METABOLIC RATE INDICATORFOR CELLULAR POPULATIONS
TECHNICAL FIELD
This invention relates generally to a system and method of determining a metabolic rate
of cellular populations. More specifically, the invention relates to a system and method of
determining the metabolic rate of cellular populations through a signal generated by a bioreporter
reaction. The invention has particular relevance to such systems and methods that are manual,
semi-automated, or fully-automated.
BACKGROUND
Biological processes appear in many different industrial settings. For example,
fermentation processes pervade the biofuels, pharmaceutical, biotechnology, food, and beverage
industries. Wastewater treatment is yet another industrial setting where microbiological processes
play an important role.
The key performance indicators of industrial bioprocesses, such as final product
concentration, process efficiency, and yield, are heavily influenced by the metabolic rates of
cellular populations throughout the industrial process (e.g., fermentation and wastewater
treatment). In the majority of industrial bioprocesses, however, only environmental parameters,
such as temperature and pH, are monitored due to the lack of a simple and rapid way to measure
the metabolic activities that are directly responsible for creating the desired product. As a result,
most processes are operated without adequate information about the physiological state of
biological catalysts used in the process.
Correctly managing cellular populations is generally recognized to be one of the most
challenging components of an industrial process or bioprocess. Inconsistent or inaccurate
management practices negatively impact the consistency and efficiency of the process. However,
the standard method of cellular population analysis by methods such as light microscopy and
viability staining presents numerous difficulties and limitations in an industrial production
setting. Such difficulties include the inherent variability in results collected by different people
due to factors such as the subjective nature of discriminating cells from non-living particles and
classifying cells as either alive or dead based on a colored stain applied to the cells. Yet another
limitation is that the microscopy method has only two levels for classifying cells: alive or dead.
This binary system fails to capture the biological reality that cells exist along a continuous scale
from alive to dead and their exact position along this continuum is determined by a multitude of
factors.
Numerous examples of methods for quantifying cell concentrations can be found in the
prior art. For example, U.S. Patent Nos. 5,445,946 and 7,527,924 disclose methods and/or
devices for cell quantification using fluorescence. U.S. Patent Application Publication
2004/015721 1 discloses methods and/or devices for cell quantification using digital microscopy.
The major shortcoming of these inventions is that they provide only alternative means of
quantifying cellular populations, and not a means of measuring the metabolic rates of cellular
populations. To measure metabolic rates, it has historically been necessary to use a method of
quantifying the production carbon dioxide and/or consumption of oxygen by cellular populations
using instruments such as a respirometer, manometer, or fermontagraph, (See e.g., Water Sci
Technol. 2007;55(10):l-9. "Respirometric assessment of biodegradation characteristics of the
scientific pitfalls of wastewaters."). Such instruments, however, are difficult to operate and
maintain partially due to the complexities of measuring gasses, which are heavily influenced by
temperature, pressure, and interactions with other matter.
There thus exists an ongoing need for improved methods of analyzing cellular
populations in industrial settings. In particular, there is a need for methods and systems capable
of determining metabolic rates of cellular populations, as opposed to concentration of the cellular
populations, while also reducing the inherent variability and difficulties associated with current
practices of managing cellular populations in industrial processes.
SUMMARY
The present invention accordingly provides a method for measuring a metabolic rate of a
cellular population. In an aspect, the method comprises providing a controller operable to track
changes in a parameter. In an embodiment, the parameter is weight and the controller is in
communication with a gravimetric device and is operable to track weights of the sample, the
bioreporter, and/or the additive and/or changes in weight of matter held within a vessel caused by
addition thereof. In another embodiment, the parameter is volume and the controller is in
communication with a volumetric device and is operable to track volumes of the sample, the
bioreporter, and/or the additive and/or changes in volume of matter within the vessel caused by
addition thereof A signal detector is also in communication with the controller and operable to
detect a metabolic signal of the cellular population. The method further utilizes a vessel capable
of holding or being associated with at least the following items: (i) a sample of the cellular
population or comprising the cellular population, (ii) a bioreporter that is capable of directly or
indirectly generating the metabolic signal, and (iii) the signal detector. The sample and the
bioreporter are combined in the vessel by adding in any order, including in phases, the sample
and the bioreporter to the vessel. One or more additives may also be added to the vessel at any
time during implementation of the method. The method also includes optionally tracking
changes in the parameter at any time during implementation of the method and optionally
recording those changes. The metabolic signal is detected with the signal detector and optionally
recorded, as well as the metabolic rate being determined and optionally recorded based upon the
detected metabolic signal. Any tracking or recording of the parameter, the metabolic signal,
and/or the metabolic rate may take place at one or more times (e.g., at one or more time points).
Alternatively, such tracking or recording may take place continuously.
In another aspect, the invention provides a system operable to determine a metabolic rate
of a cellular population. The system comprises (i) a controller; (ii) an optional gravimetric
device in communication with the controller and/or an optional volumetric device in
communication with the controller; (iii) a means of collecting a sample of the cellular population
or containing the cellular population; (iv) a bioreporter capable of reacting with a metabolic
component of the cellular population in the sample to generate a metabolic signal; (v) a signal
detector operable to detect the metabolic signal and in communication with the controller; (vi) a
vessel capable of holding or being associated with at least the following items: (a) the sample of
the cellular population or comprising the cellular population, (b) the bioreporter, and (c) the
signal detector; and (vii) a user interface in communication with the controller.
It is an advantage of the invention to provide an efficient system and method for
measuring a metabolic rate of a cellular population in an industrial process.
It is another advantage of the invention to provide a semi-automated or fully-automated
system and method for analyzing a metabolic rate of a cellular population in an industrial
process.
An additional advantage of the invention is to provide a system and method not subject to
sample preparation errors due to entrained gases, particulate matter, or other factors present in the
sample that generate imprecise volumetric dilutions.
Another advantage of the invention is to provide sample preparation improvements in the
form of an automated and self-correcting method implemented in a user interface that eliminates
operator variability in sample preparation practices.
It is a further advantage of the invention to provide a system and method for determining
through a chemical reaction with a bioreporter a metabolic rate of a cellular population in an
industrial process.
An additional advantage of the invention is to determine metabolic rates of cellular
populations in a small amount of time, such as less than ten minutes, and preferably less than
three minutes.
Another advantage of the invention is to provide a precise system and method of
determining a metabolic rate of a cellular population from an industrial process, where the
system and method are robust to varying temperatures, turbidities, and background signals in the
measured sample.
A further advantage of the invention is to provide a system and method that is capable of
transmitting metabolic rate results in real-time to other information systems within an industrial
processing plant, such as databases and control systems,
Yet another advantage of the invention is to provide a system and method with adequate
precision and a high signal to noise ratio for controlling, diagnosing, adjusting, and/or optimizing
industrial processes or bioprocesses based on metabolic rate information.
It is another advantage of the invention to measure metabolic rates with a low level of
random error, for example, the measurements should preferably have a coefficient of variation
ess than 10%, and even more preferably less than 5%.
Yet another advantage is to provide a system and method of measuring metabolic rates
that is fast and parallel so as to accommodate the needs of industrial producers with many
different concurrent biological processes to manage.
It is yet another advantage of the invention to provide a system and method of detecting
the metabolic rate of a cellular population without quantifying the number of cells in the
population, either directly or indirectly.
The foregoing has outlined rather broadly the features and technical advantages of the
present invention in order that the detailed description of the invention that follows may be better
understood. Additional features and advantages of the invention will be described hereinafter
that form the subject of the claims of the invention. It should be appreciated by those skilled in
the art that the conception and the specific embodiments disclosed may be readily utilized as a
basis for modifying or designing other embodiments for carrying out the same purposes of the
present invention. It should also be realized by those skilled in the art that such equivalent
embodiments do not depart from the spirit and scope of the invention as set forth in the appended

claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates an embodiment of the invention including a manual liquid handling
station and depicting the various components present in the system of the invention and used in
implementing the method of the invention.
Figure 2 illustrates an embodiment of the invention including an integrated controller and
weighing device with an automated liquid delivery assembly.
Figure 3 illustrates an embodiment of the invention including a self-contained unit having
automated sample collection, sample handling, liquid delivery, and analyzing features.
Figure 4 illustrates fermentation performance that was monitored by tracking cumulative
product (carbon dioxide) formation over time where the rate of product formation at any point
during batch fermentation can be obtained by the slope of the cumulative product curve.
Figure 5 illustrates an embodiment of the invention where the metabolic rate indicator
and the rate of carbon dioxide production are strongly correlated.
Figure 6 illustrates an embodiment of the invention that effectively produced consistent
results across different operators, signal detectors, and batch fermentation processes.
Figure 7 illustrates fermentation performance that was monitored by tracking cumulative
product (ethanol) formation over time where the rate of product formation at any point during
batch fermentation can be obtained by the slope of the cumulative product curve.
Figure 8 illustrates an embodiment of the invention where the metabolic rate indicator
and the rate of ethanol production are strongly correlated.
Figure 9 illustrates an embodiment of the invention where the metabolic rate indicator
consistently predicted the decay in metabolic rate during cold storage of a cellular population.
Figure 10 shows the effect of using a temperature correction algorithm to effectively
correct metabolic rates.
Figure 1 illustrates improvements for turbidity-corrected metabolic rate determinations
as compared to metabolic rates determined from metabolic signals that had not been corrected for
the influence of turbidity.
DETAILED DESCRIPTION
The disclosed and claimed invention is a robust and precise system and method for the
detection and monitoring of metabolic rates of cellular populations in industrial processes. As
compared to the currently practiced methods, the disclosed system and method improve the state
of the art in several ways as detailed herein. The final result of the cumulative improvements is a
solution for metabolic rate determinations of a cellular population that is precise and robust
regardless of operator technique and sample properties, such as turbidity, temperature, and
background signal.
The present invention has application in any industrial setting where cellular populations
are encountered. Representative industrial settings having cellular populations amenable to the
present invention include production of biofuels, wines, beers, and spirits; preparation of active
and inactive yeast intended for use in beverage, fuels, or baking industries; production of
nutritional supplements, probiotics, yeast extracts, antibiotics, recombinant proteins, ethanol, or
any other industrial yeast product; wastewater treatment systems; algal growth systems; bacterial
growth systems; thermal processing systems; cooling water systems; pulp and paper mill
activated sludge; other systems; and any combination of the foregoing.
Exemplary cell types include bacteria, archae, protists, microscopic animals, fungi,
microscopic plants, animal cells, and any combination of the foregoing. In some embodiments,
the invention is also applicable to populations of extremophiles. Nonlimiting examples of certain
cell types are given below.
Representative bacteria include (industrial purpose given in parentheses) xanthamonas
campestris (xanthan gum); pseudomonas elodea (gellan (E418)); lactobacillus delbruekii var.
bulgaricus (exopolysaccharides); lactococcus lacts var. cremoris (exopolysaccharides);
lactobacillus helveticus (exopolysaccharides); lactobacillus sake (exopolysaccharides);
rhodopseudomonas capsulate (single cell protein); corynebacterium glutamicum (L-lysine);
laminaria digitata (alginate); ascophyllum nodosum (alginate); fucus serratus (alginate);
cyamopsis tetragonolobus (guar gum); ceratonia sliqua (locust bean gum (carob)); chondrus
crispus (carageenan); gigartina stellata (carageenan); hypea muciformis (carageenan); geotrichum
candidum (single cell protein); zymomonas mobilis (ethanol); leuconostoc oenos (malolactic
fermentation); Clostridium (various); escherichia coli (various); other bacteria; related species and
cell types; the like; and any combinations of the foregoing.
Representative yeast include (industrial purpose given in parentheses) saccharomyces
cerevisiae (brewing, ethanol, winemaking); saccharomyces fructuum (baking); saccharomyces
inusitatus (baking); saccharomyces exigus (baking); Candida crusei (baking); Candida steliata
(baking); Candida milled (baking); Candida utilis (baking); torulopsis Candida (baking);
torulaspora delbruekii (baking); zygosaccharomyces rouxii (soy sauce, vinegar);
zygosaccharomyces bailii (tartaric acid metabolism); kluyveromyces thermotolerans
(frozen dough); torulaspora pretoriensis (brewing); schizosaccharomyces pombe (brewing);
schizosaccharomyces malidevorans (winemaking); aspergillus oryzae (sake); saccharomyces
sake (sake); saccharomyces kefir (kefir); Candida kefir (kefir); other yeasts; related species and
cell types; the like; and any combinations of the foregoing.
Representative fungi include (industrial purpose given in parentheses) aspergillus niger
(enzymes, citric acid, gluconic acid); sclerotium rolfsii (single cell protein); polyporus (single
cell protein); trichoderma (single cell protein); scytalidium acidophilum (single cell protein);
aureobasidium pullalans (pullulan); sclerotium glucanicum (scleroglucan); other fungi; related
species and cell types; the like; and any combinations of the foregoing.
Representative algae include (industrial purpose given in parentheses) chlorella (single
cell protein); spirulina (single cell protein, nutraceuticals); other algae; related species and cell
types; the like; and any combinations of the foregoing
In a preferred embodiment, the present invention includes a controller operable to receive
and process information and provide instructions to a user and/or to various components of the
system herein described. The term "controller" refers to an electronic device having components
such as a processor, memory device, digital storage medium, cathode ray tube, liquid crystal
display, plasma display, touch screen, or other monitor, and/or other components. In an
embodiment, the controller includes an interactive interface that guides a user, provides prompts
to the user, or provides information to the user regarding any portion of the method of the
invention. Such information may include, for example, building of calibration models, data
collection, sample placement, bioreporter and additive placement, management of resulting data
sets, etc.
The controller is preferably operable for integration and/or communication with one or
more application-specific integrated circuits, programs, computer-executable instructions or
algorithms, one or more hard-wired devices, wireless devices, and/or one or more mechanical
devices such as liquid handlers, hydraulic arms, servos, or other devices. Moreover, the
controller is operable to integrate feedback, feed-forward, or predictive loop(s) resulting from the
metabolic rate determinations of the invention. Some or all of the controller system functions
may be at a central location, such as a network server, for communication over a local area
network, wide area network, wireless network, extranet, the Internet, microwave link, infrared
link, the like, and any combinations of such links or other suitable links. In addition, other
components such as a signal conditioner or system monitor may be included to facilitate signal
transmission and signal-processing algorithms.
In one embodiment, the controller is operable to implement the method of the invention
in a semi-automated or fully-automated fashion. In another embodiment, the controller is
operable to implement the method in a manual or semi-manual fashion. Examples of these
variations of the invention are provided below in reference to the figures.
A dataset collected from a cellular population in an industrial process, for instance, may
include variables or system parameters such as bioreporter signal, oxidation-reduction potential,
H, levels of certain chemicals or ions (e.g., determined empirically, automatically, fluorescently,
electrochemically, colorimetrically, measured directly, calculated, etc.), temperature, turbidity,
pressure, process stream flow rate, dissolved or suspended solids, etc. Such system parameters
are typically measured with any type of suitable data measuring/sensing/capturing equipment,
such as pH sensors, ion analyzers, temperature sensors, thermocouples, pressure sensors,
corrosion probes, and/or any other suitable device or method. Devices capable of detecting or
sensing colorimetric, refractometric, spectrophotometric, luminometric, and/or fluorometric
signals are of particular utility for the present invention. Such data capturing equipment is
preferably in communication with the controller and, according to alternative embodiments, may
have advanced functions (including any part of the control algorithms described herein) imparted
by the controller.
Data transmission of any of the measured parameters or signals to a user, chemical
pumps, alarms, or other system components is accomplished using any suitable device, such as a
wired or wireless network, cable, digital subscriber line, internet, etc. Any suitable interface
standard(s), such as an ethernet interface, wireless interface (e.g., IEEE 802.1 a b/g n, 802.16,
Bluetooth, optical, infrared, other radiofrequency, any other suitable wireless data transmission
method, and any combinations of the foregoing), universal serial bus, telephone network, the
like, and combinations of such interfaces/connections may be used. As used herein, the term
"network" encompasses all of these data transmission methods. Any of the components, devices,
sensors, etc. herein described may be connected to one another and/or the controller using the
above-described or other suitable interface or connection.
In an embodiment, information (collectively referring to all of the inputs or outputs
generated by the method of the invention) is received from the system and archived. In another
embodiment, such information is processed according to a timetable or schedule. In a further
embodiment, such information is immediately processed in real-time/substantially real-time.
Such real-time reception may also include, for example, "streaming data" over a computer
network.
Referring now to the figures, it should be appreciated that the characteristics set forth in
relation to any of the embodiments depicted in the figures herein may be applied and
implemented in relation to any of the other embodiments depicted in the other figures or herein.
FIG 1 illustrates an embodiment of the invention including a manual liquid handling station and
depicting the various components present in the system of the invention and used in
implementing the method of the invention. In order to obtain precise measurements, the instant
invention provides a method of preparing the sample for measurement by combining the sample
and required chemical reagents in vessel 130 on weighing device 120 that is in communication
with controller 110. Controller 110 is linked to weighing device 120 and signal detector 140 via
communication links 115a and 115b, In an embodiment, controller 110 is operable to provide a
prompt to a user to place vessel 130 onto weighing device 120. Such a prompt may take the form
of a visible and/or audible prompt that the user follows. Such prompts, according to an
embodiment, include a certain order of addition into vessel 130. For example, the prompts may
suggest target weight values for each component based on previous events. Weighing device 120
then transmits to controller 110 any changes in weight due to the addition of liquids into vessel
130. Controller 110 is further operable to provide a prompt to the user to add a sample of the
cellular population taken from an industrial process. The sample may be placed into vessel 130
using any suitable means for transfer. For example, a common method of transferring a liquid
sample is through the use of a pipette or other suction-operated device.
The term "vessel" as used in the figures and herein refers to any container capable of
holding a volume of liquid and of being weighed on a weighing device. In some embodiments,
the vessel further includes a signal detector, a means of controlling the temperature of the liquid
held within the vessel, and/or a means of agitating/mixing the liquids held within the vessel. It
should be appreciated that any of the vessels or liquid chambers, containers, dispensers, etc.
herein described may include a signal detector, a temperature controlling mechanism, and/or an
agitation/mixing mechanism.
During the transfer of the sample into vessel 130, weighing device 120 is operable to
communicate weight data to controller 10 to track changes in weight of vessel 130 due to such
addition of sample. This weight tracking may take place continuously or at one or more time
points during the transfer of the sample. Controller 110 is further operable to alert the user to
insert signal detector 140 into vessel 130. In another embodiment, signal detector 140 is inserted
into vessel 130 prior to or at any time during transfer of the sample into vessel 130. In a further
embodiment, signal detector 140 is placed on the outside of vessel 130. In yet another
embodiment, signal detector 140 is an integral part of vessel 130. Controller 1 0 is further
operable to provide a prompt, for example visual or audible, to the user to transfer additive 150
(e.g., buffering agent, pH modifying agent, catalyst, coenzyme, mineral, co-substrate, and/or any
other additive needed in conjunction with bioreporter 60) and/or bioreporter 160. Weighing
device 0 continues to communicate weight change data to controller 110.
At some point during combining the sample with additive 150 and/or bioreporter 160 a
reaction takes place that generates a metabolic signal that is detectable by signal detector 140.
The metabolic signal may take the form of, for example, a colorimetric, refractometric,
spectrophotometric, luminometric, fluorometric signal, or any combinations of the foregoing. To
detect the signal, any suitable signal detector 140 may be used
The term "signal detector" refers to any suitable device capable of detecting a metabolic
activity signal as herein described. In addition, the signal detector may have the capability of
detecting more than one type of signal. For example, a particular signal detector may have the
capability of detecting more than one wavelength of a colorimetric, refractometric,
spectrophotometric, luminometric, and/or fluorometric signal, or may have the capability of
detecting a combination of such signals. In another example, the signal detector may be capable
of detecting any combination of the foregoing signals and also a turbidity and/or temperature
detection and/or measurement. In another embodiment, the term "signal detector" means a
combination of a plurality of signal detectors. For example, one signal detector may comprise a
flourometer and another may comprise a turbidity meter. It should be appreciated that the term
"signal detector" may comprise any combination of the described detectors. Furthermore, the
signal detector may be a separate component or may be combined with, for example, the vessel
and/or the measuring device as an integrated unit. Though any suitable signal detector(s) may be
used, exemplary fluorometric signal detectors are disclosed in U.S. Patent Nos. 6,369,894,
"Modular Fluorometer"; 6,670,617, "Mirror Fluorometer"; and 7,095,500, "Interchangeable Tip
Open Cell Fluorometer."
FIG 2 illustrates an embodiment of the invention including integrated controller and
weighing device assembly 200 with automated or semi-automated pump assembly 250. In this
embodiment, controller 210 is integrated with weighing device 220 and includes an internal
communication link (not shown) for communication between controller 210, weighing device
220, and other components of the system. As described above in relation to FIG 1 for controller
110, controller 210 is operable to provide prompts or other signals to a user to perform various
steps of the method of the invention. As above, controller 210 provides a visual or audible
prompt to a user to place vessel 230 onto measuring device 220. Alternatively, a mechanical arm
or other device (not shown) may place vessel 230 onto measuring device 220. In another
alternative, measuring device 220 and vessel 230 are an integrated unit.
Communication ink 215 sends signals from signal detector 240 to controller 210. Signal
detector 240 may be introduced into or associated with vessel 230 at any time during addition of
the combination of described components into vessel 230. Measuring device 220 then records
any changes in weight due to the addition of liquids into vessel 230. Controller 210 is further
operable to provide a prompt to the user to add a sample of the cellular population taken from an
industrial process. In another embodiment, a mechanical arm or other device aids in adding the
sample to vessel 230. Other common methods of transferring a liquid sample, for example,
include the use of a pipette or other suction-operated device. During the transfer of the sample
into vessel 230, measuring device 220 is operable to communicate weight data to controller 210
to track changes in weight of vessel 230 due to such addition of sample. This weight tracking
may take place continuously or at one or more time points during the transfer of the sample.
Controller 10 is further operable to alert the user to insert signal detector 240 into vessel 230. In
another embodiment, signal detector 240 is inserted into vessel 230 prior to or at any time during
transfer of the sample into vessel 230 either manually by the user or automatically through a
mechanical arm or other device (not shown). In a further embodiment, signal detector 240 is
placed on the outside of vessel 230. In yet another embodiment, signal detector 240 is an integral
part of vessel 230.
Controller 210 is further operable to provide a prompt, for example visual or audible, to
the user to initiate pump 280 to transfer an additive (e.g., buffering agent, pH modifying agent,
catalyst, coenzyme, or any other additive needed in conjunction with the bioreporter) from
additive compartment 260 and/or bioreporter compartment 270 for delivery into vessel 230 via
dispensing nozzles 285a and 285b. In another embodiment, an automated system comprising a
mechanical arm or other device initiates and/or executes delivery of the additive and/or the
bioreporter into vessel 230. The sizes of additive compartment 260 and bioreporter compartment
270 may be varied as needed for a particular application. In other embodiments, additional liquid
compartments for additional components or backup additive or bioreporter solutions may also be
included. Caps 270a and 270b are intended for refilling any needed solutions.
In further embodiments, pump assembly 250 is fully-manual, semi-automated, or fullyautomated.
Pump assembly 250 is, according to an embodiment, fully-manual where a user
initiates the pumping action in response to prompts provided by controller 210. In another
embodiment, assembly 250 is semi-automated where a user performs certain functions based on
prompts provided by controller 210 and other functions are automated. In a further embodiment,
assembly 250 is fully-automated and responds to instructions sent by controller 210 to dispense
solutions into vessel 230.
In FIG 3, an embodiment of the invention including a self-contained unit having sample
handling and analyzing features is illustrated and shown as mobile unit 300. Any of the
functions described below for mobile unit 300 may be fully-automated, or semi-automated. A
number of conduits and other channels for flow of samples, bioreporters, solutions, and the like
are shown in relation to mobile unit 300. It should be appreciated that additional pumps, valves,
or conduits may be included in mobile unit 300 as needed for any particular application. Shown
is controller 310 which in communication with several other components of mobile unit 300 and
is operable to send instructions to carry out the various described functions. Weighing device
320, vessel 330, and signal detector 340 are shown as an essentially integrated component. It
should be appreciated that these components may or may not be permanently attached to each
other. In this embodiment, signal detector 340 is shown disposed inside of vessel 330. In other
embodiments, signal detector 340 is disposed outside of vessel 330.
Sample collection port 303 may be directly connected or connected via a sample
sidestream (not shown) to process tank 302. Process tank 302 may be a main process tank, a
secondary process tank, or any suitable access line (e.g., sidestream) to a cellular population in an
industrial process. In an embodiment, sample collection port 303 includes a mechanism to create
a seal to prevent leaks before, during, and after a connection to sample line 306 is established via
joint 304. In an embodiment, joint 304 includes a means to create a fluid communication channel
between sample collection port 303 and sample line 306 so as to allow a sample of the cellular
population to be drawn into sample line 306. Joint 304 may also include an electronic
communication means to allow communication between sample collection port 303 and sample
line 306. The electronic communication may be achieved through any suitable means including,
for example, conductive pads on the mating surface of the components, one or more electrical
connectors, the like, and combinations thereof. When a connection between sample collection
port and sample line 306 is made, controller 0 is operable to send instructions to initiate a
sample of the cellular population to be drawn and sent to vessel 330 through sample line 306 and
valve 308.
In an embodiment, mobile unit 300 includes reagent containers 352, 354, and 356.
Although three reagent containers are shown in this embodiment, it should be appreciated that
any number of reagent containers may be used. Each of the reagent containers may have its own
valve or may have a combined valve that is in fluid communication with more than one of the
reagent containers. Such a combination valve is shown as valve 312 in FIG 3. The reagent
containers may house any component of a bioreporter and/or additive as described herein.
Conduits 31 a and 3 8b provide a mechanism for exhausted samples, bioreporters, additives, etc.
to be discarded. Depending on the particular application, any of the valves shown (e.g., 308,
312, 314) and other valves that may be part of mobile unit 300 actuate to cause spent liquids to
travel through conduits 318a and 318b to waste container 360. Other conduits may also be
present in mobile unit 300 as needed for a particular application.
In an embodiment, mobile unit 300 further includes wash solution container 362. In FIG
3, valve 314 is shown as operable to cause wash solution housed in wash solution container 362
to be drawn through conduit 318c. As explained above in relation to the reagent containers, it
should be appreciated that any number of wash solution containers may be used. Moreover, each
of the wash solution containers may have its own valve or may have a combined valve that is in
fluid communication with more than one of the other wash solution containers as needed for a
particular application. Ports 364a and 364b are in fluid communication with wash solution
container 362 and waster container 360, respectively. The position and number of these ports is
exemplary and it should be realized that any number of ports and positioning of ports may be
employed as needed for a particular application. Representative wash solutions include water,
mild detergent solutions, weak acids, the like, other solutions as needed, and any combinations of
such solutions.
In an embodiment, mobile unit 300 may be attached to cart 370 having wheels 372a and
372b. Cart 370 may be, for example, permanently molded into or attached to mobile unit 300 or
may be a separate component onto which the remaining components of mobile unit 300 are
placed to provide mobility. In an embodiment, mobile unit 300 further comprises a navigation
control system (not shown) operable to cause mobile unit 300 to travel automatically or semiautomatically
to various locations in an industrial facility and analyze samples of cellular
populations therein. Such autonomous mobile robotic devices are disclosed in, for example, U.S.
Patent No. 7,024,278, "Navigational Control System for a Robotic Device" and taught in
Guilherme, N. D. and C. K. Avinash (2002), "Vision for Mobile Robot Navigation: A Survey,"
IEEE Trans. Pattern Anal. Mach. Intell. 24(2): 237-267.
The order of addition of any of the described solutions (e.g., sample of cellular
population, bioreporter, additive) may occur in any order as determined by the user and/or the
controller as applicable to a particular industrial setting. For example, the sample of the cellular
population may be added first followed by the bioreporter and any needed additives, or the
bioreporter/additives may be added first followed by the sample of the cellular population. As
described herein, changes in weight of the vessel to which these components are added as well as
the generated metabolic signal may be tracked and/or recorded in real-time or substantially realtime
through communication with the controller.
While the use of a weighing device is the preferred method of sample preparation, other
adequate methods of sample preparation that do not require a weighing device are envisioned. In
one embodiment, the sample could be prepared volumetrically. For example, the process sample,
bioreporter, and any other required additives might manually be added to the vessel by means of
a measuring spoon, transfer pipette, medicine dropper, by counting drops, or by pouring. The
vessel may also have gradations or zones marked on it to indicate the approximate volume
intended for different components. The vessel may be a common laboratory item such as a
graduated cylinder, a beaker, or a tube with lines.
In another embodiment, the sample is prepared, either in whole or in part, by the action of
mechanical (manual, semi-automated, or fully-automated) pumping. One or several pumps may
be employed, and each may be designed to pump at different rates and/or frequencies to
accommodate the desired proportions of different components in the final mixture. The pumping
action may be controlled by a program that turns the pumps on and off at precisely scheduled
intervals in order to achieve precise ratios of the different components. The controller may also
receive feedback via level sensors, weight sensors, mass flow meters, volumetric flow meters,
other sensors and/or meters, or any combination of the foregoing in order to determine when to
switch the pumps on and off. In another embodiment the process sample, bioreporter, and any
other additives might be measured by filling void sections of piping or tubing prior to mixing or
at the time of mixing. The voids may be filled by a variety of means, for example manually via
pouring, by the action of gravity, or by the action of negative or positive pressure. The voids
may also be a primed process sample, bioreporter, and any other additives.
The term "bioreporter" refers generally to any molecule capable of reacting with a
component inherently present or inducible in the cellular population of the industrial process to
generate a detectable metabolic signal. A variety of approaches are available for generating a
metabolic signal. The preferred approach is to use a biochemical reporter molecule that reacts
with native enzymes to produce a detectable signal. The generated signal should preferably be a
light emitting signal, and most preferably be a fluorescent light emitting signal. In alternative
embodiments, the generated signal is detectable via methods including colorimetric,
refractometric, spectrophotometric, luminometric, fluorometric, or any combinations of the
foregoing.
In different embodiments, the bioreporter may take one or more of the following forms:
one reagent in one solution; more than one reagent in one solution; more than one reagent each
reagent in a separate solution; one reagent in one dry form; more than one reagent in one dry
form; more than one reagent each reagent in a separate dry form; the like; and any combination
of the foregoing.
In a preferred embodiment, the bioreporter is a molecule or group of molecules that can
react with a broad set of enzymes so as to create a detectable signal in a short amount of time.
Targeting a broad set of enzymes also ensures that the signal correlates with the overall
metabolic rate of the cellular population, and that the approach is feasible for use over a wide
range of conditions and metabolic rates. In an embodiment, the set of reaction enzymes is shared
almost universally among different organisms. In another embodiment, the set of reaction
enzymes is species or cell type specific to allow differentiation among such species or cell types.
In an embodiment, the bioreporter should also be capable of penetrating the cell wall and/or
plasma membrane structures to gain access to the cytoplasmic enzymes. In some cases, this
penetration may be aided by the use of other factors, such as detergents. In other embodiments,
the sample of the cellular population is subjected to chemical or mechanical shear to lyse the
cells and the bioreporter would not need to be capable of such penetration.
In an embodiment, the signal generated by the metabolic activity of the cellular
population preferably conforms to certain expected behaviors. For example, the signal should
not exhibit an extremely short signal lifetime, and should not suffer from severe dependence on
pH, temperature, ionic strength, etc. Effective bioreporters include those substances that are
consistently chemically reactive with known components in the cellular population and that do
not significantly degrade with time (i.e., have an acceptable shelf life). Preferably the signal
intensity should be substantially proportional to its concentration and not significantly quenched
or otherwise diminished by other components in the cellular population. Furthermore, the
generated signal should be at manageable wavelengths (e.g., other components in the cellular
population should not interfere with the signal properties at those wavelengths) and
excitation/emission wavelengths (in the case of a fluorescent signal) that are separate from other
fluorescent components that may be present.
Representative bioreporters capable of being converted into a molecule that reacts to
generate a fluorescent signal for use in the invention include derivatives of naphthalene
disulfonic acid; aminomethyl coumarin; pyrene; pyrene tetrasulfonic acid; 4-raethyl umbelliferyl;
cascade yellow; fluorescein; carboxyfluorescein; bodipy-FL; bodipy-TR tetramethyl rhodamine;
rhodamine 110; carboxyrhodamine; lucifer yellow; Hoechst 33342; sulforhodamine 110;
resorufin; napthafluorescein; SNARF-1; cyanine dyes; the like; other fluorophores; and any
combinations of the foregoing.
To generate a bioreporter molecule, the above-listed or other suitable fluorescent
molecules can be, for example, derivatized with a suitable leaving group. The leaving group
functions as the enzmatically labile portion of the bioreporter molecule. The leaving group a so
functions to alter the fluorescent properties of the fluorescent molecule so that the bioreporter is
in one fluorescent state when complexed with the leaving group, and in a second fluorescent state
when the leaving group is enzymatically cleaved from the core fluorescent molecule.
In another embodiment, a suitable bioreporter configuration involves separating a
fluorescent molecule and a quencher molecule by a short biopolymer of nucleic acids, proteins,
carbohydrates, or lipids. Many cellular enzymes function to degrade biopolymers. In an
embodiment, the bioreporter is a hairpin shaped DNA molecule with an internally quenched
flourophore, the fluorescence of which is restored upon degradation of the DNA molecule by
nuclease enzymes present in the cellular population. Another suitable bioreporter molecule
involves labeling a biopolymer at a high density with one fluorescent molecule that is able to
quench its own fluorescence at high local concentrations. Upon degradation of the biopolymer,
short segments would be released that fluoresce.
It should be appreciated that the set of possible combinations between leaving groups and
fluorescent core molecules, and between biopolymers and fluorophore/quencher pairs, is
extremely large and suitable combinations may be determined by one skilled in the art.
Nonlimiting examples of certain bioreporter chemistries that are useful within the context of the
instant invention are given below.
Representative bioreporter chemistries include (broad classes of target enzymes given in
parentheses) 4-methy!umbelliferyl glucopyranoside (glycosidase), 6,8-difluoro-4-
methylumbelliferyl xylanoside (glycosidase), 6 8-diftuoro-4-methylumbelliferyl glucopyranoside
(glycosidase), 4-(Trifluoromethyl)umbelliferyl galactopyranoside (glycosidase), fluorescein-di-
B-D- glucopyranoside (glycosidase), fluorescein-di-B-D- galactopyranoside (glycosidase),
naphthalimide phosphate (phospatase), 4-nitrophenyl phosphate (phospatase), 4-
methylumbelliferyl phosphate (phospatase), 6,8-difluoro-4-methyIumbelliferyl phosphate
(phospatase), di-O-phosphatidylfluorescein (phospatase), O-phosphatidylfluorescein
(phospatase), di-O-acetyl 5-chloromethylffuorescein (lipase/esterase), di-Oacelylsulfofluorescein
(lipase/esterase), di-O-acetyi -(6) carboxynaphthofluorescein
(lipase/esterase), 6,8-difluoro-4-methylumbelliferyl butyrate (lipase/esterase),
methylumbelliferyl-butyrate (lipase/esterase ), 4-(Trifluoromethyl)umbelliferyl butyrate
(lipase/esterase), di-O-propionylfluorescein (lipase/esterase), O-propionylfluorescein
(lipase/esterase), di-O-butyrylfluorescein (lipase/esterase), O-butyrylfluorescein
(lipase/esterase), di-O-acetylfluorescein (lipase/esterase), O-acetylfluorescein (lipase/esterase),
resorufin acetate (lipase/esterase), resorufm isobutyloxycarbonyloxy (lipase/esterase), 4-
methylumbelliferyl sulfate (sulfatase), 6,8-difluoro-4-methylumbelliferyl sulfate (sulfatase),
aminomethylcoumarin-L-leucine (protease), Gly-Pro-aminomethylcoumarin (protease), resazurin
(oxidoreductase), fluorophore-polypeptide-quencher (protease), fluorophore-carbohydratequencher
(glycosidase), fluorophore-lipid-quencher (lipase), fluorophore-polynucleotidequencher
(nuclease); other bioreporters; the like; and any combinations of the foregoing.
An exemplary embodiment of the invention is the following. In the process of preparing
the sample for measurement, the instant the bioreporter comes in contact with the cellular
population a biochemical reaction begins that produces a metabolic activity signal. The instant
invention employs several methods to eliminate variability in metabolic rate measurements due
to reaction timing. First, the controller/weighing device pairing automatically records the precise
moment that the bioreporter solution contacts the cellular population sample. This was a
shortcoming of the prior art since the user was responsible for both mixing the solutions and
starting the timer. Second, the reaction start time recorded by the controller/weighing device
pairing is used by the controller/signal detector pairing to automatically measure the signal in the
prepared sample at precisely timed intervals according the instructions operated by the controller:
By removing the burden of timing from the human user and automating the responsibility
through controller/weighing device and controller/signal detection parings, the invention
effectively enables metabolic rate measurements through precise reaction timing.
Another notable advantage of the present invention's signal detector-into-sample
embodiment is that progress of the reaction can be monitored continuously from beginning to
end, which would be difficult to impossible in the prior art due to the need to transfer the sample
between a temperature control device and the detection instrument. With a continuous reaction
monitoring system, it is possible to calculate the reaction rate as opposed to the reaction endpoint
as practiced in the prior art. Measuring reaction rates is more reproducible than measuring
reaction endpoints because the rate calculation takes into consideration the variable background
signals present at the beginning of the reaction, the dilutions performed during sample
preparation, and the reaction temperatures.
Furthermore, certain signals, especially those detected through optical means, are difficult
or impossible to accurately measure in samples with turbidity, or light scattering properties,
unless the signal detection instrument has an associated calibration model the captures the effects
of sample turbidity on signal strength. The instant invention may employ, for example, a dual
channel signal detector, where one channel is designed to measure fluorescence and the other
channel designed for measuring turbidity. In addition, the instant invention may utilize a
software procedure for building and storing calibration curves that model the signal detector's
response to increasing turbidity from the process sample. Together, these improvements allow
for accurate signal detection in sample compositions of different turbidities.
In many instances, the described system and method will require calibration to ensure
continued precision. Though any suitable method of calibration may be used, it should be
realized that calibration of the signal detection instruments in particular is important for several
reasons. First, the instruments may drift over time due to internal fluctuations in the signal
detection mechanisms. Second, when a plurality of instruments is simultaneously or sequentially
used, normalization of inherent differences in signal detection properties aids to produce
consistent results. Third, the properties of the samples being measured can change over time.
For example, with regard to fluorescence detection, many conventional systems use a quick twopoint
method of calibration. This method involves calibrating by measuring a blank sample
containing no fluorescent signal, and then measuring a second sample containing a standard
amount of fluorescent signal. While this approach can work effectively for quantifying
fluorescence in samples that contain zero turbidity, the majority of samples of cellular
populations encountered in industrial systems possess varying amounts of turbidity. Turbidity
effects the detection of fluorescent signals in unpredictable ways. For instance, turbid matter can
scatter fluorescent light away from, or towards, the signal detector. Also, turbid matter can
absorb excitation and/or emission light and prevent it from exciting the fiuorogenic molecules or
from reaching the signal detector. For these reasons, it is preferred to calibrate against a mixture
of the process sample and a standard solution rather than calibrating against a standard solution.
An exemplary approach to perform such calibration is to build a first calibration curve
using a solution with zero fluorescence, and a second curve using a solution with a known
amount of fluorescence. Each of the curves should contain three or more measurements made on
different proportions of process sample mixed with the respective solutions. Even more
preferable is to use a structured calibration work flow encoded in a user interface and to prepare
the calibration samples on a digital balance (i.e., weight measuring device) in communication
with a controller. In this way, it is possible to build and store multiple models for samples of
different compositions, to perform quality control checks as the models are being constructed,
and to capture other information such as the total suspended solids (TSS) information for the
calibration sample and to use this information to report back the TSS of an unknown sample
measured at some point in the future. Yet another advantage to the preferred approach is that it
creates a defined calibration space with respect to fluorescence and turbidity, which is useful
because samples intended for metabolic rate measurement can be prepared with some flexibility
on the digital balance and still fall within the space of the calibration model.
It is well known that the metabolic rate of cellular populations is temperature dependent.
Higher temperatures will generally produce higher metabolic rates, unless the temperature
becomes too high for the enzymes to properly function. Conversely, lower temperatures
typically result in reduced metabolic rates. If the temperature becomes too low, however, the
metabolic rate may be reduced to cause the amount of metabolic signal produced via reaction
with the bioreporter to be decreased to a level too low to be accurately detected by the signal
detector. Several other factors become important under these circumstances (e.g., background
signal) and it is generally preferable to avoid measuring rates when the metabolic signal is
insufficient. In instances where the temperature range encountered has such an effect, it is
preferable to control the temperature of the prepared sample within temperature limits. While
this approach offers the possibility of exquisite control, it typically requires specialized
equipment and attentive care from the operator to ensure that the equipment is functioning
correctly. For example, an effective temperature control scheme might involve the use of
temperature controlled reagents, or a temperature controlled environmental chamber in which the
measurements are performed, or a vessel equipped with one or more heating elements. If the
ambient climate is cold enough, or if the cellular population is small enough, it may be necessary
to artificially elevate the temperature of the prepared sample through any necessary means of
temperature control to generate a sufficiently high signal for accurate detection and rate
calculations.
The preferred approach is to allow the temperature to fluctuate within the limits of room
temperature. With this approach it is important to measure the temperature of each reaction, and
to have a reasonable correction factor for the metabolic rate of the cells in the prepared sample to
correct for fluctuations in temperature. This information would then be used by a software
program or other algorithm in the controller to calculate all metabolic rates as if they were
collected at a standard temperature. When taking this approach, the temperature normalization
algorithm wi l become increasingly important when the measured temperature is farther from the
reporting temperature. For this reason, the standard temperature for reporting metabolic rates
might be 24°C (75°F) because this is close to the average room temperature. However, the
reporting temperature can be set to other values if desired.
Previous attempts to manage the influence of temperature on biological reactions
involved the use of various strategies to control the reaction temperature. These methods were
problematic because the temperature control was difficult to manage, the temperature difference
between the desired and actual temperature was usually unknown, and because the user was
responsible for transferring the sample between a temperature control device and the signal
detection instrument. To overcome these limitations and eliminate the measurement variability
that they inherently introduce, the invention advances the state of the art on several fronts. First,
the invention, for example, may utilize a signal detector-into-sample approach as opposed to a
sample-into-cuvette-into-signal detector approach found in the prior art. The invention may also
utilize a signal detector with a built-in thermistor, or digital thermometer. The signal detectorinto-
sample approach and the built in thermometer enable direct measurement of the sample
temperature during the reaction because the signal detector is not separated from the sample by a
plastic or glass cuvette. The improved method may also utilize the reaction temperature data and
a temperature correction algorithm to report the metabolic rate at a standardized temperature each
time. As a result, the user is free from the burden of using a temperature control device or even
from knowing the temperature of the reaction.
In some cellular populations, substances that interfere with accurate metabolic signal
measurements may be present, or when the cellular population is a mixed population of more
than species. In such cases, it may be desirable to apply filtration or other means of separation
(e.g., centrifugation, coagulation, or floatation) before or after the metabolic rate measurement.
Separation of interference-causing substances or separation by species and/or ce l size may be
desirable to produce more specific and valuable measurements of metabolic rate. For example,
in a mixed population a certain species may produce an enzyme that predictably reacts with the
bioreporter to produce a precise signal for determining metabolic rate and another species may
produce an enzyme that quenches the signal or inhibits the reaction. Moreover, it may desirable
to collect a sample of the cellular population, separate one or more cell types or species (or
simply separate all cells) from the medium present in the industrial process, and re-suspend the
cells in another solution (e.g., buffer or water) to enhance the reaction with the bioreporter. In
other cases, it may beneficial to dilute the obtained sample. In these embodiments, the controller
would be further operable to correct any measurements according to a dilution factor correction.
Another significant improvement of the present invention over the prior art relates to the
benefits of using a computer software program to conduct various elements of the method. At a
basic level, the software provides the benefit of improved accuracy and reduced errors
attributable to assigning tasks to the computer that were previously assigned to the human
operator. For example, by not having to manually transfer metabolic rate information from the
instrument to a permanent storage location, such as record book, spreadsheet, or database, the
integrity of the information is conserved. The software program ensures the reliability of the
system by guiding the measurement process all the way from sample preparation, data collection,
numerical calculations, and results archiving. Such a system can be useful for improving process
operations by enabling the comparison of operators, conditions, and even performance across
plants within the same company. At a higher level, the software program enhances the
functionality of the system by enabling features that would be difficult to impossible using
methods known from the prior art. For example, each measurement may collect >100 data points
and perform >50 calculations before deriving the final metabolic rate for that sample. In
addition, the digital information resulting from the use of software driven measurement systems
allows for further value to be derived from the measurement. For example, it is now possible to
input metabolic rate data to a model-based control algorithm intended to monitor and improve the
state of the process. Likewise, data collected with the system can be viewed in real-time by all
interested parties using intranet, extranet, or internet connectivity.
Using any of the embodiments herein described, or any combinations of various elements
within those embodiments, the detected metabolic signal is analyzed by the controller. The
controller is operable to execute an algorithm, program, executable digital instruction, or the like
to determine the metabolic rate of the cellular population based upon the metabolic signal. The
metabolic rate is used to implement methods to control and/or manage the cellular population (or
any one or more species within the cellular population) for any purpose that is the objective of
the particular industrial process being tested.
An example of the basic steps implemented by a controller for processing a metabolic
signal to produce a metabolic rate indicator can be understood from the following outline of
events. The method includes a first step of converting the arbitrary units of the measured signal
into units of defined signal concentration. This conversion is conducted by reference to the
calibration model, which defines the amount of signal expected from the signal detector when
measuring solutions with known concentrations of signal mixed with varying proportions of
process sample at a known temperature. The result of this first step is an accurate measurement
of signal concentration that accounts for the interference of turbidity and temperature on signal
strength. Converting the measured signal units into units of defined signal concentration occurs
at least twice during the measurement procedure, although there is no limit to the number of
times that these measurements and subsequent conversions can be conducted.
The second step of processing metabolic signals to produce a metabolic rate indicator
involves calculating the magnitude in signal concentration change between the initial
measurement time and the final measurement time. The change in signal concentration is
divided by the change in time between the initial and final measurement to achieve an average
rate of signal change. However, in order to produce a metabolic rate indicator it may be
necessary to further process the data to adjust for the influence of sample dilution and sample
temperature. Accordingly, the third step may be to multiply the rate of change calculated for the
prepared sample by the magnitude of dilution used to prepare the sample. The magnitude of
dilution may be determined according to the information transmitted from the weighing device to
the controller during sample preparation. The fourth and final step may be to adjust the
metabolic rate to account for the difference between the actual temperature at which the reaction
was performed and the standard temperature used for reporting. In practice, this may involve
converting the metabolic rate measured at the actual reaction temperature into the metabolic rate
that would have been observed had the reaction been performed at a different temperature. The
metabolic rate at a particular temperature can be calculated using a suitable mathematical
equation (e.g., a two-point form of the Arrhenius equation) and a reasonable correction factor if it
is known for some other temperature.
The foregoing may be better understood by reference to the following examples, which
are intended for illustrative purposes and are not intended to limit the scope of the invention.
Example 1
Metabolic Rate of Carbon Dioxide Formation
In this example, it is demonstrated that the metabolic rate indicator of the instant
invention correlates with the actual rate of product (carbon dioxide) formation during batch
fermentation. A batch fermentation of liquefied corn mash (32% total solids) was performed
with newly propagated yeast. Fermentation performance was monitored by tracking cumulative
product formation over time with a continuous carbon dioxide off-gas analyzer (FIG 4), The rate
of product formation at any point during batch fermentation can be obtained by the slope of the
cumulative product curve. As shown in FIG 5, the rate of product formation and the metabolic
rate indicator of the instant invention will show a strong correlation using the bioreporter i-Opropionylfluorescein
and a pH 7.6 buffer will show a strong correlation.
Example 2
Independent Signal Detectors. Operators, and Batch Fermentations
This example illustrates the consistent performance of the metabolic rate indicator across
different operators, signal detectors, and batch fermentations. Over a one month period, five
different operators collected a total of 300 metabolic rate measurements with the system of the
invention using the bioreporter di-0-acetylfluorescein and a pH 7.6 buffer across 25 different
batch fermentations in an industrial ethanol plant. Each sample was tested once with a first
signal detector unit and then re-tested with a second signal detector unit. The signal detectors
were periodically calibrated against a mixture of the process sample and standard solutions
during the one month trial. As shown in FIG 6, the method produced consistent results across
different operators, signal detectors, and batches.
Example 3
Metabolic Rate of Ethanol Formation
In this example, it is demonstrated that the metabolic rate indicator of the instant
invention correlates with the rate of ethanol formation during batch fermentation. An industrial
batch fermentation of liquefied corn mash was performed with newly propagated yeast.
Fermentation performance was monitored by tracking cumulative product formation over time
through intermittent high performance liquid chromatography analysis (FIG 7). The rate of
product formation at any point during batch fermentation can be obtained by the slope of the
cumulative product curve. As shown in FIG 8, the rate of ethanol formation and the metabolic
rate indicator of the instant invention using the bioreporter O-acetylfluorescein and a pH 7.6
buffer will show a strong correlation.
Example 4
Metabolic Rate Decay of a Cellular Population during Storage
In some circumstances, cellular populations are added directly into a fermentation vessel
from a storage environment. This practice is common in the brewing and baking industries, as
we l as in some fuel ethanol processes where cell recycling occurs. This situation also occurs
when the propagation step is skipped and cells in dry form are pitched directly into a
fermentation vessel. Under all of these circumstances, it is desirable to know how much ce l
matter to add. This example demonstrates that the method of the instant invention can be used to
predict decaying metabolic rates of a stored population of the bacterium xanthamonas campestris
using the bioreporter 6,8-difluoro-4-methylumbelliferyl butyrate and pH 7.6 buffer (FIG 9). This
information can be used to determine the amount of cells to add into the next batch rather than
adding based on the amount of solids or on the amount of viable cells.
Example 5
Temperature Normalized Metabolic Rates
This example demonstrates that the method and system of the instant invention can be
used to effectively normalize metabolic rates measured at different temperatures for a cellular
population of algae measured over the range of normal room temperatures using the bioreporter
resorufin acetate and a p 8.7 buffer (FIG 10).
Example 6
Turbidity Corrected Metabolic Rates
This example demonstrates effective turbidity corrected metabolic rates with the method
and system of the instant invention. Metabolic rates for a cellular population of yeast were
measured with the method and system of the invention using the bioreporter di-Oacetylfluorescein
and a pH 7.6 buffer in the presence of increasing amounts of turbidity
contributed by the non-cellular matter in a solution of spent sugar cane extract (FIG 11). A first
set of measurements were made in reference to a calibration model that was constructed in the
absence of any non-cellular turbidity (uncorrected data). A second set of metabolic rate
determinations were made from the same samples but in reference to the appropriate calibration
models constructed in the presence of 0%, 50%, or 100% relative sample turbidity.
All of the compositions and methods disclosed and claimed herein can be made and
executed without undue experimentation in light of the present disclosure. While this invention
may be embodied in many different forms, there are described in detail herein specific preferred
embodiments of the invention. The present disclosure is an exemplification of the principles of
the invention and is not intended to limit the invention to the particular embodiments illustrated.
Any ranges given either in absolute terms or in approximate terms are intended to
encompass both, and any definitions used herein are intended to be clarifying and not limiting.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the
invention are approximations, the numerical values set forth in the specific examples are reported
as precisely as possible. Any numerical value, however, inherently contains certain errors
necessarily resulting from the standard deviation found in their respective testing measurements.
Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges
(including all fractional and whole values) subsumed therein.
Furthermore, the invention encompasses any and all possible combinations of some or all
of the various embodiments described herein. Any and all patents, patent applications, scientific
papers, and other references cited in this application, as well as any references cited therein, are
hereby incorporated by reference in their entirety. It should also be understood that various
changes and modifications to the presently preferred embodiments described herein will be
apparent to those skilled in the art. Such changes and modifications can be made without
departing from the spirit and scope of the invention and without diminishing its intended
advantages. It is therefore intended that such changes and modifications be covered by the
appended claims.
CLAIMS
The claimed invention is:
1. A method for measuring a metabolic rate of a cellular population, the method
comprising:
(a) providing a controller, wherein the controller is operable to track
changes in a parameter;
(b) providing a signal detector in communication with the controller and
operable to detect a metabolic signal;
(c) providing a vessel capable of holding or being associated with at least
the following items: (i) a sample containing the cellular population, (ii) a
bioreporter that is capable of directly or indirectly generating the metabolic signal,
and (iii) the signal detector;
(d) combining the sample and the bioreporter in the vessel by adding in any
order, including in phases, the sample and the bioreporter to the vessel, wherein
adding the sample and/or the bioreporter to the vessel causes the parameter to
change;
(e) optionally combining an additive with the sample and/or the bioreporter
at any time before or during step (d), including in phases, wherein combining the
additive with the sample and/or the bioreporter causes the parameter to change;
(f) optionally tracking changes in the parameter during or after steps (d) to
(e);
(g) detecting the metabolic signal with the signal detector at least once
during step (d) or step (e) and/or at least once after step (d) or step (e);
(h) determining the metabolic rate of the cellular population based upon the
metabolic signals detected in step (g);
(i) optionally recording the parameter changes tracked in step (f); and
( ) optionally recording the metabolic signal detected in step (g); and
(k) optionally recording the metabolic rate determined in step (h)
2. The method of Claim 1, wherein (i) the parameter and/or changes thereto is
tracked and/or recorded in real-time or substantially real-time and/or (ii) the metabolic
signal and/or changes thereto is detected, tracked, and/or recorded in real-time or
substantially real-time and/or (iii) the metabolic rate and/or changes thereto is tracked
and/or recorded in real-time or substantially real-time.
3. The method of Claim 1, wherein (i) the parameter is weight and the
controller is in communication with a gravimetric device and is operable in step (f) to
track weights of the sample, the bioreporter, and/or the additive and/or changes in weight
of matter held within the vessel caused by addition thereof and/or (ii) the parameter is
volume and the controller is in communication with a volumetric device and is operable
in step (f) to track volumes of the sample, the bioreporter, and/or the additive and or
changes in volume of matter within the vessel caused by addition thereof.
4. The method of Claim 1, wherein step (d) further comprises, in order: (i)
adding the sample containing the cellular population to the vessel while recording
changes in the parameter and (ii) adding the bioreporter to the vessel while tracking
changes in the parameter and detecting the metabolic signal; or (i) adding the bioreporter
to the vessel while tracking changes in the parameter and (ii) adding the sample to the
vessel while tracking changes in the parameter and detecting the metabolic signal.
5. The method of Claim 1, wherein the cellular population is selected from the
group consisting of: bacteria, archae, protists, microscopic animals, fungi, yeast,
microscopic plants, animal cells, and any combination of the foregoing.
6. The method of Claim 1, further comprising an interactive user interface in
communication with the controller and operable to guide a user to initiate and/or perform
any one or more of steps (a) to (k) in any order, through a series of visual and/or audible
prompts.
7. The method of Claim 1, wherein the controller is operable to initiate and/or
perform any one or more of steps (a) to (k) on-line and/or automatically.
8. The method of Claim 1 wherein the vessel and the signal detector are part of
an integral unit.
9. The method of Claim 1, wherein the bioreporter is selected from the group
consisting of: one reagent in one solution; more than one reagent in one solution; more
than one reagent each reagent in a separate solution; one reagent in one dry form; more
than one reagent in one dry form; more than one reagent each reagent in a separate dry
form; and any combination of the foregoing.
10. The method of Claim 1, wherein the metabolic signal is detectable using
methods selected from the group consisting of: colorimetry, refractometry,
spectrophotometry, luminometry, fluorometry, and any combination of the foregoing,
1 . The method of Claim 1, wherein the signal detector is further operable to
detect turbidity and/or temperature and determining the metabolic rate of the cellular
population based upon the metabolic signal includes a corrective function for turbidity
and/or temperature.
2. The method of Claim 1, further comprising operating any one or more of the
steps of the method over a network.
13. A system operable to determine a metabolic rate of a cellular population, the
system comprising:
(a) a controller;
(b) an optional gravimetric device in communication with the controller
and/or an optional volumetric device in communication with the controller;
(c) a means of collecting a sample of the cellular population;
(d) a bioreporter capable of reacting with a metabolic component of the
cellular population in the sample to generate a metabolic signal;
(e) a signal detector operable to detect the metabolic signal and in
communication with the controller;
(f) a vessel capable of holding or being associated with at least the
following items: (i) the sample of the cellular population, (ii) the bioreporter, and
(iii) the signal detector; and
(g) a user interface in communication with the controller
14. The system of Claim 13, wherein the gravimetric device is operable to track
changes in weight of the vessel and its contained matter and/or changes in weight of
containers that hold the sample, the bioreporter, and/or the additive and their contained
matter.
15. The system of Claim 13, wherein the volumetric device is operable to track
changes in volume of matter within the vessel and/or changes in volumes of matter in
containers holding the sample, the bioreporter, and/or the additive.