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In the biofilms representing periodontitis, the total counts of bacteria (log₁₀ CFU) differed significantly between the initial pH values at 24 and 48 h (each p < 0.001; Fig. 3a). At 24 h, the lowest values were counted at pH 5 and 5.5 with approximately 5.8–5.9 log₁₀ CFU, while the highest counts were detected at pH 7–8 with a mean of 9.3–9.5 log₁₀ CFU. At 48 h, the lowest CFU counts were 6.3 log₁₀ when the initial pH was 5, and the highest values were 9.3–9.4 log₁₀ (pH 7–8). At both time points, post hoc analysis showed differences between each pH 5, 5.5, 6, and 6.5 on the one hand, and pH 7 on the other (each p < 0.01).

At 24 h, the percentages of T. forsythia and T. denticola were higher at pH 5 and 5.5 in comparison with the initial pH 7 (each p < 0.001). At 48 h, the percentage of both species remained high at pH 5 (both p < 0.001). In contrast, the percentage of P. gingivalis increased with increasing pH. The differences for pH 5–6.5 were all statistically significant versus pH 7 (p < 0.001). Apart from P. gingivalis, T. forsythia, and T. denticola, the other bacteria were present at higher percentages at pH 5 (p = 0.004), pH 5.5 (p = 0.017), pH 6 (p = 0.002), and pH 6.5 (p = 0.012) in comparison with the initial pH 7 (Fig. 3b).

The metabolic activity also showed a dependency to the pH level, increasing with elevated pH values at 24 and at 48 h (Fig. 3c). At both time points, the activities were lowest at pH 5 and 5.5, with each being statistically significantly different to pH 7 (each p < 0.001). At 24 h, the biofilm metabolic activity was higher for the initial pH level of 8 versus pH 7 (p < 0.001).

At 24 h, the biofilm mass was higher for the initial pH level of 6.5 versus pH 7 (p < 0.001). At 48 h, there was a decreased mass for pH 5 and 5.5 in comparison with pH 7 (both p < 0.001; Fig. 3d).

The pH measurements were performed at 24 and 48 h. The gradients were from pH 5.21 and 5.24 (initially pH 5) to pH 7.45 and 7.48 (initially pH 8) at 6 and 24 h. It is of interest to note that the initial pH 7 changed to 7.08 (Table 4).

Table 4. Initial pH values and values (mean ± SD) in the surrounding media after 24 and 48 h of formation of the eight-species biofilm representing “periodontitis”