Comparison of optical density, total cell protein, and number of viable cells (via fluorescent staining) as measures of microbial growth kinetics in suspended and biofilm cultures during biodegradation of naphthalene

Most studies on bacterial growth kinetics have been dependent on theoretical modeling with general biomass measurements using either dry weight or optical density (OD), without distinguishing live from dead bacteria or debris. As a result, there remains considerable uncertainty in reliably predicting rates of biodegradation for design of treatment processes for environmental pollutants.; This research focused on measurement of bacterial growth rates and activities in suspended cultures and biofilms using Pseudomonas putida (ATCC 17484) for biodegradation of naphthalene. As expected, the rates of biodegradation differed between suspended and immobilized cultures. A comparison was made of the impact of three biomass measures: optical density, total cell protein, and living cell number on the calculated rate of naphthalene disappearance. Living cell number was determined by a fluorescent staining technique and use of epifluorescence microscopy. More than 90% of total cells remained viable over the course of each experiment (35 to 54 hours).; All three techniques experienced difficulties reconciling calculated values of biomass growth and naphthalene disappearance. This was considered to be a consequence of the production of intermediate products detected in the chromatograms, and possibly adsorption and subsequent release of naphthalene, which resulted in a lag time between the disappearance of naphthalene and the appearance of biomass. Inclusion of a lag time in the integrated Monod expression improved the agreement between experimental and calculated values of biomass and naphthalene concentrations. However, further improvements will require more detailed kinetics of the actual biochemical pathway.