A new tool in the toolbox - Anaerobic Benzene Bioremediation. Presented by: Sandra Dworatzek, SiREM 22 March 2017

Transcription

1 A new tool in the toolbox - Anaerobic Benzene Bioremediation Presented by: Sandra Dworatzek, SiREM 22 March 2017

2 Outline Introduction and background Aerobic vs anaerobic degradation Anaerobic benzene degrading cultures Treatability case study Biomarkers Conclusions 2

3 U.S. Petroleum Distribution Industry 144 refineries 200,000 miles of crude oil and refined petroleum product pipelines 38 Jones Act vessels (U.S. flag ships that move products between U.S. ports) 3,300 coastal, Great Lakes and river tank barges 200,000 rail tank cars 1,400 petroleum product terminals 100,000 tanker trucks >150,000 retail fueling sites (Source: American Fuel & Petrochemical Manufacturers) 3

4 Petroleum Hydrocarbon Compounds Total petroleum hydrocarbon compounds (TPH) include gas range (C2-C5) and diesel range (C10-C28) Aromatic or C6 refers to those that contain the benzene ring PHCs of primary concern in groundwater are benzene, toluene, ethylbenzene, and the xylenes (the BTEX compounds) 4

5 / Benzene Potent carcinogen Particularly mobile in groundwater due to low sorption & high water solubility Most difficult BTEX compound to degrade anaerobically (unsubstituted ring structure) 5

6 Bioremediation of Petroleum Hydrocarbon Compounds Redox zones of a typical contaminant plume (Source: Parsons 2004). 6

7 Aerobic BTEX Bioremediation Aerobic bioremediation approaches rely on availability or delivery of oxygen (e.g., peroxides, bioventing) When contamination is deep or under established reducing conditions, aerobic bioremediation can be difficult to establish and maintain. Intrinsic microbial populations (e.g., Pseudomonas) are often capable of performing aerobic biodegradation - bioaugmentation not generally required 7

8 Aerobic BTEX Bioremediation (cont d) Microbial catalyzed oxidation reaction between dissolved oxygen (DO) and BTEX is thermodynamically favored BTEX compounds are highly reduced and the preferred terminal electron acceptor is oxygen. oxidation of BTEX compounds requires 3.1 milligrams per liter (mg/l) of dissolved oxygen (DO) to 1 mg/l of a BTEX compound (Aronson and Howard, 1997). 8

9 / Aerobic Benzene Degradation Aerobic Benzene Degradation - O 2 Required Dioxygenase O 2 Dioxygenase O 2 Krebs Cycle CO 2 + H 2 O 9

10 Anaerobic BTEX Bioremediation Biodegradation of BTEX occurs under anaerobic conditions: Methanogenic Nitrate reducing Sulfate reducing Microbial populations may be present at low concentration but growth is slow bioaugmentation may accelerate 10

11 Anaerobic BTEX Bioremediation (cont d) Requires alternate electron acceptor - Methanogenic conditions - CO 2 naturally present serves as electron acceptor Other electron acceptors: Sulfate - highly soluble and naturally present in many aquifers Nitrate challenges related secondary water quality regulations TEX degraders more often found, benzene becomes a bottleneck 11

12 In Search of Elusive Anaerobic Benzene Activation Mechanisms Hydroxylation to phenol Methylation to toluene Carbonylation to benzaldehyde Carboxylation to benzoate Benzoyl- CoA is a central metabolite Other???? 12

13 Anaerobic Benzene Degrading Cultures Microcosm Enrichment Cultures Electron Acceptors NO 3 - DGG-1 CH 4 SO 4 2- Denitrifying Pepto Methanogenic ORM2 Sulfur-reducing Relative abundance, % Peptococcaceae spp. Putative functional gene abcda Deltaproteobacteria ORM2 Novel mechanism Unknown functional gene Credit to Dr. Fei Luo, University of Toronto for the slide and metagenomics work 13

14 Introducing DGG-1 Dunja Grbic-Galic Stanford University Environmental Engineering and Science PhD supervisor of Elizabeth Edwards Anaerobic microbiology Microbial transformation of organic pollutants, with an emphasis on ground water and soil habitats. 14

15 Anaerobic Benzene Culture DGG-1 Currently scaling up DGG-1 to field-scale volumes DGG-1 seed culture Edwards and Grbic-Galic,

16 Culture Development and Use for Bioaugmentation Culture Enrichment & Characterization Contaminated Sites Biomarkers Field Injection - bioaugmentation Treatability Testing Culture Scale Up 16

17 copies/ml culture Introducing ORM2 Deltaproteobacterium benzene specialist derived from DGG-1 Not yet isolated in pure culture Slow grower, ~ 30 day doubling time 10 7 Benzene ORM fed starved Time (days) 17 0

18 Benzene concentration (μmol/l) DGG-1 Culture Performance Negative control Full strength culture Benzene amendment (~30 mg/l) Time (days) 18

19 Anaerobic Biotreatability Case Study Anaerobic conditions maintained during set up, incubation and sampling in glove bags filled with N 2 /CO 2 / H 2 gas mixture B T E X Degradation of BTEX monitored by GC under various conditions 19

20 Batch Treatability Study Design Features Sterile Control autoclaved and poisoned to inhibit microbes measure possible abiotic losses Active Control unamended Biostimulation addition of electron acceptor Bioaugmentation+ Biostimulation addition of known degrading populations Treatability studies are custom designed for each site 20

21 Biotreatability Case Study Ontario Site Sandy aquifer Benzene concentration of 7 mg/l ORM2 negative prior to DGG-1 bioaugmentation Evaluated degradation under either sulfate or methanogenic conditions with and without DGG-1 bioaugmentation Evaluated benzene only as well as a BTEX + naphthalene mixture 21

22 Microcosm Study Results: Benzene as substrate, Methanogenic conditions 22

23 Benzene as substrate, Sulfate-reducing conditions 23

24 BTEX + Naphthalene as substrates, Methanogenic conditions 24

25 BTEX + Naphthalene as substrate, Sulfate-reducing conditions 25

26 Concentration (mmol/bottle) Methane (mmoles/bottle) BTEX+Naphthalene as substrate, Sulfatereducing conditions Days Benzene Toluene Ethyl Benzene o-xylene p,m-xylene Bioaugmented with DGG-1 Methane Napthalene 26

27 Development of Molecular Tools for Monitoring Anaerobic BTEX Degradation Targets Available Relevant to Anaerobic Benzene Gene-Trac ORM2 specific test for DGG-1 anaerobic benzene degrader Gene-Trac SRB - sulfate reducing organisms Anaerobic BTEX Targets (in development) ORM2 more general test for entire group abca - Benzene carboxylase - Peptococcaceae bssa - Benzyl succinate synthase toluene degradation Benzene fermenting gene ORM2 discovery work currently underway at UofT 27

28 Anaerobic/Aerobic Degradation of BTEX Spill Site Bioaugmentation with Anaerobic Benzene Culture + electron acceptor (e.g., Nutrisulfate TM ) Optional O 2 addition (e.g., isoc or peroxides) Groundwater PLUME SO 4 - O 2 - Flow Direction Illustration Tersus Environmental Anaerobic BTEX Degradation in Source Zone Aerobic BTEX Degradation in Lower Concentration Downgradient Zone 28

29 Ongoing Work Additional treatability studies Determining geochemical ranges of conditions where effective (e.g., concentrations, ph, interactions with other contaminants) Biomarkers adding new tests, evaluate ubiquity of anaerobic benzene degraders in the field Please let us know if you have BTEX sites where anaerobic bioremediation could be of benefit! 29

30 Acknowledgements Jennifer Webb, Jennifer Wilkinson, Peter Dollar Professor Elizabeth Edwards, Dr. Fei Luo, Nancy Bawa, Shen Guo, Elisse Magnuson, Johnny Xiou and Sean Caffrey Kris Bradshaw, Rachel Peters Funding: 30

31 Thank you for attending! Further Information Sandra Dworatzek siremlab.com