CN116589102A

CN116589102A - Method for degrading phenol and method for synthesizing polyhydroxyalkanoate

Info

Publication number: CN116589102A
Application number: CN202310455260.7A
Authority: CN
Inventors: 石岩; 冉茵茵; 陈健欣; 张可菁; 谭晓倩; 李欣月; 柴立元; 林璋
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2023-04-25
Filing date: 2023-04-25
Publication date: 2023-08-15

Abstract

The application provides a degradation method of phenol, which comprises the following steps: inoculating copper barceli bacteria into a first culture medium with phenol concentration of 200-800 mg/L, culturing at a temperature of 26-36 ℃ at a rotating speed of 120-150 rpm, and realizing degradation of phenol through pyrolysis growth of the copper barceli bacteria; wherein the initial volume ratio of the copper bazedox bacteria to the first culture medium is 5-10%, and the pH value of the first culture medium is 5-11. The application can degrade phenol efficiently and in high quantity, has remarkable effect and is worth popularizing.

Description

Method for degrading phenol and method for synthesizing polyhydroxyalkanoate

Technical Field

The application belongs to the field of microorganism application, and particularly relates to a degradation method of phenol and a synthesis method of polyhydroxyalkanoate.

Background

Phenol is a phenolic volatile organic molecule and is mainly produced in coking, petrochemical, printing and dyeing, pharmaceutical and other industrial wastes. Phenol has the chemical formula C ₆ H ₅ OH, purified phenol is a colorless needle-like crystal with a unique odor. Phenol is sparingly soluble in water and is highly toxic and presents a risk of carcinogenesis to humans-when proteins are exposed to phenol, changes in protein properties can be caused.

Phenol is used in a wide variety of chemical branches as one of the most widely distributed chemicals, and as the chemical sector expands, a large amount of phenol-containing coking wastewater is produced, which is released into the environment, resulting in water pollution. Such as: if the phenol concentration in the water body is more than 1.5mg/L, the red blood cell count of the fish is reduced; if the phenol concentration is between 6.5 and 9.3mg/L, the gills and throat of the fish are damaged, resulting in abdominal bleeding. Expansion of the spleen gland disrupts the normal life process of fish; phenol concentrations between 5 and 20mg/L are toxic to fish.

Phenol is also not negligible to human toxicity: early poisoning is characterized by hyperexcitability, increased osmotic pressure associated with the body, shortness of breath, and acceleration of heart beat. The immediate response is excitation, followed by an increase in blood glucose and plasma concentration, paralysis, inability to breathe, and eventual death.

Currently, physical, chemical and biological treatments are the most common industrial techniques for remediation of wastewater containing phenolic organic contaminants. Physical treatment mainly utilizes physical action to remove or separate suspended solids and insoluble materials from water. However, after physical treatment, the toxicity of phenol in the wastewater is not reduced, and the problem of phenol pollution in the wastewater cannot be completely solved.

Chemical treatment is usually the extraction separation of phenol-containing organic waste water using organic solvents. However, even if the extraction separation is performed using the solvent extraction separation, the concentration of the phenolic compounds in the wastewater discharge cannot meet the national regulation level. Chemical treatments also include physical adsorption and chemical precipitation, and adsorbents generally include activated carbon, resins, and sulfided coal. However, the research of adsorbing phenol in wastewater by using activated carbon fibers is still in a starting stage, the combined industrial processing effect is not known, and the applicability is limited. The activated carbon has low price, large adsorption capacity and excellent effect, but is not easy to recycle. Chemical precipitation methods involve adding specific compounds to the wastewater, thereby precipitating phenol. Such as: phenol in the paper industry is treated by ion precipitation, and phenolic compounds are extracted and recovered. This method consumes a large amount of precipitation in the process of using precipitation, and may also cause secondary pollution.

The biological treatment cost is low, the economic benefit is high, the disposable treatment capacity is large, the ecological environment is not further damaged, and the biological treatment method is nontoxic and harmless and has no side effect. Therefore, it is widely used in the production and life of human beings. Such distribution is commonly used for treating industrial wastewater and is one of the important methods commonly used in wastewater treatment technologies. The high concentration and toxicity threatens the life of microorganisms, hampers the treatment efficiency of biological methods, and conversely reduces the degradation efficiency of traditional biological methods. That is, the biological treatment method cannot treat phenol waste liquid with high concentration and high toxicity.

Disclosure of Invention

Aiming at solving the technical problem that the prior art cannot treat high-concentration phenol waste liquid, the application provides a phenol degradation method, which comprises the following steps:

inoculating copper barceli bacteria into a first culture medium with phenol concentration of 200-800 mg/L, culturing at a temperature of 26-36 ℃ at a rotating speed of 120-150 rpm, and realizing degradation of phenol through pyrolysis growth of the copper barceli bacteria;

wherein the initial volume ratio of the copper bazedox bacteria to the first culture medium is 5-10%, and the pH value of the first culture medium is 5-11.

Further, the method further comprises the step of activating the copper bazier before inoculating the copper bazier, and the step of activating the copper bazier comprises the following steps: placing the copper bazedox bacteria in a second culture medium, and culturing for 12-96 h at the temperature of 29-32 ℃; wherein the volume ratio of the copper bazedox bacteria to the second culture medium is 5-10%.

Further, the composition of the first culture medium comprises 1-1.5 g/L KH ₂ PO ₄ 、2～2.5g/L (NH ₄ ) ₂ SO ₄ 、1～1.5g/L K ₂ HPO ₄ 、0.01～0.02g/L CaCl ₂ 、0.2～0.3g/L MgSO ₄ 、0.015～0.02g/L FeSO ₄ 、0.01～0.02g/L MnSO ₄ 。

Further, sodium chloride is added into the first culture medium, and the mass-volume ratio of the sodium chloride to the first culture medium is 10-50 g/L.

Further, in the first culture medium, the cultivation time of the copper barceli bacteria is 12-96 hours.

Further, the second medium comprises LB broth.

The application also provides a method for synthesizing the polyhydroxyalkanoate, which comprises the following steps:

inoculating copper barceli bacteria into a first culture medium with phenol concentration of 200-800 mg/L, culturing at a speed of 120-150 rpm and a temperature of 26-36 ℃, and degrading phenol by the cracking growth of the copper barceli bacteria to obtain the phenol degradation liquid; wherein the initial volume ratio of the copper bazedox bacteria to the first culture medium is 5-10%, and the pH value of the first culture medium is 5-11;

and extracting polyhydroxyalkanoate from the phenol degradation liquid.

Further, the step of extracting polyhydroxyalkanoate from the phenol degradation liquid comprises the following steps:

extracting the phenol degradation liquid, centrifuging at 12000rpm for 20min, and collecting precipitate; wherein the temperature of the centrifugation is 4-10 ℃;

lyophilizing the precipitate to obtain lyophilized product, homogenizing in chloroform, and diluting to obtain organic mixture;

and (3) placing the organic mixture in a 60-100 temperature environment, incubating for 12-96 hours at a rotating speed of 100-150rpm, and sequentially separating, concentrating and precipitating the polyhydroxyalkanoate-containing organic phase in the organic mixture to obtain the polyhydroxyalkanoate.

Further, the concentration of nitrogen element in the first culture medium is 25-75 mg/L.

Further, the nitrogen element in the first culture medium is derived from ammonium sulfate.

Compared with the prior art, the application at least comprises the following advantages:

the application creatively selects the copper barceli to treat the phenol waste liquid as the only carbon source in the cracking growth process of the copper barceli, and the phenol is efficiently and highly degraded in the growth process of the copper barceli. In the process of incubating the copper barceli, the concentration of phenol can reach 800mg/L, the tolerance of the copper barceli to phenol is utilized to the greatest extent, the inactivation threat of high-concentration and high-toxicity phenol to microorganisms is broken, and the synergistic progress of microorganism growth and high-concentration phenol degradation is realized.

The application strictly regulates and controls parameters such as temperature, rotating speed and the like in the growth process of the copper barceli bacteria, provides a good environment for the growth of the copper barceli bacteria, and effectively ensures the survival and incubation of the copper barceli bacteria.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present application, and other drawings may be obtained according to the structures shown in these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing the effect of different concentrations of phenol on bacterial growth in example 1 of the present application;

FIG. 2 is a graph showing the effect of the addition of 10g/L salt and phenol concentration on bacterial growth and phenol degradation rate for example 1 of the present application;

FIG. 3 is a plot of the effect of pH on dephenolization degradation capacity and copper Pasteur growth in accordance with the present application;

FIG. 4 is a plot of the effect of temperature on the rate of phenol degradation by copper bazedox in the present application;

FIG. 5 is a fluorescence microscopy image of intracellular PHA accumulated using a Nile red staining method in example 2 of the present application, and a schematic diagram showing the effect of nitrogen limitation on PHA accumulation by copper Basil by using phenol as a carbon source;

FIG. 6 is a graph showing the effect of phenol concentration on PHA accumulation in example 2 of the present application;

FIG. 7 is a graphical representation of a comparison of FTIR spectra of PHA produced by copper greedy bazerland and standard PHB in example 3 of the present application;

FIG. 8 is an H-NMR spectrum of PHA produced by copper greedy barsaint in example 3 of the present application;

FIG. 9 is a graph showing the thermal decomposition properties of PHA produced by thermogravimetric analysis in comparison to standard PHB in example 3 of the present application;

FIG. 10 is a graph showing the results of thermal analysis of PHB isolated from copper greedy bus and standard PHB by DCS analysis in example 3 of the present application;

FIG. 11SEM image and EDX pattern, EDX data illustrates the elements present in PHA, which were obtained from the SEM-EDX elemental pattern, carbon, oxygen for PHB produced by copper-bulimia barsaint in example 2.

Detailed Description

The following description of the embodiments of the present application will be made in detail and with reference to the accompanying drawings, wherein it is apparent that the embodiments described are only some, but not all embodiments of the present application. All other embodiments, based on the embodiments of the application, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the application.

Moreover, the technical solutions of the embodiments of the present application may be combined with each other, but it is necessary to be based on the fact that those skilled in the art can implement the embodiments, and when the technical solutions are contradictory or cannot be implemented, it should be considered that the combination of the technical solutions does not exist, and is not within the scope of protection claimed by the present application.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs and to which this application belongs, and any method, apparatus, or material of the prior art similar or equivalent to the methods, apparatus, or materials described in the examples of this application may be used to practice the application.

The application provides a degradation method of phenol, which comprises the following steps:

inoculating copper barceli bacteria into a first culture medium with phenol concentration of 200-800 mg/L, culturing for 12-96 hours at a temperature of 26-36 ℃ at a rotating speed of 120 rpm-150, and realizing degradation of phenol through pyrolysis growth of the copper barceli bacteria;

wherein the volume ratio of the copper bazedox bacteria to the first culture medium is 5-10%.

The copper barceli bacteria in the application can be lignin degradation bacteria cuprimavadusbasilensisB-8 with the preservation number of CGMCC No.4240, and can be particularly seen in patent document with the publication number of CN 102093971A.

In some embodiments, the method further comprises activating the copper barceli bacteria prior to inoculating the copper barceli bacteria, the activating the copper barceli bacteria comprising the steps of: placing the copper bazedox bacteria in a second culture medium, and culturing for 12-96 h at the temperature of 29-32 ℃; wherein the initial volume ratio of the copper bazedox bacteria to the second culture medium is 5-10%.

Illustratively, the second medium comprises an LB broth medium.

Preferably, the copper bazedox bacteria can be cultivated overnight at 29-32℃in LB broth on a rotating shaker at a speed of 120-150 rpm. Further, the activation of copper bazedox bacteria can be achieved by dilution in LB broth medium for 2h to reach logarithmic growth phase.

Preferably, after the cultivation is completed, the copper barceli bacterial liquid is centrifuged at 10000-12,000 rpm for 10-20 min, and then 5% of the inoculum is transferred to the first culture medium for providing phenol concentration.

Preferably, the seed transfer process may be performed under aerobic conditions.

The first medium may, for example, comprise a composition of 1 to 1.5g/L KH ₂ PO ₄ 、2～2.5g/L (NH ₄ ) ₂ SO ₄ 、1～1.5g/L K ₂ HPO ₄ 、0.01～0.02g/L CaCl ₂ 、0.2～0.3g/L MgSO ₄ 、0.015～0.02g/LFeSO ₄ 、0.01～0.02g/L MnSO ₄ 。

The pH of the first medium may be 5 to 11.

In some embodiments, sodium chloride may be added to the first medium prior to inoculation of copper bazedox bacteria, the mass to volume ratio of sodium chloride to the first medium being 10 to 50g/L. The addition of sodium chloride can effectively regulate the salinity of the first culture medium, so that the growth rate of the copper barceli bacteria under different salinity conditions and the degradation performance of the copper barceli bacteria on phenol can be measured. The copper bazedox bacteria can be applied to wastewater with high phenol concentration and high salinity.

The application provides an application of the phenol degradation method in phenol sewage treatment.

s1, inoculating copper barceli into a first culture medium with phenol concentration of 200-800 mg/L, culturing at a speed of 120-150 rpm and a temperature of 26-36 ℃, and degrading phenol by the cracking growth of the copper barceli to obtain a phenol degradation liquid; wherein the initial volume ratio of the copper bazedox bacteria to the first culture medium is 5-10%, and the pH value of the first culture medium is 5-11.

Ammonium sulfate is added into the first culture medium, and the concentration of nitrogen element in the first culture medium is controlled to be 25-75 mg/L.

The application makes the copper barceli strain in stress state by limiting the nitrogen concentration in the first culture medium.

Specifically, ammonium sulfate with the concentration of 0.125-0.5 g/L can be added into the first culture medium, the fermentation is carried out for 7 days at the speed of 150rpm at the temperature of 30 ℃, and in the process, the tin dyeing and the dyeing are carried out on the biological synthesized PHA by adopting a Nile red method, and the accumulation time is detected by a fluorescence microscope. During seven days of incubation, 2ml samples were withdrawn every 12 h. The supernatant of the sample was removed and the cells in the sample were resuspended with 150. Mu.l deionized water and 50. Mu.l dimethyl sulfoxide to give a suspension.

Subsequently, 40 μl of nile red (80 μg/ml dissolved in acetone) was added to the suspension to give a final concentration of 3.1 μg nile red per ml of suspension, and incubated for 30min at room temperature. Aliquots of the suspension were pipetted into 96-well microwell plates. Fluorescence was then read at excitation and emission wavelengths 535 and 605nm, respectively, using a Wallac EnVision Manager 1.12.1.12 software program in an EnVision multi-label plate reader using a monochromator.

By way of example, the characteristics of polyhydroxyalkanoates and their biopolymers can be characterized by fourier transform infrared spectroscopy, HNMR spectroscopy, thermogravimetric analysis, differential scanning calorimetry, and the like.

S2, extracting polyhydroxyalkanoate from the phenol degradation liquid.

The method specifically comprises the following steps:

s10, extracting the phenol degradation liquid, centrifuging for 15-20 min at a rotating speed of 10000-12000 rpm, and collecting precipitate; wherein the temperature of the centrifugation is 4-10 ℃;

s20, freeze-drying the bacterial precipitate to obtain a freeze-dried product, and homogenizing and diluting the freeze-dried product in chloroform to obtain an organic mixture;

s30, placing the organic mixture in a 60-100 temperature environment, incubating for 8-12 hours at a rotating speed of 100-150rpm, and sequentially separating, concentrating and precipitating the polyhydroxyalkanoate-containing organic phase in the organic mixture to obtain the polyhydroxyalkanoate.

Further refining: PHA (i.e., polyhydroxyalkanoate, the same applies hereinafter) was extracted by chloroform-methanol method. 100ml of copper barceli bacteria solution (namely the phenol degradation solution, the same as described below) after the reaction in the first culture medium is extracted, and after centrifugation at 12000rpm for 20min at 4 ℃, the precipitate is collected and washed with 10ml of deionized water for more than two times to obtain bacteria. The bacterial particles are frozen and lyophilized to obtain a lyophilized product. The lyophilisate was ground and then homogenized in 1ml chloroform. To re-suspend the dried biomass, the slurry is vigorously vortexed. Then, more chloroform was added until a concentration of 1g of stem cells in 25ml of chloroform was reached in the slurry, to obtain an organic mixture.

To extract the polymer formed in the bacteria, the organic mixture was incubated overnight at 60℃in a water bath at 100-150 rpm. After adding 2ml of deionized water, the organic mixture was stirred well for 5min. Then, the liquid was separated from the organic mixture using centrifugation, the organic phase containing the polyhydroxyalkanoate soluble in chloroform was collected, and nitrogen was discharged through a syringe filter having a 0.45m PTFE membrane, and the polyhydroxyalkanoate organic phase was concentrated to 1ml. The precipitate was separated by adding ten times the amount of cooled methanol, stirring and mixing the methanol with the polyhydroxyalkanoate organic phase for 10 to 30min, centrifuging at 2500rpm and 4℃for 15min, and washing the precipitate twice with methanol. The precipitate containing polyhydroxyalkanoate was dried at room temperature with a nitrogen flow. The dried product was then dissolved in chloroform, filtered again, and added dropwise to rapidly mixing methanol. As previously described, the precipitate was separated by centrifugation prior to drying to yield PHA.

Plastic is an important material in modern life, consuming/producing more than 1.4 million tons of plastic per year. Producing such large quantities of plastics requires the use of about 1.5 hundred million tons of chemical fossil fuel and generates large amounts of waste, which can take thousands of years to depolymerize. However, the impact of plastic products on the environment is increasing and petroleum resources are gradually depleted.

As a substitute for plastics, studies on the production of polyhydroxyalkanoates (namely PHAs described below) have meant extremely high economic and environmental benefits. Polyhydroxyalkanoate is an intracellular polyester synthesized by copper bazier, and exists mainly as a carbon source and a storage substance of energy source in copper bazier, and has many excellent properties similar to the physicochemical properties of synthetic plastics and biodegradability, biocompatibility, optical activity, piezoelectricity, gas-barrier property and the like which are not possessed by synthetic plastics. The polyhydroxyalkanoate has wide application prospect in the aspects of biodegradable packaging materials, tissue engineering materials, slow-release materials, electrical materials and medical materials. However, PHA is difficult to be applied on a large scale due to the production cost.

The application directionally regulates and controls the phenol degradation products of the copper barceli to generate a large amount of polyhydroxyalkanoate products with high application value, can be applied to various fields instead of plastics, and effectively reduces the production cost and application threshold of polyhydroxyalkanoate.

To facilitate a further understanding of the application by those skilled in the art, reference is now made to the accompanying drawings, in which:

it should be noted that, the English definitions appearing in the drawings are as follows:

phenol degradation percent phenol degradation; time-time; DCW & PHA-copper bescens dry weight and PHA weight; PHA content PHA-percentage; fluorescence microscopy image of intracellular accumulated PHA-fluorescence microscopy images of intracellular accumulated PHA; transmissittance-transmittance; wavelength-wavelength; chemical shift; DTG-derivative thermogravimetry; TG-thermogravimetry; temperature-temperature; DCS differential scanning calorimetry; temperature, melting enthalpy of mering enthalpy; decomposition enthalpy decomposition; exothermic is exothermic.

Example 1

Copper barceli was inoculated under sterile conditions into 100ml of LB broth (including tryptone 10g/l, yeast extract 5g/l and NaCl 10g/l, which can be abbreviated as LB broth, the same shall apply hereinafter) and activated by culture at 30℃for 18h on a rotary shaker (150 rpm). The activated copper bazedox was centrifuged at 12000rpm for 10min, washed and resuspended in mineral salt medium (i.e., first medium, the same applies hereinafter) providing phenol at different concentrations of 200-800 mg/L to expand the culture and incubated on a rotary shaker (150 rpm).

Wherein, the copper barceli may be lignin degradation bacteria cuprimavadusbasilensisB-8 with a preservation number of CGMCC No. 4240.

NaCl (10-50 g/L) with different concentrations is added into the first culture medium, and phenol degradation under different salinity conditions is studied. After anaerobic incubation at 30℃for 3 days, the phenol concentration of phenol in the medium was monitored every 12 h. The culture was centrifuged at 12000rpm for 20min and the phenol concentration in the supernatant was monitored using the 4-aminoantipyrine method.

The degradation properties of the strains were determined under different temperature conditions. In mineral salt medium, the concentration of phenol is changed by 200mg/L, the pH value is 7, the rotating speed of the vibrating screen is 150rpm, and the temperature is 26-34 ℃. After 12h of incubation, the phenol content remaining in the medium was determined.

By combining FIGS. 1-4, FIG. 1 shows a comparison of the effect of different concentrations of phenol on the growth of copper Basil at pH 7, temperature 30, and rotational speed 150rpm, it can be seen that as the concentration of phenol increases, the degradation time also increases, and the strain can degrade phenol up to 800mg/L in 96 hours, indicating that copper Basil has very strong tolerance to phenol.

FIG. 2 illustrates the effect of varying concentrations of phenol and 10g/LNaCl on degradation rate and growth of copper Pasteur at pH 7, temperature 30, and rotational speed 150rpm. The phenol concentration of 400mg/L was completely degraded after 144 hours, indicating salt tolerance of the copper Basil strain.

FIG. 3 shows the effect of pH on dephenolization and degradation capacity of copper Pasteur bacteria growth. The degradation properties of the copper bazedox bacteria were determined under different initial pH conditions. In mineral salt medium, the concentration of phenol is 200mg/L, the pH value of the medium is respectively adjusted to 5-11, the temperature is 30 ℃, and the rotating speed is 150rpm. After 12h of incubation in a shaker, the phenol content remaining in the mineral salt medium was determined. It can be seen that, at a pH between 6 and 9, the copper bazedox species can utilize phenol as a carbon source and degrade it; for best results, the ideal pH is 7. When the pH value is between 7 and 8, the degradation performance of the strain is improved, and the degradation rate can reach 99.9% after 24 hours.

Example 2

In this example, the ability to produce copper polyhydroxyalkanoate, basel, using phenol as the sole carbon source was explored. The overnight activated bacteria were centrifuged at 12000rpm for 10min. Copper bescens barceli bacteria were resuspended in mineral salt medium providing phenol concentration and limited to nitrogen. Accumulation of PHA was monitored by fluorescence microscopy.

In the experiment, ammonium sulfate with different concentrations is added into the first culture medium, and the concentration is 0.125-0.5 g/L. The concentration of phenol is 200-800 mg/L, the pH value is 7, the temperature is 30 ℃, and the rotation/min is 150. Fermentation time exceeds 7 days, and in the process, the biological synthesized PHA is subjected to tin dyeing by using a Nile red method, and the accumulation time is detected by using a fluorescence microscope. During seven days of incubation, 2ml samples were withdrawn every 12 h. The supernatant was removed and the cells resuspended with 150. Mu.l deionized water and 50. Mu.l dimethyl sulfoxide.

Subsequently, 40. Mu.l of nile red (80. Mu.g/ml dissolved in acetone) was added to the suspension, giving a final concentration of 3.1 micrograms of nile red per ml of suspension, and incubated for 30min at room temperature. Fluorescence was then read at excitation and emission wavelengths 535 and 605nm, respectively, using a Wallac EnVision Manager 1.12.1.12 software program in an EnVision multi-label plate reader using a monochromator.

Extracting PHA by chloroform methanol method. 100ml of copper barceli bacteria liquid (namely the phenol degradation liquid) which is reacted in the first culture medium is extracted, and the mixture is centrifuged at 12000rpm for 20min at 4 ℃. After washing twice with 10ml of deionized water, the precipitate was collected. The precipitated bacterial particles were then frozen and lyophilized, the lyophilized material was ground and then homogenized in 1ml chloroform. To re-suspend the dried biomass, the slurry was vortexed vigorously and more chloroform was added until a concentration of 1g stem cells in 25ml chloroform was reached in the slurry, yielding an organic mixture.

To extract the polymer formed in the copper bazedox bacteria, the organic mixture is incubated overnight at 60℃in a water bath at 100-150 rpm. After adding 2ml of deionized water, the organic mixture was stirred well for 5min. The liquid is then separated from the cell debris using centrifugation. The organic phase containing the chloroform soluble polyhydroxyalkanoate was collected and passed through a syringe filter with a 0.45m PTFE membrane. The nitrogen was vented and the organic solution was concentrated to 1ml. The extracted solvent was precipitated by adding ten times the amount of previously cooled methanol and mixing it with a rotating machine for 10 to 30min. Centrifugation was used to separate the precipitate from the liquid at 2500rpm and 4 ℃ for 15min to remove the supernatant, which was washed twice with methanol. The polymer-containing particles were dried at room temperature with a nitrogen stream. The final product was then dissolved in chloroform, filtered again, and added dropwise to rapidly mixing methanol.

The first culture medium comprises the following components: KH (KH) ₂ PO ₄ 、(NH ₄ ) ₂ SO ₄ 2 g/L、K ₂ HPO ₄ 1g/L、CaCl ₂ 0.01g/L、MgSO ₄ 0.2 g/L、FeSO ₄ 0.015 g/L、MnSO ₄ 0.01 g/L。

FIG. 5 shows that a decrease in nitrogen concentration from 100mg/L to 25mg/L significantly affected PHA production and a significant increase in fluorescence, PHA content of 9.9% and 4.8%, respectively. In addition, with time, PHA content showed a significant upward trend, reaching a maximum at 36h, when the nitrogen concentration was 25 mg/L; at this time, PHA content was 12.34% of dry weight of the copper-bulbus barsaint.

FIG. 6 shows the effect of phenol concentration on PHA production. When the cells were cultured in the presence of 400mg/L phenol, the maximum yield of PHA was 124mg. At 800 mg/L. Phenol concentrations were 600mg/L and 200mg/L, with yields of 34.5, 98 and 56mg PHA per liter, respectively.

Example 3

In this example, characterization of cumulative PHA produced by copper greedy Basil using phenol as the sole carbon source was explored. Copper bazedox was inoculated 1 night in LB medium and then diluted 5-fold in fresh LB medium. When the logarithmic phase was reached, the culture was centrifuged at 12000rpm for 20min, then resuspended in mineral salt medium containing 25mg/L nitrogen and 400mg/L phenol and inoculated at 30℃in a rotary shaker at 150rpm for 36h. After extraction by chloroform, PHA was characterized as described in the previous example.

FIG. 7 shows the FTIR spectrum results of PHA isolated for copper greedy barceli. About 1277cm in FTIR spectrum in the presence of 400mg/L phenol ^-1 The absorbance peak at this point shows saturated ester bonds of the C-O group. At 1378 and 1452cm ^-1 The absorption peaks at the sites respectively indicate methyl (-CH) ₃ ) Stretching and bending modes of radical vibration. The unique absorption peaks of carbonyl (c=o) and methyl (-CH) groups are located at 1720, 2932cm ^-1 . FIG. 7 shows the IR spectrum of PHB (one type of PHA) produced by copper greedy barsaint and the IR spectrum of standard PHB; the similarity of these two products is apparent.

FIG. 8 shows the H-NMR spectrum of PHA produced by copper Pasteur species using phenol as the sole carbon source. Characteristic peaks of PHB are detected, e.g., -CH bimodal delta=5.21 and 5.23ppm, -CH ² δ=2.50 and 2.61ppm, -CH for multiple peaks ³ Bimodal δ=1.21. The large peak of δ=7.3 ppm represents solvent (CHCl) ₃ ) While the small peaks at δ=1.61 and 1.63ppm are due to slight H-O contamination of the solvent. These findings are identical to the results obtained using the PHB standard.

FIG. 9 shows the thermal decomposition properties of the PHA produced by thermogravimetric analysis compared to standard PHB. The thermal stability of PHB produced by copper Pasteur was studied by thermogravimetric analysis. The decomposition of the polymer was completed in one stage and occurred at a temperature of 273.41 ℃, the polymer was completely degraded, which was very close to the decomposition temperature of standard PHB.

FIG. 10 shows a comparison of the thermal analysis of isolated PHB from copper Pasteur form and standard PHB by DCS analysis. The melting temperature, glass transition temperature and heat associated with melting of PHB were analyzed by DSC. T of PHB _m 182.61 ℃and a first-peak heat of fusion of 45.09J/g. The second peak appears at 292.4℃and is related to a heat of 47.6J/g, which is also similar to the DCS results of standard PHB.

In the above technical solution of the present application, the above is only a preferred embodiment of the present application, and therefore, the patent scope of the present application is not limited thereto, and all the equivalent structural changes made by the description of the present application and the content of the accompanying drawings or the direct/indirect application in other related technical fields are included in the patent protection scope of the present application.

Claims

1. A method for degrading phenol, comprising the steps of:

2. The degradation method according to claim 1, further comprising the step of activating the copper bazedox bacteria before inoculating the copper bazedox bacteria, said activating the copper bazedox bacteria comprising the steps of: placing the copper bazedox bacteria in a second culture medium, and culturing for 12-96 h at the temperature of 29-32 ℃; wherein the volume ratio of the copper bazedox bacteria to the second culture medium is 5-10%.

3. The degradation method according to claim 1, wherein the composition of the first medium comprises 1 to 1.5g/LKH ₂ PO ₄ 、2～2.5g/L(NH ₄ ) ₂ SO ₄ 、1～1.5g/LK ₂ HPO ₄ 、0.01～0.02g/LCaCl ₂ 、0.2～0.3g/L MgSO ₄ 、0.015～0.02g/LFeSO ₄ 、0.01～0.02g/LMnSO ₄ 。

4. The degradation method according to claim 1, wherein sodium chloride is added to the first medium, and the mass-volume ratio of the sodium chloride to the first medium is 10 to 50g/L.

5. The degradation method according to claim 1, wherein the culturing time of the copper barceli in the first medium is 12 to 96 hours.

6. The degradation method according to claim 2, wherein the second medium comprises LB broth.

7. A method for synthesizing polyhydroxyalkanoate, which is characterized by comprising the following steps:

inoculating copper barceli bacteria into a first culture medium with phenol concentration of 200-800 mg/L, culturing for 12-96 hours at a temperature of 26-36 ℃ at a rotating speed of 120-150 rpm, and degrading phenol by the pyrolysis growth of the copper barceli bacteria to obtain a phenol degradation liquid; wherein the initial volume ratio of the copper bazedox bacteria to the first culture medium is 5-10%, and the pH value of the first culture medium is 5-11;

and extracting polyhydroxyalkanoate from the phenol degradation liquid.

8. The method of synthesizing according to claim 7, wherein the extracting polyhydroxyalkanoate in the phenol degradation liquid comprises the steps of:

extracting the phenol degradation liquid, centrifuging at a rotating speed of 10000-12000 rpm for 15-20 min, and collecting precipitate; wherein the temperature of the centrifugation is 4-10 ℃;

and (3) placing the organic mixture in a 60-100 temperature environment, incubating for 8-12 hours at a rotating speed of 100-150rpm, and sequentially separating, concentrating and precipitating the polyhydroxyalkanoate-containing organic phase in the organic mixture to obtain the polyhydroxyalkanoate.

9. The method according to claim 7, wherein the concentration of nitrogen element in the first medium is 13 to 75mg/L.

10. The method of claim 9, wherein the nitrogen element in the first medium is derived from ammonium sulfate.