Umeå University's logo

The replacement of fossil-based plastics with biobased and biodegradable alternatives has become an important research challenge in recent years, aiming to eliminate the negative environmental impact of persistent plastics in nature. In this report, design of experiments was successfully exploited to develop an efficient and sustainable method for extracting intracellular PHA from Photobacterium ganghwense C2.2 using dihydrolevoglucosenone (Cyrene) and ethanol as biobased solvents obtainable from sustainable sources. The extraction conditions were studied and optimized against the yield and molecular weight. The temperature range for the extraction was scouted by using differential scanning calorimetry, while size exclusion chromatography coupled to refractive index and multiangle light scattering detectors was used to assess the molecular weights of the extracted polymers. The examined ranges in the model were, respectively, 1.6–8.4% (w/v) of lyophilized cells content per 10 mL of solvent, 3–17 min extraction time, and temperatures from 116 to 144 °C. Time and temperature strongly affected the extraction yields and molecular weights of the obtained polymers while the concentration of bacterial biomass only effected the molecular weight. Several quadratic and interaction coefficients were significant in the well-fit partial least-squares regression models (R² > 0.8, Q² > 0.6) indicating that nonlinear effects and interacting parameter contributed to the optimization targets. The optimized extraction should be performed at 130 °C for 15 min with 2% loading of bacterial biomass. The predicted yield and molecular weight of the polymer matched the values obtained from the real experiment under the optimized conditions. The method setup provided similar yield and higher molecular weight in much shorter time compared to overnight Soxhlet extraction with CHCl₃. The clean ¹H nuclear magnetic resonance spectra of polymers extracted from bacteria indicate that high purity materials can be obtained using an optimized extraction scheme. Additionally, the Cyrene solvent could be recycled at least five times and still performed the extraction equally well as the fresh solvent. Finally, the current method demonstrated a high potential for scalability using a HP4750 stirred filtration cell. Three different filtration conditions were tested, achieving up to 97.4% recovery at 80 °C using a 0.3 μm glass fiber membrane, with a flux of 312.5 LMH.