A process for synthesis of poly-γ-glutamic acid

Abstract

The present invention relates to a field of biopolymers and microbial fermentation. Specifically, the present invention relates to a microbial process for industrial production of a biopolymer. More particularly, the present invention relates to a process for synthesis of poly-γ-glutamic acid from maltose and sugarcane bagasse. The process of the present invention is devoid of pre- treating of raw materials (biomass) thereby eliminating environmental and cost concerns.

Description

[0001] A PROCESS FOR SYNTHESIS OF POLY -y- GLUTAMIC ACID

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention relates to a microbial process for industrial production of a biopolymer. Particularly, the present invention relates to a process for synthesis of poly-y-glutamic acid from maltose and sugarcane bagasse. More particularly, present invention relates to a process for synthesis of poly-y-glutamic acid which devoid of pre-treating of raw materials (biomass) thereby eliminating environmental and cost concerns.

[0004] BACKGROUND OF THE INVENTION

[0005] Poly-y-glutamic acid (y-PGA) is a natural and biodegradable polymer. Reference may be made to the Journal “Biotechnology for Biofuels 9.1, 2016 (1-12)” which discloses the microbial synthesis of poly-y-glutamic acid, current progress, challenges, and future perspectives. Chemically y-PGA is an anionic homopolyamide composed of L- and D-glutamic acid linked through amide linkages.

[0006] Reference may be made to the Journal “Foods, 11(5) 739, 2022” which discloses recent advances in microbial synthesis of poly-y-glutamic acid and describes that owing to its specific biological properties, the y-PGA biopolymer is widely implicated in various applications, including agricultural and food processing, medical treatments, cosmetics, and others. The y-PGA biopolymer is very demanding due to its functional properties. However, the lower yield and high production costs limit its application. Currently, its production is carried out using four main methods: peptide synthesis, biotransformation, chemical synthesis, and microbial-based production.

[0007] Microbial-based production of y-PGA is one of the most advantageous compared to other processes as it involves lesser environmental concerns, employs cheaper raw substrates, and has higher production with mild reactions.

[0008] Reference may be made to the Journal “World Journal of Microbiology and Biotechnology, 33, 1-8, 2017” which discloses that the Bacillus species are generally reported as one of the highest producers of y-PGA. Most Bacillus strains, such as Bacillussubtilis, Bacillus paralicheniformis and Bacilluslicheniformis reported for y-PGA, are generally regarded as safe (GRAS) organisms. They are widely implicated in its production. Although microbial fermentation for y-PGA is well-established with ample genetic and metabolic pathway information, cost yield is still the major bottleneck for its industrial production. The microbial strains and culture medium constitute a significant chunk of y-PGA production cost. Thus, the process requires cheaper feedstock serving as a carbon or complete medium source and an efficient microbial strain for utilizing complex feedstock for y-PGA production.

[0009] Researchers globally, in conjunction with genetic engineering, have optimized various synthetic and waste-based media for y-PGA production.Notable achievements include solid-state fermentation using Bacillus velezensis, resulting in yields as high as 158 g / kg of guar gum.

[0010] Reference may be made to the Journal “Biomass Conversion and Biorefinery, 13(6), 4555-4573, 2023a” which discloses the Poly-gamma-glutamic acid biopolymer as a sleeping giant with diverse applications and unique opportunities for commercialization and several other highly reported strains include Bacillus paralicheniformis producing 284 g / L, Bacillus subtilis producing 101 g / 1, Bacillus licheniformis producing 41.6g / l; and Bacillus siamensis, producing 25.22 g / L using synthetic medium. In order to minimize the cost of production, several studies have explored different wastes ranging from hydrolysate of lignocellulosic biomass to food wastes, including tomato, and reported as high as 100 g / L of y-PGA. However, the significant limitation of the waste -based approach is a requirement of pretreatment process for releasing monosaccharide sugars from complex polysaccharides such as cellulose and hemicellulose.

[0011] However, aforesaid known synthetic or biological media-based methods possess disadvantages / limitations i) produce higher amounts of impurities, and separation cause more additional costs, ii) lesser selectivity and productivity; iii) requires multiple substrates / components to have effective conversion, iv) requires higher temperature, and v) requires high cost feedstock and their pretreatment before using in the media.

[0012] Therefore, there is an unmet need to provide a process for y-PGA production directly from raw materials (agro-waste), without the need for any pretreatment. OBJECTIVES OF THE INVENTION

[0013] Main object of the present invention is to develop a cost-effective process for the industrial production of poly-y-glutamic acid (y-PGA) by utilizing untreated sugarcane bagasse as a raw substrate.

[0014] Another object of the present invention is to optimize the synthetic medium with maltose as a carbon source for y-PGA production, resulting in higher yields compared to previous reports.

[0015] Yet another object of the present invention is to demonstrate feasibility of using Bacillus velezensis WAI 1 for y-PGA production from sugarcane bagasse, a readily available agro-waste.

[0016] Yet another object of the present invention is to reduce the cost of production associated with raw substrates and their pre-treatment by utilizing sugarcane bagasse directly without any pretreatment process.

[0017] Yet another objective of the present invention is to contribute to the advancement of the biopolymer industry by addressing the challenges related to cost and sustainability in y-PGA production, thus making it more accessible for various applications in food, cosmetics, and other industries.

[0018] SUMMARY OF THE INVENTION

[0019] Accordingly, present invention provides a process for synthesis of poly-y-glutamic acid, comprising the steps of: i. incubating and treating a nutrition media with Bacillus velezensis for a duration of 100 to 140 hours, wherein the nutrition media comprises: a) a carbon source selected from 20 to 30 wt. % of maltose or 1 to 5 wt. % of non-treated sugarcane bagasse, b) 0.5 to 1.5 wt. % of yeast extract as a nitrogen source, and c) 5 to 7 wt. % of L-glutamic acid as an external source.

[0020] In an embodiment of the present invention, the nutrition media comprises 25% of maltose as the carbon source.

[0021] In another embodiment of the present invention, the nutrition media comprises 3% of the sugarcane bagasse as the carbon source. In yet another embodiment of the present invention, the nutrition media comprises 1 wt.% yeast extract as the nitrogen source.

[0022] In yet another embodiment of the present invention, the nutrition media comprises 6% of L- glutamic acid as the external source.

[0023] In yet another embodiment of the present invention, the nutrition media further comprises 0.05 to 0.2 % of Na2HPO4, 0.05 to 0.2 % of NaH2PO4and 0.01 to 0.05 % of MgSO4.7H2O.

[0024] In yet another embodiment of the present invention, the pH of the nutrition media is maintained in a range of 6.5 to 7.5 throughout the incubation period.

[0025] In yet another embodiment of the present invention, the process is carried out in a flask method.

[0026] In yet another embodiment of the present invention, the sugarcane bagasse is washed to remove dirt and oven dried at 50°C.

[0027] In yet another embodiment of the present invention, the oven-dried sugarcane bagasse is ground to a powder form, and used without any pre-treatment for synthesis of the poly-y-glutamic acid.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 shows enzyme activities in bacterial isolate WA11.

[0030] Figure 2 shows 16S rRNA gene -based molecular phylogeny analysis using the Neighbour Joining method.

[0031] Figure 3 shows optimization of media and conditions for y-PGA production from synthetic media A and B. Temperature and pH optimization; C and D. Carbon and Nitrogen source optimization; E,F, and G. Yeast extract, Maltose, and L-Glutamic acid concentration optimization; H. Inoculum concentration optimization; I. Production using optimized conditions; and J. Shaking speed standardization (in Revolution per minute RPM). Statistical tests were performed using One-way Anova with Tukey’s multiple comparison test.

[0032] Figure 4 shows characterization of y-PGA produced from synthetic medium A. TLC characterization, B and C. FTIR analysis of standard y-PGA (Sigma) and crude y-PGA, and D and E. 1H NMR of standard and crude y-PGA.

[0033] Figure 5 shows y-PGA production using sugarcane bagasse as a carbon source. DETAILED DESCRIPTION OF THE INVENTION

[0034] The PGGA is a naturally occurring biopolymer based on anionic homopolyamide.

[0035] The present invention relates to a microbial process for industrial production of a biopolymer. The present invention relates to a process for synthesis of poly-y-glutamic acid from maltose and sugarcane bagasse. The process of the present invention is devoid of pre-treating of raw materials (biomass) thereby eliminating environmental and cost concerns.

[0036] The present invention relates to a process for synthesis of poly-y-glutamic acid, comprising of: a) incubating and treating nutrition media with Bacillus velezensis for a duration of 120 hours, wherein the nutrition media comprises, one of 20 to 30% of maltose or 1 to 5% of non-treated sugarcane bagasse as a carbon source, 0.5 to 1.5 wt% yeast extract as a nitrogen source, and 5 to 7% of L-glutamic acid as an external source etc.

[0037] The nutrition media preferably comprises 25% of maltose as the carbon source for production of the poly-y-glutamic acid.

[0038] The nutrition media preferably comprises 3% of the sugarcane bagasse as the carbon source for production of the poly-y-glutamic acid.

[0039] The nutrition media preferably comprises 1 wt% yeast extract as the nitrogen source, and 6% of L-glutamic acid as the external source.

[0040] The nutrition media further comprises 0.05 to 0.2 % of Na2HPO4, 0.05 to 0.2 % of NaH2PO4 and 0.01 to 0.05 % of MgSO4.7H2O. pH of the nutrition media is maintained in a range of 6.5 to 7.5, preferably 7, throughout the incubation period.

[0041] The process is carried out in a flask method.

[0042] The sugarcane bagasse is washed to remove dirt, followed by oven drying at 50°C and grinding to a powder form and used without any pre-treatment for production of the poly-y-glutamic acid. The process provided by the present invention utilizes lignocellulose biomass (LCB) i.e., sugarcane bagasse for producing high value biopolymer PGA without involving any pretreatment steps that potentially reduces cost of production and environmental concerns.

[0043] The process includes incubating and treating nutrition media containing 25% of maltose as a carbon source, 1 wt% yeast extract as a nitrogen source, and 6% of L-glutamic acid as an external source, with Bacillus velezensis for a duration of 120 hours. In another exemplary embodiment, the process includes incubating and treating nutrition media containing 3% of non-treated sugarcane bagasse as a carbon source, 1 wt% yeast extract as a nitrogen source, and 6% of L-glutamic acid as an external source, with Bacillus velezensis for duration of 120 hours.

[0044] The present invention provides a method of preparation of poly-y-glutamic acid from maltose and sugarcane bagasse powder, comprising incubating and treating nutrition media comprising 20 to 30 wt. % of maltose or 2 to 4 wt. % of non-treated sugarcane bagasse as carbon source, 0.5- 1.5 wt.% of yeast extract as nitrogen source, pH in range of 6.5-7.5, 5-7 wt. % of L-glutamic acid as external source and rest is water or DI water; with bacillus species in a flask method wherein the sugarcane bagasse powder is obtained from bagasse waste which is washed, dried and grinded into said powder and used as it is without any chemical pre-treatment.

[0045] The bacterial strain used herein provides lignocellulolytic activity, which is confirmed in Figure 1 where the qualitative screening of strain on different media plates are carried out, which included 0.5%CMC, 0.2% Veratryl Alcohol, 0.2% Guaiacol, and 0.5% Xylan. The formation of zones indicates potential of lignocellulolytic activity.

[0046] The carbon source material used herein in the process can be waste / degraded sugarcane juice, molasses or untreated LCB wastes. LCB is Lignocellulose biomass. The inventors have carried out experiments using commercially available cellulose (4%) (Himedia, Cat.No.RM126) and got around 15 g / 1 of PGA. Further, inventors have used different LCB waste (other than sugarcane bagasse), which includes untreated Rice bran and Water Hyacinth.

[0047] It is further submitted that the literature known studies have always involved pre-treatment (mostly acidic treatment) of LCB for production of PGA. Liu et al. Food Bioscience, 46, 101575, 2022, reported use of Guar gum as carbon source along with pre-treatment, which is not a case in present invention. The present invention utilizes LCB for producing high value biopolymer PGA without involving any pretreatment steps that could potentially reduce cost of production and environmental concerns.

[0048] In a nutshell, the present invention relates to a process of y-PGA production using Bacillus velezensis WA11, which was isolated from Water Hyacinth plant. y-PGA is one of the costliest biopolymers of the current industry trend, which is involved in various applications. Inventors have provided a method by which 104 g / L of crude dried y-PGA is obtained from the optimized synthetic medium containing maltose as a carbon source with a productivity of 1.09 g / L / h. Moreover, inventors have also provided alternative, better and efficient method in terms of cost, ease and carbon source using waste materials, by replacing the synthetic carbon source with e. g. sugarcane bagasse, to address the feed substrate and its treatment cost where it provided 11 to 12 g / L of crude dried y-PGA from sugarcane bagasse. This is the first report on producing y-PGA using lignocellulosic biomass, and that to the requirement of any without pre-treatment (process steps). Additionally, inventors have also reported 2.5-fold higher y-PGA production than the previous best reported study utilizing maltose as a carbon source. The present invention may help in reduction of costs associated with substrate for y-PGA production.

[0049] EXAMPLES

[0050] Following examples are given by way of illustration and therefore should not be construed to limit the scope of the invention.

[0051] Materials

[0052] Cellulose (GRM126, Himedia, Supplier: Omkar Traders, India, Maharashtra, Katraj, Pune), Bushnell-Hass broth (M350, Himedia, Supplier: Omkar Traders, India, Maharashtra, Katraj, Pune), Carboxymethylcellulose sodium salt (MB 138, Himedia, Supplier: Omkar Traders, India, Maharashtra, Katraj, Pune), Beechwood Xylan (RM10398, Himedia, Supplier: Omkar Traders, India, Maharashtra, Katraj, Pune), Veratryl alcohol (RM2779, Himedia, Omkar Traders, India, Maharashtra, Katraj, Pune), Guaiacol (RM 118, Himedia, Omkar Traders, India, Maharashtra, Katraj, Pune), Congo Red (GRM508, Himedia, Omkar Traders, India, Maharashtra, Katraj, Pune), Dextrose anhydrous (GRM016, Himedia, Omkar Traders, India, Maharashtra, Katraj, Pune), Yeast Extract (RM027, Himedia, Omkar Traders, India, Maharashtra, Katraj, Pune), Peptone (RM667, Himedia, Omkar Traders, India, Maharashtra, Katraj, Pune), Sodium Chloride (MB023, Himedia, Omkar Traders, India, Maharashtra, Katraj, Pune), L-Glutamic acid monosodium salt monohydrate, Hi-AR (GRM681, Himedia, Omkar Traders, India, Maharashtra, Katraj, Pune), di-Sodium hydrogen phosphate anhydrous (TC051, Himedia, Omkar Traders, India, Maharashtra, Katraj, Pune), Sodium dihydrogen orthophosphate (GRM1255, Himedia, Omkar Traders, India, Maharashtra, Katraj, Pune), Magnesium sulphate heptahydrate, isi.piHi- AR™ / ACS (GRM684, Himedia, Omkar Traders, India, Maharashtra, Katraj, Pune), Ligation Sequencing Kit (SQK-LSK109, Oxford Nanopore Technologies, Genotypic, India), DNeasy PowerSoil Pro Kit (47014, Qiagen, Xydor). Example 1: Screening for cellulolytic, ligninolytic, and xylanolytic activities

[0053] The screening for lignocellulolytic potential was carried out as described by Falade et al., (2017). Peroxidase production and ligninolytic potentials of fresh water bacteria Raoultella ornithinolytica and Ensifer adhaerens Biotechnology reports, 16, 12-17. The bacterium WA11 isolated from Water Hyacinth was inoculated in nutrient broth and incubated for 12 hours at 37 °C. After the incubation, one loopful of overnight grown culture was spot inoculated in the Bushnell Hass agar medium supplemented with different carbon sources (0.5% Carboxymethyl cellulose (CMC), 0.5% Beechwood xylan, 0.2% Veratryl alcohol (VA) and Guaiacol (GA)) and yeast extract (0.1%) for 48 hours at 37 °C. The activity was confirmed by observing the zone of degradation obtained after following staining procedures: For CMC and Xylan-containing plates: the plate was stained with 0.2% Congo Red for 15 minutes, followed by two times washing with 5M Sodium chloride for 15 minutes; For VA and GA plates, staining was carried out using 1:2 diluted Grams Iodine for 15 minutes followed by washing with distilled water. All the chemicals were procured from Himedia and Sigma Chemicals.

[0054] The cellulolytic, ligninolytic, and xylanolytic activities are observed in bacterial isolate WA11 (Figure 1). Further, screening suggested WA11 is positive for y-PGA production. Therefore further optimization was carried out for y-PGA using WA11 bacterial isolate. The screening for ligninolytic activity is carried out using 0.2% Veratryl Alcohol (VA) and Guaiacol, cellulolytic activity using 0.5% of Carboxymethyl cellulose (CMC), and xylanolytic activity using Beechwood xylan 0.5%. Note: The bacterium WAI 1 isolated culture is submitted in IDA facility of National Centre for Microbial Resource (NCMR) at National Center for Cell Science (NCCS), Pune, India on 31stJuly 2023, having accession number MCC 0264.

[0055] Example 2: Screening for y-PGA producers and molecular characterization

[0056] Briefly, a colony of WA11 isolate was inoculated in seed medium (Glucose 2%, Yeast extract, Peptone, Sodium Chloride, and Beef extract 0.5%, pH 7.0) at 37 °C overnight. 5% of the overnight grown isolates were inoculated in a fermentation medium (Glucose 5%, E-Glutamic acid monosodium salt monohydrate 4%, Na2HPO4 0.1%, NaH2POr 0.7%, MgSO4-7H2O 0.02%, Yeast extract 1%, pH 7.0). Following incubation for 24 hours at 28 °C, the broth was centrifuged at 10,000 rpm for 20 minutes. The resultant supernatant was mixed with 4X chilled methanol volume and was observed for fibrous precipitate. The molecular characterization of the y-PGA- producing isolate was carried out using 16S rRNA sequencing. The 16S rRNA gene was amplified using 27F (5’ CCAGAGTTTGATCMTGGCTCAG 3’) and 1492R (5’ TACGGYTACCTTGTTACGACTT 3’) universal primers and sequenced using a 3500x1 genetic analyzer (Thermofisher, USA). The NCBI accession number for the 16S rRNA sequence is OR263461. The phylogenetic analysis was conducted using MEGA vl l software using the Neighbour Joining Method.

[0057] The 16S rRNA sequence (1412 bp) was analyzed for closest type strain similarity using EZtaxon. The top-hit strain was Bacillus velezensis CR-502(T), with 100% similarity and 95.4% completeness. Further, phylogenetic investigation using the Neighbour Joining method with 1000 bootstrap suggested close phylogeny to Bacillus velezensis (Figure 2).

[0058] Example 3: Optimization of y-PGA production and characterization

[0059] The optimization of medium components, their concentration, and physical parameters (pH, Temperature, and rotation speed) was carried out using the One Factor at a Time (OFAT) approach. The seed medium composition was (Glucose 2%, Yeast extract, Peptone, Sodium Chloride, and Beef extract 0.5%, pH 7.0) for all the optimization experiments. The initial fermentation medium consisted of Glucose 5%, L-Glutamic acid monosodium salt monohydrate 4%, Na2HPO40.1%, NaH2PO40.7%, MgSO4.7H2O 0.02%, Yeast extract 1%, pH 7.0. The produced crude y-PGA was characterized using Thin Layer Chromatography using solvents n- Butanol: Acetic Acid: H2O in the ratio of 12:5:3, Fourier Transform Infrared (FTIR) Spectroscopy (ATR mode using a Bruker Tensor II spectrophotometer equipped with a diamond crystal probe detector. Absorbance spectra were recorded from 500-4000 cm-1with a resolution of 4 cm”11), and Nuclear Magnetic Resonance (HI NMR, lOmg / mL sample was dissolved in D2O (deuterium oxide) and recorded 1H NMR spectra using Bruker AV 500 MHz). All the statistical analysis was performed in Graphpad PRISM v8. The OFAT strategy is used to optimize the media and physical parameters for y-PGA production. Higher production was obtained at 28°C and pH 7.0. Maltose and yeast extract were the favourable carbon and nitrogen sources, respectively. Further, concentration optimization suggested 25%, 1%, and 6% as the optimum maltose, yeast extract, and L-glutamate concentrations for y-PGA production by Bacillus velezensis WAI 1. Upon final validation, 104.3 g / L of dried crude y-PGA is obtained with productivity of 1.09 g / L / h from synthetic medium, respectively (Figure 3 A to J) from optimized synthetic medium. The OFAT strategy was utilized to optimize the media and physical parameters for y-PGA production. Higher production was obtained at 28°C and pH 7.0 in 24 hours. Since the glutamic acid uptake system is pH- dependent, the pH is a very crucial factor for production of y-PGA, reports have suggested pH 6.5 to 7.0 as the favourable pH for most of the Bacillus species [Luo, Z. et al., (2016). Microbial synthesis of poly-y-glutamic acid: current progress, challenges, and future perspectives. Biotechnology for Biofuels, 9, 1-12; Cromwick et al., (1996). Effects of pH and aeration on y-poly (glutamic acid) formation by Bacillus licheniformis in controlled batch fermentor cultures. Biotechnology and Bioengineering, 50(2), 222-227], The optimization for carbon sources for the production of y-PGA showed that maltose is the most effective source for the Bacillus velezensis, whereas sucrose was the secod-most effective sugar for production. Unlike other bacillus species, the isolate used in the present study was providing significant enhancements in the production of biopolymer (Wang, D. et al., (2020). High-level production of poly-y-glutamic acid from untreated molasses by Bacillus siamensis IR10. Microbial Cell Factories, 19, 1-13; Elbanna, K. et al., (2024). Poly (y) glutamic acid: a unique microbial biopolymer with diverse commercial applicability. Frontiers in microbiology, 15, 1348411). The yeast extract outperformed the other nitrogen sources used in the present study, however, increasing its concentration did not yield linear production of y-PGA. 1 % was thus used for further optimizations. In comparison to the initial media, the OFAT -based medium and process parameter optimizations resulted in more than 5 -fold increase in the y-PGA production by Bacillus velezensis WA11.

[0060] The optimized synthetic media consisted of Maltose- 25%, E-Glutamic acid- 6%, Na2HPO4 - 0.1%, NaH2PO4- 0.7%, MgSO4.7H2O- 0.02%, Yeast Extract- 1%, pH- 7.0, RPM of 180. The medium components Na2HPO4, NaH2PO4 and MgSO4.7H2O are generally used in smaller quantities but have a very significant role in the overall microbial growth and product formation. Generally, the Na2HPO4 (0.05 to 0.2 %) and NaH2PO4 (0.05 to 0.2 %) are used as a buffering agent facilitating the maintenance of pH during fermentation. The MgSO4-7H2O (0.01 to 0.05 %) provides magnesium ions that serve as an essential cofactors for several enzymatic reactions occurring in bacterial cells, thus supporting growth and metabolism.

[0061] Further, the y-PGA was characterized using different methods (Figure 4 A to E). Fig 4A shows the Primary characterization of crude y-PGA was carried out using Thin-Layer chromatography (TLC). For TLC, the mobile-phase composition was N-Butanol : Acetic Acid : H2O in the ratio of 12:5:3. Following the TLC run, the chromatography layer was sprayed with 20 ml of 0.2% Ninhydrin made in Acetone. This was followed by drying in the oven at 60 degree Celsius, till the colour was developed. 4B and 4C shows the Fourier Transform Infrared (FTIR) Spectroscopy (ATR mode using a Bruker Tensor II spectrophotometer equipped with a diamond crystal probe detector) was used for characterization of y-PGA. Absorbance spectra were recorded at 500-4000 / cm with a resolution of 4 cm-11. Fig 4D and 4E shows the final characterization was done using Nuclear Magnetic Resonance (1H NMR, lOmg / mL sample was dissolved in D2O (deuterium oxide) and recorded 1H NMR spectra using Bruker AV 500 MHz). The highest y-PGA production reported by using maltose as a carbon source from Bacillus subtilis was 35 g / 1 with a productivity of 0.36 g / L / h ((Ogawa et al., (1997). Efficient production of y-polyglutamic acid by Bacillus subtilis (natto) in jar fermenters. Bioscience, biotechnology, and biochemistry, 67(10), 1684-1687)). In the present study, 104.3 g / L y-PGA production with productivity of 1.09 g / L / h, 2.9-fold higher than the previous study using maltose as a carbon source is reported.

[0062] Example 4: Poly- y-glutamic acid production from Bagasse

[0063] The sugarcane bagasse was collected from a local shop in Pune, India, and was immediately transferred to the laboratory. Further, the sugarcane bagasse was washed twice with tap water to remove dirt, followed by Oven drying at 50°C until it was dried completely. The dried sugarcane bagasse was grounded to coarse powder form using a home-based grinder. The resultant sugarcane bagasse was added with an optimized medium (except maltose as a carbon source) and incubated further for y-PGA production.

[0064] The dried sugarcane bagasse was added with optimized medium components (except Maltose C- source) at different concentrations (0%, 1%, 2%, 3%) along with L-Glutamic acid- 6%, Na2HPO4- 0.1%, NaH2PO4- 0.7%, MgSO4.7H2O- 0.02%, Yeast Extract- 1%, pH- 7.0. Different concentrations of bagasse were used to evaluate y-PGA production. The highest production of 12.5 g / L in 3% bagasse-containing media is observed. Further, time optimization suggested 120 hours as the best time for harvesting crude y-PGA (11.1 g / L) (Figure 5). The structure was confirmed using NMR.

[0065] In the present study, y-PGA production was carried out using Bacillus velezensis WA11 isolated from Water Hyacinth. y-PGA is one of the costliest biopolymers involved in various applications. 104 g / L of crude dried y-PGA from the optimized synthetic medium containing maltose as a carbon source with a productivity of 1.09 g / L / h is obtained. However, the synthetic carbon source was replaced with sugarcane bagasse to address the feed substrate and its treatment cost. 11 to 12 g / L of crude dried y-PGA is obtained from sugarcane bagasse. This is the first report on producing y-PGA using lignocellulosic biomass without pre-treatment. Additionally, 2.5-fold higher y-PGA production is reported than the previous best reported study utilizing maltose as a carbon source. The study helps reduce costs associated with substrates for y-PGA production.

[0066] ADVANTAGES OF THE INVENTION

[0067] • By utilizing untreated sugarcane bagasse as a raw substrate, the invention significantly reduces the production costs associated with raw materials and their pre-treatment. This makes the production of poly-y-glutamic acid (y-PGA) more economically viable.

[0068] • The use of sugarcane bagasse as a raw substrate is a sustainable approach as it utilizes an agro-waste material, reducing the environmental impact associated with waste disposal.

[0069] • The process provides a scalable and efficient process for the industrial production of y- PGA, making it more accessible for diverse applications.

[0070] • The direct utilization of sugarcane bagasse without the need for pretreatment simplifies the production process. This eliminates the requirement for additional steps and resources, making the process more streamlined and efficient.

Claims

We Claim1. A process for synthesis of poly-y-glutamic acid, comprising the steps of: a) incubating and treating a nutrition media with Bacillus velezensis for a duration of 100 to 140 hours, wherein the nutrition media comprises: i. a carbon source selected from 20 to 30 wt. % of maltose or 1 to 5 wt. % of non-treated sugarcane bagasse, ii. 0.5 to 1.5 wt. % of yeast extract as a nitrogen source, and iii. 5 to 7 wt. % of L-glutamic acid as an external source.

2. The process as claimed in claim 1, wherein the nutrition media comprises 25% of maltose as the carbon source.

3. The process as claimed in claim 1, wherein the nutrition media comprises 3% of the sugarcane bagasse as the carbon source.

4. The process as claimed in claim 1, wherein the nutrition media comprises 1 wt.% yeast extract as the nitrogen source.

5. The process as claimed in claim 1, wherein the nutrition media comprises 6% of L- glutamic acid as the external source.

6. The process as claimed in claim 1, wherein the nutrition media further comprises 0.05 to 0.2 % of Na2HPO4, 0.05 to 0.2 % of NaH2PO4and 0.01 to 0.05 % of MgSO4.7H2O.

7. The process as claimed in claim 1, wherein the pH of the nutrition media is maintained in a range of 6.5 to 7.5 throughout the incubation period.

8. The process as claimed in claim 1, wherein the process is carried out in a flask method.

9. The process as claimed in claim 1, wherein the sugarcane bagasse is washed to remove dirt and oven dried at 50°C.

10. The process as claimed in claim 9, wherein the oven-dried sugarcane bagasse is ground to a powder form, and used without any pre-treatment for synthesis of the poly-y-glutamic acid.

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