**3. Results**

Intestinal sulfate-reducing bacteria, *D. piger* Vib-7, showed the highest rate (biomass accumulation, sulfate and lactate consumption, and sulfide and acetate production), both increasing and decreasing trends, until the 60th h of cultivation in the control (3.5 mM of sulfate and 17.3 mM of lactate) medium (Figure 1). The stationary growth phase was achieved after 60 h of cultivation and the following percentage decreases and increases in contents were measured: biomass (increased by 87%), sulfate (decreased by 95%), sulfide (increased by 83%), lactate (decreased by 88%) and acetate (increased by 91%). Relative growth and survival of *D. piger* Vib-7 achieved the highest percentages at 7 mM of sulfate and 35.6 mM of lactate. Higher concentrations than these resulted in the stability of relative growth and it stayed at the same level during 12 to 48 h. Lower concentrations of sulfate (<3.5 mM) and lactate (<17.8 mM) were not enough for the achievement of maximum growth parameters.

**Figure 1.** The growth of *D. piger* Vib-7 and their sulfate reduction.

As can be seen in Figure 2, the consumption of sulfate was highly dependent on its di fferent concentrations in cultivation medium, time of cultivation, and the presence of lactate donors (it was constant at 17.8 mM of lactate).

**Figure 2.** Growth of *D. piger* Vib-7, and their survival and sulfate reduction parameters during 12, 24, 36, and 48 h of cultivation: the effect of electron acceptor (sulfate)/donor (lactate) at different concentrations (columns: first = 12 h, second = 24 h, third = 36 h, fourth = 48 h).

After 12 h, 54% of the sulfate was consumed in medium with lower sulfate concentrations (0.87 mM), although after 48 h, sulfate was almost consumed (98%) at the lowest concentration (0.87 mM) and only 28% at the highest sulfate concentration, where 72% was not used during this time period. Under other conditions, the following changes occurred: di fferent lactate concentrations (4.45 mM, 8.9 mM, 17.8 mM, 35.6 mM, 53.4 mM, or 89 mM) were added in the cultivation medium and the consumption of the sulfate was measured.

As can be seen in Figure 2, sulfate consumption depended not only on its concentration, but was also strongly correlated with the concentration of an electron donor (lactate). Within this environment 14% of the sulfate was used at the lowest lactate concentration (4.45 mM) and 50% at 89 mM of lactate in the medium after 12 h of cultivation. The time of cultivation and lactate concentration increased the sulfate reduction in the medium. After 48 h, sulfate was used only 39–55% at the lowest concentrations of lactate (4.45–8.9 mM) because not enough electron donor was present. However, increasing the lactate concentrations from 35.6 to 89 mM induced 91–98% consumption of sulfate. The same trend was noticed with the lactate consumption. It could be seen that the production of sulfide was not very much influenced by the concentration of electron acceptor (0.87 mM to 17.5 mM), or the electron donor (4.45 mM to 89 mM), in the time interval from 24 to 48 h. The hydrogen sulfide production during this time period was stable. The highest production (78%) of sulfide was accumulated during the first 12 h and gradually decreased to 39%, 29%, and 22%, after 24, 36, and 48 h, respectively, under the conditions of 3.5 mM sulfate and 17.8 mM lactate. A similar trend was noticed in acetate production, although acetate production was more influenced by the sulfate and lactate concentration in the medium, as well as by the cultivation time. The highest production of acetate was until the 36th h of cultivation and after this period it decreased (Figure 2).

Based on di fferent concentrations of electron acceptor and donor, PCA was carried out (Figure 3) that included the separate parameters of biomass, sulfate and lactate consumption, and H2S and acetate production, as well as PCA that included all mentioned parameters.

**Figure 3.** Principal component analysis of the *D. piger* Vib-7 growth and the parameters of sulfate reduction under the e ffect of electron acceptor (sulfate)/donor (lactate) at di fferent concentrations.

PCA that included separate parameters did not show clusters that would indicate a trend observed in Figure 2, but PCA that included all parameters showed that concentrations of 53 mM lactate and 0.87 mM sulfate, 1.75 mM sulfate and 35.6 mM lactate, and 3.5 mM sulfate and 17.3 mM lactate formed separated clusters. These findings indicated that lower concentrations of sulfate were prevailing in an environment with higher concentrations of lactate.

To observe side shifts in the process of sulfate reduction in the intestinal environment, including different concentrations of sulfate and lactate, cross correlation analysis was carried out between the following parameters: biomass and sulfate, biomass and sulfide, biomass and lactate, biomass and acetate, sulfate and sulfide, sulfate and lactate, sulfate and acetate, sulfide and lactate, sulfide and acetate, and lactate and acetate (Figure 4).

**Figure 4.** Cross-correlation analysis between growth (biomass) and sulfate reduction parameters under the effect of electron acceptor (sulfate)/donor (lactate) at different concentrations.

The higher sulfate concentrations resulted in a shift to the left or right side on the Y axis, in comparison to the control sample (red line in Figure 4A), by all parameters, although more significantly by the following parameters: biomass and lactate, biomass and acetate, sulfate and lactate, and sulfate and acetate. Oppositely, lactate concentration effect did not cause similar shifting on the Y axis (Figure 4B).

PCA of the *D. piger* Vib-7 growth and the parameters of sulfate reduction based on cross-correlation analysis clearly showed an isolated cluster of the highest sulfate consumption (17.5 mM) in comparison with other concentrations. This means that bacteria were not able to fully consume these high sulfate concentrations during 48 h of cultivation (Figure 5).

**Figure 5.** Principal component analysis of the *D. piger* Vib-7 growth and the parameters of sulfate reduction based on cross-correlation analysis.

The kinetic parameters of *D. piger* Vib-7 growth under the effect of electron acceptor (sulfate)/donor (lactate) at different concentrations are shown in Table 1.

**Table 1.** Kinetics of *D. piger* Vib-7 growth under the effect of electron acceptor/donor.


Under sulfate concentrations of 10.5 mM the shortest lag phase was measured and specific maximum rate of growth (μmax) was the fastest at 7.0 mM of sulfate. Under electron donor (lactate) concentrations the shortest lag phase and the fastest specific maximum rate of growth were detected at 53.4 mM and 35.6 mM of lactate, respectively.
