**1. Introduction**

The destination of food remains from the small intestine, together with microbial biomass, is the large intestine, which represents an open system of the small–large intestine axis [1]. This means that the large intestine is a reactor for constant microorganism cultivation [2]. This fact is supported by the calculation that 200 g of digestive material is present in the large intestine of an adult human [2,3]. The intestinal lumen biomass includes almost 55% microorganisms, which are present in the total fecal content [1,4,5]. The microbial mass in the large intestine is 1011–1012 cells/g feces of the following dominant genera: *Bifidobacterium*, *Bacteroides*, *Lactobacillus*, *Escherichia*, *Enterococcus*, *Atopobium*, *Faecalibacterium*, *Clostridium*, and 40 other bacterial species that represent 99% of the colon microbiota [1,4,6,7].

The majority of these bacteria are able to cleave complex organic compounds in the fermentation process and they produce molecular hydrogen, di fferent acids including acetate and lactate, and other compounds. The production of lactate depends on the fermentative properties of lactic acid bacteria (e.g., *Bifidobacterium*, *Lactobacillus*, and *Streptococcus*) [4]. This means that lactate and acetate can be also used by other groups of microorganisms. These compounds can be used as electron donors and carbon sources in the metabolic processes of microorganisms [7–10]. Intestinal microbiota is especially involved in the digestion processes of short-chain fatty acids [4]. The physiology and metabolism of humans is highly dependent on intestinal microorganisms and consequently a ffects human physiological functions and health [1–3,11,12]. On the other hand, another important component of human physiological status is the capability of the intestinal system to absorb sulfate for amino acid development, such as cysteine and methionine, and its regular involvement in assimilation processes. Concentrations of sulfate in the intestine are dependent on human diet since sulfate is present di fferently in di fferent food commodities [13–16]; another factor is that sulfate absorption is done individually in each human, meaning that the total sulfate content in the intestine is highly influenced by eating habits. The importance of daily sulfate intake can be emphasized by the fact that staple food commodities (such as some breads) represent high sulfate sources (>10 μmol/g) as do popular beverages such as beers and wines (>2.5 μmol/g) [13].

The remnants of sulfate that are not absorbed by the intestines and the presence of lactate make a good environment for sulfate-reducing bacteria (SRB) that are regularly found in human and animal intestines [1,4,17–21]. SRB use sulfate as the final electron acceptor in the process of dissimilatory sulfate reduction and form the end product of hydrogen sulfide [22–27]. Di fferent organic compounds, including lactate, can be exogenic electron donors for this process and can be oxidized to acetate [18,28]. *Desulfovibrio* genus is the dominant SRB in the human intestine [5,22]. Previous studies indicated a correlation between the SRB intestinal presence and ailments, such as cholecystitis, brain abscesses, and abdominal cavity ulcerative enterocolitis, making *Desulfovibrio* species an important factor during both mono- and poly-microbial infections of the gastrointestinal tract [2–4,12]. Consequently, the prevalence of SRB in the intestines is dependent on the occurrence of sulfate and lactate presence in the gut. It is also important to stress that the intestinal environment should be monitored due to its influence on SRB since a connection with these bacterial strains and inflammatory bowel diseases (IBD) has been found [1–3,11]. The e ffects of sulfate and lactate at di fferent concentrations on intestinal *Desulfovibrio* species growth and their sulfate reduction parameters have not been well studied.

The aim of this research was to find correlations between di fferent sulfate and lactate concentrations and *Desulfovibrio piger* Vib-7 growth parameters and their dissimilatory sulfate reduction in the small–large intestinal environment.

## **2. Experimental Section**

#### *2.1. Bacterial Culture and Cultivation*

The sulfate-reducing bacteria of the *Desulfovibrio piger* strain Vib-7 was used as the object of the study. This strain was isolated from the human large intestine and identified based on physiological and biochemical properties and sequence analysis of the 16S rRNA gene. The accession number in GenBank is KT881309.1. The strain of SRB was kept in the collection of microorganisms at the Laboratory of Anaerobic Microorganisms of the Department of Experimental Biology at Masaryk University (Brno, Czech Republic).

The bacterial culture was grown in modified liquid Postgate C medium [23] for 72 h at 37 ◦C under anaerobic conditions [29]. The following sodium sulfate concentrations were prepared in medium: 0.87 mM, 1.75 mM, 3.5 mM, 7 mM, 10.5 mM, and 17.5 mM. Di fferent concentrations of electron donors and their e ffect in the medium were determined in the presence of sodium lactate (4.45 mM, 8.9 mM, 17.8 mM, 35.6 mM, 53.4 mM, or 89 mM). The control medium consisted of 3.5 mM sulfate and 17.8 mM lactate. The determination of biomass, sulfate, hydrogen sulfide, lactate, and acetate concentrations were determined after 12, 24, 36, 48, 60, and 72 h.

#### *2.2. Bacterial Biomass Determination*

In total, 1 mL of liquid medium without Mohr's salt in a plastic cuvette was measured in a biophotometer (Eppendorf BioPhotometer ®D30, Hamburg, Germany) as a blank. The same procedure with the bacterial suspension was performed. The optical density (OD340) was always measured before the experiments to provide approximately the same amount of SRB in each experiment [7].

## *2.3. Sulfate Determination*

The sulfate concentration in the liquid medium was measured by turbidimetric method after 12 h intervals of cultivation. In total, 40 mg/<sup>L</sup> BaCl2 solution was prepared in 0.12 M HCl and mixed with glycerol in a 1:1 ratio. The supernatant of the sample was obtained by centrifugation at 5000× *g* at 23 ◦C and 1 mL was added to 10 mL of BaCl2:glycerol solution and carefully mixed. The absorbance of the mixed solution was measured after 10 min at 520 nm (Spectrosonic Genesis 5, Ecublens, Switzerland). A cultivation medium without bacteria growth was used as a control [30].

#### *2.4. Hydrogen Sulfide Determination*

The concentration of hydrogen sulfide was determined in cultivation medium after di fferent time intervals. In total, 1 mL of the sample was added to 10 mL ofa5g/L solution of zinc acetate and 2 mL of 0.75 g/mL p-aminodimethylaniline in a solution of sulfuric acid (2 M). The mixture stood for 5 min at room temperature. After that, 0.5 mL of 12 g/<sup>L</sup> solution of ferric chloride dissolved in 15 mM sulfuric acid was added. After standing another 5 min at room temperature, the mixture was centrifuged 5000× *g* at 23 ◦C. The absorbance of the mixture was determined at a wavelength of 665 nm by a spectrophotometer (Cecil Aquarius CE 7200 Double Beam Spectrophotometer, London, UK) [31,32].

#### *2.5. Lactate and Acetate Determination*

The measurement was repeated in the same manner using a cultivation medium and it served as the control sample. Measurements of lactate concentration using a lactate assay kit (Sigma-Aldrich, Catalog Number MAK064, Prague, Czech Republic) were carried out. Accumulation of acetate ions in the process of bacterial growth in the medium was determined using the acetate assay kit (Abnova, Colorimetric, Catalog Number KA3764, Prague, Czech Republic).

## *2.6. Statistical Analysis*

Using the experimental data, the basic statistical parameters (M—mean, m—standard error, M ± m) were calculated. The accurate approximation was when *p* ≤ 0.0533 [33]. Statistical significance was measured with the use of principal component analysis (PCA) that gave overall di fferences among compared groups. Statistical analysis was done by SPSS 20 statistical software (IBM Corporation, Armonk, NY, USA). Plots were built by software package Origin7.0 (Northampton, UK).
