Soil samples (0.5 g each) taken from 360 places were spread on agar plates containing 20% NaCl, with pH adjusted to 5.0, 7.0 and 9.0 respectively. The pH of the soil samples ranged between 5.0 and 6.0. After incubation of plates for 3 weeks at 37°C, colony formations were observed in 132 soil samples (red circles in Fig. 1), at least on one of the three agar plates. Numbers of colonies per plate ranged from 1 on 51 plates to 40 on 1 plate. The sum of colonies amounted to 49 from the medium of pH 9.0, 534 from the pH 7.0 medium, and 61 from the medium of pH 5.0. By inspecting each plate, representative colonies were picked up and transferred to fresh plates and purified by plating out of serial dilutions. A few isolates gradually failed to form colonies on fresh plates. When colonies failed to grow on fresh 20% NaCl plates, concentration of NaCl was decreased to 15 or 10%. It was observed that 26 strains failed to grow in the presence of 20% NaCl. According to our definition of the present paper, these strains are not 'halophilic bacteria', but these strains were included in the further characterization (see discussion).

Finally, 176 strains were obtained (Table 1): 27 strains from 23 samples on alkaline medium (pH 9.0), 139 strains from 120 samples on neutral medium (pH 7.0), and 10 strains from 9 samples on acidic medium (pH 5.0). Endospores were observed by microscope after spore staining 17. These strains have been kept at 5°C on agar plates of 10% NaCl.

On the other hand, from 228 soil samples (black circles in Fig. 1), about two thirds of the 360 samples collected, no colonies appeared on any plates of the three different pH values. There was no distinct bias in the distribution of black and red circles. In order to estimate if those soil samples contain indeed no microorganisms able to grow at 20% NaCl, two soil samples were randomly picked up, and 0.5 g each was spread on to 10 agar plates of pH 7.0. The colony numbers per plate ranged from 0 on 3 plates to 5 in 1 plate, amounting to 14 in sample 1. From another sample the numbers were from 0 on 3 plates to 4 on 1 plate, amounting to 14 colonies. These data may suggest that halophilic bacteria able to grow at 20% NaCl inhabit any soil samples, at least at a frequency of 1 c.f.u. (colony forming unit)/g soil, in the area we investigated.

Five soil samples which gave considerable numbers of colonies on the aerobic cultures were spread on agar plates (pH 7.0) and incubated in anaerobic jar for 3 weeks. No colonies were observed at all, while 30 to 40 colonies appeared from the same soil samples incubated aerobically.

Of the 27 strains isolated on the alkaline medium, 21 strains did not grow in media without NaCl, and all except one (strain No. 31) showed optimal growth at alkaline pH, 8.5–9.5 in the presence of 10% NaCl. On the other hand, about 78% (116/149 strains) of the strains isolated on neutral and acidic media were shown to be able to grow without added NaCl, and all strains showed optimal growth at pH 6.5–7.5 (Table 1).

Altogether, 176 strains were divided into 3 groups. Strains of group I and group II may be classified as moderately halophilic bacteria, according to the classification proposed by Kushner et al. 6.

Group I (54 isolates) showed optimal growth between 1.7 and 2.6 M NaCl, and no growth in the absence of added NaCl.

Group II (62 isolates) showed optimal growth between 0.9 and 1.7 M NaCl, and growth in the absence of added NaCl.

Group III (60 isolates) showed optimal growth between 0 and 0.9 M NaCl, and growth in the absence of added NaCl.

Sequences of PCR-amplified partial 16S rRNA genes were determined (about 500 nucleotides), and the 176 strains were tentatively identified by comparing to sequences deposited in databases (Table 1). Summaries of tentative identifications are given in Table 2.

The numbers of total bacteria (c.f.u. on plates with no NaCl) and halophilic bacteria were determined in six inland soil samples; three samples had 30–40 colonies on the 20% NaCl agar plates (pH 7.0), and three samples had just 1 colony on the same medium. As shown in Table 1, the number of c.f.u. on the plates ranged from 340 × 1,000 to 28 × 1,000, thus the total bacteria of inland soil samples were in a range from 1.4 × 10⁷/g (340,000 × 20 × 2) to 1.1 × 10⁶/g (28,000 × 20 × 2). Roughly speaking, one tenth of the total bacteria were occupied by endospore-forming bacteria, and only very few of the endospore-forming bacteria, roughly 1 out of 20,000 or more cells, are halophilic bacteria. Table 3(A) also suggests that most, if not all, of halophilic bacteria are surviving as endospores in the soil samples, in a range of less than 1 to about 500/g soil.

In a separate experiment, 0.5 g of a soil sample was suspended in 2 ml of sterile 10% NaCl, and heated at 80°C. After incubation for 0, 5, 10, 30, and 60 min, three 0.1 ml aliquots were taken, spread on 20% NaCl agar medium (pH 7.0), and incubated for 2 weeks. Data in Table 4 clearly indicated that numbers of c.f.u. (halophilic bacteria) showed little decrease upon heating for 60 min, indicating again that the most of halophilic bacteria were surviving as endospores in soil.

Outdoor accumulations (dust, fine sands etc.) were collected from places like roofs of buildings, veranda, cars, and barks of trees, where heavy rainfall would wash away the previous ones. Numbers of bacterial cells and endospores present ranged from 0.8 × 10⁶ to 7.6 × 10⁶/g and from 89 × 10³ to 812 × 10³/g, respectively. No colonies, however, were observed on any 20% NaCl plates (pH 5.0, 7.0, and 9.0) from 0.5 g samples, even after 8 weeks incubation. Repeated incubations of outdoor accumulations collected 2 weeks after rainfall gave no colonies at all.

Six samples were collected from three spots of seashore in Tateyama of Chiba prefecture, a city confronting Tokyo Bay (Fig. 1). Samples were suspended, heated at 80°C, and subjected to colony counting. Table 3(B) showed clearly that the total numbers of bacteria and endospores were roughly 1000 time smaller than those of inland soil samples. Numbers of halophilic bacteria, however, were almost the same as those of inland soil samples.

Analyses of Cl content of soil samples suggested that NaCl contents of soil samples taken from near seashore were as high as 15–20 mg NaCl/g, whereas the 360 inland soil samples contained less than 1 mg NaCl/g.

The agar plate with 20% NaCl used in this study was based on the medium No. 168 recommended by JCM (Japan Collection of Microorganisms) for the cultivation of haloarchaea. All of the pink to brown colonies that might be haloarchaea were picked up, but they all belonged of the family Bacillaceae. To ascertain that haloarchaea are not present in the soil samples, at least to the limit of detection, soil samples were spread on agar plates of 20% NaCl, pH 7.0 and 9.0, supplemented with 30 μg/ml ampicillin. Generally, numbers of colonies decreased to less than one tenth of those obtained on plates without ampicillin. From five soil samples out of 107 samples tested, four colonies were obtained on plates of pH 7.0, and 12 colonies on those of pH 9.0. No colony appeared from seven samples from seashore at Tateyama. The colonies were transferred to liquid media containing ampicillin, and 10 strains that grew were subjected DNA extraction and PCR amplification using both bacterial and archaeal 16S rRNA gene primer sets. All of them yielded amplification bands only with bacterial primers, suggesting they were not haloarchaea but halophilic bacteria harboring plasmids with β-lactamase genes.

Since halophilic bacteria have been recognized to live in the Dead Sea 18, numerous halophilic and halotolerant microorganisms, both aerobic and anaerobic, both Bacteria and Archaea, have been isolated from saline environments. Thanks to the enthusiastic devotion of A. Oren on halophilic microorganisms 7, we know that halophilic Bacteria are not restricted to the class Bacilli (Bacillus sensu lato) but distributed through classes of Cyanobacteria, α-, β-, γ-, and δ-proteobacteria, Clostridia, Actinobacteria, Flavobacteria, etc. Although Oren defined the "halophilic" as tolerance to 10% (100 g/L) NaCl in his book 7, some of the halophilic microorganisms are able to grow in the presence of 20% NaCl. We also know that all microorganisms that were intentionally isolated as halophiles are inhabitants of saline environments. On the other hand, some bacteria isolated from soil are able to tolerate high NaCl concentrations. For example, Bacillus clarkii, B. agaradhaerens, and B. pseudofirmus are tolerant up to 16% or 17% NaCl 13. To the best knowledge of the authors of this paper, no reports have been published on the isolation of microorganisms able to grow at 20% or higher NaCl concentrations from ordinary non-saline soil samples. It has tacitly been believed that habitats of halophiles able to grow in media containing more than 20% are restricted to saline environments 1415.

In the present study, we have demonstrated that halophilic bacteria that are able to grow in the presence of 20% NaCl are inhabiting almost everywhere in non-saline environments such as ordinary garden soils, yards, fields and roadways in an area surrounding Tokyo. We isolated 176 strains, and analysis of partial sequences of their 16S rRNA genes showed that some of them possessed similarities higher than 94.8% with those of Bacillus haloalkaliphilus 20, Filobacillus milosensis 21, Gracilibacillus halotolerans 22, Halobacillus karajensis 23, Halobacillus litoralis 24, Halobacillus trueperi 24, Lentibacillus salicampi 25, Paraliobacillus ryukyuensis 26, Virgibacillus halodenitrificans 27, Virgibacillus marismortui 28 and Virgibacillus picturae 29. Most of the strains of these species have been isolated from saline environments, and were reported to be halophilic, capable of growth at 20% NaCl. All strains of species of the genera of the family Bacillacea were endospore formers, except Bacillus saliphilus 30. Sequences of 15 isolates (14 isolates from alkaline medium, and one isolated from neutral medium) showed similarities less than 92% to any deposited sequences, thus they may represent novel taxa within the family Bacillaceae. For unknown reason(s), cells of some colonies on the initial isolation plates failed to grow when transferred to fresh plates, but grew on plates with lower NaCl concentrations. Some growth factors present in soil might be responsible for this phenomenon 31.

A large number of halophilic bacteria of group I (no grow without added NaCl) were shown to be alkaliphilic, and most of the group II and III, which grew without added NaCl, were neutrophilic. The haloalkaliphiles, halophilic and alkaliphilic bacteria 2, have been found mainly in extremely alkaline and saline environments, which were distributed in the Rift Valley lakes of East Africa, soda lakes of the United States and Inner Mongolia of China, etc.

Quite interesting is the fact that none of the isolates of halophilic bacteria showed similarities with any halophilic microorganisms of the classes mentioned above other than the endospore-forming Bacillus sensu lato. There remains a possibility, however, that colonies of halophilic bacteria other than Bacilli on agar plates unfortunately escaped from being picked up for purification. Another possibility is that those halophilic bacteria lost the ability to form colonies on the particular agar plates we used during repeated transfers in the purification procedures, or that they simply did not form colonies because of unsuitableness of the composition of agar plates to them.

Spore-formers of the family Bacillaceae were easily isolated from a number of environments by suspending a sample in water and heating at 80°C for 10 to 30 min, even from environments unrelated to their growth requirements. For example, a thermophile Geobacillus stearothermophilus was isolated from ordinary soil, and many of the alkaliphilic Bacillus species have been isolated from soils that were not particularly alkaline 32. Now we know that roughly one tenth of the culturable total bacteria present in ordinary soil were endospore-forming bacteria, only very few of them were halophilic bacteria, and most of the halophilic bacteria were surviving as endospores. Also, the exact distribution of so called "soil microorganisms" of each taxon may fluctuate depending on the soil and also on season, number of cells of alkaliphiles and methanogens have been shown to be an order of 10⁴ to 10⁵/g of neutral soil 24. The numbers of endospores of the halophilic bacteria ranged from less than 1 to 500/g in our experiments. A question is "Are the endospores of halophilic bacteria indigenous to soil, and if not, where did they come from?" Here we remember that (i) NaCl contents of soil samples taken from near seashore contained as high as 15–20 mg NaCl/g, whereas the 360 inland soil samples contained less than 1 mg NaCl/g, and that (ii) numbers of endospores of halophilic bacteria were almost the same in the inland soil samples and the seashore sand samples. These facts strongly suggest that the endospores of halophilic bacteria are neither from the sea nor from minute highly saline niche produced by evaporation of seawater on seashore. Then, where are they from?

Although we have no confirming data at present, we may speculate that the halophilic bacteria have been transported by westerlies either as vegetative cells or as endospores from the indigenous highly saline environments, such as salt lakes in Inner Mongolia or salterns confronting Yellow Sea or East Sea (Japan Sea) in Korea. In fact, several novel genera and species have been isolated from these areas recently. It has been realized that Aeolian dust (mineral dust) and sand storms are plaguing North-East Asia 33. The storms originate in the arid inland parts of China and Mongolia and blow across the Korean peninsula and Japan. It is believed that cold air masses from Siberia whip deserts and soils eastward after the dry continental winter. The dusts kicked up into the jet stream are carried by the prevailing westerlies across mainland Asia, over the Sea of Japan and Pacific Ocean and reach into the main land United States in just five days. It is demonstrated that the dusts reach even to Hawaii, which is over 6,000 km away 3435. China's sand storms are referred to as Huangsha in China, "Asian dust" or Whangsa in Korea, and "Yellow sand" or Kosa event in Japan. The authors of this study believe that bacteria, at least their endospores, thriving in salt lakes, soda lakes, and the surrounding saline soils in the arid region have been kept carried to Japan for thousands of years together with the mineral dusts. A long-distance transport of fungal spores by winds across the English Channel was demonstrated by Hirst et al. 36

Cells of non-endospore-forming halophilic bacteria and halophilic archaea (see below) thriving in the saline environments would be flown to Japan by westerlies together with the endospores, but they would die sooner or later after they arrive at soil, at least because of the hypotonic conditions caused by rainfall.

If the above speculation is correct, the next question is "At what density are there the endospores of halophilic bacteria in the yellow sand and how long do they survive in soil?" The fact that dusts accumulated for several days after the last rainfall gave no colonies on 20% NaCl agar plates suggests that frequencies of the presence of endospores of halophilic bacteria transported by the westerlies are very low, and 40 colonies that appeared on the agar plate are the result of precipitation of the endospores for a very long time span. Endospores are known to be able to survive for quite a long time, at least several decades, or even thousands of years. There is a famous case that a endospore of bacteria closely related to extant Bacillus sphaericus was revived and cultured from the abdominal contents of extinct bees preserved for 25 to 40 million years in buried Dominican amber 37. An even more spectacular claim was made that a bacteria closely related to Virgibacillus marismortui was isolated from fluid inclusions in rock salt crystals of Permian age, over 250 million years old 38. Although there is no well-documented data on the longevity of endospores in the soil, we may expect they can survive at least for decades.

Recently, several moderately halophilic bacteria, representing novel species of the genus Halobacillus (97.1 to 98.4% similarities to Halobacillus litoralis in the 16S rRNA gene sequences) were isolated by enrichment culture in a medium containing 20% NaCl from damaged medieval wall paintings and building materials in Austria 39. To the best knowledge of authors of this paper, this is the only exception of the isolation of halophilic bacteria from non-saline environments. Virgibacillus carmonensis, V. necropolis and V. picturae isolated from samples of biofilm formation on the mural paintings in Spain 28 are moderately halophilic with optimal growth at NaCl concentration of 5 to 10%, but it is not clear if they are able to grow at 20% NaCl.

A total of 360 soil samples were collected in an area (103 km by 126 km) surrounding Tokyo, Japan (Tokyo, Saitama-, Chiba-, Kanagawa- and Ibaraki- prefecture) (Fig. 1). Each soil sample was taken from surfaces such as gardens, fields, yards and roadways, which was separated each other by at least 1 km. There exist no highly saline environments in this region such as salterns and salt lakes. On the other hands, 6 accumulation samples were taken from roof of a car, roofs of three buildings, and veranda of two rooms of building in the campus of Toyo University or nearby cities. Several samples were also taken from seashore of Tateyama, a city southern part of Chiba-prefecture confronting Tokyo-Bay (Fig. 1). Each soil sample was collected into 50 ml sterile FALCON tubes (Becton Dickinson) with sterile spatula, and kept at room temperature until use.

A complex growth medium contained the following ingredients (per liter). 5.0 g casamino acids (Difco), 5.0 g yeast extract (Difco), 1.0 g sodium glutamate·H₂O, 3.0 g trisodium citrate·2H₂O, 2.0 g KCl, 0.2 g MgSO₄·7H₂O, 36 mg FeCl₂·4H₂O, 200 g (3.4 M) NaCl and 20 g Bacto-agar (Difco), pH 7.2. After autoclaving, pH was adjusted to 5.0, 7.0 or 9.0 by adding pre-calculated amounts of diluted sterile H₂SO₄ or Na₂CO₃ solutions. Soil samples (0.5 g each) were placed directly on the three agar plates of different pH, spread with spatula, and incubated at 37°C for 3 weeks in plastic bags to prevent desiccation. Colonies were picked up, transferred to fresh agar plates of the same pH, and pure cultures were obtained by plating serial dilutions and repeated transfers on agar plates of the same medium. In some experiments, the agar plates of pH 7.0 and 9.0 were supplemented with 30 μg/ml ampicillin for the selective isolation of haloarchaeal strains.

Growth was determined by inoculating pre-cultures of purified strains into 30 ml test tubes each containing 3 ml liquid media with varying NaCl concentrations (0, 0.9, 1.7, 2.6, 3.4, 4.3 and 5.2 M, pH 7 or 9) and pH (5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 and 10.0, in the presence of 1.7 M NaCl) and shaken at 37°C with 120 rpm. Growth was monitored by taking 0.1 ml culture broth periodically and measuring absorbance at 660 nm.

For the estimation of the numbers of colony forming unit and halophilic bacteria present in the soil samples, 0.5 g samples were suspended in 2 ml of distilled water or sterile 10% NaCl solution, diluted serially with the same solution, and 0.1 ml each was spread on agar plates without added NaCl or of 20% NaCl, respectively, pH 7.0, followed by incubation at 37°C for 2 weeks. Separate experiments on 9 soil samples have shown that increasing concentrations of NaCl of agar plates for colony counting (0, 5, 10, 15 and 20%) just decreased the number of c.f.u. The numbers of total endospore-forming bacteria were determined after heat treatment of the soil suspension at 80°C for 60 min, followed by dilution and spreading on the plates without added NaCl. Heat resistance of endospores is known to differ in different strains of Bacillaceae and depends on the NaCl concentration during heating 4041.

Endospore formations of the isolates were examined with a phase-contrast microscope (×1,000) or after spore-staining according to the method of Wirtz-Conklin 17.

For the detection of anaerobic halophilic bacteria, 5 soil samples were spread on the agar plates as described above, and incubated at 37°C in an anaerobic jar using a deoxygenation reagent, Anaero Pauch Anaero, and Anaero Pack Rectangular Jar (Mitsubishi Gas Chemical Co., Inc., Tokyo).

Total DNAs were extracted by the method of Cline et al. 42. The 16S rRNA genes were amplified by PCR and directly sequenced by using the ABI PRISM^® BigDye Terminator v3.1 Cycle Sequencing Kits (Applied Biosystems) with the following forward and reverse primers for Bacteria: 5'-AGAGTTTGATCCTGGCTCAG-3' (positions 8–27 according to Escherichia coli numbering) and 5'-GACTACCAGGGTATCTAATC-3' (positions 805–786) on the ABI PRISM^® 310 Genetic Analyzer (Applied Biosystems). In some experiments, the complete 16S rRNA genes were amplified by PCR with the forward primer and a reverse primer: 5'-GGCTACCTTGTTACGACTT-3' (positions 1510–1492). Archaeal primer sets were 5'-ATTCCGGTTGATCCTGCCGG-3' (positions 6–25 according to E. coli numbering) and 5'-AGGAGGTGATCCAGCCGCAG-3' (positions 1540–1521). The closest known relatives of the sequenced organisms were determined by sequence database searches. These sequences and those of known related strains retrieved from the DNA Data Bank of Japan 434445 were aligned using the CLUSTAL W Multiple Sequence Alignment Program 46. The phylogenetic tree was reconstructed by the neighbour-joining method 47 and was evaluated by bootstrap sampling 48.

Ten grams of each soil sample was suspended in 100 ml of distilled water, shaken for 1 h, and supernatants were obtained by centrifugation. NaCl content in soil samples was calculated by determining Cl content of the soil extract by the method of Mohr 49. The pH of the soil extract was determined with a pH meter. Water content was determined by measuring the decrease of weight after heating 1 g soil sample at 120°C for 1, 2 and 3 hours.