Alfalfa is difficult to ensile because of its low water soluble carbohydrate content, low dry matter content, and high buffering capacity. Therefore, the isolation of mutants of L. plantarum was undertaken to develop a strain(s) which could improve the fermentation of alfalfa silage when used as an inoculum.

Efforts to isolate starch- or hemicellulose-utilizing strains proved unsuccessful as did the isolation of high temperature resistant mutants and oxygen resistant mutants. Mutants resistant to plumbagin (an intracellular O₂- generator) and citrate were isolated. Of these mutants, some were found to be more sensitive to cadmium than the parent strain. Though these three characteristics point to an enhanced manganese accumulation since O₂- is scavenged by manganese, citrate will chelate manganese, making it unavailable to the organism, and cadmium is transported by the manganese transport system, the rates of 54_Mn transport were no greater than the parent. When grown aerobically on a glucose-limited medium, the parent strain consumed oxygen and produced high concentrations of acetic acid and low concentrations of lactic acid. By contrast, one mutant (strain MC226), produced high concentrations of lactic acid and demonstrated low rates of O₂ consumption and low levels of acetic acid.

The parent strain and two plumbagin- and citrate-resistant mutants were used to inoculate alfalfa for silage production. The inoculated silages showed a lower pH and higher titratable acidity than that of an uninoculated control. Silages inoculated with the parent strain exhibited high levels of acetic acid and low levels of lactic acid, indicative of oxygen consumption by the parent strain. Less acetic acid was evident in silages inoculated with the mutant strains. All silages contained a high level of butyric and isobutyric acids and propionic and isovaleric acids were also detected, indicative of clostridial growth.