Predominance of Roseobacter, Sulfitobacter, Glaciecola and Psychrobacter in seawater collected off Ushuaia, Argentina, Sub-Antarctica.

Bacterial diversity in sub-Antarctic seawater, collected off Ushuaia, Argentina, was examined using a culture independent approach. The composition of the 16S rRNA gene libraries from seawater and seawater contaminated with the water soluble fraction of crude oil was statistically different (P value 0.001). In both libraries, clones representing the Alphaproteobacteria, Gammaproteobacteria, the Cytophaga-Flavobacterium-Bacteroidetes group and unculturable bacteria were dominant. Clones associated with the genera Roseobacter, Sulfitobacter, Staleya, Glaciecola, Colwellia, Marinomonas, Cytophaga and Cellulophaga were common to both the libraries. However, clones associated with Psychrobacter, Arcobacter, Formosa algae, Polaribacter, Ulvibacter and Tenacibaculum were found only in seawater contaminated with hydrocarbons (Table 1). Further, the percentage of clones of Roseobacter, Sulfitobacter and Glaceicola was high in seawater (43%, 90% and 12% respectively) compared to seawater contaminated with hydrocarbons (35%, 4% and 9% respectively). One of the clones F2C63 showed 100% similarity with Marinomonas ushuaiensis a bacterium identified by us from the same site.

Effects of nutrient and temperature on degradation of petroleum hydrocarbons in contaminated sub-Antarctic soil.

Mesocosm studies using sub-Antarctic soil artificially contaminated with diesel or crude oil were conducted in Kerguelen Archipelago (49 degrees 21' S, 70 degrees 13' E) in an attempt to evaluate the potential of a bioremediation approach in high latitude environments. All mesocosms were sampled on a regular basis over six months period. Soils responded positively to temperature increase from 4 degrees C to 20 degrees C, and to the addition of a commercial oleophilic fertilizer containing N and P. Both factors increased the hydrocarbon-degrading microbial abundance and total petroleum hydrocarbons (TPH) degradation. In general, alkanes were faster degraded than polyaromatic hydrocarbons (PAHs). After 180 days, total alkane losses of both oils reached 77-95% whereas total PAHs never exceeded 80% with optimal conditions at 10 degrees C and fertilizer added. Detailed analysis of naphthalenes, dibenzothiophenes, phenanthrenes, and pyrenes showed a clear decrease of their degradation rate as a function of the size of the PAH molecules. During the experiment there was only a slight decrease in the toxicity, whereas the concentration of TPH decreased significantly during the same time. The most significant reduction in toxicity occurred at 4 degrees C. Therefore, bioremediation of hydrocarbon-contaminated sub-Antarctic soil appears to be feasible, and various engineering strategies, such as heating or amending the soil can accelerate hydrocarbon degradation. However, the residual toxicity of contaminated soil remained drastically high before the desired cleanup is complete and it can represent a limiting factor in the bioremediation of sub-Antarctic soil.

Psychrobacter salsus sp. nov. and Psychrobacter adeliensis sp. nov. isolated from fast ice from Adelie Land, Antarctica.

Nine psychrotolerant bacteria were isolated from fast ice in the middle of Geologie Archipelago, Adelie Land, Antarctica and were categorized into two groups, based on their SDS-PAGE profiles. Representatives from each of the two groups, namely strains DD 48T and SJ 14T exhibited phenotypic and chemotaxonomic characteristics confirming to the genus Psychrobacter. The 16S rRNA gene sequence indicated that the two isolates are closely related to each other and to the already reported fifteen species of Psychrobacter. Detailed studies on the phenotypic characteristics, chemotaxonomic properties and phylogenetic analysis of strains DD 48T and SJ 14T indicated that they are distinctly different from each other and the reported species of Psychrobacter. At the DNA-DNA hybridisation level, the two species exhibit less than 70% similarity. Thus, strains DD 48T and SJ 14T are identified as new species of the genus Psychrobacter for which the names Psychrobacter salsus sp. nov. and Psychrobacter adeliensis sp. nov. respectively are proposed.

Degradation of petroleum hydrocarbons in two sub-antarctic soils: influence of an oleophilic fertilizer.

In order to determine the long-term effects of fertilizer on the degradation rate and the toxicity of hydrocarbons in sub-Antarctic soils contaminated by petroleum hydrocarbons, a field study was initiated in December 2000 on two different soils of the Kerguelen Islands (69 degrees 42'E, 49 degrees 19'S). The number of hydrocarbon-degrading bacteria (HDB) increased greatly after crude-oil and diesel-fuel contamination, and the fertilizer addition had a favorable effect on HDB growth and activity. Hydrocarbon-degrading bacteria counts remained high until the end of the experiment although the total hydrocarbon content in all contaminated soils was reduced to 80 to 90% of their initial value after 330 d. Degradation of n-alkanes was enhanced significantly in the presence of the fertilizer, while the degradation of polycyclic aromatic hydrocarbons (PAHs) was only barely enhanced. Toxicity results showed a noticeable reduction with time, although toxicity remained present and important in both soils at the end of the experiment. In addition, fertilized plots showed a toxic signal greater than unfertilized ones. Overall results clearly show that fertilizer addition improves the rate of degradation of both oil contaminants. However, remaining toxic residues may constitute a drawback of the fertilizer-assisted biodegradation process at low temperatures.

Effects of temperature and fertilization on total vs. active bacterial communities exposed to crude and diesel oil pollution in NW Mediterranean Sea.

The dynamics of total and active microbial communities were studied in seawater microcosms amended with crude or diesel oil at different temperatures (25, 10 and 4 degrees C) in the presence/absence of organic fertilization (Inipol EAP 22). Total and hydrocarbon-degrading microbes were enumerated by fluorescence microscopy and Most Probable Number (MPN) method, respectively. Total (16S rDNA-based) vs. active (16S rRNA) bacterial community structure was monitored by Capillary-Electrophoresis Single Strand Conformation Polymorphism (CE-SSCP) fingerprinting. Hydrocarbons were analyzed after 12 weeks of incubation by gas chromatography-mass spectrometry. Total and hydrocarbon-degrading microbial counts were highly influenced by fertilization while no important differences were observed between temperatures. Higher biodegradation levels were observed in fertilized microcosms. Temperature and fertilization induced changes in structure of total bacterial communities. However, fertilization showed a more important effect on active bacterial structure. The calculation of Simpson's diversity index showed similar trends among temperatures whereas fertilization reduced diversity index of both total and active bacterial communities.

Gallaecimonas pentaromativorans gen. nov., sp. nov., a bacterium carrying 16S rRNA gene heterogeneity and able to degrade high-molecular-mass polycyclic aromatic hydrocarbons.

A Gram-negative, rod-shaped, halotolerant bacterium, designated strain CEE_131(T), which degraded high-molecular-mass polycyclic aromatic hydrocarbons of four and five rings, was isolated from intertidal sediment of Corcubion Ria in Cee, A Coruña, Spain. Direct sequencing showed ambiguities and suggested heterogeneity. Cloned 16S rRNA gene sequence PCR products yielded five different sequences varying at five positions. Strain CEE_131(T) showed rather distant relationships to its phylogenetically closest neighbours, including the genera Rheinheimera and Serratia , exhibiting 91 % sequence similarity with Rheinheimera perlucida BA131(T) and Serratia proteamaculans subsp. quinovora DSM 4597(T). The major fatty acids were C(16 : 1 )omega7c, C(16 : 0) and C(18 : 1)omega7c. The DNA G+C content was 41.7 mol%. On the basis of these distinct phenotypic and genotypic characteristics, strain CEE_131(T) is considered to represent a novel species in a new genus in the class Gammaproteobacteria, for which the name Gallaecimonas pentaromativorans gen. nov., sp. nov. is proposed. The type strain is CEE_131(T) (=DSM 21945(T)=CECT 7479(T)).

Comparative mesocosm study of biostimulation efficiency in two different oil-amended sub-antarctic soils.

Biological treatment has become increasingly popular as a remediation method for soils and groundwater contaminated with petroleum hydrocarbon, chlorinated solvents, and pesticides. Bioremediation has been considered for application in cold regions such as Arctic and sub-Arctic climates and Antarctica. Studies to date suggest that indigenous microbes suitable for bioremediation exist in soils in these regions. This paper reports on two case studies at the sub-Antarctic Kerguelen Island in which indigenous bacteria were found that were capable of mineralizing petroleum hydrocarbons in soil contaminated with crude oil and diesel fuel. All results demonstrate a serious influence of the soil properties on the biostimulation efficiency. Both temperature elevation and fertilizer addition have a more significant impact on the microbial assemblages in the mineral soil than in the organic one. Analysis of the hydrocarbons remaining at the end of the experiments confirmed the bacterial observations. Optimum temperature seems to be around 10 degrees C in organic soil, whereas it was higher in mineral soil. The benefit of adding nutrients was much stronger in mineral than in the organic soil. Overall, this study suggests that biostimulation treatments were driven by soil properties and that ex situ bioremediation for treatment of cold contaminated soils will allow greater control over soil temperature, a limiting factor in cold climates.

Marinomonas polaris sp. nov., a psychrohalotolerant strain isolated from coastal sea water off the subantarctic Kerguelen islands.

Two aerobic, psychrohalotolerant, motile bacterial isolates, CK13T and CK16, isolated from sea-water samples collected off the subantarctic Kerguelen island, were characterized by using a polyphasic taxonomic approach. On the basis of 16S rRNA gene sequence data, the strains were 99.6% similar and exhibited 93-97% similarity with the seven recognized species of Marinomonas. The most closely related species were Marinomonas pontica and Marinomonas primoryensis, with 97 and 96 % similarity at the 16S rRNA gene sequence level, respectively. DNA-DNA hybridization values between strain CK13T and M. pontica and M. primoryensis were only 58 and 40%, respectively. The major fatty acids present in strain CK13T were iso-C(16:0), C(16:0), C(16:1)omega7c and C(18:1)omega7c. The DNA G+C content of strain CK13T was 41.2 mol%. Phosphatidylethanolamine and phosphatidylglycerol were identified as the predominant phospholipids. All the above characteristics support the affiliation of strain CK13T to the genus Marinomonas. Phylogenetic analysis and phenotypic and genotypic distinctiveness confirmed that strains CK13T and CK16 are members of a novel species of the genus Marinomonas, for which the name Marinomonas polaris sp. nov. is proposed. The type strain is CK13T (=MTCC 6645T=DSM 16579T=JCM 12522T).

Marinobacter maritimus sp. nov., a psychrotolerant strain isolated from sea water off the subantarctic Kerguelen islands.

A psychrotolerant, Gram-negative, motile bacterium, designated CK 47(T), was isolated from sea water off the subantarctic Kerguelen islands (50 degrees 40' S 68 degrees 25' E). The isolate grew optimally at 22 degrees C and minimum and maximum temperature of growth were 4 and 37 degrees C, respectively. It required Na(+) for growth and exhibited optimum growth at pH 8.5 and 4% NaCl. It utilized hexane, heptane and petroleum ether as sole sources of carbon. Strain CK 47(T) had Q9 as the major respiratory quinone and C(16 : 0) (21.7%), C(17:0) (21.3%), C(18:0) (5.7%), C(18:1)omega7c (9.0%) and C(18:1)omega9c (31.4%) as predominant fatty acids. The G+C content of the DNA was 58 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that CK 47(T) formed a coherent cluster within the genus Marinobacter. It exhibited highest 16S rRNA gene sequence similarity of 96.8% with Marinobacter lipolyticus. However, the level of DNA-DNA relatedness between strain CK47(T) and M. lipolyticus was only 55%. On the basis of phenotypic characteristics, and phylogenetic and genotypic distinctiveness, strain CK 47(T) is considered to represent a novel species of the genus Marinobacter. The name Marinobacter maritimus sp. nov. is proposed, with CK 47(T) (=JCM 12521(T)=MTCC 6519(T)) as the type strain.

Marinomonas ushuaiensis sp. nov., isolated from coastal sea water in Ushuaia, Argentina, sub-Antarctica.

A Gram-negative, rod-shaped, psychrophilic, motile, non-spore-forming bacterium, strain U1T, was isolated from Ushuaia located at the southernmost tip of Argentina. On the basis of 16S rRNA gene sequence similarity, strain U1T was found to be closely related to Marinomonas communis (DSM 5604T) and Marinomonas primoryensis (IAM 15010T). At the DNA-DNA level, however, the values for similarity were 41 and 25 %, respectively. The major fatty acids present were iso-C(16 : 0), C(16 : 1)omega7c, iso-C(17 : 1) and C(18 : 1)omega7c and the G+C content of the DNA was 43.6 mol%. All of the above characteristics support the affiliation of strain U1T to the genus Marinomonas. Furthermore, on the basis of phenotypic features, chemotaxonomic characteristics and phylogenetic analysis of the 16S rRNA gene sequence, it appears that strain U1T is distinct from the four Marinomonas species with validly published names. Strain U1T, therefore, represents a novel species, for which the name Marinomonas ushuaiensis sp. nov. is proposed. The type strain of M. ushuaiensis is U1T (=MTCC 6143T=DSM 15871T=JCM 12170T).

Halomonas glaciei sp. nov. isolated from fast ice of Adelie Land, Antarctica.

Eleven psychrophilic bacteria were isolated from a solid layer of fast ice in the middle of Pointe-Geologie Archipelago, Adelie Land, Antarctica. The 11 isolates based on the phenotypic characteristics, chemotaxonomic and phylogenetic analysis have been identified as members of the genus Halomonas. All the isolates at the 16S rDNA sequence level were identical, possessed the 15 conserved nucleotides of the family Halomonadaceae and four nucleotides of the genus Halomonas. Therefore, the 16S rDNA sequence of DD 39 was used for calculating the evolutionary distances and for phylogenetic analysis. It was observed that DD 39 formed a robust cluster with H. variabilis, from which it differed by 0.7%. Further DNA-DNA hybridization studies indicated low DNA-DNA homology (15%) between H. variabilis and DD 39. Between the 11 Antarctic isolates the homology was >85%. In addition it was observed that DD 39 was different from H. variabilis in that it was psychrophilic, could tolerate only up to 15% sodium chloride, could not hydrolyse esculin, could not reduce nitrate, was urease negative, could not utilize glycerol as a carbon source, and was resistant to ampicillin and erythromycin and sensitive to nalidixic acid. In addition, it also exhibited distinct differences with respect to high content of C(16:1) and low levels of cyclo-C(17:0) and cyclo-C(19:0). DD 39 also differed from all the other reported species of Halomonas with respect to many phenotypic characteristics. It is proposed therefore that DD 39 should be placed in the genus Halomonas as a new species that is Halomonas glaciei. The type strain of H. glaciei is DD 39(T) (MTCC 4321; JCM 11692).