CN109762756A - It is a kind of efficient photosynthetic bacteria to be inhibited to become the method for the adherent Liquid Culture photosynthetic bacteria of light - Google Patents
It is a kind of efficient photosynthetic bacteria to be inhibited to become the method for the adherent Liquid Culture photosynthetic bacteria of light Download PDFInfo
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Abstract
Photosynthetic bacteria is efficiently inhibited to become the method for the adherent Liquid Culture photosynthetic bacteria of light the invention discloses a kind of, that photosynthetic bacteria is difficult to be utilized, dispersed favorable solubility, the mounting medium that media flow is mutually good, nontoxic in a liquid are added in photosynthetic bacteria liquid culture medium process for preparation, mounting medium includes one of xanthan gum, carragheen and gelatin or a variety of, is then sterilized, is inoculated with and illumination cultivation to culture medium.There are good dissolution characteristics using xanthan gum, carragheen and gelatin in the present invention; they are dispersed in photosynthetic bacteria culture medium; form colloidal solution; culture medium has preferable mobility; photosynthetic bacterial thallus is adsorbed on xanthan gum, carragheen and gelatin in suspension growth during the cultivation process; to inhibit the adherent effect of photosynthetic bacteria, it is conducive to scale high-efficient culture.
Description
Technical field
The present invention relates to a kind of technical field of microbiology, a kind of efficiently inhibition in photosynthetic bacteria production process is particularly belonged to
Photosynthetic bacteria becomes the method for the adherent Liquid Culture photosynthetic bacteria of light.
Background technique
Currently, Anoxygenic photosynthetic bacteria (Anoxygenic Phototrophic Bacteria, APB) is one kind in anaerobism
Under the conditions of, photosynthesis is carried out as electron donor using various organic matters or inorganic matter but does not discharge the prokaryotic micro-organisms of oxygen
General name, habit be known as " photosynthetic bacteria (PSB) ".
Existing research shows that photosynthetic bacteria monoid has more than more than 80 categories 200, photosynthetic bacteria is as a kind of important microorganism
Resource, in culture fishery (bait, additive of bait and breeding water body cleanser etc.), agricultural, (microbial manure and plant are raw
Long regulator etc.), animal husbandry (additive for microbe feedstuff), pollution environment biological prosthetic and administer, medicines and health protection and energy
The fields such as source (producing hydrogen) have further investigation and application, and in the side such as microorganism resource, production equipment, preparation and product and application
Face achieves major progress, but is directed to the demand of production practices and development, it is still necessary to put forth effort to develop new characteristic photosynthetic bacteria strain
Resource, large-scale production specificity is strong, effect is obvious, product and preparation of high quality, high stability, and wherein large-scale production is
Meet key link and the basis of production development demand.
The development of bioreactor be the key that solve photosynthetic bacteria pilot scale culture, and in reactor " light " it is reasonable
Distribution and efficient an important factor for being developed using the core for being bioreactor and limitation enlargement.It has designed and has opened at present
The bioreactor of diversified forms is sent out.From the appearance, sum up mainly includes column (tank) formula, plate (box) formula, pipe
The combination and deformation of formula (culture tube coils in many ways) and this 3 class formation are (such as: multiple field, circulation pattern and coil type disk
Around shape etc.) bioreactor.From the point of view of the illumination methods of light source, bioreactor includes external light source, built-in light source
3 seed types such as combine with built-in external light source.Such as: publication number is respectively CN101402915A (2008), CN2199988
In the Chinese patents such as (1994), CN201301313 (2008), the light source of reactor design is reactor central in-house light source
And/or external light source, but due to being limited by light source irradiation light path, the volume of this kind of bioreactor is not too large, difficult
To reach the requirement of ability for mass production.Multi-point source and array light source design especially inside bioreactor, such as: public
The number of opening is respectively CN 2918431Y (2006), CN 1088893 (2005), CN 2301448 (1997), CN
202415537U (2012) etc. and Yang Suping patent (CN 201210480354.1) etc. are provided with multiple spot in culture apparatus
Multi-point source is evenly distributed in reactor by light source or array light source, design feature, and lighting is uniform, if pot type is reacted
Device increases the quantity of light source, and reactor inside diameter can increase substantially, if do not consider using sunlight, receiving can be used
The higher metal material of pressure resistance and according to yield needs, realizes the design and manufacture of the large-tonnage of reactor, therefore, this multiple spot light
The design of source or array light source meets the direction of bioreactor large-scale development.It is built-in in Photoreactor operational process
Light source reactor encounters a bottleneck problem again.Photosynthetic bacteria has phototaxis or even near-wall air curtain extremely serious, photosynthetic bacteria
It is grown in Photoreactor inner wall and the veneer of light source pipe outer wall, has severely impacted the penetrance of light, constrained photosynthetic bacteria
Growth rate.Solve the problems, such as that this strategy mainly devises the washing and brushing device of adherent photosynthetic bacteria in the reactor at present,
Such as (CN101402915A, 2008), Zhang Wendi (CN such as Huanghai Sea intelligence (CN2616547Y, 2003), yellow rising sun heros
202415537U, 2012) and Chinese patents such as Yang Suping etc. (CN 201210480354.1), their photoproduction in design
Cleaning brush is driven using the mechanically or magnetically modes such as power in object reactor, removes adherent thallus.Problem is this with cleaning patch
The photosynthetic bacteria bioreactor volume of wall device is not easy too big, it is difficult to accomplish scale production as non-photosynthetic bacterium.
To find out its cause, mainly photosynthetic organism is in reaction operational process, if bioreactor tonnage is larger, it is difficult to will be in reactor
The removal of the photosynthetic bacteria of wall and numerous light source table face paste walls.Thus it analyzes, that designs at present has adherent thallus cleaning device
Reactor is difficult to realize the efficient large-scale culture of photosynthetic bacteria.
By literature search, photosynthetic bacteria becomes the method for the adherent removal of light at present, and point of penetration is primary concern is that in reactor
The adherent cleaning device of photosynthetic bacteria is installed in upper design, and opposite have ignored inhibits photosynthetic from improving the consideration of starting with of bacteria culture media angle
Bacterium becomes the adherent effect of light.Although Chinese patent (CN102477406A, 2011), which once disclosed one kind, prevents Purple Non-sulfur
Bacterium adhere-wall culture base, inventor adds 0.3%~0.5% spaonin powder in conventional medium can inhibit purple nonsulfur bacteria
Adherent growth, but the description in relation to inhibiting bacterium adherent is had not seen from the embodiment of the patent.Furthermore spaonin powder is numerous objects
The mixture of matter has sterilization and a variety of effects such as surfactant, has to many bacteriums, fungi and inhibits growth and sterilization
Effect, in the conventional cognitive of technical staff, adding spaonin powder in the medium should be inhibited to the growth of bacterium.
Have using the microorganism remediation technologies such as microorganism formulation purifying aquatic water and control culture diseases unique excellent
Gesture is widely used to culture fishery, and has played important function.Anoxygenic photosynthetic bacteria bacterial strain multiplicity, especially marsh
Red pseudomonas does not strive oxygen since its oxygen availability is low with aquiculture animal, and can efficiently utilize or catabolism water body in
Noxious material, such as ammonia nitrogen, nitrite nitrogen, hydrogen sulfide etc., secondly, its thallus is rich in protein, carotenoid, vitamin, auxiliary
The nutrition such as enzyme Q, growth promotion and the ingredients such as disease-resistant have been widely applied always but also as bait, therefore since the eighties in last century
In culture fishery, the effect of its important regulating and controlling is played.But photosynthetic bacteria currently used in the market is derived mainly from fresh water or Lu Yuan
Strain, and source Yu Haiyang and seldom for the strain (strain) of sea-farming hypersaline environment water remediation.Inventor was once
Separation obtains the purple sulfur bacteria YL28 of one plant of salt tolerant from Quanzhou, Fujian Luoyang Bridge mangrove environment, is accredited as ocean chomophoric bacterium
(Marichromatium gracile), deposit number CGMCC 7.313, the strain growth is fast, adaptable, can efficiently remove
Inorganic tri-state nitrogen in Marine water, and can be grown using nitrite and nitrate as only nitrogen source, and have to nitrite nitrogen
There is efficient removal ability.But the bacterium, during illumination cultivation, the light that becomes is adherent serious, and adherent rate is even as high as in reaction vessel
The 70% of total Fungal biodiversity, is unfavorable for scale efficient liquid culture.Therefore, the invention patent for photosynthetic bacteria it is adherent this
One is universal and serious problems, photosynthetic carefully with ocean chomophoric bacterium (salt tolerant marine species) and this two plants of Rhodopseudomonas palustris (fresh water species)
Bacterium is culture bacterial strain, starts in terms of improvement bacteria culture media inhibition photosynthetic bacteria becomes the adherent effect of light and is studied.
Summary of the invention
In view of this, the purpose of the present invention is to provide the adherent effects that one kind does not influence thalli growth, inhibition photosynthetic bacteria
The one kind answered efficiently inhibits photosynthetic bacteria to become the method for the adherent Liquid Culture photosynthetic bacteria of light.
In order to achieve the above objectives, the technical scheme is that
It is a kind of it is efficient inhibit photosynthetic bacteria to become the method for the adherent Liquid Culture photosynthetic bacteria of light, in photosynthetic bacteria liquid culture
Add during basigamy system it is that photosynthetic bacteria is difficult to be utilized, in a liquid dispersed favorable solubility, media flow it is mutually good,
Nontoxic mounting medium, mounting medium includes one of xanthan gum, carragheen and gelatin or a variety of, then to culture medium
It sterilized, be inoculated with and illumination cultivation.
Further, in photosynthetic bacteria liquid culture medium added with mass-volume concentration range be 0.05% (m/v)~
The mounting medium of 0.5% (m/v).
Further, in photosynthetic bacteria liquid culture medium illumination cultivation photosynthetic bacteria, intensity of illumination be 1000lx~
5000lx。
It further, is 0.25% (m/v)~0.35% added with mass-volume concentration in photosynthetic bacteria liquid culture medium
(m/v) gelatin or 0.25% (m/v)~0.30% (m/v) carragheen of xanthan gum, 0.30% (m/v)~0.5% (m/v).
Further, in photosynthetic bacteria liquid culture medium process for preparation, first mounting medium is dissolved in water, consolidate
Determine medium solution, then use mounting medium solution as the solvent for preparing culture medium, according to conventional photosynthetic bacteria culture medium formula and
Preparation method carries out the preparation of the culture medium containing mounting medium.
Further, mounting medium is xanthan gum, when dissolving xanthan gum, under agitation, by powdered xanthan
Glue is added in the room temperature water of 80~85% final volumes in a small amount of multiple mode, it is stirred at room temperature 10~after twenty minutes, it is heated to
It 50~60 DEG C, persistently stirs 10~20 minutes and sufficiently dissolves to get xanthan gum solution is arrived.
Further, mounting medium is carragheen or gelatin, and carragheen or gelatin are added to the room of 20~30% final volumes
In warm water, 80~90 DEG C are heated with stirring to, lasting stirring heat preservation sufficiently dissolution in 10~20 minutes stops heating, adds while hot
Room temperature water, and be stirred continuously, it is diluted to amount of liquid and accounts for 80~85% final volumes, be cooled to 30~45 DEG C, obtain carragheen
Solution or gelatin solution.
Further, the photosynthetic bacteria liquid culture medium is suitble to fresh water species photosynthetic bacteria or salt tolerant marine species photosynthetic thin
The culture of bacterium.
Further, the fresh water monoid photosynthetic bacteria is Rhodopseudomonas palustris, the salt tolerant marine species photosynthetic bacteria
For ocean chomophoric bacterium.
The adherent Liquid Culture photosynthetic bacteria of light after adopting the above technical scheme, a kind of efficiently inhibition photosynthetic bacteria of the present invention becomes
Method, inhibit the adherent principle of photosynthetic bacteria to be: xanthan gum, carragheen and gelatin have good dissolution characteristics, they
It is dispersed in photosynthetic bacteria culture medium, forms colloidal solution, culture medium has preferable mobility, photosynthetic thin during the cultivation process
Bacterium thallus is adsorbed on xanthan gum, carragheen and gelatin in suspension growth, to inhibit the adherent effect of photosynthetic bacteria.With existing skill
Art is compared, and the present invention has the advantage of following five aspect:
(1) the adherent method of photosynthetic bacteria is solved at present mainly on bioreactor equipped with dress of scrubbing or rub
It sets, is scrubbed adherent photosynthetic bacteria in bacteria suspension by mechanical friction mode, and the present invention inhibits the adherent side of photosynthetic bacteria
Method, cost is lower, and the operation is more convenient.
(2) photosynthetic bacteria has many application values such as photosynthetic-hydrogen-production and wastewater treatment, limits the key technology of its application
One of be photosynthetic bacteria large-scale culture.The design concept of built-in multi-point source or built-in array light source, photo-biological are installed
Reactor is easily achieved designing and manufacturing for large-tonnage, but since the photosynthetic bacteria light that becomes is adherent serious, is difficult to realize photosynthetic thin
The large-scale culture of bacterium.Currently, equipped with scrubbing or the bioreactor of rubbing device, it is difficult due to the limitation of technology and cost
To realize the manufacture of large-tonnage, and the present invention obviously inhibits photosynthetic bacteria adherent growth, safe and non-toxic, is more advantageous to scale height
Effect culture.
(3) Chinese patent (CN102477406A), which once disclosed one kind, prevents purple nonsulfur bacteria adhere-wall culture base, hair
Bright person adds 0.3%~0.5% spaonin powder in conventional medium can inhibit the adherent growth of purple nonsulfur bacteria, but specially from this
The description in relation to inhibiting bacterium adherent is had not seen in the embodiment of benefit.Furthermore document report spaonin powder has sterilization and surface living
Property a variety of effects such as agent, have the function of inhibiting growth and sterilization to many bacteriums, fungi, thus speculate, add in the medium
It, should be inhibited to photosynthetic bacterium growth when high concentration spaonin powder.We cultivate by test in purple sulfur bacteria
Spaonin powder is added in base, ocean chomophoric bacterium YL28 is significantly inhibited, and adds 0.3% and 0.5% spaonin powder, YL28
The inhibiting rate of growth is respectively 37.8% and 85.4%, this may be that the bacterial strain is more sensitive to spaonin powder.And the present invention uses
Xanthan gum, carragheen and gelatin etc. be food grade materials, it is safe and non-toxic, do not inhibit bacterial growth.The present invention be also xanthan gum,
The application of carragheen and gelatin provides a kind of new purposes, i.e., photosynthetic bacteria is inhibited to become light patch in photosynthetic bacteria incubation
Wall, therefore can be applied to photosynthetic bacteria product production field.
(4) the method for the present invention is easy to operate, is applicable in the rule of the Different groups photosynthetic bacteria such as Yu Haiyang's Facultative Halophiles and fresh water bacterium
Modelling and high density liquid pure culture.
(5) it inhibits the photosynthetic bacteria light that becomes adherent, reduces block effect of the adherent thallus to light, thus the method for the present invention
The light transmittance of bioreactor can be effectively improved.
Detailed description of the invention
Fig. 1 is that xanthan gum is adherent to ocean chomophoric bacterium YL28 thallus in the present invention, suspend and the influence of total biomass is illustrated
Figure；
Fig. 2 influence schematic diagram adherent to ocean chomophoric bacterium YL28 thallus, suspension and total biomass for gelatin in the present invention；
Fig. 3 is that carragheen is adherent to ocean chomophoric bacterium YL28 thallus in the present invention, suspend and the influence of total biomass is illustrated
Figure.
Specific embodiment
In order to further explain the technical solution of the present invention, being explained in detail below by specific embodiment the present invention
It states.
One, culture medium is prepared
(1) purple sulfur bacteria conventional medium formula (formula 1) and preparation
Formula: sodium chloride: 30.0g/L；Anhydrous sodium acetate: 2.0g/L；Potassium chloride: 0.34g/L；Magnesium chloride: 1.50g/L；
Ammonium chloride: 0.34g/L；Yeast powder: 2.0g/L；Sodium bicarbonate: 1.5g/L；Calcium chloride: 0.2g/L；Sodium thiosulfate: 0.2g/L；
Trace element solution: 1.0mL/L；Phosphorus liquid 100mL/L.
Trace element solution: sodium molybdate 20.0mg/L；Nickel chloride 20.0mg/L；Copper sulphate 1.0mg/L；Ferrous sulfate heptahydrate
0.8g/L；Boric acid 300.0mg/L；Zinc sulfate 30.0mg/L；Manganese chloride 40.0mg/L.
Phosphorus liquid: taking potassium dihydrogen phosphate 3.4g, is dissolved in 1L water and is configured to 3.4g/L potassium dihydrogen phosphate, and 121 DEG C go out
Bacterium 30 minutes, cooling was spare.
Preparation method (for preparing 1L culture medium): the room temperature water (solvent) of about 800mL is taken to remove under agitation
Outside phosphorus liquid, successively each reagent is added in the room temperature water of 800mL by formula ratio and sequence, after a kind of dissolution of reagent again plus another kind
Reagent, finally plus room temperature water complements to 900mL, moist heat sterilization 30 minutes at 121 DEG C, cooling, then plus 100mL phosphorus liquid, shake up
Purple sulfur bacteria conventional medium is obtained, the pH value of the culture medium is about 7.0~7.2.
Purple sulfur bacteria conventional medium belongs to high salt culture medium, can be used for salt tolerant marine species ocean chomophoric bacterium YL28 bacterial strain
The culture of (deposit number CGMCC 7.313).The YL28 bacterial strain is separated by this room and is identified, being detailed in document, " plant height contains rhodopin
Mangrove ocean purple sulfur bacteria separation identification and characteristic, microorganism journal, 2011,51 (10): 1318-1325 "
(2) purple nonsulfur bacteria conventional medium formula (formula 2) and preparation
Formula: anhydrous sodium acetate: 2.46g/L；Ammonium chloride: 0.54g/L；Yeast powder: 1.0g/L；Anhydrous calcium chloride:
0.075g/L；Magnesium sulfate: 0.2g/L；Ferrous sulfate 28.0m g/L；Trace element solution: 0.1mL/L；Phosphorus liquid: 100mL/L.
The formula of trace element solution: boric acid 280.0mg/L；Copper sulphate 0.65g/L；Sodium molybdate 6.0g/L；Manganese sulfate
9.8g/L；Zinc sulfate 2.4g/L.
Phosphorus liquid is prepared: being configured to containing 6.0g/L potassium dihydrogen phosphate and 9.0g/L dipotassium hydrogen phosphate phosphoric acid solution, at 121 DEG C
Moist heat sterilization 30 minutes, cooling was spare.
Purple nonsulfur bacteria conventional medium preparation method (for preparing 1L): taking about 800mL room temperature water (solvent),
Under stirring condition, each reagent outside dephosphorization liquid is sequentially added by formula ratio and requirement, it is again plus another after a kind of reagent dissolution
Kind reagent, finally plus room temperature water complements to 900mL, moist heat sterilization 30 minutes at 121 DEG C, cooling, adds 100mL phosphorus liquid,
It shakes up, obtains purple nonsulfur bacteria conventional medium, the pH value of the culture medium is about 7.0~7.2.In the present embodiment, phosphorus liquid
It is eventually adding, avoids the temperature in sterilization process very high, phosphate and calcium salt easily form precipitating.
Purple nonsulfur bacteria conventional medium formula belongs to less salt culture medium, can be used for fresh water species Rhodopseudomonas palustris
The culture of CQV97 bacterial strain (deposit number MCCC1I00117) or other Rhodopseudomonas palustris bacterial strains (such as CGMCC 1.2180).
(3) it is prepared containing 0.3% carragheen photosynthetic bacteria culture medium
For preparing final volume 1.0L culture medium: weighing 3.0g carragheen, be added to 200mL (account for final volume 20%)
In room temperature water, 80 DEG C~90 DEG C are heated with stirring to, heat preservation is persistently stirred 10~20 minutes, and mass-volume concentration 1.5% is configured to
(w/v) the first carrageenan solutions are added room temperature distilled water under agitation while hot, the first carrageenan solutions are diluted to
800mL obtains the second carrageenan solutions, and the amount of liquid of 800mL solution accounts for 80% final volume, and the second carrageenan solutions are cooled to 30
~45 DEG C.In the present invention, the first carrageenan solutions of 1.5% (w/v) indicate to contain 1.5g carragheen in 100mL solvent.
It is illustrated for using 1 progress culture medium preparation of formula below, using the second carrageenan solutions of 800mL as matching
The solvent of culture medium processed is added by above-mentioned purple sulfur bacteria conventional medium formula (formula 1) and requirement under agitation
Enter each reagent, after completely dissolution, with room temperature water deficiency to 900mL, then conventional sterilant, i.e., the moist heat sterilization at 121 DEG C
30min is then cooled to room temperature, and adds the sterilized phosphorus liquid of 100mL, and the card of mass-volume concentration 0.3% (w/v) is made
Glue photosynthetic bacteria culture medium is drawn, the pH value of the culture medium is about 7.0~7.2.In the present invention, 0.3% (w/v) carragheen is photosynthetic thin
Bacterium culture medium indicates to contain 0.3g carragheen in 100mL culture medium.
(4) it is prepared containing 0.3% xanthan gum photosynthetic bacteria culture medium
For preparing final volume 1.0L culture medium: 3.0g xanthan gum is weighed, it is under agitation, slowly (i.e. a small amount of more
Secondary mode) it is added to 800mL room temperature water, the water of 800mL room temperature water accounts for 80% final volume, is stirred at room temperature after ten minutes, adds
Heat stirs 10~20 minutes persistently to 50 DEG C~60 DEG C to get xanthan gum solution.
It is illustrated for using 2 progress culture medium preparation of formula below, using xanthan gum solution obtained as preparation
The solvent of culture medium.800mL xanthan gum solution is taken, under agitation, outside dephosphorization liquid, by above-mentioned purple nonsulfur bacteria culture medium
Each reagent is added in formula (formula 2), after completely dissolution, complements to 900mL with room temperature water, cold routinely after 121 DEG C of sterilizing 30min
But to room temperature, sterilized phosphorus liquid 100mL is added, the photosynthetic bacteria of the xanthan gum of mass-volume concentration 0.3% (w/v) is made
Culture medium, pH value are about 7.0~7.2.In the present invention, 0.3% (w/v) xanthan gum photosynthetic bacteria culture medium indicates 100mL culture
Contain 0.3g xanthan gum in base.
It should be noted that (one) above-mentioned xanthan gum and carragheen are photosynthetic bacteria (such as ocean chomophoric bacterium YL28 bacterial strain
Or Rhodopseudomonas palustris CQV97 bacterial strain) be difficult to be utilized, dispersed favorable solubility, media flow phase in a liquid
Well, nontoxic mounting medium, the present invention in, mounting medium can also be gelatin；(2) concentration of mounting medium (is used
Amount) it can be adjusted according to actual needs；(3) formula 1 can be used in photosynthetic bacteria culture medium containing mounting medium or formula 2 adds
Add one of gelatin, carragheen and gelatin or a variety of is prepared.
Two, photosynthetic bacteria is cultivated
Embodiment one
It adds xanthan gum using formula 1 to be prepared, with reference to above-mentioned " preparing containing 0.3% xanthan gum photosynthetic bacteria culture medium "
Method, addition reagent 2 become formula 1 by formula, adjust xanthan gum dosage prepare containing 0.05% (w/v), 0.25% (w/v),
The purple sulfur bacteria culture medium of 0.35% (w/v) and 0.50% (w/v) xanthan gum are accordingly dispensed into the blue lid that specification is 500mL
In culture bottle (it is 530mL that blue lid culture bottle, which fills the total measurement (volume) of liquid), per bottled 500mL culture medium, sterilizing is cooled to room temperature；
It is inoculated with ocean chomophoric bacterium YL28 (deposit number CGMCC7.313) bacteria suspension, strain is inoculated into the indigo plant containing 500mL culture medium
In lid bottle, inoculum concentration accounts for 3.0% (v/v) of total measurement (volume) (530mL), then fills blue lid culture bottle with same medium, covers close
Envelope, shakes up, total measurement (volume) 530mL.Each processing repeats 3 bottles, (is prepared using formula 1 with the culture medium without xanthan gum
Culture medium) be control.
Each culture bottle of inoculation is placed in 32 DEG C, is cultivated under 3000lx intensity of illumination, it is sooner or later each to shake once, it is continuous to cultivate
5 days.The bacteria suspension of each culture bottle is poured into other container, for measuring suspended biomass in bacteria suspension.Each culture bottle is then
Mass concentration 3.0%NaCl solution is added, scrubs adherent thallus into NaCl solution with hairbrush, 530mL is settled to, for surveying
Fixed adherent biomass, the sum of suspended biomass and adherent biomass are total biomass.Before measurement, sample collects bacterium through high speed centrifugation
Body, the remaining xanthan gum with 3.0%NaCl solution washing thalline 2 times, in removal system.With the absorbance value at 660nm
(OD660) biomass of each culture bottle is represented, absorbance value is bigger, and it is more to represent biomass.Not cultivate the training of thallus accordingly
Supporting base or 3.0%NaCl solution is control, measures each culture bottle culture solution or adherent bacterium respectively on spectrophotometer in 660nm
Absorbance value (the OD at place660).Adherent rate (r%) and suspensibility (r%) are respectively that adherent biomass and suspended biomass account for culture
The percentage of total biomass in bottle, the percentage of the total biomass and no xanthan gum control bottle total biomass of each xanthan gum concentration processing
Than representing influence of the xanthan gum to total biomass.
Xanthan gum is adherent to ocean chomophoric bacterium YL28 thallus, suspend and the influence of total biomass is as shown in Figure 1, without xanthan gum
It compares in culture bottle, the adherent of thalli growth, suspension and total biomass (OD660) it is respectively 1.781,0.805 and 2.586, thallus
Adherent rate and suspensibility are respectively 68.87% and 31.13%, it is seen that the ocean chomophoric bacterium YL28 bacterial strain is adherent serious.Xanthan gum
In 0.05%~0.5% range, thallus adherent rate is substantially reduced concentration, and suspension thalline is significantly raised, and total biomass is not affected by
It significantly affects.Although solution is still in good mobility, base in 0.25%~0.35% range when xanthan gum concentration is up to 0.5%
Originally reach minimum adherent rate, xanthan gum concentration is lower, and cost is also low, and broth viscosity is lower, and mobility and translucency are more preferable.It is comprehensive
It closes and considers selection xanthan gum optimum concentration range 0.25%~0.35%.
Embodiment two
It adds gelatin using formula 1 to be prepared, with reference to above-mentioned containing " preparation of 0.3% carragheen photosynthetic bacteria culture medium " side
Method, mounting medium become gelatin by carragheen, the dosage for adjusting gelatin prepare containing 0.05% (w/v), 0.3% (w/v) and
The purple sulfur bacteria culture medium of 0.50% (w/v) gelatin；
It is inoculated with ocean chomophoric bacterium YL28 bacteria suspension, is control with the culture bottle without gelatin.Inoculum concentration, cultivation temperature, when
Between and intensity of illumination according to embodiment one correlation method carry out, the measurement of adherent biomass, suspended biomass and total biomass,
And the calculation method of the percentage (r%) of adherent rate (r%), suspensibility (r%) and total biomass is accordingly retouched by embodiment one
State method progress.
Gelatin is adherent to ocean chomophoric bacterium YL28 thallus, suspend and the influence of total biomass is as shown in Fig. 2, in gelatin-free
It compares in culture bottle, thallus adherent rate and suspensibility are respectively 68.87% and 31.13%.Gelatin concentration is 0.05%~0.5%
In range, thallus adherent rate is substantially reduced, and suspension thalline is significantly raised, is influenced total biomass less than 10%, and culture medium
Mobility and light transmittance are good.But adherent rate basically reaches minimum in 0.3%~0.50% range, from gelatin dosage and Cheng Benfang
Face considers, selects gelatin optimum concentration range 0.3%~0.50%.
Embodiment three
Prepare using the addition carragheen of formula 1, " match containing 0.3% carragheen photosynthetic bacteria culture medium with reference to above-mentioned
System " method, adjusts the dosage of carragheen, prepares and contains 0.05% (w/v), 0.10% (w/v), 0.25% (w/v), 0.3% (w/
And the purple sulfur bacteria culture medium of 0.50% (w/v) carragheen v)；
It is inoculated with ocean chomophoric bacterium YL28 bacteria suspension, is control with the culture bottle without carragheen.Inoculum concentration, cultivation temperature,
Time and intensity of illumination are carried out according to the correlation method of embodiment one, the survey of adherent biomass, suspended biomass and total biomass
The calculation method of fixed and adherent rate (r%), suspensibility (r%) and total biomass percentage (r%) is corresponding by embodiment one
Description method carries out.
Carragheen is adherent to ocean chomophoric bacterium YL28 thallus, suspend and the influence of total biomass is as shown in figure 3, in no OK a karaoke club
Glue compares in culture bottle, and thallus adherent rate and suspensibility are respectively 68.87% and 31.13%.Carrageenan concentrations 0.05%~
In 0.5% range, thallus adherent rate is substantially reduced, and suspension thalline is significantly raised, but when carrageenan concentrations reach 0.30% or more,
Total biomass is influenced by about 10% or more, and biomass decreases, and media flow decreases.But 0.25%~
Minimum adherent rate is basically reached in 0.3% range, total biomass is influenced less than 10%.Comprehensively consider selection carragheen optimum concentration
Range 0.25%~0.3%.
Example IV
It adds xanthan gum using formula 1 to be prepared, with reference to above-mentioned " preparing containing 0.3% xanthan gum photosynthetic bacteria culture medium "
Method, addition reagent become formula 1 by formula 2, prepare the purple sulfur bacteria culture medium for containing 0.3% (w/v) xanthan gum, inoculation sea
Foreign chomophoric bacterium YL28 bacteria suspension is control with the culture bottle without xanthan gum.Inoculum concentration, cultivation temperature, time are according to embodiment
One method carries out, and observes light intensity in the case where intensity of illumination is respectively 1000lx~5000lx respectively and inhibits photosynthetic bacteria to xanthan gum
Become the adherent influence of light.The measurement of adherent biomass, suspended biomass, total biomass is carried out by the corresponding description method of embodiment one
And the calculating of adherent rate (r%).When 1000,2000,3000,4000 and 5000lx is set separately in intensity of illumination, do not adding
Add in the culture bottle of xanthan gum, ocean chomophoric bacterium YL28 thallus adherent rate is respectively 75.2%, 74.3%, 68.5%, 49.1%
With 41.8%, as light intensity increases, thallus adherent rate is reduced, but when light intensity reaches 4000lx, adherent rate is although lower, but thallus
Growth is obviously suppressed.When adding 0.30% xanthan gum in culture medium, ocean chomophoric bacterium YL28 thallus is adherent to be respectively
24.1%, 22.4%, 20.7%, 14.0% and 11.5%, even if light intensity reaches 4000lx~5000lx, thalli growth is also simultaneously
It is not affected by obvious inhibition.It can be seen that add 0.3% xanthan gum compared with being not added with xanthan gum, in culture medium, 1000lx~
Within the scope of 5000lx, thallus adherent rate is substantially reduced, and as light intensity increases, adherent rate is reduced.In addition, addition xanthan gum also has
Have and alleviates high light intensity (being greater than 4000lx) to the inhibiting effect of thalli growth.
Embodiment five
Xanthan gum is added using formula 2 and prepares the purple sulfur bacteria culture medium for containing 0.25% (w/v) xanthan gum, using matching
Side 2 adds gelatin and prepares the purple sulfur bacteria culture medium for containing 0.30% (w/v) gelatin, and number is respectively xanthan gum group and gelatin
Group.Xanthan gum group and components of gelatin not Jie Zhong Rhodopseudomonas palustris CQV97 suspension, with the culture bottle without xanthan gum or gelatin
For control group, three repetitions of every group of setting.Inoculum concentration is 3% (v/v), and in 32 DEG C, 3000lx illumination stationary culture is respectively shaken sooner or later
It is dynamic primary, it cultivates 5 days.The measurement of adherent biomass, suspended biomass, total biomass is carried out by the corresponding description method of embodiment one
And the calculating of adherent rate (r%).Control group total biomass, adherent rate and suspensibility are respectively 3.45,35.68% and
64.32%, xanthan gum group is respectively 3.31,15.87% and 84.13%, and components of gelatin is not 3.21,16.30% and
83.70%.It can be seen that adding 0.25% (w/v) xanthan gum and 0.30% (w/v) gelatin in culture medium, hence it is evident that inhibit marsh
The light that becomes of red pseudomonas is adherent, improves the biomass in bacteria suspension, and total biomass decreases, but influences smaller.
Above-described embodiment and non-limiting method of the invention, any person of an ordinary skill in the technical field do it
Appropriate changes or modifications, all should be regarded as not departing from patent category of the invention.
Claims (9)
- A kind of method of the adherent Liquid Culture photosynthetic bacteria of light 1. efficiently inhibition photosynthetic bacteria becomes, it is characterised in that: photosynthetic thin That photosynthetic bacteria is difficult to be utilized, dispersed favorable solubility, culture medium in a liquid are added in bacteria liquid culture medium process for preparation Good, the nontoxic mounting medium of mobile phase, mounting medium include one of xanthan gum, carragheen and gelatin or a variety of, Then it sterilized, be inoculated with and illumination cultivation to culture medium.
- The method of the adherent Liquid Culture photosynthetic bacteria of light 2. a kind of efficiently inhibition photosynthetic bacteria as described in claim 1 becomes, Be characterized in that: it is 0.05% (m/v)~0.5% (m/v) that mass-volume concentration range is added in photosynthetic bacteria liquid culture medium Mounting medium.
- The method of the adherent Liquid Culture photosynthetic bacteria of light 3. a kind of efficiently inhibition photosynthetic bacteria as described in claim 1 becomes, Be characterized in that: in photosynthetic bacteria liquid culture medium when illumination cultivation photosynthetic bacteria, intensity of illumination is 1000lx~5000lx.
- The method of the adherent Liquid Culture photosynthetic bacteria of light 4. a kind of efficiently inhibition photosynthetic bacteria as described in claim 1 becomes, It is characterized in that: the Huang for being 0.25% (m/v)~0.35% (m/v) added with mass-volume concentration in photosynthetic bacteria liquid culture medium Virgin rubber, the gelatin of 0.30% (m/v)~0.5% (m/v) or 0.25% (m/v)~0.30% (m/v) carragheen.
- The method of the adherent Liquid Culture photosynthetic bacteria of light 5. a kind of efficiently inhibition photosynthetic bacteria as described in claim 1 becomes, It is characterized in that: in photosynthetic bacteria liquid culture medium process for preparation, mounting medium being dissolved in water first, it is molten to obtain mounting medium Liquid, then use mounting medium solution as the solvent for preparing culture medium, according to conventional photosynthetic bacteria culture medium formula and preparation method Carry out the preparation of the culture medium containing mounting medium.
- The method of the adherent Liquid Culture photosynthetic bacteria of light 6. a kind of efficiently inhibition photosynthetic bacteria as claimed in claim 5 becomes, Be characterized in that: mounting medium is xanthan gum, when dissolving xanthan gum, under agitation, by powdered xanthan gum with a small amount of Multiple mode is added in the room temperature water of 80~85% final volumes, it is stirred at room temperature 10~after twenty minutes, 50~60 DEG C are heated to, It persistently stirs 10~20 minutes and sufficiently dissolves to get xanthan gum solution is arrived.
- The method of the adherent Liquid Culture photosynthetic bacteria of light 7. a kind of efficiently inhibition photosynthetic bacteria as claimed in claim 5 becomes, it is special Sign is: mounting medium is carragheen or gelatin, and carragheen or gelatin are added in the room temperature water of 20~30% final volumes, stirring 80~90 DEG C are heated to, lasting stirring heat preservation sufficiently dissolution in 10~20 minutes stops heating, adds room temperature water while hot, not Disconnected stirring, is diluted to amount of liquid and accounts for 80~85% final volumes, be cooled to 30~45 DEG C, obtain carrageenan solutions or gelatin Solution.
- The method of the adherent Liquid Culture photosynthetic bacteria of light 8. a kind of efficiently inhibition photosynthetic bacteria as described in claim 1 becomes, it is special Sign is: the photosynthetic bacteria liquid culture medium is suitble to the culture of fresh water species photosynthetic bacteria or salt tolerant marine species photosynthetic bacteria.
- The method of the adherent Liquid Culture photosynthetic bacteria of light 9. a kind of efficiently inhibition photosynthetic bacteria as claimed in claim 8 becomes, Be characterized in that: the fresh water monoid photosynthetic bacteria is Rhodopseudomonas palustris, and the salt tolerant marine species photosynthetic bacteria is ocean Color bacterium.
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