Abstract
The determination of the monomer fractions in polyhydroxyalkanoates is of great importance for research on microbial-produced plastic material. The development of new process designs, the validation of mathematical models, and intelligent control strategies for production depend enormously on the correctness of the analyzed monomer fractions. Most of the available detection methods focus on the determination of the monomer fractions of the homopolymer poly(3-hydroxybutyrate). Only a few can analyze the monomer content in copolymers such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate), which usually require expensive measuring devices, a high preparation time or the use of environmentally harmful halogenated solvents such as chloroform or dichloromethane. This work presents a fast, simple, and inexpensive method for the analysis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with high-performance liquid chromatography. Samples from a bioreactor experiment for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with Cupriavidus necator H16 were examined regarding their monomer content using the new method and gas chromatography analysis, one of the most frequently used methods in literature. The results from our new method were validated using gas chromatography measurements and show excellent agreement.Key points∙ The presented HPLC method is an inexpensive, fast and environmentally friendly alternative to existing methods for quantification of monomeric composition of PHBV.∙ Validation with state of the art GC measurement exhibits excellent agreement over a broad range of PHBV monomer fractions.
Highlights
Non-degradable waste is polluting our planet more and more
For all steps up to the analysis of the hydrolysis products with highperformance liquid chromatography (HPLC), approx. 1 h and 15 min is required per sample
Correct analysis of PHAs is essential for the design of competitive microbial polymer production processes
Summary
Non-degradable waste is polluting our planet more and more. In particular, the accumulation of plastic in marineAppl Microbiol Biotechnol (2021) 105:4743–4749 variety of microorganisms and plants as storage compounds under limited conditions with inexpensive and renewable substrates (Koller et al 2005, 2010; Brigham and Riedel 2019; Riedel et al 2014; Koller and Braunegg 2018). There are a number of analytical methods in literature for determining the monomer content of PHAs (Koller and Rodrıguez-Contreras 2015; Tan et al 2014). Some methods, such as analysis with a transmission electron microscope (TEM) (Thomson et al 2011) or Nile red staining (Gorenflo et al 1999; Wu et al 2003; Pick and Rachutin-Zalogin 2012; Zuriani et al 2013), focus on the detection of the intracellular granules that contain the PHAs. Beside using harmful chemicals for TEM fixation, absolute quantification after staining with Nile red is often a problem. The monomers from hydrolyzed heteropolymers overlap and other hydrolysis products from the cell have an absorption maximum at 235 nm, which makes error-free analysis even more difficult (Law and Slepecky 1960a)
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