| Polycyclic aromatic hydrocarbons (PAHs) are a class of organic pollutants whichattract increasing attention in recent years for their toxic, mutagenic and carcinogenic, andwill produce great harm to ecological environment and human health. Bioremediation isconsidered as a cost-effective and environment-friendly process for removing PAHs and is amajor way in the environment. Although microbial remediation has been the principalmeans of PAHs contaminated site remediation, there are still some problems: the main focusof microbial degradation is more concentrated on pollutants degradation and degradationefficiency, etc., and usually only understand the status of the degradation process andmicrobial growth from the macroscopic angle, they cannot reflect how the characteristics ofbiodegradation microorganism cells change during the process. The physiological state ofthe microorganism cells, metabolism and reproductive capacity has a decisive role for theefficiency of pollutants degradation. Therefore, it is necessary to investigate the state ofindividual bacteria cells during degradation process, and this will provide a better way forunderstanding microbial degradation mechanism, so as to give full play to the degradationability of microorganism.Thus, this study attempts to combine flow cytometry and fluorescent dyes to detect themembrane properties, bacterial activity and the growth state of efficient phenanthrenedegrading bacteria GY2B (Sphingomonas sp. GY2B) during the process of degradation. Atthe same time, we also analyzed the functional groups and zeta potential of bacterial cells’surface, investigated the changes of individual GY2B cell cultivated under differentsubstrate condition as well as detected the effects of phenanthrene degradation products acton its degradation bacteria GY2B. This helped understanding the effects which acted ondegradation bacteria by pollutants during the degradation process and provided bettertheoretical fundament for microbial bioremediation. The main results were as follows:1) Flow cytometry instrument combining with fluorescent dyes can be widely appliedin the field of environmental microbiology. The application of bacterial cell membrane permeability and the cell’s state detection provides the more in-depth research on thesingle-cell level of bacteria, so as to better understand the degradation process of thebacteria.2) As the degradation process of phenanthrene, it continued to damage the GY2Bbacterial cell membrane surface, which resulted in increased cell membrane permeability,made a certain damage to membrane structure. When cultivated under differentconcentrations of phenanthrene, GY2B cell membrane permeability increased as thedegradation of phenanthrene.3) When the initial concentrations of phenanthrene is in a certain range, the increase ofmembrane permeability will be good for GY2B to degrade the phenanthrene. But with theincrease of phenanthrene concentration, the impact which acted on the membranepermeability of degrading bacteria by phenanthrene will gradually deepened, the higherconcentration of phenanthrene can induce a swelling phenomenon of GY2B membrane, thismade Zeta potential value reduced and increased the proton permeability permeability.When concentration accumulated to a certain extent is instant pore structure, cell membraneintegrity was badly damaged and GY2B bacterial cells dissolved and dead.4）When the initial concentration is low, as the degradation process the phenanthrenecan produce intermediate metabolites1-hydroxy-2-naphthalene acid and salicylic acid, butits concentration is low, and the two kinds of metabolites were eventually fully biodegradedduring the degradation process. Under the condition of low concentration of intermediatemetabolites, these two kinds of intermediate metabolite can be used as carbon source topromote GY2B breeding rather than affect reproductive capacity of bacteria GY2B, andalso greatly increase the proportion of active cells during the process of degradation.5） At the condition of higher phenanthrene initial concentration, as the degradationprocess,1-hydroxy-2-naphthalene acid accumulation in great quantities, while the salicylicacid was accumulated at a lower concentration. High concentrations of intermediatemetabolites of Sphingomonas sp. GY2B had toxic effect, they can restrain the growth of thebacteria GY2B. Although GY2B bacteria can make better use of1-hydroxy-2-naphthalene acid, in1-hydroxy-2-naphthalene acid system active cells accounted for a small proportion,suggesting that1-hydroxy-2-naphthalene acid has a stronger toxicity.The accumulation of1-hydroxy-2-naphthalene acid in the degradation process will lead to the large numbersdeath action of Sphingomonas sp. GY2B. The accumulation of salicylic acid concentration,although less than1-hydroxy-2-naphthalene acid, but it also can produce certain toxic effectand kill bacteria GY2B, making the degradation efficiency reduced. |
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