CN111378597B

CN111378597B - Manganese oxidizing bacterium capable of being used for demanganization and application thereof

Info

Publication number: CN111378597B
Application number: CN202010052135.8A
Authority: CN
Inventors: 王进; 李平; 岳正波; 张林义; 陈明珠; 邵锐; 许庆升
Original assignee: Hefei University of Technology
Current assignee: Hefei University of Technology
Priority date: 2020-01-17
Filing date: 2020-01-17
Publication date: 2022-07-12
Anticipated expiration: 2040-01-17
Also published as: CN111378597A

Abstract

The invention discloses a manganese oxidizing bacterium capable of being used for efficient demanganization and application thereof, wherein the bacterium is bacillus, is classified and named as Brevibacillus sp.MM1, is preserved in Guangdong province microorganism strain preservation center, and has the preservation number of GDMCC NO: 60818, preservation date of 2019, 10 months and 23 days. The bacillus (Brevibacillus sp. MM1) has higher manganese oxidation capacity, and can efficiently remove Mn in a water body or a solid matrix under acidic and neutral conditions²⁺Meanwhile, other heavy metals including iron, copper, zinc, chromium, cadmium and arsenic can be removed, and the application prospect is wide.

Description

Manganese oxidizing bacterium capable of being used for demanganization and application thereof

Technical Field

The invention belongs to the field of environmental engineering, and particularly relates to a manganese oxidizing bacterium capable of being used for efficient demanganization and application thereof.

Background

Because the manganese ion concentration in water and soil exceeds the standard and seriously harms human health due to industrial activities such as mining, electrolysis, steel making and the like, the demanganization technology becomes a research hotspot of all countries in the world.

In the manganese compound, the lower the valence of manganese, the more toxic, so Mn²⁺The threat to human beings is greatest. Manganese oxidation is a highly active mineralization stage, and manganese oxides are natural strong oxidizers, which play an important role in the biogeochemical cycle. The oxidation rate of the manganese oxidation process mediated by microorganisms is far higher than that of non-biological oxidation, and the generated biological manganese oxide can oxidize various organic and inorganic compounds in the nature, remove heavy metals and serve as an electron acceptor for anaerobic respiration while removing manganese in the environment, and has high application value in the treatment and restoration of heavy metal and organic matter polluted environments. Therefore, the screening of manganese-resistant demanganizing microorganisms and the development of biological demanganizing technology have far-reaching significance in the treatment of manganese-polluted soil and the restoration of manganese-polluted water.

The manganese oxidizing bacteria are widely distributed in the environment and commonly exist in seawater, fresh water, soil and various sediments. Typical p-Mn that has been isolated so far²⁺Bacteria with oxidizing ability are mainly classified into the following groups: aeromonas (Aeromonas), Metallobacterium (Metallogenin), Pseudomonas (Pseudomonas), Bacillus (Bacillus), Cellulus (Leptothrix), Microbacterium (Pedomicrobium), Vibrio (Vibrio), Microthrix (Clothrix), Arthrobacter (Arthrobacter), Microbacterium (Hyphomicium), and Spirospirium (Oceanospirillum).

In the research of treating manganese pollution by microorganisms at the near stage, the method for treating manganese pollution by microorganisms is applied to engineering practice and has good effect. However, the manganese oxidizing bacteria have been published and reported to be mainly used under the condition of neutral pH, and reports of screening for obtaining the manganese oxidizing bacteria in an extremely acidic environment are relatively limited. On the other hand, since acidic mine wastewater and industrial wastewater generally contain a plurality of metal ions at the same time, it is necessary to screen out acid-resistant manganese-oxidizing bacteria capable of removing a plurality of heavy metals at the same time for treating acidic manganese-containing mine wastewater or other acidic manganese-containing industrial wastewater.

Disclosure of Invention

The invention aims to provide a strain of manganese oxidizing bacteria for efficient demanganization and application thereof in a water body or a solid matrix.

In order to realize the purpose of the invention, the following technical scheme is adopted:

in a first aspect, the invention provides a manganese oxidizing bacterium which can be used for efficient demanganization, wherein the bacterium is bacillus, is classified and named as Brevibacillus sp, is deposited in Guangdong province microorganism culture collection center, and has a deposit number of GDMCC NO: 60818, the preservation date is 2019, 10 and 23 days, and the preservation address is History No. 59, 5 building of the Jiedu Zhonglu No. 100 prefecture in Guangzhou city.

Bacillus (Brevibacillus sp.) MM1 is a strain of acid-resistant facultative anaerobic manganese oxidizing bacteria screened from acidic mine wastewater in Maanshan, and the capacity of removing manganese of the strain under acidic conditions is improved through domestication, so that a strain of manganese oxidizing bacteria capable of efficiently removing manganese is obtained, and other heavy metals can be removed by the strain.

The bacillus (Brevibacillus sp.) MM1 is a gram-positive bacterium, is in the shape of a short rod (0.6-1.2 mu m), has motility, cannot hydrolyze starch, is oxidized and fermented by oxidized glucose, is positive in catalase, negative in oxidase, negative in V-P experiment, negative in methyl red experiment, negative in indole experiment and positive in nitrate reduction experiment.

Further, the Bacillus (Brevibacillus sp.) MM1 can grow under the condition that the pH value is 4-7 and has Mn²⁺The oxidation activity is preferably 5.5 to 7 in terms of pH.

Further, the Bacillus (Brevibacillus sp.) MM1 is at Mn²⁺At a concentration of not more than 30mM, capable of growing and having Mn²⁺And (4) oxidation activity.

In a second aspect, the present invention provides a microbial agent for manganese removal, comprising the bacillus (Brevibacillus sp.) MM1 as an active ingredient. Appropriate auxiliary materials can be added into the microbial agent according to needs.

In a third aspect, the invention provides the bacillus (Brevibacillus sp.) MM1 for removing Mn in water body or solid matrix²⁺The use of (1). The bacillus (Brevibacillus sp.) MM1 can be used for killing bacillusThe Mn exists and develops in the water body or the solid matrix of the bacteria²⁺Oxidation activity and thus can be used for removing Mn from water or solid matrix²⁺。

In a fourth aspect, the invention provides a method for removing Mn from a water body or a solid matrix²⁺The method comprises the steps of inoculating the bacillus (Brevibacillus sp.) MM1 or a microbial agent containing the bacillus (Brevibacillus sp.) MM1 into a water body or a solid matrix to be treated, and culturing under the condition that the pH is 4-7 (preferably 5.5-7), namely, finishing Mn²⁺And (4) removing.

Further, the culture time varies depending on the activity at the time of bacterial addition and the conditions of the water body or the solid substrate.

Further, Mn can be performed using MM1 of Bacillus (Brevibacillus sp.) of the present invention²⁺The removed water body includes but is not limited to acid mine wastewater, acid industrial wastewater, domestic wastewater, underground water or tap water, and Mn can be performed by using the bacillus (Brevibacillus sp.) MM1 of the invention²⁺The solid substrate removed includes, but is not limited to, soil or sediment.

In addition, the bacillus (Brevibacillus sp.) MM1 and the microbial agent containing the bacillus (Brevibacillus sp.) MM1 can remove Mn²⁺In addition, other heavy metals (e.g., iron, copper, zinc, chromium, cadmium, arsenic) can be removed.

The invention has the beneficial effects that:

1. the bacillus (Brevibacillus sp.) MM1 has higher manganese oxidation capacity, and can efficiently remove Mn in a water body or a solid matrix under acidic and neutral conditions²⁺Meanwhile, other heavy metals including iron, copper, zinc, chromium, cadmium and arsenic can be removed, and the application prospect is wide.

2. The bacillus (Brevibacillus sp.) MM1 has acid resistance, is facultative anaerobe, has good activity in the presence of low dissolved oxygen, has strong environmental adaptability, can be used for removing heavy metals in acid mine wastewater or acid industrial wastewater, can adapt to wastewater under different dissolved oxygen conditions, and is also suitable for biological in-situ remediation technology in an anoxic environment.

3. Bacillus sp (Brevibacillus sp.) MM1 of the invention is in Mn²⁺Can grow at a concentration of 30mM and has Mn²⁺Oxidation activity, the concentration is far higher than Mn in common water body and solid matrix²⁺And (4) concentration.

4. According to the invention, the bacillus (Brevibacillus sp.) MM1 can be prepared into a biological material by an immobilization technology, and the biological material has the advantages of low cost, strong adaptability to different polluted water bodies, no secondary pollution and good application prospect in the biological treatment process of manganese-containing wastewater and underground water compared with a physical and chemical method.

Drawings

FIG. 1 is a xps scan of a solid-phase product generated by oxidizing manganese ions with Bacillus (Brevibacillus sp.) MM1 in example 1 of the present invention (in FIG. 1, Raw represents detected Raw Peak data without processing, Background represents instrument Background, Peak 1-3 represents a manganese valence Peak obtained after processing Raw data, and Sum represents the Sum of all data after processing, which is used for comparing with Raw to ensure that data after processing are not distorted).

FIG. 2 is a scanning electron micrograph of Bacillus (Brevibacillus sp.) MM1 in example 2 of the present invention.

FIG. 3 shows the gram identification result of Bacillus (Brevibacillus sp.) MM1 in example 2 of the present invention.

FIG. 4 is a phylogenetic tree diagram of Bacillus sp MM1 according to example 2 of the present invention.

FIG. 5 is a diagram showing the change of the bacterial concentration of Bacillus (Brevibacillus sp.) MM1 under different pH conditions (upper panel in FIG. 5) and the removal rate of manganese element (lower panel in FIG. 5) in example 3 of the present invention.

FIG. 6 is a graph showing the removal rate of Bacillus (Brevibacillus sp.) MM1 at different initial manganese ion concentrations in example 4 of the present invention.

Detailed Description

The following examples are given for the detailed implementation and the specific operation procedures, but the scope of the present invention is not limited to the following examples.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The formulations of the media and solutions used in the following examples are as follows:

the LB culture medium has the formula: 10g/L tryptone, 10g/L NaCl, 5g/L yeast extract, autoclaving at 121 deg.C for 20 min.

The formula of the manganese-containing liquid culture medium is as follows: 10g/L tryptone, 10g/L NaCl, 5g/L yeast extract, 0.755g/LMnSO₄And carrying out autoclaving at 121 ℃ for 20min under the protection of nitrogen.

The formula of the Postage liquid culture medium is as follows (the concentration of each component in the liquid A and the liquid B is the concentration in the final culture medium):

solution A: 0.5g/L KH₂PO₄、1.0g/LNH₄Cl、1.0g/LNa₂SO₄、0.1g/LCaCl₂·2H₂O、2.0g/LMgSO₄·7H₂O, 1.0g/L yeast extract and 3.5g/L sodium lactate, after all the medicines are fully dissolved, adjusting the pH value of a culture medium to 7, subpackaging the obtained solution in 50mL serum bottles, filling excessive nitrogen into each bottle, and sterilizing the bottle for 20min at 121 ℃ in a high-pressure steam sterilization pot under the protection of nitrogen, wherein the final volume of the culture medium is 40 mL;

and B, liquid B: 0.1g/L ascorbic acid, 0.5g/L (NH)₄)₂Fe(SO₄)₂·6H₂O；

Cooling the solution A to below 60 deg.C, and injecting the solution B sterilized by filtration with 0.22 μm filter tip in sterile environment with sterile disposable syringe.

The formula of the Postage solid culture medium is as follows (the concentration of each component in the solution A and the solution B is the concentration in the final culture medium):

solution A: 0.5g/L KH₂PO₄、1.0g/LNH₄Cl、1.0g/LNa₂SO₄、0.1g/LCaCl₂·2H₂O、2.0g/LMgSO₄·7H₂O, 1.0g/L yeast extract, 3.5g/L sodium lactate and 15g/L agar powder, adjusting the pH value of a culture medium to 7 after all the medicines are fully dissolved, subpackaging the medicines into 500mL wide-mouth bottles, and sterilizing for 20min at 121 ℃ in an autoclave under the protection of nitrogen;

Cooling the solution A to below 60 deg.C, adding the solution B sterilized by filtration with 0.22 μm filter head, and keeping the temperature at 60 deg.C.

Example 1 screening isolation and purification of Bacillus (Brevibacillus sp.) MM1

The procedure for the selection of Bacillus (Brevibacillus sp.) MM1 in this example was as follows:

(1) preparation of a culture medium: an LB culture medium, a manganese-containing liquid culture medium, a Postage liquid culture medium and a Postage solid culture medium are prepared according to the formula.

Preparing a double-layer Postage solid plate: pouring a layer of Postage solid plate, and pouring a layer of plate to cover immediately after the inoculation operation is finished, so that the thallus can grow in a facultative anaerobic environment.

(2) Enrichment of a bacterial source: inoculating the bacterial liquid extracted from the acid mine wastewater of Maanshan in a Postage liquid culture medium according to an inoculation ratio of 5 percent (volume ratio), performing light-shielding culture on a constant-temperature shaking table at 35 ℃ and 120rpm for about 15 days, selecting a serum bottle with obviously turbid culture medium, repeating the steps for several times, and obtaining the high-concentration bacterial liquid.

(3) Separation and purification of manganese oxidizing bacteria: diluting the high-concentration bacterial liquid obtained in the step (2) in sterile water according to multiples of 10, 100, 1000 and 10000, uniformly coating 20 mu L of diluted bacterial liquid on a double-layer Postage solid plate in a sterile environment, culturing for 3d in a constant-temperature light-proof incubator at 35 ℃, selecting a solid plate with a moderate colony number, selecting a single colony and scribing on the double-layer Postage solid plate, and repeating the step until the colony forms are uniform to obtain the bacterial strains to be screened.

(4) Primary screening of manganese oxidizing bacteria: and (4) selecting the strain to be screened obtained in the step (3), and inoculating the strain to an LB culture medium. After culturing for 48h at 35 ℃ and 120rpm with a constant temperature shaking table, inoculating the manganese-containing liquid culture medium with the inoculum size of 1 percent (volume ratio). After shaking culture at 35 deg.C and 120rpm for 72h, the residual manganese concentration in the solution was measured by flame atomic absorption method, and the pH of the culture was measured, which was compared with the sterile blank test group cultured under the same conditions. The result shows that the strain with the number of MM1 in the initial screening stage shows strong removal capacity to manganese, and the removal rate to manganese can reach 93.1% after 3d of culture at the initial manganese concentration of 5 mM.

(5) Re-screening of manganese oxidizing bacteria: inoculating the strain with the number of MM-1 obtained by primary screening into a manganese-containing liquid culture medium, carrying out shake culture at the constant temperature of 35 ℃ and 120rpm for 72h, taking bacterial liquid, centrifuging at the rotating speed of 8000xg in a low-temperature high-speed centrifuge to obtain a precipitate, carrying out vacuum drying on the precipitate, grinding into powder, and carrying out xps scanning, wherein as shown in figure 1, the peak value of manganese in an oxidation state in the precipitate is detected, and the manganese oxide capacity of the strain is indicated. The strain with manganese oxidation activity (namely the strain with the number of MM1 obtained by screening in the invention) is inoculated in a glycerol tube and stored in a refrigerator at the temperature of minus 80 ℃. A strain with the number of MM1 is obtained through the separation of the above route, and the strain is proved to have the capacity of manganese oxidation.

Example 2 identification of manganese-oxidizing bacteria

Performing morphological identification, biochemical identification and 16S rRNA sequencing analysis on the obtained strain with the number of MM 1:

1. the physiological growth form of the strain is as follows: the liquid culture medium is dispersed without forming a mycoderm; the bacterial colony on the solid culture medium is white, opaque, smooth in surface and regular in edge;

2. the bacterium is a coryneform bacterium, and the scanning electron microscope results are shown in FIG. 2.

3. As shown in fig. 3, the gram-positive bacteria were identified as gram-positive bacteria: gram-positive bacteria are known to be bluish purple and gram-negative bacteria are known to be red, and gram-positive bacteria are known to be bluish purple after gram staining in fig. 3.

4. The biochemical identification result is as follows: the product is facultative anaerobic, is in a short rod shape (0.6-1.2 mu m), has motility, can not hydrolyze starch, is oxidized and fermented by oxidized glucose, is positive by catalase, negative by oxidase, negative by V-P experiment, negative by methyl red experiment, negative by indole experiment and positive by nitrate reduction experiment.

5. Bacterial genome DNA extraction and 16S rRNA sequence analysis:

(1) the bacterial genome DNA extraction, PCR amplification and sequencing experiments are all completed by the agency of biological engineering (Shanghai) corporation, and the obtained gene sequence is shown as SEQ ID NO. 1.

(2) The obtained sequences were compared for nucleotide homology in GeneBank by the BLAST program on NCBI (national Center for Biotechnology information). The result showed that the strain MM1 in GeneBank has 68% similarity with the accession number NR040981.1, and is identified as Bacillus and named Brevibacillus sp.MM1. The phylogenetic tree is shown in fig. 4.

Example 3 Bacillus (Brevibacillus sp.) MM1 for treating manganese-containing wastewater with different pH values

The cryopreserved strain was thawed and inoculated into filter-sterilized LB medium, and cultured at 35 ℃ at 120rpm for 1d to restore its activity.

Quantitative manganese sulfate was previously added to LB medium to an initial manganese ion concentration of 10mM, and initial pH was adjusted to 4, 5.5, 7 and 8, respectively. The inoculum size was adjusted to give an initial inoculum OD600 value of 0.1 in the medium, and a blank set was set. Culturing at 35 deg.C and 120rpm, sampling at 1d, 2d and 3d, centrifuging, collecting supernatant, filtering, testing manganese content (flame atomic absorption spectrometry), comparing with blank group, and determining bacterial concentration change and manganese removal efficiency of strain under different pH conditions, with the results shown in upper graph of FIG. 5 and lower graph of FIG. 5.

When bacillus sp MM1 was adapted for a period of time, higher concentrations could still be achieved than at an initial pH of 7, and finally for Mn, when the initial pH was 5.5 (Brevibacillus sp.)²⁺The ions have certain removal capacity; while when the pH was 4, Bacillus (Brevibacillus sp.) MM1 showed characteristics of not being well adapted to the culture environment and was resistant to Mn²⁺The ion-removing ability was rather low, so that it was presumed that the strain was weak toThe acidic environment has better adaptability, can treat the waste water with weak acidity, but can not adapt to the environment with strong acidity. When the pH is 8, the strain also shows characteristics that it does not well adapt to the culture environment, and it is resistant to Mn²⁺The removal of ions is weak, indicating that the strain is not suitable for growth under more alkaline initial conditions.

Therefore, the strain is suitable for repairing weakly acidic manganese-containing acidic mine wastewater or acidic industrial wastewater.

Example 4 different Mn²⁺Bacillus (Brevibacillus sp.) MM1 vs. Mn at concentration²⁺Removal of ions

Manganese sulfate with different concentrations is added in advance into LB culture medium and the inoculation amount is adjusted to ensure that the initial Mn in the culture medium²⁺The concentrations were 1, 5, 10, 20 and 30mM, respectively, the OD600 value of the initial bacterial suspension was 0.1, and a blank set was set. The culture was carried out at 35 ℃ and 120rpm, and samples were taken at the time of 1d, 2d and 3d of culture, respectively. Centrifuging the bacterial liquid, collecting supernatant, filtering, testing manganese content (flame atomic absorption spectrometry), comparing blank groups, and determining strain Mn at different Mn²⁺The removal efficiency of manganese element at the concentration is shown in fig. 6. When Mn is present²⁺When the concentration is less than or equal to 10mM, the strains Brevibacillus sp.MM1 are all aligned to Mn²⁺The removal rate of the catalyst is as high as more than 90 percent, and when Mn is used²⁺When the concentration reaches 30mM, 20 percent of Mn can still be removed² ⁺. The strain can be used for efficiently repairing manganese-containing underground water and treating high-concentration manganese-containing wastewater.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

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aattccacgt gtagcggtga aatgcgtaga gatgtggagg aacaccagtg gcgaaggcgg 720

ctttctggtc tgtaactgac gctgaggcgc gaaagcgtgg ggagcaaaca ggattagata 780

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Claims

1. A strain of manganese oxidizing bacteria for demanganization, characterized in that: the bacteria are bacillus, are classified and named as Brevibacillus sp, are preserved in Guangdong province microorganism strain preservation center, and have the preservation number of GDMCC NO: 60818, preservation date of 2019, 10 months and 23 days.

2. The manganese oxidizing bacterium according to claim 1, wherein: the bacterium can grow under the condition that the pH value is 4-7 and has Mn²⁺And (4) oxidation activity.

3. The manganese oxidizing bacterium according to claim 1, wherein: the bacterium is in Mn²⁺At a concentration of not more than 30mM, capable of growing and having Mn²⁺And (4) oxidation activity.

4. The manganese oxidizing bacterium according to claim 1, wherein: the bacteria are facultative anaerobes.

5. The manganese oxidizing bacterium according to claim 1, 2, 3 or 4, wherein: the bacteria can be in non-sterile water body or solidSurvival and development of Mn in the matrix²⁺And (4) oxidation activity.

6. The manganese oxidizing bacterium according to claim 5, wherein: the water body comprises at least one of acid industrial wastewater, domestic wastewater, underground water and tap water; the solid substrate includes at least one of soil and sediment.

7. A microbial agent for removing manganese, which is characterized in that: the microbial agent comprises the manganese-oxidizing bacterium according to claim 1 as an active ingredient.

8. Use of the manganese-oxidizing bacteria of claim 1, wherein: for removing Mn in water body or solid matrix²⁺。

9. Method for removing Mn in water body or solid matrix²⁺The method of (2), characterized by: inoculating the manganese-oxidizing bacteria of claim 1 or the microbial agent of claim 7 into a water body or a solid substrate to be treated, and culturing at a pH of 4-7.