A Sphingobacterium sp. RL, microbial inoculum and its application in degrading phenanthrene in water

Abstract

The present invention belongs to the field of environmental microbiological technology, and discloses a Sphingobacterium RL, a microbial agent and its application in degrading phenanthrene in water. The strain is Sphingobacterium Sphingobacterium sp. RL , which was deposited in the Guangdong Provincial Microbial Strain Preservation Center on April 11, 2024, with the deposit number of 64518. This strain belongs to facultative anaerobic strains, can tolerate short-term ultraviolet irradiation, and has strong survival and reproduction abilities in natural water environments under natural sunlight, low oxygen or anoxic conditions. The present invention also provides a microbial agent with the Sphingobacterium RL as the active ingredient, and its application in enhancing the degradation of phenanthrene in water. Based on its new biological characteristics, after being prepared into a microbial agent, this strain can continuously carry out biological enhanced degradation of phenanthrene in various natural waters, and can meet the needs of low-cost and large-scale popularization and application.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental microorganisms and water pollution treatment, and particularly relates to a preferred Sphingobacterium RL, a microbial agent and their application in degrading phenanthrene in water bodies. Background Art

[0002] In the prior art, phenanthrene is widely used, including in industrial production, oil refining, dye manufacturing and other processes. It is also used as a raw material for manufacturing certain resins. There is a certain concentration of phenanthrene pollution in the water bodies of certain environments, which pollutes both the environment and organisms. Phenanthrene is a polycyclic aromatic hydrocarbon containing three benzene rings, with the chemical formula C14H10, CAS number 85-01-8, and exists in coal tar. The centers of the three rings of phenanthrene are not on a straight line. It is an isomer of anthracene, a white crystalline powder that can sublime. It is insoluble in water, slightly soluble in ethanol, and soluble in ether, glacial acetic acid, benzene, carbon tetrachloride, carbon disulfide, etc. The solution has blue fluorescence. It is used in the synthesis of resins, plant growth hormones, vat dyes, tannins, etc. It is usually used in the manufacture of dyes and pesticides, etc., and is used as a stabilizer for smokeless gunpowder; phenanthrene is oxidized to phenanthraquinone, and the biphenyl acid obtained by oxidation can be used to make polyester resins and alkyd resins; phenanthrene oxidation can obtain phthalic anhydride, cyclohexanone, and phenol; in papermaking, phenanthrene can be used as an anti-fog agent for pulp; in medicine, phenanthrene can be synthesized into alkaloids; in the dye industry, phenanthrene can be used to prepare 2-aminophenanthraquinone, benzanthrone, sulfurized vat dyes (blue BO, black BB and brown), etc.; phenanthrene can be hydrogenated under high temperature and high pressure to obtain hydrophenanthrene, which is a fuel for high-level jet aircraft.

[0003] Phenanthrene is an important organic pollutant in the water environment and can accumulate in water bodies, soil and sediments. It can enter water bodies through various channels, including industrial emissions, oil spills, etc. In water bodies, phenanthrene can be adsorbed by suspended solids and sediments, and can also be transformed and removed through processes such as volatilization, photolysis, oxidation and biodegradation. As a polycyclic aromatic hydrocarbon, phenanthrene can act as a sex hormone analogue in the water environment and have important effects on the morphology, behavior and physiological functions of fish. Research shows that phenanthrene can induce the activation of antioxidant enzymes and may disrupt the metabolism of sex hormones by interfering with the aromatase system.

[0004] Due to the stability and bioaccumulation of phenanthrene in water bodies, it may pose health risks to humans and aquatic organisms, presenting environmental and health risks. Long-term exposure or ingestion of water containing phenanthrene may cause harm to the human body. On October 27, 2017, the International Agency for Research on Cancer of the World Health Organization released a preliminary list of carcinogens for reference. Phenanthrene is included in the list of Group 3 carcinogens. Therefore, phenanthrene is a common organic pollutant in water bodies, with specific physical and chemical properties and biological impacts. In order to protect water bodies and human health, it is necessary to closely monitor the pollution status of phenanthrene and take corresponding prevention and control measures.

[0005] In the prior art, Sphingobacterium is a Sphingobacterium genus of microorganisms, and its origin is China. Conventional Sphingobacterium strains require sufficient oxygen for their metabolic activities. They have a similarity of 99.323% with the type strain Sphingobacterium siyangense SY1(T) EU046272 (Diff / Total nt5 / 739); when cultured on LB medium at 25°C for 5 days, the colonies are circular, milky yellow and opaque, with a smooth and moist surface, slightly convex in the center, regular edges, no halo, and a diameter of 3-5 mm. Their main use is for research, and the specific use is as an offshore bacterium. The Sphingobacterium strain W144 disclosed in Chinese patent document CN104046580A for degrading polycyclic aromatic hydrocarbon organic pollutants in soil is described in its specification as a Gram-negative bacterium, spherical ( Figure 1 ), and this strain is an aerobic strain that can bioremediate soil or environmental polycyclic aromatic hydrocarbon organic pollutants under aerobic conditions. However, when degrading the above organic pollutants, this strain usually requires sufficient oxygen to participate in order to grow rapidly, and it cannot tolerate natural sunlight (including short-term ultraviolet irradiation). Therefore, this strain cannot maintain growth for a long time and intensively degrade the above organic pollutants in natural sunlight conditions and low-oxygen (or anoxic) water environments such as natural rivers in the wild.

[0006] Therefore, it is necessary to study a new strain and microbial agent and be able to apply them to the intensive degradation of phenanthrene in natural river water under natural sunlight and low-oxygen or anoxic conditions. Summary of the Invention

[0007] To solve the above problems existing in the prior art, the object of the present invention is to provide a strain of Sphingobacterium RL obtained through special collection, cultivation and screening. After synchronous induced gene mutation and environmental tolerance optimization treatment, the finally obtained strain belongs to a facultative anaerobic strain. Most of the single bacteria in its colonies are ellipsoidal and a small part are short rod-shaped. It has unique biological genetic characteristics, enhancing its long-term survival and reproduction ability in water bodies under natural sunlight and low-oxygen (or anoxic) environments such as rivers, and being able to continuously bioremediate phenanthrene in various wild water bodies under natural conditions; the present invention also provides a microbial agent using this strain and its application. This strain has good environmental adaptability, can tolerate short-term ultraviolet irradiation and anoxic and other harsh natural water body environments, has a high degradation efficiency, is environmentally friendly and low-cost, can meet the needs of large-scale popularization and application, and has good application prospects in the field of sewage treatment in natural water bodies.

[0008] The above object of the present invention is achieved by adopting the following technical solutions:

[0009] A strain of Sphingobacterium RL, which is Sphingobacterium Sphingobacterium sp. RL, it was deposited in the Guangdong Provincial Microbial Strain Preservation Center on April 11, 2024. The deposit address is the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province. The deposit number is GDMCC No: 64518. It has been synchronously treated with induced gene mutation and environmental tolerance, belongs to facultative anaerobic strain, can tolerate short-term ultraviolet irradiation, and has strong survival and reproduction ability in natural water environment under natural sunlight, low oxygen or anoxic conditions.

[0010] The Sphingobacterium RL was obtained by selecting the soil of a coal chemical industry site with a relatively high phenanthrene content and a large number of strains as the inoculated soil, drying it to obtain the inoculated soil powder; continuously heating the inoculated soil powder to increase its temperature from 25°C to 55°C, and simultaneously inducing microbial gene mutation by short-term ultraviolet irradiation, and repeatedly turning the inoculated soil powder to obtain some strains with the ability to degrade phenanthrene that continue to survive and have strong environmental stress resistance as the original strains, and then through cultivation, enrichment, separation and screening.

[0011] A microbial inoculum uses the above-mentioned Sphingobacterium RL as the active ingredient; specifically, it is a suspension bacterial liquid or a humic acid-based agglomerate solidified bacterial agent; the humic acid-based agglomerate solidified bacterial agent includes Sphingobacterium RL and a humic acid-based agglomerate carrier, and the above-mentioned Sphingobacterium RL is colonized and immobilized on the humic acid-based agglomerate carrier.

[0012] The application of the above-mentioned Sphingobacterium RL or microbial inoculum in degrading phenanthrene in water. The conditions for the microbial inoculum to enhance the degradation of phenanthrene in water are: in the field natural environment, the water temperature is 25-35°C, and the pH value is 6-9.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] (1) Through the collection at specific locations, and through steps of inducing gene mutations, treating and cultivating for environmental tolerance, and screening, the Sphingobacterium RL strain selected has unique biological genetic characteristics different from conventional strains of this type. It belongs to facultative anaerobic strains, has strong survival and reproduction abilities under low-oxygen or anoxic conditions, has a wide temperature and pH adaptability, can tolerate short-term ultraviolet irradiation or long-term low-level ultraviolet irradiation in natural sunlight, and has strong survival and reproduction abilities in natural water environments under natural sunlight, low-oxygen or anoxic conditions; most single bacteria in the colonies of this strain are ellipsoidal, and a small part are short rod-shaped, and their shapes are significantly smaller than those of conventional Sphingobacterium strains; it belongs to aerobic or facultative anaerobic strains and can grow rapidly in natural waters under low-oxygen conditions such as without aeration and in wild rivers; based on its new biological characteristics, after being prepared into a microbial agent, it can biostimulate the degradation of phenanthrene in water; this strain can survive and reproduce whether it is suspended in water or in a state of being fixed or colonized on a carrier, and both have good degradation ability of phenanthrene in water and can biostimulate the degradation of phenanthrene in water.

[0015] (2) The humic acid-based aggregate solidified microbial agent provided by the present invention is to RL colonize the strain on the surface of the humic acid aggregate carrier. In the wild natural water environment, the humic acid aggregate carrier provides a carbon source and a local growth microenvironment for the RL strain, enabling the degradation reaction to proceed rapidly and continuously, improving the degradation efficiency and duration of phenanthrene in natural waters, and at the same time improving the utilization rate of the strain and reducing the input amount of the microbial agent; it can grow rapidly within 12 hours, and the degradation rate can reach more than 50%. As the degradation time extends, the degradation rate also gradually increases, and ultimately a degradation rate of more than 99% of phenanthrene in water in the natural environment can be achieved.

[0016] (3) For the microbial agent provided by the present invention, the RL strain in the humic acid-based aggregate solidified microbial agent cooperates with the humic acid-based aggregates. Humic acid, as a nutrient substance, can provide energy and a carbon source for the strain, promote its metabolic process, and is beneficial to the long-term degradation of low-concentration phenanthrene in natural waters by the strain; at the same time, the solidified microbial agent can protect the strain from being less affected by the external environment and helps the bacteria survive in a complex natural water environment (the solidified microbial agent can usually remain in a suspended state in water); compared with free bacteria (suspended bacterial liquid), the solidified microbial agent prepared by culturing on AlAWC-HA humic acid-based aggregates at an addition amount of 10 wt% at pH 7 and a temperature of 30 °C for 12 hours has a degradation rate of low-concentration phenanthrene in water increased by about 20% compared with the suspended microbial agent.

[0017] (4)The strain, microbial inoculum and their applications provided by the present invention have strong survival and reproduction abilities in natural water bodies under low-oxygen or anoxic conditions in the wild natural environment, can tolerate sunlight irradiation, have good environmental adaptability, can survive in water bodies in the natural environment for a long time, have high degradation efficiency, are environmentally friendly and low in cost, can meet the needs of large-scale popularization and application, and have good application prospects in the fields of sewage treatment and the like. Description of the Drawings

[0018] The present invention is further illustrated by the accompanying drawings, but the embodiments in the drawings do not constitute any limitation to the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the following drawings.

[0019] Figure 1 SEM image of the bacterial morphology of Sphingobacterium RL in the embodiment of the present invention;

[0020] Figure 2 Schematic diagram of the comparison of the degradation rates of phenanthrene in water by different preferred strains in the embodiment of the present invention;

[0021] Figure 3 Schematic diagram of the comparison of the growth curves of different preferred strains (including RL) in the embodiment of the present invention in water with 0.1 mg / L phenanthrene;

[0022] Figure 4 Schematic diagram of the comparison of the growth effects of strain RL in the embodiment of the present invention at different temperatures;

[0023] Figure 5 Schematic diagram of the comparison of the growth effects of strain RL in the embodiment of the present invention at different pH values;

[0024] Figure 6 Morphology schematic diagram of different humic acid-based aggregate materials prepared in the embodiment of the present invention;

[0025] Figure 7 Fourier transform infrared spectroscopy (FTIR) schematic diagram of different humic acid-based aggregate materials prepared in the embodiment of the present invention;

[0026] Figure 8 X-ray diffraction (XRD) schematic diagram of different humic acid-based aggregate materials prepared in the embodiment of the present invention;

[0027] Figure 9 X-ray photoelectron spectroscopy XPS characterization diagram of different humic acid-based aggregate materials prepared in the embodiment of the present invention;

[0028] Figure 10 Schematic diagram of the comparison of the strain loading amounts of the microbial inoculum prepared in the embodiment of the present invention at different fixed temperatures;

[0029] Figure 11 Schematic diagram of the comparison of phenanthrene degradation rates of the microbial inoculum prepared in the embodiments of the present invention at different fixed temperatures;

[0030] Figure 12 Schematic diagram of the comparison of strain loadings of the microbial inoculum prepared in the embodiments of the present invention at different strain addition amounts;

[0031] Figure 13 Schematic diagram of the comparison of phenanthrene degradation rates of the microbial inoculum prepared in the embodiments of the present invention at different strain addition amounts;

[0032] Figure 14 Schematic diagram of the comparison of degradation rates of the suspended bacterial liquid and the solidified bacterial agent prepared in the embodiments of the present invention. Detailed implementation manners

[0033] The following further elaborates on the present invention in detail in conjunction with the attached Figures 1-13 figures and multiple specific embodiments, but the protection scope of the present invention is not limited thereto.

[0034] The "Viability Report" related to the strain in the present invention application records the following content: A sample of the biomaterial of Sphingobacterium RL, which is the strain applied for preservation by the applicant, is named Sphingobacterium sp. RL , and its taxonomic name is Sphingobacterium sp. . This sample was preserved in the Guangdong Provincial Microbial Strain Preservation Center (GDMCC) on April 11, 2024. The preservation address is on the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province. The preservation number is CGMCC No. 64518, and the test result on April 11, 2024 is that it is viable.

[0035] Example 1

[0036] In this example, in order to enhance the degradation and removal of phenanthrene in water, the original strain was collected from the contaminated soil sample of the coal chemical industry site. After synchronous treatment of induced gene mutation and environmental tolerance, and then through enrichment, separation and screening, a preferred strain of Sphingobacterium was obtained. This strain of Sphingobacterium RL is Sphingobacterium sp. RL Sphingobacterium, which was preserved in the Guangdong Provincial Microbial Strain Preservation Center on April 11, 2024. The preservation address is on the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province. The preservation number is GDMCC No: 64518. It was found through testing that it belongs to facultative anaerobic strain, can tolerate short-term ultraviolet irradiation, and has strong survival and reproduction abilities in the natural water environment under natural sunlight, low oxygen or anoxic conditions.

[0037] See the attached Figure 1, the biological characteristics of the colonies of the RL strain are as follows: the colonies are circular and slightly raised, with a smooth surface, regular edges, and a diameter of 4-6 mm; most of the single bacteria in the colonies are ellipsoidal, and a few are short cylindrical with smooth ends. They have no flagella, a length of about 2-4 μm, and a diameter of about 1-2 μm. They are smaller in size than conventional Sphingobacterium strains and belong to facultative anaerobic strains. They can tolerate short-term ultraviolet irradiation, and their survival and reproduction abilities in natural water bodies such as rivers under natural sunlight, low-oxygen or anoxic conditions are significantly enhanced; the length of the 16s rDNA sequence of the RL strain is 1417 bp, and the nucleotide sequence is as shown in the attached sequence list.

[0038] The Sphingobacterium RL is obtained by selecting the soil of a coal chemical industry site with a relatively high phenanthrene content and a large number of strains as the inoculated soil, and drying it to obtain the inoculated soil powder; then continuously heating the inoculated soil powder to slowly increase its temperature from 25 °C to 55 °C and then maintaining it; during this process, short-term ultraviolet irradiation is simultaneously used to induce microbial gene mutations, and the inoculated soil powder is repeatedly turned over to obtain a strain mutagenized soil sample (as the subsequent inoculated soil sample); the part of the strain mutagenized soil sample that continues to survive and has strong environmental stress resistance and the ability to degrade phenanthrene is used as the original strain, and then the preferred strain is obtained through cultivation, enrichment, separation, and screening.

[0039] The preferred strain of the Sphingobacterium RL is obtained by the following method:

[0040] Weigh 10 g of the inoculated soil sample (in the strain mutagenized soil sample) after synchronous treatment of induced gene variation and environmental tolerance and add it to 100 mL of phenanthrene-containing water. After shaking cultivation and standing, take 10 mL of the inoculum and transfer it to 90 mL of fresh water containing 0.1 mg / L of phenanthrene, shake and cultivate until the 7th day, and repeat this process 5 times, gradually increasing the phenanthrene concentration to 20 mg / L. Then, streak the obtained solution on a solid medium containing 20 mg / L of phenanthrene and incubate it upside down at 30 °C, and observe the colony growth status;

[0041] Pick a single colony and inoculate it into 50 mL of a medium containing 0.1 mg / L of phenanthrene. The pH of the medium is 7. After culturing at 30 °C and 160 rpm until the 7th day, measure the residual phenanthrene concentration. Streak the culture solution with removal effect on the solid medium again for purification, and finally obtain multiple preferred strain single bacteria with the activity of removing phenanthrene;

[0042] The described phenanthrene-containing water body is a water body containing 0.1 mg / L of phenanthrene, which is prepared by adding 1 g of Na2HPO4, 0.5 g of KH2PO4, 1 g of NH4Cl, 2.2 mg of MnSO4·H2O, 20 mg of CuSO4, 8 mg of MgSO4, 2 mg of FeSO4·7H2O, and 2.6 mg of CaCl2·H2O, then adding phenanthrene to 0.1 mg / L, making up the volume to 1000 mL, and adjusting the pH to 7.

[0043] The described Sphingobacterium RL is obtained by further adopting the following enrichment, separation, and screening steps based on different preferred strains:

[0044] S1: For each of the obtained multiple preferred single strains, use a nutrient medium for resuscitation and amplification culture, then separate and pick 4 strains with stronger degradation ability, and name them F-1, F-2, F-3, and F-4 respectively;

[0045] The nutrient medium is prepared by adding 3 g of beef extract, 10 g of peptone, and 5 g of NaCl, making up the volume to 1000 mL, and adjusting the pH to 7.2;

[0046] S2: After amplifying the 4 strains to an OD600 of 0.45 respectively, inoculate them into the water body containing 0.1 mg / L of phenanthrene at an inoculation amount of 10 wt%;

[0047] S3: Cultivate under the conditions of a constant temperature of 30 °C, a pH of 7, and a constant shaking speed of 160 r / min until the 7th day, detect the residual concentration of phenanthrene in the water body, screen out the strain F-4 with the best properties and the highest phenanthrene degradation rate, and name it Sphingobacterium sp . RL.

[0048] A microbial inoculant uses the described Sphingobacterium RL as an active ingredient, and specifically is a suspension bacterial liquid or a humic acid-based agglomerate solidified bacterial agent; the humic acid-based agglomerate solidified bacterial agent includes Sphingobacterium RL and a humic acid-based agglomerate carrier, and the described Sphingobacterium RL is immobilized on the humic acid-based agglomerate carrier; the described humic acid-based agglomerate is an agglomerate formed by filtering the filtrate collected after pretreatment and composting of agricultural and forestry waste, collecting the solid humic acid, and freeze-drying.

[0049] The humic acid-based agglomerate carrier of the described humic acid-based agglomerate solidified bacterial agent is one of CM-HA, AW-HA, AcAWC-HA, AlAWC-HA, and HTAWC-HA;

[0050] The temperature for immobilizing Sphingobacterium RL on the humic acid-based agglomerate carrier is 25 - 35 °C;

[0051] The addition amount of the humic acid-based agglomerate carrier and Sphingobacterium RL is 5 - 15 wt%.

[0052] The humic acid-based aggregate solidifying bactericide is prepared by the following steps:

[0053] A1: Prepare a humic acid-based aggregate carrier;

[0054] A2: Prepare Sphingobacterium sp. RL bacterial liquid;

[0055] A3: Add the humic acid-based aggregate carrier and Sphingobacterium sp. RL bacterial liquid at an addition amount of 10 wt% each, culture at a rotation speed of 130 r / min, a temperature of 30 °C and a pH of 7 for 12 hours, so that Sphingobacterium sp. RL bacteria colonize on the humic acid-based aggregate carrier;

[0056] A4: Filter, wash with phosphate buffer solution, and prepare to obtain a humic acid-based aggregate carrier immobilized bactericide for standby.

[0057] The application of the aforementioned Sphingobacterium sp. RL or microbial bactericide in degrading phenanthrene in water, and the conditions for the microbial bactericide to enhance the degradation of phenanthrene in water are: in the field natural environment, the water temperature is 25-35 °C, and the pH value is 6-9.

[0058] Example 2

[0059] A strain of Sphingobacterium sp. RL, a microbial bactericide and their application in degrading phenanthrene in water provided by the present invention are further refined on the basis of Example 1. The specific processes of collection, treatment, breeding, separation and identification of the Sphingobacterium sp. RL strain in the embodiment of the present invention are as follows:

[0060] 1. Collection of original strain samples

[0061] Collection of original strain soil samples: The samples are collected according to the "Mixed Sample Collection Method" in the "Technical Specification for Soil Environmental Monitoring" (HJ / T 166-2004), and the fixed-point sampling is carried out by the plum-blossom point sampling method. The present invention pre-collects soil samples from multiple different scenarios, specifically including soil samples from coal chemical industrial sites, electronic waste dismantling areas, main traffic roadsides, and agricultural lands. By detecting the phenanthrene concentration in each sample soil, the soil of the coal chemical industrial site with a relatively high phenanthrene concentration is preliminarily selected as the strain mutagenesis soil sample for cultivation; the original strain soil sample is dried and prepared into a powder for standby;

[0062] The original strain soil sample of Sphingobacterium sp. RL in the embodiment of the present invention is collected from the polluted soil of a coal chemical industrial site in Ordos City, Inner Mongolia Autonomous Region, China.

[0063] 2. Preparation of strain mutagenesis soil samples

[0064] Select the original strain sample soil collected from a coal chemical industry site with a relatively high phenanthrene content and a large number of strains, and perform synchronous induced gene mutation and environmental tolerance treatment:

[0065] Heat the original strain soil sample, and slowly increase its temperature from low to high, from 25 °C to 55 °C, and then maintain it at 55 °C for a total duration of 30 - 45 min; during this process, irradiate the sample with ultraviolet light synchronously; in this example, ultraviolet light with a wavelength of 253.7 nm and a power of 40 W is used for irradiation (sterilization), irradiating for 2 minutes each time, irradiating again after an interval of 3 minutes, with a total duration of 30 - 45 min. During the short-term irradiation period, the soil sample is flipped multiple times. The method of synchronous short-term ultraviolet irradiation and heating is used to induce gene mutation in the strain and improve its adaptability to the environmental temperature, obtaining a strain-mutated soil sample; among the strain-mutated soil samples, a small number of strains that can finally survive are those with strong stress resistance in the natural environment and the ability to degrade phenanthrene at the same time.

[0066] 3. Preparation of the culture medium

[0067] The phenanthrene-containing water body is a water body containing 0.1 mg / L of phenanthrene, which is prepared from 1 g of Na2HPO4, 0.5 g of KH2PO4, 1 g of NH4Cl, 2.2 mg of MnSO4·H2O, 20 mg of CuSO4, 8 mg of MgSO4, 2 mg of FeSO4·7H2O, and 2.6 mg of CaCl2·H2O. Then, phenanthrene is added to 0.1 mg / L, and the volume is fixed to 1000 mL, with a pH of 7.

[0068] Nutrient culture medium: 3 g of beef extract, 10 g of peptone, 5 g of NaCl, fixed volume to 1000 mL, pH 7.2.

[0069] All the culture media used in the above experiments are sterilized by high-pressure steam at 121 °C for 20 min before use.

[0070] 4. Preliminary screening of the preferred strains of the degrading strains

[0071] Take the strain-mutated soil sample as the inoculated soil sample, and in the laboratory environment, supplement the aqueous solution containing phenanthrene multiple times for cultivation to screen out more strains with the ability to degrade phenanthrene. After the inoculated soil community is stable, conduct preliminary screening. Specifically:

[0072] Weigh 10 g of the strain-mutated soil sample into 100 mL of the phenanthrene-containing water body, shake and cultivate, then let it stand. Then, take 10 mL of the inoculation liquid and transfer it into 90 mL of a new water body containing 0.1 mg / L of phenanthrene, shake and cultivate until the 7th day, repeat 5 times, gradually increase the phenanthrene concentration to 20 mg / L, streak the obtained solution on a solid medium containing 20 mg / L of phenanthrene, and incubate it upside down at 30 °C, and observe the growth status of the colonies;

[0073] Pick a single colony and inoculate it into 50 mL of a medium containing 0.1 mg / L phenanthrene. The pH of the medium is 7. After culturing at 30 °C and 160 rpm for 7 days, measure the residual amount of phenanthrene. Re-streak the culture broth with removal effect on a solid medium for purification, and finally obtain a single bacterium with the activity of removing phenanthrene. Re-streak the culture broth with removal effect on a solid medium for purification, and finally obtain multiple preferred single bacteria with the activity of removing phenanthrene.

[0074] 5. Enrichment, isolation and screening of preferred highly efficient strains

[0075] For the multiple preferred single bacteria obtained, resuscitate and expand them respectively using a nutrient medium, and perform the following enrichment, isolation and screening:

[0076] S1: From the multiple preferred single bacteria obtained, screen, isolate, and pick 4 strains with stronger degradation ability, and name them F-1, F-2, F-3, F-4 (RL) respectively. Resuscitate and expand them respectively using a nutrient medium.

[0077] S2: After amplifying the 4 strains to an OD600 of 0.45 respectively, inoculate them into a water body containing 0.1 mg / L phenanthrene at an inoculation amount of 10 wt%.

[0078] S3: Cultivate under the conditions of constant temperature oscillation at pH 7, temperature 30 °C, and 160 r / min until the 7th day, detect the residual concentration of phenanthrene in the water body, screen out the strain F-4 with the best properties and the highest phenanthrene degradation rate, and name it Sphingobacterium sp. RL . The degradation rate of the RL (F-4) strain is the highest, and the degradation rate of phenanthrene in the water body is greater than 48.97%; if the continuous degradation time is longer, the degradation rate can reach more than 99%.

[0079] S4: The OD600 of the RL (F-4) strain in a water body containing 0.1 mg / L phenanthrene rises from 0.054 to 0.138. This strain can be applied to the water body. Compared with conventional strains, this strain can grow continuously and rapidly without adding aeration (low oxygen), and a higher phenanthrene degradation rate is obtained with a smaller amount of the strain added, which can effectively reduce the amount of the strain applied during use and reduce the difficulty and cost of expansion culture.

[0080] The specific degradation rate comparison chart of each preferred strain can be seen in the appendix Figure 2 .

[0081] The growth curve comparison chart of each preferred strain (including RL) in a water body containing 0.1 mg / L phenanthrene can be seen in the appendix Figure 3 .

[0082] From Figure 2 , Figure 3It can be seen that the RL strain (F-4) is a facultative anaerobic strain, with a stable growth rate under low-oxygen or anoxic conditions, the highest enhanced degradation rate of phenanthrene ultimately, and the highest enhanced degradation efficiency within the same time.

[0083] 6. Identification of Sphingobacterium RL

[0084] (1) Morphological identification: See Appendix Figure 1 , after the above screening and isolation, a strain of Sphingobacterium RL was obtained. Its colony is round and slightly raised, with a smooth surface, neat edges, difficult to pick up with an inoculation loop, round and light yellow, 4-6 mm in diameter, without a special smell. Under a scanning electron microscope, most single bacteria are ellipsoidal and a few are short cylindrical, without flagellar structures, about 2-4 μm in length and about 1-2 μm in diameter, smaller in shape than conventional Sphingobacterium strains, belonging to facultative anaerobic strains, and its survival ability under low-oxygen or anoxic conditions is significantly enhanced.

[0085] (2) Molecular biological identification:

[0086] Obtain the 16s rDNA sequence list (see the attached table), log in to the NCBI website to upload the RL strain for BLAST comparison, and it is known that the RL strain is Sphingobacterium sp. strain.

[0087] 7. Optimization of the culture growth conditions of Sphingobacterium RL strain

[0088] Optimize the enlarged culture conditions for the strain with stronger degradation ability Sphingobacterium sp. RL(F-4) strains.

[0089] After amplifying the Sphingobacterium sp. RL(F-4) strain to an OD600 of 0.45, inoculate it into the nutrient medium at an inoculation amount of 10 wt%, and incubate it at a constant temperature of pH 7, 130 r / min, and temperatures of 25, 30, and 35 °C respectively. When the temperature is 30 °C, the growth rate is the fastest, and the OD600 is 1.602. At the set temperatures of 25-35 °C, the OD600 is 1.398-1.602. This strain is suitable for culturing at 25-35 °C. The growth comparison effect diagram at different temperatures can be seen in Appendix Figure 4 .

[0090] After amplifying the Sphingobacterium sp. RL(F-4)After the strain was amplified to an OD600 of 0.45, it was inoculated into the nutrient medium at an inoculation amount of 10 wt%, and cultured at a temperature of 30 °C, 130 r / min, and constant temperatures with pH values of 5, 6, 7, 8, and 9 respectively. When the pH was 7, the growth rate was the fastest, and the OD600 was 1.546 at the 12th hour. When the set pH was 6 - 9, the OD600 at the 12th hour could reach 1.233 - 1.546. When the pH was 5, it decreased rapidly, and within 36 hours of the culture process, the highest OD600 was only 0.403. This strain is suitable for culturing at a pH of 6 - 9. The growth comparison effect diagrams at different pH values are shown in the appendix Figure 5 。

[0091] Therefore, the optimal conditions for enlarged culture are 30 °C and pH 7. This RL strain has a wide adaptation range during the enlarged culture stage, with a temperature adaptation range of 25 - 35 °C and a pH adaptation range of 6 - 9.

[0092] Example 3

[0093] Based on the Sphingobacterium RL provided in Example 1 of the present invention, a microbial agent using this Sphingobacterium RL as an active ingredient is provided in this embodiment of the present invention.

[0094] This microbial agent includes two types: a suspension bacterial liquid and a solidified bacterial agent.

[0095] For the preparation of the suspension bacterial liquid, the Sphingobacterium RL strain was added to the solution at an addition amount of 10 wt%.

[0096] The solidified bacterial agent is specifically a humic acid - type agglomerate solidified bacterial agent, which includes Sphingobacterium RL and a humic acid - type agglomerate carrier. Sphingobacterium RL is colonized and immobilized on the humic acid - type agglomerate carrier, and this Sphingobacterium RL is used as the active ingredient.

[0097] The solidified bacterial agent is prepared by the following steps:

[0098] (I) Preparation of humic acid

[0099] 1. Raw material preparation

[0100] Fresh cow dung was collected from an intensive dairy farm, and then the fresh cow dung was passed through a solid - liquid separator to reduce the moisture content to about 70%; apple wood came from the branch waste of apple trees, and the apple wood was crushed into 2 - mm wood chips for subsequent experiments.

[0101] 2. Pretreatment of agricultural and forestry waste

[0102] Acid treatment: Mix apple wood with 0.5 mol / L HCl at a mass ratio of 1:10 and soak for 12 hours at 25 °C. Then repeatedly rinse the apple wood in the solution until neutral to wash away the HCl on the surface of the apple wood. Finally, air-dry the apple wood to a moisture content of about 20% for subsequent composting.

[0103] Alkali treatment: Mix apple wood with 0.5 mol / L NaOH at a mass ratio of 1:10 and soak for 12 hours at 25 °C. Then repeatedly rinse the apple wood in the solution until neutral to wash away the NaOH on the surface of the apple wood. Finally, air-dry the apple wood to a moisture content of about 20% for subsequent composting.

[0104] High-temperature treatment: Mix apple wood with water at a ratio of 1:10 and conduct high-temperature treatment at 110 °C for 2 hours. Then repeatedly rinse the apple wood in the solution until neutral to wash away the substances on the surface of the apple wood. Finally, air-dry the apple wood to a moisture content of about 20% for subsequent composting.

[0105] 3 Composting methods

[0106] Conduct separate composting of pure cow dung; mix untreated apple wood, acid-pretreated apple wood, alkali-pretreated apple wood, and high-temperature-pretreated apple wood in a box according to a dry matter ratio of cow dung to apple wood of 2:1. That is: (1) pure cow dung (CM), (2) cow dung:apple wood = 2:1 (AW); (3) cow dung:acid-treated apple wood = 2:1 (AcAW); (4) cow dung:alkali-treated apple wood = 2:1 (AlAW); (5) cow dung:high-temperature-treated apple wood = 2:1 (HTAW);

[0107] All composting is carried out by static composting, and manual turning is performed once every 10 days during the composting process.

[0108] 4 Humic acid extraction

[0109] Weigh 2 g of the frozen and freeze-dried sample and add it to a 50 mL centrifuge tube, then add 35 mL of alkaline extraction solution. Oscillate in a water bath constant temperature oscillator at 240 r / min for 10 min, then heat at 60 °C for 2 h. Centrifuge at 4000 r / min for ten minutes in a centrifuge and then filter to collect the filtrate. Add 5 mL of 1M hydrochloric acid to acidify the pH to below 2, keep it in a constant temperature water bath at 60 °C for 1 h, then centrifuge at 4000 r / min for 10 min, filter the supernatant, and collect the solid as humic acid. The aggregates formed after freeze-drying are humic acid-like aggregates, which are used after passing through a 100-mesh sieve.

[0110] The product schematic diagrams of different humic acid-like aggregate materials prepared in this example are shown in the appendix Figure 6, where A is the cow dung treatment group (CM-HA); B is the apple wood group (AW-HA); C is the acid treatment group (AcAWC-HA); D is the alkali treatment group (AlAWC-HA); E is the high temperature treatment group (HTAWC-HA).

[0111] (II) Immobilization and optimization of RL strain

[0112] 1. Optimization of immobilization materials

[0113] Different humic acid aggregates (CM-HA, AW-HA, AcAWC-HA, AlAWC-HA, HTAWC-HA) prepared were used as the carrier materials for strain immobilization, and Sphingobacterium sp. The RL (F-4) strain was added to the medium at an addition amount of 10 wt%, and cultured for 12 hours at a rotation speed of 130 r / min, a temperature of 30 °C, and a pH of 7; the AL strain was colonized on the surface of the humic acid aggregate carrier to prepare the humic acid aggregate solidified bacterial agent, which was rinsed with sulfate buffer and reserved.

[0114] The Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) characterization diagrams of different immobilized carrier materials prepared in the examples of the present invention are shown in the appendix Figures 7-9 .

[0115] 2. Optimization of immobilization temperature

[0116] The prepared AlAWC-HA humic acid aggregate carrier material and the RL strain were added to the medium at an addition amount of 10 wt%, and cultured for 12 hours at a rotation speed of 130 r / min, a pH of 7, and temperatures of 25, 30, and 35 °C respectively. The microbial strain was colonized on the surface of the humic acid aggregate carrier to prepare the humic acid aggregate solidified bacterial agent, which was rinsed with phosphate buffer and reserved.

[0117] Take 5 mg of the AlAWC-HA humic acid aggregate carrier and 10 wt% of the RL strain, add them to 50 mL of water containing 0.1 mg / L of phenanthrene, and shake them in a constant temperature oscillator at 25 °C, 30 °C, 35 °C, 130 r / min, and a pH of 7 until the 7th day, and test the strain loading amount and the degradation rate of phenanthrene at different temperatures. See the appendix Figures 10-11 , and it was observed that the degradation rate of the solidified bacterial agent with an immobilization temperature of 30 °C was the best, which was 67.94%, and the amount of loaded strain was 1.72×10 7 cells / g.

[0118] 3. Optimization of strain addition amount

[0119] The prepared AlAWC-HA humic acid aggregates and RL strains were added at 5, 10, and 15 wt% respectively, and cultured for 12 hours at a rotation speed of 130 r / min, pH of 7, and temperature of 30 °C. The microorganisms were colonized on the humic acid aggregates to prepare the humic acid aggregate immobilized microbial agent, which was rinsed with sulfate buffer and reserved for use.

[0120] Take 5 mg of the AlAWC-HA humic acid aggregate carrier and add it to 50 mL of water containing 0.1 mg / L of phenanthrene. Shake it in a constant temperature oscillator at 30 °C, 130 r / min, and pH of 7 until the 7th day.

[0121] See Figures 12-13 , and it was observed that among them Sphingobacterium sp. The degradation rate of the RL strain added at 10 wt% was the best, which was 68.11%, and the amount of loaded strain was 1.79×10 7 cells / g.

[0122] Example 4

[0123] This example provides the specific application of the microbial agent on the basis of Examples 1-3.

[0124] After laboratory tests, the microbial agent prepared from Sphingobacterium RL, including the suspension bacterial liquid or the immobilized bacterial agent, can be applied to enhance the degradation of phenanthrene in water. The conditions for the microbial agent to enhance the degradation of phenanthrene in water are: water temperature 25-35 °C, pH value 6-9; the optimal conditions are water temperature 30 °C, pH 6, and the addition amount of the bacterial agent can be selected according to the actual situation.

[0125] In this example, the following steps were used to test the loading capacity of the immobilized bacterial agent for the RL strain and its degradation performance for phenanthrene:

[0126] 1. Take 5 mg of the immobilized bacterial agent prepared from different humic acid aggregate carriers respectively, add it to 50 mL of water containing 0.1 mg / L of phenanthrene, and shake it in a constant temperature oscillator at 30 °C, 130 r / min, and pH of 7 until the 7th day.

[0127] It was observed that the immobilized bacterial agent with AlAWC-HA as the carrier had the best degradation rate, which was 67.94%, and the amount of loaded strain was 1.72×10 7 cells / g.

[0128] 2. Compare the removal efficiency of the suspension bacterial liquid and the immobilized bacterial agent

[0129] 5 mg of the immobilized bacterial agent and the suspended bacterial liquid with AlAWC-HA as the carrier were respectively taken and added to 50 mL of water containing 0.1 mg / L of phenanthrene. Under the conditions of 30 °C, 130 r / min, and pH 7, it was shaken in a constant temperature oscillator until the 7th day.

[0130] See the appendix Figure 14 , compared with the free bacteria in the suspended bacterial liquid, in an anoxic or hypoxic water environment without aeration, after culturing for 12 hours at a pH of 7 and a temperature of 30 °C with a 10 wt% addition amount, the growth condition of the suspended bacterial liquid was good and the degradation rate was about 50%. After immobilizing the strain on the AlAWC-HA humic acid aggregate carrier, the degradation rate of the immobilized bacterial agent was about 70%. Thus, the degradation rate of the immobilized bacterial agent for phenanthrene increased by 19.41%. This is mainly due to the fact that on the one hand, the humic acid aggregate carrier, as a kind of nutrient, can provide a carrier, energy, and carbon source for the strain, promote its metabolic process, and is beneficial to the degradation of phenanthrene by the strain. On the other hand, it can provide a microscopic local growth environment for the strain, supplement oxygen, protect the strain, reduce its influence from the external environment, and help the RL strain survive and reproduce in a complex environment and maintain a high degradation efficiency.

[0131] Example 5

[0132] This example is based on Example 4 and provides the specific application of the microbial immobilized bacterial agent in natural outdoor waters.

[0133] The immobilized bacterial agent with AlAWC-HA as the carrier prepared in Example 4 was added to the water body of an open-air chemical continuous sewage treatment pond of a certain chemical enterprise in the south with a phenanthrene concentration of about 0.05 mg / L at an addition amount of 0.1 wt%. The water temperature varied in the range of 25 - 30 °C, the pH value ranged from 6 to 8, the water body was in a slow-flowing and sun-exposed environment, without aeration and stirring. The immobilized bacterial agent was naturally suspended in the water body or attached to the surface of the pool wall and the upper surface of the sediment. The degradation rate of phenanthrene gradually increased with the increase of the degradation time: after 12 hours of dosing, the degradation rate was about 55%, and after 24 hours, it reached more than 65%. After continuously dosing 0.05 wt% of the dosing amount every day for 7 days, the degradation rate could reach more than 80%. After 10 days of dosing, the degradation rate could reach more than 99%. If you want to improve the degradation efficiency (speed), you can appropriately increase the dosing amount of the immobilized bacterial agent, including the initial dosing amount and the subsequent supplementary dosing amount.

[0134] It was found in tests of more other examples that the degradation rate of phenanthrene by this immobilized bacterial agent in slow-flowing water is higher than that in static water; the degradation rate of high-concentration phenanthrene in the water body is higher than that of low-concentration phenanthrene; this immobilized bacterial agent can tolerate natural sunlight and can carry out long-term microbial enhanced removal of phenanthrene in various natural water bodies under natural outdoor conditions.

[0135] In each of the above embodiments of the present invention, by collecting, processing, cultivating, and screening the optimal RL strain, which has unique microbial genetic characteristics and belongs to facultative anaerobic strains, has strong survival and reproduction abilities under low-oxygen or anoxic conditions, has a wide temperature and pH adaptability, can tolerate short-term ultraviolet irradiation or long-term low-level ultraviolet irradiation (natural sunlight exposure) in natural water bodies, and has strong survival and reproduction abilities in natural water environments under natural sunlight exposure, low-oxygen or anoxic conditions; the prepared suspension bacterial liquid and humic acid-based agglomerate solidified bacterial agent can both survive in water bodies for a long time under natural conditions and have an enhanced degradation ability for phenanthrene in water bodies. The environmental adaptability of this strain and the microbial bacterial agent is good, with high degradation efficiency, environmental friendliness, and low cost, which can meet the needs of large-scale popularization and application, has good economic and environmental benefits, and has good application prospects in the field of sewage treatment and other fields.

[0136] Finally, it should be noted that although the content of the present invention has been introduced in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present invention. After those skilled in the art have read the above content, various modifications and substitutions to the present invention will be obvious. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims

1. A strain of Sphingobacillus RL, characterized in that: Sphingobacterium Sphingobacterium sp. RL , was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 11, 2024. The preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province. The preservation number is GDMCC No: 64518. It is a facultative anaerobic strain that can tolerate short-term ultraviolet radiation and has strong survival and reproduction ability in natural water environment under natural sunlight, low oxygen or hypoxia conditions.

2. A microbial agent, characterized in that: The active ingredient is the Sphingobacillus RL according to claim 1.

3. The microbial agent according to claim 2, characterized in that: It is a suspended bacterial liquid or humic acid aggregate solidifying bacterial agent; the humic acid aggregate solidifying bacterial agent comprises sphingobacillus RL and a humic acid aggregate carrier, and the sphingobacillus RL is immobilized on the humic acid aggregate carrier; the humic acid aggregate is an aggregate formed by cold-drying the filtrate collected after filtering agricultural and forestry wastes, pre-treating and composting, and the collected solid humic acid.

4. The microbial agent according to claim 3, characterized in that: The humic acid aggregate carrier is one of CM-HA, AW-HA, AcAWC-HA, AlAWC-HA, and HTAWC-HA; Among them, CM-HA is the humic acid aggregate carrier obtained from the cow dung treatment group; AW-HA is a humic acid aggregate carrier obtained from the apple wood group; AcAWC-HA is the humic acid aggregate carrier obtained from the acid treatment group; AlAWC-HA is the humic acid aggregate carrier obtained from the alkali treatment group; HTAWC-HA) is the humic acid aggregate carrier obtained from the high temperature treatment group; The temperature for immobilizing Sphingobacillus RL on the humic acid aggregate carrier is 25-35°C; The addition amount of humic acid aggregate carrier and Sphingobacillus RL is 5~15wt%.

5. The microbial agent according to claim 3, characterized in that: The humic acid aggregate solidifying agent is prepared by the following steps: A1: Preparation of humic acid aggregate carrier; A2: Preparation of Sphingobacillus RL bacterial solution; A3: The humic acid aggregate carrier and the Sphingobacillus RL bacterial solution were added at a rate of 10 wt%, and cultured for 12 hours at a speed of 130 r / min, a temperature of 30°C and a pH of 7, so that the Sphingobacillus RL bacteria were colonized on the humic acid aggregate carrier; A4: Filter and rinse with phosphate buffer to prepare the humic acid aggregate carrier-immobilized bacterial agent for use.

6. Use of the Sphingobacillus RL according to claim 1 or the microbial agent according to any one of claims 2 to 5 in degrading phenanthrene in water.

7. The use according to claim 6, characterized in that: The conditions for the microbial agent to enhance the degradation of phenanthrene in water bodies are: in the wild natural environment, the water temperature is 25~35℃ and the pH value is 6-9.

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