Molecular Genetic Tools for Monitoring Ecosystems and the Environment

Transcription

1 Molecular Genetic Tools for Monitoring Ecosystems and the Environment Loring (Larry) Nies School of Civil Engineering Purdue University West Lafayette, Indiana

2 Current and Former Students: Brett. Baldwin, Matthew Mesarch, Jennifer Nebe, Leila Nyberg, Brett Sutton, and Victoria Waranowski Collaborators: Professor Cindy H. Nakatsu Professor onald F. Turco Department of Agronomy Purdue University Funding: The National Science Foundation

3 ationale Microorganisms have a Central ole in Ecosystems Microorganisms are Sentinel Organisms

4 What do we Monitor? Community Structure Diversity Assessment Population & Biomass Phylogenetic Identification Community Function Pollution Catabolism Biogeochemical Cycling Carbon Fixation

5 Several different methods have been used to: Monitor the Dechlorination of Chlorinated Solvents Assess the Effectiveness of Bioaugmentation (e.g. MTBE) Detect Pathogenic Microorganisms Assess BTEX Bioremediation

6 ES&T, 2002

7 Discovery of genes associated with toxicity of E.coli O157:H7 (Perna et al., 2001) One scientist reports that there are only 1,407 human pathogens. This includes viruses, bacteria, parasites, protozoa and fungi.

8 16S rna Gene-Based Detection of Tetrachloroethene- Dechlorinating Desulfuromonas and Dehalococcoides Species (AEM. Löffler et al., 2002)

9 Genome Sequence of the PCE-Dechlorinating Bacterium Dehalococcoides ethenogenes (Science, Seshadri et al., 2005)

10 Question? How does one evaluate whether Sparging, Bioventing, Blowing, Injecting, Diffusing, Compressing, or eleasing OXYGEN into the Sub-Surface Enriches for Aerobic Microbial Populations?

11 Answer It is difficult! We do not have good direct Biological assessment tools emediation Scientists & Engineers can use to : Optimize Aeration Systems Characterize Natural Attenuation

12 Project Objective: Develop Tools to Assess Aeration emediation Technology & Natural Attenuation at BTEX sites

13 Tool Oxygenase Gene Detection & Enumeration Approach: 1) Gene Selection 2) Primer Design 3) Pure Culture Evaluation 4) Laboratory Microcosm Evaluation 5) Field Evaluation

14 Targeted Oxygenase Genes for Monitoring BTEX Bioremediation MO PHE NAH H H H CH3 TOL CH2 CH3 TOD CH3 H H BPH H

15 Primer Design N.1 Type D Type N N.2 D.1 D.2 N.2.A N.2.B N.2.C D.1.A D.1.C D.1.B D.2.A D.2.B D.2.C NarAa hocococcus sp. NCIMB12038 IpbA1 Pseudomonas sp. J1 CumA1 P. fluorescens IPO1 EdoA1 P. fluorescens CA-4 IpbAa P. putida E204 BphA C. testosteroni B-356 BphA1 Burkholderia sp. JB1 BphA1 Pseudomonas sp. KKS102 BphA1 Pseudomonas sp. B4 BphA B. cepacia LB400 BphA1 P. pseudoalcaligenes KF707 BphA1. erythropolis TA421 BphA1. globerulus P6 BpdC1 hodococcus sp. M5 BphA1 hodococcus sp. HA1 IpbA1. erythropolis BD2 TcbAa Pseudomonas sp. P51 BedC1 P. putida ML2 TodC1 P.putida F1 PhnAc A. faecalis AFK2 NahAc Cycloclasticus sp. 1P-32 NahAc Cycloclasticus sp. W NahAc C. pugetti PS-1 NahAc P. stutzeri AN10 PahA3 P aeruginosa PaK1 NahAc P. putida NCIB9816 NahA3 P. putida BS202 NdoC2 P. putida ATCC17484 NahAc P. putida G7 PahAc P. putida OUS82 NagAc Pseudomonas sp. U2 DntAc Burkholderia sp DNT NahAc N. naphthovorans NAG-2N-126 NahAc N. naphthovorans NAG-2N-113 PhnAc Burkholderia sp. P007 TOD BPH1 BPH2 BPH3 & BPH4 BPH4 NAH Identify Subfamilies of elated Oxygenase Biphenyl Genes Dioxygenases Subfamilies are based on Substrate Specificity Toluene Dioxygenases Detects Catabolic Function, Naphthalene NOT Phylogenetic Identity Dioxygenases

16 Primer Pseudomonas putida G7 Pseudomonas putida F1 Pure Cultures Pseudomonas putida HS1 hodococcus erythropolis TA421 Pseudomonas aeruginosa JI104 Pseudomonas sp. CF600 NAH TOD TOL BPH4 MO PHE

17 Microcosm Studies Single Substrate Genes Enriched PHE Benzene BPH H H NAH H H

18 Microcosm Studies Single Substrate Genes Enriched PHE Toluene MO

19 Microcosm Studies Single Substrate Genes Enriched PHE m-xylene CH3 TOL CH2 MO

20 Microcosm Studies Single Substrate Genes Enriched PHE p-xylene CH3 CH2 TOL

21 Microcosm Studies Single Substrate Genes Enriched PHE o-xylene MO

22 Microcosm Studies Single Substrate Genes Enriched PHE Naphthalene NAH H H

23 Microcosm Studies Single Substrate Genes Enriched PHE Biphenyl BPH H H

24 Microcosm Studies - Summary Genes Enriched Substrate(s) Present 30% 20% 10% of unique isolates had isolates had PHE had and MO two one oxygenase additional and genes TOL PHE MO TOL NAH BPH Broad Aromatic Exposure Toluene &/or Xylenes m- &/or p-xylenes Benzene &/or Naphthalene Benzene &/or Biphenyl

25 DGGE Profiles of 16S rdna Fragments PC amplify V3 region of rna gene Increasing Denaturant Products with low G+C content Products with higher G+C content Products separated by sequence composition NOT size. Provides a fingerprint of bacterial community

26 DGGE Profiles of Soil Microcosms Unamended Naphthalene M M Shifts in microbial population during enrichment with aromatic hydrocarbons.

27 Naphthaleneutilizing Isolate Naphthalene Microcosm Duplicates (week 4) Linking Community Structure to Function DGGE profile of naphthalene microcosm showed enrichment. Naphthaleneutilizing Isolate NAH detected in microcosm DNA All naphthalene isolates contained NAH Two of the dominant subpopulations contain NAH Enrichment with naphthalene selects for NAH subfamily of oxygenase genes

28 Appearance of Phenol Hydroxylase DNA & mna gene copies in response to Benzene in soil 1.E E Copies, per g soil 1.E+06 1.E+05 1.E+04 1.E+03 1.E+02 1.E Benzene, ug/l 1.E Time, h DNA mna Benzene 0

29 Evidence for MNA in the Field Log BTEX (μg/l) >4 >3 >2 >1 n.d. OW-19 OW-20 OW-23 OW-21 Groundwater Flow Direction 0 80 Scale in feet OW-24 OW-22 OW-18 W-1 OW-5 OW-17 OW-12 OW-16

30 Genotypes in the Field Number of Oxygenase Genotypes n.d. OW-19 OW-20 OW-23 OW-21 Groundwater Flow Direction 0 80 Scale in feet OW-24 OW-22 OW-18 W-1 OW-5 OW-17 OW-12 OW-16

31 DGGE Profiles at BTEX Sites Marker OW-5 OW-24 W-1 OW-18 OW-17 MW-11 OW-23 MW-5 OW-12 MW-2 MW-12 MW-3 MW-7 OW-21 OW-19 OW-20 OW-22 MW-9 MW-10 MW-4 MW-6 OW-16 Marker Decreasing BTEX levels Down gradient Up gradient Enrichment evident in contaminated wells and downgradient wells at sites compared to little selection in sentinel and upgradient wells.

32 Gene Enumeration in the Field Log(Copy number/g soil) PHE MO Log(BTX)

33 At BTEX Concentrations >1 mg/l Many Genotypes are Detected Total BTEX Log (ug/l) Genotypes BTEX = 4.9 PHE MO NAH TOL BPH4 TOD BTEX = 4.7 PHE MO NAH TOL TOD BTEX = 4.5 PHE MO NAH TOL TOD BTX = 4.0 PHE MO NAH BEX = 3.8 PHE MO NAH BPH4 BTEX = 3.6 PHE NAH TOL BEX = 3.1 PHE MO NAH TOL TOD

34 At BTEX Concentrations <1 mg/l Fewer Genotypes are Detected Total BTEX Log (ug/l) BE = 2.5 {anaerobic?} Genotypes B = 2.3 PHE MO BPH4 BEX = 2.2 PHE MO NAH B = 1.4 PHE MO NAH (X) = n.d. PHE MO NAH (X) = n.d. PHE MO NAH

35 Oxygenases are Detected Downgradient but not Upgradient Genotypes Total BTEX BTEX = n.d. PHE MO BTEX = n.d. PHE MO NAH BTEX = n.d. PHE MO NAH TOL BTEX = n.d. PHE MO BTEX = n.d. BTEX = n.d. Oxygenase BTEX = n.d. Genes BTEX = n.d. BTEX = n.d. Downgradient Wells Upgradient Wells

36 Engineered emediation TOL MO NAH PHE BPH-1 BTEX DO Gene copies 1e+13 1e+12 1e+11 1e+10 1e+9 1e+8 1e+7 1e+6 1e+5 1e+4 1e+3 1e+2 1e+1 1e+0 OC Injection J S D M J S D M BTEX (ug/l)

37 Effect of carbon-based manufactured nanoparticles on anaerobic microbial communities Microbial Community Analysis PC-DGGE Anaerobic Toxicity Assay C 60 Gas Volume (ml) Time (days)

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41 Questions?