Chemostat Growth Metabolic Profiling

Cultures were monitored every day to check for contamination by plating on BHI agar media and ziehl neelsen stain. Cultures from chemostat were regularly sampled for measuring OD and colony forming units (Beste et al, 2011 (link)). Carbon‐di‐oxide production from the cultures was monitored using Gas analyzer. The supernatant was collected, filtered using 0.22 μm unit filters and used for substrate consumption and product excretion analyses. To measure the glycerol uptake, the amounts of glycerol in the supernatant and fresh medium was measured using glycerol assay kit by a coupled enzyme assay involving glycerol kinase and glycerol phosphate oxidase, resulting in a colorimetric (570 nm) product, proportional to the glycerol present. To measure NH₄Cl uptake, the amounts of NH₄Cl were measured using ammonia assay kit by reaction of ammonia present in the samples involving L‐glutamate dehydrogenase activity. Dry weight of the cells was measured by centrifuging cultures, drying the cell pellet using freeze dryer and weighing the cells. The dried pellet was used for protein analysis using Bicinchoninic Acid Kit for Protein Determination.