DOSE-DEPENDENT EFFECT OF ELECTRON ACCEPTOR AND DONOR ON DISSIMILATORY SULFATE REDUCTION BY BACTERIA DESULFOVIBRIO PIGER VIB-7 OF HUMAN INTESTINE

Growth of Desulfovibrio piger Vib-7 bacteria of human intestine under the influence of the electron acceptor and donor in different concentrations and the parameters of dissimilatory sulfate reduction have been studied. An increased intensity of the bacterial growth and production of hydrogen sulfide by the D. piger Vib-7 bacteria was studied under influence of high concentrations of sulfate and lactate (17.4 and 35.6 mM, respec-tively). The largest quantity of hydrogen sulfide (6.06 mM) under influence of 10.5 mM of was detected on the 72 hour of cultivation; while the bacteria D. piger Vib-7 used about 58% of sulfate ion. The largest quantity of acetate (21.1 mM) is detected on the 72 the hour of growth during cultivating the D. piger Vib-7 in the medium containing 53.4 mM of lactate. Based on the obtained experimental data surface models of sulfate reduction parameters by D. piger Vib-7 under the influence of electron acceptor and donor different concentrations were constructed. These surface models have provided an opportunity to confirm and establish optimum growing points by the studied bacteria, their sulfate consumption and sulfide production, the lactate consumption and acetate accumulation. The correlation coefficients ( r ) between parameters of the dissimilatory sulfate reduction by the D. piger Vib-7 were defined. Strong negative and positive correlations between these parameters have been observed.


INTRODUCTION
Large intestine is an open system which receives the remains of food from the small intestine together with microbial biomass. It is a system of continuous cultivation of microorganisms [2,10]. Large intestine of adult human contains more than 200 g of digestive material. The average daily output of faeces is approximately 120 g [6,11].
The intestinal biomass contains up to 55% microorganisms of the total microbial content of faeces people [6,7,13]. In the large intestine microbial mass is 10 11 -10 12

MATERIAL AND METHODS
The objects of the study were sulfate-reducing bacteria of the Desulfovibrio piger strain Vib-7 obtained from the human large intestine [12]. The strain is kept in the collection of microorganisms at the Biotechnology Laboratory of the Pharmacy Faculty at the University of Veterinary and Pharmaceutical Sciences Brno (Czech Republic).
To determine the effect of different concentrations of electron acceptor in the culture medium additionally was added various amounts of Na 2 SO 4 , final media concentrations of the were 0,87 mM, 1.75 mM, 3.5 mM, 7 mM, 10.5 mM or 17.5 mM. In addition, to study the effect of different concentrations of electron donor in the medium additionally was added various amounts C 3 H 5 O 3 Na, final media concentrations of the lactate were 4.45 mM, 8.9 mM, 17.8 mM, 35.6 mM, 53.4 mM or 89 mM . Control were medium which contained 3.5 mM and 17.8 mM . Accumulation biomass of sulphate-reducing bacteria in liquid medium (without Mohr's salt) was determined by turbidity of dilute suspension of cells by photometric method [16].
The sulfate ions concentration in the medium was determined by turbidymetric method after it had been precipitated by barium chloride. To stabilize the suspension, glycerol was used [9].
Hydrogen sulfide in the culture medium was photometrically determined using spectrophotometer (λ=665 nm, cuvette with optical path 30 mm). The reaction mixture had the following composition: zinc citrate (27.3 mM) -10 ml; distilled water -1.98 ml; n-aminodimethylaniline solution (5.5 mM) -4 ml, and 20 µl of test solution. After 5 min, 1 ml of ferric chloride (0.125 M) was added and methylene blue formation was observed. The concentration of hydrogen sulfide by calibration curve was established.
Determination of lactate concentration was carried out through a dehydrogenation reaction of lactate by lactate dehydrogenase in the presence of NAD + , with formation of pyruvate and NADH. For determination of lactate content the following reagents were used: hydrazine-glycine buffer pH 9.0 (glycine -0.1 M solution containing 0.1 M hydrazine); NAD + -0.03 M solution, pH 6.0; lactate dehydrogenase solution (protein content was about 2 mg/ml). The samples of glycine and hydrazine were dissolved in a small amount of distilled water, a pH of 9.0 was maintained by a 2 N NaOH solution, then the mixture was diluted by distilled water to 100 ml. Solutions of NAD + and lactate dehydrogenase were kept on ice. The content of the tubes was thoroughly mixed and placed inside a thermostat at +25°C for 60 min. After incubation, samples were cooled and then the optical density of the samples was measured at 340 nm. The quantity of the lactic acid was subsequently calculated [17].
Accumulation of acetate ions by the bacterial cells during their growth in the medium was determined by titration [3].
Using the experimental data, the basic statistical parameters (M -mean, m -standard error, M±m) have been calculated. For the estimation of the reliability between the statistical characteristics, Student's t-test was used. The difference was reliable when P≥0.95 [1]. Statistical processing of the results was performed using packet Excel and Origin computer programs [8].
The correlation analysis of the parameters was carried using packet Excel program. The main result of a correlation is called Pearson's correlation coefficient (or "r"). It is best represents the contemporary use of the simple correlation that assesses the linear relationship between two variables. The coefficient indicates the strength of the relationship, with values ranging from 0 to 1 in absolute value. The larger the magnitude of the coefficient, the stronger the relationship between the variables. The sign of the coefficient indicates the direction of the relationship as null, positive, or negative. A null relationship between variables X and Y suggests that an increase in variable X is accompanied with both an increase and a decrease in variable Y and vice versa [4].

RESULTS AND DISCUSSION
The studied sulfate-reducing bacteria the Desulfovibrio piger Vib-7 actively reduced of sulfate and produced of hydrogen sulfide (up to 3.16 mM) as well as they assimilated of lactate and accumulated of acetate (up to 15.63 mM) in the control (3.5 mM of sulfate and 17.3 mM of lactate) medium ( Fig. 1, 2).
Increasing the concentration of sulfate ion as electron acceptor in two (7.0 mM), three (10.5 mM) or five (17.5 mM) times in the culture medium leads to actively growth and producing of hydrogen sulfide by the studied D. piger Vib-7 (Fig. 1).
The stimulation of sulfate reduction is not occurred proportional to the increase of sulfate concentration in the medium. The largest quantity of hydrogen sulfide (6.06 mM) under influence of 10.5 mM of was detected on the 72 hour of cultivation; while the bacteria D. piger Vib-7 used about 58% of sulfate ion. Perhaps, the rest (42%) of the electron acceptor was in excess and the bacteria could not assimilate such number of sulfate. Under these conditions the D. piger Vib-7 acumulated up to 5.64 g/l of biomass. The increasing of the sulfate ion concentration up to 17.5 mM (in five times) in the medium is not caused biomass increasing and stimulation of hydrogen sulfide production compared to 10.5 mM of sulfate concentration. Perhaps, the dissimilatory sulfate reduction process can limit other factors such as the presence of electron donor in the medium. Moreover, the decrease in the concentration of sulfate up to 1.75 mM and 0.87 mM caused a significant biomass decrease (up to 0.88 and 1.95 g/l, respectively) and process of sulfate reduction by the D. piger Vib-7. Possibly, the low concentrations of electron acceptor were not insufficient for the sulfate-reducing bacteria of the human intestine.
The increase of lactate concentration in two (35.6 mM), three (53.4 mM) or five (89 mM) times stimulated acetate production in the medium by the studied D. piger Vib-7 (Fig. 2). The increase of lactate concentration did not lead to a proportional increase of sulfate reduction parameters as was in the case with the increasing of sulfate concentration in the medium. The largest quantity of acetate (21.10 mM) was detected on the 72 hour of cultivation during cultivating the D. piger Vib-7 in the medium which contained 53.4 mM of lactate. The bacteria used about 41% of lactate from the medium under these conditions. Perhaps an excess of lactate in the medium has not been used by the studied bacteria. The highest biomass (7.76 g/l) was detected when bacterial culture D. piger Vib-7 grow in the medium which contained 35.6 mM of lactate. Increasing the concentrations of lactate up to 53.4 and 89 mM in the culture medium did not contribute to increasing level of hydrogen sulfide and growth of the studied sulfate-reducing bacteria.
The decrease in the concentrations of lactate up to 8.9 and 4.45 mM caused the biomass decrease and sulfate reduction parameters as was in the case with the decreasing of sulfate concentration in the medium.
Thus, increasing concentrations of the electron acceptor (sulfate) up to 10.5 mM and electron donor (lactate) up to 53.4 mM in the culture medium contributed to the activation process of the dissimilatory sulfate reduction by the studied D. piger Vib-7. ). Five main matrices for sulfate reduction parameters were obtained (Table 1). The sizes of obtained matrices were 7×7 for biomass, sulfide and acetate production and 6×7 for sulfate and lactate consumption.
Based on these matrices were constructed surface models of sulfate reduction parameters by D. piger Vib-7 under influence of electron donor and acceptor different concentrations (Fig. 3). The obtained surface models show that bacteria D. piger Vib-7 intensively grown in the media containing increased concentrations of electron acceptor and donor (sulfate and lactate, respectively). The most intense sulfate reduction process occurs at concentrations within 3.  Thus, the constructed surface models have provided an opportunity to confirm and establish the optimum growing point by the studied bacteria, their sulfate consumption and sulfide production, and the lactate consumption and acetate accumulation.
Obviously, the increased concentration of electron acceptor and donor can cause intense growth of the studied bacteria D. piger Vib-7 as well as intensive accumulation of sulfide and acetate in the human intestine. These conditions may be the cause of ulcerative colitis and in turn bowel cancer. Hydrogen sulfide has negatively affects on the intestinal mucosa, it is toxic to epithelial cells, specifically inhibits the growth of the colonocytes [2,11], phagocytosis, causes the death of intestinal bacteria [5,6,14], induces hyperproliferation and metabolic abnormalities of epithelial cells [2]. It was found that the high level of this metabolite and the presence of SRB significantly increased during inflammation of the colon [5,13,15]. Therefore, regulation of the concentration of hydrogen sulfide in the colon is very important to maintain the integrity of the colonocytes [2,11].
A correlation analysis is related in the sense that both deal with relationships among variables. The correlation coefficient is a measure of linear association between two variables [4]. Therefore, the next task of the study was perform the correlation analysis between parameters of dissimilatory sulfate reduction.
The correlation coefficients (r) between the parameters by the D. piger Vib-7 were defined ( Table 2). Between biomass and sulfate; biomass and lactate; sulfate and sulfide; sulfate and acetate; lactate and acetate; as well as lactate and sulfide a strong inversely negative correlation has been demonstrated. Between biomass and sulfide; biomass and acetate; lactate and sulfate; acetate and sulfide a strong positive correlation has been demonstrated.
The correlation coefficient ranges from -1.0 to +1.0. The closer r is to +1 or -1, the more closely the two variables are related. If r is close to 0, it means there is no relationship between the variables. If r is positive, it means that as one variable gets larger the other gets larger. If r is negative it means that as one gets larger, the other gets smaller (often called an "inverse" correlation). While correlation coefficients are normally reported as r = (a value between -1 and +1), squaring them makes then easier to understand. Values between 0.7 and 1.0 (-0.7 and -1.0) indicate a strong positive (negative) linear relationship via a firm linear rule [4].

CONCLUSIONS
Increasing the concentrations of sulfate and lactate up to 17.4 mM and 35.6 mM respectively in the culture medium leads to actively growth and producing hydrogen sulfide by the studied D. piger Vib-7. The largest quantity of hydrogen sulfide (6.06 mM) under influence of 10.5 mM of was detected on the 72 hour of cultivation; while the bacteria D. piger Vib-7 used about 58% of sulfate ion. The largest quantity of acetate (21.10 mM) was detected on the 72 hour of cultivation during cultivating the D. piger Vib-7 in the medium which contained 53.4 mM of lactate.
Based on obtained results matrices of surface models of sulfate reduction parameters by D. piger Vib-7 under influence of electron donor and acceptor used in different concentrations were constructed. These surface models have provided an opportunity to confirm and establish the optimum growing point by the studied bacteria, their sulfate consumption and sulfide production, and the lactate consumption and acetate accumulation.
The correlation coefficients (r) between the parameters of dissimilatory sulfate reduction by the D. piger Vib-7 were defined. Strong negative and positive correlations between the parameters have been demonstrated. Continued Table 2 Закінчення табл. 2