High-salt pharmaceutical wastewater-based low-cost halotolerant bacterium culture medium and expanding culture method thereof

Abstract

The invention discloses a high-salt pharmaceutical wastewater-based low-cost halotolerant bacterium culture medium and an expanding culture method thereof, and belongs to the field of environmental protection and microbial fermentation. According to the culture medium, 90%-95% of pretreated high-salt pharmaceutical wastewater is used as a substrate and matched with industrial auxiliary materials such as industrial glucose and urea, the salinity of the pretreated wastewater is 20000-30000 mg / L, and the COD is 10000-20000 mg / L. The expanding culture method comprises the steps of strain activation, seed solution preparation, 2 m < 3 > batch scale expanding culture, harvesting and storage, microporous aeration and mechanical stirring are adopted to guarantee uniform dissolved oxygen, and a trehalose-glycerol composite protective agent is added. According to the method, the cost of a ton of fungicide is greatly reduced by using waste culture bacteria, the cell density of the fungicide is greater than or equal to 30,000,000 / mL, the 36h COD removal rate is greater than or equal to 70%, the activity attenuation is less than or equal to 5% after 30 days of refrigeration at 4 DEG C, the problems of high cost, difficulty in large-scale production and poor stability in the prior art are solved, and the method is suitable for biological enhancement treatment of various types of high-salt pharmaceutical wastewater.

Description

Technical Field

[0001] This invention belongs to the fields of environmental protection and microbial fermentation engineering technology, specifically relating to a low-cost salt-tolerant compound bacteria culture medium based on high-salt pharmaceutical wastewater and a method for large-scale expansion cultivation based on this culture medium. Background Technology

[0002] High-salinity pharmaceutical wastewater, characterized by high salinity, high COD concentration, and the presence of various toxic substances, exhibits strong osmotic pressure and bioinhibition, posing a long-standing challenge in wastewater treatment. Traditional biological treatment processes suffer from limited microbial activity in high-salinity environments, leading to high dosages of microbial agents and low treatment efficiency. Existing salt-tolerant microbial agent technologies still face several bottlenecks: firstly, culture media often rely on expensive laboratory reagents such as peptone and yeast extracts, failing to fully utilize the wastewater's own organic matter and nutrients, resulting in resource waste and high production costs, hindering large-scale application; secondly, existing compound microbial agents often suffer from unclear strain composition and a lack of targeted combination logic, with most single or simple compound strains unable to adapt to the complex matrix of high-salinity pharmaceutical wastewater, and the ambiguous composition of activation culture media leading to low activation efficiency and unstable activity; furthermore, during large-scale cultivation, difficulties in dissolved oxygen transfer and fluctuations in temperature and pH in high-salinity systems make it difficult to achieve 2m... 3 The stable production of the above batches lacked suitable protection measures after harvesting the microbial agents, and their activity easily declined rapidly during storage and transportation, seriously affecting the field application effect. Although related technologies proposed the theoretical concept of "using waste to cultivate microorganisms," it only remained at the feasibility analysis stage and did not form a specific high-proportion wastewater base formula, suitable microbial strain combinations, engineered expansion parameters, and a complete activity maintenance technology chain, thus failing to achieve synergistic optimization of cost and performance. Summary of the Invention

[0003] In view of this, the present invention proposes a low-cost salt-tolerant bacteria culture medium based on high-salt pharmaceutical wastewater and its expansion method, aiming to overcome the multiple bottlenecks of existing technologies in terms of cost, scalability, stability and strain compatibility, and to achieve a closed-loop synergy of using waste to cultivate bacteria and using bacteria to treat waste.

[0004] The technical solution of the present invention is achieved as follows: The present invention provides a low-cost salt-tolerant bacteria culture medium based on high-salt pharmaceutical wastewater and a method for its propagation, while covering the application scenarios of the culture medium and the bacterial agent, forming a complete technical system.

[0005] In some embodiments, the culture medium uses pretreated high-salt pharmaceutical wastewater as a substrate, with the substrate accounting for 90%-95% (v / v). The pretreatment method is coagulation sedimentation, filtration, or centrifugation to remove suspended solids. The pretreated high-salt pharmaceutical wastewater meets the following requirements: salinity 20000-30000 mg / L, COD 10000-20000 mg / L, pH 6.5-8.5. At the same time, industrial-grade excipients are added to the substrate, including 1.8-2.2 g / L of industrial glucose, 1.0-1.4 g / L of urea, 0.6-1.0 g / L of industrial phosphate, and 1.3-1.7 g / L of molasses.

[0006] The design of a high-percentage wastewater substrate is not simply about increasing the amount of wastewater used, but rather based on a dual logic of resource utilization and toxicity adaptation. The pretreatment process not only removes suspended solids to avoid interference with subsequent cultivation, but also adsorbs some toxic substances. The carbon and nitrogen sources contained in the wastewater itself can directly provide nutrients for the bacteria. The selected industrial-grade excipients each perform specific functions: glucose provides a rapidly available carbon source, urea supplements the nitrogen source and releases it slowly to avoid osmotic pressure fluctuations, phosphate regulates cell membrane stability to enhance the salt tolerance of the bacteria, and molasses provides a complex carbon source and humic acid substances, which can further adsorb residual toxicity in the wastewater and form a synergy with the wastewater substrate. This not only counteracts the inhibitory effect of high-salt wastewater, but also eliminates the need to rely on expensive reagents, thus controlling costs from the source.

[0007] In some embodiments, the preferred culture medium is pretreated high-salt pharmaceutical wastewater comprising 90% (v / v), and the industrial-grade excipients are formulated as follows: industrial glucose 2.0 g / L, urea 1.2 g / L, industrial phosphate 0.8 g / L, and molasses 1.5 g / L.

[0008] This formulation is based on a precise match between the nutritional needs of the microbial strain and the characteristics of the wastewater substrate. It achieves the optimal state of microbial growth and metabolic activity through nutritional balance. The ratio of glucose to molasses can take into account both rapid carbon source supply and continuous nutrient supplementation. The amount of urea and phosphate is exactly matched to the nitrogen and phosphorus requirements of the microbial strain in a high-salt environment, avoiding abnormal microbial growth caused by nutrient excess or deficiency, and ultimately achieving the best balance between cost and performance.

[0009] In some implementations, the pretreated high-salt pharmaceutical wastewater has a salinity of 25,000-30,000 mg / L and a COD of 15,000-20,000 mg / L.

[0010] This index range is determined based on the salt tolerance limit and nutrient utilization capacity of the selected compound microbial strains. Higher salinity and COD concentrations can further improve the resource utilization of wastewater, but will not exceed the tolerance range of the microbial strains. At the same time, the wastewater is richer in nutrients at this concentration, which can reduce the amount of excipients added, further enhance the cost advantage, and can be adapted to the actual water quality conditions of most high-salt pharmaceutical wastewater, thus improving the universality of the technology.

[0011] In some embodiments, the propagation method includes strain activation, seed culture preparation, and 2m 3 The batch-scale expansion, harvesting, and storage steps involve a salt-tolerant compound strain composed of Halomonas (CGMCC No. 1.1776), Bacillushalophilus (CGMCC No. 1.2308), and Rhodococcus (CGMCC No. 1.15086) in a volume ratio of 40%:35%:25%.

[0012] This bacterial combination is not a random combination of existing strains, but a functional closed loop formed based on the treatment requirements of high-salt pharmaceutical wastewater. Halomonas has extremely strong salt tolerance and can maintain its metabolic stability in a high osmotic pressure environment, providing a suitable growth basis for the entire bacterial community. Halophilic Bacillus can produce salt-tolerant extracellular enzymes, which can efficiently degrade aromatic and other toxic substances in wastewater and reduce the inhibitory effect of wastewater on the bacterial community. Rhodococcus is good at tolerating high COD loads and can enhance the degradation efficiency of heterocyclic compounds and other recalcitrant organic matter. The metabolites of the three can be utilized by each other to form a synergistic effect of "salt tolerance-detoxification-degradation". The specific ratio is designed to achieve the optimal balance of the functions of each bacterial species and avoid functional shortcomings caused by an excessively high or low proportion of a single bacterial species.

[0013] In some embodiments, the activated culture medium contains 5%-10% pretreated high-salt pharmaceutical wastewater and includes 1.0 g / L industrial glucose, 0.5 g / L urea, 0.3 g / L industrial phosphate, and 0.2 g / L yeast extract.

[0014] The core design principles of activation culture medium are gradient adaptation and precise nutrition. The addition of a low proportion of wastewater allows the strains to adapt to the high-salt and complex substrate environment in advance, avoiding stress reactions when subsequently inoculated with a high proportion of wastewater culture medium. The amount of selected excipients is lower than that of expansion culture medium, which not only meets the basic nutritional needs of the strains during the activation stage, but also prevents the strains from aging prematurely due to nutrient overload. The addition of a small amount of yeast powder can provide growth factors such as vitamins, further improving activation efficiency and ensuring that the strains quickly enter the logarithmic growth phase.

[0015] In some embodiments, during the seed culture preparation process, the activated bacterial strain is inoculated into a 1L-5L seed tank at an inoculation rate of 5%-10%, using the aforementioned culture medium, and cultured at a controlled temperature of 25±2℃, dissolved oxygen of 4-6 mg / L, and pH of 7.5±0.2 until the cell density reaches ≥1×10⁻⁶. 8 per mL.

[0016] Seed culture, as the foundation for large-scale propagation, directly affects the final performance of the inoculum. The inoculum size ensures rapid proliferation without excessive nutrient competition. Temperature, dissolved oxygen, and pH parameters are set based on the optimal growth conditions for the composite strain. A stable environment promotes synchronous growth and avoids uneven growth. Once the target cell density is reached, the strain exhibits the strongest metabolic activity and adaptability, laying the foundation for subsequent 2m... 3 This provides a guarantee for the smooth progress of large-scale cultivation.

[0017] In some implementations, 2m 3 During batch-scale expansion, the seed culture is inoculated at an inoculum volume of 8%-12% into a 2m² cell. 3 The fermenter uses the above-mentioned culture medium, with the temperature controlled at 25℃, dissolved oxygen at 4mg / L, initial pH at 7.5, and expansion cycle at 14 days. Microporous aeration discs combined with mechanical stirring are used to ensure uniform dissolved oxygen. The mechanical stirring speed is 100-200rpm, and the microporous aeration pressure is 0.2-0.3MPa.

[0018] The core challenges of large-scale propagation are the low dissolved oxygen transfer efficiency and environmental stability control in high-salt systems. Microporous aeration discs can generate tiny bubbles, increasing the gas-liquid contact area and improving dissolved oxygen utilization. Combined with mechanical stirring at a specific speed, this can effectively eliminate oxygen dead zones in the fermenter, ensuring that all strains receive sufficient oxygen. Temperature, dissolved oxygen, and pH parameters are more precise than in the seed culture preparation stage because environmental fluctuations have a more significant impact on strain growth in large-scale systems. Strict parameter control can prevent cell aging or contamination. The design of specific inoculum quantities and propagation cycles is to balance inoculum yield and activity, ensuring that the strains are in optimal condition at the end of the propagation.

[0019] In some implementations, during the harvesting and storage process, after the expansion culture is completed, the mycelial sludge is collected by standing or centrifugation. The centrifugation parameters are 4000-6000 rpm and 10-20 min. After adding a compound protectant of 3%-7% trehalose and 2%-5% glycerol, it is stored at 4°C.

[0020] The centrifugation parameters can efficiently collect bacterial sludge while avoiding cell membrane rupture caused by excessive centrifugation force or time, thus protecting the integrity of the bacteria. Trehalose can protect the cell membrane structure through vitrification and resist dehydration damage during refrigeration. Glycerol can lower the freezing point of the system and reduce the damage of low temperature to the bacteria. The combined use of the two forms a synergistic protective effect, which can more effectively maintain the activity of the bacteria compared to a single protective agent. Refrigeration at 4°C can reduce the metabolic rate of the bacteria and further extend the storage period of the bacterial agent.

[0021] The present invention has the following advantages over the prior art: This invention breaks through the limitations of existing technologies in wastewater resource utilization by synergistically designing a high-proportion, high-salt pharmaceutical wastewater substrate with specific industrial excipients. Simultaneously, it forms a complete technological closed loop by combining targeted composite microbial strains, suitable activation culture media, and precise large-scale expansion parameters. Compared with existing technologies, this invention not only significantly reduces the production cost of microbial agents, avoiding reliance on traditional expensive reagents and the waste of wastewater resources, but also effectively overcomes the scale-up bottleneck in large-scale expansion, solving the problems of unclear microbial strains, low activation efficiency, and poor storage stability in existing microbial agents. Through functional synergy between microbial strains, adaptation of culture media to microbial strains, and optimization of expansion processes and equipment, this invention achieves high efficiency and stability of microbial agents in the treatment of high-salt, high-COD pharmaceutical wastewater, adapting to various types of high-salt pharmaceutical wastewater treatment scenarios. It provides a more practical and economical solution for the bio-enhanced treatment of high-salt pharmaceutical wastewater and has broad prospects for industrial application. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] Experimental high-salinity pharmaceutical wastewater raw materials and pretreatment methods: Wastewater raw material information:

[0024] Pretreatment methods: 1. Pretreatment of Sample 1 (chemical synthesis wastewater) Step 1: Take a 10m water sample 3 Transfer to the equalization tank, turn on the agitator (model JBJ-500, speed 80rpm), stir for 15 minutes to make the water quality uniform, and test the initial indicators (salinity 28000mg / L, COD 16500mg / L, pH 6.9, SS 125mg / L).

[0025] Step 2: Coagulation and sedimentation treatment: Add PAC (polyaluminum chloride, industrial grade, 30% content) in batches to the equalization tank at a dosage of 50 mg / L. First, stir at 120 rpm for 2 min (rapid mixing), then stir at 60 rpm for 15 min (slow flocculation). After standing for 30 min, add PAM (polyacrylamide, industrial grade, molecular weight 8 million) at a dosage of 5 mg / L. Stir at 40 rpm for 5 min and let stand for 2 h.

[0026] Step 3: Ceramic membrane filtration: Pump the supernatant after coagulation and sedimentation into the ceramic membrane filtration system (membrane material: α-Al2O3, membrane pore size 0.2μm, membrane area 10m²), control the operating pressure at 0.3MPa and the transmembrane flux at 80L / (m²·h), and backwash once every 30min during the filtration process (backwash pressure 0.4MPa, backwash time 30s) to remove fine flocs and some particles that adsorb toxic substances.

[0027] Step 4: Water quality testing after pretreatment: salinity 27800mg / L, COD 16200mg / L, pH 7.0, SS 32mg / L, meeting the requirements for culture medium substrate, ready for use.

[0028] 2. Pretreatment of Sample 2 (Bio-fermentation wastewater) Step 1: Take a 10m water sample 3 Transfer the water to the centrifugal feed tank, turn on the submersible mixer (model QJB-3 / 8-400 / 3-740, speed 740rpm), stir for 10 minutes to homogenize the water quality, and test the initial indicators (salinity 26000mg / L, COD 19200mg / L, pH 7.3, SS 98mg / L).

[0029] Step 2: Centrifugation: A horizontal spiral sedimentation centrifuge (model LW450×1800, drum diameter 450mm, drum length 1800mm) was used, with the rotation speed controlled at 4000rpm and the feed flow rate at 2m³ / min. 3 / h, centrifuge to separate fermentation residue (mycelium, protein clots), and collect the supernatant.

[0030] Step 3: Precision filtration: Pass the centrifuged liquid into a precision filter (the filter material is a polypropylene meltblown filter element with a pore size of 5μm and a filtration area of ​​0.5m²), and control the operating pressure to 0.2MPa to remove residual fine suspended particles.

[0031] Step 4: Water quality testing after pretreatment: salinity 25800mg / L, COD 18800mg / L, pH 7.2, SS 28mg / L, meeting the requirements for culture medium substrate, ready for use.

[0032] 3. Pretreatment of Sample 3 (Antibiotic Wastewater) Step 1: Take a 10m water sample 3 Transfer the mixture to the coagulation reaction tank, turn on the mechanical agitator (model JBT-600, speed 70 rpm), stir for 12 minutes to homogenize the water quality, and test the initial indicators (salinity 23000 mg / L, COD 13800 mg / L, pH 7.1, SS 112 mg / L).

[0033] Step 2: Coagulation and sedimentation treatment: Adjust the pH to 7.0, add PAC (industrial grade, content 30%) at a dosage of 40 mg / L, stir at 100 rpm for 3 min, then add PAM (industrial grade, molecular weight 10 million) at a dosage of 4 mg / L, stir at 50 rpm for 12 min, and let stand for 1.5 h to allow the suspended solids to settle completely.

[0034] Step 3: Plate and frame filtration: Pass the supernatant of coagulation sedimentation into a plate and frame filter press (model XMYZ200 / 1250-UB, filtration area 200m², filter cloth material is polyester needle-punched felt), control the filtration pressure to 0.6MPa and the filtration temperature to 25℃, to further remove suspended solids and some antibiotic residue adsorbed particles.

[0035] Step 4: Water quality testing after pretreatment: salinity 22800mg / L, COD 13500mg / L, pH 7.1, SS 35mg / L, meeting the requirements for culture medium substrate, ready for use.

[0036] 4. Pretreatment of Sample 4 (Mixed Pharmaceutical Wastewater) Step 1: Take a 10m water sample 3 Transfer to the equalization tank, turn on the double impeller agitator (model JBQ-800, speed 90rpm), stir for 20 minutes to homogenize the water quality, and test the initial indicators (salinity 29000mg / L, COD 18500mg / L, pH 6.8, SS 135mg / L).

[0037] Step 2: Enhanced coagulation treatment: Add PAC (industrial grade, 30% content) at a dosage of 60 mg / L, stir at 150 rpm for 2 min, then add PAM (industrial grade, molecular weight 12 million) at a dosage of 6 mg / L, stir at 60 rpm for 18 min, and let stand for 2.5 h to enhance the flocculation effect for complex pollutant types.

[0038] Step 3: Sand filtration + activated carbon filtration: First, pass the coagulated supernatant into a quartz sand filter (the filter media is quartz sand, with a particle size of 0.5-1.2 mm and a filter bed height of 1.2 m), controlling the filtration rate at 8 m / h; then pass it into an activated carbon filter (the filter media is columnar activated carbon, with a particle size of 3-5 mm and an iodine value ≥1000 mg / g), controlling the filtration rate at 5 m / h, to adsorb some toxic organic matter and odors.

[0039] Step 4: Water quality testing after pretreatment: salinity 28700mg / L, COD 18100mg / L, pH 6.9, SS 42mg / L, meeting the requirements for culture medium substrate, ready for use.

[0040] Example 1 Step 1: Preparation of bacterial strains: The salt-tolerant compound bacterial strain consists of Halomonas spp. CGMCC No. 1.1776, Bacillushalophilus CGMCC No. 1.2308, and Rhodococcus sp. CGMCC No. 1.15086 in a volume ratio of 40%:35%:25%. The three strains are inoculated from the slant medium into LB liquid medium and activated at 25°C and 180 rpm for 24 hours.

[0041] Step 2: Experimental Design: The Plackett-Burman design was used to screen for significant influencing factors. Six factors were selected: industrial glucose (A), urea (B), industrial phosphate (C), molasses (D), MgSO4·7H2O (E), and CaCl2 (F). Each factor was set with two levels (low level: A=1.5g / L, B=0.8g / L, C=0.4g / L, D=1.0g / L, E=0.05g / L, F=0.02g / L; high level: A=2.5g / L, B=1.6g / L, C=1.2g / L, D=2.0g / L, E=0.15g / L, F=0.08g / L), for a total of 12 experimental groups, with 3 replicates in each group.

[0042] Step 3: Culture medium preparation: Using the pretreated wastewater from Sample 1 as a base (accounting for 90%), add industrial-grade excipients according to the factor levels designed for each experimental group, adjust the pH to 7.5, dispense into 250mL Erlenmeyer flasks, 100mL per flask, autoclave at 121℃ for 30min, and cool to room temperature for later use.

[0043] Step 4: Inoculation and culture: Inoculate the activated compound bacterial strain into an Erlenmeyer flask at a 5% inoculation rate and culture at 25℃ and 180rpm for 14 days. After the culture is completed, the cell density is detected by plate counting method (PCA medium) as an evaluation indicator.

[0044] Step 5: Screening of significant factors: The experimental data were analyzed using Design-Expert 12.0 software. The significant influencing factors with a contribution rate >15% were industrial glucose (A), urea (B), industrial phosphate (C), and molasses (D), with contribution rates of 28.6%, 22.3%, 18.7%, and 16.2%, respectively. MgSO4·7H2O and CaCl2 were not significant factors.

[0045] Step 6: Response surface methodology optimization: Based on significant factors, a central composite design (CCD) was adopted, with cell density as the response value. Fifteen experimental groups were designed to establish a quadratic regression model. The model R² = 0.9745, indicating a good model fit.

[0046] Step 7: Optimal Formulation Validation: Solve the quadratic regression model to obtain the optimal formulation: pretreated sample 1 wastewater accounted for 90% (v / v), industrial glucose 2.0 g / L, urea 1.2 g / L, industrial phosphate 0.8 g / L, and molasses 1.5 g / L. Three parallel culture media were prepared according to this formulation. After inoculation with the compound bacterial strain, the average cell density was measured to be 34.5 million cells / mL, validating the model's reliability.

[0047] Step 8: Cost accounting: Industrial glucose (market price 3000 yuan / ton), urea (market price 2200 yuan / ton), industrial phosphate (market price 5000 yuan / ton), molasses (market price 1800 yuan / ton), the total cost of auxiliary materials per ton of bacterial solution is calculated to be 12 yuan. Adding the cost of wastewater pretreatment, energy consumption, etc., the comprehensive cost per ton of bacterial agent is 135 yuan.

[0048] Performance verification steps: COD was detected using the potassium dichromate method (GB11914-89), cell density was detected using the plate count method, and activity was tested periodically during refrigeration. Sample 1 of pretreated wastewater was taken, and the salinity was adjusted to 27800 mg / L and COD to 16200 mg / L. The bacterial agent was added (dosage: 0.8 g / L), and the COD removal rate was tested after 36 hours. The bacterial agent was then added to a 5% trehalose + 3% glycerol composite protectant and refrigerated at 4℃. The activity decay rate was tested at 7, 15, and 30 days.

[0049] Performance verification results:

[0050] Example 2 Step 1: Culture medium preparation: According to the optimal formula in Example 1, using the pretreated wastewater of Sample 2 as the base (accounting for 90%), add 2.0 g / L of industrial glucose, 1.2 g / L of urea, 0.8 g / L of industrial phosphate, and 1.5 g / L of molasses, and adjust the pH to 7.5 for later use.

[0051] Step 2: Strain activation: The compound strain was inoculated into an activation medium containing 8% of the pretreated wastewater from Sample 2 (1.0 g / L industrial glucose, 0.5 g / L urea, 0.3 g / L industrial phosphate, and 0.2 g / L yeast extract), and cultured at 25°C and 180 rpm for 36 hours until the logarithmic growth phase. The cell density was then measured to be ≥1×10⁻⁶. 8 per mL.

[0052] Step 3: Single-factor parameter optimization: A 5L fermenter (model BLBIO-5G, tank material 316L stainless steel) was used. The inoculum amount was fixed at 10% and the stirring speed was 150 rpm. The effects of temperature, dissolved oxygen, initial pH and expansion cycle on the performance of the inoculum were investigated in sequence. Five levels were set for each factor and three parallel experiments were conducted for each group.

[0053] -Temperature optimization: The temperature was set to 20℃, 22℃, 25℃, 28℃, and 30℃, while other parameters were kept constant (dissolved oxygen 4mg / L, pH 7.5, cycle 14 days). The results showed that the cell density was the highest (35 million cells / mL) and the COD removal rate was the best (73.5%) at 25℃.

[0054] - Dissolved oxygen optimization: The concentrations were set to 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, and 6 mg / L, while other parameters were kept constant (temperature 25℃, pH 7.5, cycle 14 days). The results showed that the cell metabolic activity was optimal at 4 mg / L, and the COD removal rate reached 73.5%.

[0055] - Initial pH optimization: The pH was set to 6.5, 7.0, 7.5, 8.0, and 8.5, while other parameters were kept constant (temperature 25℃, dissolved oxygen 4mg / L, cycle 14 days). The results showed that the bacterial strain grew most stably and had the smallest coefficient of variation in cell density at pH 7.5.

[0056] - Cycle optimization: 10 days, 12 days, 14 days, 16 days, and 18 days were set, with other parameters fixed (temperature 25℃, dissolved oxygen 4mg / L, pH 7.5). The results showed that the bacteria reached the growth plateau at 14 days, and further extending the cycle did not significantly improve activity.

[0057] Step 4: Optimal parameter combination verification: Three parallel experiments were conducted with the optimal parameters (temperature 25℃, dissolved oxygen 4mg / L, initial pH 7.5, cycle 14 days) to detect cell density, COD removal rate and parameter fluctuation range.

[0058] Performance verification steps: After the expansion culture was completed, the cell density was detected by plate counting method; the pretreated wastewater sample 2 was taken, the salinity was adjusted to 25800 mg / L and COD to 18800 mg / L, and bacterial agent (0.8 g / L) was added. After 36 h, the COD removal rate was detected by potassium dichromate method; the coefficient of variation of performance of the three parallel experiments was calculated.

[0059] Performance verification results:

[0060] Example 3 Step 1: Culture medium preparation: According to the optimal formulation in Example 1, prepare 2m³ of culture medium using the pretreated wastewater from Sample 3. 3 Culture medium, transfer to 2m 3 Fermentation tank (model BLBIO-2000, tank body material 316L stainless steel, stirring type is six straight blade turbine), autoclave at 121℃ for 30 minutes, cool to 25℃ for use.

[0061] Step 2: Strain activation: Follow the same strain activation steps as in Example 2 to obtain a strain in the logarithmic growth phase.

[0062] Step 3: Seed culture preparation: Inoculate the activated bacterial strain at an 8% inoculum into a 5L seed tank, using the optimal culture medium from Example 1, controlling the temperature at 25±2℃, dissolved oxygen at 5mg / L, pH at 7.5±0.2, stirring speed at 200rpm, and incubate for 24h. The cell density should be ≥1×10⁻⁶. 8 Quantity / mL, for later use.

[0063] Step 4: 2m 3 Large-scale cultivation: Inoculate 2m² seed culture solution at a 10% inoculum rate. 3 The fermenter uses a microporous aeration disc (pore size 5-10μm, air distribution uniformity ≥90%) combined with mechanical stirring, controlling the stirring speed at 150rpm and the aeration pressure at 0.25MPa to maintain dissolved oxygen at 4mg / L; the temperature is controlled at 25℃ through a water bath in the fermenter jacket, the initial pH is 7.5, and the expansion cycle is 14 days.

[0064] Step 5: Process monitoring: Samples are taken at 9:00 and 16:00 daily. Cell morphology is examined using an optical microscope (1000x magnification) to observe for contaminating bacteria (such as mold and miscellaneous bacteria) and cell aging phenomena (such as cell membrane rupture and cell aggregation). Cell density and COD removal rate are tested every 48 hours, and aeration pressure and stirring speed are adjusted in a timely manner.

[0065] Step 6: Harvesting the inoculum: After the expansion culture is completed, transfer the fermentation broth to a horizontal spiral sedimentation centrifuge (model LW650×2200), control the speed at 5000 rpm and the centrifugation time at 15 min, and collect the inoculum sludge.

[0066] Step 7: Activity maintenance: Add 5% trehalose + 3% glycerol compound protectant (w / v) to the sludge, mix for 15 minutes using a double helix conical mixer (model SHJ-200) to ensure that the protectant is in uniform contact with the sludge, and then dispense into sterile PE containers and store at 4°C.

[0067] Performance verification steps: Immediately after harvest, cell density was detected using the plate count method; Pretreated wastewater sample 3 was taken, and the salinity was adjusted to 22800 mg / L and COD to 13500 mg / L. Microbial agent (0.7 g / L) was added, and the COD removal rate was detected using the potassium dichromate method after 36 hours; the microbial agent was refrigerated at 4℃, and samples were taken at 30 and 60 days to examine cell morphology under a microscope and detect the activity decay rate; 2m... 3 Cost per ton of microbial agent and batch pass rate.

[0068] Performance verification results:

[0069] Example 4 Step 1: Preparation of inoculum: According to Example 3, 2m3 The expansion process involves preparing bacterial agents using pretreated wastewater from sample 4 as a substrate for later use.

[0070] Step 2: Construction of the simulated biochemical system: Construct an SBR reactor (model SBR-100, made of plexiglass) with an effective volume of 100L, equipped with an aeration device (microporous aeration head), a stirring device (paddle mixer), an online pH monitor, and an online DO monitor.

[0071] Step 3: System Start-up: Add pretreated wastewater from Sample 4 (salinity 28700 mg / L, COD 18100 mg / L) to the reactor, inoculate with the prepared bacterial agent at a dosage of 1.0 g / L, control the system temperature at 25℃, DO 3-5 mg / L, and pH 7.0-7.5, and operate in a cycle of "influent-aeration-sedimentation-drainage" for 12 hours per cycle (1 hour influent, 8 hours aeration, 2 hours sedimentation, and 1 hour drainage).

[0072] Step 4: Continuous operation monitoring: Run continuously for 30 days, recording parameters such as influent COD (18000-22000mg / L), salinity, pH, and DO daily; test effluent COD after daily drainage and calculate the 36-hour COD removal rate; on the 30th day of operation, sample and test the activity of the microbial agent in the sludge in the reactor; periodically test the sludge settling ratio (SVI) to determine the stability of system operation.

[0073] Performance verification steps: COD of influent and effluent was detected by potassium dichromate method, sludge settling ratio (SVI) was determined by standard method, and bacterial activity was verified by degradation experiment.

[0074] Performance verification results:

[0075] Comparative Example 1 Step 1: Wastewater pretreatment: The pretreatment process is completely consistent with that of Sample 1 in Example 1, and pretreated wastewater with the same physicochemical properties is obtained.

[0076] Step 2: Strain preparation: Select the single salt-tolerant strain (Microbacterium chrysogenum CGMCC No. 27319) disclosed in the existing patent CN113604397A, inoculate it from the slant medium to LB liquid medium, and activate it at 25℃ and 180rpm for 24h for later use.

[0077] Step 3: Culture medium preparation: Prepare the culture medium according to the optimal formula in Example 1, dispense it into a 5L fermenter, and autoclave at 121℃ for 30min.

[0078] Step 4: Expansion process: The optimal process parameters are completely consistent with those in Example 2 (temperature 25℃, dissolved oxygen 4mg / L, pH 7.5, cycle 14 days).

[0079] Step 5: Harvesting and storing the microbial agent: The harvesting and storage methods are the same as in Example 3 (centrifugation at 5000 rpm for 15 min, 5% trehalose + 3% glycerol protectant, refrigerated at 4℃).

[0080] Performance verification steps: The performance verification steps are completely consistent with those in Example 1.

[0081] Performance verification results:

[0082] Comparative Example 2 Step 1: Preparation of microbial strain: The microbial strain is completely identical to that in Example 1, and the activation method is the same.

[0083] Step 2: Preparation of conventional culture medium: Prepare conventional LB culture medium with the following formula: 10 g / L peptone, 5 g / L yeast extract, and 20 g / L NaCl. Adjust the pH to 7.5, dispense into 5L fermenters, and autoclave at 121°C for 30 min.

[0084] Step 3: Propagation process: The optimal process parameters are completely consistent with those in Example 2.

[0085] Step 4: Harvesting and storing the microbial agent: The harvesting and storage methods are the same as in Example 3.

[0086] Performance verification steps: The performance verification steps are completely consistent with those in Example 1.

[0087] Performance verification results:

[0088] Comparative Example 3 Step 1: Wastewater pretreatment: The pretreatment process is completely consistent with that of Sample 2 in Example 2.

[0089] Step 2: Preparation of bacterial strains and culture medium: completely consistent with Examples 1 and 2.

[0090] Step 3: Conventional expansion process: Using existing conventional expansion parameters, temperature 28℃, dissolved oxygen 8mg / L, conventional aeration (aeration head orifice diameter 50μm) + stirring speed 300rpm, initial pH 7.0, cycle 14 days, without process microscopic inspection monitoring.

[0091] Step 4: Harvesting and storing the microbial agent: The harvesting and storage methods are the same as in Example 3.

[0092] Performance verification steps: The performance verification steps are completely consistent with those in Example 2.

[0093] Performance verification results:

[0094] Comparative Example 4 Step 1: Wastewater pretreatment, strain preparation, culture medium preparation, and expansion process: completely consistent with Example 3.

[0095] Step 2: Harvesting and storing the inoculum: After the expansion culture is completed, centrifuge at 5000 rpm for 15 minutes to collect the inoculum sludge. Without adding any preservatives, directly dispense it into sterile PE containers and store it at 4℃.

[0096] Performance verification steps: The performance verification steps are completely consistent with those in Example 3.

[0097] Performance verification results:

[0098] Comparative Example 5 Step 1: Wastewater pretreatment: The pretreatment process is completely consistent with that of Sample 1 in Example 1.

[0099] Step 2: Culture medium preparation: The proportion of wastewater substrate is adjusted to 70% (a common proportion in existing technologies), and the proportions of other excipients (industrial glucose 2.0g / L, urea 1.2g / L, industrial phosphate 0.8g / L, molasses 1.5g / L) and pH adjustment methods are the same as in Example 1.

[0100] Step 3: Preparation of strains, propagation process, harvesting and storage of inoculum: completely consistent with Examples 1, 2 and 3.

[0101] Performance verification steps: The performance verification steps are completely consistent with those in Example 1.

[0102] Performance verification results:

[0103] Comparative Example 6 Step 1: Wastewater pretreatment: The pretreatment process is completely consistent with that of Sample 1 in Example 1.

[0104] Step 2: Culture medium preparation: Wastewater accounts for 90% (consistent with Example 1), and the excipients are replaced with conventional ratios of existing technology (industrial glucose 3.0g / L, urea 0.8g / L, industrial phosphate 0.5g / L, molasses-free), and the pH is adjusted to 7.5.

[0105] Step 3: Preparation of strains, propagation process, harvesting and storage of inoculum: completely consistent with Examples 1, 2 and 3.

[0106] Performance verification steps: The performance verification steps are completely consistent with those in Example 1.

[0107] Performance verification results:

[0108] Examples 1-4, targeting chemical synthesis, bio-fermentation, antibiotic, and mixed pharmaceutical wastewater, fully verified the feasibility and universality of the technical solution of this invention through targeted pretreatment processes, optimized culture medium formulations, customized expansion parameters, and ternary compound bacterial strain combinations. The results show that, using 90%-95% pretreated wastewater as a base, combined with specific industrial excipients, and integrating a 2m³ batch-scale expansion process with a compound protective agent, this invention can stably obtain salt-tolerant bacterial agents with a cell density exceeding 30 million cells / mL, a 36-hour COD removal rate ≥70%, and an activity attenuation rate ≤5% after 30 days of refrigeration at 4℃, with a cost of only about 135 yuan per ton of bacterial agent. Compared to the comparative examples using single bacterial strains, conventional culture media, traditional expansion processes, or methods lacking protective agents, this invention exhibits significant advantages in cost control, bacterial activity, storage stability, and reliability for large-scale production. It effectively addresses the industry pain points of high cost, difficulty in scaling up, and poor stability of bacterial agents for high-salt pharmaceutical wastewater in existing technologies, achieving extreme cost reduction and high-performance synergy through "waste-based bacterial cultivation," and providing a practical and feasible industrialization solution for the bio-enhanced treatment of high-salt pharmaceutical wastewater.

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A low-cost, salt-tolerant bacteria culture medium based on high-salt pharmaceutical wastewater, characterized in that: The substrate is pretreated high-salt pharmaceutical wastewater, and the substrate accounts for 90%-95% (v / v). The pretreatment is a method of removing suspended solids by coagulation sedimentation, filtration or centrifugation. The pretreated high-salt pharmaceutical wastewater meets the following requirements: salinity 20000-30000 mg / L, COD 10000-20000 mg / L, pH 6.5-8.

5. Industrial-grade excipients are added to the substrate, including 1.8-2.2 g / L of industrial glucose, 1.0-1.4 g / L of urea, 0.6-1.0 g / L of industrial phosphate, and 1.3-1.7 g / L of molasses.

2. The low-cost, salt-tolerant bacteria culture medium based on high-salt pharmaceutical wastewater according to claim 1, characterized in that: The pretreated high-salt pharmaceutical wastewater accounts for 90% (v / v), and the industrial-grade excipients are formulated as follows: industrial glucose 2.0 g / L, urea 1.2 g / L, industrial phosphate 0.8 g / L, and molasses 1.5 g / L.

3. The low-cost, salt-tolerant bacteria culture medium based on high-salt pharmaceutical wastewater according to claim 1, characterized in that: The pretreated high-salt pharmaceutical wastewater has a salinity of 25,000-30,000 mg / L and a COD of 15,000-20,000 mg / L.

4. A method for expanding the culture medium according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Activation of bacterial strains: The salt-tolerant compound bacterial strains were inoculated into the activation medium and cultured at 25°C and 150-200 rpm for 24-48 hours until the logarithmic growth phase was reached; the salt-tolerant compound bacterial strains were composed of Halomonas assp CGMCC No.1.1776, Bacillushalophilus CGMCC No.1.2308 and Rhodococcus sp CGMCC No.1.15086 in a volume ratio of 40%:35%:25%; the activation medium contained 5%-10% pretreated high-salt pharmaceutical wastewater and included 1.0 g / L of industrial glucose, 0.5 g / L of urea, 0.3 g / L of industrial phosphate and 0.2 g / L of yeast powder; (2) Seed culture preparation: Inoculate the activated strain into a 1L-5L seed tank at an inoculation rate of 5%-10%, using the culture medium described in any one of claims 1-3, controlling the temperature at 25±2℃, dissolved oxygen at 4-6mg / L, and pH at 7.5±0.2, and culture until the cell density is ≥1×10⁻⁶. 8 cells / mL; (3) 2m 3 Batch-scale expansion: Inoculate 2m² seed culture at an inoculum volume of 8%-12%. 3 The fermenter uses the culture medium described in any one of claims 1-3, and controls the temperature at 25°C, dissolved oxygen at 4 mg / L, initial pH at 7.5, and expansion cycle at 14 days. (4) Harvesting and storage: After the expansion culture is completed, the mycelium is collected by standing or centrifugation, and then stored at 4℃ after adding a protectant.

5. The propagation method according to claim 4, characterized in that: In step (3), a microporous aeration disc combined with mechanical stirring is used to ensure uniform dissolved oxygen. The mechanical stirring speed is 100-200 rpm and the microporous aeration pressure is 0.2-0.3 MPa.

6. The propagation method according to claim 4, characterized in that: The centrifugation parameters in step (4) are 4000-6000 rpm and 10-20 min; the protective agent is a composite system of trehalose 3%-7% and glycerol 2%-5% (w / v), and the bacterial activity decay rate is ≤5% after refrigeration at 4℃ for 30 days.

7. The propagation method according to claim 4, characterized in that: In step (3), the cell morphology is examined daily during the expansion culture process to monitor for contaminants and bacterial aging.

8. The propagation method according to any one of claims 4-7, characterized in that: The obtained salt-tolerant compound bacterial agent has a cell density of ≥30 million cells / mL, and under the conditions of high-salt pharmaceutical wastewater with salinity of 20,000-30,000 mg / L and COD of 10,000-20,000 mg / L, the COD removal rate is ≥70% after 36 hours.

9. The application of the high-salt pharmaceutical wastewater-based low-cost salt-tolerant bacteria culture medium according to any one of claims 1-3 in the production of bio-enhanced treatment agents for high-salt, high-COD pharmaceutical wastewater.

10. The application of the salt-tolerant compound bacterial agent prepared by the propagation method according to any one of claims 4-7 in the treatment of high-salt, high-COD pharmaceutical wastewater, wherein the high-salt, high-COD pharmaceutical wastewater is chemically synthesized, biologically fermented, or antibiotic-type pharmaceutical wastewater.

Images