Mechanism Of Enhanced Pyrene Biodegradation By Bacteria Immobilized In Layer-by-layer Assembly Microcapsules

Immobilized microorganism is an effective strategy to solve the bad environmental adaptability of free microbes in the actual remediation progress.Traditional microbial immobilization methods all have defects in some degrees which are adverse for microbial degradation of pollutants.As a result,the development of novel immobilized microorganism technology possessing excellent protection effect and mass transfer performance is of great significance for practical application of microorganism in the environmental remediation.This paper used polycation electrolyte（chitosan,CHI）and polyanion electrolyte（alginate,ALG）for immobilized material,and a light yellow mycobacterium（a kind of pyrene-degrading bacteria strain）CP13 as experiment microorganism species.Using the alternate deposition of the two kinds of electrolyte material on CaCO3 templates,the layers of self-assembly（layer by layer assembly,LBL）microcapsules were prepared and CP13 were encapsulated.On the base of the comparing remediation effect of pyrene in soil by CP13 immobilized using the microcapsule and traditional method,adaptive performance of the LBL microcapsule immobilized CP13 studied in four kinds of environmental factors,such as different initial pyrene concentrations,p H,temperatures and salinity,as well as the soil microbial community change during soil bioremediation were comprehensive investigated.The mechanism of promoting the pyrene remediation effect of the CP13 by the LBL microcapsule immobilization and its ecological risk for soil were also discussed.Meanwhile,the interaction between extracellular polymeric substances（EPS）of the CP13 and composition material of the LBL microcapsule（CHI and ALG）was also investigated and the mechanism of promoting adaptive performance to the environment of the CP13 by the LBL microcapsule was also disscussed.The main conclusions and innovations of this paper are as follows:1.Using the LBL self-assembly technique and the CaCO₃ template particles to preapare LBL CHI/ALG microcapsule successfully,as well as using the LBL CHI/ALG microcapsule for immobilization of the microorganism CP13.The micro-structure characteristics and basic properties of LBL microcapsule was investigated.Results show that overall shape of the microcapsule was like a flake,with 200-400 nm height and 3-4 μm length.The microcapsule has porous structure,mainly are mesoporous,and pore diameters are concentrated in the 2-50 nm.Compared with the free bacteria,LBL microcapsule immobilized CP13 showed stronger environmental adaptability to environmental factors,and could adapt to the higher initial concentration of pyrene,more wide pH and temperature range and higher concentration of salt stress environment.The reasons for these results may be as follows.One reason is the exesiting of the encapsulation microbial capsule wall material（polyelectrolyte）between the external environment and the bacteria have the effect of barriers for degrading bacteria,and the capsule wall is helpful to maintain the pH inside of bacteria,avoiding bacteria acidification and also causing the microbes to adapt to a wider range of temperature.The other reason is the capsule wall material has porous properties,causing high porosity of the prepared LBL microcapsule,so as to ensure the good delivery of oxygen and the free diffusion of the substrate and the products,allowing the substrates and products move freely in and out through the micropores in the capsule wall.So that it can avoid accumulation of the toxic products,which is advantageous to the survival,growth,reproduction and metabolism of the immobilized bacteria.2.Soil remediation experiments revealed that the LBL-immobilized cells could cause a significant increase in the biodiversity of the bacterial community,PYR removal efficient,soil enzyme activity and the number of PYR-degrading bacteria in the soil,which were good for improving the soil ecological quality.Proteobacteria and Actinobacteria were observed as the predominant groups during bioremediation in the LBL groups.The active uncultured bacteria belonged to Xanthomonadaceae,Planococcaceae,Pseudomonas,Mycobacterium,Sphingomonadaceae,Acinetobacter,Flavobacterium,Comamonadaceae,Bacillus,Sphingobacterium,Enterobacteriaceae,and Streptomyces,while the latter two classes had rarely been associated with PAH-degrading ability.Among them,the Pseudomonas、Bacillus、Sphingobacterium and Comamonadaceae are widely reported as PAH-degrading bacteria.In the LBL treatment,we also found some famous photosynthesis bacteria,such as Chloroflexus,which kind of bacteria can produced O₂,is conducive to microbial metabolism of pollutants.LBL microcapsule immobilized bacterium improved soil bacterial diversity,further confirmed that the process of removing pollutants in soil.There is a strong correlation between the pollutants degradation and bioremediation efficiency and the microbial species occurred and the soil microbial diversity.3.Appling the flow cytometry method,cell physiological characteristics of the CP13 in the forms of free bacteria and LBL microcapsule immobilized in the process of the pyrene degradation were analyzed,respectively,revealing the protection mechanism of the LBL microcapsule on the microorganisms.During degradation process of pyrene,pyrene and its products could continuously act on the cell surface of free CP13,leading to certain damage to membrane structure,increased membrane permeability,and reduced membrane potential,or even making the cell membrane ruptured,membrane integrity badly damaged and eventually killed the degrading bacteria.LBL microcapsule immobilization could effectively slow down the increase velocity of cell membrane permeability,the lower velocity and amplitude of the membrane potential,cell death rate and the number of dead cells.So,the microcapsule immobilization of the degrading bacteria exists obvious protective effect for the cells.4.Using the three dimensional fluorescence spectrum,circular dichroism spectrum,infrared spectrum and laser light scattering analysis,the interactions between the EPS of the bacteria CP13 and LBL microcapsule materials（CHI and ALG）were studied.The research results showed that there is combination ability between the CHI in the LBL microcapsule and protein components of the EPS,generating stable ground state substance,which resulted in fluorescence quenching phenomenon.This suggests that the impact by LBL microcapsule on the microorganism is mainly acted through interaction between the CHI and the protein component of the EPS,CHI-EPS compounds generated from which showed sharp increase of average molecular weight than the original EPS,expanded the Z-mean square rotating radius and the hydraulic radius of the original EPS,reduced the internal density of the EPS,and made the EPS structure stretched and configuration loosed,resulting in raising the gaiden mass transfer performance of the microbial cell and the capture efficiency of pollutants,eventually making the degrading performance of the bacteria improved.The LBL self-assembly microcapsule technology for degrading bacteria immobilization developed in this paper can provide technical support for the microbe bioremediation technology application in environmental remediation.