Biodegradation and Bioremediation of Organic Compounds by Lawrence Wackett, PhD

Biodegradation and Bioremediation of Organic Compounds by Lawrence Wackett, PhD

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00:18
good day I'm here to talk to you about bio degradation and bio remediation and I'm going to tell you why microbes are so successful at this why they are used in environment and how we can engineer them and systems to make them even better at what they do so first about bacterial properties that makes them good for bio remediation first of all
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microbes are everywhere on Earth so they're in soil water air and even in the deserts where you have very hot conditions very dry conditions they can live in the cracks and in fact if you see if you look in the desert and you see the color on rocks often that's due to the pigments of bacteria that are living in the crevices of the rock the other point is that bacteria are present at enormous quantities on earth they are really the most successful living things
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on earth so one way to look at this is just sheer numbers so it's been estimated that there are ten to the 31 prokaryotes on earth now one way to calibrate thinking about that is say to compare two grains of sand well that's not even close the number of grains of sand on earth is 10 to the 18 so let's assume that every planet in the Milky Way galaxy has a planet around it that has sand like Earth if you took all 10 quadrillion planets in the Milky Way
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galaxy multiplied that by the grains of sand on earth you'd have ten to the 31 so that's what you have to do to get to a number that large which is the number of bacterial cells on the planet now I want to give you an example of how microbes they're numbers that presents have where can really make a difference in terms of bioremediation in this case not engineered but naturally occurring by remediation so 2010 there was enormous
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oil spill off the coast of the United States at an oil platform it exploded and the oil was spewing into the Gulf of Mexico so this was something that people were very alarmed about and many people thought that this was going to cause environmental distress for years or even decades and in fact a year later there were many beaches that looked very clean and the water was much clearer so what happened well it was figured out that
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there were bacteria in the Gulf of Mexico that were naturally good at eating oil some because there were many oil seeps in the Gulf of Mexico so they had gotten adapted and evolved to handle that oil to eat that oil and then over time they in really a very short time they proliferated and ate a great preponderance of the oil that was there so we're interested in this how do they do this I mean oil does not seem very
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palatable to us but to bacteria they in fact some of them will adhere to the oil droplets basically eat the alkanes the single ring aromatics the polycyclic aromatic s-- and and they do this very efficiently and as I said this is what might look like under a microscope and in a light field image and as I said this is what many of beaches in the Gulf of Mexico looked like because of this
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enormous capacity of microbes to eat huge amounts of material so we'd like to use this so one way that humans use this is to study the biochemistry the microbiology of the organisms that that eat these both natural products like oil as well industrial chemicals and a lot of that information has been compiled over a long period of time and and we did this
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working myself and professor Linda Ellis that started eighteen years ago or nineteen years ago now the biocatalysis biodegradation database so we compiled information not just from research here at minnesota but from all around the world many researchers over many decades to better understand how microbes will degrade all different types of organic chemicals and this database has now been
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taken over and it's now maintained and Zurich Switzerland where it's called the air bog biocatalysis bio degradation database and the database is continuing to develop and we're very happy to see continuing developments and we hope this will continue for many many years now there's another feature of this database whereby we can use the accrued knowledge for many years of
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biodegradation research to then extract rules and knowledge that can be used to predict the biodegradation of chemicals that have not yet been tested now this is important think about chemicals are being new ones being made all the time by industry for all kinds of uses as pesticides drugs and and we would like to know what their fate is in the environment so we use this pathway prediction system that we've developed and many people thousands of peoples
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have used this around the world and let me show you a little bit about how it works so generally the user will enter a compound they can draw this with a an easy drawing package and then press go and the system will start predicting putting rules on to the structure and then predicting biodegradation reactions and then string those together to make pathways we've published extensively on this if you want
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more how this works you can look up some of the many papers that have been published on this now just to give you an example of you know how this is used in in one particular example I'm going to show you is with the chemical atrazine that's shown on the next slide in this case it's a chemical that's used in agriculture to kill weeds and you can see there's a lot of wines going and from different metabolites so a cuisine is at the top the parent compound then
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it makes can make various products and those in turn can make products and you can see that these these pathways are overlapping so it can get quite complex but we've been able to also put priorities you see the green lines the green lines would be the most likely pathway is based on the current knowledge so if you follow the green you'd see you just go down one pathway and and that's a pathway that's been
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well established in nature so the prediction system works quite well so let's see what what has been known from experimental work so this is computational prediction which could be applied in this case to its it's applied to a cuisine a known compound for bio degradation but it can be applied to millions of chemicals even ones that are nearly underdeveloped and and so it's used a lot by industry by government regulatory agencies to get an idea of
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the fate of chemicals in the environment let's see how a cuisine is known to be degraded this was worked out first by Marvin de sousa working in my laboratory and he showed that what bacteria do is start starting with atrazine they remove the chlorine substituent to make hydroxy atrazine that's shown here and on the right and then that in turn undergoes another hydrolytic reaction to remove
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the end the group to make this compound and isopropyl amyloid and then you see that that further reacts it was shown by Mervyn another enzyme a TCC that makes cyanuric acid and then Mervin also Betsy Martinez working in the lab in Jennifer sefa Nick showed that cyanuric acid is further metabolized ultimately it goes to ammonia carbon dioxide so the
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molecule is completely degraded now we studied this further at the biochemical level so we we looked at the first reaction purified the enzyme that's involved we've subsequently gotten the x-ray structure for one of the enzymes that does this dechlorination reaction of a cuisine two hydroxy atrazine and that's shown here the this is what you're seeing as a dimer and the highlighted is the active site region when you see the color and so we've
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learned a lot about how the the reaction works what are the key residues in the reaction so this really helps us if we want to use these enzymes as I'll show you to do by remediation to know much more about their structure their stability their their mechanism of action what might inhibit them and so forth so here's an example to where we had could define the active site this this shows you the bind bound atrazine
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analog in the active site there's a zinc atom that's nearby the zinc activates water and it's this is the water that attacks the substrate and leads to the production of hydroxy atrazine so by using these kind of tricks of using substrate analogs we can learn a lot about and then the x-ray crystallography we can learn a lot about the function of these enzymes and to be a better able to
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to use these pretty effectively alright now how can this be used in the environment how can be used in by remediation well it turns out that hydroxy a cuisine is considered non-toxic it's not regulated so even just a bacterium that can do this first reaction taking a cuisine two hydroxy a cuisine can be valuable for bioremediation so let me show you how we use that so in this case after zine is used often in cornfields so it's used to
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kill weeds and you can see this is a very wet cornfield if it rains early in the year when a cuisine is applied you could potentially get some running off the field into streams and rivers it might make its way to municipal water treatment plants and then we felt that we could use then this atrazine degradation mechanism in a biological system in an engineered system in beads that would then be put
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on to a sand bed filter the water then this is something that's in normal water treatment plants very common and so as the water goes through the filter it's also going through the beads and that's it's passing by the atrazine is degraded and so it's removed and then it's not present in the drinking water so what about these beads these are you know what are they these are not naturally occurring this is where the organism and
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the enzyme is is put into this bead so that it's not released and also it's preserved in a more stabilized form but how do we make these so let me show you what these beads are so the beads have bacteria inside them and it's made of porous silica so this is a in the middle as an electron micrograph that shows you this kind of porous spongy structure with the bacterial cells that are
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trapped so this has been broken apart normally the bacteria completely surrounded by the silica and so they can't get out but what chemicals and water get in the water then gets cleaned and we published again on this you can look up some of our several publications if you want to learn more about how we make these but I'll just tell you briefly how we make these because remember the bacteria are trapped inside they're not just stuck onto the surface so how do we make a
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structure like this well it turns out it starts out it's like it's a liquid so we make it when we make it into a gel so this would be like making a certain kind of cake right where you have a liquid material that you then cook and make it into a gel food that you might eat in a way same way we have now these silica precursors these silica and alkoxides we mix with the bacterium we may also mix some other materials polymers and
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then that liquid gels in the system that we make and then the the polymer annex in the sense forms around the bacteria so the bacteria get trapped or encapsulated within the matrix and then it can be used now the difference between the cooking example and our manufacturing example is that the cooking has to occur at high temperature and of course we want to preserve the enzymes so we use materials that we can
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encapsulate the bacteria at an at a room temperature so it's a very gentle procedure which is essential now for for keeping the organism alive all right let's see another way an application another way we can make materials like this but first let's let's say just I'm just going to tell you briefly the the values of making this type of material one it separates the organism from the environment it protects the the bacteria
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against releasing so we want to keep them contained we don't want them to to go through the filter and get diluted it allows storage and transport so we make beads there they're dry they can be shipped in containers and we've done this two different sites you provide stable biodegrading material than for four different applications there's many manufacturing options we make beads but we've also made wafers
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and fibers as you'll see and and also this material the silica material is fairly inexpensive so we can make a material that's that's useful for my remediation but but not at a very high price which most people don't want to pay they want to clean up an environment but they usually want to pay the the least amount that they can to get the job done and this helps to do that now as I sort of indicated that we make an
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another material so this is more like something that you might see this is called cotton candy some of you might have you know eaten cotton candy it's it's a basically of a fiber of sugar and then it's usually has some flavour and colorings in it and in the process of doing this there's a middle spinner that then basically jets out the sugar solution and then it forms these
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fibers well we can actually do something similar with the silica material in bacteria I'm going to show you that here on the next slide there's light microscopy images on the left or actually in the upper right I'm sorry and then the others are electron microscope images you can see how the fibers are very thin they're really nano fibers that then go out around the bacteria that get encapsulated in the
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upper right is a light micrograph and that's showing green fluorescent bacteria GFP that is inside the cell so it's now fluorescent and allows us to very nicely image the bacteria and there's sort of grow glowing green color and you can see that they're stacked in the fibers we can get a pretty high density now of bacteria in the fibers so these belike bio catalytic filters you can use these it almost looks like a cotton knee
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mesh and you could pack this into a filter cartridge you could have it for handling gases that are coming off through the filter for example or liquid so that we think there are many applications of this type of material as well and so this is something we're actively working on to promote bioremediation applications all right so this illustrates one of the benefits of
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the encapsulation process here we have bacteria in the bread with free cells you get a good activity but after about three weeks or so the cells are licen and you can you largely lose activity but you can see with the black points and the curves that in the beads the the catalyst in this case the atrazine degrading enzyme stays active for a very
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long period of time in this case four months which is about as long actually as the student doing the work took time points so you got bored after that and you can see also the top graph is at room temperature and the bottom is at four degrees centigrade and you can see that the bacteria in the beads behave and perform better while they're inside the beads and we think that this is because when the cells are encapsulated
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they crack open a little bit and they become more porous to the chemical in this case atrazine so they actually show enhanced degradation rates so this is an added benefit of the encapsulation process all right so I'm just going to tell you about a newer application of how we want to expand the use of these materials and to biodegrade a much wider class of materials so
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this just illustrates another spell so chemical spills are hopefully not common but they do occur and you know when they do we really depend on bacteria to clean it up either just bacteria and natural ecosystems if they handle it or in some cases we have to intervene and engineer systems to clean up the chemical in this case it was a coal cleaning chemical this happened in the u.s. in the state
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of West Virginia the structure of the chemical is shown and it just illustrates that this can be really damaging the the the local waterways were closed that people couldn't shower they have to bring in bottled water and so we'd really like to be able to very widely predict the biodegradation of chemicals something I told you a little bit about but also then to be able to very quickly engineer systems that could handle
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chemicals based on those predictions and let me tell you briefly what we're doing about this area so the idea would be to use something like the prediction system to in this case predict pathways but also to we're taking that further so that we could select the ideal enzymes and microorganisms that would that would degrade those chemicals and then we could put those into silica microfibers that then could be deployed and with predictions with computer work with a
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lot of knowledge built up ahead of time this is the kind of thing then we could make a prediction and make a treatment agent very very quickly and that's the goal to be able to do by remediation much more effectively and much more quickly so part of this is to understand how different chemicals is degraded and to do that we're looking at the structures of the many biodegrading enzymes to see the range of chemicals that they will biodegrade so this is one
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such chemical or one such enzyme naphthalene dioxygenase and you see the chemical naphthalene bound in the active site and this is possible due to the work David Gibson who first purified and studied these enzymes and s Ramaswamy who deduced the x-ray structure of these this enzyme and a number of related enzymes and so from there from the knowledge that's generated on the structure and the mechanism of these
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enzymes we can use now software and doct other chemicals into the active site that might fit and react by getting near the iron and then also thus that's the site where oxygen would bind and be activated for reaction with these different aromatic molecules so in fact the Nobel Prize in Chemistry was awarded last year for this type of work due to basically use computational methods for predicting chemical biochemical
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reactions so it's it's it's considered a very important tool that is being used much more widely now in chemistry and biochemistry and we seek to use this from biodegradation so this you see another chemical docked in the active site now it has the thinnest three rings instead of the two of naphthalene and this fits in and we would predict it gets oxygenated which it and it turns out experimentally that it does well what about if you now have a fourth ring
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well it turns out that would also fit into the active site of methylene dioxygen ace and would nicely get oxygenated so we can use sort of the go-between of computational prediction experimental validation so that we can over time really greatly extend the use we feel of computational predictions let me show you some of the experiments that have been done to complement this work for example with this enzyme naphthalene dioxygen ace we just published recently
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that naps on ethylene dioxygen ace will work on all of these aromatic ring structures many of them shown here in in this graphic so it's a very broad specificity enzyme it has a very nonspecific hydrophobic active site and there's quite a number of bio degradation enzymes that are like this and a number of them have x-ray structures so we feel that this approach with naphthalene dioxygenase can be greatly extended in
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order to do better bioremediation in the future to do it in a much more predictive way and to very much more quickly when there's a spill when there's a need to treat a certain chemical that we can very quickly assemble the existing knowledge make computational predictions and then go and and do something about the situation very very quickly so so that's a lot of our research now and where we think it's heading so why is this important well I
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think many of you know that water is B you know clean water especially that for drinking water for agriculture for industry for all applications is becoming harder to come by so one really beautiful way to look at this this is from the United States Geological Service website that what this person did who made this graphic was to basically calculate how much water there is on earth and then make it
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into spheres that would be proportional now to the actual amount of water and then to place that on a globe so you can really get a sense of how precious water is so you can see that the largest sphere is all water on earth completely including all the ocean water the smaller sphere in the middle is just the fresh water and then the smallest sphere which is very very tiny on a global scale represents the all the water
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that's in rivers and lakes all around the world which are the major sources of water for for many of the seven and a half billion people in the world and we need to keep this clean right we need to keep this in a state where humans can can use it safely and so I think that bioremediation is going to be a very important tool it has been in the past and I think it's going to be even more important in the future to keep our water clean not only for ourselves but for our future
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generations and just to leave you with the last thought about bioremediation is that I like to think about microbes as our best friends in this regard that by removing unwanted chemicals from the environment and I'd like to make the analogy here to the to the human immune system so many of you know that that in our bodies we produce immunoglobulins that circulate in our bloodstream and protect us so by going around and
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binding to antigens and recognizing foreign things that come into our body they can protect us from viruses and pathogenic organisms and this is very important to our health and I think just the same way if you think on a global ecosystem scale that that bacteria are everywhere as I've indicated they're huge numbers so they're in in all of these niches and they've acquired the ability to degrade not only natural
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chemicals but many of the synthetic chemicals that humans have devised and so in the process of doing this they keep our help keep our planet clean so we if we have spills we have discharges of chemicals bacteria are doing their normal thing and metabolizing them but they're doing us a big favor in doing so so just to conclude I'd like to acknowledge a lot of partners in this work also I have a disclaimer here the
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university that I work at University of Minnesota requires that I let people know that I have formed to start up a company with a colleague Alexson and so we do by remediation so many of the things that I talked about today have commercial interest although I've talked about general knowledge and bio remediation that are things that we publish on and we totally public about everything that we
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do in that regard so al al axon is a major collaborator his laboratory has worked and developed the silicon capsulation technology that we use there are many postdoctoral research associates that have contributed to the work on silicon capsulation and atrazine also graduate students some of whom I acknowledge during the course of the talk for their important contributions on atrazine bio degradation and also the
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funding sources that are important to make this all possible so with that I'll thank you very much and I hope you will be very interested in biocatalysis bioremediation and maybe conduct your own research in this area and good luck to you in your future endeavors thank you very much

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