A biological treatment method for melatonin production wastewater

Abstract

This invention provides a biological treatment method for melatonin production wastewater, which solves the technical problems of high cost of decolorizing agents and the need for further improvement in decolorization effect in traditional adsorption and flocculation decolorization methods. The method includes biological fermentation pretreatment of the melatonin production wastewater. The bacteria used in the biological fermentation pretreatment include *Shewanella decolorationis* QCA2, which was deposited on December 17, 2020, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 21391.

Description

Technical Field

[0001] This invention relates to the field of biotechnology for wastewater treatment, specifically a biological treatment method for melatonin production wastewater. Background Technology

[0002] Melatonin, also known as melatonin or melatonin hormone, has the chemical name N-acetyl-5-methoxytryptamine. Produced by the pineal gland in mammals and humans, melatonin is an endogenous hormone that induces natural sleep. It overcomes sleep disorders and improves sleep quality by regulating natural sleep patterns.

[0003] Currently, there are very few reports in literature and patents regarding the treatment of melatonin production wastewater. This wastewater is characterized by high color. Traditional decolorization methods use adsorption and flocculation, which are expensive and the decolorization effect needs further improvement. This invention uses biological fermentation pretreatment to achieve wastewater decolorization, which greatly reduces wastewater treatment costs and produces no secondary pollution, thus having great application prospects. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a biological treatment method for melatonin production wastewater, which solves the technical issues of high cost of decolorizing agents and the need for further improvement in decolorization effect in traditional adsorption and flocculation decolorization methods.

[0005] A biological treatment method for melatonin production wastewater, characterized by: pretreatment of the melatonin production wastewater by biological fermentation, wherein the bacteria used in the biological fermentation pretreatment include Shewanella decolorationis QCA2, which was deposited on December 17, 2020, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 21391.

[0006] A biological treatment method for melatonin production wastewater, characterized by comprising the following steps performed sequentially:

[0007] S1. Treat the melatonin production wastewater by conditioning;

[0008] S2. Pre-treat the melatonin production wastewater through biological fermentation.

[0009] S3. The effluent from the biological fermentation pretreatment is sequentially subjected to ABR anaerobic treatment and SBR aerobic treatment.

[0010] in,

[0011] In step S2, the residence time for bio-fermentation pretreatment is 48-96 h, the dissolved oxygen is 0.5-3 mg / L, and 5% bacterial solution is inoculated according to the tank volume. The selected bacterial strains are obtained by mixing Marinobacter, Gemmobacter, Thaurea, Sphingobacteriales, and Shewanella decolorizationis QCA2 in a live cell ratio of 10-30:5-10:10-20:2-8:5-15. The concentration of each bacterial strain before mixing is 10. 8 CFU / ml;

[0012] In step S3, the retention time for the ABR anaerobic treatment is 24-48 h, the temperature in the biological reactor is 25-35℃, the dissolved oxygen is 0.5-1 mg / L, and the pH is 6-9; the retention time for the SBR aerobic treatment is 24-50 h, the temperature in the biological reactor is 25-35℃, the dissolved oxygen is 2-6 mg / L, and the COD:N:P ratio is 300:5:1 (ammonium chloride is added for nitrogen, and potassium dihydrogen phosphate is added for phosphorus). Both the ABR anaerobic and SBR aerobic treatments are inoculated with 5% bacterial solution per tank volume. The selected bacterial species are Pseudomonas sp., Arthrobacter chlorophenolicus, Sphingonas chlorophenolica, Commonas sp., Microbacterium sp., and Ochrobactrum. Tritici was prepared by mixing bacteria in a live count ratio of 20:5:15:20:15:25, with each type of bacteria having a concentration of 10 before mixing. 8 CFU / ml.

[0013] Furthermore, the biochemical device used in ABR anaerobic treatment has a reactor with 1 / 3 (V / V) volcanic rock filter media and 1 / 3 (V / V) biological carrier carbon added by tank volume.

[0014] Furthermore, the biocarrier carbon is 1-6 mesh coal-based granular activated carbon, with an iodine adsorption value of 450-600 mg / L.

[0015] Furthermore, the biochemical device used in SBR aerobic treatment has 20% (V / V) volcanic rock and 10% (V / V) polyurethane sponge filler added to the reactor according to the tank volume.

[0016] Furthermore, the polyurethane sponge filler is a cubic sponge with dimensions of 1cm x 1cm x 1cm, and its expansion coefficient upon contact with water is 1.3-1.6 times, its porosity is 80-90%, its water holding capacity is 4-6, and its bulk density is 40-60 kg / m³. 3 .

[0017] The biological treatment method for melatonin production wastewater of the present invention has the following advantages:

[0018] (1) Shewanella decolorationis QCA2 was screened in activated sludge samples and showed good color removal effect on melatonin production wastewater. In static experimental observation, the color removal rate of QCA2 on melatonin production wastewater reached 85% after 48 hours.

[0019] (2) Based on the comparison with traditional physical and chemical pretreatment, biological fermentation pretreatment combined with ABR+SBR biochemical treatment process is selected, which is close to full biochemical treatment. Not only is there no secondary pollution, but it can also replace the traditional physical and chemical treatment process, simplify the entire process flow, and greatly reduce the cost of wastewater treatment and disposal. Attached Figure Description

[0020] Figure 1 These are photos of the melatonin production wastewater before and after decolorization in Example 2.

[0021] Figure 2 These are photos of the melatonin production wastewater before and after microelectrolysis in Example 4.

[0022] Figure 3 These are before and after photos of the Fenton treatment (liquid alkali coagulation) of melatonin production wastewater in Example 4.

[0023] Figure 4 These are before and after photos of the Fenton treatment (lime coagulation) of melatonin production wastewater in Example 4. Detailed Implementation

[0024] The present invention will be described below through specific embodiments, but the present invention is not limited thereto. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described are commercially available unless otherwise specified. The following embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of protection of this patent.

[0025] Example 1

[0026] Isolation of Shewanella decolorationis strain

[0027] You can refer to patent CN201811383781.1. The only differences are: the source is melatonin production wastewater; and the culture medium used is different.

[0028] The culture medium used during the acclimatization process is as follows: 0.2g MgSO4, 0.02g KCl, 1g KH2PO4, 2.6g K2HPO4, 30% blackening production wastewater, distilled water to make up to 1000mL, 40g water-washed agar, pH 7.5-8, autoclaved at 121℃ for 30min and ready for use.

[0029] Identification of strain morphology and physiological and biochemical levels

[0030] When the strain of this invention grows on solid LB medium, the colonies are round, with a smooth and flat surface, neat edges, and are colorless to pale pink and transparent.

[0031] Identification at the molecular biological level:

[0032] Bacterial DNA was extracted using a DNA extraction kit (Sangon) following standard extraction procedures, followed by PCR amplification of the target fragment. A pair of universal primers was designed for amplifying the 16S rDNA sequence:

[0033] 27F 5 '-AGAGTTTGATCMTGGCTCAG-3 ';

[0034] 1492R 5 '-GGTTACCTTGTTACGACTT-3 ';

[0035] Using genomic DNA as a template, Premix Tap™ was added for PCR amplification. The PCR product was detected by 1% agarose gel electrophoresis, purified using a DNA purification and recovery kit, ligated into the pGM-T vector, transformed into E. coli DH5α competent cells, plated onto LB solid medium containing ampicillin, and cultured at 37°C for 12 h. Colonies were picked and cultured in liquid LB medium, shaken at 37°C and 180 rpm overnight. Plasmids were extracted using a plasmid extraction kit and analyzed.

[0036] The PCR reaction conditions were as follows: 94℃ pre-denaturation for 5 min; followed by 30 cycles of 94℃ denaturation for 1 min; 55℃ annealing for 1 min; and 72℃ extension for 5 min. The PCR amplification products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequence is shown in SEQ ID NO. 1. The strain was identified by 16S rDNA, and the results were compared for homology using the Genebank Blast program, confirming that the strain was similar to *Shewanella decoloris*. Shewanella decolorationisThe homology with the strain reached 99%, further confirming that the strain is *Shewanella decoloris*. This strain was deposited on December 17, 2020, at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC NO. 21391.

[0037]

[0038] Example 2

[0039] Decolorization experiment of melatonin production wastewater

[0040] The decolorizing Shewanella QCA2 strain was separately inoculated into a bio-fermentation pretreatment device containing melatonin production wastewater diluted 3.6 times, resulting in a Shewanella QCA2 concentration of 10. 8 The inoculum concentration was calculated based on the tank volume, with an inoculum amount of 3% (V / V). The mixture was placed in a biological shaker and reacted for 48 hours at a temperature of 35°C and a rotation speed of 150 rpm / min.

[0041] like Figure 1 As shown, the melatonin production wastewater originated from Hubei Weitekang Pharmaceutical Co., Ltd. The raw water was a brown turbid liquid with a color intensity of 4096 times. After filtration, the raw water became a clear brown liquid. A CASS reactor was used for biological fermentation pretreatment. 5% (V / V) of the above-mentioned shaker-expanded *Shewanella catalyticosa* QCA2 bacterial solution was inoculated into the reactor. Dissolved oxygen was controlled at 3 mg / L, and the temperature was 25-35℃. The influent of the biological fermentation pretreatment (i.e., the raw water diluted 3.6 times) was a clear brownish-yellow liquid with a color intensity of 1024 times, and the effluent was a pale white clear liquid with a color intensity of 8 times. This indicates that *Shewanella catalyticosa* QCA2 has a good effect on removing color from the melatonin production wastewater. In static experimental observation, the color removal rate of QCA2 for melatonin production wastewater reached 85% after 48 hours.

[0042] Example 3

[0043] S1. Condition the wastewater from melatonin production. Specifically, dilute the wastewater to TDS < 2.5% and adjust the pH to 7-8.

[0044] S2. The conditioned melatonin production wastewater is transported to a biological pretreatment device. Based on the tank volume, 5% (V / V) of degrading bacteria is inoculated and biological fermentation pretreatment is carried out. The dissolved oxygen is controlled at 3 mg / L. The COD degradation and transformation pattern of the wastewater is observed. The COD, pH and other indicators of the effluent from the device are measured every 24 hours.

[0045] The degrading bacteria were prepared by mixing Marinobacter, Gemmobacter, Thaurea, Sphingobacteriales, and Shewanella decolorationis QCA2 in a live bacteria ratio of 20:5:20:5:10. The concentration of each bacteria before mixing was 10. 8 CFU / ml.

[0046] Table 1 shows the experimental data from the bio-fermentation pretreatment stage of melatonin production wastewater in Example 3.

[0047]

[0048] Example 4: Biological fermentation pretreatment effluent + ABR anaerobic + SBR aerobic

[0049] 1) Take the effluent from the biological pretreatment device, adjust the pH to 7-8, and then transport it to the ABR anaerobic biological treatment device (calculated according to the tank volume, filled with 1 / 3 (V / V) volcanic rock filter media, 1 / 3 (V / V) 1~6 mesh activated carbon, the iodine adsorption value of activated carbon is 450-600mg / L, and inoculated with 5% (V / V) compound bacterial culture solution) for anaerobic biological treatment. The temperature inside the ABR anaerobic biological treatment device is 30℃ and the dissolved oxygen is 1 mg / L.

[0050] The composite bacterial strain used in the ABR anaerobic treatment and SBR aerobic treatment was prepared by mixing Pseudomonas sp., Arthrobacter chlorophenolicus, Sphingonas chlorophenolica, Commonas sp., Microbacterium sp., and Ochrobactrum tritici in a ratio of 20:5:15:20:15:25. The concentration of each bacterial strain before mixing was 10. 8 CFU / ml.

[0051] Table 2 shows the experimental data for COD removal from wastewater using the ABR anaerobic biological treatment device in Example 4.

[0052]

[0053] 2) Take the effluent from the ABR anaerobic biological treatment unit, adjust the pH to 7-8, and then introduce it into the SBR aerobic biological treatment unit (calculated based on tank volume, filled with 20% (V / V) volcanic rock and 10% (V / V) polyurethane sponge packing material; the polyurethane sponge packing material is a cubic sponge with dimensions of 1cm x 1cm x 1cm, and its expansion coefficient after absorbing water is 1.3-1.6 times; the filling volume is calculated based on the volume after water absorption and expansion; the porosity is 80-90%, the water holding capacity is 4-6, and the bulk density is 40-60 kg / m³). 3 The bacteria were inoculated with 5% (V / V) compound bacterial culture solution and subjected to aerobic biochemical treatment. The temperature inside the SBR aerobic biochemical device was 30℃, the dissolved oxygen was 6mg / L, and the COD:N:P ratio was adjusted to 300:5:1.

[0054] Table 3 shows the experimental data on COD removal from wastewater using the SBR aerobic biological system in Example 4.

[0055]

[0056] Example 5: Comparison of traditional physicochemical pretreatment technologies and biological fermentation pretreatment technologies for melatonin production wastewater

[0057] Production wastewater → Micro-electrolysis → Fenton

[0058] (1) Production wastewater → micro-electrolysis

[0059] 1) Experimental procedure: Take 600 mL of production wastewater into a beaker, filter it, add about 6.63 mL of concentrated sulfuric acid to adjust its pH to about 3.0, pour it into the iron-carbon micro-electrolysis reaction device, react at room temperature for 3 hours, and after the reaction is completed, take out the water sample with pH of about 4.0, filter it, collect the filtrate and store it for later testing.

[0060] 2) Experimental phenomena: such as Figure 2 As shown, the production wastewater sample was a brown turbid liquid with a small amount of black suspended matter. After filtration, it became a brown clear liquid with a pH of about 8.0. Concentrated sulfuric acid was added to adjust the pH to about 3.0, and the water sample turned brownish-yellow. After reacting at room temperature for 3 hours, micro-electrolysis produced a brownish-yellow clear liquid.

[0061] 3) Micro-electrolysis test data record: see Table 4.

[0062] Table 4 shows the experimental data of micro-electrolysis in Example 5.

[0063]

[0064] (2) Micro-electrolysis of water → Fenton oxidation (liquid alkali coagulation)

[0065] 1) Experimental procedure: Take 500 mL of micro-electrolysis water into a beaker with a pH of about 4.0, add 2.5 g of FeSO4·7H2O, stir until completely dissolved, then add 10 mL of 30% (w / w) hydrogen peroxide and place it on a magnetic stirrer to stir. After reacting at room temperature for 2 hours, take out the water sample, add 18.65 g of 30% (w / w) liquid alkali to adjust the pH to about 9.5, add an appropriate amount of PAM, let stand for 30 minutes, filter, collect and store the filtrate for later testing.

[0066] 2) Experimental phenomena: such as Figure 3 As shown, the micro-electrolysis effluent is a clear, brownish-yellow liquid with a pH of approximately 4.0. Adding FeSO4·7H2O did not cause a significant color change. Adding hydrogen peroxide turned the effluent black. After reacting at room temperature for 2 hours, the Fenton effluent showed no significant change. Adjusting the pH to 9.5 with liquid alkali did not cause a significant color change. Adding an appropriate amount of PAM and allowing the mixture to stand for 30 minutes resulted in a darker color, making the mud-to-water ratio indistinguishable. After filtration, the mud weight was 12.32 g, and the filtered effluent was brown.

[0067] 3) Data record of Fenton small-scale experiment: see Table 5.

[0068] Table 5 shows the experimental data of the Fenton small-scale test (liquid alkali coagulation) in Example 5.

[0069]

[0070] (3) Micro-electrolysis of water → Fenton oxidation (lime coagulation)

[0071] 1) Experimental procedure: Take 500 mL of micro-electrolysis water into a beaker with a pH of about 4.0, add 2.5 g of FeSO4·7H2O, stir until completely dissolved, then add 10 mL of 30% (w / w) hydrogen peroxide and place it on a magnetic stirrer to stir. After reacting at room temperature for 2 hours, take out the water sample, add 8.83 g of lime to adjust the pH to about 9.5, add an appropriate amount of PAM, let stand for 30 minutes, filter, collect and store the filtrate for later testing.

[0072] 2) Experimental phenomena: such as Figure 4 As shown, the micro-electrolysis effluent is a clear, brownish-yellow liquid with a pH of approximately 4.0. Adding FeSO4·7H2O did not cause any significant change in the water sample. Adding hydrogen peroxide turned the color black. After reacting at room temperature for 2 hours, the Fenton effluent was brownish-brown. Adding lime to adjust the pH to 9.5 turned the water sample coffee-colored. After adding an appropriate amount of PAM, stirring, and letting it stand for 30 minutes, with a mud-to-water ratio of 200:300, the wet mud weight after filtration was 45.62 g, and the filtered water was brown.

[0073] 3) Data record of Fenton small-scale experiment: see Table 6.

[0074] Table 6 shows the experimental data of Fenton's small-scale test (lime concrete) in Example 5.

[0075]

[0076] Comparison of COD in effluent and reagent consumption between traditional physicochemical pretreatment and biological fermentation pretreatment processes: see Tables 7-9.

[0077] Table 7 shows the COD data of the effluent from the traditional physicochemical pretreatment process in Example 5.

[0078]

[0079] Table 8 shows the COD data of the effluent from the bio-fermentation pretreatment process in Example 3.

[0080]

[0081] Table 9 provides an overview of the reagents and solid waste generated in traditional physicochemical processes.

[0082]

[0083] Cost accounting: Sulfuric acid 700 yuan / ton, liquid alkali 1000 yuan / ton, hydrogen peroxide 1500 yuan / ton, hazardous waste disposal 5000 yuan / ton.

Claims

1. A biological treatment method for melatonin production wastewater, characterized in that: The method includes biological fermentation pretreatment of melatonin production wastewater. The bacteria used in the biological fermentation pretreatment include Shewanella decolorationis QCA2, which was deposited on December 17, 2020, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 21391. The biological treatment method for melatonin production wastewater includes the following steps performed sequentially: S1. Treat the melatonin production wastewater by conditioning; S2. Pre-treat the melatonin production wastewater through biological fermentation. S3. The effluent from the biological fermentation pretreatment is sequentially subjected to ABR anaerobic treatment and SBR aerobic treatment. in, In step S2, the residence time for the bio-fermentation pretreatment is 48-96 h, the dissolved oxygen is 0.5-3 mg / L, and 5% bacterial solution is inoculated according to the tank volume. The selected bacterial strains are obtained by mixing *Hymenobacterium*, *Bacillus*, *Tauridella*, *Sphingomyelin*, and *Shewanella decolori* QCA2 in a live cell ratio of 10-30:5-10:10-20:2-8:5-15. The concentration of each bacterial strain before mixing is 10. 8 CFU / ml; In step S3, the residence time for the ABR anaerobic treatment is 24-48 h, the temperature in the biological reactor is 25-35℃, the dissolved oxygen is 0.5-1 mg / L, and the pH is 6-9; the residence time for the SBR aerobic treatment is 24-50 h, the temperature in the biological reactor is 25-35℃, the dissolved oxygen is 2-6 mg / L, and the COD:N:P ratio is 300:5:

1. Both the ABR anaerobic and SBR aerobic treatments are inoculated with 5% bacterial solution per tank volume. The selected bacterial strains are Pseudomonas, Arthrobacter chlorophenate, Sphingosine chlorophenate, Trichomonas vaginalis, Microbacterium, and Aureobacterium chrysogenum, mixed in a viable count ratio of 20:5:15:20:15:25, with each strain having a pre-mixed concentration of 10. 8 CFU / ml.

2. The biological treatment method for melatonin production wastewater as described in claim 1, characterized in that: The biochemical unit used in ABR anaerobic treatment has a reactor with 1 / 3 (V / V) volcanic rock filter media and 1 / 3 (V / V) biological carrier carbon added according to the tank volume.

3. The biological treatment method for melatonin production wastewater as described in claim 2, characterized in that: The biocarrier carbon is 1-6 mesh coal-based granular activated carbon, with an iodine adsorption value of 450-600 mg / L.

4. The biological treatment method for melatonin production wastewater as described in claim 1, characterized in that: The biochemical device used in SBR aerobic treatment has a reactor with 20% (V / V) volcanic rock and 10% (V / V) polyurethane sponge filler added according to the tank volume.

5. The biological treatment method for melatonin production wastewater as described in claim 4, characterized in that: The polyurethane foam filler is a cubic sponge with dimensions of 1cm x 1cm x 1cm. Its expansion coefficient upon contact with water is 1.3-1.6 times, its porosity is 80-90%, its water holding capacity is 4-6%, and its bulk density is 40-60 kg / m³. 3 .

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