In Situ Bioreactors for In-Well Groundwater Remediation

Eric J. Raes, P.E., LSRPKerry Sublette

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

In the beginning…..

The Bio-Trap® Sampler:• Rapidly colonized by indigenous

bacteria forming active biofilms• Thousands used worldwide for

over a decade for forensic analysis of groundwater microbiology

Cell division inside of Bio-Sep® Bead within a Bio-Trap Sampler

X-Section of Bio-Sep® Bead Interior of Bio-Sep® Bead

Bio-Trap® Sampler with Bio-Sep® Beads

Nomexand PAC

• Adsorptive surface• High surface area

The Bio-Sep ISBR• Bio-Sep beads provide an incredible

surface area for microbial growth• Gas sparging (air or N2) creates an

airlift for circulation of groundwater through the bioreactor. • Contaminated groundwater is

treated as it moves through the column of Bio-Sep beads

• 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

1 ¼” PVCFits in 2” well

Air or N2 and nutrient delivery

Topside control

• Nutrient reservoirs and pumps

• Air pump• Air flow control

• Petroleum hydrocarbons• Aerobic• Anaerobic

• Chlorinated hydrocarbons• Anaerobic

• Fuel oxygenates (MTBE, TBA)• Emerging contaminants (1,4-Dioxane)

Bio-Sep ISBR Applications

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

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

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.

RT-qPCR Analysis of BR1 Bio-traps During ISBR Testing

No expression of hydrocarbon catabolic genes until the complete bioreactor system was in service.

Anaerobic ISBRs, the same but different

N2 sparger

1 ¼” PVCFits in 2” well

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

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)

Groundwater Contaminants & Microbiology

0

200

400

600

800

1000

1200

1400

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

0 1 2 3 4 5

Conc

entr

atio

n (µ

g/L)

Cells

/mL

DHC BVC VCR DHBt PCE TCE cDCE Ethene VC

Quarters

Dehalococcoides (DHC)Concentration with Depth

(Monitored by Bio-traps)

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04

-140

-105

-85

-60

Cells or gene copies/bead

Elev

atio

n (ft

)

ND

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05

-140

-105

-85

-60

Cells or gene copies/bead

Elev

atio

n (ft

)

Pre-ISBR Post-ISBR

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04

-140

-105

-85

-60

Cells or gene copies/bead

Elev

atio

n (ft

)

Vinyl Chloride RDases with DepthPost-ISBR

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04

-140

-105

-85

-60

Cells or gene copies/bead

Elev

atio

n (ft

)

ND

NDND

NDND

NDND

Pre-ISBR

ISBRs Will Transfer Degraders from One Well to Another

In situtreatment and colonization of ISBR

Inoculation and/or kick start degradation

Transfer of DHC in a TCE Plume

0

500

1000

1500

2000

2500

3000-9

00

-870

-840

-810

-720

-630

-540

-450

-360

-270

-180 -90 0 30 60 90 120

150

Time (days)

[VO

C],

ug/L TCE

cis-DCEVCEthene

nZVI/

Emulsified oil

Colonized ISBR installed

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

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

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

Eric RaesBioEnhance, LLCeric@bio-enhance.com(973)219-6185

Kerry SubletteBioEnhance, LLCksublette@microbe.com(918)691-0639

Chemical Oxidation & Aerobic ISBR

Case study: Fuel oil release

Naphthalene degraders Pre- and Post-ISCO

0

2000

4000

6000

8000

10000

1.0E+00

1.0E+02

1.0E+04

1.0E+06

1.0E+08

1.0E+10

Pre-ISCO ISCO Post-ISCOBiostimulation

Post-ISCOMNA

δ13 C

DIC

(‰)

Gen

e Co

pies

/bea

d

NAH Population (DNA) NAH Expression (RNA) DIC Del (‰)

Present but not active

Present & active Aerobic naphthalene

degraders are present but not

active

0

2000

4000

6000

8000

10000

1.0E+00

1.0E+02

1.0E+04

1.0E+06

1.0E+08

δ13 C

DIC

(‰)

Gen

e Co

pies

/bea

d

NAH Population (DNA) NAH Expression (RNA) DIC Del (‰)

ND NA

Naphthalene degraders ISBR – Post-ISCO

No ISBRPresent but not active

ISBR DeactivatedNo activity or mineralization

ISBR InstalledActive &

mineralizing

ISBR Re-installedActive &

mineralizing