CN110669716A

CN110669716A - Method for separating high-concentration phenol and heavy metal resistant and low-temperature resistant rhodococcus

Info

Publication number: CN110669716A
Application number: CN201911106876.3A
Authority: CN
Inventors: 赵天涛; 高艳辉; 刘毫; 张磊; 尹镝宁; 陈静; 张云茹; 韩斌
Original assignee: Chongqing University of Technology
Current assignee: Chongqing University of Technology
Priority date: 2019-11-13
Filing date: 2019-11-13
Publication date: 2020-01-10

Abstract

The invention relates to a method for separating high-concentration phenol and heavy metal resistant and low-temperature resistant rhodococcus, which comprises the following steps: the strain is put into an LB liquid culture medium for enrichment culture, high-concentration phenol degradation with low, medium and high phenol concentration gradients and domestication of high-concentration heavy metal tolerant functional bacteria are carried out by a sequencing batch operation method, and after domestication, the strain is separated and purified to obtain the pure strain Rhodococcus erythropolis. The method of the invention can shorten the domestication and separation period of the functional strain with high-concentration phenol degradation and high-concentration heavy metal tolerance, and the strain has fast growth and reproduction, strong phenol and heavy metal tolerance and easy formation of dominant bacteria. When the strain obtained by the method is used for treating wastewater containing high phenol and heavy metal, the strain has the advantages of high integration degree, simple process flow, low operation management difficulty and the like, and is favorable for solving the problem of biological dephenolization of wastewater containing high-concentration phenol and heavy metal.

Description

Method for separating high-concentration phenol and heavy metal resistant and low-temperature resistant rhodococcus

Technical Field

The invention belongs to the technical field of environmental microbiology, and particularly relates to a strain separation method of high-concentration phenol and heavy metal resistant and low-temperature resistant rhodococcus.

Background

With the rapid development of industries such as fiber, plastic, chemical industry, building materials, coking and the like, the discharge amount of phenol-containing wastewater is greatly increased, and phenols become one of the current main water pollution sources. Phenol and its derivatives are highly toxic environmental pollutants distributed throughout the world and are considered by the U.S. environmental protection agency as priority pollutants and toxic substances. Besides being highly toxic, phenol is easily chlorinated to produce more difficult-to-degrade chlorophenol hazardous waste. In 2017, 10 and 27, phenol is classified as a class 3 carcinogen in a carcinogen list published by the international cancer research institute of the world health organization. Phenol has toxic and killing effects on all living bodies, and can directly enter the blood circulation of human bodies through the contact of skin and mucous membranes to cause the injury of human tissues and generate carcinogenic effect. Thus, phenol has been blacklisted by our country as an environmentally preferred pollutant and 65 toxic pollutants. Phenol production, coking, oil refining, metallurgy, plastics, chemical fibers, insulating materials, phenolic resins, pharmacy, explosives, pesticides and other industries generate high-concentration phenol wastewater, a large amount of phenol enters atmospheric circulation, and directly enters soil and water through rainfall, volatilization and other modes, so that a great amount of animals and plants die and distort, and the safety of human beings is finally affected. Phenol is a common pollutant in life and production, and the removal research of phenol is always concerned by environmental workers.

The phenol-containing wastewater or soil treatment technology comprises distillation, ion exchange, liquid-liquid extraction, activated carbon adsorption, pervaporation, advanced oxidation, biological treatment and the like, and the non-biological method has many defects, such as only realizing phase state conversion or converting into substances with higher toxicity, high treatment cost and the like, separating and screening from a pollution source to obtain high-efficiency phenol-reducing bacteria, researching the degradation characteristics of the phenol-reducing bacteria, and finally applying the phenol-containing bacteria to a phenol-containing wastewater treatment system in the form of activated sludge or aerobic particles, so that the method is the most economic, efficient, green and easy-to-operate treatment method for treating high-concentration phenol-containing wastewater. However, most research results show that most of phenol-reducing bacteria separated at present have the concentration of phenol capable of being degraded within 300-1000 mg/L and lower concentration of degradable phenol, and actually, the concentration of phenol in phenol-containing wastewater discharged from factories is up to thousands or even ten thousands of milligrams per liter, so that certain difficulty exists in treating the phenol-containing wastewater with high concentration. At present, the wastewater is treated by combining multiple processes such as physical-chemical process, biological process and the like, but when the physical-chemical process is used as a front end to treat the wastewater, the process is complex, and the wastewater is generally in the defect of high operating cost if the wastewater is required to be continuously diluted to a biodegradable concentration range. The additional pretreatment cost in the early stage is high, the operability is poor, the environment is not green, in addition, the wastewater is a very complex mixed system, most of the wastewater is rich in various high-concentration toxic substances, the growth of microorganisms is inhibited, the phenolic substances in the wastewater cannot be completely mineralized by the conventional treatment process, a plurality of other toxic pollutants cannot be effectively removed, even if the effluent discharged up to the standard still has the accumulated pollution effect on the environment, and therefore, the bottleneck still exists in the treatment of the high-concentration phenol wastewater or the soil by utilizing the conventional microorganisms at present. Therefore, when the phenol-reducing bacteria are bred efficiently, the phenol-reducing efficiency can be improved by using modern biotechnology such as factor mutagenesis, gene fusion and the like and researching catalysts and the like, and the development and realization of the technology for synchronously removing various toxic substances have great research significance for breeding the green, efficient, durable, broad-spectrum and strong-adaptability phenol-reducing bacteria.

In recent years, researchers have discovered that a particular genus Rhodococcus, a class of microorganisms between mycobacteria and Nocardia, has a multifunctional metabolic pathway and resistance to xenobiotic toxicity that enables them to mineralize phenol in the presence of phenolic waste products and other toxic substances. The bacteria have the characteristic of synchronously removing various toxic substances, wherein heavy metals are common coexisting pollutants of phenol-containing wastewater, related researches mostly focus on the influence of the heavy metals on the phenol removal effect, the research of removing the heavy metals by utilizing the rhodococcus is less, the phenol and the heavy metals can be synchronously removed in one reactor under the aerobic condition, and the contradiction among the growth of phenol degrading bacteria, the phenol removal and the inhibition of the heavy metal toxicity is solved. Through analysis of a mechanism of the Rhodococcus for tolerating high phenol, the strain is found to have a close relationship with a metabolic pathway and living environment thereof in the characteristic of removing high-concentration phenol.

On one hand, the rhodococcus can realize the synchronous removal of phenol and heavy metal, thereby not only shortening the dephenolization period, but also reducing the toxic action of heavy metal accumulation on thalli;

on the other hand, the rhodococcus belongs to heterotrophic aerobic microorganisms, and the supply of oxygen and a matrix not only accelerates the proliferation and differentiation of cells to enable the cells to quickly assimilate phenol into cell components, but also enables the cells to keep higher dephenolization enzyme activity and accelerates the removal of high-concentration phenol from the enzyme activity level.

However, the phenol degradation strain obtained by the traditional separation method has poor tolerance to phenol, the phenol tolerance concentration of the phenol degradation strain is below 1500mg/L, the acclimatization and separation period of the strain is long, and the strain can not maintain efficient and stable dephenolization performance under the coexistence of high-concentration phenol, various organic pollutants and heavy metals and low-temperature conditions.

Disclosure of Invention

The invention aims to provide a method for separating high-concentration phenol and heavy metal resistant and low-temperature resistant rhodococcus so as to overcome the problems in the prior separation technology. The invention quickly carries out domestication and separation of the high-concentration phenol-resistant strain by means of the reactor, shortens the strain separation period, and adopts a point plate method to carry out heavy metal ion, low-temperature and high-temperature adaptive stimulation on the strain, so that the strain can ensure stable phenol and heavy metal ion removal effect under the coexistence of high-concentration phenol and heavy metal and low-temperature conditions.

A method for separating high-concentration phenol and heavy metal resistant and low-temperature resistant rhodococcus has the following steps:

1) taking strains, putting the strains into an LB liquid culture medium, carrying out shake culture at the constant temperature of 30-37 ℃, and carrying out enrichment culture for 1-2 days; obtaining an enriched strain, inoculating the obtained enriched strain to a separation culture medium containing 300mg/L phenol, and carrying out passage for 2-3 times under the same condition to obtain enriched bacterial liquid without impurities;

2) high concentration phenol resistant mixed strain domestication

2.1 Low concentration phenol acclimation

Inoculating the enriched strain into the simulated wastewater in the reactor, adjusting the initial PH of the simulated wastewater to 7.0-8.0 (in an aerobic environment), setting the initial phenol concentration to 300mg/L, domesticating, replacing the simulated wastewater after the phenol concentration in the simulated wastewater in the reactor is lower than 30mg/L, increasing the phenol concentration in the simulated wastewater to 400mg/L, repeating until the phenol concentration in the simulated wastewater reaches 500 mg/L. The phenol removal rate is more than or equal to 90 percent, and a low-concentration phenol domesticated bacterial liquid is obtained;

2.2 Medium concentration phenol acclimation

Inoculating the low-concentration phenol domesticated bacterial liquid obtained in the step 2.1 into simulated wastewater of a new reactor according to the inoculation amount of 10% of the volume of the simulated wastewater, gradually increasing the phenol concentration of the simulated wastewater from 500mg/L to 1000mg/L, and obtaining a medium-concentration phenol domesticated bacterial liquid, wherein the phenol removal rate is more than or equal to 90%;

2.3 high phenol concentration acclimatization

Inoculating the bacteria liquid domesticated in the medium phenol concentration in the step 2.2 into simulated wastewater of a new reactor according to the inoculation amount of 10% of the volume of the simulated wastewater, gradually increasing the phenol concentration of the bacteria liquid from 1000mg/L to 3000mg/L, and finishing domestication to obtain a high-concentration phenol-resistant mixed bacteria liquid;

3) isolation of pure bacteria

Mixing the obtained mixed bacterial liquid according to the proportion of 10 after the acclimatization is finished^-1-10^-7And (3) performing gradient dilution, coating the obtained product on an M9 solid culture medium, culturing at 30-37 ℃, after bacterial colonies grow, selecting bacterial colonies with different forms on another M9 solid culture medium, partitioning and streaking until the generated bacterial colonies are single bacterial colonies with different forms, and obtaining a plurality of high-concentration phenol resistant purified strains.

4) Screening: respectively inoculating a plurality of purified strains into a separation culture medium added with 1500-3000mg/L phenol, and screening out strains with the phenol removal rate of more than or equal to 90% when the phenol concentration is less than 2500mg/L and the phenol removal rate of more than or equal to 70% when the phenol concentration is more than or equal to 2500mg/L to obtain high-concentration phenol-resistant strains;

5) pure strain phenol tolerance domestication

Domesticating the high-concentration phenol-resistant strain obtained in the step 4) by using an M9 solid culture medium, and gradually increasing the phenol concentration until no bacterial colony grows on a plate of the M9 solid culture medium, so as to obtain the high-concentration phenol-resistant strain.

The strain in the step 1) is derived from the film edge soil of the covering layer of the refuse landfill.

Step 2), the low-concentration phenol acclimation time is 6-10 h; the acclimation time of the medium-concentration phenol is 16-18 h; the high-concentration phenol acclimation time is 24-72 h.

And step 2) the reactor consists of a bacteria filter, an air duct, a constant-temperature heating stirrer, a container and an air pump.

The step-by-step increase in the step 2) is to increase the concentration of phenol to 100mg/L each time.

Step 2.1 heavy metal ions of the simulated wastewater contain 500 mu mol/L of Cu²⁺、Cd²⁺、Pb²⁺、Cr³⁺、Ni²⁺And Mn²⁺。

The separation medium is composed of Na₂HPO₄6-8g/L、KH₂PO₄2.77-5.54g/L，NaCl 0.3-1g/L、NH₄Cl0.5-1.5g/L、CaCl₂0.01-0.05g/L、MgSO₄0.1-0.5 g/L; adding water, mixing, sterilizing and dissolving.

The M9 solid culture medium is a separation culture medium containing 15-20g/L agar powder, and the phenol concentration of the M9 solid culture medium is 1000-2000 mg/L.

The domestication method in the step 5): culturing colonies with maximum tolerance concentration of 10g/L phenol by dot plate method, culturing the colonies with LB liquid culture medium to obtain seed liquid, diluting according to gradient to obtain dot plate seed liquid with OD of 1.0, 0.5, 0.25, 0.125 and 0.0625, dropping 2 μ L of the seed liquid onto M9 solid culture medium plate containing higher concentration phenol, inducing and acclimatizing, standing and culturing at 30-37 deg.C, and observing every 24 h.

The LB liquid culture medium is prepared by adding water into yeast powder 3-8g/L and peptone 8-12g/L, Nacl8-12g/L, mixing, sterilizing and dissolving.

The method of the invention is adopted to separate pure strains from the soil on the membrane side of the covering layer of the refuse landfill, DNA sequencing is finished by Chongqing Ongqing biology company, the 16S rDNA sequence is shown in SEQ ID NO:1, and then the pure strains are named as Rhodococcus erythropolis C1. The preservation number of the strain is as follows: CCTCC NO, M2019725, preservation date: year 2019, month 9, day 17, category name: rhodococcus erythropolis C1(Rhodococcus ruber C1) depositary Unit: china center for type culture Collection, collection address: wuhan, Wuhan university.

The pure strain Rhodococcus erythropolis C1 with phenol degradation function, which is obtained by the method, can not only tolerate high-concentration phenol, but also has the function of synchronously removing a plurality of heavy metals, and can still completely remove phenol under the low-temperature culture condition.

The Rhodococcus erythropolis C1 obtained by the method of the present invention has the characteristics that: various organic matters such as phenol, glucose, sodium citrate, sodium succinate and the like can be used as carbon sources and ammonium sulfate and ammonium chloride are used as nitrogen sources for growth, the growing carbon sources and nitrogen sources are rich, and the defects of slow growth, long generation period, low biomass concentration, weak environmental adaptability and the like of the traditional phenol degrading bacteria are overcome; can degrade various toxic substances such as benzene, toluene, 2, 4-dichlorophen, 4-chlorophenol and the like; the method can realize the synchronous removal of phenol and heavy metal ions at low temperature, has high tolerance to phenol, can tolerate phenol as high as 10g/L, and overcomes various limitations of complicated pretreatment of the traditional biological phenol removal process; the method has the capability of efficiently degrading high-concentration phenol, can efficiently remove phenol in the wastewater, and has stable phenol removal performance.

The method adopts a reactor consisting of a bacteria filter, an air duct, a constant-temperature heating stirrer, a container, an air pump and the like, meets the requirements of the strains for removing phenol on dissolved oxygen and nutrient substances, and is convenient for adjusting parameters such as phenol concentration, pH, temperature, dissolved oxygen and the like. The reactor can not only continuously provide air, maintain relatively stable dissolved oxygen condition, but also automatically control temperature; after the phenol concentration is detected every 6h, phenol and other nutrients are supplemented, so that the phenol is always maintained at a relatively stable level. Compared with the traditional method for domesticating the phenol removal strains by adopting conventional utensils such as conical flasks and the like, the method ensures that the strains with stable phenol removal performance continuously keep activity on the basis of ensuring the phenol removal effect, and lays a foundation for the separation of subsequent strains. Compared with the traditional domestication period of 2-3 months, the domestication method saves nearly half of the time, the culture condition of the reactor is similar to the real wastewater treatment condition, and the practical application possibility of the strain is greatly increased compared with the environment provided by a conical flask.

Drawings

FIG. 1 is a reactor used in the process of the present invention; wherein, 1 is an air pump, 2 is a gas flowmeter, 3 is a first bacteria filter, 4 is a sampling port, 5 is a second bacteria filter, 6 is a temperature probe, 7 is a constant temperature heating stirrer, and 8 is a container;

FIG. 2 is a graph showing the removal of phenol from Rhodococcus erythropolis C1 strain obtained by the method of the present invention under different pH (A), temperature (B) and salinity (C) conditions;

FIG. 3 is a graph showing the growth curve and phenol degradation curve of a pure strain Rhodococcus erythropolis C1 cultured in phenol at different initial concentrations, obtained by the method of the present invention;

FIG. 4 is a graph showing the growth curve of Rhodococcus erythropolis C1 obtained by the method of the present invention under the cultivation conditions with different concentrations of heavy metal ions.

FIG. 5 shows the phenol removal rate of a pure strain Rhodococcus erythropolis C1 obtained by the method of the present invention under the cultivation condition of heavy metal ions of different concentrations.

FIG. 6 shows the removal rate of heavy metal ions with different concentrations by Rhodococcus ruber C1.

Table 1 shows the physiological and biochemical characteristics and the utilization of different substrates of a pure strain Rhodococcus erythropolis C1 obtained by the method of the present invention.

Detailed Description

1. Experimental Material

Composition of LB liquid medium: 5g/L yeast powder and 10g/L, Nacl10 g/10 g/L peptone are prepared by adding water, mixing, sterilizing and dissolving.

Composition of M9 solid medium: na (Na)₂HPO₄7g/L、KH₂PO4 3g/L，NaCl 0.5g/L、NH₄Cl 1g/L、CaCl₂0.02g/L、MgSO₄0.2g/L and 15g/L of agar powder; adding water, mixing, sterilizing and dissolving.

Composition of the isolation medium: na (Na)₂HPO₄7g/L、KH₂PO4 3g/L，NaCl 0.5g/L、NH₄Cl 1g/L、CaCl₂0.02g/L、MgSO₄0.2g/L, adding water, mixing, sterilizing and dissolving to obtain the product.

The reagents of the invention are all commercial analytical pure products.

The film-side soil of the covering layer of the refuse landfill is taken from the urban domestic refuse landfill of the tea garden village of the growing bridge in the south quay of Chongqing city.

2. Steps of the method of the invention

(1) Strain enrichment: taking 15g of landfill covering layer membrane edge soil, putting the landfill covering layer membrane edge soil into a 250mL conical flask filled with 100mL of sterilized LB liquid culture medium, fully shaking up, culturing at 30 ℃ and 200rpm, preserving in a refrigerator at 4 ℃ after 2d, then inoculating the enriched bacterial liquid into a 250mL conical flask filled with 100mL of phenol separation culture medium containing 300mg/L according to the inoculum size of 5%, fully shaking up, culturing at 30 ℃ and 200rpm, and carrying out passage for 3 times under the same condition to obtain the enriched bacterial liquid without soil impurities.

(2) Domesticating strains: firstly, a low-concentration phenol domestication stage, inoculating enriched strains into a reactor (see figure 1) filled with 1L of simulated wastewater according to the inoculation amount of 10%, setting the initial phenol concentration to be 300mg/L to start domestication, gradually increasing the phenol concentration in the simulated wastewater to 500mg/L (increasing the phenol concentration to 100mg/L every time) after all phenol in the reactor is removed, wherein the phenol removal rate is more than or equal to 90%, and the phenol removal rate is ensured to be more than 90%.

And then, a medium-concentration phenol acclimation stage, after the low-concentration phenol acclimation stage of the strains is finished, inoculating the bacterial liquid into a new reactor according to the inoculation amount of 10% from the reactor, gradually increasing the phenol concentration of the simulated wastewater from 500mg/L (increasing the phenol concentration to 100mg/L every time) to 1000mg/L, and ensuring that the phenol removal rate is over 90%.

And finally, a high-concentration phenol domestication stage, after the phenol domestication stage in the strains is finished, inoculating the bacterial liquid into a new reactor according to the inoculation amount of 10% from the reactor, gradually increasing the phenol concentration of the simulated wastewater from 1000mg/L (increasing the phenol concentration to 100mg/L every time) to 3000mg/L, ensuring that the phenol removal rate is over 70%, and finishing the domestication to obtain the high-concentration phenol-resistant mixed bacterial liquid.

The pH value of all stages is controlled to be 7.0-8.0, the temperature is controlled to be 30-37 ℃, and the dissolved oxygen is controlled to be 3-5 mg/L.

See fig. 1. The reactor comprises fungus filter, air duct, constant temperature heating agitator, container, air pump, and wherein air pump 1, gas flowmeter 2, first fungus filter 3 loop through the air duct connection, and the one end of air duct is stretched into in the container 8, and the upper portion of sample connection 4 is provided with the switch, and second fungus filter 6 is fixed on the pipe on container top, and temperature probe 6 is connected for constant temperature heating agitator 7 electricity.

(3) And (3) pure bacteria separation: diluting the domesticated bacteria solution with cooled sterile distilled water by 10 times to obtain 10 dilution^-1、10^-2、10^-3、10^-4、10^-5、10^-6、10^-7The dilution of (4), spreading the dilution on M9 solid medium, and culturing at 35 ℃ for several daysAfter the bacterial colony grows, selecting bacterial colonies with different forms, carrying out four-partition repeated streaking on another M9 solid culture medium until the generated bacterial colony is a single bacterial strain with different forms, completing pure bacterial separation, and obtaining a plurality of purified bacterial strains resistant to high-concentration phenol.

(4) Screening: respectively inoculating a plurality of purified strains into a separation culture medium added with 1500-3000mg/L, carrying out shake culture at 35 ℃ and 200rpm, screening to obtain a strain with a removal rate of more than or equal to 90% when the concentration of phenol is within 2500mg/L, and obtaining a pure strain resistant to high-concentration phenol, which is named as Rhodococcus ruber C1.

3. Strain identification

And (3) extracting the genome DNA of the high-concentration phenol-resistant pure strain obtained in the second step by using a bacterial genome DNA extraction kit of Life Technologies to be used as a target fragment, and performing PCR amplification. 5 mu LPCR amplification products were subjected to 1% agarose gel electrophoresis, and the desired fragment was recovered by using a cut gel to perform 16S r DNA sequencing. Sequencing of 16S r DNA was carried out by Shanghai Meiji Biomedicine science and technology Co. DNA sequencing was carried out using Seq Forward, Seq Reverse, and Seq Internal as primers. The DNA sequence of Rhodococcus erythropolis (Rhodococcus ruber) C1 is shown in SEQ ID NO: 1:

GGGACTGCCGATCCACCTTCGACGGCTCCCTCCACGAGGGGTTAGGCCACCGGCTTCGGGTGTTACCGACTTTCATGACGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCGTTGCTGATCTGCGATTACTAGCGACTCCGACTTCACGGGGTCGAGTTGCAGACCCCGATCCGAACTGAGACCGGCTTTAAGGGATTCGCTCCACCTCGCGGTATCGCAGCCCTCTGTACCGGCCATTGTAGCATGTGTGAAGCCCTGGACATAAGGGGCATGATGACTTGACGTCGTCCCCACCTTCCTCCGAGTTGACCCCGGCAGTCTCCTGCGAGTCCCCACCATTACGTGCTGGCAACACAGGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTACACCGACCACAAGGGAAACCCCATCTCTGGGGCGGTCCGGTGTATGTCAAACCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCACATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGCGCTTAATGCGTTAGCTACGGCACGGATCCCGTGGAAGGAAACCCACACCTAGCGCCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTACCCACGCTTTCGCTCCTCAGCGTCAGTTACTGCCCAGAGACCCGCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTTCACCGCTACACCAGGAATTCCAGTCTCCCCTGCAGTACTCAAGTCTGCCCGTATCGCCTGCAAGCCCGCAGTTGAGCTGCGGGTTTTCACAGACGACGCGACAAACCGCCTACGAGCTCTTTACGCCCAGTAATTCCGGACAACGCTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTGGCCGGTGCTTCTTCTGTACCTACCGTCACTTGCGCTTCGTCGGTACTGAAAGAGGTTTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGTCGTCGCCTTGGTGGGCCGTTACCCCACCAACAAGCTGATAGGCCGCGGGCCCATCCTGCACCGGAAAACCTTTCCACCCCGGAACATGCATCCCGAGGTCCTATCCGGTATTAGACCCAGTTTCCCAGGCTTATCCCGAAGTGCAGGGCAGATCACCCACGTGTTACTCACCCGTTCGCCACTAATCCACCCAGCAAGCTGGGCTTCATCGTTCGACTGCATGTGTAAGCATCGCCGCCACGCCATC16S rRNA amplification Using broad-spectrum primers F27(SEQ ID NO:2-AGAGTTTGATCATGGCTCAG) and R1492(SEQ ID NO: 3-GGTTACCTTGTTACGACTT).

Example 1 test for phenol removing ability of Mixed strains obtained by the method for separating a high concentration phenol-resistant Strain in acclimation stage

Preparing separation culture medium with phenol concentration of 2000mg/L, sterilizing 100mL of the separation culture medium in a 250mL conical flask with high temperature steam at 121 deg.C for 30min, cooling, and adding 2mL of acclimatized high concentration resistant phenol mixed bacteria liquid (OD) into the flask with a pipette_600nm1-2), sealing, placing into a constant temperature shaking table, culturing at 30 deg.C and 200rpm, and measuring OD of bacterial liquid every 6 hr_600nmThe growth condition of the thalli is determined, the content of phenol in a separation culture medium is measured at the same time, the removal effect of the phenol is determined, 2000mg/L of phenol is basically and completely removed by the mixed bacteria liquid within 72 hours, and the removal rate is up to more than 96%.

Example 2 culture Condition of Rhodococcus erythropolis C1 with different toxic substances and common saccharide substances as sole carbon sources

Inoculating Rhodococcus erythropolis C1 strain in LB liquid culture medium, placing in a constant temperature shaking table, culturing at 30 deg.C and 200rpm for 24h, taking activated bacterial liquid, and performing scanning electron microscope analysis to observe strain morphology, to find that C1 is in the form of short rod, aseptic filament, with diameter of 5 μm and length of 10 μm; according to the inoculation amount of 2 percent, the strains are respectively inoculated into a separation culture medium which takes mannitol, xylose, sodium succinate, sucrose, sodium formate, sodium acetate, sodium benzoate, methanol, ethanol, phenol, benzene, toluene, 2, 4-dichlorophenol and 4-chlorophenol as unique carbon sources, the separation culture medium is placed into a constant temperature shaking table and cultured for 72 hours under the conditions of 30 ℃ and 200rpm, and the results are shown in Table 1, and the C1 strains can grow by utilizing the carbon sources. Therefore, the separated high-concentration phenol degradation resistant strain C1 has rich carbon source for growth and wide degradation spectrum.

TABLE 1 Biochemical characteristics of Strain C1 and carbohydrate carbon source and toxic degradation substrate spectra (+, positive; -, negative)

Example 3 phenol degradation Properties of Rhodococcus erythropolis C1 under different environmental conditions

Inoculating Rhodococcus erythropolis C1 strain in LB liquid culture medium, placing in constant temperature shaking table, culturing at 30 deg.C and 200rpm for 24h, respectively taking 100mL of isolation culture medium in 250mL conical flask, sterilizing with high temperature steam at 121 deg.C for 30min, cooling, and adding 2mL of cultured Rhodococcus erythropolis C1 bacterial liquid (OD) into the flask_600nm1-2), sealing, placing into a constant temperature shaking table under the conditions of different temperatures (10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 37 ℃, 40 ℃, 43 ℃, 45 ℃ and 50 ℃), different pH (4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0), different salinity (0%, 1%, 2%, 4%, 6% and 8%) and different phenol concentrations (58, 67, 85, 109, 226.2, 400.8, 564.8, 630.8, 718.8, 908.8, 1024.8, 1224.8, 1510.8, 1618.8, 1805.8 and 2000mg/L), culturing the other optimization experiments at 40 ℃ and 200rpm except the temperature optimization experiment, obtaining the initial phenol concentration of 1800mg/L in other optimization experiments except the initial phenol concentration optimization experiment, and measuring OD (OD) at intervals of 8h_600nmThe growth of the cells was determined, the phenol content in the medium was measured, and the phenol removal effect was determined, as shown in FIG. 2A, which shows that Rhodococcus erythropolisThe C1 strain is capable of growing and degrading phenol completely at 10-50 ℃. The most suitable phenol degradation temperature is 40 ℃; FIG. 2B shows that Rhodococcus erythropolis C1 strain can tolerate a pH of 4-10 with an optimum pH of 7.0. FIG. 2C shows that Rhodococcus erythropolis C1 strain can tolerate concentrations of NaCl up to 6%. As can be seen from FIG. 3, when the concentration of phenol is less than or equal to 2000mg/L, the complete degradation of phenol can be realized, when the concentration of phenol is 58-226.2mg/L, the complete degradation time of phenol is 6-10h, when the concentration of phenol is 400-1024.8mg/L, the complete degradation time of phenol is 14-20h, and when the concentration of phenol is 1224.8-2000mg/L, the complete degradation time of phenol is 27-74.5 h.

Meanwhile, through an experiment of an M9 solid culture base plate method, the maximum phenol tolerance concentration of induced Rhodococcus erythropolis can reach 10g/L, and in a separation culture medium, the Rhodococcus erythropolis is placed in a constant-temperature shaking table and cultured under the conditions of 40 ℃ and 200rpm, the phenol removal rate is 34%, the high-concentration phenol has strong toxicity, but the Rhodococcus erythropolis C1 strain still shows a high removal effect.

Example 4 Effect of Rhodococcus erythropolis C1 on removal of heavy metal ions at various concentrations

Preparing heavy metal ions (Cu) respectively²⁺、Cd²⁺、Cr³⁺、Pb²⁺、Mn²⁺) LB liquid medium (containing phenol 500mg/L) with concentration of 0. mu. mol/L, 500. mu. mol/L, 1000. mu. mol/L, 2000. mu. mol/L, 4000. mu. mol/L, respectively, 100mL of the above medium was put in 500mL conical flask, sterilized by high temperature steam at 121 deg.C for 30min, cooled, and 2mL of Rhodococcus erythropolis bacterial liquid (OD) was added to the flask with pipette_600nm1-2), sealing with bottle sealing membrane, culturing in shaker at 40 deg.C and 200rpm, and measuring OD of bacterial liquid every 12 hr_600nmThe growth condition and the residual quantity of phenol of the thalli are determined, the content of heavy metal elements in the liquid phase of an LB liquid culture medium is determined at the same time, the removal effect is determined, as can be seen from figure 6, when the initial concentration of metal ions is 500 mu mol/L, the removal capacities of the C1 bacterial strains to different metals are different, the removal capacities are sequentially Pb & gt Mn & gt Cd & gt Cu & gt Cr & gt Ni, the removal capacity to Pb is strongest, and the Rhodococcus erythropolis C1 bacterial strain pair is cultured for 3 days under the condition of 500mg/L phenol stress, so that the removal capacity to Pb is strongestThe removal capacity of each heavy metal with different concentrations is respectively as follows: the removal rates of Pb (500 mu mol/L-4000 mu mol/L), Cd (500 mu mol/L-4000 mu mol/L), Mn (500 mu mol/L-4000 mu mol/L), Cr (500 mu mol/L-4000 mu mol/L) and Cu (500 mu mol/L-4000 mu mol/L) respectively reach 90.03-95.46%, 76.37-86.66%, 38.72-90.90%, 95%, 42.33-65.02%, 61.40-70.25% and 24.54-57.40%; 500. mu. mol/L Pb in the presence of 500mg/L phenol, compared with the blank²⁺And Mn²⁺Promotes the growth of Rhodococcus erythropolis C1 strain (FIG. 4) and the degradation of phenol (FIG. 5); in comparison with other heavy metal elements, in Cd²⁺In the presence of the Cd-Cd removal rate, the growth of the strain is inhibited to a certain extent, but the strain still slowly grows by utilizing LB, and finally, the Cd-Cd removal rate is not less than 76%. The Rhodococcus C1 strain has the capability of removing metals and phenol together under the coexistence of phenol and heavy metals, so that the Rhodococcus C1 strain is suitable for treating heavy metal-mixed phenol-containing chemical wastewater.

The above examples are merely a few typical examples of the present invention, and it is obvious that the present invention is not limited to the above embodiments, but many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Sequence listing

<110> Chongqing university of science and technology

<120> method for separating high-concentration phenol and heavy metal resistant and low-temperature resistant rhodococcus

<160>3

<170>SIPOSequenceListing 1.0

<210>1

<211>1436

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

gggactgccg atccaccttc gacggctccc tccacgaggg gttaggccac cggcttcggg 60

tgttaccgac tttcatgacg tgacgggcgg tgtgtacaag gcccgggaac gtattcaccg 120

cagcgttgct gatctgcgat tactagcgac tccgacttca cggggtcgag ttgcagaccc 180

cgatccgaac tgagaccggc tttaagggat tcgctccacc tcgcggtatc gcagccctct 240

gtaccggcca ttgtagcatg tgtgaagccc tggacataag gggcatgatg acttgacgtc 300

gtccccacct tcctccgagt tgaccccggc agtctcctgc gagtccccac cattacgtgc 360

tggcaacaca ggacaagggt tgcgctcgtt gcgggactta acccaacatc tcacgacacg 420

agctgacgac agccatgcac cacctgtaca ccgaccacaa gggaaacccc atctctgggg 480

cggtccggtg tatgtcaaac ccaggtaagg ttcttcgcgt tgcatcgaat taatccacat 540

gctccgccgc ttgtgcgggc ccccgtcaat tcctttgagt tttagccttg cggccgtact 600

ccccaggcgg ggcgcttaat gcgttagcta cggcacggat cccgtggaag gaaacccaca 660

cctagcgccc accgtttacg gcgtggacta ccagggtatc taatcctgtt cgctacccac 720

gctttcgctc ctcagcgtca gttactgccc agagacccgc cttcgccacc ggtgttcctc 780

ctgatatctg cgcatttcac cgctacacca ggaattccag tctcccctgc agtactcaag 840

tctgcccgta tcgcctgcaa gcccgcagtt gagctgcggg ttttcacaga cgacgcgaca 900

aaccgcctac gagctcttta cgcccagtaa ttccggacaa cgctcgcacc ctacgtatta 960

ccgcggctgc tggcacgtag ttggccggtg cttcttctgt acctaccgtc acttgcgctt 1020

cgtcggtact gaaagaggtt tacaacccga aggccgtcat ccctcacgcg gcgtcgctgc 1080

atcaggcttg cgcccattgt gcaatattcc ccactgctgc ctcccgtagg agtctgggcc 1140

gtgtctcagt cccagtgtgg ccggtcgccc tctcaggccg gctacccgtc gtcgccttgg 1200

tgggccgtta ccccaccaac aagctgatag gccgcgggcc catcctgcac cggaaaacct 1260

ttccaccccg gaacatgcat cccgaggtcc tatccggtat tagacccagt ttcccaggct 1320

tatcccgaag tgcagggcag atcacccacg tgttactcac ccgttcgcca ctaatccacc 1380

cagcaagctg ggcttcatcg ttcgactgca tgtgtaagca tcgccgccac gccatc 1436

<210>2

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

agagtttgat catggctcag 20

<210>3

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<212>DNA

<213> Artificial Sequence (Artificial Sequence)

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ggttaccttg ttacgactt 19

Claims

1. A method for separating high-concentration phenol and heavy metal resistant and low-temperature resistant rhodococcus is characterized by comprising the following steps:

2) high concentration phenol resistant mixed strain domestication

2.1 Low concentration phenol acclimation

Inoculating the enriched strain into the simulated wastewater in the reactor, adjusting the initial PH of the simulated wastewater to 7.0-8.0, setting the initial phenol concentration to 300mg/L, domesticating, replacing the simulated wastewater after the phenol concentration in the simulated wastewater in the reactor is lower than 30mg/L, increasing the phenol concentration in the simulated wastewater to 400mg/L, repeating until the phenol concentration in the simulated wastewater reaches 500mg/L, and the phenol removal rate is more than or equal to 90%, so as to obtain a low-concentration phenol domesticated bacterial liquid;

2.2 Medium concentration phenol acclimation

2.3 high phenol concentration acclimatization

3) isolation of pure bacteria

4) Screening: respectively inoculating a plurality of purified strains into a separation culture medium added with 1500-3000mg/L phenol, and screening out phenol removal rate more than or equal to 90% when the phenol concentration is less than 2500mg/L to obtain high-concentration-resistant phenol strains;

5) pure strain phenol tolerance domestication

2. The separation method according to claim 1, characterized in that: step 2), the low-concentration phenol acclimation time is 6-10 h; the acclimation time of the medium-concentration phenol is 16-18 h; the high-concentration phenol acclimation time is 24-72 h.

3. The separation method according to claim 1, characterized in that: and step 2) the reactor consists of a bacteria filter, an air duct, a constant-temperature heating stirrer, a container and an air pump.

4. The separation method according to claim 1, characterized in that: the step-by-step increase in the step 2) is to increase the concentration of phenol to 100mg/L each time.

5. The separation method according to claim 1, characterized in that: step 2.1 heavy metal ions of the simulated wastewater contain 500 mu mol/L of Cu²⁺、Cd²⁺、Pb²⁺、Cr³⁺、Ni²⁺And Mn²⁺。

6. The separation method according to claim 1, characterized in that: the separation medium is composed of Na₂HPO₄6-8g/L、KH₂PO₄2.77-5.54g/L，NaCl 0.3-1g/L、NH₄Cl 0.5-1.5g/L、CaCl₂0.01-0.05g/L、MgSO₄0.1-0.5 g/L; adding water, mixing, sterilizing and dissolving.

7. The separation method according to claim 1, characterized in that: the M9 solid culture medium is a separation culture medium containing 15-20g/L agar powder, and the phenol concentration of the M9 solid culture medium is 1000-2000 mg/L.

8. The separation method according to claim 1, characterized in that: the domestication method in the step 5): culturing colonies with maximum tolerance concentration of 10g/L phenol by dot plate method, culturing the colonies with LB liquid culture medium to obtain seed liquid, diluting according to gradient to obtain dot plate seed liquid with OD of 1.0, 0.5, 0.25, 0.125 and 0.0625, dropping 2 μ L of the seed liquid onto M9 solid culture medium plate containing higher concentration phenol, inducing and acclimatizing, standing and culturing at 30-37 deg.C, and observing every 24 h.

9. The separation method according to claim 8, characterized in that: the LB liquid culture medium is prepared by adding water into yeast powder 3-8g/L and peptone 8-12g/L, Nacl8-12g/L, mixing, sterilizing and dissolving.