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Title:
RENEWABLE SUGARS FROM OIL PALM WASTES
Document Type and Number:
WIPO Patent Application WO/2013/043036
Kind Code:
A1
Abstract:
The invention provides method for producing sugars from oil palm fronds wherein the steps comprises of (1) extracting oil palm frond juice from the oil palm fronds; and (2) treating the oil palm frond juice. The invention also provides a method of using the said oil palm frond juice for producing polyhydroxyalkanoates such as polymer of hydroxyalkanoic acid, hydroxybutyric acid, hydroxyvaleric acid, and a copolymer thereof, wherein the copolymers maybe poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), polymers and/or copolymers of hydroxyterminated polyhydroxybutyrate (PHB-OH), heteropolymers thereof and any other polymers. In addition to that, biofuels and organic acids such as bioethanol, biobutanol, lactic acid, succinic acid, and all products that can be produced through chemical and biological synthesis from these sugars from the OPF juice. The present invention also provides a method for producing polyhydroxyalkanoate (PHA) by incubating at least one strain of PHA-producing microorganisms in a culture medium comprising of sugars and/or a derivative thereof from the oil palm frond juice. The microorganisms used can be of bacteria, mold or yeast from the group Azotobacter, Pseudomonas, Coliform, Alcaligenes, Bacillus, Lactobacillus and genetically modified form thereof preferably Azotobacter chroococcum, recombinant Escherichia coli, Alcaligenes latus, Pseudomonas oleovorans and Cupriavidus necator.

Inventors:
HASSAN MOHD ALI (MY)
ARIFFIN HIDAYAH (MY)
MOHD ZAHARI MIOR AHMAD KHUSHAIRI (MY)
ZAKARIA MOHD RAFEIN (MY)
SALIHON JAILANI (MY)
MOKHTAR MOHD NORIZNAN (MY)
SHIRAI YOSHIHITO (JP)
Application Number:
PCT/MY2012/000218
Publication Date:
March 28, 2013
Filing Date:
July 30, 2012
Export Citation:
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Assignee:
UNIV PUTRA MALAYSIA (MY)
HASSAN MOHD ALI (MY)
ARIFFIN HIDAYAH (MY)
MOHD ZAHARI MIOR AHMAD KHUSHAIRI (MY)
ZAKARIA MOHD RAFEIN (MY)
SALIHON JAILANI (MY)
MOKHTAR MOHD NORIZNAN (MY)
SHIRAI YOSHIHITO (JP)
International Classes:
C08H8/00; C13K1/02; C12P7/08; C12P7/10; C12P19/02
Domestic Patent References:
WO2007083746A12007-07-26
WO2010067425A12010-06-17
WO2011002330A12011-01-06
WO2008143204A12008-11-27
WO2011002329A12011-01-06
Foreign References:
US20100167351A12010-07-01
Other References:
L.S. HONG, D. IBRAHIM, I.C. OMAR: "Microscopic Studies Of Oil Palm Frond During Processing For Saccharification", THE INTERNET JOURNAL OF BIOENGINEERING, vol. 4, no. 2, 2010, XP002688441, ISSN: 1937-8246, Retrieved from the Internet [retrieved on 20121130], DOI: 10.5580/1f4e
H. YAMADA ET AL.: "Old oil palm trunk: A promising source of sugarsfor bioethanol production", B I O M A S S AND B I O ENERGY, vol. 34, no. 11, 17 June 2010 (2010-06-17), pages 1608 - 1613, XP002688442, Retrieved from the Internet [retrieved on 20121130], DOI: 10.1016/j.biombioe.2010.06.011
Attorney, Agent or Firm:
KANDIAH, Geetha (Suite 8-7-2 Menara Mutiara Bangsar,Jalan Liku Off Jalan Riong, Bangsa, Kuala Lumpur ., MY)
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Claims:
CLAIMS

1 . A method for producing renewable sugars from oil palm fronds wherein the steps comprises of:

a. extracting of oil palm frond juice from the oil palm fronds; and

b. treating the oil palm frond juice.

2. The method as claimed in claim 1 wherein the oil palm frond juice is treated using sodium hydroxide, calcium chloride and sulfuric acid for precipitate removal. 3. The method as claimed in claim 2 wherein the sodium hydroxide adjusts the pH of the oil palm frond juice to between a range of pH 10 to pH 12.

4. The method as claimed in claim 2 wherein the calcium chloride is between 0 to 1% (w/v). 5. The method as claimed in claim 2 wherein the precipitates can be of solid materials, impurities, glucan, pectin, other polysaccharides or a combination thereof.

6. The method as claimed in claim 5 wherein the precipitates are further removed by centrifugation.

7. The method according to claim 1 , wherein the treated oil palm frond juice comprises of sugars selected from one or more from the group consisting of glucose, sucrose, fructose and other monosaccharides, disaccharides and oligosaccharides. 8. A sugar produced according to any claims 1 to 7.

9. A use of sugars produced according to any claims 1 to 7 for the production of microbial polymers, biofuels, organic acids or a combination thereof. 10. The use as claimed in claim 9 wherein the polymers produced are polyhydroxyalkanoates selected from the group consisting of a polymer of hydroxyalkanoic acid, hydro xybutyric acid, hydroxyvaleric acid and a copolymer thereof.

1 1. The use as claimed in claim 9 wherein the polymers produced are selected from the group consisting of poly(3-hydroxybutyrate) (PHB), a copolymer is a poly(hydroxybutrate-co- hydroxyvalerate) (PHB-co-HV), a poly(3-hydroxybutrate-co-4-hydroxybutyrate) (P3HB-co- 4HB), a copolymer of hydroxyterminated polyhydroxybutyrate (PHB-OH) and a heteropolymer thereof.

12. The use as claimed in claim 9 wherein the biofuel produced can be of bioethanol, biobutanol or their derivatives thereof.

13. The use as claimed in claim 9 wherein the organic acid produced can be of lactic acid, succinic acid or their derivatives thereof .

14. The use as claimed in claim 10 wherein the polyhydroxyalkanoates is produced by bacterial fermentation of at least one strain of polyhydroxyalkanoates-producing microorganism. 15. The use as claimed in claim 14 wherein the microorganism is bacteria, mold or yeast.

16. The use as claimed in claim 15, wherein the bacteria is selected from the group consisting Pseudomonas, Alcaligenes, Bacillus, Lactobacillus, Coliform, Rhodococcum, Methylobacterium, Cupriavidus and a genetically modified form thereof.

Description:
RENEWABLE SUGARS FROM OIL PALM WASTES

FIELD OF INVENTION

The field of invention is on a method of producing renewable sugars from oil palm wastes.

BACKGROUND OF INVENTION

The use of renewable resources such as agricultural and agro based-industry wastes as raw materials for the production of fermentable sugars may decrease the production cost and reduce the dependence on the food crops. Bioconversion of agricultural waste into sugars has been widely studied (Nguyen ef al. 2010; Tengerdy and Szakacs, 2003; Saddler and Mackie, 1990). In Malaysia, oil palm plantation and palm oil industry is the main contributor to the generation of agricultural waste. Studies have been done to utilize the waste efficiently, for example to convert the oil palm empty fruit bunch (OPEFB) into fermentable sugars (Kader et al. 1999; Ariffin et al. 2006 and 2008a; Roslan et al. 201 1 ), utilization of the OPEFB as substrate for enzyme production (Umi Kalsom et al. 1997; Ariffin ef al. 2008b) and pulp preparation from OPEFB (Rushdan 2002). OPEFB is a lignocellulose which contains about 70 - 80 % of holocellulose. Due to the high content of polysaccharide, it has great potential to be converted to fermentable sugars for the production of value-added products such as bioethanol, biobutanol, lactid acid and bioplastic.

However, the main problem with OPEFB is that it is difficult to be hydrolyzed due to its natural lignin component which is recalcitrant to degradation. Various pretreatments such as mechanical, chemical and steam pretreatments are needed in order to loosen up the lignocellulose structure. In addition to that, enzymes need to be used in order to convert the holocellulose into sugars. The overall process is not only contributing to the high cost of sugars production, but it also causes the need in proper wastewater treatment system since chemicals are being used in the pretreatment.

The most generated oil palm biomass is oil palm frond (OPF), which amounted to 83 million tonnes (wet weight) (MPOC, 2010). OPF is obtained during pruning for harvesting fresh fruit bunch (FFB), and therefore, it is available daily. OPF is currently under-utilized as the plantation owners believe that OPF is beneficial for nutrient recycling and soil conservation (Wan Zahari et al. 2008). Hence, pruned fronds are just discarded in the plantation. Our study shows that OPF does not contain high metallic nutrients as expected, but contain high carbohydrates. Therefore, OPF can be utilized for other purpose without disturbing the nutrient recycling process. Since OPF is available daily, sugars from OPF could be potential feedstock candidate in the production of value-added products such as polyhydroxyalkanoates (PHA), bioethanol, biobutanol, lactic acid, succinic, and etc.

Polyhydroxyalkanoates (PHAs) and more specifically, poly(3-hydroxybutyrate) (PHB), a short side chain length polymer, have been known for years as being naturally synthesized biodegradable, biocompatible thermoplastics. These are bacterial polyesters used as energy storage when microorganisms are submitted to adverse growth conditions. The polymers are then formed as intracellular granules that can accumulate to 80 percent of the cell mass. So far, PHAs have been produced through fermentation processes followed by extraction and purification methods. In P(3HB) production, the cost of raw materials is about 40 to 50% from the total production cost, whereby the cost of carbon source alone accounts for 70 to 80% of the total raw material cost (Cavalheiro et al., 2009; Koller et al., 2010). In order to address this drawback, it is necessary to make use of cheap carbon sources that are also abundant.

SUMMARY OF THE INVENTION

The invention provides method for producing sugars from oil palm fronds wherein the steps comprises of (1 ) extracting oil palm frond juice from the oil palm fronds; and (2) treating the oil palm frond juice. The invention also provides a method of using the said oil palm frond juice for producing polyhydroxyalkanoates such as polymer of hydroxyalkanoic acid, hydroxybutyric acid, hydroxyvaleric acid, and a copolymer thereof, wherein the copolymers maybe poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV), poly(3-hydroxybutyrate-co-4- hydroxybutyrate) (P3HB4HB), polymers and/or copolymers of hydroxyterminated polyhydroxybutyrate (PHB-OH), heteropolymers thereof and any other polymers. In addition to that, biofueis and organic acids such as bioethanol, biobutanol, lactic acid, succinic acid, and all products that can be produced through chemical and biological synthesis from these sugars from the OPF juice.

The present invention also provides a method for producing polyhydroxyalkanoate (PHA) by incubating at least one strain of PHA-producing microorganisms in a culture medium comprising of sugars and/or a derivative thereof from the oil palm frond juice. The microorganisms used can be of bacteria, mold or yeast from the group Azotobacter, Pseudomonas, Coliform, Alcaligenes, Bacillus, Lactobacillus and genetically modified form thereof preferably Azotobacter chroococcum, recombinant Escherichia coli, Alcaligenes latus, Pseudomonas oleovorans and Cupriavidus necator.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 refers to the schematic diagram of fresh oil palm fronds (OPF) without leaves (X, Y, and Z representing the different section of OPF from the oil palm trunk). FIG. 2 refers to material balance for the extraction of OPF juice.

FIG. 3 refers to OPF juice, sugars content and composition at different section of fresh OPF. FIG. 4 refers to Fourier transform infrared (FTIR) spectra for precipitate of OPF juice after treatment using calcium chloride (CaCI 2 ) and sodium hydroxide (NaOH).

FIG. 5 refers to 500 MHz 1 H nuclear magnetic resonance (NMR) spectrum of poly-3- hydroxybutyrate (P(3HB)) produced by Cupriavidus necator (CCUG52238 1" ) when supplemented with 30% (v/v) OPF juice.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides renewable sugars obtained from oil palm wastes in particular oil palm fronds and the production thereof. Hereinafter, this specification will describe the present invention according to the preferred embodiments of the present invention. However, the following example 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.

This invention was carried out by characterizing and utilizing the juice from oil palm fronds as alternative renewable sugars for the production of value added products. 25 kg offresh OPF (without leaves) were collected from the oil palm plantation at Universiti Putra Malaysia, Serdang, Selangor, Malaysia, and cut into 1.0 m length at three different sections; initial (X), middle (Y), and edge (Z) as shown in FIG. 1. The OPF juice was extracted by pressing the frond using a conventional sugarcane press machine. The OPF juice was centrifuged at 15000xg for 15 minutes at 4°C and the supernatant was filtered using a mixed cellulose ester membrane filter with the pore size between 3 to 5μιη (Cole Parmer). The filtrate was stored at -20°C before use. FIG. 2 shows the material balance for the extraction of the OPF juice. By using the sugarcane pressing machine, we managed to get 17 wt% of OPF juice (4.2 kg) from 25 kg of fresh OPF. The pH of the OPF juice was approximately 5.0.

The moisture content was determined by drying the fresh OPF at 105°C for 48 hour. The proximate analysis for the determination of ash content, fat, fiber and crude protein was determined according to the standard methods (APHA 1985). CNHS analyzer and Inductively Coupled Plasma (ICP) (Perkin Elmer, USA) were used to determine the carbon, nitrogen, nutrients and heavy metal elements in the OPF juice. The sugars content in the OPF juice was determined by a high performance liquid chromatography (HPLC) (Agilent Series 1200, USA) using the Supelcosil LC-NH2 column (Sigma Aldrich) (25cm x 4.6mm ID, 5 μιη particles) with a Rl detector operated at 30°C. The mobile phase was acetonitrile: water (75%:25%) at a flow rate of 1 .0 ml/min. The components were identified by comparing their retention times with those of authentic standards under analytical conditions and quantified by external standard method (Kafkas er a/. 2006).

Table 1 : Proximate and chemical analysis of fresh OPF and OPF juice *

Analysis Unit Fresh OPF OPF juice

Moisture 69.17 90.01

Crude protein (N x 6.25) 0.32 0.13

Ash 1 .10 0.94

Total fat >- g/100g 1.51 ND

Crude fiber 49.52 1 .28

Dietary fiber 20.76 2.18

Carbohydrate* s 27.90 8.92

Energy kcal/1 OOg 167.99 40.56

Vitamin C mg/100g ND 1 16.67

Vitamin A (as β-carotene) Mg/100g ND 1 157.99

* Data is the mean of duplicate samples

" Carbohydrate is calculated by subtracting the sum of the moisture, crude protein, total fat and ash from 100%

ND = Non detectable

Table 2: Amount of sugars contained in the OPF juice from different section of fresh oil palm frond.

Fresh Sugars (g/l) b

OPF OPF Juice, OPF juice Total sugars

OPF,

Section 3 weight (g) (wt %) Fructose

(g) Glucose Sucrose (g/i) weight

XA 925.05 195.55 21 .14 1 .91 61 .17 16.95 80.03

YA 595.13 128.23 21 .55 0.78 57.48 19.89 78.15

Z A 281 .68 65.24 23.16 1 .10 52.98 22.99 77.07

XB 926.01 182.44 19.70 1 .26 54.66 16.47 72.39

YB 596.06 139.36 23.34 1 .42 51 .94 20.18 73.54

Z B 295.06 67.04 22.72 1 .30 49.66 21 .16 72.12

Xc 1038.12 214.94 20.70 1 .97 54.51 19.60 76.08

Yc 606.06 135.35 22.33 2.00 52.34 22.10 76.44

Zc 391 .38 80.82 20.65 3.35 50.83 24.77 78.95

Average 1884.85 402.99 21 .38 1 .68 53.95 20.46 76.09

Values are means of duplicate samples

A, B, and C determined the three (3) different OPF from different oil palm tree

Determined by HPLC

Table 3: Nutrient and heavy metals content in OPF and OPF juice.

Analysis Fresh OPF OPF juice

N (%) 0.9 0.8

C (%) 49 39

C/N 56 50

* OC (%) 37 29

Composition of nutrients and metal elements

S (%) 0.2 0.4

P (%) 0.02 0.02

K (%) 0.2 2.3

Ca (%) 1 .4 2.9

Mg (%) 0.2 0.5

B (ppm) 4 2

Mn (ppm) 61 152

Cu (ppm) 2 2

Fe (ppm) 100 66

Zn (ppm) 3 9

Organic carbon

Data is the mean of duplicate samples

The OPF juice is further treated to remove precipitate formed after extraction process. The step of removing precipitates by exposing OPF juice to NaOH and CaCI 2 . The amount of sulfuric acid and NaOH used in the step of removing of precipitates produces a pH between pH 10 to 12. The amount of CaCI 2 added is between 0 to 1% (w/v). The precipitates formed were glucan, pectin, and other polysaccharides. Precipitates were removed by centrifugation at 15000xg for 15 minutes at a range between 4°C to 30 °C, with 4 °C being the optimum temperature and analyzed using FTIR. Sugars content and composition before and after pre- treatment were also determined by using HPLC (Agilent Series 1200, USA).

Table 4: Sugars content and composition before and after treatment of OPF juice.

Before treatment After treatment

Sugars

Concentration (g/l) Concentration (g/l)

Fructose 5.36 6.48

Glucose 43.33 43.33

Sucrose 5.92 4.80

Total sugars 54.61 54.61

For dewatering of OPF juice, different temperature for evaporation were used which are in the range of 60°C to 100°C and vacuum pressure between 0 mbar to 50 mbar. Concentrations of OPF juice for storage were in the range of 20% to 80% and for storage at temperature range between -20°C to 30°C.

The sugars from the OPF juice can be used for various applications including the production of various products which includes polyhydroxyalkanoates such as polymer of hydroxyalkanoic acid, hydroxybutyric acid, hydroxyvaleric acid, and a copolymer thereof, wherein the copolymers maybe poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV), poly(3- hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), polymers and/or copolymers of hydroxyterminated polyhydroxybutyrate (PHB-OH), heteropolymers thereof and any other polymers. In addition to that, biofuels and organic acids such as bioethanol, biobutanol, lactic acid, succinic acid, and all products that can be produced through chemical and biological synthesis from these sugars from the OPF juice.

The polyhydroxyalkanoate (PHA) produced using the OPF can be conducted by incubating at least one strain of a PHA-producing microorganism in a culture medium comprising of the said sugars and/or a derivative thereof. The microorganisms used can be of bacteria, mold or yeast from the group Azotobacter, Pseudomonas, Coliform, Alcaligenes, Bacillus, Lactobacillus and genetically modified form thereof. More specifically, microorganisms maybe Azotobacter chroococcum, recombinant Escherichia coli, Alcaligenes latus, Pseudomonas oleovorans and Cupriavidus necator.

For example, the sugars can be utilized to produce poly(3-hydroxybutyrate) [P(3HB)] through bacterial fermentation of the OPF juice by Cupriavidus necator CCUG 52238 T . The pre- grown cells of C. necator (CCUG 52238 T ) (10%v/v) were transferred into a 180 mL mineral salt medium (MSM) in 500 mL flasks containing (per liter of distilled water) 6.7 g of potassium dihydrogen phosphate (KH 2 P0 4 ), 1 .5 g of dipotassium hydrogen phosphate (K 2 HP0 4 ), 1 .0 g of ammonium sulphate ((NH^SO^, 0.2 g of magnesium sulphate (MgS0 4 ) and 1 mL microelements solution (Hassan et al. 1997). The OPF juice was sterilized in an autoclave separately and added into the medium at various volumes to vary the concentrations ranging from 10% to 50% (v/v). A comparison study using a mixture of technical grade sugars comprising glucose (12 g/L), sucrose (3g/L) and fructose (1 g/L), with total sugar concentration of 16 g/L, was also carried out in order to mimic the sugar composition in the OPF juice at 30% (v/v) concentration. The initial pH of the medium was adjusted to 7.0 with 2M sodium hydroxide (NaOH) before the addition of OPF juice. The cultures were incubated at 30°C under aerobic condition with agitation speed of 200rpm and the samples were harvested after 48 hours for the determination of cell dry weight (CDW) and P(3HB) content in the cells. The samples from the bacterial fermentations were taken at the end of the cultivation period to measure the total dry weight and P(3HB) content. Each sample was centrifuged at 11000xg for 5 minutes at 4°C and the solids were washed with distilled water and centrifuged for two consecutive times. Dry weight measurements were carried out by drying the solids at 50°C and cooling in a desiccator to constant weight. The P(3HB) content and composition in the lyophilized cell were determined using the gas chromatography (Shimadzu GC-2014). Approximately, 20mg of lyophilized cells were subjected to methanolysis in the presence of methanol and sulfuric acid [85%:15% (v/v)]. The organic layer containing the reaction products was separated, dried over Na 2 S0 4 , and analyzed by GC according to the standard method (Braunegg et al. 1978) using an ID-BP1 capillary column, 30 m x 0.25 mm x 0.25 μιη film thickness (SGE).

Table 5: Biosynthesis of P(3HB) by Cupriavidus necator (CCUG52238 T )

concentrations of OPF juice and mixture of technical grade sugars 3

a Fermentation was conducted at 30°C, 200 rpm for 48 hours

b Determined by HPLC analysis

c Determined by GC from lyophilized cells For the extraction and characterization of P(3HB), solvent extraction method was carried out in order to extract the P(3HB) from the freeze-dried cells (Rahayu et al. 2008; Zakaria ef al. 2010b). Approximately, 3.0g of the freeze dried cells was stirred in 600 mL chloroform for 24 hours at 30°C. The extracts were filtered to remove cell debris and the chloroform was concentrated to about 15 mL using a rotary evaporator (Rotavapor 220, Buchi, Switzerland). The concentrated solution was then added drop-wise to 150 ml of rapidly stirred cold methanol to precipitate the P(3HB). The precipitated P(3HB) was recovered by filtration using a Whatman filter paper No. 1 and dried overnight in vacuum to completely eliminate the solvent. Gel permeation chromatography (GPC) and proton ( 1 H) NMR analysis were carried out in order to characterize the biopolymer produced from the OPF juice and the mixture of technical grade sugars. The average molecular weight (M w ), the number of average molecular weight (M n ) and the polydispersity index (M M n ) of the biopolymer were determined by using the size exclusion chromatography (SEC) on a TOSOH HLC-8120 GPC system with a refractive index (Rl) detector at 40°C utilizing the TOSOH TSKgel Super HM-M column and chloroform eluent (0.6 ml/min) (Ariffin et al. 2008). The calibration curves for the GPC analysis were obtained using the polystyrene standards with a low polydispersity as described by Ariffin et al. (2008). 12 mg sample was dissolved in 2 mL chloroform and the solution was filtered through a membrane filter with 0.45 \im pore size.

Proton ( 1 H) NMR spectra were recorded on a 500 MHz JEOL JNM-ECP500 FT NMR system. Chloroform-d was used as the solvent. Chemical shifts were reported as ό " values (ppm) relative to internal tetramethylsilane (TMS) in CDCI 3 . The expected 1 H NMR chemical shifts were predicted using a ChemNMR program in a CS ChemDraw Ultra version 6.0 (Ariffin et al. 2008). Table 6: Molecular weight and polydispersity index of purified P(3HB) recovered after fermentation by Cupriavidus necator (CCUG 52238 T ) using various concentrations of OPF juice and technical grade sugars.

a Determined by GPC analysis

Table 6 displays the results for the number of average molecular weight {M n ) and the average molecular weight (M w ) of P(3HB) produced from the OPF juice and the mixture of technical grade sugars. It can be observed that the molecular weight and polydispersity index {MJM n ) obtained were almost comparable with the P(3HB) obtained from the mixture of technical grade sugars when 30% (v/v) of the OPF juice concentration was used as a carbon source. It was interesting to note that; good polydispersity values lower than 2 were obtained from the experiments supplemented with 30% to 40% (v/v) of OPF juice, indicating the uniform formation of P(3HB) within the cell cytoplasm (Yezza et al. 2007). It was reported that, the M n oi the biopolymer produced can be varied and dependent on the type of strain, type and concentration of substrate used, pH culture and temperature (Zakaria er a/. 2010b). In our study, the highest M w (812 kDa) was obtained when the medium was supplemented with 30% (v/v) of the OPF juice and corresponded well to those reported by other authors (Anderson and Dawes, 1990; Doi, 1990; Yezza et al. 2007; Cavalheiro er a/. 2009). In this invention, it was expected that the type of biopolymer produced from the cells was P(3HB) due to the feedstock used during the fermentation. The identity of the biopolymer produced was confirmed by 1 H NMR and the spectrum is shown in FIG. 5. The figure shows that, the ratio of integrated values of peaks was 1 : 0.64 : 0.32, approximately 3 : 2 : 1 , which revealed the presence of 3, 2 and 1 protons at chemical shifts of 1.2, 2.5 and 5.2, respectively. Likewise, similar assignments of P(3HB) signals have also been reported previously by other authors (Pal and Paul, 2002; De Rooy ef al. 2007). Based on the reports and the estimation of 1 H NMR chemical shifts using a ChemNMR program in a CS ChemDraw Ultra version 6.0, the peaks as shown in FIG. 5 were assigned. The assignments of the 1 H NMR signals revealed that P(3HB) was produced by C. necator (CCUG 52238 T ) using the OPF juice hence, this confirmed the speculation.