CN107129934A - Quick screening aerobic microbiological optimal pH conditioning agent and the method for determining the most suitable growth pH value - Google Patents
Quick screening aerobic microbiological optimal pH conditioning agent and the method for determining the most suitable growth pH value Download PDFInfo
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Abstract
The invention belongs to microorganism field, and in particular to quick screening aerobic microbiological optimal pH conditioning agent and the method for determining above-mentioned aerobic microbiological the most suitable growth pH value.The method of the present invention includes following steps：（1）Select the suitable alkalescence of aerobic microbiological culture and acidic ph modifier；（2）Verify optimal pH conditioning agent and determine the growth stationary phase of aerobic microbiological；（3）Screen the most suitable growth pH value；（4）Verify the most suitable growth pH value of the microorganism.By the method screening of the present invention and determine, obtained aerobic microbiological optimal pH conditioning agent and and growth of aerobic microorganisms most suitable pH value is determined, had using the method for the present invention it is simple to operate, quick, and the degree of accuracy it is high the characteristics of.
Description
Technical field
The invention belongs to microorganism field, and in particular to quick screening aerobic microbiological optimal pH conditioning agent, further relate to
The method for the aerobic microbiological the most suitable growth pH value stated.
Background technology
PH is the key parameter in microculture, and pH levels can influence the activity of desmoenzyme, reaction rate, Yi Jixi
Intracellular growth speed etc., and then influence cell concentration and purpose product yield.Meanwhile, the ion concentration in pH adjusting agent can influence carefully
Physiological status, the reaction rate of born of the same parents.From suitable pH adjusting agent and the suitable pH levels of maintenance, the growth of cell can be promoted,
Realize the high density fermentation of microorganism.
At present, it is general using being cultivated in triangular flask or fermentation tank, maintain certain pH by using different pH adjusting agents
Level, the influence grown according to different pH adjusting agents to microorganism determines the optimal pH conditioning agent of microorganism；By using triangle
Cultivated in bottle or fermentation tank, different pH levels are maintained using pH adjusting agent, microorganism is given birth to according to different pH controlled levels
Long influence, screens the most suitable growth pH value of microorganism.The microorganism optimal pH conditioning agent and the operation of the screening technique of pH levels
Complicated, workload is big, waste time and energy and accuracy is low.
The content of the invention
In order to solve above-mentioned technical problem, the invention provides one kind is simple to operate, quick, and the high screening of the degree of accuracy
Go out the method for the optimal pH conditioning agent and the most suitable growth pH value of aerobic microbiological strain.
The method of the present invention is the technical problem that the above is solved by following technical schemes：
Quick screening aerobic microbiological optimal pH conditioning agent and the method for determining the most suitable growth pH value, including following steps
Suddenly：
(1) aerobic microbiological is cultivated, the inorganic salts containing pH adjusting agent ion of various concentrations, root are added into culture medium
The optimal pH conditioning agent of aerobic microbiological is filtered out according to microorganism growing state；
(2) pH value of aerobic microbiological is adjusted using optimal pH conditioning agent in step (1), it is steady according to growth of aerobic microorganisms
Oxygen dissolving value draws the most suitable growth that pH value when oxygen dissolving value is minimum is aerobic microbiological with the situation of change of pH levels in periodically
PH value.
Aerobic microbiological is cultivated in the full-automatic growth analysis instrument of microorganism or triangular flask.
It is preferred that, above-mentioned quick screening aerobic microbiological optimal pH conditioning agent and the method for determining the most suitable growth pH value, bag
Include following step：
(1) selection aerobic microbiological culture suitable alkaline pH adjusting agent and acidic ph modifier：
In the full-automatic growth analysis instrument of microorganism or triangular flask, adjusted by being added into culture medium containing alkaline pH
Agent cation or the inorganic salts containing acidic ph modifier anion, according to different inorganic salt concentrations to growth of aerobic microorganisms
Influence, draws the most suitable growth alkaline pH adjusting agent A classes liquid for promoting aerobic microbiological significantly to grow, it is grown not made significant difference
Alkaline pH adjusting agent B class liquid, the acidic ph modifier C class liquid not made significant difference is grown to it；
(2) the growth stationary phase of checking optimal pH conditioning agent and determination aerobic microbiological：
Aerobic microbiological culture is carried out using different alkaline solutions as pH adjusting agent, A is further verified according to growing state
Liquid is optimal pH conditioning agent；The constant cultivation temperature of maintenance and dissolved oxygen level, pH value is adjusted using A liquid, carries out microculture,
Its growth curve is drawn, growth stationary phase of the microorganism under this condition of culture is determined；Above-mentioned alkaline solution is included most
Suitable pH adjusting agent A class liquid；
(3) the most suitable growth pH value is screened：
Cultivated under conditions of step (2), before microorganism enters growth stationary phase, pH value is adjusted using A liquid；When
Into after stationary phase, pH is down to the minimum that the microorganism grows pH scopes using C liquid, maintained after 8-12min, stream plus B liquid
PH value is improved, until reaching that the microorganism grows the peak of pH scopes, the pH elevation process is stable in the growth of the microorganism
Completed in phase, according to the situation of change of oxygen dissolving value in pH change procedures, pH value when oxygen dissolving value is minimum is drawn, for the microorganism
The most suitable growth pH value；
(4) the most suitable growth pH value of the microorganism is verified：
Under conditions of step (2), different pH value are set, described pH value includes the most suitable growth pH of aerobic microbiological
Value, is cultivated, the influence grown according to different pH levels to microorganism further determines that micro- life using A liquid regulation pH value
The most suitable growth pH value of thing.
PH adjusting agent includes conventional alkaline pH adjusting agent and acidic ph modifier；
Alkaline pH adjusting agent is：Any of ammoniacal liquor, sodium hydroxide, potassium hydroxide；
Acidic ph modifier is：Any of hydrochloric acid, sulfuric acid, nitric acid.
Aerobic microbiological passes through seed culture, using the full-automatic growth analysis instrument of microorganism or triangular flask fermented and cultured,
The influence grown according to the addition concentration containing pH adjusting agent cationic inorganic salt to microorganism, screens optimal pH conditioning agent；Using
Fermentation tank culture, according to situation of change of the oxygen dissolving value in stationary phase with pH levels, determines the most suitable growth pH value of microorganism.
According to the growth characteristics of aerobic microbiological, the suitable seed culture condition of selection and fermentation culture conditions, wherein：Kind
Sub- condition of culture is triangular flask culture, fermentation culture conditions include the full-automatic growth analysis instrument of microorganism or triangular flask and
The condition of culture of fermentation tank.
Aerobic microbiological strain is ox source Escherichia coli BXDC-N03, pig circular ring virus Cap protein expression bacterial strain
E.coliBZCP, ST171 plants of pig septic streptococcus, haemophilus parasuis BZXM-F05, avian pasteurella multocida BZXM-Q03, L- color
Any of propylhomoserin production bacterial strain E.coli BZTRP.
The inventive concept of the present invention is, by seed culture, in the full-automatic growth analysis instrument of microorganism or triangular flask
The inorganic salts that addition various concentrations contain pH adjusting agent ion carry out fermented and cultured, according to the aerobic micro- life of the ion pair of various concentrations
The influence of thing growth, rationally infers the influence grown containing different ions pH adjusting agent to the microorganism, show that optimal pH is adjusted
Agent is saved, and adjusts the pH value in the microbial cultivation process to verify that the optimal pH is adjusted by using different pH adjusting agents
Agent and its accuracy of screening technique.In fermentation tank top fermentation culture, determine with optimal pH conditioning agent and carry out aerobic microbiological
The stationary phase of culture, improved using the alkaline pH adjusting agent that does not make significant difference and acidic ph modifier is grown to the microorganism or
PH value in person's reduction stationary phase, according to situation of change of the oxygen dissolving value in stationary phase with pH levels, determines pH when oxygen dissolving value is minimum
It is worth the most suitable growth pH value for the microorganism, and carries out the microculture by setting different pH value and verifies that this is most suitable
Grow pH value and its accuracy of screening technique；The application microorganism is grown the alkaline pH adjusting agent that does not make significant difference and
Acidic ph modifier improves or reduced pH value in stationary phase, can exclude in pH value change procedure pH adjusting agent in itself to molten
The influence of oxygen value, it is the single factor for causing dissolved oxygen value changes in stationary phase to ensure pH value change.
The beneficial effects of the present invention are to the first seed culture of aerobic microbiological strain, be then respectively adopted microorganism complete
Automatic growth analyzer or triangular flask fermented and cultured screen the optimal pH conditioning agent of the aerobic microbiological；Ferment tank culture
Screen the optimum pH of the aerobic microbiological.In the fermentation process of the present invention, in the full-automatic growth analysis instrument of microorganism or three
In the bottle of angle, into culture medium, addition various concentrations contain conventional alkaline pH adjusting agent cation or acidic ph modifier anion
Inorganic salts, add the influence grown to the microorganism by inorganic salts, show that alkaline pH adjusting agent cation and acid pH are adjusted
The influence that section agent anion grows to the microorganism, then rationally infer that the pH adjusting agent containing different ions grows to the microorganism
Influence, so as to accurately screen the optimal pH conditioning agent of the microorganism；
It is certain value based on growth of aerobic microorganisms stationary phase oxygen consumption level in ferment tank culture, by using
Alkaline pH adjusting agent of the oxygen dissolving value without influence and acidic ph modifier are improved or pH levels in reduction stationary phase, according to dissolved oxygen
It is worth the change with pH levels, draws pH levels when oxygen dissolving value is minimum, then quickly determine the most suitable growth pH value of the microorganism.Gram
The fermenting experiment for adjusting pH pH levels different with setting using different pH adjusting agents in commonsense method has been taken to adjust to screen optimal pH
Workload that section agent and growth pH value method are present is big, waste time and energy, the defect that accuracy is low.Sieved by the method for the present invention
Choosing, obtained aerobic microbiological optimal pH conditioning agent and and be determined growth of aerobic microorganisms most suitable pH value, it is simple to operate,
Quickly, and the degree of accuracy is high.
Brief description of the drawings
The influence figure that Fig. 1 is cultivated ox source Escherichia coli for inorganic salts addition in embodiment 1；
Fig. 2 is influence figure of the pH adjusting agent in embodiment 1 to ox source Escherichia coli Growth；
Fig. 3 is variation diagram of the oxygen dissolving value in ox source Escherichia coli stationary phase in embodiment 1 with pH levels；
Fig. 4 is influence figure of the pH levels in embodiment 1 to ox source Escherichia coli Growth；
The influence figure that Fig. 5 is cultivated E.coli BZCP for inorganic salts addition in embodiment 2；
The influence figure that Fig. 6 grows for pH adjusting agent in embodiment 2 to E.coli BZCP；
Fig. 7 is variation diagram of the oxygen dissolving value in E.coli BZCP stationary phases in embodiment 2 with pH levels；
The influence figure that Fig. 8 grows for pH levels in embodiment 2 to E.coli BZCP；
The influence figure that Fig. 9 is cultivated pig septic streptococcus ST171 for inorganic salts addition in embodiment 3；
The influence figure that Figure 10 grows for pH adjusting agent in embodiment 3 to pig septic streptococcus ST171；
Figure 11 is variation diagram of the oxygen dissolving value in pig septic streptococcus ST171 stationary phases in embodiment 3 with pH levels；
Figure 12 is the influence figure of pH Level On Pigs septic streptococcus ST171 growths in embodiment 3；
Figure 13 is influence figure of the inorganic salts addition to haemophilus parasuis culture in embodiment 4；
The influence figure that Figure 14 grows for pH adjusting agent in embodiment 4 to haemophilus parasuis；
Figure 15 is variation diagram of the oxygen dissolving value in haemophilus parasuis stationary phase in embodiment 4 with pH levels；
The influence figure that Figure 16 grows for pH levels in embodiment 4 to haemophilus parasuis；
Figure 17 is influence figure of the inorganic salts addition to avian pasteurella multocida culture in embodiment 5；
The influence figure that Figure 18 grows for pH adjusting agent in embodiment 5 to avian pasteurella multocida；
Figure 19 is variation diagram of the oxygen dissolving value in avian pasteurella multocida stationary phase in embodiment 5 with pH levels；
The influence figure that Figure 20 grows for pH levels in embodiment 5 to avian pasteurella multocida；
The influence figure that Figure 21 is cultivated E.coli BZTRP for inorganic salts addition in embodiment 6；
The influence figure that Figure 22 grows for pH adjusting agent in embodiment 6 to E.coli BZTRP；
Figure 23 is variation diagram of the oxygen dissolving value in E.coli BZTRP stationary phases in embodiment 6 with pH levels；
The influence figure that Figure 24 grows for pH levels in embodiment 6 to E.coli BZTRP.
Embodiment
The present invention is further limited with reference to the accompanying drawings and detailed description, so as to those skilled in the art
The present invention is known more about, but the present invention is not limited with this.
Embodiment 1
Title：The optimal pH conditioning agent and growth pH value of quick screening ox source Escherichia coli
Bacterial strain：Ox source Escherichia coli BXDC-N03, is provided by Shandong Province Binzhou animal and veterinary research institute.
Culture medium：Seed culture medium is that (Shandong Yanggu moistens prosperous biological products to be had microbiological culture media with fermentation medium
Limit company).Condition of culture：
Seed culture：Picking single bacterium colony, is seeded in the 250mL triangular flasks equipped with 60mL culture mediums, pH 7.0, temperature 37
℃.Rotating speed 180rpm, incubation time 12h.
Fermented and cultured：
The full-automatic growth analysis instrument of microorganism：Inoculum concentration 2%, the initial pH 7.0 of culture medium, 37 DEG C of temperature, amplitude moderate,
Frequency moderate, cultivates 18h.
Fermentation tank：Inoculum concentration 5%, pH maintains 7.0, and 37 DEG C of temperature, dissolved oxygen level maintains 30%, cultivates 20h.
Operating procedure
(1) screening of pH adjusting agent
In the full-automatic growth analysis instrument of microorganism, NaCl, KCl, NH of various concentrations are added into culture medium4Cl、
NaNO3And Na2SO4To investigate Na+、K+、NH4 +、Cl-、NO3 -And SO4 2-Influence (Fig. 1) to ox source Escherichia coli Growth.By Fig. 1
(a, b, c) is learnt, addition NaCl does not make significant difference to ox source Escherichia coli Growth, then Na+Ion pair ox source Escherichia coli Growth
Do not make significant difference, therefore deduction NaOH does not make significant difference to ox source Escherichia coli Growth and is B liquid from NaOH；Add KCl and
NH4Cl can improve ox source Bacillus coli cells concentration, and addition NH4When Cl cell concentration is higher than addition KCl, then NH4 +From
Son infers NH most useful for ox source Escherichia coli Growth3·H2O is most useful for ox source Escherichia coli Growth and selects NH3·H2O is A
Liquid.Learnt by Fig. 1 (a, d, e), addition NaCl does not make significant difference to ox source Escherichia coli Growth, then Cl-Ion pair ox source large intestine
Bacillus growth does not make significant difference, therefore deduction HCl does not make significant difference to ox source Escherichia coli Growth and is B liquid from HCl；NaNO3
And Na2SO4Addition can improve the cell concentration of ox source Escherichia coli, then HNO3And H2SO4The life of ox source Escherichia coli can be promoted
It is long.
(2) checking of optimal pH conditioning agent and ox source Escherichia coli stationary phase determine
Ox source Escherichia coli are cultivated in fermentation tank, respectively with NH3·H2O, KOH and NaOH solution regulation pH value (Fig. 2).
Learnt by Fig. 2, with NH3·H2O, KOH are above using NaOH as pH adjusting agent as cell concentration during pH adjusting agent, and
With NH3·H2O then proves A liquid NH as cell concentration highest during pH adjusting agent3·H2O is the optimal pH of ox source Escherichia coli
The accuracy of screening pH adjusting agent in conditioning agent and step (1), and then prove NaOH and HCl as the accuracy of B liquid and C liquid；
It is determined that with NH3·H2When O solution is as pH adjusting agent, the stationary phase of ox source Escherichia coli is 12~17h.
(3) screening of the most suitable growth pH value
Ox source Escherichia coli are cultivated in fermentation tank, before culture during 13h, pH value is adjusted with A liquid；When in 13h, stream plus C
PH is down to 6.0 (process takes 10min) by liquid, stable 10min, after after dissolved oxygen value stabilization, starting stream plus B liquid, by pH value by
6.0 rise to 9.0 (process takes 30min).PH value is risen to during 9.0 by 6.0, and respective change (Fig. 3) occurs for oxygen dissolving value,
When pH value is 7.2, oxygen dissolving value is minimum, then shows the horizontal highest of oxygen consumption of now ox source Escherichia coli, illustrate ox source Escherichia coli
Growth in pH value 7.2 is most vigorous, it is thus determined that the most suitable growth pH value of ox source Escherichia coli is 7.2.
(4) checking of the most suitable growth pH value
Ox source Escherichia coli are cultivated in fermentation tank, using A liquid by pH value be respectively maintained at 6.8,7.0,7.2,7.4 and
7.6, cultivate 17h, viewing test result (Fig. 4).When pH maintains 7.2, cell concentration highest then shows ox source Escherichia coli
The most suitable growth pH value is 7.2, so as to prove that step (3) screens the accuracy of the most suitable growth pH value method.
Embodiment 2
Title：The optimal pH conditioning agent and growth pH value of quick screening recombination bacillus coli
Bacterial strain：Pig circular ring virus Cap protein expresses bacterial strain E.coli BZCP, by Shandong Province Binzhou animal and veterinary research institute
There is provided.
Culture medium：Seed culture medium and fermentation medium are engineering bacteria special culture media (the prosperous biological system of Shandong Yanggu profit
Product Co., Ltd).
Condition of culture：
Seed culture：Picking single bacterium colony, is seeded in the 250mL triangular flasks equipped with 60mL culture mediums, pH 7.0, temperature 37
℃.Rotating speed 180rpm, incubation time 12h.
Fermented and cultured：
Triangular flask：Inoculum concentration 2%, the initial pH 7.0 of culture medium, 37 DEG C of temperature, rotating speed 150rpm cultivates 18h.Fermentation tank：
Inoculum concentration 2%, pH maintains 7.0, and 37 DEG C of temperature, dissolved oxygen level maintains 30%, cultivates 18h.
Operating procedure
(1) screening of pH adjusting agent
Cultivated in triangular flask, NaCl, KCl, NH of various concentrations are added into culture medium4Cl、NaNO3And Na2SO4Come
Investigate Na+、K+、NH4 +、Cl-、NO3 -And SO4 2-The influence (Fig. 5) grown to E.coli BZCP.Learnt by Fig. 5 (a, b, c),
NaCl addition does not make significant difference to E.coli BZCP growths, then Na+Ion pair E.coli BZCP growth does not make significant difference,
Therefore deduction NaOH does not make significant difference to E.coli BZCP growths and is B liquid from NaOH；KCl and NH4Cl addition can be improved
E.coli BZCP cell concentrations, and addition NH4When Cl cell concentration is higher than addition KCl, then NH4 +Ion is most useful for E.coli
BZCP grows, therefore infers NH3·H2O grows most useful for E.coliBZCP and selects NH3·H2O is A liquid.Obtained by Fig. 1 (a, d, e)
Know, NaCl addition does not make significant difference to E.coli BZCP growths, then Cl-Ion pair E.coli BZCP are grown without notable shadow
Ring, therefore deduction HCl does not make significant difference to E.coli BZCP growths and is B liquid from HCl；NaNO3And Na2SO4Addition can carry
High E.coli BZCP cell concentration, then HNO3And H2SO4E.coli BZCP growth can be promoted.
(2) checking of optimal pH conditioning agent and E.coli BZCP stationary phases determine
E.coli BZCP are cultivated in fermentation tank, respectively with NH3·H2O, KOH and NaOH solution regulation pH value (Fig. 6).By
Fig. 6 is learnt, with NH3·H2O, KOH are above using NaOH as pH adjusting agent as cell concentration during pH adjusting agent, and with
NH3·H2O then proves A liquid NH as cell concentration highest during pH adjusting agent3·H2O adjusts for E.coli BZCP optimal pH
The accuracy of screening pH adjusting agent in agent and step (1) is saved, and then proves NaOH and HCl as the accuracy of B liquid and C liquid；Really
Determine with NH3·H2When O solution is as pH adjusting agent, E.coliBZCP stationary phase is 12~17h.
(3) screening of the most suitable growth pH value
E.coli BZCP are cultivated in fermentation tank, before culture during 13h, pH value is adjusted with A liquid；When in 13h, stream plus C liquid
PH is down to after 6.0 (process takes 10min), stable 10min, dissolved oxygen value stabilization, starts stream plus B liquid, by pH value by 6.0 liters
To 9.0 (process takes 30min).PH value is risen to during 9.0 by 6.0, and respective change (Fig. 7) occurs for oxygen dissolving value, in pH value
For 7.1 when, oxygen dissolving value is minimum, then shows the now E.coli BZCP horizontal highest of oxygen consumption, illustrate E.coli BZCP in pH value
Growth when 7.1 is most vigorous, it is thus determined that E.coliBZCP the most suitable growth pH value is 7.1.
(4) checking of the most suitable growth pH value
In fermentation tank cultivate E.coli BZCP, using A liquid by pH value be respectively maintained at 6.7,6.9,7.1,7.3 and
7.5, cultivate 16h, viewing test result (Fig. 8).When pH maintains 7.1, cell concentration highest then shows E.coli BZCP's
The most suitable growth pH value is 7.1, so as to prove that step (3) screens the accuracy of the most suitable growth pH value method.
Embodiment 3
Title：The optimal pH conditioning agent and growth pH value of quick ST171 plants of screening pig septic streptococcus
Bacterial strain：ST171 plants of pig septic streptococcus, is provided by Shandong Province Binzhou animal and veterinary research institute.
Culture medium：Seed culture medium and fermentation medium are that (Shandong Yanggu moistens prosperous biological products to be had microbiological culture media
Limit company).
Condition of culture：
Seed culture：Picking single bacterium colony, is seeded in the 250mL triangular flasks equipped with 60mL culture mediums, pH 7.0, temperature 37
℃.Rotating speed 150rpm, incubation time 8h.
Fermented and cultured：
The full-automatic growth analysis instrument of microorganism：Inoculum concentration 2%, the initial pH 7.0 of culture medium, 37 DEG C of temperature, amplitude moderate,
Frequency moderate, cultivates 12h.
Fermentation tank：Inoculum concentration 2%, pH maintains 7.2, and 37 DEG C of temperature, dissolved oxygen level maintains 30%, cultivates 12h.
Operating procedure：
(1) screening of pH adjusting agent
In the full-automatic growth analysis instrument of microorganism, NaCl, KCl, NH of various concentrations are added into culture medium4Cl、
NaNO3And Na2SO4To investigate Na+、K+、NH4 +、Cl-、NO3 -And SO4 2-Influence to the ST171 plants of growths of pig septic streptococcus
(Fig. 9).Learnt by Fig. 9 (a, b, c), NaCl addition does not make significant difference to the ST171 plants of growths of pig septic streptococcus, then Na+
The growth of ST171 plants of ion pair pig septic streptococcus does not make significant difference, therefore infers NaOH to ST171 plants of pig septic streptococcus
Growth does not make significant difference and is B liquid from NaOH；NH4Cl addition reduction pig ST171 plants of cell concentrations of septic streptococcus, then
NH4 +Ion is unfavorable for ST171 plants of pig septic streptococcus and grown, therefore NH3·H2O cannot function as pig septic streptococcus ST171
The pH adjusting agent of strain culture；KCl addition improves pig ST171 plants of cell concentrations of septic streptococcus, then K+Ion loses beneficial to pig
Courageous and upright streptococcus ST171 plants of growths, therefore KOH can promote the ST171 plants of growths of pig septic streptococcus and be A liquid from KOH.By scheming
1 (a, d, e) learns that NaCl addition does not make significant difference to the ST171 plants of growths of pig septic streptococcus, then Cl-Ion pair pig loses
Courageous and upright streptococcus ST171 plants of growths do not make significant difference, therefore infer HCl to the ST171 plants of growths of pig septic streptococcus without notable shadow
Ring and select HCl to be B liquid；NaNO3And Na2SO4Addition can improve the cell concentration of ST171 plants of pig septic streptococcus, then
HNO3And H2SO4The growth of ST171 plants of pig septic streptococcus can be promoted as pH adjusting agent.
(2) ST171 plants of stationary phases of the checking of optimal pH conditioning agent and pig septic streptococcus determine
Pig septic streptococcus ST171 is cultivated in fermentation tank, respectively with KOH, NaOH and NH3·H2O solution adjusts pH
It is worth (Figure 10).Learnt by Figure 10, cell concentration highest during using KOH as pH adjusting agent, and with NH3·H2O is used as pH adjusting agent
When cell concentration it is minimum, then prove A liquid KOH in the optimal pH conditioning agent and step (1) of ST171 plants of pig septic streptococcus
The accuracy of pH adjusting agent is screened, and then proves NaOH and HCl as the accuracy of B liquid and C liquid；It is determined that using KOH solution as
During pH adjusting agent, the stationary phase of ST171 plants of pig septic streptococcus is 8~10h.
(3) screening of the most suitable growth pH value
Pig septic streptococcus ST171 is cultivated in fermentation tank, before culture during 9h, pH value is adjusted with A liquid；When in 9h,
PH is down to after 6.0 (process takes 10min), stable 10min, dissolved oxygen value stabilization by stream plus C liquid, starts stream plus B liquid, by pH value
9.0 (process takes 30min) are risen to by 6.0.PH value is risen to during 9.0 by 6.0, and respective change (figure occurs for oxygen dissolving value
11), when pH value is 7.5, oxygen dissolving value is minimum, then shows the horizontal highest of oxygen consumption of now ST171 plants of pig septic streptococcus, say
The bright ST171 plants of growths in pH value 7.5 of pig septic streptococcus are most vigorous, it is thus determined that ST171 plants of pig septic streptococcus
The most suitable growth pH value be 7.5.(4) checking of the most suitable growth pH value
Pig septic streptococcus ST171 is cultivated in fermentation tank, pH value is respectively maintained at 7.1,7.3,7.5 using A liquid
And 7.7, cultivate 10h, viewing test result (Figure 12).When pH maintains 7.5, cell concentration highest then shows pig septic
The most suitable growth pH value of ST171 plants of streptococcus is 7.5, so as to prove that step (3) screens the accuracy of the most suitable growth pH value method.
Embodiment 4
Title：The optimal pH conditioning agent and growth pH value of quick screening haemophilus parasuis
Bacterial strain：Haemophilus parasuis BZXM-F05, is provided by Shandong Province Binzhou animal and veterinary research institute.
Culture medium：Seed culture medium and fermentation medium are TSB culture mediums (Beijing bispin microculture based articles
Factory).
Condition of culture：
Seed culture：Picking single bacterium colony, is seeded in the 250mL triangular flasks equipped with 60mL culture mediums, pH 7.0, temperature 37
℃.Rotating speed 100rpm, incubation time 16h.
Fermented and cultured：
Triangular flask：Inoculum concentration 2%, the initial pH 7.0 of culture medium, 37 DEG C of temperature, rotating speed 120rpm cultivates 24h.Fermentation tank：
Inoculum concentration 2%, pH maintains 7.0, and 37 DEG C of temperature, dissolved oxygen level maintains 10%, cultivates 24h.
Operating procedure：
(1) screening of pH adjusting agent
In triangular flask culture, NaCl, KCl, NH of various concentrations are added into culture medium4Cl、NaNO3And Na2SO4Come
Investigate Na+、K+、NH4 +、Cl-、NO3 -And SO4 2-The influence (Figure 13) grown to haemophilus parasuis.Learnt by Figure 13 (a, b, c),
NaCl addition does not make significant difference to haemophilus parasuis growth, then Na+The growth of ion pair haemophilus parasuis is without notable shadow
Ring, therefore deduction NaOH does not make significant difference to haemophilus parasuis growth and is B liquid from NaOH；NH4The Cl secondary pig of addition reduction
The cell concentration of haemophilus, then NH4 +Ion is unfavorable for haemophilus parasuis and grown, therefore NH3·H2It is bloodthirsty that O cannot function as secondary pig
The pH adjusting agent of bacillus culture；KCl addition improves haemophilus parasuis cell concentration, then K+Ion is conducive to the bloodthirsty bar of secondary pig
Bacteria growing, therefore KOH can promote haemophilus parasuis to grow and select KOH to be A liquid.Learnt by Fig. 1 (a, d, e), NaCl addition
Haemophilus parasuis growth is not made significant difference, then Cl-The growth of ion pair haemophilus parasuis does not make significant difference, therefore infers HCl
To haemophilus parasuis growth do not make significant difference and from HCl be B liquid；NaNO3And Na2SO4Addition can improve the bloodthirsty bar of secondary pig
The cell concentration of bacterium, then HNO3And H2SO4The growth of haemophilus parasuis can be promoted as pH adjusting agent.
(2) checking of optimal pH conditioning agent and haemophilus parasuis stationary phase determine
Haemophilus parasuis is cultivated in fermentation tank, with KOH, NaOH and NH3·H2O solution regulation pH value (Figure 14).By
Figure 14 learns, cell concentration highest during using KOH as pH adjusting agent, and with NH3·H2O is dense as cell during pH adjusting agent
Degree is minimum, then proves A liquid KOH to screen the accurate of pH adjusting agent in the optimal pH conditioning agent and step (1) of haemophilus parasuis
Property, and then prove NaOH and HCl as the accuracy of B liquid and C liquid；It is determined that during using KOH solution as pH adjusting agent, secondary pig is bloodthirsty
The stationary phase of bacillus is 16~20h.
(3) screening of the most suitable growth pH value
Haemophilus parasuis is cultivated in fermentation tank, before culture during 17h, pH value is adjusted with A liquid；When in 17h, stream plus C
PH is down to after 6.0 (process takes 10min), stable 10min, dissolved oxygen value stabilization by liquid, starts stream plus B liquid, by pH value by 6.0
Rise to 9.0 (process takes 30min).PH value is risen to during 9.0 by 6.0, and respective change (Figure 15) occurs for oxygen dissolving value, in pH
Be worth for 7.2 when, oxygen dissolving value is minimum, then shows the horizontal highest of oxygen consumption of now haemophilus parasuis, illustrate that haemophilus parasuis exists
Growth during pH value 7.2 is most vigorous, it is thus determined that the most suitable growth pH value of haemophilus parasuis is 7.2.
(4) the most suitable growth pH value is tested
Haemophilus parasuis is cultivated in fermentation tank, using A liquid by pH value be respectively maintained at 6.8,7.0,7.2,7.4 and
7.6, cultivate 20h, viewing test result (Figure 16).When pH maintains 7.2, cell concentration highest then shows the bloodthirsty bar of secondary pig
The most suitable growth pH value of bacterium is 7.2, so as to prove that step (3) screens the accuracy of the most suitable growth pH value method.
Embodiment 5
Title：The optimal pH conditioning agent and growth pH value of quick screening avian pasteurella multocida
Bacterial strain：Avian pasteurella multocida BZXM-Q03, is provided by Shandong Province Binzhou animal and veterinary research institute.
Culture medium：Seed culture medium and fermentation medium are that (Shandong Yanggu moistens prosperous biological products to be had microbiological culture media
Limit company).
Condition of culture：
Seed culture：Picking single bacterium colony, is seeded in the 250mL triangular flasks equipped with 60mL culture mediums, pH 7.0, temperature 37
℃.Rotating speed 100rpm, incubation time 10h.
Fermented and cultured：
The full-automatic growth analysis instrument of microorganism：Inoculum concentration 2%, the initial pH 7.0 of culture medium, 37 DEG C of temperature, amplitude moderate,
Frequency moderate, cultivates 12h.
Fermentation tank：Inoculum concentration 2%, pH maintains 7.0, and 37 DEG C of temperature, dissolved oxygen level maintains 20%, cultivates 12h.
Operating procedure：
(1) screening of pH adjusting agent
In the full-automatic growth analysis instrument of microorganism, NaCl, KCl, NH of various concentrations are added into culture medium4Cl、
NaNO3And Na2SO4To investigate Na+、K+、NH4 +、Cl-、NO3 -And SO4 2-The influence (Figure 17) grown to avian pasteurella multocida.By Figure 17
(a, b, c) is learnt, NaCl addition does not make significant difference to avian pasteurella multocida growth, then Na+The growth of ion pair avian pasteurella multocida
Do not make significant difference, therefore deduction NaOH does not make significant difference to avian pasteurella multocida growth and is B liquid from NaOH；NH4Cl addition drop
The cell concentration of low avian pasteurella multocida, then NH4 +Ion is unfavorable for avian pasteurella multocida and grown, therefore NH3·H2O cannot function as fowl Pasteur
The pH adjusting agent of bacillus culture；KCl addition improves avian pasteurella multocida cell concentration, then K+Ion is conducive to avian pasteurella multocida to give birth to
It is long, therefore KOH can promote avian pasteurella multocida to grow and select KOH to be A liquid.Learnt by Fig. 1 (a, d, e), NaCl addition is to fowl bar
The growth of family name bacillus does not make significant difference, then Cl-The growth of ion pair avian pasteurella multocida does not make significant difference, therefore infers HCl to fowl Pasteur's bar
Bacteria growing does not make significant difference and is B liquid from HCl；NaNO3And Na2SO4Addition can improve the cell concentration of avian pasteurella multocida,
Then HNO3And H2SO4The growth of avian pasteurella multocida can be promoted as pH adjusting agent.
(2) checking of optimal pH conditioning agent and avian pasteurella multocida stationary phase determine
Avian pasteurella multocida is cultivated in fermentation tank, respectively with KOH, NaOH and NH3·H2O solution regulation pH value (Figure 18).
Learnt by Figure 18, cell concentration highest during using KOH as pH adjusting agent, and with NH3·H2O is used as cell during pH adjusting agent
Concentration is minimum, then proves the accuracy of screening pH adjusting agent in the optimal pH conditioning agent and step (1) of A liquid KOH avian pasteurella multocidas,
And then prove NaOH and HCl as the accuracy of B liquid and C liquid；It is determined that during using KOH solution as pH adjusting agent, avian pasteurella multocida
Stationary phase be 8~10h.
(3) screening of the most suitable growth pH value
Avian pasteurella multocida is cultivated in fermentation tank, before culture during 8.5h, pH value is adjusted with A liquid；When in 8.5h, stream plus C
PH is down to after 6.0 (process takes 10min), stable 10min, dissolved oxygen value stabilization by liquid, starts stream plus B liquid, by pH value by 6.0
Rise to 9.0 (process takes 30min).PH value is risen to during 9.0 by 6.0, and respective change (Figure 19) occurs for oxygen dissolving value, in pH
Be worth for 7.2 when, oxygen dissolving value is minimum, then shows the horizontal highest of oxygen consumption of now avian pasteurella multocida, illustrate avian pasteurella multocida in pH value
Growth when 7.2 is most vigorous, it is thus determined that the most suitable growth pH value of avian pasteurella multocida is 7.2.
Then the most suitable growth pH value of avian pasteurella multocida is 7.2.
(4) checking of the most suitable growth pH value
Avian pasteurella multocida is cultivated in fermentation tank, pH value is respectively maintained at 6.8,7.0,7.2,7.4 and 7.6 using A liquid,
Cultivate 20h, viewing test result (Figure 20).When pH maintains 7.2, cell concentration highest then shows avian pasteurella multocida most
Suitable growth pH value is 7.2, so as to prove that step (3) screens the accuracy of the most suitable growth pH value method.
Embodiment 6
Title：The optimal pH conditioning agent and growth pH value of quick screening L-Trp production bacterial strain
Bacterial strain：L-Trp produces bacterial strain E.coli BZTRP, is provided by Shandong Province Binzhou animal and veterinary research institute.
Culture medium：Seed culture medium and fermentation medium are：Glucose 10g/L, dusty yeast 2g/L, peptone 3g/L,
KH2PO4 1.5g/L、MgSO4 2g/L、(NH4)2SO4 2g/L、pH 7.0。
Condition of culture：
Seed culture：Picking single bacterium colony, is seeded in the 250mL triangular flasks equipped with 60mL culture mediums, pH 7.0, temperature 37
DEG C, rotating speed 180rpm, incubation time 12h.
Fermented and cultured：
Triangular flask culture：Inoculum concentration 10%, the initial pH 7.0 of culture medium, 37 DEG C of temperature, rotating speed 180rpm cultivates 40h.
Fermentation tank：Inoculum concentration 10%；PH maintains 7.0；37 DEG C of temperature；Dissolved oxygen level maintains 20%；Initial glucose
When exhausting, glucose solution is added using dissolved oxygen feedback supplement strategy；Cultivate 40h.
Operating procedure：
(1) screening of pH adjusting agent
In the full-automatic growth analysis instrument of microorganism, NaCl, KCl, NH of various concentrations are added into culture medium4Cl、
NaNO3And Na2SO4To investigate Na+、K+、NH4 +、Cl-、NO3 -And SO4 2-The influence (Figure 21) grown to E.coli BZTRP.By scheming
21 (a, b, c) learn that NaCl addition does not make significant difference to E.coli BZTRP growths, then Na+Ion pair E.coli BZTRP
Growth do not make significant difference, therefore infer NaOH to E.coli BZTRP growth do not make significant difference and from NaOH be B liquid；KCl and
NH4Cl addition can improve E.coli BZTRP cell concentrations, and addition NH4When Cl cell concentration is higher than addition KCl, then
NH4 +Ion grows most useful for E.coli BZTRP, therefore infers NH3·H2O grows most useful for E.coli BZTRP and selects NH3·
H2O is A liquid.Learnt by Fig. 1 (a, d, e), NaCl addition does not make significant difference to E.coli BZTRP growths, then Cl-Ion pair
E.coli BZTRP growths do not make significant difference, therefore deduction HCl does not make significant difference to E.coli BZTRP growths and is B from HCl
Liquid；NaNO3And Na2SO4Addition can improve E.coli BZTRP cell concentration, then HNO3And H2SO4E.coli can be promoted
BZTRP growth.
(2) checking of optimal pH conditioning agent and E.coli BZTRP stationary phases determine
E.coli BZTRP are cultivated in fermentation tank, respectively with NH3·H2O, KOH and NaOH solution regulation pH value (figure
22).Learnt by Figure 22, with NH3·H2O, KOH are above being used as pH adjusting agent using NaOH as cell concentration during pH adjusting agent
, and with NH3·H2O then proves A liquid NH as cell concentration highest during pH adjusting agent3·H2O is E.coli BZTRP's
The accuracy of screening pH adjusting agent in optimal pH conditioning agent and step (1), and then prove NaOH and HCl as the standard of B liquid and C liquid
True property；It is determined that with NH3·H2When O solution is as pH adjusting agent, E.coli BZTRP stationary phases are 26~36h.
(3) screening of the most suitable growth pH value
E.coli BZTRP are cultivated in fermentation tank, before culture during 28h, pH value is adjusted with A liquid；When in 28h, stream plus C
PH is down to after 5.5 (process takes 15min), stable 10min, dissolved oxygen value stabilization by liquid, starts stream plus B liquid, by pH value by 5.5
Rise to 8.5 (process takes 30min).PH value is risen to during 8.5 by 5.5, and respective change (Figure 23) occurs for oxygen dissolving value, in pH
Be worth for 7.0 when, oxygen dissolving value is minimum, then shows the now E.coli BZTRP horizontal highest of oxygen consumption, illustrate that E.coli BZTRP exist
Growth during pH value 7.0 is most vigorous, it is thus determined that E.coli BZTRP the most suitable growth value pH is 7.0.
(4) checking of the most suitable growth pH value
In fermentation tank cultivate E.coli BZTRP, using A liquid by pH value be respectively maintained at 6.4,6.8,7.0,7.2 and
7.4, cultivate 36h, viewing test result (Figure 24).When pH maintains 7.0, cell concentration highest then shows E.coli
BZTRP the most suitable growth pH value is 7.0, so as to prove that step (3) screens the accuracy of the most suitable growth pH value method.
Claims (7)
1. quickly screening aerobic microbiological optimal pH conditioning agent and the method for determining the most suitable growth pH value, including following steps：
（1）Aerobic microbiological is cultivated, the inorganic salts containing pH adjusting agent ion of various concentrations are added into culture medium, according to micro-
Biological growth situation filters out the optimal pH conditioning agent of aerobic microbiological；
（2）Utilize step（1）Middle optimal pH conditioning agent adjusts the pH value of aerobic microbiological, according to growth of aerobic microorganisms stationary phase
Interior oxygen dissolving value draws the most suitable growth pH that pH value when oxygen dissolving value is minimum is aerobic microbiological with the situation of change of pH levels
Value.
2. quick screening aerobic microbiological optimal pH conditioning agent as claimed in claim 1 and the side for determining the most suitable growth pH value
Method, it is characterised in that cultivate aerobic microbiological in the full-automatic growth analysis instrument of microorganism or triangular flask.
3. quick screening aerobic microbiological optimal pH conditioning agent as claimed in claim 1 and the side for determining the most suitable growth pH value
Method, including following steps：
（1）The alkaline pH adjusting agent and acidic ph modifier for selecting aerobic microbiological culture suitable：
In the full-automatic growth analysis instrument of microorganism or triangular flask, by being added into culture medium containing alkaline pH adjusting agent sun
Ion or the inorganic salts containing acidic ph modifier anion, according to shadow of the different inorganic salt concentrations to growth of aerobic microorganisms
Ring, draw the most suitable growth alkaline pH adjusting agent A classes liquid for promoting aerobic microbiological significantly to grow, what is do not made significant difference is grown to it
Alkaline pH adjusting agent B class liquid, the acidic ph modifier C class liquid not made significant difference is grown to it；
（2）Verify optimal pH conditioning agent and determine the growth stationary phase of aerobic microbiological：
Aerobic microbiological culture is carried out using different alkaline solutions as pH adjusting agent, further verifies that A liquid is according to growing state
Optimal pH conditioning agent；The constant cultivation temperature of maintenance and dissolved oxygen level, utilize A liquid to adjust pH value, carry out microculture, draw
Its growth curve, determines growth stationary phase of the microorganism under this condition of culture；Above-mentioned alkaline solution includes optimal pH
Conditioning agent A class liquid；
（3）Screen the most suitable growth pH value：
In step（2）Under conditions of cultivated, microorganism enter growth stationary phase before, utilize A liquid regulation pH value；Work as entrance
After stationary phase, pH is down to the minimum that the microorganism grows pH scopes using C liquid, maintained after 8-12 min, stream plus B liquid are improved
PH value, until reaching that the microorganism grows the peak of pH scopes, the pH elevation process is within the growth stationary phase of the microorganism
Complete, according to the situation of change of oxygen dissolving value in pH change procedures, pH value when oxygen dissolving value is minimum is drawn, for the most suitable of the microorganism
Grow pH value；
（4）Verify the most suitable growth pH value of the microorganism：
In step（2）Under conditions of, different pH value are set, and described pH value includes the most suitable growth pH value of aerobic microbiological,
Cultivated using A liquid regulation pH value, the influence grown according to different pH levels to microorganism further determines that the microorganism
The most suitable growth pH value.
4. quick screening aerobic microbiological optimal pH conditioning agent as claimed in claim 1 and the side for determining the most suitable growth pH value
Method, it is characterised in that pH adjusting agent includes conventional alkaline pH adjusting agent and acidic ph modifier；
Alkaline pH adjusting agent is：Any of ammoniacal liquor, sodium hydroxide, potassium hydroxide；
Acidic ph modifier is：Any of hydrochloric acid, sulfuric acid, nitric acid.
5. quick screening aerobic microbiological optimal pH conditioning agent as claimed in claim 1 and the side for determining the most suitable growth pH value
Method, it is characterised in that aerobic microbiological passes through seed culture, is fermented using the full-automatic growth analysis instrument of microorganism or triangular flask
Optimal pH conditioning agent is screened in culture, the influence grown according to the addition concentration containing pH adjusting agent cationic inorganic salt to microorganism；
Using fermentation tank culture, according to situation of change of the oxygen dissolving value in stationary phase with pH levels, the most suitable growth pH value of microorganism is determined.
6. quick screening aerobic microbiological optimal pH conditioning agent as claimed in claim 4 and the side for determining the most suitable growth pH value
Method, it is characterised in that according to the growth characteristics of aerobic microbiological, the suitable seed culture condition of selection and fermentation culture conditions,
Wherein：Seed culture condition is triangular flask culture, and fermentation culture conditions include the full-automatic growth analysis instrument of microorganism or triangle
The condition of culture of bottle and fermentation tank.
7. quick screening aerobic microbiological optimal pH conditioning agent as claimed in claim 1 and the side for determining the most suitable growth pH value
Method, it is characterised in that aerobic microbiological strain is ox source Escherichia coli BXDC-N03, pig circular ring virus Cap protein expression bacterial strainE. coli BZCP, ST171 plants of pig septic streptococcus, haemophilus parasuis BZXM-F05, avian pasteurella multocida BZXM-Q03, L-
Tryptophan-producing Strain strainE. coliAny of BZTRP.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100434509C (en) * | 2002-03-26 | 2008-11-19 | 有限会社新世纪发酵研究所 | Method of continuous culture of anaerobic bacterium |
| US20160304376A1 (en) * | 2015-04-20 | 2016-10-20 | Arizona Board Of Regents On Behalf Of Arizona State University | Methods and Systems for pH Treatment and Extraction of Leachable Resources and Pollutants from Sludge |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100434509C (en) * | 2002-03-26 | 2008-11-19 | 有限会社新世纪发酵研究所 | Method of continuous culture of anaerobic bacterium |
| US20160304376A1 (en) * | 2015-04-20 | 2016-10-20 | Arizona Board Of Regents On Behalf Of Arizona State University | Methods and Systems for pH Treatment and Extraction of Leachable Resources and Pollutants from Sludge |
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| Title |
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| 刘吉华: "《中药生物技术[M]》", 31 December 2015 * |
| 张虎成，齐贺: "《发酵原料药生产[M]》", 31 December 2014 * |
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