Method for degrading phenol and method for synthesizing polyhydroxyalkanoate

Abstract

The application provides a phenol degradation method, comprising the following steps: inoculating Cupriavidus basellens in a first culture medium with a phenol concentration of 200-800 mg / L, culturing at a temperature of 26-36 DEG C under a rotating speed of 120-150 rpm, and realizing phenol degradation through lysis growth of the Cupriavidus basellens; wherein the initial volume ratio of the Cupriavidus basellens to the first culture medium is 5-10%, and the pH value of the first culture medium is 5-11. The application can efficiently and highly degrade phenol, has a remarkable effect, and is worth promoting.

Description

Technical Field

[0001] This invention belongs to the field of microbial applications, specifically relating to a method for the degradation of phenol and a method for the synthesis of polyhydroxy fatty acid esters. Background Technology

[0002] Phenol is a volatile phenolic organic molecule, mainly produced in industrial waste from coking, petrochemical, printing and dyeing, and pharmaceutical industries. The chemical formula of phenol is C6H5OH. Purified phenol is a colorless, needle-like crystal with a distinctive odor. Phenol is slightly soluble in water, highly toxic, and poses a carcinogenic risk to humans—when proteins are exposed to phenol, their properties are altered.

[0003] As one of the most widely distributed chemical substances, phenol is used in various branches of chemistry. With the expansion of the chemical industry, large amounts of phenol-containing coking wastewater have been generated, which is released into the environment, causing water pollution. For example, if the concentration of phenol in water exceeds 1.5 mg / L, the red blood cell count in fish will decrease; if the concentration is between 6.5 and 9.3 mg / L, it will damage the gills and throat of fish, leading to abdominal bleeding. The expansion of the spleen disrupts the normal life processes of fish; phenol concentrations between 5 and 20 mg / L are toxic to fish.

[0004] The toxicity of phenol to humans should not be ignored: early signs of poisoning include over-excitement, increased osmotic pressure related to the body, rapid breathing, and accelerated heartbeat. The immediate reaction is excitement, followed by elevated blood sugar and plasma concentrations, paralysis, respiratory failure, and ultimately death.

[0005] Currently, physical, chemical, and biological treatments are the most common industrial technologies for remediating wastewater containing phenolic organic pollutants. Physical treatment mainly utilizes physical processes to remove or separate suspended solids and insoluble substances from water. However, physical treatment does not reduce the toxicity of phenol in the wastewater, and the phenol pollution problem in wastewater cannot be completely solved.

[0006] Chemical treatment typically utilizes organic solvent extraction to separate phenol-containing organically damaged wastewater. However, even with solvent extraction, the concentration of phenolic compounds in the wastewater discharge often fails to meet national standards. Chemical treatment also includes physical adsorption and chemical precipitation, with adsorbents typically including activated carbon, resin, and coal sulfide. However, research on using activated carbon fibers to adsorb phenol from wastewater is still in its early stages, and the effectiveness of combined industrial processing is unknown, limiting its applicability. Activated carbon is inexpensive, has a large adsorption capacity, and is highly effective, but it is difficult to recycle. Chemical precipitation methods involve adding specific compounds to the wastewater to precipitate phenol. For example, ion precipitation treats phenol in the paper industry, simultaneously extracting and recovering phenolic compounds. This method not only consumes a large amount of precipitation during the utilization of rainwater but may also generate secondary pollution.

[0007] Biological treatment is low-cost, highly economical, and can process large volumes at a time without further damage to the ecological environment. It is non-toxic, harmless, and has no side effects. Therefore, it has been widely used in human production and daily life. This method is commonly used to treat industrial wastewater and is one of the most important and frequently used wastewater treatment technologies. However, high concentrations and toxicity threaten the life of microorganisms, hindering the treatment efficiency of biological methods; in fact, the degradation efficiency of traditional biological methods may actually decrease. That is, biological treatment methods cannot treat high-concentration, highly toxic phenol wastewater. Summary of the Invention

[0008] To address the technical problem that existing technologies cannot handle high-concentration phenol wastewater, this invention provides a method for degrading phenol, comprising the following steps:

[0009] Basellus spp. was inoculated into a first culture medium with a phenol concentration of 200–800 mg / L and cultured at a speed of 120–150 rpm and a temperature of 26–36 °C. The degradation of phenol was achieved through the lytic growth of Basellus spp.

[0010] The initial volume ratio of the *Basellium clavatum* to the first culture medium is 5-10%, and the pH value of the first culture medium is 5-11.

[0011] Furthermore, before inoculating *Basellus salvia*, the method includes activating the *Basellus salvia*, which includes the steps of: placing the *Basellus salvia* in a second culture medium and culturing it at a temperature of 29–32°C for 12–96 hours; wherein the volume ratio of the *Basellus salvia* to the second culture medium is 5–10%.

[0012] Furthermore, the first culture medium comprises 1–1.5 g / L KH₂PO₄, 2–2.5 g / L (NH₄)₂SO₄, 1–1.5 g / L K₂HPO₄, 0.01–0.02 g / L CaCl₂, 0.2–0.3 g / L MgSO₄, 0.015–0.02 g / L FeSO₄, and 0.01–0.02 g / L MnSO₄.

[0013] Furthermore, sodium chloride is added to the first culture medium, wherein the mass-to-volume ratio of sodium chloride to the first culture medium is 10–50 g / L.

[0014] Furthermore, in the first culture medium, the culture time of the *Basellium clavatum* is 12–96 h.

[0015] Furthermore, the second culture medium includes LB broth medium.

[0016] This invention also provides a method for synthesizing polyhydroxy fatty acid esters, comprising the following steps:

[0017] Basellus coccidioides was inoculated into a first culture medium with a phenol concentration of 200–800 mg / L and cultured at 26–36°C with a rotation speed of 120–150 rpm. The degradation of phenol was achieved through the lytic growth of Basellus coccidioides to obtain the phenol degradation solution. The initial volume ratio of Basellus coccidioides to the first culture medium was 5–10%, and the pH of the first culture medium was 5–11.

[0018] Polyhydroxy fatty acid esters were extracted from the phenol degradation solution.

[0019] Furthermore, the extraction of polyhydroxy fatty acid esters from the phenol degradation solution includes the following steps:

[0020] The phenol degradation solution was extracted, centrifuged at 12,000 rpm for 20 min, and the precipitate was collected; wherein the centrifugation temperature was 4–10 °C.

[0021] The precipitate was freeze-dried to obtain a freeze-dried product, which was then homogenized and diluted in chloroform to obtain an organic mixture.

[0022] The organic mixture is placed in a temperature environment of 60℃~100℃ and incubated at a speed of 100-150rpm for 12~96h. The organic phase containing polyhydroxy fatty acid ester in the organic mixture is then separated, concentrated, and precipitated sequentially to obtain the polyhydroxy fatty acid ester.

[0023] Furthermore, the nitrogen concentration in the first culture medium is 25–75 mg / L.

[0024] Furthermore, the nitrogen element in the first culture medium is derived from ammonium sulfate.

[0025] Compared with the prior art, the present invention has at least the following advantages:

[0026] This invention innovatively selects *Basella albopictus* to treat phenol wastewater, using it as the sole carbon source during the pyrolytic growth process of *Basella albopictus*. Phenol is efficiently and massively degraded during the growth of *Basella albopictus*. During the incubation process of *Basella albopictus*, the phenol concentration can reach as high as 800 mg / L, maximizing the tolerance of *Basella albopictus* to phenol. This overcomes the threat of inactivation of microorganisms by high concentrations and high toxicity of phenol, achieving synergistic progress in microbial growth and high-concentration phenol degradation.

[0027] This invention strictly controls parameters such as temperature and rotation speed during the growth of *Basella affinis*, providing an excellent environment for its growth and effectively ensuring its survival and incubation. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0029] Figure 1 This is a schematic diagram comparing the effects of different concentrations of phenol on bacterial growth in Example 1 of this application;

[0030] Figure 2 This is a schematic diagram comparing the effects of adding 10 g / L salt and phenol concentration on bacterial growth and phenol degradation rate in Example 1 of this application.

[0031] Figure 3 This is a line graph showing the effect of pH on the dephenolization capacity and the growth of *Bacillus basellii* in this application.

[0032] Figure 4 Line graph showing the effect of temperature on the rate of phenol degradation by *Bacillus basellii* in this application;

[0033] Figure 5 is a fluorescence microscope image of PHA accumulated in cells stained with Nile Red in Example 2 of this application, and a schematic diagram of the effect of nitrogen limitation on PHA accumulation in Bacillus basellii by using phenol as a carbon source.

[0034] Figure 6 This is a schematic diagram illustrating the effect of phenol concentration on PHA accumulation in Example 2 of this application;

[0035] Figure 7 This is a schematic diagram comparing the FTIR spectra of PHA produced by *Bacillus baseneri* and standard PHB in Example 3 of this application;

[0036] Figure 8 The image shows the 1H-NMR spectrum of PHA produced by *Bacillus basellae* in Example 3 of this application.

[0037] Figure 9 In Example 3 of this application, the thermal decomposition characteristics of the produced PHA were compared with those of standard PHB by thermogravimetric analysis.

[0038] Figure 10 This is a thermal analysis result diagram comparing PHB isolated from *Bacillus basellae* and standard PHB using DCS analysis in Example 3 of this application.

[0039] Figure 11SEM images and EDX spectra, with EDX data illustrating the elements present in PHA, obtained from SEM-EDX elemental spectra, for the carbon and oxygen of PHA produced by *Basella alba* in implementation 2. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0042] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention, as well as the prior art known to those skilled in the art and the description of this invention, may be implemented using any prior art methods, devices, and materials similar to or equivalent to those described, used, or made of materials in the embodiments of this invention.

[0043] This invention provides a method for degrading phenol, comprising the following steps:

[0044] Basellus spp. was inoculated into a first culture medium with a phenol concentration of 200–800 mg / L and cultured at 120–150 rpm and 26–36 °C for 12–96 h. Phenol degradation was achieved through the lytic growth of Basellus spp.

[0045] The volume ratio of the *Basellium clavatum* to the first culture medium is 5-10%.

[0046] The copper-degrading bacteria in this application can be Cupriavidus basilensis B-8, which has the accession number CGMCC No.4240. For details, please refer to the patent document with publication number CN 102093971 A.

[0047] In some embodiments, the inoculation of *Basella affinis* further includes activating the *Basella affinis*, wherein activating the *Basella affinis* includes the steps of: placing the *Basella affinis* in a second culture medium and culturing it at a temperature of 29°C to 32°C for 12 to 96 hours; wherein the initial volume ratio of the *Basella affinis* to the second culture medium is 5 to 10%.

[0048] For example, the second culture medium includes LB broth medium.

[0049] Preferably, *Basellium clavatum* can be cultured overnight in LB broth medium on a rotary shaker at 29–32°C and a rotation speed of 120–150 rpm. Further, it can be diluted 5-fold in LB broth medium and allowed to grow for 2 hours to reach the logarithmic growth phase, thereby activating *Basellium clavatum*.

[0050] Preferably, after cultivation, the *Basella alba* bacterial suspension can be centrifuged at 10,000–12,000 rpm for 10–20 min, and then 5% of the inoculum can be transferred to a first culture medium providing phenol concentration.

[0051] Preferably, the transinoculation process can be carried out under aerobic conditions.

[0052] For example, the composition of the first culture medium may include 1-1.5 g / L KH2PO4, 2-2.5 g / L (NH4)2SO4, 1-1.5 g / L K2HPO4, 0.01-0.02 g / L CaCl2, 0.2-0.3 g / L MgSO4, 0.015-0.02 g / L FeSO4, and 0.01-0.02 g / L MnSO4.

[0053] The pH of the first culture medium can be 5 to 11.

[0054] In some embodiments, sodium chloride can be added to the first culture medium before inoculation with *Basella alba*, wherein the mass-to-volume ratio of sodium chloride to the first culture medium is 10–50 g / L. The addition of sodium chloride can effectively adjust the salinity of the first culture medium, thereby allowing for the determination of the growth rate of *Basella alba* and its degradation performance for phenol under different salinity conditions. The *Basella alba* of this invention can be applied to wastewater with high phenol concentration and high salinity.

[0055] This invention innovatively selects *Basella albopictus* to treat phenol wastewater, using it as the sole carbon source during the pyrolytic growth process of *Basella albopictus*. Phenol is efficiently and massively degraded during the growth of *Basella albopictus*. During the incubation process of *Basella albopictus*, the phenol concentration can reach as high as 800 mg / L, maximizing the tolerance of *Basella albopictus* to phenol. This overcomes the threat of inactivation of microorganisms by high concentrations and high toxicity of phenol, achieving synergistic progress in microbial growth and high-concentration phenol degradation.

[0056] This invention strictly controls parameters such as temperature and rotation speed during the growth of *Basella affinis*, providing an excellent environment for its growth and effectively ensuring its survival and incubation.

[0057] This invention provides the application of the phenol degradation method described in any one of the above claims in phenol wastewater treatment.

[0058] This invention also provides a method for synthesizing polyhydroxy fatty acid esters, comprising the following steps:

[0059] S1. Inoculate *Basella cordata* into a first culture medium with a phenol concentration of 200–800 mg / L, and culture at a speed of 120–150 rpm and a temperature of 26–36°C. The degradation of phenol is achieved through the lytic growth of *Basella cordata*, yielding a phenol degradation solution. The initial volume ratio of *Basella cordata* to the first culture medium is 5–10%, and the pH of the first culture medium is 5–11.

[0060] Add ammonium sulfate to the first culture medium to control the nitrogen concentration in the first culture medium to be 25-75 mg / L.

[0061] This invention puts *Basellus* in a state of stress by limiting the nitrogen concentration in the first culture medium.

[0062] Specifically, ammonium sulfate at a concentration of 0.125–0.5 g / L can be added to the first culture medium, and fermentation can be carried out at 30°C and 150 rpm for 7 days. During this process, the biosynthesized PHA is stained with Nile Red using tin staining, and its accumulation time is detected by fluorescence microscopy. During the seven-day incubation period, 2 ml of sample is drawn every 12 hours. The supernatant of the sample is removed, and the cells in the sample are resuspended in 150 μl of deionized water and 50 μl of dimethyl sulfoxide to obtain a suspension.

[0063] Subsequently, 40 μl of Nile Red (dissolved in acetone at 80 μg / mL) was added to the suspension to obtain a final concentration of 3.1 μg Nile Red per mL of suspension, and the mixture was incubated at room temperature for 30 min. Aliquots of the suspension were then pipetted into 96-well microplates. Fluorescence was then read at excitation and emission wavelengths of 535 nm and 605 nm using a Wallac EnVision multi-label plate reader on a monochromator with Wallac EnVision Manager 1.12 software.

[0064] For example, the characteristics of polyhydroxyalkanoates and their properties as biopolymers can be characterized by methods such as Fourier transform infrared spectroscopy, ¹H NMR spectroscopy, thermogravimetric analysis, and differential scanning calorimetry.

[0065] S2. Extract polyhydroxy fatty acid esters from the phenol degradation solution.

[0066] The specific steps include:

[0067] S10. Extract the phenol degradation solution, centrifuge at 10000-12000 rpm for 15-20 min, and collect the precipitate; wherein the centrifugation temperature is 4-10℃;

[0068] S20. Freeze-dry the bacterial precipitate to obtain a freeze-dried product, then homogenize and dilute the freeze-dried product in chloroform to obtain an organic mixture;

[0069] S30. The organic mixture is placed in a temperature environment of 60℃~100℃ and incubated at a speed of 100-150rpm for 8~12h. The organic phase containing polyhydroxy fatty acid ester in the organic mixture is then separated, concentrated, and precipitated sequentially to obtain the polyhydroxy fatty acid ester.

[0070] Further refinement: PHA (polyhydroxyalkanoates, hereinafter the same) was extracted using the chloroform-methanol method. 100 ml of the *Basella alba* bacterial culture (i.e., the phenol degradation solution mentioned above, hereinafter the same) after the reaction in the first culture medium was extracted, and centrifuged at 12000 rpm for 20 min at 4°C. The precipitate was collected and washed at least twice with 10 ml of deionized water to obtain bacteria. The bacterial particles were frozen and lyophilized to obtain lyophilized material. The lyophilized material was ground and then homogenized in 1 ml of chloroform. To resuspend the dried biomass, the slurry was vigorously vortexed. Then, more chloroform was added until the slurry reached a concentration of 1 g of stem cells per 25 ml of chloroform, yielding an organic mixture.

[0071] To extract the polymer formed in the bacteria, the organic mixture was incubated overnight in a water bath at 60°C and 100–150 rpm. After adding 2 ml of deionized water, the organic mixture was stirred thoroughly for 5 min. The liquid was then separated from the organic mixture by centrifugation, collecting the organic phase containing chloroform-soluble polyhydroxyalkanoates (PHA). The PHA organic phase was concentrated to 1 ml by purging nitrogen through a syringe filter with a 0.45 μm PTFE membrane. The PHA organic phase was then mixed with ten times the volume of cooled methanol for 10–30 min, followed by centrifugation at 2500 rpm and 4°C for 15 min to separate the precipitate. The precipitate was washed twice with methanol. The precipitate containing PHA was dried at room temperature using a nitrogen stream. The dried product was then dissolved in chloroform, filtered again, and added dropwise to rapidly mixed methanol. The precipitate was separated by centrifugation before drying, as previously described, to obtain PHA.

[0072] Plastic, as an essential material in modern life, is consumed / produced at a rate exceeding 140 million tons annually. Producing such a large quantity of plastic requires approximately 150 million tons of chemical fossil fuels and generates substantial amounts of waste, which may take thousands of years to depolymerize. However, the environmental impact of plastic products is increasing daily, while petroleum resources are gradually depleting.

[0073] As a substitute for plastics, the research and production of polyhydroxyalkanoates (PHAs) represent significant economic and environmental benefits. PHAs are intracellular polyesters synthesized by *P. basellae*, which primarily function as carbon and energy storage substances within the bacteria. They possess physicochemical properties similar to synthetic plastics, but also exhibit many superior properties not found in synthetic plastics, such as biodegradability, biocompatibility, optical activity, piezoelectricity, and gas barrier properties. PHAs have broad application prospects in biodegradable packaging materials, tissue engineering materials, sustained-release materials, electrical materials, and medical materials. However, due to production costs, large-scale application of PHAs is difficult.

[0074] This application describes the targeted regulation of phenol degradation products by *Bacillus basellae* to generate a large number of high-value polyhydroxyalkanoates (PHA) that can replace plastics in various fields, effectively reducing the production cost and application threshold of PHAs.

[0075] To facilitate a further understanding of the present invention by those skilled in the art, the following examples are provided:

[0076] It should be noted that the English translations appearing in the attached image are as follows:

[0077] Phenol degradation - percentage of phenol degradation; time - time; DCW & PHA - dry weight of *Bacillus basellariae* and PHA weight; PHA content - percentage; fluorescence microscopy image of intracellular accumulated PHA - fluorescence microscopy image of intracellular accumulated PHA; transmittance; wavelength; chemical shift; DTG - derivative thermogravimetric analysis; TG - thermogravimetric analysis; temperature; DCS - differential scanning calorimetry; temperature; melting enthalpy; decomposition enthalpy; exothermic.

[0078] Example 1

[0079] Under aseptic conditions, *Bacillus basellii* was inoculated into 100 ml of LB broth (containing 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl, hereinafter referred to as LB broth) and incubated at 30°C for 18 h on a rotary shaker (150 rpm). The activated *Bacillus basellii* was then centrifuged at 12000 rpm for 10 min, washed, and resuspended in mineral salt medium (i.e., the first medium, hereinafter referred to as the first medium) containing different concentrations of phenol (200–800 mg / L) to expand the culture, and incubated on a rotary shaker (150 rpm).

[0080] The copper-degrading bacteria mentioned can be Cupriavidusbasilensis B-8, which has the preservation number CGMCC No.4240.

[0081] Different concentrations of NaCl (10–50 g / L) were added to the first culture medium to study the degradation of phenol under different salinity conditions. After anaerobic culture at 30 °C for 3 days, the phenol concentration in the culture medium was monitored every 12 h. The culture was centrifuged at 12000 rpm for 20 min, and the phenol concentration in the supernatant was monitored using the 4-aminoantipyrine method.

[0082] The degradation performance of the strain was determined under different temperature conditions. In a mineral salt medium, the phenol concentration varied by 200 mg / L, the pH was 7, the vibrating screen speed was 150 rpm, and the temperature ranged from 26 to 34 °C. After 12 hours of cultivation, the remaining phenol content in the culture medium was measured.

[0083] Will Figures 1-4 Combine, Figure 1The study compared the effects of different concentrations of phenol on the growth of *Bacillus basellae* under conditions of pH 7, temperature 30°C, and rotation speed 150 rpm. It can be seen that the degradation time increases with the increase of phenol concentration. The strain can degrade up to 800 mg / L of phenol within 96 h, indicating that *Bacillus basellae* has a strong tolerance to phenol.

[0084] Figure 2 The effects of different concentrations of phenol and 10 g / L NaCl on the degradation rate and growth of *Bacillus basellariae* were investigated under the conditions of pH 7, temperature 30°C, and rotation speed 150 rpm. The complete degradation of 400 mg / L phenol after 144 h demonstrated the salt tolerance of *Bacillus basellariae*.

[0085] Figure 3 This study demonstrated the effect of pH on the phenol degradation ability of *Bacillus basenelii*. The degradation performance of this bacterium was measured under different initial pH conditions. In a mineral salt medium with a phenol concentration of 200 mg / L, the pH was adjusted to 5–11, the temperature was 30℃, and the rotation speed was 150 rpm. After 12 h of incubation in a shaker, the remaining phenol content in the mineral salt medium was measured. It can be seen that *Bacillus basenelii* can utilize phenol as a carbon source and degrade it when the pH is between 6 and 9; the ideal pH is 7 for optimal results. When the pH is between 7 and 8, the degradation performance of the strain is improved, and the degradation rate reaches 99.9% after 24 h.

[0086] Example 2

[0087] In this embodiment, the ability to produce polyhydroxyalkanoates (PHA) using phenol as the sole carbon source was explored. Overnight activated bacteria were centrifuged at 12,000 rpm for 10 min. The PHA bacteria were resuspended in a mineral salt medium providing phenol concentration and limited to nitrogen. PHA accumulation was monitored using fluorescence microscopy.

[0088] In the experiment, ammonium sulfate at different concentrations (0.125–0.5 g / L) was added to the first culture medium. Phenol concentration ranged from 200–800 mg / L, pH was 7, temperature was 30℃, and the fermentation speed was 150 rpm. Fermentation lasted for more than 7 days. During this process, biosynthesized PHA was stained with Nile Red using tin staining, and its accumulation time was detected using fluorescence microscopy. During the seven-day incubation period, 2 ml of sample was collected every 12 hours. The supernatant was removed, and the cells were resuspended in 150 μl of deionized water and 50 μl of dimethyl sulfoxide.

[0089] Subsequently, 40 μl of Nile Red (80 μg / ml dissolved in acetone) was added to the suspension to obtain a final concentration of 3.1 μg Nile Red per ml of suspension, and the mixture was incubated at room temperature for 30 min. Fluorescence was then read at excitation and emission wavelengths of 535 and 605 nm using the Wallac EnVision Manager 1.12 software program on the EnVision multi-label reader of the monochromator.

[0090] PHA was extracted using the chloroform-methanol method. 100 ml of the *Bacillus baseneriifolius* bacterial culture (i.e., the phenol degradation solution mentioned above) from the first culture medium was extracted and centrifuged at 12000 rpm for 20 min at 4 °C. After washing twice with 10 ml of deionized water, the precipitate was collected. The precipitated bacterial particles were then frozen and lyophilized. The lyophilized material was ground and homogenized in 1 ml of chloroform. To resuspend the dried biomass, the slurry was vigorously vortexed, and more chloroform was added until the slurry reached a concentration of 1 g of stem cells per 25 ml of chloroform, yielding an organic mixture.

[0091] To extract the polymer formed in *Bacillus basellae*, the organic mixture was incubated overnight in a water bath at 60°C and 100–150 rpm. After adding 2 ml of deionized water, the organic mixture was stirred thoroughly for 5 min. The liquid was then separated from the cell debris using centrifugation. The organic phase containing the chloroform-soluble polyhydroxyalkanoate was collected and filtered through a syringe filter with a 0.45 mPTFE membrane. Nitrogen gas was purged, and the organic solution was concentrated to 1 ml. The solvent was precipitated by adding ten times the volume of previously cooled methanol and mixing it with a rotary mixer for 10–30 min. The precipitate was separated from the liquid by centrifugation at 2500 rpm and 4°C for 15 min to remove the supernatant, which was then washed twice with methanol. The polymer-containing particles were dried at room temperature under a nitrogen stream. The final product was then dissolved in chloroform, filtered again, and added dropwise to rapidly mixed methanol.

[0092] The composition of the first culture medium is as follows: KH2PO4, (NH4)2SO4 2 g / L, K2HPO4 1 g / L, CaCl2 0.01 g / L, MgSO4 0.2 g / L, FeSO4 0.015 g / L, MnSO4 0.01 g / L.

[0093] Figure 5 shows that decreasing the nitrogen concentration from 100 mg / L to 25 mg / L significantly affected PHA production, and fluorescence increased markedly, with PHA contents of 9.9% and 4.8%, respectively. Furthermore, the PHA content showed a significant upward trend over time, reaching its maximum at 36 h when the nitrogen concentration was 25 mg / L; at this point, the PHA content accounted for 12.34% of the dry weight of *Bacillus basellae*.

[0094] Figure 6 The effect of phenol concentration on PHA production was shown. The maximum PHA yield was 124 mg / L when cells were cultured in the presence of 400 mg / L phenol. At 800 mg / L, phenol concentrations of 600 mg / L and 200 mg / L yielded yields of 34.5, 98, and 56 mg / L PHA, respectively.

[0095] Example 3

[0096] In this embodiment, the characterization of cumulative PHA produced by *Basella alba* using phenol as the sole carbon source was explored. *Basella alba* was inoculated in LB medium overnight and then diluted 5-fold in fresh LB medium. When the logarithmic phase was reached, the culture was centrifuged at 12,000 rpm for 20 min, then resuspended in mineral salt medium containing 25 mg / L nitrogen and 400 mg / L phenol, and inoculated at 30°C in a rotary shaker at 150 rpm for 36 h. PHA was characterized after extraction using the chloroform method as described in the previous embodiment.

[0097] Figure 7 The FTIR spectra of PHA isolated from *P. basellae* are shown. In the presence of 400 mg / L phenol, it appears at approximately 1277 cm⁻¹ in the FTIR spectrum. -1 The absorption peaks at 1378 and 1452 cm⁻¹ indicate the saturated ester bond of the CO group. -1 The absorption peaks at these locations indicate the stretching and bending modes of the methyl (-CH3) group vibration, respectively. The unique absorption peaks for the carbonyl (C=O) and methyl (-CH) groups are located at 1720 and 2932 cm⁻¹, respectively. -1 . Figure 7 The infrared spectra of PHB (a type of PHA) produced by *Bacillus basellae* and standard PHB were shown; the similarity between the two products is obvious.

[0098] Figure 8 The 1H-NMR spectrum of PHB produced by *Phytosporum basellariae* using phenol as the sole carbon source is shown. Characteristic peaks of PHB, such as the –CH doublet at δ = 5.21 and 5.23 ppm, were detected. 2 The multiplets have δ = 2.50 and 2.61 ppm, –CH 3 The double peak has a δ value of 1.21. The large peak at δ = 7.3 ppm indicates the solvent (CHCl3), while the smaller peaks at δ = 1.61 and 1.63 ppm are due to slight HO contamination from the solvent. These findings are consistent with those obtained using the PHB standard.

[0099] Figure 9Thermogravimetric analysis (TGA) revealed the thermal decomposition characteristics of the produced PHA compared to standard PHA. The thermal stability of PHA produced by *P. basellae* was also investigated using TGA. The polymer decomposition was completed in one stage, occurring at 273.41 °C, indicating complete polymer degradation, which is very close to the decomposition temperature of standard PHA.

[0100] Figure 10 This paper presents a comparison of the thermal analysis of isolated PHB from *Bacillus basellae* and standard PHB using DCS analysis. The melting temperature, glass transition temperature, and heat associated with melting of PHB were analyzed using DSC. The TH of PHB... m The first peak has a fusion heat of 45.09 J / g at 182.61℃. The second peak appears at 292.4℃ and is associated with a heat of 47.6 J / g, which is similar to the DCS results for standard PHB.

[0101] The above technical solutions of the present invention are merely preferred embodiments and do not limit the patent scope of the present invention. All equivalent structural transformations made under the technical concept of the present invention using the contents of the specification and drawings of the present invention, or direct / indirect applications in other related technical fields, are included in the patent protection scope of the present invention.

Claims

1. A method for degrading phenol, characterized by, Including the following steps: The *Cupriavidus basilensis* B-8 (CGMCC No. 4240) was inoculated into a first culture medium with a phenol concentration of 200-800 mg / L and cultured at 30°C with a rotation speed of 120-150 rpm. Phenol degradation was achieved through the lytic growth of the *Cupriavidus basilensis*. The first culture medium consisted of 1-1.5 g / L KH₂PO₄, 2-2.5 g / L (NH₄)₂SO₄, 1-1.5 g / L K₂HPO₄, 0.01-0.02 g / L CaCl₂, 0.2-0.3 g / L MgSO₄, 0.015-0.02 g / L FeSO₄, and 0.01-0.02 g / L MnSO₄. The initial volume ratio of the *Basellium clavatum* to the first culture medium is 5-10%, and the pH value of the first culture medium is 7-8.

2. The degradation method of claim 1, wherein, Before inoculating *Basellus salvia*, the method further includes activating *Basellus salvia*, which includes the steps of: placing *Basellus salvia* in a second culture medium and culturing it at a temperature of 29-32°C for 12-96 hours; wherein the volume ratio of *Basellus salvia* to the second culture medium is 5-10%.

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

4. The degradation method of claim 1, wherein, In the first culture medium, the culture time of the *Basellium clavatum* is 12-96 hours.

5. The degradation method of claim 2, wherein, The second culture medium includes LB broth medium.

6. A method for synthesizing polyhydroxyalkanoates, characterized by, Includes the following steps: Inoculate the culture medium with a phenol concentration of 200-800 mg / L with preservation number CGMCC No. The *Cupriavidus basilensis* B-8 strain (.4240) was cultured at 30°C and a rotation speed of 120-150 rpm for 12-96 hours. Phenol was degraded through the lytic growth of the *Cupriavidus basilensis*, yielding a phenol degradation solution. The initial volume ratio of the *Cupriavidus basilensis* to the first culture medium was 5-10%, the pH of the first culture medium was 7-8, and the composition of the first culture medium included 1-1.5 g / L KH₂PO₄, 2-2.5 g / L (NH₄)₂SO₄, 1-1.5 g / L K₂HPO₄, 0.01-0.02 g / L CaCl₂, 0.2-0.3 g / L MgSO₄, 0.015-0.02 g / L FeSO₄, and 0.01-0.02 g / L MnSO₄. Polyhydroxy fatty acid esters were extracted from the phenol degradation solution.

7. The method of synthesis of claim 6, wherein, The extraction of polyhydroxy fatty acid esters from the phenol degradation solution includes the following steps: extracting the phenol degradation solution, centrifuging at 10,000-12,000 rpm for 15-20 min, and collecting the precipitate; wherein the centrifugation temperature is 4-10℃; lyophilizing the precipitate to obtain a lyophilized product, homogenizing and diluting the lyophilized product in chloroform to obtain an organic mixture; placing the organic mixture in a 60℃-100℃ environment and incubating at 100-150 rpm for 8-12 h, and then sequentially separating, concentrating, and precipitating the polyhydroxy fatty acid ester-containing organic phase in the organic mixture to obtain the polyhydroxy fatty acid ester.

8. The method of synthesis of claim 6, wherein, The nitrogen concentration in the first culture medium is 13~75 mg / L.

9. The method of synthesis of claim 8, wherein, The nitrogen in the first culture medium comes from ammonium sulfate.

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