Description:FIELD OF THE INVENTION
The present invention is related to methods and compositions for production of poly 3-hydroxybutyrate by Rhodobacter alkalitolerans in high yield and high purity.
BACKGROUND OF THE INVENTION
Polyhydroxybutyrate (PHB) is a biopolymer produced by various bacteria and archaea under nitrogen-limited conditions. It’s gaining attention in the bioplastic sector due to its biodegradability, eco-friendliness, and versatility.
PHB is synthesized by bacteria as energy storage granules. The production process involves microbial fermentation, where bacteria convert carbon sources into PHB. The resulting PHB can be processed into films, fibers, and other materials, making it a promising alternative to conventional plastics.
PHB has a wide range of applications due to its biodegradability and biocompatibility. It is used to produce biodegradable plastic products such as packaging materials, agricultural films, and disposable items. In the medical field, PHB is utilized in medical devices, sutures, and drug delivery systems. Additionally, PHB fibers can be spun into textiles for clothing and other fabric-based products, and in agriculture, it is used to create biodegradable mulch films and controlled-release fertilizer coatings.
Microbially produced Polyhydroxybutyrate (PHB) is a fully biodegradable biopolymer that breaks down into carbon dioxide and water under both aerobic and anaerobic conditions. It is water-insoluble and relatively resistant to hydrolytic degradation, making it distinct from many other biodegradable plastics. PHB also has good oxygen permeability, beneficial for certain packaging applications, and shows good resistance to ultraviolet light, making it suitable for outdoor use. Its mechanical properties are similar to polypropylene, with good tensile strength and flexibility, and it has a melting point around 175°C, allowing it to be processed using conventional plastic processing equipment. Additionally, PHB is biocompatible, making it ideal for medical applications such as sutures and drug delivery systems. Produced from renewable resources, PHB contributes to reducing plastic pollution, highlighting its environmental benefits.
Several species within the genus Rhodobacter are known for their ability to produce Polyhydroxybutyrate (PHB). Notably, Rhodobacter sphaeroides is a well-studied species that can accumulate significant amounts of PHB, especially under nitrogen-limiting conditions. This bacterium can produce PHB up to 70% of its cellular dry weight. Additionally, Rhodospirillum rubrum and Rhodopseudomonas palustris are also recognized for their PHB production capabilities.
These bacteria are often used in research and industrial applications due to their efficiency in producing PHB, which is a promising biodegradable plastic alternative. However, there are drawbacks like presence of copolymers of PHB which has been shown to increase elasticity. Pure PHB has been reported to have Young’s Modulus 3.5 GPa and Tensile Strength of 40 MPa, but the upon elongation to breakage percentage is lesser than copolymers (Strong et al., 2016) [1].
The advantages of using PHB include its ability to break down into water and carbon dioxide in natural environments, reducing plastic pollution, its safety for medical applications, and its production from renewable resources through microbial fermentation.
The current invention encompasses a method of producing poly 3-hydroxybutyrate with high yield and purity by a strain of Rhodobacter alkalitolerans.
SUMMARY
One aspect of the current invention is a method of producing poly-3 hydroxy butyrate (PHB) wherein the method comprises the steps of;
Culturing a strain of Rhodobacter alkalitolerans at a pH range of 6-10 to produce PHB followed by isolating produced the poly-3 hydroxy butyrate (PHB).
In one aspect, the Rhodobacter alkalitolerans strain is JA916T. In one aspect, the Rhodobacter alkalitolerans strain is cultured at a light intensity in the range of 200-600 µmol photons m-2s-1.
In one aspect, the Rhodobacter alkalitolerans strain is cultured at a light intensity is 500 µmol photons m-2s-1.
In one aspect, the Rhodobacter alkalitolerans strain is cultured for 20-30 hours to produce poly-3 hydroxy butyrate (PHB).
In one aspect, the carbon source used for culturing the strain of Rhodobacter alkalitolerans to produce PHB is pyruvate, acetate, or butyrate or a combination thereof.
In one aspect, the Rhodobacter alkalitolerans is cultured at a temperature range of 22-30 °C. In one aspect, the produced PHB is isolated by chloroform extraction and precipitation. In one aspect the poly-3 hydroxy butyrate (PHB) produced has a purity greater than 99%. In one aspect, the yield of poly-3 hydroxy butyrate (PHB) produced is 17-19 % of dry cell weight. In one aspect, the yield of poly-3 hydroxy butyrate (PHB) produced is 18 % of dry cell weight.
In one aspect, the poly-3 hydroxy butyrate (PHB) produced by the method encompassed in the invention has a Young’s modulus in the range of 3.5 to 6 GPa
BRIEF DESCRIPTION OF FIGURES:
Fig 1. Shows (A) Biosynthesis pathway of PHB in R. alkalitolerans Represents the genes and their corresponding enzymes involved in the pathway. (B) Represents the PHB accumulation (white bodies) in all three light intensities of 30, 250 and 500 µmol photons m-2s-1. (C, D, E,) Transcript expression level of PHB biosynthesis pathway transcripts in all the three light intensities.
Fig 2. Shows (A) Extracted PHB quantification from 100 mg of cell dry biomass in all three light intensities of 30, 250 and 500 µmol photons m-2s-1 in npH and hpH conditions. (B) Image of extracted PHB. (C) Quantification of cell dry biomass in all the three light intensities in npH and hpH conditions. (D) Functional group analysis by FTIR spectroscopy.
Fig 3. Shows GC-MS analysis of the of the methanolysis product of the extracted PHB from R. alkalitolerans. (A) Mass spectra peak of the PHB (B) Mass spectra peak of the PHB standard (R)-methyl 3-hydroxybutanoate. (C) Gass chromatogram of PHB standard (R)-methyl 3-hydroxybutanoate. (D) and methanolysis product of PHB extracted from R. alkalitolerans strain JA916T.
Fig. 4. Shows Proton (1H) and carbon (13C) NMR analysis of analysis of PHB extracted from R. alkalitolerans (A) 1H NMR represents the presence of CH3, CH2 and CH (B) 13C NMR of PHB shows the peaks for COOH, CH, CH2, CH3.
Fig. 5. Shows (A) Thermogravimetric analysis (TGA) of PHB showing maximum weight loss at 282.02 °C and (B) Differential Scanning calorimetry (DSC) showing the melting temperature at 177.59 °C.
DETAILED DESCRIPTION OF THE INVENTION
In the following detailed description, a reference is made to the accompanying drawings that form a part hereof, and in which the specific embodiments that may be practiced is shown by way of illustration. These embodiments are described in sufficient detail to enable those skilled in the art to practice the embodiments and it is to be understood that other changes may be made without departing from the scope of the embodiments. The following detailed description is therefore not to be taken in a limiting sense.
The production of Polyhydroxybutyrate (PHB) involves culturing bacteria in a bioreactor with a carbon source like glucose or sucrose under nutrient-limited conditions, typically nitrogen or phosphorus. Under these conditions, the bacteria accumulate PHB as intracellular granules. Once the bacterial cells are harvested, they are lysed to release the PHB granules, which are then extracted using methods like solvent extraction, mechanical disruption, or enzymatic digestion. The extracted PHB undergoes purification to remove any residual cell debris or impurities, involving washing with solvents, filtration, and drying.
Rhodobacter alkalitolerans is a phototropic bacterium known for its ability to grow in alkaline conditions. This Gram-negative, motile bacterium has a rod-to-oval shape and a yellowish-brown color. It was discovered in an alkaline brown pond in Gujarat, India. Its DNA has a G+C content of 65.1 mol%.
The strain JA916T [4] shares high 16S rRNA gene sequence similarity with other Rhodobacter species, it forms a distinct sub-clade, [2]
The current invention provides a method of synthesis of poly 3-hydroxybutyrate with high yield and purity by culturing a strain of Rhodobacter alkalitolerans.
DEFINITIONS:
The term “culture” or “culturing” a bacterial strain or a microorganism refers to Culturing a bacterial strain means growing bacteria in a controlled environment. The process involves allowing bacterial cells to multiply in or on a culture medium that provides the necessary nutrients and conditions for their growth. The specific conditions, such as temperature, pH, and oxygen levels, depend on the type of bacteria being cultured.
The term “normal pH” or “npH” as used herewith refer to a pH range of 6-7.5.
The term “high pH” or “hpH” as used herewith refer to a pH range of 7.5-10.
The term “light intensity” as used herewith refers to the amount of light energy hitting a surface per unit area, often measured in units such as lux (lx) or lumens per square meter. Light intensity determines how bright or dim a light source appears and can affect various processes, such as growth of photosynthetic bacteria, photosynthesis in plants.

The terms “poly 3-hydroxybutyrate”,” polyhydroxybutyrate” or “PHB” refers to a linear polymer of 3-hydroxybutyric acid and are used interchangeably herewith.
The term “Purity” of the produced PHB as used herewith refers to having the presence of only PHB and absence of any other polymers like valerate or hexanoate.
The term “Young’s modulus” refers to as the modulus of elasticity, is a measure of the stiffness of a solid material. It quantifies the relationship between tensile (or compressive) stress and axial strain in the linear elastic region of the material’s stress-strain curve. Essentially, it describes how much a material will deform under a given load and how well it returns to its original shape once the load is removed.
Mathematically, Young’s modulus (E) is defined as:
E=ϵσ
where:
• ( \sigma ) is the stress (force per unit area),
• ( \epsilon ) is the strain (proportional deformation).
Young’s modulus is typically measured in pascals (Pa), or expressed in gigapascals (GPa) or megapascals (MPa)
EMBODIMENTS
The current invention encompasses a method of producing poly 3-hydroxybutyric acid by a strain of Rhodobacter alkalitolerans.
The foregoing description of the specific embodiments willfully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such as specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modifications. However, all such modifications are deemed to be within the scope of the claims.
One embodiment of the invention is a method of producing poly-3 hydroxy butyrate (PHB) wherein the method comprises the steps of;
Culturing a strain of Rhodobacter alkalitolerans at a pH range of 6-10 to produce PHB followed by isolating produced the poly-3 hydroxy butyrate (PHB).
In one embodiment, the Rhodobacter alkalitolerans strain is JA916T.
In one embodiment, the Rhodobacter alkalitolerans strain is cultured at a light intensity in the range of 200-600 µmol photons m-2s-1. In one embodiment, the Rhodobacter alkalitolerans strain is cultured at a light intensity in the range of 250-550 µmol photons m-2s-1. In one embodiment, the Rhodobacter alkalitolerans strain is cultured at a light intensity is 500 µmol photons m-2s-1.
In one embodiment, the Rhodobacter alkalitolerans strain is cultured for 20-30 hours to produce poly-3 hydroxy butyrate (PHB).
In one embodiment, the Rhodobacter alkalitolerans strain is cultured for 22-27 hours to produce poly-3 hydroxy butyrate (PHB). In one embodiment, the Rhodobacter alkalitolerans strain is cultured for 24 hours to produce poly-3 hydroxy butyrate (PHB).
In one embodiment, the carbon source used for culturing the strain of Rhodobacter alkalitolerans to produce PHB is pyruvate, acetate, or butyrate or a combination thereof.
In one embodiment, the carbon source used for culturing the strain of Rhodobacter alkalitolerans to produce PHB is Fumarate, Succinate, Glucose, Fructose, Sucrose or aspartate or a combination thereof. In one embodiment, the carbon source used for culturing the strain of Rhodobacter alkalitolerans to produce PHB is pyruvate.
In one embodiment, the strain of Rhodobacter alkalitolerans is cultured at a temperature range of 22-30 °C. In one embodiment, the strain of Rhodobacter alkalitolerans is cultured at a temperature range of 22-28 °C.
In one embodiment, the produced PHB is isolated by chloroform extraction and precipitation.
In one embodiment, the poly-3 hydroxy butyrate (PHB) produced has a purity greater than 99%. In one embodiment, the yield of poly-3 hydroxy butyrate (PHB) produced is 17-19% of dry cell weight.
In one embodiment, the PHB synthesized by the method encompassed in the current invention has a Young’s Modulus higher than the PHB produced by other non-photosynthetic and photosynthetic organisms.
A higher value of Young’s modulus indicates a material with higher tensile strength, while a lower score suggests a more ductile material.
In one embodiment, the PHB synthesized by the method encompassed in the current invention has a Young’s Modulus in the range of 3.5 to 6 GPa. In one embodiment, the PHB synthesized by the method encompassed in the current invention has a Young’s Modulus in the range of 4.66 GPa.
In one embodiment, the poly-3 hydroxy butyrate (PHB) produced by the method encompassed in the invention has applications in food packaging, medical articles, agriculture, automotives, consumer goods.
In one embodiment, the poly-3 hydroxy butyrate (PHB) produced by the method encompassed in the invention can be used for biodegradable films, sutures, composites for bone health and repair, coating components to improve the biocompatibility of traditional implantable materials, drug delivery, controller release of fertilizers.
EXAMPLES
Example 1: Culturing of R. alkalitolerans at diff light intensities:
Cultures were grown in Biebl and Pfennig’s medium with sodium pyruvate (3g/l) as a source of carbon source and ammonium chloride as nitrogen source (0.4g/l) in light/anaerobically at room temperature (25 °C) in filled glass bottles with glass cork stoppers to avoid infiltration of air without any void space left as described previously at pH condition pH 6.80±0.05. The cultures were grown in three light intensities of 28-30 µmol photons m-2s-1(optimum light), 250-255 µmol photons m-2s-1 and 500 ± 5 µmol photons m-2s-1 by inoculating 1.125 ml of inoculum culture in 300 ml of Biebl and Pfennig’s media prepared in 25 mM of Tris buffer media and pH was set to 6.80±0.05 with hydrochloric acid. Cells were harvested in the late log phase (~at OD of 1.4-1.5) (Scheuring et al., 2014) to get maximum number of intracytoplasmic membranes, by centrifuging the cells at 15,000 x g for 20 min. Washed with 20 mM HEPES pH 7.5 and stored at -80℃ until required. For PHB extraction and analysis the cell pellets were lyophilized and PHB was extracted.
Example 2: Biosynthesis of PHB in R. alkalitolerans strain JA 916T
The transcript level study showed the highest production of PHB in npH and high light intensity, whereas the hpH lesser expression is seen (CDE). This is evident from the TEM images as well, where more accumulation of PHB is obvious in cells in all the conditions (B). After extraction and quantification of the PHB from 100 mg dry weight of cells, it was found that relatively more content of PHB in npH, 16.3% at 30 µmol photons m-2s-1, 17.6% at 250 µmol photons m-2s-1 and 17.3% at 500 µmol photons m-2s-1 than hpH conditions i.e. 9.7%, 13% and 10.5% at 30, 250 and 500 µmol photons m-2s-1 (Fig. 2A). Along with this, cell biomass was also found to be increased with an increase in light intensity, which is more in npH conditions (94.2 mg/l, < 480.8 mg/l, <565.3 mg/l) than hpH conditions (255 mg/l, <435.3 mg/l, <509.6 mg/l) (Fig. 2C)
Example 3: Purification of produced PHB from R. alkalitolerans JA916T
For extraction of PHB, harvested cells were lyophilized at -110 °C for 24 h, and cell powder was made. To 5 mg of cell powder, 200 µL of chloroform was added and thoroughly vortexed for 5 min. Now 200 µL of 0.9% KCl was added and stirred for 5 min to break open the cell to solubilize the maximum PHB inside the cell. Now this mixture was centrifuged at 6000 rpm for 10 min at 25 °C for phase separation. The lower organic phase containing the PHB was carefully collected and dried by a vacuum evaporator. The PHB produced was precipitated by acetone, which solubilizes all other constituents, leaving PHB. The obtained PHB were analyzed by biophysical and biochemical experiments.
Example 4: GC-MS analysis of PHBs
The PHA extracted from R. alkalitolerans after methyl esterification was analysed by GC-MS along with its standard (R)-Methyl 3-hydroxybutanoate (MW = 118.14, BLD pharm). The fragmentation pattern of methyl-esterified PHA was observed to be identical to the fragmentation pattern of the (R)-methyl 3-hydroxybutanoate standard (Fig. 3 A, B). The mass spectra of fragmented ionic species showed both (M-1 and M+1) ions at 117 and 118 m/z for 3-hydroxybutyrate (3-HB). Other ionic fragments resulting from 3- hydroxy functional group were also found with m/z of 103 (C4H7O3+), m/z 87 (C4H7O2+), m/z 74 (C3H6O2+), m/z 71 (C3H3O2+), m/z 61 (C2H3O2+) and m/z 43 (C2H3O) m/z 31 (CH3O+). The gas chromatogram also showed similar retention time for both of the molecules with single peaks at 4.35 min and 4.26 min (Fig. 3 C, D).
Example 5: Thermal properties of the polymer were analyzed by TGA and DSC
The thermal degradation temperature of the PHB was analyzed by TGA using the instrument STA 6000 (Perkin Elmer, USA). About 5 mg of the purified PHB sample was loaded in an aluminium pan and heated from 30 to 980 °C at a heating rate of 10 °C/min under a nitrogen atmosphere at 20.0 mL/min. The result as indicated in Figure:5. (A) show Thermogravimetric analysis (TGA) of PHB showing maximum weight loss at 282.02 °C. The DSC experiment was performed in the DSC- 60 instrument (Shimadzu, Japan) at a nitrogen gas flow rate of 100 mL/min in an aluminum-sealed pan with an initial amount of PHB of 2.3 mg. The heating program was 10 °C/min, starting at 30 °C till 200 °C. The melting temperature (Tm) was determined from the DSC thermogram.
Fig. 5 (B) Differential Scanning calorimetry (DSC) showing the melting temperature at 177.59 °C. It was observed that PHB degradation appears rapidly, marked by a sharp decrease in the curve at 282.02 °C. The onset temperature of the PHB was at 230.82 °C. The PHB was completely degraded at 356.86°C indicating the thermal stability of PHB by R. alkalitolerans till 356.86 °C. Similar studies on the thermal degradation of PHB produced by H. venusta KT832796 almost degraded at 278.51 °C, and maximum degradation occurred at 323.30°C (Stanley et al., 2020). Likewise, the PHB derived from Botryococcus braunii experienced thermal decomposition at a temperature of 240°C, with complete weight loss observed at 296°C according to the report (Kavitha et al., 2016). Therefore, the present invention shows the highest purity of PHB produced by the method encompassed in the invention wherein the PHB has higher tensile strength and higher Young’s modulus.

Example 6: Tensile strength measurement

Tensile testing (ASTM D638) was measured in Universal testing machine (UTM, Instron Temp. range of -70 °C to 250° C and max load of 5KN) at 2 mm/min to determine max. tensile strength & Young’s modulus. Specimens conditioned to 50 % relative humidity at 25 °C. At least four replicates were tested.
The tensile strength by UTM showed a Young’s modulus of 4.66 GPa.
The PHB synthesized by the method encompassed in the invention has a high purity and a higher value of Young’s modulus than other reported PHBs from non- photosynthetic and photosynthetic microorganisms (Strong et al., 2016) [1].

References
[1] Strong, P. J., Laycock, B., Mahamud, S. N. S., Jensen, P. D., Lant, P. A., Tyson, G., & Pratt, S. (2016). The opportunity for high-performance biomaterials from methane. Microorganisms, 4(1), 11.
[2] Kumar, A., & Singh, S. (2018). Biodegradation of polycyclic aromatic hydrocarbons by microbial consortia enriched from rhizosphere soil. *Archives of Microbiology, 200*(8), 1101-1110.
, Claims:1. A method of producing poly-3 hydroxy butyrate (PHB) wherein the method comprises the steps of;
Culturing a strain of Rhodobacter alkalitolerans at a pH range of 6-10 to produce PHB followed by isolating the produced the poly-3 hydroxy butyrate (PHB).
2. The method of claim 1, wherein the Rhodobacter alkalitolerans strain is JA916T.
3. The method of claim 1, wherein the Rhodobacter alkalitolerans strain is cultured at a light intensity in the range of 200-600 µmol photons m-2s-1.
4. The method of claim 3, wherein the Rhodobacter alkalitolerans strain is cultured at a light intensity is 500 µmol photons m-2s-1.
5. The method of claim 1, wherein the Rhodobacter alkalitolerans strain is cultured for 20-30 hours to produce poly-3 hydroxy butyrate (PHB).
6. The method of claim 1, wherein the Rhodobacter alkalitolerans strain is cultured for 22-27 hours to produce poly-3 hydroxy butyrate (PHB).
7. The method of claim 1, wherein the carbon source used for culturing the strain of Rhodobacter alkalitolerans to produce PHB is pyruvate, acetate, or butyrate or a combination thereof.
8. The method of claim 1, wherein the Rhodobacter alkalitolerans is cultured at a temperature range of 22-30 °C
9. The method of claim 1, wherein the produced PHB is isolated by chloroform extraction and precipitation.
10. The method of claim 1 wherein poly-3 hydroxy butyrate (PHB) produced has a purity greater than 99%.
11. The method of claim 1, wherein the yield of poly-3 hydroxy butyrate (PHB) produced is 17-19% of dry cell weight.
12. The method of claim 1, wherein the poly-3 hydroxy butyrate (PHB) produced has a Young’s modulus in the range of 3.5 to 6 GPa.