CN107164231A - The screening technique of high-efficiency grease degradation bacteria - Google Patents
The screening technique of high-efficiency grease degradation bacteria Download PDFInfo
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- CN107164231A CN107164231A CN201710566203.0A CN201710566203A CN107164231A CN 107164231 A CN107164231 A CN 107164231A CN 201710566203 A CN201710566203 A CN 201710566203A CN 107164231 A CN107164231 A CN 107164231A
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Abstract
The present invention relates to a kind of screening technique of grease degrading strain, it comprises the following steps：(1) microbe inoculation：Bacterium sludge water sample will be contained to be inoculated in enriched medium；(2) microorganism is cultivated：By every 3~7 days transfer cultures of microorganism after step (1) middle inoculation to new enriched medium, continue enrichment culture 20~30 days, it is ensured that the abundant growth of required microorganism fungus kind；(3) acclimated microorganism：By the bacterium solution after step (2) middle enrichment culture under conditions of 25~30 DEG C, 150~180rpm shaking tables, it is seeded to by progress gradient domestication in the incremental enriched medium containing oil wastewater of gradient；(4) microorganism is screened：It is grease degrading strain to choose bacterial strain of transparent loop diameter (D)/colony diameter (d) ratio more than 2, compared with prior art, the invention has the advantages that：Lubricant component olive oil is added in the enriched medium of the present invention, and control pH is conducive to the growth and enrichment of grease degrading strain between 7.2 7.4, is that follow-up bacteria selection separation provides the foundation.
Description
Technical field
The invention belongs to biodegradable processing technology field, and in particular to a kind of screening technique of grease degrading strain and should
With.
Background technology
As China Living consumption is improved and regional tourist industry is developed rapidly, in sanitary sewage and catering industry
The discharge capacity of oil wastewater also gradually increases, and the problem of environmental pollution caused by China's oily wastewater in catering industry is increasingly serious.And
The oily substance for entering sewage treatment plant is wrapped in filler outer layer, makes the conduction of oxygen be obstructed, and causes to support microbial metabolism disorderly
Disorderly, the treatment effect of final influence sewage.And the oily substance for entering water body can then swim in water surface, answering for water body is hindered
Oxygen and self purification process, destroy the ecological balance of water body.
At present, the processing method of oil wastewater mainly has Physical, chemical method and bioanalysis.Physical and chemical method due to
Investment is big, it is complicated to take up an area wide, flow, and easily produces secondary pollution, and practical application is less.And Biochemical method oil wastewater is then
Grease is decomposed in the effect of the degradation enzyme systems such as the lipase produced in the main microorganism growth process using in oil wastewater to be turned
Change.Wherein, bioremediation is great development prospect come oil wastewater of degrading, using the vital movement process of microorganism to oil
Fat waste water is shifted and converted, and using the carbon source and the energy needed for grease as its growth, and is hydrolyzed into the presence of enzyme sweet
Oil and long chain fatty acids, are finally degraded to H2O and CO2Deng metabolite, and, non-secondary pollution advantage low with cost, and extremely
Favor.And China's oil-polluted water processing biological method is mainly using some conventional sanitary sewage disposal works such as activated sludge processes
Skill, but due to lacking efficient degradation microorganism in waste water, effect is not satisfactory, it is impossible to meet the processing requirement of oil wastewater, because
This, how screening high-efficiency grease degrading microorganism and being introduced into Fat wastewater treatment is the key for solving this problem.
The content of the invention
A technical problem to be solved by this invention is that the present situation for being directed to prior art provides a kind of breakneck acceleration soon
The screening technique of high-efficiency grease degradation bacteria.
Another technical problem to be solved by this invention is that the present situation offer one kind for being directed to prior art utilizes above-mentioned oil
Application of the microorganism that the screening technique of fat degradation bacteria is filtered out in degraded oil wastewater.
The present invention solve the technical scheme that is used of above-mentioned technical problem for：The screening technique of the high-efficiency grease degradation bacteria,
It is characterized in that：Comprise the following steps：
(1) microbe inoculation：Bacterium sludge water sample will be contained to be inoculated in enriched medium, be placed in 25~30 DEG C, 150~
Cultivated in 180rpm shaking table, the preparation composition and weight of the enriched medium are as follows：5.0~8.0g of NaCl, peptone
10.0~15.0g, 5.0~6.0g of beef extract, 6~8mL of olive oil, distilled water 1L；
(2) microorganism is cultivated：By every 3~7 days transfer cultures of microorganism after step (1) middle inoculation to new enrichment culture
Base, continues enrichment culture 20~30 days, it is ensured that the abundant growth of required microorganism fungus kind；
(3) acclimated microorganism：By the bacterium solution after step (2) middle enrichment culture in 25~30 DEG C, the bar of 150~180rpm shaking tables
Under part, it is seeded to by gradient domestication is carried out in the incremental enriched medium containing oil wastewater of gradient, is used as one within every 3~5 days
Individual gradient acclimation period, domestication obtains the microorganism through acclimation after 10~20 days；
(4) microorganism is screened：Microbial inoculum after step (3) middle domestication is diluted to various concentrations by gradient dilution method
After suspension bacteria liquid, it is spread evenly across in isolation medium, shape is obtained after cultivating 24~48h in 25~30 DEG C of constant incubators
Different single bacterium colonies, chooses transparent loop diameter (D)/colony diameter (d) ratio and is more than 2 bacterial strain for grease degrading strain, described point
Preparation composition and weight from culture medium is as follows：6~8mL of grease, 20~40mL of Tween-80, inorganic salts nutrient solution 1000mL,
18~25g of agar.
Further, (4) middle inorganic salts nutrient solution composition is the step：KH2PO40.3~0.5g, MgSO4·7H2O
0.12~0.15g, K2HPO41.5~1.8g, (NH4)2SO45.0~10.0g of 1.0~1.5g, NaCl, distilled water 1L.
Further, the step (1) middle enriched medium pH be 7.2~7.4.
The microorganism that the screening technique that the present invention also provides a kind of above-mentioned grease degrading strain of utilization is filtered out is in degraded grease
Application in waste water.
Compared with prior art, the invention has the advantages that：Lubricant component is added in the enriched medium of the present invention
Olive oil, and control pH is conducive to the growth and enrichment of grease degrading strain between 7.2-7.4, is follow-up bacteria selection point
From providing the foundation, this characteristic of lipase can be produced using the experiment of transparent circle method, production fat furthermore with grease degrading strain
Separation screening high-efficiency grease degradation bacteria is tested in enzyme aptitude tests, is not only operated simple and feasible, is conducive to quickly filtering out again
High-efficiency grease degradation bacteria, excludes the influence of other miscellaneous bacterias, meanwhile, the present invention is in order to which obtain can fast degradation grease and useless to grease
Water change of water quality strong adaptability high-efficiency grease degradation bacteria, the bacterium solution after enrichment is added in actual oil wastewater and tamed,
Efficiently solve and high-efficiency grease degrading microorganism is incorporated into Fat wastewater treatment environment this key issue, be at engineering practice
Manage oil wastewater and provide technical support.
Embodiment
The invention will be further described by the following examples.
Embodiment 1
The screening technique of this high-efficiency grease degradation bacteria comprises the following steps：
(1) microbe inoculation：Bacterium sludge water sample will be contained to be inoculated in enriched medium, 25 DEG C are placed in, in 150rpm shaking table
Culture, the preparation composition and weight of enriched medium is as follows：NaCl 5.0g, peptone 10.0g, beef extract 5.0g, olive oil
6mL, distilled water 1L；
(2) microorganism is cultivated：By the every 3 days transfer cultures of microorganism after step (1) middle inoculation to new enriched medium,
Continue enrichment culture 20 days, it is ensured that the abundant growth of required microorganism fungus kind；
(3) acclimated microorganism：By the bacterium solution after step (2) middle enrichment culture under conditions of 25 DEG C, 150rpm shaking tables, inoculation
Gradient domestication is carried out in the incremental enriched medium containing oil wastewater of gradient to pressing, week is tamed as a gradient within every 3 days
Phase, domestication obtains the microorganism through acclimation after 10 days；
(4) microorganism is screened：The microbial inoculum after step (3) middle domestication is made into concentration by 10 times of gradient dilution methods is
10-5, 10-6, 10-7Suspension bacteria liquid after, be spread evenly across in isolation medium, in 25 DEG C of constant incubators cultivate 24h after obtain
Variform single bacterium colony is obtained, transparent loop diameter (D)/colony diameter (d) ratio is chosen and is more than 2 bacterial strain for grease degrading strain,
Wherein, the preparation composition and weight of isolation medium are as follows：Grease 6mL, Tween-80 20mL, inorganic salts nutrient solution 1000mL,
Agar 18g, and the following KH of preparation composition and weight of inorganic salts nutrient solution2PO40.3g, MgSO4·7H2O 0.12g, K2HPO4
1.5g, (NH4)2SO41.0g, NaCl 5.0g, distilled water 1L, and with oese by colony inoculation in flat board to containing being separately cultured
In the triangular flask of base, 32 DEG C are placed in, fat is produced in 180rpm Shaking cultures, test strain nutrient solution lipase active after 48h, screening
The stronger bacterial strain of fat enzyme ability.
Embodiment 2
The screening technique of this high-efficiency grease degradation bacteria comprises the following steps：
(1) microbe inoculation：Bacterium sludge water sample will be contained to be inoculated in enriched medium, 30 DEG C are placed in, in 180rpm shaking table
Culture, the preparation composition and weight of enriched medium is as follows：NaCl 8.0g, peptone 15.0g, beef extract 6.0g, olive oil
8mL, distilled water 1L；
(2) microorganism is cultivated：By the every 7 days transfer cultures of microorganism after step (1) middle inoculation to new enriched medium,
Continue enrichment culture 30 days, it is ensured that the abundant growth of required microorganism fungus kind；
(3) acclimated microorganism：By the bacterium solution after step (2) middle enrichment culture under conditions of 30 DEG C, 180rpm shaking tables, inoculation
Gradient domestication is carried out in the incremental enriched medium containing oil wastewater of gradient to pressing, week is tamed as a gradient within every 5 days
Phase, domestication obtains the microorganism through acclimation after 20 days；
(4) microorganism is screened：The microbial inoculum after step (3) middle domestication is made into concentration by 10 times of gradient dilution methods is
10-5, 10-6, 10-7Suspension bacteria liquid after, be spread evenly across in isolation medium, in 30 DEG C of constant incubators cultivate 48h after obtain
Variform single bacterium colony is obtained, transparent loop diameter (D)/colony diameter (d) ratio is chosen and is more than 2 bacterial strain for grease degrading strain,
Wherein, the preparation composition and weight of isolation medium are as follows：Grease 8mL, Tween-80 40mL, inorganic salts nutrient solution 1000mL,
Agar 25g, and the following KH of preparation composition and weight of inorganic salts nutrient solution2PO40.5g, MgSO4·7H2O 0.15g, K2HPO4
1.8g, (NH4)2SO41.5g, NaCl 10.0g, distilled water 1L, and with oese by colony inoculation in flat board to containing being separately cultured
In the triangular flask of base, 32 DEG C are placed in, fat is produced in 180rpm Shaking cultures, test strain nutrient solution lipase active after 48h, screening
The stronger bacterial strain of fat enzyme ability.
Embodiment 3
The screening technique of this high-efficiency grease degradation bacteria comprises the following steps：
(1) microbe inoculation：Bacterium sludge water sample will be contained to be inoculated in enriched medium, 28 DEG C are placed in, in 170rpm shaking table
Culture, the preparation composition and weight of enriched medium is as follows：NaCl 7.0g, peptone 12.0g, beef extract 5.5.0g, olive oil
7mL, distilled water 1L；
(2) microorganism is cultivated：By the every 5 days transfer cultures of microorganism after step (1) middle inoculation to new enriched medium,
Continue enrichment culture 25 days, it is ensured that the abundant growth of required microorganism fungus kind；
(3) acclimated microorganism：By the bacterium solution after step (2) middle enrichment culture under conditions of 28 DEG C, 160rpm shaking tables, inoculation
Gradient domestication is carried out in the incremental enriched medium containing oil wastewater of gradient to pressing, week is tamed as a gradient within every 4 days
Phase, domestication obtains the microorganism through acclimation after 15 days；
(4) microorganism is screened：The microbial inoculum after step (3) middle domestication is made into concentration by 10 times of gradient dilution methods is
10-5, 10-6, 10-7Suspension bacteria liquid after, be spread evenly across in isolation medium, in 28 DEG C of constant incubators cultivate 30h after obtain
Variform single bacterium colony is obtained, transparent loop diameter (D)/colony diameter (d) ratio is chosen and is more than 2 bacterial strain for grease degrading strain,
Wherein, the preparation composition and weight of isolation medium are as follows：Grease 7mL, Tween-80 30mL, inorganic salts nutrient solution 1000mL,
Agar 20g, and the following KH of preparation composition and weight of inorganic salts nutrient solution2PO40.4g, MgSO4·7H2O 0.13g, K2HPO4
1.6g, (NH4)2SO41.2g, NaCl 8.0g, distilled water 1L, and with oese by colony inoculation in flat board to containing being separately cultured
In the triangular flask of base, 32 DEG C are placed in, fat is produced in 180rpm Shaking cultures, test strain nutrient solution lipase active after 48h, screening
The stronger bacterial strain of fat enzyme ability.
Embodiment 4
By without the microbe to screen mode of oil wastewater Domestication tests and the micro- life for carrying out oil wastewater Domestication tests
The contrast of thing screening mode is found：
36 kinds of microbial inoculums are finally filtered out without the infected water sample of oil wastewater Domestication tests, beginning CODcr is put into
For 1356.40mg/L, fat content is discovery in 353.65mg/L, pH 6.4 Fat wastewater treatment, and CODcr clearances are less than
30% microbial inoculum has 20 kinds, and the microbial inoculum obtained after oil wastewater Domestication tests is 20 kinds, is equally devoted reality
Found in the processing of oil wastewater, wherein the CODcr clearances for there are 12 kinds of microbial inoculums are higher than 50%, 12 kinds of microbial inoculums are respectively onion
Pseudomonad (Burkholderia cepacia), bacillus licheniformis (Bacillus licheniformis), sub- sieve of solution fat
Yeast (Yarrowia lipolytica), bacillus amyloliquefaciens (Bacillus amyloliquefaciens) expansion mould
(Penicillium expansum), Micrococcus (Micrococcus), bacillus subtilis (Bacillus subtilis),
Bite born of the same parents bacterium (Cytopaga), Bacillus alcaligenes (Alacligenes xylosoxidanssss), Yarrowia lipolytica (Yarrowia
Lipolytica), acinetobacter calcoaceticus (Acinetobacter) and Penicillium notatum (Penicillium)；Experiment can be proved, by reality
The microbial inoculum that border oil wastewater Domestication tests reach is preferable to the applicability of waste water, while in addition to cutting grease, to waste water COD
Degradation effect is also preferable, is more suitable for actual production with using.
Claims (3)
1. a kind of screening technique of high-efficiency grease degradation bacteria, it is characterised in that：Comprise the following steps：
(1) microbe inoculation：Bacterium sludge water sample will be contained be inoculated in enriched medium, and be placed in 25~30 DEG C, 150~180rpm
Cultivated in shaking table, the preparation composition and weight of the enriched medium are as follows：5.0~8.0g of NaCl, peptone 10.0~
15.0g, 5.0~6.0g of beef extract, 6~8mL of olive oil, distilled water 1L；
(2) microorganism is cultivated：By every 3~7 days transfer cultures of microorganism after step (1) middle inoculation to new enriched medium,
Continue enrichment culture 20~30 days, it is ensured that the abundant growth of required microorganism fungus kind；
(3) acclimated microorganism：By the bacterium solution after step (2) middle enrichment culture in 25~30 DEG C, the condition of 150~180rpm shaking tables
Under, it is seeded to by gradient domestication is carried out in the incremental enriched medium containing oil wastewater of gradient, is used as one within every 3~5 days
Gradient acclimation period, domestication obtains the microorganism through acclimation after 10~20 days；
(4) microorganism is screened：Microbial inoculum after step (3) middle domestication is diluted to the suspension of various concentrations by gradient dilution method
After bacterium solution, it is spread evenly across in isolation medium, is cultivated in 25~30 DEG C of constant incubators and shape difference is obtained after 24~48h
Single bacterium colony, it is grease degrading strain to choose transparent loop diameter (D)/colony diameter (d) ratio to be more than 2 bacterial strain, the separation training
The preparation composition and weight for supporting base are as follows：6~8mL of grease, 20~40mL of Tween-80, inorganic salts nutrient solution 1000mL, agar
18~25g.
2. the screening technique of high-efficiency grease degradation bacteria according to claim 1, it is characterised in that the step (1) middle enrichment
The pH of culture medium is 7.2~7.4.
3. the screening technique of high-efficiency grease degradation bacteria according to claim 1, it is characterised in that the step (4) in it is inorganic
Salt nutrient solution is constituted：KH2PO40.3~0.5g, MgSO4·7H2O 0.12~0.15g, K2HPO41.5~1.8g, (NH4)2SO45.0~10.0g of 1.0~1.5g, NaCl, distilled water 1L.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106854632A (en) * | 2016-12-28 | 2017-06-16 | 商丘师范学院 | A kind of mix bacterium agent for protein in sewage of degrading and fat and its preparation method and application |
| 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 |
| CN108911120A (en) * | 2018-07-18 | 2018-11-30 | 山西龙盘微生物科技有限公司 | A kind of acclimation method for daily use chemicals cleaning product activated sludge of degrading |
| CN111607552A (en) * | 2020-06-29 | 2020-09-01 | 重庆工商大学 | Domestication and breeding method of tetrahydrofuran efficient degradation strain |
| CN112044584A (en) * | 2020-08-27 | 2020-12-08 | 程亮 | Probiotics draws raw materials processing apparatus |
| CN112375706A (en) * | 2020-11-12 | 2021-02-19 | 扬州鑫威环保生物科技有限公司 | Culture method of microbial strain for degrading kitchen waste |
| CN113308404A (en) * | 2021-06-01 | 2021-08-27 | 中农新科(苏州)有机循环研究院有限公司 | Screening method of grease degrading strain and grease degrading product |
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| CN104862244A (en) * | 2015-03-26 | 2015-08-26 | 北京北华中清环境工程技术有限公司 | Efficient composite bacterial agent for removing mixed grease-containing wastewater COD, and applications thereof |
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Patent Citations (1)
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106854632A (en) * | 2016-12-28 | 2017-06-16 | 商丘师范学院 | A kind of mix bacterium agent for protein in sewage of degrading and fat and its preparation method and application |
| 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 |
| CN108911120A (en) * | 2018-07-18 | 2018-11-30 | 山西龙盘微生物科技有限公司 | A kind of acclimation method for daily use chemicals cleaning product activated sludge of degrading |
| CN111607552A (en) * | 2020-06-29 | 2020-09-01 | 重庆工商大学 | Domestication and breeding method of tetrahydrofuran efficient degradation strain |
| CN112044584A (en) * | 2020-08-27 | 2020-12-08 | 程亮 | Probiotics draws raw materials processing apparatus |
| CN112375706A (en) * | 2020-11-12 | 2021-02-19 | 扬州鑫威环保生物科技有限公司 | Culture method of microbial strain for degrading kitchen waste |
| CN113308404A (en) * | 2021-06-01 | 2021-08-27 | 中农新科(苏州)有机循环研究院有限公司 | Screening method of grease degrading strain and grease degrading product |
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