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Grant 1R01ES024255-01 Lisa Alvarez-Cohen UC Berkeley

Metabolic Interactions Supporting Effective TCE Bioremediation under Various Biogeochemical Conditions. Grant 1R01ES024255-01 Lisa Alvarez-Cohen UC Berkeley. Technical Background. Anaerobic microbial reductive dechlorination. PCE. TCE. cis -DCE. VC. ETH. Dehalococcoides mccartyi.

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Grant 1R01ES024255-01 Lisa Alvarez-Cohen UC Berkeley

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  1. Metabolic Interactions Supporting Effective TCE Bioremediation under Various Biogeochemical Conditions Grant 1R01ES024255-01 Lisa Alvarez-Cohen UC Berkeley

  2. Technical Background Anaerobic microbial reductive dechlorination PCE TCE cis-DCE VC ETH Dehalococcoides mccartyi Clostridium, Dehalobacter, Dehalospirillum, Desulfitobacterium, Desulfomonile, Desulfuromonas, Sulfurospirillum, Geobacter, etc • Electron acceptors: chlorinated ethenes • Electron donor: H2 • Carbon source: acetate • Some important RDase genes: • pceA, tceA, bvcA and vcrA • (all require corrinoids)

  3. Material exchanges in dechlorinating communities • D. mccartyi do not live alone in nature. Important to determine how environmental changes affect material exchanges in communities. Microbial consortia ferment organics to hydrogen, providing electron donor required for Dehalococcoides to respire TCE Organic Substrate (lactate/whey/molasses) CO2 Hydrogenotrophic Acetogens Fermenters ??? Methanogens DMB, thiamine, biotin H2 Acetate Vitamin B12 Acetate Vitamin B12 TCE Ethene Dehalococcoides mccartyi

  4. “Microorganisms do not exist in isolation but form complex ecological interaction webs” Karoline Faust & Jeroen Raes Nature Reviews Microbiology 2012 10, 538-550 

  5. Stable Isotope Probing of Enrichment growth of dechlorinating community without external cobalamin RNA-SIP Fractionation heavy light Desulfovibrio vulgaris Hildenbrough (DvH) Pelosinus fermentens (PF) PF DvH Examples of iTags analysis of heavy and light RNA-SIP fractions (HF and LF) cDNA, PCR, 35 cycles Examples of iTags analysis of heavy and light RNA-SIP fractions (HF and LF) Examples of iTags analysis of heavy and light RNA-SIP fractions (HF and LF) Examples of iTags analysis of heavy and light RNA-SIP fractions (HF and LF) HF LF cDNA, PCR, 35 cycles cDNA, PCR, 35 cycles Example of RNA-SIP Fractionation

  6. Bin-genomesRecoveredfromMetagenomicBinning Veillonellaceae Dehalococcoides Desulfovibrio Sedimentibacter Spirochaetaceae Coverage (HiTCE) Bacteroides Clostridium With nearly complete corrinoid biosynthesis pathway Coverage (HiTCEB12)

  7. Pathway compilation of Selected Genomes in Groundwater Enrichment Sequence similarity KEGG mapped Porphyrin and chlorophyll pathway (B12 generation) from Dehalococcoides, Veillonellaceae and Desulfovibrio genomes derived from metagenome

  8. Tri-Culture of D. mccartyi 195 by corrinoid salvaging and remodeling in defined tri-culture Men et al., 2014 Environ. Microbiol. DOI: 10.1111/1462-2920.12500

  9. Aim 3:Isotopomer Metabolomics

  10. CO, an obligate by-product from an imcomplete Wood-Ljungdahl Carbon Fixation Pathway of D. mccartyi Zhuang et al., 2014 PNAS 111: 6419–6424

  11. CO accumulation in Dhc195 and DvH/Dhc195 co-culture Men et al., (2012) ISME J. Zhuang et al., 2014 PNAS 111: 6419–6424

  12. CO serves as a potential energy source for Syntrophomonaswolfeigrowth SyntrophomonasWolfei/Dhcco-cultures a) CO effect on S. wolfei growth, b) CO production from S. wolfei growth, c) CO consumption by S. wolfei

  13. Technical Objectives Aim 1: Construct TCE-dechlorinating consortia of fully sequenced organisms and maintain in chemostats Aim 2: Identify changes in microbial community that occur in response to geochemical perturbations Aim 3: Elucidate networked interactions in the consortia that occur in response to geochemical perturbations

  14. qPCR Expression array Defined consortia Quantitative correlation TCE Ethene Technical Approach 1) Construct defined consortia (and inoculate chemostats) 2) Perturb chemostats (Identify changes in microbial community) Cell activity & metabolite exchange RMT analysis 3) Apply random matrix theory (RMT) and metabolomics (Elucidate networked interactions)

  15. D. vulgaris Hildenborough lactate fermentation Lactate Acetate CO2 H2 methanogenesis D. mccartyi strains ? dechlorination PCE VC, ETH Aim 1:Construct TCE-dechlorinating consortia • Begin with a lactate fermenter and two D. mccartyi strains (with different reductive dehalogenases) • Sequentially add microorganisms that represent homoacetogenic, hydrogenotrophic methanogenic and acetoclastic methanogenic functions acetogenesis

  16. D. vulgaris Hildenborough lactate fermentation Lactate Acetate CO2 H2 methanogenesis D. mccartyi strains ? dechlorination PCE VC, ETH Aim 1:Inoculate and Optimize Chemostats • Inoculate chemostats with defined consortia acetogenesis • Then optimize chemostats to retain all desired functions

  17. qPCR Aim 2:Perturb Chemostats with Geochemical Stresses • Changes in pH, salinity, acetate, sulfate, sulfide, iron species • Amendments with alternative terminal electron acceptors Steady state reactor Monitor TCE reduction, cell growth, changes in metabolite pool, etc. Apply environmental stress

  18. Aim 2: Microarray-based genome and transtricptomeanalysis Cell lysis RNA isolation DNA removal Reverse transcription Labeling and hybridization ss-cDNA Cell lysis DNA isolation Labeling and hybridization Cells Purified DNA/RNA Scanning Data Analysis

  19. Aim 2:Identify Changes in Intercellular Metabolites Phelan et al., Nature Chemical Biology 2012 8, 26-35

  20. Aim 3:Map Gene Network and Interactions Zhou et al., mBio, Sept/Oct 2010, 4.

  21. qPCR Aim 3:Validate Interrelationships • Use quantitative analysis on targeted metabolites • qPCR • 13C stable isotope labeling • Targeted metabolomics + GC/MS GC/MS

  22. Overall Project Plan

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