Complex microbial inoculant tolerant to high-salinity wastewater as well as preparation method and application of complex microbial inoculant
By mixing Halomonas nigrificans and Sphingomonas paucimobilis to form a compound bacterial agent, the problem of decreased microbial activity in high-salinity wastewater was solved, achieving efficient pollutant degradation and system stability.
Patent Information
- Application Number
- CN202511746691.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-26
- Publication Date
- 2026-02-10
AI Technical Summary
Existing technologies struggle to effectively degrade pollutants in high-salinity wastewater. Traditional biological treatment processes suffer from decreased microbial activity in high-salinity environments, and naturally salt-tolerant strains lack the ability to degrade specific industrial organic pollutants.
A compound microbial agent was formed by mixing Halomonas nigrificans and Sphingomonas paucimobilis in a certain proportion. Through co-cultivation, the microbial activity was maintained in a high-salt environment and the degradation efficiency of pollutants was improved.
The compound microbial agent significantly improved microbial activity and COD removal rate in high-salt environments, shortened system start-up time, enhanced engineering adaptability, and possessed good stability and modular potential.
Smart Images
Figure CN121495744A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biological treatment technology for high-salinity wastewater, specifically relating to a composite bacterial agent tolerant to high-salinity wastewater, its preparation method, and its application method. Background Technology
[0002] High-salinity wastewater typically refers to wastewater with a salinity exceeding 10 g / L. While traditional biological treatment processes (such as activated sludge and biofilm processes) can stably treat low- to medium-salinity wastewater, when the salinity exceeds 10 g / L, the system's osmotic pressure rises sharply, causing conventional microorganisms to experience cell dehydration, inhibited enzyme activity, or even death, resulting in a sharp reduction in system biomass and a significant decrease in treatment efficiency.
[0003] To address the aforementioned issues, existing biological treatment technologies for high-salinity wastewater mainly fall into two categories: one is to acquire salt tolerance through long-term domestication of conventional functional bacteria; the other is to directly add natural salt-tolerant bacterial strains to achieve pollutant degradation. While the former can improve salt tolerance to some extent, it has a long domestication period and poor adaptability; the latter, although it can establish bacterial communities in a short period, these strains mostly originate from extreme environments such as salt flats or oceans. Although they have good salt tolerance, they generally lack the ability to degrade specific industrial organic pollutants, making it difficult to meet the requirements for compliant discharge of high-salinity wastewater.
[0004] Recent studies have shown that some halophilic bacteria can synthesize or secrete compatible solutes (such as betaine, trehalose, and proline) to maintain cellular osmotic balance. If these compatible solutes can be absorbed and utilized by other functional bacteria in the system, osmotic stress on functional bacteria can be alleviated without additional carbon sources, restoring their metabolic activity and thus achieving a "synergistic salt tolerance" effect among microorganisms. However, a mature composite bacterial agent construction system based on this mechanism is still lacking, which can achieve efficient degradation of specific pollutants while possessing salt tolerance properties, thereby effectively shortening system start-up time and expanding the application scope of biological methods in the treatment of high-salt organic wastewater. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of the prior art and to provide a compound bacterial agent that is resistant to high salinity wastewater, its preparation method and its application method.
[0006] The specific technical solution adopted in this invention is as follows:
[0007] In a first aspect, the present invention provides a compound bacterial agent that is tolerant to high salinity wastewater. The compound bacterial agent is composed of salt-tolerant bacterial strains and functional bacterial strains mixed at a live bacteria ratio of 1:(0.5~2).
[0008] The salt-tolerant strain is Halomonas nigrificans, with accession number DSM 105749, deposited at the German Microbiological Culture Collection Center; the functional strain is Sphingomonaspaucimobilis, with accession number ATCC 10829, deposited at the American Center for Type Culture Collection.
[0009] Secondly, the present invention provides a method for preparing a composite bacterial agent tolerant to high salinity wastewater, the specific steps of which are as follows:
[0010] S1: Salt-tolerant strains and functional strains were inoculated into sterile LB liquid medium and cultured at 30-33℃ and 120-200 rpm until the logarithmic growth phase to obtain seed culture of salt-tolerant strains and seed culture of functional strains, respectively.
[0011] S2: Inoculate the salt-tolerant strain seed culture and the functional strain seed culture at a live cell ratio of 1:(0.5~2) into a new sterile LB liquid medium and incubate at 30~33℃ and 120~200 rpm for 6~8 hours to obtain a compound bacterial agent tolerant to high salinity wastewater.
[0012] The salt-tolerant strain is Halomonas nigrificans, with accession number DSM 105749, deposited at the German Microbiological Culture Collection Center; the functional strain is Sphingomonaspaucimobilis, with accession number ATCC 10829, deposited at the American Center for Type Culture Collection.
[0013] Preferably, the LB liquid culture medium comprises: 5 g / L yeast extract, 10 g / L trypsin and 10 g / L sodium chloride; the pH of the LB liquid culture medium is controlled at 7.0~7.2.
[0014] Preferably, the inoculation amounts of the salt-tolerant strain seed culture and the functional strain seed culture in step S2 are 0.5% to 1% of the LB medium, respectively.
[0015] Thirdly, the present invention provides a composite bacterial agent tolerant to high salinity wastewater obtained by the preparation method described in the second aspect.
[0016] Fourthly, the present invention provides an application of a high-salinity wastewater tolerant compound microbial agent, wherein high-salinity organic wastewater to be treated is added to a reaction vessel, and the high-salinity wastewater tolerant compound microbial agent described in the first or second aspect is added at 1% to 2% of the volume of the high-salinity organic wastewater, and the COD in the high-salinity organic wastewater is removed by culturing in an aerobic environment.
[0017] Preferably, the salinity of the high-salinity organic wastewater is 10~50 g / L.
[0018] Preferably, the COD concentration of the high-salinity organic wastewater is 300~600 mg / L.
[0019] Compared with the prior art, the present invention has the following advantages:
[0020] (1) No need for long-term domestication, can be directly added and used: The compound bacterial agent provided by the present invention is composed of salt-tolerant bacteria and functional bacteria selected by natural or conventional methods. It can be directly used for the treatment of high-salt wastewater without long-term and high-cost artificial domestication, which shortens the system start-up time and improves engineering adaptability.
[0021] (2) It exhibits synergistic degradation advantages in medium and high salinity environments: the co-culture system can still maintain high microbial activity and COD removal rate under salinity conditions of 30 g / L and above, which is significantly better than the single culture of functional bacteria, breaking through the problem of degradation failure of traditional bacterial agents in high salinity wastewater.
[0022] (3) Clear mechanism and strong stability: Salt-tolerant bacteria can alleviate osmotic pressure stress in the co-culture system by synthesizing and excreting compatible solutes such as betaine, thereby enhancing the survival and metabolic capacity of functional bacteria in a high-salt environment. The bacterial community structure is stable and the functional synergy is significant.
[0023] (4) The formulation of the microbial agent is flexible and can be extended to other functional microbial species (such as denitrifying bacteria and phosphorus-removing bacteria), and has good potential for modularization and industrialization. Attached Figure Description
[0024] Figure 1 The growth curves of the functional strains under different salinities in Example 4 are shown.
[0025] Figure 2 The growth curves of the salt-tolerant strains under different salinity conditions in Example 4 are shown.
[0026] Figure 3 The growth curves of the compound microbial agent under different salinities in Example 4 are shown. Detailed Implementation
[0027] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly, provided that there is no mutual conflict.
[0028] It should be noted that in the following specific embodiments, the salt-tolerant strain is *Halomonas nigrificans*, accession number DSM 105749, deposited at the German Microbiological Culture Collection Center; the functional strain is *Sphingomonas paucimobilis*, accession number ATCC 10829, deposited at the American Center for Type Culture Collection. Both strains are publicly available.
[0029] Example 1
[0030] This embodiment provides a method for preparing a compound bacterial agent tolerant to high salinity wastewater, the specific steps of which are as follows:
[0031] (1) Dissolve 5 g yeast extract, 10 g tryptic peptone and 10 g sodium chloride in 1 L distilled water, adjust the pH to 7.0 with NaOH solution, and then autoclave at 121℃ for 20 min to obtain sterilized LB liquid culture medium.
[0032] (2) Salt-tolerant strains and functional strains were inoculated into sterile LB liquid medium and cultured at 30°C and 200 rpm for 12 hours until the logarithmic growth phase, respectively, to obtain seed culture of salt-tolerant strains and seed culture of functional strains.
[0033] (3) Measure the viable cell count (CFU) of the seed culture of the salt-tolerant strain and the seed culture of the functional strain to ensure the predetermined inoculation concentration (approximately 10⁻⁶) for both strains. 6 ~10 7 (CFU / mL). Take 1 mL of each of the salt-tolerant strain seed culture and the functional strain seed culture, inoculate them into 200 mL of new sterile LB liquid medium, and incubate at 30℃ and 200 rpm for 8 hours to obtain a compound bacterial agent tolerant to high salinity wastewater.
[0034] Example 2
[0035] To verify the wastewater treatment effect of the high-salinity-tolerant compound microbial agent provided by this invention, this embodiment conducted a treatment experiment on 10 g / L high-salinity organic wastewater, as detailed below:
[0036] (1) Prepare simulated high-salt organic wastewater, including 468 mg / L glucose, 10 g / L NaCl, 50 mg / L NH4Cl, 50 mg / L KH2PO4 and 50 mg / L K2HPO4, with COD concentration of 500 mg / L and ammonia nitrogen concentration of 50 mg / L, and adjust pH to 7.0.
[0037] (2) In this embodiment, several 1500 mL feed bottles are used as reaction vessels, and 1000 mL of the above-mentioned simulated high-salt organic wastewater is added.
[0038] (3) The high-salinity-tolerant composite bacterial agent prepared in Example 1 was inoculated into the reaction vessel at 2% of the simulated high-salinity organic wastewater volume, as the composite bacterial agent group (G4-Low). Salt-tolerant bacterial solutions in the logarithmic growth phase were inoculated into the reaction vessel at 2%, as the salt-tolerant bacterial group (G3-Low). Functional bacterial solutions in the logarithmic growth phase were inoculated into the reaction vessel at 2%, as the functional bacterial group (G2-Low). No bacterial solution was added to the blank control group (G1-Low).
[0039] (3) All groups were cultured at 32℃ and 150 rpm for 48 hours, and samples were taken periodically. The changes in COD concentration in the wastewater were determined using the potassium dichromate method, and the results are shown in Table 1. The OD of each group's culture medium was also measured. 600 Values are used to assess the growth status of the microbial community.
[0040] Example 3
[0041] To verify the wastewater treatment effect of the high-salinity wastewater-tolerant compound bacterial agent provided by this invention, this embodiment conducted a treatment experiment on 30 g / L high-salinity organic wastewater, as detailed below:
[0042] (1) Prepare simulated high-salt organic wastewater, including 468 mg / L glucose, 30 g / L NaCl, 50 mg / L NH4Cl, 50 mg / L KH2PO4 and 50 mg / L K2HPO4, with COD concentration of 500 mg / L and ammonia nitrogen concentration of 50 mg / L, and adjust pH to 7.0.
[0043] (2) In this embodiment, several 1500 mL feed bottles are used as reaction vessels, and 1000 mL of the above-mentioned simulated high-salt organic wastewater is added.
[0044] (3) The high-salinity-tolerant composite bacterial agent prepared in Example 1 was inoculated into the reaction vessel at 2% of the simulated high-salinity organic wastewater volume, as the composite bacterial agent group (G4-Med). Salt-tolerant bacterial solutions in the logarithmic growth phase were inoculated into the reaction vessel at 2%, as the salt-tolerant bacterial group (G3-Med). Functional bacterial solutions in the logarithmic growth phase were inoculated into the reaction vessel at 2%, as the functional bacterial group (G2-Med). No bacterial solution was added to the blank control group (G1-Med).
[0045] (3) All groups were cultured at 32℃ and 150 rpm for 48 hours, and samples were taken periodically. The changes in COD concentration in the wastewater were determined using the potassium dichromate method, and the results are shown in Table 1. The OD of each group's culture medium was also measured.600 Values are used to assess the growth status of the microbial community.
[0046] Table 1 Comparison of COD removal rates in each group in Examples 2 and 3
[0047]
[0048] As shown in Table 1, at a salinity of 10 g / L, the functional bacterial group (G2-Low) exhibited a certain COD degradation capacity (38.44%), while the salt-tolerant bacterial group (G3-Low) showed a lower degradation capacity (19.02%). The compound bacterial agent group (G4-Low) showed a significantly enhanced synergistic degradation capacity, with a COD removal rate of 64.3%.
[0049] Under high-salt conditions of 30 g / L, the COD degradation capacity of functional bacteria (group G2-Med) and salt-tolerant bacteria (group G3-Med) was low (12.16% and 15.82%, respectively) when treated alone, while the composite bacterial agent (G4-Med) still maintained a COD removal rate of 39.57%, which is 2 to 3 times that of single-bacterial treatment. This indicates that the composite bacterial agent provided by the present invention can effectively maintain the metabolic capacity of functional bacteria in a high-salt environment.
[0050] Example 4
[0051] This embodiment conducts experiments to determine the growth capabilities of functional strains, salt-tolerant strains, and compound bacterial agents under different salinity gradients, as detailed below:
[0052] (1) Preparation of strains
[0053] Functional and salt-tolerant strains were inoculated into liquid LB medium and revived in a shaker at 37°C and 200 rpm for 24 hours for later use.
[0054] (2) Salinity gradient setting
[0055] To simulate different salt stress environments, six NaCl concentration gradient groups were set up, namely 10, 20, 30, 40, 50, and 60 g / L. The NaCl used in the experiment was prepared as a stock solution at a concentration of 15%, which was autoclaved and then added to the basic LB liquid medium in proportion to adjust to the required final concentration.
[0056] (3) Training system setup
[0057] The experiment used 48-well microplates as culture containers. 360 μL of LB medium corresponding to the salinity gradient was added to each well, and then bacterial culture was inoculated at a 1:100 volume ratio (initial OD). 600 (Approximately 0.1), to a final volume of 200 μL. Each treatment was configured with three parallel replicates, and blank wells without bacterial inoculation were included as a control.
[0058] (4) Co-cultivation setting
[0059] In the co-culture treatment group, the bacterial suspensions of functional strains and salt-tolerant strains were thoroughly mixed at a 1:1 volume ratio and then inoculated. Other culture conditions were the same as those in the single-strain culture group to analyze the changes in growth adaptability of the strains after co-culture.
[0060] (5) Culture conditions and data collection
[0061] After inoculation, the 96-well plate was placed in an automated microplate reader, the incubation temperature was set to 30℃, and the program was set to automatically shake and read the OD once per hour. 600 The value was continuously monitored for 24 hours to obtain dynamic growth curve data of the strain.
[0062] (6) Data processing methods
[0063] The raw OD obtained was processed using a Python script. 600 The data were smoothed (e.g., by Loess local weighted regression fitting), and growth curves of each strain under different salinity conditions were plotted. By comparing their maximum OD values, logarithmic growth rates, and differences in OD at the growth endpoint, the salt tolerance and co-culture effectiveness of the strains were comprehensively evaluated.
[0064] The results are as follows Figures 1-3 As shown.
[0065] according to Figure 1 It can be seen that functional bacteria can grow rapidly at a salinity of 10 g / L, and OD 600 The maximum value reached 0.78, and the maximum growth coefficient (μmax) was 0.069, indicating that it has good growth ability under low salinity conditions; however, its growth was significantly inhibited with increasing salinity, and the OD value dropped to 0.069 at 30 g / L and above. 600 It dropped below 0.3, and μmax dropped below 0.015, with almost no growth.
[0066] according to Figure 2 It can be seen that halophilic bacteria maintain stable growth within a salinity range of 10–60 g / L, and OD 600 The maximum value is close to or exceeds 1.0, the maximum μmax is up to 0.209, and the Lag time is controlled within 1.3 to 1.6 hours, showing its good salt tolerance and growth adaptability.
[0067] Figure 3 The growth of the compound microbial agent provided by this invention is shown. At a salinity of 30 g / L, the OD of the compound microbial agent group is... 600 The maximum value was 1.18, and the μmax was 0.157, which was significantly better than the groups of functional bacteria and salt-tolerant bacteria cultured alone, indicating that salt-tolerant bacteria and functional bacteria improved the growth rate through a synergistic mechanism.
[0068] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.
Claims
1. A compound bacterial agent tolerant to high salinity wastewater, characterized in that, The compound microbial agent is composed of salt-tolerant strains and functional strains mixed at a live cell ratio of 1:(0.5~2); The salt-tolerant strain is Halomonas nigrificans, with accession number DSM 105749, deposited at the German Microbiological Culture Collection Center; the functional strain is Sphingomonaspaucimobilis, with accession number ATCC 10829, deposited at the American Center for Type Culture Collection.
2. A method for preparing a composite bacterial agent tolerant to high salinity wastewater, characterized in that, The specific steps are as follows: S1: Salt-tolerant strains and functional strains were inoculated into sterile LB liquid medium and cultured at 30-33℃ and 120-200rpm until the logarithmic growth phase to obtain seed culture of salt-tolerant strains and seed culture of functional strains, respectively. S2: Inoculate the salt-tolerant strain seed culture and the functional strain seed culture at a live cell ratio of 1:(0.5~2) into a new sterile LB liquid medium and incubate at 30~33℃ and 120~200 rpm for 6~8 hours to obtain a compound bacterial agent tolerant to high salinity wastewater. The salt-tolerant strain is Halomonas nigrificans, with accession number DSM 105749, deposited at the German Microbiological Culture Collection Center; the functional strain is Sphingomonaspaucimobilis, with accession number ATCC 10829, deposited at the American Center for Type Culture Collection.
3. The preparation method of the high-salinity-tolerant composite microbial agent according to claim 2, characterized in that, The LB liquid culture medium comprises: 5 g / L yeast extract, 10 g / L trypsinized peptone and 10 g / L sodium chloride; the pH of the LB liquid culture medium is controlled at 7.0~7.
2.
4. The preparation method of the high-salinity-tolerant composite microbial agent according to claim 2, characterized in that, In step S2, the inoculation amounts of the salt-tolerant strain seed culture and the functional strain seed culture are 0.5% to 1% of the LB medium, respectively.
5. A composite bacterial agent tolerant to high salinity wastewater, obtained by any one of the preparation methods described in claims 2 to 4.
6. An application of a compound microbial agent tolerant to high salinity wastewater, characterized in that, Add the high-salinity organic wastewater to be treated to the reaction vessel, and add the high-salinity wastewater tolerant compound bacterial agent as described in claim 1 or 5 at 1% to 2% of the volume of the high-salinity organic wastewater. Cultivate in an aerobic environment to remove COD from the high-salinity organic wastewater.
7. The application of the high-salinity-tolerant compound microbial agent according to claim 6, characterized in that, The salinity of the high-salinity organic wastewater is 10~50 g / L.
8. The application of the high-salinity-tolerant compound microbial agent according to claim 6, characterized in that, The COD concentration of the high-salinity organic wastewater is 300~600 mg / L.