CN102154180B - Culture medium for microvesicle bacteria BS03 and preparation method thereof - Google Patents
Culture medium for microvesicle bacteria BS03 and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a culture medium for microvesicle bacteria BS03 and a preparation method thereof, relating to a microbial culture medium and providing a culture medium, which is suitable for efficient culture of the microvesicle bacteria BS03 and can obviously increase cell live weight of the microvesicle bacteria BS03 and yield of algae-killing active substances, and a preparation method of the culture medium. The culture medium comprises 1L of distilled water, 1-20g/L of peptone and 1-15g/L of cane sugar. The preparation method of the culture medium comprises the steps of: adding the peptone and the cane sugar into the distilled water, enabling a constant volume to be 1L, and sterilizing to obtain the culture medium of the microvesicle bacteria BS03. The composite culture medium suitable for high-dense culture of the microvesicle bacteria BS03 is developed by taking 2216E as a basic culture medium and adopting various methods for optimizing culturing conditions.
Description
Technical field
The present invention relates to microbiological culture media, particularly substratum of a kind of microvesicle bacterium BS03 and preparation method thereof.
Background technology
In recent years, because stretch of coastal water severe contamination and eutrophication, the red tide occurrence frequency sharply increases, scale constantly enlarges, new red tide algae kind constantly occurs, poisonous red tide algae kind ratio rises, and the region area of harm is also increasing, at present at more than 30 countries and regions frequent occurrences such as the U.S., Japan, China, Canada, France, Sweden, Norway, Philippines, India, Indonesia, Malaysia, Korea S, Hong Kong.The reduction water body eutrophication degree is sought effective red tide and the endotoxin contamination controlling way is imperative, and red-tide control becomes the outer urgent need to solve the problem of Present Domestic.
At present, the control techniques of algae can be summarized as 3 kinds, i.e. physical method, chemical process and biological method.Physical method mainly is to make the frustule flocculation sediment by adding flocculation agent or clay etc.; Chemical process is to add some chemical algicides such as copper sulfate etc. in the sensing wawter bloom water body to kill frustule, these two kinds of method costs are all higher, alternative poor, can kill together with some beneficial organisms in the treating processes, killing frustule and also can cause simultaneously the sudden outburst of intracellular toxin, environment is existed all limitation such as secondary pollution.Biological method mainly refers to utilize the mutual ecological relationship between the biology, sets up controlling elements-host's running balance system in specific body of water, with the generation that prevents algae pollution or a kind of method of administering the algae pollution that has occured.At present, both at home and abroad to the research of the Biological Mechanism of the outburst of wawter bloom and red tide and extinction also ground zero, molten algae bacterium wherein is as to the biological control of the harmful algal domestic and international focus of research especially.
Molten algae bacterium (algicidal bacteria) is that a class suppresses algal grown in direct or indirect mode, or kills the general designation of the bacterium of algae, dissolving frustule.Current, both at home and abroad the research of molten algae bacterium still is in the preliminary stage, also be just to begin for the research of the molten algae active metabolite of bacterium.Kill be seen in the algae bacterium report maximum be to lean on to discharge the outer material of born of the same parents and kill algae, and what of the outer material of secretion born of the same parents are associated with the height of cell density usually, Optimal Medium then is a kind of effective ways that improve cell density.The expansion that is used for the effective separation of follow-up active substance and kills the research work of algae mechanism in order to obtain a certain amount of Algicidal substances, it is particularly important that the Optimization Work of bacteria culture medium and culture condition just seems.
Substratum is that the people is the most important culture environment that offers microorganism growth, and being affects microorganism growth propagation and the synthetic important factor of meta-bolites.The factor that affects microorganism growth process has a lot, mainly comprises substratum (composition, concentration) and culture condition (temperature, pH value, rotating speed, inoculum size, oxygen-supply quantity) etc.Because the fermentation culture based component is numerous, and often there is interaction in each factor, so the medium optimization workload is large and complicated.Multiple optimization method in the mathematical statistics has begun to be widely used in the Optimization Work of microbiological culture media, and wherein experimental technique relatively more commonly used has the methods such as single factor method, orthogonal experimental design method, uniform design, total divisor method of experimental design and response surface design method.
Traditional optimization method such as single factor method Optimal Medium once can only be considered the impact of a factor level, and workload is large, need repeatedly experiment and long test period, also can't show the interaction relationship between each factor.Therefore, the novel culture medium optimized method in conjunction with single factor method and existing mathematical statistics method has been subject to increasing application.Uniform design be a kind of multifactor optimized method created of the calm and peaceful Wang Yuan of China mathematician Fang Kai (1, Fang Kaitai, Wang Yuan. homogeneous design and uniform designs table [M]. Beijing: Science Press, 1994,21-23).The method combines principle and the multivariate statistics of number theory, is obtaining many successful examples aspect the optimization of microbiological culture media in recent years.The basic ideas of homogeneous design are exactly to make the experimental point full and uniform dispersion as far as possible, make each test point have better representativeness, but give up simultaneously neat comparable requirement, to reduce test number (TN); Then remedy this defective by multivariate statistical method, make conclusion (of pressure testing) reliable equally.Therefore uniform design is a kind of test design method of considering test point full and uniform distribution in trial stretch, and under the identical condition of test number, the deviation of homogeneous design is little more than orthogonal design.Because homogeneous design is no longer considered the neat comparability of orthogonal test, so its test-results is carried out Quadratic Regression Fitting with method of gradual regression, the last optimum value that can obtain each factor according to fit equation with single factor rotation method.
Summary of the invention
But the object of the present invention is to provide a kind of high-efficient culture microvesicle bacterium BS03 and significantly improve its cell density and the substratum of the microvesicle bacterium BS03 of algicdal activity material output.
Another object of the present invention is to provide the preparation method of the substratum of a kind of microvesicle bacterium BS03.
Described microvesicle bacterium BS03 is preserved in Chinese Typical Representative culture collection center on September 3rd, 2010, and preservation center deposit number is CCTCC NO:M 2010218, and the address at Chinese Typical Representative culture collection center is Wuhan, China, Wuhan University.
The composition of the substratum of described microvesicle bacterium BS03 is to contain peptone 1～20g/L, sucrose 1～15g/L in the distilled water of 1L.
The composition of the substratum of described microvesicle bacterium BS03 preferably contains peptone 10.50g, sucrose 8g in the distilled water of 1L.
The preparation method of the substratum of described microvesicle bacterium BS03 is:
With being settled to 1L in Tryptones, the sucrose adding distilled water, namely get the substratum of microvesicle bacterium BS03 after the sterilization.
Described sterilization can be adopted autoclaving, and described autoclaved condition can be: 121 ℃, and 20min.
The present invention is at first take the 2216E substratum as basic medium, adopt single factor method to explore optimum culture condition, namely control other conditions constant, pH, salinity, inorganic salt are chosen respectively different gradients and type is tested, investigate it to the impact of thalli growth and algicdal activity material output, find out major influence factors and carry out homogeneous design.Because medium optimization is the work that amount is large and complicated, at first select several complex nutrients sources among the present invention, be those nutritive substances (such as peptone, yeast powder) that not only can be used as carbon source but also can be used as nitrogenous source, then add different trace ingredientss on their basis, investigate each composition to thalli growth and the extremely impact of algae efficient.Final selection peptone, sucrose, incubation time, pH value, inoculum size are important factor of influence, utilize uniform design, take bacterium liquid turbidity, dry cell weight and toxic limit medium dose (LD50) as evaluation index, carry out the design that mixes of 5 factors, 15 levels, recycling DPS7.0 software carries out Quadratic regression polynomial analysis to experimental value, sets up at last regression model.Nutrient media components after the optimization is: peptone 10.50g/L, sucrose 8g/L, incubation time 32h, inoculum size 3.00%, initial pH value 7.5.
Description of drawings
Fig. 1 be in the embodiment of the invention different culture media on microvesicle bacterium BS03 growth with kill the figure that affects of algae efficient.In Fig. 1, X-coordinate is substratum, from left to right is followed successively by PYS substratum, LBK substratum, 2216E substratum, BK substratum, B substratum, A substratum, and right ordinate zou is the optical density value OD of wavelength under 600nm
600, left ordinate zou is semilethal rate LD
50(%); Histogram is for being semilethal rate LD
50, broken line graph is OD
600
Fig. 2 be in the embodiment of the invention different carbon sources on microvesicle bacterium BS03 growth with kill the figure that affects of algae efficient.In Fig. 2, X-coordinate is carbon source, from left to right be followed successively by glucose (Glucose), maltose (Maltose), Zulkovsky starch (Solublestarch), sucrose (Cane sugar), lactose (Lactobiose), left ordinate zou is semilethal rate LD
50(%), right ordinate zou is the optical density value OD of wavelength under 600nm
600, histogram is for being semilethal rate LD
50, broken line graph is OD
600
Fig. 3 be in the embodiment of the invention different nitrogen sources on microvesicle bacterium BS03 growth with kill the figure that affects of algae efficient.In Fig. 3, X-coordinate is nitrogenous source, from left to right is followed successively by soy peptone (Soya Peptone), peptone (Peptone), extractum carnis (Beefextract), KNO
3, yeast powder (Yeast Powder), NaNO
3, left ordinate zou is semilethal rate LD
50(%), right ordinate zou is the optical density value OD of wavelength under 600nm
600, histogram is for being semilethal rate LD
50, broken line graph is OD
600
Fig. 4 be in the embodiment of the invention different nitrogen sources on microvesicle bacterium BS03 growth with kill the figure that affects of algae efficient.In Fig. 4, X-coordinate is inorganic salt, from left to right is followed successively by NaNO
3, KBr, FeSO
4, CaCl
2, K
2HPO
4,, left ordinate zou is semilethal rate LD
50(%), right ordinate zou is the optical density value OD of wavelength under 600nm
600, histogram is for being semilethal rate LD
50, broken line graph is OD
600
Fig. 5 be in the embodiment of the invention different nitrogen sources on microvesicle bacterium BS03 growth with kill the figure that affects of algae efficient.In Fig. 5, X-coordinate is the pH value, from left to right is followed successively by 5～9,, left ordinate zou is semilethal rate LD
50(%), right ordinate zou is the optical density value OD of wavelength under 600nm
600, histogram is for being semilethal rate LD
50, broken line graph is OD
600
Embodiment
Following examples are to further specify of the present invention, but the invention is not restricted to following embodiment.
Bacterial classification: microvesicle bacterium BS03 is used and environmental microorganism institute separating and preserving by Xiamen University, be initially identified as microvesicle Pseudomonas (Microbulbifer sp.), be preserved in Chinese Typical Representative culture collection center on September 3rd, 2010, preservation center deposit number is CCTCC NO:M2010218.
Algae kind: Alexandrium tamarense (AT) is without bacterial strain, and algae kind system is provided by hydrobiont institute of Ji'nan University, and the Alexandrium tamarense that obtains through the degerming of the aseptic algae technology of the applicant is without bacterial strain.The used nutrient solution of algae is the f/2 nutrient solution.Algae places indoor triangular flask to cultivate, and temperature is 20 ± 1 ℃, and illumination condition is 12h illumination, and 12h is dark.
Initial medium (2216E): peptone (Peptone) 5g, yeast extract (Yeast Extract) 1g, high ferric phosphate 0.1g, agar powder 10g (solid medium), pH7.0～7.2, the Chen Haishui constant volume is to 1L.
Tuurbidimetry: get the thalline fermented liquid of suitable stoste or dilution, do contrast with empty substratum, under wavelength 600nm, measure optical density(OD) (OD) value.
Dry cell weight (DCW): draw fermented liquid 10ml, the centrifugal 10min of 8000r/min uses deionized water wash thalline 3 times after the supernatant discarded, and 80 ℃ of constant temperature dry to constant weight, and weigh and calculate DCW (g/L).
LD
50: get after the fermentation culture without mycetocyte filtrate, according to 0.5%, 1.0%, 1.5% 3 kind of different concns be added in the test algae, fixes with Compound Iodine Solutlon behind the effect 24h, under opticmicroscope, count.Use blue cosmos LD
50Software calculates mld (LD
50).
Employing 2216E is basic medium, carries out the exploration of single factor method optimum culture condition.
The impact of temperature: setting respectively culture temperature is 20,25,28,30 and 35 ℃, pH7.0～7.2,1% inoculum sizes, and 250ml triangle shaking flask liquid amount is 50ml, after the 180r/min shaking table is cultivated 24h, gets bacterium liquid and measures OD
600And LD
50
The impact of rotating speed: set respectively shaking table concussion speed and be 120,150,180 and 210r/min, all the other conditions are same as described above, cultivate to get fermented liquid behind the 24h and measure OD
600And LD
50
The impact of initial pH: the HCl that selects respectively 2.0mol/l and the pH of the NaOH accent substratum of 2.0mol/l are 5.0,6.0,7.0,8.0 and 9.0,28 ℃ of temperature, 1% inoculum size, 250ml triangle shaking flask liquid amount is 50ml, after the 180r/min shaking table is cultivated 24h, get bacterium liquid and measure OD
600And LD
50
Effects of salinity: the salinity of setting respectively substratum with artificial seawater is 10,20,30,40 and 50 ‰, initial pH7.0～7.2,28 ℃ of temperature, 1% inoculum size, 250ml triangle shaking flask liquid amount is 50ml, after the 180r/min shaking table is cultivated 24h,, then get fermented liquid and measure OD600 and LD
50
Experimental result shows: temperature plays an important role for the biomass of bacterial strain and the secretion of active metabolite in microorganism growth process, this BS03 bacterial strain mycetocyte density between 20～30 ℃ increases along with the rising of temperature, when temperature during greater than 30 ℃, the nectar degree descends to some extent.In the temperature change process, the generation rule of thalli growth and Algicidal substances is consistent.Therefore consider that saving the energy selects 28 ℃ to be culture condition.The change of microbial cultivation process medium speed directly impact be exactly the dissolved oxygen amount of microorganism, too high or too low dissolved oxygen speed all is unfavorable for thalli growth, when rotating speed is 180r/min, this BS03 bacterial strain biomass and LD
50Be respectively 1.726,0.648, this value is the shaking speed of suitable thalli growth and product active substance.PH affects larger in microorganism growth process, and this bacterium is that its biomass increases along with the rising of pH value under 5.0～8.0 the culture condition at pH, and LD
50The pH value be 7.0 o'clock minimum, namely this moment thalline to produce the ability of active substance the strongest.When the pH value greater than 8.0 and less than 6.0 the time thalline biomass and to produce the ability of actives all restricted.Therefore this bacterium is adapted at growing under the neutral meta-alkalescence condition most.Salinity is for the growth increment important in inhibiting of marine microorganism, and by the result as can be known, the generation of BS03 growth and algicdal activity material thereof all is subject to the remarkably influenced that salt concn changes.When the substratum salinity was 10 ‰, thalli growth was slow, and the ability of producing molten algae active substance is low, at this moment LD
50Be 4.105.Along with salinity increase thalli growth is rapid, molten algae active substance secretion capacity also obviously strengthens.When salinity greater than 30 ‰, increase though the algae rate of killing of this bacterium has slightly, thalli growth obviously slows down.Therefore 30 ‰ be the salinity range that is fit to microvesicle bacterium BS03.
So the optimal culture condition of BS03 is: culture temperature is 28 ℃, and the suitable initial pH of substratum is 7.0, and salinity is 30 ‰, and rotating speed is 180r/min.
At first select in the present embodiment several complex nutrients sources be those not only can be used as carbon source but also can make the nutritive substance (such as peptone, yeast powder etc.) of nitrogenous source and two kinds inorganic nitrogen-sourced.Then add different trace ingredientss on their basis, investigate each composition to the impact (referring to Fig. 1～5) of the growth of microvesicle bacterium and product active substance.
Take 2216E as basic medium (without Carbon and nitrogen sources), be complex nutrients sources and NaNO with 5.0g/L soy peptone, peptone, extractum carnis, yeast powder
3And KNO
3For inorganic nitrogen-sourced.All the other component concentrations are same as basic medium.Culture condition is: inoculum size is 1%, 28 ℃, and 180r/min, pH are 7, and salinity is 30 ‰, measures OD behind the shake-flask culture 24h
600And LD
50Result's demonstration, the BS03 bacterial strain is more suitable for being grown in the organonitrogen.The biomass of thalline is lower in the inorganic nitrogen, KNO
3, NaNO
3OD
600Value is respectively 0.071,0.06133, and the biomass of extractum carnis condition hypothallus reaches maximum OD in the organonitrogen
600Be 1.126, peptone takes second place 1.003, but the secretion capacity of the molten algae active substance of bacterial strain BS03 this moment is not as strong under the peptone culture condition.Therefore analysis-by-synthesis BS03 strain cultures selects peptone as its optimum nitrogen source, continues follow-up experiment.
The Zulkovsky starch, maltose, lactose, glucose, the sucrose that add respectively 1g/L on the basis of 5.0g/L Tryptones serve as carbon source, and only to be added with peptone in contrast 1, all the other component concentrations are same as basic medium, and 2216E is contrast 2.Culture condition is: inoculum size is 1%, 28 ℃, and 180r/min, pH are 7, and salinity is 30 ‰, measures OD behind the shake-flask culture 24h
600And LD
50In conjunction with increment and LD
50Investigate the component that adds and whether there is promoter action in the BS03 bacterial strain.The result shows: the fermented liquid biomass of different tests group substratum BS03 is all many than contrast 2 (former substratum 2216E), but difference is little.And algicidal effect difference of each group is clearly, may since medium component and proportioning Different Effects BS03 produce the ability of active Algicidal substances.
The contrast 1 that only is added with peptone is increment or killing the algae rate all is starkly lower than contrast 2, i.e. former basic medium; Carbon source is added in the component, adds the LD of its cell of substratum born of the same parents filtrate of dextrose plus saccharose
50Be 1.021,0.965, all be lower than 1.201 of former basic medium, and the LD of its cell of the substratum of other several interpolation carbon sources born of the same parents filtrate
50The LD that all is higher than the cell born of the same parents filtrate of former basic medium
50, this shows that dextrose plus saccharose has promoter action to microvesicle bacterium BS03 secretion activity material in these carbon source compositions; Because considering that Cost Problems event subsequent experimental selection sucrose is the optimum carbon source of bacterial strain BS03 substratum.
In fermentation process, the inoculum size of thalline and fermentation time also are the important factors that affects generation and the active metabolite secretion of microbial cells biomass.Therefore follow-up experiment is listed inoculum size and the fermentation time of thalline in affect bacterial strain BS03 fermentation factor of influence, participates in the medium optimization process.
Illustrate that this equation is believable to the simulation and forecast of dry cell weight.By above uniform design, each factor all has certain impact to killing algae rate and thalli growth amount as can be known, this microvesicle bacterium BS03 the optimal medium scheme be: pH value 7.5, incubation time 32h, peptone 10.50g/L, sucrose 8.0g/L, inoculum size is 3.0%.
According to pre-stage test, selecting peptone, sucrose, pH, inoculum size, incubation time is the homogeneous design that factor of influence carries out 5 factors, 15 levels, and all the other components are same as initial medium.Five factors are respectively independent variable(s) X1, X2, X3, X4 and X5, dry cell weight and OD
600Be dependent variable Y
1, Y
2Culture condition is selected: 28 ℃, and 180r/min, salinity is 30 ‰.Each factor all arranges 15 levels, and experimental result through DPS software data treatment system quadratic polynomial stepwise regression analysis, and is carried out test of significance (referring to table 1, table 2) to this model
Table 1
Regression equation is:
Y＝5.38134906+113.60189202X1×X1-3.793014914X2×X2+2.5854730603X3×X3-28.422411492X4×X4-0.15092230997X5×X5-65.98236457X1×X3-270.25223629X1×X5-10.298424271X2×X3+20.758363788X2×X4+49.38660676X2×X5+28.185002912X3×X4+145.52421280X3×X5-135.09790077X4×X5。
Coefficient R=0.99982, F value=209.8485, conspicuous level p=0.0540, surplus standard deviation S=0.06049, the coefficient R a=0.99743 after the adjustment, Durbin-Watson statistic d=1.72.
Table 2
Regression equation is:
Y＝10.83501238-172.80147120X3+432.5156109X5+8.067639869X3×X3-0.03230489278X4×X4-0.014902212031X1×X2-0.03589177354X1×X5+2.7406271234X2×X3-6.868578732X2×X5+0.017883024404X3×X4-20.232457027X3×X5。
Coefficient R=0.98340, F value=11.7502, conspicuous level p=0.0149, surplus standard deviation S=0.25665, the coefficient R a=0.94063 after the adjustment, Durbin-Watson statistic d=2.91.
By above uniform design, must this microvesicle bacterium BS03 the optimal medium scheme be: pH7.5, incubation time 32h, peptone 10.50g/L, sucrose 8.0g/L, inoculum size is 3.0%.The result is consistent with result take dry weight as index.
Embodiment 4
By above-mentioned optimization experiment, obtained optimum medium prescription and the optimal culture condition of microvesicle bacterium BS03.Initial medium and Optimal Medium are respectively applied to the shake-flask culture of BS03, culture condition adopts optimum culture condition.Thalline fermentation obvious effect of increasing production (referring to table 3) after the optimization.Cultivate 32h in shaking flask after, the dry cell weight value of its stationary phase can be up to 4.725g/L, surpasses the 3.498g/L with the predictor of regression model, and has improved 0.4 times than the basic medium before optimizing; Bacterial strain OD value after the optimization reaches 2.807, also surpasses the predictor 2.619 of regression model.Illustrate and utilize the homogeneous design optimization experiment not only feasibility is high, and effect is remarkable.
Table 3
Claims (1)
1. the substratum of a microvesicle bacterium BS03 is characterized in that described microvesicle bacterium BS03 is preserved in Chinese Typical Representative culture collection center on September 3rd, 2010, and deposit number is CCTCC NO:M 2010218;
The composition of the substratum of described microvesicle bacterium BS03 is to contain peptone 10.50g, sucrose 8g in the distilled water of 1L;
The substratum of described microvesicle bacterium BS03 is prepared by following methods: with being settled to 1L in peptone, the sucrose adding distilled water, namely get the substratum of microvesicle bacterium BS03 after the sterilization.
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