CN110951641B

CN110951641B - Bacillus subtilis, microbial agent and application thereof

Info

Publication number: CN110951641B
Application number: CN201911268190.4A
Authority: CN
Inventors: 蒋细良; 吴蓓蕾; 李梅
Original assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Priority date: 2019-12-11
Filing date: 2019-12-11
Publication date: 2022-01-07
Anticipated expiration: 2039-12-11
Also published as: CN110951641A

Abstract

The disclosure relates to bacillus subtilis and application thereof. The preservation number of the Bacillus subtilis is CGMCC No. 16828. The present disclosure also provides a microbial agent containing the bacillus subtilis as described above as an effective ingredient. The disclosure also provides the bacillus subtilis and the application of the microbial agent in treating printing and dyeing wastewater. Through above-mentioned technical scheme, this openly can get rid of the organic matter in the printing and dyeing waste water more fast effectively, can handle the printing and dyeing waste water to safe emission standard in the short time.

Description

Bacillus subtilis, microbial agent and application thereof

Technical Field

The disclosure belongs to the technical field of microorganisms, and particularly relates to bacillus subtilis, a microbial agent and application thereof.

Background

The printing and dyeing wastewater is wastewater discharged from cotton, hemp, chemical fiber and blended products thereof, silk printing and dyeing, wool dyeing and finishing, silk factories and the like. The types and the processing techniques of the fibers are different, and the water quantity and the water quality of the printing and dyeing wastewater are also different. Wherein the amount of the wastewater in the printing and dyeing mill is large, 100-200 t of water is consumed for every 1t of textile processed by printing and dyeing, and 80-90% of the wastewater is discharged. The printing and dyeing wastewater has the characteristics of large water quantity, high organic pollutant content, large alkalinity, large water quality change and the like, belongs to one of industrial wastewater difficult to treat, and contains dye, slurry, auxiliary agent, oil agent, acid and alkali, fiber impurities, sand substances, inorganic salt and the like.

The discharge amount of the printing and dyeing wastewater is large, according to European statistics, the weight ratio of the fabric to the discharged wastewater is 1: 150-1: 200, and the weight ratio of the fabric to the discharged wastewater is about 1: 200-1: 400 in China. The textile industry wastewater in China is the sixth grade of national industrial wastewater discharge, and 80% of the wastewater belongs to printing and dyeing wastewater.

The printing and dyeing wastewater belongs to one of industrial wastewater which is difficult to treat. For technical and economic reasons, most of the currently adopted biological-physical treatment methods can only meet basic emission requirements. Although the chroma is slightly reduced, the organic substances are only decomposed into smaller substances, the properties of the decomposition products are difficult to control and control, and no harm to the environment can be ensured. And the existing treatment method has large floor area, more investment and high treatment cost, so that the production cost is high. It is estimated that the treatment cost is basically equivalent to the city tap water price when the wastewater after treatment reaches the secondary discharge standard. If the requirement of wastewater reuse is met, the treatment cost is higher, so the practical operation is quite difficult.

Disclosure of Invention

In order to overcome the defect that the existing sewage treatment method cannot sufficiently and effectively print and dye organic pollutants in wastewater, the invention provides bacillus subtilis.

The inventor of the present disclosure isolated a strain of bacillus subtilis, and found that it can efficiently degrade organic pollutants in printing and dyeing wastewater, and can perform innocent treatment of printing and dyeing wastewater, thereby obtaining the present invention.

In order to achieve the above object, the present disclosure provides, in a first aspect, a Bacillus subtilis having a accession number of CGMCC No. 16828.

The second aspect of the disclosure provides a microbial agent, which comprises thallus and a culture medium, wherein the thallus contains bacillus subtilis with the preservation number of CGMCC No. 16828.

Optionally, in each gram of the microbial agent, the viable count of the bacillus subtilis with the preservation number of CGMCC No.16828 is 10⁸～10¹⁰CFU。

Optionally, the medium is beef extract peptone medium, nutrient broth medium, or LB medium, or a combination of two or three thereof.

In a third aspect of the present disclosure, there is provided a use of the bacillus subtilis according to the first aspect of the present disclosure or the microbial agent according to the second aspect of the present disclosure in treating printing and dyeing wastewater.

Optionally, the method for treating printing and dyeing wastewater comprises the following steps: mixing bacillus subtilis or the microbial agent as claimed in any one of claims 2 to 4 with the printing and dyeing wastewater for biodegradation treatment, then adding a flocculating agent into the wastewater after biodegradation treatment for coagulating sedimentation, and obtaining purified water after solid-liquid separation.

Optionally, the flocculant is an organic flocculant and/or an inorganic flocculant; the inorganic flocculant is aluminum sulfate, aluminum chloride, alum, ferrous sulfate hydrate, ferric sulfate, ferric chloride, polyaluminum sulfate, polyferric chloride or polyferric sulfate, or a composition of two or three of the aluminum sulfate, the aluminum chloride, the alum, the ferrous sulfate hydrate, the ferric sulfate, the ferric chloride, the polyaluminum chloride and the polyferric sulfate; the organic flocculant is a polyacrylamide flocculant.

Through the technical scheme, the bacillus subtilis with the preservation number of CGMCC No.16828 can quickly and efficiently degrade organic pollutants in the printing and dyeing wastewater. The bacillus subtilis and the microbial agent containing the bacillus subtilis can treat printing and dyeing wastewater to a safe discharge standard in a short time, the treatment process is safe, environment-friendly and pollution-free, and the treatment cost is low.

Additional features and advantages of the disclosure will be set forth in the detailed description which follows.

Biological material preservation

The Bacillus subtilis is a pure culture separated from a sewage sample collected from a Beijing river channel by the inventor, the preservation number of the Bacillus subtilis is CGMCC No.16828, the preservation date of the Bacillus subtilis is 2018, 11 months and 27 days, the preservation unit is the common microorganism center of the China Committee for culture Collection of microorganisms, the address of the Bacillus subtilis is located in the microbial research institute of China academy of sciences No. 3 of West Lu No.1 Hosth of the sunward area in Beijing, and the Bacillus subtilis is named in a classified manner.

Detailed Description

The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.

The inventor of the present disclosure isolated and cultured a new strain from a sewage sample collected from Beijing river, and identified the strain to be Bacillus subtilis. The strain is preserved in the common microorganism center of China microorganism culture preservation management Committee of China preservation units appointed by the State intellectual Property office, the preservation date is 11 months and 27 days in 2018, and the preservation number is CGMCC No. 16828.

The first aspect of the disclosure provides a Bacillus subtilis with a preservation number of CGMCC No. 16828.

The bacillus subtilis disclosed by the invention can survive and grow and reproduce in a conventional bacterial culture medium. The bacillus subtilis disclosed by the invention is an excellent organic matter degrading bacterium, and can be used for treating printing and dyeing wastewater very efficiently and quickly.

Wherein the amount of thallus contained in the microbial agent can be changed within a wide range, for example, the viable count of the bacillus subtilis with the preservation number of CGMCC No.16828 can be 10 in each gram of the microbial agent⁸～10¹¹CFU, preferably, per gram of microbial agent, the viable count of Bacillus subtilis with preservation number of CGMCC No.16828 can be 10⁹～10¹⁰CFU。

According to the present disclosure, the culture medium may be various kinds of culture media that can be used for culturing bacillus subtilis, for example, a common culture medium such as beef extract peptone medium, broth medium, LB medium, etc. may be used as a seed medium for preservation of the strain; while fermentation media are generally used for production, the variety of such media is also well known to those skilled in the art. The above-mentioned medium can be commercially available or prepared according to the description of "microbiological Culture Medium Manual". For example, the seed medium may be a beef extract peptone medium containing 1-3g/L beef extract, 5-15g/L peptone, 3-8g/L sodium chloride and 15-20g/L agar. For example, the fermentation medium may contain: 40-150g/L of starch, 0.5-8g/L of sodium chloride, 0.5-5g/L of calcium carbonate, 2-10g/L of monopotassium phosphate and 1-10g/L of ferrous chloride.

Wherein, the above-mentioned various culture mediums can be sterilized according to conventional sterilization methods for use, for example, under the conditions of 115 ℃ and 125 ℃ and 1.5-2 standard atmospheric pressures for 10-30 minutes.

Among them, the method for culturing Bacillus subtilis is not particularly limited, and for example, Bacillus subtilis may be first cultured in a seed medium (LB medium) to a density OD₆₀₀The value is 0.6-0.8, obtaining bacterial liquid, inoculating 2-5 weight parts of bacterial liquid of bacillus subtilis in 100 weight parts of culture medium (15 g of glucose, 1g of starch, 25g of bean cake powder, 1g of manganese sulfate, 1.5g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, 0.2g of yeast extract, 0.1g of ferric chloride, 0.1g of calcium carbonate and pH7.0-7.2), and culturing at 37 ℃ until the viable count of the bacillus subtilis is (2-20) multiplied by 10⁷One per gram of medium.

Preferably, the viable count of the bacillus subtilis with the preservation number of CGMCC No.16828 in each gram of the microbial agent obtained by culturing can be 10⁷-10¹¹CFU, more preferably 10⁹-10¹⁰And (4) CFU. During the culturing, the concentration of viable bacteria can be obtained by a conventional method such as a hemocyte count method or an OD value observation method.

The cultured bacterial liquid can be directly used as a microbial agent, and preferably, the bacterial liquid is further processed into a microbial agent in a more convenient storage formulation by the steps of sterile filtration, freeze drying and the like. The culture conditions of the strains are not particularly limited, and can be the conditions commonly used in the process of culturing the bacillus subtilis, for example, shaking culture is adopted, the culture temperature can be 28-37 ℃, and the culture time can be 1-3 days.

The water quality of the printing and dyeing wastewater is not particularly required, for example, the BOD5 of the printing and dyeing wastewater can be 150-300 mg/L; CODcr can be 400-700 mg/L; the SS may be 200 to 500mg/L, and the chroma may be 400 to 1000 times.

According to the present disclosure, a method for treating printing and dyeing wastewater by using bacillus subtilis with the preservation number of CGMCC No.16828 or a microbial agent containing the bacillus subtilis can comprise the following steps:

introducing the printing and dyeing wastewater into a grid tank, an adjusting tank, a sedimentation tank, a hydrolysis acidification tank, an anaerobic tank, an aerobic activated sludge tank, a biological contact oxidation tank, a secondary sedimentation tank, a coagulation sedimentation tank and a biological activated carbon tank in sequence, and finally obtaining purified effluent: wherein the bacterial liquid or microbial agent containing the bacillus subtilis with the preservation number of CGMCC No.16828 can be added into a biological contact oxidation tank, and the adding amount can be 10 according to the number of viable bacteria per cubic meter of liquid⁸-10¹²CFU/day. In another embodiment, the liquid obtained from the regulating tank can be introduced into the integrated membrane bioreactor for treatment, and a microbial agent is added into a contact oxidation section of the membrane bioreactor.

In order to further improve the effect of degrading and adsorbing and removing the pollutants in the printing and dyeing wastewater, in one embodiment, the immobilized bacteria agent can be formed by fixing the bacteria solution of bacillus subtilis with CGMCC No.16828 on a carrier, for example, the immobilized bacteria agent is formed by fixing the bacteria solution of bacillus subtilis 10⁸-10¹²And (3) mixing, stirring and standing the CFU/mL bacterial liquid and activated carbon to enable the bacterial liquid to be adsorbed on the activated carbon, and after drying, feeding the activated carbon adsorbed with the bacterial liquid into a biological contact oxidation tank for contact oxidation.

Further, in order to promote the removal of pollutants from the printing and dyeing wastewater by coagulation, a flocculant may be added to the liquid after the biological oxidation treatment, for example, a flocculant may be added to a sedimentation coagulation tank. Flocculants may be of a type conventional in the art, for example inorganic flocculants such as aluminium sulphate, aluminium chloride, alum, ferrous sulphate hydrate, ferric sulphate, ferric chloride, polyaluminium chloride (PAC), polyaluminium sulphate (PAS), polyaluminium chloride (PFC) or polyferric sulphate (PFS), or a combination of two or three thereof; the organic flocculant may be an organic polymeric flocculant, for example, a polyacrylamide-based flocculant, and specifically, may include polyacrylamide, and/or a polymer obtained by graft-copolymerizing a natural polymeric substance such as starch, cellulose, chitosan, or the like with acrylamide.

The present invention will be described in further detail with reference to examples, but the scope of the present invention is not limited to the following examples.

Example 1

Inoculating Bacillus subtilis with preservation number of CGMCC No.16828 into beef extract peptone culture solution from a preservation slant, and shake-culturing at 37 deg.C and 180rpm for 12h to obtain seed solution. The beef extract peptone medium comprises the following components: beef extract 5.0g/L, peptone 10.0gL, NaCl5.0gL, pH 7.2-7.4. Adding 100mL of the seed bacterial liquid into 100L of fermentation medium (the fermentation medium contains 80g/L of starch, 3g/L of sodium chloride, 3g/L of calcium carbonate, 5g/L of potassium dihydrogen phosphate and 5g/L of ferrous chloride), culturing at 30 ℃, sampling in the culture process and observing by a microscope direct counting method until the viable count of the bacillus subtilis in each gram of the culture solution is 10¹⁰CFU, the obtained culture solution is the microbial agent of this example.

Comparative example 1

A strain with the preservation number of CGMCC No.8172, purchased from China general microbiological culture Collection center, is inoculated into a shake flask culture medium (the shake flask culture medium contains 5g/L of peptone, 5g/L of yeast extract powder, 10g/L of NaCl, 1g/L of glucose and pH7.0-7.2) from a preservation inclined plane, and is subjected to shaking culture at 35 ℃ and 180rpm until the logarithmic phase, so as to prepare a seed solution for later use. Adding 100mL of the above seed bacterial liquid into 100L of fermentation medium (the fermentation medium contains 5g/L peptone, 5g/L yeast extract powder, 10g/L NaCl, 2g/L glucose, pH7.0-7.5), culturing at 35 deg.C, sampling during the culture process, and observing by microscope direct counting method until the viable count of Rhodococcus in each gram of culture solution reaches 10¹⁰CFU, the culture solution obtained is the microbial inoculum of the comparative example.

Comparative example 2

From Biwofeng Biotech GmbH, Foshan

A CW agent.

Comparative examples 3 to 6

Respectively inoculating 4 strains of bacillus subtilis separated together with the bacillus subtilis with the preservation number of CGMCC No.16828 into beef extract peptone culture solution, carrying out shake culture on a shaker at 37 ℃ and 180rpm for 12 hours, and respectively marking the obtained bacterial solutions as seed solutions of comparative examples 3-6. Adding 100mL of the seed bacterial liquid into 100L of fermentation medium (the fermentation medium contains 80g/L of starch, 3g/L of sodium chloride, 3g/L of calcium carbonate, 5g/L of potassium dihydrogen phosphate and 5g/L of ferrous chloride), culturing at 30 ℃, sampling in the culture process and observing by a microscope direct counting method until the viable count of the bacillus subtilis in each gram of the culture solution is 10¹⁰And CFU, wherein the obtained culture solution is the microbial agent of the comparative examples 3-6.

Test example 1

Testing the capability of the microbial agent in treating printing and dyeing wastewater in the integrated membrane bioreactor; 267.4mg/L, CODcr of BOD5 in the wastewater of a certain dye mill is 325mg/L of 620.8mg/L, SS, 107.5mg/L of ammonia nitrogen and 660 times of chroma.

The treatment process comprises the following steps: the printing and dyeing wastewater is sequentially introduced into a grid tank, a regulating tank, an integrated membrane bioreactor, a coagulating sedimentation tank, a disinfection tank and effluent, microbial agents of examples and comparative examples are respectively added into a contact oxidation section of the integrated membrane bioreactor, the effluent is collected after normal operation for water quality detection, and the results are listed in table 1.

TABLE 1

Test example 2

Testing the capability of the microbial agent in treating printing and dyeing wastewater in a biological aeration tank; the BOD5 in the wastewater of a certain dye and print mill is 190mg/L, CODcr, 425mg/L, SS and 182mg/L, the ammonia nitrogen is 57mg/L and the chroma is 480 times.

The treatment process comprises the following steps: the printing and dyeing wastewater is sequentially introduced into a grid tank, an adjusting tank, a sedimentation tank, a hydrolysis acidification tank, an anaerobic tank, an aerobic activated sludge tank, a biological contact oxidation tank, a secondary sedimentation tank, a coagulation sedimentation tank, a biological activated carbon tank and effluent, and the microbial agents of example 1 and comparative examples 1-6 are respectively added into the contact oxidation tank, wherein the adding amount is 10% by volume. The effluent was collected for water quality testing and the results are listed in table 2.

TABLE 2

As can be seen from the data in tables 1 and 2, compared with comparative examples 1 to 6, the microbial agent containing bacillus subtilis with the preservation number of CGMCC No.16828 used in example 1 can rapidly degrade organic pollutants in printing and dyeing wastewater, is applicable to sewage treatment processes by a biofilm method and a biological aeration tank method, and has high treatment efficiency and good purification effect.

The preferred embodiments of the present disclosure have been described in detail above, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. In order to avoid unnecessary repetition, various possible combinations will not be separately described in this disclosure.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.

Claims

1. The Bacillus subtilis is characterized in that the preservation number of the Bacillus subtilis is CGMCC No. 16828.

2. A microbial agent contains thalli and a culture medium, and is characterized in that the thalli contains bacillus subtilis with the preservation number of CGMCC No. 16828.

3. The microbial agent according to claim 2, wherein the viable count of bacillus subtilis with the preservation number of CGMCC No.16828 in each gram of the microbial agent is 10⁸～10¹⁰CFU。

4. The microbial inoculant according to claim 2, wherein the medium is beef extract peptone medium, nutrient broth medium or LB medium, or a combination of two or three thereof.

5. Use of the bacillus subtilis of claim 1 or the microbial inoculant of any one of claims 2 to 4 in treatment of printing and dyeing wastewater.

6. The use of claim 5, wherein the method of treating printing and dyeing wastewater comprises: mixing bacillus subtilis or the microbial agent as claimed in any one of claims 2 to 4 with the printing and dyeing wastewater for biodegradation treatment, then adding a flocculating agent into the wastewater after biodegradation treatment for coagulating sedimentation, and obtaining purified water after solid-liquid separation.

7. Use according to claim 6, wherein the flocculant is an organic flocculant and/or an inorganic flocculant; the inorganic flocculant is aluminum sulfate, aluminum chloride, alum, ferrous sulfate hydrate, ferric sulfate, ferric chloride, polyaluminum sulfate, polyferric chloride or polyferric sulfate, or a composition of two or three of the aluminum sulfate, the aluminum chloride, the alum, the ferrous sulfate hydrate, the ferric sulfate, the ferric chloride, the polyaluminum chloride and the polyferric sulfate; the organic flocculant is a polyacrylamide flocculant.