Versatile Solidified Nanofibrous Cellulose-Containing Media for Growth of Extremophiles

Preparation of cellulose plate.

Plates of nanofibrous cellulose were prepared as previously described (4). Cellulose (Funacel SF from Funakoshi, Tokyo, Japan, or microcrystalline cellulose, for thin-layer chromatography, from Merck, Darmstadt, Germany) was dispersed in a saturated aqueous solution of Ca(SCN)₂·4H₂O (Wako Pure Chemical, Osaka, Japan) at a concentration of 3% (by weight). Twenty-milliliter aliquots of the dispersion were placed in glass culture dishes and heated to 121°C for 1 min in an autoclave to dissolve the cellulose. The solutions were allowed to solidify at room temperature overnight. Ca(SCN)₂ was removed from the solidified plates by washing the plates with methanol and then a copious amount of water. The cellulose plates thus obtained were highly porous materials that consisted of nanofibers (20 to 50 nm in width) of crystalline cellulose and had a typical pore size on the order of several hundred nanometers (4). The cellulose plates were autoclaved at 121°C for 20 min, and then 20 ml of a double-strength (2×) medium was laid on top of the plates. The nutrient components were allowed to diffuse into the pores of the cellulose plates with gentle agitation on an orbital shaker for 4 h, after which the excess supernatant was discarded.

Stability of cellulose plate under extreme culture conditions.

Cellulose, agar, and gellan gum (often referred to by the trade name Gelrite) plates containing Luria-Bertani (LB) broth, nutrient broth (NB), or soybean casein digest (SCD) broth at three different pH values (2, 7, and 12) were prepared. The pH was adjusted with H₂SO₄ or NaOH-Na₂CO₃. The plates were kept in an incubator set to 60, 70, or 80°C and examined by visual inspection for syneresis, softening, or melting. The stability of agar and gellan gum plates depended strongly on the type of medium, temperature, and pH. Only LB-agar and SCD-agar plates at pH 7 remained solid at 70°C. Gellan gum plates showed better stability: LB-gellan gum at pH 2, 7, and 12 and SCD-gellan gum at pH 7 and 12 remained solid at 70°C. However, none of them remained solid at 80°C. The NB-gellan gum medium did not solidify even at room temperature, probably because the concentrations of divalent cations in NB were too low to induce solidification. To develop agar- or gellan gum-supported media for these conditions, the medium formulations, such as agar concentration, had to be modified empirically to ensure solidification (14). In contrast, the cellulose plates remained solid under all conditions tested.

Culture of extremophiles on cellulose media.

Various extremophiles, including acidophilic Acidiphilium acidophilum ATCC 27807^T, alkaliphilic Bacillus clarkii DSM8720^T, thermophilic Geobacillus stearothermophilus ATCC 12016 and Thermus thermophilus JCM10941^T, acidothermophilic Sulfolobus acidocaldarius DSM639^T, and alkalithermophilic Bacillus sp. strain TX-3 (JCM9162) (13), were cultured on the cellulose plates. Culture media and culture conditions are given in Table 1. The plates were inoculated from liquid cultures and incubated for 1 to 10 days (see Table 1 for details). Conventional agar plates and/or gellan gum plates containing the same nutrients were used for control experiments. In culturing thermophilic microorganisms, care was taken to prevent water evaporation. The lid and body of the culture dish were sealed with adhesive tape, wrapped with a wet paper towel, and sealed in a zip-lock bag during incubation at high temperature.

Culture of an acidophile and an alkaliphile.

Hydrolysis of agar in acidic media and the toxicity of hydrolysates prevented growth of A. acidophilum on the agar-solidified medium (10, 12). In contrast, visible colonies of A. acidophilum were formed on the cellulose and gellan gum plates after incubation for 8 days, showing their chemical inertness in the acidic environment. In the case of alkaliphilic B. clarkii, no difference was observed in the number of colonies formed on the cellulose and agar plates.

Culture of thermophiles.

G. stearothermophilus grew as well on nutrient cellulose at pH 6.8 and 55°C as it did on nutrient agar. The cellulose plate could also be used successfully for culturing thermophilic T. thermophilus at 80°C (Fig. 1A). At this temperature, agar and gellan gum media do not remain solid (4). T. thermophilus formed colonies on a TM medium (JCM medium 273) solidified by gellan gum and a magnesium gelling aid (14), but the effect of syneresis was evident, leading to irregular colony shapes and merger of neighboring colonies (Fig. 1B).

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FIG. 1.

(A and B) Colonies of T. thermophilus formed on a cellulose plate containing TM broth (A) and a gellan gum plate containing TM broth plus 0.1% MgCl₂·6H₂O (B). Images were taken after incubation at 80°C for 2 days. Syneresis prevented formation of well-isolated colonies on the gellan gum plate. Scale bar represents 1 cm. (C and D) Colonies of T. thermophilus formed on a cellulose plate containing TM broth (C) and TM broth plus 0.1% CaCl₂·2H₂O (D). Images were taken after incubation at 80°C for 4 days. Scale bar represents 5 mm. (E and F) Optical micrographs showing colonies of S. acidocaldarius formed on the cellulose plate (E) and the gellan gum plate (F) after incubation for 7 days at pH 2 and 80°C. Scale bar represents 1 mm.

We also examined the effects of magnesium and calcium gelling aids on the growth of T. thermophilus because the addition of a gelling aid negatively affects microbial growth in some cases (14). T. thermophilus was cultured at 80°C for 4 days on cellulose plates that contained TM broth, TM broth plus 0.1% MgCl₂·6H₂O, or TM broth plus 0.1% CaCl₂·2H₂O. Culturing T. thermophilus on solidified TM medium that was free from a gelling aid was made possible only by using cellulose.

T. thermophilus formed yellow circular colonies on a TM-cellulose plate (Fig. 1C). The pigmentation was so intense that the entire plate became yellowish after incubation for 4 days. The pigmentation is due to the formation of carotenoids and is one of the important phenotypic characteristics of Thermus strains (22). Interestingly, the pigmentation became less intense when the cellulose plate was supplemented with 0.1% MgCl₂·6H₂O, although the colony shape remained unaffected (data not shown). T. thermophilus formed nonpigmented umbonate colonies (Fig. 1D) on the cellulose plate that contained 0.1% CaCl₂·2H₂O. The results show that the color and shape of T. thermophilus colonies are significantly affected by the type of gelling aid.

Culture of an acidothermophile and an alkalithermophile.

Acidothermophilic S. acidocaldarius formed colonies on both cellulose and gellan gum plates at pH 2 and 80°C, although the colonies were smaller than 300 μm in diameter and difficult to enumerate accurately (Fig. 1E and F). Close examination revealed significant effects of syneresis only on the colonies on the gellan gum plate. Alkalithermophilic Bacillus sp. strain TX-3 was cultured successfully on the cellulose plate at pH 10 and 55°C. Colony formation was also confirmed at 65°C with cellulose, whereas culture on the agar plate met with sporadic success due to syneresis. Gellan gum was unusable because the addition of a calcium or magnesium gelling aid to alkaline NB resulted in precipitation.

Conclusions.

Solidified media using nanofibrous cellulose plates can be used for culturing various extremophiles. The cellulose-supported media have several advantages over conventional solidified media using agar or gellan gum (9, 14) for culture of extremophiles, including versatility, stability, and ease of preparation. First, essentially any medium can be solidified with the cellulose plate, regardless of the culture conditions, while the solidification of conventional solidified media depends strongly on the composition of the medium, such as the concentrations of solidifying agent and gelling aids (11, 16, 17). The cellulose plate, for example, allows the preparation of solidified media for extremophiles that contain very dilute and low-nutrient fluids. Various previously uncultured microorganisms have been propagated successfully by using such dilute, low-nutrient media (3, 19, 23). However, solidifying such media with agar or gellan gum for use under extreme culture conditions is difficult. Second, the stability of the cellulose plate is unparalleled. It remains unchanged even at temperatures much higher than the known upper temperature limit for life (122°C) (20). Because oxygen does not affect the stability of the cellulose plate, it may be possible to use the plates to culture anaerobic hyperthermophiles at temperatures at or above 100°C, although this has not been tested. Third, syneresis of the cellulose plate is negligible. Finally, preparation of the cellulose medium obviates such hassles as handling very hot solutions, which is often necessary for preparing gellan gum medium (1).

One drawback may be that preparation of the cellulose plate is somewhat time-consuming compared with reported solidified media. However, unlike conventional solidified media, the cellulose plate can be reused by treating the used plate with 2% (by weight) aqueous NaOH at 120°C for 20 min (4).