| Fluoronitrobenzenes (FNBs), due to their "simulation effect", "electronic effect", "block effect", and "penetration effect", have become important intermediates for the manufacturing of synthetic pesticides, pharmaceuticals, dyes and fine organic chemicals. With their extensive use, FNBs enter into the environment, causing the air, soil and water pollution. However, less knowledge is available about their environmental behavior, fate and the control technology. In view of this,2-fluoronitrobenze (2-FNB),3-fluoronitrobenze (3-FNB),4-fluoronitrobenze (4-FNB),2,4-difluoronitrobenze (2,4-DFNB) and 2,3,4-trifluoronitrobenze (2,3,4-TFNB) were chosen as the targeted pollutants in this research work. Firstly, the anaerobic biotransformation pathways of five FNBs and the microbial community structures were studied by batch experiments. Then, aerobic biotransformation pathways of the anaerobic biotransformation products from five FNBs and microbial community structures were examined. Finally, based on the above results, the anaerobic and aerobic system was used to study FNBs removal efficiency and their effects on the stability of system. The main results obtained are as follows:(1) During about 250 d acclimation,2-FNB,3-FNB,4-FNB,2,4-DFNB and 2,3,4-TFNB could be transformed into the corresponding fluoroanilines (FAs) without any lag, but were not further transformed except 3-FNB. The kinetic tests showed that the maximum transformation rates of 2-FNB,3-FNB,4-FNB,2,4-DFNB and 2,3,4-TFNB were (21.21±1.73), (32.14± 2.33), (21.33± 2.48), (33.89± 6.87), and (10.87±0.84) mg FNB (gVSS h)-1, respectively.According to the results of GC-MS and HPLC analysis, a possible pathway for 3-FNB biotransformation was proposed:3-FNB biotransformation via 3-fluoroaniline, 4-amino-2-fluorobenzoic acid,4-amino-2-fluorobenzoyl and benzoic acid, successively. The denaturing gradient gel electrophoresis (DGGE) analysis demonstrated that Methanosaeta thermophila, Methanothrix soehngenii and Clostridium sp. possibly played the important role in 3-FNB biotransformation via defluorination.(2) Under aerobic conditions, the removal efficiencies of five main FNBs anaerobic biotransformation products, including 2-fluoroaniline (2-FA),3-fluoroaniline (3-FA),4-fluoroaniline (4-FA),2,4-difluoroaniline (2,4-DFA), and 2,3,4-trifluoroaniline (2,3,4-TFA), reached more than 85% after acclimation of 58 d,43 d,26 d,51 d, and 165 d, respectively. The kinetic tests showed that the maximum transformation rates of 2-FA,3-FA,4-FA,2,4-DFA, and 2,3,4-TFA were (21.23±0.91), (11.75±0.99), (15.27 ±2.04), and (8.84±0.93) mg FA (g VSS h)-1,respectively. The biotransformation rates were closely related with the hydrophobic parameters, electrical parameters and thermodynamic parameters.The catechol 2,3-dioxygenase (C2,3-DO) was the main dioxygenase enzyme for 2-FA,4-FA,2,4-DFA, and 2,3,4-TFA with biotransformation main amount of (80.99 ±0.07), (84.66±0.39), (73.79±8.62), and (18.69±1.37) U (mg protein min)-1, respectively. The dioxygenase enzyme for 3-FA biotransformation was catechol 1,2-dioxygenase(C1,2-DO), with the amount of (13.83± 0.44) U (mg protein min)-1. Also, the five FAs enriched cultures showed certain potential of degrading other four FAs; when FAs coexisted with aniline, the biotrasformation rates of five FAs decreased. Obviously, the dioxygenase enzyme induced by FAs has diversity feature.The DGGE analysis revealed that unique bacterial communities were formed after five FAs enrichment. The biotransformation of 2-FA could be associated with Novosphingobium sp., Bradyrhizobium sp., Aquaspirillum sp., Aminobacter sp., Ochrobactrum sp., and Labrys sp.; the biotransformation of 3-FA could be associated with Variovorax sp., Aquaspirillum sp., Bradyrhizobium sp., and Lachnobacterium sp.; the biotransformation of 4-FA could be associated with Brevundimonas sp., and Rhodanobacter sp.; the biotransformation of 2,4-DFA could be associated with Thauera sp. and Acidovorax caeni;the biotransformation of 2,3,4-TFA could be associated with Williamsia serinedens and Ralstonia sp. The Shannon-Wiener indexes in five FAs enriched culture decreased with the increase of fluorine substitution, indicating the significant effect of fluorine substitution on the microbial diversity.(3) The Upflow Anaerobic Sludge Reactor (UASB) and Moving Bed Biofilm Reactor (MBBR) combined system was used to study 2,4-DFNB removal efficiency and its effect on the stability of system. The results showed that the highest 2,4-DFNB volume loading rate reached 0.54 kg FNB m-3 d-1 when CODcr volume loading and HRT were 5.4 kg CODcr m-3 d-1 and 20h, respectively.With the gradual increase of 2,4-DFNB volume loading, the stability of system was degraded. When 2,4-DFNB volume loading reached 0.84 kg FNB m-3 d-1, the effluent CODcr increased from (100.92±14.36) mg L-1 to (418.65±43.88) mg L-1, and also the effluent 2,4-DFA concentration increased from (12.69±1.74) mg L-1 to (163.16± 13.70) mg L-1, while the nitrification was completely inhibited. The instability of UASB was due to the complex inhibitory effect of low pH value and the high 2,4-DFNB concentration in the reactor, while the MBBR instability was due to the inhibiting effect of 2,4-DFNB and its intermediate products. The microbial community structures in UASB reactor were analyzed by using High-throughput Sequencing Technology. When the stability of anaerobic system was destroyed, the percentages of Desulfovibrio genus, Smithella genus, Methanosaeta genus, Bacteroidetes phylum and Chloroflexi phylum decreased.(4) Two FAs degrading strains, designated as JF-3 and DF-3, were obtained from 3-FA and 4-FA enrichment culture by the dilution plate method, respectively. The isolates were identified and classified on the basis of 16S rRNA gene sequence analysis. The strain JF-3 belonged to the genus Rhizobium, and the strain DF-3 belonged to the genus Ralstonia. The kinetic tests showed that the strains JF-3 and DF-3 were with the largest biotransformation rates of 67.66 and 106.20 mg (g dry cell min)-1, respectively.According to the results of GC-MS detection and the type of dioxygenase enzyme, the possible pathways for 3-FA and 4-FA biotransformation were proposed:3-FA (4-FA) biotransformation via 3-aminophenol (4-aminophenol), resorcinol (hydroquinone), hydroxyhydroquinone,4-oxo-2-hexene acid,3-ketaoadipic acid and succinic acid, successively.The results made clear FNBs biotransformation pathways and mechanism under anaerobic and aerobic environment, and also provided the theoretical basis and technical support for FNBs pollution control. |
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