PAH-degrading Microbial Isolation,Characterization And Performance Of Pah Removal In Plant

Polycyclic aromatic hydrocarbons(PAHs),a category of persistent organic pollutants(POPs)that are widespread in the terrestrial environment,pose a grave concern to many countries globally.Under natural conditions,PAHs in soil can be removed by volatilization,photolysis,chemical oxidation,microorganism degradation and the absorption and accumulation by plants.PAHs have limited ability to absorb and accumulate PAHs and the PAHs accumulated would do harm to the plants.In the practical application,bioremediation is the main way is to remove the PAHs in soil.However,the biodegradation of PAHs during bioremediation is severely limited by their low bioavailability raised from low aqueous solubility and high sorption to soil particles.Microbial degradation and microbe-aided PAH removal techniques are considered as viable routes in mineralization and detoxification of PAHs.These organic pollutants have caught global attention due to their potential toxicity,carcinogenicity,teratogenicity,and mutagenicity.In microbiology,the degradation pathways of aromatic compounds are traditionally studied from a single bacterial strain that is isolated from and can degrade such pollutants.Some well-adapted microorganisms,such as the sphingomonads(a group of microbes belonging to the Family Sphingomonadaceae in a-Proteobacteria)and Mycobacterium are found in these PAH-contaminated environments.The first objective of this thesis was to investigate the characteristics of a sphingomonad isolate(Sphingobium sp.RS1),as well as analyze the metabolites involved in phenanthrene biodegradation and pyrene co-metabolism.Furthermore,the mycobacterial-aided removal of these compounds using ryegrass(Lolium multiflorum Lam)was investigated.In our research,we isolated an efficient polycyclic aromatic hydrocarbon(PAH)-degrading terrestrial bacterium RS1 from a PAH-contaminated site in Nanjing,China.With this strain,the bacterial growth characteristics,as well as the influences of environmental conditions on PAHs(phenanthrene and pyrene)degradation,were investigated.Furthermore,we inoculated Mycobacterium sp.strain ATCC PYR-GCK into the roots of Italian ryegrass(Lolium multiflorum Lam).With greenhouse experiments,the effects of PAH uptake and mycobacterial-assisted remediation of both PHE and PYR(in plant roots and shoots)on plant growth and PAHs uptake were investigated.The main results were shown as follows:(1)Based on the morphological characteristics and 16S rRNA sequence homology analysis,the phenanthrene-degrading isolate was identified as Sphingobium sp.RS1 within the a-Proteobacteria subdivision.The experiments on PAHs-degrading features and growth characteristics of this sphingomonad showed that strain RS1 can utilize phenanthrene as the sole carbon source.At 30? and 150 rpm,the degrading ratio of phenanthrene(100 mg.L-1)in the medium was 90%after 72 hours rotary culture and phenanthrene would be degraded totally within 7 days.The PAH-degrading rates increased,whereby,at 30? and 150(r/min),the Alphaproteobacterium isolate RS1 was also capable of cometabolizing high molecular weight PAH PYR supplemented with PHE,resulting in PHE mineralization and PYR degradation to minimal amounts,with the transient accumulation of catechol alone,identified using HPLC.Liquid chromatography-mass spectrometry(LC/MS)analysis indicated that PYR and PHE degraded to common intermediate and final metabolites by isolate RS1,revealing that the bacterium can take up versatile interconnected pathways.(2)At 30? and 150(r/min),the strain was also capable of cometabolizing high molecular weight PAH PYR supplemented with PHE,resulting in PHE mineralization and PYR degradation to minimal amounts,with the transient accumulation of catechol alone,identified using HPLC.Liquid chromatography-mass spectrometry(LC/MS)analysis indicated that PYR and PHE degraded to common intermediate and final metabolites by isolate RS1,revealing that the bacterium can take up versatile interconnected pathways.(3)The mycobacterial strain was tested in the endophyte-assisted remediation using ryegrass(Lolium multiflorum Lam)treatment of two polycyclic aromatic hydrocarbons(P YR and PHE)from the soil.Mycobacterium sp.strain ATCC P YR-GCK was colonized in ryegrass roots,The inoculation of plants with strain ATCC PYR-GCK reduced the risks associated with plant PAH contamination,based on observations of decreased concentration,accumulation,and translocation factors of phenanthrene and pyrene in ryegrass.The reduction of the two PAHs by the action of bacteria in the plant roots was confirmed by enzymes(polyphenyl oxidase and plant peroxidase)activity.Our results will have significant implications for the assessment of the environmental risks of PAHs and in finding ways to circumvent plant PAH contamination.This research further makes several noteworthy contributions to the existing literature especially in the areas of environmental microbiology,environmental pollution and microbial remediation in sphingomonads.This group,together with Mycobacterium,have adapted well to PAH-contaminated soil and plant environments compared to most bacterial genera that degrade them.It has equally provided some level of research validity for its theoretical review on the bioremediation and endophyte-aided phytoremediation of PAHs PHE and PYR,which has confirmed the understanding that cometabolism is a property that bacteria may portray to full effect,both in the laboratory(using sphingomonads)and pot tests(utilizing Mycobacterium).This study also made methodological contributions inLiquid Chromatography/Mass Spectrometry(LC/MS)and HPLC that will help to advance metabolite pathway research in the future.Finally,this study adds to the inadequate literature on mycobacterial phytoremediation,aimed at enhancing the knowledge on Mycobacterium interactions with plants and PAHs.This is based on the fourth chapter of this study.