FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION (See section 10, rule 13)
“A method for production of polyhydroxyalkanoates (PHAs)”
APPLICANT (S) Reliance Industries Limited
3rd Floor, Maker Chamber-IV, 222, Nariman Point, Mumbai-400021, Maharashtra, India
The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF INVENTION
The present disclosure is related to the broad field of environmental biotechnology and is particularly directed to production of polyhydroxyalkanoates (PHAs) from microorganisms. The present invention also relates to methods for growing microorganisms for producing value added products in an efficient manner using lipids, plant oils and fats, and fatty acids as carbon sources.
BACKGROUND AND PRIOR ART OF INVENTION
Polyhydroxyalkanoates (PHAs) are thermoplastic polyesters that serve as carbon and energy storage vehicles in microorganisms. PHAs are biodegradable in both aerobic and anaerobic conditions, are biocompatible with mammalian tissues, and, as thermoplastics, can be used as alternatives to fossil fuel-based plastics such as polypropylene, polyethylene, and polystyrene. In comparison to petrochemical-based plastics, which are neither biodegradable nor made from sustainable sources of carbon, PHA plastics afford significant environmental benefits.
Owing to the increasing awareness of environmental, food, health and safety issues, the growing orientation towards natural or non-synthetic products, production of microorganisms and material by microbial bioconversion or fermentation are gaining significance and increasingly importance.
Bioavailability of water insoluble plant oils or fats is a limiting factor for uptake by microorganisms as feed leading to poor growth. The fermentative production of value-added products requires supporting carbon sources including essential nutrients for culture, fermentation, or the like, which can be assimilated by the growing microorganisms. Examples of such carbon sources include carbohydrates, fats and plant oils, fatty acids, and waste or cooking oils.
Conventionally, the utilization of food crop derived sugars in genetically engineered microorganism-based aqueous fermentation systems is often regarded as the most efficient and economical platform for PHA production. Specifically, sugar based PHA production processes are capable of generating high density fermentation cultures and high PHA inclusion concentrations, and, by maximizing the cell culture density and PHA inclusion concentration therein, it is believed that carbon, chemical, and energy efficiencies are also maximized.

In the conventional methods, the carbon source are generally glucose, sucrose, and fructose etc. Fats or oils or fatty acids also have been used as carbon source, however, these materials have extremely low solubility in aqueous media, resulting low microbial assimilation rate, rendering the most significant problem associated with using such carbon sources for culturing the microorganisms. Further, the oil/fat/fatty acid tend to solidify, and form aggregates due to higher melting point of some oils and/or fats and/or fatty acids as compared to the culture temperature (such as palm oil is solid at about 25-41oC). These aggregates are larger than the liquid medium droplets, causing further decrease in the microbial assimilation rate.
Thus, there exists a major problem in the culture of the microorganisms for the microbial production of materials such as polyhydroxyalkanoates (PHAs) in an industrially efficient manner by suitably using fatty acids, fats and/or oils, which have low solubility in media, as carbon source. The present invention is an attempt to overcome aforesaid problems in the prior art by proposing a method for production of polyhydroxyalkanoates (PHAs) from microorganisms in an efficient manner using lipids, plant oils and fats, and fatty acids as carbon sources.
SUMMARY OF THE INVENTION
The present invention relates to method for producing polyhydroxyalkanoates (PHAs) from microorganisms. The method of the present invention involves microbial growth in a plant oil-based oil-in-water emulsion as carbon sources and the subsequent production of microbial metabolites. Further, the present invention also relates to a method for the extraction of the microbial metabolites which are high value-added products.
The invention also discloses a method for the production of microbial metabolites by using plant oils having low water solubility and high melting point at the temperature for culture of the microorganisms as carbon sources that can be suitably assimilated by the microorganisms.
BRIEF DESCRIPTION OF DRAWINGS:
The accompanying drawings illustrate some of the embodiments of the present invention and together with the descriptions, serve to explain the invention. These drawings have been provided by way of illustration and not by way of limitation. The components in the drawings are not necessarily drawn to scale, emphasis instead being placed upon clearly illustrating the principles of the aspects of the embodiments.

Figure 1: Comparative biomass production of Cupriavidus necator in different concentrations of guar gum.
Figure 2: Comparative biomass and PHA production by Cupriavidus necator using palm oil (1%) as carbon source. Non-emulsified palm oil alone was used as control to compare emulsified palm oil using various concentration of guar gum (GG) as mentioned in Table 2.
Figure 3: Comparative biomass production of Cupriavidus necator in emulsified plant oils
Figure 4: Comparative growth and PHA accumulation by Cupriavidus necator grown in non-emulsified and emulsified palm oil (1%) at 1L scale.
Figure 5: Comparative productivities of Cupriavidus necator grown in 1% of non-emulsified and emulsified palm oil, cottonseed oil, sunflower oil and coconut oil.
Figure 6: GC profiles of standard PHA (A) and PHAs produced using fructose (B) and guar gum emulsified palm oil (C).
DETAIL DESCRIPTION OF THE INVENTION
At the very outset of the detailed description, it may be understood that the ensuing description only illustrates a particular form of this invention. However, such a particular form is only exemplary embodiment, and without intending to imply any limitation on the scope of this invention. Accordingly, the description is to be understood as an exemplary embodiment and teaching of invention and not intended to be taken restrictively.
Unless defined otherwise, technical, and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
The present invention aims to provide methods for the production of polyhydroxyalkanoates (PHAs) from microorganisms. The method of the present invention involves microbial growth in a plant oil-based oil-in-water emulsion as carbon sources and the subsequent production of microbial metabolites. The present invention has been developed for the production of value-added products in an efficient manner using lipids, plant oils and fats, and fatty acids as carbon sources for microorganisms.

It is known that fats and plant oils, and/or mixtures thereof have very low solubility in aqueous media rendering inefficient utilization as carbon source by the growing microorganism. Taking this into account, the present inventors have envisioned method for production of PHAs by emulsifying lipids or plant oils using natural emulsifiers/surfactants and/or mixtures thereof which allow microorganisms to assimilate plant oil or fats efficiently.
The inventors of the present invention have noted the problems associated with known methods for production of PHAs and have developed a method for the emulsification of plant oils using bio-based emulsifiers to allow the microorganisms to efficiently utilize the plant oils for the productions of PHAs.
The inventors of the present invention have designed the instant invention examined methods for emulsifications and microbial production of materials in an industrially efficient manner by using plant oils and/or lipids having extremely low solubility and high melting point at normal culture temperature, as carbon source. The inventors of the present invention have found out that an emulsion containing a lipid/oil and a natural plant gum are very stable and allows the microorganism to suitably assimilate plant oils having higher melting points and low solubility in aqueous media at the cultivation temperature of the microorganism.
An aspect of the present invention relates to an emulsion for microbial growth using plant oils as carbon sources and thereby production of microbial metabolites. The emulsion for microbial growth in the present invention essentially contains a lipid/oil and natural plant gum.
The term “emulsification” as used herein refers to a state in which one of two or more mutually insoluble substances is dispersed in another substance(s). Moreover, the term “emulsification” as used herein, the two or more substances are not necessarily of the same phase, which may include a state in which the continuous phase is liquid and dispersed phase is solid. Such combinations of substances include mixtures of water and fats/oils, mixtures of water and fatty acids, and mixtures of water, fats and/oils.
The term “lipid/oil” as used herein refers to a fatty acid, a fat and/or oil, and/or a mixture thereof having low water solubility. The type of lipid/oil depends on the type of microorganism used and the type of microbial metabolite to be produced. Any type of lipid/oil can be used if the microorganism can assimilate it as carbon source.

The lipid/oil is preferably non-petroleum origin and derived from renewable sources including, plants, animals, fishes, microalgae, macroalgae and other products thereof. Typically, plant and animal oils and lipids derived from plant and animals are preferred. To avoid competition with food, it is more preferable to use non-edible plant oils.
In another aspect of the present invention relates to plant oils that include, but not limited to palm oil, corn oil, peanut oil, sesame oil, cottonseed oil, castor oil, mustard oil, jatropha oil, olive oil, avocado oil, canola oil, soybean oil, sunflower oil, safflower oil, coconut oil, date seed oil, karanja oil, rice oil, rapeseed oil, fish oil, waste cooking oil, tallow, pig oil, palm kernel oil, algae-derived oils and fats, refined products of these fats and oils, and their components such as fatty acids, salts of fatty acids and fatty acid esters.
In another aspect of the present invention relates to emulsifiers used in conventional art to emulsify fats and oils include, but not limited to glycerides such as monoglycerides and diglycerides, and organic acid monoglycerides such as acetic acid monoglycerides, lactic acid monoglycerides, citric acid monoglycerides, succinic acid monoglycerides, and diacetyl monoglycerides; polyglycerides, polyglycerol condensed ricinoleic acid ester, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, lecithin, fractionated lecithin, enzyme-treated lecithin, saponin, polysaccharides such as gum arabic and xanthan gum, and proteins such as sodium caseinate, soy proteins, and partially degraded gluten, sodium dodecyl sulphate (SDS), tween 20, tween 60, tween 80, triton-X100, polyethylene glycol (PEG) and the like.
Guar gum - a natural “plant gum” or “polysaccharide” or “galactomannan” also commercially known as “guar flour” is one of the preferred emulsifiers in the present invention. Guar gum is a natural galactomannan and non-ionic in character. In a preferred aspect of the present invention to prepare an effective emulsion, the amount of guar gum used in the range from 0.01% to 0.5%, preferably 0.02% to 0.4%, and more preferably 0.1% to 0.2%.
The emulsion of the present invention characteristically has an improved emulsion activity, emulsion stability, non-toxic and assertive for utilizing plant oils as carbon source for growing the microorganism, and the production of targeted microbial metabolites. Moreover, the emulsion may contain one or more additional emulsifier as/if needed.

The emulsion is preferably prepared as an oil-in-water emulsion before being used in microbial culture so that the emulsion has good dispersibility in the aqueous nutrient medium and can be efficiently utilized by the growing microorganisms.
With respect to the emulsion stability, the lipid/oil content used in the emulsion before used in the microbial culture is preferably 1% to 10%. In commercial production of microorganisms and microbial metabolites frequently involves intermittent or continuous feeding of lipid/oil as carbon source to the fermenter during the cultivation period. In such cases, the lipid/oil in emulsion is preferably 1% to 5%, and more preferably 1% to 2%.
In an aspect of the present invention the emulsion can be prepared by any conventionally known method for preparing emulsions, includes magnetic stirrer, stirrer with a propeller or the like, vortex, homogenizer, ultrasonic devices, microfluidizer and colloid mil. Combinations of two or more of these methods may be also used.
In an aspect of the present invention, depending on operating protocol, the oil-in-water emulsion containing a lipid/oil phase as prepared above, having low solubility in aqueous culture medium, may be utilized as carbon source by any manner for culturing microorganisms. For instance, the emulsion may be prepared previously and stored or the like before being added to the medium; or an aqueous phase and a lipid/oil phase respectively from separate feedstocks tanks may be fed into the same line for in-line emulsification, followed by adding to the medium.
In an aspect of the present invention the microorganisms can be cultured using any media, as per the purpose of culturing and/or as long as it is suitable for the microorganism, containing the emulsified lipid/oil as carbon source for microbial growth and production of microbial metabolites.
In an aspect of the present invention the type of microorganisms used for the cultivation and/or production of microbial metabolites are microorganisms of the genera Escherichia, Cupriavidus, Alcaligenes, Pseudomonas, Bacillus, Azotobacter, Aeromonas, Nocardia, Ralstonia, Wautersia, and Camomonas but are not particularly limited as long as the microorganism assimilate plant oils, fats, and/or the like, and can produce metabolites. Microorganisms isolated from nature and/or obtained by genetical modification or mutation or genome editing (e.g., CRISPR), and the like may suitably use. Examples of microorganisms include archaea, bacteria, fungi, actinomycetes, and other microbes.

In a preferred aspect of the present invention specific preferred microorganism is Cupriavidus necator H16
In a preferred aspect of the present invention the type of polyhydroxyalkanoates (PHA) is not particularly limited as long as it is produced by a microorganism.
In an aspect of the present invention examples of such microbial metabolites include, but not limited to, polyhydroxyalkanoates (PHAs), alcohols (e.g., ethanol, butanol, propanol, and 1,4-butanediol), carboxylic acids (e.g., lactic acid, succinic acid, and adipic acid), amino acids (e.g., glutamine, lysine, and threonine), lipids (e.g. docosahexaenoic acid and eicosapentaenoic acid), enzymes (e.g. lipase) proteins, and antibiotics.
In a preferred aspect of the present invention specific examples of microbial metabolites preferred in the present invention are polyhydroxyalkanoates (PHAs). Polyhydroxyalkanoates (PHAs) are produced by microorganisms and accumulated as energy storage material. Polyhydroxyalkanoates (PHAs) are biodegradable thermoplastic polyesters and can be incorporated into the natural carbon cycle process.
The polyhydroxyalkanoates (PHA) is preferably a homopolymer of a component selected from 3-hydroxyalkanoate, or a copolymer of two or more components selected from 3-hydroxyalkanoate. Examples include poly(3-hydroxybutyrate) (PHB) which is a homopolymer of a 3-hydroxyalkanoate, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] which is a copolymer of 3-hydroxyalkanoate, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] and poly(3-hydroxybutyrate-co-4-hydroxyvalerate) [P(3HB-co-4HV)] which are copolymers of 3-hydroxyalkanoate, and polymers of 3-hydroxyalkanoate. In addition, specific co¬polymers include but not limited to poly(3-hydroxy-butyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)], poly(3-hydroxy-butyrate-co-4-hydroxypropionate) [P(3HB-co-3HP)], poly(3-hydroxybutyrate-co-3-hy droxyhexanoate-co-3-hydroxy octanoate-co-3 -hydroxydecanoate-co-3-hydroxydodecanoate) [P(3HB-3HHx-3HO-3HD-3HDD)], and the like.
In an aspect of the present invention the method of cultivation is not limited to a particular growth or culture conditions (e.g., nutrient composition of the media, type of carbon source and its feeding method, scale of culture, aeration and stirring conditions, culture temperature, and duration of cultivation) as long as it includes adding plant oils as a carbon source to the medium. The culture

method more preferably include a medium need to be in a state that can be stirred in the cultivation vessel, adding emulsified carbon source at the time of inoculation and/or fed continuously or intermittently. The medium may be an aqueous or liquid medium that is separated into two phases, or a suspension medium.
In a preferred aspect of the present invention the microbial metabolites such as, PHAs can be accumulated intracellularly in the microorganisms by the cultivation method described above, which can be recovered from the cells applying any of the well-known methods.
In a preferred aspect of the present invention improved microbial assimilation of plant oils as carbon source is referred to an increase in the consumption of plant oils per hour or during the cultivation period and a consequent increase in the yield of microbial cells, microbial cell dry weight and/or the microbial metabolites produced by the cultivated microorganisms.
The yield of microbial cells may be measured by conventionally known methods, such as by periodical determination of optical density, preferably at 600 nm, or by measuring the dry cell weight (g/L). The yield of microbial metabolites produced may be measured by conventionally known methods, such as by measuring dry weight, GC, GC-MS, HPLC, TGA, or the like. In the present invention, the amount of PHA accumulated in the microbial cells was measured after extraction with organic solvent and drying in accordance with the method of Kato et al. (Appl. MicroBiol. Biotechnol., vol. 45, p.363 (1996); Bull. Chem. Soc., vol 69. p.515 (1996)).
Still further, an aspect of the present invention is to provide an oil in water emulsion for culturing microorganisms and production of microbial metabolites in an industrially efficient manner.
Accordingly, an important embodiment of the present invention is to a method for the production of polyhydroxyalkanoates (PHAs), the method comprises the step of:
a. Preparing an oil-in-water emulsion comprising guar gum and plant oils;
b. Culturing microbial cells in a nutrient medium comprising guar gum-based oil-in-
water emulsion of step (a);
c. Obtaining dry cell biomass of the microbial cells of step (b); and
d. Isolating and purifying polyhydroxyalkanoates (PHAs) from the dried cell biomass
of step (c).

Another embodiment of the present invention is to provide a method wherein step (a) for obtaining the oil-in water emulsion comprises the steps of:
a. Dissolving guar gum in water;
b. Stirring the mixture vigorously at 30 -50oC using a stirrer at 2000 - 2500 rpm for
15 – 20 mins;
c. Cooling the mixture to room temperature of about 28oC to 30oC and filtering the
cooled mixture through muslin cloth with pore size 100 – 500 µm to remove
undissolved particles;
d. Adding the mixture of step (c) to lipid/oil, followed by stirring for 2 min to 15 min
to dissolve the emulsifier to obtain the oil-in-water emulsion.
Another embodiment of the present invention is to provide a method wherein the oil-in-water emulsion obtained in step (d) is composed of 0.5-1% v/v plant oil along with guar gum in the range from 0.02% w/v to 0.4% w/v.
Another embodiment of the present invention is to provide a method wherein the plant oil is selected from palm oil, corn oil, peanut oil, sesame oil, cottonseed oil, castor oil, mustard oil, jatropha oil, olive oil, avocado oil, canola oil, soybean oil, sunflower oil, safflower oil, coconut oil, date seed oil, karanja oil, rice oil, rapeseed oil, fish oil, waste cooking oil, tallow, pig oil, palm kernel oil, algae-derived oils , refined products of these fats and oils, and their components such as fatty acids, salts of fatty acids and fatty acid esters.
Another embodiment of the present invention is to provide a method wherein the step (b) for culturing the microbial cells comprises the steps of:
a. Inoculating a glycerol stock of microbial cells into the seed culture medium and
culturing for 18-24 hours in Shaking Incubator at 30oC and 125-200 rpm;
b. Inoculating the seed culture into a chemically defined medium (CDM) preculture
medium comprising fructose as carbon source and culturing for 20-24 hours in
shaking incubator at 30oC and 125-200 rpm;
c. Inoculating the preculture into a chemically defined medium (CDM) production
medium composed of fructose and/or plant oils as carbon sources and culturing for

48 hours to 66 hours in shaking incubator at 25-30oC and 120-180 rpm to obtain cell biomass.
Another embodiment of the present invention is to provide a method wherein the step (c) for obtaining the dry cell mass comprises the steps of:
a. Terminating cultivation of cells;
b. recovering the cells from a known volume of culture suspension by centrifugation
at 5000-7000 g for 10-20 min to determine cell biomass;
c. Washing the recovered cells first with water, secondly with methanol and finally
with hexane to remove any residual/unutilised oil;
d. Drying the cells for 18-24 h in hot air oven at 50-60oC;
e. Cooling down the dried cells to room temperature in a desiccator to obtain the dried
cell mass for further isolation and purification of the PHA.
Another embodiment of the present invention is to provide a method wherein the microbial cells are selected from the group consisting of Pseudomonus aeruginosa, Bacillus cereuc, Escherichia coli, Aureobasidium melanogenum, Alkaliphilus oremlandii, Candida glabrata, Candida rugosa, Yarrowia lipolytica, Bacillus tequilensis, Bacillus safensis.
Another embodiment of the present invention is to provide a method wherein the bacteria is selected from the genus Cupriavidus or recombinant thereof.
Another embodiment of the present invention is to provide a method wherein step (d) for isolation and purification of PHA comprises the steps of:
a. Treating the dried cells obtained in step (g) in claim 6 with solvent and stirring the
mixture at 150-200 rpm for 36 h to 48 h at 50oC;
b. Concentrating the chloroform solution containing PHA after recovering the cell
residue from step (a), followed by gradual addition of ice-cold ethanol;
c. Stirring the mixture gently for 10-15 min and leaving it still for 20-30 min to
complete the precipitation of extracted PHA;
d. Filtrating or centrifuging the mixture at 5000-7000 g for 10-20 min, followed by
washing with water 2 to 3 times to completely remove the traces of ethanol from
the mixture to the PHA;

e. drying the recovered PHA at 50-55oC for 12-18 hours and the dry weight of the recovered PHA was measured to calculate the polymer content in the cells.
Another embodiment of the present invention is to provide a method wherein polyhydroxyalkanoate is selected from poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3 -hydroxyhexanoate) [P(3HB-co-3HHx)], poly(3 -hydroxybutyrate-co-3 -hydroxyvalerate) [P(3HB-co-3HV)] and poly(3-hydroxybutyrate-co-4-hydroxyvalerate) [P(3HB-co-4HV)], poly(3-hydroxy-butyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)], poly(3-hydroxy-butyrate-co-4-hydroxypropionate) [P(3HB-co-3HP)], and poly(3-hydroxybutyrate-co-3‐hydroxyhexanoate‐ co‐3‐hydroxyoctanoate‐co‐3‐hydroxydecanoate‐co‐3‐hydroxydodecanoate) [P(3HB-3HHx‐3HO‐ 3HD‐3HDD)].
Another embodiment of the present invention is to provide a method wherein the solvent of step (a) is selected from the group consisting of chloroform, dichloromethane (DCM), Acetone, cyclohexanone, ethyl methyl ketone.
Without limiting the scope of the present invention as described above in any way, the present invention has been further explained through the examples provided below.
Experimental Data: EXAMPLE 1:
i. The strain, media, and culture conditions
In the present invention Cupriavidus. necator H16 was used to produce microbial biomass and PHAs using a Chemically Defined Medium (CDM) described by Budde et.al (2011) with slight modification. CDM containing fructose (2 g/L to 20 g/L) and/or plant oils were used as sole carbon sources in the growth and PHA production medium. The seed culture was prepared using colony from plate into a culture medium for 24 h, followed by inoculation to a preculture medium for 12 h to 24 h. Finally, the microorganism from preculture medium was inoculated to the CDM containing fructose and/or plant oil for growth and PHAs production.
The composition of the seed culture medium (ATCC Medium 3) was as follows: 3 g/L Beef extract and 5 g/L Peptone (pH 6.8).

CDM with fructose (10 g/L) was used for preparing the preculture. The composition of CDM was as follows: 1g/L (NH4)2SO4, 1.5 g/L KH2PO4, 4.47 g/L Na2HPO4.2H2O, 1 g/L MgSO4.7H2O and 1 mL trace metal salt solution (1 L deionized water containing, 10 mL concentrated HCL (35%), 10 g FeSO4.7H2O, 2.25 g ZnSO4.7H2O, 1 g CuSO4.5H2O, 0.5 g MnSO4.H2O, 2 g CaCl2.2H2O, 0.23 g Na2B4O7.10H2O, 0.1 g (NH4)6Mo7O24.4H2O dissolve therein). The pH of the preculture medium was maintained at pH 7.0 +/- 0.1. The method of culturing Cupriavidus necator is described below.
First, a glycerol stock (50 µL) of Cupriavidus necator ATCC17699 procured from Hi-Media laboratories pvt. ltd, Mumbai was inoculated to into the seed culture medium (10 mL) and cultured for 24 hours in Shaking Incubator at 30oC and 125 rpm. The seed culture was then inoculated at 1.0% v/v into a 250 mL Erlenmeyer flask containing 50 mL of the preculture medium. The preculture was carried out by culturing for 20-24 hours in Shaking Incubator following and above-mentioned operating conditions.
Next, the preculture was inoculated at OD600nm 0.075 as starting OD into a 250 mL-flask containing 50-70 mL of the above mentioned CDM with fructose and/or plant oils as carbon sources. The culture was carried out for 48 hours to 66 hours in Shaking Incubator following and above-mentioned operating conditions.
Growth of the microorganism was determined by measuring optical density (OD) at 600 nm using a spectrophotometer [Shimadzu, Model No. UV1800] and/or measuring the dry cell weight at regular interval and/or at the termination of the cultivation following the method described below, unless otherwise mentioned.
For determination of cell biomass after termination of the cultivation, the cells were recovered from a known volume of culture suspension by centrifugation at 6500 g for 10 min. The recovered cells were washed with water, and/or followed by methanol and finally by hexane to remove any residual/unutilised oil. After washing, the cells were dried for 24 h in hot air oven at 60oC. The dried cells were cooled down to room temperature in a desiccator and dry cell weight was measured in gram per liter (g/L) following the equation given below:
[(A1 – A0)/Ø] x 1000 (g/L)
where,

A0 - weight of empty vessel (g)
A1 - weight of vessel with biomass (g)
Ø - volume fraction of culture suspension (ml)
ii. Preference of carbon source:
Plant oils as carbon sources used in the example include palm oil, cotton seed oil, canola oil, sunflower oil, sesame seed oil, coconut oil, mustard oil, groundnut oil, avocado oil, soybean oil, olive oil, corn oil and castor oil. Table 1 depicts the content major fatty acids in plant oils that have been chosen as carbon sources which are publicly known in the art. The plant oils mentioned in Table-1 used as sole carbon source in CDM for growing the microorganism and production PHAs in the forthcoming Examples of this invention as described below.
[Table 1. List of plant oils with major fatty acid contents]

Plant oils
tested as C-source Content of major fatty acids (%)

Lauric Myristi Palmiti Stearic Palmitole Oleic acid Linoleic Linolenic Ricinolei

acid (C12, c acid (C14, c acid (C16, acid (C18, ic acid (C16, (C18, mono- acid (C18, acid (C18, c acid (C18,
saturate saturate saturate saturate mono- unsaturat poly- poly- unsaturat
d) d) d) d) unsaturat ed) ed) unsaturat ed) unsaturat ed) ed hydroxy)
Avocado 0.04 0.05 15.23 0.47 5.69 65.06 11.25 0.84 -
oil
Canola – 0.07 4.29 2.59 0.29 65.39 16.32 7.54 -
oil
Castor - - 1.1 1.0 - 4.1 4.8 0.5 87.8
oil
Coconut 47.7 19.9 16.5 2.7 – 6.2 1.6 - -
oil

Cottonse ed oil – = 19.78 1.57 0.40 17.65 52.23 8.52 -
Corn oil - 0.1 12.0 2.0 0.2 29.1 54.5 0.9 -
Groundn ut oil - 0.05 7.5 2.1 0.07 71.1 18.2 - -
Mustard oil – – 3.5 1.6 0.1 19.7 22.2 13.4 -
Olive oil – – 16.5 2.3 1.8 66.4 16.4 1.6 -
Palm oil - - 44.2 4.5 – 39.3 9.6 0.3 -
Sesame oil – – 9.7 6.5 0.11 41.5 40.9 0.21 -
Soybean oil – – 10.3 3.8 – 24.3 52.7 7.9 -
Sunflow er oil 0.02 0.09 6.2 2.8 0.12 28.0 62.2 0.16 -
iii. Preparation of oil-in-water emulsions using natural guar gum and determination of
emulsification characteristics
Emulsions were prepared using the materials and amounts shown in Table 2. The method for preparing the emulsions is described as follows. Required amounts of the emulsifier and water were weighted, and mixed. The mixture was stirred well 2 min to 15 min to dissolve the emulsifier properly. After dissolution of the emulsifier, the solution was mixed with measured amount of plant oil, followed by vigorous mixing 10 min to 15 min using stirrer at 2000 rpm.
In case of guar gum, it is dissolved in water at room temperature (26oC to 30oC) or preferably in between 30oC to 60oC, and more preferably in the range between 40oC to 50oC. The mixture was stirred vigorously at 40oC using a stirrer at 2500 rpm for 15 – 20 mins. The mixture was cooled

down to room temperature or about 28oC to 30oC and filtered through muslin cloth, preferably with pore size 100 – 500 µm, to remove undissolved particles if any. After removing the undissolved substances, the mixture was added to the measured amount of lipid/oil, followed by mixing vigorously as described above.
As for emulsification characteristic of the thus prepared emulsions, Emulsification Activity Index (EAI) was determined according to the equation described below.
The present invention intends to determine a suitable concentration of guar gum for emulsification of a known concentration of plant oils that enables the growing microorganism to suitably assimilate plant oils as carbon sources. Oil-in-water emulsions were prepared using palm oil (1% v/v) with guar gum in the range from 0.02% w/v to 0.4% w/v as shown in Table 2. The emulsification characteristics such EAI was determined by using below equation according to Li and Xiang (PloSOne, vol.14(3), e0213189 (2019)). Comparative EAIs were determined with respect to the percentage of emulsifier used for emulsification of palm oil (1%) and indicated in Table 2.
EAI (m2/g) = (2.303 x 2 x A x N) / (C x Ø x10000)
where,
A – absorbance at 500 nm;
N – emulsion dilution factor;
C – emulsifier concentration;
Ø – volume fraction of oil phase
As shown in Table 2, the emulsions with guar gum 0.4% w/v exhibited EAI. However, from the point of operational difficulties due highly viscous solution there is limitation of applying the emulsion for growing the microorganism. Similarly, emulsion with 0.02% w/v exhibited low EAI, leading to instability of the emulsion in growth media, resulting inefficient utilization of the plant oil as carbon source, thus limiting its application for growing the microorganism. As shown in the Table 2, emulsion containing 0.2% w/v exhibited moderately high EAI with better emulsion stability and low viscosity, hence selected for further comparative growth experiments and productions of PHAs.

[Table 2. Emulsification activity of oil-in-water emulsions prepared with guar gum]

Concentration of guar gum (% Concentration of Oil (% EAI (m2/g) weight) weight)
0 1 0
0.02 1 0.0016
0.05 1 0.00387
0.1 1 0.00835
0.2 1 0.0165
0.4 1 0.0356
EXAMPLE 2: Effects of guar gum concentrations on growth of Cupriavidus necator
The same procedure as in Example 1 was followed, except that guar gum at various concentrations of Example 1 were used in CDM to test their impacts on growth or PHA accumulation or as a potential carbon source. For comparison, CDM containing fructose 2 g/L alone as sole carbon source was used for growing the microorganism. Cultures were grown for 48 hours following the same operating protocol as described in Example 1.
After termination of the cultivation at 48 hours after inoculation, the cells were recovered by centrifugation at 6500 g for 10 min. The recovered cells were processed to determine the dry cell weight following the method as described in Example 1. Results obtained are represented in Figure 1. Amounts of Guar gum higher than 0.1% in culture media showed inhibitory effects on microbial growth (p<0.05).
EXAMPLE 3: Growth of Cupriavidus necator in plant oil emulsified with guar gum at various concentrations
The same procedure as in Example 1 and 2 was followed, except that palm oil (1%) emulsified with various concentrations of guar gum from Example 2 were used as carbon source. CDM containing non-emulsified palm oil (1%) was used as controls. Cultures were grown in shaker incubator for 66 h following the same operating conditions as mentioned in Example 1. After the

termination of the culture, the dry cell weight was measured following the method as described in Example 1.
PHA accumulated in the cells was recovered using chloroform as extracting solvent and determined the dry weight of the recovered PHA to calculate the polymer content in the cells following the method as described in Example 9. The results from Example 3 are represented in Figure 2. Amounts of Guar gum around 0.1% in culture media showed significant enhancement of microbial growth (p<0.05). Non-emulsified palm oil alone was used as control to compare emulsified palm oil using various concentration of guar gum (GG) as mentioned in Table 2.
EXAMPLE 4: Growth of Cupriavidus necator in 13 emulsified plant oils
The same procedure as in Examples 1, 2 and 3 were followed, except that 13 selected plant oils as listed in Table 1 emulsified with 0.2% w/v guar gum as described in Examples 1 and 2 were used as carbon sources. Cultures were grown in shaker incubator for 66 h following the same operating conditions as mentioned in Example 3. After the termination of the culture, the dry cell weight was measured following the method as described in Example 3. The result is represented in Figure 3. Other than mustard oil and castor oil which contain certain inhibitory fatty acids, all the other oils in culture media produced similar biomass (p>0.05) in guar gum emulsified microbial culture. Guar gum showed similar emulsification properties for a range of plant oils and can be applied to a host of culture media.
EXAMPLE 5: Determination of growth and PHA accumulation with palm oil as carbon source
The same procedure as in Example 4 was followed, except that non-emulsified palm oil of Example 3 was used as carbon source for microbial growth and PHA production in scale up cultivation (up to 1L volume). The results obtained are represented in Figure 4. Addition of guar gum along with oil in culture media significantly (p<0.05) improved the biomass growth and PHA production of the microbial culture (doubling in both biomass and PHA accumulation on addition of guar gum).
Determination of residual oil remained in the culture media at the time of termination of the culture was recovered and estimated the percent oil utilization following the method of Budde et al. (Budde, et al., Appl. Microbiol. Biotechnol., 89: 1611-1619 (2011)).

EXAMPLE 6: Comparative productivities of Cupriavidus necator in four selected plant oils
The same procedure as in Examples 1, 4 and 5 were followed, except that 1% v/v non-emulsified plant oils (Palm oil, Coconut oil, Cottonseed oil and Sunflower oil) and the same plant oils emulsified using 0.1% guar gum, were used as carbon source for growth and PHA production.
Procedure for inoculum development was followed as described in Example 1 using the same seed culture medium and preculture medium.
The preculture was inoculated at OD600nm 0.075 as starting OD into a 1000 mL-Erlenmeyer flask containing 200 mL of the above mentioned CDM containing 2 g/L fructose and emulsified and non-emulsified above-mentioned plants oils (1%, v/v) as carbon source for growth and PHA accumulation.
Emulsions of plant oils using guar gum 0.1% were prepared prior to inoculation as described in Example 1 and used once in growth medium at the time of inoculation as carbon source for microbial growth and PHA production. The initial pH of the medium was in the range between pH 7.0 to 7.1.
Culture was maintained for 66 h in shaker following the same operating protocol as in Example 3, 4 and 5.
After termination of the cultivation, the cells were recovered by centrifugation and measured the dry cell weight following the method described elsewhere in Example 1.
PHA accumulated in the cells was recovered using chloroform as extracting solvent and determined the dry weight of the recovered polymer content as described below in Example 7. The results from Example 6 are represented in Figure 5. The results showed that addition of guar gum as an emulsifier along with oil in culture media significantly (p<0.05) improved the biomass growth and coconut oil combined with guar gum gave the highest biomass production.
EXAMPLE 7: Extraction and quantification of PHAs
Dry cell biomass recovered from culture broth following the method described in Example 1 were further processed to determine the PHA content accumulated in the cells. PHA accumulated in the cells was recovered using chloroform as extracting solvent. Dried cells (1 g) were treated with chloroform (100 mL) and the mixture was stirred at 200 rpm for 36 h to 48 h at 50oC. After

recovering the cell residue, the chloroform solution containing PHA was concentrated in a rotary evaporator to a total volume of 5 mL to 10 mL, followed by gradual addition of 25 mL to 60 mL ice-cold ethanol. The mixture was stirred gently for 10 min and then left for 20 min to complete the precipitation of extracted PHA. The PHA thus precipitated was recovered by filtration or centrifugation at 6500 g for 10 min, followed by washing with water 2 to 3 times to completely remove the traces of ethanol. The recovered PHA was dried at 50oC for 12 h and the dry weight of the recovered PHA was measured to calculate the polymer content in the cells.
EXAMPLE 8: Gas Chromatographic analysis of PHA
PHA extracted from Example 5 was further analyzed using gas chromatography against standard PHA (P(3HB), PHBHV and PHBHHx). Dried PHA material (20 mg) was dissolved in chloroform, hydrolysed and esterified using 3% methanolic HCl for 8 hrs at 100°C in reflux. The content was cooled at room temperature and 1 ml of water added to form bilayer. Lower chloroform layer was carefully transferred into a GC vial and injected in GC/FID. For analysis, FAME-wax column was used with the injector temperature of 80°C, oven temperature of 280°C and run time of 60 mins. The result confirming the nature of PHA is represented in Figure 6. GC analysis confirmed the produced PHA using above mentioned method is P(3HB).
The above results obtained from Examples 3, 5, 6 and 7 showed nearly 2-3 folds improved biomass and PHA production with emulsified plant oils of diverse sources. The results from examples as described above demonstrate that guar gum at low concentrations (0.02 to 0.1%) has no adverse effect on growth of Cupriavidus necator and that the emulsions prepared using guar gum, as disclosed in the present invention achieves better assimilation of plant oils as carbon source than that the use a non-emulsified oil.
Advantages of the present invention:
• Enviornmentally safe and use of non-toxic materials
• Cost efficient
• Increased production of PHA
• Increased utilization of carbon source etc.

We claim:
1. A method for the production of polyhydroxyalkanoates (PHAs), the method comprises the
step of:
a. Preparing an oil-in-water emulsion comprising guar gum and plant oils;
b. Culturing microbial cells in a nutrient medium comprising guar gum-based oil-in-
water emulsion of step (a);
c. Obtaining dry cell biomass of the microbial cells of step (b); and
d. Isolating and purifying polyhydroxyalkanoates (PHAs) from the dried cell biomass
of step (c).
2. The method as claimed in claim 1, wherein step (a) for obtaining the oil-in water emulsion
comprises the steps of:
a. Dissolving guar gum in water;
b. Stirring the mixture vigorously at 30 -50oC using a stirrer at 2000 – 2500 rpm for
15 – 20 mins;
c. Cooling the mixture to room temperature of about 28oC to 30oC and filtering the
cooled mixture through muslin cloth with pore size 100 – 500 µm to remove
undissolved particles;
d. Adding the mixture of step (c) to lipid/oil, followed by stirring for 2 min to 15 min
to dissolve the emulsifier to obtain the oil-in-water emulsion.
3. The method as claimed in claim 2, wherein the oil-in-water emulsion obtained in step (d) is composed of 0.5-1% v/v plant oil along with guar gum in the range from 0.02% w/v to 0.4% w/v.
4. The method as claimed in claim 3, wherein the plant oil is selected from palm oil, corn oil, peanut oil, sesame oil, cottonseed oil, castor oil, mustard oil, jatropha oil, olive oil, avocado oil, canola oil, soybean oil, sunflower oil, safflower oil, coconut oil, date seed oil, karanja oil, rice oil, rapeseed oil, fish oil, waste cooking oil, tallow, pig oil, palm kernel oil, algae-derived oils , refined products of these fats and oils, and their components such as fatty acids, salts of fatty acids and fatty acid esters.
5. The method as claimed in claim 1, wherein the step (b) for culturing the microbial cells comprises the steps of:

a. Inoculating a glycerol stock of microbial cells into the seed culture medium and
culturing for 18-24 hours in Shaking Incubator at 30oC and 125-200 rpm;
b. Inoculating the seed culture into a chemically defined medium (CDM) preculture
medium comprising fructose as carbon source and culturing for 20-24 hours in
shaking incubator at 30oC and 125-200 rpm;
c. Inoculating the preculture into a chemically defined medium (CDM) production
medium composed of fructose and/or plant oils as carbon sources and culturing for
48 hours to 66 hours in shaking incubator at 25-30oC and 120-180 rpm to obtain
cell biomass.
6. The method as claimed in claim 1, wherein the step (c) for obtaining the dry cell mass
comprises the steps of:
a. Terminating cultivation of cells;
b. recovering the cells from a known volume of culture suspension by centrifugation
at 5000-7000 g for 10-20 min to determine cell biomass;
c. Washing the recovered cells first with water, secondly with methanol and finally
with hexane to remove any residual/unutilized oil;
d. Drying the cells for 18-24 h in hot air oven at 50-60oC;
e. Cooling down the dried cells to room temperature in a desiccator to obtain the dried
cell mass for further isolation and purification of the PHA.
7. The method as claimed in claim 1, wherein the microbial cells are selected from the group consisting of Pseudomonus aeruginosa, Bacillus cereuc, Escherichia coli, Aureobasidium melanogenum, Alkaliphilus oremlandii, Candida glabrata, Candida rugosa, Yarrowia lipolytica, Bacillus tequilensis, Bacillus safensis.
8. The method as claimed in claim 7, wherein the bacteria is selected from the genus Cupriavidus or recombinant thereof.
9. The method as claimed in claim 1, wherein step (d) for isolation and purification of PHA comprises the steps of:
a. Treating the dried cells obtained in step (g) in claim 6 with solvent and stirring the
mixture at 150-200 rpm for 36 h to 48 h at 50oC;
b. Concentrating the chloroform solution containing PHA after recovering the cell
residue from step (a), and followed by gradual addition of ice-cold ethanol;

c. Stirring the mixture gently for 10-15 min and leaving it still for 20-30 min to
complete the precipitation of extracted PHA;
d. Filtrating or centrifugating the mixture at 5000-7000 g for 10-20 min, followed by
washing with water 2 to 3 times to completely remove the traces of ethanol from
the mixture to the PHA;
e. drying the recovered PHA at 50-55oC for 12-18 hours and the dry weight of the
recovered PHA was measured to calculate the polymer content in the cells.
10. The method as claimed in claim 1, wherein polyhydroxyalkanoate is selected from poly(3-
hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-
3HHx)], poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] and poly(3-
hydroxybutyrate-co-4-hydroxyvalerate) [P(3HB-co-4HV)], poly(3-hydroxy-butyrate-co-
4-hydroxybutyrate) [P(3HB-co-4HB)], poly(3-hydroxy-butyrate-co-4-hydroxypropionate)
[P(3HB-co-3HP)], and poly(3-hydroxybutyrate-co-3‐hydroxyhexanoate‐co‐3‐
hydroxyoctanoate‐co‐3‐hydroxydecanoate‐co‐3‐hydroxydodecanoate) [P(3HB-3HHx‐ 3HO‐3HD‐3HDD)].