CN106434422A - Preparation method of vibrio bacterial agent with contaminated seawater denitrification ability and bacterial agent thereof - Google Patents
Preparation method of vibrio bacterial agent with contaminated seawater denitrification ability and bacterial agent thereof Download PDFInfo
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02F3/00—Biological treatment of water, waste water, or sewage
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- C02F2101/166—Nitrites
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Abstract
The invention discloses a preparation method of a vibrio bacterial agent with contaminated seawater denitrification ability and a bacterial agent thereof. The method comprises a step of inoculating a culture medium with a bacterial strain to culture the strain, wherein the insulation with the bacterial strain is inoculating a beef extract-peptone medium with a vibrio test-tube strain; the culture temperature is 20-40 DEG C, the shaking speed is 120-180rpm, and shaking culture with a shake flask is performed till the logarithmic phase; and the vibrio is collected in the China General Microbiological Culture Collection Center (CGMCC), with a collection number CGMCC No.12311. The contaminated seawater denitrification vibrio is made into a microbial-cleaning bacterial agent by use of a vibrio strain and applied to marine culture wastewater treatment, and industrial application prospects are good.
Description
Technical field
The present invention relates to the processing technology field of dirty seawater, more particularly to a kind of arc with dirty seawater denitrification ability
Bacterium, and the purposes of this vibrios, and method and its microbial inoculum of biological denitrogenation microbial agent is prepared with this vibrios.
Background technology
At present, in highdensity cultivation system, in feed, about 75%～80% nitrogen can enter in breeding water body, and with
Different degrees of accumulation in ammonia nitrogen, nitrate nitrogen, the form of nitrite nitrogen, and aquiculture animal is worked the mischief, thus
Limit in the specific yield of sea-farming, and the waste water of its discharge and also contain abundant nitrogen-containing compound, seawater can be accelerated
Eutrophication, causes Disaster And Prevention Measures of Red Tides, brings harm to coastal ocean ecological environment.
Dirty seawater denitrifier is briefly that a class can carry out denitrifying bacterium under aerobic condition.Its presence makes
Nitrifying process and denitrification process be able in same reactor synchronous carry out, it is anti-that the product of nitration reaction can become denitrification
The substrate answered, accelerates reaction speed, reduces operating cost, the OH of anti-nitration reaction release simultaneously-Nitration reaction can be compensated
The alkali consuming, it is to avoid the accumulation of acidic materials, maintains stablizing of system pH value.
Known technology, such as published 201210152921.0 1 plants of cold-resistant pseudomonad Den-05 of Chinese patent application and
Its screening technique and application, filter out low-temperature aerobic denitrifying bacterium using microorganism pure culture technigne from natural habitat
PseudomonaspsychrophiliaDen-05, it is achieved that the preparation of biological denitrogenation microbial agent, provides for biological fortification system
Efficient denitrification bacterial classification, and be shortened by the anaerobism cycle and then shorten the control methods of aeration period, improve the municipal wastewater treatment plant winter
Season denitrification effect it is achieved that under aerobic condition nitrate nitrogen removal.
Further, 201410397855.2 1 plants of efficient denitrification acinetobacter calcoaceticus novel species of Chinese patent and its application, acinetobacter calcoaceticus
Acinetobacterguangzhouensis YZS-X1-1 has preferable denitrification ability, can effectively remove ammonia nitrogen and nitrous
Hydrochlorate nitrogen, thus environmental protection.
Further, a kind of desulfurization simultaneously of Chinese patent 200910213798.7, denitrogenation, the method for biologically treating wastewater of decolouring,
Disclose a kind of desulfurization simultaneously, denitrogenation, the method for biologically treating wastewater of decolouring.
Further, the salt cholerae strain of Chinese patent 201210171297.9 1 plant height salt biological denitrificaion and its in wastewater treatment
In application.
Further, the polyculture enrichment screening method of Chinese patent application 201110288612.1 ocean heterotrophic denitrifying bacteria.
The industry urgently more diversified vibrios with dirty seawater denitrification ability, to solve in dirty seawater living intensive treatment
The problem existing.
Content of the invention
Problem to be solved by this invention is, provides a kind of vibrios with dirty seawater denitrification ability and application thereof, and
Apply the only dirty microbial inoculum of this vibrios preparation, and the preparation method of this microbial inoculum.
One of this case purpose is to provide a kind of bacterial preparation process of the vibrios with dirty seawater denitrification ability.
A kind of preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability, the method has inoculation in culture
In base, the step of culture bacterial classification, wherein, inoculation is that this vibrios test tube kind is inoculated in beef-protein medium,
Cultivation temperature is 20 DEG C -40 DEG C, hunting speed is 120-180 rev/min, and shake flask is cultivated to logarithmic phase；
The described vibrios with dirty seawater denitrification ability, is preserved in China Committee for Culture Collection of Microorganisms common
Microorganism center, it is CGMCC No.12311 that culture presevation is numbered.
The preparation method of the aforesaid vibrios microbial inoculum with dirty seawater denitrification ability, also, cultured bacterial classification is accessed
Step to fermentation tank culture medium：By cultured bacterial classification, by the inoculum concentration of 0.5%-1% or 1%-2% of culture medium weight
It is linked in the fermentation tank of preset volume, have the fermentation tank culture medium of predetermined amount in fermentation tank, cultivate to exponential phase.
The preparation method of the aforesaid vibrios microbial inoculum with dirty seawater denitrification ability, also, the step of fermented and cultured：Bacterial classification
After being linked into fermentation tank, the throughput of filtrated air is 10-30 liter/min.
The preparation method of the aforesaid vibrios microbial inoculum with dirty seawater denitrification ability, mixing speed is 120 rev/min -200
Rev/min, or/and, cultivation temperature is 25 DEG C -40 DEG C.
The preparation method of the aforesaid vibrios microbial inoculum with dirty seawater denitrification ability, whole process incubation time is that 24-48 is little
When.
The preparation method of the aforesaid vibrios microbial inoculum with dirty seawater denitrification ability, nutrient solution packing after the completion of fermentation, or
Using the absorption of aseptic attachment, it is dried, make solid fungicide.
The preparation method of the aforesaid vibrios microbial inoculum with dirty seawater denitrification ability, wherein, by inoculation in culture medium
In, the step of culture bacterial classification, wherein, described beef-protein medium has：Peptone 1%, beef extract 0.3%, chlorine
Change sodium 0.5%, pH 7.0, balance of additive.
The preparation method of the aforesaid vibrios microbial inoculum with dirty seawater denitrification ability, wherein, cultured bacterial classification is accessed
Step to fermentation tank culture medium：Described fermentation tank culture based component has：Na2HPO4、KH2PO4、KNO3, glucose and seawater；
When seawater amount is 1000 milliliters, Na2HPO4For 7.9 grams, KH2PO4For 1.5 grams, KNO3For 1.0 grams, glucose is 9.4 grams, sends out
Fermentation tank Medium's PH Value is 7.0-7.5.
The preparation method of the aforesaid vibrios microbial inoculum with dirty seawater denitrification ability, wherein, by inoculation in culture medium
In, culture bacterial classification step, wherein, cultivation temperature be 20 DEG C -25 DEG C or 26 DEG C -30 DEG C or 31 DEG C -35 DEG C or 36 DEG C -37 DEG C or
38-40 DEG C of arbitrary value range.
The preparation method of the aforesaid vibrios microbial inoculum with dirty seawater denitrification ability, wherein, by inoculation in culture medium
In, the step of culture bacterial classification, wherein, hunting speed is 120-150 rev/min or 151-160 rev/min or 161-170 rev/min
Arbitrary value range of clock or 171-180 rev/min.
The preparation method of the aforesaid vibrios microbial inoculum with dirty seawater denitrification ability, wherein, the step of fermented and cultured：Bacterial classification
It is linked into fermentation tank after fermentation culture, filtrated air throughput is preferably 10 liters/min or 20 liters/min or 30 liters/min
Any value；
The preparation method of the aforesaid vibrios microbial inoculum with dirty seawater denitrification ability, wherein, mixing speed is preferably 60-90
Rev/min or 91-120 rev/min of arbitrary value range；Or/and, cultivation temperature be preferably 25 DEG C -30 DEG C or 31 DEG C -35 DEG C or
36 DEG C -40 DEG C of arbitrary value range；
The preparation method of the aforesaid vibrios microbial inoculum with dirty seawater denitrification ability, wherein, described whole process incubation time
It is preferably 24-30 hour or 31-36 hour or 37-42 hour or the arbitrary value range of 43-48 hour；
The preparation method of the aforesaid vibrios microbial inoculum with dirty seawater denitrification ability, wherein, described dirt seawater denitrogenation is only dirty
Bacterium, suitable pH value is in the arbitrary value range of 5.0-6.0 or 6.1-7.0 or 7.1-8.0 or 8.1-9.0；
The preparation method of the aforesaid vibrios microbial inoculum with dirty seawater denitrification ability, wherein, described dirt seawater denitrifier bacterium
The suitable carbon source of strain be glucose or sodium acetate or sodium succinate or sodium oxalate or sodium citrate any one.
This case further object is to provide a kind of microbial inoculum of the vibrios with dirty seawater denitrification ability, can use aforesaid method
Preparation.
The vibrios microbial inoculum with dirty seawater denitrification ability of preceding method preparation, wherein, described have dirty seawater denitrogenation energy
The vibrios of power is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and it is CGMCC that culture presevation is numbered
No.12311.
The aforesaid vibrios microbial inoculum with dirty seawater denitrification ability, wherein, the described vibrios with dirty seawater denitrification ability,
Suitable carbon source be glucose or sodium acetate or sodium succinate or sodium oxalate or sodium citrate any one；Or/and, described have dirty sea
The G+C content of the vibrios DNA of denitrogenation of water ability is 44.3mol%；Described " G+C content " refers to " guanine (G) in DNA molecular
With the Mole percent ratio shared by cytimidine (C) "；Or/and, the temperature of suitable growth is 20 DEG C -25 DEG C or 26 DEG C -30 DEG C or 31
DEG C -35 DEG C or 36 DEG C -37 DEG C or 38-40 DEG C of arbitrary value range；Or/and, the described vibrios with dirty seawater denitrification ability, fit
Close pH value in the arbitrary value range of 5.0-6.0 or 6.1-7.0 or 7.1-8.0 or 8.1-9.0.
This case further object is to provide a kind of purposes of aforementioned vibrios microbial inoculum.
The purposes of the aforesaid vibrios microbial inoculum with dirty seawater denitrification ability, for the wastewater treatment after seawater utilization, institute
Stating the waste water after seawater utilization is the marine culture wastewater containing nitrogen source pollutant.
The purposes of the aforesaid vibrios microbial inoculum with dirty seawater denitrification ability, for the wastewater treatment after seawater utilization, institute
State the waste water after seawater utilization, nitrogen source pollutant therein, with nitrate nitrogen (NO3 -- N) meter, initial concentration is 150 mg/litre.
This case further object is to provide a kind of vibrios with dirty seawater denitrification ability, and this case is also known as dirty seawater denitrogenation arc
Bacterium.
The vibrios that the present invention has dirty seawater denitrification ability takes technical scheme below to realize：
One plant of vibrios with dirty seawater denitrification ability, is preserved in China Committee for Culture Collection of Microorganisms commonly micro-
Bio-Centers, it is CGMCC No.12311 that culture presevation is numbered.
The vibrios that this case has dirty seawater denitrification ability also can be realized using following technical measures further：
The aforesaid vibrios with dirty seawater denitrification ability, wherein, described bacterial strain 16S rDNA sequence is：
This case further object is to provide a kind of aforementioned vibrios purposes.
The purposes of the aforesaid vibrios with dirty seawater denitrification ability, wherein, microorganism formulation that this bacterial strain is made, use
Wastewater treatment after seawater utilization, the waste water after described seawater utilization is the marine culture wastewater containing nitrogen source pollutant.
The purposes of the aforesaid vibrios with dirty seawater denitrification ability, wherein, this bacterial strain is made and uses after microorganism formulation
Wastewater treatment after seawater utilization, the waste water after described seawater utilization, nitrogen source pollutant therein, with nitrate nitrogen (NO3 --
N) count, initial concentration is 150 mg/litre.
Thus, dirty microbial inoculum is used for marine culture wastewater only to have the microorganism that the cholerae strain of dirty seawater denitrification ability makes
Process, process time only 24 hours, sewage water denitrification clean-up effect is with regard to highly significant, and microbial inoculum has reliable bio-safety
Property.
The present invention compared with prior art has significant advantage and beneficial effect.From above technical scheme, this
Bright under excellent structure configures, at least following advantage：
This case has vibrios of dirty seawater denitrification ability and application thereof, the vibrios MCW153 in the present invention, not only has relatively
Good denitrification ability, can effectively remove nitrate nitrogen and nitrite nitrogen, and safe and harmless, and biological safety test shows
(thalline feeds goldfish 3 months), goldfish upgrowth situation is good, and is better than control group.Vibrios MCW153 is used for making biological denitrificaion
Microbial inoculum, for solving, seawater aquaculture system total nitrogen is exceeded to wait eutrophication problem, has good industry application and market
Prospect.
This case dirt seawater denitrifier be successfully separated can solve marine culture wastewater process long felt a difficult problem, this
Case dirt seawater denitrifier separation method is simple, separates and cultural method is relatively easy to promote, with the development of culture technique, it can
So that by the use of many organic matters (including fishes and shrimps excreta, food-residue etc.) as its carbon source, therefore, dirt seawater denitrifier makees for this case
For the biomaterial of the efficient dirty microbial bacterial agent only of dirty seawater, there are excellent industry promotion and application and be worth.
Be there is by this case the microbial inoculum of the vibrios preparation of dirty seawater denitrification ability, the manufacturing cost more cheap of microbial inoculum can be made, more
It is beneficial to industry popularization and application；The microbial inoculum being obtained by the bacterial preparation process of this case especially can meet the purification of marine culture wastewater
The practical problem processing and demand, can quickly start the biological cleaning ability of newly-built saline sewage treatment facility using this microbial inoculum
With the strengthened denitrification disposal ability recovering the biosystem deteriorating.
The vibrios MCW153 that one plant of this case has dirty seawater denitrification ability has good total nitrogen removal effect, and laboratory is shaken
Bottle test result shows, manual simulation's waste water nitrogen removal rate is higher.
It can be seen that, vibrios described in this case is prepared into the microorganism formulation administered for marine culture wastewater, for current routine
Sewage disposal technology can not process the situation of marine culture wastewater (low-carbon high-nitrogen) effectively, using the biological reinforced skill of this case
Art, by adding the microbial inoculum of this case vibrios in sewage treatment facility, is substantially shorter the sludge acclimatization time, significantly improves denitrogenation
Treatment effect, can effectively reduce investment outlay and operating cost, thus solving de- present in current marine culture wastewater biological treatment
Nitrogen effect difference and the problem in the urgent need to address such as sludge acclimatization period is long.
Present invention contrast prior art have significant contribute and progressive, strictly there is novelty, creativeness, practicality
Good technology.
Brief description
Fig. 1 is vibrios thalline transmission electron microscope photo of the present invention；
Fig. 2 is vibrios optimum temperature schematic diagram of the present invention；
Fig. 3 is the most suitable rotating speed schematic diagram of vibrios of the present invention；
Fig. 4 is the most suitable inoculum concentration schematic diagram of vibrios of the present invention；
Fig. 5 is the most suitable throughput schematic diagram of vibrios of the present invention；
Fig. 6 is vibrios of the present invention optimal fermentation time schematic diagram；
Fig. 7 is vibrios optimum pH schematic diagram of the present invention；
Fig. 8 is the most suitable carbon source schematic diagram of vibrios of the present invention；
Fig. 9 is the denitrification effect schematic diagram to artificial seawater breeding wastewater for the microbial inoculum of the present invention；
Figure 10 is the biological safety test of the vibrios MCW153 that one plant of this case has dirty seawater denitrification ability.
Specific embodiment
The specific embodiment of the present invention is shown in detail in by following examples.
Below in conjunction with preferred embodiment, to the specific embodiment providing according to the present invention, step, feature and its effect,
Describe in detail as follows.
Embodiment 1
A kind of vibrios with dirty seawater denitrification ability, this case is also referred to as dirty seawater denitrifier, or claims：Dirty seawater denitrogenation arc
Bacterium, this vibrios is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and it is CGMCC that culture presevation is numbered
No.12311.
The G+C content of the described vibrios DNA with dirty seawater denitrification ability is 44.3mol%.There is dirty seawater denitrogenation
The vibrios of ability, Gram-negative (G-), cell is oval, with polar flagella motion.There is dirty seawater denitrification ability
Vibrios, oxidizing ferment, catalase, any one reduction reaction of nitrate are the positive.There is the vibrios bacterium of dirty seawater denitrification ability
Strain is carbon source using glucose, sodium acetate, sodium succinate etc..Have dirty seawater denitrification ability cholerae strain utilize glucose and
Other (galactolipin, arabinose, maltose etc.) some carbohydrates produce acid.The suitable pH value of dirty seawater denitrifier, between 5-9, is fitted
Preferably growth temperature is 30-35 DEG C.
The vibrios MCW153 that one plant of this case has dirty seawater denitrification ability has good total nitrogen removal effect, and laboratory is shaken
Bottle test result shows, manual simulation's waste water nitrogen removal rate is higher.
Embodiment 2
There is the only dirty bacterial preparation process of the vibrios of dirty seawater denitrification ability, including following technique mistake described in embodiment 1
Journey：
Step 1：Dirty seawater denitrifier test tube kind is inoculated in beef-protein medium, cultivation temperature be 30 DEG C,
Hunting speed is 160 revs/min, and shake flask is cultivated to logarithmic phase；Beef-protein medium is commercially available culture medium.
Step 2：By above-mentioned cultured bacterial classification, it is linked in fermentation tank by the inoculum concentration of culture medium weight 0.5%-2%,
There is culture medium, the culture medium used by fermentation tank has in fermentation tank：Na2HPO47.9 grams, KH2PO41.5 grams, KNO31.0 grams, grape
9.4 grams of sugar, 1000 milliliters of seawater.
Step 3：In fermentation tank production process, the throughput of filtrated air is 10-30 liter/min, and mixing speed is 160
Rev/min, cultivation temperature is 30 DEG C, and whole process incubation time is 24 hours, nutrient solution packing after the completion of fermentation, or using aseptic
Attachment adsorbs, is dried, and makes solid fungicide.
As can be seen here, dirty seawater denitrifier separation method is simple, separates and culture is relatively easy to, sending out with culture technique
Exhibition, it can be by the use of many organic matters (including fishes and shrimps excreta, food-residue etc.) as its carbon source.
Microbial inoculum, manufacturing cost more cheap are prepared by dirty seawater denitrifier, is relatively beneficial to industry popularization and application.Thus, dirty sea
The microbial inoculum that denitrogenation of water bacterial preparation process obtains especially can meet practical problem and the demand of the purified treatment of marine culture wastewater,
The biological cleaning ability of newly-built saline sewage treatment facility can quickly be started using this microbial inoculum and recover the biosystem deteriorating
Strengthened denitrification disposal ability.
Embodiment 3
A kind of purposes of the vibrios with dirty seawater denitrification ability, is that dirty microbial inoculum is used for the microorganism made of cholerae strain only
Marine culture wastewater is processed, and process time is 24 hours, and sewage water denitrification clean-up effect is notable.
Embodiment 4
Referring to accompanying drawing 1 to shown in Figure 10, this case is related to component and quotes percentage by weight, under do not repeat.
One plant of vibrios MCW153 with dirty seawater denitrification ability, this biomaterial is preserved in Chinese microorganism strain preservation
Administration committee's common micro-organisms center, it is CGMCC No.12311 that culture presevation is numbered, March 25 2016 preservation time,
Survival, is shown in that preservation proves；Fig. 1 is bacterial strain MCW153 transmission electron microscope photo；
In this case, the vibrios MCW153 with dirty seawater denitrification ability, also known as dirty seawater denitrifier, is described in detail below：
This dirty seawater denitrifier is identified as vibrio (Vibrio sp.MCW153), and Main Biological is gram
Negative (G-), cell is ellipse, amphitrichous, 0.8 × 1.2 micron of thalline size；3 are cultivated on existing LB culture medium flat plate
Graininess that it bacterium colony is cream-coloured, flattened round, surface is slight moistens, edge slight fold, and diameter is in 2.5 millimeter.
Described dirt seawater denitrogenation dirty bacterium only, oxidizing ferment, catalase, any one reduction reaction of nitrate are the positive；Its
Urease test, indole reaction are feminine gender；Produce acid using some carbohydrates such as glucose, galactolipin, arabinose, maltose.Ginseng
See dirty seawater denitrogenation dirty bacterium MCW153 physiological and biochemical property table only.Described dirt seawater denitrogenation only the suitable pH value of dirty bacterium 5-9 it
Between, preferably suitable growth temperature is 30 DEG C.The G+C content of described dirt seawater denitrogenation dirty bacterium DNA only is 44.3mol%；Wherein, " G
+ C content " is " guanine (G) and the Mole percent ratio shared by cytimidine (C) in DNA molecular " being often referred to.
Dirty seawater denitrifier MCW153 physiological and biochemical property
The Genbank number of logging in of this bacterial strain 16S rDNA is KU644134.
Carry out PCR (PCR) amplification, polymerase chain reaction to separating the bacterial strain 16S rDNA sequence obtaining
Sea raw work sequencing should be served by (PCR) product, basic sequence is as follows：
The bacterial strain 16S rDNA sequence of dirty seawater denitrogenation vibrios is：
NCBI the sequencing results show, the 16S rDNA sequence of MCW153 bacterial strain and the multiple bacterium of Vibrio sp. (vibrios)
The sequence homology of strain is more than 99%.
There is the preparation method of the vibrios microbial inoculum of dirty seawater denitrification ability, including following technical process：
Step 1：The vibrios test tube kind with dirty seawater denitrification ability is inoculated in beef-protein medium, culture
Temperature is 25 DEG C -40 DEG C, hunting speed is 120-180 rev/min, and shake flask is cultivated to logarithmic phase；Beef extract-peptone is trained
Foster base can be general commercially available product.
Step 1 further embodiment is：
Preferably beef-protein medium has：Peptone 1%, beef extract 0.3%, sodium chloride 0.5%, pH
7.0, it will not go into details for balance of usual additive；
The described vibrios with dirty seawater denitrification ability is preserved in Chinese microorganism strain preservation conservator for aforesaid
The bacterial classification of meeting common micro-organisms center, referring to Fig. 1, deposit number is CGMCC No.12311；
The described vibrios test tube kind with dirty seawater denitrification ability is inoculated in the culture temperature in beef-protein medium
Degree is preferably 20 DEG C -25 DEG C or 26 DEG C -30 DEG C or 31 DEG C -35 DEG C or 36 DEG C -37 DEG C or 38-40 DEG C of arbitrary value range；
Referring to Fig. 2, test shows, temperature be 20 DEG C～40 DEG C when, bacterial strain can well-grown, and temperature be 30 DEG C
When, biomass reaches maximum.
The described vibrios test tube kind with dirty seawater denitrification ability is inoculated in the shaking flask in beef-protein medium
The speed of vibration is preferably 120-150 rev/min or 151-160 rev/min or 161-170 rev/min or 171-180 rev/min
Arbitrary value range of clock, mixing speed rev/min also is indicated as r/min；
Referring to Fig. 3, test shows the impact to strain growth for the shaking speed, and the speed of shake flask is too low to be unfavorable for bacterial strain
The growth of MCW153, with faster rotational speed, cellular biomass increases, but too high rotating speed easily makes nutrient solution overflow, and therefore, fits
Suitable strain culturing rotating speed is 120-180 rev/min, and optimal rotating speed is 160 revs/min.
Step 2：By above-mentioned cultured bacterial classification, it is linked into default appearance by the inoculum concentration of the 0.5%-2% of culture medium weight
In long-pending fermentation tank, there is the culture medium of predetermined amount in fermentation tank, such as by above-mentioned cultured bacterial classification, the inoculum concentration by 1% accesses
To in the fermentation tank of preset volume amount, such as 10 liters of fermentation tanks, cultivate to exponential phase；
Step 2 further embodiment is：
Preferably the culture medium used by fermentation tank has：Na2HPO47.9 gram, KH2PO41.5 grams, KNO31.0 grams, glucose
9.4 grams, pH 7.0-7.5,1000 milliliters of seawater；
The percentage by weight of described dirt seawater the denitrogenation inoculum concentration of dirty bacterium and culture medium only be preferably 1%-1.5% or
Arbitrary value range of 1.5%-2%.
Referring to Fig. 4, this test indicates the inoculum concentration of the vibrios with dirty seawater denitrification ability to the impact preparing microbial inoculum.
After cultivating 24 hours, increase with inoculum concentration, less, therefore, the suitable inoculum concentration of bacteria fermentation exists the change of microbial inoculum increment
1-2%, but combine cost factor, selects 1% inoculum concentration can effective reduces cost；
Step 3：In fermentation tank production process, the throughput of filtrated air is 10-30 liter/min, and mixing speed is 120
Revs/min -200 revs/min, cultivation temperature is 25 DEG C -40 DEG C, and whole process incubation time is 24-48 hour, trains after the completion of fermentation
Nutrient solution dispenses, or is adsorbed using aseptic attachment, is dried, and makes solid fungicide.
Throughput liter/min also is indicated as L/min.
Step 3 further embodiment is：
Described filtrated air throughput is preferably 10 liters/min or 20 liters/min or 30 liters/min of arbitrary value range；
Referring to Fig. 5, test shows that the impact to strain fermentation for the throughput is larger, and wherein, throughput is 10-30 liter/min
When, microbial inoculum well-grown.
Mixing speed is preferably 60-90 rev/min or 90-120 rev/min of arbitrary value range；
Cultivation temperature is preferably 25 DEG C -30 DEG C or 31 DEG C -35 DEG C or 36 DEG C -40 DEG C of arbitrary value range；
Described whole process incubation time is preferably 24-30 hour or 30-36 hour or 36-42 hour or 42-48 is little former
One value range；Hour also is indicated as h.
Referring to Fig. 6, test shows, with the increase of fermented incubation time, thalline constantly grows, and wherein, fermentation time is
4-16 hour, microbial inoculum is in optimal growth conditions.
The culture medium of described fermentation tank includes：Na2HPO47.9 grams of (disodium hydrogen phosphate), KH2PO4(potassium dihydrogen phosphate)
1.5 grams, KNO31.0 grams of (potassium nitrate), 9.4 grams of glucose, pH 7.0-7.5,1000 milliliters of seawater；
The described vibrios with dirty seawater denitrification ability, suitable pH value is preferably in 5.0-6.0 or 6.1-7.0 or 7.1-8.0
Or the arbitrary value range of 8.1-9.0.
The described vibrios with dirty seawater denitrification ability, optimum pH is preferably in any value of 6 or 7 or 8 or 9；
Referring to Fig. 7, it is wider, in the equal energy of initial pH value 5.0-9.0 scope that test shows that bacterial strain MCW153 adapts to pH value range
Interior energy well grows.
Referring to Fig. 8, test shows carbon source (glucose, sodium acetate, sodium succinate, sodium oxalate, sodium citrate etc.) to microbial inoculum
Fermentation has large effect, and wherein, glucose is that nitrate nitrogen removal effect during carbon source is optimal, and therefore, glucose is bacterial strain
The most suitable carbon source of MCW153.
Acceptable after the completion of fermentation：Nutrient solution is directly dispensed with plastic barrel, or makes solid using charcoal absorption
Microbial inoculum.
Microbial inoculum is obtained by preceding method there is reliable biological safety.
Referring to Figure 10, biological safety test (thalline feeds goldfish) shows, vibrios MCW153 bacterial strain, safe and harmless, bacterium
Body feeds goldfish 3 months, and goldfish upgrowth situation is good, and is better than control group.
The process that microbial inoculum is used for marine culture wastewater is obtained by preceding method, sewage water denitrification clean-up effect is notable.
Marine culture wastewater is applied to by the vibrios microbial inoculum with dirty seawater denitrification ability that foregoing embodiments are obtained
Process, and constantly Measures program prepared by further microbial inoculum, can make the manufacturing cost more cheap of microbial inoculum, the industry of being relatively beneficial to pushes away
Wide apply, have following tests can illustrate that dirty microbial inoculum processes the excellent effect of marine culture wastewater only for this case dirt seawater denitrogenation.
Laboratory shake flask is tested, referring to Fig. 9：
MCW153 inoculation in nitrate nitrogen (NO3 -- N) initial concentration be 150 mg/litre artificial seawater cultivation useless
In water, 30 DEG C, 160 revs/min of shaking table cultures, timing sampling measures thalli growth, nitrate nitrogen (NO3 -- N) and nitrite nitrogen
(NO2 -- N) concentration levels.
The result of Fig. 9 shows, MCW153 can be by nitrate nitrogen (NO in 12 hours3 -- N) concentration is more than 150 mg/litre
It is down to below 0.05 mg/litre, meanwhile, nitrite nitrogen (NO2 -- N) concentration be first stepped up, then gradually reduce to 0.05
Below mg/litre, illustrate that MCW153 bacterial strain has efficient denitrification denitrogenation ability, artificial seawater breeding wastewater is had preferably
Denitrogenation clean-up effect.
In addition, by Fig. 9 it is also shown that the growth of MCW153 bacterial strain is synchronized with nitrate nitrogen (NO3 -- N) concentration reduction, and
The growth curve of MCW153 bacterial strain, nitrite nitrogen (NO2 -- N) metabolic chart in lag phase substantially all in 0-3 hour, so
Enter logarithmic phase (4-15 hour) afterwards, about reach highest thalli growth amount at 24 hours.Concentration unit " mg/litre " also illustrates that
For " mg/L ".Waste water after seawater utilization is the marine culture wastewater containing nitrogen source pollutant.
This case has vibrios of dirty seawater denitrification ability and application thereof, the vibrios MCW153 in the present invention, not only has relatively
Good denitrification ability, can effectively remove nitrate nitrogen and nitrite nitrogen, and safe and harmless, and biological safety test shows
(thalline feeds goldfish 3 months), goldfish upgrowth situation is good, and is better than control group.Vibrios MCW153 can be used for making further
Make biological denitrogenation microbial agent, seawater aquaculture system total nitrogen is exceeded to wait eutrophication problem particularly with solving, and has good
Application prospect.
The above, be only presently preferred embodiments of the present invention, and not the present invention is made with any pro forma restriction, all
It is any simple modification, equivalent variations and modification above example made according to the technical spirit of the present invention, all still fall within
In the range of technical solution of the present invention.
Claims (10)
1. a kind of preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability, the method has inoculation in culture medium
In, the step of culture bacterial classification, wherein, inoculation is that this vibrios test tube kind is inoculated in beef-protein medium, training
Foster temperature is 20 DEG C -40 DEG C, hunting speed is 120-180 rev/min, and shake flask is cultivated to logarithmic phase；
The described vibrios with dirty seawater denitrification ability, is preserved in China Committee for Culture Collection of Microorganisms's commonly micro- life
Thing center, it is CGMCC No.12311 that culture presevation is numbered.
2. there is the preparation method of the vibrios microbial inoculum of dirty seawater denitrification ability as claimed in claim 1, also, will be cultured
The step that bacterial classification is linked into fermentation tank culture medium：By cultured bacterial classification, by 0.5%-1% or 1%-2% of culture medium weight
Inoculum concentration be linked in the fermentation tank of preset volume, have the fermentation tank culture medium of predetermined amount in fermentation tank, cultivate to logarithm life
For a long time.
3. there is the preparation method of the vibrios microbial inoculum of dirty seawater denitrification ability, also, fermented and cultured as claimed in claim 2
Step：After bacterial classification is linked into fermentation tank, the throughput of filtrated air is 10-30 liter/min, or/and, mixing speed is 120 turns/
- 200 revs/min of minute, or/and, cultivation temperature is 25 DEG C -40 DEG C, or/and, whole process incubation time is 24-48 hour, or/
With, nutrient solution packing after the completion of fermentation, or adsorbed using aseptic attachment, be dried, make solid fungicide.
4. the preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability as described in one of claim 1-3, wherein, by bacterium
Strain is inoculated in culture medium, the step of culture bacterial classification, and wherein, described beef-protein medium has：Peptone 1%,
Beef extract 0.3%, sodium chloride 0.5%, pH 7.0, balance of additive.
5. the preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability as described in one of claim 2-4, wherein, will train
The step that the bacterial classification supported is linked into fermentation tank culture medium：Described fermentation tank culture based component has：Na2HPO4、KH2PO4、
KNO3, glucose and seawater；When seawater amount is 1000 milliliters, Na2HPO4For 7.9 grams, KH2PO4For 1.5 grams, KNO3For 1.0
Gram, glucose is 9.4 grams, and fermentation tank culture medium pH value is 7.0-7.5.
6. the preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability as described in one of claim 1-5, wherein, by bacterium
Strain is inoculated in culture medium, the step of culture bacterial classification, and wherein, cultivation temperature is 20 DEG C -25 DEG C or 26 DEG C -30 DEG C or 31 DEG C -35
DEG C or 36 DEG C -37 DEG C or 38-40 DEG C of arbitrary value range.
7. the preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability as described in one of claim 1-6, wherein, by bacterium
Strain is inoculated in culture medium, the step of culture bacterial classification, and wherein, hunting speed is 120-150 rev/min or 151-160 rev/min
Or 161-170 rev/min or 171-180 rev/min of arbitrary value range.
8. the preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability as described in one of claim 3-7, wherein, fermentation
The step of culture：Bacterial classification is linked into fermentation tank after fermentation culture, and filtrated air throughput is preferably 10 liters/min or 20 liters/min
Any value of clock or 30 liters/min；Or/and, mixing speed is preferably 60-90 rev/min or 91-120 rev/min of arbitrary model
Enclose value；Or/and, cultivation temperature is preferably 25 DEG C -30 DEG C or 31 DEG C -35 DEG C or 36 DEG C -40 DEG C of arbitrary value range；Or/and, institute
State whole process incubation time and be preferably 24-30 hour or 31-36 hour or 37-42 hour or the arbitrary value range of 43-48 hour；
Or/and, described dirt seawater denitrogenation dirty bacterium only, suitable pH value is in the arbitrary model of 5.0-6.0 or 6.1-7.0 or 7.1-8.0 or 8.1-9.0
Enclose value；Or/and, the suitable carbon source of described dirt seawater denitrogenation bacteria strain be glucose or sodium acetate or sodium succinate or sodium oxalate or
Sodium citrate any one.
9. as the vibrios microbial inoculum with dirty seawater denitrification ability of one of claim 1-8 methods described preparation, wherein, described tool
The vibrios having dirty seawater denitrification ability is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and bacterial classification is protected
Hiding numbering is CGMCC No.12311.
10. there is the vibrios microbial inoculum of dirty seawater denitrification ability as claimed in claim 9, wherein, described have dirty seawater denitrogenation
The vibrios of ability, suitable carbon source be glucose or sodium acetate or sodium succinate or sodium oxalate or sodium citrate any one；Or/and,
The G+C content of the described vibrios DNA with dirty seawater denitrification ability is 44.3mol%；Described " G+C content " refers to " DNA molecular
Mole percent ratio shared by middle guanine (G) and cytimidine (C) "；Or/and, the temperature of suitable growth is 20 DEG C -25 DEG C or 26
DEG C -30 DEG C or 31 DEG C -35 DEG C or 36 DEG C -37 DEG C or 38-40 DEG C of arbitrary value range；Or/and, described have dirty seawater denitrogenation energy
The vibrios of power, suitable pH value is in the arbitrary value range of 5.0-6.0 or 6.1-7.0 or 7.1-8.0 or 8.1-9.0.
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