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Microbial Dechlorinating Consortia & Brief Introduction to Metagenomics

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Microbial Dechlorinating Consortia & Brief Introduction to Metagenomics Microbial Dechlorinating Consortia & Brief Introduction to Metagenomics Elizabeth A. Edwards Department of Chemical Engineering and Applied Chemistry And Cell and Systems Biology University of Toronto Centre for Applied Bioscience and Bioengineering Co-Authors and Acknowledgments Edwards lab Ivy Yang, Courtney Toth, Katherine Picott, Olivia Bulka, Fei Luo, Nadia Morson, Olivia Molenda, Mahbod Sandra Dworatzek Hajighasemi, Laura Hug, Shuiquan Tang, Marie Phil Dennis Manchester, Luz Puentes, Camilla Nesbo, Xioaming Jeff Roberts & Jen Webb Liang, Line Lomheim, Kai Wei, Jine Jine Li, Cleo Ho, Ahsan Islam, Cheryl Devine, Alfredo Perez de Mora, Anna Zila, Sarah McRae, Laurent Laquitaine, Winnie Chan, Ariel Grostern, Melanie Duhamel, Alison Waller… Dr. David Major, Evan Cox And many more Michaye McMaster & others Collaborators Frank Löffler (U. Tenn) Krishna Mahadevan (U of Toronto) Barb Sherwood Lollar, Brent Sleep (U of Toronto) Alfred Spormann (Stanford) Ruth Richardson & Stephen Zinder (Cornell) Lorenz Adrian (UFZ); Craig Criddle (Stanford) 1 Fate of contaminants in the environment: role of Biology CF TCE spill Typically Dissolved plume of oxygen contaminant Clay lens becomes depleted Groundwater Flow Impermeable layer Bioremediation Bioremediation: the remediation (clean up) of contaminated sites (soil, sediment, groundwater) using microorganisms in an engineered system • ex situ (on-site): in above-ground bioreactors • in situ (in-place): the subsurface is the bioreactor • Biostimulation vs. Bioaugmentation Three challenges: 1) Mixing 2) Mixing Above Nutrients 3) Mixing ground and/or microbes Bioreactor Extraction Injection well Aquifer well Bioreactor Overview of Microbial Metabolism Something to eat Something to “breathe” Electron Donor or Substrate Electron Acceptor (Reduced) (Oxidized) Sugars, Proteins, Fats Nitrate (NO3) Oxygen (O2) Toluene, Benzene Sulfate (SO4); SeO4 Fe(III), CO2 H2, Fe(II), H2S, FeS Enzymes in a microbe Electron Donor or Substrate Electron Acceptor (Oxidized) (Reduced) CO 2 N , H S; Seo + o 2- Water 2 2 H , Fe(III), S , SO4 Fe(II), CH4 If energy is released (DeltaG<0) then microbe can grow Anaerobic PCE Dechlorination Cl Cl Before 1989 C=C Cl H Cl Cl C=C H H Cl Cl PCE C=C H H TCE Cl Cl C=C DCE H Cl VC Made a bad situation worse! Complete Reductive Dechlorination After 1989 Cl H C=C H H Cl Cl C=C H H Wow! TCE Cl Cl C=C H H DCE H Cl C=C VC Mediated by Microbes H H under ANAEROBIC conditions Ethene Requires electron donor Gossett and Freedman, 1989 - Cornell Anaerobic Microcosms from Site where lots of ethene was detected (1995) Sterile Active Control Donor1 Donor2 Donor3 7 TCE Dechlorination to Ethene by the “KB-1” Consortium (circa 1998) 0.40 TCE Ethene + HCl 0.35 TCE Ethene Methanol CH4+CO2 0.30 Cells 0.25 0.20 Enriched since 1997 on 0.15 VC TCE/MeOH moles/bottle) 0.10 0.05 cDCE Strictly anaerobic TCE TCE ( 0.00 0 5 10 15 20 Time (Days) 1995 – 1999: Enrichment of KB-1 Sediment/Groundwater Microcosms from Ontario site - Produced lots of Ethene Sediment Free Culture in defined Anaerobic Media 2002: Founding of Dave Major & Evan Cox, Geosyntec Phil Denis, Sandra Dworatzek, Jeff Roberts and many other fabulous staff Growth scale-up to several liters 1995 – 1999: Enrichment of KB-1 Sediment/Groundwater Microcosms from Ontario site - Produced lots of Ethene Sediment Free Culture in defined Anaerobic Media 2002: Founding of Growth scale-up to several liters Dechlorinators occupy a similar niche to methanogens Fermentable substrate (ethanol, lactate, methanol) Fermenters Dechlorinators occupy a similar niche to methanogens Fermentable substrate (ethanol, lactate, methanol) Fermenters Dehalococcoides KB-1/VC (SEM) Organohalide Respiration Something to eat Something to “breathe” Electron Donor or Substrate Electron Acceptor CO2 (Reduced) (Oxidized) H Perchloroethene 2 Trichloroethene Dichloroethene Vinyl Choride Enzymes in an Anaerobe Electron Acceptor Electron Donor or Substrate (Reduced) (Oxidized) CH4 Trichloroethene H+ Dichloroethene Vinyl Chloride, Ethene Organohalide-respiring bacteria use these reactions as electron sinks Hydrogenolysis PCE TCE Catalyzed by reductive dehalogenases Dihaloelimination 1,1,2,2-TeCA trans-DCE Research on Reductive Dechlorination and Organohalide Respiration 1989 2H HCl 2H HCl 2H HCl 2H HCl Dehalobacter Dehalococcoides 1997 Dehalospirillum Desulfitobacterium (Only some strains) Desulfuromonas Dehalogenimonas 2015 +Dehalococcoides 1993 Ethene is non-toxic, produced by many fruit to stimulate ripening What followed since ~2001 • Practical Application for • Fundamental Science Bioremediation – Organisms that dechlorinate? – How to scale up & grow faster – Microbial ecology, physiology, – Regulatory approvals evolution – How to deliver to subsurface – Growth kinetics – How to tracking organisms – Enzymes involved – Other contaminants – Mechanisms and substrates – Modelling fate and transport – Microbial community – How much culture to add? interactions – What are inhibitors? – Omics…. Fundamental Science KEY ORGANISMS (Dehalococcoides, Dehalobacter, Dehalogenimonas) ENZYMES MICROBIAL COMMUNITY Bommer, M., et al. Structural Basis for Organohalide Respiration. Science. 2014, 346:455–458. Fundamental Science KEY ORGANISMS MICROBIAL ENZYMES COMMUNITY (specific dechlorinating bacteria) (a protein) Two types of organohalide-respiring bacteria Maphosa, de Vos and Smidt, Trends in Biotechnology Vol.28 No.6, 2010 Some Good Substrates for Dechlorinators Dehalococcoides Dehalobacter Dehalobacter Desulfitobacterium Dehalogenimonas Desulfitobacterium H3C H Sulfurospirillum Geobacter H Trichloroethene 1,1,1-Trichloroethane Chloroform (TCE) (1,1,1-TCA) (CF) Dehalococcoides Dehalobacter Dehalogenimonas Dehalogenimonas Trichlorobenzene Trichloropropane H3C Two cultures: KB-1 Culture H No overlap in TCE Dhc substrates ACT-3 Geo Culture cDCE Dhc 1,1,1-TCA CF VC Dhb Dhc 1,1-DCA DCM Dhb Ethene CA Dhc = Dehalococcoides Dhb = Dehalobacter 21 Geo = Geobacter WBC-2 and KB-1 Pathway Convergence KB-1 Culture 1,1,2,2-TeCA TCE WBC-2 Culture tDCE Dhc ACT-3 Geo Culture cDCE Dhc 1,1,1-TCA CF VC Dhb Dhc 1,1-DCA DCM Dhb Ethene CA Dhc = Dehalococcoides Dhb = Dehalobacter 22 Geo = Geobacter Dehalogenimonas dechlorinates tDCE KB-1 Culture 1,1,2,2-TeCA Dhb TCE WBC-2 Culture tDCE Dhc ACT-3 Geo Culture Dhg cDCE Dhc 1,1,1-TCA CF VC Dhb Dhc 1,1-DCA DCM Dhb Ethene CA Dhc = Dehalococcoides Dhb = Dehalobacter Dhg = Dehalogenimonas Geo = Geobacter 23 Dehalogenimonas dechlorinates tDCE KB-1 Culture 1,1,2,2-TeCA Dhb TCE WBC-2 Culture tDCE Dhc1 ACT-3 Geo Culture Dhg cDCE Dhc2 1,1,1-TCA CF VC Dhb1 Dhc2 1,1-DCA DCM Dhb2 Ethene CA Dhc = Dehalococcoides Dhb = Dehalobacter Dhg = Dehalogenimonas Geo = Geobacter 24 Pathway Convergence & Substrate Interference Competition Dhb, Dhc, Other PCE 1,1,2,2-TeCA Abiotic Dhb TCE tDCE Dhb, Dhc, Other 1,1,2-TCA Dhg cDCE Other, Dhb Dhc 1,1,1-TCA CF VC Inhibition → Dhb 1,2-DCA Dhc 1,1-DCA DCM Other, Dhb, Dhc Dhb Ethene CA Dhc = Dehalococcoides Dhb = Dehalobacter Dhg = Dehalogenimonas Other = Desulfitobacterium, Sulfurospirillum, Geobacter Enzyme Assays to explore impact of inhibition Data from “Cell-Free” extracts of a Dehalobacter mixed culture Lyse all cells to release all proteins (including dehalogenases) Assay enzymes directly Adding CF+ artificial electron donor in reducing buffer e- donor CF (oxidized) e- donor DCM (reduced) 26 Genomes of Organohalide-Respiring Bacteria (OHRB) (Sequence DNA) Organohalide-respiring Bacteria Mbp rdhA genes Versatile metabolism (facultative OHRB): Halogenated and non Desulfitobacterium hafniense Y51 5.73 4 halogenated Desulfitobacterium hafniense DCB-2 5.28 7 electron Anaeromyxobacter dehalogenans 2CP-C 5.01 2 acceptors Geobacter lovleyi SZ 3.87 2 Restricted metabolism (Obligate OHRB): Dehalobacter restrictus 2.94 25 Dehalobacter strain CF 2.91 20 Only Dehalogenimonas lykanthroporepellens 1.66 19 halogenated electron Dehalococcoides mccartyi strain 195 1.47 17 acceptors Dehalococcoides mccartyi strain VS 1.41 36 Dehalococcoides mccartyi strain CBDB1 1.4 32 Dehalococcoides mccartyi strain KBVC1 1.39 22 Organohalide respiration and reductive dehalogenases (RDases) with cobamide (B12) as the cofactor Outside of the cell Inside the cell 28 Enzyme Characterization pceA Sulfurospirillum, Desulfitobacterium, Dehalobacter, Clostridium, Geobacter Multiple Dehalococcoides tceA strains vcrA Certain bvcA Dehalococcoides 1,2-Dichloroethane Process- and Organism-Specific Biomarkers Quantitative PCR based tests to detect Chlorinated Chlorinated Chlorinated Chlorinated DNA in Ethanes Ethenes Methanes Propanes Environmental Samples pceA Dhc cfrA Dhb 1 cfrA Dhb 1 dcpA Dhg pceA tceA dcrA Dhb 2 tceA bvcA Dhb-related Dhc; Dhg vcrA PCBs CO2 bvcA Dhc; Dhg RD-17 (CG-1) vcrA RD-1 (CG-4) RD-1 (CG-5) Dhc Functional Gene Biomarkers Dhc: Dehalococcoides mccartyi 16S rRNA Gene Biomarkers Dhb: Dehalobacter sp. Dhg: Dehalogenimonas sp. Slide from Frank Loeffler 30 Process- and Organism-Specific Biomarkers Chlorinated Chlorinated Chlorinated Chlorinated Chlorinated HCH & Ethanes Ethenes Methanes Propanes Benzenes Lindane pceA Dhc cfrA Dhb 1 cfrA Dhb 1 dcpA Dhg Dhb, Dhc pceA Cl Dhb tceA Dhb Dhb dcrA Dhb 2 tceA Cl bvcA Dhb-related Dhc; Dhg vcrA PCBs CO2 bvcA Dhc; Dhg RD-17 (CG-1) CO +CH vcrA RD-1 (CG-4) 2 4 RD-1 (CG-5) Dhc Functional Gene Biomarkers Dhc: Dehalococcoides mccartyi 16S rRNA Gene Biomarkers Dhb: Dehalobacter sp. Dhg: Dehalogenimonas sp. Slide from Frank Loeffler 31 Role of the Community: Dehalococcoides isolates grow slowly KB-1 Consortium Community Diversity KB-1 Consortium
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