In Situ Bioreactors for In-Well Groundwater Remediation. Eric J. Raes, P.E., LSRP Kerry Sublette

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1 In Situ Bioreactors for In-Well Groundwater Remediation Eric J. Raes, P.E., LSRP Kerry Sublette

2 The Bio-Sep In Situ Bioreactor (ISBR) Enhancement of in situ bioremediation in groundwater with compact bioreactor installed in-well Overcomes common limitations of bioremediation of groundwater Low contaminant concentrations A threshold concentration of substrate is required for growth Substrate inhibition At high concentrations some biodegradable contaminants can be toxic to the organisms that have the ability to degrade them

3 In the beginning.. Adsorptive surface High surface area Bio-Trap Sampler with Bio-Sep Beads X-Section of Bio-Sep Bead Interior of Bio-Sep Bead Nomex and PAC The Bio-Trap Sampler: Cell division inside of Bio-Sep Bead within a Bio-Trap Sampler Rapidly colonized by indigenous bacteria forming active biofilms Thousands used worldwide for over a decade for forensic analysis of groundwater microbiology

4 The Bio-Sep ISBR Bio-Sep beads provide an incredible surface area for microbial growth Gas sparging (air or N 2 ) creates an airlift for circulation of groundwater through the bioreactor. Contaminated groundwater is treated as it moves through the column of Bio-Sep beads 1 ¼ PVC Fits in 2 well Nutrient addition (N, P, electron donors, electron acceptors) support growth of desired indigenous microbes Water exiting the reactor carries contaminant-degrading microbes into the aquifer Air or N 2 and nutrient delivery

5 Topside control Nutrient reservoirs and pumps Air pump Air flow control

6 Bio-Sep ISBR Applications Petroleum hydrocarbons Aerobic Anaerobic Chlorinated hydrocarbons Anaerobic Fuel oxygenates (MTBE, TBA) Emerging contaminants (1,4-Dioxane)

7 Case Study Aerobic ISBR Low Concentrations of Hydrocarbons Fuel oil release impacting soil and groundwater beneath a private residence The bioreactor well & four monitoring wells were installed in the basement Wells spaced 1 ft. apart

8 ISBR Timeline Days -67 through -34: Air sparging only (no Bio-Sep) Days -34 through 0: Air sparging and nutrient delivery (no Bio-Sep) Day 0 onward: Complete bioreactor system operational (Bio-Sep beads added) Bio-traps (Microbial Insights, Inc) used through out testing to monitor microbiology of bioreactor well and monitoring wells

9 qpcr Analysis of BR1 Bio-traps During ISBR Testing Didn t see much difference in hydrocarbon catabolic genes with DNA. This is common observation since hydrocarbon degraders are ubiquitous in the environment.

10 RT-qPCR Analysis of BR1 Bio-traps During ISBR Testing No expression of hydrocarbon catabolic genes until the complete bioreactor system was in service.

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12 Anaerobic ISBRs, the same but different 1 ¼ PVC Fits in 2 well N 2 sparger

13 Case Study Anaerobic ISBR Chlorinated solvent impacted site Fractured bedrock aquifer Deep groundwater impacts (140 bgs) Unfavorable geochemistry Low but measureable DO DO increase with rain event

14 Case Study Anaerobic Bioreactor ISBR installed at a depth of 30 BGS Liquid carbon electron donor Groundwater monitoring Contaminant concentrations Geochemistry qpcr for Dehalococcoides and functional genes for reductive dechlorination (bio-traps and groundwater)

15 Groundwater Contaminants & Microbiology 1.0E E Cells/mL 1.0E E E Concentration (µg/l) 1.0E E DHC BVC VCR DHBt PCE TCE cdce Ethene VC 0 Quarters

16 Dehalococcoides (DHC) Concentration with Depth (Monitored by Bio-traps) Pre-ISBR Post-ISBR Elevation (ft) Elevation (ft) ND E E E E E+04 Cells or gene copies/bead 1.00E E E E E E+05 Cells or gene copies/bead

17 Vinyl Chloride RDases with Depth Pre-ISBR Post-ISBR -60 ND -60 Elevation (ft) ND ND ND ND Elevation (ft) ND ND E E E E E+04 Cells or gene copies/bead 1.00E E E E E+04 Cells or gene copies/bead

18 ISBRs Will Transfer Degraders from One Well to Another In situ treatment and colonization of ISBR Inoculation and/or kick start degradation

19 Transfer of DHC in a TCE Plume nzvi/ Emulsified oil Colonized ISBR installed [VOC], ug/l TCE cis-dce VC Ethene Time (days)

20 When to consider an ISBR Inhibitory contaminant concentrations Dilute plumes (persistent low levels of contaminants) Following ISCO Difficult situations Limited physical access Where one-time amendment injection is not feasible Where bioremediation has failed previously

21 ISBR Limitations Aerobic operation limited to low concentrations of reduced iron (fouling) Radius of influence decreases with increasing hydraulic conductivity of aquifer matrix Works best with contaminants adsorbed by activated carbon

22 ISBR O&M and Costs O&M System checks every 2-4 wks Power Nutrients Water level (ISBR must be totally submerged to function) Costs Life of project rental $10,000 for one unit (ISBR and controller) $15,000 for two units Decreasing per unit costs with addition of more units at a given site

23 Eric Raes BioEnhance, LLC (973) Kerry Sublette BioEnhance, LLC (918)

24 Chemical Oxidation & Aerobic ISBR Case study: Fuel oil release

25 1.0E E+08 Naphthalene degraders Pre- and Present but not active Post-ISCO Present & active Aerobic naphthalene degraders are present but not active Gene Copies/bead 1.0E E δ 13 C DIC ( ) 1.0E E+00 Pre-ISCO ISCO Post-ISCO Biostimulation Post-ISCO MNA NAH Population (DNA) NAH Expression (RNA) DIC Del ( ) 0

26 Naphthalene degraders ISBR Post-ISCO 1.0E E+06 No ISBR Present but not active ISBR Installed Active & mineralizing ISBR Deactivated No activity or mineralization ISBR Reinstalled Active & mineralizing Gene Copies/bead 1.0E δ 13 C DIC ( ) 1.0E ND NA 1.0E+00 0 NAH Population (DNA) NAH Expression (RNA) DIC Del ( )