The Mechanism Study Of Amino Acids Or Carbon Nanotubes Amendment On Biodegradation Activities Of An Alkane Degrading Strain GS3C

Bioremediation of petroleum-contaminated soil is the use of microorganism metabolism to remove petroleum hydrocarbons. Compared with other technologies, bioremediation is often considered as a cost-effective and no secondary pollutant technology. However, the fate of petroleum hydrocarbons in the environment is largely controlled by abiotic factors, which influence rates of microbial growth, and enzymatic activities that determine the rates of petroleum hydrocarbon utilization. Special attention should be piad on the approach that how to enhance bioremediation efficiency by abiotic factors optimization.In this study hexadecane was used as a representative petroleum alkane, and the following issues were studied: (1) enrichment, isolation and identification of hexadecane degrading microorganisms; (2) characterization of hexadecane degradation by the degrading strain, and the effect of initial hexadecane concentration, pH, salinity and hevavy metal on biodegradation activity; (3) the effect of nutrient amendment on biodegradation and mechanism of biodegradation promotion which stimulated by amino acids amendment; (4) the effect of carbon nanotubes on hexadacane biodegradation and its cytotoxicity; (5) environmental chemical behavior and cytotoxicity mechanism of carbon nanotubes.An n-alkane-degrading strain GS3C was isolated from petroleum-contaminated soil and was identified by API test and 16S rRNA gene sequencing techniques. The strain GS3C was identified to be Burkholderia cepacia. Hexadecane can be degraded by the strain form 750 mg/L to 200 mg/L in 4 days. Study on growth factor of strain GS3C indicated that strain GS3C could grow well at neutral and initial carbon source concentration from 75.8 to 18950mg/L. Biodegradation activity was inhibited at 2% salinity or higher, slightly promoted by 9 mg/L Cd²⁺, and inhibited by 200 mg/L Cu²⁺ or higher. Degradation efficiency was promoted greatly (form 51.8% of control to 99.5%) with 500 mg/L yeast extract amendment. But biodegradation acitivity was almost completely prohibited with 10 mg/L carbon nanotubes amendment.Biodegradation promotion were not significant with carbon sources, nitrogen sources or vitamins amendment. The biodegradation activity was only promoted with the addition of amino acids. Further study indicated that L-phenylalanine, L-glutamic acid, L-proline, L-lysine, L-valine, L-leucine and L-phenylalanine were active ingredient in yeast extract. And mixed amino acids were more effective on biodegradation promotion. The mechanism of biodegradation activity promotion with amino acids amendment was investigated by microbial enzymology. It was also shown that cytochrome P450 monooxygenase was promoted by amino acids amendment. The concentration of cytochrome P450 in strain GS3C was increasing promoted with higher L-phenylalanine amendment. Meanwhile, hexadecane biodegradation was promoted via terminal oxidation which stimulated by cytochrome P450. A significant positive linear relationship between cytochrome P450 activity and biodegradation efficiency of GS3C was observed. The results indicate that amino acid is the primary factor of nutrient amendment in promoting hexadecane biodegradation by promoting cytochrome P450 activity in GS3C.In the presence of NADH as electron donor, obvious DNA cleavage into form?(nicked DNA) was observed in dark condition after 4 hours incubation. The DNA cleavage intensity caused by different carbon nanotubes followed as covalent functionalized carbon nanotubes > uncovalent functionalized single walled carbon nanotubes (SWNTs). And in the same condition, DNA cleavage intensity caused by carboxylated single wall carbon nanotubes (SWNT-COOH) was higher than polyethyleneglycol-lated single wall carbon nanotubes (SWNT-PEG). The results help to understand the cytotoxicity and potential environmental risk of carbon nanotubes.Considering the DNA cleavage effect by carbon nanotubes, cytotoxicity mechanism was also studied. It was shown that carbon nanotubes were efficient intermediate to trigger electron transfer from electron donor to O₂ in dark condition which leads to superoxide radical (O₂^-·) formation. The electron transfer intensity caused by different functionalized carbon nanotubes also followed as covalent functionalized SWNTs > uncovalent functionalized SWNTs. The reason is that Stone-Wales defect sites which changed from sp²-hybridized carbon atom on side wall of carbon nanotubes were more reactive than perfect one. But electron transfer efficiency of SWNT-COOH is better than more functionalizaed SWNT-PEG. It's possible that COOH groups on side wall of SWNT-COOH have a larger amount of positive charge and are more electrophilic. The amount of O₂^-· formation was increased with increasing NADH and carbon nanotubes concentration, in which the effect of NADH dose on O₂^-· formation is more significantly. It is possible that the accumulation of O₂^-· lead to the formation of highly reactive hydroxyl radical (·OH), which eventually leads to DNA cleavage and cytotoxicity. The results help to understand the mechanisms of how biodegradation activity of strain GS3C was inhibited by carbon nanotubes.