CN108238681A - Composite biological agent for low-temperature wastewater treatment and its preparation method and application - Google Patents
Composite biological agent for low-temperature wastewater treatment and its preparation method and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
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- C12N1/20—Bacteria; Culture media therefor
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02F2209/00—Controlling or monitoring parameters in water treatment
- C02F2209/08—Chemical Oxygen Demand [COD]; Biological Oxygen Demand [BOD]
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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Abstract
The present invention discloses a kind of composite biological agent for low-temperature wastewater treatment, which contains：10 zymotic fluids of Methylotrophic bacillus methylotrophicus WSWGH and complex enzyme；Complex enzyme includes protease, cellulase, oxidoreducing enzyme and pectase；Cryogenic temperature is 0~20 DEG C.Preparation method invention additionally discloses the above-mentioned composite biological agent for low-temperature wastewater treatment and the application in low-temperature wastewater treatment.Low temperature biocontrol bacterial strain WSWGH 10 is used for sewage treatment field by the present invention for the first time, by the way that the zymotic fluid of low temperature biocontrol bacterial strain WSWGH 10 and complex enzyme is compound, the zymotic fluid and a variety of complex enzymes for making low temperature biocontrol bacterial strain WSWGH 10 act synergistically, solve thing problems, such as current microorganism, treated sewage effect is relatively low under cryogenic, organic matter that can under cryogenic in efficient degradation sewage, treated sewage.
Description
Technical field
The present invention relates to sewage disposal, particularly for composite biological agent of low-temperature wastewater treatment and preparation method thereof and
Using.
Background technology
Water exists with three kinds of liquid, gaseous state and solid-state states, is widely distributed in the earth's crust, hydrosphere, atmospheric thermodynamics and organism,
Carry out water cycle.The Wuli-Shili-Renli system approach of water determines that it is the important compound for forming Eco-Environment System, is human survival
And carry out the essential natural resources of production activity.Water resource refers to be present in rivers, lake, glacier and ground in water recycles
Under water in shallower water-bearing layer, be mainly derived from Atmospheric precipitation, recycle to be supplemented and updated by water.Water pollution is not
There is water quality type or contamination type water shortage in the area that some script shortage of water resources can only be aggravated, can also threaten health and the life of the public
Bioplasm amount seriously affects the sustainable development of human society.Under normal circumstances, there is certain dissolved oxygen amount in water, dissolved oxygen amount is not only
Ensure aquatile existence, moreover it is possible to contaminant degradation be promoted by redox reaction, promote the self-cleaning of natural water body.It is biological dirty
The discharge of water, industrial wastewater makes large amount of organic enter natural water with a large amount of nitrogen, phosphorus, potassium element, causes algae in water a large amount of
Breeding, dissolved oxygen in water declines, so that water plant, aquatic animal mortality.In addition, the pollutant in natural water can also
Human body is entered by drinking-water or food chain, makes the acute or chronic poisoning of human body.
At present, sewage disposal technology mainly includes following several：(1) chemical substance is added in into sewage to degrade or recycle
Pollutant in sewage, including the precipitation method, neutralisation, oxidation-reduction method and Chemical Physics sterilization etc.；(2) physical chemistry is utilized
Method except the pollutant in decontaminated water, including ion-exchange, absorption method and membrane separation process etc.；(3) by physical method come
Insoluble pollutant in separation, recycling sewage；(4) by the metabolism of microorganism by the organic pollutant degradation in sewage
Method for stable and harmless substance.Microbial degradation method is because with secondary pollution is small, processing is thorough, low energy consumption and efficient
The advantages that and be widely used, however, microbial activity is affected by temperature in existing microbial degradation method, decline it with temperature
Treated sewage ability declines.
Invention content
Goal of the invention：The object of the present invention is to provide a kind of microorganisms for being capable of efficient sewerage processing under cryogenic to answer
Close biological agent.
It is a further object of the present invention to provide the preparation method and application of above-mentioned composite biological agent.
Technical solution：The present invention provides a kind of composite biological agent for low-temperature wastewater treatment, the composite biological agent
Contain：Methylotrophic bacillus (Bacillus methylotrophicus) WSWGH-10 zymotic fluids and complex enzyme；It is compound
Enzyme includes protease, cellulase, oxidoreducing enzyme and pectase；Above-mentioned low temperature refers to that temperature is 0~20 DEG C.
WSWGH-10 zymotic fluids are as follows for Methylotrophic bacillus (Bacillus methylotrophicus)
It prepares：By Methylotrophic bacillus (Bacillus methylotrophicus) WSWGH-10 strain culturings to pair
It is inoculated into LB fluid nutrient mediums by volume fraction 0.5%~1.5% after the number phase, under the conditions of 0~30 DEG C, shaking table concussion 120~
180rpm cultivates 24~48 hours to get Methylotrophic bacillus (Bacillus methylotrophicus) WSWGH-
10 zymotic fluids.
Methylotrophic bacillus (Bacillus methylotrophicus) WSWGH-10 is preserved in China Microbiological
Culture presevation administration committee common micro-organisms center, deposit number are CGMCC No.11541, and the strain is in Publication No.
Disclosed in CN105670958A, the first patent application No. is 201510762018.X.
By Methylotrophic bacillus methylotrophicusWSWGH-10 strain culturings to logarithmic phase
Process culture medium and condition of culture known in the art can be used to carry out.
The present invention composite biological agent in, a concentration of 5~30U/mL of protease, cellulase a concentration of 5~
20U/mL, a concentration of 10~25U/mL of oxidoreducing enzyme, a concentration of 5~20U/mL of pectase.When complex enzyme content herein
In the range of when, complex enzyme can be with Methylotrophic bacillus methylotrophicusWSWGH-10 zymotic fluids
Coordinated, efficiently treated sewage under cryogenic.Preferably, protease is subtilopeptidase A；Cellulase comes
Derived from aspergillus niger, carrier is starch；Oxidoreducing enzyme is catalase, from cattle liver；Pectase is pectin lyase.
The enzyme activity of protease is defined as：1mL crude enzyme liquids, under the conditions of 40 DEG C, pH3.0, caseinhydrolysate generates 1 μ within 1 minute
The enzyme amount of g tyrosine is 1 enzyme activity unit, is represented with U/mL.
The enzyme activity of cellulase is defined as：1mL crude enzyme liquids, under the conditions of 40 DEG C, pH value are 4.5, hydrolysis filter paper production in 1 minute
The enzyme amount of raw 1 μ g glucose is 1 enzyme activity unit, is represented with U/mL.
The enzyme activity of oxidoreducing enzyme is defined as：1mL crude enzyme liquids, under the conditions of 25 DEG C, pH value are 8.0,1 minute 1 μ of reduction
mol K3Fe(CN)6Enzyme amount for 1 enzyme activity unit, represented with U/mL.
The enzyme activity of pectase is defined as：1mL crude enzyme liquids under the conditions of 50 DEG C, pH value are 3.5, decompose pectin in 1 minute and generate
The enzyme amount of 1 μ g galacturonic acids is an enzyme-activity unit, is represented with U/mL.
Another aspect of the present invention provide the above-mentioned composite biological agent for low-temperature wastewater treatment preparation method, including with
Lower step：
1) the Methylotrophic bacillus of activation (Bacillus methylotrophicus) WSWGH-10 bacterial strains are trained
Logarithmic phase is supported, the strain cultivated to logarithmic phase is then inoculated into LB liquid by 0.5%~1.5% inoculum concentration of culture volume
In body culture medium, at 0~30 DEG C, shaking table shakes 120~180rpm, cultivates 24~48 hours, and Methylotrophic gemma bar is made
Bacterium (Bacillus methylotrophicus) WSWGH-10 zymotic fluids；
2) protease, cellulase, oxidoreducing enzyme and pectase are added in zymotic fluid made from step 1), so as to get
Composite biological agent in, a concentration of 5~30U/mL of protease, a concentration of 5~20U/mL of cellulase, oxidoreducing enzyme
A concentration of 10~25U/mL, a concentration of 5~20U/mL of pectase.
Above-mentioned cryogenic temperature is 0~20 DEG C；Further, composite biological agent of the invention still has at 0~10 DEG C
There is the efficiency of higher treated sewage.
Advantageous effect：The present invention is for the first time by low temperature biocontrol bacterial strain WSWGH-10 for sewage treatment field, by by low temperature
The zymotic fluid of biocontrol bacterial strain WSWGH-10 and a variety of complex enzymes are compound, make the zymotic fluid of low temperature biocontrol bacterial strain WSWGH-10 with it is a variety of
Complex enzyme acts synergistically, and solve thing problems, such as current microorganism, treated sewage effect is relatively low under cryogenic, Neng Gou
Organic matter under cryogenic conditions in efficient degradation sewage, treated sewage.
Specific embodiment
Reagent source：
Subtilopeptidase A：Shanghai Industry Co., Ltd in future；
Cellulase (being starch from aspergillus niger carrier)：Aladdin reagent (Shanghai) Co., Ltd.；
Catalase (derives from cattle liver)：Aladdin reagent (Shanghai) Co., Ltd.；
Pectin lyase：Shanghai is through chemistry Science and Technology Ltd. of section.
Methylotrophic bacillus (Bacillus methylotrophicus) WSWGH-10 is preserved in China Microbiological
Culture presevation administration committee common micro-organisms center, deposit number are CGMCC No.11541, the strain and its screening and identification
Method is disclosed in Publication No. CN105670958A, the first patent application No. is 201510762018.X.
Embodiment 1
Preparation method step for the composite biological agent of low-temperature wastewater treatment is as follows：
1) Methylotrophic bacillus (Bacillus methylotrophicus) WSWGH-10 is taken, LB is inoculated in and consolidates
In body culture medium, the Methylotrophic bacillus (Bacillus activated is made in 10 DEG C of activation culture 48h
methylotrophicus)WSWGH-10；Then by the Methylotrophic bacillus (Bacillus of activation
Methylotrophicus) WSWGH-10 is inoculated in LB fluid nutrient mediums, and culture is to logarithmic phase at 10 DEG C, then by volume point
Number 1% is inoculated into another LB fluid nutrient mediums, and at 15 DEG C, the concussion of 120rpm shaking tables cultivates 36 hours, Methylotrophic is made
Bacillus (Bacillus methylotrophicus) WSWGH-10 zymotic fluids；
2) subtilopeptidase A is added in zymotic fluid made from step 1), cellulase (derives from aspergillus niger carrier
For starch), catalase (derive from cattle liver) and pectin lyase, be made for the compound bio system of low-temperature wastewater treatment
Agent.In obtained composite biological agent, a concentration of 20U/mL of subtilopeptidase A, a concentration of 10U/mL of cellulase,
A concentration of 15U/mL of catalase, a concentration of 15U/mL of pectin lyase.
Embodiment 2
Preparation method step for the composite biological agent of low-temperature wastewater treatment is as follows：
1) Methylotrophic bacillus (Bacillus methylotrophicus) WSWGH-10 is taken, LB is inoculated in and consolidates
In body culture medium, the Methylotrophic bacillus (Bacillus activated is made in 15 DEG C of activation culture 48h
methylotrophicus)WSWGH-10；Then by the Methylotrophic bacillus (Bacillus of activation
Methylotrophicus) WSWGH-10 is inoculated in LB fluid nutrient mediums, and culture is to logarithmic phase at 20 DEG C, then by volume point
Number 0.5% is inoculated into another LB fluid nutrient mediums, and at 20 DEG C, the concussion of 150rpm shaking tables cultivates 48 hours, methylotrophy is made
Type bacillus (Bacillus methylotrophicus) WSWGH-10 zymotic fluids；
2) subtilopeptidase A is added in zymotic fluid made from step 1), cellulase (derives from aspergillus niger carrier
For starch), catalase (derive from cattle liver) and pectin lyase, be made for the compound bio system of low-temperature wastewater treatment
Agent.In obtained composite biological agent, a concentration of 5U/mL of subtilopeptidase A, a concentration of 20U/mL of cellulase, mistake
A concentration of 25U/mL of hydrogen oxide enzyme, a concentration of 5U/mL of pectin lyase.
Embodiment 3
Preparation method step for the composite biological agent of low-temperature wastewater treatment is as follows：
1) Methylotrophic bacillus (Bacillus methylotrophicus) WSWGH-10 is taken, LB is inoculated in and consolidates
In body culture medium, the Methylotrophic bacillus (Bacillus activated is made in 5 DEG C of activation culture 48h
methylotrophicus)WSWGH-10；Then by the Methylotrophic bacillus (Bacillus of activation
Methylotrophicus) WSWGH-10 is inoculated in LB fluid nutrient mediums, and culture is to logarithmic phase at 0 DEG C, then by volume point
Number 1.5% is inoculated into another LB fluid nutrient mediums, and at 0 DEG C, the concussion of 180rpm shaking tables cultivates 24 hours, methylotrophy is made
Type bacillus (Bacillus methylotrophicus) WSWGH-10 zymotic fluids；
2) subtilopeptidase A is added in zymotic fluid made from step 1), cellulase (derives from aspergillus niger carrier
For starch), catalase (derive from cattle liver) and pectin lyase, be made for the compound bio system of low-temperature wastewater treatment
Agent.In obtained composite biological agent, a concentration of 30U/mL of subtilopeptidase A, a concentration of 5U/mL of cellulase, mistake
A concentration of 10U/mL of hydrogen oxide enzyme, a concentration of 20U/mL of pectin lyase.
Comparative example 1
The preparation method of Methylotrophic bacillus (Bacillus methylotrophicus) WSWGH-10 zymotic fluids
It is as follows：
Methylotrophic bacillus (Bacillus methylotrophicus) WSWGH-10 is taken, is inoculated in LB solids
In culture medium, the Methylotrophic bacillus (Bacillus activated is made in 10 DEG C of activation culture 48h
methylotrophicus)WSWGH-10；Then by the Methylotrophic bacillus (Bacillus of activation
Methylotrophicus) WSWGH-10 is inoculated in LB fluid nutrient mediums, and culture is to logarithmic phase at 10 DEG C, then by volume point
Number 1% is inoculated into another LB fluid nutrient mediums, and at 15 DEG C, the concussion of 120rpm shaking tables cultivates 36 hours, Methylotrophic is made
Bacillus (Bacillus methylotrophicus) WSWGH-10 zymotic fluids.
Comparative example 2
Preparation method step for the composite biological agent of low-temperature wastewater treatment is as follows：
1) Methylotrophic bacillus (Bacillus methylotrophicus) WSWGH-10 is taken, LB is inoculated in and consolidates
In body culture medium, the Methylotrophic bacillus (Bacillus activated is made in 10 DEG C of activation culture 48h
methylotrophicus)WSWGH-10；Then by the Methylotrophic bacillus (Bacillus of activation
Methylotrophicus) WSWGH-10 is inoculated in LB fluid nutrient mediums, and culture is to logarithmic phase at 10 DEG C, then by volume point
Number 1% is inoculated into another LB fluid nutrient mediums, and at 15 DEG C, the concussion of 120rpm shaking tables cultivates 36 hours, Methylotrophic is made
Bacillus (Bacillus methylotrophicus) WSWGH-10 zymotic fluids；
2) subtilopeptidase A is added in zymotic fluid made from step 1), is made for the compound of low-temperature wastewater treatment
Biological agent.In obtained composite biological agent, a concentration of 20U/mL of subtilopeptidase A.
Comparative example 3
Preparation method step for the composite biological agent of low-temperature wastewater treatment is as follows：
1) Methylotrophic bacillus (Bacillus methylotrophicus) WSWGH-10 is taken, LB is inoculated in and consolidates
In body culture medium, the Methylotrophic bacillus (Bacillus activated is made in 10 DEG C of activation culture 48h
methylotrophicus)WSWGH-10；Then by the Methylotrophic bacillus (Bacillus of activation
Methylotrophicus) WSWGH-10 is inoculated in LB fluid nutrient mediums, and culture is to logarithmic phase at 10 DEG C, then by volume point
Number 1% is inoculated into another LB fluid nutrient mediums, and at 15 DEG C, the concussion of 120rpm shaking tables cultivates 36 hours, Methylotrophic is made
Bacillus (Bacillus methylotrophicus) WSWGH-10 zymotic fluids；
2) cellulase (being starch from aspergillus niger carrier), hydrogen peroxide are added in zymotic fluid made from step 1)
Enzyme (deriving from cattle liver) and pectin lyase, are made the composite biological agent for low-temperature wastewater treatment.Obtained compound life
In object preparation, a concentration of 10U/mL of cellulase, a concentration of 15U/mL of catalase, pectin lyase it is a concentration of
15U/mL。
Embodiment 4
Methylotrophic bud made from composite biological agent made from Example 1~3, comparative example 2,3 and comparative example 1
Spore bacillus (Bacillus methylotrophicus) WSWGH-10 zymotic fluids, test its influence to water body indices.
The water sampling out of certain river carries out water quality improvement experiment using river model at different temperatures.It is untreated
In water sample, CODcr contents are 152mg/L, BOD5Content is 106mg/L, ammonia-nitrogen content 9.23mg/L.Composite biological agent with
The mass ratio of water is 1: 50000, in adding in Examples 1 to 3, composite biological agent made from comparative example 2,3 or right into water sample
After (Bacillus methylotrophicus) the WSWGH-10 zymotic fluids of Methylotrophic bacillus made from ratio 1 120h
Measure CODcr, BOD in water sample5And ammonia-nitrogen content.Test result is as shown in table 1.
As shown in Table 1, in composite biological agent of the invention, the zymotic fluid of low temperature biocontrol bacterial strain WSWGH-10 with it is a variety of multiple
The compound synergistic effect of synthase can effectively reduce CODcr, BOD in sewage under cryogenic5And ammonia-nitrogen content.
Table 1
Claims (7)
1. a kind of composite biological agent for low-temperature wastewater treatment, which is characterized in that the composite biological agent contains：Methyl is sought
Support type bacillus WSWGH-10 zymotic fluids and complex enzyme；The complex enzyme include protease, cellulase, oxidoreducing enzyme and
Pectase；The cryogenic temperature is 0~20 DEG C.
2. the composite biological agent according to claim 1 for low-temperature wastewater treatment, which is characterized in that the methyl battalion
Type bacillus WSWGH-10 zymotic fluids are supported to prepare as follows：Methylotrophic bacillus WSWGH-10 is cultivated
It is inoculated into LB fluid nutrient mediums by volume fraction 0.5%~1.5% after to logarithmic phase, at 0~30 DEG C, shaking table concussion 120~
180rpm cultivates 24~48 hours to get the Methylotrophic bacillus WSWGH-10 zymotic fluids.
3. the composite biological agent according to claim 2 for low-temperature wastewater treatment, which is characterized in that protease it is dense
It spends for 5~30U/mL, a concentration of 5~20U/mL of cellulase, a concentration of 10~25U/mL of oxidoreducing enzyme, pectase
A concentration of 5~20U/mL.
4. the composite biological agent according to claim 1 for low-temperature wastewater treatment, which is characterized in that the protease
For subtilopeptidase A；The cellulase derives from aspergillus niger, and carrier is starch；The oxidoreducing enzyme is hydrogen peroxide
Enzyme, from cattle liver；The pectase is pectin lyase.
5. the preparation method of the composite biological agent for low-temperature wastewater treatment in Claims 1 to 4 described in any one,
It is characterized in that, which includes the following steps：
1) the Methylotrophic bacillus WSWGH-10 of activation is cultivated to logarithmic phase, then by culture volume 0.5%~
The strain cultivated to logarithmic phase is inoculated into LB fluid nutrient mediums by 1.5% inoculum concentration, at 0~30 DEG C, shaking table concussion 120~
180rpm is cultivated 24~48 hours, and Methylotrophic bacillus WSWGH-10 zymotic fluids are made；
2) protease, cellulase, oxidoreducing enzyme and pectase are added in zymotic fluid described made from step 1), so as to get
Composite biological agent in, a concentration of 5~30U/mL of protease, a concentration of 5~20U/mL of cellulase, oxidoreducing enzyme
A concentration of 10~25U/mL, a concentration of 5~20U/mL of pectase.
6. application of the composite biological agent described in claim 1 in low-temperature wastewater treatment, the cryogenic temperature is 0~20
℃。
7. application of the composite biological agent according to claim 6 in low-temperature wastewater treatment, the cryogenic temperature for 0~
10℃。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112340856A (en) * | 2020-11-03 | 2021-02-09 | 苏州汉风科技发展有限公司 | Biological environment-friendly wastewater treatment method |
| CN113142214A (en) * | 2021-05-24 | 2021-07-23 | 黑龙江省科学院微生物研究所 | Application of antibacterial protein of bacillus methylotrophicus wswGH-10 and separation and purification method |
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