Application of the Alkaline-Digestion-HPLC Method to the Rapid Determination of Polyhydroxyalkanoate in Activated Sludge

Abstract

A new method for analyzing polyhydroxyalkanoate (PHA) in activated sludge based on alkaline digestion followed by high-performance liquid chromatography (HPLC) was evaluated. The effects of the concentration of NaOH, reaction time, and reaction temperature were examined using activatedsludge samples containing PHA and commercially purchased PHA. To determine PHA concentrations, 0.5 mL 2 N NaOH was added to 1 mL activated-sludge mixed liquor, and the resulting mixture was heated at 105°C in a drying oven for 1 h to convert 3-hydroxybutyrate units into 2-butenoate and 3-hydroxyvalerate units into 2-pentenoate. The mixture was then acidified with 0.5 mL of 2 N H 2 SO 4 , and the solids were removed and subsequently analyzed by HPLC. The present method gave results consistent with those obtained by the conventional method of methanolysis followed by gas chromatography. The present method can facilitate investigations of PHA in activated sludge by reducing the time and labor required for analysis and by eliminating the use of organic solvents.

Fast, inexpensive, and reliable HPLC method to determine monomer fractions in poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

“…In the present work, we demonstrate that neither an expensive ion exchange column (Satoh et al 2016 ) nor harmful solvents (Korotkova et al 1997 ) are necessary to measure copolymer composition, like 3HB and 3HV contents, in a short and cost-efficient way. For this purpose, culture samples from a bioreactor experiment with Cupriavidus necator H16 as well as PHBV test samples with 12% 3HV content are processed.…”

“…For the determination of hydroxyalkanoate concentration, 1 mL culture broth was alkaline digested and prepared as reported in Satoh et al (2016). Further, 10 μL of the digested and filtered samples were loaded on the reverse phase column Inertsil 100A ODS-3 (5 μm pore size, 250 × 4.6mm, MZ-Analysentechnik GmbH, Mainz, Germany) and eluted isocratically with 1 mL/min at 60 • C. The eluent consists of 92% low concentrated sulfuric acid (0.025% solution, Carl Roth, Karlsruhe, Germany) and 8% acetonitrile (Carl Roth, Karlsruhe).…”

“…The 3HB and 3HV concentrations of the samples were determined by crotonic (Carl Roth, Karlsruhe, Germany) and 2-pentenoic acid standard samples (Sigma-Aldrich, St. Louis, USA), respectively. Since the alkaline digestion does not lead to a complete conversion of 3HB to crotonic acid and 3HV to 2pentenoic acid, a PHBV sample (12% 3HV, Sigma-Aldrich /Merck, Darmstadt, Germany) with known concentration is needed to calculate the conversion yields Y 3H B and Y 3H V (Satoh et al 2016):…”

“…A good alternative is provided by liquid chromatography (LC) methods that have already been tested on high-performance liquid chromatography (HPLC) systems (Karr et al 1983 ; Hesselmann et al 1999 ; Korotkova et al 1997 ; Satoh et al 2016 ). Various detection systems can be used for analysis, such as UV detectors as commonly used in HPLC systems (Korotkova et al 1997 ).…”

“…For this purpose, culture samples from a bioreactor experiment with Cupriavidus necator H16 as well as PHBV test samples with 12% 3HV content are processed. For successful quantification of 3HV with a reverse phase column, an alkaline hydrolysis is required (Satoh et al 2016 ; Korotkova et al 1997 ; Watanabe et al 2012 ). In contrast to acidic hydrolysis, the chemical conversion of 3HV in the polymer takes place due to the higher nucleophilicity of hydroxide ions during alkaline hydrolysis.…”