CN110628590A - Fermentation method for reducing risk of bacterial contamination - Google Patents
Fermentation method for reducing risk of bacterial contamination Download PDFInfo
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- CN110628590A CN110628590A CN201810657037.XA CN201810657037A CN110628590A CN 110628590 A CN110628590 A CN 110628590A CN 201810657037 A CN201810657037 A CN 201810657037A CN 110628590 A CN110628590 A CN 110628590A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
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- C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination
- C12M37/02—Filters
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
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Abstract
The invention relates to a convenient fermentation method with low contamination risk, which comprises the following steps of using a strain culture bottle (100), a inoculation tube (300) and a pumping device (13), wherein the strain culture bottle (100) comprises a bottle body (1) and a sealable bottle cap (2), a first guide tube (3) and a second guide tube (4) are arranged on the bottle cap (2), the first guide tube (3) extends into the bottom of the bottle body (1), the outer parts of the first guide tube (3) and the second guide tube (4) are respectively connected with a first air filter (5) and a second air filter (6), one end (14) of the inoculation tube (300) is provided with an inoculation needle (15) connected with a fermentation tank (400), and the other end (16) is provided with a connecting piece (17) connected with the strain culture bottle (100), and the method comprises the following steps: 1) using a strain culture bottle (100) to culture strains; 2) in a sterile environment, removing the first air filter (5) from the strain culture flask (100) and connecting the inoculation tube (300) with the first conduit (3) through the connecting piece (17); 3) connecting the inoculating needle (15) with the fermenter (400); 4) arranging the pumping device (13) at any position of the inoculation tube (300), and starting the pumping device (13) during inoculation; and 5) fermenting in a fermenter (400).
Description
Technical Field
The present invention relates to a fermentation method with low risk of contamination, which uses a strain flask that can be conveniently and continuously used in strain culture and inoculation sections, and connects the strain flask and an inoculation tube under flame protection in a sterile environment such as a super clean bench. The invention can also connect the inoculation tube with other devices such as a strain culture bottle, a material supplementing device and the like through multiple branches at the same time, so as to complete the connection of the strain culture bottle and the material supplementing device with the fermentation tank at one time.
The invention also relates to a method for fermenting microalgae to prepare a microalgae extract, which comprises using the fermentation method to reduce the risk of bacterial contamination.
Background
In the fermentation process, if contaminated by other bacteria, the production is seriously affected, which is a fatal injury of industrial fermentation, and therefore, each operation link needs to be strictly controlled.
Prior to large scale fermentation in fermenters, it is often necessary to culture a certain number of pure microbial species in a sterile environment. When the prepared culture medium is put into the fermentation tank, high-temperature disinfection is carried out, and then the fermentation tank is cooled to the temperature required by the process, the prepared microbial strains are transferred into the fermentation tank, and the process is inoculation. The inoculation operation needs to be finished under a sterile condition, otherwise, the mixed bacteria pollution is caused.
The fermentation tank inoculation method mainly comprises a flame inoculation method and a differential pressure inoculation method. The flame inoculation method needs to add a flame around the inoculation port, quickly open the inoculation port cap after the tank pressure is set to zero, pour the seed liquid into the fermentation tank under the protection of the flame, and place the inoculation cap in the range that the flame can protect. The disadvantage of this inoculation method is that it cannot perform aseptic operation strictly, the inoculation amount is small, the inoculation port is exposed for a long time, contamination is difficult to avoid, and if the pot pressure is not well controlled during inoculation, fire is easily caused.
The differential pressure inoculation technology is to utilize the instantaneous pressure difference between the seeding tank and the inoculation bottle to press the strains in the inoculation bottle into the fermentation tank. The fermentation tank is provided with an inoculation port, which is sterilized together with the fermentation tank, and the inoculation port is sterilized or disinfected by steam or chemical reagent before the seed bottle is butted with the fermentation tank. The disadvantage of this method is that it requires the material pressure-bearing capacity of the seed bottle, and the equipment is usually complicated, and the problem of sterilization still needs to be considered when the seed bottle and the fermentation tank are butted.
In the patent application of the invention of a plurality of fermentation tank inoculation devices, an inoculation bottle with a complex design is adopted, or the traditional fermentation tank is modified, such as a steam sterilization device is added. This adds to some extent to the production costs and the retrofitting of the fermenter may affect the tightness. See CN201711122592.4, CN201721231024.3, CN201720903902.5, CN201720770058.3, etc.
CN201621227296.1 discloses a seed tank inoculation device, which is characterized in that a thin film is arranged on an inoculation port of a fermentation tank, an inoculation needle is connected with the tail end of an inoculation tube connected with a seed bottle, and a peristaltic pump is arranged on the inoculation tube. During inoculation, the inoculation needle is inserted into the film under the flame of the inoculation port, the risk of mixed bacteria pollution is reduced, the inoculation liquid is pumped by the peristaltic pump, and the operation frequency of the fermentation tank for increasing and decreasing the pressure is reduced.
In addition, during the fermentation process, feeding is usually required, and the conventional treatment in the field is to connect different feeding bottles with the fermentation tank through a plurality of feeding ports on the fermentation tank. This communication process is similar to inoculation communication and also requires protection from contamination with a flame.
In recent years, more and more microalgae have been used for production of raw materials for health products, cosmetics, and the like. For example, microalgae produce polyunsaturated fatty acids that are beneficial for cardiovascular, etc. The extract of Botryococcus braunii, which contains polysaccharides, proteins and the like, has been found to have acetylcholinesterase, butyrylcholinesterase, tyrosinase inhibitory activity, antioxidant activity, and can alleviate cell damage and the like caused by hydrogen peroxide.
Large-scale cultivation of microalgae is usually carried out in outdoor ponds, which can utilize sunlight and CO in the air2And the like, but the condition control degree is poor. To be liftedThe production efficiency of microalgae products is high, algae can be cultured in a fermentation tank, the prevention of the pollution of mixed bacteria is one of the requirements, and other parameters also need to be controlled and optimized.
Disclosure of Invention
The inventor of the invention connects the strain culture bottle and the material supplementing device with the fermentation tank at one time through the multi-branch inoculation pipe, so that the time and the frequency of exposing the content of the fermentation tank or the content of the strain culture bottle/the material supplementing device to the external environment are greatly reduced, and the contamination probability is reduced. At the same time, the inventors have also adopted a unique strain culture flask that can be conveniently accessed to an inoculating device after small-scale strain culture. By means of the device and method, operation which is more convenient and faster than that of the inoculation device/method in the prior art can be achieved, and the risk of bacterial contamination is further reduced.
In particular, in a first aspect, the present invention provides an inoculating device comprising a seed culture flask, a seeding tube, and a pumping device, wherein the strain culture bottle comprises a bottle body and a sealable bottle cap, the bottle cap is provided with a first conduit and a second conduit, wherein the first conduit extends into the bottom of the bottle body, the outside of the second conduit is connected with an air filter, one end of the inoculation tube is provided with an inoculation needle connected with the fermentation tank, the other end is divided into more than two branches at branch nodes, wherein at least one branch is connected with the strain culture bottle, and at least one other branch is connected with the feeding device, an intercepting device for realizing switching of branch pipeline switches is arranged between the branch node of the inoculation pipe and the tail end of each branch, the tail end of the branch which is not connected with any device can be sealed through the intercepting device, and the pumping device is arranged between the tail end of the inoculation pipe, which is provided with the inoculation needle, and the branch node of the inoculation pipe.
The body of the strain culture bottle can be made of any material, and the sealable bottle cap can be made of any material as long as the sealing performance can be maintained after the conduit is arranged. The second conduit of the strain culture bottle can be connected with more than one air filter. The air filter may be a filter having a pore size of 0.22 microns or less.
The inoculation tube can be an integrally formed tube, such as a silicone tube and the like.
The ends of the branches of the inoculation tube connected to the seed flasks and/or the feeding device may be provided with connectors, wherein the connectors connected to the seed flasks/feeding device may be identical or different and in one embodiment are metal straight-throughs, such as copper straight-throughs.
The feeding device can be a feeding bottle filled with feeding liquid.
The inoculating needle on the inoculating tube can be a contact pin butted with a feed supplement port of the fermentation tank. The inoculation needle of the inoculation tube is connected with the fermentation tank through a material supplementing hole of the fermentation tank or other holes with smaller openings.
The catch means may be a clip, such as a dovetail clip.
The pumping device may be a peristaltic pump.
In a second aspect, the present invention provides a convenient and reduced risk of contamination inoculation/supplementation method comprising the use of an inoculation device according to the present invention, the method comprising: 1) connecting the inoculating needle of the inoculating tube with the fermentation tank; 2) adjusting the interception device, and opening an inoculation or feed supplement branch; and 3) turning on the pumping device.
Preferably, the inoculation needle of the inoculation tube is connected to the fermentor immediately after the fermentor is sterilized.
By adopting the inoculation device and the inoculation method, the connection of the strain culture bottle and the material supplementing device with the fermentation tank can be completed at one time under the flame protection, so that the time and the times of exposing the content of the fermentation tank or the content of the strain culture bottle/the material supplementing device to the external environment are greatly reduced, and the contamination probability is reduced. In addition, due to the arrangement of the pumping device, the pressure of the fermentation tank does not need to be regulated, and the arrangement of the interception device can flexibly and conveniently switch between inoculation/feeding and control the inoculation/feeding time and amount according to the requirement.
In a third aspect, a set of cultivation/inoculation kit facilitating inoculation is provided, comprising a strain culture bottle, an inoculation tube and a pumping device, wherein the strain culture bottle comprises a bottle body and a sealable bottle cap, a first guide tube and a second guide tube are arranged on the bottle cap, the first guide tube extends into the bottom of the bottle body, the outer parts of the first guide tube and the second guide tube are respectively connected with a first air filter and a second air filter, one end of the inoculation tube is provided with an inoculation needle capable of being connected with a fermentation tank, and the other end of the inoculation tube is provided with a connecting piece connected with the strain culture bottle. In the strain culture stage, the strain culture bottle is used for culture, and gases required by strains are optionally introduced from the first conduit or the first conduit and the second conduit are closed by an interception device; before inoculation, the first air filter is dismantled in a sterile environment, a connecting piece of the inoculation pipe, which is connected with the strain culture bottle, is connected with a first guide pipe of the strain culture bottle, then an inoculation needle arranged on the inoculation pipe is connected with the fermentation tank under the protection of flame, and a pumping device is arranged between two ends of the inoculation pipe.
In some embodiments, one end of the inoculation tube is provided with an inoculation needle connected with the fermentation tank, the other end of the inoculation tube is branched into more than two branches, the tail end of at least one branch is provided with a connecting piece connected with the strain culture bottle, the tail ends of the other branches are provided with connecting pieces connected with the feeding device, and an intercepting device for realizing the switching of branch pipeline switches can be arranged between the branch node of the inoculation tube and the tail ends of the branches. In one embodiment, the arresting means may be a clip, such as a dovetail clip. In the embodiment in which the seed tube is multi-branched, the pumping device is arranged between the end of the seed tube connected to the fermenter and the branching node. Before inoculation, the connecting piece at the tail end of each branch is connected with a strain culture bottle and a material supplementing device in a sterile environment, and the branch which is not connected with any device is subjected to sealing treatment by an intercepting device.
The body of the strain culture bottle can be made of any material, and the sealable bottle cap can be made of any material as long as the sealing performance can be maintained after the guide pipe is arranged. More than one air filter can be connected to the first conduit and the second conduit of the strain culture bottle. The air filter may be a filter having a pore size of 0.22 microns or less.
The inoculation tube can be an integrally formed tube, such as a silicone tube and the like.
The connections of the inoculation tube to the seed culture flask/feeding set may be the same or different and in one embodiment are metal straight connections, such as copper straight connections.
The feeding device can be a feeding bottle filled with feeding liquid.
The inoculating needle on the inoculating tube can be a contact pin butted with a feed supplement port of the fermentation tank. The inoculation needle of the inoculation tube is connected with the fermentation tank through a material supplementing hole of the fermentation tank or other holes with smaller openings.
In one embodiment, the above-mentioned arresting means may be a clip, for example a dovetail clip.
The pumping device may be a peristaltic pump.
In a fifth aspect, the present invention provides a convenient fermentation method for reducing the risk of contamination, comprising using a culture/inoculation kit, wherein the culture/inoculation kit comprises a seed culture bottle, an inoculation tube, and a pumping device, the seed culture bottle comprises a bottle body and a sealable bottle cap, a first conduit and a second conduit are arranged on the bottle cap, wherein the first conduit extends into the bottom of the bottle body, the outer parts of the first conduit and the second conduit are respectively connected with a first air filter and a second air filter, an inoculation needle capable of being connected with a fermentation tank is arranged at one end of the inoculation tube, and a connecting piece connected with the seed culture bottle is arranged at the other end of the inoculation tube, the method comprising the following steps:
1) culturing strains by using a strain culture bottle;
2) after the strain culture is finished, in an aseptic environment, removing a first air filter on a strain culture bottle, and connecting a inoculation pipe with a first conduit of the strain culture bottle through a connecting piece;
3) under the protection of flame, the inoculation of the inoculation tube is connected with the fermentation tank;
4) arranging a pumping device on the inoculation tube, and starting the pumping device during inoculation; and
5) the fermentation is carried out in a fermenter.
Preferably, the inoculation needle of the inoculation tube is connected to the fermenter immediately after sterilization of the fermenter.
The body of the strain culture bottle can be made of any material, and the sealable bottle cap can be made of any material as long as the sealing performance can be maintained after the guide pipe is arranged. More than one air filter can be connected to the first conduit and the second conduit of the strain culture bottle. The air filter may be a filter having a pore size of 0.22 microns or less.
The inoculation tube can be an integrally formed tube, such as a silicone tube and the like.
The connector of the inoculation tube connected with the strain culture flask can be a metal straight through, such as a copper straight through.
The inoculating needle on the inoculating tube can be a contact pin butted with a feed supplement port of the fermentation tank. The inoculation needle of the inoculation tube is connected with the fermentation tank through a material supplementing hole of the fermentation tank or other holes with smaller openings.
The pumping device may be a peristaltic pump.
In a sixth aspect, the present invention provides a convenient fermentation method for reducing the risk of contamination, comprising using a culture/inoculation kit, wherein the culture/inoculation kit comprises a seed culture bottle, an inoculation tube, and a pumping device, the seed culture bottle comprises a bottle body and a sealable bottle cap, a first conduit and a second conduit are arranged on the bottle cap, wherein the first conduit extends into the bottom of the bottle body, the outer parts of the first conduit and the second conduit are respectively connected with a first air filter and a second air filter, an inoculation needle connected with a fermentation tank is arranged at one end of the inoculation tube, the other end of the inoculation tube is divided into more than two branches, wherein at least one branch is provided with a connector connected with the seed culture bottle, and at least another branch is provided with a connector connected with a feeding device, the method comprising the steps of:
1) culturing strains by using a strain culture bottle;
2) after strain culture is finished, in an aseptic environment, removing a first air filter on a strain culture bottle, connecting a seed inoculation pipe with a first guide pipe of the strain culture bottle through a connecting piece, connecting the seed inoculation pipe with a material supplementing device through the connecting piece, and sealing a branch not connected with any device;
3) under the protection of flame, the inoculation of the inoculation tube is connected with the fermentation tank;
4) arranging a pumping device between the tail end of the inoculation pipe connected with the fermentation tank and the branch node, opening an inoculation branch, closing a material supplementing branch, and starting the pumping device for inoculation; and
5) closing the inoculation branch, fermenting in a fermentation tank, and opening the feeding branch to feed materials when needed.
Preferably, the inoculation needle of the inoculation tube is connected to the fermenter immediately after sterilization of the fermenter.
The body of the strain culture bottle can be made of any material, and the sealable bottle cap can be made of any material as long as the sealing performance can be maintained after the guide pipe is arranged. More than one air filter can be connected to the first conduit and the second conduit of the strain culture bottle. The air filter may be a filter having a pore size of 0.45 microns or less.
The inoculation tube can be an integrally formed tube, such as a silicone tube and the like.
The connections of the inoculation tube to the seed culture flask/feeding set may be the same or different and in one embodiment are metal straight connections, such as copper straight connections.
The feeding device can be a feeding bottle filled with feeding liquid.
The inoculating needle on the inoculating tube can be a contact pin butted with a feed supplement port of the fermentation tank. The inoculation needle of the inoculation tube is connected with the fermentation tank through a material supplementing hole of the fermentation tank or other holes with smaller openings.
An interception device for realizing the switching of branch pipeline switches is arranged between the branch node of the inoculation pipe and the tail end of each branch so as to realize the opening, closing and sealing of the inoculation/feed supplement branch. The intercepting means may be a clip, such as a dovetail clip.
The pumping device may be a peristaltic pump.
In a seventh aspect, the present invention also relates to a method of fermenting algae to produce an algae extract, comprising:
1) seed culture and inoculation according to the method of the fifth or sixth aspect;
2) performing primary and secondary fermentation, and feeding according to the method of the sixth aspect;
3) collecting algae cells, and extracting extracellular polysaccharide.
The species may be an alga, such as Botryococcus braunii, in particular Botryococcus braunii Showa. The culture medium of Botryococcus braunii may contain glucose at a concentration of 4000-15000 mg/L. The concentration of glucose in the culture medium can be respectively maintained at 4-6 g/L, 4-10g/L and 4-15g/L in the stages of algae seed culture, primary fermentation and secondary fermentation.
The collection of algal cells can be obtained by centrifuging the algal solution. The collection of algal cells can also be obtained by centrifugation combined with filtration. The filtration of the algae cells can be carried out by a cloth bag filter, wherein the cloth bag filter is 4000 meshes.
The extraction of exopolysaccharides can be carried out in hot water. Specifically, the algal cells can be placed in hot water of 90-100 ℃ and stirred, so as to extract the exopolysaccharide. The extracted exopolysaccharides can be separated from the cell bodies by filtration means.
The exopolysaccharide extract obtained can be concentrated and further lyophilized.
In addition, the algal cells may be cold water washed prior to extraction of the exopolysaccharide.
The primary and/or secondary fermentation may be carried out under light.
By using the kit and the method for culturing and supplementing the materials, the strain culture bottle can be conveniently and continuously used in the strain culturing and inoculating links, the link that strains are transferred from the strain culture bottle to the seed bottle so as to be exposed to the external environment is avoided, and the cleaning and sterilizing burden of the culture bottle is reduced. In addition, the connection of the strain culture bottle and the inoculation tube can be carried out under the flame protection in an aseptic environment such as a super clean bench, and the inoculation needle of the inoculation tube can be rapidly connected with a smaller opening of the fermentation tank under the flame protection, so that the direct contact between the cultured strain and the external environment is avoided, and the risk of contamination is comprehensively reduced. In some embodiments, the inoculation tube is connected with the material supplementing device in an aseptic environment, so that the connection of the strain culture bottle and the material supplementing device with the fermentation tank is completed at one time under the protection of flame, the time and the times of exposing the content of the fermentation tank or the content of the strain culture bottle/material supplementing device to the external environment are greatly reduced, and the contamination probability is reduced. In addition, due to the arrangement of the pumping device, the pressure of the fermentation tank does not need to be regulated, and the arrangement of the interception device can flexibly and conveniently switch between inoculation/feeding and control the inoculation/feeding time and amount according to the requirement.
Drawings
FIG. 1 shows a schematic of a seed culture flask of the present invention.
FIG. 2 shows a schematic of an apparatus for uniformly aerating a plurality of seed culture flasks of the present invention.
Figure 3 shows a schematic view of an inoculating device of the invention.
FIG. 4 shows a schematic of an inoculation feeding apparatus of the present invention.
FIG. 5 shows a schematic diagram of the connection of the inoculation/feed apparatus of the invention to a fermenter.
FIG. 6 shows the nutrient metabolism curve during a 500L fermentation.
Detailed Description
In fermentation processes, contamination problems often exist. And the inoculation link of the strains from the strain culture bottle or the seed bottle to the fermentation tank has higher risk of contamination.
Fermentors are typically bulky, even with seed tanks, having a volume of about 50L, and are often connected to other devices that cannot enter the sterile environment of a sterile room, clean bench, etc. Therefore, in the traditional inoculation process, the possibility of bacterial contamination can be reduced only by burning the fire circle on the inoculation opening of the fermentation tank. However, the inoculation port is usually large and the flame does not provide an absolute antibacterial effect. In addition, due to the presence of fire, the material of the seed bottle is required to be heat-resistant, fire-resistant and nonflammable.
In the case of differential pressure inoculation, the sterile environment is generally created by steam sterilizing the space between the inoculation port and the seed culture bottle or the seed bottle after the inoculation port is docked with the two, however, the need to tailor or modify the fermentation tank and the seed bottle increases the cost, and the steam sterilization itself also increases the energy consumption (see, for example, CN201120260946.3 and the like).
Furthermore, feeding is generally required after the fermentation has been carried out for a certain period of time, at which time it may have been some time since the fermenter had been sterilized. Dust and the like may accumulate at the material supplementing opening. Thus, even if the flame is ignited, dust cannot be prevented from falling into the fermentation tank, thereby possibly causing a problem of contamination.
In the invention, common and universal strain culture bottles are simply transformed, so that the strain culture bottles can be conveniently used for the seed scale culture and inoculation links, the use of the bottles is reduced, and the cleaning burden is reduced. Simultaneously, contain this strain culture bottle inoculation device in through the fermentation cylinder sterilization after the jar body still send out when scalding, be connected with the less feed supplement mouth of opening under the protection of flame, reduce the contamination probability. The inoculation tube of the inoculation device can be connected with the feeding bottle, which is equivalent to feeding and inoculation, only one feeding hole of the fermentation tank needs to be opened once, and the feeding hole is opened when the tank body is still hot after the fermentation tank is just sterilized to complete connection. Therefore, the risk of contamination is further reduced by reducing the number of the used feed supplementing ports and reducing the opening times of the fermentation tank.
The apparatus and method of use of the present invention will be further described with reference to the accompanying drawings.
As shown in FIG. 1, the present invention provides a strain culture bottle 100, which comprises a bottle body 1 and a sealable bottle cap 2, wherein the bottle cap is provided with a first conduit 3 and a second conduit 4, wherein the first conduit extends into the bottom of the bottle body, and the outer portions of the first conduit and the second conduit are respectively connected with a first air filter 5 and a second air filter 6.
During the initial small-scale culturing phase of the microorganism, the first conduit 3 may be used as an air inlet pipe for introducing oxygen, carbon dioxide or other gases required by the strain culture flask, and the second conduit 5 is used as an air outlet pipe. The introduced gas is filtered through a first air filter 5 connected to the first guide duct 3. In one embodiment, the first and second air filters 5 and 6 may be further coupled to more than one air filter to enhance bacterial filtration. In the case of anaerobic culture, the entry of external gas can be prevented by any arrangement, such as clamping the first conduit 3 and the second conduit 4 with a clamp or the like.
At this stage, multiple seed flasks may be connected to a gas supply to supply the same constituent gas at the same rate to the media to achieve parallel experimental conditions in the multiple seed flasks. Specifically, as shown in FIG. 2, the gas supply apparatus 200 includes a gas generation apparatus 7 and a gas supply pipe 8, and the gas supply pipe 8 has a plurality of branches 9-1, 9-2, 9-3, etc. Each branch is provided at a distal end with a plurality of air filters 10-1, 10-2, 10-3, etc., respectively. Before the culture starts, all the used instruments are sterilized, strains and culture media are introduced into a strain culture bottle, then the first air filter 5 of the strain culture bottle 100-1 is directly connected with the air filter 10-1 of one branch 9-1 of the air supply pipe 8 or is connected with the air filter 10-1 through a section of pipe in an aseptic environment, the air filter 5 of the second strain culture bottle 100-2 is connected with the air filter 10-2, and the like, and the steps are respectively connected. If the number of the strain culture bottles is less than that of the branches of the gas supply pipe, the gas interception treatment is carried out by using a clamp 11 and the like at the branches of the gas supply pipe without connection, so as to avoid unnecessary waste.
After the first small-scale culture is completed, the inoculum from the inoculum flask 100 is introduced into the fermentor 400 through the inoculating device 300 of the present invention. As shown in FIG. 3, the inoculating device 300 comprises a seed culture bottle 100 excluding the first air filter 5, a seeding tube 12 and a peristaltic pump 13, wherein one end 14 of the seeding tube 12 is provided with a pin 15 matching with the feeding port of the fermenter, and the other end 16 is provided with a metal through 17 connected with the first conduit 3 of the seed culture bottle 100. The inoculation device is assembled in a sterile environment. Specifically, in a sterile environment such as an ultra clean bench, under flame protection, the first air filter 5 of the seed culture flask 100 is removed (e.g., pulled off or cut off), the cotton yarn and the tin foil covering the metal feedthrough 17 of the seed tube 12 are detached, and the metal feedthrough 17 is inserted into the first guide tube 3. The peristaltic pump is disposed on any portion of the inoculation tube 12.
In one embodiment, the inoculation tube 12 may be an integrally formed Y-shaped tube or a multi-branched tube, as shown in FIG. 4, one end 14 of which is provided with the above-mentioned insertion pin 15, and the branches 16-1 and 16-2 at the other end are provided with a metal through 17 connected to the first conduit 3 of the seed culture flask 100, and the other branches such as 16-3 and 16-4 may be provided with fittings 19 connected to the feeding flask 18, such as metal through. If the number of branches is greater than the number of seed culture flasks and feed flasks, the interception process may be performed at the non-connected branch ports, such as 16-4, using clamps 20 or the like. The peristaltic pump 13 is disposed between the one end 14 of the seed tube 12 and the branch port 21, and each branch tube can be intercepted by a clip 20 or the like between the branch port 21 and each branch port when the peristaltic pump is not required to provide power.
Preferably, the inoculation is performed shortly after the sterilization of the fermenter 400, and preferably while the tank 23 is still hot after the sterilization of the fermenter 400, the pin 15 of the inoculation device 300 is inserted into the feeding opening 24 under flame protection, as shown in fig. 5. And when the tank body is still hot shortly after sterilization, the dust and the thallus at the feed supplementing port are less. The connection of the fermenter and the inoculation line is carried out at this time, so that the risk of contamination is minimized. In addition, because the feed supplement opening is much smaller than the opening of the inoculation opening, the contact surface between the internal environment and the external environment of the fermentation tank is greatly reduced through the feed supplement opening inoculation. And under the condition that the inoculation pipe is connected with the material supplementing bottle, only one material supplementing opening is opened for a short time in the inoculation and material supplementing links, and compared with the condition that a plurality of material supplementing openings are used, the risk of bacterial contamination is greatly reduced.
When inoculation is needed, the branches of the inoculation pipes except the connected strain culture bottle 100 are intercepted, the peristaltic pump is started, and the bacterial liquid is led into the fermentation tank. When the material is required to be supplemented, intercepting the branches of the inoculation tube except the required material supplementing bottle, starting the peristaltic pump, and introducing the supplemented liquid into the fermentation tank.
As described above, in the invention, the strain culture bottle is used for small-scale culture and inoculation of strains at the same time, replacement is not needed, the cleaning and sterilization burden of the apparatus can be reduced, and meanwhile, the possibility of contamination is reduced because the link of transferring from the strain culture bottle to the seed bottle is avoided. In addition, when the strain culture bottle is connected with the inoculation device, the strain culture bottle can be carried out in a sterile environment such as an ultra-clean bench under the protection of flame, and the contamination probability is low. Moreover, the inoculation device can also be connected with the material supplementing bottle under the protection of flame in a sterile environment such as a super clean bench, so that the material supplementing and the inoculation only need to open one material supplementing opening of the fermentation tank once, and the opening time is short, so that the risk of bacteria infection in the fermentation is further reduced.
The above-described apparatus and its operation of the present invention are suitable for the fermentation of algae, such as botryococcus braunii.
Botryococcus braunii is commonly used for the production of hydrocarbons. In the present invention, the inventors produced exopolysaccharides, proteins and the like on a large scale by fermentation of botryococcus braunii, and found that the growth rate of botryococcus braunii is faster than that of simple autotrophy or heterotropy when the botryococcus braunii is polycultured in light and sugar-containing environments. The mixed culture is realized by adding an illumination device with adjustable intensity into a traditional fermentation tank.
The algae cells obtained by fermentation culture are generally collected by cloth bag filtration, and the efficiency is low; can also be collected by centrifugation, and has high cost. In the invention, in order to collect algae bodies to the maximum extent and extract extracellular polysaccharide as much as possible, a mode of centrifugation mainly and filtration additionally is adopted.
Exopolysaccharides produced by algal cells are essentially insoluble in the culture medium and white or clear chitin can be seen by microscopic examination. After treatment with hot water, the chitin is dissolved in water, separated from the cells, and then collected by filtration to obtain a polysaccharide shell. The operation is simple, and no organic solvent is added, so that the use safety of the product is greatly improved, and the cost is not required.
In the following examples, the large-scale algal fermentation and extracellular polysaccharide extraction using the apparatus and method of the present invention will be described in detail.
Example 1: culture of species of Showa Botryococcus braunii
1.1 preparation of Chu13 plates
A culture medium is prepared according to the formula shown in the following table 1, the pH value is adjusted to 7.5 by using 1mol/L sodium hydroxide solution and 1mol/L hydrochloric acid solution, agar powder is added according to the amount of 18g/L, moist heat sterilization is carried out at 121 ℃ for 30min, and the agar powder is cooled to prepare a bevel or a flat plate.
TABLE 1 culture Medium recipe for Chu13 plate
1.2 mother slant inoculation
Using the solid Chu13 medium slant prepared above, a Botryococcus braunii Showa (Botryococcus braunii Showa) (donation from Melvin Calvin Laboratory, UCB registration No. B85-B41) was inoculated using an inoculating spatula and incubated at 25. + -. 1 ℃ for 20 days in a light incubator with a light dark cycle of 24:0 and a light intensity of 4000 lux.
1.3 seed plating
The botryococcus cells are picked from the inclined plane and put on the prepared Chu13 solid culture medium plate, and are cultured in a light incubator at 25 +/-1 ℃ for about 20 days, the light-dark period is 24:0, and the light intensity is 4000 lux. And observing the growth condition of the flat plate, recording, culturing the flat plate with algae growing for about 20 days, storing the flat plate in a refrigerator at 2-4 ℃ for later use, and simultaneously checking the production capacity. The validity period of the low-temperature preservation of the algae seed plates is 6 months, and the algae seeds need to be preserved again after the expiration period.
The above-mentioned productivity check is specifically performed as follows: chu13 liquid culture medium is prepared according to the formula, and is sterilized by moist heat at 121 ℃ for 30min, and is packaged in a 125ml sterile ventilation triangular flask in a sterile room after being cooled. Extracting 3 sub-plates, using an inoculating loop to draw a colony loop of algae, inoculating the colony loop into an aeration bottle, placing the aeration bottle in an illumination shaking table, culturing at 25 +/-1 ℃ for about 15 days at 100r/min, and performing light-dark period of 24:0 and illumination intensity of 4000 lux. Sampling and detecting the OD value by using a microplate reader. And under the wavelength of 682nm, the OD value is more than or equal to 0.2, the algae cells are yellow green when observed under a microscope, the conglobation grows, the algae bodies are full, the algae liquid is yellow green or green when observed by naked eyes, the culture solution is clear, and the algae seeds reach the standard when the algae conglobation particles are visible. Otherwise, the activation is needed to be carried out again from the female bevel.
1.4Cultivation of algal species
A liquid medium was prepared according to the formulation shown in Table 2, sterilized by moist heat at 121 ℃ for 30min, cooled and then packed in a 125ml sterile air-vent flask in a volume of 60ml in a sterile room. Inoculating with flat plate algae, cutting algae with inoculating loop, inoculating into triangular flask, culturing at 100r/min for 20 days at 25 + -1 deg.C on illumination shaker, and stopping when the algae is in logarithmic growth phase (OD value is not less than 0.2 at 682nm wavelength) with light intensity of 4000lux at 24:0 in dark period.
Then, 100mL of culture medium is filled in a 250mL triangular flask, the algae liquid in the 125mL triangular flask is inoculated, the OD value is about 0.1 at the wavelength of 682nm after inoculation, the culture medium is placed on a light shaking table for 100r/min for 20 days, and the culture temperature, the light dark period and the light intensity are the same as above, and the culture is stopped when the algae is in the logarithmic phase (the OD value is more than or equal to 0.3 at the wavelength of 682 nm).
TABLE 2 algal species Medium formulation
1.5 mother flask seed liquid culture
A sterilized flask having a volume of 1L was used, and as shown in FIG. 1, 800mL of the medium having the formulation shown in Table 2 was placed therein. Inoculating the strain in the 250mL triangular flask, placing on a culture shelf, and introducing 1-2% CO2Mixing with air (multiple 0.22 μm filter is added on the gas pipeline to ensure air sterility, as shown in FIG. 2), culturing at 25 + -1 deg.C for about 8 days in light dark period of 24:0, and transferring microalgae in logarithmic growth phase (OD value is not less than 0.5 at 682nm wavelength) into the next-stage culture bottle.
A sterilized culture flask with a volume of 5L was used, and as shown in FIG. 1, 4L of the medium with the formulation shown in Table 2 was placed in the flask, inoculated with the algal solution in the above 1L aeration flask, placed on a culture shelf, and passed through a 1-2% CO gas2Culturing in mixed gas with air at light-dark period of 24:0 and 25 + -1 deg.C for about 10 days, and transferring into first-stage seeding tank for culturing when microalgae is in logarithmic growth phase (OD value is not less than 0.5 at 682nm wavelength).
Practice ofEXAMPLE 2 inoculation of fermenter
A5L-volume culture flask for culturing microalgae in example 2 to logarithmic phase is cut off the air filter 5 of the culture flask as shown in FIG. 1 under the protection of alcohol burner flame in a super clean bench, and the copper straight-through 17 of the sterilized inoculation tube 300 shown in FIG. 4 is rapidly inserted into the conduit 3 of the culture flask 100, and the connecting piece 19 is inserted into the butt silicone tube of the supplement bottle 18.
The seeding tank of 50L capacity was sterilized, and 35L fermentation medium was filled, the pH was adjusted to 7.0 as shown in Table 3, and then sterilized.
TABLE 3 fermentation Medium formulation
After the sterilization of the seed tank is finished and the tank body is still hot, the inoculation device connecting the culture bottle and the supplement bottle is moved out of the super clean bench and enters a fermentation chamber, and the inoculation needle 15 at one end of the inoculation tube is inserted into a supplement port of the seed tank by using a flame inoculation method.
After the seeding tank is cooled, the peristaltic pump 13 is arranged between the inoculating needle 15 and the branch node 21 of the inoculating tube, and the grape algae seed liquid is pumped into the seeding tank by closing the feed supplement pipeline, wherein the tank pressure is 0.03-0.05 Mpa.
Example 3 fermentation
In the fermentation process of the seeding tank, the temperature of the tank is controlled to be 25 +/-0.5 ℃, the pressure of the tank is controlled to be 0.03-0.05Mpa, and sterile air and 2 percent CO are introduced2Mixed gas with a ventilation rate of 0.4m3The stirring speed is 150r/min, the initial illumination intensity is 4000lux, and the OD is obtained682After reaching 0.3, the light was adjusted to the maximum (10000 lux). After the fermentation is started, the growth morphology and C, N, P metabolic change of the algae are observed until the glucose is less than or equal to 4g/L and NO is added3 -≤4mmol/L，PO4 -When the concentration is less than or equal to 0.4mmol/L, the algae seed supplementing pipeline is opened, the algae seed pipeline is closed, and the algae seed is supplemented by the peristaltic pumpRespectively adding glucose (the concentration of the feed supplement liquid is about 150g/L) and KNO3(feed solution concentration about 140mmol/L) and K2HPO4(the concentration of the feed solution is about 15mmol/L), so that the concentration of glucose is kept between 4 and 10g/L, and NO is3 -The concentration of (A) is maintained at 3-8mmol/L, PO4 -The concentration of (A) is kept at 0.3-0.8 mmol/L.
About 2 weeks in culture, OD682Reaching about 1.0, transferring into a large fermentation tank with the capacity of 500L for fermentation.
The sterilization of the large fermentor and the addition of the medium into the homozygote jar, the formulation of the fermentation broth of the large fermentor is shown in Table 3, the volume of the medium is 350L, the pH is adjusted to 7.0 and the sterilization is carried out.
Sterilizing an inoculation pipeline of the seeding tank by using 0.3 +/-0.01 Mpa steam for 90-120 minutes, and pressing the seeds into a large fermentation tank by using a differential pressure method after the sterilization is finished. After inoculation, the inoculation valve of the large fermentation tank and the discharge valve of the seed tank are closed in sequence, all small exhaust gases on the pipeline are opened, and the pipeline is flushed by steam.
In the fermentation process of large fermenter, controlling the temperature at 25 + -0.5 deg.C and the pressure at 0.03-0.05Mpa, and introducing air and 2% CO2Mixed gas with a ventilation of 4m3And h, opening an illumination system, wherein the initial illumination intensity is 4000lux, the illumination is gradually increased to the strongest intensity (about 10000lux) along with the increase of the biomass, the stirring speed is reduced to 100r/min after inoculation, and the stirring speed can be gradually increased to 200r/min along with the increase of the biomass in the later period.
The growth morphology and C, N, P metabolic change of algae are mainly observed in the early stage of fermentation culture until the nutrient substances are consumed until the glucose is less than or equal to 4g/L and the NO is reduced3 -≤4mmol/L，PO4 -Respectively supplementing glucose (feed supplement solution concentration 150g/L)), KNO when the concentration is less than or equal to 0.4mmol/L3(feed solution concentration 140mmol/L) and K2HPO4(the concentration of the feed solution is 15mmol/L), so that the concentration of the glucose is kept between 4 and 15g/L, and NO is3 -The concentration of (A) is maintained at 3-8mmol/L, PO4 -The concentration of (A) is kept at 0.3-0.8 mmol/L.
The fermentation culture period is about 4 weeks, and the OD is in the algae liquid682The value reaches about 1.8, and the fermentation is finished when the wet weight of algae is about 40 g/L. Has been fermentedThe nutrient metabolism and growth during the process are shown in FIG. 6.
Example 4 extraction and concentration of exopolysaccharides
The 350L of the algae liquid obtained by fermentation is centrifuged by a tube centrifuge (GQ105 clean type, Xinda) at 16000r/min to obtain about 10kg of algae mud. The supernatant obtained by centrifugation was passed through a cloth bag filter, which was packed with a 4000 mesh filter bag and filtered to obtain about 200g of algal cells.
Transferring the collected algae mud and algae cells into a stainless steel container, adding 50L of pure water for dilution, and centrifuging again through a tube centrifuge at 16000r/min to obtain about 10kg of algae mud. Repeating the above steps for 2 times to remove glucose and inorganic salts from the algae mud. The water content of the obtained algae mud is about 70 wt%, and the algae mud is put into a clean container for standby.
Mixing the algae mud with purified water at a volume ratio of 1:3, and placing into an extraction tank (Yiwu Vietyi machine, 50L type). Introducing steam into the tank jacket to heat the material while stirring, maintaining at 90-100 deg.C for 20min, stopping stirring, standing for 20min to allow the algae mass to settle and float naturally, and facilitating filtration. The extraction tank is provided with a 2000-mesh filter screen for the first filtration, filtrate is filtered again by a column type filter (Yiwufeiyi machine) with a 4000-mesh filter screen connected behind the cooking tank, and finally the filtrate is filtered for the third time by a filter press (Huadong, SH-300 x 300 type) with 2.0-micron filter paper, and about 30L of clear filtrate without obvious solid impurities is obtained and enters the concentration equipment. And after filtering, adding purified water into the algae residue in the cooking tank again, and performing secondary extraction, wherein the process is the same as the above. The total amount of the two extracts was about 60L.
The extract was concentrated using a single effect evaporator (Yiwu Vietyi machine, type 50L). Specifically, the extract is pumped into a concentration tank of a single-effect evaporator, the material is heated by steam through a jacket, and simultaneously the evaporator is vacuumized by a water circulation vacuum pump, and the vacuum degree is kept to be about-0.03 MPa. The material is boiled, evaporated and concentrated, and finally concentrated to about 10L. Centrifuging the obtained concentrated solution with low-speed large-capacity centrifuge (DD-5M) at 4000r/min for 5min, collecting supernatant about 9.5L, placing the supernatant into two 5L bottles, placing into a sterilizing pan, sterilizing at 121 deg.C for 20 min.
And (4) putting the sterilized supernatant into a freezing tray, freezing the supernatant into ice blocks at the temperature of-80 ℃, freezing the ice blocks into the ice blocks, and performing vacuum freeze drying treatment, wherein the finished product can be collected after drying for 72 hours generally to obtain about 270g of dry powder of the water extract. The dry powder is light yellow green flocculent solid, has special fragrance of Botryococcus, is soluble in water, and is insoluble in ethanol. The water content is less than or equal to 5 percent, the pH value is 6-8, and the polysaccharide content is more than or equal to 50 percent.
The polysaccharide content test reagents and materials were as follows:
1.1 reagents
Sulfuric acid (H2SO4), ρ 1.84 g/mL;
phenol (C6H6O), redistilled;
glucose (C)6H12O6) Before use, the mixture is dried at a constant temperature of 105 ℃ to constant weight;
80% phenol solution: weighing 80g of phenol (5.2) in a 100mL beaker, adding water to dissolve the phenol, transferring the phenol to a 100mL brown volumetric flask for constant volume, and storing the phenol in a refrigerator at 4 ℃ in a dark place;
5% phenol solution: 5mL of phenol solution (5.4) is absorbed and dissolved in 75mL of water, and the mixture is uniformly mixed and prepared on site;
100mg/L standard glucose solution: 0.100g of glucose was weighed into a 100mL beaker, dissolved in water, and stored in a refrigerator at 4 ℃ in a volume of 1000 mL.
2.1 preparation of Standard Curve
0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, and 1.0mL of the standard glucose working solution were aspirated and placed in a 25mL brown cuvette, and the volume was adjusted to 1.0mL with distilled water. To the sample solution was added 1.0mL of phenol solution, then 5.0mL of sulfuric acid (added perpendicular to the liquid surface without touching the tube wall to mix well with the reaction solution) was added rapidly, allowed to stand for 10min, vortexed, and allowed to stand at room temperature or in a 30 ℃ water bath for 20 min. The absorbance was measured at 490nm wavelength. And (4) establishing a standard curve by taking the mass concentration of the glucose as an abscissa and the absorbance value as an ordinate.
2.2 sample treatment
Weighing microalgae extract sample 0.01g (accurate to 0.0001g), dissolving with water, metering to 100mL volumetric flask, and shaking. The absorbance was measured by pipetting 1.0mL of the sample solution into a 25mL brown cuvette, following procedure 2.1.
2.3 blank test
In parallel with the measurement of the sample, the same amount of all reagents is taken and the same analytical procedure is used, but no sample is added. 2.4 calculation of results
The total sugar content X in the sample is calculated according to the formula (1), and three significant figures are reserved in the calculation result.
Total sugar content
The polysaccharide content is expressed in units of grams per hundred grams (g/100g) in terms of mass fraction ω, calculated according to formula (2):
polysaccharide content
In the formula:
m1-determining the sugar content in microgram (μ g) of the sample from the standard curve;
V1-sample volumetric volume in milliliters (mL);
V2-the volume of the removed sample measurement solution in milliliters (mL) for colorimetric measurements;
m2-sample mass in grams (g); 0.9 is a correction factor for glucose to glucan.
The present invention has been described with reference to the accompanying drawings and the above specific embodiments, but these embodiments do not limit the scope of the present invention. The scope of protection of the invention is defined by the appended claims.
Claims (10)
1. A convenient fermentation method with low contamination risk comprises the steps of using a strain culture bottle (100), a inoculation pipe (300) and a pumping device (13), wherein the strain culture bottle (100) comprises a bottle body (1) and a sealable bottle cap (2), a first guide pipe (3) and a second guide pipe (4) are arranged on the bottle cap (2), the first guide pipe (3) extends into the bottom of the bottle body (1), the outer parts of the first guide pipe (3) and the second guide pipe (4) are respectively connected with a first air filter (5) and a second air filter (6), one end (14) of the inoculation pipe (300) is provided with an inoculation needle (15) connected with a fermentation tank (400), the other end (16) is provided with a connecting piece (17) connected with the strain culture bottle (100),
the method comprises the following steps:
1) using a strain culture bottle (100) to culture strains;
2) in a sterile environment, removing the first air filter (5) from the strain culture bottle (100) and connecting the inoculation tube (300) with the first conduit (3) through the connecting piece (17);
3) connecting the inoculating needle (15) with the fermentation tank (400);
4) arranging the pumping device (13) at any position of the inoculation tube (300), and starting the pumping device (13) during inoculation; and
5) the fermentation is carried out in a fermentor (400).
2. A convenient fermentation method with low contamination risk comprises the steps of using a strain culture bottle (100), a inoculation tube (300) and a pumping device (13), wherein the strain culture bottle (100) comprises a bottle body (1) and a sealable bottle cap (2), a first guide tube (3) and a second guide tube (4) are arranged on the bottle cap (2), the first guide tube (3) extends into the bottom of the bottle body (2), the outer parts of the first guide tube (3) and the second guide tube (4) are respectively connected with a first air filter (5) and a second air filter (6), one end (14) of the inoculation tube (300) is provided with an inoculation needle (15) connected with a fermentation tank (400), the other end of the inoculation tube is branched into more than two branches at a branch node (21), a connecting piece (17) connected with the strain culture bottle (100) is arranged at the tail end (16) of at least one branch (16-1), and the end (16) of at least one other branch (16-3) is provided with a connection piece (19) connected with a feeding device (18),
the method comprises the following steps:
1) using a strain culture bottle (100) to culture strains;
2) in a sterile environment, removing the first air filter (5) from the seed culture flask (100) and connecting the inoculation tube (300) to the first conduit (3) via a connector (17) and to the feeding device (18) via a connector (19), optionally closing the branch end (16) not connected to any device;
3) connecting the inoculating needle (15) with the fermentation tank (400);
4) arranging a pumping device (13) between the tail end (14) of the inoculation pipe (300) and the branch node (21), opening an inoculation branch, closing a material supplementing branch, and starting the pumping device (13) for inoculation; and
5) the inoculation branch is closed, fermentation is carried out in the fermentation tank (400), and the feeding branch is opened for feeding when needed.
3. A fermentation process according to claim 2, wherein the opening, closing and closing of each branch is achieved by providing an interception means (20) between the branching node (21) and the end (16) of each branch.
4. A fermentation process according to claim 3, wherein the interception means (20) is a clip.
5. Fermentation process according to claim 1 or 2, wherein the inoculation needle (15) is connected to the fermenter (400) immediately after sterilization of the fermenter (400).
6. A fermentation process according to claim 1 or 2 wherein the inoculating needle (15) is a pin which abuts the refill (24) of the fermentor (400).
7. A fermentation process according to claim 1 or 2, wherein one or more air filters are connected to the first conduit (3) and/or the second conduit (4).
8. A fermentation process as claimed in claim 1 or 2, wherein the connecting piece (17) is a metal feedthrough.
9. A fermentation process as claimed in claim 1 or 2, wherein the connecting piece (19) is a metal feedthrough.
10. A fermentation process according to claim 1 or 2 wherein the pumping means (13) is a peristaltic pump.
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