Generally, extremophiles have been deemed to survive in the extreme environments to which they had adapted to grow. Recently many extremophiles have been isolated from places where they are not expected to grow. Alkaliphilic microorganisms have been isolated from acidic soil samples with pH 4.0, and thermophiles have been isolated from samples of low temperature. Numerous moderately halophilic microorganisms, defined as those that grow optimally in media containing 0.5–2.5 Molar (3–15%) NaCl, and halotolerant microorganisms that are able to grow in media without added NaCl and in the presence of high NaCl have been isolated from saline environments such as salterns, salt lakes and sea sands. It has tacitly been believed that habitats of halophiles able to grow in media containing more than 20% (3.4 M) are restricted to saline environments, and no reports have been published on the isolation of halophiles from ordinary garden soil samples.
We demonstrated that many halophilic bacteria that are able to grow in the presence of 20% NaCl are inhabiting in non-saline environments such as ordinary garden soils, yards, fields and roadways in an area surrounding Tokyo, Japan. Analyses of partial 16S rRNA gene sequences of 176 isolates suggested that they were halophiles belonging to genera of the family Bacillaceae, Bacillus (11 isolates), Filobacillus (19 isolates), Gracilibacillus (6 isolates), Halobacillus (102 isolates), Lentibacillus (1 isolate), Paraliobacillus (5 isolates) and Virgibacillus (17 isolates). Sequences of 15 isolates showed similarities less than 92%, suggesting that they may represent novel taxa within the family Bacillaceae.
The numbers of total bacteria of inland soil samples were in a range from 1.4 × 107/g to 1.1 × 106/g. One tenth of the total bacteria was occupied by endospore-forming bacteria. Only very few of the endospore-forming bacteria, roughly 1 out of 20,000, are halophilic bacteria. Most of the halophilic bacteria were surviving as endospores in the soil samples, in a range of less than 1 to about 500/g soil. Samples collected from seashore in a city confronting Tokyo Bay gave the total numbers of bacteria and endospores roughly 1000 time smaller than those of inland soil samples. Numbers of halophilic bacteria per gram, however, were almost the same as those of inland soil samples. A possible source of the halophilic endospore originating from Asian dust storms is discussed.
Extremophiles are microorganisms adapted to grow in conditions such as extreme pH, temperature, salinity and absence of oxygen . The representatives are acidophiles (Thiobacillus ferroxidans), alkaliphiles (Bacillus alcalophilus), hyperthermophiles (Thermoproteus tenax), extreme halophiles (Halobacterium salinarum) and methanogens (Methanobacterium formicicum). In general, it has been believed that they survive in the extreme environments to which they had adapted to grow. Many extremophiles, however, have been isolated from places where they are not expected to grow. Alkaliphilic microorganisms were isolated from acidic soil samples with pH 4.0 as well as from neutral and alkaline soil . Thermophiles have been isolated from environments of high temperature and also from samples of lower temperature such as soil, food, animal's excrement and seawater . For example, Bacillus stearothermophilus (now Geobacillus stearothermophilus) and Clostridium thermoautotrophicus (now Moorella thermoautotrophica) were isolated from ordinary soil. Strictly anaerobic bacteria such as methanogens, sulfate-reducers, and homoacetogens were isolated from rice paddies during dry fallow period, arable soils, and even from desert soils [4,5]. Thus, the notion that isolation of an organism from a given environment does not mean that the organism is growing in that environments, but just surviving is now generally accepted.
Halophilic microorganisms are adapted to conditions of high salinity and require a certain concentration of NaCl for their optimum growth [6,7]. They have been isolated from various saline environments such as salt lakes (eg. the Dead Sea, the Great Salt Lake), salterns, solar salts and subsurface salt formation. Extremely halophilic microorganisms require high concentration of NaCl for their growth, with optimum concentrations of 2.5–5.2 M (15–30%). Haloarcula vallismortis and Haloterrigena turkmenica for example, have been isolated from salt pool of Death Valley, California, and saline soil of Turkmenia, respectively [8,9]. Moderate halophiles are defined as those that grow optimally in media containing 0.5–2.5 M (3–15%) NaCl, such as Halomonas maura isolated from a saltern in Morocco, and Marinococcus halophilus isolated from sea sands [10,11]. Halotolerant microorganisms possess the ability to grow in media without added NaCl and also in the presence of high concentrations of NaCl. For example, Halobacillus salinus isolated from a salt lake in Korea is able to grow without added salt and in media containing up to 23% NaCl .
Are halophiles inhabiting non-saline environments such as garden soil, yards and field? Bacillus clarkii, B. agaradhaerens and B. pseudofirmus are examples of halotolerant bacteria isolated from soil samples that were shown to be tolerant up to 16% or 17% NaCl . It has, however, been tacitly believed that habitats of halophiles able to grow in media containing higher concentrations, let's say 20% (3.4 M), are restricted to saline environments [14,15], and 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. In 1980 Onishi et al.  surveyed extensively the occurrence of halophilic bacteria in more or less saline samples collected in Japan. They adopted enrichment culture in a medium containing 4 M (23.4%) NaCl, a customary concentration for the cultivation of Halobacterium spp. They isolated 168 strains finally, but no enrichment was obtained from one third of 287 samples of sea sands and seaweeds collected on seashore. They did not include ordinary garden soil samples. It should be pointed out that a non-pigmented haloarchaeon strain 172P1 (designated later as Natrialba asiatica ) was isolated during their survey from dry beach sands with granular salts attached.
In this study, we defined "halophilic bacteria", for convenience, as microorganisms that form colonies on agar plates of a complex medium with 20% added NaCl, and demonstrated that halophilic bacteria are inhabiting in non-saline environments such as ordinary garden soils, yards, fields and roadways in an area surrounding Tokyo, Japan. Phylogenetic analyses of the isolates suggested that they were halophiles belonging to genera of the family Bacillaceae.
Isolation of halophilic bacteria from soil samples
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).
Figure 1. Collection sites of the 360 soil samples. Red circles; colonies were detected from at least one of the three plates of different pH, black circles; colonies were not detected. A white double circle indicates Tokyo Station, and a white circle indicates Narita Airport.
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 . 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.
Table 1. Strains isolated from ordinary soil samples on agar plates containing 20% NaCl.
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.
Growth range of NaCl concentration and pH in liquid media
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. .
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.
Tentative identification of the isolates by partial 16S rRNA gene sequences
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.
Isolates from the alkaline medium
Ten out of 27 strains showed more than 98% sequence similarities to Bacillus haloalkaliphilus. It was noteworthy that these isolates differed considerably in the range of NaCl for growth; from 0.9–1.7 M to 1.7–4.3 M. Two isolates possessed 97.2 and 94.0% similarities to Filobacillus milosensis, and one isolate was most closely related to Gracilibacillus halotolerans (96.1% similarities). Fourteen other isolates had less than 92% sequence similarities to any deposited sequences. Eight isolates showed 86.9–88.0% similarities to 'Bacillus nitritophilus', and one isolate 87.3% similarity to 'Planococcus psychrotoleratus' but these species have not been validly published. Similarities of complete sequences of the 14 isolates (data not shown) were less than 92%, thus they may represent novel taxa. Out of these 14 isolates, 5 strains were pigmented brown and 2 isolates were yellow.
Table 2. Tentative identification of the isolates by partial 16S rRNA gene sequences.