Research On The Pahs Removal Mechanisms By Immobilized Bacillus Sp.P1 Under Heavy Metal Stress

Polycyclic aromatic hydrocarbons(PAHs)are ubiquitous persistent organic pollutions which are resistant to environmental degradation.PAHs can pose acute or chronic toxic effect on the environment and human being.More and more researchers have paid attentions on the pollution because some of them have been even identified as carcinogenic,mutagenic and teratogenic.Although PAHs may be redu ced by adsorption,volatilization,photolysis,and chemical degradation,biodegradation is the main degradation process due to the characteristics of low cost and non-secondary pollution.However,the coexisting heavy metals in the environment may affect t he degradation of PAHs by impacting the growth of microorganism,enzyme productions and characteristics of enzymatic degradation process of PAHs bidegradation.Freely suspended bacteria tend to be involved in vicious competitions with indigenous microbes,thus lost the advantages of dominant bacteria in practical applications.Besides,some cultured exogenous microbes are screened in soft conditions and turn out to be hard to adapt to natural environmental conditions.Immobilization enables bacteria entrapped in polymeric matrices,which can improve bioremediation efficiency of PAHs via enhancing microbe density,resisting to environmental impact and recovering microbes.Increasing studies have paid attention to the application of immobilized microorganisms for PAHs degradation.Mechanisms of PAHs treatment by immobilized microorganisms are still not deeply understood,especially in the presence of heavy metals.In general,toxic heavy metals and PAHs pose detrimental effect on microbes or make microbes resistant to pollutants.Detoxification of immobilized bacteria reveals the process and response of toxicological effect and oxidative damage that PAHs and heavy metals posed on microorganisms.Better detoxification effect helps keeping microbes vigorous,thus improving PAHs degradation.What’s more,the combination of microbes with biochar enables an efficiently and effectively restore of PAHs-heavy metal polluted soils,which is very important in soil remediation.Hence,PAHs-degrading Bacillus sp.P1 was selected in this research,the mechanisms of the impact of Pb(II)and Cd(II)on phenanthrene(PHE)biodegradation by freely suspended Bacillus sp.P1 were elucidated by investigating the biodegradation rate,metabolites and enzyme variations(enzymatic degrad ation ability,component,and activities).Bacillus sp.P1 was then immobilized and the differences of PAHs degradation by immobilized and suspended Bacillus sp.P1 in the presence heavy metal were investigated.The detoxification differences between immobilized bacteria and free suspended bacteria in PHE degrading process in the present of Cd(II)were explored,which helped better understanding of immobilization effect on bacteria.Bacillus sp.P1 was immobilized on biochar in the last part,the combinatio n of bacteria with biochar was used for soil polluted by phenanthrene and Cd(II).The results can provide theoretical guidance for application of microorganisms on the remediation of combined pollution of PAHs and heavy metals in soils.The study can be summarized in the following four sections:The first section focused on the impact of Pb(II)and Cd(II)on PHE biodegradation by Bacillus sp.P1 and the enzyme variations.The capability and pathway of PHE degradation by Bacillus sp.P1 were analyzed.Besides,the alterations of extracellular enzymes(EE)and intracellular enzymes(IE)concentrations were perceived through analyzing SDS-PAGE fingerprint by Gelprosoftware.Catechol 2,3-dioxygenase activities in EE and IE were also detected in this study.Enzymatic degradations of PHE by EE and IE were analyzed as well as the optimal pH and temperature.The results indicated that heavy metals could cause enzyme variation,thus affecting the phenanthrene biodegradation by Bacillus sp.P1.Pb(II)and Cd(II)inhibited PHE degradation as a whole,and Cd(II)was much more toxic to Bacillus sp.P1 than Pb(II).PHE degradation by the highly competent Bacillus sp.P1 could be inhibited by heavy metals,the inhibition effect was weaked as time went by,and inhibition rate varied with different heavy metals.Pb(II)and Cd(II)did not affect the protocatechuate metabolic pathways,they only had an effect on the concentration of degradation components.The inhibition effect of Cd(II)were stronger than Pb(II)on PHE metabolism,which can be attributed to that the toxicity of Cd(II)on microorganisms was greater than Pb(II).With the increase of Pb(II),the total contents and the protein bands with molecular mass of 33-45 kDaof EE and IE exhibited trends of firstly increase and then decrease.While w ith the increase of Cd(II),the total contents and the protein bands with molecular mass of 33-45 kDaof EE and IE all decreased sharply.Catechol 2,3-dioxygenase activities in EE and IE were remarkably changed in the presence of different concentrations of Pb(II)during the process of degrading PHE.Low concentration of Pb(II)promoted PHE degradation while high content of Pb(II)had the opposite effect.Cd(II)showed an inhibition effect on catechol 2,3-dioxygenase activities in EE and IE.Low concentrationsof Pb(II)can increase the enzymatic degradation of PHE at an optimal pH and temperature,while high Pb(II)can decrease the enzymatic degradation.Cd(II)posed an inhibition effect on PHE degradation by EE and IE.And the higher Cd(II)content was,the more remarkably inhibition effect it posed.The second section investigated the optimal experimental conditions for the immobilization of Bacillus sp.P1 using polyvinyl alcohol(PVA)and sodium alginate(SA).The reusability was detected by batch experiment for five cycles.The differences of PAHs adsorption capabilities,kinetic equations and surface properties between immobilized and suspended inactived Bacillus sp.P1 in the presence of Cd(II)were also evaluated.The shielding effect of pH,temperature,different kinds of heavy metals and PAHs metabolitics phenanthrenequinone resulted by immobilization were studied as well.The results showed that the optimal levels of PVA,SA and inoculum size for immobilization of Bacillus sp.P1 was 12%,0.3% and 10 mL,respectively.There were much more porous structures in immobilized gel beads compared to suspended cells,which increased the adsorption sites for contaminants,enhanced the bioavailability,therefore accelerating the removal rate of PAHs.The adsorption processes of PHE and pyrene(PYR)fitted pseudo-second order kinetics equation,and the conformance of PHE was better than PYR.According to the kinetic parameters,the adsorption rate of PHE was declined but the adsorption amount of PHE was ascended after immobilization.While the adsorption rate and amount of PYR both increased after immobilization,and the variations of PYR adsorption rate and amount were smaller than the changes of PHE.The immobilized bead cells could be resued for degrading PHE,which significantly reduced the costs for immobilization preparation.The environmental impact resistance was enhanced because of immobilization,which exhibited as more extensively adaptibilities of pH and temperature and strong shielding capabilities of toxic materials including heavy metals and metabolites.Overall,immobilization protected the Bacillus sp.P1 and accelerated PHE degradation.The third section studied the physiological and biochemical response and detoxification mechanisms of Bacillus sp.P1 in PHE degrading in the presence of Cd(II).The biomass,total protein,detoxification indexes including SOD,CAT and GSH of immobilized and suspended Bacillus sp.P1 were investigated.The correlation between heavy metal accumulation and detoxification indexes was analyzed.The relationship between extracellular secretions and heavy metal concentrations was also investigated to illustrate the mechanisms of immobilization on PHE degradation by Bacillus sp.P1 with Cd(II)stress.The results indicated that PAHs and heavy metals both could pose stress on Bacillus sp.P1,it would activate the antioxidant defense system to resist the oxidative damage.When the damage was too large to remit,the cells were injured or even dead.Because of immobilization,the biomass of Bacillus sp.P1 increased,and the detoxification indexes changed a lot.SOD activity,CAT activity and GSH content varied as the accumulation of Cd(II).SOD activity was the most sensible index among the three indexes.The carrier of immobilization slightly promoted efflux mechanism of Bacillus sp.P1.The carrier could immobilize exopolysaccharide and other secretions,which increased Cd(II)adsorption.As time went by,the saccharides content and extracellular protein secreted by immobilized Bacillus sp.P1 were higher than suspended Bacillus sp.P1,and Cd(II)concentration was lower in immobilized system than the suspended system.E xtracellular secretions were much more in immobilized cells,which helped with pollution restoration for both heavy metals and organic matters.In the fourth section,Bacillus sp.P1 and biochar were combined to restore the soils polluted with PHE and Cd(II).Bacillus sp.P1 was immobilized on biochar for a synergistic effect.The adsorption of PHE by biochar amended soils in the presence of Cd(II)was investigated.Kinetic equation and pH effect on this adsorption process were also detected to study solo effect of biochar on PHE-Cd(II)polluted soil.Besides,the solo and combination effects of Bacillus sp.P1 and biochar on PHECd(II)soil amendment were also analyzed.And interaction mechanisms of microbes with biochar were summarized.The results showed that the adsorption rate of PHE by biochar amended soils increased as time went by.The largest adsorption amount occurred in the fourth hour,the adsorption equilibrium could be reached in twenty-four hours.There was a competition effect between PHE and Cd(II).The adsorption processes of PHE by biochar amended soils fitted pseudo-second order kinetics equation.PHE adsorption rate decreased because of Cd(II)stress.Alkaline conditions promoted PHE adsorption in the presence of Cd(II),while the adsorption amount was the lowest in the neutral environment.Cd(II)decreased PHE adsorption amount in acidic conditions but increased PHE adsorption amount in alkaline conditions.Freely suspended Bacillus sp.P1 alone was not good at dealing with PHE-Cd(II)in soils.The combination of Bacillus sp.P1 and biochar enabled better PHE degradation than being used alone.However,biochar alone exhibited more excellent in Cd(II)adsorption and immobilization than utilizing the combination of Bacillus sp.P1 and biochar.