Bioremediation:The current and the

futureGraeme Paton

Technical Director, RemediosDiarmuid O’Sullivan

Senior Environmental Consultant

Bioremediation

“ The act of stimulating the metabolism or co-metabolism of contaminants in the natural environment. Can be ex-situ, in-situ or involve natural attenuation”

6 months - 3 years (ex-situ usually quicker)

Decomposition of biopolymers

Cell

Oxidation

Endoenzyme Exoenzyme

Dimers

Monomers

Endoenzyme Exoenzyme

Extracellular enzymes

??

½ O2 H2O

CO2

Tool Kit

3 componentsInductionConstraintsConstraintsCommunityCommunity

Induction

Catabolic biosensors enable a prediction of the bioavailability of the target molecule in that matrix

Hence if the genes for degradation are in the matrix, degradation is very likely

Tool Kit

3 componentsInductionInductionConstraintsCommunityCommunity

Procedure for lux-based assay

% o

f th

e c

on

trol lu

min

escen

ce

100 %

10 %

Increasing toxicity

Tool Kit

3 componentsInductionInductionConstraintsConstraintsCommunity

Paton et al 2003

MPN- Tetrazolium

Site Location- Oil spill 1

ToxicityToxicityToxicityToxicity

mpnmpnmpnmpn

InductionInductionInductionInduction

TPHTPHTPHTPH

Tool Kit- at oil spill

Kuwait Oil Lake- Oil spill 2

During the first Gulf war (1990-1991) 50 million tonnes of soil was contaminated

Air-borne oily material contaminated one thousand square kilometres of soil.

Systematic Grid

Can we predict?

Bioremediation (f) [TPH] (f) availability (f) degraders (f) constraints

BF= Induction x [TPH] mpn

x (1-EC)

Prediction

Once all of the data have been integrated an estimation of the likely case for degradation can be made

Iroegbu et al., 2003

TPH

Week 0 Week 15

Prediction v Actual

Prediction

There is a close relationship between the predicted value and the actual hydrocarbon concentration.

This could define the end-pointRegular monitoring confirmed the

progress of this workThis has been extended to over

twenty trials to assess the value of the data

0.1

1

10

100

1000

1 10 100 1000 10000 100000

Max specific deg rate mg TPH/ day

Bioremediation Factor

Prediction

0.1

1

10

100

1000

1 10 100 1000 10000 100000

Max specific deg rate mg TPH/ day

Bioremediation Factor

Prediction

Gaswork Site- 10,000 tonnes

Bulk chemical- 2,500 tonnes

Coupling Technologies

The Big Issues

On four occasions we have been required to seed/ augment a biological treatment process. This is a growing issue and a commercial opportunity.

Bioaugmentation Agents

Inoc. Source Pre-condition

1, 2, 3, 4

Substrate

Remedios

Nutrients

Soil Solution

Examples of Data

Production of CO2 in all samples and controls for both, Universal and Conical flasks experiments. Results given as the average of 4 replicates (mg l-1).

a and b letters represent significant differences (p ? 0.05)

Universal bottles

a

Control 0N:1P 5N:1P 10N:1P 20N:1P

Mean SD Mean SD Mean SD Mean SD Mean SD

Day 1 0.15 0.02 0.10 0.00 0.10 0.01 0.10 0.00 0.09 0.00

Day 3 0.14 0.00 0.14 0.02 0.10 0.01 0.11 0.01 0.10 0.00 Day 7 0.22 0.02 0.19 0.04 0.20 0.04 0.15 0.04 0.15 0.01

Conical flasks

b

Control 0N:1P 10N:2P 20N:2P 40N:2P

Mean SD Mean SD Mean SD Mean SD Mean SD Day 1 1.73 0.17 5.54 2.12 6.85 1.43 3.31 1.174 2.43 0.62 Day 3 0.87 0.09 2.25 0.41 4.76 2.50 2.39 0.156 1.08 0.13 Day 7 0.82 0.13 2.26 0.21 2.40 0.55 2.67 0.612 1.65 0.70

Production of CO2 in all samples and controls for both, Universal and Conical flasks experiments. Results given as the average of 4 replicates (mg l-1).

a and b letters represent significant differences (p ? 0.05)

Universal bottles

a

Control 0N:1P 5N:1P 10N:1P 20N:1P

Mean SD Mean SD Mean SD Mean SD Mean SD

Day 1 0.15 0.02 0.10 0.00 0.10 0.01 0.10 0.00 0.09 0.00

Day 3 0.14 0.00 0.14 0.02 0.10 0.01 0.11 0.01 0.10 0.00 Day 7 0.22 0.02 0.19 0.04 0.20 0.04 0.15 0.04 0.15 0.01

Conical flasks

b

Control 0N:1P 10N:2P 20N:2P 40N:2P

Mean SD Mean SD Mean SD Mean SD Mean SD Day 1 1.73 0.17 5.54 2.12 6.85 1.43 3.31 1.174 2.43 0.62 Day 3 0.87 0.09 2.25 0.41 4.76 2.50 2.39 0.156 1.08 0.13 Day 7 0.82 0.13 2.26 0.21 2.40 0.55 2.67 0.612 1.65 0.70

What does this mean?

Oil Technics- cfu= 108; mpn- 107

Supplier 1- cfu= 106; mpn- 104

Supplier 2- cfu= 106; mpn- 105

Supplier 3- cfu= 104; mpn- 103

Oil Technics and precondition- cfu= 109; mpn- 107

Supplier 1 and precondition- cfu= 108; mpn- 105

Supplier 2 and precondition- cfu= 107; mpn- 106

Supplier 3 and precondition- cfu= 105; mpn- 103

Respiration

Time (day)

0 10 20 30 40 50 60 70

Res

pira

tion

rate

(m

g g-1

of d

ry s

oil)

0.0

0.1

0.2

0.3

0.4

0.5LSD (5%)

Time (day)

0 10 20 30 40 50 60 70

Res

pira

tion

rate

(m

g g-1

of d

ry s

oil)

0.0

0.1

0.2

0.3

0.4

0.5LSD (5%)

RespirationRespiration

DaysDays

Degradation

Time (day)

0 10 20 30 40 50 60 70

% b

iolu

min

osce

nce

0

10

20

30

40

50

60

70LSD (5 %)

Time (day)

0 10 20 30 40 50 60 70

% b

iolu

min

osce

nce

0

10

20

30

40

50

60

70LSD (5 %)

TPHTPH

DaysDays

0.1

1

10

100

1000

1 10 100 1000 10000 100000

Max specific deg rate mg TPH/ day

Bioremediation Factor

Prediction

0.1

1

10

100

1000

1 10 100 1000 10000 100000

Max specific deg rate mg TPH/ day

Bioremediation Factor

Prediction

What does this mean

Augmentation is possibleSteps are required to refineImmobilisation of wood is not idealCost issues and value of application

need to be consideredNeed to focus and develop the skill-

base