ate, propionate, ethanol) along with microbial cells (Stevenson and Weimer, 2007; Weimer et al., 2009). Hydrogen and formate are produced by many microorganisms in the rumen; however, methanogens are also present in the rumen and convert H2, and CO2 to CH4 (Wright et al., 2006). Methanogenesis represents the primary H2 consumer in the rumen and energy captured as methane escapes the rumen via eructation (Boadi et al., 2004, Martin et al., 2010). Energy lost as methane represents a 2 to 7% loss in gross energy intake energy of the animal (Branine and Johnson, 1990) and a loss of 10 to 15% of the apparently digestible feed energy to the host animal (Blaxter and Clapperton, 1965). However, direct inhibition of rumen methane production also results in energy loss in the form of eructated H2 and reduced microbial protein (Chalupa, 1980). Chemo-lithoautotrophic acetogens are bacteria that utilize CO2 as their sole source of carbon and reduce it to acetate with H2 as the source of energy (Drake et al., 2008; Ragsdale, 2008). Acetogens are known to be present in the rumen but they are less numerous and considered to be less efficient than methanogens for utilization of hydrogen as a substrate (Martin et al., 2010). Replacement of methanogenesis with acetogenesis could decrease energy losses and increase the efficiency of ruminant production. Consequently, research on acetogenesis in ruminant animals has been focused toward two related areas of interest and application. First of all, since methane formed as a result of ruminal fermentation is subsequently eructated and is lost to the animal; thus, it would increase energetic efficiency of the host animal if this loss of feed energy and carbon could be minimized (Boadi et al., 2004; Martin et al., 2010). Secondly, there is increasing interest in global warming forced by the production of greenhouse gasses such as CO2, CH4, and NO2 (Boadi et al., 2004; Morrison, 2009). Reductive acetogenesis is a means for developing alternative H2 sinks away from methanogens that produce CH4 (Joblin, 1999). Acetogenesis may provide an important model to find solutions for limiting CH4 emissions from livestock and livestock wastes (Morrison, 2009). Efforts to enhance in vivo acetogenesis in the rumen have
not been as successful as in vitro studies (Fonty et al., 2007). Methanogens are thought to outcompete acetogens because methanogens have a lower hydrogen threshold (Martin et al., 2010); however, most acetogens have been isolated in batch culture in the presence of high hydrogen concentrations and have not been selected for low hydrogen thresholds. A key may be a better understanding of hydrogen use by acetogens. The objective of this study was to use H2-limited continuous culture to demonstrate that it could be used to isolate ruminal acetogenic bacteria able to grow on low threshold concentrations of H2 utilizing CO2 as their sole carbon source.
Materials and Methods Source of Organisms Acetogenic bacterial strains were isolated either from rumen contents collected either from a ruminally fistulated Angus steer fed a diet of alfalfa and orchard grass hay at maintenance or of a ruminally fistulated lactating Holstein Friesian dairy cow consuming a 60:40 percent hay and corn silage: corn grain diet at 2.6% of her body weight. Rumen contents were used to inoculate H2-limiting continuous cultures. Individual strains were isolated after at least 8 turnovers of the continuous culture.
Media and Growth Conditions All media were prepared by the anaerobic techniques of (Hungate, 1966) as modified by (Balch and Wolfe, 1976; Bryant, 1972). The basal semidefined acetogen medium used for growth and nutritional studies and the methanogen medium are listed in Table 1. The medium was boiled under a stream of oxygen-free CO2, sealed, and autoclaved (120°C, 18 Ib/in², 15 min). The pH of the medium was adjusted to 6.8 with NaOH before boiling. The cooled medium was transferred into an anaerobic glove box (Coy Laboratories, Ann Arbor, MI) containing 95% CO2: 5% H2. For all media, the reducing agents, carbonate buffer and vitamins were added separately to the medium in the anaerobic glove box as sterile
34 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011