Studies On Tobacco Wastewater Treatment As Well As Its Microbiological Mechanism

With the aims of tobacco wastewater treatment and nicotine degradation, bioaugmentation using Pseudomonas sp. HF-1 with high nicotine-degrading efficiency in sequencing batch reactor (SBR) system and Fenton oxidation was applied combinedly and the ecological safety of these treatments was evaluated. Then the ecological mechanisms of Pseudomonas sp. HF-1 for bioaugmentation in SBR system and the molecular mechanisms for nicotine degradation by strain HF-1 were discussed. The main results of this study were as follows:(1) The highly effective nicotine-degrading; bacterium Pseudomonas sp. HF-1 was augmented in an SBR system used to treat tobacco wastewater. Compared to the non-bioaugmented (non-BA) system, the bioaugmented (BA) system exhibited considerably stronger pollutant disposal abilities, with 100% nicotine degradation rate and more than 84% chemical oxygen demand (COD) removal rate within 12 h. Nicotine degradation had a significant effect on COD removal in SBRs (r=0.928, p<0.01).(2) The conditions for Fenton oxidation of SBR effluent were optimized by Box-Behnken experiment design. When pH 4.13-4.66, Fe2+concentration of 5.30-6.09 mmol/L,30% H2O2≥8.20 ml/L, the color removal rate was 96.03±2.57%, while COD decreased from 1100.33±82.80 mg/L to 73.67±19.70 mg/L, with its removal rate being 93.30±2.92%. The quality of the final effluent of tobacco wastewater after treatment had hit the State's first-class standard for the industrial discharge (<100 mg/L COD, GB8978-1996).(3) Both the contents of hydroxyl free radical during the whole process of Fenton oxidation and the toxic effects of final effluent on microorganisms were monitored. The result showed that the content of hydroxyl free radical in final effluent was 0.383±0.52 U/mL. There were no inhibition of final effluent on growth of E. coli, P. putida, Pseudomonas sp. HF-1, Acinetobacter sp. TW and Sphingomonas sp. TY. No significant differences of the contents of SOD, CAT and MDA were observed in strain HF-1 cultured in final effluent inorganic salt media (ISM) compared to that in sterile water ISM (p>0.05). Thus, the combination of strain Pseudomonas sp. HF-1 bioaugmentation in SBR and Fenton oxidation was one of the ecological safety strategies for tobacco wastewater treatment.(4) The mechanisms of bioaugmentation were systematically investigated using a combination of polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) and a toxicity assay [protein carbonyl (PC) and DNA-protein crosslinking (DPC)]. DGGE fingerprint profiles showed that the number of bands and the Shannon-Wiener index decreased at a nicotine load of 250 mg/L compared to a 40-130 mg/L nicotine load in the non-BA system. However, a stepwise increase of the Shannon-Wiener index was found during all periods in the BA system. A comparison of sequences excised from DGGE gels demonstrated there were significant differences in the dominant microbial species between the two SBRs. This result suggested that bioaugmentation with strain HF-1 could select cooperators to treat complicated tobacco wastewater, resulting in enhancing the whole disposing efficiency. The PC content and the DPC coefficient increased significantly at levels higher than 80 mg/L nicotine in the non-BA system; nevertheless, no increasing was observed in the BA system during the stepwise increasing nicotine load. This indicated that bioaugmentation using strain HF-1 resulted in the maintenance of high treatment activity by minimizing the nicotine toxicity to other microbes in the BA system. It could be seen clearly that the rapid nicotine degradation of strain HF-1 performed a vital function in SBR by influencing the microbial community structure and the dynamics and activity of the activated sludge system.(5) Nicotine degrading genes were demonstrated to locate on the plasmid but not on the chromosome of Pseudomonas sp. HF-1 via various methods including plasmid elimination, plasmid transformation, and PCR and RT-PCR amplification of key nicotine degrading gene hsp. The specific plasmid was named as plasmid pMHl, and sequenced by SEFA-PCR, showing the size of 23315bp. It was different from the well-known nicotine-degrdating plasmid pAO1 with size of 165 kb in Arthrobacter nicotinovorans. The analysis of the content of G+C mol% showed that pMH1 could be stable and permanent kept in strain HF-1, even without nicotine stress. The analysis of bioinformatics showed that genes encoding for replication and signal transduction locate on the positive strand of plamisd pMHl while that encoding for nicotine degradation and horizontal transfer do on the negative one. The nicotine degrading genes refer to the gene hsp and gene amo. Gene hsp may encode the enzyme catalyzing 6-hydroxy-3-succinoylpyridine to 2,5-dihydroxypyridne. Gene amo may encode the enzyme catalyzing pseudooxynicotine to 3-succinoyl-pyridine and methylamine. There were two fragments of gene hsp in pMHl. Therefore, it can be reasonably speculated that the relative small size, stable and permanent maintaince of plasmid pMHland repetitive nicotine degrading genes fragments in pMH1 may be the main molecular mechanisms for rapid and long-term effective degradation of nicotine by strain HF-1 in tobacco wastewater treament.