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so why methanogens first off methanogens

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can be ideal candidates for life on Mars

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as well as other bodies in the solar

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system the four strains we used in our

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lab are listed down here and like Lucy

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said earlier all of our methanogens

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utilize only carbon dioxide as a carbon

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source and hydrogen as an energy source

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and so they're anaerobic which would be

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nice and Mars also their

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non-photosynthetic so that means they

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could live under the subsurface and

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finally methanogens were arose early on

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earth and so if Mars had a similar early

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history perhaps they happen to arise on

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Mars as well so these are the four

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strains we use in our lab but in this

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experiment we only used meth a no

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thermal vector wolfie I and meth a no

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bacterium for me cecum so we did a

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freeze thaw experiment and we wanted to

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approach mimicking Mars temperature

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conditions and so these are just two

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charts from curiosity at Gale Crater and

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the first the top chart shows daily

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temperature minimum and maximum in the

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air from late winter too late spring or

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about one hat one fourth of the Martian

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air and so you can see that at late

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winter temperatures don't really get

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above zero degrees and they go down to

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about negative 80 Celsius whereas in the

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late spring temperatures can rise a tiny

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bit above zero and there's not as much

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variation there and so we use this data

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for our initial experiments yesterday's

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talk by Brandon Stackhouse write a good

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introduction to thaw features on Mars

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and so this is just background for our

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experiment that we do see evidence of

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what could be thathi such as skeleton

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rain pingos and polygons so the

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questions we wanted to ask in our

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experiment are can limit antigens that

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we work with survive these freeze-thaw

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cycles or does the stress

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happen to kill them do they go dormant

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stuff and be like something like that

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and so we wanted to oopsy initially look

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at just a variation in min and max

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temperature of 20 degrees and ultimately

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get up to 80 degrees next we want to

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know if our methanogens can actively

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grow during these freeze-thaw cycles if

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the temperature does get above freezing

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can they take that opportunity to grow

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and finally we wanted to look at the

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porosity of the soil or the regolith we

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use in the experiments to see if greater

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pore size can provide some sort of safe

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haven so we conducted four experiments

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and these are four different sets in our

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first set we looked at porosity where we

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used just one of our organisms for me

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see come in a standard anaerobic medium

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that we grow our methanogens in and we

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had two subsets where we had a mixture

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of sand and gravel and then just stand

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and we had ten military ten milliliters

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of medium in there four sets two three

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and four we were basically looking at

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how the amount of liquid medium and the

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amount of dry regolith affected the

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survival or growth of our organisms and

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so we used both for me see command mole

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feei in each of these experiments and

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then different amounts of sand and

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liquid medium and so set to has five

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grams and ten milliliters set three has

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10 grams of sand five milliliters of

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medium and set for was our saturation

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experiment where we only provided enough

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medium to saturate just above the soil

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layer so here are some of our results

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first I just want to explain what we

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would see and then I'll explain what we

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did see so here on the bottom is the

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length of the experiment in days in

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order to measure growth of methanogens

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since they're anaerobic the easiest way

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is to measure the % methane produced in

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the headspace of the test tube using a

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gas chromatograph and so when our

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organisms are producing methane that

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basically means that there

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growing and so if we did not take any

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remove any methane to test remove any

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air from the test tube to test it for

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methane then this methane would be

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constant throughout the whole trial

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because there's nowhere for that methane

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to go but when we do take measurements

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such as which all these data points are

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then the methane decreases because we're

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removing that methane from the test tube

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and so that this decrease here is due to

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sampling of the test tubes and I just

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want to explain that so um in this graph

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you can see here that these are our

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different cycles of freeze and thaw I

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guess first we initially grew our

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organism at their growth temperature

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which is 37 degrees Celsius and then we

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subjected them to different cycles of 4

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degrees Celsius and the light blue is

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negative 15 degrees Celsius and so

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what's interesting about this set is

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that we do see an increase in methane

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which means that methane was produced

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because there's nowhere for that methane

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to go and then come back basically to be

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removed when we're taking a sample and

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the only way for it to be reproduced is

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for methanogens to produce it and so

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that's an interesting aspect and then

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there's just another one here we're

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removing air from the sample didn't

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decrease the methane and this again was

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set one where we had sand and sand and

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gravel and so it does seem that maybe

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porosity could play a role in providing

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a better growth environment and so this

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is just something we're going to look

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into further in set two we had five

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grams of sand and ten milliliters of

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medium and we used our two organisms

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wolffian for me see come and I just want

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to note that all of these graphs are on

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the same axis for comparison they're all

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they all go up to forty five percent

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methane but again you can see here that

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we put them at their initial growth

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temperatures which is 37 degrees for

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for me to come and 55 degrees Celsius

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for Wolfie I and then we subjected them

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to alternating growth cycles and so what

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we wanted to look at was what was

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happening here since we aren't seeing

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any real increase in % methane did our

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methanogens die are they going into

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dormancy are they still active and so I

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performed a transfer just to new medium

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and at room temperature and at least

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Wolfie I is still viable it rebounded

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and the organisms are able to again

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produce more methane and even with for

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me cecum some methane was produced and

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hopefully I plan on continuing these

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experiments and hopefully those numbers

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will go up in set three there was 10

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grams of sand and five milliliters of

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medium this basically just shows that

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the more liquid medium ultimately

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available provides greater growth and

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inset for which is just the saturation

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experiment we had initially put the

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organisms directly into four degrees C

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we didn't allow for that initial growth

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period at the growth temperature and

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they didn't grow so we put them then

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into their growth temperatures and they

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were still viable after being exposed to

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4 degrees Celsius and so they did grow

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and then were subjected to freeze thaw

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cycles again for at least wolfie I we

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did see an increase in methane which

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means that methane was being produced

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another interesting thing that we saw

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with our experiment is this black band

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within the first sometimes it's just

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below the surface or sometimes it's

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actually at the surface but we're not

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quite sure what it is right now it could

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either be an interaction with the sodium

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sulfide that we add to our medium with

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the soil or it could be some alteration

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of the environment by the microorganisms

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something that I looked at was the %

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methane produced in each test tube

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and it does look like the tubes with

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more methane produced have wider bands

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and so this is also something we're

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going to look into further so in

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conclusion our organism Wolfie I is very

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robust organism it is a thermo file but

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is also is able to rebound quickly from

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4 degrees Celsius and negative 15

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degrees Celsius and it has shown

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production of methane at those

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temperatures as well another conclusion

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is that a greater amount of initial

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liquid medium promotes greater growth

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such as when we use 10 milliliters

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medium and only half as much sand versus

205
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when we used a saturation experiment and

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then finally our discoloration of sand

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could indicate environmental alteration

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but this is something we're going to

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look into further for the future studies

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we're going to continue these

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experiments and also repeat them just to

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make sure that those increases in

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methane are accurate and that you know

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00:10:04,910 --> 00:10:02,160
something wasn't going wrong we also

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want to explore porosity further with

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different Mars simulants oils such as

217
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JSC Mars one and the Mars Mojave

218
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simulant and we're also going to further

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00:10:15,710 --> 00:10:13,500
our temperature study by using a

220
00:10:17,480 --> 00:10:15,720
negative 80 degree freezer to more mimic

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those temperatures on Mars that we see

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and so how does this relate to Mars life

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is robust I was also going to use the

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life finds away but I didn't but life's

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robust so these are actually

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thermophiles that I'm working with but

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at least Wolfie I shows promise of being

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able to rebound from freeze-thaw cycles

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and if Mars was perhaps similar to earth

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in the past if there were dormant

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microorganisms they could be existing in

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so do we have any questions for Rebecca

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thank you for your talk I just want to

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make sure i understand correctly

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wondering your grass so um on the

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graphic showing set to for example think

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you should i seen it yeah sir sins

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through the so we're in their first red

239
00:11:31,559 --> 00:11:27,429
box where you see a methane increase

240
00:11:33,179 --> 00:11:31,569
that's at minus 15 degrees c so you're

241
00:11:34,949 --> 00:11:33,189
sampling from the headspace but at this

242
00:11:38,549 --> 00:11:34,959
temperature your medium is is frozen i

243
00:11:39,929 --> 00:11:38,559
believe correct yep um so when you start

244
00:11:42,059 --> 00:11:39,939
at the end of the four degree experiment

245
00:11:43,859 --> 00:11:42,069
you take a a time measurement and you

246
00:11:47,309 --> 00:11:43,869
have a concentration of methane and then

247
00:11:48,689 --> 00:11:47,319
after a few few days you take it after a

248
00:11:51,389 --> 00:11:48,699
few days minus fifteen and you see an

249
00:11:53,069 --> 00:11:51,399
increase in methane so does it mean like

250
00:11:55,379 --> 00:11:53,079
so the methane would have died fused

251
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through the ice and go into the

252
00:11:59,999 --> 00:11:57,549
headspace the vial and that's what you

253
00:12:04,409 --> 00:12:00,009
measure is that correct or do you melt

254
00:12:06,210 --> 00:12:04,419
your sample before they when we do take

255
00:12:08,039 --> 00:12:06,220
measurements they we are taking the

256
00:12:10,229 --> 00:12:08,049
measurements at room temperature so it's

257
00:12:12,179 --> 00:12:10,239
possible that there's some melting but

258
00:12:14,789 --> 00:12:12,189
they're basically still frozen when

259
00:12:17,249 --> 00:12:14,799
we're taking our measurements um we're

260
00:12:20,780 --> 00:12:17,259
also interested in why this is occurring

261
00:12:23,340 --> 00:12:20,790
we looked at methane solubility in

262
00:12:25,289 --> 00:12:23,350
liquid water and frozen water and it's

263
00:12:28,079 --> 00:12:25,299
has nothing to do with solubility

264
00:12:30,179 --> 00:12:28,089
because solubility is very low but we're

265
00:12:31,229 --> 00:12:30,189
interested in this as well and it's

266
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something that weren't further

267
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investigation so this is also something

268
00:12:38,699 --> 00:12:36,399
that's see in the field in permafrost

269
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during the onset of freezing you get

270
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this freeze expulsion of methane from

271
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the water phase and it gets pumped out

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so if the onset of winter comes on you

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get these enormous bursts of methane is

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where the majority or in some places of

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methane flux comes from so I'm I was

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worried about the same thing think

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what's happening is that you're freezing

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your solution your ex building all your

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methane out there so it's not active

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production okay you're expelling the

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production you've made during the four C

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times and then you're dropping back down

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again yeah right okay yeah that is um

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see that's both very good points that's

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something we would expect as well as

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methane was being produced earlier

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either

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at 4 degrees c if it was in the soil or

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in the ice and then it was released

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later maybe we can have one more

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question for Rebecca if there's one can

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you comment on the slope of your I mean

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it does that mean anything the fact that

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they have different um basically wolfie

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I is just a more robust grower in itself

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it's more robust organism in various

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environments it just shows better growth

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and survivability and so I think that's

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the difference between Wolfie I and for

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me to come oh yes that also i don't know

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i mean this is these are both very um

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this these experiments are all very

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preliminary and this is just we've only

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looked at how much % methane was

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produced over the time period and now we

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have to go back and look at everything