Effect of CO₂ on Colony Development by Bifidobacterium Species

Abstract

This report investigates the requirement for CO₂ for colony formation by Bifidobacterium species in both anoxic and oxic environments. All tested Bifidobacterium species exhibited difficulty in developing colonies in an atmosphere of 100% N₂ but developed well when 1% CO₂ was present. In the presence of CO₂, the oxygen tolerance of the tested species was not improved. In the absence of CO₂, only B. boum, a microaerophilic species, could develop colonies under an N₂-based 5% O₂ atmosphere, indicating that while CO₂ is not an essential factor for colony development, both CO₂ and O₂ have stimulatory effects on B. boum colony development.

Bifidobacterium strains, known to be part of the beneficial flora in the human intestinal tract, were originally discovered by Tissier in 1900 (13), and more than 30 species have since been recognized. Bifidobacterium species are defined as gram-positive anaerobes, which do not grow under aerobic conditions (4); however, the O₂ tolerance depends on the species (1-6, 11, 12, 14), and some species are reported to show O₂ tolerance only in the presence of CO₂ (4, 7-10). The effects of CO₂ on the growth of Bifidobacterium species were tested with several species upon isolation and characterization. For most species listed in Bergey's Manual of Systematic Bacteriology, CO₂ does not affect anaerobic growth in stab culture; exceptions include B. angulatum and B. asteroides, the growth of which was previously reported to be highly stimulated (4, 9). It was also reported that some Bifidobacterium species, such as B. indicum, B. asteroides, B. globosum, B. boum, and B. thermophilum, grow in stab cultures under air if the air is enriched with 10% CO₂ (7, 8, 10). Although CO₂ is conventionally used in Bifidobacterium species culture atmospheres, the need for CO₂ for and the effect of CO₂ concentrations on the growth of Bifidobacterium species under anoxic and oxic conditions have been essentially unknown.

In this study, we investigated the effect of CO₂ on colony development by using several Bifidobacterium species. The main objectives of the present study were to (i) determine the CO₂ effect on colony formation under anoxic conditions and (ii) investigate the effect of CO₂ on O₂ tolerance.

Requirement for CO₂ for growth of Bifidobacterium species.

Several Bifidobacterium species were selected for the observation of the CO₂ effect on colony formation. B. bifidum is the type species of the genus Bifidobacterium. B. longum, B. breve, and B. infantis are all commonly used as probiotics to benefit the human intestinal tract. B. boum, B. globosum, and B. thermophilum are reported to grow in stab culture under atmospheric conditions of 90% air-10% CO₂, without the cells' becoming catalase or pseudo-catalase positive (8, 10). B. boum and B. thermophilum were also determined in our previous study to be microaerophilic species (5). Strains were grown at 37°C in modified MRS medium (without 0.5% sodium acetate) containing 1% (wt/vol) glucose, 1% proteose peptone, 0.2% beef extract, 0.5% yeast extract, 0.2% ammonium citrate, 0.02% MgCl₂, 0.2% K₂HPO₄, and 0.005% MnSO₄ as previously described (5). Modified MRS medium supplemented with 1.5% agar (Wako Pure Chemical Inc., Osaka, Japan) was autoclaved, and 20-ml aliquots of medium were poured into plates (diameter, 9 cm). Before streaking of the individual strains, medium-containing plates were preincubated in an anaerobic chamber filled with 100% N₂ atmosphere (N₂ gas was rendered O₂ free by passing through an O₂ trapper column [Nikka Seiko, Japan]) for 24 h. The specific preparation of the Bifidobacterium species liquid cultures has been described previously (5). The tested Bifidobacterium strains were precultured using liquid medium prior to plating. When a strain had grown to an optical density at 660 nm of 1.0, 20 μl of precultured liquid medium was dropped onto the medium surface and then streaked by manipulating the sterilized loop needle. Nine to 12 plates for each strain were reproducibly streaked. After streaking, the medium plates (three plates for each gas condition) were incubated at 37°C for 48 h under various gas conditions by using an anaerobic chamber (5-liter volume; Sanshin-Kogyo, Tokyo, Japan). To obtain reliable results, we repeated the experiments two to three times on different days. The gas mixtures composed of various concentrations of N₂, CO₂, and O₂ were made using gas flow meters (Kofloc, Tokyo, Japan), and the gas composition was checked by gas chromatography as previously described (5). The pH of the modified MRS medium changed from 6.50 to 6.30 after autoclaving at 121°C for 15 min. The medium pH did not shift upon preincubation under 100% N₂ for 24 h. The medium pH decreased from 6.30 to 6.29 when the medium was incubated under 1% CO₂-99% N₂ for 48 h and from 6.30 to 6.01 upon incubation under 20% CO₂-80% N₂ for 48 h (the medium pH was measured using control liquid medium which did not contain agar and cells). Colony development was evaluated by using a number scale as described in the figure legends and Table 1 and averaging numbers from repeat experiments.

TABLE 1.

Effect of CO₂ concentration on colony formation by Bifidobacterium species

Strain	Colony formationa under:
	100% N2	10% CO2-90% N2
B. bifidum JCM1255T	+1	+2
B. boum JCM1211T	+1	+4
B. breve JCM1192T	+1	+4
B. globosum JCM7089	+1	+4
B. infantis JCM1222T	+1	+4
B. longum JCM1217T	+1	+4
B. thermophilum JCM1207T	+1	+4

^a+1, tiny colony (visible colonies were detectable [colony diameter, less than 0.1 mm]); +2, small colony (colony diameter, approximately 0.1 mm); +4, large colony (colony diameter, approximately 1 mm).

The assessment of colony formation by each species tested showed the stimulation of colony development in the presence of 10% CO₂ (Table 1). All of the tested Bifidobacterium species demonstrated difficulty in developing colonies under 100% N₂. The observed growth stimulation was attributed mainly to the enhancement of colony size but not the number of colonies.

To study the effect of CO₂ and O₂ concentrations on the stimulation of colony formation, four Bifidobacterium species were chosen for further experiments. The growth of these four species under oxic conditions in liquid shaking cultures was tested in our previous study, in which B. bifidum and B. longum showed O₂-sensitive growth profiles and B. boum and B. thermophilum showed microaerophilic growth profiles (5). As shown in Fig. 1, the presence of CO₂ strongly stimulated colony formation, and the effects were similar under 1% CO₂-99% N₂ and 20% CO₂-80% N₂.

FIG. 1.

Effect of CO₂ concentration on colony formation by Bifidobacterium species. After being streaked with Bifidobacterium species, plates were incubated for 48 h under conditions of 100% N₂, 1% CO₂-99% N₂, and 20% CO₂-80% N₂. Although B. bifidum showed difficulty in developing colonies under almost all culture conditions, it grows well in liquid MRS medium, as previously described (5). Colonies shown were scored as follows: +1, tiny colony (visible colonies were detectable [colony diameter, less than 0.1 mm]); +2, small colony (colony diameter, approximately 0.1 mm); and +4, large colony (colony diameter, approximately 1 mm).

Effect of O₂ on colony development in the absence or presence of 1% CO₂.

In the absence of 1% CO₂, B. bifidum, B. longum, and B. thermophilum did not form colonies under an N₂-based 5% O₂ atmosphere (visible colonies were undetectable). B. boum showed difficulty in developing colonies under a 100% N₂ atmosphere but developed well when 5% O₂ was present (Fig. 2). This result indicates that CO₂ is not an essential factor for B. boum colony development if 5% O₂ is present as a substitute for CO₂. O₂ must have an inhibitory influence on the growth of B. bifidum, B. longum, and B. thermophilum without acting as a growth stimulator.

FIG. 2.

Effect of CO₂ and O₂ on colony formation by Bifidobacterium species. After the medium was streaked with Bifidobacterium species, the plates were incubated for 48 h under conditions of 5% O₂-95% N₂, 5% O₂-1% CO₂-94% N₂, 1% CO₂-99% N₂, and 10% O₂-1% CO₂-89% N₂. Plates and colonies shown were scored as follows: ±, visible colonies were not detectable; +1, tiny colony (visible colonies were detectable [colony diameter, less than 0.1 mm]); +2, small colony (colony diameter, approximately 0.1 mm); +3, medium-sized colony (colony diameter, approximately 0.5 mm); +3.5, colonies evaluated as +3 and +4 in repeat experiments; +4, large colony (colony diameter, approximately 1 mm).

In the presence of 1% CO₂, B. bifidum and B. longum could not form colonies under conditions of 5% O₂ (Fig. 2). B. boum and B. thermophilum could develop colonies in the presence of 5% to 20% O₂; however, the colony development was increasingly inhibited as the O₂ concentration increased (visible colonies were detectable under 15 and 20% O₂, but colony diameters were less than 0.1 mm) (Fig. 2). These colony development profiles were almost the same in the presence of 10% CO₂ (data not shown).

Conclusions.

Several reports have mentioned that CO₂ has no significant effect on the growth of Bifidobacterium species; however, all the research was conducted by stab culture (4, 7-10). In this study, we determined that the presence of CO₂ is an essential factor for the surface growth of the tested Bifidobacterium strains under anoxic conditions. The role of CO₂ is not so much to improve O₂ tolerance as to stimulate growth. The molecular mechanism of this bifidobacterial CO₂ requirement needs to be clarified by investigating the enzyme involved in CO₂ fixation, CO₂ hydration, and carboxylation reactions. The mechanism of O₂ stimulation of B. boum colony development may in fact be similar to that of stimulation by CO₂, because CO₂ is the most oxidized form of carbon and may act as an electron acceptor, like O₂, to maintain a cellular redox balance.