Journal of Biodegradation (2013 ) Irmene Ortı´z Antonio Velasco Sylvie Le Borgne Sergio Revah

1. Biodegradation of DDT by stimulation of indigenous microbial populations in soil with cosubstrates Journal of Biodegradation (2013) Irmene Ortı´z Antonio Velasco Sylvie Le Borgne Sergio Revah Speaker: Shin-Yu, Lin & Yen-Wei, Lin

2. Introduction

3. DDT • 1874 synthesized. • 1930s large-scale worldwide production (restricted to malaria). • 1960s prohibited.

4. DDT • persistence in soils (>10 years) • lipophilic http://case.ntu.edu.tw/hs/wordpress/?p=8445

5. Biodegradation of DDT http://case.ntu.edu.tw/hs/wordpress/?p=8445

6. Biodegradation of DDT Soil solidification,flushing, incineration, absorption (Castelo-Grande et al. 2010; DallaVilla and PupoNogueira 2006; Purnomo et al. 2010;Shareefand uzZaman 2010; Tian et al. 2008; Waltersand Aitken 2001﹔Aislabie et al. 1997; Castelo-Grande et al.2010; Foght et al. 2001; Kamanavalli and Ninnekar2004; Kantachote et al. 2004; Purnomo et al. 2008;Purnomoet al. 2010; Zhao et al. 2010)

7. Cosubstrates • Single microbial sp. is hard to take DDT as a carbon source →co-metabolism • Add cosubstrate → promote microbial growth, induce participating enzymes

8. Cosubstrates trichloroethylene (TCE) toluene Co-metabolism (toluene monooxygenases ) Biodegradation

9. Objective To evaluate the degradation and mineralization of DDT, DDD and DDE in microcosm experiments using different cosubstratesto stimulate indigenous microbial populations in soils.

10. Materials and Methods

11. Soil : • Test: AddToluene, Hexane, Phenol, Acetic acid, Ethanol and Glucose, respectively • Control: without cosubstrates • Abiotic control: Sterile (All with equivalent doses of DDT, DDD and DDE, and quantified CHNS analyzer) Sample from rural areas in the state of Chiapas, Mexico.

12. Mineral medium: Triple 17, NutrimentosMinerales de Hidalgo, Me´xico • Microcosm experiments: 實驗七周，每周加入 cosubstrate 125mL flask with 5g polluted soil, 10mL medium and cosubstrate

14. Influence of the concentration • Toluene, control, abiotic control *測量Toluene 消耗與CO2產生量(扣除因分解Tolune)

15. Analytical Methods • Gas Chromatography 移動相 固定相

16. Analytical Methods • DDT, DDD, DDEextraction • Sonication 利用超音波打碎細胞，萃取其中DDT等物質 • DNAanalysis • UltraCleanSoil DNA isolation kit→PCR →pGEM-T vectorinto E.coli (電穿孔) →sequencing→CLUSTAL W →分析微生物多樣性

17. Results and Discussion

18. Effect of different cosubstrates tested on DDT degradation and CO2 production Acetic acid have the complete inhibition of DDT degradation. DDTdegradation was enhanced by the presence of cosubstrates.

19. Degradation of DDT with different cosubstrate dose 69% Control : C Phenol : P Toluene : T 25% P T C P T P T The strongest effects on DDT degradation was obtained with → the intermediate concentration of toluene → the higherconcentration of phenol

20. Carbon dioxide production as a function of different amounts of toluene added 3.32 mg Carbon/g Drysoil 1.43 mg Carbon/g Drysoil 0.79 mg Carbon/g Drysoil Higher CO2 production was observed in the treatments with higher amounts of toluene added. This higher microbial activity did not result in a higher DDT consumption.

21. DDT degradation in the presence of toluene 0.64 mg Carbon/g Drysoil 2.6 mg Carbon/g Drysoil The degradation started after 400 h. No further degradation after 900 h. The cosubstrate addition affected the activity of the microbial community.

22. DGGE analysis of bacterial communities

23. The bacterial diversity was low in this soil, probably due to the toxicity of DDT and its metabolites. • The Azoarcusgenus comprises species able to degrade toluene and other aromatic compounds in the presence of oxygen or nitrate(Song et al. 2000; Van Schie and Young 1998; Zhou et al. 1995).

24. Conclusions

25. There was a stimulatory effect of adding phenol, hexane and toluene as cosubstratesto degrade DDT, DDE and DDD. • An optimal range lie between the low and intermediate concentrations (0.64 and 2.6 mg Carbon/g Drysoil). • The process had a lag phase of approximately 400 h. • An Azoarcuslike bacterium could provide broad-specificity oxygenases enhancing the degradation of DDT.

26. Comment • 目前尚未知微生物是如何降解DDT,其過程機制為何 • 未來期望能了解輔助碳源如何參與微生物降解過程,以及了解其中的調控機制 • 未來期望可純化DDT降解酵素,以使其效能更廣泛

27. Reference • http://www.sonicator.com/ • http://www.genscript.com/dna_purification.html?src=google&gclid=CLKRh6KmubcCFUNipQod4TsA5w • http://tw.knowledge.yahoo.com/question/question?qid=1508021503835 • http://www.aandb.com.cn/script/edfile/2008/10/20081016-222221-46115394.pdf

28. Thanks for your listening~