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Fourier transform infrared (FTIR) spectroscopy is a biophysical technique used for non-destructive biochemical profiling of biological samples. It can provide comprehensive information about the total cellular biochemical profile of microbial cells. In this study, FTIR spectroscopy was used to perform biochemical characterization of twenty-nine bacterial strains isolated from the Antarctic meltwater ponds. The bacteria were grown on two forms of brain heart infusion (BHI) medium: agar at six different temperatures (4, 10, 18, 25, 30, and 37°C) and on broth at 18°C. Multivariate data analysis approaches such as principal component analysis (PCA) and correlation analysis were used to study the difference in biochemical profiles induced by the cultivation conditions. The observed results indicated a strong correlation between FTIR spectra and the phylogenetic relationships among the studied bacteria. The most accurate taxonomy-aligned clustering was achieved with bacteria cultivated on agar. Cultivation on two forms of BHI medium provided biochemically different bacterial biomass. The impact of temperature on the total cellular biochemical profile of the studied bacteria was species-specific, however, similarly for all bacteria, lipid spectral region was the least affected while polysaccharide region was the most affected by different temperatures. The biggest temperature-triggered changes of the cell chemistry were detected for bacteria with a wide temperature tolerance such Pseudomonas lundensis strains and Acinetobacter lwoffii BIM B-1558.

A novel cold-adapted bacteria Arthrobacter oryzae BIM B-1663 isolated from Antarctic green snow showed keratinase activity and efficient poultry feather degradation. A. oryzae strain degraded more than 80 % of chicken feathers within 7 days of cultivation at 25 °C. The optimal keratinase activity for A. oryzae BIM B-1663 was observed at 50 °C, both for α-keratin (44.86 U/mL) and for β-keratin (94 mU/mL). The obtained results from sulfite and thiol groups tests and Fourier transform infrared spectroscopy (FTIR) showed that A. oryzae strain has a different keratin degradation mechanism than the reference strain Bacillus licheniformis CCM 2145T. FTIR fingerprinting can be used for monitoring of feather hydrolysis as it showed distinct chemical differences in feather meal hydrolysates, retentate and permeate from A. oryzae and B. licheniformis strains.

In this study, for the first time, we report the identification and characterization of culturable fast-growing bacteria isolated from the sea-affected temporary meltwater ponds (MPs) in the East Antarctica area of the Vecherny region (−67.656317, 46.175058) of the Thala Hills Oasis, Enderby Land. Water samples from the studied MPs showed alkaline pH (from 8.0 to 10.1) and highly varied total dissolved solids (86–94,000 mg/L). In total, twenty-nine bacterial isolates were retrieved from the studied MPs. The phylogenetic analysis based on 16S rRNA gene sequence similarities showed that the isolated bacteria belong to the phyla Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes and the twelve genera Pseudomonas, Shewanella, Acinetobacter, Sporosarcina, Facklamia, Carnobacterium, Arthrobacter, Brachybacterium, Micrococcus, Agrococcus, Leifsonia, and Flavobacterium. Most of the isolated bacteria were psychrotrophs and showed the production of one or more extracellular enzymes. Lipolytic and proteolytic activities were more prevalent among the isolates. Five isolates from the Actinobacteria phylum and one isolate from the Bacteroidetes phylum had strong pigmentation. Antibiotic susceptibility testing revealed that most of the isolates are resistant to at least one antibiotic, and seven isolates showed multi-resistance.

Temperature fluctuations and nutrient composition are the main parameters influencing green snow microbiome. In this study we investigated the influence of temperature and nutrient conditions on the growth and cellular chemical profile of bacteria isolated from green snow. Chemical profiling of the green snow bacteria was done by high-throughput FTIR spectroscopy combined with multivariate data analysis. We showed that temperature and nutrients fluctuations strongly affect growth ability and chemical profile of the green snow bacteria. The size of colonies for green snow bacteria grown at higher (25 °C) and lower (4 °C and 10 °C) than optimal temperature (18 °C) was smaller. All isolates grew on rich medium, and only 19 isolates were able to grow on synthetic minimal media. Lipid and mixed spectral regions showed to be phylogeny related. FTIR fingerprinting indicates that lipids are often affected by the temperature fluctuations. Growth on different media resulted in the change of the whole chemical profile, where lipids showed to be more affected than proteins and polysaccharides. Correlation analysis showed that nutrient composition is clearly strongly influencing chemical changes in the cells, followed by temperature.

Global climate change is significantly affecting marine life off the northern tip of the Antarctic Peninsula, but little is known about microbial ecology in this area. The main goal of this study was to investigate the bacterioplankton community structure in surface waters using pyrosequencing and to determine factors influencing this community. Pelagibacterales and Rhodobacterales (Alphaproteobacteria), Oceanospirillales and Alteromonadales (Gammaproteobacteria), and Flavobacteriales (Bacteroidetes) were the core taxa in our samples, and the five most relatively abundant genera were Pelagibacter, Polaribacter, Octadecabacter, group HTCC2207 and Sulfitobacter. Although nutrients and chlorophyll a (chl a) contributed more to bacterioplankton community structure than water masses or depth, only 30.39% of the variance could be explained by the investigated environmental factors, as revealed by RDA and pRDA. No significant difference with respect to nutrients and chl a was observed among water masses or depth, as indicated by ANOVA. Furthermore, significant correlations among the dominant bacterial genera were more common than correlations between dominant genera and environmental factors, as revealed by Spearman analysis. We conclude that nutrients and chl a become homogeneous and that interpopulation interactions may have a central role in influencing the bacterial community structure in surface waters off the northern tip of the Antarctic Peninsula during the summer.

A previously uncultured cyanobacterium, strain KNUA009, was axenically isolated from a meltwater stream on Barton Peninsula, King George Island, South Shetland Islands, Antarctica. Molecular evidences showed that the isolate belongs to groups of globally distributed cryosphere cyanobacterial clones and this new isolate represents the first laboratory culture to be assigned to these groups. Strain KNUA009 was able to thrive at low temperatures ranging between 5°C and 20°C, but did not survive at temperatures of 25°C and above. As the isolate morphologically resembled Oscillatoria species, it is suggested that this cyanobacterium may represent a new species clade with cold resistance within the genus Oscillatoria. Keywords: Barton Peninsular; cryosphere cyanobacteria; King George Island; uncultured Oscillatoria species.

Affiliations of the dominant culturable bacteria isolated from Potter Cove, South Shetland Islands, Antarctica, were investigated together with their production of cold-active hydrolytic enzymes. A total of 189 aerobic heterotrophic bacterial isolates were obtained at 4°C and sorted into 63 phylotypes based on their amplified ribosomal DNA restriction analysis profiles. The sequencing of the 16S rRNA genes of representatives from each phylotype showed that the isolates belong to the phyla Proteobacteria (classes Alpha- and Gamma-proteobacteria), Bacteroidetes (class Flavobacteria), Actinobacteria (class Actinobacteria) and Firmicutes (class Bacilli). The predominant culturable group in the site studied belongs to the class Gammaproteobacteria, with 65 isolates affiliated to the genus Pseudoalteromonas and 58 to Psychrobacter. Among the 189 isolates screened, producers of amylases (9.5%), pectinases (22.8%), cellulases (14.8%), CM-cellulases (25.4%), xylanases (20.1%) and proteases (44.4%) were detected. More than 25% of the isolates produced at least one extracellular enzyme, with some of them producing up to six of the tested extracellular enzymatic activities. These results suggest that a high culturable bacterial diversity is present in Potter Cove and that this place represents a promising source of biomolecules. Keywords: Microbial enzymes; Antarctic bacteria; marine bacteria; cold enzymes; psychrophiles.

We report the isolation and identification of bacteria that produce extracellular cold-active proteases, obtained from water samples collected near the Uruguayan Antarctic Base on King George Island, South Shetlands. The bacteria belonged to the genera Pseudomonas (growth between 4 and 30°C) and Flavobacterium (growth between 4 and 18°C). In all cases, extracellular protease production was evident when reaching the stationary phase at 18 and 4ºC, but was not detected at 30ºC. The zymogram revealed the secretion of one extracellular protease per isolate, each with different relative electrophoretic mobility. The extracellular proteases produced at 4ºC showed thermal activity and stability at 30ºC. Both activity and stability at temperature higher that 10ºC have no physiological meaning because the isolates do not experience such temperatures in the Antarctic environment; however, the possible ecological value of cold-active and -stable extracellular proteases is discussed. Keywords: Antarctic, cold-active enzymes, protease.

There is a growing concern about the ability to produce enough nutritious food to feed the global human population in this century. Environmental conflicts and a limited freshwater supply constrain further developments in agriculture; global fisheries have levelled off, and aquaculture may have to play a more prominent role in supplying human food. Freshwater is important, but it is also a major challenge to cultivate the oceans in an environmentally, economically and energy-friendly way. To support this, a long-term vision must be to derive new sources of feed, primarily taken from outside the human food chain, and to move carnivore production to a lower trophic level. The main aim of this paper is to speculate on how feed supplies can be produced for an expanding aquaculture industry by and beyond 2050 and to establish a roadmap of the actions needed to achieve this. Resources from agriculture, fish meal and fish oil are the major components of pellet fish feeds. All cultured animals take advantage of a certain fraction of fish meal in the feed, and marine carnivores depend on a supply of marine lipids containing highly unsaturated fatty acids (HUFA, with ≥3 double bonds and ≥20 carbon chain length) in the feed. The availability of HUFA is likely the main constraint for developing carnivore aquaculture in the next decades. The availability of fish meal and oil will decrease, and the competition for plant products will increase. New harvested resources are herbivore zooplankton, such as Antarctic krill and red feed, and new produced resources are macroalgae, transgenic higher HUFA-producing plants and bacterial biomass. These products are to a limited extent components of the human food chain, and all these resources will help to move cultured carnivores to lower trophic levels and can thereby increase the production capacity and the sustainability of the production. Mariculture can only become as successful as agriculture in the coming century if carnivores can be produced at around Trophic Level 2, based mainly on plant resources. There is little potential for increasing the traditional fish meal food chain in aquaculture. KEYWORDS: Global aquaculture · Mariculture · Feed resources · Marine lipids · HUFA · Trophic level

Antarctic bacteria producing extracellular lipolytic enzymes with activity at low temperature were isolated, and the most promising strain, named G, was identified as a Psychrobacter species based on 16S rDNA sequence alignment. The genomic DNA of this bacterium was used to construct its plasmid genomic library into pUC118 plasmid vectors, and to screen the cold-active lipolytic enzyme genes. Two genes encoding for cold-active lipolytic enzymes, Lip-1452 (with an open reading frame of 1452 bp in length) and Lip-948 (with an open reading frame of 948 bp in length), were screened. The primary structure of the two lipases deduced from the nucleotide sequence showed a consensus pentapeptide containing the active serine (Lip-1452, GDSAG, and Lip-948, GNSMG) and a conserved His-Gly dipeptide in the N-terminal part of the enzyme. Protein sequence alignment and conserved regions analysis indicated that the two lipases probably belonged to family IV and family V of the bacterial lipolytic enzymes, respectively. The upstream and downstream sequences of the two lipolytic lipases were also obtained. The two lipase genes were cloned into the expression vector pCold III and integrated into Escherichia coli BL21 (DE3). The functional expression of both lipase genes by E. coli BL21 (DE3) cells was observed as the formation of clear haloes around colonies on a 1% (vol/vol) tributyrin plate upon induction with isopropyl-b-Dthiogalactopyranoside at 5°C. A lipase activity assay showed that the specific activity of the pCold III+Lip-948 expression system was up to 3.7 U ml-1, whereas that of pCold III+Lip-1452 was very low.

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.

Fifty-seven Antarctic marine bacteria were examined for their ability to degrade commercial diesel oil as the sole organic substrate at both 4 °C and 20 °C. Based on the preliminary screening, two isolates (B11 and B15) with high capacity to degrade diesel oil were selected and their biodegradation effi ciency was quantifi ed by gas chromatographic analysis. As expected for psychrotrophs, diesel oil biodegradation was slower at 4 °C than at 20 °C. The two strains also mineralized the C28 n-paraffi n octacosane at 20 °C and polychlorinated biphenyls (PCBs) at 4 °C and 20 °C.

Two strains of psychrotolerant Antarctic marine bacteria were isolated and characterized using biochemical and molecular techniques. Sequencing of 16S rRNA gene showed that UVvi strain belongs to the genus Arthrobacter whereas UVps strain is related to the Flexibacter-Cytophaga-Bacteroides (FCB) group. Response of the strains to solar radiation was studied during the summer of 1999 in Potter Cove, near Jubany station (South Shetland Island, Antarctica). The effect of photosynthetically available radiation (PAR, 400-700 nm), ultraviolet-A (UV-A, 320-400 nm) and ultraviolet-B radiation (UV-B, 280-320 nm) on cell viability was studied using mixed cultures in quartz bottles covered with interferential filters and exposed to solar radiation. In all experiments, four treatments were used: dark (with light screened out), PAR (with UV radiation screened out), PAR+UV-A (UV-B screened out) and PAR+UV-A+UV-B. Under the assayed conditions, PAR+UV-A and PAR+UV-A+UV-B radiation showed similar negative effects on the viability of the studied strains. However, at the end of the exposure time, mortality values in PAR+UV-A+UV-B treatments were higher than those observed under PAR+UV-A treatments. In both PAR+UV-A and PAR+UV-A+UV-B treatments we observed high levels of hydrogen peroxide compared with the dark control. The Arthrobacter UVvi strain showed significant recovery in dark conditions after exposure to the PAR+UV-A but not after the PAR+UV-A+UV-B treatment. This strain proved to be more resistant to UV radiation than the FCB group-related UVps strain. The results showed that UV radiation has a deleterious effect on these Antarctic marine bacteria and also revealed that the analysed components of the Antarctic bacterioplankton may have different responses when they are exposed to the same irradiance conditions.

Protease-producing psychrotolerant bacteria were isolated from Antarctic biotopes on casein agar plates using different incubation temperatures. Most of the isolates were non-spore-forming Gram-negative motile rods with catalase activity, 30% were pigmented and none of them were glucose fermenters. All the strains were grown in liquid cultures at 20°C and protease secretion was tested using the azocasein method. Despite their capacity for production of a clear zone of hydrolysis in agar plates, some strains did not produce detectable levels of proteolytic activity in liquid cultures. The lowest apparent optimum temperature for protease activity found in culture supernatants was 40°C. Almost all the strains showed activation energy values about 10-20 kJ-mol?1 lower than that observed for a mesophilic Subtilisin. Most of the proteases showed optimal activity at neutral or alkaline pH values and developed a multiple-band profile on gelatine-SDS-PAGE. It was observed that the lower the strain isolation temperature was, the more stongly cold-adapted–in terms of optimal temperature and activation energy–were the proteases produced by them. This dependence of the characteristics of the proteases on the isolation temperature is an important factor to take into account in the design of screening programmes directed towards the isolation of psychrotolerant bacteria able to produce proteases strongly or weakly adapted to work in the cold. The Antarctic area explored proved to be a promising source of proteolytic bacteria with potential use in industrial processes to be carried out at low or moderate temperatures.

The efficiency of physical concentration mechanisms for enrichment of algae and bacteria in newly formed sea-ice was investigated under defined conditions in the laboratory. Sea-ice formation was simulated in a 3,000 l tank under different patterns of water movement. When ice formed in an artificially generated current pattern, algal cells were substantially enriched within the ice matrix. Enrichment factors for chlorophyll a calculated from the ratio between the concentrations in ice and underlying water reached values of up to 53. Repeated mixing of ice crystals into the water column, as well as flow of water through the new ice layer, contributed to the enrichment of algae in the ice. Wave action during ice formation revealed lower phytoplankton enrichment factors of up to 9. Mixing of floating ice crystals with underlying water and pumping of water into the ice matrix by periodical expansion and compression of the slush ice layer were responsible for the wave-induced enrichment of algal cells. Physical enrichment of bacteria within the ice was negligible. Bacterial biomass within new ice was enhanced only when the concentration of algae was high. At low algal biomass, bacteria experienced substantial losses in the ice, most likely due to brine drainage, which were not observed for the microalgae. Bacterial cells are therefore not scavenged by ice crystals and the observed enrichment and sustainment of bacterial biomass within newly formed ice depend on their attachment to cells or aggregates of algae. Division rates of bacteria changed only slightly during ice formation.