CN108315287A - Photosynthetic bacteria cultivates microbial inoculum and preparation method thereof - Google Patents
Photosynthetic bacteria cultivates microbial inoculum and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to probiotics fields, and in particular to a kind of culture microbial inoculum and preparation method thereof producing photosynthetic bacteria.Photosynthetic bacteria freeze-drying after culture is prepared into freeze-dried powder microbial inoculum, then passes through process optimization with photosynthetic bacteria culture medium raw material, mixed according to a certain percentage, pack and be prepared as finished product, as produce the culture microbial inoculum of photosynthetic bacteria.When producing photosynthetic bacteria, culture microbial inoculum is added tap water and is dissolved in transparent airtight container or PE polybags according to a certain percentage, 25~40 degree, photosynthetic bacteria can be produced by cultivating under 2,000 4000 lux illumination intensity.Culture microbial inoculum prepared by the present invention is powdered, retention cycle is long, and production photosynthetic bacteria operating method is simple, quick, can disposably be prepared into the volume of needs, it solves existing photosynthetic bacteria production, first multistage culture is needed largely to prepare the tedious steps of seed fluid strain.
Description
Technical field
The present invention relates to probiotics fields, more particularly to photosynthetic bacteria culture microbial inoculum and preparation method thereof.
Background technology
Photosynthetic bacteria (PSB) is that occur that earliest, photosynthetic a kind of prokaryotes general name can be carried out, extensively on the earth
In general distribution Yu Haiyang, lake, river, activated sludge and agricultural land soil.Photosynthetic bacteria is in agricultural production and water quality improvement field
It has a wide range of applications.Studies have shown that photosynthetic bacteria own growth, which is metabolized, will produce abundant B family vitamin, carotenoid,
Therefore Co-Q10, folic acid etc. have very high nutritive value as animal feed additive.In addition, photosynthetic bacteria can have
Effect removal waste water in organic matter and nitrogen phosphate and sulfur compound, be commonly used in aquaculture field reduce the ammonia nitrogen in pool,
Nitrite and organic matter.
In recent years, the red false unit cell in the photosynthetic bacteria such as marsh of Rhodospirillaceae (Rhodospirillaceae) pseudomonas
Bacterium, capsula Rhodopseudomonas, Rhodopseudomonas spheroides, in aquaculture field using more and more extensive, production and processing side
Formula also has more report, many in particular for the research report of photosynthetic bacteria culture medium formulation optimization, training method method.
As patent 200810222313.6 disclose it is a kind of making container with cement pond, with overlay cover sealing cement pit in
Photosynthetic bacteria liquid culture is cultivated in the case where more than 20 degree sunlights irradiate, realizes large-scale industrialized production.Patent
201310147031.5 disclose using multiple transferred species, repeatedly cultivate, and with second acid for adjusting pH, continue culture and eventually become sticky purple
Red photosynthetic bacteria.The training method production photosynthetic bacteria of above-mentioned patent all first needs the seed bacterium solution of 20-25% or so,
It is first obtained when large-scale production and a large amount of seed bacterium solutions is obtained by multistage culture, operated relatively complicated, the consuming time is longer.
Currently, common photosynthetic bacteria product, predominantly photosynthetic bacteria culture medium and photosynthetic bacteria liquid in the market, culture
When bacterium solution inoculum concentration 20% or so.Due to being limited by cost and lyophilized technique technology, also seldom see at present straight
Connect the research that photosynthetic bacteria production is carried out using photosynthetic bacteria freeze-dried powder microbial inoculum and culture medium compounding.
Invention content
The object of the present invention is to provide a kind of photosynthetic bacterias to cultivate microbial inoculum.
It is yet another object of the invention to provide photosynthetic bacterias to cultivate bacterial preparation process.
It is yet another object of the invention to provide application of the photosynthetic bacteria culture microbial inoculum in producing photosynthetic bacteria.
The first aspect of the present invention, provides a kind of photosynthetic bacteria culture microbial inoculum, and the culture microbial inoculum includes：
(3) photosynthetic bacteria culture medium；
(4) photosynthetic bacteria freeze-dried powder microbial inoculum.
In another preferred example, the photosynthetic bacteria is selected from the group：Rhodospirillaceae (Rhodospirillaceae) vacation unit cell
The photosynthetic bacteria of Pseudomonas.
In another preferred example, the photosynthetic bacteria is selected from the group：Rhodopseudomonas palustris, capsula Rhodopseudomonas, ball
Shape red pseudomonas.
In another preferred example, the culture microbial inoculum is made of photosynthetic bacteria culture medium and photosynthetic bacteria freeze-dried powder microbial inoculum.
In another preferred example, in the photosynthetic bacteria culture microbial inoculum,
Photosynthetic bacteria culture medium 80wt%-99wt%；
Photosynthetic bacteria freeze-dried powder microbial inoculum 1wt%-20wt%.
In another preferred example, in the photosynthetic bacteria culture microbial inoculum,
Photosynthetic bacteria culture medium 90wt%-99wt%；
Photosynthetic bacteria freeze-dried powder microbial inoculum 1wt%-10wt%.
In another preferred example, in the photosynthetic bacteria culture microbial inoculum,
Photosynthetic bacteria culture medium 90wt%-98wt%；
Photosynthetic bacteria freeze-dried powder microbial inoculum 2wt%-10wt%.
In another preferred example, the photosynthetic bacteria culture medium contains raw material：Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, second
Sour sodium, sodium chloride, calcium chloride, magnesium chloride, yeast extract, peptone, ammonium chloride, ferrous sulfate, manganese sulfate.
In another preferred example, each raw material dosage proportioning of the photosynthetic bacteria culture medium is：
Potassium dihydrogen phosphate：10~50 parts,
Dipotassium hydrogen phosphate：5 parts,
Sodium acetate：150~450 parts,
Sodium chloride：150 parts,
Calcium chloride：10 parts,
Magnesium chloride：5 parts,
Yeast extract：25-55 parts,
Peptone：10-25 parts,
Ammonium chloride：60~280 parts；
Ferrous sulfate：1 part；
Manganese sulfate：1 part；
Wherein part is parts by weight.
In another preferred example, in the photosynthetic bacteria freeze-dried powder microbial inoculum, contain protective agent.
In another preferred example, the protective agent includes basic protective agent and synergistic protector.
In another preferred example, the protective agent is selected from the group：Sucrose, mannose, trehalose, maltodextrin, resists milk powder
Bad hematic acid, sodium glutamate, or combinations thereof.
In another preferred example, the milk powder is skimmed milk power.
In another preferred example, the protective agent contains：5-10wt% sucrose, 6-10wt% milk powder (such as defatted milk
Powder), 0.5-2wt% maltodextrins, 0.01-0.1wt% ascorbic acid.
In another preferred example, the protective agent contains：8wt% sucrose, 6wt% milk powder (such as skimmed milk power), 1wt%
Maltodextrin, 0.05wt% ascorbic acid.
In another preferred example, each raw material dosage proportioning of the photosynthetic bacteria culture medium is：
Potassium dihydrogen phosphate：10~50 parts,
Dipotassium hydrogen phosphate：5 parts,
Sodium acetate：150~450 parts,
Sodium chloride：150 parts,
Calcium chloride：10 parts,
Magnesium chloride：5 parts,
Yeast extract：25-55 parts,
Peptone：10-25 parts,
Ammonium chloride：60~280 parts；
Ferrous sulfate：1 part；
Manganese sulfate：1 part；
Wherein part is parts by weight；
In the photosynthetic bacteria freeze-dried powder microbial inoculum, contain protective agent；The protective agent is selected from the group：Sucrose, milk powder, sweet dew
Sugar, trehalose, maltodextrin, ascorbic acid, sodium glutamate, or combinations thereof.
In another preferred example, the milk powder is skimmed milk power.
The second aspect of the present invention provides a kind of preparation method of photosynthetic bacteria culture microbial inoculum, including step：
(1) photosynthetic bacteria culture medium and photosynthetic bacteria freeze-dried powder microbial inoculum are provided；
(2) photosynthetic bacteria culture medium in (1) and photosynthetic bacteria freeze-dried powder microbial inoculum are uniformly mixed；
(3) optionally packaging step.
In another preferred example, the photosynthetic bacteria culture medium preparation method is：By potassium dihydrogen phosphate, dipotassium hydrogen phosphate,
Sodium acetate, sodium chloride, calcium chloride, magnesium chloride, yeast extract, peptone, ammonium chloride, ferrous sulfate, manganese sulfate are uniformly mixed system
Obtain photosynthetic bacteria culture medium.
In another preferred example, the preparation method of the photosynthetic bacteria freeze-dried powder microbial inoculum includes step：
(i) bacterium mud is provided；
(ii) protective agent is added into the bacterium mud in (i), obtains added with protectant bacterium mud；
(iii) in (ii) added in protectant bacterium mud be added water (preferably pure water), obtain bacterium mud-protective agent it is molten
Liquid；
(iv) pre-freeze processing is carried out to bacterium mud in (iii)-protection agent solution, obtains freeze-drying pre-freeze bacterium；
(v) frozen dried is carried out to the freeze-drying pre-freeze bacterium in (iv), obtains photosynthetic bacteria freeze-dried powder microbial inoculum.
In another preferred example, the collection bacterium mud is to precipitate cultured photosynthetic bacteria liquid with flocculant, is collected
Bacterium mud.
In another preferred example, the protective agent contains：5-10wt% sucrose, 6-10wt% milk powder (such as defatted milk
Powder), 0.5-2wt% maltodextrins, 0.01-0.1wt% ascorbic acid.
In another preferred example, the protective agent contains：8wt% sucrose, 6wt% milk powder (such as skimmed milk power), 1wt%
Maltodextrin, 0.05wt% ascorbic acid.
In another preferred example, in the step (iii), according to 1:Pure water is added in 0.5~1 weight ratio.
In another preferred example, in the step (iv), pre-freeze treatment temperature is -65 degree.
In another preferred example, in the step (iv), pre-freeze processing time is 2-24 hours；Preferably 5-20h；More
Good is 10h.
In another preferred example, in the step (v), during frozen dried, freeze dryer vacuum degree 0.01- is set
0.05mba, preferably 0.02mba.
In another preferred example, in the step (v), the frozen dried time is 20-50 hours, preferably 30 hours.
The third aspect of the present invention provides the photosynthetic bacteria culture microbial inoculum described in the first aspect of the present invention in production light
Close the application in bacterium.
The fourth aspect of the present invention provides a kind of method producing photosynthetic bacteria, uses the first aspect of the present invention institute
The photosynthetic bacteria culture microbial inoculum stated.
In another preferred example, the method includes the steps：
(a) container is provided；
(b) the photosynthetic bacteria culture microbial inoculum described in the first aspect of the present invention is dissolved in the container, forms solution
1；
(c) solution 1 in (b) is cultivated.
In another preferred example, in the step (c), the culture carries out under anaerobic environment.
In another preferred example, the anaerobic environment by by the solution 1 in the step (b) be placed under closed environment into
Row.
In another preferred example, the anaerobic environment covers one layer or more by 1 surface of solution in the step (b)
Tunic is realized.
In another preferred example, one or more layers described film is clear PE plastic foil.
In another preferred example, in the step (c), the culture carries out at a temperature of 25-40, preferably 25-35 degree, more
It is preferred that 30 degree.
In another preferred example, in the step (c), the culture carries out under light illumination.
In another preferred example, the intensity of illumination is the luxs 1000-5000, and preferred luxs 2000-4000 are more excellent
Select 3000 luxs.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Description of the drawings
Fig. 1 is the purebred Rhodopseudomonas palustris of solid medium separation.
Fig. 2 is photosynthetic bacteria Rhodopseudomonas palustris freeze-dried powder microbial inoculum.
Specific implementation mode
The present inventor after extensive and in-depth study, by optimized photosynthetic bacteria culture medium and uses the technique optimized
It is prepared by (including provide bacterium mud, protective agent is added in bacterium mud, carry out pre-freeze processing after pure water is added, then carry out frozen dried)
Photosynthetic bacteria freeze-dried powder microbial inoculum (wherein containing protective agent, such as sucrose, milk powder, maltodextrin, ascorbic acid) according to certain
Ratio mixing compounding, prepare photosynthetic bacteria cultivate microbial inoculum.In use, being directly added into a certain amount of water, (example is cultivated by a batch
Such as cultivated under anaerobic environment) large-scale photosynthetic bacteria liquid is can be obtained, without in additionally access seed liquor, operation side
Just, using simple, cultivate needed for the period it is short.On this basis, the present invention is completed.
Term
Photosynthetic bacteria culture medium
Photosynthetic bacteria culture medium is the nutritional ingredient for cultivating photosynthetic bacteria, necessary to the growth according to photosynthetic bacteria
Carbon source, nitrogen source and inorganic salts etc. determine final photosynthetic bacteria culture medium by medium component optimization.
Typically, medium component of the present invention includes：Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium acetate, sodium chloride, chlorination
Calcium, magnesium chloride, yeast extract, peptone, ammonium chloride, ferrous sulfate, manganese sulfate.
Protective agent
Protective agent is that photosynthetic bacteria freeze-dried powder mixes when preparing with bacterium mud, to prevent photosynthetic bacteria being freeze-dried and storing
When microbial activity damage.Basic protective agent is to meet somatic cells, protein when being freeze-dried not damaged by extraneous factor
Basis.Synergistic protector is that the basic protective agent of cooperation is used in conjunction with, and has synergistic effect, enhances frozen-dried protective effect.
Typically, in the present invention, basic protective agent includes：Sucrose, milk powder (such as skimmed milk power), mannose, trehalose.
Typically, in the present invention, synergistic protector includes：Maltodextrin, ascorbic acid, sodium glutamate.
Photosynthetic bacteria cultivates microbial inoculum
The present invention " photosynthetic bacteria cultivates microbial inoculum " is the culture microbial inoculum of production photosynthetic bacteria, specifically includes photosynthetic bacteria freeze-drying
Powder microbial inoculum and photosynthetic bacteria culture medium；According to a certain percentage by photosynthetic bacteria culture medium after optimizing and freeze-dried powder microbial inoculum
Photosynthetic bacteria culture microbial inoculum is made in mixing.During mass production photosynthetic bacteria liquid, in culture without adding seed
Bacterium solution can be directly added into water and carry out the photosynthetic bacteria liquid that culture acquisition needs volume, simplify incubation step, shorten culture
Period.
Main advantages of the present invention are：
(1) culture microbial inoculum prepared by the present invention is powdered, and retention cycle is long；
(2) culture microbial inoculum prepared by the present invention is convenient for packaging, transport；
(3) present invention production photosynthetic bacteria operating method is simple, quick, can disposably be prepared into the volume of needs, solves
Existing photosynthetic bacteria production, needs the tedious steps for first step by step largely preparing seed fluid strain.
Following specific embodiment, the present invention is further explained.It should be understood that these embodiments be merely to illustrate the present invention and
It is not used in and limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition,
Such as (Sambrook and Russel l et al., molecular cloning：Laboratory manual (Molecular Cloning-A
Laboratory Manual) (third edition) (2001) CSHL publishing houses) described in condition, or according to proposed by manufacturer
Condition.Unless otherwise stated, otherwise percentage and number are calculated by weight.Experiment material used in following embodiment and examination
Agent can obtain unless otherwise instructed from commercially available channel.
It should be understood that embodiment of the present invention is not limited to act embodiment set forth below, further include each specific implementation mode
Between arbitrary combination.
Material and universal method
Photosynthetic bacteria described in embodiment is Rhodopseudomonas palustris.
The present invention is used as production after being mixed using mixed culture medium raw material and the photosynthetic bacteria freeze-dried powder microbial inoculum of preparation
The culture microbial inoculum of photosynthetic bacteria.
Photosynthetic bacteria culture medium raw material proportioning is：Potassium dihydrogen phosphate：10~50 parts, dipotassium hydrogen phosphate：5 parts, sodium acetate：
150~450 parts, sodium chloride：150 parts, calcium chloride：10 parts, magnesium chloride：5 parts, yeast extract：50 parts, peptone：20 parts, chlorination
Ammonium：60~280 parts；Ferrous sulfate：1 part, 1 part of manganese sulfate；The photosynthetic bacteria culture medium raw material dosage prepared is 500~1000
Part.
The preparation method of photosynthetic bacteria freeze-dried powder microbial inoculum is：Flocculant precipitation half is added in cultured photosynthetic bacteria liquid
Hour, collection bacterium mud, 8% sucrose of addition, 6% milk powder, 1% maltodextrin, 0.05% ascorbic acid, after calculating total weight, then
According to 1:Pure water is added in 0.5 ratio, stirs evenly, and -65 degree pre-freezes 10 hours place into freeze dryer, vacuum degree is arranged
0.02mba is freeze-dried 30 hours, obtains photosynthetic bacteria freeze-dried powder microbial inoculum；Photosynthetic bacteria freeze-dried powder microbial inoculum dosage 20~100
Part.
The present invention produces the culture microbial inoculum and preparation method thereof of photosynthetic bacteria, and concrete scheme is as follows：It will be proportioned photosynthetic
Bacteria Culture based raw material is mixed according to a certain percentage with the photosynthetic bacteria freeze-dried powder microbial inoculum of preparation, photosynthetic thin as producing
The culture microbial inoculum of bacterium.Then tap water is added in culture microbial inoculum according to a certain percentage and is dissolved in transparent airtight container or PE modelings
In material bag, 25~40 degree, culture production photosynthetic bacteria under 2000-4000 lux illumination intensity.
The present invention produces the culture microbial inoculum and preparation method thereof of photosynthetic bacteria, is by proportioned photosynthetic bacteria culture medium original
Material and the freeze-dried powder microbial inoculum prepared mix make solid, powdery culture microbial inoculum according to a certain percentage, then according to certain
Ratio be added to the water, can be carried out according to the cultural method of photosynthetic bacteria routine, operation it is relatively simple, it is easy to use, training
The foster period is short, and convenient for packaging and transport.
The preparation method of 1 photosynthetic bacteria freeze-dried powder microbial inoculum of embodiment
Solid medium forms (g/L)：
0.2 yeast extract of dipotassium hydrogen phosphate 0.8 sodium acetate, 1.0 magnesium sulfate, 8.0 agar 20.0
Fluid nutrient medium forms (g/L)：
0.2 yeast extract of ammonium chloride 2.0 potassium dihydrogen phosphate, 0.9 sodium acetate, 4.5 dipotassium hydrogen phosphate, 0.8 magnesium chloride 0.3
0.5 peptone of calcium chloride 0.1 manganese sulfate, 0.002 ferrous sulfate, 0.002 sodium bicarbonate, 1.0 sodium chloride, 0.5 Na2EDTA
0.02
The collection of thalline
The isolated purebred Rhodopseudomonas palustris on solid medium, solid plate culture are having 40W incandescent lamps
It is carried out in the anaerobic culture box of illumination, 30 degree of constant temperature incubations 3-5 days, sees that attached drawing 1 is purebred Rhodopseudomonas palustris.It will be purebred
Rhodopseudomonas palustris thalline scrapes off, and is seeded in respectively in the sterilized fluid nutrient medium of 6 bottles of 2.5L, in 30 degree of constant temperature
In incubator, 40W incandescent lights obtained the purebred Rhodopseudomonas palustris of 15L according to lower culture 3-5 days.
Then the purebred Rhodopseudomonas palustris of above-mentioned 15L is seeded to the larger clear PE equipped with 85L fluid nutrient mediums
Polybag, tightens sack, and polybag is placed in the square plastic drum of 120L or so.It is super with the water used in fluid nutrient medium
Drainage has reduced the pollution of miscellaneous bacteria.The intensity of illumination culture 5 days or so of 25-30 degree, the luxs 2000-4000 can be ripe.From
Cultured bacterium solution collects thalline and centrifugation, plate-frame filtering, the modes such as flocculation may be used.Above-mentioned 120L bacterium solutions are used into high-velocity tube
Formula centrifuge is separated by solid-liquid separation, and good separating effect may be implemented.High molecular polymerization polysaccharide substance has certain flocculation
Effect, and source is natural extraction, no chemical components.Cultured bacterium solution is transferred in drum, flocculant stirring is added, it is quiet
It sets half an hour, thalline is all deposited in bottom, and supernatant is outwelled, and collects bacterium mud, and the whole that bacterium mud substantially may be implemented is returned
It receives.
The optimization of lyophilized technique
The cryogenic vacuum of microorganism is freeze-dried, and is that certain protective agent is added in the bacterium mud of collection, first pre- in -70 degree
Freeze added with protectant bacterium mud, then utilizes cryogenic vacuum freeze dryer, be directly from the distillation of ice crystal state by frozen moisture
Gaseous state realizes dry process.During freeze-drying, protectant ingredient of different Institute of Micro-biology also difference,
Therefore, it is necessary to be determined by experiment best protection agent prescription to improve the survival rate of freeze-dried powder.
Inventor compounds sucrose with skimmed milk power as basic protective agent, adds synergistic protector on this basis
Maltodextrin and ascorbic acid are uniformly mixed and then according to 1:Pure water is added in 0.5 ratio, stirs evenly, -65 degree pre-freezes
It 10 hours, places into freeze dryer, vacuum degree 0.02mba is set, be freeze-dried 30 hours or so, obtain photosynthetic bacteria freeze-dried powder
Microbial inoculum is shown in that attached drawing 2 is photosynthetic bacteria freeze-dried powder microbial inoculum.It is determined by experiment protectant additional proportion, as a result such as the following table 1 institute
Show：
Table 1
The final optimization of C/C composites for obtaining freeze drying protectant, 8% sucrose, 6% milk powder, 1% maltodextrin, 0.05% Vitamin C
The survival rate of acid, photosynthetic bacteria freeze-dried powder can reach 72.8%.Be lyophilized obtained microbial inoculum need to be placed on 4 degree it is stored refrigerated, can
1 year as long as resting period.
2 photosynthetic bacteria of embodiment cultivates the preparation method of microbial inoculum
The composition of photosynthetic bacteria culture medium raw material is different, can influence the cell concentration of final photosynthetic bacteria culture solution.Simultaneously
The dosage of freeze-dried powder microbial inoculum in the feed also contributes to final cell concentration.
Weigh culture medium raw material, potassium dihydrogen phosphate：10g, dipotassium hydrogen phosphate：5g, sodium acetate：450g, sodium chloride：150g,
Calcium chloride：10g, magnesium chloride：5g, yeast extract：50g, peptone：20g, ammonium chloride：60g；Ferrous sulfate：1g；Manganese sulfate 1g；
The total weight of culture medium raw material dosage is 762g, weighs photosynthetic bacteria freeze-dried powder microbial inoculum 20g, the above material is mixed
Uniformly, the culture microbial inoculum of total weight 782g is prepared and is completed.The all solid, powdery ingredients of microbial inoculum are cultivated, convenient for packaging, transport.
The culture microbial inoculum of 782g has the liquid photosynthetic bacteria that can produce 100L altogether.
Specific implementation culture 10L photosynthetic bacteria liquids
Above-mentioned culture microbial inoculum 78.2g is taken, is added after 10L originally water dissolutions into pink liquid, packing is transparent to 4 2.5L
It in plastic bottle, being placed in constant incubator, 30 degree of temperature is set, one 40W incandescent lamp of placement provides light source in incubator,
After culture 5 days, bacterium solution color becomes peony, and can reach 1.6, pH after spectrophotometer OD600 is measured is
8.9, viable count is about 3,500,000,000/ml, can meet the needs of application.So, in this cultivation cycle, whether viable count also has
The space of promotion, in this regard, the dosage by increasing freeze-dried powder microbial inoculum, is equivalent to and improves strain initial inoculum, while to culture
The content of salt ion advanced optimizes in based raw material, to shorten cultivation cycle, improves viable bacteria content.
3 photosynthetic bacteria of embodiment cultivates the preparation method of microbial inoculum
Adjust the Related Component content in photosynthetic bacteria culture medium raw material so that entire raw material gross weight is 967g, adjustment
Raw material composition is as follows afterwards：
Weigh culture medium raw material, potassium dihydrogen phosphate：50g, dipotassium hydrogen phosphate：5g, sodium acetate：450g, sodium chloride：150g,
Calcium chloride：10g, magnesium chloride：5g, yeast extract：30g, peptone：15g, ammonium chloride：250g；Ferrous sulfate：1g；Manganese sulfate
1g；
The dosage of photosynthetic bacteria freeze-dried powder microbial inoculum increases to 100g, and the above material is uniformly mixed, the training of total weight 1067g
Bacteria agent is prepared and is completed.The liquid photosynthetic bacteria of 100L can be produced altogether.
Specific implementation culture 10L photosynthetic bacteria liquids
Above-mentioned culture microbial inoculum 106.7g is taken, is added after 10L originally water dissolutions into red liquid, packing is transparent to 4 2.5L
It in plastic bottle, being placed in constant incubator, 25 degree of temperature is set, one 40W incandescent lamp of placement provides light source in incubator,
It after culture 4 days, is watched outside bottle, bacterium solution color is peony, and obviously red drive member, pH are contained in bottle and is
9.1,1.9 can be reached after spectrophotometer OD600 is measured, viable count is about 9,100,000,000/ml.Optimize culture medium raw material dosage
After freeze-dried powder microbial inoculum dosage, incubation time can shorten one day, while thalline content is increased dramatically, and can meet completely
Application demand.
Embodiment 4 utilizes culture microbial inoculum mass production photosynthetic bacteria liquid
In the actual production of photosynthetic bacteria, to obtain large batch of bacterium solution, it is necessary first to prepare a certain amount of seed
Bacterium solution.For example, initially there was only the seed bacterium solution of 4L, need to produce 500L photosynthetic bacteria liquids, be calculated according to 20% inoculum concentration,
Firstly the need of the seed liquor for providing 100L, and the acquisition of this 100L seed liquor needs the seed bacterium solution of 20L, the bacterium of same 20L
Liquid is also required to the initial bacterium solution of 4L, it can thus be seen that the photosynthetic bacteria liquid of production 500L, needs three-level to cultivate one's ability
It is enough to obtain, it is calculated within 4 days according to a cycle, needs 12 days in total.And improved culture microbial inoculum can be at 4 days through the invention
Between, it can disposably produce the photosynthetic bacteria liquid of 500L.
Specific implementation culture 500L photosynthetic bacteria liquids
Prepare 2 300L transparent plastic pails, 2667.5g culture microbial inoculums are weighed in each bucket, 250L originally water dissolutions are added
For red liquid, one layer of polybag film is covered in liquid surface, to provide the anaerobic environment of culture, is set using Shui nationality heater
30 degree of heating of temperature are set, while the light source irradiation of 3000 luxs or so being provided using incandescent lamp.By culture in 4 days or so, 2
A plastic barrel bacterium solution has become the thick bacterium solution of laking, spectrophotometer OD600 detections 1.85 or so, pH 9.2, viable count
About 8,000,000,000/ml.
It is preserved under 5 photosynthetic bacteria of embodiment culture microbial inoculum room temperature
Photosynthetic bacteria culture microbial inoculum is sealed in aluminium foil bag, cool place is placed in, drying, stores at room temperature.Respectively to 3
Month, 6 months, the sample of 12 months holding times was cultivated, and examined whether microbial inoculum can still turn out photosynthetic bacteria liquid.
By culture, all left and right is transformed into peony to 3 months and 6 months samples on day 4, and bacterial concentration is high, and OD600 can reach
To 1.8 or so, viable count about 7,000,000,000/ml meets application demand.12 months samples need to become within 5 days or so deep by culture
Red, OD600 reach 1.7 or so, and viable count about 6,000,000,000/ml can also meet application demand.
It can be seen that the sample in half a year holding time does not substantially influence productive culture, preserve 1 year sample into
When row production photosynthetic bacteria, culture processing maturation period can be 2 days or so more, will not influence very much using effect.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (13)
1. a kind of photosynthetic bacteria cultivates microbial inoculum, which is characterized in that the culture microbial inoculum includes：
(1) photosynthetic bacteria culture medium；
(2) photosynthetic bacteria freeze-dried powder microbial inoculum.
2. photosynthetic bacteria as described in claim 1 cultivates microbial inoculum, which is characterized in that in the photosynthetic bacteria culture microbial inoculum,
Photosynthetic bacteria culture medium 80wt%-99wt%；
Photosynthetic bacteria freeze-dried powder microbial inoculum 1wt%-20wt%.
3. photosynthetic bacteria as claimed in claim 2 cultivates microbial inoculum, which is characterized in that in the photosynthetic bacteria culture microbial inoculum,
Photosynthetic bacteria culture medium 90wt%-99wt%；
Photosynthetic bacteria freeze-dried powder microbial inoculum 1wt%-10wt%.
4. photosynthetic bacteria as claimed in claim 1 or 2 cultivates microbial inoculum, which is characterized in that the photosynthetic bacteria culture medium contains
There is raw material：Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium acetate, sodium chloride, calcium chloride, magnesium chloride, yeast extract, peptone, chlorination
Ammonium, ferrous sulfate, manganese sulfate.
5. photosynthetic bacteria as claimed in claim 4 cultivates microbial inoculum, which is characterized in that each raw material of the photosynthetic bacteria culture medium
Consumption proportion is：
Potassium dihydrogen phosphate：10~50 parts,
Dipotassium hydrogen phosphate：5 parts,
Sodium acetate：150~450 parts,
Sodium chloride：150 parts,
Calcium chloride：10 parts,
Magnesium chloride：5 parts,
Yeast extract：25-55 parts,
Peptone：10-25 parts,
Ammonium chloride：60~280 parts；
Ferrous sulfate：1 part；
Manganese sulfate：1 part；
Wherein part is parts by weight.
6. photosynthetic bacteria as claimed in claim 1 or 2 cultivates microbial inoculum, which is characterized in that the photosynthetic bacteria freeze-dried powder microbial inoculum
In, contain protective agent.
7. photosynthetic bacteria as claimed in claim 6 cultivates microbial inoculum, which is characterized in that the protective agent contains：5-10wt% sugarcanes
Sugar, 6-10wt% milk powder (such as skimmed milk power), 0.5-2wt% maltodextrins, 0.01-0.1wt% ascorbic acid.
8. photosynthetic bacteria as claimed in claim 1 or 2 cultivates microbial inoculum, which is characterized in that the photosynthetic bacteria culture medium is each
Raw material dosage matches：
Potassium dihydrogen phosphate：10~50 parts,
Dipotassium hydrogen phosphate：5 parts,
Sodium acetate：150~450 parts,
Sodium chloride：150 parts,
Calcium chloride：10 parts,
Magnesium chloride：5 parts,
Yeast extract：25-55 parts,
Peptone：10-25 parts,
Ammonium chloride：60~280 parts；
Ferrous sulfate：1 part；
Manganese sulfate：1 part；
Wherein part is parts by weight；
In the photosynthetic bacteria freeze-dried powder microbial inoculum, contain protective agent；The protective agent is selected from the group：Sucrose, milk powder, mannose,
Trehalose, maltodextrin, ascorbic acid, sodium glutamate, or combinations thereof.
9. a kind of preparation method of photosynthetic bacteria culture microbial inoculum, which is characterized in that including step：
(1) photosynthetic bacteria culture medium and photosynthetic bacteria freeze-dried powder microbial inoculum are provided；
(2) photosynthetic bacteria culture medium in (1) and photosynthetic bacteria freeze-dried powder microbial inoculum are uniformly mixed；
(3) optionally packaging step.
10. method as claimed in claim 9, which is characterized in that the preparation method of the photosynthetic bacteria freeze-dried powder microbial inoculum includes
Step：
(i) bacterium mud is provided；
(ii) protective agent is added into the bacterium mud in (i), obtains added with protectant bacterium mud；
(iii) in (ii) added in protectant bacterium mud be added water (preferably pure water), obtain bacterium mud-protection agent solution；
(iv) pre-freeze processing is carried out to bacterium mud in (iii)-protection agent solution, obtains freeze-drying pre-freeze bacterium；
(v) frozen dried is carried out to the freeze-drying pre-freeze bacterium in (iv), obtains photosynthetic bacteria freeze-dried powder microbial inoculum.
11. application of the photosynthetic bacteria culture microbial inoculum as claimed in claim 1 or 2 in producing photosynthetic bacteria.
12. a kind of method producing photosynthetic bacteria, which is characterized in that cultivate bacterium using the photosynthetic bacteria described in claims 1 or 22
Agent.
13. method as claimed in claim 12, which is characterized in that the method includes the steps：
(a) container is provided；
(b) photosynthetic bacteria described in claim 1 culture microbial inoculum is dissolved in the container, forms solution 1；
(c) solution 1 in (b) is cultivated.
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