Advances in Anaerobic Benzene Bioremediation: Microbes, Mechanisms, and Biotechnologies Sandra Dworatzek, Phil Dennis and Jeff Roberts RemTech, Virtual October 15, 2020 Introduction and Acknowledgements • Sandra Dworatzek, Jennifer Webb (SiREM, Guelph, ON) • Elizabeth Edwards, Nancy Bawa, Shen Guo and Courtney Toth (University of Toronto, Toronto, ON) • Kris Bradshaw and Rachel Peters (Federated Co-operatives Ltd., Saskatoon, SK) • Krista Stevenson (Imperial Oil, Sarnia, ON) The Landscape of Hydrocarbon Bioremediation: A Lot Has Changed… Microbial bioremediation is currently the most common technology used to remediate petroleum hydrocarbons Microbial Remediation Phytoremediation Chemical Treatment Contain and Excavate Pump and Treat Other Near Cold Lake, Alberta Adapted from Elekwachi et al., 2014 (J Bioremed Biodeg 5) What Sites are Currently Being Targeted for Hydrocarbon Bioremediation? 4 Shallow Soils and Groundwater 1 2 (aerobic) Offshore Spills Ex situ Bioreactors (mostly aerobic) (mostly aerobic) 3 Tailings Ponds 5 Deeper Groundwater (aerobic and anaerobic) (intrinsically anaerobic) Groundwater Bioremediation Technologies Focusing on Anaerobic Microbial Processes Natural Attenuation Biostimulation Bioaugmentation unsaturated zone aquifer 3- PO4 sugars Plume source - 2- NO3 SO4 VFAs GW flow aquitard Bioaugmentation for anaerobic sites works! Dehalococcoides (Dhc) bioaugmentation is widely accepted to improve reductive dehalogenation of chlorinated ethenes 1 Month Post KB-1® Bioaugmentation Dhc TCE Bioaugmentation for anaerobic sites works! Dehalococcoides (Dhc) bioaugmentation is widely accepted to improve reductive dehalogenation of chlorinated ethenes 7 Months Post KB-1® Bioaugmentation Dhc TCE Bioaugmentation for anaerobic sites works! Dehalococcoides (Dhc) bioaugmentation is widely accepted to improve reductive dehalogenation of chlorinated ethenes 13 Months Post KB-1® Bioaugmentation Dhc TCE Bioaugmentation for anaerobic sites works! Dehalococcoides (Dhc) bioaugmentation is widely accepted to improve reductive dehalogenation of chlorinated ethenes 26 Months Post KB-1® Bioaugmentation Dhc TCE •/ BTEX/Benzene Challenges • Retail gas stations, refineries and fuel handling stations among potential sources • BTEX comprises ~18% of gasoline • Benzene is typically around 1% Benzene: • Potent carcinogen • Particularly mobile in groundwater due to low sorption & high water solubility • Most difficult BTEX compound to degrade anaerobically (unsubstituted ring structure) • Anaerobic conditions, bottleneck to site remediation Why Go Anaerobic for BTEX? • Hydrocarbon sites can go anaerobic - high organic loading consumes O2 • Electron acceptors (NO3/SO4/CO2) often already present in subsurface • Anaerobic electron acceptors are soluble, easier to apply/distribute compared to O2 (e.g., epsom salts (sulfate)) • Viable in situ remediation option for deep contamination Benzene as a Proof-of-Concept Benzene is the most difficult (and least understood) BTEX compound to degrade anaerobically, and is often the driver for remediation efforts Intrinsic microbial processes can bioremediate benzene anaerobically, but are often slow or even undetectable in situ. Microcosm Study of BTEX Natural Attenuation (Saskatchewan, CAN) 25 20 Benzene Benzene Toluene 15 m, p-Xylene 10 Toluene 5o-Xylene Ethylbenzene BTEX (mg/L) 0 0 100 200 300 400 500 600 Ethyl Xylene Days benzene Hydrocarbons “Burn Through” O2 While aerobic processes are far more efficient thermodynamically, anaerobic processes can be used for anoxic sites Hydrocarbon Net Reaction ΔG°’ (kJ mol-1) Benzene C6H6 + 7.5 O2 6 CO2 + 3 H2O -3202 - + C6H6 + 6 NO3 + 6 H 6 CO2 + 3 N2 + 6 H2O -3008 2- + C6H6 + 3.75 SO4 + 7.5 H 6 CO2 + 3.75 H2S + 3 H2O -214 C6H6 + 4.5 H2O 3.75 CH4 + 2.25 CO2 -135 Naphthalene C10H8 + 12 O2 10 CO2 + 4 H2O -5093 - + C10H8 + 9.6 NO3 + 9.6 H 10 CO2 + 4.8 N2+ 8.8 H2O -4782 2- + C10H8 + 6 SO4 + 12 H 10 CO2 + 6 H2S + 4 H2O -313 C10H8 + 8 H2O 6 CH4 + 4 CO2 -186 Hexadecane C16H34 + 24.5 O2 16 CO2 + 17 H2O -10392 - + C16H34 + 19.6 NO3 + 19.6 H 16 CO2 + 9.8 N2+ 26.8 H2O -9757 2- + C16H34 + 12.25 SO4 + 24.5 H 16 CO2 + 12.5 H2S + 17 H2O -632 C16H34 + 7.5H2O 12.25 CH4 + 3.75CO2 -372 Adapted from Widdel and Musat, 2010 (Handbook of Hydrocarbon and Lipid Microbiology, ed. K. N. Timmis) Key Difference Between Bioremediation of Chlorinated Solvents vs Hydrocarbons Hydrocarbons are electron donors rather than electron acceptors Adding carbon (sugars, VFAs, yeast extract) may not enhance bioremediation performance Adding electron acceptors does not always enhance bioremediation either Gieg et al., 2014 (Curr Opin Biotechnol 27) Overview of Anaerobic Benzene Degradation Fermentative Processes Benzene ? Benzoate Deltaproteobacteria ORM2 Benzoyl-CoA Thermincola spp. Fatty acids, Alcohols Sulfate-Reducing Bacteria Nitrate-reducing bacteria 2- SO4 H2, Formate, - + NO3 NH4 Methanogenic Acetate Anammox - HS - bacteria Archaea NO2 N2 CO2 CH4, CO2 CO2 Monitored using GeneTrac® ORM2 Anaerobic Benzene Degrader • Benzene specialist derived from an oil refinery site in 2003 • ORM2 is a Deltaproteobacterium • Produces enzymes that ferment benzene • Slower growing ~ 30 day doubling time DGG-B Culture – ORM2’s Home • DGG-B successfully scaled up to commercial volumes Benzene degradation rate = 0.6 mg /L/ day 1011 ORM2/L 25 20 15 10 mg/L Benzene Benzene 5 0 0 200 400 600 Anaerobic benzene degraders appear to be ubiquitous in anoxic benzene-contaminated environments, but in very low numbers 1.0E+08 ORM2 abcA Pepto-Ben 1.0E+07 1.0E+06 1.0E+05 Approx. Threshold Required for Active Degradation 1.0E+04 1.0E+03 1.0E+02 qPCR qPCR (copies/mL)Target 1.0E+01 1.0E+00 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Treatability Testing Scope BTEX-contaminated materials from multiple sites were assessed for their anaerobic benzene bioremediation potential Tested: Intrinsic bioremediation 5 8 9 10 Biostimulation (nitrate or sulfate) 6 3 4 2 DGG-B bioaugmentation 7 1 Treatability Testing Incubate and Homogenized core samples Mix monitor (60 g) 200 mL groundwater slurries 50 mL headspace (10% CO2 / 90% N2) *Aqueous BTEX concentrations ranged between 0.1 – 20 mg/L, Groundwater sample depending on site materials Current Treatability Studies Underway Treatability (Microcosm) Field Pilots Culture(s) Studies Site Location Target Substrate(s) Tested Successful Start Date Start Date Bioaug’? Saskatchewan Benzene DGG-B Dec-2016 Yes Nov-2019 Ontario BTX DGG-BTX Jul-2019 Yes Delayed North Carolina Benzene, MTBE DGG-B Jul-2019 Yes Planning Louisiana Benzene DGG-B Nov-2019 Yes Oct-2019 Yes New Jersey Benzene DGG-B Nov-2019 2+ Apr-2020 (SO4 + DGG-B) Saskatchewan BTEX DGG-B Jan-2020 NRBC, New Jersey Benzene May-2020 DGG-B Benzene, NRBC, NRBC New Jersey May-2020 Chlorobenzene DGG-B (1/3 replicates) Alberta Benzene DGG-BX Sep-2020 BTEX, DGG-BTX, Indiana -- -- Oct-2020 Chlor. solvents KB-1 Plus Intrinsic biodegradation correlates to known benzene degraders 8 1 Nanjing, China (Methanogenic) Saskatchewan, Canada 10 (Sulfate-Reducing) 8 8 6 6 4 4 2 2 Benzene (mg/L) Benzene(mg/L) 0 0 1.0E+091.0E+12-50 50 150 250 1.0E+091.0E+12 0 100 200 300 1.0E+081.0E+11 1.0E+081.0E+11 1.0E+071.0E+10 1.0E+071.0E+10 Pepto like 1.0E+061.0E+09 1.0E+061.0E+09 abcA - 1.0E+051.0E+08 1.0E+051.0E+08 1.0E+041.0E+07 1.0E+041.0E+07 ORM2 General Bacteria 1.0E+031.0E+06 General Bacteria 1.0E+031.0E+06 Target Gene Target (Copies/L) ** * * Target Gene Target (Copies/L) 0 379 0 134 260 Time (Days) Time (Days) * = below quantifiable limits Borden II – Key Highlights Methanogenic toluene degradation observed in untreated site materials. Injecting heat-killed (HK) culture did not enhance BTX bioremediation. Sterile Control Untreated DGG-BTX HK 18 18 18 16 16 16 14 14 14 12 12 12 10 10 10 Benzene 8 8 8 o-Xylene 6 6 6 BTX (µmol/bottle) 4 4 Toluene 4 2 2 2 0 0 0 0 100 200 300 400 500 0 100 200 300 400 500 0 100 200 300 400 500 Time (Days) Borden II – Key Highlights DGG-T enhanced rates of toluene degradation by up to ~ 63%. DGG-X enhanced both toluene (~ 26%) and o-xylene degradation. Untreated DGG-T only DGG-X only 18 18 18 16 16 16 14 14 14 Benzene 12 12 12 10 10 10 8 8 8 6 6 Toluene 6 BTX (µmol/bottle) 4 4 4 2 2 2 o-Xylene 0 0 0 0 100 200 300 400 500 0 100 200 300 400 500 0 100 200 300 400 500 Time (Days) 24 Borden II – Key Highlights Benzene degradation was only observed in bioaugmented bottles with active toluene and o-xylene metabolism. DGG-B only DGG-BTX DGG-BTX (sequential) 20 20 20 DGG-T DGG-B 18 18 Benzene DGG-X 16 16 15 14 14 12 12 10 10 10 8 8 6 o-Xylene 6 5 BTX (µmol/bottle) 4 Toluene 4 2 2 0 0 0 0 100 200 300 400 500 0 100 200 300 400 500 0 100 200 300 400 500 Time (Days) 25 Target concentrations (≥108 copies/L) Intrinsic Nitrate DGG-B Bioremediation Biostimulation Bioaugmentation 10 10 10 DGG-B added 5 5 5 Benzene(mg/L) 0 0 0 0 100 200 0 100 200 0 100 200 1.0E+111.0E+08 1.0E+111.0E+08 1.0E+11 1.0E+091.0E+06 1.0E+091.0E+06 1.0E+091.0E+06 1.0E+071.0E+04 1.0E+071.0E+04 1.0E+071.0E+03 1.0E+02 1.0E+02 abcA Bacteria 1.0E+05 1.0E+05 1.0E+05 ORM2 1.0E+00 1.0E+00 1.0E+00 1.0E+03 * * * * 1.0E+03 ** * ** 1.0E+03 * * * 12 109 12 109 179 12 109 179 Target Gene Target (Copies/L) Days *= below quantifiable limits Bioaugmentation also increases the abundance of other key microbes 100 3 Ontario Canada (Methanogenic) Methanomethylovorans(10 80 Methanoregula(100) Methanosaeta(100) 60 Spirochaetaceae(93) 40 Desulfovibrio(100) % Abundance % Ferribacterium(100) 20 Albidiferax(97) ORM2 0 DGG-B 14 77 109 Days FAQs: Is Anaerobic Benzene Degradation