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     

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.     

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.     

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.     

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.     

The investigation of the composition of liquid radioactive waste

In investigation the process of composition sediment of liquid unorganic radioactive waste, that are forming in cistern-selectors at PNPI RAS, it was discovered apart from great quantity of ions of different metals and radionuclides considerable maintenance of organic material (to 30% and more from volume of sediment) unknown origin. A supposition was made about its microbiological origin. Investigation shows, that the main microorganisms, setting this sediment, are the bacterious of Pseudomonas kind, capable of effectively bind in process of grow the radionuclide 90Sr, that confirms the potential posibility of using this microorganisms for bioremediation of liquid low radioactive wastes (LRW).     

Culture-dependent comparison of microbial diversity in deep granitic groundwater from two sites considered for a Swedish final repository of spent nuclear fuel

Site selection for a spent nuclear fuel (SNF) repository required analysis of microbial abundance and diversity at two Swedish sites, Forsmark and Laxemar-Simpevarp. Information about sulphate-reducing bacteria (SRB) was required, as sulphide could corrode copper SNF canisters. Total number of cells (TNC) and ATP were analysed, and plate counts and most probable number (MPN) analyses were conducted using eight media based on different electron donors and acceptors for specific microorganism physiological groups. Groundwater chemical composition and E(h) were analysed; sampling depths were 112-978 m below sea level. TNC was 5.5 × 10(3) to 4.7 × 10(5) cells mL(-1), correlating with ATP concentrations. Culturability in TNC percentage was 0.01-35.9, averaging 5.12. Culturable numbers varied greatly between sample positions and uncorrelated with depth. SRB were found in 29 samples and were below detection in three; the MPN of SRB correlated negatively with E(h), as did the MPN of acetogens. Data indicated that microbial sulphate reduction was ongoing in many sampled aquifers; published stable isotope data and modelling results supported this observation. The sites did not differ significantly, but the large data range suggested that analysis of more samples would enable detailed evaluation of microbial processes and their relationship with geochemical information.     

Mixing and sulphate-reducing activity of bacteria in swelling, compacted bentonite clay under high-level radioactive waste repository conditions

AIM: The fate of micro-organisms in the bentonite clay surrounding high-level radioactive waste (HLW)-containing copper canisters in a future Swedish underground (500 m) repository were investigated. METHODS AND RESULTS: Laboratory experiments were designed in which the mixing of various bacterial species with swelling bentonite was studied. A clear trend of fewer cultivable bacteria at depth was seen in the clay. This trend was consistent as the incubation time was increased from 8 h to 28 weeks. Sulphate-reducing bacteria were found to be active, reducing sulphate at the lowest density studied, 1.5 g cm-3, but sulphate reduction activity ceased at higher densities. CONCLUSIONS: The number of viable micro-organisms in an HLW repository bentonite clay buffer will decrease rapidly during swelling and very few viable cells will be present at full compaction. SIGNIFICANCE AND IMPACT OF THE STUDY: Sulphate-reducing bacteria will most probably not be able to induce corrosion of HLW-containing copper canisters.     

Physico-chemical characteristics of angiotensin-converting enzyme from the bovine lung

The angiotensin-converting enzyme from bovine lung was isolated by chromatography with a 25-30% yield and purified 2200-2600-fold. The active molecule concentration in the enzyme preparations was 70-100% as could be judged from titration by inhibitor SQ 20,881. The molecular mass of the enzyme according to electrophoretic data is about 132 kDa; the maximal radius of the enzyme molecule as determined by electron microscopy is 68 +/- 9 A. Five enzyme isoforms with pI of 4.85, 4.7, 4.54, 4.38 and 4.3, respectively, were identified. The kinetic parameters of hydrolysis of three synthetic peptide substrates and the constants of activation of the substrate (Z-Phe-His-Leu) hydrolysis by chloride anions were determined.     

Physico-chemical characteristics of catalases from Micrococcus sp. n

The physico-chemical properties of heme-containing and non-heme catalases isolated from the cell culture of Micrococcus sp. n. grown under intensive aeration were studied. The enzyme preparations were homogenous during polyacrylamide disc electrophoresis. The spectral and functional properties of the enzymes (e. g. specific activity, subunit molecular weight, quaternary structure, amino acid composition, immunoprecipitability, N-terminal amino acid sequences) were investigated. Monocrystals of non-heme catalase applicable for an X-ray analysis were grown and examined by X-ray spectroscopy. Both enzymes were stable upon storage in 40% ammonium sulfate for 2 months and resistant to lyophylization without any significant loss of their activity. Non-heme catalase is apparently an independent enzyme which is not derived from heme-containing catalase via dissociation, limited proteolysis or heme cleavage.     

Microbacterium isolates from the vicinity of a radioactive waste depository and their interactions with uranium

Three oligotrophic bacterial strains were cultured from the ground water of the deep-well monitoring site S15 of the Siberian radioactive waste depository Tomsk-7, Russia. They were affiliated with Actinobacteria from the genus Microbacterium. The almost fully sequenced 16S rRNA genes of two of the isolates, S15-M2 and S15-M5, were identical to those of cultured representatives of the species Microbacterium oxydans. The third isolate, S15-M4, shared 99.8% of 16S rRNA gene identity with them. The latter isolate possessed a distinct cell morphology as well as carbon source utilization pattern from the M. oxydans strains S15-M2 and S15-M5. The three isolates tolerated equal amounts of uranium, lead, copper, silver and chromium but they differed in their tolerance of cadmium and nickel. The cells of all three strains accumulated high amounts of uranium, i.e. up to 240 mg U (g dry biomass)(-1) in the case of M. oxydans S15-M2. X-ray absorption spectroscopy (XAS) analysis showed that this strain precipitated U(VI) at pH 4.5 as a meta-autunite-like phase. At pH 2, the uranium formed complexes with organically bound phosphate groups on the cell surface. The results of the XAS studies were consistent with those obtained by transmission electron microscopy (TEM) and energy dispersive X-ray analysis (EDX).     

Comparison of bacteria from deep subsurface sediment and adjacent groundwater

Samples of groundwater and the enclosing sediments were compared for densities of bacteria using direct (acridine orange direct staining) and viable (growth on 1% PTYG medium) count methodology. Sediments to a depth of 550 m were collected from boreholes at three sites on the Savannah River Site near Aiken, South Carolina, using techniques to insure a minimum of surface contamination. Clusters of wells screened at discreet intervals were established at each site. Bacterial densities in sediment were higher, by both direct and viable count, than in groundwater samples. Differences between direct and viable counts were much greater for groundwater samples than for sediment samples. Densities of bacteria in sediment ranged from less than 1.00×10⁶ bacteria/g dry weight (gdw) up to 5.01 ×10⁸ bacteria/gdw for direct counts, while viable counts were less than 1.00×10³ CFU/gdw to 4.07×10⁷ CFU/gdw. Bacteria densities in groundwater were 1.00×10³–6.31×10⁴ bacteria/ml and 5.75–4.57×10² CFU/ml for direct and viable counts, respectively. Isolates from sediment were also found to assimilate a wider variety of carbon compounds than groundwater bacteria. The data suggest that oligotrophic aquifer sediments have unique and dense bacterial communities that are attached and not reflected in groundwater found in the strata. Effective in situ bioremediation of contaimination in these aquifers may require sampling and characterization of sediment communities.     

Effect of gamma radiation on native endolithic microorganisms from a radioactive waste deposit site.

A time-course experiment was conducted to evaluate the effects of gamma radiation on the indigenous microbiota present in rock obtained from Yucca Mountain, Nevada Test Site. Microcosms were constructed by placing pulverized Yucca Mountain rock in polystyrene cylinders. Continuous exposure (96 h) at a dose rate of 1.63 Gy/min was used to mimic the near-field environment surrounding waste canisters. The expected maximum surface dose rate from one unbreached canister designed to contain spent nuclear fuels is 0.06 Gy/min. Considering the current repository packing design, multiple canisters within one vault, the cumulative dose rate may well approach that used in this experiment. The microbial communities were characterized after receiving cumulative doses of 0, 0.098, 0. 58, 2.33, 4.67, 7.01 and 9.34 kGy. Radiation-resistant microorganisms in the pulverized rock became viable but nonculturable (VBNC) after a cumulative dose of 2.33 kGy. VBNC microorganisms lose the ability to grow on media on which they have routinely been cultured in response to the environmental stress imposed (i.e. radiation) but can be detected throughout the time course using direct fluorescence microscopy techniques. Two representative exopolysaccharide-producing isolates from Yucca Mountain were exposed to the same radiation regimen in sand microcosms. One isolate was much more radiation-resistant than the other, but both had greater resistance than the general microbial community based on culturable counts. However, when respiring cell counts (VBNC) were compared after irradiation, the results would indicate much more radiation resistance of the individual isolates and the microbial community in general. These results have significant implications for underground storage of nuclear waste as they indicate that indigenous microorganisms are capable of surviving gamma irradiation in a VBNC state.     

Dewatering of phosphorus extracted from liquid swine waste

Phosphorus (P) recovery from liquid swine manure is an attractive technology when on-farm application of liquid swine manure is not an option. We developed a technology that enables separation of this P, but its high moisture content makes transportation difficult. In this work, we investigated dewatering procedures to concentrate the P product. Sludge rich in calcium phosphate (> 20% P2O5) was obtained using a field prototype, and it was further dewatered using a combination of polymer treatment and filter bags. Anionic polyacrylamide polymer treatment (> or = 20 mg/L) was effective to flocculate the P-rich sludge, which enhanced filtration and dewatering. Without polymer, filtration was incomplete due to clogging of filters. Non-woven polypropylene and monofilament filter bag fabrics with mesh size < or = 200 microm retained > 99% of suspended solids and total P. Solids content dramatically increased from about 1.5% to > 90%. These dewatered solids can be transported more economically off the farm for use as a valuable fertilizer material.     

Physico-chemical and biological characteristics of Pseudomonas aeruginosa elastase

Elastase isolated from P. aeruginosa clinical strain hydrolyzes elastin, casein, hemoglobin, ovalbumin, gelatin, fibrin, collagen. The optimum pH ensuring the activity of the enzyme is 7.8-8.0. Elastase shows maximum stability at pH 6.6-9.0. Heating at 80 degrees C for 10 minutes results in its practically complete inactivation. Elastase is a highly radiosensitive enzyme. Chelating agents and zinc, cobalt, mercury ions suppress its activity. Sodium and ammonium chlorides selectively inhibit the elastolytic, but not proteolytic activity of the enzyme. Elastase shows pronounced dermonecrotic and keratolytic action.     

Molecular epidemiology and microbiological characteristics of Cryptococcus gattii VGII isolates from China

Cryptococcus gattii (C. gattii) is a fungal pathogen that once caused an outbreak of cryptococcosis on Vancouver Island, and had spread worldwide, while few data were available in China. In this study, seven clinical isolates of C. gattii VGII were collected from 19 hospitals, Multi-locus Sequence Typing (MLST) analysis and whole-genome sequencing (WGS) was performed, combined with published data for phylogenetic analysis. In addition, in vitro antifungal susceptibility testing, phenotypic analysis, and in vivo virulence studies were performed, subsequently, histopathological analysis of lung tissue was performed. C.gattii VGII infected patients were mainly immunocompetent male, and most of them had symptoms of central nervous system (CNS) involvement. MLST results showed that isolates from China exhibited high genetic diversity, and sequence type (ST) 7 was the major ST among the isolates. Some clinical isolates showed a close phylogenetic relationship with strains from Australia and South America. All clinical isolates did not show resistance to antifungal drugs. In addition, there was no correlation between virulence factors (temperature, melanin production, and capsule size) and virulence while in vivo experiments showed significant differences in virulence among strains. Lung fungal burden and damage to lung tissue correlated with virulence, and degree of damage to lung tissue in mice may highlight differences in virulence. Our work seeks to provide useful data for molecular epidemiology, antifungal susceptibility, and virulence differences of C. gattii VGII in China.     

Gamma-cystathionase: the non-ideal gel filtration high performance liquid chromatography. Physico-chemical and immunochemical characteristics of the enzyme

The rapid method for gamma-cystathionase purification was developed. It is based on the non-ideal gel filtration HPLC. The isolated homogeneous enzyme was used for immunization and immunosorbent preparation. A monospecific polyclonal antibody was prepared. The substrate specificity of the isolated enzyme was studied.     

Reconstruction of the radionuclide spectrum of liquid radioactive waste released into the Techa river in 1949-1951

The major part of the liquid radioactive waste released by the Mayak Production Association (PA) radiochemical plant into the Techa river occurred in 1949-1951, but there is information on only one single radiochemical analysis of a water sample taken on 24 and 25 September 1951. These data are here used to assess the spectrum of radionuclides that were released between 1949 and 1951. For this purpose, details of the radiochemical methods of radionuclide extraction and radiometric measurements of beta-activity used at Mayak PA in the 1950s have been taken into account. It is concluded that the data from the radiochemical measurements agree with the theoretical composition of fission products in uranium after exposure times in the reactor (120 days) and subsequent hold times (35 days) that were typical for the procedures at that time. The results of the analysis are at variance with assumptions that underlie the current Techa river dosimetry system. They confirm the conclusion that the external doses to the Techa river residents in the critical period up to 1952 were predominantly due to short-lived fission products.