TMAH degrading bacterium and application thereof

Abstract

The invention relates to the technical field of water treatment, and provides a TMAH degrading bacterium and application thereof. The strain is a soil water microbe (Aquammicrobe) HZ-O-037, and the preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) No. 35031. The strain disclosed by the invention can be used for treating sewage containing high-concentration (10000 mg / L) TMAH, the degradation efficiency is high, and the degradation rate within 48 hours can reach 96%.

Description

Technical Field

[0001] The present invention relates to the technical field of water treatment, and in particular to TMAH-degrading bacteria and applications thereof. Background Art

[0002] Tetramethylammonium hydroxide (TMAH) is a toxic nitrogen-containing organic substance. It's often used as a developer in the manufacturing of integrated circuits, liquid crystal panels, and chips. Consequently, it frequently produces large amounts of organic wastewater containing TMAH. This wastewater is characterized by large volumes (accounting for 40% to 50% of industrial wastewater), high pollutant concentrations (TMAH concentrations reaching thousands or even tens of thousands of ppm), high pollution intensity, and strong toxicity. Direct discharge or improper treatment of this wastewater can cause serious health and ecological risks.

[0003] TMAH has a quaternary ammonium base with four methyl groups in its structural formula. It is stable and difficult to be decomposed by microorganisms. The hydroxyl groups in its structural formula make it highly alkaline and corrosive. + It is a substance similar to the neurotoxins of poisonous snails, which is inhibitory to microorganisms in sewage treatment at high concentrations. Traditional TMAH treatment methods include ion exchange, ultrafiltration and reverse osmosis, but these technologies are expensive and will produce secondary pollution after treatment. Biological treatment of TMAH is currently the most effective and pollution-free method. However, biological methods often use anaerobic microorganisms for treatment, and the TMAH degradation efficiency is low. The concentration of treated TMAH is low, and anaerobic treatment requires strict control of dissolved oxygen, which makes operation and maintenance more difficult. There are few reports on the treatment of TMAH by aerobic microorganisms. In response to these shortcomings, this case provides an aerobic TMAH-degrading bacterium that can degrade high-concentration TMAH and convert TMAH into biodegradable ammonia nitrogen and carbon sources. Summary of the Invention

[0004] In view of this, the present invention proposes a TMAH-degrading bacterium and its application, which can degrade high-concentration TMAH and convert TMAH into biodegradable ammonia nitrogen and carbon sources.

[0005] The technical solution of the present invention is achieved as follows: In the first aspect, the present invention provides a TMAH-degrading bacterium, which is a soil water microbacterium ( Aquamicrobium terrae ) HZ-O-037, the deposit number is CGMCC No. 35031.

[0006] On the basis of the above technical solution, preferably, the soil water microbes ( Aquamicrobium terrae ) The nucleotide sequence of 16S rDNA of HZ-O-037 is shown in SEQ ID No. 1.

[0007] The second aspect of the present application provides application of the TMAH degrading bacteria in sewage treatment containing tetramethylammonium hydroxide.

[0008] On the basis of the above technical solutions, preferably, the soil water microbacterium (HZ-O-037) is added into the sewage containing tetramethylammonium hydroxide, and the tetramethylammonium hydroxide is degraded into ammonia nitrogen and a carbon source. Aquamicrobium terrae

[0009] On the basis of the above technical solutions, preferably, the concentration of the tetramethylammonium hydroxide in the sewage is less than 10000 mg / L.

[0010] On the basis of the above technical solutions, preferably, the pH value of the sewage is 7-10.

[0011] The third aspect of the present application provides a bacterium agent for degrading TMAH, which comprises the soil water microbacterium (HZ-O-037) described above, and the preparation is a solid or liquid preparation. Aquamicrobium terrae

[0012] The TMAH degrading bacteria and the application thereof have the following beneficial effects relative to the prior art: The strain of the present application can treat sewage containing high concentration (10000 mg / L) of TMAH, and has high degradation efficiency, and the degradation rate can reach 96% in 48 h. BRIEF DESCRIPTION OF DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0014] Figure 1 It is a growth chart of different strains; Figure 2 It is a TMAH degradation capacity chart of different strains; Figure 3 It is a scanning electron microscope photograph of the HZ-O-037 strain; Figure 4 It is a phylogenetic tree analysis chart of the HZ-O-037 strain; Figure 5 It is a chart of the influence of different rotating speeds on the HZ-O-037 strain; Figure 6 It is a chart of the influence of initial TMAH concentration on the HZ-O-037 strain; Figure 7 It is a chart of the influence of initial pH concentration on the HZ-O-037 strain. ​​DETAILED DESCRIPTION

[0015] The technical solutions in the embodiments of the present application will be clearly and completely described in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort fall within the protection scope of the present application.

[0016] I. Screening of strains 1. Enrichment of strains The sludge in the bottom of the aerobic tank in the chip factory sewage treatment system is used for enrichment. After centrifugation of the sludge, the supernatant is removed, and the sludge sediment is added into the enrichment medium at a ratio of 5%-10% (w / v) under aerobic conditions at 28°C. The changes of TMAH and pH in the medium are detected every day. When the TMAH content in the medium decreases to 0 mg / L, the sludge is inoculated into a new medium at a ratio of 5%-10% again, and the TMAH content is increased. After domestication for 1-2 months according to this method, the TMAH degradation efficiency of the sludge is improved compared with the initial stage. At this time, the TMAH degrading bacteria enrichment liquid is obtained, and the enrichment bacteria liquid can be used for separation and purification by plate dilution coating and streaking method.

[0017] Enrichment medium formula: TMAH 2000-10000 mg, NH4Cl 0.5-2 g, K2HPO4 0.7 g, KH2PO4 0.3 g, MgSO4·7H2O 0.5 g, supplemented with distilled water to 1 L, pH 7.5-8.5.

[0018] 2. Isolation and purification of strains Dilution coating method and streaking purification: the enrichment bacteria liquid is diluted with liquid purification medium to 10 -1 ~10 -7 7 gradients, 100 ul of each gradient is coated on the solid purification medium, 5 parallel samples are prepared for each gradient, and aerobic culture is carried out at 28°C for 48-72 h. Different colonies growing on the plates are picked and streaked for purification. After 7-8 generations of streaking, 4 strains of bacteria are finally purified, Purification medium (liquid): TMAH 2000-10000 mg, NH4Cl 0.5-2 g, K2HPO4 0.7 g, KH2PO4 0.3 g, MgSO4·7H2O 0.5 g, trace elements 1 mL, supplemented with distilled water to 1 L, pH 7.5-8.5.

[0019] Microelements: H3BO3 0.6 g, CoSO4*7H2O 0.4 g, ZnSO4*7H2O 0.32 g, MnCl2*7H2O 0.082 g, Na2MoO4*2H2O 0.0648 g, NiCl2*6H2O 0.064 g, CuSO4*5H2O 0.028 g, FeSO4*7H2O 0.5 g, CaCl2*2H2O 0.2 g, and distilled water was added to 1 L.

[0020] Purified medium (solid): 1.5%-2% (w / v) agar powder was added to the liquid purified medium.

[0021] 3. Screening of the ability of TMAH degradation of the strains The four strains obtained by screening were compared in terms of TMAH degradation ability, and the strain with the strongest TMAH degradation ability was selected as the experimental strain.

[0022] 3.1 Experimental method Strain activation: The four strains were inoculated in liquid purified medium for activation, 30°C, 120 r / min, shaking culture for 48 h, and the strain concentration reached 1*10 8 cells / ml, which was the completion of activation The activated strains were inoculated in liquid purified medium at 1% (v / v), and the concentration of TMAH in the liquid purified medium was 2500 mg / L, 30°C, 120 r / min, shaking culture for 48 h, and the change of TMAH and OD 600 value was detected every 12 h.

[0023] 3.2 Experimental results OD 600 value represents the concentration of the bacteria, and the higher the value, the higher the concentration of the bacteria. It can be seen from Figure 1 that strain 3 grows the fastest and has the highest concentration; strain 1 is second, and strains 3 and 4 grow slower.

[0024] The degradation ability of the four strains to TMAH is shown in Figure 2 , and the degradation rate of strain 3 to TMAH reached 95.47%; followed by strain 1, with a degradation rate of 76%; the degradation rates of strains 4 and 2 were 60.13% and 34.94%, respectively, and strain 3 had the strongest degradation ability to TMAH.

[0025] In summary, strain 3 grows the fastest and has the strongest degradation ability to TMAH, so strain 3 is selected as the experimental strain in the future, named HZ-O-037.

[0026] The strain is classified and named as: aquaspirillum sp. Aquamicrobium terraeHZ-O-037 was deposited at China General Microbiological Culture Collection Center (CGMCC) on June 27, 2025, and the deposit number is CGMCC No. 35031. The deposit address is No. 1, Yikhinaxi Lu, Jiaoyi 3, Beijing Chaoyang Qu, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

[0027] II. Morphological and molecular biological identification of the strain 1. Morphology of the strain The strain HZ-O-037 was streaked on beef extract proteose peptone solid medium and cultured at 28°C under aerobic conditions for 24-48 h. The diameter of the colony of HZ-O-037 was 0.1-5 mm. The colony was semi-transparent white, raised, smooth and non-glossy, round, smooth and transparent in edge, odorless and tasteless. The microstructure of the strain was observed by scanning electron microscopy, and the strain was bacillus, 1-1.5 μm long and 0.3-0.5 μm wide, as shown in FIG. 1. Figure 3

[0028] 2. Molecular biological identification of the strain The genome of the strain HZ-O-037 was extracted, and 16S rDNA sequencing was performed. The obtained sequence is as follows: ​

[0029] The sequencing results were compared with the known sequences in the Genbank database, and as shown in Figure 4 Figure 1, the strain HZ-O-037 had the highest sequence homology with Aquaspirillum sp., and was identified as Aquaspirillum. Aquamicrobium terrae

[0030] III. Optimization of the expansion conditions of the strain By optimizing the three aspects of different rotation speeds (dissolved oxygen), different initial TMAH concentrations, and different initial pH in the culture medium, the strain can grow rapidly and further improve the efficiency of degrading TMAH.

[0031] 1. Effect of different rotation speeds on the strain HZ-O-037 Strain activation: The strain HZ-O-037 was inoculated into a liquid purified culture medium for activation, 28°C, 120 r / min, shaking culture for 48 h, and the strain concentration reached 1×10 8 cells / ml, which was the completion of activation.

[0032] The activated strain was inoculated into a purified culture medium containing 2500 mg / L TMAH at a ratio of 1% (v / v), pH 7.0, and cultured at 28°C for 48 h at shaking speeds of 0, 50, 100, 150, and 200 r / min, respectively. Then the OD600 (bacterial concentration) and TMAH concentration were measured, and the results are shown in Figure 5 and Table 1.

[0033] Table 1. Effect of different rotation speeds on the strain HZ-O-037

[0034] Figure 5 As shown in Table 1, when the rotation speed was 100, 150, and 200 r / min, the TMAH degradation rate could reach more than 90%. When the rotation speed was 150 r / min, the degradation efficiency of the strain was the highest, reaching 99.59%. When the rotation speed was 0, which represented low dissolved oxygen and low growth rate, the TMAH degradation efficiency was poor, and the degradation rate was only 13.24%. Therefore, the optimal expansion rotation speed of the strain was selected as 150 r / min.

[0035] 2. Effect of different initial TMAH concentrations on the strain HZ-O-037 ​The activated strain was inoculated into purified medium containing different concentrations of TMAH (1000, 2000, 5000, 7000, 10000 mg / L) at a ratio of 1% (v / v), and the initial pH was 7.0. After 48 h of culture at 28°C with a shaking speed of 150 r / min, the OD600 (bacterial concentration) and TMAH concentration were determined, respectively. The results are shown in Table 2. Figure 6 and Table 2.

[0036] Table 2 Effect of initial TMAH concentration on strain HZ-O-037

[0037] Figure 6 As shown in Table 2 and Table 3, when the initial TMAH concentration was 1000-10000 mg / L, the degradation rate of TMAH could reach more than 96%. When the initial TMAH concentration was 1000 mg / L, the carbon source was insufficient, resulting in a low bacterial concentration, and the OD600 was only 0.279. When the initial TMAH concentration was 2000-5000 mg / L, the degradation rate of TMAH could reach 99% or more. Further increasing the initial TMAH concentration to 96% might be due to the high concentration, which was too high for the strain to degrade. Therefore, the optimal initial TMAH concentration for the strain was selected as 2000 mg / L.

[0038] 3. Effect of different initial pH concentrations on strain HZ-O-037 The activated strain was inoculated into purified medium containing 2000 mg / L of initial TMAH concentration at a ratio of 1% (v / v), and the initial pH was set to 6, 7, 8, 9 and 10, respectively. After 48 h of culture at 28°C with a shaking speed of 150 r / min, the OD600 (bacterial concentration) and TMAH concentration were determined, respectively. The results are shown in Table 3. Figure 7 and Table 3.

[0039] Table 3 Effect of pH on strain HZ-O-037

[0040] Figure 7 As shown in Table 3, when the initial pH was 7-10, the degradation rate of TMAH could reach more than 90%. When the initial pH was 6.0, the low initial pH affected the growth of the strain, and thus the degradation rate of TMAH was low, which was 53.3%. When the initial pH was 7-8, the degradation rate of TMAH could reach 98% or more. Further increasing the initial pH to 10.0, the degradation rate only decreased to 91.1%. This indicated that the strain had strong adaptability to alkaline environment, and the growth and metabolism were more suitable for slightly alkaline environment. Therefore, the optimal initial pH for the strain was selected as 8.0.

[0041] IV. Effect of strain treatment on TMAH wastewater 1. Effect of treatment on high-concentration TMAH wastewater Strain activation and expansion: The strain HZ-O-037 was inoculated into a purified culture medium for activation and expansion. The initial conditions for expansion were set as pH 7.0, a shaking speed of 150 r / min, an initial TMAH concentration of 2000 mg / L, 28°C, and 120 r / min. The strain was cultured for 48 h, and the strain concentration reached 1 x 10 8 cell / ml, which indicated that the activation of high-concentration TMAH was completed. Wastewater preparation: The pH of the influent containing TMAH in the chip factory wastewater treatment system was 7.5-9.0, and the TMAH concentration was 2800 mg / L. The TMAH concentration in the influent was increased to 6000 mg / L, and subsequent experiments were performed.

[0042] The activated and expanded strain was inoculated into high-concentration TMAH wastewater at a ratio of 1% (v / v), and aeration treatment was performed at room temperature for 48 h. Sampling was performed every 12 h to detect the TMAH concentration. The experimental results are shown in Table 4.

[0043] Table 4. Effect of strain treatment on high-concentration TMAH wastewater

[0044] As shown in Table 4, after 48 h, the TMAH concentration decreased from 6128 mg / L to 114 mg / L, and the TMAH degradation rate was 98.14%. The pH of the water sample was not significantly affected, which indicated that the strain HZ-O-037 could treat high-concentration TMAH wastewater.

[0045] 2. Application example of treatment of electronic wastewater A certain electronic enterprise in Yixing has wastewater containing high-concentration TMAH, with a TMAH content of 1500-2500 mg / L and an influent pH of 8.0-9.0. Currently, the effluent nitrogen is over-standard, and it is found that TMAH in the system cannot be completely degraded, which leads to incomplete release of organic nitrogen, and thus the total nitrogen in the system is over-standard. In order to solve the problems of high cost and poor treatment effect, microbial inoculants are added to the system.

[0046] The strain HZ-O-037 was added to the system at a ratio of 1‰ (v / v). After addition, the system was stably operated for one month, and the water quality parameters of the effluent before and after the use of the inoculant are shown in Table 5.

[0047]

[0048] In summary, after the use of the HZ-O-037 strain, the effluent of the system is stable, and the indicators meet the discharge standards. Moreover, through the use of the microbial strain, the use of physical and chemical agents is reduced, which greatly reduces the cost.

[0049] The above merely provides the preferred embodiment of the present application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A TMAH-degrading bacterium, characterized in that: The bacterial species is soil water microbes ( Aquamicrobium terrae ) HZ-O-037, the deposit number is CGMCC No. 35031.

2. A TMAH-degrading bacterium according to claim 1, characterized in that: The soil water microbes ( Aquamicrobium Terrae ) The nucleotide sequence of 16S rDNA of HZ-O-037 is shown in SEQ ID No.

1.

3. The use of a TMAH-degrading bacterium in the treatment of sewage containing tetramethylammonium hydroxide according to claim 1, wherein: Soil water microbes ( Aquamicrobium terrae ) The bacterial liquid of HZ-O-037 is added to the wastewater containing tetramethylammonium hydroxide to degrade the tetramethylammonium hydroxide into ammonia nitrogen and carbon source.

4. The use according to claim 3, characterized in that: The concentration of tetramethylammonium hydroxide in the sewage is less than 10,000 mg / L.

5. The use according to claim 3, characterized in that: The pH value of the sewage is 7-10.

6. A bacterial agent for degrading TMAH, characterized in that: Including the soil water microbacteria according to claim 1 ( Aquamicrobium terrae )HZ-O-037, the preparation is a solid or liquid preparation.

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