Bioadsorption, Uptake And Biodegradation Of Polycyclic Aromatic Hydrocarbons By PH Microbial Consortium

With the increase of demands for energy, a large number of petroleumhydrocarbons enter our environment in the process of exploitation, transportation andrefining, especially oil spills accidents frequently occur, posing serious threat tomarine life, environment and human health, and destroying the ecological balancewhich may take years or even decades to recover. In the face of polycyclic aromatichydrocarbons which is difficult to degrade after weathering and has carcinogenesis,tetratogenesis and mutagenesis, and the special marine environment such as lowtemperature, lack of nutrition and high salt concentration, this paper preliminarydiscussed the mechanism of bioadsorption, uptake and degradation for petroleumhydrocarbon, in particular the polycyclic aromatic hydrocarbons, and mimicked theoil spill bioremediation process by immobilized microorganism technology toenhance the biodegradation of polycyclic aromatic hydrocarbons. All the conclusionsare as follows:(1) A group of polycyclic aromatic hydrocarbons degrading bacteria named asPH microbial consortium was isolated from oil contaminated sludge in Shenglioilfield. The degradation rate of crude oil is60%. Through the morphological,physiological and biochemical identification of bacteria and16S rDNA gene sequenceanalysis, it showed that PH-1, PH-2, PH-3and PH-4respectively were affiliated toPseudomonas sp.(KF113575), Bacillus sp.(KF113576), Ochrobactrum sp.(KF421109) and Pseudomonas sp.(KF113577), respectively.(2) The most suitable environmental conditions for PH microbial consortiumdegrading different substrates were different. The most suitable environmentalconditions for PH microbial consortium degrading NAP were NAP concentration800mg/L, pH=7.5, NaCl concentration10g/L, temperature35oC; that degrading PHEwere PHE concentration40mg/L, pH=7, NaCl concentration5g/L, temperature30oC; that degrading PYR were PYR concentration100mg/L, pH=7, NaCl concentration15g/L, temperature30oC; that degrading crude oil were crude oilconcentration3g/L, pH=8, NaCl concentration7g/L, temperature30oC. Thebiodegradation rate of NAP, PHE, PYR, and crude oil at respective appropriateconditions were80%,30%,56%and48%, respectively.(3) PH microbial consortium performed high resistance for heavy metals ion.They were tolerant to6.2mM Cu2+,2.7mM Zn2+and9.5mM Pb2+. Biodegradationrates of NAP, PHE, PYR and crude oil were53%,21%,32%and44%in the presenceof heavy metal (Cu2+,1.7mM and Zn2+,2mM), respectively.(4) In the presence of co-metabolic substrates such as glucose, α-lactose, solublestarch, yeast powder and urea, α-lactose was the best co-metabolic substrate in theprocess of degradation of petroleum hydrocarbons. The PH microbial consortiumbiodegradation rate of NAP, PHE, PYR and crude oil was95%,68%,60%and95%under co-metabolism conditions, respectively.(5) The stability of surface adsorption and cell uptake by live microbialconsortium followed a decreasing order of NAP> PHE≈PYR> crude oil. Theadsorption by heat-killed microbial consortium was constant for PAHs, whiledecreased for crude oil.(6) The hydrophobicity of PH microbial consortium was higher than35%performing strong degradation activity and emulsifying properties; the microbialhydrophobicity was associated with the substrate concentration, toxicty and microbecounts; the metabolic efficiency of periplasmic, cytoplasmic and extracellularenzymes secreted by PH microbial consortium for diverse substrates was different.(7) The cell ultrastructure of PH-3cultivating in beef extract peptone, crude oilor PAHs medium was observed by transmission electron microscope. These resultssuggested that the bacteria cell absorbed and took in crude oil and PAHs by changingthe permeability of cell membrane and cell wall, and then degrade them possibly bythe cytoplasm (including internal organelles and enzyme, etc.).(8) The biosorption processes of NAP, PHE, PYR and crude oil by walnut shelland semicoke followed the pseudo-second order kinetic formula. The smaller theconcentration was, the better it fitted. The factor importance order of influencing the preparation of immobilized microorganism by different carrier materials was diverse.Explored on the preparation of immobilized microorganism and biodegradation rateof crude oil exhibited that the preparation and importance of immobilized microbialconsortium by walnut shell were walnut shell (20%)> CaCl2(1%)> α-lactose(0.1%)> SA (6%); that by semicoke were α-lactose(0.3%)＞semicoke (20%)＞SA(7%)＞CaCl2(3%); that by activated carbon were α-lactose (0.5%)> CaCl2(5%)>activated carbon (20%)> SA (4%). Under the best degradation conditions, thedegradation rate of crude oil by the immobilized bacteria (48%) was higher than thatof free bacteria by21%after5d.(9) The functional groups on microbial consortium with different treatment andits strength were diverse. The microbial consortium cells had carboxyl, amino andphosphate etc. functional groups. The biodegradation process passed throughbiosorption and uptake of pollution stages. The structure of semicoke became biggerporous and easily adhered with microbial consortium after immobilization.(10) Custom-designed devices with artificial and natural seawater, chemicaltreatments (added oil dispersant), and free and immobilized microbial consortiumenlarging indoor flask experiments were applied to mimic marine oil-spillbioremediation. The results showed that the PH microbial consortium mainlyconsumed nitro nitrogen and phosphorus source; Comparing with free microbialconsortium, the samples of immobilized microbial consortium had more bacterialcount and higher biodegradation rate of dissolved crude oil, and needed more nutrient.PH PAHs-degrading bacteria had obvious degradation efficiency on five kinds ofalkyl aromatics; the degradation rate of NAP series in natural seawater rate byimmobilized microbial consortium was higher than50%; the degradation rates ofPHE, dibenzothiophene, fluorene and chrysene series were higher than30%, thebiodegradation rate of total aromatics reached at63%; Biodegradation efficiency inartificial seawater was better than that in natural seawater. To get excellent efficiency,adding bioremediation agents, phosphorus source and nitrate nitrogen source werenecessary. The experimental results have great significance for understanding howmicrobial consortium adsorb, take in and degrade contaminated substrates. Moreover,they provide theoretical and technical support for the feasibility of the immobilizedPAHs degrading bacteria agent applicated in oil spill bioremediation.