PHA Downstream Process

Enzymatic and Mechanical Cell Disruption Treatments

Combinations of enzymatic and mechanical cell disruption treatments have been used for recovering the intracellular PHAs [[1]]. Bacteria contain a significant proportion of their biomass as DNA, a jelly-like polymer, and disruption of the cells releases this DNA in the cell homogenate. Therefore, the homogenate can be quite viscous and difficult to process. Thus, enzymes such as benzonase (a commercial nuclease) may need to be added, for example at a concentration of 10 μL/L cell broth at pH 10, to reduce viscosity by digesting DNA and ease processing through certain mechanical cell disruption devices [[1]]. In some cases, the PHA pellet recovered from the digested cells has been further treated with ozone or peroxide to remove contaminants [[1]].

Supercritical Fluids

Supercritical fluids have attracted attention for PHA recovery [[2],[3],[4],[5]] (Table 1). Of particular interest is supercritical CO2 as it is inexpensive, readily available, does not leave behind a toxic residue, has a low reactivity, is nonflammable and has a moderate critical temperature (31°C) and pressure (7.29 MPa).

Supercritical fluids have been used to extract nearly 90% of the PHA in the biomass at purities ranging from 86% to 99% [[2][5]] (Table 1). A variety of other relatively less used methods of recovering PHA exist [[2]].

Recovery method Advantages Recovery agent Microorganism Yield*(Purity) Ref:
  Mechanical Disruption Less use of chemicals; Reduced polymer degradation;   Sonication, chloroform     Alcaligenes lata DSM1123   95%   [6]
Enzymatic Digestion (with or Without mechanical treatment Good polymer recovery; high purity; reduced use of chemicals other than enzymes   Alcalase, SDS, EDTA     Pseudomonas putida   90% (92.6%)   [7]
    Benzonase, Alcalase, lysozyme, Flavourzyme; microfluidizer   P. putida PGA1   99.2%   [8]
Supercritical fluids Low toxicity; low cost; high polymer purity CO2 Bacterial Cells 90% (99%) [9]
  • [1] Horowitz DM, Brennan EM (2010) Methods for the separation and purification of Biopolymers. European Patents 1,070,135,
  • [2] Jacquel N, Lo C-W, Wei Y-H, Wu H-S, Wang SS (2008) Isolation and purification of bacterial poly(3-hydroxyalkanoates). Biochem Eng J 39 (1):15-27.
  • [3] Darani KK, Reza Mozafari M (2010) Supercritical fluids technology in bioprocess industries: A review. J Biochem Technol 2 (1):144-152
  • [4] Khosravi-Darani K (2010) Research activities on supercritical fluid science in food biotechnology. Crit Rev Food Sci Nutr 50 (6):479-488
  • [5] Posada JA, Naranjo JM, López JA, Higuita JC, Cardona CA (2011) Design and analysis of poly-3-hydroxybutyrate production processes from crude glycerol. Process Biochem 46 (1):310-317. doi:10.1016/j.procbio.2010.09.003
  • [6] López-Cuellar M, Alba-Flores J, Rodríguez J, Pérez-Guevara F (2011) Production of polyhydroxyalkanoates (PHAs) with canola oil as carbon source. Int J Biol Macromol 48 (1):74-80
  • [7] Lo C-W, Wu H-S, Wei Y-H (2011) High throughput study of separation of poly(3-hydroxybutyrate) from recombinant Escherichia coli XL1 blue. J Taiwan Inst Chem E 42 (2):240-246. doi:10.1016/j.jtice.2010.08.001
  • [8] Kathiraser Y, Aroua MK, Ramachandran KB, Tan IKP (2007) Chemical characterization of medium chain length polyhydroxyalkanoates (PHAs) recovered by enzymatic treatment and ultrafiltration. J Chem Technol Biot 82 (9):847-855
  • [9] Tamer IM, Moo-Young M, Chisti Y (1998) Disruption of Alcaligenes latus for recovery of poly (ß-hydroxybutyric acid): Comparison of high-pressure homogenization, bead milling, and chemically induced lysis. Ind Eng Chem Res 37 (5):1807-1814