Humin-mediated 2, 4, 6-tribromophenol biodegradation preparation and application thereof

Abstract

The invention relates to the field of organic matter degradation, and discloses a humin-mediated 2, 4, 6-tribromophenol biodegradation preparation and application thereof. The biodegradation preparation takes an anaerobic mineral salt culture medium as a matrix, and the matrix contains 25-35 g / L of humin, 1-2 mL / L of an organic acid salt electron donor, 1-2 mL / L of a vitamin solution and 1.5-2.5 mg / L of anaerobic microorganisms. The biodegradation preparation disclosed by the invention contains humin, so that the degradation efficiency of anaerobic microorganisms on 2, 4, 6-tribromophenol can be greatly improved. Humin has the advantages of being environmentally friendly, free of toxicity, wide in source and good in stability in a degradation system.

Description

Technical Field

[0001] The present invention relates to the field of organic matter degradation, and particularly to a humin-mediated 2,4,6-tribromophenol biodegradation preparation and its application. Background Art

[0002] 2,4,6-Tribromophenol (TBP) is one of the most widely produced brominated phenols and is an intermediate in the synthesis of brominated flame retardants. It is also used as an insecticide and a preservative for wood and paper. The structure of 2,4,6-tribromophenol is stable and it is not easily degraded naturally. It can diffuse and migrate in environments such as water bodies, the atmosphere, and soil, directly endangering ecological safety and human health. In recent decades, the extensive use of brominated phenol compounds has led to their widespread presence in environmental media such as water bodies and soil, and they have also been found in mammals and even the human body. Therefore, it is urgent to develop practical remediation technologies to solve the environmental pollution caused by TBP.

[0003] Currently, the methods for degrading bromophenol pollution generally include physical treatment (landfill sealing, high-temperature incineration, adsorption extraction, etc.), chemical treatment (oxidation-reduction method), and biodegradation. Through physical treatment methods, the pollution problem generally cannot be fundamentally solved. For example, the landfill method is only suitable for disposing of a small amount of pollutants that are not easily leaked. Even so, attention must be paid to preventing leakage, and at the same time, the leachate must be monitored and controlled at both ends to improve the long-term safety of the landfill. Highly efficient chemical treatment methods have high requirements for the process, and generally, the chemical reagent consumption is large and the cost is expensive. Therefore, biodegradation treatment is currently considered the most promising and potential method as the cleanest and most economical treatment method. This technology does not produce secondary pollution, has a low economic cost, and is easy to implement. However, the current biodegradation method for 2,4,6-tribromophenol has low efficiency and the effect is still not ideal.

[0004] In summary, it is necessary to develop an efficient biodegradation method specifically for 2,4,6-tribromophenol. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a humin-mediated 2,4,6-tribromophenol biodegradation preparation and its application. The biodegradation preparation of the present invention contains humin, which can greatly improve the degradation efficiency of anaerobic microorganisms on 2,4,6-tribromophenol. Humin has the advantages of environmental friendliness, non-toxicity, wide sources, and good stability in the degradation system.

[0006] The specific technical solution of the present invention is as follows: In a first aspect, the present invention provides a humin-mediated biodegradation preparation for 2,4,6-tribromophenol, using an anaerobic mineral salt medium as a substrate, and the substrate contains: 25-35 g / L of humin, 1-2 mL / L of organic acid salt electron donor, 1-2 mL / L of vitamin solution, and 1.5-2.5 mg / L of anaerobic microorganisms.

[0007] In the above formula of the biodegradation preparation for 2,4,6-tribromophenol of the present invention, anaerobic microorganisms can biodegrade 2,4,6-tribromophenol under anaerobic conditions. However, under ordinary conditions (without humin), the biodegradation efficiency of most anaerobic microorganisms for 2,4,6-tribromophenol is relatively low, so it is difficult to achieve efficient degradation, which limits its large-scale application. The present invention accidentally discovered that in the presence of humin, the degradation efficiency of anaerobic microorganisms for 2,4,6-tribromophenol can be greatly improved.

[0008] Through further research, the present invention found that the mechanism of action of humin is as follows: The anaerobic degradation of tribromophenol is an electron-consuming process. Microorganisms use organic acid salts (such as sodium lactate) as electron donors and tribromophenol as electron acceptors through respiration, thereby causing degradation. Humin can act as a solid electron mediator in the above degradation system to promote the biodegradation ability of anaerobic microorganisms for tribromophenol.

[0009] In addition, compared with existing solid electron mediators, humin also has the following advantages: (1) It is naturally made, environmentally friendly and non-toxic, and widely exists in the natural environment; (2) Humin is a solid insoluble substance, which is insoluble in both acids and bases. Compared with the currently common water-soluble electron mediators, it is not easily lost during biodegradation, has good pH adaptability to the environment, and its chemical structure is extremely stable and can maintain its activity for a long time.

[0010] The present invention also found through subsequent experiments that the above biodegradation preparation of the present invention has a certain specificity for 2,4,6-tribromophenol. When attempting to use this biodegradation preparation to biodegrade other organic pollutants (such as tetrachloroethylene, etc.), it was found that there was no obvious degradation effect.

[0011] In addition, the role of the anaerobic mineral salt medium in the present invention is: in anaerobic culture, anaerobic mineral salts are essential nutrient components for cells to maintain life activities, mainly including Na + , K + , Ca 2+ , Mg 2+ , Cl － , PO4 3- , SO4 2- , HCO3 －etc. Their main function is to maintain the osmotic pressure balance of the cell culture medium and participate in the cell's metabolic activities. In addition, by providing elements such as sodium, potassium, and calcium, they help the cells regulate the cell membrane function. For example, Na + is the most important cation in the extracellular fluid and is crucial for maintaining the constancy of the osmotic pressure.

[0012] The role of vitamins in the present invention is as follows: Vitamins are a class of organic compounds required for the growth of microorganisms. They play the role of coenzymes or cofactors in the process of cell metabolism and are crucial for the growth and development of microorganisms. In the culture medium of anaerobic microorganisms, vitamins are usually one of the essential nutrients, which helps the growth and metabolic activities of microorganisms. Vitamins are also crucial for the growth and metabolism of anaerobic microorganisms, and they may participate in various biological processes such as the energy metabolism and cell synthesis of microorganisms.

[0013] Preferably, the anaerobic microorganism is selected from one of the following three compositions: F0: Clostridium drakei with a ratio of (9 - 11):(3 - 4):(2.6 - 3.2):(2.4 - 2.8):(2.2 - 2.6):(2 - 2.4):(2 - 2.4):(1.5 - 1.9), Halomicrobium sp. LC1Hm, Anaerohalosphaera lusitana, Dermatophilus congolensis, Candidatus Enterovibrio luxaltus, Jeotgalibaca dankookensis, Halodesulfurarchaeum formicicum, and Halomicrobium mukohataei.

[0014] F25: Streptomyces sp. SAT1, an endophytic actinomycete of plants, Megasphaera hexanoica, Megasphaera massiliensis, Streptomyces sp. Sge12, Dermabacter jinjuensis, Halomicrobium sp. LC1Hm, Anaerohalosphaera lusitana, and Halodesulfurarchaeum formicicum in a ratio of (8 - 10) : (4 - 6) : (3.5 - 5.5) : (3 - 5) : (2 - 4) : (2 - 4) : (2 - 3) : (1 - 3).

[0015] F150: Streptomyces sp. Sge12, Halomicrobium sp. LC1Hm, Jeotgalibaca dankookensis, Halodesulfurarchaeum formicicum, Dermabacter jinjuensis, Denitratisoma sp. DHT3, and Halomicrobium mukohataei in a ratio of (4 - 5) : (3 - 5) : (2 - 4) : (2 - 3) : (1 - 3) : (1 - 3) : (1 - 3).

[0016] Preferably, the method for preparing humin is as follows: Take soil sediment, perform alkali extraction with alkali, centrifuge and discard the supernatant; repeat the alkali extraction until the color of the supernatant is light tea color; treat the obtained precipitate with HF, centrifuge, wash the precipitate until it is neutral, centrifuge, wash with water, and collect the precipitated humin.

[0017] Preferably, the organic acid salt electron donor is sodium lactate.

[0018] Preferably, the composition of the anaerobic mineral salt medium includes: 5 - 15 mL / L of salts stock solution, 0.05 - 0.15 mL / L of Se / W stock solution, 0.1 - 1 mL / L of trace element solution, 2 - 2.5 g / L of Tris - ethylsulfonic acid, 0.1 - 1 mL / L of resazurin, and the balance is water.

[0019] Preferably, the composition of the salts stock solution includes: 70 - 80 g / L of NH4Cl, 1 - 1.5 g / L of CaCl2•2H2O, 45 - 55 g / L of MgCl2•6H2O, 15 - 25 g / L of K2HPO4, 90 - 110 g / L of NaCl, 25 - 35 g / L of KCl, and the balance is water.

[0020] Preferably, the composition of the Se / W solution includes: 3 - 7 g of NaOH, 0.04 - 0.08 g of NaSeO3•5H2O, 0.06 - 0.1 g of Na2WO4•2H2O, and the balance is water.

[0021] Preferably, the composition of the trace element solution includes: 15 - 25 mL of 20 - 30 wt% HCl, 2 - 4 g of FeCl2•4H2O, 0.3 - 0.4 g of CoCl2•6H2O, 0.1 - 0.3 g of MnCl2•4H2O, 0.1 - 0.2 g of ZnCl2, 0.01 - 0.02 g of H3BO3, 0.002 - 0.006 g of CuCl2•2H2O, 0.02 - 0.06 g of NiCl2•6H2O, 0.07 - 0.08 g of Na2MoO4•2H2O, and the balance is water.

[0022] Preferably, the composition of the vitamin solution includes: 0.06 - 0.1 g of biotin, 0.1 - 0.3 g of nicotinic acid, 0.1 - 0.3 g of +D - calcium pantothenate, 0.002 - 0.006 g of VB 12 0.002 - 0.006 g, 0.1 - 0.3 g of p - aminobenzoic acid, 0.1 - 0.3 g of lipoic acid, 0.1 - 0.3 g of thiamine, 0.3 - 0.5 g of pyridoxal hydrochloride, 0.1 - 0.3 g of + nicotinamide, and the balance is water.

[0023] In a second aspect, the present invention provides the application of the above - mentioned biodegradation preparation in degrading 2,4,6 - tribromophenol: Mix 2,4,6 - tribromophenol with the 2,4,6 - tribromophenol biodegradation preparation and carry out degradation in a dark environment.

[0024] Preferably, the concentration of 2,4,6 - tribromophenol in the 2,4,6 - tribromophenol biodegradation preparation is 20 - 30 μmol / L.

[0025] Preferably, the temperature of the degradation is 25 - 35 °C.

[0026] Compared with the prior art, the beneficial effects of the present invention are: (1) The biodegradation preparation of the present invention contains humin, which can significantly improve the degradation efficiency of anaerobic microorganisms on 2,4,6 - tribromophenol.

[0027] (2) Humins are naturally made, environmentally friendly and non-toxic, and widely present in the natural environment. Therefore, they are safe, harmless and low-cost.

[0028] (3) Humins are a kind of solid insoluble substances, which are insoluble in both acids and bases. Compared with the currently common water-soluble electron mediators, they are not easily lost during biodegradation, have good pH adaptability to the environment, and their chemical structure is extremely stable, and can maintain activity for a long time.

[0029] (4) The biodegradation preparation of the present invention has a certain specificity for 2,4,6-tribromophenol. When using this biodegradation preparation to biodegrade other organic pollutants (such as tetrachloroethylene, etc.), it is found that there is no obvious degradation effect. Description of the Drawings

[0030] Figure 1 It is the degradation result diagram of Example 1.

[0031] Figure 2 It is the degradation result diagram of Example 2.

[0032] Figure 3 It is the degradation result diagram of Example 3.

[0033] Figure 4 It is the degradation result diagram of Comparative Example 1.

[0034] Figure 5 It is the degradation result diagram of Comparative Example 2.

[0035] Figure 6 It is the degradation result diagram of Application Example 2. Detailed Embodiments

[0036] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following examples are all conventional methods in the art unless otherwise specified.

[0037] General Embodiment First aspect, the present invention provides a humin-mediated 2,4,6-tribromophenol biodegradation preparation, using anaerobic mineral salt medium as the matrix, and the matrix contains: humins 25 - 35 g / L, organic acid salt electron donor 1 - 2 mL / L, vitamin solution 1 - 2 mL / L, anaerobic microorganisms 1.5 - 2.5 mg / L.

[0038] In some specific implementation cases, the anaerobic microorganisms include Clostridium drakei, Halomicrobium sp. LC1Hm, Anaerohalosphaera lusitana, Dermatophilus congolensis, Candidatus Enterovibrio luxaltus, Jeotgalibaca dankookensis, Halodesulfurarchaeum formicicum, and Halomicrobium mukohataei in a ratio of (9-11):(3-4):(2.6-3.2):(2.4-2.8):(2.2-2.6):(2-2.4):(2-2.4):(1.5-1.9).

[0039] In some specific implementation cases, the anaerobic microorganisms include Streptomyces sp.SAT1, Megasphaera hexanoica, Megasphaera massiliensis, Streptomyces sp. Sge12, Dermabacter jinjuensis, Halomicrobiumsp. LC1Hm, Anaerohalosphaera lusitana, and Halodesulfurarchaeum formicicum in a ratio of (8-10):(4-6):(3.5-5.5):(3-5):(2-4):(2-4):(2-3):(1-3).

[0040] In some specific implementation cases, the anaerobic microorganisms include Streptomyces sp. Sge12, Halomicrobium sp.LC1Hm, Jeotgalibaca dankookensis, Halodesulfurarchaeum formicicum, Dermabacter jinjuensis, Denitratisoma sp. DHT3, and Halomicrobium mukohataei in a ratio of (4-5):(3-5):(2-4):(2-3):(1-3):(1-3):(1-3).

[0041] In some specific embodiments, the preparation method of the humin is as follows: soil sediment is taken, alkali extraction is carried out with alkali, centrifuged and the supernatant is discarded; the alkali extraction is repeated until the color of the supernatant is light tea color; the obtained precipitate is treated with HF, centrifuged, the precipitate is washed to neutral, centrifuged, washed with water, and the precipitated humin is collected.

[0042] In some specific embodiments, the organic acid salt electron donor is sodium lactate.

[0043] In some specific embodiments, the composition of the anaerobic mineral salt medium includes: 5 - 15 mL / L of salts stock solution, 0.05 - 0.15 mL / L of Se / W stock solution, 0.1 - 1 mL / L of trace element solution, 2 - 2.5 g / L of Tris-ethanesulfonic acid, 0.1 - 1 mL / L of resazurin, and the balance is water.

[0044] In some specific embodiments, the composition of the salts stock solution includes: 70 - 80 g / L of NH4Cl, 1 - 1.5 g / L of CaCl2•2H2O, 45 - 55 g / L of MgCl2•6H2O, 15 - 25 g / L of K2HPO4, 90 - 110 g / L of NaCl, 25 - 35 g / L of KCl, and the balance is water.

[0045] In some specific embodiments, the composition of the Se / W solution includes: 3 - 7 g of NaOH, 0.04 - 0.08 g of NaSeO3•5H2O, 0.06 - 0.1 g of Na2WO4•2H2O, and the balance is water.

[0046] In some specific embodiments, the composition of the trace element solution includes: 15 - 25 mL of 20 - 30 wt% HCl, 2 - 4 g of FeCl2•4H2O, 0.3 - 0.4 g of CoCl2•6H2O, 0.1 - 0.3 g of MnCl2•4H2O, 0.1 - 0.2 g of ZnCl2, 0.01 - 0.02 g of H3BO3, 0.002 - 0.006 g of CuCl2•2H2O, 0.02 - 0.06 g of NiCl2•6H2O, 0.07 - 0.08 g of Na2MoO4•2H2O, and the balance is water.

[0047] In some specific embodiments, the composition of the vitamin solution includes: 0.06 - 0.1 g of biotin, 0.1 - 0.3 g of nicotinic acid, + 0.1 - 0.3 g of calcium D-pantothenate, 0.002 - 0.006 g of VB 12 0.002 - 0.006 g, 0.1 - 0.3 g of p-aminobenzoic acid, 0.1 - 0.3 g of lipoic acid, 0.1 - 0.3 g of thiamine, 0.3 - 0.5 g of pyridoxal hydrochloride, + 0.1 - 0.3 g of nicotinamide, and the balance is water.

[0048] In a second aspect, the present invention provides the application of the above biodegradation preparation in degrading 2,4,6-tribromophenol: mixing 2,4,6-tribromophenol with a 2,4,6-tribromophenol biodegradation preparation and carrying out degradation in a dark environment.

[0049] In some specific implementation cases, the concentration of 2,4,6-tribromophenol in the 2,4,6-tribromophenol biodegradation preparation is 20 - 30 μmol / L.

[0050] In some specific implementation cases, the temperature of the degradation is 25 - 35 °C.

[0051] Specific examples and comparative examples The technical solution of the present invention will be further specifically described below through specific examples in conjunction with the accompanying drawings.

[0052] Example 1 A humin-mediated 2,4,6-tribromophenol biodegradation preparation is prepared by mixing the following components: 1 L of anaerobic mineral salt medium, 2 mg of anaerobic microorganisms, 33.33 g of humin, 1.532 mL of sodium lactate electron donor, and 1.67 mL of vitamin solution.

[0053] The above anaerobic microorganisms are a mixed flora enriched and cultured from the sediment at the 0 cmbsf layer of the columnar sediment at a depth of 1290 - 1295 m in the Haima cold seep area (110°28ʹE, 16°43ʹN) of the Qiongdongnan Sea, and it includes Clostridium drakei, Halomicrobium sp. LC1Hm, Anaerohalosphaera lusitana, Dermatophiluscongolensis, Candidatus Enterovibrio luxaltus, Jeotgalibaca dankookensis, Halodesulfurarchaeum formicicum, and Halomicrobium mukohataei in a ratio of 10.2:3.5:2.9:2.6:2.4:2.2: 2.2:1.7.

[0054] The formulation of the above anaerobic mineral salt medium is as follows: for every 1 L of the medium, add 10 mL of salts stock solution (100×), 0.1 mL of Se / W stock solution (10000×), 0.5 mL of trace elements (2000×), 2.292 g of Tris-ethanesulfonic acid, 0.5 mL of resazurin, and make up the volume with water.

[0055] The composition of each liter of salts stock solution (100×) is: NH4Cl 74 g / L, CaCl2•2H2O 1.20 g / L, MgCl2•6H2O 50 g / L, K2HPO4 20 g / L, NaCl 100 g / L, KCl 30 g / L, and make up the volume with water.

[0056] The composition of each liter of Se / W solution is: NaOH 5 g, NaSeO3•5H2O 0.06 g, Na2WO4•2H2O 0.08 g, and make up the volume with water.

[0057] The composition of each liter of trace element solution (2000×) is: HCl (25%, ω) 20 mL, FeCl2•4H2O 3 g, CoCl2•6H2O 0.38 g, MnCl2•4H2O 0.20 g, ZnCl2 0.14 g, H3BO3 0.012 g, CuCl2•2H2O 0.004 g, NiCl2•6H2O 0.04 g, Na2MoO4•2H2O 0.072 g, and make up the volume with water.

[0058] The composition of each liter of vitamin solution (1000×) is: biotin 0.08 g, nicotinic acid 0.20 g, +D-calcium pantothenate 0.20 g, VB 12 0.004 g, p-aminobenzoic acid 0.20 g, lipoic acid 0.20 g, thiamine 0.20 g, pyridoxal hydrochloride 0.40 g, +nicotinamide 0.20 g, and make up the volume with water.

[0059] The preparation method of the above humin is as follows: Weigh 1.5 kg of soil sediment into 6 250 mL centrifuge bottles, add 150 mL of 0.1 M NaOH solution, place it in a shaker for 24 h for alkaline extraction. After 24 h, centrifuge and discard the supernatant. Alkaline treatment should be carried out at least 4 times (the shaking time for the subsequent times can be extended to 48 h and 72 h respectively) until the color of the supernatant of alkaline extraction becomes light tea color. Centrifuge, discard the supernatant, treat the precipitate with 2% HF acid twice, and discard the supernatant after centrifugation. Wash the precipitate to neutral (it can be slowly adjusted to neutral with dilute hydrochloric acid during washing, and after adjusting the pH each time, place it in a shaker to make it react and mix thoroughly), centrifuge, wash with water, collect the precipitate, which is humin. The centrifugation conditions are: 5000 rpm, 15 min, shaker incubator: 170 rpm, 30°C.

[0060] Example 2 A humin-mediated biodegradation preparation for 2,4,6-tribromophenol, which is made by mixing the following components: 1 L of anaerobic mineral salt medium, 2 mg of anaerobic microorganisms, 33.33 g of humin, 1.532 mL of sodium lactate electron donor, and 1.67 mL of vitamin solution.

[0061] The above-mentioned anaerobic microorganisms are a mixed flora obtained by enrichment culture of the sediment at the 25 cmbsf layer of the columnar sediment at a depth of 1290 - 1295 m in the hippocampal cold seep area of the Qiongdongnan Sea (110°28ʹE, 16°43ʹN). It includes Streptomyces sp. SAT1, an endophytic streptomycete of plants, Megasphaera hexanoica, Megasphaera massiliensis, Streptomyces sp. Sge12, Dermabacter jinjuensis, Halomicrobium sp. LC1Hm, Anaerohalosphaera lusitana, and Halodesulfurarchaeum formicicum in a ratio of 9.4:4.9:4.5:4:2.7:2.6:2.5:2.1.

[0062] The formula of the above-mentioned anaerobic mineral salt medium is: add 10 mL of salts stock solution (100×), 0.1 mL of Se / W stock solution (10000×), 0.5 mL of trace elements (2000×), 2.292 g of Tris ethyl sulfonate, 0.5 mL of resazurin, and the balance is water to each 1 L of the medium.

[0063] The composition of each liter of salts stock solution (100×) is: NH4Cl 74 g / L, CaCl2•2H2O 1.20 g / L, MgCl2•6H2O 50 g / L, K2HPO4 20 g / L, NaCl 100 g / L, KCl 30 g / L, and the balance is water.

[0064] The composition of each liter of Se / W solution is: NaOH 5 g, NaSeO3•5H2O 0.06 g, Na2WO4•2H2O 0.08 g, and the balance is water.

[0065] The composition of each liter of trace element solution (2000×) is as follows: 20 mL of HCl (25%, ω), 3 g of FeCl2•4H2O, 0.38 g of CoCl2•6H2O, 0.20 g of MnCl2•4H2O, 0.14 g of ZnCl2, 0.012 g of H3BO3, 0.004 g of CuCl2•2H2O, 0.04 g of NiCl2•6H2O, 0.072 g of Na2MoO4•2H2O, with the balance being water.

[0066] The composition of each liter of vitamin solution (1000×) is as follows: 0.08 g of biotin, 0.20 g of nicotinic acid, + 0.20 g of D-calcium pantothenate, 0.004 g of VB 12 0.20 g of p-aminobenzoic acid, 0.20 g of lipoic acid, 0.20 g of thiamine, 0.40 g of pyridoxal hydrochloride, + 0.20 g of nicotinamide, with the balance being water. The vitamin stock solution needs to be added through a 0.22 μm sterile filter membrane.

[0067] The preparation method of the above-mentioned humin is as follows: Weigh 1.5 kg of soil sediment into 6 250 mL centrifuge bottles, add 150 mL of 0.1 M NaOH solution, place it in a shaker for 24 h for alkali extraction, and after 24 h, centrifuge and discard the supernatant. The alkali treatment is carried out at least 4 times (the shaker oscillation time for the subsequent times can be extended to 48 h and 72 h respectively) until the color of the supernatant of the alkali extraction becomes light tea color. Centrifuge, discard the supernatant, and treat the precipitate with 2% HF acid twice, and discard the supernatant after centrifugation. Wash the precipitate to neutral (it can be slowly adjusted to neutral with dilute hydrochloric acid during washing, and after adjusting the pH each time, place it in a shaker to make it fully react and mix evenly), centrifuge, wash with water, collect the precipitate, which is humin. The centrifugation conditions are: 5000 rpm, 15 min, and the shaking incubator: 170 rpm, 30°C.

[0068] Example 3 A humin-mediated 2,4,6-tribromophenol biodegradation preparation is made by mixing the following components: 1 L of anaerobic mineral salt medium, 2 mg of anaerobic microorganisms, 33.33 g of humin, 1.532 mL of sodium lactate electron donor, and 1.67 mL of vitamin solution.

[0069] The above-mentioned anaerobic microorganisms are a mixed bacterial community enriched from the sediment at the 150 cmbsf layer of the columnar sediment at a depth of 1290 - 1295 m in the hippocampal cold seep area (110°28ʹE, 16°43ʹN) in the Qiongdongnan Sea. It includes Streptomyces sp. Sge12, Halomicrobium sp. LC1Hm, Jeotgalibaca dankookensis, Halodesulfurarchaeum formicicum, Dermabacter jinjuensis, Denitratisoma sp. DHT3, and Halomicrobium mukohataei in a ratio of 4.5:3:2.5:2:2:2:1.8.

[0070] The formula of the above-mentioned anaerobic mineral salt medium is as follows: for every 1 L of the medium, add 10 mL of salts stock solution (100×), 0.1 mL of Se / W stock solution (10000×), 0.5 mL of trace elements (2000×), 2.292 g of Tris ethyl sulfonic acid, 0.5 mL of resazurin, and the balance is water.

[0071] The composition of each liter of salts stock solution (100×) is: 74 g / L of NH4Cl, 1.20 g / L of CaCl2•2H2O, 50 g / L of MgCl2•6H2O, 20 g / L of K2HPO4, 100 g / L of NaCl, 30 g / L of KCl, and the balance is water.

[0072] The composition of each liter of Se / W solution is: 5 g of NaOH, 0.06 g of NaSeO3•5H2O, 0.08 g of Na2WO4•2H2O, and the balance is water.

[0073] The composition of each liter of trace element solution (2000×) is: 20 mL of HCl (25%, ω), 3 g of FeCl2•4H2O, 0.38 g of CoCl2•6H2O, 0.20 g of MnCl2•4H2O, 0.14 g of ZnCl2, 0.012 g of H3BO3, 0.004 g of CuCl2•2H2O, 0.04 g of NiCl2•6H2O, 0.072 g of Na2MoO4•2H2O, and the balance is water.

[0074] The composition of each liter of vitamin solution (1000×) is: 0.08 g of biotin, 0.20 g of nicotinic acid, +0.20 g of D - calcium pantothenate, VB 120.004 g, 0.20 g of p-aminobenzoic acid, 0.20 g of lipoic acid, 0.20 g of thiamine, 0.40 g of pyridoxal hydrochloride, 0.20 g of nicotinamide, and the balance being water. The vitamin stock solution needs to be added through a 0.22 μm sterile filter membrane.

[0075] The preparation method of the above-mentioned humin is as follows: Weigh 1.5 kg of soil sediment into 6 250 mL centrifuge bottles, add 150 mL of 0.1 M NaOH solution, place it in a shaker for 24 h for alkali extraction, and after 24 h, centrifuge and discard the supernatant. The alkali treatment is carried out at least 4 times (the shaking time in the later times can be extended to 48 h and 72 h respectively) until the color of the supernatant of the alkali extraction becomes light tea color. Centrifuge, discard the supernatant, and treat the precipitate with 2% HF acid twice, and discard the supernatant after centrifugation. Wash the precipitate to neutrality (it can be slowly adjusted to neutrality with dilute hydrochloric acid during washing, and after adjusting the pH each time, place it in a shaker to make it fully react and mix evenly), centrifuge, wash with water, and collect the precipitate, which is humin. The centrifugation conditions are: 5000 rpm, 15 min, and the shaking incubator: 170 rpm, 30 °C.

[0076] Control Example 1 (without anaerobic microorganisms) A humin-mediated 2,4,6-tribromophenol biodegradation preparation, which is made by mixing the following components: 1 L of anaerobic mineral salt medium, 33.33 g of humin, 1.532 mL of sodium lactate electron donor, and 1.67 mL of vitamin solution.

[0077] The formula of the above-mentioned anaerobic mineral salt medium is: Add 10 mL of salts stock solution (100×), 0.1 mL of Se / W stock solution (10000×), 0.5 mL of trace elements (2000×), 2.292 g of Tris ethylsulfonic acid, 0.5 mL of resazurin to each 1 L of the medium, and the balance being water.

[0078] The composition of each liter of salts stock solution (100×) is: 74 g / L of NH4Cl, 1.20 g / L of CaCl2•2H2O, 50 g / L of MgCl2•6H2O, 20 g / L of K2HPO4, 100 g / L of NaCl, 30 g / L of KCl, and the balance being water.

[0079] The composition of each liter of Se / W solution is: 5 g of NaOH, 0.06 g of NaSeO3•5H2O, 0.08 g of Na2WO4•2H2O, and the balance being water.

[0080] The composition of each liter of trace element solution (2000×) is as follows: 20 mL of HCl (25%, ω), 3 g of FeCl2•4H2O, 0.38 g of CoCl2•6H2O, 0.20 g of MnCl2•4H2O, 0.14 g of ZnCl2, 0.012 g of H3BO3, 0.004 g of CuCl2•2H2O, 0.04 g of NiCl2•6H2O, 0.072 g of Na2MoO4•2H2O, with the balance being water.

[0081] The composition of each liter of vitamin solution (1000×) is as follows: 0.08 g of biotin, 0.20 g of nicotinic acid, + 0.20 g of calcium D-pantothenate, 0.004 g of VB 12 0.20 g of p-aminobenzoic acid, 0.20 g of lipoic acid, 0.20 g of thiamine, 0.40 g of pyridoxal hydrochloride, + 0.20 g of nicotinamide, with the balance being water.

[0082] The preparation method of the above-mentioned humin is as follows: Weigh 1.5 kg of soil sediment into 6 250 mL centrifuge bottles, add 150 mL of 0.1 M NaOH solution, place it in a shaker for 24 h for alkali extraction, and after 24 h, centrifuge and discard the supernatant. The alkali treatment is carried out at least 4 times (the shaking time for the latter several times can be extended to 48 h and 72 h respectively) until the color of the supernatant of the alkali extraction becomes light tea color. Centrifuge, discard the supernatant, treat the precipitate with 2% HF acid twice, and discard the supernatant after centrifugation. Wash the precipitate to neutral (it can be slowly adjusted to neutral with dilute hydrochloric acid during the water washing process, and after adjusting the pH each time, place it in a shaker to make it react fully and mix evenly), centrifuge, wash with water, collect the precipitate, which is humin. The centrifugation conditions are: 5000 rpm, 15 min, and the shaking incubator: 170 rpm, 30 °C.

[0083] Comparative Example 2 (without humin) A 2,4,6-tribromophenol biodegradation preparation is made by mixing the following components: 1 L of anaerobic mineral salt medium, 2 mg of anaerobic microorganisms, 1.532 mL of sodium lactate electron donor, and 1.67 mL of vitamin solution.

[0084] The above-mentioned anaerobic microorganisms are a mixed flora obtained by enrichment culture of the sediments at the 0 cmbsf, 25 cmbsf, and 150 cmbsf horizons of the columnar sediments at a depth of 1290 - 1295 m in the hippocampal cold seep area (110°28ʹE, 16°43ʹN) in the Qiongdongnan Sea, and it includes: Comparative Example 2-1: Clostridium drakei, Halomicrobium sp. LC1Hm, Anaerohalosphaera lusitana, Dermatophilus congolensis, Candidatus Enterovibrio luxaltus, Jeotgalibaca dankookensis, Halodesulfurarchaeum formicicum, and Halomicrobium mukohataei with a ratio of 10.2:3.5:2.9:2.6:2.4:2.2: 2.2:1.7.

[0085] Comparative Example 2-2: Streptomyces sp. SAT1, Megasphaera hexanoica, Megasphaera massiliensis, Streptomyces sp. Sge12, Dermabacter jinjuensis, Halomicrobium sp. LC1Hm, Anaerohalosphaera lusitana, and Halodesulfurarchaeum formicicum with a ratio of 9.4:4.9:4.5:4:2.7:2.6:2.5:2.1.

[0086] Comparative Example 2-3: Streptomyces sp. Sge12, Halomicrobium sp. LC1Hm, Jeotgalibaca dankookensis, Halodesulfurarchaeum formicicum, Dermabacter jinjuensis, Denitratisoma sp. DHT3, and Halomicrobium mukohataei with a ratio of 4.5:3:2.5:2:2:2:1.8.

[0087] The formulation of the above anaerobic mineral salt medium is as follows: for every 1 L of the medium, add 10 mL of salts stock solution (100×), 0.1 mL of Se / W stock solution (10,000×), 0.5 mL of trace element solution (2000×), 2.292 g of Tris ethyl sulfonic acid, 0.5 mL of resazurin, and the balance is water.

[0088] The composition of each liter of salts stock solution (100×) is: 74 g / L of NH4Cl, 1.20 g / L of CaCl2•2H2O, 50 g / L of MgCl2•6H2O, 20 g / L of K2HPO4, 100 g / L of NaCl, 30 g / L of KCl, and the balance is water.

[0089] The composition of each liter of Se / W solution is: 5 g of NaOH, 0.06 g of NaSeO3•5H2O, 0.08 g of Na2WO4•2H2O, and the balance is water.

[0090] The composition of each liter of trace element solution (2000×) is: 20 mL of HCl (25%, ω), 3 g of FeCl2•4H2O, 0.38 g of CoCl2•6H2O, 0.20 g of MnCl2•4H2O, 0.14 g of ZnCl2, 0.012 g of H3BO3, 0.004 g of CuCl2•2H2O, 0.04 g of NiCl2•6H2O, 0.072 g of Na2MoO4•2H2O, and the balance is water.

[0091] The composition of each liter of vitamin solution (1000×) is: 0.08 g of biotin, 0.20 g of nicotinic acid, + 0.20 g of D-calcium pantothenate, VB 12 0.004 g, 0.20 g of p-aminobenzoic acid, 0.20 g of lipoic acid, 0.20 g of thiamine, 0.40 g of pyridoxal hydrochloride, + 0.20 g of nicotinamide, and the balance is water. The vitamin stock solution needs to be added through a 0.22 μm sterile filter membrane.

[0092] Application Example 1 (Degradation test of 2,4,6-tribromophenol) Add 2,4,6-tribromophenol at an addition amount of 25 μmol / L to the biodegradation preparations of Examples 1-3 and Comparative Examples 1-2 respectively, and conduct a degradation test under a dark environment at 30 ± 1 °C as follows: First, prepare 1 L of anaerobic mineral medium and place it in a 2-L three-necked flask. Add 1.532 ml of sodium lactate. Connect the left side of the three-necked flask to a gas device to purge nitrogen (purity 99.99%) to remove oxygen. Connect the upper side to a condenser to prevent liquid evaporation, and seal the right side with a rubber stopper. Place the three-necked flask on a heating mantle, heat it to boiling for 20 min, and then cool it in a nitrogen stream. After the medium has cooled, add 0.0242 g of L-cysteine hydrochloride, 0.048 g of Na2S•9H2O, 0.0771 g of dithiothreitol, and 2.52 g of NaHCO3 (L-cysteine hydrochloride, Na2S•9H2O, dithiothreitol, and NaHCO3 are all used as reducing agents to ensure anaerobic conditions in the medium). Prepare 60 mL serum bottles, add 30 mL of anaerobic mineral salt medium and 1 g of humin (humin is not added in Comparative Example 2). Use a syringe to draw 30 ml of the solution from the three-necked flask, quickly dispense it into the serum bottles, and continuously blow nitrogen for 8 min. Quickly plug with a Teflon-coated butyl rubber stopper (inner diameter 13 mm, produced by Wheaton, USA). Then cover with an aluminum cap and seal. Finally, autoclave at 121 °C for 20 minutes. The degradation of 2,4,6-tribromophenol is completed under anaerobic conditions.

[0093] When conducting the degradation experiment, use a syringe to add 0.05 mL of vitamin solution to the above-sterilized bottle through a 0.2-μm filter membrane, and then use a microsyringe to add 6 μL of 2,4,6-tribromophenol with a concentration of 100 μmol / mL. The final concentration of TBP in the system is 25 μmol / L. 2,4,6-Tribromophenol needs to be prepared in the form of a stock solution before being added to the medium. The preparation method is as follows: Dissolve the purchased 2,4,6-tribromophenol (0.03308 g) in 1 ml of acetone, sonicate until completely dissolved, prepare a 100 μmol / mL stock solution, seal the cap, seal with a sealing film, and store in a 4 °C refrigerator.

[0094] Inoculate the above system with anaerobic microorganisms (except for Comparative Example 1), and then statically culture in an incubator at 30 °C in the dark. After 0, 5, 15, and 20 days of culture, use a disposable syringe to take 1 ml of sample from the culture system, and use isooctane (chromatographic grade 99%) with the same volume as the extractant to extract TBP and its metabolites. Vortex for 1 h, transfer to a 2-mL centrifuge tube, centrifuge at 15,000 ×g for 2 min, transfer the supernatant to a brown injection vial, and use a gas chromatography-mass spectrometer (GCMS)-QP2020 (Shimadzu, Kyoto, Japan), equipped with an SH-Rxi-5Sil MS capillary column (30 m × 0.25 mm inner diameter × 0.25 μm) for instrument detection.

[0095] The concentration of 2,4,6-tribromophenol was determined by gas chromatography-mass spectrometry (Shimadzu QP 2010 Ultra gas chromatography-mass spectrometer GC-MS). Chromatographic conditions: Rtx-5MS chromatographic column (30 m × 0.25 mm, 0.25 μm); injection port temperature 250 °C. Temperature programming: initial temperature 50 °C, hold for 2 min; then increase the temperature at a rate of 10 °C·min -1 to 160 °C at a rate and hold for 2 min; then increase the temperature at a rate of 15 °C·min -1 to 250 °C at a rate and hold for 3 min. The carrier gas was high-purity helium (purity greater than 99.999%), and the carrier gas flow rate was 1.2 ml·min -1 ; splitless injection; injection volume 1 μL. Mass spectrometry conditions: electron impact ionization source (EI), electron energy 70 eV; ion source temperature 250 °C, chromatograph-mass spectrometry interface temperature 250 °C; solvent delay 3 min; qualitative analysis by full ion scan (SCAN), quantitative analysis by selected ion scan (SIM). Calibration curves for TBP and its degradation products (i.e., 2,6-DBP, 2,4-DBP, 4-BP, phenol) were established, including 6 different concentrations: 0, 5, 10, 20, 30, and 40 μM.

[0096] The degradation of 2,4,6-tribromophenol in the culture was determined by a semi-quantitative method of relative quantification. By calculating the change in the molar percentage of different 2,4,6-bromophenols before and after degradation, the debromination reaction was judged.

[0097] Figure 1 Showing the degradation results of Example 1, after culturing for 15 days in the above anaerobic culture system, 2,4,6-TBP was basically completely degraded and gradually converted into 2,4-DBP, 4-BP, and phenol.

[0098] Figure 2 Showing the degradation results of Example 2, after culturing for 10 days in the above anaerobic culture system, 2,4,6-TBP was basically completely degraded and gradually converted into 2,4-DBP, 4-BP, and phenol.

[0099] Figure 3 Showing the degradation results of Example 3, after culturing for 10 days in the above anaerobic culture system, 2,4,6-TBP was completely degraded, and the intermediate products 2,4-DBP and 4-BP were also completely degraded into phenol.

[0100] Figure 4It shows the degradation result of Comparative Example 1. Humicins were added and anaerobic microorganisms were not added. 2,4,6-Tribromophenol was hardly degraded compared with the standard sample, indicating that the anaerobic degradation of 2,4,6-tribromophenol must involve anaerobic microorganisms.

[0101] Figure 5 It shows the degradation result of Comparative Example 2. Humicins were not added and only anaerobic microorganisms were added. 2,4,6-Tribromophenol was also hardly degraded, indicating that the degradation of 2,4,6-tribromophenol must involve humicins. Humicins can replace soil as an electron mediator to mediate the reductive degradation experiment of 2,4,6-tribromophenol.

[0102] The above experimental results show that the biodegradation preparations of Examples 1-3 can efficiently degrade 2,4,6-tribromophenol, while single anaerobic microorganisms and single humicins do not have obvious 2,4,6-tribromophenol degradation ability.

[0103] Application Example 2 (Degradation test of tetrachloroethylene) 2 μL of pure tetrachloroethylene (PCE) solution (Aladdin) was added to the biodegradation preparations (Examples 1-3) respectively, and the final concentration of PCE in the system was 0.665 mmol / L (other steps refer to Application Example 1).

[0104] Detection method: Headspace-gas chromatography (GC-ECD) was used to quantitatively detect PCE, 1,1,2-TCA and their degradation products in the sample to be tested. The headspace sampling system (Agilent 7697A headspace Sampler) adopted the high-speed oscillation mode. The sample equilibrium time was 15 min, the pressurization time was 0.1 min, the time for filling the quantitative tube was 0.15 min, the equilibrium time of the quantitative tube was 0.10 min, and the injection time was 1.0 min; the transfer line pressure was 73 kPa, and the headspace bottle pressure was 74 kPa; the furnace temperature was set at 70 °C, the temperature of the quantitative tube was 80 °C, and the transfer line temperature was 90 °C.

[0105] The setting conditions of the gas chromatograph (Agilent 8890) are as follows: The temperature programming starts from 40 °C, holds for 8 min, then rises to 100 °C at a rate of 10 °C per minute, then rises to 220 °C at a rate of 25 °C per minute, and then holds for 5 min; the injection port temperature is set at 250 °C; the detector temperature is set at 300 °C; the injection mode adopts constant flow injection, the carrier gas is high-purity nitrogen, the flow rate is set at 1 ml / min, and the split ratio is 50:1; the chromatographic column is selected as DB-624 U1, with a specification of 30 m × 250 μm × 0.25 μm.

[0106] As Figure 6As shown, the results show that the biodegradation preparation of the present invention has almost no degradation effect on tetrachloroethylene, and the tetrachloroethylene PCE has not undergone degradation. The contents of its products TCE, cis-DCE, and trans-DCE are all 0. It can be seen from this that the biodegradation preparation of the present invention does not have an obvious degradation effect on other organic pollutants such as tetrachloroethylene, but has a certain degradation specificity for 2,4,6-tribromophenol.

[0107] The raw materials and equipment used in the present invention are all common raw materials and equipment in the art without special instructions; the methods used in the present invention are all conventional methods in the art without special instructions.

[0108] The above are only the preferred embodiments of the present invention, and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent transformations made to the above embodiments based on the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A humin-mediated biodegradation preparation for 2,4,6-tribromophenol, characterized in that: Using anaerobic mineral salt medium as the substrate, and the substrate contains: Humin 25-35 g / L, Organic acid salt electron donor 1-2 mL / L, Vitamin solution 1-2 mL / L, Anaerobic microorganisms 1.5-2.5 mg / L.

2. The biodegradable preparation according to claim 1, wherein: The anaerobic microorganisms include: Clostridium drakei, Halomicrobium sp. LC1Hm, Anaerohalosphaera lusitana, Dermatophilus congolensis, Candidatus Enterovibrio luxaltus, Jeotgalibacadankookensis, Halodesulfurarchaeum formicicum, and Halomicrobium mukohataei in a ratio of (9-11):(3-4):(2.6-3.2):(2.4-2.8):(2.2-2.6):(2-2.4):(2-2.4):(1.5-1.9); or Streptomycessp.SAT1, Megasphaera hexanoica, Megasphaera massiliensis, Streptomyces sp.Sge12, Dermabacter jinjuensis, Halomicrobium sp. LC1Hm, Anaerohalosphaeralusitana, and Halodesulfurarchaeum formicicum in a ratio of (8-10):(4-6):(3.5-5.5):(3-5):(2-4):(2-4):(2-3):(1-3); or Streptomyces sp. Sge12, Halomicrobium sp.LC1Hm, Jeotgalibaca dankookensis, Halodesulfurarchaeumformicicum, Dermabacter jinjuensis, Denitratisoma sp. DHT3, and Halomicrobiummukohataei in a ratio of (4-5):(3-5):(2-4):(2-3):(1-3):(1-3):(1-3).

3. The biodegradable preparation according to claim 1, characterized in that: The preparation method of the humin is: taking soil sediment, adding alkali for alkali extraction, centrifuging and discarding the supernatant; repeating the alkali extraction until the color of the supernatant is light tea color; treating the obtained precipitate with HF, centrifuging, washing the precipitate to neutral, centrifuging, washing with water, and collecting the precipitated humin.

4. The biodegradable preparation according to claim 1, characterized in that: The composition of the anaerobic mineral salt medium includes: 5 - 15 mL / L of salts stock solution, 0.05 - 0.15 mL / L of Se / W stock solution, 0.1 - 1 mL / L of trace element solution, 2 - 2.5 g / L of Tris-ethanesulfonic acid, 0.1 - 1 mL / L of resazurin, and the balance is water.

5. The biodegradable preparation according to claim 4, wherein: The composition of the salts stock solution includes: 70 - 80 g / L of NH4Cl, 1 - 1.5 g / L of CaCl2•2H2O, 45 - 55 g / L of MgCl2•6H2O, 15 - 25 g / L of K2HPO4, 90 - 110 g / L of NaCl, 25 - 35 g / L of KCl, and the balance is water.

6. The biodegradable preparation according to claim 4, wherein: The composition of the Se / W solution includes: 3 - 7 g of NaOH, 0.04 - 0.08 g of NaSeO3•5H2O, 0.06 - 0.1 g of Na2WO4•2H2O, and the balance is water.

7. The biodegradable preparation according to claim 4, wherein: The composition of the trace element solution includes: 15 - 25 mL of 20 - 30 wt% HCl, 2 - 4 g of FeCl2•4H2O, 0.3 - 0.4 g of CoCl2•6H2O, 0.1 - 0.3 g of MnCl2•4H2O, 0.1 - 0.2 g of ZnCl2, 0.01 - 0.02 g of H3BO3, 0.002 - 0.006 g of CuCl2•2H2O, 0.02 - 0.06 g of NiCl2•6H2O, 0.07 - 0.08 g of Na2MoO4•2H2O, and the balance is water.

8. The biodegradable preparation according to claim 1, characterized in that: The composition of the vitamin solution includes: biotin 0.06 - 0.1 g, niacin 0.1 - 0.3 g, +calcium D-pantothenate 0.1 - 0.3 g, VB 12 0.002 - 0.006 g, p-aminobenzoic acid 0.1 - 0.3 g, lipoic acid 0.1 - 0.3 g, thiamine 0.1 - 0.3 g, pyridoxal hydrochloride 0.3 - 0.5 g, +nicotinamide 0.1 - 0.3 g, and the balance is water.

9. Use of the biodegradation preparation according to any one of claims 1 - 8 in degrading 2,4,6 - tribromophenol.

10. The application according to claim 9, characterized in that: Mix 2,4,6 - tribromophenol with the 2,4,6 - tribromophenol biodegradation preparation and carry out degradation in a dark environment.

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