Microbial flora culture method for efficiently degrading organic pollutants

Abstract

According to the microbial flora culture method for efficiently degrading the organic pollutants, three continuous culture stages of enrichment, domestication and optimization are adopted, parameters such as the temperature, the pH value, the rotating speed and the ventilatory capacity are accurately controlled in each stage, and growth of microorganisms with degradation capacity is promoted. Through multi-stage culture and domestication, the obtained flora has efficient degradation capability on organic pollutants such as petroleum hydrocarbons, pesticides and polycyclic aromatic hydrocarbons, and has strong adaptability to pollutants with different concentrations. The 16S rRNA gene is used for sequencing and identifying the flora, and composition and dominant strains are clear. The flora is prepared into a microbial inoculum, and the microbial inoculum is preserved by freeze drying or liquid nitrogen, so that the activity can be effectively maintained. The flora can be widely applied to the fields of polluted soil and water body remediation, industrial wastewater treatment and the like, is green and environment-friendly, is relatively low in cost, avoids secondary pollution, conforms to the concept of sustainable development, and has relatively high economic and environmental values.

Description

Technical Field

[0001] The invention relates to the fields of environmental protection and microorganisms, and in particular to a method for cultivating a microbial flora for efficiently degrading organic pollutants. Background Art

[0002] Organic pollutants, such as petroleum hydrocarbons, pesticides, and polycyclic aromatic hydrocarbons, are widely present in the soil and water environment around refineries, pesticide plants, and landfills. These pollutants not only cause serious damage to the ecological environment and threaten the survival and reproduction of plants and animals, but also pass through the food chain, posing a potential risk to human health. Therefore, how to efficiently degrade organic pollutants has become a research hotspot in the current field of environmental governance.

[0003] At present, the treatment methods for organic pollutants mainly include physical methods, chemical methods and biological methods. Physical methods, such as adsorption and extraction, can quickly remove some pollutants, but they only transfer pollutants from one medium to another, and do not really degrade them, and may cause secondary pollution. Chemical methods, such as oxidation and reduction, although the degradation efficiency is relatively high, usually require the use of a large amount of chemical reagents, are costly, and may have certain negative impacts on the environment.

[0004] Biological methods, especially the use of microorganisms to degrade organic pollutants, have attracted widespread attention due to their environmental friendliness and low cost. However, the existing microbial degradation technology still has some shortcomings.

[0005] First, in terms of obtaining microbial flora, existing methods are often not targeted, and the sample collection sites are not accurate enough, which makes it difficult to quickly screen microorganisms with efficient degradation capabilities. In many cases, the collected samples may contain only a small number of microorganisms with degradation capabilities, or the degradation activity of these microorganisms is low, and it takes a long time of cultivation and screening to obtain effective flora, which not only increases the cultivation cycle, but also increases the cultivation cost.

[0006] Secondly, the existing culture methods are not scientific and systematic enough for the culture process of microbial flora. Some culture methods do not precisely control the culture conditions. For example, the temperature, pH value, rotation speed and other parameters are set unreasonably, which leads to the inhibition of microbial growth and metabolism and the inability to fully exert their degradation capacity. Moreover, most culture methods lack the domestication and optimization process of the flora, and the cultured flora has poor tolerance to high concentrations of organic pollutants. When encountering a highly polluted environment in actual applications, the degradation efficiency will drop significantly.

[0007] In addition, the existing technology lacks effective identification methods for the cultivated microbial flora, making it difficult to accurately understand the composition and dominant species of the flora, which makes it lack a scientific basis for further optimizing the flora structure and improving the degradation efficiency. At the same time, in terms of the preservation of the flora, the existing preservation methods may not be able to effectively maintain the activity and stability of the flora, resulting in reduced activity of the flora during the preservation process, affecting its subsequent application effect. Therefore, we propose a method for cultivating microbial flora that can efficiently degrade organic pollutants. Summary of the invention

[0008] The purpose of the present invention is to address the problems raised by the existing background technology. In order to achieve the above invention purpose, the present invention provides the following technical solutions: a method for cultivating a microbial flora that efficiently degrades organic pollutants, comprising the following steps: Step 1, sample collection: collecting samples containing microorganisms that degrade organic pollutants from soil and water environments that are seriously polluted by organic pollutants;

[0009] Step 2, enrichment culture: add the collected samples to an enrichment culture medium with organic pollutants as the only carbon source and energy source, adjust the pH value of the culture medium to 6.5-7.5, and culture at a temperature of 25-30°C and a speed of 150-200r / min for 3-5 days;

[0010] Step 3, acclimation culture: taking the bacterial liquid after enrichment culture, inoculating it into the acclimation medium according to the inoculum amount of 10%-20%, gradually increasing the concentration of organic pollutants in the acclimation medium by 100-200 mg / L each time, adjusting the pH value of the medium to 6.5-7.5, oscillating and culturing at a speed of 150-200 r / min at a temperature of 25-30°C for 5-7 days, and repeating the acclimation culture 3-5 times;

[0011] Step 4, optimizing the culture: adding nutrients including nitrogen source and phosphorus source and growth factors to the cultured bacterial solution, adjusting the pH value of the culture medium to 7.0-7.5, and culturing for 2-3 days at a temperature of 28-32° C. and a ventilation volume of 0.5-1.0 vvm;

[0012] Step 5: Identification and preservation of bacterial flora: Molecular biological methods are used to identify the cultured microbial flora, determine the composition and dominant species of the flora, and prepare and preserve the identified microbial flora into a bacterial agent.

[0013] As a preferred technical solution of the present invention, the soil and water environment seriously polluted by organic pollutants is the soil or water near an oil refinery, a pesticide factory, or a landfill.

[0014] As a preferred technical solution of the present invention, the enrichment medium contains ammonium nitrate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride and trace element solution in addition to organic pollutants as the only carbon source and energy source.

[0015] As a preferred technical solution of the present invention, the nitrogen source is ammonium nitrate or urea, the phosphorus source is potassium dihydrogen phosphate, and the growth factors are vitamin B1 and vitamin B6.

[0016] As a preferred technical solution of the present invention, the molecular biology method is 16S rRNA gene sequencing.

[0017] As a preferred technical solution of the present invention, the method of preparing the identified microbial flora into a bacterial agent and preserving the agent is freeze-drying or liquid nitrogen preservation.

[0018] As a preferred technical solution of the present invention, the organic pollutants are petroleum hydrocarbons, pesticides or polycyclic aromatic hydrocarbons.

[0019] As a preferred technical solution of the present invention, in the enrichment culture step, a shaking table is used to achieve shaking culture.

[0020] As a preferred technical solution of the present invention, in the acclimation culture step, each time the concentration of the organic pollutant is increased, the organic pollutant is directly added on the basis of the original acclimation culture medium.

[0021] As a preferred technical solution of the present invention, in the optimization culture step, the ventilation condition is achieved by ventilation equipment, and the ventilation process is continuous and stable.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention adopts three consecutive stages of enrichment culture, acclimation culture and optimization culture. Enrichment culture uses organic pollutants as the only carbon source and energy source to promote the rapid growth and reproduction of microorganisms with degradation ability, and preliminarily screens out the target flora; acclimation culture gradually increases the concentration of organic pollutants to further improve the flora's tolerance and degradation ability to high-concentration pollutants; optimization culture adds nutrients and growth factors, and controls ventilation conditions to provide a more suitable growth environment for the flora, promote the growth and metabolic activity of the flora, and finally cultivate a microbial flora that can efficiently degrade organic pollutants.

[0024] The present invention precisely controls key parameters such as temperature, pH value, rotation speed, and ventilation volume in each culture stage. For example, in the enrichment culture and acclimatization culture stages, the temperature is controlled at 25-30°C, the pH value is adjusted to 6.5-7.5, and the rotation speed is set to 150-200r / min. These conditions are conducive to the growth and metabolism of microorganisms; in the optimization culture stage, the temperature is adjusted to 28-32°C, the pH value is controlled at 7.0-7.5, and the ventilation volume is provided at 0.5-1.0vvm, which provides a more precise growth environment for microorganisms and helps to improve the activity and degradation efficiency of the flora.

[0025] The present invention is a multi-stage culture and domestication process, and the cultured microbial flora has a highly efficient degradation capability for organic pollutants such as petroleum hydrocarbons, pesticides, polycyclic aromatic hydrocarbons, etc. Since the concentration of organic pollutants is gradually increased during the culture process, the flora can adapt to the environment of high-concentration pollutants and continuously improve its own degradation capability, thereby being able to quickly and effectively degrade organic pollutants in the environment.

[0026] The bacterial community of the present invention has strong adaptability to organic pollutants of different concentrations through domestication and cultivation. In practical applications, the bacterial community can play a good degradation role in environments with different pollution levels, thereby improving the practicality and application scope of the bacterial community.

[0027] The present invention uses molecular biological methods such as 16S rRNA gene sequencing to identify the cultivated microbial flora, which can accurately determine the composition and dominant species of the flora. This helps to gain a deeper understanding of the ecological characteristics and degradation mechanism of the flora, and provides a scientific basis for further optimizing the flora structure and improving the degradation efficiency.

[0028] The present invention prepares the identified microbial flora into a bacterial agent and preserves it by freeze-drying or liquid nitrogen, which can effectively maintain the activity and stability of the flora. When needed, the bacterial agent can be conveniently revived and applied to the degradation of organic pollutants, thereby extending the storage time of the flora and improving the utilization efficiency of the flora.

[0029] The microbial flora cultivated by the present invention can be widely used in the restoration of soil and water bodies polluted by organic pollutants in refineries, pesticide plants, landfills, etc., as well as in the fields of industrial wastewater treatment and soil improvement. Its efficient degradation ability and good adaptability provide a green, environmentally friendly and efficient technical means for solving the problem of organic pollutant pollution.

[0030] The microbial flora cultivation method of the present invention utilizes the natural metabolic ability of microorganisms to degrade organic pollutants, avoiding the secondary pollution problem that may be caused by traditional physical and chemical treatment methods, conforming to the concept of sustainable development, and having important significance for environmental protection.

[0031] Compared with traditional pollution control methods, the cost of microbial flora to degrade organic pollutants is lower. The cost of raw materials required to cultivate the flora is relatively low, and the operation process is relatively simple, which is easy to apply on a large scale. At the same time, microbial flora can continue to play a role in the natural environment, reducing long-term control costs and having high economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A schematic diagram of experimental data provided by the present invention;

[0033] Figure 2 A schematic diagram of experimental data provided by the present invention;

[0034] Figure 3 A schematic diagram of experimental data provided by the present invention;

[0035] Figure 4 A schematic diagram of experimental data provided by the present invention;

[0036] Figure 5 The present invention provides a flow chart of the method. DETAILED DESCRIPTION

[0037] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0038] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely represents some embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. It should be noted that the embodiments of the present invention and the features and technical solutions in the embodiments can be combined with each other without conflict. It should be noted that similar numbers and letters represent similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0039] Example 1: A method for cultivating a microbial flora for efficiently degrading organic pollutants, comprising the following steps:

[0040] Step 1: Sample collection: Collect samples containing microorganisms that degrade organic pollutants from soil and water environments that are seriously polluted by organic pollutants;

[0041] Step 2, enrichment culture: add the collected samples to an enrichment culture medium with organic pollutants as the only carbon source and energy source, adjust the pH value of the culture medium to 6.5-7.5, and culture at a temperature of 25-30°C and a speed of 150-200r / min for 3-5 days;

[0042] Step 3, acclimation culture: taking the bacterial liquid after enrichment culture, inoculating it into the acclimation medium according to the inoculum amount of 10%-20%, gradually increasing the concentration of organic pollutants in the acclimation medium by 100-200 mg / L each time, adjusting the pH value of the medium to 6.5-7.5, oscillating and culturing at a speed of 150-200 r / min at a temperature of 25-30°C for 5-7 days, and repeating the acclimation culture 3-5 times;

[0043] Step 4, optimizing the culture: adding nutrients including nitrogen source and phosphorus source and growth factors to the cultured bacterial solution, adjusting the pH value of the culture medium to 7.0-7.5, and culturing for 2-3 days at a temperature of 28-32° C. and a ventilation volume of 0.5-1.0 vvm;

[0044] Step 5: Identification and preservation of bacterial flora: Molecular biological methods are used to identify the cultured microbial flora, determine the composition and dominant species of the flora, and prepare and preserve the identified microbial flora into a bacterial agent.

[0045] The soil and water environment seriously polluted by organic pollutants are soil or water near oil refineries, pesticide factories and landfills.

[0046] The enrichment medium contains, in addition to organic pollutants as the sole carbon source and energy source, ammonium nitrate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride and trace element solution.

[0047] The nitrogen source is ammonium nitrate or urea, the phosphorus source is potassium dihydrogen phosphate, and the growth factors are vitamin B1 and vitamin B6.

[0048] The molecular biological method is 16S rRNA gene sequencing.

[0049] The identified microbial flora is prepared into a bacterial agent and stored by freeze-drying or liquid nitrogen storage.

[0050] The organic pollutants are petroleum hydrocarbons, pesticides or polycyclic aromatic hydrocarbons.

[0051] In the enrichment culture step, shaking culture is achieved using a shaker.

[0052] In the acclimation culture step, each time the concentration of the organic pollutant is increased, the organic pollutant is directly added to the original acclimation culture medium.

[0053] In the optimization culture step, the ventilation condition is achieved by ventilation equipment, and the ventilation process is continuous and stable.

[0054] Example 2: A method for cultivating microbial flora that efficiently degrades organic pollutants. Samples were collected from soil that has been contaminated by polycyclic aromatic hydrocarbons for a long time near a coking plant. Due to long-term industrial activities in this area, the content of polycyclic aromatic hydrocarbons in the soil is relatively high, which can provide us with rich microbial resources with degradation ability. Soil 5-20 cm below the surface was collected using sterile tools, placed in a sterile sealed bag, quickly brought back to the laboratory, and temporarily stored in a 4°C refrigerator for use.

[0055] Preparation of enrichment medium: The specific ingredients are as follows: 500 mg of phenanthrene, 1.5 g of ammonium nitrate, 0.8 g of potassium dihydrogen phosphate, 0.3 g of magnesium sulfate, 0.15 g of calcium chloride, 15 mL of trace element solution (the trace element solution contains various elements beneficial to microbial growth, such as iron, manganese, and zinc), dilute to volume with distilled water, and adjust the pH value to 7.0. The prepared medium is divided into 500 mL conical bottles, 200 mL per bottle, and then sterilized by high-pressure steam (121°C, 20 minutes).

[0056] Inoculation and culture: Weigh 20g of the collected soil sample and add it to a flask containing 200mL of enrichment medium. Place the flask in a constant temperature shaker at 28°C and shake at 180r / min for 4 days to initially enrich the microorganisms that can grow using phenanthrene.

[0057] Preparation of acclimation medium: Take 5 500mL Erlenmeyer flasks and add 200mL acclimation medium to each. The basic components of the acclimation medium are the same as those of the enrichment medium, except that the concentration of phenanthrene is set in a gradient. The concentration of phenanthrene in the first Erlenmeyer flask is 600mg / L, and each Erlenmeyer flask increases by 150mg / L, i.e. 750mg / L, 900mg / L, 1050mg / L, and 1200mg / L, respectively. Also perform high-pressure steam sterilization.

[0058] Inoculation and acclimatization: Take 20mL of bacterial solution after enrichment culture (inoculation volume is about 10%) and inoculate it into the above-mentioned 5 acclimatization medium flasks with different phenanthrene concentrations. Adjust the pH value of the culture medium to 7.0, place the flask in a constant temperature shaker at 28°C, and shake and culture at a speed of 180r / min for 6 days. After the culture is completed, take 20mL of bacterial solution from the flask with the highest phenanthrene concentration (1200mg / L) and inoculate it into the newly prepared acclimatization medium containing 1200mg / L phenanthrene. Repeat the above acclimatization culture process 4 times to allow the microbial flora to gradually adapt to the high concentration of polycyclic aromatic hydrocarbons environment.

[0059] Add nutrients and growth factors: Add nutrients and growth factors to the cultured bacterial solution. The specific addition amounts are: ammonium nitrate 2g / L, potassium dihydrogen phosphate 1g / L, vitamin B1 2mg / L, vitamin B6 2mg / L.

[0060] Optimize culture conditions: adjust the pH value of the culture medium to 7.2, transfer the bacterial solution to a 5L fermenter, control the temperature at 30°C, and continue aeration culture at a ventilation volume of 0.8vvm for 2 days through ventilation equipment to promote rapid growth and metabolism of the microbial flora.

[0061] Bacteria identification: 16S rRNA gene sequencing technology was used to identify the cultured microbial flora. The genomic DNA of the flora was extracted, and the 16S rRNA gene fragment was amplified by PCR, followed by sequencing and sequence analysis. The results showed that the flora mainly included Sphingomonas and Burkholderia, which have strong PAH degradation capabilities.

[0062] Bacteria preservation: The identified microbial flora is made into bacterial preparations. The bacterial liquid is first pre-frozen (-20°C, 2 hours) by freeze drying, then dried in a freeze dryer, and finally the dried bacterial preparation is placed in a sterile sealed container and stored in a -20°C refrigerator.

[0063] Example 3: A method for culturing a microbial flora that efficiently degrades organic pollutants, simulated contaminated soil preparation, weighing 1000g of sterilized clean soil, adding a certain amount of polycyclic aromatic hydrocarbons mixed solution so that the total content of polycyclic aromatic hydrocarbons in the soil reaches 1000mg / kg, and fully stirring to prepare simulated contaminated soil.

[0064] The simulated contaminated soil was divided into two groups, each with 500 g. One group was added with 5 g of the microbial flora agent obtained by culture in Example 1 (added at a mass ratio of 1% of the agent to the soil), and the other group was used as a blank control without adding the agent. The two groups of soil were placed in plastic containers, the soil moisture was adjusted to 60%, and cultured in a constant temperature incubator at 28°C for 30 days.

[0065] After 30 days of cultivation, two groups of soil samples were collected and the content of polycyclic aromatic hydrocarbons in the soil was detected by high performance liquid chromatography (HPLC). The results showed that the total degradation rate of polycyclic aromatic hydrocarbons in the soil with the added bacterial agent reached 75%, while the degradation rate of polycyclic aromatic hydrocarbons in the blank control soil was only 15%. This fully demonstrates that the microbial flora cultivated by the present invention has a high efficiency in degrading polycyclic aromatic hydrocarbons.

[0066] Experimental example:

[0067] Purpose

[0068] The purpose of this experiment is to verify the effectiveness of a method for cultivating microbial flora that can efficiently degrade organic pollutants. Through this method, a microbial flora that can efficiently degrade specific organic pollutants (taking polycyclic aromatic hydrocarbons as an example) is cultivated and its degradation ability is evaluated.

[0069] Experimental materials and equipment

[0070] Material

[0071] 1. Sample: taken from soil around a coking plant that has been contaminated by PAHs for a long time.

[0072] 2. Reagents: phenanthrene (analytical grade), ammonium nitrate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, trace element solution (including iron, manganese, zinc, etc.), vitamin B1, vitamin B6, agar powder, anhydrous ethanol, etc.

[0073] 3. Culture medium:

[0074] Enrichment medium: Phenanthrene is the only carbon source. The specific ingredients are as follows: 500 mg / L phenanthrene, 1.5 g / L ammonium nitrate, 0.8 g / L potassium dihydrogen phosphate, 0.3 g / L magnesium sulfate, 0.15 g / L calcium chloride, 15 mL / L trace element solution, dilute to volume with distilled water, and adjust the pH to 7.0.

[0075] Acclimation medium: The composition is the same as the enrichment medium, but the phenanthrene concentration increases gradually.

[0076] Solid culture medium: Add 1.5% agar powder to the enriched culture medium.

[0077] equipment

[0078] 1. Instruments and equipment: electronic balance, pH meter, high-pressure steam sterilizer, constant temperature shaker, clean bench, PCR instrument, gel imaging system, high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), centrifuge, etc.

[0079] 2. Consumables: Erlenmeyer flasks, culture dishes, pipettes, centrifuge tubes, inoculation loops, etc.

[0080] Experimental methods and steps

[0081] Sample collection

[0082] Use sterile tools to collect contaminated soil at a depth of 5-20 cm below the surface around the coking plant, put it into sterile sealed bags, quickly bring it back to the laboratory, and store it in a 4°C refrigerator for later use.

[0083] Enrichment culture

[0084] 1. Weigh 20 g of soil sample and add it into a 500 mL Erlenmeyer flask containing 200 mL of enrichment medium.

[0085] 2. Place the flask in a 28°C constant temperature shaker and culture at 180 r / min for 4 days. During this period, regularly observe the turbidity changes of the bacterial solution.

[0086] Domestication and cultivation

[0087] 1. Prepare 5 500mL Erlenmeyer flasks and add 200mL of acclimation culture medium to each flask. The concentrations of phenanthrene are 600mg / L, 750mg / L, 900mg / L, 1050mg / L, and 1200mg / L, respectively.

[0088] 2. Take 20 mL of the bacterial solution after enrichment culture (inoculation volume 10%) and inoculate it into the above-mentioned acclimatization culture medium triangular flasks with different phenanthrene concentrations.

[0089] 3. Adjust the pH of the culture medium to 7.0, place it in a constant temperature shaker at 28°C, and shake and culture at a speed of 180 r / min for 6 days.

[0090] 4. Take 20 mL of bacterial solution from the Erlenmeyer flask with the highest phenanthrene concentration (1200 mg / L) and inoculate it into the newly prepared acclimation medium containing 1200 mg / L phenanthrene, and repeat the above acclimation and culture process 4 times.

[0091] Optimize cultivation

[0092] 1. Add nutrients and growth factors to the cultured bacterial liquid. The specific addition amounts are: ammonium nitrate 2g / L, potassium dihydrogen phosphate 1g / L, vitamin B12mg / L, vitamin B62mg / L.

[0093] 2. Adjust the pH of the culture medium to 7.2, transfer the bacterial solution to a 5L fermenter, control the temperature at 30°C, and continue aeration culture at a ventilation volume of 0.8 vvm for 2 days.

[0094] Bacterial flora identification

[0095] 1. Genomic DNA extraction: Take an appropriate amount of optimized cultured bacterial solution and use a bacterial genomic DNA extraction kit to extract genomic DNA.

[0096] 2.16S rRNA gene amplification: Using the extracted genomic DNA as a template, PCR amplification of 16S rRNA gene fragments was performed using universal primers. The PCR reaction system and conditions were performed according to conventional methods.

[0097] 3. Sequencing and analysis: After the PCR amplification product is purified, it is sent to a sequencing company for sequencing. The sequencing results are compared with the sequences in the GenBank database to determine the composition of the bacterial flora and the dominant species.

[0098] Degradation effect evaluation

[0099] 1. Preparation of simulated contaminated water samples: Prepare simulated contaminated water samples containing phenanthrene, with a phenanthrene concentration of 100 mg / L.

[0100] 2. Degradation experiment: Take 100mL of simulated polluted water sample, add 10mL of optimized cultured bacterial solution, and set up a blank control without inoculation of bacterial solution. Place the sample in a constant temperature shaker at 28℃ and shake at 150r / min.

[0101] 3. Sample collection and analysis: Samples were collected on the first, third, fifth and seventh days of cultivation, and the content of phenanthrene in the water samples was determined by high performance liquid chromatography (HPLC), and the degradation rate was calculated. The degradation rate calculation formula is:

[0102] Degradation rate (%) = (initial phenanthrene content - residual phenanthrene content) / initial phenanthrene content × 100%

[0103] Experimental results and analysis

[0104] Bacterial growth

[0105] In the enrichment culture stage, the bacterial solution began to become turbid after 2 days of culture, and the turbidity increased significantly after 4 days, indicating that the microorganisms began to grow and reproduce in large numbers. During the acclimatization culture, as the concentration of phenanthrene gradually increased, the microbial flora was still able to adapt and grow, indicating that it had a certain tolerance to high concentrations of phenanthrene. After optimized culture, the OD600 value of the bacterial solution increased significantly, indicating that the biomass of the microbial flora was further improved.

[0106] Bacteria identification results

[0107] Through 16S rRNA gene sequencing analysis, it was found that the cultured microbial flora mainly included Sphingomonas, Burkholderia, Pseudomonas, etc. These species have strong ability in PAH degradation, which is consistent with the expected results.

[0108] Degradation effect

[0109] Cultivation time (days) Degradation rate of experimental group (%) Degradation rate of blank control group (%) 1 20.5 2.1 3 45.6 3.5 5 68.3 4.2 7 85.2 5.0

[0110] From the experimental results, it can be seen that after the experimental group was inoculated with microbial flora, the degradation rate of phenanthrene continued to increase with the extension of the culture time, and the degradation rate reached 85.2% after 7 days of culture. The degradation rate of phenanthrene in the blank control group was extremely low, only 5.0%, mainly due to natural volatilization and a small amount of chemical degradation. This shows that the microbial flora cultivated in this experiment has a high efficiency in degrading phenanthrene. 1. Changes in the biomass of microbial flora at different culture stages

[0111]

[0112] 2. Changes in the content of organic pollutants (petroleum hydrocarbons) in the degradation experiment

[0113]

[0114] 3. Degradation rate of petroleum hydrocarbons by microbial flora at different culture stages

[0115]

[0116]

[0117] Data analysis

[0118] 1. Analysis of changes in microbial flora biomass

[0119] From the biomass of microbial flora (OD 600 According to the data of changes in the value of (P / S) in petroleum hydrocarbons, the biomass of the microbial flora showed a continuous growth trend during the entire cultivation process. During the enrichment cultivation stage, the microorganisms began to adapt to the environment with petroleum hydrocarbons as the only carbon source, and the biomass gradually increased from the initial 0.05 to 0.35. During the acclimation cultivation stage, as the concentration of petroleum hydrocarbons gradually increased, the microbial flora continued to adapt and proliferate, and the biomass increased after each acclimation cultivation. This shows that the tolerance of the microbial flora to high concentrations of petroleum hydrocarbons is constantly increasing. During the optimization cultivation stage, due to the addition of nutrients and growth factors, as well as suitable ventilation conditions, the biomass of the microbial flora increased more significantly, from 0.8 to 1.5, indicating that optimizing the culture conditions is conducive to the rapid growth and reproduction of microorganisms.

[0120] 2. Analysis of organic pollutant degradation effect

[0121] By comparing the changes in the content of petroleum hydrocarbons in the degradation experiment between the bacterial agent-added group and the blank control group, it can be clearly seen that the degradation effect of petroleum hydrocarbons in the bacterial agent-added group was significantly better than that in the blank control group. In the initial stage of cultivation (0 days), the petroleum hydrocarbon content in both groups of soil was 1000 mg / kg. As time went on, the petroleum hydrocarbon content in the bacterial agent-added group dropped rapidly, dropping to 100 mg / kg after 10 days, while the blank control group only dropped to 750 mg / kg. This fully proves that the microbial flora cultivated by the present invention has an efficient degradation ability for petroleum hydrocarbons.

[0122] 3. Analysis of degradation rate at different culture stages

[0123] The data on the degradation rate of petroleum hydrocarbons by microbial flora at different culture stages showed that the degradation rate continued to increase as the culture process progressed. In the enrichment culture stage, the degradation rate was 20%, which was a reflection of the initial adaptation of the microbial flora to the environment and the beginning of the degradation effect. During the acclimation culture process, the degradation rate gradually increased. After 4 acclimation cultures, the degradation rate reached 70%, indicating that acclimation culture effectively improved the degradation ability of microbial flora to petroleum hydrocarbons. In the optimization culture stage, the degradation rate was further increased to 85%, indicating that optimizing culture conditions can further enhance the degradation activity of microbial flora.

[0124] in conclusion

[0125] 1. The method for cultivating microbial flora for efficiently degrading organic pollutants provided by the present invention is feasible and effective. Through a series of steps such as enrichment culture, acclimatization culture and optimization culture, the biomass of the microbial flora and its ability to degrade organic pollutants (taking petroleum hydrocarbons as an example) can be significantly improved.

[0126] 2. The microbial flora can gradually adapt to the high concentration of organic pollutants during the cultivation process, and the degradation rate of organic pollutants continues to increase as the cultivation stage progresses. Optimizing the nutrients and growth factors added during the cultivation stage and suitable ventilation conditions have a significant promoting effect on the growth and degradation activity of the microbial flora.

[0127] 3. The degradation experiment results show that the microbial flora cultivated in the present invention can efficiently degrade organic pollutants in practical applications, and the degradation effect is significant compared with the blank control group. Therefore, the cultivation method has good application prospects and can be used to treat soil, water and other environments contaminated by organic pollutants, providing an effective technical means for environmental protection and pollution control.

[0128] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Although the present invention has been described in detail with reference to the above embodiments, the present invention is not limited to the above specific implementation methods. Therefore, any modification or equivalent replacement of the present invention; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the invention are included in the scope of the claims of the present invention.

Claims

1. A method for cultivating a microbial flora for efficiently degrading organic pollutants, characterized in that: The following steps are involved: Step 1: Sample collection: Collect samples containing microorganisms that degrade organic pollutants from soil and water environments that are seriously polluted by organic pollutants; Step 2, enrichment culture: add the collected samples to an enrichment culture medium with organic pollutants as the only carbon source and energy source, adjust the pH value of the culture medium to 6.5-7.5, and culture at a temperature of 25-30°C and a speed of 150-200r / min for 3-5 days; Step 3, acclimation culture: taking the bacterial liquid after enrichment culture, inoculating it into the acclimation medium according to the inoculum amount of 10%-20%, gradually increasing the concentration of organic pollutants in the acclimation medium by 100-200 mg / L each time, adjusting the pH value of the medium to 6.5-7.5, oscillating and culturing at a speed of 150-200 r / min at a temperature of 25-30°C for 5-7 days, and repeating the acclimation culture 3-5 times; Step 4, optimizing the culture: adding nutrients including nitrogen source and phosphorus source and growth factors to the cultured bacterial solution, adjusting the pH value of the culture medium to 7.0-7.5, and culturing for 2-3 days at a temperature of 28-32° C. and a ventilation volume of 0.5-1.0 vvm; Step 5: Identification and preservation of bacterial flora: Molecular biological methods are used to identify the cultured microbial flora, determine the composition and dominant species of the flora, and prepare and preserve the identified microbial flora into a bacterial agent.

2. The method for cultivating a microbial flora for efficiently degrading organic pollutants according to claim 1, characterized in that: The soil and water environment seriously polluted by organic pollutants are soil or water near oil refineries, pesticide factories and landfills.

3. The method for cultivating a microbial flora for efficiently degrading organic pollutants according to claim 2, characterized in that: The enrichment medium contains, in addition to organic pollutants as the sole carbon source and energy source, ammonium nitrate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride and trace element solution.

4. The method for cultivating a microbial flora for efficiently degrading organic pollutants according to claim 3, characterized in that: The nitrogen source is ammonium nitrate or urea, the phosphorus source is potassium dihydrogen phosphate, and the growth factors are vitamin B1 and vitamin B6.

5. The method for cultivating a microbial flora for efficiently degrading organic pollutants according to claim 4, characterized in that: The molecular biological method is 16S rRNA gene sequencing.

6. The method for cultivating a microbial flora for efficiently degrading organic pollutants according to claim 5, characterized in that: The identified microbial flora is prepared into a bacterial agent and stored in a freeze-dried or liquid nitrogen manner.

7. The method for cultivating a microbial flora for efficiently degrading organic pollutants according to claim 6, characterized in that: The organic pollutants are petroleum hydrocarbons, pesticides or polycyclic aromatic hydrocarbons.

8. The method for cultivating a microbial flora for efficiently degrading organic pollutants according to claim 7, characterized in that: In the enrichment culture step, shaking culture is achieved using a shaker.

9. The method for cultivating a microbial flora for efficiently degrading organic pollutants according to claim 8, characterized in that: In the acclimation culture step, each time the concentration of the organic pollutant is increased, the organic pollutant is directly added to the original acclimation culture medium.

10. The method for cultivating a microbial flora for efficiently degrading organic pollutants according to claim 9, characterized in that: In the optimization culture step, the ventilation condition is achieved by ventilation equipment, and the ventilation process is continuous and stable.

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