CN110157639A - A kind of preparation method and application being resistant to denitrifying bacterium and its microbial inoculum with high salt - Google Patents
A kind of preparation method and application being resistant to denitrifying bacterium and its microbial inoculum with high salt Download PDFInfo
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Abstract
The invention discloses a kind of preparation methods and application for being resistant to denitrifying bacterium and its microbial inoculum with high salt, belong to field of environment microorganism, the bacterium is named as Pseudomonas stutzeri (Pseudomonas stutzeri), belongs to anaerobic denitrifying bacteria strain, and deposit number is CGMCC No.15310.The Pseudomonas stutzeri salt tolerant nitrogen removal characteristics with higher, denitrification percent, can be by water body middle and high concentration nitrate nitrogens under conditions of salinity is less than or equal to 6% up to 99% or more
Description
Technical field:
The invention belongs to field of environment microorganism, and in particular to a kind of salt tolerant denitrifying bacterium and the preparation method and application thereof.
Background technique:
With the development and the improvement of people's living standards of industrial and agricultural production, the discharge amount of China's nitrogen-containing pollutant increases rapidly
Add, nitrogen-containing pollutant is one of main source of water eutrophication.According to rough Statistics, there are about 30,000,000 people to drink high nitre in China
Hydrochlorate water, azotate pollution have become one of the essential environmental factors of China's cancer generation.Nitrate is ingested humans and animals body
After interior, it can partially be reduced into nitrite.Hemoglobin oxygen in blood can be turned to ferrihemoglobin by nitrite anions, after
Person does not have the ability in conjunction with oxygen, and when ferrihemoglobin content increases in blood, blood conveys the ability decline of oxygen, seriously
Person leads to tissue purple sore, clinically claims high-speed rail hemalbumin disease.Whole nation urban wastewater treatment has generallyd use at three-level at present
Reason, good denitrification dephosphorization effect, and most sewage effluents can reach level-one emission standard A, effectively contain
The water pollution continuous worsening impetus.
But the high concentration nitrate waste liquid that industrial activity generates, there is water-quality constituents complexity, contain organic pollutant, salt
Divide the features such as high, bio-toxicity is strong, increases the difficulty of waste liquid denitrogenation.Such as chemical fertilizer manufacture, gunpowder manufacture, gold of part industrial activity
Nitrate and nitrous containing high concentration in the waste liquids of industrial discharges such as metal surface pickling processes, plating and the etching of route galley
Hydrochlorate.Nitrate content reaches 2640mg/L in the waste liquid discharged such as Production of Potassium Nitrate factory, 640mg/L containing nitrite.Such as electricity
Plating industry generates the nitrate concentration contained in waste liquid when carrying out hanger strip and is up to 100000mg/L.
For waste liquid denitrogenation, it is most economical thorough improvement skill that nitrate nitrogen is converted nitrogen by bioanalysis denitrification denitrogenation
Art.But the biological osmolality that the high salinity waste liquid of industrial production discharge will lead to microorganism in conventional biological treatment system is inclined
Height, the rupture of microbial cell film；Microbial enzymatic activities are suppressed；Sludge settling effect variation etc..So under high salt conditions often
The biological denitrification process of rule cannot effectively carry out denitrification denitrogenation, and industrial at present for high concentration nitrate liquid waste processing normal
Using techniques such as ion exchange, evaporative crystallization, reverse osmosis and catalysis reduction, but there are pollution transportations and secondary pollution for these methods
The problems such as, it can not achieve the final disposal of pollutant.Therefore, the high-efficiency strain for screening salt tolerant is to solve high concentration under high salt conditions
One of the effective way of nitrate denitrification denitrogenation.
As the patent of invention of 108118008 A of Publication No. CN disclose a kind of Efficient salt-tolerant microbial bacterial agent and its
Preparation method and application, utilize compounding microbial inoculum: secondary coccus FSTB-2, north are shown in microbacterium FSTB-4, Pseudomonas stutzeri FSTB-5
At least one of, contain marsh cock Salmonella F S D N-A and Staphylococcus cohnis FSDN-C simultaneously, and contain arthrobacterium FDN-
At least one of 1 and Shui Shi Flavobacterium FDN-2.The invention also discloses the preparation method and application of above-mentioned microbial inoculum.The microorganism
Microbial inoculum is used directly for total nitrogen and COD in processing high slat-containing wastewater, can also be added in all kinds of biochemical reaction structures
Improve microorganism group at the salt resistant character of microorganism system, improves the total nitrogen and COD removal efficiency of system in optimization wastewater treatment.
But it can also be seen that the invention utilizes microbial inoculum combined treatment waste liquid, virtually improve the cost and microbial inoculum of microbial inoculum raw material
The time of preparation and human cost.
Summary of the invention:
In order to solve the problems, such as high concentration nitrate liquid waste processing is not thorough in the prior art, there are secondary pollution and often
Advise denitrification process salt tolerant effect is poor, complex treatment process, it is at high cost the problems such as, the present invention provide it is a kind of efficiently, be resistant to it is with high salt
Denitrifying bacterium with high concentration nitrate degradation and preparation method thereof and purposes.
A kind of tolerance denitrifying bacterium with high salt, specially Pseudomonas stutzeri (Pseudomonas stutzeri), preservation are compiled
Number be CGMCC No.15310.
The Pseudomonas stutzeri is preserved in China Committee for Culture Collection of Microorganisms on January 25th, 2018
Common micro-organisms center, deposit number are CGMCC No.15310, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number, Institute of Microorganism, Academia Sinica, postcode 100101.
The Pseudomonas stutzeri is derived from the sludge in certain industrial wastes anaerobic pond, and after up to domestication in 400 days
Separation screening obtains, and the salinity and nitrate concentration of anaerobic sludge substrate are stepped up during domestication in 400 days.
The feature of the Pseudomonas stutzeri are as follows: Gram-negative, colony colour is faint yellow, and bacterium colony is in for single raised bacterium
It falls, there is fold on surface, and edge is irregular；The form of the projection electricity thallus under the microscope be it is rod-shaped, it is straight or bend to arcuation.
Preferably, the suitable cultivation temperature of the anaerobic denitrifying bacterium is 20 DEG C -40 DEG C, pH value 6.6-8.0.
Tolerance sodium chloride salt concentration of the Pseudomonas stutzeri in waste liquid is 1-6%, the nitrate ion of toleranceConcentration is 100-30000mg/L.
It is a further object of the present invention to provide the microbial inoculum of above-mentioned tolerance denitrifying bacterium with high salt, the microbial inoculum is specially that thallus is dry
Powder or bacterium vigor are 103-104The bacteria suspension of cfu/L.
It is a further object of the present invention to provide the preparation methods of above-mentioned tolerance denitrifying bacterium microbial inoculum with high salt: specific steps are such as
Under:
(1) fermented and cultured: by the Pseudomonas stutzeri it is activated after, by 3%-10% inoculum concentration access fermentation medium
In, control fermentation temperature: 20 DEG C -40 DEG C, dissolved oxygen < 0.2mg/L, tank pressure: when 0.03-0.10Mpa, 30-50rpm shake culture
Between 72-96h, obtain fermentation liquid；The composition of the fermentation medium are as follows: peptone 6g/L-12g/L, yeast extract 3g/L-6g/L,
Sodium chloride 8-12g/L, surplus are distilled water, 7.5,121 DEG C of sterilizing 20min of pH value；
(2) fermentation liquid that the step (1) obtains is centrifuged, is lyophilized to obtain the thalli dry powder or is centrifuged, diluted
Thallus obtains the bacteria suspension.
Preferably, in the step (1), the activation method step of the Pseudomonas stutzeri are as follows: the Amur is false single
Born of the same parents are inoculated in seed culture medium, control 30 DEG C -38 DEG C of cultivation temperature, 30-50rpm shake culture 48-72h.
Preferably, the seed liquid culture medium composition are as follows: KNO32g/L-4g/L, MgSO4·7H2O 0.2g/L-0.4g/
L, K2HPO40.5g/L-0.8g/L, sodium potassium tartrate tetrahydrate 20g/L-30g/L, sodium chloride 8-12g/L, surplus are water, pH value 7.2,
121 DEG C of sterilizing 20min.
Preferably, in the step (2), centrifugal condition are as follows: centrifuging temperature: 4 DEG C, revolving speed 6000-7500rpm, centrifugation
Time is 10-15min.
Preferably, in the step (2), bacteria suspension is specially the bacterial sediment for obtaining fermentation, through the fermented and cultured
Base dilution or the salt water dilution of the sodium chloride ionic strength such as warp and fermentation medium obtain bacteria suspension.
The equal sodium chloride ionic strength dilution is specially to utilize sodium chloride concentration for 8-12g/L salt the bacterial sediment
Water dilution obtains bacteria suspension.
It is highly preferred that bacteria suspension is specially the bacterial sediment for obtaining fermentation in the step (2), trained through the fermentation
It supports base dilution and obtains bacteria suspension.
It is a further object of the present invention to provide use of the above-mentioned tolerance denitrifying bacterium with high salt in the processing of waste liquid denitrification denitrogenation
On the way.
Preferably, the sodium chloride salt concentration in the waste liquid is 1-6%, the nitrate ion of toleranceConcentration is
100-30000mg/L。
Preferably, in the waste liquid,For (0.7-2.5): 1.
It is highly preferred that in the waste liquidFor (1-2.5): 1.
Preferably, the liquid waste processing temperature is 20-40 DEG C, waste liquor PH 4.5-8.0.
It is highly preferred that the liquid waste processing temperature is 25-35 DEG C, pH 6.5-8.0.
Preferably, the method that the Pseudomonas stutzeri is applied in the processing of waste liquid denitrification denitrogenation is as follows:
It is 10 that the fermented rear dilution of the Pseudomonas stutzeri, which is become bacterium vigor,3-104The bacteria suspension of cfu/L, will be described
Bacteria suspension is added in waste liquid as inoculum according to mixing with sludge volume ratio 5-15:100；When the hydraulic retention of the waste liquid
Between be 12-22h.
Wherein, the dilution process of thalli dry powder and the preparation method of bacteria suspension are identical, i.e., using fermentation medium or with containing
Having sodium chloride concentration is that 8-12g/L salt water dilutes thalli dry powder, and obtaining bacterium vigor is 103-104The bacteria suspension of cfu/L.
It is highly preferred that the Pseudomonas stutzeri is in the processing of waste liquid denitrification denitrogenation using specific step is as follows:
It is 10 that the fermented rear dilution of the Pseudomonas stutzeri, which is become bacterium vigor,3-104The bacteria suspension of cfu/L, will be described
Bacteria suspension is added into reactor according to mixing with sludge volume ratio 5-15:100, carries out hybrid reaction, when the hybrid reaction
Between be 6-12 hour, inject waste liquid into reactor, the hydraulic detention time of the waste liquid is 12-22h, and gained denitrification percent reaches
99% or more.
It is highly preferred that the Pseudomonas stutzeri is applied in the processing of waste liquid denitrification denitrogenation method particularly includes:
The waste liquid uses two stages anaerobic treatment method, is acidified first with anaerobic acidification flora to the waste liquid
Processing quickly converts organic matter to organic matter progress open loop chain rupture difficult to degrade to the VFA of good biodegradability, controls anaerobism
Acidification reaction time 4-8h, nitrate recirculation ratio 100-300% after the reaction was completed mix processed waste liquid with nitric acid bisulfate waste liquor,
Be discharged into after regulating water quality includes that anaerobic denitrifying rank is carried out in the reactor of Pseudomonas stutzeri thallus and mud mixture
Section, Pseudomonas stutzeri make electron donor using nitrate nitrogen as electron acceptor, with the VFA that anaerobic acidification generates, carry out the anti-nitre of anaerobism
Change reaction treatment sewage, controls nitrate recirculation ratio 100-300%.Gained denitrification percent is up to 99% or more, the Pseudomonas stutzeri bacterium
Suspension and mud mixture are obtained according to above-mentioned preparation method hybrid reaction 6-12 hours.
The nitrate recirculation ratio is reflux waste liquid flow and water inlet waste liquid flow-rate ratio.
In the waste liquidFor concentration ratio.
It should be noted that sludge described above is the resulting conventional excess sludge of Normal wastewater biochemical treatment system.
The utility model has the advantages that
Pseudomonas stutzeri and its application of the invention has following advantage compared with prior art:
(1) significant strain properties: Pseudomonas stutzeri of the invention is strong to tolerance with high salt, can be in high salt conditions
Lower realization denitrification denitrogenation solves the limitation with high salt to conventional biological treatment so that high concentration nitrate waste liquid have it is a kind of it is economical,
Efficient stable and thorough processing method.
Pseudomonas stutzeri salt tolerant nitrogen removal characteristics with higher, denitrification percent are less than or equal to 6% up to 99% or more, in salinity
Under the conditions of can be by water body middle and high concentration nitrate nitrogenIt is reduced to harmless nitrogen and without nitrite
Accumulation applies in denitrification denitrogenation reactor denitrogenation efficient stable, without secondary pollution, can shorten the starting period.
(2) present invention can complete the synchronous removal of carbon nitrogen, use this for the organic liquid waste removal nitrate of high concentration
Invention has good economic benefit and environmental benefit.
(3) utilization feature of the strain for nitrogen, carbon source:
Strain of the present invention can utilize several kinds of carbon source, have good adaptability.
In the present invention, Pseudomonas stutzeri can efficiently use the nitrate in waste liquid as nitrogen source, efficient-decomposition.
Pseudomonas stutzeri of the invention can efficiently use the produced tunning after the acidification of anaerobic acidification microflora fermentation
VFA (volatile fatty acid) etc. carries out anti-nitration reaction as electron donor, reaches denitrogenation and removal organic liquid waste waste liquid is organic
The effect of carbon.(4) it is added using culture medium of the present invention by the microbial inoculum that fermented and cultured is prepared anti-to anaerobic denitrifying
Device is answered, the domestication starting efficiency for handling nitric acid bisulfate waste liquor with high salt can be accelerated.
(5) bacterial strain denitrification denitrogenation principle: Pseudomonas stutzeri provided by the present invention can be under high salt conditions, to have
Machine object is electron donor,For electron acceptor, it is reduced to nitrogen.It is disposed waste liquid simple process using the bacterial strain, denitrogenation
Thoroughly, effect stability saves operating cost.In practical applications, bacterial strain can be placed in high nitric acid bisulfate waste liquor with high salt and is realized instead
Nitrify the purpose of denitrogenation.
(6) denitrification parameter setting: 1. in the present inventionRatio setting is effectively guaranteed the completely anti-nitre of microbial inoculum
Organic matter electron donor needed for changing, effectively control BOD/NO3 -> 0.6, prevent nitric efficiency from reducing.
2. temperature and pH state modulator can effectively ensure that bacterial strain metabolic response is normally carried out and keeps to show in the present invention
The microbial activity of work, when exceeding this range, functional microorganism activity will inhibit, and treatment effect can deteriorate.
3. microbial inoculum mixing sludge is added in waste water by the preferred solution of the invention, on the one hand pure bacterium attachment sludge growth has
Conducive to avoiding pure bacterium from being lost from reactor, on the other hand start successfully that the reactor pure bacterium in the inside is as dominant bacteria, its in sludge
His microbiological paper method is formed cometabolism symbiosis with pure bacterium by developing, and enhances the metabolism speed of functional microorganism
Rate.
4. effective attachment life of the strain on sludge has been effectively ensured in the control of microbial inoculum and sludge incorporation time in the present invention
It is long；In addition, hydraulic detention time has the effect of increasing significantly for the effect of denitrification denitrogenation rate in the present invention.
Detailed description of the invention:
Fig. 1 is Pseudomonas stutzeri (Pseudomonas stutzeri) phylogenetic tree；
Fig. 2 is the transmission electron microscope photo of Pseudomonas stutzeri (Pseudomonas stutzeri)
Specific embodiment:
The present invention is described below by specific embodiment.In addition, embodiment be interpreted as it is illustrative, rather than limit
The scope of the present invention processed, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art,
Various changing under the premise of without departing substantially from spirit and scope of the present invention, to material component and dosage progress in these embodiments
Become or change also belongs to protection scope of the present invention.The present invention is further explained in the light of specific embodiments, but this hair
It is bright to be not limited to following embodiment.
Embodiment 1: the separation identification of bacterial strain Pseudomonas stutzeri:
(1) it is enriched with, the composition of isolation medium and the culture medium that spreads cultivation:
Enriched medium: potassium nitrate 2g, epsom salt 0.2g, dipotassium hydrogen phosphate 1.76g, sodium potassium tartrate tetrahydrate 20g, 7 water
Ferrous sulfate 0.005g, calcium chloride 0.02g, ammonium chloride 0.63g, NaCl 5%, distilled water 1000mL, pH value 7.5.
Isolation medium: potassium nitrate 2g, epsom salt 0.2g, dipotassium hydrogen phosphate 0.5g, sodium potassium tartrate tetrahydrate 20g, NaCl
5%, distilled water 1000mL, pH value 7.0-7.2, solid medium separately add agar 1.5% and 1mL bromine Moschus thymol blue solution.
Fermentation medium: peptone 8g, yeast extract 4g, sodium chloride 10g, distilled water 1000mL pH value 7.5.
(2) separation, purifying of Pseudomonas stutzeri:
It weighs denitrification sludge of the 5g by domestication to be placed in 250mL anaerobism bottle, adds 100mL sterilizing enriched medium and nitrogen
It is sealed after blowing deoxygenation, is placed in 30 DEG C of constant incubator cultures 4 days, 2ml mixed bacteria liquid is then taken to be seeded in anaerobic glove box
Selective agar medium operates 3 times repeatedly.1mL bacteria suspension is drawn to 9mL dilution (sterile anaerobic from Selective agar medium later
Water), obtain 10-2Dilution suspension is successively diluted to 10 by 10 times of dilution methods-7, each dilution bacteria suspension is thus made.
It is drawn respectively in anaerobic glove box on each dilution suspension 0.1mL to denitrifying bacterium separation solid medium uniformly
Coating.Plate is inverted later, is put into 30 DEG C of constant incubators, culture is to growing obvious bacterium colony.Then picking single strain, flat
Repeatedly scribing line purifies the Pseudomonas stutzeri in the present invention on plate.
(3) PCR amplification and sequencing of 16S rRNA:
It is extracted using Full-automatic magnetic beads instrument for extracting nucleic acid Smart LabAssist-16 and MO-BIO PowerSoil DNA
Kit (Taiwan dot nanometer technical concern Co., Ltd) extracts and the DNA of the obtained Pseudomonas stutzeri of purifying.Take 5 μ L
DNA sample carries out electrophoresis detection under the conditions of 120V, 20-25min, and observation quality inspection is carried out under gel-electrophoretic apparatus.
Select bacterium universal primer F16S-27 (5`-AGAGTTTGATCCTGGCTCAG-3`) and R16S-1492 (5`-
CGGTTACCTTGTTACGACTTC-3` PCR amplification) is carried out.PCR reaction condition are as follows: 94 DEG C of initial denaturation 3min, 94 DEG C of denaturation
30s, 54 DEG C of annealing 30s, 72 DEG C of extension 90s recycle 30 periods；Then 72 DEG C of extension 10min；Last 4 DEG C of preservations.
Successively Beijing Ao Weisen is entrusted by PCR product purifying and quality inspection and purified product, pcr amplification product after the recovery
Gene Tech. Company Limited is sequenced.
The 16S rDNA length that bacterial strain is obtained after sequencing is the sequence of 1430bp, be submitted to Genbank and other bacterial strains into
Row compares, and connects (Neighbour Joining) method phylogenetic tree construction using Phylogenetic Analysis (MEGA3.1) software ortho position
The evolutionary distance of (referring to attached drawing 1) discovery bacterial strain and Pseudomonas sp. is closest, determines that it belongs to Pseudomonas stutzeri
Belong to, be named as Pseudomonas stutzeri (Pseudomonas stutzeri), is shone referring to the transmission electron microscope that Fig. 2 is Pseudomonas stutzeri
Piece.
(4) the denitrogenation test of Pseudomonas stutzeri
It is 2000mg/L by the sterilized nitrate concentration of 100mL, the enriched medium that salt is divided into 3% is packed into 250mL tri-
In the bottle of angle, seed bacterium solution, stationary culture are accessed according to 10% inoculum concentration.Within the scope of 25-35 DEG C, Amur in three days time
Pseudomonad can nitrogen removal rate up to 85% or more, nitrogen removal rate is up to 99%.Preceding 48h of the bacterial strain of the present invention in culture
There is nitrite accumulation phenomenon, nitrite concentration gradually decreases later, and total nitrogen is reduced to 20mg/L or less.
Embodiment 2: the preparation of Pseudomonas stutzeri microbial inoculum
After the activated culture of Pseudomonas stutzeri, by 5% inoculum concentration access fermentation medium, fermentation temperature is controlled:
30 DEG C, dissolved oxygen < 0.2mg/L, tank pressure: 0.03-0.10Mpa, 35rpm shake culture time 72h obtain fermentation liquid, through 4 DEG C,
7500rpm, centrifugal concentrating 10min discard supernatant liquid, and precipitating is diluted with no bacteria fermentation culture medium, and obtaining bacterium vigor is 103-
104The filling formation liquid Pseudomonas stutzeri agent product of bacteria suspension is used for high concentration nitrate with high salt by the bacteria suspension of cfu/L
The processing of waste liquid denitrification denitrogenation.
The composition of the fermentation medium are as follows: peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, surplus are distillation
Water, 7.5,121 DEG C of sterilizing 20min of pH value, is cooled to room temperature after sterilizing.
Embodiment 3: the microbial inoculum preparation of Pseudomonas stutzeri
The Pseudomonas stutzeri is inoculated in seed culture medium, controls 32 DEG C of cultivation temperature, 35rpm shake culture
52h obtains seed liquor.
By Pseudomonas stutzeri seed liquor, accessed in fermentation medium by 10% inoculum concentration, fermentation culture conditions are as follows: in 35
DEG C, dissolved oxygen < 0.2mg/L, under the conditions of tank presses 0.03-0.10Mpa, incubation time 72h；
Fermentation liquid is placed in refrigerated centrifuge in 4 DEG C, 7500rpm, centrifugal concentrating 10min, discards supernatant liquid, and precipitating uses chlorine
Change the Sterile Saline that na concn is 10g/L to dilute, obtaining bacterium vigor is 103The bacteria suspension of cfu/L, by the filling formation liquid of bacteria suspension
Body Pseudomonas stutzeri agent product, the processing for high concentration nitrate waste liquid denitrification denitrogenation with high salt.
The seed liquid culture medium composition are as follows: KNO33g/L, MgSO4·7H2O 0.3g/L, K2HPO40.6g/L, winestone
Sour potassium sodium 25g/L, sodium chloride 10g/L, surplus are water, 7.2,121 DEG C of sterilizing 20min of pH value.
The composition of the fermentation medium are as follows: peptone 8g, yeast extract 4g, sodium chloride 10g, distilled water 1000mL, pH value
7.5,121 DEG C of sterilizing 20min, are cooled to room temperature after sterilizing.
It is prepared by the microbial inoculum of 4 Pseudomonas stutzeri of embodiment
The Pseudomonas stutzeri is inoculated in seed culture medium, controls 30 DEG C of cultivation temperature, 30rpm shake culture
60h obtains seed liquor.
Pseudomonas stutzeri seed liquor is accessed in fermentation medium by 3% inoculum concentration, control fermentation temperature: 20 DEG C DEG C,
Dissolved oxygen < 0.2mg/L, tank pressure: 0.03-0.10Mpa, 30rpm shake culture time 72-96h obtain fermentation liquid.
After the fermented culture of the Pseudomonas stutzeri, by fermentation liquid after low-temperature centrifugation, liquid is discarded supernatant, obtains bacterium
Body precipitating, the bacterial sediment is diluted with fermentation medium, and obtaining bacterium vigor is about 104The bacteria suspension of cfu/L, is filled
Dress, finally obtains microbial inoculum product.
The centrifugal condition are as follows: centrifuging temperature: 4 DEG C, revolving speed 7500rpm, centrifugation time 10min.
The seed liquid culture medium composition are as follows: KNO324g/L, MgSO4·7H2O 0.2g/L, K2HPO40.5g/L, winestone
Sour potassium sodium 20g/L, sodium chloride 10g/L, surplus are water, 7.2,121 DEG C of sterilizing 20min of pH value.
The composition of the fermentation medium are as follows: peptone 6g/L, yeast extract 3g/L, sodium chloride 10g/L, surplus are distillation
Water, 7.5,121 DEG C of sterilizing 20min of pH value, is cooled to room temperature after sterilizing.
It is prepared by the microbial inoculum of 5 Pseudomonas stutzeri of embodiment
The Pseudomonas stutzeri is inoculated in seed culture medium, controls 38 DEG C of cultivation temperature, 50rpm shake culture
48h obtains seed liquor.
By Pseudomonas stutzeri seed liquor by 10% inoculum concentration access fermentation medium, fermentation temperature is controlled: 40 DEG C, molten
Oxygen < 0.2mg/L is solved, tank pressure: 0.10Mpa, 50rpm shake culture time 72h obtain fermentation liquid.
After the fermented culture of the Pseudomonas stutzeri, by fermentation liquid after low-temperature centrifugation, liquid is discarded supernatant, obtains bacterium
Body precipitating, the bacterial sediment is diluted with no bacteria fermentation culture medium, and obtaining bacterium vigor is 5 × 103The bacteria suspension of cfu/L, into
Row is filling, finally obtains microbial inoculum product.
The centrifugal condition are as follows: centrifuging temperature: 4 DEG C, revolving speed 7500rpm, centrifugation time 10min.
The seed liquid culture medium composition are as follows: KNO34g/L, MgSO4·7H2O-0.4g/L, K2HPO40.8g/L, winestone
Sour potassium sodium 30g/L, sodium chloride 10g/L, surplus are water, 7.2,121 DEG C of sterilizing 20min of pH value.
The composition of the fermentation medium are as follows: peptone 12g/L, yeast extract 6g/L, sodium chloride 10g/L, surplus are distillation
Water, 7.5,121 DEG C of sterilizing 20min of pH value, is cooled to room temperature after sterilizing.
It is prepared by the microbial inoculum of 6 Pseudomonas stutzeri of embodiment
The Pseudomonas stutzeri is inoculated in seed culture medium, controls 36 DEG C of cultivation temperature, 40rpm shake culture
48h obtains seed liquor.
By Pseudomonas stutzeri seed liquor by 5% inoculum concentration access fermentation medium, fermentation temperature is controlled: 35 DEG C, molten
Oxygen < 0.2mg/L is solved, tank pressure: 0.03-0.10Mpa, 35rpm shake culture time 72h obtain fermentation liquid.
After the fermented culture of the Pseudomonas stutzeri, by fermentation liquid after low-temperature centrifugation, liquid is discarded supernatant, obtains bacterium
Body precipitating, the bacterial sediment is diluted with no bacteria fermentation culture medium, and obtaining bacterium vigor is 8 × 103The bacteria suspension of cfu/L, into
Row is filling, finally obtains microbial inoculum product.
The centrifugal condition are as follows: centrifuging temperature: 4 DEG C, revolving speed 7500rpm, centrifugation time 10min.
The seed liquid culture medium composition are as follows: KNO32g/L-4g/L, MgSO4·7H2O 0.2g/L-0.4g/L, K2HPO4
0.5g/L-0.8g/L, sodium potassium tartrate tetrahydrate 20g/L-30g/L, sodium chloride 10g/L, surplus are water, 7.2,121 DEG C of pH value sterilizings
20min。
The composition of the fermentation medium are as follows: peptone 10g/L, yeast extract 4g/L, sodium chloride 11g/L, surplus are distillation
Water, 7.5,121 DEG C of sterilizing 20min of pH value, is cooled to room temperature after sterilizing.
It is prepared by the microbial inoculum of 7 Pseudomonas stutzeri of embodiment
According to the Spawn incubation method fermented and cultured Pseudomonas stutzeri of embodiment 6, Pseudomonas stutzeri fermentation liquid is obtained,
4 DEG C, revolving speed 7500rpm, centrifugation time 10min discard supernatant liquid, obtain bacterial sediment, freeze-dried acquisition microbial inoculum dry powder.
8 Pseudomonas stutzeri of embodiment is used for the processing of high nitric acid bisulfate waste liquor with high salt
The microbial inoculum product that the embodiment of the present invention 3 is prepared: the bacteria suspension of 1.5g/L is pressed and conventional denitrification reactor
The ratio mixing of interior sludge volume amount ratio 1:10 is added into reactor, stops avoiding microbial inoculum from being lost into waste liquid, guarantees that microbial inoculum exists
The residence time is 7 hours in reactor, then into waste liquid, controls reactor and enters waste liquidInstead
Answer treatment temperature control at 30-35 DEG C, pH 6.5-7.5, wait strides to step up into water salinity and nitrate concentration, finally into
Sodium chloride salt concentration is improved by 0.5% to 6% in water, and nitrate ion concentration is improved by 500mg/L to 20000mg/L, is given up
The hydraulic detention time of liquid is 16 hours, is discharged nothingAccumulation.After processing, nitrate nitrogen is reduced in aqueous waste solution out
4.5mg/L, water outletConcentration is maintained at 20mg/L or less.
9 Pseudomonas stutzeri of embodiment is used for the processing of high nitric acid bisulfate waste liquor with high salt
The microbial inoculum product that the embodiment of the present invention 4 is prepared is pressed and sludge volume amount in conventional denitrification reactor
Ratio mixing than 1:10 is added into reactor, stops water inlet and microbial inoculum is avoided to be lost, when guaranteeing that microbial inoculum stops in reactor
Between be 12 hours, then into waste liquid, control reactor into waste liquid water Reaction treatment temperature control
System waits strides to step up into water sodium chloride salinity and nitrate concentration at 35 DEG C, pH 5.5-6.0, in final water inlet, chlorination
Sodium salt concentration is improved by 0.5% to 6%, and nitrate ion concentration is improved by 100mg/L to 30000mg/L, the waterpower of waste liquid
Residence time is 22 hours, is discharged nothingAccumulation.After processing, nitrate nitrogen is reduced to 20mg/L in waste liquid in water outlet, water outlet
's Concentration is maintained at 100mg/L or less.
10 Pseudomonas stutzeri of embodiment is used for the processing of high nitric acid bisulfate waste liquor with high salt
The microbial inoculum product that the embodiment of the present invention 5 is prepared is pressed and sludge volume amount ratio in conventional denitrification reactor
The ratio mixing of 15:100 is added into reactor, stops water inlet and microbial inoculum is avoided to be lost, when guaranteeing that microbial inoculum stops in reactor
Between 8 hours, then into waste liquid, control reactor into waste liquid water The control of reaction treatment temperature
At 30-35 DEG C, pH 7.5-8.0, strides is waited to step up into water sodium chloride salinity and nitrate concentration, in final water inlet, chlorine
Change sodium salt concentration to be improved by 0.5% to 6%, nitrate ion concentration is improved by 100mg/L to 25000mg/L, the water of waste liquid
The power residence time is 17.5 hours, is discharged nothingAccumulation.After processing, in water outlet, nitrate nitrogen is reduced to 15mg/ in waste liquid
L, water outletConcentration is maintained at 50mg/L or less.
11 Pseudomonas stutzeri of embodiment is used for the processing of high nitric acid bisulfate waste liquor with high salt
The microbial inoculum product that the embodiment of the present invention 6 is prepared is pressed and sludge volume amount ratio in conventional denitrification reactor
The ratio mixing of 12:100 is added into reactor, stops water inlet and microbial inoculum is avoided to be lost, when guaranteeing that microbial inoculum stops in reactor
Between be 9 hours, then into waste liquid, control reactor into waste liquid water The control of reaction treatment temperature
At 35-40 DEG C, pH 4.5-5.5, strides is waited to step up into water sodium chloride salinity and nitrate concentration, in final water inlet, chlorine
Change sodium salt concentration to be improved by 0.5% to 6%, nitrate ion concentration is improved by 100mg/L to 28000mg/L, the water of waste liquid
The power residence time is 12 hours, nothingAccumulation.After processing, in water outlet, nitrate nitrogen is reduced to 18mg/L in waste liquid, water outlet
'sConcentration is maintained at 70mg/L or less.
12 Pseudomonas stutzeri of embodiment is used for the processing of high nitric acid bisulfate waste liquor with high salt
It is 10 that microbial inoculum dry powder prepared by the embodiment of the present invention 7, which is diluted to bacterium vigor with no bacteria fermentation culture medium,4Cfu/L's
Bacteria suspension, by mycelium dilution liquid and sludge volume ratio 15:100, mixing is added into reactor, is stopped water inlet and is avoided microbial inoculum stream
It loses, guarantees that microbial inoculum residence time in reactor is 9 hours, then into waste liquid, control reactor into waste liquid waterReaction treatment temperature is controlled at 35 DEG C, pH 5.0-5.5, and strides is waited to step up into water sodium chloride salt
Point and nitrate concentration, in final water inlet, sodium chloride salt concentration is improved by 0.5% to 6%, nitrate ion concentration by
100mg/L is improved to 30000mg/L, and the hydraulic detention time of waste liquid is 12 hours, is discharged nothingAccumulation.After processing, out
In water, nitrate nitrogen is reduced to 20mg/L in waste liquid, water outletConcentration is maintained at 100mg/L or less.
13 Pseudomonas stutzeri of embodiment is used for the processing of high nitric acid bisulfate waste liquor with high salt
It is 10 that Pseudomonas stutzeri thallus, which is diluted to bacterium vigor with no bacteria fermentation culture medium,4The bacteria suspension of cfu/L, by bacterium
Body dilution and sludge volume ratio 1:10, mixing are added into reactor, stop water inlet and microbial inoculum is avoided to be lost, and guarantee microbial inoculum anti-
Answering the residence time in device is 9 hours.
The waste liquid uses two stages anaerobic treatment method, is acidified first with anaerobic acidification flora to the waste liquid
Processing quickly converts organic matter to organic matter progress open loop chain rupture difficult to degrade to the VFA of good biodegradability, controls anaerobism
Acidification reaction time 4-8h, nitrate recirculation ratio 100-300% after the reaction was completed mix processed waste liquid with nitric acid bisulfate waste liquor,
Be discharged into after regulating water quality includes that the anti-nitre of anaerobism is carried out in the reactor of above-mentioned Pseudomonas stutzeri thallus and mud mixture
The change stage controls reactor into waste liquid waterReaction treatment temperature, pH water inlet sodium chloride salinity and nitrate anion are most dense
It spends, the hydraulic detention time of waste liquid is the same as embodiment 12.
Pseudomonas stutzeri makees electron donor using nitrate nitrogen as electron acceptor, with the VFA that anaerobic acidification generates, and carries out anaerobism
Anti-nitration reaction handles sewage, controls nitrate recirculation ratio 100-300%.Nitrate nitrogen is reduced to 20mg/L in waste liquid, water outletConcentration is maintained at 100mg/L or less.
Processing of the conventional sludge of comparative example 1 for high nitric acid bisulfate waste liquor with high salt
Liquid waste processing is carried out according to the processing method of embodiment 8, adds same amount of identical sludge eventually to reactor, and press
According to the facts apply 8 steps to be operated, be with the difference of embodiment 8: sludge is not outer to access microbial inoculum, and furthermore difference also resides in most
Eventually in water inlet, sodium chloride salt concentration is improved by 0.5% to 2%, and nitrate ion concentration is improved by 500mg/L to 5000mg/
L, the hydraulic detention time of waste liquid are similarly 16 hours, finally measure water outletConcentration is 50mg/L, andAccumulation is obvious,Concentration is 580mg/L, and ORP is increased to+150mv, causes treatment effect continuous worsening.According to the experiment effect data, already
It cannot continue to improve sodium chloride salt concentration by conventional Treatment of Sludge and the waste liquid of nitrate ion concentration reaches good
Denitrification effect.
Claims (8)
1. a kind of tolerance denitrifying bacterium with high salt, it is characterised in that: the tolerance denitrifying bacterium with high salt is specially Pseudomonas stutzeri
(Pseudomonas stutzeri), deposit number are CGMCC No.15310.
2. the microbial inoculum containing tolerance denitrifying bacterium with high salt described in claim 1, it is characterised in that: the microbial inoculum is specially that thallus is dry
Powder or bacterium vigor are 103-104The bacteria suspension of cfu/L.
3. the preparation method of tolerance denitrifying bacterium microbial inoculum with high salt described in claim 2, it is characterised in that: specific step is as follows:
(1) fermented and cultured: by the Pseudomonas stutzeri it is activated after, by 3%-10% inoculum concentration access fermentation medium in,
Control fermentation temperature: 20 DEG C -40 DEG C, dissolved oxygen < 0.2mg/L, tank pressure: 0.03-0.10Mpa, 30-50rpm shake culture time
72-96h obtains fermentation liquid；The composition of the fermentation medium are as follows: peptone 6g/L-12g/L, yeast extract 3g/L-6g/L, chlorine
Change sodium 8-12g/L, surplus is distilled water, 7.5,121 DEG C of sterilizing 20min of pH value；
(2) fermentation liquid that the step (1) obtains is centrifuged, is lyophilized to obtain thalli dry powder or is centrifuged, dilutes thallus and obtain
Bacteria suspension.
4. being resistant to the fermentation culture method of denitrifying bacterium microbial inoculum with high salt as claimed in claim 3, it is characterised in that:
The activation step of the Pseudomonas stutzeri are as follows: the Amur vacation unit cell is inoculated in seed culture medium, control culture
30 DEG C -38 DEG C of temperature, 30-50rpm shake culture 48-72h.
5. being resistant to the fermentation culture method of denitrifying bacterium with high salt as claimed in claim 4, it is characterised in that: the seed liquor culture
Base composition are as follows: KNO32g/L-4g/L, MgSO4·7H2O 0.2g/L-0.4g/L, K2HPO40.5g/L-0.8g/L, potassium tartrate
Sodium 20g/L-30g/L, sodium chloride 8-12g/L, surplus are water, 7.2,121 DEG C of sterilizing 20min of pH value.
6. purposes of the tolerance denitrifying bacterium with high salt in the processing of waste liquid denitrification denitrogenation described in claim 1.
7. being resistant to purposes of the denitrifying bacterium with high salt in the processing of waste liquid denitrification denitrogenation as claimed in claim 6, it is characterised in that:
In the waste liquid sodium chloride salt concentration be 1-6%, nitrate ion concentration 100-30000mg/L,For (0.7-
2.5): 1, the liquid waste processing temperature is 20-40 DEG C, pH 4.5-8.0.
8. purposes of the tolerance denitrifying bacterium with high salt in the processing of waste liquid denitrification denitrogenation described in claim 7, it is characterised in that: will
It is 10 that dilution, which becomes bacterium vigor, after the Pseudomonas stutzeri is fermented3-104The bacteria suspension of cfu/L, by the bacteria suspension according to
It mixes with sludge volume ratio 5-15:100 and is added in waste liquid as inoculum；The hydraulic detention time of the waste liquid is 12-
22h。
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| CN114395505A (en) * | 2021-12-21 | 2022-04-26 | 江苏南资环保科技有限公司 | Low-temperature denitrifying bacterium and application thereof |
| CN114395505B (en) * | 2021-12-21 | 2023-08-18 | 江苏南资环保科技有限公司 | Low-temperature denitrifying bacterium and application thereof |
| CN114703093A (en) * | 2022-03-18 | 2022-07-05 | 曲阜师范大学 | Facultative anaerobic complete denitrification aerogenic bacterium Y23 and application thereof |
| CN114703093B (en) * | 2022-03-18 | 2023-08-22 | 曲阜师范大学 | Facultative anaerobic complete denitrification gas producing bacterium Y23 and application thereof |
| CN114752545A (en) * | 2022-03-23 | 2022-07-15 | 北京工业大学 | High-salt-tolerance denitrifying bacterium flora screening and culturing method |
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