Journal of Biotechnology 239 (2016) 98–105 Contents lists available at ScienceDirect Journal of Biotechnology j ourna l ho me pa ge: www.elsevier.com/locate/jbiotec A new biological recovery approach for PHA using mealworm, Tenebrio molitor Paramasivam Murugan a , Lizhu Han a , Chee-Yuen Gan b , Frans H.J. Maurer c , Kumar Sudesh a,∗ a Ecobiomaterial Research Laboratory, School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia b Analytical Biochemistry Research Centre, Universiti Sains Malaysia, 11800 Penang, Malaysia c Department of Chemistry, Polymer & Materials Chemistry, Lund University, Box 124, Lund SE-221 00, Sweden a r t i c l e i n f o Article history: Received 19 July 2016 Received in revised form 5 October 2016 Accepted 12 October 2016 Available online 13 October 2016 Keywords: Cupriavidus necator Polyhydroxyalkanoates Biological recovery Mealworm a b s t r a c t Bacterial polyhydroxyalkanoates (PHA) are expensive partly due to the recovery and purification pro- cesses. Thus, many studies have been carried out in order to minimize the cost. Here we report on the use of mealworm, which is the larva of mealworm beetle (Tenebrio molitor) to recover PHA granules from Cupriavidus necator. Mealworms were shown to readily consume the freeze-dried C. necator cells and excrete the PHA granules in the form of whitish feces. Further purification using water, detergent and heat resulted in almost 100% pure PHA granules. Comparison with chloroform extraction showed no signs of reduction in the molecular weight and dispersion of the PHA molecules. Scanning electron microscopy and dynamic light scattering measurements revealed that the biologically recovered PHA granules retained their native spherical morphology. The PHA granules were subjected to a battery of tests to determine their purity and properties in comparison to the chloroform extracted PHA. This study has demonstrated the possibility of using mealworms as a biological agent to partially purify the PHA granules. © 2016 Elsevier B.V. All rights reserved. 1. Introduction In this modern age, plastics are ubiquitous and human life without plastic materials is unimaginable. Although plastics are beneficial for human’s day to day life, dumping of plastic waste products into the natural environment has led to plastic pollu- tion. This has negatively affected the land and marine wildlife (Eriksen et al., 2014; Nelms et al., 2015). Recent revelations regard- ing microplastics have aggravated the problem and revealed the greater dangers of plastic materials (Law and Thompson, 2014; Lönnstedt and Eklöv, 2016). A possible solution for these problems Abbreviations: PHA, polyhydroxyalkanoate; C.necator, Cupriavidus necator; P(3HB-co-3HHx), Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)-; SDS, sodium dodecyl sulphate; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel elec- trophoresis; GC, gas chromatography; CME, caprylic methyl ester; Mw, weight average molecular weight; Mn, number average molecular weight; TGA, thermo gravimetric analysis; SEM, scanning electron micrograpy; ATR-FTIR, attenuated total reflectance- fourier transform infrared spectroscopy; CDW, cell dry weight; NMR, nuclear magnetic resonance spectroscopy; DSC, differential scanning calorimeter; Tm, melting temperature. ∗ Corresponding author. E-mail address: ksudesh@usm.my (K. Sudesh). is the production and use of biodegradable plastics. Polyhydrox- yalkanoates (PHAs) are biological polyesters produced by certain bacteria as cell inclusion bodies when there is a limitation of essential nutrients but in the presence of excessive carbon source (Anderson and Dawes, 1990; Pohlmann et al., 2006). PHAs are known to be degradable by soil bacteria and fungi (Boyandin et al., 2013) thus making them environmentally friendly plastics. There- fore, one can expect the PHA-based microplastics to be completely mineralized in the environment. Among the various types of PHAs that can be synthesized by bacteria, poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) [P(3HB-co-3HHx)] is a thermoplastic that has similar mechanical and physical properties comparable to some commodity plastics of petrochemical origin (Doi et al., 1995). How- ever, the cost of PHAs is more than petrochemical plastics. The production and use of PHAs at industrial level is still at its infancy due to the high cost (Li et al., 2016). One of the reasons for the high cost of PHAs is the recovery process. Since PHAs are accumulated in the bacterial cell cytoplasm, it is necessary to lyse the cells in order to recover the PHA granules. In addition, the PHA granules are closely associated with several types of proteins which are located on the surface of the granules (Bresan et al., 2016; Sudesh et al., 2004). Most of the PHA extraction processes are developed using organic solvents such as chloroform and dichloromethane (Choi http://dx.doi.org/10.1016/j.jbiotec.2016.10.012 0168-1656/© 2016 Elsevier B.V. All rights reserved.