Hydiitzia sp. c2 and application thereof

Abstract

This invention relates to a strain of Hedizella ( Dietzia maris The strain C2, with its strain preservation number CCTCC NO: M2025275, is also described in this invention. This invention relates to the application of the above-mentioned strain in the biological treatment of organic pollution from petroleum and petrochemical industries. This strain, isolated from core samples from the Daqing Oilfield, can simultaneously secrete denitrification reductase and monooxygenase under aerobic conditions, allowing for simultaneous oxidation and reduction. AlkB hydroxylase is responsible for the degradation of long-chain alkanes (C5-C16), P450 monooxygenase is responsible for the oxidative degradation of benzene compounds and low-cyclic aromatic hydrocarbons, and denitrification reductase is responsible for the dechlorination of low-chlorinated organic compounds. It can significantly improve the degradation rate of recalcitrant components in petroleum, laying the foundation for the treatment of oily sludge, petroleum-contaminated soil, petroleum wastewater, and petrochemical wastewater, and providing a new avenue for the green and low-carbon remediation of petroleum and petrochemical wastewater and petroleum-contaminated soil in my country.

Description

Technical Field

[0001] This invention belongs to the field of environmental remediation and relates to a strain of Hedizella C2 and its applications. Background Technology

[0002] Since the advent of the Industrial Revolution, petroleum has become the lifeblood of industry, deeply permeating every sector. The enormous demand for petroleum and its derivatives has led to large quantities of petroleum and its processed products entering soil and water bodies, causing environmental pollution. Among these, petroleum components such as benzene compounds, heterocyclic compounds, and polycyclic aromatic hydrocarbons possess carcinogenic, teratogenic, and mutagenic properties, seriously threatening food safety, drinking water safety, and human health. Severe pollution can even lead to ecosystem imbalance and biodiversity crises. Therefore, solving the problem of petroleum pollution has always been a focus of attention for the petroleum and petrochemical industries, and it is also the greatest challenge for countries in building a green, low-carbon, and sustainable economic and social development system.

[0003] The nation urgently needs green, safe, practical, and efficient technologies for the remediation of petroleum pollution. In particular, the in-situ remediation of construction site soil and groundwater presents a significant challenge for the entire industry. Currently, the environmental protection industry primarily employs chemical oxidation and thermal desorption / desorption technologies to remediate petroleum-contaminated soil. Chemical oxidation technology uses alkali-activated sodium persulfate or advanced chemical reactions such as Fenton / Fenton-like reactions to generate free radicals that destroy the molecules of organic pollutants, achieving a maximum degradation rate of 30-40% for crude oil. This method is effective in oxidizing low-molecular-weight hydrocarbons and is stable with moderate operational risks. However, a drawback is that carbonate, phosphate, and nitrate ions in the soil can interfere with and annihilate hydroxyl and persulfate free radicals, making complete removal of petroleum hydrocarbons difficult. Thermal desorption / desorption technology heats the soil to 320℃-560℃, causing hydrocarbons to evaporate and separating organic pollutants through negative pressure gas-phase extraction. Its advantages include good and stable treatment results. The main drawback is its high cost, with unit operating costs ranging from 600 to 1100 yuan / ton, along with high equipment investment and depreciation costs. Furthermore, high temperatures kill soil microorganisms and enzymes, destroy humus and organic matter, leading to decreased soil fertility and severely damaging soil structure, rendering it unusable for cultivation. In addition, the released gases are complex in composition and have low economic value, requiring incineration to treat them, thus causing air pollution.

[0004] Petroleum hydrocarbons have diverse compositions, and their combustion releases large amounts of uncontrollable carcinogens, such as dioxins, exacerbating smog and secondary pollution. Compared to physical and chemical technologies, microbial technology shows broad application prospects and significant advantages in petroleum pollution remediation. Microorganisms, due to their strong vitality, diverse metabolic pathways, large specific surface area, and rapid reproduction capacity, are playing an increasingly important role in global environmental remediation. In nature, petroleum ultimately degrades through microorganisms, eventually transforming into carbon dioxide and water—a process resulting from millions of years of natural evolution. Therefore, using microbial technology to remediate petroleum pollution is the most scientifically sound choice. Microbial remediation technology has been recognized by the international academic community as an effective method for green treatment of petroleum-contaminated soil, becoming one of the most promising technologies for soil remediation. With its advantages of high efficiency, low cost, safety, and no secondary pollution, bio-environmental protection technology is booming. However, due to the diverse soil types, complex structures, large climate differences, and the presence of a large amount of biomass in my country, microbial remediation technology faces technical bottlenecks such as long remediation cycles, low degradation efficiency, and poor tolerance to highly toxic substances. Therefore, developing efficient petroleum treatment microbial agents to address the above-mentioned pain points and improve biodegradation efficiency is key to solving organic pollution from petroleum and petrochemicals.

[0005] For C-type compounds such as pterane (Pr) and phytane (Ph) in petroleum 32 -C 44 Traditional biotechnologies have not been very effective in degrading large molecular weight n-alkanes, heterocyclic components such as quinolines, indoles, and pyrazoles, and recalcitrant components such as halogenated hydrocarbons, especially polycyclic aromatic hydrocarbons such as benzene, naphthalene, anthracene, phenanthrene, and fluorene. Therefore, it is particularly urgent to develop functional environmental microorganisms by utilizing my country's abundant microbial resources. Summary of the Invention

[0006] The primary objective of this invention is to provide a strain of Hedizs bacterium C2 to improve biodegradation efficiency and overcome the problems of long remediation cycles, low degradation efficiency, and poor tolerance to highly toxic substances in microbial remediation technology.

[0007] A second objective of this invention is to provide the application of the aforementioned Hedizella C2.

[0008] This invention is achieved through the following technical solution:

[0009] I. A strain of Hedizil ( Dietzia maris C2, whose strain preservation number is CCTCC NO: M 2025275.

[0010] II. The above-mentioned Hedizella Dietzia maris Application of C2 in biological treatment of organic pollution in petroleum and petrochemical industries.

[0011] Furthermore, the treatment of petroleum and petrochemical organic pollution involves the efficient degradation of petroleum substances under conditions of compensated tryptophan or compound symbiotic synergistic bacteria.

[0012] Furthermore, the highly efficient degradation of petroleum-based substances is petroleum-based C5-C. 44 Components.

[0013] Furthermore, the aforementioned petroleum and petrochemical organic pollution includes the following scenarios:

[0014] (1) Industrial wastewater: oil extraction water, fracturing flowback fluid, tank washing water, petrochemical wastewater, coking wastewater, printing and dyeing wastewater, papermaking wastewater, and oil-containing metallurgical wastewater;

[0015] (2) Surface water: Surface water bodies polluted by petroleum or petrochemical industries, sewage ponds, seepage pits, and black and odorous water bodies;

[0016] (3) Oil-contaminated soil: Oil-contaminated farmland, grassland, forest land, mines, coal mines and oil mining areas, especially soil near oil wells;

[0017] (4) Hazardous waste: oily hazardous waste, drilling mud, oily sludge and other solid waste;

[0018] (5) Industrial contaminated sites: sites of petroleum and petrochemical contaminated factories and gas stations.

[0019] The positive effects of adopting the above technical solution are as follows: This invention is a strain isolated from the core of Daqing Oilfield, which can simultaneously secrete denitrification reductase and monooxygenase under aerobic conditions, with oxidation and reduction occurring simultaneously. AlkB hydroxylase is responsible for the C5-C of long-chain alkanes. 16 Degradation: P450 monooxygenase is responsible for the oxidative degradation of benzene series compounds and low-cyclic aromatic hydrocarbons. It can also synergistically degrade polycyclic aromatic hydrocarbons and ultra-long-chain alkanes in low-oxygen or complex hydrocarbon environments, reaching up to C450. 44 It can significantly improve the degradation rate of recalcitrant components in petroleum, laying the foundation for the treatment of oily sludge, petroleum-contaminated soil, petroleum wastewater, and petrochemical wastewater, and providing a new track for the green and low-carbon remediation of petroleum and petrochemical wastewater and petroleum-contaminated soil in my country. Attached Figure Description

[0020] Figure 1 It is Hedizella ( Dietzia maris C2 developmental tree;

[0021] Figure 2 It is Hedizella with added tryptophan ( Dietzia maris C2 pure culture colonies;

[0022] Figure 3 Hedizella is a bacterium that uses benzo[a]anthracene as its sole carbon source. Dietzia maris C2 streaked colonies;

[0023] Figure 4 Hedizella species that use benzo(b)fluoranthene as their sole carbon source Dietzia maris C2 streaked colonies;

[0024] Figure 5 Hedizella is a bacterium that uses benzo(a)pyrene as its sole carbon source. Dietzia maris C2 streaked colonies;

[0025] Figure 6 Hedizella species that use dibenzo(a,h)anthracene as their sole carbon source ( Dietzia maris C2 streaked colonies;

[0026] Figure 7 Hedizella is a bacterium that uses quinoline as its sole carbon source. Dietzia maris C2 streaked colony.

[0027] The Hedizella bacteria involved in this invention ( Dietzia maris C2 was deposited on February 21, 2025, in a depository recognized by the Chinese Patent Office or an international patent organization. The depository is the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China. The accession number is CCTCC NO: M2025275. Detailed Implementation

[0028] The technical solution of the present invention will be further described below with reference to specific embodiments, but it should not be construed as a limitation of the present invention:

[0029] Example 1

[0030] This example illustrates Hedizella ( Dietzia maris Acquisition of C2.

[0031] Using core samples from Daqing Oilfield as experimental material and fermentation medium as acclimatization medium, the culture was carried out at 30℃ and 140 r / min for 5 days. Then, 1 mL of fermentation broth was transferred to fresh medium for continued fermentation, repeated 5 times. Aseptic operation was performed using the extreme dilution method to obtain a single orange-colored, highly efficient petroleum-degrading bacterium. This strain could only be cultured symbiotically. After a preliminary experiment with the addition of 0.2 g / L of a complex amino acid (containing 18 amino acids), it could be cultured independently, confirming it as an amino acid-deficient strain. The predicted C2 16S rRNA sequence was compared with the NCBI 16S database using Blast+ with the parameter `identify>95`. The top 19 16S rRNA sequences with the highest `identify` were selected, and multiple sequence alignment and splicing were performed using MEGA11 software. A phylogenetic tree was constructed using the neighbor-joining (NJ) method, identifying the bacterium as *Hydizella*. Dietzia Maris Named C2, construct a phylogenetic tree as follows: Figure 1 The following culture media were used as the basal medium, with 0.2 g / L of different commercially available amino acid standards added to prepare amino acid selection plates. The results showed that the strain was effectively proliferated on the culture medium plates supplemented with tryptophan. The experimental results are as follows: Figure 2 This indicates that Hedizella ( Dietzia maris C2 is a tryptophan-deficient strain, possibly due to the loss of the tryptophan synthesis gene.

[0032] Basic culture medium: KNO3 10.0 g / L; petroleum 4 g / L; MgSO4·7H2O 2.5 g / L; FeCl3·6H2O 0.125 g / L; KH2PO4 2.5 g / L; CaCl2·2H2O 0.5 g / L; 1% BTB ethanol solution 0.5 mL; trace elements 2 mL; water 1000 mL, adjusted to pH 7.5.

[0033] Trace element solution composition: EDTA 50.0 g / L; ZnSO4 2.2 g / L; CaCl2 5.5 g / L; MnCl2·4H2O 5.06 g / L; FeSO4·7H2O 5.0 g / L; (NH4)6Mo7O2·4H2O 1.1 g / L; CuSO4·5H2O 1.57 g / L; CoCl2·6H2O 1.61 g / L; water 1000 mL

[0034] Example 2

[0035] This example illustrates Hedizella ( Dietzia maris C2 degradation capacity.

[0036] Using benzo(a)pyrene, benzo(b) fluoranthene, dibenzo(a,h) anthracene, quinoline, indole, and carbazole pyridine as the sole carbon source to replace petroleum in the basal medium, the streak plate method was used to preliminarily verify the presence of Hedizella (…). Dietzia maris The C2 strain exhibited a broad-spectrum ability to degrade polycyclic aromatic hydrocarbons (PAHs). It was found that this strain could survive using benzo(a)pyrene, benzo(b) fluoranthene, dibenzo(a,h)anthene, and quinoline as its sole carbon source, but could not utilize indole, carbazole, or pyridine. This result suggests that its metabolic pathway may target specific PAH structures (such as pentacyclic or tetracyclic compounds), while the degradation of nitrogen-containing heterocyclic compounds (such as pyridine, indole, and carbazole) typically depends on tryptophan or carbazole dioxygenases. (Heidiszolium) Dietzia maris The C2 genome may not encode these enzymes. The results are as follows: Figure 3-Figure 7 .

[0037] Example 3

[0038] This embodiment illustrates the claims regarding Hedizella (…). Dietzia maris C2 is used to treat oily wastewater such as fracturing flowback fluid and oil extraction water. Flocculation can be omitted when treating oil extraction water.

[0039] Step 1: Flocculation. Adjust the pH of the fracturing flowback fluid to 7.5-8.5 with hydrochloric acid, add 200 mg / L of polyaluminum chloride, stir at 30 rpm for 1 min, and transport the mixed liquid to a parallel inclined plate sedimentation tank. After standing for 30 minutes, the supernatant is transported out sequentially from the upper corridor of different sedimentation tanks, and the lower layer of flocculents is pumped away through pipelines, dewatered by pressure filtration, and the water is returned to the sedimentation tank. The filter residue is treated separately.

[0040] Step 2: Air flotation. The water from the previous flocculation is transferred to the air flotation chamber and nano-air flotation is used for 15 minutes. The air flotation nozzle is set at the bottom layer to physically scrape away the foam.

[0041] Step 3: Ozone oxidation. Ozone oxidation is catalyzed by 254nm ultraviolet light, using multiple vertically arranged transparent glass spiral tube units connected in series. Water is aerated with ozone at a concentration ≥30mg / L. Ozone-rich water flows dynamically within the tubes. A 254nm ultraviolet light source is positioned at the center of the spiral, and the spiral height depends on the length of the ultraviolet lamp. The oxidation process lasts for 120 minutes before the ozone is removed (after complete decomposition). Multiple oxidation units ensure further breakdown of residual soluble polymers such as guar gum and polyacrylamide in the water.

[0042] Step 4: Biodegradation. The water is transferred to a submerged biological filter using immobilized Hedizella bacteria. Dietzia maris C2 +Microbacterium foliorum CRH Microspheres undergo deep biodegradation. First, adjust the pH to 7.5-8.5, then mechanically stir at 59 rpm for 1-3 minutes. Residual dissolved oil and organic matter such as guar gum in the water are then eliminated by Hedizella bacteria. Dietzia maris The C2 immobilization treatment unit adsorbs and completely degrades the wastewater. Biodegradation conditions: temperature 30~35℃, pH 7.5~8.0, ensuring C:N=8~12:1, three submerged biological filters are connected in series, with a hydraulic retention time of 6 hours per batch for each biological filter, and the treated water quality meets the national oilfield wastewater treatment standards.

[0043] Example 4

[0044] This embodiment illustrates the Hedizella strain of the claims (…). Dietzia maris C2 is used in the remediation of persistent non-point source polluted water bodies such as seepage pits or polluted rivers.

[0045] Seepage pits and industrially polluted rivers contain large amounts of persistent organic pollutants (mineral oil, benzene compounds, chlorinated hydrocarbons, polycyclic aromatic hydrocarbons, and pesticide residues), resulting in severe water pollution.

[0046] Step 1: Dredging. A cutter suction dredger is used for rapid dredging. The sludge is then dewatered and treated separately by filter pressing.

[0047] Step 2, oxidation treatment, utilizes a commercially available integrated nano-ozone generator for flotation oxidation pretreatment. Nanoscale bubbles have a large adsorption area (larger bubbles have a larger specific surface area) and a slow rising speed, ensuring sufficient reaction between ozone and suspended and dissolved organic matter in the water. Batch treatment time is 15-60 minutes, which can be flexibly adjusted according to the degree of water pollution. The flotation unit is fixed at the bottom (near the bottom layer) of the floating island's plastic frame. The floating island uses a hollow mesh design with a mesh depth of 0.5-1m and a mesh spacing of 1m. Foam and oil collectors are installed within the mesh, primarily for collecting suspended solids in the water.

[0048] Step 3: Biological treatment. After 12 hours of ozone treatment, the ozone is completely decomposed. Hedizella bacteria are then directly sprayed into the water. Dietzia maris C2 , Microbacterium foliorum CRH A 1:1 ratio of biological dry powder preparation was added to the freeze-dried bacterial powder preparation at a concentration of 0.1‰ of the polluted water mass. Water remediation was completed in 7-14 days. Water transparency increased significantly, persistent organic pollutants decreased to safe levels, and the natural ecological capacity of the water body was significantly restored.

[0049] Example 5

[0050] This embodiment illustrates the Hedizella strain of the claims (…). Dietzia maris C2 is a method for remediating petroleum and petrochemical organic contaminated soil.

[0051] Step 1: Land preparation. Add 0.01% nano iron powder to the oil-contaminated soil to be repaired and mix it thoroughly by rotary tillage.

[0052] Step 2: Add the treatment and remediation agent, adding 0.1% Hedizella bacteria (based on the mass percentage of the soil to be remediated). Dietzia maris C2 dry powder, Penicillium oxalicum Ba03 The strain preservation number is CCTCC M2025262. Apply 0.1‰ of the biological agent, mix thoroughly, and maintain soil moisture at 20-25%. Add agricultural fertilizer to ensure a carbon-nitrogen-phosphorus ratio of 100:6-8:1, with the carbon source calculated based on 65% of petroleum pollutants, and ammonium nitrate being the preferred nitrogen source.

[0053] Step 3: Cover with non-woven fabric curtains. A 2-3 cm layer of non-woven fabric curtain, filled with sawdust or straw powder and 0.1% Hedizella bacteria, is attached to the soil surface of each experimental plot. Dietzia maris C2 bacterial agent, humidity 25%.

[0054] Step 4: Control temperature and humidity, maintaining the optimal soil moisture at 25-40% and the temperature at 25-33℃. During in-situ remediation, cover with agricultural film and then a shade net. Turn the soil every 3-7 days to ensure sufficient contact between the microorganisms and pollutants. Bioremediation is complete in 30 days, at which point the degradation rate of petroleum hydrocarbons in the soil will be ≥90%, and the soil's ecological function will be restored. Trees and grass can be planted in the same year to restore green space.

[0055] Example 6

[0056] This embodiment illustrates the Hedizella strain of the claims (…). Dietzia maris C2 Repair of Oily Sludge HW08 Usage Instructions

[0057] Step 1, oil sludge fine sorting, uses high-temperature bacteria + glycolipid and lipopeptide compound biological surfactants to clean oily sludge, separating plastic products, clothing, gloves and other debris. High-pressure water gun is used to spray the above-mentioned high-temperature bacteria preparation to clean the debris. The muddy water enters the water tank below through the holes in the inner wall of the spiral drum, and is pumped into the hot washing unit by high pressure.

[0058] Step 2, crude oil recovery, uses high-temperature emulsifying bacteria at 50-60 degrees Celsius to break down the emulsion, achieving three-phase separation of oil, cement, and sludge. The crude oil is then physically scraped off, and the sludge enters the biodegradation unit.

[0059] Step 3, add Hedizella ( Dietzia maris C2, in combination with other degrading bacteria and non-specific peroxidases, deeply degrades recalcitrant petroleum components remaining in the soil. The oil sludge is adjusted to 32-35℃, with a liquid-to-solid ratio of 2:1 and a pH of 5.0-6.0. 0.01-1% of a non-specific petroleum heavy component degrading enzyme product (lignin peroxidase, manganese peroxidase) is added. The reaction is stirred for 3 hours, and calcium hydroxide is added to adjust the pH to 7.5. Then, *Hydizella* (…) is added. Dietzia maris C2 liquid bacterial agent, liquid-to-solid ratio 3-4:1, aerobic aeration and stirring for 20 minutes, standing for 30 minutes, solid-water-oil three-phase separation, oil skimming for 10 minutes, one cycle per hour, petroleum hydrocarbon degradation rate can reach over 95% in 8-12 hours, total petroleum hydrocarbons can be degraded to below 3‰, Hedizella ( Dietzia maris C2 is a key strain for the deep degradation of petroleum and is widely used in the field of oilfield environmental protection.

[0060] This invention relates to a bacterial strain isolated from core samples of the Daqing Oilfield. This strain can simultaneously secrete denitrification reductase and monooxygenase under aerobic conditions, allowing oxidation and reduction to occur concurrently. The AlkB hydroxylase is responsible for the C5-C6 phases of long-chain alkanes. 16The P450 monooxygenase is responsible for the oxidative degradation of benzene series compounds and low-cyclic aromatic hydrocarbons, while the denitrification reductase is responsible for the dechlorination of low-chlorine organic compounds. It can significantly improve the degradation rate of recalcitrant components in petroleum, laying the foundation for the treatment of oily sludge, petroleum-contaminated soil, petroleum wastewater, and petrochemical wastewater, and providing a new track for the green and low-carbon remediation of petroleum and petrochemical wastewater and petroleum-contaminated soil in my country.

Claims

1. A strain of Hedizil ( Dietzia maris C2, whose strain preservation number is CCTCC NO: M 2025275.

2. The Hedizella bacteria according to claim 1 ( Dietzia maris Application of C2 in biological treatment of organic pollution in petroleum and petrochemical industries.

3. The application according to claim 2, characterized in that: The treatment of petroleum and petrochemical organic pollution involves the efficient degradation of petroleum substances under conditions of compensated tryptophan or compound symbiotic synergistic bacteria.

4. The application according to claim 2, characterized in that: The highly efficient degradation of petroleum-based substances is petroleum-based C5-C. 44 Components.

5. The application according to claim 2, characterized in that: The aforementioned petroleum and petrochemical organic pollution includes the following scenarios: (1) Industrial wastewater: oil extraction water, fracturing flowback fluid, tank washing water, petrochemical wastewater, coking wastewater, printing and dyeing wastewater, papermaking wastewater, and oil-containing metallurgical wastewater; (2) Surface water: Surface water bodies polluted by petroleum or petrochemical industries, sewage ponds, seepage pits, and black and odorous water bodies; (3) Oil-contaminated soil: Oil-contaminated farmland, grassland, forest land, mines, coal mines and oil mining areas, especially soil near oil wells; (4) Hazardous waste: oily hazardous waste, drilling mud, oily sludge and other solid waste; (5) Industrial contaminated sites: Oil and petrochemical pollution sites and gas stations.

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