Impact of Low Herbicide Concentrations on Microbial Communities in the Vejen Aquifer

1. de Lipathayet al. 2003. In situ exposure to low herbicide concentrations affects microbial population composition and catabolic gene frequency in an aerobic shallow aquifer. Appl. Environ. Microbiol. 69: 461-467.

2. Objective • To evaluate how the in situ exposure of the Vejen aquifer to low phenoxyacid herbicide concentrations affected the indigenous microbial community composition Aquifer = an underground bed or layer of permeable rock, sediment, or soil that contains water and releases it in appreciable amounts; water-bearing zone.

3. 2,4-dichlorophenoxyacetic acid (2,4-D) Mecoprop = 2-(2-methyl-4-chlorophenoxy)propionic acid Dichlorprop = 2-(2,4-dichlorophenoxy)propionic acid Aquifer contaminated with a mixture of mecoprop, dichlorprop, 2-,ethyl-4,6-dinitrophenol (DNOC), bentazone, isoproturon, 2,4-dichlorbenzamide continuously for 216 days, bromide as tracer

4. 40 µg/L of each herbicideinjected for 216 days, samples in Fig. 3 taken 40 days later

5. Techniques used • Enumeration • Metabolism

6. Techniques used • Enumeration • Cultivation: • Non-cultivation: • Metabolism • Degradation: • Mineralization:

7. Techniques used • Enumeration • Cultivation: CFUs, MPN • Non-cultivation: DAPI, molecular biology techniques 16S rDNA (MPN-PCR, general bacteria (PRBA338f and PRUN518r), Pseudomonas (PSMGf and 785r) • Metabolism • Degradation: MPN (25% contaminant disappearance), 16S rDNA(population with tdf gene via RFLP) • Mineralization: MPN (25% 14C-CO2)

10. Strengths and weaknesses of techniques • Microscopic – DAPI • Cultivation techniques • Water agar, Gould’s S1 • Phenoxyacid degraders • Molecular biology techniques • Bacteria, Pseudomonas, tdf A, B, C • Whole community DNA was extracted • Primers for ….

11. 40 days later

12. MPN-PCR

13. No Pseudomonas on Gould’s but detected with 16S rDNA Pseudomonas in acclimated sediment Role of Pseudomonas in degradation? Mineralization study?

14. MPN-25% degradation

15. MPN-25% degradation - RFLP patterns in B2 different from others - tdf no homology with known sequences

16. Restriction endonucleases Specific recognition sites 4-6 basepairs

17. To evaluate how the in situ exposure of the Vejen aquifer to low phenoxyacid herbicide concentrations affected the indigenous microbial community composition • Was objective achieved? • Continuous exposure can markedly affect composition of bacterial community of a subsurface aquifer • Increase in culturability, Pseudomonas, phenoxyacid degraders, bacteria with tdf genes

18. Phenoxyacid herbicides • 2,4-D = 2,4-dichlorophenoxyacetic acid • Mecoprop= 2-(2-methyl-4-chlorophenoxy) propionicacid • Dichlorprop = 2-(2,4-dichlorophenoxy)propionic acid

19. Stainless steel piston sampler