Isolation and Purification of Sulfate-Reducing Bacteria

*Ivan Kushkevych*

*This chapter, I dedicate to all scientists who have interests in the research on the physiology and biochemistry of this promising and heterogeneous group of microorganisms, the sulfate-reducing bacteria.*

*Author.*

## **Abstract**

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.

**Keywords:** sulfate-reducing bacteria, sulfate, sulfite agar, hydrogen sulfide, isolation, identification

### **1. Introduction**

Sulfate-reducing bacteria (SRB) are a heterogeneous group of microorganisms which is widespread in anaerobic places where sulfate-containing compounds are present [1–5]. 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, 6–8]. 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 [11]. The SRB can also use ammonium salts as nitrogen sources [3, 11]. SRB species can assimilate molecular nitrogen [3]. So, SRB are widespread in the following areas as lake depths, soils, swamps [1, 3], and biogas plant [12–14] and also present in the human and animal intestines [15–19]. The main species of intestinal SRB, *Desulfovibrio* genus, are often isolated from patients with inflammatory bowel disease (IBD) and healthy subjects [15, 20–25]. Other species of SRB, *Desulfomicrobium*, *Desulfobulbus*, *Desulfobacter*, *Desulfomonas*, and *Desulfotomaculum* were also seldom isolated from human and animal feces [1, 23, 26].

An increased number of SRB are often detected in patients with periodontitis [18]; inflammatory bowel diseases, including ulcerative colitis; and many other diseases [27–31]. 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:

