1. INTRODUCTION:

Polyhydroxyalkanoates (PHA), is a family of biopolyesters with diverse structural units (Fig-1). PHAs, synthesized by many Gram positive and Gram-negative bacteria as storage compounds, are deposited as insoluble inclusions in the cytoplasm¹. Nearly 150 different hydroxyalkanoates are identified, as constituents of bacterial storage polyesters ^[2]. Polyhydroxyalkanoate (PHAs) aliphatic polymer are being used as biodegradable plastic due to their potentially hydrolysable ester bonds ³.

Fig-1: General chemical structure of Polyhydroxyalkalonates (PHAs), where R represents an alkyl group, value of x varies from 1 – 4 and n can be 1000 to 10,000. In case of PHB x =1 and R is CH_3.

The homopolymer Poly 3-hydroxy butyrate (PHB) (Fig-1) is the first member of PHAs family to be discovered in microbes and most common member of PHA ^[4]. PHB have a high degree of polymerization; are highly crystalline, optically active, isotactic, piezoelectric and insoluble in water with other features which make them highly competitive with polypropylene and other synthetic plastics (Table-1).

Table-1. Physical properties of poly (3-hydroxybutyrate) P (3HB) compared to polypropylene ⁵.

Properties	P (3HB)	PP
Crystalline melting point, °C	175	176
Crystallinity, %	80	70
Molecular weight, Daltons	5 x 10⁵	2 x 10⁵
Glass transition temperature, °C	15	- 10
Density, g/cm³	1.25	0.905
Young’s modulus, GPa	3.5	1.7
Tensile strength, MPa	40	38
Extension to break, %	6	400
UV resistance	Good	Poor

PHAs, along with their mechanical properties, owing to their other imperious properties such as, biocompatibility, high immunotolerance, biodegradability and being less toxic, they are favorably being used as in various biomedical applications (Table-2).

Table-2. Medical Applications of Polyhydroxybutyrate (PHB) and its copolymers

Medical Applications of Polyhydroxybutyrate (PHB) and its copolymers

References (Miscellaneous)

· Sutures, repair patches, slings, Nerve repair devices

· Cardiovascular patches, cardiovascular devices

· Orthopedic pins, Adhesion barriers, stents

· Guided tissue repair/regeneration devices

· Articular cartilage repair devices,

· Nerve guides, tendon repair devices,

· Bone-marrow scaffolds,

· Tissue engineering and wound dressings

· Drug Delivery etc.

[6, 7, 8, 9, 10, 11, 12, 13, 14]

The medical industry has mostly been utilizing PHAs obtained by chemical synthesis. Production cost, commercially available PHA is relatively higher than that of traditional synthetic plastics. Therefore, bacterial strains which are able to produce large quantities of intracellular PHA using low-cost substrates are always in demand.

In fact, to commercialize naturally produced PHAs, widespread effort has been dedicated to reducing the production cost by the development of bacterial strains that utilize waste bye product and more competent fermentation/recovery processes because the price of the substrate has the largest influence on the production cost of PHA^15,16,17.

In the present study, soil and sewage samples were collected from different region of Meerut, Uttar Pradesh, India. Several PHB accumulating bacteria were isolated from these soil and sewage samples. The most promising bacteria among those were further characterized using morphological, biochemical techniques. The isolated bacterium is predicted belonging to the genus Bacillus and tested for the potential of production and accumulation of PHB using different waste bye products.

2. MATERIAL AND METHODS:

Investigations were carried out at the Department of Botany J.V. College Baraut Baghpat on the isolation of poly -3-hydroxy butyrate (PHB) producing bacteria from different sources and their screening for maximum PHB production. The efficient strains were selected, culture parameters were optimized. The general procedure and techniques adopted are as follows:

2.1 Collection of samples and Isolation of Bacteria:

Soil and sewage samples were collected from various places of Meerut. The collected samples were serially diluted and plated on nutrient agar medium ^[18]. The characteristic bacterial colonies were, chosen, picked up, purified and preserved on nutrient agar slants till further use.

2.2 Rapid screening of bacterial isolates positive for PHB production:

Viable colony method of screening using Sudan Black-B dye was used for testing the PHB production ability of all the isolates^[19]. For rapid screening of PHB producers, nutrient agar medium supplemented with 1 per cent glucose was sterilized by autoclaving at 121°C for 20 minutes and cooled to 45°C. The medium was poured into sterile petri plates and allowed to solidify. The petri plate was divided in four equal parts and a bacterial isolate was spotted in each part then the plates were incubated at 30°C for 24 hours. Ethanolic solution of (0.02%) Sudan Black B was spread over the colonies. The plates kept undisturbed for 30 minutes. Thereafter washed with ethanol (96%), the excess stain was removed from the colonies. The colonies with dark blue color were considered as positive for PHB production. All the positive isolates were given some code numbers based on their source of isolation.

2.3 Quantification of PHB production and selection of isolates:

All the Sudan Black-B positive isolates were subjected to quantification of PHB production ^[20]. The bacterial cells containing the polymer were centrifuged at 10,000 rpm for 10 min. and the pellet washed with acetone and ethanol to remove the unwanted materials. The pellet was re-suspended in equal volume of 4 per cent sodium hypochlorite and incubated at room temperature for 30 min. Then the mixture was again centrifuged. Supernatant was discarded while the cell pellet containing PHB was washed with acetone and ethanol again. Lastly, hot chloroform was used to dissolve the polymer (PHB) granules.

The chloroform was filtered and to the filtrate, concentrated 10 ml hot H₂SO₄ was added. The addition of sulfuric acid converts the polymer into crotonic acid which is brown colored. After cooling down the solution, the absorbance was taken at 235nm using sulfuric acid as blank. PHB concentration is read against the standard curve of PHB prepared via pure PHB (Sigma, USA). PHB concentration is expressed in terms of g/l of culture.

2.4 Batch Fermentation and culture conditions:

Studies were carried out in batch fermentations in 250 ml Erlenmeyer flasks containing 100 ml of different wastes to be used as carbon source viz., soya extract, corn extract, molasses, milk whey, and distillery waste liquor was taken without any pretreatment after simple filtration. The medium was sterilized with autoclave medium (i.e., organic source). The flasks were inoculated with selected bacterial isolate and maintained at 30ºC and 120rpm for 48 h.

2.5 Optimization of different parameters for maximum PHB Production:

In the course of study, various parameters have also been optimized with the selected isolate to get the maximized PHB production. Different set of experiments were performed adding different nitrogen source viz., nitrogen source Peptone, Beef extract, Yeast extract, ammonium sulphate and ammonium chloride (@ conc. of 1%) to the medium, similarly impact of different pH (5.0 to 9.0), trace elements viz., ZnSO₄, CuSO₄, FeCl₃, CaCl₂ and MgSO₄, and temperature (28 – 45) was studied.

2.6 Characterization of most efficient PHB producing bacterial isolate:

The selected, most efficient PHB producing bacterial isolate was subjected to a set of morphological, physiological and biochemical tests for the purpose of identification. Biochemical tests i.e., citrate utilization, catalase activity, motility, indole production, methyl red, utilization of different sugars (glucose, fructose, sucrose xylose, sorbitol etc.), Vogues Proskauer etc. were carried out with 24 hrs old cultures²¹. The data thus obtained was compared Bergey’s Manual of Determinative Bacteriology to come to some conclusion^22,23.

3. RESULTS:

From the total of 8 samples (6 soil samples, 2 sewage water samples) collected from different regions of Meerut, India, a total of 28 representative bacteria were isolated, purified and maintained as pure cultures.

Out of total 28 isolates 10 isolates were found to accumulate PHB. Out of 10 isolates, 8 were from the soils samples and 2 were from the waste sewage water collected from different regions of Meerut accumulated PHB (Table-3).

Table-3: Description of sample collected and the isolates with their label

S. No.	Sample collected	No. of Representative Isolates with the given Code number	Sudan Black B positive isolates
1.	6 Soil samples	17 (SM-1, to SM-17)	SM-1, SM-4, SM-5, SM-7, SM-8, SM-11, SM-12, and SM-13
2.	2 Sewage water	11 (WM-1, to WM-11)	WM-1, and WM-3

Out of Sudan Black-B positive isolates, 5 isolates were strongly stained i.e., SM-5, SM-8, SM-11, SM-12, WM-3 and hence tested for PHB yield. SM-11 was found the highest PHB producing isolate among them (Fig-2). Hence SM-11 isolate was selected for further study.

Fig-2: Preliminary comparison of PHB production among different isolates

3.1 PHB yield with different organic waste by SM-11:

Among the different organic sources tested to evaluate their effects on PHB yield, milk whey was found to be the best organic source. It yielded a mean PHB of 1.45 g/l. This was followed by distillery waste with a mean PHB of 1.25g/l and the lowest yield is by molasses 1.1 g/l.

3.2 Effect of different nitrogen sources on PHB yield:

As a result of using different nitrogen sources with milk whey as an organic source on PHB yield by the selected isolate SM-11, yeast extract resulted to be the best source. It yielded a mean of 2.375g/l, followed by peptone with the mean PHB yield of 2.3g/l. next was from ammonium chloride with a mean PHB yield of 2.125g/l. Then was ammonium nitrate with a yield of 2.05g/l and the lowest PHB yield was by beef extract with a mean PHB yield of 1.7 g/l.

3.3 Effect of trace elements:

In the presence of CaCl₂, the isolate produced 2.5g/l PHB, while in presence of CuSO_4,MgSO_4,ZnSO₄, and FeCl₃ the isolate resulted in the production of 1.3g/l, 1.75g/l, 1.45g/l and 1.2g/l PHB respectively.

3.4 Effect of pH on PHB yield by SM-11:

Keeping other conditions and media, as optimized earlier, the effect of pH was studied from 5 to 9. It was found that at the pH 6, the bacterial culture resulted in maximum PHB production (Fig-3).

Fig-3: With optimized composition of media, comparison of PHB production by the isolate SM-11 at different pH

3.5 Effect of incubation temperature on PHB yield by SM-11:

With all other conditions and media composition as optimized earlier, the effect of temperature was studied keeping incubation temperature 28, 30, 35, 37, 40, and 45ºC. Incubation temperature of 37°C was found to be best for PHB production, as it resulted in maximum yield (Fig-4).

Fig-4: Effect of temperature on PHB production by the isolate SM-11, with other parameters optimized

Though the highest PHB production was shown to be at 45ºC, but as we can see there is not that much significant difference between 35ºC, 37ºC and 45ºC. While maintaining that much high incubation temperature will be a matter of significant cost, hence the PHB production and incubation temperature can be decided accordingly.

3.6 Characterization and Identification of isolate SM-11

The morphological and biochemical characteristics of SM-11 are summarized in the Table-4. As per the results morphological and biochemical test of isolate SM-11 obtained, shown in the table-3, according to Bergey’s Manual of Systematic Bacteriology ^[23], preliminary identification indicates that SM-11 isolate was most probably a Bacillus sp.

Table-4: Morphological and biochemical characteristics of SM-11

Test	Result/Reaction
Shape, Gram Staining	Rod Shaped, Gram Positive
Spore Staining	Spore Forming
Carbohydrate Fermentation
Lactose	+
Fructose	+
Maltose	-
Sucrose	+
Mannitol	-
Biochemical Tests
Casein Hydrolysis	+
Citrate Utilization	+
Urease	+
Catalase	+
Amylase	+
Voges Proskauer	-ve

4. DISCUSSION:

Plastic is undoubtedly causing a serious environment pollution and termed as cancer of nature ^[24]. Synthetic plastic being almost non-degradable. Public has been made aware about the ill effects of plastics on health and environment ^{25, 26}.

However, in modern days, plastic has become an indispensable part of life, because of its various application in different sectors of our life. Therefore, to minimize the harmful effects on environment, biodegradable polymers are produced, which are analogous in properties with synthetic plastic, and hence termed as biodegradable plastic or bioplastic ^{27, 28}. Biodegradable plastic waste can be degraded in newly designed biodigesters resulting in production of gas (60% to 65% methane), ammonia and manure ²⁹.

Poly (ß-hydroxybutyrate) (PHB) is a biodegradable plastic that has numerous applications in medical sector and belongs to the family of polyhydroxyalkanoates (PHA).

Present work was aimed to isolate and identify PHB producing bacteria by utilization of inexpensive alternate carbon sources preferably the waste byproducts of other industry. The isolate SM-11 from the study found to be promising in that way. Using milk whey as an alternate carbon source and other optimized conditions the isolate was able to produce as much as 3.53g/l of PHB, which is quite improved in comparison of studies in the past (Table-5).

Hence, the bacterial isolate resulted from the present study is expected to be promising. Though further molecular studies are required to identify the species and strain of isolate. Further studies at bioreactor level and optimization of parameters are endorsed to analyze the competence of the isolate at commercial level.

5. ACKNOWLEDGEMENT:

The authors are thankful to the Principal, Janta Vedic College, Baraut, Dr. Manav Kumar Choudhary, Department of Chemistry, JV College, Baraut for providing facilities and being helpful to carry out this work.

6. CONFLICT OF INTEREST:

The authors declare no conflict of interest, financial or otherwise

7. REFERENCES:

1. Steinbuchel A. Polyhydroxyalkanoic acids. In Biomaterials: Novel Materials from Biological Sources, Edited by Byrom D. Macmillan Publishers, Basingstoke, United Kingdom. 1991; pp.123-213

2. Steinbuchel A, Valentin HE. Diversity of bacterial polyhydroxyalkanoic acids. FEMS Microbiology Letters 1995; 128: 219–228.

3. Singh P, Parmar N. Isolation and characterization of two novel polyhydroxybutyrate (PHB)-producing bacteria. African Journal of Biotechnology. 2011; 10 (24): 4907-4919.

4. Jackson DE, Srienc F. Novel methods to synthesize Polyhydroxyalkanoates. Biochemical Engineering Annals of the New York Academy of Sciences. 1994; 8 (745):134-148.

5. Khanna S, Srivastava AK. Recent advances in microbial polyhydroxyalkanoates. Process Biochemistry 2005; 40: 607–619.

6. Hasirci V, Kok FN. (2003). Polyhydroxybutyrate and Its Copolymers: Applications in the Medical Field. In Tissue Engineering and Novel Delivery Systems. Edited by Yaszemski MJ, Trantolo DJ, Lewandrowski K-U, Hasirci V, Altobelli DE and Wise DL Taylor & Francis, 2004; 1-19.

7. Brigham CJ, Sinskey AJ. Applications of Polyhydroxyalkanoates in the Medical Industry. International Journal of Biotechnology for Wellness Industries. 2012; 1: 53-60

8. Shrivastav A, Kim HY, Kim YR. Advances in the Applications of Polyhydroxyalkanoate Nanoparticles for Novel Drug Delivery System. BioMed Research International. 2013; Article ID 581684, A review. Available from: URL: http://dx.doi.org/10.1155/2013/581684

9. Luef KP, Stelzer F, Wiesbrock F. Poly(hydroxy alkanoate)s in Medical Applications. Chemical and Biochemical Engineering Quarterly 2012; 29(2): 287–297.

10. Ali I, Jamil N. Polyhydroxyalkanoates: Current applications in the medical field. Frontiers in Biology. 2016; 11: 19–27.

11. Balgun-Agbaje OA, Odeniyi OA, Odeniyi MA. Applications of Polyhydroxyalkanoates in Drug Delivery. Fabad Journal of Pharmaceutical Sciences. 2019; 44(2):147-158.

12. Bonartsev AP, Bonartseva GA, Reshetov IV, Kirpichnikov MP, Shaitan KV. Application of Polyhydroxyalkanoates in Medicine and the Biological Activity of Natural Poly(3-Hydroxybutyrate). Acta Naturae. 2019; 11(2):4-16.

13. Elmowafy E, Abdal-Hay A, Skouras A, Tiboni M, Casettari L, Guarino V. Polyhydroxyalkanoate (PHA): applications in drug delivery and tissue engineering, Expert Review of Medical Devices, 2019; 16 (6): 467-482.

14. Babos G, Rydz J, Kawalec M, Klim M, Fodor-Kardos A, Trif L, Feczko T. Poly(3-Hydroxybutyrate)-Based Nanoparticles for Sorafenib and Doxorubicin Anticancer Drug Delivery. International Journal of Molecular Science. 2020; 21(19): 7312

15. Pozo C, Toledo, MVM, Rodelas B, Lopez JG. Effects of culture conditions on the production of polyhydroxyalkanoates by Azotobacter chroococcum H23 in media containing a high concentration of alpechin (wastewater from olive oil mills) as primary carbon source. Journal of Biotechnology. 2002; 97:125-131.

16. Van-Thuoc D, Quillaguaman J, Mamo G, Mattiasson B (2008). Utilization of agricultural residues for poly(3-hydroxybutyrate) production by Halomonas boliviensis LC1. Journal of Applied Microbiology. 2008; 104(2): 420–428

17. Troschl C, Meixner K, Drosg B. Cyanobacterial PHA Production—Review of Recent Advances and a Summary of Three Years’ Working Experience Running a Pilot Plant. Bioengineering. 2017; 4 (2): 26.

18. Anon. Mannual of microbiological methods. Mc-Graw Hill Book Co., New York. 1957 (p157)

19. Juan ML, Gonzalez LW, Walker GC. A Novel Screening Method for Isolating Exopolysaccharide deficient Mutants. Applied and Environmental Microbiology. 1998; 64: 4600-4602.

20. Slepecky RA, Law JH. Assay of poly-βhydroxybutyric acid. Journal of Bacteriology. 1961; 82(1): 37-42.

21. Cappuccino TG, Sherman N. Microbiology, a laboratory manual. The Benjamin/cummings publishing, Company Inc., California. 1992

22. Krieg NR, Holt JG. Bergey’s Manual of Systematic Bacteriology, Williams and Wilkins, Baltimore, MD 1984.

23. Holt JG, Krieg NR, Sneath PHA, Stanley JT, Williams ST. Bergey’s manual of determinative bacteriology, 9th edn. Lippincot, Williams and Wilkins, Baltimore 2000.

24. Purushothama KV. Plastic a cancer in Nature: Trends, Problems and Policies in India. Research J. Humanities and Social Sciences 2015; 6(3): 209-212. doi: 10.5958/2321-5828.2015.00026.1

25. Antony A, George A, Jose J, Paul L, Babu MP, Sikha P S, Paul L, Sr. Naveena CMC, Joseph A. A Study to evaluate the effectiveness of Planned Teaching Programme on knowledge regarding the Ill effects of plastics on health and environment among housewives in selected community at Aluva. Int. J. Nur. Edu. and Research. 2018; 6(4):335-337. doi: 10.5958/2454-2660.2018.00081.9

26. Rani U. A Study to Assess the Effectiveness of Structured Programme on Knowledge regarding Hazards of Plastic usage among Housewives in selected Community Area, Bangalore. Int. J. of Advances in Nur. Management. 2019; 7(3):255-257. doi: 10.5958/2454-2652.2019.00058.1

27. Thiruchelvi. R, Kavitha K, Shankari K. New Biotechnological Routes for Greener Bio-plastics from Seaweeds. Research J. Pharm. and Tech 2020; 13(5):2488-2492. DOI: 10.5958/0974-360X.2020.00444.8

28. Saini RD. New Age Biodegradable and Compostable Plastics. Asian J. Research Chem. 2018; 11(1):179-188. doi: 10.5958/0974-4150.2018.00037.8

29. Thangavel S, Sujin David Rajan S., Kevin B.C. A Novel Approach to treat Bio Wastes Using Biodigesters and Microorganisms Employed to Increase Plastic Degradation. Research J. Engineering and Tech. Oct.-Dec., 2013; 4(4):221-225.

30. Yilmaz M, Beyatli Y. Poly-β-hydroxybutyrate (PHB) production by a Bacillus cereus MS strain in sugarbeet molasses. Sugar industry. 2005; 130(2):109-112.

31. Ataei SA, Vasheghani‐Farahani E, Shojaosadati SA, Tehrani HA. Isolation of PHA–Producing Bacteria from Date Syrup Waste. Macromolecular Symposia. 2008; 269 (1): 11-16.

32. Reddy AR, Kumar RB, Prabhakar KV. Isolation and Identification of PolyHydroxyButyrate (PHB) producing bacteria from Sewage sample. Research Journal of Pharmacy and Technology. 2017; 10(4): 1065-1069. DOI: 10.5958/0974-360X.2017.00193.7

33. Hire D, Pawar N, Kate S. Isolation and Screening of Poly β-Hydroxybutyrate Producing Microbes. International Journal of Research and Analytical Reviews. 2019; 6 (2):634-637.

34. Tehmina A, Shagufta S., Muhammed T, Huma M, Ali RA, Sehrish F, AbuSaeed H. Bioconversion of Agricultural Wastes to Polyhydroxybutyrate by Azotobacter vinelandii. Pakistan Journal of Zoology. 2020; 52 (6): 2227-2231.

35. Alagunachiyar M, Jeevitha RAJ, Handique M, S. Sudha Sri Kesavan. Isolation and Screening of Poly Hydroxy Butyrate (PHB) Producing Bacteria from Jeppiaar Salt Pane, Chennai. Research J. Pharm. and Tech. Sept, 2015; 8(9):1276-1280. doi: 10.5958/0974-360X.2015.00231.0

36. Jaiganesh R, Tattwaprakash C, Rahul KG, Iyappan S, Jaganathan MK. Characterization and Optimization of process parameter for maximum Poly (-β-) hydroxyl butyrate production by bacteria isolated from the soil. Research J. Pharm. and Tech. 2019; 12(10):4926-4930. doi: 10.5958/0974-360X.2019.00854.0

37. Deepa S, Bhuvanehswari P, Kanimozhi K, Panneerselvam A. Utilization of Industrial Waste for the Production of Poly- Beta- Hydroxy Butyrate (Phb) by Alcaligenes eutrophus. Research J. Science and Tech Oct.- Dec., 2013; 5(4):421-424.

Received on 09.02.2021 Modified on 21.06.2021

Research J. Pharm. and Tech. 2022; 15(5):2053-2058.

DOI: 10.52711/0974-360X.2022.00339

Concentration of PHB produced by the best isolate in the study (units are as reported in concerned research paper)	Carbon Source	Bacteria	Reference
0.3g/L	4% Beet molasses	B. cereus M5 strain	[30]
2.2g/L	% date syrup added with mineral salt medium	Not mentioned	[31]
0.88g/L	Nutrient Broth	Isolate-2 (not identified)	[32]
2.10 g/L	Nutrient Agar	Isolate (not identified)	[33]
285mg/100ml	wheat brawn as a carbon source with 0.2% peptone	Azotobacter vinelandii.	[34]
0.225g/100ml	Nutrient Agar	Isolate AJ11 (not identified)	[35]
14.44µg/ml	Nutrient Agar	Bacterial isolate from soil (not identified)	[36]
48µg/10ml	Seasame oil	Alcaligenes eutrophus	[37]
3.53g/L	Milk Whey with 1% yeast extract	Bacillus sp.	Present study