FIELD OF INVENTION

The present invention relates to a process for the extraction and purification of polyester granules, particularly polyhydroxyalkanoates (PHAs) from cell biomass. In more particular, the present invention provides a method for extracting and purifying PHA and other polyester granules by a natural and environmentally friendly process which applies the concept of digestive system of suitable animals.

BACKGROUND OF THE INVENTION

Plastics are undoubtedly one of the most useful materials invented by humans. Because of its diverse material properties and durability, lightweight and ease of processing, low cost and inertness, the application of plastics is widespread in our modern life. Commodity plastics are produced from petrochemical sources, which are non-renewable and will one day be depleted. In addition, petrochemical plastics are non-biodegradable and therefore cannot be assimilated by our natural ecosystem. In response to these issues and with the full awareness of the importance of plastics in our modern lifestyle, there has been a constantly growing interest in biodegradable plastics that can be produced from renewable resources.

PHAs and polylactic acid (PLA) are two examples of biodegradable and bio-based polyesters with thermoplastic properties. Many studies have been conducted on both of these bio-based polyesters. Many types of naturally occurring microorganisms produce PHAs under conditions of excess carbon source. The PHAs are accumulated in the form of water-insoluble granules in the cell cytoplasm. The use of genetic engineering techniques has enabled the construction of recombinant microorganisms and transgenic plants with the ability to produce various types of PHAs. Recently, it has also been demonstrated that recombinant bacterial cells can produce both PHAs and PLA.

A major challenge in the production of PHAs is the extraction and purification of the PHAs from cell biomass. Conventionally, the cell biomass can be directly subjected to PHA extraction by using a mixture of sodium hyperchloride, sodium hydroxide (or potassium hydroxide) and acids (such as hydrochloric acid or sulfuric acid) with or without enzyme pretreatment to hydrolyze the cell wall. The PHA granules released from the cells are then recovered by centrifugation and dried. Alternatively, the cell biomass can also be subjected to PHA extraction using a cocktail of solvents that specifically dissolves the PHA. The solvent solution containing the dissolved PHA is then concentrated and added into a non-solvent that results in the precipitation of the PHA, which can then be recovered and dried. The latter solvent extraction process results in a fairly high molecular weight PHA while the former aqueous extraction method using sodium hyperchloride results in PHA with lower molecular weights because the acidic and alkaline conditions breaks down the PHA molecules. Both these processes are not only tedious and costly they are also not environmentally friendly. These extraction processes cause the potentially eco-friendly PHAs higher in cost compared to petrochemical plastics. In addition, these processes also require much energy and results in carbon dioxide emission and other environmental pollutants such as wastewater contaminated with acids, bases, chemicals and solvents.

There are some technologies disclosed by the prior arts relating to methods for extracting or recovering PHAs from biomass. All of the technologies reported thus far in the prior arts rely on the use of solvents and/or chemicals, which causes the PHAs obtained not environmentally friendly.

There is a U.S. Patent No. US2006105440 relating to a method for producing PHA by extracting, separating and purifying PHA from biomass containing PHA having a weight average molecular weight of more than 2,000,000. The biomass is heated at 4O ⁰ C to 500 ⁰ C before an aprotic organic solvent is added therein. It can be further heated at 40 ⁰ C to 200 ⁰ C. The aprotic solvent can also be used together with water and/or alcohol. This method is capable of producing PHA with good operability, however a treatment of the aprotic organic solvent is required.

Another U.S. Patent No. US2005222373 also relates to a method for producing PHA crystal. This method comprises mixing a solution of PHA in a good solvent with a poor solvent at 50 ⁰ C to 13O ⁰ C to precipitate PHA. PHA crystal with fluidity which is conventionally difficult to obtain can be produced by this method. However, this method still applies a solvent extraction concept in which specific chemicals are required.

U.S. Patent No. US5918747 also discloses a process for recovering PHAs using centrifugal fractionation from a biological source material. The process includes the steps of comminuting the biological source material in a fluid, partitioning the PHA from the other components of the biological source material by centrifugal fractionation to form a solid-solid separation and recovering the PHA. Even though a centrifugation step is added to facilitate the solid-solid separation, mere is still a need for various chemicals such as chlorinated carbon solvents and organic solvents as the fluid.

A process for recovering PHA using air classification is also disclosed in U.S. Patent No. US5849854. This method comprises comminuting the biological source material, air-classifying the biological source material such that the PHA particles are separated from other components of the biological source material, and recovering the PHA. A suitable fluid is still required for this process and a few fractionation steps are also required to be carried out before the PHA is obtained.

A PCT publication No. WO2006103699 also relates to a process for the extraction of PHA from bacteria using commercially available cell lytic actinomycetes culture. The PHA producing bacteria is cultured for 2 to 3 days and heated at 50 ⁰ C to 80 ⁰ C, before the actinomycetes culture is added into the bacterial culture. The PHA is recovered by a water immiscible solvent or a surfactant and a chelating agent.

Most of the technologies disclosed in the prior arts are based on the concept of solvent extraction and requires various complicated techniques. Based on the foregoing, there is clearly a need for a simple, natural and economical process for recovering PHAs and other polyester materials from biomass.

SUMMARY OF INVENTION

The primary object of the present invention is to provide a simple and innovative method for extraction and purification of PHAs and other polyesters from biological sources.

Another object of the present invention is to develop a method for extracting and purifying polyester granules, particularly PHAs from bacterial cells without using organic solvents, which is completely natural, environmentally friendly and cost- saving.

Still another object of the present invention is to provide a method for recovering polyester granules which applies the concept of digestive system of suitable animals.

At least one of the preceding objects is met, in whole or in part, by the present invention, in which one of the embodiments of the present invention describes a process for recovering polyesters from polyester-containing biomass comprising feeding the biomass to an animal which excretes fecal pellets containing the polyesters; and separating the polyesters from the fecal pellets. In one of the preferred embodiments of the present invention, the polyesters are particularly PHAs or PLA. Preferably, the PHAs are poly(3-hydroxybutyrate), poly(3- hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxybutyrate-co-3- hydroxyhexanoate), poly(3-hydroxybutyrate-co-4-hydroxybutyrate), poly(3- hydroxybutyrate-co-S-hydroxyvalerate-co-S-hydroxyhexanoate), poly(3- hydroxyhexanoate-co-S-hydroxyoctanoate-co-S-hydroxydecanoate -co-S- hydroxydodecanoate-co-3-hydroxytetradecanoate) or a combination of any two or more thereof.

Another preferred embodiment of the present invention discloses that the biomass is derived from an organism containing polyesters, particularly PHAs. Preferably, the biomass is derived from a plant, an algae, a cyanobacterium, a bacterium or a combination of any two or more thereof.

Still another preferred embodiment of the present invention discloses that the animal is a rodent, a goat, a sheep, a cow, a bird or an aquatic organism.

Further embodiment of the present invention discloses a process for recovering polyesters from polyester-containing biomass which further comprises a step of formulating the biomass into an animal feed before feeding it to the animal. Preferably, the biomass is subjected to a heat treatment before being formulated into the animal feed.

Another further embodiment of the present invention is a process for recovering polyesters from polyester-containing biomass which further comprises a step of purifying the polyesters from the fecal pellets. These polyesters can be purified by removing water-soluble components from water-insoluble polyester-containing components within the fecal pellets.

Yet another further embodiment of the present invention is a process for recovering polyesters from polyester-containing biomass which further comprises a step of drying the polyesters recovered. A step of pelletizing the polyesters after drying can also be carried out according to the preferred embodiment of the present invention.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The embodiments described herein are not intended as limitations on the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of facilitating an understanding of the invention, there is illustrated in the accompanying drawing the preferred embodiments from an inspection of which when considered in connection with the following description, the invention, its construction and operation and many of its advantages would be readily understood and appreciated.

Figure 1 shows the morphology of Cupriavidus necator cells, one of the example of bacterial cells used for the the production of PHAs as described by one of the preferred embodiments of the present invention, in the early stages of cultivation (a), just before harvesting (b) and after freeze-drying (c). The images of cultivation and harvesting are phase contrast light microscopy images as seen using 1000 times magnification, whereas the image of the freeze-dried cells is taken using a digital camera.

Figure 2 is the morphology and PHA content in fecal pellets of rats, one of the example of animal employed for recovering the PHAs as described by one of the preferred embodiments of the present invention, that have consumed the freeze-dried cells shown in Fig.l.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a process for the extraction and purification of PHA as well as other polyester granules from cell biomass. In more particular, the present invention provides a method for extracting and purifying PHA and other polyester granules by a natural and environmentally friendly process which applies the concept of digestive system of suitable animals.

Hereinafter, the invention shall be described according to the preferred embodiments of the present invention and by referring to the accompanying description and drawings. However, it is to be understood that limiting the description to the preferred embodiments of the invention and to the drawings is merely to facilitate discussion of the present invention and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim.

The present invention discloses a process for recovering polyesters from polyester- containing biomass comprising feeding the biomass to an animal which excretes fecal pellets containing the polyesters; and separating the polyesters from the fecal pellets.

The process disclosed in the present invention is a completely natural biological process useful for the extraction and purification of water-insoluble polyesters from cell biomass. This process applies merely the natural digestive system of a suitable animal without using any chemicals or solvents for the recovery of polyesters. Therefore, the PHAs obtained are truly bio-based and capable of satisfying the need for an environmentally friendly and economically viable polyester material.

One skilled in the art shall appreciate the fact that the biomass employed is derived from an organism containing polyesters, particularly PHAs. These organisms are capable of producing and accumulating PHAs naturally. Preferably, the organism is a plant, an algae, a cyanobacterium, a bacterium or a combination of any two or more thereof. The production of PHAs in commercial scale involves the cultivation of large quantities of microorganisms, such as bacterial or cyanobacterial cells in bioreactors. The cells are preferably fed with growth nutrients as well as carbon sources for the synthesis of PHAs. In a typical fermentation process, the quantity of PHAs accumulated by the cells can amount up to 80 wt% of the cell dry weight. The carbon sources that can be used for the synthesis of PHAs include sugars (such as fructose), vegetable oils (such as soybean oil and palm oil) and agroindustry byproducts among other substrates. The fermentation process takes approximately 36 to 60 hours after which the cells are harvested by centrifugation or allowed to sediment naturally into a concentrated cell biomass.

hi the present invention, the polyester-containing cell biomass can be fed to the suitable animal directly, hi accordance with the preferred embodiment of the present invention, the process disclosed can further comprise a step of formulating the biomass into an animal feed before feeding it to the animal. Initially, the cell biomass, which can be a wet cell paste obtained at the end of a typical fermentation process of the organism is used as the component to be formulated into the animal feed. Preferably, the cell biomass should be a source of nutrient (protein and lipid) for the animals. Ideally, this should be single cell protein (SCP) containing PHAs. Preferably, the biomass is subjected to a heat treatment before being formulated into the animal feed. Besides the concentrated wet cell paste, the formulated animal feed can also comprise starch and soybean meal. This mixture can then be extruded into the form of dry pellets before feeding to animals.

In accordance with the preferred embodiment of the present invention, the suitable animal employed is a rodent, a goat, a sheep, a cow, a bird or an aquatic organism. However, the present invention does not intend to limit the use of other suitable animals available, providing that the animal is capable of excreting PHAs or other polyester granules in its fecal pellets after consuming PHAs- or polyesters-containing biomass from their daily dietary or formulated animal feed.

The digestive system of the suitable animals is capable of hydrolyzing the biomass material using various enzymes available in the digestive tract. These enzymes can specifically breakdown or hydrolyze proteins, lipids and carbohydrates into small water-soluble molecules including amino acids, fatty acids, glucose that are eventually absorbed as nutrients. Those compounds that cannot be hydrolyzed such as PHAs will be excreted. The digestive tract of the animals may contain microorganisms that possess specific enzymes for the hydrolysis of PHAs. However, the retention time of the cells with PHAs in the digestive tract of animals is too short for significant hydrolysis of PHA to take place.

The hydrolysis process releases the water-insoluble polyester granules from the cell biomass. The lipid and protein components of cells are naturally hydrolyzed and absorbed by the animal's digestive tract. Meanwhile, the water-insoluble polyester granules such as PHAs are left intact as there is no suitable enzyme in the animal's digestive tract that is capable of hydrolyzing the granules. As the granules pass through the animal's digestive tract, the granules are naturally concentrated and excreted in the form of fecal pellets. The fecal pellets are therefore rich in PHA or other polyester granules.

Subsequently, the fecal pellets can be collected from the suitable animals as early as the following day after the feed that contains the PHA or polyester granules is consumed. Depending on the type of animal and the environment where the fecal pellets are distributed, the subsequent process of purifying the PHA or polyester granules from the fecal pellets can be varied from 1 day to more than 6 months. The fecal pellets of rats for example with a PHA content of more than 80 wt% can be kept for several years before subsequent processing steps. The collected pellets can also be used directly as plastic resin for certain applications such as for example mulching films and seedling bags. For higher purity PHAs or other polyester granules, the pellets are subjected to a further purification process in which they can be homogenized and washed with cold or hot water. This washing process removes water-soluble components from the PHA granules. Finally, the washed PHA granules that are white and pure can be recovered by various methods such as filtration, centrifugation and/or sedimentation. This washing process can be modified depending on the type of animals employed and the concentration of PHA granules in their feces, as well as the final application of the PHA.

According to a further embodiment of the present invention, a step of drying the polyesters recovered can also be carried out. This is followed by a step of pelletizing the polyesters after drying. The entire extraction and purification process can be carried out at ambient conditions. This process can also be further combined with the conventional solvent and/chemical extraction and purification process to remove traces of proteins and lipids that may still coexist with the PHA granules. In this case, the quantity of solvents and/or chemicals will be greatly reduced compared to the methods that rely solely on solvents and/or chemicals.

The major type of polyesters recovered or obtained from the present process are PHAs and PLAs. Preferably, the PHAs are poly(3-hydroxybutyrate), poly(3- hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxybutyrate-co-3- hydroxyhexanoate), poly(3-hydroxybutyrate-co-4-hydroxybutyrate), poly(3- hydroxybutyrate-co-S-hydroxyvalerate-co-S-hydroxyhexanoate), poly(3- hydroxyhexanoate-co-3-hydroxyoctanoate-co-3-hydroxydecanoate -co-3- hydroxydodecanoate-co-3-hydroxytetradecanoate) or a combination of any two or more thereof.

The analysis conducted after the PHAs extraction and purification shows that the process disclosed is capable of achieving a presence of 50% to 95% of PHA content by weight.

The present disclosure includes as contained in the appended claims, as well as that of the foregoing description. Although this invention has been described in its preferred form with a degree of particularity, it is understood that the present disclosure of the preferred form has been made only by way of example and that numerous changes in the details of construction and the combination and arrangements of parts may be resorted to without departing from the scope of the invention.

EXAMPLE

Examples are provided below to illustrate different aspects and preferred embodiments of the present invention. The example is given solely for the purpose of illustration, and is not to be construed as limitations of the present invention since many variations thereof are possible without departing from its spirit and scope.

Example 1 The bacterial strain used for producing PHAs was C. necator Hl 6. C. necator was chosen because it is a strain that has been widely used and shows no signs of pathogenicity. In addition, this strain was originally developed as a source of single cell protein (SCP) in the 1970s. C. necator was grown for 24 hour in nutrient-rich (NR) medium at 30 ⁰ C. The inoculum preparation medium is tabulated in Table 1. Table 1

Precultures were grown in NR medium for 12 to 24 hour at 30 ⁰ C and 3% (v/v) of the inoculum was transferred into mineral salts medium (MM), which was incubated at 30 ⁰ C. 1 L of MM was prepared by adding 2.8 g Of KH ₂ PO ₄ , 3.32 g OfNa ₂ HPO ₄ and 0.54 g of urea into distilled water. The medium used for the biosynthesis of poly(3- hydroxybutyrate) [P(3HB)] is shown in Table 2.

Table 2

Trace elements solution as shown in Table 3 was added to the MM. The stock composition of the trace elements solution is measured by g/L in 0.1 N HCl. Oils were then autoclaved separately and added into the MM. At the end of cultivation (60 h), cells were harvested by centrifugation at 10,000 x g for 10 min and freeze-dried. Table 3

Table 4 shows the production of P(3HB) by C. necator cells from two types of palm oil products. The cells were incubated for 60 hours at 30 ⁰ C, freeze-dried and used directly as an animal feed. The content of P(3HB), which is the type of PHAs recovered from the freeze-dried cells in this example, is also demonstrated in Table 4.

Table 4

Figure 1 shows the transformation of the C. necator cells before and after the accumulation of PHA granules. The cells containing large quantities of PHAs appear fatter and reveal transparent granules in the cells. The freeze-dried cells have a flaky morphology with a fruity smell when the cells were cultivated in the mineral medium with palm oil as the sole carbon source.

The freeze-dried cells were then quantified and known amounts were left in a petridish to be consumed voluntarily by house rats. A web cam was mounted to monitor and confirm that the freeze-dried cells were actually eaten by rats. This experiment was continued over a period of 6 to 12 months. It was found that the rats showed high preference for the freeze-dried cells.

The fecal pellets of the rats were then collected from various places in the vicinity of the feeding location. These fecal pellets were easily found, which were usually white or grayish in color. The collected fecal pellets were subjected to various analyses, which confirmed the presence of 50-100% by weight PHA. Figure 2 illustrates the content in fecal pellets of rats that have consumed the P(3HB)-containing freeze-dried cells.