Strain BND22^T was cultivated from the coastal saline soil sample of the intertidal zone located in Qingdao, PRChina (36°12′36″ N, 120°22′10″ E; elevation, 0 m) in 2019. The pH, salt concentration, and organic matter content of the saline soil were 7.9, 5.3 g/kg, and 1.3 g/kg, respectively. Strains ZY10^T, ZY41^T, ZY58^T, and ZY43^T were isolated from the natural sea salt produced from Huanghua marine solar saltern in Hebei, PRChina, in 2018. These samples were diluted with liquid NHM, spread on NHM agar plates, and incubated aerobically at 37°C for 30 days (Sun et al., 2022). Single colonies with a shade of red were selected and streaked on fresh NHM agar slants, and the pure colonies were formed after three-time repeated. For further use, the purified strains were preserved in liquid NHM with 15% (w/v) glycerol at -20°C. Halobacterium salinarum CGMCC 1.2367, Haloglomus irregulare JCM 33318^T, Halosegnis longus JCM 33319^T, Natronomonas gomsonensis KCTC 4088^T, Natronomonas halophila C90^T, Natronomonas salina YPL13^T, and Salinirubellus salinus ZS-35-S2^T were used as reference strains and routinely grown aerobically in NHM liquid medium.

The genomic DNA of strains BND22^T, ZY10^T, ZY41^T, ZY58^T, and ZY43^T was prepared using a DNA extraction kit for bacteria (Jiangsu Cowin Biotech Co., Ltd.). The PCR amplification of the 16S rRNA genes and the rpoB′ (RNA polymerase subunit B′) genes were carried out by two pairs of primers, 20F (5’-ATTCCGGTTGATCCTGCCGG-3’) and 1452R (5’-AGGAGGTGATCCAGCCGCAG-3’), and HrpoB2 1420F and HrpoA 153R, respectively (Cui et al., 2009; Minegishi et al., 2010). The sequencing of purified PCR amplicons was performed by SinoGenoMax (Beijing). The 16S rRNA and rpoB′ genes of related species of the Haloarculaceae were downloaded from the NCBI database. Phylogenetic trees were constructed with MEGA 6 (Tamura et al., 2013) based on three different algorithms, ML (maximum-likelihood) (Felsenstein, 1981), MP (maximum-parsimony) (Fitch, 1971) and NJ (neighbor-joining) (Saitou and Nei, 1987). The calculation of pairwise sequence similarities of 16S rRNA and rpoB′ gene between strains BND22^T, ZY10^T, ZY41^T, ZY58^T, ZY43^T, and related species was performed by using the online pairwise sequence alignment tool of EzBioCloud (Yoon et al., 2017).

For microscopical examination, these five isolates were grown in liquid NHM for 7–10 days. The cells of these haloarchaea were observed under a phase-contrast microscope (Eclipse Ci-L, Nikon). Gram staining was performed according to an improved technique developed by Dussault (Dussault, 1955). The pigmentation of the colonies was examined on NHM agar plates after aerobic incubation for 10–15 days at 37°C. The ranges of NaCl and MgCl₂ concentrations, temperature, and pH for growth were determined following the methodology described previously (Sun et al., 2022). The remaining phenotypic features of the five isolates and related members were based on the minimal criteria for the description of halophilic archaeal new taxa (Oren et al., 1997). Each test was carried out in triplicates.

Polar lipids of strains BND22^T, ZY10^T, ZY41^T, ZY58^T, and ZY43^T were extracted with chloroform and methanol mixture, then precipitated by ten volumes of cold acetone after concentration. The polar lipid profiles of isolated halophilic archaea were analyzed by one-dimensional and two-dimensional thin-layer chromatography (TLC) (Cui et al., 2010). The phosphate stain reagent was prepared to detect phospholipids (Vaskovsky and Kostetsk, 1968). Glycolipids and phospholipids were shown with sulfuric acid-ethanol (1:2, v/v) followed by heating at 150 °C for 3 min. The polar lipids of these halophilic archaea were characterized by MALDI-TOF/MS (Xin et al., 2022).

Strains BND22^T, ZY10^T, ZY41^T, ZY58^T, and ZY43^T all had a single copy of the 16S rRNA gene (1,467 bp, 1,467 bp, 1,472 bp, 1,472 bp, and 1,466 bp, respectively). These sequences are identical to the same genes in their genomes. Based on the 16S rRNA gene similarity, the closest relatives of strains BND22^T, ZY10^T, ZY41^T, ZY58^T, and ZY43^T were Salinirubellus salinus ZS-35-S2^T (95.2%), Halosegnis rubeus F17-44^T (95.1%), Haloglomus irregulare F16-60^T (98.1%), Haloglomus irregulare F16-60^T (98.2%), Natronomonas salina YPL13^T (98.0%), respectively ( Table S1 ). Except for 99.6% gene similarity between strains ZY41^T and ZY58^T, those among strains BND22^T, ZY10^T, and ZY43^T were 92.9–96.8%, less than 98.65%, the suggested threshold for separating different archaeal or bacterial species (Kim et al., 2014). The 16S rRNA gene phylogeny indicated that strains BND22^T and ZY10^T formed two distinct clades separated from Salinirubellus salinus ZS-35-S2^T and Halosegnis members, strains ZY41^T and ZY58^T clustered with Haloglomus irregulare F16-60^T, and strain ZY43^T gathered with the current members of Natronomonas ( Figure 1A ).

The full length of the rpoB′ genes from strains BND22^T, ZY10^T, ZY41^T, ZY58^T, and ZY43^T were 1,827 bp. The gene similarities among these five strains were 84.9–95.2%, and they shared 81.1–91.7% sequence similarities with related members of the family Haloarculaceae ( Table S2 ). Strains BND22^T, ZY10^T, ZY41^T, ZY58^T, and ZY43^T shared maximum sequence similarities with Sala cibi CBA1133^T (88.3%), Halosegnis longus F12-1^T (89.4%), Haloglomus irregulare F16-60^T (91.4%), Haloglomus irregulare F16-60^T (91.6%), Natronomonas salina YPL13^T (91.7%), respectively. The rpoB’ gene phylogeny revealed that strains BND22^T and ZY10^T clustered together and separated from the genus Halosegnis, strains ZY41^T and ZY58^T formed a monophyletic branch closely clustered with Haloglomus irregulare F16-60^T, strain ZY43^T was a close relative of Natronomonas salina YPL13^T within Natronomonas ( Figure 1B ).

The complete genomes of strains BND22^T, ZY10^T, ZY41^T, ZY58^T, and ZY43^T were de novo assembled in a total of 2, 5, 3, 1, and 2 contigs, respectively. The complete genome size of new strains ranged from 4.0 to 4.4 Mb, within those of the current Haloarculaceae members (2.6–5.1 Mb). These five strains had one ribosomal RNA operon in their genomes and had different numbers (47, 47, 45, 43, and 48, respectively) of tRNA genes. The DNA G+C content of strains BND22^T, ZY10^T, ZY41^T, ZY58^T, and ZY43^T are 66.6%, 66.8%, 67.9%, 68.5%, and 66.9%, respectively ( Table S3 ). These values are higher than those of Halosegnis members (64.7–66.0 mol%) and Haloglomus (68.0 mol%), within the range of the genus Natronomonas (63.2–67.4 mol%), and less than that of Salinirubellus salinus ZS-35-S2^T (69.0 mol%). Other genomic features were detailed in Table S3 .

The ML phylogenomic tree based on 122 concatenated archaeal protein genes indicated that strain BND22^T gathered with ZY10^T and distinctly separated from Halosegnis and Sala, strains ZY41^T and ZY58^T formed a tight clade with Haloglomus irregulare F16-60^T, and strain ZY43^T tightly clustered with the current Natronomonas members ( Figure 1C ).

The ANI, AAI, and DDH values among these five strains and Haloarculaceae members were 70–91%, 56–92%, and 18–45% ( Tables S4 - S6 ), significantly below the cut-off values, 95–96% (ANI), 95% (AAI), and 70% (DDH), for species delineation (Goris et al., 2007; Richter and Rosselló-Móra, 2009; Luo et al., 2014). This suggested that strains BND22^T, ZY10^T, ZY41^T, ZY58^T, and ZY43^T represent five different species. The AAI values were proved to be useful for genera demarcation and a cut-off value (≤ 76%) was proposed to differentiate genera within the family Natrialbaceae (de la Haba et al., 2021); but the value was inappropriate to be used in grouping the current Haloarculaceae members. If the cut-off value was used to separate the genera within the family Haloarculaceae, the current six Natronomonas species should represent six different genera ( Table S6 ). A cut-off value of < 69% is appropriate to distinguish genera within Haloarculaceae according to the AAI values and evolutionary relationships among the current members of this family. Thus, strains BND22^T and ZY10^T represent two novel species of a new genus, strains ZY41^T and ZY58^T two novel different species of Haloglomus, and strain ZY43^T a new species of Natronomonas based on the AAI cut-off (< 69%).

It must be noted that the ANI, AAI, and DDH values between Halosegnis longus F12-1^T and Sala cibi CBA1133^T described recently are 99%, 94%, and 98%, much more than the threshold values of the species boundary. This close relatedness was supported by the 16S rRNA, rpoB’, and 122 conserved archaeal protein marker genes -based phylogenies between Halosegnis longus F12-1^T and Sala cibi CBA1133^T ( Figure 1 ). According to these results Sala cibi (Song et al., 2022) should be proposed as a later heterotypic synonym of Halosegnis longus (Durán-Viseras et al., 2021).

The subsystem category distribution of novel isolates was annotated by the RAST server ( Figure S1 ). The amino acids and derivatives, and carbohydrates and protein metabolism were the richest RAST subsystems of these five isolates, indicating their possibility to metabolize diverse proteins and carbohydrates. Based on the KEGG database, strains BND22^T and ZY58^T, ZY41^T and ZY43^T contained the genes involved in both glycolysis and pyruvate oxidation pathways, respectively, corresponding to their capability to utilize glucose and pyruvate for growth. In contrast, the genes related to fructose utilization, casein, starch, or Tween 80 hydrolysis were not found in all these strains, which was confirmed by the results of phenotypic tests. Additionally, none of the four key enzymes involved in complete denitrification, NarG (nitrate reductase), NirK (copper-containing nitrite reductase), Nor (nitric oxide reductase), and Nos (nitrous-oxide reductase), was found in the genomes of these five strains, this confirmed by their inability to produce nitrogen growing with nitrate anaerobically.

Total of 3,788 orthologous clusters, 566 core clusters, 3,077 accessory clusters, and 145 unique clusters were found in the genomes of strains BND22^T, ZY10^T, Salinirubellus salinus ZS-35-S2^T, Halosegnis longus F12-1^T, and Halosegnis rubeus F17-44^T. The isolates BND22^T and ZY10^T contained 62 and 61 unique clusters, while the related members only contained 9, 9, and 4 unique clusters, respectively ( Figure S2A ). Strains ZY41^T, ZY58^T, and Haloglomus irregulare F16-60^T had 3,850 OCs, including 2,427 core OCs, 1,320 accessory OCs and 103 unique clusters, and contained 22, 13, and 68 unique clusters, respectively ( Figure S2B ). Strain ZY43^T, Natronomonas moolapensis 8.8.11^T, Natronomonas pharaonis DSM 2160^T, Natronomonas salina YPL13^T and Natronomonas salsuginis F20-122^T had 3,472 clusters, including 1,427 core OCs, 1,851 accessory OCs and 194 unique clusters. These strains contained 63, 24, 16, 70, and 21 unique clusters, respectively ( Figure S2C ). The patterns of shared and unique OCs differentiated these novel strains from their relatives.

Many previous studies reported that haloarchaea could produce valuable products of biotechnological aspects, such as haloenzymes, bacteriorhodopsin, biodegradable polyhydroxyalkanoates (PHAs), and carotenoids (Han et al., 2010; Amoozegar et al., 2017; Ashwini et al., 2017; Serrano et al., 2022). Strains ZY10^T, ZY41^T, ZY58^T, and ZY43^T possessed bacteriorhodopsin protein genes, indicating their capacity for light-mediated ATP synthesis. Strain BND22^T contained the gene cluster encoding for PHA synthesis regulatory protein (PhaR), PHA granule binding protein (PhaP), and type IIIA PHA synthase (PhaE and PhaC subunits), and strain ZY41^T contained the gene cluster encoding for acetoacetyl CoA reductase (PhaB), PhaC, PhaE and PhaR ( Figure S3 ), indicating the probable synthesis of PHAs by the new isolates. Additionally, the genes encoded the enzymes involved in the production of β-carotene, phytoene synthase (CrtB), phytoene desaturase (CrtI), and lycopene cyclase (CrtY), were detected in strains ZY41^T and ZY58^T (Serrano et al., 2022). Moreover, one β-carotene monooxygenase (Brp) was detected in the vicinity of the crtY gene, suggesting the possibility of strain ZY41^T to produce retinal from β-carotene ( Figure S4 ). These results indicate that these novel halophilic archaeal strains have much potential in biotechnological applications.

Cells of strains BND22^T, ZY10^T, ZY41^T, ZY58^T, and ZY43^T were motile and Gram-stain-negative. Except for strains BND22^T and ZY58^T were pleomorphic, while other strains were coccoid ( Figure S5 ). The colonies of all strains were red and their cells lysed in distilled water. The growth of strains BND22^T, ZY10^T, ZY41^T, ZY58^T, and ZY43^T occurred in NHM with NaCl concentrations of 1.7–4.8 M, 3.1–5.1 M, 2.1–4.8 M, 2.6–4.3 M, and 2.1–4.8 M, respectively; the five strains grew best at 3.1–4.8 M NaCl. The optimum magnesium concentration varied between 0.05 and 0.7 M, even if magnesium was not necessary for their growth. The growth temperature ranges for these five strains were 20–55 °C, 30–50 °C, 25–60 °C, 25–55 °C, and 30–60 °C, respectively, with optima between 35 and 40°C. These five strains are mainly neutrophilic with pH optima at 6.5–7.5. These strains could not grow with arginine or DMSO anaerobically. Reduction of nitrate to nitrite was found in strains BND22^T, ZY10^T, ZY41^T, and ZY43^T but not in strain ZY58^T. Indole and H₂S were not produced by these strains, and none of the casein, gelatin, starch, or Tween 80 was found to be hydrolyzed. Of the 19 antibiotics tested, only bacitracin (0.04 IU per disc), nitrofurantoin (300 µg), novobiocin (30 µg per disc), nystatin (100 µg), rifampin (5 µg), and trimethoprim (5) could inhibit growth. The remained antibiotics, ampicillin (10 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), erythromycin (15 µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid (30 µg), neomycin (30 µg), norfloxacin (10 µg), penicillin G (10 IU), streptomycin (10 µg), tetracycline (30 µg), or vancomycin (30 µg), were not found to be effective. The distinct phenotypic characteristics differentiating isolates BND22^T and ZY10^T from the related species of the family Haloarculaceae (Salinirubellus salinus ZS-35-S2^T, Halosegnis longus JCM 33317^T, Halosegnis rubeus JCM 33319^T, and Haloglomus irregulare JCM 33318^T) were optimum NaCl, utilization of specific carbon sources, and presence of PGS. Strains ZY41^T and ZY58^T could be differentiated from Haloglomus irregulare JCM 33318^T based on cell motility, gelatin hydrolysis and presence of PGS. The difference between strain ZY43^T and the current members of the Natronomonas included H₂S formation, cell morphology, catalase, and oxidase activity ( Tables 1 , 2 ) (Kamekura et al., 1997; Burns et al., 2010; Kim et al., 2013; Durán-Viseras et al., 2020a; Durán-Viseras et al., 2020b; Yin et al., 2020; Durán-Viseras et al., 2021).

The common phospholipids, phosphatidylglycerol (PG) and phosphatidylglycerol phosphate methyl ester (PGP-Me), were detected in strains BND22^T, ZY10^T, ZY41^T, and ZY58^T. It was remarkable that the absence of phosphatidylglycerol sulfate (PGS) in these four strains compared with the Halosegnis and Haloglomus members. The phospholipid profile of strain ZY43^T was PG, PGP-Me, and PGS, similar to that of the Natronomonas members ( Figure S6 ). Diverse glycolipids were detected in these five strains, three glycolipids (GL1, GL-PL1, and GL-PL2) in strain BND22^T, two glycolipids (GL1, GL2) in strain ZY10^T, one glycolipid (GL1) in strains ZY41^T and ZY58^T, and two different glycolipids (GL01, GL02) in strain ZY43^T. The TLC mobilities of GL1, GL2 (GL-PL1), and GL02 were similar to those of sulfated mannosyl glucosyl diether (S-DGD-1), mannosyl glucosyl diether (DGD-1), and sulfated galactosyl mannosyl glucosyl diether (S-TGD-1), respectively, based on one-dimensional TLC ( Figure S6 ).

The MALDI-TOF/MS patterns of the polar lipids of strains BND22^T, ZY10^T, ZY41^T, ZY58^T, and ZY43^T were obtained based on the mode of negative ion ( Figure S7 ). The signals at m/z 731.6, 805.7, 875.7, 885.6, 899.6, 955.7, and 969.6 represented PA, PG, PG (C₂₀C₂₅), PGS, PGP-Me, PGS (C₂₀C₂₅), and PGP-Me (C₂₀C₂₅), respectively. Except for strains ZY41^T and ZY58^T, the ion of m/z 991.7 appearing in others represented DGD-1. Strains BND22^T, ZY10^T, ZY41^T, and ZY58^T showed S-DGD-1 signals at m/z 1055.7, 1071.7, and 1125.7. The ion at m/z 1217.7 detected in strain ZY43^T corresponded to S-TGD-1. In addition, several glycosyl-cardiolipins, DGD-PA, S-DGD-PA, and S-DGD-PA (+Na) were detected and judged by the signals of m/z 1690.4, 1770.3, and 1792.3, respectively (Lobasso et al., 2015). These results were mostly in accordance with those detected by TLC.

The polyphasic and genome-based classification indicated that strains BND22^T (= CGMCC 1.18780^T = JCM 34966^T) and ZY10^T (= CGMCC 1.17475^T = JCM 34319^T), represent two different new species of a novel genus within Haloarculaceae, Halorarius litoreus gen. nov., sp. nov. Halorarius halobius sp. nov.; strains ZY41^T (= CGMCC 1.17030^T = JCM 34161^T) and ZY58^T (= CGMCC 1.17216^T = JCM 34163^T) represent two different new species of Haloglomus, Haloglomus halophilum sp. nov. and Haloglomus salinum sp. nov.; and strain ZY43^T (= CGMCC 1.17202^T = JCM 34162^T) represents a novel species of Natronomonas, Natronomonas marina sp. nov.

We propose Sala cibi Song et al., 2022 as a later heterotypic synonym of Halosegnis longus (Durán-Viseras et al., 2021). (type strain F12-1^T = CECT 9685^T = JCM 33317^T; another reference strain of this species, CBA1133 = KACC 22148 = JCM 34265).

Halorarius (Hal.o.ra’ri.us. Gr. masc. n. hals halos, salt; L. masc. adj. orarius, belonging to the seashore; N.L. masc. n. Halorarius salt microorganism isolated from the seashore).

Cells are Gram-stain-negative, motile, pleomorphic and coccoid under optimal growth conditions. Cells are found to be lysed in distilled water. Colonies on NHM agar plate are red, moist and round. Growth was found at 20–55°C, 1.7–5.1 M NaCl, 0–1.0 M MgCl₂ and pH 6.5–8.5. The activity of catalase was positive while that of oxidase was negative. Some sugars can be utilized for growth and with the production of acids. The cultures contain PA, PG, PGP-Me, S-DGD-1, and DGD-1. The type species is Halorarius litoreus. Recommended three-letter abbreviation: Hor.

Halorarius litoreus (li.to’re.us. L. masc. adj. litoreus, of or belonging to the seashore).

The cells of type strain are pleomorphic (1.0–1.2×1.0–3.0 μm), motile, and Gram-negative. Colonies are red and circular, about 5 mm in diameter following incubation for 14 days at 37°C. The type strain could grow in NHM with NaCl concentrations of 1.7–4.8 M and grew best at 3.4 M NaCl. The optimum magnesium concentration was 0.03 M, even if magnesium was not necessary for growth. The growth temperature range for the type strain was 20–55 °C, with optimum at 35°C. The type strain is neutrophilic with pH optima at 7.5. The type strain can grow anaerobically with nitrate but not with arginine or DMSO. Reduction of nitrate to nitrite was found but gas was not produced. Diverse substrates, _D-glucose, sucrose, _D-mannitol, _D-sorbitol, acetate, succinate, _L-aspartate, _L-glutamate, _L-lysine and _L-ornithine, can be utilized for growth, while _D-fructose, _D-galactose, lactose, maltose, _D-mannose, _D-ribose, _L-sorbose, _D-xylose, glycerol, citrate, fumarate, _DL-lactate, _L-malate, pyruvate, _L-arginine, glycine or _L-alanine do not support growth. The catalase test is positive while the oxidase is negative. Indole and H₂S formation are not detected. None of casein, gelatin, starch or Tween 80 was found to be hydrolyzed. The type strain contains PA, PG, PGP-Me, DGD-1 (DGD-PA), and S-DGD-1 (S-DGD-PA).

The type strain, BND22^T (= CGMCC 1.18780^T = JCM 34966^T), was isolated from the coastal saline soil sample of the intertidal zone located in Qingdao, PRChina. The DNA G+C content of the type strain is 66.6% (genome). The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and genome of strain BND22^T are MW338920 and CP101454–CP101455, respectively.

Halorarius halobius (ha.lo’bi.us. Gr. masc. n. hals, halos, salt; Gr. masc. n. bios, life; N.L. masc. adj. halobius, growing in salt).

The cells of type strain are coccoid (0.5–2.0 μm), motile, and Gram-negative. Colonies are red and circular, about 0.5 mm in diameter following incubation for 14 days at 37°C. The type strain could grow in NHM with NaCl concentrations of 3.1–5.1 M and grew best at 4.8 M NaCl. The optimum magnesium concentration was 0.5 M, even if magnesium was not necessary for growth. The growth temperature range for the type strain was 30–50 °C, with optimum at 37°C. The type strain is neutrophilic with pH optima at 7.5. The type strain can grow anaerobically with nitrate but not with arginine or DMSO. Reduction of nitrate to nitrite was found but gas was not produced. Diverse substrates, _D-mannose, sucrose, glycerol, _D-mannitol, _D-sorbitol, fumarate, _DL-lactate, _L-malate, pyruvate, succinate, glycine, _L-aspartate, _L-glutamate and _L-lysine, can be utilized for growth, while _D-fructose, _D-galactose, _D-glucose, lactose, maltose, _D-ribose, _L-sorbose, _D-xylose, acetate, citrate, _L-arginine, _L-alanine or _L-ornithine do not support growth. Positive for catalase and oxidase activity. Indole and H₂S formation are not detected. None of casein, gelatin, starch or Tween 80 was found to be hydrolyzed. The type strain contains PG, PGP-Me, DGD-1 and S-DGD-1.

The type strain, ZY10^T (= CGMCC 1.17475^T = JCM 34319^T), was isolated from the natural sea salt produced from Huanghua marine solar saltern in Hebei, PRChina. The DNA G+C content of the type strain is 66.8% (genome). The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and whole genome of strain ZY10^T are MN589739 and CP101161–CP101165, respectively.

Haloglomus halophilum (ha.lo’phi.lum. Gr. masc. n. hals, halos salt; Gr. masc. adj. philos loving; N.L. neut. adj. halophilum salt-loving, concerning the requirement for salt).

The cells of type strain are coccoid (0.5–2.0 μm), motile, and Gram-negative. Colonies are red and circular, about 0.5 mm in diameter following incubation for 14 days at 37°C. The type strain could grow in NHM with NaCl concentrations of 2.1–4.8 M and grew best at 3.1 M NaCl. The optimum magnesium concentration was 0.7 M, even if magnesium was not necessary for growth. The growth temperature range for the type strain was 25–60 °C, with optimum at 40°C. The type strain is neutrophilic with pH optima at 7.5. The type strain cannot grow anaerobically with arginine, DMSO, or nitrate. Reduction of nitrate to nitrite was found but gas was not produced. Diverse substrates, glycerol, _D-mannitol, _D-sorbitol, acetate, _DL-lactate, pyruvate, succinate, _L-ornithine and _L-lysine, can be utilized for growth, while _D-fructose, _D-galactose, _D-glucose, lactose, maltose, _D-mannose, _D-ribose, _L-sorbose, sucrose, _D-xylose, citrate, fumarate, _L-alanine, _L-arginine, _L-aspartate, _L-glutamate, glycine, or _L-malate do not support growth. Catalase positive and oxidase negative. Indole and H₂S are not produced. None of casein, gelatin, starch or Tween 80 was found to be hydrolyzed. The type strain contains PG, PGP-Me, and S-DGD-1.

The type strain, ZY41^T (= CGMCC 1.17030^T = JCM 34161^T), was isolated from the natural sea salt produced from Huanghua marine solar saltern in Hebei, PRChina. The DNA G+C content of the type strain is 67.9% (genome). The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and whole genome of strain ZY41^T are MK721000 and CP101166–CP101168, respectively.

Haloglomus salinum (sa.li’num. N.L. neut. adj. salinum belonging to salt works)

The cells of type strain are pleomorphic (0.8–1.0×1.0–2.0 μm), motile, and Gram-negative. Colonies are red and circular, about 0.5 mm in diameter following incubation for 14 days at 37°C. The type strain could grow in NHM with NaCl concentrations of 2.6–4.3 M and grew best at 3.1 M NaCl. The optimum magnesium concentration was 0.03 M, even if magnesium was not necessary for growth. The growth temperature range for the type strain was 25–55 °C, with optimum at 37°C. The type strain is neutrophilic with pH optima at 7.0. The type strain cannot grow anaerobically with arginine, DMSO, or nitrate. Reduction of nitrate to nitrite was not found and gas was not produced. Diverse substrates, _D-galactose, _D-glucose, _D-mannose, sucrose, acetate, pyruvate, _DL-lactate, can be utilized for growth, while _D-fructose, lactose, maltose, _D-ribose, _L-sorbose, _D-xylose, glycerol, _D-mannitol, _D-sorbitol, citrate, fumarate, _L-malate, succinate, _L-aspartate, _L-alanine, _L-arginine, _L-glutamate, glycine, _L-lysine, or _L-ornithine do not support growth. Catalase positive and oxidase negative. Indole and H₂S production are not detected. None of casein, gelatin, starch or Tween 80 was found to be hydrolyzed. The type strain contains PG, PGP-Me, and S-DGD-1.

The type strain, ZY58^T (= CGMCC 1.17216^T = JCM 34163^T), was isolated from the natural sea salt produced from Huanghua marine solar saltern in Hebei, PRChina. The DNA G+C content of the type strain is 68.5% (genome). The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and whole genome of strain ZY58^T are MK920153 and CP101153, respectively.

Natronomonas marina (ma.ri’na. L. fem. adj. marina of the marine).

The cells of type strain are coccoid (0.5–2.0 μm), motile, and Gram-negative. Colonies are red and circular, about 0.5 mm in diameter following incubation for 14 days at 37°C. The type strain could grow in NHM with NaCl concentrations of 2.1–4.8 M and grew best at 3.1 M NaCl. The optimum magnesium concentration was 0.05 M, even if magnesium was not necessary for growth. The growth temperature range for the type strain was 30–60 °C, with optimum at 40°C. The type strain is neutrophilic with pH optima at 6.5. The type strain cannot grow anaerobically with arginine, DMSO, or nitrate. Reduction of nitrate to nitrite was found but gas was not produced. Diverse substrates, glycerol, _D-sorbitol, acetate, _DL-lactate, pyruvate, succinate, _L-aspartate, _L-alanine, _L-lysine, and _L-ornithine can be utilized for growth, while _D-fructose, _D-galactose, _D-glucose, lactose, maltose, _D-mannose, _D-ribose, _L-sorbose, sucrose, _D-xylose, _D-mannitol, acetate, citrate, fumarate, _L-arginine, _L-glutamate glycine or _L-malate do not support growth. The catalase test is positive while the oxidase is negative. Indole and H₂S production are not detected. None of casein, gelatin, starch or Tween 80 was found to be hydrolyzed. The type strain contains PG, PGP-Me, PGS, DGD-1, and S-TGD-1.

The type strain, ZY43^T (= CGMCC 1.17202^T = JCM 34162^T), was isolated from the natural sea salt produced from Huanghua marine solar saltern in Hebei, PRChina. The DNA G+C content of the type strain is 66.9% (genome). The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and whole genome of strain ZY43^T are MK920152 and CP101154–CP101155, respectively.