Research On Performance And Mechanism Of Composite Microorganism Agent And Its High-Efficiency Microbial Strains Degrading BTX

In previous work, a solid composite microorganism agent (CMA) was successfully prepared through inoculating mixed culture on a specific carrier via fermentation process for removal of benzene, toluene, and o-xylene (BTX) compounds. The mixed microbial consortia proved to be efficient in decomposing BTX compounds according to the foregoing investigation. In the present work, the solid CMA and traditional activated sludge were compared for inoculating the BTFs. Two sets of BTF were started up in parallel. The start-up period and performance of the two sets of BTFs were investigated in detail. More importantly, the feasibility and potential of using the solid CMA as the mixed inocula of BTFs were evaluated through investigating the long-term performance of BTX-fed BTF inoculated with the CMA. The experimental results show that the start-ups were accomplished within 7days and 24 days for the CMA-BTF and AS-BTF, respectively. In addition, the growth of the biofilm attached on the carrier in MA-BTF was faster than that in AS-BTF. These results indicated that the start-up periond of CMA-BTF could be shorten greatly as compared to AS-BTF. The main parameters that affect the BTF's performance including inlet concentration, inlet loading rate, nutrient, empty bed retention time (EBRT), and mass transfer etc. At LR below 60.0g/m³Â·h the BTF can degrade all the three compounds effectively, for EBRTs of 90, 60, 45 and 30 s, respectively. The maximum EC of the BTF was 55.3, 73.6, 81.0, and 97.7g/m³Â·h at inlet BTo-X loads of 60.8, 92.4, 122.1, and 146.4g/m³Â·h , respectively. the results presented indicated that effective treatment of BTo-X mixtures could be obtained in the BTF system inoculated with the CMA.The optimum pH of the system was 6⁷; The carbon mineralization in the BTX -degrading BTF inoculated with the CMA was 77.5-78.4%, indicating that these compounds were eliminated mainly by aerobic degradation; An increase in the CO₂ production percentages of different sections was accompanied by increases in the EC. No various increases in pressure drop were observed during the whole operation, indicating that the BTF can obtain long-term stable operation. The BTX-degrading process of the system followed the Michaelis-Menten kinetic model, the rmax (specific maximum degradation rate per volum) values for benzene, toluene, and o-xylene were 140.4, 0.032, and 0.018g·m^-3·h^-1, respectively. Accordingly, the gas saturation constant were 1.021, 0968, and 0.601 g·m^-3.The strain byf-4 capable of degrading BTEX was isolated from CMA. Based on the previous identification data, this isolate was identified as Mycobacterium cosmeticum, which was a new strain able to degrade BTEX as individual compounds or a mixture effectively. This isolate could completely degrade B, T, E, and X as individual growth substrate. B and T were preferred to be degraded by byf-4 as compared to E and X . Studies to evaluate substrate degradation patterns in all possible BTEX combinations revealed that, in each of these mixtures, B was degraded fastest, followed by T, E and then X. Competition inhibition interactions were observed in multiple-substrate studies. The biodegradation rate of each BTEX in a mixture was lower than that of single substrate experiments. It was noted that o-xylene was inhibited by other substrate in a mixture seriously. In multiple-substrate studies, each of BTE compounds in binary or ternary mixtures was completely consumed, and a carbon recovery rate of more than 90.5% was obtained for these possible combinations, indicating all components of the mixture were simultaneously degraded and further mineralized or incorporated into cells by this isolate. However, carbon recovery values in other multiple-substrate combinations containing o-xylene were in the range of 78.6-81.2%. It seems that the presence of any of BTE compounds led to the incomplete metabolism of o-xylene in a mixture.The degrading process of the strain byf-4 followed the Haldane kinetic model. The maximum specific degradation rate for degrading benzene, toluene, ethyl benzene, and o-xylene were 0.518, 0.491, 0.443 and 0.422h^-1, respectively. Accordingly, the maximum specific growth rate was 0.352, 0.278, 0.172 and 0.136h^-1, respectively. The decay codfficient degrading benzene, toluene, ethyl benzene, and o-xylene were 0.003, 0.0036, 0.0042, and 0.0063h^-1, respectively; The yield coefficient was 0.6626, 0.5801, 0.5415, 0.5512mg/mg, respectively. The experimental data on the yield coefficient of the strain was consistent with that of the specific growth rate. The BTEX-degrading capacity of this isolate was higher than that previously reported. The excellent ability of byf-4 to degrade and mineralize a wide range of substrates displayed its potential use in bioremediation and waste treatment.The degrading enzyme genes of the strain byf-4 were also investigated in the present work through designing degenerate primers. A 351bp gene fragments were obtained by PCR. The identification analysis of the gene fragments shows that it may be the toluene dioxygenase (todC1) genes; furthermore, a 1609bp gene fragments were obtained by nested PCR and sequence recombinant, which was homologous to 90% with todC1 of Sphingomonas sp. Therefore, Presumably the todC1 was successfully cloned from strain byf-4. A pair of RT-PCR primers were designed and synthesized according to the known gene sequences in order to further verify the todC1 gene by RT-PCR. The experimental results show that the genes can be expressed on BTEX as growth substrates, further verifying the obtained genes were toluene dioxynase genes. These results indicated that the toluene dioxynase was possibly one of the key enzymes during the degradation of BTEX by strain byf-4.