CN106085906A - A kind of Grouper cultivating sewage improvement complex microorganism preparations and preparation method thereof - Google Patents
A kind of Grouper cultivating sewage improvement complex microorganism preparations and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of Grouper cultivating sewage improvement complex microorganism preparations and preparation method thereof, containing weight ratio in described preparation is the bacillus subtilis of 2:2～4:1～2:1～2, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria.And the viable bacteria amount of bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria is respectively as follows: 6.0 × 10 in the preparation (including complex microorganism powderous preparations and complex microorganism liquid preparation) finally prepared9～9.0 × 1010CFU/ml、6.0×109～9.0 × 1010CFU/ml、3.0×108～9.0 × 109CFU/ml and 3.0 × 107～9.0 × 109CFU/ml.This complex microorganism preparations viable bacteria content is high, moisture is low, and stability is high, it is possible to effectively absorb the harmful substances such as the ammonia nitrogen in breeding water body and nitrite nitrogen, it is possible to effectively improve sea-farming sewage.
Description
Technical field
The invention belongs to microbial technology field, relate to a kind of microorganism formulation, be specifically related to Grouper cultivating sewage and change
Good complex microorganism preparations, also relates to the preparation method of complex microorganism preparations, including powderous preparations and liquid preparation
Preparation method.
Background technology
Along with socioeconomic development and the increase of the market demand, China's aquaculture industry is in fast development rank
Section.But, while aquatic products industry fast development, degenerating and water pollution day occurs in the breed variety that culture fishery causes
Becoming serious, particularly any discharge of high-density breeding, industrial wastewater and the sanitary sewage of culture zone, territory, coastal waters, ocean, makes cultivation
The self-cleaning in waters reduces with regulating power, and water environment deteriorates, and frequent occurrence, ecological balance and bio-diversity are destroyed.
The deteriorating water quality of Cultivated water not only causes serious pollution to environment, and has fatal threat to organism, to China
Fishery economic brings massive losses.Therefore process aquiculture sewerage and to reach discharge standard be the task of top priority.Reason
Saying on Lun, many conventional physics, chemical and biochemical method of wastewater treatment may be used for cultivating wastewater purification, but and live
Sewage is compared, and the waste water that sea water aquaculture produces has that two obvious features, the i.e. content of potential pollutant are low and the water yield
Greatly, the main component of pollutant, structure and the difference of common Lu Yuan sewage in seawater salinity effect and breeding wastewater, increase in addition
Add the intractability of sea water aquiculture waste water.
Cabrilla delicious meat, processing dressing percentage may be up to 67%, and suitable temperature range (18-32 DEG C) is wide, growth, suitable salt model
Enclosing (1-40 ‰) also wide, growth is fast, and resistance against diseases is strong, has become one of south China Main Economic cultivation fingerling.In recent years
Coming, the Fish high yield healthy breading system advocated has promoted the fast development of China's Grouper cultivating especially so that fish culture
Total output is climbed to a higher point successively, creates huge economic benefit and social benefit.But, in cabrilla production practices, still suffer from
Some problems.Producing substantial amounts of metabolite in Grouper cultivating production process, Excreta, remnants including cultivated animals raise
Material, plankton residuum etc..In China, the cultivation of cabrilla is the same to the cultivation of fish species with other, basic employing " three
Pond is unified ", i.e. the ingesting of cultured fishes, drain and the decomposition of metabolite completes in same pond.Every day in breeding production
Throwing something and feeding feedstuff, cultured fishes are ingested every day, and excretion every day, metabolite constantly accumulate, and improve water quality condition if irrational,
Self-pollution degree will gradually increase the weight of, and causes breeding water body Quality Down, and water quality factor changes, and aggravates sending out of all kinds of disease
Raw.Ammonia nitrogen and cultured water are the major pollutants in aquiculture waste water, are also the materials being difficult to most remove.Research at present is relatively
Many is the physical processing techniques such as foam separation, filtration.These physical treatment facilities have cost and the advantage such as operating cost is low,
But dissolubility pollutant can not be removed, particularly ammonia nitrogen can not be removed effectively.In recent years, people use for reference livestock and poultry cultivation warp
Test, attempt in breeding water body, use microorganism formulation and strengthen animal immunizing power, suppress pathogenic microorganism, reduce sending out of disease
Raw, improve breeding ecological environment, improve output of aquatic products and quality, achieve remarkable result.
As far back as 1981, the epoch after just having scholar to point out antibiotic will be the epoch of microorganism formulation, microorganism system
Agent is always domestic and international biology and the emphasis of medical domain research.Domestic and international market is prosperous to the demand of microorganism formulation at present
Containing, supply falls short of demand.China is large agricultural country, and aquaculture occupies sizable proportion, consumes substantial amounts of microorganism system every year
Agent, market prospect is the most vast.At present, the microorganism formulation product of advanced country the most all contains 5 sections 10 and belongs to 80 in the world
Remaining kind, and the viable count of its product is up to 1010Individual/gram.Since the eighties in 20th century, the research of China's microecological health-care food
With exploitation fast development, but no matter yet suffer from many problems from research field or application and need to solve.With regard to Aquatic product
For cultivation water improvement, first, the microorganism formulation of single culture can not reduce ammonia nitrogen and cultured water；Secondly, on the market
Sold complex microorganism preparations, because strain proportion compatibility is undesirable or viable count content is low, causes these products can not be abundant
Play different strain compatibility and reduce the effect of the aspect such as ammonia nitrogen and cultured water；Again, it is adaptable to the bacterium of aquatic microorganisms preparation
Planting and lack, screening technique is the simplest, it is adaptable to the microorganism formulation of sea water aquaculture of aquatic animal is the most few；Finally,
Aquatic microorganisms preparation stability is poor, and the processing Techniques of preserving of product has much room for improvement etc..
Summary of the invention
For situation about presently, there are, the present invention provides a kind of Grouper cultivating sewage improvement complex microorganism preparations,
Presently commercially available aquiculture sewerage can be overcome single or the shortcoming and defect of complex microorganism preparations.
Present invention also offers the preparation method of Grouper cultivating sewage improvement complex microorganism preparations, prepared preparation
Viable bacteria content is high, moisture is low, and stability is high.
To achieve these goals, the present invention provides following technical scheme:
A kind of Grouper cultivating sewage improvement complex microorganism preparations, containing weight ratio in described preparation is 2:2～4:1
～the bacillus subtilis of 2:1～2, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria.
Preferably, in aforementioned Grouper cultivating sewage improvement complex microorganism preparations, described bacillus subtilis, lichens
The weight ratio of bacillus cereus, denitrifying bacteria and lactic acid bacteria is 2:2:1:1.
Or, it is preferable that the weight ratio of described bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria
For 2:4:2:2.
The preparation method of aforementioned Grouper cultivating sewage improvement complex microorganism preparations, comprises the steps:
(1) bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria are taken respectively in respective culture medium
Cultivating activation, be separately added into 5ml sterilized water in often pipe bacterium, the bacteria suspension made, respectively as first order seed, the most respectively will
Cultured first order seed be by mass percentage 5～8% inoculum concentration transfer to containing 200ml liquid seed culture medium
In 1000ml triangular flask, then triangular flask is placed in shaking table, temperature be 28～37 DEG C, hunting speed be the condition of 250rpm
Lower shaken cultivation 16～20 hours, respectively obtain bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria two grades
Seed liquor, standby；
(2) compound, Xiang Ji based on wheat bran, soybean cake powder and the Semen arachidis hypogaeae dregs that mass ratio is 6～7:2～2.5:1～1.5
Plinth compound adds and accounts for glucose, 0.5～the CaCO of 2% that base mix mass percent is 5.0%3, 0.2～1%
MnSO4·H2The NaCl of O and 0.3～1% obtains solid butt；Then in solid butt, add the nothing of 1～2 times of weight
Bacterium water, pH value is adjusted to 6.5～7.2, again by its sterilizing 15～30min under 120～130 DEG C of hot conditionss after stirring, treats
It is cooled to room temperature, can be used as solid fermentation culture medium and use；
(3) by above-mentioned secondary seed solution be the most by mass percentage respectively 5～10% inoculum concentration be inoculated into above-mentioned solid
In fermentation medium, fully mix, in airtight solid-state fermentation tank, humidity be 28～37%, temperature be 36～37 DEG C of conditions
Bottom fermentation, fermentation time is 30～48 hours, obtains tunning；
(4) adding the corn starch of 15～20 times of weight in above-mentioned tunning, fully mix, temperature is 50～65 DEG C
Under the conditions of vacuum condensation be dried 8～15 hours, obtain being dried tunning, then pulverize, cross 40～60 mesh, obtain complex microorganism
Powderous preparations.
Wherein, described step (4) can also replace by following step:
Using collective media as diluent, tunning is diluted 2～200 times；Filter, it is ensured that viable bacteria content in filtrate
1.0 × 107～109CFU/ml, then filtrate is concentrated, use rotating speed be the centrifuge 1 of 10000rpm～2 minutes or
Use 4000～5000rpm centrifuge 10～30 minutes, collect centrifugal after precipitate, be dissolved in again outstanding for precipitate
In collective media, it is thus achieved that concentrated liquid, it is ensured that viable bacteria content is 1.0 × 109～1012CFU/ml, obtains complex microorganism liquid
Body preparation；
Described collective media is solid medium, and its formula is: bacteriological peptone 5g, yeast powder 5g, glucose 5g,
Dipotassium hydrogen phosphate 4g and agar 18g, its pH are 7.4；
Or fluid medium, its formula is: sodium carboxymethyl cellulose 20g, peptone 5g, yeast extract 5g, sodium chloride 5g and
Potassium dihydrogen phosphate 1g, its pH are 7.4.
Wherein, described bacillus subtilis and the culture medium prescription used by Bacillus licheniformis are: tryptone 10g,
Yeast powder 5g, sodium chloride 5g and agar powder 15g.It is dissolved in appropriate distilled water, at electric furnace after above-mentioned each reagent precise
Upper heated and boiled ensures that it is completely dissolved, and adds distilled water and is settled to 1000ml, adjusts its PH=7.2 ± 0.2, in 121 DEG C of high pressure
Sterilizing is standby.
Culture medium used by described denitrifying bacteria is LB culture medium, and its formula is: tryptone 10g, yeast extract 5g, chlorine
Change sodium 5g, sodium nitrate 0.85g and agar powder 20g.It is dissolved in appropriate distilled water, at electric furnace after above-mentioned each reagent precise
Upper heated and boiled ensures that it is completely dissolved, and adds distilled water and is settled to 1000ml, adjusts its PH=7.0 ± 0.2, high at 121 DEG C
Pressure sterilizing is standby.
Culture medium used by described lactic acid bacteria is MRS culture medium, and its formula is: peptone 10g, Carnis Bovis seu Bubali cream 10g, yeast extract
5g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, glucose 20g, Tween 80 1ml, dibasic ammonium citrate 2g, magnesium sulfate 0.58g, manganese sulfate
0.25g and agar powder 15g.Being dissolved in appropriate distilled water after above-mentioned each reagent precise, on electric furnace, heated and boiled is protected
Demonstrate,proving it to be completely dissolved, then add distilled water and be settled to 1000ml, adjust its PH=6.2 ± 0.2,121 DEG C of autoclavings are standby.
The method using coated plate determines bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria concentration.Will
Above-mentioned each strain respectively takes 1 gram and puts in 150ml conical flask, is added thereto to 100ml sterilized water, and this can be considered as strain and be diluted
The bacterium colony mother solution of 100 times.Mother solution is placed on the shaking table of 200r/min vibration 30min, makes strain mix homogeneously.After vibration
Strain mother solution move in aseptic operating platform, and take its supernatant 0.5ml in 10ml centrifuge tube, be then added thereto to
4.5ml sterilized water, closes the lid centrifuge tube, vibrates 10 seconds so that it is mix homogeneously, this is mother solution and is diluted 10 on whirlpool instrument
Times.Repeat this operation, stepwise dilution, be diluted to 1010 always, take the diluent of each gradient of 0.1ml in the culture medium of corresponding strain
In, to smear uniformly with glass slicker, each Concentraton gradient takes 3 repetitions.Culture medium inversion is put in 30 DEG C after completing by coated plate
Constant incubator in cultivate 24 hours.Observe the growing state of bacterium colony in culture medium, if antibacterial growing way is failed to understand after 24 hours
Showing can proper extension incubation time.Until the bacterium colony on culture medium flat plate is high-visible.
Hay spore in the final preparation (including complex microorganism powderous preparations and complex microorganism liquid preparation) prepared
The viable bacteria amount of bacillus, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria is respectively as follows: 6.0 × 109～9.0 × 1010CFU/ml、
6.0×109～9.0 × 1010CFU/ml、3.0×108～9.0 × 109CFU/ml and 3.0 × 107～9.0 × 109CFU/ml。
The invention has the beneficial effects as follows:
(1) present invention is by using 4 kinds of different microorganism fungus kinds to be used in mixed way, and uses suitable ratio, it is possible to
Effectively sea-farming sewage is improved, be experimentally confirmed and there is preferable improved effect；
(2) this sea-farming sewage improvement complex microorganism preparations viable bacteria content is high, moisture is low, and stability is high,
It is the multiple prebiotic of the prebiotic functions such as harmful substance such as a kind of ammonia nitrogen that can utilize and absorb in breeding water body and nitrite nitrogen
Bacterium and the complex microorganism preparations of the beneficial agents such as the enzyme of its secretion, amino acid bio element；
(3) all there are sale, steady sources, low price in the raw materials used domestic market of this complex microorganism preparations, produces work
Skill is simple, workable；
(4) this complex microorganism preparations answers the market demand, can be made into powder dose-type and liquid dosage form.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
By specific embodiment, the present invention will be described in detail below.These embodiments are provided to be able to more
Thoroughly understand the present invention, and complete for the scope of the present invention can be conveyed to those skilled in the art.
" comprising " or " including " as mentioned by the middle of description and claim in the whole text is an open language, therefore should
It is construed to " comprise but be not limited to ".Description subsequent descriptions is to implement the better embodiment of the present invention, and right described description is
For the purpose of the rule of description, it is not limited to the scope of the present invention.Protection scope of the present invention is when regarding appended power
Profit requires that defined person is as the criterion.
Embodiment 1
A kind of Grouper cultivating sewage improvement complex microorganism preparations, containing weight ratio in described preparation is 2:2:1:1
Bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria.
The preparation method of Grouper cultivating sewage improvement complex microorganism preparations, comprises the steps:
(1) bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria are taken respectively in respective culture medium
Cultivating activation, be separately added into 5ml sterilized water in often pipe bacterium, the bacteria suspension made, respectively as first order seed, the most respectively will
Cultured first order seed be by mass percentage 5% inoculum concentration transfer to the 1000ml containing 200ml liquid seed culture medium
In triangular flask, then triangular flask is placed in shaking table, temperature be 28 DEG C, hunting speed be 250rpm under conditions of shaken cultivation
20 hours, respectively obtain bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria secondary seed solution, standby；
Wherein, described bacillus subtilis and the culture medium prescription used by Bacillus licheniformis are: tryptone 10g,
Yeast powder 5g, sodium chloride 5g and agar powder 15g.It is dissolved in appropriate distilled water, at electric furnace after above-mentioned each reagent precise
Upper heated and boiled ensures that it is completely dissolved, and adds distilled water and is settled to 1000ml, adjusts its PH=7.2 ± 0.2, in 121 DEG C of high pressure
Sterilizing is standby.
Culture medium used by described denitrifying bacteria is LB culture medium, and its formula is: tryptone 10g, yeast extract 5g, chlorine
Change sodium 5g, sodium nitrate 0.85g and agar powder 20g.It is dissolved in appropriate distilled water, at electric furnace after above-mentioned each reagent precise
Upper heated and boiled ensures that it is completely dissolved, and adds distilled water and is settled to 1000ml, adjusts its PH=7.0 ± 0.2, high at 121 DEG C
Pressure sterilizing is standby.
Culture medium used by described lactic acid bacteria is MRS culture medium, and its formula is: peptone 10g, Carnis Bovis seu Bubali cream 10g, yeast extract
5g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, glucose 20g, Tween 80 1ml, dibasic ammonium citrate 2g, magnesium sulfate 0.58g, manganese sulfate
0.25g and agar powder 15g.Being dissolved in appropriate distilled water after above-mentioned each reagent precise, on electric furnace, heated and boiled is protected
Demonstrate,proving it to be completely dissolved, then add distilled water and be settled to 1000ml, adjust its PH=6.2 ± 0.2,121 DEG C of autoclavings are standby；
(2) compound based on mass ratio wheat bran, soybean cake powder and Semen arachidis hypogaeae dregs as 6:2:1, adds in base mix
Add and account for glucose, the CaCO of 0.5% that base mix mass percent is 5.0%3, the MnSO of 1%4·H2O's and 1%
NaCl obtains solid butt；Then adding the sterilized water of 1 times of weight in solid butt, pH value is adjusted to 6.5, after stirring
Again by its sterilizing 30min under 120 DEG C of hot conditionss, it is cooled to room temperature, can be used as solid fermentation culture medium and use；
(3) by above-mentioned secondary seed solution be the most by mass percentage respectively 10% inoculum concentration be inoculated into above-mentioned solid fermentation
In culture medium, fully mixing, in airtight solid-state fermentation tank, humidity is 28%, temperature is fermented, during fermentation under the conditions of being 37 DEG C
Between be 30 hours, obtain tunning；
(4) adding the corn starch of 15 times of weight in above-mentioned tunning, fully mix, temperature is true under the conditions of being 50 DEG C
Air cooling is solidifying to be dried 8 hours, obtains being dried tunning, then pulverizes, crosses 40 mesh, obtain complex microorganism powderous preparations.
Bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and the viable bacteria amount of lactic acid bacteria in the final preparation prepared
It is respectively as follows: 6.0 × 109CFU/ml、2.0×1010CFU/ml、3.0×108CFU/ml and 9.0 × 109CFU/ml。
Embodiment 2
A kind of Grouper cultivating sewage improvement complex microorganism preparations, containing weight ratio in described preparation is 2:3:1:2
Bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria.
The preparation method of above-mentioned Grouper cultivating sewage improvement complex microorganism preparations, comprises the steps:
(2) bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria are taken respectively in respective culture medium
Cultivating activation, be separately added into 5ml sterilized water in often pipe bacterium, the bacteria suspension made, respectively as first order seed, the most respectively will
Cultured first order seed be by mass percentage 8% inoculum concentration transfer to the 1000ml containing 200ml liquid seed culture medium
In triangular flask, then triangular flask is placed in shaking table, temperature be 37 DEG C, hunting speed be 250rpm under conditions of shaken cultivation
16 hours, respectively obtain bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria secondary seed solution, standby；
Wherein, described bacillus subtilis and the culture medium prescription used by Bacillus licheniformis are: tryptone 10g,
Yeast powder 5g, sodium chloride 5g and agar powder 15g.It is dissolved in appropriate distilled water, at electric furnace after above-mentioned each reagent precise
Upper heated and boiled ensures that it is completely dissolved, and adds distilled water and is settled to 1000ml, adjusts its PH=7.2 ± 0.2, in 121 DEG C of high pressure
Sterilizing is standby.
Culture medium used by described denitrifying bacteria is LB culture medium, and its formula is: tryptone 10g, yeast extract 5g, chlorine
Change sodium 5g, sodium nitrate 0.85g and agar powder 20g.It is dissolved in appropriate distilled water, at electric furnace after above-mentioned each reagent precise
Upper heated and boiled ensures that it is completely dissolved, and adds distilled water and is settled to 1000ml, adjusts its PH=7.0 ± 0.2, high at 121 DEG C
Pressure sterilizing is standby.
Culture medium used by described lactic acid bacteria is MRS culture medium, and its formula is: peptone 10g, Carnis Bovis seu Bubali cream 10g, yeast extract
5g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, glucose 20g, Tween 80 1ml, dibasic ammonium citrate 2g, magnesium sulfate 0.58g, manganese sulfate
0.25g and agar powder 15g.Being dissolved in appropriate distilled water after above-mentioned each reagent precise, on electric furnace, heated and boiled is protected
Demonstrate,proving it to be completely dissolved, then add distilled water and be settled to 1000ml, adjust its PH=6.2 ± 0.2,121 DEG C of autoclavings are standby；
(2) compound based on mass ratio wheat bran, soybean cake powder and Semen arachidis hypogaeae dregs as 7:2.5:1.5, to base mix
Middle interpolation accounts for the CaCO of the glucose 2% that base mix mass percent is 5.0%3, the MnSO of 0.2%4·H2O and 0.3%
NaCl obtain solid butt；Then adding the sterilized water of 2 times of weight in solid butt, pH value is adjusted to 7.2, stirs
After again by its sterilizing 15min under 130 DEG C of hot conditionss, be cooled to room temperature, can be used as solid fermentation culture medium use；
(3) by above-mentioned secondary seed solution be the most by mass percentage respectively 10% inoculum concentration be inoculated into above-mentioned solid fermentation
In culture medium, fully mixing, in airtight solid-state fermentation tank, humidity is 37%, temperature is fermented, during fermentation under the conditions of being 36 DEG C
Between be 48 hours, obtain tunning；
(4) adding the corn starch of 20 times of weight in above-mentioned tunning, fully mix, temperature is true under the conditions of being 65 DEG C
Air cooling is solidifying to be dried 8 hours, obtains being dried tunning, then pulverizes, crosses 60 mesh, obtain complex microorganism powderous preparations.
Hay spore in the final preparation (including complex microorganism powderous preparations and complex microorganism liquid preparation) prepared
The viable bacteria amount of bacillus, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria is respectively as follows: 5.0 × 1010CFU/ml、5.0×
1010CFU/ml、1.0×109CFU/ml and 3.0 × 107CFU/ml。
Embodiment 3
A kind of Grouper cultivating sewage improvement complex microorganism preparations, described bacillus subtilis, Bacillus licheniformis,
The weight ratio of denitrifying bacteria and lactic acid bacteria is 2:4:2:2.
The preparation method of above-mentioned Grouper cultivating sewage improvement complex microorganism preparations, comprises the steps:
(3) bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria are taken respectively in respective culture medium
Cultivating activation, be separately added into 5ml sterilized water in often pipe bacterium, the bacteria suspension made, respectively as first order seed, the most respectively will
Cultured first order seed be by mass percentage 6% inoculum concentration transfer to the 1000ml containing 200ml liquid seed culture medium
In triangular flask, then triangular flask is placed in shaking table, temperature be 30 DEG C, hunting speed be 250rpm under conditions of shaken cultivation
18 hours, respectively obtain bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria secondary seed solution, standby；
Wherein, described bacillus subtilis and the culture medium prescription used by Bacillus licheniformis are: tryptone 10g,
Yeast powder 5g, sodium chloride 5g and agar powder 15g.It is dissolved in appropriate distilled water, at electric furnace after above-mentioned each reagent precise
Upper heated and boiled ensures that it is completely dissolved, and adds distilled water and is settled to 1000ml, adjusts its PH=7.2 ± 0.2, in 121 DEG C of high pressure
Sterilizing is standby.
Culture medium used by described denitrifying bacteria is LB culture medium, and its formula is: tryptone 10g, yeast extract 5g, chlorine
Change sodium 5g, sodium nitrate 0.85g and agar powder 20g.It is dissolved in appropriate distilled water, at electric furnace after above-mentioned each reagent precise
Upper heated and boiled ensures that it is completely dissolved, and adds distilled water and is settled to 1000ml, adjusts its PH=7.0 ± 0.2, high at 121 DEG C
Pressure sterilizing is standby.
Culture medium used by described lactic acid bacteria is MRS culture medium, and its formula is: peptone 10g, Carnis Bovis seu Bubali cream 10g, yeast extract
5g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, glucose 20g, Tween 80 1ml, dibasic ammonium citrate 2g, magnesium sulfate 0.58g, manganese sulfate
0.25g and agar powder 15g.Being dissolved in appropriate distilled water after above-mentioned each reagent precise, on electric furnace, heated and boiled is protected
Demonstrate,proving it to be completely dissolved, then add distilled water and be settled to 1000ml, adjust its PH=6.2 ± 0.2,121 DEG C of autoclavings are standby；
(2) compound based on mass ratio wheat bran, soybean cake powder and Semen arachidis hypogaeae dregs as 7:2.2:1.2, to base mix
Middle interpolation accounts for glucose, the CaCO of 1% that base mix mass percent is 5.0%3, the MnSO of 0.5%4·H2O and
The NaCl of 0.5% obtains solid butt；Then adding the sterilized water of 1.5 times of weight in solid butt, pH value is adjusted to 7, stirs
Again by its sterilizing 20min under 125 DEG C of hot conditionss after mixing uniformly, it is cooled to room temperature, can be used as solid fermentation culture medium
Use；
(3) by above-mentioned secondary seed solution be the most by mass percentage respectively 8% inoculum concentration be inoculated into above-mentioned solid fermentation
In culture medium, fully mixing, in airtight solid-state fermentation tank, humidity is 30%, temperature is fermented, during fermentation under the conditions of being 36 DEG C
Between be 36 hours, obtain tunning；
(4) with collective media (solid medium, its formula is: bacteriological peptone 5g, yeast powder 5g, glucose 5g,
Dipotassium hydrogen phosphate 4g and agar 18g, its pH are 7.4；) as diluent, tunning is diluted 100 times；Filter, it is ensured that filtrate
Middle viable bacteria content is 1.0 × 107～109CFU/ml, then filtrate is concentrated, the centrifuge 2 points using rotating speed to be 10000rpm
Clock or use the centrifuge 10 minutes of 5000rpm, collect centrifugal after precipitate, by precipitate again outstanding be dissolved in general
In culture medium, it is thus achieved that concentrated liquid, it is ensured that viable bacteria content is 1.0 × 109～1012CFU/ml, obtains complex microorganism liquid system
Agent.
Hay spore in the final preparation (including complex microorganism powderous preparations and complex microorganism liquid preparation) prepared
The viable bacteria amount of bacillus, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria is respectively as follows: 4.0 × 1010CFU/ml、9.0×
1010CFU/ml、9.0×109CFU/ml and 5.0 × 108CFU/ml。
Embodiment 4
A kind of Grouper cultivating sewage improvement complex microorganism preparations, containing weight ratio in described preparation is 2:4:1:2
Bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria.
The preparation method of above-mentioned Grouper cultivating sewage improvement complex microorganism preparations, comprises the steps:
(4) bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria are taken respectively in respective culture medium
Cultivating activation, be separately added into 5ml sterilized water in often pipe bacterium, the bacteria suspension made, respectively as first order seed, the most respectively will
Cultured first order seed be by mass percentage 7% inoculum concentration transfer to the 1000ml containing 200ml liquid seed culture medium
In triangular flask, then triangular flask is placed in shaking table, temperature be 35 DEG C, hunting speed be 250rpm under conditions of shaken cultivation
18 hours, respectively obtain bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria secondary seed solution, standby；
Wherein, described bacillus subtilis and the culture medium prescription used by Bacillus licheniformis are: tryptone 10g,
Yeast powder 5g, sodium chloride 5g and agar powder 15g.It is dissolved in appropriate distilled water, at electric furnace after above-mentioned each reagent precise
Upper heated and boiled ensures that it is completely dissolved, and adds distilled water and is settled to 1000ml, adjusts its PH=7.2 ± 0.2, in 121 DEG C of high pressure
Sterilizing is standby.
Culture medium used by described denitrifying bacteria is LB culture medium, and its formula is: tryptone 10g, yeast extract 5g, chlorine
Change sodium 5g, sodium nitrate 0.85g and agar powder 20g.It is dissolved in appropriate distilled water, at electric furnace after above-mentioned each reagent precise
Upper heated and boiled ensures that it is completely dissolved, and adds distilled water and is settled to 1000ml, adjusts its PH=7.0 ± 0.2, high at 121 DEG C
Pressure sterilizing is standby.
Culture medium used by described lactic acid bacteria is MRS culture medium, and its formula is: peptone 10g, Carnis Bovis seu Bubali cream 10g, yeast extract
5g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, glucose 20g, Tween 80 1ml, dibasic ammonium citrate 2g, magnesium sulfate 0.58g, manganese sulfate
0.25g and agar powder 15g.Being dissolved in appropriate distilled water after above-mentioned each reagent precise, on electric furnace, heated and boiled is protected
Demonstrate,proving it to be completely dissolved, then add distilled water and be settled to 1000ml, adjust its PH=6.2 ± 0.2,121 DEG C of autoclavings are standby；
(2) compound based on mass ratio wheat bran, soybean cake powder and Semen arachidis hypogaeae dregs as 6:2.5:1, in base mix
Add and account for glucose, the CaCO of 1.5% that base mix mass percent is 5.0%3, the MnSO of 0.8%4·H2O and
The NaCl of 0.8% obtains solid butt；Then adding the sterilized water of 2 times of weight in solid butt, pH value is adjusted to 6.8, stirs
Again by its sterilizing 25min under 130 DEG C of hot conditionss after mixing uniformly, it is cooled to room temperature, can be used as solid fermentation culture medium
Use；
(3) by above-mentioned secondary seed solution be the most by mass percentage respectively 8% inoculum concentration be inoculated into above-mentioned solid fermentation
In culture medium, fully mixing, in airtight solid-state fermentation tank, humidity is 35%, temperature is fermented, during fermentation under the conditions of being 37 DEG C
Between be 42 hours, obtain tunning；
(4) with collective media, (fluid medium, its formula is: sodium carboxymethyl cellulose 20g, peptone 5g, yeast extract
5g, sodium chloride 5g and potassium dihydrogen phosphate 1g, its pH is 7.4) as diluent, tunning is diluted 200 times；Filter, it is ensured that
In filtrate, viable bacteria content is 1.0 × 107～109CFU/ml, then by filtrate concentrate, use rotating speed be 10000rpm centrifuge from
The centrifuge of the heart 1 minute or employing 4000rpm 30 minutes, collects the precipitate after being centrifuged, is again hanged by precipitate molten
In collective media, it is thus achieved that concentrated liquid, it is ensured that viable bacteria content is 1.0 × 109～1012CFU/ml, obtains complex microorganism
Liquid preparation.
Hay spore in the final preparation (including complex microorganism powderous preparations and complex microorganism liquid preparation) prepared
The viable bacteria amount of bacillus, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria is respectively as follows: 9.0 × 109CFU/ml、7.0×
1010CFU/ml、4.0×109CFU/ml and 6.0 × 108CFU/ml。
In order to prove effectiveness of the invention, applicant has also carried out following test:
Experiment one: complex microorganism preparations (complex microorganism powderous preparations or complex microorganism liquid preparation) is to simulation stone
The impact of speckle fish culture sewage and effect
1. test equipment:
(1) instrument
Constant incubator, culture dish, whirlpool instrument, aseptic operating platform, shaking table, conical flask, spectrophotometer and supporting colorimetric
Ware, 25ml color-comparison tube, volumetric flask, liquid-transfering gun etc..
(2) reagent
Sulfanilamide, sodium nitrite, hydrochloride naphthodiamide, ammonium sulfate, sodium citrate, sodium hydroxide, phenol solution, nitrous ferrum ferrum
Various reagent etc. required for Cyanogran., liquor natrii hypochloritis and preparation culture medium.
2. test water body: the preparation of indoor cabrilla high-elevation breeding pond simulation water
Weigh 0.2463g sodium nitrite (in 110 DEG C of drying) and 0.4716g ammonium sulfate (in 110 DEG C of drying) is dissolved in few
It is settled to 1000ml after amount pure water, obtains nitrite concentration respectively 50mg/L and ammonia nitrogen concentration is the storing solution of 100mg/L.
Solution used in experiment needs to dilute 50 times on this basis, obtains nitrite nitrogen and the water sample of ammonia nitrogen concentration such as table 1.
Each content of material in front simulation water sample tested by table 1
3. test method: (quadrature factor-water-glass, represent respectively in table is the addition of the various strains that table 2 represents
Four kinds of bacterium are added to and shake in 150ml water, are added followed by experimental group by four kinds of strain addition g, and often group simulates water
Amount is 5L) and table 3 (L9 (34) orthogonal table, the ratio between the various strains that in table, each data represent), if 1 blank group
(without strain), totally 10 groups, often group 3 is parallel, and every morning 10 carries out ammonia nitrogen and the mensuration of nitrite record, even
Continuous record 6 days.Wherein, the most corresponding numbering 1-3 of table 3,4-6,7-9 of the level 1,2,3 in table 2.The hay of the most numbered 1
Bacillus cereus, Bacillus licheniformis, denitrifying bacteria, the ratio of lactic acid bacteria are 1:1:1:1, then corresponding weight be respectively 2g,
2g, 1g, 1g, the bacillus subtilis of the most numbered 2, Bacillus licheniformis, denitrifying bacteria, the ratio of lactic acid bacteria are 1:
2:2:2, then corresponding weight is respectively 2g, 4g, 2g, 2g.
Factor-the water-glass of table 2 orthogonal experiment
Table 3 L9(34) orthogonal
4. result of the test
Lab simulation is cultivated by complex microorganism preparations (complex microorganism powderous preparations or complex microorganism liquid preparation)
As shown in table 4, nitrite nitrogen changes of contents is as shown in table 5 in the ammonia-nitrogen content change of sewage.Before adding complex microorganism preparations,
Ammonia-nitrogen content in simulation water is 2.40mg/L, and nitrite nitrogen content is 1.04mg/L.
It can be seen that add in the water sample that bacterium processes from table 4 and table 5, the content of its ammonia nitrogen and nitrite nitrogen has bright
Aobvious reduction.For ammonia nitrogen, the fall ammonia nitrogen effect of the 1st group is the most obvious, after adding complex microorganism preparations 48 hours,
The peak value that ammonia-nitrogen content declines has reached 97.5%.Ammonia nitrogen degradation rate in water body is taken second place by the 3rd group of microorganism formulation, is adding
After microorganism formulation 84 hours, ammonia nitrogen degradation in water body 74.2%.Ammonia nitrogen degradation effect is least apparent that the 9th group
Microorganism formulation, after adding microorganism formulation 84 hours, in water body, the degradation rate of ammonia nitrogen only has 21%.Adding in simulation water
After entering microorganism formulation, the content of its nitrite nitrogen is declining always, carries out water sample nitrite nitrogen content at the 6th time and surveys
Regularly, the content of nitrite nitrogen reduces 93.3%.
Thus can draw, all four strain (bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria)
The complex microorganism preparations of compatibility gained all has obvious degradation effect, different proportion compatibilities to the nitrite nitrogen in water body
Microorganism formulation the degradation effect of nitrite nitrogen is not had notable difference.
In sum, the 1st group of microorganism formulation (i.e. bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid
Bacterium is formed by weight 2:2:1:1 compatibility) the most obvious to the impact effect of ammonia nitrogen in water body and nitrite nitrogen.Can be seen
Work is under simulation water experiment condition, one group of complex microorganism preparations that the compatibility obtained is optimum.
The change of table 4 simulated water sample ammonia-nitrogen content (mg/L)
Table 5 simulated water sample nitrite nitrogen content (mg/L) changes
Experiment two: complex microorganism preparations (complex microorganism powderous preparations or complex microorganism liquid preparation) is to outdoor stone
The impact of speckle fish high-elevation breeding pond sewage and effect
1. test water body: cultivation water water sample picks up from Grouper cultivating field, Yu Hong village, Sanya, and its salinity is about 35 ‰.
The container of sampling is the white plastic bucket of 25L, and sampled point is four diverse locations on the same shrimp pool, gathers water sample 75L altogether.Adopt
Within after backwater sample 12 hours, measuring ammonia nitrogen and the content of nitrite nitrogen in water body, its Nitrite Nitrogen and ammonia-nitrogen content content divide
Not Wei 0.13mg/L and 3.12mg/L, be then dispensed in the beaker of 2000ml and test.Experiment is divided into 10 groups, wherein 1-
9 groups is experimental group, and the 10th group is blank group, contrast identical under conditions of carry out, its water body of identical time interval determination
In ammonia nitrogen and the change of nitrite nitrogen content.
2. test method: with experiment one, complex microorganism preparations addition is identical with experiment 1.
3. result of the test
The ammonia-nitrogen content of Penaeus vannamei high-elevation breeding pond sewage is changed as shown in table 6 by complex microorganism preparations, nitrous
The change of hydrochlorate nitrogen content is as shown in table 7.Before addition complex microorganism preparations, the ammonia-nitrogen content in water body is 3.12mg/L, nitrous acid
The content of salt nitrogen is 0.133mg/L.
Ammonia-nitrogen content (mg/L) change in table 6 aquaculture wastewater
Table 7 aquaculture wastewater Content of Nitrite Nitrogen (mg/L) changes
* note: "-" represents that water sample does not develops the color when colorimetric
As can be seen from Table 6, the ammonia-nitrogen content of cultivation water sample is always in reducing trend.2nd group of experiment is compound in addition
Behind 84 hours of microorganism formulation, in water body, the degradation rate of ammonia nitrogen has reached 93.2%, is that in 9 experimental grouies ammonia nitrogen degradation rate is
High one group.In other experimental grouies, the ammonia nitrogen degradation rate of only the 6th group is a little the lowest, but is adding microorganism formulation
After 84 hours, its ammonia nitrogen degradation rate is still up to 84.0%.Visible complex microorganism preparations is to the ammonia nitrogen in actual aquaculture wastewater
Degradation effect is obvious, and before and after adding microorganism formulation, ammonia-nitrogen content reduces 84.0%～93.2%.
What table 7 represented is in aquaculture wastewater before and after addition complex microorganism preparations, the change of its nitrite nitrogen content, from
It can be seen that after adding microorganism formulation 24 hours, during the 1st water-quality determination, the nitrite nitrogen in its water body contains in table
Measure the most extremely low.In the determination experiment of water quality several times afterwards, after adding sulfanilamide and hydrochloric acid naphthalene sodium oxalate in liquid to be measured, treat
Survey liquid the most no longer to develop the color, can approximate and regard as that water body do not had nitrite nitrogen or its content is atomic, negligible
Disregard.
In sum, the 2nd group of microorganism formulation (bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria
Compatibility is carried out according to its weight ratio 2:4:2:2) the most obvious on the impact of ammonia nitrogen in water body, adding microorganism formulation 84 hours
After, its ammonia-nitrogen content have dropped 93.2%.
The foregoing is only embodiments of the invention, not thereby limit the scope of the claims of the present invention, every utilize this
Equivalent structure or equivalence flow process that bright description is made convert, or are directly or indirectly used in other relevant technology necks
Territory, is the most in like manner included in the scope of patent protection of the present invention.
Claims (9)
1. a Grouper cultivating sewage improvement complex microorganism preparations, it is characterised in that containing weight ratio in described preparation
Bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria for 2:2～4:1～2:1～2.
Grouper cultivating sewage improvement complex microorganism preparations the most according to claim 1, it is characterised in that described withered
The weight ratio of grass bacillus cereus, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria is 2:2:1:1.
Grouper cultivating sewage improvement complex microorganism preparations the most according to claim 1, it is characterised in that described withered
The weight ratio of grass bacillus cereus, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria is 2:4:2:2.
4. according to the system of the Grouper cultivating sewage improvement complex microorganism preparations described according to any one of claims 1 to 3
Preparation Method, it is characterised in that described preparation method comprises the steps:
(1) take bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria respectively to cultivate in respective culture medium
Activation, is separately added into 5ml sterilized water in often pipe bacterium, and the bacteria suspension made, respectively as first order seed, will be cultivated the most respectively
Good first order seed be by mass percentage 5～8% inoculum concentration transfer to the 1000ml tri-containing 200ml liquid seed culture medium
In the bottle of angle, then triangular flask is placed in shaking table, temperature be 28～37 DEG C, hunting speed be 250rpm under conditions of vibration training
Support 16～20 hours, respectively obtain bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria secondary seed solution,
Standby；
(2) compound based on wheat bran, soybean cake powder and the Semen arachidis hypogaeae dregs that mass ratio is 6～7:2～2.5:1～1.5, mixed to basis
Close material adds and account for glucose, 0.5～the CaCO of 2% that base mix mass percent is 5.0%3, 0.2～1%
MnSO4·H2The NaCl of O and 0.3～1% obtains solid butt；Then in solid butt, add the aseptic of 1～2 times of weight
Water, pH value is adjusted to 6.5～7.2, again by its sterilizing 15～30min under 120～130 DEG C of hot conditionss after stirring, treats cold
But to room temperature, can be used as solid fermentation culture medium and use；
(3) by above-mentioned secondary seed solution be the most by mass percentage respectively 5～10% inoculum concentration be inoculated into above-mentioned solid fermentation
In culture medium, fully mix, in airtight solid-state fermentation tank, humidity be 28～37%, temperature be that 36～37 DEG C of conditions issue
Ferment, fermentation time is 30～48 hours, obtains tunning；
(4) adding the corn starch of 15～20 times of weight in above-mentioned tunning, fully mix, temperature is 50～65 DEG C of conditions
Lower vacuum condensation is dried 8～15 hours, obtains being dried tunning, then pulverizes, crosses 40～60 mesh, obtain complex microorganism powdery
Preparation.
The preparation method of Grouper cultivating sewage improvement complex microorganism preparations the most according to claim 4, its feature
Being, described step (4) can also replace by following step:
Using collective media as diluent, tunning is diluted 2～200 times；Filter, it is ensured that in filtrate, viable bacteria content exists
1.0×107～109CFU/ml, then filtrate concentrated, uses rotating speed to be the centrifuge 1 of 10000rpm～2 minutes or adopt
Centrifuge 10 with 4000～5000rpm～30 minutes, collect centrifugal after precipitate, by precipitate again outstanding be dissolved in logical
With in culture medium, it is thus achieved that concentrated liquid, it is ensured that viable bacteria content is 1.0 × 109～1012CFU/ml, obtains complex microorganism liquid
Preparation；
Described collective media is solid medium, and its formula is: bacteriological peptone 5g, yeast powder 5g, glucose 5g, phosphoric acid
Hydrogen dipotassium 4g and agar 18g, its pH are 7.4；
Or fluid medium, its formula is: sodium carboxymethyl cellulose 20g, peptone 5g, yeast extract 5g, sodium chloride 5g and phosphoric acid
Potassium dihydrogen 1g, its pH are 7.4.
The preparation method of Grouper cultivating sewage improvement complex microorganism preparations the most according to claim 4, its feature
Being, the culture medium prescription used by bacillus subtilis and Bacillus licheniformis is: tryptone 10g, yeast powder 5g, chlorination
Sodium 5g and agar powder 15g.
The preparation method of Grouper cultivating sewage improvement complex microorganism preparations the most according to claim 4, its feature
Being, the culture medium used by denitrifying bacteria is LB culture medium, and its formula is: tryptone 10g, yeast extract 5g, sodium chloride 5g,
Sodium nitrate 0.85g and agar powder 20g.
The preparation method of Grouper cultivating sewage improvement complex microorganism preparations the most according to claim 4, its feature
Being, the culture medium used by lactic acid bacteria is MRS culture medium, and its formula is: peptone 10g, Carnis Bovis seu Bubali cream 10g, yeast extract 5g, phosphoric acid
Hydrogen dipotassium 2g, sodium acetate 5g, glucose 20g, Tween 80 1ml, dibasic ammonium citrate 2g, magnesium sulfate 0.58g, manganese sulfate 0.25g and
Agar powder 15g.
9., according to the preparation method of the Grouper cultivating sewage improvement complex microorganism preparations described in claim 4 or 5, it is special
Levying and be, in described preparation, the viable bacteria amount of bacillus subtilis, Bacillus licheniformis, denitrifying bacteria and lactic acid bacteria is respectively as follows:
6.0×109～9.0 × 1010CFU/ml、6.0×109～9.0 × 1010CFU/ml、3.0×108～9.0 × 109CFU/ml and
3.0×107～9.0 × 109CFU/ml。
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| CN106635893A (en) * | 2016-11-28 | 2017-05-10 | 中国水产科学研究院珠江水产研究所 | Method for preparing and applying ammonia-nitrogen reducing protective material |
| CN107867757A (en) * | 2017-09-07 | 2018-04-03 | 中山大学 | A kind of water quality improvement method for cultivating lithosporic fry |
| CN109825458A (en) * | 2019-03-20 | 2019-05-31 | 浙江省淡水水产研究所 | A kind of compound micro-ecological preparation and preparation method thereof |
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- 2016-06-16 CN CN201610424849.0A patent/CN106085906A/en active Pending
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| CN106635893A (en) * | 2016-11-28 | 2017-05-10 | 中国水产科学研究院珠江水产研究所 | Method for preparing and applying ammonia-nitrogen reducing protective material |
| CN107867757A (en) * | 2017-09-07 | 2018-04-03 | 中山大学 | A kind of water quality improvement method for cultivating lithosporic fry |
| CN107867757B (en) * | 2017-09-07 | 2020-10-30 | 中山大学 | Water quality improvement method for cultivating grouper fries |
| CN109825458A (en) * | 2019-03-20 | 2019-05-31 | 浙江省淡水水产研究所 | A kind of compound micro-ecological preparation and preparation method thereof |
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