Method for treating cell-broken-wall wastewater and application thereof in fermentation of halophilic bacteria

Abstract

The present application relates to the technical field of biological fermentation, and particularly relates to a cell wall breaking wastewater treatment method and application thereof in halophilic bacteria fermentation, wherein the cell wall breaking wastewater is obtained by cell wall breaking of halophilic bacteria fermentation liquor, and the treatment method comprises the following steps: performing rough filtration on the cell wall breaking wastewater to remove solid suspensions and insoluble impurities in the cell wall breaking wastewater, and then performing membrane filtration on the cell wall breaking wastewater by using a nanofiltration membrane with a cut-off average molecular weight of 700 Da or less, so as to obtain a salt-containing clear liquid; further, the salt-containing clear liquid can be partially or entirely used as a solvent of a fermentation culture medium for halophilic bacteria fermentation process. Through the specific wastewater treatment method, the cell wall breaking wastewater in the halophilic bacteria fermentation process can be recycled, and the PHA yield can be effectively ensured, and even the growth rate of the production strain can be improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of wastewater treatment, and particularly relates to a method for treating cell wall-broken wastewater and application of the method in halophilic bacteria fermentation. BACKGROUND

[0002] Polyhydroxyalkanoate (PHA) is a kind of high molecular polyester synthesized by microorganisms, which has high biodegradability and biocompatibility compared with traditional petroleum-based plastic products. PHA is a new type of biocompatible material, which can help solve the increasingly serious plastic pollution problem and has important significance for the construction of "green water and green mountains". At present, PHA is mainly produced by fermentation of different engineering bacteria. For example, the patent CN202210353439.7 realizes the production of PHA by engineering modification of Rothia. The patent CN201010578858.8 proposes a strain of Halomonas (a kind of halophilic bacteria), which is applied to the production of PHA. Even though halophilic bacteria have great potential as the next generation of industrial microorganisms, they have the advantages of open fermentation and resistance to other bacteria under the external conditions of high salt concentration. Moreover, the large-scale production of PHA by halophilic bacteria, represented by Halomonas, can avoid the huge energy consumption of aseptic fermentation and effectively reduce costs. However, the production cost of PHA is still relatively high, which hinders its commercial development. Specifically, in the extraction process of PHA, in order to improve the purity of PHA products, the bacterial cell precipitate is usually washed 1-5 times with a washing liquid such as water, physiological saline and various buffers (such as PBS buffer). However, after washing, there are still a large amount of fermentation metabolites in the bacterial cells. In order to further extract PHA from the bacterial cells, the cells need to be broken, such as using organic solvent extraction, physical and mechanical crushing, surfactant method or enzyme method, etc. to break the cell wall. And the broken bacterial cells are washed again with a washing liquid. Therefore, the extraction process of PHA consumes a large amount of water resources, and the wastewater treatment and discharge generated by the extraction process of PHA will greatly increase the production cost of enterprises.

[0003] To solve the above problems, patent CN111362445B proposes a polyhydroxyalkanoate fermentation broth treatment method. The method separates the polyhydroxyalkanoate fermentation broth by first solid-liquid separation, then adds an adsorbent to the obtained fermentation supernatant for adsorption treatment, and further performs plate and frame filtration on the treated fermentation supernatant. Finally, the salt-containing fermentation waste liquid is recycled to the polyhydroxyalkanoate fermentation stage for reuse. The method can realize the reuse of high-salinity wastewater generated during PHA fermentation by halophilic bacteria, and can also ensure the PHA yield. However, this method is only suitable for the fermentation supernatant after solid-liquid separation of the polyhydroxyalkanoate fermentation broth. That is, the method provides an idea for how to treat the wastewater generated during washing of the bacterial cells in the PHA extraction process. However, how to solve the problem of large water consumption and difficult treatment during cell wall breaking and subsequent washing is still very serious.

[0004] Therefore, the present application is proposed. SUMMARY

[0005] In patent CN111362445B, it is mentioned that if the generated high-salinity wastewater is directly reused for fermentation, the insoluble bacterial cell fragments, denatured proteins, cell toxins, pigments and other difficult-to-utilize substances in the fermentation wastewater will cause the viscosity of the fermentation system to increase, the dissolved oxygen and mass transfer to deteriorate, thereby affecting the fermentation efficiency of the fermentation strain. With the recycling of the fermentation wastewater, these substances will continue to accumulate, causing difficulties in subsequent separation, thereby reducing the PHA yield and purity. However, the present application also encounters the above problems during the resource utilization of the wastewater generated during cell wall breaking and subsequent washing. In particular, compared with the fermentation supernatant in patent CN111362445B, the wastewater generated during cell wall breaking and subsequent washing in the present application has more complex components, including the above-mentioned insoluble bacterial cell fragments, denatured proteins, cell toxins, pigments and other substances, and has the characteristics of high salinity, high alkalinity and high viscosity. Using general water treatment methods, including the method in patent CN111362445B, even if the impurities and harmful substances can be removed to a large extent, the treated supernatant cannot be directly used for the fermentation stage of polyhydroxyalkanoate, and it is difficult to ensure the fermentation efficiency of the fermentation strain and the subsequent PHA yield.

[0006] Therefore, the present application provides a cell wall breaking wastewater treatment method and its application in halophilic bacteria fermentation. By using a specific nanofiltration membrane to filter the cell wall breaking wastewater, the wastewater generated during cell wall breaking and subsequent washing is directly reused, and the PHA yield is effectively ensured during the reuse process. Furthermore, the growth rate of the halophilic bacterial strain is also improved, and the production cycle is shortened.

[0007] Specifically, the method for treating cell wall broken wastewater, which is obtained by cell wall breaking of a fermentation broth of halophilic bacteria, comprises: performing rough filtration on the cell wall broken wastewater to remove solid suspensions and insoluble impurities therein, and then performing membrane filtration on the cell wall broken wastewater by using a nanofiltration membrane with a cut-off average molecular weight of 700 Da or less, so as to obtain a salt-containing clear liquid.

[0008] The nanofiltration membrane with the specific structure selected in the application has good cut-off effect on substances that are not conducive to PHA production fermentation in the cell wall broken wastewater, in particular, the nanofiltration membrane has a removal rate of divalent ions and multivalent ions in water of more than 95%, and can effectively remove multivalent ions such as calcium, magnesium, carbonate and sulfate, thereby avoiding the influence of trace elements remaining in the cell wall broken wastewater on subsequent fermentation and avoiding the imbalance of trace elements caused by subsequent trace element supplement; and the nanofiltration membrane can also retain components conducive to the growth of halophilic bacteria, such as part of inorganic salts and nutrient components, so that the salt-containing clear liquid after membrane filtration can be directly reused in the fermentation medium of halophilic bacteria, and the method greatly simplifies the process flow, saves the use of inorganic salts, ingredients and water resources, reduces the treatment pressure and cost of wastewater, and significantly reduces the production cost of halophilic bacteria for PHA production.

[0009] Preferably, in the method for treating cell wall broken wastewater provided by the application, the salt content in the salt-containing clear liquid is greater than or equal to 20 g / L, and preferably ranges from 20 g / L to 30 g / L.

[0010] In order to improve the utilization value of the salt-containing clear liquid, it is found that the salt-containing clear liquid with different concentrations has a significant influence on the fermentation system of halophilic bacteria when the salt-containing clear liquid obtained by membrane filtration of the nanofiltration membrane is directly reused in the fermentation process of halophilic bacteria, and if the components conducive to the growth of halophilic bacteria are too low or too high, the indicators of the fermentation system will be unstable, the monitoring difficulty and operation complexity will be increased, and in particular, the influence of the salt content is particularly prominent, and the salt-containing clear liquid often needs to be supplemented with salt substances (such as sodium chloride) to be used for normal PHA production.

[0011] Preferably, the method for treating the cell disruption wastewater provided by the present application comprises: treating the salt-containing supernatant by a reverse osmosis membrane to a salt content higher than 20 g / L.

[0012] It is found by comparison that, compared with other concentration methods such as heat concentration, membrane evaporation concentration, vacuum concentration, etc., the reverse osmosis membrane method of the present application can not only efficiently concentrate the salt-containing supernatant, but also retain a large amount of nutrient components in the concentrated salt-containing supernatant, which are suitable for reuse in the fermentation process of the halophilic bacteria, especially for improving the growth rate of the halophilic bacteria. The reverse osmosis membrane in the present application can be obtained by commercial purchase.

[0013] Preferably, when the pressure during the membrane filtration is 0.1-1 MPa, for example, 0.1 MPa, 0.2 MPa, 0.3 MPa, 0.4 MPa, 0.5 MPa, 0.6 MPa, 0.7 MPa or 0.8 MPa, the effect of membrane filtration can be significantly improved while ensuring that the obtained salt-containing supernatant can be directly reused in the fermentation of the halophilic bacteria, and more preferably 0.3-0.6 MPa.

[0014] Preferably, the method for treating the cell disruption wastewater provided by the present application is a flocculation sedimentation coarse filtration, wherein the flocculating agent is an anionic polyacrylamide.

[0015] It is also found in the present application that the anionic polyacrylamide molecular chain contains a certain amount of polar groups that can adsorb suspended solid particles in water, so as to bridge the particles to form large flocs. Therefore, it can significantly accelerate the sedimentation of particles in the cell disruption wastewater, very obviously accelerate the clarification of the cell disruption wastewater, effectively promote the membrane filtration efficiency and ensure the growth of the halophilic bacteria when the salt-containing supernatant is reused, etc. When the amount of the anionic polyacrylamide used is 0.01-10 ppm, it can fully play a role, and preferably the amount of the anionic polyacrylamide used is 2-6 ppm.

[0016] The present application also provides a salt-containing supernatant obtained by the method for treating the cell disruption wastewater as described above.

[0017] The present application also provides a fermentation process of the halophilic bacteria as described above, comprising: using part or all of the salt-containing supernatant as described above as a solvent of the fermentation medium of the halophilic bacteria to promote the growth of the halophilic bacteria.

[0018] Preferably, in the fermentation process of the halophilic bacteria provided by the present application, the halophilic bacteria include halophilic bacteria and / or halophilic archaea, preferably the halophilic bacteria are halophilic bacteria, further preferably bacteria of the genus Halomonas and their derived strains or combinations thereof, more preferably any species of the genus Halomonas, such as any of Halomonas bluephagenesis, Halomonas campaniensis, Halomonas aydingkolgenesis, Halomonas aerodenitrificans, Halomonas halocynthiae.

[0019] Further preferably, the halophilic bacteria are Halomonas bluephagenesis TD01, with the accession number of CGMCC No. 4353, obtained from Tsinghua University.

[0020] The above-mentioned salt-containing supernatant is used in whole or in part to promote the growth of halophilic algae.

[0021] Preferably, in the fermentation process of the halophilic bacteria provided by the present application, the cell wall broken wastewater is obtained by enzymatic cell wall breaking of the fermentation broth of the halophilic bacteria.

[0022] Compared with other methods for obtaining cell wall broken wastewater, such as organic solvent extraction, physical and mechanical crushing, and surfactant method, etc., it is found in experiments that enzymatic cell wall breaking is more preferable, and the salt-containing supernatant obtained by enzymatic cell wall breaking and the above-mentioned treatment is more conducive to promoting the fermentation of the halophilic bacteria.

[0023] Preferably, in the fermentation process of the halophilic bacteria provided by the present application, the fermentation medium is obtained by adding each material into a solvent containing 20-100% (mass percentage) of the salt-containing supernatant, preferably the fermentation medium is obtained by directly adding each material into the salt-containing supernatant (i.e. 100% salt-containing supernatant as a preparation solvent of the fermentation medium).

[0024] The mass concentration of salt in the fermentation medium is not less than 0.2%, preferably 0.2-10%. Preferably, the fermentation medium mainly includes, in terms of mass percentage: 0.1-20% glucose, 0-5% yeast powder, 0.1-5% urea, 0-10% peptone, 0.2-10% sodium chloride, 0.1-10% other inorganic salts, 1.2-15% trace element component I, and 0.1-1.2% trace element component II.

[0025] The other inorganic salt is one or more of magnesium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate and ammonium chloride; the trace element component I is obtained by adding ammonium ferric citrate and calcium chloride into the salt-containing liquid, and the mass percentage of ammonium ferric citrate in the trace element component I is 0.2-4.5%, and the mass percentage of calcium chloride is 0.3-2%; the trace element component II includes one or more of zinc sulfate heptahydrate, manganese chloride tetrahydrate, boric acid, manganese sulfate, cobalt chloride hexahydrate, copper sulfate pentahydrate, nickel chloride hexahydrate and sodium molybdate dihydrate.

[0026] Further preferably, the fermentation process comprises:

[0027] The bacteria used for production are inoculated into a slant bottle for slant culture to obtain a slant strain;

[0028] The slant strain is subjected to shake flask culture until OD 600 is 2.5-5.0 to obtain a primary seed; wherein the fermentation time of the primary seed is 12-24 h;

[0029] The primary seed is further subjected to shake flask culture until OD 600 is 2.5-5.0 to obtain a secondary seed; wherein the fermentation time of the secondary seed is 8-24 h;

[0030] The secondary seed is inoculated into the fermentation medium for fermentation culture to obtain a halophilic bacteria fermentation liquor; wherein the inoculation amount of the secondary seed is 2-10%, and the fermentation culture time is 8-48 h; the fermentation culture is carried out under the conditions of aeration and stirring; during the fermentation culture, the initial stirring speed is 200-300 rpm, and after 6-10 h of fermentation culture, the speed is stabilized at 600-800 rpm; the aeration amount of the aeration is 3-5 L / min; the fermentation culture temperature is 20-45°C; the initial pH value of the fermentation medium is 4.5-10; and the initial dissolved oxygen of the fermentation medium is 10-100%.

[0031] The cell wall breaking wastewater treatment method and application thereof in halophilic bacteria fermentation provided by the present application is that the cell wall breaking wastewater is filtered by using a specific nanofiltration membrane, most of the organic impurities harmful to the bacteria in the cell wall breaking wastewater are removed, and the cell wall breaking wastewater after treatment becomes salt-containing liquid which has no toxic and harmful effects on the bacteria, and is directly used in the preparation of the fermentation medium of the halophilic bacteria to replace the solvent, which can reduce the cost of auxiliary materials, reduce the loss of water resources, reduce the treatment pressure of wastewater, and reduce the discharge of three wastes, so that the method provided by the present application is a low-cost and high-efficient and feasible treatment method. In addition, the salt-containing liquid obtained by the cell wall breaking wastewater treatment method of the present application is used for fermentation instead of pure water source, the residual nutrients are used for the growth of the halophilic bacteria and the production of PHA, and it is verified that the yield of PHA can be effectively ensured, and the growth rate of the halophilic bacteria strain is further improved, and the production cycle is shortened. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the present application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0033] Figure 1 The process flow chart of the cell wall breaking wastewater treatment method provided by the present application is shown in the figure.

[0034] Figure 2 The process flow chart of the fermentation recycling process of the cell wall breaking wastewater provided by the present application is shown in the figure.

[0035] Figure 3 The growth curve of Halomonas sp TD01 in Example 7, Example 10 and Comparative Example 5 when producing PHB by fermentation of different cell wall breaking wastewater is shown in the figure. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the present application more clear, the technical solutions in the present application will be described clearly and completely in combination with the drawings in the present application. Obviously, the described embodiments are some embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0037] Unless otherwise specified in the examples, the techniques or conditions described in the literature or according to the product specification are used. Unless otherwise specified, the reagents or instruments used are conventional products that can be purchased through regular channels. The nanofiltration membrane used in the present application can be purchased from Zhejiang Jianmembrane Technology Co., Ltd.

[0038] The cell wall broken wastewater in the embodiments of the present application is obtained in the process of extracting polyhydroxyalkanoate after enzymatic cell wall breaking from the fermentation broth obtained by using the conventional fermentation process of Halomonas bluephagenesis TD01 (the preservation registration number is CGMCC No. 4353, which can be obtained by the public from Tsinghua University) for preparing polyhydroxyalkanoate. For specific methods, refer to the description in paragraphs 【0046-0048】 of patent CN111362445B.

[0039] Among them, the polyhydroxyalkanoate (PHA) can be PHB, PHV, P3HP, PHO, PHN, PHBV, P34HB or PHBHHx. In specific embodiments, PHB is taken as an example to explain the method of the present application in detail.

[0040] Example 1: Cell wall broken wastewater treatment method

[0041] A cell wall broken wastewater treatment method, as shown in Figure 1 , the steps are as follows:

[0042] (1) The above cell wall broken wastewater is introduced into a flocculation sedimentation tank containing 5 ppm of anionic polyacrylamide, and flocculation sedimentation filtration is performed to remove the solid suspended matter and insoluble impurities therein, thereby obtaining wastewater supernatant.

[0043] (2) The wastewater supernatant obtained in step (1) is subjected to membrane filtration at a pressure of 0.3 MPa through a nanofiltration membrane with a weight average molecular weight cutoff of 200 Da to remove small molecular impurities and large molecular organic matter therein, thereby obtaining a salt-containing supernatant.

[0044] (3) The salt-containing supernatant obtained in step (2) is temporarily stored in a nanofiltration wastewater storage tank, which is equipped with a salinity measuring probe. When the capacity of the nanofiltration wastewater storage tank reaches 80%, automatic salinity detection is performed. After detection, the salt content of the salt-containing supernatant is 15 g / L.

[0045] (4) The salt-containing supernatant with a salt content of 15 g / L is subjected to reverse osmosis membrane treatment to concentrate it to a salt content of 20 g / L, and then transferred to a cell disruption wastewater reuse storage tank.

[0046] Example 2: Cell wall broken wastewater treatment method

[0047] A method for treating cell wall breaking wastewater is basically the same as that in Embodiment 2, except that the reverse osmosis membrane process in step (4) is adjusted to further concentrate the salt-containing supernatant to a salt content of 30 g / L.

[0048] Fermentation medium for halophilic bacteria fermentation

[0049] A fermentation medium for halophilic bacteria fermentation is obtained by directly adding each material into the salt-containing supernatant obtained in Embodiment 1, and the mass concentration of salt in the obtained fermentation medium is 2%, and the fermentation medium is mainly composed of 2.5% glucose, 1% yeast powder, 0.5% peptone, 0.5% sodium chloride, 0.5% magnesium sulfate, 0.3% ammonium chloride, 0.5% urea, 2% trace element component I, 0.1% zinc sulfate heptahydrate, 0.2% manganese chloride tetrahydrate, 0.1% copper sulfate pentahydrate and 0.1% sodium molybdate dihydrate; wherein the trace element component I is obtained by adding ferric ammonium citrate and calcium chloride into the salt-containing supernatant, and the mass percentage of ferric ammonium citrate in the trace element component I is 0.6%, and the mass percentage of calcium chloride is 1%.

[0050] Fermentation medium for halophilic bacteria fermentation

[0051] A fermentation medium for halophilic bacteria fermentation is basically the same as that in Embodiment 3, except that the salt-containing supernatant obtained in Embodiment 1 is replaced by an equal amount of the salt-containing supernatant obtained in step (3) in Embodiment 1.

[0052] Fermentation medium for halophilic bacteria fermentation

[0053] A fermentation medium for halophilic bacteria fermentation is basically the same as that in Embodiment 3, except that the salt-containing supernatant obtained in Embodiment 1 is replaced by an equal amount of the salt-containing supernatant obtained in Embodiment 2.

[0054] Fermentation medium for halophilic bacteria fermentation

[0055] A fermentation medium for halophilic bacteria fermentation is basically the same as that in Embodiment 3, except that the salt-containing supernatant obtained in Embodiment 1 is diluted by 1 times with common solvent water before the fermentation medium is prepared.

[0056] Halophilic bacteria fermentation process

[0057] A process for fermentative production of poly-3-hydroxybutyrate (PHB) is shown in the process flow diagram as Figure 2 , and the specific steps are as follows:

[0058] (1) Halophilic bacteria TD01 is inoculated from a frozen tube into a slant bottle for slant culture to obtain a slant strain;

[0059] (2) the slant strain is transferred to a primary seed bottle for shake flask culture for 16 h to OD 600 is 3.0, to obtain a primary seed;

[0060] (3) the primary seed is transferred to a secondary seed bottle for shake flask culture for 18 h to OD 600 is 3.5, to obtain a secondary seed;

[0061] (4) the secondary seed is inoculated into a fermentation medium in a fermenter at an inoculation amount of 6% for fermentation culture for 36 h to obtain a halophilic bacterial fermentation liquor; the fermentation medium is obtained in Example 3, and the fermentation culture is carried out under the conditions of aeration and stirring; during the fermentation culture, the initial stirring speed is 250 rpm, and the stirring speed is stabilized at 700 rpm after 8 h of fermentation culture; the aeration amount of the aeration is 4 L / min; the fermentation culture temperature is 35°C; the initial pH value of the fermentation medium is 6; and the initial dissolved oxygen of the fermentation medium is 30%.

[0062] (5) the halophilic bacterial fermentation liquor is subjected to centrifugation and washing to obtain a bacterial body;

[0063] (6) the bacterial body is subjected to wall-breaking lysis and enzymatic extraction of PHB therein, and after washing, a cell wall-breaking wastewater is obtained;

[0064] (7) the cell wall-breaking wastewater is treated by using the method of enzymatic wall breaking, and the treatment method refers to steps (1)-(4) in Example 1, to obtain a salt-containing clear liquor;

[0065] (8) the salt-containing clear liquor is used to prepare a fermentation medium by using the method in Example 3, and is reused in the fermenter in the above step (4).

[0066] Example 8 Halophilic bacterial fermentation process

[0067] A fermentation reuse process for PHB fermentation production is basically the same as that in Example 7, except that the fermentation medium in step (4) is replaced by an equal amount of the fermentation medium obtained in Example 4, and the treatment method in step (7) refers to steps (1)-(3) in Example 1 to obtain a salt-containing clear liquor.

[0068] Example 9 Halophilic bacterial fermentation process

[0069] A fermentation reuse process for PHB fermentation production is basically the same as that in Example 7, except that the fermentation medium in step (4) is replaced by an equal amount of the fermentation medium obtained in Example 5, and the treatment method in step (7) refers to Example 2 to obtain a salt-containing clear liquor.

[0070] Example 10 Halophilic bacterial fermentation process

[0071] A fermentation reuse process for PHB fermentation production, which is basically the same as Example 7, except that the fermentation medium is replaced with the fermentation medium obtained in Example 6, and the treatment method in step (7) is replaced with the salt-containing liquid obtained in Example 1.

[0072] Example 11 Halomonas fermentation process

[0073] A fermentation reuse process for PHB fermentation production, which is basically the same as Example 7, except that Halomonas bluephagenesis WZY254 (a derivative of TD01, obtained from Example 1 in CN 113564193 A) is used instead of Halomonas TD01.

[0074] Comparative Example 1

[0075] A method for treating cell wall broken wastewater using a nanofiltration membrane with a molecular weight cut-off of 800 Da, which is basically the same as Example 1, except that the sodium filter membrane with a molecular weight cut-off of 200 Da in step (2) is replaced with a nanofiltration membrane with a molecular weight cut-off of 800 Da.

[0076] A fermentation reuse process for PHB fermentation production, the specific steps are:

[0077] (1) Halomonas TD01 is inoculated from a frozen tube into a slant bottle for slant culture, and a slant strain is obtained;

[0078] (2) The slant strain is transferred to a primary seed bottle for shake flask culture for 16 h to OD 600 is 3.0, and a primary seed is obtained;

[0079] (3) The primary seed is transferred to a secondary seed bottle for further shake flask culture for 18 h to OD 600 is 3.5, and a secondary seed is obtained;

[0080] (4) The secondary seed is inoculated into the fermentation medium in the fermentation tank at an inoculation amount of 6% for fermentation culture for 36 h, and a Halomonas fermentation liquor is obtained;

[0081] Wherein the fermentation medium is basically the same as Example 3, except that the salt-containing liquid obtained in Example 1 is replaced with the salt-containing liquid obtained by the method for treating cell wall broken wastewater using a nanofiltration membrane with a molecular weight cut-off of 800 Da;

[0082] and the fermentation culture is carried out under conditions of aeration and stirring; during the fermentation culture, the initial rotation speed of the stirring is 250 rpm, and after 8 h of fermentation culture, the rotation speed is stabilized at 700 rpm; the aeration amount of the aeration is 4 L / min; the temperature of the fermentation culture is 35°C; the initial pH value of the fermentation medium is 6; and the initial dissolved oxygen of the fermentation medium is 30%.

[0083] (5) The halophilic bacteria fermentation liquor is subjected to centrifugation and washing to obtain bacterial cells;

[0084] (6) The bacterial cells are subjected to wall-breaking lysis and enzymatic extraction of PHB therein, and after washing, cell wall-breaking wastewater is obtained.

[0085] (7) The cell wall-breaking wastewater is treated by using an enzymatic wall-breaking method, and the treatment method refers to the method of treating the cell wall-breaking wastewater by using the nanofiltration membrane with a molecular weight cut-off of 800 Da described above, to obtain a salt-containing clear liquid.

[0086] (8) The salt-containing clear liquid is used to prepare the fermentation medium by using the method in step (4), and is reused in the fermenter in step (4) described above.

[0087] Comparative Example 2

[0088] A fermentation medium for halophilic bacteria fermentation, which is basically the same as that in Example 3, except that the salt-containing clear liquid therein is replaced by water in equal mass.

[0089] The fermentation medium is used to replace the fermentation medium in Example 7 in equal mass, and is used in the fermentation reuse process for PHB fermentation production in Example 7.

[0090] In the halophilic bacteria fermentation processes in Examples 7-11, Comparative Example 1 and Comparative Example 2, the fermentation stage is carried out according to the conventional fermentation requirements, and the OD 600 is measured, and the growth curve is plotted, as shown in Figure 3 After the fermentation is completed, each fermentation liquor is freeze-dried, and PHB is extracted and prepared. The cell dry weight and PHB yield are measured, and the test results are shown in Table 1 below.

[0091] Table 1

[0092]

[0093]

[0094] From the above Table 1 and Figure 3As can be seen, the high-salinity wastewater supernatant of different concentrations is used to replace the solvent water and sodium chloride in the culture medium to carry out the PHB production fermentation. The growth state of the strain in Example 7 in the first 12 hours is similar to that in Comparative Example 2, and the growth state is gradually superior to that in Comparative Example 2 as the fermentation proceeds. The OD600 value of the PHB production strain cultured in Example 7 is higher than 400 at the end of the fermentation. Therefore, it can be seen that the salt-containing supernatant obtained by treating the cell wall-broken wastewater according to the present application can replace the solvent water and inorganic salt in the fermentation medium, so as to be used for the fermentation of halophilic bacteria. The normal yield of PHB can be ensured, the use of water resources and inorganic salt can be reduced, and the production cycle can be shortened, which has very important technical significance. In addition, as can be seen from the test index of Comparative Example 1, the molecular weight cut-off of the nanofiltration membrane has a significant influence on the growth degree of the halophilic bacteria during membrane filtration, which directly affects the yield of the final PHB.

[0095] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A fermentation process of halophilic bacteria, characterized in that, The application relates to a method for preparing a fermentation medium for halophilic bacteria, and belongs to the technical field of fermentation medium preparation. The method comprises the following steps: using a salt-containing clear liquid obtained by treating cell wall-broken wastewater as a solvent of the fermentation medium for the halophilic bacteria; the halophilic bacteria are halomonas or a derivative strain thereof or a combination thereof; the cell wall-broken wastewater is obtained by enzymatically breaking the cell wall of the fermentation liquid of the halophilic bacteria; the treatment method of the cell wall-broken wastewater comprises the following steps: after the cell wall-broken wastewater is coarsely filtered to remove solid suspensions and insoluble impurities, the cell wall-broken wastewater is subjected to membrane filtration by using a nanofiltration membrane with a cut-off average molecular weight of 700 Da or below, so as to obtain the salt-containing clear liquid; the pressure during the membrane filtration is 0.3-0.6 MPa; the salt content in the salt-containing clear liquid is 20-30 g / L; the fermentation medium is obtained by adding various materials into the solvent containing 20-100% of the salt-containing clear liquid. The coarsely filtering is flocculation and sedimentation coarsely filtering, wherein the flocculating agent is an anionic polyacrylamide. The fermentation medium is obtained by directly adding various materials into the salt-containing clear liquid. ​ ​ 2. The fermentation process of halophilic bacteria according to claim 1, characterized in that, ​ 3. The fermentation process according to claim 1 or 2, characterized in that, ​

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