Effect of CO2 on Colony Development by Bifidobacterium Species

This report investigates the requirement for CO₂ for colonyformation by Bifidobacterium species in both anoxic and oxicenvironments. All tested Bifidobacterium species exhibited difficultyin developing colonies in an atmosphere of 100% N₂ but developedwell when 1% CO₂ was present. In the presence of CO₂, the oxygentolerance of the tested species was not improved. In the absenceof CO₂, only B. boum, a microaerophilic species, could developcolonies under an N₂-based 5% O₂ atmosphere, indicating thatwhile CO₂ is not an essential factor for colony development,both CO₂ and O₂ have stimulatory effects on B. boum colony development.

INTRODUCTION

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INTRODUCTION

Bifidobacterium strains, known to be part of the beneficialflora in the human intestinal tract, were originally discoveredby Tissier in 1900 (13), and more than 30 species have sincebeen recognized. Bifidobacterium species are defined as gram-positiveanaerobes, which do not grow under aerobic conditions (4); however,the O₂ tolerance depends on the species (1-6, 11, 12, 14), andsome species are reported to show O₂ tolerance only in the presenceof CO₂ (4, 7-10). The effects of CO₂ on the growth of Bifidobacteriumspecies were tested with several species upon isolation andcharacterization. For most species listed in Bergey's Manualof Systematic Bacteriology, CO₂ does not affect anaerobic growthin 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, andB. thermophilum, grow in stab cultures under air if the airis enriched with 10% CO₂ (7, 8, 10). Although CO₂ is conventionallyused in Bifidobacterium species culture atmospheres, the needfor CO₂ for and the effect of CO₂ concentrations on the growthof Bifidobacterium species under anoxic and oxic conditionshave been essentially unknown.

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

Requirement for CO₂ for growth of Bifidobacterium species.

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Requirement for CO₂...

Several Bifidobacterium species were selected for the observationof the CO₂ effect on colony formation. B. bifidum is the typespecies of the genus Bifidobacterium. B. longum, B. breve, andB. infantis are all commonly used as probiotics to benefit thehuman intestinal tract. B. boum, B. globosum, and B. thermophilumare reported to grow in stab culture under atmospheric conditionsof 90% air-10% CO₂, without the cells' becoming catalase orpseudo-catalase positive (8, 10). B. boum and B. thermophilumwere also determined in our previous study to be microaerophilicspecies (5). Strains were grown at 37°C in modified MRSmedium (without 0.5% sodium acetate) containing 1% (wt/vol)glucose, 1% proteose peptone, 0.2% beef extract, 0.5% yeastextract, 0.2% ammonium citrate, 0.02% MgCl₂, 0.2% K₂HPO₄, and0.005% MnSO₄ as previously described (5). Modified MRS mediumsupplemented with 1.5% agar (Wako Pure Chemical Inc., Osaka,Japan) was autoclaved, and 20-ml aliquots of medium were pouredinto plates (diameter, 9 cm). Before streaking of the individualstrains, medium-containing plates were preincubated in an anaerobicchamber filled with 100% N₂ atmosphere (N₂ gas was renderedO₂ free by passing through an O₂ trapper column [Nikka Seiko,Japan]) for 24 h. The specific preparation of the Bifidobacteriumspecies liquid cultures has been described previously (5). Thetested Bifidobacterium strains were precultured using liquidmedium prior to plating. When a strain had grown to an opticaldensity at 660 nm of 1.0, 20 µl of precultured liquidmedium was dropped onto the medium surface and then streakedby manipulating the sterilized loop needle. Nine to 12 platesfor each strain were reproducibly streaked. After streaking,the medium plates (three plates for each gas condition) wereincubated at 37°C for 48 h under various gas conditionsby using an anaerobic chamber (5-liter volume; Sanshin-Kogyo,Tokyo, Japan). To obtain reliable results, we repeated the experimentstwo to three times on different days. The gas mixtures composedof various concentrations of N₂, CO₂, and O₂ were made usinggas flow meters (Kofloc, Tokyo, Japan), and the gas compositionwas checked by gas chromatography as previously described (5).The pH of the modified MRS medium changed from 6.50 to 6.30after autoclaving at 121°C for 15 min. The medium pH didnot shift upon preincubation under 100% N₂ for 24 h. The mediumpH decreased from 6.30 to 6.29 when the medium was incubatedunder 1% CO₂-99% N₂ for 48 h and from 6.30 to 6.01 upon incubationunder 20% CO₂-80% N₂ for 48 h (the medium pH was measured usingcontrol liquid medium which did not contain agar and cells).Colony development was evaluated by using a number scale asdescribed in the figure legends and Table 1 and averaging numbersfrom repeat experiments.

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TABLE 1. Effect of CO₂ concentration on colony formation by Bifidobacterium species

The assessment of colony formation by each species tested showedthe stimulation of colony development in the presence of 10%CO₂ (Table 1). All of the tested Bifidobacterium species demonstrateddifficulty in developing colonies under 100% N₂. The observedgrowth stimulation was attributed mainly to the enhancementof colony size but not the number of colonies.

To study the effect of CO₂ and O₂ concentrations on the stimulationof colony formation, four Bifidobacterium species were chosenfor further experiments. The growth of these four species underoxic conditions in liquid shaking cultures was tested in ourprevious study, in which B. bifidum and B. longum showed O₂-sensitivegrowth profiles and B. boum and B. thermophilum showed microaerophilicgrowth profiles (5). As shown in Fig. 1, the presence of CO₂strongly stimulated colony formation, and the effects were similarunder 1% CO₂-99% N₂ and 20% CO₂-80% N₂.

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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.

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Effect of O₂ on...

In the absence of 1% CO₂, B. bifidum, B. longum, and B. thermophilumdid not form colonies under an N₂-based 5% O₂ atmosphere (visiblecolonies were undetectable). B. boum showed difficulty in developingcolonies under a 100% N₂ atmosphere but developed well when5% O₂ was present (Fig. 2). This result indicates that CO₂ isnot an essential factor for B. boum colony development if 5%O₂ is present as a substitute for CO₂. O₂ must have an inhibitoryinfluence on the growth of B. bifidum, B. longum, and B. thermophilumwithout acting as a growth stimulator.

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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 notform colonies under conditions of 5% O₂ (Fig. 2). B. boum andB. thermophilum could develop colonies in the presence of 5%to 20% O₂; however, the colony development was increasinglyinhibited as the O₂ concentration increased (visible colonieswere detectable under 15 and 20% O₂, but colony diameters wereless than 0.1 mm) (Fig. 2). These colony development profileswere almost the same in the presence of 10% CO₂ (data not shown).

Conclusions.

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Conclusions.

Several reports have mentioned that CO₂ has no significant effecton the growth of Bifidobacterium species; however, all the researchwas conducted by stab culture (4, 7-10). In this study, we determinedthat the presence of CO₂ is an essential factor for the surfacegrowth of the tested Bifidobacterium strains under anoxic conditions.The role of CO₂ is not so much to improve O₂ tolerance as tostimulate growth. The molecular mechanism of this bifidobacterialCO₂ requirement needs to be clarified by investigating the enzymeinvolved in CO₂ fixation, CO₂ hydration, and carboxylation reactions.The mechanism of O₂ stimulation of B. boum colony developmentmay in fact be similar to that of stimulation by CO₂, becauseCO₂ is the most oxidized form of carbon and may act as an electronacceptor, like O₂, to maintain a cellular redox balance.

ACKNOWLEDGMENTS

We thank Tohru Kodama and Junichi Nakagawa for valuable discussions.We also thank Mitsunori Todoroki and Shingo Tamaru for helpfultechnical assistance at Tokyo University of Agriculture.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biosciences, Tokyo University of Agriculture, 1-1-1 Setagaya-ku, Tokyo 156-8502, Japan. Phone and fax: 81-3-5477-2764. E-mail: kawashin{at}nodai.ac.jp

Published ahead of print on 5 October 2007.

REFERENCES

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