ELECTROFERMENTATION : How to combine microbial electro-chemistry and fermentation principles to optimize the production of biohydrogen and biobased molecules from waste
Eric TRABLY et Nicolas BERNET
Laboratoire de Biotechnologie de l’Environnement, INRAE Narbonne, FRANCE
Nowadays, Hydrogen is considered as one of the most serious alternatives to fossil fuels in the transportation sector. The development of green technologies to produce renewable H2 is crucial to ensure the sustainability of these systems. The conversion of raw biomass or organic waste by biological processes is very promising as these processes present the lowest environmental impacts and hydrogen can be concomitantly produced along with valuable biobased molecules. However, bioprocesses are often based on mixed culture fermentation that presents several disadvantages such as a high variability together with thermodynamic limitations. To overcome these issues, coupling dark fermentation and bioelectrochemical technologies has been intensively investigated over the past decade and some examples will be here presented. By extension, a new method of bioprocess control so-called electrofermentation has recently been proposed combining the fields of fermentation and electromicrobiology. The fundamentals of electrofermentation, including interspecies electron transfer (IET) as core mechanism, will be presented as well as some experimental evidence of a better control of the microbial metabolic pathways towards the production of biohydrogen and other valuable bio-based molecules.
thank you again so my name is Eric try so I’m researcher at the laboratory of environmental biotechnology in France in nbor and uh I would like to thank the organiz organizing committee and a for having invited me to present our work about electr fermentation so I’m not going to talk about um micropolitan remediation Al some excellent groups in the world are working in Electro remediation so I will focus today on electr fermentation and I’ve have prepared this presentation with my colleague Nicolas ber uh we’re working together uh on this aspect so what is electr fermentation is how to combine fermentation and microbial electrochemistry principle uh to optimize so the gradation but also the uh to optimize the production of biohydrogen and biob molecules so the content of my talk uh is in three parts first is a bit of the context few words about the context of the research and a focus on biohydrogen second uh part is about dark fermentation and MEC microbial electrolysis cells so I will I will give some principles and also some interesting results we got by coupling these two processes and finally uh more fundamental research uh I will present more fundamental research on electr fermentation is it a step forward in the degradation or the optimization of these of these processes so why hydrogen we have all heard about the hydrogen energy um so it has some advantages first is highly abundant U uh it’s it’s high abundant everywhere and he has a high energy uh density it’s about twice the energy density of methane so 122 m per kilogram so it’s very interesting in terms it’s a energetic molecule and it produces um low greenos gases emission from his combustion only water and is it can be an important Vector for the energy transition in the near future for us for us but he has some main limits and the main limits is a low volumetric density of these molecules it’s only 10 K per liter meaning that is 1/4 of the methane content in terms of energy per liter so it creates some problems with the storage and transportation that are really energy consuming so we have to store hydrogen at 350 or 700 bars and atus 200° celus to to to store these molecules so it it consume a lot of energy and there is a strict regulation for production of storage of course because it’s a dangerous molecule and is mainly present in complex form that’s important because it’s not natural resource is really always complex with other molecules so for that we need hydrogen processes to extract and recover from different types of resources that I we present so that’s the main point of this energy is not uh is not a natural energy so but however it’s not only question of Transportation uh hydrogen Market already exist in the world and you can see here the world production of hydrogen is already about 75 74 million tons per year in EU about 10 in France about 1 million ton per year and is mainly used in the refinery for the hydrogenation of sulfur compounds when we produce gasoline so it’s Alo used in the abbush process to produce ammonia and to to produce some solvents or or air and other chemicals and is also used in food industry however if you look at the transportation here is almost nothing nothing on the market and if we um consider the hydrogen available for transportation is also almost nothing is about 9% of the hydrogen that can be commercialized and used for transportation so we need to find new way of prod production and is transportation a reality so I I provided few examples so there are lot of example now in the world but few examples around here so if you go to Paris you can take a hype taxi so there are there is a float of about around 700 Vehicles light vehicles in Paris all running so they are blue like this running with hydrogen so hydrogen station is about 700 bars and fling time is quite short is only 5 minutes uh and autonomy is is uh as high as the best electric car 600 kilm so for some lines also when you don’t have electricity like here so there are few example in Germany in France and Europe uh you have trains I think that’s I mean they plan to be the future e e Vehicles trains trucks rather than light vehicles and they plan also your you may have heard about that the Airbus planes by 2035 to to to be to use hydrogen as as kerosin so we need a lot of huge amount of energy linked to hydrogen and um the hydrogen conceal which is lot of expert in the domain they produce they they um uh calculated the amount of hydrogen expected by 2050 regarding the global energy demand and if you look at the demand it will increase so we are around here by a factor of eight in total by 2050 so it will be a huge demand but it’s not only for transportation as I mentioned is also for power generation and buffering when you have a overflow of electricity which can buffer with hydrogen uh and of course electrolyzes here it’s also a huge amount of hydrogen for the decarbonization of the industry is very important uh so for the industrial energy and building heat and power for for for for everyone so we need new sources of hydrogen and as I explained we need to extract and and convert some natural resources and here this is a current production of hydrogen uh in Europe and if you look at the resources is natural gas mainly oil and coal so at 90% is produced from fossil fuels and only 10% from electrolyzes from water and also electricity can be produced from fossil Fu also so the envir environmental impact can be quite important here so in interestingly the we have divided the production of hydrogen in two type uh of uh Technologies one is from the the resources of energy so one is the fossil fuel energy here with hydrocarbon reforming so is what we’re talking about currently the hydrogen production it comes from this way and now if we are interested in renewable sources meaning that power electrical power windmill or or solar panels we can produce by electrolyzes and water splitting the hydrogen so it can be a green hydrogen and even Greener if we are interested in a biomass and including waste the subject of the talk today waste and influence we can produce by two sets of Technologies one are thermochemical processes by gasification or or or thermal gasification and biological processes and within the biological processes we have four sets of technologies that I will detail here so one is a water bio photolysis is welln technology where micro are able to do water splitting by using light so the advantage is a direct production of hydrogen but the limits are the light and also uh we need to control the medium composition so it’s not really um uh adapted to to treat influent from the industry or waste water the second type of technology is really linked is a photofermentation so it’s carried by purpose nonsulfur bacteria in nitrogen starvation they produce hydrogen so it can be used with eent uh but it requires light and a control of the nitrogen content in the eant so today if we focus on eent and we do not really control the composition of this effluent there are two type of technologies that can with this projection is dark fermentation which is a classical fermentation where we use fermentative bacteria at low ph and it fits with a waste effluent uh uh degradation and conversion however as you will see conversion needs are limited quite limited and the last technology or the microbial electrolyzes cells mecs here the main support catalyzers are electroactive bacteria that are able to convert the organic matter to electrons and then we we we do and I will explain water in this process so effluence or kind of effluence can be used is quite intensive however we need to add a little bit of power in the system uh that the reaction can occur so this is the main contest so in terms of life cycle analysis dark fermentation or biological processes in general have the lowest uh um impact uh in almost all criteria than other Technologies so it’s quite interesting in terms of environmental impact however you will see maybe the the intensity or the conversion efficiency are not that high so let’s go in detail in the dark fermentation and microbial electroly cells so principle and challenges what are the main uh uh aspect on this Technologies so first a few years ago we did did a literature survey of all articles and patents uh dealing with these two type of Technologies so here you have um summary of the of the Articles and patterns so we started with 2,600 articles with so it is quite more complex that only four words like this anerobic fermentation in Mis culture so we we we used a quite complex um research however it was around anerobic fermentation in Mis culture and we removed all the papers dealing with meane only pure culture and also review paper we we wanted to focus on technical papers and here are the results in term of distribution of the papers more than 75% of the papers of course were dealing with dark fermentation 10% on dark fermentation and anerobic digestion because it’s part of the anerobic digestion and only few amount of papers around 5% on MEC only we are quite surprised and 1% on coupling dark fermentation and mec what I’m going to present today and also uh about 10% with uh photofermentation and coupling these two processes that could be interesting as well and if we compare this with the patent it’s quite interesting here the dark fermentation and dark fermentation coupled to anerobic digestion we have less patent so Dynamics were more more towards MC and coupling dark fermentation and MC so there’s a strong Dynamics on developing this kind of process to improve the hydrogen efficiency so now in details more in details we made some calculations so what is the dark fermentation representing 75% of the articles in this domain so it’s part of the anerobic digestion pathway so from carbohydrates we have four steps hydrolysis acetogenesis acetogenesis and finally methanogenesis so if we stop the pro degradation process to this step we can accumulate vfa acetate berate and also hydrogen so we need to stop here the methanogenesis so hydrogen is is found in every natural ecosystem and mainly because hydrogen is electron vector and microorganisms like to use this electron Vector for their own respiration and finally in dark fermentation reactor we stop the methanogenesis is quite easy because we practice each shock treatment so it’s not the main factor each shock treatment we stop we kill the methanogens and then playing with the HRT low HRT only a few hours to one day and low PH one 5.5 methanogenesis cannot occur however as you will see we have other type of interactions it’s just because mainly the the main producers of hydrogen are Clum species this type of species so they stop here they are acidogenic bacteria able to accumulate hydrogen as a release of Sur of electrons in their metabolism and through the acetate and mate pathway and often if we have overlow of this macro of the electrons inside the microorganism they they shift the metabolic Pathway to lactate or to the ab Pathways so it’s it’s called Santo Genesis so as soon as we accumulate vfa too much vfa or hydrogen they switch here to another type of nonproducing pathway but this these are the metabolic limitation on top of that in Mis culture so when we treat eent and waste we have also ecological limitations and mainly the the most important are lactic acid bacteria they out compete quite easily Clum species they are growing uh under the same condition and they are using carbohydrates to produce lactate and all these arrows are pathway that can use hydrogen as a source of electrons for their own pathway so they produce propionate we can produce propionate caate and so on and it can be a source also of electrons to produce new type of molecules sorry uh so just remember that it’s not only metabolic but also ecological limitation that that we have to play with so in 2000 in 2000 around 2000 a paper uh they they reach the high yield fermentation here I did Dark fermentation using enzymes so 12 moles thank you har 12 moles of 12 mes of hydrogen per mole of glucose here so this is a Max maximum we can reach but in in reality what happens is we have the acetate pathway we produce four moles of hydrogen and acetate berate pathway two moles of hydrogen and berate and a mixed pathway acetate ethanol passway two moles of hydrogen and in Mis culture the maximum we assume the maximum we can reach is about 2.5 moles of hydrogen per mole of glucose and this is important because because you can see here you have different vfa that are con concomitantly produced with hydrogen and this represent around 20% of the Cod per gram of cod of glucose only so 80% of the energy remains in the vfa so if we look at back to the literature uh the what can affect the metabolic Network or or the metabolic population mostly is a substrate so in iterature we found that the substrate is very important with of course higher production with symol sugars and then it can decrease U regarding the complexity of the organic waste so simple sugar indutrial influence but overall we are around 10% only if we consider all the papers 10% of the Cod converted to hydrogen of course you we have outliers here but the maximum is 33% but one good point if we look at the operating parameters in all papers you can see here is quite a robust process because dark fermentation is about 10% all along whatever the condition mesophilic thermophilic whatever the volume of the reactor and the mode of operation badge fed batch or continuous RoR so that means uh in one way is it’s limited converstion yield but also it’s quite a robust process that can be used for any type or any operating conditions as long as is suitable with hydrogen produces so in summary for the dark fermentation process you have organic matter we produce hydrogen and organic acids some alcohols some biomass undegraded organic matter the maximum thetical we which is four mol of hydrogen per mole of glucose represent only 33% of conversion and in the literature the average he we we measured we found was 10% so here we have quite an increase we we should reach this uh this this 33% however the energetical conversion efficiency is is very high because we don’t provide a lot of energy I mean it’s just a fermentation the Delta G is very low is minus 200s mainly so bacteria are able to convert the organic matter bys and the main advantages are here so high organic clothes well proof technology for a large operating conditions I showed you so and is well adapted therefore for waste and Wastewater treatments however there are some metabolic limitations and a limited y of hydrogen so we need to cou this to other processes so the most obvious process is anerobic digestion that I will I won’t present today I don’t have time but at Short Term it’s ready to C out these two but what we focused on is all to convert the vfa so 80% of the remaining Cod to hydrogen so for this one technology interesting technology uh representing 5% of the Publications articles is the Mec microbial electr cells you will have a talk this afternoon in details of this technology and here organic acid and alcohols can can be efficiently converted to CO2 by respiration and we use the capacity of electroactive bacteria to transfer their electrons from their respiration to the electrode air so electroactive bacteria able to transfer electrons to a material and then by applying here of voltage we’re able to reduce the protons here at the cathode to produce uh hydrogen so there are some sorry advantages so how is it working how is it working is bacteria able to convert organic matter here and you can have mediators here so or non electroactive bacteria as well but mediators that can be reduced or oxidized on the surface of the electron you can have a direct transfer through cytochromes which are small proteins or through cytochromes and nanowire so this all these pathway are well known depending on the type of electroactive bacteria it’s important for for the electr fermentation part and MC the benefit is if you start from water you split water to produce hydrogen is quite a huge amount of difference of potential but if you start from organic compound like I the difference is lower so if you compare the potential difference between MEC here and a water electrolyzer you can see that the difference of potential you we should apply is around 0.2 volt compared to uh 1.22 volts in in the electrolyzer so it’s 10 times lower more or less in MEC because the electrons comes from the organ I acids so and what I I wanted to highlight also is a maximum the thetical Y is about 100% all organic acids can be converted to electrons and the average Yi in publication is 50% so it’s a high hogen production yield High energetical conversion yield uh and also hydrogen is almost purified here at the cathode so in in a pilot we reach about 99.5% of pure hydrogen at the cathodic compartment however the limits are high material cost and low organic load in the system so I I wanted to show you some results practical results on coupling dark fermentation and M because it’s quite obvious that we can use organic acid in this system so for that uh we perform we try to apply this concept on Agro Industrial Waste Waters a set of agroindustrial waste Waters by coupling dark fermentation and MEC and comparing the efficiency of the systems so in the first step in dark fermentation first it it really depends on the type of substrate and here you can see the hydrogen yield compared to the type of substrate so from uh cheese Way Fruit uh juice uh wastewater treatment plant frood processing Wastewater uh sugar uh waste water here and some other ago industrial so depending on the type of waste water we have reached different hydrogen yield in the second step we produced hydrogen I I will show you the results but what is important we made an energy uh balance in this system and you can see here that the Energy Applied and for all uh eent we recovered uh in brown here up to 700% of energy recovered in form of hydrogen than the energy we applied so it’s quite efficient process here at La scale and the Cod removal in this system uh range between 40 to 60% so it’s also efficient in terms of cood or vfa removal coming from fermentation so here’s the total results of the system so hydrogen yield in fermentation you can compare the hydrogen wield found in MEC and there is a factor between six to 16 uh times more hydrogen in the second compartment is more efficient in terms of yield and the over yield so here is excuse me uh a volume but if you look at if you convert this volume to moles of hydrogen per gram or per mole of glucose we reach around 10 moles of hydrogen per mole of glucose which is quite uh close to the maximum limit of 12 so it it was really efficient on this kind of a fluent and we reach a 70% C removal in aage in this by coupling these two processes so it is quite interesting in terms of efficiency uh however we can we can still think that maybe dark fermentation is not efficient enough to produce hydrogen so I wanted just to to show you a new approach uh we are developing is focusing on the fermentation to produce a molecule so different set of uh vfa so in this project a biotech EU biote biotane sorry project we try to convert the fit stock different fit stock waste to a set of jet fuels with a calibrated type of jet fuel for that the first step here we are focusing on is a coupling dark fermentation and MEC and then carboxylic acid propionic acid and hydrogen will be converted in the Second Step uh with uh some uh input from uh Thermo gasification here in the second step we be converted to butan Di and acetoin and and so on to to to jet fuel and I wanted to focus on this aspect because fermentation here we won’t produce any hydrogen it won’t be the purpose but it will be to focus on metabolite production and then a new aspect of the Mec it’s also to polish to degrade nonusable uh uh unwanted metabolites to produce hydrogen and like this we can have an infent enrich in what we want as a metabolite and also hydrogen that can be further converted so it’s Al also another aspect of the M that can be used as a polishing process that that be uh that would be interesting so now we can have this question and if we put MEC and dark fermentation together because operating conditions are quite similar and finally uh we can process the fermentation and the Mec process in the same compartment and so what we developed uh in the last years is the electr fermentation uh approach so is it a step forward uh than simple MEC or micro electrochemical conversion so electr fermentation what is this it consist in operating the fermentation of energ Rich substrate such as carbohydrate or alcohol in presence here of an electrode or electroactive bacteria to provide an an external or supplementary electron Source or electron sync and like this the thermodynamic limitation can be overcome in some aspect and to control the metabolic pathways what I’m I’m going to show you so there is a difference between electrosynthesis and electr fermentation electrosynthesis is when electrons in in terms of amount of electrons are directly converted to a product so we have a certain amount of electrons it goes directly to a product this is what we call electrosynthesis electr fermentation is not the same um purpose it’s only to control here the metabolic pathway of reacting bacteria by changing the NAD nadh uh uh ratio in the cells and like this metabolic pathway can be uh could be control by playing with the orp uh the Redux potential so the idea was to uh trigger some metabolic pathway in this aspect or if it’s not directly with the electroactive bacteria it can be a support here of the electroactive bacteria as electron sync or source of the fermentative bacteria so for Electro fermentation I’m sorry I think I miss I missed the slide yeah sorry so electr fermentation the reaction are not supported not only by the electrical current but mainly by the presence of an energy reach substrate so electrical current is not the product of the interet or the main energy source and as I I said electric current is just a trigger allowing a fermentation that normally is not thod thermodynamically not feasible and the main difference between electr fermentation here and microb electrosynthesis is it doesn’t require high curent density to occur so low energy and is just a trigger of the the metabolic pathway so now what are the example uh so we did some electr fermentation of glucose in Mis culture in presence of electrodes so polarized electrodes in batch test so here you can see the metabolite distribution so in the control is an open circuit no electrodes it was a classical batch so you can see here a lot of lactate in this system with this kind of Consortium ethanol and acetate and little bit of hydrogen on top of that and we apply different type of um uh different uh type of uh potential so minus 0.9 volt up to plus 0.9 volt on the working electrode and what happened here you can see a shift a clear shift of the metabolic distribution from lactate to ethanol in this case or berate and acetate but we didn’t find any relation between the voltage here and the shift uh the metabolic shift so far so what we did a microbial characterization of this is system and we found that in the control we had Anor bacteria in red and strepto Cass so lactic acid bacteria in Blue by applying difference of potential with a low current we observed here we we showed a clear shift towards anob bacteria which are also hydrogen producers not as efficient at Clum but also hydrogen producers so a clear shift of bacteria in all conditions and then uh other tpia here and some remaining strepto cocac so we had a mixture of metabolite pathway due to the selection of and and clear selection of ano bacteria in this system at the end of the batch fermentation so here with a PCA analyis you can see that is clear and is well known also we confirm this with that strepto Co Cass as lactic acid bacteria correlated with the lactic acid accumulation with hydrogen bate and other anob bacteria with acetate ethanol succinate accumulation which is quite obvious but what was important in this system is that we selected the bacteria specifically by applying a very uh small um difference of part between is very small electrical density current density so hypothesis we made was about the microbal diversity shift so the presence of electrode caused clearly the microbal diversity shift but the metabolic shift uh linked to the microbi was linked to the presence of the electrode in The Bu or to the presence of electroactive bacteria that was the question that’s is the question so what we did then is we only use electroactive bacteria joter suf reduc is the most known uh electroactive bacteria in batch test with glucose and acetate um at low ph and temperature so like this fermentation uh fermentative bacteria can can grow and what we found is on the right you have the metabolic distribution so with the mix cure we have this distribution mostly in this case buten Di and buty rate and when we had a geobacter we found shift again by just adding no electrod just adding a geob fruc a clear shift towards Beauty rate here in light green oh sorry light green here buty rate less ethanol and but di Bey rate fermentation pathway and if we look at the microbal community um I’m sorry I didn’t put the names here but we turn the micrel from red and blue uh species to more blue species related to clas species and geobacter Su distance was only a tiny a minority abundant here species in brown so meaning that it’s not only the electrode but in in our opinion one hypothesis is like electron transfer between this electroactive bacteria and other bacteria in the system can trigger the metabolic pathway of the fermentative bacteria so it’s is what I’m I’m trying I’m going to show you is the presence only of geobacter sufur reduc because of shift INF fermentation product and microbial population so hypothesis again is is there interspecies electron transfer between the the bacteria and can it occur in mixed culture fermentation so we looked at Old results with a new uh eye um so these results were performed uh in chemat with glucose and eight different inoculum and after a steady state so it was really chemat glucose was sterilized with chemat we got eight different uh steady state uh ecosystems so unsurprisingly on the left you have the result so with eight different inocular we found eight different performances because it’s really linked to the microbal Comm community so on R uh the hydrogen yield from 1 to 2.5 and blue the productivity from from 9 to 5 here for the lowest however at that time we had we we performed the DNA fingerprinting and you can see here some DNA fingerprints with one abundant bacteria always the same so was that was surprising and subdominant bacteria here so the hypothesis is what the minority species was very important to uh control the metabolic pathway of the fermentative bacteria the fermentative bacteria was Clum P mon almost in all cases and minority species where eoli ocus or lactobacillus so the hypothesis at that time is was a key role of this minority bacteria and you will see that it can be linked to interspecies electron transfer as well so what we did also we tried to reintroduce the species directly in the ecosystem in the mic community so we try to reintroduce the species we identify previously in the microbial community and what we found is different behaviors the first one is no significant no significative effect we add the species nothing happens different so it was the the case of anus species in another type of behavior it’s a classical microbial competition in this case we added again Clum panum in the system so the fermentative bacteria and what happened is between EA and Clum panum a competition between these two and with average hydrogen yield between these two ecosystem the third Behavior bit more interesting is a synerg synergetic uh effect where when we added eoli here you can see the hydrogen yield was increased by 2.5% but at that time uh some other groups are working on adding uh fa an anerobic facultative microbes to degrade the traces of oxygen in the medium like this Clum can can work more easily towards hydrogen production so in that time it can be due to oxygen Deion at start of the growth but what we didn’t explain what this effect with ronia tra now know his name is cidus Nik which is a strict aerobic bacteria so we we tried anyway this bacterial and you can see here the ecosystem was the performances were multiplied by 3.5 so quite a lot and if we look at the metabolite we observed again a shift between the bate lactate fermentation towards acetate and berate which is clearly hyrogen producing Pathways so the presence of a non-growing uh aerobic bacteria in this system was favoring was triggering the metabolic pathway so finally with a group here they focus non because the problem is oxygen consuming bacteria we can have traces of oxygen so they focus more on sulfate there something we can control sulfate reducing bacteria so they did a co-culture of Clum acetum so with a beep marel here Clum acetum fermentated bacteria with a sulfate reducing bacteria interestingly in that case with no sulfate so not able to grow so alone in pure Clum acetum was able to grow andri vgis not able of course but in Co culture you can see here cleto with no sulfate again we’re able to grow together you should I should mention here that if you look at the so here is expressed in genome copies if you look at the genome cop copies we have a bit lower genome copies in Co culture than in pure culture for Clum aceto it’s quite important because we will I will present some hypothesis on parasitism of this kind of bacteria andaris is growing well here the effect on the metabolism so we did some metat transcriptomic analysis and what we found is clearly a shift in close ponum from a from lactate passway here in culture it was multipli by eight times higher acetate Beauty rate and then hydrogen was multiplied so amount of hydrogen by 2.5 so it’s quite consistent what what we observed previously and finally with a picture uh we found that it was a close interaction direct interspecies electron transfer here between Clum in green and here in Red dgis so in Co culture you can see that some of them were totally yellow meaning that they are exchanging some material in in their system so without an electron acceptor in the system Theo vgis was able the hypothesis was this mcrm was able to provide electrons or to provide electrons to Clum acetum so we perform or or we did this hypothesis and then uh we did some theoretical approach on this interaction and one question is is it electr electronic parasitism of Theo or Rona or neor Sor or other electroactive bacteria then when they are not in favorable conditions for growing they’re able to use another microorganisms as electron acceptor or electron donor so here you have a conceptual um graph of what we think can occur in this system the electroactive bacteria normally are able to uh provide electrons to the anode to minerals to to to a material uh to grow and in some aspect if they don’t have any electron acceptor they can use electron accepting microbes here so that was the hypothesis uh and especially fermentative bacteria so it can be used as a trigger also of a fermentative bacteria so so to go a little bit um deeper in this aspect what we did is we focus on one specific fermentative bacteria again close rum pan and one welln electroactive bacteria geobacter suur reduc so what we did in that case it was more to focus on the simpler metabolic pathway the glycerol fermentation because there is a direct link here between glycerol and propan diol so it can be directly converted to propan diol by reduction and GAC Su reduc so the hypothesis was to use acetate in the medium but with no uh electron acceptor in the system so what uh could doac is only provide the electrons to Clum only in absence of electrod so in pure culture Clum pum is able to convert glycerol in different set of different types of metabolites in mixture mainly propane one three propenal and in presence of geobacter su reduc acetate is oxidized and electrons are provided to Clum pum and if we look at the carbon distribution uh so carbon dioxide is not provided here with pure culture in blue you have the propen dial and in Orange the butanol accumulation and in in co- culture what happened again triggered the metabolic pathway towards propen dial meaning that we have a clear excuse me a clear and obvious observation that electrons coming from geaa were used by Clum panum to another pathway here however it’s the same as p and the biomass the total biomass decreases meaning that it’s kind of electronic parasitism during this process but geobacter sufur distance was able to grow with acetate only as electron donor in this system so it’s a way for them to to survive in this system and finally to finish um what we did is a metat transcriptomic analysis we we try to understand what were the mechanisms of behind this transfer of electrons was it direct transfer of electrons or through cytochromes or with mediators and what we found here is with a metat transcriptomic analysis we found two potential pathway so one through a mediator so some kind of cobamide that can transfer from Joba redance to Clum panum and way through cytochromes here that were overexpressed in culture and the electrons can go through this pathway also so we are still not sure of the pathway because these two pathway were uh improved during the culture so the main question now we have is is uh at which extent this interspecies electron transfer uh is generated can occur with all type of fermentative or electroactive bacteria and the effect of electr fermentation we form is it a direct effect on the microbial population or is just a selection of electroactive bacteria and then this type of uh um reaction can occur as well so there is still a lot of question in this aspect but I showed you that the fermentation pathway in culture or in presence of electrod can be very important in terms of control or even improving some metabolic pathway quite efficiently so the main conclusion of my talk today is about the environmental bioener for biohydrogen and also fermentation so first coupling that fermentation and microbi electrolyzes we think is a promising way to produce hydrogen in this case or uh to polish uh uh fermentation effluent so up to 10 moles of hydrogen per mole of glucose I I should say that a clap scale is quite efficient and now the most important is the upscaling process and we we have to focus on this aspect uh all groups on the upscaling and the limitation of upscaling in this process and electr fermentation in our opinion is one way to better control control the population selection and also the fermentation process when we want to optimize uh a fermentation pathway and there is still a large field of Investigation of interspecies electron transfer so there are many um groups working also in electr microbiology in culture we we we assume that it has a great potential to to overcome some thermodynamics limitation and then better control the metabolic pathway so thank you very much for for your attention and thank you for all my colleagues in the lab working in this on this field thank you [Applause]