Isothermal microcalorimetry for bacterial activity.

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Markos

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Apr 14, 2015, 2:59:49 PM4/14/15
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Hi,

I've been thinking to experiment common temperature sensors (Eg: NTC) to
measure microbial activity.

And today I found this paper:

http://blogs.nature.com/spoonful/2011/08/researchers_measure_bacterias.html

Would be possible to use a NTC thermistor, and Arduino for data
acquisition, for detecting temperature changes in a medium containing
bacteria and nutrients in a DIY approach?

Has anyone had any practical experience with this technique?

Thanks,
Markos

Simon Quellen Field

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Apr 14, 2015, 4:03:57 PM4/14/15
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The instrument they use is described here:
"http://www.tainstruments.com/pdf/brochure/MicrocalorimetryBrochure.pdf"

The specs are pretty impressive to a DIY hacker: The TAM48 keeps the temperature constant to within 0.0001 degrees Celsius. That might be a little hard to do with a thermistor and an Arduino.

They use thermoelectric modules (that sounds like the Peltier coolers used for cooling CPU chips, but presumably much smaller. Maybe we could take apart a CPU cooler and remove just one of the dozens of Peltier junctions in it and reconnect the junction to new leads.)

One side of the thermoelectric module is attached to a heat sink in a thermostat (a water bath? Mercury bath?) that has that 0.0001 degree Celsius spec. But because they expect the thermostat to have some variation, they use two thermoelectric modules -- one for the sample ampoule, and a second for a reference (say pure water or sand, to match the heat capacity of the sample) ampoule. Then they monitor the difference between the two.

The "isothermal" part of the name comes from the way they use the thermoelectric modules to pump heat away from the sample to keep the temperature constant. They measure the heat flow by monitoring how much power is required to keep the temperature constant.

Building a DIY version sounds like great project. You could tell if you were on the right track by using the same potassium carbonate / hydrochloric acid reaction they use to characterize the professional devices. Operational amplifiers can be used to amplify the tiny voltage differences between the sensors. The results could then be sent to a 16 bit A/D converter for monitoring by the microcontroller, which could then use a DAC going to the thermoelectric modules to offset the heat flow.

​

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Nathan McCorkle

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Apr 14, 2015, 6:13:25 PM4/14/15
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Sounds like the inverse of:

In that case, they measure how much POWER the heater/cooler requires to keep the temperature constant. So you might be able to get away with a very sensitive comparator for the temperature sensor, and then monitor the power using a current-sense-resistor... the possible advantage is that converting power to temperature-change will be inefficient so big changes in power induce smaller changes in temperature, so if you monitor the heater/cooler power you need less sensitivity than if you were monitoring the temperature sensor directly. If an exact and precise input is needed for the comparator, then this might be a moot point.

I have a feeling you'd need a good heat-stable/heat-calibrated voltage reference:



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Markos

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Apr 15, 2015, 1:43:59 PM4/15/15
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Hi Simon and Nathan,

Thanks for your attention.

The article inspired me to test a more general idea of ​​using temperature to monitor biological processes and use this information to quantify (biodegradable) organic load in wastewater.

Whitout to pump heat away from the sample.

A standard method for measuring the organic load in effluent is BOD (Biochemical Oxygen Demand) and is basically a measure cell respiration.

I did some work to automate respirometry measurement with Arduino and a commercial DO sensor. (http://www.c2o.pro.br/en/automation/x73.html)

My idea now is try to measure the heat dissipated by bacterial metabolic processes in an adiabatic environment using thermistors, and use this technique to estimate the organic load in wastewater.

Imagine keeping an aerated bioreactor containing bacteria fed by domestic effluents.

To make a measurement I take a sample of sludge from bioreactor and let some time only with aeration but no effluent to go into starvation state.

Imagine also two adiabatic cells (vial) (A and B), each with two thermistors mounted in a Wheatstone bridge.

_V+_
|     |
R1 R2
|     |
p1 p2
|     |
R3 R4
|     |
Gnd


R2 and R3 are installed in the reference cell B, and R1 and R4 in measurement cell A.

In compartment A I add a sample of starved sludge diluted with the effluent to be analyzed.

In compartment B (reference) I put the same starved sludge volume but diluted only with water.

Note: It is important that the sludge, the test effluent and water, are all at the same initial temperature.

It would be possible to amplify, with an OpAmp circuit, the voltage difference between p1 and p2, and measure this value with an Arduino?

The measurement time interval should be standardized and temperature variations should be initially calibrated with samples of known nutrient concentration.

Would bo possible detect temperature variations under these conditions with conventional thermistors?

What do you think about this idea?

At the moment I am dedicating to a project with LEDs. But I intend to explore the possibilities of (bio)thermal measurements in the future.

Thanks for the comments.
Markos


On 14-04-2015 17:03, Simon Quellen Field wrote:
The instrument they use is described here:
"http://www.tainstruments.com/pdf/brochure/MicrocalorimetryBrochure.pdf"

The specs are pretty impressive to a DIY hacker: The TAM48 keeps the temperature constant to within 0.0001 degrees Celsius. That might be a little hard to do with a thermistor and an Arduino.

They use thermoelectric modules (that sounds like the Peltier coolers used for cooling CPU chips, but presumably much smaller. Maybe we could take apart a CPU cooler and remove just one of the dozens of Peltier junctions in it and reconnect the junction to new leads.)

One side of the thermoelectric module is attached to a heat sink in a thermostat (a water bath? Mercury bath?) that has that 0.0001 degree Celsius spec. But because they expect the thermostat to have some variation, they use two thermoelectric modules -- one for the sample ampoule, and a second for a reference (say pure water or sand, to match the heat capacity of the sample) ampoule. Then they monitor the difference between the two.

The "isothermal" part of the name comes from the way they use the thermoelectric modules to pump heat away from the sample to keep the temperature constant. They measure the heat flow by monitoring how much power is required to keep the temperature constant.

Building a DIY version sounds like great project. You could tell if you were on the right track by using the same potassium carbonate / hydrochloric acid reaction they use to characterize the professional devices. Operational amplifiers can be used to amplify the tiny voltage differences between the sensors. The results could then be sent to a 16 bit A/D converter for monitoring by the microcontroller, which could then use a DAC going to the thermoelectric modules to offset the heat flow.

​

-----
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On Tue, Apr 14, 2015 at 12:02 PM, Markos <mar...@c2o.pro.br> wrote:
Hi,

I've been thinking to experiment common temperature sensors (Eg: NTC) to measure microbial activity.

And today I found this paper:

http://blogs.nature.com/spoonful/2011/08/researchers_measure_bacterias.html

Would be possible to use a NTC thermistor, and Arduino for data acquisition, for detecting temperature changes in a medium containing bacteria and nutrients in a DIY approach?

Has anyone had any practical experience with this technique?

Thanks,
Markos


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Nathan McCorkle

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Apr 15, 2015, 3:47:06 PM4/15/15
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On Wed, Apr 15, 2015 at 10:47 AM, Markos <mar...@c2o.pro.br> wrote:

It would be possible to amplify, with an OpAmp circuit, the voltage difference between p1 and p2, and measure this value with an Arduino?
 
Googling 'wheatstone bridge arduino instrumentation amplifier' gives this for top result:

Looks like the other search results to the arduino forums could be helpful too.

 
The measurement time interval should be standardized and temperature variations should be initially calibrated with samples of known nutrient concentration.

Would bo possible detect temperature variations under these conditions with conventional thermistors?

I can't say. It all depends on signal-to-noise, the detector will have some efficiency of converting heat (vibration/phonons or IR-type radiation) to signal (some number of electrons, some amount of voltage on those electrons)... and the amplification required and the scheme you amplify with will have their own noises associated. Some tricks can be done to reduce certain types of noise, while other kinds of noise might be unavoidable because of the detector or other project/application related constraints (i.e. does it work underwater, in a dry environment, carried around, attached to batteries or AC power, operated next to a heavy-duty motor, etc...).
 

What do you think about this idea?

You're on the right track! If you have a good straight-forward lab/bio protocol to follow, and you know what the results are, then I would recommend getting to the prototyping stage very soon. Don't over-analyze too much... get some results and then re-analyze again later, but that time you will have some facts to help guide your next decisions.

I have definitely let over-analysis cripple progress... overall I am OK with it because my long-term goals are quite complex from an engineering perspective... but along the way I've learned that I spent a lot more time thinking than doing, and these things should really go hand in hand. Come up with a hypothesis (i.e. "I think normal thermistors, an arduino, an intrumentation amp and wheatstone bridge will work to detect the K2CO3 + HCl reaction's heat") and test it... then when it fails you're no longer speculating... but hey, maybe it works and you don't waste tons of time 'rat-holing' and never actually experimenting.

:)

I will continue to answer electrical questions as much as I can!

Regards,
-Nathan

John Griessen

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Apr 15, 2015, 4:15:04 PM4/15/15
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On 04/14/2015 03:03 PM, Simon Quellen Field wrote:
> Would be possible to use a NTC thermistor, and Arduino for data acquisition, for detecting temperature changes in a medium
> containing bacteria and nutrients in a DIY approach?
>
> Has anyone had any practical experience with this technique?

I've used thermistors. Not like you want to.

They vary from part to part a lot. The sensitivity might be OK. What strikes me
is your aim is to measure very dilute solutions and I wonder what "containing bacteria" means?


" heat dissipated by bacterial metabolic processes in an adiabatic environment measured with thermistors to estimate the organic
load in wastewater. Imagine keeping an aerated bioreactor containing bacteria fed by domestic effluents."

Are the bacteria fixed, or do they wash away? If they are fixed on a surface, there's a chance.

Insulation against heat flow is a very big thing in this setting because the thermal mass of the
whole experiment is huge compared to the bacteria mass in aggregate.

How do you reconcile adiabatic and aerated? How do you reconcile adiabatic and "fed by"?
Is the aeration all just recycled gas inside the reactor with no ins or outs of gases?
Do you seal up the system and watch its temperature and pressure change for a while -- that's
what we usually call adiabatic...

Here's an idea: Reduce the heat sink mass surrounding the supposedly fixed bacteria. Aeration can stir a vat of
solution, but for exposing it to the bacteria flow a small trickle of it over them in a thin sheet so
it does not carry away much heat. Measure differential temperature of the in and out drip of solution
flowing over the bacteria surface.

If the bacteria are not fixed, just washing around in solution, then the above is no help, and bioreactor
insulation is almost everything.

(You still need the nice stable temp sensor system to measure heat generation...)

Jeff Backstrom

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Apr 16, 2015, 5:30:49 AM4/16/15
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If you're looking for a field-able method for mycobacterium detection, you might look at improvising from the existing MGIT:


Thermal differences on the scale needed for mycobacteria would be very difficult to put into the field. If you insisted on going this route, you could probably fish up something similar to what is used in thermal conductivity detectors for gas chromatography.


In other words, back to the ol' Wheatstone bridge.



On Tue, Apr 14, 2015 at 12:02 PM, Markos <mar...@c2o.pro.br> wrote:


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John Griessen

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Apr 16, 2015, 10:08:27 AM4/16/15
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On 04/16/2015 04:30 AM, Jeff Backstrom wrote:
> In other words, back to the ol' Wheatstone bridge.
>

A bridge with a thermistor in one leg could be very sensitive...but...

That just moves the place you need high accuracy from the volt reference
to the resistor temp coefficients of the bridge resistors... Something has to be accurate over
the temperature range of interest. Comparing your thermistor voltage to a good
voltage reference with a high res. A2D converter can be as good, and has the possibility
of correction factors built into a look up table for the A2D conversion numbers.
With a wheatstone bridge, more components need high accuracy, low drift.
With a wheatstone bridge, ambient temperature affects your readings and there's no
correcting that or linearity of the A2D in code.

When you're done, you probably want to
put the temperature back to near a standard starting point, so you want cooling
that has a heat flow path that is small when turned off. Thermoelectrics are not like that --
they have a big heat flow path on or off, and need a big heatsink to ambient to get rid of
all their excess heat of operating. So a heat exchanger connected by small diameter
PE tubing, with a valve near the bioreactor wall would do well. A microliter sized TE
cooled and TE monitored chamber would be much easier to do than a "pint sized" one that runs
continuously and uses its own characterized power flow as the heat flow sensor. Just from scale reasons.

Markos

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Apr 16, 2015, 1:56:50 PM4/16/15
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Hi Nathan,

Thanks for the tips.

I really spend some time planning the project and I agree that I should test some ideas with prototypes.

I've bought some NTC sensors and I will do some initial tests.

I'm thinking to use mixtures of ethanol and water and try to detect the heat released by the mixtures of ethanol and water in different proportions at different compartments.

In this work:
http://jdesbonnet.blogspot.com.br/2010/10/computer-interface-to-low-cost.html
he uses a technique of polarity reversal to remove the common mode interference.

Best Regards,
Markos

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Markos

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Apr 16, 2015, 2:31:44 PM4/16/15
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Hi John,

>What strikes me is your aim is to measure very dilute solutions and I
wonder what "containing bacteria" means?

A sample of sludge (flocs of sludge).

The type of sludge formed in activated sludge system for wastewater
treatment (http://www.iwa100as.org/history.php).

This sludge is composed of bacteria, protozoa and other microorganisms
(http://www.engitech.com/asm.htm).

>Are the bacteria fixed, or do they wash away? If they are fixed on a
surface, there's a chance.

In principle I was not planning on using sessile bacteria in biofilms.
But only flocs of sludge in suspension.

> How do you reconcile adiabatic and aerated? How do you reconcile
adiabatic and "fed by"?

The aeration would be maintained only in bioreactor to keep a stock of
biomass for analysis.

The analysis would be done only with a sample of sludge injected in an
adiabatic compartment that would be maintained without aeration during
the measurements and diluted with the areated efluent to be analysed.

The same amount of sludge would be injected in other adiabatic
compartmente diluted with aerated water.

The dissolve oxygen in efluent and water (both previously aerated) would
keep the metabolism during the analysis time. (I expect :^)

I supose that in the reference compartment (with water and no
nutrients*) would happen only endogenous respiration.

*As nutrients I mean the organic load in the effluent that would be
analyzed.

I do not plan to measure absolute values of heat, but only "temperature
differences" between the two compartments (with nutrients and no nutrients).

You think it might be more appropriate to use biomass fixed on a solid
support?

But in this case how I could provide the same amount of biomass in the
two compartments (with nutrients and without nutrients)?

To control the analysis the difference in heat released in compartments
should be the result only of the difference in the concentration of
nutrients (organic load in the effluent) and not by the difference in
biomass.

Thanks for your comments.

Best Regards,
Markos

Markos

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Apr 16, 2015, 2:42:26 PM4/16/15
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Hi Jeff,

Thanks for the link.

  In the case of Thermal Conductivity Detector (TCD) the filaments (resistor) are heated intentionally by an electric current and the system for monitoring biological activity the heating would result only from the energy released by the bioprocesses.

Yes you are right.

I'll have to cross that bridge. :^)

Best Regards,
Markos



On 16-04-2015 06:30, Jeff Backstrom wrote:

If you're looking for a field-able method for mycobacterium detection, you might look at improvising from the existing MGIT:


Thermal differences on the scale needed for mycobacteria would be very difficult to put into the field. If you insisted on going this route, you could probably fish up something similar to what is used in thermal conductivity detectors for gas chromatography.


In other words, back to the ol' Wheatstone bridge.


On Tue, Apr 14, 2015 at 12:02 PM, Markos <mar...@c2o.pro.br> wrote:
Hi,

I've been thinking to experiment common temperature sensors (Eg: NTC) to measure microbial activity.

And today I found this paper:

http://blogs.nature.com/spoonful/2011/08/researchers_measure_bacterias.html

Would be possible to use a NTC thermistor, and Arduino for data acquisition, for detecting temperature changes in a medium containing bacteria and nutrients in a DIY approach?

Has anyone had any practical experience with this technique?

Thanks,
Markos


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Jeff Backstrom

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Apr 16, 2015, 3:39:05 PM4/16/15
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On Thu, Apr 16, 2015 at 11:45 AM, Markos <mar...@c2o.pro.br> wrote:

  In the case of Thermal Conductivity Detector (TCD) the filaments (resistor) are heated intentionally by an electric current and the system for monitoring biological activity the heating would result only from the energy released by the bioprocesses.

Depends upon what you're looking for. In the instance of mycobacteria (and many other potential pathogens), it will need to be heated above ambient anyway. In that context, it could be constructed in a fashion similar to the differential scanning calorimeter, and see whether the sample needs to be heated more or less than the control in order to maintain ~37C or whatever.


 

John Griessen

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Apr 16, 2015, 8:33:18 PM4/16/15
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On 04/16/2015 01:34 PM, Markos wrote:
> You think it might be more appropriate to use biomass fixed on a solid support?

No opinion. Not enough info.

I was simply idea generating for you.

"To control the analysis the difference in heat released in compartments should be the result only of the difference in the
concentration of nutrients (organic load in the effluent) and not by the difference in biomass. "

Sounds like an assumption rather than a measurement.

John Griessen

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Apr 16, 2015, 8:35:13 PM4/16/15
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On 04/16/2015 02:38 PM, Jeff Backstrom wrote:
> see whether the sample needs to be heated more or less than the control in order to maintain ~37C or whatever.
>


This strike me as the finding of the "most sensitive" sweet spot of the reaction.

Sounds like a good approach with such tiny heat flows to detect....
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