Polyhydroxyalkanoate quantification in organic wastes and pure cultures using a single-step extraction and 1H NMR analysis.
In this study, a proton nuclear magnetic resonance (1H NMR) method was developed to quantitatively analyze polyhydroxyalkanoate (PHA) content in Cupriavidus necator H16, Azotobacter vinelandii AvOP, and mixed microbial cultures from the effluent of an agricultural waste treatment anaerobic digester. In contrast to previous methods, a single-step PHA extractive method using deuterated chloroform was established, thereby facilitating direct 1H NMR analysis. The accuracy of the method was verified through comparison with well-established gas chromatography (GC) methanolysis techniques. Nile blue fluorescence staining was also carried out to serve as an independent and qualitative indicator of intracellular PHA content. The results indicate that the 1H NMR method is appropriate for rapid and non-destructive quantification of overall PHA content and determination of PHA copolymer composition in a variety of cultures. Notably, this technique was effective in measuring PHA content in full-strength waste samples where high concentrations of background impurities and organic compounds are present. The straightforward procedures minimize error-introducing steps, require less time and materials, and result in an accurate method suitable for routine analyses.
Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT 84322-4105, USA.
Elisabeth
E
Linton
Asif
A
Rahman
Sridhar
S
Viamajala
Ronald C
RC
Sims
Charles D
CD
Miller
D000693:Anaerobiosis; D016948:Azotobacter vinelandii / Q000737:chemistry / Q000378:metabolism; D019149:Bioreactors; D020118:Cupriavidus necator / Q000737:chemistry / Q000378:metabolism; D004784:Environmental Monitoring; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D054813:Polyhydroxyalkanoates / Q000737:chemistry*; D014865:Waste Disposal, Fluid / Q000379:methods; D014874:Water Pollutants, Chemical / Q000737:chemistry*
D054813:Polyhydroxyalkanoates; D014874:Water Pollutants, Chemical