CN109402107A - A kind of immobilization of efficient denitrification bacterium and the preparation method and application of microbial inoculum packet - Google Patents
A kind of immobilization of efficient denitrification bacterium and the preparation method and application of microbial inoculum packet Download PDFInfo
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Abstract
The present invention relates to the preparation methods and application of a kind of immobilization of efficient denitrification bacterium and microbial inoculum packet.It is first to filter out efficient heterotrophic nitrification aerobic denitrifying bacteria that microbial inoculum, which packs standby, and salt tolerance is then carried out to it and tames to obtain salt tolerant heterotrophic nitrification aerobic denitrifying bacteria；With modified zeolite adsorption of immobilization salt tolerant heterotrophic nitrification aerobic denitrifying bacteria, immobilization salt tolerant heterotrophic nitrification aerobic denitrifying bacteria zeolite is obtained；It is laid in cloth bag again to obtain the final product.Zeolite granular bacterial density in it is 1 × 109~5×109CFU/mL.Manufactured immobilized microbial inoculum packet is widely used in fish-farming in shallow marine water field and aquaculture pond, and efficiently removal is realized in hypersaline environment；And it can be used repeatedly for microbial inoculum packet; greatly reduce cost; it effectively overcomes and is directly easily flowed with seawer tide using existing for free state microbial bacteria; it cannot be accurately positioned and come into full contact with bacterium; also external microorganism is avoided to introduce the threat to aquaculture products quality; it protects aquaculture area water ecology safe, is expected to be used widely.
Description
Technical field
The invention belongs to water pollution control technologies, and in particular to a kind of immobilization of efficient denitrification bacterium and the preparation of microbial inoculum packet
Method and application.
Background technique
The mariculture industry in China rapidly develops, and is meeting people to having played important function in marine product demand.However,
In production process, a large amount of pollutants that aquaculture area, farm generate are directly entered marine environment, and long-term accumulation leads to water quality
Deteriorate, marine ecosystems are unbalance.Nitrogen source pollution in aquaculture process mostlys come from artificial bait throwing in, animal wastes, residual
Bait and dead animals and plants, exist in the form of nitrate nitrogen and ammonia nitrogen etc..It induces gill rot in the presence of the cheek tissue that can destroy fish
Disease inhibits blood oxygen carrying capability, declines breathing function, cause cultivated animals anoxic or be poisoned to death.For this purpose, control cultivation water
Nitrogen content is particularly important to guarantee marine product safety and marine ecology balance in body.
For the control method of ammonia nitrogen in sea water culture environment, physical method, chemical method and bioanalysis are mainly used at present.Object
Logos changes the processing methods such as water, aeration, zeolite powder or chitosan absorption；Chemical method includes using appropriate quick lime, ozone, mistake
The chemical reagent such as hydrogen oxide, chlorine dioxide reduce ammonia nitrogen concentration；Bioanalysis includes microorganism, plant and aquatic animal or more
The joint repair etc. of kind biology.Wherein bioanalysis has compared with conventional physical chemical method and invests low, Environmental compatibility
It is good, without secondary pollution, do not endanger the advantages that breeding function, be river system repair technology most with prospects at present.
Existing patent Integral synchronous nitration denitrification sewage-treatment plant (CN201510843362.1) proposes integration
Synchronous nitration and denitrification sewage-treatment plant, this method remove ammonia nitrogen and nitrate nitrogen in breeding water body simultaneously, need micro- life
The nitrification of object and denitrification cooperate simultaneously, need the synergy of multiple-microorganism.But this method treatment process
Complexity, cost increase.Meanwhile, it is capable to which the autotrophic ammonia oxidizing bacterium for removing ammonia nitrogen is very fragile, it is easy the variation by environmental condition
(such as: salinity increases) can directly result in its normal ammonia nitrogen metabolic function and lose.
Therefore the bacterial strain of prior art tends not to reach preferable removal effect in high salt conditions.Especially supported in seawater
It grows in area.A kind of existing domestication of heterotrophic nitrification aerobic denitrifying bacteria of patent and screening technique (CN201510077694.3) are public
Domestication and the screening technique of a kind of heterotrophic nitrification aerobic denitrifying bacteria are opened, but cannot be with high salt using the bacterial strain that this method obtains
Condition reaches preferable removal effect.Especially in aquaculture area, due to ammonia nitrogen and pollution of nitrate nitrogen be primarily present as
Lower two parts: (1) organic pollutants such as remaining bait, excrement pass through gravitational settling to breeding environment bottom, then longer
Time in, the decomposition product ammonia nitrogen in high density and nitrate nitrogen ammonia nitrogen and nitrate nitrogen of layer waters diffusion bottom-up again；
(2) ammonia nitrogen and nitrate nitrogen to dissociate present in water body.
Meanwhile directly easily being flowed with seawer tide using free state microbial bacterial agent, cannot be accurately positioned etc. makes to utilize effect
Rate substantially reduces, and is unable to satisfy removal effect and causes to waste, and can not recycle, reuse；Aquaculture area water body is sensitive,
External microorganism, which introduces, can jeopardize aquaculture products quality, or even it is made not survive normally.
Summary of the invention
The purpose of the present invention is to provide a kind of immobilized microbial inoculum packet preparation methods of efficient denitrification bacterium and application, to overcome
Problem of the existing technology.
Another object of the present invention is to existing efficient denitrification bacterium Zhuo Beier Salmonella (ZobellellaSp. it) carries out
Domestication makes it play high pollution in seawater and fresh water with high salt to obtain salt tolerant heterotrophic nitrification aerobic denitrifying bacteria
Object removal efficiency.
Further object of the invention, which is to overcome to have, directly to be used in free state microbial bacterial agent use process with water
Stream diffusion, microbial inoculum usage amount is big, at high cost, utilization efficiency is low and irretrievable shortcomings.
The present invention considers the salinity of marine culture wastewater compared with surface water body height first, and general function microorganism can not play
Effect and aquaculture area water body are sensitive, and external microorganism, which introduces, can jeopardize aquaculture products quality, or even make its nothing
Method is normally survived；And be unable to reach preferable repairing effect using existing conventional physical, chemistry and biological method, and administer at
This height.Therefore, from the present invention comes from the salt tolerant heterotrophic nitrification aerobic denitrifying bacteria for screening, taming in the deposit of aquaculture area
Breeding water body and ammonia nitrogen and nitrate nitrogen in deposit are managed, not only high treating effect, expense are low to realize, and because of ground system
Preferably, it will not constitute a threat to original ecological environment.
Also, in view of in the seawater of flowable state, due to the flowing of water body, directly use free state microbial bacterial agent easily with
Seawer tide is flowed and cannot be accurately positioned, therefore utilization efficiency is low, is unable to satisfy removal effect etc..So the present invention is for the first time
A kind of immobilized microbial inoculum packet removed for breeding water body ammonia nitrogen and nitrate nitrogen is proposed, so as to guarantee the microorganism in it
Not only while enough microbial biomass with unit volume will not have been broken up by water flow, but also can be to salt tolerant heterotrophic nitrification aerobic denitrifying
Bacterium has protective effect, so that the major issue that high-effect bacterial effectively adds in the seawater is realized, to reach ammonia nitrogen and nitre
The practical efficient removal of hydrochlorate nitrogen.
Immobilized microbial inoculum packet of the invention is that be evenly equipped with several immobilization salt tolerants different for tiling in corrosion resistant, permeable geotechnological cloth bag
Support nitrification aerobic denitrifying bacteria zeolite granular, wherein modified zeolite partial size between 3-8mm range, in bacterial density be
1×109~5×109CFU/mL, the bacterium are the heterotrophic nitrification aerobic denitrifying bacteria tamed.
Above-mentioned immobilized microbial inoculum packet preparation method is to filter out efficient heterotrophic nitrification aerobic denitrifying bacteria first, then right
Heterotrophic nitrification aerobic denitrifying bacteria carries out salt tolerance and tames to obtain salt tolerant heterotrophic nitrification aerobic denitrifying bacteria；And then with modification
Zeolite is carrier, and it is anti-to obtain immobilization salt tolerant heterotrophic nitrification aerobic for adsorption of immobilization salt tolerant heterotrophic nitrification aerobic denitrifying bacteria
Nitrobacteria zeolite；Finally, above-mentioned immobilization salt tolerant heterotrophic nitrification aerobic denitrifying bacteria zeolite is laid in rectangular geotextiles
To get the immobilized microbial inoculum packet for having arrived heterotrophic nitrification aerobic denitrifying bacteria in bag.
The screening technique of above-mentioned efficient heterotrophic nitrification aerobic denitrifying bacteria, includes the following steps:
(1) enrichment culture: weighing the bottom sediment of dry weight 10g-20g, is added to one and fills 100mL enriched medium
In 250mL conical flask, supernatant is taken extremely to divide after shake culture a couple of days under conditions of 150r/min-200r/min, 28 DEG C -30 DEG C
From collecting in culture medium, in above-mentioned condition shake culture a couple of days.Then, take 10 mL of supernatant to fresh isolation medium, in
150r/min-200r/min, repeats the above steps 3 times so to improve heterotrophism at shake culture a couple of days under conditions of 28 DEG C -30 DEG C
The concentration for nitrifying aerobic denitrifying bacteria, finally obtains heterotrophic nitrification aerobic denitrifying bacteria pregnant solution；Above-mentioned enriched medium
Are as follows: NaCl 5g；Yeast extract 5g；Tryptone 10g, distilled water 1000mL adjust pH=8.0,121 DEG C of sterilizing 30min；It is above-mentioned
Isolation medium are as follows: NaCl 5g, KH2PO41.5g, MgSO4﹒ 7H2O 0.01g, Na2HPO47.9g, sodium citrate 5.96g,
NaNO30.4268g, NH4Cl 0.2686g, trace element solution 2mL, deionized water 1000mL adjust pH=8.0, and 121 DEG C go out
Bacterium 30min；Above-mentioned trace element solution are as follows: ZnSO4 2.2g, CaCl2 5.5g, Na2EDTA 63.70g, MnCl2·4H2O
5.06g FeSO4·7H2O 5.0g, CuSO4·5H2O 1.57g, Na2MoO4·4H2O 1.1g, CoCl2·6H2O 1.61g, goes
Ionized water 1000mL.
(2) above-mentioned heterotrophic nitrification aerobic denitrifying bacteria pregnant solution purifies and separates: is pressed 10-1-10-7Gradient is diluted,
It is coated on solid medium, in 28 DEG C of -30 DEG C of culture a couple of days, after bacterium colony grows up to, selects the bacterium colony of different shape another
It is crossed repeatedly on a solid medium, it is isolated more until the single bacterium that the bacterium colony of generation is single form not of the same race
The purebred heterotrophic nitrification aerobic denitrifying bacteria of strain, above-mentioned culture medium are as follows: NaCl 5g, KH2PO41.5g, MgSO4﹒ 7H2O
0.01g, Na2HPO47.9g, sodium citrate 5.96g, NaNO30.4268g, NH4Cl 0.2686g, trace element solution 2mL,
Agar 20g, distilled water 1000mL adjust pH=7.2,121 DEG C of sterilizing 30min.
The upper heterotrophic nitrification aerobic denitrifying bacteria salt tolerance acclimation method, which is characterized in that separated above-mentioned
To more plants of purebred feeding nitrification aerobic denitrifying bacteria pickings into a new enriched medium in 150r/min-200r/min,
Shake culture a couple of days under conditions of 28 DEG C -30 DEG C, respectively obtain single strain pregnant solution.To above-mentioned each single strain pregnant solution entrance pair
When the number rise period, another 100mL salinity is inoculated in successively with the inoculum concentration of each single strain pregnant solution and new enriched medium 5%
(in terms of NaCl, g/L) is 5,15,25,35,45,55,65,75,85 and 95, and initial ammonia nitrogen and nitrate nitrogen additive amount are
In the enriched medium of 70mg/L, in 150r/min-200r/min, 28 DEG C -30 DEG C condition shake culture a couple of days, filter out pair
Ammonia nitrogen and nitrate nitrogen degradation effect are not less than 90% heterotrophic nitrification aerobic denitrifying bacteria, the as salt tolerant of good degrading effect
Heterotrophic nitrification aerobic denitrifying bacteria.
Above-mentioned heterotrophic nitrification aerobic denitrifying bacteria process for fixation, which is characterized in that select partial size modified for 3-8mm
For zeolite granular after fixation support, to take OD600=0.6-1.5, density is 1 × 109~5×109CFU/mL bacterium solution 50-100mL,
Every gram of modified zeolite being added in 10-20mL bacterium solution, 28-30 DEG C, 150-300 rpm oscillation absorption 12-24h discards supernatant liquid,
With sterile purified water rinse modified zeolite for several times to get arrive immobilization salt tolerant heterotrophic nitrification aerobic denitrifying bacteria zeolite.
Above-mentioned modified zeolite, which is characterized in that be the 5-10ml phosphoric acid solution that pH=5 are added to by every gram of zeolite, 28-
30 DEG C, 150-300rpm vibrates 30-90min, after standing 12-24h, is rinsed for several times using sterile purified water；Then 0.5- is used
The FeCl of 1mol/L3After impregnating 12-24h, then for several times with sterile purified water rinse, remove extra FeCl3, using high-pressure sterilizing pot
In 121 DEG C, the 30min that sterilizes is to get modified zeolite.
The immobilized microbial inoculum packet of above-mentioned heterotrophic nitrification aerobic denitrifying bacteria, which is characterized in that coat immobilized microbial inoculum
It is placed at water/sediment interface for deposit ammonia nitrogen removal；It accumulates or sheds and be placed in surface water body, breeding wastewater, big face
In product sea area, removed for ammonia nitrogen and nitrate nitrogen, and Reusability can be activated.
Salt tolerant heterotrophic nitrification aerobic obtained in above-mentioned heterotrophic nitrification aerobic denitrifying bacteria salt tolerance acclimation method is anti-
Nitrobacteria both can be applied to fresh water, surface water body ammonia nitrogen and pollution of nitrate nitrogen water body purification, can also be applied to breeding wastewater,
Large area sea area, salinity are higher than ammonia nitrogen and pollution of nitrate nitrogen water body purification under the conditions of 30.
The preparation method of above-mentioned immobilized microbial inoculum packet, it is characterised in that the above-mentioned anti-nitre of immobilization salt tolerant heterotrophic nitrification aerobic
Change immobilization salt tolerant heterotrophic nitrification aerobic denitrifying bacteria zeolite obtained in bacterium zeolite preparation method, both can be applied to light
Water, surface water body ammonia nitrogen and pollution of nitrate nitrogen water body purification can also be applied to breeding wastewater, wide sea area salinity is higher than 30
Ammonia nitrogen and pollution of nitrate nitrogen water body purification under part.
A kind of immobilized microbial inoculum packet preparation method of above-mentioned heterotrophic nitrification aerobic denitrifying bacteria, which is characterized in that institute
The salt tolerant heterotrophic nitrification aerobic denitrifying bacteria stated is the salt resistant function that has tamed, and synchronizes removal ammonia nitrogen and nitrate
The Zhuo Beier Salmonella of nitrogenZobellella sp.。
Obviously, the present invention satisfactorily solves heterotrophic nitrification aerobic denitrifying bacteria and loses it in hypersaline environment and play and makees
Use effect；Manufactured immobilized microbial inoculum packet is widely used in fish-farming in shallow marine water field and aquaculture pond, is effectively overcomed existing straight
It connects using free state microbial bacterial agent, it is existing easily to be flowed with seawer tide, it cannot be accurately positioned and utilization efficiency is low, increase greatly
Breeding wastewater and the heterotrophic nitrification aerobic denitrifying bacteria enough residence times for having added flowing, it is come into full contact with, realized high
Effect removal；And immobilized microbial inoculum packet Reusability can be activated, greatly reduces cost, therefore not only protect sea-farming
Area's water ecology safety, and avoid threat of the external microorganism introducing to aquaculture products quality.
Specific embodiment
Below by embodiment, invention is further described in detail.
Embodiment 1
To certain aquaculture area, deposit is sampled, and reaches 238mg/L by detection deposit ammonia nitrogen concentration, nitrate concentration is
254mg/L, uses that immobilized microbial inoculum packet carries out ammonia nitrogen to deposition interface under conditions of salinity is 35 ‰ and nitrate nitrogen inhibits
Experiment, comprises the following steps that
Firstly, filtering out efficient heterotrophic nitrification aerobic denitrifying bacteria: weighing the bottom sediment of dry weight 10g, be added to one
It fills in the 250mL conical flask of 100mL enriched medium, is taken after shake culture a couple of days under conditions of 150r/min, 28 DEG C
Clear liquid is into isolation medium, in above-mentioned condition shake culture a couple of days.Then, 10 mL of supernatant is taken to be separately cultured to fresh
Base, shake culture a couple of days under conditions of 150r/min, 28 DEG C repeat the above steps 3 times so to improve heterotrophic nitrification aerobic
The concentration of denitrifying bacteria finally obtains heterotrophic nitrification aerobic denitrifying bacteria pregnant solution；By the anti-nitre of above-mentioned heterotrophic nitrification aerobic
Change Enrichment of bacteria liquid and presses 10-1-10-7Gradient is diluted, and is coated on solid medium, long to bacterium colony in 28 DEG C of culture a couple of days
Cheng Hou, the bacterium colony for selecting different shape are crossed repeatedly on another solid medium, until the bacterium colony of generation is difference
The single bacterium of the single form of kind, isolated more plants of purebred heterotrophic nitrification aerobic denitrifying bacterias
Then, salt tolerance domestication is carried out to heterotrophic nitrification aerobic denitrifying bacteria: by above-mentioned isolated more plants of purebred feeding nitrifications
Aerobic denitrifying bacteria picking shake culture a couple of days under conditions of 150r/min, 28 DEG C into an enriched medium, respectively
Obtain single strain pregnant solution.When above-mentioned each single strain pregnant solution enters increased logarithmic phase, with each single strain pregnant solution and enrichment
The inoculum concentration of culture medium 5% be successively inoculated in another 100mL salinity (in terms of NaCl, g/L) be 5,15,25,35,45,55,65,
75,85 and 95, initial ammonia nitrogen and nitrate nitrogen additive amount are in the enriched medium of 70mg/L, in 150r/min-200r/
Min, is filtered out to the anti-nitre of the heterotrophic nitrification aerobic of ammonia nitrogen and nitrate nitrogen good degrading effect 28 DEG C condition shake culture a couple of days
Changing bacterium is salt tolerant heterotrophic nitrification aerobic denitrifying bacteria.The bacterium of better performances is picked out, the salt tolerant heterotrophic nitrification aerobic
Denitrifying bacteria be tamed Zhuo Beier Salmonella (Zobellellasp)。
Again, zeolite is modified: zeolite is added the ratio that 100g zeolite is placed in 500ml phosphoric acid solution (pH=5)
To phosphoric acid solution, 30 DEG C, 150rpm vibrates 30min, and after standing for 24 hours, zeolite for several times, removes extra phosphorus with sterile purified water rinse
Acid；Then the FeCl of 1mol/L is used3After impregnating zeolite for 24 hours, is washed 5 times with sterile purified water, remove extra FeCl3, use
High-pressure sterilizing pot is in 121 DEG C, and sterilize 30min, obtains modified zeolite.
Modified zeolite is carried out microbial immobilized: selecting 5mm modified zeolite for fixation support, take 500mL bacterium solution
(OD600=1.5,1.05 × 109CFU/mL), by 30g(dry weight) modified zeolite be added bacterium solution in, 30 DEG C, 150rpm oscillation inhale
It is attached to discard supernatant liquid for 24 hours, it is rinsed modified zeolite 3 times with sterile purified water, to remove, to obtain immobilization after unadsorbed bacterium resistance to
Salt heterotrophic nitrification aerobic denitrifying bacteria zeolite.
Corrosion resistant, permeable non-woven geotextile is selected to make pouch, 10g immobilization salt tolerant heterotrophic nitrification aerobic denitrifying is thin
Bacterium zeolite is laid in the geotechnological cloth bag of 10cm × 10cm to get the immobilized microbial inoculum for having arrived heterotrophic nitrification aerobic denitrifying bacteria
Packet, is covered at water/sediment interface, to be used for deposit ammonia nitrogen removal.
The present invention carries out ammonia to breeding water body deposition interface using heterotrophic nitrification aerobic denitrifying bacteria immobilized microbial inoculum packet
Nitrogen and nitrate Degrading experiment: taking 1 immobilized microbial inoculum packet to be placed in 0.25 square of reactor, the environment for being 25 DEG C in temperature
Under be removed experiment, as a result, it has been found that: immobilized microbial inoculum packet can significantly inhibit the production of ammonia nitrogen and nitrate nitrogen in water in 9 days
It is raw, 73% ± 17% is reached to the control rate of ammonia nitrogen；To the inhibiting rate of nitrate nitrogen up to 68% ± 6%.
Embodiment 2
To certain aquaculture area, deposit is sampled, and reaches 46mg/L by detection deposit ammonia nitrogen concentration, nitrate concentration is
39mg/L, use immobilized microbial inoculum packet under conditions of salinity is 35 ‰ in water body ammonia nitrogen and nitrate nitrogen be removed reality
It tests, comprises the following steps that
Firstly, filtering out efficient heterotrophic nitrification aerobic denitrifying bacteria: weighing the bottom sediment of dry weight 20g, be added to one
It fills in the 250mL conical flask of 100mL enriched medium, is taken after shake culture a couple of days under conditions of 200r/min, 30 DEG C
Clear liquid is into disjoint set culture medium, in above-mentioned condition shake culture a couple of days.Then, take 10 mL of supernatant to fresh separation training
Support base, shake culture a couple of days under conditions of 200r/min, 30 DEG C, so repeat the above steps 3 times it is good to improve heterotrophic nitrification
The concentration of oxygen denitrifying bacteria finally obtains heterotrophic nitrification aerobic denitrifying bacteria pregnant solution；Above-mentioned heterotrophic nitrification aerobic is anti-
Nitrobacteria pregnant solution presses 10-1-10-7Gradient is diluted, and is coated on solid medium, in 28 DEG C of culture a couple of days, to bacterium colony
After growing up to, the bacterium colony for selecting different shape is crossed repeatedly on another solid medium, until the bacterium colony of generation is not
The single bacterium of single form of the same race, isolated more plants of purebred heterotrophic nitrification aerobic denitrifying bacterias
Then, salt tolerance domestication is carried out to heterotrophic nitrification aerobic denitrifying bacteria: by above-mentioned isolated more plants of purebred feeding nitrifications
Aerobic denitrifying bacteria picking shake culture a couple of days under conditions of 200r/min, 30 DEG C into an enriched medium, respectively
Obtain single strain pregnant solution.When above-mentioned each single strain pregnant solution enters increased logarithmic phase, with each single strain pregnant solution and enrichment
The inoculum concentration of culture medium 5% be successively inoculated in another 100mL salinity (in terms of NaCl, g/L) be 5,15,25,35,45,55,65,
75,85 and 95, initial ammonia nitrogen and nitrate nitrogen additive amount are in the enriched medium of 70mg/L, in 150r/min-200r/
Min, is filtered out to the anti-nitre of the heterotrophic nitrification aerobic of ammonia nitrogen and nitrate nitrogen good degrading effect 28 DEG C condition shake culture a couple of days
Changing bacterium is salt tolerant heterotrophic nitrification aerobic denitrifying bacteria.The bacterium of better performances is picked out, the salt tolerant heterotrophic nitrification aerobic
Denitrifying bacteria be tamed Zhuo Beier Salmonella (Zobellellasp)。
Again, zeolite is modified: zeolite is added the ratio that 100g zeolite is placed in 500ml phosphoric acid solution (pH=5)
To phosphoric acid solution, 30 DEG C, 150rpm vibrates 30min, and after standing for 24 hours, zeolite is rinsed 5 times with sterile purified water, removes extra phosphorus
Acid；Then the FeCl of 1mol/L is used3After impregnating zeolite for 24 hours, for several times with sterile purified water rinse, extra FeCl is removed3, use
High-pressure sterilizing pot is in 121 DEG C, and sterilize 30min, obtains modified zeolite.
Modified zeolite is carried out microbial immobilized: selecting 8mm modified zeolite for fixation support, take 200mL bacterium solution
(OD600=1.5,1.05 × 109CFU/mL), by 20g(dry weight) modified zeolite be added bacterium solution in, 30 DEG C, 150 rpm oscillation inhale
Attached to discard supernatant liquid for 24 hours, rinsing zeolite 3 times with sterile purified water, to obtain immobilization salt tolerant after unadsorbed bacterium different to remove
Support nitrification aerobic denitrifying bacteria zeolite.10g immobilization salt tolerant heterotrophic nitrification aerobic denitrifying bacteria zeolite is laid in 10cm
Up to immobilized microbial inoculum packet in the geotechnological cloth bag of × 10cm, it is covered at water/sediment interface for deposit ammonia nitrogen removal.
Ammonia nitrogen and nitrate Degrading experiment are carried out to breeding water body deposition interface: 1 immobilized microbial inoculum packet being taken to be placed in 100mL
Reactor in, be removed experiment in the environment of temperature is 25 DEG C, as a result, it has been found that: immobilized microbial inoculum packet it is interior for 24 hours can
The generation for significantly removing ammonia nitrogen and nitrate in water removal, reaches 96% ± 3% to the control rate of ammonia nitrogen；The inhibiting rate of nitrate nitrogen is reached
92%±5%.Therefore it is expected to widely apply breeding water body.
Claims (10)
1. a kind of immobilized microbial inoculum packet of efficient denitrification bacterium is evenly equipped with it is characterized in that tiling in corrosion resistant, permeable geotechnological cloth bag
Several immobilization salt tolerant heterotrophic nitrification aerobic denitrifying bacteria zeolite granulars, the modified zeolite partial size between 3-8mm range,
Interior bacterial density is 1 × 109~5×109CFU/mL, the bacterium are the heterotrophic nitrification aerobic denitrifying bacteria tamed.
2. the preparation method of immobilized microbial inoculum packet described in claim 1, it is characterised in that filter out efficient heterotrophic nitrification first
Aerobic denitrifying bacteria, then to the heterotrophic nitrification aerobic denitrifying bacteria carry out salt tolerance tame to obtain salt tolerant heterotrophic nitrification it is good
Oxygen denitrifying bacteria；And then immobilizing is to adsorb salt tolerant heterotrophic nitrification aerobic denitrifying bacteria using modified zeolite as carrier,
Obtain immobilization salt tolerant heterotrophic nitrification aerobic denitrifying bacteria zeolite；Finally, above-mentioned immobilization salt tolerant heterotrophic nitrification aerobic is anti-
Nitrobacteria zeolite is laid in the immobilized bacterium that heterotrophic nitrification aerobic denitrifying bacteria is arrived in a rectangular geotechnological cloth bag
Agent packet.
3. the preparation method of immobilized microbial inoculum packet as claimed in claim 2, it is characterized in that above-mentioned efficient heterotrophic nitrification aerobic is anti-
The screening technique of nitrobacteria, includes the following steps:
(1) enrichment culture: weighing the bottom sediment of dry weight 10-20g, is added to one and fills 100mL enriched medium
In 250mL conical flask, supernatant to disjoint set is taken to train after shake culture a couple of days under conditions of 150-200r/min, 28-30 DEG C
It supports in base, in above-mentioned condition shake culture a couple of days；Then, take 10 mL of supernatant to fresh isolation medium, in 150r/
Min-200r/min, repeats the above steps 3 times so to improve heterotrophic nitrification at shake culture a couple of days under conditions of 28 DEG C -30 DEG C
The concentration of aerobic denitrifying bacteria finally obtains heterotrophic nitrification aerobic denitrifying bacteria pregnant solution；Above-mentioned enriched medium are as follows:
NaCl 5g；Yeast extract 5g；Tryptone 10g, distilled water 1000mL adjust pH=8.0,121 DEG C of sterilizing 30min；Above-mentioned separation
Culture medium are as follows: NaCl 5g, KH2PO41.5g, MgSO4﹒ 7H2O 0.01g, Na2HPO47.9g, sodium citrate 5.96g, NaNO3
0.4268g, NH4Cl 0.2686g, trace element solution 2mL, deionized water 1000mL adjust pH=8.0,121 DEG C of sterilizings
30min；Above-mentioned trace element solution are as follows: ZnSO4 2.2g, CaCl2 5.5g, Na2EDTA 63.70g, MnCl2·4H2O
5.06g FeSO4·7H2O 5.0g, CuSO4·5H2O 1.57g, Na2MoO4·4H2O 1.1g, CoCl2·6H2O 1.61g, goes
Ionized water 1000mL；
(2) above-mentioned heterotrophic nitrification aerobic denitrifying bacteria pregnant solution purifies and separates: is pressed 10-1-10-7Gradient is diluted, coating
On solid medium, in 28 DEG C of -30 DEG C of culture a couple of days, after bacterium colony grows up to, the bacterium colony for selecting different shape is solid at another
It is crossed repeatedly on body culture medium, until the single bacterium that the bacterium colony of generation is single form not of the same race, isolated more plants pure
Kind heterotrophic nitrification aerobic denitrifying bacteria, above-mentioned solid medium are as follows: NaCl 5g, KH2PO41.5g, MgSO4﹒ 7H2O
0.01g, Na2HPO47.9g, sodium citrate 5.96g, NaNO30.4268g, NH4Cl 0.2686g, trace element solution 2mL,
Agar 20g, distilled water 1000mL adjust pH=7.2,121 DEG C of sterilizing 30min.
4. the preparation method of immobilized microbial inoculum packet as claimed in claim 2, it is characterized in that above-mentioned heterotrophic nitrification aerobic denitrifying
Bacterial Salt Tolerance acclimation method is that above-mentioned more plants of isolated purebred feeding nitrification aerobic denitrifying bacteria pickings are new to one
In fresh enriched medium, shake culture a couple of days under conditions of 150r/min-200r/min, 28-30 DEG C, single bacterium is respectively obtained
Strain pregnant solution, when above-mentioned each single strain pregnant solution enters increased logarithmic phase, with each single strain pregnant solution and enriched medium 5%
Inoculum concentration to be successively inoculated in another 100mL salinity be respectively 5,15,25,35,45,55,65,75,85 and 95, initial ammonia nitrogen
It is in the new enriched medium of 70mg/L with nitrate nitrogen additive amount, in 150r/min-200r/min, 28 DEG C -30 DEG C
Condition shake culture a couple of days filters out the heterotrophic nitrification aerobic denitrifying to ammonia nitrogen and nitrate nitrogen degradation effect not less than 90%
Bacterium is the salt tolerant heterotrophic nitrification aerobic denitrifying bacteria of good degrading effect.
5. the preparation method of immobilized microbial inoculum packet as claimed in claim 2, it is characterised in that the process for fixation is selection
Partial size is fixation support in 3-8mm modified zeolite particle, takes OD600=0.6-1.5, and density is 1 × 109~5×109 CFU/mL
Bacterium solution 50-100mL, every gram of modified zeolite are added in 10-20mL bacterium solution, and 28-30 DEG C, 150-300 rpm oscillation absorption 12-24h,
Liquid is discarded supernatant, rinses modified zeolite for several times to get thin to immobilization salt tolerant heterotrophic nitrification aerobic denitrifying with sterile purified water
Bacterium zeolite.
6. the preparation method of immobilized microbial inoculum packet as claimed in claim 5, it is characterised in that the modified zeolite is by every gram
Zeolite is added to the 5-10ml phosphoric acid solution of pH=5, and 28-30 DEG C, 150-300rpm vibrates 30-90min, after standing 12-24h, connects
With sterile purified water rinse for several times；Then the FeCl of 0.5-1mol/L is used3After impregnating 12-24h, then moistened with sterile purified water
It washes for several times, removes extra FeCl3, using high-pressure sterilizing pot in 121 DEG C, the 30min that sterilizes is to get modified zeolite.
7. the immobilized microbial inoculum packet of heterotrophic nitrification aerobic denitrifying bacteria as described in claim 1, it is characterized in that by immobilization
Microbial inoculum cladding is placed at water/sediment interface for deposit ammonia nitrogen removal；It sheds and is placed in surface water body, breeding wastewater and big
In area sea area, removed for ammonia nitrogen and nitrate nitrogen, and Reusability can be activated.
8. salt tolerant heterotrophic nitrification obtained in heterotrophic nitrification aerobic denitrifying bacteria salt tolerance acclimation method described in claim 4 is good
Oxygen denitrifying bacteria can be applied to fresh water, surface water body ammonia nitrogen and pollution of nitrate nitrogen water body purification, and is applied to cultivation and gives up
Water and large area sea area salinity are higher than ammonia nitrogen and pollution of nitrate nitrogen water body purification under the conditions of 30.
9. the preparation method of immobilized microbial inoculum packet described in claim 5, it is characterised in that above-mentioned immobilization salt tolerant heterotrophic nitrification
Immobilization salt tolerant heterotrophic nitrification aerobic denitrifying bacteria zeolite obtained, is applied in aerobic denitrifying bacteria zeolite preparation method
Fresh water, surface water body ammonia nitrogen and pollution of nitrate nitrogen water body purification, and be higher than applied to breeding wastewater, large area sea area salinity
Ammonia nitrogen and pollution of nitrate nitrogen water body purification under the conditions of 30.
10. immobilized microbial inoculum packet as described in claim 1, which is characterized in that the salt tolerant heterotrophic nitrification aerobic denitrifying
Bacterium be tamed and there is salt resistant function, and the Zhuo Beier Salmonella of synchronous removal ammonia nitrogen and nitrate nitrogenZobellella
sp.。
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