Effect of oxygen, inoculum composition and flow rate on development of mixed-culture oral biofilms

Abstract

The effect of aeration on the development of a defined biofilm consortium of oral bacteria was investigated in a two-stage chemostat system. An inoculum comprising 10 species, including both facultatively anaerobic and obligately anaerobic bacteria, and species associated with oral health and disease, was inoculated into an anaerobic first-stage chemostat vessel. The effluent from this chemostat was linked to an aerated [200 ml CO2/air (5:95, v/v) min-1] second-stage vessel, in which removable hydroxyapatite discs were inserted to allow biofilm formation. Comparisons were made of planktonic and biofilm communities in the aerated second-stage vessel by means of viable counts. Both planktonic and early biofilm communities were dominated by Neisseria subflava, comprising > 40% of total c.f.u. in the fluid phase, and > 80% of c.f.u. in 2 h biofilms. Obligate anaerobes persisted in this mixed culture, and succession in biofilms led them to predominate only after 7 d. Despite the continuous addition of air, the dissolved oxygen tension (dO2) within the culture remained low (< 5% of air saturation), and the redox potential (Eh) was -275 mV. In order to assess the significance of the presence of N. subflava in community development, a subsequent experiment omitted this aerobe from the inoculum, to produce a nine-species culture. The planktonic phase was predominated by three streptococcal species, Prevotella nigrescens and Fusobacterium nucleatum. Biofilms again underwent successional changes, with anaerobes increasing in proportion with time. In contrast to the culture including N. subflava, dO2 was 50-60% of air saturation, and the Eh was +50 mV. In the final experiment, the rate of addition of first-stage culture was reduced to 1/10 of that in the previous experiment, in order to determine whether anaerobes were growing, rather than merely persisting in the aerated culture. The data for the planktonic phase indicated that the anaerobes were growing in aerated (dO2 40-50%, Eh +100 mV) conditions. Once again, anaerobes increased in proportion in older biofilms. The study indicates that mixed cultures can protect obligate anaerobes from the toxic effects of oxygen, both in the biofilm and planktonic modes of growth.