Biosynthesis of polyhydroxyalkanoates from styrene by Enterobacter spp. isolated from polluted environment

Abstract

BACKGROUND: Styrene and its metabolites are known to have serious adverse effects on human health and hence, strategies to prevent its release, eradicate it from the environment, and understand its route of degradation are being considered.

METHODS: A total of 18 strains were isolated from 4 samples of diesel contaminated soils. Among them 5 strains were selected for their ability to degrade styrene and use it as a sole carbon source to produce PHA. These strains were identified as Enterobacter spp. on the basis of 16S rRNA gene sequencing. Bacteria were screened for their ability to produce PHA by utilizing glucose and styrene as a carbon sources. Screening for PHA production was done by Nile blue A, Sudan black B, and phase contrast microscopy and the selected 3 strains showed positive results. Growth kinetics along with time profiling of PHA was performed for glucose and styrene as carbon sources.

RESULTS:PHA extraction was done at equal intervals of 12 h by sodium hypochlorite method which showed that these strains accumulate maximum amount of PHA after 48 h in glucose (30.60%). FTIR analysis of PHA was done which revealed homopolymer PHB and copolymer (PHB-co-PHV) production in strains by utilizing glucose and styrene. Gas chromatography mass spectrometry was carried out to identify the metabolites produced by bacterial strains grown on styrene. Metabolites of styrene degradation included propyne and phenylalanine. Genomic DNA isolation was carried out to amplify phaC gene which encodes PHA synthase enzyme.

CONCLUSIONS: The conversion of styrene to polyhydroxyalkanoates (PHA) provides a new and unique link between an aromatic environmental pollutant and aliphatic PHA accumulation.

Keywords

biodegradable polymers / environmental pollutants / PHA / FTIR / recycling / bacteria

References

[1] Ali I, Jamil N (2014). Enhanced biosynthesis of poly(3-hydroxybutyrate) from potato starch by Bacillus cereus strain 64-INS in a laboratory-scale fermenter. Prep Biochem Biotechnol, 44(8): 822–833

[2] Ali I, Jamil N (2016). Polyhydroxyalkanoates: Current applications in the medical field. Front Biol, 11(1): 19–27

[3] Ali I, Jamil N ( 2016).Biosynthesis and characterization of poly3-hydroxyalkanote (PHA) from newly isolated bacterium Bacillus sp. AZR-1. Iran J Sci Technol Trans Sci, pp. 1–8

[4] Beltrametti F, Marconi A M, Bestetti G, Colombo C, Galli E, Ruzzi M, Zennaro E (1997). Sequencing and functional analysis of styrene catabolism genes from Pseudomonas fluorescens ST. Appl Environ Microbiol, 63(6): 2232–2239

[5] Chanprateep S (2010). Current trends in biodegradable polyhydroxyalkanoates. J Biosci Bioeng, 110(6): 621–632

[6] Chaudhry W N, Jamil N, Ali I, Ayaz M H, Hasnain S (2011). Screening for polyhydroxyalkanoate (PHA)-producing bacterial strains and comparison of PHA production from various inexpensive carbon sources. Ann Microbiol, 61(3): 623–629

[7] Choi J, Lee S Y (1999). Factors affecting the economics of polyhydroxyalkanoate production by bacterial fermentation. Appl Microbiol Biotechnol, 51(1): 13–21

[8] Hartmans S, van der Werf M J, de Bont J A (1990). Bacterial degradation of styrene involving a novel flavin adenine dinucleotide-dependent styrene monooxygenase. Appl Environ Microbiol, 56(5): 1347–1351

[9] Hindré T, Brüggemann H, Buchrieser C, Héchard Y (2008). Transcriptional profiling of Legionella pneumophila biofilm cells and the influence of iron on biofilm formation. Microbiology, 154(Pt 1): 30–41

[10] Hollmann F, Lin P C, Witholt B, Schmid A (2003). Stereospecific biocatalytic epoxidation: the first example of direct regeneration of a FAD-dependent monooxygenase for catalysis. J Am Chem Soc, 125(27): 8209–8217

[11] Hussain Z, Khan K M, Hussain K (2010). Microwave–metal interaction pyrolysis of polystyrene. J Anal Appl Pyrolysis, 89(1): 39–43

[12] Luhana K, Patel V(2013). Quantitative extraction and analysis of bioplastic (PHA) accumulated in bacterial isolates of paint industry effluent. International Journal of Chemtech Applications (INTJCA), 2(3): 52–62

[13] Michael P, Loganayagi R, Nancy D, Ranandkumar S G, Indra  A P(2012). Isolation and characterization of indigenous Ralstonia strain, YRF1 for high Polyhydroxy Alkanoates (PHA) production. Elixir Appl Biol, 48(2012): 9424–9427

[14] Miskolczi N, Borsodi N, Buyong F, Angyal A, Williams P T (2011). Production of pyrolytic oils by catalytic pyrolysis of Malaysian refuse-derived fuels in continuously stirred batch reactor. Fuel Process Technol, 92(5): 925–932

[15] Mohammadi M, Hassan M A, Shirai Y, Man H C, Ariffin H, Yee L N, Mumtaz T, Chong M L, Phang L Y (2012). Separation and purification of polyhydroxyalkanoates from newly isolated Comamonas sp. EB172 by simple digestion with sodium hydroxide. Sep Sci Technol, 47(3): 534–541

[16] Moita R, Lemos P C (2012). Biopolymers production from mixed cultures  and  pyrolysis   by-products.  J  Biotechnol, 157(4): 578–583

[17] O’Leary N D, O’Connor K E, Dobson A D (2002). Biochemistry, genetics and physiology of microbial styrene degradation. FEMS Microbiol Rev, 26(4): 403–417

[18] Obruca S, Marova I, Svoboda Z, Mikulikova R (2010). Use of controlled exogenous stress for improvement of poly(3-hydroxybutyrate) production in Cupriavidus necator. Folia Microbiol (Praha), 55(1): 17–22

[19] Otari S, Ghosh J (2009). Production and characterization of the polymer polyhydroxy butyrate-copolyhydroxy valerate by Bacillus megaterium NCIM 2475. Current Research Journal of Biological Sciences, 1(2): 23–26

[20] Paladino L P A ( 2009) .Screening, optimization and extraction of polyhydroxyalkanoates and peptidoglycan from Bacillus megaterium. 

[21] Park M S, Han J H, Yoo S S, Lee E Y, Lee S G, Park S (2005). Degradation of styrene by a new isolate Pseudomonas putida SN1. Korean J Chem Eng, 22(3): 418–424

[22] Peplinski K, Ehrenreich A, Döring C, Bömeke M, Reinecke F, Hutmacher C, Steinbüchel A (2010). Genome-wide transcriptome analyses of the ‘Knallgas’ bacterium Ralstonia eutropha H16 with regard to polyhydroxyalkanoate metabolism. Microbiology, 156(Pt 7): 2136–2152

[23] Pötter M, Steinbüchel A (2005). Poly(3-hydroxybutyrate) granule-associated proteins: impacts on poly(3-hydroxybutyrate) synthesis and degradation. Biomacromolecules, 6(2): 552–560

[24] Rosazza J P, Huang Z, Dostal L, Volm T, Rousseau B (1995). Review: biocatalytic transformations of ferulic acid: an abundant aromatic natural product. J Ind Microbiol, 15(6): 457–471

[25] Rossi G, Monticelli L, Puisto S R, Vattulainen I, Ala-Nissila T (2011). Coarse-graining polymers with the MARTINI force-field: polystyrene as a benchmark case. Soft Matter, 7(2): 698–708

[26] Sambrook J, Russell D W (2001). Molecular cloning: a laboratory manual (3-volume set). Vol. 999. Cold spring harbor laboratory press Cold Spring Harbor, New York

[27] Sathesh Prabu C, Murugesan A (2010). Effective utilization and management of coir industrial waste for the production of poly-β-hydroxybutyrate (PHB) using the bacterium Azotobacter beijerinickii. Int J Environ Res, 4(3): 519–524

[28] Spiekermann P, Rehm B H, Kalscheuer R, Baumeister D, Steinbüchel A (1999). A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch Microbiol, 171(2): 73–80

[29] Sudesh K, Abe H, Doi Y (2000). Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Prog Polym Sci, 25(10): 1503–1555

[30] Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013). MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol, 30(12): 2725–2729

[31] Velasco A, Alonso S, García J L, Perera J, Díaz E (1998). Genetic and functional analysis of the styrene catabolic cluster of Pseudomonas sp. strain Y2. J Bacteriol, 180(5): 1063–1071

[32] Xiao N, Jiao N (2011). Formation of polyhydroxyalkanoate in aerobic anoxygenic phototrophic bacteria and its relationship to carbon source and light availability. Appl Environ Microbiol, 77(21): 7445–7450

[33] Yang C, Zhang W, Liu R, Zhang C, Gong T, Li Q, Wang S, Song C (2013). Analysis of polyhydroxyalkanoate (PHA) synthase gene and PHA-producing bacteria in activated sludge that produces PHA containing 3-hydroxydodecanoate. FEMS Microbiol Lett, 346(1): 56–64

[34] Younas T, Ali I, Jamil N (2015). Polyhydroxyalkanotes (PHAs) production by using canola oil as carbon source from bacteria isolated near paper pulp industry. Kuwait Journal of Science, 42(2)

[35] Zhu C, Nomura C T, Perrotta J A, Stipanovic A J, Nakas J P (2010). Production and characterization of poly-3-hydroxybutyrate from biodiesel-glycerol by Burkholderia cepacia ATCC 17759. Biotechnol Prog, 26(2): 424–430

[36] Zinn M, Witholt B, Egli T (2001). Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate. Adv Drug Deliv Rev, 53(1): 5–21

Acknowledgment

Authors are thankful to University of the Punjab, Pakistan, International foundation of science, IFS and HEC funded NRPU-3443, Pakistan to support this research

Conflict of interest

Authors declare no conflict of interest.

RIGHTS & PERMISSIONS

2017 Higher Education Press and Springer-Verlag Berlin Heidelberg