soil clean-up by bioremediation

1 Laboratory of Microbial Ecology and Technology

Soil clean-up by Soil clean-up by bioremediationbioremediation

Prof. dr. ir. W. VerstraeteDr. ir. N. Boonir. W. Ossieur

([email protected])

Laboratory of Microbial Ecology and Technology (LabMET)

Faculty of BioengineeringGhent UniversityLabMET.Ugent.be


Topics of discussionTopics of discussion

General considerations

Ex situ clean-up by micro-organisms

In situ clean-up by micro-organisms

Future perspectives






Future perspectives


1.1. General ConsiderationsGeneral Considerations The 33 synthetic organic contaminants reported to be

most frequently found in drinking water wells (Rittman et al.,

1994)

Tetrachloroethene (PCE) Dichloromethane Benzene

Trichloroethene (TCE) Chloromethane Ethyl benzene

1,1-Dichloroethene (DCE) Bromoform Isopropyl benzene

1,2-Dichloroethene Ethylene dibromide Toluene

Vinyl Chloride Dibromochloropropane Xylene

1,1,1-Trichloroethane Dibromochloromethane Cyclohexane

1,1,2-Trichloroethane Trifluorotrichloroethane Acetone

1,2-Dichloroethane Gamma-BHC (lindane) Dioxane

1,1-Dichloroethane Alpha-BHC Di-n-butyl-phtalate

Carbon tetracloride Delta-BHC Butyl-benzyl-phtalate

Chloroform Parathion Bis-(2-ethylhexyl)-phtalate


Many of these toxic compounds serve as food to some types of microbes.

Microbes can eliminate or neutralize many toxic

compounds in the environment

1.1. General considerationsGeneral considerations


1.1. General considerationsGeneral considerations

The application of micro-organisms to clean-up contaminated sites is considered to be a sustainable solution because it is based on:

• the natural degradation capacity of the environment

• a minimal impact on the environment Firms and environmental consulting are the

interested parties for this application


1.1. General ConsiderationsGeneral Considerations

The application of micro-organisms to realize the clean-up of contaminated soils can be done by 3 strategies:

– Natural Attenuation

– Biostimulation

– Bioaugmentation



Monitored Natural Attenuation (MNA):– In situ degradation by ‘indigenous’ bacteria under

natural conditions without intervention of human actions

– Actions: Monitoring of the degradation products produced by the indigenous populations

– Example:Perchloroethylene ethene, Dover (VS), Dehalobacter and Desulfitobacterium, Davis et al. (2002) Journal of Contaminant Hydrology, 57, 41-59.

Cl

Cl

Cl

Cl

Cl

Cl

H

Cl

H

Cl

H

Cl

H

Cl

H

HPCE TCE cis-DCE VC ethene



Principal criteria for the use of MNA– There must be solid indications that clean-up

can be achieved in a reasonable time-span (< 30 years);

– The processes must be aimed at the protection of the site and its surroundings;

– There must be transparent agreements on financial responsibilities over long-term periods, also if certain goals are not reached or unforeseen events occur;

– There must be proper geohydrological monitoring



Biostimulation:– Accelerated natural attenuation by human

intervention– Actions: in situ supplementation of

nutrients to the soil + monitoring of the degradation products produced by the stimulated indigenous population

– Example:Oil, Houston (VS), + inorganic nutrients and alternative electron acceptor, Mills et al. (2004) Marine Pollution Bulletin, 49, 425-435


1.1. General ConsiderationsGeneral ConsiderationsBioaugmentation:

– The inoculation of bacteria into the soil to improve the specific biological activity

– If the degradation by indigenous populations delivers harmful products ordemands too much time

– Actions: in situ supplementation of bacteria and nutrients in the soil + monitoring of the degradation products of the inoculated and stimulated indigenous bacteria

– Example:Tetrachloromethane, Schoolcraft (VS), Pseudomonas stutzeri KC + acetate and nutrients, Dybas et al., (2002) Environmental Science and Technology, 36, 3635-3644.






Future perspectives


2.2. EEx situx situ clean-up by m.o. clean-up by m.o.



Griftpark (Utrecht, 1996)– 10 ha, surrounded by bentonite clay wall

down to 50m into the clay layer– Time scale : 1-3 centuries!– Pump 10 m3/h water (~netto precipitation) to

1) Activated sludge

2) Decantor

3) Sandfilters

4) Activated carbon filter

+ biofilter


2.2. EEx situx situ clean-up by m.o. clean-up by m.o.Griftpark (Utrecht, 1996)

– Time scale : 1-3 centuries!– Price :

• Investment: 150 x 106 EUR or 15 x 106 EUR/ha• Operation: 0,5 EUR/M3 water or 4000 EUR/ha.yr

In (µg/L) Out

COD 120 000

90-95% removal

CN 50

Phenols 250

BTEX 8 000

PAKs 5

Oil 14 000



Membrane-aerated biofilm reactor– No undesired coagulation of iron oxides– No stripping of 1,2 dichloroethane (DCA)

(Hage et al., AMB 64, 718-725, 2004)



Example: Pd-PCB

Biopalladium byBiopalladium byShewanella oneidensisShewanella oneidensis MR-1 MR-1



Catalytic activity in solution?

PCB 21

IntermediatesBiphen

yl

ClCl

Cl

Cl

ClCl

ClCl

Cl

Cl Cl

Cl Cl

PCB 173


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time (min)

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ClCl

Cl

ClCl

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ClCl ClCl

ClCl


Catalytic activity in solution


2.2. EEx situx situ clean-up by m.o. clean-up by m.o. Example: Budelco Groundwater containing:

several mg Zn2+/Lup to 1g SO4

2-/L

Groundwater to discharge

Biopaq – UASB reactor

Aerobic reactor with limited oxygen supply

SO42- S2-

Zn2+ + S2- ZnS To re-use

Excess of S2- S0 To re-use






Future perspectives


3.3. IIn situn situ clean-up by m.o. clean-up by m.o. Several in situ bioremediation strategies (MNA,

biostimulation, bioaugmentation), and several concepts to reach clean-up goals

None of them will be the solution for the total clean-up of a contaminated site (source versus plume)

None of them is the only answer for the problem


Cl

ClCl

Cl ClCl ClCl

Cl

Cl

Cl

ClCl ClCl

ClCl

+ll

VCcis-1,2-DCETCE etheen

1,1,2-TCA 1,2-DCA

propeen1,2-DCP

Ethenes

Ethanes

Propanes

+l 0 -1/2 -l -3/2 -llOx. Degree C

resistance against reductive degradation

FAST DEGRADATION

UNDER ANAEROBIC CONDITIONS

PCE

SLOW DEGRADATION UDNER ANAEROBIC

CONDITIONS

3.3. IIn situn situ clean-up by m.o. clean-up by m.o.


3.3. IIn situn situ clean-up by m.o. clean-up by m.o. Challenges for the anaerobic remediation of

chlorinated ethenes in groundwater– DNAPL formation (inaccessibility)– Microbial degradation mechanism:

reductive dehydrochlorination, but– Accumulation of recalcitrant and carcinogenic

intermediates: cis-DCE and VC due to:• Oxidation degree of the intermediates• Competition for hydrogen

– Complete degradation from PCE to ethene seems to be attainable if Dehalococcoides species are present, but it is not a guarantee!


3.3. IIn situn situ clean-up by m.o. clean-up by m.o. Challenges for the anaerobic remediation of

chlorinated ethenes in groundwater (competition for H2) (required hydrogen pressure)

Complexfermententable

substrates

Simplefermententable

substrates

H2

PCE

TCE

TCE

cis-DCE

cis-DCE

VC

VC

C2H4

CO2 CH4 SO42- HS-

0,6-0,9 0,1-2,5

2-24

5-100 1-10

FBFB

DHB DHB MB SRB


3.3. IIn situn situ clean-up by m.o. clean-up by m.o. The biodegradation of 1,2-dichloroethane by the

anaerobic halorespiring bacteria:Desulfitobacterium dichloroeliminans strain DCA1

The anaerobic strain respires 1,2-DCA and this process delivers energy!

Complete and fast degradationof high concentrations

No toxic intermediates like VC

1 mCl

Cl


3.3. IIn situn situ clean-up by m.o. clean-up by m.o.In situ pilot test in contaminated aquifer

inoculum


inoculuminoculum

Wolk Stam

DCA1

Wolk Stam

DCA1


3.3. IIn situn situ clean-up by m.o. clean-up by m.o.In situ pilot test in contaminated aquifer

Injectionwell

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Time (day)

Co

nce

ntr

atio

n (

µm

ol/L

)

1,2-dichloorethaneEthene


3.3. IIn situ n situ clean-up by m.o.clean-up by m.o. Intensive monitoring of:

• Physico-chemical parameters:pH, T, redox potential, D.O., conductivity

• Chemical parameters:decrease 1,2-DCA and increase etheneno detection of VC and CH4

• Molecular parameters:concentration strain DCA1 through specific molecular techniques

Goal: in situ biodegradation and to obtain data for the simulation of the transport and the activity of strain DCA1 with MOCBAC-3D


3.3. IIn situ n situ clean-up by m.o.clean-up by m.o. Molecular confirmation of the transport of

strain DCA1 from the injection well towards the monitoring well

Activity of the robust strain DCA1 in the injection well and monitoring well confirmed by:– Strong decrease in the 1,2-DCA concentration (e.g. from

1142 to 1µM in a time interval of 36 days)– Increase ETHENE concentration– NO production of vinyl chloride and CH4

Excellent biodegradation capacity of strain DCA1 in reduced groundwater conditions


3.3. IIn situ n situ clean-up by m.o.clean-up by m.o. The degradation of carbon tetrachloride

(CT): field evaluation of a full-scale bioaugmentation technique in a CT- and nitrate impacted aquifer (MI)

Inoculation of Pseudomonas stutzeri KC, a denitrifying bacterium that co-metabolically degrades CT without producing chloroform (CF)

Goal: to establish and maintain a “biocurtain” for CT degradation through– the intermittent addition of base to create

favorable pH conditions;

– inoculation strain KC;

– weekly addition of acetate (electron donor), alkali, and phosphorus.


3.3. IIn situ n situ clean-up by m.o.clean-up by m.o.


3.3. IIn situ n situ clean-up by m.o.clean-up by m.o. The degradation of CT:

– High CT removal efficiencies (median of 98-99.9%)

– Uniform removal efficiencies over a significant vertical depth (15 m), despite significant variability in hydraulic conductivity

– Similar levels of strain KC colonization(>105 strain KC/g)

– Sustained and efficient (98%) removal of CT has been observed over 4 yr

– Low levels of CF (<20 ppb) and H2S (<2 ppm)


3.3. IIn situ n situ clean-up by m.o.clean-up by m.o. Approximately 18,600 m3

of contaminated groundwater was treated during the project

Closely spaced wells and intermittent substrate addition were effective means of delivering organisms and substrates to subsurface environments.

Dybas et al. (2002) Environ. Sci. Technol. 3635-3644



Exxon Valdez (March 1989): spill of33.000 tons of crude oil in Alaska

3500-5500 sea otters out of a total population in the region of approximately 35.000

300000-675000 seabirds perished



Oil degraders:– A lot of bacteria can degrade

environmental pollutants such as oil– Three modes of microbial uptake:

• Utilization of solubilized organic compound• Direct contact of cells (e.g. fimbrae)• Direct contact with fine substrate droplets

– Enhanced uptake by the production of biosurfactans


3.3. IIn situn situ clean-up by m.o. clean-up by m.o.No biosurfactans => cells do not clump and do not stick to oil droplets

oil


Biosurfactans cells stick to oil droplets







Future perspectives


4.4. Future PerspectivesFuture Perspectives A wide variety of bioremediation strategies can be

offered, based on the great diversity of ‘genetic capacity’ and ‘biological know-how’ present in the microbial ecosystem in the soil

These sustainable techniques can and will be applied in the future if:– They are proven to be safe– They are proven to offer protection to the

environment over a long-term periods (monitoring)– They are studied by interdisciplinary research– They are integrated with other clean-up strategies

to achieve a complete answer to the problem


Take-home messageTake-home message

Microbial communities bring about a wide variety of powerfull processes.

We are able to use a number of these processes for soil clean-up purposes and to develop sustainabale and safe biotechnology-techniques.

soil clean-up by bioremediation

Documents

microbial ecology

situ degradation

soil cleanup

organismsin situ

cleanup contaminated

cleanup of contaminated

natural degradation

natural conditions