CN117467580A - Rhodococcus PD10 and application thereof - Google Patents
Rhodococcus PD10 and application thereof Download PDFInfo
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- CN117467580A CN117467580A CN202311737763.XA CN202311737763A CN117467580A CN 117467580 A CN117467580 A CN 117467580A CN 202311737763 A CN202311737763 A CN 202311737763A CN 117467580 A CN117467580 A CN 117467580A
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- 241000316848 Rhodococcus <scale insect> Species 0.000 title claims abstract description 17
- 239000002351 wastewater Substances 0.000 claims abstract description 12
- XVMSFILGAMDHEY-UHFFFAOYSA-N 6-(4-aminophenyl)sulfonylpyridin-3-amine Chemical compound C1=CC(N)=CC=C1S(=O)(=O)C1=CC=C(N)C=N1 XVMSFILGAMDHEY-UHFFFAOYSA-N 0.000 claims abstract description 8
- 230000015556 catabolic process Effects 0.000 claims abstract description 7
- 238000006731 degradation reaction Methods 0.000 claims abstract description 7
- 238000004321 preservation Methods 0.000 claims abstract description 3
- 150000003839 salts Chemical class 0.000 claims description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 abstract description 64
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 abstract description 32
- 239000003344 environmental pollutant Substances 0.000 abstract description 4
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 abstract description 3
- 238000009629 microbiological culture Methods 0.000 abstract description 3
- 229910017053 inorganic salt Inorganic materials 0.000 description 15
- 230000001580 bacterial effect Effects 0.000 description 13
- 239000001963 growth medium Substances 0.000 description 13
- 238000000034 method Methods 0.000 description 12
- 230000012010 growth Effects 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 239000000725 suspension Substances 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 241000187562 Rhodococcus sp. Species 0.000 description 5
- 230000015784 hyperosmotic salinity response Effects 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 230000000593 degrading effect Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 238000004065 wastewater treatment Methods 0.000 description 4
- 241001052560 Thallis Species 0.000 description 3
- 230000003044 adaptive effect Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 238000010170 biological method Methods 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000004939 coking Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- -1 nitrogen-containing heterocyclic compound Chemical class 0.000 description 2
- 238000000053 physical method Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
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- 238000002798 spectrophotometry method Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 241000186073 Arthrobacter sp. Species 0.000 description 1
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 1
- 241000186146 Brevibacterium Species 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 241000589774 Pseudomonas sp. Species 0.000 description 1
- 208000013738 Sleep Initiation and Maintenance disease Diseases 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 208000031320 Teratogenesis Diseases 0.000 description 1
- 206010043275 Teratogenicity Diseases 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 125000006615 aromatic heterocyclic group Chemical group 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
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- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
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- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
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- 239000003205 fragrance Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 239000004434 industrial solvent Substances 0.000 description 1
- 239000010842 industrial wastewater Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 206010022437 insomnia Diseases 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
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- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000003079 shale oil Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
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- 239000011269 tar Substances 0.000 description 1
- 231100000211 teratogenicity Toxicity 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
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- 231100000419 toxicity Toxicity 0.000 description 1
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- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The invention belongs to the technical field of biological treatment of environmental pollutants, and particularly relates to the technical field of efficient degradation of pyridine in a high-salt environment. The invention provides a Rhodococcus sp PD10 which is preserved in China general microbiological culture collection center (CGMCC) in the 10 th month 9 of 2023, wherein the center address is the No.1 North West road No. 3 of the Korean region of Beijing city, and the preservation number of the strain is CGMCC No.28593. The strain PD10 provided by the invention can grow and degrade pyridine in high-salt wastewater with the highest salinity of 10% by taking pyridine as the sole carbon-nitrogen source, and the pyridine removal rate within 132h is 99.2%.
Description
Technical Field
The invention belongs to the technical field of biological treatment of environmental pollutants, and particularly relates to the technical field of efficient degradation of pyridine in a high-salt environment.
Background
Aromatic heterocyclic compounds are major environmental pollutants of interest due to their recalcitrance, persistence, toxicity and teratogenicity, which are often difficult to degrade naturally in the environment and create potential hazards to human health and ecosystems.
Pyridine is a typical nitrogen-containing heterocyclic compound, has good water solubility, is colorless or yellowish liquid, has malodor, and mainly exists in tar, coal gas and shale oil. Pyridine and its derivatives are widely used as industrial solvents and are also important raw materials or intermediates for some agricultural chemicals, medicines, commodities, dyes, fragrances and the like, and therefore, a large amount of pyridine can be detected in many industrial wastewaters such as coking wastewater, pharmaceutical wastewater, dye wastewater and the like.
Pyridine is toxic, can be used for cancerogenesis and teratogenesis, is easy to enrich in organisms, and can generate a great deal of harm when being inhaled, ingested or directly exposed to skin for a long time, such as anesthesia of the central nervous system, and causes bad symptoms such as dizziness, headache, insomnia, digestive tract dysfunction and the like.
At present, the method for treating pyridine pollutants in wastewater is mainly divided into three major types, namely a chemical method (a high-grade oxidation method), a physical method (adsorption, rectification and incineration) and a biological method. The chemical method and the physical method have high energy consumption and high cost, have relatively high requirements on equipment, can generate secondary waste flow in the wastewater treatment process, and are difficult to meet the environmental protection requirement. In contrast, the biological method relies on the characteristics of pyridine and derivatives thereof to degrade the pyridine in the wastewater by virtue of few aerobic or anaerobic microorganisms existing in natural environment, has the advantages of low cost, large treatment capacity, mild conditions, low equipment requirement and thorough degradation, and is a more economic and environment-friendly treatment method.
At present, several pyridine degrading strains have been reported successively, including Arthrobacter sp, bacillus sp, corynebacterium sp, brevibacterium sp, pseudomonas sp, rhodococcus sp, streptomyces sp, etc., which exhibit their potential for use in wastewater treatment to varying degrees. However, the actual wastewater treatment process is often accompanied by a number of complex environmental constraints, one of which is the wastewater salinity.The industrially produced waste water generally contains a large amount of salt substances such as Na + 、NO 3 - 、Cl - And the like, the too high salt concentration can inhibit and poison the growth of microorganisms, so that the microorganism cannot play full potential in wastewater treatment. Therefore, the method for screening the microorganism which is high-salt resistant and can efficiently degrade the pyridine has far-reaching significance for the effective treatment of industrial high-salt pyridine wastewater.
Disclosure of Invention
In view of this, the present invention provides a Rhodococcus sp PD10 which has been deposited in the China general microbiological culture Collection center, called CGMCC, at the address of Hospital No. 3 of North Chen West road No.1 in the Korean region of Beijing, and at the date of 10/9 of 2023, and has the accession number of CGMCC No.28593.
The strain PD10 provided by the invention can grow and degrade pyridine in high-salt wastewater with the highest salinity not exceeding 10% by taking pyridine as the sole carbon-nitrogen source, and the pyridine removal rate within 132h is 99.2%.
Drawings
FIG. 1 shows the growth curves of Rhodococcus PD-4 at different salt concentrations.
FIG. 2 shows the degradation of pyridine by Rhodococcus sp. PD-4 at various salt concentrations.
FIG. 3 shows a pyridine map of growth degradation of Rhodococcus PD10 at a salt concentration of 10%.
Detailed Description
The following examples will provide those skilled in the art with a more complete understanding of the invention, but are not intended to limit the invention in any way.
The experimental methods described in the examples below are conventional methods unless otherwise specified. In the examples, the pyridine concentration was measured at 254nm by ultraviolet spectrophotometry and OD 600 The measurement was carried out at 600nm by means of a visible spectrophotometer.
The instruments, materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
Screening and identification of rhodococcus (rhodococcus)
Rhodococcus sp is a gram-positive bacterium obtained by domestication and separation of industrial pyridine-containing wastewater collected from a certain coking plant in Shandong in 2023 in month 3.
The method comprises the following specific steps:
the soil sample suspension was enriched in colonies using an inorganic salt medium with pyridine as the sole carbon nitrogen source. The collected soil suspension samples were inoculated at an inoculum size of 10% into 250mL shake flasks containing 90mL of mineral salt medium and enriched at 30℃and 200 rpm. After 48h, passage was performed, i.e. 10% of the inoculum size was transferred to fresh mineral salts medium and enrichment culture was continued for 48h, and the procedure was repeated three times in total. After enrichment is completed, the bacterial liquid is taken for 10 -1 ～10 -9 Gradient dilution, respectively taking 10 -4 ～10 -8 100. Mu.L of each diluted bacterial liquid was spread on a solid plate of an inorganic salt medium containing 500mg/L of pyridine, and cultured in an incubator at 30℃for 7 days. And selecting single bacterial colony with good growth vigor, and carrying out streak culture for multiple times to obtain the purified bacterial strain. After sequencing by 16SrDNA, the sequence is shown as a sequence table SEQ ID NO.1, named PD-4 and submitted to BLAST alignment in NCBI, the strain is identified as Rhodococcus sp.
In the method, the culture medium is an inorganic salt culture medium which takes pyridine as the only carbon and nitrogen source, and comprises the following components: k (K) 2 HPO 4 ·3H 2 O 0.30g/L、NaH 2 PO 4 ·2H2O 0.20g/LMnSO 4 ·4H 2 O 0.01g/L、MgSO 4 ·7H 2 O 0.05g/L、FeSO 4 ·7H 2 O0.01 g/L, naCl-100.00 g/L, pH 7.0. The theoretical concentration of pyridine in the inorganic salt culture medium is 500mg/L.
Example 2
Performance of Rhodococcus PD-4 to degrade pyridine at different salt concentrations
The strain obtained by the separation is inoculated into a salt-free inorganic salt culture medium (containing 500mg/L pyridine), shake cultivation is carried out at 30 ℃ until the growth log later stage, the bacterial cells are collected by centrifugation at 5000rpm of a centrifuge for 10min, and the bacterial cells are washed for three times, and a small amount of supernatant is reserved to resuspend the bacterial cells to prepare bacterial suspension.
Inoculating the bacterial suspension into inorganic salt culture medium containing 500mg/L pyridine with salinity (NaCl mass concentration) of 0%, 2%, 4%, 6%, 8% and 10%, respectively, to obtain initial bacterial body OD 600 About 0.1, culturing in a sterile anaerobic bottle at 30deg.C for several days, sampling every 24 hr, and measuring OD of bacteria by spectrophotometry 600 Values.
As shown in FIGS. 1 and 2, in the inorganic salt culture medium using pyridine as the sole carbon and nitrogen source at 30℃and 200rpm, the growth of the strain is hardly affected when the strain is cultured in the inorganic salt culture medium with the salinity of less than 2%, and the OD is within 72 hours 600 All reach about 0.60 and can degrade pyridine basically and completely; when the salinity is raised to 4%, the growth speed of the strain is obviously slowed down, the strain still grows rapidly after a 48-hour hysteresis period and completely degrades pyridine, and the final OD is 120 hours 600 Can reach 0.51; when the salinity is continuously increased to 6% -10%, the bacteria cannot grow.
Example 3
Salt tolerance laboratory adaptive evolution of Rhodococcus (Rhodococcus sp.) PD-4
The salt concentration in the culture condition is gradually increased by adopting a laboratory adaptive evolution mode so as to obtain pyridine degrading bacteria with high salt tolerance.
The strain cultured in the inorganic salt culture medium at 4% salinity is used as an initial strain, and is continuously transferred into a fresh inorganic salt culture medium for subculture, and the inoculation amount of each time is the initial OD 600 The domestication process was performed in an anaerobic jar, gradually increasing the salt concentration in the inorganic salt medium, and finally obtaining pyridine degrading bacteria with higher salt tolerance, named rhodococcus (rhodococcus.) PD10, which can rapidly grow and degrade pyridine in inorganic salt medium with a salinity of 10% (NaCl mass concentration). The strain is preserved in China general microbiological culture collection center (CGMCC) at 10 and 9 days of 2023, the center address is CGMCC No.1 and 3 of North Chen West Lu of the Korean area of Beijing, and the preservation number of the strain is CGMCC No.28593.
Example 4
Adaptive evolution function validation of Rhodococcus (Rhodococcus sp.) PD10
And (3) diluting and coating the bacterial liquid with improved salt tolerance to obtain a single colony capable of growing and degrading pyridine on an inorganic salt solid culture medium with 10% salinity. Subsequently, salt tolerance verification was performed on the strain in a shake flask reaction system, and the salt concentration was set to 10%, specifically comprising the steps of: the domesticated Rhodococcus sp. PD10 is first cultivated in an LB medium, wherein the LB medium consists of: and 5g/L of yeast extract, 5g/L of tryptone and 10g/L of NaCl, centrifuging at 5000rpm for 10min to collect the thalli when the thalli grow to the late logarithmic growth phase, washing three times with 10% inorganic salt culture medium, and taking a small amount of supernatant for re-suspending the thalli to prepare bacterial suspension.
Respectively adding appropriate amount of bacterial suspension into 10% inorganic salt culture medium to make initial OD 600 =0.1, and pyridine was added at a final concentration of about 500mg/L, incubated on a shaker at 30 ℃,200rpm, sampled at 12h intervals, and a control group without any strain added was set. As shown in FIG. 3, the domesticated strain PD10 enters a rapid growth stage through a hysteresis period of 84 hours in a culture medium with a salinity of 10%, pyridine is rapidly degraded, and the strain PD10 grows to OD after 132 hours 600 Maximum value is 0.74, and the pyridine content is finally reduced from the initial 527.5mg/L to the minimum value of 6.5mg/L, and the degradation efficiency is as high as 99.2%.
Although the present invention has been described with reference to the above embodiments, it should be understood that the invention is not limited to the embodiments described above, but is capable of modification and variation without departing from the spirit and scope of the present invention.
Claims (3)
1. Rhodococcus sp PD10 with a preservation number of CGMCC No.28593.
2. Use of Rhodococcus sp PD10 according to claim 1 for the degradation of pyridine in high salt wastewater.
3. The use according to claim 2, wherein the high salt is NaCl at a mass concentration of not more than 10%.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311737763.XA CN117467580A (en) | 2023-12-15 | 2023-12-15 | Rhodococcus PD10 and application thereof |
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| CN202311737763.XA CN117467580A (en) | 2023-12-15 | 2023-12-15 | Rhodococcus PD10 and application thereof |
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- 2023-12-15 CN CN202311737763.XA patent/CN117467580A/en active Pending
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