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okay cool so yeah today I will be

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telling you about in vitro and in situ

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iron geochemistry and microbial activity

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in chocolate pots hot spring to start

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with I'll show you all where my field

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site is a yellow stone if you have not

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been there first of all I recommend you

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go there but Yellowstone is in the

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northwest corner of the state of Wyoming

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and my field site is just below the

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norris geyser basin about five

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kilometers south along the given river

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and on the northern edge of Yellowstone

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Caldera here we have a face on view of

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my hot spring indicated in blue is the

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flow path of the main hot spring there

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are two satellite vents but I did not

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study those in this line of research and

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these two arrows indicate the sample

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sites from my Master's research one at

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the hot spring vent one about midway

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down the flow path and this is a little

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different from some of the other hot

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springs in Yellowstone perhaps some of

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the more famous ones this is a circum-

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tral ph hot spring and i like to say

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it's more of a warm spring because the

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event is about 50 degrees Celsius and

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cools off a little bit midway down the

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flow path and like I said circum neutral

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very slightly acidic at the event this

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is a result of hydrothermal acid

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sulphate groundwater is mixing with

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Lucille ground waters and you get the

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slightly acidic vent water and up in the

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corner just some of the properties of

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the water there's a lot of ferrous iron

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in the swatter near saturation with

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silica and there's also an appreciable

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amount of sulphate and this will come up

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a little bit later so just go over

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briefly the initial study that I did for

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my masters so this was more of a culture

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based study on the main goal was to

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determine the potential for iron

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reducing organisms in materials taken

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from these two sample sites the first

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thing I did was a most probable number

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enumeration and this is a technique to

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get an estimated number of the organisms

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in in these communities and following

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that I did a series of enrichment

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cultures and in these these cultures

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they were able to sustain their level of

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activity for more than 30 transfers well

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over 400 days and I also did some 16s

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and metagenomic microbial community

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analyses and this identified well known

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and potentially novel dissimilatory iron

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reducing organisms so here we have the

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activity for the cultures for like I

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said well over 3d transfers well over

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400 days they just passed their

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thousandth day of existence little while

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ago so I'm really excited about that

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that I've kept them alive and happy for

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this long and like I said there's this

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sustained level of activity call it

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about 30 millimoles per liter of ferrous

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iron being produced and if you're not

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familiar with metagenomics don't be

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scared by this this is the one of the

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meta-genome accrue salts from my vent

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community this picture here each one of

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the colors represents a different

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organism and the shapes don't really

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mean anything but they all clustered

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together each of those dots clustering

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together those are all part of all the

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DNA coming from one organism so this is

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perhaps more intuitive way of looking at

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it these correspond it's a way of

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teasing apart the number of organisms

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that you have in your meta-genome and

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from the meta-genome identified eight

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taxonomic groups call it eight different

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organisms that are dominating this

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community four of these I was able to

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recover nearly complete reconstruct

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genome so I have almost the entire

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complement

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DNA from those organisms and for four of

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those I had hits for the PCC porin what

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is this PCC porin PCC stands for poor

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and cytochrome complex and this has

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recently been identified by liang sheet

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from pnnl as a gene complex that has

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been found in all geobacter species

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which are well-known iron reducers and

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also in some other species one in

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particular ignazio a curium album if you

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haven't heard of it that's okay but what

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is cool about that organism while it has

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not been implicated as an iron reducer

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this guy here miele re back to roseus is

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a relative of it a cousin of this

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Ignacio acti reham and I identified this

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organism in my enrichment cultures and

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it also has this porn cytochrome complex

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which is exciting because it has some

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implications that it's involved in the

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iron reduction cycle moving on to the

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current study so the main goal here is

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to look at the Institute community my

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previous study was looking at cultures I

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want to know what's actually going on at

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the hot spring so this is looking at the

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using the endogenous electron donor pool

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and again determining that iron

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reduction potential and once again

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characterize the microbial community so

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far looking only at 16s sequencing again

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we have hot spring here is the flow path

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again and these symbols in red are spots

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that we took core samples this was from

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summer two summers ago six spots along

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the flow path here we have some pretty

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field shots here's the flow path from

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the top one of the core samples we

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collected and then this is the pool that

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forms at the top of hot spring so

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there's a couple different treatments

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for this experiment there were I added

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there were treatments with and without

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additional electron donor and treatments

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with

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molybdate edition was to knock out any

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sulfate-reducing bacteria which might be

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contributing to the amount of iron being

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reduced from sulfate producers

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by-product sulfide which can chemically

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react with iron here's the results from

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that set so this is site 1 that means

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the hot spring vent site 3 is the third

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core site which is about 2 meters away

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and the main takeaway from these these

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figures is within without additional

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electron donor and with and without

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molybdate there's not a whole lot of

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difference at site 1 however you move

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just the short distance way the purple

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and green symbols are the ones that were

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amended with additional electron donor

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and there's a much increased level of

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activity but again there is not much

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difference but with with and without

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molybdate so the takeaway from that is

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there's not a significant influence from

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sulfate reducers this is another measure

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of microbial activity this is the

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measure of fe 2 over fe total the more

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fe 2 that's produced the more active

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they are so once again you have a high

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level have activity at the vent

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diminished you when you move a little

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bit away and at site five there's almost

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no activity so what does this mean we're

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still interested in where the electrons

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excuse me where the carbon source is

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coming from this is by no means a closed

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system there is plenty of influx of

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other plant and animal debris flowing

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into the system but you see this

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decrease in activity and my thought is

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if the only source of carbon is this

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exogenous material coming in you would

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see a more similar level of yes in

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similar level of activity throughout hot

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spring but you don't see that so to me

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this would suggest that there is a

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source of carbon coming from the hot

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spring vent and to support this

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from the core samples that were

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relatives of sitter or accidents which

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is an autotrophic iron oxidizer so

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there's good indication that this carbon

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might be being produced in situ so the

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vent is the hot spot of activity so

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comparing these different communities

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this is a principal components analysis

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plot each one of these symbols

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represents an entire microbial community

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and this is a way of very quickly

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looking at how related all of these

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communities are so in blue we have

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everything from site 1 again this is the

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hot spring vent this is the t0 of my in

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vitro reduction assay this is day 20 all

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of those treatments cluster very very

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closely together this would mean that

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there is not much difference between the

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microbial community and what it also

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shows is that activity was stimulated by

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the addition of electron donor but that

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the microbial community was not changed

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as a result of it and I would also like

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to point out that within these

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communities there is also that organism

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I mentioned earlier eggnog bacterium i

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did get hits for this organism in here

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so again this is all leading up to what

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is potentially the in situ iron reducer

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so my my latest line of research is a

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stable isotope probing experiment this

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is using carbon 13 labeled acetate to

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probe the microbial community and again

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look for that active those active

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organisms so this looks fairly similar

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to the results from my insta to their

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excuse me in vitro reduction assay the

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activity is stimulated by the addition

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of electron donor but in green here

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there is still an appreciable amount of

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activity without additional electron

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donor and some conclusions there's an

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active community at the vent

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um the community appears to be electron

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donor limited within a short distance of

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the vent there are organisms related to

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a dissimilatory iron reducers that I

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identified in my enrichment culture and

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I did not have time to go into the iron

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isotope fractionation but we are also

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looking at that to see if there is a bio

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signature of the microbial activity with

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distance and depth from the vent and

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that thanks to everyone who funded me

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and organized this conference and with

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that we'll take questions okay we have

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time for a few questions em okay last

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month I was in Yellowstone and I saw

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warning signs all over Hot Springs and

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how acidic they could be and why why

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they are so a city that you cannot even

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touch them uh yeah so like I said with

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my hat spring it's a little anomalous

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from the rest in Yellowstone but yeah

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the extremely acidic ones that is

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there's a lot of sulfide in the ground

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waters so I think that's probably the

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yeah yeah sulfuric acid is probably the

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most of the source of acidity and hot

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and you want else okay um I guess we