Method for recycling treatment of salt water and application thereof in halophilic bacteria fermentation

Abstract

The present application relates to wastewater treatment technical field, especially to a kind of salt water's reuse processing method and its application in halophilic bacteria fermentation.The salt water's reuse processing method includes: removing bicarbonate ion in the salt water;Wherein, the salt water is obtained by the following method: after cell wall breaking of halophilic bacteria fermentation liquor, cell wall breaking wastewater is obtained, and then the cell wall breaking wastewater is coarsely filtered to remove solid suspended matter and insoluble impurities therein, and then membrane filtration is carried out using nanofiltration membrane with average molecular weight cut-off within 700Da, to obtain the salt water.The reuse salt water obtained by the salt water's reuse processing method of the present application is used to replace fresh culture medium for fermentation to grow halophilic bacteria and produce PHA, which can improve cell dry weight and PHA content.

Description

Technical Field

[0001] This invention relates to the field of wastewater treatment technology, and in particular to a method for the reuse of brine and its application in halophilic bacteria fermentation. Background Technology

[0002] Currently, when using microorganisms to produce a wide variety of products on a large scale in industry, the nutrients required by microorganisms in the culture medium (such as nitrogen source urea, phosphorus source dipotassium hydrogen phosphate, carbon source glucose, etc.) are often added by liquid feeding. The preparation of the substrate culture medium also requires a large amount of water to dissolve. Furthermore, the environment required for microbial growth has high requirements for water purity. Therefore, the cost of water accounts for a considerable proportion of the raw material cost in the production process.

[0003] Halomonas Halomonas sp This is a type of moderately halophilic bacterium isolated from the Ayding Salt Lake in Xinjiang Uygur Autonomous Region. Its optimal growth salt concentration is 6%, and its optimal growth pH is 9.0. During growth, it consumes a large amount of glucose to synthesize its dominant product, PHA. After fermentation, the entire fermentation system still contains more than 3% inorganic salts. To meet wastewater treatment standards, a large amount of process water is required to dilute the fermentation wastewater before treatment. Therefore, wastewater treatment constitutes a significant portion of the fermentation process using halophilic bacteria.

[0004] The existing solution involves first removing insoluble bacterial fragments, denatured proteins, cytotoxins, pigments, and other difficult-to-utilize substances from the fermentation waste liquid. Simultaneously, some monovalent metal ions (sodium ions, potassium ions, etc.) are removed. Based on the residual salt concentration in the resulting brine, new inorganic salts and nutrients are added to prepare the substrate culture medium for the next batch of fermentation. For example, patent CN117361699A discloses a method for treating cell wall disruption wastewater and its application in halophilic bacteria fermentation. This method uses the treated saline solution as the solvent for preparing the fermentation culture medium, and various materials need to be added subsequently.

[0005] It is evident that the brine treated as described above cannot completely replace fresh culture medium for fermentation. New inorganic salts and nutrients need to be added, and there is still room for further improvement in its fermentation effect.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The present invention has found that the above-mentioned problem arises from the fact that, due to the complexity of organisms, some cations or anions that inhibit the growth of halophilic bacteria may still remain during the fermentation process. Therefore, if the pretreated brine is used directly for the next batch of fermentation, the fermentation efficiency and yield will be reduced due to the presence of these inhibitory substances.

[0008] Subsequent extensive research led to an unexpected discovery that the main ion affecting the production of PHA by halophilic bacteria is the bicarbonate ion. If the bicarbonate ions are removed from the pretreated brine, the resulting recycled brine can achieve a fermentation effect comparable to that of fresh culture medium.

[0009] Based on this, the present invention has the following technical solution: In a first aspect, the present invention provides a method for reusing brine, comprising: removing bicarbonate ions from the brine; The brine is obtained by the following method: After cell wall disruption of the halophilic bacteria fermentation broth, cell wall disruption wastewater is obtained. The cell wall disruption wastewater is then coarsely filtered to remove suspended solids and insoluble impurities. Finally, it is filtered using a nanofiltration membrane with an average molecular weight cutoff of less than 700 Da to obtain the brine.

[0010] In practice, those skilled in the art can select the most suitable method to remove bicarbonate ions from the brine according to specific conditions and requirements, such as by adjusting the temperature and pH of the brine, ion exchange, or precipitation.

[0011] Preferably, the bicarbonate ions in the brine are removed by heating it to 100-121°C, for example, 100°C, 102°C, 104°C, 105°C, 106°C, 108°C, 110°C, 112°C, 114°C, 115°C, 116°C, 118°C, 120°C, or 121°C.

[0012] The present invention further discovers that when using a heating method, controlling the brine within the aforementioned range is more conducive to removing bicarbonate ions from the brine; if the temperature is too low, the bicarbonate ions will not decompose sufficiently. If the temperature is too high, ammonium ions and other ions in the recycled brine will undergo decomposition reactions or Maillard reactions, affecting the quality of the recycled brine.

[0013] Preferably, the heating time is 5 to 20 minutes.

[0014] Preferably, bicarbonate ions in the brine are removed by adjusting the pH of the brine to 5-7.

[0015] In this invention, the pH of the salt water can be adjusted to the above-mentioned range by adding acid or alkali to the salt water.

[0016] Preferably, the concentration of bicarbonate ions in the saline solution is less than or equal to 100 mmol / L; more preferably, the concentration of bicarbonate ions in the saline solution is less than or equal to 20 mmol / L.

[0017] In this invention, the above-mentioned heating treatment and pH adjustment methods are more effective in removing bicarbonate ions within the above concentration range.

[0018] Preferably, the pressure during membrane filtration is 0.1~1MPa; more preferably 0.3~0.6MPa.

[0019] Preferably, when the pressure during membrane filtration is 0.1~1MPa, for example, it can be 0.1MPa, 0.2MPa, 0.3MPa, 0.4MPa, 0.5MPa, 0.6MPa, 0.7MPa or 0.8MPa, it can significantly improve the membrane filtration effect while ensuring that the obtained saline solution can be directly reused for halophilic bacteria fermentation. More preferably, it is 0.3~0.6MPa.

[0020] Preferably, the coarse filtration is a flocculation sedimentation coarse filtration, wherein the flocculant is anionic polyacrylamide; more preferably, the amount of anionic polyacrylamide used is 2~6 ppm.

[0021] Secondly, the present invention provides a reusable brine solution, which is prepared by the above-described reusable treatment method.

[0022] Thirdly, the present invention provides a fermentation process for halophilic bacteria, wherein the halophilic bacteria fermentation culture medium includes the recycled brine.

[0023] In this invention, the halophilic bacteria include halophilic bacteria and / or the halophilic archaea. Preferably, the halophilic bacteria are halophilic bacteria, and more preferably, they are genus *Halomonas*. Halomonas Bacteria and their derived strains or combinations thereof, more preferably any species of the genus *Bacillus*, such as Halomonas bluephagenesis , Halomonas campaniensis , Halomonas aydingkolgenesis , Halomonas aerodenitrificans , Halomonas halocynthiae Any of the following.

[0024] Further preferred Halomonas bluephagenesis TD01, with accession number CGMCC No.4353, was obtained from Tsinghua University.

[0025] Further preferred Halomonas campaniensis LS21 was obtained from Tsinghua University under the CGMCC No. 6593 deposit registration number.

[0026] Preferably, the cell wall disruption wastewater is obtained by enzymatically disrupting the cell wall of the halophilic bacteria fermentation broth.

[0027] Preferably, the fermentation process includes: The bacteria used in production are inoculated into slant bottles for slant culture to obtain slant bacterial strains; The slant culture was cultured in shake flasks until OD reached. 600 The concentration is 2.5~5.0, and the fermentation time of the primary seed is 12~24h; The primary seeds were then cultured in shake flasks until OD (dose retardation). 600 The concentration of the sample is 2.5~5.0, and the fermentation time of the secondary seed is 8~24h. The secondary seed is inoculated into the recycled brine for fermentation culture to obtain a halophilic bacteria fermentation broth; wherein the inoculation amount of the secondary seed is 2-10%, and the fermentation culture time is 8-48 hours; the fermentation culture is carried out under aeration and stirring conditions; during the fermentation culture, the initial stirring speed is 200-300 rpm, and after 6-10 hours of fermentation culture, the stirring speed is stabilized at 600-800 rpm; the aeration rate is 3-5 L / min; the fermentation culture temperature is 20-45℃; the initial pH value of the fermentation culture medium is 4.5-10; and the initial dissolved oxygen value of the fermentation culture medium is 10-100%.

[0028] In this invention, the fermentation medium can be any existing culture medium, preferably one suitable for the growth and metabolism of *Haloxylon ammodendron*. Examples include, but are not limited to, one or more of MM medium, MMG medium, LB medium, TB medium, beef extract peptone medium, or SB medium. Of course, it can also be a modified medium based on the above-mentioned media according to the desired purpose. In other words, those skilled in the art can routinely select a suitable culture medium, as long as it allows for the growth and / or metabolism of the microorganisms.

[0029] Based on the above technical solution, the beneficial effects of the present invention are as follows: The brine reuse treatment method provided by this invention and its application in halophilic bacteria fermentation achieves fermentation effects comparable to fresh culture medium by removing bicarbonate ions from the brine. This not only reduces auxiliary material costs and water resource consumption but also alleviates wastewater treatment pressure and reduces emissions of waste gas, wastewater, and solid waste. Therefore, the method provided by this invention is a low-cost, efficient, and feasible treatment method. Furthermore, the reused brine obtained by this invention can replace fresh culture medium for fermentation to promote the growth of halophilic bacteria and the production of PHA, thereby increasing cell dry weight and PHA content. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0031] Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels. The nanofiltration membrane used in this invention can be purchased from Zhejiang Jianmo Technology Co., Ltd.

[0032] The polyhydroxyalkanoate (PHA) can be P3HB, PHB, PHV, P3HP, PHO, PHN, PHBV, P34HB, or PHBHHx, all of which have comparable fermentation effects. In a specific embodiment, P3HB is used as an example to illustrate the method of the present invention in detail.

[0033] The following examples of Halomonas Halomonas bluephagenesis TD01 was deposited on November 19, 2010, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 4353, and classified as *Halomonas*. Halomonas sp. TD01, also known as Halomonas bluephagenesis TD01; it is described in patent application publication number CN102120973A, and the public can obtain this bacterium from Tsinghua University. *Haloxymonas* Halomonas campaniensis LS21 is disclosed in "Engineering self-flocculating Halomonas campaniensis for wastewaterless open and continuous fermentation" (Chen, Ling, Guan-Qing, et al. Biotechnology and Bioengineering, 2018. DOI: 10.1002 / bit.26897.). Halomonas Halomonas bluephagenesisWZY254 is disclosed in "Hyperproduction of PHA copolymers containing high fractions of 4-hydroxybutyrate (4HB) by outer membrane-defected Halomonas bluephagenesis grown in bioreactors" (Wang Z, Zheng Y, Ji M, et al. Microbial Biotechnology. DOI:10.1111 / 1751-7915.13999.). Halomonas Halomonas bluephagenesis TD68-194 was disclosed in "Stimulus response-based fine-tuning of polyhydroxyalkanoate pathway in Halomonas" (CJYAB, ADH, FJY, et al. Metabolic Engineering, 2020, 57:85-95.). *Halomonas* Halomonas bluephagegenesis TDH4 is disclosed in Effective production of Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by engineered Halomonas bluephagenesis grown on glucose and1,4-Butanediol (Zhang L, Ye JW, Zhang X, et al. Bioresource technology, 2022, 355:127270.DOI:10.1016 / j.biortech.2022.127270.).

[0034] Example 1 This embodiment provides a method for reusing brine, including the following steps: The brine is heated to 110°C and held for 15 minutes to obtain recycled brine; the concentration of bicarbonate ions in the recycled brine is 0.2 mmol / L and the pH is 6; thus, recycled brine is obtained.

[0035] The specific steps of the method for preparing the brine (before reuse treatment) include: (1) Inoculate Halomonas TD01 from the cryovial into a slant bottle for slant culture to obtain slant culture; (2) The slant culture was transferred to a primary inoculum bottle and cultured in a shake flask for 16 hours until the OD value reached 16. 600 A value of 3.0 is obtained, resulting in a level 1 seed; (3) Transfer the primary seeds to the secondary seed bottle and continue shaking culture for 18 hours until OD. 600 A value of 3.5 is obtained, resulting in a secondary seed; (4) The secondary seeds were inoculated into the fermentation medium of the fermenter at an inoculation amount of 6% and fermented for 36 hours to obtain halophilic bacteria fermentation broth; The specific formula for the fermentation medium (MM medium) used to cultivate halophilic microorganisms is as follows: Urea 0.5 g / L; MgSO4 0.2 g / L; KH2PO4 1.5 g / L; and a total of <0.1 g / L of Fe(III)-NH4-Citrate, CaCl2·2H2O, ZnSO4·7H2O, MnCl2·4H2O, H3BO3, CoCl2·6H2O, CuSO4·5H2O, NiCl2·6H2O, and NaMoO4·2H2O.

[0036] Using the aforementioned MM medium as the basal medium, and adding 20 g / L glucose and 10 g / L sodium chloride, a fermenter experiment was conducted. The fermenter used was a 7 L fermenter with a fermentation liquid volume of 3 L, a pH set at 9.0, and a temperature maintained at 37 °C.

[0037] The fermentation culture was carried out under aeration and stirring conditions; during the fermentation culture, the initial stirring speed was 250 rpm, and after 8 hours of fermentation, the stirring speed stabilized at 700 rpm; the aeration rate was 4 L / min; the fermentation culture temperature was 35℃; the initial pH value of the fermentation medium was 6; and the initial dissolved oxygen value of the fermentation medium was 30%.

[0038] (5) The halophilic bacteria fermentation broth is centrifuged and washed to obtain bacterial cells; (6) The bacterial cells were subjected to cell wall disruption and enzymatic hydrolysis to extract P3HB, and the cells were washed to obtain cell wall disruption wastewater; (7) The cell wall disruption wastewater is introduced into a flocculation sedimentation tank containing 5 ppm of anionic polyacrylamide for flocculation sedimentation filtration to remove solid suspended matter and insoluble impurities, thus obtaining clear wastewater.

[0039] (8) The wastewater solution obtained in step (7) is filtered through a nanofiltration membrane with a weight-average molecular weight cutoff of 200 Da at a pressure of 0.3 MPa to remove small molecule impurities and large molecule organic matter, thereby obtaining the brine.

[0040] This embodiment further provides a P3HB fermentation process, which includes: reusing the recycled brine described in Example 1 into the fermenter of step (4) above to replace the fresh fermentation medium.

[0041] Example 2 This embodiment provides a method for recycling brine, which differs from Embodiment 1 only in that the brine is heated to 100°C to obtain recycled brine; the concentration of bicarbonate ions in the recycled brine is 0.3 mmol / L and the pH is 5.5.

[0042] This embodiment further provides a P3HB fermentation process, which includes: reusing the recycled brine described in Example 2 into the fermenter of step (4) above to replace the fresh fermentation medium.

[0043] Example 3 This embodiment provides a method for recycling brine, which differs from Embodiment 1 only in that the brine is heated to 121°C to obtain recycled brine; the concentration of bicarbonate ions in the recycled brine is 0.24 mmol / L and the pH is 6.5.

[0044] This embodiment further provides a P3HB fermentation process, which includes: reusing the recycled brine described in Example 3 into the fermenter of step (4) above to replace the fresh fermentation medium.

[0045] Example 4 This embodiment provides a method for reusing brine, which differs from Embodiment 1 only in that: the brine is heated to 110°C, and 0.02M sodium hydroxide is added to adjust the pH of the brine to obtain reusable brine; the concentration of bicarbonate ions in the reusable brine is 0.42 mmol / L, and the pH is 6.5.

[0046] This embodiment further provides a P3HB fermentation process, which includes: reusing the recycled brine described in Example 4 into the fermenter of step (4) above to replace the fresh fermentation medium.

[0047] Example 5 This embodiment provides a method for recycling brine, which differs from Embodiment 1 only in that: the brine is heated to 130°C to obtain recycled brine; the concentration of bicarbonate ions in the recycled brine is 0.62 mmol / L, and the pH is 6.5.

[0048] This embodiment further provides a P3HB fermentation process, which includes: reusing the recycled brine described in Example 5 into the fermenter of step (4) above to replace the fresh fermentation medium.

[0049] Example 6 This embodiment provides a method for recycling brine, which differs from Embodiment 6 only in that: no heating treatment is performed, and only 0.02M sodium hydroxide is added to the brine to adjust the pH of the brine to obtain recycled brine; the concentration of bicarbonate ions in the recycled brine is 0.72 mmol / L, and the pH is 7.5.

[0050] This embodiment further provides a P3HB fermentation process, which includes: reusing the recycled brine described in Example 7 into the fermenter of step (4) above to replace the fresh fermentation medium.

[0051] Example 7 This embodiment provides a P3HB fermentation process, which differs from Embodiment 1 only in that: *Haloxylon ammodendron* TD01 is replaced with... Halomonas campaniensis LS21.

[0052] Example 8 This embodiment provides a P3HB fermentation process, which differs from Embodiment 1 only in that: *Haloxylon ammodendron* TD01 is replaced with... Halomonas bluephagenesis WZY254.

[0053] Example 9 This embodiment provides a P3HB fermentation process, which differs from Embodiment 1 only in that: *Haloxylon ammodendron* TD01 is replaced with... Halomonas bluephagenesis TD68-194.

[0054] Example 10 This embodiment provides a P3HB fermentation process, which differs from Embodiment 1 only in that: *Haloxylon ammodendron* TD01 is replaced with... Halomonas bluephagenesis TDH4.

[0055] Comparative Example 1 This embodiment provides a P3HB fermentation process, which differs from Embodiment 1 only in that the recycled brine is replaced with an equal amount of untreated brine, wherein the concentration of bicarbonate ions in the brine is 20 mmol / L.

[0056] Comparative Example 2 This embodiment provides a P3HB fermentation process, which differs from Embodiment 8 only in that the recycled brine is replaced with an equal amount of untreated brine, wherein the concentration of bicarbonate ions in the brine is 20 mmol / L.

[0057] Comparative Example 3 This embodiment provides a P3HB fermentation process, which differs from Embodiment 9 only in that the recycled brine is replaced with an equal amount of untreated brine, wherein the concentration of bicarbonate ions in the brine is 20 mmol / L.

[0058] Comparative Example 4 This embodiment provides a P3HB fermentation process, which differs from Embodiment 10 only in that the recycled brine is replaced with an equal amount of untreated brine, wherein the concentration of bicarbonate ions in the brine is 20 mmol / L.

[0059] Comparative Example 5 This embodiment provides a P3HB fermentation process, which differs from Embodiment 11 only in that the recycled brine is replaced with an equal amount of untreated brine, wherein the concentration of bicarbonate ions in the brine is 20 mmol / L.

[0060] Test case After fermentation, the fermentation broths were freeze-dried and P3HB was extracted and prepared. The cell dry weight and P3HB yield were determined, and the test results are shown in Table 1 below.

[0061] Methods for determining P3HB content by gas chromatography: The furnace temperature was set to 80℃, the injector temperature to 200℃, the detector temperature to 220℃, and the column head pressure to 0.25 MPa. The programmed temperature rise conditions were: 80℃ for 1.5 minutes, then ramped up to 140℃ at a rate of 30℃ / min, followed by ramping up to 220℃ at a rate of 40℃ / min and holding at this temperature for 0.5 minutes. The sample injection volume was 1 μl, using a microsyringe manufactured by Agilent Technologies.

[0062] Gas chromatograph sample preparation: Take 40-60 mg of stem cells from the sample to be tested (centrifuge the bacterial culture at 10000 rpm at room temperature for 10 minutes, wash the resulting cell pellet once with water, and then freeze-dry to obtain stem cells; the homopolymer is produced in the cells), add 2 ml of chloroform and 2 ml of esterification solution (pure methanol containing 3% (v / v) concentrated sulfuric acid and 1 g / L benzoic acid as an internal standard) to an esterification tube, seal the tube, and heat at 100℃ for 4 hours. After cooling, add 1 ml of distilled water, shake thoroughly, and let stand until the chloroform phase and aqueous phase completely separate. Inject 1 μl of the lower chloroform phase into the gas chromatograph (HP Hewlett Packard 6890) for chromatographic analysis. Operate the gas chromatograph according to the HP Hewlett Packard 6890 gas chromatograph instruction manual.

[0063] Preparation of standard samples: Take 10~20 mg of standard sample into an esterification tube, add 2 ml of chloroform and 2 ml of esterification solution, seal the tube, and carry out esterification at 100℃.

[0064] Results analysis: Using the standard sample as a control, if the esterified sample of the cells to be tested (the sample to be tested) has a significant peak at the standard sample, the mass of each monomer can be calculated based on the peak area, and then the molar ratio can be calculated based on the mass fraction of each monomer; the proportion of polymer in the dry weight of the cells (wt%) can be calculated based on the amount of sample added.

[0065] The dry cell weight (CDW, g / L) mentioned below is the ratio of the mass of the dried cell to the volume of the fermentation product.

[0066] The P3HB content (wt%) mentioned below refers to the mass ratio of P3HB to the dried bacterial cells.

[0067] Table 1 Halomonas bluephagenesis TD01, Halomonas campaniensis LS21, Halomonas bluephagenesis WZY254, Halomonas bluephagenesis TD68-194, and Halomonas bluephagenesis TDH4 were fermented using fresh culture medium (bicarbonate ion concentration of 0 mmol / L). The results were compared with those of fermentation using recycled brine in Examples 1 and 8-11 above. The results are shown in Table 2. Table 2 The results showed that using the recycled brine obtained in the example for fermentation during the fermentation process of Haloxylon ammodendron can slightly increase cell dry weight and PHA content compared to untreated brine, significantly reduce production labor costs, and slightly increase sugar conversion rate, which has great industrial application value.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fermentation process for halophilic bacteria, characterized in that, The halophilic bacteria fermentation medium includes recycled brine, and the method for preparing the recycled brine includes: heating the brine to 100~121℃ for 5~20min and adjusting the pH of the brine to 5~7 to remove bicarbonate ions from the brine; The brine is obtained by the following method: cell wall disruption is performed on the fermentation broth of halophilic bacteria to obtain cell wall disruption wastewater, and then the cell wall disruption wastewater is coarsely filtered to remove solid suspended matter and insoluble impurities, and then membrane filtered using a nanofiltration membrane with an average molecular weight cutoff of less than 700 Da to obtain the brine. The concentration of bicarbonate ions in the saline solution is less than or equal to 100 mmol / L; The pressure during membrane filtration is 0.1~1MPa; and / or the coarse filtration is flocculation sedimentation coarse filtration, wherein the flocculant is anionic polyacrylamide; The halophilic bacteria are Halomonas or their derivatives or combinations thereof; The recycled brine is used instead of fresh culture medium for fermentation to promote the growth of halophilic bacteria and the production of PHA, thereby increasing cell dry weight and PHA content.

2. The fermentation process according to claim 1, characterized in that, The pressure during membrane filtration is 0.3~0.6MPa.

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