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thank you so I am starting off the

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isotope section but I want to let you

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know that i am not talking at all about

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isotopes i actually asked the organizers

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but to put me on wednesday in case of

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travel problems on monday so anyway no

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isotopes sorry feel free to leave if you

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want so background why are we looking at

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low pressure well we try to mimic

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Martian conditions and Mars pressure is

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much much lower than that on earth it's

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about seven millibar well as Earth

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surface pressures about a thousand

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millibar and nowhere on earth can we

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actually get to seven millibar even at

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the top of Mount Everest and so we're

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looking to see you know there's no life

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on Earth here that evolved at low

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pressure so we're actually very

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interested in this topic and what's

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interesting is that few experiments

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actually test the effect of low pressure

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on organism metabolism and growth

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however if you were here Monday you

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heard Sam waters talk about low pressure

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and so she gave a great introduction to

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why we look at low pressure and her lap

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she worked in the Nicholson lab and

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Andrew sugar who is also at the

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University of Florida has looked at 22

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different bacterial species that are

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commonly found on earth some are found

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in spacecraft clean rooms and he's

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looked at survival and growth under low

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pressure and so sugar and Nicholson

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found that there's this 25 millibar

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limit to growth where most species are

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able to grow and metabolize down to 25

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millibar but then they reach this limit

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where many species either died off or

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they simply can't grow anymore and so

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we're interested in figuring out maybe

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why this limit occurs and looking more

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into this limit so in my lab we use

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methanogens and they're members of the

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domain Archaea they're anaerobic and

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they use molecular hydrogen as an energy

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source in carbon

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side as a carbon source which is found

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on Mars and we use for species with a no

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thermo vector wolfy I nathanael star

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Cena Barker I Nathanael bacterium for me

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sikkim methanococcus Meeropol udhas so

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in the parenthesis you'll see their

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optimum growth temperature which it's

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obviously very high and not very Mars

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like but the reason that we don't use a

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cycra file or a cold loving with antigen

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is because they just grow so slowly that

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I would be here for another 20 years and

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my advisor would be like just just leave

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so we do use a thermophilic methanogens

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but they can survive low pressure and i

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have other experiments that show that

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but these four species are they

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basically show the breadth of the

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methanogenic archaea and that's why we

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use these for so in this talk i will

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talk to you about six experiments i ran

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from at varying pressure and the

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pressures are listed here and you can

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see that the first experiment was up to

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143 millibar and experiment 5 & 6

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arranged between six and twenty mellow

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bar and the reason we have arranged is

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because we are still developing our

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vacuum chamber and right now our Val are

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open and closed valve isn't as precise

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as we would like it to be and that's why

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we have that range there but we're going

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to fix that in the future but in a in

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each experiment I had 20 test tubes

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total which included five test tubes for

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each of the four species so there are

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five replicates and then these

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methanogens survive in liquid medium and

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so that is a problem at low pressure

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which I'll get to but each tube is ten

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milliliters of liquid medium prepared

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anaerobically and so it contains carbon

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dioxide its pressurized with hydrogen

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gas which is their energy source and

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growth is monitored by methane

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action so once I make these test tubes

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with the myth antigens in them I put

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them in the Pegasus planetary simulation

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chamber at the University of Arkansas

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and before I sew these these tubes are

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sealed and before I puncture them i

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evacuate the chamber and then fill it

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with hydrogen and carbon dioxide gas

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then evacuated again and I cycle this

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three times in order to get rid of all

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that residual oxygen and atmosphere in

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there and then I let the chamber

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stabilize for about a day due to

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outgassing from the walls and whatnot

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then with this specialized device you

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see here which I have nicknamed the

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pokey device I'm able to turn a crank

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and it will either lower or raise and

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I'm able to puncture the tubes within

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the chamber at the pressure that I

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desire and so then the tubes remain

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punctured and they can équilibre twith

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the atmosphere and then on the second to

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last day I fill up the chamber with

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carbon dioxide otherwise there'd be a

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suction effect in the tubes and if I

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open the chamber it would allow oxygen

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in which would kill them and then on I

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unplug sure the tube so that they're

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full of carbon dioxide and then I take

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them out the methods were the same for

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all six experiments except for

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experiment six where I had a cotton ball

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right above the liquid and six or five

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grams of JSC Mars one to act as a

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diffusion barrier to slow the

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evaporation of water so here are my

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results for one of our organisms and

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it's very there's a lot of information

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here so I'm going to go over it slowly

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on the left is methane production which

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is how we measure the growth in our of

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our methanogens and on the bottom are

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the experiments so there's two columns

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for each of the six experiments and the

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pressures are also listed there and each

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column the light gray is the original

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tube and that corresponds to the tube

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that was actually in the chamber under

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low pressure

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and then the dark great column is the

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tube that I transferred point five

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milliliters from the original tube to

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the transfer tube okay so these all this

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methane production is actually following

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exposure and following an incubation

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period so i had the tubes in the chamber

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exposed to low pressure i took them out

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I'd performed a transfer and then I put

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all the tubes at their ideal incubation

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temperature and so what we can say is

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that initially when I fill the chamber

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up with carbon dioxide the methane

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content is about zero because the tubes

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are being filled with carbon dioxide and

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so we can compare these post-exposure

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methane values to the zero points of the

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actual experiment and so you'll see that

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there's growth or methane production in

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each of the six experiments even at even

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if it's just a few percent at six Mel

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bar and so the reason there are no

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original tubes for experiments five and

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six is because there was so much

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evaporation that there wasn't after

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performing the point five milliliter

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transfer there wasn't anything left in

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those tubes so I just discarded them so

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this is for Barker I wolfie I and

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Meeropol udhas you see the same thing

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there's methane production following

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exposure to low pressure and for me

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cecum the reason I show this one

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separately is because you can see that

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the methane production in both the

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original and the transfer tubes is very

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similar and so if you were considering

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that oh well you know you transfer them

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with antigens and they have new media

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and new growth so maybe they went

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dormant and then you know we're able to

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regrow again maybe the transfer media is

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different maybe the low pressure you

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know affected their media the growth

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between the original tubes and the

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transfer tubes are the same so in

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conclusion the low pressure exposure is

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not lethal to actively met

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rising cells so right now we can't

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measure in situ growth of of them

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advantage ins during the exposure

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because it's actually fairly difficult

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to take a sample out of a low pressure

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vacuum but so there's sort of a black

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box of during the exposure what is

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actually happening but we know that we

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had actively metabolizing methanogens

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before the experiment and then we had

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actively metabolising with antigens

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after the experiment the limiting factor

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is currently the evaporation of liquid

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media this is not something we can get

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rid of considering we're working at low

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pressure if we lowered the temperature

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20 degrees the methanogens wouldn't grow

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anyway even if we had a psycho Phillip

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meth antigen it would just grow so

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slowly that even the slowly rate of

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evaporation of water might be faster

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than the actual slow growth rate of a

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psycho Phillip organism and then the

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methane production and optical density

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measurements that I haven't shown do

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indicate growth at least following

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exposure but what we really want to know

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is are the cells capable of active

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growth at low pressure institute during

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the experiment and so this fall we've

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achieved or obtained a device that we're

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able to put an actual whole system

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within our chamber to measure real time

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methane production and hopefully we're

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going to see if it works and it should

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but you know science so we'll see and

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then we're also going to attempt to

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lengthen our experiments using various

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diffusion barriers such as different

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soils or salts in the media or even just

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adding a hydrophobic filter on top of

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our syringe needles in the chamber just

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to slow that evaporation down and so I'm

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going to be that guy and I do have a

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call for help I've spoken to many of you

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on Monday and greatly appreciate your

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input and probably be emailing you in

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the future but you know we're trying to

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set up our low pressure vacuum chamber

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if anyone has

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the information about any of these

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topics it'd be greatly appreciated but

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you know competition as well so I'm

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working on it yeah we're all friends

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here but other than that thank you all

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right do we have any questions either

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online or in person so you're growing

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these cells on hydrogen and you're

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growing them at Steven millibars total

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preciate it was a t20 h202 so may it

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just be that that low-pressure there's

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just not enough hydrogen for them to

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grow yeah and that that's definitely a

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possibility other studies have shown

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that they can grow on very very limited

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amounts of hydrogen but that's something

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that we definitely need to take into

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account in our our false we should talk

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about Steven phallic hydrogen limitation

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okay so I know at least a seat of orange

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can use acetate as an electron donor can

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you use that instead of hydrogen a

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fascinating factory um yes that's

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possible where we use these species just

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because they don't require any organic

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compounds and hydrogen and carbon

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dioxide well carbon dioxide Minos on

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Mars but it's also postulated that

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hydrogen is available and they're the

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simplest compounds so that's why we use