Use of a continuous-flow bioreactor to evaluate nitrate reduction rate of Halomonas desiderata in cementitious environment relevant to nuclear waste deep repository

The redox level of repositories can influence the mobility of the waste components stored in them (i.e. radionuclides) and thus the related safety assessments. Microbial activity is known to impact the control of redox reactions, the mechanisms and kinetics of which must be evaluated. This study investigates the denitrification rates of a model bacterium Halomonas desiderata (Hd) in cementitious environment with alkaline and anoxic conditions comparable to those found in radioactive waste repository cells. The growth and the total oxidized nitrogen (TON) reduction rates of Hd was determined in a continuous bioreactor with several feeding solutions with or without solid cement paste. Temporary nitrite accumulation and reduced denitrification rates are correlated with diminished bacterial growth. When the system was fed by optimal culture medium supplemented with acetate and nitrate, the TON reduction rates varied between 0.082 mM TON/h and 0.063 mM TON/h, depending on whether solid cement paste was present in the reactor or not. When the culture medium was replaced with pure cement leachate, the reaction rates increased to 0.137 mM TON/h with solid cement paste and dropped to 0.023 mM TON/h without. In these conditions at pH 10, solid cement paste had no negative influence on Hd activity

Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals

After closure of a waste disposal cell in a repository for radioactive waste, resaturation is likely to cause the release of soluble species contained in cement and bituminous matrices, such as ionic species (nitrates, sulfates, calcium and alkaline ions, etc.), organic matter (mainly organic acids), or gases (from steel containers and reinforced concrete structures as well as from radiolysis within the waste packages). However, in the presence of nitrates in the near-field of waste, the waste cell can initiate oxidative conditions leading to enhanced mobility of redox-sensitive radionuclides (RN). In biotic conditions and in the presence of organic matter and/or hydrogen as electron donors, nitrates may be microbiologically reduced, allowing a return to reducing conditions that promote the safety of storage. Our work aims to analyze the possible microbial reactivity of nitrates at the bitumen – concrete interface in conditions as close as possible to radioactive waste storage conditions in order (i) to evaluate the nitrate reaction kinetics; (ii) to identify the by-products (NO2−, NH4+, N2, N2O, etc.); and (iii) to discriminate between the roles of planktonic bacteria and those adhering as a biofilm structure in the denitrifying activity. Leaching experiments on solid matrices (bitumen and cement pastes) were first implemented to define the physicochemical conditions that microorganisms are likely to meet at the bitumen-concrete interface, e.g. highly alkaline pH conditions (10 < pH < 11) imposed by the cement matrix. The screening of a range of anaerobic denitrifying bacterial strains led us to select Halomonas desiderata as a model bacterium capable of catalyzing the reaction of nitrate reduction in these particular conditions of pH. The denitrifying activity of H. desiderata was quantified in a batch bioreactor in the presence of solid matrices and/or leachate from bitumen and cement matrices. Denitrification was relatively fast in the presence of cement matrix (<100 h) and 2–3 times slower in the presence of bituminous matrix (pH 9.7). The maximal rate of denitrification was approximately 0.063 mM h−1 and some traces of nitrite were detected for a few hours (<2%). Overall, the presence of solid cement promoted the kinetics of denitrification. The inspection of the solid surfaces at the end of the experiment revealed the presence of a biofilm of H. desiderata on the cement paste surface. These attached bacteria showed a comparable denitrifying activity to planktonic bacterial culture. However, no colonization of bitumen was observed either by SEM or by epifluorescence microscopy

Coupled iron-microbial catalysis for CO 2 hydrogenation with multispecies microbial communities

The hydrogenation of carbon dioxide offers a large range of possible reactions for converting hydrogen to chemical compounds that can be easily stored, transported and used as fuels or platform molecules. In this study, CO2 hydrogenation was biocatalysed by multispecies microbial communities to produce formate, butyrate and acetate. A hybrid metal/microbial catalysis was pointed out in the presence of iron. Addition of FeCl3 10 mM increased the production of acetate by 265% and butyrate by 73%, to 5.26 and 14.19 g/L, respectively. A stable acetate production rate of 830 mg/L/d was thus sustained for more than 20 days. The presence of iron promoted the selection of Firmicutes and the best performances were linked to the growth of a restricted number of dominant species of two genera: Clostridium and Megasphaera. Various possible catalysis mechanisms are discussed and guidelines are proposed for further development and scale-up of the process

Impact des facteurs biotiques sur le réseau métabolique des écosystèmes producteurs d’hydrogène par voie fermentaire en culture mixte

Nowadays, mixed cultures are considered as a serious alternative to pure cultures for biotechnology processes. Indeed, mixed cultures can be efficient under non-sterile conditions, and can use a wide variety of organic compounds as substrate. Their main limitation is instability due to the presence of unwanted metabolic pathways resulting from complex microbial interactions. In particular, the role of bacteria in low abundance remains to be elucidated. This work has therefore consisted to determine the role of minority bacteria in the hydrogen production using fermentative. Seven inocula have been grown in a continuous way in the same operating conditions. Six times on seven, the same bacterium was found to be the dominant species of the ecosystem, despite differences in the hydrogen production. Considering the seven ecosystems obtains, only the nature and the diversity of the low abundance species differed, showing that the bacteria in low abundance play a key role by guiding the overall ecosystem metabolism. In a second step, this work consisted in using some of these minority species as ecological engineers of microbial ecosystem. In order to study this aspect, a hydrogen-producing microbial community has been artificially modified by adding exogenous bacterial strains with redundant functions and/or complementary native strains. Results in batch reactors have shown that the hydrogen production performances could be improved by the addition of certain strains. Results obtained cannot be explained by simple trophic interactions and suggest the presence of interaction mechanism of cooperation among microorganisms. Moreover, under more favorable operating conditions; the addition of certain species in low abundance could stabilize the metabolism of microbial ecosystem without affecting the hydrogen production. In all cases, competitive interactions were not favorable for hydrogen production. Trials were then realized in continuous reactors. These trials have shown that the method used to implant strains in reactors could be a key factor for using the ecological engineers.Les cultures mixtes sont aujourd’hui considérées comme une sérieuse alternative aux cultures pures dans le cadre des biotechnologies du fait leur capacité à traiter une large variété de substrats organiques et ce en conditions non stériles. La principale restriction à leur utilisation réside toutefois dans une instabilité du procédé liée à la présence de voies métaboliques non désirées résultant d’interactions microbiennes complexes. Notamment, le rôle des bactéries de faible abondance dans les écosystèmes reste à être élucidé. Ce travail a donc consisté à déterminer le rôle des bactéries minoritaires dans la production d’hydrogène par voie fermentaire. Dans un premier temps, sept inocula ont été cultivés en réacteurs continus, dans les mêmes conditions opératoires. La même espèce dominante a été observée six fois sur sept mais les performances de production d’hydrogène différaient. Seules la nature et la diversité des espèces minoritaires variaient d’un écosystème à l’autre prouvant ainsi que les bactéries en proportion minoritaires jouent un rôle clé en orientant le métabolisme global de l’écosystème. Dans un second temps, certaines de ces espèces minoritaires ont été utilisées comme perturbateurs biotiques. Pour cela, un écosystème producteur d’hydrogène a été modifié artificiellement en introduisant des souches bactériennes exogènes aux fonctions redondantes et/ou complémentaires des souches indigènes. Les résultats en réacteur batch ont montré que les performances de production d’hydrogène pouvaient ainsi être améliorées. Globalement, les résultats obtenus ne peuvent être expliqués par de simples interactions trophiques et suggèrent la présence de mécanismes d’interactions de coopération entre microorganismes. De plus, sous des conditions opératoires plus favorables, l’insertion de certaines espèces minoritaires a permis de stabiliser l’écosystème microbien, sans pour autant en affecter la production d’hydrogène. Dans tous les cas, les interactions compétitives n'ont pas été favorables à la production d'hydrogène. Enfin, des essais en réacteur continu ont montré que le mode d’implantation des souches peut être un facteur primordial

Impact of biotic factors on the metabolic network of fermentative hydrogen-producing ecosystems in mixed culture

De nos jours, les cultures mixtes sont considérées comme une sérieuse alternative aux cultures pures pour les procédés de biotechnologie. En effet, les cultures mixtes peuvent fonctionner en réacteur continu, dans des conditions non-stériles et traiter une grande variété de substrats organiques. La principale restriction de l'utilisation de ces bioprocédés en cultures mixtes réside dans leur instabilité liée à la présence de voies métaboliques non désirées résultant d'interactions microbiennes complexes. Notamment, le rôle des bactéries de faible abondance reste à être élucidé. Ce travail a donc consisté, dans un premier temps à déterminer le rôle des bactéries minoritaires dans la production d'hydrogène par voie fermentaire en utilisant un chémostat alimenté en continu avec un milieu à base de glucose. Sept inocula ont été cultivés dans les mêmes conditions opératoires. De façon remarquable, Clostridium pasteurianum a été retrouvé comme espèce dominante de l'écosystème six fois sur sept. Seules la nature et la diversité des espèces minoritaires variaient d'un écosystème à l'autre. Ainsi, il a été montré que la structure des communautés microbiennes a une influence significative sur la production de bio-hydrogène. Au sein de ces communautés, les bactéries en proportion minoritaires jouent un rôle clé en orientant le métabolisme globale de l'écosystème. La deuxième étape de ce travail a consisté à utiliser certaines de ces espèces minoritaires comme Ingénieurs Ecologiques des Ecosystèmes microbiens (IEEM). Pour cela, la structure d'une communauté microbienne productrice d'hydrogène a été modifiée artificiellement en introduisant des souches bactériennes exogènes aux fonctions redondantes et/ou complémentaires des souches indigènes. Les résultats en réacteur batch ont montré que les performances de production d'hydrogène pouvaient être améliorées jusqu'à un facteur 3,5 par l'ajout de certaines souches. Dans l'ensemble, les résultats obtenus ne peuvent être expliqués par de simples interactions trophiques et suggèrent la présence de mécanismes d'interactions de coopération entre microorganismes. De plus, sous des conditions opératoires plus favorables (inoculum, milieu), l'insertion de certaines espèces minoritaires a permis plutôt de stabiliser le métabolisme de l'écosystème microbien sans pour autant en affecter favorablement la production d'hydrogène. Dans tous les cas, les interactions compétitives n'ont pas été favorables à la production d'hydrogène. Enfin, des essais en réacteur continu ont montré que le mode d'implantation des souches peut être un facteur primordial pour l'utilisation d'IEEM. En conclusion, ce travail a montré la potentialité d'utiliser des bactéries exogènes, en proportions minoritaires, comme facteurs biotiques pour stabiliser et/ou orienter les métabolismes microbiens vers des fonctions d'intérêt au sein des cultures mixtes microbiennes.Nowadays mixed cultures are considered as a serious alternative to pure cultures in biotechnological processes. Mixed cultures can be operated continuously, under unsterile conditions and from various organic substrates. One of the most constraints remains the chronic instability of the mixed culture processes due to the presence of unwanted metabolic pathways resulting from complex microbial interactions. More particularly the role of bacteria in low abundance remains to be elucidated. Therefore this work consisted initially to determine the contribution of sub-dominant bacteria to fermentative hydrogen production using a chemostat continuously fed with a glucose-based medium. Seven inocula were grown under the same operating conditions. Interestingly, Clostridium pasteurianum was found as dominant in six assays on seven at steady state. Only the minority bacterial population differed with regards to their identity and diversity. Acting as true keystone species, these minority bacteria impacted substantially the metabolic network of the overall ecosystem despite their low abundance. In a second step, this work consisted in using some of these minority species as Ecological Engineers of Microbial Ecosystem (EEME). In order to study this aspect, the structure of a hydrogen-producing microbial community has been artificially modified by adding exogenous bacterial strains with redundant functions and/or complementary native strains. Results in batch reactors have shown that the hydrogen production performances could be improved to a 3.5 factor by the addition of certain strains. Results obtained can not be explained by simple trophic interactions and suggest the presence of interaction mechanism of cooperation among microorganisms. Moreover, under more favourable operating conditions (inoculum, culture medium), the addition of certain species in low abundance could stabilize the metabolism of microbial ecosystem without necessarily favourably affect the hydrogen production. In all cases, competitive interactions were not favourable for hydrogen production. Trials were then realised in continuous reactors. These trials have shown that the method used to implant strains in reactors could be a key factor for using the EEME.As a conclusion, this study has shown the potential to use exogenous bacteria, in minority proportions, as biotic factors to stabilised and/or guides microbial metabolisms to functions of interest within microbial mixed cultures

Impact des facteurs biotiques sur le réseau métabolique des écosystèmes producteurs d'hydrogène par voie fermentaire en culture mixte

De nos jours, les cultures mixtes sont considérées comme une sérieuse alternative aux cultures pures pour les procédés de biotechnologie. En effet, les cultures mixtes peuvent fonctionner en réacteur continu, dans des conditions non-stériles et traiter une grande variété de substrats organiques. La principale restriction de l'utilisation de ces bioprocédés en cultures mixtes réside dans leur instabilité liée à la présence de voies métaboliques non désirées résultant d'interactions microbiennes complexes. Notamment, le rôle des bactéries de faible abondance reste à être élucidé. Ce travail a donc consisté, dans un premier temps à déterminer le rôle des bactéries minoritaires dans la production d'hydrogène par voie fermentaire en utilisant un chémostat alimenté en continu avec un milieu à base de glucose. Sept inocula ont été cultivés dans les mêmes conditions opératoires. De façon remarquable, Clostridium pasteurianum a été retrouvé comme espèce dominante de l'écosystème six fois sur sept. Seules la nature et la diversité des espèces minoritaires variaient d'un écosystème à l'autre. Ainsi, il a été montré que la structure des communautés microbiennes a une influence significative sur la production de bio-hydrogène. Au sein de ces communautés, les bactéries en proportion minoritaires jouent un rôle clé en orientant le métabolisme globale de l'écosystème. La deuxième étape de ce travail a consisté à utiliser certaines de ces espèces minoritaires comme Ingénieurs Ecologiques des Ecosystèmes microbiens (IEEM). Pour cela, la structure d'une communauté microbienne productrice d'hydrogène a été modifiée artificiellement en introduisant des souches bactériennes exogènes aux fonctions redondantes et/ou complémentaires des souches indigènes. Les résultats en réacteur batch ont montré que les performances de production d'hydrogène pouvaient être améliorées jusqu'à un facteur 3,5 par l'ajout de certaines souches. Dans l'ensemble, les résultats obtenus ne peuvent être expliqués par de simples interactions trophiques et suggèrent la présence de mécanismes d'interactions de coopération entre microorganismes. De plus, sous des conditions opératoires plus favorables (inoculum, milieu), l'insertion de certaines espèces minoritaires a permis plutôt de stabiliser le métabolisme de l'écosystème microbien sans pour autant en affecter favorablement la production d'hydrogène. Dans tous les cas, les interactions compétitives n'ont pas été favorables à la production d'hydrogène. Enfin, des essais en réacteur continu ont montré que le mode d'implantation des souches peut être un facteur primordial pour l'utilisation d'IEEM. En conclusion, ce travail a montré la potentialité d'utiliser des bactéries exogènes, en proportions minoritaires, comme facteurs biotiques pour stabiliser et/ou orienter les métabolismes microbiens vers des fonctions d'intérêt au sein des cultures mixtes microbiennes.Nowadays mixed cultures are considered as a serious alternative to pure cultures in biotechnological processes. Mixed cultures can be operated continuously, under unsterile conditions and from various organic substrates. One of the most constraints remains the chronic instability of the mixed culture processes due to the presence of unwanted metabolic pathways resulting from complex microbial interactions. More particularly the role of bacteria in low abundance remains to be elucidated. Therefore this work consisted initially to determine the contribution of sub-dominant bacteria to fermentative hydrogen production using a chemostat continuously fed with a glucose-based medium. Seven inocula were grown under the same operating conditions. Interestingly, Clostridium pasteurianum was found as dominant in six assays on seven at steady state. Only the minority bacterial population differed with regards to their identity and diversity. Acting as true keystone species, these minority bacteria impacted substantially the metabolic network of the overall ecosystem despite their low abundance. In a second step, this work consisted in using some of these minority species as Ecological Engineers of Microbial Ecosystem (EEME). In order to study this aspect, the structure of a hydrogen-producing microbial community has been artificially modified by adding exogenous bacterial strains with redundant functions and/or complementary native strains. Results in batch reactors have shown that the hydrogen production performances could be improved to a 3.5 factor by the addition of certain strains. Results obtained can not be explained by simple trophic interactions and suggest the presence of interaction mechanism of cooperation among microorganisms. Moreover, under more favourable operating conditions (inoculum, culture medium), the addition of certain species in low abundance could stabilize the metabolism of microbial ecosystem without necessarily favourably affect the hydrogen production. In all cases, competitive interactions were not favourable for hydrogen production. Trials were then realised in continuous reactors. These trials have shown that the method used to implant strains in reactors could be a key factor for using the EEME.As a conclusion, this study has shown the potential to use exogenous bacteria, in minority proportions, as biotic factors to stabilised and/or guides microbial metabolisms to functions of interest within microbial mixed cultures.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Biological Methanation of H2 and CO2 with Mixed Cultures: Current Advances, Hurdles and Challenges

International audienceIn order to take action against global warming and ensure a greater energy independence, countries around the world are expected to drastically increase the proportion of renewable energy in their energy mix. However, the intermittent production of energy explains why energy supply and demand do not match. In this context, biomethanation, coupled with anaerobic digestion, could be an interesting approach to transform the extra amount of produced electricity by converting hydrogen (produced by electrolysis) and carbon dioxide (present in biogas) into methane. In this review, we have summarized several recently published results which involve biological methanation processes performed by mixed cultures, with an emphasis on microbiological as well as process aspects. In particular, the different microorganisms involved in the process, as well as the used metabolic pathways, along with their kinetic and thermodynamic specificities, are described. Furthermore, the influence of process parameters such as the type of reactor, the type of diffuser and the choice of H-2 injection (in situ or ex situ) or the different operating conditions are presented. Explanations of the different performances observed in literature are assumed, technical bottlenecks are listed, and possible solutions to overcome these issues are presented. Finally, the current commercial deployment of this technology is discussed through the example of three companies offering different biomethanation solutions

Biological Methanation of H2 and CO2 with Mixed Cultures: Current Advances, Hurdles and Challenges

International audienceIn order to take action against global warming and ensure a greater energy independence, countries around the world are expected to drastically increase the proportion of renewable energy in their energy mix. However, the intermittent production of energy explains why energy supply and demand do not match. In this context, biomethanation, coupled with anaerobic digestion, could be an interesting approach to transform the extra amount of produced electricity by converting hydrogen (produced by electrolysis) and carbon dioxide (present in biogas) into methane. In this review, we have summarized several recently published results which involve biological methanation processes performed by mixed cultures, with an emphasis on microbiological as well as process aspects. In particular, the different microorganisms involved in the process, as well as the used metabolic pathways, along with their kinetic and thermodynamic specificities, are described. Furthermore, the influence of process parameters such as the type of reactor, the type of diffuser and the choice of H-2 injection (in situ or ex situ) or the different operating conditions are presented. Explanations of the different performances observed in literature are assumed, technical bottlenecks are listed, and possible solutions to overcome these issues are presented. Finally, the current commercial deployment of this technology is discussed through the example of three companies offering different biomethanation solutions

Surface and bacterial reduction of nitrate at alkaline pH: Conditions comparable to a nuclear waste repository

International audienceThis study investigates the reactivity of nitrates in abiotic and biotic conditions at alkaline pH in the context of a repository for long-lived inter-mediate-level radioactive waste. The work, carried out under environmental conditions comparable to those prevailing in the repository: alkaline pH, no oxygen, solid materials (cementitious material, steel), aims to identify the by-products of the nitrate reduction and to evaluate reaction kinetics of these reactions with and without the presence of denitrifying microorganisms. This paper demonstrates that, even at the high pH characteristic of nuclear waste repositories, nitrate reduction may be a likely scenario, with biotic catalysis in the presence of microorganisms and surface catalysis in the presence of steel and/or corrosion products

Power to Gas - Biological methanation key technology for energy transition

Power to Gas - Biological methanation key technology for energy transition. WORSHOP TRANSENE