Edgardo A. Hernández

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.

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.

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)).

Small-scale studies towards a rational use of bioaugmentation in an Antarctic hydrocarbon-contaminated soil

Abstract Bioaugmentation is a controversial strategy. In this work, the effect of the inoculum size and the absence of natural microflora on the efficiency of hydrocarbon removal were studied. Two levels of inoculum (106 and 109 CFU g-1) were applied to soil microcosms containing sterile (S6 and S9) and non-sterile (NS6 and NS9) oil contaminated Antarctic soil. Community controls (CC) and biostimulated autochthonous microflora (BAM) were also included. Total heterotrophic aerobic (THAB) and hydrocarbon degrading (HDB) bacteria as well as total petroleum hydrocarbons (TPH) were analysed. At day 0, THAB and HDB counts (CFU g-1) showed no differences among CC, BAM and NS6 but significantly higher values were observed in NS9 and S9. At day 60, three different levels of HDB were observed. The lower level was represented by CC (106 CFU g-1), a second group (5 x 107 CFU g-1) was represented by BAM, NS6, NS9 and S6, and the third level was constituted by S9 (1 x 109 CFU g-1). TPH values at day 60 decreased significantly in all systems excluding the controls. NS6, NS9, S6 and S9 were not different from those corresponding to BAM. Results suggest that the bioaugmentation of a chronically diesel fuel-contaminated Antarctic soil is unlikely to be profitable or beneficial.

Response of two Antarctic marine bacteria to different natural UV radiation doses and wavelengths

The aim of this work was to investigate the effect of different fractions of UVR on two Antarctic marine bacteria (Arthrobacter UVvi and FCB-related UVps strains) and to study the relationship between the bacterial viability and the UVB dose. Ten experiments exposing strains to natural solar radiation were conducted in Potter Cove, South Shetland Islands. The effect of different UVR wavelengths on viability was analysed by using cultures in quartz bottles covered with interferential filters. Six treatments were performed: DARK, PAR (with UVR shielded off), UVA360, UVA320, UVB305 and UVB280. In all UVR treatments, strains showed significant losses of viability under high and moderate irradiance and no differences were observed between UV treatments. Under high UVB dose (15.0 kJ m−2 received in only two hours), the effect of UVB treatments was significantly higher than that observed under UVA treatments. However, UVA caused a significant reduction on bacterial viability. Survival rates were negatively correlated with integrated UVB dose, FCB-related UVps being more sensitive than Arthobacter UVvi. The similar values observed in viability when the same dose was received in different time periods suggested that DNA repair mechanisms are not much effective in these strains. The different response to the UV wavelength ranges studied here suggests that changes in the spectral composition of natural radiation could differentially affect the components of Antarctic marine bacterial communities.

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.

Pelagic and benthic communities of the Antarctic ecosystem of Potter Cove: Genomics and ecological implications

Molecular technologies are more frequently applied in Antarctic ecosystem research and the growing amount of sequence-based information available in databases adds a new dimension to understanding the response of Antarctic organisms and communities to environmental change. We apply molecular techniques, including fingerprinting, and amplicon and metagenome sequencing, to understand biodiversity and phylogeography to resolve adaptive processes in an Antarctic coastal ecosystem from microbial to macrobenthic organisms and communities. Interpretation of the molecular data is not only achieved by their combination with classical methods (pigment analyses or microscopy), but furthermore by combining molecular with environmental data (e.g., sediment characteristics, biogeochemistry or oceanography) in space and over time. The studies form part of a long-term ecosystem investigation in Potter Cove on King-George Island, Antarctica, in which we follow the effects of rapid retreat of the local glacier on the cove ecosystem. We formulate and encourage new approaches to integrate molecular tools into Antarctic ecosystem research, environmental conservation actions, and polar ocean observatories.

Element concentrations of environmental concern in surface sediment samples from a broad marine area of 25 de Mayo (King George) Island, South Shetland Islands

Western Antarctica (WA) constitutes the area with the highest human presence in the white continent and also the region where the effects of global warming are more evident worldwide. Such human presence represents a potential risk of pollution with both, organic and inorganic contaminants. Global warming also could modify dynamics and transport of the pollutants, increasing summer water runoff, ice melting and iceberg scouring. Under this fast-changing scenario, knowledge about the concentration of contaminants is essential to evaluate the environmental status of this ecologically relevant area. In this work, we performed the first regional-scale monitoring of 9 trace elements (Cr, Co, Ni, Cu, Zn, As, Pb, Cd and Hg), as well as Fe and Mn, in surface sediment from 64 sites comprising six different areas in Maxwell Bay, 25 de Mayo (King George) Island. Target elements were quantified in surface sediment samples (20-30 m depth) obtained during two summer Antarctic expeditions: 2010/11 and 2011/12 by inductively coupled plasma linked to a quadrupole mass spectrometer (ICP-MS). Based on the average values observed for the reference areas, baseline values were defined for the studied region. A regional enrichment in Cu (compared with the global mean upper crust) was observed and related to the widespread mineralization of volcanic rocks. The most anthropized area (South Fildes) mainly showed sediment class 3 (moderately polluted) for Pb, Cd and Hg with a number samples revealing some highly contaminated hot spots. Although elemental contamination in some samples close to scientific stations or sites where logistic operations were evidenced, a pollution pattern was not clearly identified. The present work represents the first regional-scale attempt to define the baseline values and the anthropic impacts in this region of the WA and also provides the first data about Hg concentration in surface sediment of the study area.

Sources and distribution of biomarkers in surficial sediments from a polar marine ecosystem (Potter Cove, King George Island, Antarctica)

Sedimentary organic matter (OM) represents the energy supply for the shelf benthos at the Antarctic Ocean, and has yet to be properly characterized in terms of sources and composition for the Potter Cove region, King George/25 de Mayo Island. This energy input occurs mainly during the brief summer and provides the majority of available energy for the year, in a region with high endemism and limited source variety of sedimentary OM. Thus, the aim of this study is to identify the OM origin and degradation degree based on the spatial distribution and type of organic biomarkers. Twelve surficial sediment samples were collected and analyzed for the presence of n-alkanols and sterols. The different spatial patterns between the analyzed compounds indicated distinct OM sources and degradation degrees. First, relatively fresh phytoplankton organic matter and an enhanced bacterial activity were associated with the occurrence of seaweeds detritus and represent the source of n-alkanols. Second, relatively fresh material mainly associated with seaweeds debris were identified as the source of macroalgae sterols. Our results shed some light into the base of the Potter Cove trophic benthic chain and increase our understanding on the region’s biogeochemical processes relating to OM recycling. It also provides a baseline for assessing future changes in the structure of the benthic food web in this environment, which is subject to noticeable glaciers retreat.

Microbial and viral-like rhodopsins present in coastal marine sediments from four polar and subpolar regions

ABSTRACT Rhodopsins are broadly distributed. In this work, we analyzed 23 metagenomes corresponding to marine sediment samples from four regions that share cold climate conditions (Norway; Sweden; Argentina and Antarctica). In order to investigate the genes evolution of viral rhodopsins, an initial set of 6224 bacterial rhodopsin sequences according to COG5524 were retrieved from the 23 metagenomes. After selection by the presence of transmembrane domains and alignment, 123 viral (51) and non‐viral (72) sequences (>50 amino acids) were finally included in further analysis. Viral rhodopsin genes were homologs of Phaeocystis globosa virus and Organic lake Phycodnavirus. Non‐viral microbial rhodopsin genes were ascribed to Bacteroidetes, Planctomycetes, Firmicutes, Actinobacteria, Cyanobacteria, Proteobacteria, Deinococcus‐Thermus and Cryptophyta and Fungi. A rescreening using Blastp, using as queries the viral sequences previously described, retrieved 30 sequences (>100 amino acids). Phylogeographic analysis revealed a geographical clustering of the sequences affiliated to the viral group. This clustering was not observed for the microbial non‐viral sequences. The phylogenetic reconstruction allowed us to propose the existence of a putative ancestor of viral rhodopsin genes related to Actinobacteria and Chloroflexi. This is the first report about the existence of a phylogeographic association of the viral rhodopsin sequences from marine sediments. &NA; Graphical Abstract Figure. Information in this manuscript is the first study on microbial rhodopsins present in coastal marine sediments. Also it reported a previously undescribed allopatric cladism for the viral rhodopsins sequences.