CN107858313A - Culture medium and cultural method for Rhodopseudomonas photosynthetic bacteria apposition growth - Google Patents
Culture medium and cultural method for Rhodopseudomonas photosynthetic bacteria apposition growth Download PDFInfo
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Abstract
The invention discloses the culture medium and cultural method for Rhodopseudomonas photosynthetic bacteria apposition growth, configures first by absolute ethyl alcohol, Ca (H2PO4)2·H2O、MgSO4·7H2O, Triammonium citrate, KH2PO4, whiting, yeast extract, glutamic acid, organic matter rot liquid and pure water composition culture medium；Again by culture medium it is unsterilised under conditions of be directly placed at opening transparent vessel in, ensure to provide suitable temperature to culture medium, illumination condition, and carry out appropriate aeration, inoculation Rhodopseudomonas photosynthetic bacteria, which is cultivated to salt crystalloid bond area on container inner wall, to increase, the thickness that red pseudomonas group is grown in the salt crystalloid of attachment can be up to 1~3mm, scrape down and obtain slimy red pseudomonas, above-mentioned cultural method is reliable and stable, raw material is easy to get, it is convenient to carry out, economic benefit is big, slimy red pseudomonas facilitates the harvest of photosynthetic bacteria and further recycling.
Description
Technical field
The invention belongs to microbial technology field, is related to microbiological culture media and cultural method, and in particular to for red vacation
The culture medium and cultural method of zygosaccharomyces photosynthetic bacteria apposition growth.
Background technology
Photosynthetic bacteria especially Rhodopseudomonas photosynthetic bacteria at present treatment of Organic Wastewater (such as soybean processing waste water,
Poultry feces, cow dung sewage, pig-farm wastewater, food and drink waste water etc.), administer heavy metal wastewater thereby, aquaculture, the neck such as livestock and poultry cultivation
Using more, it has multiple functions in domain, such as the absorption of ammoniacal nitrogen, hydrogen sulfide and other harmful substances is purified water, as
Cultivate the bait of zooplankter and as feed addictive, the biological coenzyme of production etc.；And photosynthetic bacteria can produce hydrogen,
So that in terms of new energy research highly significant.The fluid nutrient medium master of the Rhodopseudomonas photosynthetic bacteria utilized extensively at present
It is sodium acetate, yeast extract, magnesium sulfate, ammonium chloride, potassium dihydrogen phosphate and water etc. to want composition, and its solid medium main component is ox
Meat extract, peptone, sodium chloride, agar and water etc., these culture mediums for the raised growth of red pseudomonas provide sufficient carbon,
The basic elements such as nitrogen, phosphorus and other trophic factors.
The reason for Rhodopseudomonas photosynthetic bacteria is used to handle organic wastewater and mechanism also have substantial amounts of research.Conventional
Research shows that the material such as caused low molecular organic acidses, amino acid, carbohydrate can after being degraded by heterotrophicy bacteria for high molecular organic matter
To be utilized by red pseudomonas；Either under aerobic or anaerobic condition, many photosynthetic bacterias, which can be resistant to, at a relatively high to be had
Machine thing concentration, and further degradation of organic substances in the sour environment that can be formed after organic matter degradation, realize the place to organic matter
The growth of reason and thalline itself.But conventional research is substantially based on the red pseudomonas for state growth of swimming, although some are literary
The adherent growth (i.e. apposition growth) that red pseudomonas is reported in data is offered, but how red pseudomonas is realized without research
A large amount of apposition growths.
Because red pseudomonas has multiple functions, in the market has a large amount of red pseudomonas can purchase, but these red vacations
Monad is the liquid existence form containing a large amount of water, rather than is done as bacillus subtilis thalline together with related substances
Into solid-state microbial inoculum, this liquid existence form of red pseudomonas causes the concentration of wherein bacterium to reach higher, also limits
Its Conservation environment, preservation condition and how easily amount transport etc..Although can by centrifugation or other concentration techniques
To concentrate red pseudomonas to a certain extent, but it needs to consume substantial amounts of energy and time, and economic value added is not high；And
The red pseudomonas for state growth of swimming can only also be turned out by being commercially for the culture medium of rapid, high volume culture red pseudomonas, and
It is unable to the red pseudomonas of mass propgation attached state growth.So, if non-liquid form presence or mud can be directly obtained
The red pseudomonas of shape, then the concentration of red pseudomonas can be greatly enhanced, also can be the transport of red pseudomonas and enter one
Step utilizes and provides facility.
The content of the invention
To overcome the drawbacks described above of prior art, it is an object of the invention to provide for Rhodopseudomonas photosynthetic bacteria
The culture medium of apposition growth, on the other hand, the present invention also aims to provide above-mentioned to be used for Rhodopseudomonas photosynthetic bacteria
The cultural method of the culture medium of apposition growth, further, the present invention also aims to be provided with above-mentioned cultural method to prepare
Obtained Rhodopseudomonas photosynthetic bacteria.
Inventor has found a variety of large-scaled culture methods of red pseudomonas on the basis of early-stage Study, such as utilizes meal
Drink raw material mass propgation red pseudomonas based on rubbish or mixed feed, wherein be based on the thalline for state growth of swimming,
The red pseudomonas of attached state growth also occurs simultaneously；Inventor continues research and finds that by culture medium apposition growth can be obtained
Muddy Rhodopseudomonas photosynthetic bacteria, cell concentration far above swim state growth, toxigenic capacity is low, and culture side
Method is reliable and stable, raw material is easy to get, convenient to carry out, is easy to implement the further recycling of red pseudomonas.
The purpose of the present invention is achieved through the following technical solutions：
For the culture medium of Rhodopseudomonas photosynthetic bacteria apposition growth, by absolute ethyl alcohol, Ca (H2PO4)2·H2O、
MgSO4·7H2O, Triammonium citrate, KH2PO4, rot liquid and pure water of whiting, yeast extract, glutamic acid, organic matter mix
Conjunction forms；Wherein,
The addition of the absolute ethyl alcohol is 50~100mL/L,
Ca (the H2PO4)2·H2O addition is 1.4~2.8g/L,
The MgSO4·7H2O addition is 0.4~1.0g/L,
The addition of the Triammonium citrate is 5~20g/L,
The KH2PO4Addition be 0.7~3.5g/L,
The addition of the whiting is 1~5g/L,
The addition of the yeast extract is 2~5g/L,
The addition of the glutamic acid is 1~3g/L,
The addition of the rotten liquid of the organic matter is 80~100mL/L,
Surplus is pure water；And pass through H3PO4The initial pH value for adjusting the culture medium is 5.5~6.5.
The rotten liquid of the organic matter includes food garbage, animal excrements, planktonic algae wawter bloom, cereal crops, animal and matched somebody with somebody
Feed, the one or more in soybean processing waste are closed, are distributed after being mixed with water by 40~60 days putrid fermentation to water bodys smelly
Taste, and it is 1000~2500mg/L to control the total nitrogen content of the wherein non-liquid part for sinking to the bottom thing.
For the cultural method of the culture medium of Rhodopseudomonas photosynthetic bacteria apposition growth, comprise the following steps：
S1, by the good culture medium of above-mentioned configuration under conditions of unsterilised, be directly placed at opening transparent vessel in, and will
Said vesse is placed in glasshouse, ensures the intensity of illumination on daytime in 5000~60000lx；
S2, the culture medium in step S1 in container is aerated, partial air is input in culture medium, formed local
Aerobic environment；
S3, the culture medium inoculated Rhodopseudomonas photosynthetic bacteria into step S2 after aeration, the amount for being inoculated with mother liquor are 50
~100mL/L, and the cell concentration for being inoculated with instantaneous Rhodopseudomonas photosynthetic bacteria in wild Oryza species is 5.0 × 1011~2.0 ×
1012cells/L；
S4, the medium culture 35 days after being inoculated with step S3 is following, precipitation salt crystalloid on container inner wall, with salt
Crystalloid is separated out on container inner wall, adhered to, and red pseudomonas is grown on the crystal that inwall separates out, the crystal color
Gradually it is changed into kermesinus from colourless；When the red pseudomonas of apposition growth peaks, the thickness adhered on inwall is 1~3mm,
In muddy, while also growth has the Rhodopseudomonas photosynthetic bacteria for state of swimming in culture medium water body；Finally by the mud
The attachment of shape, which scrapes down, produces Rhodopseudomonas photosynthetic bacteria.
Temperature is 22~42 DEG C in opening transparent vessel described in step S1.
Aeration mode described in step S2 is to carry out local aeration using gas stone, and starts to expose when starting culture
Gas, stop aeration when culture was by 20~25 days.
Further, the salt crystalloid described in step S4 includes oxygen, hydrogen, phosphorus, magnesium, carbon, nitrogen.
Further, the precipitation salt crystalloid described in step S4 is Mg (HCO3)2(NH4)2HPO4Mixture.
Further, medium culture described in step S4 is after 1 day, is separated out on container inner wall in small salt crystalloid makes
Wall is roughening.
Further, after slimy Rhodopseudomonas photosynthetic bacteria scrapes down described in step S4, continue to add
The culture medium configured described in step S1, and it is no more than the addition described in step S1, meeting continued propagation is attached on container inner wall
The Rhodopseudomonas photosynthetic bacteria group of state.
The component and addition explanation of culture medium：The effect of absolute ethyl alcohol is except the life for Rhodopseudomonas photosynthetic bacteria
It is long to provide outside organic carbon source, it is crucial that being slightly soluble in using plurality of inorganic salt or the characteristic insoluble in ethanol, be advantageous to nothing
Reacted between machine salt generation new inorganic salts and with crystal form separate out on container inner wall；Ca(H2PO4)2·H2O is provided
Calcium, P elements；MgSO4·7H2O provides magnesium, element sulphur；Triammonium citrate is to provide inorganic nitrogen in acid condition；KH2PO4
Potassium, P elements are provided；Whiting is provided with the calcium constituent and DIC of deposit property；Yeast extract and glutamic acid provide micro-
The growth factor such as secondary element and amino acid；The rotten liquid of organic matter can be not only provided with beneficial to the micro of photosynthetic bacterium growth needs
Growth factors such as element, low molecular organic acidses and amino acid, but also contain certain microorganism, but these microorganisms with point
Based on the heterotrophicy bacteria for solving macromolecule organic, effective competition can not be formed with red pseudomonas in basal culture medium,
But engender red pseudomonas advantage.The addition scope of said components is mainly from culture effect, maintenance Medium's PH Value<
Multiple angles such as 7 and control culture medium cost are set out, when addition is on the low side, it is impossible to effectively facilitate the precipitation of inorganic salt crystal；
When addition is excessive, some of which salt reacts to each other and directly generates precipitation of salts not soluble in water, it is impossible to by photosynthetic bacteria
Effectively utilize.
In the present invention, the precipitation of salt crystalloid is committed step on container inner wall.Can not be molten using some inorganic salts
In or be slightly soluble in the characteristic of ethanol, be advantageous to precipitation, the attachment of inorganic salts crystal.Meeting between inorganic salts ingredients in culture medium
Some occur to react to each other, some salts of generation separate out crystal in the aqueous solution containing ethanol and are attached on container inner wall,
Contain the elements such as oxygen, hydrogen, phosphorus, magnesium, carbon, nitrogen by testing and analyzing the salt crystalloid, and contain ammonium ion and phosphate anion,
Concrete form is Mg (HCO3)2(NH4)2HPO4Mixture；Also have simultaneously in container in medium liquid containing trace element
And trophic factors, possess the illumination of the high concentration growth of red pseudomonas, temperature, nutriment condition on container inner wall, so as to
It is high density attached state flora to realize a large amount of apposition growths of Rhodopseudomonas photosynthetic bacteria.Salt crystalloid is gradual on container inner wall
Become pink or shallow kermesinus, be because the knot that Rhodopseudomonas photosynthetic bacteria grows on the salt crystalloid of attachment
Fruit, final Rhodopseudomonas photosynthetic bacteria form one layer of dark red color substance on container inner wall, are scraped in culture early stage with finger
It can substantially feel wherein there is coarse particles thing when taking, the salt crystalloid as separated out.
On the basis of common sense in the field is met, it is each preferably real that above-mentioned each optimum condition can produce the present invention in any combination
Example；The raw material used in the present invention and reagent are commercially available or be conventional selection in addition to having specified otherwise in addition.
Compared with prior art, the present invention has the advantages that：
1st, reliable and stable provided by the present invention for the culture medium of Rhodopseudomonas photosynthetic bacteria, incubation is simple,
Condition of culture is easily obtained and convenient to carry out, and economic benefit is big.
2nd, in breeding method of the invention, Rhodopseudomonas photosynthetic bacteria apposition growth is on container inner wall, in mud
Shape, it can directly scrape down and be far above the red pseudomonas for state growth of swimming without concentration, the wherein concentration of red pseudomonas
Cultivating system, significantly facilitate the harvest of photosynthetic bacteria and further recycling.
Brief description of the drawings
Fig. 1 is that the glass inside wall of cylinder adheres to the photo after photosynthetic bacteria is scraped with hand in embodiment 1；
Photo when left side glass jar is a large amount of apposition growths of red pseudomonas in embodiment 2 in Fig. 2；
Photo when kermesinus wide mouth glass bottle is a large amount of apposition growths of red pseudomonas in embodiment 3 in Fig. 3；
Photo when Fig. 4 is wide mouth glass bottle inwall photosynthetic bacteria apposition growth in embodiment 4；
Photo when kermesinus conical flask is a large amount of apposition growths of red pseudomonas in embodiment 5 in Fig. 5.
Embodiment
Following examples will be helpful to those skilled in the art and further understand the present invention, but not limit in any form
The present invention.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, also
It can make certain adjustments and improvements.These belong to protection scope of the present invention.
Embodiment 1
50L is configured by absolute ethyl alcohol, Ca (H2PO4)2·H2O、MgSO4·7H2O, Triammonium citrate, KH2PO4, powder formed carbon
The culture medium of the rotten liquid of sour calcium, yeast extract, glutamic acid, organic matter and pure water composition；Wherein, absolute ethyl alcohol adds in the culture medium
Measure as 100mL/L, Ca (H2PO4)2·H2O additions are 2.8g/L, MgSO4·7H2O additions are 1g/L, Triammonium citrate adds
Dosage is 20g/L, KH2PO4Addition is 3.5g/L, the addition of whiting is 5g/L, yeast extract addition is 5g/
L, glutamic acid addition be 3g/L, organic matter rot liquid addition be 100mL/L, surplus is pure water；And pass through H3PO4Regulation is just
Beginning pH value is 6.5；Wherein, organic matter rots liquid to be mixed by planktonic algae wawter bloom, mouldy flour and soybean processing waste with water
Formed afterwards by 2 months putrid fermentations, water body gives out heavier stink, and water colour is brown, and controls and therein non-sink to the bottom thing
Liquid part total nitrogen content in 2500mg/L.
In 7~August part, it is 70L water white transparencies that cumulative volume is directly placed at after unsterilised to the good culture medium of above-mentioned configuration
In glass jar, glass jar is placed in glasshouse, to ensure the intensity of illumination on daytime in 5000~60000lx, culture period
Between in container water temperature at 25~42 DEG C.2 long 4cm, diameter 2cm gas stone progress air aeration are placed in glass jar, by portion
Divide air to be input in culture medium, ensure the anaerobic environment being not all of in culture medium, form local aerobic environment；Then it is right
The commercially available Rhodopseudomonas photosynthetic bacteria of inoculation of medium, the amount for being inoculated with mother liquor are 100mL/L, and are cultivated after ensureing inoculation
The cell concentration of instantaneous red pseudomonas is 2.0 × 10 in base12cells/L。
There is small salt crystalloid after 1 day and separated out in culture on the visible flint glass inside wall of cylinder so that container inner wall becomes
It is relatively rough；Continuity over time, the salt crystalloid adhered on inwall increase.The salt crystalloid separated out on scraping container inner wall
Carry out X-ray fluorescence spectra detection and analysis, it is known that its mixture being made up of elements such as oxygen, phosphorus, magnesium, carbon, nitrogen, and obtain
The mass ratio composition of each element；The salt crystalloid of precipitation is dissolved in the laggard field headquarters of pure water and supports salt component analysis, can obtain and wherein contain
There are ammonium ion and phosphate anion；And crystal can become white by colorless solid after the crystal is placed into a period of time in atmosphere
Color solid, and the white solid is insoluble in water, and the salt crystalloid for determining whether out apposition growth on container inner wall is
Mg(HCO3)2(NH4)2HPO4Mixture.
During culture 5 days, the area increase of salt crystalloid attachment on container inner wall, the salt crystalloid of attachment gradually becomes pink
Color；When cultivating 11 days, attachment is more and more on container inner wall, and color gradually becomes kermesinus, and dark red color substance gradually expands
It is scattered on whole container inner walls below liquid level, microbiological analysis is carried out to dark red color substance using high-throughout Miseq platforms
It is determined that the kermesinus microorganism of generation is Rhodopseudomonas photosynthetic bacteria.
When culture was to the 25th day, stop aeration；When cultivating 35 days, the Rhodopseudomonas photosynthetic bacteria of above-mentioned apposition growth
It may proceed to grow into peak on container inner wall, the local thickness for adhering to red pseudomonas group reaches 3mm；And in container water body face
Color also becomes sepia, i.e., also raised growth swims the red pseudomonas of state in culture medium water body；Finally by the red of apposition growth
Pseudomonad group, which scrapes down, produces slimy attached state red pseudomonas.
After the red pseudomonas group of apposition growth is scraped down, continue the nutrient media components in appropriate addition step S1,
The amount of addition is the half in step S1, continues to obtain the attached state red pseudomonas of continued propagation.
Embodiment 2
50L is configured by absolute ethyl alcohol, Ca (H2PO4)2·H2O、MgSO4·7H2O, Triammonium citrate, KH2PO4, powder formed carbon
The culture medium of the rotten liquid of sour calcium, yeast extract, glutamic acid, organic matter and pure water composition；Wherein, absolute ethyl alcohol adds in the culture medium
Measure as 80mL/L, Ca (H2PO4)2·H2O additions are 2.3g/L, MgSO4·7H2O additions are 0.8g/L, Triammonium citrate adds
Dosage is 10g/L, KH2PO4Addition is 2.1g/L, the addition of whiting is 3g/L, yeast extract addition is 3g/
L, glutamic acid addition be 2g/L, organic matter rot liquid addition be 100mL/L, surplus is pure water；And pass through H3PO4Regulation is just
Beginning pH value is 6.0；Wherein, organic matter rots liquid to be mixed by planktonic algae wawter bloom, mouldy flour and soybean processing waste with water
Formed afterwards by 40 days putrid fermentations, water body gives out heavier stink, water colour is crineous, and controls the wherein non-thing that sinks to the bottom
Total nitrogen content in liquid is in 1800mg/L.
In 8~September part, cumulative volume 70L water white transparency glass is directly placed at after unsterilised to the good culture medium of above-mentioned configuration
In glass cylinder, glass jar is placed in glasshouse, to ensure the intensity of illumination on daytime during 5000~60000lx, culture
Water temperature is at 25~42 DEG C in container.Gas stone progress air aeration to placing 1 long 4cm, diameter 2cm in glass jar, by part
Air is input in culture medium, ensures the anaerobic environment being not all of in culture medium, but form local aerobic environment；Then
The Rhodopseudomonas photosynthetic bacteria commercially available to inoculation of medium, the amount for being inoculated with mother liquor are 100mL/L, and are trained after ensureing inoculation
The cell concentration for supporting instantaneous red pseudomonas in base is 1.0 × 1012cells/L。
There is small salt crystalloid after 1 day and separated out in culture on visible glass jar inwall so that container inner wall becomes to compare
It is coarse；Continuity over time, the salt crystalloid adhered on inwall increase.The salt crystalloid separated out on scraping container inner wall is carried out
X-ray fluorescence spectra tests and analyzes, it is known that its mixture being made up of elements such as oxygen, phosphorus, magnesium, carbon, nitrogen, and obtain each member
The mass ratio composition of element；The salt crystalloid of precipitation is dissolved in the laggard field headquarters of pure water and supports salt component analysis, can obtain and wherein contain ammonium
Radical ion and phosphate anion；And crystal can be become white admittedly by colorless solid after the crystal is placed into a period of time in atmosphere
Body, and the white solid is insoluble in water, and the salt crystalloid for determining whether out apposition growth on container inner wall is Mg
(HCO3)2(NH4)2HPO4Mixture.
During culture 7 days, the area increase of salt crystalloid attachment on container inner wall, the salt crystalloid of attachment gradually becomes shallow dark
It is red；When cultivating 11 days, attachment is more and more on container inner wall, and color gradually becomes kermesinus, and dark red color substance is gradual
It is diffused on whole container inner walls below liquid level, microorganism point is carried out to dark red color substance using high-throughout Miseq platforms
Analysis determines that the kermesinus microorganism of generation is Rhodopseudomonas photosynthetic bacteria.
When culture was to the 20th day, stop aeration；When cultivating 35 days, the Rhodopseudomonas photosynthetic bacteria of above-mentioned apposition growth
It may proceed to grow into peak on container inner wall, the thickness of local attachment red pseudomonas group reaches 3mm；And mixture in container
Color also become kermesinus, i.e., also raised growth swims the red pseudomonas of state in culture medium water body；Finally by apposition growth
Red pseudomonas group scrape down and produce slimy attached state red pseudomonas.
After the red pseudomonas group of apposition growth is scraped down, continue the nutrient media components in appropriate addition step S1,
The amount of addition is the half in step S1, continues to obtain the attached state red pseudomonas of continued propagation.
Embodiment 3
10L is configured by absolute ethyl alcohol, Ca (H2PO4)2·H2O、MgSO4·7H2O, Triammonium citrate, KH2PO4, powder formed carbon
The culture medium of the rotten liquid of sour calcium, yeast extract, glutamic acid, organic matter and pure water composition；Wherein, absolute ethyl alcohol adds in the culture medium
Measure as 70mL/L, Ca (H2PO4)2·H2O additions are 2g/L, MgSO4·7H2O additions are 1g/L, Triammonium citrate addition
For 10g/L, KH2PO4Addition is 2g/L, the addition of whiting is 3g/L, yeast extract addition is 3g/L, paddy ammonia
Sour addition is 2g/L, organic matter rots, and liquid addition is 100mL/L, and surplus is pure water；And pass through H3PO4Adjust initial pH value
For 6.0；Wherein, the rotten liquid of organic matter is by 40 after being mixed by food garbage, animal excrements and animal mixed feed with water
Its putrid fermentation forms, and water body gives out heavier stink, and water colour is yellowish-brown, and controls the non-liquid for sinking to the bottom thing therein
Total nitrogen content is in 2000mg/L.
It is directly placed at after unsterilised to the good culture medium of above-mentioned configuration in the colourless wide mouth glass bottles of 10L, May by glass
Glass bottle is placed in glasshouse, and to ensure the intensity of illumination on daytime, water temperature exists in container during 5000~60000lx, culture
22~33 DEG C.Gas stone progress air aeration to placing 1 long 4cm, diameter 2cm in vial, training is input to by partial air
Support in base, ensure the anaerobic environment being not all of in culture medium, form local aerobic environment；Then to inoculation of medium city
The Rhodopseudomonas photosynthetic bacteria sold, the amount for being inoculated with mother liquor are 80mL/L, and ensure to be inoculated with instantaneous red false single in wild Oryza species
The cell concentration of born of the same parents bacterium is 6.3 × 1011cells/L。
There is small salt crystalloid after 1 day and separated out in culture on the visible flint glass inside wall of cylinder so that container inner wall becomes
It is relatively rough；The salt crystalloid adhered to after cultivating 5 days on inwall increases.The salt crystalloid separated out on scraping container inner wall carries out X
Ray fluorescence spectra tests and analyzes, it is known that its mixture being made up of elements such as oxygen, phosphorus, magnesium, carbon, nitrogen, and obtain each member
The mass ratio composition of element；The salt crystalloid of precipitation is dissolved in the laggard field headquarters of pure water and supports salt component analysis, can obtain and wherein contain ammonium
Radical ion and phosphate anion；And crystal can be become white admittedly by colorless solid after the crystal is placed into a period of time in atmosphere
Body, and the white solid is insoluble in water, and the salt crystalloid for determining whether out apposition growth on container inner wall is Mg
(HCO3)2(NH4)2HPO4Mixture.
During culture 11 days, the area increase of salt crystalloid attachment on container inner wall, the salt crystalloid of attachment gradually becomes shallow
Kermesinus；When culture was to the 20th day, stop aeration；When cultivating 22 days, attachment is more and more on container inner wall, and color gradually becomes
Into kermesinus, and dark red color substance is gradually diffused on whole container inner walls below liquid level, utilizes high-throughout Miseq platforms
Microbiological analysis is carried out to dark red color substance and determines that the kermesinus microorganism of generation is Rhodopseudomonas photosynthetic bacteria.
When cultivating 35 days, the thickness of local attachment red pseudomonas group reaches 2mm；And the color of mixture also becomes in container
Into kermesinus, i.e., also raised growth swims the red pseudomonas of state in culture medium water body；Finally by the red false unit cell of apposition growth
Flora, which scrapes down, produces slimy attached state red pseudomonas.
After the red pseudomonas group of apposition growth is scraped down, continue the nutrient media components in appropriate addition step S1,
The amount of addition is the half in step S1, continues to obtain the attached state red pseudomonas of continued propagation.
Embodiment 4
10L is configured by absolute ethyl alcohol, Ca (H2PO4)2·H2O、MgSO4·7H2O, Triammonium citrate, KH2PO4, powder formed carbon
The culture medium of the rotten liquid of sour calcium, yeast extract, glutamic acid, organic matter and pure water composition；Wherein, absolute ethyl alcohol adds in the culture medium
Measure as 50mL/L, Ca (H2PO4)2·H2O additions are 1.4g/L, MgSO4·7H2O additions are 0.4g/L, Triammonium citrate adds
Dosage is 5g/L, KH2PO4Addition is 0.7g/L, the addition of whiting is 1g/L, yeast extract addition be 2g/L,
Glutamic acid addition is 1g/L, organic matter rots, and liquid addition is 80mL/L, and surplus is pure water；And pass through H3PO4Adjust initial pH
It is worth for 5.5；Wherein, organic matter rot liquid for after being mixed by food garbage, animal excrements and animal mixed feed with water by
Putrid fermentation forms within 50 days, and water body gives out heavier stink, and water colour is brown, and controls the non-liquid for sinking to the bottom thing therein
Total nitrogen content is in 1000mg/L.
It is directly placed at after unsterilised to the good culture medium of above-mentioned configuration in the colourless wide mouth glass bottles of 10L, will October
Wide mouth glass bottle is placed in glasshouse, to ensure the intensity of illumination on daytime during 5000~60000lx, culture in container
Water temperature is at 22~38 DEG C.Gas stone progress air aeration to placing 1 long 4cm, diameter 2cm in vial, partial air is defeated
Enter into culture medium, ensure the anaerobic environment being not all of in culture medium, form local aerobic environment；Then in culture medium
Commercially available Rhodopseudomonas photosynthetic bacteria is inoculated with, the amount for being inoculated with mother liquor is 50mL/L, and ensures to be inoculated with instantaneous in wild Oryza species
The cell concentration of red pseudomonas is 5.0 × 1011cells/L。
There is small salt crystalloid after 1 day and separated out in culture on visible flint glass bottle inwall so that container inner wall becomes
It is relatively rough；The salt crystalloid adhered to after cultivating 10 days on inwall increases.The salt crystalloid separated out on scraping container inner wall carries out X
Ray fluorescence spectra tests and analyzes, it is known that its mixture being made up of elements such as oxygen, phosphorus, magnesium, carbon, nitrogen, and obtain each member
The mass ratio composition of element；The salt crystalloid of precipitation is dissolved in the laggard field headquarters of pure water and supports salt component analysis, can obtain and wherein contain ammonium
Radical ion and phosphate anion；And crystal can be become white admittedly by colorless solid after the crystal is placed into a period of time in atmosphere
Body, and the white solid is insoluble in water, and the salt crystalloid for determining whether out apposition growth on container inner wall is Mg
(HCO3)2(NH4)2HPO4Mixture.
When cultivating 11 days, the area of salt crystalloid attachment increases on container inner wall, is gradually become in the salt crystalloid of attachment
Pink；When culture was to the 20th day, stop aeration；When cultivating 22 days, attachment is more and more on container inner wall, and color gradually becomes
Into kermesinus, and dark red color substance is gradually diffused into below liquid level on whole container inner walls, utilizes high-throughout Miseq platforms pair
Dark red color substance carries out microbiological analysis and determines that the kermesinus microorganism of generation is Rhodopseudomonas photosynthetic bacteria.
When cultivating 30 days, the Rhodopseudomonas photosynthetic bacteria of above-mentioned apposition growth may proceed to grow on container inner wall,
The thickness in thickness reaches 1mm；And the color of mixture also becomes kermesinus in container, i.e., also raised growth floats in culture medium water body
Swim the red pseudomonas of state；Finally the red pseudomonas group of apposition growth is scraped down and produces the red false list of slimy attached state
Born of the same parents bacterium.
After the red pseudomonas group of apposition growth is scraped down, continue the nutrient media components in appropriate addition step S1,
The amount of addition is the half in step S1, continues to obtain the attached state red pseudomonas of continued propagation.
Embodiment 5
6L is configured by absolute ethyl alcohol, Ca (H2PO4)2·H2O、MgSO4·7H2O, Triammonium citrate, KH2PO4, powder formed carbon
The culture medium of the rotten liquid of sour calcium, yeast extract, glutamic acid, organic matter and pure water composition；Wherein, absolute ethyl alcohol adds in the culture medium
Measure as 60mL/L, Ca (H2PO4)2·H2O additions are 1.8g/L, MgSO4·7H2O additions are 0.5g/L, Triammonium citrate adds
Dosage is 10g/L, KH2PO4Addition is 2g/L, the addition of whiting is 2g/L, yeast extract addition be 4g/L,
Glutamic acid addition is 2g/L, organic matter rots, and liquid addition is 90mL/L, and surplus is pure water；And pass through H3PO4Adjust initial pH
It is worth for 5.8；Wherein, organic matter rot liquid for after being mixed by food garbage, animal excrements and animal mixed feed with water by
Putrid fermentation forms within 50 days, and water body gives out heavier stink, and water colour is crineous, and controls the non-liquid for sinking to the bottom thing therein
Total nitrogen content in 1500mg/L.
Cumulative volume 6.5L colourless transparent glass conical flask is directly placed at after unsterilised to the good culture medium of above-mentioned configuration
In, conical flask is placed in glasshouse in August part, to ensure the intensity of illumination on daytime in 5000~60000lx, culture period
Between in container water temperature at 22~42 DEG C.Gas stone progress air aeration to placing 1 long 4cm, diameter 2cm in conical flask, by portion
Divide air to be input in culture medium, ensure the anaerobic environment being not all of in culture medium, form local aerobic environment；Then it is right
The commercially available Rhodopseudomonas photosynthetic bacteria of inoculation of medium, the amount for being inoculated with mother liquor are 60mL/L, and are cultivated after ensureing inoculation
The cell concentration of instantaneous red pseudomonas is 1.0 × 10 in base12cells/L。
There is small salt crystalloid after 1 day and separated out in culture on visible colourless conical flask inwall so that container inner wall becomes
It is relatively rough；The salt crystalloid adhered to after cultivating 8 days on inwall increases.The salt crystalloid separated out on scraping container inner wall carries out X
Ray fluorescence spectra tests and analyzes, it is known that its mixture being made up of elements such as oxygen, phosphorus, magnesium, carbon, nitrogen, and obtain each member
The mass ratio composition of element；The salt crystalloid of precipitation is dissolved in the laggard field headquarters of pure water and supports salt component analysis, can obtain and wherein contain ammonium
Radical ion and phosphate anion；And crystal can be become white admittedly by colorless solid after the crystal is placed into a period of time in atmosphere
Body, and the white solid is insoluble in water, and the salt crystalloid for determining whether out apposition growth on container inner wall is Mg
(HCO3)2(NH4)2HPO4Mixture.
When cultivating 11 days, the area of salt crystalloid attachment increases on container inner wall, is gradually become in the salt crystalloid of attachment
Shallow kermesinus；When cultivating 20 days, stopping aeration, attachment is more and more on container inner wall, and color gradually becomes kermesinus, and secretly
Red material is gradually diffused into below liquid level on whole container inner walls, and dark red color substance is entered using high-throughout Miseq platforms
Row microbiological analysis obtains, and the kermesinus microorganism of generation is Rhodopseudomonas photosynthetic bacteria.
When cultivating 30 days, the Rhodopseudomonas photosynthetic bacteria of above-mentioned apposition growth may proceed to grow on container inner wall,
The thickness in thickness reaches 1.5mm；And the color of mixture also becomes kermesinus in container, i.e., also raised growth in culture medium water body
The red pseudomonas for state of swimming；Finally the red pseudomonas group of apposition growth is scraped down and produces the red vacation of slimy attached state
Monad.
After the red pseudomonas group of apposition growth is scraped down, continue the nutrient media components in appropriate addition step S1,
The amount of addition is the half in step S1, continues to obtain the attached state red pseudomonas of continued propagation.
Described above is presently preferred embodiments of the present invention, but the present invention should not be limited to disclosed in the embodiment
Content.So every do not depart from the lower equivalent or modification completed of spirit disclosed in this invention, the model that the present invention protects is both fallen within
Enclose.
Claims (10)
1. the culture medium for Rhodopseudomonas photosynthetic bacteria apposition growth, it is characterised in that by absolute ethyl alcohol, Ca
(H2PO4)2·H2O、MgSO4·7H2O, Triammonium citrate, KH2PO4, whiting, yeast extract, glutamic acid, organic matter it is rotten
Rotten liquid and pure water mix；Wherein,
The addition of the absolute ethyl alcohol is 50~100mL/L,
Ca (the H2PO4)2·H2O addition is 1.4~2.8g/L,
The MgSO4·7H2O addition is 0.4~1.0g/L,
The addition of the Triammonium citrate is 5~20g/L,
The KH2PO4Addition be 0.7~3.5g/L,
The addition of the whiting is 1~5g/L,
The addition of the yeast extract is 2~5g/L,
The addition of the glutamic acid is 1~3g/L,
The addition of the rotten liquid of the organic matter is 80~100mL/L,
Surplus is pure water；And pass through H3PO4The initial pH value for adjusting the culture medium is 5.5~6.5.
2. it is used for the culture medium of Rhodopseudomonas photosynthetic bacteria apposition growth according to claim 1, it is characterised in that institute
Stating the rotten liquid of organic matter includes food garbage, animal excrements, planktonic algae wawter bloom, cereal crops, animal mixed feed, soybean
One or more in processing waste mix with water, give an offensive smell and obtain to water body by 40~60 days putrid fermentations, control
The total nitrogen content of the wherein non-liquid part for sinking to the bottom thing is 1000~2500mg/L.
3. the cultural method of the culture medium according to claim 1 or claim 2 for Rhodopseudomonas photosynthetic bacteria apposition growth,
It is characterised in that it includes following steps：
S1, by the good culture medium of above-mentioned configuration under conditions of unsterilised, be directly placed in opening transparent vessel, and will be above-mentioned
Container, which is placed in glasshouse, ensures the intensity of illumination on daytime in 5000~60000lx；
S2, the culture medium in step S1 in container is aerated, partial air is input in culture medium, formed local aerobic
Environment；
S3, be aerated into step S2 after culture medium inoculated Rhodopseudomonas photosynthetic bacteria, be inoculated with the amount of mother liquor for 50~
100mL/L, and the cell concentration for being inoculated with instantaneous Rhodopseudomonas photosynthetic bacteria in wild Oryza species is 5.0 × 1011~2.0 ×
1012cells/L；
It is S4, the medium culture 35 days after being inoculated with step S3 is following, salt crystalloid is separated out on container inner wall, with salt crystalline substance
Body is separated out on container inner wall, adhered to, and red pseudomonas is grown on the crystal that inwall separates out, and the crystal color is gradual
It is changed into kermesinus from colourless；When the red pseudomonas of apposition growth peaks, the thickness adhered on inwall is 1~3mm, in mud
Pulpous state, while also growth has the Rhodopseudomonas photosynthetic bacteria for state of swimming in culture medium water body；Finally will be described slimy
Attachment, which scrapes down, produces Rhodopseudomonas photosynthetic bacteria.
4. it is used for the cultural method of the culture medium of Rhodopseudomonas photosynthetic bacteria apposition growth according to claim 3, its
It is characterised by, the temperature being open described in step S1 in transparent vessel is 22~42 DEG C.
5. it is used for the cultural method of the culture medium of Rhodopseudomonas photosynthetic bacteria apposition growth according to claim 3, its
It is characterised by, the aeration mode described in step S2 is to carry out local aeration using gas stone, and starts to expose when starting culture
Gas, stop aeration when culture was by 20~25 days.
6. it is used for the cultural method of the culture medium of Rhodopseudomonas photosynthetic bacteria apposition growth according to claim 3, its
It is characterised by, salt crystalloid described in step S4 includes oxygen, hydrogen, phosphorus, magnesium, carbon, nitrogen.
7. it is used for the cultural method of the culture medium of Rhodopseudomonas photosynthetic bacteria apposition growth according to claim 6, its
It is characterised by, the precipitation salt crystalloid described in step S4 is Mg (HCO3)2(NH4)2HPO4Mixture.
8. it is used for the cultural method of the culture medium of Rhodopseudomonas photosynthetic bacteria apposition growth according to claim 6, its
It is characterised by, medium culture described in step S4 separates out small salt crystalloid after 1 day, on container inner wall makes inwall thicker
It is rough.
9. it is used for the cultural method of the culture medium of Rhodopseudomonas photosynthetic bacteria apposition growth according to claim 3, its
It is characterised by, after slimy Rhodopseudomonas photosynthetic bacteria scrapes down described in step S4, continues to add step S1 institutes
The culture medium configured is stated, and is no more than addition described in step S1, the red false unit cell of continued propagation attached state on container inner wall
Pseudomonas photosynthetic bacteria group.
10. a kind of Rhodopseudomonas photosynthetic bacteria, it is characterised in that as the cultural method described in any one of claim 3~9
Culture obtains.
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