Identification of polyhydroxyalkanoate (PHA)-producing Bacillus spp. using the polymerase chain reaction (PCR)

Abstract

Aims: The aim of the work was to develop efficient method to identify polyhydroxyalkanoate (PHA)-producing species of Bacillus from numerous soil isolates of bacteria. Identification of the isolates and characterization of the PHA produced by strains positive on the polymerase chain reaction (PCR) was envisaged. Methods and Results: Different bacteria isolated from soil were screened by PCR using two sets of primers designed for Bacillus megaterium. Amongst 23 isolates examined, the DNA of 12 isolates reacted positively with the primers giving amplicons identical in size to that obtained from B. megaterium. The isolates which were identified as strains of B. sphaericus, B. circulans, B. brevis and B. licheniformis, produced 11-41% of PHA in biomass, in sucrosecontaining medium, over a growth period of 24-72 h. The nature of the PHA thus produced was analyzed by Fourier transform infrared spectroscopy, gas chromatography and by nuclear magnetic resonance (NMR) and found to contain polyhydroxy butyrate and polyhydroxyvalerate. Conclusions:The results indicate that most of our isolates from different species contained the B. megaterium type of PHA synthase. Bacillus licheniformis appeared to belong to another group as it did not react with both sets of primers. Significance and Impact of the Study: This study shows the universality of the B. megaterium type of PHA synthase in soil isolates of Bacillus. Some variations were also found.

Bacterial polyhydroxyalkanoates-eco-friendly next generation plastic: Production, biocompatibility, biodegradation, physical properties and applications

“…Although these staining methods are quite sensitive, but it is rather time-consuming and labor-intensive work to screen a large number of environmental isolates. Moreover, prior to identification and isolation of PHA-producing bacteria by phenotypic methods, it is necessary to provide appropriate carbon sources, nutrient limitation conditions and a long culture time is required for PHA granule accumulation to the bacterial cells (78,79). Sudan black B is nonspecific to PHA as it also stains other lipid bodies.…”

“…Sudan black B is nonspecific to PHA as it also stains other lipid bodies. Nile blue A and Nile red are reported to be more specific than Sudan black B for detection (79). In addition, these methods cannot distinguish between bacteria that accumulate PHA granules and those that accumulate lipid compounds (78).…”

“…Genotypic-based screening method is specific, reliable and quick which circumvents the major drawbacks inherent in phenotypic detection methods (78,(81)(82)(83)(84). For genotypic-based screening, the degenerate or and specific oligo nucleotide primers are designed based on multiple sequence alignment results and are used as PCR primers to detect PHA synthase genes (78,79,81,(83)(84)(85). For rapid detection, colony PCR technique is employed for screening PHA producers from the environment (78,80).…”

Extractability of polyhydroxyalkanoate synthesized by Bacillus flexus cultivated in organic and inorganic nutrient media

“…Biomass obtained after centrifugation of fermented broth was dried to a constant weight [9]. Various methods were used for hydrolysis of cell wall and PHA extraction: (a) sodium hypochlorite solution was used for cell hydrolysis and PHA was solubilised in chloroform [12, at 40°C, for 2 h. The chloroform layer was separated by hexane in 1:2 proportions and the precipitated polymer was dried at 45°C to a constant weight; and (c) dried biomass (100 mg) was suspended in water (5 ml) and pH was set to 12 by ammonia solution (25% v/v).…”

“…As an alternative, enzymatic methods have been evolved for extraction [5,6]. Various Bacillus species are known to produce PHA [7][8][9]. But the extensive cross-linking and the thick peptidoglycan layer present in the gram-positive Bacillus species enables the bacterium to retain cell wall even in the presence of various hydrolytic enzymes intracellular biomolecules.…”