Rhodococcus ruber and its use

Abstract

The application relates to the field of microorganisms and discloses a Rhodococcus ruber and application thereof. The Rhodococcus ruber has a preservation number of CGMCC NO. 31356. The Rhodococcus ruber can effectively degrade alkanes and remove COD in sewage.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to Rhodococcus ruber and its applications. Background Technology

[0002] As a crucial pillar of my country's economy, the petrochemical industry inevitably generates various types of wastewater during its processing and production. With increasing national emphasis on environmental protection, the Beijing-Tianjin-Hebei region has implemented stricter requirements for suspended solids, COD, and ammonia nitrogen in its wastewater discharge. Wastewater undergoing advanced treatment must meet the Class IV surface water standard, making existing wastewater treatment processes in refining and chemical enterprises insufficient to meet these new environmental demands. To satisfy the needs of water resource protection, reclaimed water utilization, and continuous pollutant reduction, the advanced removal of organic pollutants has become a key focus in the water treatment field.

[0003] Wastewater treatment in my country's refining and chemical enterprises is mainly achieved through source reduction, process control, and end-of-pipe treatment. Early wastewater treatment primarily involved a "physicochemical pretreatment + biological organic matter removal" process before discharge. With increasingly stringent wastewater discharge standards, it is necessary to add technologies such as biological total nitrogen removal and advanced oxidation for organic matter removal to the existing processes to achieve deep removal of organic matter and total nitrogen. Deep organic matter removal often employs advanced oxidation processes, first improving the biodegradability of wastewater through oxidation, followed by further biological treatment. Currently, the most researched advanced oxidation technologies include ozone catalytic oxidation, Fenton oxidation, and electrochemical oxidation.

[0004] While ozone oxidation has a strong ability to decolorize and remove organic pollutants, it suffers from high ozone consumption, low utilization rate, long hydraulic retention time, high operating costs, and the risk of ozone emission polluting the atmosphere. Fenton oxidation technology has low investment and relatively low direct operating costs, but the generated sludge is hazardous waste and produced in large quantities, with disposal costs exceeding 20 yuan / ton of water. Electrochemical oxidation is highly effective at removing organic matter from wastewater, but its disadvantages include very high investment costs, high energy consumption, rapid electrode wear, and high operating costs. Although the aforementioned advanced oxidation methods can effectively remove organic matter from wastewater, their generally high treatment costs and the risk of secondary pollution limit their widespread application.

[0005] Biochemical methods for removing organic matter are low-cost and stable, making them the primary technology used in industrial wastewater treatment. However, wastewater typically exhibits low residual pollutant concentrations after two stages of biochemical treatment, resulting in an imbalanced nutrient ratio and persistent, difficult-to-degrade organic matter, thus increasing the difficulty of advanced wastewater treatment. Therefore, conventional microbial treatment methods are insufficient to meet the requirements for advanced removal of recalcitrant organic pollutants from wastewater. It is necessary to develop efficient technologies for removing recalcitrant organic pollutants, and to achieve advanced removal of these pollutants from wastewater by screening for microorganisms with specific degradation effects. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems existing in the prior art and to provide a strain of Rhodococcus and its application, which can effectively degrade alkanes and remove COD from wastewater.

[0007] To achieve the above objectives, the first aspect of the present invention provides a strain of Rhodococcus rubrum, the Rhodococcus rubrum having the accession number CGMCC NO.31356.

[0008] A second aspect of the present invention provides a bacterial agent containing Rhodococcus rubrum as described above; preferably, the bacterial agent is a liquid bacterial agent.

[0009] A third aspect of the present invention provides the use of Rhodococcus as described above or the bacterial agent as described above in the degradation of alkane.

[0010] The fourth aspect of the present invention provides the application of Rhodococcus as described above or the bacterial agent as described above in wastewater treatment.

[0011] The fifth aspect of the present invention provides a method for treating wastewater, the method comprising: contacting the wastewater with Rhodococcus as described above or with the bacterial agent as described above.

[0012] The Rhodococcus redissus described in this invention can effectively utilize alkanes as a carbon source, and furthermore, can effectively remove COD from petrochemical wastewater in a short period of time at room temperature.

[0013] Biological Preservation

[0014] The Rhodococcus ruber of this invention, with accession number BHYAG-6, was deposited on July 18, 2024, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101) (abbreviation of depositary institution: CGMCC), with accession number CGMCC No. 31356. Attached Figure Description

[0015] Figure 1The colony morphology of Rhodococcus bHYAG-6 of this invention is described;

[0016] Figure 2 This is a scanning electron microscope image of Rhodococcus b. BHYAG-6 of the present invention;

[0017] Figure 3 This is the sealed glass bottle used in the degradation experiment of this invention. Detailed Implementation

[0018] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0019] The first aspect of this invention provides a strain of Rhodococcus, the preservation number of which is CGMCC NO: 31356. In this invention, it is designated as BHYAG-6.

[0020] According to the present invention, the 16S rDNA sequence of the Rhodococcus is shown in SEQ ID NO: 1.

[0021] SEQ ID NO: 1:

[0022]

[0023] The optimal growth pH for *Rhodococcus rubrum* described in this invention is 6-8, and the optimal growth temperature is 28-35℃. On ISP-2 integrated potato medium, it exhibits orange-yellow, round, opaque, rough-surfaced, dried colonies. Figure 1 As shown.

[0024] A second aspect of the present invention provides a microbial agent containing Rhodococcus as described above.

[0025] Preferably, the bacterial agent is a liquid bacterial agent.

[0026] In this invention, the preparation method of the bacterial agent may include: culturing Rhodococcus as described above in a liquid culture medium and resuspending it in a buffer solution.

[0027] Preferably, the viable count of Rhodococcus rubrum in the bacterial agent is not less than 10. 10 CFU / mL, preferably 10 10 -10 12 CFU / mL.

[0028] A third aspect of the present invention provides the use of Rhodococcus as described above or the bacterial agent as described above in the degradation of alkane.

[0029] Preferably, the alkane is C9-C. 34 At least one of the alkanes.

[0030] The fourth aspect of the present invention provides the application of Rhodococcus as described above or the bacterial agent as described above in wastewater treatment.

[0031] In this invention, the wastewater is petrochemical wastewater.

[0032] Furthermore, the pollutants in the wastewater include at least one of the alkanes.

[0033] Preferably, the wastewater contains C9-C 34 At least one of the alkanes.

[0034] Preferably, the alkane may be provided in the form of diesel oil and / or paraffin.

[0035] Preferably, the alkane content in the wastewater is 50-1000 μg / L, more preferably 100-400 μg / L.

[0036] Preferably, the COD of the wastewater is 30-500 mg / L, and more preferably 50-200 mg / L.

[0037] The fifth aspect of the present invention provides a method for treating wastewater, the method comprising: contacting the wastewater with Rhodococcus as described above or with the bacterial agent as described above.

[0038] Preferably, the contact method may be to contact the liquid bacterial agent with the wastewater to be treated.

[0039] Preferably, the contact conditions may include a temperature of 25-35°C and a time of 12-48 hours.

[0040] According to the present invention, the amount of Rhodococcus can be determined based on the degree of pollution of the wastewater.

[0041] In some specific embodiments of the present invention, the amount of Rhodococcus rubrum, based on the viable count, is not less than 10 per liter of wastewater. 12 CFU, preferably 10 13 -10 15 CFU. The wastewater can be as described above.

[0042] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0043] Unless otherwise specified, all reagents and materials used in the following examples were purchased from reputable chemical reagent suppliers and were of analytical purity.

[0044] The culture medium used in this invention is as follows:

[0045] Inorganic salt liquid culture medium: ammonium chloride 0.67g, sodium nitrate 0.06g, magnesium sulfate heptahydrate 0.1g, calcium chloride 0.1g, dipotassium hydrogen phosphate trihydrate 1.5g, potassium dihydrogen phosphate 0.5g, ferric chloride hexahydrate 0.1g, deionized water 1L, pH 7-7.2.

[0046] Enrichment medium: Add 1 g / L liquid paraffin to the inorganic salt liquid medium.

[0047] Inorganic salt solid culture medium: Add 20g of purified agar to the inorganic salt liquid culture medium.

[0048] Paraffin screening solid culture medium: 1 g / L liquid paraffin was added to the inorganic salt solid culture medium.

[0049] LB liquid medium: 10g tryptone, 10g NaCl, 5g yeast extract, 1L deionized water, pH 7.2.

[0050] Composition of LB plate medium: 20g of purified agar is added to LB liquid medium.

[0051] The composition of ISP-2 integrated potato culture medium is as follows: 4g yeast extract, 10g malt extract, 4g glucose, 20g agar, 1L deionized water, pH 7.2-7.4.

[0052] Alkane removal rate = (Alkane content before degradation - Alkane content after degradation) / Alkane content before degradation × 100%.

[0053] The COD test method refers to the rapid digestion spectrophotometric method for the determination of chemical oxygen demand in water quality (HJ / T 399-2007).

[0054] Example 1

[0055] This embodiment illustrates the enrichment, screening, and purification of Rhodococcus bacillus BHYAG-6 as described in this invention.

[0056] (1) Enrichment: Take 15 ml of the mixture of sludge and water from the aerobic pool of the saline wastewater biochemical treatment plant of Tianjin Petrochemical and add it to 85 ml of enrichment medium for culture. Incubate at 35℃ and 180 rpm for 7 days.

[0057] Take 15 ml of the mixed culture medium and transfer it to 85 ml of enrichment medium for further culture. Repeat this process three times to complete the enrichment culture.

[0058] (2) Screening and purification: The enriched culture medium from step (1) was serially diluted, and the dilution factor was 10. 7 10 8 and 10 9 The bacterial suspension was evenly spread on LB agar plates and incubated at 35°C for 24 hours. Colony morphology was observed. The isolated colonies were then isolated again using LB agar until a single *Rhodococcus rubrum* strain was obtained. This *Rhodococcus rubrum* strain was then deposited at a cultural heritage site with the accession number CGMCC No. 31356 and designated BHYAG-6. A scanning electron microscope image of *Rhodococcus rubrum* BHYAG-6 is shown below. Figure 2 As shown, the bacterial cells are short rod-shaped and approximately 0.6 μm × 1 μm in size.

[0059] Example 2

[0060] This embodiment is used to illustrate the degradation ability of Rhodococcus b. BHYAG-6 of the present invention on alkanes.

[0061] The glycerol-preserved Rhodococcus bacillus BHYAG-6 culture was inoculated onto paraffin solid selection medium using a disposable inoculation loop and cultured at 30°C for 7 days in a biochemical incubator. It was able to grow on paraffin solid selection medium with paraffin as the sole carbon source, so it can be considered that this strain has the ability to degrade alkanes.

[0062] Example 3

[0063] This embodiment is used to illustrate the bacterial agent and its preparation method described in this invention.

[0064] After activating and culturing Rhodococcus rubrum BHYAG-6 slant for 24 hours, one loopful was added to LB liquid medium. 100 mL of LB liquid medium was placed in a 250 mL Erlenmeyer flask, and the flask was incubated at 30°C and 180 rpm on a shaker to obtain a bacterial suspension with an OD600 of 5 (LB liquid medium as a blank control). The suspension was centrifuged at 5000 rpm for 5 min, the supernatant was removed, and the bacterial cells were retained. The bacterial cells were resuspended in sterile 0.01 mol / L PBS buffer to obtain the bacterial agent described in this invention. The bacterial agent had an OD600 of 3.5 and a viable count of 3.2 × 10⁻⁶. 11 CFU / ml.

[0065] Example 4

[0066] This embodiment illustrates the degradation effect of Rhodococcus bacillus BHYAG-6 on alkanes as described in this invention.

[0067] (1) Take 100mL of inorganic salt liquid culture medium and place it in a 250mL Erlenmeyer flask. Seal the flask with a breathable sealing film and sterilize it in an autoclave at 121℃ for 20min. After cooling, add 1g / L of paraffin to the Erlenmeyer flask.

[0068] (2) Add the above-mentioned microbial agent to make the viable bacteria count in the system 1.5 × 10⁻⁶. 10 CFU / mL, such as Figure 3 As shown, the samples were sealed with breathable sealing film and treated on a shaker at 30°C and 180 rpm for 3 days, 5 days, and 7 days to obtain experimental group samples. In step (1), paraffin was added to the inorganic salt culture medium, but no bacterial agent was added. The samples were treated in the same way for 3 days, 5 days, and 7 days to obtain blank control group.

[0069] (3) The experimental group and blank control group samples obtained in the above steps were respectively added to n-hexane at a volume ratio of 1:10, extracted for 20 min, and allowed to stand until complete separation. The upper organic phase was taken, and the C9-C content in the blank control group and experimental group was measured by gas chromatography-mass spectrometry. 34 The alkane content is shown in Table 1.

[0070] Table 1

[0071]

[0072] As can be seen from Table 1, the Rhodococcus bacillus BHYAG-6 provided by the present invention can fully decompose alkanes.

[0073] Example 5

[0074] This embodiment illustrates the treatment effect of Rhodococcus bacillus BHYAG-6 on petrochemical wastewater as described in this invention.

[0075] Rhodococcus bacillus BHYAG-6 was used to treat the biochemical effluent containing saline wastewater from a petrochemical enterprise. The wastewater had a conductivity of 2800 μS / cm and a COD of 69 mg / L.

[0076] 100 mL of the above-mentioned saline wastewater from a petrochemical plant was placed in a 250 mL Erlenmeyer flask, and the above-mentioned bacterial agent was added to make the viable bacteria count in the reaction system 2.8 × 10⁻⁶. 10 CFU / mL, sealed with a breathable sealing film, and treated on a shaker at 30℃ and 180rpm for 1 day to obtain the experimental group sample. 110mL of biochemical effluent containing saline from a petrochemical plant was treated without adding any bacterial agent and treated in the same way for 1 day to obtain the blank control group.

[0077] Equal amounts of samples from the control and experimental groups were taken, and the samples from the experimental and control groups were extracted with dichloromethane, dried with anhydrous sodium sulfate, purged with nitrogen, and analyzed by GC-MS. The C content in the control group sample was measured. 12 -C 34 The content of mixed alkanes was 120.73 μg / L, and the C content in the experimental group sample was... 12 -C 34 The mixed alkane content is 42.67 μg / L.

[0078] Equal amounts of samples from the experimental group and the control group were taken, centrifuged at 10000 r / min for 3 min, and the supernatant was filtered through a 0.22 micrometer membrane to obtain the COD results, as shown in Table 2.

[0079] Table 2

[0080] sample COD (mg / L) Blank control group 69 Experimental group samples 30

[0081] The Rhodococcus bacillus BHYAG-6 provided by this invention has the ability to degrade mixed alkanes in petrochemical wastewater, with a 24-hour degradation rate of 64.7%. As shown in Table 2, Rhodococcus bacillus BHYAG-6 also has the ability to degrade COD in petrochemical wastewater, with a 24-hour COD removal rate of 56.5%.

[0082] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A strain of Rhodococcus ruber, characterized in that, The preservation number of the Rhodococcus is CGMCC NO.31356.

2. The Rhodococcus rubescens according to claim 1, wherein, The 16S rDNA sequence of the Rhodococcus is shown in SEQ ID NO:

1.

3. A microbial agent, wherein, The bacterial agent contains Rhodococcus rubrum as described in claim 1 or 2; Preferably, the bacterial agent is a liquid bacterial agent.

4. The microbial agent according to claim 3, wherein, The viable count of Rhodococcus rubrum in the bacterial agent is not less than 10. 10 CFU / mL, preferably 10 10 -10 12 CFU / mL.

5. The application of the Rhodococcus rubrum according to claim 1 or 2 or the bacterial agent according to claim 3 or 4 in the degradation of alkanes.

6. The application according to claim 5, characterized in that, The alkane is C9-C. 34 At least one of the alkanes.

7. The application of the Rhodococcus rubrum according to claim 1 or 2 or the bacterial agent according to claim 3 or 4 in wastewater treatment.

8. The application according to claim 7, wherein, The wastewater is petrochemical wastewater.

9. The application according to claim 7, wherein, The pollutants in the wastewater include at least one alkane, preferably C9-C6. 34 At least one of the alkanes; Preferably, the alkane content in the wastewater is 50-1000 μg / L, more preferably 100-400 μg / L; Preferably, the COD of the wastewater is 30-500 mg / L, and more preferably 50-200 mg / L.

10. A method for treating wastewater, characterized in that, The treatment method includes contacting the Rhodococcus rubrum of claim 1 or 2 or the bacterial agent of claim 3 or 4 with the wastewater.

11. The processing method according to claim 10, wherein, The amount of Rhodococcus bacteria used, based on viable count, is not less than 10 per liter of wastewater. 12 CFU, preferably 10 13 -10 15 CFU.

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