Hespellia stercorisuis gen. nov., sp. nov. and Hespellia porcina sp. nov., isolated from swine manure storage pits Terence R. Whitehead, 1 Michael A. Cotta, 1 Matthew D. Collins 2 and Paul A. Lawson 2 Correspondence Terence R. Whitehead whitehtr@ncaur.usda.gov 1 Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, USDA, Agricultural Research Service, 1815 N. University Street, Peoria, IL 61604, USA 2 School of Food Biosciences, University of Reading, Reading, RG6 6AP, UK Four Gram-positive-staining, strictly anaerobic, non-spore-forming, rod-shaped organisms were isolated from a pig manure storage pit. Comparative 16S rRNA gene sequence analysis revealed that the isolates belonged to two related but distinct groups. Sequence analysis showed that the two groups of isolates were highly related to each other (approx. 97 % 16S rRNA gene sequence similarity), forming a distinct cluster within the Clostridium coccoides suprageneric rDNA grouping. Biochemical and physiological studies confirmed the division of the isolates into two related, albeit distinct, groups. Based on both phenotypic and phylogenetic evidence, it is proposed that the unidentified rod-shaped isolates from pig manure should be classified in a novel genus, Hespellia gen. nov., as Hespellia stercorisuis sp. nov. and Hespellia porcina sp. nov. The type species of the novel genus is H. stercorisuis (type strain, PC18 T =NRRL B-23456 T =CCUG 46279 T =ATCC BAA-677 T ) and the type strain of H. porcina is PC80 T (=NRRL B-23458 T =ATCC BAA-674 T ). INTRODUCTION Intensive modern livestock-farming practices have resulted in the concentration of generated waste products into increasingly smaller locations. Lagoon treatment or deep- pit storage are among the more favoured methods that are used to handle liquid swine manure. Storage of swine manure is associated with the production of a variety of odorous chemicals, many of which are due to the incom- plete digestion processes that are associated with anaerobic systems (Yasuhara & Fuwa, 1979; Spoelstra, 1980; Yasuhara et al., 1984; Zahn et al., 1997). In addition, production of gaseous emissions (such as ammonia) within confined facilities, such as those used with swine, can pose potential health problems to both animals and human workers. Although production of these chemicals is the result of microbiological activity, little is known about the types of micro-organisms that are responsible for their production. During the course of an ongoing study into the microbial diversity that is present within manure storage pits (Whitehead & Cotta, 2000; Cotta et al., 2003), we charac- terized four strictly anaerobic, Gram-positive, asporogenous, rod-shaped organisms of uncertain taxonomic position. Based on the results of a polyphasic taxonomic study, it is proposed that the isolates should be assigned to a novel genus, Hespellia gen. nov., as Hespellia stercorisuis sp. nov. and Hespellia porcina sp. nov. METHODS Isolates PC17, PC18 T , PC80 T and PC82 were recovered from a manure storage pit from a swine facility near Peoria, IL, USA, where feeder pigs were fed a corn/soybean-based diet. Samples (50– 100 ml) from manure storage pits were collected by using a tank sampler and transferred to Whirl-Pak sampling bags (both from NASCO). Samples were kept on ice until they were returned to the laboratory. Isolations and enumerations were performed by plating samples that were diluted serially in anaerobic buffer onto habitat- simulating media, which contained either 40 % (v/v) substrate- depleted rumen fluid (RF medium) (Dehority & Grubb, 1976; Leedle & Hespell, 1980) or 80 % (v/v) clarified swine manure slurry (Slurry medium; 8000 g, 20 min, 4 uC) (Cotta et al., 2003). Media used in these experiments were prepared anaerobically by using the method of Hungate, as modified by Bryant (1972). Basic media contained macro- and microminerals, buffers, reducing agents and other components, as in RGM medium described by Hespell et al. (1987) or anaerobic BHI medium as described by Whitehead & Flint (1995). No additional volatile fatty acids (FAs) were added to Abbreviations: ALD, aldehyde; DMA, dimethylacetal; FA, fatty acid; NRRL, Northern Regional Research Laboratory. Published online ahead of print on 1 August 2003 as DOI 10.1099/ ijs.0.02719-0. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains NRRL B-23456 T and NRRL B-23458 T are AF445264 and AF445239, respectively. 02719 G 2004 IUMS Printed in Great Britain 241 International Journal of Systematic and Evolutionary Microbiology (2004), 54, 241–245 DOI 10.1099/ijs.0.02719-0