CN111349550A - Device and method for fermenting aerobic strains at high flux and application of device and method - Google Patents
Device and method for fermenting aerobic strains at high flux and application of device and method Download PDFInfo
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Abstract
The invention relates to the technical field of strain breeding, and discloses a device and a method for fermenting aerobic strains with high flux and application thereof, wherein the device for fermenting aerobic strains with high flux comprises a high-flux screening pore plate and a pore plate cover, at least one baffle plate perpendicular to the side wall is arranged on the inner side wall of a micropore of the high-flux screening pore plate, and the relationship between the length A of the baffle plate and the diameter D of the micropore of the high-flux screening pore plate satisfies the following requirements: 0 < A.ltoreq. 2/7D, preferably: 1/35D is less than or equal to A is less than or equal to 1/7D; the method comprises the step of inoculating the aerobic bacteria into the device filled with the culture medium to perform fermentation culture on the aerobic bacteria, and the method and the device can effectively solve the problems of low strain screening flux, low screening efficiency, high evaporation rate, poor pellet shape and poor fermentation effect of the conventional shake flask fermentation aerobic strains.
Description
Technical Field
The invention relates to the technical field of strain breeding, in particular to a device and a method for fermenting aerobic bacteria in high flux and application thereof.
Background
The high-throughput screening technology is a method for processing tens of millions of samples by using a deep-well plate as a culture or reaction medium and an automatic device instead of manpower at a DNA level or a cell level and sensitively and quickly acquiring data, and has the characteristics of trace amount, rapidness, sensitivity and the like. Currently, two main aspects are focused on the research of a high-throughput parallel cell culture instrument: 1) the throughput of culture is improved by minimizing the volume of the reactor, namely a micro (milliliter or even microliter) bioreactor is adopted; 2) based on the rich experience of the cell culture process, the conditions of the industrial reactor are simulated as much as possible in the high-flux culture process, namely, the process control and the parameter control of the microreactor are realized.
In the traditional strain breeding process, strains with better fermentation effect are selected from the strains through primary screening and secondary screening in a shake flask and then are subjected to a small fermentation tank test. For the screening of aerobic strains, the shake flask culture is easy to implement, the oxygen transfer rate is high, oxygen required by the growth and metabolism of the aerobic strains can be provided, and the method is widely used for early research on fermentation.
With the development of biotechnology, the construction of strains, breeding and related research of scale enlargement of fermentation engineering, high flux is required to be achieved to improve efficiency, the shake flask has low flux and high evaporation rate, consumes a large amount of manpower and material resources, consumes long time and cannot meet the current work requirement, the high-flux screening pore plate circulating in the market at present can achieve the purpose of improving efficiency by high flux, but due to the particularity of growth and metabolism of aerobic strains and the requirement of detection quantity of metabolites of the aerobic strains (the total volume of the current pore plate is smaller), the high-flux screening pore plate cannot meet the requirements of growth and metabolism of the aerobic strains and the detection requirement of the aerobic strains.
Therefore, the problems of low strain screening flux, low screening efficiency, high evaporation rate, poor pellet morphology and poor fermentation effect existing in the conventional shake flask fermentation aerobic strain are urgently needed to be solved in the field.
Disclosure of Invention
The invention aims to overcome the problems of low strain screening flux, low screening efficiency, high evaporation rate, poor pellet morphology and poor fermentation effect of the traditional shake flask in aerobic strain fermentation in the prior art, and provides a device and a method for screening aerobic bacteria at high flux and application thereof.
In order to achieve the above object, a first aspect of the present invention provides an apparatus for fermenting aerobic bacteria at high throughput, the apparatus for fermenting aerobic bacteria at high throughput includes a high throughput screening pore plate and a pore plate cover, at least one baffle perpendicular to the sidewall is disposed on the inner sidewall of the micropores of the high throughput screening pore plate, wherein a relationship between a length a of the baffle and a diameter D of the micropores of the high throughput screening pore plate satisfies: 0 < A.ltoreq. 2/7D, preferably: 1/35D is less than or equal to A is less than or equal to 1/7D.
In a second aspect, the invention provides a method for fermenting aerobic bacteria, wherein the aerobic bacteria are inoculated into the device filled with a culture medium to perform fermentation culture on the aerobic bacteria.
In a third aspect, the invention provides the use of the apparatus or fermentation method described above in high throughput screening of aerobic bacterial species.
By adopting the technical scheme, the device and the method for screening the aerobic bacteria at high flux are adopted to carry out fermentation culture on the aerobic bacteria, and the device and the method can effectively solve the problems of low strain screening flux, low screening efficiency, high evaporation rate, poor bacteria ball shape and poor fermentation effect in the traditional shake flask fermentation of the aerobic bacteria. For example, in example 1, the screening flux of aspergillus niger could reach 132 colonies, the liquid evaporation rate was only 1.5%, the acidity was 15.67%, and the pellet morphology was in a cluster chrysanthemum shape, and the morphology was better.
Drawings
FIG. 1 is a top view of a high throughput screening well plate of example 1
FIG. 2 is a schematic view of the fastening device opened;
FIG. 3 is a microscopic image of Aspergillus niger obtained by fermentation culture in example 1.
Description of the reference numerals
1 high flux screening orifice plate 11 micropore 12 baffle
2 fastening device 21 lock cover 22 lock catch 23 lock hook
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
In the present invention, the term "upper" is used to refer to the position of the sandwich plate lid in direct contact with air, and "lower" refers to the position of the sandwich plate lid in direct contact with the high throughput screening plate, unless otherwise stated.
The invention provides a device for fermenting aerobic strains in high flux, which can comprise a high flux screening pore plate 1 and a pore plate cover, wherein the inner side wall of micropores 11 of the high flux screening pore plate 1 is provided with at least one baffle plate 12 vertical to the side wall, and the relationship between the length A of the baffle plate and the diameter D of the micropores of the high flux screening pore plate satisfies the following conditions: 0 < A.ltoreq. 2/7D, preferably: 1/35D is less than or equal to A is less than or equal to 1/7D.
In the invention, the relationship between the thickness T of the baffle plate 12 and the diameter D of the micropores of the high-flux screening pore plate satisfies the following conditions: 0 < T.ltoreq. 1/7D, preferably 1/35 D.ltoreq.T.ltoreq. 3/35D.
In the present invention, the shape of the micropores of the high-throughput screening pore plate is not particularly limited, and may be, for example, a square (rectangle or square) or circular hole, and when the shape of the micropores is a square, the diameter D refers to the diameter D of an inscribed circle of the square.
In the present invention, the shape of the baffle 12 is not limited, and may be, for example, a rectangle or a square, and those skilled in the art can set the shape according to the circumstances.
According to an embodiment of the present invention, the baffle 12 is rectangular, a side of a shorter side of the rectangle is connected to a bottom wall of the microwell 11 of the high-throughput screening well plate 1, a side of a longer side of the baffle 12 may be vertically connected to a side wall of the microwell 11 of the high-throughput screening well plate 1, and another side of the longer side is not in contact with the side wall of the microwell 11 of the high-throughput screening well plate 1.
In addition, according to another embodiment of the present invention, the rectangle is connected to the bottom wall of the microwell 11 of the high-throughput screening well plate 1 only through the shorter side, and the longer sides are not in contact with the side wall of the microwell 11 of the high-throughput screening well plate 1.
In the present invention, the relationship between the height H of the baffle 12 and the depth S of the micropores 11 of the high-throughput screening plate 1 may satisfy: H.ltoreq.S 0, preferably 1/2 S.ltoreq.H.ltoreq.S.
The inventor of the present invention found in research that when the relationship between the length a of the baffle and the diameter D of the micro-pores of the high-throughput screening orifice plate satisfies: 1/35D is less than or equal to A is less than or equal to 1/7D, and the relation between the thickness T of the baffle plate 12 and the diameter D of the micropores of the high-flux screening pore plate satisfies that: 1/35D ≦ T ≦ 3/35D, the relationship between the height H of the baffle 12 and the depth S of the microwells 11 of the high throughput screening well plate 1 may satisfy: 1/2S is less than or equal to H and less than or equal to S, the evaporation rate can be further reduced, the fermentation effect is improved, and a better bacterium ball shape is obtained.
In the present invention, the length a of the baffle 12 refers to the length of the baffle 12 along the diameter direction of the micropores 11 of the high throughput screening plate 1.
In the present invention, the depth S refers to a distance from the bottom wall of the microwell 11 of the high throughput screening well plate 1 to the aperture.
In the present invention, the height H is a length of the baffle 12 in the direction of the depth S.
In the present invention, the thickness T is a length in a direction perpendicular to the length a of the baffle 12.
According to the invention, the orifice plate cover is a sandwich-type orifice plate cover, and the sandwich-type orifice plate cover sequentially comprises a stainless steel orifice plate, a filtering membrane and a silica gel orifice plate from top to bottom.
In the present invention, the filtration membrane is not particularly limited, and may be, for example, a polytetrafluoroethylene filtration membrane, a filter paper, or a hollow fiber filtration membrane.
In the present invention, the high-throughput screening well plate 1 may be selected from a 6-well plate, a 24-well plate, a 48-well plate, a 96-well plate or a 384-well plate, and preferably is a 6-well plate (as shown in fig. 1).
In the present invention, the well plate lid may be selected from a 6-well plate lid, a 24-well plate lid, a 48-well plate lid, a 96-well plate lid or a 384-well plate lid, preferably a 24-well plate lid.
In the present invention, the well plate lid may be selected from a 6-well plate lid, a 24-well plate lid, a 48-well plate lid, a 96-well plate lid or a 384-well plate lid, meaning that the number of wells of a stainless steel well plate in the well plate lid is 6 wells, 24 wells, 48 wells, 96 wells or 384 wells, respectively.
In the invention, the number of the holes of the silica gel pore plate in the pore plate cover can be larger than, smaller than or equal to the number of the holes of the pore plate cover, and preferably, the number of the holes of the silica gel pore plate in the pore plate cover is equal to the number of the holes of the stainless steel pore plate. According to a specific embodiment of the present invention, the number of the holes of the silica gel hole plate in the hole plate cover is equal to the number of the holes of the stainless steel hole plate, and each of the micropores 11 on the high-flux screening hole plate 1 can correspond to four holes in the silica gel hole plate.
In the invention, the diameter of the hole on the hole plate cover (stainless steel hole plate) can be 1-4mm, the diameter of the silica gel hole plate can be 1-3mm, and particularly, when the diameter of the hole on the hole plate cover (stainless steel hole plate) is 2.5-4mm, the diameter of the silica gel hole plate is 1-2mm, the evaporation capacity of liquid can be further reduced under the condition of not influencing the fermentation effect of aerobic bacteria.
According to a preferred embodiment of the present invention, the high-throughput screening well plate 1 is a 6-well plate (as shown in fig. 1), the number of the wells of the stainless steel well plate is 24, and the number of the wells of the silica gel well plate is 24.
In the present invention, the total volume of the high-throughput screening well plate 1 may be 30-360mL, and particularly, in the present invention, when the high-throughput screening well plate 1 is a 6-well plate (as shown in fig. 1), a better growth environment may be provided for aerobic bacteria, the screening throughput of aerobic bacteria is increased, and the screening efficiency is increased, and preferably, the total volume of the high-throughput screening well plate 1 may be 240 mL and 300 mL.
In the present invention, the screening flux refers to the number of colonies of the aerobic bacteria that can be inoculated in the high-throughput screening pore plate or the shake flask that can be placed on the same shaking table (i.e., the primary culture amount of the high-throughput screening pore plate and the shake flask on the shaking table) in the primary aerobic bacteria culture process.
In the present invention, the apparatus may further include a jig for fixing the high-throughput screening well plate 1 on a shaker and fixing the well plate cover on the high-throughput screening well plate 1.
As shown in fig. 2, according to a preferred embodiment of the present invention, the fixture may include a base, a fastening device 2 for fastening the well cover to the high-throughput screening well plate, and a fixing mechanism for fixing the base to the rocking platform, wherein the high-throughput screening well plate 1 may be placed in the base in the present invention.
In the present invention, the base is not particularly limited, and the base may include two sidewalls or four sidewalls, and in the case of two sidewalls, the two sidewalls are parallel to each other, and in the case of four sidewalls, the positions where the four sidewalls are connected to each other are perpendicular to each other.
In the invention, the fastening device 2 may include a lock cover 21, a lock 22 and a lock hook 23, the lock 22 is disposed on the lock cover 21, the lock cover 21 is disposed on the side wall of the base, the lock hook 23 is disposed on the orifice plate cover, and the fastening device fixes the orifice plate cover on the high-flux screening orifice plate by locking the lock and the lock hook.
In the present invention, the fixing means is not particularly limited, and may be any means that can be used for fixing in the prior art, and for example, the fixing means may be a bolt or may be welded.
In the present invention, regarding a specific embodiment of the clamp, the base is fixed on the shaker by bolts, and the base includes two parallel side walls, a lock cover 21 in the fastening device 2 is respectively disposed on the two side walls, a lock catch 22 is disposed on the lock cover 21, lock hooks 23 of the fastening device 2 are disposed on two side edges of the aperture plate cover corresponding to the two side walls of the base, the lock cover 21 of the fastening device 2 is hinged on the side walls of the base, the lock catch 22 is hinged on the lock cover 21, the lock catch 22 can be snapped into the lock hooks 23, and a schematic diagram when the fastening device 2 is loosened is shown in fig. 2.
The second aspect of the present invention provides a fermentation method of aerobic bacteria, which may include inoculating the aerobic bacteria into the micropores 11 of the high-throughput screening well plate 1 filled with a culture medium, and performing fermentation culture on the aerobic bacteria.
In the present invention, the aerobic bacteria may be any aerobic bacteria, and are preferably selected from the group consisting of rhizopus, mucor, neurospora, aspergillus, and penicillium, and more preferably aspergillus.
In the present invention, the aspergillus may be selected from aspergillus flavus, aspergillus niger or aspergillus fumigatus, according to a preferred embodiment of the present invention, the aspergillus is aspergillus niger.
In the present invention, in the high-throughput screening well plate 1, the inoculation amount of the aerobic bacterial strain is such that the number of spores of the aerobic bacterial strain is 5 to 40 ten thousand spores based on one milliliter of the culture medium, and according to a preferred embodiment of the present invention, the inoculation amount of the aerobic bacterial strain is such that the number of spores of the aerobic bacterial strain is 15 to 30 ten thousand spores based on one milliliter of the culture medium.
In the present invention, the culture medium is further sterilized before the aerobic bacteria are inoculated into the culture medium, and the sterilization conditions are not particularly limited, and may be conventional and usable for sterilization of the culture medium, and for example, the sterilization conditions may be: sterilizing at 121-125 deg.C under 0.1-0.15MPa for 20-30 min.
According to the fermentation method of an aerobic bacterial strain of the present invention, there is no particular requirement for the composition of the culture medium as long as it can be used for the fermentation medium of an aerobic bacterial strain. Preferably, when the aerobic bacterial strain is aspergillus niger, the culture medium contains an enzymolysis product obtained by enzymolysis of a starchy raw material, and the amount of the enzymolysis product obtained by enzymolysis of the starchy raw material accounts for 80-100 wt% of the total amount of the fermentation culture medium. Generally, a product obtained by enzymolysis of a starchy raw material is called a liquefied liquid, the liquefied liquid is subjected to solid-liquid separation to obtain enzymolysis residues and a liquefied clear liquid, and the liquefied clear liquid can be used for preparing a culture medium generally or can be mixed with the liquefied clear liquid to prepare the culture medium. Therefore, in the invention, the enzymolysis product obtained by enzymolysis of the starchy raw material comprises the liquefied clear solution obtained by solid-liquid separation, the liquefied liquid without solid-liquid separation and the mixture of the two. The culture medium is preferably prepared by mixing liquefied liquid and liquefied clear liquid with water or not, wherein the total sugar of the liquefied liquid and liquefied clear liquid is 10-20%, and the weight of the liquefied liquid and liquefied clear liquid can be (0.1-0.3): 1.
in the present invention, the starch liquefaction clear solution can be prepared by various methods, for example, by the following method: pulverizing starchy raw materials, performing enzymolysis on the pulverized product, performing solid-liquid separation on the product obtained by enzymolysis to obtain liquefied clear liquid and enzymolysis residues, wherein the solid content of the enzymolysis residues is 45-55 wt%, preferably 49-51 wt% under the condition of solid-liquid separation.
The starchy material can be various starchy materials which are known in the art and can be used for preparing citric acid through enzymolysis and fermentation, for example, the starchy material can be one or more selected from corn, potatoes (such as cassava) and wheat, and preferably, the starchy material is corn.
In the present invention, in the high-throughput screening well plate 1, the total amount of the culture medium may be 30 to 120g, and the culture medium is equally dispensed into each microwell 11 of the high-throughput screening well plate 1.
In the present invention, the fermentation culture is performed under stirring conditions, and the conditions of the fermentation culture may include: the temperature is 30-40 ℃, the time is 36-96h, the stirring speed is 200-400rpm, and preferably, the conditions of the fermentation culture can comprise: the temperature is 35-40 ℃, the time is 50-80h, and the stirring speed is 200-300 rpm.
In a third aspect, the invention provides an application of the device or the fermentation method in high-throughput screening of aerobic strains.
In the invention, the aerobic bacteria are fermented by adopting the device or the method, the evaporation rate of liquid in the high-flux screening pore plate 1 can be effectively reduced, the mycelium pellet shape of the obtained aerobic bacteria is in a cluster chrysanthemum shape, the mycelium pellet shape is better, compared with the traditional shake flask culture, the mycelium length for fermenting the aerobic bacteria by adopting the device and the method is shortened by 5-10%, the length of the obtained mycelium is shorter, the air circulation among the aerobic bacteria is facilitated, and even in the fermentation process of the aerobic bacteria, an excellent fermentation environment can be provided for the aerobic bacteria.
The present invention will be described in detail below by way of examples.
In the following examples of the present invention,
the screening flux refers to the primary culture amount of a high-throughput screening pore plate or a shake flask capable of being placed on a shaking table with the same size for colonies, 22 high-throughput screening pore plates or 40 shake flasks can be placed on the same shaking table (the length of × is 60cm × 40cm), the screening flux of the high-throughput screening pore plates is 132 (the high-throughput screening pore plates are 6 pore plates, each micropore is inoculated with one colony), and the screening flux of the shake flasks is 40 (each shake flask is inoculated with one colony).
Loading factor (%) — × 100 (total volume of material added to reactor (mL)/volume of reactor (mL));
liquid evaporation (%) — × 100% (total weight of material added to the reactor before fermentation (g) -total weight of material in the reactor after fermentation (g)/total weight of material added to the high throughput screening well plate before fermentation (g)) comprising high throughput screening well plate and shake flask, said material comprising medium and aspergillus niger spore suspension;
the acidity parameter is measured by NaOH titration method, and is measured by referring to the national standard GBT 8269-;
conversion (%) — (concentration of fermentation broth × volume of fermentation broth/weight of total sugars) × 100% wherein concentration of the obtained fermentation broth is abbreviated as acidity;
the shape of the bacterial ball is measured by a computer-based biomicroscope of XSP-16C (Shanghai Tian instruments Co., Ltd., model number), and the grade of the shape of the bacterial ball is excellent if the shape of the bacterial ball is clustered chrysanthemum (shown in figure 3); the fungus balls are clustered and chrysanthemum-shaped, but if broken hypha fragments exist, the fungus balls are good; the mushroom ball morphology is relatively scattered (non-clustered), and broken hypha fragments exist, so the grade of the mushroom ball morphology is poor;
the Aspergillus niger is purchased from China center for preservation of microorganism strains, and the type of the strain is Aspergillus niger CO 827.
Example 1
This example illustrates the apparatus and method for high throughput screening of aerobic bacteria provided by the present invention
(1) Adding culture medium into micropores of a high-throughput screening pore plate of a 6-pore plate (the volume of each micropore is 50mL, the specification of baffles in the micropore is that A is 2/35D, H is S, T is 1/35D, the number of the baffles is 4 (the baffles are arranged in an equidistant vertical mode)), sterilizing at 0.15MPa and 121 ℃ for 30min, cooling to 35 ℃ for standby application, wherein the culture medium is corn liquefied liquid and corn liquefied clear liquid, the total sugar is 16.15 wt%, and the filling amount of the culture medium in each micropore is 10 mL;
(2) inoculating aspergillus niger (aspergillus niger spore suspension) into the culture medium in the step (1), wherein the inoculation amount of each milliliter of the culture medium is 20 ten thousand spores;
(3) covering a sandwich type pore plate cover of 24 pore plate covers (the number of pores on the silica gel pore plate is 24, the diameter on the stainless steel pore plate is 2.5mm, and the diameter of the silica gel pore plate is 1mm) on the high-flux screening pore plate;
(4) fixing the high-flux screening pore plate on a shaking table through a clamp, fixing a pore plate cover on the high-flux screening pore plate (a fastening device of the clamp is shown in figure 2), and performing fermentation culture under the conditions that: the temperature was 35 ℃ and the time 72h, the stirring speed 300 rpm.
The screening flux, evaporation rate, acidity, conversion rate and pellet morphology were measured, respectively, and the results are shown in table 1.
Example 2
This example illustrates the apparatus and method for high throughput screening of aerobic bacteria provided by the present invention
(1) Adding a culture medium into micropores of a high-throughput screening pore plate of a 6-pore plate (the volume of each micropore is 40mL, the specification of baffles in the micropore is that A is 3/35D, H is 3/4S, T is 2/35D, and the number of the baffles is 4 (the baffles are arranged in an equidistant vertical mode)), sterilizing at 0.12MPa and 123 ℃ for 25min, cooling to 37 ℃ for standby application, wherein the culture medium comprises a corn liquefaction liquid and a corn liquefaction clear liquid, the total sugar is 18 wt%, and the filling amount of the culture medium in each micropore is 10 mL;
(2) inoculating Aspergillus niger (Aspergillus niger spore suspension) into the culture medium in the step (1), wherein the inoculation amount of each milliliter of the culture medium is 22 ten thousand spores;
(3) covering a sandwich type pore plate cover of 24 pore plate covers (the number of pores on the silica gel pore plate is 24, the diameter on the stainless steel pore plate is 3mm, and the diameter of the silica gel pore plate is 1.5mm) on the high-flux screening pore plate;
(4) the high-flux screening pore plate is fixed on a shaker through a clamp (shown in figure 2), a pore plate cover is fixed on the high-flux screening pore plate (a fastening device of the clamp is shown in figure 2), and fermentation culture is carried out under the following conditions: the temperature was 37 ℃ and the time 72h, the stirring speed 250 rpm.
The screening flux, evaporation rate, acidity, conversion rate and pellet morphology were measured, respectively, and the results are shown in table 1.
Example 3
This example illustrates the apparatus and method for high throughput screening of aerobic bacteria provided by the present invention
(1) Adding a culture medium into micropores of a high-throughput screening pore plate of a 6-pore plate (the volume of each micropore is 45mL, the specification of baffles in the micropore is that A is 1/14D, H is 1/2S, T is 3/35D, and the number of the baffles is 4 (the baffles are arranged in an equidistant vertical mode)), sterilizing at 0.13MPa and 124 ℃ for 22min, cooling to 40 ℃ for standby application, wherein the culture medium comprises a corn liquefaction liquid and a corn liquefaction clear liquid, the total sugar is 16.5 wt%, and the filling amount of the culture medium in each micropore is 10 mL;
(2) inoculating Aspergillus niger (Aspergillus niger spore suspension) into the culture medium in the step (1), wherein the inoculation amount per milliliter of the culture medium is 18 ten thousand spores;
(3) covering a sandwich type pore plate cover of 24 pore plate covers (the number of pores on the silica gel pore plate is 24, the diameter on the stainless steel pore plate is 3.5mm, and the diameter of the silica gel pore plate is 2mm) on the high-flux screening pore plate;
(4) fixing the high-flux screening pore plate on a shaking table through a clamp, fixing a pore plate cover on the high-flux screening pore plate (a fastening device of the clamp is shown in figure 2), and performing fermentation culture under the conditions that: the temperature was 40 ℃ and the time 72h, the stirring speed 200 rpm.
The screening flux, evaporation rate, acidity, conversion rate and pellet morphology were measured, and the results are shown in table 1.
Example 4
This example illustrates the apparatus and method for high throughput screening of aerobic bacteria provided by the present invention
The procedure of example 1 was followed except that in step (1), the total sugar was 20% by weight, and in step (2), Aspergillus niger (Aspergillus niger spore suspension) was inoculated into the medium of step (1) in an amount of 25 ten thousand spores per ml of the medium.
The screening flux, evaporation rate, acidity, conversion rate and pellet morphology were measured, and the results are shown in table 1.
Example 5
This example illustrates the apparatus and method for high throughput screening of aerobic bacteria provided by the present invention
The procedure of example 1 was followed except that the volume of each microwell was 35 mL.
The screening flux, evaporation rate, acidity, conversion rate and pellet morphology were measured, and the results are shown in table 1.
Example 6
This example illustrates the apparatus and method for high throughput screening of aerobic bacteria provided by the present invention
The procedure of example 1 was followed except that the sandwich well plate lid of the 24-well plate lid (the number of wells on the silica gel well plate lid was 24) was replaced with the sandwich well plate lid of the 96-well plate lid (the number of wells on the silica gel well plate lid was 96).
The screening flux, evaporation rate, acidity, conversion rate and pellet morphology were measured, and the results are shown in table 1.
Example 7
This example illustrates the apparatus and method for high throughput screening of aerobic bacteria provided by the present invention
Following the procedure of example 1 except that 6-well high-throughput screening plates were replaced with 24-well high-throughput screening plates, specifically:
(1) adding a culture medium into micropores of a high-throughput screening pore plate of a 6-pore plate (the volume of each micropore is 12.5mL, the specifications of baffles in the micropores meet that A is 2/35D, H is S, T is 1/35D, and the number of the baffles is 4 (the baffles are arranged in an equidistant vertical mode)), sterilizing at 0.15MPa and 121 ℃ for 30min, cooling to 35 ℃ for standby, wherein the culture medium comprises a corn liquefaction liquid and a corn liquefaction clear liquid, the total sugar is 16.15 wt%, and the filling amount of the culture medium in each micropore is 2.5 mL;
(2) inoculating Aspergillus niger (Aspergillus niger spore suspension) to the medium of step (1) with an inoculum size of 20 ten thousand spores per ml of medium.
The screening flux, evaporation rate, acidity, conversion rate and pellet morphology were measured, and the results are shown in table 1.
Example 8
This example illustrates the apparatus and method for high throughput screening of aerobic bacteria provided by the present invention
The process of example 1 was followed except that the baffles in the micropores satisfied: a is 2/7D, H is 1/3S, T is 1/7D, and the number of baffles is 4 (equidistantly vertically aligned)).
The screening flux, evaporation rate, acidity, conversion rate and pellet morphology were measured, and the results are shown in table 1.
Example 9
This example illustrates the apparatus and method for high throughput screening of aerobic bacteria provided by the present invention
The procedure of example 1 was followed except that the diameter of the 24-well plate cap on the stainless steel well plate was 2mm and the diameter of the silica gel well plate was 2 mm.
The screening flux, evaporation rate, acidity, conversion rate and pellet morphology were measured, and the results are shown in table 1.
Example 10
This example illustrates the apparatus and method for high throughput screening of aerobic bacteria provided by the present invention
The procedure of example 1 was followed except that the stirring speed was 400 rpm.
The screening flux, evaporation rate, acidity, conversion rate and pellet morphology were measured, and the results are shown in table 1.
Comparative example 1
This comparative example is illustrative of the prior art apparatus and method for aerobic bacterial growth
The procedure of example 1 was followed except that Aspergillus niger was fermented using a shake flask, specifically,
(1) adding a culture medium into 40 250mL shake flasks, sterilizing at the temperature of 121 ℃ and 0.15MPa for 30min, cooling to 35 ℃ for later use, wherein the culture medium comprises a corn liquefaction liquid and a corn liquefaction clear liquid, the total sugar content is 16.15 wt%, and the filling amount of the culture medium in the shake flasks is 40 mL;
(2) inoculating Aspergillus niger (Aspergillus niger spore suspension) into the culture medium in the step (1), wherein the inoculation amount of each milliliter of the culture medium is 20 ten thousand spores;
(3 fixing the shake flask on a shaking table through a clamp, and carrying out fermentation culture on the Aspergillus niger under the conditions of 35 ℃ of temperature, 72 hours of time and 300rpm of stirring speed.
The screening flux, evaporation rate, acidity, conversion rate and pellet morphology were measured, and the results are shown in table 1.
TABLE 1
From examples 1 to 10, comparative example 1 and table 1, it can be seen that under the same fermentation conditions, compared with the conventional fermentation method of aerobic bacterial (comparative example 1), the device and method for fermenting aerobic bacterial with high flux of the invention effectively improve the screening efficiency and fermentation effect (higher acidity) of aspergillus niger, reduce the evaporation rate and have good bacterial pellet shape;
in particular, as can be seen from examples 1 to 3, when the relationship between the length a of the baffle and the diameter D of the microwells of the high throughput screening well plate satisfies: 1/35D is less than or equal to A is less than or equal to 1/7D, and the relation between the thickness T of the baffle plate 12 and the diameter D of the micropores of the high-flux screening pore plate satisfies that: 1/35D ≦ T ≦ 3/35D, the relationship between the height H of the baffle 12 and the depth S of the microwells 11 of the high throughput screening well plate 1 may satisfy: 1/2S is not less than H and not more than S, and the evaporation rate can be further reduced, the fermentation effect can be improved and better fungus ball shape can be obtained under the condition of adopting a 6-pore plate high-flux screening pore plate.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (12)
1. The device for fermenting the aerobic strains in high flux is characterized by comprising a high-flux screening pore plate and a pore plate cover, wherein at least one baffle perpendicular to the side wall is arranged on the inner side wall of a micropore of the high-flux screening pore plate;
wherein the relationship between the length A of the baffle and the diameter D of the micropores of the high-throughput screening pore plate satisfies: 0 < A.ltoreq. 2/7D, preferably: 1/35D is less than or equal to A is less than or equal to 1/7D.
2. The apparatus of claim 1, wherein the relationship between the height H of the baffle and the depth S of the microwells of the high throughput screening well plate is such that: H.ltoreq.S 0, preferably: 1/2S is less than or equal to H and less than or equal to S.
3. The device of claim 1, wherein the orifice plate cover is a sandwich orifice plate cover comprising, from top to bottom, a stainless steel orifice plate, a filtration membrane, and a silica gel orifice plate.
4. The device of claim 1, wherein the high throughput screening well plate is selected from a 6-well plate, a 24-well plate, a 48-well plate, a 96-well plate, or a 384-well plate, preferably a 6-well plate;
the well plate lid is selected from a 6-well plate lid, a 24-well plate lid, a 48-well plate lid, a 96-well plate lid, or a 384-well plate lid, preferably a 24-well plate lid.
5. The device according to any of claims 1-4, wherein the total volume of the high throughput screening well plate is 30-360mL, preferably 240-300 mL.
6. The apparatus of any one of claims 1-4, wherein the apparatus further comprises a clamp for securing the high throughput screening well plate to a shaker and the well plate cover to the high throughput screening well plate.
7. The apparatus of claim 6, wherein the fixture comprises a base, a fastening device, and a securing mechanism to secure the base to the shaker, wherein the fastening device is configured to secure the well plate lid to the high throughput screening well plate.
8. The apparatus of claim 7, wherein the fastening device comprises a lock cover, a lock catch and a lock hook, the lock catch is disposed on the lock cover, the lock cover is disposed on the side wall of the base, the lock hook is disposed on the orifice plate cover, and the fastening device fixes the orifice plate cover on the high flux screening orifice plate through locking of the lock catch and the lock hook.
9. A method for fermenting aerobic bacteria, comprising inoculating said aerobic bacteria into the micro-pores of a high throughput screening well plate of the apparatus of any one of claims 1 to 8 filled with a culture medium to ferment and culture the aerobic bacteria.
10. The process according to claim 9, wherein the aerobic bacteria are selected from rhizopus, mucor, neurospora, aspergillus or penicillium, preferably aspergillus;
the Aspergillus is selected from Aspergillus flavus, Aspergillus niger or Aspergillus fumigatus, preferably Aspergillus niger.
11. The method according to claim 9, wherein the fermentation culture is performed under stirring conditions, wherein the stirring speed is 200-400rpm, preferably 200-300 rpm.
12. Use of the apparatus of any one of claims 1-8 or the fermentation process of any one of claims 9-11 for high throughput screening of aerobic bacterial species.
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