Purification of Polyhydroxyalkanoates

Jacquel and co-workers [5] gave an overview of the various PHA extraction methods used, along with the respective purity and recovery, from 1978 to 2006. Furthermore, Roller and co-workers [3], and Kunasundari and Sudesh [27] gave a thorough review up to 2010. In this section, the subsequent updates on PHA recovery are discussed. Table 6.1 shows a summary of the recovery methods with respect to the influencing factors in each study reported from 2011 to 2015. 

Recovery methods Bacterial strain Comments Results Reference 

NPCM digestion (alkaline) Recombinant Cupriavidus necator 0.1 M NaOH, polished with 20% (v/v) ethanol Purity and recovery in the range of 80-90 wt% [2] 

Non-halogenated solvent extraction (antisolvent method) Haloferax mediterranei Acetone Purity 98.4% recovery 91.4% [3] 

NPCM digestion (detergent) Ralstonia eutropha HI 6 Escherichia coli SDS, alkylbenzene sulfonate, AOS-40, ES 702, NaOH Purity 86% recovery 81% [28] 

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Pieper-Fiirst, U., Madkour, M.H., Mayer, F., and Steinhtichel, A. (1994) Purification and characterization of a 14-kDa protein that is hound to the surface of polyhydroxyalkanoic acid granules in Rhodococus ruber. J. Bacteriol., 176, 4328-4337. 

Gerngross, T.U., Snell, K.D., Peoples, O.P., Sinskey, A.J., Csuhai, E., Masamune, S., and Stubbe, J. (1994) Overexpression and purification of the soluble polyhydroxyalkanoate synthase from Alcaligenes eutrophus evidence for a required posttranslational modification for catalytic activity. Biochemistry, 33, 9311-9320. 

Qi, Q., Steinbtichel, A., and Rehm, B.H.A. (2000) In vitro synthesis of poly(3 -hydroxydecanoate) purification of type II polyhydroxyalkanoate synthases PhaCl and PhaC2 from Pseudomonas aeruginosa and development of an enzyme assay. Appl. Microbiol. Biotechnol., 54, 37-43. 

Ruth, K., Gmbelnik, A., Hartmann, R. et al. (2007) Efficient production of (R)-3-hydioxycaiboxylic acids by biotechnological conversion of polyhydroxyalkanoates and their purification. Biomacromolecules. 8, 279-286. 

Sudesh K, Abe H, Doi Y (2000) Synthesis, structure and properties of polyhydroxyalkanoates biological polyesters. Prog Polym Sd 25 1503-1555 Sugiyama A, Shiraki M, Kobayashi T, Morikawa G, Yamamoto M, Yamaoka M, Saito T (2002) Cloning and sequencing of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase from Acidovorax sp. strain SAl and purification of the enzyme. Curr Microbiol 45 123-127... 

Figure 2.9 Purification strategy for the polyhydroxyalkanoates. The recovery of polyhydroxyalkanoates is composed of three steps pre-treatment, extraction and purification.

C. H. Lu, Purification and separation of polyhydroxyalkanoates from bacteria, Master Thesis, Yuan Ze University, Taiwan, 2006. 

Kapritchkoff FM,Viotti AP, AUi RCP, Zuccolo M, PradeUaJGC, Maiorano AE, et al. Enzymatic recovery and purification of polyhydroxybutyrate produced by Ralstonia eutropha.J Biotechnol 2006 122 453-62. Padermshoke A, Katsumoto Y, Sato H, Ekgasit S, Noda I. Surface melting and crystaUization behavior of polyhydroxyalkanoates studied by attenuated total PHB reflection infrared method spectroscopy. Polymer 2004 45 6547-54. 

Papaneophytou CP, Velah EE, Pantazaki AA (2011) Purification and characterization of an extracellular medium-chain length polyhydroxyalkanoate depolymerase from Thermus thermophilus HB8. Polym Degrad Stab 96 670-678... 

Kim HM, Ryu KE, Bae KS, Rhee YH (2000c) Purification and characterization of extracellular medium-chain-length polyhydroxyalkanoate depolymerase from Pseudomonas sp. RY-1. J Biosci Bioeng 90 416-421... 

Miyazaki S, Takahashi K, Shiraki M, Saito T, Tezuka Y, Kasuya K (2000) Properties of a poly(3-hydroxybutyrate) depolymerase from Penicillium funiculosum. J Polym Environ 8 175-182 Mukai K, Yamada K, Doi Y (1992) Extracellular polyhydroxyalkanoate depolymerases and their inhibitor from Pseudomonas lemoignei. Int 1 Biol Macromol 14 235-239 MiiUer B, Jendrossek D (1993) Purification and properties of poly(3-hydroxyvaleric acid) depolymerase from Pseudomonas lemoignei. Appl Microbiol Biotechnol 38 487-492 Murase T, Iwata T, Doi T (2001a) Direct observation of enzymatic degradation behavior of poly[(R)-3-hydroxybutyrate] lamellar single crystals by atomic force microscopy. Macromolecules 34 5848-5853... 

Zinn M, Witholt B, Egli T (2001) Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate. Adv Dmg Deliv Rev 53 5-21 Zribi-Maaloul E, Trabelsi I, Elleuch L, Chouayekh H, Ben Salah R (2013) Purification and characterization of two polyhydroxyalcanoates from Bacillus cereus. Int J Biol Macromol 61 82-88... 

Microbial polymers (e.g. poly(3)-hydroxybutyrate-hydroxyvalerate) are excreted or stored by micro organisms cultivated on starch hydrolysates or lipidic mediums. Isolation and purification costs could be high for those products that are obtained Ifom complex mixtures. Monsanto stopped the commercialisation of its product Biopol in 1999. Since then, production has been low but some new producers are entering the market (e.g. Coopeazucar in Brazil which has built new facilities for a pilot plant production of these polyhydroxyalkanoates). 

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