Mixed strain for bioremediation of benzene and petroleum hydrocarbon co-polluted site and application thereof

Abstract

The invention discloses a mixed strain for bioremediation of a benzene and petroleum hydrocarbon co-polluted site and application of the mixed strain. The mixed bacterial strain is prepared by mixing the aerobic rhodococcus biphenyl with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No. 1.12975 and the sphingobacterium yanoikuyae with the preservation number of CGMCC No. 29277, and the mixed bacterial strain is prepared by mixing the aerobic rhodococcus biphenyl with the preservation number of CGMCC No. 29277 and the sphingobacterium yanoikuyae with the preservation number of CGMCC No. 29277. The aerobic rhodococcus biphenyl is mainly used for degrading benzene series, the sphingosine vector is mainly used for degrading petroleum hydrocarbon, a synergistic effect is achieved through mixed culture, and the degradation efficiency is improved. The mixed strain is applied to remediation of a benzene and petroleum hydrocarbon co-polluted site, and after the mixed strain is added to the polluted site, the highest benzene removal rate can reach 99% within 16 days, and the highest petroleum hydrocarbon removal rate can reach 89%. The method is green, environment-friendly, economical, efficient and suitable for groundwater and soil combined pollution remediation.

Description

Technical Field

[0001] This invention belongs to the field of environmental engineering, specifically relating to a mixed bacterial strain for bioremediation of sites co-contaminated with benzene and petroleum hydrocarbons and its application. Background Technology

[0002] Benzene compounds and petroleum hydrocarbons are common environmental pollutants, widely present in the production and use processes of industries such as petroleum, chemicals, automobiles, and plastics. These pollutants pose a serious threat to the environment and human health, especially benzene compounds, which have high volatility and toxicity; long-term exposure can cause acute and chronic poisoning, and even cancer. Petroleum hydrocarbons accumulate in water bodies and soil, affecting biological growth and even damaging ecosystems. However, traditional physicochemical remediation methods, such as chemical oxidation, adsorption, and combustion, are often costly and pose a risk of secondary pollution, making them ineffective in addressing high concentrations of benzene compounds and petroleum hydrocarbons.

[0003] With the continuous development of bioremediation technology, utilizing microorganisms to degrade organic pollutants has gradually become a green, environmentally friendly, and economically feasible remediation method. Many microorganisms, especially specific anaerobic or aerobic strains, have been proven to degrade pollutants such as benzene compounds and petroleum hydrocarbons. Bioremediation not only effectively transforms pollutants into harmless products but also offers low cost and good environmental compatibility. However, the degradation capacity of a single strain is limited, especially in environments with combined pollution, where a single strain often cannot effectively degrade both benzene compounds and petroleum hydrocarbons simultaneously.

[0004] In recent years, the application of mixed bacterial strains has gradually become a research hotspot in the field of bioremediation. By combining strains with different degradation characteristics, their synergistic effects can be leveraged, thereby significantly improving the remediation efficiency for complex pollution. For example, some strains can decompose benzene compounds and produce intermediate products, which happen to provide the necessary nutrient source for another type of strain, thus enhancing the latter's degradation capacity.

[0005] Furthermore, existing bioremediation technologies still face certain challenges in large-scale application. Most research remains limited to small-scale laboratory trials, lacking validation and experience for large-scale field applications. For the remediation of sites co-contaminated with benzene series compounds and petroleum hydrocarbons, improving the adaptability and degradation capacity of mixed bacterial strains in complex contaminated environments remains a pressing issue. Summary of the Invention

[0006] The purpose of this invention is to provide a mixed bacterial strain for the remediation of sites co-contaminated with benzene and petroleum hydrocarbons and its application, wherein the mixed bacterial strain is composed of aerobic Rhodococcus biphenyls (… Rhodococcus biphenylivorans ) and Yano sphingosine mononitrate ( Sphingomonas yanoikuyaeThe mixture consists of two strains: *Rhodococcus biphenyle*, an aerobic bacterium that degrades benzene compounds, and *Sphingosine monocytogenes*, a strain that degrades petroleum hydrocarbons. By fermenting the mixture and then applying it to the contaminated site, the growth concentration of the strains, benzene degradation efficiency, and petroleum hydrocarbon degradation efficiency can be significantly increased, achieving highly efficient bioremediation.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a mixed bacterial strain for the remediation of sites co-contaminated with benzene and petroleum hydrocarbons, consisting of aerobic Rhodococcus biphenyls (Rhodococcus biphenyls). Rhodococcus biphenylivorans ) and Yano sphingosine mononitrate ( Sphingomonas yanoikuyae It is a mixture of bacteria, in which *Rhodococcus biphenyle* represents strains that degrade benzene compounds, and *Sphingosine mononitrate* represents strains that degrade petroleum hydrocarbons.

[0008] The aerobic Rhodococcus biphenyl is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 1.12975, and the Sphingosine monocytogenes is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29277. The address of the collection is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0009] In complex polluted environments, aerobic Rhodococcus biphenyleukaryotes and Sphingosine mononitrate bacteria achieve overall improved degradation performance through metabolic complementarity, avoiding substrate competition; cross-trophic interactions promote bacterial growth; and signal molecule communication enhances enzyme expression.

[0010] Sphingosine mononitrate bacteria enhance the solubility and bioavailability of hydrophobic pollutants in the aqueous phase by secreting biosurfactants, promoting their transfer from soil particles or non-aqueous liquids to the microbially available phase. The membrane-associated dioxygenase system of aerobic Rhodococcus biphenyls initially activates benzene. The two bacteria work together to transform key intermediates through metabolic specialization and cross-feeding. Sphingosine mononitrate bacteria promptly eliminates potentially toxic intermediates, while aerobic Rhodococcus biphenyls introduces the remaining carbon flow into the tricarboxylic acid cycle, achieving complete mineralization of pollutants into CO2, water, and biomass, thus forming a stable and efficient synergistic degradation network.

[0011] Secondly, the present invention provides a method for preparing the mixed bacterial strain, the specific steps of which are: reviving and subculturing *Rhodococcus biphenyle* and *Sphingosine monocytogenes* from cryovials, mixing them at a volume ratio of 1:1 to 1:5 and inoculating them into LB medium; culturing at 30°C and 150 rpm with shaking for 24-48 hours until OD... 600 A mixed strain was prepared by observing a logarithmic growth phase with a pH of approximately 1-1.5; trace element solution was added during the culture process, and the pH was maintained in the range of 6.8-7.2.

[0012] Furthermore, the trace element solution contains 0.5 mg / L FeSO4·7H2O, 0.1 mg / L MnCl2·4H2O, 0.2 mg / L ZnSO4·7H2O, and 0.05 mg / L CuSO4·5H2O.

[0013] Thirdly, the present invention provides a liquid bacterial agent obtained by fermentation of the aforementioned mixed bacterial strains, wherein the effective viable count of the liquid bacterial agent is 10. 9 -10 10 CFU / mL, viable count 85-95%, contamination rate <1%, pH 6.8-7.2.

[0014] Fourthly, the present invention provides a method for preparing the liquid bacterial agent, the specific steps of which are: 0.5L of the mixed bacterial strain is fermented in LB medium in a stepwise manner, and cultured in 5 L, 50 L and 500 L fermenters for 48 h, 72 h and 96 h respectively, at a fermentation temperature of 30 ℃ and a stirring speed of 180–200 rpm; a trace element solution is added to the culture medium.

[0015] Fifthly, the present invention provides the application of the mixed bacterial strains or liquid bacterial agents in the bioremediation of sites co-contaminated with benzene and petroleum hydrocarbons, wherein the application specifically includes: The mixed bacterial strains or liquid bacterial agent were injected into the contaminated site to achieve a final concentration of 1×10⁻⁶. 5 CFU / mL, and add 450 g / m³ according to the volume of the contaminated site. 3 Long-lasting carbon source soybean oil, 36g / m 3 Dispersants Tween 80, 120 g / m 3 Sodium acetate, a fast-acting carbon source, 22.5 g / m³ 3 Nitrogen source yeast extract powder and 50g / m 3 The colonization auxiliary material is 200-500 mesh activated carbon, which removes benzene and petroleum hydrocarbons.

[0016] The beneficial effects of this invention are as follows: This invention provides a bacterial agent prepared by mixing *Sphingosine monocytogenes* and *Rhodococcus biphenyl*, which has a significant removal effect on benzene and petroleum hydrocarbons in groundwater, with removal rates reaching up to 99% and 89% respectively after 16 days. This bacterial agent, after laboratory-scale testing and field verification, demonstrates good environmental adaptability and engineering scalability in actual groundwater environments, making it suitable for in-situ bioremediation of similar complex contaminated sites.

[0017] The strains used are derived from the natural environment, are non-pathogenic, pose no risk of secondary pollution, grow rapidly, are highly tolerant, and require simple cultivation conditions. They can be prepared on a large scale using conventional aerobic fermentation processes. For on-site application, they can be directly injected into the aquifer without complex equipment, resulting in low energy consumption and operating costs, and simplified construction and management. The two strains are functionally complementary, simultaneously enhancing the removal of benzene and petroleum hydrocarbons. The overall remediation effect and economic advantages are significant, demonstrating good practical value and promising prospects for widespread application. Attached Figure Description

[0018] Figure 1 The three bacterial agents OD in the embodiments of the present invention 600 The value changes over time.

[0019] Figure 2 This invention demonstrates the removal efficiency of three fermentation agents on benzene in simulated groundwater.

[0020] Figure 3 This invention demonstrates the removal effect of three fermentation agents on petroleum hydrocarbons in simulated groundwater.

[0021] Figure 4 The graph shows the effect of the mixed bacterial agent in this invention on the removal of benzene in actual groundwater areas, with the horizontal axis representing different site names.

[0022] Figure 5 The graph shows the effect of the mixed bacterial agent in this invention on the removal of petroleum hydrocarbons in actual groundwater areas, with the horizontal axis representing different site names. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to the accompanying drawings. These descriptions are for illustrative purposes only and not for limiting the scope of the invention. Unless otherwise specified, the methods used in the following embodiments are conventional methods, and the reagents used can be commercially available products.

[0024] The two microbial strains involved in this invention are both existing preserved strains. The applicant has previously deposited them as biological materials, and this has been described in detail in the published Chinese patent application. Specific preservation information is as follows: the depository is the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the deposit date is October 8, 2014. Among them, *Rhodococcus biphenyl* (aerobic biphenyl-coccus) is a pre-existing microbial strain. Rhodococcus biphenylivorans The accession number for ) is CGMCC No. 1.12975, abbreviated as TG9; *Sphingosine monocytogenes* ( Sphingomonas yanoikuyae The accession number of the present invention is CGMCC No. 29277. The following specific embodiments further illustrate the content of the present invention, but the scope of protection of the present invention is not limited thereto.

[0025] Example 1 Preparation of mixed strains.

[0026] aerobic Rhodococcus biphenylophilus and Sphingosine monocytogenes were thawed separately from cryovials. LB medium was prepared with the following formula: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH adjusted to 7.0. The medium was then autoclaved at 121°C for 15 minutes. 1 ml of each of the aerobic Rhodococcus biphenylophilus and Sphingosine monocytogenes was inoculated into 100 mL of LB medium, and 0.5 ml of each was mixed and inoculated into another 100 mL of LB medium. The medium was incubated in a constant temperature shaking incubator at 30°C and 150 rpm for 24-72 hours. When OD... 600 When the pH reaches 1.0-1.5, subculture is performed under the same conditions as described above. During the culture process, trace element solutions are added according to the volume of the culture medium, with final concentrations of FeSO4·7H2O 0.5 mg / L, MnCl2·4H2O 0.1 mg / L, ZnSO4·7H2O 0.2 mg / L, and CuSO4·5H2O 0.05 mg / L, to enhance the metabolic activity of the strain. The pH is monitored and maintained within the range of 6.8-7.2. If the pH deviates, it is adjusted using 1M NaOH or HCl. Single-culture bacterial suspensions and mixed-strain suspensions are obtained and verified by plate counting to ensure the total bacterial concentration is controlled at 10. 8 -10 9 CFU / mL. Gently shake the obtained single-culture bacterial suspension and mixed strain suspension to avoid the formation of air bubbles, and store at 4°C for short-term use. For long-term storage, add 15% glycerol by volume of the bacterial suspension and freeze at -80°C.

[0027] Example 2 Prepare mineral salt culture medium (MSM) containing 1 g / L NH4Cl, 0.5 g / L KH2PO4, 1 g / L Na2HPO4, 0.2 g / L MgSO4·7H2O, 0.01 g / L CaCl2, and an appropriate amount of trace element solution (with the same formula as described in Example 1). The pH value is 7.0. Autoclave the medium.

[0028] A simulated benzene and petroleum hydrocarbon co-contamination system was prepared in a 250 mL Erlenmeyer flask: analytical grade benzene was added to the above-mentioned mineral salt medium MSM to a working concentration of 500 mg / L, and the working concentration of petroleum hydrocarbon standards was 50 mg / L. Analytical grade benzene and petroleum hydrocarbon standards were used as the sole carbon source. Three parallel experiments were set up, with three replicates for each group: the first group was treated with a suspension of *Sphingosine mononitrate*, the second group was treated with a suspension of *Rhodococcus biphenyl*, and the third group was treated with a mixed bacterial suspension. The initial concentration of the bacterial suspensions in the three groups was 10 mg / L. 7CFU / mL. All groups were incubated with shaking at 30℃ and 150 rpm. OD was measured using a spectrophotometer during the incubation period. 600 Viable bacterial concentration and contaminant concentration were determined using the plate count method and the plate count method. Benzene was determined using gas chromatography-mass spectrometry (GC-MS) with a column temperature program of initial 50℃ for 2 min, increasing to 250℃ at a rate of 10℃ / min and holding for 5 min. Petroleum hydrocarbons were determined using infrared spectrophotometry at a wavelength of 2930 cm⁻¹. -1 .

[0029] like Figure 1-3 The results showed that the OD of the mixed strain group was... 600 The value reached 2.35, indicating a culture time of 64 hours, which is higher than the OD value of the single sphingosine monoculture group. 600 The value was 1.97, and the OD value of the single aerobic Rhodococcus biphenyl group was also high. 600 The value was 2.14, indicating a culture time of 64 hours. The best degradation effect was observed with a culture time of 72 hours: the aerobic Rhodococcus biphenylein group showed a benzene degradation rate of 83%, while the mixed strain group showed a degradation rate of 90%; the Sphingosine monocytogenes group showed a petroleum hydrocarbon degradation rate of 54%, while the mixed strain group showed a degradation rate of 64%.

[0030] Example 3 The mixed bacterial strains prepared by the above method were used for in-situ remediation of groundwater co-contaminated with benzene and petroleum hydrocarbons.

[0031] The site was selected from the actual groundwater contaminated by benzene and petroleum hydrocarbons in the project. The site investigation showed that the average concentration of benzene was 180 mg / L, ranging from 30 to 400 mg / L, and the average concentration of petroleum hydrocarbons was 9 mg / L, ranging from 2 to 30 mg / L. They were mainly distributed in the aquifer at a depth of 5-15m.

[0032] An in-situ bioremediation strategy was adopted: First, groundwater samples were extracted from monitoring wells (6 wells spaced 10m apart) to verify the adaptability of the mixed bacterial strains. Preliminary laboratory experiments showed a survival rate >85%. A mixed bacterial strain suspension was prepared: The mixed bacterial strain suspension described in Example 1 was used as the seed culture for stepwise fermentation and expansion. LB medium (with the same composition as in Example 1) was added to a 5L fermenter, followed by inoculation with the seed culture at an initial volume of 0.5L and a bacterial concentration of 10-1. 9 CFU / mL, cultured at 30℃ and 200 rpm for 48 hours, and OD was monitored. 600 The value reaches 2.0 or higher, and the bacterial concentration reaches 2×10⁻⁶. 9 CFU / mL; then, 5 L of culture medium was transferred to a 50 L fermenter, and fresh LB medium was added to a total volume of 50 L. The culture was then continued under the same conditions for 72 hours until the bacterial concentration reached 3 × 10⁻⁶. 9CFU / mL; finally, transfer 50L of culture medium to a 500L fermenter, add fresh LB medium to a total volume of 500L, and incubate under the same conditions for 96 hours, achieving a final bacterial concentration of 4×10⁻⁶. 9 CFU / mL was used to obtain a large-scale mixed bacterial suspension. The entire expansion process was maintained at pH 6.8-7.2, and trace elements (as described in Example 1) were added to optimize growth.

[0033] Microbial agent (final concentration after addition to contaminated site: 1×10⁻⁶) 5 CFU / mL) and long-lasting carbon source soybean oil (450g / mL) 3 ), dispersant Tween 80 (36g / m³) 3 ), fast-acting carbon source sodium acetate (120g / m 3 ), nitrogen source yeast extract powder (22.5g / m 3 Colonization auxiliary material: 200-500 mesh activated carbon (50g / m³) 3 The solution is added to the mixing tank of the integrated injection equipment and stirred for at least 10 minutes until fully mixed. Then, it is injected into the well on-site via an injection pump with a power of 3 kW, a head of up to 112 m, and an injection pressure of 1.1 MPa. Samples are periodically collected from each well and sent back to the laboratory to determine the concentrations of benzene and petroleum hydrocarbons in the water (the determination method is as described in Example 2). Figure 4-5 As shown, after 16 days, the average removal rate of benzene in groundwater was over 82%, with a maximum removal rate of 99%; the average removal rate of petroleum hydrocarbons was 88%, with a maximum removal rate of 89%.

[0034] The embodiments described above are merely preferred implementations and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and variations without departing from the core spirit and scope of protection of the present invention. Therefore, any technical solutions achieved through equivalent substitution or equivalent change should be considered to fall within the protection scope of the present invention.

Claims

1. A mixed bacterial strain for bioremediation of sites co-contaminated with benzene and petroleum hydrocarbons, characterized in that, The mixed strain consisted of aerobic Rhodococcus biphenyle ( Rhodococcus biphenylivorans ) and Yano sphingosine mononitrate ( Sphingomonasyanoikuyae The mixture is composed of the following: the aerobic Rhodococcus biphenyleukaryote has the accession number CGMCC No. 1.12975 and the sphingosine mononitrate has the accession number CGMCC No. 29277, both of which are deposited at the China General Microbiological Culture Collection Center.

2. The method for preparing mixed strains according to claim 1, characterized in that, The specific steps are as follows: Aerobic Rhodococcus biphenylepiploicus and Sphingosine monocytogenes were thawed from cryovials, passaged, and inoculated into LB medium at a volume ratio of 1:1 to 1:5; the mixtures were then cultured at 30°C and 150 rpm with shaking for 24–48 hours until OD was reached. 600 A mixed strain was prepared by observing a logarithmic growth phase with a pH of approximately 1-1.5; trace element solution was added during the culture process, and the pH was maintained in the range of 6.8-7.

2.

3. The preparation method according to claim 2, characterized in that, The trace element solution contains 0.5 mg / L FeSO4·7H2O, 0.1 mg / L MnCl2·4H2O, 0.2 mg / L ZnSO4·7H2O, and 0.05 mg / L CuSO4·5H2O.

4. A liquid microbial agent for bioremediation of sites co-contaminated with benzene and petroleum hydrocarbons, characterized in that, It is obtained by fermentation using the mixed strain described in claim 1.

5. The method for preparing the liquid bacterial agent according to claim 4, characterized in that, The specific steps are as follows: 0.5L of the mixed strain is fermented in LB medium in a stepwise manner, and cultured in 5 L, 50 L and 500 L fermenters for 48 h, 72 h and 96 h respectively, with a fermentation temperature of 30 ℃ and a stirring speed of 180–200 rpm; a trace element solution is added to the culture medium.

6. The liquid bacterial agent according to claim 4, characterized in that, The effective viable bacteria count of the liquid bacterial agent is 10. 9 -10 10 CFU / mL, viable count 85-95%, contamination rate <1%, pH 6.8-7.

2.

7. The application of the mixed bacterial strain according to claim 1 or the liquid bacterial agent according to claim 6 in the bioremediation of sites co-contaminated with benzene and petroleum hydrocarbons.

8. The application according to claim 7, characterized in that, The specific application is as follows: The mixed bacterial strains or liquid bacterial agent were injected into the contaminated site to achieve a final concentration of 1×10⁻⁶. 5 CFU / mL, and add 450 g / m³ according to the volume of the contaminated site. 3 Long-lasting carbon source soybean oil, 36g / m 3 Dispersants Tween 80, 120 g / m 3 Sodium acetate, a fast-acting carbon source, 22.5 g / m³ 3 Nitrogen source yeast extract powder and 50g / m 3 The colonization auxiliary material is 200-500 mesh activated carbon, which removes benzene and petroleum hydrocarbons.

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