Poly(3-hydroxybutyrate) by recombinant phaeodactylum tricornutum via light-emitting diode cultivation and microwave-assisted extraction

Abstract

Bioplastic such as poly(3-hydroxybutyrate) (PHB) is an alternative approach to replace petroleum-derived plastic. However, high substrate cost and slow extraction process have hindered wide application of PHB. In this research, genetically modified Phaeodactylum tricornutum was used as an alternative PHB producer because it is able to utilize carbon dioxide. Ammonium and nitrate were used as nitrogen sources in the cultivation to suppress and activate PHB synthesis of this strain, respectively. The first phase of this study aimed to simplify the cultivation process by substituting the ammonium at its complete depletion point on day 5 with nitrate and enhance PHB productivity by different light wavelengths strategy. In the simplified cultivation method or known as one-step cultivation (OSC) for PHB synthesis, the steps in cell harvesting were removed when switching the nitrogen sources. Findings revealed that OSC is feasible as no sign of PHB synthesis suppression was present. In fact, the PHB productivity has improved to 9.75±0.64 (117.02±7.73 mg/L) from 7.40±0.52 mg/L/day (85.26±1.83 mg/L) which was achieved via multiple-step cultivation. In different light wavelength studies, red light was determined as better wavelength where the culture revealed higher specific growth rate and approximately 1.45-fold higher PHB productivity against white light culture. In the second phase of the study, PHB was extracted from wet biomass using microwave-assisted extraction (MAE) and propylene carbonate/isopropanol (PC/IPA). PC/IPA has high PHB solubility of 94.8±1.5%, boiling point of 99.1 oC, good dielectric properties, and miscible with water. Two-level full factorial design was used to evaluate the effect of the parameters that were A: extraction temperature (65-85 oC), B: extraction duration (5-15 min), and C: solvent-to-biomass ratio (5-15 mL/g) in PHB MAE. The results revealed that factor A and C significantly influenced the PHB recovery. The PHB MAE was optimized using central composite design. Based on prediction, the optimum PHB recovery of 97.89% can be achieved at 88 oC for 15 min with solvent-to-biomass ratio of 6.4 mL/g. The experimental PHB recovery of 95.63±0.70% with purity of 75±4% achieved by MAE method were significantly higher than conventional heating extraction (CHE) using chloroform (recovery: 79.53±2.87%, purity: 97±2%). The recovered PHB by MAE has high molecular weight of 1.4 x 106 Da. However, the melting point, melting enthalpy, and crystallinity were lower than PHB recovered from CHE using chloroform. The outcomes revealed the MAE system was excellent for PHB extraction as it offers high PHB recovery, cell breaking feature, safe processing conditions, wet biomass extraction and less hazardous compared to chloroform.