CN113549569B - Lysis strain, microbial agent and application thereof - Google Patents
Lysis strain, microbial agent and application thereof Download PDFInfo
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- 230000009089 cytolysis Effects 0.000 title claims abstract description 42
- 230000000813 microbial effect Effects 0.000 title claims abstract description 11
- 239000010802 sludge Substances 0.000 claims abstract description 83
- 238000000034 method Methods 0.000 claims abstract description 27
- 241000827781 Geobacillus sp. Species 0.000 claims abstract description 19
- 230000009467 reduction Effects 0.000 claims abstract description 19
- 241000626621 Geobacillus Species 0.000 claims abstract description 15
- 238000012216 screening Methods 0.000 claims abstract description 13
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 10
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 230000001580 bacterial effect Effects 0.000 claims description 21
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 238000005273 aeration Methods 0.000 claims description 13
- 239000001963 growth medium Substances 0.000 claims description 13
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 241001052560 Thallis Species 0.000 claims description 8
- 238000009630 liquid culture Methods 0.000 claims description 8
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- 239000008223 sterile water Substances 0.000 claims description 7
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 5
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- 238000007664 blowing Methods 0.000 claims description 4
- 239000002054 inoculum Substances 0.000 claims description 4
- 239000010801 sewage sludge Substances 0.000 claims description 4
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- 229940041514 candida albicans extract Drugs 0.000 description 3
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 3
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 3
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 3
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
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- 235000019319 peptone Nutrition 0.000 description 3
- 239000011684 sodium molybdate Substances 0.000 description 3
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
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- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 3
- 229910000368 zinc sulfate Inorganic materials 0.000 description 3
- 239000011686 zinc sulphate Substances 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
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- 238000003794 Gram staining Methods 0.000 description 2
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- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 2
- 239000002351 wastewater Substances 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108010076119 Caseins Proteins 0.000 description 1
- 101710089384 Extracellular protease Proteins 0.000 description 1
- 102000004366 Glucosidases Human genes 0.000 description 1
- 108010056771 Glucosidases Proteins 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
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- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
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- 239000004021 humic acid Substances 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
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- 239000010841 municipal wastewater Substances 0.000 description 1
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- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 239000010815 organic waste Substances 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 230000007065 protein hydrolysis Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- OARRHUQTFTUEOS-UHFFFAOYSA-N safranin Chemical compound [Cl-].C=12C=C(N)C(C)=CC2=NC2=CC(C)=C(N)C=C2[N+]=1C1=CC=CC=C1 OARRHUQTFTUEOS-UHFFFAOYSA-N 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
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- 238000010561 standard procedure Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F11/00—Treatment of sludge; Devices therefor
- C02F11/02—Biological treatment
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2303/00—Specific treatment goals
- C02F2303/06—Sludge reduction, e.g. by lysis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/20—Sludge processing
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Treatment Of Sludge (AREA)
Abstract
THE invention relates to a lysis strain, a microbial agent and application thereof, wherein THE lysis strain is Geobacillus sp (Geobacillus sp.) THE-5, and THE preservation number is CCTCC M2021513. THE method is obtained by separating and screening THE high-temperature domesticated mature sludge, THE lysis strain and THE sludge reduction treatment agent containing THE lysis strain can be applied to sludge reduction treatment, THE separation and screening of THE lysis strain is that Geobacillus sp THE-5 can utilize organic matters in THE sludge to grow and reproduce, and enzyme produced in THE metabolic process of THE strain can accelerate hydrolysis of extracellular polymers and lysis of sludge cells, so that THE purpose of sludge lysis is achieved.
Description
Technical Field
The invention belongs to the field of sludge biological treatment, and particularly relates to a lysis strain, a microbial agent and application thereof.
Background
Biological wastewater treatment plants (WWTPs) have been used worldwide for treating municipal wastewater. Although the organic matter in the wastewater can be effectively removed, a large amount of surplus sludge is generated. With the increasing popularity of sewage treatment plants, the population is increasing, and the construction of new sewage treatment plants and the stricter sewage treatment requirements are such that the sludge yield will continue to increase. Thus, the treatment and disposal of sludge has become an increasingly significant challenge in the field of environmental engineering.
In conventional sewage treatment plants, there are mainly two methods for reducing the residual amount of sludge, namely a wastewater line and a sludge line. The in-situ sludge reduction technology is an important means for disposing sludge from the source, the common method is to recycle the backflow sludge after lysis to the main flow bioreactor for further biodegradation, and the physical, chemical and biological means are utilized to minimize the amount of sludge discharged outwards by the whole sewage treatment system, so that the method has good application prospect. Physical and chemical methods can result in additional energy consumption, high costs, secondary pollution, and other economic costs. In contrast, biological pretreatment shows absolute advantages in accelerating the sludge hydrolysis process.
Extracellular Polymers (EPS) are an important component of the matrix of sludge flocs, consisting of a number of organic substances such as organic macromolecules like polysaccharides, proteins, humic acids, uronic acids, lipid compounds, etc. The sludge reduction by adopting the biological pretreatment method is to add hydrolase into the sludge, accelerate the hydrolysis of extracellular polymers under the catalysis of the hydrolase, destroy the microbial cell structure and dissolve insoluble substances in the sludge. However, the direct addition of hydrolase has the problems of higher cost and more severe reaction conditions, and the actual large-scale application is difficult. Therefore, in order to improve the industrial application of the biological pretreatment method, it is necessary to separate and screen out a strain capable of reducing the sludge, and the hydrolysis of extracellular polymers is accelerated by enzymes generated by the strain in the metabolic process of the sludge, so as to achieve the purpose of sludge reduction.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a lysis strain, a microbial agent and application thereof, wherein the lysis strain can utilize organic matters in sludge to grow and reproduce, and enzyme generated in the metabolic process of the strain can accelerate hydrolysis of extracellular polymers and lysis of sludge cells, so that the purpose of sludge lysis is achieved.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
The technical scheme is as follows:
A lysis strain, geobacillus sp.) th-5 belonging to THE genus Geobacillus (Geobacillus sp.) deposited at THE chinese collection of typical cultures, deposit address: the preservation date is 2021, 5 months and 11 days at the university of Wuhan in China, and the preservation number is CCTCC M2021513.
Further, THE 16S rDNA sequence of THE Geobacillus sp.) THE-5 is shown in SEQ ID: 1.
Further, the strain is obtained by separating and screening from high-temperature domesticated mature sludge.
Furthermore, the high-temperature domesticated mature sludge is obtained by domesticating raw sludge under the aeration condition at 55-80 ℃.
Further, in the domestication process, the aeration rate is 0.1-1.0vvm, and the domestication time is 5-60 days.
The second technical scheme is as follows:
A microbial agent comprising the lysis strain.
The technical scheme is as follows:
use of said lysed strain in sludge reduction treatment.
The technical scheme is as follows:
An application of the microbial agent in sludge reduction treatment.
The fifth technical scheme is that:
a method for sludge reduction treatment of the lysis strain, comprising the following steps:
step one, activation culture of the lysate:
Inoculating THE activated dominant strain of Geobacillus (Geobacillus sp.) THE-5 into a liquid culture medium, performing constant temperature shaking culture for activation to obtain seed liquid with OD 600 of 1.0-1.2, centrifuging, and collecting thalli;
step two, uniformly blowing the collected thalli with sterile water to obtain bacterial liquid;
Step three, sludge reduction treatment
Adding bacterial liquid into the organic sludge to be treated, adjusting pH to 6-10, and performing lysis reaction under aeration and stirring conditions.
Further, in THE first step, THE inoculum size of THE Geobacillus (Geobacillus sp.) THE-5 in THE liquid culture medium is 1-5%; the temperature of shaking culture is 50-85 ℃, the shaking speed is 40-200rpm, the times of shaking culture at constant temperature is 2-3 times, and the time of shaking culture each time is 6-20h;
In the second step, the OD 600 of the bacterial liquid is 0.4-0.8;
In the third step, the adding amount of the bacterial liquid is 10-15% of the volume of the organic sludge to be treated, the temperature of the lysis reaction is 50-85 ℃, the aeration rate is 0.1-1.0vvm, the stirring rate is 50-200rpm, and the time of the lysis reaction is 1-36h.
Compared with the prior art, the invention has the following beneficial effects:
1. According to THE invention, THE mature sludge is domesticated at 55-80 ℃ to separate and screen THE lysis strains, and THE lysis strains of THE Geobacillus (Geobacillus sp.) THE-5 separated by THE method can carry out invisible growth in THE sludge to be treated, secrete extracellular enzymes, promote hydrolysis of macromolecular organic matters in THE sludge, so that organic matters in a solid phase of THE sludge can be released into a liquid phase for subsequent utilization.
2. Compared with the traditional physicochemical reduction technology, the method for the lysis of the sludge has the characteristics of economy, high efficiency, lower energy consumption, high efficiency of the lysis and easy control of reaction conditions, and in the sewage treatment process, the lysed sludge can continuously flow back to a biochemical tank to provide a carbon source for the denitrification process of the sewage, and simultaneously, a large amount of dissolved organic matters released after the lysis can be used for anaerobic digestion of the sludge, so that the resource utilization rate is improved.
Drawings
FIG. 1 is a phylogenetic tree of lytic strains;
FIG. 2 is a graph showing THE change in THE activity of THE Bacillus terreus (Geobacillus sp.) THE-5 protease in example 2;
Detailed Description
The invention is further illustrated below with reference to examples.
The sewage sludge used for separation and screening of the invention is derived from: a sewage treatment plant (A/O) process of Jiangxin Africa in Nanjing city; index of sewage sludge: TSS is 10147 +/-210 mg/L; VSS 7416+ -170 mg/L; TCOD 5476+ -180 mg/L; SCOD is 75+/-10 mg/L; the pH is 6.8+/-0.2.
The sludge used in the lysis test of the invention is derived from: the A 2/O process runs the surplus sludge of the sewage plant, and the sludge properties are as follows: total Suspended Solids (TSS) of 14685+ -146 mg/L, volatile Suspended Solids (VSS) of 6021+ -25 mg/L, total Chemical Oxygen Demand (TCOD) of 4350+ -88 mg/L, dissolved chemical oxygen demand of 65+ -5 mg/L, pH of 7.0-7.3. Sieving sludge to remove particles with particle size of more than 1mm, and storing in refrigerator at 4deg.C for no more than 1 week.
The control strain YL-1 belongs to the genus Geobacillus (Geobacillus sp.) and is deposited with the China center for type culture Collection, with the deposit address being the university of Wuhan in China and the deposit time being No. 2021, 5 months and 8; the preservation number is: cctccc AB 2021109; the control strain YL-1 is a publicly available strain.
Liquid medium: macroelements: nitrilotriacetic acid 100mg,NaCl 8mg,KH 2PO4 111mg,MgSO4·7H2 O100 mg, peptone 1g, yeast extract 1g; adding 1L deionized water into microelements ：Na2MoO4·2H2O 0.025mg,FeCl3 0.28mg,CuSO4 0.16mg,MnSO4·H2O 2.2mg,H3BO3 0.5mg,ZnSO4·7H2O 0.5mg,CoCl2·6H2O 0.046mg,CaSO4 60mg., adjusting pH to 7-7.5, placing into autoclave, and sterilizing at 121deg.C for 20min.
Example 1: isolation, screening, identification and application of dominant lysozyme
The method mainly comprises the following steps of firstly separating and screening dominant strains from domesticated sludge, and secondly identifying the dominant strains; determining the species relation of dominant strains; 3. the optimal process for carrying out sludge reduction treatment by utilizing the screened dominant strain is obtained through process optimization.
1. Separating and screening the acclimatized sludge to obtain dominant strains, wherein the method specifically comprises the following steps:
step 1, domesticating sludge: domesticating sewage sludge under aeration condition of 0.1-1.0vvm at 55-80deg.C for 5-60 days;
step 2, performing gradient dilution on the domesticated and mature sludge, separating and purifying by adopting a dilution pouring method and a three-zone streaking method to obtain single-colony bacterial strains, and naming and numbering the bacterial strains respectively;
Step 2.1, plate pouring: solid medium: macroelements: nitrilotriacetic acid 100mg, naCl 8mg, KH 2PO4111mg,MgSO4·7H2 O100 mg, peptone 1g, yeast extract 1g; 20g of agar; adding 1L deionized water into microelements ：Na2MoO4·2H2O0.025mg,FeCl3 0.28mg,CuSO4 0.16mg,MnSO4·H2O 2.2mg,H3BO3 0.5mg,ZnSO4·7H2O 0.5mg,CoCl2·6H2O 0.046mg,CaSO4 60mg., adjusting pH to 7-7.5, placing into autoclave, and sterilizing at 121deg.C for 20min. And (3) after cooling to 50-55 ℃, pouring the agar into an aseptic culture dish in sequence, ensuring that the bottom of the aseptic culture dish is about 2/3 of that of the aseptic culture dish, and reversely buckling the flat plate on an aseptic table after agar is solidified.
Step 2.2, separation: taking a plurality of sterilized EP pipes, respectively adding 0.9ml of sterile water, taking 0.1ml of sludge domesticated and matured at 65 ℃, adding the sludge into the sterile water of a first pipe, fully mixing and shaking uniformly, taking 0.1ml of diluent from the first pipe into the next sterilized EP pipe, mixing and shaking uniformly, diluting to a fourth pipe and a fifth pipe in such a way that the dilution factors are respectively as follows from the first pipe: 10-1, 10 -2、10-3、10-4、10-5; two tubes of the dilutions 10 -4 and 10 -5, each 0.1ml, were added dropwise, and the dilutions were plated onto corresponding plates and applied uniformly with sterilized applicator bars. Placing the smeared flat plate on a sterile table for 20-25 min, and inverting the flat plate in an incubator for 12h when bacteria liquid permeates into the culture medium, wherein the culture temperature is 50-85 ℃. Selecting single bacterial colony by using a sterilized inoculating loop, streaking on a flat plate by adopting a three-area streaking method, repeating for 3-4 times to obtain purified bacterial colony, and separating 15 bacterial strains in the embodiment;
step 3, primarily screening the separated strains with single colonies through a hydrolysis circle test, further screening the strains with larger hydrolysis circles by adopting a sludge lysis test and an extracellular enzyme activity test, and retaining the strains with higher sludge VSS degradation rate and higher extracellular enzyme activity, namely the dominant strains; THE dominant strain obtained by screening is THE-5;
hydrolysis circle test: inoculating the lysis strain separated from the domesticated mature sludge into a skimmed milk powder solid culture medium, placing the skimmed milk powder solid culture medium into a constant temperature incubator, and observing the change condition of hydrolysis circles after each strain is 12 hours.
The solid culture medium of the skim milk powder comprises the following components: macroelements: nitrilotriacetic acid 100mg,NaCl 8mg,KH 2PO4111mg,MgSO4·7H2 O100 mg, peptone 1g, yeast extract 1g, agar 20g, skimmed milk powder 20g, distilled water 1L; the pH of the mixture is regulated to 7.0-7.5 by microelements ：Na2MoO4·2H2O 0.025mg,FeCl3 0.28mg,CuSO4 0.16mg,MnSO4·H2O 2.2mg,H3BO30.5mg,ZnSO4·7H2O 0.5mg,CoCl2·6H2O 0.046mg,CaSO4 60mg..
Preliminary sludge dissolution test: taking a plurality of conical flasks and respectively adding sludge with the solid content of 1.2% -1.5%; after the strains subjected to primary screening are respectively cultivated for 12-15 hours, sterile water is used for blowing and beating uniformly, then the strains are respectively inoculated into corresponding conical flasks, constant-temperature shaking table cultivation is carried out at the temperature of 50-85 ℃ and the oscillating speed of 100rpm, meanwhile, ultrapure water is used as blank control, the Volatile Suspended Solid (VSS) content of the sludge in each conical flask after 24 hours is measured, and the reduction rate of the VSS of the sludge is calculated.
Extracellular enzyme Activity assay: the strains after primary screening are respectively inoculated into a liquid culture medium according to the inoculum size of 1 percent, and the activity of protease is measured by adopting an azo casein method.
2. Identifying dominant strains and determining the species relationship of the dominant strains; specifically comprises physiological and biochemical identification and 16S rDNA identification:
1) Gram staining microscopy: THE dominant strain is THE-5, which is subjected to gram-staining microscopic examination, oxalic acid crystal violet dye liquor is sequentially subjected to dip-staining for 1min, iodine liquor is subjected to dip-staining for 1min,95% ethanol is decolorized for 30s, safranine liquor is subjected to dip-staining for 2-3min, and a cover glass is placed under a microscope for observation, wherein THE observation results are as follows: THE THE-5 cells appeared dark purple, rod-like, and were gram positive.
2) 16S rDNA identification: THE nucleotide sequence of THE 16S rDNA of THE dominant strain THE-5 is shown as SEQ ID: 1.
THE dominant strain THE-5 belongs to THE genus Geobacillus and THE phylogenetic tree constructed by adopting THE adjacency method is shown in figure 1.
3. And obtaining the optimal process for sludge reduction treatment by using the screened dominant strain through process optimization.
In this example, THE optimal process for sludge reduction treatment using THE dominant strain Geobacillus sp.) THE-5:
step 1, activating and culturing dominant strains:
Inoculating THE dominant strain THE-5 into a liquid culture medium according to THE inoculation amount of 1% -5%, then carrying out shake culture for 2-3 times at 50-85 ℃ to activate, obtaining seed liquid with OD 600 of 1.0-1.2, and then centrifuging to collect thalli; the time of each shaking culture is 12-15h, and the shaking speed is 100-120rpm.
Step 2, uniformly blowing the collected thalli with sterile water to obtain a bacterial liquid for later use; OD 600 of the bacterial liquid is 0.4-0.8;
step 3, the lysis treatment of the sludge
Adding bacterial liquid into the organic sludge to be treated according to the volume of 10-15%, adjusting the pH value to 6-10, and carrying out a lysis reaction for 1-36h under the conditions of stirring and aeration at 50-85 ℃; the aeration rate is 0.1-1.0vvm, and the stirring rate is 40-200rpm.
Example 2: extracellular enzyme Activity analysis of THE dominant Strain Geobacillus sp THE-5
The residual sludge is organic waste water containing suspended solids with higher concentration, and the main components of the residual sludge comprise small molecular soluble organic matters (monosaccharides, amino acids and the like) and refractory macromolecular organic matters (mainly including proteins, polysaccharides and lipid compounds), wherein the carbohydrate accounts for 20% of the total organic matters. It was found that during the dissolution of excess sludge, glucosidase, protease and alpha-amylase are closely related to the degradation of extracellular polymers of sludge, and that protein hydrolysis is a more critical factor. Therefore, the activity of the extracellular protease secreted by bacteria is of paramount importance.
THE dominant strain of THE invention, geobacillus sp. THE-5, was inoculated into THE corresponding liquid medium at an inoculum size of 1% and THE protease activity was determined by azocasein method. Bacterial solutions at different time points are taken, centrifuged in a desk centrifuge (4 ℃ C., 10 min, 14,000Xg), the supernatant and a substrate azocasein are taken to fully react for 30min at 50-85 ℃, 5% trichloroacetic acid (TCA) is immediately added, the mixture is fully mixed for 5 seconds by a vortex oscillator and then is placed under ice cubes to balance for 10 min, naOH is dripped into the supernatant after centrifugation until the concentration is 0.4M, and an absorbance value at 440nm is measured by using a UV/VIS spectrophotometer. 1 protease activity unit is defined as the amount of enzyme required to hydrolyze soluble casein to 1 micromole of tyrosine per unit time under certain experimental conditions. The results are shown in fig. 2, and the research conclusion shows that: THE average enzyme activity of THE THE-5 in 14h is 1000U/L; THE enzyme activity of THE THE-5 reaches a peak value after 6 hours of culture and is 1500U/L; between 6 and 14 hours, the extracellular enzyme activity of the strain begins to exhibit a reduced state due to rapid consumption of nutrients.
Example 3: THE dominant strain Geobacillus sp. THE-5 was investigated for its sludge dissolving performance
THE dominant strain of THE invention, namely THE Geobacillus sp, THE-5 and THE control strain YL-1 are respectively inoculated into corresponding solid culture media, activated and cultured for 12 hours at 50-85 ℃, THE activated strain is inoculated into a 250ml conical flask containing 50ml of liquid culture media by using a sterile gun head, cultured for 12 hours at 50-85 ℃, THE shaking speed is 120rpm, and THE activation is repeated for 3 times. After activated and matured thalli are centrifuged by a desk top centrifuge (6000 rpm,10 min), the thalli are collected and are blown by sterile water uniformly. The volume of the dissolved sludge in each batch is 360ml, the volume of the added bacterial liquid is 40ml, and a non-inoculated blank control group is additionally arranged, and 40ml of distilled water is added into the non-inoculated blank control group, so that the bacterial liquid volume accounts for 10% of the total reaction system. Then stirring each group for reaction for 36h at 50-85 ℃ and pH6.8-7.0 under aeration condition; the aeration rate was 0.2vvm and the stirring rate was 60.+ -.10 rpm. And measuring the VSS concentration in each sample by adopting a national standard method, and calculating the degradation rate of the sludge VSS. The results are shown in Table 1, and the study conclusion shows that: compared with a blank control group VSS, THE degradation rate of THE Geobacillus (Geobacillus sp.) THE-4 is improved by 19.8%, and THE degradation rate is improved by 17.2% compared with a control strain YL-1.
TABLE 1
| THE-5 | Control strain YL-1 | Blank control group | |
| Degradation rate of VSS (%) | 24.5±0.25 | 7.3±0.03 | 4.7±0.05 |
Example 4: effect of temperature on lysis
THE dominant strain of Geobacillus sp.) THE-5 is cultivated for 15-18 h with liquid culture medium at 60 ℃,65 ℃ and 70 ℃ respectively, activated repeatedly for 3 times, THE OD 600 of THE strain is between 1.0 and 1.2, centrifuged (600 rpm,10 min), and THE thallus is collected for subsequent lysis experiments. The temperature of each reactor was 60 ℃,65 ℃ and 70 ℃, respectively. The volume of the dissolved sludge is 360ml, the volume of the added bacterial liquid is 40ml, and the bacterial liquid volume accounts for 10% of the total reaction system. THE degradation rate of THE Bacillus terreus (Geobacillus sp.) THE-5 at different temperatures with VSS is shown in Table 2, wherein THE best sludge dissolving effect of THE Bacillus terreus (Geobacillus sp.) THE-5 at 65 ℃ is up to 24.5%.
TABLE 2
| 60℃ | 65℃ | 70℃ | |
| Degradation rate of VSS (%) | 22.9±0.25 | 24.5±0.27 | 19.8±0.27 |
The above described embodiments are only preferred examples of the invention and are not exhaustive of the possible implementations of the invention. Any obvious modifications thereof, which would be apparent to those skilled in the art without departing from the principles and spirit of the present invention, should be considered to be included within the scope of the appended claims.
Sequence listing
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<120> A lysis strain, microbial agent and use thereof
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tagagtttga tcatggctca ggacgaacgc tggcggcgtg cctaatacat gcaagtcgag 60
cggaccgaac gggagcttgc ttctgttcgg ttagcggcgg acgggtgagt aacacgtggg 120
taacctgccc gtaagaccgg gataactccg ggaaaccggg gctaataccg gataacacca 180
aagaccgcat ggtctttggt tgaaaggtgg cttttgctac cacttacgga tgggcccgcg 240
gcgcattagc tagttggtga ggtaacggct caccaaggcg acgatgcgta gccggcctga 300
gagggtgacc ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt 360
agggaatctt ccgcaatgga cgaaagtctg acggagcgac gccgcgtgag cgaagaaggt 420
cttcggatcg taaagctctg ttgttaggga agaagaagta ccgttcgaat agggcggtac 480
ggtgacggta cctaacgaga aagccccggc taactacgtg ccagcagccg cggtaatacg 540
tagggggcga gcgttgtccg gaattattgg gcgtaaagcg cgcgcaggcg gtcccttaag 600
tctgatgtga aagcccacgg ctcaaccgtg gagggtcatt ggaaactggg ggacttgagt 660
gcagaagagg agagcggaat tccacgtgta gcggtgaaat gcgtagagat gtggaggaac 720
accagtggcg aaggcggctc tctggtctgt aactgacgct gaggcgcgaa agcgtgggga 780
gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct aagtgttaga 840
ggggttaaac cctttagtgc tgtagctaac gcgttaagca ctccgcctgg ggagtacggc 900
cgcaaggctg aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt 960
taattcgaag caacgcgaag aaccttacca ggtcttgaca tcccctgaca accctggaga 1020
cagggcgttc ccccttcggg gggacagggt gacaggtggt gcatggttgt cgtcagctcg 1080
tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac cctcgcccct agttgccagc 1140
attcagttgg gcactctagg gggactgccg gctaaaagtc ggaggaaggt ggggatgacg 1200
tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatggg cggtacaaag 1260
ggctgcgaac ccgcgagggg gagcgaatcc caaaaagccg ctctcagttc ggattgcagg 1320
ctgcaactcg cctgcatgaa gccggaatcg ctagtaatcg cggatcagca tgccgcggtg 1380
aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagcttg caacacccga 1440
agtcggtgag gtaacccttt aagggagcca gccgccgaag gtggggcaag tgattggggt 1500
gaagtcgtaa caaggtaacc a 1521
Claims (7)
1. A lysis strain is characterized in that THE strain is Geobacillus sp THE-5, and is preserved in China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC M2021513;
THE 16S rDNA sequence of THE Geobacillus (Geobacillus sp.) THE-5 is shown in SEQ ID:1 is shown in the specification;
The strain is obtained by separating and screening from high-temperature domesticated mature sludge;
The high-temperature domesticated mature sludge is obtained by domesticating sewage sludge at 55-80 ℃ under the aeration condition.
2. The lysis strain of claim 1, wherein aeration rate is 0.1-1.0 vvm and acclimation time is 5-60 days.
3. A microbial agent comprising the lysis strain of any of claims 1-2.
4. Use of a lysis strain according to any of claims 1-2 in sludge reduction treatment.
5. Use of the microbial agent according to claim 3 in sludge reduction treatment.
6. A method for sludge reduction treatment using the lysis strain according to any one of claims 1 to 2, comprising the steps of:
step one, activation culture of the lysate:
Inoculating THE activated strain Geobacillus sp THE-5 into a liquid culture medium, performing constant-temperature shaking culture for activation to obtain seed liquid with OD 600 of 1.0-1.2, centrifuging, and collecting thalli;
step two, uniformly blowing the collected thalli with sterile water to obtain bacterial liquid;
Step three, sludge reduction treatment
Adding bacterial liquid into the organic sludge to be treated, adjusting pH to 6-10, and performing lysis reaction under aeration and stirring conditions.
7. The method for sludge reduction treatment by using a lysate strain according to claim 6,
In THE first step, THE inoculum size of THE Geobacillus (Geobacillus sp.) THE-5 in THE liquid culture medium is 1-5%; the temperature of shaking culture is 50-85 ℃, the shaking speed is 40-200rpm, the times of shaking culture at constant temperature is 2-3 times, and the time of shaking culture each time is 6-20h;
In the second step, the OD 600 of the bacterial liquid is 0.4-0.8;
In the third step, the adding amount of the bacterial liquid is 10-15% of the volume of the organic sludge to be treated, the temperature of the lysis reaction is 50-85 ℃, the aeration rate is 0.1-1.0vvm, the stirring rate is 50-200 rpm, and the time of the lysis reaction is 1-36h.
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| CN103193368A (en) * | 2013-04-22 | 2013-07-10 | 哈尔滨工业大学 | Method for promoting excess sludge microorganisms to hydrolyze by using thermophilic geobacillus sp |
| CN111138054A (en) * | 2020-01-09 | 2020-05-12 | 东南大学 | Biological lysis treatment method for sludge |
| KR102175728B1 (en) * | 2019-12-06 | 2020-11-06 | 동의대학교 산학협력단 | Microbial preparations for the decomposition of organic wastes including novel geobacilli strains and excipients |
| WO2020251113A1 (en) * | 2019-06-11 | 2020-12-17 | 동의대학교 산학협력단 | Novel geobacillus sp. strain and use thereof |
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| CN103193368A (en) * | 2013-04-22 | 2013-07-10 | 哈尔滨工业大学 | Method for promoting excess sludge microorganisms to hydrolyze by using thermophilic geobacillus sp |
| WO2020251113A1 (en) * | 2019-06-11 | 2020-12-17 | 동의대학교 산학협력단 | Novel geobacillus sp. strain and use thereof |
| KR102175728B1 (en) * | 2019-12-06 | 2020-11-06 | 동의대학교 산학협력단 | Microbial preparations for the decomposition of organic wastes including novel geobacilli strains and excipients |
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