Production, optimization and characterization of polyhydroxybutyrate, a biodegradable plastic by <i>Bacillus</i> spp.

<div><p>Poly-β-hydroxybutyrate (PHB) is the intracellular lipid reserve accumulated by many bacteria. The most potent terrestrial bacterium <i>Bacillus cereus</i> SE-1 showed more PHB accumulating cells (22.1 and 40% after 48 and 72 h) than that of the marine <i>Bacillus</i> sp. CS-605 (5 and 33% after 48 and 72 h). Both the isolates harbored <i>phbB</i> gene and the characteristics C=O peak was observed in the extracted PHB by Fourier transformed infrared spectroscopy analysis. Maltose was found to be the most suitable carbon source for the accumulation of PHB in <i>B. cereus</i> SE-1. The extracted PHB sample from <i>B. cereus</i> SE-1 was blended with a thermoplastic starch (TS) and an increased thermoplasticity and decreased crystallinity were observed after blending in comparison to the standard PHB. The melting temperature (<i>T</i>_m), melting enthalpy (∆Hf), and crystallinity (<i>X</i>_c) of the blended PHB sample were found to be 109.4 °C, 64.58 J/g, and 44.23%, respectively.</p></div

Kinetics of utilization of DBT by <i>Chelatococcus</i> sp. in liquid BSM as the sole source of sulfur.

<p>Residual DBT concentration was determined by RP-HPLC analysis of extract from the culture medium (⬛) inoculated with <i>Chelatococcus</i> sp. and uninoculated controls (▲). Values are mean ± SD of triplicate determinations.</p

Proteomics and Metabolomics Analyses to Elucidate the Desulfurization Pathway of <i>Chelatococcus</i> sp.

<div><p>Desulfurization of dibenzothiophene (DBT) and alkylated DBT derivatives present in transport fuel through specific cleavage of carbon-sulfur (C-S) bonds by a newly isolated bacterium <i>Chelatococcus</i> sp. is reported for the first time. Gas chromatography-mass spectrometry (GC-MS) analysis of the products of DBT degradation by <i>Chelatococcus</i> sp. showed the transient formation of 2-hydroxybiphenyl (2-HBP) which was subsequently converted to 2-methoxybiphenyl (2-MBP) by methylation at the hydroxyl group of 2-HBP. The relative ratio of 2-HBP and 2-MBP formed after 96 h of bacterial growth was determined at 4:1 suggesting partial conversion of 2-HBP or rapid degradation of 2-MBP. Nevertheless, the enzyme involved in this conversion process remains to be identified. This production of 2-MBP rather than 2-HBP from DBT desulfurization has a significant metabolic advantage for enhancing the growth and sulfur utilization from DBT by <i>Chelatococcus</i> sp. and it also reduces the environmental pollution by 2-HBP. Furthermore, desulfurization of DBT derivatives such as 4-M-DBT and 4, 6-DM-DBT by <i>Chelatococcus</i> sp. resulted in formation of 2-hydroxy-3-methyl-biphenyl and 2-hydroxy –3, 3^/- dimethyl-biphenyl, respectively as end product. The GC and X-ray fluorescence studies revealed that <i>Chelatococcus</i> sp. after 24 h of treatment at 37°C reduced the total sulfur content of diesel fuel by 12% by per gram resting cells, without compromising the quality of fuel. The LC-MS/MS analysis of tryptic digested intracellular proteins of <i>Chelatococcus</i> sp. when grown in DBT demonstrated the biosynthesis of 4S pathway desulfurizing enzymes viz. monoxygenases (DszC, DszA), desulfinase (DszB), and an NADH-dependent flavin reductase (DszD). Besides, several other intracellular proteins of <i>Chelatococcus</i> sp. having diverse biological functions were also identified by LC-MS/MS analysis. Many of these enzymes are directly involved with desulfurization process whereas the other enzymes/proteins support growth of bacteria at an expense of DBT. These combined results suggest that <i>Chelatococcus</i> sp. prefers sulfur-specific extended 4S pathway for deep-desulphurization which may have an advantage for its intended future application as a promising biodesulfurizing agent.</p></div

GC-MS analysis of metabolites of 4-M- DBT produced by <i>Chelatococcus</i> sp.

<p><b>A</b>: GC chromatogram of the culture extract showing DBTO, 2-hydroxy-3’ methyl–biphenyl and 4-M- DBT; <b>B</b>: Mass spectrum of DBTO (molecular mass, 200); <b>C</b>: Mass spectrum of 2-hydroxy-3’ methyl—biphenyl (molecular mass, 184); <b>D</b>: Mass spectrum of 4-M- DBT (molecular mass, 198).</p

GC-MS analysis of DBT metabolites produced by C<i>helatococcus</i> sp.

<p><b>A</b>: GC profile of the culture extract (96 h post inoculation) showing formation of 2- HBP, 2-MBP, and DBTO; <b>B</b>: Mass spectrum of 2-HBP (molecular mass, 170); <b>C</b>: Mass spectrum of 2-MBP (molecular mass, 184).</p

GC–FID chromatogram of desulphurization of diesel oil by <i>Chelatococcus</i> sp.

<p><b>(A)</b> Control diesel fuel; <b>(B)</b> Diesel fuel treated with C<i>helatococcus</i> sp. for 24 h at 37°C.</p

Phylogenetic relationships of isolated NBTU-06 and other closely related <i>Chelatococcus</i> species based on 16S rRNA sequencing.

<p>The tree was generated using the neighbor-joining method and the sequence from <i>Bacillus</i> sp. HSCC 1649 T (<i>Accession no</i>. <i>AB045097</i>) was considered as out-group. The data set was resampled 1,000 times by using the bootstrap option, and percentage values are given at the nodes.</p

GC-MS analysis of metabolites of 4, 6 -DM- DBT produced by <i>Chelatococcus</i> sp.

<p><b>A</b>: GC chromatogram of the culture extract showing, 2-hydroxy-3, 3’ dimethyl–biphenyl and 4, 6 -M- DBT; <b>B</b>: Mass spectrum of 2-hydroxy-3, 3’ dimethyl—biphenyl (molecular mass, 198); <b>C</b>: Mass spectrum of 4, 6-DM- DBT (molecular mass, 212).</p

A scheme for extended 4s pathway of biocatalytic desulfurization of DBT by <i>Chelatococcus</i> sp.

<p>A scheme for extended 4s pathway of biocatalytic desulfurization of DBT by <i>Chelatococcus</i> sp.</p

Bacterial growth and protein content of culture medium inoculated with <i>Chelatococcus</i> sp. in presence or absence of DBT (0.5 mM).

<p>A: growth in presence of DBT, B: growth in absence of DBT. Values are mean ± S.D of triplicate determinations. Significance of difference with respect to growth of bacteria in presence of 0.5 mM DBT.</p