Characteristics, Mechanism And Process Of Mtbe Degradation By Methylibium Petroleiphilum PM1

Methyl tert-butyl ether （MTBE）, a gasoline additive, has been widely used in the past 30 years to enhance the octane level of gasoline and improve air quality. However, the contamination of ground and surface water with MTBE has evoked substantial attention due to the frequent occurrence of storage tank leakage. With its high water solubility, poor adsorption, and recalcitrance to biodegradation, MTBE pollution spreads widely in the groundwater. Biodegradation of MTBE by Methylibium petroleiphilum PM1 was investigated in this paper, and the method to efficiently degrade MTBE was explored based on the characteristics and pathway of MTBE metabolism. Moreover, cells were immobilized in gel beads and subsequently operated in a packed-bed reactor to degrade MTBE continuously.The feasibility of MTBE degradation by M. petroleiphilum PM1 in poor nutrition solution was discussed. The results showed that cells with high concentration could degrade MTBE in poor nutrition solution. MTBE degradation was significantly promoted by the addition of K⁺ or Ba²⁺. The optimum pH was 7.0 for PM1, and phosphate buffer exhibited negative effect on MTBE degradation. Oxygen was essential to the degradation, and the value of K_m, K_s and V_max were 0.73mmol/L, 7.0mmol/L and 0.14mmol/（L·h）, respectively. The success of MTBE degradation by PM1 cells in real contaminated groundwater demonstrated its feasibility to biodegrade MTBE under poor environment.MTBE could be mineralized by PM1 growing cells in mineral salts medium （MSM） with a low biomass yield （0.21g/g MTBE）. The intermediates such as tert-butyl alcohol （TBA）, 2-hydroxy isobutyrate （HIBA） and formic acid were detected during MTBE degradation. The metabolic pathway of MTBE degradation by PM1 was not via the pathway involving isopropanol and acetone on the basis of the results that isopropanol showed absolute resistance to biodegradation by PM1 and that MTBE degradation was inhibited completely by the addition of isopropanol. The cell concentration played an important role in the TBA accumulation which was affected by the MTBE and TBA degradation rate. There were some differences in electrophoresis of proteins and MTBE degradation rates of cells grown on MTBE, TBA and ethanol, respectively.The degradation of MTBE could be enhanced by the addition of readily metabolizable organic substrates. The addition of most of the substrates had no or negative effect on MTBE degradation, whereas yeast extract, beef extract and tryptone showed positive effect. The supplemented yeast extract might be used as a substrate to enhance the propagation of cells harbouring the related degrading enzymes, whereas an inducing period was required to initiate the enhancement of MTBE degradation. The mixture of yeast extract, beef extract and tryptone had a better effect, and the proportion was optimized by response surface methodology. The optimum concentration of the above substrate was 48.7mg/L, 49.6mg/L and 39.2mg/L, respectively. As a result, MTBE degradation rate of 1.90mg/（L·h） was achieved, in comparison with 1.11mg/（L·h） when carried out in medium without addition. Strain Staphylococcus aureus, which could not degrade MTBE as the sole carbon source, were able to cometabolized MTBE with 25.5% removal efficiency when yeast extract was present.Alginate immobilized M. petroleiphilum PM1 could degrade MTBE efficiently. The adaptable range of immobilized cells on pH value, temperature and initial MTBE concentration was widened. The storage stability of MTBE degradation activity was increased by immobilization, and the half-life for the encapsulated cells stored at 28℃was about 120h, which was obviously longer than that of free cells （36h）. Efficient reusability of the beads up to 30 batches was achieved in micro-nutrition solution （MNS） as compared to only 6 batches in MSM. The gel bead immobilized with calcium alginate was stabilized to improve its structural strength and reusability. The optimum concentrations of Ca²⁺ in the crosslinking solution and in the reaction medium were 3% （v/w） and 2mM, respectively. Powdered activated carbon, diatomite, and glutaraldehyde were not suitable to work as the hardening agents. Beads treated with polyethyleneimine （PEI） could degrade MTBE at a rate of 5.79mg/（L·h）, and could be proceeded for more than 50 cycles. The biochemical reaction rather than intraparticle diffusion resistance was considered as the key step by the kinetic analysis.The packed-bed reactor with the PEI stabilized beads to degrade MTBE continuously was developed. It could be operated for 50d with high removal efficiency （more than 96%） at an influent concentration of 10mg/L, hydraulic retention time of 3200s, and a dissolved oxygen concentration of 4mg/L. The result of microbial community DNA profiling determined by denaturing gradient gel electrophoresis （DGGE） indicated that PM1 was the predominant bacterium during the whole stable operation.