CN106434424B - Vibrios and application thereof with dirty seawater denitrification ability - Google Patents
Vibrios and application thereof with dirty seawater denitrification ability Download PDFInfo
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- CN106434424B CN106434424B CN201610723504.5A CN201610723504A CN106434424B CN 106434424 B CN106434424 B CN 106434424B CN 201610723504 A CN201610723504 A CN 201610723504A CN 106434424 B CN106434424 B CN 106434424B
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Abstract
One plant of vibrios and application thereof with dirty seawater denitrification ability, the present invention relates to the processing technology field of dirty seawater, which is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and culture presevation number is CGMCC No.12311.For dirty microbial inoculum for marine culture wastewater processing, industrial application prospect is good only in order microorganism is made with cholerae strain for the purposes of dirty seawater denitrogenation vibrios.
Description
Technical field
The present invention relates to the processing technology fields of dirty seawater, more particularly to a kind of arc with dirty seawater denitrification ability
The purposes of bacterium and the vibrios, and the method for preparing biological denitrogenation microbial agent with the vibrios.
Background technique
Currently, about 75%~80% nitrogen can enter in breeding water body in feed in highdensity cultivation system, and with
Ammonia nitrogen, nitrate nitrogen, nitrite nitrogen form there is different degrees of accumulation, cause damages to aquiculture animal, thus
The specific yield of sea-farming, and also nitrogenous compound rich in the waste water of its discharge are limited, 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 one kind can carry out denitrifying bacterium under aerobic condition.Its presence makes
Nitrifying process is able to the synchronous progress in the same reactor with denitrification process, and it is anti-that the product of nitration reaction can become denitrification
The substrate answered, accelerates reaction speed, reduces operating cost, while the OH of anti-nitration reaction release-Nitration reaction can be compensated
The alkali of consumption avoids the accumulation of acidic materials, maintains the stabilization of system pH value.
Known technology, such as the cold-resistant pseudomonad Den-05 of 201210152921.0 1 plants of published 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 realizes the preparation of biological denitrogenation microbial agent, provides for biology fortification system
Efficient denitrification strain, and the control methods by shortening anaerobism period shortening aeration period, improve the municipal wastewater treatment plant winter
Season denitrification effect, realize the removal of nitrate nitrogen under aerobic condition.
In addition, 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, to protect environment.
In addition, the method for biologically treating wastewater of a kind of desulfurization simultaneously of Chinese patent 200910213798.7, denitrogenation, decoloration,
Disclose the method for biologically treating wastewater of a kind of while desulfurization, denitrogenation, decoloration.
In addition, the salt cholerae strain of 201210171297.9 1 plant height salt biological denitrificaion of Chinese patent and its in wastewater treatment
In application.
In addition, the polyculture enrichment screening method of 201110288612.1 ocean heterotrophic denitrifying bacteria of Chinese patent application.
The industry urgently more diversified vibrios with dirty seawater denitrification ability, to solve in dirty seawater living intensive treatment
There are the problem of.
Summary of the invention
The problem to be solved by the present invention is that a kind of vibrios and application thereof with dirty seawater denitrification ability is provided, and
Using the net dirty microbial inoculum of vibrios preparation and the preparation method of the microbial inoculum.
This case first purpose is to provide a kind of vibrios with dirty seawater denitrification ability, and this case is also known as dirty seawater denitrogenation arc
Bacterium.
There is the present invention vibrios of dirty seawater denitrification ability to take following technical scheme to realize:
One plant of vibrios with dirty seawater denitrification ability, it is commonly micro- to be preserved in China Committee for Culture Collection of Microorganisms
Bio-Centers, culture presevation number is CGMCC No.12311.
There is the vibrios of dirty seawater denitrification ability can also be further realized using following technical measures for this case:
The vibrios with dirty seawater denitrification ability above-mentioned, wherein the bacterial strain 16S rDNA sequence are as follows:
This case further object is to provide a kind of aforementioned vibrios purposes.
The purposes of the vibrios with dirty seawater denitrification ability above-mentioned, wherein by microorganism formulation made of the bacterial strain, use
Wastewater treatment after seawater utilization, the waste water after the seawater utilization are the marine culture wastewater containing nitrogen source pollutant.
The purposes of the vibrios with dirty seawater denitrification ability above-mentioned, wherein used after microorganism formulation is made in the bacterial strain
Wastewater treatment after seawater utilization, the waste water after the seawater utilization, nitrogen source pollutant therein, with nitrate nitrogen (NO3 --
N it) counts, initial concentration is 150 mg/litres.
The purposes of the vibrios with dirty seawater denitrification ability above-mentioned, wherein used after microorganism formulation is made in the bacterial strain
Wastewater treatment after seawater utilization, wherein having the suitable carbon source of the cholerae strain of dirty seawater denitrification ability is glucose or second
Sour sodium or sodium succinate or sodium oxalate or sodium citrate are any.
The purposes of the vibrios with dirty seawater denitrification ability above-mentioned, wherein after microorganism formulation made of the bacterial strain
For the wastewater treatment after seawater utilization, wherein have the suitable pH value of the cholerae strain of dirty seawater denitrification ability in 5.0-6.0
Or any value range of 6.1-7.0 or 7.1-8.0 or 8.1-9.0.
The purposes of the vibrios with dirty seawater denitrification ability above-mentioned, wherein used after microorganism formulation is made in the bacterial strain
Wastewater treatment after seawater utilization, wherein have the suitable pH value of the cholerae strain of dirty seawater denitrification ability 5.0 or 6.0
Or 7.0 or 8.0 or 9.0 any value.
Having microorganism made of the cholerae strain of dirty seawater denitrification ability as a result, dirty microbial inoculum is used for marine culture wastewater only
Processing is handled the time only 24 hours, and sewage water denitrification clean-up effect is with regard to highly significant, and microbial inoculum has reliable bio-safety
Property.
This case further object is to provide a kind of microbial inoculum of vibrios with dirty seawater denitrification ability.
A kind of vibrios microbial inoculum with dirty seawater denitrification ability, wherein the vibrios with dirty seawater denitrification ability protects
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center, culture presevation number is CGMCC No.12311.
The aforementioned vibrios microbial inoculum with dirty seawater denitrification ability, wherein the vibrios with dirty seawater denitrification ability is fitted
Closing carbon source is that glucose or sodium acetate or sodium succinate or sodium oxalate or sodium citrate are any；
The vibrios microbial inoculum with dirty seawater denitrification ability above-mentioned, wherein the vibrios with dirty seawater denitrification ability
The G+C content of DNA is 44.3mol%；" the G+C content " refers to " in DNA molecular shared by guanine (G) and cytimidine (C)
Mole percent ratio ".
The vibrios microbial inoculum with dirty seawater denitrification ability above-mentioned, wherein the temperature of suitable growth is 20 DEG C -25 DEG C or 25
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 any value range.
The aforementioned vibrios microbial inoculum with dirty seawater denitrification ability, wherein the vibrios with dirty seawater denitrification ability is fitted
PH value is closed in any 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 bacterial preparation process of vibrios with dirty seawater denitrification ability.
A kind of preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability, this method have strain inoculated in culture
In base, cultivate strain the step of, wherein strain inoculated is that the vibrios test tube species are inoculated in beef-protein medium,
Cultivation temperature is 20 DEG C -40 DEG C, hunting speed is 120-180 revs/min, shake flask culture to logarithmic phase；
The vibrios for having dirty seawater denitrification ability, it is common to be preserved in China Committee for Culture Collection of Microorganisms
Microorganism center, culture presevation number is CGMCC No.12311.
The preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability above-mentioned, in addition, cultured strain is accessed
The step of to fermentation tank culture medium: by cultured strain, by the inoculum concentration of the 0.5%-1% or 1%-2% of culture medium weight
It is linked into the fermentor of preset volume, there is the fermentation tank culture medium of predetermined amount, culture to logarithmic growth phase in fermentor.
The preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability above-mentioned, in addition, the step of fermented and cultured: strain
After being linked into fermentor, the ventilatory capacity of filtrated air is 10-30 liters/min；
The preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability above-mentioned, mixing speed are 120 rev/min -200
Rev/min, or/and, cultivation temperature is 25 DEG C -40 DEG C；
The preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability above-mentioned, whole process incubation time are that 24-48 is small
When；
The preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability above-mentioned, culture solution dispenses after fermentation, or
Using the absorption of sterile attachment, drying, solid fungicide is made.
The preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability above-mentioned, wherein by strain inoculated in culture medium
In, cultivate strain the step of, wherein the beef-protein medium includes peptone 1%, beef extract 0.3%, chlorine
Change sodium 0.5%, pH 7.0, surplus is additive.
The preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability above-mentioned, wherein access cultured strain
The step of to fermentation tank culture medium: the fermentation tank culture based component includes Na2HPO4、KH2PO4、KNO3, glucose and seawater；
When seawater amount is 1000mL, Na2HPO4For 7.9g, KH2PO4For 1.5g, KNO3For 1.0g, glucose 9.4g, fermentor training
Supporting base pH value is 7.0-7.5.
The preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability above-mentioned, wherein by strain inoculated in culture medium
In, cultivate strain the step of, wherein cultivation temperature be 20 DEG C -25 DEG C or 25 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 any value range.
The preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability above-mentioned, wherein by strain inoculated in culture medium
In, cultivate strain the step of, wherein hunting speed be 120-150 revs/min or 151-160 revs/min or 160-170 revs/min
Clock or 171-180 revs/min of any value range.
The preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability above-mentioned, wherein the step of fermented and cultured: strain
It is linked into fermentor post-fermentation culture, filtrated air ventilatory capacity is preferably 10 liters/min or 20 liters/min or 30 liters/min
Any value；
The preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability above-mentioned, mixing speed is preferably 60-90 revs/min
Clock or 91-120 revs/min of any value range；
The preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability above-mentioned, 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 any value range；
The preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability above-mentioned, the whole process incubation time are preferably
24-30 hours or 31-36 hours or 37-42 hours or 43-48 hours any value ranges；
The preparation method of the vibrios microbial inoculum with dirty seawater denitrification ability above-mentioned, the dirt seawater denitrogenation dirty bacterium only, is fitted
PH value is closed in any 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 vibrios microbial inoculum with dirty seawater denitrification ability above-mentioned, the dirt seawater denitrogenation bacteria strain are fitted
Closing carbon source is that glucose or sodium acetate or sodium succinate or sodium oxalate or sodium citrate are any.
The present invention has significant advantages and beneficial effects compared with prior art.From the above technical scheme, this hair
It is bright under excellent structure configuration, at least following advantage:
This case has a vibrios and application thereof of dirty seawater denitrification ability, the vibrios MCW153 in the present invention, not only have compared with
Good denitrification ability, can effectively remove nitrate nitrogen and nitrite nitrogen, and safe and harmless, biological safety experiments have shown that
(thallus feeds goldfish 3 months), goldfish upgrowth situation is good, and is better than control group.Vibrios MCW153 is for making biological denitrificaion
Microbial inoculum, equal eutrophication problems exceeded for solution seawater aquaculture system total nitrogen, has the application of good industry and market
Prospect.
This case dirt seawater denitrifier is successfully separated the problem that can solve marine culture wastewater processing long felt, this
Case dirt seawater denitrifier separation method is simple, and separation and cultural method are relatively easy to promote, and with the development of culture technique, it can
Using using many organic matters (including fishes and shrimps excreta, food-residue etc.) as its carbon source, therefore, this case dirt seawater denitrifier makees
For the biomaterial of the efficiently net dirty microbial bacterial agent of dirty seawater, it is worth with excellent industry promotion and application.
There is the microbial inoculum of the vibrios preparation of dirty seawater denitrification ability by this case, the manufacturing cost more cheap of microbial inoculum can be made, more
It is beneficial to industry popularization and application；The microbial inoculum obtained by the bacterial preparation process of this case is especially able to satisfy the purification of marine culture wastewater
The practical problem and demand of processing are capable of the biological cleaning ability of the newly-built saline sewage treatment facility of quick start using this microbial inoculum
With the strengthened denitrification processing capacity for the biosystem for restoring to deteriorate.
One plant of vibrios MCW153 with dirty seawater denitrification ability of this case has good total nitrogen removal effect, and laboratory is shaken
Bottle test the result shows that, manual simulation's waste water nitrogen removal rate is higher.
As 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 cannot effectively handle the case where marine culture wastewater (low-carbon high-nitrogen), using the biological reinforced skill of this case
Art is substantially shorter the sludge acclimatization time, significantly improves denitrogenation by adding the microbial inoculum of this case vibrios into sewage treatment facility
Treatment effect, can effectively reduce investment outlay and operating cost, to solve to take off present in current marine culture wastewater biological treatment
The problems in the urgent need to address such as nitrogen effect difference and sludge acclimatization period are too long.
The present invention compares the prior art significant contribution and a progress, strictly with novelty, creativeness, practicability
Good technology.
Detailed description of the invention
Fig. 1 is vibrios thallus 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 revolving 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 ventilatory capacity schematic diagram of vibrios of the present invention；
Fig. 6 is the best fermentation time schematic diagram of vibrios of the present invention；
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 denitrification effect schematic diagram of the microbial inoculum of the present invention to artificial seawater breeding wastewater；
Figure 10 is the biological safety test of one plant of this case vibrios MCW153 with dirty seawater denitrification ability.
Specific embodiment
A specific embodiment of the invention is shown in detail by following embodiment.
In conjunction with the preferred embodiment, to specific embodiment, step, feature and its effect provided according to the present invention,
Detailed description are as follows.
Embodiment 1
A kind of vibrios with dirty seawater denitrification ability, this case are also referred to as dirty seawater denitrifier, and it is micro- which is deposited in China
Biological inoculum preservation administration committee common micro-organisms center, culture presevation number is CGMCC No.12311.
The G+C content for having the vibrios DNA of dirty seawater denitrification ability is 44.3mol%.With dirty seawater denitrogenation
The vibrios of ability, Gram-negative (G-), cell ellipse is moved with polar flagella.With dirty seawater denitrification ability
Vibrios, oxidizing ferment, catalase, nitrate any reduction reaction be the positive.Vibrios bacterium with dirty seawater denitrification ability
Strain is carbon source using glucose, sodium acetate, sodium succinate etc..Cholerae strain with dirty seawater denitrification ability using glucose and
Other (galactolipin, arabinose, maltose etc.) some carbohydrates produce acid.The suitable pH value of dirty seawater denitrifier is fitted between 5-9
Suitable growth temperature is 30-35 DEG C.
One plant of vibrios MCW153 with dirty seawater denitrification ability of this case has good total nitrogen removal effect, and laboratory is shaken
Bottle test the result shows that, manual simulation's waste water nitrogen removal rate is higher.
Embodiment 2
There is the net dirty bacterial preparation process of the vibrios of dirty seawater denitrification ability, including following technique mistake described in embodiment 1
Journey:
Step 1: will there are the vibrios test tube species of dirty seawater denitrification ability to be inoculated in beef-protein medium, cultivate
Temperature is 30 DEG C, hunting speed is 160 revs/min, shake flask culture to logarithmic phase；Beef-protein medium is commercially available
Culture medium.
Step 2: above-mentioned cultured strain is linked into fermentor by the inoculum concentration of culture medium weight 0.5%-2%,
There is culture medium in fermentor；Culture medium used in fermentor includes Na2HPO47.9 grams, KH2PO41.5 grams, KNO31.0 grams, grape
9.4 grams, 1000 milliliters of seawater of sugar.
Step 3: in fermentor production process, the ventilatory capacity of filtrated air is 10-30 liters/min, mixing speed 160
Rev/min, cultivation temperature is 30 DEG C, and whole process incubation time is 24 hours, and culture solution dispenses after fermentation, or using sterile
Attachment absorption, drying, are made solid fungicide.
It can be seen that having the vibrios separation method of dirty seawater denitrification ability simple, separation and culture are relatively easy to, with
The development of culture technique, it can use many organic matters (including fishes and shrimps excreta, food-residue etc.) as its carbon source.
Vibrios by having dirty seawater denitrification ability prepares microbial inoculum, and manufacturing cost more cheap is relatively beneficial to industry popularization and answers
With；
The bacteria agent that there is the vibrios bacterial preparation process of dirty seawater denitrification ability to obtain as a result, is especially able to satisfy sea
The practical problem and demand of the purified treatment of water breeding wastewater, using this microbial inoculum can quick start create saline sewage processing set
The strengthened denitrification processing capacity for the biosystem that the biological cleaning ability and recovery applied deteriorate.
Embodiment 3
A kind of purposes of the vibrios with dirty seawater denitrification ability is that dirty microbial inoculum is used for sea to microorganism made of vibrios strain only
Water cultivating wastewater purification, processing time are 24 hours, and sewage water denitrification clean-up effect is significant.
Embodiment 4
Referring to attached drawing 1 to shown in Figure 10, this case is related to component reference weight percent, under do not repeat.
One plant of vibrios MCW153 with dirty seawater denitrification ability, the biomaterial are preserved in Chinese microorganism strain preservation
Administration committee's common micro-organisms center, culture presevation number is CGMCC No.12311, 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, there is the vibrios MCW153 of dirty seawater denitrification ability to be also known as dirty seawater denitrifier, be described in detail below:
The dirt seawater denitrifier is identified as vibrio (Vibrio sp.MCW153), and Main Biological is gram
Feminine gender (G-), cell is oval, amphitrichous, and 0.8 × 1.2 micron of thallus size；3 days bacterium are cultivated on LB culture medium flat plate
Fall as cream-coloured, flattened round, surface is slight graininess is wet, edge slight fold, diameter is at 2.5 millimeters or so.
The vibrios that there is dirty seawater denitrification ability, oxidizing ferment, catalase, nitrate any reduction reaction be
It is positive；Its urease test, indole reaction are feminine gender；Utilize some carbohydrates such as glucose, galactolipin, arabinose, maltose
Produce acid.Referring to dirty seawater denitrogenation dirty bacterium MCW153 physiological and biochemical property table only.The vibrios for having dirty seawater denitrification ability is suitable
Suitable pH value is between 5-9, and preferably suitable growth temperature is 30 DEG C.The G+C of the vibrios DNA that there is dirty seawater denitrification ability
Content is 44.3mol%；Wherein, " G+C content " is often referred to " in DNA molecular shared by guanine (G) and cytimidine (C)
Mole percent ratio ".
Dirty seawater denitrifier MCW153 physiological and biochemical property
The Genbank number of logging in of bacterial strain 16S rDNA is KU644134.
Polymerase chain reaction (PCR) amplification, polymerase chain reaction are carried out to isolated bacterial strain 16S rDNA sequence
(PCR) product is answered to serve the raw work sequencing in sea, base sequence is as follows:
The bacterial strain 16S rDNA sequence of dirty seawater denitrogenation vibrios are as follows:
NCBI the sequencing results show 16S rDNA sequence and Vibrio sp. (vibrios) multiple bacterium of MCW153 bacterial strain
The sequence homology of strain is 99% or more.
The preparation method of vibrios microbial inoculum with dirty seawater denitrification ability, including following technical process:
Step 1: will there are the vibrios test tube species of dirty seawater denitrification ability to be inoculated in beef-protein medium, cultivate
Temperature is 25 DEG C -40 DEG C, hunting speed is 120-180 revs/min, shake flask culture to logarithmic phase；Beef extract-peptone training
Feeding base can be general commercially available product；
Step 1 further embodiment is:
Preferred beef-protein medium includes peptone 1%, beef extract 0.3%, sodium chloride 0.5%, pH
7.0, surplus is that it will not go into details for usual additive；
The vibrios for having dirty seawater denitrification ability is preserved in Chinese microorganism strain preservation conservator to be above-mentioned
The strain of meeting common micro-organisms center, referring to Fig. 1, deposit number is CGMCC No.12311；
The vibrios test tube species for having dirty seawater denitrification ability are inoculated in the culture temperature in beef-protein medium
Any value range that 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 degree；
Referring to fig. 2, experiments have shown that, 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 vibrios test tube species for having dirty seawater denitrification ability are inoculated in the shaking flask in beef-protein medium
The speed of oscillation is preferably 120-150 revs/min or 151-160 revs/min or 161-170 revs/min or 171-180 revs/min
Any value range of clock, mixing speed rev/min also are indicated as r/min；
Referring to Fig. 3, experiments have shown that influence of the shaking speed to strain growth, 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 excessively high revolving speed is easy to overflow culture solution, therefore, fits
Suitable strain culturing revolving speed is 120-180 revs/min, and optimal revolving speed is 160 revs/min.
Step 2: by above-mentioned cultured strain, being linked into default appearance by the inoculum concentration of the 0.5%-2% of culture medium weight
There is the culture medium of predetermined amount in long-pending fermentor, in fermentor, such as by above-mentioned cultured strain, is accessed by 1% inoculum concentration
Into the fermentor of preset volume amount, such as 10 liters of fermentors, culture to logarithmic growth phase；
Step 2 further embodiment is:
Culture medium used in preferred fermentor includes Na2HPO47.9 gram, KH2PO41.5 grams, KNO31.0 grams, glucose
9.4 grams, pH 7.0-7.5,1000 milliliters of seawater；
The inoculum concentration of vibrios for having dirty seawater denitrification ability and the weight percent of culture medium are preferably 1%-
Any value range of 1.5% or 1.5%-2%；
Referring to fig. 4, this experiments have shown that vibrios with dirty seawater denitrification ability influence of the inoculum concentration to microbial inoculum is prepared.
After when culture 24 is small, as inoculum concentration increases, less, therefore, the suitable inoculum concentration of bacteria fermentation exists the variation of microbial inoculum increment
1-2%, but cost factor is combined, select 1% inoculum concentration to can effectively reduce cost；
Step 3: in fermentor production process, the ventilatory capacity of filtrated air is 10-30 liters/min, mixing speed 120
Revs/min -200 revs/min, cultivation temperature is 25 DEG C -40 DEG C, and whole process incubation time is 24-48 hours, is trained after fermentation
Nutrient solution packing, or using the absorption of sterile attachment, drying, solid fungicide is made.
Ventilatory capacity liter/min also is indicated as L/min.
Step 3 further embodiment is:
The filtrated air ventilatory capacity is preferably any value range of 10 liters/min or 20 liters/min or 30 liters/min；
Referring to Fig. 5, experiments have shown that ventilatory capacity is affected to strain fermentation, wherein ventilatory capacity is 10-30 liters/min
When, microbial inoculum well-grown.
Mixing speed is preferably 60-90 revs/min or 91-120 revs/min of any value range；
Cultivation temperature is preferably any value range of 25 DEG C -30 DEG C or 31 DEG C -35 DEG C or 36 DEG C -40 DEG C；
The whole process incubation time is preferably 24-30 hours or 31-36 hours or 37-42 hours or 43-48 small former
One value range；Hour also is indicated as h.
Referring to Fig. 6, experiments have shown that, with the increase of fermented incubation time, thallus is constantly grown, wherein fermentation time is
4-16 hours, microbial inoculum was in optimal growth conditions.
The culture medium of the fermentor 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 vibrios for having dirty seawater denitrification ability, is suitble to pH value preferably in 5.0-6.0 or 6.1-7.0 or 7.1-8.0
Or any value range of 8.1-9.0.
The vibrios that there is dirty seawater denitrification ability, optimum pH preferably 6 or 7 or 8 or 9 any value；
Referring to Fig. 7, experiments have shown that bacterial strain MCW153 adaptation pH value range is wider, in the equal energy of initial pH value 5.0-9.0 range
It is interior well to grow.
Referring to Fig. 8, experiments have shown that carbon source (glucose, sodium acetate, sodium succinate, sodium oxalate, sodium citrate etc.) is to microbial inoculum
Fermentation has large effect, wherein nitrate nitrogen removal effect when glucose is carbon source is best, and therefore, glucose is bacterial strain
The most suitable carbon source of MCW153.
After fermentation can be with: culture solution be directly dispensed with plastic barrel, or solid is made using activated carbon adsorption
Microbial inoculum.
Microbial inoculum is made with reliable biological safety by preceding method.
Referring to Figure 10, biological safety test (thallus 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.
Processing of the microbial inoculum for marine culture wastewater is made by preceding method, sewage water denitrification clean-up effect is significant.
There is the vibrios microbial inoculum of dirty seawater denitrification ability to be applied to marine culture wastewater as made from foregoing embodiments
Processing, and constantly further microbial inoculum prepares Measures program, can make the manufacturing cost more cheap of microbial inoculum, the industry of being relatively beneficial to pushes away
Wide application has following tests that can illustrate the excellent effect of this case dirt seawater denitrogenation dirty microbial inoculum processing marine culture wastewater only.
Laboratory shake flask test, referring to Fig. 9:
MCW153 strain inoculated in nitrate nitrogen (NO3 -- N) initial concentration be 150 mg/litres artificial seawater cultivation it is useless
In water, 30 DEG C, 160 revs/min of shaking table cultures, timing sampling measure thalli growth, nitrate nitrogen (NO3 -- N) and nitrite nitrogen
(NO2 -- N) concentration levels.
Fig. 9's the result shows that, MCW153 can be in 12 hours by nitrate nitrogen (NO3 -- N) concentration is more than 150 mg/litres
0.05 mg/litre is down to hereinafter, simultaneously, nitrite nitrogen (NO2 -- N) concentration be first stepped up, then gradually reduce to 0.05
Mg/litre has artificial seawater breeding wastewater preferable hereinafter, illustrating that MCW153 bacterial strain has efficient denitrification denitrogenation ability
Denitrogenation clean-up effect.
In addition, being also shown that the growth of MCW153 bacterial strain is synchronized with nitrate nitrogen (NO by Fig. 93 -- N) concentration reduction, and
Growth curve, the nitrite nitrogen (NO of MCW153 bacterial strain2 -- N) metabolic chart in lag phase substantially all at 0-3 hours, so
Enter logarithmic phase (4-15 hours) afterwards, highest thalli growth amount was about reached 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 a vibrios and application thereof of dirty seawater denitrification ability, the vibrios MCW153 in the present invention, not only have compared with
Good denitrification ability, can effectively remove nitrate nitrogen and nitrite nitrogen, and safe and harmless, biological safety experiments have shown that
(thallus feeds goldfish 3 months), goldfish upgrowth situation is good, and is better than control group.Vibrios MCW153 may further be used to make
Make biological denitrogenation microbial agent, particularly with the exceeded equal eutrophication problems of solution seawater aquaculture system total nitrogen, has good
Application prospect.
The above described is only a preferred embodiment of the present invention, be not intended to limit the present invention in any form, it is all
It is any simple modification, equivalent change and modification to the above embodiments according to the technical essence of the invention, still falls within
In the range of technical solution of the present invention.
Claims (4)
1. one plant of vibrios with dirty seawater denitrification ability, is preserved in China Committee for Culture Collection of Microorganisms's commonly micro- life
Object center, culture presevation number is CGMCCNo.12311；
The bacterial strain 16SrDNA sequence are as follows:
The G+C content of the vibrios DNA for having dirty seawater denitrification ability is 44.3mol%；The G+C content refers to DNA points
Mole percent ratio shared by guanine and cytimidine in son.
2. having the purposes of the vibrios of dirty seawater denitrification ability as described in claim 1, which is characterized in that the bacterial strain is made micro-
Biological agent, for the wastewater treatment after seawater utilization, the waste water after the seawater utilization is the seawater containing nitrogen source pollutant
Breeding wastewater；
The nitrogen source pollutant, with nitrate nitrogen (NO3 -- N) meter, initial concentration is 150 mg/litres；
In the microorganism formulation, have the suitable pH value of the cholerae strain of dirty seawater denitrification ability in 5.0-6.0 or 6.1-7.0
Or any value range of 7.1-8.0 or 8.1-9.0.
3. the purposes of the vibrios with dirty seawater denitrification ability according to claim 2, which is characterized in that by the bacterial strain system
At microorganism formulation after for the wastewater treatment after seawater utilization, wherein having the suitable of the cholerae strain of dirty seawater denitrification ability
Closing carbon source is that glucose or sodium acetate or sodium succinate or sodium oxalate or sodium citrate are any.
4. the purposes of the vibrios with dirty seawater denitrification ability according to claim 2, which is characterized in that by the bacterial strain system
At microorganism formulation after for the wastewater treatment after seawater utilization, wherein there is the cholerae strain of dirty seawater denitrification ability
It is suitble to pH value in 5.0 or 6.0 or 7.0 or 8.0 or 9.0 any values.
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