| In this work, novel strains of Lactobacillus plantarum were engineered to provide enzymatic pretreatment to lignocellulosic biomass during ensiling. This pretreatment was catalyzed by the in situ production of ferulic acid esterases. The substrates that these novel microorganisms were used to ensile were winter rye and corn stover. On winter rye, the genetically modified inoculants were able to significantly increase the enzymatic digestibility of the silage by 11.1% +/- 3.2% (P=0.05) compared to a control treatment. If combined with a subsequent thermochemical pretreatment, the benefit of inoculation with FAE producing strains of L. plantarum increased to 18.7% +/- 5.0% (P=0.05). On corn stover modified strains produced no benefit for the raw silage, but after thermochemical pretreatment, the biologically treated silage had an increase in digestibility of over 30% (P=0.05) when compared to a control treatment.;In addition, this work focused on the balance between heterologous expression and the ability to maintain a robust, competitive organism in a natural system. It was found that strains producing a high amount of heterologous esterase achieved a lower final silage population than strains producing lower amounts of esterase. In addition, this work found that using acid-inducible promoters to drive heterologous expression enabled the strain to achieve higher final populations than similar organisms using constitutive promoters.;In final, this work presents a mathematical model for optimizing the balance between enzymatic production and population development to deliver the maximum amount of enzyme using a given organism in a given environment. This model illustrates many key parameters that are important for good silage management, and is also of import to anyone seeking to enable genetically engineered microorganisms to thrive and function in the natural environment. |
