The yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 °C and 52 °C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by high-performance liquid chromatography, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2.

No abstract

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.

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.

Antarctica offers a range of extreme climatic conditions, such as low temperatures, high solar radiation and low nutrient availability, and constitutes one of the harshest environments on Earth. Despite that, it has been successfully colonized by 'cold-loving' fungi, which play a key role in decomposition cycles in cold ecosystems. However, knowledge about the ecological role of yeasts in nutrient or organic matter recycling/ mineralization remains highly fragmentary. The aim of this work was to study the yeast microbiota in samples collected on 25 de Mayo/King George Island regarding the scope of their ability to degrade polyphenolic substrates such as lignin and azo dyes. Sixty-one yeast isolates were obtained from 37 samples, including soil, rocks, wood and bones. Molecular analyses based on rDNA sequences revealed that 35 yeasts could be identified at the species level and could be classified in the genera Leucosporidiella, Rhodotorula, Cryptococcus, Bullera and Candida. Cryptococcus victoriae was by far the most ubiquitous species. In total, 33% of the yeast isolates examined showed significant activity for dye decolorization, 25% for laccase activity and 38% for ligninolytic activity. Eleven yeasts did not show positive activity in any of the assays performed and no isolates showed positive activity across all tested substrates. A high diversity of yeasts were isolated in this work, possibly including undescribed species and conspicuous Antarctic yeasts, most of them belonging to oligotrophic, slow-growing and metabolically diverse basidiomycetous genera.

No abstract

Several studies have shown that biostimulation can promote hydrocarbon bioremediation processes in Antarctic soils. However, the effect of the different nutrient sources on hydrocarbon removal heavily depends on the nutrients used and the soil characteristics. In this work, using a sample of chronically contaminated Antarctic soil that was exposed to a fresh hydrocarbon contamination, we analyzed how a complex organic nutrient source such as fish meal (FM) and a commercial fertilizer (OSEII) can affect hydrocarbon biodegradation and bacterial community composition. Both amended and unamended (control) biopiles were constructed and controlled at Carlini Station and sampled at days 0, 5, 16, 30 and 50 for microbiological, chemical and molecular analyses. FM caused a fast increase in both total heterotrophic and hydrocarbon degrading bacterial counts. These high values were maintained until the end of the assay, when statistically significant total hydrocarbon removal (71 %) was detected when compared with a control system. The FM biopile evidenced the dominance of members of the phylum Proteobacteria and a clear shift in bacterial structure at the final stage of the assay, when an increase of Actinobacteria was observed. The biopile containing the commercial fertilizer evidenced a hydrocarbon removal activity that was not statistically significant when compared with the untreated system and exhibited a bacterial community that differed from those observed in the unamended and FM-amended biopiles. In summary, biostimulation using FM in biopiles significantly enhanced the natural hydrocarbon-degradation activity of the Carlini station soils in biopile systems and caused significant changes in the bacterial community structure. The results will be considered for the future design of soil bioremediation protocols for Carlini Station and could also be taken into account to deal with dieselcontaminated soils from other cold-climate areas.

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.