Salt-tolerant and acid-tolerant high-efficiency benzene series-degrading yeast fungus and application thereof

Abstract

The application discloses a salt-tolerant and acid-tolerant high-efficiency benzene series degrading yeast fungus and application thereof, and the yeast fungus is Barnettozyma hawaiiensis with a preservation number of CCTCC M 20241391.The yeast fungus can maintain activity in a high-salinity and acidic environment, significantly reduces the organic matter content in high-salinity wastewater, and exhibits high-efficiency degradation capacity for benzene series, especially benzaldehyde, in high-salinity and acidic wastewater.The yeast fungus is particularly suitable for treating high-salinity or acidic organic wastewater and refractory industrial organic wastewater containing benzene series, such as shale gas flowback fluid, and provides an effective bacterial source for biochemical treatment of high-salinity, acidic and refractory industrial organic wastewater containing benzene series.

Description

Technical Field

[0001] This invention belongs to the field of high-salt organic wastewater treatment and relates to microbial degradation technology, particularly a yeast fungus with salt and acid tolerance. This strain is especially suitable for degrading benzene compounds in high-salt acid wastewater. Background Technology

[0002] my country's chemical, pharmaceutical, pesticide, coking, and oil and gas industries discharge large amounts of high-salt organic wastewater annually. Besides high salt content and high COD, this wastewater is also characterized by strong acidity / alkalinity, high toxicity, complex chemical composition, and poor biodegradability. Treating this type of wastewater significantly increases both economic costs and treatment difficulty. If high-salt organic wastewater is discharged directly into natural water bodies without effective treatment, its soluble inorganic salts and persistently toxic organic compounds will severely impact soil and water bodies, causing irreversible environmental damage.

[0003] For the treatment of dissolved salts in high-salinity organic wastewater, commonly used methods include electrodialysis, membrane distillation, RO membrane concentration, and evaporation crystallization. Electrodialysis, membrane distillation, and RO membrane concentration primarily concentrate salts, and the concentrated solutions require further evaporation and crystallization. Evaporation commonly uses single-effect evaporation, multi-effect evaporation, and MVR (Multi-Reactive Vapor Regulator), but due to their high operating costs, they are often used in conjunction with RO membrane concentration. Since high-concentration organic matter in high-salinity organic wastewater will clog and foul the RO membrane if directly introduced, and will accumulate in the evaporator if introduced, affecting the stable operation of the evaporator and the crystallization of dissolved salts, the organic matter content must be reduced before desalination.

[0004] Currently, the main methods for removing organic matter from high-salinity organic wastewater include advanced oxidation methods such as Fenton oxidation, ozone, electrocatalysis, and microelectrolysis, as well as biochemical methods. Advanced oxidation methods degrade organic matter by generating highly reactive free radicals; however, the high salinity of the wastewater quenches hydroxyl radicals, limiting efficiency under high salinity conditions and making it difficult to achieve the desired purification effect. Furthermore, these methods involve large investments and high operating costs. Biochemical methods utilize microorganisms to degrade organic matter in wastewater, offering a more economical and thorough treatment approach. Therefore, the use of biological methods for treating this type of wastewater remains a key research focus both domestically and internationally.

[0005] High-salt organic wastewater mainly contains Cl-. - SO4 2- Na + Ca 2+Salts and other ions are essential nutrients for microbial growth, playing a crucial role in promoting enzyme reactions, maintaining membrane balance, and regulating osmotic pressure. However, excessively high concentrations of these ions can inhibit and toxicize the growth and metabolic activities of conventional microorganisms. Halophilic and halophilic bacteria, which can tolerate or require certain salt concentrations to survive in high-salinity wastewater, are a key focus of research in the treatment of organic matter in high-salinity organic wastewater. In halophilic biological treatment systems, fungi, in addition to bacteria, also demonstrate unique application potential.

[0006] The types and chemical properties of organic matter in high-salinity organic wastewater vary greatly depending on the production process. Benzene ring compounds, as recalcitrant and toxic organic compounds, are often found in high-salinity organic wastewater. They have poor biodegradability, are difficult to biodegrade, and are toxic to conventional microorganisms. Treating this type of high-salinity organic wastewater is extremely difficult. Advanced oxidation is typically used for pretreatment, followed by a coupled biochemical process using salt-tolerant bacteria. However, this approach involves high investment and operating costs. If salt-tolerant bacteria that can directly degrade benzene ring compounds can be screened out, investment and operating costs can be significantly reduced. Summary of the Invention

[0007] Therefore, the purpose of this invention is to provide a novel salt- and acid-tolerant yeast strain, *Barnettozyma hawaiiensis*, capable of degrading benzene compounds, and to explore its application potential in practical wastewater treatment. Through this invention, the inventors not only screened a salt- and acid-tolerant yeast strain with highly efficient benzene compound degradation capabilities, but also optimized the environmental conditions for its growth and degradation performance, providing a new and effective strain resource for the biochemical treatment of highly hydrochloric and acidic recalcitrant organic wastewater.

[0008] Through long-term exploration and experimentation, and continuous reform and innovation, the inventors have provided the following technical solution to address the aforementioned technical problems: a salt- and acid-resistant, highly efficient yeast / fungus that degrades benzene compounds, *Barnettozyma hawaiiensis*. The strain's accession number is CCTCC M 20241391. The accession date is June 27, 2024. The depositary institution is the China Center for Type Culture Collection (CCTCC), located at Wuhan University.

[0009] The present invention also provides the application of the yeast fungus for the biochemical treatment of organic matter in wastewater.

[0010] According to a specific embodiment of the application of the present invention, the wastewater is one or more of high-salt organic wastewater, acidic organic wastewater, and organic wastewater containing recalcitrant benzene compounds.

[0011] According to a specific embodiment of the application described in this invention, the wastewater is benzaldehyde-containing wastewater and shale gas backflow liquid wastewater.

[0012] According to a specific embodiment of the application of the present invention, the pH value of the wastewater is 3 to 10, preferably 3 to 8, and more preferably 4 to 7.

[0013] According to a specific embodiment of the application described in this invention, the mass fraction of sodium chloride in the wastewater is 0% to 12%, preferably 0% to 8%, and more preferably 1% to 4%.

[0014] According to a specific embodiment of the application of the present invention, the temperature of the wastewater is 20-45°C, preferably 30-45°C, and more preferably 35-40°C.

[0015] According to a specific embodiment of the application described in this invention, the benzaldehyde concentration in the wastewater is 100-1000 mg / L, and the TOC concentration in the wastewater is 50 mg / L-800 mg / L.

[0016] According to a specific embodiment of the application described in this invention, after the benzaldehyde wastewater is inoculated with the yeast Barnettozyma hawaiiensis, the preferred TOC removal load of the yeast is 0.325 kg / (kg·d), the benzaldehyde removal load is 0.47 kg / (kg·d), the amount of bacteria produced per kg of TOC removed is 8.28 kg / (kg·d), and the amount of bacteria produced per kg of benzaldehyde removed is 5.68 kg / (kg·d).

[0017] The present invention also provides a method for degrading benzene compounds using the aforementioned yeast fungus, comprising the following steps:

[0018] The strain was inoculated into wastewater containing benzaldehyde, and cultured by stirring or shaking to carry out the degradation process;

[0019] Nutrients, including glucose, may or may not be added during the degradation process.

[0020] This invention also provides a method for preserving the yeast fungus, comprising inoculating the purified strain into a liquid inorganic salt preservation medium containing benzaldehyde, and storing it under preset refrigeration conditions, with periodic transfer. The liquid inorganic salt preservation medium is MSM medium, pH=5, with NaCl at 20 g / L.

[0021] Compared with the prior art, one of the above technical solutions has the following advantages:

[0022] a) The yeast fungus in this invention exhibits a highly efficient degradation ability for benzene compounds, especially benzaldehyde, in wastewater, significantly reducing the organic matter content in the wastewater.

[0023] b) This yeast strain can maintain its activity in high salinity and acidic environments, making it particularly suitable for the treatment of industrial organic wastewater that is high in salinity, acidic, and contains benzene compounds and other substances that are difficult to biodegrade.

[0024] c) This yeast strain can grow and degrade within a wide pH range of 3 to 10 and at sodium chloride concentrations of 0 to 12%, thus optimizing the adaptability to environmental conditions during wastewater treatment.

[0025] d) Wastewater treatment using the yeast strain of this invention is simple to operate, easy to apply industrially, and requires no complex equipment or operating procedures. The application of this invention improves the total organic carbon (TOC) removal rate in wastewater treatment, helping to meet stricter wastewater discharge standards.

[0026] e) Compared with traditional physicochemical treatment methods, biological treatment methods are less expensive, and the strains of this invention are easy to cultivate and preserve, reducing the long-term operating costs of wastewater treatment. Biodegradation methods do not produce secondary pollution and are a clean and environmentally friendly technology that aligns with current environmental requirements and the trend of sustainable development.

[0027] f) The application of this yeast strain is not limited to the treatment of benzaldehyde wastewater, but is also suitable for the treatment of other industrial wastewater with high salt and high organic content, and has broad market application potential. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0029] Figure 1 These are scanning electron microscope images of the mycelium and spores of strain H1.

[0030] Figure 2 It is the phylogenetic tree of strain H1.

[0031] Figure 3 This relates to the effect of pH on the mycelial diameter of strain H1. Figure 3 In the table, a represents the initial pH value, and b represents the inoculation time.

[0032] Figure 4 This relates to the effect of NaCl on the mycelial diameter of strain H1. Figure 4 In the table, a represents the NaCl salt concentration, and b represents the inoculation time.

[0033] Figure 5 The effect of different temperatures on the mycelial diameter of strain H1.

[0034] Figure 6 This relates to the effect of benzaldehyde on the mycelial diameter of strain H1. Figure 6 In the table, a represents the initial concentration of benzaldehyde, and b represents the inoculation time.

[0035] Figure 7 This relates to the effect of oxygen on the mycelial diameter of strain H1. Figure 7 In the diagram, 'a' represents air and 'b' represents the inoculation time.

[0036] Figure 8 The effect of different pH values ​​on the degradation performance of strain H1. Figure 8 In the figure, a represents the TOC removal rate, and b represents the benzaldehyde removal rate.

[0037] Figure 9 This describes the effect of different salt concentrations on the degradation performance of strain H1. Figure 9 In the figure, a represents the TOC removal rate, and b represents the benzaldehyde removal rate.

[0038] Figure 10 This describes the effect of different temperatures on the degradation performance of strain H1. Figure 10 In the figure, a represents the TOC removal rate, and b represents the benzaldehyde removal rate.

[0039] Figure 11 The effect of different initial benzaldehyde concentrations on the degradation performance of strain H1. Figure 11 In the figure, a represents the TOC removal rate, and b represents the benzaldehyde removal rate.

[0040] Figure 12 This relates to the effect of nutrient addition on the degradation performance of strain H1. Figure 12 In the figure, a represents the TOC removal rate, and b represents the benzaldehyde removal rate.

[0041] Figure 13 The effect of different inoculum amounts on the degradation performance of strain H1. Figure 13 In the figure, a represents the TOC removal rate, and b represents the benzaldehyde removal rate.

[0042] Figure 14 The TOC removal rate of strain H1 on high-salt wastewater from shale gas flowback is shown.

[0043] Figure 15 These are three-dimensional fluorescence images of high-salt wastewater from shale gas flowback at different treatment stages. Figure 15 In the diagram, a represents the original water, and b represents the original water inoculated with strain H1 for 6 days. Detailed Implementation

[0044] The following description, in conjunction with the accompanying drawings and specific embodiments, will be provided.

[0045] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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, 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. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention.

[0046] Example 1

[0047] The strain H1 described in this embodiment is the yeast / fungus *Barnettozyma hawaiiensis*, with accession number CCTCC M 20241391. The accession date was June 27, 2024, and the depositary institution is the China Center for Type Culture Collection (CCTCC), located at Wuhan University.

[0048] Shale gas backflow fluid contains a large amount of suspended solids and a large number of microorganisms. Using these as a source of microbial strains, salt-tolerant bacteria were isolated, screened, and identified.

[0049] Selective solid inorganic salt medium (MSM medium) and liquid inorganic salt medium (MSM medium) with benzaldehyde as the sole carbon source, pH=3, and NaCl concentration of 20 g / L were prepared for strain screening.

[0050] Shale gas flowback fluid was coated onto this selective solid inorganic culture medium, inverted and placed in a 35°C incubator. On the third day of cultivation, white filamentous bacteria grew and entered the solid culture medium, with black dots appearing on the surface of the solid substrate. The solid culture medium containing the bacteria was then inoculated into a liquid selective culture medium and placed in a shaker at 35°C (120 rpm) for enrichment. After 3 days of cultivation, white flocculent bacteria grew in the liquid culture medium, and spore-like black objects appeared on the surface of the bacteria.

[0051] After dilution and spreading, the black spores of the dominant colony were repeatedly inoculated and purified until a single colony was isolated, tentatively named strain H1. A portion of the single strain H1 colony, along with a portion of the solid culture medium, was inoculated onto liquid inorganic salt preservation medium and stored in a refrigerator for later use. The liquid inorganic salt preservation medium was MSM medium, pH=5, with 20 g / L NaCl added. A portion was inoculated onto liquid selective medium and cultured in a shaker at 35°C (120 r / min) for enrichment and subsequent experiments.

[0052] The MSM medium is a salt medium. In developing this invention, the inventors compared the components and amounts of several different culture media and ultimately selected a specific MSM medium provided in this embodiment. The components are: MgSO4 0.2 g / L, CaCl2 0.02 g / L, KH2PO4 1 g / L, MnSO4 0.03 g / L, K2HPO4 1 g / L, Fe2(SO4)3 0.2 g / L, NH4Cl 0.2 g / L, pH 7.0-7.2, and 1 mL of trace elements. The solvent is distilled water. The trace elements are of type SL-4, with the following components: EDTA 0.5 g / L, FeSO4·7H2O 0.2 g / L, ZnSO4·7H2O 0.01 g / L, MnCl2·4H2O 0.003 g / L, H3BO3 0.03 g / L, and CoCl2·6H2O. 0.02 g / L, CuCl2·2H2O 0.001 g / L, NiCl2·6H2O 0.002 g / L, Na2MoO4·2H2O 0.003 g / L, pH 3.4, solvent is distilled water.

[0053] Colonies selected from solid agar plates were observed. The colonies were round, composed of hyphae growing on the solid medium, with neat edges. The colony diameter increased with increasing incubation time until the hyphae covered the entire plate. The colonies were white in color, and black spores appeared on the surface of the solid medium on the third day of incubation. Scanning electron microscopy was used to observe the bacterial cells; the results are shown below. Figure 1 .

[0054] from Figure 1 As seen in images a, c, and e, the fungal body contains numerous hyphae, ranging from 10 to 40 μm in diameter, with branches and nodes. The small black dots on the surface are sporangia, approximately 600 μm in diameter, containing a large number of spores approximately 20 μm in diameter.

[0055] The selected bacterial cells were subjected to Gram staining, oxidase, peroxidase, catalase and superoxide dismutase experiments, and the test results were all positive.

[0056] According to the Handbook of Fungal Identification, strain H1 is preliminarily identified as a fungus.

[0057] After collecting strain H1 in the logarithmic growth phase, 18S rRNA gene sequencing was performed to identify the strain species XI. The entire genome of strain H1 was then sent to Megibio for Denovo sequencing to determine the mechanism by which strain H1 degrades benzaldehyde and exhibits salt, acid, and temperature tolerance at the genetic level.

[0058] DNA was extracted from the activated bacterial strain using a bacterial total DNA kit. The PCR products were amplified using the Sanger method with universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). Sequencing was performed using unidirectional primers; if no overlapping peaks were found, the other end of the sequence was detected. The sequencing results were then assembled using DNAMAN software, and the assembly result is shown in Seq ID NO.1.

[0059] The sequences were uploaded to the NCBI database and compared with their related homologous sequences. A phylogenetic tree of the strains was constructed using the Neighbor Joining Method with MEGA 7.0 software (see [link to database]). Figure 2 ).

[0060] The ITS sequence of strain H1 was correlated with Barnettozyma hawaiiensis CBS 8760 (or related strains) in NCBI.

[0061] The coding gene testing results showed that the total length of all genome sequences was 64,877,420 bp, and the total number of genomic sequences (scaffolds) was 307; the average GC content of all genes was 49.39%; the total number of genes was 19,688; the number of genes that could be matched to specific pathways in the KEGG database was 7,323; the number of genes that could be annotated with COG information in the EggNOG database was 7,923; the total number of tRNAs was 472; and the total number of rRNAs was 64.

[0062] Based on the protein sequence alignment of strain H1, its gene sequence was compared with various databases to obtain corresponding gene function annotation information. The predicted coding genes of strain H1 were then compared with the GO database for basic functional annotation and analysis to identify the characteristics of strain H1.

[0063] Strain H1 possesses numerous genes related to organic matter metabolism, bioregulation, catalysis, transport, antioxidation, and thermosusceptibility. According to GO annotations, strain H1 has 5573 metabolic process genes, primarily related to metabolic processes within the organism, including the breakdown of macromolecules and the oxidation of food molecules, indicating a strong capacity for organic matter metabolism. It also has 1155 bioregulation genes, representing processes within the organism that maintain a relatively constant internal environment through various physiological mechanisms, including neural and endocrine regulation. This demonstrates that strain H1 possesses a strong regulatory function in response to changes in the external environment, adapting to changes in pH, salinity, temperature, and the type and concentration of organic matter. After a 1-2 day lag period, strain H1 can rapidly grow and degrade organic matter. Finally, it has 6739 catalytic activity genes. The presence of catalytic activity increases the chemical reaction rate, indicating that strain H1 accelerates the decomposition of organic matter, thus exhibiting a high efficiency in organic matter removal. The strain H1 possesses 1245 transporter activity genes, which transport external organic matter to the internal environment for degradation, indicating its excellent ability to utilize external carbon sources. It also has 67 antioxidant activity genes, demonstrating its ability to resist oxidants and withstand harsh external environments. Furthermore, it contains 43 protein folding chaperone genes. Heat shock proteins (HSPs) from the 70 family act as chaperones, aiding in the folding of newly synthesized proteins under high temperature and salt conditions, preventing protein polymerization, and enhancing the heat and salt tolerance of microbial cells. This explains why strain H1 can still grow well in a high-salt environment at 45℃.

[0064] The above gene function annotations explain, at the gene level, that strain H1 possesses acid and salt tolerance, efficient organic matter degradation capabilities, and the ability to resist and adapt to harsh external environments.

[0065] 1. Growth characteristics of strain H1

[0066] 1.1 Effect of pH

[0067] The screened and purified strain H1 was inoculated into benzaldehyde-containing inorganic salt solid culture media with different pH values ​​(3–11). The TOC of the culture media was 138.56 mg / L, the mass concentration of benzaldehyde was 175.17 mg / L, and the mass concentration of NaCl was 20 g / L. The media were inverted and placed in an incubator at 35℃ to evaluate the effect of pH value on the growth of strain H1. The results are as follows. Figure 3 As shown.

[0068] The results showed that strain H1 is acid-resistant and can grow at pH values ​​of 3–10, with a more suitable growth pH value of 3–8 and an optimal growth pH value of 4–7, demonstrating strong regulatory adaptability.

[0069] 1.2 Effect of Salt Concentration

[0070] Inorganic salt solid culture media containing benzaldehyde at concentrations ranging from 0% to 12% (mass fraction) of NaCl were prepared. The TOC of the media was 136.24 mg / L, the mass concentration of benzaldehyde (ρ) was 172.24 mg / L, and the pH was 5. A portion of the bacterial strain with a pH of 5 was inoculated into each culture medium, and the media were inverted and placed in an incubator at 35°C to evaluate the effect of sodium chloride concentration on the growth of strain H1. The results are as follows: Figure 4 As shown.

[0071] The results showed that strain H1 could grow in salt concentrations of 0–12%, with a more suitable salt concentration of 0–8% and an optimal salt concentration of 1%–4%. High salinity inhibited the growth of strain H1, and the higher the salt concentration, the slower the growth rate. At a salt concentration of ≤2%, strain H1 grew fastest from day 2 to day 4 after inoculation and entered the stationary phase on day 5.

[0072] 1.3 Effect of Temperature

[0073] Strain strain H1 with a salinity of 2% was inoculated into an inorganic salt solid medium containing benzaldehyde. The medium had a TOC of 158.70 mg / L, a benzaldehyde concentration of 200 mg / L, a NaCl concentration of 20 g / L, and a pH of 5. The medium was inverted and cultured in incubators at different temperatures (20–45 °C). The effect of temperature on the growth of strain H1 was evaluated. The results are as follows: Figure 5 As shown.

[0074] The results showed that strain H1 is a mesophilic bacterium that can grow at temperatures ranging from 20 to 45°C, with a suitable temperature range of 30 to 45°C and an optimal growth temperature range of 35 to 40°C.

[0075] 1.4 Effect of initial benzaldehyde concentration

[0076] Strain strain H1, grown at 35℃, was inoculated into inorganic salt solid culture media containing different concentrations of benzaldehyde (100–1000 mg / L). The media contained 20 g / L NaCl and had a pH of 5. The media were inverted and cultured in a 35℃ incubator. The effect of the initial benzaldehyde concentration on the growth of strain H1 was evaluated. The results are as follows: Figure 6 As shown.

[0077] Initial benzaldehyde concentrations of 300 mg / L and above inhibit bacterial growth, but after a lag phase, strain H1 can grow well at initial benzaldehyde concentrations of 100–1000 mg / L. The higher the concentration, the longer the lag phase, and the fastest growth occurs 2–4 days after inoculation.

[0078] 1.5 The Effect of Oxygen

[0079] Strain H1 was inoculated into an inorganic solid medium containing benzaldehyde. The medium had a TOC of 248.01 mg / L, a benzaldehyde concentration of 313.54 mg / L, a NaCl concentration of 20 g / L, and a pH of 5. The medium was inverted and placed in an incubator at 35°C under vacuum and aeration conditions, respectively. Growth was observed daily, mycelial diameter was measured, and the effect of oxygen on the growth of strain H1 was evaluated. The results are as follows: Figure 7 As shown.

[0080] The results showed that strain H1 is an aerobic bacterium with slow growth. Day 1 was the lag phase, days 2 to 4 were the logarithmic growth phase, and day 5 was the stationary phase.

[0081] Example 2

[0082] This embodiment is an example of the application or method of strain H1 described in Example 1 in the degradation of benzaldehyde.

[0083] The degradation of benzaldehyde requires halophilic bacteria to secrete certain degrading enzymes. The growth of halophilic bacteria is affected by environmental factors, as is the secretion of these enzymes. Therefore, this example primarily explores the influence of environmental factors on the benzaldehyde degradation characteristics of strain H1.

[0084] 2.1 Effect of pH

[0085] Three Φ2cm solid culture media containing bacterial cells, grown at different pH values, were inoculated into liquid inorganic culture media containing benzaldehyde at different pH values ​​(3–11). The liquid inorganic culture media contained TOC 138.56 mg / L, ρ(benzaldehyde) 175.17 mg / L, and ρ(NaCl) 20 g / L. The media were placed in a constant temperature shaker (35℃, 120 r / min) for degradation. The effect of pH value on the degradation performance of benzaldehyde and TOC by strain H1 was evaluated. The results are as follows: Figure 8 As shown.

[0086] Based on the combined results of TOC and benzaldehyde degradation by strain H1, the suitable pH range for degradation is 3–8, with the optimal pH range being 4–6. The fastest degradation rate was observed between days 2 and 4. At unsuitable pH values, the degradation period was longer, and the degradation efficiency was lower. The best pH value was 5, and after 4 days of degradation, the TOC removal rate was 64.45%, and the benzaldehyde removal rate was 99.48%. pH value 5 was selected for subsequent examples.

[0087] 2.2 Effect of Salt Concentration

[0088] Three Φ2cm solid culture media containing bacterial cells, grown at different salt concentrations (1.2), were inoculated into benzaldehyde-containing liquid inorganic culture media with corresponding NaCl salt concentrations (0%–8% by mass). The liquid inorganic culture media had a TOC of 136.24 mg / L, a benzaldehyde concentration (ρ) of 172.24 mg / L, and a pH of 5. The media were placed in a constant-temperature shaker for degradation. The effect of sodium chloride concentration on the degradation performance of benzaldehyde and TOC by strain H1 was evaluated. The results are as follows: Figure 9 As shown.

[0089] Based on the combined results of strain H1's degradation of TOC and benzaldehyde, strain H1 showed good removal efficiency for both TOC and benzene / toluene within a salt concentration range of 0%–8%. The optimal salt concentration for degradation was 0%–4%. Higher salt concentrations resulted in lower degradation rates. The highest degradation rate was observed at salt concentrations ≤2%, achieving a TOC removal rate of over 70.25% and a benzaldehyde removal rate of over 96.90% after 4 days. Subsequent examples used a salt concentration of 2%.

[0090] 2.3 Effect of Temperature

[0091] Three Φ2cm solid culture media containing bacterial cells, grown at different temperatures (1.3°C), were inoculated into liquid inorganic medium containing benzaldehyde. The liquid inorganic medium had a TOC concentration of 158.70 mg / L, a benzaldehyde concentration of ρ(benzaldehyde) of 200.63 mg / L, and a NaCl concentration of ρ(NaCl) of 20 g / L. The media were placed in shakers (120 r / min) at different temperatures (25–40°C) to investigate the degradation reaction. The effect of temperature on the degradation performance of benzaldehyde and TOC by strain H1 was explored. The results are shown in […]. Figure 10 .

[0092] Based on the combined results of strain H1's degradation of TOC and benzaldehyde, strain H1 showed good removal rates of both TOC and benzaldehyde at temperatures ranging from 25 to 40℃. The optimal degradation temperature was 30–35℃. After 5 days of degradation, the TOC removal rate was 66.13%–81.91%, and after 4 days, the benzaldehyde removal rate was 98.07%–99.39%. The highest removal rate was observed at 35℃, and this temperature was subsequently set for subsequent experiments. The highest degradation rate was observed between the 2nd and 4th days at the optimal temperature.

[0093] 2.4 Effect of initial benzaldehyde concentration

[0094] Three Φ2cm solid culture media containing bacterial cells, grown at different benzaldehyde concentrations, were inoculated into liquid inorganic culture media with corresponding benzaldehyde concentrations (100–1000 mg / L). The liquid culture media had a mass concentration ρ(NaCl) = 20 g / L and pH = 5. The media were then placed in a constant-temperature shaker (120 r / min) for degradation. The effect of the initial benzaldehyde concentration on the degradation performance of benzaldehyde and TOC by strain H1 was evaluated. The results are shown in […]. Figure 11 .

[0095] Based on the combined results of TOC and benzaldehyde degradation by strain H1, the removal rate of organic matter at different initial concentrations was related to the inoculum quantity of strain H1. For this experiment, the optimal inoculum quantity and inoculation time showed the best match between a benzaldehyde concentration of 400 mg / L and the inoculum quantity. After 5 days of degradation, the TOC removal rate was 78.87%, and the benzaldehyde removal rate was 97.04%. Within the benzaldehyde concentration of 400 mg / L and below, the higher the benzene concentration, the lower the benzaldehyde degradation rate.

[0096] 2.5 Effects of Nutrient Addition

[0097] Strain H1, with an initial benzaldehyde concentration of 500 mg / L, was inoculated into a solid medium containing benzaldehyde and cultured at 35°C for 3 days. The cultured solid medium containing bacteria was then inoculated into two liquid media containing benzaldehyde, one without glucose and the other with 5000 mg / L glucose. The liquid media had a TOC of 401.84 mg / L, a benzaldehyde concentration of ρ(benzaldehyde) of 650 mg / L, a NaCl concentration of 20 g / L, and a pH of 5. The media were placed in a shaker (120 r / min) for degradation. The effects of nutrient addition on the degradation performance of benzaldehyde and TOC by strain H1 were investigated. The results are as follows: Figure 12 As shown.

[0098] The results showed that adding glucose could improve the removal rates of benzaldehyde and TOC.

[0099] 2.6 Effects of different inoculation amounts

[0100] To facilitate the calculation of cell weight and load, experiments with different inoculum amounts used cells cultured in liquid medium. After centrifugation, the supernatant of the cells cultured in liquid medium was discarded. The wet cell water content was 98.6%, and the density was 0.878 mL / g. The centrifuged wet cells were diluted to 18 mL, resulting in a cell concentration of 3071.43 mg / L. Different cell amounts were added to 50 mL of inorganic medium containing 60 mg / L benzaldehyde, with inoculum concentrations of 30.71 mg / L, 61.43 mg / L, 122.86 mg / L, and 184.29 mg / L, respectively. The medium contained TOC 53.38 mg / L, ρ(benzaldehyde) 60 mg / L, ρ(NaCl) 20 g / L, and pH = 5. The culture was placed in a shaker (120 r / min) at 35℃ for degradation. The effects of these effects on benzaldehyde and TOC degradation performance are shown below. Figure 13 As shown in the figure, the degradation load of strain H1 can be seen in Table 1.

[0101] Liquid bacterial degradation reaches its maximum concentration after 1 day, and autolysis begins on the second day. Therefore, the TOC and benzaldehyde removal rates are mainly based on the data from the first day of degradation.

[0102] Table 1. Bacterial cell degradation load

[0103]

[0104] After adding liquid bacterial solution, degradation reached the stable period within 1 day. For the experimental water sample with a concentration of 60 mg / L benzaldehyde, the experimental group with 122.86 mg / L bacterial solution showed the highest TOC removal rate, with TOC and benzaldehyde removal rates of 74.76% and 98.28%, respectively. The bacterial TOC removal load was 0.325 kg / (kg·d), the bacterial benzaldehyde removal load was 0.474 kg / (kg·d), the bacterial production per gram of TOC removed was 8.284 kg / (kg·d), and the bacterial production per gram of benzaldehyde removed was 5.676 kg / (kg·d).

[0105] Example 3

[0106] This embodiment demonstrates the specific application of strain H1 described in Example 1 in degrading organic matter in actual shale gas flowback fluid. The organic matter in this shale gas flowback fluid is mainly composed of benzene series organic matter (51.70%), with a mineralization of 10–50 g / L, classifying it as typical high-salt, recalcitrant organic wastewater.

[0107] TOC removal effect

[0108] Shale gas flowback fluid and shale gas flowback fluid containing foam were inoculated with strain H1 and placed in a constant temperature shaker (35℃, 120 r / min) for degradation. The degradation effect of strain H1 was verified. See [link to relevant documentation]. Figure 14 The water quality of the experiment is shown in Table 2.

[0109] Table 2 Experimental water quality

[0110]

[0111] Strain H1 can effectively degrade organic matter in shale gas flowback fluid and raw water containing foam, with the maximum TOC removal rate reaching 2 days. Figure 14 As shown in Figure a, after inoculating the raw water of shale gas flowback liquid and foam-containing flowback liquid with strain H1, the TOC removal rate reached its maximum (29.8% and 37.73%) after 2 days of degradation. This may be because the actual shale gas flowback liquid has a complex composition, and strain H1 is not adapted to it, resulting in a relatively low TOC removal rate. Strain H1 has a large number of bioregulatory genes, and the removal rate of organic matter in actual high-salt organic wastewater can be improved by increasing the concentration of inoculated bacteria and strengthening the cultivation and acclimatization of strain H1.

[0112] Fluorescent organic matter removal effect

[0113] The three-dimensional fluorescence images of strain H1 inoculated with shale gas flowback fluid before and after degradation were tested. Figure 15 The test results showed that strain H1 had a significant removal effect on fluorescent organic matter.

[0114] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A salt- and acid-tolerant yeast strain that degrades benzene compounds Barnettozyma hawaiiensis H1, characterized in that, The strain H1 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20241391.

2. The application of the yeast fungus according to claim 1, characterized in that, Used for the biochemical treatment of organic matter in wastewater.

3. The application according to claim 2, characterized in that, The wastewater is one or more of the following: high-salt organic wastewater, acidic organic wastewater, and organic wastewater containing recalcitrant benzene compounds.

4. The application according to claim 2 or 3, characterized in that, The wastewater consists of benzaldehyde-containing wastewater and shale gas backflow liquid wastewater.

5. The application according to claim 2, characterized in that, The pH value of the wastewater is 3 to 10.

6. The application according to claim 2, characterized in that, The wastewater has a sodium chloride mass fraction of 0% to 12%.

7. The application according to claim 2, characterized in that, The temperature of the wastewater is 20–45 °C.

8. The application according to claim 2, characterized in that, The wastewater has a benzaldehyde concentration of 100–1000 mg / L and a TOC concentration of 50 mg / L–800 mg / L.

9. A method for degrading benzene compounds using the yeast fungus described in claim 1, characterized in that, Includes the following steps: The strain was inoculated into wastewater containing benzaldehyde, and cultured by stirring or shaking to carry out the degradation process; Nutrients, including glucose, may or may not be added during the degradation process.

10. A method for preserving yeast fungi as described in claim 1, characterized in that, The purified strain was inoculated into a liquid inorganic salt preservation medium containing benzaldehyde and stored under pre-set refrigeration conditions, and transferred periodically.

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