Salt-resistant and acid-resistant yeast fungus capable of efficiently degrading benzene series and application of yeast fungus

Abstract

The invention discloses a salt-resistant and acid-resistant yeast fungus capable of efficiently degrading benzene series and application of the yeast fungus, the yeast fungus is Barnettozyma hawaiiensis, and the preservation number is CCTCC (China Center for Type Culture Collection) M 20241391. The yeast strain can keep activity in a high-salinity and acidic environment, obviously reduces the content of organic matters in high-salinity wastewater, and shows efficient degradation capacity on benzene series, especially benzaldehyde, in the high-salinity acidic wastewater. The method is especially suitable for treating high-salt or acidic organic wastewater and refractory industrial organic wastewater containing benzene series such as shale gas flowback fluid. An effective bacterium source is provided for biochemical treatment of high-salt acidic refractory industrial organic wastewater containing benzene series and the like.

Description

Technical Field

[0001] The invention belongs to the field of high-salt organic wastewater treatment, relates to microbial degradation technology, and particularly to a yeast fungus with salt-resistance and acid-resistance characteristics. The strain is particularly suitable for degrading benzene series in high-salt acid wastewater. Background Art

[0002] my country's chemical, pharmaceutical, pesticide, coking, petroleum and natural gas industries discharge a large amount of high-salt organic wastewater every year. In addition to the characteristics of high salt content and high COD, high-salt organic wastewater also has the characteristics of strong acidity and alkalinity, high toxicity, complex chemical composition, and poor biodegradability. The economic consumption and treatment difficulty of this type of wastewater are greatly increased during the treatment process. If the high-salt organic wastewater is directly discharged into natural water bodies without effective treatment, the soluble inorganic salts and difficult-to-degrade toxic organic matter in it will have a serious impact on the soil and water bodies, causing irreversible damage to the environment.

[0003] The commonly used treatment methods for dissolved salt in high-salinity organic wastewater are electrodialysis, membrane distillation, RO membrane concentration and evaporation crystallization. Among them, electrodialysis, membrane distillation and RO membrane concentration are mainly for salt concentration, and the concentrated solution needs further evaporation and crystallization. Single-effect evaporation, multi-effect evaporation and MVR are commonly used for evaporation. Due to their high operating costs, they are often combined with RO membrane concentration. Since the high concentration of organic matter in high-salinity organic wastewater directly enters the RO membrane, it will clog and pollute the membrane, and entering the evaporator will cause organic matter to accumulate, affecting the stable operation of the evaporator and the crystallization of dissolved salts. Therefore, organic matter must be reduced before desalination.

[0004] At present, the main methods for removing organic matter from high-salinity organic wastewater include advanced oxidation methods such as Fenton, ozone, electrocatalysis and micro-electrolysis, and biochemical methods. Advanced oxidation methods degrade organic matter by producing highly active free radicals, but the high salt in the wastewater has a quenching effect on hydroxyl radicals. The efficiency is limited under high salinity conditions, and it is difficult to achieve the expected purification effect. In addition, the investment is large and the operating costs are high. The biochemical method uses microorganisms to degrade organic matter in wastewater. It is a more economical and thorough method for treating organic matter. Therefore, the use of biological methods to treat such wastewater is still a focus of research at home and abroad.

[0005] The salt substances contained in high-salinity organic wastewater are mostly Cl - 、SO4 2- 、Na + , Ca 2+Salt substances such as salts. Although these ions are essential nutrients for the growth of microorganisms, they play an important role in promoting enzyme reactions, maintaining membrane balance and regulating osmotic pressure during the growth of microorganisms. However, if the concentration of these ions is too high, it will inhibit and poison the growth and metabolic activities of conventional microorganisms. Halophiles and halophiles can survive in high-salinity wastewater because they can tolerate or require a certain salt concentration. Therefore, the screening and application of halophiles and halophiles are the research focus of organic matter treatment in high-salinity organic wastewater. In the halotolerant biological treatment system, in addition to bacteria, fungi also show their unique application potential.

[0006] The types and chemical properties of organic matter in high-salt organic wastewater vary greatly depending on the production process. Benzene ring organic matter, as a difficult-to-degrade toxic organic matter, often exists in high-salt organic wastewater. It has poor biodegradability, is difficult to biodegrade, and is toxic to conventional microorganisms. This type of high-salt organic wastewater is extremely difficult to treat. Advanced oxidation is usually used for pretreatment, and then coupled with a salt-tolerant bacteria biochemical method, but there are problems of large investment and high operating costs. If salt-tolerant bacteria that can directly degrade benzene series can be screened out, investment can be greatly reduced and operating costs can be reduced. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide a new type of salt-tolerant and acid-tolerant yeast fungus Barnettozyma hawaiiensis that can degrade benzene series, and to explore its application potential in actual wastewater treatment. Through the present invention, the inventors not only screened out a salt-tolerant and acid-tolerant yeast fungus with high efficiency in degrading benzene series, but also optimized the environmental conditions for its growth and degradation performance, thus providing a new and effective strain resource for the biochemical treatment of high-salinity and acidic refractory organic wastewater.

[0008] The inventor has continuously reformed and innovated through long-term exploration and attempts, as well as multiple experiments and efforts, to solve the above technical problems. The technical solution provided by the present invention is to provide a salt-resistant and acid-resistant yeast fungus Barnettozyma hawaiiensis that efficiently degrades benzene series. The deposit number of the strain is CCTCC M 20241391. The deposit date of the strain is June 27, 2024, the deposit unit is China Center for Type Culture Collection, and the deposit address is Wuhan University.

[0009] The invention also provides application of the yeast fungus for 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 difficult-to-degrade benzene series.

[0011] According to a specific implementation of the application of the present invention, the wastewater is benzaldehyde-containing wastewater and shale gas flowback liquid wastewater.

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

[0013] According to a specific implementation of the application of the present 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 implementation of the application of the present invention, the benzaldehyde concentration of the wastewater is 100-1000 mg / L, and the TOC concentration of the wastewater is 50 mg / L-800 mg / L.

[0016] According to a specific embodiment of the application of the present invention, after the benzaldehyde wastewater is inoculated with the yeast fungus Barnettozyma hawaiiensis, the TOC removal load of the bacteria is preferably 0.325 kg / (kg·d), the benzaldehyde removal load is 0.47 kg / (kg·d), the bacterial production per kg of TOC removed is 8.28 kg / (kg·d), and the bacterial production per kg of benzaldehyde removed is 5.68 kg / (kg·d).

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

[0018] The strain is inoculated into wastewater containing benzaldehyde, and the culture is stirred or shaken to carry out the degradation process;

[0019] Nutrients are added or not added during the degradation process, and the nutrients include glucose.

[0020] The present invention also provides a method for preserving the yeast fungus, wherein the purified strain is inoculated into a liquid inorganic salt preservation medium containing benzaldehyde, and is stored under a preset refrigeration condition and transferred regularly. The liquid inorganic salt preservation medium is an MSM medium with a pH of 5 and a NaCl content of 20 g / L.

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

[0022] a) The yeast fungi in the present invention exhibits a highly efficient degradation ability for benzene series compounds, especially benzaldehyde, in wastewater, and significantly reduces the organic matter content in the wastewater.

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

[0024] c) The yeast strain can grow and degrade in a wide range of pH values ​​of 3 to 10 and a sodium chloride concentration of 0 to 12%, thereby optimizing the adaptability to environmental conditions during wastewater treatment.

[0025] d) The yeast strain of the present invention is used for wastewater treatment, which is simple to operate and easy to apply industrially without complicated equipment and operating procedures. The application of the present invention improves the total organic carbon (TOC) removal rate of wastewater treatment and helps to achieve more stringent wastewater discharge standards.

[0026] e) Compared with traditional physical and chemical treatment methods, biological treatment methods have lower costs, and the strains of the present invention are easy to culture and preserve, reducing the long-term operating costs of wastewater treatment. The biodegradation method does not produce secondary pollution and is a clean environmentally friendly technology that meets current environmental protection requirements and the trend of sustainable development.

[0027] f) The application of the 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 matter content, and has broad market application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 This is a scanning electron microscope image of the mycelium and spores of strain H1.

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

[0031] Figure 3 The effect of pH on the mycelium diameter of strain H1. Figure 3 In the figure, a is the initial pH value and b is the inoculation time.

[0032] Figure 4 The effect of NaCl on the mycelium diameter of strain H1. Figure 4 In the figure, a is the NaCl salt concentration and b is the inoculation time.

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

[0034] Figure 6 The effect of benzaldehyde on the mycelium diameter of strain H1. Figure 6 In the figure, a is the initial concentration of benzaldehyde and b is the inoculation time.

[0035] Figure 7 The effect of oxygen on the mycelium diameter of strain H1. Figure 7 In the figure, a is air and b is 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 is the TOC removal rate, and b is the benzaldehyde removal rate.

[0037] Fig. 9 The effect of different salt concentrations on the degradation performance of strain H1. Fig. 9 In the figure, a is the TOC removal rate, and b is the benzaldehyde removal rate.

[0038] Fig.10 The effect of different temperatures on the degradation performance of strain H1. Fig.10 In the figure, a is the TOC removal rate, and b is the benzaldehyde removal rate.

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

[0040] Fig.12 It is the effect of adding nutrients on the degradation performance of strain H1. Fig.12 In the figure, a is the TOC removal rate, and b is the benzaldehyde removal rate.

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

[0042] Fig.14 It is the TOC removal rate of strain H1 on high-salinity wastewater from shale gas flowback.

[0043] Fig.15 It is a three-dimensional fluorescence image of different treatment stages of shale gas flowback high-salinity wastewater. Fig.15 In the figure, a is the raw water, and b is the raw water inoculated with strain H1 and degraded for 6 days. DETAILED DESCRIPTION

[0044] The following is a description with reference to the accompanying drawings and specific embodiments.

[0045] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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 drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

[0046] Example 1

[0047] The strain H1 described in this example is a yeast fungus Barnettozyma hawaiiensis, and the deposit number of the strain is CCTCC M 20241391. The deposit date of the strain is June 27, 2024, the deposit unit is China Center for Type Culture Collection, and the deposit address is Wuhan University.

[0048] The high-salinity wastewater from shale gas flowback contains a large amount of suspended matter, which contains a large number of microorganisms. This is used as the source of bacteria to separate, screen and identify the salt-tolerant bacteria.

[0049] A selective solid inorganic salt medium (MSM medium) and a 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] The shale gas return fluid was applied to the selective solid inorganic culture medium, and then placed upside down in a 35°C incubator. On the third day of culture, white filamentous bacteria grew, and the bacteria grew into the solid culture medium, and black dot-like substances appeared on the surface of the solid base. The solid culture medium with bacteria was inoculated into the liquid selective culture medium, and placed in a 35°C shaker (120r / min) for enrichment. After 3 days of culture, white flocculent bacteria grew in the liquid culture medium, and spore-like black objects grew from the bacteria on the liquid surface.

[0051] After the dilution solution was applied, the black spores of the dominant colony were inoculated and purified multiple times, and finally a single colony was isolated, which was temporarily called strain H1. A portion of the single strain H1 colony with solid culture medium was inoculated into a liquid inorganic salt preservation medium and placed in a cold storage room for standby use. The liquid inorganic salt preservation medium was MSM medium, pH = 5, with NaCl 20g / L added. A portion was inoculated into a liquid selective medium, placed in a 35°C shaker (120r / min) for culture enrichment, and used for subsequent experiments.

[0052] The MSM culture medium is a salt culture medium. In the process of completing the present invention, the inventors compared the components and amounts of several different culture media, and finally selected a specific MSM culture medium provided in this embodiment, the components of which are: MgSO4 0.2g / L, CaCl2 0.02g / L, KH2PO4 1g / L, MnSO4 0.03g / L, K2HPO4 1g / L, Fe2(SO4)3 0.2g / L, NH4Cl0.2g / L, pH 7.0-7.2, trace elements 1mL, and the solvent is distilled water; wherein the trace elements adopt SL-4 type trace elements, the components of which are: EDTA 0.5g / L, FeSO4·7H2O 0.2g / L, ZnSO4·7H2O 0.01g / L, MnCl2·4H2O 0.003g / L, H3BO3 0.03g / L, CoCl2·6H2O 0.02g / L, CuCl2·2H2O0.001g / L, NiCl2·6H2O 0.002g / L, Na2MoO4·2H2O 0.003g / L, pH 3.4, the solvent is distilled water.

[0053] The colonies screened from the solid plate substrate were observed. The colonies were round in shape and consisted of hyphae growing in the solid culture medium. The edges were neat and the diameter of the colonies increased with the increase of culture time until the hyphae covered the entire plate. The color was white. On the third day of culture, black spores grew on the surface of the solid substrate. The bacteria were observed using a scanning electron microscope. The results are shown in Figure 1 .

[0054] from Figure 1 In a, c, and e, we can see that there are a large number of hyphae in the fungus body, with a diameter of 10 to 40 μm, and there are branches and nodes on the hyphae. The small black dots on the surface are sporangia, with a diameter of about 600 μm, and there are a large number of spores with a diameter of about 20 μm in the sporangia.

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

[0056] According to the Manual of Fungal Identification, it was preliminarily determined that strain H1 was similar to fungi.

[0057] The strain H1 in the logarithmic growth phase was collected and sequenced for 18S rRNA gene to identify the strain XI. It was then sent to Meiji Biotech for whole genome Denovo sequencing to find out the mechanism of the strain H1's ability to degrade benzaldehyde and its salt, acid and temperature resistance at the genetic level.

[0058] The activated strain was used to extract DNA using a bacterial total DNA kit, and the Sanger method was used to amplify the PCR product with universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The PCR product was sequenced, and unidirectional primer sequencing was performed first. If there was no overlapping peak in the unidirectional sequencing, the sequence at the other end was detected. The sequencing results were spliced ​​using DNAMAN software, and the splicing result is shown in Seq ID NO.1.

[0059] The strains were uploaded to the NCBI database and aligned with related homologous sequences. The strain phylogenetic tree was constructed using the neighbor joining method using MEGA7.0 software (see Figure 2 ).

[0060] The ITS sequence of strain H1 was aligned to Barnettozyma hawaiiensis CBS 8760 (or the same genus) in NCBI.

[0061] The results of the coding gene test showed that the total length of all sequence genomes was 64877420bp, and the total number of genome sequences (Scaffold) was 307; the average GC content of all genes was 49.39%; the total number of genes was 19688; the number of all genes that could be mapped to specific pathways in the KEGG database was 7323; the number of all genes that could be annotated with COG information in the EggNOG database was 7923; the total number of tRNAs was 472; and the total number of rRNAs was 64.

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

[0063] Strain H1 has a large number of genes for metabolizing organic matter, biological regulation function genes, catalytic function genes, transport genes, antioxidant genes and high temperature resistance genes. According to GO annotation, strain H1 has 5573 metabolic process function genes, mainly metabolic processes in organisms, including decomposition of macromolecules and oxidation of food molecules, indicating that strain H1 has a strong ability to metabolize organic matter. There are 1155 biological regulation function genes, which are the processes in which organisms maintain a relatively constant internal environment through various physiological mechanisms, including neural regulation and endocrine regulation, indicating that strain H1 has a strong regulatory function to adapt to the changes in the external environment. Therefore, when the pH value, salinity, temperature, organic matter type and concentration change, after a 1-2d delay period, strain H1 can grow and degrade rapidly. There are 6739 catalytic function genes. When the catalytic function exists, the chemical reaction rate increases, indicating that strain H1 has the function of accelerating the decomposition of organic matter, and thus has a high efficiency in organic matter removal. There are 1245 transporter genes (Transporter activity), which transport external organic matter to the internal degradation, indicating that strain H1 has a good function of utilizing external carbon sources. There are 67 antioxidant functional genes (Antioxidant activity), indicating that strain H1 has the ability to resist antioxidants and can resist the external harsh environment. There are 43 protein folding chaperone functional genes, and heat shock protein (Heat shock protein 70family, HSP) can serve as a chaperone for protein molecules, helping newly synthesized proteins to fold and avoid protein aggregation in high temperature and high salt environments, which can enhance the heat and salt resistance of microbial cells. Therefore, strain H1 can still grow well in a high salt environment of 45°C.

[0064] The above annotations of gene functions explain from a genetic level that strain H1 has the ability to resist acid and salt, efficiently degrade organic matter, and 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 an inorganic salt solid culture medium containing benzaldehyde at different pH values ​​(3-11). The culture medium contained TOC 138.56 mg / L, mass concentration ρ (benzaldehyde) 175.17 mg / L, ρ (NaCl) 20 g / L, and was placed upside down in a 35°C incubator to evaluate the effect of pH value on the growth of strain H1. The results are as follows: Figure 3 shown.

[0068] The results showed that strain H1 was acid-resistant and could grow at pH values ​​of 3-10, with a more suitable pH value of 3-8 and an optimal pH value of 4-7, and had a strong ability to adjust and adapt.

[0069] 1.2 Effect of salt concentration

[0070] Prepare an inorganic salt solid medium containing benzaldehyde with a NaCl salt concentration of 0% to 12% (mass fraction), with a TOC of 136.24 mg / L, a mass concentration of ρ (benzaldehyde) of 172.24 mg / L, and a pH of 5. Inoculate the bacteria in 1.1 with a pH value of 5 in each medium, invert and place in an incubator at 35°C to evaluate the effect of sodium chloride salt concentration on the growth of strain H1. The results are as follows Figure 4 shown.

[0071] The results showed that the strain H1 could grow at a salt concentration of 0-12%, and the most suitable salt concentration was 0-8%; the optimal growth salt concentration was 1%-4%; high salinity would inhibit the growth of the strain H1, and the higher the salt concentration, the slower the growth rate; at a salt concentration of ≤2%, the strain H1 grew fastest on the 2nd to 4th day after inoculation and entered the stable period on the 5th day.

[0072] 1.3 Effect of temperature

[0073] The 1.2 salinity 2% strain H1 was inoculated into an inorganic salt solid medium containing benzaldehyde. In the medium, TOC was 158.70 mg / L, mass concentration ρ (benzaldehyde) was 200 mg / L, ρ (NaCl) was 20 g / L, pH = 5, and it was inverted and placed in an incubator at different temperatures (20-45°C) to evaluate the effect of temperature on the growth of strain H1. The results are as follows Figure 5 shown.

[0074] The results showed that strain H1 was a mesophilic bacterium and could grow at temperatures between 20 and 45°C, with a suitable temperature between 30 and 45°C and an optimal growth temperature between 35 and 40°C.

[0075] 1.4 Effect of initial benzaldehyde concentration

[0076] The strain H1 in 1.3 was inoculated into an inorganic salt solid culture medium containing different benzaldehyde concentrations (100-1 000 mg / L) at 35°C. The culture medium contained ρ(NaCl) 20 g / L and pH 5. The medium was inverted and placed in a 35°C incubator to evaluate the effect of the initial concentration of benzaldehyde on the growth of the strain H1. The results are as follows: Figure 6 shown.

[0077] Initial benzaldehyde concentrations of 300 mg / L and above will inhibit bacterial growth, but after a lag period, strain H1 can grow well at initial benzaldehyde concentrations of 100-1 000 mg / L. The higher the concentration, the longer the lag period, and the fastest growth occurs 2-4 days after inoculation.

[0078] 1.5 Effect of oxygen

[0079] The strain H1 was inoculated into an inorganic solid culture medium containing benzaldehyde. In the culture medium, TOC was 248.01 mg / L, ρ(benzaldehyde) was 313.54 mg / L, ρ(NaCl) was 20 g / L, and pH was 5. The medium was placed upside down in a 35°C incubator under vacuum and air conditions, and its growth was observed every day. The mycelial straightness was measured to evaluate the effect of oxygen on the growth of the strain H1. The results are as follows: Figure 7 shown.

[0080] The results showed that strain H1 was an aerobic bacterium and grew slowly. The first day was a lag phase, the second to fourth days were a logarithmic growth phase, and the fifth day entered a stable phase.

[0081] Example 2

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

[0083] The degradation of benzaldehyde requires that the salt-tolerant bacteria secrete certain degradation enzymes. The growth of salt-tolerant bacteria is affected by environmental factors, and the secretion of salt-tolerant bacteria enzymes is also affected by environmental factors. Therefore, this example mainly discusses the influence of environmental factors on the benzaldehyde degradation characteristics of strain H1.

[0084] 2.1 Effect of pH

[0085] The solid culture medium containing bacteria grown at different pH values ​​in 1.1 was inoculated with 3 Φ2 cm solid substrates containing bacteria in liquid inorganic culture medium containing benzaldehyde at corresponding different pH values ​​(3-11). In the liquid inorganic culture medium, TOC was 138.56 mg / L, ρ(benzaldehyde) was 175.17 mg / L, and ρ(NaCl) was 20 g / L. The solid culture medium was placed in a constant temperature shaker (35°C, 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 shown in the figure. Figure 8 shown.

[0086] Based on the results of strain H1 degradation of TOC and benzaldehyde, the most suitable degradation pH value for strain H1 is 3-8, the best degradation pH value is 4-6, and the degradation rate is fastest in the 2nd to 4th day. Under unsuitable pH values, the lag period is longer and the degradation efficiency is lower. The best pH value is 5, and after 4 days of degradation, the TOC removal rate is 64.45%, and the benzaldehyde removal rate is 99.48%. The pH value is selected as 5 in the subsequent examples.

[0087] 2.2 Effect of salt concentration

[0088] The solid culture medium containing bacteria grown in 1.2 at different salt concentrations was inoculated with 3 Φ2 cm solid substrates containing bacteria in the corresponding liquid inorganic culture medium containing benzaldehyde at different NaCl salt concentrations (mass fraction 0% to 8%). In the liquid inorganic culture medium, TOC was 136.24 mg / L, mass concentration ρ (benzaldehyde) was 172.24 mg / L, pH = 5, and it was placed in a constant temperature shaker for degradation. The effect of sodium chloride salt concentration on the performance of strain H1 in degrading benzaldehyde and TOC was evaluated. The results are shown in the figure. Fig. 9 shown.

[0089] The results of strain H1 degradation of TOC and benzaldehyde show that strain H1 has good removal effects on TOC and benzene at salt concentrations of 0% to 8%. The optimal degradation salt concentration is 0% to 4%. The higher the salt concentration, the lower the degradation rate. The rate is highest when the salt concentration is ≤2%. After 4 days of degradation, the TOC removal rate is more than 70.25%, and the benzaldehyde removal rate reaches more than 96.90%. The salt concentration in the subsequent examples is 2%.

[0090] 2.3 Effect of temperature

[0091] The solid culture medium containing bacteria grown at different temperatures in 1.3 was inoculated with 3 Φ2 cm solid substrates containing bacteria in a liquid inorganic culture medium containing benzaldehyde. In the liquid inorganic culture medium, TOC = 158.70 mg / L, mass concentration ρ (benzaldehyde) = 200.63 mg / L, ρ (NaCl) = 20 g / L, and placed in a shaker (120 r / min) at corresponding different temperatures (25-40°C) for degradation reaction to explore the effect of temperature on the performance of strain H1 in degrading benzaldehyde and TOC. The results are shown in the figure. Fig.10 .

[0092] The results of strain H1 degradation of TOC and benzaldehyde showed that strain H1 had good TOC and benzaldehyde removal rates at 25-40℃, and the optimal degradation temperature was 30-35℃. After 5 days of degradation, the TOC removal rate was 66.13%-81.91%, and the benzaldehyde removal rate was 98.07%-99.39% after 4 days of degradation. The removal rate was the highest at 35℃, and the subsequent experimental temperature was set at 35℃. The degradation rate was the highest at the optimal temperature from 2 to 4 days.

[0093] 2.4 Effect of initial benzaldehyde concentration

[0094] The solid culture medium containing bacteria grown at different benzaldehyde concentrations was inoculated with 3 Φ2 cm solid substrates containing bacteria in liquid inorganic culture medium with corresponding benzaldehyde concentrations (100-1 000 mg / L). The liquid culture medium had a mass concentration of ρ(NaCl) = 20 g / L and a pH of 5. The solid culture medium was placed in a constant temperature shaker (120 r / min) for degradation. The effect of the initial concentration of benzaldehyde on the degradation performance of benzaldehyde and TOC by strain H1 was evaluated. The results are shown in Table 1. Fig.11 .

[0095] Based on the results of strain H1 degradation of TOC and benzaldehyde, the removal rate of organic matter at different initial concentrations is related to the inoculation amount of strain H1. For the amount of bacteria inoculated and the inoculation time in this experiment, the concentration of 400 mg / L benzaldehyde matches the inoculation amount best. After 5 days of degradation, the TOC removal rate is 78.87% and the benzaldehyde removal rate is 97.04%. For benzaldehyde 400 mg / L and below, the higher the benzene concentration, the lower the benzaldehyde degradation rate.

[0096] 2.5 Effect of nutrient addition

[0097] The strain H1 with an initial benzaldehyde concentration of 500 mg / L was inoculated into a solid culture medium containing benzaldehyde and placed in a 35°C incubator for 3 days. The cultured solid culture medium containing bacteria was inoculated into two liquid culture media containing benzaldehyde, one without glucose and the other with 5 000 mg / L glucose. In the liquid culture medium, TOC was 401.84 mg / L, mass concentration ρ(benzaldehyde) = 650 mg / L, ρ(NaCl) = 20 g / L, pH = 5, and placed in a shaker (120 r / min) for degradation reaction to explore the effect of adding nutrients on the degradation performance of benzaldehyde and TOC by strain H1. The results are shown in the figure. Fig.12 shown.

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

[0099] 2.6 Effect of different inoculum sizes

[0100] In order to facilitate the calculation of bacterial weight and load, different inoculation experiments used bacteria cultured in liquid culture medium. After centrifugation of the bacteria cultured in liquid culture medium, the supernatant was discarded. The moisture content of the wet bacteria was 98.6%, and the density was 0.878mL / g. The wet bacteria collected by centrifugation were diluted to 18mL, and the bacterial concentration was 3071.43mg / L. Different amounts of bacteria were added to 50mL of inorganic culture medium containing 60mg / L benzaldehyde. The inoculation bacterial concentrations were 30.71mg / L, 61.43mg / L, 122.86mg / L and 184.29mg / L, respectively. In the culture medium, TOC was 53.38mg / L, ρ(benzaldehyde) was 60mg / L, ρ(NaCl) was 20g / L, pH=5, and the degradation reaction was placed in a shaker (120r / min) at 35°C. The results of the degradation of benzaldehyde and TOC performance are shown in the figure. Fig.13 As shown, and the degradation load of strain H1 can be seen in Table 1.

[0101] After 1 day of degradation, the bacterial concentration reached the maximum and autolysis began on the 2nd day. Therefore, the TOC and benzaldehyde removal rates mainly refer to the data of 1 day of degradation.

[0102] Table 1 Bacterial degradation load table

[0103]

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

[0105] Example 3

[0106] This example is a specific application of the strain H1 described in Example 1 in degrading organic matter in actual shale gas flowback fluid. The organic matter in the shale gas flowback fluid is mainly 51.70% benzene-based organic matter, with a mineralization of 10-50 g / L, which is a typical high-salinity, difficult-to-degrade organic wastewater.

[0107] TOC removal effect

[0108] The shale gas flowback fluid and shale gas foam flowback fluid were inoculated with strain H1 and placed in a constant temperature shaker (35°C, 120 r / min) for degradation to verify the degradation effect of strain H1. Fig.14 , the experimental water quality 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 flowback fluid, and the TOC removal rate reaches the maximum after 2 days of degradation. Fig.14 From figure a, we can see that after the shale gas flowback fluid and the raw water containing foam flowback fluid were inoculated with strain H1, the TOC removal rate reached the maximum (29.8%, 37.73%) after 2 days of degradation. It may be that the actual shale gas flowback fluid has complex components, strain H1 is not adapted, and the TOC removal rate is relatively low. Strain H1 has a large number of biological regulatory genes, and the removal rate of organic matter in actual high-salt organic wastewater can be improved by increasing the inoculation bacterial concentration and strengthening the cultivation and domestication of strain H1.

[0112] Fluorescent organic matter removal effect

[0113] The three-dimensional fluorescence images of shale gas flowback fluid before and after inoculation of strain H1 were tested ( Fig.15 ), the test results showed that strain H1 had a significant removal effect on fluorescent organic matter.

[0114] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, 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 regarded as the protection scope of the present invention.

Claims

1. A salt- and acid-tolerant yeast fungus Barnettozyma hawaiiensis that is highly efficient in degrading benzene series, characterized in that: The deposit number of this strain is CCTCC M 20241391.

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

3. The application according to claim 2, characterized in that: The wastewater is one or more of high-salt organic wastewater, acidic organic wastewater, and organic wastewater containing difficult-to-degrade benzene series.

4. The use according to claim 2 or 3, characterized in that: The wastewater is wastewater containing benzaldehyde and shale gas flowback liquid wastewater.

5. The use according to claim 2, characterized in that: The pH value of the wastewater is 3-10, preferably 3-8, and more preferably 4-7.

6. The use according to claim 2, characterized in that: The mass fraction of sodium chloride in the wastewater is 0% to 12%, preferably 0% to 8%, and more preferably 1% to 4%.

7. The use according to claim 2, characterized in that: The temperature of the wastewater is 20-45°C, preferably 30-45°C, more preferably 35-40°C.

8. The use according to claim 2, characterized in that: The benzaldehyde concentration of the wastewater is 100-1000 mg / L, and the TOC concentration of the wastewater is 50 mg / L-800 mg / L.

9. A method for degrading benzene derivatives using the yeast fungus according to claim 1, characterized in that: The following steps are involved: The strain is inoculated into wastewater containing benzaldehyde, and the culture is stirred or shaken to carry out the degradation process; Nutrients are added or not added during the degradation process, and the nutrients include glucose.

10. A method for preserving the yeast fungus according to claim 1, characterized in that: The purified strain was inoculated into a liquid inorganic salt preservation medium containing benzaldehyde, stored under preset refrigeration conditions, and transferred regularly.

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