Nitrate and nitrite bacterial reduction at alkaline pH and high nitrate concentrations, comparison of acetate versus dihydrogen as electron donors.

This study assesses bacterial denitrification at alkaline pH, up to 12, and high nitrate concentration, up to 400 mM. Two types of electron donors organic (acetate) and inorganic (dihydrogen) were compared. With both types of electron donors, nitrite reduction was the key step, likely to increase the pH and lead to nitrite accumulation. Firstly, an acclimation process was used: nitrate was progressively increased in three cultures set at pH 9, 10, or 11. This method allowed to observe for the first time nitrate reduction up to pH 10 and 100 mM nitrate with dihydrogen, or up to pH 10 and 400 mM nitrate with acetate. Nitrate reduction kinetics were faster in the presence of acetate. To investigate further the impact of the type of electron donor, a transition from acetate to dihydrogen was tested, and the pH evolution was modelled. Denitrification with dihydrogen strongly increases the pH while with acetate the pH evolution depends on the initial pH. The main difference is the production of acidifying CO2 during the acetate oxidation. Finally, the use of long duration cultures with a highly alkaline pH allowed a nitrate reduction up to pH 11.5 with acetate. However, no reduction was possible in hydrogenotrophy as it would have increased the pH further. Instead, bacteria used organic matter from inoculum to reduce nitrate at pH 11.5. Therefore, considering bacterial denitrification in a context of alkaline pH and high nitrate concentration an organic electron donor such as acetate is advantageous.

Adaptation of neutrophilic Paracoccus denitrificans to denitrification at highly alkaline pH.

Bacterial denitrification is widely documented at neutral pH in order to improve the removal of nitrate in wastewater treatment processes. However, certain industrial contexts generate alkaline waste and effluent containing nitrate that must be denitrified. To obtain more information on denitrification at alkaline pH, this study evaluated the possibility of adapting a neutrophilic denitrifying strain, Paracoccus denitrificans, to alkaline pH. Firstly, P. denitrificans' denitrifying activity was evaluated without acclimation in batch bioreactors at pH 7.0, 8.0, 9.0 and 10.0. Then, two acclimation methods using successive batch bioreactors and a continuous bioreactor allowed P. denitrificans to be gradually exposed to alkaline pH: from 8.5 to 11.2 in 26 and 72 days respectively. Results showed that P. denitrificans could grow and catalyse nitrate reduction (i) at pH 9.0 without acclimation, (ii) at pH 10.5 in successive batch cultures with progressively increasing pH and (iii) at pH 10.8 in continuously fed culture with a hydraulic retention time (HRT) of 8 days. It was shown that denitrification affected the pH despite the presence of carbonate buffering of the P. denitrificans growth medium. With acetate as an electron donor, the pH of a carbonate buffered medium tends towards pH 10 during the process of denitrification. Graphical abstract.

Influence of Hydrogen Electron Donor, Alkaline pH, and High Nitrate Concentrations on Microbial Denitrification: A Review.

Bacterial respiration of nitrate is a natural process of nitrate reduction, which has been industrialized to treat anthropic nitrate pollution. This process, also known as "microbial denitrification", is widely documented from the fundamental and engineering points of view for the enhancement of the removal of nitrate in wastewater. For this purpose, experiments are generally conducted with heterotrophic microbial metabolism, neutral pH and moderate nitrate concentrations (<50 mM). The present review focuses on a different approach as it aims to understand the effects of hydrogenotrophy, alkaline pH and high nitrate concentration on microbial denitrification. Hydrogen has a high energy content but its low solubility, 0.74 mM (1 atm, 30 °C), in aqueous medium limits its bioavailability, putting it at a kinetic disadvantage compared to more soluble organic compounds. For most bacteria, the optimal pH varies between 7.5 and 9.5. Outside this range, denitrification is slowed down and nitrite (NO2-) accumulates. Some alkaliphilic bacteria are able to express denitrifying activity at pH levels close to 12 thanks to specific adaptation and resistance mechanisms detailed in this manuscript, and some bacterial populations support nitrate concentrations in the range of several hundred mM to 1 M. A high concentration of nitrate generally leads to an accumulation of nitrite. Nitrite accumulation can inhibit bacterial activity and may be a cause of cell death.

Development and assessment of a simple ecological model (TRIPS) for forests contaminated by radiocesium fallout.

The management of vast forested zones contaminated by radiocesium (rCs) following the Chernobyl and Fukushima fallout is of great social and economic concern in affected areas and requires appropriate dynamic models as predictive or questioning tools. Generally, the existing radio-ecological models need less fragmented data and more ecological realism in their quantitative description of the rCs cycling processes. The model TRIPS ("Transfer of Radionuclide In Perennial vegetation Systems") developed in this study privileged an integrated approach which makes the best use of mass balance studies and available explicit experimental data for Scots pine stands. A main challenge was the differentiation and calibration of foliar absorption as well as root uptake in order to well represent the rCs biocycling. The general dynamics of rCs partitioning was simulated with a relatively good precision against an independent series of observed values. In our scenario the rCs biological cycling enters a steady-state about 15 years after the atmospheric deposits. At that time, the simulations showed an equivalent contribution of foliage and root uptake to the tree contamination. But the root uptake seems not sufficient to compensate the activity decline in the tree. The initial foliar uptake and subsequent internal transfers were confirmed to have a great possible impact on the phasing of tree contamination. An extra finding concerns the roots system acting as a buffer in the early period. The TRIPS model is particularly useful in cases where site-specific integrated datasets are available, but it could also be used with adequate caution to generic sites. This development paves the way for simplification or integration of new modules, as well as for a larger number of other applications for the Chernobyl or Fukushima forests once the appropriate data become available. According to the sensitivity analysis that involves in particular reliable estimates of net foliar uptake as well as root uptake not disconnected from rCs exchange reactions in soil.

Impact of the electron donor on in situ microbial nitrate reduction in Opalinus Clay: results from the Mont Terri rock laboratory (Switzerland).

At the Mont Terri rock laboratory (Switzerland), an in situ experiment is being carried out to examine the fate of nitrate leaching from nitrate-containing bituminized radioactive waste, in a clay host rock for geological disposal. Such a release of nitrate may cause a geochemical perturbation of the clay, possibly affecting some of the favorable characteristics of the host rock. In this in situ experiment, combined transport and reactivity of nitrate is studied inside anoxic and water-saturated chambers in a borehole in the Opalinus Clay. Continuous circulation of the solution from the borehole to the surface equipment allows a regular sampling and online monitoring of its chemical composition. In this paper, in situ microbial nitrate reduction in the Opalinus Clay is discussed, in the presence or absence of additional electron donors relevant for the disposal concept and likely to be released from nitrate-containing bituminized radioactive waste: acetate (simulating bitumen degradation products) and H2 (originating from radiolysis and corrosion in the repository). The results of these tests indicate that-in case microorganisms would be active in the repository or the surrounding clay-microbial nitrate reduction can occur using electron donors naturally present in the clay (e.g. pyrite, dissolved organic matter). Nevertheless, non-reactive transport of nitrate in the clay is expected to be the main process. In contrast, when easily oxidizable electron donors would be available (e.g. acetate and H2), the microbial activity will be strongly stimulated. Both in the presence of H2 and acetate, nitrite and nitrogenous gases are predominantly produced, although some ammonium can also be formed when H2 is present. The reduction of nitrate in the clay could have an impact on the redox conditions in the pore-water and might also lead to a gas-related perturbation of the host rock, depending on the electron donor used during denitrification.

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.

Evaluation of biotechnology-derived novel proteins for the risk of food-allergic potential: advances in the development of animal models and future challenges.

Increasing concern from the public about the safety of genetically modified food has made critical to have suitable methods for recognizing associated potential hazards. Hierarchical approaches to allergenicity determination were proposed, and these include evaluation of the structural and sequence homology and serological identity of novel proteins with existing allergens, measuring the resistance to proteolytic digestion and assessment of sensitizing potential using animal models. Allergic individuals have a predisposed (i.e. atopic) genetic background, and a close resemblance to this setup is therefore desirable in animal models, which is possible by using a strain of an animal species that is prone for allergic disorders. So far, none of the animal model has been validated for the purpose of hazard identification in the context of safety assessment. However, the available knowledge suggests that the judicious use of an appropriate animal model could provide important information about the allergic potential of novel proteins. This paper provides an up-to-date review of the progress made in the field of development of in vivo models in this direction and the further goals that have to be achieved.

Extension of sensitivity and uncertainty analysis for long term dose assessment of high level nuclear waste disposal sites to uncertainties in the human behaviour.

Biosphere dose conversion factors are computed for the French high-level geological waste disposal concept and to illustrate the combined probabilistic and deterministic approach. Both (135)Cs and (79)Se are used as examples. Probabilistic analyses of the system considering all parameters, as well as physical and societal parameters independently, allow quantification of their mutual impact on overall uncertainty. As physical parameter uncertainties decreased, for example with the availability of further experimental and field data, the societal uncertainties, which are less easily constrained, particularly for the long term, become more and more significant. One also has to distinguish uncertainties impacting the low dose portion of a distribution from those impacting the high dose range, the latter having logically a greater impact in an assessment situation. The use of cumulative probability curves allows us to quantify probability variations as a function of the dose estimate, with the ratio of the probability variation (slope of the curve) indicative of uncertainties of different radionuclides. In the case of (135)Cs with better constrained physical parameters, the uncertainty in human behaviour is more significant, even in the high dose range, where they increase the probability of higher doses. For both radionuclides, uncertainties impact more strongly in the intermediate than in the high dose range. In an assessment context, the focus will be on probabilities of higher dose values. The probabilistic approach can furthermore be used to construct critical groups based on a predefined probability level and to ensure that critical groups cover the expected range of uncertainty.

The significance of agricultural vs. natural ecosystem pathways in temperate climates in assessments of long-term radiological impact.

Recent developments in performance assessment biosphere models have begun to emphasise the importance of natural accumulation pathways. In contrast to the agricultural pathways, the database for natural ecosystem pathways is less well developed, leading to a mismatch in quality of representations of the two types of system. At issue is the lack of reliable soil-plant and animal ingestion transfer factors for key radionuclides in natural ecosystems. The relative importance of the agricultural vs. natural ecosystem pathways is investigated here, in the context of a temperate site in present day, Eastern France. The BIOMASS Candidate Critical Group (CCG) methodology has been applied to map a set of eight candidate critical groups derived from the present-day societal context onto physical locations within a simple model of a river catchment system. The overall assessment model has been implemented using the Aquabios code. Annual individual dose to each of the CCGs has been calculated for each of the key radionuclides (79Se, 94Nb, 99Tc, 129I, 135Cs and 237Np) released to the valley aquifer and river. In addition to the traditional agricultural pathways, lifestyle groups exploiting natural habitats are explicitly addressed. Results show the susceptibility of different candidate critical groups to different radionuclides. A reference database typical of those employed in long-term performance assessment models is employed. Doses from external exposure (94Nb) and dust inhalation (237Np) are shown to dominate agricultural food consumption by factors of more than six, but, with the reference data set, foodstuffs obtained from natural ecosystems do not contribute significantly to critical group dose and, at most, show similar exposures to the agricultural pathways. This may lead to the conclusion that natural food can be ruled out of consideration in performance assessment models. However, systematic parametric sensitivity studies carried out on soil-plant and animal ingestion transfer factors restrict the validity of this observation and demonstrate the limitations of existing databases. Remaining uncertainties can be reduced by improving structural models for performance assessment and by better characterisation of sources of locally obtained foods. Improved characterisation of radionuclide accumulation in natural ecosystems in temperate as well as alternative future climate states should complement the modelling approach.

Validating riverine transport and speciation models using nuclear reactor-derived radiocobalt.

Risk assessment of intentional or accidental discharges of toxic substances into river systems requires combined hydraulic and chemical modeling. Periodic discharges of known volumes with low radioactivity by the Beznau nuclear reactor (Switzerland) serve as validation tracers for both river flow and chemical speciation simulation. Validation of the former has been achieved by comparison of modeled and measured arrival times of radiocobalt along a 65 km transect with a maximum reaction period of 24 hours. Modeled breakthrough curves coincide well with measurements collected during three field campaigns, in spite of the fact that sorption and sedimentation processes were not activated during simulation. This gives indirect evidence of inefficient cobalt sorption. Particle/solution distribution measurements carried out during breakthrough allow further validation of our speciation approach, which is based on Tableau setup of inorganic reactions combined with sorption and organic complexation. Modeled and measured speciation results confirm recent observations of enhanced complexation of cobalt with dissolved organic substances, which significantly reduces particle sorption. The large variability of conditional stability constants for sorption and complexation reactions, for sorption site densities, and for organic ligand concentrations explains the variability of published particle-solution distribution coefficients.

Incorporating soil structure and root distribution into plant uptake models for radionuclides: toward a more physically based transfer model.

Most biosphere and contamination assessment models are based on uniform soil conditions, since single coefficients are used to describe the transfer of contaminants to the plant. Indeed, physical and chemical characteristics and root distribution are highly variable in the soil profile. These parameters have to be considered in the formulation of a more realistic soil-plant transfer model for naturally structured soils. The impact of monolith soil structure (repacked and structured) on Zn and Mn uptake by wheat was studied in a controlled tracer application (dye and radioactive) experiment. We used Brilliant Blue and Sulforhodamine B to dye flow lines and 65Zn and 54Mn to trace soil distribution and plant uptake of surface-applied particle-reactive contaminants. Spatial variation of the soil water content during irrigation and plant growth informs indirectly about tracer and root location in the soil profile. In the structured monolith, a till pan at a depth of 30 cm limited vertical water flow and root penetration into deeper soil layers and restricted tracers to the upper third of the monolith. In the repacked monolith, roots were observed at all depths and fingering flow allowed for the fast appearance of all tracers in the outflow. These differences between the two monoliths are reflected by significantly higher 54Mn and 65Zn uptake in wheat grown on the structured monolith. The higher uptake of Mn can be modelled on the basis of radionuclide and root distribution as a function of depth and using a combination of preferential flow and rooting. The considerably higher uptake of Zn requires transfer factors which account for variable biochemical uptake as a function of location.