Determinants of Microbial-Derived Dissolved Organic Matter Diversity in Antarctic Lakes.

Identifying drivers of the molecular composition of dissolved organic matter (DOM) is essential to understand the global carbon cycle, but an unambiguous interpretation of observed patterns is challenging due to the presence of confounding factors that affect the DOM composition. Here, we show, by combining ultrahigh-resolution mass spectrometry and nuclear magnetic resonance spectroscopy, that the DOM molecular composition varies considerably among 43 lakes in East Antarctica that are isolated from terrestrial inputs and human influence. The DOM composition in these lakes is primarily driven by differences in the degree of photodegradation, sulfurization, and pH. Remarkable molecular beta-diversity of DOM was found that rivals the dissimilarity between DOM of rivers and the deep ocean, which was driven by environmental dissimilarity rather than the spatial distance. Our results emphasize that the extensive molecular diversity of DOM can arise even in one of the most pristine and organic matter source-limited environments on Earth, but at the same time the DOM composition is predictable by environmental variables and the lakes' ecological history.

Chasing Waterborne Pathogens in Antarctic Human-Made and Natural Environments, with Special Reference to Legionella spp.

Waterborne pathogenic diseases are public health issues, especially for people staying in remote environments, such as Antarctica. After repeated detection of Legionella by PCR from the shower room of Syowa Station, the Japanese Antarctic research station, we wanted to understand the occurrence of waterborne pathogens, especially Legionella, in the station and their potential sources. In this study, we analyzed water and biofilm samples collected from the water facilities of Syowa Station, as well as water samples from surrounding glacier lakes, by 16S rRNA gene-based amplicon sequencing. For Legionella spp., we further attempted to obtain a detailed community structure by using genus-specific primers. The results showed that potentially pathogenic genera were mostly localized in the station, while Legionella spp., Pseudomonas spp., and Mycobacterium spp. were also widely distributed in lakes. Genus-specific analysis of Legionella spp. within the lake environments confirmed the presence of diverse Legionella amplicon sequence variants (ASVs) that were distinctly different from the Legionella ASVs detected in the station. The majority of the Legionella ASVs inhabiting Antarctic lake habitats were phylogenetically distinct from known Legionella species, whereas the ASVs detected in the human-made station tended to contain ASVs highly similar to well-described mesophilic species with human pathogenicity. These data suggest that unexpected Legionella diversity exists in remote Antarctic cold environments and that environmental differences (e.g., temperature) in and around the station affect the community structure.IMPORTANCE We comprehensively examined the localization of potential waterborne pathogens in the Antarctic human-made and natural aquatic environment with special focus on Legionella spp. Some potential pathogenic genera were detected with low relative abundance in the natural environment, but most detections of these genera occurred in the station. Through detailed community analysis of Legionella spp., we revealed that a variety of Legionella spp. was widely distributed in the Antarctic environment and that they were phylogenetically distinct from the described species. This fact indicates that there are still diverse unknown Legionella spp. in Antarctica, and this genus encompasses a greater variety of species in low-temperature environments than is currently known. In contrast, amplicon sequence variants closely related to known Legionella spp. with reported pathogenicity were almost solely localized in the station, suggesting that human-made environments alter the Legionella community.

Dissolved Organic Matter Processing in Pristine Antarctic Streams.

Accelerated glacier melt and runoff may lead to inputs of labile dissolved organic matter (DOM) to downstream ecosystems and stimulate the associated biogeochemical processes. However, still little is known about glacial DOM composition and its downstream processing before entering the ocean, although the function of DOM in food webs and ecosystems largely depends on its composition. Here, we employ a set of molecular and optical techniques (UV-vis absorption and fluorescence spectroscopy, 1H NMR, and ultrahigh-resolution mass spectrometry) to elucidate the composition of DOM in Antarctic glacial streams and its downstream change. Glacial DOM consisted largely of a mixture of small microbial-derived biomolecules. 1H NMR analysis of bulk water revealed that these small molecules were processed downstream into more complex, structurally unrecognizable molecules. The extent of processing varied between streams. By applying multivariate statistical (compositional data) analysis of the DOM molecular data, we identified molecular compounds that were tightly associated and moved in parallel in the glacial streams. Lakes in the middle of the flow paths enhanced water residence time and allowed for both more DOM processing and production. In conclusion, downstream processing of glacial DOM is substantial in Antarctica and affects the amounts of biologically labile substrates that enter the ocean.

A comparative study of wavelength-dependent photoinactivation in photosystem II of drought-tolerant photosynthetic organisms in Antarctica and the potential risks of photoinhibition in the habitat.

Background and Aims: All photosynthetic organisms are faced with photoinhibition, which would lead to death in severe environments. Because light quality and light intensity fluctuate dynamically in natural microenvironments, quantitative and qualitative analysis of photoinhibition is important to clarify how this environmental pressure has impacted ecological behaviour in different organisms. Methods: We evaluated the wavelength dependency of photoinactivation to photosystem II (PSII) of Prasiola crispa (green alga), Umbilicaria decussata (lichen) and Ceratodon purpureus (bryophyte) harvested from East Antarctica. For evaluation, we calculated reaction coefficients, Epis, of PSII photoinactivation against energy dose using a large spectrograph. Daily fluctuation of the rate coefficient of photoinactivation, kpi, was estimated from Epis and ambient light spectra measured during the summer season. Key Results: Wavelength dependency of PSII photoinactivation was different for the three species, although they form colonies in close proximity to each other in Antarctica. The lichen exhibited substantial resistance to photoinactivation at all wavelengths, while the bryophyte showed sensitivity only to UV-B light (<325 nm). On the other hand, the green alga, P. crispa, showed ten times higher Epi to UV-B light than the bryophyte. It was much more sensitive to UV-A (325-400 nm). The risk of photoinhibition fluctuated considerably throughout the day. On the other hand, Epis were reduced dramatically for dehydrated compared with hydrated P. crispa. Conclusions: The deduced rate coefficients of photoinactivation under ambient sunlight suggested that P. crispa needs to pay a greater cost to recover from photodamage than the lichen or the bryophyte in order to keep sufficient photosynthetic activity under the Antarctic habitat. A newly identified drought-induced protection mechanism appears to operate in P. crispa, and it plays a critical role in preventing the oxygen-evolving complex from photoinactivation when the repair cycle is inhibited by dehydration.

Ideal osmotic spaces for chlorobionts or cyanobionts are differentially realized by lichenized fungi.

Lichens result from symbioses between a fungus and either a green alga or a cyanobacterium. They are known to exhibit extreme desiccation tolerance. We investigated the mechanism that makes photobionts biologically active under severe desiccation using green algal lichens (chlorolichens), cyanobacterial lichens (cyanolichens), a cephalodia-possessing lichen composed of green algal and cyanobacterial parts within the same thallus, a green algal photobiont, an aerial green alga, and a terrestrial cyanobacterium. The photosynthetic response to dehydration by the cyanolichen was almost the same as that of the terrestrial cyanobacterium but was more sensitive than that of the chlorolichen or the chlorobiont. Different responses to dehydration were closely related to cellular osmolarity; osmolarity was comparable between the cyanolichen and a cyanobacterium as well as between a chlorolichen and a green alga. In the cephalodium-possessing lichen, osmolarity and the effect of dehydration on cephalodia were similar to those exhibited by cyanolichens. The green algal part response was similar to those exhibited by chlorolichens. Through the analysis of cellular osmolarity, it was clearly shown that photobionts retain their original properties as free-living organisms even after lichenization.

Comparative analysis of milk fat decomposition activity by Mrakia spp. isolated from Skarvsnes ice-free area, East Antarctica.

Milk fat curdle is difficult to remove from sewage. In an attempt to identify an appropriate agent for bio-remediation of milk fat curdle, Mrakia strains were collected from the Skarvsnes ice-free area of Antarctica. A total of 27 strains were isolated and tested for their ability to decompose milk fat at temperatures ranging from 4°C to 15°C. All strains could decompose milk fat at 4°C and 10°C. Phylogenetic analysis and comparison of the decomposition ability of milk fat (DAMF) revealed that the DAMF may be useful for predicting the outcome of phylogenetic analysis based on ITS sequences.

Improvement of direct ethanol fermentation from woody biomasses by the Antarctic basidiomycetous yeast, Mrakia blollopis, under a low temperature condition.

The Antarctic basidiomycetous yeast Mrakia blollopis SK-4 can quite uniquely ferment various sugars under low temperature conditions. When strain SK-4 fermented lignocellulosic biomass using the direct ethanol fermentation (DEF) technique, approximately 30% to 65% of the theoretical ethanol yield was obtained without and with the addition of the non-ionic surfactant Tween 80, respectively. Therefore, DEF from lignocellulosic biomass with M. blollopis SK-4 requires the addition of a non-ionic surfactant to improve fermentation efficiency. DEF with lipase converted Eucalyptus and Japanese cedar to 12.6g/l, and 14.6g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80 and 5U/g-dry substrate lipase, ethanol concentration increased about 1.4- to 2.4-fold compared to that without Tween 80 and lipase. We therefore consider that the combination of M. blollopis SK-4 and DEF with Tween 80 and lipase has good potential for ethanol fermentation in cold environments.

Characterization of the far-red light absorbing light-harvesting chlorophyll a/b binding complex, a derivative of the distinctive Lhca gene family in green algae.

Prasiola crispa, an aerial green alga, exhibits remarkable adaptability to the extreme conditions of Antarctica by forming layered colonies capable of utilizing far-red light for photosynthesis. Despite a recent report on the structure of P. crispa's unique light-harvesting chlorophyll (Chl)-binding protein complex (Pc-frLHC), which facilitates far-red light absorption and uphill excitation energy transfer to photosystem II, the specific genes encoding the subunits of Pc-frLHC have not yet been identified. Here, we report a draft genome sequence of P. crispa strain 4113, originally isolated from soil samples on Ongul Island, Antarctica. We obtained a 92 Mbp sequence distributed in 1,045 scaffolds comprising 10,244 genes, reflecting 87.1% of the core eukaryotic gene set. Notably, 26 genes associated with the light-harvesting Chl a/b binding complex (LHC) were identified, including four Pc-frLHC genes, with similarity to a noncanonical Lhca gene with four transmembrane helices, such as Ot_Lhca6 in Ostreococcus tauri and Cr_LHCA2 in Chlamydomonas reinhardtii. A comparative analysis revealed that Pc-frLHC shares homology with certain Lhca genes found in Coccomyxa and Trebouxia species. This similarity indicates that Pc-frLHC has evolved from an ancestral Lhca gene with four transmembrane helices and branched out within the Trebouxiaceae family. Furthermore, RNA-seq analysis conducted during the initiation of Pc-frLHC gene induction under red light illumination indicated that Pc-frLHC genes were induced independently from other genes associated with photosystems or LHCs. Instead, the genes of transcription factors, helicases, chaperones, heat shock proteins, and components of blue light receptors were identified to coexpress with Pc-frLHC. Those kinds of information could provide insights into the expression mechanisms of Pc-frLHC and its evolutional development.

Influence of initial pH on ethanol production by the Antarctic basidiomycetous yeast Mrakia blollopis.

The Antarctic basidiomycetous yeast Mrakia blollopis SK-4 fermented ethanol between pH 5.0 and pH 10.0 with optimum pH at 8.0-10.0. Knowledge of ethanol fermentability as to the genus Mrakia remains incomplete. Further experiments are required to elucidate the ethanol fermentability of genus e.g., as to optimum fermentation pH, optimum fermentation temperature, and cell viability during fermentation.

Direct ethanol fermentation from lignocellulosic biomass by Antarctic basidiomycetous yeast Mrakia blollopis under a low temperature condition.

Antarctic basidiomycetous yeast Mrakia blollopis SK-4 has unique fermentability for various sugars under a low temperature condition. Hence, this yeast was used for ethanol fermentation from glucose and also for direct ethanol fermentation (DEF) from cellulosic biomass without/with Tween 80 at 10°C. Maximally, 48.2 g/l ethanol was formed from 12% (w/v) glucose. DEF converted filter paper, Japanese cedar and Eucalyptus to 12.2 g/l, 12.5 g/l and 7.2 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80, ethanol concentration increased by about 1.1-1.6-fold compared to that without Tween 80. This is the first report on DEF using cryophilic fungi under a low temperature condition. We consider that M. blollopis SK-4 has a good potential for ethanol fermentation in cold environments.

Genome Sequence of Basidiomycetous Yeast Mrakia gelida MGH-2, Isolated from Skarvsnes Ice-Free Area, East Antarctica.

Basidiomycetous yeast Mrakia gelida MGH-2 has been reported from Surikogi Ike in the Skarvsnes ice-free area, East Antarctica. Here, we report on the high-quality genome sequence of the Mrakia gelida MGH-2 strain analyzed by PacBio Sequel and HiSeq 2500 instruments.

Microbial community composition of terrestrial habitats in East Antarctica with a focus on microphototrophs.

The Antarctic terrestrial environment harbors a diverse community of microorganisms, which have adapted to the extreme conditions. The aim of this study was to describe the composition of microbial communities in a diverse range of terrestrial environments (various biocrusts and soils, sands from ephemeral wetlands, biofilms, endolithic and hypolithic communities) in East Antarctica using both molecular and morphological approaches. Amplicon sequencing of the 16S rRNA gene revealed the dominance of Chloroflexi, Cyanobacteria and Firmicutes, while sequencing of the 18S rRNA gene showed the prevalence of Alveolata, Chloroplastida, Metazoa, and Rhizaria. This study also provided a comprehensive assessment of the microphototrophic community revealing a diversity of cyanobacteria and eukaryotic microalgae in various Antarctic terrestrial samples. Filamentous cyanobacteria belonging to the orders Oscillatoriales and Pseudanabaenales dominated prokaryotic community, while members of Trebouxiophyceae were the most abundant representatives of eukaryotes. In addition, the co-occurrence analysis showed a prevalence of positive correlations with bacterial taxa frequently co-occurring together.

Uphill energy transfer mechanism for photosynthesis in an Antarctic alga.

Prasiola crispa, an aerial green alga, forms layered colonies under the severe terrestrial conditions of Antarctica. Since only far-red light is available at a deep layer of the colony, P. crispa has evolved a molecular system for photosystem II (PSII) excitation using far-red light with uphill energy transfer. However, the molecular basis underlying this system remains elusive. Here, we purified a light-harvesting chlorophyll (Chl)-binding protein complex from P. crispa (Pc-frLHC) that excites PSII with far-red light and revealed its ring-shaped structure with undecameric 11-fold symmetry at 3.13 Šresolution. The primary structure suggests that Pc-frLHC evolved from LHCI rather than LHCII. The circular arrangement of the Pc-frLHC subunits is unique among eukaryote LHCs and forms unprecedented Chl pentamers at every subunit‒subunit interface near the excitation energy exit sites. The Chl pentamers probably contribute to far-red light absorption. Pc-frLHC's unique Chl arrangement likely promotes PSII excitation with entropy-driven uphill excitation energy transfer.

Complete Genome Sequence of Polynucleobacter sp. Strain TUM22923, Isolated from Antarctic Lake Sediment.

Here, we report the complete genome sequence of Polynucleobacter sp. strain TUM22923, isolated from Antarctic lake sediment. This strain has a genome of 1,860,127 bp, comprising 1,848 protein-coding sequences. These sequence data could contribute to the elucidation of genome streamlining and low-temperature adaptation in members of Polynucleobacter, a cosmopolitan group of ultramicrobacteria.

Whole-Genome Sequences of Three Psychrotolerant Mycolicibacterium sp. Strains Isolated from Antarctic Soil.

We report the whole-genome sequences of three psychrotolerant Mycolicibacterium strains, TUM20983, TUM20984, and TUM20985, isolated from Antarctic soils. Taxonomic analyses indicate that these strains are putative new species. These genome sequences may provide insight into the cold adaptation mechanisms of Mycolicibacterium spp. through future comparative genomic studies.

Measurement of solar UV radiation in Antarctica with collagen sheets.

Collagen sheets were used in a unique evaluation method to examine skin damage caused by ultraviolet (UV) light of short wavelength during a season of the Antarctic ozone hole. The collagen sheets were exposed outdoors for 25 and 50 d, in the spring when the ozone hole was formed and in the ozone-hole-free autumn. Extracts from the exposed collagen sheets were analyzed for total protein and terminal amino acid concentrations as an index of collagen fragmentation. The results show that the amount of extractable collagen and terminal amino acid concentration in the spring exposure were approximately double and five times higher, respectively, when compared with those in the autumn exposure. During the ozone hole occurrence, the terminal amino acid concentration of the extracted collagen was about five times higher when exposure lasted 50 d from mid-September to the end of October compared to when exposure lasted 25 d from mid-September to early October. This result could be attributed to a limited amount of short-wavelength UV radiation reaching the ground surface as a result of the low height of the sun in September, when the ozone hole occurred. In fact, UV radiation measurements taken at Syowa Station indicate that short-wavelength UV radiation in the range 290-295 nm was not detected until approximately 1-2 months after the beginning of the ozone hole occurrence.

Basidiomycetous Yeast, Glaciozyma antarctica, Forming Frost-Columnar Colonies on Frozen Medium.

The basidiomycetous yeast, Glaciozyma antarctica, was isolated from various terrestrial materials collected from the Sôya coast, East Antarctica, and formed frost-columnar colonies on agar plates frozen at -1 °C. Thawed colonies were highly viscous, indicating that the yeast produced a large number of extracellular polysaccharides (EPS). G. antarctica was then cultured on frozen media containing red food coloring to observe the dynamics of solutes in unfrozen water; pigments accumulated in frozen yeast colonies, indicating that solutes were concentrated in unfrozen water of yeast colonies. Moreover, the yeast produced a small quantity of ice-binding proteins (IBPs) which inhibited ice crystal growth. Solutes in unfrozen water were considered to accumulate in the pore of frozen colonies. The extracellular IBPs may have held an unfrozen state of medium water after accumulation in the frost-columnar colony.

Characterization of the First Cultured Psychrotolerant Representative of Legionella from Antarctica Reveals Its Unique Genome Structure.

Culture-independent analysis shows that Legionella spp. inhabit a wide range of low-temperature environments, but to date, no psychrotolerant or psychrophilic strains have been reported. Here, we characterized the first cultivated psychrotolerant representative, designated strain TUM19329T, isolated from an Antarctic lake using a polyphasic approach and comparative genomic analysis. A genome-wide phylogenetic tree indicated that this strain was phylogenetically separate at the species level. Strain TUM19329T shared common physiological traits (e.g., Gram-negative, limited growth on buffered charcoal-yeast extract α-ketoglutarate [BCYEα] agar with l-cysteine requirements) with its relatives, but it also showed psychrotolerant growth properties (e.g., growth at 4°C to 25°C). Moreover, this strain altered its own cellular fatty acid composition to accumulate unsaturated fatty acid at a lower temperature, which may help maintain the cell membrane fluidity. Through comparative genomic analysis, we found that this strain possessed massive mobile genetic elements compared with other species, amounting to up to 17% of the total genes. The majority of the elements were the result of the spread of only a few insertion sequences (ISs), which were spread throughout the genome by a "copy-and-paste" mechanism. Furthermore, we found metabolic genes, such as fatty acid synthesis-related genes, acquired by horizontal gene transfer (HGT). The expansion of ISs and HGT events may play a major role in shaping the phenotype and physiology of this strain. On the basis of the features presented here, we propose a new species-Legionella antarctica sp. nov.-represented by strain TUM19329T (= GTC 22699T = NCTC 14581T). IMPORTANCE This study characterized a unique cultivated representative of the genus Legionella isolated from an Antarctic lake. This psychrotolerant strain had some common properties of known Legionella species but also displayed other characteristics, such as plasticity in fatty acid composition and an enrichment of mobile genes in the genome. These remarkable properties, as well as other factors, may contribute to cold hardiness, and this first cultivated cold-tolerant strain of the genus Legionella may serve as a model bacterium for further studies. It is worth noting that environmentally derived 16S rRNA gene phylotypes closely related to the strain characterized here have been detected from diverse environments outside Antarctica, suggesting a wide distribution of psychrotolerant Legionella bacteria. Our culture- and genome-based findings may accelerate the ongoing studies of the behavior and pathogenicity of Legionella spp., which have been monitored for many years in the context of public health.

Investigating Algal Communities in Lacustrine and Hydro-Terrestrial Environments of East Antarctica Using Deep Amplicon Sequencing.

Antarctica has one of the most extreme environments on Earth, with low temperatures and low nutrient levels. Antarctica's organisms live primarily in the coastal, ice-free areas which cover approximately 0.18% of the continent's surface. Members of Cyanobacteria and eukaryotic algae are important primary producers in Antarctica since they can synthesize organic compounds from carbon dioxide and water using solar energy. However, community structures of photosynthetic algae in Antarctica have not yet been fully explored at molecular level. In this study, we collected diverse algal samples in lacustrine and hydro-terrestrial environments of Langhovde and Skarvsnes, which are two ice-free regions in East Antarctica. We performed deep amplicon sequencing of both 16S ribosomal ribonucleic acid (rRNA) and 18S rRNA genes, and we explored the distribution of sequence variants (SVs) of these genes at single nucleotide difference resolution. SVs of filamentous Cyanobacteria genera, including Leptolyngbya, Pseudanabaena, Phormidium, Nodosilinea, Geitlerinama, and Tychonema, were identified in most of the samples, whereas Phormidesmis SVs were distributed in fewer samples. We also detected unicellular, multicellular or heterocyst forming Cyanobacteria strains, but in relatively small abundance. For SVs of eukaryotic algae, Chlorophyta, Cryptophyta, and Ochrophyta were widely distributed among the collected samples. In addition, there was a red colored bloom of eukaryotic alga, Geminigera cryophile (Cryptophyta), in the Langhovde coastal area. Eukaryotic SVs of Acutuncus antarcticus and/or Diphascon pingue of Tardigrada were dominant among most of the samples. Our data revealed the detailed structures of the algal communities in Langhovde and Skarvsnes. This will contribute to our understanding of Antarctic ecosystems and support further research into this subject.

Ammonia Oxidation Potentials and Ammonia Oxidizers of Lichen-Moss Vegetated Soils at Two Ice-free Areas in East Antarctica.

The maximum ammonia oxidation potential (AOP) of a topsoil in Langhovde, East Antarctica was 22.1±2.4| |ng N g-1 dry soil h-1 (2| |mM ammonium, 10°C, n=3). This topsoil exhibited twin AOP peaks (1 and 2| |mM ammonium) at 10°C, but not at 20°C. Six and ten operational taxonomic units (OTUs) were identified for ammonia-oxidizing bacteria (AOB) and archaea (AOA) amoA, respectively. AOB were classified into Nitrosospira; the two dominant OTUs corresponded to the Mount Everest cluster. AOA were classified into three clusters; Nitrososphaera and Nitrosocosmicus were the two dominant clusters.