Abstract:
Polyhydroxyalkanoates (PHA) is an intracellular polymer that is biologically synthesized by microorganisms as carbon and energy reserve material. This biodegradable material has properties similar to petrochemical based thermoplastics. Currently methods of PHA extraction and purification from bacterial suspension use solvent or halogenated-based approaches which are costly and pose severe environmental damage. This invention describes a combined treatment of alkaline and homogenization for extracting and purifying PHA, with higher PHA purity and recovery.

Description:
FIELD OF INVENTION 
       [0001]    The present invention relates to a method of extracting and purifying polyhydroxyalkanoate (PHA), a biodegradable plastic produced by bacterial cells. 
       BACKGROUND OF INVENTION 
       [0002]    PHA is a biodegradable, biocompatible, microbial thermoplastic which has the potential to replace petroleum-derived thermoplastics. The molecular weight of PHA is in the range of 50-1000 kDa with polymer characteristics that are similar to conventional plastics such as polypropylene. Moreover, PHA is produced from various renewable resources such as carbohydrates, organic acids, carbon dioxide and organic wastes. 
         [0003]    However, the use of biologically produced polymers is currently limited because of high production costs. P (3HB-co-3HV), a co-polymer that is commercially available currently costs more than US$5 per kilogram and is more expensive than polypropylene which is only US$1-2 per kilogram. Significant contributors to the cost of production are the productivity of PHA by the bacterial strain, carbon source and downstream processing. 
         [0004]    The difficulty of PHA recovery from microorganisms has been the primary obstacle to its commercial exploitation. The majority of separation processes that had been carried out involved the extraction of PHA from cells is not only costly but also not environmentally-friendly. Hence, a simpler, economical and environmentally friendly method of recovering intracellular PHA from microorganisms is needed to provide alternative to the conventional methods. 
       SUMMARY OF THE INVENTION 
       [0005]    This invention describes a method of extracting and purifying the bioplastic polyhydroxyalkanoate (PHA) from PHA-rich bacterial cells. The method includes the steps of (a) providing dried pellets containing PHA-rich bacterial cells, (b) pulverizing the dried pellets, (c) partially dissolving the ground pellets obtained from step (b) with an alkaline solution, (d) homogenizing the PHA-enriched alkaline solution obtained from step (c), (e) separating the non-PHA cellular material (NPCM) from the PHA-enriched alkaline solution and (f) recovering the PHA granules from the PHA-enriched alkaline solution. 
         [0006]    The present invention consists of several novel features and a combination of parts hereinafter fully described and illustrated in the accompanying description, it being understood that various changes in the details may be made without departing from the scope of the invention or sacrificing any of the advantages of the present invention. 
     
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
       [0007]    The present invention relates to a method of extracting and purifying PHA. Hereinafter, this specification will describe the present invention according to the preferred embodiments of the present invention. However, it is to be understood that limiting the description to the preferred embodiments of the invention is merely to facilitate discussion of the present invention and it is envisioned that those skilled in the art may devise various modifications and equivalents without departing from the scope of the appended claims. 
         [0008]    The embodiments disclosed herein relate to novel methods for the extraction and recovery of PHA polymer from biomass materials, wherein the biomass materials are derived from PHA-producing microorganisms. 
         [0009]    There have been a number of reports describing the solvent or halogenated extraction of PHA from biomass, primarily from PHA-producing microorganisms. The most common chemical used are chloroform, methylene chloride or 1,2-dichloroethane. Through this recovery method, more than 90% of the polymer in cells can be obtained. However, due to its chemical property that is hazardous to the environmental and costly operation, we have found that the methods are economically not feasible, particularly under conditions desired for commercial scale processing and extraction of PHA. 
         [0010]    The methods of the present invention are applicable to the recovery of PHA polymers produced by microorganisms either naturally or through genetic engineering, or PHAs that are synthetically produced. PHA is a general class of microbial polyester which are biodegradable and biocompatible properties. The physical properties can be regulated by varying the composition of the copolymers. PHA appears as discrete granules in the bacterial cells. PHAs are intracellular carbon and energy reserve materials accumulated by a wide range of bacteria. The PHA compounds are polyesters of hydroxyalkanoic acids. The monomers in the polymer are all in the D(−) configuration, implying the specificity in the biosynthetic route. PHA has molecular weights in the range of 200 to 3000 kDa, depending on the source of material. 
         [0011]    Owing to its location which is intracellular, the first step in extraction of PHA is cell disruption. Cell disruption comprised of mechanical and non-mechanical methods. 
         [0012]    Complete destruction of the wall and release of all intracellular components require destruction of the strength-providing components of the wall, i.e. peptidoglycan in Gram-negative bacteria. The cell disruption techniques with the best industrial potential are the mechanical methods. The characteristic monitored during the mechanical disruption include the efficiency of disruption (measured by amount of protein released, activity of enzymes or number of surviving cells) as well as physical properties, very important for downstream processing. Different characteristics of the disintegrated cells caused by different mechanical devices (e.g. size of cell debris, particle size distribution, viscosity) influence the efficiency of separation techniques used for the isolation of a desired product. 
         [0013]    It has been identified in the present invention by combining the chemical and mechanical processes can improve the recovery of PHA. Chemical treatment such as alkaline solution increases the permeability of the cells, causing partial protein released. While this may suffice in the partial separation of NPCM, more complete rupture may be required for the release of granular products such as bacterial inclusion bodies and PHA. Mechanical disruption poses high energy requirements and frequently causes the micronization (particle size reduction) of cell debris, thereby hindering further separations. On the combination of a chemical pretreatment such as alkaline solution, increased monovalent cation concentration with subsequent high pressure homogenization, it has been shown that the disruption is greatly enhanced. Maximum protein released is approached on a single pass, at reduced operating pressure. 
         [0014]    In a further embodiment of the present invention, different concentrations of NaOH were tested on the effectiveness of intracellular PHA recovery at 30° C. for 1 hour. For 0.1 molar NaOH treatment, protein released was 0.26 g/g biomass. Protein released increased to 0.42 g/g biomass at 0.5 molar NaOH. Besides, PHA purity obtained achieved 46% after treatment with 0.1 molar NaOH. Further increment in NaOH molarity up to 1 molar increased the protein released and PHA purity up to 67% and 0.54 g/g biomass, respectively. Thus, protein released and purity of PHA increased with the increase of NaOH concentration. On the other hand, the yield of PHA decreased with increased NaOH concentration from 0.26 g/g biomass or 80% yield at 0.1 molar NaOH to 0.20 g/g biomass or 64% yield at 1 molar NaOH. 
       EXAMPLE 
       [0015]    A culture medium containing PHA-rich cells and growth broth was separated using centrifugation method at 3500 rpm for 10 minutes. Pellet produced was rinsed with distilled water twice to remove residual fatty acids such as acetic and propionic acids. The supernatant was then discarded. The wet-pellet was dried in oven for 24 hours at 60° C. The dried biomass then was grounded by using mortar and pestle to make fine cells for further processing or stored at 4° C. 
         [0016]    5 g/L of biomass containing PHA was treated with 0.2 molar of NaOH for 60 minutes at 30° C. Homogenization was then carried out immediately. Homogenization was done by using rotor-stator type homogenizer at a fixed speed of 8000 rpm, the lowest speed to conserve energy and cost. The temperature of the sample was restricted to not exceed 35° C., thus cooling of sample with ice or cold water was required to prolong homogenization time. The final PHA obtained was rinsed twice with deionized water, centrifuged at 3500 rpm for 10 minutes and air-dried.