Metabolic Interactions Supporting Effective TCE Bioremediation under Various
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
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) Material exchanges in dechlorinating communities D. mccartyi do not live alone in nature. Important to determine
how environmental changes affect material exchanges in communities. Organic Substrate (lactate/whey/molasses) CO2 Hydrogenotrophic Acetogens Vitamin B12 Acetate TCE Fermenters H2 Acetate Microbial consortia ferment organics to hydrogen, providing
electron donor required for Dehalococcoides to respire TCE ??? Methanogens Vitamin B12 Dehalococcoides mccartyi DMB, thiamine, biotin Ethene Microorganisms do not exist in isolation but form complex ecological interaction webs Karoline Faust & Jeroen Raes Nature Reviews Microbiology 2012 10, 538-550
Stable Isotope Probing of Enrichment growth of dechlorinating community without external cobalamin RNA-SIP Fractionation heavy light PF DvH cDNA, PCR, 35 cycles Desulfovibrio vulgaris Hildenbrough (DvH) Pelosinus fermentens (PF) 5 Bin-genomes Recovered from Metagenomic Binning Veillonellaceae
Dehalococcoides Coverage (HiTCE) Desulfovibrio Sedimentibacter Spirochaetaceae Bacteroides Clostridium With nearly complete corrinoid biosynthesis pathway Coverage (HiTCEB12) 6 Pathway compilation of Selected Genomes in Groundwater Enrichment
KEGG mapped Porphyrin and chlorophyll pathway (B12 generation) from Dehalococcoides, Veillonellaceae and Desulfovibrio genomes derived from metagenome Sequence similarity 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 Aim 3: Isotopomer Metabolomics CO, an obligate byproduct from an imcomplete WoodLjungdahl Carbon Fixation
Pathway of D. mccartyi Zhuang et al., 2014 PNAS 111: 64196424 CO accumulation in Dhc195 and DvH/Dhc195 co-culture Men et al., (2012) ISME J. Zhuang et al., 2014 PNAS 111: 64196424 CO serves as a potential energy source for Syntrophomonas wolfei growth Syntrophomonas Wolfei/Dhc co-cultures 1011 0.10 B A
20 C pure S. wolfei co-culture control 0.6 mol 109 1.4 mol 3.2 mol 6.4 mol 108 8.0 mol 0 5
10 Time (day) 15 20 0.06 0.04 live 15 10 5 0.02 0.00
neg CO (mol/bottle) 1010 CO (mol/bottle) cell number/bottle 0.08 abiotic 0 4 8 12
16 0 day 0 day 24 time (day) a) CO effect on S. wolfei growth, b) CO production from S. wolfei growth, c) CO consumption by S. wolfei day 42 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 Technical Approach 1) Construct defined consortia (and inoculate chemostats) 2) Perturb chemostats (Identify changes in microbial community) Defined consortia Cell activity & metabolite exchange TCE
Expression array RMT analysis Quantitative correlation qPCR 3) Apply random matrix theory (RMT) and metabolomics (Elucidate networked interactions) Ethene Aim 1: Construct TCE-dechlorinating consortia Begin with a lactate fermenter and two D. mccartyi strains (with different reductive dehalogenases) Sequentially add microorganisms that D. vulgaris Hildenborough
represent lactate fermentation homoacetogenic, Lactate hydrogenotrophic methanogenesis methanogenic and acetoclastic methanogenic functions CO2 H2 Acetate acetogenesis D. mccartyi strains dechlorination PCE VC,
ETH Aim 1: Inoculate and Optimize Chemostats Inoculate chemostats with defined consortia D. vulgaris Hildenborough lactate fermentation Lactate CO2 methanogenesis H2 Acetate acetogenesis D. mccartyi strains
dechlorination PCE VC, ETH Then optimize chemostats to retain all desired functions Aim 2: Perturb Chemostats with Geochemical Stresses Changes in pH, salinity, acetate, sulfate, sulfide, iron species Amendments with alternative terminal electron acceptors qPCR Steady state reactor Apply environmental stress
Monitor TCE reduction, cell growth, changes in metabolite pool, etc. Aim 2: Microarray-based genome and transtricptome analysis Cell lysis Reverse transcription RNA isolation DNA removal Labeling and hybridization ss-cDNA Cell lysis
DNA isolation Cells Purified DNA/RNA Labeling and hybridization Scanning BAV1 PCE vs TCE TCE Expression 10000 1000 Data Analysis
100 10 10 100 1000 PCE Expression 10000 Aim 2: Identify Changes in Intercellular Metabolites Phelan et al., Nature Chemical Biology 2012 8, 26-35 Aim 3: Map Gene Network and Interactions
Zhou et al., mBio, Sept/Oct 2010, 4. Aim 3: Validate Interrelationships Use quantitative analysis on targeted metabolites GC/MS qPCR 13C stable isotope labeling Targeted metabolomics + GC/MS qPCR Overall Project Plan Aim 1: Construct consortia D. mccartyi strains and fermenters Methanogens and homoacetogens Inoculate chemostats Aim 2: Identify changes due to stress Apply environmental stresses Genomic and transcriptomic analysis
metabolomic analysis Aim 3: Define networked interactions Map gene networks Validate identified relationships Investigate engineered solutions YEAR 1 YEAR 2 YEAR 3 YEAR 4
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