatment S except the treatment SPNC; the fungal PLFA level at 168 days of sampling was expressed as SPNC > SPNW > SPNB > SP > SPN > S, and the treated SPNC fungal PLFA level was highest with no significant difference from the control treatment S except for the treated SPNW and SPNC. At the time of sampling on day 0, the quantity of arbuscular mycorrhizal fungi PLFA for processing SPNC is obviously different from that of other processing; the PLFA amount of the arbuscular mycorrhizal fungi is expressed by processing SPNC > SPNB > S > SPN > SPNW > SP; at day 168, the amount of arbuscular mycorrhizal fungi PLFA appeared as: SPNW > SPNB > SPNC > SPN > SP > S, and the processing was not significantly different from the comparison processing S in each case except for the processing of SPNW and SPNB. In conclusion, the addition of organic matters can increase the quantity of arbuscular mycorrhizal fungi PLFA in the soil to a certain extent.

Discussion of 3

The experimental result shows that the number of nematodes treating SPNC is the largest at the initial culture stage; at day 56, the number of treated SPNC nematodes decreased. Probably because the vegetable cake is easy to decompose in a short period, the nutrients stimulate the growth of microorganisms and bacteria-eating nematodes, and in the later period of the experiment, because the vegetable cake is greatly consumed, and insufficient nutrients support the growth and the propagation of the microorganisms and the bacteria-eating nematodes, the number of the bacteria-eating nematodes is reduced. At the end of the experimental culture, the number of nematodes appeared as: the treatments SPNW > SPN > SPNC > SPNB showed that the addition of different organic substances had different effects on nematode numbers, the treatments SPNC and SPNB were significantly different from the control treatment SPN, and in the organic substance treatment, the number of nematodes was consistently lower throughout the incubation period for the treated SPNB than for the treatments SPNW and SPNC, probably because the charcoal provided less active organic substances, which was detrimental to the growth of the bactophageal nematodes.

The experimental result shows that the addition of the organic matters and the bacterium-eating nematodes is beneficial to the petroleum remediation of the polluted soil and can reduce the petroleum concentration of the polluted soil. Compared with the sampling on the day 0, the petroleum concentration of the treated FSP, SP, SPN, SPNW, SPNC and SPNB is respectively reduced by about 8.26%, 41.15%, 56.79%, 61.89%, 68.66% and 58.91% when the sampling is carried out on the day 168, which shows that the indigenous microorganisms in the soil have certain influence on the petroleum degradation, and the petroleum concentration is basically unchanged compared with the day 0 when the FSP treatment is a sterilization treatment, namely the culture period is ended; at the sampling day 168, the oil concentration of the oil residue after the organic matter addition treatment is reduced by about 35.11%, 52.48%, 58.58%, 65.70% and 58.91% compared with that of the oil residue after the FSP treatment; the oil concentrations of the treated SPNW, SPNC and SPNB were reduced by about 12.83%, 27.81% and 4.77%, respectively, compared to the treated SPN. The result shows that the organic matter and the bacteria-eating nematode influence the petroleum repair of the polluted soil to a certain extent, and the addition of the organic matter can bring nutrients to microorganisms in the soil, stimulate the growth and the propagation of the microorganisms and have certain influence on the growth and the propagation of the bacteria-eating nematode.

The microbial activity of the soil can be improved by adding the organic matters, which is consistent with the research results of Wangbui and other people, and the microbial biomass can be obviously improved after the organic matters are added into the soil. The research shows that the microbial biomass carbon for processing the SPNW and the SPNC is increased at the initial stage of experimental culture, and the straw and the vegetable cake can increase the microbial biomass carbon in the soil; the reason may be that the straws and the vegetable cakes added in the soil release nutrient components, improve the soil environment, reduce the stress of the environment on microorganisms and promote the growth of the microorganisms; the microbial numbers show a trend of decreasing in the middle and later culture periods due to the fact that organic matters are decomposed too much and insufficient nutrients stimulate the growth and reproduction of microorganisms, and the biomass carbon of soil microorganisms in the SPNW and the SPNC is reduced. Experimental results show that the microbial activity of soil can be changed to a certain extent by adding the straws and the vegetable cakes, and the growth and the propagation of bacteria-eating nematodes are promoted. The microbial biomass carbon of SPNB increased from the initial stage to decreased at the late stage of culture, which is consistent with the studies of octoquine, indicating that biochar can increase microbial biomass carbon in soil in a short time, but gradually decrease as culture time increases. In summary, the carbon biomass of the soil microorganisms added with the straws and the vegetable cakes is higher than that of other treatments.

The basal respiration of the soil showed a tendency to rise first and then fall over the entire culture period, similar to the study in king dawn. During the whole culture period, the soil basic respiration of the SPNW and SPNC treatment is obviously different from that of other treatments in six sampling, probably because the straws and vegetable cakes added in the treatment are easy to decompose, the growth of microorganisms in the soil is stimulated, the increase of the number of bacteria-eating nematodes is facilitated, namely, the respiration in the initial culture period is enhanced, and a large amount of CO is released₂(ii) a The later culture period may be caused by the decrease of organic matter available to the microbes, the decrease of petroleum concentration in the polluted soil, the decrease of microbial stress, and CO in the soil₂The amount of emissions is reduced.

In general, organic matters added into soil have influence on the respiration entropy of microorganisms. When sampling is carried out on day 0, except for SPNC treatment, the treatment respiration entropy of nematodes is far greater than that of SP treatment and S treatment, and it is possible that the rape cake added by SPNC treatment can quickly release nutrients required for the growth of microorganisms, the stress on nematodes and microorganisms is small, and the stress on nematodes and microorganisms in SPN, SPNW and SPNB treatment is caused by the soil environment, namely the respiration entropy is increased rapidly; in the middle stage of experimental culture, the respiration entropy of microorganisms in soil is reduced, on one hand, the reason may be that nematodes and microorganisms in the soil are adapted to the soil environment, and on the other hand, the reason may be that certain microorganisms are not adapted to the soil environment in the early stage of culture, so that the death and the reduction of the number of the microorganisms are caused; at the sampling day 168, the respiratory entropy of the treated SPNC microorganisms is the largest, and is obviously different from that of other treatments, and possibly in the later culture period, wheat straws and biochar are gradually decomposed in soil to improve the soil environment, namely in the later culture period, the respiratory entropy of the treated SPNW and the respiratory entropy of the treated SPNB are smaller than that of the treated SPNC. At the end of the whole experimental culture period, the microbial respiration entropy is expressed as SPNC > SP > S > SPNB > SPNW > SPN, which shows that the addition of organic matters has certain influence on the metabolic activity of soil microbes, and the biochar and straws can improve the polluted soil environment in a certain culture period.

Throughout the incubation period, the treated SPNW and the treated SPNC had stronger fluorescein hydrolase activity than the other treatments. The reason may be that the addition of straw and vegetable cake brings a lot of nutrient substances to the microorganisms, which causes the mass propagation of some microorganisms in the soil, i.e. the activity of the fluorescein hydrolase is increased rapidly in a short time. At day 7, the FDA hydrolase activity of both SPNW and SPNC was significantly higher than control treatment S, probably because the added organic substances stimulated microbial proliferation and nematode growth during the initial phase of the experimental culture; in the later period of culture, as organic matters are gradually decomposed or absorbed along with the increase of time, nutrients available for microorganisms in soil are reduced, and the activity of FDA hydrolase is reduced in the later period. The addition of organic matters and bacteria-feeding nematodes can promote the activity of the fluorescein hydrolase in the petroleum-polluted soil, and particularly, the addition of straws and vegetable cakes can improve the activity of FDA hydrolase in the petroleum-polluted soil.

The dehydrogenase activity of the petroleum pollution treatment added was generally higher than that of the control treatment S. In the later period of experimental culture, the dehydrogenase activity of the treatment added with organic matters is higher than that of other treatments. The experimental result shows that the dehydrogenase activity for processing SPNC is strongest when sampling is carried out on

days

0, 7, 14, 28, 56 and 112; the dehydrogenase activity was strongest when SPNB was treated at day 168. The rape-seed cake is easy to decompose at the initial culture stage, so that the mass propagation of microorganisms in soil is stimulated, the oxidation and the conversion of petroleum hydrocarbon are realized, and the dehydrogenase activity for treating SPNC is increased; in the later period of culture, the hydrogen atom conversion amount of petroleum hydrocarbon in the soil is reduced, so that the activity of dehydrogenase is in a downward trend in the later period. The reason may be, on the one hand, the result of substantial oxidation and conversion of readily decomposable hydrogen ions in the contaminated soil and, on the other hand, the reduction in nutrients which microorganisms in the soil can decompose and absorb, resulting in a reduction in dehydrogenase activity. At the end of the whole incubation period, it can be concluded that the addition of organic matter favours the increase of dehydrogenase activity.

The research result shows that the addition of organic substances can improve the activity of catalase, which is consistent with the Wangbai research, and after different organic substances are added into the soil, the activity of catalase in the soil can be obviously improved. At the time of sampling on day 112, the catalase activity is shown as SPNB > SPN > SP > S > SPNW > SPNC, and SPNB has stronger catalase activity in the whole culture period, which is similar to the quiet study of plum, and the addition of biochar can improve the soil catalase activity. Experiments show that in the initial culture stage, the added organic matter can enhance the activity of catalase, so that soil microorganisms can better adapt to the environment. This is consistent with the results of the Suzuicar study, where the initial organic phase enhanced catalase activity.

When sampling is carried out on day 0, the urease activity of the added organic matter treatment is obviously different from that of other treatments; in the middle culture period, the urease activities of the SPNW, the SPNC and the SPNB are high, probably due to the addition of organic matters, the change of exogenous environment is caused, the growth and the propagation of microorganisms are stimulated, and the urease activities of the SP treatment and the SPN treatment are reduced. Urease activity was shown on day 168 when sampled as: SPNW > SPNC > SPN > SPNB > SP > S, the urease activities of the treated SPNW, SPNC and SPN are significantly different from those of the control treated S, and the addition of organic matters has certain influence on the urease activity of the petroleum-polluted soil.

The higher the activity of the sucrase, the better the soil fertility, and the activity of the sucrase is an important index for reflecting the soil fertility. The sucrase activity of SPNC is at the highest level throughout the culture period. The sucrase activity was SPNC at day 56>SPNW>SPN>SPNB>SP>S, it can be obtained that the addition of organic substances can increase the activity of the sucrase in the soil. When sampling is carried out on day 0, except for the treatment of SPNC, the activity of each treatment sucrase is lower than that of the control treatment S, which shows that the activity of sucrase can be influenced by adding petroleum into soil, and the result is consistent with the research result of Zhang Feng: the petroleum concentration is 0-30000 mg.kg^-1In this case, the sucrase activity is stimulated, and beyond this range, the sucrase activity is inhibited. The experimental result shows that the organic matter is added to increase the activity of the sucrase in the soil and improve the soil fertility. Throughout the culture period, the sucrase activity was higher for treatments SPNW and SPNB than for other treatments, indicating that straw and tortillas are beneficial for increasing sucrase activity.

Through sampling analysis on the 0 th day and the 168 th day, the phospholipid fatty acid in the soil is counted, and the statistics shows that the addition of the organic matters can stimulate the number and the activity of microorganisms in the soil, change the relative abundance degree of the microbial population in the soil, and obviously increase the total amount of bacteria by adding the organic matters. The reason may be that organic matter may act as a carbon source for microbial growth, stimulating microbial growth. At day 168, the highest amount of fungal PLFA was treated in both SPNW and SPNC, probably because straw and tortillas stimulated fungal growth and reproduction. When the sample is sampled at 168 days, the quantity of the arbuscular mycorrhizal fungi PLFA is increased, which shows that the quantity of the arbuscular mycorrhizal fungi PLFA can be influenced by the addition of organic matters. The experimental result shows that the ratio of soil fungi to bacteria in the SPNW and SPNC is large, which reflects that the soil environment can be improved by adding straws and vegetable cakes.

4 conclusion

The treatment of adding organic matters increases the activity of FDA hydrolase, dehydrogenase, catalase, urease and sucrase on the whole, which shows that the addition of organic matters can improve the microbial environment of soil, and in the whole culture period, the basic respiration of soil and microbial biomass carbon for treating SPNW and SPNC are at a higher level, so that organic matters can promote the growth of microorganisms and influence the regulation and control of edible bacteria nematodes on soil microorganisms. The research finds that the addition of organic matters can obviously increase the total amount of bacteria and arbuscular mycorrhizal fungi in soil, and the fungus/bacteria ratio of the SPNW and the SPNC is higher, which indicates that the straw, the vegetable cake and the bacteria-eating nematode act together to change the microbial community structure.

At the end of the culture, the oil concentrations of treatments FSP, SP, SPN, SPNW, SPNC and SPNB were reduced by about 8.26%, 41.15%, 56.79%, 61.89%, 68.66% and 58.91% from the initial concentrations; oil concentrations were reduced by about 35.11%, 52.48%, 58.58%, 65.70% and 58.91% for treatment SP, SPN, SPNW, SPNC and treatment SPNB compared to treatment FSP; the oil concentrations of the treated SPNW, SPNC and SPNB were reduced by about 12.83%, 27.81% and 4.77%, respectively, compared to the treated SPN. The inoculated nematodes can promote the degradation of petroleum in the polluted soil, the treatment with organic matters can better promote the degradation of the petroleum, and the efficiency of treating the petroleum in the polluted soil by SPNC with rape cake is highest.

The culture time is prolonged, or the method can degrade petroleum in the soil to a greater extent, effectively play a role in purifying the soil, and realize the recycling of the polluted soil.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the foregoing description only for the purpose of illustrating the principles of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims, specification, and equivalents thereof.

Claims

1. The method for accelerating the remediation of the petroleum-polluted soil is characterized by comprising the following steps:

(1) inoculating and culturing bacteria-eating nematodes on a nematode growth medium plate with gradually increased petroleum concentration to obtain domesticated bacteria-eating nematodes, wherein the bacteria-eating nematodes are model nematodes Caenorhabditis elegans, and the obtained domesticated bacteria-eating nematodes have the petroleum concentration of 10 g.L^-1Bacteriophagous nematodes growing on the medium;

(2) adding organic matters into the petroleum-polluted soil, and stirring and mixing uniformly; the organic matter comprises one of straw, vegetable cake or biochar, and is prepared by crushing and sieving with a 2mm sieve, and the addition amount of the organic matter is 0.5-1% of the mass of the petroleum-polluted soil;

(3) inoculating the domesticated bacteria-eating nematodes into petroleum-polluted soil added with organic matters, and allowing the bacteria-eating nematodes to enter the petroleum-polluted soil to repair the soil;

(4) and (3) measuring relevant parameters of the repaired soil, and verifying the repair capability of the bacterium-eating nematode on the petroleum-polluted soil and the repair state of the soil.

2. The method for accelerating the remediation of soil contaminated with petroleum according to claim 1, wherein said soil contaminated with petroleum is supplemented with 5 to 20 bacteriophagous nematodes per gram of dry soil.

3. The method for accelerating the remediation of oil contaminated soil according to claim 2, wherein said oil contaminated soil is supplemented with 5 grazing bacteria nematodes per gram of dry soil.

4. The method for accelerating the remediation of soil contaminated with petroleum according to claim 1, wherein the organic matter is added in an amount of 1% by weight of the soil contaminated with petroleum.