WO2011108916A2

WO2011108916A2 - A method for recovering an intracellular polyhydroxyalkanoate (pha)

Info

Publication number: WO2011108916A2
Application number: PCT/MY2010/000333
Authority: WO
Inventors: Mohd Ali Hassan; Mitra Mohammadi; Nor Asma Ab Razak; Mei Ling Chong; Suraini Abd Aziz; Amirul Al-Ashraf Abdullah; Hasfalina Che Man; Siti Nor Syairah Anis; Lai Yee Phang; Yoshihito Shirai; Wan Md Zin Wan Yunus
Original assignee: Universiti Putra Malaysia
Priority date: 2010-03-01
Filing date: 2010-12-30
Publication date: 2011-09-09
Also published as: WO2011108916A3; MY153891A

Abstract

The present invention relates to a method to recover an intracellular polyhydroxyalkanoates (PHA). The method includes the steps of (a) separating culture medium containing PHA-rich cells using centrifugation, (b) rinsing pellet obtained from step (a) with distilled water, (c) freeze- drying the pellet obtained in step (b), (d) adding distilled water to the freeze-dried sample, (e) separating non-PHA cellular material (NPCM) from PHA granules, (f) washing the pellet containing PHA from step (e) by distilled water and centrifuging for producing final pellet, and (g) freeze-drying the pellet produced from step (f).

Description

A METHOD FOR RECOVERING AN INTRACELLULAR POLYHYDROXYALKANOATE (PHA) FIELD OF INVENTION

The present invention relates to a method to recover an intracellular polyhydroxyalkanoates (PHA).

BACKGROUND OF INVENTION

PHA is an energy and/or carbon storage material synthesized and accumulated intracellular^ by numerous microorganisms, usually when essential nutritional elements such as nitrogen, phosphorous, oxygen, sulphur, or potassium is limited in the presence of excess carbon source. PHA provides the only renewable source of truly biodegradable plastic and the component elements are readily liberated as C0₂ and water, by degradation of microorganisms.

To produce PHA, a culture of microorganism is placed in a suitable medium and being fed with appropriate nutrients so that it multiplies rapidly. Once the population has reached a substantial level, the nutrient composition is changed to enable the microorganism to synthesize PHA. Depending on the species, carbon source, nutrients and culture conditions, the PHA biopolymers may account for up to 80 wt % of dry cell mass. Polyesters are deposited in the form of highly refractive granules in the cells. Depending upon the microorganism and the cultivation conditions, homo- or copolyesters with different hydroxyalkanic acids are generated. The PHA biopolymers are stored inside of the cells as discrete granules of about 0.2-0.6 μιτι in diameter. The inclusions contain about 5 to 10 wt % of water, and are largely amorphous. Each granule is surrounded by a phospholipid monolayer membrane in which proteins, including the PHA synthase are located. Other proteins (phasins) are presumed to be involved in stabilization of the amorphous hydrophobic PHA inclusions suspended in cell cytoplasm. The physical properties of PHAs are highly dependent upon their monomer units, and therefore, biodegradable polymers having a wide range of properties can be made by incorporating different monomer units.

Physico-chemical properties of PHAs are that higher surface energy, dyeability, printability, chemical digestibility in hot-alkaline medium and barrier properties. In general, the physical property of PHAs is similar to that of polypropylene. But, as its biodegradability, biocompatibility, piezoelectricity and optical activity are characteristics not possessed by common petrochemical resins.

Biological properties of PHAs are that they made from renewable sources and are aerobically and anaerobically biodegradable. Thermo-mechanical properties of PHAs are that strength, flexibility, toughness, exhibit (springy) elasticity and similarity to polyethylene and polypropylene. They are UV stable, in contrast to other bioplastics from polymers such as polylactic acid, partial ca. temperatures up to 180 °C and show a low permeation of water. The crystallinity can lie in the range of a few to 70%. Their average molecular size ranges from 1 ,000 to 2,000 kDa.

More than 150 different monomers can be combined within this family to give materials with extremely different properties. Polyhydroxyalkanoates (PHAs) are homopolymers or copolymers of hydroxyalkanoates, such as 3-hydroxybutyrate (3HB), 3-hydroxyvalerate =3HV), 4-ydroxyvalerate (4HV) and 3-hydroxyhexonate (3HH).

Poly(3-hydroxybutyrate) [P(3HB)] homopolymer, which the most extensively studied biopolyester, is rather stiff and highly crystalline. The material property of P(3HB) is similar to polypropylene, however, its high melting temperature of ca. 170 °C makes the processing of P(3HB) difficult. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymer is less stiff and tougher. P(3HB-co-3HV) copolymer shows higher elongation to break and reduced melting point ranging from 160 °C to 100 °C, depending on polymer composition (0-25 mol% 3HV).

Due to the similar mechanical properties to synthetic plastics, complete biodegradability and potential chemical pools for useful chiral compounds, PHAs have been drawn much attention for commercial application. But large scale industrialized production of PHAs has not been realized internationally. Actually, important contributors to cost of production are the fermentation substrate and downstream processing. Therefore, upstream to downstream strategies including development of better bacterial strains, efficient fermentation and recovery processes have been devoted to lower the production cost of PHAs.

Besides that, it is essential that a cheaper energy-saving technology, which is known as green technology is developed in order to protect our environment and contribute to the sustainable development. Green technology is the application of the environmental science to conserve the natural environment and resources and therefore it can help to curb the negative impacts of human involvement. The focus areas of this green technology include sustainability, reduction of waste and pollution, innovation and viability. Therefore, developing a process that allows a simple, efficient and less polluted recovery of PHAs is an attractive proposition.

SUMMARY OF INVENTION

Accordingly, the present invention provides a method for recovering an intracellular polyhydroxyalkanoate (PHA), the method includes the steps of (a) separating culture medium containing PHA-rich cells using centrifugation, (b) rinsing pellet obtained from step (a) with a liquid, preferably distilled water, (c) freeze-drying the pellet obtained in step (b), (d) adding liquid, preferably distilled water, to the freeze-dried sample, (e) separating non-PHA cellular material (NPCM) from PHA granules, (f) washing the pellet containing PHA from step (e) by distilled water and centrifuging for producing final pellet and (g) freeze-drying the pellet produced from step (f). 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

The present invention relates to a method to recover an intracellular 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.

The embodiments disclosed herein relate to novel method for the extraction and recovery of PHA polymer from biomass materials, wherein the biomass materials are derived from bacterial cell. PHAs are accumulated inside the cell and hence their extraction from the cell biomass is a critical step for economic production. The main objective is to produce a consistent, usable, clean and purified product while keeping the recovery process free of halogenated solvent.

The methods of the present invention are applicable to the recovery of PHA polymers produced by bacterial cell either naturally or through genetic engineering, or PHAs that are synthetically produced. PHA is a general class of microbial polyester which is 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. 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. Release of cell contents is vital to many investigations of bacterial metabolism. In any case, the cell disruption is a necessary operation for isolation or recovery of intracellular products. Improved bioseparation techniques are increasingly important for biotechnology because separation is often the limiting factor for the success of biological processes. The recovery portion of a process usually begins with the separation of cells and particles from the fermentation broth or conditioned cell culture medium. The choice of method depends primarily on whether the product to be recovered is contained inside the cell or is secreted into the medium. 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. 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. Selecting the best cell disruption method depends on the factors such as kind of cells and their history, sample volume and number of samples, distribution time, possible scale-up potential, effect on downstream purification processes, economics of disruption. The nature of the cell disruption process may influence the extent of product recovery, the ease of the subsequent purification steps, the nature of the suspensions to be processed and the form and quality of the final product. The characteristic monitored during the disruption process 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.

It has been identified in the present invention by using water extraction can improve the recovery of PHA from freeze-dried cell. In downstream processing, lyophilized biomass is usually employed since this prevents the separated product from changing its nature. Recently, transmission electron microscope examination of freeze-dried PHA granules revealed that the dry granules have a noncrystalline core and crystalline shell morphology.

In some cases, it may be desirable to add some water to liquid to partially depolymerize the PHA to facilitate the recovery and separation of the PHA depolymerization product from the non-PHA waste. The presence of the small amount of water at the depolymerizing/solubilizing temperature produces a liquid phase that can readily be separated from the insolubles at room temperature by filtration or the like. In general, PHA can be separated and recovered by using distilled water. Through centrifuge, cell debris and PHA materials in suspension being settled as pellet. Then, use water washing to remove remaining cell debris. Therefore, the PHA with high yield and purity can be obtained.

The results showed that by increasing the incubation time from 60 to 300 min, there is no significant difference on purity of PHA recovered. Therefore, it concludes that incubation time of 60 min is sufficient. In average, PHA purity of 92.6% and recovery yield of 93.4% was achieved by the proposed method. The proposed method is economically viable and comparable with other recovery methods in terms of the high percentage of PHA recovery and the least time- consuming. The process does not adversely affect the quality of PHA and is an environmentally suitable approach for recovery. Generally, it seems that this treatment can replace the conventional extraction for the recovery of PHA as it is more environmental friendly, non-toxic, cheap, simple, efficient and easy to handle. Therefore, this present invention system can be considered as a green technology.

EXAMPLE A culture medium containing PHA-rich cells and growth broth from Ralstonia sp. was separated using a centrifugation method such as a two-time centrifugation. Pellet produced was rinsed with distilled water twice to remove residual broth and again was centrifuged in the same condition. The supernatant was then discarded. The pellet was freeze-dried. Then it was stored at room temperature. 20 g/l of freeze-dried biomass containing PHA was treated with distilled water for 60-300 minutes at room temperature. For separating the non-PHA cellular material (NPCM) from the PHA granules, was used centrifugation method. The supernatant was then discarded and the pellet obtained was washed with distilled water, centrifuged and freeze-dried.

Claims

1. A method for recovering an intracellular polyhydroxyalkanoate (PHA), the method includes the steps of:

(a) separating culture medium containing PHA-rich cells using centrifugation;

(b) rinsing pellet obtained from step (a) with a liquid, preferably distilled water;

(c) freeze-drying the pellet obtained in step (b);

(d) adding liquid, preferably distilled water, to the freeze-dried sample;

(e) separating non-PHA cellular material (NPCM) from PHA granules;

(f) washing the pellet containing PHA from step (e) by distilled water and centrifuging for producing final pellet;

(g) freeze-drying the pellet produced from step (f).

2. The method as claimed in claim 1 wherein the PHA is extracted from Ralstonia sp. bacterial cells.

3. The method as claimed in claim 1 wherein the pellet is produced from bacterial cells after separation by centrifugation, preferably two-time centrifugation.

4. The method as claimed in claim 1 wherein the water extraction is conducted for about 60-300 minutes at room temperature without any mixing.

5. The method as claimed in claim 1 , wherein the NPCM is separated from PHA granules by centrifugation.

6. The method as claimed in claim 1 , wherein the PHA granules obtained from step (e), is rinsed with a liquid, preferably distilled water and mixed.

7. The method as claimed in claim 1 , wherein the NPCM is separated from PHA granules by centrifugation.

8. The method as claimed in claim 1 , wherein the final PHA granules are produced by freeze-drying the pellets.