This pathway has been observed in thermophilic reactors (Zinder & Koch, 1984), mesophilic reactors (Schnürer et al., 1994) and natural environments Selumetinib purchase (Nazina et al., 2006; McInerney et al., 2008). In mesophilic digestors, high ammonia levels can cause syntrophic acetate oxidation (Schnürer & Nordberg, 2008). The first syntrophic acetate-oxidizing bacterium isolated was a thermophilic homoacetogen (Lee & Zinder, 1988), but its phylogenetic position could not be established. Subsequently, three syntrophic acetate-oxidizing bacteria have been isolated and described thoroughly: the mesophilic Clostridium ultunense (Schnürer et al., 1996), the thermophilic

Thermacetogenium phaeum (Hattori et al., 2000) and Thermotoga lettingae (Balk et al., 2002). This paper describes the isolation and identification of a new syntrophic acetate-oxidizing bacterium, the mesophilic strain Sp3T, and a bacterium closely related to C. ultunense, strain Esp. Strains Sp3T and Esp were isolated from sludge

from an upflow anaerobic filter treating wastewater from a fishmeal factory. The reactor operated at 37 °C at an organic loading rate of 20–35 g COD day−1 and contained a high ammonium concentration (6 g NH4+-N L−1). Methane production was demonstrated to proceed through syntrophic acetate oxidation (Schnürer et al., 1999). As growth medium, bicarbonate-buffered basal medium (BM) was prepared by mixing solutions A–I described by Zehnder et al. (1980) with some modifications (g L−1): (A) KH2PO4, 0.41; (B) Na2HPO4, 0.43; (F) Na2SeO3·5H2O, Ferrostatin-1 research buy 0.3; and Na2WO4·2H2O, 0.3. Solution G was modified by (g L−1): pyridoxamine, 0.25;

nicotinic acid, 0.1; nicotinamide, 0.1; dl-panthothenic acid, 0.05; vitamin B12, 0.05; p-aminobenzoic acid, 0.05; pyridoxine hydrochloride, 0.1; biotin, 0.02; thioctic acid, 0.05; folic acid, 0.02; riboflavin, 0.05; and thiamine hydrochloride, 0.1. In preparing the medium, 15 mL 4��8C of solution A, 15 mL of solution B, 1 mL of solution F and 5 mL of solution I were made up with >1 L of distilled water. Unless otherwise stated, the medium was complemented with yeast extract (0.2 g L−1), boiled for 20 min and cooled under flushing with N2 to a final volume of 900 mL. The medium was dispensed into vials under flushing with N2/CO2 (80/20 v/v). The vials were sealed and autoclaved for 20 min at 121 °C. Subsequently, mixture C1, containing 1 mL of trace metal solution E, 1 mL of vitamin solution G, 12.5 mL of solution C and 34.5 mL distilled water, and mixture C2, containing 49 mL of solution D, 1 mL of solution H and 0.5 g of cysteine-HCl, were prepared separately and sterile-filtered (0.2 μm) into closed autoclaved vials filled with N2. One milliliter of each mixture was transferred by a syringe to vials containing 18 mL medium, yielding a final pH of 6.9–7.2. Unless otherwise stated, cultures were incubated in the dark at 37 °C without shaking.