Evaluation and optimization of FTIR spectroscopy to quantify PHA production by municipal wastewater sludge

Polyhydroxyalkanoate (PHA) is a family of naturally-occurring biopolymers synthesized by more than 300 microorganisms in the environment. These biopolymers have been investigated as a source material to substitute fossil fuel-based polymers; hence the synthesis of biopolymers and their characterization is a critical step in optimizing the process. Because of this, the biological production of PHA using PHA-producing microorganisms is currently the dominating process; however, the use of microbial mixed culture (MMC), such as wastewater sludge, is gaining attention. Different than pure cultures, MMC has higher culturing condition tolerance since the complex species composition and is easily obtained from wastewater treatment plants, which shortens the culturing time, lowers the cost, and promotes the application. The main constraint in MMC-based PHA is the extraction and quantification of PHA from the more complex matrix. In this paper, Fourier-transform infrared (FTIR) spectroscopy is evaluated to be used as a quantification method of PHA in MMC systems. Firstly, commercially available analytical standards, which consist of PHA/PHB, and two different solvents (chloroform and dichloromethane), were used and tested by this method, with KBr card and liquid cell methods, and the results are validated by gas chromatography mass spectrometry (GC/MS). The method was then tested using 12 samples from wastewater treatment plants. The PHA content in biomass varied from 3.42 w/w% to 1.22 w/w% following extraction with chloroform as solvent as determined by this method. In the four different combination standards, the best one is consisted of PHB and chloroform, and FTIR-liquid cell showed higher promise for PHA quantification in complex matrices.