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Frontiers in Biology

ISSN 1674-7984

ISSN 1674-7992(Online)

CN 11-5892/Q

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Front. Biol.    2018, Vol. 13 Issue (4) : 297-308    https://doi.org/10.1007/s11515-018-1509-z
RESEARCH ARTICLE
Comparative evaluation of physico-chemical characteristics of biopolyesters P(3HB) and P(3HB-co-3HV) produced by endophytic Bacillus cereus RCL 02
Rituparna Das1, Nayan Ranjan Saha2, Arundhati Pal3, Dipankar Chattopadhyay2, Amal Kanti Paul1()
1. Microbiology Laboratory, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Kolkata 700019, West Bengal, India
2. Department of Polymer Science and Technology, University of Calcutta, 92, A. P. C. Road, Kolkata 700009, West Bengal, India
3. Department of Botany, Serampore College, 9, William Carey Road, Serampore, Hooghly 712201, West Bengal, India
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Abstract

BACKGROUND: Bacteria endogenously residing within the plant tissues have attracted significant attention for production of biopolyester, polyhydroxyalkanoates (PHAs). Bacillus cereus RCL 02 (MCC 3436), a leaf endophyte of oleaginous plant Ricinus communis L. accumulates 81% poly(3-hydroxybutyrate) [P(3HB)] of its cell dry biomass when grown in mineral salts (MS) medium.

METHODS: The copolymer production efficiency of B. cereus RCL 02 was evaluated in valeric acid supplemented MS medium under biphasic cultivation condition. The copolymer so produced has been compared with the P(3HB) isolated from RCL 02 in terms of thermal, mechanical and chemical properties.

RESULTS: Valeric acid supplementation as co-substrate in the medium has led to the production of copolymer of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) [P(3HB-co-3HV)] with 14.6 mol% 3HV. The identity of the polymers has been confirmed by X-ray diffraction (XRD) analysis, Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopic studies. Thermogravimetric analysis (TGA) revealed that P(3HB) and P(3HB-co-3HV) films degraded at 278.66°C and 273.49°C, respectively. The P(3HB-co-3HV) showed lower melting temperature (165.03°C) compared to P(3HB) (170.74°C) according to differential scanning calorimetry (DSC). Incorporation of 3HV monomers decreased the tensile strength (21.52 MPa), tensile modulus (0.93 GPa), storage modulus (E′) (0.99 GPa) and increased % elongation at break (12.2%) of the copolyester. However, P(3HB) showed better barrier properties with lower water vapor transmission rate (WVTR) of 0.55 g-mil/100 in2/24 h.

CONCLUSION: These findings emphasized exploration of endophytic bacterial strain (RCL 02) to produce biodegradable polyesters which might have significant potential for industrial application.
Keywords poly(3-hydroxybutyrate)      poly(3-hydroxybutyrate-co-3-hydroxyvalerate)      biodegradable polyester      Bacillus cereus      endophytic bacteria     
Corresponding Author(s): Amal Kanti Paul   
Online First Date: 30 July 2018    Issue Date: 10 September 2018
 Cite this article:   
Rituparna Das,Nayan Ranjan Saha,Arundhati Pal, et al. Comparative evaluation of physico-chemical characteristics of biopolyesters P(3HB) and P(3HB-co-3HV) produced by endophytic Bacillus cereus RCL 02[J]. Front. Biol., 2018, 13(4): 297-308.
 URL:  
https://academic.hep.com.cn/fib/EN/10.1007/s11515-018-1509-z
https://academic.hep.com.cn/fib/EN/Y2018/V13/I4/297
Fig.1  Time course of growth, PHA content (A), and composition (B) of the copolymer accumulated by the endophytic bacterial isolate B. cereus RCL 02 cells grown in glucose containing mineral salts medium supplemented with valeric acid during biphasic cultivation.
Valeric acid,
% (v/v) a		 Growth
CDW, g/L b		 PHA,
% CDW c		 PHA,
g/L		 PHA composition, mol % d
3HB 3HV
0.05 10.15±0.08 78.89±2.21 8.00±0.07 91.6 8.4
0.1 8.54±0.11 72.98±1.15 6.23±0.08 87.5 12.5
0.2 8.05±0.14 72.34±1.42 5.82±0.11 85.4 14.6
0.3 7.65±0.07 70.93±1.18 5.43±0.05 85.6 14.4
0.4 1.28±0.03 29.04±2.16 0.37±0.04 97.3 2.7
0.5 0.62±0.05 15.53±1.14 0.10±0.05 98.5 1.5
Tab.1  Influence of valeric acid on growth, PHA content and monomer composition of polyester accumulated by the endophytic B. cereus RCL 02 during biphasic cultivation condition
Fig.2  TGA (A) and DTA (B) curves of P(3HB) and P(3HB-co-3HV) isolated from B. cereus RCL 02 as a function of temperature.
Fig.3  DSC curves of P(3HB) and P(3HB-co-3HV) isolated from B. cereus RCL 02 showing melting temperature (Tm).
Fig.4  Mechanical properties of P(3HB) and P(3HB-co-3HV) films; stress vs strain curve (A), tensile strength (B), tensile modulus (0.01% strain) (C) and elongation at break (D).
Fig.5  Storage modulus of P(3HB) and P(3HB-co-3HV) isolated from B. cereus RCL 02 as a function of temperature.
Fig.6  Water vapor transmission rate (WVTR) of P(3HB) and P(3HB-co-3HV) films prepared from the polyesters isolated from B. cereus RCL 02.
Fig.7  XRD analysis of P(3HB) and P(3HB-co-3HV) isolated from B. cereus RCL 02.
Fig.8  FTIR spectral analysis of purified P(3HB) (A) and P(3HB-co-3HV) (B) isolated from B. cereus RCL 02.
Fig.9  1H NMR spectra of purified P(3HB) (A) and P(3HB-co-3HV) (B) isolated from B. cereus RCL 02.
Fig.10  13C NMR spectra of purified P(3HB) (A) and P(3HB-co-3HV) (B) isolated from B. cereus RCL 02.
1 Anjum A, Zuber M, Zia K M, Noreen A, Anjum M N, Tabasum S (2016). Microbial production of polyhydroxyalkanoates (PHAs) and its copolymers: A review of recent advancements. Int J Biol Macromol, 89: 161–174
https://doi.org/10.1016/j.ijbiomac.2016.04.069 pmid: 27126172
2 Bayari S, Severcan F (2005). FTIR study of biodegradable biopolymers: P(3HB), P(3HB-co-4HB) and P(3HB-co-3HV). J Mol Struct, 744: 529–534
https://doi.org/10.1016/j.molstruc.2004.12.029
3 Caballero K P, Karel S F, Register R A (1995). Biosynthesis and characterization of hydroxybutyrate-hydroxycaproate copolymers. Int J Biol Macromol, 17(2): 86–92
https://doi.org/10.1016/0141-8130(95)93522-Y pmid: 7547720
4 Castro-Sowinski S, Burdman S, Matan O, Okon Y (2010). Natural functions of bacterial polyhydroxyalkanoates, in: Chen GQ (Eds.), Plastics from bacteria.Springer, Berlin, Heidelberg: 39–61
5 Catalan A I, Ferreira F, Gill P R, Batista S (2007). Production of polyhydroxyalkanoates by Herbaspirillum seropedicae grown with different sole carbon sources and on lactose when engineered to express the lacZ lacY genes. Enzyme Microb Technol, 40(5): 1352–1357
https://doi.org/10.1016/j.enzmictec.2006.10.008
6 Chen G Q, König K H, Lafferty R M (1991). Occurrence of poly-D(-)-3-hydroxyalkanoates in the genus Bacillus. FEMS Microbiol Lett, 68(2): 173–176
pmid: 1778440
7 Choi M H, Yoon S C, Lenz R W (1999). Production of poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) and poly(4-hydroxybutyric acid) without subsequent degradation by Hydrogenophaga pseudoflava. Appl Environ Microbiol, 65(4): 1570–1577
pmid: 10103252
8 Das R, Dey A, Pal A, Paul A K (2016). Influence of growth conditions on production of poly(3-hydroxybutyrate) by Bacillus cereus HAL 03 endophytic to Helianthus annuus L. J Appl Biol Biotechnol, 4: 75–84
9 Das R, Pal A, Paul A K (2017). Production of biopolyester poly(3-hydroxybutyrate) by Bacillus cereus RCL 02, a leaf endophyte of Ricinus communis L. J Microbiol Biotechnol Res, 7: 32–41
10 Doi Y, Kawaguchi Y, Nakamura Y, Kunioka M (1989). Nuclear magnetic resonance studies of poly(3-hyroxybutyrate) and polyphosphate metabolism in Alcaligenes eutrophus. Appl Environ Microbiol, 55(11): 2932–2938
pmid: 16348055
11 Grewal R, Sweesy W, Jur J S, Willoughby J (2012). Moisture vapor barrier properties of biopolymers for packaging materials in Functional materials from renewable sources. ACS Symp Ser, 1107(15): 271–296
https://doi.org/10.1021/bk-2012-1107.ch015
12 Kumar P, Patel S K, Lee J K, Kalia V C (2013). Extending the limits of Bacillus for novel biotechnological applications. Biotechnol Adv, 31(8): 1543–1561
https://doi.org/10.1016/j.biotechadv.2013.08.007 pmid: 23954286
13 Labuzek S, Radecka I (2001). Biosynthesis of PHB tercopolymer by Bacillus cereus UW85. J Appl Microbiol, 90(3): 353–357
https://doi.org/10.1046/j.1365-2672.2001.01253.x pmid: 11298229
14 Law J H, Slepecky R A (1961). Assay of polyhydroxybutyric acid. J Bacteriol, 82: 32–36
15 Masood F, Hasan F, Ahmed S, Chen P, Hameed A (2012b). Biosynthesis and characterization of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) from Bacillus cereus S10. J Polym Environ, 20(3): 865–871
https://doi.org/10.1007/s10924-012-0457-y
16 Masood F, Hasan F, Ahmed S, Hameed A (2012a). Biosynthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from Bacillus cereus FA11 isolated from TNT-contaminated soil. Ann Microbiol, 62(4): 1377–1384
https://doi.org/10.1007/s13213-011-0386-3
17 Mitomo H, Morishita N, Doi Y (1993). Composition range of crystal phase transition of isodimorphism in poly(3-hydroxybutyrate-co-3-hydroxyvalerate). Macromol, 26(21): 5809–5811
https://doi.org/10.1021/ma00073a041
18 Mizuno K, Ohta A, Hyakutake M, Ichinomiya Y, Tsuge T (2010). Isolation of polyhydroxyalkanoate-producing bacteria from a polluted soil and characterization of the isolated strain Bacillus cereus YB-4. Polym Degrad Stabil, 95(8): 1335–1339
https://doi.org/10.1016/j.polymdegradstab.2010.01.033
19 Ramsay B A, Lomaliza K, Chavarie C, Dubé B, Bataille P, Ramsay J A (1990). Production of poly-(b-hydroxybutyric-co-b-hydroxyvaleric) acids. Appl Environ Microbiol, 56(7): 2093–2098
pmid: 2117877
20 Reddy S V, Thirumala M, Mahmood S K (2009). Production of PHB and P(3HB-co-3HV) biopolymers by Bacillus megaterium strain OU303A isolated from municipal sewage sludge. World J Microbiol Biotechnol, 25(3): 391–397
https://doi.org/10.1007/s11274-008-9903-3
21 Ryan R P, Germaine K, Franks A, Ryan D J, Dowling D N (2008). Bacterial endophytes: recent developments and applications. FEMS Microbiol Lett, 278(1): 1–9
https://doi.org/10.1111/j.1574-6968.2007.00918.x pmid: 18034833
22 Saharan B S, Grewal A, Kumar P (2014). Biotechnological production of polyhydroxyalkanoates: A review on trends and latest developments. Zhongguo Shengwuzhipinxue Zazhi, 2014 (1) :1–18
23 Sato H, Ando Y, Mitomo H, Ozaki Y (2011). Infrared spectroscopy and X-ray diffraction studies of thermal behavior and lamella structures of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(HB-co-HV)) with PHB-type crystal structure and PHV-type crystal structure Macromol, 44: 2829–2837
24 Shamala T R, Divyashree M S, Davis R, Kumari K S, Vijayendra S V N, Raj B (2009). Production and characterization of bacterial polyhydroxyalkanoate copolymers and evaluation of their blends by fourier transform infrared spectroscopy and scanning electron microscopy. Indian J Microbiol, 49(3): 251–258
https://doi.org/10.1007/s12088-009-0031-z pmid: 23100778
25 Sharma P, Bajaj B K (2015a). Production and characterization of poly-3-hydroxybutyrate from Bacillus cereus PS 10. Int J Biol Macromol, 81: 241–248 PMID:26257381
https://doi.org/10.1016/j.ijbiomac.2015.08.008
26 Sharma P, Bajaj B K (2015b). Production of poly-b-hydroxybutyrate by Bacillus cereus PS 10 using biphasic-acid-pretreated rice straw. Int J Biol Macromol, 79: 704–710
https://doi.org/10.1016/j.ijbiomac.2015.05.049 pmid: 26047898
27 Sindhu R, Silviya N, Binod P, Pandey A (2013). Pentose-rich hydrolysate from acid pretreated rice straw as a carbon source for the production of poly-3-hydroxybutyrate. Biochem Eng J, 78: 67–72
https://doi.org/10.1016/j.bej.2012.12.015
28 Tajima K, Igari T, Nishimura D, Nakamura M, Satoh Y, Munekata M (2003). Isolation and characterization of Bacillus sp. INT005 accumulating polyhydroxyalkanoate (PHA) from gas field soil. J Biosci Bioeng, 95(1): 77–81
https://doi.org/10.1016/S1389-1723(03)80152-4 pmid: 16233370
29 Tanasea E E, Popaa M E, Rapa M, Popaa O (2015). PHB/Cellulose fibers based materials: physical, mechanical and barrier properties. Agric Agric Sci Procedia, 6: 608–615
https://doi.org/10.1016/j.aaspro.2015.08.099
30 Urtuvia V, Villegas P, González M, Seeger M (2014). Bacterial production of the biodegradable plastics polyhydroxyalkanoates. Int J Biol Macromol, 70: 208–213
https://doi.org/10.1016/j.ijbiomac.2014.06.001 pmid: 24974981
31 Valappil S P, Misra S K, Boccaccini A R, Keshavarz T, Bucke C, Roy I (2007). Large-scale production and efficient recovery of PHB with desirable material properties, from the newly characterised Bacillus cereus SPV. J Biotechnol, 132(3): 251–258
https://doi.org/10.1016/j.jbiotec.2007.03.013 pmid: 17532079
32 Valappil S P, Rai R, Bucke C, Roy I (2008). Polyhydroxyalkanoate biosynthesis in Bacillus cereus SPV under varied limiting conditions and an insight into the biosynthetic genes involved. J Appl Microbiol, 104(6): 1624–1635
https://doi.org/10.1111/j.1365-2672.2007.03678.x pmid: 18194257
33 Winnacker M, Rieger B (2017). Copolymers of polyhydroxyalkanoates and polyethylene glycols: recent advancements with biological and medical significance. Polym Int, 66(4): 497–503
https://doi.org/10.1002/pi.5261
34 You J W, Chiu H J, Shu W J, Don T M (2003). Influence of hydroxyvalerate content on the crystallization kinetics of poly(hydroxybutyrate-co-hydroxyvalerate). J Polym Res, 10(1): 47–54
https://doi.org/10.1023/A:1023958014221
[1] Tayyaba Naeem, Naima Khan, Nazia Jamil. Synthesis of scl-poly (3-hydroxyalkanoates) by Bacillus cereus found in freshwater, from monosaccharides and disaccharides[J]. Front. Biol., 2018, 13(1): 63-69.
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