CN106635871B - Complex microbial inoculant and application thereof - Google Patents

Complex microbial inoculant and application thereof Download PDF

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CN106635871B
CN106635871B CN201610878211.4A CN201610878211A CN106635871B CN 106635871 B CN106635871 B CN 106635871B CN 201610878211 A CN201610878211 A CN 201610878211A CN 106635871 B CN106635871 B CN 106635871B
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wsh
microbial inoculum
streptococcus
pseudomonas aeruginosa
candida
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CN106635871A (en
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崔明虎
徐彬
郑展望
陈坚
周景文
曹晓梅
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Zhe Jiang Shuangliang Sunda Environment Protection Co ltd
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Zhejiang Shuangliang Shangda Environmental Protection Co Ltd
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    • 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
    • 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
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor

Abstract

The invention discloses a complex microbial inoculant and application thereof, and belongs to the technical field of industrial microorganisms. The composite microbial inoculum provided by the invention comprises pseudomonas aeruginosa, streptococcus and candida, and the strains are respectively cultured in respective corresponding culture media to OD600Mixing the bacterial liquid according to the volume ratio of 5:3:2, wherein the volume ratio is 1. After the microbial inoculum is inoculated into grease sewage for reaction for 48 hours in an inoculation amount of 5-10%, the grease degradation rate reaches 90.2%, the microbial inoculum can be industrially applied in a large scale, and the microbial inoculum has the advantages of small investment, high efficiency, no secondary pollution and the like.

Description

Complex microbial inoculant and application thereof
Technical Field
The invention relates to a complex microbial inoculum and application thereof, belonging to the technical field of industrial microorganisms.
Background
In China, water pollution is still a serious problem. The discharge amount of industrial wastewater and urban domestic wastewater is very large. Groundwater nationwide is polluted in a dotted or planar manner. The grease wastewater is mainly discharged from industrial, agricultural, transportation and domestic wastewater. The grease covers the surface of the water body and blocks oxygen in the air from diffusing to the water body, so that the self-cleaning capacity of the water body is reduced, and the ecological balance in the water is damaged. Currently, the current practice is. The method for treating the grease wastewater mainly comprises a physical method, a chemical method and a biological method. The physical method mainly comprises the steps of dispersing the water and the oil by using gravity and centrifugal force according to the incompatibility and different densities of the water and the oil, such as a gravity separation method, a coarse granulation method, a filtration adsorption method, a membrane separation method and the like. The chemical method mainly utilizes chemical substances to carry out chemical decomposition on organic matters in the wastewater, so that loss of useful organic substances is caused, and secondary pollution is easily caused. The common methods include chemical demulsification, oil removal, chemical oxidation and the like. The biological method for treating the grease wastewater mainly comprises the steps of hydrolyzing grease into glycerol and fatty acid by using microorganisms, and finally decomposing and oxidizing the glycerol and fatty acid into products such as water, carbon dioxide and the like. The biological method for treating the grease in the wastewater has the advantages of low investment, high efficiency, no secondary pollution and the like.
Disclosure of Invention
The first purpose of the invention is to provide a composite microbial inoculum, which comprises pseudomonas aeruginosa, streptococcus and candida and is cultured to OD respectively in a volume ratio of 5:3:2600Mixing the bacterial solutions 1-1.2.
In one embodiment of the invention, the composite microbial inoculum consists of pseudomonas aeruginosa BBE, streptococcus WSH-24 and candida WSH-Z06.
In one embodiment of the invention, the Pseudomonas aeruginosa BBE, the Streptococcus WSH-24 and the Candida WSH-Z06 bacteria are domesticated; the domestication is carried out in a domestication culture medium; the domestication culture medium comprises the following components: (NH)4)2SO40.5g/L，NaCl 0.5g/L，K2HPO40.3g/L，MgSO4·7H20.2g/L of O, 1.0g/L of peptone and 1-4 g/L of soybean oil.
The second purpose of the invention is to provide the preparation method of the microbial inoculum, which is to inoculate pseudomonas aeruginosa BBE, streptococcus WSH-24 and candida WSH-Z06 to a culture medium respectively, and culture the pseudomonas aeruginosa BBE, the streptococcus WSH-24 and the candida WSH-Z06 at the temperature of 30 ℃ until the concentration of the bacteria is OD6000.1; then, the cell sap of pseudomonas aeruginosa BBE, streptococcus WSH-24 and candida WSH-Z06 are mixed according to the proportion of 5:3: 2.
In one embodiment of the invention, the pseudomonas aeruginosa BBE is inoculated into LB medium for culture.
In one embodiment of the invention, the streptococcus WSH-24 is inoculated into potato sucrose medium for culture.
In one embodiment of the invention, the Candida WSH-Z06 is inoculated into a culture medium containing 5g/L yeast powder, 10g/L peptone and 20g/L anhydrous glucose for culture.
The third purpose of the invention is to provide a method for degrading grease in grease wastewater, which is to add the composite microbial inoculum into the grease wastewater and react for 10-60 h at 30 ℃.
In one embodiment of the invention, the addition amount of the complex microbial inoculum is 5-10% by volume ratio.
The invention also provides application of the microbial inoculum in sewage treatment.
The invention has the beneficial effects that: the invention provides a composite microbial inoculum which can effectively degrade grease in grease wastewater. After the microbial inoculum is inoculated into grease wastewater for reaction for 48 hours, the degradation rate reaches 90.2 percent.
Detailed Description
And (3) measuring the degradation rate of the grease:
and (3) measuring the content of the oil in the reaction liquid by using a UV-2450 type ultraviolet-visible spectrophotometer. Taking 10mL of reaction solution, adding 1.5mL of sulfuric acid for acidification, transferring the reaction solution into a separating funnel, taking 5mL of toluene, adding the toluene into the separating funnel, shaking, standing, layering and extracting. And (4) returning the extracted water sample to the original separating funnel, and adding 5mL of toluene to repeatedly extract once. The extract was diluted with toluene and the absorbance of the extract was measured at 324 nm. And calculating the concentration of the grease according to the standard curve of the grease and the light absorption value.
Culture medium:
acclimatization medium (g/L): (NH)4)2SO40.5，NaCl 0.5，K2HPO40.3，MgSO4·7H2O0.2, peptone 1.0, soybean oil concentration increased from 1g/L to 4g/L in sequence.
Assay medium (g/L): (NH)4)2SO45.0，Na2HPO42.0，K2HPO42.0，NaCl 2.0g，MgSO40.5, and 20mL of soybean oil.
LB medium (g/L): 5 parts of yeast powder, 10 parts of peptone and 5 parts of sodium chloride, and is used for culturing pseudomonas aeruginosa.
Potato sucrose medium (g/L): potato 200, sucrose 20, for the cultivation of streptococcus WSH-24.
Candida WSH-Z06 basal medium (g/L): yeast powder 5, peptone 10 and anhydrous glucose 20.
Example 1 preparation of Complex microbial inoculum
1. Domestication of the strain: pseudomonas aeruginosa BBE (preservation number CCTCC NO: M2011185), streptococcus WSH-24 (purchased from ATCC, the number is ATCC 35246) and Candida WSH-Z06 (the preservation number is CCTCC NO: M20714) are respectively inoculated into an acclimation culture medium without soybean oil, the soybean oil concentration in the acclimation culture medium is sequentially increased to 1g/L, 2g/L, 3g/L and 4g/L for acclimation by taking 5d as a culture period, bacterial liquids before and after acclimation are respectively reacted in a detection culture medium, and the oil degradation effect is measured, and the results are shown in Table 1. Through domestication, the grease degradation rate of candida WSH-Z06 is improved to 78.2%, and the grease degradation rates of pseudomonas aeruginosa BBE and streptococcus WSH-24 are respectively improved to 50.1% and 43.9%.
2. Respectively inoculating the domesticated pseudomonas aeruginosa BBE, streptococcus WSH-24 and candida WSH-Z06 bacteria to corresponding basal culture media for culture to ensure that the bacteria OD600＝1～1.2。
3. The culture solution of pseudomonas aeruginosa BBE, streptococcus WSH-24 and candida WSH-Z06 bacteria is mixed in a ratio of 5:3:2, and preparing the complex microbial inoculum.
TABLE 1 oil and fat degradation ratio (%)
Example 2 composite bacterial agent degradation of grease wastewater
A complex microbial inoculum was prepared by the procedure of example 1, except that the strain was not acclimatized. Inoculating the microbial inoculum into the grease wastewater by 5 percent of addition amount, reacting for 48h at 30 ℃, and calculating the degradation rate. The result shows that the degradation rate of the microbial inoculum prepared without acclimatization on the grease reaches 63.5%.
Example 3 composite bacterial agent degradation of grease wastewater
The microbial inoculum prepared in example 1 was inoculated into grease wastewater at different addition levels, reacted at 30 ℃ for 48 hours, and the degradation rate was calculated, with the results shown in table 2. The microbial inoculum is inoculated according to the volume ratio of 5-10%, the oil degradation rate reaches more than 80%, and the microbial inoculum has a remarkable effect.
TABLE 2 degradation of oils under different conditions
Figure BDA0001126005890000032
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (3)

1. The composite microbial inoculum for degrading grease in grease wastewater is characterized by comprising pseudomonas aeruginosa, streptococcus and candida, wherein the microbial inoculum is prepared by respectively culturing domesticated pseudomonas aeruginosa, streptococcus and candida to OD600= 1-1.2, and mixing the bacterial liquid according to the volume ratio of 5:3: 2; the pseudomonas aeruginosa is pseudomonas aeruginosaPseudomonas aeruginosa) BBE, the preservation number of which is CCTCC NO: m2011185; the streptococcus is streptococcus (Streptococcus equi subsp. zooepidimicus) WSH-24, accession number ATCC 35246; the Candida is Candida (C)Yarrowia lipolytica) The preservation number of the WSH-Z06 is CCTCC NO: m20714; specifically, the complex microbial inoculum is prepared by the following method:
domesticating the pseudomonas aeruginosa BBE, the streptococcus WSH-24 and the candida WSH-Z06 bacteria; the domestication is carried out in a domestication culture medium; the domestication culture medium comprises the following components: (NH)4)2SO40.5 g/L，NaCl 0.5 g/L，K2HPO40.3 g/L，MgSO4·7H2O0.2 g/L, peptone 1.0g/LTaking 5d as a culture period, sequentially increasing the concentration of soybean oil in the domestication culture medium to 1g/L, 2g/L, 3g/L and 4g/L for domestication, respectively inoculating the domesticated pseudomonas aeruginosa BBE, streptococcus WSH-24 and candida WSH-Z06 bacteria to corresponding basal culture media for culture, and enabling OD to be OD6001 to 1.2; and then, culturing the domesticated culture solution of pseudomonas aeruginosa BBE, streptococcus WSH-24 and candida WSH-Z06 bacteria in a ratio of 5:3:2, and preparing the complex microbial inoculum.
2. A method for degrading grease in grease wastewater is characterized in that the composite microbial inoculum according to claim 1 is added into the grease wastewater and reacts for 10-60 hours at 30 ℃, and the addition amount of the composite microbial inoculum is 5% by volume.
3. The use of the microbial inoculum according to claim 1 in the treatment of grease waste water.
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CN108359617B (en) * 2017-12-29 2021-03-02 浙江双良商达环保有限公司 Acinetobacter CL05 and application thereof in village and town sewage dephosphorization treatment
CN108823141A (en) * 2018-06-11 2018-11-16 佛山皖阳生物科技有限公司 A kind of preparation method of oil smoke rubbish microbiological garbage treatment agent
CN109464375A (en) * 2018-12-11 2019-03-15 深圳合民生物科技有限公司 A kind of pet deodorant and its preparation method and application
CN109666602B (en) * 2018-12-19 2021-07-16 中国石油天然气集团有限公司 Composite bacteria for degrading oily sewage and preparation method thereof

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Address after: 9 floor, block A, Lianhua business center, Lianhua street, Lianhua, Hangzhou,, Xihu District, Zhejiang

Patentee after: Zhe JIANG Shuangliang Sunda Environment Protection Co.,Ltd.

Address before: 9 floor, block A, Lianhua business center, Lianhua street, Lianhua, Hangzhou,, Xihu District, Zhejiang

Patentee before: ZHEJIANG SHUANGLIANG SHANGDA ENVIRONMENTAL PROTECTION Co.,Ltd.