CN104830715A - Marine photosynthetic bacteria scale-production method - Google Patents
Marine photosynthetic bacteria scale-production method Download PDFInfo
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Abstract
The present invention discloses a marine photosynthetic bacteria scale-production method, which comprises two stages such as an aerobic fermentation process without light and a light anaerobic fermentation process, wherein in the aerobic fermentation process without light, marine photosynthetic bacteria and a culture medium used in the first stage are mixed according to a volume of 1:(5-20) and then placed into a fermentation container to ferment, and the fermentation is stopped when the dissolved oxygen partial pressure of the fermentation broth is 50-100% saturated dissolved oxygen partial pressure and the cell density OD660 achieves more than or equal to 0.8, and in the light anaerobic fermentation process, the fermentation broth of the first stage is introduced into a transparent and sterile pipeline according to a volume ratio of 1:(3-15), a light anaerobic culture medium is used, the light intensity during the fermentation process requires 1200-8000 Lux, the illumination time is 8-24 h each day, and the fermentation is stopped when the bacterial concentration OD660 is greater than 1.2 and the daily increase amount of the OD660 is less than 0.1. The method of the present invention has characteristics of short fermentation time, simple equipment, low investment, high concentration of the fermented photosynthetic bacteria liquid, good quality of the fermented photosynthetic bacteria liquid, controllable process, and easy mass production.
Description
Technical field
The present invention relates to the cultural method of a kind of bacterium, particularly a kind of marine photosynthetic bacteria used efficient large scale production method, belongs to biological technical field.
Background technology
Photosynthetic bacterium (photosynthetic bacteria, be called for short PSB) typically refer to the photosynthetic a group microorganism that a large class can not put oxygen in the environment having illumination anoxic, a photosystem and PSI is only had in photosynthetic bacteria cell, be different from green plants, their photosynthesis is non-oxygen-production.The photosynthetic original hydrogen donor of photosynthetic bacterium is not water, but some organism, and its photosynthetic result is can decomposing organic matter, and the physiological property of photosynthetic bacterium uniqueness makes their status in the ecosystem seem very important.Especially in water surrounding sewage disposal and aquaculture because it has the function and extensively by use purification of water quality and water surrounding ecological modifying agent of purifying waste water more by force.
Photosynthetic bacterium also has extremely important nutrition and nourishing function in addition, and photosynthetic bacterium is one of mushroom that nutrition is very abundant, and its protein content is up to 65%, and amino acid composition is abundant; Photosynthetic bacterium is also containing the abundant necessary vitamin B group class of VITAMIN, particularly humans and animals paedomorphosis, and as B12, folic acid etc., the content of photosynthetic bacterial thallus vitamin H is very high, is cereuisiae fermentum and chlorella more than 20 to 60 times.In addition containing various trace elements, somatomedin, the important physiologically active substance such as promoting immunity Summing Factor ubiquinone, 5-ALA (5-ALA), porphyrin, Yeast Nucleic Acid (RNA) and carotenoid.The photosynthetic bacteria liquid adding different specific weight is had in the kind of some external healthcare products.
Due to these features, in recent years in organic sewage process, aquaculture, bio-bacterial manure, agricultural planting, food, the fields such as medical treatment and health care have started the upsurge using photosynthetic bacterium.
Relative to the great demand of photosynthetic bacterium, China is also very immature at present in the production technology of photosynthetic bacterium, in large-scale production etc.Especially in marine photosynthetic bacteria used large-scale production, technology is immature, is wherein mainly manifested in the following aspects:
1, the conventional culture methods of photosynthetic bacterium depends on illumination, and scale operation is limited to the cost of investment of expensive photoreactor, the extensive smooth static gas wave refrigerator method of domestic many many employings at present, utilize the containers such as clear-glass bottle, Plastic Bottle, glass jar, plastics bag or plastic tank to carry out illumination and leave standstill small volume cultivation, slot type is used once in a while in large volume culture, semi-open or the open cultivation such as pond, but there is small scale in what these training methods carried out photosynthetic bacterium production, or culture efficiency is low, cell concentration and purity low, subject to living contaminants.
2, because current marine photosynthetic bacteria used production adopts illumination quiescent culture more, photosynthetic bacterium carries out light autotrophy or photoheterotrophic growth, and the doubling time of bacterium is long, causes the production cycle long, usually need 7-20 days.Especially in large-scale production, when from a small amount of bacterial classification expanding production to the scale of tens cubes, conventional light quiescent culture needs long time, and the production cycle is long, reduces plant factor, adds production cost.
3, marine photosynthetic bacteria used at present large-scale production uses the semi-open and open mode of production usually, and the easy microbiological contamination of substratum, the yield of photosynthetic bacterium is low, and cell concentration is usually less than 10
7cfu/ml, and in open production, miscellaneous bacteria rate is very high, affects product quality.
Fresh water photosynthetic bacterium training method is followed in the substratum of 4, current seawater photosynthetic bacterium, excessive use phosphoric acid salt, calcium ions and magnesium ions easily and in seawater is formed and precipitates, cause reagent waste on the one hand, and the transparency of substratum can be affected, interference process of growth is to the detection of thalli growth density, and simultaneously muddy substratum also can reduce effective intensity of illumination.
Due to the existence of these problems above-mentioned, strongly limit the marine photosynthetic bacteria used large-scale production of China and application thereof.
Summary of the invention
For Problems existing in marine photosynthetic bacteria used large-scale production; the object of this invention is to provide a kind of production efficiency high; the marine photosynthetic bacteria used large-scale method for producing that production cost is low, to promote that marine photosynthetic bacteria used product is in the application in the fields such as aquatic products, food, medicine and environment protection.
The present invention is according to the metabolic characteristic of photosynthetic bacterium self: namely under the condition of aerobic not light requirement, can increase cell quantity by quick heterotrophic growth, can high-level efficiency low molecular organism be utilized to grow under the condition of light anaerobism again, and synthesize the feature of some biologically active substances, by the production technique that the dark reaction and light anaerobically fermenting two benches that design aerobic not light requirement are coupled, and according to the feature that photosynthetic bacterium can grow fast under aerobic heterotrophism condition, devise the nutritious substratum in this stage applicable, promote photosynthetic bacterium ramp, the quantity of quick increase cell, this stage production equipment only needs the fermentor tank of general aerobic fermentation, do not need expensive photoresponse fermentor tank, and subordinate phase can not produce precipitation by utilizing in the photosynthetic pipeline of anaerobism, low nutrition fermention medium carries out light anaerobically fermenting, the industrial scale in this stage can flexibly by increasing and decreasing photosynthetic pipeline to arrange, and batch fermentation can be realized by applying different feed profile in light anaerobically fermenting, fed-batch, and the mode such as continuous feeding is produced, and has less investment, high degree of flexibility, can adopt the different mode of production and difference production-scale feature according to specific circumstances.
Concrete technical scheme of the present invention is as follows: a kind of marine photosynthetic bacteria used large-scale method for producing, comprises aerobic not light requirement fermenting process and two stages of light anaerobic fermentation process; First in the fermenting process of aerobic not light requirement, marine photosynthetic bacteria used bacterial classification and aerobic not light requirement fermentation stage substratum are by 1: the volume ratio of (5-20) mixes the common micro-organisms fermentation cylinder for fermentation being placed on aerobic not light requirement, and nutritive ingredient and the mass percent of aerobic not light requirement fermentation stage substratum are: sodium acetate: 0.15-0.5%, yeast powder: 0.0001-0.5%, peptone: 0.001-0.5%, ammonium chloride: 0.01-0.2%, sugar (glucose or other conventional carbon sources such as sucrose, maltose): 0.0001-2%, H
2pO
4 -: 0.001-1%, Fe
3+: 0.00001-0.01%; All the other compositions of substratum are free of contamination seawater or artificial seawater, and substratum passes through acetic acid and the 1-5 M sodium hydroxide adjustment potential of hydrogen of 1-5 M, and the pH of final substratum is between 6.0-7.5, and substratum uses after 115-121 DEG C of high-temperature sterilization.This stage fermentation container can be test tube, Erlenmeyer flask or fermentor tank, and to illumination not requirement, have that light is unglazed all can ferment, fermenting container or equipment requirements require can high pressure 115-121 DEG C sterilizing, anti-seawater corrosion, requires temperature-controllable and aseptic ventilation oxygen-supplying in fermenting process.Aerobic fermentation after photosynthetic bacterium seed liquor proportionally adds substratum in this stage Fermentation by Photosynthetic Bacteria process, between yeast phase, temperature controls at 20-40 DEG C, and during fermentation, the dissolved oxygen dividing potential drop of fermented liquid is the saturated dissolved oxygen dividing potential drop of 50%-100%.Ferment tank process pH controls between 6.3-8.0; The fermentation time of the fermentation stage of aerobic not light requirement is 24-48 hour, optical density value (i.e. OD under the photosynthetic bacterium concentration in fermentor tank reaches wavelength 660nm
660) fermentation can be stopped when being greater than 0.8, and by the fermented liquid in fermentor tank according to 1:(3-15) the Cemented filling of volume ratio sterilizing carry out light anaerobically fermenting to subordinate phase: the medium nutrient content of this process is lower than the substratum of the process of aerobic not light requirement, and not containing glucose, the carbon source such as sucrose or maltose composition, light anaerobically fermenting medium component and mass percent are: sodium acetate: 0.15-0.5%, yeast powder: 0.0001-0.1%, peptone: 0.0001-0.1%, ammonium chloride: 0.0001-0.1%, H
2pO
4 -: 0.0001-0.01%, Fe
3+: 0.00001-0.001%, all the other compositions of substratum are free of contamination seawater or artificial seawater, and substratum potential of hydrogen passes through acetic acid or the adjustment of 1-5M sodium hydroxide of 1-5M, and final pH should between 6.0-7.5.Substratum wants degerming sterilization, the method of degerming sterilization can by boiling 30-60 minute in airtight stainless steel vessel, or through autoclaving equipment 110-121 DEG C of sterilizing 20-30 minute, the sterilizing agent that perchloric acid or dioxide peroxide etc. also can be utilized chloride is degerming, ensure degerming after substratum in miscellaneous bacteria should lower than 3 cfu/ml, and residual free chlorine should lower than 0.06 mg/L.In light anaerobic fermentation process, leavening temperature is 15-40 DEG C, by heliogreenhouse, or temperature adjustment is carried out in water-bath, light source can utilize natural lighting or human assistance light source, but sunlight should be avoided to be exposed to the sun, ensure the 8-24 h light time every day, light intensity should between 1200 Lux-8000 Lux; The human assistance light sources such as usual use fluorescent lamp can increase light application time, and then accelerate the growth velocity of light anaerobic stages photosynthetic bacterium, shorten fermentation period.Marine photosynthetic bacteria used for light anaerobic fermentation stage ferments in transparent pipeline, as the concentration OD of thalline after 2-15 days
660when being greater than 1.2, and the concentration continuing to extend fermentation time thalline increases slowly, i.e. OD
660when the increment of every day is less than 0.1, stop fermentation.
Marine photosynthetic bacteria used after fermentation can direct filling or centrifugal concentrating liquid or make the photosynthetic bacteria preparation that can be used for the fields such as food, medicine, aquatic products, plantation, cultivation, environmental protection by other technique such as carrier adsorption, drying packaging.
The transparent pipeline that light anaerobic fermentation stage uses can adopt transparent glass, transparent organic glass, or other transparent plasticss are as polyethylene, polypropylene, polycarbonate, urethane (Polyurethane, PU), thermoplastic polyurethane (Thermoplastic Urethane, the material such as TPU), can be transparent hard pipeline or soft pipeline, requirements for pipes can tolerate the hydrostaticpressure being greater than 0.15 MPa, the diameter of pipeline between 90mm-600mm, and can tolerate the hydrochloric acid of 3M or the caustic soda process more than 100 hours of 5M.Pipeline and pipeline directly can be connected by steel flange or clip.The length of pipeline specifically can be determined according to need of production and production site, and general single conduit length several meters is to upper km.The parallel connection that this stage can carry out many pipelines is simultaneously fermented, and expands fermentation-scale.The flow direction and the flow of the stainless Valve controlling pipeline liquid of corresponding bore can be set between pipeline.The pump of acid-alkali-corrosive-resisting can also be connected by threeway or valve, for pipeline sterilization or pipeline cleaning, substratum fermented liquid mixing etc.
The present invention has the following advantages:
1, facility investment is few, does not need expensive photosynthetic response fermentor tank, is convenient to amplification and carries out large-scale production;
2, the aerobic not light requirement-light anaerobism coupled fermentation technique that can meet photosynthetic bacterium fast breeding is devised according to photosynthetic bacterium growth metabolic characteristic;
3, in the substratum of the fermentation stage of aerobic not light requirement, with the addition of the fermentable carbon sources such as glucose, and the organic nitrogen source improved in light anaerobic culture medium is as peptone, yeast powder and the inorganic nitrogen-sourced concentration as ammonium chloride or ammonium sulfate, improve photosynthetic bacterium aerobic heterotrophic growth proliferative speed high, the biomass of thalline can be increased fast, greatly can shorten fermentation time 2-8 doubly relative to the illumination static gas wave refrigerator of routine, and viable count improves 2-10 doubly;
4, smooth anaerobic fermentation stage of the present invention uses transparent pipeline, by adjusting the length production control scale of pipeline, and light anaerobic fermentation stage also can be used alone, and carry out batch fermentation according to the mode of supplemental medium, continuously ferment or fed-batch cultivation, there is very high handiness and controllable degree; Transparent pipeline light anaerobically fermenting can utilize lamp or source of artificial light to improve the growth rate of photosynthetic bacterium.
Embodiment
The present invention is further described below by specific embodiment.
embodiment 1:
Materials and methods
1.1 bacterial strain
Photosynthetic bacterium strain GH1, GH2 that from ooze, photosynthetic bacterium enriched and separation and purification goes out, for experiment, are defined as Rhodopseudomonas palustris through identification of morphology, Physiology and biochemistry feature in conjunction with 16S rDNA sequential analysis and little red oomycetes belongs to thiophilic little red oomycetes.
1.2 substratum
Photosynthetic bacteria enrichment culture medium composition (mass percent) contains: 0.2% sodium acetate, 0.001% potassium primary phosphate 0.03% ammonium chloride, 0.005% iron trichloride, and all the other compositions are the Chen Haishui preparation of filtering, and the second acid for adjusting pH with 5% is 7.0.Substratum loads through 115 DEG C in transparent Erlenmeyer flask or clear-glass bottle, autoclaving 30min.
The dark fermentation medium components (mass percent) of the aerobic not light requirement of photosynthetic bacterium contains: 0.3% sodium acetate, 0.005% potassium primary phosphate, 0.1% ammonium chloride, 0.1% yeast leaching powder, 0.3% glucose, 0.005% iron trichloride, all the other compositions are Chen Haishui, and with the old seawater preparation of filter paper filtering removing impurity, the acetic acid condition pH of 5% is 6.8, substratum loads through 115 DEG C in Erlenmeyer flask, autoclaving 30 min.
Photosynthetic bacteria used for light anaerobically fermenting medium component (mass percent) contains: 0.3% sodium acetate, 0.03% ammonium chloride, 0.01% yeast leaching powder, 0.005% iron trichloride, all the other compositions are Chen Haishui, Chen Haishui preparation is filtered with 300 order filter bags, regulate the pH of substratum in 6.5 with the acetic acid of 2M, substratum through 115 DEG C, autoclaving 30 min.
1.3 marine photosynthetic bacteria used fermentations
Marine photosynthetic bacteria used fermentation is divided into dark fermentation and two stages of light anaerobically fermenting of aerobic not light requirement.
1.3.1 the dark fermentation stage of aerobic not light requirement
The substratum that this stage uses is the dark fermention medium of aerobic not light requirement, aseptically by single colony inoculation of photosynthetic bacterium in the test tube that 5 ml liquid nutrient mediums are housed, 30 DEG C shake cultivation 30 hours, continue by the photosynthetic bacterium in test tube according to 10% inoculation kind amount be inoculated in 250 ml Erlenmeyer flasks of 50 ml substratum, 30 DEG C of fermentations, 150 turns of concussion cultivations, after 24 hours, continue to be inoculated into and are equipped with in the 2 L fermented liquids of 450 ml, 30 DEG C of fermentations, 150 turns shake cultivation 30 hours; Continue photosynthetic bacteria liquid to be inoculated in jumbo Erlenmeyer flask, 30 DEG C of fermentations, 150 turns of concussion cultivations are after 24 hours, and now Fermentation by Photosynthetic Bacteria liquid is long-pending reaches 5 L, the OD of bacterium
660between 1.6-2.8; Fermented liquid is inoculated into 50 L photosynthetic bacteriums and cultivates seeding tank fermentation, sterile air is passed into during fermentation, air flow is 0.1 cubic meters per minute, mixing speed is 150 turns, 30 DEG C ferment 30 hours, continue to access larger seeding tank according to the inoculum size of 10 % or produce in tank to carry out 30 DEG C of fermentations 24-48 hour, now OD of fermented liquid
660value is between 1.6-3.0.
1.3.2 light anaerobic fermentation stage
The substratum in this stage is light anaerobically fermenting substratum.By the bacterium liquid of aerobic not light requirement fermentation by aseptic pipeline according to the ratio of 1:5 be inoculated in containing substratum in the transparent pipeline of aseptically process.Transparent pipeline in this experiment is polyurethane PU transparent hose, thickness of soft tube 0.3 μm, and pipe diameter is 25 cm, and pipeline and pipeline are directly by connections such as the steel flange clips of same bore.Pipeline first cleans up through tap water after connecting before use, control solid carbon dioxide divides, be pipeline overall volume 1/20(volume ratio by total amount again) the caustic soda of 3M, alkali lye is drained after 30 minutes by external anti-corrosive water pump forced circulation wash cycles, continuing to add total amount is pipeline overall volume 1/20(volume ratio) acetic acid of 3M carries out circulation sterilization after 30 minutes, drain acetic acid, with total amount be pipeline overall volume 1/10 in density containers, boil 30 minutes after be cooled to the tap water of 50 ± 5 DEG C, clean latter 30 minutes by pump.
In transparent pipeline, the sterilizing of substratum within 30 minutes, is added after high-temperature sterilization by 110 DEG C in fermentor tank, or other chemical chemical process sterilizings available, as added hypochlorous acid or dioxide peroxide sterilizing, aseptic hypo solution should be added after sterilizing and carry out neutralization and prevent residual free chlorine >0.05 mg/L.
The leavening temperature of light anaerobic stages is 25-30 DEG C, is 1500 Lux-5000 Lux according to intensity, every day minimum light application time 8 hours, fermentation time is 3-4 days, works as OD
660>1.5 completes fermentation.
The quality control of 1.4 Fermentation by Photosynthetic Bacterias: the purity of Fermentation by Photosynthetic Bacteria liquid and growth concentration measure
The purity detecting of Fermentation by Photosynthetic Bacteria liquid, adopts the agar photosynthetic bacterium rich solids of interpolation 2% to cultivate and detects, will dilute 10 in stroke-physiological saline solution
4, 10
5, 10
6fermentation by Photosynthetic Bacteria liquid 100 μ l is doubly coated on photosynthetic bacterium rich solids substratum, through 30 DEG C, the cultivation of 3-5 days, the quantity detecting photosynthetic bacterium and miscellaneous bacteria calculates bacterial contamination rate, bacterial contamination rate=miscellaneous bacteria number/(miscellaneous bacteria number+photosynthetic bacterium bacterium number) %, in the fermenting process of whole photosynthetic bacterium, bacterial contamination rate is no more than 2%.
The growth concentration of photosynthetic bacterium measures, and utilizes spectrophotometer to detect Fermentation by Photosynthetic Bacteria liquid at OD
660optical density value, the substratum thinking the sterilizing of fermentation is contrast.The OD of the photosynthetic bacteria culture solution after fermentation ends
660should 1.2 be greater than.
Experimental result:
Experiment is carried out during 6 the end of month to 9 the end of month, has fermented 7 batches altogether in front and back, in contrast with traditional training method simultaneously.
The condition of the marine photosynthetic bacteria used fermentation of table 1 and the concentration miscellaneous bacteria rate of bacterium liquid
Note: indoor temperature is regulated by well heater and ventilating fan.
The measurement result that table 1 is cultivated for different batches photosynthetic bacterium.Marine photosynthetic bacteria used through aerobic not light requirement-light anaerobism coupled fermentation technique, higher bacterial concentration can be reached at 3-6 days.Find the growth of leavening temperature 30 DEG C of ratios 25 DEG C hurry up in addition, so suitably control leavening temperature contributes to the fermentation of photosynthetic bacterium, shorten fermentation time.The fermentation time of aerobic not light requirement in experiment-light anaerobism coupled fermentation technique is the shortest can shorten to 3 days, and the concentration OD of traditional illumination static gas wave refrigerator method photosynthetic bacterium
660just can reach 1.2 after 10 days.
embodiment 2:
By the marine photosynthetic bacteria used seed liquor of 100 ml according to 10% rate of vaccination be inoculated into dark fermentation containing carrying out aerobic not light requirement in 1L substratum 3L Erlenmeyer flask; The concrete composition of substratum is: the sodium acetate of 0.3%, the ammonium chloride of 0.005% potassium primary phosphate 0.05%, 0.05% yeast leaching powder, 0.3% glucose, and 0.005% iron trichloride, configures with substratum seawater, regulates pH=6.5 by the acetic acid of 2M; Substratum through 115 DEG C, 30 min autoclavings.Fermentation by Photosynthetic Bacteria condition is: temperature 28 DEG C, and 150 revs/min shake cultivation 24 hours, and the concentration of bacterium liquid reaches OD
660=2.83.This fermented liquid is linked into 7 liters according to 10% inoculum size and continues fermentation containing fermentation in sterilising medium Minitype seed tank, fermentation condition is: temperature 30 DEG C, stir speed (S.S.) 150 revs/min, regulates air flow to be 10 L/min, the OD of fermentation 24 hours secondary fermentation liquid
660be 2.15.Fermented liquid continues the 100 L fermentor tanks being inoculated into the substratum containing 70 L sterilizings, and leavening temperature is 28 DEG C, and stir speed (S.S.) is 150 revs/min, and the amount passing into sterile air is 0.1m
3/ min; The concentration of 24 hours secondary fermentation liquid is OD
660=1.86.Fermented liquid is passed in the transparent PU pipeline of aseptic diameter 25 centimetres and carries out photosynthetic Anaerobic culturel, pass into photosynthetic bacteria used for light Anaerobic culturel liquid according to the ratio of 9:1 simultaneously, the composition that light anaerobically fermenting culture medium is concrete is: 0.3% sodium acetate, 0.02% ammonium chloride, 0.01% yeast leaching powder, 0.0002% iron trichloride, regulates the pH of substratum in 6.5 with the acetic acid of 2 M.Substratum in fermentor tank 115 DEG C, autoclaving 30 minutes.Transparent pipeline is with this sodium-hydroxide treatment 30 minutes through 5 M.In light anaerobically fermenting in photosynthetic pipeline, room temperature 25 ± 4 DEG C cultivation, the intensities of illumination such as daylight control at 2000 ± 150 Lux, and the ducted bacteria concentration after 3 days that ferments reaches OD
660the concentration OD of the=1.54, four day photosynthetic bacteria liquid
660=1.65, color is red in purplish red, detects miscellaneous bacteria rate lower than 0.1%.In experiment from the marine photosynthetic bacteria used seed liquor of 100 ml expand the fermentation-scale of 70 L to, only experienced by four days, substantially reduce fermentation time.And the illumination static gas wave refrigerator method of employing routine needs the time more than two weeks.
embodiment 3:
Ferment 50 liters of aerobic not light requirements high density photosynthetic bacterium (its thalline OD obtained
660=2.0), with light anaerobically fermenting substratum according to 1:4,1:5, the diameter that the ratio of 1:6 joins light anaerobically fermenting is respectively in transparent urethane (PU) pipeline of 30 centimetres; Pipeline cleans, after the acetic acid sterilizing of 10%, through aseptic seawater flushing through the NaOH of 5% in advance.
Photosynthetic bacterium is natural lighting on daytime under room temperature 25 ± 5 DEG C of conditions in photosynthetic pipeline, and night is irradiated by fluorescent lamp, ferments after 5 days, detects the concentration of thalline under spectrophotometer 660nm, not inoculate the substratum of photosynthetic bacterium for blank.Result is as shown in table 2.
The impact that table 2 different vaccination Dilution ratio is fermented separately on marine photosynthetic bacteria used for light anaerobism
Can find out from above-mentioned result, the inoculum size of different ratios is to fermentation or influential, and the ratio of dilution is less, and the fermented liquid concentration after 5 days is higher.
Carry out following experiments: the NO.2 pipeline of the light anaerobically fermenting of fermentation after 5 days is proceeded continuously fermenting of semi-continuous fed-batch, and concrete steps are simultaneously: 1, add every day 30 L through autoclaved smooth Anaerobic culturel based in transparent pipeline.By the nutrient solution newly added with close on 5 mitron roads Fermentation by Photosynthetic Bacteria liquid mix.Containing 0.3% sodium acetate, 0.02% ammonium chloride in substratum, 0.01% yeast leaching powder, 0.002% iron trichloride, regulates the pH=6.5 of substratum with the acetic acid of 2 M.
2, release the Fermentation by Photosynthetic Bacteria liquid fermented of 30 L every day from the end of pipeline simultaneously.
3, the concentration of the Fermentation by Photosynthetic Bacteria liquid sent is detected every day.
4, operating process will ensure aseptic, and the alcohol by 75% is to pipeline and inoculating facility sterilization.
Continuously ferment 30 days, the concentration monitor result of the bacterium liquid of the fermented liquid of every day is as shown in table 3.
In table 3 independent light anaerobically fermenting, No. NO.2 photosynthetic pipeline is through the photosynthetic bacterium concentration of the semicontinuous fermentation of L feed supplement every day 30
| Fermentation number of days | OD 660 | Fermentation number of days | OD 660 | Fermentation number of days | OD 660 | Fermentation number of days | OD 660 | Fermentation number of days | OD 660 | Fermentation number of days | OD 660 |
| 1 | 2.30 | 6 | 2.05 | 11 | 1.96 | 16 | 1.77 | 21 | 1.86 | 26 | 1.76 |
| 2 | 2.33 | 7 | 2.06 | 12 | 1.87 | 17 | 1.77 | 22 | 1.82 | 27 | 1.73 |
| 3 | 2.33 | 8 | 1.98 | 13 | 1.92 | 18 | 1.78 | 23 | 1.68 | 28 | 1.76 |
| 4 | 2.28 | 9 | 1.86 | 14 | 1.83 | 19 | 1.90 | 24 | 1.71 | 29 | 1.72 |
| 5 | 2.20 | 10 | 1.92 | 15 | 1.75 | 20 | 1.75 | 25 | 1.73 | 30 | 1.72 |
Can find out from experimental result, carry out in the light anaerobism Semi-continuous cultivation of continuous month utilizing separately photosynthetic pipeline, the Fermentation by Photosynthetic Bacteria liquid of 30 L can be gathered in the crops every day, in whole process, the light anaerobically fermenting of photosynthetic bacterium can keep a good running status, and finally photosynthetic bacteria concentration can reach OD
660be greater than 1.5.
Those of ordinary skill in the art can understand, and in protection scope of the present invention, modifies for above-described embodiment, and it is all possible for adding and replacing, and it does not all exceed protection scope of the present invention.
Claims (10)
1. a marine photosynthetic bacteria used large-scale method for producing, is characterized in that comprising aerobic not light requirement fermenting process and two stages of light anaerobic fermentation process; First be in the fermenting process of aerobic not light requirement, marine photosynthetic bacteria used bacterial classification and aerobic not light requirement fermentation stage substratum are by 1: the volume ratio of (5-20) mixes to be placed in fermenting container ferments, leavening temperature is 20-40 DEG C, fermentation time is 24-48 hour, the dissolved oxygen dividing potential drop of fermented liquid is the saturated dissolved oxygen dividing potential drop of 50%-100%, fermenting process pH controls between 6.3-8.0, cell density OD
660reach after more than 0.8 and stop fermentation; In the light anaerobic fermentation process of subordinate phase, by first stage fermented liquid according to 1:(3 – 15) volume ratio be passed in transparent aseptic pipeline and carry out light anaerobic fermentation process, fermentation condition is: temperature is 15-40 DEG C, fermentation time is 2-15 days, adopt light anaerobic culture medium, fermenting process needs illumination not need oxygenation of ventilating, and between light intensity requirement 1200-8000lux, the light application time of every day is at 8-24 hour; As the concentration OD of thalline
660be greater than 1.2, and OD
660when the increment of every day is less than 0.1, stop fermentation.
2. marine photosynthetic bacteria used large-scale method for producing according to claim 1, is characterized in that the nutritive ingredient of described aerobic not light requirement fermentation stage substratum and mass percent are: sodium acetate: 0.15-0.5%, yeast powder: 0.0001-0.5%, peptone: 0.001-0.5%, ammonium chloride: 0.01-0.2%, sugar: 0.0001-2%, H
2pO
4 -: 0.001-1%, Fe
3+: 0.00001-0.01%, all the other compositions of substratum are free of contamination seawater or artificial seawater, and the pH of substratum, between 6.0-7.5, needs through 115-121 DEG C of high-temperature sterilization 20-30 minute before culture medium inoculated fermentation.
3. marine photosynthetic bacteria used large-scale method for producing according to claim 1, is characterized in that described aerobic not light requirement fermenting process is having light or no light condition bottom fermentation.
4. marine photosynthetic bacteria used large-scale method for producing according to claim 1, is characterized in that the fermenting container of described aerobic not light requirement fermenting process is test tube, Erlenmeyer flask or fermentor tank.
5. marine photosynthetic bacteria used large-scale method for producing according to claim 1, is characterized in that the composition of described smooth anaerobic culture medium and mass percent are: sodium acetate: 0.15-0.5%, yeast powder: 0.0001-0.1%, peptone: 0.0001-0.1%, ammonium chloride: 0.0001-0.1%, H
2pO
4-: 0.0001-0.01%, Fe
3+: 0.00001-0.001%, all the other compositions of substratum are free of contamination seawater or artificial seawater, and medium pH is between 6.0-7.5.
6. marine photosynthetic bacteria used large-scale method for producing according to claim 1 or 5; it is characterized in that the substratum of described smooth anaerobic culture medium needs sterilization; and ensureing that the miscellaneous bacteria in the substratum after sterilizing should lower than 3 cfu/ml, residual free chlorine should lower than 0.06 mg/L.
7. marine photosynthetic bacteria used large-scale method for producing described according to claim 6; it is characterized in that the method for described sterilization is boil 30-60 minute in airtight stainless steel vessel; or through autoclaving equipment 110-121 DEG C of sterilizing 20-30 minute, or utilize chloride sterilizing agent sterilizing.
8. marine photosynthetic bacteria used large-scale method for producing described according to claim 1; the material that it is characterized in that the transparent pipeline that described smooth anaerobic fermentation stage adopts is transparent glass, transparent organic glass or transparent polyethylene, polypropylene, polycarbonate, urethane.
9. marine photosynthetic bacteria used large-scale method for producing described according to claim 1 or 8, is characterized in that described transparent pipeline is hard or soft transparent pipeline, is withstand voltagely greater than 0.15 MPa.
10. marine photosynthetic bacteria used large-scale method for producing described according to claim 8, it is characterized in that described transparent pipeline needs sterilizing, its method is the caustic soda pipe blow-through 30-60 minute of the 3-5M using conduit volume 1/5-1/20 volume, again through the acetic acid washing and sterilizing 30-60 minute of the 3-5M of 1/5-1/20 volume, finally use the sterile water wash 30-60 minute of 1/5-1/10 volume; Or the caustic soda pipe blow-through 30-60 minute of the 3-5M by 1/5-1/20 volume, then clean sterilizing in 30-60 minute through the hydrogen peroxide of the 3%-5% of 1/5-1/20 volume.
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