Characterization Of Environmentally Friendly Hexabromocyclododecane Degradation And Stress Alleviation By A Novel Isolated Strain Bacillus Sp. HBCD-sjtu

Environmental pollution caused by the release of industrial chemicals is currently one of the most important environmental harms.Hexabromocyclododecane?HBCD?,a non-aromatic brominated flame retardant,is a toxic compound that consists of a cycloaliphatic ring of 12 carbon atoms to which six bromine atoms are attached.It is formed by bromination of cis–trans–trans-1,5,9-cyclododecatriene,but its use is now restricted in several countries because it is an environmental pollutant.HBCD?1,2,5,6,9,10-hexabromocyclododecane?is a widely distributed environmental pollutant.Toxicological studies have shown that HBCD may induce cancer and affect human thyroid hormones.Hexabromocyclododecanes?HBCD?,an additive brominated flame retardant,is having a great concern as a result of the extensive use and raising volumes in different environmental systems.The basic toxicities of hexabromocyclododecane?HBCD?and its toxic properties have a severe risk for the ecosystem and human beings.Soil-plant ecosystem has greatly influenced by the toxic effect of this serious pollutant?HBCD?.However,little information about HBCD degradation has been reported.One efficient method to remove these pollutants is biodegradation using bacteria.Firstly,a bacterial strain named Bacillus sp.HBCD-sjtu was isolated from a wastewater sample taken from a landfill in Shanghai,People's Republic of China,using enrichment culture techniques.Based on morphological,biochemical and phylogenetic analysis this isolate was categorized as Bacillus cereus and named strain HBCD-sjtu.Maximum growth and HBCD-degrading activity were observed when this strain was grown at 30^oC,pH 7.0 and 200 RPM in mineral salt medium containing 0.5 mM HBCD.The genome of strain HBCD-sjtu,which consists of only one circular chromosome,was sequenced on the Pacific Biosciences?PacBio?RS II sequencer?Pacific Biosciences,USA?.The coding region contained 4,414,071 bp,and 5,360 open reading frames?ORFs?.The genome encodes total rRNAs make up was 1.7%of the total genome.The whole genome contains ten genes responsible for the degradation of aromatic compounds and eighty genes associated with motility and chemotaxis.Genes encoding eleven dehalogenases were also annotated in the strain HBCD-sjtu whole genome sequence.Many other important genes responsible for oxygenases,CAZy genes?Carbohydrate active enzymes?,clustered regularly interspaced short palindromic repeats genes?CRISPRs?,comprehensive antibiotic resistance genes were also annotated in the whole genome sequence of strain Bacillus sp.HBCD-sjtu.This whole genome sequence will be crucial for illuminating the molecular mechanisms of HBCD degradation.Secondly,the classification of the strain Bacillus sp.HBCD-sjtu was performed by neighbor-joining?NJ?method using MEGA 4.1 and a phylogenetic tree was bulid.The determination of physiological and biochemical features such as the use of altered sources of carbon and enzymatic properties were conducted.Optimal conditions for the growth of Bacillus sp.HBCD-sjtu were detected at 30oC,pH 7,and 200 rev/min with 1.5 mM maximum HBCD-tolerating ability.The HBCD degradation by strain HBCD-sjtu,was optimized at different culture temperatures,pH values,HBCD concentrations,and rotation rates.The degradation rates,cell growth and HBCD transformation were checked at these optimized conditions.The salt tolerance activity of strain HBCD-sjtu was checked at different concentrations of NaCl.Maximum growth rate of strain HBCD-sjtu was noticed at 0.5%NaCl.Increase in salt concentration greatly reduced the growth rate.Four different intermediates,1,5,9-cyclododecatriene?CDT?,1,5-cyclododecadiene?CDD?,cyclododecene,and 3,9-dodecadiene were recognized based on gas chromatography-mass spectrometry analyses.The metabolite CDT was degraded by strain HBCD-sjtu using resting cell reaction.Same metabolites were identified from CDT degradation,compared to HBCD metabolites.These results provide innovative understandings into the bacterial degradation of HBCD and an environmentally friendly method of HBCD biodegradation.Thirdly,the HBCD degrading gene dehHZ1 was identified from strain HBCD-sjtu by homology modelling of enzyme DehHZ1 sequence with LinB from strain Sphingobium indicum B90A,an HBCD degrading enzyme.The three-dimensional?3D?model structures of enzyme DehHZ1 were obtained.Structure and functional relationship of the enzyme DehHZ1 with compound HBCD was performed using active sites.The HBCD degrading ability of the transformant containing the gene dehHZ1 was observed at 30oC,pH 7.0 and 200 RPM,in mineral salt medium?MSM?with 0.1 mM HBCD.An intermediate 1,5,9-cyclododecatriene?CDT?was recognized based on gas chromatography mass spectrometry analyses after HBCD degradation.These findings may provide new insights into the environment friendly route of molecular HBCD degradation and its conversion into new metabolite.Finally,the strain Bacillus sp.HBCD-sjtu was analyzed to study its effects on HBCD degradation in soil and the morphophysiological traits of wheat under different HBCD-stress conditions?0,0.2,0.4 and 0.6 mM HBCD?.Wheat plants inoculated with strain HBCD-sjtu revealed a considerable improvement in growth traits comprising shoot height,root length,shoot fresh weight,shoot dry weight,leaf number,leaf area and leaf total chlorophyll contents as compared to uninoculated plants.Due to HBCD-stress the elevated levels of malondialdehyde?MDA?,hydrogen peroxide?H₂O₂?,free proline and total soluble sugars?TSS?of leaves were found balanced in plants inoculated with strain HBCD-sjtu.Formation of new intermediate 1,5,9-cyclododecatriene?CDT?after HBCD degradation,was identified on the basis of gas chromatography mass spectrometry analyses.Strain HBCD-sjtu was also analyzed for their phosphate solubilisation and nitrogen fixation activities applying accessible standardized protocols.Indole acetic acid?IAA?production potential of strain HBCD-sjtu was studied,which stated to make IAA in large quantities.Results indicated that wheat growth can be improve by inoculation of strain HBCD-sjtu in both HBCD and non HBCD conditions.This study might be extended to other plants for growth enhancement and to find out novel environment friendly ways of HBCD degradation and its transformation into other metabolites.