Background

Polyhydroxyalkanoates (PHAs) are bacterial-origin polymers that share several characteristics with hydrocarbon-derived plastics. Among PHAs, polyhydroxybutyrates (PHBs) are the most common type, known for their biocompatibility and biodegradability. PHAs were first described in 1927 by Lemoigne [1] in bacteria from the genus Bacillus, and their presence in the cell was associated with the observation of Sudan Black–stained lipid inclusions. As early as 1958, Williamson and Wilkinson [2] described a method still widely used for the extraction and purification of PHAs from cells—treatment with an alkaline solution of sodium hypochlorite (NaClO). When Bacillus cells were suspended in such a solution, they were almost completely dissolved, including spores if present. Williamson and Wilkinson determined the best conditions (pH, temperature, incubation time) for the hypochlorite treatment and observed that the turbidity of the cell suspension started to decrease from the beginning of the treatment and reached a constant value when the cell lysis was complete. Moreover, the residual turbidity was proportionally higher in cell suspensions containing larger amounts of lipid inclusions as judged by microscopic observation, and they were able to show that the residual turbidity was strongly correlated with the concentration of PHA (µg/mL) remaining after the treatment. Hypochlorite treatment of Halomonas titanicae KHS3 cells that accumulated PHAs, followed by two washes with distilled water and one wash with ethanol, rendered a material that, upon NMR analysis, was found to be pure polyhydroxybutyrate [3]. PHB content could then be quantified by measuring the dry weight of hypochlorite-resistant material relative to the total dry biomass or, more precisely, through complete acid hydrolysis of the PHB material for spectrophotometric determination. However, neither of those possibilities allowed a rapid and continuous evaluation of the onset of the accumulation stage or the moment at which the cells reach a certain level of PHB content. It was then considered that the old turbidimetric measurements could be used to standardize a rapid, simple, and inexpensive method for the estimation of PHB content in growing cultures of PHB-accumulating strains. In the study conducted by Williamson and Wilkinson in 1958 [2], it was found that high cell density suspensions resulted in excessive residual turbidity, possibly due to incomplete cell lysis. As part of our protocol, we set a maximum cell density of OD_600nm = 2 for the assay. By using a simple spectrophotometric measurement of the remaining turbidity after NaClO hydrolysis, our fast protocol resulted in a semiquantitative estimation of the percentage of PHB accumulation expressed as weight/weight in the marine microorganisms Halomonas titanicae KHS3, Alteromonas sp., and Cobetia sp. This estimation exhibited a reliable estimation of the accumulation, providing values that correlated to PHB weight/dry cell weight or even with the accumulation measured by crotonic acid and HPLC quantifications. Moreover, when propionic acid was added to the culture media, Halomonas titanicae KHS3 accumulated the co-polymer poly-hydroxy-butyrate-valerate (PHBV), and it was also possible to estimate PHBV accumulation using the described protocol. Therefore, although not tested here, it is highly probable that this protocol will also work for other PHAs and bacterial species.