Polyhydroxyalkanoates (PHA)
C. Downstream process
Innovative downstream process of PHA production – Biological Recovery
Downstream process is an important aspect of PHA production carried out to disrupt the microbial cells, extract and purify the PHA from other cell components.
Stages in PHA Downstream Processing (Zainab-L et al., 2018)
Biomass Pre-treatment
The main objective of biomass pre-treatment is to simplify PHA retrieval from cell biomass. The first physical step in the PHA recovery process is the dilution of the cultures (fermentation broth) with water at a ratio of 1:1 (v/v) for the removal of any impurities and residual salts.
Cells are separated from the extracellular fluid through concentration/centrifugation at 4 °C, 8000 rpm for 10 min. The concentrated cells are subsequently dried by either lyophilisation (freeze drying) at − 44 °C, thermal drying at ˂ 100 °C or mechanical/physical methods to a moisture content of ~ 5%.
PHA extraction
The extraction step uses different techniques ranging from solvent, chemical, enzymes (protease and lysozyme) to biological method.
The solvent extraction method was first demonstrated by Lemoigne (1926) and remains the most commonly used method of PHA recovery in the laboratory. It involves the usage of chloroform to break the cell membrane and subsequent dissolution of the PHA. The unwanted cellular components and proteins are at this stage removed (filtered out). Although effective, but unsuitable for industrial applications due to the need for large volumes of hazardous solvents or costly enzymes.
In the biological method of PHA extraction (novel method from our laboratory), the protein layer of the cells is utilised by animals and insects and the PHA recovered from their feaces. Biological methods of PHA extraction may result in a cost-effective and environment-friendly process (Kunasundari et al., 2013; Murugan et al., 2016; Zainab-L and Sudesh 2019).
Biological recovery of PHA using (A) Laboratory rats (Sprague Dawley) (B) Mealworms
PHA Purification
The PHA is further purified by precipitation, washing, drying, and polishing. Methanol/ethanol are used for the precipitation of highly purified PHA with no reduction in molecular weight. Water and sodium hydroxide solution are used for the purification of biologically recovered PHA. The pure PHA is finally dried at room temperature.
References
- Kunasundari B, Murugaiyah V, Kaur G, Maurer FH, Sudesh K (2013). Revisiting the single cell protein application of Cupriavidus necator H16 and recovering bioplastic granules simultaneously. PLoS One 8(10): e78528.
- Lemoigne M (1926). Products of dehydration and of polymerization of β-hydroxybutyric acid. Bulletin de la Société de Chimie Biologique, 8, 770-782.
- Murugan P, Han L, Gan CY, Maurer FH, Sudesh K (2016). A new biological recovery approach for PHA using mealworm. Tenebrio molitor. Journal of Biotechnology, 239, 98-105.
- Zainab-L I, Ong SY, and Sudesh K (2018). “Polyhydroxyalkanoates,”in Kirk-Othmer Encyclopedia of Chemical Technology, no. May 2018, Hoboken, NJ, USA: John Wiley & Sons, Inc., 1-26.
- Zainab-L I, Sudesh K (2019). High cell density culture of Cupriavidus necator H16 and improved biological recovery of polyhydroxyalkanoates using mealworms. Journal of Biotechnology, 305, 35-42.
