Studies On Characteristics Of The Secretion Of Extracellular Small Active Compounds By P.aeruginosa NY3 And Their Promotion Mechanisms To The Efficiency Of Biodegradation Of Hydrocarbon

Pseudomonas aeruginosa,with a high capacity of degradation of hydrocarbons,is ubiquitous in petroleum contaminated environment.P.aeruginosa NY3 was once reputed to be oil-degraded microorganism because of its ability to metabolize concentrated hydrocarbons and other hydrophobic pollutants.P.aeruginosa NY3 has been found to secret abundant pigment substances in their extracellular liquid under certain conditions,especially when hydrocarbons or other persistent organic pollutants were used as growth carbon sources.And also it was observed that the removal efficiency of the target contaminants by strain NY3 was closely related to the production of these extracellular pigments.Until now,studies on hydrocarbon degradation genes and enzymes have been well documented.While the effects of secretion of extracellular active substances on the biodegradation of pollutants were rarely studied.This thesis pays close attentions to the characteristic of the secretion of small active substances by P.aeruginosa NY3 and have studied their promotion mechanisms of degradation of hydrocarbons.The petroleum hydrocarbon and some of its components were selected as tested contaminants.Liquid-liquid and liquid-solid extraction was employed as the samples pretreatment method.And with the help of such measuring methods as electron spin resonance(EPR),LCMS-IT-TOF,gas chromatography(GC),scanning electron microscope(SEM),UV-Vis spectroscopy,infrared spectrum(FTIR),as well as modern molecular biology methods,we carried out our experiments.The main achievements of our studies could be summarized as follows.(1)Under the conditions of hydrocarbons or their transformed metabolites as growth carbon sources,P.aeruginosa NY3 could secrete four phenezines,including pyocyanin(Pyo),1-Hydroxyphenazine(HPE),phenazine-1-carboxylic acid(PCA),and phenazine-1-amide(PCN)simultaneously.Both the rate of cell growth and the degradation efficiency of hexadecane were improved by the secretion of these phenezines.For the first time,the mechanism of promotion of the hydrocarbon degradation by extracellular secreted phenezines was revealed as: extracellular phenezines reacted with NADH or GSH could produce superoxide anion radical(·O2-)and hydroxyl radical(·OH)or hydroxyl radical(·OH),which in turn attacked and degraded hydrocarbons.And appropriate ferric ion could increase the degradation efficiency of hexadecane by 20-29 %.Such process of hydrocarbon degradation was proved to be through free radical oxidation,similar to Fenton reaction except that here the pH value was neutral or weak alkaline.(2)P.aeruginosa NY3 secreted two kinds of stereoisomers of pyochelin(PCH),which is a double-edged sword for hydrocarbon degradation.In culture medium and under aerobic conditions,PCH,coupling or chelating with ferric ion,was found to be able to degrade hexadecane.The degradation was proved to initiate with the hydroxyl radical(·OH)produced by the reaction of PCH and Fe3+,and the degradation was also through free radical mechanism.However,excessive secretion of PCH was unfavorable for the degradation efficiency of hydrocarbon because of its depressing effects on the strain NY3 cell growth.(3)Small organic acids could be produced as intermediates from hydrocarbon degradation by P.aeruginosa NY3.All the extracellular acids,like glutaric acid,succinic acid,malonic acid,and acetic acid,was found to be able to increase the growth rates of P.aeruginosa NY3 and concomitantly to promote the hexadecane degradation efficiency,while extracellular oxalic acid was with contradiction effects.It could inhibit the cell growth,but promote the degradation efficiency of hexadecane.Further study indicated that,promotion mechanisms for co-existance small organic acids on hydrocarbon degradation were different.The promotion effects of glutaric acid were through up-regulated the expression levels of alkB1 and alk B2 gens.In this way,activities of intracellular monooxygenase was increased,and in turn,theintracellular oxidation of hydrocarbons was promoted.At the same time,co-existing glutaric acid could increase the secretion amount of phenazines which could promote the extracellular hydrocarbon degradation through free radical pathways.While oxalic acid increased the degradation efficiency of hydrocarbon by improving the secretion of phenezines,especially PCA,to promote the extracellular hydrocarbon degradation through free radical pathway.Secretion of phenazines under the condition of co-existance of oxalic acid may result in additional 26-28% degradation rates of hexadecane by extracellular liquid.(4)The formation of biofilms of P.aeruginosa NY3 on the surface of polyurethane foam(PUF)was quickly and easily.Under the conditions of 12 h hydraulic retention time,the average removal rate of oil contaminates of 2 g/L in wastewater by the biofilms could maintained above 87 % within the running period of40 d.After the degradation,and means of by aeration,the activity of used biofilms could be recovered rapidly with the help of glycerol.(5)The new processing method for treatment of oil sludge from petrochemical enterprise by P.aeruginosa NY3 was established.By improving mass transfer manner of hydrophobic contaminants and by increasing the synthesis of intracellular lipopolysaccharide,Rhamnolipid could increase the activity of cells of strain NY3,and in turn,improve the efficiency of degradation of hydrocarbons,especially for the long chain alkanes.Under conditions of rapid oil degradation,part of oils could be converted to a kind of un-harmful sticky biopolymer being composed by sugar and protein.This glycoprotein existing in the system of strain NY3 could in turn promote the degradation of oil contaminants.Briefly,in this thesis,two classes of free radical producing substances from the extracellular liquid of P.aeruginosa NY3 were extracted and identified.And the free radical mechanisms of the oxidation-degradation of oil contaminants outside cells of strain NY3 caused by the secreted active substances were revealed.The two different promotion effects of small dicarboxylic acids intermediates on alkane degradation were pronounced.The treatment methods of concentrated oil-containing wastewater with biofilms of P.aeruginosa NY3 and of oil sludge with free cells of P.aeruginosa NY3 were establish.Meanwhile,the promotion mechanism of extracellularrhamnolipds on hydrocarbon degradation was studied.This study has revealed the diverse mechanisms of degradation of oil contaminants by P.aeruginosa NY3.