**4. Isolation and purification of positive SRB samples**

As already mentioned above, SRB are in close interactions with other microorganisms and can form biofilms in which they may be in a symbiotic relationship [34, 37]. Such microorganisms cooperating with SRB are often called satellite microorganisms [3]. Among the intestinal microorganisms, the species of the *Bacteroides*, *Pseudomonas*, *Clostridium*, and *Escherichia* genera are most often detected. Phototrophic green sulfur bacteria can make consortium with SRB [38]. On one hand, SRB produce hydrogen sulfide, and on the other hand, green sulfur bacteria oxidize hydrogen sulfide to molecular sulfur in the process of anoxygenic photosynthesis. Molecular sulfur may subsequently be oxidized to sulfate, in which SRB can be used as a final electron acceptor. Such an example of interaction can be consortium *Pelochromatium roseo-viridae* [11]. That is why it is important to purify the mixed cultures of SRB from satellite microorganisms which are very difficult to remove of during this process.

**27**

**Figure 2.**

*Isolation and Purification of Sulfate-Reducing Bacteria DOI: http://dx.doi.org/10.5772/intechopen.86786*

have sulfite reductase activity [39].

perature (+40°C) at a constant level.

according to the sample from where it was isolated.

*The scheme of dilution of positive SRB samples (mixed cultures).*

For obtaining pure cultures of SRB colonies, positive SRB samples (mixed

modified Postgate liquid medium. The scheme of the series of tubes is presented in **Figure 2**. Before it, the modified Postgate agar medium of the same composition like liquid should be prepared but in this case adds to the medium additional compounds: Na2SO3 (7.5 g/l) and microbiological agar (12 g/l). Sterilize it by autoclaving like Postgate liquid medium. Sodium sulfite in high concentration in medium inhibits most of intestinal species of *Enterobacteriaceae* family, including *Bacteroides*, *Pseudomonas*, and *Clostridium*, *Escherichia*, which can be satellites of SRB. The species of SRB are resisted to sulfite ions and can be used as an alternative electron acceptor [11] in the process of dissimilatory sulfate reduction since they

The modified Postgate agar medium containing sodium sulfite (Na2SO3) after sterilization in autoclave should be cooled to +40°C and 10 ml/l of sterile Mohr's salt solution, 0.05 ml/l of sterile solution of sodium sulfide and ascorbic acid (0.1 g/l) added to the medium. These components must be thoroughly mixed in the flask and a sterile 10 M solution of NaOH added to provide accordingly a pH depending on the samples. To prevent the medium solidation, use a water bath to keep the tem-

In total, 20 ml of warm modified Postgate agar medium spill in Petri palates and add to the medium 100 μl of each diluted suspension of a positive sample, thoroughly mix the suspension with the warm medium. The temperature should be

Filled with medium and suspension Petri plates introduce into an anaerobic box with oxygen uptake generators for anaerobiosis. Mohr's salt in the agar medium allows to detect black colonies of SRB since as a result, FeS was formed by hydrogen sulfide bacterial production that caused black-colored colonies. Cultivate in the thermostat at the appropriate temperature. The black colonies will be visible in 1–5 days in the deep of agar medium depending on sample and its dilution.

The black colonies obtained from Petri palates cut from agar and suspend in modified liquid Postgate medium. Cultivate in the thermostat at the appropriate temperature. The formation of black sediment (FeS precipitate) will be visible in the tube (about in 1–3 days). This sediment confirms sulfate reduction and

) containing the

SRB cultures) should be diluted (1,9) in a series of tubes (to 10<sup>−</sup><sup>5</sup>

*Microorganisms*

autoclaving.

Mohr's salt.

NaOH.

The modified liquid Postgate medium and solutions of Mohr's salt, sodium sulfide, and sodium hydroxide should be sterilized in autoclave (20 min, at 1 atm.). The sterilization provides sterile conditions and partial release of oxygen from the medium. The solution of sodium sulfide is hydrolyzed to hydrogen sulfide during

After sterilization, 10 ml/l of sterile Mohr's salt solution and 0.05 ml/l of sterile solution of sodium sulfide must be added to the medium. The addition of a small quantity (one drop) of sodium sulfide solution to the medium makes visible a black ring which confirms interactions of hydrogen sulfide and free Fe2+ released from

A sterile ascorbic acid solution also must be added to the medium, but it cannot be sterilized by autoclaving because it may partially decompose and lose its properties for redox potential. So, 20% ascorbic acid solution should be filtrated through membrane filters (0.2 μm) and added directly to the medium after sterilization. The final concentration of ascorbic acid in the medium should be 0.1 g/l, and the redox potential of the medium must be around −100 mV. Solution of hydrogen sulfide

The redox and anaerobic conditions can be controlled by sodium resazurin as an indicator. In addition, FeS reduced and Na2S contained in the medium provides the necessary redox conditions for SRB cultures. The discoloration of sodium resazurin (redox potential of discoloration Eh = −100 mV) confirms the decrease of redox potential. A pH medium (7.5) provides by the addition of a sterile 10 M solution of

The temperature of the media should be +25…+30°C for environmental samples,

The tubes with samples must be completely filled up to the edges of the test tube with completed medium and closed with rubber stoppers. In another case, tubes can be filled up incompletely, but 1 ml of sterile liquid paraffin must be filled up to

As a control of the quality of the medium, known pure culture of SRB from

Cultivate in the thermostat at +25…+30°C, +37°C, or +40°C, depending on the origin of the sample, during for 1–5 days under anaerobic conditions. SRB from birds, animals, and humans mostly grow faster than environmental species. Positive growth of SRB is indicated by observing a black FeS precipitate

As already mentioned above, SRB are in close interactions with other microorganisms and can form biofilms in which they may be in a symbiotic relationship [34, 37]. Such microorganisms cooperating with SRB are often called satellite microorganisms [3]. Among the intestinal microorganisms, the species of the *Bacteroides*, *Pseudomonas*, *Clostridium*, and *Escherichia* genera are most often detected. Phototrophic green sulfur bacteria can make consortium with SRB [38]. On one hand, SRB produce hydrogen sulfide, and on the other hand, green sulfur bacteria oxidize hydrogen sulfide to molecular sulfur in the process of anoxygenic photosynthesis. Molecular sulfur may subsequently be oxidized to sulfate, in which SRB can be used as a final electron acceptor. Such an example of interaction can be consortium *Pelochromatium roseo-viridae* [11]. That is why it is important to purify the mixed cultures of SRB from satellite microorganisms which are very difficult to

added to medium can also decrease a redox potential [3].

the top of the medium and closed with rubber stoppers.

**4. Isolation and purification of positive SRB samples**

occurred in the bottom of the tube.

remove of during this process.

and + 37°C for intestinal samples (+40°C for samples from birds).

some collections of microorganisms is recommended to also be used.

**26**

For obtaining pure cultures of SRB colonies, positive SRB samples (mixed SRB cultures) should be diluted (1,9) in a series of tubes (to 10<sup>−</sup><sup>5</sup> ) containing the modified Postgate liquid medium. The scheme of the series of tubes is presented in **Figure 2**. Before it, the modified Postgate agar medium of the same composition like liquid should be prepared but in this case adds to the medium additional compounds: Na2SO3 (7.5 g/l) and microbiological agar (12 g/l). Sterilize it by autoclaving like Postgate liquid medium. Sodium sulfite in high concentration in medium inhibits most of intestinal species of *Enterobacteriaceae* family, including *Bacteroides*, *Pseudomonas*, and *Clostridium*, *Escherichia*, which can be satellites of SRB. The species of SRB are resisted to sulfite ions and can be used as an alternative electron acceptor [11] in the process of dissimilatory sulfate reduction since they have sulfite reductase activity [39].

The modified Postgate agar medium containing sodium sulfite (Na2SO3) after sterilization in autoclave should be cooled to +40°C and 10 ml/l of sterile Mohr's salt solution, 0.05 ml/l of sterile solution of sodium sulfide and ascorbic acid (0.1 g/l) added to the medium. These components must be thoroughly mixed in the flask and a sterile 10 M solution of NaOH added to provide accordingly a pH depending on the samples. To prevent the medium solidation, use a water bath to keep the temperature (+40°C) at a constant level.

In total, 20 ml of warm modified Postgate agar medium spill in Petri palates and add to the medium 100 μl of each diluted suspension of a positive sample, thoroughly mix the suspension with the warm medium. The temperature should be according to the sample from where it was isolated.

Filled with medium and suspension Petri plates introduce into an anaerobic box with oxygen uptake generators for anaerobiosis. Mohr's salt in the agar medium allows to detect black colonies of SRB since as a result, FeS was formed by hydrogen sulfide bacterial production that caused black-colored colonies. Cultivate in the thermostat at the appropriate temperature. The black colonies will be visible in 1–5 days in the deep of agar medium depending on sample and its dilution.

The black colonies obtained from Petri palates cut from agar and suspend in modified liquid Postgate medium. Cultivate in the thermostat at the appropriate temperature. The formation of black sediment (FeS precipitate) will be visible in the tube (about in 1–3 days). This sediment confirms sulfate reduction and

**Figure 2.**

*The scheme of dilution of positive SRB samples (mixed cultures).*

#### **Figure 3.**

*The scheme of the confirmation that the isolates belong to the SRB.*

production of hydrogen sulfide, which interact with Fe2+ from Mohr's salt, and FeS precipitate is formed. However, hydrogen sulfide can also be produced by species of *Clostridium*, *Salmonella*, and other intestinal bacteria. Moreover, some sulfurreducing bacteria in the same case can also use sulfate as an electron acceptor [11]. To be sure that the selected microorganisms are not sulfur reducers or other bacteria capable of hydrogen sulfide production, the liquid media following the composition should be prepared: first modified liquid Postgate medium with sulfate (concentration 3.5 mM), second the same medium but without sulfate, and third the same medium but without sulfate containing molecular sulfur (0.5 g/l). The scheme of the confirmation that the isolates belong to the SRB is presented in **Figure 3**.

The grown black sample should be mixed and 100 μl of bacterial suspension pipetted into Eppendorf tubes (volume 1.5 ml) with 900 μl of liquid media by the scheme (**Figure 3**). Pipette 200 μl of sterile liquid paraffin on the surface of the media with suspension, and close a cap of Eppendorf tubes. Cultivate in thermostat.

If the sample after cultivation forms a black sediment in the modified liquid Postgate medium without sulfate ions that contained molecular sulfur, it means that isolates in a positive sample can belong to the sulfur-reducing bacteria (not SRB).

If the sample after cultivation does not form black sediment (FeS precipitate) in modified liquid Postgate medium without sulfate and the same medium without sulfate ions that contained molecular sulfur, but bacterial growth is observed in the medium with sulfate, it means that isolates in a positive sample belong to the SRB.

The positive sample with SRB culture should be diluted in the modified liquid Postgate medium and again seed each dilution in agar medium containing sodium sulfite (see **Figure 2**). This procedure must be repeated 3–5 times for full purification of SRB from other bacterial satellites.

After that, to check the purity of the SRB cultures from satellites, other additional tests are necessary. These additional tests are bacterial growth on the growth on different nonselective media (meat peptone agar; wort agar; starch-andammonia agar; Giltay's, Baalsrud's, and modified Postgate medium). Growth of SRB should be positive only in modified Postgate medium.

## **5. Morphological diversity: physiological and biochemical properties**

The SRB cells are spherical, oval, rod-shaped, spiral, or vibrio-shaped with a diameter of 0.4–3.0 μm. The cells can be either single or in pairs or aggregates also may form a single row of multicellular filaments [1, 3]. Most cells of SRB genera are Gram-negative, although the filamentous and spore-forming microorganisms are Gram-positive. The SRB genera are anaerobes [11]. Morphology of SRB cells can be studied by using the light microscope, phase-contrast microscopy, or electronic microscopy.

**29**

**Figure 4.**

*Isolation and Purification of Sulfate-Reducing Bacteria DOI: http://dx.doi.org/10.5772/intechopen.86786*

staining procedure and in increasing its apparent size.

gas production, and desulfoviridin test (**Figure 4**).

*The scheme of other physiological and biochemical characteristics.*

Some species of SRB have single flagellum or more flagella depending on the genus. A simple, qualitative, and rapid method for detecting bacterial flagella and their shape, length, curvature, arrangement, and number on the cell is Hardy Diagnostics Flagella Stain (HDFS) [40, 41]. In 1937, Ryu developed this method, and later Kodaka et al. further described it [42, 43]. This test is especially useful in taxonomy and identifying characteristic about SRB motile, and more recently, anaerobic bacteria. Due to their narrow diameter, SRB flagella cannot be seen with a light microscope. The method of flagella stain can provide viewing SRB flagella by employing a crystal violet in an alcoholic solution as the primary stain. The alcoholic solution evaporates and leaves a precipitate around the flagella during the

In addition to the cell morphology and the presence of flagella, the following physiological characteristic, which is no less important, is also the formation of spores. However, among the heterogeneous quantity of SRB, the species of *Desulfotomaculum* genus can sporulate. To determine the ability of the SRB cells to sporulate, 1 ml of 72 h pure culture of SRB grown in modified liquid Postgate medium should be heated at +80°C for 15 min and then 100 μl of bacterial suspension pipetted into epindorph (volume 1.5 ml) with 900 μl of liquid media, and add 200 μl of sterile liquid paraffin on the surface of the media with suspension, and close the cap of Eppendorf tubes. Cultivate in thermostat. Thermoresistant forms of the *Desulfotomaculum* genus can be observed by FeS precipitate in the Eppendorf tubes. The SRB spores can be also additionally detected by a staining method for endospore. This method was published by Dorner [44] and later modified by Schaeffer and Fulton [45]. The modified process is simpler and faster and commonly used to differentiate bacterial endospores from other vegetative cells. It is also used to differentiate spore-forming bacteria from nonspore-forming [45]. Other physiological and biochemical characteristics which are important for identification are the determination of SRB growth at various pH and temperature, biomass accumulation, sulfate/lactate consumption, hydrogen sulfide and acetate production, catalase test, indole test, nitrate reduction, carbohydrate fermentation,

The effect of acidity (pH) is one of many important environmental factors which can be used for physiological characteristics of new SRB strains. The decreasing and

*Microorganisms*

**Figure 3.**

thermostat.

tion of SRB from other bacterial satellites.

SRB should be positive only in modified Postgate medium.

production of hydrogen sulfide, which interact with Fe2+ from Mohr's salt, and FeS precipitate is formed. However, hydrogen sulfide can also be produced by species of *Clostridium*, *Salmonella*, and other intestinal bacteria. Moreover, some sulfurreducing bacteria in the same case can also use sulfate as an electron acceptor [11]. To be sure that the selected microorganisms are not sulfur reducers or other bacteria capable of hydrogen sulfide production, the liquid media following the composition should be prepared: first modified liquid Postgate medium with sulfate (concentration 3.5 mM), second the same medium but without sulfate, and third the same medium but without sulfate containing molecular sulfur (0.5 g/l). The scheme of the confirmation that the isolates belong to the SRB is presented in **Figure 3**. The grown black sample should be mixed and 100 μl of bacterial suspension pipetted into Eppendorf tubes (volume 1.5 ml) with 900 μl of liquid media by the scheme (**Figure 3**). Pipette 200 μl of sterile liquid paraffin on the surface of the media with suspension, and close a cap of Eppendorf tubes. Cultivate in

*The scheme of the confirmation that the isolates belong to the SRB.*

If the sample after cultivation forms a black sediment in the modified liquid Postgate medium without sulfate ions that contained molecular sulfur, it means that isolates in a positive sample can belong to the sulfur-reducing bacteria (not SRB). If the sample after cultivation does not form black sediment (FeS precipitate) in modified liquid Postgate medium without sulfate and the same medium without sulfate ions that contained molecular sulfur, but bacterial growth is observed in the medium with sulfate, it means that isolates in a positive sample belong to the SRB. The positive sample with SRB culture should be diluted in the modified liquid Postgate medium and again seed each dilution in agar medium containing sodium sulfite (see **Figure 2**). This procedure must be repeated 3–5 times for full purifica-

After that, to check the purity of the SRB cultures from satellites, other additional tests are necessary. These additional tests are bacterial growth on the growth on different nonselective media (meat peptone agar; wort agar; starch-andammonia agar; Giltay's, Baalsrud's, and modified Postgate medium). Growth of

**5. Morphological diversity: physiological and biochemical properties**

The SRB cells are spherical, oval, rod-shaped, spiral, or vibrio-shaped with a diameter of 0.4–3.0 μm. The cells can be either single or in pairs or aggregates also may form a single row of multicellular filaments [1, 3]. Most cells of SRB genera are Gram-negative, although the filamentous and spore-forming microorganisms are Gram-positive. The SRB genera are anaerobes [11]. Morphology of SRB cells can be studied by using the light microscope, phase-contrast microscopy, or electronic

**28**

microscopy.

Some species of SRB have single flagellum or more flagella depending on the genus. A simple, qualitative, and rapid method for detecting bacterial flagella and their shape, length, curvature, arrangement, and number on the cell is Hardy Diagnostics Flagella Stain (HDFS) [40, 41]. In 1937, Ryu developed this method, and later Kodaka et al. further described it [42, 43]. This test is especially useful in taxonomy and identifying characteristic about SRB motile, and more recently, anaerobic bacteria. Due to their narrow diameter, SRB flagella cannot be seen with a light microscope. The method of flagella stain can provide viewing SRB flagella by employing a crystal violet in an alcoholic solution as the primary stain. The alcoholic solution evaporates and leaves a precipitate around the flagella during the staining procedure and in increasing its apparent size.

In addition to the cell morphology and the presence of flagella, the following physiological characteristic, which is no less important, is also the formation of spores. However, among the heterogeneous quantity of SRB, the species of *Desulfotomaculum* genus can sporulate. To determine the ability of the SRB cells to sporulate, 1 ml of 72 h pure culture of SRB grown in modified liquid Postgate medium should be heated at +80°C for 15 min and then 100 μl of bacterial suspension pipetted into epindorph (volume 1.5 ml) with 900 μl of liquid media, and add 200 μl of sterile liquid paraffin on the surface of the media with suspension, and close the cap of Eppendorf tubes. Cultivate in thermostat. Thermoresistant forms of the *Desulfotomaculum* genus can be observed by FeS precipitate in the Eppendorf tubes. The SRB spores can be also additionally detected by a staining method for endospore. This method was published by Dorner [44] and later modified by Schaeffer and Fulton [45]. The modified process is simpler and faster and commonly used to differentiate bacterial endospores from other vegetative cells. It is also used to differentiate spore-forming bacteria from nonspore-forming [45].

Other physiological and biochemical characteristics which are important for identification are the determination of SRB growth at various pH and temperature, biomass accumulation, sulfate/lactate consumption, hydrogen sulfide and acetate production, catalase test, indole test, nitrate reduction, carbohydrate fermentation, gas production, and desulfoviridin test (**Figure 4**).

The effect of acidity (pH) is one of many important environmental factors which can be used for physiological characteristics of new SRB strains. The decreasing and

**Figure 4.**

*The scheme of other physiological and biochemical characteristics.*

increasing acidity of the medium can lead to the decrease of the SRB growth rate and hydrogen sulfide production [25]. Furutani and Schindler reported that the process of dissimilatory sulfate reduction was significantly slowed at low pH [46]. The increasing of the pH medium until 9.0–10.0 also caused growth inhibition of the studied bacteria [25]. To test the pH effect on the SRB growth, the modified liquid Postgate medium (*without Mohr's salt*) with different pH 3, 4, 5, 6, 7, 8, 9, and 10 is necessary to be prepared. Inoculation (initial concentration) of bacterial cells should be not less than 10%. After cultivation in the thermostat (24–36 h), biomass accumulation can be determined and compared in which the value of pH is optimum for SRB growth.

Most of the species of SRB are mesophilic microorganisms and live at a temperature from +20 to +40°C. Some SRB species can be also thermophilic microorganisms, e.g., *Thermodesulfobacterium* genus (*T. thermophilum*, *T. hveragerdense*, *T. commune,* and others) [11]. However, this chapter is focused on isolation and purification of mesophilic SRB. Similar to the case with test pH effect, the optimum of temperature for SRB growth is necessary to be determined. Inoculate bacterial cells (10%) in the modified liquid Postgate medium (*without Mohr's salt*), and cultivate at different temperatures (+4, +14, +20, +35, +45°C). After cultivation (24–36 h), biomass accumulation can be determined and compared in which the value of the temperature is optimum for SRB growth.

Biomass accumulation of the SRB cells in liquid medium can be measured by the photometric method by using a spectrophotometer, but the medium cannot contain Mohr's salt, since FeS precipitate makes it impossible [26, 47].

The cultivation of SRB in anaerobic, microaerophilic, or aerobic conditions allows testing their viability and resistance to molecular oxygen. However, SRB are anaerobes, but some of them may have high activity of antioxidant enzymes, catalase, and superoxide dismutase [1, 3].

Sulfate consumption as a terminal acceptor and determination of its concentration in the medium during SRB growth is important for observing and understanding more the process of dissimilatory sulfate reduction. The sulfate concentration in the medium (*without Mohr's salt*) can be assayed by the turbidimetric method by precipitation with barium chloride. For stabilizing the suspension, glycerol should be used [48].

The final product of the dissimilatory sulfate reduction process is hydrogen sulfide, which can be measured in the culture medium (*without Mohr's salt*) by a photometric method based on the reaction of sulfide and n-aminodimethylaniline with the methylene blue formation [49]. The concentration of hydrogen sulfide is determined by calibration curve. The data on the concentration of hydrogen sulfide, produced by the isolates, is supposed to help in establishing and assessing a toxicity effect of hydrogen sulfide on the epithelial cells of the human intestine. Such studies might help in predicting the development of diseases in the gastrointestinal tract, by providing further details on the etiology of bowel diseases which are very important for the clinical diagnosis of these disease types.

In the dissimilatory sulfate reduction process, SRB use exogenous electron donors. Molecular hydrogen is a universal electron donor for intestinal SRB [23, 37]. These bacteria are in close interaction with each other. It was established that SRB can completely displace methanogenic microorganisms of the intestine in the process of H2 competition [9]. This competition for molecular hydrogen between SRB and methanogens largely depends on the presence and quantity of sulfate in the gut [9]. Adding sulfate and sulfated mucopolysaccharides to fecal suspensions which contain metabolically active products of the SRB stimulates the formation of hydrogen sulfide and inhibits the intensity of the methanogenesis [1, 14]. Except H2, the second important electron donor is lactate, which SRB can oxidize incompletely to acetate or completely to CO2.

**31**

*Isolation and Purification of Sulfate-Reducing Bacteria DOI: http://dx.doi.org/10.5772/intechopen.86786*

(Colorimetric, Catalog Number KA3764) or by titration.

nitric acid and isoamylic alcohol reagents (Salkowski's reaction).

and alcohol, the strains can also assimilate some amino acids.

**sequence analysis of the 16S rRNA gene**

subgroup where this genus belongs (see **Figure 4**).

into four subgroups (**Figure 5**).

Bergey's manuals.

culture is catalase positive, the bubbles are formed.

acid reagent on 24-h cultures.

NAD+

The determination of lactate concentration can be carried out through dehydrogenation of lactate reaction by lactate dehydrogenase in the presence of

of lactate concentration is the use of lactate assay kit (Sigma-Aldrich, Catalog Number MAK064). Acetate accumulated during lactate incompletely oxidizing in the process of bacterial growth can be determined by using the acetate assay kit

, with formation of pyruvate and NADH. Another method for measurement

Simple catalase test on modified Postgate surface agar cultures can be carried out by adding a drop of 10% H2O2 solution over the colonies. Another way is adding 5 drops of 10% H2O2 solution in 1 ml of a modified liquid Postgate medium. If the

The indole production test can be carried out by using a 24-h liquid culture with

Adding sodium nitrate (5%) to modified liquid Postgate medium can be used for testing nitrate reduction. Nitrites can be tested by using a naphthylamine-sulfanilic

The ability of SRB strains to metabolize except lactate or H2 other electron donors and a carbon source is also necessary to test. With this purpose, formate, propionate, pyruvate, fumarate, malate, methanol, citrate, ethanol, acetate, glycerol, glucose, oleate, stearate, and benzoate should be added separately in modified liquid Postgate medium but without electron donor (lactate) and carbon source. A final concentration of each compound should be 1%. Glucose and pyruvate fermentation in the liquid medium can be analyzed by acidity (pH reaction) and pH indicators. This test confirms that SRB isolated strains are capable to *chemolithoheterotrophic* growth. In addition to organic acids with different carbon chain lengths

Gas production can be observed in deep culture Postgate agar in the tubes. The desulfoviridin production is a very important factor for identification of *Desulfovibrio* and *Desulfomonas* genera. The presence of this protein in bacterial cells can be examined by using ultraviolet (UV) light on Postgate agar surface cultures after treatment with a 1 N NaOH solution. Desulfoviridin will be green in UV light.

**6. Identification based on physiological and biochemical properties and** 

Identification of the SRB by morphological, physiological, and biochemical characteristics can be conducted according to Bergey's Manual of Determinative Bacteriology (ninth edition, 1994), where SRB belong to the seventh group and are called "dissimilatory sulfate- or sulfur-reducing bacteria" [11]. This group is divided

However, more modern and complex classification of SRB is published in Bergey's Manual of Systematic Bacteriology (2005), where SRB are divided into different classes, for example, class IV, *Deltaproteobacteria*, including order II, *Desulfovibrionales*; family I, *Desulfovibrionaceae* (genus I. *Desulfovibrio*); or family II, *Desulfomicrobiaceae* (genus I. *Desulfomicrobium*) [10]. For details identification based on physiological and biochemical characteristics is necessary to use both

As was mentioned above, the representatives of *Desulfovibrio* genus are often found in the animal and patients with IBD and healthy subjects, because it is necessary to pay attention to the more detailed steps for identification of the second

#### *Isolation and Purification of Sulfate-Reducing Bacteria DOI: http://dx.doi.org/10.5772/intechopen.86786*

*Microorganisms*

temperature is optimum for SRB growth.

catalase, and superoxide dismutase [1, 3].

Mohr's salt, since FeS precipitate makes it impossible [26, 47].

important for the clinical diagnosis of these disease types.

increasing acidity of the medium can lead to the decrease of the SRB growth rate and hydrogen sulfide production [25]. Furutani and Schindler reported that the process of dissimilatory sulfate reduction was significantly slowed at low pH [46]. The increasing of the pH medium until 9.0–10.0 also caused growth inhibition of the studied bacteria [25]. To test the pH effect on the SRB growth, the modified liquid Postgate medium (*without Mohr's salt*) with different pH 3, 4, 5, 6, 7, 8, 9, and 10 is necessary to be prepared. Inoculation (initial concentration) of bacterial cells should be not less than 10%. After cultivation in the thermostat (24–36 h), biomass accumulation can be determined and compared in which the value of pH is optimum for SRB growth. Most of the species of SRB are mesophilic microorganisms and live at a temperature from +20 to +40°C. Some SRB species can be also thermophilic microorganisms, e.g., *Thermodesulfobacterium* genus (*T. thermophilum*, *T. hveragerdense*, *T. commune,* and others) [11]. However, this chapter is focused on isolation and purification of mesophilic SRB. Similar to the case with test pH effect, the optimum of temperature for SRB growth is necessary to be determined. Inoculate bacterial cells (10%) in the modified liquid Postgate medium (*without Mohr's salt*), and cultivate at different temperatures (+4, +14, +20, +35, +45°C). After cultivation (24–36 h), biomass accumulation can be determined and compared in which the value of the

Biomass accumulation of the SRB cells in liquid medium can be measured by the photometric method by using a spectrophotometer, but the medium cannot contain

Sulfate consumption as a terminal acceptor and determination of its concentration in the medium during SRB growth is important for observing and understanding more the process of dissimilatory sulfate reduction. The sulfate concentration in the medium (*without Mohr's salt*) can be assayed by the turbidimetric method by precipitation with

The cultivation of SRB in anaerobic, microaerophilic, or aerobic conditions allows testing their viability and resistance to molecular oxygen. However, SRB are anaerobes, but some of them may have high activity of antioxidant enzymes,

barium chloride. For stabilizing the suspension, glycerol should be used [48].

The final product of the dissimilatory sulfate reduction process is hydrogen sulfide, which can be measured in the culture medium (*without Mohr's salt*) by a photometric method based on the reaction of sulfide and n-aminodimethylaniline with the methylene blue formation [49]. The concentration of hydrogen sulfide is determined by calibration curve. The data on the concentration of hydrogen sulfide, produced by the isolates, is supposed to help in establishing and assessing a toxicity effect of hydrogen sulfide on the epithelial cells of the human intestine. Such studies might help in predicting the development of diseases in the gastrointestinal tract, by providing further details on the etiology of bowel diseases which are very

In the dissimilatory sulfate reduction process, SRB use exogenous electron donors. Molecular hydrogen is a universal electron donor for intestinal SRB [23, 37]. These bacteria are in close interaction with each other. It was established that SRB can completely displace methanogenic microorganisms of the intestine in the process of H2 competition [9]. This competition for molecular hydrogen between SRB and methanogens largely depends on the presence and quantity of sulfate in the gut [9]. Adding sulfate and sulfated mucopolysaccharides to fecal suspensions which contain metabolically active products of the SRB stimulates the formation of hydrogen sulfide and inhibits the intensity of the methanogenesis [1, 14]. Except H2, the second important electron donor is lactate, which SRB can oxidize incompletely

**30**

to acetate or completely to CO2.

The determination of lactate concentration can be carried out through dehydrogenation of lactate reaction by lactate dehydrogenase in the presence of NAD+ , with formation of pyruvate and NADH. Another method for measurement of lactate concentration is the use of lactate assay kit (Sigma-Aldrich, Catalog Number MAK064). Acetate accumulated during lactate incompletely oxidizing in the process of bacterial growth can be determined by using the acetate assay kit (Colorimetric, Catalog Number KA3764) or by titration.

Simple catalase test on modified Postgate surface agar cultures can be carried out by adding a drop of 10% H2O2 solution over the colonies. Another way is adding 5 drops of 10% H2O2 solution in 1 ml of a modified liquid Postgate medium. If the culture is catalase positive, the bubbles are formed.

The indole production test can be carried out by using a 24-h liquid culture with nitric acid and isoamylic alcohol reagents (Salkowski's reaction).

Adding sodium nitrate (5%) to modified liquid Postgate medium can be used for testing nitrate reduction. Nitrites can be tested by using a naphthylamine-sulfanilic acid reagent on 24-h cultures.

The ability of SRB strains to metabolize except lactate or H2 other electron donors and a carbon source is also necessary to test. With this purpose, formate, propionate, pyruvate, fumarate, malate, methanol, citrate, ethanol, acetate, glycerol, glucose, oleate, stearate, and benzoate should be added separately in modified liquid Postgate medium but without electron donor (lactate) and carbon source. A final concentration of each compound should be 1%. Glucose and pyruvate fermentation in the liquid medium can be analyzed by acidity (pH reaction) and pH indicators. This test confirms that SRB isolated strains are capable to *chemolithoheterotrophic* growth. In addition to organic acids with different carbon chain lengths and alcohol, the strains can also assimilate some amino acids.

Gas production can be observed in deep culture Postgate agar in the tubes.

The desulfoviridin production is a very important factor for identification of *Desulfovibrio* and *Desulfomonas* genera. The presence of this protein in bacterial cells can be examined by using ultraviolet (UV) light on Postgate agar surface cultures after treatment with a 1 N NaOH solution. Desulfoviridin will be green in UV light.
