Joint project: Umwandlungsmechanismen in Bentonitbarrieren - Subproject B: Einfluss von mikrobiellen Prozessen auf die Bentonitumwandlung

Concerning the deep geological disposal of high-level radioactive waste (HLW), bentonite can be used because of its high swelling capacity and its low hydraulic conductivity as geo-technical barrier and buffering material in between the waste-containing canister (technical barrier) and the surrounding host rock (geological barrier). There are still many gaps in process understanding of bentonite transformations, especially in dependence of different temperatures and pore waters. Within the joint-project UMB (“Umwandlungsmechanismen in Bentonitbarrieren”), the co-operation partner Gesellschaft für Anlagen- und Reaktorsicherheit (GRS) mbH (Repository Safety Analysis), the University of Greifswald (Institute for Geography and Geology), the Federal Institute for Geosciences and Natural Resources (BGR, section of technical mineralogy), the Technical University of Munich (TUM; chair of theoretical chemistry, quantum chemistry) and the Helmholtz-Center Dresden-Rossendorf (HZDR, Institute of Resource Ecology) are supposed to define criteria which facilitate the selection of suitable bentonites in order to use them in the deep geological repository of high-level radioactive waste. HZDR analyzed two different bentonites (B36 and SD80) regarding their microbial diversity and potential microbial activity. In dependence of repository-relevant parameters (temperature, pore water, presence of substrates), microcosm experiments were set up at the GRS, containing the respective bentonites and Opalinus Clay pore water or cap rock solution, respectively. The long-term batches were incubated one year and two years at different temperatures (25 °C, 60 °C and 90 °C) in gastight bottles. Additionally, HZDR set up B36 short-term microcosms with Opalinus Clay pore water, which incubated for three month at 30 °C with six sampling points monitoring the microbial diversity and geochemical parameters.Für die tiefengeologische Lagerung von Wärme-entwickelnden, hoch-radioaktiven Abfällen kommen Bentonite aufgrund ihrer hohen Quellfähigkeit und ihrer geringen hydraulischen Leitfähigkeit als geo-technische Barriere in Betracht, welche sich zwischen der technischen Barriere (Behälter mit Abfall) und der geologischen Barriere (Wirtsgestein) befindet. Im Rahmen des Verbundprojektes „UMB“ (Umwandlungsmechanismen in Bentonitbarrieren) der Kooperationspartner Gesellschaft für Anlagen- und Reaktorsicherheit (GRS) mbH (Fachbereich Endlagersicherheitsforschung), der Universität Greifswald (Institut für Geographie und Geologie), der Bundesanstalt für Geowissenschaften und Rohstoffe (BGR, Arbeitsbereich Technische Mineralogie), der Technischen Universität München (TUM; Fachgebiet Theoretische Chemie, Quantenchemie) und dem Helmholtz-Zentrum Dresden Rossendorf (HZDR Institut für Ressourcenökologie) sollen abgesicherte, objektive Kriterien zur Auswahl geeigneter Bentonite für den Einsatz in Endlagern für wärmeentwickelnde Abfälle in Tonformationen entwickelt werden. Das HZDR analysierte hierfür die Entwicklung der mikrobiellen Diversität in den Bentoniten B36 und SD80 in Abhängigkeit von verschiedenen Parametern (Porenlösung, Temperatur, Anwesenheit von Substraten) um den möglichen Einfluss von Mikroorganismen auf die Umwandlungsprozesse im Bentonit zu erfassen. Die Bentonite wurden hierfür bei der GRS (Gesellschaft für Anlagen- und Reaktorsicherheit gGmbH) mit Opalinuston-Porenlösung bzw. verdünnter Gipshut-Lösung versetzt. Die Ansätze inkubierten in gasdichten Glasflaschen bei 25 °C, 60 °C und 90 °C für jeweils ein und zwei Jahre („Langzeit“). Des Weiteren wurden am HZDR B36 Mikrokosmen mit Opalinustonporenlösung angesetzt, welche für drei Monate bei 30 °C inkubierten („Kurzzeit“). Über die drei Monate verteilt wurden sechs Probenahmen durchgeführt, und die mikrobielle Diversität sowie ausgewählte geochemische Paramater bestimmt

Proteomic analysis of the hydrogen and carbon monoxide metabolism of Methanothermobacter marburgensis

Hydrogenotrophic methanogenic archaea are efficient H2 utilizers, but only a few are known to be able to utilize CO. Methanothermobacter thermoautotrophicus is one of the hydrogenotrophic methanogens able to grow on CO, albeit about 100 times slower than on H2 + CO2. In this study we show that the hydrogenotrophic methanogen Methanothermobacter marburgensis, is able to perform methanogenic growth on H2/CO2/CO and on CO as a sole substrate. To gain further insight in its carboxydotrophic metabolism, the proteome of M. marburgensis, grown on H2/CO2 and H2/CO2/CO, was analysed. Cultures grown with H2/CO2/CO showed relative higher abundance of enzymes involved in the reductive acetyl-CoA pathway and proteins involved in redox metabolism. The data suggest that the strong reducing capacity of CO negatively affects hydrogenotrophic methanogenesis, making growth on CO as a sole substrate difficult for this type of methanogens. M. marburgensis appears to partly deal with this by upregulating co-factor regenerating reactions and activating additional pathways allowing for formation of other products, like acetate.Research of AS is supported by an ERC grant (project 323009) and a Gravitation grant (project024.002.002)of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO)

Pathways and bioenergetics of anaerobic carbon monoxide fermentation

Carbon monoxide can act as a substrate for different modes of fermentative anaerobic metabolism. The trait of utilizing CO is spread among a diverse group of microorganisms, including members of bacteria as well as archaea. Over the last decade this metabolism has gained interest due to the potential of converting CO-rich gas, such as synthesis gas, into bio-based products. Three main types of fermentative CO metabolism can be distinguished: hydrogenogenesis, methanogenesis, and acetogenesis, generating hydrogen, methane and acetate, respectively. Here, we review the current knowledge on these three variants of microbial CO metabolism with an emphasis on the potential enzymatic routes and bio-energetics involved.The authors involved were financially supported by an ERC grant (project 323009) and the Gravitation grant (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO)

Genetic analysis of MA4079, an aldehyde dehydrogenase homolog, in Methanosarcina acetivorans

When Methanosarcina acetivorans grows on carbon monoxide (CO), it synthesizes high levels of a protein, MA4079, homologous to aldehyde dehydrogenases. To investigate the role of MA4079 in M. acetivorans, mutants lacking the encoding gene were generated and phenotypically analyzed. Loss of MA4079 had no eVect on methylotrophic growth but led to complete abrogation of methylotrophic growth in the presence of even small amounts of CO, which indicated the mutant’s inability to acclimate to the presence of this toxic gas. Prolonged incubation with CO allowed the isolation of a strain in which the eVect of MA4079 deletion is suppressed. The strain, designated Mu3, tolerated the presence of high CO partial pressures even better than the wild type. Immunological analysis using antisera against MA4079 suggested that it is not abundant in M. acetivorans. Comparison of proteins diVerentially abundant in Mu3 and the wild type revealed an elevated level of methyl-coenzyme M reductase and a decreased level of one isoform of carbon monoxide dehydrogenase/acetyl-coenzyme A synthase, which suggests that pleiotropic mutation(s) compensating for the loss of MA4079 aVected catabolism. The data presented point toward a role of MA4079 to enable M. acetivorans to properly acclimate to CO

Presence of Bradyrhizobium sp. under Continental Conditions in Central Europe

Soil samples from different locations with varied soybean cultivation histories were taken from arable fields in 2018 in East Germany and Poland (Lower Silesia) to evaluate the specific microsymbionts of the soybean, Bradyrhizobium japonicum, one to seven years after inoculation. Soybeans were grown in the selected farms between 2011 and 2017. The aim of the experiment was to investigate whether there is a difference in rhizobia contents in soils in which soybeans have been recultivated after one to seven years break, and whether this could lead to differences in soybean plant growth. The obtained soil samples were directly transferred into containers, then sterilized soybean seeds were sown into pots in the greenhouse. After 94 days of growth, the plants were harvested and various parameters such as the nodular mass, number of nodules, and dry matter in the individual plant parts were determined. In addition, the relative abundances of Bradyrhizobium sp. in soil samples were identified by sequencing. No major decline in Bradyrhizobium sp. concentration could be observed due to a longer interruption of soybean cultivation. Soil properties such as pH, P, and Mg contents did not show a significant influence on the nodule mass or number, but seem to have an influence on the relative abundance of Bradyrhizobium sp. The investigations have shown that Bradyrhizobium japonicum persists in arable soils even under Central European site conditions and enters into an effective symbiosis with soybeans for up to seven years.:Introduction Materials and Methods Results Discussion Supplementary Materials Author Contributions Funding Acknowledgments Conflicts of Interest Reference

Presence of Bradyrhizobium sp. under Continental Conditions in Central Europe

Soil samples from different locations with varied soybean cultivation histories were taken from arable fields in 2018 in East Germany and Poland (Lower Silesia) to evaluate the specific microsymbionts of the soybean, Bradyrhizobium japonicum, one to seven years after inoculation. Soybeans were grown in the selected farms between 2011 and 2017. The aim of the experiment was to investigate whether there is a difference in rhizobia contents in soils in which soybeans have been recultivated after one to seven years break, and whether this could lead to differences in soybean plant growth. The obtained soil samples were directly transferred into containers, then sterilized soybean seeds were sown into pots in the greenhouse. After 94 days of growth, the plants were harvested and various parameters such as the nodular mass, number of nodules, and dry matter in the individual plant parts were determined. In addition, the relative abundances of Bradyrhizobium sp. in soil samples were identified by sequencing. No major decline in Bradyrhizobium sp. concentration could be observed due to a longer interruption of soybean cultivation. Soil properties such as pH, P, and Mg contents did not show a significant influence on the nodule mass or number, but seem to have an influence on the relative abundance of Bradyrhizobium sp. The investigations have shown that Bradyrhizobium japonicum persists in arable soils even under Central European site conditions and enters into an effective symbiosis with soybeans for up to seven years

Joint project: Umwandlungsmechanismen in Bentonitbarrieren - Subproject B: Einfluss von mikrobiellen Prozessen auf die Bentonitumwandlung

Concerning the deep geological disposal of high-level radioactive waste (HLW), bentonite can be used because of its high swelling capacity and its low hydraulic conductivity as geo-technical barrier and buffering material in between the waste-containing canister (technical barrier) and the surrounding host rock (geological barrier). There are still many gaps in process understanding of bentonite transformations, especially in dependence of different temperatures and pore waters. Within the joint-project UMB (“Umwandlungsmechanismen in Bentonitbarrieren”), the co-operation partner Gesellschaft für Anlagen- und Reaktorsicherheit (GRS) mbH (Repository Safety Analysis), the University of Greifswald (Institute for Geography and Geology), the Federal Institute for Geosciences and Natural Resources (BGR, section of technical mineralogy), the Technical University of Munich (TUM; chair of theoretical chemistry, quantum chemistry) and the Helmholtz-Center Dresden-Rossendorf (HZDR, Institute of Resource Ecology) are supposed to define criteria which facilitate the selection of suitable bentonites in order to use them in the deep geological repository of high-level radioactive waste. HZDR analyzed two different bentonites (B36 and SD80) regarding their microbial diversity and potential microbial activity. In dependence of repository-relevant parameters (temperature, pore water, presence of substrates), microcosm experiments were set up at the GRS, containing the respective bentonites and Opalinus Clay pore water or cap rock solution, respectively. The long-term batches were incubated one year and two years at different temperatures (25 °C, 60 °C and 90 °C) in gastight bottles. Additionally, HZDR set up B36 short-term microcosms with Opalinus Clay pore water, which incubated for three month at 30 °C with six sampling points monitoring the microbial diversity and geochemical parameters.Für die tiefengeologische Lagerung von Wärme-entwickelnden, hoch-radioaktiven Abfällen kommen Bentonite aufgrund ihrer hohen Quellfähigkeit und ihrer geringen hydraulischen Leitfähigkeit als geo-technische Barriere in Betracht, welche sich zwischen der technischen Barriere (Behälter mit Abfall) und der geologischen Barriere (Wirtsgestein) befindet. Im Rahmen des Verbundprojektes „UMB“ (Umwandlungsmechanismen in Bentonitbarrieren) der Kooperationspartner Gesellschaft für Anlagen- und Reaktorsicherheit (GRS) mbH (Fachbereich Endlagersicherheitsforschung), der Universität Greifswald (Institut für Geographie und Geologie), der Bundesanstalt für Geowissenschaften und Rohstoffe (BGR, Arbeitsbereich Technische Mineralogie), der Technischen Universität München (TUM; Fachgebiet Theoretische Chemie, Quantenchemie) und dem Helmholtz-Zentrum Dresden Rossendorf (HZDR Institut für Ressourcenökologie) sollen abgesicherte, objektive Kriterien zur Auswahl geeigneter Bentonite für den Einsatz in Endlagern für wärmeentwickelnde Abfälle in Tonformationen entwickelt werden. Das HZDR analysierte hierfür die Entwicklung der mikrobiellen Diversität in den Bentoniten B36 und SD80 in Abhängigkeit von verschiedenen Parametern (Porenlösung, Temperatur, Anwesenheit von Substraten) um den möglichen Einfluss von Mikroorganismen auf die Umwandlungsprozesse im Bentonit zu erfassen. Die Bentonite wurden hierfür bei der GRS (Gesellschaft für Anlagen- und Reaktorsicherheit gGmbH) mit Opalinuston-Porenlösung bzw. verdünnter Gipshut-Lösung versetzt. Die Ansätze inkubierten in gasdichten Glasflaschen bei 25 °C, 60 °C und 90 °C für jeweils ein und zwei Jahre („Langzeit“). Des Weiteren wurden am HZDR B36 Mikrokosmen mit Opalinustonporenlösung angesetzt, welche für drei Monate bei 30 °C inkubierten („Kurzzeit“). Über die drei Monate verteilt wurden sechs Probenahmen durchgeführt, und die mikrobielle Diversität sowie ausgewählte geochemische Paramater bestimmt

Friend or Foe? Microbial impact of Calcigel bentonite on metal materials used for nuclear waste repository

Multi-barrier concept is a favorable option to store high-level nuclear waste (HLW) in a deep geological repository. Bentonites are processed clay materials that are considered as a geotechnical barrier for metal containers storing HLW. To understand the impact of indigenous microorganisms from bentonites on these metal materials, anaerobic microcosms incubating Calcigel bentonite, synthetic Opalinus clay (OPA) porewater, lactate (one of the organic acids in natural OPA porewater) or H2 gas (product from anaerobic metal corrosion) with or without cast iron metal plates were conducted for up to 9 months in triplicates for each condition and time point (sampling every 3 months). The amplicon sequencing targeting V4 region of 16S rRNA genes showed that microbial communities of raw Calcigel bentonites mainly comprised phyla Acidobacteria, Actinobacteria, Chloroflexi, Firmicutes, Proteobacteria and Methylomirabilota. In the microcosms with lactate, enrichment of Bacillaceae (Firmicutes) and uncultured MB-A2-108 (Actinobacteriota) were observed; whereas in the presence of both lactate and cast iron, genera of Firmicutes, namely Desulfotomaculum, Desulfitobacterium and Desulfallas-Sporotomaculum, were highly enriched (relative abundance ranged from 60% to 95%) associating with large decrease in sulfate and lactate concentration. These bacteria appeared to be driven by H2 gas generated from metal corrosion. Moreover, SEM-EDX analyses showed that the metal surface was corroded and covered by a carbonate passivation layer. In this layer, FeS appeared to be formed, further suggesting the influence on cast iron corrosion and formation of secondary minerals induced by sulfate-reducing bacteria.On the other hand, we supplied N2 gas mixed with H2 and CO2 (80:10:10) to stimulate growth of H2-oxidizing sulfate reducers. GC analyses showed that in the microcosms without cast iron, the content of H2 gas in the headspace decreased accompanying with decrease in sulfate concentration (measured via IC). However, in the microcosms with cast iron we noted large accumulation of H2 gas (~ 5 times more than initial value) and greater decrease in sulfate concentration. Similarly, surface corrosion was visible by SEM-EDX, and thre carbonate passivation layer with possible FeS precipitates was formed on the metal surface but in a shorter timeframe (3 months). Hence, we speculated that certain autotrophic H2-oxidizing sulfate reducers also corroded cast iron metal, and their taxonomy and mechanisms will be identified using metagenomic approaches.Altogether we concluded that microbial communities in Calcigel bentonites lead to microbially induced corrosion for cast iron under certain conditions, yet interestingly, the formation of passivation layer enhances the resistance for further metal corrosion. The actual impact of indigenous microorganisms in different bentonites, either disadvantageous or beneficial, on metal containers for HLW requires comprehensive investigations

Experimental Data: Endocytosis is a means of uranium(VI) uptake in tobacco (Nicotiana tabacum) BY-2 cells

The interaction of tobacco (Nicotiana tabacum) BY-2 cells with uranyl(VI) nitrate in phosphate-deficient medium was investigated. The hypothesis was that endocytosis is a means of uranium uptake in these cells. Analysis was in the form of physiological studies (growth and viability), electron microscopy, proteomics and biochemical studies

Development of -Lactamase as a Tool for Monitoring Conditional Gene Expression by a Tetracycline-Riboswitch in Methanosarcina acetivorans

The use of reporter gene fusions to assess cellular processes such as protein targeting and regulation of transcription or translation is established technology in archaeal, bacterial, and eukaryal genetics. Fluorescent proteins or enzymes resulting in chromogenic substrate turnover, like -galactosidase, have been particularly useful for microscopic and screening purposes. However, application of such methodology is of limited use for strictly anaerobic organisms due to the requirement of molecular oxygen for chromophore formation or color development. We have developed -lactamase from Escherichia coli (encoded by bla) in conjunction with the chromogenic substrate nitrocefin into a reporter system usable under anaerobic conditions for the methanogenic archaeon Methanosarcina acetivorans. By using a signal peptide of a putative flagellin from M. acetivorans and different catabolic promoters, we could demonstrate growth substrate-dependent secretion of -lactamase, facilitating its use in colony screening on agar plates. Furthermore, a series of fusions comprised of a constitutive promoter and sequences encoding variants of the synthetic tetracyclineresponsive riboswitch (tc-RS) was created to characterize its influence on translation initiation in M. acetivorans. One tc-RS variant resulted in more than 11-fold tetracycline-dependent regulation of bla expression, which is in the range of regulation by naturally occurring riboswitches. Thus, tc-RS fusions represent the first solely cis-active, that is, factor-independent system for controlled gene expression in Archaea