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good afternoon everyone my name is

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sabrina elkasis and i am excited to talk

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to you today about my research studying

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sub-seafloor chemolithoautotrophs as

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models for understanding the potential

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there are several different ocean worlds

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in our solar system some of which are

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shown here you have probably heard of

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enceladus and

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europa which are both very heavily

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here is a profile of enceladus one of

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the main ocean worlds that i study which

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i'll use as an example

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ocean worlds and our solar system

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have thick and icy exteriors with

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liquid water below

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this means that sunlight cannot

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penetrate to the depths of the liquid

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water so survival of life here would be

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dependent entirely on chemical energy

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just like at hydrothermal vent systems

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going a bit deeper into detail enceladus

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has a chondritic core which means that

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it's undifferentiated

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it's covered in two to sixty kilometers

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of ice

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there are no plate tectonics but the

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water beneath the ice is heated by tidal

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deformation

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circulation of this water

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or flow which is key for rock water

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interactions that create energy for life

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this water and accompanying compounds

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are released as plumes in the south

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polar region of enceladus

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where the cassini probe

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detected that the plume contains

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chemicals that microbes here on earth

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use for their metabolisms at

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so a key question that we kind of were

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investigating is how is axial seamount

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our hydrothermal vent study site and

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first deep sea hydrothermal vent systems

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are where heated water flows through

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crustal rocks

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this process alters the composition of

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both the fluid and the rocks it flows

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through creating a chemical

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disequilibrium

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that provides impetus for chemical

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reactions to occur

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life at these depths without sunlight

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like ocean worlds can capitalize on

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these reactions to power their

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in our lab we specifically focus on

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sampling diffusely flowing vents

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they are a mix of vent fluids and sea

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water at

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less than 120 degrees celsius so much

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lower in temperature than the black

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smoker you can see in the picture there

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they are the largest source of new

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carbon in the vent ecosystem containing

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5 to 10 times more cells than deep water

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and holding microbial assemblages at the

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crustal interface

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thus by sampling the water from these

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vents we also get a window into life

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our study site axial seamount is a

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hydrothermal sea mount situated on top

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of the juan de fuca ridge spreading axis

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the three diffuse fence sites of

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interest within axial were anemone

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marker 33 and marker 113.

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each of these sites was sampled each

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year from 2013 to 2015.

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this gave us the opportunity to conduct

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not only spatial analyses but also

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from the different chemical measurements

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of

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see that the vents are similar in both

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temperature and ph

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the largest difference is seen with the

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hydrogen concentration which is much

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higher at anemone and below the limit of

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detection at macro 113

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we'll see later that this is because

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there are a large number of methanogens

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at marker 113

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which draws down the hydrogen level and

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increases the methane

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which you can also see in the table

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there

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the oxygen at marker 113 is half the

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concentration of the other vents

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and the lowest measured across all vents

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sampled

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i've highlighted hydrogen and methane

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again as a reminder that both of these

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compounds were detected by the cassini

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probe in the enceladus plume so

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a lot of studies now focus on studying

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methanogens which use hydrogen and

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carbon dioxide

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in this cross your study a combination

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of approaches was used to determine the

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active sub-seafloor autotrophs over

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space time and geochemical gradients and

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i am currently still analyzing that data

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metagenomics was used to understand the

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potential functions and taxonomy in the

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sampled populations

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metatranscriptomics was used to identify

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active functions within the sampled

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population

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to further tease out the autotrophic

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signal

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rna stable isotope probing was used

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in this experimental technique

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isotopic incubations label only active

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autotrophs in the sample then further

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metatranscriptomic sequencing allows us

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to determine the identity of those

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members of the microbial population

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the goal is to construct full metabolic

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and functional profiles at all sites in

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the next few slides i'll go into detail

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about how rna stable isotope probing

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works and why it is such a powerful tool

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rna stable isotope probing

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starts with an incubation of vent fluid

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which you can see in the upper right

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along with a labeled substrate in this

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case we use 12 or 13c labeled

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bicarbonate

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the controls and experiment are labeled

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at different temperatures and we use

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three different incubation temperatures

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30 55 and 80.

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then the rna is extracted quantified and

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fractionated using an ultracentrifuge

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and

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in the ultracentrifuge tube after

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spinning it for three days

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the heavier fractions are at the bottom

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which are the microbial populations that

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took up the 13c label and the lighter

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ones are at the top which are not the

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active autotrophs

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12 to 16 fractions are collected and

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then the rna is precipitated and

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quantified

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graphs like that shown on the bottom

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left are made to understand which

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fractions to take through

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metatranscriptome sequencing which we'll

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slide from the density fractionation and

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subsequent rna precipitation we create

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these graphs the green line shows the

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control with the 12c labeled bicarbonate

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and 12l means that it's light in in the

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12c labeled bicarbonate

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the orange line shows the result of the

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13c incubation and the 13h means that

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it's heavy in the 13c label

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and as you can see the peak buoyant

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density is shifted as compared to the

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control in this orange line from 1.78 to

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around 1.81

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so

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we then sequence the metatranscriptome

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of the 13c heavy fraction to identify

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the active autotrophs which are those

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which took up the labeled 13c

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so now i created these graphs

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to compare results over space time and

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different geochemical gradients

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so this one

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is

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over space

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across all three vent sites at a single

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temperature of 80 degrees c

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during the year 2013.

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marker 113

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which is in the middle is heavily

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dominated by methanocaldococcus which is

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a methanogen and which is why earlier

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when i showed the drawdown of h2 and the

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increase in methane it's due to this

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domination of this methanogen

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marker 33 has feud to none

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methanocaldococcus

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likewise thermo vibrio is present at

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marker 33 and anemone

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but does not appear at all in marker

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so next i compared

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rna sip results again over space however

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this time it was for adc so the same

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temperature as before during 2014. this

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allows us to compare across both space

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and time because we can compare it to

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the 2013 results as well

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first all of the methanocaldococcus we

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saw at marker 113 is gone again which is

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it's in the middle

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um the middle bar

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now all three event sites have a similar

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composition of d sulfurobacterium

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thermovibrio and an unclassified member

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of the family

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d-sulfurobacteriaceae

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this shows that hydrothermal vents can

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vary quite a lot in their microbial

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community composition in just one year

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this should be kept in mind when we

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capture snapshots of the geochemical

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composition of hydrothermal vents on

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other ocean worlds just because we

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measure something once doesn't mean it

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can't change drastically in a very short

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next i looked across time

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at one vent site marker 113 and at one

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temperature of 55 degrees celsius unlike

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at 80 degrees celsius where we saw

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methanocaldocus one year and did not

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seed in the next here the microbial

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community composition remains fairly

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constant over the two years with a

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slight decrease in diversity in 2014 as

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finally i looked across temperature at

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anemone event site in 2014. the

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rightmost bar represents the entire meta

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transcriptome at this site which you can

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see contains many many members however

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at each temperature we see not only a

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distillation of the members in this meta

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transcriptome but also that just a few

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families dominate at each temperature

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i did not show it by genus this time

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since the meta transcriptome becomes

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even harder to distinguish

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at 30 and 55 degrees c members of the

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family natalie aca dominate where as

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they are absent at adc

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at adc we see members of the diesel

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feral bacteriaceae and aquificacia

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the key takeaway from this talk is that

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both axial and enceladus or other ocean

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worlds have similar environments lack of

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light rock water interactions at

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hydrothermal vents chemical energy for

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life and similar temperatures with this

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study we were able to create profiles of

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active autotrophs at variable chemical

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regimes which lends us predictive power

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of what autotrophs would exist in a

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given environmental regime on other

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ocean worlds

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this will help inform efforts to

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understand and detect life on other