U.S. patent application number 10/771958 was filed with the patent office on 2004-08-12 for multi-sample fermentor and method of using same.
This patent application is currently assigned to IRM, LLC. Invention is credited to Downs, Robert Charles, Lesley, Scott Allan, Mainquist, James Kevin, McMullan, Daniel Terence, Meyer, Andrew J., Nasoff, Marc.
Application Number | 20040157322 10/771958 |
Document ID | / |
Family ID | 25120024 |
Filed Date | 2004-08-12 |
United States Patent
Application |
20040157322 |
Kind Code |
A1 |
Downs, Robert Charles ; et
al. |
August 12, 2004 |
Multi-sample fermentor and method of using same
Abstract
A fermentation apparatus is constructed to produce a known and
repeatable amount of untainted fermentation product using multiple
fermentation vessels. To facilitate further processing compatible
with other product processing steps, the fermentation apparatus has
an array of sample vessels arranged in a container frame. The
container frame is configured to hold the sample vessels during
fermentation and to transport the vessel array to or from another
processing station. Corresponding to the number of sample vessels
in the sample vessel array, a cannula array is configured such that
each cannula may be placed inside a sample vessel. The cannula
array is attached to a gas distributor that delivers oxygen and/or
one or more other gases from a gas source through the cannula into
the sample vessel. Because the fermentation volume for each
individual sample vessel is smaller than a bulk fermentation
apparatus, the fermentation product yields are predictable and cell
growth rates can be effectively optimized.
Inventors: |
Downs, Robert Charles; (La
Jolla, CA) ; Lesley, Scott Allan; (San Diego, CA)
; Mainquist, James Kevin; (San Diego, CA) ;
McMullan, Daniel Terence; (San Diego, CA) ; Meyer,
Andrew J.; (San Diego, CA) ; Nasoff, Marc;
(San Diego, CA) |
Correspondence
Address: |
QUINE INTELLECTUAL PROPERTY LAW GROUP, P.C.
P O BOX 458
ALAMEDA
CA
94501
US
|
Assignee: |
IRM, LLC
|
Family ID: |
25120024 |
Appl. No.: |
10/771958 |
Filed: |
February 3, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10771958 |
Feb 3, 2004 |
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10071842 |
Feb 8, 2002 |
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6723555 |
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10071842 |
Feb 8, 2002 |
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09780591 |
Feb 8, 2001 |
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6635441 |
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Current U.S.
Class: |
506/26 ;
435/295.1; 506/40 |
Current CPC
Class: |
C12M 23/48 20130101;
B01J 2219/00495 20130101; Y10S 435/818 20130101; C12M 23/08
20130101; B01J 2219/0072 20130101; C12M 41/18 20130101; B01J
2219/00283 20130101; C12M 23/42 20130101; B01J 2219/0074 20130101;
B01J 2219/00493 20130101; Y10S 435/813 20130101; B01J 2219/00585
20130101; C12M 23/58 20130101; Y10S 435/808 20130101; B01L 3/0289
20130101; C12M 23/40 20130101; C40B 60/14 20130101; Y10S 435/802
20130101; B01J 2219/00418 20130101; B01J 2219/00308 20130101; B01L
3/5085 20130101; C12M 29/00 20130101 |
Class at
Publication: |
435/295.1 |
International
Class: |
C12M 001/02 |
Claims
What is claimed is:
1. A fermentation apparatus comprising: (a) a container frame
configured to contain a plurality of sample vessels; and, (b) a gas
distribution arrangement that is configured to provide gas to a
plurality of sample vessels when the sample vessels are positioned
in the container frame.
2. The fermentation apparatus of claim 1, wherein the gas
distribution arrangement comprises a gas inlet configured to
deliver gas to a plurality of cannulas, which cannulas are
configured to provide gas to the sample vessels when the sample
vessels are positioned in the container frame.
3. The fermentation apparatus of claim 1, wherein the gas
distribution arrangement comprises: (a) a dispensing plate that
comprises a top portion and a bottom portion, wherein the bottom
portion and the top portion are joined together such that a hollow
space exists between the top portion and the bottom portion; (b) an
array of sample vessel areas located in a bottom surface of the
bottom portion, which sample vessel areas each comprise a recess
and are positioned to correspond to an array of sample vessels; (c)
an array of cannulas that are in fluid communication with the
hollow space and protrude from a bottom surface of the dispensing
plate through the sample vessel areas; and (d) a gas inlet in fluid
communication with the hollow space for delivering gas into a
plurality of sample vessels via the cannulas during
fermentation.
4. The fermentation apparatus of claim 3, wherein each of the
cannulas comprises a plurality of passages.
5. The fermentation apparatus of claim 4, wherein each of the
cannulas comprises at least three passages.
6. The fermentation apparatus of claim 3, wherein the gas
distribution arrangement is configured to allow delivery of one or
more reagent to the sample vessels.
7. The fermentation apparatus of claim 1, wherein the container
frame is configured to contain an array of sample vessels.
8. The fermentation apparatus of claim 7, wherein the container
frame is configured to contain an 8 by 12 array of sample
vessels.
9. The fermentation apparatus of claim 7, wherein the container
frame is configured to contain at least 96 sample vessels.
10. The fermentation apparatus of claim 9, wherein the container
frame is configured to contain 96, 384, or 1536 sample vessels.
11. The fermentation apparatus of claim 1, wherein the container
frame is transportable.
12. The fermentation apparatus of claim 11, wherein the container
frame is configured for transport to a post-fermentation processing
station.
13. The fermentation apparatus of claim 1, wherein the container
frame is configured for placement within a temperature controlled
area, wherein a temperature controller is coupled to the container
frame and/or to the plurality of sample vessels.
14. The fermentation apparatus of claim 13, wherein the temperature
controlled area comprises a water bath or a temperature controlled
room.
15. The fermentation apparatus of claim 1, wherein the container
frame is autoclavable.
16. The fermentation apparatus of claim 1, wherein the container
frame and the gas distribution arrangement are autoclavable.
17. The fermentation apparatus of claim 1, further comprising a
plurality of sample vessels.
18. The fermentation apparatus of claim 17, wherein each of the
sample vessels has a volume of 50 to 100 ml.
19. The fermentation apparatus of claim 17, wherein each sample
vessel comprises a sample.
20. The fermentation apparatus of claim 19, wherein each sample is
80 mls or less.
21. The fermentation apparatus of claim 19, wherein the samples
each have substantially the same composition.
22. The fermentation apparatus of claim 19, wherein the samples
each have a different composition.
23. The fermentation apparatus of claim 17, wherein the sample
vessels comprise glass, plastic, metal, polycarbonate, and/or
ceramic.
24. The fermentation apparatus of claim 17, wherein one or more of
the sample vessels comprises a vent.
25. The fermentation apparatus of claim 17, further comprising a
sensor in contact with one or more of the samples in the sample
vessels.
26. The fermentation apparatus of claim 1, wherein the gas
distribution arrangement comprises a gas source which gas source
provides oxygen or a mixture of oxygen and at least one other gas
to each sample vessel during operation of the apparatus.
27. The fermentation apparatus of claim 1, further comprising a
process controller operably coupled to the gas distribution
arrangement.
28. The fermentation apparatus of claim 1, further comprising a
dispenser for dispensing one or more reagents into the plurality of
sample vessels.
29. The fermentation apparatus of claim 28, wherein the dispenser
is configured to dispense the reagents into the plurality of sample
vessels via a plurality of apertures that correspond to the sample
vessels.
30. A fermentor head for multiple sample fermentation, the
fermentor head comprising: (a) a dispensing plate that comprises a
top portion and a bottom portion, wherein the bottom portion and
the top portion are joined together such that a hollow space exists
between the top portion and the bottom portion; (b) an array of
sample vessel areas located in a bottom surface of the bottom
portion, which sample vessel areas each comprise a recess and are
positioned to correspond to an array of sample vessels; (c) an
array of cannulas that are in fluid communication with the hollow
space and protrude from a bottom surface of the dispensing plate
through the sample vessel areas; and (e) a gas inlet in fluid
communication with the hollow space for delivering gas into a
plurality of sample vessels via the cannulas during
fermentation.
31. The fermentor head of claim 30, wherein the dispensing plate
further comprises an array of apertures for accessing samples
during fermentation.
32. The fermentor head of claim. 30, wherein the array of cannulas
comprises an 8 by 12 array.
33. The fermentor head of claim 30, wherein the array of cannulas
comprises at least 96 cannulas.
34. The fermentor head of claim 33, wherein the array of cannulas
comprises 96, 384, or 1536 cannulas.
35. The fermentor head of claim 30, wherein the cannulas extend 15
to 16 centimeters below the bottom surface of the first plate.
36. The fermentor head of claim 30, wherein the sample vessels have
a volume of 50 to 200 ml.
37. The fermentor head of claim 30, wherein the sample vessels have
a volume of 50 to 100 ml.
38. The fermentor head of claim 30, wherein the cannulas deliver
gas adjacent to a bottom of the sample vessels.
39. The fermentor head of claim 30, wherein the gas inlet delivers
oxygen or nitrogen into the interior space of the second plate,
thereby providing oxygen or nitrogen to the sample vessels via the
cannulas during fermentation.
40. The fermentor head of claim 30, wherein each of the cannula
comprises at least three passages.
41. The fermentor head of claim 30, wherein the cannulas are
adapted to deliver gas, deliver fluid, or aspirate fluid from the
sample vessels during fermentation.
42. A method of fermenting a plurality of samples, the method
comprising: (a) providing a plurality of sample vessels in a
container frame, wherein each of the sample vessels contains a
sample; (b) fermenting the samples in the plurality of sample
vessels, which fermenting comprises simultaneously delivering gas
to each of the sample vessels via a plurality of cannulas
associated with the sample vessels.
43. The method of claim 42, wherein each sample has a volume of
less than 100 ml.
44. The method of claim 42, further comprising pre-processing or
post-processing the samples in the sample vessels.
45. The method of claim 44, wherein the pre-processing or
post-processing is performed in a different location than step
(b).
46. The method according to claim 44, wherein the pre-processing
and/or post-processing are performed robotically.
47. The method according to claim 44, wherein the pre-processing
and/or post-processing comprises centrifugation, aspiration, or
dispensing of one or more reagent.
48. The method of claim 42, wherein delivering gas comprises
delivering oxygen, air, and/or, nitrogen to the samples.
49. The method of claim 42, wherein delivering gas comprises
delivering air and oxygen to the samples over a period of time,
during which period of time, the ratio of air to oxygen
changes.
50. The method of claim 49, wherein the ratio changes linearly over
time or in a stepwise manner over time.
51. The method of claim 42, further comprising configuring the
sample vessels into a rectangular array, a honeycomb array, or a
linear array within the container frame.
52. The method of claim 42, further comprising transferring the
sample vessels into a centrifuge rotor.
53. The method according to claim 42, further comprising detecting
one or more fermentation conditions with a sensor coupled to one or
more sample vessels and adjusting the fermentation conditions in
the sample vessels.
54. The method according to claim 53, comprising detecting and
adjusting at pre-determined time intervals.
55. The method according to claim 53, wherein the adjusting the
fermentation conditions comprises adding a feed solution to the
sample vessels.
56. The method according to claim 53, wherein the detecting
comprises: measuring a pH of one of the samples; measuring a redox
potential of one of the samples; measuring an optical density of
one of the samples; and/or measuring a light emission from one of
the samples.
57. The method of claim 42, further comprising autoclaving the
sample vessels in the container frame.
58. The method of claim 57, further comprising autoclaving the
plurality of cannulas simultaneously with the sample vessels in the
container frame.
59. A method of fermenting a plurality of samples, the method
comprising: (a) positioning a plurality of sample vessels into a
transportable container frame, which container frame maintains the
sample vessels in an array; (b) placing the plurality of samples
into the plurality of sample vessels; (c) attaching a fermentor
head to the container frame, which fermentor head comprises an
array of cannulas, wherein the array of cannulas corresponds to the
array of sample vessels and is inserted into the sample vessels;
(d) fermenting the samples in the sample vessels, which fermenting
comprising simultaneously delivering a gas to the samples via the
array of cannulas.
60. The method of claim 59, wherein step (c) is performed prior to
step (b).
61. The method of claim 59, wherein step (b) is performed prior to
step (a).
62. The method of claim 59, wherein delivering a gas comprising
delivering oxygen, nitrogen, and/or air to the sample vessels
during step (d).
63. The method of claim 59, wherein step (d) is an anaerobic
fermentation comprising delivering an inert gas to maintain
anaerobic fermentation conditions in the sample vessels.
64. The method of claim 59, wherein the sample vessels each have a
volume between 50 and 200 ml.
65. The method of claim 59, wherein the sample vessels have a
volume between 80 and 100 ml.
66. The method of claim 59, wherein each sample has a volume less
than 200 ml.
67. The method of claim 59, wherein each sample has a volume of
less than 100 ml.
68. The method of claim 69, comprising robotically transporting the
sample vessels in the container frame.
69. The method of claim 59, further comprising simultaneously
transporting the plurality of sample vessels in the container frame
to a processing station.
70. The method of claim 69, wherein the processing station
comprises a centrifuge, an aspirator, and/or a dispenser.
71. The method of claim 70, wherein the sample container is
compatible with the centrifuge.
72. The method of claim 70, wherein the sample vessels are
compatible with the centrifuge.
73. The method of claim 70, further comprising removing the sample
vessels from the container frame and introducing the sample vessels
into the centrifuge.
74. The method of claim 70, wherein the aspirator comprises an
aspirator head which corresponds to the array of sample vessels
within the container frame, the method further including operably
attaching the aspirator head to the sample vessels and
simultaneously aspirating the samples within the sample
vessels.
75. The method of claim 70, the method further dispensing one or
more materials into the sample vessels.
76. The method of claim 70, wherein the dispenser comprises a
dispensing head corresponding to the array of sample vessels, the
method further including operably attaching the dispenser head to
the sample vessels and simultaneously dispensing one or more
materials into the sample vessels.
77. The method of claim 59, wherein the array comprises an 8 by 12
array.
78. The method of claim 59, wherein the array comprises 96, 384, or
1536 sample vessels.
79. The method of claim 59, further comprising positioning the
sample vessels in the container frame in a water bath during the
fermenting step in order to control the fermentation
temperature.
80. A method of processing a plurality of fermentation samples, the
method comprising: (a) fermenting a plurality of fermentation
samples in a plurality of sample vessels, resulting in a plurality
of fermented samples; (b) robotically transporting the sample
vessels containing the fermented samples to a centrifuge head; and
(c) centrifuging the fermented samples in the same sample vessels
in which the fermentation was performed.
81. The method of claim 80, the method further including isolating
a supernatant from the sample vessels after centrifuging the
fermentation samples.
82. The method of claim 80, wherein at least 4 sample vessels are
robotically transported to the centrifuge head at the same
time.
83. The method of claim 80, wherein at least 10 sample vessels are
robotically transported to the centrifuge head at the same
time.
84. The method of claim 80, wherein each sample vessel contains
less than 100 mL of fermentation sample.
85. The method of claim 80, wherein the plurality of sample vessels
are held in an 8 by 12 array.
Description
COPYRIGHT NOTIFICATION
[0001] Pursuant to 37 C.F.R. 1.71(e), a portion of this patent
document contains material which is subject to copyright
protection. The copyright owner has no objection to the facsimile
reproduction by anyone of the patent document or the patent
disclosure, as it appears in the Patent and Trademark Office patent
file or records, but otherwise reserves all copyright rights
whatsoever.
CROSS REFERENCE TO RELATED APPLICATIONS
[0002] Pursuant to 35 U.S.C. .sctn. 120, and any other applicable
statute or rule, the present application is a continuation-in-part
of and claims benefit of and priority to U.S. patent application
Ser. No. 09/780,591, filed Feb. 8, 2001 entitled "Multi-Sample
Fermentor and Method of Using Same," the disclosure of which is
incorporated herein by reference in its entirety for all
purposes.
BACKGROUND OF THE INVENTION
[0003] Fermentation is a key technology in many fields and
industries and is performed both on a mass production scale and on
an experimental, bench top scale. For example, fermentation systems
are used for the production of a large number of products such as
antibiotics, vaccines, synthetic biopolymers, synthetic amino
acids, and proteins. Fermentation technology is integral in the
production of recombinant proteins using biological organisms, such
as E. coli, and many other cell cultures. For example, production
of commercial pharmaceuticals such as recombinant insulin (Eli
Lilly), erythropoietin (Amgen), and interferon (Roche) all involve
fermentation as an essential step.
[0004] In addition, the recent identification of the tens of
thousands of genes comprising the human genome highlight an
important use of fermentation, namely the production of the
proteins encoded by those genes. The determination of each gene's
function is of paramount importance and therefore, the proteins
encoded by those genes must be produced, e.g., by fermentation
methods. Because each gene encodes at least one protein, tens of
thousands of proteins must be produced and isolated. However,
fermentation and isolation of the resulting protein products
typcially requires several labor intensive and time-consuming
procedures. Fermentation systems that can produce tens of thousands
of different proteins, e.g., in amounts sufficient for analysis are
therefore needed. An additional advantage would be fermentation
systems that are amenable to high throughput processes and the
microtiter plate format used in many biotechnolgy applications.
[0005] Although, rapid advances in biotechnology have enabled the
development of high throughput alternatives to traditional
laboratory bench top processes, fermentation methods have not been
amenable to automation. For example, limits in current fermentation
technology prevent the uninterrupted processing flow that
characterizes automated high throughput systems. Existing
fermentation systems typically involve multiple handling steps by
either a batch processing method or a continuous processing
method.
[0006] Fermentations are typically carried out in batch mode or
continuous mode. Batch mode processes are those in which a
fermentor is filled with a medium in which cells are grown and the
fermentation is allowed to proceed with the entire contents removed
from the fermentor at the end for downstream or post-processing.
The fermentor is then cleaned, re-filled, and inoculated for the
fermentation process to be performed again. For example, current
production scale batch processes involve first fermenting in large
scale, bulk fermentation vessels, then processing the fermentation
medium to isolate the desired fermentation product, followed by
transferring this product into the production stream for further
processing, and finally cleaning the fermentation apparatus for the
next batch. In a large scale batch culture, it is generally
necessary to provide a high initial concentration of nutrients in
order to sustain cell growth over an extended time. As a result,
substrate inhibition may occur in the early stages of cell growth
and then may be followed by a nutrient deficiency in the late
stages of fermentation. These disadvantages result in sub-optimal
cell growth rates and fermentation yields. Another disadvantage of
this method lies in the need to individually dispense the
fermentation products from the bulk fermentation apparatus into
separate sample vessels for further processing. Thus, by producing
the fermentation product on a bulk scale, the fermentation product
is not immediately available for automated processing. Further
disadvantages include the decreased efficiency of both transferring
the material to another sample vessel, as well as cleaning and
sterilizing the fermentation apparatus for the next batch. These
disadvantages result in increased production costs, inefficient
production times and decreased yields.
[0007] Continuous batch processes involve siphoning off the
fermentation product from the bulk fermentation vessel and
continuously adding nutrients to the fermentation medium according
to a calculated exponential growth curve. This curve, however, is
merely an approximation that does not accurately predict cell
growth in large, industrial scale quantities of fermentation
medium. Consequently, due to the unpredictable nature of large
scale fermentation environments, experienced personnel are required
to monitor the feeding rate very closely. Changes in the
fermentation environment may result in either poisoned fermentation
products being siphoned off into the production stream or
sub-optimal production yields due to starved fermentation mediums.
As a further disadvantage, unpredictable fermentation product
yields affect the accuracy of subsequent processing steps. For
example, when the fermentation yield decreases, the amount of
aspirating, the amount of reagent dispensed, or the centrifuge time
is no longer optimized, or even predictable. Frequent or continuous
monitoring of the fermentation process and adjustment of the
fermentation conditions is often not practicable or efficient in a
production scale process.
[0008] Neither of the current processes provides an efficient,
automated production scale fermentation. However, fermentation
remains a key processing step in a number of industries,
particularly in biotechnology industries, and thus a need exists
for incorporating fermentation processes into automated high
throughput systems. A process that produces a precise, known, and
repeatable amount of untainted fermentation product with limited
human interaction or sample vessel transfer is essential to
integrating fermentation into modern production processes. The
present invention meets these as well as other needs that will be
apparent upon review of the following detailed description and
figures.
SUMMARY OF THE INVENTION
[0009] The present invention provides methods and apparatuses for
simultaneously fermenting a plurality of samples, e.g., small
samples in an 8 by 12 array. For example, the present invention
provides a fermentation apparatus comprising a container frame
configured to contain a plurality of sample vessels and a gas
distribution arrangement coupled to the container frame. The
fermentor provides for fermentation of large numbers of samples,
e.g., to produce a large number of proteins. Alternatively, the
fermentors of the invention provide a more efficient route for
production scale fermentations.
[0010] In one aspect the invention provides a container frame
configured to contain a plurality of sample vessels, e.g., in an
array; and, a gas distribution arrangement configured to provide
gas to a plurality of sample vessels, e.g., when the sample vessels
are positioned in the container frame. The container frame is
typically configured to contain an array of sample vessels, e.g.,
an 8 by 12 array, e.g., holding at least about 96, 384, or 1536
samples. The gas distribution arrangement typically comprises a gas
inlet configured to deliver gas to a plurality of cannulas, which
are configured to provide gas to the sample vessels.
[0011] In one embodiment, the container frame is a transportable
container frame, e.g., configured for transport to a
post-fermentation processing station. In addition, the container
frame is optionally configured for placement within a temperature
controlled area, e.g., water bath or a temperature controlled room,
wherein a temperature controller is coupled to the container frame
and/or to one or more sample vessels within the container
frame.
[0012] In other embodiments, the container frame is autoclavable.
For example, the container frame is autoclavable on its own or in
combination with the gas distribution arrangement and/or the sample
vessels.
[0013] The sample vessels typically comprise glass, plastic, metal,
polycarbonate, ceramic, or the like. Each sample vessel typically
has a volume of about 50 to 100 ml, e.g., and is used to hold a
sample comprising less than about 80 mls, more typically about 65
mls. The samples in the plurality of sample vessels each have
substantially the same composition or different compositions, e.g.,
to produce a large quantity of a single protein, or to produce
multiple proteins simultaneously.
[0014] In other embodiments, the sample vessels optionally comprise
a vent, e.g., for releasing built up pressure during fermentation.
Sensors are also optionally placed in contact with one or more of
the samples in the sample vessels, e.g., for monitoring
temperature, pH, and the like.
[0015] In one embodiment, the gas distribution arrangement
comprises a dispensing plate, an array of sample vessel areas, an
array of cannulas, and a gas inlet. The dispensing plate typically
comprises a top portion and a bottom portion that are joined
together such that a hollow space exists between them. The array of
sample vessel areas is typically located in a bottom surface of the
bottom portion. Each sample vessel area comprises a recess and is
positioned to correspond to the array of sample vessels. The array
of cannulas are typically in fluid communication with the hollow
space and protrude from a bottom surface of the dispensing plate
through the sample vessel areas, e.g., to provide gas flow to the
sample vessels. In some embodiments, the cannulas comprises a
plurality of passages, e.g., at least three passages. The gas inlet
is typically in fluid communication with the hollow space for
delivering gas into a plurality of sample vessels via the cannulas
during fermentation. For example, the gas distribution arrangement
in some embodiments comprises a gas source that provides oxygen or
a mixture of oxygen and at least one other gas to each sample
vessel during operation of the apparatus.
[0016] In other embodiments, the gas distribution arrangement is
optionally configured to allow delivery of one or more reagents to
the sample vessels. For example, a dispenser is optionally coupled
to the gas distribution arrangement, e.g., for dispensing one or
more reagents into the plurality of sample vessels. The dispenser
is typically configured to dispense reagents into the plurality of
sample vessels, e.g., via a plurality of apertures corresponding to
the sample vessels.
[0017] In addition, a process controller is operably coupled to the
gas distribution arrangement, e.g., for controlling and/or
monitoring gas flow to the plurality of sample being fermented.
[0018] In another aspect, the present invention provides a
fermentor head for multiple sample fermentation. A typical
fermentor head comprises a dispensing plate that comprises a top
portion and a bottom portion, an array of sample vessels areas, an
array of cannulas and a gas inlet. The bottom portion and the top
portion of the dispensing plate are joined together such that a
hollow space exists between them. The array of sample vessel areas
is typically located in a bottom surface of the bottom portion of
the dispensing plate, which sample vessel areas each comprise a
recess and are positioned to correspond to an array of sample
vessels. The array of cannulas are typically in fluid communication
with the hollow space and protrude from a bottom surface of the
dispensing plate through the sample vessel areas, e.g., 15 to 16
cm; with the gas inlet in fluid communication with the hollow space
for delivering gas into a plurality of sample vessels via the
cannulas during fermentation. Typically, the cannulas deliver gas
adjacent to a bottom of the sample vessels. In some embodiments,
the dispensing plate further comprises an array of apertures for
accessing samples during fermentation. Alternatively, the cannulas
are adapted to deliver gas, deliver fluid, or aspirate fluid from
the sample vessels during fermentation. The vessels and samples
used with the fermentor head typically correspond to those
described above.
[0019] In another aspect, the present invention provides a method
of fermenting a plurality of samples. The method typically
comprises providing a plurality of sample vessels in a container
frame, wherein each of the sample vessels contains a sample. The
samples in the plurality of sample vessels are typically fermented,
which fermenting comprises simultaneously delivering gas, e.g.,
oxygen, air, and/or, nitrogen, to each of the sample vessels via a
plurality of cannulas associated with the sample vessels. Each
sample typically has a volume of less than 100 ml, e.g., using
sample vessels and a container frame as described above. In some
embodiments, delivering gas to the samples comprises delivering air
and oxygen to the samples over a period of time, during which
period of time, the ratio of air to oxygen changes, e.g., linearly
over time or in a stepwise manner over time.
[0020] In some embodiments, the methods further comprise detecting
one or more fermentation conditions with a sensor coupled to one or
more sample vessels and adjusting the fermentation conditions in
the sample vessels, e.g., at pre-determined time intervals. For
example, adjusting the fermentation conditions optionally comprises
adding a feed solution to the sample vessels. Detecting optionally
comprises measuring a pH of one of the samples; measuring a redox
potential of one of the samples; measuring an optical density of
one of the samples; and/or measuring a light emission from one of
the samples.
[0021] In some embodiments, the methods further comprise
pre-processing or post-processing the samples in the same set of
sample vessels, e.g., in the same or a different location as the
fermentation step. In some embodiments, the pre-processing and/or
post-processing are performed robotically. Pre-processing and/or
post-processing steps include, but are not limited to,
centrifugation, aspiration, and/or dispensing of one or more
reagent. For example, the methods optionally comprise transferring
the sample vessels into a centrifuge rotor after fermentation or
autoclaving the sample vessels, e.g., in the container frame prior
to fermentation. In addition, the cannulas are also optionally
autoclavable with the container frame.
[0022] In another aspect, the methods of the invention comprise
positioning a plurality of sample vessels into a transportable
container frame, which container frame maintains the sample vessels
in an array. The plurality of samples is optionally placed into the
plurality of sample vessels, e.g., before or after the vessels are
positioned in the frame. A fermentor head is typically attached to
the container frame, e.g., prior to or after the samples have been
added to the vessels. The fermentor head typically comprises an
array of cannulas, e.g., as described above. The cannulas typically
correspond to the array of sample vessels and are inserted into the
sample vessels when the fermentor head is attached. The samples in
the sample vessels are then fermented, e.g., by simultaneously
delivering a gas, e.g., oxygen, nitrogen, and/or air, to the
samples via the array of cannulas. In some embodiments, the
fermentation is an anaerobic fermentation comprising delivering an
inert gas to maintain anaerobic fermentation conditions in the
sample vessels. The methods optionally comprise robotic steps
pre-processing steps, and/or post processing steps as described
above. For example, the sample vessels and/or the sample container
used in the above methods are optionally configured to be
compatible with a centrifuge, wherein the method further comprises
transporting the container frame and/or sample vessels to the
centrifuge for centrifugation.
[0023] In some embodiments, the methods comprise transportation to
an aspirator or dispenser, wherein the aspirator typically
comprises an aspirator head which corresponds to the array of
sample vessels within the container frame, in which case, the
method further including operably attaching the aspirator head to
the sample vessels and simultaneously aspirating the samples within
the sample vessels. In other embodiments, a dispensing step is
included, wherein the dispenser comprises a dispensing head
corresponding to the array of sample vessels and the method further
includes operably attaching the dispenser head to the sample
vessels and simultaneously dispensing one or more materials into
the sample vessels.
[0024] In another embodiment, the present invention provides a
method of processing a plurality of fermentation samples. The
method comprises fermenting a plurality of fermentation samples in
a plurality of sample vessels, resulting in a plurality of
fermented samples; robotically transporting the sample vessels
containing the fermented samples to a centrifuge head; and
centrifuging the fermented samples in the same sample vessels in
which the fermentation was performed. For example, about 4 to about
10 sample vessels are optionally robotically transported to the
centrifuge head at the same time. The method also optionally
includes isolating a supernatant from the sample vessels after
centrifuging the fermentation samples.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1 is a schematic showing a perspective view of a
fermentation apparatus in accordance with the present
invention.
[0026] FIG. 2 is a schematic showing a top view of a fermentation
apparatus in accordance with the present invention.
[0027] FIG. 3 is a schematic illustrating a perspective view of an
individual fermentation sample vessel in accordance with the
present invention.
[0028] FIG. 4 is a block diagram of a fermentation method in
accordance with the present invention.
[0029] FIG. 5 is a block diagram showing the use of a fermentation
system within a multiple process procedure in accordance with the
present invention.
[0030] FIG. 6 is a schematic illustrating a bottom view of a gas
arrangement in accordance with the present invention.
[0031] FIG. 7 is an automated fermentation assembly in accordance
with the present invention.
[0032] FIG. 8 is a cross sectional view of a cannula in accordance
with the present invention.
[0033] FIG. 9 is schematic showing a bottom view of a sample vessel
area of a dispensing plate shown in FIG. 6 in accordance with the
present invention.
[0034] FIG. 10 is a schematic showing a cross sectional view of the
sample vessel area shown in FIG. 9 taken along the line E-E in
accordance with the present invention.
[0035] FIG. 11 is a schematic showing a cross sectional view of the
sample vessel area shown in FIG. 9 taken along line F-F in
accordance with the present invention.
[0036] FIG. 12 is a schematic showing a perspective view of a
fermentation sample vessel employing a dispensing plate in
accordance with the present invention.
[0037] FIG. 13 is a schematic drawing that illustrates a container
frame for maintaining a plurality of sample vessels in an array
configuration.
[0038] FIG. 14 is a schematic drawing that illustrates the
container frame of FIG. 13 coupled to a gas distribution
arrangement.
[0039] FIG. 15 is a schematic drawing that illustrates the
container frame of FIG. 13 coupled to an alternative gas
distribution arrangement configured for liquid additions.
[0040] FIG. 16 is a schematic drawing that illustrates the gas
distribution manifold with a liquid addition capacity of FIG.
15.
[0041] FIG. 17 is a schematic drawing that illustrating a
cross-sectional view taken along line A-A of FIG. 16.
[0042] FIG. 18 is a schematic drawing that illustrates a bottom
view of gas distribution arrangement as shown in FIG. 14.
[0043] FIG. 19 is a detail illustration from FIG. 18.
[0044] FIG. 20 is a schematic drawing illustrating a
cross-sectional view of a gas distribution arrangement including
top and bottom plates taken along line B-B of FIG. 19.
[0045] FIG. 21 is a schematic drawing that provides a side view of
the gas distribution arrangement as shown in FIG. 14.
DETAILED DISCUSSION OF THE INVENTION
[0046] The present invention provides a fermentation apparatus and
methods of fermentation. The fermentors and methods presented
herein provide production scale fermentation, e.g., automated high
throughput fermentation, as described below. For example, the
present invention provides a multi-sample fermentor comprising a
transportable container frame. The fermentor is configured to
simultaneously ferment a plurality of samples held in an array of
sample vessels within a container frame. The sample vessels provide
relatively small volume batch fermentations, e.g., about 50 ml to
80 ml of sample in each sample vessel, more typically 65 ml. In
addition, the transportable container frame provides a high
throughput aspect to the system that has been absent in previous
fermentation systems. The container frame is used to provide
processing, e.g., upstream or downstream processing in the same
sample vessels.
[0047] The present invention provides a novel fermentor apparatus
that allows batch mode fermentation using a plurality of small
samples. For example, small sample sizes overcome the disadvantage
of sub-optimal growth rates and yields that exist in large batch
mode processes. In addition, the present system eliminates the need
for sample handling for post-fermentation processing. The sample
vessels in the present invention are used directly in any
post-processing steps, which eliminates many cleaning and
sterilizing steps as well, thereby providing a less expensive, more
efficient, and faster fermentation process.
[0048] The present invention also overcomes the disadvantages of
the continuous feed systems, e.g., with the small sample sizes
used. For example, the estimated growth curves used in large scale
continuous feed processes are unnecessary in the present invention.
Therefore, the unpredictable results and frequent monitoring of
continuous feed processes are not a problem in the present
invention.
[0049] The present invention provides a fermentation apparatus that
solves the above problems, e.g., by using small sample sizes and
fermenting the multiple samples simultaneously. By simultaneously
performing multiple fermentations, e.g., small scale fermentations,
in batch mode, optimal mixing is achieved, optimal temperature and
pH can be maintained as well as many other advantages that will be
apparent upon further reading of the present description.
I. A multi-sample Fermentation Apparatus
[0050] The present invention provides a multi-sample fermentation
apparatus. Typically, the apparatuses of the invention comprise a
sample holder or container frame and a gas distribution system. For
example, in one embodiment, a container frame is used to hold
and/or transport an array of sample vessels for fermentation. A
fermentor head, e.g., comprising an array of cannulas corresponding
to the array of sample vessels, is coupled, e.g., directly, to the
container frame and/or sample vessels. Gas is distributed into the
multiple sample vessels via the cannulas and fermentor head
providing multi-sample fermentation. Various components of the
apparatuses are described in more detail below followed by methods
of using the apparatuses and example fermentors.
[0051] A Container Frame is used to Hold a Plurality of Sample
Vessels
[0052] A "container frame" as used herein refers to an arrangement
that holds and/or maintains a plurality of sample vessels in a
desired arrangement. Typically, the container frames of the
invention are transportable and autoclavable. In addition, they
typically have no movable parts. A transportable container frame is
one that is easily transported or moved while holding the sample
vessels in the desired arrangement. For example, a container frame
of the invention optionally has handles for transportation to a
processing station, e.g., after fermentation is complete. An
autoclavable container frame is one that can be placed directly in
an autoclave for sterilization, e.g., including the sample vessels
and samples if desired.
[0053] By using a transportable container frame, the entire array
of sample vessels is optionally transported to and from one
fermentation processing station to another processing station in a
multiple process production. For example, a transportable container
frame is optionally used to transport an array of sample vessels
into a temperature controlled area such as a water bath, e.g., a
water bath controlled by a temperature controller and temperature
coil immersed in the water bath. Other forms of temperature control
are also optionally used, such as temperature controlled gel baths,
ovens, glove boxes, or air chambers.
[0054] Typically, the container frame maintains the sample vessels
in an array, e.g., a rectangular array. In an embodiment shown in
FIG. 1, individual sample vessels 15 are configured in a
rectangular array, but the array is optionally configured in any
physical construct that is conducive to fermentation or that is
compatible with other processing steps. For example, a honeycomb,
circular, triangular, or linear configuration may be more efficient
in other contemplated applications of the present invention.
[0055] The container frames of the present invention typically have
a plurality of placement wells for positioning the plurality of
sample vessels, e.g., in an array. For example, the placement wells
optionally comprise indentations in the bottom of a container
frame, into which sample tubes are optionally placed. In addition
to the indentations or wells in the bottom of the container frame,
the container frames optionally include an upper portion, e.g., for
supporting the tops of the sample tubes and maintaining their
position. Example container frames are shown in FIGS. 1 (container
frame 250) and FIG. 13 (container frame 1300).
[0056] For example, the bottom of each individual sample vessel is
typically positioned within a placement well, e.g., placement well
257 in FIG. 1 or placement well 1350 in FIG. 13. The array of
placement wells preferably mirrors the configuration of the sample
vessel array and is embedded in the transportable container frame.
Placement wells may, however, be arranged in alternative
configurations. For example, placement wells may be arranged as
linear troughs, each holding a row of sample vessels. In another
embodiment, placement wells are absent from the transportable
container frame. For example, the container frame optionally has a
solid bottom surface with no indentations or wells. The sample
vessels are then positioned in the frame, e.g., tightly packed
against the sides of the frame to maintain the array
configuration.
[0057] Sample vessels of the present invention typically comprise
test tubes, other sample tubes, jars, flasks or any other container
for holding a sample. Typically, the sample vessels have a volume
of about 50 to about 200 milliliters, more typically about 80 to
about 100 ml. The sample vessels are typically placed in an array
of placement wells in a container frame, e.g., for autoclaving,
processing, fermentation, and the like.
[0058] In some embodiments, the sample vessels are constructed of
Pyrex glass or polycarbonate, but other suitable materials are
optionally used to construct the sample vessels. For example,
plastic, ceramic, metal, e.g., aluminum, or any other material is
optionally used that is non-reactive to fermentation medium or to
other materials involved in additional processes contemplated in a
multiple process production, such as in a high throughput system.
It will further be appreciated that the fermentation medium may be
the same medium in each individual sample vessels or,
alternatively, the array of sample vessels optionally includes a
combination of different fermentation mediums. For example,
fermentation medium may be the same in each individual sample
vessel and contain the same fermentation broth for a bulk synthetic
process. Alternatively, each sample vessel in an array may have a
slightly different fermentation broth in order to optimize the
production yield of a certain component.
[0059] In some embodiments, sample vessels with gripper surfaces
are optionally used. In this embodiment, the container frame
typically comprises a corresponding gripper surface, e.g., for
maintaining the vessels in the desired configuration or to aid in
transporting the array of sample vessels to and from a fermentation
station and/or processing station.
[0060] In other embodiments, sensors are optionally included in the
sample vessels of the invention. For example, a pH or temperature
sensor is optionally positioned proximal to or within a sample
vessel to monitor the fermentation reaction.
[0061] Fermentation samples are optionally placed in the sample
vessels prior to their placement in the container frame or after
such placement. In one embodiment, colonization of bacteria and
other preparative steps are performed within the sample vessels,
e.g., while they are contained in the container frame. For example,
bacteria and initial nutrients are dispensed into each sample
vessel at a prior processing station. Being able to prepare
bacteria directly in each individual sample vessel eliminates the
need to inoculate a culture and initiate colonization in a separate
container before transferring the sample to the fermentation
apparatus. Using the container frame arrangement of the present
invention to colonize the fermenting bacteria decreases costs by
eliminating a separate colonization arrangement. Once bacteria are
colonized, sample vessels are conveniently transported, e.g.,
within the container frame, to a fermentation station, e.g., a
water bath or any other temperature controlled area, such as a
heated room. At the fermentation station or any time prior, a gas
distribution arrangement is attached to the container frame to
bubble gas into each sample vessel for fermentation. The gas
distribution arrangements are described in more detail below.
[0062] A Gas Distribution Arrangement is used to Provide Gas to a
Plurality of Sample Vessels
[0063] The gas distribution arrangement is used to provide gas flow
to the sample vessels during fermentation. The gas distribution
system typically comprises a gas inlet which is configured to flow
gas from a gas source into a plurality of sample vessels in a
container frame. Typically, the gas distribution arrangement is
attached to the container frame, e.g., placed on top or screwed
down. For example, the gas distribution arrangement typically
comprises or is coupled to a plurality of cannulas through which
the gas is flowed. The cannulas extend into each sample vessel for
delivery of gas, e.g., to the bottom of the sample vessel. Such
cannulas also optionally provide agitation of the sample within the
sample vessel.
[0064] A gas source typically comprises a source of one or more
gases, for example, air and oxygen. For example, in one embodiment
the gas source contains an inlet for N.sub.2 gas and an inlet for
02 gas. The ratio of each gas can be controlled either manually or
remotely by using a process controller. The ability to adjust gas
ratios enables the present invention to optimize the amount of gas,
e.g., oxygen, needed as the growing conditions change throughout
the fermentation. For example, a process controller is optionally
used to linearly change the ratio of air/oxygen over the course of
a fermentation. Alternatively, the ratio is changed stepwise as
fermentation proceeds. Any type, mixture, or number of gases are
optionally introduced and mixed through the gas sources of the
invention and provided to fermentation samples contained in one or
more sample vessels, e.g., through a set of cannulas.
[0065] A cannula is a small tube for insertion into a duct or
vessel, e.g., a fermentation sample vessel or tube as provided
herein. In the present application, the cannulas are positionable
inside the plurality of sample vessels, e.g., they typically
comprise flexible or rigid tubes that are inserted into sample
vessels for the delivery of various gases into the sample vessels.
In one embodiment, the cannulas are arranged into an array, which
array typically corresponds to an array of sample vessels. An
example array of the invention comprises an 8 by 12 member array of
sample vessels each having an associated rigid cannula. Typically,
a cannula extends substantially to the bottom of each individual
sample vessel in order to increase aeration and mixing. For
example, the cannula optionally extend about 15 to about 16 cm from
the bottom surface of a gas distribution arrangement. In some
embodiments, two or more cannulas are provided in each sample
vessel.
[0066] In the embodiment illustrated in FIG. 8, gas flows through
cannula 22 through three passages. Gas flow through passages are
optionally individually or collectively regulated. Smaller gas
bubbles are obtained with multiple small passages than with a
single, larger passage through the cannula. As a result, gas
bubbles formed from these multiple passages have more surface area
than bubbles formed from a single passage. In a preferred
embodiment, passages are precision drilled in order to more
accurately adjust gas flow within each passage and to ensure
uniform gas delivery across the set of sample vessels. Fewer or
more passages may be used according to the specific application of
the present invention. For example, the cannulas typically have
about 1 to about 5 passages, more typically, 2 or 3 passages.
Passages are optionally the same or different sizes and may be
circular or any non-circular shape, such as rectangular, oval, or
triangular.
[0067] In one embodiment cannula are included in a cannula assembly
comprised of an array of individual cannulas corresponding to the
plurality of sample vessels. Each individual cannula is optionally
connected by a fastener which couples the cannula to a gas
distribution arrangement.
[0068] Gas, e.g., oxygen or an oxygen/air mixture, is delivered,
e.g., from a manifold or other distribution system, to the sample
vessels via the cannulas, thus oxygenating, if desired, the entire
array of sample vessels within the container frame. For example, a
gas source is optionally coupled directly to the gas distribution
arrangement, e.g., with or without the use of a manifold, as
illustrated in FIGS. 6, 12, and 14.
[0069] In this manner, the exact mixture of gases delivered from
the gas source is uniformly distributed to each individual cannula
assembly. Any gas distribution arrangement is optionally employed
that uniformly delivers oxygen, an oxygen containing mixture, or
another gas or gas mixture capable of fermenting the sample, from a
gas source into the plurality of sample vessels. Example gas
distribution arrangements are provided in FIGS. 1, 3, 12, and 14,
which are described in more detail in the examples provided
below.
[0070] In some embodiments, the gas distribution arrangement is
comprised of one or more plates attached to an array of cannula,
e.g., using a manifold, and a gas inlet, which delivers oxygen, an
oxygen containing gas mixture, or another gas or gas mixture
capable of fermenting the sample, to the sample vessels via the
cannula.
[0071] Typically, the plates are aligned and fastened together,
e.g., to form an air-tight, liquid-tight seal. A hollow space or
interior space typically exists between the plates or within one of
the plates through which gases are uniformly distributed to the
associated cannula array. Any suitable fastener may be used. For
example, guide pins, rivets, nails, nut/bolt combinations, or
magnets may be used. A releasable fastener, such as a screw or
nut/bolt combination, is used in a preferred embodiment, although
permanent type fasteners, such as adhesives, may be desired in some
applications. Vertical supports are optionally attached to the gas
distribution arrangement, thus supporting the arrangement on an
array of sample vessels.
[0072] The plates are optionally composed of any suitable material
that maintains the structural integrity of the plate during
fermentation. For example, a plate is optionally composed of metal,
plastic, ceramic, or any suitable composite. In one example, the
plates comprise Teflon.TM.-coated aluminum, thus enabling the
plates to undergo autoclave sterilization procedures along with the
container frame and sample vessels as described above.
[0073] In one embodiment, the gas distribution arrangement
comprises two plates. The first plate, e.g., the bottom plate,
typically comprises a plurality of sample vessel areas or
indentations on the bottom surface. The indentations correspond to
the array of sample vessels held in the container frame and serve
to cap the sample vessels. FIGS. 9-11 illustrate features
encompassed by the indentations, e.g., sample vessel area or
indentation 625 on bottom portion 646. The indentations or recesses
are also used, e.g., to immobilize the sample vessel within the
container frame. Although the indentations are illustrated as
circular, they are optionally any shape, e.g., to correspond to a
variety of sample vessels.
[0074] One or more vents are typically positioned on the
circumference of the sample vessel area, cap, or recess to allow
gases and built up pressure to escape the sample vessel. FIG. 11
illustrates one embodiment of a venting space. However, other
configurations of venting spaces and recesses are optionally
constructed such that built-up pressure within sample vessels can
escape without contaminating other sample vessels.
[0075] When the top surface of a sample vessel abuts the bottom
surface of the gas distribution arrangement, gases, liquids,
emulsions, or excess pressure built up in the sample vessel escape
through a recess and/or venting space created in the gas
distribution arrangement. Cross-contamination of these escaping
elements is significantly reduced because a vertical edge in the
bottom surface of the gas distribution arrangement separates each
sample vessel from an adjacent sample vessel. Moreover, gas flow
from the cannulas maintains a positive pressure within the sample
vessel such that contaminants outside a particular sample vessel
are not drawn in through the vent.
[0076] In some embodiments, the first plate comprises the plurality
of cannulas that deliver gas to the sample vessels. The cannulas
typically extend from the top surface of the plate, through the
plate, and below the bottom surface of the plate. The cannulas are
generally of sufficient length to reach within about 1 cm to about
0.1 cm of the bottom of the sample vessels. The cannulas open to
the top surface of the plate, e.g., for gas to be distributed
through the cannulas into the sample vessels. The cannulas are
configured to be positionable in an array of sample vessels, e.g.,
held in a container frame.
[0077] In addition to the cannulas, the first plate optionally
includes a plurality of apertures that correspond to the array of
sample vessels. For example, the apertures optionally provide an
opening through the first plate, through which fluids may be added
into the sample vessels when the gas distribution arrangement is
attached to a container frame.
[0078] The first plate is typically attached to a second plate,
e.g., with screws or adhesives, which second plate typically
comprises one or more gas inlets for providing gas flow into the
cannulas of the first plate. The gas inlet opens into an interior
space created between the second plate and the first plate, which
interior space provides gas flow to the cannulas.
[0079] In addition, the second plate also comprises a plurality of
apertures, e.g., to provide liquid access to the sample vessels.
The apertures of the second plate typically align with or match the
apertures on the first plate when the two plates are coupled. The
apertures provide openings through which liquid can be added into
the sample vessels in a container frame attached to the gas
distribution arrangement. The apertures also serve as openings for
an array of aspirators or dispensers that can be used to aspirate
or dispense liquid into the sample vessels. In other embodiments,
pipettes or syringes are used to draw samples or add nutrients,
water, etc, e.g., through the apertures. The gas distribution
arrangement also optionally comprises a lid for covering the
apertures when a sealed environment is desired. The first plate and
second plate together comprise a fermentor head or manifold for
delivering gas or fluid to a plurality of sample vessels. More
detailed examples are provided below.
[0080] A process controller is also optionally coupled to the
fermentation apparatus of the invention, e.g., for controlling gas
flow to the cannulas, for altering ratios of air to oxygen that are
bubbled through the cannulas, for monitoring and controlling
temperature, for directing the addition of various reagents, and
the like. An automated process using a process controller is
described in more detail in the examples below.
[0081] Other devices are also optionally coupled to the fermentor
apparatus of the present invention. For example, dispensers,
aspirators, centrifuges, and other processing devices are
optionally coupled to the fermentor or configured for use with a
container frame, e.g., so that samples can be processed in the same
vessel in which fermentation is carried out. For example, a
dispenser is optionally configured to dispense liquid into a
plurality of sample vessels held in a container frame, e.g.,
through a plurality of apertures in a gas distribution arrangement.
Aspirators are likewise optionally configured to coordinate with
the container frame and gas distribution manifolds of the present
invention.
[0082] A centrifuge is also optionally used in processing
fermentation samples. For example, a centrifuge is optionally
configured to accept the sample vessels as centrifuge tubes to
avoid transferring of samples prior to centrifugation. For more
information on centrifugation systems for use in the present
invention, see, e.g., U.S. Ser. No. 09/780,589, entitled "Automated
Centrifuge and Method of Using Same," by Downs et al, filed Feb. 8,
2001.
II. Methods of Fermenting Samples in a Multi-sample Fermentor
[0083] The multi-sample fermentors described above are used for
simultaneously fermenting a plurality of samples, e.g., in a
container frame that is transportable, e.g., to a processing
station. The present invention also provides methods of using such
fermentors, e.g., in conjunction with one or more processing steps.
For example, the methods provided typically comprise providing a
plurality of sample vessels in a container frame, each of which
sample vessels contains a sample of about 50 to about 100
milliliters, more typically 65 ml. The samples are fermented in the
sample vessels within the container frame.
[0084] Fermentation is used herein to refer generally to any
process in which cells are used to convert raw materials, e.g.,
water, air, sugars, mineral salts, nitrogen sources, and the like,
or enzyme substrates into desired products, e.g., proteins. Types
of cells used include, but are not limited to, animal cells, yeast
cells, and bacterial cells, e.g., E. coli, Bacillus, and the like.
The cells are typically grown in a growth medium and then products
are harvested. Fermenting typically involves simultaneously
delivering gas to each of the sample vessels through a plurality of
cannulas associated with the sample vessels, e.g., to aid growth of
the cells. For example, the methods typically comprise attaching a
fermentor head as described herein to a container frame containing
the plurality of samples to be fermented. Once fermented, the
samples are transferred to a post-processing station, e.g., a
centrifuge. Typically, the post-processing station is configured to
accept the same sample vessels in which the samples were fermented.
In addition, some processing stations are configured to receive the
container frame containing the sample vessels, e.g., a dispensing
or aspirating station. An example method is described below and in
FIGS. 4 and 5.
[0085] FIG. 4 describes fermentation method 300 practiced in
accordance with the present invention. Block 310 provides for a
plurality of sample vessels 15. By providing a number of smaller
volume fermentation vessels, this method is more advantageous than
production scale fermentation methods that use bulk fermentation
vessels, in that smaller volumes of growth medium are more
predictable in their yield and nutrient needs than are standard
production scale volumes that are utilized in bulk fermentation
methods. The number of sample vessels that may be fermented at any
one time is unlimited by the present invention, and instead is only
limited either by the configurational practicalities of any one
fermentation apparatus or by the number of sample vessels that may
be handled by further processing steps in the production.
[0086] Block 315 arranges a plurality of sample vessels into an
array, e.g., a rectangular 8 by 12 array. However, the array is
optionally configured in any shape that is practicable for the
fermentation apparatus. For example, sample vessels are optionally
arranged in a rectangular array, a honeycomb configuration, or a
linear array.
[0087] Block 320 arranges a plurality of cannula into an array
corresponding to the sample vessels. According to the present
invention, each cannula in this cannula array corresponds to an
individual sample vessel in the sample vessel array, which are
arranged in block 315. In one embodiment, the plurality of cannula
is limited by the number of sample vessels arranged in block
315.
[0088] Block 325 creates a gas distribution arrangement for
delivering oxygen and/or one or more other gases to a fermentation
media in the sample vessels. For example, one embodiment fastens a
cannula array to a gas distributor, which is connected to a
manifold. The cannula array may be fastened by any means achieving
a liquid-tight seal. For example, cannula are optionally connected
via a union connector to a gas distributor. Alternatively, cannula
are pneumatically connected to the distributor, or the cannula
array and gas distributor are optionally molded as a single unit.
In another embodiment, the distributor connects directly to a gas
source without using a manifold. The methods of creating a gas
distribution arrangement are optionally achieved using any method
of uniformly delivering oxygen and/or one or more other gases from
a gas source to a gas distributor such that gas is delivered to
each individual sample vessel selectively or collectively by way of
a corresponding cannula.
[0089] Block 330 transports the container frame containing the
plurality of sample vessels to a temperature controlled area. Other
methods known to those of skill in the art for controlling
temperature are also contemplated within the present invention. For
example, the container frame is optionally transported to a heated
gel bath or a controlled temperature room used to maintain a
constant temperature.
[0090] Block 335 positions the gas distribution arrangement created
in block 330 on top of the container frame, e.g., using screws or
by merely being placed on top and held in position by a groove
assembly as shown in FIG. 14. From this configuration, the array of
sample vessels is fermented in block 340.
[0091] Once fermentation is complete, block 345 removes the gas
distribution arrangement from the container frame. The sample
vessels are optionally transferred from the container frame
directly to a post-fermentation processing station in block 350,
e.g., by manipulating a gripping surface located on each sample
vessel. This post-fermentation processing station includes any
processing step where the fermentation product may be processed
directly from the sample vessel. For example, the array of sample
vessels may be transferred, either manually or robotically, from
the container frame directly to an automated centrifuge.
Alternatively, sample vessels may be transferred to an aspirating
station or detecting station. In other embodiments, the sample
vessels are not removed from the container frame but remain in it
for further processing, such as dispensing or aspirating, using a
dispenser or aspirator configured to coordinate with the array of
sample vessels in the container frame.
[0092] In block 350, the fermentation product in the sample vessels
is directly transferred into a post-fermentation processing station
and in block 355 the fermentation product is directly processed in
the sample vessels themselves. For example, in one embodiment,
sample vessels are transferred directly to a centrifuge station in
which the sample vessels are positioned directly inside the
centrifuge such that the sample vessels act as centrifugation tubes
and the fermentation product is centrifuged according to methods
known in the art. Further processing steps such as aspirating,
reagent dispensing, or detecting also optionally occur directly in
the sample vessel used in the fermentation process. In this way,
the fermentation vessel provides a sample vessel that holds the
sample throughout the entire production process, thereby
eliminating excess waste from transferring sample material from
sample vessel to sample vessel as well as decreasing the cost of
washing and sterilizing a fermentation apparatus in addition to
sample vessels from each production process step. Other multiple
process productions or analyses may also be practiced in accordance
with the present invention.
[0093] In FIG. 5, block diagram 400 shows how the present invention
is integrated into a multiple step, multiple process production.
Block 410 depicts a processing station prior to fermentation. In
one embodiment, fermentation broth and fermentation nutrients are
added to sample vessels at prior processing station 410. Other
processing steps involved in a multiple step production or analysis
are also contemplated in accordance with the present invention. For
example, bacteria colonization may occur in sample vessels at prior
processing station 410. Example preprocessing steps include, but
are not limited to, deionization, e.g., of solvents, pasteurization
of materials, and mixing, e.g., of cell nutrient broths and the
like. Such steps are typically used to process the raw materials,
such as water, cell broths, sugars, nitrogen sources, and the like,
used for the fermentation. Transporter 420, e.g., a robot, a
technician, a conveyor belt, or the like, is optionally used to
transfer the sample vessels from processing station 410 to a
fermentation apparatus such as fermentation apparatus 100. Other
embodiments of a fermentation apparatus practiced in accordance
with this invention may also be used. For example, the fermentation
apparatus shown in FIG. 14 or in FIG. 1 is optionally used.
[0094] It will further be appreciated that transporter 420 may
transfer the sample vessels individually, in groups, or in an array
configured for the fermentation apparatus. For example, in one
embodiment, a container frame transports the sample vessel array to
fermentation apparatus 100. Similarly, after fermentation,
transporter 430 transports sample vessels from a fermentation
apparatus to a post-fermentation processing station 410. In one
embodiment, transporter 430 transports a container frame holding an
array of sample vessels to a centrifuge processing station 410.
Post-processing station 410 is optionally any other processing step
occurring in a multiple process or analysis, such as an aspirating
step, a dispensing step, or a detecting step. Example
post-processing steps include, but are not limited to,
precipitation, deionization, chromatography, evaporation,
filtration, centrifugation, crystallization, drying, and the like.
These steps are generally directed to purification, retrieval, and
concentration of materials produced in the fermentation. In this
manner, multiple processing steps are executed on each sample
contained in the same sample vessel, thus enabling fermentation
processes to be incorporated into high throughput or other multiple
process systems. Example fermentation conditions are described
below.
[0095] The present invention preferably uses fermentation
conditions that lead to high level production of soluble proteins.
These fermentation conditions may employ the use of high levels of
yeast extract and bactotryptone (rich media, referred to as
terrific broth or TB). Secondly, this media is optionally
supplemented with 1% glycerol (additional carbon source). Lastly,
the media preferably is typically buffered with 50 mM MOPS.
Alternatively, a defined media comprising amino acids and 50 mM
phosphate as opposed to MOPS is used. The first two additions allow
the cells to be grown for up to about 10 hours without apparent
loss of nutrients. The highly buffered media prevents the cells
from being exposed to high levels of acid (low pH) which routinely
occurs during fermentation.
[0096] Surprisingly less than 5% of human proteins expressed in
normal Luria Broth or LB media, are typically found to be soluble.
However, using the above media, 15-20% of human proteins expressed
in E. coli now appear to be soluble.
[0097] In a preferred embodiment, the fermentation media is
prepared as follows. TB media is prepared in 7 L batches.
Antibiotics are not added to TB media until the day it will be used
for a fermentation run. To prepare the 7 L bath, the following
steps are performed: (1) Fill a clean 10 L pyrex bottle with
.about.4 L DI H.sub.2O or 18 megohm water, add a large stirbar; (2)
Add 168 g Yeast Extract; (3) Add 84 g Tryptone; (4) Add 70 ml
Glycerol; (5) Stir on stirplate until completely dissolved; (6) QS
to 6.3L, e.g., with 18 megohm water; (7) Autoclave on the longest
liquid cycle. Remove TB media from the autoclave as soon as
possible, e.g., to prevent carmelization or burning of the carbon
source and/or to allow for a quick cool down; (8) Store TB media at
room temperature; and (9) Record process. TB Media is the same for
all fermentor runs. However, Fermentor Media is not necessarily the
same for all runs. For example, one difference in media is the
antibiotic(s) added just before fermentation. On the same day of a
fermentation run, the following may be added to TB media: (1) 350
mls of 1 M MOPS pH 7.6; (2) 7 ml Antifoam; (3) 7 ml 20 mg/ml
Chloramphenicol; (4) 7 ml 100 mg/ml Ampicillin; (5) Add enough 18
megohm H.sub.2O to bring the volume up to 7 L; (6) Write everything
added to TB media on its label; (7) Cap tightly and shake bottle
well; and (8) Record process. The above medium is only one of many
possible choices known to those of skill in the art, which are
optionally used with the present fermentors and methods.
[0098] When fermentation is complete, the sample vessels are
transferred to a post-processing unit as described above, e.g., in
the container frame, either manually or robotically. For example, a
robot optionally removes the sample vessels from the container
frame and places them, e.g., in a centrifuge.
III Examples Fermentation Systems
[0099] Example Fermentor #1
[0100] In accordance with the present invention, an example
fermentation apparatus is provided in FIG. 1. Fermentation
apparatus 10 generally comprises sample holder arrangement 255,
cannula assembly 80 and gas distribution arrangement 270. The
illustrated fermentation apparatus 10 is configured to separately
and simultaneously ferment multiple batch samples in sample vessels
that are compatible with direct pre- and post-fermentation
processing as described above.
[0101] Sample holder arrangement 255 is comprised of gripping
surfaces 17, individual sample vessels 15, which typically form an
array of sample vessels, such as array 110, a transportable
container frame 250, and an array of placement wells 260
corresponding to array 110. Gripping surfaces 17 are optionally
located on each individual sample vessel 15, which collectively
form sample vessel array 110. It is preferable that gripping
surface 17 resides on the bottom of each sample vessel, but
gripping surface 17 is optionally located on any surface of the
sample vessel that enables sample vessel 15 to be transferred to or
from container frame 250 or another processing station.
[0102] The bottom of each individual sample well 15 is positioned
within a placement well,.e.g., placement well 257. The array of
placement wells 260 preferably mirrors the configuration of array
110 and is embedded in transportable container frame 250.
[0103] By using transportable container frame 250, the entire array
of sample vessels 110 is optionally transported to and from one
fermentation processing station to another processing station in a
multiple process production. In this illustrated example,
transportable container frame 250 transports array of sample
vessels 110 into a temperature controlled area 210 such as a water
bath. In this embodiment, temperature controlled area 210 is
comprised of water bath 240 in water bath container 215, which is
controlled by water bath temperature controller 220 and temperature
coil 230 immersed in water bath 240.
[0104] In FIGS. 1-3, an example gas distribution arrangement is
shown. Gas distribution arrangement 270 is comprised of gas source
85 connected to manifold 75. Conduit 70 connects manifold 75 to
connector 65. Connector 65 connects manifold 75 to gas distributor
55.
[0105] In the embodiment illustrated in FIGS. 1 and 3, cannula
assembly 80 is comprised of cannula array 120, which is composed of
individual cannulas 22 that correspond to sample vessel array 110.
Each individual cannula 22 is optionally connected by a fastener
35, which couples cannula 22 to a gas distribution arrangement 270.
Cannula 22 preferably extends substantially to the bottom of each
individual sample vessel 15 in order to increase aeration and
mixing.
[0106] In another embodiment, each individual cannula is attached
directly to gas distribution arrangement 270 in an airtight,
liquid-tight manner. Eliminating the need for a fastener, this
embodiment directly integrates cannula 22 into gas distribution
arrangement 270, thereby decreasing the number of surfaces,
grooves, and/or pockets available for possible bacterial
contamination, and thus decreasing the opportunities for
fermentation spoilage. Likewise, cannula 22, when integrated into a
gas distribution arrangement 270 are optionally autoclaved with gas
distribution arrangement 270, thereby eliminating the need to
unfasten each cannula 22 separately before cleaning and
sterilization. This convenience saves both time and money as well
as adding to the uniformity of each batch. For example, the
possibility for human error is minimized, because each cannula 22
does not have to be fastened individually before each fermentation
run or unfastened individually prior to cleaning and sterilization.
Also any non-uniformities in any one cannula 22 will be immediately
apparent as an individual cannula 22 will be constantly associated
with the same sample vessel in each run. Integrated cannula are
shown in FIG. 14.
[0107] Referring to FIG. 3, gas, e.g., oxygen, is delivered from
manifold 75 to all parts of distributor 55 through a hollow space
60 of distributor 55, thus oxygenating, if desired, the entire
array of sample vessels 110. Oxygen and/or one or more other gases
is delivered from distributor 55 through individual cannula 22,
which is connected to distributor 55 by way of cannula assembly
80.
[0108] In one embodiment, cannula assembly 80 is comprised of a
connector 45 on an inside face of distributor 55 as well as
connector 40 on an outside face of distributor 55. Fastener 35
attaches individual cannula 22 to connector 40 on distributor 55.
Arrows 25 depict oxygen and/or one or more other gases flowing from
cannula 22 into fermentation medium 20 and producing gas bubbles
30. For example, gas source 85 is optionally coupled directly to
dispensing plate 645 without the use of manifold 75, as illustrated
in FIGS. 6 and 12. Likewise, cannula assembly 80 may be constructed
by alternative methods. For example, as shown in FIG. 12, cannula
22 is attached directly to dispensing plate 645.
[0109] In this manner, the exact mixture of gases delivered from
gas source 85 is uniformly distributed to each individual cannula
assembly 80. Any gas distribution arrangement is optionally
employed that uniformly delivers oxygen, an oxygen containing
mixture, or another gas or gas mixture capable of fermenting the
sample, from gas source 85 into sample vessel 15.
[0110] FIGS. 6 and 12 illustrate another embodiment of a gas
distribution arrangement. Gas distribution arrangement 270 is
comprised of a dispensing plate 645 directly attached to an array
of cannula 120, that is configured without a manifold, manifold
conduit, or manifold connector. In this embodiment, dispensing
plate 645 is comprised of a bottom portion 646 and a top portion
647 (not shown). Inlet 630 delivers oxygen, an oxygen containing
gas mixture, or another gas or gas mixture capable of fermenting
the sample, to dispensing plate 645 from gas sources 85 (not
shown).
[0111] Bottom portion 646 and top portion 647 are aligned and
fastened together through apertures 640, e.g., to form an
air-tight, liquid-tight seal. A hollow space exists between
portions 646 and 645 through which gases are uniformly distributed
to cannula array 120. Apertures 635 are used to fasten vertical
supports to dispensing plate 645 that allow dispensing plate 645 to
rest adjacent to array of sample vessels 110. Any suitable fastener
may be used. In the illustrated example, screws connect upper
portion 647 and bottom portion 646 to form dispensing plate 645.
Screws also fasten aluminum legs to dispensing plate 645 as
vertical supports.
[0112] FIGS. 9-11 illustrate yet another embodiment of a gas
distribution arrangement. In this embodiment, cannula 22 is
directly attached to bottom portion 646. Aperture 620 holds a
dispensing tube 760 (not shown) for dispensing nutrients and other
solutions into sample vessel 15. Aperture 620 is optionally used to
access samples during the fermentation process, using, e.g.,
pipettes or syringes to draw samples or add nutrients, water,
and/or the like into the sample vessels. Fastening groove 650
enables dispensing tube 760 to be fastened to dispensing plate 645.
Indentation 655 and vertical edge 665 create a circular recess that
helps immobilize sample vessel 15 within sample vessel area 625.
Although in this embodiment, indentation 655 is circular and
corresponds to the shape of sample vessel 15, other suitable shapes
may be used.
[0113] Vent 610 is positioned on the circumference of sample vessel
area 625 and allows gases and built up pressure to escape sample
vessel 15. Referring to FIG. 11, vent 610 creates venting space
675. Because vertical edge 670 is larger than vertical edge 665,
venting space 675 occupies a deeper recess than recess 655. The
difference in height between vertical edges 670 and 665 is equal to
the height of vertical edge 680 and determines the depth of venting
space 675. Other configurations of venting space 675 and recess 655
(and, accordingly, vertical edges 665, 670, and 680) may be
constructed such that built-up pressure within sample vessel 15 can
escape through venting space 675 without contaminating other sample
vessels.
[0114] When the top surface of sample vessel 15 abuts surface 660,
gases, liquids, emulsions, or excess pressure built up in sample
vessel 15 may escape through recess 655 and venting space 675.
Cross-contamination of these escaping elements is significantly
reduced because vertical edge 670 separates sample vessel 15 from
an adjacent sample vessel 15. Moreover, gas flow from cannula 22
maintains a positive pressure within sample vessel 15 such that
contaminants outside sample vessel 15 are not drawn in through
venting space 675 into sample vessel 15 by way of recess 625, 655,
or 675. Other vents 610 may be configured such that excess gases,
liquids, emulsions, or excess pressure may escape through vent 610
without cross-contaminating other sample vessels 15.
[0115] In another embodiment of gas distribution arrangement 270,
illustrated in FIG. 2, array 110 is configured such that gas
distribution arrangement 270 oxygenates, for example, each
individual sample vessel 15 as opposed to utilizing a dispensing
plate 645. Thus, array of sample vessels 110 is optionally
oxygenated (or provided with other appropriate gas) collectively or
individually by adjusting cannula assembly 80 for any individual
sample vessel 15. For example, in one application, section A may be
oxygenated (or provided with other appropriate gas) twice as long
as section B.
[0116] In the illustrated example, cannula array 120 corresponds to
sample vessel array 110, which is composed of individual sample
vessels 15. Each individual sample vessel 15 also corresponds to an
individual cannula assembly 80 which is connected to distributor
55. Oxygen and/or one or more other gases are delivered to
distributor 55 through manifold connector 65. Oxygen and/or one or
more other gases may be delivered through each cannula assembly 80,
or selectively to certain assemblies 80. For example, cannula
assemblies 80 in sections A and B may be utilized, while no gases
flow to sections C and D.
[0117] Referring to FIGS. 3 and 12, gripping surface 17 allows for
automated or manual transfer of sample vessel 15 to and from the
fermentation apparatus or another processing station, e.g., upon
conclusion of fermentation. In one embodiment, gripping surface 17
is magnetic such that a magnet attracts gripping surface 17 and
transfers the sample vessel to another processing station. In
another embodiment, a gripping mechanism grips the outer sides of
the sample vessel to effect transfer. In yet another embodiment,
gripping surface 17 is a lip at the top of the sample vessel. Other
surfaces that may be gripped in order to transport the sample
vessel to or from the fermentation processing station are within
the scope of the present invention. For example, gripping surface
17 is optionally on the inside, outside, top or bottom of sample
vessel 15. In other embodiments, the samples are held in place and
transported with the aid of a gripper structure.
[0118] FIG. 12 illustrates one embodiment of a gas distribution
arrangement. Gas distribution arrangement 270 and cannula 22 are
used together to provide gas to a sample vessel. In this example,
oxygen, a mixture of oxygen and other gases, or another gas or gas
mixture is introduced into dispensing plate 645 through inlet 630.
Fasteners such as screws connect and align upper portion 647 to
bottom portion 646 through apertures 640. Dispensing tube 760 and
cannula 22 are directly attached to dispensing plate 645 and can be
replaced by unfastening portions 646 and 647, replacing either or
both dispensing tube 760 or cannula 22, and refastening portions
646 and 647. It is preferable for dispensing tube 760, cannula 22,
inlet 630, and portions 646 and 647 to remain fastened together
such that these elements are autoclaved as one unit. This allows
for significant sterilization without the time and cost expense of
dismantling arrangement 270 after each fermentation in order to
separately sterilize each element.
[0119] In the illustrated example, a top surface of individual
sample vessel 15 abuts directly onto surface 660 within sample
vessel area 625. The top surface of sample vessel 15 is positioned
within recess 655. Surface 660 preferably is not in contact with
the entire circumference of the top surface of sample vessel 15.
Also preferably, vent 610 is positioned adjacent to surface 660
such that a gap 672 exists between surface 660 and the vertical
edge of sample vessel 15, thereby creating a passage for excess
gases, emulsions, or pressure to escape from sample vessel 15
through venting space 675. Gas flow through cannula 22 provides
sufficient pressure such that contaminants are not drawn into
sample vessel 15 through venting space 675.
[0120] Example Fermentor #2
[0121] FIGS. 13-21 illustrate another embodiment of the fermentor
apparatus of the present invention. Generally, the apparatus
comprises a container frame comprising placement wells, and a gas
distribution arrangement comprising a cannula array. Each piece is
described in more detail below and by reference to the figures.
[0122] Container frame 1300, as shown in FIG. 13, comprises bottom
1310 and top portion 1320 connected by side portions 1325 and 1330.
The container is easily transportable, e.g., by grasping handles
1335 and 1340 which are attached to sides 1325 and 1330. Each side
1325 and 1330 has two grooves 1345 which can each receive a pin for
securing a gas distribution arrangement, such as that shown in FIG.
16, e.g., using pins 1480. Top portion 1320 and bottom portion 1310
together form an array of placement wells 1350. Bottom portion 1310
of the container frame has a plurality of indentations that serve
as bottoms for the placement wells, in which sample vessels are
placed. For example, container frame 1300 comprises an 8 by 12
array of placement wells. Top portion 1320 comprises a matching
array of holes 1360 which holes receive the sample vessels into the
container frame and hold them in position within the container
frame. Together holes 1360 and indentations 1355 in container frame
1300 form a rack for holding a plurality of sample vessels, e.g.,
tubes. Although holes 1360 are shown as circles, the shape is
optionally configured to receive any desired sample vessel.
[0123] FIG. 14 illustrates a gas distribution arrangement coupled
to container frame 1300. The gas distribution arrangement comprises
four pins 1480 which slide into grooves 1345 to hold the gas
distribution arrangement in place over the container frame. As
shown in FIG. 14, the gas distribution arrangement comprises first
plate 1465 and second plate 1470, which are typically fastened
together, e.g., using screws or pins. An optional lid, e.g., lid
1460, is also shown. In addition, the gas distribution arrangement
comprises handles 1410 and 1420 attached to second plate 1470 for
easy positioning and removal of the gas distribution
arrangement.
[0124] Inlets 1430 and 1440 provide gas inlets to the gas
distribution arrangement, which gas inlets typically receive gas
from a gas source and deliver it, e.g., to a plurality of cannulas.
Typically, the plurality of cannulas is attached to the gas
distribution arrangement, e.g., as part of the first plate. For
example, in the illustrated embodiment, cannula 1450 is part of
first plate 1465 and extends from the top of the first plate,
through the first plate and below, such that the cannula is
positionable inside a placement well, e.g., well 1350, or inside a
sample vessel positioned within placement well 1350.
[0125] Typically, first plate 1465 comprises the cannula array and
a plurality of apertures. The apertures of the first plate align
with a set of apertures on the second plate to provide access to
the sample vessels within the placement wells. The cannula array is
optionally molded as part of the first plate or separately formed
and then attached to the first plate. For example, an additional
set of apertures is optionally present in the first plate to accept
the array of cannula, e.g., which are received into the aperture
and secured using o-rings.
[0126] FIG. 18 illustrates the bottom surface of first plate 1465.
For example, on the bottom surface of the first plate, an array of
sample vessel areas 1810 or indentations are used to cap the sample
vessels and provide venting space as described above in Example 1.
Each sample vessel area comprises an aperture to provide access to
the sample vessel positioned with the associated placement well, a
cannula associated with each placement well for delivering gas into
each sample vessel positioned within the well, and a vent for
relieving pressure build up during fermentation. In addition, FIG.
18 illustrates apertures 1830 and 1840, which are used, e.g., to
attach the second plate to the first plate, e.g., via a set of
screws. FIG. 19 provides a detail drawing of a portion of FIG. 18
illustrating aperture 1920, vent 1930, and cannula 1940. In
addition, FIG. 19 illustrates gasket or o-ring 1950 that serves to
provide a seal between the first and second plates.
[0127] Second plate 1470 typically comprises a set of apertures as
described above, which correspond to the set of apertures in plate
1465. These apertures are used, e.g., for liquid dispensing and/or
venting. The apertures in the two plates connect to form a
passageway that extends through both plates for access to placement
wells 1350. The apertures are closed off from the interior space
and can be capped using a lid as shown in FIG. 14 when a sealed
system is desired. In addition, second plate 1470 typically
comprises the gas inlet, e.g., inlet 1430, and an interior space
through which gas is flowed. FIG. 21 provides a side view of the
gas distribution arrangement as shown in FIG. 14. For example, FIG.
21 shows cannulas 1450 extending below the first plate into the
placement wells and apertures 1920 extending through the first
plate and the second plate.
[0128] FIG. 20 illustrates a cross-sectional view of the gas
distribution arrangement of FIG. 14, which comprises a first and a
second plate. Top plate 1470 is attached to bottom plate 1465,
e.g., using screws positioned through apertures 1830, and 1840. The
first plate, which is on the bottom, comprises apertures 2010 and
cannulas 2020. The apertures are open holes in first plate 1465,
which align with similar apertures in second pate 1470, the top
plate. The cannula are inserted into the first plate through
another set of apertures secured with 0-rings, e.g., to form a seal
between the top and bottom plates. The cannulas extend from the top
surface of plate 1475 into placement wells 1350 such that they are
easily positioned in an array of sample vessels held in the
placement wells. Cannula 2020 does not extend into plate 1470, but
abuts it. Adjacent to where cannula 2020 abuts plate 1470 is
venting space 2030 which couples the cannula to interior space 2040
of the top plate through which interior space gas flows in through
an inlet, e.g., inlet 1430.
[0129] FIG. 15 illustrates a container frame with a liquid addition
manifold assembly coupled to it. Container frame 1300 is shown with
first plate 1465 positioned on top using pins 1480. Second plate
1470 is positioned on top of the first plate and liquid addition
manifold 1510 is shown on top of the second plate of the gas
distribution system. The liquid addition manifold is optionally
used to add liquid into the sample vessels, e.g., through
corresponding sets of apertures in the first and second plate. FIG.
16 illustrates liquid addition manifold 1510 in more detail, e.g.,
apertures 1620, which align with apertures on the first and second
plates of the gas distribution arrangement. Apertures 1620 are used
to deliver liquid reagents into the sample vessels contained in the
apparatus. Manifold 1510 is placed, e.g., using pins, on top of the
gas distribution system. In addition, FIG. 17, a cross-sectional
view of the liquid addition manifold along line A-A, illustrates
how pipettes or additional cannulas are used to dispense liquid
into the sample vessels.
[0130] Example System 3--an Automated System
[0131] FIG. 7 illustrates an example of an automated fermentation
apparatus. Process controller 705 monitors and controls various
components of apparatus 700 and preferably is a programmable
computer with an operator interface. Alternatively, process
controller 705 is any suitable processor that coordinates multiple
components of apparatus 700, such as timing mechanisms, adding
solutions, adjusting temperature, adjusting gas flow rates and gas
mixtures, detecting measurements, and/or sending an alarm or
notification prompting operator intervention. Electronic couples
710, 755, and 795 connect various components of fermentation
apparatus 700 to process controller 705. For example electronic
couple 710 enables controller 705 to start, stop, and monitor
solution flow from feed solutions 720, 735, and 745. Likewise,
electronic couple 775 enables controller 705 to start, stop and
monitor reagent dispensing into sample vessels 15. Electronic
couple 795 also enables controller 705 to transmit and receive
information from sensors 790 as well as monitor and adjust
temperature controlled areas. Other coupling devices are also
optionally used in the present invention.
[0132] In one embodiment of fermentation apparatus 700, feed
solutions 720, 735, and 745 are pumped (either singly, in
combination, sequentially, or collectively) from individual feed
tubes 725 into dispensing tube 715. Selecting the appropriate
solenoid determines which feed solution is pumped through
dispensing tube 715. For example, solenoid 730 controls flow from
feed solution 720 through feed tube 725. In another application, a
mixture of feed solutions 720 and 735 are simultaneously pumped
into dispensing tube 715. In another application, feed solution 720
is fed into dispensing tube first, followed by an incubation period
(directed by controller 705), followed by feed solution 735 being
pumped into dispensing tube 715. Different combinations of feed
solutions are optionally used and more or fewer feed solutions may
be used with apparatus 700 according to any desired
application.
[0133] Using pump 710, which is optionally a peristaltic pump,
dispensing tube 715 transfers feed solution to an individual
dispensing tube 760. Each individual dispensing tube 760
corresponds to an individual sample vessel 15 and tube 760 is
positioned such that feed solution 720, for example, is transferred
volumetrically from dispensing tube 760 into its corresponding
sample vessel 15 once solenoid 765 is opened. Each solenoid 765
corresponds to an individual sample vessel 15. Volumetric
dispensing of feed solutions is controlled by process controller
705 which preferably controls the amount, the rate and the time of
dispensing. Dispensing tube 760 is optionally composed of plastic,
metal, or any material that is non-reactive to the feed solution
being dispensed.
[0134] In one embodiment; delivery solenoids 765 work in
conjunction with pump 710 and controller 705 to deliver multiple
feed solutions such as feed solutions 720, 735, and 745 into
individual sample vessels 15. Each solenoid 765 corresponds to a
sample vessel 15 and the solenoids 765 are manifolded together and
fed by the output of a single peristaltic pump 710. Each solenoid
765 preferably opens sequentially in order to dispense a volumetric
amount of feed solution 720. However, parallel addition is also
contemplated within the present invention.
[0135] In one embodiment, feed solution 720 introduces nutrients
into fermentation medium 20 through dispensing tube 715 using pump
710 and solenoid 765 to deliver solution 720 to individual
dispensing tube 760. After addition of feed solution 720, solenoid
730 is closed and solenoid 740 corresponding to rinse solution 745
opens. Pump 710 delivers rinse solution 745 through dispensing tube
715, thereby rinsing dispensing tube 715 with solution 745, which
is then flushed into waste container 785. Solenoid 780 controls
flow from dispensing tube 715 into waste container 785. Feed
solution 735 is then pumped through dispensing tube 715 and
dispensed through tube 760. Dispensing tube 715 is rinsed again
with rinse solution 745 before another addition. Solenoids 765 are
preferably located very near to dispensing tube 760 in order to
minimize dead volume downstream. In this way, dispensing tube 715
accurately delivers a known amount of feed solution 720 and 735
without cross contaminating or fouling the next or different
addition of feed solution .through dispensing tube 715.
Accordingly, each addition is volumetrically precise with a
minimal, known amount of feed solution from a previous addition
diluting the next addition. In this way, feed solutions such as
additional nutrients, trace minerals, vitamins, sugars,
carbohydrates, nitrogen containing compounds, evaporating liquids,
pH balancing compounds, buffers, and other liquids may be added to
fermentation media 20 in an automated, yet highly precise
manner.
[0136] Coordinated by process controller 705, various components
may be activated either at pre-determined time intervals or in
response to the measurement of some physical property within sample
vessel 15. For example, in one embodiment, an operator programs
process controller 705 to incubate sample vessels 15 for a
pre-determined time period at a particular temperature, add a
desired amount of feed solution 720, and incubate further for
another pre-determined time period at a different temperature. Any
suitable combination of fermentation conditions may be programmed
into process controller 705, which optionally comprises a computer,
computer network, other data input module, or the like.
[0137] In a preferred embodiment, process controller 705
coordinates temperature control, the addition of feed solutions,
adjustment of gas rates and gas mixtures, incubation periods, and
rinsing in response to data received from sensors 790. Sensors 790
are optionally located inside or outside of individual sample
vessels 15. Sensors 790 can detect color changes
spectrophotometrically, monitor evaporation rates, measure changes
in optical density, detect light changes photometrically, detect pH
changes, electrolytically measure redox potentials, monitor
temperature fluctuations, or detect other physical changes and
transmit this data to process controller 705. In response, process
controller 705 accordingly adjusts various components of apparatus
700. For example, by measuring the redox potential, sensors 790
detect when a fermentation sample is being over-oxygenated or
over-provided with another gas and process controller 705
accordingly adjusts the gas flow or gas mixture ratio. As another
example, process controller 705 can respond to a change in pH, as
detected by sensors 790, by adding a pH buffer from feed solution
720. In one embodiment, maximum protein expression may be detected
by monitoring light emission, at which point fermentation is halted
to minimize wasting fermentation resources after optimum
fermentation yield has been reached.
[0138] Because of the uniformity of each fermentation medium 20,
cannula 22, and dispensing of feed solutions 720, very few, for
example, one, sensor 790 is all that is necessary to monitor the
entire array of sample vessels 110. Alternatively, when sample
vessels 15 contain different fermentation media 20 or undergo
different fermentation conditions, numerous sensors 790 are
optionally employed.
[0139] The above automated process is optionally used in
conjunction with any fermentor apparatus or method to known to
those of skill in the art. In particular, it is useful when
practicing fermentation using the fermentors presented herein.
However, it is noted that the examples presented herein are
provided for purposes of illustration and not of limitation. While
the foregoing invention has been described in some detail for
purposes of clarity and understanding, it will be clear to one
skilled in the art from a reading of this disclosure that various
changes in form and detail can be made without departing from the
true scope of the invention. For example, all the techniques and
apparatus described above may be used in various combinations and
other uses for the present invention are also contemplated. It is
also noted that equivalents for the particular embodiments
discussed in this description may be used in the invention as
well.
[0140] All publications, patents, patent applications, or other
documents cited in this application are incorporated by reference
in their entirety for all purposes to the same extent as if each
individual publication, patent, patent application, or other
document were individually indicated to be incorporated by
reference for all purposes.
* * * * *