Note: Descriptions are shown in the official language in which they were submitted.
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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,
.~
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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.
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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.
* TRADE MARK
22~
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.
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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
1 1 78225
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.
