Application of a mixed bacterial fermentation broth in decolorization of triphenylmethane dyes and strains

Abstract

The present invention discloses an application of a mixed bacterial fermentation broth in the decolorization of triphenylmethane dyes. The mixed bacterial fermentation broth comprises a mixture of fermentation broths of one or more of Bacillus subtilis, Pseudomonas or Actinomycetes. The mixed bacterial fermentation broth constructed in the present invention for the efficient decolorization of triphenylmethane dyes can decolorize more than 70% of water-soluble aniline blue under both static and shaking conditions, and can tolerate a pH of about 4-8 and a high concentration of water-soluble aniline blue. In addition, the bacterial fermentation broth also has a good decolorization effect on other triphenylmethane dyes. It can decolorize more than 90% of basic fuchsin and malachite green within 48 hours, and also has a certain decolorization effect on three triphenylmethane dyes, namely methyl violet, acid blue 1 and acid red 94. The types of triphenylmethane dyes decolorized are significantly more than those of the reported microbial strains, showing high broad-spectrum property, and having the possibility of being actually applied to solve triphenylmethane dye wastewater.

Description

(1) Technical Field

[0001] The present invention relates to an application of a mixed bacterial fermentation broth in decolorizing triphenylmethane dyes and strains. (2) Background Art

[0002] A key factor for human survival and social development is water source. Limited water resources and uncontrollable pollution are major problems in current water resource management. How to effectively and reasonably allocate and utilize water resources to meet the growing agricultural and industrial needs of humans is a question worthy of deep consideration. The main strategy for solving the water shortage problem lies in the rational use and recycling of water resources. Facing this situation, industrial sewage should be recycled.

[0003] According to the discharge volume and composition of industrial sewage, the dye industry and industries related to dyes (textile, printing, leather, etc.) are considered to be one of the most polluted industries. Direct discharge of dye sewage into water bodies will cause an increase in water body chromaticity, affect the aesthetic feeling of water bodies, and at the same time block the sunlight irradiation, inhibit the photosynthesis of aquatic plants, and lead to water body hypoxia, thus affecting the water body ecological balance; the most important thing is that the toxic and harmful substances in dye sewage have carcinogenic and teratogenic effects, and direct discharge into water bodies will pose a great threat to the life and health of humans, animals and plants.

[0004] Various dyes and their degradation products are the most toxic and harmful substances in dye sewage. These dyes can be roughly divided into azo, anthraquinone and triphenylmethane types according to their structures. Among them, azo dyes are the most, accounting for about 60%-70%, followed by anthraquinone dyes, accounting for about 15%, and triphenylmethane dyes are classified as the third major type of dyes. The parent structure of triphenylmethane dyes is triphenylmethane, and it can be further classified according to the types of substituents on its benzene ring, roughly including aniline blue, malachite green, methyl violet, basic fuchsin, etc. This type of dye has the advantages of low production cost, bright color, high coloring power and wide chromatographic range, so this dye is still widely used. At present, domestic and foreign researchers have conducted more research on azo and anthraquinone dyes, and relatively less research on triphenylmethane dyes.

[0005] Regarding the environmental pollution problems caused by triphenylmethane dyes and their degradation products, many scientific researchers have adopted physical or chemical methods for treatment, such as membrane filtration, material adsorption, chemical flocculation, advanced oxidation, etc. These methods are usually costly, ineffective in removing the color of dyes, and can produce a large amount of activated sludge leading to secondary pollution due to the excessive use of chemical substances. In contrast, the environmentally friendly biological method has been increasingly favored by people. The biological method mainly relies on the interaction of various microorganisms to achieve the mineralization of dyes and their degradation products. These microorganisms include bacteria, fungi, algae, etc. Bacteria have been widely concerned because of their fast growth rate, strong environmental adaptability, and ability to produce various degradation enzyme systems, and have a high degradation efficiency for dyes and their degradation products. Currently, bacteria with the ability to decolorize and degrade triphenylmethane dyes include Bacillus, Sphingomonas, Pseudomonas, etc. These bacteria can often only decolorize and degrade one or several triphenylmethane dyes, and it is difficult for a single bacterium to adapt to the impact of the natural environment. Facing this problem, the present invention aims to construct a mixed bacterial fermentation broth with strong environmental adaptability, high degradation efficiency for triphenylmethane dyes, and a wide range of dye degradation, providing relevant microbial strain resources for solving the difficult problem of treating triphenylmethane dye wastewater. (III) Summary of the Invention

[0006] The object of the present invention is to provide an application of a mixed bacterial fermentation broth in the decolorization of triphenylmethane dyes and strains, providing three microbial strains with high efficiency in degrading textile wastewater, and constructing the three strains into a mixed fermentation broth with high efficiency in degrading triphenylmethane dyes through a mixture design; compared with a single microbial strain, the mixed fermentation broth in the present invention has a higher degradation efficiency, a wider range, and stronger environmental adaptability for triphenylmethane dyes, has a very broad application prospect, and has the possibility of being practically applied to solve triphenylmethane dye wastewater. It solves the difficult problem of treating triphenylmethane dye wastewater at present.

[0007] The technical solution adopted by the present invention is:

[0008] The present invention provides an application of a mixed bacterial fermentation broth in the decolorization of triphenylmethane dyes, and the mixed bacterial fermentation broth includes a mixture of fermentation broths of one or more of Bacillus subtilis, Pseudomonas, or Actinomycetes.

[0009] Preferably, the Bacillus subtilis is Bacillus subtilis SX-6, deposited in the China Center for Type Culture Collection (CCTCC), the deposit date is: March 15, 2023, and the deposit number is: CCTCC NO: M2023336, address: Wuhan University, Wuhan, China, postal code: 430072.

[0010] Preferably, the Pseudomonas is Pseudomonas sp. SX-10, which is deposited in the China Center for Type Culture Collection (CCTCC). The deposition date is May 19, 2022, and the deposition number is CCTCC NO: M 2022670.

[0011] Preferably, the actinomycetes is Georgenia sp. SY-1, which is deposited in the China Center for Type Culture Collection (CCTCC). The deposition date is March 15, 2023, and the deposition number is CCTCC NO: M2023335.

[0012] Preferably, the mixed bacterial fermentation broth is a mixture of the fermentation broths of Bacillus subtilis SX-6, Pseudomonas sp. SX-10, and Georgenia sp. SY-1 in a volume ratio of 1:3:1.

[0013] Preferably, the fermentation broths of Bacillus subtilis SX-6, Pseudomonas sp. SX-10, and Georgenia sp. SY-1 are obtained by inoculating Bacillus subtilis SX-6, Pseudomonas sp. SX-10, and Georgenia sp. SY-1 onto an LB solid medium and culturing at 37°C for 20 - 24 h to obtain activated strains; then inoculating the activated strains into an LB liquid medium and culturing on a shaker at 37°C and 180 rpm for 48 h to obtain the single-bacterial fermentation broths. The composition of the LB solid medium is: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract, 15 - 20 g / L agar, and the solvent is water; the composition of the LB liquid medium is: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract, and the solvent is water.

[0014] Preferably, the application is carried out as follows: inoculate the mixed bacterial fermentation broth into an inorganic salt culture solution containing triphenylmethane dyes at an inoculation amount of 5% by volume concentration, and culture at 20 - 37°C, pH 3 - 11, statically or at 150 rpm until the dyes are degraded to achieve the purpose of decolorization; the triphenylmethane dyes include water-soluble aniline blue, basic fuchsin, methyl violet, acid blue 1, acid red 94, or malachite green, preferably basic fuchsin or malachite green.

[0015] Preferably, the concentration of the triphenylmethane dyes added to the inorganic salt culture solution is between 10 mg / L - 500 mg / L (preferably 200 - 500 mg / L, more preferably 200 mg / L).

[0016] Preferably, the culture conditions are 37°C, pH 6 - 7, static.

[0017] Preferably, the composition of the inorganic salt culture solution is as follows: 0.5 - 60 g / L NaCl, 1 g / L NaH₂PO₄, 1 g / L Na₂HPO₄, 0.5 g / L MgSO₄, 0.1 g / L CaCl₂, 0 - 3 g / L yeast extract, and the solvent is water. Preferably, the composition of the inorganic salt medium is: 10 g / L NaCl, 1 g / L NaH₂PO₄, 1 g / L Na₂HPO₄, 0.5 g / L MgSO₄, 0.1 g / L CaCl₂, 1 g / L yeast extract, and the solvent is water.

[0018] The present invention also relates to a Bacillus subtilis SX - 6 for degrading triphenylmethane dyes, which is preserved in the China Center for Type Culture Collection (CCTCC). The preservation date is March 15, 2023, and the preservation number is CCTCC NO: M 2023336. The address is Wuhan University, Wuhan, China, and the postal code is 430072.

[0019] The present invention also relates to a Pseudomonas sp SX - 10 for degrading triphenylmethane dyes, which is preserved in the China Center for Type Culture Collection (CCTCC). The preservation date is May 19, 2022, and the preservation number is CCTCC NO: M 2022670.

[0020] The present invention also relates to a Georgenia sp SY - 1 for degrading triphenylmethane dyes, which is preserved in the China Center for Type Culture Collection (CCTCC). The preservation date is March 15, 2023, and the preservation number is CCTCC NO: M 2023335.

[0021] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0022] The mixed bacterial fermentation broth constructed in the present invention for the efficient decolorization of triphenylmethane dyes can decolorize more than 70% of water - soluble aniline blue (200 mg / L) under both static and shaking (150 rpm) conditions, and can tolerate a pH of about 4 - 8 and a high concentration of water - soluble aniline blue (500 mg / L). In addition, the bacterial fermentation broth also has a good decolorization effect on other triphenylmethane dyes. It can decolorize more than 90% of basic fuchsin (20 mg / L) and malachite green (50 mg / L) within 48 h, and also has a certain decolorization effect on three triphenylmethane dyes, namely methyl violet, acid blue 1, and acid red 94. The types of triphenylmethane dyes decolorized are significantly more than those of the reported microbial strains, showing high broad - spectrum properties, and having the possibility of practical application in solving triphenylmethane dye wastewater. (IV) BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Degradation effects of each primary screening single bacterium on textile wastewater.

[0024] Figure 2 Decolorization effects of different bacteria and mixed bacteria fermentation broth on water-soluble aniline blue of triphenylmethane dyes.

[0025] Figure 3 Decolorization effects of mixed bacteria fermentation broth BPA-3 on water-soluble aniline blue of triphenylmethane dyes under different dissolved oxygen levels.

[0026] Figure 4 Decolorization effects of mixed bacteria fermentation broth BPA-3 on water-soluble aniline blue of triphenylmethane dyes under different yeast extract concentrations.

[0027] [[ID=I6]] Figure 5 Decolorization effects of mixed bacteria fermentation broth BPA-3 on water-soluble aniline blue of triphenylmethane dyes at different temperatures.

[0028] Figure 6 Decolorization effects of mixed bacteria fermentation broth BPA-3 on water-soluble aniline blue of triphenylmethane dyes at different pH values.

[0029] Figure 7 Decolorization effects of mixed bacteria fermentation broth BPA-3 on water-soluble aniline blue of triphenylmethane dyes at different salinities.

[0030] Figure 8 Decolorization effects of mixed bacteria fermentation broth BPA-3 on water-soluble aniline blue of triphenylmethane dyes at different concentrations.

[0031] Figure 9 Decolorization effects of mixed bacteria fermentation broth BPA-3 on different triphenylmethane dyes. (V) Specific implementation manners

[0032] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0033] The culture media involved in the present invention are as follows:

[0034] 1. Composition of LB liquid medium: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract, and the solvent is water.

[0035] 2. Composition of LB solid medium: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract, 20 g / L agar, and the solvent is water.

[0036] 3. Composition of inorganic salt medium: 10 g / L NaCl, 1 g / L NaH2PO4, 1 g / L Na2HPO4, 0.5 g / L MgSO4, 0.1 g / L CaCl2, 1 g / L yeast extract, with water as the solvent, pH 6 - 7.

[0037] 4. Acclimation medium 1: 1 g / L Na2HPO4, 1 g / L NaH2PO4, 1 g / L (NH4)2SO4, 0.2 g / L MgSO4·7H2O, 0.02 g / L CaCl2, 1 mL / L trace element solution, with a mixture of sterile water and textile wastewater in a volume ratio of 7:3 as the solvent.

[0038] 5. Acclimation medium 2: 1 g / L Na2HPO4, 1 g / L NaH2PO4, 1 g / L (NH4)2SO4, 0.2 g / L MgSO4·7H2O, 0.02 g / L CaCl2, 1 mL / L trace element solution, with a mixture of sterile water and textile wastewater in a volume ratio of 4:6 as the solvent.

[0039] 6. Acclimation medium 3: 1 g / L Na2HPO4, 1 g / L NaH2PO4, 1 g / L (NH4)2SO4, 0.2 g / L MgSO4·7H2O, 0.02 g / L CaCl2, 1 mL / L trace element solution, with a mixture of sterile water and textile wastewater in a volume ratio of 1:9 as the solvent.

[0040] 7. Solid selection medium: 1 g / L Na2HPO4, 1 g / L NaH2PO4, 1 g / L (NH4)2SO4, 0.2 g / L MgSO4·7H2O, 0.02 g / L CaCl2, 1 mL / L trace element solution, 20 g / L agar, with textile wastewater as the solvent;

[0041] 8. Trace element solutions are all: 2.5 g / L FeSO4·7H2O, 0.3 g / L MnSO4·H2O, 0.5 g / L (NH4)6Mo7O 24 ·4H2O, 1 g / L ZnSO4·7H2O, with sterile water as the solvent.

[0042] The textile wastewater is taken from Shaoxing Jierui Textile Company, with a COD of 800 - 1000 mg / L.

[0043] Example 1: Screening and identification of Bacillus subtilis SX - 6, Pseudomonas SX - 10 and Actinomyces SY - 1

[0044] (1) Collection of bacterial sources

[0045] Collect activated sludge from the oxidation ditch process section of the third-phase sewage treatment in Shaoxing Water Treatment Development Co., Ltd., and collect soil contaminated by textile wastewater from textile enterprises such as Shaoxing Yongfeng and Jierui. A total of six different samples are stored in a 4°C refrigerator for later use.

[0046] (2) Enrichment and domestication of bacterial sources

[0047] The domestication is carried out in three times. First, add 5 g of different bacterial source samples to 1 L of domestication medium 1 respectively, and treat them at room temperature (30 - 37°C) with aeration (2 mg / L) for 7 days. After 7 days, transfer the once-domesticated bacterial source samples to domestication medium 2 in time, and treat them under the same conditions for 7 days. After 7 days, transfer the twice-domesticated bacterial source samples to domestication medium 3, and treat them under the same conditions for 7 days.

[0048] (3) Primary screening of strains

[0049] Take the mixed solution of the medium after the third domestication, centrifuge it at 8000×g for 5 min, discard the supernatant, collect the thalli and dilute them with sterile water to 10 -1 、10 -2 、10 -3 、10 -4 and 10 -5 Five gradients. Coat the dilution solutions of different gradients on the solid selection medium plates, place them in a 37°C constant temperature incubator for cultivation, observe the growth of the strains on the plates every day, select colonies with different morphologies and colors, and transfer them to the LB plates by streaking multiple times until the colony morphologies of the strains on the plates are consistent. Finally, 44 strains are initially screened from the above samples.

[0050] (4) Re-screening of strains

[0051] Inoculate the initially screened strains after isolation and purification into LB liquid medium respectively, and culture them in a constant temperature shaker at 180 rpm and 37°C for 48 h. After the cultivation is completed, take 1 mL of the culture solution and inoculate it into a conical flask containing 100 mL of textile wastewater respectively, and then place them in a constant temperature shaker at 150 rpm and 37°C for 2 days. After 2 days, judge the degradation performance of each strain according to the COD removal effect, and re-screen the strains with better textile wastewater degradation effect.

[0052] The results are as Figure 1 shown. Among the 44 initially screened strains, 15 strains have a COD removal rate of more than 30% for textile wastewater, and 6 strains have a COD removal rate of more than 40%. In addition, strain SX-10 has a better COD removal effect on textile wastewater, and its COD removal rate reaches 61.3% within 2 days; secondly, the COD removal rates of strains SX-6 and SY-1 for textile wastewater can reach 53.8% and 55.9% respectively. Therefore, SX-6, SX-10 and SY-1 are used as re-screened strains for subsequent research.

[0053] (5) Strain identification and preservation

[0054] The rescreened strains SX-6, SX-10 and SY-1 were sent to Hangzhou Qingke Biotechnology Co., Ltd. for 16S rDNA sequencing. The 16S rDNA nucleotide sequence of strain SX-6 is shown in SEQ ID NO.1, the 16S rDNA nucleotide sequence of strain SX-10 is shown in SEQ ID NO.2, and the 16S rDNA nucleotide sequence of strain SY-1 is shown in SEQ ID NO.3. It was detected that the full lengths of the sequences of strains SX-6, SX-10 and SY-1 were 1378 bp, 1345 bp and 1377 bp respectively. By BLAST alignment with the NCBI database, it was found that the rescreened strain SX-6 belongs to Bacillus subtilis, SX-10 belongs to Pseudomonas sp., and SY-1 belongs to Georgenia sp. In addition, Bacillus subtilis SX-6, Pseudomonas sp. SX-10 and Georgenia sp. SY-1 were preserved in the China Center for Type Culture Collection (CCTCC), and the preservation numbers were CCTCC M NO: 2023336, CCTCC M NO: 2022670 and CCTCC M NO: 2023335 respectively.

[0055] 16S rDNA sequence of Bacillus subtilis SX-6

[0056] CCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCG

[0057] GGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCG

[0058] AGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTC

[0059] GCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATT

[0060] TGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAA

[0061] TGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGAC

[0062] TTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCC

[0063] GAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCG

[0064] TTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTT

[0065] CAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGC

[0066] GGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTG

[0067] TTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACT

[0068] GGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGC

[0069] ACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACT

[0070] TAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTA

[0071] TTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCG

[0072] CCCTATTCGAACGGTACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATC

[0073] ACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTC

[0074] CCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTAC

[0075] GCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTA

[0076] AGTGGTAGCCGAAGCCACCTTTTATGTTTGAACCATGCGGTTCAAACAACCATCCGGTATTA

[0077] GCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCAT。

[0078] Pseudomonas sp.SX-10 16S rDNA sequence

[0079] CCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGACATTC

[0080] TGATTCACGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATCCGGACT

[0081] ACGATCGGTTTTATGGGATTAGCTCCACCTCGCGGCTTGGCAACCCTTTGTACCGACCATTGT

[0082] AGCACGTGTGTAGCCCTGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCC

[0083] GGTTTGTCACCGGCAGTCTCCTTAGAGTGCCCACCATAACGTGCTGGTAACTAAGGACAAGG

[0084] GTTGCGCTCGTTACGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCA

[0085] GCACCTGTGTCTGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTCAGCATGTCAAG

[0086] GCCAGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCC

[0087] CCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCAACTTAATGCGT

[0088] TAGCTGCGCCACTAAGTTCTCAAGGAACCCAACGGCTAGTTGACATCGTTTACGGCGTGGAC

[0089] TACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTCAGTGTCAGTATCAGTCCA

[0090] GGTGGTCGCCTTCGCCACTGGTGTTCCTTCCTATATCTACGCATTTCACCGCTACACAGGAAA

[0091] TTCCACCACCCTCTACCGTACTCTAGCTCGCCAGTTTTGGATGCAGTTCCCAGGTTGAGCCCG

[0092] GGGCTTTCACATCCAACTTAACGAACCACCTACGCGCGCTTTACGCCCAGTAATTCCGATTAA

[0093] CGCTTGCACCCTTCGTATTACCGCGGCTGCTGGCACGAAGTTAGCCGGTGCTTATTCTGTCGG

[0094] TAACGTCAAAACACTAACGTATTAGGTTAATGCCCTTCCTCCCAACTTAAAGTGCTTTACAAT

[0095] CCGAAGACCTTCTTCACACACGCGGCATGGCTGGATCAGGCTTTCGCCCATTGTCCAATATTC

[0096] CCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGACTGATCATCCT

[0097] CTCAGACCAGTTACGGATCGTCGCCTTGGTGAGCCATTACCTCACCAACTAGCTAATCCGAC

[0098] CTAGGCTCATCTGATAGCGCAAGGCCCGAAGGTCCCCTGCTTTCTCCCGTAGGACGTATGCG

[0099] GTATTAGCGTTCCTTTCGGAACGTTATCCCCCACTACCAGGCAGATTCCTAGGCATTACTCACCCGTCCGCCGCTAAATCAAGGAGCAAGCTCCTCT。 [[ID=!16]]

[0100] 16S rDNA sequence of Georgenia sp. SY-1

[0101] It should be noted that there seems to be an error in the original text where the tag ID jumps from 16 to 18 without a proper 17. I have translated it as best as possible while maintaining the integrity of the provided content.

[0102] Example 2: Construction of Mixed Bacterial Fermentation Broth BPA-3

[0103] Bacillus subtilis SX-6, Pseudomonas SX-10, and Actinomyces SY-1 were respectively inoculated onto LB solid medium and cultured at 37 °C for 24 h to obtain activated strains; the activated strains were inoculated into LB liquid medium and cultured on a shaker at 37 °C and 180 rpm for 48 h to obtain single-bacterial culture broths, denoted as SX-6 culture broth (OD 600 = 2.2 - 2.3), SX-10 culture broth (OD 600 = 2.2 - 2.3), and SY-1 culture broth (OD 600 = 2.2 - 2.3).

[0104] The SX-6 culture broth, SX-10 culture broth, SY-1 culture broth, SX-6 culture broth:SX-10 culture broth with a volume ratio of 1:1, SX-6 culture broth:SY-1 culture broth with a volume ratio of 1:1, SX-10 culture broth:SY-1 culture broth with a volume ratio of 1:1, SX-6 culture broth:SX-10 culture broth:SY-1 culture broth with a volume ratio of 1:1:1, SX-6 culture broth:SX-10 culture broth:SY-1 culture broth with a volume ratio of 3:1:1, SX-6 culture broth:SX-10 culture broth:SY-1 culture broth with a volume ratio of 1:3:1, and SX-6 culture broth:SX-10 culture broth:SY-1 culture broth with a volume ratio of 1:1:3 were respectively inoculated into 100 mL of inorganic salt medium containing 200 mg / L of water-soluble aniline blue at an inoculation amount of 5% by volume and cultured under static conditions at 37 °C for 30 h, and samples were taken every 6 h. The collected samples were centrifuged at 8000 xg for 5 min, the supernatant was taken, and the absorbance at the maximum absorption wavelength of 598 nm was measured with a UV spectrophotometer and the decolorization rate was calculated. The decolorization rate was calculated according to formula (1). The specific decolorization results are shown in Figure 2 as follows.

[0105] Decolorization rate (%) = (A0 - A t ) / A t Formula (1)

[0106] A0 is the absorbance of the dye before treatment, and A t is the absorbance of the dye after treatment for t h.

[0107] Figure 2 It was shown that finally, when the volume ratio of the strains was Bacillus subtilis SX-6:Pseudomonas SX-10:Actinomyces SY-1 = 1:3:1 (denoted as mixed bacterial fermentation broth BPA-3), the decolorization effect on water-soluble aniline blue, a triphenylmethane dye, was the best, and more than 75% of water-soluble aniline blue could be decolorized within 30 h. Therefore, mixed bacterial fermentation broth BPA-3 was used for subsequent experiments.

[0108] Example 3: Decolorization of water-soluble aniline blue, a triphenylmethane dye, by mixed bacterial fermentation broth BPA-3 under different dissolved oxygen levels

[0109] The mixed bacterial fermentation broth BPA-3 from Example 2 was inoculated into an inorganic salt medium containing 200 mg / L water-soluble aniline blue. The dissolved oxygen conditions were set to static and shaking at 150 rpm, respectively. After culturing at 37°C for 30 h, samples were taken and centrifuged at 8000×g for 5 min. The supernatant and the bacterial cells were taken separately. The absorbance of the supernatant was measured at 598 nm using a UV spectrophotometer, and the decolorization rate was calculated according to the method in Example 2. The bacterial cells were diluted with an equal volume of sterile water for OD measurement 600 , and the results are shown in Figure 3 .

[0110] Finally, it was found that the mixed bacterial fermentation broth BPA-3 had a better decolorization effect on water-soluble aniline blue, a triphenylmethane dye, under static conditions, and its decolorization rate could reach 76.05% within 30 h. At the same time, its decolorization rate was also 71.27% under the condition of shaking at 150 rpm, indicating that the mixed bacterial fermentation broth BPA-3 has the potential to treat triphenylmethane dye wastewater under microaerobic and aerobic conditions

[0111] Example 4: Decolorization of water-soluble aniline blue, a triphenylmethane dye, by mixed bacterial fermentation broth BPA-3 under different yeast extract concentrations

[0112] The mixed bacterial fermentation broth BPA-3 from Example 2 was inoculated into inorganic salt media with different yeast extract contents containing 200 mg / L water-soluble aniline blue, a triphenylmethane dye. The yeast extract concentrations in the inorganic salt media were set to 0 g / L, 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, and 3 g / L, respectively. After culturing statically at 37°C for 30 h, samples were taken and centrifuged at 8000×g for 5 min. The supernatant and the bacterial cells were taken separately. The absorbance of the supernatant was measured at 598 nm using a UV spectrophotometer, and the decolorization rate was calculated according to the method in Example 2. The bacterial cells were diluted with an equal volume of sterile water for OD measurement 600 , and the results are shown in Figure 4 .

[0113] Finally, it was found that as the yeast extract concentration (0 g / L - 1 g / L) gradually increased, the decolorization rate of the mixed bacterial fermentation broth BPA-3 for water-soluble aniline blue, a triphenylmethane dye, also gradually increased. When the yeast extract concentration exceeded 1 g / L, its decolorization rate hardly increased anymore. Therefore, subsequent experiments were all carried out in an inorganic salt medium with 1 g / L yeast extract

[0114] Example 5: Decolorization of water-soluble aniline blue, a triphenylmethane dye, by mixed bacterial fermentation broth BPA-3 at different temperatures

[0115] The mixed bacterial fermentation broth BPA-3 of Example 2 was inoculated into an inorganic salt medium containing 200 mg / L of the water-soluble aniline blue of triphenylmethane dyes. The temperatures were set at 20 °C, 25 °C, 30 °C, and 37 °C respectively, and cultured for 30 h under static conditions. Samples were taken every 6 h, centrifuged at 8000×g for 5 min, the supernatant was taken, and its absorbance at 598 nm was measured with a UV spectrophotometer, and the decolorization rate was calculated according to the method of Example 2. The results are shown in Figure 5 shown below.

[0116] Finally, it was found that the decolorization efficiency of the mixed bacterial fermentation broth BPA-3 for the water-soluble aniline blue of triphenylmethane dyes increased with the increase of temperature, and the optimal decolorization temperature was 37 °C, and the decolorization rate within 30 h could reach over 75%.

[0117] Example 6: Decolorization of the water-soluble aniline blue of triphenylmethane dyes by the mixed bacterial fermentation broth BPA-3 at different pH values

[0118] The mixed bacterial fermentation broth BPA-3 of Example 2 was inoculated into an inorganic salt medium containing 200 mg / L of the water-soluble aniline blue of triphenylmethane dyes. The pH values of the inorganic salt medium were set at 3, 4, 5, 6, 7, 8, 9, 10, and 11 respectively. After culturing statically at 37 °C for 30 h, samples were taken and centrifuged at 8000×g for 5 min. The supernatant and the bacterial cells were taken respectively. The absorbance value of the supernatant at 598 nm was measured with a UV spectrophotometer, and the decolorization rate was calculated according to the method of Example 2. The bacterial cells were diluted with an equal volume of sterile water for measuring OD 600 , and the results are shown in Figure 6 shown below.

[0119] Finally, it was found that the optimal decolorization pH of the mixed bacterial fermentation broth BPA-3 for the water-soluble aniline blue of triphenylmethane dyes was 6 - 7, and the decolorization rate within 30 h could reach about 75%.

[0120] Example 7: Decolorization of the water-soluble aniline blue of triphenylmethane dyes by the mixed bacterial fermentation broth BPA-3 at different salinities

[0121] The mixed bacterial fermentation broth BPA-3 of Example 2 was inoculated into an inorganic salt medium containing 200 mg / L of the water-soluble aniline blue of triphenylmethane dyes with different NaCl contents. The NaCl concentrations in the inorganic salt medium were set at 0.5 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, and 60 g / L respectively. After culturing statically at 37 °C for 30 h, samples were taken and centrifuged at 8000×g for 5 min. The supernatant and the bacterial cells were taken respectively. The absorbance value of the supernatant at 598 nm was measured with a UV spectrophotometer, and the decolorization rate was calculated according to the method of Example 2. The bacterial cells were diluted with an equal volume of sterile water for measuring OD 600 , and the results are shown inFigure 7 as shown

[0122] Finally, it was found that the optimal decolorization salt concentration of the mixed bacterial fermentation broth BPA-3 for the triphenylmethane dye water-soluble aniline blue was 10 g / L, and its decolorization rate reached 84.34% after 30 h.

[0123] Example 8: Decolorization of water-soluble aniline blue of triphenylmethane dyes by mixed bacterial fermentation broth BPA-3

[0124] The mixed bacterial fermentation broth BPA-3 in Example 2 was inoculated into an inorganic salt medium containing water-soluble aniline blue of triphenylmethane dyes at different concentrations. The concentrations of water-soluble aniline blue in the inorganic salt medium were set to 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L respectively, and cultured for 30 h under static conditions at 37 °C. Samples were taken every 6 h, centrifuged at 8000×g for 5 min, the supernatant was taken, and its absorbance at 598 nm was measured with a UV spectrophotometer, and the decolorization rate was calculated according to the method in Example 2. The results are shown Figure 8 as shown <�

[0125] Finally, it was found that with the increase of the dye concentration, the decolorization rate of the mixed bacterial fermentation broth BPA-3 for water-soluble aniline blue gradually decreased. When the dye concentration was 200 mg / L, its decolorization rate reached 76.93%; while when the dye concentration increased to 500 mg / L, its decolorization rate was also 55.57%.

[0126] Example 9: Decolorization of different triphenylmethane dyes by mixed bacterial fermentation broth BPA-3

[0127] The mixed bacterial fermentation broth BPA-3 in Example 2 was inoculated into an inorganic salt medium containing different triphenylmethane dyes, and after static culture at 37 °C for 48 h, samples were taken and centrifuged at 8000×g for 5 min. The absorbance values at the maximum absorption wavelengths of the supernatant of each group were measured with a UV spectrophotometer and the decolorization rate was calculated. At the same time, the precipitated bacterial solution was transferred to a plate with a pipette gun and photographed. The results are shown Figure 9 as shown

[0128] The triphenylmethane dyes include basic fuchsin (20 mg / L), methyl violet (10 mg / L), acid blue 1 (20 mg / L), acid red 94 (20 mg / L), and malachite green (50 mg / L), and their maximum absorption wavelengths are 543 nm, 590 nm, 638 nm, 548 nm, and 617 nm respectively. Finally, it was found that the mixed bacterial fermentation broth BPA-3 had a certain decolorization effect on these triphenylmethane dyes. Among them, the decolorization rates for basic fuchsin and malachite green both reached over 90%, and the decolorization rates for methyl violet, acid blue 1, and acid red 94 were 37.42%, 24.47%, and 42.78% respectively. <�

[0129] The present invention has been described in connection with the above embodiments, but those skilled in the art should understand that the embodiments mentioned herein can be changed without departing from the spirit of the present invention. The above embodiments are merely exemplary and should not be used to limit the scope of the rights of the present invention.

Claims

1. Application of a mixed bacterial fermentation broth in decolorization of triphenylmethane dyes, characterized in that, The mixed bacterial fermentation broth is a mixture of the fermentation broths of Bacillus subtilis ( Bacillus subtilis ), Pseudomonas sp. ( Pseudomonas ), and Actinomycetes sp. ( Georgenia ) SY-1 in a volume ratio of 1:3:1; Bacillus subtilis ( Bacillus subtilis ), SX-6, was deposited at the China Center for Type Culture Collection on March 15, 2023, with the deposit number of CCTCC NO: M 2023336, and the address is Wuhan University, Wuhan, China, with the postal code of 430072; Pseudomonas ( Pseudomonas sp.) SX-10, deposited in the China Center for Type Culture Collection on May 19, 2022, with the deposit number of CCTCC NO: M 2022670, address: Wuhan University, Wuhan, China, Zip Code: 430072; Actinomycetes ( Georgenia sp.) SY-1, which is deposited in the China Center for Type Culture Collection. The deposit date is March 15, 2023, and the deposit number is CCTCC NO: M 2023335. The address is Wuhan University, Wuhan, China, and the postal code is 430072.

2. The application according to claim 1, wherein The application is carried out according to the following steps: inoculating the mixed bacterial fermentation broth into an inorganic salt culture medium containing triphenylmethane dyes at an inoculum concentration of 1-10% by volume, and culturing at 20-37°C, pH 3-11, standing or 150 rpm until the dye is degraded, thereby achieving the purpose of decolorization; the triphenylmethane dyes include water-soluble aniline blue, basic fuchsin, methyl violet, acid blue 1, acid red 94 or malachite green.

3. The application according to claim 2, characterized in that The concentration of the triphenylmethane dye added to the inorganic salt culture solution is 200 mg / L-500 mg / L; the inorganic salt culture solution is composed of: 0-6 g / L NaCl, 1 g / L NaH2PO4, 1 g / L Na2HPO4, 0.5 g / L MgSO4, 0.1 g / L CaCl2, 0-3 g / L yeast extract, and the solvent is water.

4. A Bacillus subtilis ( Bacillus subtilis ) SX-6 used in the application as claimed in claim 1, which is deposited in the China Center for Type Culture Collection. The deposit date is March 15, 2023, and the deposit number is CCTCC NO: M2023336. The address is Wuhan University, Wuhan, China, with the postal code 430072.

5. An actinomycetes ( Georgenia sp.) SY-1 used in the application according to claim 1, which is deposited in the China Center for Type Culture Collection. The deposit date is March 15, 2023, and the deposit number is CCTCC NO: M 2023335. The address is Wuhan University, Wuhan, China, and the postal code is 430072.

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