The plastics have threatened natural environment worldwide since long time In response to problems associated with plastic waste and its effect on the environment, there has been a considerable interest in the development and production of an alternative, biodegradable plastics or bioplastics. Expansion of conventional petrochemical plastics production, consumption and improper disposal are having a significant impact both visibly and invisibly on the environment and society [1]. Plastics are recalcitrant to microbial degradation and accumulate in the environment at a rate of 28 million tons per year recently, technologies are heading for the development of bio-green materials that cause negligible side effects on the environment [2]. The biologically-synthesized plastics, Polyhydroxybutyrate (PHBs) are promising alternatives to synthetic plastics as they are biodegradable and biocompatible. These polymers are utilized in packaging, agriculture and medicine, as they are immunologically compatible with human tissue and can be used as biofuel [3,4]. The advantage of PHB is that it is a biological origin and it is degraded naturally and completely to carbon dioxide and water under natural environment by the enzymatic activities of microbes. In organisms, PHB’s are synthesized as a survival response in stressful environmental condition in the presence of excess carbon and limited supply of nitrogen and phosphorus PHBs are naturally produced by diverse groups of prokaryotic microorganisms, including bacteria. Endophytes are endosymbiotic microorganisms that colonize the plants [5]. Mangrove endophytes are abundant and known for their numerous activities with diverse biologically active natural products used in different categories of biotechnology. The endophytic microbes of the mangrove of Mandvi in Kachchh has not known so far since bioplastics are gaining commercial importance, the production of PHBs by the halophilic and halotolerant bacterial species are very much essential. The findings will emphasize and uncover the novel endophytic bacterial strains producing PHB which might have significant potential for industrial application.

Collection of Samples

The samples of Avicennia marina (Grey mangrove) were collected from Mandvi (22°4927’N, 69°21’49’E) in Kachchh, Gujarat during 2014.The plant samples were gathered from their natural habitat, placed into sterile polythene bags and brought back directly to the laboratory. The samples were processed within 24 h of collection.

Isolation of Endophytic Bacteria from A. Marina

Healthy samples of A. marina leaves, stem and pneumatophores were collected and washed thoroughly under running tap water to remove the surface adhering debris. These samples were cut into small pieces of about 0.5 cm². The protocol for sterilization of the plant pieces with 70% ethanol for 2 minutes were done and rinsed thoroughly with sterile distilled water and again with 3% sodium hypochlorite for 3 minutes. The sterile segments were rinsed again with sterile water to remove ethanol and sodium hypochlorite from the surface. The excess moisture was blotted in a sterile filter paper [6]. The efficacy of the sterilization procedure was ascertained with the method [7]. All the segments were plated on Nutrient agar (NA) medium in triplicate, sealed using parafilm™ and incubated at 37ºC for 24 to 72 hrs. The colonies representing different morphologies were selected at random and pure cultures were made by restreaking on the same medium. Pure cultures of the isolates were preserved at -80°C in 30% glycerol. Isolates were identified based on the Bergey's Manual of Determinative Bacteriology, followed by molecular characterization of the potent strain after screening for PHB [8].

Screening and Identification of Isolates for PHB Production

Each of the purified isolates were examined microscopically. A loopful of each of these endophytic bacterial strains were smeared on a clean glass slide, air dried and heat fixed and washed it with ethanol and petroleum ether (1:1) solution. The slides were flooded with 0.3% Sudan Black B for 15 to 20 minutes until the stain turns into greenish blue. Drained off the excess stain from the slide and rinsed it with water and air dried. Finally, counter stained the glass slide with (0.5% w/v) aqueous safranin for 10s. The slide was then rinsed with water and blot dried. The slides were examined under oil immersion microscope at 100X magnification to detect the presence of PHB granules. Positive isolates of PHBs have the appearance of blue violet colour and development of yellow-brown colour indicates negative [9]. Morphological and biochemical characterization was made on the basis of the morphological features of PHB producing strain following the standard protocol described in Bergey’s Manual of Systematic Bacteriology [10,11].

Estimation of Nitrogen

A fully grown colony of the chosen endophytic bacterial isolates having the ability to produce PHB were inoculated in both Nutrient agar broth (Hi-media) and Basal mineral broth medium and kept at 37°C in orbital shaker at 250 rpm for 48 h [12]. After incubation, the broth was centrifuged at 2500 rpm for 20 minutes to get the pellet and the supernatant were used for N₂ estimation. Observation of faint yellow colour indicates the minimum production and transition of deep yellow to brownish colour indicates the maximum production of ammonia [13,14].

Extraction of PHB by Sodium Hypochlorite Method

After the fermentation, the biomass of the bacterial cells was harvested by centrifugation at 3000 rpm (acc ≈ 1000g) for 20 minutes. The residual sludge pellet was washed twice with distilled water and freeze dried at -4°C. The biomass powder was treated with 6% sodium hypochlorite and chloroform. The mixture was agitated in a shaker at 3000 rpm at 37 ? C for 3 hours. Separation of PHB-enriched solvent from centrifuge tube was achieved by using syringe. Then, the PHB material was precipitated by mixing methanol with the concentrated chloroform (9:1). Finally, the PHB-precipitate was filtered by simple filtration and then dried by evaporation at 60 ? C [15].

Quantification of PHB

The residual biomass was estimated as the difference between dry cell weight and dry weight of extracted PHB [16]. This was calculated to determine the cellular weight and accumulation other than PHB. The percentage of intracellular PHB accumulation was estimated as the percentage composition of PHB present in the dry biomass.

PHB Accumulation (%) = Dry weight of extracted PHB (mg/L) / (Dry cell weight (mg/L) × 100%.

Molecular Sequencing

The positive strains that showed maximum PHB production were considered for molecular characterization. The rRNA extraction, amplification and sequencing were done by using partial 16S rRNA sequencing from the isolated strain was accomplished by Gujarat State Biotechnology Mission (GSBTM), Gujarat, India. The acquired sequences were aligned against GenBank data base using Basic Local Alignment Search Tool (BLAST) (www.ncbi.nlm.nih.gov/BLAST) from the National Center for Biotechnology Information (NCBI) to identify sequences with high similarity [17]. The nucleotide sequences were submitted to the GenBank database and acquired the accession numbers.

Characterization of PHB

The extracted PHB were identified and characterized by employing the following analytical methods,

Uv-Vis Spectrophotometry

The extracted PHB were dissolved in chloroform and scanned in the range of 200–320 nm against chloroform blank and the spectrum was analyzed [18].

Ft-Ir Analysis

The extracted PHB were analyzed by FT-IR spectroscopy (JASCO FT/IR). The samples were mixed with 2% KBr in a sufficient ratio, and then it was ground. Then the mixtures were compressed to get translucent sample discs to form pellet and fixed followed by scanning from 400 to 4000 cm-¹ to confirm the functional groups of the extracted polymer [19].

Gas Chromatography Mass Spectroscopy (Gc-Ms)

The extracted PHB were subjected to gas chromatography mass spectrometry (GC-MS) instrument (Shimadzu, GCMS QP 2010) with column (HP5MS), 30 m x 0.25 mm. with DB-5, 0.25 µm film thickness; column oven temperature of 60°C at the rate of 32.7mL/min., injection port temperature 250°C, constant pressure of carrier gas (helium) 72.8kPa, flow rate1.20mL/min, acquisition parameters full scan, scan range30 to 500 amu. The sample were dissolved in chloroform, evaporated and dried and the extracts were dissolved in n-hexane and 1µl of this sample was dispensed into the GC-MS vial for analysis [20].

Preparation of Bio-Polymer Film

Totally 250mg of PHB solution was made by casting the polymer solution into glass petridishes. The solutions were then left to air dry at room temperature for one week followed by freeze drying [21].

Statistical Analysis

The experiments were conducted in completely randomized block design with three replications to evaluate the polyhydroxybutyrate production by the mangrove endophytic bacteria. Analysis of variance (ANOVA) was performed using Statistical Package for Social Sciences (SPSS) version 13.0. The data were presented as the mean ± SE for each treatment. Means were compared using the least significant difference (LSD) test at the 5% probability level.

This study demonstrated the occurrence and diversity of endophytic bacteria from A. marina. In Gujarat, only countable number of the reports showed the diversity of endophytic bacteria. There is no report on endophytic bacteria from the mangrove of Kachchh, Gujarat. This work may be the first report on endophytic bacterial isolates from A. marina of Kachchh. During the preliminary screening, only 28 endophytic bacterial isolates were considered for PHB production, of which ten showed positive results based on visualization by staining. Among these, the potent strain showing positive towards PHB is represented in Fig1. PHB is reported to be synthesized by more than 300 bacterial strains [22]. The lipophilic dye Sudan black B were used to stain and distinguish PHB accumulating and non-accumulating strains. The isolates appear as black blue dark spot under microscope indicative of intracellular lipid granules and the capacity to produce biopolymer [23].

Fig 1: Endophytic Bacterial Strain JJ07 Isolated from Mangrove Showing the Presence of PHB.

Accordingly, 10 endophytic bacterial isolates showed positive for PHB and designated as JA, JB, JG, JC, JK, JH, JJ, JI, JD and JE. Out of these, 70% were Gram-negative and 30% were Gram-positive bacteria. Most of the bacteria were fast growers and about 50% were motile and the remaining were non-motile. A total of three endophytic bacteria such as JB, JC and JE were gram-positive whereas JA, JG, JK, JH, JJ, JI and JD were confirmed to be Gram negative (Data not shown). A few isolates showed positive results for motility, catalase and oxidase enzyme [24]. The result on morphological characterization of isolated endophytic bacteria exhibited varied shapes, margins, texture and pigmentation and different biochemical characteristics like catalase, oxidase, citrate, methyl red, Voges-Proskauer, nitrate reduction and H₂S production (Data not shown). These results are in agreement with the findings and the isolates were determined on the basis of morphological and biochemical characteristics [25].

The isolated potent JJ07 strain was submitted to Gujarat State Biotechnology Mission (GSBTM), Gandhinagar for DNA sequencing. The acquired sequences were subjected to BLAST using NCBI BLAST tools. Based on the 16s RNA gene sequencing it was confirmed that the PHB positive isolate JJ07 was Pseudomonas nitritireducens having 1221 bp and deposited the sequence in the National Center for Biotechnology Information (NCBI) and acquired the accession number as MF351832. The PHB productions were extensively studied in the genus like Pseudomonas spp [26,27].

All the 10 strains which showed positive results for biopolymers were ammonia producers. In the metabolic pathway to transform a carbon source into PHB, the limitation of nitrogen triggers the production of PHB, due to a physiologically induced stress in the bacterial metabolism [28]. Nutrient broth medium has more capacity to produce PHB than the mineral salt medium. As it enhances the multiplicity of polyester synthesizers (bacteria) during their growth and polymer accumulation processes [29]. The solvent extraction by using sodium hypochlorite is widely used to recover PHB with high purity as this method facilitates the elimination of Non-PHB Cellular Material (NPCM) during the lysis of cells [30]. The process for PHB recovery using a dispersion of sodium hypochlorite and chloroform has been adopted in the present study [15]. The protocols followed were most efficient in retaining the maximum percentage of intracellular PHB accumulation with respect to dry cell weight. Further, the extracted PHB was an ivory white colored powder and sparingly soluble in water.

The extracted PHB was quantified and compared with the cell biomass of the isolated organisms. It was found that PHB accumulation was in proportion to the dry cell weight which was similar to the reports [16,28]. It was also concluded that sodium hypochlorite method is the most efficient method to produce bio- polymer and it is represented as percentage of intracellular PHB of the dry cell weight. Residual biomass was estimated as difference between the dry cell weight and dry extract of PHB. The PHB produced by the 10 selected strains JA01, JB02, JG03, JC04, JK05, JH06, JJ07, JI08, JD09 and JE10. Strain JJ07 produced maximum yield of PHB, 72.37% in Nutrient Broth (NB) and 48.29% in Mineral Salt Broth (MSB) medium. These results are agreement with the findings from other studies which show the difference in the yield of PHB by various strains [31].

Fig 2:  UV-Vis Spectrum of PHB Isolated from Pseudomonas nitroreducens JJ07.

Fig 3: FT-IR Spectrum of PHB Isolated from Pseudomonas nitroreducens JJ07.

Fig 4: GC-MS of PHB Isolated from Strain Pseudomonas nitroreducens JJ07.

The UV–Vis spectrophotometer scanning revealed that the absorbance of peaks were maximum at 235 nm is represented in Fig 3. In the present study, it was observed that FTIR spectra of PHBs pelletized by KBr showed variations in band patterns. All the functional groups of the polymer are described in Fig 4. The transmittance bands located at 1701 cm–1 are attributed to the stretching vibration of the C=O group (ester carbonyl) in the PHB polyester. The stretch of bands ranging from 1160 showed C-O bonding. Accompanying bands of the C-O-C groups appeared in the spectral region from 1165 to 1238 cm–1. Transmittance region from 2706–3080 cm–1 corresponds to stretching vibration of C-H bonds of methyl (CH3), and methylene (CH₂) groups. Other characteristic bands present for scl PHB were 2970, 2886, 1238 (CH₃ bend), 1165 and 821. The band Scl-co-mcl PHB had the strongest methylene -C-H- vibrations near 2970 cm–¹. The results were also correlated with the report confirming that the isolated compound was PHB and also the FT-IR spectrum obtained was in accordance with data reported in the literature [32-34]. The relative abundance of an ion is plotted against the m/z value (Fig. 5). GC-MS analysis also elucidated the characteristic fragmentation patterns, suggesting the presence of PHB. The carbonyl and hydroxyl end of the corresponding hydroxybutyrate were identified from the specific peaks in the mass spectra. The peak at m/z 100 in the mass spectrum of methyl 3- hydroxybutyrate represented its hydroxyl end. The fragmentation patterns were in concordance with the results [35].

The peak at m/z 85 in the mass spectrum represented butanoic acid. Accordingly, the peak at m/z 71 in the mass spectrum was similar to the finding which is represented as CH₃-CH₌CH-C₌O [M^•+ -H₂O-OH] and the elemental analysis also agrees well with the molecular formula for PHB. The bioplastic film was fabricated into transparent and composite films by the solvent casting method within 7 days exhibited high flexibility and elastomeric material in nature [36,37].

The present study revealed the production of environmentally and economically important PHB by the endophytic bacterial strain Pseudomonas nitritireducens isolated from the mangroves Avicennia marina, from Kachchh region. To the best of our knowledge, this is the first attempt to isolate the endophyte from A. marina which produces PHB. For large scale production of PHB, studies are needed for optimization of the medium, recombinant microbial strains, more efficient fermentation process and efficient extraction. PHB productions from microbial strains of mangrove environment are due to the fact that, marine microorganisms are more evolved by de novo to overcome various stress conditions. Hence continuous exploration of untapped marine microbes may reveal many new molecular entities with better biodegrading potentials. This sort of research will confront the emergence of eco-friendly environment in the near future.

Data Availability Statement

The authors confirm that the data supporting the finding of this study are available within the article. Raw data that support the findings of this study are available from the corresponding author upon reasonable request.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement             

All the authors thank Gujarat Institute of Desert Ecology, Bhuj for providing the facilities to conduct the experiments.

Funding Declaration

The first author thank University Grants Commission, New Delhi, and Government of India for awarding Post Doctoral Women fellowship to conduct the study.

1. Webb HK, Arnott J, Crawford RJ, Ivanova EP. Plastic Degradation and its environmental implications with special reference to poly (ethylene terephthalate). Polymers. 2013; 5: 1-18.
2. Kalaivani R, Sukumaran V. Isolation and identification of new strains to enhance the production of biopolymers from marine sample in Karankura, Tamil Nadu. European Journal of Experimental Biology. 2013; 3.
3. Gao X, Chen JC, Wu Q, Chen GQ. Polyhydroxyalkanoates as a source of chemicals, polymers, and biofuels. Curr Opin Biotechnol. 2011; 22: 768-774.
4. Hazer B, Steinbüchel A. Increased diversification of polyhydroxyalkanoates by modification reactions for industrial and medical applications. Appl Microbiol Biotechnol. 2007; 74: 1-12.
5. Kourmentza C, Placido J, Venetsaneas N, Burniol-Figols A, Varrone C, Gavala HN, et al. Recent advances and challenges towards sustainable polyhydroxyalkanoate (PHA) production. Bioengineering (Basel, Switzerland). 2017; 4: 55.
6. Gayathri S, Saravanan D, Radhakrishnan M, Balagurunathan R, Kathiresan K. Bioprospecting potential of fast growing endophytic bacteria from leaves of mangrove and salt-marsh plant species. Indian Journal of Biotechnology. 2010; 9: 397-402.
7. Schulz B, Wanke U, Draeger S, Aust HJ. Endophytes from herbaceous plants and shrubs: effectiveness of surface sterilization methods. Mycological Research. 1993; 97: 1447-1450.
8. Holt JG, Krieg NR, Sneath PHA, Stanley JT, William ST. Bergey’s Manual of determinative bacteriology. williams and wilikins, Baltimore. 1994; 786-788.
9. Santhanam A, Sasidharan S. Microbial production of polyhydroxyalkanotes (PHA) from alcaligens spp and pseudomonas oleovorans using different carbon sources. 2010; 9: 3144-3150.
10. Krieg NR, Holt JG. Bergey’s manual of systematic bacteriology. Williams and Wilkins, Baltimore, London. 1984.
11. Ngoma L, Mogatlanyane K, Babalola OO. Screening of Endophytic Bacteria towards the development of cottage industry: An in Vitro Study. J Hum Ecol. 2014; 47: 45-63.
12. Manangan T, Shawaphun S. Quantitative extraction and determination of polyhydroxyalkanoate accumulated in Alcaligenes latus dry cells. Science Asia. 2010; 36: 199-203.
13. Cappuccino JG, Sherman N. Microbiology: a laboratory manual.3rd ed. Benjamin-Cummings Pub Co (B.c.P); New York, NY, USA. 1992; 125-179.
14. Khanna S, Srivastava AK. Recent advances in microbial polyhydroxyalkanoates. Process Biochemistry. 2005; 40: 607-619.
15. Hahn SK, Chang YK, Kim BS, Chang HN. Optimization of microbial poly (3-hydroxybutyrate) recover using dispersions of sodium hypochlorite solution and chloroform. Biotechnology and bioengineering. 1994; 44: 256-261.
16. Zakaria MR, Ariffin H, Johar NAM, Aziz SA, Nishida H, Shirai Y, et al. Biosynthesis and characterization of poly (3-hydroxybutyrate-co-3-hydroxybutyrate) copolymer from wild type Comamonas sp. EB172. Polym. Degrad. Stab. 2010; 95: 1382-1386.
17. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990; 215: 403-410.
18. Selvakumar K, Srinivasan G, Baskar V, Madhan R. Production and isolation of polyhydroxyalkanoates from haloarcula marismortui MTCC 1596 using cost effective osmotic lysis methodology. European Journal of Experimental Biology. 2011; 1: 180-187.
19. Kansiz M, Billman-Jacobe H, McNaughton D. Quantitative determination of the biodegradable polymer Poly (beta-hydroxybutyrate) in a recombinant escherichia coli strain by use of mid-infrared spectroscopy and multivariative statistics. Appl Environ Microbiol. 2000; 66: 3415-3420.
20. Angelova N, Hunkeler D. Rationalizing the design of polymeric biomaterials. Trends Biotechnol. 1999; 17: 409-421.
21. Gamal RF, Abdelhady HM, Khodair TA, El-Tayeb TS, Hassan EA, Aboutaleb KA. Semi-scale production of PHAs from waste frying oil by pseudomonas fluorescens Braz J Microbiol. 2013; 44: 539-549.
22. Grigore ME, Grigorescu RM, Iancu L, Ion RM, Zaharia C, Andrei ER. Methods of synthesis, properties and biomedical applications of polyhydroxyalkanoates: a review. Journal of biomaterials science. Polymer edition. 2019; 30: 695-712.
23. Chen BY, Shiau TJ, Wei YH, Chen WM, Yu BH, Yen CY, et al. Feasibility study of polyhydroxyalkanote production for materials recycling using naturally occurring pollutant degraders. J Taiwan Inst Chem Eng. 2012; 43: 455-458.
24. Anjum N, Chandra R. Endophytic Bacteria: Optimizaton of Isolation Procedure from various medicinal Plants and their preliminary characterization. Asian Journal of Pharmaceutical and clinical Research. 2015; 8: 233-238.
25. Danial AW, Hamdy SM, Alrumman SA, Gad El-Rab SMF, Shoreit AAM, Hesham AE. Bioplastic Production by Bacillus wiedmannii AS-02 OK576278 using different agricultural wastes. Microorganisms. 2021; 9: 2395.
26. Brandl H, Gross RA, Lenz RW, Fuller RC. Pseudomonas oleovorans as a Source of Poly (beta-Hydroxyalkanoates) for Potential Applications as Biodegradable Polyesters. Appl Environ Microbiol. 1988; 54: 1977-1982.
27. Sujatha K, Mahalakshmi A, Shenbagarathai R. A study on accumulation of PHB in native pseudomonas isolates LDC-5 and LDC-25. Indian Journal of Biotechnology. 2005; 4: 216-221.
28. Du G, Chen J, Yu J, Lun S. Continuous production of poly-3-hydroxybutyrate by ralstonia eutropha in a two-stage culture system. J Biotechnol. 2001; 88: 59-65.
29. Braunegg G, Lefebvre G, Genser KF. Polyhydroxyalkanoates, biopolyesters from renewable resources: physiological and engineering aspects. J Biotechnol. 1998; 65: 127-161.
30. Jacquel N, Lo CW, Wei YH, Wu HS, Wang SS. Isolation and purification of bacterial poly (3-hydroxyalkanoates). Biochemical Engineering Journal. 2008; 39: 15-27.
31. Alshehrei F. Production of polyhydroxybutyrate (PHB) by bacteria isolated from soil of saudi arabia, J Pure Appl Microbiol. 2019; 13: 897-904.
32. Arun A, Arthi R, Shanmugabalaji V, Eyini M. Microbial production of poly-beta-hydroxybutyrate by marine microbes isolated from various marine environments. Bioresource technology. 2009; 100: 2320-2323.
33. Shamala TR, Divyashree MS, Davis R, Kumari KS, Vijayendra SV, Raj B. Production and characterization of bacterial polyhydroxyalkanoate copolymers and evaluation of their blends by fourier transform infrared spectroscopy and scanning electron microscopy. Indian J Microbiol. 2009; 49: 251-258.
34. Tanikkul P, Sullivan GL, Sarp S, Pisutpaisal N. Biosynthesis of medium chain length polyhydroxyalkanoates (mcl-PHAs) from palm oil. Case Stud Chem Environ Eng. 2020; 2: 100045.
35. Venkata MS, Reddy MV, Subhash GV, Sarma PN. Fermentative effluents from hydrogen producing bioreactor as substrate for poly (beta-OH) butyrate production with simultaneous treatment: an integrated approach. Bioresour Technol. 2010; 101: 9382-9386.
36. Hamdy SM, Danial AW, Gad El-Rab SMF, Shoreit AAM, Hesham AE. Production and optimization of bioplastic (Polyhydroxybutyrate) from Bacillus cereus strain SH-02 using response surface methodology. BMC microbiology. 2022; 22: 183.
37. Anbukarasu P, Sauvageau D, Elias A. Tuning the properties of polyhydroxybutyrate films using acetic acid via solvent casting. Sci Rep. 2016; 5: 17884.