U.S. patent application number 14/571489 was filed with the patent office on 2015-04-09 for method and system for use in monitoring biological material.
The applicant listed for this patent is PHYSICAL LOGIC AG. Invention is credited to Noel Axelrod, David Nuttman, Moria Shimoni.
Application Number | 20150099274 14/571489 |
Document ID | / |
Family ID | 52777246 |
Filed Date | 2015-04-09 |
United States Patent
Application |
20150099274 |
Kind Code |
A1 |
Axelrod; Noel ; et
al. |
April 9, 2015 |
METHOD AND SYSTEM FOR USE IN MONITORING BIOLOGICAL MATERIAL
Abstract
An optical system for determining the concentration of a
metabolic gas in a container sealed to biological contamination and
enclosing a biological material. The optical system has a broadly
tunable coherent infrared light source, a detection module, and a
control system connected to the light source and detection module
and operates the light source, to receive and analyze the data
provided by said detection module, and to process the data
indicative of the concentration of said metabolic gas in said
sealed container.
Inventors: |
Axelrod; Noel; (Jerusalem,
IL) ; Nuttman; David; (Ness Ziona, IL) ;
Shimoni; Moria; (Petah Tikva, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PHYSICAL LOGIC AG |
Zurich |
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CH |
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|
Family ID: |
52777246 |
Appl. No.: |
14/571489 |
Filed: |
December 16, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/IL2013/050520 |
Jun 17, 2013 |
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14571489 |
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13674034 |
Nov 11, 2012 |
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PCT/IL2013/050520 |
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61660744 |
Jun 17, 2012 |
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Current U.S.
Class: |
435/39 ;
435/288.7; 702/19 |
Current CPC
Class: |
C12M 41/46 20130101;
C12M 41/34 20130101; G01N 33/49 20130101; G01N 21/3504 20130101;
G01N 2033/4977 20130101; G01N 21/39 20130101; C12Q 1/04 20130101;
G01N 33/497 20130101; G01N 33/02 20130101 |
Class at
Publication: |
435/39 ;
435/288.7; 702/19 |
International
Class: |
C12Q 1/06 20060101
C12Q001/06; G01N 21/3504 20060101 G01N021/3504 |
Claims
1. An optical system for determining the concentration of a
metabolic gas in a container sealed to biological contamination and
enclosing a biological material, comprising: (i) a broadly tunable
coherent infrared light source adapted to emit (a) a first
substantially monochromatic infrared light beam with wavelengths
overlapping with an absorption peak of said metabolic gas; and (b)
a second substantially monochromatic infrared light beam with
wavelengths overlapping with a transmission peak of said metabolic
gas or being outside the absorption spectrum of said metabolic gas;
(ii) a detection module configured tor detecting said first and
said second substantially monochromatic infrared light beams
following their passage through a region of interest being part of
said sealed container or in fluid communication with it, wherein
said region of interest is free of said biological material; and
further configured for generating data indicative of transmission
of said region of interest to said first and said second
substantially monochromatic infrared light beams; and (iii) a
control system connectable to said light source and said detection
module and suitable to operate said light source, to receive and
analyze the data provided by said detection module, and to process
the data indicative of the concentration of said metabolic gas in
said sealed container.
2. The optical system of claim 1, wherein said broadly tunable
coherent infrared light source has a tunability range of at least 2
cm.sup.-1.
3. The optical system of claim 1, wherein said broadly tunable
coherent infrared light source is adapted to emit substantially
monochromatic infrared light beam with a spectral width of about
0.7 cm.sup.-1.
4. The optical system of claim 1, wherein said broadly tunable
coherent infrared light source is adapted to emit substantially
monochromatic infrared light beam with a spectral width of about
0.01 cm.sup.-1.
5. The optical system of claim 1, wherein said broadly tunable
coherent infrared light source is a quantum cascade laser
(QCL).
6. The optical system of claim 1, wherein said detection module
comprises an infrared detector and a lock-in amplifier for
improving the signal to noise ratio of said optical system.
7. The optical system of claim 1, characterized by a detection
sensitivity of said metabolic gas of about 1-10 ppm and a dynamic
range of 0-100% relative gas concentration.
8. The optical system of claim 1, which is adapted to detect
isotopologues of said metabolic gas.
9. A method for in-situ real-time non-invasive estimation of the
level of living cells proliferation and/or growth in a biological
material present in a container sealed to biological contamination,
said method comprising measuring the concentration of at least one
metabolic gas emitted by said living cells according to the
following steps: (i) providing a container sealed to biological
contamination and enclosing a biological material; (ii) providing
an optical system according to claim 1; (iii) measuring the
concentration of said at least one metabolic gas emitted by said
living cells, which is present in a region of interest, being part
of said container or in fluid communication with it, wherein said
region of interest is free of said biological material, by: (a)
positioning said region of interest between the broadly tunable
coherent infrared light source and the detection module of said
optical system; (b) applying a first substantially monochromatic
infrared light beam with wavelengths overlapping with an absorption
peak of said at least one metabolic gas and measuring the signal
with said detection module; (c) applying a second substantially
monochromatic infrared light beam with wavelengths, overlapping
with a transmission peak of said at least one metabolic gas or
being outside the absorption spectrum of said at least one
metabolic gas and measuring the signal with said detection module;
(d) measuring the concentration of said at least one metabolic gas
by processing the results obtained in (b) and (c); and (e)
optionally repeating steps (b) to (d) at least one more time;
wherein the concentration of said at least one metabolic gas is an
indication of the level of living cells proliferation and/or growth
in said sealed container.
10. The method of claim 9, wherein the concentration c of said
metabolic gas is determined in step (ii)(d) from n measured values
of the signal Si (i=1, 2, . . . , n) at different wavelengths
.lamda.i by utilizing nonlinear minimization of a model S(x,
.lamda.i) as provided by function s(x) below: S ( x ) = i = 1 n - 1
[ log ( S ( x , .lamda. i ) + .epsilon. ) S ( x , .lamda. n ) ) -
log ( S i + .epsilon. S n ) ] 2 ##EQU00007## where .epsilon. is a
noise level at the detector, and S(x, .lamda.i) is provided by the
following equation: S(x, .lamda.i)=b
.intg..sub..lamda..sub.min.sup..lamda..sup.max
f(.lamda.-.lamda..sub.i)e.sup.-.alpha..sup.(x+.sup.0.sup.t.sup.0.sup.)d.l-
amda. where b is a constant f(.lamda.-.lamda.1) is the laser
spectral distribution function around the central wavelength
.lamda.1, .alpha..lamda. is the absorption coefficient, x=ci
wherein c is the gas concentration inside the container, l is the
pathlength inside the container, c0 is the concentration of the
probed gas outside the container and l0 is die pathlength outside
the container between the infrared source and the detector.
11. The method of chum 9, wherein said biological material present
in a container sealed to biological contamination is selected from
the group consisting of blood components, cell cultures, and
microorganisms in a fermentation process.
12. The method of claim 9, wherein the distance between the central
wavelengths of said first substantially monochromatic infrared
light beam and said second substantially monochromatic infrared
light beam is below the distance between, two absorption peaks of
said at least one metabolic gas.
13. The method of claim 9, wherein the spectral width of said first
substantially monochromatic infrared light beam is wider than that
of the absorption peak of said at least one metabolic gas but
narrower than the distance between two absorption peaks of said at
least one metabolic gas.
14. The method of claim 9, wherein the concentration of said
metabolic gas is measured with a sensitivity of about 1-10 ppm and
a dynamic range of 0-100% relative gas concentration.
15. The method of claim 9, wherein said at least one metabolic gas
is selected from the group consisting of carbon dioxide, oxygen,
ammonia, hydrogen sulfide, methane, ethane, butane, ethylene,
sulfur dioxide, carbonyl sulfide and nitric oxide and isotopologues
thereof.
16. The method of claim 9, wherein said sealed container to
biological contamination is permeable to said at least one
metabolic gas and wherein the emission rare of said at least one
metabolic gas is determined by applying the following formula:
W(t)=(.rho.(t)-.rho..sup.0)vA where W(t) is the metabolic gas
emission rate of enclosed biological material in units kg/s,
.rho.(.epsilon.) is the mass concentration of the metabolic gas in
units kg/m.sup.3 at time t, determined in step (iii)(d),
.rho..sup.0 is the ambient mass concentration of the gas, v is the
membrane permeability coefficient to metabolic gas in units m/s and
A is the membrane surface area.
17. The method of claim 9, wherein said sealed container to
biological contamination is not permeable to said at least one
metabolic gas and wherein the concentration of said at least one
metabolic gas is determined in step (iii)(d) by applying the
following formula: W(t)=(.rho.(t)=.rho.(t-.tau.))V/t where W(t) is
the metabolic gas emission rate of enclosed biological material
averaged over time interval .tau. and V is the volume of the
container and .rho.(t) is the mass concentration of the metabolic
gas at time t, determined in step (iii)(d).
18. A method for detecting a microorganism, contamination in a
storage container tor platelets sealed to biological contamination,
comprising applying a method according to claim 9 for measuring the
concentration of carbon dioxide emitted by said microorganism in
said sealed storage container.
19. A method for monitoring a fermentation process in a fermenter
enclosing microorganisms and sealed to biological contamination,
comprising monitoring the amount of said microorganisms by applying
a method according to claim 9 for measuring the concentration of
carbon dioxide emitted by said microorganisms in said sealed
fermenter.
20. A method for monitoring the concentration of living cells in a
bioreactor sealed to biological contamination, comprising applying
a method according to claim 9 for measuring the concentration of
carbon dioxide emitted by said living cells in said bioreactor and
optionally correlating said concentration of carbon dioxide to as
amount of biomass of said living cells via a linear or robust
regression mathematical model
Description
TECHNOLOGICAL FIELD
[0001] The present invention in the field of monitoring the
condition/status of a biological material, and relates to a method
and system for detection of microorganisms and living cells in a
biological material by optical measurements of metabolic gases.
BACKGROUND
[0002] Monitoring the live biological activity in a biological
material is needed in various industries, for example in the
medical field for monitoring microorganisms contaminants in
blood/blood-components, in food and beverages (F&B) industries,
and in pharmaceutical industries for example for monitoring
fermentation processes.
[0003] Conventional techniques for monitoring biological activity
in a biological material generally include direct techniques such
as: viable count in which a diluted samples are grown on agar
medium dish; staining or microscopy; pH and glucose measurements;
swirling; and optical density (OD) measurements in which a sample
of the biological material taken in to a cuvette and the level of
microorganisms is determined optically based on turbidity of the
sampled biological material itself. Other known techniques utilize
monitoring the biological activity indirectly, for example based on
analysis of gases consumption (such as dissolved oxygen--dO.sub.2)
or accumulated (such as carbon dioxide--CO.sub.2) in a sample of
the biological material. In those techniques, a sample of the
biological material is incubated for a period of time to allow
consumption or accumulation of gases by live microorganisms
contained in the sample and then the metabolic gases are analyzed
chemically and/or by utilizing spectroscopic measurements. However,
conventional direct measurements made on samples of the biological
material are invasive (thus increasing the risk of contamination of
the biological material), time-consuming, and do not enable
real-time monitoring of the growing population. Direct measurements
such as for dO.sub.2 may be also inaccurate. For instance, in
fermentation processes involving fungi, the diffusion of oxygen
through the substrate does not occur at a uniform rate and
therefore measurements with dO.sub.2 electrodes when performed at
lower oxygen diffusion rate may be misleading. Also, oxygen is a
decreasing parameter that is limited by zero. Other, conventional,
IR based, indirect techniques based on gas analysis in the
container including the biological material are often influenced by
the local conditions inside the container (e.g. temperature,
pressure, humidity) and may lead to inaccurate measurements.
GENERAL DESCRIPTION
[0004] There is a need in the art for a novel technique for
in-situ, real-time, noninvasive and accurate monitoring of
biological materials, enabling the detection, monitoring and/or
controlling of microorganisms and living cells in a biological
material, such as blood components, food products and/or biological
materials used in fermentation processes, for example those used in
the pharmaceutical and/or food and beverages (F&B) industry.
Specifically there is a need in the art for an in-situ real-time
non-invasive accurate technique for detection of microorganisms in
a culture media utilizing measurement of gaseous products generated
during living cells growth/proliferation.
[0005] The known techniques for detecting metabolic gas
concentrations in-situ are generally not sensitive and precise
enough for correlate with bio growth of the microorganisms and
living cells in biological material. The known techniques for
detecting microorganisms' contaminations and/or grow in biological
material by spectroscopic measurements of metabolic gas
concentrations are generally not suited and/or are in-capable of
in-situ real time operation. This is mainly because these
techniques are invasive with respect to stored biological material
that has to be inspected, i.e. they require sampling and incubation
of the biological material in a separate sealed incubation
container (sampling vial) which is impermeable for gases and
possibly contains certain growth media. To this end, the
conventional techniques utilize sampling/transferring certain
amount of the biological material from a sealed container in which
it is stored/maintained into a suitable sampling vial/container,
which is specifically designed/selected to facilitate the
spectroscopic measurements of metabolic gas(es). Conventional
sampling vials used for this purpose are generally non-permeable to
the metabolic gas in order to enable accumulation of high
concentrations of the gas in the sampling vial. The conventional
sampling vials are also specifically configured for the
spectroscopic measurements (e.g. formed with specifically selected
materials having high transitivity to wavelengths used in
measurements). The sampled biological material is maintained in the
sampling vial for sufficient time (the detection limit is only
after incubation time of between 18 to 48 hours) for consumption or
accumulation of relatively low or high concentrations,
respectively, of gas consumed or accumulated by microorganisms
contained in the biological material. As indicated above, many of
the known techniques for growing biological material use incubation
of a test sample while providing suitable growth conditions (e.g.
providing growth medium such as agar, and/or incubating
conditions/temperatures, and/or sufficient time for growth) to
accelerate the microorganisms growth and accordingly accelerate
production of the metabolic gases by the microorganisms contained
in the sample. This is in fact because the sensitivity and/or
accuracy of the known spectroscopic metabolic gas detection
techniques require consumption or accumulation of relatively low or
high concentrations, respectively, of the gas for the detection
thereof.
[0006] The known techniques are thus not suited for in-situ real
time non-invasive, accurate monitoring of biological
activity/microorganisms in a biological material mainly because (1)
they are invasive, i.e. require sampling of the biological
material, (2) require specific equipment, i.e. the sampled
biological material is transferred into a separate, specifically
designed, sealed sampling vial (e.g. non-permeable to metabolic
gases, highly transmitting for wavelengths used in spectroscopic
measurements, e.g. containers made of glass/quartz), as well as
typically require use of specific growth media, and (3) they are
time consuming as they typically require relatively long incubation
periods (e.g. 18-48 hours) of the sampled biological material for
accumulation of sufficient (measurable/detectable) concentrations
of the metabolic gases. Non-invasive gas monitoring methods
available are not sensitive enough. In fact, in-situ, real time and
non-invasive high resolution monitoring of biological activity in
biological substances are needed in various fields. Specifically,
such traits are needed in medical fields for handling blood and
blood components for monitoring the blood/components thereof (e.g.
prior to blood transfusions). Also such traits are highly needed in
various fermentation processes in which sensitive, real-time
monitoring of the biological activity may provide substantial
increase in the yield of the fermentation process. This would allow
the control/monitoring of cells growth of a wide variety of
microorganisms (including non-transparent and pathogenic ones),
within various medium (including high turbidity/viscosity medium),
early stage detection of cell growth, shorter R&D cycle time,
and real time monitoring of biomass production process for
increasing the yield (e.g. by determining optimal seed transfer and
induction times, controlling the growth-media/biological-material
composition utilizing controlled-depletion of nutrients, etc).
[0007] Specifically, one application of the invention is to detect
bacterial contamination of a blood or blood-components, such as red
blood cells, plasma and platelets, which are commonly used for
transfusions. Particularly the invention provides for real time in
situ non-invasive monitoring of microbiological contaminants in
blood platelets which are a component of blood that is involved in
blood clotting.
[0008] Allogenic blood/blood-components for transfusion are a
potential source of infection by a variety of known and unknown
transmissible agents. Over the last three decades, the risk of
transfusion-related transmission of viral diseases such as human
immunodeficiency virus (HIV) I/II, hepatitis C virus (HCV),
hepatitis B virus (HBV) and human T-lymphotropic virus (HTLV) I/II
has decreased dramatically. With blood products now being routinely
screened by ultrasensitive techniques to minimize the risk of
transmitting viruses to recipients, the known risk of transmission
of bacteria has emerged as the greatest residual threat of
transfusion-transmitted disease.
[0009] Bacterial contamination has proved more difficult to address
than viral contamination, and remains the most prevalent
transfusion-associated infectious risk.
[0010] This is especially true for platelets, which are stored at
room temperature (20-24.degree. C.) for up to five days (rather
than the previous practice of storage for up to seven days), in
bags that are permeable to oxygen and carbon dioxide, and under
sufficient constant agitation to provide adequate oxygenation, to
prevent platelet aggregation and to maintain optimal platelet
viability and functional properties.
[0011] Storage of the platelets at optimal metabolic conditions, at
room temperatures, and with agitation in bags/containers permeable
to O.sub.2 and CO.sub.2, promote ongoing bacterial proliferation
throughout the storage period and thus increase the risk of
transmitted bacteria and bacteremia in the patient. The risk of
bacterial contamination in platelets is estimated to be one in
1500, which is 50 to 250 times higher than the combined risk of
viral infections.
[0012] As described above, the conventional techniques for testing
platelets for contaminants prior to transfusion are invasive
(require opening of the sealed blood storage container for sampling
thus increasing the risk of inadvertent contamination of the
remaining platelets), require specific equipment and possibly
growth media, and time consuming. Since the conventional techniques
do not provide real time--pre transfusion reliable testing for
contamination in platelet bag, the practical shelf life of
platelets is decreased (from seven to five days) to avoid an
increase in bacteria concentration to a levels that can cause
sepsis in recipient.
[0013] In this connection, the present invention allows for in-situ
non-invasive (without opening the container and withdrawing a
portion of the biological material) real time and sensitive
monitoring of a biological material, such as (but not limited to) a
blood component, that can serve, intentionally or unintentionally,
as a growth medium for the growth of microorganisms such as
bacteria, and that is contained within a container sealed with
respect to the biological material. The present invention provides
for monitoring any biological material, such as food, human or
animal tissues, and cell cultures, with particular application to
blood components such as platelets. The technique of the present
invention allows for detecting bacterial contamination in platelets
contained/stored in conventional platelet storage bags (e.g.
plastic bags) based on quantitative analysis of metabolic gases,
such as CO.sub.2, released by bacteria inside the platelet plastic
storage bag which is permeable to metabolic gases, while the
plastic storage bag with the platelets remains sealed to
contaminates. Furthermore, as it will be explained in more details
below, the detection of metabolic gas in the sealed container when
performed according to the method of the present invention does not
require a control/reference sample.
[0014] The concentration of the metabolic gas in a dead space
associated with the storage bag is monitored by
optical/spectroscopic measurements performed according to the
technique of the present invention as described in more details
below. The dead space, for the purposes of the invention, is a
space/region which is free of the biological material under
inspection and is in fluid communication therewith. In some
embodiments, the dead space is defined by a region/portion of the
container above a portion thereof containing the biological
material. In some other embodiments, the dead space is defined by a
gas chamber/reservoir/pipe connectable to the container (e.g. in a
manner maintaining the sealing of the container), so as to be in
the fluid communication with biological material in the container.
Such reservoir/pipe may be formed by a separate cavity configured
as an extension of the dead space in the container.
[0015] The measurements of metabolic gas concentration may for
example utilize spectroscopic measurements in mid-IR spectrum of
light at wavelength(s) overlapping with strong absorption line(s)
of a metabolic gas (e.g. CO.sub.2 or other metabolic gases). The
light is transmitted through a part (dead space) of the plastic bag
that is above the stored platelets, and is appropriately detected
by an IR detector. The spectroscopic technique of the invention
provides for determining the concentration of CO.sub.2 or other
metabolic gas by measuring light absorption within the plastic bag,
while allows for discarding/discriminating the absorbance of the
plastic bag itself, thus enabling in-situ monitoring of the
metabolic gases contained in the dead space of the bag.
[0016] The technique of the invention allows the detection of
different transfusion-relevant contaminating bacterial species.
This approach provides on-line measurement of respiratory gases
such as CO.sub.2 at ambient atmospheric concentrations without the
need for any pre-concentration or gas separation. The method is
non-invasive since it does not require opening the plastic platelet
bag for examination. This non-invasive bacterial detection method
represents a new approach to prevent the transmission of bacterial
contamination of platelets with an advantage of the method is that
all measurements can be performed in real time, until right up to
the time of transfusion and therefore the risk for sample errors is
reduced to a minimum and the platelets' storage time is extended.
Also, unlike conventional methods, the method of the present
invention can be used with containers that are sealed with respect
to the biological fluid, and are either impermeable or are
permeable to the metabolic gas(es) being monitored. Some other
possible applications of the invention include real-time, precise
monitoring of fermentation processes. Owing to the specific
features of the method and device of the invention, the monitoring
is made possible for any kind of microorganisms (even pathogen) and
any kind of culture medium (even those with high turbidity and/or
viscosity). This allows the standardization of the monitoring
procedure for any kind of fermentation process. In fermentation,
the biological material generally added to the growth medium.
Specifically for example, fermentation processes are used in the
pharmaceutical industry for generating various biological
substances such as: microbial cells (such as E. coli); microbial
enzymes (catalase, amylase, protease, etc); primary metabolites
(ethanol, citric acid, glutamic acid, etc); secondary metabolites
(antibiotic, recombinant products: insulin, hepatitis B vaccine,
interferon, etc).
[0017] The technique of the invention allows the detection of
different microorganism's species including: aerobic &
anaerobic; transparent & non-transparent; and pathogenic
microorganisms. This approach provides on-line high resolution
measurement of respiratory gases such as CO.sub.2 at ambient
atmospheric concentrations without the need for any incubation, gas
separation, drying system or calibration. The detection method and
system of the invention can apply qualitative and/or quantitative
analysis and estimation of a level of biological activity of
microorganisms, for example in agar plates, and monitoring of
biological activity such as in the case of measuring living cells
growth in the production of pharmaceutical products or proteins.
Microorganisms are used commercially to produce foods (such as
vinegar, yogurt, cause beer and wine spoilage), antibiotics and
chemicals such as ethanol. Production of some of the most important
and complex pharmaceuticals such as insulin, hormones, antibodies,
or other proteins is carried out using microorganisms (such as E.
coli) that have been modified genetically using recombinant DNA
technology. From the early stages of commercial production of
recombinant proteins and other pharmaceutical material, the
handling of cultures has been subject to challenges. One of these
challenges is how to cope with the problem of instability of
production processes as in the case of recombinant organisms and
induction. Commercial production of products on a large scale,
especially in the pharmaceutical industry using fermenters, depends
heavily on the stable maintenance of the organisms during
production and harvesting time. The fermentation process of
recombinant bacteria needs to be precise and the cells
concentration has to be monitored.
[0018] For clarity, in the following description, monitoring of
pharmaceutical fermentation processes for generating proteins is
specifically described. However it should be understood that the
present invention can be used for real time monitoring of other
fermentation processes in the pharmaceutical industry and/or in the
F&B industries.
[0019] In fermentation, specific microorganism species are
deliberately introduced into a fermentation container containing a
biological material serving as growth medium. The fermentation
container is kept at suitable conditions (e.g. glucose, yeast,
agitation, temperature) encouraging the production of the desired
biological substances/proteins by microorganism. Typically, a gas
inlet is coupled to a fermentation container to supply suitable
atmospheric conditions for the microorganisms' metabolism (e.g.
supply of ambient air) and a gas outlet is also coupled to the
fermentation container to evacuate gas which is richer in metabolic
gases generated by the microorganisms' metabolism. In many cases,
fermentation processes are monitored by occasionally collecting a
sample from the biological material in the container and analyzing
that sample (such as OD measurements) in order to determine data
indicative of the amounts of microorganisms and/or the amount of
the produce material or proteins in the sample, and utilize that
data for controlling the fermentation process.
[0020] The amount of the biological substance, which is to be
produced in the fermentation, is generally correlated with the
amount/the rate of change of the amount of microorganisms in the
biological material in the container. To this end, an accurate
real-time monitoring of the amount of microorganisms enables
accurate control over the fermentation process and provides for
significantly increasing the yield of the product to be
produced.
[0021] For example, the production of recombinant protein is
correlated with an optimal induction process and microorganisms'
amount. During the first stage, log (logarithmic) phase, the
microorganisms is being grown to certain, very specific, well
define amount in which an inducer added to the culture media. Then
at a later/second stage, the recombinant bacteria stops
proliferating and use their "cell energy" for the production of the
recombinant protein. Problems that may occur during bacteria log
phase (due to problems with nutrients, for example), lack of
precision during induction and/or over grown of the bacteria may
lead to lower yield. Thus, by monitoring a time profile of the
amount of microorganisms and/or changes thereof, the fermentation
process can be controlled to improve the yield of the generated
product and harvesting time (see FIG. 9).
[0022] However, the conventional techniques for monitoring biomass
in fermentation processes are not performed continuously and are
incapable of being carried out with high sensitivity in real time,
and typically involve occasional collection of samples from the
fermentation container and examining these samples by techniques
such as optical/critical density measurement, viable counts, and by
measuring the produced material. These results, inter alia, in that
the optimal time points for collection of fermentation yield are
often missed.
[0023] The present invention provides for an accurate real time and
continuous/periodical monitoring of fermentation process by
continuous/periodical monitoring of the atmospheric conditions in
the container to determine data indicative of a rate of metabolic
gas production by microorganisms contained therein, or a change in
such rate of production, and thereby determining the amount of the
microorganisms, biomass, or the rate of change in this amount. The
later are processed to control the fermentation process accurately
in time such as to improve the yield. For example the monitored
amount of microorganisms and/or rate of change thereof may be
compared with a reference data/model to control the fermentation
conditions (e.g. temperatures of the fermenter, gaseous atmosphere
therein, nutrients or other materials supplied during the
fermentation process etc), and/or identify a time at which the
fermentation process transit from the "first" (production stage) to
the "second" ("proliferation stage") stages, for stopping,
harvesting or managing the process at this time.
[0024] The system of the present invention may be configured to
continuously and/or periodically/repeatedly monitor the
gas/atmosphere in the fermentation container and may be in optical
communication with the gas within the container itself (e.g. above
the biological material) and/or in optical communication with a gas
flowing out from the fermentation container (e.g. gas being in
fluid communication with the inside of the container, for example
gas flowing through the gas outlet of the container).
[0025] Yet another attractive application of the present invention
is related to detection of isotopologues of metabolic gases.
Isotopologues are molecules that are identical except for their
isotopic composition. Examples of the isotopologues of carbon
dioxide are .sup.12C.sub.16O.sub.2, .sup.13C.sup.16O.sub.2,
.sup.16O.sup.12C.sup.18O, .sup.16O.sup.13C.sup.18O. The natural
abundance of isotopologues that contain a rare isotope is
negligible in comparison to the common molecule. For example, the
natural abundance of .sup.13C.sup.16O.sub.2 is 0.0111%, and the
natural abundance of .sup.18O.sup.13C.sup.18O is 10-8. Different
isotopologues of the same molecules have different vibration
frequencies, and thus different absorption spectra in the IR
region. For example, molecules of .sup.13C.sup.16O.sub.2 have a
strong absorption at 2270.29 cm.sup.-1, while the absorption
strength of the nearest absorption line of .sup.12C.sup.16O.sub.2
at 2277.427 cm.sup.-1 is weaker by a factor of about 30 than that
of .sup.13C.sup.16O.sub.2. This provides means for discrimination
between different isotopologues of the same molecules by means of
infrared absorption spectroscopy. In particular, a typical tunable
QCL operating in continuous wavelength (CW) mode can have a beam
spectral width as narrow as 0.01 cm.sup.-1. That provides means for
unambiguous measurement of concentrations of isotopologues of a
molecule under study in a setup as described above. Isotopologues
can serve as biomarkers to trace particular metabolic processes.
One example of such an application is the use of
D-glucose-.sup.13C.sub.6 as a carbon based nutrition source for
bacteria for checking specific metabolic processes, that in turn
can be used for example to study the efficiency of the fermentation
reaction of glucose for ethyl alcohol production at different
stages of the fermentation process.
[0026] Thus, the present invention provides a novel system and
method for in-situ nondestructive (non-invasive) real time
detection of microorganisms in biological materials.
[0027] It should be understood that the term biological material
herein relates to any material that can serve as culture media for
growth of microorganisms and/or living cells. In this regards, this
term includes and is not limited to blood and blood components such
as platelets, F&B products and biological materials used in
fermentation processes (e.g. in the F&B and/or pharmaceutical
industries). Also the term microorganisms relates to any living
organisms such as bacteria, fungi etc. The term metabolic gas
relates to one or more gases produced or consumed by
microorganism's metabolism, and may include for example gases such
as carbon dioxide (CO.sub.2) produced during respiration. Some not
limiting examples of gases that are specifically included in this
definition are carbon dioxide, oxygen, ammonia, hydrogen sulfide,
methane, ethane, butane, ethylene, sulfur dioxide, carbonyl sulfide
and nitric oxide. Examples of gases that are specifically excluded
from this definition include argon and inert gases such as helium
or nitrogen which do not participate in the metabolic process. For
clarity, in the following description, the present invention is
described specifically in relation to the carbon dioxide metabolic
gas. Nevertheless it should be understood that the technique of the
present invention is not limited to carbon dioxide and can also be
applied to detection microorganisms based on other metabolic
gases.
[0028] It should further be noted that the term in situ in the
context of the current disclosure refers to detection of
microorganisms being performed directly on metabolic gas(es) formed
in the original storage/fermentation container (at times termed
herein as biological material (BM) container), without sampling or
opening the BM container. To this end, the detection is performed
without a need to expose the biological material in the container
to external microorganisms. In this regards, the term in-situ
measurement should be interpreted broadly, as including analysis of
the gas(es) directly inside the storage container/bag of the
biological material, where the term directly signifies applied to
gas(es) in a dead space being in fluid communication with the
biological fluid in the storage container, namely a portion of the
container itself or a reservoir/pipe non-invasively connectable to
said portion of the container. For example, metabolic gas detection
may be performed at the gas outlet of a fermentation container
and/or at a certain gas reservoir connectable to a BM container of
blood/blood-components (e.g. bag/vial) by a fluid connection that
does not permit external microorganism contaminant into the BM
container (e.g. utilizing a transfusion needle to connect the
gas-chamber/reservoir to the BM container to allow gas flow to the
reservoir without opening the BM container.
[0029] In this regards, the detection is non-invasive and
nondestructive in the sense that measurement procedure does not
destroy or affect in any way the biological material, and thus the
biological material can still be used for its original purpose,
after the detection procedure. The detection is real-time in the
sense that no incubation period is required and the results of the
detection can be obtained within a relatively short time scale
(seconds or minutes).
[0030] The goal of the detection is to determinate the presence of
microorganisms such as bacteria (e.g. bacteria contamination) in
the biological material. Yet it can be quantitative analysis and
estimation of level of biological activity of microorganisms for
example in agar plates and also monitoring of biological activity
of microorganisms such as in case of fermentation process.
[0031] Thus, according to one broad aspect of the invention, there
is provided a method for use in detection of microorganisms in a
biological material, the method comprising:
[0032] (i) applying non-invasive in-situ optical measurements to a
region of interest being a dead space free of a biological material
and in a fluid communication with a portion of a container
containing the biological material, wherein said optical
measurements comprise illuminating said region of interest with
light of a including at least first and second predetermined
wavelengths of substantially narrow spectrum corresponding to
respectively an absorption peak of at least one metabolic gas and a
spectral region outside the absorption peak of said at least one
metabolic gas, and measuring transmission of said first and second
wavelengths through said dead space; and
[0033] (ii) analyzing measured data of said transmission and
generating data indicative of a concentration of the at least one
metabolic gas in said dead space which is in the fluid
communication with the biological material, said generated data
being thereby indicative of microorganisms in the biological
material.
[0034] The measurements of metabolic gas concentration may
generally be performed utilizing infrared and/or visible portions
of electromagnetic radiation.
[0035] According to some embodiments of the present invention the
method further includes processing the data indicative of the
concentration of the at least one metabolic gas utilizing
equilibrium condition between a rate of generation or consumption
of the at least one metabolic gas by the microorganisms and a rate
of flow of the at least one metabolic gas into and/or out of the
container. Then utilizing the equilibrium condition data about the
microorganisms in the biological material is
obtained/determined.
[0036] According to some embodiments of the present invention the
spectral width of the substantially narrow spectrum of the first
wavelength overlaps and exceeds a spectral width of an absorption
line of said metabolic gas. Also, in some cases, the spectral width
of the first wavelength is less than a spectral distance between
two spectrally adjacent absorption lines of the metabolic gas.
[0037] Moreover in some cases the spectral separation between the
first and second wavelengths of the light source is substantially
small such that the first and second wavelengths are characterized
by same or similar transmission through predetermined materials
used for containers of the biological material. In this connection,
according to some embodiments the transmission of the second
predetermined wavelength (being in the spectral region outside the
absorption peak of the metabolic gas), is indicative of absorbance
of the first wavelength by materials in the region of interest,
other than the metabolic gas. These other materials typically have
a spectral absorbance band substantially wider than that of the
absorption peak of the metabolic gas. Thus analyzing the measured
data of the transmission may include utilizing the measured
transmission of the second predetermined wavelength to process the
measured data of the transmission of the first predetermined
wavelength, which overlaps the absorption peak. This increases the
sensitivity in determination of the concentration of the metabolic
gas and allows detection of the microorganisms.
[0038] The high sensitivity of detection enables determination of
the concentration of the metabolic gas based on equilibrium
condition between a rate of generation or consumption of the at
least one metabolic gas by the microorganisms and a rate of flow of
the at least one metabolic gas into and/or out of the container.
Also, in cases where the region of interest at which measurements
are conducted is the dead space above the portion with the
biological material in the container, the high sensitivity and the
use of the above mentions first and second types of wavelengths may
provide for eliminating a need for a-priory knowledge of optical
properties of the container.
[0039] The at least one metabolic gas may include one or more of
the following: carbon dioxide, oxygen, ammonia, hydrogen sulfide,
methane, ethane, butane, ethylene, sulfur dioxide, carbonyl sulfide
and nitric oxide. In some embodiments the metabolic gas includes
carbon dioxide and the at least first and second wavelengths are in
a spectral regime of high absorbance by carbon dioxide.
Specifically the first wavelength overlaps one of absorption peaks
of carbon dioxide in that regime, and the second wavelength
overlaps a transmission peak in the carbon dioxide spectrum. For
example the spectral regime may be in a mid-IR regime (e.g.
spectral regime of high absorbance by CO.sub.2 is in the vicinity
of 4.3 microns). According to some embodiments of the present
invention the optical measurements include spectroscopic
measurements. According to some embodiments of the present
invention, the illuminating the dead space (the region of interest
to be measured) includes operating a broadly tunable coherent IR
light source, for producing light of the above mentioned at least
first and second wavelengths. The light is directed to propagate
along a path of a certain predetermined optical path (length)
through the dead space and, the method includes operating a
detection module for detecting the light transmitted through the
dead space. In some cases, the broadly tunable coherent IR light
source is a Quantum Cascade Laser (QCL). Also in some cases,
operating of the broadly tunable coherent IR light source includes
modulating light intensity in the at least first and second
wavelengths, and operating a lock-in amplifier (associated with the
detection module) to determine the transmission of the region of
interest to the modulated first and second wavelengths with high
signal to noise ratio signal detection based on the modulation.
[0040] According to some embodiments the optical measurements are
applied to the dead space of the container, while the container is
remained sealed with respect to the biological material under
measurements. To this end, in some cases the container may be
permeable to the at least one metabolic gas and data about the
microorganisms in the biological material are determined based on
an equilibrium condition defined by diffusion of the at least one
metabolic gas through walls of the container. To this end the
container may be a storage container for platelets, such as a
conventional platelets storage container.
[0041] According to some embodiments the optical measurements are
applied to the dead space of the container, where the dead space
may be defined by one or more of the following: (i) a portion of
the container above the portion containing the biological material;
(ii) a gas chamber configured to be connectable to the container so
as to be in the fluid communication with the closed container (e.g.
the gas chamber may be a gas outlet of the container; (iii) an
extension of the dead space of the container by an attached
reservoir transparent to the at least first and second
wavelengths.
[0042] In some embodiments of the present invention the container
is configured for use in a process of fermentation. The optical
measurements may be performed through one or more optical windows
optically coupled to the dead space of the container. In some
cases, for monitoring the fermentation process, the optical
measurements and the data analysis are performed continuously or
periodically. Accordingly the determined gas concentration data
being thereby indicative of at least one of: (a) amount of
microorganisms in said container as a function of time; and (b) a
rate of change in amount of microorganisms in said container as a
function of time. Thus method further includes processing the gas
concentration data to monitor the fermentation process.
[0043] According to some embodiments the method of the present
invention for determining the concentration of at least one
metabolic gas, includes:
[0044] (i) measuring IR transmission through the dead space in two
or more wavelengths comprising the above noted first and second
wavelengths. The measuring comprises: tuning a central wavelength
of illuminating light each one of the two or more wavelengths;
detecting IR light in the two or more wavelengths transmitted
through the dead space; and generating measured data indicative of
two or more intensity values comprising first and second intensity
values corresponding to the light transmitted through the dead
space in the first and second wavelengths for a given optical path
defined by the optical system and the dead space; and
[0045] (ii) processing the measured data based on an absorption
model of the at least one metabolic gas. The processing comprises
determining a best fit between intensity values obtained from the
absorption model and the measured intensity values, and thereby
determining the concentration of the at least one metabolic
gas.
[0046] In some embodiments the method further includes utilizing
the concentration of the metabolic gas for estimating a degree of
microbial contamination of the biological material.
[0047] In another broad aspect of the present invention there is
provided a system for use in detection of microorganisms in a
biological material. The system includes:
[0048] (a) an optical system including: a broadly tunable coherent
IR light source and a detection module that includes a detector
sensitive in the IR wavelength regime. The broadly tunable coherent
IR light source is configured and operable for producing light in a
predetermined spectrum including at least first and second
predetermined wavelengths of substantially narrow spectra
corresponding to respectively an absorption peak of at least one
metabolic gas and a spectral region outside the absorption peak of
said at least one metabolic gas. The detection module is configured
for detecting light of the first and second wavelengths passing
through a region of interest (e.g. being a region located in
between the light source and the detection module). The detection
module is configured for generating data indicative of transmission
of that region of interest to the at least first and second
wavelengths; and
[0049] (b) a control system connectable to the light source and to
the detection module and configured and operable for carrying out
the following: [0050] operating the light source to produce the
light of at least the first and second wavelengths. The first
wavelength is selected such that the detected transmission for the
first wavelength provides measured data indicative of absorbance by
the at least one metabolic gas in the region of interest. The
second wavelength is selected such that the detected transmission
for the second wavelength provides detection of measured reference
data indicative of absorbance of the first wavelength by materials
in the region of interest other than said at least one metabolic
gas; [0051] receiving and analyzing the measured data and the
measured reference data, and generating data indicative of the
concentration of the metabolic gas in the region of interest. This
thereby enables non-invasive in-situ detection of microorganisms in
a biological material when located in fluid communication with the
region of interest. According to some embodiments of the present
invention the light source is broadly tunable light source having a
tunability range of at least 2 cm.sup.-1. The broadly tunable light
source may be a Quantum Cascade Laser (QCL) with a tunability range
exceeding 30 cm.sup.-1. In some embodiments, the detection module
includes a lock-in amplifier, and the control system is adapted for
operating the broadly tunable IR light source for modulating light
intensity of the at least first and second wavelengths, and
operating the lock-in amplifier to determine the transmission of
the region of interest to the at least first and second wavelengths
with high signal to noise ratio based on the modulation. To this
end the optical system is configured to define illumination and
detection paths for the light of said at least first and second
wavelengths intersecting with the region of interest for placement,
in the region of interest, a dead space, which is free of the
biological material and is in fluid communication with a portion of
a container containing said biological material. In this connection
in some embodiments the system of the present invention may further
include a mechanism for positioning the container of the biological
material with respect to the optical system, such that the
illumination and detection paths intersect with the dead space of
the container and traverses a predetermined optical path (length)
through the dead space. It should be understood that in various
embodiments of the present invention, the system of the invention
and/or its controller (control system) may be configured and
operable for carrying out various operations of the method
described above and more also described more specifically
below.
[0052] According to another broad aspect of the present invention
there is provided a method for use in detection of microorganisms
in a biological material. The method includes:
[0053] (i) applying non-invasive in-situ optical measurements to a
region of interest being a dead space free of a biological material
and in a fluid communication with a portion of a container
containing the biological material. The optical measurements
include illuminating the region of interest with light of a
predetermined substantially narrow spectrum including two or more
predetermined wavelengths and measuring transmission of said two or
more wavelengths through the dead space;
[0054] (ii) analyzing measured data of said transmission and
generating data indicative of a concentration of the at least one
metabolic gas in the dead space which is in fluid communication
with the biological material; and
[0055] (iii) processing the gas concentration data based on an
equilibrium condition between a rate of generation or consumption
of the at least one metabolic gas by said microorganisms and a rate
of flow of said at least one metabolic gas into and out of the
container, thereby generating data indicative of microorganisms in
the biological material.
[0056] According to another broad aspect of the present invention
there is provided a container including: a sealable main body for
containing a biological materials and being permeable to at least
one metabolic gas; and a reservoir being configured for fluid
communication with the sealed main body, and having at least a
portion thereof at least partially transparent to one or more
wavelengths corresponding to at least one absorption peak of the at
least one metabolic gas.
[0057] According to yet another broad aspect of the present
invention there is provided a reservoir for use in inspection of
biological material contained in a sealed platelets storage
container permeable to at least one metabolic gas. The reservoir is
connectable to the container, so as to be in fluid communication
with a dead space in the container (above a portion thereof where
the biological material is contained) while the container is
maintained sealed with respect to the biological material and
contaminates/microorganisms. Also the reservoir has at least a
portion thereof which is at least partially transparent to one or
more wavelengths corresponding to at least one absorption peak of
at least one metabolic gas.
[0058] The present invention thus provides novel, effective and
simple technique for accurate in-situ real time non-invasive
monitoring of a biological material for detection microorganisms
therein. Additional features and elements of the present invention
are described in more details in the detailed description of
embodiments below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0059] In order to better understand the subject matter that is
disclosed herein and to exemplify how it may be carried out in
practice, embodiments will now be described, by way of non-limiting
example only, with reference to the accompanying drawings, in
which:
[0060] FIG. 1A is a block diagram schematically illustration a
system for detecting microorganisms in biological material
according to some embodiments of the present invention;
[0061] FIG. 1B is a graph exemplifying absorption cross section of
metabolic carbon dioxide (CO.sub.2) gas at ambient conditions;
[0062] FIGS. 1C and 1D respectively illustrate two examples of the
spectrum of light beams of first and second wavelengths suitable
for use in the invention, where the first and second wavelengths
are respectively superposed to overlap/cover an absorption peak
(absorption line) and absorption valley (transmission line) of the
CO.sub.2 absorption spectrum in the mid-IR wavelengths band near
4.3 microns;
[0063] FIGS. 1E and 1F respectively show logarithmic scale graphs
illustrating the transmittance of CO.sub.2 for the first and second
type wavelengths of FIGS. 1C and 1D, and a ration of the CO.sub.2
transmission in these wavelengths;
[0064] FIGS. 2A and 2B exemplify a system for detecting
microorganisms in biological material according to some embodiments
of the present invention, utilizing a separate closed gas chamber
connected to a container of biological material;
[0065] FIG. 3 shows the experimental results for a measured plot of
% CO.sub.2 vs. bacterial concentration;
[0066] FIG. 4 shows simulated absorption versus wavelength and
CO.sub.2 concentrations of the beam of the QCL in the IR spectral
range 2355 cm.sup.-1-2410 cm.sup.-1;
[0067] FIGS. 5A and 5B show experimental set ups of the system of
the present invention used for detection of metabolic CO.sub.2
inside a platelets product in a plastic bag utilizing direct
measurement through the bag containing the platelets (FIG. 5A) and
utilizing a separate gas chamber connected to the platelets
container (FIG. 5B);
[0068] FIGS. 6A to 6D show experimental results for a fermentation
process/run of recombinant protein production utilizing Escherichia
coli E. Coli fermentation;
[0069] FIGS. 7A and 7B show experimental results for the monitoring
of plant cells growth by CO.sub.2 online measurements performed
with the device and method of the present invention versus standard
routine measurements of fresh weight (FIG. 7A) or conductivity
(FIG. 7B);
[0070] FIG. 8 shows experimental results regarding the detection of
carbon dioxide (.sup.12CO.sub.2) and an isotopologue thereof
(.sup.13CO.sub.2), vs time, with very high sensitivity (1 ppm
order); and
[0071] FIG. 9 shows experimental results comparing real-time and
continuous monitoring of carbon dioxide performed according to the
present invention versus manual sample measurements of biomass via
optical density measurements (OD)
DETAILED DESCRIPTION OF EMBODIMENTS
[0072] The principles and operation of in-situ real-time and
non-invasive detection of microorganisms in biological materials
according to the present invention may be better understood with
reference to the drawings and the accompanying description.
[0073] The inventive technique is based on measuring the absorption
of illuminating light (typically in the infrared spectrum)
transmitted through a gaseous atmosphere in fluid communication
with the biological material, e.g. in a portion of a storage
container above the biological material. Living microorganisms
produce metabolic gases such as carbon dioxide (CO.sub.2) during
respiration. By means of infrared absorption, the concentration of
metabolic gases can be measured inside the storage container.
[0074] Reference is made to FIG. 1A illustrating schematically in a
block diagram a system 10 according to some embodiments of the
present invention for detection of microorganisms in a biological
material. The system 10 includes an optical system which includes a
tunable broadband IR light source 12 and a detection module 15. The
tunable broadband IR light source 12 is configured and operable for
emitting light in a predetermined substantially narrow spectrum.
The tunable broadband IR light source 12 is controllably operated
for emitting light in at least a first and a second predetermined
wavelengths, wherein the first wavelength corresponds to an
absorption peak of at least one metabolic gas to be detected, and
the second predetermined wavelength is in a spectral region outside
the absorption peak of the at least one metabolic gas. The
detection module 15 includes a detector 14 sensitive in the IR
wavelength regime. The light source and the detection module are
arranged to form respectively illumination and detection paths,
e.g. being in optical communication with one another, intersecting
a region of interest. The detection module is configured and
operable for detecting light in the first and second wavelengths,
such as light passing through the region of interest located in
between the light source 12 and the detector 14, and generating
intensity data/signals indicative of the intensity of detected
light in the first and second wavelengths. This data is therefore
indicative of the transmittance of the region of interest to the at
least said first and second wavelengths.
[0075] Further provided in the system 10 is a control system 30
(e.g. controller), which is connectable to the optical system, i.e.
to the light source 12 and to the detection module 15. The
controller 30 is configured and operable for operating the light
source 12 to emit light in the selected at least first and second
wavelengths, and for receiving and analyzing measured/detected
data/signals from the detection module and generating data
indicative of a concentration of the metabolic gas in the region of
interest.
[0076] In some embodiments, the light source 12 and the detector 14
are arranged in space-apart relationship defining the region of
interest therebetween for spectroscopic measurements. To this end,
light source 12 and the detector 14 are arranged such that a
suitable container 24 of a biological material 26 and/or more
specifically a dead space 28 associated with and being in fluid
communication with such container 24 can be placed. The biological
material is that in which microorganisms should be detected by
optical/spectroscopic measurements performed by the system 10 of
the present invention. As indicated above, the dead space 28 of the
container is actually any space being in fluid communication with
the atmosphere in the container above biological material 26. This
may include any one of the following: the portion 28 of the
container 24 above the biological material as illustrated for
example in FIG. 1A, and/or any suitable gas-chamber such as a
reservoir and/or an outlet pipe/tube connected to the container and
being in fluid communication with its atmosphere, as will be
described below with reference to FIGS. 2A-2B and 5B.
[0077] The analysis of the measured data is generally based on the
principles of spectroscopy. However, in the present invention, the
first and second wavelengths are particularly selected to enable
accurate and high-sensitivity measurements of the concentrations of
one or more metabolic gases, even in noisy environment, possibly
having a wide range of unknown parameters. The first wavelengths is
selected to be highly affected by absorbance by the at least one
metabolic gas in the region of interest, i.e. to overlap with the
absorption line (at times termed herein as "peak"). The second
wavelength is on the other hand selected to be less affected by
absorbance of the metabolic gas, but nevertheless it is selected to
be spectrally close to the first wavelength, such that it provides
reference data indicative of absorbance of the first wavelength by
other materials in the region of interest. For example the distance
between the first and second wavelengths may in some embodiments be
about half of distance between two absorption lines of the
metabolic gas, or in some embodiments the second wavelength may be
located outside the spectral range including intense absorption
lines of the metabolic gas. This is exemplified in and described in
more details below with reference to FIGS. 1C and 1D.
[0078] In this regards, it should be noted that the inventors have
found that some metabolic gases are associated with spectral
regimes of high absorbance in which sharp absorbance peaks exist in
the vicinity of sharp transmission peaks (absorbance valleys). On
the contrary, the absorption spectra of various materials (e.g.
polymer, plastic of which various containers are made as well as
vapors such as water vapors), which are in many cases located in
the optical path of the measured gaseous media and impede accurate
spectroscopic measurements of such metabolic gases, are typically
associated with relatively broad and non-volatile spectral profile
which does not have sharp peaks. Taking advantage of this finding,
some embodiments of the invention utilize the so-selected second
wavelength to provide reference to the absorbance of the first
wavelength by materials other than the metabolic gas in the
vicinity of the region of interest. This can be done because the
spectral distance between the first and second wavelengths is
selected such, or is sufficiently small, such that the first and
second wavelengths exhibit same or nearly similar absorption by
various materials possibly located in the optical path.
[0079] Thus, the at least two wavelengths are specifically selected
such that the one of them is highly absorptive by the metabolic gas
to be detected (which concentration is to be measured) and the
second is selected to be less absorbed by the metabolic gas but
absorbed to similar level (as the first wavelength) by other
materials conventionally/typically located in the optical path of
measurements. This allows for in situ non-invasive
optical/spectroscopic measurements of the metabolic gas
concentration with high accuracy and/or high sensitivity while not
reducing the effects of unknown materials/condition in the vicinity
and/or in the region of interest of the measurement.
[0080] In this connection it should be understood that in some
embodiments the system and method of the present invention are
adapted for conducting optical measurements in more than two
wavelengths; e.g. with one or more wavelengths of the first
type--highly absorbed by the metabolic gas, and/or with one or more
wavelengths of the second type--less/negligibly absorbed by the
metabolic gas. More specifically a coherent broadly tunable light
source (e.g. Mid-IR light source) is used to emit light at first
type wavelength overlapping with absorption line(s) of the
metabolic gas and having a spectral width wider than that of the
absorption line. In the description below the term overlapping is
used also to denote that the spectral width of the light source
exceeds the spectral width of metabolic gas (CO.sub.2) lines. This
ensures robustness and high repeatability of the measurements. More
precisely, the spectral width of the light source is preferably
more than the spectral width of the absorption line, but less than
the distance between two adjacent spectral absorption lines. Thus,
during a wavelength scan of the laser/light-source, the scanning
accuracy with spectral resolution comparable or higher than the
spectral width of the light source in order to enable to tune the
light source to overlap/cover the absorption line. The use of light
source with spectral width exceeding the spectral width of
absorption lines provides that spectral resolution of tuning needs
not to be higher than the width of the gas absorption line, which
may be very small (e.g. wave-number below 0.05 cm.sup.-1) and which
spectral resolution of tuning is hard to achieve. Also, the
spectral width of the absorption line is a function of gas pressure
and temperature. Hence utilizing a light source having spectral
width exceeding the width of absorption line, decreases the
sensitivity of the variations of the spectral width of the
absorption lines thus improving the robustness of the measurements
under varying conditions (temperatures and pressures). Also the
condition that the spectral width of the light source is less than
the distance between two adjacent spectral absorption lines ensures
high contrast and/or resolution when measuring at the second type
wavelength, away from the maximum absorption.
[0081] In fact, in some embodiments the light source 12 is a
broad-band tunable light source with sufficiently narrow spectral
peak. The control system 30 is in communication (by wires or
wireless signal communication) with the light source 12 and
configured for swiping the wavelength emitted by the light source
12 (continuously or discretely) over a certain wavelength range
including several absorption peaks and/or valleys of absorption in
the absorption spectra of the metabolic gas to be detected. The
control system is also in communication (by wires or wireless
signal communication) with the detection module (e.g. for operating
it) for receiving measured data indicative of transmission of the
several wavelengths in this spectral ranges such that one or more
of the wavelengths correspond to the first type wavelength and one
or more of them correspond to the second type wavelength. This
procedure, in which data on transmission of more than two such
wavelengths is acquired, is used in some cases, to further improve
the accuracy and sensitivity of the system and method of the
present invention event in very noisy scenarios.
[0082] It should be understood, although not specifically
illustrated, that the control system is typically a computer system
including inter alia digital or analog input and output ports,
memory, data processor, and may be implemented as hardware and/or
software modules. Such a computer system may be at least partially
integral with the detection module.
[0083] In some embodiments of the present invention, the light
source 12 is configured for emitting substantially monochromatic
light, which narrow spectral width is in the order of the width of
certain spectral peaks/features in the absorbance profile of the
metabolic gas to be detected. For example, in some cases the light
source 12 is a tunable IR light source/laser. In particular the
light source may be selected to be tunable within a certain
wavelength band in the mid-IR regime (the term mid-IR is used
herein to designate wavelengths of light in the spectral range of 3
to 30 microns. In some particular embodiments of the system 10 of
the present invention the tunable quantum cascade laser (QCL) is
used as the light source 12, since it provides wide wavelength
tunability and sufficiently narrow spectral width (sufficiently
monochromatic light emission). Alternatively or additionally, the
light source 12 can also be a broadband source equipped with
suitable narrow-band spectral filters in the mid-IR regime. The use
of a tunable light source instead of the fixed wavelength source
allows for determining the metabolic gas concentration within the
container without using any etalon.
[0084] The detection module 15 may include an IR detector 14 whose
output is connected to an electronic signal processor/amplifier 13.
In some embodiments, the electronic signal processor 13 includes or
is constituted by a lock-in amplifier.
[0085] In some cases, the at least one first wavelength is selected
to be in a spectral regime of high absorbance of carbon dioxide
(being the probed metabolic gas) such that first wavelength
overlaps an absorption peak/line of carbon dioxide in this regime.
For example the at least one first wavelength may be in the
wavelength band in the vicinity of 4.3 microns which corresponds to
spectral regime of high absorbance by CO.sub.2. Indeed, in some
cases also the second wavelength is selected close to the first
wavelength and in the similar regime of high absorbance by the
metabolic gas. However the second wavelength is specifically
selected/tuned to fall outside of an absorption line (e.g. in an
absorption valley) of the carbon-dioxide/metabolic gas such that it
only provides a reference to the absorbance by other material in
this regime.
[0086] In this connection, reference is now made to FIGS. 1B, 1C
and 1D, in which FIG. 1B shows a graph of the absorption cross
section of CO.sub.2 at ambient conditions (namely pressure of 1
atmosphere and temperature of 25.degree. C.) as a function of wave
number (taken from HITRAN database), and FIGS. 1C and 1D show in
more details a part of this absorption cross section of CO.sub.2
superposed with two examples of spectral emission profile of the
light source for the first and second wavelengths produced by a QCL
light source. These profiles correspond to substantially
monochromatic light beams of first and second narrow wavelength
spectra. In the example of FIG. 1C the emission profile of the
light source for the first and second wavelengths respectively,
overlap/cover an absorption peak (absorption line) of the CO.sub.2
and overlap/cover an absorption valley (transmission line) of
CO.sub.2 in the mid-IR spectrum near 4.3 microns. In FIG. 1D, as in
FIG. 1C the emission profile of the first wavelength, overlaps an
absorption line of the metabolic gas, while the second wavelength
is located outside a spectral range of intense absorption lines of
the metabolic gas.
[0087] More specifically FIG. 1B shows the features of the CO.sub.2
absorption spectrum in wave-number range of near about 2300 to 2380
cm.sup.-1 (i.e. for mid-IR wavelength range of about 4.2 to 4.35
microns).
[0088] FIG. 1C shows in more details the absorption spectrum (graph
Gco.sub.2) of CO.sub.2 in the wave-number range of about 2359 to
2364 cm.sup.-1 (i.e. for wavelengths ranging between 4.230 to 4.239
microns) and illustrates the typical width of CO.sub.2 absorption
lines of about 0.07 cm.sup.-1 (namely in the order of about 0.15 nm
in that wavelength range).
[0089] FIGS. 1C and 1D present narrow spectral profile (graphs
G.sub.1 in these figures) of a first light beam (first type
wavelength) produced by a tunable QCL light source 12 used in some
embodiments of the present invention. The typical spectral width of
the QCL emission is of full-width-half-maximum (FWHM) of about
FWHM=0.7 cm.sup.-1 (i.e. about 1.25 nm), and the figures show said
spectrum of the light-beam being tuned to a central wave-number of
2361.4 cm.sup.-1 (wavelength of about 4234.8 nm microns)
corresponding to the maximum of one of the absorption lines of the
CO.sub.2 gas.
[0090] In the example of FIG. 1C the second wavelength presented by
graph G.sub.2 was tuned to central wavenumber of 2362 cm.sup.-1
(wavelength of about 4233.7 nm) which is in the middle between the
two absorption lines of the metabolic gas. Thus, in this case the
CO.sub.2absorption at the second wavenumber will be much lower,
than at the first one.
[0091] In the example of FIG. 1D the second wavelength presented by
graph G.sub.2 was tuned to central wavenumber of 2385 cm.sup.-1
(4192.9 nm). Therefore in this example the second wavenumber is
outside the spectral range/band of high absorbance by the metabolic
gas (e.g. outside the so called 4.3 micron absorbance band of
CO.sub.2 at which main CO.sub.2absorption lines are present).
Accordingly the CO.sub.2 absorption at 2385 cm.sup.-1 wavenumber is
much lower, than its absorbance at both at the 2361.4 cm.sup.-1 and
2362 cm.sup.-1 wave-numbers.
[0092] The transmittance of CO.sub.2 for the above exemplified
wave-numbers of the first and second type wavelengths is
illustrated in logarithmic scale in FIG. 1E for various
concentrations of the metabolic gas CO.sub.2. The transmittance was
simulated for a container with transparent walls filled with mixed
N.sub.2 and CO.sub.2 gases at different CO.sub.2 concentrations, at
normal pressure, normal/room temperature of 300 K, and with optical
absorption path of 8 cm. The transmission of the first type
wavelength (graph G.sub.1 in FIGS. 1C and 1D corresponding to
wave-number 2361.4 cm.sup.-1) is illustrated in graph T1. The
transmission of the second type wavelengths (graphs G.sub.2 in
FIGS. 1C and 1D) corresponding to wave-numbers 2362 cm.sup.-1 and
2385 cm.sup.-1 are illustrated in graphs T2 and T3 respectively. As
shown from these graphs, the absorbance of the first wave-number in
the CO.sub.2 (graph T1) is substantially higher than the absorbance
of the second type wave-numbers (graphs T2 and T3).
[0093] FIG. 1F shows in self-explanatory manner the transmittance
ratios T1/T3 and T2/T3. These ratios are monotonically decreasing
functions in both cases. Accordingly in some embodiments of the
present invention it may be sufficient measure the CO.sub.2
absorbance to obtain the value of one such ratio which is
sufficient to for unambiguous determination of the CO.sub.2
concentration in the container. However, in some embodiments of the
present invention the reliability of the measurements are further
improved by utilizing more than two wavelengths (e.g. to obtain
more than one such ratio). That is especially relevant for high
concentrations of CO.sub.2 gas (exceeding few per cent level) where
absorption at certain wavelengths could be very strong and measured
signal very weak.
[0094] To this end, according to the invention the controller
operates/tunes the light source to emit light in the first
substantially monochromatic wavelength(s) (e.g. as illustrated in
graph G.sub.1) corresponding and overlapping/covering the high
absorbance peaks/lines of the metabolic gas such as those of
CO.sub.2 illustrated in the figure, thereby providing accurate
measurement of the CO.sub.2 absorbance. Additionally, as noted
above, the controller operates/tunes the light source to emit light
in second substantially monochromatic wavelength(s) (e.g. as
illustrated in graphs G.sub.2 of FIGS. 1C and 1D) corresponding to
regions/valleys of low absorbance of the metabolic gas (e.g.
transmission lines between the absorption lines which spacing is in
the order of about 1 cm.sup.-1 to 1.5 cm.sup.-1 --about 1.8 nm-2.5
nm in wavelength, and/or regions outside the high absorbance band
of the gas). These second monochromatic wavelength(s), while being
narrow and tuned so as to substantially not overlap with the
absorption lines are provided for reference to the absorption of
materials in the optical path other than the probed metabolic gas.
Further, these second wavelengths are in the general
neighborhood/band of the first wavelengths such that they provide
reference data being indicative, with good accuracy, of the
absorption of the first optical wavelength(s) in the optical path,
in case the probed metabolic gas was absent there. In other words,
the second predetermined wavelength is selected such that measured
data of the transmission thereof provides reference indicative to
the optical absorbance of the first wavelength by materials other
than the metabolic gas.
[0095] For example, in some cases a spectral distance between a
"first type" wavelength of the emitted light and a "second type"
wavelength serving for the reference measurement (to which the
metabolic gas is substantially transmitting) is in the order of 0.5
cm.sup.-1-0.75 cm.sup.-1 that corresponds to the half of distance
between two absorption lines of CO.sub.2 gas. Yet, in other case
the separation distance can be of order 30 cm.sup.-1 for the second
absorption line to be outside the whole spectral range with intense
absorption lines. In that case this wavelength could be used as
reference wavelength transmittance of light beam at this wavelength
does not depends on the concentration of CO.sub.2, but rather
reflects absorptions of the light beam on other optical components
such as container walls transmittance. Hence, the spectral distance
between the first and second wavelengths is substantially small
such that the first and second wavelengths are characterized by
same or similar transmission through predetermined/conventional
materials used for conventional containers of biological
material.
[0096] The use of such first type wavelength(s) for the measurement
of absorbance by the metabolic gas and the second type
wavelength(s) close thereto for reference measurements allows to
detect small/minute changes in the metabolic gas concentration with
high sensitivity and accuracy. For example changes of 10 ppm (or
0.001%), and even as low as 1 ppm (0.0001%) in the gas
concentration can be detected even in relatively noisy environments
in which various other materials such as those of the walls of the
storage-bag and/or fermentation container and/or vapors (e.g. water
vapors) are located in the region of interest. The effects of the
later can be discarded with accuracy based on the reference
measurements of the second wavelengths being close to the first
wavelengths. Consequently, the present invention allows the
detection of relative concentrations of metabolic gas from 0 to
100% (full dynamic range) using one single device having a
sensitivity range of 1-100 ppm, preferably 1-10 ppm.
[0097] As a result of the above, in some cases the technique of the
present invention is employed to measure metabolic gas
concentrations in conventional containers such as conventional
storage bags/vials for platelets. This is achieved by taking
advantage of the above described technique utilizing the "first
type" and "second type" (measurement and reference wavelengths) in
the spectroscopic measurements. Thus, the detection of various
microorganisms in-situ in such conventional and generally arbitrary
containers is made possible although the materials of the container
is not a-priory known, and although the container's materials may
include materials such as polymers and/or other materials, which
may be relatively opaque to the IR wavelengths used by the system
of the invention (e.g. opaque to wavelengths in the mid-IR).
[0098] As noted above in some embodiments the detection module 15
includes an electronic signal processor/lock-in amplifier 13 that
receives the signal from the IR detector 14. Use of the lock-in
amplifier enables to even further improve the signal to noise ratio
(SNR) provided by the system thus further improving the sensitivity
and accuracy of the measurements relating to the concentration(s)
of metabolic gases and consequently detection of microorganisms. To
this end, in such embodiments the control system 30 is adapted for
operating the tunable broadband IR light source 12 for applying
time modulation to intensity of light emitted in one or more (e.g.
in each) of the at least two (first and second) wavelengths, and
also operating the lock-in amplifier 13 to determine/measure the
detected intensity(ies) of the emitted light with high accuracy
based on that modulation. Accordingly, transmittance of the region
of interest to the first and second wavelengths (e.g. to all
wavelengths used in the measurement) can be determined with high
accuracy based on the intensity modulation, while noise is mostly
discarded as it is generally not modulated in the same way. It
should be noted that the configuration and operation of various
lock-in amplifiers are generally known in the art of signal
processing and are therefore not specifically described herein. A
person versed in this art would readily appreciate the various
possible configurations of such lock-in amplifier with appropriate
modulation to the emitted illumination to be used in the system of
the invention.
[0099] In some embodiments, the system 10 is configured and
operable for utilizing its ability to accurately detect small
changes in the metabolic gas concentration, for operation in-situ
and/or in real time to non-invasively detect microorganisms in the
containers/bags permeable to the metabolic gas and/or in
association with one or more openings (inlets/outlets) through
which in/out flow of gases may occur. Specifically, in some
embodiments, after analyzing the concentrations of the metabolic
gas in the dead space 28, the controller 30 is adapted for further
processing the concentration of metabolic gas to detect
microorganisms in the biological material 26.
[0100] In some cases, the detection is merely qualitative to
identify whether significant levels/amounts of such microorganisms
exist in the biological material 26. In other cases, the detection
is qualitative and is aimed at estimating the levels (e.g.
amounts/concentrations) of the microorganisms in the biological
material 26.
[0101] In some embodiments the system is adapted/configured for
operating in closed containers, sealed and non-permeable to the
probed metabolic gas(es). In such cases, the concentration of the
metabolic gas in the container is a function of the time the
container is sealed and the amount of microorganisms therein during
that time. Specifically, in sealed container the concentration of
CO.sub.2 is increasing function with time as long as there is
biological activity inside of container responsible for CO.sub.2
emission. Thus, the concentration of living cells in that case
depends on the growth history of the cells from the beginning of
incubation. The correspondence between the number of cells and
measured CO.sub.2 concentration could be find out in that case by
computing rate of changes of CO.sub.2concentration and correlating
it with respiration rate of a single cells and the number of
cells.
[0102] Alternatively or additionally, in some embodiments the
container is not sealed with respect to the metabolic gas. For
example, in cases of monitoring a biological material stored in
bags or storage vials for blood components, which are permeable to
the metabolic gas, and/or in cases of monitoring a biological
material in fermentation containers associated with gas inlet(s)
and/or outlet. Advantageously, the present invention allows for
detecting microorganisms also in such containers in-situ and in
real time without taking and sealing a sample of biological
material from the containers and without incubating the sample
(i.e. non-invasive detection). This is performed by taking
advantage of the high sensitivity and accuracy of the technique of
the invention for detecting small changes in the metabolic gas
concentration.
[0103] To this end, the controller 30 system may operate to measure
the metabolic gas concentration in a dead space associated with the
container of biological material, while the dead space is non
sealed to the metabolic gas, and to utilize the measured
concentration of the metabolic gas to detect the microorganisms
qualitatively and/or qualitatively based on an equilibrium
condition (e.g. balance/difference) between a rate of escape/flow
of the metabolic gas from/into the container and a rate of
generation or consumption of the metabolic gas by the
microorganisms.
[0104] In cases the container is permeable to the metabolic gas,
processing may be performed by computing/estimating the
microorganisms level in the biological martial 26 based on the
measured concentration of the metabolic gas diffusion of that gas
through walls of the container 24. In this case, the container may
be a conventional storage container for platelets or other blood
components.
[0105] Additionally or alternatively system 10 of the invention
allows real-time in-situ detection of microorganisms in biological
material 26 contained in fermentation container 24 (e.g. in a
container of a fermentation system). To this end, the controller 30
may be configured and operable to determine the level of
microorganisms based on the equilibrium condition (e.g.
balance/difference) between the rate of escape/flow of the
metabolic gas from/into the container 24 through an outlet thereof,
and a rate of generation or consumption of the metabolic gas by the
microorganisms. For example that balance may be determined based on
a difference between concentrations of the metabolic gas in a gas
inlet to the container 24 (e.g. the concentration in the external
atmosphere) and a concentration of that metabolic gas in the
atmosphere in the dead space 28 of the container, which may be a
dead space of the container itself (e.g. above the biological
material 26) or a dead space in fluid communication therewith, for
example located at a gas outlet of the container. The difference in
the metabolic gas concentrations corresponds to the amount of
microorganisms in the container. More particularly, this is the
difference that may be computed from the difference in the amount
of metabolic gas flowing in and out of the container (e.g. computed
by the controller 30 as the difference between the products of the
flow rates in the gas inlet and outlets multiplied by the metabolic
gas concentration thereat respectively).
[0106] The controller 30 may utilize predetermined data of the
concentration of the probed metabolic gas in the gas inlet (e.g. in
the external atmosphere of the container. Also the
spectroscopic/optical measurements may be applied to a region of
interest associated with the cavity/dead-space of the container
itself and/or at a gas outlet from the container. The light source
12 and the detector 14 may be optically coupled to one or more
optical windows exposing the dead-space to be inspected (e.g.
optical windows coupled to the fermentation container and/or to gas
outlet).
[0107] In some embodiments of the present invention, the system 10
is specifically configured for real time and continuous/periodical
monitoring of fermentation processes. In such embodiments the
controller 30 may be adapted to apply continuous/periodic detection
of microorganisms and/or their level in the fermentation container.
In this connection, the controller 30 may be adapted to repeatedly
operate the optical system in the manner described above to obtain,
in real time, and/or repeatedly within predetermined time
intervals, data indicative of the metabolic gas concentration in
the container and/or in its outlet. Repeatedly/continuously
obtaining such gas concentration during a period of time provides
indication to the amount of microorganisms in the container as a
function of time and/or indication to the change/rate of change in
this amount. The controller may be adapted to process the data
indicative of the amount of microorganisms, or the change thereof,
as function of time for monitoring and/or controlling the
fermentation process occurring in the container. For example,
reference data/model relating to the fermentation process in a
container may be utilized by the controller to determine
actions/operations to be carried out for controlling the
fermentation (e.g. stopping the fermentation and/or changing some
of the fermentation conditions such as temperature and/or other
conditions). This reference data may for example be stored in the
form of a lookup table (LUT) and/or a set of one or more
functions/models relating to the amount of micro-organisms and/or
rate of change in their amount with certain actions to be carried
out and/or certain fermentation conditions to be
applied/maintained.
[0108] The fermentation process monitoring/controlling can be
realized using the a model (e.g. mathematical-model/formula and/or
data) relating the amount of biomass in the fermentor with measured
concentration of CO.sub.2. The model may be pre-determined and
predefined in advance and loaded to memory and/or other storage
device of the controller 30 in the form of data/LUT and/or as a set
of instructions soft/hard coded.
[0109] Such a model relating the amount of biomass in fermentor
with the concentration of CO.sub.2 may be obtained/determined in
advance by utilizing various techniques. For example, the amount of
biomass may be measured by optical density techniques (OD) and/or
viable counts and/or other run parameters such as pH, RPM, TEMP,
and the total volume (TV) of the metabolic gas CO.sub.2
emitted/consumed by the microorganisms from the beginning of the
run (seeding time). The model may be a mathematical model based for
example on multivariable robust regression analysis. Verification
of the mathematical model may be performed by number of
fermentation processes performed under same conditions, which are
optimal for the high product yield in the batch fermentor.
[0110] A RUN protocol, such as executable instructions and/or LUT,
is then used by the controller 30 for estimating, the amount of
biomass in the fermentor based on the model's parameters and the
real-time measured concentration of CO.sub.2 gas and possibly other
run parameters. The RUN protocol is based on estimated values of
the biomass and may include data indicative of different
aspects/actions to be taken during the monitoring of the
fermentation process (e.g. conditions for adding nutrients for cell
in fermentation, determining optimal inducing time (for recombinant
protein production) and harvesting time, controlling pH level and
other run aspects.
[0111] As noted above, in cases of biological containers which are
not-sealed to the metabolic gas (e.g. permeable containers or
containers of fermentation), the bacterial growth will be reflected
in real time in changes in the metabolic gas concentration. For
example, in case the bacteria are no longer alive, the carbon
dioxide concentration will be substantially equal to equilibrate
with that in the air outside the storage tank.
[0112] The concentration of metabolic gases inside a gas permeable
container is determined by equilibrium conditions between release
and rate of diffusion of metabolic gases through the walls of the
container. It should be noted that in the following description
containers permeable to the metabolic gas(es) are considered as an
example of not-sealed containers with respect to the metabolic
gas(es). Also, in the description below such permeability and
diffusion of the gas from and into the container is exemplified as
relating to the permeability of the container's walls. However, it
should be understood that the technique described below is
applicable to any other type of containers no sealed with respect
to metabolic gases, wherein the flow in and out of the container
may be additionally or alternatively through gas inlets and/or
outlets of the container. Further, in connection with the diffusion
equations used in the description below, it should be noted that in
case of non-permeable non-sealed containers these can be
substituted by proper equations taking into account other
"diffusion" paths such as flow through inlet/outlet pipes and
concentration of the metabolic gas therein. In other words,
although the examples in the description below mainly refers to
permeable platelets bags, it should be understood that the
technique can easily be generalized by a person versed in the art
for other types of cavities where metabolic gas may be
concentrated, including the dead space in the container itself
and/or that of the inlet/outlet pipes of a fermentation container
for example.
[0113] The equation for gas (such as CO.sub.2) production and
transport through the walls of a permeable container states:
.differential. m CO 2 .differential. t = - JA + W ( 1 )
##EQU00001##
where m.sub.CO2 is the mass of CO.sub.2 gas inside the container, J
is the diffusion flux from the walls of the container in units
kg/(s.m.sup.2), A is the surface of the walls exposed to the gas
exchange and W is the source term that describe total rate of
CO.sub.2 production inside the container. W has units of kg/s. The
diffusion flux is given by equation 2:
J=(.rho..sub.CO2(t)-.rho..sub.CO2.sup..differential.)v (2)
where, .rho..sub.CO2(t) is the mass concentration of the CO.sub.2
gas in units kg/m.sup.3, measured at time t,
p.sub.CO2.sup..differential. is the ambient mass concentration of
CO.sub.2 gas, v is the membrane permeability coefficient in units
m/s.
[0114] In equilibrium,
.differential. m CO 2 .differential. t = W ( 4 ) ##EQU00002##
meaning that the CO.sub.2 emission rate of enclosed biological
material is equal to the total diffusion rate through the container
walls:
W(t)=(.rho..sub.CO2(t)-.rho..sub.CO2.sup.0)vA (3)
[0115] If the container is sealed, then this equation is
inapplicable, since no gas exchange can undergo through the
container walls. In this case, the concentration of CO.sub.2 is
gathered by Eq. (1) with the first term in the right side equal to
zero:
.differential. m CO 2 .differential. t = 0 , ##EQU00003##
where W(t) is the CO.sub.2 emission rate of enclosed biological
material in units kg/s measured at time t. Taking into account that
mass concentration is defined as the mass of a constituent divided
by the volume .rho..sub.CO2=m.sub.CO2/V, the following expression
is obtained for .rho..sub.CO2 by integrating Eq. (4):
.rho. CO 2 ( .tau. ) = 1 V .intg. 0 .tau. W ( t ) t ( 5 )
##EQU00004##
where V is the volume of the container. Since, W(t) is nonnegative,
.rho..sub.CO2(.tau.) is monotonically increasing function with
time. Time t=0 in the integral corresponds to the beginning of the
run (seed time in the fermentation process).
[0116] From the above equation, the following expression can be
obtained for mean (W(t)) CO.sub.2 emission of enclosed biological
material averaged over time interval .tau.:
( W ( t ) ) = ( .rho. CO 2 ( t ) - .rho. CO 2 ( t - .tau. ) ) V
.tau. ( 6 ) ##EQU00005##
[0117] The change of concentration n.sub.CO2 can be measured by
means of IR absorption of beam of tunable IR light source such as
Quantum Cascade Laser directed through the container walls. As
described above, the use of the tunable source instead of the fixed
wavelength source allows direct measurement of CO.sub.2
concentration inside the container regardless of container material
and without use of etalon container.
[0118] The following are some examples of the technique of the
present invention for detection of metabolic gas(es) concentration,
assume that the container walls are at least partially transparent
at the mid-IR frequency range where strong absorption of CO.sub.2
occurs (around 2260 cm.sup.-1-2390 cm.sup.-1), and that the path
for optical beam in the gaseous atmosphere inside the container is
provided.
[0119] The dependence of % CO.sub.2 level on the increase of
bacterial contamination was studied experimentally. Staphylococcus
epidermidis obtained from the American Type Culture Collection
(ATCC) were used to contaminate a bag of platelets that were
collected from a single donorby apheresis. The bacterially
inoculated apheresis platelets were agitated at 22.degree. C. and
measurements were performed using QCL spectroscopy. The platelet
container was measured before and during bacterial contamination.
Samples were taken from the contaminated platelet bag and a
standard culture plate count was used for determining bacterial
concentration [colony forming unit (CFU)/mL] in the platelet
medium.
[0120] Referring now to the drawings, FIG. 3 is a plot of % CO2 vs.
bacterial concentration. The bacterial concentration that was
measured at the point where % CO.sub.2 started to rise was between
1*10.sup.6 CFU/mL to 6*10.sup.6 CFU/mL. The Y-axis shows % CO.sub.2
level and the X-axis shows bacterial concentration measured using
standard titration analysis.
[0121] Turning back to FIG. 1A, system 10 of the present invention
may be used for measuring the concentration of carbon dioxide in
the dead space 28 above the platelets 26 in a gas-permeable bag 24
that has been removed temporarily from storage and agitation for
the purpose of measuring the concentration of carbon dioxide in
dead space 28. The tunable infrared laser 12 (for example a QCL)
and an infrared detector 14 are positioned so that the light beam
20 from laser 12 is aimed at detector 14. In this example, the
light beam 20 is focused on detector 14 by a calcium fluoride lens
18. Bag 24 is positioned between laser 12 and detector 14 so that
light beam 20 traverses dead space 28. Controller 30 tunes laser 12
to emit light beam 20 at selected wavelengths in the vicinity of
4.3 microns at a pulse repetition rate of 5 KHz, receive the
corresponding response signals from detector 14, and analyze those
signals to estimate the concentration of carbon dioxide in dead
space 28. As noted above, the signal reception and analysis portion
of the controller 30 may be implemented by a lock-in amplifier that
locks onto the 5 KHz signal from detector 14 and displays the
amplitude and phase of that signal. For an accurate measurement of
the concentration of carbon dioxide in dead space 28 the path
length of light beam 20 across the interior of bag 24 is preferably
at least several centimeters.
[0122] In practice, a sufficiently long optical path through bag 24
may not be available, and/or the walls of bag 24 may not be
sufficiently transparent at the relevant wavelengths to allow an
accurate measurement of the concentration of carbon dioxide in dead
space 28. In this connection, reference is made to FIGS. 2A and 2B
illustrating two modified configurations of system 10 that deal
with these problems. System 10 is configured generally similar to
that of FIGS. 1A. As shown in FIG. 2A, the bag 24 may be held in
place by two vertical walls 16. In distinction to the system of
FIG. 1A, in FIG. 2B a separate closed (e.g. cylindrical) gas
chamber (pipe/reservoir) 40 is used and is connected to the bag 24
using a connecting tube 50 which is by its one end connected by
fusion with a heating instrument to the bag, as is routinely done
to platelet bags in blood banks, for various reasons of their own.
Such procedures are performed routinely without damaging the
platelet bags or introducing contamination. The other end of the
connecting tube 50 is connected to the chamber 40 that is at least
partially transparent in at least a portion thereof to the relevant
wavelengths and that is sufficiently rigid and long enough to
provide a predetermined fixed optical path (e.g. of several
centimeters) for light beam 20. In this specific but not limiting
example, a filter 52 is used in tube 50, that is permeable to gases
but not to liquids, and thus keeps liquids from bag 24 out of
reservoir 40 but allows the gaseous contents of reservoir 40 to
equilibrate with the gaseous contents of dead space 28 in the bag
24 so that the concentration of carbon dioxide in reservoir 40 is
substantially identical to the concentration of carbon dioxide in
dead space 28. The equilibration of the concentration of carbon
dioxide between reservoir 40 and dead space 28 occurs sufficiently
fast such that no special steps are needed to hasten this
equilibration. Effectively, the interior of reservoir 40 is an
extension of dead space 28. Such connection to the separate gas
chamber may be used in case the required optical path (for
detection of the specific metabolic gas) inside the container is
unavailable and/or in case the container's walls totally block the
IR radiation.
[0123] In a general case, irrespectively of whether the container
is sealed or permeable with respect to the metabolic gases to be
detected, the following technique may be carried out by the
controller 30 for accurate, in-situ real time determination of the
metabolic gas(es) concentration. IR transmission through the dead
space in two or more wavelengths (comprising the above described
first and second wavelengths) is measured as described above. The
measured data is processed by the controller 30 for example by
utilizing an absorption model of the at least one metabolic gas. To
this end, a best fit between intensity values obtained from the
absorption model and the measured intensity values is performed to
thereby determine the concentration of the at least one metabolic
gas.
[0124] The following is a not limiting example of the mathematical
description of the measurement procedure of metabolic gas
concentration inside a container using tunable IR light source.
[0125] The transmitted laser light intensity I(.lamda..sub.0)
measured on the detector is given by at the laser central
wavelength .lamda..sub.0 is given by
I(.lamda..sub.0)=.eta.I.sub.0.intg..sub..lamda.min.sup..lamda.maxf(.lamd-
a.-.lamda..sub.0)e.sup.-a.lamda.(cl+c.sup.0.sup.I.sup.0.sup.)d.lamda.
(7)
[0126] where I.sub.0 is the laser intensity, .eta. is the total
intensity loss that are not related to optical gas absorption, ax
is the absorption coefficient (in cm.sup.-1) at the given
wavelength of the light .lamda., c is the probed gas concentration
(by volume) inside the container, c.sub.0 is the concentration of
the probed gas outside the container in the atmosphere, l is the
pathlength inside the container, lo is the pathlength outside the
container between IR source and detector, f(.lamda.-.lamda..sub.0)
is the laser spectral distribution function around the central
wavelength .lamda..sub.0.
[0127] The integration limits .lamda..sub.min and .lamda..sub.max
with .lamda..sub.min<.lamda..sub.0<.lamda..sub.max are
assumed to be such that f(.lamda.) is nearly zero outside the
integration domain.
[0128] The absorption coefficient .alpha..sub..alpha. can be
calculated as:
.alpha..sub..lamda.=n.sigma.(.lamda.) (8)
[0129] where n=P/k.sub.BT is the concentration of molecules and
.sigma.(.lamda.) is the absorption cross section in cm.sup.2. The
signal on the detector is assumed to be proportional to the
transmittance intensity. In case of tunable laser the central
wavelength can be changed within certain range.
[0130] Thus, a model S(x, .lamda.i) for a signal on the detector
can be written as
S(x, .lamda.i)=b
.intg..sub..lamda..sub.min.sup..lamda..sup.maxf(.lamda.-.lamda..sub.i)e.s-
up.-.alpha..sup..lamda..sup.(x+c.sup.0.sup.l.sup.0.sup.)d.lamda.
(9)
[0131] where x=cl and b is a constant, x (and therefore c ) can be
found from equation (11) if the measurement is done at two or more
wavelengths of light .lamda.. In that case the unknown constant b
can be excluded from the set of equations.
[0132] The concentration c can be determined from n measured values
of the signal S.sub.i i=1, . . . , n at different wavelengths
.lamda. by utilizing nonlinear minimization of the model S(x,
.lamda.i) as provided by function s(x) below:
S ( x ) = i = 1 n - 1 [ log ( S ( x , .lamda. i ) + .epsilon. ) S (
x , .lamda. n ) ) - log ( S i + .epsilon. S n ) ] 2 ( 10 )
##EQU00006##
[0133] where .epsilon. is a noise level at the detector. s(x) is
essentially least squire norm of the logarithm of the ratio between
measured and theoretical signals Si and S(x, .lamda.i) at
wavelength .lamda.i, i=1, . . . , n-1 and the signal at
.lamda..sub..eta.. Thus, .lamda..sub..eta. is used as a reference
wavelength for .lamda.i, i=1, . . . , n-1. The parameter e insures
that function s(x) is not singular if one of Si=0. From equation
(9) the concentration c can be determined, provided that the
optical path length l is known.
[0134] The following is a specific example for using the technique
of the invention for CO.sub.2absorption simulations and evaluation.
The simulations of CO.sub.2 absorption within plastic bags were
performed using HITRAN database of CO.sub.2 line intensities at
ambient conditions and are described above with reference to FIGS.
1B and 1C.
[0135] FIG. 4 shows simulated absorption versus wavelength and
CO.sub.2 concentrations of the beam of the QCL calculated using
equation (7) in the IR spectral range 2355 cm.sup.-1-2410
cm.sup.-1. The graphs H.sub.1, H.sub.2, H.sub.3 and H.sub.4
correspond to the absorbed intensities for respectively 4%, 1%,
0.08% and 0% of the CO.sub.2 concentration. As shown, the graphs
have smooth behavior of the spectra on wavelength and the
transmittance increases when light frequency changes from 2360
cm.sup.-1-2410 cm.sup.-1
[0136] Reference is made to FIGS. 5A and 5B showing experimental
set ups of the system of the present invention for detection of
metabolic CO.sub.2 inside a platelets product in a plastic bag
utilizing respectively direct measurement through the bag
containing the platelets (FIG. 5A) and utilizing a separate gas
chamber connected to the platelets container (FIG. 5B). As shown in
the figures in a self-explanatory manner, the set up utilizes
tunable QCL, IR detector (e.g. equipped with a CaF2 plano-convex
lens), lock-in amplifier detector and a controller. The QCL
operates in the pulse mode with repetition frequency of 5 kHz and
pulse width 500 nsec. The tunability range of the QCL includes the
measurement range that was from 2361.4 cm.sup.-1 till 2391
cm.sup.-1. The procedure for determination CO.sub.2concentration
within container may thus be as follows: the light beam is
transmitted from the QCL through a small container connected to the
plastic bag, container as shown in FIG. 2B described above. Signal
on IR detector is measured at different wavelengths of light
.lamda..sub.i i=1, . . . , n in the range from 2361.4 cm.sup.-1
till 2391 cm.sup.-1. The concentration c of CO.sub.2 gas inside the
container is estimated using equation (10) for different using the
nonlinear minimization of the function s(x).
[0137] As noted above in embodiments of the present invention
configured for monitoring and/or controlling fermentation
processes, the monitoring and controlling of the fermentation may
be based on a model such as formula and/or reference data relating
various parameters of the fermentation including metabolic gas
concentration with an estimation of the biomass (i.e. it amount) in
the fermentation. In some cases a Linear Regression Model is used
for estimating the biomass during the fermentation process.
[0138] In this connection, the regression model may be pre-computed
model calculated utilizing statistical regression analysis for
modeling the relationship between one or more parameters, also
considered as to as predictor x and/or regressor variable(s), and
one or more other parameters, also referred to as response
variable(s) y.
[0139] For the case of estimating of biomass in the fermentation,
the regressor/predictor variable(s) (generally denoted herein
x.sub.i) include the concentration C of CO.sub.2 measured in the
gases emitted from the fermenter, and/or TV being the Total Volume
of CO.sub.2 emitted from the beginning of the run (seeding time).
The response variable (generally denoted herein y) may include the
optical density OD of the biological material in the container
and/or the viable count VC (namely a measure of the count of
microorganisms as typically obtained via microscope count). In
cases that multiple linear regression model is used the response
variables--generally noted y are related to k repressor's, x.sub.1,
x.sub.2, . . . , x.sub.k according to the following formula:
y=.beta..sub.0+.beta..sub.1x.sub.1+.beta..sub.2x.sub.2+. . .
.beta..sub.kx.sub.k+.epsilon. (11)
where .beta.i, i=0, . . . , k, are model parameters (e.g.
coefficients of the model), x.sub.i are the regressor/predictor
variable(s) (e.g. parameters of the fermentation process which are
independent from the response variable (OD in this case)), and s is
the random components with supposed mean zero and variance
.sigma..sup.2.
[0140] The regressor/predictor variable(s) x.sub.i may be measured
in real-time during the fermentation process (e.g. without
sampling/extracting the material from fermentor and probing). For
example, the total volume (TV) of CO.sub.2 gas emitted by
microorganisms vs. time may be calculated using the formula:
TV(t)=.intg..sub.o.sup.trate.times.c(t)dt (12)
[0141] where TV (t) (in liters) is the total volume of CO.sub.2 gas
emitted by the species since the beginning of the run (from the
beginning of the fermentation process), rate is the aeration rate
(e.g. measured in liters/min), and c(t) is the concentration of
CO.sub.2 (in volume fraction) measured in emission gases from the
fermentor.
[0142] In this connection, it should be understood that according
to some embodiments of the present invention, in addition to the
CO.sub.2 concentration predictor parameter c(t), in some cases
other run/predictor parameters/variable(s), may also be measured
(e.g. continuously) to provide estimation of the response
parameter(s) y with improved accuracy. To this end the generalized
formula (13) above may be used as a model defining the relation
between the predictor parameters measured/considered x.sub.i and
the response parameters y. For example the predictor parameters
x.sub.i may include:
[0143] RPM--agitation rate in fermentor (e.g. provided as an input
from a controller controlling the operation of the fermentation
system);
[0144] RATE--aeration rate measured in liters/min (e.g. measured at
the inlet and/or outlets from the fermenter);
[0145] pH--acidity level in fermentor (e.g. measured by pH
electrode in the fermenter)
[0146] DO--dissolved oxygen dO.sub.2 (e.g. measured by dissolved
oxygen probe in the fermenter)
[0147] Temp--temperature in fermentor,
[0148] FIG. 6A is a table illustrating several of the above
predictor parameters measured/obtained during an experiment of a
fermentation process/run of recombinant protein production
utilizing Escherichia coli E. Coli fermentation. The recombinant
proteins are widely used in biotechnology and medical applications
as vaccines and protein therapeutics, and as industrial enzymes for
detergents and fuel ethanol production. Such recombinant protein
products are made by inserting the gene that encodes the desired
protein into a host cell (bacteria, yeast, insect, or animal cells)
capable of producing this protein. FIG. 6B shows graphs OD.sub.g
and TV.sub.g illustrating respectively the measured optical
density--OD and the total volume of CO.sub.2 emission--TV, as a
function of time. OD and TV were measured during a fermentation
experiment. As shown the OD is increased from 1 to about 55 during
the first 10 hours of the experiment. Then it gradually continues
to grow up to values of 75 during the next 12 hours of experiment.
TV was measured continuously while OD at 15 time points as
specified in the table in FIG. 6A. The correlation coefficient
between OD and TV is 94.4%.
[0149] FIG. 6C is a graphical illustration showing two regression
and reference plots, RG and RF, corresponding to the optical
density of a biological material as a function of time. Plot RF is
a reference plot obtained by direct measurements of the optical
density taken during the experimental fermentation run. The
regression plot RG is obtained during the experimental fermentation
run by processing the measured CO.sub.2 concentration based on the
regression model which is used according to the present invention.
As shown in the graphs the OD results obtained by the regression
model based on the CO.sub.2 concentrations are similar and almost
same as those obtained by direct OD measurements.
[0150] The parameters of the regression model are estimated by
means of the robust regression algorithm based on equations 11 and
12 as described above. More specifically, the robust regression
operates by assigning a weight to each data point. Weighting is
done automatically and iteratively using a process known as
iteratively reweighted least squares. In the first iteration, each
point is assigned equal weight and model coefficients (.beta.i in
equation 11 above) are estimated using ordinary least squares. At
subsequent iterations, weights are recomputed, so that points,
which were farther from model predictions in the previous
iteration, are given lower weight. The model coefficients are then
recomputed using weighted least squares. The process continues
until the values of the coefficient estimates converge within a
specified tolerance.
[0151] The regression model used in this experiment utilizes the
following regression coefficients pi between response variable OD
(y in equation 11 above) and the regressor/predictor variables
(x.sub.i in equation 11 above) including: C, TV, RPM, pH and TEMP.
To this end the following regression model was used for the
estimated model parameters for prediction of OD from the measured
values of CO.sub.2 concentration and additional measured parameters
of the fermentation process (the estimated OD being indicative of
the biomass amount):
OD=0.38+0.17*TV+2.06*C+0.0013*(RPM-50)+22.07*(pH-7.0)-0.95*(TEMP-37)
(13)
[0152] FIG. 6D shows the experimental results for a measured plot
P1 of CO.sub.2 concentration (left y axis) and the plot P2 of OD
(right y axis) as a function of time. As shown in the figure there
is a correlation between the OD and CO.sub.2 measurements through
during first 325 minutes of the experiment. Then, due to stress in
carbon source, the bacteria/microorganisms keep growing with
alternative metabolic cycles, as seen in CO.sub.2concentration plot
P1. When the biomass/microorganisms die, the CO.sub.2 decreases
while the OD parameter stays constant.
[0153] FIGS. 7A and 7B show plant cells growth monitoring by
CO.sub.2 online measurement made with the device and method of the
invention versus fresh weight or conductivity measurements. The
measurements have been conducted for plant cells in disposable
fermenters. The high resolution of the device of the invention (1
ppm sensitivity) and continuous (real time) CO.sub.2 measurements
show a very high correlation with cell proliferation as measured by
standard methodologies (fresh weight or conductivity). FIG. 7A
shows the total volume of CO.sub.2 gas emitted by plant cells vs
time, during the whole experimental run correlates with fresh
weight. Time=0 is the seeding time. The total CO.sub.2is correlated
with the biomass and growth rate. This quantity was calculated
using formula (14):
V(t)=rate.times..intg..sub.o.sup.ts(t)dt (14)
[0154] where V(t) (in L) is the total volume of CO.sub.2 gas
emitted by the cells since the beginning of the run, rate is the
aeration rate (in L/min), s(t) is the concentration of CO.sub.2 (in
volume fraction) measured in emission gases from the bioreactor.
s(t) does not contain the initial concentration measured at the
beginning of the run prior to the seeding time, which was
subtracted from s(t). FIG. 7B shows the % concentration of CO.sub.2
gas emitted by plant cells vs time, and correlation with
conductivity, during the whole experimental run.
[0155] FIG. 8 shows experimental results demonstrating that the
device and method of the invention can be employed for detecting
isotopologues of a metabolic gas, such as carbon dioxide, with a
very high sensitivity. In this experiment the presence of people in
an office was monitored during five days based on the concentration
of CO.sub.2 and CO.sub.2. The results show that late afternoon,
when people were leaving, the total concentration of CO.sub.2 was
decreasing while, in the morning, the total concentration of
CO.sub.2 was increasing. It also shows that the device of the
invention was able to detect variations of CO.sub.2 concentration
in the air between 0 and 0.07% (below 700 ppm), but also very
slight variations of CO.sub.2 concentration in the ppm order,
namely between 0 and 7 ppm (about 1% of the total concentration of
CO.sub.2 is composed of isotopologue CO.sub.2). For the sake of
clarity, the curve showing the concentration of .sup.13CO.sub.2 has
been represented with a .times.100 scale.
[0156] FIG. 9 shows experimental results demonstrating the
advantages of the online and continuous monitoring of bacteria
growth via detection of carbon dioxide concentration by the method
and device of the invention (continuous line) versus periodic
optical density manual measurements (dashed line). The lag phase
(first 1.5 h) and log phase (1.5 h-3 h) can be clearly seen with
the method of the invention but not with OD measurements.
Furthermore, IPTG induction at h=3 and its blocking effect on
bacterial replication can be clearly monitored (time during which
the protein of interest is produced). Routine OD measurements were
taken 4 times to monitor the fermentation process (at about h=1.75,
2, 16.25 and 17.75) while the method and device of the invention
enable a continuous monitoring of the bacterial population. Thanks
to the present method, it has been shown that bacterial replication
restarted at h=10 and that a further IPTG induction would have been
possible to optimize the production process.
[0157] Thus, the present invention provides novel, effective and
simple techniques for accurate in-situ real time non-invasive
monitoring of a biological material by monitoring metabolic gases
in the dead space associated with the biological material. The
biological material that can be monitored/inspected utilizing the
invention includes but is not limited to sugars, proteins, or
nucleic acids, or a combination of these substances. They may also
be living entities, such as cells and tissues. They may be made
from a variety of natural resources--human, animal, plant and other
microorganism--and may be produced by biotechnology methods.
Example of biological product is blood transfusion products such as
RBC, platelets and plasma. The biological materials may include
food product (food microbiology products). Bacterial viability
determination is one of the major concerns in the food industry
because injured bacteria cause a significant health threat if they
revive during food distribution and storage and it is important to
examine the efficacy of various intervention treatments used in
food processing. Also, the invention provides for effective
monitoring of a fermentation process, where micro-organisms are
exploited to produce a wide variety of products such as dairy
products (cheese, yogurt), beverages (beer, wine), single cell
proteins (SCP), antibiotics, chemicals (citric and acetic acid,
amino acids, enzymes, vitamins), fuels (ethanol, methanol,
methane). Those skilled in the art will readily appreciate that
various modifications and changes can be applied to the embodiments
of the invention as hereinbefore exemplified without departing from
the scope thereof defined in and by the appended claims.
* * * * *