CN115820493A - Method for enriching azotobacter and degrading dye by culturing azotobacter with cellulose - Google Patents
Method for enriching azotobacter and degrading dye by culturing azotobacter with cellulose Download PDFInfo
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Abstract
The invention discloses a method for enriching azotobacter and degrading dye by culturing azotobacter with cellulose. The method for enriching azotobacter and degrading dye by culturing azotobacter with cellulose comprises the following steps: 1. collecting a soil sample as an inoculum, and culturing by using a culture medium; 2. inoculating the mixed bacteria in the step 1 into a culture bottle containing a culture medium, introducing nitrogen, culturing under a proper temperature condition, and enriching azotobacter; 3. centrifugally collecting the azotobacteria enriched in the step 2, and suspending in a carbon source-free culture medium to obtain a high-concentration bacterial suspension; 4. inoculating the bacterial suspension obtained in the step 3 into a culture medium containing cellulose and dye, and determining the change of the concentrations of sugar, protein and dye in the culture medium and the cellulase activity of azotobacter. The invention provides a brand new technical route for treating dye (such as azo dye methyl orange) wastewater by nitrogen fixation mixed bacteria, has the advantages of low cost, environmental protection and the like, and has wide application prospect in the aspects of waste paper cellulose utilization and dye degradation.
Description
Technical Field
The invention relates to an enrichment culture method of microorganisms, in particular to a method for enriching azotobacter and degrading dyes by culturing azotobacter with cellulose.
Background
The worldwide paper and paperboard production in 2021 is reported to 12105 million tons, which is 7.50% higher than 2020. Due to the quality and specification of paper products and the mixing of waste paper and other wastes, the recycling of paper fibers into new paper is very difficult, the recycling rate of the waste paper in 2021 year is 51.3%, and the utilization rate of the waste paper is 54.1%. Besides recycling, other waste paper treatment modes such as landfill and incineration can release harmful substances, cause secondary pollution to the environment and have low economic benefit. The waste paper regenerated fiber technology can reduce the production cost of the fiber and save raw materials. But the waste paper recycling industry is not formed in China, and the waste paper recycling quality is uneven. Multiple recoveries can result in fiber loss and reduced bonding strength between cellulose macromolecules. The anaerobic digestion of the waste paper can effectively utilize the waste paper resource and convert the waste paper resource into various organic acids, alcohols, methane and the like. But is limited by the higher carbon to nitrogen ratio and requires co-digestion with a substrate having a high nitrogen content to improve its digestibility. As a microorganism capable of fixing, converting and utilizing nitrogen, azotobacter is an ideal method for resisting high carbon-nitrogen ratio by decomposing waste paper by using azotobacter capable of releasing cellulase.
In order to obtain vivid color paper, a papermaking factory uses azo dyes for dyeing, and a large amount of dye waste water is generated. The azo dye wastewater has toxicity, can cause DNA damage and cancer, and its discharge is harmful to aquatic organisms and humans. Biological decolorization is an environment-friendly technology for treating azo dye wastewater, but the wastewater treatment with high carbon-nitrogen ratio needs additional nitrogen source. The nitrogen-fixing bacteria are utilized to treat the wastewater, so that the increase of a nitrogen source can be avoided, and the cost is reduced.
Disclosure of Invention
The invention aims to provide a method for enriching azotobacter and degrading dye by culturing azotobacter with cellulose.
In order to achieve the aim, the invention provides a method for enriching azotobacter and degrading dye by culturing azotobacter with cellulose, which comprises the following steps:
(1) Collecting a soil sample as an inoculation source, suspending the soil sample in sterile water, and uniformly mixing; inoculating the suspension into a culture bottle, adding a culture medium to make the volume of a culture system reach 50mL, introducing nitrogen, sealing, and performing shake culture in a constant-temperature incubator at 30-32 ℃ for 48-72h;
(2) Adding 2.5-5mL of the culture solution obtained in the step (1) into a new culture bottle, adding 45-47.5mL of culture medium (total system is 50 mL), introducing nitrogen, sealing, and performing shake culture at 30-32 ℃ in a constant temperature incubator;
(3) After culturing for 48-72h, centrifuging the culture solution, removing the supernatant, suspending the thallus in a new 50mL culture medium, inoculating into a culture bottle, introducing nitrogen, sealing, and continuously performing shaking culture at 30-32 ℃;
(4) Repeating the step (3) for 10-15 times;
(5) Inoculating 2.5-5mL of enriched azotobacteria culture solution into a culture bottle, adding 45-47.5mL of culture medium (total system 50 mL) into the culture bottle, introducing nitrogen, sealing, and performing shake culture at 30-32 deg.C for 20-24h;
(6) Centrifuging the azotobacter culture solution in the step (5), discarding supernatant, and suspending thalli in a carbon source-free culture medium to obtain high-concentration bacterial suspension; taking 5mL of the high-concentration bacterial suspension, inoculating the high-concentration bacterial suspension into a culture bottle, adding a carbon-source-free culture medium, cellulose materials and dyes, finally introducing nitrogen into the culture bottle, sealing, and performing shaking culture in a constant-temperature incubator at 30-32 ℃; sampling every day, and measuring the concentration change of sugar, protein and dye for 1-5 days; on day 5, cellulase (CMC enzyme) and beta-glucosidase activities of azotobacteria were measured.
Preferably, the medium does not contain a nitrogen source.
The culture medium contains KH 2 PO 4 2.3g/L,K 2 HPO 4 5.75g/L,MgSO 4 ·7H 2 O 0.025g/L,CaCl 2 ·2H 2 O0.025 g/L, glucose 10g/L, H 3 BO 3 0.124mg/L,FeSO 4 ·7H 2 O 0.1068mg/L,CoSO 4 ·7H 2 O 0.0512mg/L,CuSO 4 ·5H 2 O 0.0044mg/L,MnCl 2 ·4H 2 O 0.00384mg/L,Na 2 MoO 4 ·2H 2 O 0.1068mg/L,ZnSO 4 ·7H 2 O 0.0512mg/L。
The carbon source-free medium in the step (6) is a medium obtained by removing glucose from the medium.
Preferably, step (4) is capable of enriching for nitrogen-fixing bacteria.
Preferably, the OD of the high-concentration bacterial suspension in step (6) 600 ＝1.8-2.0。
In the present invention, the cellulosic material is paper, preferably toilet paper; the dye is an azo dye, preferably methyl orange. The toilet paper can be utilized by azotobacter, and the methyl orange can be degraded.
Further, the final content of the toilet paper in the step (6) is 1.8-2g/L, and the final content of methyl orange is 20-25mg/L.
Preferably, the sealing is carried out after nitrogen is introduced for 10-15 min.
Preferably, the shaking culture rotation speed is 120-130rpm.
Preferably, the centrifugation conditions are: centrifuging at 7000-8000rpm for 6-10min.
Preferably, the culture temperature in steps (1) to (6) is 30 ℃.
The invention provides a brand new technical route for treating dye wastewater (such as azo dye methyl orange) by nitrogen fixation mixed bacteria, has the advantages of low cost, environmental protection and the like, and has wide application prospect in the aspects of waste paper cellulose utilization and dye degradation.
Drawings
FIG. 1 is a graph showing the effect of enriched nitrogen-fixing bacteria genus level diversity (A) and temperature on the growth of nitrogen-fixing bacteria (B) in a preferred embodiment of the present invention.
FIG. 2 is a diagram showing the content changes of sugar (A) and protein (B) and CMC enzyme activity and beta-glucosidase enzyme activity (C) of azotobacter in the process of culturing azotobacter by using toilet paper as a carbon source in the preferred embodiment of the present invention.
FIG. 3 is a graph showing the content change of methyl orange in the process of degrading methyl orange by culturing azotobacter using toilet paper as a carbon source according to the preferred embodiment of the present invention.
Detailed Description
In order to solve the problems of pollution of methyl orange dye and waste toilet paper, the invention provides a method for degrading methyl orange by culturing azotobacter by using toilet paper.
The invention adopts the following technical scheme:
(1) Taking soil as an inoculation source, suspending the soil in sterile water, and fully and uniformly mixing; and (3) inoculating 5mL of the suspension into a serum bottle, adding 45mL of culture medium, introducing nitrogen for 10min, and sealing by using a butyl rubber plug and an aluminum cap. The cells were incubated at 30 ℃ and 120rpm for 72h in a thermostatted incubator.
(2) 5mL of the culture solution obtained in (1) was put into a fresh serum bottle, 45mL of the culture medium was added, nitrogen was introduced for 10min, and the mixture was sealed with a butyl rubber stopper and an aluminum cap, and cultured in a constant temperature incubator at 30 ℃ and 120 rpm. After culturing for 72h, centrifuging the culture solution for 10min at 8000rpm by using a centrifuge, discarding the supernatant, and keeping the thallus; the above thalli are resuspended in a new 50mL culture medium, a serum bottle is inoculated, nitrogen is introduced for 10min, a butyl rubber plug and an aluminum cap are used for sealing, and the shaking culture is continued at 30 ℃. The steps are repeated for 10 to 15 times.
(3) Inoculating 5mL of the enriched azotobacteria culture solution into a 120mL serum bottle, adding 45mL of the culture medium into the serum bottle, exposing to nitrogen for 10min, sealing with a butyl rubber plug and an aluminum cap, and culturing at 30 ℃ and 120rpm for 24h.
(4) And (4) centrifuging the azotobacter culture solution in the step (3) by using a centrifugal machine at 8000rpm for 10min, discarding supernatant, and resuspending thalli in a culture medium without a carbon source to obtain high-concentration bacterial suspension. And (3) inoculating 5mL of the high-concentration bacterial suspension into a serum bottle, adding a culture medium without a carbon source, toilet paper and methyl orange, finally introducing nitrogen for 10min, and sealing by using a butyl rubber plug and an aluminum cap, wherein the volume of the culture solution in the serum bottle is 50mL, the content of the toilet paper is 2g/L, and the content of the methyl orange is 25mg/L. Culturing the serum bottle in a constant temperature incubator at 30 deg.C and 120rpm, sampling every day, and measuring the concentration change of sugar, protein and methyl orange for 5 days; and measuring the activities of CMC enzyme and beta-glucosidase enzyme of azotobacter on the 5 th day.
Preferably, the medium component in step (1) is KH 2 PO 4 2.3g/L,K 2 HPO 4 5.75g/L,MgSO 4 ·7H 2 O0.025g/L,CaCl 2 ·2H 2 O0.025 g/L, glucose 10g/L, H 3 BO 3 0.124mg/L,FeSO 4 ·7H 2 O 0.1068mg/L,CoSO 4 ·7H 2 O 0.0512mg/L,CuSO 4 ·5H 2 O 0.0044mg/L,MnCl 2 ·4H 2 O 0.00384mg/L,Na 2 MoO 4 ·2H 2 O 0.1068mg/L,ZnSO 4 ·7H 2 O 0.0512mg/L.
The carbon source-free medium in the step (4) is a medium obtained by removing glucose in the medium.
Preferably, the medium in step (1) is nitrogen-free.
Preferably, step (2) is capable of enriching for nitrogen-fixing bacteria.
Preferably, the concentration of the high-concentration azotobacter suspension in the step (4) is OD 600 ＝1.8-2.0。
Preferably, the amount of the azotobacteria inoculum in step (4) is 10% (v/v).
Preferably, the final content of the toilet paper in the step (4) is 2g/L, and the final content of methyl orange is 25mg/L.
Preferably, the hygiene paper in the step (4) can be absorbed by azotobacter, and the methyl orange can be degraded.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1 enrichment of the azotobacter group
The soil sample is taken from grassland under the integrated midwestern department downstairs of the agricultural university of Anhui, 0.1g of soil sample is added into 5mL of culture medium to be sufficiently and uniformly vibrated, 5mL of the suspension is inoculated into a serum bottle, 45mL of culture medium is added, and a butyl rubber plug and an aluminum cap are used for sealing after nitrogen is introduced for 10min. The cells were incubated at 30 ℃ and 120rpm for 72h in a thermostatted incubator.
Adding 5mL of the above culture solution into a new serum bottle, adding 45mL of culture medium, introducing nitrogen for 10min, sealing with a butyl rubber plug and an aluminum cap, and culturing at 30 deg.C and 120rpm in a constant temperature incubator. After culturing for 72h, centrifuging the culture solution for 10min at 8000rpm by using a centrifuge, discarding the supernatant, and keeping the thallus; the above thalli are resuspended in a new 50mL culture medium, a serum bottle is inoculated, nitrogen is introduced for 10min, a butyl rubber plug and an aluminum cap are used for sealing, and the shaking culture is continued at 30 ℃. The steps are repeated for 10 to 15 times. And (3) analyzing the diversity of the enriched nitrogen-fixing flora by high-throughput sequencing.
The culture medium comprises the following components: KH (Perkin Elmer) 2 PO 4 2.3g/L,K 2 HPO 4 5.75g/L,MgSO 4 ·7H 2 O 0.025g/L,CaCl 2 ·2H 2 O0.025 g/L, glucose 10g/L, H 3 BO 3 0.124mg/L,FeSO 4 ·7H 2 O 0.1068mg/L,CoSO 4 ·7H 2 O 0.0512mg/L,CuSO 4 ·5H 2 O 0.0044mg/L,MnCl 2 ·4H 2 O 0.00384mg/L,Na 2 MoO 4 ·2H 2 O 0.1068mg/L,ZnSO 4 ·7H 2 O 0.0512mg/L。
The enrichment resulted in a diversity of azotobacter genus levels as shown in fig. 1A, which consisted mainly of Klebsiella (80.77%), clostridium 12 (17.16%) and Clostridium 1 (1.88%).
Example 2 optimization of enrichment temperature conditions
And adding 5mL of bacterial liquid into a serum bottle, adding 45mL of culture medium, introducing nitrogen for 10min, and sealing by using a butyl rubber plug and an aluminum cap. The temperature rotation speed of the constant temperature incubator is 120rpm, the temperature is respectively set to be 25 ℃,30 ℃ and 35 DEG CCulturing at the above three temperatures for 48 hr, and measuring bacterial liquid OD 600 And determining the optimal growth temperature.
As shown in FIG. 1B, the growth of nitrogen-fixing bacteria was the best at 30 ℃.
Example 3 method for degrading methyl orange by using azotobacter cultured on toilet paper
Inoculating 5mL of the enriched azotobacter culture solution into a 120mL serum bottle, adding 45mL of the culture medium into the serum bottle, exposing to nitrogen for 10min, sealing with a butyl rubber plug and an aluminum cap, and culturing at 30 ℃ and 120rpm for 24h. After the completion of the culture, the suspension was centrifuged, washed with 0.9% sodium chloride solution and resuspended in a medium without a carbon source (i.e., the medium contained no glucose) to obtain a high-concentration azotobacter suspension.
Absorbing 5mL of high-concentration azotobacter suspension, inoculating the suspension into a serum bottle, adding toilet paper, methyl orange and a culture medium without a carbon source, finally exposing the serum bottle to nitrogen for 10min, sealing the bottle by using a butyl rubber plug and an aluminum cap, and culturing for 5 days at 30 ℃ and 120rpm, wherein the volume of the culture solution in the serum bottle is 50mL, the content of the toilet paper is 2g/L, and the content of the methyl orange is 25mg/L.
Sampling every day to determine the content change of sugar, protein and methyl orange in the sample and the CMC enzyme activity and the beta-glucosidase activity of azotobacter on the 5 th day, wherein the specific steps are as follows:
the sample in the serum bottle was aspirated by syringe, centrifuged at 10000rpm for 10min and filtered through a 0.45 μm filter.
(1) Determination of sugar content variation by anthrone-sulfuric acid method: 1mL of the filtered sample is put into a test tube, 4mL of anthrone-sulfuric acid reagent is added, and after uniform mixing, the mixture is heated in a water bath at 100 ℃ for 10min. After cooling, the absorbance was measured at a wavelength of 540 nm.
(2) And (3) measuring the protein content of the sample: the BCA miniprotein concentration assay kit describes the method for detecting protein concentration.
(3) Measuring the concentration change of methyl orange by a spectrophotometric method: 0.3mL of the sample was diluted with water to 3mL, and the absorbance was measured at a wavelength of 465 nm.
(4) CMC enzyme activity: 0.5mL of the centrifuged sample was added to 1.5mL of a 1% CMC-Na solution as an enzyme solution. Taking the inactivated enzyme solution as a blank control, mixing uniformly and covering, and reacting in a water bath at 50 ℃ for 1 hour. Taken out and added into 1.5mL rapidlyDNS reagent, shake in boiling water and react for 5 minutes. Taking out, cooling to room temperature, adding 6.5mL of water, shaking up and measuring OD 540 。
(5) Beta-glucosidase activity: 0.5ml of the centrifuged sample was used as a crude enzyme solution, and 1.5ml of a 0.5% salicin solution was added. Water bath at 50 ℃ for 1 hour. After taking out, 1.5mL of DNS reagent is rapidly added, and the mixture is shaken up in boiling water to react for 5 minutes. Taking out, cooling to room temperature, adding 2.5ml of water, shaking up and measuring OD 540 。
The situation that the azotobacteria decomposes the toilet paper and is converted into sugar after 5 days is shown in figure 2A, and the sugar content in the culture medium reaches 6.7mg/L after 5 days of culture; the protein content in the medium is shown in FIG. 2B, increasing the protein content to 69.1mg/L; the CMC enzyme activity and the beta-glucosidase activity of azotobacteria at day 5 are shown in figure 2C, the CMC enzyme activity reaches 0.080U/mL, and the beta-glucosidase activity reaches 0.069U/mL. The content of methyl orange is changed as shown in fig. 3, the content of methyl orange rapidly decreases from 48h to 120h, and the result shows that the azotobacter group can degrade methyl orange while decomposing toilet paper.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (8)
1. A method for enriching azotobacter and degrading dye by culturing azotobacter with cellulose is characterized by comprising the following steps:
(1) Collecting a soil sample as an inoculation source, suspending the soil sample in sterile water, and mixing uniformly; inoculating the suspension into a culture bottle, adding a culture medium, introducing nitrogen, sealing, and performing shake culture at 30-32 deg.C for 48-72h in a constant temperature incubator, wherein the volume of the culture system is 50 mL;
(2) Adding 2.5-5mL of the culture solution obtained in the step (1) into a new culture bottle, adding 45-47.5mL of a culture medium, introducing nitrogen, sealing, and performing shake culture at 30-32 ℃ in a constant-temperature incubator;
(3) After culturing for 48-72h, centrifuging the culture solution, removing the supernatant, suspending the thallus in a new 50mL culture medium, inoculating into a culture bottle, introducing nitrogen, sealing, and continuously performing shaking culture at 30-32 ℃;
(4) Repeating the step (3) for 10-15 times;
(5) Inoculating 2.5-5mL of enriched azotobacteria culture solution into a culture bottle, adding 45-47.5mL of culture medium into the culture bottle, introducing nitrogen, sealing, and performing shake culture at 30-32 deg.C for 20-24h;
(6) Centrifuging the azotobacter culture solution in the step (5), discarding supernatant, and suspending thalli in a carbon source-free culture medium to obtain high-concentration bacterial suspension; taking 5mL of the high-concentration bacterial suspension, inoculating the high-concentration bacterial suspension into a culture bottle, adding a carbon-source-free culture medium, cellulose materials and dyes, finally introducing nitrogen into the culture bottle, sealing, and performing shaking culture in a constant-temperature incubator at 30-32 ℃; sampling every day, and measuring the concentration change of sugar, protein and dye for 1-5 days; measuring the activity of cellulase and beta-glucosidase of azotobacter on the 5 th day;
preferably, the medium component is KH 2 PO 4 2.3g/L,K 2 HPO 4 5.75g/L,MgSO 4 ·7H 2 O 0.025g/L,CaCl 2 ·2H 2 O0.025 g/L, glucose 10g/L, H 3 BO 3 0.124mg/L,FeSO 4 ·7H 2 O 0.1068mg/L,CoSO 4 ·7H 2 O 0.0512mg/L,CuSO 4 ·5H 2 O 0.0044mg/L,MnCl 2 ·4H 2 O 0.00384mg/L,Na 2 MoO 4 ·2H 2 O 0.1068mg/L,ZnSO 4 ·7H 2 O 0.0512mg/L；
The carbon-source-free medium in the step (6) is a medium obtained by removing glucose from the medium.
2. The method of claim 1, wherein the OD of the high concentration bacterial suspension in step (6) 600 ＝1.8-2.0。
3. The method of claim 1, wherein the cellulosic material is toilet paper and the dye is methyl orange.
4. The method according to claim 4, wherein the final content of the toilet paper in the step (6) is 1.8-2g/L, and the final content of methyl orange is 20-25mg/L.
5. The method according to any one of claims 1 to 4, wherein the sealing is carried out after 10 to 15min of introducing nitrogen gas.
6. The method according to any one of claims 1 to 4, wherein the shaking culture rotation speed is 120 to 130rpm.
7. The method according to any one of claims 1 to 4, wherein the centrifugation conditions are: centrifuging at 7000-8000rpm for 6-10min.
8. The method according to any one of claims 1 to 4, wherein the culture temperature in steps (1) to (6) is 30 ℃.
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