Production and Characterization of Bioplastic by Polyhydroxybutyrate Accumulating Erythrobacter aquimaris Isolated from Mangrove Rhizosphere

Abstract

The synthesis of bioplastic from marine microbes has a great attendance in the realm of biotechnological applications for sustainable eco-management. This study aims to isolate novel strains of poly-β-hydroxybutyrate (PHB)-producing bacteria from the mangrove rhizosphere, Red Sea, Saudi Arabia, and to characterize the extracted polymer. The efficient marine bacterial isolates were identified by the phylogenetic analysis of the 16S rRNA genes as Tamlana crocina, Bacillus aquimaris, Erythrobacter aquimaris, and Halomonas halophila. The optimization of PHB accumulation by E. aquimaris was achieved at 120 h, pH 8.0, 35 °C, and 2% NaCl, using glucose and peptone as the best carbon and nitrogen sources at a C:N ratio of 9.2:1. The characterization of the extracted biopolymer by Fourier-transform infrared spectroscopy (FTIR), Nuclear magnetic resonance (NMR), and Gas chromatography-mass spectrometry (GC-MS) proves the presence of hydroxyl, methyl, methylene, methine, and ester carbonyl groups, as well as derivative products of butanoic acid, that confirmed the structure of the polymer as PHB. This is the first report on E. aquimaris as a PHB producer, which promoted the hypothesis that marine rhizospheric bacteria were a new area of research for the production of biopolymers of commercial value.

Keywords: 16S rRNA gene; Erythrobacter aquimaris; FTIR; GC-MS; NMR; mangrove; poly-β-hydroxybutyrate.

Figure 1

Screening of promising marine bacterial isolate, KKU-MD13, for poly-β-hydroxybutyrate (PHB) production. (a) Mangaroves in Al-Madhaya coast; (b) bacterial culture; (c) plate staining with Sudan Black-B; (d) slide staining with Sudan Black-B; (e) slide staining with Acridine orange; (f) powder of extracted PHB.

Figure 2

Phylogenetic tree based on 16S rRNA nucleotide sequences of the bacterial isolates Tamlana crocina, Bacillus aquimaris, Erythrobacter aquimaris, and Halomonas halophila, with other sequences of published strains generated by the neighbor-joining method using MEGA 5.0 software. The scale bar corresponds to a 0.05 nucleotide substitution per sequence position. The numbers at the nodes indicate the levels of bootstrap support (%) based on 1000 resampled data sets. The number in parentheses represents the accession number in GenBank.

Figure 3

Quantitative screening of promising marine bacterial isolates; Halomonas halophila (KKU-MD12), Erythrobacter aquimaris (KKU-MD13), Tamlana crocina (KKU-MR35), and Bacillus aquimaris (KKU-MR42) for PHB production. The values set by oneself letter(s) on the same column were not significantly different.

Figure 4

Effect of fermentation period on the PHB production by E. aquimaris. The values set by oneself letter(s) on the same line were not significantly different.

Figure 5

Effect of initial pH on the PHB production by E. aquimaris. The values set by oneself letter(s) on the same line were not significantly different.

Figure 6

Effect of incubation temperature on the PHB production by E. aquimaris. The values set by oneself letter(s) on the same line were not significantly different.

Figure 7

Effect of NaCl on the PHB production by E. aquimaris. The values set by oneself letter(s) on the same line were not significantly different.

Figure 8

Effect of carbon source on the PHB production by E. aquimaris. The values set by oneself letter(s) on the same column were not significantly different.

Figure 9

Effect of nitrogen source on the PHB production by E. aquimaris. The values set by oneself letter(s) on the same column were not significantly different.

Figure 10

Effect of C:N ratio on the PHB production by E. aquimaris. The values set by oneself letter(s) on the same column were not significantly different.

Figure 11

Fourier-transform infrared spectrum of PHB extracted from E. aquimaris. The absorption bands at 3440, 2960, 2932, 1724, and 1280 cm⁻¹ corresponding to OH, CH₃, CH₂, C=O, and C–O groups.

Figure 12

Nuclear Magnetic Resonance (NMR) analysis of PHB extracted from E. aquimaris. (a) ¹H NMR spectrum of PHB shows signals at chemical shift δ 1.31 (d, CH₃,), 2.472 (q, CH₂,), and 5.26–5.30 ppm (m, CH,). (b) ¹³C NMR spectrum of PHB shows signals at 169.13, 67.61, 40.81, and 19.77 ppm for carbon atom of CO, CH, CH₂, and CH₃ groups.

Figure 13

Gas chromatography–mass spectrometry chromatogram of PHB extracted from E. aquimaris shows three monomeric compositions of PHB; Butanoic acid, 4-Iodo-, methyl ester; Hexanoic acid, 2-(1-methylethyl)-5-oxo-, methyl ester; and Decanoic acid, 8-methyl-, methyl ester.

Figure 14

Mass spectra of (a) Butanoic acid, 4-Iodo-, methyl ester; (b) Hexanoic acid, 2-(1-methylethyl)-5-oxo-, methyl ester; (c): Decanoic acid, 8-methyl-, methyl ester.