CN104862257B - A kind of method of indigenous nitrogen microbial enrichment culture and its application in ammonia nitrogen pollution in water body improvement - Google Patents
A kind of method of indigenous nitrogen microbial enrichment culture and its application in ammonia nitrogen pollution in water body improvement Download PDFInfo
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Abstract
Application the invention discloses a kind of method of indigenous nitrogen microbial enrichment culture and its in ammonia nitrogen pollution in water body improvement, belongs to environmental microbiology and field of environment protection.Environmental sample is gathered from the water body for needing to administer, is cultivated to obtain indigenous nitrogen microbial enrichment culture species with specific enriched medium；Enrichment culture species, enriched medium are added in water sample in situ and are enlarged culture, then enrichment culture species that will be enlarged by cultivating is administered to water body, to administer ammonia nitrogen pollution in water body.The present invention obtains efficiently indigenous nitrogen microbial by sample in situ be enriched with the method for amplification, more more targeted than general merchandise microbial inoculum, it also avoid adaptability and ecological safety hidden danger of the external microbial inoculum to the water body.The present invention is enriched with by the condition of human controllable, ensure that the fast-growth of efficient indigenous nitrogen microbial, more more stable than directly to water body addition growth-promoting agent effect, also without the risk of secondary pollution.
Description
Technical field
The invention belongs to environmental microbiology and field of environment protection, is related to a kind of indigenous nitrogen microbial enrichment culture
Method and its ammonia nitrogen pollution in water body improvement in application.
Background technology
Industrial and agricultural production and live to the substantial amounts of nutritive salt of discharge into water（Such as nitrogen, phosphorus）, so as to cause body eutrophication.
This is most one of Tough questions that China's water environment faces.
When monitoring and administering eutrophication problem, ammonia nitrogen is an important monitoring and administers index.China city at present
It is 5mg/L to the emission request of ammonia nitrogen in the most severe standard of town sewage treatment plant-one-level A discharge standard, and China's earth's surface
The standard of ammonia nitrogen is 1.5mg/L in water IV water.This means under no successive depths treatment measures, handled through sewage treatment plant
If the in line environment of the waste water crossed, water body ammonia nitrogen will be caused exceeded.The reparation of natural water ammonia and nitrogen pollution mainly passes through biology
Repair.Microorganism is first elected in biological prosthetic：The main path that nitrogen departs from water body environment is the nitration denitrification by microorganism
The rate-limiting step of approach, wherein most critical is the nitrifying process of ammonia nitrogen.The currently used microorganism remediation technology in China has two classes.
One kind is the dispensing commercialization microbial bacterial agent directly into polluted river channel water body.The microorganism formulation of application mainly includes the U.S.
Clear-Flo series microbial inoculum, LLMO biological active liquids, the efficacious microbial colony of Japan, the photosynthetic bacteria of China, nitrobacteria
Deng, and achieve certain regulation effect.Shanghai City water sector is using water-bed aeration and the method for adding microbial combination
Western Shuan Jing river courses are administered, realizes and removes purpose that is black smelly and reducing ammonia nitrogen.Shanghai Yulei Environmental Biotechnology Co., Ltd. makes
Sediment of Shanghai Suzhou River is repaired with " Dong Jiang actinomyces ", also has significant treatment effect to soap river eutrophication water.These
Although the application of microbial inoculum has a successful story, but the problem of there is also being difficult to overcome：These microbial inoculums are all the external microorganisms of environment,
The problem of not acclimatized be present, its using effect is not also sufficiently stable；The even ecology peace of these microorganisms also biotic intrusion
Full blast danger.
To overcome the shortcomings of microbial bacterial agent, researcher develops another kind of technology：Micro- life is added to polluted river channel water body
Thing growth-promoting agent（Nutriment）, promote the growth of " original inhabitants " microorganism and the metabolism to pollutant, reach what is purified water
Purpose.General los biosynthesis technology（Shanghai）Co., Ltd, Environmental science and technology system of East China Normal University and Xuhui District of Shanghai ring
Guarantor office is using the Bioenergizer water body purifications growth-promoting agent of Probidic Solution companies of the U.S. on the Xuhui District Shang Ao pools
Tested in one section of river course.As a result show, add growth-promoting agent for eliminating black and odorous water, increase Dissolved Oxygen in Water（DO）Make
With obvious and rapid, rate of nitrification also significantly improves.Compared with microbial inoculum technology, ecological safety risk substantially reduces this technology,
But there is also deficiency：Bio-stimulant easily causes the secondary pollution of environment, and its growth-promoting effect is to environmental condition requirement
Harshness, it is difficult to promote on a large scale, therefore limit its application.
The content of the invention
The defects of it is an object of the invention to overcome prior art and deficiency, there is provided a kind of indigenous nitrogen microbial enrichment
Application of the method and this method of culture in ammonia nitrogen pollution in water body improvement.The present invention utilizes enriched medium, passes through interior
Enrichment needs the river course or lake environment sample administered, and obtains efficient indigenous nitrogen microbial enrichment culture species, so
Adaptable culture is obtained by amplifying culture afterwards, for accelerating water body nitrogen conversion process, to administer ammonia nitrogen pollution in water body.
The purpose of the present invention is achieved through the following technical solutions：
A kind of method of indigenous nitrogen microbial enrichment culture, comprises the following steps：Adopted from the water body for needing to administer
Collect sediments, be transferred in enriched medium, after shaken cultivation, culture is transferred to fresh enriched medium
In, continue shaken cultivation；Indigenous nitrogen microbial enrichment culture species is obtained after continuous several times switching culture.It is described
Enriched medium formula it is as follows：
| Nutritive salt | Dosage（Every liter） |
| (NH4)2SO4 | 0.035-0.06g |
| K2HPO4 | 0.16-0.32g |
| MgSO4·7H2O | 0.0225-0.0492g |
| NaCl | 0.275-0.35g |
| CaCl2·2H2O | 0.06-0.083g |
| Trace element | 0.5-2mL |
5% Na of above-mentioned enriched medium2CO3Solution adjusts pH so that pH value 7.3-7.8；Wherein, it is micro-
Form and be（g/L）：
| FeCl2·4H2O | 2.75 | CuSO4·5H2O | 0.045 | ZnSO4·7H2O | 0.3 |
| CoCl2·6H2O | 0.35 | MnCl2·2H2O | 0.3 | EDTA | 0.5 |
| NaMoO4·2H2O | 0.22 | NaSeO4·10H2O | 0.01 | NiCl2·6H2O | 0.1 |
| H3BO4 | 0.014 | Na3C6H5O7 | 0.64 | Vitamin C | 0.15 |
The ratio that described sediments are transferred to enriched medium is preferably 20-50g/200-300mL.
The condition of described shaken cultivation is preferably：100-160rpm shaken cultivations 2-3 days at room temperature.
The ratio that described culture is transferred to fresh enriched medium is preferably 1:5-100（Volume ratio）.
Described culture switching number is preferably 3-5 times.
A kind of method that indigenous nitrogen microbial enriched substance expands culture, comprises the following steps：Gather in the above method
Sediments source place water body overlying water 30-50L, add the above-mentioned enriched mediums of 0.1-1L, be inoculated with above-mentioned enrichment culture thing
Seed 100-400mL, aeration culture.Aeration is passed through air through Bubbled stone using mini air compressor from bucket bottom, controls dissolved oxygen
Level is in 2-4mg/L.Culture can use professional fermentation tank, can also use common plasticses bucket and cement pit, but single culture body
For system's control in the range of 30-60L, administering large body can use multiple cultivating systems high-volume to cultivate.
The condition of described aeration culture preferably cultivate 2-5 days at room temperature by aeration.
The method of above-mentioned indigenous nitrogen microbial enrichment culture or indigenous nitrogen microbial enriched substance expand culture
Application of the method in ammonia nitrogen pollution in water body improvement.
A kind of method for administering ammonia and nitrogen pollution water body, comprises the following steps：The enrichment of culture will be expanded by the above method
Culture seed by volume 1:10000-100000 splashes into the water body for needing to administer.Water body dissolved oxygen is kept in governance process
Horizontal more than 2mg/L, artificial aeration is needed during dissolved oxygen deficiency.
The time that enrichment culture species of described expansion culture is splashed is preferably the morning of fair weather.
It is of the invention to be relative to the major advantage of prior art：
1st, the present invention obtains efficiently indigenous nitrogen microbial by sample in situ be enriched with the method for amplification, than one
As commodity microbial inoculum it is more targeted, it also avoid adaptability and ecological safety hidden danger of the external microbial inoculum to the water body；
2nd, the present invention is enriched with by the condition of human controllable, ensure that the fast fast-growing of efficient indigenous nitrogen microbial
It is long, it is more more stable than directly to water body addition growth-promoting agent effect, also without the risk of secondary pollution, there is efficient and broad spectrum activity.
Brief description of the drawings
Fig. 1 is to patrol department's river original inhabitants' nitrogen microbial enrichment and expand culture effect detection figure, and ordinate unit is in figure
×104copy/mL。
Fig. 2 is to patrol ammonia nitrogen concentration result of variations figure in department's river enclosure ecosystem water sample.
Fig. 3 is to patrol nitrite nitrogen change in concentration result figure in department's river enclosure ecosystem water sample.
Fig. 4 is to patrol nitrate nitrogen change in concentration result figure in department's river enclosure ecosystem water sample.
Fig. 5 is the enrichment of South Lake original inhabitants' nitrogen microbial and expands culture effect detection figure, in figure ordinate unit for ×
105copy/mL。
Fig. 6 is ammonia nitrogen concentration result of variations figure in South Lake enclosure ecosystem water sample.
Fig. 7 is nitrite nitrogen change in concentration result figure in South Lake enclosure ecosystem water sample.
Fig. 8 is nitrate nitrogen change in concentration result figure in the enclosure ecosystem water sample of South Lake river.
Embodiment
The present invention is described in further detail with reference to embodiment and Figure of description, but the implementation of the present invention
Mode not limited to this.
The Wuhan City Xun Si rivers enclosure water correction of embodiment 1
With the gloomy bottom sampler collection Wuhan City Xun Si rivers of Peter（Longitude：114 ° 18 ' 0 ", latitude：30°29’21”）Sediments
Sample 100g, load valve bag, and transport the nitrogen microbial enrichment culture of indoor progress back rapidly.
Enrichment culture based formulas used is：(NH4)2SO40.035g, K2HPO40.16g, MgSO4•7H2O 0.0225g,
NaCl 0.275g, CaCl2•2H2O 0.083g, micro- 0.5mL, distilled water 1L, 5% Na2CO3Solution adjusts pH so that
PH value is 7.3.Wherein, trace element formula is：FeCl2•4H2O 2.75g, CuSO4•5H2O 0.045g, ZnSO4•7H2O
0.3g, CoCl2•6H2O 0.35g, MnCl2•2H2O 0.3g, EDTA 0.5g, NaMoO4•2H2O 0.22g, NaSeO4•10H2O
0.01g, NiCl2•6H2O 0.1g, H3BO40.014g, Na3C6H5O70.64g, Vitamin C 0.15g, distilled water 1L.
The bed mud 50g of fresh collection is inoculated into 250mL enriched mediums, room temperature is shaken in the case of shaking speed 120rpm
Culture is swung, 50mL cultures of being transferred after 3 days continue culture into the fresh enriched mediums of 250mL and carry out next round enrichment again in 3 days
Switching.By 3 wheel enrichment switching cultures, indigenous nitrogen microbial enrichment culture species is obtained.Ammonia oxidizing bacteria is in ammonia nitrogen
Play the part of important function in nitrifying process, therefore pass through fluorescence quantitative PCR detection wherein ammonia oxidizing bacteria quantity in enrichment process
Change, to indicate indigenous nitrogen microbial enrichment culture effect.
Quantitative fluorescent PCR the primer is the primer pair for ammonia oxidizing bacteria amoA genes（amoA-1F：5’-
GGGGTTTCTACTGGTGGT-3 ', amoA-2R：5’-CCCCTCKGSAAAGCCTTCTTC-3’）, and by ammonia oxidizing bacteria amoA
Gene cloning（Cloning primer is identical with fluorescence quantification PCR primer）Artificial plasmid, which is built, on to pMD18-T carriers is used as standard specimen system
Make quantitation curves.Fluorescent quantitative PCR result is shown in Fig. 1, shows concentration effect clearly, is enriched with by 3 wheels, in culture
The abundance of ammonia oxidizing bacteria is from initial 0.13 × 104Copy/mL increases to 6.73 × 109copy/mL。
Take 30L to patrol department's river overlying water water sample to be attached in 50L Plastic Drum, and add the above-mentioned enriched mediums of 0.2L simultaneously.
Above-mentioned enrichment culture species of 100mL, at room temperature aeration culture are inoculated with into bucket.Aeration passes through gas using mini air compressor
Afrodite is filled with air from bucket bottom, maintains dissolved oxygen 3mg/L or so in bucket.Aeration culture obtains expanding the enriched product of culture after 3 days,
And by fluorescence quantitative PCR detection wherein ammonia oxidizing bacteria number change, to indicate to expand culture effect.Quantitative fluorescent PCR knot
Fruit sees Fig. 1, shows to expand culture effect clearly, expand the abundance of ammonia oxidizing bacteria in cultured products from 2.17 ×
107Copy/mL increases to 2.31 × 109copy/mL。
4 2m × 2m enclosure is established patrolling department river, mean depth 0.7m in enclosure.4 treatment groups of Setup Experiments：A groups
Stand（Control group）；B groups are aerated；The inoculation of C groups expands the enriched product of culture but is not aerated；D groups are aerated and are inoculated with expansion culture
Enriched product；C, D groups inoculum concentration is all 1:10000（Volume ratio）.Expand the morning of the enriched product selection fair weather of culture
Morning adds；Aeration is filled with air by Bubbled stone using mini air compressor from the bottom, is uninterruptedly aerated, and maintains enclosure
Middle dissolved oxygen 2mg/L.NH in detection enclosure daily4 +-N（Ammonia nitrogen）、NO2 --N（Nitrite nitrogen）、NO3 --N（Nitrate nitrogen）Change, detection knot
Fruit sees Fig. 2-4.As a result show that the conversion ratio of ammonia nitrogen is significantly higher than other groups in D group enclosures in the case of uninterrupted aeration；C
Nitrate nitrogen and nitrous nitrogen accumulation are all significantly higher than other groups in group and D group enclosures；B groups are compared with C groups, although in mineralized nitrogen
In rate relatively, but nitrate nitrogen and nitrous nitrogen accumulation are then substantially low, and indication aeration is limited to nitrifying process stimulation, and ammonia nitrogen can
It can be assimilated by other aerobic microbiologicals.
The Nanhu Lake, Wuhan enclosure ecosystem of embodiment 2
Lake Nanhu, Wuhan, China peripheral cell, colleges and universities gather, densely populated, and point, pollution of area source source are numerous；Dragon King's mouth sewage disposal
The tail water of factory also in line South Lake；Therefore South Lake water body total nitrogen and ammonia nitrogen are horizontal long-term high, and water body is bad V classes water quality.
With the gloomy bottom sampler collection South Lake of Peter（Longitude：114 ° 21 ' 56.83 ", latitude：30°28’43.45”）Sediments
Sample 200g, loads valve bag, and the nitrogen microbial enrichment culture of indoor progress is transported in refrigeration back.
Enrichment culture based formulas used is：(NH4)2SO40.06g, K2HPO40.32g, MgSO4•7H2O 0.0492g,
NaCl 0.35g, CaCl2•2H2O 0.06g, micro- 2mL, distilled water 1L, 5% Na2CO3Solution adjusts pH so that pH value
For 7.8.Wherein, trace element formula is：FeCl2•4H2O 2.75g, CuSO4•5H2O 0.045g, ZnSO4•7H2O 0.3g,
CoCl2•6H2O 0.35g, MnCl2•2H2O 0.3g, EDTA 0.5g, NaMoO4•2H2O 0.22g, NaSeO4•10H2O 0.01g,
NiCl2•6H2O 0.1g, H3BO4 0.014g, Na3C6H5O70.64g, Vitamin C 0.15g, distilled water 1L.
The bed mud 30g of fresh collection is inoculated into 250mL enriched mediums, room temperature is shaken in the case of shaking speed 120rpm
Culture is swung, 20mL cultures of being transferred after 2 days continue culture into the fresh enriched mediums of 250mL and carry out next round enrichment again in 2 days
Switching.By 3 wheel enrichment switching cultures, indigenous nitrogen microbial enrichment culture species is obtained.Ammonia oxidizing bacteria is in ammonia nitrogen
Play the part of important function in nitrifying process, therefore pass through quantitative fluorescent PCR in enrichment process（With embodiment 1）Detect wherein ammoxidation
The change of bacterial number, to indicate indigenous nitrogen microbial enrichment culture effect.Fluorescent quantitative PCR result is shown in Fig. 5, shows richness
Collect effect clearly, be enriched with by 3 wheels, the abundance of ammonia oxidizing bacteria is from initial 0.95 × 10 in culture4Copy/mL increases
It is added to 7.60 × 1010copy/mL。
Take 30L South Lakes overlying water water sample to be attached in 50L Plastic Drum, and add the above-mentioned enriched mediums of 0.1L simultaneously.To
200mL enrichment cultures species is inoculated with bucket, at room temperature aeration culture.Aeration using mini air compressor by Bubbled stone from
Bucket bottom is filled with air, maintains dissolved oxygen 4mg/L or so in bucket.Aeration culture obtains expanding the enriched product of culture after 3 days.And pass through
Fluorescence quantitative PCR detection wherein ammonia oxidizing bacteria number change, to indicate to expand culture effect.Fluorescent quantitative PCR result is shown in figure
5, show to expand culture effect clearly, expand the abundance of ammonia oxidizing bacteria in cultured products from 4.67 × 108Copy/mL increases
It is added to 4.43 × 1010copy/mL。
4 3m × 3m enclosure, enclosure mean depth 1.6m are established in this experiment in South Lake.4 treatment groups of Setup Experiments：A
Group is stood（Control group）；B groups are aerated；The inoculation of C groups expands the enriched product of culture but is not aerated；D groups are aerated and are inoculated with expansion training
Foster enriched product；C, D groups inoculum concentration is all 1:100000（Volume ratio）.Expand the enriched product selection fair weather of culture
Add in the morning；Aeration is filled with air by Bubbled stone using mini air compressor from the bottom, is uninterruptedly aerated, maintains to enclose
Every middle dissolved oxygen 4mg/L.NH in detection enclosure daily4 +-N、NO2 --N、NO3 -- N change, testing result are shown in Fig. 6-8.As a result show
In the case of uninterrupted aeration, relatively, but the two is all for the conversion ratio of ammonia nitrogen, nitrous nitrogen accumulation in C groups and D group enclosures
It is significantly higher than A, B group enclosure；All treatment group nitrate nitrogen accumulations are significantly higher than control group, but with D group enclosure highests.This shows
The nitrifying process for effectively promoting ammonia nitrogen in enclosure that adds of product is trained in enrichment, and product is trained in addition enrichment and combination aeration can be more
Effectively improve transformation efficiency.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>Hubei University Of Technology
<120>A kind of method of indigenous nitrogen microbial enrichment culture and its answering in ammonia nitrogen pollution in water body improvement
With
<130> 1
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> amoA-1F
<400> 1
ggggtttcta ctggtggt 18
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> amoA-2R
<400> 2
cccctckgsa aagccttctt c 21
Claims (10)
- A kind of 1. method of indigenous nitrogen microbial enrichment culture, it is characterised in that comprise the following steps：Administered from needs Sediments are gathered in water body, are transferred in enriched medium, after shaken cultivation, culture is transferred to fresh enrichment In culture medium, continue shaken cultivation；Indigenous nitrogen microbial enrichment culture species are obtained after continuous several times switching culture Son；The formula of described enriched medium is：(NH4)2SO40.035-0.06g, K2HPO40.16-0.32g, MgSO4•7H2O 0.0225-0.0492g, NaCl 0.275-0.35g, CaCl2•2H2O 0.06-0.083g, micro- 0.5-2mL, distilled water 1L, pH 7.3-7.8；Wherein, trace element formula is：FeCl2•4H2O 2.75g, CuSO4•5H2O 0.045g, ZnSO4•7H2O 0.3g, CoCl2•6H2O 0.35g, MnCl2•2H2O 0.3g, EDTA 0.5g, NaMoO4•2H2O 0.22g, NaSeO4•10H2O 0.01g, NiCl2•6H2O 0.1g, H3BO40.014g, Na3C6H5O70.64g, Vitamin C 0.15g, distilled water 1L.
- 2. the method for indigenous nitrogen microbial enrichment culture according to claim 1, it is characterised in that：Described top layer The ratio that bed mud is transferred to enriched medium is 20-50g/200-300mL.
- 3. the method for indigenous nitrogen microbial enrichment culture according to claim 1, it is characterised in that：Described vibration The condition of culture is：100-160rpm shaken cultivations 2-3 days at room temperature.
- 4. the method for indigenous nitrogen microbial enrichment culture according to claim 1, it is characterised in that：Described culture The volume ratio that thing is transferred to fresh enriched medium is 1:5-100；Culture switching number is 3-5 times.
- 5. a kind of method that indigenous nitrogen microbial enriched substance expands culture, it is characterised in that comprise the following steps：Collection power Profit requires the sediments source place water body overlying water 30-50L in 1 methods described, adds side described in 0.1-1L claims 1 Enriched medium in method, the indigenous nitrogen microbial enrichment that inoculation is obtained using any one of claim 1-4 methods described Culture seed 100-400mL, aeration culture.
- 6. the method that indigenous nitrogen microbial enriched substance according to claim 5 expands culture, it is characterised in that：Expand Cultivating system is 30-60L.
- 7. the method that indigenous nitrogen microbial enriched substance according to claim 5 expands culture, it is characterised in that：It is described Aeration culture condition at room temperature aeration culture 2-5 days, water body dissolved oxygen level is 2-4mg/L.
- 8. application of the method in ammonia nitrogen pollution in water body improvement described in claim any one of 1-7.
- A kind of 9. method for administering ammonia and nitrogen pollution water body, it is characterised in that comprise the following steps：Will be any by claim 5-7 Method described in expands enrichment culture species by volume 1 of culture:10000-100000 splashes to the water for needing to administer In body；More than water body dissolved oxygen level 2mg/L is kept in governance process.
- 10. the method according to claim 9 for administering ammonia and nitrogen pollution water body, it is characterised in that：In the morning of fair weather,Enrichment culture species that will be enlarged by culture is splashed into the water body for needing to administer.
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| CN102531201A (en) * | 2012-02-06 | 2012-07-04 | 宁波天韵生态治理工程有限公司 | Method and device for utilizing microorganism in-situ circulation cultivation to treat water body pollution |
| CN102633369A (en) * | 2012-04-17 | 2012-08-15 | 西安建筑科技大学 | Method for repairing micro-polluted water body by utilizing bottom mud biological agent |
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2015
- 2015-06-02 CN CN201510293258.XA patent/CN104862257B/en not_active Expired - Fee Related
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| CN1348987A (en) * | 2001-12-02 | 2002-05-15 | 重庆和润实业(集团)有限公司 | Prepn of microbial colony sludge for efficient treatment of sewage |
| EP2067540A1 (en) * | 2007-11-29 | 2009-06-10 | Cleanfield ApS | Underground in situ bioremediation using site-specific microorganisms |
| CN102531201A (en) * | 2012-02-06 | 2012-07-04 | 宁波天韵生态治理工程有限公司 | Method and device for utilizing microorganism in-situ circulation cultivation to treat water body pollution |
| CN102633369A (en) * | 2012-04-17 | 2012-08-15 | 西安建筑科技大学 | Method for repairing micro-polluted water body by utilizing bottom mud biological agent |
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