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[Music]

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[Applause]

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all right thank you all for thank you to

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the convenience Guinea's for having me

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in this session and for all of you for

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sticking doll of the very end pretty

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much the second last talk is pretty much

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the end I'm going to switch gears and

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move to laboratory investigations from

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from theoretical conversations in the

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previous talk and talk about

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physiological mechanisms and mineral

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transformations of hyperthermophilic ion

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reduction so this is probably a gross

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oversimplification but I want to remind

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and reiterate that on earth we know that

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biology greatly influences geology and

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it's this coevolution of life and Earth

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that yields organic geochemical and

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mineral clues to trace and describe some

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of the earliest life on our planet now

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among the mineral clues that we turn to

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our iron minerals such as iron oxides

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that we know today have been central to

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some of the earliest biological

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metabolisms yet when we look at the rock

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record from a mineral perspective we

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don't know if we can discern a truly

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microvilli altered mineral so one

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approach to doing this would be perhaps

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to examine one specific micro mineral

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process to really get at identifying its

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mineral signatures and the mechanisms by

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which they form so I'm gonna make the

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argument that we can explore and better

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understand a micro mineral process such

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as hyperthermophilic ion reduction to do

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this now a hyperthermophiles just to

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revisit this fer of people in this

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audience who may not be a microbiologist

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a hypothermia file is an organism that

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grows optimally above 80 degrees Celsius

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and an iron reducer is an organism that

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couples the oxidation of organics or

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hydrogen with the extra cellular

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reduction of an iron oxide source to

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gain energy now this type of microbial

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metabolism occurs widely in in

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terrestrial hot springs in deep sea

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hydrothermal vents yet our understanding

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of its physiology as well as mineral

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transformations is extremely limited so

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we're actually probing and trying to

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understand this process a little closely

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using a model system a model

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crenarchaeota called podrick pardek Tim

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Delaney a type strain su o6

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now I want to highlight a few key

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characteristics about this organism

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before going any further so by reading

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Tim Delaney I is a isolate that was

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taken from an actively venting

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hydrothermal chimney and that grows

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optimally at 90 degrees Celsius and it's

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hydrogen atrophic and when it comes to

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electron acceptors it can only really

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use an insoluble iron oxide such as

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Farah hydride and nitrate but it is

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unable to use any soluble iron such as

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ferric citrate or any macro particulate

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crystalline oxides such as birth I'd

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hematite or magnetite now when it comes

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to looking at its genome we find that

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its genome encodes for these 17

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predicted C type cytochromes and many of

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these C type cytochromes are actually

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Multi heme proteins which we know from

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model mesophilic bacteria are are

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essential for extracellular electron

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transfer so we started off by asking

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that given this organism only given that

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this organism only grows on these

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insoluble iron oxides but not on any

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soluble iron does it use some of these

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other iron mineral phases and to really

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get at this question we decided to

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synthesize a range of nano phase

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minerals these several of these

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different types of iron oxides to test

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for growth and we found overall that the

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organism grows on a wide range of these

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minerals when they're presented as these

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nano particulate mineral phases I'll be

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to varying degrees depending on the

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oxide it grew to the highest cell

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concentration at the fastest growth rate

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producing the most Fe to using Farah

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hydride it grew to moderate cell

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concentrations growth rates and Fe to

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production using AK again a deliberate

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across I'd shown in green and orange and

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to the lowest cell concentrations growth

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rates and Fe to production using

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goethite and hematite now these results

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are not unsurprising given the expected

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crystallinity and thermodynamic

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stability of these phases but what is a

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key finding here is that the organism

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can use this wide range of minerals when

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we think about mineral parameters such

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now next we wanted to know given that

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this organism can use this wide range of

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mineral phases does it form mineral

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different mineral end products as a

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result of reduction and to really get at

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this question we do try to examine the

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mineral end products using a range of

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bulk and reflectance spectroscopy sa-2

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of which I will primarily be focusing on

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today to show you results this

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mid-infrared attenuated total

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reflectance and mossbauer spectroscopy

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x' in addition you will notice that many

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of these blots have a range of

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conditions and in spectra and the reason

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why this is is because we're looking at

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biogenic mineral transformations and

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comparing them to a suite of a biogenic

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or abiotic mineral transformations that

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may be plausible accounting for any

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transformations that may result from

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heat non growing cells or the growth

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medium itself what we find over all is

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that when Farrah hydrate transformations

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are considered that the organism

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primarily makes magnetite which is seen

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with the two prominent absorptions here

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for for the mineral phase and in the

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attenuated total reflectance and in the

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moss power as well we see the magnetite

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but in addition to the magnetite we also

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see minor amounts of ferrous phosphate

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and ferrous carbonate form relative to

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abiotic controls with lipid across side

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instead of magnetite we see the

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formation primarily of a ferrous

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carbonate phase such a siddha right in

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the attenuated total reflectance

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spectroscopy and in the Moskva we see

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that same phase but also in addition to

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that phase we see minor amounts of

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ferrous phosphate forming with AK again

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eh we see minor amounts of a ferrous

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phosphate phase as well as magnetite

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minor amount of magnetite form relative

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to the end to the abiotic controls now

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when we put together there is also

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showed you with regards to the rates of

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reduction and the spectral

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characteristics together we find that

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the minerals that form are really

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affected by the type of oxide that we we

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start and we think about growing these

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organisms with as well as the FE two

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flux

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now with Farah hydride we find which

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shows this fast rate of reduction in

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high Fe to accumulation magnetite

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nucleation and crystal growth is favored

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whereas and also in addition to that we

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see minor amounts of ferrous phosphate

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in carbonate forming with a lipid agro

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side which shows slow rates of reduction

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and low fe2 accumulation we see Sid

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right and ferrous phosphate forming with

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AK again yet which shows fast rates of

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reduction but significantly lower if we

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do accumulation relative to Farah

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hydride we see that minor amounts of

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magnetite and Affairs phosphate vivvy

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night can accumulate so context here can

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really make a difference in thinking

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about the different types of mineral

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phases that can form so from a

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physiological perspective we wanted to

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further probe and understand does this

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organism require cell mineral contact

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for reduction and to do this we really

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perform these barrier experiments that

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you that grew this organism with the

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mineral that was either in case in the

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dialysis barrier so as to prevent any

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cell mineral contact and these were

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compared to conditions where the mineral

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was available as free suspensions and in

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addition to this we also compared the

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compared these two conditions to another

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condition where we had an empty dialysis

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barrier added to free suspensions of

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Farah hydrate and this condition

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primarily served the purpose of ensuring

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that the the barrier itself did not pose

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any toxicity for growth now all of these

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conditions we examined as is but also in

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the presence of an artificial electron

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shuttle a QD s an artificial iron key

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later NTA and also cell free spent

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supernatant supposed to account for any

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endogenously produced shuttles of he

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laters so what we found from these

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results were that be delaney I was

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unable to grow without any mineral

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contact and that you'll notice we're

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looking at number of cells over time and

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that when the mineral was available as a

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free suspension regardless of whether a

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shuttle key later or cell free spent

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supernatant was added to the medium that

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the

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cells grew rapidly over time and showed

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no differences in the rates of growth

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whereas when the menorah was encased in

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a barrier that cells rapidly died or

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could barely sustain any growth now when

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we look at the FE 2 production over time

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for these experiments particularly for

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the experiments where free suspensions

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of Farrah hydride were made available

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with the addition of a shuttle Oki later

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or self-respond supernatant that only a

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few - production was stimulated upon

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addition of the shuttle a QD s so next

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we wanted to know given that this

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organism has these 17 C type cytochromes

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which c type cytochromes may be involved

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in iron reduction and so to do this we

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actually looked at we extracted the

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proteins from Farrah hydrate grown cells

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and compared them to the protein

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extracts of nitrate grown cells and we

241
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stained specific stains specifically for

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the cytochromes using a heme stain and

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here we found differential protein bands

244
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in in these two conditions which we

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excised digested and sent for mass spec

246
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and found that an ATM C Drive cytochrome

247
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was uniquely produced in the Farrah

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hydride grown cultures and 3 C type

249
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cytochromes 1/8 heme mono him and died

250
00:10:39,019 --> 00:10:35,010
him C type cytochrome were produced in

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either Farrah hydrate or nitrate grown

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cultures or both cultures rather so what

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are these C type cytochromes and I'm

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primarily going to focus on these two

255
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highlighted here in this next slide when

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we look think about these c TF

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cytochromes in the context of the genome

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of this organism we find that 11 of

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these 17 total c type cytochromes in

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this organism are these cytochromes are

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highlighted as yellow circles here and

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the number is the number of hims heme

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groups the the cytochromes have and the

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these eleven of these see that sairam's

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up

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are these for punitive operands depicted

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in these four colors here that encode

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these membrane reductase complexes for

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which we have really no homologues in

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other iron reducing general such as

271
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those of para baculum ferrell Globo

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Sergio Globus and more interestingly the

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two cytochromes that we found on the

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previous slide are actually the farah

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hydride condition see dep cytochrome is

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encoded by this gene right here

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the 7-8 form and just part of this

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period of operon that also in contains a

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gene that encodes a 13 heme c type

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cytochrome so and and here we see that

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this is the farah hydrate and nitrate

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culture where we see another a-team see

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that cytochrome that's part of this

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operon that has offs related to solve

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for reductases so i would be interesting

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to know what which genes are

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differentially expressed as well and

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also quantitatively determine the

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abundances of these proteins that are

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produced and that's part of future work

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for us

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so I want to quickly summarize what I've

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been able to share today with you and

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that that is that a wide range of nano

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phase iron oxides can be used for iron

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reduction at high temperatures and the

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key to recognizing what I want to

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emphasize here is that the part the the

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nano phase part of this is that you know

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we need to think about environmentally

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relevant phases in that the nano

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particular it phases are likely more

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environmentally relevant and that growth

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of reduction rates inversely correlated

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with oxide crystallinity in

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thermodynamic stability this is not

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surprising but that farah hydride a

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Cugini and lipid accrue site make

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interesting phases to further explore

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the end products of reduction depend

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upon oxide in Fe to flux and that

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magnetite fares phosphates and first

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carbonates can form the latter two of

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these to the best of our knowledge have

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not been reported for high-temperature

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iron reducers and so we'd be interested

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to see if this is a phenomenon that

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occurs overall additionally from a

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physiological standpoint cell mineral

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contact is required for reduction

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and that potentially novelty type

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cytochromes may be produced with Farah

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hydride relative to nitrate and these

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are looking into further using

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differential gene expression and

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quantitative proteomics experiments and

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with that I'd like to thank folks at

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UMass Amherst Mount Holyoke College as

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well as Brooker optics and also funding

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without which none of this work would be

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possible so thank you I think we have a

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question over here sorry I misunderstood

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something because you you studied the

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growth of the tombola microbe of the

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microorganisms on ferric minerals and

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then you showed most power spectra

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biotic and abiotic so what does that

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these two different spectra where do

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they correspond so the so the biotic

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spectra are taken from exponentially

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grown cells the minerals that were

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transformed after cells had been cells

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grew on those minerals and the abiotic

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abiotic condition is where the there

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were no cells added to the same exact

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set up okay so it is the same descent

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mineral just before the growth and after

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the growth no no it is it's actually

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heat reacted so it's so these are grown

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these are cultures grown at 90 degrees

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Celsius in anaerobic cultures and so the

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abiotic in the most power spectra

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reflect heat transformations that may be

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occurring for the same duration that we

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see any sort of biogenic transformation

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does that make sense yes she we have

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time for maybe one quick question if

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there's others all right thank you thank