Biogeochemical Controls Over Organohalide-Respiring Chloroflexi - PowerPoint PPT Presentation

Biogeochemical Controls Over Organohalide-Respiring Chloroflexi Frank Löffler Center for Environmental Biotechnology Department of Microbiology Department of Civil & Environmental Engineering Bioscience Division, Oak Ridge Na@onal Laboratory !

Contaminated Sites in the U.S. 1,322 Superfund sites 3,747 RCRA sites >450,000 Brownfields 53% 55% OF ALL of a Superfund or a 166 M ILLIO N 3 Live RCRA Correc@ve Including OF THE U.S. C HILDREN P EO M ILES Within PLE P O PULATIO N UNDER A GE 5 Ac@on Site, Equal to ... and in Europe Majority of sites impacted EPA, 2013 with chlorinated compounds 2

Reductive Dechlorination: A Process that Leads to Contaminant Detoxification In Situ 2[H] 2[H] 2[H] 2[H] HCl HCl HCl HCl Energy Energy Energy Energy Organohalide Respiration Freedman, D. L., and J. M. Gossett. 1989. Appl. Environ. Microbiol. 55:2144-2151 He et al. 2003. Nature. 424:62-65 3

Populations Involved in Reductive Dechlorination of CEs 2[H] 2[H] 2[H] 2[H] HCl HCl HCl HCl Dehalococcoides mccartyi Geobacter lovleyi, Dehalobacter, Sulfurospirillum, Desulfuromonas, Desulfitobacterium Sung et al. 2006 Löffler et al. 2013 AEM, 72:2775 4 IJSEM, 63:625

PopulaMons Involved in ReducMve DechlorinaMon of Chlorinated Ethenes 1,173 GW samples, 111 sites 849 samples: Dhc & Dhgm 65%: Dhgm outnumber Dhc Yang et al. ISME J. 2017. 11:2767-2780 5 He et al. 2003, Nature, 424:62

Populations Involved in Reductive Dechlorination of Chlorinated Ethenes He et al. 2003, Nature, 424:62 Löffler et al. 2013, IJSEM, 63:625 1,173 GW samples, 111 sites Dehalococcoides (Dhc) 849 samples: Dhc & Dhgm 65%: Dhgm outnumber Dhc Dehalogenimonas (Dhgm) Yang et al. ISME J. 2017. 11:2767-2780 6 He et al. 2003, Nature, 424:62

Structural Basis of Organohalide RespiraMon Dimer of PceA Sulfurospirillum mul0vorans 2 FeS Clusters Bommer et al. 2014. Science, 346:455 Corrinoid Payne et al. 2015. Nature, 517:513 Cofactor 7

5’deoxyadenosyl Adenosylcobalamin Corrin ring Upper ligand Methyl Methylcobalamin Cyanocobalamin Cyano Lower base Vitamin B 12 Dimethylbenzimidazole (DMB) 8

Lower Bases Benzimidazole (Bza) type OH OCH 3 OCH 3 N N N N N N CH 3 Corrin ring CH 3 CH 3 N N N N N N CH 3 Nucleobase type N N N CH 3 N SCH 3 N N N N N N N N N N N N NH 2 OH NH 2 NH 2 O O N SCH 3 N SCH 3 N NH 2 N N N O Purine N N NH N N N NH 2 NH 2 O Yan et al. 2018. Phenol type Naphthimidazole Nat. Chem. Biol. 14:8-14. N O O CH 3 N 9

De novo Biosynthesis of Corrinoids Bacteria Archaea (n = 56,902) (n = 1,362) Geobacter lovleyi • Corrinoid prototroph Complete Incomplete Complete or missing Incomplete or missing Dehalococcoides mccartyi • Corrinoid auxotroph 10

Dhc & Dhgm : Strict Requirement for Corrinoid Dhc strain BAV1 cDCE No No B 12 No B 12 dechlorination VC ETH cDCE VC Ethene Limited B 12 [1 µg/L] VC stall Complete Sufficient B 12 Dechlorination [25 µg/L] (Detoxification) 11

Specific Aims • Aim 1: Explore the specific cobamide requirements of organohalide- No B 12 respiring Dhc relevant for detoxifica@on of chlorinated ethenes • Aim 2: Demonstrate that geochemical condi@ons affect the specific cobamide pool, and hence Dhc ac@vity • Aim 3: Iden@fy community and Dhc biomarkers that indicate when cobamide and/or lower base bioavailability limit Dhc reduc@ve dechlorina@on ac@vity 12

Simplified Model H 2 of Organohalide 2 H + Respiration Dehalococcoides mccartyi strain BAV1 2 e - H + Dehalococcoides mccartyi • Corrinoid auxotroph R-Cl + H + ATP Corrinoid' Cofactor' Geobacter lovleyi • Corrinoid prototroph R-H + Cl - H + 13

Who Supplies Corrinoid to Dhc and Dhgm ? Dehalococcoides mccartyi Geobacter lovleyi ? Corrinoid + Corrinoid – 14

Co-Culture Experiments: Corrinoid Producer / Dhc mccartyi Corrinoid auxotroph Cobamide Dhc producing mccartyi microbe Geobacter spp. Strain BAV1 Strain FL2 Dhc ? dechlorination activity? 15

Summary of Co-Culture Experiments Corrinoid auxotroph Cobamide Dhc Co-Cultures producing mccartyi microbe Dhc Geobacter spp. Strain BAV1 Growth Strain FL2 Corrinoid Producer Dhc Strains Dhc ? Geobacter lovleyi BAV1, FL2 + dechlorination activity? Geobacter sulfurreducens BAV1, FL2 - Sporomusa sp. BAV1, FL2, GT - Acetobacterium sp. BAV1, FL2 - Clostridium aceticum BAV1, FL2 - Methanosarcina barkeri BAV1, FL2, GT - 16

Summary of Co-Culture Experiments Corrinoid auxotroph Cobamide Dhc Co-Cultures producing Dhc mccartyi microbe Dhc Growth Geobacter spp. Strain BAV1 Growth Strain FL2 Corrinoid Producer Dhc Strains with DMB Dhc ? Geobacter lovleyi BAV1, FL2 + + dechlorination activity? Geobacter sulfurreducens BAV1, FL2 - + Sporomusa sp. BAV1, FL2, GT - + Acetobacterium sp. BAV1, FL2 - + Clostridium aceticum BAV1, FL2 - + Methanosarcina barkeri BAV1, FL2, GT - + Yan et al. 2012. Appl. Environ. Microbiol. 78:6630-6636 Yan et al. 2013. Phil. Trans. R. Soc. B. 368, 20120320 17

Co-Culture Experiments Corrinoid auxotroph Cobamide Dhc producing mccartyi microbe Co-Cultures Dhc Geobacter spp. Strain BAV1 Strain FL2 Growth Corrinoid Producer Dhc Strains Dhc ? dechlorination activity? Geobacter lovleyi BAV1, FL2 + Geobacter sulfurreducens Wildtype BAV1, FL2 - Geobacter sulfurreducens + pNJ052 BAV1, FL2 + Acetobacterium sp. BAV1, FL2 - Clostridium aceticum BAV1, FL2 - BAV1, FL2, Methanosarcina barkeri - GT Yan et al. 2012. Appl. Environ. Microbiol. 78:6630 6636 Yan et al. 2013. Phil. Trans. R. Soc. B. 368, 20120320 - 18

Key Dhc RDases pceA p eA / t tc eA Dhc str. 195 PceA/PteA TceA Dhc str. FL2 tc TceA eA Dhc str. BAV1 BvcA bv cA Dhc str. VS vc VcrA rA Dhc str. GT 19

Lower Base Affects Dechlorination Activity Dechlorination rates [mmoles Cl - L -1 d -1 ] Strain BAV1 (BvcA) cDCE, VC and ethene [µmoles/bottle] [DMB]Cba [Ben]Cba Time (days) Yan et al. 2016. ISME J. 10:1092–1101 20

Lower Base Affects Dechlorination Activity Dechlorination rates [mmoles Cl - L -1 d -1 ] Strain GT (VcrA) cDCE, VC and ethene [µmoles/bottle] [DMB]Cba [Ben]Cba Time (days) Lower base affects dechlorination rates and endpoints Yan et al. 2016. ISME J. 10:1092–1101 21

Corrinoid Production Under Different Redox Conditions Third Creek Site Knoxville, TN • Metal-manufacturing Sediment • Chlorinated solvents Nitr Su Me Ir l Ferme on f a at t te h - e Re a - - Re Re n d n o u t d d g i c u u e n i c c n g n GW i i i g n n c g g Commerce Street Superfund Site MW 7 - Williston, Vermont • Mul@-tenant industrial park • TCE, cis -DCE, petroleum hydrocarbons, metals (chromium, cadmium, nickel) Corrinoid Extraction and Identification 22

Corrinoids Produced by the Community Under Different Redox Conditions Corrinoids(( Lactate( Iron(( Sulfate( Nitrate( Glucose fermen2ng( Methanogenic( produced( reducing( reducing( reducing( - + - + + + + - - - - - - - + - - - - - + + - - - - + - - - - + - - - 23

Redox Conditions Affect Corrinoid Type(s) and Quantity OH N 5-OHBza-Cba N ������������� �� � �������� � CH 3 N � MeBza-Cba 5-Hyd Dimet N � � CH 3 N � DMB-Cba � N CH 3 � Phe-Cba ����������� O � CH 3 Cre-Cba O � Glucose CH 4 Nitrate Sulfate Iron ng fermenting reducing reducing reducing 24 Seus et al. 2019. In Preparation

Corrinoid Quantity and Quality Determine Dhc Activity cDCE No B 12 VC ETH Limited B 12 [1 µ g/L] Sufficient B 12 [25 µ g/L] 25

NaBH 4 N 2 O Co(III) Co(I) Co(II) Banks et al. 1968. J. Chem. Soc. A, 2886 Impact of N 2 O on corrinoid-dependent reductive dechlorination? 26

N 2 O Inhibits Corrinoid-Dependent Reductive Dechlorination N 2 O [µM] Geobacter lovleyi N 2 O [µM] Geobacter lovleyi (Fumarate) (PCE) Dechlorination (% Cl - released) PCE, TCE, cDCE (µmol/bottle) Fumarate, succinate (mM) 0 0 PCE 10 TCE 20 20 cDCE 100 60 Time (days) Time (days) 27

Impact of Increased N 2 O in Environmental Systems N 2 O (µM) 0.3 K i, N2O = 40.8 ± 3.8 Yongchao Yin µM 12.5 37.4 75 K i, N2O = 21.2 ± 3.5 µM 84 Yin et al. 2019. Environ. Sci. Technol. 143 Nitrous Oxide is a Potent Inhibitor of Bacterial Reductive Dechlorination. 65.7 K i, N2O = 9.6 ± 0.4 µM 53:692-701 Jurado et al. 2017, Sci. Total Environ. 584–585:207-218 28