Polyhydroxyalkanoate biosynthesis and optimisation of thermophilic Geobacillus stearothermophilus strain K4E3_SPR_NPP.

Polyhydroxyalkanoates (PHA) can be used to combat the challenges associated with plastic because it is biodegradable and can be produced from renewable resources. Extremophiles are considered to be potential PHA producers. An initial screening for the PHA synthesizing ability of a thermophilic bacteria Geobacillus stearothermophilus strain K4E3_SPR_NPP was carried out using Sudan black B staining. Nile red viable colony staining was used to further verify that the isolates produced PHA. Crotonic acid assays were used to determine the concentrations of PHA. The bacteria showed 31% PHA accumulation per dry cell weight (PHA/DCW) when glucose was used as a carbon source for growth. The molecule was identified to be medium chain length PHA, A copolymer of PHA containing poly(3-hydroxybutyrate)-poly(3-hydroxyvalerate)-poly(3-hydroxyhexanoate) (PHB-PHV-PHHX) using 1H-NMR. Six carbon sources and four nitrogen sources were screened for the synthesis of maximum PHA content, of which lactose and ammonium nitrate showed 45% and 53% PHA/DCW respectively. The important factors in the experiment are identified using the Plackett-Burman design, and optimization is performed using the response surface method. Response surface methodology was used to optimize the three important factors, and the maximum biomass and PHA productions were discovered. Optimal concentrations yielded a maximum of 0.48 g/l biomass and 0.32 g/l PHA, measuring 66.66% PHA accumulation. Dairy industry effluent was employed for the synthesis of PHA, yielding 0.73 g/l biomass and 0.33 g/l PHA, measuring 45% PHA accumulation. These findings add credibility to the possibility of adopting thermophilic isolates for PHA production using low-cost substrates.

Identification of Quorum Quenching N-Acyl Homoserine Lactonases from Priestia aryabhattai J1D and Bacillus cereus G Isolated from the Rhizosphere.

Several pathogenic bacteria communicate using N-acyl homoserine lactone (AHL) as a quorum sensing (QS) molecule. The process of interfering with the QS system is known as quorum quenching (QQ), it is an effective tool to control QS-dependent virulence in pathogens. In the present study, rhizosphere bacterial isolates were screened for their ability to produce AHL lactonase enzyme as QQ molecules, which hydrolyses AHL signalling molecules and consequently blocks the QS system. Potent N-hexanoyl-l-homoserine lactone (C6HSL) hydrolytic QQ activity was detected in rhizosphere isolates namely Bacillus cereus G and Priestia aryabhattai J1D. The cell-free supernatant of the bacterial isolates indicated a reduction in biofilm formation in the human pathogens Vibrio cholerae, Pseudomonas aeruginosa, and Staphylococcus aureus without inhibiting cells, signifying their biocontrol property. Furthermore, liquid chromatography high resolution mass spectrometry analysis confirmed C6HSL hydrolytic activity by AHL lactonase produced by these rhizosphere isolates. Also, the aiiA homologous gene from the bacterial isolates showed similarity with the aiiA lactonase gene from Bacillus species, which was further confirmed by homology modelling. In silico structure analysis by comparing with the structure of Bacillus revealed the similarity in the active site, indicating the same degradation pattern. Based on available reported data, the present study indicates the first report of the presence of the aiiA lactonase gene in P. aryabhattai.