welcome to lecture nine on the course on bioreactors under the nptel online certification
program let us in lecture eight we looked at the solution to the practice problem two
point two we are in module two which looks at the two major outcomes from bioreactors
the bio mass in the products and some enzyme related aspects the products from enzyme reactions
let us move forward in that module most likely we would complete the module in this lecture
the um last aspect that we are going to see in this lecture is the concentrations
of biomass or cells substrates and products the measurement methods we saw how crucial
the information on biomass the concentration substrate concentration and product concentrations
were in terms of kinetics in turn the design and operation of a relevant actors
therefore we need to have good measurement methods to find out the concentrations of
biomass cells the cells or biomass substrates and products we would look at some of the
common measurement methods that are used for these there are two kinds of methods one
online and the other off line online means that the
bioreactor is not disturbed it is measured online as the process goes on without any
disturbance off line we take a sample from bioreactors and then measure it away from
the line or the away from the bioreactor and that is why it is called off line method we
will look at some online methods they are of course preferred because they are
measured at the point of interest without disturbing the bioreactor so the best view
or the best information comes from online methods but few are available for reliable
measurements in that way comes the difficulty with online methods for example if we look
at measuring cell concentration there are a few online methods there is fluorescence
method that measures the fluorescence of a molecule inside the cell that's called NADH
you all know NADH at NADH dinucleotide the hydrogenated form of it NADH is fluorescent
n a d is not and the level of NADH is a direct indication of the cell concentration
higher they may be a fluorescence higher the cell concentration the relationship is known
is is has been established so you could follow the increase in cell concentration through
the NADH fluorescence or it's also called the culture fluorescence there may be a it's
fluorescence change what is typically done is the fluorescence measurement a probe is
placed in a special well in the bioreactor this is an optical probe before that i
need to tell you that NADH the optimal wave length pair for NADH is let's say three
forty four sixty nanometres so the excitation wavelength needs to at three forty nanometres
the emission wavelength needs to be measured at four sixty nanometres the um in a spectra
of fluorimeter you have a nice quet or a some other well kind of a measurement you have
a controlled way of sending in three forty and measuring it at four sixty where as in
the bioreactor typically what is done is fiber optic probe is interfaced with a
spectra fluorimeter the fiberoptic bundle the some bundles carry the excitation wavelength
from the spectra fluorimeter and projected in an open ended geometry in to the bioreactor
and then the emission wave length at four sixty is collected through the same fibre
optic probe taken back to the spectra fluorimeter and measured this is called an open ended
geometry of measurement well those are the details the main points here is that the
cell concentration can be measured by following the culture fluorescence or the NADH fluorescence
the conductivity of cells can also be measured there are some probes for it the reliability
not so sure may be that can also be used it's an online method for the substrate and product
concentrations there are a few online methods as i mentioned earlier the optical fibre
is integrated with a spectro photometer or a fluorimeter or you could take a sample
from the bioreactor as it is operating but bring the sample back in to the bioreactor
after it flows through what is called a flow cell which is placed in the fluorimeter for
measurement ok this is another way of doing that
the um you could used absorbance fluorescence for some
substrate some products what is recently of interest it is what is called near infra
red spectroscopic interact we have paper on this which was the work was a done by sanjay
tiwari it was developed as a part of species and was also applied in the industry the
advantage here let me talk of this as specific case so that you will understand the advantage
here this industry was a looking at using low levels of substrate because the substrates
was toxic to the cells for their one a product commercial product so the substrate needs
to be maintain at a certain low concentration now the order of about millimolar and so on
i think it's about two point five to five millimolar so if hold on it is a
it substrate concentrations need to be measured need to kept at a low level and also the
product concentration needs to be maintained at a certain level otherwise it would become
toxic so let us concentrate on the product here the older method that was followed
was something like this they would take a sample from the bioreactor this is the production
reactor the measurement such as h p l c and so on are available in the lab which is at
a distance from the production unit the production unit is large therefore they need to take
a sample from here come there measure it it takes time for measurement may be about twenty
twenty five minutes and then bring it back by that time the product concentration would
have gone up and they would have lost the batch it would have become toxic to the cells
and they would have lost the batch so to avoid that they definitely needed an online measurement
which would immediately tell them what their concentration was so that they could take
a appropriate action to maintain the product levels at acceptable levels so to do that
sanjay i had worked on n i r spectroscopy and developed a method for doing that and
that obviously here was very beneficial to the industry that's a paper there it was
published in biochemical engineering journal if you are interested you can take a look
at there also the gas that comes out of the bioreactor now we provide air for most aerobic
bioreactor systems the exit gas which consists of c o two and o two and of course nitrogen
and so on that can also be measured to provide some input into what is happening in the bio
reactor so those are the available online methods a few are available but off line
are the most used although there are difficulties with off line system such as the one that
we talked about just now let us look at off line measurements let us look at measuring
the total cell concentration take one at a time ya i think that is ok the total cell
concentration if you are considering a bunch of cells consists of both live cells
and dead cells if we look at live cells alone because the viable cells are the once
that are contributing to product formation and so on we would want to follow the viable
cell concentration sometimes the viability percentages are very high ninety seven ninety
eight percent
that it doesn't really make a difference whether you follow viable cell concentration or total
cell concentration but there is a difference in principle and it can come in it can
turn out to be important the difference can turn out to be important so we need to know
that there is a total cell concentration measurement and a viable cell concentration measurement
let us first look at the methods to measure total cell concentration the first one is
what is called OD are optical density OD is stands for optical density it's a historic
term it doesn't represent what it measures but we will talk about you could also measure
dry mass take sample largin of sample dry it out completely and then measure the mass
that is the you know the mass and that mass came from a certain volume if a mass
of the cells by the volume gives you the total cell concentration you could also use something
called packed cell volume this is the volume that is occupied by the cells in a rather
concentrated solution of cells typically in the industry ok so this is packed cell volume
is typically use in some industries as a measure of the total cell concentration the fraction
of a volume that is occupied by the cells or you could also use cell contents measurement
especially for molds we said if we remember we said the molds were organisms that grow
by extension of high faith so the number of cells may not be an indicator just one
cell but increasing in mass through extension of high faith for though such kind of
organisms we measured the contents of the cell so those are the methods for total cell
concentration measurement let us see the principles one by one the optical density the so called
optical density is measured by using a spectrophotometer but it is actually a measure of a cell scatter
it is not a measure of absorbance by the cell this is something that you need to remember
very clearly we have cuvette here that is place let us say that the cuvette has a cell
suspension here fill till the measurement level then there is a certain wave length
that is a shown on the cuvette the cells you know they are micron sized so the mie scattering
rains comes in to effect the scatter cells and whatever light is not scattered is sent
to the detector that is measured so that is measured and related to the incoming light
to get the extent of light that has been scattered the light that is been scattered is proportional
to the concentration of cells ok make this difference very clearly set in your minds
this is cell scatter and not cell absorbance although the same instrument is used and something
called a optical density the term is used to which is the complete but any way in
in terms of the principles it's a well accepted term term but keep this in mind it is a
measurement of scattering by the cells and the cells scatter is the amount scattered
is proportional to the cell concentration that's a reason why we used about six hundred
nanometres the principle is scatter the scattering occurs maximally when things are not when
the wavelengths are not absorbed by the cell so we would like to work with wavelengths
that are not absorbed by the cell so anything in the range of five hundred to six hundred
is actually fine infact i have even used four fifty in certain cases so that's perfectly
fine because you are measuring the extent of lights scatter six hundred is a very
typical number you could use that any thing between five hundred and six hundred is perfectly
fine then what is done is using a calibration curve between the dry cell concentration remember
we did talk about dry cell concentration measurement you take a largin of sample you tried on the
cells completely using a vacuum over night and then you measure the mass of cells that
was present in that particular volume therefore you have a dry cell concentration before you
are going to dry that you take a sample and measure the o d and thereby you can relate
dry cell concentrations various values of them to the o d and you would get a straight
line in a certain range and that is the calibration curve you could a measure o d and get the
relevant
dry cell concentration using this calibration curve and that was it typically done the linear
regime is a important while measuring you need to pay attention to that the linearity
there are two aspects there we if you plot cell dry cell concentration versus o d it
is going to be linear till a certain point and above a certain dry cell concentration
the linearity is going to break down that's that's a law of nature so it is going to
be linear here and above a certain dry cell concentration it is actually going to saturate
we should not operate in this regime of course we measure but we limit are range of operation
to this linear region in other words we need to measure only in this linear region if it
happens to be in a higher o d region we should actually dilute the sample to get the o d
reading and this range and then get the raisol concentration corresponding to the
measured only and multiplied by the dilution factor to get the actual od if you follow
all the this it's fine otherwise this keep this in mind whenever there is a need for
measurement you go through this in detail or look at some reference book you can always
ask me to there is another reason for the loss of linearity that is because the detector
range if a the amount of light reaching the detector goes below a certain or if the amount
of light that is scattered is very high then the detector saturation happens then also
there is a problem so we don't work in the region of detector saturation depending on
the spectrophotometer i would feel very comfortable working only with od values say
between point one and about one or so that way i am pretty much in the linear regime
and in the normal detector region of course this depends on the spectrometer the maximum
value and so on so for you need to worry about in during this measurement interference by
salts and substrates also because your we are taking a sample here drying out the whole
thing when we dry out whatever it's a soluble in the broth is going to contribute to
the mass measurement so the mass would contain the mass that is measured would contain
dry cells plus the salts plus you unutilise substrate plus the other things so we need
to have an appropriate control by which we can account for the mass of salts and substrates
and so on so that we need to take in to account while measuring od this is a finer point i
just start elements in this here during the measurement there is a video here let me show
you the link which can give you some more details in my want to take a look at it's
a what is given in number fourteen od measurement you can go and take a look at this video
ok let's move forward with packed cell volume the percent of total thus we mentioned
earlier now this is going to be a cell suspension you spin it down using a centrifuge
and the cells are going to occupy this volume here the total volume is this so this
volume by the total volume is going to give you the percentage packed cell volume this
is used in the industry the next is cell contents measurement especially for mold which grows
by extension of high fair not by divisional cells the amount of DNA the amount of total
nitrogen the amount of protein the amount of amino acid in a cells could be measured
and if we assume that the percentage DNA per cell does not vary too much then this becomes
a direct measure of the cell concentration it'self ok this is the bases for the measurement
of cell concentration through the measurement of it's contents the typical contents that
are measured are DNA total nitrogen protein an amino acids in that order of importance
DNA is usually preferred because doesn't vary too much during the various parts of a
cell cycle and so on before a before it's split up so total nitrogen could be measured
protein could also be measured but there would be variations amino acids there are some variations
but these are all accepted methods for cell contents measured that those were the methods
for measuring total cell concentration now let us look at the methods for measuring the
concentration of cells that are live that are viable is the viable cells are the once
that are going to actually contribute to the functioning of the bioreactor the methods
are dye exclusion plating and others we just look at these two methods there are videos
here before get in to the videos let me just tell you the principle of dye exclusion
and plating the dye exclusion means or is based on the principle that's certain dyes
which can be visualized are excluded by living cells so in the presence of dye the cell is
go into appear un dye it you know uncoloured so by counting the number of cells that
are not coloured and by counting total of cells you can have percentage viablity or
the viable cell concentration it'self from direct measurements there are slides specialized
slides that can be use for these measurements they are called bar chambers let say
called and there are base by which you could reliably measure the number of a cells
in a particular volume and therefore the cell concentration that is dye exclusion method
principle the plating principle is that you have an agar plate a solid medium agar
it contains agar and then you take a certain low concentration of cells and plate them
here and assuming when you plate them here the cells are going to multiply and they
multi when the multiply to a certain extent the colonies that arrives from each cell become
visible you know one cell becomes two cells become four cells each cells in so on as and
as long as there are imbalance of cells that would form a colony ok and at that stage
the colonies become visible to the naked eye and you can count the number of colonies assuming
that each colony arises from a single cell you could count the number of colonies and
relate them to the number of cells that were that were there in at the time when the
plating was done ok these are methods take time does the plating method takes time you
will have to keep at over night an so on even for bacteria which grow fast
but that is a method that can be used because colonies will arise only from live cells
so as long as you make sure that the cell concentration is measurable by counting
the number of colonies you could use this the videos here let me show you the details
of the videos you may want to look at them for the trypan the number fifteen for the
trypan blue exclusion method and sixteen for plating for viable cell concentration
measurement these are nice videos that you can look at for the details of the measurement
for the substrate and product concentrations spectroscopy many kinds of spectroscopy
is can be used absorbance spectroscopy which we normally know the absorbance at a particular
wavelength which is particular for that molecule is used to measured the concentration
fluorescence we looked at any fluorescence and so on they could be fluorescence substrates
fluorescence products that can utilise this method iR spectroscopy infra red spectroscopy
we did talk of something related to that near iR NMR nuclear magnetic reference spectroscopy
EPR electron paramagnetic reference spectroscopy it can all be used spectroscopic methods can
be used for off line measurement of concentration of substrates and products chromatography
matographic methods can be used gascromatography high pressure liquid chromatography HPLC so
on they can be used and there are many such methods that can be effectively i think we
have come to the end of a module two very briefly summarizing what we diden module two
we looked at biomass and bio products is two important outcomes of a bio process that
is that was the main theme of module two we looked at the details of these products
and then the models for the rate of cell formation simple models as well as some detail models
and then how what are substrates and how they are converted and how the substrate effects
the growth rate we had seen that some models for that and also when we looked at that
we showed that monad model gives us the effect of substrate on growth rate that's
a simplest representation there are other representations that are given here and then
for the product formation we have the luedeking piret model r p equals alpha r x plus beta
x the growth dependent parameter the growth independent parameter and so on then we talked
about a few concepts such as yield coffiecients and maintenance then we looked at enzymes
some enzymes that are used in the industry enzymes that can be used for bio conversions
in a bio reactor and we said that we need to know the kinetics of the enzymes we looked
at michaelis menten kinetics in detail we went through the derivation then we also looked
at innovation kinetics the competitive non competitive and the uncompetitive innovation
kinetics we worked out a couple of problems based on those and in this lecture we looked
at methods to measure the concentrations of biomass or cells substrates and products we
said that they were two different kinds the online and the offline methods online a preferred
but the reliability of the methods available are to a certain extent very few methods are
highly reliable then we looked at the principles we looked at methods for total cell concentration
measurement such as o d measurement the pack cell volume measurement the cell contents
measurement and methods for viable cell concentration such as dye exclusion and plating
finally some methods for substrate and product concentrations we also saw this concludes
module two and this is lecture nine when we meet in lecture ten we will take things
forward with module number three see you