FIELD OF INVENTION The present invention relates to a method for producing biodegradable resin from a sustainable resource through microorganism fermentation. In more particular, the disclosed method capable of producing biodegradable resin without employing any hazardous solvent for extracting the resin content from the microorganisms.

BACKGROUND OF THE INVENTION

Polyhydroxyalkanoate (PHA) is a storage polymer accumulated naturally in the cytoplasm of many microorganisms. Studies on the biotechnological production of PHA are being pursued because of its biodegradable and thermoplastic properties. Poly(3-hydroxybutyrate) [P(3HB)] is the most common type of PHA. However, pure P(3HB) has limited application as a thermoplastic material due to its unfavorable properties. The mechanical properties of P(3HB), i.e., the Young's Modulus (3.5 GPa) and the tensile strength (about 40 MPa) is comparable to those of polypropylene (PP). However, the elongation needed to break for P(3HB) (6 %) is poor compared to that of PP (400 %). Therefore, P(3HB) is suffer and more brittle in comparison to PP. Although natural P(3HB) is stiff and brittle, the P(3HB) film prepared using cold- drawing procedures have improved mechanical properties. In addition, the fibers of ultrahigh molecular weight P(3HB) produced by a recombinant bacterial strain also showed superior properties with a high tensile strength. P(3HB) is therefore a material with significant commercial potential. Nonetheless, conventional production of PHA using microorganisms requires an extraction step to separate the accumulated PHA or any other derivatives from the cellular mass of the microorganisms. The extraction step is not only tizne consuming as multiple solvents may be used but also recurs additional cost to the process. Moreover, the solvents used in the extraction method are organic based and can be environmental pollutant once discharged to the surrounding environment. In Japan patent publication no. 2008086238, a method for producing polyhydroxyalkanoate is disclosed. The claimed method employs Cupriavidus necator in fermentation for producing the PHA in a culture medium containing specifically butyric acid and/or butanol.

Another Japan patent publication no. 2000189183 provides a method to treat organic component in a vegetable oil waste without generating methane gas during the disposal process utilizing a microorganism species, while the microorganism used can generate PHA from the vegetable oil waste at the same time. Specifically, the sludge of the oil waste is subjected to an anaerobic treatment which is stopped at the acid fermentation step before proceeding to methane generation. Then fermented organic acid is separated from the sludge using centrifugation and the organic acid is further concentrated using ion exchange resin or by heating prior to adding the microorganisms species to the concentrated organic acid for producing PHA.

Further Japan patent publication no. 2002306190 offers a process to extract PHA from bacteria mass. In this application, the claimed process first crush the bacterial cell wall to release the PHA content thus forming a water soluble fraction and water insoluble fraction. Later, the water insoluble fraction is treated with an oxidizing agent to obtain the PHA.

Yu has filed an United State application no. 7141400 regarding a system and method for converting organic waste to thermoplastic material. The disclosed method first treats the organic waste through a first type of bacteria to convert the organic waste to organic acid followed by treating the formed organic acid with PHA-producing bacteria to polymerise the organic acid to form PHA.

A method for directly separating and purifying PHA in cells from a bacterial fermentation liquid is filed for an United Patent application with publication no. 2007072276. The fermentation liquid is first treated with physical force to break the cell wall of the bacteria to release PHA content followed by pH adjustment to alkaline condition and adding of anionic surfactant to precipitate the PHA content. SUMMARY OF THE INVENTION

The present invention aim to provide a process to produce PHA, particularly PoIy(S- hydroxybutyrate, from vegetable oil and/or animal fat or waste of vegetable oil and/or waste of animal fat utilizing a microbial approach.

Another object of the present invention is to provide a PHA producing process where the PHA can be used directly for manufacturing of biodegradable resin free from the need of solvent purification. Hence, the disclosed approach is much less hazardous then the available conventional process.

Further object of the present invention is to disclose a cost effective approach to produce PHA as the PHA produced can be used for plastic extrusion without tedious and expensive purification step as well as the necessary labor cost to carry out the purification step.

At least one of the preceding objects is met, in whole or in part, by the present invention, in which one of the embodiment of the present invention includes a method for producing biodegradable resins comprising the steps of culturing polyhydroxyalkanoate (PHA)-producing microorganisms in a culture medium containing plant oil as carbon source for a duration of 24 to 96 hours; harvesting the cultured PHA-producing microorganisms from the culture medium; drying the harvested PHA-producing microorganisms to form dried cell mass; and pulverizing the dried cell mass to a predetermined size forming resins powder.

In order to increase versatility of the produced biodegradable resin for different application, preferably the produced resin powder is mixed with at least one additive rendering the plastic produced thereon with the desired characteristic. The additive can be any one or combination of a filler/stabilizer or polymerization aid additive.

Pursuant to the preferred embodiment, the disclosed method may include an extruding step of the resin powder to produce biodegradable resin for various of application. It is in the preferred embodiment of the present invention that the PHA-producing microorganisms is any one or combination of Burkholderia sp. Alcaligenes sp. Pseudomonas sp. Cupriavidus necator Hl 6 in order to achieve high yield of PHA within the cellular matrix of the microorganism used thus the PHA can be used directly without the extraction method. Preferably, the culture medium is maintained within a temperature of 30 to 4O ⁰ C at apH of 6 to 8.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 showing the cultivation of the Cupriavidus necator Hl 6 in the culture medium at different stage of time (a) at 6 hours, (b) at 12 hours, (c) at

24 hours, (d) at 36 hours and (e) at 48 hours; and

Figure 2 is a flowchart depicting the work flow of the disclosed method.

DETAILED DESCRIPTION OF THE 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 embodiment describes herein is not intended as limitations on the scope of the invention.

Starter cultures were grown in NR medium for 24 h 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. hi order to prepare 1 L of MM, 2.8 g Of KH ₂ PO ₄ , 3.32 g Of Na ₂ HPO ₄ and 0.54 g of urea were added into 500 mL of distilled water. The medium was stirred thoroughly. Then the mixture was topped up to 1 L. The pH of the medium was adjusted to 7.0 before autoclaving. Compositions of trace elements (g/L) were FeCl ₃ .6H ₂ O (20), CaCl ₂ -H ₂ O (10), CuSO ₄ .5H ₂ O (0.03), MnCl ₂ .4H ₂ O (0.05), ZnSO ₄ .7H ₂ O (0.1). These solution was added to the MM at a final concentration of 10 mL/L. Oils were autoclaved separately and added into the MM. Sodium propionate was fed (2.5 gL ^'1 ) into the culture medium at 48 h and 60 h of cultivation in the case of synthesizing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co3HV)j. At the end of cultivation (72 h), cells were harvested by centrifugation at 10,000 x g for 10 min and freeze-dried.

The present invention includes a method for producing biodegradable resins comprising the steps of culturing polyhydroxyalkanoate (PHA)-producing microorganisms in a culture medium containing plant oil as carbon source for a duration of 24 to 96 hours; harvesting the cultured PHA-producing microorganisms from the culture medium; drying the harvested PHA-producing microorganisms to form dried cell mass; and pulverizing the dried cell mass to a predetermined size forming resins powder. One skilled in the art shall appreciate the fact that the culturing of the PHA-producing microorganism mentioned herein throughout the description can be started with a starter culture, preferably in a nutrient rich medium, for 16 to 48 hours at a temperature of 20 ⁰ C to 40 ⁰ C to acquire a pure and active PHA- producing microorganism prior to mass culturing the PHA producing microorganism in a fermentation tank. In the most preferred embodiment of the present invention, a •mineral salt medium is employed for fermentation containing 2.0 to 4.0 g Of KH ₂ PO ₄ , 2.0 to 4.Og Of Na ₂ HPO ₄ and 0.25 to 1.5g of urea in 500 mL of distilled water. Other trace element may be used in the mineral salt medium are FeCl ₃ .6H ₂ O, CaCl ₂ -H ₂ O, CuSO ₄ .5H ₂ O, MnCl ₂ .4H ₂ O, and ZnSO ₄ JH ₂ O which the concentration of the trace element can range from 0.001 to 20 g/L. Then, vegetable oil and/or animal fat to be used as the carbon source for culturing the PHA-producing microorganism is mixed with the culture medium and followed by inoculation of the starter culture into the culture medium. Nonetheless, both the culture medium (mineral salt medium) and the vegetable oil and/or animal fat are both autoclaved to rid off any other microorganism possibly presented in these medium which can potentially spoiled the PHA-producing process.

Apart from that, it is apparent to one skilled in the art that it is possible to employ different approach to harvest the cultured PHA-producing microorgasnism such as filtration, centrifugation and so on. La the most preferred embodiment, centrifugation is used in the present invention that the PHA-producing microorganism is harvested via centrifugation at 8000 to 15000 g for 3 to 15 minutes, m the preferred embodiment, the drying step is conducted through lyophilization to reduce the moisture or water content in the harvested cell mass though heat drying can be used as well.

According to another preferred embodiment, Sodium propionate may be added into the culture medium at a specified interval within 32 hours to 72 hours, more preferably at 48 hours and/or 60 hours, to stimulate production of poly(3- hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-cσ-3HV)] in the PHA-producing microorganism. Through this embodiment, the PHA accumulated in the microbial cell mass shall possess the specifically property desired by the user of the present invention.

Furthermore, in the preferred embodiment, the PHA-producing microorganisms can be any one or combination of Burkholderia sp., Alcaligenes sp., Pseudomonas sp. Yet, it is Cupriavidus necator Hl 6 most preferably employed in the present invention. It was found by the inventors of the present invention that utilization of vegetable oil and/or animal fat can greatly enhance the yield of the PHA ₅ specifically poly(3- hydroxybutyrate), in the PHA-producing microorganism. Moreover, vegetable oil waste and/or animal fat waste can be used as well in the present invention. Thus it avoids the need of solvent extraction to remove the cellular mass as amount of the non-PHA cellular mass is negligible comparing the high yielded PHA content available. Preferably, the vegetable oil can be any one or combination of Coconut oil, soybean oil, sunflower oil. Yet, palm oil and Jathropha oil are used in the most preferred embodiment.

To attain the optimal yield from the present invention, the culture medium with the added oil is maintained within a temperature of 30 to 40 ⁰ C at a pH of 6 to 8 in one of the preferred embodiments. Not only this condition favors cultivation of the PHA- producing microorganism but also increase yield of the PHA in the cell of microorganism. In respect to another embodiment, the resins powder may further mix with at least one additive and/or other types of resin powder in order to produce a biodegradable plastic or resin. For example, other resin such as, but not limited to, poly(lactic acid) (PLA) or poly(butylenes adipate/butylenes terephthalate) aliphatic-aromatic copolyester or polycaprolactone (PCL) can be used in combination with the resin powder produced via the disclosed method. Similarly, the additive used in the present invention any one or combination of a filler/stabilizer or polymerization aid additive. For example, the filler material can be cellulose or other fibers which are naturally derived or chemically derived. Further, the polymerization aid additive can be plasticizer such as glycerol to be added for rendering the produced resin with the desired characteristics.

In another embodiment, the resin powder acquired or mixture of the resin acquired through the above mentioned method can be further process by extruding the resin powder to produce resin or plastic which is preferably biodegradable.

Still another embodiment of the present invention includes resin derives from the process described throughout this specification.

The following example is intended to further illustrate the invention, without any intent for the invention to be limited to the specific embodiments described therein.

EXAMPLE 1

The bacterial strain used was Cupriavidus necator Hl 6. C. necator was grown for 24 h in nutrient rich (NR) medium containing 10 gL ^"1 meat extract, 10 gL ^"1 peptone and 2 gL ^'1 yeast extract at 30 ⁰ C.

Starter cultures were grown in NR medium for 24 h at 30 ⁰ C and 3% (v/v) of the inoculum was transferred into mineral salts medium (MM), which was incubated at 30 °C. In order to prepare 1 L of MM, 2.8 g Of KH ₂ PO ₄ , 3.32 g OfNa ₂ HPO ₄ and 0.54 g of urea were added into 500 mL of distilled water. The medium was stirred thoroughly. Then the mixture was topped up to 1 L. The pH of the medium was adjusted to 7.0 before autoclaving. Compositions of trace elements (g/L) were FeCl ₃ .6H ₂ O (20), CaCl ₂ -H ₂ O (1O) ₅ CuSO ₄ .5H ₂ O (0.03), MnCl ₂ .4H ₂ O (0.05), ZnS O ₄ .7H ₂ O (0.1). These solution was added to the MM at a final concentration of 10 mL/L. Oils were autoclaved separately and added into the MM. Sodium propionate was fed (2.5 gL ^"1 ) into the culture medium at 48 h and 60 h of cultivation in the case of synthesizing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]. At the end of cultivation (72 h), cells were harvested by centrifugation at 10,000 x g for 10 min and freeze-dried.

The following are examples of experimental data obtained:

Results obtained from shake flask culture

Table 1: Biosynthesis of P(3HB) from different plant oils by C. necator ^a

Incubated for 72 h at 30 ⁰ C, initial pH 7.0, 200 rpm. bPHA content in freeze-dried cells 3HB, 3-hydroxybutyrate

Table 2: Biosynthesis of PHBV from a mixture of plant oils and sodium propionate ⁸

aIncubated for 72 h at 30 °C, initial pH 7.O ₅ 200 rpm. Sodium propionate was added at

48 h (2.5 gU ¹ ) and 60 h (2.5 gl/ ¹ )

⁵ PHBV content in freeze-dried cells

3HB, 3-hydroxybutyrate; 3HV, 3-hydroxyvalerate

Based on the preliminary results obtained in small-scale experiments, the biosynthesis of PHA was scaled-up to 1OL and IOOL fermenters. P(3HB) accumulation of up to 85 wt% of the cell dry weight was obtained when C. necator cells were grown on CPKO.

The resulting cell dry weight was approximately 70 g/L.

EXAMPLE 2

PHAs are accumulated in the microbial cells in the form of water insoluble granules.

The existing technologies rely on the PHAs that have been extracted and purified from the microbial cells. This process involves the use of solvents, chemicals and the mixtures of solvents and chemicals. In a typical example, the cells that contain the

PHAs are subjected to hydrolysis by using a combination of enzymes and chemicals.

Once the cells are hydrolyzed, the PHA granules are released from the cells. The PHA granules are then separated from the hydrolyzed cell material by centrifugation.

Several washing steps are necessary in order to remove most of the cellular materials that may be attached to the PHA granules. Finally, the washed granules are dried and the final product appears as white or slightly brownish powder.

In the current invention, the cells obtained from the fermentation process were dried without any further treatment. The cells were filled PHA granules as can be seen in Figure 1. The dried cells that contain the PHA granules were then subjected to grinding to a predetermined size. Chunks of freeze dried cells were converted into powder form that was later mixed with other components to make the bioplastic resin.

EXAMPLE 3 The generalized formulation developed for the preparation of bioplastic resin is based on the following steps:

1) Defining the formulations

The following shows one example of the formulation of preparing bioplastic resin:

Table 3

2) Pre-mixing the materials and extrusion process

Typically biopolymers and other additives would be pre-mixed and homogenized in a mixer or homogenizer in many polymer manufacturing facilities. Then, it was relayed on gravimetric or volumetric feeders to deliver the correct formulation of polymers and additives to the extruder. However, the present invention used separated hoppers acted as an in-line mixed for the pre-mixing the material before the blending materials are delivered into the extruder. This ensures a constant flow rate to the extruder on a minute-by- minute second-by-second basis and ensures accuracy is maintained throughout the production run. Changes therein and other uses will occur to those skilled in the art which are encompassed within the scope of the invention as defined by the scope of the claims.