A strain of Bacillus zanthoxylum ZM2-4 and its bacterial agent and its application in degradation of oily wastewater

Abstract

The present invention relates to a strain of Bacillus zanthoxylum ZM2‑4 and its bacterial agent and application in degrading oily wastewater, belonging to the technical field of microorganisms. Bacillus zanthoxylum ZM2‑4 is deposited in the General Microbiological Center of China Microbiological Culture Collection Committee, with a deposit number of CGMCC No.32386, a deposit date of October 28, 2024, and a deposit address of the depository institution: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The present invention also provides an application of Bacillus zanthoxylum ZM2‑4 in degrading oily wastewater. The diesel degradation rate of Bacillus zanthoxylum ZM2‑4 of the present invention can reach more than 70%. After being made into a solid preparation, it has the advantages of easy preservation and slow decay. It solves the problem that there is currently no Bacillus zanthoxylum for treating oily petrochemical wastewater on the market.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Bacillus zanthoxylum ZM2-4 and a bacterial agent thereof and application of the same in degrading oily wastewater. Background Art

[0002] Oil is a mixture of different hydrocarbons. With the development of social economy, the use of oil is becoming more and more widespread, and the environmental pollution caused by its oil is becoming more and more serious. The current treatment methods include mechanical method, physical method (gravity method, membrane separation method, centrifugal method, flotation method, adsorption method), chemical method (chemical coagulation method, chemical oxidation method, electrochemical method) and biological method. The biological method has become a research hotspot because of its superiority in reducing the pollution of oil to the environment. Biological method refers to a controlled or spontaneous process that uses organisms to catalyze the degradation of organic pollutants into other pollutants to eliminate pollution. The biological method has the advantages of low production cost, no secondary pollution, and can be repaired in situ. It is a new technology with development prospects. It is known that there are many microorganisms in nature that can use petroleum or its chemical products as carbon sources and energy sources, and they can degrade petroleum into minerals. Vecchio Hi GI reported that the ability to degrade petroleum was enhanced by adding indigenous microorganisms. Sorkhon NA et al. separated Bacillus sp, Enterbacleriaceae, Pseudomonas sp, Nocardia sp, Sireptomyces sp and Rhodococcus sp from the oil-contaminated soil and beaches in Kuwait, among which Rhodococcus sp had the highest content and the strongest oil removal ability. According to statistics, more than 200 microorganisms that can degrade petroleum have been found so far, including bacteria such as Pseudomonas sp, Corynebacterium sp, Micrococcis sp, Alcaligenes sp and other non-spore bacteria. Most of these research works are based on the separation and identification of microbial species contained in oil-contaminated soil, and lack further research on the ability of the selected microorganisms to degrade petroleum and practical applications.

[0003] However, there is no related report on the use of Bacillus zanthoxylum for treating oil-containing petrochemical wastewater in the prior art. Summary of the invention

[0004] In view of the problem that Bacillus zanthoxylum has not been used in the existing biological treatment of oily wastewater, the present invention provides a strain of Bacillus zanthoxylum ZM2-4 and its bacterial agent and application in degrading oily wastewater to solve the above problem.

[0005] In a first aspect, the present invention provides a strain of Bacillus zanthoxylum ZM2-4, wherein the Bacillus zanthoxylum ( Bacillus Zanthoxyli ) ZM2-4 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.32386, the deposit date is October 28, 2024, and the address of the deposit institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] In a second aspect, the present invention provides a solid bacterial agent containing Bacillus zanthoxylum ZM2-4.

[0007] A method for preparing the above solid bacterial agent comprises the following steps:

[0008] 6% calcium carbonate and 2% corn starch carrier, based on the weight of the fermentation liquid, were added to the fermentation liquid containing Bacillus zanthoxylum ZM2-4, and the feed pressure of the pressure spray drying tower was controlled to be 25MPa, the inlet air temperature to be 175°C, the outlet air temperature to be 75°C-80°C, the negative pressure to be 50Pa, and the moisture content to be controlled within 5%, and the dry process was performed to obtain a solid bacterial agent containing Bacillus zanthoxylum ZM2-4.

[0009] Furthermore, the number of spores in the solid bacterial agent is ≥ 2.0×10 10 CFU / g.

[0010] Furthermore, the spore rate in the fermentation broth is ≥90%, the maturity is ≥70%; when pH=8.0, the spore count is ≥2×10 9 CFU / ml.

[0011] Furthermore, the preparation method of the fermentation broth is as follows:

[0012] 5L of LB culture solution of Bacillus zanthoxylum ZM2-4 was inoculated into a fermentation medium, and a fully automatic fermentation equipment was used to control the fermentation temperature to 37±1°C, the rotation speed to 200rpm, the ventilation ratio to 1:1, the dissolved oxygen to 20%, and the air volume to 1.5:1 until the fermentation was completed; the fermentation medium was: 10ml / L diesel, 1g / L ammonium sulfate, 1g / L dipotassium hydrogen phosphate, 1g / L magnesium sulfate, 0.5g / L sodium chloride, 20g / L peptone, 5g / L sucrose, and natural pH.

[0013] In a third aspect, the present invention provides an application of Bacillus zanthoxylum ZM2-4 in degrading oily wastewater.

[0014] The beneficial effects of the present invention are:

[0015] The Zanthoxylum ZM2-4 provided by the present invention has a strong ability to degrade oil in sewage, wherein the bacterial powder is added to sewage containing diesel at a ratio of 0.05‰ to 0.1‰, and the diesel degradation rate can reach more than 70%. After being made into a solid preparation, it decays by 8.33% when stored at a constant temperature of 37°C for 14 months, and decays by 2.78% when stored at room temperature, and has the advantages of easy storage and slow decay. The problem that there is currently no Zanthoxylum Zanthoxylum for treating oil-containing petrochemical sewage on the market is solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 This is a growth diagram of Bacillus zanthoxylum ZM2-4 in Example 1 of the present invention in LB medium.

[0018] Figure 2 This is the Gram staining image of Bacillus zanthoxylum ZM2-4 in Example 1 of the present invention.

[0019] Figure 3 This is a trend chart of the attenuation verification experiment of the solid bacterial agent containing Bacillus zanthoxylum ZM2-4 in Example 3 of the present invention.

[0020] Figure 4 It is a curve diagram of the diesel standard solution of Example 4 of the present invention.

[0021] Figure 5 This is a diagram showing the oil removal effect of different inoculation amounts of Bacillus zanthoxylum ZM2-4 on simulated sewage in Example 4 of the present invention.

[0022] Figure 6 It is a trend chart of the oily wastewater treatment experiment in the workshop of Example 5 of the present invention. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0024] Example 1

[0025] Isolation, Screening and Identification of Bacillus zanthoxylum ZM2-4

[0026] 1. Sampling

[0027] On May 16, 2023, oily wastewater from the biochemical pool was retrieved at Sinopec Shanghai Gaoqiao Petrochemical Co., Ltd.

[0028] 2. Separation

[0029] Prepare oil culture medium: 10g / L diesel, 2.0g / L ammonium sulfate, 2.0g / L potassium dihydrogen phosphate, 4ml trace element solution, adjust pH to 7.0, the trace element solution is as follows: 4.0g magnesium sulfate heptahydrate, 1g copper sulfate pentahydrate, 0.5g manganese sulfate monohydrate, 1g anhydrous calcium chloride, dilute to 1000ml for use. Take another 10ml of oily wastewater and add it to a 90ml sterile water triangle bottle with glass beads, shake at 37℃, 180rpm for 30min, and dilute the dispersed bacterial suspension to 10 -5 , 10 -6 , 10 -7 and 10 -8 , 0.1 ml was taken from the above gradient and added to the petroleum culture medium respectively, each gradient was repeated 3 times, evenly spread, and cultured at 37°C for 24 h. A single colony was selected from the gradient with colonies grown, and inoculated into a sterilized LB solid culture medium (10 g tryptone, 5 g yeast extract, 10 g NaCl, 15-20 g agar, distilled water to 1000 ml, pH = about 7.0, sterilized at 121°C for 20 min) for single bacteria purification, and then the purified single bacteria were returned to the petroleum culture medium for acclimation 3 times to obtain 4 purified petroleum-degrading strains (numbered ZM2-1, ZM2-2, ZM2-3, and ZM2-4 in sequence). The target bacteria were cultured with sterile LB liquid culture medium (10 g tryptone, 5 g yeast extract, NaCl 10g, distilled water to 1000ml, pH = about 7.0, sterilized at 121℃ for 20min) for expansion, and then stored in 50% glycerol tubes at -80℃.

[0030] 3. Enzyme production test verification

[0031] (1) Activation culture medium: 0.2 g / L yeast extract, 1 g / L sodium chloride, 3.5 g / L disodium hydrogen phosphate, 1.5 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 10 ml diesel, distilled water to 1000 ml, divided into 250 ml Erlenmeyer flasks, each containing 50 ml of liquid, sterilized at 121°C for 20 min and set aside.

[0032] (2) Rhodamine B medium: 0.5 g ammonium sulfate, 4 g sodium chloride, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 15 g agar powder, 100 ml tributyrin emulsion (4% PVA: tributyrin = 3:1, ultrasonic emulsification), dilute to 1000 ml with distilled water, sterilize at 121°C for 20 min, add 1 ml rhodamine B solution (mass concentration <10 mg / ml) when the temperature drops to 60°C, mix well, and pour into a plate for later use.

[0033] (3) Enzyme production medium: 5.0 g glucose, 2.0 g soy peptone, 6.0 g urea, 1.0 g potassium phosphate dihydrate, 1.0 g ammonium sulfate, 0.5 g magnesium sulfate heptahydrate, 10 ml tributyrin, dilute to 1000 ml with distilled water, pH natural, divide into 1000 ml baffled flasks, fill with 250 ml, sterilize at 115°C for 15 min and set aside.

[0034] (4) Enzyme production verification steps: The strains preserved in glycerol tubes were first inoculated into activation medium and cultured at 37°C for 24 h. Then, they were inoculated into enzyme production medium at a 10% inoculation rate and cultured for 28-30 h. 50 ul of the supernatant of the enzyme production medium was spread on a rhodamine B medium plate. Rhodamine B can specifically bind to the fatty acids produced by lipase degradation of oils and fats, and a rose-red color will be generated around the enzyme-producing colonies. The larger the color circle, the higher the enzyme production. Observation of the structure revealed that only strain ZM2-4 produced lipase.

[0035] 4. Identification

[0036] The colonies of strain ZM2-4 on LB plate medium are milky white, slightly raised and opaque, with rough edges and easy to pick up. Figure 1 As shown. Gram staining is positive, such as Figure 2 The cultured LB plate culture medium was sent to the Sanger sequencing laboratory of Beijing Nosai Genome Research Center Co., Ltd. for identification. During the identification process, the gene sequence of strain ZM2-4 was obtained as shown in SEQ ID NO.1. The identification result was Bacillus zanthoxylum Bacillus Zanthoxyli Bacillus zanthoxylum ZM2-4 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.32386 and the deposit date October 28, 2024. The address of the deposit institution is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0037] Example 2

[0038] Preparation of solid bacterial agent containing Bacillus zanthoxylum ZM2-4

[0039] 1. Fermentation medium: 10L diesel, 1kg ammonium sulfate, 1kg potassium dihydrogen phosphate, 1kg magnesium sulfate, 0.5kg sodium chloride, 20kg peptone, 5kg sucrose, natural pH, add water to make up to 850L and homogenize (equipment manufacturer: Ningbo Xinzhi Biotechnology Co., Ltd., model: Xinzhi SCIENTZ-207A ultra-high pressure homogenizer), sterilize at 121℃ for 20min, and then add water to make up to 1000L.

[0040] 2. Take 5L of LB culture solution of Bacillus zanthoxylum ZM2-4 and inoculate it into the fermentation medium. Use fully automatic fermentation equipment (manufacturer: Zhenjiang Jiangda Technology Co., Ltd.) with a temperature of 37±1℃, a rotation speed of 200rpm, a ventilation ratio of 1:1, and reduce the dissolved oxygen to 20%, and increase the air volume to 1.5:1 until the fermentation is completed.

[0041] 3. Criteria for judging the end of fermentation: spore rate ≥ 90%, maturity ≥ 70%, OD no longer increases, pH rises to 8.0, spore count ≥ 2×10 9 CFU / ml, fermentation period 44~48h.

[0042] 4. Preparation of solid preparations: According to the weight of the fermentation liquid, 6wt% calcium carbonate and 2wt% corn starch carrier were added to fix the bacteria and lipase. The feed pressure of the pressure spray drying tower (equipment manufacturer: Zhejiang Erle Drying Equipment Co., Ltd., model: YPG200) was controlled to be 25MPa, the inlet temperature was 175℃, the outlet temperature was 75℃~80℃, the negative pressure was 50Pa, and the moisture was controlled within 5%. Drying was performed, and 91kg of bacterial powder was collected and mixed with a mixer (equipment manufacturer: Jiangsu Fast Machinery Co., Ltd., model: DZ100) to obtain a solid bacterial agent containing Bacillus zanthoxylum ZM2-4. After the sample was water bathed at 80℃ for 20min, the spore count was detected to be 2.16×10 10 CFU / g (tested in accordance with GB20287-2006).

[0043] Example 3

[0044] Attenuation verification experiment

[0045] The solid bacterial agent containing Bacillus zanthoxylum ZM2-4 prepared in Example 2 was placed in a transparent ziplock bag in a 37°C constant temperature incubator for accelerated storage experiment. The control group was placed at room temperature. The verification period was 14 months (August 13, 2023 to October 12, 2024). The number of spores was detected on the 10th of each month. The decay rate was as follows: Figure 3 The spore count decayed by 8.33% at 37°C for 14 months and by 2.78% at room temperature. It was more stable when stored at room temperature, meeting the on-site storage conditions and the acceptable range.

[0046] Example 4

[0047] Artificial wastewater simulation experiment

[0048] 1. Simulated culture medium: 0.8g ammonium sulfate, 0.8g dipotassium hydrogen phosphate, 0.25g magnesium sulfate, 0.03g calcium chloride, 0.2g potassium dihydrogen phosphate, 0.09g ferrous sulfate heptahydrate, 2g emulsified diesel, dilute to 1000ml with distilled water, initial pH=7.2.

[0049] 2. Detection Method

[0050] 2.1. Plotting the diesel concentration and absorbance curve

[0051] (1) Prepare a series of diesel standard solutions with concentrations of 0, 0.005, 0.01, 0.03, 0.05, 0.07 and 0.1 (unit: ml diesel / 25 ml petroleum ether) in a 25 ml volumetric flask;

[0052] (2) Take a solution with a concentration of 0.07 ml diesel / 25 ml petroleum ether, fill it to three-quarters of the height of the cuvette, put it into the sample cell of the spectrophotometer, and scan it from 200 nm to 500 nm. The maximum absorbance value is measured at a wavelength of 220 nm, so the maximum absorption wavelength k = 220 nm.

[0053] (3) Drawing of working curve: At the wavelength of maximum absorption, using pure petroleum ether as reference, measure the absorbance values ​​of the diesel standard solutions of the above series of concentrations respectively, and draw the standard curve of the diesel standard solution based on the obtained data, such as Figure 4 shown.

[0054] (4) Experimental steps: Add bacterial powder at a ratio of 0.05‰~0.1‰ (the bacterial amount in the culture medium increases by 1.08×10 6 ~2.16×10 6 CFU / ml), compared with the blank without bacteria, aeration treatment was carried out at 37℃, and the diesel degradation rate was calculated every 12h.

[0055] (5) Calculation: Pour the liquid in the conical flask into a separatory funnel, then add 20 ml of petroleum ether to the original conical flask. After shaking and washing, transfer the solution in the conical flask to the separatory funnel and mix it thoroughly. Pay attention to degassing. After standing for 5 minutes, release the lower layer of liquid to the original conical flask. Use a funnel plugged with a small amount of absorbent cotton to filter the upper petroleum ether extract. Place the filtrate in a 50 ml volumetric flask, then add 20 ml of petroleum ether to the original conical flask for extraction again. Finally, dilute the upper organic phase obtained from the two extractions to 50 ml with petroleum ether. Take 0.5 ml of the solution and dilute it to 25 ml with petroleum ether. Then measure the absorbance at OD = 220 nm.

[0056] Diesel degradation rate 100%;

[0057] Experimental data such as Figure 5 As shown in the figure, group A is the control group without bacteria; group B is the group with 0.05‰ bacterial powder; group C is the group with 0.10‰ bacterial powder.

[0058] from Figure 5 It can be seen that the diesel degradation rate of the experimental group with solid bacterial agent containing ZM2-4 was significantly higher than that of the control group. The agent began to work immediately after it was added, and lipase should play a role first. With the proliferation of ZM2-4, there was a rapid degradation peak period from 24h to 60h, indicating that ZM2-4 and lipase were working at the same time. After 72h, the degradation tended to be stable and fixed at about 73%. According to the changes in the degradation curve, it is recommended to add bacterial agent every 72h to ensure the continuity of degradation. The diesel content of the simulated sewage is 0.2%, about 2000ppm, which is 40 times higher than that of the actual petrochemical biochemical pool sewage. Therefore, it can be inferred that ZM2-4 can survive in oily wastewater with lower concentrations.

[0059] Example 5

[0060] On-site oily wastewater experiment

[0061] This experiment was carried out in the first workshop of the refinery water service of Sinopec Qilu Branch on September 19, 2024. The workshop discharges 280~300m3 of oily wastewater per hour. 3 Up to 60000m 3 The sewage treatment process is to filter the oily wastewater through a screen machine, then flotate to remove the scum, and then enter the primary and secondary aeration tanks after homogenization. The oil content in this section is 40~45mg / L. According to the plan, the solid bacterial agent containing Bacillus Zanthoxylum ZM2-4 is added to the biochemical pool for the first time at 0.3‰ of the total mass of the sewage, and 0.1‰ is added every 48h. The monitoring is continued for 96h. The data of the online monitoring system are shown in Table 1 and Figure 6 As shown in Table 1 and Figure 6 From the data trend, it can be seen that in the case of low oil concentration, the degradation is more thorough, and the residual oil value after 72 hours is less than 1.8mg / L, which is far lower than the industry standard of ≤10mg / L. The inflection point of the on-site degradation curve is almost the same as that of the simulated sewage experimental curve. The degradation peak will appear in 24-60 hours, that is, the degradation will be accelerated after 24 hours of addition. If the peak is to continue to appear, adding every 48 hours is more in line with the company's requirements.

[0062] Table 1- Monitoring results of oily wastewater

[0063]

[0064] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.

Claims

1. A strain of Bacillus zanthoxylum ZM2-4, characterized in that: The Bacillus zanthoxylum ( Bacillus Zanthoxyli ) ZM2-4 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.32386, the deposit date is October 28, 2024, and the address of the deposit institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The Bacillus zanthoxylum ZM2-4 according to claim 1, characterized in that The 16SrDNA sequence of Bacillus zanthoxylum ZM2-4 is shown in SEQ ID NO.

1.

3. A bacterial agent containing Bacillus zanthoxylum ZM2-4 as claimed in claim 1, characterized in that: The bacterial agent is a solid bacterial agent.

4. The bacterial agent according to claim 3, characterized in that The preparation method of the solid bacterial agent comprises the following steps: 6% calcium carbonate and 2% corn starch carrier, based on the weight of the fermentation liquid, were added to the fermentation liquid containing Bacillus zanthoxylum ZM2-4, and the feed pressure of the pressure spray drying tower was controlled to be 25MPa, the inlet air temperature to be 175°C, the outlet air temperature to be 75°C-80°C, the negative pressure to be 50Pa, and the moisture content to be controlled within 5%, and the dry process was performed to obtain a solid bacterial agent containing Bacillus zanthoxylum ZM2-4.

5. The bacterial agent according to claim 4, characterized in that The number of spores in the solid bacterial agent is ≥2.0×10 10 CFU / g.

6. The bacterial agent according to claim 4, characterized in that The spore rate in the fermentation liquid is ≥90%, the maturity is ≥70%; when pH=8.0, the spore number is ≥2×10 9 CFU / ml.

7. The bacterial agent according to claim 6, characterized in that The preparation method of the fermentation liquid is as follows: 5L of LB culture liquid of Bacillus zanthoxylum ZM2-4 is inoculated into a fermentation medium, and a fully automatic fermentation device is used to control the fermentation temperature to 37±1°C, the rotation speed to 200rpm, the ventilation ratio to 1:1, the dissolved oxygen is reduced to 20%, and the air flow is increased to 1.5:1 until the fermentation is completed; the fermentation medium is: 10ml / L diesel, 1g / L ammonium sulfate, 1g / L dipotassium hydrogen phosphate, 1g / L magnesium sulfate, 0.5g / L sodium chloride, 20g / L peptone, 5g / L sucrose, and natural pH.

8. Use of the Bacillus zanthoxylum ZM2-4 as claimed in claim 1 in degrading oily wastewater.

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