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

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

3. TCEEthene Dehalococcoides mccartyi Methanogens Vitamin B 12 H 2 Acetate CO 2 Hydrogenotrophic Acetogens Fermenters Organic Substrate (lactate/whey/molasses) Vitamin B 12 Acetate ??? Microbial consortia ferment organics to hydrogen, providing electron donor required for Dehalococcoides to respire TCE Material exchanges in dechlorinating communities D. mccartyi do not live alone in nature. Important to determine how environmental changes affect material exchanges in communities. DMB, thiamine, biotin

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 5 Examples of iTags analysis of heavy and light RNA-SIP fractions (HF and LF) Example of RNA-SIP Fractionation RNA-SIP Fractionation HFLF cDNA, PCR, 35 cycles cDNA, PCR, 35 cycles heavylight cDNA, PCR, 35 cycles Desulfovibrio vulgaris Hildenbrough (DvH) Pelosinus fermentens (PF) DvH PF

6. 6 Bin-genomes Recovered from Metagenomic Binning Coverage (HiTCEB12) Coverage (HiTCE) Dehalococcoides Veillonellaceae Desulfovibrio Spirochaetaceae Bacteroides Clostridium Sedimentibacter With nearly complete corrinoid biosynthesis pathway

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

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. a) CO effect on S. wolfei growth, b) CO production from S. wolfei growth, c) CO consumption by S. wolfei CO serves as a potential energy source for Syntrophomonas wolfei growth Syntrophomonas Wolfei/Dhc co-cultures

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

15. Aim 1: Construct TCE-dechlorinating consortia Begin with a lactate fermenter and two D. mccartyi strains (with different reductive dehalogenases) LactateAcetate H2H2 CO 2 D. vulgaris Hildenborough D. mccartyi strains PCEVC, ETH lactate fermentation dechlorination methanogenesis Sequentially add microorganisms that represent homoacetogenic, hydrogenotrophic methanogenic and acetoclastic methanogenic functions acetogenesis

16. Aim 1: Inoculate and Optimize Chemostats Inoculate chemostats with defined consortia Then optimize chemostats to retain all desired functions LactateAcetate H2H2 CO 2 D. vulgaris Hildenborough D. mccartyi strains PCEVC, ETH lactate fermentation dechlorination methanogenesis acetogenesis

17. 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 Apply environmental stress qPCR Monitor TCE reduction, cell growth, changes in metabolite pool, etc.

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

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

22. Aim 1: Construct consortia ● D. mccartyi strains and fermenters ● Methanogens and homoacetogens ● Inoculate chemostats YEAR 1YEAR 2YEAR 3YEAR 4 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 Overall Project Plan