Polyhydroxybutyrates (PHBs) are macromolecules synthesized by bacteria. They are inclusion bodies accumulated as reserve materials when the bacteria grow under different stress conditions. Because of their fast degradability under natural environmental conditions, PHBs are selected as alternatives for production of biodegradable plastics. The aim of this work was to isolate potential PHB producing bacteria, evaluate PHB production using agro-residues as carbon sources. Among fifty bacterial strains isolated from different localities, ten PHB accumulating strains were selected and compared for their ability to accumulate PHB granules inside their cells. Isolate Arba Minch Waste Water (AWW) identified as Bacillus spp was found to be the best producer. The optimum pH, temperature, and incubation period for best PHB production by the isolate were 7, 37 °C, and 48 h respectively at 150 rpm. PHB production was best with glucose as carbon source and peptone as nitrogen source. The strain was able to accumulate 55.6, 51.6, 37.4 and 25% PHB when pretreated sugar cane bagasse, corn cob, teff straw (Eragrostis tef) and banana peel were used as carbon sources respectively. Fourier transform-infrared authentication results of the extracted and purified PHB identified its functional units as C–H, CH2, C=O and C–O groups. UV–Vis spectrophotometric analysis and biodegradability test confirmed the similarity of the extract with standard PHB and its suitability for bioplastic production. The isolated Bacillus sp can be used for feasible production of PHB using agro-residues especially sugarcane bagasse which can reduce the production cost in addition to reducing the disposal problem of these substrates. The yield of PHB can further be boosted by optimization of production parameters as substrates.

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Production of biodegradable plastic by polyhydroxybutyrate (PHB) accumulating bacteria using low cost agricultural waste material

The microbially derived polyhydroxyalkanoates biopolymers could impact the global climate scenario by replacing the conventional non-degradable, petrochemical-based polymer. The biogenesis, characterization and properties of PHAs by Bacillus species using renewable substrates have been elaborated by many for their wide applications. On the other hand Bacillus species are advantageous over other bacteria due to their abundance even in extreme ecological conditions, higher growth rates even on cheap substrates, higher PHAs production ability, and the ease of extracting the PHAs. Bacillus species possess hydrolytic enzymes that can be exploited for economical PHAs production. This review summarizes the recent trends in both non-growth and growth associated PHAs production by Bacillus species which may provide direction leading to future research towards this growing quest for biodegradable plastics, one more critical step ahead towards sustainable development.

Bacillus and biopolymer: Prospects and challenges.

Biobased degradable plastics have received significant attention owing to their potential application as a green alternative to synthetic plastics. A dye-based procedure was used to screen poly-3-hydroxybutyrate (PHB)-producing marine bacteria isolated from the Red Sea, Saudi Arabia. Among the 56 bacterial isolates, Pseudodonghicola xiamenensis, identified using 16S rRNA gene analyses, accumulated the highest amount of PHB. The highest PHB production by P. xiamenensis was achieved after 96 h of incubation at pH 7.5 and 35 °C in the presence of 4% NaCl, and peptone was the preferred nitrogen source. The use of date syrup at 4% (w/v) resulted in a PHB concentration of 15.54 g/L and a PHB yield of 38.85% of the date syrup, with a productivity rate of 0.162 g/L/h, which could substantially improve the production cost. Structural assessment of the bioplastic by Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy revealed the presence of methyl, hydroxyl, methine, methylene, and ester carbonyl groups in the extracted polymer. The derivative products of butanoic acid estimated by gas chromatography-mass spectrometry [butanoic acid, 2-amino-4-(methylseleno), hexanoic acid, 4-methyl-, methyl ester, and hexanedioic acid, monomethyl ester] confirmed the structure of PHB. The present results are the first report on the production of a bioplastic by P. xiamenensis, suggesting that Red Sea habitats are a potential biological reservoir for novel bioplastic-producing bacteria.

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Bioplastic (poly-3-hydroxybutyrate) production by the marine bacterium Pseudodonghicola xiamenensis through date syrup valorization and structural assessment of the biopolymer.

Municipal sewage triggers a stress prone environment to accumulate polyhydroxyalkanoates (PHAs) in the cytosol of bacteria. In view of that, different Bacillus species were isolated from municipal sewage and screened for evaluating their efficacy of PHA production. Growth parameters such as temperature, pH, glucose concentration and carbon nitrogen combinations were optimized with respect to higher biomass production as it is analogous to PHA accumulation. Under optimized conditions, the Bacillus species produced 3.09 g/L of PHAs which was estimated as a higher yield in comparison to other similar strains. Fourier transform infrared spectroscopic analysis of the extracted polyhydroxybutyrate confirmed the distinct peak corresponding to C=O group, whereas proton nuclear magnetic resonance (1H NMR) and differential scanning colorimetric analysis exhibited detailed insight of its chemical structure and properties by reflecting monomeric unit. The high yielding bacterial isolate was identified by 16S rDNA sequencing and the sequence was confirmed as Bacillus subtilis with an accession no. KP172548 after submission to NCBI data base. The potential bacterium may be further exploited for cost effective and mass scale production of biopolymer.

Production of Polyhydroxyalkanoates (PHAs) by Bacillus Strain Isolated from Waste Water and Its Biochemical Characterization

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.

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

Polyhydroxyalkanoates (PHAs) are polyesters naturally synthesized by numerous bacteria as intracellular energy storage materials during unbalanced growth. These polyesters find many applications in medicine, veterinary practice, tissue engineering, food packaging, etc. In the present study, spent wash and oil mill soil samples were screened for the isolation of bacteria producing polyhydroxyalkanoates. Different bacterial isolates were screened by PCR using primers designed for Bacillus megaterium. Isolate-4 reacted positively giving amplicons similar in size to that of Bacillus megaterium. Based on 16S rRNA gene sequences, isolate-4 was identified as Paenibacillus durus. Nitrogen limited mineral salt medium with fructose as chief carbon source was used for production of Polyhydroxybutyrate (PHB) in shake flask studies. The effect of various carbon and nitrogen sources on PHB production was evaluated, with respect to yield and PHB content. The study revealed that fructose and peptone gave better PHB accumulation and growth rates for the isolate and the reference strain, and hence, were the best among the carbon and nitrogen sources used. Further studies are needed to optimize the production conditions.

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Arabi Mohammed Saleh

Papers

Production of Polyhydroxyalkanoate from Paenibacillus durus BV-1 Isolated from Oil Mill Soil