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you

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[Music]

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thank you for having me my name is

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Lawrence Tyler I'm a postdoc in match

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strikes lab at Michigan State University

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and I've been working on metabolomics as

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a means of mapping metabolic networks in

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microbial cultures and also an

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environmental sample since I started in

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this lab about a year and a half ago and

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so today I'm going to talk to you about

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a particular site that I've been working

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at the coast range ophiolite in

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California oh look it's the same slide

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that Katrina had it's almost like we

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worked in the same lab so you've already

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seen this you're already familiar with

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the reactions that are associated with

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repentance ation but what I really want

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to draw your attention to is the part of

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this reaction that interests me the most

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and that's this production of methane

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and these small organics and what's

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interesting about that to me is that

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while microbes can utilize methane and

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these small organics that are produced

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by this reaction they're also producing

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these things themselves

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we know that microbes can produce

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methane they produce these small

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organics and so it creates this really

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amazing and confounding grey area

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between geochemistry and biochemistry

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which i think is really at the crux of

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not only astrobiology but Originals life

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questions as well and it creates this

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beautiful loving relationship between

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microbes in rock and so as I mentioned

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I'm working specifically at the coast

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range ophiolite it's just one of many

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surprising environments that our lab is

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studying and this site is about three

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hours north of San Francisco or an hour

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north of Napa really nice drive up the

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coast and stop through wine country and

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there's two particular sites at the

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coast range ophiolite microbial

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Observatory which is shortened as chromo

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and that's Quarry Valley and the core

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shed well and each one of those sites

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has a cluster of well there's three at

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Quarry Valley and then there's another

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five plus an older well that was drilled

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before this project at korshack well and

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this is what the wells look like not

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very exciting to look at on the surface

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and I took these samples in May in June

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of last year

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and this is some work that Katrina did

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while she was still a PhD student in

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Matt's lab looking at 16s diversity in

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some of these wells and what I want to

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point out this is a figure from her

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latest paper what I want to point out

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here is the incredible decrease in alpha

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diversity that occurs as you go from

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these lower pH wells like CS w14 down to

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these very high alkaline walls like QV 1

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1 and c SW 1 1 so as you get into these

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higher alkaline well these deeper wells

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we really see a decrease in the amount

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of diversity that we see in these

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communities become very very simple and

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that's why I decided to focus on these

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1:1 wells because they are hyper alkalyn

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and they are very simple communities and

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I thought that they best reflected this

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relationship between microbes and mr.

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Penton izing system and so if we just

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want to look at the 1:1 wells at a

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glance these are the deepest wells at

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these sites with the exception of csw

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old and they are the highest pH as I

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mentioned they're the least diverse and

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so there you have the simplest

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communities they're also uncased which

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means that they can be influenced by

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water coming from the surface rain water

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which there isn't much of in this area

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but it is a concern and so we can have

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this percolation of organic material

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from the surface down into the swell

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water and if we just look really quickly

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at some of the chemical parameters of

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these wells they're about the same depth

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the pH ranges from 11.5 to 12.5 the

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highly reducing very low dissolved

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oxygen and relatively low biomass

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compared to something like seawater for

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example or surface strains in the area

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but again I want to draw your attention

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to the concentration of methane in these

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wells and some of these simple organic

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acids like acetate in forming in the QV

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wells in particular these organic acids

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are sometimes below detection limits but

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that doesn't necessarily mean that

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they're not being produced they may just

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be cycled very very rapidly so we're not

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really sure if the microbes are

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producing methane or if they're

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consuming methane or if they're

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producing something else entirely

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if we look at the meta genomic data

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which is provided by Billy Brazelton

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who's now professor at University of

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Utah we see a lot of pathways for things

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that we would expect and well that's

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influenced by groundwater percolating

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down into the wall so genes associated

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with the hydrolysis of cellulose from

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plants for example or the degradation of

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poly aromatic hydrocarbons fatty acid

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catabolism we also see a ton of gene

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pathways associated with fermentation

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which makes sense because it's a very

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low oxygen environment so the production

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of acetone

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for example which permit ativ cells used

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to store excess acetate and this makes

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sense when we look again at the chemical

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parameters of the wells you can see this

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production of formate and acetate

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through all of these fermentated

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processes so that's interesting but what

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about the methane well if we start

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looking at the genes associated with

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methane production and consumption we

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start to get a really interesting story

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here so the reductive acetyl co a

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pathway or the wood long draw pathway

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isn't really prevalent in these wells

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and neither are the genes for Masana

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genesis which makes sense because we

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weren't really able to find any evidence

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of mismanage ends in the 16s data either

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that comes from these meta-genome so it

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doesn't really look like methanogenesis

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is happening in these well there is

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Mehsana Genesis from acetate here in a

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small amount but if we can't find the

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misc antigens and that could be there

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because of a horizontal gene transfer or

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maybe it's been mislabeled any number of

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things we do find them quite a bit of

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acetate formation from acetyl co a and

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then all of these available genes and

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gene pathways for the consumption of

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methane from methyl a trophy and not

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quite as much methane oxidation to co2

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as I would have expected so there's all

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of these potential pathways for

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consuming methane and using it up but

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not necessarily pushing it to co2 and

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perhaps that's because co2 precipitates

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out so readily in these systems I'm not

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really sure

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so we have this very complex metabolic

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network here that we're trying to tease

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apart and while we have all this

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metagenomic data all that's really

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telling us is that these cells have the

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potential or they have the knowledge for

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these processes not necessarily that

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these processes are actively occurring

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and that's where metabolomics comes into

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play so for those of you in the audience

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who are not biochemist or not microbial

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ecologists like me a metabolite is a

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small organic molecule it's a product or

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an intermediate of a metabolic process

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and the metabolomic election of all the

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metabolites produced by an organism a

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population or a community so metabolomic

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this is really a snapshot of metabolism

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as it's occurring we have genomics which

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will tell us that a cell has the

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knowledge of a process proteomics will

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tell us that the cells producing the

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tools to perform that process but

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metabolomics is evidence of the process

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in action of course there are some

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limitations and as I mentioned before

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there's not a lot of biomass in these

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wells so in order to really get at some

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of the rarer metabolites that are like

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the smoking gun of metabolism we need

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quite a bit of carbon on these on these

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filters if we assume that each cell

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contains about 30 50 grams of carbon

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that means I would need to filter about

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50 litres of water that's a huge amount

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and while the one on wells have a pretty

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high fluid output that's not true for

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all of the wells at this site and

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there's also limitations just to

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filtration the filters can clog because

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as this highly alkaline water comes in

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contact with the atmosphere carbonates

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precipitate out clog the filter it's

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difficult to keep this water cold to

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prevent all kinds of metabolic

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interactions from occurring as you're

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filtering so the data shown here is for

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about 12 liters of water so it's really

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a two-step process first a filter the

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water through a vacuum filtration and a

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glass stand like this through a PTFE

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filter and then yes that's the front

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seat of a car this is the best picture I

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had of me actually doing this in the

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field so and then that filtered water is

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run again through an SP II cartridge

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which stands for solid phase extraction

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which captures all of the dissolved

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carbon in the sample so that can be run

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separately so we're looking at two pools

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metabolites here whatever trapped on the

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filter which represents intracellular

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metabolites and then all of the

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dissolved organic matter that's present

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in the well water and if we look at that

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and compare those samples on a PCA plot

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we see that the fluid samples are sort

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of similar to each other but drastically

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different from everything else what's

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happening it's not going over there goes

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sorry

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and then the biomass samples are very

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very similar to each other and there's

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our blank and our biomass like so if we

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look at all of the different compounds

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that we've observed which are analyzed

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on a tandem liquid chromatography mass

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spec we had a total of about 6,800

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compounds when we subtracted out

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whatever ones were also found in the

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blanks that was about 4500 and only 79

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of those samples or those compounds were

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found across all the samples so there

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were a lot of compounds that were very

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unique to the extracellular fluid and

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only a few that were unique to the

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biomass and the CSW well had a lot more

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unique compounds from the QV well for

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whatever reason there's also a ton of

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possible identifications for all of

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these compounds we searched database and

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compared with the mass-to-charge ratio

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retention time fragmentation and try to

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figure out what all of these compounds

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are and there are 50,000 possible IDs in

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a human metabolomic data base which we

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were able to narrow down to about 3600

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in the CAG database so some of the

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common compounds that we found here are

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some compounds of interest lots of

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different vitamins including a lot of B

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vitamins and the metabolites of those

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vitamins quinolones which are used in

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respiration and for different metabolic

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processes aromatic hydrocarbons acetone

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acetate which I mentioned is linked to

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fermentated processes and antibiotics

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and the products of the degradation of

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antibiotics all very interesting but

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what was most interesting to me is that

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I found all of the compounds that are

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associated with this are ump

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formaldehyde fixation

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halfway associated with methyl a trophy

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all of the compounds associated with the

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cesarean pathway for formaldehyde

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fixation some of the ones associated

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with tetrahydrofolate pathway and some

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of the ones that were associated with a

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glutathione pathway as well so there's

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plenty of evidence here from methyl a

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trophy occurring in these wells not only

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in the meta genome but also in the

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metabolome and very little evidence I

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believe for Mehsana Genesis at least in

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this particular site we still need to

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use some more sensitive methods to

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measure some of these small

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intermediates

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because lc/ms doesn't really pick up the

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very small metabolites especially

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uncharged ones and we have a ton of an

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unidentified compounds that may also be

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used as possible biomarkers for for

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looking for microbes associated with

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serpentine izing environment potentially

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on other worlds and they may be very

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very useful for for those kinds of

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studies so I'm running out of time but

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the next steps that we're going to do in

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this project is we're going to construct

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metabolic networks in our using the

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metabolome and the meta-genome i'm going

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to do some further work in chromo and

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including some work at c SW old because

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it is such a deep well and it is so

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interesting it's got very low amount of

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biomass there's a lot of potential there

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I've also been using these same

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techniques in the Oman drilling project

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and the picture from the front seat of a

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car was some problems from that project

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that's an entirely separate funny story

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I'm also using these techniques on

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microcosms and enrichments in the

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laboratory we're talking about

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potentially using them on micro cosmos

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from lost city and definitely if you're

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interested in this work and particularly

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the work that we're doing at chromo you

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should check out Marisa Buddha's and

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Lindsey Williams's talk this afternoon

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in this room and hopefully we'll be able

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to use some of this work to eventually

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find a microbe in space and so with that

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I'd like to thank you for your attention

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and I welcome your questions if there's

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we have time for a question or two for

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Lord oh yeah in the back if you could

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either speak loudly or come up to a

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microphone so I'm still working very

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preliminary on some of these enrichment

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experiments and microcosms but if you're

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interested in enrichment experiments

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from from this site in particular

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definitely check out Mary's talk this

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afternoon but we've been basically

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providing a reductant to to these

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microcosms and seeing if we can observe

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methyl a trophy in the production of co2

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in our lab hi hi Jamie Foster University

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of Florida I'm really curious about the

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metabolomic so did you see any cycling

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any Dial cycling within your community

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or you is this just a single time point

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what's the reproducibility of your

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signatures in your community

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so this is a single time point I

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initially took triplicates samples but

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the signal was so low that I had to pull

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all of them and that's why I was a 12

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leader sample because originally it was

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four in samples that I've done in

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culturing these results are very

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reproducible and triplicate samples look

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very very alike as far as

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reproducibility at this particular site

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this is still very preliminary and we're

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still working through the methodology

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and so I was only able to take one

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sample from each well but we'll be going

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back and hopefully doing this at a

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larger scale in order to show that

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reproducibility and maybe see some

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differences seasonally or with other