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Enzymes are essential to many industries such as food, pharmaceuticals, textiles, and paper. Many enzyme production processes commonly utilize sugars such as glucose and sucrose as feedstocks for microbial hosts. However, the agricultural methods to produce these sugars can negatively impact the environment through land use changes and pollution from fertilizer use. Therefore, using alternative feedstocks that are not dependent on agriculture (such as acetate produced from CO2 by acetogens) for large-scale enzyme production is a desirable goal. Bacillus licheniformis has shown promise as a major producer of alkaline protease which has a variety of uses in industry and household products. In an effort to steer away from using conventional carbon sources, we focus on developing a bioprocess using B. licheniformis to produce a protease using the non-conventional carbon source acetate. We began with media optimization where various media components (phosphate concentration, nitrogen source, and salt concentration) were altered to determine the acetate medium recipe that would result in fastest bacterial growth to the highest optical density (OD). Results showed that higher phosphate concentrations (68 mM) led to the highest OD, while ammonium was a superior nitrogen source compared to nitrate. For salinity, 2% (w/v) proved best for bacterial growth. Growth curves were measured for B. licheniformis in LB, glucose, and acetate media to assess growth trends and compare carbon sources. To calculate the amount of protease the bacteria produced, we employed a colorimetric assay involving the digestion of casein to release tyrosine, which, when reacted with Folin–Ciocâlteu reagent, creates a blue chromophore. We compared protease production in media with the different carbon sources and discuss the environmental implications of these processes.