**3. Results**

The vibrio shape was a dominant shape of the cells, as expected. Though they are very small and thin that makes them very often hard to be observed. These cells were marked as *Desulfovibrio* sp. Due to their characteristic shape, gram negativity and flagellar motility (Figure 2). Very abundant were also cells, oval form. Chain and cluster shaped had cocci that were larger than vibrios, same as some rod shape cells were observed too. Rods have almost similar characteristics as cocci. Not abundantly spirilloid forms of bacteria were present too. They had long shape and were very thin, curved multiple times (maximum twelve curves) (Figure 2A). They had long, polar flagella that are responsible for rapid movement. Gram-negative bacteria only were not only present in SRB cultures isolated from rodents (Figure 2B).

**Figure 2.** Sulfate-reducing bacteria (SRB) mixed culture: native slide ( **A**), Gram staining (**B**), capsule staining ( **C**), DAPI staining ( **D**).

*Desulfotomaculum* is rod-shaped (stained Gram-positive) (representing non-SRB genera in the gut) can be seen in Figure 2C since it has a short rod oval shape. According to the previous microscopic technique, cocci can be encapsulated or not. More often encapsulated cocci are present in pairs. The formation of capsules occurs probably due to a non-favorable environment, such as high hydrogen sulfide concentrations due to sulfate-reducing bacteria presence. It is important to stress out that capsule formation is not defined as SRB characteristic. DAPI (4,6-diamidino-2-phenylindole) staining is compliant with the observations made by the previous technique (Figure 2D). The most abundant was vibrio cell-shape. SRB present in the gu<sup>t</sup> isolate was probably *Desulfovibrio* sp., according to literature data that is describing them as the most frequently isolated species in the intestinal inflammation environment. Cocci were confirmed by DAPI staining since they are significantly brighter and larger than other cells. The findings that DAPI cultures bind to DNA molecules indicate that some oval-shaped have more DNA than others, meaning that they are unrelated to each other. Di fferent sizes of cocci, gained by previous techniques, is supporting this interpretation. These cells were found in multiple isolates because thin rods of exceeding length were found by DAPI staining. These cells represent a common microbiome in the intestines that are capable to survive in conditions designed for SRB cultivation.

The fastest bacterial growth and viability, measured spectrophotometrically OD430 (Figure 3), was detected after 24 h of cultivation at 37 ◦C and pH from 8.0 to 9.0. A significant drop in viability was observed at pH 10. The absence of black precipitate was observed in tubes with Mohr's salt and pH > 10 (Figure 3A). This result is indicating a threshold limit pH ≥ 10 both for sulfate-reducers and other (contaminating) species. The values did not reach zero value but were stabilized at around 30–40% of maximum bacterial growth. It means that bacteria were capable to survive and divide at this pH, reaching an optical density of 0.3. Black precipitate occurred at all pH values, meaning that bacteria can survive a longer time period before starting to metabolize and produce hydrogen sulfide. The changing of color in the tubes at pH 11 and 12 occurred due to basic conditions. It means that the measured values of optical density can be explained by the extreme pH e ffect.

**Figure 3.** Various pH ( **A**) and temperature (**B**) influence on relative viability of SRB cultures.

After 72 h of cultivation bacterial growth of all samples was observed. SRB cultures can grow at various ranges of temperature conditions, not only at 37 ◦C, though the fastest growth occurred at temperature ranges from 37 ◦C to 45 ◦C. Another observation was that cells survived for three days at 50 ◦C and died on the temperatures higher than 60 ◦C and at the temperature of 5 ◦C (no bacterial growth, no hydrogen sulfide production, black precipitate not occurred and low OD430 values were measured. The growth was slow at a temperature of 25 ◦C. The relative viability values of SRB are shown in Figure 3B.

The concentrations of H2S in time change according to cell number, same as their metabolic activity rate. The maximum measured hydrogen sulfide concentrations were measured after 48 h of cultivation (Figure 4). After 48 h of cultivation H2S concentrations decreased due to the decrease in relative substrate concentration in the medium, though H2S can clear out from the medium. H2S is present in a soluble form in the medium and can be released as the gaseous phase (the presence of a bubble under the lid, accompanied by hydrogen sulfide sharp smell) into the environment. Consequently, sulfide concentrations dropped at the beginning of the cultivation. After six hours of cultivation, soluble sulfide was eliminated into gaseous phase and it was a point where the lowest H2S levels were detected. Mixed cultures from mice with ulcerative colitis had 1.39 times higher production of H2S in comparison with samples from healthy mice. The maximal difference was 20 μmol/L after 48 h of cultivation.

**Figure 4.** Amount of hydrogen sulfide in cultivation medium in 72 h.

It should be noted that sulfate-reducing microbial communities from healthy mice and mice with colitis were used only as of the model objects for confirmation of morphology distribution and hydrogen sulfide production in different groups of animals (healthy and with ulcerative colitis). Another part of the study consisted of a literature data overview that was conducted by meta-analysis. This method was used for comparing SRB prevalence in healthy individuals and people with developed inflammatory bowel disease. The occurrence of SRB in a group of healthy people and patients with ulcerative colitis (UC) was studied (Figure 5). The location of the square on the right side means that not healthy people are more likely to experience SRB. A significant difference in the occurrence of SRB in healthy people can be observed in the first study [34]. The other two studies [5,35] already touch the zero effect line at a 95% confidence interval, so there is no significant difference. The diamond can then be seen on the right side. Summary of the studies found that SRB is less common in healthy people than in people with UC.


**Figure 5.** The occurrence of SRB in a group of healthy people and patients with UC.

The production of hydrogen sulfide occurs in the process of dissimilatory sulfate reduction, where tree main enzymes are involved. Since the species of *Desulfovibrio* genus were dominant among SRB in both mice and people with ulcerative colitis, the activity of the enzymes involved in the processes of sulfate reduction in *Desulfovibrio* and other intestinal SRB *Desulfomicrobium* was compared (Figure 6). In the case of enzyme activity in cell-free extracts, it was found that in all cases it had the lower enzymatic activity of *Desulfomicrobium* sp. phosphotransacetylase and pyruvate-ferredoxin activity was more or less the same in *Desulfovibrio* bacteria. Thus, it can be argued that the activity of Na+/K<sup>+</sup> ATPase is the highest of the investigated enzymes in the cell-free extracts of *Desulfovibrio*. Similar results were observed in soluble fractions. The activity of Na+/K<sup>+</sup> ATPase is highest in *Desulfovibrio* than *Desulfomicrobium* in all enzymes examined. In the case of sediment fractions, higher Na+/K<sup>+</sup> ATPase activity was again found in *Desulfovibrio* bacteria and no activity was observed in both *Desulfovibrio* and *Desulfomicrobium* in the other investigated enzymes, phosphotransacetylase, and pyruvate-ferredoxin oxidoreductase.

**Figure 6.** Enzyme activity in *Desulfovibrio* and *Desulfomicrobium*.

Thus, the contribution of sulfate-reducing microbial communities, especially of the *Desulfovibrio* genus, in both groups of healthy people and patients with UC and enzymatic activities of bacterial cells is based on a meta-analysis is obvious. Though, the number of studies is certainly not enough for a stronger conclusion.
