2.2. Acid pretreatment and ensiling
Dilute acid pretreatment was carried out similar to that described by Digman et al. (2010). Briefly, substrate was rehydrated to meet the moisture content (MC) targets of 35%, 50% or 65% (w.b.) prior to pretreatment. Sulfuric acid was applied as a 9 M solution at ambient (room) temperature by manually mixing for individual samples to reach 35 or 75 g acid/kg DM, resulting in an acid concentration of
1.8–14.9% in water of samples by weight. Next, alfalfa stems were inoculated with lactic acid bacteria inoculant (Ecosyl MTD/1, Ecosyl Products Ltd., Stokesley, UK) at a rate of 105 colony-forming units/g alfalfa, according to manufacturer’s specification. The inoculated material was ensiled by sealing it in a 1 liter Tulpen-form borosilicate glass canning jars (Weck-Rundrandglas 100, J. Weck GmbH and Co., Öflingen, Germany). After ensiling for about 10 months, samples were taken from each jar, and the remaining silages were frozen at -20 °C.