Determination and distribution of diesel components in igneous rock surrounding underground diesel storage facilities in Sweden.

In Sweden, a preliminary investigation of the contamination situation of igneous rock surrounding underground storage facilities of diesel showed that the situation was severe. The diesel was believed to have penetrated into the rock as far as 50 m from the walls of the vaults. Consequently, the risk for contamination of groundwater and recipients could not be neglected. To be able to assess the fate of diesel components in rock, both a suitable drilling method and a method for the determination of a wide range of diesel components were needed. The analytical method presented made it possible to quantify a number of hydrocarbons in rock samples collected with triple-tube core drilling. The samples were dissolved in hydrofluoric acid (HF) with hexane in Teflon centrifuge tubes. After digestion of the rock, extraction of the analytes with hexane was performed. Determination of the individual hydrocarbons present was done with gas chromatography-mass spectrometry (GC-MS). The method was used to study the environmental impact of the underground storage of diesel. The drilling method enabled sampling without contamination risks. Our data show that the major transport of diesel components in rock occurs through fracture systems and that diffusion of diesel through the rock is of minor importance. The results have drastically changed the view of the contamination situation of diesel in the vicinity of storage facilities in hard rock in Sweden.

Occurrence and identification of microorganisms in compacted clay-based buffer material designed for use in a nuclear fuel waste disposal vault.

A full-scale nuclear fuel waste disposal container experiment was carried out 240 m below ground in an underground granitic rock research laboratory in Canada. An electric heater was surrounded by buffer material composed of sand and bentonite clay and provided heat equivalent to what is anticipated in a Canadian nuclear fuel waste repository. During the experiment, the heat caused a mass transport of water and moisture content gradients developed in the buffer ranging from 13% closest to the heater to 23% at the rock wall of the deposition hole. Upon decommissioning after 2.5 years, microorganisms could be cultured from all samples having a moisture content above 15% but not from samples with a moisture content below 15%. Heterotrophic aerobic and anaerobic bacteria were found in numbers ranging from 10(1) to 10(6) cells/g dry weight buffer. Approximately 10(2), or less, sulphate-reducing bacteria and methanogens per gram of dry weight buffer were also found. Identification of buffer population members was performed using Analytical Profile Index (API) strips for isolated bacteria and 16S rRNA gene sequencing for in situ samples. A total of 79 isolates from five buffer layers were identified with API strips as representing the beta, gamma and delta groups of Proteobacteria and Gram-positive bacteria. Sixty-seven 16S rRNA clones that were obtained from three buffer layers were classified into 21 clone groups representing alpha and gamma groups of Proteobacteria, Gram-positive bacteria, and a yeast. Approximately 20% of the population comprised Gram-positive bacteria. Members of the genera Amycolatopsis, Bacillus, and Nocardia predominated. Among Gram-negative bacteria, the genera Acinetobacter and Pseudomonas predominated. Analysis of lipid biomarker signatures and in situ leucine uptake demonstrated that the buffer population was viable. The results suggest that a nuclear fuel waste buffer will be populated by active microorganisms only if the moisture content is above a value where free water is available for active life.

Diversity and distribution of subterranean bacteria in groundwater at Oklo in Gabon, Africa, as determined by 16S rRNA gene sequencing.

This paper describes how ground water was sampled, DNA extracted, amplified and cloned and how information available in the ribosomal 16S rRNA gene was used for mapping diversity and distribution of subterranean bacteria in groundwater at the Bangombé site in the Oklo region. The results showed that this site was inhabited by a diversified population of bacteria. Each borehole was dominated by species that did not dominate in any of the other boreholes; a result that probably reflects documented differences in the geochemical environment. Two of the sequences obtained were identified at genus level to represent Acinetobacter and Zoogloea, but most of the 44 sequences found were only distantly related to species in the DNA database. The deepest borehole, BAX01 (105 m), had the highest number of bacteria and also total organic carbon (TOC). This borehole harboured only Proteobacteria beta group sequences while sequences related to Proteobacteria beta, gamma and delta groups and Gram-positive bacteria were found in the other four boreholes. Two of the boreholes, BAX02 (34 m) and BAX04 (10 m) had many 16S rRNA gene sequences in common and also had similar counts of bacteria, content of TOC, pH and equal conductivity, suggesting a hydraulic connection between them.

Benefits associated with the stalk of Gallionella ferruginea, evaluated by comparison of a stalk-forming and a non-stalk-forming strain and biofilm studies in situ.

Factors that regulate and induce stalk formation by the iron-oxidizing and stalk-forming bacterium Gallionella ferruginea were studied in laboratory cultures and in situ. A stalk-forming strain, Sta(+), and a non-stalk-forming strain, Sta(-), were used for comparative studies of the benefits associated with the stalk. Two different growth media were used: a ferrous sulfide medium (FS-medium), with slow oxidation of iron giving high concentrations of toxic oxygen radicals and a ferrous carbonate medium (FC-medium), with fast iron oxidation giving low concentration of the toxic oxygen radicals. It was found that Sta(+) cells grown in the FS-medium survived 3 weeks longer than Sta(-) cells grown in the FS-medium. When each strain was grown in the FC-medium, the Sta(-) cells had an advantage and survived 8 weeks longer than the Sta(+) cells. No difference in survival was found for Sta(+) cells grown in FS-medium compared to growth in FC-medium. In laboratory cultures, the average stalk length per cell values were 7-2.5 times higher (92 h and 150-300 h growth, respectively) in a medium with 620 µM iron than in a medium with 290 µM iron. Gallionella ferruginea Sta(+) outcompeted Sta(-) cells when inoculated as mixed populations in FC-medium. It has previously been suggested that stalk formation in vitro is induced by oxygen. To confirm this observation, biofilm development in natural waters was studied in two wells, one with trace amounts of oxygen (LH) and one without (TH). A dense biofilm developed on surfaces exposed to flowing well LH water, but no biofilm developed in well TH. Stalks were formed in water samples from both wells when allowed to make contact with air. This work demonstrates for the first time that the stalk has a protecting function against the toxic oxygen radicals formed during the chemical iron oxidation. It also shows that it is the oxidation rate of the ferrous iron and not its concentration that is harmful to the cells. The stalk gives G. ferruginea a unique possibility to colonize and survive in habitats with high contents of iron, inaccessible for bacteria without a defense system against the oxidation of iron.

Phylogeny and phenotypic characterization of the stalk-forming and iron-oxidizing bacterium Gallionella ferruginea.

The 16S rRNA gene of Gallionella ferruginea was amplified by polymerase chain reaction and sequenced by direct double-stranded sequencing. The phylogenetic analysis placed G. ferruginea in the beta-group of the Proteobacteria, with 90.0% similarity to Nitrosolobus multiformis and 88.6% to Rhodocyclus purpureus. The published phenotypic characteristics of G. ferruginea were compiled and supplemented with growth experiments using ferrous iron, thiosulphate and sulphide as electron donor, and nitrate as nitrogen source. G. ferruginea is a Gram-negative, curved bacterium with one polar flagellum. It grows auto- and mixotrophically with CO2, glucose, fructose and sucrose as carbon sources, ferrous iron as an electron donor and ammonium or nitrate as nitrogen sources. Two G. ferruginea specific oligonucleotide probes are suggested. An iron-oxidizing bacterium without stalk-forming ability, but with the same growth pattern as G. ferruginea, was identified as G. ferruginea by comparison of highly variable parts of the 16S rRNA gene. This indicates that the stalk is not essential for growth.