A humic acid-degrading bacterium and its application

Abstract

The present invention provides a humic acid degrading bacterium, which is a Bacillus firmus HA-X4 strain, which is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee with a deposit number of CGMCC 24269. The present invention also provides an application of the strain in degrading humic acid. The Bacillus firmus HA-X4 CGMCC 24269 strain of the present invention can effectively degrade humic acid in wastewater. When the amount of the humic acid degrading bacterial solution added is 4% by volume, the effect of degrading humic acid is best.

Description

Technical Field

[0001] The invention relates to a humic acid degrading bacterium and application thereof. Background Art

[0002] In order to curb the increasingly serious trend of urban water pollution, it is increasingly important to study the causes of black and smelly water bodies.

[0003] The blackening and odor formation of water bodies is the result of the combined effects of physics, chemistry, biology and other factors. Different water quality, environmental factors, hydraulic conditions, etc. will lead to different mechanisms of black and odor formation in water bodies. Fe, Mn, and S are considered to be the main causes of blackening and odor formation: after a large amount of organic matter enters the water body, an oxygen-deficient environment is quickly formed, and odorous substances such as hydrogen sulfide, amines, ammonia, and thiols and black substances such as FeS and MnS are produced during the anaerobic degradation process, causing the water body to turn black and smelly. In addition to H2S, NH3, amines, and thiols, scholars have also found the presence of a large amount of humic acid in the suspended matter of the polluted water body.

[0004] Humic acid is mainly composed of carbon, hydrogen, oxygen, nitrogen, sulfur and other elements. It is a polycondensate with no fixed composition structure. Due to the presence of functional groups such as carboxyl, alcohol hydroxyl, phenolic hydroxyl, quinone, semiquinone, methoxy, carbonyl, etc., humic acid has unique physical and chemical properties such as solubility, colloidal adsorption, good hydrophilicity, thermal decomposition, weak acidity, and photolysis. Humic acid in aquatic systems, like the "white blood cells" in human blood, cannot be unbalanced. An appropriate amount of humic acid can play a "green" effect and promote the carbon cycle, metal ion complexation, and migration and transformation of organic compounds in the water environment. When the humic acid content is too high, it will produce a strange color and odor, and combine with the chlorine produced by water treatment to generate carcinogenic chlorine-containing compounds; it will also complex with heavy metals and biocides, making their movement in the water body stronger; in addition, it will reduce the adsorption rate and adsorption equilibrium capacity of low-molecular organic chemicals and inorganic pollutants.

[0005] Humic acid has a complex structure and is one of the main components of water pollution. The presence of humic acid affects the taste of drinking water. During the chlorination process of drinking water, humic acid reacts with residual chlorine in the water to produce toxic disinfection byproducts such as trichloromethane (THMs) and haloacetic acids (HAAs), which have adverse effects on human health.

[0006] In recent years, people have paid more and more attention to the drinking water safety issues caused by the increase of natural organic pollution load in water, such as color and odor, and THMs produced by chlorine disinfection.

[0007] The use of advanced oxidation technology to treat humic acid has attracted much attention. Although it can effectively remove humic acid, it costs a lot. The main methods for controlling HA in water are physical methods and chemical oxidation methods. The physical method removes HA from water through processes such as coagulation, flocculation and adsorption, but this method only transfers HA to the solid phase and still requires subsequent solid waste treatment. The chemical oxidation method has attracted much attention because it can quickly decompose mineralized HA. Common chemical oxidation methods include photocatalysis, Fenton oxidation and electrochemical oxidation, but they generally have disadvantages such as harsh reaction conditions and complex operations. Microbial degradation has unique advantages such as low cost, high efficiency and no secondary pollution. At the same time, microbial degradation of organic matter also has the advantages of wide sources, convenient selection and simple operation. Therefore, it is of great practical significance to separate and screen humic acid efficient degradation bacteria in nature, and to explore the factors such as the initial concentration of humic acid, pH, and inoculation amount that affect the degradation performance of efficient humic acid degradation bacteria, so as to provide an application basis for establishing a technology that makes efficient humic acid degradation bacteria the dominant bacteria on biological activated carbon. Summary of the invention

[0008] In order to solve the problems existing in the prior art, the present invention provides a humic acid-degrading bacterium, and also provides the application of the strain in degrading humic acid.

[0009] The invention provides a humic acid degrading bacterium, which is a Bacillus firmus HA-X4 strain, preserved in the General Microbiological Center of China National Committee for Microbiological Culture Collection, with a preservation address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a preservation number of CGMCC No. 24269.

[0010] The invention also provides a humic acid-degrading bacterial liquid, which is obtained by expanding and culturing the Bacillus firmus HA-X4 strain.

[0011] Preferably, the expanded culture is specifically as follows: inoculating the Bacillus firmus strain HA-X4 into a beef extract peptone liquid culture medium and culturing it in a shaking incubator at 30° C. for 18-30 hours.

[0012] The present invention also provides the use of the above-mentioned humic acid-degrading bacteria or humic acid-degrading bacterial liquid in the preparation of products for degrading humic acid.

[0013] Preferably, when degrading humic acid, the humic acid-degrading bacterial liquid is added to the humic acid-containing material for degradation.

[0014] Preferably, the degradation temperature is 30°C.

[0015] Preferably, when degrading humic acid, the amount of the humic acid-degrading bacterial solution added is 2-6% by volume.

[0016] As a further preference, when degrading humic acid, the amount of the humic acid-degrading bacterial solution added is 4% by volume.

[0017] Preferably, the initial pH is 8 when degrading humic acid.

[0018] Preferably, when degrading humic acid, the degradation time is 70-75 hours, and the preferred degradation time is 72 hours.

[0019] The Bacillus firmus HA-X4 CGMCC 24269 strain of the present invention can effectively degrade humic acid in wastewater. When the added amount of the humic acid degrading bacterial liquid is 4% by volume, the effect of degrading humic acid is the best. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0021] Figure 1 This is the removal rate of humic acid by the five bacterial strains obtained in the initial screening.

[0022] Figure 2 This is the morphology of the humic acid-degrading bacteria HA-X4 obtained by repeated screening on PDA solid culture medium.

[0023] Figure 3 This is the phylogenetic tree of strain HA-X4.

[0024] Figure 4 This is the effect of the initial concentration of humic acid on the degradation performance of humic acid.

[0025] Figure 5 This is the effect of initial pH on the degradation performance of humic acid.

[0026] Figure 6 This is the effect of bacterial solution dosage on the humic acid degradation performance. DETAILED DESCRIPTION

[0027] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0028] Example 1

[0029] 1 Materials and methods

[0030] 1.1 Sampling

[0031] Soil samples were collected from the soil 5 cm below the rotten wood and the mud at the bottom of the artificial lake at the Pengjiaping Campus of Lanzhou University of Technology.

[0032] 1.2 Culture medium

[0033] (1) Enrichment medium: beef extract peptone medium: 5 g beef extract, 10 g peptone, 5 g NaCl, 1 L distilled water, 20 g agar, pH = 7.0-7.2.

[0034] (2) Acclimation medium 1: 0.5 g beef extract, 1 g peptone, 0.5 g NaCl, 0.005 g humic acid, 1 L distilled water, 20 g agar, pH = 7.0-7.2.

[0035] (3) Acclimation medium 2: 0.1 g beef extract, 0.1 g peptone, 0.05 g NaCl, 0.015 g humic acid, 1 L distilled water, 20 g agar, pH = 7.0-7.2.

[0036] (4) Acclimation medium 3: 0.03 g humic acid, 1 L distilled water, 20 g agar, pH = 7.0-7.2.

[0037] 1.3 Preparation of humic acid solution

[0038] Weigh 1g of humic acid, add it to 900mL of deionized water, stir it thoroughly to make it completely dissolved, first add deionized water to dilute to 1L, then adjust the pH to 7, and store it in a brown bottle for later use.

[0039] 1.4 Experimental instruments and equipment

[0040] Electronic balance; high-pressure steam sterilizer; sterile operating table; electric constant temperature incubator; UV-visible spectrophotometer; pH meter; optical microscope; ordinary refrigerator; adjustable electric heating plate; constant temperature air bath oscillator; scanning electron microscope; portable colorimeter.

[0041] 1.5 Choice of soil dilution

[0042] Take 10g of the collected soil sample, put it into a conical flask filled with 90mL of water, add 2-3 glass beads, shake it at 150r / min at room temperature for 1 hour, let it stand for 20min, and take the supernatant to get 10 -1 The soil suspension of dilution was diluted successively to obtain 10 -2 ,10 -3 , 10 -4 ,10 -5Dilution of soil suspension was performed, and 100 μL of each dilution of bacterial suspension was taken and spread on beef extract peptone medium. Three parallel samples were set for each dilution and cultured at 30°C for 3 days. The appropriate dilution was selected according to the growth of the bacteria so that different colonies could be easily picked out and purified. -3 At this dilution, the colonies in the culture medium are evenly distributed and it is easy to pick out a single colony, so 10 is selected. -3 The diluted soil suspension was used for subsequent bacterial screening experiments.

[0043] 1.6 Preliminary screening of humic acid-degrading bacteria

[0044] 10 -3 The bacteria on the beef extract peptone solid medium with a dilution degree were separated and purified to obtain 20 strains of bacteria. The separated and purified strains were inoculated in the acclimation medium 1, cultured at 30°C for a certain period of time, and the grown colonies were transferred to the acclimation medium 2 for gradient acclimation. After the colonies in the acclimation medium 2 grew out, they were transferred to the acclimation medium 3 for transfer screening and culture. Five humic acid-degrading bacteria with larger colonies were obtained by preliminary screening, and they were numbered HA-X1, HA-X2, HA-X3, HA-X4, and HA-X5, respectively.

[0045] 1.7 Rescreening of Humic Acid Degrading Bacteria

[0046] The five humic acid-degrading bacteria, HA-X1, HA-X2, HA-X3, HA-X4, and HA-X5, were prepared into OD 600 The bacterial suspension of 0.6 was added into the humic acid wastewater with an initial concentration of 25 mg / L and an initial pH of 7 at a volume ratio of 2%. The culture was carried out in a shaking table at 150 r / min and 30°C. A control group and three parallel samples were set up. The UV 254 (UV 254 It indicates the absorption value of organic pollutants such as aromatic hydrocarbons in water at an ultraviolet wavelength of 253.7nm. These compounds are usually the main components of natural organic matter with complex structures, and the absorbance at this wavelength can be used to indirectly indicate the concentration of these compounds. The experimental results show that the degradation rate of humic acid is the highest at 72h. At this time, the degradation rates of humic acid by HA-X1, HA-X2, HA-X3, HA-X4, and HA-X5 are 43.18%, 28.21%, 31%, 47.59%, and 19.69%, respectively. Therefore, HA-X4 with a degradation rate of more than 45% was finally selected, and the measurement time of subsequent experiments was set to 72h.

[0047] Figure 1 This is the removal rate of humic acid by the five bacterial strains obtained in the initial screening.

[0048] 1.8 Preparation of bacterial solution

[0049] The strain HA-X4 in step 1.6 was inoculated into beef extract peptone liquid medium and cultured in a shaking incubator at 30°C. After 24 hours, the fresh culture was added into a centrifuge tube filled with sterile saline and the bacterial solution was diluted to a concentration of 10 8 CFU·mL -1 , and store in a 4℃ refrigerator for later use.

[0050] 1.9 Morphological observation and identification of humic acid-degrading bacteria

[0051] 1.9.1 Morphological observation

[0052] It can be seen from observation that the colonies of HA-X4 on the beef extract peptone medium are in the shape of a wheel, with a smooth apricot-colored surface and a translucent middle.

[0053] Figure 2 This is the morphology of the humic acid-degrading bacteria HA-X4 obtained by repeated screening on PDA solid culture medium.

[0054] 1.9.2 Identification

[0055] HA-X4 was measured, and the 16S rDNA homology of the strain "HA-X4" with multiple strains was 100.00%. According to the literature, the 16S rDNA sequences of Bacillus firmus and Bacillus oceanisediminis are very similar, so further morphological differentiation is required, and HA-X4 is finally identified as Bacillus firmus. The strain Bacillus firmus HA-X4 was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on January 10, 2022, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 24269.

[0056] Figure 3 This is the phylogenetic tree of strain HA-X4.

[0057] The 16S rDNA gene sequence of HA-X4 is:

[0058]

[0059] 2 Degradation characteristics of humic acid-efficient degradation bacteria Bacillus firmus CGMCC 24269

[0060] 2.1 Degradation experiment of humic acid-degrading bacteria Bacillus firmus CGMCC 24269

[0061] 2.1.1 Initial concentration of humic acid

[0062] Humic acid wastewater with concentrations of 5 mg / L, 15 mg / L, 25 mg / L, 35 mg / L, 45 mg / L and 55 mg / L were prepared respectively, the pH was adjusted to 7, 2% volume of CGMCC 24269 bacterial solution was added respectively, and the mixture was cultured in a shaking incubator at 30°C and 150 r / min. UV254, VIS400 and colorimetry were measured after 72 hours.

[0063] 2.1.2 Initial pH

[0064] Humic acid wastewater with an initial humic acid concentration of 25 mg / L and an initial pH of 3, 4, 5, 6, 7, 8, 9, and 10 was prepared respectively, and 2% volume of CGMCC 24269 bacterial solution was added respectively. The mixture was cultured in a shaking incubator at 30°C and 150 r / min. UV254, VIS400 and colorimetry were measured for 72 hours.

[0065] 2.1.3 Bacterial solution dosage

[0066] Humic acid wastewater with an initial humic acid concentration of 25 mg / L and an initial pH of 8 was prepared, and 0.5%, 1%, 2%, 3%, 4%, 5%, and 6% of the volume of CGMCC 24269 bacterial solution were added respectively. The water was cultured in a shaking incubator at 30°C and 150 r / min. The UV254, VIS400 and colorimetry were measured for 72 hours.

[0067] 2.2 Degradation experimental results of humic acid highly efficient degradation bacteria Bacillus firmus CGMCC 24269.

[0068] 2.2.1 Initial concentration of humic acid

[0069] When the initial concentration of humic acid was 25 mg / L, the degradation rate of humic acid by HA-X4 reached the highest, and the removal rates of UV254, VIS400 (using ultraviolet-visible spectrophotometry with ultrapure water as blank control to measure the absorbance of the sample solution at 400nm. VIS400 is used to characterize the content of chromogenic groups in complex organic matter) and chromaticity were 43.26%, 52.02% and 62.56%, respectively.

[0070] Figure 4 This is the effect of the initial concentration of humic acid on the degradation performance of humic acid.

[0071] 2.2.2 Initial pH

[0072] When the initial pH is acidic, the removal rates of UV254, VIS400 and chromaticity are high, and when the initial pH is 3, the removal rates of UV254, VIS400 and chromaticity are 81.4%, 90.32% and 97.63% respectively, but the removal rate of TCOD (total chemical oxygen demand) is only 30.28%, which is basically not removed. Therefore, when the pH is acidic, the high removal rates of UV254, VIS400 and chromaticity are caused by the precipitation of humic acid under acidic conditions, not the degradation of humic acid by strain HA-X4. When the initial pH is 8, the removal rates of UV254, VIS400 and chromaticity are 47.34%, 54.39% and 65.29% respectively, and the removal rate of TCOD reaches the highest, which is 52.79%. Therefore, the suitable pH for the growth of strain HA-X4 is 8.

[0073] Figure 5 This is the effect of initial pH on the degradation performance of humic acid.

[0074] 2.2.3 Bacterial solution dosage

[0075] When the bacterial solution dosage was 4% by volume, the degradation rate of humic acid by HA-X4 reached the highest, and the removal rates of UV254, VIS400, and chromaticity were 54.67%, 61.86%, and 72.83%, respectively.

[0076] Figure 6 This is the effect of bacterial solution dosage on the humic acid degradation performance.

[0077] 2.3 Conclusion

[0078] Prepare 25 mg / L humic acid wastewater, adjust the pH to 8, add 4% volume of Bacillus firmus CGMCC24269 bacterial solution, and culture at 30°C and 150 r / min in a shaking table. Under the above conditions, the degradation rate of humic acid is optimal.

[0079] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. Sequence Listing <110> Lanzhou University of Technology <120> A humic acid-degrading bacterium and its application <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1378 <212> DNA <213> Bacillus firmus <400> 1 tgggagcttg ctccctgaag tcagcggcgg acgggtgagt aacacgtggg caacctgcct 60 gtaagactgg gataactccg ggaaaccggg gctaataccg gataattctt tccctcacat 120 gagggaaagc tgaaagatgg tttcggctat cacttacaga tgggcccgcg gcgcattagc 180 tagttggtga ggtaacggct caccaaggca acgatgcgta gccgacctga gagggtgatc 240 ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt agggaatctt 300 ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag tgatgaaggt ttcggatcg 360 taaaactctg ttgttaggga agaacaagta ccggagtaac tgccggtacc ttgacggtac 420 ctaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag 480 cgttgtccgg aattattggg cgtaaagcgc gcgcaggcgg ttccttaagt ctgatgtgaa 540 agccccggc tcaaccgggg agggtcattg gaaactgggg aacttgagtg cagaagagaa 600 gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg tggaggaaca ccagtggcga 660 aggcgactct ttggtctgta actgacgctg aggcgcgaaa gcgtggggag caaacaggat 720 tagataccct ggtagtccac gccgtaaacg atgagtgcta agtgttagag ggtttccgcc 780 ctttagtgct gcagcaaacg cattaagcac tccgcctggg gagtacggcc gcaaggctga 840 aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc 900 aacgcgaaga accttaccag gtcttgacat ctcctgacaa ccctagagat agggcgttcc 960 ccttcggggg acaggatgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt 1020 tgggttaagt cccgcaacga gcgcaaccct tgatcttagt tgccagcatt cagttgggca 1080 ctctaaggtg actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg 1140 ccccttatga cctgggctac acacgtgcta caatggatgg tacaaagggc tgcgagaccg 1200 cgaggttaag cgaatcccat aaaaccattc tcagttcgga ttgcaggctg caactcgcct 1260 gcatgaagcc ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg 1320 gccttgtaca caccgcccgt cacaccacga gagtttgtaa cacccgaagt cggtgggg 1378

Claims

1. A humic acid-degrading bacterium, characterized in that: The humic acid degrading bacteria is Bacillus firmus HA-X4 strain, which is preserved in the General Microbiological Center of China Microbiological Culture Collection Administration, with the preservation number of CGMCC 24269.

2. Humic acid degradation bacterial liquid, characterized by: The invention is obtained by expanding and culturing the Bacillus firmus HA-X4 strain described in claim 1.

3. The humic acid-degrading bacterial liquid according to claim 2, characterized in that: The expanded culture specifically comprises: inoculating the Bacillus firmus strain HA-X4 according to claim 1 into a beef extract peptone liquid culture medium and culturing it in a shaking table at 30° C. for 18-30 hours.

4. Use of the humic acid-degrading bacteria according to claim 1 or the humic acid-degrading bacteria liquid according to claim 2 in preparing a product of degrading humic acid.

5. The use according to claim 4, characterized in that: When degrading humic acid, the humic acid-degrading bacterial liquid is added to the humic acid-containing material for degradation.

6. The use according to claim 5, characterized in that: The degradation temperature is 30°C.

7. The use according to any one of claims 4 to 6, characterized in that: When degrading humic acid, the added amount of the humic acid-degrading bacterial solution is 2-6% by volume.

8. The use according to any one of claim 7, characterized in that: When degrading humic acid, the added amount of the humic acid-degrading bacterial solution is 4% by volume.

9. The use according to any one of claims 4 to 6, characterized in that: When degrading humic acid, the initial pH is 8.

10. The use according to any one of claims 4 to 6, characterized in that: When degrading humic acid, the degradation time is 70-75h.

11. The use according to claim 10, characterized in that: When degrading humic acid, the degradation time is 72h.

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