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Ten strains of psychrotolerant methylotrophic bacteria were isolated from the samples collected in Larsemann and Bunger Hills (Antarctica). Most of the isolates are assigned to the genus Pseudomonas, representatives of the genera Janthinobacterium, Massilia, Methylotenera and Flavobacterium were also found. Majority of isolates were able to grow on a wide range of sugars, methylamines and other substrates. Optimal growth temperatures for the isolated strains varied from 6 °C to 28 °C. The optimal concentration of NaCl was 0.5-2.0%. The optimal pH values of the medium were 6-7. It was found that three strains synthesized indole-3-acetic acid on a medium with L-tryptophan reaching 11-12 μg/ml. The values of intracellular carbohydrates in several strains exceeded 50 μg/ml. Presence of calcium-dependent and lanthanum-dependent methanol dehydrogenase have been shown for some isolates. Strains xBan7, xBan20, xBan37, xBan49, xPrg27, xPrg48, xPrg51 showed the presence of free amino acids. Bioprospection of Earth cryosphere for such microorganisms has a potential in biotechnology., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

When cells are exposed to freezing temperatures, high concentrations of cryoprotective agents (CPA) prevent ice crystal formation, thus enhancing cell survival. However, high concentrations of CPAs can also cause cell toxicity. Exopolysaccharides (EPSs) from polar marine environments exhibit lower toxicity and display effects similar to traditional CPA. In this study, we sought to address these issues by i) selecting strains that produce EPS with novel cryoprotective activity, and ii) optimizing culture conditions for EPS production. Sixty-six bacteria producing mucous substances were isolated from the Ross Sea (Antarctic Ocean) using solid marine agar plates. Among them, Pseudoalteromonas sp. RosPo-2 was ultimately selected based on the rheological properties of the produced EPS (p-CY02). Cryoprotective activity experiments demonstrated that p-CY02 exhibited significantly cryoprotective activity at a concentration of 0.8% (w/v) on mammalian cells (HaCaT). This activity was further improved when combined with various concentrations of dimethyl sulfoxide (DMSO) compared to using DMSO alone. Moreover, the survival rate of HaCaT cells treated with 5% (v/v) DMSO and 0.8% (w/v) p-CY02 was measured at 87.9 ± 2.8% after freezing treatment. This suggests that p-CY02 may be developed as a more effective, less toxic, and novel non-permeating CPA. To enhance the production of EPS with cryoprotective activity, Response Surface Methodology (RSM) was implemented, resulting in a 1.64-fold increase in production of EPS with cryoprotective activity.

Production of biomass and lipids in batch cultures of the Antarctic thraustochytrid Oblongichytrium sp. RT2316-13, is reported. The microorganism proved capable of producing nearly 67% docosahexaenoic acid (DHA) and 15% eicosapentaenoic acid (EPA) in its total lipid fraction. Biomass with a maximum total lipid content of 33.5% (wt/wt) could be produced at 15°C in batch culture using a medium containing glucose (20 g/L), yeast extract (10.5 g/L), and other minor components. A lower culture temperature (5°C) reduced biomass and lipid productivities compared to culture at 15°C, but enhanced the DHA and EPA content of the lipids by 6.4- and 3.3-fold, respectively. Both a simple minimally structured mathematical model and a more complex genome-scale metabolic model (GEM) allowed the fermentation profiles in batch cultures to be satisfactorily simulated, but the GEM provided much greater insight in the biochemical and physiological phenomena underlying the observed behavior. Unlike the simpler model, the GEM could be interrogated for the possible effects of various external factors such as oxygen supply, on the expected outcomes. In silico predictions of oxygen effects were consistent with literature observations for DHA producing thraustochytrids., (© 2020 Wiley Periodicals LLC.)

Natural red pigments have been widely used as food and cosmetics additives. However, due to toxic byproducts or allergen issues, it is still necessary to look for some other red pigment products. This study proposed combinatorial strategies to improve production of a new kind of red pigments from the fungus Geomyces WNF-15A, isolated from Antarctica. A high-production medium was developed by statistical experimental design, which was further simplified for industrial use by single-factor experiments. Strain breeding by atmospheric room temperature plasma mutagenesis generated a mutant, Geomyces sp. WNF-15A-M210, which increased production of red pigments by 24.4% and shortened culture phase by 33.3% comparing with the wild-type. The production of red pigments by this mutant favored a weak alkaline condition but required only mild dissolved oxygen tension. Control of initial pH 8.5 (process pH around 7.5) increased red pigments production by 19% comparing with natural condition. Precursor and inhibitor addition experiments indicated that the red pigments were synthesized by polyketide pathway, and feeding 6 mmol/L precursor of sodium acetate by three aliquots at days 3 to 5 improved biosynthesis of red pigments by 27%. Finally, the developed culture process was verified in a 5-L stirred tank bioreactor. The red pigments production of the pH regulation group reached 1.11-fold of the control and 1.95-fold of the precursor regulation group, respectively. This study provides high-production strain, optimized medium, and bioprocess for the possible industrial production of Antarctic Geomyces red pigments in future. PRACTICAL APPLICATION: Antarctic Geomyces red pigments showed high color value, nontoxic characteristic, and good water solubility. It holds potential for industrial use and is under development for food additive in China currently. This study provides an optional manufacturing process for this new kind of red pigments., (© 2020 Institute of Food Technologists®.)

Objective: Oleaginous yeasts are a renewable and alternative source of oil for third-generation biodiesel. This work aimed to evaluate the effects of glucose concentration (30-100 g L -1 ) on growth, lipid synthesis, and fatty acids (FA) profile of three Rhodotorula spp. (R. glacialis R15, R. glutinis R4, and R. glutinis R48) isolated from Antarctica, and estimate the key quality parameters of the biodiesel produced by yeasts to confirm their potential as feedstocks for third-generation biodiesel synthesis., Results: Yeasts accumulated 50-69.5% of lipids (w/w) under nitrogen-limitation and glucose-excess (C/N = 40-133). Glucose concentration increase influenced positively lipid accumulation (69.5% w/w) and FA profile of R. glacialis R15. Lipid accumulation (53% on average) of R. glutinis strains was not significantly affected by glucose concentration; content of saturated (~ 30%) and polyunsaturated FA (~ 29-30%) was slightly influenced. FA profiles of lipids synthesized by R15, R4, and R48 are similar to vegetable oils used in biodiesel industry with C16 and C18 FA (95-99%) as the major components, and contain mainly oleic (C18:1), palmitic (C16:0), and linoleic (C18:2) acids, which are suitable for biodiesel synthesis. Estimated fuel properties for biodiesel produced by R15, R4, and R48 satisfied all the criteria established by ASTM D6751 and EN 14214 with good cetane number, iodine value, and oxidation stability. An improvement in biodiesel quality of R15 was observed with the glucose increase. The best global properties of biodiesel from R4 were obtained with 30 g L -1 of glucose., Conclusions: Rhodotorula spp. from Antarctica are promising candidates for third-generation biodiesel synthesis.

Lower temperature biohydrogen production has always been attractive, due to the lower energy requirements. However, the slow metabolic rate of psychrotolerant biohydrogen-producing bacteria is a common problem that affects their biohydrogen yield. This study reports on the improved substrate synthesis and biohydrogen productivity by the psychrotolerant Klebsiella sp. strain ABZ11, isolated from Antarctic seawater sample. The isolate was screened for biohydrogen production at 30°C, under facultative anaerobic condition. The isolate is able to ferment glucose, fructose and sucrose with biohydrogen production rate and yield of 0.8 mol/l/h and 3.8 mol/g, respectively at 10 g/l glucose concentration. It also showed 74% carbohydrate uptake and 95% oxygen uptake ability, and a wide growth temperature range with optimum at 37°C. Klebsiella sp. ABZ11 has a short biohydrogen production lag phase, fast substrate uptake and is able to tolerate the presence of oxygen in the culture medium. Thus, the isolate has a potential to be used for lower temperature biohydrogen production process., (© 2018 Abdullahi Mohammed et al.)

The production, purification, and characterization of an extracellular protease released by Rhodotorula mucilaginosa L7 were evaluated in this study. This strain was isolated from an Antarctic marine alga and previously selected among others based on the capacity to produce the highest extracellular proteolytic activity in preliminary tests. R. mucilaginosa L7 was grown in Saboraud-dextrose medium at 25 °C, and the cell growth, pH of the medium, extracellular protease production and the glucose and protein consumption were determined as a function of time. The protease was then purified, and the effects of pH, temperature, and salt concentration on the catalytic activity and enzyme stability were determined. Enzyme production started at the beginning of the exponential phase of growth and reached a maximum after 48 h, which was accompanied by a decrease in the pH as well as reductions of the protein and glucose concentrations in the medium. The purified protease presented optimal catalytic activity at pH 5.0 and 50 °C. Finally, the enzyme was stable in the presence of high concentrations of NaCl. These characteristics are of interest for future studies and may lead to potential biotechnological applications that require enzyme activity and stability under acidic conditions and/or high salt concentrations., (Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Microalgal lipids are promising alternative feedstocks for biodiesel production. Scenedesmus sp. NJ-1, an oil-rich freshwater microalga isolated from Antarctica, was identified to be a suitable candidate to produce biodiesel in this study. This strain could grow at temperatures ranging from 4 to 35 °C. With regular decrease in nitrate concentration in the medium, large quantities of triacylglycerols accumulated under batch culture conditions detected by thin layer chromatography and BODIPY 505/515 fluorescent staining. Scenedesmus sp. NJ-1 achieved the average biomass productivity of 0.105 g l⁻¹ d⁻¹ (dry weight) and nearly the highest lipid content (35 % of dry cell weight) was reached at day 28 in the batch culture. Neutral lipids accounted for 78 % of total lipids, and C18:1 (n-9), C16:0 were the major fatty acids in total lipids, composing 37 and 20 % of total fatty acids of Scenedesmus sp. NJ-1 grown for 36 days, respectively. These results suggested that Scenedesmus sp. NJ-1 was a good source of microalgal oils for biodiesel production.

This study demonstrated the potential of microbial isolates from Antarctic soils to produce hydrolytic enzymes by using specific substrates. The results revealed potential of the strains to produce a broad spectrum of hydrolytic enzymes. Strain A-1 isolated from soil samples in Casey Station, Wilkes Land, was identified as Nocardioides sp. on the basis of morphological, biochemical, physiological observations and also chemotaxonomy analysis. Enzymatic and antimicrobial activities of the cell-free supernatants were explored after growth of strain A-1 in mineral salts medium supplemented with different carbon sources. It was found that the carbon sources favored the production of a broad spectrum of enzymes as well as compounds with antimicrobial activity against Gram-positive and Gram-negative bacteria, especially Staphylococcus aureus and Xanthomonas oryzae. Preliminary analysis showed that the compounds with antimicrobial activity produced by the strain A-1 are mainly glycolipids and/or lipopeptides depending on the used carbon source. The results revealed a great potential of the Antarctic Nocardioides sp. strain A-1 for biotechnological, biopharmaceutical and biocontrol applications as a source of industrially important enzymes and antimicrobial/antifungal compounds.

It is believed that the exposure of organisms to harsh climate conditions may select for differential enzymatic activities, making the surviving organisms a very promising source for bioprospecting. Soil bacteria play an important role in degradation of organic matter, which is mostly due to their ability to decompose cellulose-based materials. This work focuses on the isolation and identification of cellulolytic bacteria from soil found in two environments with stressful climate conditions (Antarctica and the Brazilian semi-arid caatinga). Cellulolytic bacteria were selected using enrichments at high and low temperatures (4 or 60°C) in liquid media (trypic soy broth-TSB and minimum salt medium-MM) supplemented with cellulose (1%). Many of the isolates (119 out of 254-46.9%) displayed the ability to degrade carboxymethyl-cellulose, indicating the presence of endoglucolytic activity, while only a minority of these isolates (23 out of 254-9.1%) showed exoglucolytic activity (degradation of avicel). The obtained isolates revealed a preferential endoglucolytic activity according to the temperature of enrichments. Also, the identification of some isolates by partial sequencing of the 16S rRNA gene indicated that the Bacteroidetes (e.g., Pedobacter, Chryseobacterium and Flavobacterium) were the main phylum of cellulolytic bacteria isolated from soil in Antarctica; the Firmicutes (e.g., Bacillus) were more commonly isolated from samples from the caatinga; and Actinobacteria were found in both types of soil (e.g., Microbacterium and Arthrobacter). In conclusion, this work reports the isolation of bacteria able to degrade cellulose-based material from soil at very low or very high temperatures, a finding that should be further explored in the search for cellulolytic enzymes to be used in the bioenergy industry.

The sequencing and the annotation of the marine Antarctic Pseudoalteromonas haloplanktis TAC125 genome has paved the way to investigate on the molecular mechanisms involved in adaptation to cold conditions. The growing interest in this unique bacterium prompted the developing of several genetic tools for studying it at the molecular level. To allow a deeper understanding of the PhTAC125 physiology a genetic system for the reverse genetics in this bacterium was developed. In the present work, we describe a practical technique for allelic exchange and/or gene inactivation by in-frame deletion and the use of a counterselectable marker in P. haloplanktis. The construction of suitable non-replicating plasmid and methods used to carry out a two-step integration-segregation strategy in this bacterium are reported in detail.Furthermore two examples, in which the developed methodology was applied to find out gene function or to construct genetically engineered bacterial strains, were described.

Cultivation techniques were used to study the heterotrophic bacterial diversity in two microbial mat samples originating from the littoral zone of two continental Antarctic lakes (Forlidas Pond and Lundström Lake) in the Dufek Massif (within the Pensacola Mountains group of the Transantarctic Mountains) and Shackleton Range, respectively. Nearly 800 isolates were picked after incubation on several growth media at different temperatures. They were grouped using a whole-genome fingerprinting technique, repetitive element palindromic PCR and partial 16S rRNA gene sequencing. Phylogenetic analysis of the complete 16S rRNA gene sequences of 82 representatives showed that the isolates belonged to four major phylogenetic groups: Actinobacteria, Bacteroidetes, Proteobacteria and Firmicutes. A relatively large difference between the samples was apparent. Forlidas Pond is a completely frozen water body underlain by hypersaline brine, with summer thaw forming a slightly saline littoral moat. This was reflected in the bacterial diversity with a dominance of isolates belonging to Firmicutes, whereas isolates from the freshwater Lundström Lake revealed a dominance of Actinobacteria. A total of 42 different genera were recovered, including first records from Antarctica for Albidiferax, Bosea, Curvibacter, Luteimonas, Ornithinibacillus, Pseudoxanthomonas, Sphingopyxis and Spirosoma. Additionally, a considerable number of potential new species and new genera were recovered distributed over different phylogenetic groups. For several species where previously only the type strain was available in cultivation, we report additional strains. Comparison with public databases showed that overall, 72% of the phylotypes are cosmopolitan whereas 23% are currently only known from Antarctica. However, for the Bacteroidetes, the majority of the phylotypes recovered are at present known only from Antarctica and many of these represent previously unknown species.

Enterobacter hormaechei and Brevibacterium antarcticum strains isolated from ornithogenic soils of Galindez Island (West Antarctica) were investigated for their resistance to Cu2+ cations and for their capacity to Cu2+ uptake from the environment. The studied strains are capable to grow in the concentration range of copper 100-1100 mg/l and to extract 11-75% of Cu2+ from the environment depending on cultivation parameters and copper output concentration in the culture medium.

A chemically defined minimal medium has been developed for growing the Antarctic psychrotrophic bacterium Pseudomonas syringae Lz4W, a model system for studying cold adaptation. This natural isolate from Antarctica has an absolute requirement for two branched chain amino acids, isoleucine and valine, in addition to low osmolality of the growth medium. The bacterium contains threonine deaminase but lacks acetohydroxyacid synthase suggesting that a defect lies in the isoleucine and valine biosynthetic pathway causing auxotrophy. Succinate was found to be preferred carbon source over glucose as it could suppress the glucose metabolizing enzymes in the cells, like in other pseudomonads. The development of the minimal medium (MM Lz) for growing the Antarctic P. syringae Lz4W strain would be useful for investigation of the catabolite repression control mechanism at a very low temperature (below 5 degrees C), which is predominant in vast area of our global ecosystems., ((c) 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nutrient enrichment is known to increase bacterioplankton population density in a variety of Antarctic freshwater lakes. However, relatively little is known about the associated changes in species composition. In this study, the bacterioplankton community composition of one such lake was studied following natural nutrient enrichment to investigate the resistance of the system to environmental change. Heywood Lake is an enriched freshwater maritime Antarctic lake, with nitrogen and phosphorus concentrations significantly higher than its more oligotrophic neighbours (by at least an order of magnitude). This major change in lake chemistry has occurred following large increases in the fur seal population over the last 30 years. Using analysis of 16S rRNA gene fragments, fatty acid methyl ester analysis, denaturing gradient gel electrophoresis and fluorescence in situ hybridization, significant changes are reported in lake microbiology which have resulted in a distinct bacterioplankton community. In comparison to its more oligotrophic neighbours, nutrient-enriched Heywood Lake has a high bacterioplankton population density, reduced species richness and an increasing evenness among key groups. Only 42.3 % of the clones found with > or =97 % similarity to a named genus were also present in adjacent oligotrophic lakes, including three of the dominant groups. Critically, there was an apparent shift in dominance with trophic status (from the beta-Proteobacteria to the Actinobacteria). Other key observations included the absence of a dominant group of Cyanobacteria and the presence of marine bacteria. The significant impact of natural nutrient enrichment on the microbiology of Heywood Lake, therefore, suggests that low-temperature oligotrophic freshwater lake systems might have low resistance to environmental change.

A haloalkalophilic Halomonas strain CRSS, isolated from salt sediments in Antarctica, produced exocellular polysaccharides (EPS) up to 2.9 g g(-1) dry cells. Acetate was the most efficient carbon source for EPS production. The composition of media strongly affected the nature of the polymers; a mannan and a xylo-mannan, were obtained when cells were grown on complex media. Acetate was the most efficient carbon source for EPS production and in presence of this substrate, a new polysaccharide, a fructo-glucan, was produced. The EPS fraction was composed by glucose, fructose, glucosamine and galactosamine in relative proportions of 1:0.7:0.3:trace.

Six Gram-positive, non-motile, UV- and draught-tolerant bacteria were isolated from antarctic soil and rock samples. The pink to orange cocci grew well on oligotrophic medium PYGV (pH 7.5) at 9-18 degrees C. They tolerated 0-10% NaCl, were aerobic to facultatively anaerobic and contained ornithine in their cell wall (type A3beta, Orn-Gly2). The lipid profiles of four strains were found to be typical for those of D. radiodurans. Major fatty acids were 16:1cis9, 15:1cis9, 17:1cis9 and i17:1cis9, the respiratory quinone of three strains was MK-8. Comparative 16S rDNA gene sequencing revealed phylogenetic relationships to the Deinococcus clade, especially to D. radiopugnans. The levels of 16S rRNA gene sequence similarity and DNA-DNA hybridisation data showed the six isolates represented new taxa. Phenotypic properties supported the description of three new species which were different from the eight known Deinococcus species and particularly from D. radiopugnans. Soil isolate AA-692T (DSM 12807T) is the type strain of Deinococcus frigens sp. nov., with AA-752 (DSM 15993) and AA-829 (DSM 15994) as additional strains from soil. The endolithic isolate AA-1444T, Deinococcus saxicola sp. nov., (DSM 15974T) came from antarctic sandstone, and Deinococcus marmoris sp. nov. (isolate AA-63T [DSM 12784T]) as well as AA-69 (DSM 15951) were isolated from antarctic marble.

Xylanase production by the Antarctic psychrophilic yeast Cryptococcus adeliae was increased 4.3 fold by optimizing the culture medium composition using statistical designs. The optimized medium containing 24.2 g l(-1) xylan and 10.2 g l(-1) yeast extract and having an initial pH of 7.5 yielded xylanase activity at 400 nkat (nanokatal) ml(-1) after 168-h shake culture at 4 degrees C. In addition, very little endoglucanase, beta-mannanase, beta-xylosidase, beta-glucosidase, alpha-L-arabinofuranosidase, and no filter paper cellulase activities were detected. Among 12 carbon sources tested, maximum xylanase activity was induced by xylan, followed by lignocelluloses such as steamed wheat straw and alkali-treated bagasse. The level of enzyme activity produced on other carbon sources appeared to be constitutive. Among the complex organic nitrogen sources tested, the xylanase activity was most enhanced by yeast extract, followed by soymeal, Pharmamedia (cotton seed protein), and Alburex (potato protein). A batch culture at 10 degrees C in a 5-1 fermenter (3.5-1 working volume) using the optimized medium gave 385 nkat at 111 h of cultivation. The crude xylanase showed optimal activity at pH 5.0-5.5 and good stability at pH 4-9 (21 h at 4 degrees C). Although the enzyme was maximally active at 45 degrees - 50 degrees C, it appeared very thermolabile, showing a half-life of 78 min at 35 degrees C. At 40 degrees - 50 degrees C, it lost 71%-95% activity within 5 min. This is the first report on the production as well as on the properties of thermolabile xylanase produced by an Antarctic yeast.

The growth kinetics of two psychrotolerant Antarctic bacteria, Hydrogenophaga pseudoflava CR3/2/10 (2/10) and Brevibacterium sp. strain CR3/1/15 (1/15), were examined over a range of temperatures in both batch culture and glycerol-limited chemostat cultures. The maximum specific growth rate (mu max) and Ks values for both bacteria were functions of temperature, although the cell yields were relatively constant with respect to temperature. The mu max values of both strains increased up to an optimum temperature, 24 degrees C for 2/10 and 20 degrees C for 1/15. Strain 1/15 might therefore be considered to be more psychrophilic than strain 2/10. For both bacteria, the specific affinity (mu max/Ks) for glycerol uptake was lower at 2 than at 16 degrees C, indicating a greater tendency to substrate limitation at low temperature. As the temperature increased from 2 to 16 degrees C, the specific affinity of 1/15 for glycerol increased more rapidly than it did for 2/10. Thus 1/15, on the basis of this criterion, was less psychrophilic than was 2/10. The steady-state growth kinetics of the two strains at 2 and 16 degrees C imply that 1/15 would be able to outgrow 2/10 only at relatively low substrate concentrations (< 0.32 g of glycerol.liter-1) and high temperatures (> 12 degrees C), which suggests that 1/15 has a less psychrotolerant survival strategy than does 2/10. Our data were compared with other data in the literature for bacteria growing at low temperatures. They also showed an increase of substrate-specific affinity with increasing temperature.(ABSTRACT TRUNCATED AT 250 WORDS)