The microbiology programme for UK Nirex

Summary The Nirex Safety Assessment Research Programme (NSARP) considers the effect of microbial action on the repository near field. The upper limit of growth for natural soil organisms appears to be pH 12.25. Environmental conditions in the repository will probably allow slow growth particularly on damp wastes. Experiments using packed columns of waste and concrete have shown that an extremely high pH is not conducive to rapid microbial growth. However, viable populations can exist within regions adjacent to the concrete, e.g. where a surface film coats the concrete. Carbon dioxide and methane will be produced by microbial action within the repository but actual rates of production will be lower than that in a domestic landfill. The cellulosic fraction of waste is the main determinant of cell growth. It appears to be the sugar acids arising from alkaline degradation of cellulose which cause enhanced plutonium solubility. The potentially beneficial reduction of chemically derived polyhydroxy acids by the microorganisms is possible. A mathematical model has been constructed to describe the main features of biological action in the repository.     

Modelling of microbial degradation processes: The behaviour ofmicroorganisms in a waste repository

Summary In a repository for radioactive disposal the waste material is kept in place by several shells and boundaries to prevent a long term recycling of the material into the environment. Present investigations on various chemical and biological processes can be extrapolated into future centuries only with great uncertainty. Models may therefore be a good tool to forecast processes which may occur within the repository and to estimate whether the barriers present will prevent the leaching of waste material within a given time span. A mathematical model is described based on an experimental laboratory setup, a microcosm described by West et al.^19–22 simulating in a laboratory system repository conditions for a Swiss L/ILW repository. It includes microbial as well as physico-chemical processes. These simulations indicate that biological processes such as gas formation or proton release should also be included into the safety assessment of the repositories.     

Disposal of low- and intermediate-level waste in Switzerland: Basic aspects of potential relevance to microbial effects

Summary Current projects for the disposal of low-and intermediate-level radioactive waste in Switzerland are based on the concept of a horizontally accessed repository under a hillside. Most of the waste to be disposed of in such a repository is operational and decommissioning waste from nuclear power plants and wastes from medicine, industry and research. This waste is generally solidified in cement and placed in steel drums or concrete containers. Once the by the anaerobic corrosion of steel and by microbial degradation of organic material, to escape from the near field. Valanginian marl, which is one of three envisaged options for the host rock, is characterized by a high carbonate content, up to 75% in some locations. The organic content of the marl is between 1% and 2%, while pyrite is present in concentrations up to 5%. The groundwater is reducing, and its pH tends to lie in the neutral to slightly alkaline range. Potentially important microbial effects on the long-term performance of the system are microbial degradation of barrier materials and organics, the effect of microorganisms on sorption, and their role as catalysts.     

Consideration of microbiology in modelling the near field of a L/ILW repository

Summary The long-term safety of proposed repositories for nuclear waste is demonstrated by the use of chains of mathematical models describing the performance of the various barriers to radionuclide mobilisation, transport, release into the biosphere and eventual uptake by man. Microbial contamination of such repositories is to be expected, and hence the extent and consequences of microbial activity must also be quantified. This paper describes a modelling approach to determine the maximum microbial activity in the near field of a repository, which can thus be related to maximum possible degradation of performance. The approach is illustrated by application to a proposed Swiss repository for low- and intermediate-level waste (L/ILW), which is immobilised in concrete and emplaced in a marl host rock.     

The potential significance of microbial activity in radioactive waste disposal

Summary Active microorganisms can exist in any proposed environment if the basic requirements for life are satisfied, i.e. a suitable temperature and pH, the presence of the necessary nutrients and water. If conditions are not favourable microbes may survive in a dormant state until a change will allow activity. In local pockets microenvironments may become established where microbial activity may increase leading to altered environmental conditions and to changes in the near-field, e.g. degradation and breakdown of barriers, gas generation and/or uptake and transport of nuclides.     

Microbial energetics applied to waste repositories

Summary Through their catalytic abilities microbes can increase rates of chemical reactions which would take a very long time to reach equilibrium under abiotic conditions. Microbes also alter the concentration and composition of chemicals in the environment, thereby creating new conditions for further biological and chemical reactions. Rates of degradation and possible indirect consequences on leaching rates in waste repositories are a function of the presence or absence of microbes and of the conditions which allow them to become catalytically active.Microbially mediated reactions are no exception to the rule that all chemical processes are basically governed by thermodynamic laws. Naturally occurring processes proceed in the direction that leads to the minimal potential energy level attained when equilibrium is reached. A continuous supply of energy to an ecosystem in the form of biochemically unstable compounds maintains non-equilibrium conditions, a prerequisite for all chemotrophic life. Energy is released as a chemical reaction progresses towards equilibrium. Microbes scavenge that portion of the free energy of reaction (G_r) which can be converted into biochemically usable forms during the chemical oxidation processes. As electrontransfer catalysts, the microorganisms mediate reactions which are thermodynamically possible thereby stimulating reaction rates. Decomposition and mineralization in systems without a continuous supply of substrates and oxidants will lead to equilibria with minimal free energy levels at which point further microbial action would cease. The differences in the free energy levels of reactions (G_r), represent the maximal energy which is available to microorganisms for maintenance and growth. How much of the released free energy will be conserved in energy-rich bonds, compounds (e.g. ATP), and chemical potentials (e.g. emf) useful for biosynthesis and biological work is characteristic for the microbes involved and the processes and metabolic routes employed.Materials whose elements are not present in the most oxidized form attainable in the oxic environment of our planet are potentially reactive. Microbial activities are associated only with chemical reactions whose free energy changes are exergonic. This should be kept in mind for all investigations related to the role of microbes in repositories or in the layout of proper waste storage conditions. Rigorous application of thermodynamic concepts to environmental microbiology allows one to develop models and design experiments which are often difficult to conceive of in complex natural systems from physiological information alone. Thermodynamic considerations also aid in selecting proper deposition conditions and in carrying out thoughtful experiments in areas related to microbial ecology of waste repositories.     

Microbial dissolution and stabilization of toxic metals and radionuclides in mixed wastes

Summary Microbial activity in mixed wastes can have an appreciable effect on the dissolution or precipitation of toxic metals and radionuclides. Fundamental information on microbial dissolution and stabilization (immobilization) of toxic metals and radionuclides, in particular actinides and fission products, in nuclear wastes under various microbial process conditions, e.g., aerobic, denitrifying, iron-reducing, fermentative, sulfate-reducing, and methanogenic conditions is very limited. Microbial transformations of typical waste components such as metal oxides, metal coprecipitates, naturally occurring minerals, and metal organic complexes are reviewed. Such information can be useful in the development of 1) predictive models on the fate and long-term transport of toxic metals and radionuclides from waste disposal sites, and 2) biotechnological applications of waste treatment leading to volume reduction and stabilization as wall as recovery and recycling of radionuclides and toxic metals.     

Microbial potential in deep-sea sediments

Summary In spite of high pressures and low temperatures in abyssal sediments of the North Atlantic Ocean, bacterial activity is evident and highest in the top 10 cm. At these locations the input of degradable organic material to the deep-sea bottom is low. Oxygen, therefore, remains the dominant oxidant in surface sediments. Although alternative electron acceptors like nitrate, oxidized manganese and sulfate are present in large amounts, they are not utilized in this natural habitat. In sediment cores which were collected from the site for laboratory perturbation studies, it was possible to stimulate microbially mediated processes which are dormant in situ. When the oxygen supply was cut off, nitrate and manganese reduction occurred. Denitrification was the major process observed in the upper anoxic layers, while nitrate-ammonification and manganese reduction occurred in deeper sediment strata (4–8 cm). This is evidence for the presence of a variety of different bacteria and of an anaerobic heterotrophic potential. Most of the activity is located in the top 10 cm of these sediments. The shift to anaerobiosis initiates microbial activities through which metals are converted into their mobile species at the lowered redox potential. Evaluation of the suitability of the deep sea as a repository for waste materials will have to account for the large dormant potential of microbial activities and the consequences of their release by changing the environmental conditions at the sea floor.     

燕山中元古界大红峪组微生物席碎片的发现及初步研究

元古代碳酸盐岩沉积孕育了大量与微生物活动有关的沉积构造,但是对于相同时期的碎屑岩沉积环境中微生物引起的沉积构造的研究却很少。在元古代碎屑岩沉积环境中发现的最常晃的与微生物活动有关的沉积构造是皱饰构造和变余波痕,前人研究结果已经证明它们为微生物引起的沉积构造。在华北地台中元古界长城系大红峪组砂岩层面上发育一些与变余波痕和皱饰构造共生的一种特殊的沉积现象,即纺锤状砂质碎片。通过研究证实这些砂质碎片是微生物与物理营力相互作用形成的,也就是微生物席碎片。其发现有益于以后对类似沉积构造的研究;也有助于认识和了解中元古代生物群演化、古生命环境。     

Heavy metal accumulation by bacteria and other microorganisms

Summary Bacteria, and other microorganisms, exhibit a number of metabolism-dependent and-independent processes or the uptake and accumulation of heavy metals and radionuclides. The removal of such harmful substances from effluents and waste waters by microbe-based technologies may provide an alternative or additional means of metal/radionuclide recovery for economic reasons and/or environmental protection. Both living and dead cells as well as products derived from or produced by microorganisms can be effective metal accumulators and there is evidence that some biomass-based clean-up processes are economically viable. However, many aspects of metal-microbe interactions remain unexploited in biotechnology and further development and application is necessary, particularly to the problem of radionuclide release into the environment.     

Psychrophilic and psychrotrophic microorganisms

Psychrophilic and psychrotrophic microorganisms have the ability to grow at 0 degree C. Psychrotrophic microorganisms have a maximum temperature for growth above 20 degrees C and are widespread in natural environments and in foods. Psychrophilic microorganisms have a maximum temperature for growth at 20 degrees C or below and are restricted to permanently cold habitats. This ability to grow at low temperature may be correlated with a lower temperature characteristic than that of the mesophiles, an increasing proportion of unsaturated fatty acids in the lipid phase of the cell membrane, which makes it more fluid, and a protein conformation functional at low temperature. The relatively low maximum temperature of growth for these microorganisms is often considered to be due to the thermolability of one or more essential cellular components, particularly enzymes, while some degradative activities are enhanced, resulting in an exhaustion of cell energy, a leakage of intracellular substances or complete lysis. Psychrotrophic microorganisms are well-known for their degradative activities in foods. Some are pathogenic or toxinogenic for man, animals or plants. However in natural microbial ecosystems psychrotrophic and psychrophilic microorganisms can play a large role in the biodegradation of organic matter during cold seasons.     

The Swedish final repository and the possible risk of interactions by microbial activities

Summary The final Swedish repository for low- and intermediate-level nuclear waste is described, and some of the possible problems caused by microbial activity during storage are discussed. Microbial degradation of bitumen constitutes one of the greatest risks in the silo part of a repository. The production of carbon dioxide due to both aerobic and anaerobic processes might lead to a decrease in the pH of the water, inducing corrosion of the metal construction and storage containers, with large amounts of hydrogen gas being produced. A risk assessment for the repository must thus take into account the various activities of microbes.     

Evaluating the fate of genetically modified microorganisms in the environment: Are they inherently less fit?

Genetically modified microorganisms hold great promise for environmental applications. Nonetheless, some may have unintended adverse effects. Of particular concern for risk assessment is the simple fact that microorganisms are self-replicating entities, so that it may be impossible to control an adverse effect simply by discontinuing further releases of the organism. It has been suggested, however, that genetically modified microorganisms will be poor competitors and therefore unable to persist in the wild due to energetic inefficiency, disruption of genomic coadaptation, or domestication. Many studies support the hypothesis that genetically modified microorganisms are less fit than their progenitors, but there are a few noteworthy counter-examples in which genetic modifications unexpectedly enhance competitive fitness. Furthermore, subsequent evolution may eliminate the maladaptive effects of some genes, increasing the likelihood that a modified organism or its engineered genes will persist. Evaluating the likelihood that a genetically modified microorganism or its engineered genes will persist is a complex ecological and evolutionary problem. Therefore, an efficient regulatory framework would require such evaluations only when there are plausible scenarios for significant adverse environmental effects.     

Microbial methylation of benzenethiols and release of methylthiobenzenes

Three phylogenetically diverse microorganisms methylated several different chloro- and nitro-substituted benzenethiols to yield the corresponding methylthiobenzenes. These products were identified by gas chromatography-mass spectrometry. In several cases large percentages of the methylthio products were released by intact cells into the medium, suggesting that microbial methylation of xenobiotic thiols may be a significant biotransformation in many ecosystems.     

Microbial legradation of bitumen

Summary Bitumen is commonly employed as a matrix for the long-term storage of low and intermediate level radioactive waste. As bitumen can be degraded by microbial activity, it is of great significance to determine the rates at which it may occur in nuclear waste repositories.Experiments have been carried out under optimal culture conditions using bitumen with a highly increased surface area. The potential of different microbial consortia to degrade bitumen has been examined. The investigations showed clearly that bitumen-degrading organisms are ubiquitous. In general the organisms formed biofilms on the accessible substrate surface area. Under oxic culture conditions a bitumen degradation rate of 20–50 g bitumen · m^–2· y^–1 leading to a CO₂ liberation of 15–40 l was observed. Anoxic conditions yielded a 100 times smaller degradation rate of 0.2–0.6 g bitumen · m^–2 · y^–1 and a CO₂ production of 0.15–0.45 l.Based on linear extrapolation the experimentally determined degradation rates would lead to a 25–70% deterioration of the bitumen matrix under oxic and 0.3–0.8% under anoxic conditions within 1000 years.     

Plant growth stimulation by inoculation with symbiotic and associative rhizosphere microorganisms

SelectedRhizobium bacteria, arbuscular mycorrhiza-forming (AM) fungi and associative bacteria have been shown to stimulate the growth of legumes, gramineae and cruciferae in field experiments on different soil types in temperate regions. A combination of microorganisms with different metabolic capacities (N₂-fixation, P-mobilization; production of phytohormones and antibiotics) can partly surpass the effect of single inoculations, or can produce a positive effect where single inoculations are ineffective. Growth stimulation by inoculation requires microorganisms with phytoeffective metabolic characteristics and the ability to survive in the rhizosphere during the growth period. Another prerequisite is an adequate supply of plant assimilates for the production of microbial phytoeffective metabolites. Type of inoculum, method of inoculation and agricultural measures can influence the effect of the inoculation. Research is necessary to extend our knowledge both of basic principles, and about using microorganisms in practice.     

Microbial degradation of bitumen

Summary A blown bitumen Mexphalte R 90/40 with a high content of saturated hydrocarbons was degraded by several microorganisms to the same extent. In batch cultures ofSaccharomycopsis lipolytica, maximal biodegradation was estimated to be about 9% w/w, 3.2·10^–3 g/cm² and 3.1·10^–3 cm of degraded bitumen. The Mexphalte R 90/40 degradation rate was closely coupled to biofilm formation. The microbial activity concerned predominantly the oxidation of saturated hydrocarbons. A direct distillation bitumen 80/100 with a low content of saturated hydrocarbons and a high content of aromatic hydrocarbons and resins was more resistant to biodegradation.     

Microbial methylation of benzenethiols and release of methylthiobenzenes

Summary Three phylogenetically diverse microorganisms methylated several different chloro- and nitro-substituted benzenethiols to yield the corresponding methylthiobenzenes. These products were identified by gas chromatography-mass spectrometry. In several cases large percentages of the methylthio products were released by intact cells into the medium, suggesting that microbial methylation of xenobiotic thiols may be a significant biotransformation in many ecosystems.Acknowledgments. This investigation was supported by research grant ES 02639 from the National Institute for Environmental Health Sciences, and an equipment grant (RR07013) from the Biomedical Research Support grant program. Division of Research Resource, NIH. We thank Steve Hawthorne for assistance in performing GC-MS analyses.