Lucas Ruberto

Effectiveness of the natural bacterial flora, biostimulation and bioaugmentation on the bioremediation of a hydrocarbon contaminated Antarctic soil

Abstract Microcosms systems ( 250 g soil in 1 l flasks) were performed in Jubany Station (King George Island, South Shetland Islands) to analyse biodegradation of gas-oil in Antarctic soils under natural conditions. Abiotic loss of hydrocarbons, biodegradation activity of indigenous microflora and biostimulation with N and P were studied. In addition, biaugmentation with a previously isolated psychrotolerant strain (B-2-2) was analysed. Hydrocarbon concentration, heterotrophic and hydrocarbon-degrading bacterial counts and predominant bacterial groups were evaluated during 51 days. A significant loss of hydrocarbons was observed in abiotic control. Indigenous microflora showed increased heterotrophic counts and hydrocarbon-degrading/heterotrophic ratio. This fact was associated with a significant degrading activity (35% higher than the control). Bioaugmentation with B-2-2 strain improved the bioremediation efficiency (75% of the hydrocarbon was removed). High levels of N and P produced an initial inhibition of bacterial growth. Finally, bacterial diversity was reduced in contaminated soil. Our results showed that autochthonous bacterial flora from Antarctic soils is able to degrade an important fraction of the gas-oil and that bioaugmentation represents a valuable alternative tool to improve bioremediation.

Bacterial Community Dynamics during Bioremediation of Diesel Oil-Contaminated Antarctic Soil

The effect of nutrient and inocula amendment in a bioremediation field trial using a nutrient-poor Antarctic soil chronically contaminated with hydrocarbons was tested. The analysis of the effects that the treatments caused in bacterial numbers and hydrocarbon removal was combined with the elucidation of the changes occurring on the bacterial community, by 16S rDNA-based terminal restriction fragment length polymorphism (T-RFLP) typing, and the detection of some of the genes involved in the catabolism of hydrocarbons. All treatments caused a significant increase in the number of bacteria able to grow on hydrocarbons and a significant decrease in the soil hydrocarbon content, as compared to the control. However, there were no significant differences between treatments. Comparison of the soil T-RFLP profiles indicated that there were changes in the structure and composition of bacterial communities during the bioremediation trial, although the communities in treated plots were highly similar irrespective of the treatment applied, and they had a similar temporal dynamics. These results showed that nutrient addition was the main factor contributing to the outcome of the bioremediation experiment. This was supported by the lack of evidence of the establishment of inoculated consortia in soils, since their characteristic electrophoretic peaks were only detectable in soil profiles at the beginning of the experiment. Genetic potential for naphthalene degradation, evidenced by detection of nahAc gene, was observed in all soil plots including the control. In treated plots, an increase in the detection of catechol degradation genes (nahH and catA) and in a key gene of denitrification (nosZ) was observed as well. These results indicate that treatments favored the degradation of aromatic hydrocarbons and probably stimulated denitrification, at least transiently. This mesocosm study shows that recovery of chronically contaminated Antarctic soils can be successfully accelerated using biostimulation with nutrients, and that this causes a change in the indigenous bacterial communities and in the genetic potential for hydrocarbon degradation.

Psychrotolerant hydrocarbon-degrading Rhodococcus strains isolated from polluted Antarctic soils

Three hydrocarbon-degrading Rhodococcus strains isolated from polluted Antarctic soils proved to be closely related despite their different origins. Strains had a similar hydrocarbon degradation pattern and optimum growth temperature ranged between 25ºC and 30ºC, showing that strains are psychrotolerant but not psychrophiles. Specific growth rate on rich media ranged between 0.12 and 0.21 h−1, higher than those observed on hydrocarbons as carbon source. Results suggest that in Antarctic contaminated soils, closely related Rhodococcus strains are present and could play an important role in decontamination. Microcosm systems showed that, although the natural microflora respond significantly to the pollutants, bioaugmentation with Rhodococcus strain (ADH), improved biodegradation either alone or mixed with a hydrocarbon-degrading Acinetobacter strain. In comparison with microcosm where only ADH was inoculated, a non-significant decrease in hydrocarbon concentration was observed when ADH was inoculated as mixed culture with a previously tested strain. Pollutants dramatically reduced bacterial groups in soils resulting in a dominance of Pseudomonas. Microcosms showed that when natural microflora has no previous history of exposure to the pollutants, bioaugmentation with autochthonous strains improves degradation of the contaminants. The positive response of the native bacteria to the pollutants leaves the question open as to whether bioaugmentation is necessary when soils have a long previous exposure to hydrocarbons.

Influence of nutrients addition and bioaugmentation on the hydrocarbon biodegradation of a chronically contaminated Antarctic soil.

Complexity involved in the transport of soils and the restrictive legislation for the area makes on‐site bioremediation the strategy of choice to reduce hydrocarbons contamination in Antarctica. The effect of biostimulation (with N and P) and bioaugmentation (with two bacterial consortia and a mix of bacterial strains) was analysed by using microcosms set up on metal trays containing 2·5 kg of contaminated soil from Marambio Station. At the end of the assay (45 days), all biostimulated systems showed significant increases in total heterotrophic aerobic and hydrocarbon‐degrading bacterial counts. However, no differences were detected between bioaugmented and nonbioaugmented systems, except for J13 system which seemed to exert a negative effect on the natural bacterial flora. Hydrocarbons removal efficiencies agreed with changes in bacterial counts reaching 86 and 81% in M10 (bioaugmented) and CC (biostimulated only) systems. Results confirmed the feasibility of the application of bioremediation strategies to reduce hydrocarbon contamination in Antarctic soils and showed that, when soils are chronically contaminated, biostimulation is the best option. Bioaugmentation with hydrocarbon‐degrading bacteria at numbers comparable to the total heterotrophic aerobic counts showed by the natural microflora did not improve the process and showed that they would turn the procedure unnecessarily more complex.

Phenanthrene Biodegradation in Soils Using an Antarctic Bacterial Consortium

ABSTRACT Biodegradation of polycyclic aromatic hydrocarbons (PAHs) in Antarctic soils is limited by low temperatures, lack of adequate levels of nutrients, low number of PAH-tolerant members in the autochthonous microbiota and low bioavailability of contaminants. In the present work, microcosms systems (performed in 1-L glass flasks containing Antarctic soil supplemented with 1744 ppm of phenanthrene) were used to study (i) the effect of biostimulation with a complex organic source of nutrients (fish meal) combined with a surfactant (Brij 700); (ii) the effect of bioaugmentation with a psychrotolerant PAH-degrading bacterial consortium (M10); (iii) the effect of the combination of both strategies. The authors found that combination of biostimulation and bioaugmentation caused a significant removal (46.6%) of phenanthrene after 56 days under Antarctic environmental conditions. When bioaugmentation or biostimulation were applied separately, nonsignificant reduction in phenanthrene concentration was observed. Microtox test showed a low increase in toxicity only in the most efficient system. Results proved that “in situ” bioremediation process of phenanthrene-contaminated soils is possible in Antarctic stations. In addition, inoculation with a psychrotolerant PAH-degrading bacterial consortium in association with a mix of fish meal and a high-molecular-weight surfactant improved phenanthrene removal and should be the selected strategy when the number of hydrocarbons degrading bacteria in the target soil is low.

Bizionia argentinensis sp. nov., isolated from surface marine water in Antarctica.

A marine bacterial strain, designated strain JUB59(T), was isolated from surface seawater in Antarctica and subsequently characterized. Cells were found to be Gram-negative, non-motile rods forming butyrous, shiny, yellowish orange colonies on marine agar. Growth occurred at 2-28 degrees C (optimally at 22-25 degrees C) but not at 30 degrees C; Na+ ions were required, but 9 % NaCl (w/v) was not tolerated. Phylogenetic analysis, based on comparisons of the complete 16S rRNA gene sequence of the novel isolate with the sequences of closely related strains, showed that strain JUB59(T) belonged to the family Flavobacteriaceae, representing a novel species of the genus Bizionia. The highest levels of sequence similarity were found with respect to Bizionia myxarmorum ADA-4(T) (97.4 %) and Bizionia algoritergicola APA-1(T) (97.1 %). However, the DNA-DNA relatedness of strain JUB59(T) with respect to these two strains was low (15.9-17.3 and 19.3-22.1 %, respectively). The predominant fatty acids of strain JUB59(T) were iso-15 : 1omega10c (18.1 %), iso-15 : 0 (17.3 %), anteiso-15 : 0 (13.9 %), iso-17 : 0 3-OH (9.2 %), 15 : 0 (6.0 %) and iso-16 : 0 3-OH (5.3 %). The main polar lipids were phosphatidylethanolamine, an aminolipid, an amino-positive phospholipid and two unidentified lipids. MK-6 was the major respiratory quinone (>90 %) and the DNA G+C content was 34 mol%. On the basis of the data obtained, strain JUB59(T) represents a novel species of the genus Bizionia, for which the name Bizionia argentinensis sp. nov. is proposed. The type strain is JUB59(T) (=DSM 19628(T)=CCM-A-29 1259(T)).

Bacteriology of Extremely Cold Soils Exposed to Hydrocarbon Pollution

The study of bacterial communities present in natural ecosystems has been the object of attention of several research groups during the past 15 years, driven mainly by the development of DNA-based methodologies (Amman et al. 1995; Holben 1997). Taking advantage of these methods, it was shown that most of the individual components of natural bacterial communities are incapable of growth on standard culture media. In this sense, it was evident that more than 99% of the bacterial cells present in a soil sample were not able to grow on standard culture media (Roszak and Colwell 1987; Torsvik et al. 1990). Soil represents an extremely complex matrix . Soils considered as habitat are characterized by physical, chemical, and temporal heterogeneities at any scale from km to nm (Young and Ritz 2000). Each field, forest, tundra, or cold desert has a unique soil food web with a particular proportion of organisms, and a particular level of complexity within each group of organisms. These differences are the result of soil, vegetation, and climate factors. Detailed studies on soil micro-organisms demonstrated that even within soil habitats deemed homogeneous at the plot scale, distribution of soil bacteria was highly structured, with different bacterial communities located in specific sites, probably in response to the heterogeneities imposed by the habitat (Franklin and Mills 2003). Although the above-cited references dealt with agricultural soils, the concept of soil heterogeneity is a general rule (Young and Crawford 2004) and must be kept in mind in all descriptive or analytical studies of soil bacterial communities. Bacterial populations located in individual soil microhabitats perform several biophysical and biochemical processes, turning the soil into the most complex and biodiverse ecosystem on Earth. As was mentioned above, local climate conditions are among the main factors conditioning the number and type of micro-organisms present in a soil. In Antarctica (as well as in the Arctic , the Alps , the Puna, and other high-altitude soils), low

The water column as an attenuating factor of the UVR effects on bacteria from a coastal Antarctic marine environment

The effect of UVR on the viability of the culturable bacterial community fraction (CBC), and two of their isolated components (Arthrobacter-UVvi and Bizionia-UVps), was studied in the top few metres of the water column at Potter Cove, King George Island, Antarctica. Quartz flasks containing CBC from surface waters were exposed to solar radiation at depths of 0, 1 and 3 m. Similar experiments using UVps and UVvi isolates were performed. In some experiments interferential filters were used to discriminate photosynthetic active radiation (PAR), UV-A and UV-B. CBC from depths of 0, 10 and 30 m were also exposed to surface solar radiation. The deleterious effect of UVR was observed at the surface and at a depth of 1 m, but not at a depth of 3 m. Studies with interferential filters showed low bacterial viability values at depths of 0 and 1 m under both UVR treatments. However, under low radiation doses the effect attributed to UV-B was higher than that caused by UV-A. The surface CBC was more resistant to UVR compared with CBC from a depth of 30 m. The results showed that CBC inhabiting waters above the pycnocline (located at a depth of 5–10 m) are more efficiently adapted to UVR than are those from below the pycnocline. The impact of UVR on the marine bacterioplankton studied was only detected in the first metre of the stratified water column of Potter Cove, which has high levels of suspended particulate matter. These results support the evidence for a significant UVR-attenuating effect in the water column of this coastal Antarctic water.

Bioprospection of cold-adapted yeasts with biotechnological potential from Antarctica

The aim of this study was to investigate the ability to produce extracellular hydrolytic enzymes at low temperature of yeasts isolated from 25 de Mayo island, Antarctica, and to identify those exhibiting one or more of the evaluated enzymatic activities. A total of 105 yeast isolates were obtained from different samples and 66 were identified. They belonged to 12 basidiomycetous and four ascomycetous genera. Most of the isolates were ascribed to the genera Cryptococcus, Mrakia, Cystobasidium, Rhodotorula, Gueomyces, Phenoliferia, Leucosporidium, and Pichia. Results from enzymes production at low temperatures revealed that the Antarctic environment contains metabolically diverse cultivable yeasts, which represent potential tools for biotechnological applications. While most the isolates proved to produce 2‐4 of the investigated exoenzymes, two of them evidenced the six evaluated enzymatic activities: Pichia caribbica and Guehomyces pullulans, which were characterized as psycrotolerant and psycrophilic, respectively. In addition, P. caribbica could assimilate several n‐alkanes and diesel fuel. The enzyme production profile and hydrocarbons assimilation capacity, combined with its high level of biomass production and the extended exponential growth phase make P. caribbica a promising tool for cold environments biotechnological purposes in the field of cold‐enzymes production and oil spills bioremediation as well.

Rich bacterial assemblages from Maritime Antarctica (Potter Cove, South Shetlands) reveal several kinds of endemic and undescribed phylotypes.

Bacterial richness in maritime Antarctica has been poorly described to date. Phylogenetic affiliation of seawater free-living microbial assemblages was studied from three locations near the Argentinean Jubany Station during two Antarctic summers. Sixty 16S RNA cloned sequences were phylogenetically affiliated to Alphaproteobacteria (30/60 clones), Gammaproteobacteria(19/60 clones), Betaproteobacteria and Cytophaga-Flavobacteriia-Bacteroides (CFB), which were (2/60) and (3/60) respectively. Furthermore, six out of 60 clones could not be classified. Both, Alphaproteobacteria and Gammaproteobacteria, showed several endemic and previously undescribed sequences. Moreover, the absence of Cyanobacteria sequences in our samples is remarkable. In conclusion, we are reporting a rich sequence assemblage composed of widely divergent isolates among themselves and distant from the most closely related sequences currently deposited in data banks.