CN104480224B - A kind of control method of fermentation process pH value - Google Patents
A kind of control method of fermentation process pH value Download PDFInfo
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- CN104480224B CN104480224B CN201410761614.1A CN201410761614A CN104480224B CN 104480224 B CN104480224 B CN 104480224B CN 201410761614 A CN201410761614 A CN 201410761614A CN 104480224 B CN104480224 B CN 104480224B
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Abstract
The invention provides the control method of a kind of fermentation process pH value, belong to microorganism fermentation process and control technical field.The control method of pH value of the present invention, carries out sterilizing by pretreated resin, is then added in fermentation liquid carry out the regulation of pH value.In particular by anion exchange resin, the pH value of microbial fermentation solution is adjusted, the pH value of fermentation liquid is controlled in a rational numerical value or scope the most quickly and accurately, avoid the unnecessary intermediate product using above-mentioned pH value control method to be brought, simplify the technological process of entirety, time saving and energy saving, and the pH value of whole sweat is easily controllable.
Description
Technical field
The invention belongs to microorganism fermentation process and control technical field, be specifically related to the control method of a kind of fermentation process pH value.
Background technology
In sweat, the pH value of culture fluid is microorganism aggregative indicator of metabolic activity under the conditions of certain environment, is an important fermentation parameter.The growth of thalline and the accumulation of product are had a great impact by it.The change of pH value can cause the change of various enzyme activity, affects thalline and substrate utilizes the structure of speed and cell, so that affecting the production of thalline and the synthesis of product.PH value also can affect somatic cells membrane charge situation, causes permeability of the membrane to change, thus affects the thalline absorption to nutrition and the formation etc. of its metabolite.
During the fermentation, due to the metabolism of microorganism, fermentation liquid typically has the existence of the organic acid such as lactic acid, acetic acid.In this process, if not carrying out the regulation of pH value, then very likely can cause fermenting suppressed and making productivity reduce.If adding alkaline matter to be adjusted, the salts substances that on the one hand can increase fermentation liquid affects being smoothed out of downstream extraction, on the other hand can increase the osmotic pressure of fermentation liquid, also influence whether the normal growth of microorganism.Therefore, it is necessary to the Changing Pattern of pH value in grasp sweat, monitor in time and be controlled by, making it be in optimal state.
Difference according to different microorganisms sweat, currently known pH value control method mainly has: adds aqueous alkali (sodium hydroxide solution, potassium hydroxide solution, ammonia etc.), adds alkalescence material (calcium hydroxide, sodium carbonate etc.) and use buffer solution etc., the acid produced in sweat can be neutralized by the addition of these materials effectively, but also can produce various salts substances simultaneously.The existence of these salts substances, on the one hand adds the osmotic pressure in sweat, on the other hand directly increases the burden of downstream extraction and product purification；And the collection method of these salts substances all can additionally increase production cost and affect the production technology of entirety.So, explore a kind of pH value control method that can overcome drawbacks described above one of can yet be regarded as and effectively reduce cost, improve the shortcut of productivity.
Known, ion exchange resin is a class with can the regenerating of functional group, Reusability and insoluble inert polymer material, not for organism absorption.According to the difference of exchangeable ion, ion exchange resin is divided into cation exchange resin and the big class of anion exchange resin two.Cation exchange resin is divided into storng-acid cation exchange resin and weak-acid cation-exchange resin, wherein, the dissociation capability of storng-acid cation exchange resin is very strong, all can dissociate and produce ion exchange in any acidity or alkaline solution, and its effect PH scope is between 1 ~ 14；Otherwise, the dissociation capability of weak-acid cation-exchange resin is the most weak, can only dissociate and produce ion exchange in faintly acid to alkaline solution, and its effect PH scope is only between 5 ~ 14.Anion exchange resin is generally divided into strong-base anion-exchange resin and weak-base anion-exchange resin two kinds, wherein strong-base anion-exchange resin can adsorb all of anion, can use under different PH environment, and weak-base anion-exchange resin can only adsorber acid radical ion, can only use in neutral or sour environment.And, the release of the hydroxide ion of weak-base anion-exchange resin is one and discharges process slowly.In resin regeneration process, hydroxide ion can be adsorbed on the basic group of anion exchange resin, and anion exchange resin can dissociate hydroxide ion in water and make solution alkaline, and the anion in water can be adsorbed onto on the basic group of anion exchange resin, thus complete exchange process.At present, ion exchange resin is mainly used in water and processes, also material purification, concentrate, separate, the aspect such as transformation, the decolouring of material and the catalyst of substance ion composition has purposes widely.
Summary of the invention
The technical problem to be solved is, for the deficiencies in the prior art, it is provided that a kind of easy and simple to handle, to the subsequent treatment of fermentation liquid without the pH value control method of added burden.
For solving above-mentioned technical problem, the technical solution adopted in the present invention is: the control method of a kind of fermentation process pH value, including sterilizing and the regulation of pH value of resin, concretely comprise the following steps and pretreated resin is carried out sterilizing, be then added in fermentation liquid carry out the regulation of pH value.
As the further optimization to the present invention, described resin is 0.5:1 (g:L) ~ 30:1(g:L with the mass volume ratio of described fermentation liquid).
As the further optimization to the present invention, the addition of described resin is once to add in the either phase of fermentation process.
As the further optimization to the present invention, described resin is macroporous resin or gel resin.
As the further optimization to the present invention, described resin is macropore negative resin Lsi-363b, macropore negative resin Lsa-900C or gel negative resin 201 × 7.
Beneficial effects of the present invention is described further as follows below in conjunction with the mechanism of action:
Microbial fermentation solution is a complicated system, prolongation along with fermentation time, its composition constantly changes, generally speaking, fermentation liquid exists other complex components beyond a large amount of mycelium, strain metabolite, culture medium, target product and target product thereof, Just because of this, fermentation liquid to be made maintain in whole sweat one suitably in the range of be extremely difficult, but fermentating liquid PH value is but the key parameter controlling target product yield, if pH value is improper, or it is excessive to fluctuate, the output of target product all can be had a strong impact on.The control method of pH value in prior art, it is adjusted for example with PH regulators such as sodium hydroxide solution, potassium hydroxide solution, ammonia, calcium hydroxide or sodium carbonate, in the range of being all only capable of maintaining pH value to be in suitably at short notice, after PH regulator metabolite in fermentation liquid or existing material are combined, the maintenance ability of its pH value weakens rapidly, even reduces to zero.At this time it is accomplished by repeating aforesaid PH and regulates operation, again regulate in the range of suitably, then proceed to fermentation, when improper, then be adjusted, go round and begin again, waste time and energy, and it is the most bad to control effect, and processing ease microbiological contamination frequently.Even continuous print PH control method, it is also desirable to the situation of change of monitor fermentation liquid pH value in real time, prepare suitable PH regulator, the most still can introduce product incoherent with target product so that follow-up is refined procedure complicated.Based on this, the control method of pH value of the present invention, according to the characteristic of microbial fermentation solution, uses resin to carry out the regulation of microbial fermentation solution pH value, being adjusted the pH value of microbial fermentation solution in particular by anion exchange resin, anion exchange resin constantly discharges OH in fermentation liquid-With the H in environment+Neutralize, thus the pH value of fermentation liquid is controlled in a rational numerical value or scope the most quickly and accurately, it is to avoid the unnecessary intermediate product that employing above-mentioned pH value control method is brought, simplify the technological process of entirety, time saving and energy saving, and the pH value of whole sweat is easily controllable.
Resin employed in the control method of fermentation process pH value of the present invention is any one in the mixture of macropore negative resin, macropore positive resin and the mixture of macropore negative resin, gel negative resin and gel positive resin and gel negative resin.Anion exchange resin is in resin regeneration process, hydroxide ion can be adsorbed on its basic group, and anion exchange resin can dissociate hydroxide ion in water and make solution alkaline, and the anion in water can be adsorbed onto on the basic group of anion exchange resin, thus complete exchange process.While exchange process completes, hydroxide ion carries out reaction with the hydrion of generation in sweat and generates H2O, thus complete the regulation process of pH value.Obviously, the regulation process of this pH value need not given either continuously or intermittently add alkali liquor and is adjusted, the regulation process of whole pH value is a process being automatically performed, simplify the cumbersome process of pH value regulation, alleviate operation easier, add after resin, can automatically, be accurately finished the regulation of fermentating liquid PH value.
The control method of fermentation process pH value of the present invention is particularly suitable for the regulation of the microbial fermentation solution pH value of acidity or weak acid environment, controls effective, simplifies production technology, alleviates the operation easier of operating personnel.
Detailed description of the invention
In order to be further elucidated with rather than limit the present invention in more detail, following embodiment is only limitted to explanation of the invention.
The control method of fermentation process pH value of the present invention, including sterilizing and the regulation of pH value of resin, concretely comprises the following steps and pretreated resin is carried out sterilizing, be then added in fermentation liquid carry out the regulation of pH value.
Described resin is 0.5:1 (g:L) ~ 30:1(g:L with the mass volume ratio of described fermentation liquid).
The addition of described resin is once to add in the either phase of fermentation process.
Described resin is macroporous resin or gel resin.
As the further optimization to the present invention, described resin is macropore negative resin Lsi-363b, macropore negative resin Lsa-900C or gel negative resin 201 × 7.
It should be noted that the pretreatment of ion exchange resin is conventional processing method, belongs to known prior art, repeat no more.
Embodiment one
As a example by oleaginous yeast glucose fermentation Lipid-producing, 500mL fermentation liquid, shaking table is cultivated 24 hours, and the w/v adding gel negative resin 201 × 7 0.5g(resin after sterilizing and fermentation liquid is 1:1(g:L)), recording the mass volume ratio of glucose in fermentation liquid is 2.69%, cell number is 0.6 hundred million/mL, PH is 5.98, and after cultivating 48 hours, recording the mass volume ratio of glucose in fermentation liquid is 1.03%, cell number be 2.31 hundred million/mL, PH be 5.68.
Comparative example one: unlike embodiment one, after shaking table is cultivated 24 hours, the PH using sodium hydroxide solution aseptically to regulate fermentation liquid is 5.98, and carbon source concentration now is identical with embodiment one with cell number；After cultivating 48 hours, recording the mass volume ratio of glucose in fermentation liquid is 2.05%, cell number be 1.22 hundred million/mL, PH be 3.98.
Comparative example two: unlike embodiment one, after shaking table is cultivated 24 hours, the PH using calcium carbonate soln aseptically to regulate fermentation liquid is 5.98, and carbon source concentration now is identical with embodiment one with cell number；After cultivating 48 hours, recording the mass volume ratio of glucose in fermentation liquid is 1.69%, cell number be 1.85 hundred million/mL, PH be 4.83.
The present embodiment one, in oleaginous yeast fermentation liquid, adds the gel negative resin 201 × 7 after sterilizing, and after cultivating 48 hours, the consumption rate of glucose is 61.7%, and cell number increases by 1.71 hundred million/mL, and pH value is basicly stable；In comparative example one, the consumption rate of glucose is 23.8%, and cell number increases by 0.62 hundred million/mL, and pH value fluctuation is very big, differs 2.0 with initial p H；In comparative example one, the consumption rate of glucose is 37.2%, and cell number increases by 1.25 hundred million/mL, and pH value fluctuation is relatively big, differs 1.11 with initial p H.This shows, utilize the pH value of gel negative resin 201 × 7 regulation oleaginous yeast fermentation liquid, pH value can remain basicly stable within the longer time, so may advantageously facilitate the conversion of carbon source, the conversion rate making carbon source doubles, increase the reproduction speed of cell, thus shorten fermentation period, save corresponding cost and labor cost.
Embodiment two
As a example by oleaginous yeast glucose fermentation Lipid-producing, 500mL fermentation liquid, shaking table is cultivated 24 hours, and the w/v adding gel negative resin 201 × 7 5g(resin after sterilizing and fermentation liquid is 10:1(g:L)), recording the mass volume ratio of glucose in fermentation liquid is 2.56%, cell number is 0.58 hundred million/mL, PH is 5.66, and after cultivating 48 hours, recording the mass volume ratio of glucose in fermentation liquid is 1.37%, cell number be 2.72 hundred million/mL, PH be 6.26.
Comparative example one: after shaking table is cultivated 24 hours, the PH using sodium hydroxide solution aseptically to regulate fermentation liquid is 5.66, and carbon source concentration now is identical with embodiment two with cell number；After cultivating 48 hours, recording the mass volume ratio of glucose in fermentation liquid is 1.95%, cell number be 1.82 hundred million/mL, PH be 3.18.
Comparative example two: unlike embodiment two, after shaking table is cultivated 24 hours, the PH using calcium carbonate soln aseptically to regulate fermentation liquid is 5.66, and carbon source concentration now is identical with embodiment two with cell number；After cultivating 48 hours, recording the mass volume ratio of glucose in fermentation liquid is 1.62%, cell number be 2.01 hundred million/mL, PH be 4.56.
The present embodiment two is essentially identical with embodiment one with the contrast effect of comparative example one and comparative example two, repeats no more.
Embodiment three
As a example by oleaginous yeast glucose fermentation Lipid-producing, 500mL fermentation liquid, shaking table is cultivated 24 hours, and the w/v adding gel negative resin 201 × 7 0.05g(resin after sterilizing and fermentation liquid is 0.5:1(g:L)), recording the mass volume ratio of glucose in fermentation liquid is 2.72%, cell number is 0.62 hundred million/mL, PH is 6.02, and after cultivating 48 hours, recording the mass volume ratio of glucose in fermentation liquid is 1.12%, cell number be 2.32 hundred million/mL, PH be 5.87.
Comparative example one: unlike embodiment three, after shaking table is cultivated 24 hours, the PH using sodium hydroxide solution aseptically to regulate fermentation liquid is 6.02, and carbon source concentration now is identical with embodiment three with cell number；After cultivating 48 hours, recording the mass volume ratio of glucose in fermentation liquid is 1.93%, cell number be 1.78 hundred million/mL, PH be 3.78.
Comparative example two: unlike embodiment three, after shaking table is cultivated 24 hours, the PH using calcium carbonate soln aseptically to regulate fermentation liquid is 6.02, and carbon source concentration now is identical with embodiment three with cell number；After cultivating 48 hours, recording the mass volume ratio of glucose in fermentation liquid is 1.53%, cell number be 2.10 hundred million/mL, PH be 5.02.
The present embodiment three is essentially identical with embodiment one with the contrast effect of comparative example one and comparative example two, repeats no more.
Embodiment four
Produce as a example by 1,3-PD by Klebsiella pneumoniae fermentation glycerol, 500mL fermentation liquid, shaking table is cultivated 20 hours, the w/v adding aseptic macropore negative resin Lsi-363b 1g(resin and fermentation liquid is 2:1(g:L)), recording the mass volume ratio of glycerol in fermentation liquid is 2.29%, 1, ammediol (PDO) is 0.5%(w/v), PH is 4.98, and after cultivating 28 hours, recording the mass volume ratio of glycerol in fermentation liquid is 0.03%, PDO is 1.5%(w/v), PH is 5.68.
Comparative example one: unlike embodiment four, with aseptic sodium hydroxide solution regulation and control PH for 4.98, glycerol now is identical with embodiment four with the concentration of PDO, after cultivating 28 hours, recording the mass volume ratio of glycerol in fermentation liquid is 0.83%, and PDO is 1.05%(w/v), PH is 7.25.
Comparative example two: unlike embodiment four, with aseptic calcium oxide suspension regulation and control PH for 4.98, glycerol now is identical with embodiment four with the concentration of PDO, after cultivating 28 hours, recording the mass volume ratio of glycerol in fermentation liquid is 0.23%, and PDO is 1.28%(w/v), PH is 6.94.
Can be seen that from above-described embodiment four and comparative example, from the point of view of the fluctuation range of PH, the effect utilizing macropore negative resin Lsi-363b regulation PH is best, next to that the regulation of calcium oxide suspension, it it is finally sodium hydroxide solution regulation, comparing comparative example one and the control method of comparative example two, the fluctuation range of embodiment four PH is less, within the identical time, the conversion ratio of glycerol is faster, being conducive to the generation of product, the yield of 1,3-PD is higher.
Embodiment five
Produce as a example by 1,3-PD by Klebsiella pneumoniae fermentation glycerol, 500mL fermentation liquid, shaking table is cultivated 20 hours, the w/v adding aseptic macropore negative resin Lsi-363b 4g(resin and fermentation liquid is 8:1(g:L)), recording the mass volume ratio of glycerol in fermentation liquid is 2.13%, 1, ammediol (PDO) is 0.54%(w/v), PH is 4.62, and after cultivating 28 hours, recording the mass volume ratio of glycerol in fermentation liquid is 0.08%, PDO is 2.1%(w/v), PH is 6.93.
Comparative example one: unlike embodiment five, with aseptic sodium hydroxide solution regulation and control PH for 4.62, glycerol now is identical with embodiment five with the concentration of PDO, after cultivating 28 hours, recording the mass volume ratio of glycerol in fermentation liquid is 0.79%, and PDO is 1.13%(w/v), PH is 7.05.
Comparative example two: unlike embodiment five, with aseptic calcium oxide suspension regulation and control PH for 4.62, glycerol now is identical with embodiment five with the concentration of PDO, after cultivating 28 hours, recording the mass volume ratio of glycerol in fermentation liquid is 0.25%, and PDO is 1.32%(w/v), PH is 6.84.
In the present embodiment five, although after adding macropore negative resin Lsi-363b regulation PH and cultivating 28 hours, the PH fluctuation of fermentation liquid is slightly larger compared to using the regulation and control of aseptic calcium oxide suspension, but it is high the most not change its carbon source conversion ratio, the advantage that Product yields is high；Reason is probably what the characteristic of resin itself was caused.Due in whole sweat, in fermentation system, product formation speed is fast, its PH amplitude of variation certainly will be caused bigger, although resin moderated PH is a dynamic process, the most do not weaken the increase of pH value, but being exactly conversion and the generation of product of such dynamic changing process, beneficially nutrient substance, this also shows that the advantage place with macropore negative resin Lsi-363b regulation Klebsiella pneumoniae fermentation liquid PH.
Embodiment six
Produce as a example by 1,3-PD by Klebsiella pneumoniae fermentation glycerol, 500mL fermentation liquid, shaking table is cultivated 20 hours, the w/v adding aseptic macropore negative resin Lsi-363b 8g(resin and fermentation liquid is 16:1(g:L)), recording the mass volume ratio of glycerol in fermentation liquid is 2.02%, 1, ammediol (PDO) is 0.52%(w/v), PH is 4.68, and after cultivating 28 hours, recording the mass volume ratio of glycerol in fermentation liquid is 0.09%, PDO is 2.26%(w/v), PH is 6.23.
The enforcement of the comparative example of the present embodiment is essentially identical with embodiment five, repeats no more；And contrast effect, essentially identical with embodiment five, repeat no more.
Embodiment seven
As a example by the cultivation of heterophytic chlorella, 500mL fermentation liquid, shaking table cultivation 7 days, extra-nutrition material, it is subsequently adding aseptic macropore negative resin Lsa-900C
15g(resin is 30:1(g:L with the w/v of fermentation liquid)), now, in fermentation liquid, the mass volume ratio of glucose is 2.29%, cell number is 3.06 hundred million/mL, PH is 4.58, and after cultivating 4 days, in fermentation liquid, the mass volume ratio of glucose is 0.53%, cell number be 6.57 hundred million/mL, PH be 5.33.
Comparative example one: unlike embodiment seven, pH value is regulated with sodium hydroxide solution and hydrochloric acid solution, in fermentation liquid now, the mass volume ratio of glucose is identical with embodiment seven with cell number, after cultivating 4 days, in fermentation liquid, the mass volume ratio of glucose is 1.08%, cell number be 5.50 hundred million/mL, PH be 7.33.
By embodiment seven and comparative example one this it appears that, utilizing sodium hydroxide solution and hydrochloric acid solution regulation pH value, after cultivating 4 days, the obvious Billy of its pH value amplitude of variation is much bigger with macropore negative resin Lsa-900C regulation, the few 1.07 hundred million/mL of cell number, hence it is evident that be unfavorable for the growth of chlorella.
Embodiment eight
As a example by the cultivation of heterophytic chlorella, 500mL fermentation liquid, shaking table cultivation 7 days, then supplement the nutrients material, adds aseptic macropore negative resin Lsa-900C
8g(resin is 12:1(g:L with the w/v of fermentation liquid)), in fermentation liquid, the mass volume ratio of glucose is 2.29%, cell number is 3.238 hundred million/mL, PH is 4.62, after cultivating 5 days, in fermentation liquid, the mass volume ratio of glucose is glucose 0.38%, cell number be 7.62 hundred million/mL, PH be 6.36.
The enforcement of the comparative example of the present embodiment is essentially identical with embodiment seven, repeats no more；And contrast effect, essentially identical with embodiment seven, repeat no more.
Embodiment nine
As a example by the cultivation of heterophytic chlorella, 30L fermentation liquid, fermentor cultivation 11 days, the w/v being subsequently adding aseptic macropore negative resin Lsa-900C 240g(resin and fermentation liquid is 8:1(g:L)), now, in fermentation liquid, cell number is 20.24 hundred million/mL, PH is 4.11, after cultivating 1 day, in fermentation liquid cell number be 21.37 hundred million/mL, PH be 5.04；After cultivating 2 days, in fermentation liquid cell number be 22.0 hundred million/mL, PH be 4.52.
Comparative example one: unlike embodiment nine, regulates pH value with sodium hydroxide solution and hydrochloric acid solution, and the cell number in fermentation liquid now is identical with embodiment nine, after cultivating 1 day, in fermentation liquid cell number be 20.48 hundred million/mL, PH be 6.24；After cultivating 2 days, in fermentation liquid cell number be 20.64 hundred million/mL, PH be 6.78.
After comparative example nine and comparative example one are it can be seen that cultivate 1 day, the former cell number increases more than the latter, and the fluctuation of PH is also less than the latter；After cultivating 2 days, essentially identical with afore-mentioned, in the case of PH fluctuation is relatively big, being unfavorable for the growth of chlorella, the growth of its cell will be compared slowly, therefore, the pH value utilizing macropore negative resin Lsa-900C regulation fermentation liquid can preferably maintain stablizing of pH value, reduces fluctuating margin, advantageously in growth and the breeding of microorganism, make yield higher, shorten fermentation period simultaneously.
It should be noted that, described fermentation liquid refers not only to the culture fluid that initial PH is faintly acid or acidity, it is also included within the fermentation liquid of microorganism not pregnancy ceased acid in sweat, described resin can add in the either phase of fermentation, the PH of fermentation liquid when i.e. scalable adds resin, it is also possible to be the PH being continually changing in the sweat after adding resin.
In sum, clearly, all any amendments made according to the technical support essence of the present invention and change still fall within the range of the technology of the present invention supports for every claim that the control method of the present invention a kind of fermentation process pH value is addressed and technical support.
Claims (2)
1. the control method of a fermentation process pH value, sterilizing and the regulation of pH value including resin, it is characterized in that: pretreated resin is carried out sterilizing, then any time period at fermentation process once joins the regulation carrying out pH value in fermentation liquid, described microorganism is oleaginous yeast or heterophytic chlorella, and described resin is macropore negative resin Lsa-900C or gel negative resin 201 × 7.
2. the control method of fermentation process pH value as claimed in claim 1, it is characterised in that: described resin is 0.5:1 ~ 30:1 with the mass volume ratio g:L of described fermentation liquid.
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