CA1178225A - Hollow fiber membrane microbiological reactors - Google Patents
Hollow fiber membrane microbiological reactorsInfo
- Publication number
- CA1178225A CA1178225A CA000402564A CA402564A CA1178225A CA 1178225 A CA1178225 A CA 1178225A CA 000402564 A CA000402564 A CA 000402564A CA 402564 A CA402564 A CA 402564A CA 1178225 A CA1178225 A CA 1178225A
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- Prior art keywords
- cells
- lumen
- housing
- reactor
- pores
- Prior art date
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Abstract
FOLLOW FIBER MEMBRANE MICROBIOLOGICAL REACTORS
ABSTRACT OF THE DISCLOSURE
Apparatus and methods for microbiological proces-sing of organic materials, particularly for production of valuable products. Asymmetric hollow fibers are employed in a flow reactor, where the hollow fibers have a semipermeable membrane surrounding a lumen, where the semipermeable mem-brane is supported by a sponge structure. The pores of the sponge structure serve as a housing for microorganisms or cells with high density packing of the microorganisms or cells in the pores. Nutrient medium continuously flowing through the lumen provides nutrients to the microorganisms or cells as well as any substrates to be processed by the micro-organisms or cells. The nutrients and substrates diffuse through the semipermeable membrane into the pores, where they are processed, and the metabolic products diffuse into the lumen. The lumen effluent is then processed for the desired products. Optionally, oxygen is provided external to the hollow fiber to enhance the amount of oxygen available to the microorganisms and cells.
ABSTRACT OF THE DISCLOSURE
Apparatus and methods for microbiological proces-sing of organic materials, particularly for production of valuable products. Asymmetric hollow fibers are employed in a flow reactor, where the hollow fibers have a semipermeable membrane surrounding a lumen, where the semipermeable mem-brane is supported by a sponge structure. The pores of the sponge structure serve as a housing for microorganisms or cells with high density packing of the microorganisms or cells in the pores. Nutrient medium continuously flowing through the lumen provides nutrients to the microorganisms or cells as well as any substrates to be processed by the micro-organisms or cells. The nutrients and substrates diffuse through the semipermeable membrane into the pores, where they are processed, and the metabolic products diffuse into the lumen. The lumen effluent is then processed for the desired products. Optionally, oxygen is provided external to the hollow fiber to enhance the amount of oxygen available to the microorganisms and cells.
Description
~ 7~5 This invention relates to a microbiological flow reactor and to a method for continuously transforming a substrate to a product in such a reactor.
Although the catalytic properties of microorganisms have been exploited in various biochemical processes for years, the techniques generally employed to carry out these transformations have their origins in traditional batch-fermentation methods, and have undergone little change since their original initiation. With the relatively recent appear--ance of recombinant DNA techniques for genetically alteringcellular function and metabolism, there is an increasing need to improve the exploitation of microorganisms to produce valuable products or process effluent streams. There is little known about the dynamics of cell growth. The ablli-ty to supply nutrients to the cell organisms, the manner in whlch the organisms become distributed in a reactor, -the effect on such distribution of the supply of nutrients to the organisms and the removal of excretion products from the organisms remains a matter of uncertain-ty. In addition to the concerns about distribution of nutrients and removal of excretion products, the fragile nature of the cells limits the manner in which the cells may be handled during the processing. Techniques which have found application include fermenting involving mechanical agitation and a flowing stream through a reactor for supplying nutrient and removing product, air-lift fermentors; fluidized-bed fermentors; immobilized cells and the like.
In order to maximize the benefits of using micro-organisms, substantial improvements are required in the yields of product obtained employing microorganisms where the yield is based on per unit of reactant as well as per ~mit volume of reactor, the packing density of the microorganisms, .~
~ ~82~
the rate of production, the viabili-ty of the oryanisms, and the like.
In the prior art U.S. Patent No. 3,580,8~0 describes a method and apparatus using microorganisms for sewage treatment employing a porous membrane. U~S. Patent No.
3,7~7,790 teaches microorganism entrapment: for controlled release. See also U.S. Patent No. 3,860,'190. U.S. Patent No.
3,875,008 teaches microorganisms encapsulation in a hollow filament. U.S. Patent No. 4,148,689 teaches entrapment of microorganisms in a gelled sol.
According to the invention method and apparatus are provided for microbiological transformation of a nutri~nt stream. The appara-tus employs at least one asymmetric hollow fiber having an internal membrane surrounding a lumen and a porous supporting wall. A nutrien-t medium flows through the lumen providing nutrient for the microorganisms in the pores of the wall and removing microbiological products. Optlonally, oxygen is provided external of the hollow fiber to enhance oxygen availability. The apparatus provides for high packing densities of microorganisms in the pores with good viability providing for enhanced efficiency in metabolizing substrates.
Thus, more specifically, in a first aspect the invention is a method for continuously transforming a sub-strate to a product by microbiological means employing cells in a flow reactor, said flow reactor comprising: a housing;
at least one hollow fiber in said housing, said hollow fiber having an inlet port and an outlet port and characterized by having a lumen, a porous membrane surrounding said lumen and having orifices smaller than said cells, and a spongy supporting wall having pores internally communicating through said orifices with said lumen and externally communicating with the volume enclosed by said housing through openings of a size greater than said cells; and a nutrient medium pervading ~a said housing; said method comprising: growing cells in said housing so as to substantially fill said wall pores, while continually passing substrate containing nutrient medium into said lumen through said inlet por-t, whereby nutrients S and substrate flow into said pores and substrate is trans-formed to product by said cells and diffuses through said orifices into said lumen; and continuously removing nutrient medium containlng product from said lumen through said exit port.
In a second aspect the invention is a microbiologi-cal flow reactor comprising: a housing; at least one hollow fiber enclosed in said housing, said hollow fiber having an inlet port and an outlet port communicating outside sai.d housing and urther characterized by having a lumen; said lumen enclosed by a membrane having orifices smaller than cells to be employed in said microb.iological reactor; and a spongy supportlng wall ha~ing pores internally communicating through said orifices with said lumen and externally communi-cating with the volume enclosed by said housing -through open-ings of a size greater than said cells; a nutrient medium prevading said housing; and cells filling at least 60~ of the available volume of said pores.
The invention is illustrated, by way of example, in the drawings in which:
Figure 1 is a schematic view of a single fiber reactor;
Fi.gure 2 is a flowchart of a single fiber reactor providing for monitoring the streams entering and exiting from the reactor; and Figure 3 is a cross-sectional view of a multifiber reactor.
3 117822~
Novel reactors and method employing the reactor are provided for microbiological transformations. The reactors employ at least one, normally a plurality of asymmetric hollow fibers which are conveniently mounted in parallel in a closed housing. For the purposes of this invention, micro-organisms will be used as illustrative of single cells which can be cultured ln vitr_. It should be appreciated that the subject invention is applicable to single cell lines, par-ticularly proliferative single cell lines.
The microorganisms are inoculated into the fluid in ` ~ the space surrounding the hollow fibers, while a nutrientmedium is directed to the lumen of the hollow fibers. The nutrients and substrates pass, by flowing or diffusing into the pores of the hollow fiber wall containing the micro-organisms, while the microbiological products flow or diffuse back into the lumen and into the interfiber spaces. In this way, nutrient continuously washes the microorganisms in the pores and products are removed from the pores to prevent inhibition of the microorganism metabolism.
The hollow fibers which are employed are asymmetric hollow fibers having a thin internal porous membrane sup-ported by a relatively thick porous wall. The orifices of the inner membrane will ~enerally have molecular weight cut-offs of less than about 200,000, preferably less than about 100,000, and may be 50,000 or less, usually not less than about 5,000, more usually not less than about 10,000 molecular weight. The choice of molecular weight cut-off will be determined by the degree to which microorganisms are inhibited from entering the lumen, while allowing for diffu-sion or flow of desired materials between the lumen and wall pores of the hollow fiber.
The purpose of the inner membrane is to inhibit cell leakage into the lumen and to provide molecular separa-tion capability, while permitting a relatively rapid ra-te of diffusion and flow of solutes between the lumen and wall pores. Generally, the thickness of the inner membrane will be not less than about 0.01~ and not more than about 1~, more 4 1 17~2~
usually not more than about 0.5~. The diameter of the orifices of the inner membrane will generally be from about one to two orders of ma~nitude smaller than the smallest dimension of the microorganism being culti~ated. For bacter-ia, this will usually be from about 0.001~ to about 0.005~,while for larger cells, larger orifices will be acceptable.
The porous supporting wall surrounds the inner membrane and supports the inner membrane, with the pores or cavities of the wall communicating through the orifices of the inner membrane with the lumen. The thickness of the wall is not critical to this invention, although beyond a certain thickness, providing for nutrients throughout the pores may become difficult. The outer wall will -therefore be of from about 50 to 500microns thick, more usually from about 75 to ~OOmicrons thick and preferably of from about 100 to 200microns thick. Outer diameters of the fiber will ~eneral-ly vary ~rom about 0.25mm to about 2.5mm.
The porous wall or outer region of the fiber will be mostly void space, there being at least SO~ void space, more usually at least 60~ void space and usuall~ not more than about 90% void space, more usually from about 65 to 85%
void space. This region is normally termed the sponge region~ The pores of the wall will have relatively free access to the outside, the openings generally being at least about 5~l and may be 10~ or greater, usually being not greater than about 50~ on the average. The openings are large enough for the microorganism of interest to enter the pore. The volume of individual pores will be sufficient to house at least about 102 cells, usually at least about 103 cells.
The length of the fiber in the reactor can be varied widely depending upon the rate at which the fluid flows through the lumen, the potential for further transfor-mation of the desired product, the efficiency and rate at which the desired substrate is transformed, the pressure drop across the lumen and other process considerations. Lengths will usually be at least about lcm, more usually at least about 5cm, and may be 50cm or longer.
~ 1~82~5 The diameter of the lumen may vary widely depending upon the desired rate of flow, the rate of flow and diffusion of nutrients into the pores, the efficiency of utilization of the nutrients in the nutrient medium and the desired concen-tration of product. The ratio of the diameter of the lumento the diameter of the fiber will vary widely, llsually heing at least about 20% and generally not more than about 90%, the above considerations affecting the ratio. The significant factor in the ratio is the greatest path lenyth nutrient must flow to feed all of the cell population and the ability to provide adequate amounts of nutrients across that path length. Therefore, the wall and the cell nutrient require-ments will play a role in the hollow fiber design.
A wide variety of materials are employed for the production of asymmetrical hollow fibers. The particular material is not a critical part of this invention, so long as it does not deleteriously affect the growth o the micro-organisms nor react with the nutrients and products. Various inert polymeric materials can be employed, both organic and ~0 inorganic, and a numbers of hollow fibers are commercially available. Illustrative hollow fiber membranes include polysulfone membranes, terpolymers of vinyl chloride, vinylidene chloride and acrylonitrile (available as Dynel ) polypropylene membranes and cellulosic membranes (available as Cuprophan ). The materials may be hydrophilic or hydro-phobic or combinations thereof. If desired, the various materials may be further modified to introduce functionali-ties onto the fiber.
While a reactor having a single hollow fiber may be employed, for the most part a plurality of fibers will be employed in a single housing or shell. The housing will enclose the hollow fibers so that the fibers are washed in the nutrient medium which flows out of the pores of the hollow fiber. One or more ports may be provided in the shell for introducing materials external to the fibers, for sam-pling, for removal of gases, for removal of the product containing spent nutrient stream for isolation of product and recycling of nutrients, for adding nutrients, or the like.
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The housing may also be used for maintaining a pressure diferential between the lumen and the outside o~ the hollow fiber. The packing of the hollow fiber6 in the shell will vary depending upon the desirability of having microorganisms ~rown outside the pores of the shell, t~le ability for diffu-sion ~etween the hollow fibers and the volume outside the fibers in the ~hell, and the ease with which oxygen can be diffused through the medium. For the most part, the packing will be determined empirically and will vary with the nature of the microorganism, as well as the purpose of the reactor.
A wide variety of microorganisms and cells may be grown in the reactor. Particularly, bacteria, yeast and fungi can be effectively grown. Not only can naturally occurring microorganisms and cells be employed, but also microorganisms and cells which have been modi~ied by conjuga-tion or genetic engineering techni~ues, such a~ transforma-tion, DNA inser~ions, transduction, fu~ion and the like.
~mong cells which may be grown in the reactor are various mammalian cells which can be cultured ln vitro, particularly tl1mor cells and hybridomas.
Cells can be employed in which DNA replication is substantially inhi~ited or terminated, but metabolism con-tinues for relatively long periods of time. The cells con tinue to express genes, other than the blocked genes involved with DNA replication. Where the cells have been transformed with exogenous yenes, these genes will be expressed to pro-vide the desired product.
By preventing DNA replication, the nutrients are used more efficiently for the functioning of ~he microbio-logical reactor. The inhibition of DNA replication can be achieved in a variety of ways, ~uch as chemical inhibitors, temperature sensitive mutants, mutants lacking an inter-mediate in the bio~ynthetic pathway to DNA replication, or ~he like.
The nutrient medium employed will be dependent upon the microorganism or cell involved, and the product desired or purpose for the reactor. For example, the nutrient medium will be adapted to the particulAr microorganism or cell.
~ 1~82~5 Besides nutrients, other substances may be included to sup-port growth and/or cell differentiation. By contrast, the product may be a natural product such as an excreted protein e.g. enzvmes, hormones, lymphokines, toxins, immunoglobulins, or ~he like or a non-proteinaceous organic compound resulting from transformation of a substrate, such as by epoxidation, hydroxylation, esterification e.g. acetate, phosphate, uronate or sulfate, reduction, methylation, etherification with sugars, or the like. Thus, the reactor can act as a source of a wide variety of compounds, either as the natural product, such as a polypeptide or protein, or for transform-ing a synthetic substrate. Alternatively, the reactor may be used with a wide variety of effluents from various corNmercial processing sources, such as chemical processinq plants, sewage plants, water treatment plants, or the like.
Besides nutrients provided in the lumen, additional nutrients may be provided in the shell space. Particularly, because of the low solubility of oxygen in water, additional oxygen may be provided into the fluid surrounding the hollow fibers. To further enhance oxygen content, the fluid and shell space may be pressurized so that the concentration of oxygen in the nutrient solution is increased.
During operation, the cells substantially fill the wall pores to greater than 50~ of the available vol~me, usually greater than 60% and cell densities filling greater than 80% of the void volume are achievable. The high cell packing density is realized because of the efficiency of introduction of nutrients and oxygen into the wall pores as well as the efficient removal of product from the wall pores.
For further understanding of the invention, the drawings will now be considered. The reactor 10 is comprised of a single hollow fiber 12 which is centrally extended in a glass tube 14 ~nd sealed at its ends in the tube 14 by seals 16 and 20. Seals 16 and 20 enclose the space 22 in tube 14.
The fiber extends to the ends of seals 16 and 20 so as to provide inlet port 24 and exit port 26 for introduction and removal respectively of the nutrient medium. To provide for the opportunity for additional oxygen supply to the shell
Although the catalytic properties of microorganisms have been exploited in various biochemical processes for years, the techniques generally employed to carry out these transformations have their origins in traditional batch-fermentation methods, and have undergone little change since their original initiation. With the relatively recent appear--ance of recombinant DNA techniques for genetically alteringcellular function and metabolism, there is an increasing need to improve the exploitation of microorganisms to produce valuable products or process effluent streams. There is little known about the dynamics of cell growth. The ablli-ty to supply nutrients to the cell organisms, the manner in whlch the organisms become distributed in a reactor, -the effect on such distribution of the supply of nutrients to the organisms and the removal of excretion products from the organisms remains a matter of uncertain-ty. In addition to the concerns about distribution of nutrients and removal of excretion products, the fragile nature of the cells limits the manner in which the cells may be handled during the processing. Techniques which have found application include fermenting involving mechanical agitation and a flowing stream through a reactor for supplying nutrient and removing product, air-lift fermentors; fluidized-bed fermentors; immobilized cells and the like.
In order to maximize the benefits of using micro-organisms, substantial improvements are required in the yields of product obtained employing microorganisms where the yield is based on per unit of reactant as well as per ~mit volume of reactor, the packing density of the microorganisms, .~
~ ~82~
the rate of production, the viabili-ty of the oryanisms, and the like.
In the prior art U.S. Patent No. 3,580,8~0 describes a method and apparatus using microorganisms for sewage treatment employing a porous membrane. U~S. Patent No.
3,7~7,790 teaches microorganism entrapment: for controlled release. See also U.S. Patent No. 3,860,'190. U.S. Patent No.
3,875,008 teaches microorganisms encapsulation in a hollow filament. U.S. Patent No. 4,148,689 teaches entrapment of microorganisms in a gelled sol.
According to the invention method and apparatus are provided for microbiological transformation of a nutri~nt stream. The appara-tus employs at least one asymmetric hollow fiber having an internal membrane surrounding a lumen and a porous supporting wall. A nutrien-t medium flows through the lumen providing nutrient for the microorganisms in the pores of the wall and removing microbiological products. Optlonally, oxygen is provided external of the hollow fiber to enhance oxygen availability. The apparatus provides for high packing densities of microorganisms in the pores with good viability providing for enhanced efficiency in metabolizing substrates.
Thus, more specifically, in a first aspect the invention is a method for continuously transforming a sub-strate to a product by microbiological means employing cells in a flow reactor, said flow reactor comprising: a housing;
at least one hollow fiber in said housing, said hollow fiber having an inlet port and an outlet port and characterized by having a lumen, a porous membrane surrounding said lumen and having orifices smaller than said cells, and a spongy supporting wall having pores internally communicating through said orifices with said lumen and externally communicating with the volume enclosed by said housing through openings of a size greater than said cells; and a nutrient medium pervading ~a said housing; said method comprising: growing cells in said housing so as to substantially fill said wall pores, while continually passing substrate containing nutrient medium into said lumen through said inlet por-t, whereby nutrients S and substrate flow into said pores and substrate is trans-formed to product by said cells and diffuses through said orifices into said lumen; and continuously removing nutrient medium containlng product from said lumen through said exit port.
In a second aspect the invention is a microbiologi-cal flow reactor comprising: a housing; at least one hollow fiber enclosed in said housing, said hollow fiber having an inlet port and an outlet port communicating outside sai.d housing and urther characterized by having a lumen; said lumen enclosed by a membrane having orifices smaller than cells to be employed in said microb.iological reactor; and a spongy supportlng wall ha~ing pores internally communicating through said orifices with said lumen and externally communi-cating with the volume enclosed by said housing -through open-ings of a size greater than said cells; a nutrient medium prevading said housing; and cells filling at least 60~ of the available volume of said pores.
The invention is illustrated, by way of example, in the drawings in which:
Figure 1 is a schematic view of a single fiber reactor;
Fi.gure 2 is a flowchart of a single fiber reactor providing for monitoring the streams entering and exiting from the reactor; and Figure 3 is a cross-sectional view of a multifiber reactor.
3 117822~
Novel reactors and method employing the reactor are provided for microbiological transformations. The reactors employ at least one, normally a plurality of asymmetric hollow fibers which are conveniently mounted in parallel in a closed housing. For the purposes of this invention, micro-organisms will be used as illustrative of single cells which can be cultured ln vitr_. It should be appreciated that the subject invention is applicable to single cell lines, par-ticularly proliferative single cell lines.
The microorganisms are inoculated into the fluid in ` ~ the space surrounding the hollow fibers, while a nutrientmedium is directed to the lumen of the hollow fibers. The nutrients and substrates pass, by flowing or diffusing into the pores of the hollow fiber wall containing the micro-organisms, while the microbiological products flow or diffuse back into the lumen and into the interfiber spaces. In this way, nutrient continuously washes the microorganisms in the pores and products are removed from the pores to prevent inhibition of the microorganism metabolism.
The hollow fibers which are employed are asymmetric hollow fibers having a thin internal porous membrane sup-ported by a relatively thick porous wall. The orifices of the inner membrane will ~enerally have molecular weight cut-offs of less than about 200,000, preferably less than about 100,000, and may be 50,000 or less, usually not less than about 5,000, more usually not less than about 10,000 molecular weight. The choice of molecular weight cut-off will be determined by the degree to which microorganisms are inhibited from entering the lumen, while allowing for diffu-sion or flow of desired materials between the lumen and wall pores of the hollow fiber.
The purpose of the inner membrane is to inhibit cell leakage into the lumen and to provide molecular separa-tion capability, while permitting a relatively rapid ra-te of diffusion and flow of solutes between the lumen and wall pores. Generally, the thickness of the inner membrane will be not less than about 0.01~ and not more than about 1~, more 4 1 17~2~
usually not more than about 0.5~. The diameter of the orifices of the inner membrane will generally be from about one to two orders of ma~nitude smaller than the smallest dimension of the microorganism being culti~ated. For bacter-ia, this will usually be from about 0.001~ to about 0.005~,while for larger cells, larger orifices will be acceptable.
The porous supporting wall surrounds the inner membrane and supports the inner membrane, with the pores or cavities of the wall communicating through the orifices of the inner membrane with the lumen. The thickness of the wall is not critical to this invention, although beyond a certain thickness, providing for nutrients throughout the pores may become difficult. The outer wall will -therefore be of from about 50 to 500microns thick, more usually from about 75 to ~OOmicrons thick and preferably of from about 100 to 200microns thick. Outer diameters of the fiber will ~eneral-ly vary ~rom about 0.25mm to about 2.5mm.
The porous wall or outer region of the fiber will be mostly void space, there being at least SO~ void space, more usually at least 60~ void space and usuall~ not more than about 90% void space, more usually from about 65 to 85%
void space. This region is normally termed the sponge region~ The pores of the wall will have relatively free access to the outside, the openings generally being at least about 5~l and may be 10~ or greater, usually being not greater than about 50~ on the average. The openings are large enough for the microorganism of interest to enter the pore. The volume of individual pores will be sufficient to house at least about 102 cells, usually at least about 103 cells.
The length of the fiber in the reactor can be varied widely depending upon the rate at which the fluid flows through the lumen, the potential for further transfor-mation of the desired product, the efficiency and rate at which the desired substrate is transformed, the pressure drop across the lumen and other process considerations. Lengths will usually be at least about lcm, more usually at least about 5cm, and may be 50cm or longer.
~ 1~82~5 The diameter of the lumen may vary widely depending upon the desired rate of flow, the rate of flow and diffusion of nutrients into the pores, the efficiency of utilization of the nutrients in the nutrient medium and the desired concen-tration of product. The ratio of the diameter of the lumento the diameter of the fiber will vary widely, llsually heing at least about 20% and generally not more than about 90%, the above considerations affecting the ratio. The significant factor in the ratio is the greatest path lenyth nutrient must flow to feed all of the cell population and the ability to provide adequate amounts of nutrients across that path length. Therefore, the wall and the cell nutrient require-ments will play a role in the hollow fiber design.
A wide variety of materials are employed for the production of asymmetrical hollow fibers. The particular material is not a critical part of this invention, so long as it does not deleteriously affect the growth o the micro-organisms nor react with the nutrients and products. Various inert polymeric materials can be employed, both organic and ~0 inorganic, and a numbers of hollow fibers are commercially available. Illustrative hollow fiber membranes include polysulfone membranes, terpolymers of vinyl chloride, vinylidene chloride and acrylonitrile (available as Dynel ) polypropylene membranes and cellulosic membranes (available as Cuprophan ). The materials may be hydrophilic or hydro-phobic or combinations thereof. If desired, the various materials may be further modified to introduce functionali-ties onto the fiber.
While a reactor having a single hollow fiber may be employed, for the most part a plurality of fibers will be employed in a single housing or shell. The housing will enclose the hollow fibers so that the fibers are washed in the nutrient medium which flows out of the pores of the hollow fiber. One or more ports may be provided in the shell for introducing materials external to the fibers, for sam-pling, for removal of gases, for removal of the product containing spent nutrient stream for isolation of product and recycling of nutrients, for adding nutrients, or the like.
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The housing may also be used for maintaining a pressure diferential between the lumen and the outside o~ the hollow fiber. The packing of the hollow fiber6 in the shell will vary depending upon the desirability of having microorganisms ~rown outside the pores of the shell, t~le ability for diffu-sion ~etween the hollow fibers and the volume outside the fibers in the ~hell, and the ease with which oxygen can be diffused through the medium. For the most part, the packing will be determined empirically and will vary with the nature of the microorganism, as well as the purpose of the reactor.
A wide variety of microorganisms and cells may be grown in the reactor. Particularly, bacteria, yeast and fungi can be effectively grown. Not only can naturally occurring microorganisms and cells be employed, but also microorganisms and cells which have been modi~ied by conjuga-tion or genetic engineering techni~ues, such a~ transforma-tion, DNA inser~ions, transduction, fu~ion and the like.
~mong cells which may be grown in the reactor are various mammalian cells which can be cultured ln vitro, particularly tl1mor cells and hybridomas.
Cells can be employed in which DNA replication is substantially inhi~ited or terminated, but metabolism con-tinues for relatively long periods of time. The cells con tinue to express genes, other than the blocked genes involved with DNA replication. Where the cells have been transformed with exogenous yenes, these genes will be expressed to pro-vide the desired product.
By preventing DNA replication, the nutrients are used more efficiently for the functioning of ~he microbio-logical reactor. The inhibition of DNA replication can be achieved in a variety of ways, ~uch as chemical inhibitors, temperature sensitive mutants, mutants lacking an inter-mediate in the bio~ynthetic pathway to DNA replication, or ~he like.
The nutrient medium employed will be dependent upon the microorganism or cell involved, and the product desired or purpose for the reactor. For example, the nutrient medium will be adapted to the particulAr microorganism or cell.
~ 1~82~5 Besides nutrients, other substances may be included to sup-port growth and/or cell differentiation. By contrast, the product may be a natural product such as an excreted protein e.g. enzvmes, hormones, lymphokines, toxins, immunoglobulins, or ~he like or a non-proteinaceous organic compound resulting from transformation of a substrate, such as by epoxidation, hydroxylation, esterification e.g. acetate, phosphate, uronate or sulfate, reduction, methylation, etherification with sugars, or the like. Thus, the reactor can act as a source of a wide variety of compounds, either as the natural product, such as a polypeptide or protein, or for transform-ing a synthetic substrate. Alternatively, the reactor may be used with a wide variety of effluents from various corNmercial processing sources, such as chemical processinq plants, sewage plants, water treatment plants, or the like.
Besides nutrients provided in the lumen, additional nutrients may be provided in the shell space. Particularly, because of the low solubility of oxygen in water, additional oxygen may be provided into the fluid surrounding the hollow fibers. To further enhance oxygen content, the fluid and shell space may be pressurized so that the concentration of oxygen in the nutrient solution is increased.
During operation, the cells substantially fill the wall pores to greater than 50~ of the available vol~me, usually greater than 60% and cell densities filling greater than 80% of the void volume are achievable. The high cell packing density is realized because of the efficiency of introduction of nutrients and oxygen into the wall pores as well as the efficient removal of product from the wall pores.
For further understanding of the invention, the drawings will now be considered. The reactor 10 is comprised of a single hollow fiber 12 which is centrally extended in a glass tube 14 ~nd sealed at its ends in the tube 14 by seals 16 and 20. Seals 16 and 20 enclose the space 22 in tube 14.
The fiber extends to the ends of seals 16 and 20 so as to provide inlet port 24 and exit port 26 for introduction and removal respectively of the nutrient medium. To provide for the opportunity for additional oxygen supply to the shell
2 5 space 22, as well as for monitoring gas production in the shell space 22, conduits ~8 and 30 are connected to the tube 14 in fluid transfer relationship internal to the seals 16 and 20. A manometer 32 is attached to conduit 30 for moni-toring the pressure of the gas supply o:c if desired, thepressure in space 22. Connected to the inlet port 24 is inlet conduit 34 eguipped with pressure gauge 36 for monitor-ing the pressure of the inlet nutrient stream. Outlet con-duit 40 is connected to outlet port 26 :in fluid receivin~
relationship and a pressure gauge 42 is mounted on the outlet conduit 40 to provide for monitoring the pressure of the lumen effluent. In addition to providing for the introduc-tion of gas or other materials into the shell space 22, conduits 28 and 30 also provide the opportunity to i~noculate the reactor with microorganisms or cells.
Figure 2 is a diagram of the eguipment used in a nu~ber of tests. The reactor 10a has single fiber 12a which is sealed in the tube 14a by seals 16a and 20a. Pressurized oxygen is provided by gas cylinder 50a, which is connected by lines 52a and 54a to nutrient media reservoir 56a. Pressure regulator 60a mounted in line 52a controls the oxygen pres-sure in line 52a. The oxygen pressure forces the nutrient media in reservoir 56a into line 62a in which is moun-ted three-way valve 64a, the remaining arm being fitted with syringe 66a. Line 62a connects with peristaltic pump 70a which controls ~he flow of the nutrient medium ~hrough line 7~a to inlet port 24a of hollow ~iber 12a. Line 72a has a series of coils 78a to allow for temperature control of the nutrient medium fed to hollow fiber 12a. Side arm 30a of tube 14a is connected by a conduit 74a to shell space sam-pling conduit 76a and hl~idifier 80a. The humidifier 80a is connected by means of co~duit 82a to line 52a to permit humidified oxygen to be introduced into the reactor shell space 22a. Side arm 28a is connected by means of line 84a to three-way valve 86a which serves to pass the effluent from the shell space 22a into sample collection tube 90a or by means of line 92a to shell-space effluent reservoir 94a.
~ 1~822~
g The nutrient media fed into inlet port 24a by means of line ~2a are monitored through line 96a, while the lumen effluent exiting exit port 26a is monitored through line lQOa. Lines 76a, 96a, and lOOa are all connected to line 102a which is connected to a manometer 104a for monitoring the pressure in the reactor. The nutrient medium of the lumen exiting through exit 26a is connected by line 106a to three-way valve llOa which serves to connect the effluent to sample collection tube 112a or lumen effluent reservoir 114a.
For temperature control, the reactor and portions of the components connected to the reactor may be maintained in an incubator 116a indicated by the broken lines.
Figure 3 depicts a multihollow fiber reactor 120 having a plurality of hollow fibers 122 in a housing or shell 1~4. The hollow fibers 122 are mounted on manifold discs 126 and 130 which serve to hold the hollow fib~rs in position while allowing access between the hollow fibers 1~2 and ~hambers 132 and 134. Chamber 134 has inlet port 136 while chambex 132 has outlet port 140. Gas inlet conduit 142 connects to g~s manifold 144 which distributes the gas evenly about the periphery of the housing 124. Gas outlet 146 is provided to control the pressure in the reactor 120. The reactor is provided with a septum 150 mounted on side arm 15~. The septum permits the innoculation of the reactor with cells and removal of samples without disturbing the reactor.
In studying the subject reactor, reactors having from 20 to 40 fibers were studied. The asymmetric hollow fibers employed were obtained from Amicon Corporation. The fibers are resistant to acids, alkalies and water-organic solvent mixtures with organic solvent concentrations of up to 50%. The fibers have a relatively dense inner wall which serves as a semipermeable membrane, being approximately 0.1-1.0~ thick. The hollow fibers employed have molecular weight cut-offs for the membranes between lO,OOOdaltons and 60,000daltons. The maximum pore diameter for the upper range of molecular weight cut-off is about O.01~, which is about 1-2 orders of magnitude less than ~he minimum dimension of most microbial cells. The remainder of the wall-thickness I~7822.~
provides support for the inner membrane and is approximately 100-200~ thick, with 70-80% of the volume in the outer region void space. The fiber wall has pore sizes of the order of 1~. The fi~er studied had outer diamet:ers ranging from about 0.5mm to 1.~mm and fiber lengths were about 25cm.
The organism studied was the bacterial strain E.
coli C600 transformed with pBR322. For the purpose of the subject study, the production of ~-lact~mase was studied.
The E. coli strain propagates at extremely high rates, undergoing cell division about every 20-30mins. The transformed ~acteria produce ~-lactamase at a rate approxi-mately 50 times greater than the wild type strain.
The reactor employed is depicted in Figure 2. The cultures were maintained at a temperature of 37C and a pressure of approximately latm. The fiber employed was Amicon PW-60/ ~5cm long, mounted in a 5mm O.D. glass tube.
The culture growth medium was a rich medium containing lOg tryptone, lOg NaCl, and 5g yeast extract per liter of water, pH6.5-7.5. In addition, 20~g/ml of thymine was added. The nutrient medium was saturated with pure oxygen at latm before perfusion through the reactor and humidified oxygen gas at latm was continuously passed through the reactor shell space ~ollowing the inoculation procedure. Besides following the production of ~-lactamase, the cell density in the reactor was also monitored. For the E. coli cultures, cell densities ~ ,. _ of 2 x loL cells/ml of void space in the sponge region were observed. This density corresponds to the situation in which the volume of the cells accounts for 60-70% of the available space within the fiber wall. In conventional fermentation processes where significant attempts have been made to attain hiyh cell densities, the highest densities reported are between 1 x 101 and 1 x lOllcells/ml of suspending medium.
If the productivity of the reactor system is the same per cell, the subject reactor provides a significant reduction in the reactor volume for a given reactor production rate. In a few instances, cell packing densities were ~bserved which were nearly 100% of the available space, with the hollow-fiber culture appearing as a tissue cell mass analoyous to the situation seen in the body where blood capillaries supply the body~s tissue cells.
~ -Lactamase production by E. coli cultures was obtained from dead cells, rather than by excretion, and continued at significant levels for at least three weeks and no fall off in enzyme productivity was obse:rved at the time of termination. The ~-lactamase production r~te, expressed in terms of units enzyme activity/cell-hr was only 1% of khat measured in a comparable batch shaker-flask culture conducted for comparison. ~owever, if the ~-lactamase productivity is expressed in terms of units enzyme activity/volume of reactor hr., the hollow fibex reactor is producing at: a rate of 100 kimes higher than the productivity measured in the shaker-flask culture. Therefore, while the reactor under relatively non-optimum conditions was not performing as well as a shaker-~lask culture on a cell basis, on a reactor volume basis, the culture was approximately two orders of magnitude more productive than tha comparable shaker-flask culture. Enzyme concentrations of 0.2-0.4 units/ml were achieved.
It is evident from the above results, that the subject invention provides a highly efficient compact reac-tor, where extremely high cell densities can be achieved. By providing optimum conditions for diffusing the nutrients into the pores of the hollow fibers, substantially all of the void space of the hollow fiber walls can be filled with cells and rapid and efficient metabolism of substrates in the nutrient medium and the lumen achieved. By virtue of the effective nutrient distribution good viability of the cells is main-tained for long periods of time, so that the reactor main-tains a high efficiency. By recycling, enhanced conversion of substrates can be achieved. Furthermore, relatively few cells pass into the lumen, so that the removal of the cells from the nutrient medium stream can be efficiently achieved.
By employing the subject reactors with microorganisms or other cells, high yields may be obtained of a wide varieky of naturally occurring compoun~s or of enzymatically transformed products.
I ~J~225 Although the foregoing invention has been described in some detail by way of illustration and example for pur-poses of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
relationship and a pressure gauge 42 is mounted on the outlet conduit 40 to provide for monitoring the pressure of the lumen effluent. In addition to providing for the introduc-tion of gas or other materials into the shell space 22, conduits 28 and 30 also provide the opportunity to i~noculate the reactor with microorganisms or cells.
Figure 2 is a diagram of the eguipment used in a nu~ber of tests. The reactor 10a has single fiber 12a which is sealed in the tube 14a by seals 16a and 20a. Pressurized oxygen is provided by gas cylinder 50a, which is connected by lines 52a and 54a to nutrient media reservoir 56a. Pressure regulator 60a mounted in line 52a controls the oxygen pres-sure in line 52a. The oxygen pressure forces the nutrient media in reservoir 56a into line 62a in which is moun-ted three-way valve 64a, the remaining arm being fitted with syringe 66a. Line 62a connects with peristaltic pump 70a which controls ~he flow of the nutrient medium ~hrough line 7~a to inlet port 24a of hollow ~iber 12a. Line 72a has a series of coils 78a to allow for temperature control of the nutrient medium fed to hollow fiber 12a. Side arm 30a of tube 14a is connected by a conduit 74a to shell space sam-pling conduit 76a and hl~idifier 80a. The humidifier 80a is connected by means of co~duit 82a to line 52a to permit humidified oxygen to be introduced into the reactor shell space 22a. Side arm 28a is connected by means of line 84a to three-way valve 86a which serves to pass the effluent from the shell space 22a into sample collection tube 90a or by means of line 92a to shell-space effluent reservoir 94a.
~ 1~822~
g The nutrient media fed into inlet port 24a by means of line ~2a are monitored through line 96a, while the lumen effluent exiting exit port 26a is monitored through line lQOa. Lines 76a, 96a, and lOOa are all connected to line 102a which is connected to a manometer 104a for monitoring the pressure in the reactor. The nutrient medium of the lumen exiting through exit 26a is connected by line 106a to three-way valve llOa which serves to connect the effluent to sample collection tube 112a or lumen effluent reservoir 114a.
For temperature control, the reactor and portions of the components connected to the reactor may be maintained in an incubator 116a indicated by the broken lines.
Figure 3 depicts a multihollow fiber reactor 120 having a plurality of hollow fibers 122 in a housing or shell 1~4. The hollow fibers 122 are mounted on manifold discs 126 and 130 which serve to hold the hollow fib~rs in position while allowing access between the hollow fibers 1~2 and ~hambers 132 and 134. Chamber 134 has inlet port 136 while chambex 132 has outlet port 140. Gas inlet conduit 142 connects to g~s manifold 144 which distributes the gas evenly about the periphery of the housing 124. Gas outlet 146 is provided to control the pressure in the reactor 120. The reactor is provided with a septum 150 mounted on side arm 15~. The septum permits the innoculation of the reactor with cells and removal of samples without disturbing the reactor.
In studying the subject reactor, reactors having from 20 to 40 fibers were studied. The asymmetric hollow fibers employed were obtained from Amicon Corporation. The fibers are resistant to acids, alkalies and water-organic solvent mixtures with organic solvent concentrations of up to 50%. The fibers have a relatively dense inner wall which serves as a semipermeable membrane, being approximately 0.1-1.0~ thick. The hollow fibers employed have molecular weight cut-offs for the membranes between lO,OOOdaltons and 60,000daltons. The maximum pore diameter for the upper range of molecular weight cut-off is about O.01~, which is about 1-2 orders of magnitude less than ~he minimum dimension of most microbial cells. The remainder of the wall-thickness I~7822.~
provides support for the inner membrane and is approximately 100-200~ thick, with 70-80% of the volume in the outer region void space. The fiber wall has pore sizes of the order of 1~. The fi~er studied had outer diamet:ers ranging from about 0.5mm to 1.~mm and fiber lengths were about 25cm.
The organism studied was the bacterial strain E.
coli C600 transformed with pBR322. For the purpose of the subject study, the production of ~-lact~mase was studied.
The E. coli strain propagates at extremely high rates, undergoing cell division about every 20-30mins. The transformed ~acteria produce ~-lactamase at a rate approxi-mately 50 times greater than the wild type strain.
The reactor employed is depicted in Figure 2. The cultures were maintained at a temperature of 37C and a pressure of approximately latm. The fiber employed was Amicon PW-60/ ~5cm long, mounted in a 5mm O.D. glass tube.
The culture growth medium was a rich medium containing lOg tryptone, lOg NaCl, and 5g yeast extract per liter of water, pH6.5-7.5. In addition, 20~g/ml of thymine was added. The nutrient medium was saturated with pure oxygen at latm before perfusion through the reactor and humidified oxygen gas at latm was continuously passed through the reactor shell space ~ollowing the inoculation procedure. Besides following the production of ~-lactamase, the cell density in the reactor was also monitored. For the E. coli cultures, cell densities ~ ,. _ of 2 x loL cells/ml of void space in the sponge region were observed. This density corresponds to the situation in which the volume of the cells accounts for 60-70% of the available space within the fiber wall. In conventional fermentation processes where significant attempts have been made to attain hiyh cell densities, the highest densities reported are between 1 x 101 and 1 x lOllcells/ml of suspending medium.
If the productivity of the reactor system is the same per cell, the subject reactor provides a significant reduction in the reactor volume for a given reactor production rate. In a few instances, cell packing densities were ~bserved which were nearly 100% of the available space, with the hollow-fiber culture appearing as a tissue cell mass analoyous to the situation seen in the body where blood capillaries supply the body~s tissue cells.
~ -Lactamase production by E. coli cultures was obtained from dead cells, rather than by excretion, and continued at significant levels for at least three weeks and no fall off in enzyme productivity was obse:rved at the time of termination. The ~-lactamase production r~te, expressed in terms of units enzyme activity/cell-hr was only 1% of khat measured in a comparable batch shaker-flask culture conducted for comparison. ~owever, if the ~-lactamase productivity is expressed in terms of units enzyme activity/volume of reactor hr., the hollow fibex reactor is producing at: a rate of 100 kimes higher than the productivity measured in the shaker-flask culture. Therefore, while the reactor under relatively non-optimum conditions was not performing as well as a shaker-~lask culture on a cell basis, on a reactor volume basis, the culture was approximately two orders of magnitude more productive than tha comparable shaker-flask culture. Enzyme concentrations of 0.2-0.4 units/ml were achieved.
It is evident from the above results, that the subject invention provides a highly efficient compact reac-tor, where extremely high cell densities can be achieved. By providing optimum conditions for diffusing the nutrients into the pores of the hollow fibers, substantially all of the void space of the hollow fiber walls can be filled with cells and rapid and efficient metabolism of substrates in the nutrient medium and the lumen achieved. By virtue of the effective nutrient distribution good viability of the cells is main-tained for long periods of time, so that the reactor main-tains a high efficiency. By recycling, enhanced conversion of substrates can be achieved. Furthermore, relatively few cells pass into the lumen, so that the removal of the cells from the nutrient medium stream can be efficiently achieved.
By employing the subject reactors with microorganisms or other cells, high yields may be obtained of a wide varieky of naturally occurring compoun~s or of enzymatically transformed products.
I ~J~225 Although the foregoing invention has been described in some detail by way of illustration and example for pur-poses of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
Claims (16)
1. A method for continuously transforming a substrate to a product by microbiological means employing cells in a flow reactor, said flow reactor comprising:
a housing;
at least one hollow fiber in said housing, said hollow fiber having an inlet port and an outlet port and characterized by having a lumen, a porous membrane sur-rounding said lumen and having orifices smaller than said cells, and a spongy supporting wall having pores internally communicating through said orifices with said lumen and externally communicating with the volume enclosed by said housing through openings of a size greater than said cells;
and a nutrient medium pervading said housing;
said method comprising:
growing cells in said housing so as to substan-tially fill said wall pores, while continually passing sub-strate containing nutrient medium into said lumen through said inlet port, whereby nutrients and substrate flow into said pores and substrate is transformed to product by said cells and diffuses thorugh said orifices into said lumen; and continuously removing nutrient medium containing product from said lumen through said exit port.
a housing;
at least one hollow fiber in said housing, said hollow fiber having an inlet port and an outlet port and characterized by having a lumen, a porous membrane sur-rounding said lumen and having orifices smaller than said cells, and a spongy supporting wall having pores internally communicating through said orifices with said lumen and externally communicating with the volume enclosed by said housing through openings of a size greater than said cells;
and a nutrient medium pervading said housing;
said method comprising:
growing cells in said housing so as to substan-tially fill said wall pores, while continually passing sub-strate containing nutrient medium into said lumen through said inlet port, whereby nutrients and substrate flow into said pores and substrate is transformed to product by said cells and diffuses thorugh said orifices into said lumen; and continuously removing nutrient medium containing product from said lumen through said exit port.
2. A method according to claim 1, wherein oxygen is introduced into said housing in the volume surrounding said hollow fiber.
3. A method according to claim 1, wherein said cells are prokaryotic.
4. A method according to claim 3, wherein said product is a polypeptide.
5. A method according to claim 1, wherein said cells are eukaryotic single cells.
6. A method according to claim 5, wherein said cells are yeast cells.
7. A method according to claim 5, wherein said cells are fungi.
8. A method according to claim 1, wherein said cells fill the volume of said pores to greater than about 60%.
9. A microbiological flow reactor comprising:
a housing;
at least one hollow fiber enclosed in said housing, said hollow fiber having an inlet port and an outlet port communicating outside said housing and further characterized by having a lumen; said lumen enclosed by a membrane having orifices smaller than cells to be employed in said micro-biological reactor; and a spongy supporting wall having pores internally communicating through said orifices with said lumen and externally communicating with the volume enclosed by said housing through openings of a size greater than said cells;
a nutrient medium prevading said housing; and cells filling at least 60% of the available volume of said pores.
a housing;
at least one hollow fiber enclosed in said housing, said hollow fiber having an inlet port and an outlet port communicating outside said housing and further characterized by having a lumen; said lumen enclosed by a membrane having orifices smaller than cells to be employed in said micro-biological reactor; and a spongy supporting wall having pores internally communicating through said orifices with said lumen and externally communicating with the volume enclosed by said housing through openings of a size greater than said cells;
a nutrient medium prevading said housing; and cells filling at least 60% of the available volume of said pores.
10. A reactor according to Claim 9, wherein said cells fill at least about 80% of the available volume of said pores.
11. A reactor according to Claim 9, wherein said cells are prokaryotic.
12. A reactor according to Claim 10, wherein said cells axe eukaryotic single cells.
13. A reactor according to Claim 12, wherein said cells are yeast.
14. A reactor according to Claim 12, wherein said cells are fungi.
15. A reactor according to Claim 9, wherein the membrane has a molecular weight cut-off of less than about 200,000.
16. A reactor according to Claim 9, having at least one entry port and at least one exit port to the space in said housing about said hollow fiber.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000402564A CA1178225A (en) | 1982-05-10 | 1982-05-10 | Hollow fiber membrane microbiological reactors |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000402564A CA1178225A (en) | 1982-05-10 | 1982-05-10 | Hollow fiber membrane microbiological reactors |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1178225A true CA1178225A (en) | 1984-11-20 |
Family
ID=4122736
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000402564A Expired CA1178225A (en) | 1982-05-10 | 1982-05-10 | Hollow fiber membrane microbiological reactors |
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| Country | Link |
|---|---|
| CA (1) | CA1178225A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109153954A (en) * | 2016-05-05 | 2019-01-04 | 泰尔茂比司特公司 | Automated production and collection |
-
1982
- 1982-05-10 CA CA000402564A patent/CA1178225A/en not_active Expired
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109153954A (en) * | 2016-05-05 | 2019-01-04 | 泰尔茂比司特公司 | Automated production and collection |
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