阅读说明：本技术 一株泛菌及菌锰混合物及在降解孔雀石绿中的应用 (Pantoea and bacterium manganese mixture and application thereof in degrading malachite green ) 是由 赵国琰 孙元凯 王文静 于 2021-04-30 设计创作，主要内容包括：本发明涉及环境微生物技术领域,具体涉及一株泛菌及菌锰混合物及在降解孔雀石绿中的应用。该菌株已于2021年3月24日保藏于中国典型培养物保藏中心(简称CCTCC,地址为：湖北省武汉市武昌区八一路珞珈山),保藏编号为CCTCC M 2021491,所述菌锰混合物由泛菌Pantoea eucrina SS01及其锰氧化产物混合而成,将泛菌Pantoea eucrinaSS01及其培养液与氯化锰溶液混合培养,将培养得到的产物烘干即得到菌锰混合物。所述菌锰混合物能够快速降解高浓度孔雀石绿,且能够持续性降解高浓度孔雀石绿,降解产物无毒无害,使用成本低,生态恢复好,应用前景极为广阔,能够解决目前三苯甲烷类染料污染水体难以治理的实践难题。(The invention relates to the technical field of environmental microorganisms, and particularly relates to a pantoea and bacterium manganese mixture and application thereof in degrading malachite green. The strain is preserved in China center for type culture Collection (CCTCC for short, the address is eight Lopa Jia mountain in Wuchang district, Wuhan city, Hubei province) at 24 months at 2021, the preservation number is CCTCC M2021491, the bacteria-manganese mixture is prepared by mixing Pantoea eutroprina SS01 and manganese oxidation products thereof, the Pantoea eutropha aSS01 and culture solution thereof are mixed with manganese chloride solution for culture, and the cultured products are dried to obtain the bacteria-manganese mixture. The bacterium-manganese mixture can rapidly degrade high-concentration malachite green, can persistently degrade the high-concentration malachite green, has the advantages of nontoxic and harmless degradation products, low use cost, good ecological restoration and extremely wide application prospect, and can solve the practical problem that the water body polluted by the existing triphenylmethane dyes is difficult to control.)

1. A Pantoea eutropha SS01 strain is preserved in China center for type culture Collection (CCTCC, address: eight Lopa of Lopa nationality in Wuchang district, Wuhan city, Hubei province) 24 months at 2021, with the preservation number of CCTCC M2021491.

2. The bacterium manganese mixture is characterized by being prepared by mixing Pantoea eutropha SS01 and manganese oxidation products thereof.

3. The bacterial manganese mixture of claim 2, prepared by the following method: mixing Pantoea eutropha SS01 and culture solution thereof with a divalent manganese compound solution for culture, and drying the cultured product to obtain a bacteria-manganese mixture; preferably, the divalent manganese compound is selected from manganese chloride or manganese sulfate.

4. The bacteria-manganese mixture according to claim 2, wherein said Pantoea eutropha SS01 is cultured by inoculating in LB liquid medium to obtain a large amount of bacteria as seed liquid;

preferably, the culture conditions of Pantoea eucrina SS01 in LB liquid medium are: culturing overnight in a shaker at 25-35 ℃ and 150-200 rpm.

5. The bacteria-manganese mixture according to claim 2, wherein the concentration of the manganese chloride solution is 1-3M, preferably 2M;

preferably, the manganese chloride solution is filtered and sterilized prior to use.

6. The mixture of bacteria and manganese of claim 2, wherein said culture medium is a K's medium and the culture conditions are: shake culturing at constant temperature of 28-32 ℃.

7. The bacteria-manganese mixture according to claim 2, wherein the manganese chloride solution is added at the following timing: measurement of OD of Pantoea eutrina SS01 and its culture solution₆₀₀Value, to OD₆₀₀When the value is 0.7-0.9, adding manganese chloride solution to make the final concentration of manganese chloride solution be 15-25 mM, preferably 20 mM.

8. The bacteria-manganese mixture according to claim 2, wherein the time of mixed culture is 2 to 4 days, preferably 3 days;

or, the drying condition is 50-60 ℃ and the time is 10-12 h.

9. Use of the mixture of bacterial manganese according to any one of claims 2 to 8 for degrading malachite green.

10. The application of claim 9, wherein the method of applying is: adding a bacterium-manganese mixture into the malachite green solution, adjusting the pH to 4.0-7.0 and the temperature to 25-35 ℃, and reacting for 12-24 hours to degrade the malachite green;

preferably, the mass ratio of the bacteria-manganese mixture to the malachite green is 1-1.5: 1;

preferably, the concentration of malachite green in the malachite green solution is 1000mg/L or less;

preferably, the malachite green is repeatedly added to the reaction solution of the malachite green solution and the mixture of bacteria and manganese at a concentration of 600mg/L or less, and the number of times of repeated addition is 5 or less.

Technical Field

The invention relates to the technical field of environmental microorganisms, and particularly relates to a pantoea and bacterium manganese mixture and application thereof in degrading malachite green.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Malachite Green (MG) is a basic triphenylmethane dye, is very soluble in water, and the aqueous solution thereof is blue-green. On one hand, malachite green is used as a dye to dye leather, paper, acrylic fibers and the like, and on the other hand, malachite green is used as a food additive and a colorant to be applied to industries of food, health care and the like. In addition, malachite green has broad-spectrum bactericidal action, is widely applied to the industries of aquatic product culture and preservation and the like, and has very obvious effects of killing fish endoparasites, preventing and treating saprolegniasis, gill rot and the like. However, subsequent studies have found that malachite green has high toxicity, high residue, carcinogenic and mutagenic hazard, and poses serious threat to human health through food chain, so that malachite green has been regarded as a forbidden drug in many countries including china. But because of the advantages of low price, good effect and the like, the method is still illegally used in some areas, and the content of the detected aquatic products exceeds the standard.

At present, the malachite green treatment method mainly comprises a physical method and a chemical method. The method for treating the wastewater by a physical method mainly comprises an adsorption method, a membrane filtration method, an osmosis method, an extraction technology and the like; the chemical method for treating the organic pollutants mainly comprises an electrochemical treatment method and a chemical oxidation method. However, these methods have high cost and low efficiency in sewage treatment, and are prone to secondary pollution, which greatly limits their wide application. Therefore, it is highly desirable to find a method which is low in cost, high in efficiency, free from secondary pollution and environmentally friendly.

The microorganism has the advantages of high propagation speed, strong adaptability to severe environment, easy generation of a large amount of degradation-related enzymes and the like, so that the microorganism treatment technology is favored by researchers, and the screening of the strain with high-efficiency degradation capability has important practical significance. Many researches show that pseudomonas, bacillus, saccharomycetes and the like have a decoloring function on triphenylmethane dyes, but the inventor finds that the single microbial degradation efficiency is low, and the purification enzymatic degradation increases the cost.

Disclosure of Invention

Aiming at the technical problems of high cost, low efficiency and easy occurrence of secondary pollution in malachite green sewage treatment in the prior art, one of the purposes of the invention is to provide a Pantoea eucrina SS01 strain, a bacteria-manganese mixture formed by mixing Pantoea eucrina SS01 and manganese oxidation products thereof, and application of the bacteria-manganese mixture in degrading malachite green. The invention can rapidly degrade high-concentration malachite green, can continuously degrade the high-concentration malachite green, and has high degradation rate and accumulated degradation rate.

In order to achieve the above object, the technical solution of the present invention is as follows:

in the first aspect of the invention, a Pantoea eutrina SS01 strain is provided, which has been preserved in China center for type culture Collection (CCTCC for short, address: Bayinyao mountain in Wuchang district, Wuhan City, Hubei province) 24 months at 2021, with the preservation number of CCTCC M2021491.

In a second aspect of the present invention, there is provided a microbial manganese mixture, which is prepared by mixing Pantoea eutropha SS01 and manganese oxidation products thereof;

specifically, the bacterial manganese mixture is prepared by the following method: mixing Pantoea eutropha SS01 and culture solution thereof with manganese chloride solution, culturing, and drying the cultured product to obtain the manganese mixture.

In a third aspect of the invention, there is provided a use of the manganese mixture of the second aspect in degrading malachite green.

The specific embodiment of the invention has the following beneficial effects:

the bacterium-manganese mixture in the embodiment of the invention can rapidly degrade high-concentration malachite green, and degrade 1000mg/L of malachite green to 38.10mg/L within 24h, wherein the degradation rate is 96.19%;

the bacterium-manganese mixture in the embodiment of the invention can continuously degrade high-concentration malachite green, 600mg/L of malachite green is added every 24 hours in continuous feeding degradation, the addition is performed for five times in an accumulated way, the final concentration of the remained malachite green is 89.54mg/L, and the accumulated degradation rate is 97.02%;

toxicity tests on soybean and blue algae prove that the manganese mixture has no influence on the growth and development of organisms on degradation products of malachite green, and the degradation products are proved to be non-toxic and harmless;

compared with the traditional expensive physical and chemical method, the method has the advantages of low use cost, good ecological restoration and the like, has extremely wide application prospect, and can solve the practical problem that the water body polluted by the triphenylmethane dye is difficult to control at present.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a graph showing the result of HPLC-MS analysis of degradation products of a manganese mixture; wherein fig. 1a is a bacterial manganese mixture degradation product HPLC diagram, fig. 1b is a partial HPLC enlargement of a bacterial manganese mixture degradation product, fig. 1c is a mass spectrum of 4-vinyl-N, N-dimethylaniline (2.550 min, m/z 147), fig. 1d is a mass spectrum of (4-dimethylamino-phenyl) -phenyl-methanone (2.649 min, m/z 226), fig. 1e is a mass spectrum of dimethyl- (4-oxo-cyclohexa-2, 5 dienylidene) ammonium (3.567 min, m/z 136), fig. 1f is a mass spectrum of 4-dimethylaminobenzaldehyde (4.209 min, m/z 150), fig. 1g is a mass spectrum of 4-methylaminobenzoic acid (6.476 min, m/z 152), fig. 1h is a mass spectrum of demethyl malachite green (17.791 min, m/z 315);

FIG. 2 is a graph showing the effect of bacteria manganese mixture degrading malachite green of different concentrations in example 3 of the present invention;

FIG. 3 is a graph showing the effect of the manganese mixture of example 4 of the present invention on the sustained degradation of malachite green;

FIG. 4 is a graph of the effect of malachite green and its degradation products on soybean growth of example 5 of the present invention; wherein MG is malachite green, S is malachite green decolorization liquid processed by bacteria, SM is malachite green degradation product processed by bacteria-manganese mixture, and Water is control group.

FIG. 5 is a Pantoea eutropha SS01 NJ clade of example 1 of the present invention.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

In one embodiment of the present invention, there is provided a Pantoea eutropha SS01 strain, which has been preserved in China center for type culture Collection (CCTCC for short, address: eight Lopa of Wuhan city, Hubei province) at 24 months 3 in 2021, with the preservation number of CCTCC M2021491.

The Pantoea eutrina SS01 is separated from Suaeda salsa leaves, the bacterial colony is thick and yellow, has odor and smooth surface, and the optimal growth temperature is 30 ℃ and the pH value is 7.

The invention also protects a microbial inoculum with the active ingredient of Pantoea eucrina SS 01.

In one embodiment of the invention, the bacteria-manganese mixture is prepared by mixing Pantoea eutropha SS01 and manganese oxidation products thereof;

specifically, the bacterial manganese mixture is prepared by the following method: mixing Pantoea eutropha SS01 and its culture solution with divalent manganese compound solution, culturing, and oven drying to obtain bacteria-manganese mixture.

Preferably, the divalent manganese compound is selected from manganese chloride or manganese sulfate;

in one or more embodiments, the Pantoea eucrina SS01 is cultured by inoculating in LB liquid medium to obtain a large amount of cells as a seed solution;

further, the culture condition of the pantoea in the LB liquid culture medium is that the pantoea is cultured in a shaking table with the temperature of 25-35 ℃ and the rotating speed of 150-200 rpm for overnight;

in one or more embodiments, the concentration of the manganese chloride solution is 1-3M, preferably 2M.

Further, the manganese chloride solution is filtered and sterilized before use;

in one or more embodiments, the medium is K's medium and the culture conditions are: shake culturing at a constant temperature of 28-32 ℃;

in one or more embodiments, the manganese chloride solution is added at the timing of: measurement of OD of Pantoea eutrina SS01 and its culture solution₆₀₀Value, to OD₆₀₀When the value is 0.7-0.9, adding manganese chloride solution to make the final concentration of the manganese chloride solution be 15-25 mM, preferably 20mM, and obtaining the bacteria manganese mixture.

In one or more embodiments, the time of mixed culture is 2-4 days, preferably 3 days;

in one or more embodiments, the drying is performed at 50-60 ℃ for 10-12 h.

The bacteria with manganese oxidation activity are called manganese oxidizing bacteria, and the species of the manganese oxidizing bacteria are various and widely distributed. Manganese-oxidizing bacteria that have been isolated in the prior art are mainly concentrated in the phylum Firmicutes, actinomycetes and Proteobacteria alpha, beta and gamma. Oxides of manganese produced by the oxidation of manganese-oxidizing bacteria are referred to as bacterial oxides of manganese. Biological manganese oxides comprise the form of Mn (III/IV) oxide, Mn (ii) carbonate, which occur on the surface of bacterial extracellular or endospores.

The bacteria-manganese mixture is formed by mixing Pantoea eutropha SS01 and manganese oxidation products thereof, compared with manganese oxidation bacteria or manganese oxidation products, the efficiency of the bacteria-manganese mixture is obviously improved when organic pollutants are degraded, Mn (II) generated by degrading organic matters can be oxidized into Mn (III/IV) again by Pantoea eutropha SS01, and the generated Mn (III/IV) oxide can be continuously oxidized and degraded into organic pollutants, namely, the Pantoea eutropha SS01 and the manganese oxidation products thereof have synergistic effect.

In one embodiment of the invention, the application of the manganese mixture in degrading malachite green is provided;

in one or more embodiments, the method of applying is: adding a bacterium-manganese mixture into the malachite green solution, adjusting the pH to 4.0-7.0 and the temperature to 25-35 ℃, and reacting for 12-24 hours to degrade the malachite green;

preferably, the mass ratio of the bacteria-manganese mixture to the malachite green is 1-1.5: 1.

preferably, the concentration of malachite green in the malachite green solution is 1000mg/L or less;

preferably, the malachite green is repeatedly added to the reaction solution of the malachite green solution and the mixture of bacteria and manganese at a concentration of 600mg/L or less, and the number of times of repeated addition is 5 or less.

The invention will be further explained and illustrated with reference to specific examples.

Example 1

Isolation and characterization of Pantoea eutropha SS01

1. Screening of Pantoea eutropha SS01

The Pantoea eutropha SS01 is separated from suaeda salsa leaves, and the separation method comprises the following steps:

1) the overground part of the collected suaeda salsa plant sample is selected to be about 5g of complete leaves, washed by clean water, wiped dry and weighed for recording.

2) And (3) placing the mixture in a sterile operating platform, soaking the mixture in Tween 80 for 5 minutes, washing the mixture with sterile water for 3 times, soaking the mixture in Tween 80 for 3 minutes, washing the mixture with sterile water for 3 times, and repeating the steps for 3-5 times. Then in 0.1% of HgCl₂Soaking in the solution for 1 min, soaking in 75% ethanol for 3 min, and washing with sterile water. And finally, coating the flat plate with water for the first time, and detecting whether the disinfection is thorough.

3) The sterilized test specimens were transferred to a sterilized mortar using sterile forceps and ground.

4) Adding the homogenate into MnCl containing heavy metal with concentration of 5mmol/L₂The LB liquid medium of (5) was cultured for 48 hours on a shaker at 150 rpm.

5) The cells grown in LB liquid medium were isolated and cultured by streaking.

6) Selecting colony numbers of different forms, selecting single colony, and further culturing in LB liquid culture medium at 30 deg.C and 150rpm for 48 h.

2. Identification of strains

Microbiological characterization:

the Pantoea eutropha SS01 colony is viscous and yellow, has odor, smooth surface, and optimal growth temperature of 30 deg.C and pH of 7.

Molecular biological characteristics:

the strain is Pantoea eucrina SS01 through the sequence comparison analysis of the strain 16S rDNA gene, an NJ evolutionary tree is made based on the 16S rDNA gene sequences of the manganese-oxidizing bacteria separated out in the experiment and other reported manganese-oxidizing bacteria (figure 5), Sulfolobus acidocaldarius strain ATCC 33909 is used as an outer group, the Bootstrap value is 1000, and only the self-expansion value higher than 50% is shown. Bar, 0.05.

Example 2

Preparing a bacterium-manganese mixture:

(1) carrying out streak culture on a strain SS01, selecting a single colony, inoculating the single colony in an LB liquid culture medium, and carrying out overnight culture in a shaking table with the rotation speed of 150-200 rpm at the temperature of 25-35 ℃ to obtain a large amount of thalli as a seed solution; (2) weighing MnCl₂125.84g is dissolved in 500mL deionized water, and stirred and dissolved at normal temperature to obtain MnCl with the final concentration of 2M₂And filtering for sterilization; (3) inoculating 1% of SS01 bacterial liquid in the step (1) in a K culture medium, placing the culture medium in a constant temperature shaking table at 30 ℃ and 120rpm for shake culture, and measuring OD once per hour₆₀₀Value, to OD₆₀₀At a value of 0.8, the 2M MnCl of step (2) is added to the flask₂The final concentration is 20mM, and the culture is continued for 3 days to obtain the manganeseMixing; (4) and (4) centrifuging the bacteria-manganese mixture obtained in the step (3) at 4000rpm for 15min, discarding the supernatant, washing twice with sterile deionized water, centrifuging again, precipitating the bacteria-manganese mixture in an oven at 60 ℃, and continuously baking for 12 h.

Example 3

The dried bacterium-manganese mixture dry powder obtained in the example 1 is weighed by balance, 10mg of each part is respectively resuspended by 200mg/L, 300mg/L, 500mg/L and 1000mg/L of malachite green solution, the mixture is put in a shaking table with the temperature of 30 ℃ and the rpm to be shaken, the residual content of the malachite green is measured after 24 hours, three parallel experiments are arranged in each experiment, the measurement is carried out three times, and the average value is taken for use. The whole process keeps sterile operation. And centrifuging the degraded sample at 12000rpm for 5min, taking the supernatant, measuring the light absorption value at 620nm by using an ultraviolet-visible spectrophotometer, and calculating the degradation rate.

The degradation rate calculation mode is as follows: measuring the light absorption value of the solution at 620nm by using an ultraviolet visible spectrophotometer, and calculating the degradation rate:

the degradation rate (%) - (a-B)/ax100; in the formula, A is the OD value before degradation, and B is the OD value after degradation.

The degradation rates of the bacterial manganese mixture on 200mg/L, 300mg/L, 500mg/L and 1000mg/L are respectively 84.99%, 89.40%, 92.38% and 96.19%, as shown in figure 2.

Degradation products of malachite green were analyzed by HPLC-MS as shown in fig. 1 to give typical malachite green degradation products such as 4-vinyl-N, N-dimethylaniline (2.550 min, m/z 147, fig. 1c), (4-dimethylamino-phenyl) -phenyl-methanone (2.649 min, m/z 226, fig. 1d), dimethyl- (4-oxo-cyclohexa-2, 5 dienylidene) ammonium (3.567 min, m/z 136, fig. 1e), 4-dimethylaminobenzaldehyde (4.209 min, m/z 150, fig. 1f), 4-methylaminobenzoic acid (6.476 min, m/z 152, fig. 1g), demethylmalachite green (17.791 min, m/z315, fig. 1 h).

Example 4

Weighing the bacteria-manganese mixture dry powder prepared in example 1 by using balance, wherein each 10mg dry powder is obtained by resuspending the dry powder by 600mg/L malachite green, placing the powder in a shaking table with the temperature of 30 ℃ and the rpm of 120 for shaking and decoloring, measuring the residual content of the malachite green once every 24h, supplementing the malachite green with the content of 600mg/L, repeating the whole process five times, setting three parallel experiments in each experiment, measuring three times for each time, and taking an average value for use. The whole process keeps sterile operation. And centrifuging the degraded sample at 12000rpm for 5min, taking the supernatant, measuring the light absorption value at 620nm by using an ultraviolet-visible spectrophotometer, and calculating the degradation rate.

The manganese mixture showed sustained degradation capacity, with a final cumulative degradation rate of 97.02% and a cumulative degraded malachite green concentration of 2920mg/L, as shown in FIG. 3.

Example 5

The experiments of soybean growth influence are respectively carried out by using malachite green MG, malachite green destaining solution S after bacteria treatment, malachite green degradation product SM after bacteria and manganese mixture treatment and Water of a control group:

soybean: weighing 10MG of dried powder of mixture of bacteria powder and manganese obtained by drying with balance, preparing 1000MG/L MG by using sterile deionized water, suspending the dried powder, placing the powder in a shaking table with the temperature of 30 ℃ and the rpm of 120 for shaking and decoloring, centrifuging for 10min at the rpm of 12000 after 24h, discarding the precipitate, filtering and sterilizing the supernatant, and using the supernatant as water for soybean germination for later use.

Mixing 90 soybean seeds with 3-5% H₂O₂Sterilizing the solution for 5min, washing with sterile water for 3-5 times, placing into sterilized large plate with double-layer filter paper, adding 20mL of malachite green metabolite (1000mg/L malachite green) or 1000mg/L malachite green solution, setting sterile water group (sterile water instead of malachite green) as control group, culturing in 30 deg.C incubator in dark place, measuring soybean germination number in the second day, and measuring root length.

The experimental results are shown in fig. 4, and it can be seen from fig. 4 that:

the germination rate of the soybeans is not influenced by malachite green (figure 4), and the germination conditions of malachite green groups are similar to those of a control group and are all about 93 percent; the malachite green has a great influence on the development of soybean root tips, the soybean root stems of the control group grow about 18mm on the next day, while the size of the malachite green treatment group is only about 7mm (figure 4), and the difference is obvious. According to the figure 4, the degradation products have no obvious influence on the soybean germination condition, which is about 93 percent, the supernatant liquid decolorized by the strain SS01 still has influence on the subsequent growth of the soybean, the condition is similar to that of malachite green group, which influences the normal growth of the soybean rhizome (figure 4S), the degradation products degraded by the bacterial manganese compound have obvious influence on the soybean, the condition is close to that of a control group (figure 4SM), the rhizome length in the next day is 3mm smaller than that of the control group on the average, but the rhizome length is larger than 0.05 through T test, so that the significant difference does not exist, the product degraded by the bacterial manganese compound is completely detoxified, and the degradation products do not have substances with stronger toxicity.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.