BTEX facultative anaerobic degrading bacterium and application thereof

Abstract

The invention relates to the technical field of microorganisms and environmental protection, and discloses a BTEX facultative anaerobic degrading bacterium and application thereof, the BTEX facultative anaerobic degrading bacterium is Rhodococcus sp. HB3, and the preservation number of the BTEX facultative anaerobic degrading bacterium is GDMCC No: 63282. The rhodococcus HB3 can efficiently degrade benzene, methylbenzene, dimethylbenzene and ethylbenzene, and is good in universality; the rhodococcus HB3 can rapidly grow in a micro-aerobic (2-8%) environment at 10-37 DEG C, the salt tolerance can reach 3.0%, and the rhodococcus HB3 can adapt to a low-oxygen and salinized oil sludge polluted soil environment, so that the strain has a good removal effect on benzene series in oil sludge polluted soil and has an engineering application prospect.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms and environmental protection technology, and in particular to a BTEX facultative anaerobic degrading bacterium and an application thereof. Background Art

[0002] During the oil extraction process, two types of sludge are produced: produced liquid sludge and ground sludge. Due to accidents, maintenance, improper operation, leakage, etc., the petroleum hydrocarbon pollutants contained in the sludge enter the soil, change the physical and chemical properties of the soil, and cause pollution and damage to the ecological environment. In addition, the sludge with high water content contains demulsifiers, flocculants, and a large amount of inorganic salts, such as Cl - 、SO4 2- 、Na + , Ca 2+ etc., affecting the survival rate of plants and causing ecological deterioration.

[0003] Benzene series (BTEX) include benzene, toluene, xylene, and ethylbenzene. Due to their stable presence in the environment due to their benzene rings, their resistance to degradation, their "three-hazard" nature, and their high toxicity, BTEX has long been a key challenge in the remediation of petroleum hydrocarbon pollutants and has garnered significant attention. Common physical and chemical remediation techniques for BTEX-contaminated soils include vapor phase extraction, enhanced desorption, and chemical oxidation. However, these techniques suffer from various technical limitations, including damage to soil structure, high treatment costs, and the risk of secondary pollution. Microbial degradation, on the other hand, has garnered widespread attention due to its cost-effectiveness, ease of operation, environmental friendliness, and the absence of secondary pollution.

[0004] However, current microbial remediation is subject to the following conditions: (1) Oil sludge pollution decomposes quickly in the surface soil, but as BTEX migrates, the concentration in the lower and middle layers of the soil aeration zone is higher (18-90 mg / L), and the environmental oxygen content in this location is low (oxygen concentration 2-8%). Conventional BTEX-degrading bacteria products are aerobic bacteria and cannot treat BTEX pollution in a microaerobic environment; (2) Oil sludge-contaminated soils are all salinized to varying degrees, with a salinity of 0.5-2.5%. Conventional BTEX-degrading bacteria products are not salt-tolerant and cannot treat BTEX pollution in salinized environments. Summary of the Invention

[0005] The purpose of the present invention is to provide a BTEX facultative anaerobic degrading bacterium and its application, which solves the problem that microorganisms in BTEX bioremediation technology of oil sludge contaminated soil cannot grow in microaerobic and salinized environments, significantly improves the removal efficiency of benzene series in oil sludge contaminated soil, and improves soil quality.

[0006] In order to achieve the above object, the present invention provides a BTEX facultative anaerobic degrading bacterium on one hand. The BTEX facultative anaerobic degrading bacterium is Rhodococcus sp. HB3, and its deposit number is GDMCC No: 63282.

[0007] The second aspect of the present invention provides a method for using the BTEX facultative anaerobic degrading bacteria in the degradation of benzene series.

[0008] Preferably, the benzene series compound is one or more of benzene, toluene, ethylbenzene and xylene.

[0009] Preferably, the method is used in bioremediation of oil sludge contaminated sites.

[0010] A third aspect of the present invention provides a bacterial agent containing the BTEX facultative anaerobic degrading bacteria described above.

[0011] Preferably, the bacterial agent further contains a fixed carrier.

[0012] Preferably, the fixed carrier is selected from one or more of an organic material carrier, a plant material carrier and a mineral material carrier;

[0013] Preferably, the organic material carrier is selected from one or more of sodium alginate, chitosan, polyacrylamide, polyvinyl alcohol and polyglycol;

[0014] Preferably, the plant material carrier is corn flour and / or starch;

[0015] Preferably, the mineral material carrier is selected from one or more of clay, talc, kaolin and zeolite.

[0016] Preferably, the bacterial agent is in the form of a liquid, emulsion, suspension, powder or granule.

[0017] A fourth aspect of the present invention provides a method for preparing a bacterial agent, the method comprising: fermenting and culturing the BTEX facultative anaerobic degrading bacteria as described above.

[0018] Preferably, the fermentation conditions include: 26-32° C., pH 6.5-7.5, and time 18-24 h.

[0019] Preferably, the method further comprises: fixing the fermentation liquid obtained by fermentation culture on a fixed carrier.

[0020] A fifth aspect of the present invention provides a bacterial agent prepared by the method described above.

[0021] A sixth aspect of the present invention provides a method for remediating an oil sludge contaminated site, the method comprising: adding the BTEX facultative anaerobic degrading bacteria or the bacterial agent described above to the oil sludge contaminated site.

[0022] The BTEX facultative anaerobic degrading bacteria provided by the present invention are Rhodococcus sp. HB3, which can efficiently degrade benzene, toluene, xylene, and ethylbenzene and has good versatility. The strain can grow rapidly in a microaerobic environment (2-8%) and at 10-37°C. It has a salinity tolerance of up to 3.0% and can adapt to low-oxygen and salinized oil sludge-contaminated soil environments. Therefore, the strain has a good removal effect on benzene series in oil sludge-contaminated soil and has a promising prospect for engineering applications.

[0023] Biological Deposits

[0024] The BTEX facultative anaerobic degrading bacteria provided by the present invention have been deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on March 20, 2023, with a deposit number of GDMCC No: 63282, a classification name: Rhodococcus sp. HB3, and a deposit address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a diagram of the screening and colony morphology of Rhodococcus HB3 provided by the present invention;

[0026] Figure 2 This is an electron microscope scan of the strain Rhodococcus HB3 provided by the present invention;

[0027] Figure 3 This is a 16S rRNA phylogenetic tree diagram of Rhodococcus HB3 provided by the present invention;

[0028] Figure 4 This is a growth curve diagram of Rhodococcus HB3 provided by the present invention;

[0029] Figure 5 This is a graph showing the growth characteristics of Rhodococcus HB3 at different temperatures provided by the present invention;

[0030] Figure 6 This is a graph showing the growth characteristics of Rhodococcus HB3 at different pH values ​​provided by the present invention;

[0031] Figure 7 This is a graph showing the growth characteristics of Rhodococcus HB3 at different salinities provided by the present invention;

[0032] Figure 8 This is a diagram of the microbial agent product prepared in Example 7 of the present invention;

[0033] Figure 9This is a graph showing the benzene degradation test results of Rhodococcus HB3 in Test Example 1 of the present invention;

[0034] Figure 10 This is a graph showing the results of a toluene degradation test using Rhodococcus sp. HB3 in Test Example 2 of the present invention;

[0035] Figure 11 This is a graph showing the results of an ethylbenzene degradation test using Rhodococcus sp. HB3 in Test Example 3 of the present invention;

[0036] Figure 12 This is a graph showing the results of a xylene degradation test by Rhodococcus HB3 in Test Example 4 of the present invention;

[0037] Figure 13 This is the BTEX degradation test of Rhodococcus HB3 in Test Example 5 of the present invention in a simulated actual soil environment. DETAILED DESCRIPTION

[0038] The following describes the specific embodiments of the present invention in detail with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0039] The endpoints of the ranges and any values ​​disclosed herein 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 endpoints of each range, the endpoints of each range and 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 to be specifically disclosed herein.

[0040] In the present invention, the unit of "salinity": % means "weight %"; the unit of "oxygen concentration": % means "volume %".

[0041] The present invention provides a BTEX facultative anaerobic degrading bacterium, wherein the BTEX facultative anaerobic degrading bacterium is Rhodococcus sp. HB3, whose preservation number is GDMCC No: 63282 and the preservation date is March 20, 2023.

[0042] Oily sludge pollution is mostly concentrated in the middle and lower layers of the soil aeration zone. Conventional BTEX-degrading bacteria cannot adapt to microaerobic environments (oxygen concentration 2-8%), while the Rhodococcus HB3 provided by the present invention has good adaptability in microaerobic environments; oily sludge-contaminated soils are all salinized to varying degrees, and conventional BTEX-degrading bacteria products cannot treat BTEX pollution in salinized environments, while the Rhodococcus HB3 provided by the present invention has a high salinity tolerance (up to 3.0%); at the same time, the Rhodococcus HB3 provided by the present invention has high degradation efficiency for benzene series (benzene, toluene, xylene and ethylbenzene) and good versatility.

[0043] The Rhodococcus HB3 provided by the present invention can adapt to the low-oxygen, salinized petroleum-contaminated soil environment. When the strain is used for harmless bioremediation of petroleum-contaminated soil, it has a good removal rate of benzene series substances, good economy, good versatility, and is suitable for engineering applications.

[0044] The Rhodococcus sp. HB3 described in this invention was screened and purified from oily sludge from the North China oil and gas fields. Specifically, the method employed a multivariate gradient method for enrichment and acclimation. Using an anaerobic hypoxic workstation to establish an oxygen concentration gradient, the system simultaneously applied an increasing gradient of mixed benzene, a decreasing gradient of yeast powder, and an increasing gradient of sodium salt. After 5 to 7 cycles, the strain was screened and purified to be a facultative anaerobic BTEX-degrading bacterium capable of rapid growth in a microaerobic environment at 10 to 40°C, with a salinity tolerance of up to 3.0%, and the ability to efficiently degrade benzene derivatives.

[0045] In a specific embodiment, the screening method for Rhodococcus HB3 comprises the following steps:

[0046] (1) Oily sludge samples were collected from the North China oil and gas fields using a 1 L plastic container, crushed, and passed through a 20-mesh sieve to obtain oily sludge samples.

[0047] (2) Prepare culture medium.

[0048] Enrichment medium formula: ammonium sulfate 2g / L, dipotassium hydrogen phosphate 0.5g / L, potassium dihydrogen phosphate 0.5g / L, calcium chloride 0.1g / L, sodium chloride 0.1g / L, magnesium sulfate 0.2g / L, ferrous sulfate 0.2g / L, balance deionized water, pH 7.0;

[0049] Trace element solution formula: 0.01g / L CaCl2, 0.5g / L FeCl2, 0.38g / L CuSO4, 1.1g / L ZnSO4, 1.58g / L MnSO4;

[0050] Acclimation medium formula: Add trace element solution, mixed benzene, and optional yeast powder and sodium salt to the enrichment medium. Mixed benzene is a mixture of benzene, toluene, ethylbenzene, and xylene in a volume ratio of 1:1:1:1;

[0051] Purified solid culture medium formula: yeast extract 3g / L, trypsin 10g / L, sodium chloride 5g / L, agar 25g / L.

[0052] (3) Add 10 g of the oil sludge sample to 100 ml of enrichment medium. After incubation at 30-37°C and 150 rpm on a shaker for 5-7 days, take 10 ml of the supernatant and add it to 100 ml of fresh enrichment medium. Repeat the enrichment culture 3-5 times to obtain the enrichment solution.

[0053] (4) A multivariate gradient method was used to screen BTEX-degrading bacteria. The acclimation medium contained a trace element solution, and the content of the trace element solution in the acclimation medium was 0.1% v / v (i.e., volume %). Four gradient variables, including mixed benzene, yeast powder, oxygen content, and salinity, were set. First, 10 ml of the enrichment solution was added to 100 ml of the acclimation medium. The variable mixed benzene was increased step by step from 40 mg / L to 200 mg / L; yeast powder was decreased step by step from 100 mg / L to 0 mg / L; salinity was increased step by step from 1.0% to 3.0%, and the salts included NaCl and / or Na2SO4;

[0054] Culture at 30-37°C, 135 rpm, in a shaking incubator for five cycles, each lasting 7-10 days. Specific parameters are shown in Table 1 below (in Table 1, the amount of each component added refers to the amount in the acclimation medium);

[0055] Table 1

[0056]

[0057] Secondly, the dilution plate method was used for separation, and the obtained F5 acclimation culture fluid was diluted to 10 -3 , 10 -4 , 10 -5 , 10 -6 Afterwards, 50 μl of bacterial solution was diluted and spread on fresh purified solid culture medium. The oxygen content gradient was adjusted to 20%, 15%, 10%, 5%, and 2% using the three-gas (carbon dioxide, nitrogen, and compressed air) hypoxic mode of the anaerobic hypoxic workstation. The culture conditions for each gradient were 30-37°C and 60-85% RH, and the cycle was 3 days.

[0058] (5) After colonies grow on the plate, select single colonies of different colors and morphologies, inoculate them onto purified solid culture medium using the plate streak method, and repeat the above streak separation process until purified colonies with a single morphology are formed. Store at 4°C.

[0059] (6) The purified colonies were inoculated back into the acclimation medium (100 mg / L mixed benzene, without yeast powder and sodium salt) for growth, and the benzene degradation rate was measured after 96 h. That is, the formula of the acclimation medium was: 0.1% v / v trace elements, 100 mg / L mixed benzene, and the remainder was enrichment medium.

[0060] (7) The strain with the highest degradation rate was screened out and strain HB3 was obtained.

[0061] In the present invention, in step (4), when preparing the acclimation medium, the mixed benzene is first dissolved in a cosolvent (N,N-dimethylformamide) and then added to the enrichment medium. The cosolvent content is 0.2-1.0% v / v of the liquid content of the acclimation medium.

[0062] In the present invention, the strain HB3 has a long rod-like morphology.

[0063] In the present invention, the colony characteristics of the strain HB3 are: the colonies are round, orange-red, smooth and moist on the surface, and have neat edges.

[0064] Furthermore, the 16S rRNA gene sequencing result of the strain HB3 is shown in SEQ ID NO: 1.

[0065] Based on the comprehensive physiological and biochemical characteristics and molecular biological characteristics, the strain HB3 was determined to belong to the genus Rhodococcus (Rhodococcus sp.) and was named Rhodococcus sp. HB3 (i.e. Rhodococcus HB3).

[0066] The present invention also provides a use of the BTEX facultative anaerobic degrading bacteria described above in degrading benzene series.

[0067] Specifically, the benzene series compound is one or more of benzene, toluene, ethylbenzene and xylene.

[0068] In a preferred embodiment, the invention is used in bioremediation of oil sludge contaminated sites.

[0069] The present invention also provides a bacterial agent, which contains the BTEX facultative anaerobic degrading bacteria described above.

[0070] The present invention does not limit the specific ingredients in the bacterial agent, as long as it contains the bacterial suspension, fermentation broth, or culture concentrate of the above-mentioned facultative anaerobic BTEX-degrading bacteria as the active ingredient. It is understood that the bacterial agent may also contain other strains used in combination with the facultative anaerobic BTEX-degrading bacteria to enhance the degradation of benzene series.

[0071] In a preferred embodiment, the bacterial agent contains not only the above-mentioned active ingredients but also a fixed carrier.

[0072] The present invention is not limited to the specific type of the fixed carrier, and can be a carrier commonly used in the field of microbial agents and biologically inert. Specifically, for example, it can be one or more of an organic material carrier, a plant material carrier, and a mineral material carrier.

[0073] In a preferred embodiment, the fixed carrier is an organic material carrier, a plant material carrier, a mineral material carrier, a novel material composited with an organic material carrier and a plant material carrier, or a novel material composited with an organic material carrier and a mineral material carrier.

[0074] Further preferably, the organic material carrier is selected from one or more of sodium alginate, chitosan, polyacrylamide, polyvinyl alcohol and polyglycol.

[0075] Further preferably, the plant material carrier is corn flour and / or starch.

[0076] Further preferably, the mineral material carrier is selected from one or more of clay, talc, kaolin and zeolite.

[0077] The present invention does not limit the specific dosage form of the bacterial agent, which can be selected according to actual needs. Specifically, it can be a liquid, emulsion, suspension, powder or granule.

[0078] The present invention also provides a method for preparing a bacterial agent, which comprises: fermenting and culturing the BTEX facultative anaerobic degrading bacteria (ie, Rhodococcus HB3) as described above.

[0079] In a preferred embodiment, the fermentation conditions include: 26-32° C., pH 6.5-7.5, and time 18-24 h.

[0080] In a preferred embodiment, the method further comprises: fixing the fermentation broth on a fixed carrier. The fixation method for the bacterial agent can be adsorption, embedding, cross-linking, covalent bonding, etc. For ease of operation, adsorption is preferred.

[0081] When the fermentation liquid is fixed on the fixed carrier by the adsorption method, the method for preparing the bacterial agent specifically includes: fermenting and culturing the BTEX facultative anaerobic degrading bacteria, and then mixing the obtained fermentation liquid with the fixed carrier.

[0082] In a specific embodiment, the method for preparing the bacterial agent (using adsorption as the immobilization method) comprises the following steps:

[0083] (1) Using high-silicon zeolite powder as a solid carrier, wherein the particle size of the high-silicon zeolite powder is

[0084] ≤0.08mm, pH value is 6.5~8.5, specific surface area is 1000~1500m 2 / g, total pore volume of 0.5 to 0.7 cm 3 / g, and an average pore size of 1.5 to 2.5 nm; the high-silicon zeolite powder was sterilized at 121°C under high pressure for 20 minutes.

[0085] (2) Activate the Rhodococcus spp. strain (OD 600 =2.0) was inoculated into a seed tank containing a culture medium at a volume ratio of 10%, and cultured at 28-30° C., pH 7.0, with aeration and stirring for 18-24 hours; the culture solution obtained from the seed tank was inoculated into a large fermentation tank containing a culture medium at a volume ratio of 10% v / v, and fermented (at 28-30° C., pH 7.0) for 18-24 hours to obtain a fermentation liquid;

[0086] The culture medium formula includes: 4-6 wt% carbon source, 4-8 wt% nitrogen source, and 0.1-0.3 wt% inorganic salt, with the balance being water; wherein the carbon source is a compound of cereal starch and glucose in a mass ratio of 3:1, wherein the cereal starch is selected from one or more of corn flour, potato flour, and cassava flour; the nitrogen source is organic nitrogen, which is selected from one or more of corn steep liquor, soybean meal, and yeast extract; and the inorganic salt is phosphate.

[0087] (3) The fermentation broth was concentrated at a volume ratio of 1:3, fully mixed with high-silicon zeolite powder, and then dried to obtain a bacterial agent.

[0088] The present invention also provides a use of the above-mentioned bacterial agent in the bioremediation of oil sludge contaminated sites.

[0089] In the present invention, the bacterial agent can be used alone or in combination with other bioremediation technologies such as chemical oxidation / reduction technology, gas phase extraction technology, enhanced desorption technology, etc.

[0090] In a preferred embodiment, the microbial agent is compounded with compound fertilizer and then used to repair oil sludge contaminated sites.

[0091] Further preferably, the compound fertilizer is one or more of potassium phosphate, ammonium phosphate, ammonium chlorophosphate, potassium nitrate and potassium ammonium nitrate.

[0092] Further preferably, the mass ratio of the microbial agent to the compound fertilizer is 50 to 100:10, specifically, for example, 50:10, 60:10, 70:10, 80:10, 90:10 or 100:10.

[0093] The present invention will be described in detail below through examples, but the protection scope of the present invention is not limited thereto.

[0094] Example 1

[0095] This example is used to illustrate the screening of Rhodococcus sp. HB3 of the present invention.

[0096] (1) Oily sludge samples were collected from the North China oil and gas fields using a 1 L plastic container, crushed, and passed through a 20-mesh sieve to obtain oily sludge samples.

[0097] (2) Prepare culture medium.

[0098] Enrichment medium formula: ammonium sulfate 2g / L, dipotassium hydrogen phosphate 0.5g / L, potassium dihydrogen phosphate 0.5g / L, calcium chloride 0.1g / L, sodium chloride 0.1g / L, magnesium sulfate 0.2g / L, ferrous sulfate 0.2g / L, balance deionized water, pH 7.0;

[0099] Purified solid culture medium formula: yeast extract 3g / L, trypsin 10g / L, sodium chloride 5g / L, agar 25g / L;

[0100] Trace element solution formula: 0.01g / L CaCl2, 0.5g / L FeCl2, 0.38g / L CuSO4, 1.1g / L ZnSO4, 1.58g / L MnSO4;

[0101] Acclimation medium formula: Add mixed benzene, trace element solution, and optional yeast powder and sodium salt to the enrichment medium, wherein the mixed benzene is a mixture of benzene, toluene, ethylbenzene and xylene in a volume ratio of 1:1:1:1;

[0102] Among them, F1 acclimation medium: mixed benzene 40mg / L, yeast powder 100mg / L, sodium salt 1.0wt%, 0.1vol% trace element solution, the rest is enrichment medium;

[0103] F2 acclimation medium: 80 mg / L benzene, 75 mg / L yeast extract, 1.5 wt% sodium salt, 0.1 vol% trace element solution, and the remainder is enrichment medium;

[0104] F3 acclimation medium: 120 mg / L benzene, 50 mg / L yeast extract, 2.0 wt% sodium salt, 0.1 vol% trace element solution, and the remainder is enrichment medium;

[0105] F4 acclimation medium: 160 mg / L benzene, 25 mg / L yeast extract, 2.5 wt% sodium salt, 0.1 vol% trace element solution, and the remainder is enrichment medium;

[0106] F5 acclimation medium: 200 mg / L benzene, 0 mg / L yeast powder, 3.0 wt% sodium salt, 0.1 volume% trace element solution, and the remainder is enrichment medium.

[0107] (3) Add 10 g of the oil sludge sample to 100 ml of enrichment medium. After incubation at 30°C and 150 rpm on a shaker for 7 days, take 10 ml of the supernatant and add it to 100 ml of fresh enrichment medium. Repeat the enrichment culture three times to obtain the enrichment solution.

[0108] (4) First, 10 ml of the enrichment solution was added to 100 ml of F1 acclimation medium and cultured at 30°C and 135 rpm for 7 days to obtain F1 acclimation culture medium;

[0109] Add 10 ml of F1 acclimation culture medium to 100 mL of F2 acclimation medium, and culture at 30°C and 135 rpm on a shaker for 10 days to obtain F2 acclimation culture medium;

[0110] Add 10 ml of F2 acclimation culture medium to 100 mL of F3 acclimation medium, and culture at 30°C, 135 rpm on a shaker for 10 days to obtain F3 acclimation culture medium;

[0111] Add 10 ml of F3 acclimation culture medium to 100 mL of F4 acclimation medium and culture at 30°C and 135 rpm on a shaker for 10 days to obtain F4 acclimation culture medium;

[0112] Add 10 ml of F4 acclimation culture medium to 100 mL of F5 acclimation medium and culture at 30°C and 135 rpm on a shaker for 10 days to obtain F5 acclimation culture medium;

[0113] Secondly, the dilution plate method was used for separation, and the obtained F5 acclimation culture fluid was diluted to 10 -3 , 10 -4 , 10 -5 , 10 -6 Afterwards, 50 μl of bacterial solution was diluted and spread on fresh purified solid culture medium. The oxygen content gradient was adjusted to 20%, 15%, 10%, 5%, and 2% using the anaerobic hypoxic workstation's three-gas (carbon dioxide, nitrogen, and compressed air) hypoxic mode. The culture conditions for each gradient were 30°C and 68% RH, with a 3-day / cycle period.

[0114] (5) After colonies grow on the plate, select single colonies of different colors and morphologies, inoculate them onto purified solid culture medium using the plate streak method, and repeat the above streak separation process until purified colonies with a single morphology are formed. Store at 4°C.

[0115] (6) The purified colonies were inoculated back into acclimation medium (formula: 0.1% v / v trace elements, 100 mg / L mixed benzene, and the remainder was enrichment medium) for growth, and the benzene degradation rate was measured after 96 h.

[0116] (7) The strain with the highest degradation rate was screened out and strain HB3 was obtained.

[0117] Figure 1 Schematic diagram of the morphology of strain HB3 in step (5), wherein: Figure 1 a is the presence of strain HB3 in the mixed bacteria state during the screening process, Figure 1 b is a purified colony of strain HB3.

[0118] Example 2

[0119] This example is used to illustrate the identification of Rhodococcus sp. HB3 of the present invention.

[0120] 1. Morphological identification

[0121] (1) By Figure 1b It can be seen that the colonies of strain HB3 are round, orange-red in color, with a smooth and moist surface and neat edges.

[0122] (2) The strain HB3 was observed under a scanning electron microscope. The results are as follows Figure 2 As shown. Figure 2 It can be seen that strain HB3 is in the shape of a long rod.

[0123] 2. Molecular Biology Identification

[0124] (1) The strain HB3 obtained in Example 1 was streaked and cultured in a purified solid medium. After culture, the whole genome was extracted using a DNA kit, and PCR amplification was performed using primers 27-F and 1492-R. The PCR reaction system consisted of 25 μL of the following: 1 μL each of the upstream and downstream primers (10 μmol / L), 1 μL of DNA template (10 ng / μL), 12.5 μL of 2× Taq Master Mix, and ultrapure water to 25 μL. The PCR reaction conditions were: 94°C for 5 min; 35 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min 30 s; and a final extension at 72°C for 7 min.

[0125] (2) The PCR amplification products were sequenced by Shanghai Sangon Biotechnology Co., Ltd., and the obtained gene sequences were compared with GenBank for homology to determine their taxonomic status.

[0126] (3) The 16S rRNA gene sequencing results of strain HB3 are shown in SEQ ID NO: 1. After comparison with the database, it was determined that strain HB3 belongs to the genus Rhodococcus sp. and was named Rhodococcus sp. HB3 (i.e., Rhodococcus HB3).

[0127] Among them, the 16S rRNA phylogenetic tree of strain HB3 is shown in Figure 3 .

[0128] Among them, the sequence of SEQ ID NO: 1 is as follows:

[0129] GTTTGATTCAGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACA

[0130] TGCAAGTCGAACGATGAAGCCCAGCTTGCTGGGTGGATTAGTGGCGAA

[0131] CGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTTCGGGATAAGCCT

[0132] GGGAAACTGGGTCTAATACCGGATAGGACCTCGGGATGCATGTTCCGG

[0133] GGTGGAAAGGTTTTCCGGTGCAGGATGGGCCCGCGGCCTATCAGCTTG

[0134] TTGGTGGGGTAACGGCCCACCAAGGCGACGACGGGTAGCCGGCCTGA

[0135] GAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTAC

[0136] GGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGC

[0137] AGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTC

[0138] AGTACCGACGAAGCGCAAGTGACGGTAGGTACAGAAGAAGCACCGGC

[0139] CAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTC

[0140] CGGAATTACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGTCT

[0141] GTGAAAACCCGCAGCTCAACTGCGGGCTTGCAGGCGATACGGGCAGA

[0142] CTTGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATG

[0143] CGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCA

[0144] GTAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGA

[0145] TACCCTGGTAGTCCACGCCGTAAACGGTGGGCGCTAGGTGTGGGTTTC

[0146] CTTCCACGGGATCCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGG

[0147] GGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCG

[0148] CACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCT

[0149] TACCTGGGTTTGACATACACCGGACCGCCCCAGAGATGGGGTTTCCCT

[0150] TGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTG

[0151] AGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTGTGTTG

[0152] CCAGCACGTAATGGTGGGGACTCGCAGGAGACTGCCGGGGTCAACTC

[0153] GGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCCAGG

[0154] GCTTCACACATGCTACAATGGCCGGTACAGAGGGCTGCGATACCGCGA

[0155] GGTGGAGCGAATCCCTTAAAGCCGGTCTCAGTTCGGATCGGGGTCTGC

[0156] AACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAAC

[0157] GCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTC

[0158] ATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCCTCGTGGGA

[0159] GGGAGCCGTCGAAGGTGGGATCGGCGATTGGGACGAAGTCGTAA.

[0160] Example 3

[0161] This example is used to illustrate the growth curve determination of Rhodococcus HB3 described in the present invention.

[0162] Use an inoculating loop to pick the strain from the slant containing Rhodococcus HB3 and mix it in 100 ml of sterile water. Inoculate it at a 1% volume ratio into sterile nutrient medium (5 g / L yeast extract, 10 g / L trypsin, 5 g / L sodium chloride, pH 7.0) and incubate at 37°C with a shaker at 135 rpm. Sampling is performed every 4 hours, and the OD value is measured by UV spectrophotometer. 600 Value, the result is Figure 4 shown.

[0163] Depend on Figure 4 It can be seen that 0 to 4 hours is the lag phase of Rhodococcus HB3, 4 to 24 hours is the logarithmic growth phase of the bacteria, 24 to 40 hours is the stable phase of the bacteria, and after 40 hours the bacteria enters the decay phase.

[0164] Example 4

[0165] This example is used to illustrate the determination of the optimal growth temperature of Rhodococcus HB3 described in the present invention.

[0166] Take 1 mL of the seed solution of strain HB3 (OD 600 The culture medium was inoculated with a 50 mL nutrient medium (value = 0.5). Under the same other experimental conditions, the temperature of the nutrient medium was set to 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, and 45°C, respectively. After shaking culture at a speed of 135 rpm for 24 h, samples were taken from the bacterial liquid to measure the OD 600 Value, the result is Figure 5 shown.

[0167] Depend on Figure 5 It can be seen that when the temperature is 15-40℃, after culturing for 24h, OD 600 A value of 1.3 or above indicates that Rhodococcus HB3 can grow rapidly at temperatures between 15 and 40°C, making it suitable for engineering applications in soil aeration zone remediation. The optimal growth temperature for Rhodococcus HB3 is 20 to 30°C.

[0168] Example 5

[0169] This example is used to illustrate the determination of the optimal growth pH of Rhodococcus HB3 described in the present invention.

[0170] Take 1 mL of the seed solution of strain HB3 (OD 600 The pH value = 0.5) was inoculated into 50 mL of nutrient medium. Under the same experimental conditions, the initial pH of the nutrient medium was set to 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0, respectively. After shaking culture at 135 rpm and 30 ° C for 24 h, samples were taken from the bacterial liquid to measure the OD 600 Value, the result is Figure 6 shown.

[0171] Depend on Figure 6 It can be seen that the optimum pH of Rhodococcus HB3 is 6.0-9.0.

[0172] Example 6

[0173] This example is used to illustrate the salt tolerance test of Rhodococcus sp. HB3 described in the present invention.

[0174] Take 1 mL of the seed solution of strain HB3 (OD 600 The initial salt concentration of the nutrient medium was set to 1.0 wt%, 1.5 wt%, 2.0 wt%, 2.5 wt%, 3.0 wt%, and 3.5 wt% under the same experimental conditions. After shaking culture at 135 rpm and 30°C for 24 h, samples were taken from the bacterial culture to measure the OD value. 600 Value, the result is Figure 7 shown.

[0175] Depend on Figure 7 It can be seen that the optimum salt tolerance of Rhodococcus HB3 can reach 3.0%.

[0176] Example 7

[0177] This example is used to illustrate the bacterial agent of the present invention (the immobilized bacterial agent is prepared by the immobilization adsorption method) and its preparation method.

[0178] (1) High-silicon zeolite powder is used as the adsorbent, wherein the particle size of the high-silicon zeolite powder is ≤0.08 mm, the pH value is 7.5, and the specific surface area is 1200 m 2 / g, total pore volume of 0.6cm 3 / g, and an average pore size of 1.8nm; the high-silicon zeolite powder was sterilized at 121°C for 20min and set aside.

[0179] (2) Activate the Rhodococcus genus strain HB3 (OD 600=2.0) was inoculated into a seed tank containing a culture medium at 10% by volume, and cultured with aeration and stirring at 30° C. and pH 7.0 for 18 h; the culture solution in the seed tank was inoculated into a large fermentation tank at an inoculum amount of 10% by volume, and fermented and cultured at 30° C. and pH 7.0 for 18 h to obtain a fermentation liquid.

[0180] The culture medium formula is: 4wt% carbon source (cereal starch and glucose), 8wt% nitrogen source (organic nitrogen), 0.1wt% inorganic salt (phosphate), and the balance is water, wherein the mass ratio of cereal starch to glucose is 3:1, the cereal starch is corn flour, and the organic nitrogen is corn steep liquor;

[0181] (3) After 50L of fermentation liquid was concentrated at a volume ratio of 1:3, it was dried at 70℃ and then fully mixed with high-silicon zeolite powder at a ratio of 1:1 to form a microbial agent product (see Figure 8 ).

[0182] Test Example 1

[0183] This test example is used to illustrate the benzene degradation test of Rhodococcus sp. HB3 described in the present invention.

[0184] 1. Test method:

[0185] (1) The experiment was divided into 5 groups: blank control group, benzene test group 1 (50 mg / L), benzene test group 2 (100 mg / L), benzene test group 3 (150 mg / L), and benzene test group 4 (200 mg / L).

[0186] Among them, blank control group: enriched culture medium + 0.1 volume% trace element solution;

[0187] Benzene test group 1: enriched medium + 50 mg / L benzene + 0.1 vol% trace element solution;

[0188] Benzene test group 2: enriched medium + 100 mg / L benzene + 0.1 vol% trace element solution;

[0189] Benzene test group 3: enriched medium + 150 mg / L benzene + 0.1 vol% trace element solution;

[0190] Benzene test group 4: enriched medium + 200 mg / L benzene + 0.1 vol% trace element solution;

[0191] (2) Inoculate 1 mL of seed solution (OD 600 The samples were shaken at 30°C, pH 7.0, and 135 rpm for 96 h, and the benzene degradation rate of each group was calculated and the OD of each group was measured. 600 .

[0192] 2. Test results:

[0193] After calculation, the benzene degradation rates in benzene test group 1, benzene test group 2, benzene test group 3 and benzene test group 4 were 99.3%, 98.5%, 92.6% and 85.7% respectively. 600 like Figure 9 shown.

[0194] The results showed that the optimal benzene degradation concentration for Rhodococcus HB3 could reach 150 mg / L, among which the degradation rate of the benzene content group of 100 mg / L, which was close to the actual working conditions, reached 98.5%.

[0195] Test Example 2

[0196] This test example is used to illustrate the toluene degradation experiment of Rhodococcus sp. HB3 described in the present invention.

[0197] The test was performed according to the method described in Test Example 1, except that benzene was replaced by toluene (specifically: the test was divided into 5 groups, namely blank control group, toluene test group 1, toluene test group 2, toluene test group 3, and toluene test group 4; among them, in toluene test group 1, the toluene content was 50 mg / L; in toluene test group 2, the toluene content was 100 mg / L; in toluene test group 3, the toluene content was 150 mg / L; in toluene test group 4, the toluene content was 200 mg / L).

[0198] After testing, the toluene degradation rates of toluene test group 1, toluene test group 2, toluene test group 3, and toluene test group 4 were 93.8%, 93.2%, 91.7%, and 90.5%, respectively. At the same time, the OD values ​​of each group were measured. 600 , toluene degradation rate and OD 600 like Figure 10 shown.

[0199] The results showed that the degradation rate of Rhodococcus HB3 reached 93.2% in the toluene content group of 100 mg / L.

[0200] Test Example 3

[0201] The test was performed according to the method of Test Example 1, except that benzene was replaced by ethylbenzene (specifically: the test was divided into 5 groups, namely blank control group, ethylbenzene test group 1, ethylbenzene test group 2, ethylbenzene test group 3, and ethylbenzene test group 4; among them, in ethylbenzene test group 1, the ethylbenzene content was 50 mg / L; in ethylbenzene test group 2, the ethylbenzene content was 100 mg / L; in ethylbenzene test group 3, the ethylbenzene content was 150 mg / L; in ethylbenzene test group 4, the ethylbenzene content was 200 mg / L).

[0202] After testing, the ethylbenzene degradation rates of ethylbenzene test group 1, ethylbenzene test group 2, ethylbenzene test group 3, and ethylbenzene test group 4 were 87.5%, 86.0%, 80.7%, and 72.8%, respectively. At the same time, the OD values ​​of each group were measured. 600 , toluene degradation rate and OD 600 like Figure 11 shown.

[0203] The results showed that the degradation rate of Rhodococcus HB3 reached 86.0% in the ethylbenzene content group of 100 mg / L.

[0204] Test Example 4

[0205] The test was performed according to the method of Test Example 1, except that benzene was replaced by xylene (specifically: the test was divided into 5 groups, including blank control group, xylene test group 1, xylene test group 2, xylene test group 3, and xylene test group 4; among them, in xylene test group 1, the xylene content was 50 mg / L; in xylene test group 2, the xylene content was 100 mg / L; in xylene test group 3, the xylene content was 150 mg / L; in xylene test group 4, the xylene content was 200 mg / L).

[0206] After testing, the xylene degradation rates of xylene test group 1, xylene test group 2, xylene test group 3, and xylene test group 4 were 80.1%, 77.9%, 75.8%, and 61.5%, respectively. At the same time, the OD values ​​of each group were measured. 600 , xylene degradation rate and OD 600 like Figure 12 shown.

[0207] The results showed that the degradation rate of Rhodococcus HB3 reached 77.9% in the xylene content group of 100 mg / L.

[0208] Test Example 5

[0209] This test example is used to illustrate the BTEX degradation test of the Rhodococcus sp. HB3 described in the present invention in a simulated actual soil environment.

[0210] 1. Test method:

[0211] (1) The experiment was divided into two groups: a blank control group and a BTEX test group (the volume ratio of benzene, toluene, ethylbenzene and xylene was 1:1:1:1, and the mixed benzene was 100 mg / L).

[0212] Among them, blank control group: enriched culture medium + 0.1 volume% trace element solution;

[0213] BTEX test group: enriched medium + 100 mg / L mixed benzene + 0.1% trace element solution by volume;

[0214] (2) Inoculate 1 mL of seed solution (OD 600 The cells were cultured in a shaking incubator at 18°C, pH 7.5, 6% oxygen concentration, 2% salinity and 135 rpm for 10 days, and the BTEX degradation rate of each group was calculated.

[0215] 2. Test results:

[0216] After calculation, compared with the control group, the BTEX degradation rates in the benzene test group after 10 days were 99.5% for benzene, 94.6% for toluene, 80.4% for xylene, and 88.7% for ethylbenzene. Figure 13 shown.

[0217] Test Example 6

[0218] This test case is used to illustrate the remediation of contaminated soil using Rhodococcus HB3.

[0219] Test method:

[0220] (1) A BTEX-contaminated site in an oil and gas field in North China was selected. Three experimental groups were selected: a control group (same plot, same soil properties, similar pollutant content, and no statistically significant difference), a commercial microbial product group (aerobic microorganisms), and an HB3 microbial product group (prepared in Example 7).

[0221] (2) The on-site pollution depth was 2 to 3 m, the pH was 7.8, the salinity was 2%, the dissolved oxygen was 4.5%, and the concentrations of pollutants in the vadose zone were 92.8 mg / L of benzene, 759.4 mg / L of toluene, 713.2 mg / L of xylene, and 191.7 mg / L of ethylbenzene. According to the Soil Environmental Quality Standard for the Risk Control of Soil Pollution in Construction Land (GB36600-2018), the pollutants benzene and xylene were found to be above the standard.

[0222] (3) The HB3 microbial agent product group and the commercial microbial agent product group were activated with water (wherein the mass ratio of the microbial agent product group to water was 1:9) to obtain activated bacterial solution, which was injected with a low-pressure (0.5 MPa) injection device at a pressure of 16.4 kg / m 3 (Bacteria solution / soil) Activated bacteria solution was injected into the 2m and 3m soil layers, respectively, and the changes in soil BTEX content were measured after 40 days.

[0223] Test results:

[0224] After testing, compared with the control group (degradation rate 5.2%), the BTEX degradation rates of the commercial bacterial product group were 75.0% for benzene, 68.5% for toluene, 54.2% for xylene, and 66.1% for ethylbenzene, while the BTEX degradation rates of the HB3 bacterial product group were 98.8% for benzene, 93.5% for toluene, 75.9% for xylene, and 86.3% for ethylbenzene, indicating that the HB3 bacterial product provided by the present invention has a significant effect in degrading BTEX in the aeration zone.

[0225] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A facultative anaerobic BTEX-degrading bacterium, characterized in that, The facultative anaerobic BTEX-degrading bacterium is Rhodococcus sp. HB3, and its preservation number is GDMCC No: 63282.

2. Application of the facultative anaerobic BTEX-degrading bacterium according to claim 1 in degrading BTEX.

3. The application according to claim 2, wherein The BTEX is one or more of benzene, toluene, ethylbenzene, and xylene.

4. The application according to claim 2, characterized in that It is an application in bioremediation of oil sludge-polluted sites.

5. A bacterial agent, characterized in that, The microbial agent contains the facultative anaerobic BTEX-degrading bacterium according to claim 1.

6. The microbial agent according to claim 5, characterized in that, The microbial agent also contains a fixed carrier.

7. The microbial agent according to claim 6, wherein The fixed carrier is selected from one or more of organic material carriers, plant material carriers, and mineral material carriers; Preferably, the organic material carrier is selected from one or more of sodium alginate, chitosan, polyacrylamide, polyvinyl alcohol, and polyethylene glycol; Preferably, the plant material carrier is corn flour and / or starch; Preferably, the mineral material carrier is selected from one or more of clay, talc, kaolin, and zeolite.

8. The bacterial agent according to claim 5, characterized in that, The dosage form of the microbial agent is liquid, emulsion, suspension, powder, or granule.

9. A method for preparing a microbial agent, characterized in that, The method includes: fermenting and culturing the facultative anaerobic BTEX-degrading bacterium according to claim 1.

10. The method according to claim 9, characterized in that, The conditions of the fermentation include: 26-32 °C, pH 6.5-7.5, and time 18-24 h.

11. The method according to claim 9 or 10, characterized in that The method also includes: fixing the fermentation broth obtained by fermentation and culture on the fixed carrier.

12. A microbial agent prepared by the method according to any one of claims 9-11.

13. A method for repairing an oil sludge contaminated site, characterized in that, The method includes: injecting the facultative anaerobic BTEX-degrading bacterium according to claim 1 or the microbial agent according to any one of claims 5-8 and 12 into the oil sludge-polluted site.

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