Abundance and function of bacteria in the Southern Ocean.

The very low water temperatures existing in polar oceans that experience seasonal advance and retreat of pack ice do not inhibit the presence of large bacterial populations. Bacteria may contribute significantly to the energy transfers within the Southern Ocean. In the last decades, notable progress has been made in the knowledge of the role of marine bacteria in the Southern Ocean. A short overview of the abundance and function ofAntarctic marine bacteria is given, with respect to metabolic activity. The importance of spatial and temporal variability is described. The ecological function of Antarctic marine bacterioplankton is discussed. Depending on food web structure, bacteria may be either a link in food webs supporting metazoan production, or a sink where bacterial production is metabolised by microorganisms. In the more oligotrophic areas and during certain periods of the year bacterial biomass dominates phytoplankton. The microbial food web is therefore the dominant pathway for carbon and energy flow in Antarctic seawater.

A perspective on cold enzymes: current knowledge and frequently asked questions.

Studies on psychrophilic enzymes to determine the structural features important for cold-activity have attracted increased attention in the last few years. This enhanced interest is due to the attractive properties of such proteins, i.e. a high specific activity and a low thermal stability, and thus, these enzymes constitute a tremendous potential for fundamental research and biotechnological applications. This review examines the impact of low temperatures on life, the diversity of adaptation to counteract these effects and gives an overview of the features proposed to account for low thermal stability and cold-activity, following the chronological order of the catalytic cycle phases. Moreover, we present an overview of recent techniques used in the analysis of the flexibility of a protein structure which is an important concept in cold-adaptation; an overview of biotechnological potential of psychrophilic enzymes and finally, a few frequently asked questions about cold-adaptation and their possible answers.

Arthrobacter gangotriensis sp. nov. and Arthrobacter kerguelensis sp. nov. from Antarctica.

Two coryneform bacteria were isolated from a penguin rookery soil sample collected in Antarctica, near the Indian station Dakshin Gangotri (strain Lz1y(T)), and from sea water from Kerguelen island, Antarctica (strain KGN15(T)). They have morphological and chemotaxonomic properties (peptidoglycan A4alpha type; major menaquinones MK-8, MK-9 and MK-10; predominant fatty acids anteiso-C(15 : 0) and anteiso-C(17 : 0)) that are characteristic of members of the genus Arthrobacter. The isolates shared 97.8 % 16S rRNA gene sequence similarity to each other and were most closely related to Arthrobacter sulfureus (about 98.5 % sequence similarity). DNA-DNA hybridization experiments revealed 50 % relatedness between the isolates, while the levels of DNA-DNA relatedness between strains Lz1y(T) and KGN15 (T) and their phylogenetic relative, A. sulfureus, were respectively 54 and 12 %. Based on the above data and distinct phenotypic differences between the isolates and A. sulfureus, two novel species are proposed, Arthrobacter gangotriensis sp. nov. (type strain Lz1y(T)=DSM 15796(T)=JCM 12166(T)) and Arthrobacter kerguelensis sp. nov. (type strain KGN15(T)=DSM 15797(T)=JCM 12165(T)).

Crude oil bioremediation in sub-Antarctic intertidal sediments: chemistry and toxicity of oiled residues.

The effectiveness of fertilizers for crude oil bioremediation in sub-Antarctic intertidal sediments was tested over a one-year period in a series of ten (10) experimental enclosures. Chemical, microbial and toxicological parameters demonstrated the effectiveness of various fertilizers in a pristine environment where hydrocarbon degrading bacteria (HDB) had not been stimulated by previous accidental spills or human activities. The low temperature of seawater (3-4 degrees C) had no obvious effects on the HDB community and the bioremediation process. Over 90% of n-alkanes were degraded in the first six months and most light aromatics (2-3 rings) disappeared during the first year of observation. The toxicity of oiled residues (Microtox(R) SP) was significantly reduced in the first 6 months of the process, but it increased again in the last months of the experiment. One of the fertilizers containing fishbone compost enriched with urea, inorganic phosphorus and a lipidic surfactant reduced significantly the toxicity of oil residues in the last 3 months of the experiment. Interstitial waters collected below the oil slicks during the remediation showed no toxicity, and even stimulated Vibrio fischeri. When comparing all fertilizers to the control plots, a good correlation (r(2)=0.82) was found between the growth rate of HDB and the degradation rate of n-alkanes in the first 90 days of the experiment only indicating that fertilizers were efficient for at least 3 months but their beneficial effects were lost after 6 months.

Biostimulation of natural microbial assemblages in oil-amended vegetated and desert sub-Antarctic soils.

A field study was initiated in December 2000 in two selected soils of The Grande Terre (Kerguelen Archipelago) with the objective of determining the long-term effects of fertilizer addition on the biodegradation rate and the toxicity of oil residues under severe sub-Antarctic conditions. Two soils were selected. The first site supports an abundant vegetal cover; the second one was desert soil, devoid of plant material. These two soils were located in the vicinity of the permanent station of Port-aux-Français (69 degrees 42'E; 49 degrees 19'S). A series of five experimental plots (0.75 x 0.75 m) were settled firmly into each of the studied soils. Each plot received 500 mL of diesel or Arabian light crude oil, and some of them were treated with a bioremediation agent: slow-release fertilizer Inipol EAP-22 (Elf Atochem). All the plots were sampled on a regular basis over a 1 year period. Heterotrophic and hydrocarbon-degrading microorganisms increased by two orders of magnitude during the first month of the experimentation in all treated enclosures, but differences appeared between the different plots. The microbial response was improved by bioremediation treatments. However, fertilizer addition had a greater impact on the desert soil when compared to the vegetated one. All chemical indices show a reduction of alkanes and light aromatics. Toxicity results show a high variability between treatments and environmental conditions. As a conclusion, it is clear that the microbial response was rapid and efficient in spite of the severe weather conditions, and the rate of degradation was improved by bioremediation treatments. However, after 1 year of treatment, the signal of a relatively high toxicity of oiled residues remained present in the two studied soils.

Distribution of enteric bacteria in Antarctic seawater surrounding the Port-aux-Français permanent station (Kerguelen Island).

Untreated sewage has been released from Port-aux Français station, Kerguelen Island, into the Southern Ocean for more than 50 years. We investigated the spatial distribution of faecal bacteria indicators during a one-year survey conducted in seawater off Morbihan Bay near the French permanent station of the Kerguelen Island (49 degrees 21(')S, 70 degrees 30(')E). Seawater samples were taken bimonthly from nine stations evenly distributed around the sewage outfalls of the station. Escherichia coli and enterococci were estimated using specific microplates (Miniaturized method for the enumeration of E. coli or enterococci in surface and waste waters, "MU/EC or MU/SF methods", BIO-RAD( Copyright)). In order to evaluate the role of seasonal changes of environmental parameters on the survival of enteric bacteria, total and saprophytic bacterial abundances were also estimated in all seawater samples. High densities of faecal bacteria (maximum 10(4) cells 100 ml(-1)) were found in seawater surrounding the sewage outfall. However, enteric bacterial counts decreased rapidly with increasing distance from the outfall. In all samples collected further than 2 km from the outfall, the bacterial indicators were absent or present in small numbers (<10 cells 100 ml(-1)). Faecal coliforms were not detected in samples collected at pristine sites located 10 km from Port-aux-Français. Despite these low contamination levels, faecal bacteria were always detected in the vicinity of the sewage outfall during the seasonal survey. The concentration of faecal bacteria may be related to the number of people inhabiting the station.

Inelastic electron scattering observation using energy filtered transmission electron microscopy for silicon -- germanium nanostructures imaging.

This paper presents a new technique using energy filtered TEM (EFTEM) for inelastic electron scattering contrast imaging of Germanium distribution in Si-SiGe nanostructures. Comparing electron energy loss spectra (EELS) obtained in both SiGe and Si single crystals, we found a spectrum area strongly sensitive to the presence of Ge in the range [50-100 eV]. In this energy loss window, EELS spectrum shows a smooth steeply shaped background strongly depending on Ge concentration. Germanium mapping inside SiGe can thus be performed through imaging of the EELS background slope variation, obtained by processing the ratio of two energy filtered TEM images, respectively, acquired at 90 and 60 eV. This technique gives contrasted images strongly similar to those obtained using STEM Z-contrast, but presenting some advantages: elastic interaction (diffraction) is eliminated, and contrast is insensitive to polycrystalline grains orientation or specimen thickness. Moreover, since the extracted signal is a spectral signature (inelastic energy loss) we demonstrate that it can be used for observation and quantification of Ge concentration depth profile of SiGe buried layers.

Convergent beam electron diffraction extinction distance measurements for quantitative analysis of si(1-x),Ge(x).

A new method to determine the concentration of germanium in Si(1-x) Ge(x) single crystals is presented. It is based on extinction distance measurements by means of convergent beam electron diffraction (CBED). The two-beam condition CBED intensity oscillation (the so-called rocking curve) is measured for the 004 diffracted beam and compared with a numerical simulation. Using the two-beam dynamical diffraction approximation theory, this approach yields very precise values for both specimen thickness and effective extinction distance (Ultramicroscopy 87 (2001) 5). First a theoretical extinction distance zetag(x) for strain relaxed Si(1-x)Ge(x) is calculated assuming a solid solution and using tabulated atomic scattering factors of silicon and germanium atoms. It is found that for single crystals zetag(x) decreases from 156 nm in pure silicon to 90 nm in pure germanium. Measurements on calibrated strain relaxed SiGe layers with variable germanium concentrations show an excellent agreement between experimental and calculated extinction distances zetag(x). As a consequence the experimental extinction distance zetag(x) becomes an indirect measure of the germanium concentration with a 1-2 at % sensitivity. The method turns out to be insensitive to strain as experimental zetag(x) values obtained on strained SiGe layers fit the theoretical extinction distance curve calculated for strain relaxed SiGe.

Effectiveness of bioremediation of crude oil contaminated subantarctic intertidal sediment: the microbial response.

A field study was initiated in February 1996 in a remote sandy beach of The Grande Terre (Kerguelen Archipelago, 69 degrees 42 degrees E, 49 degrees 19 degrees S) with the objective of determining the long-term effects of some bioremediation agents on the biodegradation rate and the toxicity of oil residues under severe subantarctic conditions. A series of 10 experimental plots were settled firmly into sediment. Each plot received 2L of Arabian light crude oil and some of them were treated with bioremediation agents: slow release fertilizer Inipol EAP-22 (Elf Atochem) or fish composts. Plots were sampled on a regular basis over a 3-year period. A two-order of magnitude increase of saprophytic and hydrocarbon-utilizing microorganisms occurred during the first month of the experiment in all treated enclosures, but no clear differences appeared between the plots. Very high microbial populations were present during the experiment. Biodegradation within treated spots was faster than within the untreated ones and appeared almost complete after 6 months as indicated by the degradation index of aliphatic hydrocarbons within all plots. The analysis of interstitial water collected below the oily residues presented no toxicity. However, a high toxicity signal, using Microtox solid phase, appeared for all oiled sand samples with a noticeable reduction with time even if the toxicity signal remained present and strong after 311 days of oil exposition. As a conclusion, it is clear that the microbial response was rapid and efficient in spite of the severe weather conditions, and the rate of degradation was improved in presence of bioremediation agents. However, the remaining residues had a relatively high toxicity.

Did psychrophilic enzymes really win the challenge?

Organisms living in permanently cold environments, which actually represent the greatest proportion of our planet, display at low temperatures metabolic fluxes comparable to those exhibited by mesophilic organisms at moderate temperatures. They produce cold-evolved enzymes partially able to cope with the reduction in chemical reaction rates and the increased viscosity of the medium induced by low temperatures. In most cases, the adaptation is achieved through a reduction in the activation energy, leading to a high catalytic efficiency, which possibly originates from an increased flexibility of either a selected area of or the overall protein structure. This enhanced plasticity seems in return to be responsible for the weak thermal stability of cold enzymes. These particular properties render cold enzymes particularly useful in investigating the possible relationships existing between stability, flexibility, and specific activity and make them potentially unrivaled for numerous biotechnological tasks. In most cases, however, the adaptation appears to be far from being fully achieved.

Crystal thickness and extinction distance determination using energy filtered CBED pattern intensity measurement and dynamical diffraction theory fitting.

A new method for measuring thickness and extinction distance of single crystals based on computed adjustment of measured and calculated CBED pattern intensity profiles is presented and discussed. The experimental beam intensity distribution is measured from an energy filtered CBED pattern recorded on a CCD camera. The calculated profile is based on dynamical diffraction theory, and with the two-beam approximation the analytical expression contains only two free parameters: specimen thickness t and extinction distance xig. Parameter refinement through minimization of the difference between experimental and calculated intensity profiles is carried out using Origin 5.0 software from Microcal. The iterative procedure always converges to a unique solution in a few seconds, yielding an accurate value for both thickness and extinction distance. The method is extensively tested on silicon using the (0 0 4) Bragg reflection. On specimens in the usual TEM thickness range, the method gives result similar to the conventional (P.M. Kelly et al., Phys. Stat. Sol. A31 (1975) 771; S.M. Allen, Philos. Mag. A 43 (1981) 325) graphical methods, both based on the measurement of fringe spacing. Moreover, it is shown that the calculation matches perfectly both the positions of the minimums and maximums as well as the amplitude of maximums. For any single intensity profile, specimen thickness and extinction distance can be determined with a precision of about 0.2%. A statistical comparison of our method with the Kelly and Allen techniques, based on more than 50 experiments, shows an improvement in measured extinction distance dispersion. Using 197 keV electrons, and liquid-nitrogen cryo-holder, the new technique yields an experimental value of 161+/-3 nm for the extinction distance for silicon with the (0 0 4) Bragg reflection. The equivalent tabulated value at 0 K is about 156 nm. Using the Kelly and Allen methods, the extinction distance is found to be 162+/-6 nm. The improvement in precision is a direct consequence of matching the intensity profile envelope, which contains information on the extinction distance. Also the accuracy of thickness determination is improved and is around 0.5 to 1% for common specimen thickness. The minimum measurable sample thickness is shown to be two to three times thinner than with the Kelly and Allen methods (0.3 xig, as opposed to 0.8 xig). With no independent calculation of the extinction distance needed, the method is also applicable on unknown crystals. The method is fast, simple and can be easily automated.

Response of Antarctic Soil Bacterial Assemblages to Contamination by Diesel Fuel and Crude Oil.

The effects of diesel fuel and "Arabian light" crude oil addition on Antarctic bacterial assemblages were studied in four contaminated soils during 1 year in the Terre Adelie land area. Monthly sampling allowed a regular survey of the bacterial changes occurring in the contaminated soils. All samples were analyzed for total bacteria, heterotrophic culturable microbiota, and hydrocarbon-utilizing microbiota. Crude oil contamination induced an initial increase of all bacterial parameters in all contaminated soils. Diesel oil contamination had a more complex effect. Hydrocarbon degrading bacterial abundance increases occurred after diesel oil addition. In contrast, general heterotrophic bacterial abundance could significantly decrease in the same conditions. In all cases the stimulatory effects of oil addition disappeared after several months of contamination.

Field observations on the variability of crude oil impact on indigenous hydrocarbon-degrading bacteria from sub-Antarctic intertidal sediments.

Oil pollution of the oceans has been a problem ever since man began to use fossil fuels. Biodegradation by naturally occurring populations of micro-organisms is a major mechanism for the removal of petroleum from the environment. To examine the effects of crude oil pollution on intertidal bacteria, we repeated the same contamination experiments on nine different sub-Antarctic intertidal beaches using specifically built enclosures (PVC pipe, 15 cm in inner diameter and 30 cm in height). Despite the pristine environmental conditions, significant numbers of indigenous hydrocarbon-degrading bacteria were observed in all the studied beaches. Introduction of oil into these previously oil-free environments resulted in several orders of magnitude of increase in hydrocarbon-degrading micro-organisms within a few days in some of the studied sites but has no obvious effects on two others. The physical environment of the bacterial assemblage seems to play a major role in the biodegradation capacities. After 3 months of contamination, both remaining oil concentrations and biodegradation indexes differ strongly between the different stations. Thus, chemical and biological parameters reveal a strong heterogeneity of biodegradation capacities between the different sites.

Effect of dispersed oil on heterotrophic bacterial communities in cold marine waters.

Mesocosm studies were conducted to evaluate the effect of dispersed oil on total and heterotrophic bacterial communities of under-ice seawater from the St. Lawrence Estuary. A regular survey of bacterial changes in the oil-contaminated seawater was performed during a two week period. The bacterial community structure was investigated by carrying out 27 morphological and biochemical tests on 168 isolated strains. The results show a detectable but transient response of the bacterial community to crude oil addition. While total bacterial counts were approximately constant during the experiment, dispersed oil induced an increase in heterotrophic bacterial microflora (from 10(4) to 10(5) bacteria ml(-1) after two weeks of contamination). The dispersed oil appeared to have an inhibitory effect on some components of the bacterial community. A decrease of most probable number values was observed just after addition of crude oil in the most polluted tanks and one day later in the less polluted tank. However, except for the most polluted tank, this adverse effect disappeared rapidly. While the dispersed oil induced a total disappearance of some components of the bacterial community in the most polluted tank, the structure of the bacterial community in the less polluted tank appeared relatively unchanged after 14 days of contamination.

Influence of temperature on the growth potential of Southern polar marine bacteria.

Regular surveys of heterotrophic microflora from seawater were conducted in the subantarctic (Kerguelen archipelago) and Antarctic (Terre Adélie area). Although a predominance of psychrophilic bacteria could be expected for such polar marine environments, there were no significant differences between results obtained after incubation at two different temperatures (4°C for 21 days or 18°C for 6 days). To investigate this further, four sets of bacterial strains were isolated from the subantarctic area (early fall, late fall, spring, and summer) and one set of Antarctic bacteria was isolated in summer. The growth rates of the 143 strains collected were determined at four different temperatures (4, 7, 20, and 30°C). The results clearly indicated that a large majority of the isolated bacteria must be considered psychrotrophic and not truly psychrophilic strains.

Short-term variations of bacterioplankton in Antarctic zone: Terre Adelie area.

Previous studies on Antarctic seawater have demonstrated the presence of significant numbers of bacteria, but their in situ activity has not been demonstrated. In order to demonstrate this hypothetical activity, a scheduled survey was conducted from January to February 1986 in a coastal area of Adelie Land. Seawater samples were collected in a selected station every day or every hour during a 17 hour period. Bacterial communities in each sample were studied by measuring direct and viable counts, frequency of dividing cells estimation, taxonomic analysis, and heterotrophic potential. Complementary studies used batch cultures with artificial nutrient supplements. The results clearly suggest a strong potential activity of the natural Antarctic bacterial microflora.