Rapid Method for Estimating Polyhydroxybutyrate Accumulation in Bacteria Using Sodium Hypochlorite

Abstract

This protocol outlines the use of the previously described sodium hypochlorite extraction method for estimating the accumulation of polyhydroxybutyrate (PHB) in bacteria. Sodium hypochlorite (NaClO) is widely used for PHB extraction as it oxidizes most components of the cells except PHB. We assessed the feasibility of using NaClO extraction for the estimation of PHB accumulation in bacterial cells (expressed as a percentage w/w). This allowed us to use a simple spectrophotometric measurement of the turbidity of the PHB extracted by NaClO as a semiquantitative estimation of PHB accumulation in the marine microorganisms Halomonas titanicae KHS3, Alteromonas sp., and Cobetia sp. However, this fast and easy protocol could be used for any bacterial species as long as some details are considered. This estimation exhibited a good correlation with the accumulation measured as dry cell weight or even with the accumulation measured by crotonic acid and HPLC quantifications. The key advantage of this protocol is how fast it allows an estimation of PHB accumulation in Halomonas , Alteromonas , and Cobetia cultures (results are available in 50 min), enabling the identification of the appropriate moment to harvest cells for further extraction, polymer characterization, and accurate quantification using more reliable and time-consuming methods. This protocol is very useful during bacterial cultivation for a quick evaluation of PHA accumulation without requiring (i) large volumes of cultures, (ii) a long time for analysis compared to dry cell weight, (iii) preparation of standard curves with sulfuric acid hydrolysis for crotonic acid quantification, or (iv) specific equipment and/or technical services for HPLC quantification. Key features • Fast and easy method for bacterial PHB content estimation in cultures of different marine microorganisms. It can be used in other PHB-accumulating bacteria. • Useful to explore culture conditions to achieve maximal PHB accumulation. • Useful to follow the kinetics of both PHB accumulation and mobilization throughout culture development. • In cultures with high (50%–70% dry cell weight) or very low (<15%) PHB accumulation, differences are visible by the naked eye before spectrophotometric measurement.