CN102965313A - Method for screening denitrifying bacteria - Google Patents
Method for screening denitrifying bacteria Download PDFInfo
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- CN102965313A CN102965313A CN 201210466988 CN201210466988A CN102965313A CN 102965313 A CN102965313 A CN 102965313A CN 201210466988 CN201210466988 CN 201210466988 CN 201210466988 A CN201210466988 A CN 201210466988A CN 102965313 A CN102965313 A CN 102965313A
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- denitrifying bacterium
- bacterium
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Abstract
The invention belongs to the field of environmental biology, and particularly relates to a method for screening denitrifying bacteria with high degradation rate in constructed wetlands, which comprises the steps of: firstly, mixing the source of the denitrifying bacteria obtained by preliminary screening in the constructed wetlands with a culture medium, and then carrying out shake cultivation; then, putting bacteria liquid obtained by shake cultivation into an enrichment medium for enrichment cultivation to obtain denitrifying bacteria liquid; inoculating the denitrifying bacteria liquid onto a flat-plate separation medium; after that, culturing the flat-plate separation medium at the temperature of 25-30 DEG C until bacterial colony grows well; and finally, picking the inclined plane strain of denitrifying bacterial strains, inoculating the inclined plane strain into an activation culture medium for shake cultivation, washing by stroke-physiological saline solution in a way of centrifugation, diluting and refrigerating for later use. The screened denitrifying bacterial strain is strong in degradation ability and rapid in degradation speed for nitrite in waste water. Furthermore, the strain is higher in stability and low in input cost in a microecosystem of pond culture.
Description
Technical field
The invention belongs to the environmental organism field, be specifically related to the method for a kind of denitrifying bacterium with high degradation rate that from artificial swamp, filters out.
Background technology
For a long time, pond culture is the mainstay of China's freshwater aquaculture industry, and aqiuculrue output accounts for more than 50% of national freshwater fish annual amount.But in recent years, pond culture has also run into many problems in development, as: residual bait, fish, the shrimps movement, dead algae is on the increase, the continuous accumulation of bottom organism, these organism are under anoxia condition, through fermentation, decompose and produce a large amount of ammonia nitrogens, hydrogen sulfide and nitrite etc., nitrogen in water, after the elements such as phosphorus reach certain numerical value, cause the pond waters eutrophication, thereby pond water quality is changed, the pH value descends, the probiotics self-purification capacity weakens in the water body, destroy the balance of microorganisms in water, have a strong impact on fish, shrimps metabolism and growing, even cause its death.
The control of China's Aquacultural water environment and the development of the ecotechnology of reparation are quick and diversified, such as the waterplant recovery technique, biological floating bed system, little ecotechnology etc., still, little ecotechnology is significantly adopted by everybody because of easy to use, with low cost, effect.Little ecotechnology is the denitrification by the bacterial strain of denitrifying bacterium, and the nitrite in the reduction water body makes it to generate harmless nitrogen, removes the harm of nitrite; Consume nitrogen nutrition, suppress algae excessive propagation, purifying water body.
Bacterial strain stability in the microecosystem of pond culture of screening, isolated denitrifying bacterium is stronger from artificial swamp, decomposition effect to the ammonia nitrogen in the aquaculture water, nitrite nitrogen is better, input cost is low, has simultaneously also established the basis theoretical for the biological restoration of breeding environment.
Summary of the invention
The objective of the invention is from artificial swamp, to filter out the denitrifying bacterium that a strain has high degradation efficiency, and it is screened and separation and purification, then be applied in the pond culture, reach the effect that reduces water body in nitrite, the purifying pond.
In order to realize above technique effect, the present invention comprises as follows:
The bacterium source of the primary dcreening operation denitrifying bacterium that the present invention is used is from ecological engineering research centre, INST OF FIESHERY MACHINES AND pond, and outdoor artificial vertical subsurface flow wetland top layer is the filtrate obtained of 30 centimeters and the compound sample of water down.This artificial swamp is mainly used in purifying treatment pond culture wastewater and recycle.
A kind of method of screening denitrifying bacterium, its step comprises:
(1) enrichment culture of denitrifying bacterium: rear shaking table cultivation is mixed in the denitrifying bacterium bacterium source that primary dcreening operation in the artificial swamp obtains with substratum; Again shaking table is cultivated the bacteria suspension that obtains and place the enrichment medium enrichment culture, obtain denitrifying bacterium bacterium liquid;
(2) solid culture of denitrifying bacterium bacterial strain: the denitrifying bacterium bacterium liquid in the step (1) is inoculated on the plate isolation base, then the plate isolation base is placed 25 ℃ of-30 ℃ of condition underscores to cultivate, until colony growth is good;
The slant strains of the denitrification bacterial strain that (3) obtains in the picking step (2) is inoculated in shaking table cultivation in the activation medium, and is for subsequent use with refrigerating after stroke-physiological saline solution centrifuge washing and the dilution again.
In the described step (1), denitrifying bacterium bacterium source and substratum are to mix rear shaking table at 1: 10 to cultivate 7-10 days according to volume ratio, and culture temperature is 30 ℃, and the rotation rotating speed is 75 rev/mins
In the described step (1), the composition of enrichment medium comprises the raw material of following ratio: Trisodium Citrate, MgSO
47H
2O, K
2HPO
43H
2O, KNO
3, KH
2PO
4And the amount ratio of distilled water is 5.0g: 0.2g: 1.0g: 2.0: 1.0g: 1000mL, the pH value of described enrichment medium is to sterilize 15 minutes under 7.2～7.5,121 ℃ of conditions
In the described step (2), the plate isolation base is nutrient agar medium, and the pH that separates base is 7.0-7.2, and sterilization is 15 minutes under 121 ℃ of conditions.
In the described step (3), the shaking table culture temperature is 25 ℃-30 ℃, and cultivating incubation time is 18-25 hour, the bacterial strain behind the centrifuge washing is diluted to () mL bacterium liquid with stroke-physiological saline solution and preserves under 0 ℃ of-4 ℃ of condition.
The authentication method of described denitrifying bacterium, step comprises:
(a) with the denitrifying bacterium bacterium liquid for subsequent use that obtains in the step (3) according to 10% inoculum size, be inoculated in the breeding wastewater, shaking table was cultivated 24 hours under 28 ℃ of-32 ℃ of conditions;
(b) in the shaking table culturing process, every sampling in 2 hours 1 time, each sampling amount is 10mL, and ammonia nitrogen, nitrite nitrogen content in the working sample.
The invention has the beneficial effects as follows: the primary dcreening operation from artificial swamp of denitrification bacterial strain of the present invention obtains, again through further cultivating, screening, become a strain for the extremely strong denitrifying bacterium bacterial strain of nitrite nitrogen degradation capability, then be inoculated in the breeding wastewater according to 10% inoculum size, this waste water at the content of the bacterium liquid inoculation nitrite nitrogen in the water after three hours more than 75%, so be applied in the effect that can well play purifying water body in the pond culture, suppress algae excessive propagation.The bacterial strain of described denitrifying bacterium stability in the microecosystem of pond culture is stronger, and input cost is low.
Description of drawings
The bacterium colony figure of the denitrifying bacterium that Fig. 1 obtains for this screening method.
The denitrifying bacterium that Fig. 2 obtains for this screening method is inoculated in the breeding wastewater, the variation of nitrite nitrogen content.
Embodiment
Below in conjunction with embodiment, the invention will be further described:
Consider that microbe species is abundant and similar with bottom environment with aquaculture water in the trapped substance on the filter material for artificial wetland, so the basic medium that the suspended substance that filtrate is held back in consideration screens as function yeast.Original microflora on the filtrate as the screening object, through continuous shake-flask culture with constantly after the switching enrichment, is filtered out the flora that is adapted to high organic substrate and nitrite nitrogen concentration and has the efficient degradation effect.
Concrete grammar is as follows:
1, the separation and Culture of denitrifying bacterium: down 30 centimeters are got the compound sample of filtrate and water from the artificial swamp top layer, are the anaerobic environment of artificial swamp herein, are conducive to filter out anaerobic bacteria flora.Sampling point distributions is at artificial swamp middle part, adopts three samples and mixes under rear 4 ℃ of conditions and refrigerate.Get the upper strata compound sample in the compound sample of refrigeration, this compound sample is divided into two portions, aly be the screening flora, another part be substratum, and substratum is placed on high-pressure sterilizing pot inner high voltage sterilization 15 minutes, and then 4 ℃ of conditions refrigerate for subsequent use.Unpasteurized muddy water compound sample was mixed with sterilized water by 10: 1, make the bacterium source.
Be to mix rear shaking table at 1: 10 to cultivate 7 days with bacterium source and substratum according to volume ratio, culture temperature is 30 ℃, and the rotation rotating speed is 75 rev/mins, and then transfers and obtain the bacteria suspension of denitrifying bacterium after three times.
2, the enrichment culture of denitrifying bacterium: the bacterial suspension inoculation of the denitrifying bacterium that obtains in the above-mentioned steps is cultivated 1-2 week in enrichment medium.Used enrichment medium is by 5.0g Trisodium Citrate, 0.2gMgSO
47H
2O, 1.0g K
2HPO
43H
2O, 2.0g KNO
3, 1.0g KH
2PO
4And 1000mL distilled water composition, the pH value of described enrichment medium is to sterilize 15 minutes under 7.2～7.5,121 ℃ of conditions.
3, the solid culture of denitrifying bacterium bacterial strain:
At first make the plate isolation base, according to the prescription on the nutrient agar medium bottle, take by weighing the triangular flask that the 32g nutrient agar medium places 1000mL, and adding ionized water stirring and dissolving, when stirring, measure the pH value of substratum with pH meter, pH value with NaOH solution and HCl solution are regulated substratum makes the pH value between 7.0-7.2, and carries out sterilising treatment.
Then be down flat plate: while hot nutrient agar is down flat plate (aseptic technique), keeps flat, solidify rear for subsequent use until nutrient agar medium.
At last, will be streak culture as Object of Development through the denitrifying bacterium bacterium liquid of enrichment culture.Draw denitrifying bacterium bacterium liquid 100uL and place the flat board of carrying out mark, treat that bacteria suspension has all been drawn after, again with smearing bacterium liquid through the L-type spreading rod of sterilizing.Flat board places worktable after all being coated with and wiping, and after the evaporation of bacterium liquid is slightly dried, is inverted flat board and cultivates in incubator.Used incubator is biological culturing room CO2gas incubator, and design temperature is 30 ℃, the growing state of the bacterium colonies such as the form of observed and recorded bacterium colony, color after 2 days.Through repeatedly plate isolation and purifying, obtain the bacterial strain of comparative advantage again.The form of obtained strains is that opaque in oyster white, the smooth of the edge, the substratum, liquid is transparent.Gained colonial morphology figure as shown in Figure 1.
4, the slant strains of the above-mentioned denitrifying bacterium bacterial strain of picking is inoculated in the activation medium shaking table and cultivates, and shaking table was cultivated after 18-20 hour, and again with stroke-physiological saline solution centrifuge washing 3 times, become bacterium liquid with normal saline dilution at last, 4 ℃ of refrigerations are for subsequent use.
5, the degradation effect of denitrifying bacterium is identified: the denitrifying bacterium bacterium liquid that makes according to 10% inoculum size, is inoculated in the breeding wastewater, and shaking table was cultivated 24 hours under 28 ℃ of-32 ℃ of conditions.In the shaking table culturing process, every sampling in 2 hours 1 time, each sampling amount is 10mL, and ammonia nitrogen, nitrite nitrogen content in the working sample.Take the time as X-coordinate, ammonia nitrogen, nitrite nitrogen content are ordinate zou mapping, concrete outcome as shown in Figure 2.
Among Fig. 2, control group is the nitrite nitrogen content value in the breeding wastewater that does not add denitrifying bacterium bacterium liquid, experimental group is to have added nitrite nitrogen value in the breeding wastewater of the denitrifying bacterium bacterium liquid that this screening method obtains, after two groups of numerical value contrasts, breeding wastewater at the content of the bacterium liquid inoculation nitrite nitrogen in the water after three hours more than 75%, degradation effect is obvious, and degradation speed is very fast.
Claims (6)
1. method of screening denitrifying bacterium, its step comprises:
(1) enrichment culture of denitrifying bacterium: rear shaking table cultivation is mixed in the denitrifying bacterium bacterium source that primary dcreening operation in the artificial swamp obtains with substratum; Again shaking table is cultivated the bacteria suspension that obtains and place the enrichment medium enrichment culture, obtain denitrifying bacterium bacterium liquid;
(2) solid culture of denitrifying bacterium: the denitrifying bacterium bacterium liquid in the step (1) is inoculated on the plate isolation base, then the plate isolation base is placed under 25 ℃ of-30 ℃ of conditions and cultivate, until colony growth is good;
The slant strains of the denitrification bacterial strain that (3) obtains in the picking step (2) is inoculated in shaking table cultivation in the activation medium, and is for subsequent use with refrigerating after stroke-physiological saline solution centrifuge washing and the dilution again.
2. a kind of method of screening denitrifying bacterium according to claim 1, it is characterized in that: in the described step (1), denitrifying bacterium bacterium source and substratum are to mix rear shaking table at 1: 10 to cultivate 7-10 days according to volume ratio, culture temperature is 30 ℃, and the rotation rotating speed is 70～80 rev/mins.
3. a kind of method of screening denitrifying bacterium according to claim 1, it is characterized in that: in the described step (1), the composition of enrichment medium comprises the raw material of following ratio: Trisodium Citrate, MgSO
47H
2O, K
2HPO
43H
2O, KNO
3, KH
2PO
4And the amount ratio of distilled water is 5.0g: 0.2g: 1.0g: 2.0: 1.0g: 1000mL, the pH value of described enrichment medium is to sterilize 15 minutes under 7.2～7.5,121 ℃ of conditions.
4. a kind of method of screening denitrifying bacterium according to claim 1, it is characterized in that: in the described step (2), the plate isolation base is nutrient agar medium, and the pH that separates base is 7.0-7.2, and sterilization is 15 minutes under 121 ℃ of conditions.
5. a kind of method of screening denitrifying bacterium according to claim 1, it is characterized in that: in the described step (3), the shaking table culture temperature is 25 ℃-30 ℃, cultivating incubation time is 18-25 hour, with the bacterial strain behind the centrifuge washing with the stroke-physiological saline solution dilution and under 0 ℃ of-4 ℃ of condition, preserve.
6. the method for a screening as claimed in claim 1 denitrifying bacterium, it is characterized in that: the authentication method of described denitrifying bacterium may further comprise the steps:
(a) with the denitrifying bacterium bacterium liquid for subsequent use that obtains in the step (3) according to 10% inoculum size, be inoculated in the breeding wastewater, shaking table was cultivated 24 hours under 28 ℃ of-32 ℃ of conditions;
(b) in the shaking table culturing process, every sampling in 2 hours 1 time, each sampling amount is 10mL, and ammonia nitrogen, nitrite nitrogen content in the working sample.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110452820A (en) * | 2019-08-16 | 2019-11-15 | 鹭滨环保科技（上海）股份有限公司 | A kind of screening of autotrophic denitrification bacterium and identification method |
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2012
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110452820A (en) * | 2019-08-16 | 2019-11-15 | 鹭滨环保科技（上海）股份有限公司 | A kind of screening of autotrophic denitrification bacterium and identification method |
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Application publication date: 20130313 |