OIL OR WASTE PALM OIL

1. TECHNICAL FIELD OF THE INVENTION

The present invention relates to a process for the production of biopolymer such as poly(3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) and derivatives thereof by using waste fish oil or waste palm oil as carbon feedstock for the biosynthesis process.

2. BACKGROUND OF THE INVENTION

As environmental awareness is increasing, more concerns have been raised on problems due to using of non-biodegradable polymers which tend to affect ecological systems by their long term presence. The cost for handling disposal of conventional petrochemical-based plastics such as polyvinyl chloride (PVC) is increasing and release of hazards from waste incineration such as dioxin emission makes synthetic waste management an arduous task. Thus there is a growing interest to source for a method of producing inexpensive alternatives such as producing of biodegradable polymers or biocompatible polymers. Among those biodegradable polymers, polyhydroxyalkanoates (PHAs) are of great interest and have resulted in an interesting new source of commodity polymers. PHAs are produced by microorganisms and stored in the cell cytoplasm as water-insoluble inclusions. Different structural analogs of PHA polymer can be produced, depending on the microorganism and substrate used as feedstock. Poly (3-hydroxybuty rate), i.e. P(3HB) is the most abundant polymer produced in nature, a linear unbranched polymer built up of (R)-3 hydroxybutyric acid monomers.

In the past, PHA was produced from microorganisms using simple sugars, free fatty acids and triacylglycerols. The cost of the carbon substrate reportedly contributes to more than 50% of the production cost of most bioproducts. Therefore, there is a need to source for inexpensive renewable materials such as agriculture and industrial coproducts as feedstocks for PHA production.

It would hence be advantageous if the above shortcomings can be alleviated by having waste fish oil or waste palm oil as a promising alternative carbon feedstock for the production of PHA. The present invention uses microorganisms of genus Cupriavidus necator for biosynthesis of PHAs or any other biodegradable polymers production. Fish oil has high amount of palmitic acid and oleic acid, while Cupriavidus necator is known to well utilize these fatty acids for PHA conversion. 3. SUMMARY OF THE INVENTION

Accordingly, it is the primary aim of the present invention to provide a process for the production of biopolymer from waste fish oil or waste palm oil wherein waste fish oil or waste palm oil is used as the carbon feedstock in the production of biodegradable polymer.

It is yet another object of the present invention to provide a process for the production of biopolymer from waste fish oil or waste palm oil wherein the biopolymer produced resembles the characteristic of a thermoplastic.

It is yet a further object of the present invention to provide a process for the production of biopolymer from waste fish oil or waste palm oil to replace conventional petrochemical-based plastics so as to reduce non-biodegradable solid wastes.

Yet another object of the present invention is to provide a process for the production of biopolymer from waste fish oil or waste palm oil to reduce cost for handling disposal of conventional petrochemical-based plastics such as polyvinyl chloride (PVC).

Yet another object of the present invention is to provide a process for the production of biopolymer from waste fish oil or waste palm oil as another alternative carbon feedstock to reduce the cost of PHAs production. Other and further objects of the invention will become apparent with an understanding of the following detailed description of the invention or upon employment of the invention in practice.

According to a preferred embodiment of the present invention there is provided,

A process for the production of biopolymer comprising the steps of, i. culruring biopolymer producing bacteria in nutrient medium; ii. forming and accumulating the biopolymer by adding carbon feedstock to said culture; iii. recovering the biopolymer from said culture; characterised in that said added carbon feedstock is waste fish oil or waste palm oil.

4. BRIEF DESCRIPTION OF THE DRAWINGS

Other aspect of the present invention and their advantages will be discerned after studying the Detailed Description in conjunction with the accompanying drawings in which:

FIG. 1 shows the fatty acids compositions of waste fish oil used in the present invention. FIG. 2- A shows the composition of nutrient rich medium.

FIG. 2-B shows the composition mineral medium broth.

FIG. 2-C shows the composition of trace element solution.

FIG. 3 shows a flow chart of a method of producing PHA resin. FIG. 4 shows the results obtained from two different concentration of waste fish oil on the biosynthesis of P(3HB).

FIG. 5-A shows the appearance of waste fish oil that is in liquid form at 37 ° C. FIG. 5-B shows the appearance of waste fish oil that crystallizes at 30 ° C.

FIG. 6 shows the effects of two different method of carbon source preparation on the biosynthesis of P(3HB).

FIG. 7 shows the results obtained when various concentration of waste fish oil are used.

FIG. 8-A and FIG. 8-B show the results obtained when various concentration of sodium valerate are used. 5. DETAILED DESCRIPTION OF THE DRAWINGS

In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those of ordinary skill in the art that the invention may be practised without these specific details. In other instances, well known methods, procedures and/ or components have not been described in detail so as not to obscure the invention. The invention will be more clearly understood from the following description of the embodiments thereof, given by way of example only with reference to the accompanying drawings, which are not drawn to scale.

The present invention describes a process for the production of biopolymer by using waste fish oil or waste palm oil as carbon feedstock for bacterial cell growth and polyhydroxylalkanoate (PH A) assimilation.

The microorganisms of the present invention is not subject to limitation, as long as it is a microorganism of the genus Cupriavidus, Burklialderia, Alcaligenes or Pseudomonas, which is capable of synthesizing the polymer as described above. Examples thereof include Cupriavidus necator H16 (formerly known as Ralstonia eutropha, Alcaligenes eutrophus and Wautersia eutroplia) for the biosynthesis of poly(3hydroxybutyrate), i.e. P(3HB) homopolymer and poly(3- hydroxybutyrate-co-3-hydroxyvalerate), i.e. P(3HB-co-3HV) copolymer and poly(3-hydroxybutyrate-co-4- hydroxybutyrate), i.e. P(3HB-co-4HB) copolymer and derivatives thereof. Numerous other strains such as Bacillus cereus SPV, Sinorhizobium meliloti, Azotobacter chroococcum G-3, Pseudomonas putida KT2440 and Metylobacterium sp V49 also gaining attention for the PHA production. Hereinafter, Cupriavidus necator H16 is an example of microorganism used in the present invention.

The maintenance of bacterial strains can be carried out by using of mineral medium (MM) agar plate and 20 % glycerol. The strain is consistently streaked on a MM agar plate with 10 g/L of fructose as the carbon source and 0.54 g/L of ammonium chloride (NH4CI) as the nitrogen source and incubated at 30 C for 72 hours before being kept in 4 ° C chiller. The strain can be maintained in a 20 % v/v glycerol stock solution and kept in -20 ⁰ C freezer for a longer period of storage. Referring now to FIG. 1, there is shown the fatty acids compositions of waste fish oil. The waste fish oil generally contains 36.8 % oleic acid, 31.3 % palmitic acid, 8.1 % stearic acid and 7.6 % linoleic acid. Alternatively, waste palm oil with free fatty acid (FFA) in the range of 50% - 80% also can be used as carbon feedstock for bacterial cell growth. The oil is sterilized by autoclave procedure before it is used in the PHA biosynthesis process.

The production of biopolymer of the present invention is generally being carried out by culturing microorganisms in a medium using one-stage cultivation, forming and accumulating the biopolymer of the present invention in the microorganism or in the culture and thereafter recovering the biopolymer from cultured microorganism or from the culture. The PHA biosynthesis for the production of biopolymer is carried out by inoculating an amount of bacterial cells into nutrient rich (NR) medium and cultured for approximately 5 to 12 hours at 25 ° C to 35 ⁰ C on a rotary shaker at a speed of 150 - 250 rotation per minute (rpm) for inoculum preparation. FIG. 2- A shows the composition of NR medium (Doi et al., 1995b) whereby the pH of NR medium is adjusted to 7.0 prior to autoclave. Said NR medium may be composed of 10 g/L peptone (enzymatic digest from gelatin), 10 g/L meat extract and 2 g/L yeast extract. Said NR medium is used to activate and to enrich the bacterial cells so that sufficient cell biomass or inoculums is prepared for the subsequent culturing step in PHA production. Before the prepared inoculums is proceeded to subsequent culturing step, the optical density (OD) of bacterial cultures is then determined with UV/ Visible spectrophotometer at the wavelength of 600 nm. This can be carried out by using non-inoculated NR broth as blank, aliquot approximately 500 μί of the bacterial inoculums into a clean cuvette for OD reading (ODeoo).

Upon reaching an ODeoo of 4.0 - 5.0, the prepared inoculum is then cultured in mineral medium (MM) broth with nitrogen limitation to initiate PHA production. FIG. 2-B shows the composition MM broth (Doi et al., 1995b). Said MM is prepared by dissolving approximate 2.8 g/L of monopotassium phosphate (KH ₂ PO ₄ ), 3.32 g/L of disodium phosphate (Na ₂ HPO ₄ ) and 0.54 g/L of ammonium chloride (NH ₄ C1) as nitrogen source in distilled water and the pH of the medium is adjusted to 7.0. Meanwhile, stock solution of magnesium sulphate heptahydrate (MgSO ₄ · 7H ₂ O) at 50 % (w/v) is prepared and autoclaved separately. Trace elements solution with the composition as shown in FIG. 2-C (Kahar et al., 2004) is prepared by dissolving in 0.1 N hydrochloric acid (HC1) and filter-sterilized with sterilized cellulose acetate membrane filter (pore size of 0.2 μτ ^η ). Approximately 0.25 g/L of MgSO · 7H ₂ 0, 1 mL/L of trace elements and 10 g/L of fructose are added aseptically into the MM solution.

During the cultivation, a desired concentration of waste fish oil or waste palm oil and approximately 3% v/v of the prepared inoculum are added aseptically into the MM broth in an inoculated flask. Said inoculated flask is then incubated at 25 - 35 ° C, 150 - 250 rpm for 36 - 72 hours. Additionally, sodium valerate and sodium-4-hydroxybutyrate (Na4HB) which are the precursors for the 3HV and 4HB biosynthesis respectively are added into the culture medium at specific interval within 32 hours to 72 hours at various concentrations. 20 % w/v of stock solutions of said both precursors are prepared and autoclaved separately. Said stock solution of sodium valerate can be converted from valeric acid. 40 g of sodium hydroxide (NaOH) was dissolved in 1 L of (95%) absolute ethanol and stirred. 102 ml of valeric acid is added slowly into the NaOH solution. The salt precipitate of valeric acid is then recovered and dried completely in the oven at 45 C until a constant weight is achieved. The sodium salt of 4-hydroxybutyrate is prepared by the reaction of γ- butyrolactone with NaOH solution. A total of 60 g NaOH is stirred in 1 L of (95%) absolute ethanol until complete dissolution. Approximately 120 mL γ- butyrolactone is poured into the NaOH solution. The precipitated sodium salt of 4-hydroxybutyrate is placed in 45 ⁰ C oven until constant weight. After the drying process, the Na4HB is kept in an airtight container. In the present invention, said precursors are added into MM broth at 48 hours and / or 60 hours to produce copolymer of P(3HB-co-3HV) and P(3HB-co-4HB) respectively and derivatives thereof.

Upon completion of the cultivation process, the PHA compositions produced in the present invention can be recovered from the PHA-producing microorganism by harvesting the cultured cells through centrifugation such as tubular centrifugation. After centrifugation, the supernatant is decanted. Cell pellet is washed and vortexed with hexane to remove the residual oil. The washed cell pellets are resuspended in distilled water, transferred into Bijoux bottles and frozen at -20 ⁰ C for 24 hours prior to lyophilisation.

During the lyophilisation process, the harvested cell pellets are subjected to freeze drying for approximately 2 days by using of freeze dryer. Said harvested cell pellets also can be dried by means of oven drying to obtain dried cells. The lyophilized cells are then prepared for gas chromatography (GC) analysis or other characterization studies.

Meanwhile, the dried cells are pulverized to a predetermined size by using grinder to obtain cell powder. The obtained cell powder is then mixed with 3 - 5 % w/v detergent (cells : detergents = 1 : 2.5 w/w) for approximately 3 - 5 hours at 50 - 60 C water bath. Detergents such as linear alkylbenzene sulfonic acid (LAS-99), Triton x-100, and sodium dodecyl sulfate (SDS) can be used in the mixing process. Upon completion of mixing, the mixture is subjected to centrifugation and washing with distilled water for 10 - 15 minutes by means of tubular centrifuge to remove unwanted impurities. Said mixture is then transferred for oven drying to obtain PHA powder and followed by pelletize the PHA in resin form by extruder. FIG. 3 shows a flow chart of a method of producing PHA resin. By having the above mentioned method of producing PHA resin, the purity of said produced PHA resin can be determined and therefore also able to determine the biodegradability of said resin to be used in particular application such as the application of said resin in slow release fertilizer.

Hereinafter, the present invention is described in more details with reference to the following examples which should not be construed to limit the scope of the present invention. Example 1 - PHA biosynthesis by Cupriavidus necator

The PHA biosynthesis is carried out by inoculating substantially two loopfull of bacterial cells into 50 mL nutrient rich (NR) medium as described above and cultured for approximately 5 hours at 30 C on a rotary shaker at a speed of 200 rotation per minute (rpm) for inoculum preparation. Before the prepared inoculum is proceeded to subsequent culturing step, the optical density (OD) of bacterial cultures is determined with UV/ Visible spectrophotometer at the wavelength of 600 nm. This can be carried out by using non-inoculated NR broth as blank, aliquot approximately 500 μΙ_ of the bacterial inoculums into a clean cuvette for OD reading (ODeoo).

Upon reaching an ODeoo of 4.0 - 5.0, the prepared inoculum is then cultured in mineral medium (MM) broth as described above with nitrogen limitation to initiate PHA production. During the cultivation, waste fish oil with concentration range of 2.5 - 12.5 g/L or waste palm oil with concentration range of 5 - 10 wt% and approximately 3% v/v of the prepared inoculum are added aseptically into the MM broth in an inoculated flask. Said inoculated flask is then incubated at 30 ⁰ C, 200 rpm for 48 hours. Meanwhile, sodium valerate at concentration range of 1 - 13 g/L and sodium-4-hydroxybut rate (Na4HB) at concentration range of 1 - 13 g/L which are the precursors for the 3HV and 4HB biosynthesis respectively are added into the culture medium at specific intervals within 32 hours to 72 hours. Said precursors are added into MM broth at 48 hours and / or 60 hours to produce copolymer of P(3HB-co-3HV) and P(3HB-co-4HB) respectively.

Upon completion of the cultivation process, the cultured cells are harvested by centrifugation (8000 rpm, 7 minutes, 4 ⁰ C). After centrifugation, the supernatant is decanted. Cell pellet is washed and vortexed with approximately 20 mL of hexane to remove the residual oil. The cell pellet is then re-centrifuged and the hexane is discarded. The cell pellet is resuspended with about 50 mL of distilled water, centrifuged and decanted to remove the remaining hexane. The washed cell pellets are resuspended in 1 mL of distilled water, transferred into Bijoux bottles and frozen at -20 ° C for 24 hours prior to lyophilisation.

During the lyophilisation process, the harvested cells are subjected to freeze drying for approximately 2 days by using of freeze dryer. The lyophilized cells can be prepared for gas chromatography (GC) analysis.

Meanwhile, polymers can be extracted and purified by having the lyophilized cells to be stirred for 5 days in chloroform with ratio of cells to chloroform, lg : lOOmL. The cells are then filtered with filter paper to remove cell debris and the filtrate is concentrated tc _f about 20 mL. The polymer is precipitated and purified by dripping the concentrated solution into 100 mL of vigorously stirred cool methanol. The purified polymer is obtained after removal of the excessive methanol and dried in oven. The molecular weights of the purified polymers are determined by using gel permeation chromatography (GPC). Example 2 - Characterization of PHAs

Measurement of cell dry weight (CDW)

The dry Bijoux bottles are pre-weighed before being used to store the harvested cell pellets. After lyophilisation, said Bijoux bottles are weighed to acquire the CDW. The CDW is calculated as follow: CDW (g/L) = (weight of bottle with lyophilized cells - pre-weighed empty bottle) x 1000

Volume of culture (mL)

Determination of PHA content and composition by Gas Chromatography

The PHA content is determined by subjecting approximately 18-21 mg of the obtained lyophilised cells to methanolysis by means of heating at 100 °C for 140 minutes in 2 mL of methanolysis solution [compose of mixture of methanol and concentrated sulphuric acid at a ratio of 85 : 15 (v/v)] and 2 mL of chloroform. Upon completion of the heating process, the reaction mixture is cooled to room temperature and then followed by adding 1 mL of distilled water. The solution is vortexed for 1 minute to separate the mixture into two heterogeneous layers: hydrophilic layer (water dissolvable material) with cell debris on the top and hydrophobic layer (chloroform with resulting methyl esters) at the bottom. The hydrophobic layer is transferred with Pasteur pipette to a clean universal bottle containing sodium sulphate anhydrous to remove excess water. Then 0.5 mL of the chloroform with resulting methyl esters is added with 0.5 mL of internal standard, i.e. caprylic acid methyl ester (CME) solution (CME : chloroform at a ratio of 1 : 500) for GC analysis. The calculation of PHA content and monomer composition are based on the comparison of their peak areas of certain retention time to those of the internal standard. The CDW is needed for the calculation of total PHA and residual biomass. The calculations are as follows: P(3HB) homopolymer

Total P(3HB) (g/L) = P(3HB) content (wt%) x CDW (g/L) P(3HB-co-3HV) copolymer

Total PHA (g/L) = PHA content (wt%) x CDW (g/L) P(3HB-co-4HB) copolymer

Total PHA (g/L) = PHA content (wt%) x CDW (g/L) Determination of molecular weight

The molecular weights of the extracted and purified polymers are determined by using gel permeation chromatography (GPC) system connected to a refractive index detector. Polymers are dissolved in chloroform at concentration of 1 mg/mL and filtered through 0.45 μιη PTFE membrane. Chloroform is used as the eluent with a flow rate of 0.8 mL/min at 40 °C.

Example 3 - Effects of waste fish oil homogenized with gum Arabic on P(3HB) biosynthesis Referring now to FIG. 4, there is shown the results obtained from two different concentration of waste fish oil on the biosynthesis of P(3HB). It was found that the results obtained for CDW is considered low when compared with plant oil such as crude palm kernel oil with 67 wt% of PHA content and 6 g/L CDW with just 5 g/L of crude palm kernel oil (CPKO) (Lee at al., 2008). The low CDW could be contributed by the solid state of the oil as the waste fish oil crystallizes at 30 °C. The bacterium is unable to completely utilize the clumped oil for bioconversion into P(3HB). FIG. 5-A and FIG. 5-B show that the appearance of waste fish oil that is in liquid form at 37 ° C but crystallizes at 30 C. However, the bacterium grows optimally at 30 ° C and therefore, gum Arabic is added as emulsifier to homogenize with the waste fish oil. Cultivation is conducted by having incubation at 30 ° C, 200 rpm for a period of 48 hours. FIG. 6 shows the effects of two different method of carbon source preparation on the biosynthesis of P(3HB). In first method, the fish oil is homogenized with 2.5 g/L of gum Arabic using homogenizer. In second method, the gum Arabic is added separately into the MM medium before addition of oil. Results showed that no significant different between the two methods employed. However, PHA content increased from 48 wt% to 70 wt% when comparing with results without using of gum Arabic. Therefore, the use of gum Arabic as emulsifier aid the bacterium in utilization of the waste fish oil or waste palm oil for bioconversion into polyhydroxyalkanoate. In the present invention, gum Arabic at concentration of 2.5 g/L - 20 g/L can be used.

Example 4 - Effects of adding different concentration of waste fish oil on P(3HB) biosynthesis

Referring now to FIG. 7, there is shown the results obtained when various concentration of waste fish oil are used. The cultivation is carried out by having incubation at 30 ° C, 200 rpm for a period of 48 hours. It was found that the P(3HB) accumulation and CDW increased as waste fish oil concentration increased from 2.5 g/L to 12.5 g/L. This indicated that the oil was consumed by the cells for cell growth and P(3HB) accumulation. The CDW (3.05 g/L) was the highest when 15 g/L of waste fish oil was used. The P(3HB) content (74 wt%) and total PHA (2.1 g/L) was the highest when 12.5 g/L of waste fish oil was used. The PHA content and total P(3HB) accumulation was constant after the 12.5 g/L concentration.

Example 5 - Biosynthesis of P(3HB-co-3HV) copolymer from mixtures of waste fish oil with different concentration of sodium valerate

Referring now to FIG. 8-A and FIG. 8-B, there is shown the results obtained when various concentration of sodium valerate are used. The cultivation is carried out by having incubation at 30 ° C, 200 rpm for a period of 48 hours. It was found that the PHA content (wt%) accumulation trend was high at the lowest concentration of sodium valerate at lg/L and decreased as the concentration of the precursor was increased. The 3HV monomer composition increased gradually with the increase in the concentration of sodium valerate with 13 g/L of sodium valerate producing 63 mol% of 3HV. The waste fish oil was fed at a lower concentration of 5 g/L. This was done to force the bacterium to uptake the precursor for second monomer assimilation rather than using the waste fish oil to produce 3HB homopolymer. Example 6 - Biosynthesis of P(3HB-co-4HB) copolymer from mixtures of waste fish oil with different concentration of sodium 4-hydroxybutyrate

The cultivation is carried out at various concentration of sodium 4- hydroxybutyrate (Na4HB) by having incubation at 30 ⁰ C, 200 rpm for a period of 48 hours. The PHA content (wt%) accumulation tends to be decreased considerably as concentration of sodium 4-hydroxybutyrate is increased. While the 4HB monomer composition will increase gradually with increasing concentration of sodium 4-hydroxybutyrate.

While the preferred embodiment of the present invention and its advantages has been disclosed in the above Detailed Description, the invention is not limited thereto but only by the scope of the appended claim.