CN105132303B - A kind of application of bacillus fusiformis in handling leather-making waste water coloration - Google Patents

Abstract

The invention discloses an application of Bacillus fusiformis in treating the chromaticity of tannery wastewater. The treatment of the chromaticity of tannery wastewater by using Bacillus fusiformis comprises the following steps: (1) culture medium preparation; (2) fermentation: Spindle bacillus strains are inoculated into shake flasks equipped with medium, and fermented to obtain a fermentation liquid; (3) extraction of bioflocculants: the fermentation liquid is centrifuged, extracted, re-centrifuged, and vacuum freeze-dried to obtain bioflocculant products; ( 4) Wastewater treatment: add the biological flocculant into the beaker containing the tannery wastewater, and carry out stirring treatment on a magnetic stirrer. The invention uses a new biological flocculant to treat the chroma in tannery wastewater, and the removal efficiency can reach 79.8%. The unique performance of the biological flocculant, such as green environmental protection and no secondary pollution, is used to remove high-concentration chroma in tannery wastewater. It provides a new method, broadens the application of the function of Bacillus fusiformis, and has strong application value.

Description

Application of a kind of Bacillus fusiformis in treating the chromaticity of tannery wastewater

technical field

The invention relates to the field of biotechnology, in particular to the application of Bacillus fusiformis in treating the chromaticity of tanning wastewater.

Background technique

The leather industry is a high-pollution industry. With the increase of the output of the leather industry, the pollutants discharged by the leather industry are also increasing, and the environmental pollution of leather wastewater is becoming increasingly prominent.

Due to the characteristics of the tanning process, the composition of the tanning wastewater is complex, the color is dark, and the chroma is relatively high. The large chroma is mainly caused by dyeing, chrome tanning, vegetable tanning and gray alkali waste liquid. For a long time, people have paid more attention to the treatment of chroma in tannery wastewater. In the "Discharge Standard of Water Pollutants for Tannery and Fur Processing Industry" (GB 30486-2013) officially released by the Ministry of Environmental Protection on December 27, 2013, the discharge limit value of chroma in tannery wastewater is stipulated. The direct discharge limits of chroma in existing leather enterprises, new enterprises and special protected areas are 50, 30, and 20 times respectively. emission standard is 50 times), and the new standard is more stringent. But as we all know, chroma is difficult to deal with, and it is a major problem that plagues the treatment of leather wastewater. At present, fly ash-based coagulation method, nano- _TiO2 photocatalytic oxidation method, physicochemical method mainly using catalytic iron internal electrolysis method, etc. are usually used to remove chroma in tannery wastewater. However, it still exists in that the treatment of high-concentration leather water is not highly targeted, the decolorization efficiency is not high, and it is easy to cause secondary pollution to the environment.

Contents of the invention

In order to solve the above technical problems, the present invention provides an application of Bacillus fusiformis . The preservation number of Bacillus fusiformis is CCTCC M 2014514, which is preserved in China Center for Type Culture Collection, and the preservation date is October 26, 2014. In Japan, by extracting the bio-flocculant from the strain of Bacillus fusiformis, and using the bio-flocculant to treat the chroma in the tannery wastewater, it is green and environmentally friendly, does not produce secondary pollution, etc., and eliminates the high-concentration color produced in the tannery production. To reduce the pollution of waste water to the environment, and build a new technology for clean tanning waste water color treatment.

The application of a spindle bacillus that solves the above technical problems is characterized in that: the preservation number is CCTCC M2014514, and the spindle spindle bacillus is used to reduce the chroma in tanning wastewater.

The spindle bacillus is cultivated and domesticated from the activated sludge of a tannery.

Described cultivation and domestication step are as follows:

(1) Enrichment culture of strains:

Collect the activated sludge from the tannery and inoculate it into the sterilized and cooled LB medium. The culture ratio of the sludge to the medium is about 1:8-12 by volume. Cultivate on a shaking table at 28-32°C for 46-50h , LB medium is prepared as follows: peptone 8-12g, yeast extract 4-7g, NaCl 8-12g, add distilled water to 1000mL, pH6.8-7.2, sterilize at 115-125°C for 15-25min; shaker speed is 100r/ min.

(2) Domestication of strains:

Inoculate 5mL of the enriched culture into 100mL of LB medium containing tannery waste water and culture it with shaking at 28-32°C for 45-50h, and gradually increase the amount of tannery waste water to 40 mL, 60 mL, and 80 mL, and acclimatize step by step ; Step by step domestication, the purpose is to make the cultured strains adapt to the high concentration chroma of tannery wastewater.

The activated sludge cultivated through step (1) is the enriched culture solution.

Whether the colony is successfully domesticated during the domestication process is determined by detecting whether the color removal rate of the LB medium containing tannery wastewater before and after cultivation at each level of domestication is always maintained at a high level. If the color removal rate is always maintained at At a higher level, the next level of domestication can be carried out; the domestication is carried out in an aerobic reactor; the domestication medium is LB medium.

③Separation of strains:

By dilution plate method, using solid LB medium as the separation medium, various bacteria were isolated from the acclimatized bacterial liquid, and each strain was purified.

Dilution plate method, that is: use a sterile pipette to draw 20mL of domesticated activated sludge, put it into a Erlenmeyer flask with glass beads, oscillate, and fully disperse the cells of various bacteria (observed with a microscope, the cells are Unicellular). Use a sterile pipette to draw 1mL of bacterial suspension into a large test tube containing 9mL and mix well, then use a sterile pipette to draw 1mL from this test tube and add it to another test tube containing 9mL of sterile water, mix evenly, and By analogy, 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^-7 different dilutions of bacterial cell suspensions were made. Then use a sterile pipette to draw dilutions of different dilutions into sterile petri dishes, and then pour LB solid medium that has been sterilized and cooled to about 45-50°C into each sterile petri dish. Pour about 15 mL of culture medium. Afterwards, gently rotate the petri dish back and forth, left and right on the aseptic operating table, so that the diluted bacterial suspension and the melted agar medium are evenly mixed, and then allowed to stand after mixing. After the plate is cooled, place the plate upside down in a 37°C incubator and incubate for 1-2 days. Finally, pick a few cells from the single colony grown after culture and inoculate them on the slant of the test tube of LB medium.

Purification steps: the strains inoculated in each slope are separated by the aforementioned dilution plate method, and the strains are repeatedly separated until the isolated strains are pure culture, that is, after the bacterial lawn grows, the characteristics of the colonies are consistent.

In the optimization scheme, there is also the screening of bioflocculant producing bacteria in the present invention, and the cultivation and domestication also include the screening of bioflocculant producing bacteria, such as the following steps:

(1) Primary screening

The isolated and purified Bacillus fusiformis strain was inoculated in the flocculation medium for cultivation, the culture medium was centrifuged, and the supernatant was taken for preliminary screening of flocculation activity, that is, 2 mL of the culture medium was added to 100 mL of 4 g/L kaolin to suspend At the same time, the kaolin suspension without culture medium was used as a control to observe whether it has flocculation activity to judge whether it has flocculation activity, and then conduct a preliminary screening. If it is flocculation, it is preliminarily judged that it has the ability to produce flocculants, and further testing is required. The flocculation activity of the flocculant in the culture medium; the flocculation medium is prepared as follows: NaNO ₃ 1-3 g, KC1 0-11 g, K ₂ HPO ₄ 0.5-1.5 g, MgSO ₄ 0.3-0.7 g, FeSO ₄ 0.01 g, Sucrose 27-32 g and distilled water 1 L. Except FeSO ₄ , all other raw materials were sterilized at 115-125°C for 15-25 min. After the sterilization was completed and cooled, FeSO ₄ was dissolved in sterilized distilled water and filtered. Then the filtered FeSO ₄ solution is added in the medium;

(2) Re-screening

Inoculate the strains with flocculation ability that were screened out respectively in the flocculation medium and cultivate them. After 45-50 hours, use the flocculation rate as a measure of the flocculant-producing ability of the strains, and select spindle spores with biological flocculant production. bacilli. The screened Bacillus fusiformis can not only produce flocculant, but also have the ability to remove pollutants.

The application of a kind of spindle bacillus among the present invention is characterized in that: utilize spindle spindle bacillus to process the method for tanning waste water chromaticity to comprise the following steps:

(l) Medium preparation: NaNO ₃ 1.5-2.5g, KC1 0-11g, K ₂ HPO ₄ 0.6-1.2g, MgSO ₄ 0.3-0.7g, FeSO ₄ 0.01g, sucrose 28-32g and distilled water 1L, except Except for FeSO ₄ , the rest of the raw materials were sterilized at 115-125°C for 15-25min. After the sterilization was completed and cooled, FeSO ₄ was dissolved in sterilized distilled water, and the dissolved FeSO ₄ was filtered with a sterile filter. Bacteria pipette sucks the filtered _FeSO solution into the culture medium;

(2) Fermentation: Inoculate the strain of Bacillus fusiformis into a 250mL shaker flask with 50mL medium, place it in a shaker with a rotation speed of 180r/min, and shake it at 32-38°C for 45-50h; after cultivation Then inoculate a 250mL Erlenmeyer flask with 100mL of culture medium, and then vibrate at 32-38°C for 24-68 hours to obtain a fermentation broth. 12. Fermentation liquid refers to the culture liquid that has been cultured for 24-68 hours in step (2).

(3) Extraction of biological flocculants: Put the fermentation broth into a centrifuge with a rotating speed of 5000-10000r/min, centrifuge for 10min, collect the supernatant, and then add 2-4 times the volume of ethanol to the supernatant to obtain Let the mixture stand at 3-5°C for 5-7h, then put the mixture into a centrifuge with a rotation speed of 5000-10000r/min, centrifuge for 8-12min, collect the precipitate, dissolve it in distilled water for 3h, and put the solution into a centrifuge with a rotation speed of In a centrifuge at 5000-10000r/min, centrifuge for 8-12min, then dissolve the obtained sediment in distilled water for 2-3h, and repeat dialysis for 1-2 times until the nose can no longer smell ethanol, so as to remove small molecules and residual organic matter. solvent, and finally freeze-dry the centrifuged precipitate in a vacuum freeze dryer to obtain the finished bioflocculant;

(4) Wastewater treatment: add the biological flocculant into the beaker containing tannery wastewater, and react on a magnetic stirrer; it can be used at a temperature of 45-55°C, a pH of 7.8-8.2, and a stirring speed of 180-220r/ The wastewater treatment reaction is carried out under the condition of 25 minutes, and the reaction time can be 25-50 minutes, or longer.

The pH of the flocculation medium or culture medium is 6.7-7.2.

The ethanol in the step (3) is pre-cooled ethanol at a temperature of 4°C.

The rotational speed of the centrifuge in the step (3) is 8000r/min.

The spindle bacillus in the present invention is adapted to the environment containing high-concentration chroma wastewater through enrichment and domestication, and can secrete a metabolite when cultured in the medium, which is a biological flocculant; the biological flocculant is mainly Containing macromolecular components such as protein, sugar, glycoprotein, sugar amine and fat, it combines with suspended solids in wastewater through ionic bonds, hydrogen bonds, etc., and because it has some active groups, it overcomes inorganic macromolecules and The inherent defects of synthetic organic polymer flocculants can react with pollutants in wastewater to achieve the purpose of removing pollutants. Proteins, sugars, glycoproteins, glycosamines and fats, all of which are biodegradable, non-toxic and non-secondary pollution.

The present invention uses Bacillus fusiformis and corresponding biological flocculants to treat the chroma in tannery wastewater. When the reaction time is 30 minutes, the removal efficiency can reach 79.8%, the degradation speed is fast (this efficiency is achieved in the reaction time of 30 minutes), and the biological The flocculant is environmentally friendly, does not produce secondary pollution and other unique properties, eliminates the environmental pollution of high-concentration chroma wastewater produced in tannery production, and builds a clean new technology for chroma treatment in tannery wastewater, so as to remove The high-concentration chroma in wastewater provides a new method; in addition, it also broadens the application of the function of the spindle bacillus, making it have a strong application value.

The invention uses the biological flocculant produced by the spindle bacillus to treat waste water, has good coagulation effect and unique decolorization effect, wide application range, easy biodegradation, can eliminate secondary pollution, is safe and reliable, and belongs to the green environmental protection product.

Detailed ways

The present invention will be described in further detail below through examples, but the protection scope of the present invention is not limited to this example.

Example 1

(1) Screening of Bacillus fusiformis

① Enrichment culture of strains

The activated sludge taken from the tannery was inoculated into the sterilized and cooled LB medium (all the mediums mentioned in the present invention were sterilized and cooled), and cultured on a shaking table at 30°C for 48h. LB medium was prepared as follows: peptone 10g, yeast extract 5g, NaCl 10g, distilled water 1000mL, pH 7.0. Sterilized at 121°C for 20min.

② Domestication of strains

Inoculate 5 mL of the enriched culture solution into 100 mL of LB medium containing tannery wastewater (20 mL of tannery wastewater, chromaticity of 2000 degrees (the chromaticity is measured by a colorimeter)) and shake at 30°C for 48 hours. And gradually increase the amount of tannery wastewater to 40mL, 60 mL, 80 mL, domesticate step by step. Its purpose is to make the cultured bacteria adapt to the high concentration chroma of tannery wastewater.

Whether the colony is successfully domesticated during the domestication process is determined by detecting whether the color removal rate of the LB medium containing tannery wastewater before and after cultivation at each level of domestication is always maintained at a high level. If the color removal rate is always maintained at At a higher level, the next level of domestication can be carried out; the domestication is carried out in an aerobic reactor; the domestication medium is LB medium.

③Separation of strains

By dilution plate method, using solid LB medium as the separation medium, various bacteria were isolated from the acclimatized bacterial liquid, and each strain was purified.

Dilution plate method. That is: Use a sterile pipette to draw 20mL of domesticated activated sludge, put it into a Erlenmeyer flask with glass beads, oscillate, and fully disperse the cells of various bacteria (observed with a microscope, the cells are single cells). Use a sterile pipette to draw 1mL of bacterial suspension into a large test tube containing 9mL and mix well, then use a sterile pipette to draw 1mL from this test tube and add it to another test tube containing 9mL of sterile water, mix evenly, and By analogy, 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^-7 different dilutions of bacterial cell suspensions were made. Then use a sterile pipette to draw dilutions of different dilutions into sterile petri dishes, and then pour LB solid medium that has been sterilized and cooled to about 45-50°C into each sterile petri dish. Pour about 15 mL of culture medium. Afterwards, gently rotate the petri dish back and forth, left and right on the aseptic operating table, so that the diluted bacterial suspension and the melted agar medium are evenly mixed, and then allowed to stand after mixing. After the plate is cooled, place the plate upside down in a 37°C incubator and incubate for 1-2 days. Finally, pick a few cells from the single colony grown after culture and inoculate them on the slant of the test tube of LB medium.

Purification steps: the strains inoculated in each slope are separated by the aforementioned dilution plate method, and the strains are repeatedly separated until the isolated strains are pure culture, that is, after the bacterial lawn grows, the characteristics of the colonies are consistent.

④ Screening of bioflocculant-producing bacteria

a. primary screening

The separated and purified strains were inoculated in the flocculation medium for culture, the culture solution was centrifuged, and the supernatant was taken for the primary screening of the flocculation activity, that is: take 2 mL of the culture solution and add 100 mL of 4 g/L kaolin suspension At the same time, the kaolin suspension without culture medium was used as a control to observe the phenomenon, and to judge whether it had flocculation activity by whether flocculation occurred, so as to carry out preliminary screening. If it is flocculated, it is preliminarily judged that it has the ability to produce biological flocculants, and it is necessary to further test the flocculation activity of the flocculants in the culture medium. The flocculation medium was prepared as follows: NaNO ₃ 2 g, KC1 0.5 g, K ₂ HPO ₄ 1 g, MgSO ₄ 0.5 g, FeSO ₄ 0.01 g, sucrose 30 g and distilled water 1 L, pH natural, sterilized.

b. Double screening

The strains with flocculation ability screened out were inoculated in the flocculation medium and cultivated respectively. After 48 hours, the flocculation rate was used as the measure of the flocculant production ability of the strain, and a bioflocculant strain with the best flocculation effect was selected from it. produce bacteria.

⑤ Identification of bioflocculant-producing bacteria

Analyze the physiological and biochemical characteristics of the screened bioflocculant-producing bacteria with high flocculation activity, and obtain the 16S rRNA gene sequence of each strain through molecular biology techniques, and then obtain its classification information through database comparison. It was determined that its species was Bacillus fusiformis .

(2) Medium preparation: NaNO ₃ 2 g, KC1 0.5 g, K ₂ HPO ₄ 1 g, MgSO ₄ 0.5 g, FeSO ₄ 0.01 g, sucrose ₃₀ g and distilled water 1L. Sterilize at ℃ for 20 min, and dissolve FeSO ₄ in distilled water after sterilization. , filter the dissolved FeSO ₄ with a sterile filter, and add the filtered FeSO ₄ solution into the culture medium with a sterile pipette.

(3) fermentation

Inoculate the Bacillus fusiformis isolated and screened in this laboratory into a 250mL shaker flask containing 50mL of medium, place it in a shaker with a rotation speed of 180r/min, shake it at 35°C for 48h, and then inoculate it until A 250mL Erlenmeyer flask with 100mL of culture medium was shaken at 35°C for 24h.

(4) Extraction of bioflocculant

Put the fermented liquid into a centrifuge with a rotating speed of 8000r/min, centrifuge for 10min, and collect the supernatant. Then 3 times the volume of pre-cooled ethanol (4° C.) was added to the supernatant, and the resulting mixture was left standing at 4° C. for 6 h. Put the mixture into a centrifuge with a rotating speed of 10000r/min, centrifuge for 10min, and collect the precipitate. Dissolve in an appropriate amount of distilled water for 3 hours, put the mixture into a centrifuge with a rotating speed of 8000r/min, and centrifuge for 10 minutes, then dissolve the obtained precipitate in distilled water for 3 hours, and repeat dialysis once until the nose can no longer smell ethanol, so as to remove small molecules and residual organic solvents. Finally, the centrifuged precipitate is freeze-dried in a vacuum freeze dryer to obtain the finished biological flocculant.

(5) Wastewater treatment

Add 1.1 g of biological flocculant into a beaker containing 100 mL of tanning wastewater, and react on a magnetic stirrer. The reaction conditions are as follows: the temperature is 50° C., the pH is 8, and the stirring speed is 200 r/min. When the initial chromaticity value in the tannery wastewater is 1500 degrees (the colorimeter is used to measure the chromaticity), the bioflocculant can remove the chromaticity of 1197 degrees, the removal efficiency reaches 79.8%, the reaction time is 30min, and the degradation rate Fast, no secondary pollution.

Example 2

(1) Screening of Bacillus fusiformis

① Enrichment culture of strains

The activated sludge taken from the tannery was inoculated into the sterilized and cooled LB medium (all the mediums mentioned in the present invention were sterilized and cooled), and cultured on a shaking table at 30°C for 48h. LB medium was prepared as follows: peptone 10g, yeast extract 5g, NaCl 10g, distilled water 1000mL, pH 7.0. Sterilized at 121°C for 20min.

② Domestication of strains

Inoculate 5 mL of the enriched culture solution into 100 mL of LB medium containing tannery wastewater (20 mL of tannery wastewater, chromaticity of 2000 degrees (the chromaticity is measured by a colorimeter)) and shake at 30°C for 48 hours. And gradually increase the amount of tannery wastewater to 40mL, 60 mL, 80 mL, domesticate step by step. Its purpose is to make the cultured bacteria adapt to the high concentration chroma of tannery wastewater.

③Separation of strains

By dilution plate method, using solid LB medium as the separation medium, various bacteria were isolated from the acclimatized bacterial liquid, and each strain was purified.

(2) Medium preparation: NaNO ₃ 2 g, KC1 0.5 g, K ₂ HPO ₄ 1 g, MgSO ₄ 0.5 g, FeSO ₄ 0.01 g, sucrose ₃₀ g and distilled water 1L. Sterilize at ℃ for 20 min, and dissolve FeSO ₄ in distilled water after sterilization. , filter the dissolved FeSO ₄ with a sterilized filter, and add the filtered FeSO ₄ solution into the medium with a sterile pipette;

(3) fermentation

Inoculate the Bacillus fusiformis isolated and screened in this laboratory into a 250mL shaker flask containing 50mL of medium, place it in a shaker with a rotation speed of 180r/min, shake it at 35°C for 48h, and then inoculate it until A 250mL Erlenmeyer flask with 100mL of culture medium was shaken at 35°C for 24h.

(4) Extraction of bioflocculant

Put the fermented liquid into a centrifuge with a rotating speed of 8000r/min, centrifuge for 10min, and collect the supernatant. Then 3 times the volume of pre-cooled ethanol (4° C.) was added to the supernatant, and the resulting mixture was left standing at 4° C. for 6 h. Put the mixture into a centrifuge with a rotating speed of 10000r/min, centrifuge for 10min, and collect the precipitate. Dissolve in an appropriate amount of distilled water for 3 hours, put the mixture into a centrifuge with a rotating speed of 8000r/min, and centrifuge for 10 minutes, then dissolve the obtained precipitate in distilled water for 3 hours, and repeat dialysis once until the nose can no longer smell ethanol, so as to remove small molecules and residual organic solvents. Finally, the centrifuged precipitate is freeze-dried in a vacuum freeze dryer to obtain the finished biological flocculant.

(5) Wastewater treatment

Add 1.1 g of biological flocculant into a beaker containing 100 mL of tanning wastewater, and react on a magnetic stirrer. The reaction conditions are as follows: the temperature is 50° C., the pH is 8, and the stirring speed is 200 r/min.

When the initial chromaticity value in the tannery wastewater is 1200 degrees (the chromaticity is measured by a colorimeter), the bioflocculant can remove the chromaticity of 903.6 degrees, the removal efficiency reaches 75.3%, the reaction time is 30 minutes, and the degradation rate Fast, no secondary pollution.

Example 3

Screening of Bacillus fusiformis

(1) Enrichment culture of strains:

Collect the activated sludge from the tannery and inoculate it into the sterilized and cooled LB medium. The culture ratio of the sludge to the medium is about 1:8 by volume. Cultivate on a shaker at 28°C for 46 hours. The LB medium is prepared as follows : Peptone 8g, yeast extract 4g, NaCl 8g, add distilled water to 1000mL, pH 6.8, sterilize at 115°C for 25min; shaker speed is 100r/min.

(2) Domestication of strains:

Inoculate 5 mL of enriched culture bacteria into 100 mL of LB medium containing tannery wastewater and culture at 28°C for 50 hours with shaking, and gradually increase the amount of tannery wastewater to 40 mL, 60 mL, and 80 mL, and acclimatize step by step; , the purpose of which is to adapt the cultured strains to the high concentration chroma of tannery wastewater.

The activated sludge cultivated through step (1) is the enriched culture solution.

③Separation of strains:

By dilution plate method, using solid LB medium as the separation medium, various bacteria were isolated from the acclimatized bacterial liquid, and each strain was purified.

Screening of Bacteria Produced by Bioflocculant

(1) Primary screening

The isolated and purified Bacillus fusiformis strain was inoculated in the flocculation medium for cultivation, the culture medium was centrifuged, and the supernatant was taken for preliminary screening of flocculation activity, that is, 2 mL of the culture medium was added to 100 mL of 4 g/L kaolin to suspend At the same time, the kaolin suspension without culture medium was used as a control to observe whether it has flocculation activity to judge whether it has flocculation activity, and then conduct a preliminary screening. If it is flocculation, it is preliminarily judged that it has the ability to produce flocculants, and further testing is required. The flocculation activity of the flocculant in the culture medium; the flocculation medium was prepared as follows: NaNO ₃ 1 g, KC1 0 g, K ₂ HPO ₄ 0.5 g, MgSO ₄ 0.3 g, FeSO ₄ 0.01 g, sucrose 27 g and distilled water 1 L, except Except for FeSO ₄ , the rest of the raw materials were sterilized at 115°C for 25 min. After the sterilization and cooling, FeSO ₄ was dissolved in sterilized distilled water, filtered, and then the filtered FeSO ₄ solution was added to the culture medium. Flocculation medium pH 6.7;

(2) Re-screening

Inoculate the strains with flocculation ability that were screened out respectively in the flocculation medium and cultivate them. After 45-50 hours, use the flocculation rate as a measure of the flocculant-producing ability of the strains, and select spindle spores with biological flocculant production. bacilli. The screened Bacillus fusiformis can not only produce flocculant, but also have the ability to remove pollutants.

The method for processing the chromaticity of tannery wastewater by using Spindle bacillus comprises the following steps:

(l) Medium preparation: NaNO ₃ 1.5g, KC1 0g, K ₂ HPO ₄ 0.6 g, MgSO ₄ 0.3 g, FeSO ₄ 0.01 g, sucrose 28 g and distilled water 1L, except FeSO ₄ , the rest of the raw materials were at 115°C Sterilize for 25 min. After the sterilization is completed and cool down, dissolve FeSO ₄ in sterilized distilled water, filter the dissolved FeSO ₄ with a sterile filter, and add the filtered FeSO ₄ solution with a sterile pipette. In the medium, the pH of the medium is 6.7;

(2) Fermentation: Inoculate the strain of Bacillus fusiformis into a 250mL shaker flask with 50mL medium, place it in a shaker with a rotation speed of 180r/min, and shake it at 32-38°C for 45h; the cultured bacteria The seed was inoculated into a 250mL Erlenmeyer flask containing 100mL medium, and then shaken and cultured at 32°C for 24 hours to obtain a fermentation broth. The inoculum ratio of the strain to the medium was 1:8 by volume; 2) The culture medium after culturing for 24 hours.

(3) Extraction of bioflocculant: Put the fermentation broth into a centrifuge with a rotating speed of 10000r/min, centrifuge for 10min, collect the supernatant, then add 4 times the volume of pre-cooled ethanol to the supernatant, and the temperature is 4°C , the resulting mixture was left standing at 5°C for 5 hours, and then the mixture was put into a centrifuge with a speed of 10000r/min, centrifuged for 12min, the precipitate was collected, dissolved in distilled water for 3h, and the solution was put into a centrifuge with a speed of 10000r/min Centrifuge for 12 minutes, then dissolve the precipitate in distilled water for 3 hours, and dialyze twice until the nose can no longer smell the smell of ethanol, so as to remove small molecules and residual organic solvents, and finally place the centrifuged precipitate in a vacuum freeze dryer Freeze-drying to obtain the finished bioflocculant;

(4) Wastewater treatment: add biological flocculant into a beaker containing tanning wastewater, and react on a magnetic stirrer;

Add 1.1 g of biological flocculant into a beaker containing 100 mL of tanning wastewater, and react on a magnetic stirrer. The reaction conditions are as follows: the temperature is 55° C., the pH is 8.2, and the stirring speed is 200 r/min.

When the initial chromaticity value in the tannery wastewater is 2500 degrees (the chromaticity is measured by a colorimeter), the bioflocculant can remove the chromaticity of 1562.5 degrees, the removal efficiency reaches 62.5%, and the reaction time is 40-50min. The degradation rate is fast and there is no secondary pollution.

Example 4

Screening of Bacillus fusiformis

(1) Enrichment culture of strains:

Collect the activated sludge from the tannery and inoculate it into the sterilized and cooled LB medium. The culture ratio of the sludge to the medium is about 1:12 by volume. Cultivate on a shaker at 32°C for 46 hours. The LB medium is prepared as follows : Peptone 12g, yeast extract 7g, NaC 12g, add distilled water to 1000mL, pH7.2, sterilize at 125°C for 25min; shaker speed is 100r/min.

(2) Domestication of strains:

Inoculate 5mL of the enriched culture into 100mL of LB medium containing tannery waste water and culture it with shaking at 28-32°C for 45-50h, and gradually increase the amount of tannery waste water to 40 mL, 60 mL, and 80 mL, and acclimatize step by step ; Step by step domestication, the purpose is to make the cultured strains adapt to the high concentration chroma of tannery wastewater.

The activated sludge cultivated through step (1) is the enriched culture solution.

③Separation of strains:

By dilution plate method, using solid LB medium as the separation medium, various bacteria were isolated from the acclimatized bacterial liquid, and each strain was purified.

Screening of Bacteria Produced by Bioflocculant

(1) Primary screening

The isolated and purified Bacillus fusiformis strain was inoculated in the flocculation medium for cultivation, the culture medium was centrifuged, and the supernatant was taken for preliminary screening of flocculation activity, that is, 2 mL of the culture medium was added to 100 mL of 4 g/L kaolin to suspend At the same time, the kaolin suspension without culture medium was used as a control to observe whether it has flocculation activity to judge whether it has flocculation activity, and then conduct a preliminary screening. If it is flocculation, it is preliminarily judged that it has the ability to produce flocculants, and further testing is required. The flocculation activity of the flocculant in the culture medium; the flocculation medium was prepared as follows: NaNO ₃ 3 g, KC1 1 g, K ₂ HPO ₄ 1.5 g, MgSO ₄ 0.7 g, FeSO ₄ 0.01 g, sucrose 32 g and distilled water 1 L, except Except for FeSO ₄ , the rest of the raw materials were sterilized at 125°C for 25 min. After the sterilization and cooling, FeSO ₄ was dissolved in sterilized distilled water, filtered, and then the filtered FeSO ₄ solution was added to the culture medium. Flocculation medium pH 7.2;

(2) Re-screening

The initially screened strains with flocculation ability were inoculated in the flocculation medium and cultured respectively. After 50 hours, the flocculation rate was used as a measure of the flocculant-producing ability of the strains, and Bacillus spindleis with biological flocculant production was selected from them. The screened Bacillus fusiformis can not only produce flocculant, but also have the ability to remove pollutants.

The method for processing the chromaticity of tannery wastewater by using Spindle bacillus comprises the following steps:

(l) Medium preparation: NaNO ₃ 2.5g, KC1 1g, K ₂ HPO ₄ 1.2 g, MgSO ₄ 0.7 g, FeSO ₄ 0.01 g, sucrose 32 g and distilled water 1L, except FeSO ₄ , the rest of the raw materials were at 125°C Sterilize for 25 min. After the sterilization is completed and cool down, dissolve FeSO ₄ in sterilized distilled water, filter the dissolved FeSO ₄ with a sterile filter, and add the filtered FeSO ₄ solution with a sterile pipette. In the medium, the pH of the medium is 7.2;

(2) Fermentation: Inoculate the strain of Bacillus fusiformis into a 250mL shaker flask containing 50mL of medium, place it in a shaker with a rotation speed of 180r/min, and shake it at 38°C for 50h; Inoculate into a 250mL Erlenmeyer flask with 100mL medium, and then shake and culture at 38°C for 68 hours to obtain a fermentation broth. The inoculum ratio of the strain to the medium is 1:12 by volume; the fermentation broth refers to step (2) After 68h culture medium.

(3) Extraction of bioflocculant: Put the fermentation broth into a centrifuge with a rotation speed of 5000r/min, centrifuge for 10min, collect the supernatant, then add 2 times the volume of pre-cooled ethanol to the supernatant, and the temperature is 4°C , the resulting mixture was left to stand at 3°C for 5-7h, then the mixture was put into a centrifuge with a speed of 5000r/min, centrifuged for 8min, the precipitate was collected, dissolved in distilled water for 3h, and the solution was put into a centrifuge with a speed of 5000r/min In a centrifuge, centrifuge for 8 minutes, then dissolve the obtained precipitate in distilled water for 2 hours, and repeat the dialysis once until the nose can no longer smell the smell of ethanol, so as to remove small molecules and residual organic solvents, and finally place the centrifuged precipitate in vacuum freeze-drying Freeze-drying in the machine can get the finished product of biological flocculant;

(4) Wastewater treatment: add biological flocculant into a beaker containing tanning wastewater, and react on a magnetic stirrer;

Add 1.1 g of biological flocculant into a beaker containing 100 mL of tanning wastewater, and react on a magnetic stirrer. The reaction conditions are as follows: the temperature is 45° C., the pH is 7.8, and the stirring speed is 180 r/min.

When the initial chromaticity value in the tannery wastewater is 4000 degrees (the chromaticity is measured by a colorimeter), the bioflocculant can remove the chromaticity of 2408 degrees, the removal efficiency reaches 60.2%, and the reaction time is about 50 minutes. Fast speed, no secondary pollution.

The present invention is not limited to the foregoing specific embodiments. The present invention extends to any new feature or any new combination disclosed in this specification, and any new method or process step or any new combination disclosed.

Claims (6)

1. An application of Bacillus fusiformis, characterized in that: the preservation number of the Bacillus fusiformis is CCTCC M 2014514, and it is applied to reduce chroma in tannery wastewater; the Bacillus fusiformis is produced by a tannery It is cultivated and domesticated in activated sludge. 2. The application of Bacillus fusiformis according to claim 1, characterized in that: the cultivation and domestication steps are as follows: (1) Enrichment culture of strains: Collect the activated sludge from the tannery and inoculate it into the sterilized and cooled LB medium. The culture ratio of the sludge to the medium is about 1:8-12 by volume. Cultivate on a shaking table at 28-32°C for 46-50h , LB medium is prepared as follows: peptone 8-12g, yeast extract 4-7g, NaCl 8-12g, add distilled water to 1000mL, pH6.8-7.2, sterilize at 115-125°C for 15-25min; (2) Domestication of strains: Inoculate 5mL of the enriched culture into 100mL of LB medium containing tannery waste water and culture it with shaking at 28-32°C for 45-50h, and gradually increase the amount of tannery waste water to 40 mL, 60 mL, and 80 mL, and acclimatize step by step ; (3) Separation of strains: Through the dilution plate method, using solid LB medium as the separation medium, various bacteria were isolated from the acclimatized bacterial liquid, and each strain was purified to obtain the strain of Bacillus fusiformis. 3. According to the application of a kind of Bacillus fusiformis described in claim 2, it is characterized in that: said cultivation and domestication also include the screening of bioflocculant producing bacteria, such as the following steps: (1) Primary screening The isolated and purified Bacillus fusiformis strain was inoculated in the flocculation medium for cultivation, the culture medium was centrifuged, and the supernatant was taken for preliminary screening of flocculation activity, that is, 2 mL of the culture medium was added to 100 mL of 4 g/L kaolin to suspend At the same time, it is compared with the kaolin suspension without culture medium, and observed to judge whether it has flocculation activity by whether flocculation occurs, so as to conduct preliminary screening. If flocculation, it is initially determined that it has the ability to produce flocculants; The preparation is as follows: NaNO ₃ 1-3g, KC1 0-11 g, K ₂ HPO ₄ 0.5-1.5 g, MgSO ₄ 0.3-0.7 g, FeSO ₄ 0.01 g, sucrose 27-32 g and distilled water 1L, except FeSO ₄ , The rest of the raw materials were sterilized at 115-125°C for 15-25 min. After the sterilization was completed and cooled, FeSO ₄ was dissolved in sterilized distilled water, filtered, and then the filtered FeSO ₄ solution was added to the culture medium; The pH of the flocculation medium is 6.7-7.2; (2) Re-screening Inoculate the strains with flocculation ability that were screened out respectively in the flocculation medium and cultivate them. After 45-50 hours, use the flocculation rate as a measure of the flocculant-producing ability of the strains, and select spindle spores with biological flocculant production. bacilli. 4. The application of a kind of Bacillus fusiformis ( Bacillus fusiformis ) according to any one of claims 1-3, characterized in that: the method of using Bacillus fusiformis to treat the chromaticity of tannery wastewater comprises the following steps: (l) Medium preparation: NaNO ₃ 1.5-2.5g, KC1 0-11g, K ₂ HPO ₄ 0.6-1.2g, MgSO ₄ 0.3-0.7g, FeSO ₄ 0.01g, sucrose 28-32g and distilled water 1L, except Except for FeSO ₄ , the rest of the raw materials were sterilized at 115-125°C for 15-25min. After the sterilization was completed and cooled, FeSO ₄ was dissolved in sterilized distilled water, filtered, and then the filtered FeSO ₄ solution was added to the culture In the base; pH 6.7-7.2 of the culture medium; (2) Fermentation: Inoculate the strain of Bacillus fusiformis into a 250mL shaker flask with 50mL medium, place it in a shaker with a rotation speed of 180r/min, and shake it at 32-38°C for 45-50h; after cultivation Then inoculate a 250mL Erlenmeyer flask with 100mL of culture medium, and then vibrate at 32-38°C for 24-68 hours to obtain a fermentation broth. 12; (3) Extraction of biological flocculants: Put the fermentation broth into a centrifuge with a rotating speed of 5000-10000r/min, centrifuge for 10min, collect the supernatant, and then add 2-4 times the volume of ethanol to the supernatant to obtain Let the mixture stand at 3-5°C for 5-7h, then put the mixture into a centrifuge with a rotation speed of 5000-10000r/min, centrifuge for 8-12min, collect the precipitate, dissolve it in distilled water for 2.5-3.5h, put the solution in Put it into a centrifuge with a rotating speed of 5000-10000r/min, centrifuge for 8-12min, dissolve the obtained precipitate in distilled water for 2-3h, repeat dialysis for 1-2 times, and then place the centrifuged precipitate in a vacuum freeze dryer for freeze-drying , to obtain the finished bioflocculant; (4) Wastewater treatment: add biological flocculant into a beaker containing tannery wastewater, and react on a magnetic stirrer. 5. The application of Bacillus fusiformis according to claim 4, characterized in that the ethanol in the step (3) is pre-cooled ethanol at a temperature of 3-5°C. 6. The application of a kind of Bacillus fusiformis according to claim 4, characterized in that: the rotational speed of the centrifuge in the step (3) is 8000r/min.