Flavobacterium psychraerolatum sp. nov., Flavobacterium aeripolare sp. nov. and Flavobacterium gelidicaeli sp. nov. isolated from Antarctic air

Flavobacterium psychraerolatum sp. nov., Flavobacterium aeripolare sp. nov. and Flavobacterium gelidicaeli sp. nov. isolated from Antarctic air | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Flavobacterium psychraerolatum sp. nov., Flavobacterium aeripolare sp. nov. and Flavobacterium gelidicaeli sp. nov. isolated from Antarctic air Eliana V. Machin, Jitka Vives, Rodolfo Javier Menes This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8347779/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Four aerobic, Gram-stain-negative, non-motile, rod-shaped, psychrophilic and yellow to light orange-pigmented bacterial strains, designated 7Aᵀ, 14Aᵀ, 28Aᵀ and 7E, were isolated from air samples collected at Fildes Peninsula, King George Island, Antarctica. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the novel strains were related to members of the genus Flavobacterium , and revealed that strain 7A T shared 98.40% similarity to F. faecale WV33 T , while strains 14A T , 28A T and 7E showed 98.88%, 98.74% and 98.61% similarity, respectively, to F. frigidarium A2i T . The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between these novel isolates and their closest relatives were below the cut-off values used for species delineation of 95-96% and 70%, respectively. Moreover, the ANI and dDDH values between strains 28A T and 7E were 97.62 and 78.30%, respectively, indicating that these strains represented the same species. All four strains contained menaquinone-6 as the sole respiratory quinone. The DNA G+C content of all strains ranged from 32.8 to 34.5 mol%. Based on phylogenetic, genomic and phenotypic data, we propose that strains 7Aᵀ, 14Aᵀ and 28Aᵀ represent three novel species of the genus Flavobacterium , for which the names Flavobacterium psychraerolatum (7A T =CCM 8976 T =DSM 111734 T ), Flavobacterium aeripolare (14A T =CCM 8972 T =CGMCC 1.18502 T ) and Flavobacterium gelidicaeli (28A T =CCM 8973 T =CGMCC 1.18503 T ) are proposed. Taxonomy Bacteriology Flavobacterium sp airborne bacteria Antarctica psychrophiles Figures Figure 1 INTRODUCTION The genus Flavobacterium , proposed by Bergey et al. [ 1 ], belongs to the phylum Bacteroidota , and is the type genus of the family Flavobacteriaceae [ 2 ]. At the time of writing, the genus comprises 334 species with validly published names ( https://lpsn.dsmz.de/genus/flavobacterium , accessed 10 October 2025). Members of this genus are aerobic, Gram-stain-negative, rod-shaped bacteria, non-motile or motile by gliding, and typically produce yellow to orange-pigmented colonies. Menaquinone-6 (MK-6) is the sole or predominant respiratory quinone [ 2 , 3 ]. Flavobacterium species are widely distributed in nature, and have been isolated from temperate and cold freshwater habitats [ 4 – 6 ], freshwater fish tissues [ 3 ], seawater [ 7 , 8 ], sediment [ 9 – 11 ], temperate and cold soils [ 12 , 13 ], glaciers [ 14 , 15 ], lakes, activated sludge [ 16 ], and Antarctic microbial mats [ 17 ], as well as from the atmosphere [ 18 ]. In the sea ice of both the Arctic and Antarctic regions, Flavobacterium has been reported as a dominant genus within the bacterial community [ 19 ]. These species are of ecological and biotechnological interest due to their ability to produce cold-active enzymes [ 3 ]. The present study was undertaken to determine the taxonomic position of four novel Flavobacterium strains isolated from Antarctic air, using a polyphasic approach consistent with the minimal standards for describing new taxa within the family Flavobacteriaceae [ 20 ]. ISOLATION AND ECOLOGY To investigate the diversity of cultivable bacteria from the air in the Fildes Peninsula, King George Island (62°08'-62°14'S and 59°02'-58°51'W, Antarctica), air samples were collected in February 2018 using R2A agar as the growth medium. Plates were incubated at 5°C for up to 30 days. Colonies displaying distinct morphologies were selected, purified, and subjected to further characterization. Four isolates, designated 7A T , 14A T , 28A T and 7E were subcultured periodically on R2A agar and maintained at 4°C for short-term preservation. For long-term storage, strains were preserved at -80°C in TSB supplemented with 30 % (v/v) glycerol or lyophilized. The reference strain Flavobacterium frigidarium DSM 17623 T was obtained from German Collection of Microorganisms and Cell Cultures (DSMZ) and included in this study for comparative purposes. All strains were routinely cultivated on R2A agar at 15°C for 48–72 h. For exploring the cultivable bacterial diversity belonging to Flavobacterium genus in King George Island, 16S rRNA gene sequences of isolates were retrieved from GenBank. These strains were obtained from a variety of niches (different type of soils, sea and lake water, sea sediment, penguin faeces, moraine, algae, microbial mats). The strains 27B (MZ613136), 17C2 (MZ613133), 7C (MZ613121), H2 (MZ613117), Ac7 (MZ613114), Ab1 (MZ613110), and Aa2 (MZ613107) were isolated by our group from air collected in 2017 and 2018 as explained previously [ 21 ]. To reveal their phylogenetic relationships, a phylogenetic tree (Fig. S1) was constructed as described below. The results showed that the Antarctic isolates were distributed into multiple distinct clades, which suggests they have a long evolutionary history of divergence. 16S rRNA PHYLOGENY Genomic DNA was extracted from all strains as described previously [ 21 ]. Nearly full-length 16S rRNA gene fragments were amplified using the universal primers 27F and 1492R [ 22 ]. The resulting sequences were compared with publicly available type strain sequences with validly published names retrieved from GenBank. Pairwise sequence similarities were calculated using the global alignment algorithm implemented on the EzBioCloud server [ 23 ]. The 16S rRNA gene sequences of the four strains and those of closely related species were aligned using the CLUSTAL W tool [ 24 ] implemented in MEGA version 11 [ 25 ]. Phylogenetic trees were reconstructed using both the maximum-likelihood (ML) [ 26 ] and the neighbour-joining (NJ) [ 27 ] methods in MEGA 11. Evolutionary distances were calculated using Kimura’s two-parameter model [ 28 ]. The robustness of the inferred tree topologies was evaluated using bootstrap analysis with 1000 replications [ 29 ]. The EzBioCloud analysis of the nearly complete 16S rRNA gene sequences revealed that all four isolates shared the highest sequence similarities with members of the genus Flavobacterium . Specifically, strain 7Aᵀ showed 98.40 % similarity to F. faecale WV33ᵀ, 98.12% to F. algicola TC2ᵀ, and 96.73 % to F. ovatum W201Eᵀ. Strain 14Aᵀ exhibited 98.88 % similarity to F. frigidarium A2iᵀ, 97.88 % to F. kayseriense F-47ᵀ, and 97.56 % to F. fryxellicola DSM 16209ᵀ. Strains 28Aᵀ and 7E shared the highest similarity with F. frigidarium A2iᵀ (98.74 % and 98.61%, respectively), followed by F. muglaense F-60ᵀ (98.26 % and 98.41 %, respectively), F. crassostreae LPB0076ᵀ (97.97% and 98.12 %, respectively), and F. fryxellicola DSM 16209ᵀ (97.69 % and 97.70%, respectively). Phylogenetic trees based on evolutionary distances calculated using Kimura’s two-parameter model and constructed using the ML method (Fig. S2) showed that all four isolates were affiliated with the genus Flavobacterium . Strain 7Aᵀ clustered in a clade with F. faecale WV33ᵀ with moderate bootstrap support (77%). Strain 14Aᵀ formed a clade with F. frigidarium A2iᵀ and F. crassostreae LPB0076ᵀ, though with a low bootstrap support (< 70 %). Strains 28Aᵀ and 7E clustered together in the same clade that 14A T . Strain 7A T showed a similar tree topology using the NJ method (Fig. S3), and strain 14A T clustered with F. frigidarium A2iᵀ, whereas 28A T and 7E clustered with F. crassostreae LPB0076ᵀ and F. muglaense F-60ᵀ. GENOME FEATURES AND PHYLOGENOMICS The draft genome assembly of strains 7A T , 14A T , 28A T and 7E was generated at the DOE Joint Genome Institute (JGI) using Illumina technology [ 30 ] as described previously [ 21 ]. An Illumina standard shotgun library was constructed and sequenced using the Illumina NovaSeq S4 platform which generated 9,564,230 − 13,730,208 reads totaling 1,444,198,730-2,073,261,408 bp for all strains. The authenticity of the final assembly genome was verified by comparing the 16S rRNA gene sequences obtained from the genome and by the PCR amplification method, using Align Sequences Nucleotide blast tool ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ) optimized for highly similar sequences (megablast) and with default algorithm parameters.16S rRNA gene sequences extracted from the genome shared 100% identity with the partial sequences obtained from PCR obtained as explained above and sequenced by the Sanger method. For further comparative analyses, genome annotations were performed using the Rapid Annotation using Subsystem Technology (RAST) pipeline [ 31 – 33 ]. Venn diagrams were constructed to compare coding sequences (CDSs) among genomes based on their annotated functions and to identify unique and shared features, using the VennDiagram R package (v1.7.3) in RStudio (v1.4.1717). Only CDSs with defined product annotations were included; genes lacking such annotation (e.g. hypothetical proteins, tRNA, rRNA, etc.) were excluded. To assess genome relatedness, average nucleotide identity (ANI) values between strains 7Aᵀ, 14Aᵀ, 28Aᵀ and 7E and their closest Flavobacterium relatives were calculated using the OrthoANIu algorithm via the EzBioCloud web server [ 34 ]. Additionally, digital DNA–DNA hybridization (dDDH) values were obtained using the Genome-to-Genome Distance Calculator (GGDC) 3.0 ( https://ggdc.dsmz.de/ggdc.php# ) with formula 2 [ 35 ]. Genomes retrieved from NCBI were processed with the Genome Taxonomy Database Toolkit (GTDB-Tk v2.1.0) to generate concatenated alignments of 120 bacterial single-copy marker genes [ 36 ]. The phylogenomic tree was constructed in MEGA 11 using the Poisson correction model [ 37 ] with 1000 bootstrap replicates. To further evaluate whether strains 7Aᵀ, 14Aᵀ, 28Aᵀ and 7E represent novel species, genome sequence data of these strains were uploaded to the Type (Strain) Genome Server (TYGS) ( https://tygs.dsmz.de ; accessed 2025-10-10) for whole-genome-based phylogenetic analyses. Branch support was inferred from 100 pseudo-bootstrap replicates. Biosynthetic gene clusters potentially involved in the production of secondary metabolites were identified using the AntiSMASH online tool (version 8.0.1) [ 38 ]. Assembly statistics for all strains are presented in Table S1. CheckM analysis (v1.2.2) [ 39 ] indicated high genome completeness (98.28–99.22%) and low contamination levels (0.20–0.77%) across the isolates (Table S1). The G + C content, calculated from the whole genome sequences, ranged from 32.8 to 34.5 mol%, consistent with the reported range for members of the genus Flavobacterium (30–41 mol%) [ 3 ]. The ANI and dDDH values between strains 7Aᵀ, 14Aᵀ, 28Aᵀ and validly published Flavobacterium species names were below 84.94% and 30.80%, respectively (Table S2), supporting their designation as novel species. However, ANI and dDDH values between strains 28Aᵀ and 7E were 97.62% and 78.30%, respectively (Table S2), exceeding the commonly accepted thresholds for species delineation (95–96% ANI and 70% dDDH) [ 40 – 42 ], indicating that they belong to the same species. Phylogenomic analysis based on 120 single-copy marker genes revealed that the novel strains clustered into distinct clades (Fig. 1 ). Strain 7Aᵀ grouped in a clade with F. faecale WV33ᵀ and F. algicola MBE-1 (bootstrap support 83% and 100% respectively). Strain 14Aᵀ clustered with F. frigidarium DSM 17623ᵀ with high bootstrap support (100%). Similarly, strains 28Aᵀ and 7E clustered in a clade with F. frigidarium DSM 17623ᵀ, supported by a bootstrap value of 83%. Similar results were observed with the TYGS phylogenomic tree (Fig. S4). According to RAST annotation results, the genome of strain 7Aᵀ contained 3,683 coding sequences (CDSs) and 53 RNA genes, while the genome of strain 14Aᵀ comprised 3,349 CDSs and 55 RNA genes. Strain 28Aᵀ possessed 3,626 CDSs and 53 RNA genes, whereas strain 7E contained 3,995 CDSs and 51 RNA genes (Table S1). The genomes of all four novel isolates included genes encoding cold shock proteins (CSP family), which are known to be essential for cellular responses to cold stress. In addition, strains 28Aᵀ and 7E harbored genes involved in carotenoid biosynthesis, including phytoene synthase and phytoene desaturase. These genes are associated with membrane fluidity regulation under low temperatures and have been linked to increased resistance to environmental stressors such as freeze–thaw cycles and solar radiation, particularly in Antarctic bacteria [ 43 – 45 ]. Furthermore, the genomes encoded enzymes involved in the biosynthesis of straight-chain and branched-chain fatty acids (e.g. β-ketoacyl-ACP synthase II and III [KAS-II, KAS-III]), which are important for maintaining membrane fluidity and may contribute to cold adaptation [ 12 , 45 ]. Notably, all four strains contained genes for toxin–antitoxin (TA) replicon stabilization systems, which were absent in their closest phylogenetic relatives. Comparative genomic analysis showed that the novel isolates possessed distinct gene content relative to F. algicola MBE-1, F. faecale WV33ᵀ, and F. frigidarium DSM 17623ᵀ (Fig. S5; Table S3), supporting their classification as novel taxa. The Venn diagram analysis revealed that the genome of strain 7Aᵀ contains 112 unique coding sequences (CDSs) not found in F. algicola MBE-1 and F. faecale WV33ᵀ, while 1,456 CDSs are shared among the three strains (Fig. S5). Notably, strain 7Aᵀ harbors genes associated with the nitrosative stress response that are absent in the reference strains. In contrast, genes involved in lactose and galactose uptake and metabolism are conserved across all three genomes (Table S3). Similarly, strains 14Aᵀ, 28Aᵀ, and 7E possess55, 48, and 71 unique coding sequences (CDSs), respectively, and share a core genome of1219 CDSs with F. frigidarium DSM 17623ᵀ and F. crassostreae LPB0076 T (Fig. S5) For instance, genes associated with sucrose utilization were identified exclusively in strain 14Aᵀ, while genes encoding mercury reductase were shared among strains 14A T , 28A T and 7E. Furthermore, variation in the presence of genes involved in resistance to toxic compounds was observed, highlighting functional diversification among the strains. Analysis of the genomes using antiSMASH v8.0 identified several putative biosynthetic gene clusters (BGCs) associated with secondary metabolite production in the novel isolates (Table S4). Specifically, strain 7Aᵀ harbored four BGCs, including one predicted to encode a terpene compound involved in carotenoid biosynthesis. Strain 14Aᵀ also contained four BGCs, two of which were associated with the biosynthesis of carotenoids and bacillomycin D, a known antifungal agent. In strains 28Aᵀ and 7E, five and six BGCs were detected, respectively, including one predicted to encode arylpolyene/resorcinol biosynthesis, potentially responsible for the production of flexirubin-like pigments. These findings are consistent with the pigmentation phenotypes observed in these strains (Table 1 ). Table 1 Differential characteristics of the novel strains with respect to closely related Flavobacterium species. Strains: 1, 7A T ; 2, F . algicola TC2 T ; 3, F . faecale KCTC 32457 T ; 4, 14A T ; 5, 28A T ; 6, 7E; 7, F . frigidarium DSM 17623 T ; 8, F. crassostreae LPB0076 T . All data, unless otherwise indicated, were from this study. +, Positive; –, negative; ND, not determined. Characteristic 1 2 § 3* 4 5 6 7 8ˁ Growth at 0°C - - - - - - + † - % NaCl range for growth (w/v) 0–5 0–3 0.5-8 0–6 0–6 0–6 0–5 † 0–4 pH range for growth 6.0–9.0 5.5-8.0 5.0–9.0 6.0–9.0 6.0–8.0 6.0–8.0 5.6–8.4 † 5.5-8.0 Congo red adsorption - + - - - - + - Flexirubin-type pigments - - - - + + - - Hydrolysis of aesculin + + - + + - + - agar + - + - - - - casein + - - + + + + + CM-cellulose + - - - - - - - DNA - - + + + + - - gelatin - - - + + + + - L-tyrosine - - - - + + - - starch + + + + - + - - Acid from fructose + ND ND + + + - ND glucose + - - + + + - - L-arabinose - - - - + - ND - mannitol + + - + + + - + melibiose ND - - + + - ND - Enzyme activity (API ZYM) β-Galactosidase + - + - - - - - Alkaline phosphatase + + + + + + - + Esterase C4 - - + - - - - - Valine arylamidase + - - + + + + + α-Glucosidase + - - - - - - - N-Acetyl-β -glucosaminidase - + - + - - - - Acid phosphatase + + - + + + - + Lipase C14 - - + - - - - - DNA G + C content (mol%) 33.8 ¶ 33.9 37.0 34.5 ¶ 32.8 ¶ 32.8 ¶ 35.0 † 35.9 ¶ § Data from [ 50 ] * Data from [ 51 ] †Data from [ 52 ] ˁ Data from [ 53 ] ¶ Data from the genome sequence PHYSIOLOGY AND CHEMOTAXONOMY Growth on/in several media including R2A broth and agar (Merck), PCA (Difco), TSA (Difco), TSB (Difco), NA (Oxoid), NB (Oxoid), MacConkey agar (Difco), Cetrimide agar (Merck) and Marine agar (HiMedia) was evaluated at 15°C after 7 days of incubation. Anaerobic growth was assessed on R2A agar at 15°C for 7 days using the Anaerocult A Mini gas generator system (Merck). Cell morphology was examined by Transmission Electron Microscopy (TEM). Cells grown in R2A broth for 48 h were fixed in an aqueous solution of 2.5% (w/v) glutaraldehyde for four hours at 4°C. Each sample was mounted on a 200-mesh copper grid coated with a 90 nm LR White (London Resin) membrane and incubated for 10 min. Excess liquid was removed by capillary action, and the grids were air-dried for an additional 10 min. Negative staining was performed using a 2% (w/v) aqueous uranyl acetate solution for one minute, followed by drying via capillarity. Subsequently, the samples underwent three washes with ultrapure water, with drying steps between each wash. The grids were then left to rest for 10 min prior to imaging. Samples were observed using a Zeiss EM 109T Transmission Electron Microscope equipped with a Gatan ES1000W digital camera as shown in Fig. S6. The presence of flexirubin-type pigments was tested by flooding the colonies grown on NA with 20 % (w/v) KOH solution [ 2 ]. Growth at different temperatures (from − 5 to 30°C in ~ 5°C intervals) was assessed in R2A broth (Oxoid) with 5 % (v/v) glycerol supplementation at − 5 and 0 °C. The pH growth range (pH 5.0–11.0, in 1.0 unit increments) was determined in R2A broth buffered with K₂HPO₄–KH₂PO₄ (pH 5.0–8.0), NaHCO 3 /Na 2 CO 3 (pH 9.0–10.0) and NaHCO 3 –NaOH (pH 11.0). The initial pH was adjusted using HCl or NaOH as required, and media were sterilized by filtration. Tolerance to NaCl was tested in R2A broth prepared without NaCl and supplemented with a final NaCl concentration of 0.5 to 7% (w/v) within aproximately 1% interval. Growth under different temperature, pH, and salinity conditions was monitored by measuring optical density at 660 nm (OD₆₆₀) over time. Slopes of OD₆₆₀ versus time curves were averaged from triplicate runs for each condition to determine the optimal growth. [ 46 ]. Catalase activity was determined by bubble production upon application of 3 % (v/v) hydrogen peroxide, and oxidase activity using Bactident oxidase strips (Merck). The following biochemical tests were conducted as described by Barrow and Feltham [ 47 ], using NA as basal medium and incubated at 15°C for 7 days: ONPG (2-nitrophenyl-β-D-galactopyranoside) hydrolysis, Voges–Proskauer reaction, nitrate reduction and denitrification, and hydrolysis of Tween 20, Tween 60, Tween 80, starch, aesculin, L-tyrosine, carboxymethyl cellulose (CM-cellulose), casein and urea. Additional assays, including DNA hydrolysis, gelatin hydrolysis (method 2), citrate utilization (method 1), indole production, H₂S production from cysteine, phenylalanine deaminase and lecithinase activities, were performed following standard protocols [ 48 ], at 15°C for 7 days. Agar degradation was tested on R2A agar at 15°C by observing colony-associated agar softening. Motility (by hanging drop technique) and cell morphology were examined by phase-contrast microscopy (Zeiss Axioplan, Germany) in exponentially growing cultures in R2A broth at 15°C. Acid production from carbohydrates was assessed in Ammonium Salt Sugars (ASS) medium [ 47 ] with the following composition (L − 1 ): 1.0 g (NH 4 ) 2 HPO 4 , 0.2 g KCl, 0.2 g MgSO 4 .7H 2 O, 0.2 g yeast extract, 20 g agar and 0.04 g bromothymol blue, with each carbohydrate added aseptically from sterile stock solutions to a final concentration of 1 % (w/v). For strain 7Aᵀ, ASS medium was prepared without agar. To determine the range of substrates used for growth as carbon and energy source the following basal media was prepared (per liter): 6.24 g NaH 2 PO 4 , 2.0 g (NH 4 )SO 4 , 1.5 g KCl /L, 10 mL of Wolin's vitamin solution, and 10 mL trace element solution (according to DSMZ medium 318), final pH 7.2. Additional phenotypic characterization was carried out using API ZYM and API 20NE test kits (bioMérieux), following the manufacturer’s protocols, but incubated at 15°C. For API 20NE results were read after 7 days. Strains 7Aᵀ, 14Aᵀ, 28Aᵀ and 7E were catalase- and oxidase-positive, and formed yellow to light orange-pigmented colonies (Fig. S7). The isolates exhibited phenotypic traits that differentiated them from the reference species (Table 1 ). Although strains 28Aᵀ and 7E represented the same species based on genomic data, they showed several phenotypic differences. For example, strain 28Aᵀ hydrolysed aesculin, whereas strain 7E did not. Strain 28Aᵀ also produced acid from arabinose and melibiose and was able to utilize raffinose as a sole carbon and energy source, unlike strain 7E. Flexirubin-type pigments were detected in both strains 28Aᵀ and 7E, but were absent in strains 7Aᵀ and 14Aᵀ. Growth occurred on NA, PCA, R2A agar and broth, and in NB and TSB in 48–72 h with similar performance, but no growth was obtained on cetrimide agar, MacConkey agar or marine agar or under anaerobic conditions. Physiological tests, showed that all four strains grew over a temperature range of 5–20°C. Strain 28Aᵀ was able to grow up to 25°C. Strains 7Aᵀ and 14Aᵀ grew within a pH range of 6.0–9.0, whereas strains 28Aᵀ and 7E grew within a pH range of 6.0–8.0. Strain 7Aᵀ tolerated NaCl concentrations up to 5.0 % (w/v), whereas strains 14Aᵀ, 28Aᵀ and 7E grew in the presence of up to 6.0 % (w/v) NaCl. Detailed phenotypic traits specific to each novel strain are summarized in the species descriptions. Isoprenoid quinones were extracted and analysed as described previously [ 22 ]. Menaquinone-6 (MK-6) was the sole respiratory quinone detected in all strains, consistent with other members of the genus Flavobacterium [ 3 ]. Fatty acid methyl ester (FAME) profiles were analysed according to the standard protocol of the Sherlock Microbial Identification System (MIDI, version 6.2; RTSBA 6.21 database) [ 49 ]. Cultures were grown on R2A agar at 20°C until late exponential phase. FAMEs were extracted and analysed using a 7890B gas chromatograph (Agilent Technologies). The most abundant fatty acids identified in all strains are presented in Table S5, which are consistent with previously described species of Flavobacterium and with the genus description [ 2 ]. Despite this general similarity, species differentiation was supported by quantitative differences in the composition of both major and minor fatty acids. The major fatty acids of 7A T were summed feature 3 (C 16:1 ω 7c /C 16:1 ω 6c ), iso-C 15:0, iso-C 15:0 3-OH, and anteiso-C 15:0 . Strain 14Aᵀ showed a profile dominated by summed feature 3 (C 16:1 ω 7c /C 16:1 ω 6c ), iso-C 15:0 , and C 15:1 ω 6c . In strain 28Aᵀ, the predominant components were C 15:1 ω 6c , summed feature 3 (C 16:1 ω 7c /C 16:1 ω 6c ), and iso-C 15:0 . CONCLUSION Psychrophilic or psychrotolerant Flavobacterium type species have been reported from King George Island and nearby Antarctic environments, such as F. antarcticum [ 54 ] (isolated from soil near a penguin habitat, F. faecale [ 51 ] (isolated from penguin faeces, F. kingsejongi [ 55 ] (isolated from penguin faeces), F. ardleyense [ 56 ] (obtained from soil on Ardley Island), F. azizsancarii [ 57 ] (isolated from lake water on Ardley Island) F. collinsense [ 58 ] (isolated from a till sample near the Collins Glacier front), and F. weaverense and F. segetis [ 59 ] (both isolated from terrestrial Antarctic samples). A recent study [ 60 ] revealed some surprising phylogenetic diversity among Flavobacterium strains isolated from microbial mats in ponds and streams on King George Island. The research identified 20 distinct species among 50 isolates. This highlights how much we have underestimated the biodiversity of Flavobacterium in Antarctic freshwater ecosystems. The novel strains 7Aᵀ, 14Aᵀ, 28Aᵀ, and 7E, isolated from the atmosphere, greatly expand our understanding of the Flavobacterium genus in these unique ecosystems. To our knowledge, these are the first Flavobacterium species formally described from Antarctic air samples, suggesting their ability to disperse through the air and withstand extreme atmospheric conditions. Their presence in the atmosphere suggests they might play important roles in long-distance microbial transport and colonization in polar environments, complementing previous studies from terrestrial and aquatic habitats. According to the results obtained, strains 7A T , 14A T , 28A T and 7E exhibited distinct genotypic and phenotypic characteristics compared to previously described species of the genus Flavobacterium . At the genomic level, all strains displayed ANI and dDDH values below the established thresholds for species delineation when compared with validly published Flavobacterium species names. Phylogenomic analysis further supported their taxonomic distinctiveness, revealing that the novel isolates formed well-separated clades within the genus. Several phenotypic differences were also observed. For example, strain 7Aᵀ produced acid from glucose and mannitol, and hydrolysed CM-cellulose, distinguishing it from F. faecale KCTC 32457ᵀ. Strains 14Aᵀ, 28Aᵀ and 7E hydrolysed DNA and starch, and produced acid from fructose, mannitol, glucose and xylose, traits not observed in F. frigidarium DSM 17623ᵀ. Additionally, strains 28Aᵀ and 7E hydrolysed L-tyrosine, in contrast to F. frigidarium DSM 17623ᵀ. On the other hand, strains 14Aᵀ, 28Aᵀ and 7E hydrolysed gelatin whereas F. crassostreae LPB0076 T did not. Strain 28A T produced acid from L-arabinose unlike F. crassostreae LPB0076 T . Additionally, strains 14A T and 28A T produced acid from melibiose, characteristic absent in F. crassostreae LPB0076 T . Taken together, the genotypic, phylogenomic, phenotypic and chemotaxonomic data clearly indicate that strains 7Aᵀ, 14Aᵀ, and 28Aᵀ represent novel species within the genus Flavobacterium , for which the names Flavobacterium psychraerolatum sp. nov., Flavobacterium aeripolare sp. nov. and Flavobacterium gelidicaeli sp. nov., respectively, are proposed. PROTOLOGUE Description of Flavobacterium psychraerolatum sp. nov. Flavobacterium psychraerolatum (psychr.a.e.ro.la’tum. Gr. masc. adj. psychros , cold; Gr. masc. n. aer , air; L. masc. perf. part. latus , carried; N.L. neut. part. adj. psychraerolatum , transported by cold-air). Cells are Gram-stain-negative, non-motile, non-spore-forming rods occurring singly or in pairs, measuring 0.2–0.3 µm in width and 1.0–1.8 µm in length. Strictly aerobic. Colonies grown on R2A agar are circular, smooth, flat, glistening, yellow-pigmented with entire margins and measure 1–2 mm in diameter after 48 h incubation at 15°C. Flexirubin-type pigments are not produced. Growth occurs on NA, PCA, R2A agar and broth, and in NB and TSB at 15°C, but not on cetrimide agar, MacConkey agar or marine agar. No growth occurs under anaerobic conditions. Growth occurs at 5–20°C (optimum 15–20°C), pH 6.0–9.0 (optimum pH 6.0), and in the presence of 0–5.0 % (w/v) NaCl (optimum 2 %). Positive for catalase and oxidase activities. Hydrolysis of aesculin, agar, CM-cellulose, Tween 20, and starch is positive. Hydrolysis of DNA, gelatin, lecithin, L-tyrosine, Tween 60, Tween 80, and urea is negative. The ONPG test is positive. Citrate utilization, indole production, nitrate reduction and denitrification, H₂S production from cysteine, L-phenylalanine deaminase, L-tyrosine pigment production, and Voges–Proskauer reaction are negative. Acid is produced from D-fructose, D-galactose, D-glucose, D-mannitol, D-xylose, and weakly from glycerol. No acid is produced from L-arabinose, D-raffinose, or rhamnose. Utilization of the following compounds as sole carbon and energy sources is observed: D-arabitol (weak reaction), D-cellobiose, D-fructose, D-galactose, glycerol, maltose, D-mannitol, melibiose, and D-xylose. D-raffinose, rhamnose, and D-trehalose are not utilized. Positive reactions are observed for β-galactosidase, assimilation of D-glucose, D-maltose, D-mannitol, and weakly D-mannose. Negative reactions are recorded for aesculin hydrolysis (note: this contradicts the result obtained on solid medium), arginine dihydrolase, gelatinase, glucose fermentation under anaerobic conditions, indole production, nitrate reduction, urease activity, and assimilation of L-arabinose, adipic acid, capric acid, citrate, malic acid, N-acetylglucosamine, phenylacetic acid, and potassium gluconate. Positive reactions are observed for alkaline phosphatase, acid phosphatase, α-glucosidase, leucine arylamidase, and weakly for β-galactosidase, valine arylamidase, and naphthol-AS-BI-phosphohydrolase. Negative reactions are recorded for α-chymotrypsin, esterase (C4), esterase lipase (C8), lipase (C14), α-fucosidase, α-galactosidase, β-glucuronidase, β-glucosidase, α-mannosidase, N-acetyl-β-glucosaminidase, cystine arylamidase, and trypsin. The sole menaquinone is MK-6. The major fatty acids are summed feature 3 (C 16:1 ω 7c /C 16:1 ω 6c ), iso-C 15:0 , iso-C 15:0 iso 3-OH, and anteiso-C 15:0 . The type strain is 7Aᵀ (= CCM 8976ᵀ =DSM 111734ᵀ), isolated from air collected at Artigas Antarctic Scientific Base (62°11′04″S 58°51′07″W), Fildes Peninsula, King George Island, Antarctica. The DNA G + C content of the type strain is 33.8 mol%. The GenBank accession coder for the 16S rRNA gene sequence is MN007163. The Whole Genome Shotgun project has been deposited under the accession JAOQMX000000000; the version described in this paper is JAOQMX010000000.1. Description of Flavobacterium aeripolare sp. nov. Flavobacterium aeripolare (ae.ri.po.la´re. L. masc. n. aer , air; M.L. masc. adj. polaris , of, or pertaining to a pole; N.L. neut. adj. aeripolare , pertaining to polar air). Cells are Gram-stain-negative, non-motile, non-spore-forming rods, occurring singly or in pairs, measuring 0.2–0.3 µm in width and 1.0–4.0 µm in length. Strictly aerobic. Colonies on R2A agar are circular, flat, smooth, glistening, yellow-pigmented, with entire margins, and measure 1.5–2.5 mm in diameter after 48 h of incubation at 15°C. Flexirubin-type pigments are not produced. Growth occurs on marine agar, NA, PCA, R2A agar and broth, and in NB and TSB at 15°C, but not on cetrimide agar or MacConkey agar. No growth occurs under anaerobic conditions. Growth is observed at 5–20°C (optimum 15–20°C), pH 6.0–9.0 (optimum pH 8.0), and in the presence of 0–6.0 % (w/v) NaCl (optimum 1.5 %). Positive for catalase and oxidase activities. Hydrolysis of aesculin, casein, gelatin, Tween 20, Tween 60, Tween 80, and starch is positive. Hydrolysis of agar, CM-cellulose, DNA, lecithin, L-tyrosine, and urea is negative. The ONPG test, citrate utilization, H₂S production from cysteine, nitrate reduction and denitrification, L-phenylalanine deaminase activity, pigment production from L-tyrosine, and the Voges–Proskauer reaction are negative. Acid is produced from D-fructose, D-galactose, D-glucose, glycerol, maltose, D-mannitol, melibiose, D-trehalose (weak reaction), and D-xylose. No acid is produced from L-arabinose, D-raffinose, or rhamnose. The following compounds are utilized as sole carbon and energy sources: D-arabitol (weak reaction), D-cellobiose, D-fructose, D-galactose, glycerol, maltose, D-mannitol, melibiose (weak reaction), D-raffinose, and D-xylose. L-arabinose, rhamnose, and D-trehalose are not utilized. Positive reactions are observed for assimilation of D-glucose, D-mannose, D-mannitol, and D-maltose. Negative reactions are recorded for aesculin hydrolysis and gelatinase (note: these differ from solid media results), nitrate reduction, indole production, glucose fermentation under anaerobic conditions, arginine dihydrolase, urease, β-galactosidase, and assimilation of L-arabinose, N-acetylglucosamine, potassium gluconate, capric acid, adipic acid, malic acid, citrate, and phenylacetic acid. Positive enzyme activities are detected for alkaline phosphatase, leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, and N-acetyl-β-glucosaminidase. Esterase lipase (C8) is weakly positive. Negative reactions are obtained for esterase (C4), lipase (C14), cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, α-mannosidase, and α-fucosidase. The sole menaquinone is MK-6. The major fatty acids are summed feature 3 (C 16:1 ω 7c /C 16:1 ω 6c ), iso-C 15:0 , iso-C 15:0 3-OH, and C 15:1 ω6c . The type strain is 14A T (= CCM 8972 T = CGMCC 1.18502 T ), isolated from the air collected at Antarctic Scientific Base Artigas (62°11′04″S 58°51′07″W), Fildes peninsula, King George Island, Antarctica. The DNA G + C content is 34.5%. The GenBank accession code for the 16S rRNA gene sequence 14A T is MN007165. The Whole Genome Shotgun project has been deposited at GenBank under the accession JABSNL000000000. The version described in this paper is version JABSNL000000000.1. Description of Flavobacterium gelidicaeli sp. nov. Flavobacterium gelidicaeli (ge.li.di.cae´li. L. masc. adj. gelidus , icy, frozen; L. neut. n. caelum , sky, air; N.L. gen. n. gelidicaeli , of cold air). Cells are Gram-stain-negative, non-motile, non-spore-forming rods, occurring singly or in pairs, measuring 0.2–0.3 µm in width and 1.0–3.0 µm in length. Strictly aerobic. Colonies on R2A agar are circular with entire margins, flat, smooth, glistening, yellow to light orange-pigmented, and measure 1–2 mm in diameter after 48 h of incubation at 15°C. Flexirubin-type pigments are produced (positive KOH test). Growth occurs on R2A agar and broth, TSA, PCA, NA, and in TSB and NB at 15°C, but not on MacConkey agar or cetrimide agar. No growth occurs under anaerobic conditions on R2A. Growth is observed at 5–25°C (optimum 15–20°C), pH 6.0–8.0 (optimum pH 6.0), and in the presence of 0–6.0 % (w/v) NaCl (optimum 1 %). Positive for catalase and oxidase activities. Hydrolysis of casein, DNA, gelatin, Tween 20, Tween 60, Tween 80, and L-tyrosine is positive. Hydrolysis of aesculin and starch is variable. Hydrolysis of agar, CM-cellulose, lecithin, and urea is negative. Citrate utilization, hydrogen sulfide production from cysteine, nitrate reduction, denitrification, ONPG test, phenylalanine deaminase, pigment production from L-tyrosine, and the Voges–Proskauer reaction are all negative. Acid is produced from D-fructose, D-glucose, glycerol, maltose, D-mannitol, D-raffinose, and D-xylose. A weak reaction is observed with D-galactose. Acid production from L-arabinose and melibiose is variable, and no acid is produced from rhamnose. The following carbohydrates are utilized as carbon and energy sources: D-fructose, D-galactose, glycerol, maltose, D-mannitol, melibiose (weak reaction), and D-xylose. L-arabinose, rhamnose, and D-trehalose are not utilized. Utilization of D-raffinose is variable. The strain is positive for gelatinase, D-glucose, D-mannose, D-mannitol, and D-maltose, and negative for nitrate reduction, indole production, glucose fermentation (anaerobic), arginine dihydrolase, urease, β-galactosidase, L-arabinose, N-acetyl-glucosamine, potassium gluconate, capric acid, adipic acid, malic acid, citrate, and phenylacetic acid. Aesculin hydrolysis yields variable results. Positive reactions are observed for alkaline phosphatase, leucine arylamidase, valine arylamidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase. Negative reactions are obtained for esterase (C4), esterase lipase (C8), lipase (C14), cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase. The sole menaquinone is MK-6. The major fatty acids are C 15:1 ω6c , summed feature 3 (C 16:1 ω7c /C 16:1 ω6c ) and iso-C 15:0 . The type strain is 28A T (= CCM 8973T = CGMCC 1.18503 T ), collected from the air sampled at Antarctic Scientific Base Artigas (62°11′04″S 58°51′07″W), Fildes peninsula, King George Island, Antarctica. The DNA G + C content is 32.8%. The GenBank accession code for the 16S rRNA gene sequence is MN007166. The Whole Genome Shotgun project has been deposited at GenBank under the accession JABSNK000000000. The version described in this paper is version JABSNK000000000.1. All characteristics of the type strain 28Aᵀ are consistent with the species description of Flavobacterium gelidicaeli , except for the following: growth at 25°C, positive results for aesculin hydrolysis, acid production from melibiose and L-arabinose, assimilation of D-raffinose, and a negative result for starch hydrolysis. Declarations Conflicts of interest The authors declare that there are no conflicts of interest. Funding information The authors received no specific grant from any funding agency. Acknowledgements RJM thank the Uruguayan Antarctic Institute and the staff of the Artigas Antarctic Scientific Base for their support during the summer 2018 campaign. The authors are also grateful to Professor Aharon Oren (The Hebrew University of Jerusalem) for his assistance in the species names. JV would like to thank the Czech Antarctic Research Programme and the Masaryk University Grant Agency (CAREER RESTART Muni/R/1443/2024) for their support. References Bergey DH, Harrison FC, Breed RS, Hammer BW, Huntoon FM (1923) Bergey's Manual of Determinative Bacteriology, 1st edn. The Williams & Wilkins Co, Baltimore Bernardet J-F, Bowman JP (2006) The Genus Flavobacterium . In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E (eds) The Prokaryotes: Volume 7: Proteobacteria: Delta, Epsilon Subclass. Springer New York, New York, NY, pp 481–531 Bernardet J-F, Bowman JP (2015) Flavobacterium . In: Bergey’s Manual of Systematics of Archaea and Bacteria. (eds M.E. Trujillo, S. 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Int J Syst Evol Microbiol 56:1239–1244. 10.1099/ijs.0.64164-0 Górniak D, Świątecki A, Kowalik J, Grzesiak J, Jastrzębski J, Zdanowski MK (2025) High antagonistic activity and antibiotic resistance of flavobacteria of polar microbial freshwater mats on King George Island in maritime Antarctica. Sci Rep 15:13615. 10.1038/s41598-025-97205-x Additional Declarations The authors declare no competing interests. Supplementary Files SupplementaryinformationFlavobacteriumV2.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8347779","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":559535334,"identity":"db85f765-dc44-4d05-8d0f-eac3b3810fef","order_by":0,"name":"Eliana V. Machin","email":"","orcid":"https://orcid.org/0000-0003-0252-4601","institution":"Laboratorio de Ecología Microbiana y Microbiología Ambiental, Facultad de Química y Unidad Asociada del Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Uruguay","correspondingAuthor":false,"prefix":"","firstName":"Eliana","middleName":"V.","lastName":"Machin","suffix":""},{"id":559535396,"identity":"53e3eced-1f96-4cda-92e3-1e5249561357","order_by":1,"name":"Jitka Vives","email":"","orcid":"","institution":"Department of Experimental Biology, Czech Collection of Microorganisms, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic","correspondingAuthor":false,"prefix":"","firstName":"Jitka","middleName":"","lastName":"Vives","suffix":""},{"id":559535397,"identity":"a1145fd4-b5f6-4307-9013-27d375dc345b","order_by":2,"name":"Rodolfo Javier Menes","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYBACxgYwZcMPIiVI0ZIm2UC0Fig4TIIW5mmHjz348Oe8BN8B5oO3eRgOJzYQdNjstHTDmW23JSQPsCVbE6klx0yat+F2ncEBHjNpIrXkf5P+8+echMEB/m/Easlhk2ZgOwDUwsNGrJY0M8netmQJycNsxpZzDNKNCWoxnJ38TOLHHzsJvuPND2+8qbCWJawFruIwiDBgcCSoRR7OOgCh7AnpGAWjYBSMgpEHANaCO+AQHUNkAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-8968-6149","institution":"Laboratorio de Ecología Microbiana y Microbiología Ambiental, Facultad de Química y Unidad Asociada del Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Uruguay","correspondingAuthor":true,"prefix":"","firstName":"Rodolfo","middleName":"Javier","lastName":"Menes","suffix":""}],"badges":[],"createdAt":"2025-12-12 16:55:09","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-8347779/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8347779/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":98198471,"identity":"c16b24bf-7a5c-46dc-8fd1-10a040de705b","added_by":"auto","created_at":"2025-12-15 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07:13:33","extension":"html","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":182857,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8347779/v1/7b1176bd82f031a0db29b28c.html"},{"id":98198467,"identity":"e983f55a-75dd-489c-8aa1-e59ca22c8e1a","added_by":"auto","created_at":"2025-12-15 07:13:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":104802,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenomic tree based on 120 single-copy marker genes concatenated showing the relation of the strains 7A\u003csup\u003eT\u003c/sup\u003e, 14A\u003csup\u003eT\u003c/sup\u003e, 28A\u003csup\u003eT\u003c/sup\u003e and 7E and their closely related \u003cem\u003eFlavobacterium \u003c/em\u003especies. \u003cem\u003eChryseobacterium angstadtii\u003c/em\u003e KM\u003csup\u003eT\u003c/sup\u003e was used as the outgroup. Maximum-likelihood tree was created using Poisson correction model and filled circles indicate branches that were also recovered in tree generated with neighbour-joining method. Bootstrap percentages based on 1000 replicates are listed in the nodes (only values \u0026gt;70% are shown). Bar, 0.050 substitutions per nucleotide position.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8347779/v1/a0789b77114e5aae265e5ee6.png"},{"id":98444877,"identity":"66682136-88c2-40e0-880a-80a21109299a","added_by":"auto","created_at":"2025-12-17 17:17:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":926542,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8347779/v1/7833e347-b8b9-4f3d-ba71-e4082e5a6747.pdf"},{"id":98431833,"identity":"d821c29a-9e73-404f-bdd3-a1b3fa376516","added_by":"auto","created_at":"2025-12-17 16:48:28","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1420023,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryinformationFlavobacteriumV2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8347779/v1/4fdecf0c9f3989578efcea45.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cem\u003eFlavobacterium psychraerolatum \u003c/em\u003esp. nov., \u003cem\u003eFlavobacterium aeripolare\u003c/em\u003e sp. nov. and \u003cem\u003eFlavobacterium gelidicaeli\u003c/em\u003e sp. nov. isolated from Antarctic air\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe genus \u003cem\u003eFlavobacterium\u003c/em\u003e, proposed by Bergey et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], belongs to the phylum \u003cem\u003eBacteroidota\u003c/em\u003e, and is the type genus of the family \u003cem\u003eFlavobacteriaceae\u003c/em\u003e [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. At the time of writing, the genus comprises 334 species with validly published names (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://lpsn.dsmz.de/genus/flavobacterium\u003c/span\u003e\u003cspan address=\"https://lpsn.dsmz.de/genus/flavobacterium\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed 10 October 2025). Members of this genus are aerobic, Gram-stain-negative, rod-shaped bacteria, non-motile or motile by gliding, and typically produce yellow to orange-pigmented colonies. Menaquinone-6 (MK-6) is the sole or predominant respiratory quinone [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. \u003cem\u003eFlavobacterium\u003c/em\u003e species are widely distributed in nature, and have been isolated from temperate and cold freshwater habitats [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], freshwater fish tissues [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], seawater [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], sediment [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], temperate and cold soils [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], glaciers [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], lakes, activated sludge [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and Antarctic microbial mats [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], as well as from the atmosphere [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In the sea ice of both the Arctic and Antarctic regions, \u003cem\u003eFlavobacterium\u003c/em\u003e has been reported as a dominant genus within the bacterial community [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These species are of ecological and biotechnological interest due to their ability to produce cold-active enzymes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The present study was undertaken to determine the taxonomic position of four novel \u003cem\u003eFlavobacterium\u003c/em\u003e strains isolated from Antarctic air, using a polyphasic approach consistent with the minimal standards for describing new taxa within the family \u003cem\u003eFlavobacteriaceae\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e"},{"header":"ISOLATION AND ECOLOGY","content":"\u003cp\u003eTo investigate the diversity of cultivable bacteria from the air in the Fildes Peninsula, King George Island (62°08'-62°14'S and 59°02'-58°51'W, Antarctica), air samples were collected in February 2018 using R2A agar as the growth medium. Plates were incubated at 5°C for up to 30 days. Colonies displaying distinct morphologies were selected, purified, and subjected to further characterization. Four isolates, designated 7A\u003csup\u003eT\u003c/sup\u003e, 14A\u003csup\u003eT\u003c/sup\u003e, 28A\u003csup\u003eT\u003c/sup\u003e and 7E were subcultured periodically on R2A agar and maintained at 4°C for short-term preservation. For long-term storage, strains were preserved at -80°C in TSB supplemented with 30 % (v/v) glycerol or lyophilized. The reference strain \u003cem\u003eFlavobacterium frigidarium\u003c/em\u003e DSM 17623\u003csup\u003eT\u003c/sup\u003e was obtained from German Collection of Microorganisms and Cell Cultures (DSMZ) and included in this study for comparative purposes. All strains were routinely cultivated on R2A agar at 15°C for 48–72 h.\u003c/p\u003e \u003cp\u003eFor exploring the cultivable bacterial diversity belonging to \u003cem\u003eFlavobacterium\u003c/em\u003e genus in King George Island, 16S rRNA gene sequences of isolates were retrieved from GenBank. These strains were obtained from a variety of niches (different type of soils, sea and lake water, sea sediment, penguin faeces, moraine, algae, microbial mats). The strains 27B (MZ613136), 17C2 (MZ613133), 7C (MZ613121), H2 (MZ613117), Ac7 (MZ613114), Ab1 (MZ613110), and Aa2 (MZ613107) were isolated by our group from air collected in 2017 and 2018 as explained previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. To reveal their phylogenetic relationships, a phylogenetic tree (Fig. S1) was constructed as described below. The results showed that the Antarctic isolates were distributed into multiple distinct clades, which suggests they have a long evolutionary history of divergence.\u003c/p\u003e \u003cp\u003e \u003cb\u003e16S rRNA PHYLOGENY\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGenomic DNA was extracted from all strains as described previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Nearly full-length 16S rRNA gene fragments were amplified using the universal primers 27F and 1492R [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The resulting sequences were compared with publicly available type strain sequences with validly published names retrieved from GenBank. Pairwise sequence similarities were calculated using the global alignment algorithm implemented on the EzBioCloud server [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The 16S rRNA gene sequences of the four strains and those of closely related species were aligned using the CLUSTAL W tool [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] implemented in MEGA version 11 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Phylogenetic trees were reconstructed using both the maximum-likelihood (ML) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and the neighbour-joining (NJ) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] methods in MEGA 11. Evolutionary distances were calculated using Kimura’s two-parameter model [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The robustness of the inferred tree topologies was evaluated using bootstrap analysis with 1000 replications [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The EzBioCloud analysis of the nearly complete 16S rRNA gene sequences revealed that all four isolates shared the highest sequence similarities with members of the genus \u003cem\u003eFlavobacterium\u003c/em\u003e. Specifically, strain 7Aᵀ showed 98.40 % similarity to \u003cem\u003eF. faecale\u003c/em\u003e WV33ᵀ, 98.12% to \u003cem\u003eF. algicola\u003c/em\u003e TC2ᵀ, and 96.73 % to \u003cem\u003eF. ovatum\u003c/em\u003e W201Eᵀ. Strain 14Aᵀ exhibited 98.88 % similarity to \u003cem\u003eF. frigidarium\u003c/em\u003e A2iᵀ, 97.88 % to \u003cem\u003eF. kayseriense\u003c/em\u003e F-47ᵀ, and 97.56 % to \u003cem\u003eF. fryxellicola\u003c/em\u003e DSM 16209ᵀ. Strains 28Aᵀ and 7E shared the highest similarity with \u003cem\u003eF. frigidarium\u003c/em\u003e A2iᵀ (98.74 % and 98.61%, respectively), followed by \u003cem\u003eF. muglaense\u003c/em\u003e F-60ᵀ (98.26 % and 98.41 %, respectively), \u003cem\u003eF. crassostreae\u003c/em\u003e LPB0076ᵀ (97.97% and 98.12 %, respectively), and \u003cem\u003eF. fryxellicola\u003c/em\u003e DSM 16209ᵀ (97.69 % and 97.70%, respectively). Phylogenetic trees based on evolutionary distances calculated using Kimura’s two-parameter model and constructed using the ML method (Fig. S2) showed that all four isolates were affiliated with the genus \u003cem\u003eFlavobacterium\u003c/em\u003e. Strain 7Aᵀ clustered in a clade with \u003cem\u003eF. faecale\u003c/em\u003e WV33ᵀ with moderate bootstrap support (77%). Strain 14Aᵀ formed a clade with \u003cem\u003eF. frigidarium\u003c/em\u003e A2iᵀ and \u003cem\u003eF. crassostreae\u003c/em\u003e LPB0076ᵀ, though with a low bootstrap support (\u0026lt; 70 %). Strains 28Aᵀ and 7E clustered together in the same clade that 14A\u003csup\u003eT\u003c/sup\u003e. Strain 7A\u003csup\u003eT\u003c/sup\u003e showed a similar tree topology using the NJ method (Fig. S3), and strain 14A\u003csup\u003eT\u003c/sup\u003e clustered with \u003cem\u003eF. frigidarium\u003c/em\u003e A2iᵀ, whereas 28A\u003csup\u003eT\u003c/sup\u003e and 7E clustered with \u003cem\u003eF. crassostreae\u003c/em\u003e LPB0076ᵀ and \u003cem\u003eF. muglaense\u003c/em\u003e F-60ᵀ.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"GENOME FEATURES AND PHYLOGENOMICS","content":"\u003cp\u003eThe draft genome assembly of strains 7A\u003csup\u003eT\u003c/sup\u003e, 14A\u003csup\u003eT\u003c/sup\u003e, 28A\u003csup\u003eT\u003c/sup\u003e and 7E was generated at the DOE Joint Genome Institute (JGI) using Illumina technology [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] as described previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. An Illumina standard shotgun library was constructed and sequenced using the Illumina NovaSeq S4 platform which generated 9,564,230 − 13,730,208 reads totaling 1,444,198,730-2,073,261,408 bp for all strains. The authenticity of the final assembly genome was verified by comparing the 16S rRNA gene sequences obtained from the genome and by the PCR amplification method, using Align Sequences Nucleotide blast tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e optimized for highly similar sequences (megablast) and with default algorithm parameters.16S rRNA gene sequences extracted from the genome shared 100% identity with the partial sequences obtained from PCR obtained as explained above and sequenced by the Sanger method.\u003c/p\u003e\u003cp\u003eFor further comparative analyses, genome annotations were performed using the Rapid Annotation using Subsystem Technology (RAST) pipeline [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e–\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Venn diagrams were constructed to compare coding sequences (CDSs) among genomes based on their annotated functions and to identify unique and shared features, using the VennDiagram R package (v1.7.3) in RStudio (v1.4.1717). Only CDSs with defined product annotations were included; genes lacking such annotation (e.g. hypothetical proteins, tRNA, rRNA, etc.) were excluded. To assess genome relatedness, average nucleotide identity (ANI) values between strains 7Aᵀ, 14Aᵀ, 28Aᵀ and 7E and their closest \u003cem\u003eFlavobacterium\u003c/em\u003e relatives were calculated using the OrthoANIu algorithm via the EzBioCloud web server [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Additionally, digital DNA–DNA hybridization (dDDH) values were obtained using the Genome-to-Genome Distance Calculator (GGDC) 3.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ggdc.dsmz.de/ggdc.php#\u003c/span\u003e\u003cspan address=\"https://ggdc.dsmz.de/ggdc.php#\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with formula 2 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Genomes retrieved from NCBI were processed with the Genome Taxonomy Database Toolkit (GTDB-Tk v2.1.0) to generate concatenated alignments of 120 bacterial single-copy marker genes [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The phylogenomic tree was constructed in MEGA 11 using the Poisson correction model [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] with 1000 bootstrap replicates. To further evaluate whether strains 7Aᵀ, 14Aᵀ, 28Aᵀ and 7E represent novel species, genome sequence data of these strains were uploaded to the Type (Strain) Genome Server (TYGS) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://tygs.dsmz.de\u003c/span\u003e\u003cspan address=\"https://tygs.dsmz.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed 2025-10-10) for whole-genome-based phylogenetic analyses. Branch support was inferred from 100 pseudo-bootstrap replicates.\u003c/p\u003e\u003cp\u003eBiosynthetic gene clusters potentially involved in the production of secondary metabolites were identified using the AntiSMASH online tool (version 8.0.1) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Assembly statistics for all strains are presented in Table S1. CheckM analysis (v1.2.2) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] indicated high genome completeness (98.28–99.22%) and low contamination levels (0.20–0.77%) across the isolates (Table S1). The G + C content, calculated from the whole genome sequences, ranged from 32.8 to 34.5 mol%, consistent with the reported range for members of the genus \u003cem\u003eFlavobacterium\u003c/em\u003e (30–41 mol%) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The ANI and dDDH values between strains 7Aᵀ, 14Aᵀ, 28Aᵀ and validly published \u003cem\u003eFlavobacterium\u003c/em\u003e species names were below 84.94% and 30.80%, respectively (Table S2), supporting their designation as novel species. However, ANI and dDDH values between strains 28Aᵀ and 7E were 97.62% and 78.30%, respectively (Table S2), exceeding the commonly accepted thresholds for species delineation (95–96% ANI and 70% dDDH) [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e–\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], indicating that they belong to the same species. Phylogenomic analysis based on 120 single-copy marker genes revealed that the novel strains clustered into distinct clades (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Strain 7Aᵀ grouped in a clade with \u003cem\u003eF. faecale\u003c/em\u003e WV33ᵀ and \u003cem\u003eF. algicola\u003c/em\u003e MBE-1 (bootstrap support 83% and 100% respectively). Strain 14Aᵀ clustered with \u003cem\u003eF. frigidarium\u003c/em\u003e DSM 17623ᵀ with high bootstrap support (100%). Similarly, strains 28Aᵀ and 7E clustered in a clade with \u003cem\u003eF. frigidarium\u003c/em\u003e DSM 17623ᵀ, supported by a bootstrap value of 83%. Similar results were observed with the TYGS phylogenomic tree (Fig. S4). According to RAST annotation results, the genome of strain 7Aᵀ contained 3,683 coding sequences (CDSs) and 53 RNA genes, while the genome of strain 14Aᵀ comprised 3,349 CDSs and 55 RNA genes. Strain 28Aᵀ possessed 3,626 CDSs and 53 RNA genes, whereas strain 7E contained 3,995 CDSs and 51 RNA genes (Table S1). The genomes of all four novel isolates included genes encoding cold shock proteins (CSP family), which are known to be essential for cellular responses to cold stress. In addition, strains 28Aᵀ and 7E harbored genes involved in carotenoid biosynthesis, including phytoene synthase and phytoene desaturase. These genes are associated with membrane fluidity regulation under low temperatures and have been linked to increased resistance to environmental stressors such as freeze–thaw cycles and solar radiation, particularly in Antarctic bacteria [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e–\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Furthermore, the genomes encoded enzymes involved in the biosynthesis of straight-chain and branched-chain fatty acids (e.g. β-ketoacyl-ACP synthase II and III [KAS-II, KAS-III]), which are important for maintaining membrane fluidity and may contribute to cold adaptation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Notably, all four strains contained genes for toxin–antitoxin (TA) replicon stabilization systems, which were absent in their closest phylogenetic relatives. Comparative genomic analysis showed that the novel isolates possessed distinct gene content relative to \u003cem\u003eF. algicola\u003c/em\u003e MBE-1, \u003cem\u003eF. faecale\u003c/em\u003e WV33ᵀ, and \u003cem\u003eF. frigidarium\u003c/em\u003e DSM 17623ᵀ (Fig. S5; Table S3), supporting their classification as novel taxa.\u003c/p\u003e\u003cp\u003eThe Venn diagram analysis revealed that the genome of strain 7Aᵀ contains 112 unique coding sequences (CDSs) not found in \u003cem\u003eF. algicola\u003c/em\u003e MBE-1 and \u003cem\u003eF. faecale\u003c/em\u003e WV33ᵀ, while 1,456 CDSs are shared among the three strains (Fig. S5). Notably, strain 7Aᵀ harbors genes associated with the nitrosative stress response that are absent in the reference strains. In contrast, genes involved in lactose and galactose uptake and metabolism are conserved across all three genomes (Table S3). Similarly, strains 14Aᵀ, 28Aᵀ, and 7E possess55, 48, and 71 unique coding sequences (CDSs), respectively, and share a core genome of1219 CDSs with \u003cem\u003eF. frigidarium\u003c/em\u003e DSM 17623ᵀ and \u003cem\u003eF. crassostreae\u003c/em\u003e LPB0076\u003csup\u003eT\u003c/sup\u003e (Fig. S5) For instance, genes associated with sucrose utilization were identified exclusively in strain 14Aᵀ, while genes encoding mercury reductase were shared among strains 14A\u003csup\u003eT\u003c/sup\u003e, 28A\u003csup\u003eT\u003c/sup\u003e and 7E. Furthermore, variation in the presence of genes involved in resistance to toxic compounds was observed, highlighting functional diversification among the strains.\u003c/p\u003e\u003cp\u003eAnalysis of the genomes using antiSMASH v8.0 identified several putative biosynthetic gene clusters (BGCs) associated with secondary metabolite production in the novel isolates (Table S4). Specifically, strain 7Aᵀ harbored four BGCs, including one predicted to encode a terpene compound involved in carotenoid biosynthesis. Strain 14Aᵀ also contained four BGCs, two of which were associated with the biosynthesis of carotenoids and bacillomycin D, a known antifungal agent. In strains 28Aᵀ and 7E, five and six BGCs were detected, respectively, including one predicted to encode arylpolyene/resorcinol biosynthesis, potentially responsible for the production of flexirubin-like pigments. These findings are consistent with the pigmentation phenotypes observed in these strains (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDifferential characteristics of the novel strains with respect to closely related \u003cem\u003eFlavobacterium\u003c/em\u003e species. Strains: 1, 7A\u003csup\u003eT\u003c/sup\u003e; 2, \u003cem\u003eF\u003c/em\u003e. \u003cem\u003ealgicola\u003c/em\u003e TC2\u003csup\u003eT\u003c/sup\u003e; 3, \u003cem\u003eF\u003c/em\u003e. \u003cem\u003efaecale\u003c/em\u003e KCTC 32457\u003csup\u003eT\u003c/sup\u003e; 4, 14A\u003csup\u003eT\u003c/sup\u003e; 5, 28A\u003csup\u003eT\u003c/sup\u003e; 6, 7E; 7, \u003cem\u003eF\u003c/em\u003e. \u003cem\u003efrigidarium\u003c/em\u003e DSM 17623\u003csup\u003eT\u003c/sup\u003e; 8, \u003cem\u003eF. crassostreae\u003c/em\u003e LPB0076\u003csup\u003eT\u003c/sup\u003e. All data, unless otherwise indicated, were from this study. +, Positive; –, negative; ND, not determined.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003csup\u003e§\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3*\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8ˁ\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrowth at 0°C\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003csup\u003e†\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e% NaCl range for growth (w/v)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0–5\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0–3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5-8\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0–6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0–6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0–6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0–5\u003csup\u003e†\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0–4\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH range for growth\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.0–9.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.5-8.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.0–9.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.0–9.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.0–8.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.0–8.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5.6–8.4\u003csup\u003e†\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5.5-8.0\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCongo red adsorption\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlexirubin-type pigments\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHydrolysis of\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eaesculin\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eagar\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecasein\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCM-cellulose\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDNA\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003egelatin\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-tyrosine\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003estarch\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAcid from\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003efructose\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eglucose\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-arabinose\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emannitol\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emelibiose\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEnzyme activity (API ZYM)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-Galactosidase\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlkaline phosphatase\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEsterase C4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eValine arylamidase\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eα-Glucosidase\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN-Acetyl-β -glucosaminidase\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcid phosphatase\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLipase C14\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDNA G + C content (mol%)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33.8\u003csup\u003e¶\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.9\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34.5\u003csup\u003e¶\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e32.8\u003csup\u003e¶\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e32.8\u003csup\u003e¶\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e35.0\u003csup\u003e†\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e35.9\u003csup\u003e¶\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003e§ Data from [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003e* Data from [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003e†Data from [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003eˁ Data from [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003e¶ Data from the genome sequence\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e"},{"header":"PHYSIOLOGY AND CHEMOTAXONOMY","content":"\u003cp\u003eGrowth on/in several media including R2A broth and agar (Merck), PCA (Difco), TSA (Difco), TSB (Difco), NA (Oxoid), NB (Oxoid), MacConkey agar (Difco), Cetrimide agar (Merck) and Marine agar (HiMedia) was evaluated at 15\u0026deg;C after 7 days of incubation. Anaerobic growth was assessed on R2A agar at 15\u0026deg;C for 7 days using the Anaerocult A Mini gas generator system (Merck). Cell morphology was examined by Transmission Electron Microscopy (TEM). Cells grown in R2A broth for 48 h were fixed in an aqueous solution of 2.5% (w/v) glutaraldehyde for four hours at 4\u0026deg;C. Each sample was mounted on a 200-mesh copper grid coated with a 90 nm LR White (London Resin) membrane and incubated for 10 min. Excess liquid was removed by capillary action, and the grids were air-dried for an additional 10 min. Negative staining was performed using a 2% (w/v) aqueous uranyl acetate solution for one minute, followed by drying via capillarity. Subsequently, the samples underwent three washes with ultrapure water, with drying steps between each wash. The grids were then left to rest for 10 min prior to imaging. Samples were observed using a Zeiss EM 109T Transmission Electron Microscope equipped with a Gatan ES1000W digital camera as shown in Fig. S6. The presence of flexirubin-type pigments was tested by flooding the colonies grown on NA with 20 % (w/v) KOH solution [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Growth at different temperatures (from \u0026minus;\u0026thinsp;5 to 30\u0026deg;C in ~\u0026thinsp;5\u0026deg;C intervals) was assessed in R2A broth (Oxoid) with 5 % (v/v) glycerol supplementation at \u0026minus;\u0026thinsp;5 and 0 \u0026deg;C. The pH growth range (pH 5.0\u0026ndash;11.0, in 1.0 unit increments) was determined in R2A broth buffered with K₂HPO₄\u0026ndash;KH₂PO₄ (pH 5.0\u0026ndash;8.0), NaHCO\u003csub\u003e3\u003c/sub\u003e/Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (pH 9.0\u0026ndash;10.0) and NaHCO\u003csub\u003e3\u003c/sub\u003e\u0026ndash;NaOH (pH 11.0). The initial pH was adjusted using HCl or NaOH as required, and media were sterilized by filtration. Tolerance to NaCl was tested in R2A broth prepared without NaCl and supplemented with a final NaCl concentration of 0.5 to 7% (w/v) within aproximately 1% interval. Growth under different temperature, pH, and salinity conditions was monitored by measuring optical density at 660 nm (OD₆₆₀) over time. Slopes of OD₆₆₀ versus time curves were averaged from triplicate runs for each condition to determine the optimal growth. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Catalase activity was determined by bubble production upon application of 3 % (v/v) hydrogen peroxide, and oxidase activity using Bactident oxidase strips (Merck).\u003c/p\u003e \u003cp\u003eThe following biochemical tests were conducted as described by Barrow and Feltham [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], using NA as basal medium and incubated at 15\u0026deg;C for 7 days: ONPG (2-nitrophenyl-β-D-galactopyranoside) hydrolysis, Voges\u0026ndash;Proskauer reaction, nitrate reduction and denitrification, and hydrolysis of Tween 20, Tween 60, Tween 80, starch, aesculin, L-tyrosine, carboxymethyl cellulose (CM-cellulose), casein and urea. Additional assays, including DNA hydrolysis, gelatin hydrolysis (method 2), citrate utilization (method 1), indole production, H₂S production from cysteine, phenylalanine deaminase and lecithinase activities, were performed following standard protocols [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], at 15\u0026deg;C for 7 days.\u003c/p\u003e \u003cp\u003eAgar degradation was tested on R2A agar at 15\u0026deg;C by observing colony-associated agar softening. Motility (by hanging drop technique) and cell morphology were examined by phase-contrast microscopy (Zeiss Axioplan, Germany) in exponentially growing cultures in R2A broth at 15\u0026deg;C. Acid production from carbohydrates was assessed in Ammonium Salt Sugars (ASS) medium [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] with the following composition (L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): 1.0 g (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 0.2 g KCl, 0.2 g MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO, 0.2 g yeast extract, 20 g agar and 0.04 g bromothymol blue, with each carbohydrate added aseptically from sterile stock solutions to a final concentration of 1 % (w/v). For strain 7Aᵀ, ASS medium was prepared without agar. To determine the range of substrates used for growth as carbon and energy source the following basal media was prepared (per liter): 6.24 g NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 2.0 g (NH\u003csub\u003e4\u003c/sub\u003e)SO\u003csub\u003e4\u003c/sub\u003e, 1.5 g KCl /L, 10 mL of Wolin's vitamin solution, and 10 mL trace element solution (according to DSMZ medium 318), final pH 7.2. Additional phenotypic characterization was carried out using API ZYM and API 20NE test kits (bioM\u0026eacute;rieux), following the manufacturer\u0026rsquo;s protocols, but incubated at 15\u0026deg;C. For API 20NE results were read after 7 days. Strains 7Aᵀ, 14Aᵀ, 28Aᵀ and 7E were catalase- and oxidase-positive, and formed yellow to light orange-pigmented colonies (Fig. S7). The isolates exhibited phenotypic traits that differentiated them from the reference species (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Although strains 28Aᵀ and 7E represented the same species based on genomic data, they showed several phenotypic differences. For example, strain 28Aᵀ hydrolysed aesculin, whereas strain 7E did not. Strain 28Aᵀ also produced acid from arabinose and melibiose and was able to utilize raffinose as a sole carbon and energy source, unlike strain 7E. Flexirubin-type pigments were detected in both strains 28Aᵀ and 7E, but were absent in strains 7Aᵀ and 14Aᵀ.\u003c/p\u003e \u003cp\u003eGrowth occurred on NA, PCA, R2A agar and broth, and in NB and TSB in 48\u0026ndash;72 h with similar performance, but no growth was obtained on cetrimide agar, MacConkey agar or marine agar or under anaerobic conditions. Physiological tests, showed that all four strains grew over a temperature range of 5\u0026ndash;20\u0026deg;C. Strain 28Aᵀ was able to grow up to 25\u0026deg;C. Strains 7Aᵀ and 14Aᵀ grew within a pH range of 6.0\u0026ndash;9.0, whereas strains 28Aᵀ and 7E grew within a pH range of 6.0\u0026ndash;8.0. Strain 7Aᵀ tolerated NaCl concentrations up to 5.0 % (w/v), whereas strains 14Aᵀ, 28Aᵀ and 7E grew in the presence of up to 6.0 % (w/v) NaCl. Detailed phenotypic traits specific to each novel strain are summarized in the species descriptions.\u003c/p\u003e \u003cp\u003eIsoprenoid quinones were extracted and analysed as described previously [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Menaquinone-6 (MK-6) was the sole respiratory quinone detected in all strains, consistent with other members of the genus \u003cem\u003eFlavobacterium\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Fatty acid methyl ester (FAME) profiles were analysed according to the standard protocol of the Sherlock Microbial Identification System (MIDI, version 6.2; RTSBA 6.21 database) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Cultures were grown on R2A agar at 20\u0026deg;C until late exponential phase. FAMEs were extracted and analysed using a 7890B gas chromatograph (Agilent Technologies). The most abundant fatty acids identified in all strains are presented in Table S5, which are consistent with previously described species of \u003cem\u003eFlavobacterium\u003c/em\u003e and with the genus description [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite this general similarity, species differentiation was supported by quantitative differences in the composition of both major and minor fatty acids. The major fatty acids of 7A\u003csup\u003eT\u003c/sup\u003e were summed feature 3 (C\u003csub\u003e16:1\u003c/sub\u003e ω\u003cem\u003e7c\u003c/em\u003e/C\u003csub\u003e16:1\u003c/sub\u003e ω\u003cem\u003e6c\u003c/em\u003e), iso-C\u003csub\u003e15:0,\u003c/sub\u003e iso-C\u003csub\u003e15:0\u003c/sub\u003e 3-OH, and anteiso-C\u003csub\u003e15:0\u003c/sub\u003e. Strain 14Aᵀ showed a profile dominated by summed feature 3 (C\u003csub\u003e16:1\u003c/sub\u003e ω\u003cem\u003e7c\u003c/em\u003e/C\u003csub\u003e16:1\u003c/sub\u003e ω\u003cem\u003e6c\u003c/em\u003e), iso-C\u003csub\u003e15:0\u003c/sub\u003e, and C\u003csub\u003e15:1\u003c/sub\u003e ω\u003cem\u003e6c\u003c/em\u003e. In strain 28Aᵀ, the predominant components were C\u003csub\u003e15:1\u003c/sub\u003e ω\u003cem\u003e6c\u003c/em\u003e, summed feature 3 (C\u003csub\u003e16:1\u003c/sub\u003e ω\u003cem\u003e7c\u003c/em\u003e/C\u003csub\u003e16:1\u003c/sub\u003e ω\u003cem\u003e6c\u003c/em\u003e), and iso-C\u003csub\u003e15:0\u003c/sub\u003e.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003ePsychrophilic or psychrotolerant \u003cem\u003eFlavobacterium\u003c/em\u003e type species have been reported from King George Island and nearby Antarctic environments, such as \u003cem\u003eF. antarcticum\u003c/em\u003e [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] (isolated from soil near a penguin habitat, \u003cem\u003eF. faecale\u003c/em\u003e [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] (isolated from penguin faeces, \u003cem\u003eF. kingsejongi\u003c/em\u003e [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] (isolated from penguin faeces), \u003cem\u003eF. ardleyense\u003c/em\u003e [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] (obtained from soil on Ardley Island), \u003cem\u003eF. azizsancarii\u003c/em\u003e [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] (isolated from lake water on Ardley Island) \u003cem\u003eF. collinsense\u003c/em\u003e [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] (isolated from a till sample near the Collins Glacier front), and \u003cem\u003eF. weaverense\u003c/em\u003e and \u003cem\u003eF. segetis\u003c/em\u003e [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] (both isolated from terrestrial Antarctic samples). A recent study [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] revealed some surprising phylogenetic diversity among \u003cem\u003eFlavobacterium\u003c/em\u003e strains isolated from microbial mats in ponds and streams on King George Island. The research identified 20 distinct species among 50 isolates. This highlights how much we have underestimated the biodiversity of \u003cem\u003eFlavobacterium\u003c/em\u003e in Antarctic freshwater ecosystems. The novel strains 7Aᵀ, 14Aᵀ, 28Aᵀ, and 7E, isolated from the atmosphere, greatly expand our understanding of the \u003cem\u003eFlavobacterium\u003c/em\u003e genus in these unique ecosystems. To our knowledge, these are the first \u003cem\u003eFlavobacterium\u003c/em\u003e species formally described from Antarctic air samples, suggesting their ability to disperse through the air and withstand extreme atmospheric conditions. Their presence in the atmosphere suggests they might play important roles in long-distance microbial transport and colonization in polar environments, complementing previous studies from terrestrial and aquatic habitats.\u003c/p\u003e \u003cp\u003eAccording to the results obtained, strains 7A\u003csup\u003eT\u003c/sup\u003e, 14A\u003csup\u003eT\u003c/sup\u003e, 28A\u003csup\u003eT\u003c/sup\u003e and 7E exhibited distinct genotypic and phenotypic characteristics compared to previously described species of the genus \u003cem\u003eFlavobacterium\u003c/em\u003e. At the genomic level, all strains displayed ANI and dDDH values below the established thresholds for species delineation when compared with validly published \u003cem\u003eFlavobacterium\u003c/em\u003e species names. Phylogenomic analysis further supported their taxonomic distinctiveness, revealing that the novel isolates formed well-separated clades within the genus. Several phenotypic differences were also observed. For example, strain 7Aᵀ produced acid from glucose and mannitol, and hydrolysed CM-cellulose, distinguishing it from \u003cem\u003eF. faecale\u003c/em\u003e KCTC 32457ᵀ. Strains 14Aᵀ, 28Aᵀ and 7E hydrolysed DNA and starch, and produced acid from fructose, mannitol, glucose and xylose, traits not observed in \u003cem\u003eF. frigidarium\u003c/em\u003e DSM 17623ᵀ. Additionally, strains 28Aᵀ and 7E hydrolysed L-tyrosine, in contrast to \u003cem\u003eF. frigidarium\u003c/em\u003e DSM 17623ᵀ. On the other hand, strains 14Aᵀ, 28Aᵀ and 7E hydrolysed gelatin whereas \u003cem\u003eF. crassostreae\u003c/em\u003e LPB0076\u003csup\u003eT\u003c/sup\u003e did not. Strain 28A\u003csup\u003eT\u003c/sup\u003e produced acid from L-arabinose unlike \u003cem\u003eF. crassostreae\u003c/em\u003e LPB0076\u003csup\u003eT\u003c/sup\u003e. Additionally, strains 14A\u003csup\u003eT\u003c/sup\u003e and 28A\u003csup\u003eT\u003c/sup\u003e produced acid from melibiose, characteristic absent in \u003cem\u003eF. crassostreae\u003c/em\u003e LPB0076\u003csup\u003eT\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTaken together, the genotypic, phylogenomic, phenotypic and chemotaxonomic data clearly indicate that strains 7Aᵀ, 14Aᵀ, and 28Aᵀ represent novel species within the genus \u003cem\u003eFlavobacterium\u003c/em\u003e, for which the names \u003cem\u003eFlavobacterium psychraerolatum\u003c/em\u003e sp. nov., \u003cem\u003eFlavobacterium aeripolare\u003c/em\u003e sp. nov. \u003cem\u003eand Flavobacterium gelidicaeli\u003c/em\u003e sp. nov., respectively, are proposed.\u003c/p\u003e"},{"header":"PROTOLOGUE","content":"\u003cp\u003e \u003cb\u003eDescription of\u003c/b\u003e \u003cb\u003eFlavobacterium psychraerolatum\u003c/b\u003e \u003cb\u003esp. nov.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eFlavobacterium psychraerolatum\u003c/em\u003e (psychr.a.e.ro.la\u0026rsquo;tum. Gr. masc. adj. \u003cem\u003epsychros\u003c/em\u003e, cold; Gr. masc. n. \u003cem\u003eaer\u003c/em\u003e, air; L. masc. perf. part. \u003cem\u003elatus\u003c/em\u003e, carried; N.L. neut. part. adj. \u003cem\u003epsychraerolatum\u003c/em\u003e, transported by cold-air).\u003c/p\u003e \u003cp\u003eCells are Gram-stain-negative, non-motile, non-spore-forming rods occurring singly or in pairs, measuring 0.2\u0026ndash;0.3 \u0026micro;m in width and 1.0\u0026ndash;1.8 \u0026micro;m in length. Strictly aerobic. Colonies grown on R2A agar are circular, smooth, flat, glistening, yellow-pigmented with entire margins and measure 1\u0026ndash;2 mm in diameter after 48 h incubation at 15\u0026deg;C. Flexirubin-type pigments are not produced. Growth occurs on NA, PCA, R2A agar and broth, and in NB and TSB at 15\u0026deg;C, but not on cetrimide agar, MacConkey agar or marine agar. No growth occurs under anaerobic conditions. Growth occurs at 5\u0026ndash;20\u0026deg;C (optimum 15\u0026ndash;20\u0026deg;C), pH 6.0\u0026ndash;9.0 (optimum pH 6.0), and in the presence of 0\u0026ndash;5.0 % (w/v) NaCl (optimum 2 %). Positive for catalase and oxidase activities. Hydrolysis of aesculin, agar, CM-cellulose, Tween 20, and starch is positive. Hydrolysis of DNA, gelatin, lecithin, L-tyrosine, Tween 60, Tween 80, and urea is negative. The ONPG test is positive. Citrate utilization, indole production, nitrate reduction and denitrification, H₂S production from cysteine, L-phenylalanine deaminase, L-tyrosine pigment production, and Voges\u0026ndash;Proskauer reaction are negative. Acid is produced from D-fructose, D-galactose, D-glucose, D-mannitol, D-xylose, and weakly from glycerol. No acid is produced from L-arabinose, D-raffinose, or rhamnose. Utilization of the following compounds as sole carbon and energy sources is observed: D-arabitol (weak reaction), D-cellobiose, D-fructose, D-galactose, glycerol, maltose, D-mannitol, melibiose, and D-xylose. D-raffinose, rhamnose, and D-trehalose are not utilized. Positive reactions are observed for β-galactosidase, assimilation of D-glucose, D-maltose, D-mannitol, and weakly D-mannose. Negative reactions are recorded for aesculin hydrolysis (note: this contradicts the result obtained on solid medium), arginine dihydrolase, gelatinase, glucose fermentation under anaerobic conditions, indole production, nitrate reduction, urease activity, and assimilation of L-arabinose, adipic acid, capric acid, citrate, malic acid, N-acetylglucosamine, phenylacetic acid, and potassium gluconate. Positive reactions are observed for alkaline phosphatase, acid phosphatase, α-glucosidase, leucine arylamidase, and weakly for β-galactosidase, valine arylamidase, and naphthol-AS-BI-phosphohydrolase. Negative reactions are recorded for α-chymotrypsin, esterase (C4), esterase lipase (C8), lipase (C14), α-fucosidase, α-galactosidase, β-glucuronidase, β-glucosidase, α-mannosidase, N-acetyl-β-glucosaminidase, cystine arylamidase, and trypsin. The sole menaquinone is MK-6. The major fatty acids are summed feature 3 (C\u003csub\u003e16:1\u003c/sub\u003e ω\u003cem\u003e7c\u003c/em\u003e/C\u003csub\u003e16:1\u003c/sub\u003e ω\u003cem\u003e6c\u003c/em\u003e), iso-C\u003csub\u003e15:0\u003c/sub\u003e, iso-C\u003csub\u003e15:0 iso\u003c/sub\u003e 3-OH, and anteiso-C\u003csub\u003e15:0\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThe type strain is 7Aᵀ (=\u0026thinsp;CCM 8976ᵀ =DSM 111734ᵀ), isolated from air collected at Artigas Antarctic Scientific Base (62\u0026deg;11\u0026prime;04\u0026Prime;S 58\u0026deg;51\u0026prime;07\u0026Prime;W), Fildes Peninsula, King George Island, Antarctica. The DNA G\u0026thinsp;+\u0026thinsp;C content of the type strain is 33.8 mol%. The GenBank accession coder for the 16S rRNA gene sequence is MN007163. The Whole Genome Shotgun project has been deposited under the accession JAOQMX000000000; the version described in this paper is JAOQMX010000000.1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDescription of\u003c/b\u003e \u003cb\u003eFlavobacterium aeripolare\u003c/b\u003e \u003cb\u003esp. nov.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eFlavobacterium aeripolare\u003c/em\u003e (ae.ri.po.la\u0026acute;re. L. masc. n. \u003cem\u003eaer\u003c/em\u003e, air; M.L. masc. adj. \u003cem\u003epolaris\u003c/em\u003e, of, or pertaining to a pole; N.L. neut. adj. \u003cem\u003eaeripolare\u003c/em\u003e, pertaining to polar air).\u003c/p\u003e \u003cp\u003eCells are Gram-stain-negative, non-motile, non-spore-forming rods, occurring singly or in pairs, measuring 0.2\u0026ndash;0.3 \u0026micro;m in width and 1.0\u0026ndash;4.0 \u0026micro;m in length. Strictly aerobic. Colonies on R2A agar are circular, flat, smooth, glistening, yellow-pigmented, with entire margins, and measure 1.5\u0026ndash;2.5 mm in diameter after 48 h of incubation at 15\u0026deg;C. Flexirubin-type pigments are not produced. Growth occurs on marine agar, NA, PCA, R2A agar and broth, and in NB and TSB at 15\u0026deg;C, but not on cetrimide agar or MacConkey agar. No growth occurs under anaerobic conditions. Growth is observed at 5\u0026ndash;20\u0026deg;C (optimum 15\u0026ndash;20\u0026deg;C), pH 6.0\u0026ndash;9.0 (optimum pH 8.0), and in the presence of 0\u0026ndash;6.0 % (w/v) NaCl (optimum 1.5 %). Positive for catalase and oxidase activities. Hydrolysis of aesculin, casein, gelatin, Tween 20, Tween 60, Tween 80, and starch is positive. Hydrolysis of agar, CM-cellulose, DNA, lecithin, L-tyrosine, and urea is negative. The ONPG test, citrate utilization, H₂S production from cysteine, nitrate reduction and denitrification, L-phenylalanine deaminase activity, pigment production from L-tyrosine, and the Voges\u0026ndash;Proskauer reaction are negative. Acid is produced from D-fructose, D-galactose, D-glucose, glycerol, maltose, D-mannitol, melibiose, D-trehalose (weak reaction), and D-xylose. No acid is produced from L-arabinose, D-raffinose, or rhamnose. The following compounds are utilized as sole carbon and energy sources: D-arabitol (weak reaction), D-cellobiose, D-fructose, D-galactose, glycerol, maltose, D-mannitol, melibiose (weak reaction), D-raffinose, and D-xylose. L-arabinose, rhamnose, and D-trehalose are not utilized. Positive reactions are observed for assimilation of D-glucose, D-mannose, D-mannitol, and D-maltose. Negative reactions are recorded for aesculin hydrolysis and gelatinase (note: these differ from solid media results), nitrate reduction, indole production, glucose fermentation under anaerobic conditions, arginine dihydrolase, urease, β-galactosidase, and assimilation of L-arabinose, N-acetylglucosamine, potassium gluconate, capric acid, adipic acid, malic acid, citrate, and phenylacetic acid. Positive enzyme activities are detected for alkaline phosphatase, leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, and N-acetyl-β-glucosaminidase. Esterase lipase (C8) is weakly positive. Negative reactions are obtained for esterase (C4), lipase (C14), cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, α-mannosidase, and α-fucosidase. The sole menaquinone is MK-6. The major fatty acids are summed feature 3 (C\u003csub\u003e16:1\u003c/sub\u003e ω\u003cem\u003e7c\u003c/em\u003e/C\u003csub\u003e16:1\u003c/sub\u003e ω\u003cem\u003e6c\u003c/em\u003e), iso-C\u003csub\u003e15:0\u003c/sub\u003e, iso-C\u003csub\u003e15:0\u003c/sub\u003e 3-OH, and C\u003csub\u003e15:1\u003c/sub\u003e \u003cem\u003eω6c\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe type strain is 14A\u003csup\u003eT\u003c/sup\u003e (=\u0026thinsp;CCM 8972\u003csup\u003eT\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;CGMCC 1.18502\u003csup\u003eT\u003c/sup\u003e), isolated from the air collected at Antarctic Scientific Base Artigas (62\u0026deg;11\u0026prime;04\u0026Prime;S 58\u0026deg;51\u0026prime;07\u0026Prime;W), Fildes peninsula, King George Island, Antarctica. The DNA G\u0026thinsp;+\u0026thinsp;C content is 34.5%. The GenBank accession code for the 16S rRNA gene sequence 14A\u003csup\u003eT\u003c/sup\u003e is MN007165. The Whole Genome Shotgun project has been deposited at GenBank under the accession JABSNL000000000. The version described in this paper is version JABSNL000000000.1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDescription of\u003c/b\u003e \u003cb\u003eFlavobacterium gelidicaeli\u003c/b\u003e \u003cb\u003esp. nov.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eFlavobacterium gelidicaeli\u003c/em\u003e (ge.li.di.cae\u0026acute;li. L. masc. adj. \u003cem\u003egelidus\u003c/em\u003e, icy, frozen; L. neut. n. \u003cem\u003ecaelum\u003c/em\u003e, sky, air; N.L. gen. n. \u003cem\u003egelidicaeli\u003c/em\u003e, of cold air).\u003c/p\u003e \u003cp\u003eCells are Gram-stain-negative, non-motile, non-spore-forming rods, occurring singly or in pairs, measuring 0.2\u0026ndash;0.3 \u0026micro;m in width and 1.0\u0026ndash;3.0 \u0026micro;m in length. Strictly aerobic. Colonies on R2A agar are circular with entire margins, flat, smooth, glistening, yellow to light orange-pigmented, and measure 1\u0026ndash;2 mm in diameter after 48 h of incubation at 15\u0026deg;C. Flexirubin-type pigments are produced (positive KOH test). Growth occurs on R2A agar and broth, TSA, PCA, NA, and in TSB and NB at 15\u0026deg;C, but not on MacConkey agar or cetrimide agar. No growth occurs under anaerobic conditions on R2A. Growth is observed at 5\u0026ndash;25\u0026deg;C (optimum 15\u0026ndash;20\u0026deg;C), pH 6.0\u0026ndash;8.0 (optimum pH 6.0), and in the presence of 0\u0026ndash;6.0 % (w/v) NaCl (optimum 1 %). Positive for catalase and oxidase activities. Hydrolysis of casein, DNA, gelatin, Tween 20, Tween 60, Tween 80, and L-tyrosine is positive. Hydrolysis of aesculin and starch is variable. Hydrolysis of agar, CM-cellulose, lecithin, and urea is negative. Citrate utilization, hydrogen sulfide production from cysteine, nitrate reduction, denitrification, ONPG test, phenylalanine deaminase, pigment production from L-tyrosine, and the Voges\u0026ndash;Proskauer reaction are all negative. Acid is produced from D-fructose, D-glucose, glycerol, maltose, D-mannitol, D-raffinose, and D-xylose. A weak reaction is observed with D-galactose. Acid production from L-arabinose and melibiose is variable, and no acid is produced from rhamnose. The following carbohydrates are utilized as carbon and energy sources: D-fructose, D-galactose, glycerol, maltose, D-mannitol, melibiose (weak reaction), and D-xylose. L-arabinose, rhamnose, and D-trehalose are not utilized. Utilization of D-raffinose is variable. The strain is positive for gelatinase, D-glucose, D-mannose, D-mannitol, and D-maltose, and negative for nitrate reduction, indole production, glucose fermentation (anaerobic), arginine dihydrolase, urease, β-galactosidase, L-arabinose, N-acetyl-glucosamine, potassium gluconate, capric acid, adipic acid, malic acid, citrate, and phenylacetic acid. Aesculin hydrolysis yields variable results. Positive reactions are observed for alkaline phosphatase, leucine arylamidase, valine arylamidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase. Negative reactions are obtained for esterase (C4), esterase lipase (C8), lipase (C14), cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase. The sole menaquinone is MK-6. The major fatty acids are C\u003csub\u003e15:1\u003c/sub\u003e \u003cem\u003eω6c\u003c/em\u003e, summed feature 3 (C\u003csub\u003e16:1\u003c/sub\u003e \u003cem\u003eω7c\u003c/em\u003e/C\u003csub\u003e16:1\u003c/sub\u003e \u003cem\u003eω6c\u003c/em\u003e) and iso-C\u003csub\u003e15:0\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThe type strain is 28A\u003csup\u003eT\u003c/sup\u003e (=\u0026thinsp;CCM 8973T\u0026thinsp;=\u0026thinsp;CGMCC 1.18503\u003csup\u003eT\u003c/sup\u003e), collected from the air sampled at Antarctic Scientific Base Artigas (62\u0026deg;11\u0026prime;04\u0026Prime;S 58\u0026deg;51\u0026prime;07\u0026Prime;W), Fildes peninsula, King George Island, Antarctica. The DNA G\u0026thinsp;+\u0026thinsp;C content is 32.8%. The GenBank accession code for the 16S rRNA gene sequence is MN007166. The Whole Genome Shotgun project has been deposited at GenBank under the accession JABSNK000000000. The version described in this paper is version JABSNK000000000.1. All characteristics of the type strain 28Aᵀ are consistent with the species description of \u003cem\u003eFlavobacterium gelidicaeli\u003c/em\u003e, except for the following: growth at 25\u0026deg;C, positive results for aesculin hydrolysis, acid production from melibiose and L-arabinose, assimilation of D-raffinose, and a negative result for starch hydrolysis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of interest\u003c/h2\u003e \u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding information\u003c/h2\u003e \u003cp\u003eThe authors received no specific grant from any funding agency.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eRJM thank the Uruguayan Antarctic Institute and the staff of the Artigas Antarctic Scientific Base for their support during the summer 2018 campaign. The authors are also grateful to Professor Aharon Oren (The Hebrew University of Jerusalem) for his assistance in the species names. JV would like to thank the Czech Antarctic Research Programme and the Masaryk University Grant Agency (CAREER RESTART Muni/R/1443/2024) for their support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBergey DH, Harrison FC, Breed RS, Hammer BW, Huntoon FM (1923) Bergey's Manual of Determinative Bacteriology, 1st edn. The Williams \u0026amp; Wilkins Co, Baltimore\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBernardet J-F, Bowman JP (2006) The Genus \u003cem\u003eFlavobacterium\u003c/em\u003e. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E (eds) The Prokaryotes: Volume 7: Proteobacteria: Delta, Epsilon Subclass. Springer New York, New York, NY, pp 481\u0026ndash;531\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBernardet J-F, Bowman JP (2015) \u003cem\u003eFlavobacterium\u003c/em\u003e. In: Bergey\u0026rsquo;s Manual of Systematics of Archaea and Bacteria. (eds M.E. Trujillo, S. Dedysh, P. DeVos, B. Hedlund, P. K\u0026auml;mpfer, F.A. Rainey and W.B. Whitman). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/9781118960608.gbm00312\u003c/span\u003e\u003cspan address=\"10.1002/9781118960608.gbm00312\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoung Y, Jang H-J, Song J, Cho J-C (2019) \u003cem\u003eFlavobacterium hydrophilum\u003c/em\u003e sp. nov. and \u003cem\u003eFlavobacterium cheongpyeongense\u003c/em\u003e sp. nov., isolated from freshwater. 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Sci Rep 15:13615. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-025-97205-x\u003c/span\u003e\u003cspan address=\"10.1038/s41598-025-97205-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Facultad de Química y Unidad Asociada del Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Uruguay","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Flavobacterium sp, airborne bacteria, Antarctica, psychrophiles","lastPublishedDoi":"10.21203/rs.3.rs-8347779/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8347779/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFour aerobic, Gram-stain-negative, non-motile, rod-shaped, psychrophilic and yellow to light orange-pigmented bacterial strains, designated 7Aᵀ, 14Aᵀ, 28Aᵀ and 7E, were isolated from air samples collected at Fildes Peninsula, King George Island, Antarctica. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the novel strains were related to members of the genus \u003cem\u003eFlavobacterium\u003c/em\u003e, and revealed that strain 7A\u003csup\u003eT\u003c/sup\u003e shared 98.40% similarity to\u003cem\u003e F. faecale\u003c/em\u003e WV33\u003csup\u003eT\u003c/sup\u003e, while strains 14A\u003csup\u003eT\u003c/sup\u003e, 28A\u003csup\u003eT\u003c/sup\u003e and 7E showed 98.88%, 98.74% and 98.61% similarity, respectively, to \u003cem\u003eF. frigidarium\u003c/em\u003e A2i\u003csup\u003eT\u003c/sup\u003e. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between these novel isolates and their closest relatives were below the cut-off values used for species delineation of 95-96% and 70%, respectively. Moreover, the ANI and dDDH values between strains 28A\u003csup\u003eT\u003c/sup\u003e and 7E were 97.62 and 78.30%, respectively, indicating that these strains represented the same species. All four strains contained menaquinone-6 as the sole respiratory quinone. The DNA G+C content of all strains ranged from 32.8 to 34.5 mol%. Based on phylogenetic, genomic and phenotypic data, we propose that strains 7Aᵀ, 14Aᵀ and 28Aᵀ represent three novel species of the genus \u003cem\u003eFlavobacterium\u003c/em\u003e, for which the names \u003cem\u003eFlavobacterium psychraerolatum \u003c/em\u003e(7A\u003csup\u003eT\u003c/sup\u003e=CCM 8976\u003csup\u003eT\u003c/sup\u003e=DSM 111734\u003csup\u003eT\u003c/sup\u003e),\u003cem\u003e Flavobacterium aeripolare\u003c/em\u003e (14A\u003csup\u003eT\u003c/sup\u003e=CCM 8972\u003csup\u003eT\u003c/sup\u003e=CGMCC 1.18502\u003csup\u003eT\u003c/sup\u003e)\u003cem\u003e \u003c/em\u003eand\u003cem\u003e Flavobacterium gelidicaeli\u003c/em\u003e (28A\u003csup\u003eT\u003c/sup\u003e=CCM 8973\u003csup\u003eT\u003c/sup\u003e=CGMCC 1.18503\u003csup\u003eT\u003c/sup\u003e) are proposed.\u003c/p\u003e","manuscriptTitle":"Flavobacterium psychraerolatum sp. nov., Flavobacterium aeripolare sp. nov. and Flavobacterium gelidicaeli sp. nov. isolated from Antarctic air","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-15 07:13:28","doi":"10.21203/rs.3.rs-8347779/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"75745147-9309-40cb-af3e-cccfe9eaa588","owner":[],"postedDate":"December 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59571269,"name":"Taxonomy"},{"id":59571270,"name":"Bacteriology"}],"tags":[],"updatedAt":"2025-12-15T07:13:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-15 07:13:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8347779","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8347779","identity":"rs-8347779","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}