Rhodopyridium and application thereof

Abstract

The present application relates to the field of microbial technology, and particularly relates to a pyridine-loving Rhodococcus and application thereof.The strain is preserved in China General Microbiological Culture Collection Center on January 24, 2022, and the preservation number is CGMCC NO.24287.Experiments show that the strain can still have significant degradation effect on pyridine under high salt, can effectively inhibit harmful substances, can utilize pyridine in a biochemical system as a unique carbon and nitrogen source, completely and harmlessly degrade pyridine, ensure normal operation of the biochemical system, avoid problems such as high cost and secondary pollution caused by physical and chemical treatment, lay a material foundation for the research on biological treatment of wastewater containing pyridine compounds, and have high application value.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Rhodococcus pyridostigmine and its applications. Background Technology

[0002] Wastewater from industries such as oil refining, leather, and pharmaceuticals is complex in composition, containing phenols, cyanides, high concentrations of ammonia nitrogen, and various other recalcitrant organic compounds, such as aromatics, heterocyclic compounds, and polycyclic aromatic hydrocarbons. Pyridine is a typical nitrogen-containing heterocyclic compound in wastewater from these industries; it is highly toxic, teratogenic, carcinogenic, and difficult to biodegrade, making traditional biological treatment methods ineffective and posing potential hazards to the ecological environment and human health.

[0003] Currently, the main technologies for treating pyridine wastewater include physical, chemical, and biological methods. Biological methods offer advantages such as no secondary pollution and low cost, making them an ideal method for pyridine degradation in wastewater. With the deepening research into the degradation process of organic pollutants, biodegradation caused by microorganisms is attracting increasing attention.

[0004] In recent years, scholars at home and abroad have successively screened out some microorganisms that can degrade pyridine. However, the degradation efficiency of pyridine under high salt concentration is not high in 24 hours, and no bacterial agent has been developed to simultaneously degrade pyridine compounds other than pyridine. Therefore, the field still needs to seek a bacterial strain that is highly salt-tolerant, has high degradation efficiency, and good environmental tolerance for degrading pyridine. Summary of the Invention

[0005] In view of this, the present invention provides a strain of Rhodococcus pyridostigmine and its application. This strain is tolerant to high salt and can rapidly degrade pyridine even in the absence of carbon and nitrogen sources and under high salt conditions. The degradation rate of pyridine at an initial concentration of 100-500 mg / L can reach 90%-100%.

[0006] This invention isolated strain LZST2022-01 from activated sludge of a wastewater treatment plant in Binzhou City, Shandong Province. Its morphological characteristics are: rod-shaped or short rod-shaped; it forms yellow colonies on ordinary nutrient agar medium, is non-spore-forming, opaque, with a smooth, moist surface, neat edges, and is easily picked up. The nucleotide sequence of the strain's 16S rDNA is shown in SEQ ID NO: 1. Morphological and ITS rRNA identification confirmed it as *Klebsiella oxytoca*, with accession number CGMCC NO. 24287.

[0007] This invention also provides a microbial inoculant, the raw material of which includes Rhodococcus pyridostigmine with accession number CGMCC NO. 24287. The microbial inoculant can be Rhodococcus pyridostigmine cells, or its culture, lysate, or extract.

[0008] The present invention also provides a method for preparing the aforementioned microbial inoculant, wherein Rhodococcus pyridostigmine with accession number CGMCCNO.24287 is activated to prepare a seed liquid, which is then inoculated into a fermentation medium for fermentation. The fermented cells are collected and subjected to adsorption, centrifugation and drying treatment in sequence.

[0009] The activation of the bacterial strain specifically includes: inoculating the bacterial strain into a test tube containing LB liquid medium and culturing it at 30-37°C and 150-180 rpm for 18-24 hours.

[0010] The preparation of the seed culture includes: inoculating the activated strain into 200 mL of LB liquid medium and culturing at 30–37 °C and 150–180 rpm for 18–24 h.

[0011] The fermentation process involves inoculating a seed culture of Rhodococcus pyridostigmine at a volume percentage of 5%–10% into a fermentation medium for fermentation. The fermentation conditions are as follows: temperature 30℃–37℃, pH 7.0–7.5, fermenter pressure 0.05 MPa, initial rotation speed 200 rpm, initial aeration rate 10–15 L / min, maximum rotation speed 500 rpm, maximum aeration rate 30 L / min (1:2.6), DO ≥ 20 mg / L, and fermentation cycle 10–18 h.

[0012] The fermentation medium comprises water and the following components at the following concentrations: KH2PO4 1.7 g / L, K2HPO4 0.22 g / L, NaH2PO4 0.26 g / L, MgSO4·7H2O 0.23 g / L, CaCl2 0.28 g / L, FeCl3 0.003 g / L, (NH4)2SO4 1.0 g / L, pH 6.8-7.2.

[0013] In this invention, the fermentation broth is adsorbed after fermentation. There are no particular limitations on the specific adsorption method; commonly used adsorption materials and procedures in the art can be employed. In a specific embodiment of this invention, diatomaceous earth at a mass ratio of 4–10% and wheat bran at a mass ratio of 1–2% are preferentially used to adsorb the fermentation broth. The mass percentages of the diatomaceous earth and wheat bran refer to their respective mass percentages relative to the fermentation broth.

[0014] The microbial agent prepared according to the method of the present invention has an effective viable count of 2.0 × 10⁻⁶. 9 ~5×10 10 cfu / g.

[0015] The present invention also provides the application of the aforementioned Rhodococcus pyridostigmine, the aforementioned microbial agent, or the microbial agent prepared by the aforementioned method in the treatment of wastewater, wherein the wastewater is high-salt pyridine wastewater.

[0016] Experiments show that the Rhodococcus pyridostigma LZST2022-01 of this invention can rapidly degrade pyridine under conditions of no carbon source, no nitrogen source and high salt. It has a significant degradation effect on wastewater with salt concentration ≤10000mg / L and initial pyridine concentration ≤600mg / L. Among them, the degradation rate of pyridine with an initial concentration of 100-500mg / L reaches 90%-100%.

[0017] The present invention also provides a wastewater treatment method, wherein the aforementioned Rhodococcus pyridostigmine or the aforementioned microbial agent is added to the wastewater.

[0018] Experiments show that for wastewater with a salt concentration ≤10000 mg / L and an initial pyridine concentration ≤500 mg / L, adding the microbial agent described in this invention at a ratio of 1-5‰ of the wastewater mass can achieve a pyridine degradation rate of 90%-100%. This indicates that the strain of this invention still has a significant degradation effect on pyridine under high salt conditions.

[0019] In a specific embodiment of the present invention, the microbial agent is activated before being added, and the activation specifically includes: inoculating the microbial agent into the nutrient salt at an inoculation amount of 1-5 g / L for a first activation;

[0020] Add activated sludge to dissolve and carry out secondary activation;

[0021] Add 100–500 mg / L of pyridine for third activation;

[0022] The conditions for the first, second, and third activations are: dissolved oxygen 2–4 mg / L; pH 7.0–7.5; temperature 28–37°C; and aeration for 4–6 hours.

[0023] The nutrients comprise water and the following components: KH₂PO₄ 0.05–0.1 g / L, K₂HPO₄ 0.1–0.5 g / L, NaH₂PO₄ 0.1–0.5 g / L, MgSO₄·7H₂O 0.1–0.5 g / L, CaCl₂ 0.1–0.5 g / L, and FeCl₃ 0.001–0.01 g / L.

[0024] The *Rhodococcus pyridostigmine* LZST2022-01 provided by this invention can rapidly degrade pyridine under conditions of no carbon or nitrogen source and high salt concentration. It has a significant degradation effect on wastewater with salt concentration ≤10000 mg / L and initial pyridine concentration ≤600 mg / L. Specifically, the degradation rate of pyridine with an initial concentration of 100-500 mg / L reaches 90%-100%. It can effectively inhibit harmful substances and can utilize pyridine in the biochemical system as the sole carbon and nitrogen source to thoroughly and harmlessly degrade pyridine, ensuring the normal operation of the biochemical system. This avoids the problems of high cost and secondary pollution caused by physicochemical treatment methods. It lays a material foundation for the research on biological treatment of wastewater containing pyridine compounds and has high application value.

[0025] Biological Preservation Instructions

[0026] LZST2022-01, classified and named: Rhodococcus pyridinovorans, was deposited on January 24, 2022, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCCNo. 24287. Attached Figure Description

[0027] Figure 1 Morphological characteristics of strain LZST2022-01;

[0028] Figure 2 The effect of pyridine concentration on the pyridine degradation efficiency of strain LZST2022-01 in Example 2;

[0029] Figure 3 This illustrates the effect of temperature on the growth of strain LZST2022-01 in Example 2.

[0030] Figure 4 The effect of pH on the growth of strain LZST2022-01 is shown in Example 2. Detailed Implementation

[0031] This invention discloses a strain of Rhodococcus pyridostigmine and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0032] The present invention will be further illustrated below with reference to the embodiments:

[0033] Example 1: Isolation and Identification of Strains

[0034] 1. Obtaining the strain

[0035] The *Rhodococcus pyridostigmine* strain was obtained from activated sludge samples taken from a wastewater treatment plant in Binzhou City, Shandong Province, through primary and secondary screening. The specific steps are as follows:

[0036] Pyridine gradient acclimation was performed using activated sludge at concentrations of 200 mg·L⁻¹. -1 500mg·L -1 700mg·L -1 and 800 mg·L -1 The data shows that in the pyridine degradation experimental system, the experimental group at 200 mg·L⁻¹ -1 At the pyridine concentration, strains capable of degrading pyridine were found, with good degradation efficiency and a degradation rate of 93.94%. Further acclimatization culture was conducted, and when the pyridine concentration was increased to 500 mg / L... -1 At various concentrations, turbidity was observed during 72 hours of cultivation, and the data showed a degradation effect with a degradation rate exceeding 80%. Further increasing the pyridine concentration gradient for acclimatization and shortening the treatment time, with pyridine gradient concentrations of 700 mg·L⁻¹, was also observed. -1 and 800 mg·L -1 At 72 hours of incubation, the experimental group was able to completely degrade the material; at the highest acclimatization concentration of 800 mg·L⁻¹, the degradation was complete. -1 When the culture time was shortened to 48 hours, the degradation rate of pyridine in the experimental groups reached 100%.

[0037] The bacterial suspension was spread onto culture dishes containing solid inorganic salt medium of corresponding pyridine concentrations. After 48 hours of incubation, each culture dish was covered with colonies. The colonies were similar in morphology, yellow in color, non-spore-forming, opaque, with smooth and moist surfaces, neat edges, and easy to pick up. (See...) Figure 1 The samples were stored separately and further purified to verify the degradation effect. By measuring the change in pyridine content before and after fermentation, it was determined that the strain could degrade pyridine, and the LZST2002-02 strain with good degradation effect and stable inheritance was isolated and screened.

[0038] The inorganic salt culture medium consists of: 0.09 g KH2PO4, 0.22 g K2HPO4, 0.26 g NaH2PO4, 0.23 g MgSO4·7H2O, 0.28 g CaCl2, and 0.003 g FeCl3, with a pH of 7.0–7.2 and 1000 mL distilled water. The medium is sterilized at 121°C for 20 min.

[0039] Selective culture medium: KH2PO4 0.09g, K2HPO4 0.22g, NaH2PO4 0.26g, MgSO4·7H2O 0.23g, CaCl2 0.28g, FeCl3 0.003g, agar 15g, pyridine concentration 800mg / L, pH 7.0~7.2, distilled water 1000mL, sterilized at 121℃ for 20min.

[0040] 2. Identification of pyridine-degrading bacterial strains

[0041] The 16S rDNA gene of pyridine-degrading bacteria was cloned, sequenced, and then BLAST-aligned in GenBank. Results are as follows: Figure 1 The results showed that the pyridine-degrading strain was identified as Rhodococcus pyridinovorans.

[0042] Example 2 Degradation characteristics of the strain

[0043] 1. Effect of pyridine concentration on the degradation of pyridine by Rhodococcus pyridostigminophilus

[0044] The initial pyridine concentration was selected as 100–800 mg / L, which allows Rhodococcus pyridoxae to degrade pyridine. The salt concentration was ≤10000 mg / L. The bacteria were cultured at 30–37℃ and 150–180 r / min for 24 h to study the effect of pyridine concentration on the degradation of pyridine by Rhodococcus pyridoxae.

[0045] (1) Seed culture preparation: The Rhodococcus pyridostigmine strain was inoculated into 100 mL of LB medium and cultured in a shaker at 32–37 °C and 150–180 r / min for 16–24 h to prepare the seed culture for later use.

[0046] (2) The seed culture obtained in step (1) was inoculated into 100 mL of inorganic salt medium (the formula of which is 0.09 g KH2PO4, 0.22 g K2HPO4, 0.26 g NaH2PO4, 0.23 g MgSO4·7H2O, 0.28 g CaCl2, 0.003 g FeCl3, 1 L distilled water, pH 7.0-7.2) at an inoculation rate of 2-5% (V / V), with pyridine concentrations of 100 mg / L and 200 mg / L respectively. 300 mg / L, 400 mg / L, 500 mg / L, 600 mg / L, 700 mg / L, and 800 mg / L were cultured in a shaker at 30–37℃ and 150–180 r / min for 24 h. 10 mL of the cultured bacterial solution under different pyridine concentrations was taken and centrifuged at 12000 r / min for 10 min. 1 mL of the supernatant was taken from each solution. The determination of pyridine was performed according to GB / T14672-1993, "Determination of Pyridine in Water - Gas Chromatography".

[0047] (3) When the initial concentration of pyridine is 100–500 mg / L and the salt concentration is ≤10000 mg / L, the *Rhodococcus pyridostigma* strain can completely degrade pyridine within 24 hours. When the initial concentration of pyridine is 600 mg / L and the salt concentration is ≤10000 mg / L, the degradation rate is about 70% within 24 hours. As the concentration of pyridine increases, the degradation rate gradually decreases. Figure 2 As shown.

[0048] 2. Effect of temperature on the degradation of pyridine by Rhodococcus pyridostigminophilus

[0049] (1) The maximum concentration of pyridine that Rhodococcus pyridosteophyte can completely degrade is 500 mg / L as the initial pyridine concentration. Under the condition of pH 7.0 in the culture medium, the effect of temperature on the degradation of pyridine by Rhodococcus pyridosteophyte is studied.

[0050] (2) In order to explore the optimal temperature for the growth of the target strain in inorganic salt medium, the growth of the strain was tested at 25, 30, 32, 35, 37, 40, 42 and 45℃ respectively. The target strain was fermented for 24h in inorganic salt liquid medium (containing 500mg·L-1 pyridine) with an inoculum of 5% at a pH of about 7.0. The growth of the target strain was monitored.

[0051] (3) The results show that the cell concentration of the target strain increases with increasing temperature, and the cell concentration is highest at 40℃. Therefore, the optimal growth temperature for the target strain is 40℃. The results are as follows: Figure 3 show.

[0052] 3. Effect of pH on the degradation of pyridine by Rhodococcus pyridostigminophilus

[0053] The initial pyridine concentration was set at 500 mg / L, the maximum concentration at which Rhodococcus pyridoxae can completely degrade pyridine. The effect of pH on the degradation of pyridine by Rhodococcus pyridoxae was studied under culture conditions of 30℃ to 37℃.

[0054] (1) Seed culture preparation: The Rhodococcus pyridostigmine strain was inoculated into 100 mL of LB medium and cultured at 30–37 °C and 150–180 r / min for 16–24 h to prepare the seed culture for later use.

[0055] (2) The seed culture obtained in step (1) was inoculated at a rate of 2-5% (V / V) into 100 mL of inorganic salt culture medium with a pyridine concentration of 500 mg / L (the formula of which is 0.09 g KH2PO4, 0.22 g K2HPO4, 0.26 g NaH2PO4, 0.23 g MgSO4·7H2O, 0.28 g CaCl2, 0.003 g FeCl3, and 1 L distilled water). The culture was placed in a shaker at 150-180 r / min at 25, 30, 35, 37, and 40 °C for 24 h. 10 mL of the cultured bacterial solution under different temperature conditions was centrifuged at 12000 r / min for 10 min, and 1 mL of the supernatant was taken. The determination of pyridine was performed in accordance with GB / T14672-1993 Determination of Pyridine in Water by Gas Chromatography.

[0056] (3) Under the conditions of an initial pyridine concentration of 500 mg / L and a culture temperature of 37℃, the *Rhodococcus pyridostigmine* strain can effectively degrade pyridine within a pH range of 6.5–8.0, and can completely degrade pyridine at pH 7.5. When the pH is greater than or less than pH 7.5, the degradation rate of pyridine by this strain decreases to varying degrees, as shown in the results below. Figure 4 show.

[0057] Example 3: Preparation of a highly efficient pyridine-degrading bacterial agent

[0058] (1) Activation of strain: Take 1 mL of the frozen pyridine-degrading bacteria obtained in Example 1 and inoculate it into an Erlenmeyer flask containing 100 mL of LB liquid sterile medium (the formula of which is 10 g / L tryptone, 5 g / L yeast extract, 20 g / L NaCl, pH 7.0-7.2), and place it in an incubator at 37°C and 170 rpm for shaking culture until the logarithmic phase.

[0059] (2) Liquid seed preparation: 100 mL of activated strain was transferred to 1000 mL of LB liquid sterile medium (the formula of which is 10 g / L tryptone, 5 g / L yeast extract, 20 g / L NaCl, pH 7.0-7.2) and cultured in an incubator at 37°C and 170 rpm with shaking until the logarithmic phase.

[0060] (3) Seed tank fermentation: The prepared liquid seeds were inoculated into a 50L seed tank with a liquid volume of 70% at an inoculation rate of 5% (V / V) for expansion culture.

[0061] (4) Fermentation in production tank: Inoculate the seed liquid into the culture medium of the production tank with a liquid volume of 70% at an inoculation rate of 8% (V / V). After fermentation, the number of bacteria reaches more than 10 billion / mL. After fermentation, the culture medium is discharged from the tank to obtain liquid bacterial agent.

[0062] (5) Preparation of solid inoculum: After fermentation, the liquid inoculum was concentrated at a ratio of 3:1 (V / V), and 8% diatomaceous earth and 2% corn cob were added by mass to adsorb the bacteria. Then, the mixture was centrifuged and dried to obtain solid inoculum. The moisture content of the microbial inoculum obtained by the above method was controlled to be below 10%.

[0063] The culture conditions for the seed tank fermentation in step (3) and the production tank fermentation in step (4) of the above process are as follows: the air-to-air ratio of sterile air is 1:1.2, the stirring speed is 200 rpm, the culture temperature is 37℃, and the fermentation cycle is 20h.

[0064] In the above steps, the fermentation medium formula used in the seed tank and production tank is as follows: glucose 1.0%, corn starch 1.0%, soybean meal 2.0%, yeast powder 0.3%, KH2PO4 1.7g / L, K2HPO4 0.22g / L, NaH2PO4 0.26g / L, MgSO4·7H2O 0.23g / L, CaCl2 0.28g / L, FeCl3 0.003g / L, (NH4)2SO4 1.0g / L, initial pyridine concentration 500mg / L, water balance, pH 6.8-7.2.

[0065] Example 4: Application of highly efficient pyridine-degrading bacteria in the treatment of high-salt pyridine wastewater from sewage treatment plants

[0066] (1) Activation of microbial agents

[0067] The microbial agent obtained in Example 3 was activated by inoculating it into nutrient salt at a weight-to-volume ratio of 3 g / L. The activation conditions were: dissolved oxygen 2-4 mg / L.

[0068] Activation was performed at pH 7.0–7.2 and temperature 28–37℃ with aeration for 4–6 hours. The nutrient composition was as follows: KH₂PO₄ 0.09 g / L, K₂HPO₄ 0.22 g / L, NaH₂PO₄ 0.26 g / L, MgSO₄·7H₂O 0.23 g / L, CaCl₂ 0.28 g / L, FeCl₃ 0.003 g / L. Activated sludge was used for dissolution and activation. In the above activation system, 200 mg / L of pyridine was added as the sole carbon and nitrogen source for activation treatment.

[0069] (2) Application of highly efficient pyridine-degrading bacteria in high-salt pyridine wastewater

[0070] The bacterial agent obtained according to the above steps was applied on-site at a wastewater treatment plant of an environmental engineering company in Binzhou City. The bacterial agent was added to wastewater containing pyridine at a dosage of 2‰. The initial pyridine concentration in the wastewater was 400 mg / L, and the salt concentration was 9500 mg / L. After aeration at room temperature for 24 hours, the residual amount of pyridine was determined by gas chromatography according to GB / T14672-1993, the determination of pyridine in water quality. According to the formula pyridine degradation rate = (pyridine content in initial wastewater - pyridine content in final wastewater) / pyridine content in initial wastewater × 100%, the pyridine degradation rate was found to be above 95%, indicating that the pyridine-degrading bacterial agent can efficiently degrade pyridine in wastewater and meet the application requirements under on-site conditions.

[0071] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Rhodococcus pyridinovorans with accession number CGMCC NO. 24287; the Rhodococcus pyridinovorans, under conditions of salt concentration ≤10000 mg / L, using pyridine as the sole carbon and nitrogen source, achieves a degradation rate of 90%~100% of pyridine at an initial concentration of 100~500 mg / L within 24 h.

2. The *Rhodococcus pyridostigmine* according to claim 1, characterized in that, Its nucleotide sequence of 16S rDNA is shown in SEQ ID NO:

1.

3. A microbial inoculant, characterized in that, Its raw materials include Rhodococcus pyridostigmine with accession number CGMCC NO. 24287.

4. The method for preparing the microbial inoculant according to claim 3, characterized in that, The Rhodococcus pyridostigmine with accession number CGMCC NO.24287 was activated to prepare a seed culture, which was then inoculated into a fermentation medium for fermentation. The fermented cells were collected and subjected to adsorption, centrifugation and drying treatment in sequence.

5. The preparation method according to claim 4, characterized in that, The activation of the strain specifically includes: inoculating the strain into a test tube containing LB liquid medium and culturing it at 30~37℃ and 150~180rpm for 18h~24h; The preparation of the seed culture includes: inoculating the activated strain into 200 mL of LB liquid medium and culturing at 30-37°C and 150-180 rpm for 18-24 hours.

6. The preparation method according to claim 4, characterized in that, The fermentation process involves inoculating a seed culture of Rhodococcus pyridostigmine at a volume percentage of 5% to 10% into a fermentation medium. The fermentation conditions are as follows: temperature 30℃ to 37℃, pH 7.0 to 7.5, fermenter pressure 0.05 MPa, initial rotation speed 200 rpm, initial aeration rate 10 to 15 L / min, maximum rotation speed 500 rpm, maximum aeration rate 30 L / min (1:2.6), DO ≥ 20 mg / L, and fermentation cycle 10 to 18 hours.

7. The preparation method according to claim 4, characterized in that, The fermentation medium comprises water and the following components at the following concentrations: KH2PO4 1.7 g / L, K2HPO4 0.22 g / L, NaH2PO4 0.26 g / L, MgSO4·7H2O 0.23 g / L, CaCl2 0.28 g / L, FeCl3 0.003 g / L, (NH4)2SO4 1.0 g / L, pH 6.8-7.

2.

8. The application of the Rhodococcus pyridostigmine of claim 1 or 2, the microbial agent of claim 3, or the microbial agent prepared by any one of claims 4 to 7 in the treatment of wastewater; wherein the wastewater is high-salt pyridine wastewater.

9. A method for treating wastewater, characterized in that, Add the Rhodococcus pyridostigmine of claim 1 or 2, the microbial agent of claim 3, or the microbial agent prepared by any one of claims 4 to 7 to the wastewater.

10. The processing method according to claim 9, characterized in that, The microbial agent is activated before being added, and the activation specifically includes: inoculating the microbial agent into the nutrient salt at an inoculation amount of 1~5g / L for the first activation; Add activated sludge to dissolve and carry out secondary activation; Add 100~500 mg / L of pyridine for third activation; The conditions for the first, second, and third activations are: dissolved oxygen 2~4 mg / L; pH 7.0~7.5; temperature 28~37℃; and aeration for 4~6 hours. The nutrients comprise water and the following components: KH₂PO₄ 0.05~0.1 g / L, K₂HPO₄ 0.1~0.5 g / L, NaH₂PO₄ 0.1~0.5 g / L, MgSO₄·7H₂O 0.1~0.5 g / L, CaCl₂ 0.1~0.5 g / L, and FeCl₃ 0.001~0.01 g / L.

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