| Polycyclic aromatic hydrocarbons (PAHs) are a class of organic pollutants which attractincreasing attention in recent years for their widespread occurrence in the environment. PAHsare of great environmental and human health concerns since many PAHs have been shown tobe toxic, mutagenic and carcinogenic, and can be accumulated and transferred throughout thefood chain. Bioremediation is considered as a cost-effective and environment-friendly processfor removing PAHs and is a major way in the environment. Though a large number ofbacteria capable of biodegrading PAHs have been isolated from the natural environment,there are still some problems. The bacteria utilizing one kind of PAHs as the sole carbonsource isolated from environment usually have high selectivity for compounds of PAHs andare not very flexible to the changes of their environmental variables. In addition, thehigh-efficiency bacteria can be constructed by mixing the dominant bacteria simply. However,the optimum combination of a variety of bacteria is a very complex and difficult subject. Forexample, it is difficult to achieve the maximum efficiency because of the interspecificinhibition, and some metabolites produced during the degradation process are more toxic thanPAHs themselves. Therefore, it is urgent to obtain a high-efficiency PAHs-degradingbacterium with high environmental adaptability and by which less or even non toxicmetabolites are produced during the degradation process.The purpose of this study was to construct a high-efficiency PAHs-degrading bacteriumby protoplast fusion between Sphingomonas sp. GY2B and Pseudomonas sp. GP3A, which isflexible to the changes of their environmental variables, and capable of degrading multiplePAHs simultaneously. To improve the formation and regeneration frequency of protoplasts forGramnegative bacteria, the effects of some factors on protoplast isolation and regenerationwere investigated in present study. The degradation capability and environmental adaptabilityof the fusant strain were also tested by comparing with its parents and other strains. Finally,the PAHs degradation mechanism by the fusant strain was analyzed and discussed. The mainresearch results are as the followings:(1) Pretreatment with penicillin G sodium salt and EDTA enhanced the protoplastformation of Bacterial strains of GY2B and GP3A. The optimal condition for GY2B andGP3A protoplast formation were in5mg/ml lysozyme at37°C for80min and100min in ashaking culture, respectively. The protoplast formation frequency was95.2%and99.6%forGP3A and GY2B in shaking cultures, while only28.1%and34.2%in the static culture. Areasonable concentration of calcium ions, magnesium ions and L-serine increased the regeneration frequency of protoplasts. Double layers culture (soft-agar overlayers) methodcould also significantly increase the regeneration frequency as compared with single or mixedculture method.(2) The study on antibiotic resistance showed that fusants of GP3A and GY2B could bescreened using regeneration culture containing80g/ml piperacillin and80g/ml ceftazidine,or80g/ml piperacillin and erythrocin (100~150g/ml). Fusants were screened successfullythrough this method. Then the selected strains were inoculated in solid mineral mediumconsisting of single phenanthrene and pyrene. Subsequently, the strains with obvious clearzone were inoculated in liquid mineral medium containing phenanthrene and pyrene forscreening. A fusant strain with the highest degradation ability of phenanthrene and pyrene wasselected and named F14. The results of colony morphology, electron microscope, scanningelectron microscope (SEM) and molecular technology (PCR-RFLP) indicated that fusantstrain F14was different from its parents and was the fusant strain of GP3A and GY2B.(3) Phenanthrene (100mg/L) could be almost completely degraded within24hours by thefusant strain F14, which was much quicker than GY2B and other strains. The fusant strainF14had a wider range of temperature (25-40°C) and pH value (6.5-9.0). Non-competitivesubstrate inhibition kinetics was found to be fit for phenanthrene biodegradation and thebiodegradation rate constant k was134.77mg phenanthrene g/X/h the saturation constant KSwas77.50mg/L and inhibition constant KSIwas742mg/L. A larger k and KSIvalue indicatesthat the culture could endure high concentration of phenanthrene. F14could degrade about46%,37.1%and18%pyrene after10d inoculation at the initial concentration of15,50and100mg/L, respectively. The degradation of phenanthrene and pyrene in mixture compoundsystems showed that phenanthrene degradation was clearly delayed in the presence of pyreneand pyrene degradation was appears enhanced in the presence of phenanthrene.F14has different degradation pathway with GY2B. There are two pathway of degradingphenanthrene by F14: under the effect of dioxygenase, phenanthrene was cleaved andsubsequently metabolized to cis-3,4-phenanthrenedihydrodiol andcis-1,2-phenanthrenedihydrodiol, then transformed to phenanthrene-3,4-diol andphenanthrene-1,2-diol. Phenanthrene-3,4-diol was then cleaved to1-hydroxy-2-naphthoicacid, which was further decarboxylated to1-naphthol.1-Naphthol was cleaved toacetylsalicylic acid. Phenanthrene-1,2-diol was cleaved to methyl2-hydroxy-1-naphthoic acid,which was transformed to acetylsalicylic acid. Acetylsalicylic acid was transformed tosalicylic acid, which was further transformed to catechol and then accessed TCA-cycle andfinally exhaustive degraded to CO2and H2O. The toxicity of2-hydroxy-1-naphthoic acid was less than1-hydroxy-2-naphthoic acid indicating that phenanthrene was metabolized through apathway having less accumulation of potentially toxic metabolites by F14. An intermediateproduct4,5-dihydropyrene was detected during the degradation of mixture of phenanthreneand pyrene by F14.The degradation tests of substrates diversity were evidenced that strain F14could usenaphthalene, salicylic acid,2-hydroxy-1-naphthoic acid and catechol as the sole carbon sourceexcept o-phthalate and quinol. The experiment of degrading mixed PAHs showed that thepresence of pyrene or mixture of fluorene, anthracene, and phenanthrene could enhance thedegradation of acenaphthene. The presence of other three PAHs could promote thedegradation of anthracene and fluorene, respectively. However, the presence of pyrene andother six PAHs could inhibit the degradation of anthracene and fluorene, respectively. Pyrenedegradation was enhanced in the presence of low-ring PAHs. F14could degrade the mixtureof naphthalene, phenanthrene and pyrene, the mixture of acenaphthene, anthracene,phenanthrene and fluorene, the mixture of naphthalene, acenaphthene, anthracene, fluorene,phenanthrene, fluoranthene and pyrene. |
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