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today i'm going to talk about microbial

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bowel signatures in the deep terrestrial

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subsurface and i'll be specifically

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focusing on the approaches of plfa

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analysis and carbon isotope analyses for

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so when we talk about life in the deep

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terrestrial subsurface we're talking

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specifically about life in the

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continental crust so these are

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microorganisms that live several

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kilometers deep

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in natural fractures within crystalline

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rock

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and we call these systems extreme

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environments due to the high

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temperatures and pressures as well as

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low nutrient availability in these

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systems and despite these conditions we

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still see that microbial communities are

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surviving for geological time scales

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within these environments

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so of course for astrobiologists this is

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an important question or important

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system to look at just because we're

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wondering is there a possibility that

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there's life in the subsurface of other

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planets or of moons and how could they

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so this diagram is a representation of

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the carbon cycle in the deep terrestrial

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subsurface

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so in yellow boxes we have the carbon

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pools and in the black boxes we have the

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organisms involved

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so the earth's surface and the shallow

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subsurface are ultimately dependent on

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so we have photosynthesis up here

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producing oxygen and organic carbon and

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this organic carbon is either buried or

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it's utilized by heterotrophic organisms

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such as sulfate reducing bacteria and

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as we go deeper in the subsurface this

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organic carbon may become unavailable

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for heterotrophs and organisms are going

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to have to rely on an alternative

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mechanism to fix carbon

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so one hypothesis is a hydrogen-driven

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chemolitho-autotrophic community

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and this is based on

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the concept that hydrogen is produced by

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abiotic processes such as outgassing

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water rock interaction and radiolytic

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decomposition of water

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and this hydrogen can be utilized by

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acetogens and methanogens to produce

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acetate and methane

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and acetoclastic methanogens can utilize

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this acetate and produce methane

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and

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all this methane that is produced can be

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utilized by methanotrophic bacteria

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which produce hydrogen and co2 as a

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result so

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evidently methane is an important uh

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important point in this in this

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so in this study we're using

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phospholipid fatty acids as

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biosignatures for viable microbial

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communities

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so phospholipids are a major component

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of bacterial and eukaryotic cell

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membranes and they're known to degrade

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rapidly upon cell death

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so as a result we can use these as

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indicators for microbial life that is

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actually alive

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secondly we can look at the

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concentrations of the plfa to estimate

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the cell densities within these

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environments

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and thirdly we can look at the

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composition of the plfas to get an idea

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of what kind of microbial communities or

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what kind of microorganisms are present

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so

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in addition to plfa analysis we've used

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two carbon isotope analyses and the

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first one is delta c13

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which we heard a little bit about

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yesterday

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and what we're interested in for this

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technique is uh c12 and c13 only not c14

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and to illustrate this concept i've

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included these

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these three

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carbon sources dissolved in organic

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carbon dissolved organic carbon and

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methane

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and autotrophy heterotrophy and

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methanotrophy are the processes by which

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microorganisms will uptake this carbon

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from these sources

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and they may use this carbon to produce

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plfa

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so the important point for delta c13

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analysis is that microorganisms

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preferentially use the lighter carbon

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isotope c12

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so

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if we have a

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ratio

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or a particular delta c13 value for the

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carbon source

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which is basically the ratio of c13 to

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c12

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if a microorganism uptakes that carbon

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it'll preferentially use c12 so that

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ratio will decrease and we'll see a more

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negative delta c13

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and then when they produce plfa you'll

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also see that fractionation so the plfa

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will be even more depleted

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and the extent of this fractionation

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depends on the metabolism so

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we can look at these delta c13 values to

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think about what kind of metabolisms are

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occurring in these systems

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the second carbon isotope analysis or

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technique that we use is radiocarbon

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and this is based on the idea that c14

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is radioactive and it decays over time

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in this case if we have a certain amount

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of c14

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in your carbon source

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we won't see that fractionation effect

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due to the equations that we're using uh

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for delta c

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delta c14

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so in this case if a microorganism is

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using

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one particular carbon source that has a

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specific delta 14c value we should see

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that same value in the microbial biomass

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and again in the plfa so we can look at

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the microbial carbon sources using this

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technique

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so

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sampling from the deep subsurface is

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difficult just because of the

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inaccessibility of these

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of these systems

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but luckily some of the deepest mines in

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the world are located in south africa

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and they provide us with access to these

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systems

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up to 3.5 kilometers depth

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so

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basically the exploratory boreholes that

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they drill into the walls of the tunnels

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can sometimes tap into reservoirs of

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water which may contain microbial

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communities

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and what we do is go down into these

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mines and filter this water and then

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do analyses such as plfa analysis or

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other people are doing like dna analysis

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just to gain insight into who's there

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so in this system or in this study we

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are looking at six samples from four

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different minds and these four minds are

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drifantene

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kluth and beatrix

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and the depths of these samples come

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from about one kilometer depth to about

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3.5

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so we looked at we extracted plfa from

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the six samples

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and used those concentrations of plfa to

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estimate cell densities within these

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within these environments

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so

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as you can see the cell densities are

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actually very low in all all across the

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six samples

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at less than 10 to the five cells per

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milliliter of water

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and this is consistent with direct cell

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counts via microscopy as well as

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previous investigations of other

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in the case of kluth this is an

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extremely low cell density

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it's actually estimated at about 20

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cells per mil

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so for those of you who are

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microbiologists you'll probably

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appreciate that this is extremely low

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and slightly or very interesting

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because

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this raises the question for us as to

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whether there's actually anything living

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there perhaps this is actually just

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so we also looked at the

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composition of the plfa to identify

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different microbial groups and this uh

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diagram here just represents

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the differences between the different

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systems so what we see when we look at

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this is that they do the composition of

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the communities varies

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and we see

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some similar some similarities between

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the two beatrix samples uh drifontaine

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contained evidence for gram-positive

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bacteria and sulfate reducers tautona 1

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and 2 and beatrix 1 and 2

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contain these cyclic

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plfa which are

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indicators for

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microbial responses to environmental

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stressors such as nutrient

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but

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overall what we find from this is that

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we're seeing differences in the

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microbial communities so we're probably

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going to see differences in metabolisms

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and this is in fact what we see

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we see

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here we're looking at the delta c13

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values of plfa which are

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indicated

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by the orange boxes and then we also

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have three potential carbon sources

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dissolved in organic carbon dissolved

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organic carbon and methane

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and

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for drifontaine we saw very depleted

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plfa

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and the negative offset from methane is

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indicative of some utilization of

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methane as a carbon source so

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methanotrophy

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for tautona one and tautomeno two and

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beatrix

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uh the negative offset from the

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dic to plfa is indicative of autotrophic

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communities

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and then for beatrix we actually see we

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we think that there's a combination of

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methanotropic activity as well as

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autotrophy

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in terms of cliff this is the sample

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with the very low cell density um we

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didn't have enough plfa for delta c13

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analysis

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but these values for dic and methane are

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consistent with a lack of methanogenesis

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and abiotic methane so that's

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interesting in terms of

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and we also used radiocarbon analysis

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delta c14

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and to confirm the carbon sources so

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for dry fontaine the plfa were slightly

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more enriched than the dissolved

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inorganic carbon and methane

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so it looks like there is some influence

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from these two carbon pools but perhaps

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there's slightly younger carbon coming

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in as well

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for tautona 1 it's consistent with what

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we saw with delta c13 they're utilizing

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dic as a carbon source so autotrophic

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processes

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and

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for tautona 2 same same thing um

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consistent with autotrophic processes

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and beatrix again it looks like there's

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they're utilizing dic and methane so

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autotrophy and methanotrophy and as

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you'll notice we haven't we don't have

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measurements for doc here so organic

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carbon just because in these systems

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organic carbon is usually very low and

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so it's in all these cases it was very

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difficult to measure

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radiocarbon

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so we have to keep that in mind

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so basically

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my overall conclusions from this is that

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possible fatty acid analysis is a useful

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tool for looking at viable microbial

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communities in the deep terrestrial

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subsurface

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carbon isotopes can provide insight into

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the microbial metabolisms that are

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occurring in these systems

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and

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in the case of our samples

297
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we observed plfa in all of our samples

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extremely low cell densities and some of

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them particularly in the one sample

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clues

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and we also observed a range of

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microbial metabolisms including

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00:11:37,190 --> 00:11:35,279
methanotrophy and autotrophy so the

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presence of these or the observation of

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these two metabolisms is consistent with

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perhaps the chemolithoautotrophic

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community

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so again as astrobiologists i'm just

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highlighting the implications of this

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study

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worth wondering is there life on the

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subsurface of other planets and moons

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so could in if we had a system um or a

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planet that you know the the surface of

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the planet or moon was inhospitable to

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life could there be life surviving

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independent of the photosphere

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and could those microbial communities

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survive over geological time skills

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and so this point is

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very interesting for some of you may

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have heard about a month ago there was a

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paper in

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nature by holland at all and there they

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actually are looking in a different mine

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site in canada and they have found

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water that is about 2.7 billion years

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old

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and

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in our systems we are seeing very old

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water like

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approximately 20 million years old but

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to see microbial communities living in a

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system that is 2.7 billion years old is

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pretty incredible so um just a note for

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you guys to

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look at that and then also just as

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another point survival through the late

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heavy bombardment if

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this is kind of an origins question but

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in the early earth maybe the surface of

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the earth could have been sterilized by

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that 3.8 billion years ago there's the

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meteorite impacts and

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um could life have survived through that

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by living in the subsurface

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so overall there's lots of

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astrobiological implications

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and i just want to thank the south

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africa gold mines for providing us with

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access to these systems because

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otherwise we would never be able to look

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at them and also my collaborators tell

354
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us onstop barbara sherwood lawler

355
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kenneth wilkie

356
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eric womack and eric sakowski and my lab

357
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group including my supervisor greg

358
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i'm particularly interested where you're

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looking at the carbon-14 and the site

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where you had the highest

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percentage of uh carbon-14 incorporated

362
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in your organic material yeah

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the fact that yeah it looks like your

364
00:14:02,470 --> 00:14:00,959
metabolisms were doing doc the fact that

365
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there's any carbon-14 at all do you

366
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think that you're still getting just

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telegraphed stuff from the surface i

368
00:14:09,670 --> 00:14:07,360
mean if it's been relic for a million

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years there shouldn't be any carbon 14

370
00:14:13,509 --> 00:14:11,199
really exactly

371
00:14:15,509 --> 00:14:13,519
um it depends on the system so i haven't

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included exactly the ages of these

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systems and i can't remember them all

374
00:14:23,030 --> 00:14:20,720
part um you know by heart but i um

375
00:14:24,550 --> 00:14:23,040
yes you know we have to think about

376
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um

377
00:14:27,269 --> 00:14:26,480
where is this organic carbon coming from

378
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and

379
00:14:32,389 --> 00:14:29,600
um in some cases we did measure a

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00:14:35,910 --> 00:14:32,399
dissolved or organic carbon

381
00:14:38,150 --> 00:14:35,920
radiocarbon measurement and

382
00:14:40,550 --> 00:14:38,160
it's actually very difficult to do

383
00:14:42,230 --> 00:14:40,560
because we use these resin columns

384
00:14:44,790 --> 00:14:42,240
i actually don't do it personally but

385
00:14:46,790 --> 00:14:44,800
our collaborators do and um you know

386
00:14:49,269 --> 00:14:46,800
sometimes we have to think about whether

387
00:14:51,509 --> 00:14:49,279
we're introducing young organic carbon

388
00:14:53,269 --> 00:14:51,519
to that pool so

389
00:14:55,110 --> 00:14:53,279
um

390
00:14:56,790 --> 00:14:55,120
yeah so

391
00:14:57,990 --> 00:14:56,800
i don't know i

392
00:14:59,750 --> 00:14:58,000
yeah it's something to think about

393
00:15:01,110 --> 00:14:59,760
whether like what how much influence

394
00:15:03,110 --> 00:15:01,120
there is from the surface which is

395
00:15:04,710 --> 00:15:03,120
exactly what what we're looking at is

396
00:15:10,790 --> 00:15:04,720
can you know are they completely

397
00:15:15,030 --> 00:15:12,629
i'm just wondering whether either you or

398
00:15:16,389 --> 00:15:15,040
any of your collaborators uh have done

399
00:15:18,069 --> 00:15:16,399
or are thinking about doing any

400
00:15:19,670 --> 00:15:18,079
culture-based work with this system

401
00:15:21,189 --> 00:15:19,680
trying to actually grow up anything

402
00:15:23,750 --> 00:15:21,199
that's down there

403
00:15:26,470 --> 00:15:23,760
i you know we have such a like such a

404
00:15:28,389 --> 00:15:26,480
huge group of collaborators i'm doing

405
00:15:30,310 --> 00:15:28,399
everything you know dna analysis i i

406
00:15:33,670 --> 00:15:30,320
believe we do have some doing culture

407
00:15:49,430 --> 00:15:33,680
work um but i'm not entirely sure

408
00:15:55,910 --> 00:15:52,710
so with respect to the methanol trophy

409
00:15:59,269 --> 00:15:55,920
is your system totally loxic or

410
00:16:01,350 --> 00:15:59,279
do you get can the plfa analysis hint uh

411
00:16:04,069 --> 00:16:01,360
towards which type of methanol trophy

412
00:16:05,110 --> 00:16:04,079
this uh these uh communities are

413
00:16:06,870 --> 00:16:05,120
undergoing

414
00:16:08,150 --> 00:16:06,880
yeah so

415
00:16:11,590 --> 00:16:08,160
that's that's a good question because

416
00:16:13,430 --> 00:16:11,600
methanotrophy is typically a um aerobic

417
00:16:16,710 --> 00:16:13,440
process but then you have the anaerobic

418
00:16:19,030 --> 00:16:16,720
process of methanogen methanotropes and

419
00:16:19,829 --> 00:16:19,040
the animoxon right

420
00:16:22,470 --> 00:16:19,839
so

421
00:16:24,509 --> 00:16:22,480
um with which is a kind of a

422
00:16:27,829 --> 00:16:24,519
consortium of uh

423
00:16:29,269 --> 00:16:27,839
methanotrophic bacteria and um sulfate

424
00:16:31,509 --> 00:16:29,279
reducers i believe

425
00:16:32,389 --> 00:16:31,519
archaea but yeah archaea

426
00:16:33,990 --> 00:16:32,399
um

427
00:16:36,230 --> 00:16:34,000
so yeah it's an interesting question

428
00:16:37,590 --> 00:16:36,240
because we actually for that one sample

429
00:16:40,389 --> 00:16:37,600
that was always on the left of the

430
00:16:41,670 --> 00:16:40,399
graphs where we saw methanotrophy or

431
00:16:43,350 --> 00:16:41,680
you know it

432
00:16:45,590 --> 00:16:43,360
the carbon isotopes definitely say

433
00:16:47,990 --> 00:16:45,600
methanotrophy um we're not actually

434
00:16:50,470 --> 00:16:48,000
seeing the biomarkers for methanotrophy

435
00:16:52,470 --> 00:16:50,480
so there's a couple plfa biomarkers that

436
00:16:53,509 --> 00:16:52,480
are indicative of methanotrops um

437
00:16:55,990 --> 00:16:53,519
aerobic

438
00:16:57,430 --> 00:16:56,000
methanotropes so we're not seeing those

439
00:16:59,670 --> 00:16:57,440
so that's what i'm starting i'm trying

440
00:17:01,829 --> 00:16:59,680
to think about is like you know how what

441
00:17:04,309 --> 00:17:01,839
is actually occurring here because or

442
00:17:06,309 --> 00:17:04,319
you know maybe not all methanotrops have

443
00:17:07,350 --> 00:17:06,319
those biomarkers so

444
00:17:09,029 --> 00:17:07,360
um

445
00:17:10,470 --> 00:17:09,039
but yeah i mean if i could see those

446
00:17:13,669 --> 00:17:10,480
biomarkers then i would know for sure

447
00:17:16,069 --> 00:17:13,679
that it is aerobic um but we do we do

448
00:17:18,069 --> 00:17:16,079
have oxygen in some of these systems so

449
00:17:20,150 --> 00:17:18,079
yeah do you have any uh dead on the

450
00:17:22,230 --> 00:17:20,160
nitrate present because that's a

451
00:17:25,110 --> 00:17:22,240
alternative pathway for bacterial

452
00:17:27,510 --> 00:17:25,120
methanotrophy under anoxic conditions uh

453
00:17:29,270 --> 00:17:27,520
you know what actually i can't really

454
00:17:33,270 --> 00:17:29,280
think offhand of what we have yeah for

455
00:17:37,750 --> 00:17:36,390
last question i think

456
00:17:39,430 --> 00:17:37,760
i'm just out of curiosity how do you

457
00:17:43,430 --> 00:17:39,440
date water

458
00:17:45,190 --> 00:17:43,440
find something that's 2.7

459
00:17:47,029 --> 00:17:45,200
yeah just out of somebody might know

460
00:17:49,990 --> 00:17:47,039
this better than me but i believe it's

461
00:17:52,310 --> 00:17:50,000
i'm having to do with um oxygen isotopes

462
00:17:54,789 --> 00:17:52,320
and hydrogen isotopes yeah okay i see

463
00:17:57,510 --> 00:17:54,799
nodding heads

464
00:17:59,270 --> 00:17:57,520
yeah yeah