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Recent field observations have identified the widespread occurrence of fluid seepage through the eastern Mediterranean Sea floor in association with mud volcanism or along deep faults. Gas hydrates and methane seeps are frequently found in cold seep areas and were anticipated targets of the MEDINAUT/MEDINETH initiatives. The study presented herein has utilized a multi-disciplinary approach incorporating observations and sampling of visually selected sites by the manned submersible Nautile and by ship-based sediment coring and geophysical surveys. The study focuses on the biogeochemical and ecological processes and conditions related to methane seepage, especially the anaerobic oxidation of methane (AOM), associated with ascending fluids on Kazan mud volcano in the eastern Mediterranean. Sampling of adjacent box cores for studies on the microbiology, biomarkers, pore water and solid phase geochemistry allowed us to integrate different biogeochemical data within a spatially highly heterogeneous system. Geophysical results clearly indicate the spatial heterogeneity of mud volcano environments. Results from pore water geochemistry and modeling efforts indicate that the rate of AOM is similar to 6 mol m(-2) year(-1), which is lower than at active seep sites associated with conditions of focused flow, but greater than diffusion-dominated sites. Furthermore, under the non-focused flow conditions at Kazan mud volcano advective flow velocities are of the order of a few centimeters per year and gas hydrate formation is predicted to occur at a sediment depth of about 2 m and below. The methane flux through these sediments supports a large and diverse community of micro- and macrobiota, as demonstrated by carbon isotopic measurements on bulk organic matter, authigenic carbonates, specific biomarker compounds, and macrofaunal tissues. Because the AOM community appears to be able to completely oxidize methane at the rate it is seeping through the sediments, ultimate sinks for methane in this environment are authigenic carbonates and biomass. Significant differences in organic geochemical data between this site and those of other cold seep environments, even within the eastern Mediterranean mud volcanoes, indicate that the microbiological communities carrying out AOM varies depending on specific conditions such as methane flux and salinity. (C) 2004 Elsevier B.V. All rights reserved.

Although abundant geochemical data indicate that anaerobic methane oxidation occurs in marine sediments, the linkage to specific microorganisms remains unclear. In order to examine processes of methane consumption and oxidation, sediment samples from mud volcanoes at two distinct sites on the Mediterranean Ridge were collected via the submersible Nautile. Geochemical data strongly indicate that methane is oxidized under anaerobic conditions, and compound-specific carbon isotope analyses indicate that this reaction is facilitated by a consortium of archaea and bacteria. Specifically, these methane-rich sediments contain high abundances of methanogen-specific biomarkers that are significantly depleted in 13C (13C values are as low as 95). Biomarkers inferred to derive from sulfate-reducing bacteria and other heterotrophic bacteria are similarly depleted. Consistent with previous work, such depletion can be explained by consumption of 13C-depleted methane by methanogens operating in reverse and as part a consortium of organisms in which sulfate serves as the terminal electron acceptor. Moreover, our results indicate that this process is widespread in Mediterranean mud volcanoes and in some localized settings is the predominant microbiological process.

Indigenous heterotrophic marine bacteria are of great importance to global nutrient cycling. Predominant native bacteria are of ultramicrobacterial dimensions, are not associated with aggregates and must have truly remarkable abilities for substrate capture. Agar media are unsuited for the isolation of the dominant oceanic bacterioplankton. In contrast, the dilution culture technique [Button et al. (1993) Appl. Environ. Microbiol. 59, 881-891; Schut et al. (1993) Appl. Environ. Microbiol. 59, 2150-2160] leads to a successful enrichment of the dominant cell types. Up to 50% of the indigenous bacterial population in water obtained from Resurrection Bay was able to grow in dilution tubes containing only filtered, autoclaved natural sea water (FAS). Ultramicrobacteria (UMB), very small bacteria with small genomes, predominate in such cultures. Generally, dilution factors that resulted in inocula of approximately 2 cells per tube were optimal and prevented outgrowth of atypical large bacteria. Strain RB2256 [Schut et al. (1993) Appl. Environ. Microbiol. 59, 2150-2160], one of the UMB isolated by this dilution culture technique and tentatively identified as a marine Sphingomonas sp., was investigated in more detail. Although reverted from obligately oligotrophic to facultatively oligotrophic upon isolation, this strain possessed a number of traits assigned to a 'model oligotroph' and some unpredicted novel properties. The cells showed no miniaturisation upon starvation but consistently exhibited low cell volumes. They had a very low DNA content, were rich in protein, and contained only one copy of the rRNA operon. The cells were well adapted to the simultaneous utilisation of mixed substrates. A constitutive, high-affinity and binding protein-dependent uptake system for mixed amino acids was found that would allow realistic in situ generation times at the prevailing amino acid concentrations. Further studies on this same organism revealed that the cells appeared to be extremely resistant to various stress-inducing agents. High survival rates were observed after high-intensity heat shocks, treatments with H2O2 or with ethanol. Moreover, no marked differences were observed between starved or actively growing cells in this respect, particularly when cells were grown in chemostat. Application of the dilution culture technique to the field of subsurface microbiology could be adopted to study the occurrence of UMB in groundwater with a comparable and stable 'low-nutrient-conditioned' phenotype.

To simulate growth conditions experienced by microbiota at O-2- limited interfaces of organic matter in compost, an experimental system capable of maintaining dual limitations of oxygen and carbon for extended periods, i.e., a pO(2)-auxostat, has been used. N-15 tracer studies on thermophilic (53 degrees C) decomposition processes occurring in manure-straw aggregates showed the emission of dinitrogen gas from the reactor as a result of simultaneous nitrification and denitrification at low pO(2) values (0.1 to 2.0%, vol/vol). The N loss was confirmed by nitrogen budget studies of the system, Depending on the imposed pO(2), 0.6 to 1.4 mmol of N/day (i.e., 20 to 40% of input N) was emitted as N-2. When the pO(2) was raised, the rates of both nitrification and denitrification increased instantaneously, indicating that ammonia oxidation was limited by oxygen. In auxostats permanently running at pO(2) greater than or equal to 2% (vol/vol), the free ammonium pool was almost completely oxidized and was converted to nitrite plus nitrate and N-2 gas. Labelling of the auxostat with [C- 13]carbonate was conducted to reveal whether nitrification was of autotrophic or heterotrophic origin, Incorporation of (CO2)- C-13 into population-specific cellular compounds was evaluated by profiling the saponifiable phospholipid fairy acids (FAs) by using capillary gas chromatography and subsequently analyzing the C-13/C-12 ratios of the individual FAs, after their combustion to CO2, by isotope ratio mass spectrometry. Apart from the observed label incorporation into FAs originating from a microflora belonging to the genus Methylococcus (type X group), supporting nitrification of a methylotrophic nature, this analysis also corroborated the absence of truly autotrophic nitrifying populations, Nevertheless, the extent to which ammonia oxidation continued to exist in this thermophilic community suggested that a major energy gain could be associated with it. [KEYWORDS: Methane oxidation; nitrous-oxide; sewage-sludge; microbial activity; methylococcus; bacteria; nitrate; nitrite; methylosinus; temperature]

A non-colony-forming axenic strain of Phaeocystis globosa (Harlot) Lagerheim was shown to produce a water-soluble beta-D-glucan. This glucan consisted of about 20 glucose units, mainly (1 --> 3)-linked, with branching at position 6. Therefore, it can be classified as a chrysolaminaran. Glucan production occurred mainly during the stationary growth phase and resulted in concentrations as high as 76 pg glucose per cell. When cultures were deprived of light the glucans were consumed, which supports their possible role as compounds used for temporary storage of energy.

The aim of this project was to establish the minimal inhibitory concentration (MIC) of lactic acid for growth of Clostridium tyrobutyricum. A pH-auxostat was used to maintain a constant pH and to allow continuous growth at the highest possible rates at fixed, but adjustable concentrations of lactate. By raising the concentration of lactic acid and keeping the pH constant, the growth rate was shown to decrease linearly with increasing lactic acid concentration. The pK(a) of lactic acid, measured in the actual growth medium at 37 degrees C, was 3.40 (+/- 0.03). Based on this value, the MIC(undiss) values for each pH were estimated. The MIC of total lactic acid (MIC(tot)) ranged from 150 mmol l(-1) to 1510 mmol l(-1) at pH 4.6-6.25, respectively. The corresponding MIC values of undissociated lactic acid (MIC(undiss)) ranged from 8.9 to 2.1 mmol l(-1) at the same pH values. These results emphasize the importance of a rapid pH decrease and an equally rapid initial lactic acid fermentation of the ensilage, in order to sufficiently suppress clostridial growth.

Four closely related strains of thermophilic bacteria were isolated via enrichment in batch and continuous culture with inulin as the sole source of carbon and energy by using inoculations from various sources. These new strains were isolated from beet pulp from a sugar refinery, soil around a Jerusalem artichoke, fresh cow manure, and mud from a tropical pond in a botanical garden. The cells of this novel species of strictly anaerobic, gram-positive bacteria were rod shaped and nonmotile. Growth on inulin was possible between 40 and 65-degrees-C, with optimum growth at 58-degrees-C. All strains were capable of fermenting a large number of sugars. Formate, acetate, ethanol, lactate, H2, and succinate were the main organic fermentation products after growth on fructose, glucose, or inulin. Synthesis of inulinase in batch culture closely paralleled growth, and the enzyme was almost completely cell bound. Strain IC is described as the type strain of a new species, Clostridium thermosuccinogenes sp. nov., with a G+C content of 35.9 mol%.

The degradation of the following amino acids was investigated in mixed cultures obtained from a waste water purification plant: aspartate, glutamate, serine, alanine, valine and leucine. Inhibition of sulfate-reducing bacteria in these mixed cultures by molybdate was found to inhibit amino acid degradation. The degradation of serine, alanine, valine and leucine was accelerated considerably by active sulfate reduction. The fermentation of aspartate and glutamate was not stimulated by the presence of sulfate-reducing bacteria. The existence of species which are able to ferment valine and leucine by coupling their oxidation to the reduction of exogenous acetate to butyrate was demonstrated