🦠Unlocking the Potential of Microbes: Dive into the world of microbial innovation with our latest YouTube video! Explore how microbes are revolutionizing the circular economy by integrating plastics and converting lignin into bio-based plastics using cutting-edge synthetic biology techniques. Learn about the transformative role microbes play in sustainable solutions, shaping the future of eco-friendly materials
📣Speakers:
Dr. Jose Jimenez- Reader at Imperial College London. Department of Life Sciences
🧪Title: Microbial integration of plastics in the circular economy
Prof. Tim Bugg – Professor of Biological Chemistry. The University of Warwick
🧬Title: Microbial conversion of lignin to monomers for bio-based plastics using synthetic biology
#Sustainability #bioplastics #bioreactor #environment #lignin
@GreenERAHub
Good morning ER everyone Welcome to The Greener Hub webinar series my name is Evelyn suniga and I’m one of the communications officers for the greena Hop project uh for those who haven’t been here before uh The Greener hop project officially starting in September 2022 as a coordination support action of
The European Commission on the horizon Europe program it currently represents 15 aets co- funds and self- sustained initiatives in the field of Agri food and biotechnology H we started this breakfast Series last year in October H addressing the topic of circularity in agriculture H if you’re interested on
Those topics related to mixed farming systems mitigation of greenhouse house gas emissions sustainable farming H circular Food Systems among others all our recordings are now in YouTube and now and for these three months we are going to focus on a different topic which is going to be biotechnology to advance Sustainable Solutions for
Biotechnology and here we’re going to have topics related to plastic um degradation and production of chemicals in a sustainable Way Green chemicals biofuel production protein engineering among others and today we’re gonna have two panelists Dr Jose Jimenez and Professor Tim bug we’re gonna open our presentation with h Dr Jimenez who is
Currently let me share one second my screen um this um Prof Dr himz is currently a a reader in synthetic biology at the at Imperial col College London his research focuses on a number of topics at the interface H between synthetic biology and evolution his group is interested on understanding
How Evolution shapes H the properties of protein exhibiting in quantum mechanics in VI in trade-offs for governing gene expression and in the D dynamics of complex microbial communities today Prof Dr Jimenez talk is about is microbial conversion to leing monoverse for biobased plastic using in synthetic
Biology H thank you very much H Dr Jimenez for accepting our invitation to present your project H I’m going to H stop sharing my screen and and the floor is yours and you can you can start showing your presentation thanks so much okay yeah thanks a lot thanks a lot for the
Invitation I have to say that I think you’ve got the titles wrong because mine is mine is about Plastics oh I okay I’m so sorry yes it’s it’s all right I have my title here so I’m sorry yes I’ve mixed up okay microbial integration of Plastics in the secular economy yes
Perfect that’s it yeah thank you very much it’s a great pleasure being here today um so something that you that I probably need to let you know is that we got a project awarded by the erob biotech one of the the second call of the aob biotech office actually uh in
Which uh what we were uh proposing to do is to use microorganisms to try to valorize uh plastic waste so I’m today as the project coordinator here presenting some of the work that we did so give a summary of the pro and highlighting some socas some of the
Results that we got so it’s a project that started at the beginning of the pandemic and early 2020 uh and finished uh last year so around spring uh depending on the partner or so of 2023 so uh in the project we had five Partners so it’s one of a relatively
Small uh Consortium for a European project uh apart from US based in Imperial College London we had Partners in Germany in aen and and leig um and then we had Partners um at the University of Valencia in Spain as well and a company based in France and they specialize in the production of
Polyurethan for for different applications um so the overarching idea of the what you have on the screen at the moment okay uh so we wanted to Target Plastics and then use microorganisms either whole microorganisms or enzymes to break down the Plastics into constituent monomers um and then I’m not sure if you
Can see my uh my pointer now um and then uh the idea is to take those constituent monomers and either use them as they are for making fresh plastic or transform them by using in engineer microorganisms again into molecules with a certain other value in our case we looked for bifunctional
Molecules silic polyols Etc that could be put together or combined with different chemicals in order to make new materials and the material that we were U the materials a family of materials actually that we were targeting are polyurethanes again because of the interest of the company and we call them
Biop polyurethan because some of the constituent monomers come from renewable or bio renewable sources and they can potentially be biodegradable all right uh the Plastics that we target for hydrolysis that we targeted for hydrolysis are pet and mainly polyamide based polyurethanes uh for the purposes of today’s talk I’m going to focus mainly
On pet uh so pet as you all know is a polyester of um talate and an ethylin glycol those are the con between monomers now there are two main ideas in this project that I want to discuss separately one of them is the idea of up cycling so we take
Something and then uh we con convert it into something different uh that could have a different functionality uh the other idea is uh what we call Pet trophy is the breaking down of pet by using uh microorganisms and then they have both of them are challenging but one is more challenging
Than the other so I’m going to start talking about the easier of these ideas which is um the easiest of these ideas which is the idea of up cycling uh that you have Illustrated here as I said we take something that is been used for one particular purpose and then we
Convert into something with a possibly a different functionality so we give it a second life okay um so this is something so the idea then is to take these uh monomers that come from pet degradation talate and ethylen glycol and then transform into something else uh and
This is some work done by uh two people in my lab Dr bank and Dr Abdul mutalib um and you may have seen some things like this in the news it’s it’s all of been used constantly um the idea is basically to engineer microorganisms to use draal mainly and then make all the
Molecules of value uh what we decided to do is a molecule called violing that you have on the screen right now uh we like it a lot so it has anti-tumoral and antimicrobial properties but uh what we like of it is that it’s purple so it’s
For us is super easy to screen if something’s working or not and and what Alice and Umar did in this project and actually most of the results that are going to show throughout the presentation come from the work that they’ve done in the lab and what they
Did is to plug the Biol lasing pathway that is involved um so the the genes needed for transforming um for allowing microorganisms to produce bioline into different strains of pseudomonas we like ponas because uh they’re very good at degrading different types of molecules and in particular
They can grow using teral as the S carbon SCE um and then when those gen are transfering to the microorganism then they accumulate they start accumulating this purple molecule you have some additional results here now um there are different strings that we can use we went into the environment and we
Actually isolated a few many of them happen to be to the Monas sorry I don’t know why the slides are switching all the time um and and what you can see is that um basically when we have the violation pathway in the microorganism we can see a decrease in teral
Concentration that correlates with the accumulation in in the molecule of value in particular this this PES uh is particularly good at at doing that um okay that’s good you you you may have seen other experiments like this in the news as I said people have made vanilline uh and they’ve made all
Different things coming out of talate or coming out of what comes of of the products of pet hydrolysis uh some of our partners in the project uh in particular Dr Tio and Professor plank in aen uh and their team they’ve demonstrated that it’s possible to turn talate into one of the target
Molecules uh like hydroxy alcano acid so this was one of the target molecules of the because they can be incorporated for for making different types of Po in the structure of different types of polyurethanes um and once again you have an example the green line here is the
Formation of this molecule and then you can see that both substr terest and E glycol go down uh this process has been improved throughout the period and now it’s been scaled up and we can get much larger quantities of of this molecule this is just one of the examples we
Managed to do a few of the others um this is the biopolyurethane that can be made incorporating these hydroxy alcano acids and you have one example here um this is now at a scale that is not very big but it’s big enough to test the properties of these materials other
Molecules that can be produced uh are buttin diodes in particular one4 uh this is used commercially already for the synthesis of standard polyurethanes um for making insulation panels actually uh and we we are trying to do is to replace the buttin dial that comes from from pet from oil uh and
Replace it with the bio version that we can obtain from recycling Plastics um other molecules that we obtain in the project like different types of butenes like two and three uh are interesting and so Prema uh in collaboration with the University of Strasburg uh checked the properties of
Some of the resulting materials and it turns out that they are very specific so depending on which is the isomer of this butin dial that we can incorporate into the structure of the material we can have pure things that could be useful or that are complete throw away um are not
Useful at all um and this is uh kind of ongoing research um that we are very excited about okay um as I said even though the upcycling side of um working with with plastics is uh fible and there are several examples that have been you may have be familiar with already
Something that I wanted to discuss today is this idea of pety this is something else that we try to do in the per and here the what we try to do basically is to engineer microorganisms so they they could fit directly into plast with of plastic waste um and in particular we
Made um different we tried to make different ex of pseudomonas able to produce the enzymes that are needed for breaking down pads so that they can Feit on the monomers so today the only organism that we know of that is able to feed on pet it’s called idon
Sensis Uh it was isolated in 2016 is the one that you you’ve probably seen in the news as well because it also happens to be uh the source of the enzym some of the enzymes that we use for pet hydrolysis okay um so this organism is
Able to make the pet Tes and also some an enzyme called meas doesn’t really matter but instead of breaking down the polymer breaks down one of the o that make the polymer to release the ethyl and glycon Tate and it also has a a pathway for talate assimilation now the problem with this
Organism is that it’s it’s a bit picky it’s difficult to work with not really very good uh for not really tractable from a molecular biology point of view and it doesn’t grow very well so we we you know there are several reasons why we wanted
To make this process fter um so at the beginning of the pro we were very very happy because we were able to go into the environment and then scream for better enzy so we thought one of the things that we can do better is the hydrolysis of of the plastic so our
Collaborators in in Li led by Professor Zimmerman went into different locations including some composting hips and the composting beit is important because as you all know during compost um a high temperature is achieved so compost hips are a great source of uh thermophilic enzymes uh and in this case having
Enzymes that can operate at temperatures higher than normal is very good because they tend to be to do very well dealing with plastic waste so the higher the temperature the closer the polymer is to the something called the glass transition and it becomes more accessible for the degradation for
Enatic degradation so ther having thermophilic enzymes that are able to tolerate those temperatures is something that makes the plastic degradation better and again if you follow the news recently or maybe not that recently there are some industrial processes in which some thermophilic enzymes are involved in particular one called LCC is
The gold standard for for pet enzimatic recycling so our collaborators went into the environment they were able to isolate a number of uh genes producing lightly producing enzymes able to degrade pth uh a number of them and there’s one of them in particular I want to highlight today is called
Phl7 uh because it’s faster uh it more pet and this is a hydrolysis as assay using pet film as a substrate and what you can see in the blue bars is the phl7 outperforms the gold the standard of the field that is called LCC so this is excellent news and we
Were very excited about it and then the idea is very simple so well actually know before getting into that I want to show you something that you probably haven’t seen that is um the possibility so I want you to see how these enzymes uh perform in real time actually well
This is a um this is an accelerated video and what you can see here in this vessel is a clam um uh tray that you can get from the supermarket so sometimes when you go to the supermarket and you buy the fruit they tend to come in these
Um uh containers that have a a clam shell shape uh so this is one of those containers that have been immersed in this solution of salts uh some enzymes are going to be added to it and then we are going to check the hydrolysis in
Real time uh this is a video prepared by our collaborators in liik so that’s the plastic being hydrolized this is the time in hours so about a day after hydrolysis uh the tray has been completely degraded and then on top of that we can actually hydrolyze a
Second and what you can see is that basically dissolves completely okay I’m going to stop it here I mean we can try or our collaborators try different types of plastics not only the for Consumer trade that you can get from the supermarket but is just to give you an
Idea of how efficient uh these enzymes are in V okay now uh Beyond hydrolysis in vual that is what’s been done in Industry these days um we thought that there might be a case uh for trying hydrolysis in Vivo this is with whole microorganisms and part of the reasoning
Be behind that is uh that we also conducted some life cycle assessment of these processes and in particular we looked at what is the Environmental cost of doing this enzimatic hydrolysis in v and for that we take into account four processes so there are four processes in
Which we can divide that enatic hydris so one of them is the uh production of the enzymes what we call the fermentation uh another one is the process of hydrolysis itself and then there are some uh Downstream processes that we need to conduct in order to recover the monom from the hydrolysis
And we also need to take into account the energy or the sustainability cost of pre-treating the pet plastic okay now uh those are the four sections that you see in all these parts and I want to highlight a few of them so for example if we look at what is the global warming
Associated to all these processes what we can see is that the process of producing the enzymes account for about one quarter of it um together with the pre-treatment uh that’s about half of the cost uh we have some others uh in the land use for example um most of it corresponds to uh
The process of generating the enzymes that we need for the hydrolysis uh if we look at fre consumption um once again if we look at what is the cost of the fermentation and the pet pre treatment that accounts for close to half of the process again with
All of this what I’m trying to say is that there are this is already telling us that we could have we could make this uh process of enzimatic hydrolysis way more sustainable if we were to address some of these points and in particular what we thought of doing is if we were
To use whole microorganisms that can produce the enzymes in C2 in the presence of the Plastics everything should become way more sustainable because we could save lots of of this environmental cost especially the cost of fermentation um okay so um the idea is very simple so what we can do is to take
Our favorite microorganism and as I said we work a lot with Sedonas are very good for the removal of uh different molecules from the environment and in particular aromatic substrates and what we did was to plug to put inside them the genes making different types of enzymes uh and you
Have some examples here we have more actually but but just in these pictures where you are seen is a colony of Sedonas PDA that is able to express and secrete one of these enzymes and the colon is growing in uh on plates that have a pet nanop particles in them so
These Halos that you see around the colony corresponds to the clearance of some of that plastic okay so this shows us that these microorganisms when they grow they are able to produce the enzymes and the enzymes are active and are able to to break down the plastic
That is uh surrounding them not all of them are equally effective actually we have here negative control that is not supposed to do anything um these are thermophilic and this one is mesophilic comes from the sanis and what you can see is that the hydrolysis rate is very
Very low another way of looking at these results is a project that was conducted by Charlotte Bosworth in my lab is to look at the activity of these enzymes when they once they are produced and secreted into the superat um once again there’s no purification here all we did was to grow
The microorganisms take the supernatant as they are so they contain the enzymes that have been secreted by the engineer bacteria and then test those the activity of the enzymes present in those supernatant against different types of pet Plastics and we tried a bunch of them um including films prom this is the
Standard pretreatment that is done by in Industry these days for pet hydrolysis we tried some uh Brown pet that we had much smaller size we call the microplastics and then we also tried a pet um formulation in which the pet postc consumer pet films have been pre-treated in a specific way that I’m
Not able to share today because of uh confidentiality issues this is being considered for for patenting at the moment but just to say that is a different type of pre treatment that we give to pet and then we tested the activity as I said of those uh bacterial supernatant again those Plastics and
What you can see here is that out of all the possible combinations that we tested only the new pre-treatment produces a plastic that is really suitable for hydrolysis using the different types of enzymes that we have in here LCC is the gold standard for enatic recycling uh then we tried some some mesophilic
Enzymes including uh fast petas which is a PES that comes from Ella that has been optimized using artificial intelligence uh on our new phl7 that we identified throughout the project so we detected activity in some of the superans being the best one this phl7 in mesophilic conditions um again so this shows that
These engineer strings are able to produce Petes that are functional now um a different example uh so it’s great that we can grow microorganisms produce enzymes and then use them for initro hydris but the question is can we engineer microorganisms so that they can fit directly on the pl plastic and that
Be fantastic so that’s what we’ve been testing for quite uh some time now uh and you have some results on the screen here so we’ve tried a couple of things so the first one is use microorganisms that have been engineer to produce mesophilic enzymes so they grow at 30
Degrees okay uh so here what we are testing is whether the microorganism is able to grow on pet or not and this is all the pre-treated pet the one that is the most accessible that I presented in the previous screen and what you can see
Is that in the presence of pet we have a little bit of growth now this is a very tricky experiment because in addition to the plastic which is meant to become the carbon Source we need to help the microbes to grow a little bit previously so that they can accumulate enough of
The enzymes in the superet and so that they can produce the monomers and that they can the microbs can use carbon so we need to grow them in the presence of an additional carbon Source in our case we tested some of them the best results
We get with 10% of LV of Rich medium diluted into the minimal medium in which we have the plastic okay so a little bit of growth not impressive then we decided to do a slightly different experiment in which uh what we do is uh why don’t we take the why don’t we take
The microorganisms uh we grow them in Rich medium in the presence of the plastic so enzymes get generated the plastic gets hydrolized the rich medium eventually disappear because of the microbial growth then we purify those resulting cultures and we use them so we remove the cells and then we use
Them to inoculate fresh cells again to see if they’re able to feed uh on the resulting pet this gives us the advantage of being able to incubate the initial culture at a much higher temperature uh for a number of days so the way this this experiment works is we
Inoculate with our cultures in Rich medium they produce use enzymes so they grow normal let’s say uh then we take that culture and we increase the temperature to 65 degrees the original organisms that we have in the culture die because of the high temperature um and then after three days
We recover the superan remove the cells and then use the resulting medium to uh inoculate with a new mic with a fresh microorganism able to use the monomers and in this case what you can see is that after a few hours uh after a day or
So um we have microbial growth which is much higher than what we can detect um in the previous assay note the very different scale so here we incubate for three weeks here we only incubate for a couple of days or so or three days maximum uh and we see microbial growth
So the bottom line of this experiment is that it is possible to detect P trophy so microbial growth but the problem is is if we use the standard conditions that you would expect to achieve in the lab uh the growth is negligible if on the contrary we try to use conditions in
Which we promote the faster degradation because of the plastic because of the high temperature we kill the C at the expense of being able to produce something that can be used as a as a a growth substrate so it’s a bit more elaborated experiment uh but we can get
A better growth chills and still the growth is is not impressive what we had in mind when we started the project was that we were able to grow anything on pet and it turns out that is not that is okay um in addition to the experimental work in in this project
We’ve also explored the responsible research and Innovation side of the project we’ve been talking to lots of people conducting interviews with different stakeholders in the plastic production plastic recycling uh chain uh and including industry NGS Etc uh and we’ve realized that the there are some aspects of this enzimatic recycling of
Plastics uh that are maybe not as good as we thought they were uh but there are some other um Silver Linings in which we think these biobased approaches could have an impact so for example uh something that that is pretty obvious is that so far everything that you’ve probably seen in
The news and that presented today relate to pet uh so pet is the only enzimatic uh plastic recycling process that is at trl6 um obviously be great to have similar technologies that can Target other other polymers and we are working on that things like you know being able to degrade or hydroly polyethylene
Polypropylene polystyrene Etc um it turns out that pet is out of all the Plastics out there is the best the easiest one to collect uh the best one collected sorted and recycled by other methods uh so probably not the best example of use of biology so we
Have technologies that are very good at dealing with pet uh and we don’t think we are going to be we can do better than than those Technologies um part of the problem is that the scale of biodegradation is very small uh as you can see even though the
Hydrolysis takes place in a day the chemical hydrolysis even faster than that um in addition we know that pet trophy you know the growth that I just presented is is not great so increasing the sustainability of the process is is going to be hard as I said there are
Some Silver Linings as as well so pet might be well enzimatic degradation of plastics might be the only option uh for dealing with plastics that are actually not recovered so these Plastics that accumulate in the environment for example in wastewater treatment plants or even in environmental releases okay
Uh so maybe having enzymes that are able to degrade Plastics in the environment is the way to go um about this um even though the rates are not great the sustainability is definitely greater compared to other other process of recycling of plastics uh and in particular a very good selling point for uh
Biological degradation or recycling of plastics is that enzymes are very specific of of their substrates that they don’t really care too much about additives um so for example dealing with mixed waste or with plastics that are contaminated with additives Etc should be easier using uh biological approaches compared to other processes because
We don’t have the problem of having to care so much about uh possible contaminants okay with this um I just want to finish um and I want to thank everyone that took part in the project or the project partners and everyone that contributed you have them listed
Here if you wanted to get in touch with us you with us you have my email address on the screen as well as the project manager’s email address um we have a web page that I invite you to check uh we are on Twitter uh uh and of course I
Wanted to thank the sponsors for the opportunity of conducting This research and and you for your attention today thank you very much thank you Dr Jose Jimenez um I was just looking at some H questions that I have but I think you answered all of them um if people have questions in the
In the Q&A section you’re welcome to put the questions and also uh on the chat H I wanted to ask what is What is the involvement of the industry partner in the in in your project like what’s what do they kind of they take from which
Point yeah so they they were in charge of taking the monomers that we comp produce biologically and make new materials with them and and obviously test them um some of some of those some of these uh processes can be scaled up so that’s something that we were
Investigating as well uh but some others are uh still at a very low TRL so we haven’t been able to produce enough amounts of the of these building blocks so that they can be put together uh in order to make enough of the material to
Be tested but for some of them it works very well okay and then is there a way to so they already know like with the composition of the monomers if these monomers are going to be biodegradable or composed like the material will be compostable somehow uh so the rule of f is all
Polyamides and polyesters that they can make should be biodegradable polyesters are going to be much harder to to biodegrade but we we do not know um so it’s something that we need to test uh in principle they should be uh but we haven’t we didn’t have the time to test
That yeah okay and we have a a question here more questions here coming and so it says do you think that the enzimatic approach could be useful for treating pla based Plastics yes uh pla it’s an interesting problem because it’s solved as biodegradable but is it’s only well as
Compostable but it’s only industrially compostable okay so it’s compostable so it has to be collected first and then in industry and the specific conditions it can be compostable but something that we’ve been looking into uh pla is a polyester U it’s polylactic acid and and the some of the esterases that are
Active against other polyesters including pet are active against again are active uh also against pla so yeah enatic treatment of pla is definitely an option okay fantastic we have another question it says very interesting talk so much to learn about plastic biodegradation I have a question about the environmental and human impact of
Releasing the enzymes in the environment because in the graph with the cost of this technology the fermentation process presented a high carcinogenic impact for humans if I understood correctly uh not carcinogenic uh it was the so it was more like an environmental cost so that that life cycle assessment
We conducted trying to mimic the current industrial process of of pet hydrolysis okay um and out of the several compartments that we analyzed we realized that the enzyme production generates a high sustainability cost and actually even high economic cost because depending on the cost of oil prod using
The enzymes can be more expensive uh than making fresh new pet plastic out of oil okay but in terms of sustainability we what we wanted to see is whether there are some aspects of the process in which we can improve the sustainability by maybe addressing or trying to make
Those processes more efficient and in particular we think that the fermentation is one of them so it is true that if we wanted to if we wanted to make enzymes to treat plastic in the environment we would just need to produce them somewhere else uh through a fermentation process and then purify and
Then treat to you know in order to treat the Plastics in the environment so therefore the cost still is going to be high now the question is whether the sustainability cost is going to could be somehow compensated by the cost of removing microplastics which we know are bad for
For the environment and for health right uh that’s one important that’s I think is one important the other aspect is that it might be possible to reduce the environmental cost of making these enzymes by maybe for example produce them in situ um this could potentially involve
GMO so it’s it’s a different type of problem that I not that probably we need to have a separate discussion but one of the possibilities I guess is if we could have engineering microorganisms that we could use in a contain environment that could produce the enzymes on the spot
That would save the environmental cost Prov Ed that we are not able to release those those organisms into the environment and what I’m thinking and these are conversations that we are having for example with waste water companies is to try to to use some of the infrastructure that is already
Available for treating Urban waste water so most of the microplastics that that end up in the environment actually come from textiles from you know from from the laundry from the Wasing machines uh could we get rid of those microplastics by implementing this type of process in in a container environment so that we
Can kill the microorganism after so you know there are different aspects of the technology that are worth considering but but the point of that that part of the presentation was to say that making there is a lot of room for improvement especially if we address specifically the fermentation
Process okay yeah it’s there’s a lot of things to think about when it comes to like what different like problems we can face when we like okay yeah this GMO but yeah we have to contain them we have’t going to take this last question because we we’re going to have
To move to the next talk so it says what are the challenges to treat Composites can the filler interfere interfere with enatic activity so the short answer is with don’t no okay uh but in principle so in principle I would say it shouldn’t really be a problem I mean you need to
Think about how these enzymes or these microorganisms operate in the environment in which they’re meant so none of these enzymes has Evol to deal with plastics okay we can evolve them artificially or or for example the Petes that we have isolated so they have probably evolved to degrade other
Polyesters that are present naturally present in nature in particular some of the enzymes that we use we know degrade so are able to degrade cutin so it’s it’s a polyester made by plants um so but you need to think that in nature everything’s complex everything is a
Super complex mixture with lots of uh different molecules uh contaminating the others uh think about you know how plants eventually bi degrade or L biod degrades um and enzymes eventually you know they find the right substrate and they’re able to degrade it despite despite being in a super complex uh
Mixture of many different substrates so I don’t see why um having mixed Plastics uh could be a problem or Composites you know Plastics that have different actually none of the Plastics that we use actually is pure it has a blend of plastics different layers etc etc so I
Think one of the good things of biology is that they should be able to deal with with complex substrate okay great Dr Jimenez thank you very much for accepting and again I apologize for the mix up in the title um there are some other questions
In the in the Q&A section that you can like answer if you if you like so and now we’re gonna move to uh Professor Tim buck and thank you very much for for joining us Professor Buck I’m just going to share my screen uh for a second for a short
Presentation all right yeah sorry I was problems logging on yeah that’s no no no problem so one sec and professor professor Tim bug is a professor of biological chemistry at the University of Warwick and he research focuses on the study of bacterial enzymes for degradation of the leing
Biopolymer found in plant cell walls including metabolic engineering for the conversion of Ling into renewable aromatic chemicals and the enzyme involvement in bacterial cell um cell wall peptidoglycan biosynthesis as targets for the development of noble antibacterial agents H Professor um B talk today is about microbial bi microbial conversion of liing monomers
For biobased plastic using synthetic biology and this is part of me of the my mimo project and professor bug thank you very much I’m just going to stop sharing my screen have to find the button there you go and thank you very much for much for joining us and then you can start
Sharing your presentation thanks so much okay and this one yes can you see that yes perfect yes okay right good morning everyone so my name’s Tim bug from the University of Warick in the UK I’m going to talk about the uh the Milo project that I was coordinator for this is
Another era to biotech project and so I think you will all know there’s a lot of interest in trying to find renewable sources to make uh fuels and chemicals as Society we have to move away from using petrochemicals um to make um fuels and chemicals so one of the possible
Solutions for doing this is to use renewable plant biomass and I’m sure most of you will know about the the bior refiner concept whereby you can take plant biomass you can take the food part of the crop and then you can use the nonfood part of the crop uh which is
Essentially agricultural waste and convert that into fuels and chemicals so if you do a therm chemical pre-treatment on ligos cellulose you can fractionate it into cellulose hemicellulose and ligin which are the three components of plant biomass so the cellulose can be converted through glucose into ethanol bioethanol which is
Biofuel hemicellulose can also be converted into ethanol by different organisms or it can be fermented into organic acids um such as lactic acid succinic acid and for example lactic acid acid can then be converted into polylactic acid however lignin is the the big unsolved problem in the biorefinery
Concept uh it’s a very refractive polymer which I’ll talk about in a moment and in practice it’s often burnt for energy because it does have calorific value but clearly it would be much better if you could convert lignin into useful chemicals and potentially some high value chemicals I’ve shown a couple there
So why is lignin so difficult to degrade um so it’s a high molecular weight aromatic polymer uh it contains sorry it contains these arc3 units which are linked together via different kinds of linkages uh you get e this is the the beta arther is the major uh component that has an ether linkage
Here and then other uh components have got carbon carbon bonds carbon carbon bonds ether bonds so these are are not susceptible to hydrolic cleavage very difficult to break and it’s also heterogeneous um so it’s a mixture and that’s because in the plant is made via radical cyclization however lignin is uh the the
Biggest potential source of renewable aromatic carbon in the biosphere so if we could convert lignin then it’s a great source of aromatic chemicals across different plants there are small differences in substitution patterns So Soft Woods contain mainly G units hard woods also contain s units and grasses also contain each H units
But they all contain these kind of uh components so technology that works for one should work for other plant types so I haven’t got time to explain all the work that we’ve been doing over the last 15 years but one of the approaches is to try and use synthetic
Biology to engineer bacterial lignin degraders to break down lignin but also produce useful products and we’ve discovered several uh bacteria that can break down ligning I’m going to talk about two of them rocus josi and pamon ptia which last we also talked about and this project uh is in collaboration with
Biion bioplastics based in the UK and they are interested in making um substit substitutes for talic acid so talic acid as as you heard is involved in making uh P which is used to make drinks bottles all sorts of things um p is um not really biodegradable or or slowly
Biodegr rable biome are more interested in pbat which is biodegradable uh Al contains talic acid also butane dial and adipic acid so these other components can be sourced from renewable sources but talic acid comes from oil comes from oxidation of of parazine so they were interested in uh trying to make aromatic
DIC carboxilic acids from lignin so how are we going to do that uh so ligan degradation the pathways are not fully understood but we do know that one pathway comes through vanilic acid and protu acid and normally this is degraded via oxidative cleavage between the three and four
Position um in rocus and pseudomonas and other organisms down this beta keto adipate pathway and um my first collaboration with biome um we came came up with a a strategy I know that in there are other enzymes in other organisms that can cleave protu acid either between the 23
Position or the 4 five position here and that generates uh these extra dial Ring cleavage products and it’s known in the in the literature that if you treat those with ammonia or ammonium chloride they can cyclize to make Pines so therefore potentially we could make these pyodine dicarboxylic acids and you
Think okay great this looks nice on paper but it’s not going to work in practice well actually remarkably it did work um so if we insert the either the liab or the pr a genes into rocus ji using an inducible expression Vector then we see the production of
These compounds as bioproducts in use in a microbial biot transformation in seven to nine days so this takes time it’s hard to to break down ligan um but we saw Titus of about 100 migs per liter which is a quite exciting uh demonstration that this is possible and we published this back in
2015 so we need commercially if this is going to be feasible we need to make grams per liter multiple grams per liter and so we need to improve the tighter so by 2021 we’d improve to about 300 M Pita by knocking out the competing pathway in ricus
Ji um so the aims of the milimo project um firstly were to try and improve the titer in rocus josi by overexpressing lignin oxidizing genes uh can we make this organism better at breaking down lignin but also we interested in sud PDA so this organism can break down
Polymeric ligin but it really is better at degrading low molecular weight um ligin fragments and so the aim here was to try and engineer this organism to convert parakum aric acid which can be released from lignin or lignocellulose by an alkaline pre-treatment and to use this organism to make um paradine dicarboxylic
Acids and then finally to try and scale up this process to develop a a process that biome could use um to produce pdca so these are the partners uh in the project uh myself Eduardo Diaz uh at CSC in in Madrid and his Co cooworker Helena Stephanie bber at inra in
Versailes in Paris who’s an expert on liing characterization sorry I said Eduardo is an expert on on pudos PDA uh Ralph tor’s group at University of stutgart who’s an expert in biochemical engineering and Yan is it was his PhD student who worked on this project biome bioplastics was John and Amy from uh
Biome and uh Nova who also did some life cycle analysis on the process so we started in March 2020 exactly the wrong time to start so exactly when the pandemic started um and but we were finally able to meet uh towards the end of the project so I’m going to talk about Ricos
Jostii and then sud sudon PTA so the first aim was to try and uh improve the tier in ronus jostii by overexpressing ligin oxidizing genes so we’d shown in 2011 that this peroxidase dip B was involved in ligant degradation but also on the Genome of this organism there are three
Multicopper oxidases MCO ABC which we really don’t know anything about and they we’ve tried to express some of these they don’t express very well as recombinant enzymes so what happens if we overexpress the genes so we overexpressed each of these in rocus jostii and we saw improvements in pdca
Release so dip B gave us about a 1.4 fold increase but these multicopper oxidases gave us a 2.5 and 3.5 fold increase um which is interesting that the multicopper oxidases seem to be more effective uh in this kind of application uh than the peroxidase so
That gets our titer up to about 500 MX per liter which is an improvement it’s also interesting that they’re more effective than overexpressing um exogenous genes so we also have expressed genes from streptomyces uh and a mopis but using overexpressing the endogenous genes worked better uh so that was published
Recently the other thing that we’ve done in ricus jostii is to to see whether we can engineer this to also degrade cellulose so if we use ligna cellulose as a feed stock we’re using the ligning fraction but if we could also use the cellulose fraction then we could improve the Tighter and
We knew we we noticed that there are cobos hydrolases present in ricus ji and it can grow on celios which is the disaccharide and if we express um two different endocellulases in rcus jostii then that on an inducible plasmid that does permit growth on carboxymethyl cellulose which is a soluble uh cell
Substrate and also ricus jti to my surprise contains this dehydro shikimate dehydratase so I should have explained here the idea is to go along the shikimate pathway which makes aromatic amino acids and there is a link from dehydroshikimate to protu acid so this uh enzyme 3 dehydr chikate dehydratase
Which we were planning to import into this organism but in fact it already has it um so there is potential we haven’t yet managed to put all of these things together but there is potential to engineer this organism also to degrade the cellulose fraction um of lignocellulose which would improve the titer
Further so that’s rocus josi uh sudon PDA um Eduardo Diaz’s group wanted to investigate this um Gene called Pepe which was discovered in a in a previous project This is highly induced in the presence of lignin it’s induced about 20 fold in the presence of lignin and it’s
An extracellular peroxidase and it has its own type one secretion system which is quite remarkable uh it’s a very large polypeptide and we were hoping that this might actually increase might have activity for ligin oxidation and it turns out that this Pepe it’s a manganese oxidase so it can
Generate manganese 3 manganese 4 oxides which enhance the consumption of monoaromatics but it turns out there’s no change in the structure of the polymeric ligning this was done with uh Stephanie B’s group AD inra so an interesting enzyme although it turns out it’s not active on polymeric lignin but Eduardo’s group have also
Engineered um pudos ptito kt2440 to convert par taric acid into uh 24 pdca so this is also by knocking out the beeto adipate pathway the competing pathway and overexpressing this PO monooxygenase and resting cells then quite rapidly convert parakum Maric acid into 24 pdca it’s strange that P sudon PDA only
Does this in resting cells rather than in growing cells and there’s some evidence from Eduardo’s group that’s because this ring fishing product the CHMS seems to be quite toxic uh for pudos PTA but this process is faster than rocus jti so it has the advantage that if you can make aromatic monomers you
Can convert them faster into the pdca so the other thing we wanted to do on the project was to see if we could combine these two so can you use combine the ability of the rocus josi to De degrade High molecular weight ligdin and then the ability of P sudon PDA to more
Quickly convert the monomers into the pdca so to do a co-culture and this was done at uh University of stutgart so you can see that Pon PDA when you give it high molecular ligin um this was a an alkal um a soda ligin those of you who who know about different types of
Lignins you get a lower tighter of pdca uh because sudos PDA doesn’t degrade High molecular weight ligin so well but when we do a co-culture with rocus josi we can improve um the tighter of pdca production uh and then this one was with a third organism from my group that’s another bacterial ligin
Degrader and the productivity of production is improved quite dramatically uh by doing this co-culture so there is potential to use co-culture to um use the advantages of the of the two organisms together and then finally trying to do scaleup um this was work at University of stutgart so the padin Ty carboxilic
Acids turn out to be a rather difficult molecule to extract out of fermentation proc because it exists as a spitter iion so you can’t just extract it into organic solvent you can do ion exchange chromatography but that’s not great on a really big scale so they have this rather nice method called reactive
Extraction uh where they form a complex between the pdca and an extractant which is trioctyl amine and that allows the pdca to be extracted into oxonol and the pdca can then be released through um change in PH and that works nicely so we get about 95% recovery of the pdca and you can
Then crystallize that out um at low temperature under acidic conditions and they’ve scaled this technology up using a centrifugal extractor so this can be done on on a laboratory scale um and they have uh scaled up the pdca production at 1 and a half ler and uh 30 ler fermenters so we’ve we’ve
Improved the ability to produce the pdca at scale and it’s it’s really that up to to bio whether they want to um take this further I think the tighter is not quite high enough at the moment but we’ve certainly made some progress during the project and then biome are also
Interested in finding new markets for their products so just to mention they have a new collaboration with um company called sha green um using their Plastics so they currently they sell first generation Plastics which are starch-based Plastics uh but they’re biodegradable and so one application is as uh tree
Shelters which otherwise would just make litter in the environment so a new product is is on the market and you’re welcome to look at that website so in conclusion uh we can make padin dioic acids from lignins uh but there’s two different organisms um which have their advantages and disadvantages
So rocus jostii is better at degrading High molecular weight ligning um but it’s a relatively slow conversion and it’s also difficult to do the genetic modification and if anyone wants to ask me about that I can say more about that pudos PDA is much easier to do genetic modification um and it
Works well with uh aromatic monomers it doesn’t work so well with um polymeric liing um and we’ve made some progress in trying to develop um a scalable process for PDC production and um it was nice to finally meet towards the end of the project we met in vesi in in April uh
2023 and I’d like to thank uh EU for sponsorship and thank you for your attention thank you very much Professor bog it was very interesting talk and like these two talks really like blow my mind I like it I love it and we have a question here uh in the Q&A it says
Thanks Professor Buck for the intrinsic talk do you think ER that I ionic liquids might be an option for the pdca extraction could be yeah that I think that’s that’s a technology we haven’t thought of about I think um this was Ralph tor’s um group at stutgart and they they
Already had this reactive extraction technology that they wanted to apply um I think I’d have to ask him about that um there is interest in using iuid yet okay um but we haven’t tried it okay um you you were you mentioned that that the the genetic modification of Roc
Cocos is H challenging so in how how is it that interests to me why so it’s just hard yeah so whereas with PUD PTA you can do this in about a week and it works reliably and I’ve even had project students do that with ricus jti it it has a fairly low transformation
Efficiency you need to use a lot of DNA and it also has a low rate of recombination so use recombination based methods so you have to then use counter selection sucr counter selection and you have to just keep plating it and plating it and hoping um so there’s no crisper
Available okay in Ric caucus may probably one day someone will will work out Ric cus uh crisper in rocus but um someone who is in my group actually in Brazil she spent half of her PhD trying to develop crisper in ricus Jost ey and it failed yeah so
It’s just um yeah it’s a challenging organism but it does have some real advantages and then so talking about like designing and producing GMO um what is the policy landscape at the moment in terms of GMOs that are going to be um produced for plastic or kind of
Contained what is the regulation that they required no yeah it would have to be contained uh in in a fermentor but you can do that yeah so that can be done um okay so if you scale it up they can just be like there fermenting and yeah
Okay uh so that it can be done as long as it’s contained yes okay um I have a I have another question here um where is it so is there any um because like sometimes other types of microbes fungi or maybe protans can work in microbial communities to maybe degrade like in a
More efficient way is there like a a part of the project or other projects maybe working on on this kind of uh approaches like microbial communities to try to degrade Lan yeah so that’s interesting um and I’ve been involved collaboratively in one or two pieces of work so there is
There’s interest in the sort of microbial Ecology of soil uh and um so whereas there’s a big difference between fungi and bacteria here so fungi essentially basically a single organism that does the whole thing whereas in soil it’s very much a community a bacteria and and yeah it’s
Complicated um and there’s also interest in environments like termites wood eating termites for example um and you see some of the same sort of organisms appearing but also different organisms so there’s and it is quite hard to unpick exactly what each organism is doing so I think it’s for
Microbiology it’s it’s quite fascinating subject but if you want to use it for technology then you probably need to know what what each of your organisms is doing yeah because the if you use um communities fermenters they can change with time and so you can lose activity
And you don’t know why you’ve lost activity you know so you need to control it if you’re going to use it industrially okay we have another question here uh it says what are the sources from the Ling using your work and the molecular weight can you extrapolate you work to other types of
Ling like the sub products from paper industry so that’s yeah very question so there’s uh lots of different types of lignin and um there’s a big question really for us and for biome is what feed stock are you going to use um and you have to think very carefully about the
Life cycle analysis and the technoeconomic analysis and so we’ve we tend to use either uh just wheat straw Lig no cellulos which is just chopped wheed straw which works uh with Roc caucus or this um it’s a green value protobind lignin which is a soda lignin and the advantage of that for us
Is that it has some water solubility so one of the problems with liing one of the many problems with liing is that many preparations of it are not water soluble so if you’re going to do a a microbial conversion you need some kind of solubility in water so um yeah can
You apply this to other kinds of lignin uh maybe um so for example organosol legin has no water solubility so that’s a bit of a problem craft ligning everyone would like to use craft ligning because it comes out of the pulp paper uh industry craft ligning because of its condensed
Structure is more difficult to convert um now interestingly the rotor caucus does have some activity with craft ligin but we get lower tits so it’s putting all of these things together to try and come up with a process that is actually commercially viable yeah okay great there’s lots there’s lots of
Craft ligging around if if someone can crack that problem yeah yeah um okay so I think I’m going to close for now we just past 10 seven minutes H I would like to thank to Professor B and Dr Jimenez for two fantastic talks thank you very much for uh providing us great
Overview of your project is very interesting and I hope people in the audience learn like a lot of more a lot more about um linking and plastic uh degradation um thank you so much H for for my panelist and I would like to remind you all that H to register to our
Second Breakfast Club H which is going to be on January uh 25th at 10: a.m. H I will be sending the invitation uh to everyone and we going to have uh another again two panelists and Professor Frank delin who’s going to be talking about control of yeast H bacteria co-cultivations in bioreactors
And H Dr Alan Gard we’ll be talking about biotechnology at the cell membrane um and thank you very much everyone for your questions very insightful very interesting H and I’m going to H close this webinar here and thank you um my panelists again thank you very much bye bye see you in another
Opportunity thanks