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

Removal efficiency of Fenton oxidation process on wastewater containing tetracycline resistance genes

13th IWA Specialized Conference on Small Water and Wastewater Systems 5th IWA Specialized Conference on Resources-Oriented Sanitation

Dr Ping Zeng

Chinese Research Chinese Research Academy of Environmental Academy of Environmental Sciences Sciences

2016.09.16

Introduction Materials and Methods Results and Discuss

Content Content

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.

Attention for tet genes 2000 2004-2006 now time

WHO Annual Report on Infectious Disease: Overcoming Antimicrobial Resistance Pruden and Rysz defined ARGs as “contaminants” 。 Being detected in various environments

• ver the

worldwide scale

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.

UV disinfection :bacteria may recover replication activity under visible light by the DNA repair enzyme photolyase

Ozone: shifting supercoiled plasmid DNA to open circular plasmid DNA chlorine :the possible leakage of resistance carrier elements and produce the resistance to chlorine

Available technology

Wastewater treatment technology :unsteable,

tet genes accumulation Fenton: disrupt of double- and

single-stranded nucleic acids,

• xidation of proteins (key enzyme

inhibition) and amino acids and have inefficient knowledge

Introduction

Materials and Methods

Initial conditions of SBR effluent

Parameters Value TOC(mg/L) 14.216 16SrRNA(log(copies/mL) 7.68 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

SBR/MBR 出水 膜组件 (MBR) 曝气 进水泵 进水桶 出水泵

Information of target genes and 16SrRNA

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

Single factor experiment TOC,DOM and DGGE for the

• ptimal

parameters Q-pcr for ARGs Results and discussion

Fenton oxidation process

Results and Discussion

Operation Conditions of Fenton Oxidation

Serious number Reaction time pH H2O2 dosage(mol/L) FeSO4

.7H2O dosage(mmol/L)

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

Fenton reaction on TOC and DOM

0.0 0.2 0.4 0.6 4 6 8 10 12 14 16 TOC and TOC removal rate

(a)Time (min)

TOC TOC removal rate raw water 1 5 10 30 60 50 100 150 200 340 350 360 370 PeakB PeakT PeakB and PeakT fluorensence intensity 0.0 0.2 0.4 0.6 4 6 8 10 12 14 16 TOC TOC removal rate TOC and TOC removal rate

(b)pH

raw water 2 3 5 8 50 100 150 200 350 360 370 PeakB PeakT PeakB and PeakT fluorensence intensity 0.0 0.2 0.4 0.6 0.8 4 6 8 10 12 14 16 TOC TOC removal rate TOC and TOC removal rate

(c) H2O2 dosage(mol/L)

raw water 0.05 0.1 0.2 0.25 0.3 50 100 150 200 250 350 360 370 PeakB PeakT PeakB and PeakT fluorensence intensity 0.0 0.2 0.4 0.6 6 8 10 12 14 16 TOC TOC removal rate TOC and TOC removal rate

(d)Fe2+ dosage (mmol/L)

raw water 20 25 30 35 40 50 100 150 200 340 350 360 370 PeakB PeakT PeakB and PeakT fluorensence intensity

Optimum conditions for organic removal: t=5 min, pH=8, Fe2+ dosage=20mmol/L, H2O2dosage=0.1mol/L

13

Analysis of DGGE imagine

Sample similarity（CS（%）） A1 88.7 A2 78.0 A3 49.5 A4 79.6 A5 77.2 A6 84.5 A7 50.0 A8 76.5 A9 66.6 Raw water 100.0

The lower similarity, the more DNA was destroyed

Analysis of DGGE imagine

Samples Similarity（CS（%）） B1 49.6 B2 45.7 B3 66.9 B4 57.7 B5 55.7 B6 53.4 B7 46.0 B8 50.8 B9 49.6 B10 87.7

Optimum conditions: t=10min, pH=5, Fe2+ dosage=30mmol/L, H2O2dosage=0.2mol/L

Effect of Fenton reaction on target genes,16SrRNA

16S tet(A) tet(C) tet(M) tet(G) tet(X) 1 2 3 4 5 6 7 8 raw water treated Genes concentration（ log(copies/ml)）

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
• xidize genes conformation.

Effect of Fenton reaction on different resistant mechanisms

26% 6% 22% 24% 22% 68%

target genes

log（tetX） log（tetM） log(tetA) log(tetC） log（tetG） raw water treated

23% 12% 27% 18% 20% 65%

Antibiotic mechanism:

• Efflux pump proteins:

tet(A)、tet(C) 与tet(G),

• Eencoding ribosomal

protection proteins (RPPs): tet(M)

• Encoding an inactivating

enzyme: tet(X)

• 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

18

Conclusion

At the optimal parameters of pH=3, 0.2 mol/L H2O2 with 30 mmol/L FeSO4

.7H2O reacting for 30 min, 99% of absolute gen was

removed, DGGE files have illustrated bacterial community diversity and equitability had decreased after Fenton oxidation, the similarity is

• nly 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.

Thank you!

zengping@craes.org.cn