In situ bacterial colonization of compacted bentonite under deep geological high-level radioactive waste repository conditions

Subsurface microorganisms are expected to invade, colonize, and influence the safety performance of deep geological spent nuclear fuel (SNF) repositories. An understanding of the interactions of subsurface dwelling microbial communities with the storage is thus essential. For this to be achieved, experiments must be conducted under in situ conditions. We investigated the presence of groundwater microorganisms in repository bentonite saturated with groundwater recovered from tests conducted at the Äspö underground Hard Rock Laboratory in Sweden. A 16S ribosomal RNA and dissimilatory bisulfite reductase gene distribution between the bentonite and groundwater samples suggested that the sulfate-reducing bacteria widespread in the aquifers were not common in the clay. Aerophilic bacteria could be cultured from samples run at ≤55°C but not at ≥67°C. Generally, the largely gram-negative groundwater microorganisms were poorly represented in the bentonite while the gram-positive bacteria commonly found in the clay predominated. Thus, bentonite compacted to a density of approximately 2 g cm^?3 together with elevated temperatures might discourage the mass introduction of the predominantly mesophilic granitic aquifer bacteria into future SNF repositories in the long run.     

Analysis of copper corrosion in compacted bentonite clay as a function of clay density and growth conditions for sulfate‐reducing bacteria

Aims: To investigate the relationships between sulfate‐reducing bacteria (SRB), growth conditions, bentonite densities and copper sulfide generation under circumstances relevant to underground, high‐level radioactive waste repositories. Methods and Results: Experiments took place 450 m underground, connected under in situ pressure to groundwater containing SRB. The microbial reduction of sulfate to sulfide and subsequent corrosion of copper test plates buried in compacted bentonite were analysed using radioactive sulfur (³⁵SO₄^2?) as tracer. Mass distribution of copper sulfide on the plates indicated a diffusive process. The relationship between average diffusion coefficients (D_s) and tested density (ρ) was linear. D_s (m² s^?1) = ?0·004 × ρ (kg m^?3) + 8·2, decreasing by 0·2 D_s units per 50 kg m^?3 increase in density, from 1·2 × 10^?11 m² s^?1 at 1750 kg m^?3 to 0·2 × 10^?11 m² s^?1 at 2000 kg m^?3. Conclusions: It is possible that sulfide corrosion of waste canisters in future radioactive waste repositories depends mainly on sulfide concentration at the boundary between groundwater and the buffer, which in turn depends on SRB growth conditions (e.g., sulfate accessibility, carbon availability and electron donors) and geochemical parameters (e.g., presence of ferrous iron, which immobilizes sulfide). Maintaining high bentonite density is also important in mitigating canister corrosion. Significance and Impact of the Study: The sulfide diffusion coefficients can be used in safety calculations regarding waste canister corrosion. The work supports findings that microbial activity in compacted bentonite will be restricted. The study emphasizes the importance of growth conditions for sulfate reduction at the groundwater boundary of the bentonite buffer and linked sulfide production.     

Distribution and activity of microorganisms in the deep repository for liquid radioactive waste at the Siberian Chemical Combine

The physicochemical conditions, composition of microbial communities, and the rates of anaerobic processes in the deep sandy horizons used as a repository for liquid radioactive wastes (LRW) at the Siberian Chemical Combine (Seversk, Tomsk oblast), were studied. Formation waters from the observation wells drilled into the production horizons of the radioactive waste disposal site were found to be inhabited by microorganisms of different physiological groups, including aerobic organotrophs, anaerobic fermentative, denitrifying, sulfate-reducing, and methanogenic bacteria. The density of microbial population, as determined by cultural methods, was low and usually did not exceed 10(4) cells/ml. Enrichment cultures of microorganisms producing gases (hydrogen, methane, carbon dioxide, and hydrogen sulfide) and capable of participation in the precipitation of metal sulfides were obtained from the waters of production horizons. The contemporary processes of sulfate reduction and methanogenesis were assayed; the rates of these terminal processes of organic matter destruction were found to be low. The denitrifying bacteria from the underground repository were capable of reducing the nitrates contained in the wastes, provided sources of energy and biogenic elements were available. Biosorption of radionuclides by the biomass of aerobic bacteria isolated from groundwater was demonstrated. The results obtained give us insight into the functional structure of the microbial community inhabiting the waters of repository production horizons. This study indicates that the numbers and activity of microbial cells are low both inside and outside the zone of radioactive waste dispersion, in spite of the long period of waste discharge.     

Distribution and activity of microorganisms in the deep repository for liquid radioactive waste at the Siberian Chemical Combine

The physicochemical conditions, composition of microbial communities, and the rates of anaerobic processes in the deep sand horizons used as a repository for liquid radioactive wastes (LRW) at the Siberian Chemical Combine (Seversk, Tomsk oblast), were studied. Formation waters from the observation wells drilled into the horizons used for the radioactive waste disposal were found to be inhabited by microorganisms of different physiological groups, including aerobic organotrophs, anaerobic fermentative, denitrifying, sulfate-reducing, and methanogenic bacteria. The density of microbial population, as determined by cultural methods, was low and usually did not exceed 10⁴ cells/ml. Enrichment cultures of microorganisms producing gases (hydrogen, methane, carbon dioxide, and hydrogen sulfide) and capable of participation in the precipitation of metal sulfides were obtained from the waters of the disposal site. The contemporary processes of sulfate reduction and methanogenesis were assayed; the rates of these terminal processes of organic matter destruction were found to be low. The denitrifying bacteria from the deep repository were capable of reducing the nitrates contained in the wastes, provided sources of energy and biogenic elements were available. Biosorption of radionuclides by the biomass of aerobic bacteria isolated from groundwater was demonstrated. The results obtained give us insight into the functional structure of the microbial community inhabiting the waters of repository horizons. This study indicates that the numbers and activity of microbial cells are low both inside and outside the zone of radioactive waste dispersion, in spite of the long period of waste discharge.     

Radionuclide transport model for risk evaluation of high-level radioactive waste in Northwestern China

The proper disposal of high-level radioactive waste (HLRW) is highly challenging. Numerical simulations are helpful methods of evaluating the risks of radionuclide transport. This paper examines the hydrogeological conditions of a prospective HLRW repository in China. A regional radionuclide transport model is first constructed using the TOUGH2-MP/EOS7R module at a site in northwestern China to evaluate radionuclide transport behavior. A flow model calibration under a steady state shows that the simulated and observed hydraulic heads match well. Hypothetical radionuclides (⁹⁰Sr, ¹³⁷Cs, ²³⁵U and ²³⁹Pu) are assumed to be instantaneously released at three locations with large groundwater velocities. Transport modeling shows that ²³⁵U is the most sensitive element and has the plume size (2400 m) after 10,000 years. Sensitivity analyses of parameters, including the permeability, distribution coefficient and diffusion coefficient, are carried out. The results show that the distribution coefficient is the most critical parameter for nuclide transport. These findings will provide a preliminary reference for the radionuclide migration process at a prospective HLRW disposal site, which could be informative for safety analyses and further transport in and around the repository system.     

Effects of hydrogen and acetate on benzene mineralisation under sulphate-reducing conditions

Syntrophic mineralisation of benzene, as recently proposed for a sulphate-reducing enrichment culture, was tested in product inhibition experiments with acetate and hydrogen, both putative intermediates of anaerobic benzene fermentation. Using [(13)C(6)]-benzene enabled tracking the inhibition of benzene mineralisation sensitively by analysis of (13)CO(2). In noninhibited cultures, hydrogen was detected at partial pressures of 2.4 × 10(-6) ± 1.5 × 10(-6) atm. Acetate was detected at concentrations of 17 ± 2 μM. Spiking with 0.1 atm hydrogen produced a transient inhibitory effect on (13)CO(2) formation. In cultures spiked with higher amounts of hydrogen, benzene mineralisation did not restart after hydrogen consumption, possibly due to the toxic effects of the sulphide produced. An inhibitory effect was also observed when acetate was added to the cultures (0.3, 3.5 and 30 mM). Benzene mineralisation resumed after acetate was degraded to concentrations found in noninhibited cultures, indicating that acetate is another key intermediate in anaerobic benzene mineralisation. Although benzene mineralisation by a single sulphate reducer cannot be ruled out, our results strongly point to an involvement of syntrophic interactions in the process. Thermodynamic calculations revealed that, under in situ conditions, benzene fermentation to hydrogen and acetate yielded a free energy change of ΔG'=-83.1 ± 5.6 kJ mol(-1). Benzene mineralisation ceased when ΔG' values declined below -61.3 ± 5.3 kJ mol(-1) in the presence of acetate, indicating that ATP-consuming reactions are involved in the pathway.     

Physico-chemical and microbiological characteristics of groundwater from observation boreholes of a deep radioactive liquid waste repository

A radioactive liquid waste repository was found to be a habitat of the rich microbial community with a high catabolic potential. Groundwater from a depth of 162-189 m contained aerobic saprotrophic and anaerobic fermentative, sulfate-reducing, and denitrifying bacteria. Nitrate-reducing bacteria residing in this groundwater were isolated in pure cultures. Based on the results of their physiological studies, 16S rRNA sequencing, and phylogenetic analysis, the microorganisms isolated were ascribed to one phylogenetic branch, the gamma-subclass of gram-negative bacteria. Among six isolates, four belonged to the genus Acinetobacter, whereas two others belonged to the genera Comamonas and Aeromonas. The data obtained indicate that the microflora of the repository can exert a certain effect on the chemical composition of the formation fluids and bearing rocks, as well as on the migration of radionuclides.     

Physicochemical and microbiological characteristics of groundwater from observation wells of a deep radioactive liquid waste repository

A radioactive liquid waste repository was found to be the habitat of a rich microbial community with a high catabolic potential. Groundwater from a depth of 162–189 m contained aerobic saprotrophic and anaerobic fermentative, sulfate-reducing, and denitrifying bacteria. Nitrate-reducing bacteria residing in this groundwater were isolated in pure cultures. Based on the results of their physiological studies, 16S rRNA sequencing, and phylogenetic analysis, the microorganisms isolated were ascribed to one phylogenetic branch, the γ-subclass of gram-negative bacteria. Among six isolates, four belonged to the genusAcinetobacter, whereas two others belonged to the generaComamonas andAeromonas. The data obtained indicate that the microflora of the repository can exert a certain effect on the chemical composition of the formation fluids and bearing rocks, as well as on the migration of radionuclides     

Population profiles of a stable, commensalistic bacterial culture grown with toluene under sulphate-reducing conditions.

BACKGROUND: Most bacteria present in nature are not culturable in pure culture by means of classic cultivation methods (Pace NR, 1997, Science 276:734-740; Amann RI et al., 1995, Microbiol Rev 59:143-169.). However, it was recently shown that most aerobic heterotrophic bacteria could grow only on artificial media when other micro-organisms are present (Kaeberlein T et al., 2002, Science 296:1127-1129). Because the sulphate reducer Desulfobacula toluolica DSM 7467 and a bacterium (strain MV1) identified as Cellulosimicrobium sp. were not culturable unaccompanied, flow cytometry was used to highlight the strains' relation within the consortium. METHODS: DNA patterns were used to provide strain-specific information about population proliferation dynamics. Cells were grown anaerobically and fed with toluene under sulphate-reducing conditions. RESULTS: Oxidation of toluene occurred only in association with sulphate reduction and growth of D. toluolica. A characteristic chromosomal pattern, with at least six subpopulations of D. toluolica, appeared during the stationary phase, and asymmetric cell division was detected. The accompanying strain MV1 grew repeatedly to a high percentage of the culture only in certain growth phases of D. toluolica independently of the feeding substrate toluene. CONCLUSIONS: A commensalistic relation between the two strains is suggested. The repeated rapid and frequent changes of the quantities within the community subsets are indicative of very flexible adaptations to changing environmental conditions, reflecting the need for modulated cell states and the ability to use every available source of carbon and energy for survival.     

Impact assessment of ionizing radiation on human and non-human biota from the vicinity of a near-surface radioactive waste repository

This work describes the radiological assessment of the near-surface Maisiagala radioactive waste repository (Lithuania) over the period 2005–2012, with focus on water pathways and special emphasis on tritium. The study includes an assessment of the effect of post-closure upgrading, the durability of which is greater than 30 years. Both human and terrestrial non-human biota are considered, with local low-intensity forestry and small farms being the area of concern. The radiological exposure was evaluated using the RESRAD-OFFSITE, RESRAD-BIOTA and ERICA codes in combination with long-term data from a dedicated environmental monitoring programme. All measurements were performed at the Lithuanian Institute of Physics as part of this project. It is determined that, after repository upgrading, radiological exposure to humans are significantly lower than the human dose constraint of 0.2 mSv/year valid in the Republic of Lithuania. Likewise, for non-human biota, dose rates are below the ERICA/PROTECT screening levels. The potential annual effective inhalation dose that could be incurred by the highest-exposed human individual (which is due to tritiated water vapour airborne release over the most exposed area) does not exceed 0.1 μSv. Tritium-labelled drinking water appears to be the main pathway for human impact, representing about 83 % of the exposure. Annual committed effective dose (CED) values for members of the public consuming birch sap as medical practice are calculated to be several orders of magnitude below the CEDs for the same location associated with drinking of well water. The data presented here indicate that upper soil-layer samples may not provide a good indication of potential exposure to terrestrial deep-rooted trees, as demonstrated by an investigation of stratified ³H in soil moisture, expressed on a wet soil mass basis, in an area with subsurface contamination.     

Nonsporeforming bacteria in the armyworm,Pseudaletia unipuncta,under gnotobiotic conditions

Several common species of bacteria were added singly or in pairs to axenic cultures of fifth instar, Pseudaletia unipuncta larvae. Significant host mortality occurred in monoxenic and dixenic tests involving Serratia marcescens and Pseudomonas aeruginosa. Dixenic combination aided infection by S. marcescens but inhibited infection by P. aeruginosa. Investigations with the various bacteria failed to reveal any invasive mechanisms. Bacteriophage infection of S. marcescens did not measurably influence its effect on the xenic host. Infected host insects usually died at molt or at pupation. This may be related to bacterial activity noted opposite the pyloric sphincter and possible weakening of the gut or breakage at that site. An egg blackening condition was also noted but its etiology was not clarified. Most of the bacteria tested were not pathogenic under monoxenic or dixenic conditions, and some were apparently useful to the host nutritionally. Deficient diets complicated the bacteria-host relationships. The physical and chemical environment of the gut, plant-derived antibacterial substances, and enteric bacterial interactions are all important factors in determining the influence of the gut flora on the survival of the armyworm.     

Relative contributions of archaea and bacteria to microbial ammonia oxidation differ under different conditions during agricultural waste composting

The aim of this study was to compare the relative contribution of ammonia-oxidizing archaea (AOA) and bacteria (AOB) to nitrification during agricultural waste composting. The AOA and AOB amoA gene abundance and composition were determined by quantitative PCR and denaturing gradient gel electrophoresis (DGGE), respectively. The results showed that the archaeal amoA gene was abundant throughout the composting process, while the bacterial amoA gene abundance decreased to undetectable level during the thermophilic and cooling stages. DGGE showed more diverse archaeal amoA gene composition when the potential ammonia oxidation (PAO) rate reached peak values. A significant positive relationship was observed between the PAO rate and the archaeal amoA gene abundance (R2=0.554; P<0.001), indicating that archaea dominated ammonia oxidation during the thermophilic and cooling stages. Bacteria were also related to ammonia oxidation activity (R2=0.503; P=0.03) especially during the mesophilic and maturation stages.     

Viability staining and detection of metabolic activity of sourdough lactic acid bacteria under stress conditions

World Journal of Microbiology and Biotechnology - Forty-one strains of lactic acid bacteria isolated from wheat sourdoughs were exposed to acid, osmotic and oxidative stresses. Live/Dead®...     

Low- and high-level expressions of heme oxygenase-1 in cultured cells under uninduced conditions

Heme oxygenase-1 (HO-1) degrades heme into biliverdin, iron, and CO. The enzyme participates in adaptive and protective responses to oxidative stress and various inflammatory stimuli. We examined the regulation of HO-1 expression in culture cells under uninduced conditions. Observations by in situ hybridization and immunostaining showed that in cultured mouse fibroblast Balb/3T3 cells not subjected to treatment, 10-15% of cells highly expressed HO-1. The similar pattern of the expression of HO-1 was observed with mouse embryo liver BNL-CL2 cells and Chinese hamster ovary cells. The marked expression of HO-1 was related to the activation of stress-activated protein kinase and to the expression of cyclooxygenase (Cox)-2. When the cells were treated with arachidonic acid, a precursor of prostaglandin, induction of HO-1 in the HO-1-expressing cells but not in the low-expressing cells occurred. This increase was abrogated by the treatment with the Cox inhibitors, indomethacin, and dexamethasone. Neither prostaglandin H2, E2 nor F2a induced HO-1 expression. These results suggest that some cells respond to the cellular stress and intermediates of prostaglandin biosynthesis may act as endogenous stressors to induce HO-1.     

Effect of exogenous siderophores on iron uptake activity of marine bacteria under iron-limited conditions

More than 60% of species examined from a total of 421 strains of heterotrophic marine bacteria which were isolated from marine sponges and seawater were observed to have no detectable siderophore production even when Fe(III) was present in the culture medium at a concentration of 1.0 pM. The growth of one such non-siderophore-producing strain, alpha proteobacterium V0210, was stimulated under iron-limited conditions with the addition of an isolated exogenous siderophore, N,N'-bis (2,3-dihydroxybenzoyl)-O-serylserine from a Vibrio sp. Growth was also stimulated by the addition of three exogenous siderophore extracts from siderophore-producing bacteria. Radioisotope studies using (59)Fe showed that the iron uptake ability of V0210 increased only with the addition of exogenous siderophores. Biosynthesis of a hydroxamate siderophore by V0210 was shown by paper electrophoresis and chemical assays for the detection of hydroxamates and catechols. An 85-kDa iron-regulated outer membrane protein was induced only under iron-limited conditions in the presence of exogenous siderophores. This is the first report of bacterial iron uptake through an induced siderophore in response to exogenous siderophores. Our results suggest that siderophores are necessary signaling compounds for growth and for iron uptake by some non-siderophore-producing marine bacteria under iron-limited conditions.     

Mixing ability analysis of wheat cultivar mixtures under diseased and nondiseased conditions

Summary Mixing ability analyses, adapted from combining ability analyses used in plant breeding, were performed on yield and stripe rust (Puccinia striiformis) severity data for two-way mixtures among either four or five club wheat (Triticum aesitivum) cultivars grown in five environments. Initially, two statistics were calculated for each trait: general mixing ability (GMA), the average performance of a cultivar over all of the mixtures, and specific mixing ability (SMA), the deviation of a mixture from the estimated performance of the pair based on its average performance in mixtures. General mixing ability was further divided into two components: genotype performing ability (GPA), the innate ability of a cultivar to yield and resist disease in pure stand, and true general mixing ability (TGMA), the average ability of a cultivar to influence yield and disease when mixed with other cultivars. Significant mean squares for genotypes, GMA, SMA, and TGMA were found for all of the traits in most environments. Examination of TGMA and SMA revealed cultivars and cultivar combinations that were statistically better mixers than the others. Some of the significant effects were probably due to the use of cultivars that differed in height and stripe rust resistance, but for other combinations there was no apparent explanation for enhanced mixing ability.Paper No. 9132 of the Oregon Agricultural Experiment Station. Supported in part by USDA Grants 88-34106-3631 and 88-37151-3662     

High overall diversity and dominance of microdiverse relationships in salt marsh sulphate-reducing bacteria

The biogeochemistry of North Atlantic salt marshes is characterized by the interplay between the marsh grass Spartina and sulphate-reducing bacteria (SRB), which mineralize the diverse carbon substrates provided by the plants. It was hypothesized that SRB populations display high diversity within the sediment as a result of the rich spatial and chemical structuring provided by Spartina roots. A 2000-member 16S rRNA gene library, prepared with delta-proteobacterial SRB-selective primers, was analysed for diversity patterns and phylogenetic relationships. Sequence clustering detected 348 16S rRNA sequence types (ribotypes) related to delta-proteobacterial SRB, and it was estimated that a total of 623 ribotypes were present in the library. Similarity clustering showed that approximately 46% of these sequences fell into groups with < 1% divergence; thus, microheterogeneity accounts for a large portion of the observable genetic diversity. Phylogenetic comparison revealed that sequences most frequently recovered were associated with the Desulfobacteriaceae and Desulfobulbaceae families. Sequences from the Desulfovibrionaceae family were also observed, but were infrequent. Over 80% of the delta-proteobacterial ribotypes clustered with cultured representatives of Desulfosarcina, Desulfococcus and Desulfobacterium genera, suggesting that complete oxidizers with high substrate versatility dominate. The large-scale approach demonstrates the co-existence of numerous SRB-like sequences and reveals an unexpected amount of microdiversity.     

Variability in survival of Pectinatus cerevisiiphilus, strictly anaerobic bacteria, under different oxygen conditions

Survival of Pectinatus cerevisiiphilus DSM 20466 in pure culture at variable temperatures under different oxygen concentrations was measured. Survival of P. cerevisiiphilus in co-culture with Saccharomyces cerevisiae under both saturated oxygen and brewing conditions was also studied. The survival of strictly anaerobic bacteria to oxygen seems to follow the classical laws of heat resistance. The D(oxy) values of P. cerevisiiphilus , calculated as a function of oxygen level, shows that the oxygen level is important for the survival duration of the bacteria. The temperature greatly influences the oxygen resistance of P. cerevisiiphilus, which increases when the temperature decreases. P. cerevisiiphilus resists better in co-culture than in pure culture under saturated oxygen conditions. Therefore, the oxygenation of the wort does not totally eliminate the risk of beer contamination by this bacterium. Under brewing conditions in co-culture at 8 degrees C, P. cerevisiiphilus grows slowly to reach a final cell concentration up to 10(6) cells/mL in beer, which is undrinkable. Pectinatus is a strictly anaerobic bacterium; however, it is resistant under certain oxygen conditions of incubation. This resistance is considerably higher in the presence of Saccharomyces cerevisiae .     

Interactions among Glomus irregulare, arbuscular mycorrhizal spore-associated bacteria, and plant pathogens under in vitro conditions

Arbuscular mycorrhizal (AM) fungi interact with bacteria (AM fungi-associated bacteria, AMB) in the mycorrhizosphere. We previously identified a set of AMB that enhance AM fungal colonization, plant growth, and inhibit pathogens. Here, we used transformed carrot root cultures in a two-compartment plate system for further in vitro studies on interactions taking place among Glomus irregulare (syn.Glomus intraradices), AMB, and plant pathogens. We found that exudates of G. irregulare stimulated growth of all ten AMB isolates tested in multi-well plates. AMB growth stimulation was observed also during co-cultivation of three of these AMB with G. irregulare in the hyphal compartment. In addition, co-cultivation stimulated growth of G. irregulare hyphae and spore production, as well as G. irregulare root colonization. GC/MS analysis in a preliminary screening of metabolites revealed differences in concentrations of several identified but also unidentified compounds in G. irregulare hyphal exudates. Exudates in presence of three different AMB isolates co-cultivated with G. irregulare contained several additional compounds that differed in amount compared with G. irregulare alone. The results indicate that G. irregulare exudates contain carbohydrates, amino acids, and unidentified compounds that could serve as a substrate to stimulate AMB growth. With regard to effects on plant pathogens, growth inhibition of Rhizoctonia solani, Verticillium dahliae, and Pectobacterium carotovorum ssp. carotovorum was evident in the presence of the AMB isolates tested together with the G. irregulare exudates. These in vitro studies suggest that G. irregulare and AMB stimulate growth of each other and that they together seem to provide an additive effect against growth of both fungal and bacterial pathogens.