Bioremediation material, application and bioremediation method of polycyclic aromatic hydrocarbon contaminated soil

Abstract

The invention belongs to the technical field of microbial degradation of polycyclic aromatic hydrocarbons, and relates to a bioremediation material and application thereof, and a bioremediation method for soil polluted by polycyclic aromatic hydrocarbons. The bioremediation material is prepared from a microbial agent, small molecular fatty acid and a co-metabolism substrate, the microbial agent contains lactobacillus paracasei, and the preservation number of the lactobacillus paracasei is CGMCC (China General Microbiological Culture Collection Center) No.24463. The bioremediation material is prepared from the microbial agent, the small molecular fatty acid and the co-metabolism substrate. The bioremediation material disclosed by the invention has an excellent degradation effect on soil polycyclic aromatic hydrocarbon. The polycyclic aromatic hydrocarbon contaminated soil bioremediation method has the advantages of being easy to operate, good in remediation effect, low in remediation cost, free of secondary pollution and the like, and can be widely applied to remediation of polycyclic aromatic hydrocarbon contaminated soil.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial degradation of polycyclic aromatic hydrocarbons, and specifically relates to a bioremediation material, application of the bioremediation material in degradation of polycyclic aromatic hydrocarbons, and a bioremediation method for polycyclic aromatic hydrocarbon-contaminated soil based on the bioremediation material. Background Art

[0002] During the process of oil exploration, mining, transportation, and refining, some oil spills into the soil due to improper operation or accidental leakage, causing petroleum hydrocarbon pollution in the soil. Every year, about 8 million petroleum pollutants are discharged into the environment through crude oil landing and accidental leakage. 6 t, petroleum hydrocarbon pollution has become the focus of world attention. In the 1980s, the United States had serious petroleum hydrocarbon soil pollution problems. About half of its 450,000 brownfields were contaminated with petroleum hydrocarbons. During the same period, the number of sites contaminated by petroleum hydrocarbons in Canada was as high as 60%. Many areas and factories in the United Kingdom also had serious oil pollution. Germany, the Netherlands, the United Kingdom and other countries have formulated relevant remediation project plans to strengthen the remediation and treatment of petroleum hydrocarbon-contaminated soil.

[0003] Since my country became one of the world's top ten oil-producing countries in 1978, more and more oil and gas reservoirs and fields have been explored and developed, and the number of oil companies has increased. About 700,000 tons of crude oil are produced each year, of which about one-tenth enters the soil environment, resulting in about 100,000 tons of newly contaminated soil in my country each year. The situation of soil petroleum hydrocarbon pollution is serious, which has hindered the process of sustainable development in my country.

[0004] Polycyclic aromatic hydrocarbons refer to aromatic hydrocarbons containing two or more benzene rings, referred to as PAHs. According to the different ways of connecting the benzene rings, they can be mainly divided into two categories. One category is the non-condensed ring type, which includes biphenyl, biphenyls and polyphenylated aliphatic hydrocarbons. The compound formed by the σ bond between benzene rings is called biphenyl. Biphenyl derivatives and biphenyl compounds are named after biphenyl as the parent. Polyphenylated aliphatic hydrocarbons are compounds formed by several benzene rings replacing hydrogen atoms in aliphatic hydrocarbons. This type of compound is named after phenyl as a substituent and aliphatic hydrocarbon as a parent. The structure and properties of non-condensed ring polycyclic aromatic hydrocarbons such as biphenyl, biphenyls and polyphenylated aliphatic hydrocarbons are similar to those of single-ring aromatic hydrocarbons. The other category is the condensed ring type, that is, two carbon atoms are shared by two benzene rings. For example, naphthalene, the molecular formula of naphthalene is C 10 H8, it is formed by two benzene rings sharing two adjacent carbon atoms. Naphthalene is a white flaky crystal with a melting point of 80°C and a boiling point of 218°C. It is insoluble in water but easily soluble in hot alcohol, ether and other organic solvents. It is volatile, easy to sublimate, and has a special smell. Naphthalene is the compound with the highest content in coal tar, accounting for about 10% in high-temperature coal tar. The molecular formula of anthracene is C 14 H 10, which is composed of three benzene rings fused together. Anthracene exists in coal tar, with a content of about 0.25%. Phenanthrene also exists in coal tar and is an isomer of anthracene.

[0005] The sources of PAHs are divided into natural sources and anthropogenic sources. Natural sources mainly come from the biosynthesis process of terrestrial and aquatic plants and microorganisms. In addition, PAHs also exist in natural fires in forests and grasslands, volcanic eruptions, fossil fuels, lignin and sediments; anthropogenic sources are mainly formed by incomplete combustion of various mineral fuels (such as coal, oil and natural gas, etc.), wood, paper and other hydrocarbons, or pyrolysis under reducing conditions.

[0006] Unlike other persistent organic pollutants that are globally banned from production and use, the main sources of release of most PAHs are usually closely related to human activities. Therefore, the presence of PAHs in the environment is currently of great concern. PAHs are ubiquitous in various environments. They mainly enter surface water directly through atmospheric deposition, river runoff, urban and industrial wastewater, and leakage during transportation. Water is an important medium for the migration of PAHs in the environment. Surface runoff, atmospheric deposition, soil-sediment and air-water interface input exchange are all effective ways for their diffusion. This pollutant has extremely low solubility in water and has strong affinity and biochemical persistence. It has a strong adsorption effect on marine sediments and can often exist stably in different media in nature. However, when sediments are subjected to external forces such as waves, tides and ocean currents, trawling, ship traffic and water conservancy project operations, the pollutants in the sediments are released, exacerbating the pollution.

[0007] Most PAHs have strong toxic side effects on organisms, including immunotoxicity and carcinogenicity. Among them, benzo[a]pyrene has been classified as Group 1 by the International Atomic Energy Agency and is carcinogenic to humans. Other PAHs are divided into Group 2A and Group 2B. At the same time, PAHs have high bioaccumulation, which makes the adverse effects more serious. Even trace amounts can cause various diseases and cause serious harm to the human body. There is increasing evidence that long-term exposure to PAHs can not only cause certain specific cancers, but also certain other diseases, such as obesity and type 2 diabetes (T2D).

[0008] PAHs pollution has traditionally been treated using physical and chemical methods. Conventional methods using temperature, electrokinetics and redox have the advantages of short cycles and quick results, but their high energy consumption and obvious damage to the original site cannot be ignored. Microbial degradation has gradually become the main research direction for PAHs degradation because it has the advantages of lower cost and more convenient operation than other degradation methods.

[0009] In the process of studying the microbial degradation of difficult-to-degrade PAHs, the physiological metabolic pathways of bacteria have become the focus of attention. According to literature reports, bacteria can gradually oxidize PAHs into carbon dioxide and water as the only carbon source, and the speed of this process is usually inversely proportional to the number of rings of PAHs. Bacteria have a significant degradation effect on polycyclic aromatic hydrocarbons with less than four rings, but it is more difficult to degrade polycyclic aromatic hydrocarbons with four rings or more. Therefore, it is necessary to develop a microbial agent that can degrade polycyclic aromatic hydrocarbons with a higher number of rings. Summary of the invention

[0010] The inventor of the present invention has developed a Lactobacillus paracasei (preservation number CGMCC No. 24463), and found that: the Lactobacillus paracasei (preservation number CGMCC No. 24463) has good degradation activity on polycyclic aromatic hydrocarbons; a bacterial agent containing Lactobacillus paracasei (preservation number CGMCC No. 24463) is added to polycyclic aromatic hydrocarbon contaminated soil to repair the polycyclic aromatic hydrocarbon pollution of the soil; and the addition of co-metabolism substrates and small molecular fatty acids at the same time can significantly improve the degradation effect of microorganisms on soil polycyclic aromatic hydrocarbons. Thus, the present invention is completed.

[0011] The first aspect of the present invention provides a bioremediation material, comprising a bacterial agent, a small molecule fatty acid and a cometabolism substrate, wherein the bacterial agent comprises Lactobacillus paracasei, and the preservation number of the Lactobacillus paracasei is CGMCC No.24463.

[0012] The second aspect of the present invention provides the use of the bioremediation material in the degradation of polycyclic aromatic hydrocarbons.

[0013] The third aspect of the present invention provides a method for bioremediation of soil contaminated by polycyclic aromatic hydrocarbons, comprising the following steps: contacting the soil contaminated by polycyclic aromatic hydrocarbons with the bioremediation material.

[0014] The bioremediation material of the present invention has an excellent degradation effect on soil polycyclic aromatic hydrocarbons. The bioremediation method of polycyclic aromatic hydrocarbon-contaminated soil of the present invention has the advantages of simple operation, good remediation effect, low remediation cost, no secondary pollution, etc., and can be widely used in the remediation of polycyclic aromatic hydrocarbon-contaminated soil.

[0015] Other features and advantages of the present invention will be described in detail in the following detailed description.

[0016] Biological Deposit Description

[0017] The biological material Lactobacillus paracasei F1 of the present invention is classified and named Lactobacillus paracasei, and was deposited in the China General Microbiological Culture Collection Center (CGMCC) of the China Microbiological Culture Collection Committee on March 2, 2022, with the address being: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number being CGMCC NO.24463. DETAILED DESCRIPTION

[0018] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0019] The invention provides a bioremediation material, comprising a bacterial agent, a small molecule fatty acid (also referred to herein as a small molecule acid) and a cometabolism substrate. The bacterial agent comprises Lactobacillus paracasei, and the preservation number of the Lactobacillus paracasei is CGMCC No.24463.

[0020] The Lactobacillus paracasei (preservation number CGMCC No. 24463) used in the present invention is a facultative anaerobic bacterium, Gram-positive, and is obtained by screening and separation from polycyclic aromatic hydrocarbon-contaminated soil in Tianjin.

[0021] The specific screening and separation steps are:

[0022] (1) Domestication

[0023] Take 3-5g of soil and add it to the sterilized inorganic salt culture medium. The volume of the culture medium is 150mL / 250mL Erlenmeyer flask. Then add phenanthrene as the only carbon source, shake well, and culture in a shaking cabinet at 30℃ and 150r / min. After 5 days, take 1mL of the bacterial solution in the culture bottle for transfer, and repeat at least 3 times.

[0024] (2) Separation

[0025] After the acclimated bacterial solution is diluted to an appropriate dilution, the bacterial solution is streaked using the plate streak method. Three parallel samples are made for each concentration gradient. After streaking, the culture dish is placed in an incubator at 30°C and cultured for 3 days.

[0026] (3) Purification

[0027] Observe the various colonies growing on the surface of the agar plate, pay attention to the differences in size, shape, edge, surface structure, transparency, color, properties, etc., take photos and save them. Repeatedly purify the colonies that have not formed single bacteria until the size and shape of the colonies on the same plate are uniform.

[0028] The purified Lactobacillus paracasei (named F1) was sent to Beijing Meiji Sanger Biopharmaceutical Technology Co., Ltd. for 16S rDNA strain identification. The results of the spliced ​​gene sequence of the strain and the NCBI comparison showed that the strain had 100% homology with the gene sequence of Lactobacillus paracasei strain. The isolated strain F1 was identified as Lactobacillus paracasei.

[0029] The lactobacillus paracasei F1 colonies provided by the invention are milky white, round, mostly with a diameter of more than 1.0 mm, a smooth and raised surface, and neat edges.

[0030] The inventors have found that in the process of PAH degradation, the presence of co-metabolism substrates increases the amount of microorganisms, thereby increasing the amount of enzymes necessary for PAHs degradation, and these enzymes can improve the degradation efficiency and degradation effect of PAHs. The co-metabolism substrate is preferably at least one of pyrogallic acid, phthalic acid, and catechol.

[0031] Small molecule acids generally refer to fatty acids with no more than 8 carbon atoms, preferably no more than 6 carbon atoms, which have strong chelating properties for multivalent cations (such as Fe 3+ 、Al 3+ ) ability, thus significantly affecting the mobility and solubility of pollutants in nature. The existence of PAHs on land is mainly attached to the soil. PAHs will undergo a series of physical and chemical changes in the soil, such as adsorption and "aging", and these changes are related to the characteristics of the soil itself. The inventors have found that small molecule acids affect the presence of PAHs in the soil by changing the content and quality of soil organic matter (SOM), the aggregate structure in the soil, and the biological activity of microorganisms, thereby strengthening the contact between PAHs and microorganisms and improving the degradation efficiency of PAHs. The small molecule acid used in the repair material of the present invention is preferably selected from at least one of citric acid, tartaric acid, L-malic acid, and succinic acid.

[0032] The bacterial agent in the bioremediation material of the present invention can be the solid component after centrifugation of bacterial culture, that is, the culture obtained after bacterial culture is centrifuged, solid-liquid separated, and the remaining part after the supernatant is discarded, which contains a small amount of water and is a wet bacterial agent.

[0033] In the bioremediation material of the present invention, controlling the amount of small molecule acid and co-metabolism substrate added can achieve a better compounding effect. Preferably, relative to the total number of microorganisms of 10 7 ~10 9The content of the small molecule acid is 0.5-3 mg, and the content of the co-metabolism substrate is 0.1-1.5 mg.

[0034] The bioremediation material of the present invention can be used for degrading polycyclic aromatic hydrocarbons, in particular for degrading polycyclic aromatic hydrocarbons in soil containing polycyclic aromatic hydrocarbons or in groundwater containing polycyclic aromatic hydrocarbons.

[0035] Experiments have shown that the Lactobacillus paracasei of the present invention has good degradation activity on various types of polycyclic aromatic hydrocarbons.

[0036] The polycyclic aromatic hydrocarbons refer to aromatic hydrocarbons containing two or more benzene rings, including both condensed-ring polycyclic aromatic hydrocarbons and non-condensed-ring polycyclic aromatic hydrocarbons.

[0037] The condensed-ring polycyclic aromatic hydrocarbons include but are not limited to naphthalene, anthracene, phenanthrene, pyrene, At least one of benzopyrene, benzanthracene, benzofluoranthene and indenopyrene. The benzopyrene includes benzo[a]pyrene and benzo[e]pyrene. The benzanthracene includes benzo[a]anthracene and dibenzo[a,h]anthracene. The indenopyrene includes indeno[1,2,3-cd]pyrene.

[0038] The non-condensed-ring polycyclic aromatic hydrocarbons include, but are not limited to, at least one of biphenyl (i.e., biphenyl), bipolyphenyl, and polyphenylated aliphatic hydrocarbons. The bipolyphenyl may be biphenyl terphenyl, etc., and the polyphenylated aliphatic hydrocarbons may be diphenylmethane, diphenylethane, etc.

[0039] The present invention also provides a bioremediation method for polycyclic aromatic hydrocarbons-contaminated soil, comprising the following steps: contacting the polycyclic aromatic hydrocarbons-contaminated soil with the bioremediation material.

[0040] According to a preferred embodiment of the present invention, the bacterial agent is based on the total number of microorganisms 10 7 ~10 9 The amount of the bacterial strain is 0.5-3 mg / g soil; the amount of the co-metabolism substrate is 0.1-1.5 mg / g soil.

[0041] The contact conditions are usually natural environmental conditions. The bacterial agent of the present invention can play a good role in degrading polycyclic aromatic hydrocarbons at natural environmental temperature and in a wide pH range (5-10).

[0042] The present invention will be further described below in conjunction with embodiments, but the scope of the present invention is not limited to these embodiments.

[0043] The quality of bioremediation methods for petroleum hydrocarbon contamination in soil is ultimately evaluated by the degradation effect of petroleum hydrocarbons in the soil. Therefore, a soil model evaluation method for bioremediation methods for petroleum hydrocarbon contaminated soil was established. The model evaluation method is as follows: ① Enrich the bacterial agent in an inorganic salt culture medium with the target pollutant as the only carbon source; ② Centrifugal separation to obtain the enriched bacterial agent; ③ Put the bacterial agent, small molecule acid, co-metabolism substrate, water retaining agent, soil conditioner, and nutrients into the soil together; ④ Add the target pollutant to the soil; ⑤ Regularly test the soil petroleum hydrocarbon content and the total number of soil bacteria, and calculate the degradation effect of petroleum hydrocarbons.

[0044] The specific steps are:

[0045] 1. Take 1.5mL of bacterial solution and place it in a conical flask containing 150mL of inorganic salt culture medium, then add the target pollutant, shake at 150r / min for 5-10 days (depending on the amount of bacteria) to obtain enriched bacterial solution.

[0046] 2. The obtained enriched bacterial liquid was centrifuged at 8000r / min for solid-liquid separation, the upper culture medium solution was discarded, 100mL of inorganic salt culture medium was added and shaken thoroughly, and then centrifuged again at 8000r / min for solid-liquid separation, the upper solution was discarded, and the bottom solid was retained for later use.

[0047] 3. Add water retaining agent, soil conditioner, nutrients and bottom solids to the soil, add small molecule acid and co-metabolism substrate, stir well, and add deionized water to maintain the soil moisture content within a certain range.

[0048] 4. Add the target pollutant to the soil and stir well.

[0049] 5. Test the content of target pollutants in soil and the total number of soil bacteria every week.

[0050] 6. Add water every two days to keep the soil moisture content within a certain range, and turn the soil 1-3 times a week.

[0051] Inorganic salt culture medium: NH4Cl 0.67g, NaNO3 1.06g, MgSO4·7H2O 0.1g, CaCl2 0.1g, K2HPO4·3H2O 1.5g, KH2PO4 0.5g, FeCl3·6H2O 0.1g, deionized water 1L, pH 7.0-7.5.

[0052] Analytical methods

[0053] The method for determining heterotrophic bacteria refers to the standard plate counting method of "Industrial Circulating Cooling Water Quality Analysis Method" GB / T14643.1-1993.

[0054] The determination method of PAHs in soil is based on the "Gas Chromatography-Mass Spectrometry for the Determination of Semivolatile Organic Compounds in Soil and Sediment" HJ 834-2017. A dichloromethane-acetone mixed solution is used for pressurized fluid extraction of PAHs. When the constant volume is 1.0 mL and the injection volume is 1.0 μL, the detection limit of the PAHs determination method is 0.06-0.3 mg / kg, and the lower limit of determination is 0.24-1.20 mg / kg.

[0055] Method for determination of total soil bacteria count

[0056] The total number of bacteria in the soil was determined by the plate culture colony counting method, and the culture medium for soil bacteria was meat extract peptone medium.

[0057] The specific measurement steps are:

[0058] Weigh 10g of soil sample, put it into a triangular flask containing 90mL of sterile deionized water, and place it on a shaker at room temperature for 4 hours at a speed of 160 times / min to fully mix the soil sample with water, fully disperse the microbial cells in the soil, and separate them from the soil. -1 Soil suspension: pipette 1 mL of the soil suspension into 9 mL of sterile water containing 8.5 g / L NaCl, pipette and pump 3 times with a sterile pipette to mix well, and make 10 -2 Soil suspension. And so on to make 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 Soil suspensions with different dilutions. After selecting the appropriate dilution, the total bacterial count of the soil suspension was determined by the mixed plate count method.

[0059] The total number of bacteria in the soil, x (individuals / g), is calculated according to the following formula.

[0060]

[0061] Where:

[0062] x——total number of soil bacteria, pieces / g;

[0063] e——Total number of bacteria in soil suspension, counts / mL;

[0064] V——volume of soil suspension, mL;

[0065] m——sampling volume, g;

[0066] w——soil moisture content, %.

[0067] Meat extract peptone culture medium: NaCl 10g, peptone 10g, beef extract 5g, agar 18g, deionized water 1L, pH 7.2-7.4.

[0068] The following examples and comparative examples all adopt the following test methods:

[0069] The soil used was dry soil sterilized at 250℃ for 24h. Sawdust was added to improve soil air permeability; polyacrylic acid was added to improve soil water retention; urea was added to supplement soil N source. Mix well. The solid part of bacterial solution, small molecular acid, and co-metabolism substrate were fully mixed with deionized water and added to the soil. Finally, the target pollutant polycyclic aromatic hydrocarbons were dissolved in 3mL acetone and added to the soil. The soil environment temperature range was 18-23℃.

[0070] The soil was turned over once a week and deionized water was added until the soil moisture content reached the initial value. No other intervention measures were taken.

[0071] The method for obtaining the solid part of the bacterial solution comprises: (1) taking 1.5 mL of the bacterial solution into a conical flask containing 150 mL of inorganic salt culture medium, adding the target pollutant, and shaking at 150 r / min for 5-10 days to obtain the OD 600 The enriched bacterial liquid is 1.0. (2) The obtained enriched bacterial liquid is centrifuged at 8000r / min for solid-liquid separation, the upper culture medium solution is discarded, 100mL of inorganic salt culture medium is added, and after sufficient shaking, the solid-liquid separation is centrifuged again at 8000r / min, and the upper solution is discarded to obtain the solid part of the bacterial liquid. The inorganic salt culture medium composition: NH4Cl 0.67g, NaNO3 1.06g, MgSO4·7H2O 0.1g, CaCl2 0.1g, K2HPO4·3H2O1.5g, KH2PO4 0.5g, FeCl3·6H2O 0.1g, deionized water 1L, pH value 7.0-7.5.

[0072] In the following examples and comparative examples:

[0073] Biphenyl was purchased from Beijing Inokai Technology Co., Ltd.

[0074] Phenanthrene was purchased from Beijing Inokai Technology Co., Ltd.

[0075] Benzo[a]pyrene was purchased from Beijing Inokai Technology Co., Ltd.

[0076] Dietrichia coli was purchased from China General Microbiological Culture Collection Center, with the collection number CGMCC1.6332 T .

[0077] Example 1

[0078] Weigh 6g of polyacrylic acid and dissolve it in 200g of deionized water to form aqueous solution 1, weigh 0.96g of urea and dissolve it in 100g of deionized water to form aqueous solution 2, take Lactobacillus paracasei and mix it in 100g of deionized water to form aqueous solution 3, take 6g of citric acid and dissolve it in 100g of deionized water to form aqueous solution 4, take 3g of pyrogallic acid and dissolve it in 100g of deionized water to form aqueous solution 5, weigh 45g of sawdust and mix it in 2400g of soil, then add aqueous solutions 1, 2, 3, 4, and 5 and mix them evenly. Add 150mg of phenanthrene to the evenly mixed soil.

[0079] Example 2

[0080] Weigh 6g of polyacrylic acid and dissolve it in 200g of deionized water to form aqueous solution 1, weigh 0.96g of urea and dissolve it in 100g of deionized water to form aqueous solution 2, take Lactobacillus paracasei and mix it in 100g of deionized water to form aqueous solution 3, take 1.8g of tartaric acid and dissolve it in 100g of deionized water to form aqueous solution 4, take 0.6g of phthalic acid and dissolve it in 100g of deionized water to form aqueous solution 5, weigh 45g of sawdust and mix it in 2400g of soil, then add aqueous solutions 1, 2, 3, 4, and 5 and mix well. Add 150mg of phenanthrene to the well-mixed soil.

[0081] Example 3

[0082] Weigh 6g of polyacrylic acid and dissolve it in 200g of deionized water to form aqueous solution 1, weigh 0.96g of urea and dissolve it in 100g of deionized water to form aqueous solution 2, take Lactobacillus paracasei and mix it in 100g of deionized water to form aqueous solution 3, take 8.5g of L-malic acid and dissolve it in 100g of deionized water to form aqueous solution 4, take 4.2g of catechol and dissolve it in 100g of deionized water to form aqueous solution 5, weigh 45g of sawdust and mix it in 2400g of soil, then add aqueous solutions 1, 2, 3, 4, and 5 to mix well. Add 150mg of phenanthrene to the mixed soil.

[0083] Example 4

[0084] Weigh 6g of polyacrylic acid and dissolve it in 200g of deionized water to form aqueous solution 1, weigh 0.96g of urea and dissolve it in 100g of deionized water to form aqueous solution 2, take Lactobacillus paracasei and mix it in 100g of deionized water to form aqueous solution 3, take 6g of citric acid and dissolve it in 100g of deionized water to form aqueous solution 4, take 3g of pyrogallic acid and dissolve it in 100g of deionized water to form aqueous solution 5, weigh 45g of sawdust and mix it in 2400g of soil, then add aqueous solutions 1, 2, 3, 4, and 5 and mix them evenly. Add 150mg of benzo[a]pyrene to the evenly mixed soil.

[0085] Example 5

[0086] Weigh 6g of polyacrylic acid and dissolve it in 200g of deionized water to form aqueous solution 1, weigh 0.96g of urea and dissolve it in 100g of deionized water to form aqueous solution 2, take Lactobacillus paracasei and mix it in 100g of deionized water to form aqueous solution 3, take 1.8g of tartaric acid and dissolve it in 100g of deionized water to form aqueous solution 4, take 0.6g of phthalic acid and dissolve it in 100g of deionized water to form aqueous solution 5, weigh 45g of sawdust and mix it in 2400g of soil, then add aqueous solutions 1, 2, 3, 4, and 5 and mix them evenly. Add 150mg of benzo[a]pyrene to the evenly mixed soil.

[0087] Example 6

[0088] Weigh 6g of polyacrylic acid and dissolve it in 200g of deionized water to form aqueous solution 1, weigh 0.96g of urea and dissolve it in 100g of deionized water to form aqueous solution 2, take Lactobacillus paracasei and mix it in 100g of deionized water to form aqueous solution 3, take 8.5g of L-malic acid and dissolve it in 100g of deionized water to form aqueous solution 4, take 4.2g of catechol and dissolve it in 100g of deionized water to form aqueous solution 5, weigh 45g of sawdust and mix it in 2400g of soil, then add aqueous solutions 1, 2, 3, 4, and 5 and mix them evenly. Add 150mg of benzo[a]pyrene to the evenly mixed soil.

[0089] Example 7

[0090] Weigh 6g of polyacrylic acid and dissolve it in 200g of deionized water to form aqueous solution 1, weigh 0.96g of urea and dissolve it in 100g of deionized water to form aqueous solution 2, take Lactobacillus paracasei and mix it in 100g of deionized water to form aqueous solution 3, take 6g of citric acid and dissolve it in 100g of deionized water to form aqueous solution 4, take 3g of pyrogallic acid and dissolve it in 100g of deionized water to form aqueous solution 5, weigh 45g of sawdust and mix it in 2400g of soil, then add aqueous solutions 1, 2, 3, 4, and 5 and mix them evenly. Add 150mg of biphenyl to the evenly mixed soil.

[0091] Comparative Example 1

[0092] 6 g of polyacrylic acid was weighed and dissolved in 200 g of deionized water to form aqueous solution 1, 0.96 g of urea was weighed and dissolved in 100 g of deionized water to form aqueous solution 2, Dietrichia was mixed in 300 g of deionized water to form aqueous solution 3, 45 g of sawdust was weighed and mixed in 2400 g of soil, and then aqueous solutions 1, 2, and 3 were added and mixed evenly. 150 mg of phenanthrene was added to the evenly mixed soil.

[0093] Comparative Example 2

[0094] Weigh 6 g of polyacrylic acid and dissolve it in 200 g of deionized water to form aqueous solution 1, weigh 0.96 g of urea and dissolve it in 100 g of deionized water to form aqueous solution 2, take Dietrichia and mix it in 300 g of deionized water to form aqueous solution 3, weigh 45 g of sawdust and mix it in 2400 g of soil, then add aqueous solutions 1, 2, and 3 and mix them evenly. Add 150 mg of benzo[a]pyrene to the evenly mixed soil.

[0095] Comparative Example 3

[0096] 6 g of polyacrylic acid was weighed and dissolved in 200 g of deionized water to form aqueous solution 1, 0.96 g of urea was weighed and dissolved in 100 g of deionized water to form aqueous solution 2, Lactobacillus paracasei was mixed in 300 g of deionized water to form aqueous solution 3, 45 g of sawdust was weighed and mixed in 2400 g of soil, and then aqueous solutions 1, 2, and 3 were added and mixed evenly. 150 mg of phenanthrene was added to the evenly mixed soil.

[0097] Comparative Example 4

[0098] 6 g of polyacrylic acid was weighed and dissolved in 200 g of deionized water to form aqueous solution 1, 0.96 g of urea was weighed and dissolved in 100 g of deionized water to form aqueous solution 2, Lactobacillus paracasei was mixed in 300 g of deionized water to form aqueous solution 3, 45 g of sawdust was weighed and mixed in 2400 g of soil, and then aqueous solutions 1, 2, and 3 were added and mixed evenly. 150 mg of benzo[a]pyrene was added to the evenly mixed soil.

[0099] Comparative Example 5

[0100] 6 g of polyacrylic acid was weighed and dissolved in 200 g of deionized water to form aqueous solution 1, 0.96 g of urea was weighed and dissolved in 100 g of deionized water to form aqueous solution 2, Lactobacillus paracasei was mixed in 100 g of deionized water to form aqueous solution 3, 9 g of citric acid was dissolved in 200 g of deionized water to form aqueous solution 4, 45 g of sawdust was weighed and mixed in 2400 g of soil, and then aqueous solutions 1, 2, 3, and 4 were added and mixed evenly. 150 mg of phenanthrene was added to the evenly mixed soil.

[0101] Comparative Example 6

[0102] 6 g of polyacrylic acid was weighed and dissolved in 200 g of deionized water to form aqueous solution 1, 0.96 g of urea was weighed and dissolved in 100 g of deionized water to form aqueous solution 2, Lactobacillus paracasei was mixed in 100 g of deionized water to form aqueous solution 3, 9 g of pyrogallic acid was dissolved in 200 g of deionized water to form aqueous solution 4, 45 g of sawdust was weighed and mixed in 2400 g of soil, and then aqueous solutions 1, 2, 3, and 4 were added and mixed evenly. 150 mg of phenanthrene was added to the evenly mixed soil.

[0103] Test Example 1

[0104] Soil samples of the above embodiments and comparative examples were taken regularly to measure the residual PAH content in the soil samples and investigate the degradation of PAHs. The results are shown in Table 1.

[0105] Table 1 Polycyclic aromatic hydrocarbons content of soil samples in Examples and Comparative Examples

[0106] initial 7d 14d 21d 28d 35d 42d 49d Example 1 50.0 46.1 42.0 28.9 15.7 7.6 3.3 1.7 Example 2 50.0 48.0 45.5 33.6 19.9 10.5 5.6 2.3 Example 3 50.0 44.7 39.9 26.1 13.0 5.2 2.9 1.5 Example 4 50.0 48.2 46.8 42.1 35.0 28.9 23.4 19.7 Example 5 50.0 49.0 48.1 45.7 38.2 31.1 26.6 21.0 Example 6 50.0 48.0 45.0 39.4 33.2 26.7 21.5 17.9 Example 7 50.0 44.1 38.7 21.4 11.0 6.4 2.9 1.1 Comparative Example 1 50.0 49.6 48.9 46.7 42.5 41.8 40.7 39.9 Comparative Example 2 50.0 50.0 49.8 48.7 47.6 47.1 47.0 47.1 Comparative Example 3 50.0 48.4 46.2 35.6 21.5 13.5 9.8 2.6 Comparative Example 4 50.0 49.8 49.4 46.2 41.0 33.4 28.4 22.6 Comparative Example 5 50.0 48.2 45.9 34.9 20.8 12.4 8.2 2.5 Comparative Example 6 50.0 48.3 46.0 34.9 20.9 12.3 8.1 2.5

[0107] Note: The unit of PAH content is mg / kg.

[0108] It can be seen from the results of the examples in Table 1 that the bioremediation material of the present invention has an obvious degradation effect on various polycyclic aromatic hydrocarbons in the soil.

[0109] The degradation effects of using other strains, only using the Lactobacillus paracasei of the present invention, or selecting a small molecule acid or a cometabolism substrate to cooperate with Lactobacillus paracasei are all worse than those of the embodiments, indicating that the Lactobacillus paracasei of the present invention has a good PAH degradation effect, and that the simultaneous addition of the cometabolism substrate and the small molecule acid can significantly improve the degradation effect of microorganisms on soil PAHs.

[0110] Test Example 2

[0111] Soil samples of the above embodiments and comparative examples were taken regularly to measure the total number of bacteria in the soil samples and to investigate the growth of microorganisms. The results are shown in Table 2.

[0112] Table 2 Growth of soil microorganisms in the examples and comparative examples

[0113] initial 7d 14d 21d 28d 35d 42d 49d Example 1 35 213 782 1578 1234 841 569 288 Example 2 34 145 521 1107 722 591 401 213 Example 3 34 339 980 1890 1390 1007 798 452 Example 4 37 123 340 763 512 401 257 142 Example 5 32 82 255 709 433 275 245 101 Example 6 38 161 468 894 604 469 316 165 Example 7 36 234 801 1583 1341 897 604 311 Comparative Example 1 38 116 452 1002 458 325 278 117 Comparative Example 2 38 74 213 644 398 245 216 69 Comparative Example 3 37 192 704 1420 1111 757 512 259 Comparative Example 4 38 111 306 687 461 361 231 128 Comparative Example 5 38 200 732 1491 1155 795 538 269 Comparative Example 6 38 202 739 1477 1167 781 532 272

[0114] Note: The total number of bacteria is 10 6 Pieces / g.

[0115] It can be seen from the results in Table 2 that with the increase of treatment time, the growth of Lactobacillus paracasei in the soil reached a peak at 21 days and dropped to a level equivalent to 7 days at 49 days. During this period, the content of polycyclic aromatic hydrocarbons in the soil samples continued to decrease, indicating that the reduction in the content of polycyclic aromatic hydrocarbons in the soil was due to the degradation of Lactobacillus paracasei.

[0116] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

[0117] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

Claims

1. A bioremediation material, comprising a bacterial agent, a small molecule fatty acid and a cometabolism substrate, wherein the bacterial agent comprises Lactobacillus paracasei, and the preservation number of the Lactobacillus paracasei is CGMCC No.24463.

2. The bioremediation material according to claim 1, wherein The small molecule fatty acid is a fatty acid with no more than 8 carbon atoms, preferably a fatty acid with no more than 6 carbon atoms; more preferably, it is at least one selected from citric acid, tartaric acid, L-malic acid, and succinic acid.

3. The bioremediation material according to claim 1, wherein: The co-metabolism substrate is selected from at least one of pyrogallic acid, phthalic acid and catechol.

4. The bioremediation material according to claim 1, wherein The bacterial agent is the solid component after centrifugation of the bacterial culture.

5. The bioremediation material according to any one of claims 1 to 4, wherein: In the bioremediation material, relative to the total number of microorganisms, 7 ~10 9 The content of the small molecule fatty acid is 0.5-3 mg, and the content of the cometabolic substrate is 0.1-1.5 mg.

6. Use of the bioremediation material according to any one of claims 1 to 5 in the degradation of polycyclic aromatic hydrocarbons.

7. The use according to claim 6, wherein: The bioremediation material is used for degrading polycyclic aromatic hydrocarbons in soil containing polycyclic aromatic hydrocarbons or in groundwater containing polycyclic aromatic hydrocarbons.

8. The use according to claim 6 or 7, wherein: The polycyclic aromatic hydrocarbons are condensed-ring polycyclic aromatic hydrocarbons or non-condensed-ring polycyclic aromatic hydrocarbons; The condensed-ring polycyclic aromatic hydrocarbons are selected from naphthalene, anthracene, phenanthrene, pyrene, At least one of benzopyrene, benzanthracene, benzofluoranthene and indenopyrene; The non-condensed-ring polycyclic aromatic hydrocarbon is preferably selected from at least one of biphenyl, bipolyphenyl and polyphenylated aliphatic hydrocarbons.

9. The use according to claim 8, wherein: The biphenyl is terphenyl; the polyphenylated aliphatic hydrocarbon is at least one of diphenylmethane and diphenylethane.

10. A bioremediation method for polycyclic aromatic hydrocarbons contaminated soil, comprising the following steps: The polycyclic aromatic hydrocarbon contaminated soil is contacted with the bioremediation material according to any one of claims 1 to 5.

11. The bioremediation method according to claim 10, wherein: The total number of microorganisms is 10 7 ~10 9 The amount of the bacterial strain is 0.5-3 mg / g soil; the amount of the small molecule fatty acid is 0.5-3 mg / g soil; and the amount of the co-metabolism substrate is 0.1-1.5 mg / g soil.

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