CN112481315B - Method for preparing citric acid by continuous liquid-changing fermentation - Google Patents
Method for preparing citric acid by continuous liquid-changing fermentation Download PDFInfo
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Abstract
The invention relates to the field of microbial fermentation, and discloses a method for preparing citric acid by continuous liquid-changing fermentation. Wherein the method comprises the following steps: (I) Pumping fermentation liquor in the fermentation tank into a membrane filtration system through a pump to be separated through a filtration membrane; (II) collecting the fermentation supernatant that has passed through the filtration membrane and returning the liquid containing the bacterial balls that has not passed through the filtration membrane to the fermenter; (III) continuously feeding fresh fermentation medium into the fermenter, and the feeding rate of the fresh fermentation medium is equal to the collection rate of the fermentation broth; and (IV) extracting the fermentation clear liquid to prepare the citric acid. The method can reduce product inhibition, maintain bacterial activity, and prolong acid production period.
Description
Technical Field
The invention relates to the field of microbial fermentation, in particular to a method for preparing citric acid by continuous liquid-changing fermentation.
Background
Citric acid, also known as citric acid, is an important tricarboxyl organic acid product, and currently commercially available products are mainly anhydrous citric acid and citric acid monohydrate, and are mainly used as acidulants, flavoring agents, preservatives and antistaling agents in the food and beverage industries, and are called as first edible acidulants. Citric acid is a product of the overflow metabolism (overflow metabolism) of aspergillus niger and can only accumulate in large quantities under conditions of unbalanced metabolic pathways during fermentation.
Currently, liquid submerged fermentation is the mainstream technology of the industrial production of citric acid. The fermentation method mainly comprises the following steps:
and (5) material supplementing and fermentation. In recent years, a large number of feed supplement processes for citric acid fermentation are reported, but most of citric acid fermentation enterprises still adopt stable batch fermentation processes, and feed supplement fermentation with higher production strength is required by workers, so that most of reports at present are at laboratory level.
Semi-continuous liquid-changing fermentation. The method has simple operation, can prolong the acid production period and improve the production strength, but requires short liquid changing operation time, and can thoroughly change liquid (update the culture medium) at one time, thereby being easy to have great influence on the activity of the thalli.
And (5) fixed fermentation. CN107022541a discloses an immobilization method of aspergillus niger, which realizes continuous fermentation of citric acid produced by aspergillus niger fermentation, takes pretreated fiber material as immobilization medium, and cultures the activated aspergillus niger in a fermentation vessel filled with immobilization medium, so that the strain is adsorbed on the fiber material in the culture process. In addition, the method is used for reusing the strain, fresh culture medium is supplemented by one-time liquid replacement, fermentation is carried out for 600 hours, 8 cycles of culture are repeated, the seed production period is saved, and the mycelium immobilization treatment can increase additional cost and period.
It follows that the existing fermentation processes exist: the feeding fermentation has high requirements on workers, the semi-continuous liquid-changing fermentation has short operation time, the updated culture medium has influence on the activity of the thalli, and the fixed fermentation cost has high period.
Therefore, developing a simple continuous fermentation mode for effectively maintaining the activity of the thalli is the most direct means for realizing cost-controllable and stable culture.
Disclosure of Invention
The invention aims to overcome the problems of the prior fermentation method, and provides a method for preparing citric acid by continuous liquid-changing fermentation. The method can prolong fermentation period and increase citric acid accumulation rate.
In order to achieve the above object, the present invention provides a method for preparing citric acid by continuous liquid-changing fermentation, wherein the method comprises:
(I) Pumping fermentation liquor in a fermentation tank 1 into a membrane filtration system 3 through a pump 2 to be separated through a filtration membrane;
(II) collecting the fermentation clear liquid a which has passed through the filtration membrane and returning the liquid b containing the fungus balls which has not passed through the filtration membrane to the fermentation tank 1;
(III) continuously feeding fresh fermentation medium into the fermenter 1, and the feeding rate of the fresh fermentation medium being equal to the collection rate of the fermentation broth a; and
(IV) extracting the fermentation clear liquid a to prepare the citric acid.
According to the technical scheme, the fermentation tank-membrane filtration system coupling device is adopted, in the process of producing the citric acid by fermenting the aspergillus niger deep clear liquid, the sterile membrane filtration closed-loop circulation system is adopted, so that the fermentation clear liquid is permeated and discharged, fresh fermentation culture medium is synchronously supplemented, continuous liquid-changing culture is realized, the fermentation can be carried out in a whole-process sterile state, the product inhibition is reduced, the activity of thalli is kept, the fermentation period is prolonged to 84-100h, and the citric acid accumulation rate reaches 0.81-1.32 g/(L.h).
Drawings
FIG. 1 is a schematic diagram of an apparatus and method for producing citric acid coupled to a fermenter-membrane filtration system of the present invention.
Description of the reference numerals
1. Fermentation tank 2, pump 3 and membrane filtration system
a. Fermentation clear liquid b, liquid c containing fungus balls and fermentation culture medium
M, agitating unit
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
The invention provides a method for preparing citric acid by continuous liquid-changing fermentation, which comprises the following steps:
(I) Pumping fermentation liquor in a fermentation tank 1 into a membrane filtration system 3 through a pump 2 to be separated through a filtration membrane;
(II) collecting the fermentation clear liquid a which has passed through the filtration membrane and returning the liquid b containing the fungus balls which has not passed through the filtration membrane to the fermentation tank 1;
(III) continuously feeding fresh fermentation medium into the fermenter 1, and the feeding rate of the fresh fermentation medium being equal to the collection rate of the fermentation broth a; and
(IV) extracting the fermentation clear liquid a to prepare the citric acid.
According to the invention, the term "continuous change" refers to a continuous replenishment of fresh fermentation medium.
According to the invention, before step (I), the membrane filtration system 3 and the pipeline connected with the membrane filtration system 3 are washed and soaked in alcohol, acid or alkali in advance, and the washed and soaked membrane filtration system 3 and the pipeline communicated with the membrane filtration system 3 can form a sterile membrane filtration closed-loop circulation system with the fermentation tank 1; wherein, when the alcohol is adopted for washing and soaking, the concentration of the alcohol is more than 75%, and the sterile purified water is used for washing the pipeline at least twice, preferably the alcohol with the concentration of 75-90%, more preferably the alcohol with the concentration of 75-80% is used for saving cost; wherein, when the alkali is adopted for washing and soaking, the alkali is selected from one or more of sodium hydroxide, potassium hydroxide and ammonia water, and the pipe is washed by sterile purified water until the pH value of the solution in the pipe is neutral; in addition, the pH value of the alkali is 12-14; wherein when the washing and soaking are carried out by using acid, the acid is selected from one or more of citric acid, phosphoric acid and acetic acid, preferably citric acid, especially when the washing and soaking are carried out by using phosphoric acid and acetic acid, and the pipeline is washed by sterile purified water until the pH value of the solution in the pipeline is neutral, and in addition, the special description is that the washing by sterile water is not needed when the washing and soaking are carried out by using citric acid; wherein the pH of the acid is 2-4, preferably pH 3. In the present invention, the number of times of the rinsing is not particularly limited, and preferably, the soaking time is 4 to 8 hours, preferably 5 to 7 hours, more preferably 6 hours.
According to the invention, the membrane filtration system 3 further comprises a vibrating device, in which the filter membrane is arranged; the vibration device is not particularly limited as long as the vibration device can drive the filtering membrane to vibrate, and preferably, the vibration device is a shaking table, and the shaking table can be obtained through commercial purchase, for example, the shaking table is purchased from Shanghai Zhi City analytical instrument manufacturing company, and the model is ZWF-100.
According to the invention, the parameters of the vibration device may be: the rotary amplitude is 20-50mm, and the vibration speed is 150-250r/min; preferably, the rotary amplitude is 30-40mm, and the vibration speed is 180-220r/min; in the invention, the vibration direction of the vibration device is perpendicular to the flowing direction of the filtering fluid in the filtering membrane, so that the turbulence degree of the fermentation liquid fluid can be increased, and the pollution of the filtering membrane can be reduced.
According to the invention, the filtering mode adopted by the filtering membrane is tangential flow filtration, namely, the flowing direction of liquid is perpendicular to the filtering direction, so that the pollution of the filtering membrane can be relieved; in the invention, the vibration device is combined with the filtering membrane, so that the filtering mode of the filtering membrane is different from that of a conventional membrane, and when the conventional membrane is adopted for filtering, the turbulence degree of fluid is mainly determined by the flow rate of the fluid.
According to the invention, the filtering membrane is an ultrafiltration membrane and/or a microfiltration membrane, and preferably, the filtering membrane can be one or more of a roll-type membrane, a tubular membrane and a hollow fiber membrane, wherein the roll-type membrane is a roll-type ultrafiltration membrane, and the tubular membrane is a tubular ceramic membrane. In the invention, the filtering membrane is limited to the ultrafiltration membrane and/or the microfiltration membrane, so that normal working operation can be ensured, the filtering membrane cannot be used as a nanofiltration membrane, and otherwise, the blocking phenomenon can occur. In addition, in the present invention, the type of membrane and the membrane area selected are different depending on the fermentation systems of different specifications. The material of the filtration membrane may be an organic membrane or an inorganic membrane. In the invention, the molecular weight cut-off of the filtering membrane is limited to 1K-100K Da, and the molecular weight of the citric acid is 192.14Da, so that small molecules such as the citric acid can be randomly penetrated, so that macromolecules such as proteins can be better cut-off, and the citric acid is enriched; in the present invention, da is a unit of daltons and molecular weight. In the invention, the smaller the molecular weight cut-off of the filtering membrane, the more proteins and oligosaccharides are cut-off, and the fewer impurities are in the clear citric acid solution; in addition, in the present invention, the permeation rate of the citric acid clear liquid is also related to the resistance, and the greater the resistance, the slower the permeation rate of the citric acid clear liquid.
In the present invention, the molecular weight cut-off (MWCO: molecular weight cutoff) is the cut-off property of an ultrafiltration membrane and/or a microfiltration membrane represented by the molecular weight, and is also called a cut-off molecular weight.
According to the invention, the pressure of the filtering fluid in the filtering membrane is not higher than 0.1MPa, preferably 0.05-0.1MPa; in the invention, the pressure of the filtering fluid in the filtering membrane is limited to be within the range, so that the mechanical damage to the bacterial balls in the liquid containing the bacterial balls is small, the relative activity of the bacterial balls can be kept, the acid production rate can be maintained in a certain period, and the acid production period can be prolonged.
According to the invention, the effective filtration area of the filtration membrane is 0.05-0.7m relative to a 5L fermentation system 2 . In addition, in the present invention, the roll type membrane, the tubular type membrane and the hollow fiber membrane are all commercially available, for example, they are available from the manufacturers of Xiamen Sanda environmental technologies, inc., the model is tubular ceramic membrane SPPM-C-10, and they are also available from Tianjin Dawa technology development Co., ltd, the model is hollow fiber ultrafiltration membrane DC-4K.
According to the invention, the end of the feeding pipe of the fresh fermentation medium is arranged above the liquid level of the fermentation tank 1; the end of the suction pipe of the pump 2 is arranged below the liquid level of the fermentation tank 1, preferably, the end of the suction pipe of the pump 2 is arranged in the middle area of the bottom air distributor and the bottom stirring paddle of the fermentation tank 1, more preferably, at 1/4 to 3/4 of the middle area of the bottom air distributor and the bottom stirring paddle, and most preferably, at 1/2 of the middle area of the bottom air distributor and the bottom stirring paddle, so as to ensure that the residence time of fresh fermentation medium in the fermentation tank 1 is maximum, as shown in fig. 1. In addition, in fig. 1, the air distributor is not labeled.
According to the invention, the membrane surface transmission rate of the fermentation liquor is 0.005-5.0m/s, and the acidity of the fermentation liquor is 5-15g/dL, in the invention, the bodyVolumetric flow rate (m) 3 /s) is equal to the membrane surface transmission rate (m/s) x the membrane area (m 2 ) The method comprises the steps of carrying out a first treatment on the surface of the Preferably, the collection rate of the fermentation clear liquid a is 1-10ml/min and the acidity is 8-12g/dL relative to a 5L fermentation system; in the invention, the fermentation clear liquid a is the fermentation clear liquid rich in citric acid. In the present invention, the fermentation clear liquid a is the liquid that permeates the membrane filtration system 3 after the separation.
According to the invention, the parameters of the liquid b containing the bacterial balls include: the average grain size of the fungus balls is 100-200 mu m, and in addition, the loose degree of the fungus balls is moderate, and the fungus balls have no obvious broken hyphae. In the present invention, the liquid b containing the bacterial cells is the concentrated liquid after separation.
According to the invention, the pump 2 is a gear pump or a centrifugal pump, a pump driven circulation is used, wherein the volumetric flow rate of the fermentation broth pumped by the pump 2 into the membrane filtration system 3 is 300-750ml/min and the acidity is 5-15g/dL relative to a 5L fermenter, preferably the volumetric flow rate of the fermentation broth pumped by the pump 2 into the membrane filtration system 3 is 450-600ml/min and the acidity is 8-12g/dL relative to a 5L fermenter. In the present invention, the pump 2 is commercially available, for example, from Boschig constant flow pump Co., ltd, model number WT3000-1JA.
According to the invention, the fermentation broth may be prepared by the following method:
(I-1) preparing fermentation seed liquid from spore suspension by seed activation in advance, and placing the fermentation seed liquid in the fermentation tank 1;
(I-2) contacting the liquefied and saccharified starch product with corn steep liquor and phosphate to prepare a fresh fermentation medium;
(I-3) sterilizing and cooling the fresh fermentation medium, and then feeding the fresh fermentation medium into the fermentation tank 1 to be contacted with the fermentation seed liquid for fermentation.
According to the invention, in step (I-1), the seed transfer process is aseptic. In addition, seed activation is performed in a seed tank.
According to the present invention, in step (I-3), the conditions of the fermentation may include: the ventilation is 0.5-1.5vvm, the stirring speed is 500-1000r/min, and the fermentation time is 24-32h; preferably, the ventilation is 0.8-1.2vvm, the stirring speed is 600-900r/min, and the fermentation time is 26-30h. In addition, in the present invention, the aeration rate in the fermenter is generally expressed in vvm, i.e., the ratio of aeration per minute to the actual volume of the tank, volume/volume/min.
According to the invention, the volume ratio of the amount of the fermentation seed liquid to the fresh fermentation medium is 1: (4-19), preferably 1: (9-14), more preferably 1:10.
According to the invention, fresh bran koji is resuspended to a spore suspension and activated overnight in a 1L seed tank to produce a fermentation seed liquid.
According to the invention, starch raw materials are liquefied and saccharified to obtain clear liquid, and corn steep liquor and phosphate are added to prepare a fermentation medium, wherein the total sugar content can be 12-18 wt%, preferably 18 wt%, and the corn steep liquor addition amount is 1-2 wt%, preferably 1/60 (i.e. 1.667 wt%), based on the total weight of the fermentation medium; so that the carbon to nitrogen ratio is 80:1, controlling the total phosphorus content to be 200-400ppm, preferably 400ppm, inoculating the fermentation seed liquid according to 10% of inoculum size after sterilizing and cooling the fermentation culture medium, and under the defined ventilation and stirring conditions, the adaptation period is about 12 hours, the fermentation is a rapid acid production period for 24-32 hours, and the corresponding acid production rate per unit time reaches 0.3-0.4 g/(dL.h).
According to the invention, the fermentation tank-membrane filtration system coupling device is adopted, and in the process of producing the citric acid by fermenting the aspergillus niger deep clear liquid, the sterile membrane filtration closed-loop circulation system is adopted, and preferably, the membrane filtration system is in an axial vibration state, so that the fermentation clear liquid is permeated and discharged, and fresh fermentation culture medium is synchronously supplemented, thereby realizing continuous liquid-changing culture, being capable of being carried out in a whole aseptic state, reducing product inhibition, maintaining the activity of thalli and prolonging the acid production period.
The present invention will be described in detail by examples. In the following examples, the rate of citric acid accumulation was measured by the acid-base neutralization titration method; the starch and corn steep liquor are commercial products of biochemical energy corn starch factories of Jilin, wherein the brand of the biochemical energy corn starch factories is edible corn starch.
Example 1
This example is intended to illustrate the preparation of citric acid using the apparatus shown in figure 1 and the method of the present invention.
A fermenter-membrane filtration system coupling as shown in fig. 1.
(I) Fermenting and culturing Aspergillus niger in a 5L fermentation tank 1: fresh bran koji is resuspended to a spore suspension and activated overnight in a 1L seed tank to produce a fermentation seed liquid which is then placed in the fermenter 1. Liquefying and saccharifying starch raw materials to obtain clear liquid, taking the total weight of a fermentation culture medium as a reference, preparing a fresh fermentation culture medium by using corn steep liquor and phosphate with the total sugar content of 18 percent and 1.667 weight percent, so that the carbon nitrogen ratio is 80:1, the total phosphorus content is 400ppm, sterilizing and cooling the fresh fermentation culture medium, then inoculating fermentation seed liquid (namely, the volume ratio of the fermentation seed liquid to the fresh fermentation culture medium is 1:9) according to the inoculation amount of 10 weight percent, introducing air flow of 1vvm, stirring at the speed of 1000r/min for about 12 hours, fermenting for 32 hours as a rapid acid production period, and starting a sterile membrane filtration closed-loop circulation system according to the corresponding acid production rate per unit time of 0.4g/dL for 32 hours;
(II) the membrane filtration system 3 and its connection pipe are previously rinsed and soaked with 1g/L citric acid solution (ph=3) for at least 6 hours, and then the fermentation broth in the fermenter 1 is pumped into the membrane filtration system 3 via the pump 2; wherein the filtering membrane is a hollow fiber membrane with the molecular weight cutoff of 4kDa, and the effective filtering area is 1.0m 2 The method comprises the steps of carrying out a first treatment on the surface of the The vibration direction of the shaking table is perpendicular to the flowing direction of the filtered fluid in the filtering membrane; the tail end of the feeding pipeline of the fermentation medium is arranged above the liquid level of the fermentation tank 1; the tail end of the suction pipeline of the pump 2 is positioned at 1/2 of the middle area between the bottom air distributor of the fermentation tank 1 and the bottom stirring paddle; wherein the gear pump drives the fermentation liquor to flow, the maximum flow speed is 750ml/min, the acidity of the fermentation liquor is 8.9g/dL, the pressure of the filtering fluid in the filtering membrane is 0.1MPa, and the fermentation of the citric acid-rich liquid which permeates the filtering membraneThe flow rate of the clear liquid a is 0.3ml/min, the acidity in the clear liquid is 5.3g/dL, and the liquid b containing the bacteria balls is returned to the fermentation tank 1;
(III) continuously feeding fresh fermentation medium into the fermenter 1, and the feeding rate of the fresh fermentation medium being equal to the collection rate of the fermentation broth a; that is, the collection rate of the fermentation clear liquid a is 10ml/min and the acidity is 8g/dL relative to a 5L fermentation system; and
(IV) purifying the fermentation clear liquid a to prepare the citric acid.
Results: the fermentation period is prolonged to 96 hours, and the accumulation rate of the citric acid reaches 1.11 g/(L.h).
Example 2
This example is intended to illustrate the preparation of citric acid using the apparatus shown in figure 1 and the method of the present invention.
Citric acid was prepared in the same manner as in example 1 except that: the filtering membrane is a tubular ceramic membrane with the molecular weight cut-off of 10K-40K Da, and is provided with a cooling temperature control jacket, and the effective filtering area is 0.5m 2 The method comprises the steps of carrying out a first treatment on the surface of the Wherein, fix on the speed governing oscillation equipment surface of the whirl amplitude 22mm, vibration speed 150 r/min.
Results: the fermentation period is prolonged to 96 hours, and the accumulation rate of the citric acid reaches 1.03 g/(L.h).
Example 3
This example is intended to illustrate the preparation of citric acid using the apparatus shown in figure 1 and the method of the present invention.
Citric acid was prepared in the same manner as in example 1 except that: the filtering membrane is a hollow fiber membrane with the molecular weight cutoff between 6K and 20K Da, and the effective filtering area is 1.0m 2 The method comprises the steps of carrying out a first treatment on the surface of the Wherein, the vibration plate is fixed on the surface of a shaking table with a rotary amplitude of 30mm and a vibration speed of 250 r/min.
Results: the fermentation period is prolonged to 100h, and the accumulation rate of the citric acid reaches 1.32 g/(L.h).
Example 4
This example is intended to illustrate the preparation of citric acid using the apparatus shown in figure 1 and the method of the present invention.
Citric acid was prepared in the same manner as in example 1 except that: the filtering membrane is a tubular ceramic membrane with the molecular weight cut-off of 60K-100K Da, and is provided with a cooling temperature control jacket, and the effective filtering area is 0.3m 2 。
Results: the fermentation period is prolonged to 84 hours, and the accumulation rate of citric acid reaches 0.92 g/(L.h).
Example 5
This example is intended to illustrate the preparation of citric acid using the apparatus shown in figure 1 and the method of the present invention.
Citric acid was prepared in the same manner as in example 1 except that: the filter membrane is a coiled ultrafiltration membrane with the molecular weight cutoff of 1K-10kDa, and the effective filtration area is 0.24m 2 。
Results: the fermentation period was prolonged to 84 hours, and the citric acid accumulation rate reached 1.21 g/(L.h).
Example 6
This example is intended to illustrate the preparation of citric acid using the apparatus shown in figure 1 and the method of the present invention.
Citric acid was prepared in the same manner as in example 1 except that: the membrane filtration system 3 and its connection tubing were previously rinsed with 0.1mol/L sodium hydroxide for 6 hours and rinsed with sterile purified water to pH neutral.
Results: the fermentation period is prolonged to 90h, and the accumulation rate of the citric acid reaches 1.04 g/(L.h).
Example 7
This example illustrates the preparation of citric acid using the fermenter-membrane filtration system coupling shown in FIG. 1 and the method of the present invention.
Citric acid was prepared in the same manner as in example 1 except that: the volume ratio of the dosage of the fermentation seed liquid to the dosage of the fresh fermentation medium is 1:19.
results: the fermentation period is prolonged to 96 hours, and the accumulation rate of citric acid reaches 0.97 g/(L.h).
Example 8
This example is intended to illustrate the preparation of citric acid using the apparatus shown in figure 1 and the method of the present invention.
Citric acid was produced in the same manner as in example 1 except that the end of the suction pipe of the pump 2 was located at 1/4 of the middle area of the bottom air distributor and the bottom stirring paddle of the fermenter 1; and the end of the feeding pipe of the fermentation medium is arranged at a distance of 1/4 of the distance above the liquid level of the fermentation tank 1.
Results: the fermentation period is prolonged to 84 hours, and the accumulation rate of citric acid reaches 0.81 g/(L.h).
Example 9
This example is intended to illustrate the preparation of citric acid using the apparatus shown in figure 1 and the method of the present invention.
Citric acid was prepared in the same manner as in example 1 except that: the collection rate of the fermentation clear liquid is 5ml/min, and the acidity is 10g/dL.
Results: the fermentation period is prolonged to 84 hours, and the accumulation rate of citric acid reaches 0.88 g/(L.h).
Example 10
This example is intended to illustrate the preparation of citric acid using the apparatus shown in figure 1 and the method of the present invention.
Citric acid was prepared in the same manner as in example 1 except that: the membrane filtration system 3 and its connection tubing were pre-soaked with a 75% alcohol rinse for at least 6 hours and rinsed twice with sterile purified water.
Results: the fermentation period is prolonged to 90h, and the accumulation rate of the citric acid reaches 1.12 g/(L.h).
Comparative example 1
Citric acid was prepared in the same manner as in example 1 except that: instead of using the fermenter-membrane filtration system coupling of the present invention shown in FIG. 1, a plate and frame filter as is conventional in the art is used.
Results: as a result, the effective circulation of the pellets was not achieved, and as a result, the fermentation time was 60 hours, and the citric acid accumulation rate reached 0.1 g/(L.h).
Comparative example 2
Citric acid was prepared in the same manner as in example 1 except that: the pressure of the filtered fluid in the filtering membrane is 0.5MPa.
Results: as the environmental pressure changes, the formation of the fungus balls is uneven, the fungus balls have more broken hyphae, the fermentation time is 60h, and the accumulation rate of citric acid reaches 0.52 g/(L.h).
Comparative example 3
Citric acid was prepared in the same manner as in example 1 except that: the vibration direction of the shaking table is parallel to the flowing direction of the filtering fluid in the filtering membrane.
Results: the membrane pollution is aggravated due to the attachment of the bacteria balls, the product permeability is reduced, and as a result, the fermentation time is 60h, and the citric acid accumulation rate reaches 0.57 g/(L.h).
Comparative example 4
Citric acid was prepared in the same manner as in example 1 except that: the feeding rate of the fresh fermentation medium is not equal to the collection rate of the fermentation broth, i.e. the feeding rate of the fresh fermentation medium is higher than the collection rate of the fermentation broth.
Results: as a result of too high a concentration of limiting nutrients, the fermentation time was 60 hours, and the citric acid accumulation rate reached 0.67 g/(L.h).
Comparative example 5
Citric acid was prepared in the same manner as in example 1 except that: no citric acid is used for pre-flushing and soaking the membrane filtration system 3 and its connecting pipes.
Results: the external circulation pipeline is not subjected to aseptic treatment, so that acid-resistant mixed bacteria such as lactic acid bacteria are caused to grow, the content of lactic acid is high, the fermentation time is 60 hours, and the accumulation rate of citric acid reaches 0.62 g/(L.h).
Comparative example 6
Citric acid was prepared in the same manner as in example 1 except that: nanofiltration membranes are used.
Results: because the nanofiltration membrane has small pores, the nanofiltration membrane is easy to block and can not work normally.
As can be seen from the results of examples 1-10 and comparative examples 1-6, in the process of producing citric acid by fermenting Aspergillus niger deep clear liquid by adopting a fermenter-membrane filtration system coupling device, fermentation clear liquid is permeated and discharged by adopting a sterile membrane filtration closed-loop circulation system, fresh fermentation culture medium is synchronously supplemented, continuous liquid-changing culture is realized, the fermentation period can be prolonged to 84-100h (normal fermentation time is 60-72 h), and the citric acid accumulation rate reaches 0.81-1.32 g/(L.h).
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (10)
1. A method for preparing citric acid by continuous liquid-changing fermentation, which is characterized by comprising the following steps:
(I) Pumping fermentation liquor in a fermentation tank (1) into a membrane filtration system (3) through a pump (2) to be separated through a filtration membrane;
the filtering membrane is an ultrafiltration membrane and/or a microfiltration membrane; the molecular weight cut-off of the filtering membrane is 1K-100kDa; the pressure of the filtering fluid in the filtering membrane is 0.05-0.1MPa;
the membrane filtration system (3) further comprises a vibration device, and the filtration membrane is arranged on the vibration device; the vibration direction of the vibration device is perpendicular to the flowing direction of the fermentation liquid; the parameters of the vibration device are as follows: the rotary amplitude is 20-50mm, and the vibration speed is 150-250r/min;
the filtration mode adopted by the membrane filtration system (3) is tangential flow filtration;
(II) collecting the fermentation clear liquid (a) which has passed through the filtration membrane and returning the liquid (b) which has not passed through the filtration membrane and contains the fungus balls to the fermenter (1);
(III) continuously feeding fresh fermentation medium into the fermenter (1) at a feed rate equal to the collection rate of the fermentation broth (a); and
(IV) extracting the fermentation supernatant (a) to prepare citric acid.
2. The method according to claim 1, wherein prior to step (I), the membrane filtration system (3) and the piping in communication with the membrane filtration system (3) are rinsed and soaked with alcohol, acid or base.
3. The method of claim 1, wherein the filtration membrane is one or more of a roll membrane, a tubular membrane, and a hollow fiber membrane.
4. The method according to claim 1, wherein the end of the feed pipe of fresh fermentation medium is arranged above the liquid level of the fermenter (1); the tail end of the suction pipeline of the pump (2) is arranged below the liquid level of the fermentation tank (1).
5. The method of claim 1, wherein the fermentation broth has a membrane side transmission rate of 0.005-5m/s and an acidity of 5-15g/dL.
6. The method according to claim 1, wherein the average particle size of the pellet is 100-200 μm.
7. The method according to claim 1, wherein the volumetric flow rate of the fermentation broth pumped by the pump (2) into the membrane filtration system (3) is 300-750ml/min and the acidity is 5-15g/dL relative to a 5L fermenter.
8. The method according to claim 1 or 7, wherein the fermentation broth is prepared by:
(I-1) preparing fermentation seed liquid from spore suspension by seed activation in advance, and placing the fermentation seed liquid in the fermentation tank (1);
(I-2) contacting the starch liquefied saccharification product with corn steep liquor and phosphate to prepare a fresh fermentation medium;
and (I-3) sterilizing and cooling the fresh fermentation medium, feeding the fresh fermentation medium into the fermentation tank (1), and contacting the fresh fermentation medium with the fermentation seed liquid for fermentation to obtain fermentation liquid.
9. The method of claim 8, wherein in step (I-3), the fermentation conditions comprise: the ventilation is 0.5-1.5vvm, the stirring speed is 500-1000r/min, and the fermentation time is 24-32h.
10. The method of claim 8, wherein the volume ratio of the fermentation seed liquid to the amount of fresh fermentation medium is 1: (4-19).
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| CN102864082B (en) * | 2012-09-19 | 2014-04-30 | 中粮生物化学（安徽）股份有限公司 | Method for culturing citric acid fermenting seeds and method for preparing citric acid by fermenting |
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| CN103290070B (en) * | 2013-06-20 | 2015-03-11 | 南通凯赛生化工程设备有限公司 | Method for producing citric acid through continuous batch feeding fermentation |
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