ualitative and quantitative evaluation of bacterial polyhydroxyalkanoates: a critical review of existing methodologies

Abstract

Polyhydroxyalkanoates (PHAs) are non-toxic aliphatic polyester biopolymers that are gaining traction as bio-based and biodegradable materials. To support the growing body of research on this subject, scientists and other stakeholders require insight into the synthesis, structure and quantity of PHA produced by bacterial isolates. To address the challenges that researchers face when seeking to evaluate bacterial PHA production, this manuscript presents a cohesive collection and critical evaluation of the methods described in literature, highlighting the applications, advantages, and disadvantages of each. Environmental samples are typically screened for PHA production based on uptake of lipophilic dyes. Thereafter, quantification of polyhydroxybutyrate and/or polyhydroxyvalerate monomers is typically performed using gas chromatography or high-performance liquid chromatography. These methods are robust with good specificity, but their application range is limited by non-availability of commercial standards. Methods based on lipophilic fluorescent dye emissions measured by spectrofluorometry or flow cytometry present simple, cost effective and efficient alternatives, but there are issues related to lack of specificity. For identification and quantification of monomers other than polyhydroxybutyrate and/or polyhydroxyvalerate, and to elucidate monomeric spatial arrangements within the polymer structure requires more sophisticated equipment and skilled analyses including gas chromatography-mass spectrometry, high performance liquid chromatography-mass spectrometry, and finally nuclear magnetic resonance. These methods are presented and discussed, along with other ancillary methods including Raman spectroscopy and Fourier-transform infrared spectroscopy, and methods based on molecular biology principles.