The differences between features of the second subgroup SRB genera according to Bergey’s manual of determinative bacteriology .
Sulfate-reducing bacteria (SRB) are a widespread group of microorganisms that are often isolated from the anoxygenic environments (lake depths, soil, or swamps), and they are also present in the human and animal intestines. This group is often detected in patients with inflammatory bowel disease, including ulcerative colitis. That is why new rapid methods for their isolation, purification, and identification are important and necessary. In this chapter, the methods of mesophilic SRB isolation from various environments are described. Particular attention is paid to the purification of mesophilic SRB since they can be in close interaction with other microorganisms (Clostridium, Bacteroides, Pseudomonas, etc.), which are their frequent satellites. Moreover, the main methods of mesophilic SRB identification based on their morphological, physiological, biochemical, and genetical characteristics are presented.
- sulfate-reducing bacteria
- sulfite agar
- hydrogen sulfide
Sulfate-reducing bacteria (SRB) are a heterogeneous group of microorganisms which is widespread in anaerobic places where sulfate-containing compounds are present [1, 2, 3, 4, 5]. These microorganisms use sulfate ions, which are reduced to hydrogen sulfide in the process called “dissimilatory sulfate reducing” or “sulfate respiration.” In this process, sulfate is a terminal electron acceptor [1, 2, 6, 7, 8]. For the implementation of dissimilatory sulfate reduction, exogenous electron donors are necessary [3, 4].
Molecular hydrogen is the main electron donor for all SRB, but commonly used electron donors are also lactate, acetate, pyruvate, ethanol, fatty acids, amino acids, dicarboxylic acids, and other organic compounds [9, 10]. Depending on the species of SRB, organic compounds can be oxidized completely to carbon dioxide or incompletely with the formation of acetate . The SRB can also use ammonium salts as nitrogen sources [3, 11]. SRB species can assimilate molecular nitrogen . So, SRB are widespread in the following areas as lake depths, soils, swamps [1, 3], and biogas plant [12, 13, 14] and also present in the human and animal intestines [15, 16, 17, 18, 19]. The main species of intestinal SRB,
An increased number of SRB are often detected in patients with periodontitis ; inflammatory bowel diseases, including ulcerative colitis; and many other diseases [27, 28, 29, 30, 31]. Some scientists also suggest that SRB may be the cause of some forms of colon cancer, given the fact that these microorganisms produce hydrogen sulfide affecting the intestinal cell metabolism causing various diseases [32, 33]. That is why the isolation of SRB new strains, their purification from other microorganisms, and study of SRB cultural, physiological, biochemical, and genetical properties in detail are necessary.
It should be also noted that many species may be uncultured, so it is important to apply molecular and genetic methods such as Illumina sequencing. This method can give a clear picture of SRB diversity in the detected sample. However, in this chapter, the focus will be on isolation, purification, and cultivation of cultured mesophilic SRB species.
The goal of chapter is to describe:
Methods of sample selections from water, soil, swamp, and feces of human or animals and from biopsy material
Media, isolation, purification, and cultivation conditions
Morphological diversity and physiological and biochemical properties
Identification based on physiological and biochemical properties and sequence analysis of the 16S rRNA gene
Generalization of this research
2. Selection of samples
As was noted, the SRB can be present in a sulfate-rich environment. The samples selected from the different ecotopes should be directly placed in anoxic modified Postgate liquid medium . The composition of the medium and conditions of selections is described in Section 3.
2.1 Samples from environment (water, soil, swamp, and environmental surfaces)
One milliliter of water (or 1 g of swamp, soil, and metal rust) should be suspended in 9 ml of anoxic Postgate liquid medium. The tubes should be brim-filled with medium and closed to provide anaerobic conditions. Another option to provide anaerobic conditions is to add in tube 1 ml sterile liquid paraffin. The schema of sampling is presented in Figure 1.
2.2 Samples from feces of human or animals
It is thought that the species of SRB, their composition, and the number found in the intestinal lumen differ from that of the composition and number of microorganisms on the surface of the intestinal mucosa [2, 9, 28, 34]. Similar to environmental samples (see Figure 1), fecal samples from human or animals should be fresh and directly suspended in anoxic modified Postgate liquid medium (pH 7.5,
2.3 Samples from intestine (biopsy or sections of the large intestine of animals)
Intestinal SRB with other intestinal bacteria can form biofilms on the surface of the epithelial cells of the large intestine . These biofilms include species of
For isolation of SRB from biofilms, 10−5 M EDTA (ethylenediaminetetraacetic acid) should be added to the modified Postgate liquid medium for releasing SRB from a biofilm. A fresh piece of biopsy should be weighed, and its approximate square (in cm2) must be calculated and added to 9 ml of the modified Postgate liquid medium (pH 7.5,
The same procedure can be applied for isolation of SRB from sections of the large intestine of animals.
3. Medium and cultivation conditions
The composition of modified liquid Postgate medium [3, 35] is the following (g/l): Na2SO4 (0.5); KH2PO4 (0.3); K2HPO4 (0.5); (NH4)2SO4 (0.2); NH4Cl (1.0); CaCl2 × 6H2O (0.06); MgSO4 × 7H2O (0.1); lactate, C3H5O3Na (2.0); yeast extract (1.0); FeSO4 × 7H2O (0.004); sodium citrate, C6H5O7Na3 × 2H2O (0.3); and distilled water (1 l).
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 autoclaving.
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 Mohr’s salt.
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 added to medium can also decrease a redox potential .
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 NaOH.
The temperature of the media should be +25…+30°C for environmental samples, and + 37°C for intestinal samples (+40°C for samples from birds).
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 the top of the medium and closed with rubber stoppers.
As a control of the quality of the medium, known pure culture of SRB from some collections of microorganisms is recommended to also be used.
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 occurred in the bottom of the tube.
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 . Among the intestinal microorganisms, the species of the
For obtaining pure cultures of SRB colonies, positive SRB samples (mixed SRB cultures) should be diluted (1,9) in a series of tubes (to 10−5) 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
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 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
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-and-ammonia 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 . Morphology of SRB cells can be studied by using the light microscope, phase-contrast microscopy, or electronic 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
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 increasing acidity of the medium can lead to the decrease of the SRB growth rate and hydrogen sulfide production . Furutani and Schindler reported that the process of dissimilatory sulfate reduction was significantly slowed at low pH . The increasing of the pH medium until 9.0–10.0 also caused growth inhibition of the studied bacteria . To test the pH effect on the SRB growth, the modified liquid Postgate medium (
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.,
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 (
The final product of the dissimilatory sulfate reduction process is hydrogen sulfide, which can be measured in the culture medium (
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 . This competition for molecular hydrogen between SRB and methanogens largely depends on the presence and quantity of sulfate in the gut . 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.
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
Gas production can be observed in deep culture Postgate agar in the tubes.
The desulfoviridin production is a very important factor for identification of
6. Identification based on physiological and biochemical properties and sequence analysis of the 16S rRNA gene
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” . This group is divided into four subgroups (Figure 5).
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,
As was mentioned above, the representatives of
The second subgroup includes
|Spiral or vibrio-shaped cells||+||—||—||—||—|
|Oval or rod-shaped cells||—||+||+||+||+|
|Movement with the polar flagella||+/−||+/−||+||+/−|
|Optimal temperature range|
|Ability of bacteria to grow in the presence of sulfate|
|H2 + CO2 + acetate as a carbon source||+||+||+||+||+|
Other SRB genera can be identified by Bergey’s manuals [10, 11]. However, for complete identification based on morphological, physiological, and biochemical properties, the molecular methods, in particular the sequence analysis of 16S rRNA gene, are also necessary to be applied . Except sequence analysis of 16S rRNA gene, it is important to confirm the SRB species by using primers of functional genes of dissimilatory sulfate-reduction, such as
|Functional genes||Primer sequence||Amplicon length (pb)|
Further on the example of one isolate of intestinal SRB, identification based on sequence analysis of 16S rRNA gene by using the universal primers will be described. The schema of this identification is presented in Figure 6.
|Primers||Sequence||Amplicon length (pb)|
|8FPL||5ʼ-AGTTTGATCCTGGCTCAG-3ʼ position 8–27||Approximately 1500|
|1492RPL||5ʼ-GGTTACCTTGTTACGACTT-3ʼ position 1510–1492|
|806R||5ʼ-GGACTACCAGGGTATCTAAT-3ʼ position 806–787||Approximately 800|
|SRB strains||Acc. No||Identities||Identity (%)|
The amplicons were amplified by a preliminary incubation at 94°C for 5 min (initial denaturation) and then 34 cycles of 94°C for 1 min (denaturation), 55°C for 1 min (annealing of primers), and 72°C for 2 min (polymerization), using a thermocycler (model MJ Research PTC-200, USA). After the last amplification cycle, the samples were incubated further at 72°C for 2 min for complete elongation of the final PCR products and cooled at 10°C.
The 16S rRNA gene amplicons which were used for sequence analysis were obtained by using the PCR method. The PCR products were separated by electrophoresis (Figure 7). Before sequence analysis the absorbance of amplicons (8FPL/806R, amplicon I about 800 bp; 8FPL/1492RPL, amplicon I about 1500 bp; 8FPL/806R, amplicon II about 800 bp; 8FPL/1492RPL, amplicon II about 1500 bp) was determined .
By comparison of individual sequencing data from the amplicons 1–5, the following gene for 16S rRNA sequence of the total length 1370 bp was completed:
The obtained sequence results of SRB isolated colony were also compared by BLASTN analysis with the nucleotide sequences of 16S rRNA gene of other strains (Table 4).
Thus, the nucleotide sequence of the 16S rRNA gene of SRB has the highest homology (99%) compared to deposited nucleotide sequence
Moore W.E. found SRB for the first time in people’s feces and identified it as
7. Generalization of the research
Taking into consideration all research described in the chapter, it is necessary to generalize that isolation of mesophilic SRB from environmental samples (water, soil, swamp, etc.) and intestinal samples can be similar, although swamps and feces are required to determine dry matter of the samples. It is important to purify a positive sample of SRB from other satellite microorganisms such as
For identifications of SRB based on morphological, physiological, and biochemical characteristics, two Bergey’s Manuals [10, 11] are recommended. Moreover, all isolated SRB species should be confirmed by the sequence analysis of the 16S rRNA gene by using universal primers or primers of functional genes of dissimilatory sulfate-reduction, such as
The methods of sample selections from water, soil, swamp, and feces of humans or animals and from biopsy material and the process of SRB isolation and purifications are similar, although cultivation conditions may differ. Identification based on physiological and biochemical properties is a complex process, and many other factors must be considered. For this identification, Bergey’s Manuals are recommended to be used. The sequence analysis of the 16S rRNA gene should confirm the identification process based on physiological and biochemical properties.
It is of vital importance to obtain new strains of the SRB from various ecotopes and identify them and study their growth and physiological and biochemical properties. Aside from that, the process of dissimilatory sulfate reduction by SRB and the production of hydrogen sulfide should be investigated in order to clarify the etiological role of these bacteria in the nature and in the development of various diseases.
This study was supported by Grant Agency of the Masaryk University (MUNI/A/0902/2018).
Conflict of interest
The authors declare no conflict of interest.