Effect of growth conditions on VSC production by oral biofilms

Objectives: To investigate the effect of media composition and growth conditions on VSC production by a perfused model system (sorbarod) comprising a tongue microcosm biofilm.

Methods: Inoculum was generated from 1 subject using a sterile toothbrush brushed firmly against the posterior dorsum of the tongue. Sterile sorbarod filters were inoculated and incubated for 24 hours anaerobically to allow bacterial attachment. Sorbarod filters were then attached to the model system and biofilms cultivated for a further 24 hours with continuous perfusion of gas (50ml/hour) and media (36ml/hour). Media comprised 1/5^th strength BHI supplemented with 0.001%w/v dithiothreitol, 0.01%w/v L-cysteine and 0.0001%w/v haemin. The effect on VSC generation of decreasing L-cysteine and changing gas source from anaerobic gas (80% nitrogen, 10% carbon dioxide, and 10% hydrogen) to sterile lab air was investigated. Sorbarods were run in triplicate. Biofilm gas samples were analysed for VSCs using gas chromatography with flame photometric detection.

Results: Under normal growth conditions, VSCs generated by 48h biofilms were: 13025±2460ppb for hydrogen sulfide and 6144±3114ppb for methyl mercaptan. Omitting L-cysteine from the growth medium approximately halved hydrogen sulfide production (to 6959±2674ppb). In contrast, methyl mercaptan production increased almost 3-fold (to 17609±3789ppb). Changing the gas source from sterile anaerobic gas to filtered lab air resulted in large reductions in both hydrogen sulfide (to 3739±2370ppb) and methyl mercaptan (to 2893±1705ppb) production.

Conclusion: Changes in growth conditions caused dramatic alterations to VSC production probably through effects on VSC precursor (cysteine) levels and/or the microcosm species composition (gas sparging). Careful control of growth conditions is essential for development of the model as an anti-malodour screening tool.