CN114292797B - Physarum viscosum and application of microbial flocculant thereof in sewage treatment - Google Patents
Physarum viscosum and application of microbial flocculant thereof in sewage treatment Download PDFInfo
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- Separation Of Suspended Particles By Flocculating Agents (AREA)
Abstract
The invention discloses a strain of slime mold and application of a microbial flocculant thereof in sewage treatment, and relates to the technical field of environmental microorganisms. Said Campylobacter adhesionEnsifer adhaerens Ea01 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2022019. Subjecting said Physarum viscosum toEnsifer adhaerens Ea01 is inoculated on the fermentation medium, and the fermentation liquid is the microbial flocculant. The microbial flocculant can effectively solve the problem of suspended pollutants in water. Adding 2% (v/v) and 5% (w/v) CaCl into the microbial flocculant2The flocculation rate of 5g/L kaolin is 89.5 percent by adding 2.5 percent (v/v). The strain Ea01 can be applied to various sewage treatments, strengthens the flocculation capacity of suspended matters in water, promotes the separation of mud and water, and has remarkable flocculation effect.
Description
Technical Field
The invention belongs to the technical field of environmental microorganisms, and particularly relates to a strain of slime mold and application of a microbial flocculant thereof in sewage treatment.
Background
The flocculation process is an indispensable preposed unit operation technology in the water treatment process, and determines the operation condition, the final effluent quality and the operation cost of the subsequent process, so that the flocculation process becomes one of important research contents in the field of environmental engineering. At present, flocculation technology has been widely used in the water treatment industry. Traditional flocculation mainly adopts chemical flocculants such as aluminium salt, molysite, for traditional flocculation, microbial flocculation has advantages such as high efficiency, safety, nontoxic, biodegradable, no secondary pollution, has overcome traditional flocculating agent and has been difficult for the biological decomposition, can harm the shortcoming of human health, increase environmental risk, has better broad-spectrum nature in the aspect of sewage treatment, can also reach fine effect simultaneously. Therefore, the novel microbial flocculant is prepared, the purposes of efficient flocculation and sedimentation and reduction of suspended particulate matters in water are achieved, and the method has important research significance for sewage treatment and restoration.
Disclosure of Invention
Aiming at the problems, the invention aims to provide a strain of corynebacterium mucosae to achieve the aims of effectively settling suspended particles in sewage and reducing the turbidity of a water body.
In order to achieve the above object, the first aspect of the present invention provides a bioflocculating bacterium identified as a strain of CampylobacterEnsifer adhaerens Ea01, preserved in China Center for Type Culture Collection (CCTCC), and the name of the strain:Ensifer adhaerens ea011 and the preservation number is CCTCC NO: m2022019, the preservation date is 2022, 01 month and 05 days, and the preservation address is Wuhan university in China.
Said Physarum viscosumEnsifer adhaerens Ea01 is gram-negative bacteria, and is in the shape of short rod, and the shape of bacteria growing on LB is round, white, transparent, glossy, smooth surface, convex lens shape, and regular edge.
Said Physarum viscosumEnsifer adhaerens Ea01, containing the gene sequence shown in SEQ ID NO. 1.
In a second aspect of the invention, there is provided a microorganism belonging to the genus CampylobacterEnsifer adhaerens Ea01 microbial flocculant preparation method. The method comprises the following steps:
(1) the said Physarum viscosumEnsifer adhaerensEa01, inoculating in beef extract peptone culture medium, culturing to obtain seed liquid;
(2) and (2) inoculating the seed liquid obtained in the step (1) to a fermentation medium according to a certain inoculation amount, and performing fermentation culture to obtain a fermentation liquid with flocculation activity, namely the microbial flocculant.
Further, the fermentation medium comprises the following components: 7g/L potassium dihydrogen phosphate, 20g/L glucose, 0.1g/L sodium chloride, 0.2g/L ammonium sulfate, 0.5g/L urea, 0.5g/L yeast powder and 0.2g/L magnesium sulfate heptahydrate.
Further, the fermentation culture conditions in the step (2) are as follows: the temperature is 30 ℃, the pH is 6-8, the rotating speed is 150r/min, and the time is 2-3 d.
The microbial flocculant has high-efficiency flocculation capacity, and in a test of 5g/L of kaolin, the flocculation rate of the microbial flocculant on the kaolin reaches 89.5%.
In a third aspect of the present invention, there is provided a microorganism belonging to the genus CampylobacterEnsifer adhaerensEa01 for use in sewage treatment.
The fourth aspect of the invention provides a sewage flocculation and precipitation method, which comprises the following steps:
(1) expanding and culturing the microbial flocculant;
(2) adding a certain amount of 5% (w/v) coagulant aid CaCl2And then adding a certain amount of the microbial flocculant.
Further, when SS (suspended matter concentration) is more than 1000mg/L, the adding amount of the coagulant aid is 0.5-4% (v/v), and the adding amount of the microbial flocculant is 1-5% (v/v); and when the SS (suspended matter concentration) is less than 1000mg/L, the adding amount of the coagulant aid is 0.5-4 per mill (v/v), and the adding amount of the microbial flocculant is 1-5 per mill (v/v).
Compared with the prior art, the invention has the following advantages:
the microbial flocculant is not limited by water, can be applied to various sewage treatments, forms obvious alum flocs, quickly precipitates and has a remarkable flocculation effect. The microbial flocculant disclosed by the invention is simple to prepare, good in effect and low in cost, and has great engineering application potential and wide application prospect.
Drawings
The invention is further illustrated by the following examples in conjunction with the drawings.
FIG. 1 is the colony morphology of the strain Ea01 on a plate in example 1
FIG. 2 is the growth morphology of the strain Ea01 in example 1 in a fermentation medium;
FIG. 3 is a phylogenetic tree of strain Ea01 in example 2.
Detailed Description
Example 1: screening and performance determination of biological flocculation bacteria
The flocculating agent is obtained by separating and screening the activated sludge of a certain sewage treatment plant in Shanghai city. The separation method comprises the following steps:
primary screening: 5mL of the bottom sludge is taken to be put into 45 mL of sterilized beef extract peptone enrichment medium, and after the culture is carried out for 1 d under constant temperature shaking at 30 ℃ and 150rpm, 1 mL of enrichment culture solution is taken to be put into a triangular flask containing 50 mL of enrichment medium, and the culture is carried out for 1 d under constant temperature shaking at 30 ℃ and 150 rpm. Carrying out gradient dilution on the enrichment solution, and taking 10-4、10-5、10-6Three gradients were plated on solid plates of enrichment medium (agar 15g/L, the remaining components were in beef extract peptone enrichment medium). Culturing in 30 deg.C constant temperature incubator until single colony grows out, selecting single clones with different shapes and sizes, streaking, purifying, numbering, preserving, separating, purifying to obtain multiple strains, and primarily screening.
Re-screening: inoculating the single colony obtained by primary screening into an enrichment medium for activation, further inoculating into a fermentation medium for culture at 30 ℃ and 150rpm for 2d to obtain a fermentation liquid, and performing flocculation activity determination. The determination method comprises the following steps: after being sieved by 5000 meshes of kaolin, 5g/L of kaolin suspension is prepared to be used as simulated suspended substance to pollute the water body. In that3mL of fermentation broth and 2.5mL of 5% (w/v) CaCl were added to 100mL of the simulated water sample2As a coagulant aid, NaOH and HCl are used for adjusting the pH value to 7, a magnetic stirrer is adopted for uniformly mixing and then standing for 30min, and meanwhile, a water sample without any microbial inoculum is used as a blank control under the same conditions. Respectively measuring the absorbance of the supernatant of the sample to be measured and the absorbance of the supernatant of the blank solution at 550nm by using an ultraviolet-visible spectrophotometer, and calculating the flocculation activity by using the following formula:
flocculation rate (%) = (B-a)/B100
In the formula, A-measuring the absorbance of the supernatant of the sample; b-absorbance of supernatant of blank solution
The strain Ea01 with the highest flocculation rate can be obtained. The obtained strain Ea01 was streaked on an LB plate, and the morphology of the colonies was observed as shown in FIG. 1.
Example 2: molecular biological characterization of strains
Extracting the genome DNA of the strain Ea01, and using the genome DNA as a template to amplify 16S rDNA, a primer: 16S rDNA-8F: AGAGTTTGATCCTGGCTCA, 1492R: GGTTACCTTGTTACGACTT. PCR reaction (20. mu.L): 5 mu L of template DNA, 10 mu L of PCR Taqmix, 0.6 mu L of each upstream primer and downstream primer, and ddH2O to the reaction system was 20. mu.L. PCR procedure: pre-denaturation at 94 deg.C for 5 min, denaturation at 94 deg.C for 30 s, annealing at 50 deg.C for 30 s, extension at 72 deg.C for 1 min, circulating for 30 times, extension at 72 deg.C for 10 min, and cooling at 16 deg.C for 10 min. The purification and sequencing of the PCR product was performed by Shanghai Jilie biotechnology, Inc., and the sequencing sequence was registered in GenBank for homology sequence alignment analysis, and MEGA 7.0 software was used to construct phylogenetic tree of the strain (FIG. 2).
The effective nucleotide sequence length of Ea01 is 1317bp, and the sequence is shown in a gene sequence table SEQ ID NO. 1. Through comparison, the sequence has 100% homology with NCBI database (NCBI accession number: MT 431909.1), and belongs to the species CladosporiumEnsifer adhaerensIs named asEnsifer adhaerens Ea01。
Example 3: flocculation capability verification of microbial flocculant
The microbial flocculant has high flocculation capacity. To verify the effect, the following experiments were performed: the volume of the bacterial liquidInoculating 2% of the inoculum size in a fermentation medium, and performing constant temperature shaking culture at 30 ℃ and 150rpm for 2d to obtain a fermentation liquid. After being sieved by 5000 meshes of kaolin, 5g/L of kaolin suspension is prepared to be used as simulated suspended substance to pollute the water body. 3mL of fermentation broth and 2.5mL of 5% (w/v) CaCl were added to 100mL of simulated water sample2Stirring and mixing evenly, standing for 30min, and measuring the light absorption value of the supernatant at 550nm to obtain the flocculation rate of 89.5%.
Example 4: application effect of microbial flocculant in sewage treatment
The microbial flocculant can be widely applied to flocculation of various water bodies such as aquaculture water bodies, livestock and poultry breeding wastewater, black and odorous river water bodies and the like, and has important significance for treatment and restoration of water bodies in different environments. To verify the effect, the following experiments were performed:
firstly, the application of the microbial flocculant in aquaculture water. 100mL of aquaculture water is taken, the pH value is 7.52, and the turbidity is 500 NTU. 250uL of 5% (w/v) CaCl was added2Then, 300uL of fermentation liquor is added, and the mixture is stirred uniformly and then stands for 30 min. Compared with a control group without the microbial flocculant, the water body is obviously clarified, suspended particles are settled to the bottom, and the turbidity is reduced by 67.2%.
And secondly, the application of the microbial flocculant in the pig raising wastewater. Taking 100mL of oxidation pond wastewater of a certain pig farm, wherein the pH value is 8.05, and the SS is 5000 mg/L. 2.5mL of 5% (w/v) CaCl was added2Then 3mL of fermentation broth is added, stirred uniformly and then kept stand for 30 min. The SS was reduced by 75.9% compared to the control without microbial flocculant.
And secondly, the application of the microbial flocculant in the black and odorous water body of the river. Taking 100mL of black and odorous water in a river channel, wherein the pH value is 7.69, and the turbidity is 100 NTU. 250uL of 5% (w/v) CaCl was added2Then, 300uL of fermentation liquor is added, and the mixture is stirred uniformly and then stands for 30 min. The turbidity decreased by 59.3% compared to the control without microbial flocculant.
The microbial flocculant is not limited by water, can be widely used for repairing and treating various water bodies, and can obviously remove suspended matters in the water bodies.
Sequence listing
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<120> Physarum viscosum and application of microbial flocculant thereof in sewage treatment
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Claims (6)
1. Physarum viscosumEnsifer adhaerens Ea01, which is preserved in China center for type culture Collection, and the preservation number of the strain is CCTCC NO: m2022019, preservation date 2022, 01 month and 05 days.
2. The preparation method of the microbial flocculant is characterized by comprising the following steps of:
(1) the method of producing the microorganism of claim 1Ensifer adhaerens Ea01, inoculating into beef extract peptone culture medium, culturing to obtain seed liquid;
(2) and (2) inoculating the seed solution obtained in the step (1) to a fermentation medium according to a certain inoculation amount, and performing fermentation culture to obtain a fermentation liquid with flocculation activity, namely the microbial flocculant.
3. The method of claim 2, wherein the fermentation medium comprises the following components: 7g/L potassium dihydrogen phosphate, 20g/L glucose, 0.1g/L sodium chloride, 0.2g/L ammonium sulfate, 0.5g/L urea, 0.5g/L yeast powder and 0.2g/L magnesium sulfate heptahydrate.
4. The method according to claim 2, wherein the fermentation conditions in step (2) are as follows: the temperature is 30 ℃, the pH is 6-8, the rotating speed is 150r/min, and the time is 2-3 d.
5. A microbial flocculant, wherein the microbial flocculant is produced by the method according to claims 2 to 4.
6. Use of a microbial flocculant according to claim 5 in the treatment of wastewater, comprising the steps of:
(1) expanding and culturing the microbial flocculant;
(2) adding a certain amount of 5% (w/v) coagulant aid CaCl2Then adding a certain amount of the microbial flocculant; when the concentration of suspended matters is more than 1000mg/L, the adding amount of the coagulant aid is 0.5-4% (v/v), and the adding amount of the microbial flocculant is 1-5% (v/v); when the concentration of suspended matters is less than 1000mg/L, the adding amount of the coagulant aid is 0.5-4 per mill (v/v), and the adding amount of the microbial flocculant is 1-5 per mill (v/v).
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