Preparation method and application of biochar loaded with rhodococcus erythropolis

Abstract

The invention discloses a preparation method and application of biochar loaded with rhodococcus erythropolis, relates to the technical field of soil petroleum pollution remediation, and aims at solving the problem that the effect of soil petroleum hydrocarbon remediation by an existing biological method is poor. The method comprises the following steps: 1, air-drying straws, crushing, and carbonizing to obtain biochar; 2, activating Rhodococcus erythropolis KB1, then transferring the activated bacterial liquid into an LB liquid culture medium for culturing, and centrifugally collecting thalli; resuspending the thalli by using sterile normal saline to prepare rhodococcus erythropolis liquid; and 3, mixing the rhodococcus erythropolis liquid with the biochar, and carrying out vibration culture to obtain the rhodococcus erythropolis-loaded biochar. The biochar loaded with rhodococcus erythropolis has a remarkable removal effect on soil petroleum hydrocarbon, and the influence of petroleum hydrocarbon on soil is reduced. The method is used for repairing the petroleum-contaminated soil.

Description

Technical Field

[0001] The invention relates to the technical field of soil petroleum pollution remediation, and in particular to a preparation method and application of biochar loaded with Rhodococcus erythropolis. Background Art

[0002] Petroleum hydrocarbon compounds pose a serious threat to human health, the environment and ecosystems, and are carcinogenic, genotoxic and neurotoxic. Petroleum hydrocarbons can damage the physical and chemical properties of soils, such as reducing soil permeability, changing soil structure and reducing soil fertility. Petroleum hydrocarbon pollution can also disrupt soil microbial communities and affect nutrient cycles in the soil, and cause biological toxicity in aquatic environments, leading to the destruction of ecosystems and the reduction of biodiversity.

[0003] Soil petroleum hydrocarbon remediation technology mainly includes three methods: physical, chemical and biological. Physical remediation usually uses heating to decompose petroleum hydrocarbons, and chemical remediation usually adds chemical oxidants to the soil. Although these two methods are effective, they are costly and not suitable for in-situ soil remediation. Plant and microbial remediation technology is the mainstream technology for in-situ remediation of petroleum-contaminated soil, but studies have found that the removal effect of soil petroleum hydrocarbons by local plants and single microbial agents is not good, and new technology research and development is urgently needed. Summary of the invention

[0004] The present invention aims to solve the problem that the existing biological method for soil petroleum hydrocarbon remediation has a poor effect, and provides a preparation method and application of biochar loaded with Rhodococcus erythropolis.

[0005] The method for preparing biochar loaded with Rhodococcus erythropolis of the present invention comprises the following steps:

[0006] 1. Air-dry the straw, crush it, and then carbonize it to obtain biochar;

[0007] 2. Inoculate Rhodococcus erythropolis KB1 in 5 mL of sterile LB liquid culture medium and culture overnight to obtain an activated bacterial solution; then transfer the activated bacterial solution to 100 mL of sterile LB liquid culture medium, culture at 25°C for 7 days, and collect the bacterial cells by centrifugation at 4°C; resuspend the bacterial cells in sterile physiological saline to prepare a Rhodococcus erythropolis solution;

[0008] 3. Mix the Rhodococcus erythropolis liquid with biochar, and culture them by vibration to obtain biochar loaded with Rhodococcus erythropolis.

[0009] Furthermore, the Rhodococcus erythropolis KB1 has been deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit date is October 17, 2024, and the deposit number is CCTCC NO: M 20242230.

[0010] Furthermore, the carbonization conditions in step 1 are: carbonization temperature 450-500° C., anaerobic conditions, and carbonization time 2-3 hours.

[0011] Further, the concentration of the Rhodococcus erythropolis solution in step 2 is 10 8 ~10 9 CFU·mL -1 .

[0012] Furthermore, in step three, the liquid ratio of biochar to Rhodococcus erythropolis liquid is 1 g:1 mL.

[0013] Furthermore, the conditions for the vibration culture in step three are: at 25°C to 30°C, at a vibration speed of 120rpm to 140rpm, and for 24h to 28h.

[0014] The present invention also provides the use of biochar loaded with Rhodococcus erythropolis in repairing petroleum-contaminated soil.

[0015] Furthermore, the amount of biochar loaded with Rhodococcus erythropolis is 1 to 3 t / hm 2 .

[0016] Beneficial effects of the present invention:

[0017] The present invention provides a biochar loaded with Rhodococcus erythropolis. The present invention uses Rhodococcus erythropolis KB1 screened from petroleum-contaminated soil. Rhodococcus erythropolis KB1 can effectively multiply and degrade petroleum hydrocarbon substances in a polluted environment. It can decompose complex petroleum hydrocarbon compounds, has strong adaptability, and can survive and function in extreme environments.

[0018] The biochar improves the biological activity of Rhodococcus erythropolis and increases the removal effect of petroleum hydrocarbons in petroleum-contaminated soil. The biochar loaded with Rhodococcus erythropolis has a significant effect on removing petroleum hydrocarbons from soil and reduces the impact of petroleum hydrocarbons on soil.

[0019] The present invention utilizes biological restoration means to efficiently degrade petroleum hydrocarbon pollutants, thereby reducing dependence on chemical treatment and having positive significance for environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Phylogenetic tree for molecular identification of strains;

[0021] Figure 2 The growth of the strains after six days of cultivation under different pH conditions;

[0022] Figure 3 The growth of the strains after six days of cultivation at different temperature values;

[0023] Figure 4 is the degradation rate of petroleum hydrocarbons by the strain in six days;

[0024] Figure 5 The removal effect of biochar loaded with Rhodococcus erythropolis on soil petroleum hydrocarbons. DETAILED DESCRIPTION

[0025] The embodiments of the present invention are described in detail below. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0026] Embodiment 1:

[0027] The method for obtaining Rhodococcus erythropolis KB1 in this embodiment is:

[0028] Initial screening process: Using the plate streak method, select the oil-contaminated soil (0-10 cm) from the Liaohe River estuary, add 10 grams of soil sample to a 250 ml conical flask containing 100 ml of sterile 0.85% NaCl solution. Oscillate at 180 rpm for 30 minutes at 25°C, then let stand for 1 hour. Take 1 ml of supernatant and add it to 50 ml of fresh OMSM medium, and further enrich and culture at 180 rpm in a dark environment at 25°C.

[0029] Liquid enrichment medium: Liquid enrichment medium consists of mineral salt medium (MSM) containing 1.0 g NaCl, 1.0 g K2HPO4, 1.0 g KH2PO4, 0.2 g MgSO4·7H2O, 0.02 g CaCl2 and 1.0 g NH4NO3 per liter. The enrichment medium is used at a concentration of 1 g·L -1 Crude oil was used as the sole carbon source in crude mineral salts medium (OMSM).

[0030] Enrichment process: After 7 days of enrichment, 5 ml of bacterial solution was transferred to a 15 ml centrifuge tube, centrifuged at 5000 rpm for 10 minutes at 4°C, washed twice with 0.85% NaCl solution, and then added with 50 ml of fresh OMSM for continuous enrichment culture. The enrichment time for each passage was 7 days, and the total enrichment process included 6 passages.

[0031] Purification process: After 6 generations of continuous enrichment, the bacterial solution was diluted in gradients and inoculated onto LB solid medium. Colonies with different morphologies were repeatedly streaked to isolate pure cultures.

[0032] Strain selection: At 25°C, the strain with the highest growth rate and crude oil degradation rate in OMSM was selected. The strain was molecularly identified, and the genomic DNA of strain BJ2 was extracted using a genome extraction kit (FastDNASpin Kit for Soil) to amplify the 16S rRNA gene. The universal primers for PCR were: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′), and the reaction system was 25 μL, including Premix version 2.0 12.5 μL, 27F (10 nM) 1 μL, 1492R (10 nM) 1 μL, DNA template 1 μL, and sterile water to 25 μL. The reaction conditions were: 94°C pre-denaturation for 4 minutes; 94°C denaturation for 30 seconds, 55°C annealing for 50 seconds, 72°C extension for 1 minute, a total of 35 cycles; 72°C final extension for 10 minutes. After PCR amplification, the product was detected by 1.5% agarose gel electrophoresis, and the result showed a clear characteristic band at about 1500 bp. The PCR amplification product was sequenced, and the sequencing result is shown in SEQ ID NO: 1 in the sequence table. The obtained sequence was uploaded to GenBank. The homology was compared with the sequence in the gene library by BLAST analysis, and the Neighbor-Joining method in MEGA5.1 software was used to construct a phylogenetic evolutionary tree, as shown in Figure 1. Figure 1 The phylogenetic tree is shown. The strain has the highest homology with Rhodococcus erythropolis, reaching 99%. By combining the identification results such as bacterial morphological characteristics and growth conditions, the strain of this embodiment is determined to be Rhodococcus erythropolis, and is named Rhodococcus erythropolis KB1.

[0033] Rhodococcus erythropolis KB1 has been deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China. The deposit date is October 17, 2024, and the deposit number is CCTCC NO: M20242230.

[0034] Example 2: An experiment was conducted to optimize the growth conditions of Rhodococcus erythropolis KB1.

[0035] The LB medium was divided into several triangular flasks, each with 100 mL. Different pH values ​​(5.0, 6.0, 7.0, 8.0, 9.0) were set and cultured at 25°C. Different temperatures (4°C, 15°C, 25°C, 35°C) were set and cultured at pH 7. The logarithmic growth phase bacterial suspension of strain KB1 was inoculated into the medium at a concentration of 10 5 Each pH value and temperature condition was repeated three times. The culture flask was placed in a shaking incubator under the corresponding temperature conditions and cultured at a speed of 150 rpm for 6 days. 1 ml of sample was taken every day, and the optical density (OD600) of the culture solution was measured at 600 nm using a spectrophotometer to evaluate the growth of the bacteria. The growth curve of Rhodococcus erythropolis KB1 under different pH conditions is shown in the figure. Figure 2 The growth curves under different temperature conditions are as follows Figure 3 The results showed that Rhodococcus erythropolis KB1 grew best in the pH range of 6-7 and the temperature range of 15-35℃.

[0036] Example 3: Degradation of petroleum by Rhodococcus erythropolis KB1.

[0037] The KB1 strain of Rhodococcus erythropolis was cultured to the logarithmic growth phase. A liquid culture medium containing 1% petroleum (volume ratio) was prepared and the culture medium was divided into several triangular flasks, with 100 mL of culture medium added to each bottle. The concentration of the KB1 suspension of Rhodococcus erythropolis was adjusted to 10 7 bacteria / mL, add 1mL of bacterial suspension to each culture bottle to make the final concentration of bacterial suspension 10 5 bacteria / mL. The control group was not inoculated with bacterial suspension, but only added an equal amount of sterile water. Each group of experiments was repeated 3 times. The culture bottle was placed in an shaking incubator at 25°C and cultured at a speed of 150rpm for 6 days. 1mL of sample was taken every day, and the optical density (OD600) of the culture solution was measured at 600nm by a spectrophotometer to estimate the growth of bacteria. The degradation products of petroleum were analyzed by gas chromatography-mass spectrometry (GC-MS), and the types and concentrations of degradation products were specifically detected. Calculate the degradation rate of petroleum: degradation rate (%) = (initial petroleum concentration-remaining petroleum concentration) / initial petroleum concentration × 100.

[0038] The degradation of petroleum by Rhodococcus erythropolis KB1 Figure 4 As shown in the figure, by the sixth day, the degradation rate of oil by Rhodococcus erythropolis KB1 was 86.89%, and the Rhodococcus erythropolis KB1 strain had a significant effect on the degradation of oil within six days.

[0039] Embodiment 4:

[0040] In this embodiment, reed straw is used as raw material to prepare biochar.

[0041] 1. Crush the air-dried reed straw and place it in a carbonization furnace. Control the temperature of the carbonization furnace to 500°C, anaerobic conditions, and time for 3 hours. After the carbonization process is completed, cool it to 25°C to obtain reed biochar.

[0042] 2. Inoculate Rhodococcus erythropolis KB1 in 5 mL of sterile LB liquid medium and culture overnight to obtain an activated bacterial solution; then transfer the activated bacterial solution to 100 mL of sterile LB liquid medium, culture at 25°C for 7 days, and collect the cells by centrifugation at 4°C; resuspend the cells in sterile saline to prepare a concentration of 10 9 CFU·mL -1 of Rhodococcus erythropolis liquid;

[0043] 3. Mix the Rhodococcus erythropolis liquid with biochar, with the solid-liquid ratio of biochar to Rhodococcus erythropolis liquid being 1 g:1 mL, and culture the mixture at 25°C and a vibration speed of 140 rpm for 24 h to obtain biochar loaded with Rhodococcus erythropolis.

[0044] The biochar loaded with Rhodococcus erythropolis prepared in this example is used to repair petroleum-contaminated soil:

[0045] Test soil: Soil from the Liaohe River estuary that passed through a 2 mm sieve. According to the local petroleum pollution characteristics, petroleum-contaminated soil 1000 mg / kg was prepared by adding crude oil.

[0046] The test container was a PVC tube with a diameter of 5 cm and a height of 13 cm. 150 g of the prepared petroleum-contaminated soil was added into the PVC tube to obtain a soil column. The soil columns were divided into four groups: (1) the soil columns of the untreated group were not treated; (2) the microbial agent group: the soil columns were added with a concentration of 10 9 CFU·mL -1 (3) Biochar group: biochar was added to the soil column, the amount of biochar was 5.0 g / soil column; (4) Biochar loaded bacterial agent group: biochar loaded with Rhodococcus erythropolis prepared in this example was added to the soil column, the amount of biochar added was 7.5 g / soil column, and 6 replicates were set for each group.

[0047] A reed seedling was transplanted into each soil column, and all the soil columns were placed in the original soil of Liaohe River Estuary, maintaining the same environmental conditions as in the wild, and were left in the wild for 90 days.

[0048] Data determination:

[0049] The soil samples were freeze-dried in the dark and passed through a 2 mm sieve for later use. The petroleum hydrocarbons in the soil were determined (gas chromatography) according to the environmental protection standard HJ 1021-2019.

[0050] like Figure 5 As shown, during the entire cultivation process, both bacterial agents and organisms significantly promoted the removal of soil petroleum hydrocarbons, but bacterial agents loaded with petroleum hydrocarbons had the best effect on the removal of soil petroleum hydrocarbons.

[0051] In summary, the biochar loaded with Rhodococcus erythropolis of the present invention has a significant effect on removing petroleum hydrocarbons in the soil and can reduce the impact of petroleum hydrocarbons on the soil.

Claims

1. A method for preparing biochar loaded with Rhodococcus erythropolis, characterized in that: The method comprises the following steps:

1. Air-dry the straw, crush it, and then carbonize it to obtain biochar; 2. Inoculate Rhodococcus erythropolis KB1 in 5 mL of sterile LB liquid culture medium and culture overnight to obtain an activated bacterial solution; then transfer the activated bacterial solution to 100 mL of sterile LB liquid culture medium, culture at 25°C for 7 days, and collect the bacterial cells by centrifugation at 4°C; resuspend the bacterial cells in sterile physiological saline to prepare a Rhodococcus erythropolis solution; 3. Mix the Rhodococcus erythropolis liquid with biochar, and culture them by vibration to obtain biochar loaded with Rhodococcus erythropolis.

2. The method for preparing biochar loaded with Rhodococcus erythropolis according to claim 1, characterized in that: The Rhodococcus erythropolis KB1 has been deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit date is October 17, 2024, and the deposit number is CCTCC NO: M20242230.

3. The method for preparing biochar loaded with Rhodococcus erythropolis according to claim 1, characterized in that: The carbonization conditions in step 1 are: carbonization temperature 450°C to 500°C, anaerobic conditions, and carbonization for 2 to 3 hours.

4. The method for preparing biochar loaded with Rhodococcus erythropolis according to claim 1, characterized in that: The concentration of the Rhodococcus erythropolis solution in step 2 is 10 8 ~10 9 CFU·mL -1 .

5. The method for preparing biochar loaded with Rhodococcus erythropolis according to claim 1, characterized in that: In step three, the liquid ratio of biochar to Rhodococcus erythropolis liquid is 1g:1mL.

6. The method for preparing biochar loaded with Rhodococcus erythropolis according to claim 1, characterized in that: The conditions for the vibration culture in step three are: at 25° C. to 30° C., at a vibration speed of 120 rpm to 140 rpm, for 24 h to 28 h to obtain biochar loaded with Rhodococcus erythropolis.

7. Use of biochar loaded with Rhodococcus erythropolis prepared by the method according to claim 1 in remediation of petroleum-contaminated soil.

8. The use according to claim 7, characterized in that: The amount of biochar loaded with Rhodococcus erythropolis is 1-3 t / hm 2 .

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