process on wastewater containing tetracycline resistance genes Dr - PowerPoint PPT Presentation

13th IWA Specialized Conference on Small Water and Wastewater Systems 5th IWA Specialized Conference on Resources-Oriented Sanitation Removal efficiency of Fenton oxidation process on wastewater containing tetracycline resistance genes Dr Ping Zeng Chinese Research Chinese Research Academy of Environmental Academy of Environmental Sciences Sciences 2016.09.16

Content Content Introduction Materials and Methods Results and Discuss Conclusions

Background for ARGs Recently, the abuse of tetracycline for human, veterinary and agricultural purposes has resulted in the development of tet resistance bacteria and tet genes (tets), which is a sever threat to human health. WHO Annual Pruden and Attention for tet genes Rysz defined Report on Infectious ARGs as “contaminants” Disease: Being 。 Overcoming detected in Antimicrobial various Resistance environments over the worldwide scale 2000 now time 2004-2006

Introduction Introduction Tetracycline is one of the most commonly used therapeutics in human and veterinary medicine. There are at least 45 different tetracycline resistance genes(tet) classes.

Introduction Available technology Wastewater treatment Ozone: shifting technology :unsteable, supercoiled plasmid tet genes accumulation DNA to open circular plasmid DNA UV disinfection :bacteria chlorine :the possible may recover replication leakage of resistance activity under visible light by carrier elements and the DNA repair enzyme produce the resistance to photolyase chlorine Fenton: disrupt of double- and single-stranded nucleic acids, oxidation of proteins (key enzyme inhibition) and amino acids and have inefficient knowledge

Materials and Methods

Initial conditions of SBR effluent SBR/MBR 出水 膜组件 出水泵 进水泵 (MBR) Parameters Value 曝气 TOC(mg/L) 14.216 16SrRNA(log(copies/mL) 7.68 进水桶 tet(A)(log(copies/mL) 5.76 tet(C) (log(copies/mL) 6.29 tet(M) (log(copies/mL) 1.56 tet(G) (log(copies/mL) 5.85 tet(X) (log(copies/mL) 6.72

Information of target genes and 16SrRNA Primer Sequence （ 5’ - 3’ ） Target Genes Melting Amplicon Size Temperature （ o C ） （ bp ） 16SrRNA CCTACGGGAGGCAGCAG 53 169 ATTACCGCGGCTGCTGG （ F357/R518 ） tet （ A ） GCTACATCCTGCTTGCCTTC 55 210 CATAGATCGCCGTGAAGAGG tet （ C ） CTTGAGAGCCTTCAACCCAG 55 418 ATGGTCGTCATCTACCTGCC tet （ G ） 55 468 GCTCGGTGGTATCTCTGCTC AGCAACAGAATCGGGAACAC tet （ M ） GTGGACAAAGGTACAACGAG 55 406 CGGTAAAGTTCGTCACACAC tet （ X ） CAATAATTGGTGGTGGACCC 55 468 TTCTTACCTTGGACATCCCG

Fenton oxidation process Q-pcr for ARGs Single factor experiment TOC,DOM and Results and DGGE for the discussion optimal parameters

Results and Discussion

Operation Conditions of Fenton Oxidation Reaction . 7H 2 O dosage(mmol/L) Serious number pH H 2 O 2 dosage(mol/L) FeSO 4 time A1 1 3 0.2 30 A2 5 3 0.2 30 A3 10 3 0.2 30 A4 30 3 0.2 30 A5 60 3 0.2 30 A6 10 2 0.2 30 A7 10 3 0.2 30 A8 10 5 0.2 30 A9 10 8 0.2 30 B1 10 3 0.05 30 B2 10 3 0.1 30 B3 10 3 0.2 30 B4 10 3 0.25 30 B5 10 3 0.3 30 B6 10 3 0.2 20 B7 10 3 0.2 25 B8 10 3 0.2 30 B9 10 3 0.2 35 B10 10 3 0.2 40 Raw water 0 0 0 0

Fenton reaction on TOC and DOM 16 370 16 370 TOC TOC TOC removal rate 14 14 TOC removal rate 360 PeakB 360 PeakB 12 12 PeakT PeakT PeakB and PeakT fluorensence intensity PeakB and PeakT fluorensence intensity 350 10 10 350 TOC and TOC removal rate 8 TOC and TOC removal rate 8 340 200 6 6 200 4 4 0.6 0.6 150 150 0.4 0.4 100 100 0.2 0.2 0.0 50 0.0 50 raw water 1 60 5 10 30 raw water 2 3 5 8 (a)Time (min) (b)pH 16 370 16 370 TOC TOC TOC removal rate 14 14 TOC removal rate 360 PeakB PeakB 12 12 PeakT 360 PeakT 10 PeakB and PeakT fluorensence intensity PeakB and PeakT fluorensence intensity 350 10 8 TOC and TOC removal rate TOC and TOC removal rate 350 8 340 6 200 250 6 4 0.8 200 0.6 150 0.6 150 0.4 0.4 100 100 0.2 0.2 0.0 50 0.0 50 raw water 0.05 0.2 0.25 0.1 0.3 25 20 30 40 raw water 35 (c) H 2 O 2 dosage(mol/L) (d)Fe 2+ dosage (mmol/L) Optimum conditions for organic removal: t=5 min, pH=8, Fe 2+ dosage=20mmol/L, H 2 O 2 dosage=0.1mol/L

Analysis of DGGE imagine similarity （ CS （ % ）） Sample A1 88.7 A2 78.0 The lower similarity, the more A3 49.5 DNA was destroyed A4 79.6 A5 77.2 A6 84.5 A7 50.0 A8 76.5 A9 66.6 13 Raw water 100.0

Analysis of DGGE imagine Similarity （ CS （ % ）） Samples B1 49.6 B2 45.7 Optimum conditions: B3 66.9 B4 57.7 t=10min, pH=5, B5 55.7 Fe 2+ dosage=30mmol/L, B6 53.4 H 2 O 2 dosage=0.2mol/L B7 46.0 B8 50.8 B9 49.6 B10 87.7

Effect of Fenton reaction on target genes,16SrRNA raw water Genes concentration （ log(copies/ml) ） 8 treated 7 6 5 4 3 2 1 0 16S tet(A) tet(C) tet(M) tet(G) tet(X) target genes • The removal rate of target genes absolute concentration almost excessed 99%, except tet(M) of 48%. • In log(copies/ml), tet(A), tet(C), tet(X) and tet(G) all have been eliminated 2.4-4.5logs. • Fenton regent not only can disinfect antibiotic resistance bacteria inactive, also can oxidize genes conformation.

Effect of Fenton reaction on different resistant mechanisms Antibiotic mechanism : target genes • Efflux pump proteins: tet(A) 、 tet(C) 与 tet(G), log （ tetX ） log （ tetM ） log(tetC ） log （ tetG ） log(tetA) • Eencoding ribosomal protection proteins (RPPs): tet(M) 6% • Encoding an inactivating 22% 65% 12% enzyme: tet(X) 27% 24% 68% 26% 18% 20% 23% 22% raw water treated • The tatol DNA reduced indicated by the ring • Efflux pump proteins: tet(A) 、 tet(C) 与 tet(G), the ring and the ratio reduced • Encoding an inactivating enzyme: tet(X), the ring and the ratio reduced

Conclusion

Conclusion  At the optimal parameters of pH=3, 0.2 mol/L H 2 O 2 with 30 mmol/L . 7H 2 O reacting for 30 min, 99% of absolute gen was FeSO 4 removed,  DGGE files have illustrated bacterial community diversity and equitability had decreased after Fenton oxidation, the similarity is only 49.5% after treatment to raw water.  Comparing different mechanism, Fenton regent have stronger damage on that genes encode efflux pump proteins and modified enzyme and have relative week damage effect on that genes that encode ribosomal protection proteins. 18

Thank you! zengping@craes.org.cn