Cyanobacterial Polyhydroxybutyrate (PHB): Screening, Optimization and Characterization

<div><p>In modern life petroleum-based plastic has become indispensable due to its frequent use as an easily available and a low cost packaging and moulding material. However, its rapidly growing use is causing aquatic and terrestrial pollution. Under these circumstances, research and development for biodegradable plastic (bioplastics) is inevitable. Polyhydroxybutyrate (PHB), a type of microbial polyester that accumulates as a carbon/energy storage material in various microorganisms can be a good alternative. In this study, 23 cyanobacterial strains (15 heterocystous and 8 non-heterocystous) were screened for PHB production. The highest PHB (6.44% w/w of dry cells) was detected in <i>Nostoc muscorum</i> NCCU- 442 and the lowest in <i>Spirulina platensis</i> NCCU-S5 (0.51% w/w of dry cells), whereas no PHB was found in <i>Cylindrospermum sp</i>., <i>Oscillatoria sp</i>. and <i>Plectonema sp</i>. Presence of PHB granules in <i>Nostoc muscorum</i> NCCU- 442 was confirmed microscopically with Sudan black B and Nile red A staining. Pretreatment of biomass with methanol: acetone: water: dimethylformamide [40: 40: 18: 2 (MAD-I)] with 2 h magnetic bar stirring followed by 30 h continuous chloroform soxhlet extraction acted as optimal extraction conditions. Optimized physicochemical conditions viz. 7.5 pH, 30°C temperature, 10:14 h light:dark periods with 0.4% glucose (as additional carbon source), 1.0 gl^-1 sodium chloride and phosphorus deficiency yielded 26.37% PHB on 7^th day instead of 21^st day. Using FTIR, ¹H NMR and GC-MS, extracted polymer was identified as PHB. Thermal properties (melting temperature, decomposition temperatures etc.) of the extracted polymer were determined by TGA and DSC. Further, the polymer showed good tensile strength and young’s modulus with a low extension to break ratio comparable to petrochemical plastic. Biodegradability potential tested as weight loss percentage showed efficient degradation (24.58%) of PHB within 60 days by mixed microbial culture in comparison to petrochemical plastic.</p></div

GC MS analysis.

<p>(a) GC spectra of isolated PHB from <i>Nostoc muscorum</i> NCCU- 442 (b) Comparision of the peak (Rt-10.297) with mass spectra MS library (NIST 11).</p

DSC analysis of isolated PHB from <i>Nostoc muscorum</i> NCCU- 442.

<p>(a) during first heating scan (b) Cooling scan (c) Second heating scan.</p

TGA analysis.

<p>(a) TGA thermogram and (b) DTG curve of isolated PHB from <i>Nostoc muscorum</i> NCCU- 442.</p

A typical stress-strain curve of PHB film.

<p>A typical stress-strain curve of PHB film.</p

Biodegradation potentiality of conventional plastic and PHB film with mixed microbial culture.

<p>Biodegradation potentiality of conventional plastic and PHB film with mixed microbial culture.</p

Screening of cyanobacterial strains for PHB (%).

<p>Screening of cyanobacterial strains for PHB (%).</p

(a-e). Optimization of PHB extraction process in <i>Nostoc muscorum</i> NCCU- 442.

<p>(a). Effect of addition of pre-treatment solvents, (b) shaking/ stirring during pre-treatment, (c) duration of pre-treatment, (d) solvent nature during soxhlet extraction (e) Duration of soxhlet extraction (using chloroform).</p

(a-e). Optimization of culture conditions of <i>Nostoc muscorum</i> NCCU- 442 for PHB yield.

<p>(a) Effect of pH, (b) temperature, (c) NaCl-addition, (d) P- deficiency (e) Duration of light and dark periods.</p

¹H NMR spectrum of isolated PHB from <i>Nostoc muscorum</i> NCCU- 442.

<p>¹H NMR spectrum of isolated PHB from <i>Nostoc muscorum</i> NCCU- 442.</p