master thesis ppt slide

ACTIVITY AND SPATIAL DYNAMICS OF PESTICIDE DEGRADERS IN

UNSATURATED POROUS MEDIA By

Kishor Acharya Master in Water Science

University of Duisburg-Essen, Germany kishra@env.dtu.dk

Supervisor: Arnaud Dechesne DTU Environment

arde@env.dtu.dk

25/09/2013

The vadose zone: Unsaturated soil

U.Szewzyk, TU Berlin,Lecture Soil &Sediments

•Host immense density and diversity of prokaryotes

•Receives pollutant (eg., pesticides) and interacts with them

Arrangement of water in soil pores and its effect on microbial processes!!!

The vadose zone: Unsaturated soil

F.E. Moyano et al. (2013)

Wet

co

nd

itio

ns

•Exist as thin liquid film adsorbed to solid soil particles

- discontinuous and fragmented

•Variation in availability of water and nutrients

Soil water- critical for processes in unsaturated soil

• Water transport substrate /pollutant

• Water support bacterial life

Soil water can impose limitations on degrader activity (and pesticide degradation)

Quantifying soil water is important !!!

Quantifying water in unsaturated medium

1. Water content (θ)

-Volumetric water content (θv)

=V water/Vsoil

-Gravimetric water content (θm)

=(g wet soil – g oven dried soil)/ g oven dried soil

-Total porosity

=V pores/ Vsoil

-% water filled pore space

=(θv/ porosity) * 100

2. Water potential (Y)

-measure energy state and biological availability of water

Ytot = Ym + Ys + Yb

Ym : matric potential ( kPa, J/kg)

Ys : osmotic potential

Yb : gravitational and other forces

Link between θ and Y

• Soil water characteristics curve varies accros soil types

• Depends on the soil porosity

M Tuller & D Or (2005)

Soil water status and degraders activity • Dimension of liquid film varies with θ and Y. - degraders activities influenced

• How the varied water saturation state influence the spatial dynamics and activity of pesticide degraders in unsaturated porous medium?

Research Hypothesis

Higher soil water content will enhance the degraders spatial dynamics and activity within the unsaturated porous

medium.

Pesticide Degrader: Sphingomonas sp.erg5

Strictily aerobic, oligotropic, gram negative alpha proteobacteria.

Isolated from ground water sediments

erg5 strain able to degrade MCPA (2-methyl-4-chlorophenoxyacetic acid)

Tagged with Green Fluorescence Protein

Study in unsaturatd soil

• Challenges -Soil complex and spatially heterogenous

-Precise humidity control is difficult

-Soil is opaque

An answer: Simplified experimental systems

- Porous Surface Model: 2-D analogue

- Sand Microcosm: 3-D analogue

Objective of study

• To quantify erg5 surface colonization rate in PSM (2-D) surface at ranges of imposed matric potentials.

• To estimate the dispersion rate of bacteria in sand microcosoms(3-D) of various particle size distributions and moisture content( liquid film thickness).

• To explore the correlation between 2-D and 3-D unsaturated systems on the basis of erg5 dispersion results in these systems.

• To understand the effect of varied water content on MCPA mineralization within sand microcosms with different water contents.

Motility test

Pseudomonas putida KT2440 Sphingomonas sp. erg5

Erg5 is non-motile

Porous Surface Model (PSM): 2D analogue of unsaturated soil pores

Observation of erg5 colony expansion : -0.2 to -2.5 kPa

Dechesne et al. 2008

10% R2B + MCPA (25 mg/L)

Effect of matric potential on erg5 colony diameter

•Diameter of colony increased with time at every matric potential

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

0.00 50.00 100.00 150.00 200.00

Co

lon

y D

iam

ete

r (m

m)

Time (hours)

- 1.1 KPA - 0.2 KPa -2.5 KPa Slopes: 18.4 µm/hr

12.1 ± 2.8 µm/hr 7.5 ± 1.3 µm/hr

Colonization 2.5 × faster at

-0.2 than at -2.5 kPa

For Pseudomonas putida KT 2440 colonization

60 × faster at – 0.5 than at -3.5 kPa (Dechesne et al. 2008 )

Kinetics of surface colonization as a function of matric potentail

y = -6.6633x + 18.462

y = 0.0943x + 6.3582

0

5

10

15

20

25

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8

Rate

of

co

lon

izati

on

(μ

m/h

r)

Matric Potential(-kPa)

•No significant influence on colonization at intermediate and lower matric potential

Surface colonization Cell Growth

+ Random cell dispersion

Sand Microcosm (3-D system)

• Artifical sytem for experimental work • Initial condition can be controlled - substrate amount, water content, matric potentail, innoculation

point

• Estimation of erg5 dispersion rate and MCPA mineralization at different water saturation status.

Types of Sand Matrix in Microcosms • Fine sand (clean quartz sand) • Mixture of fine and rough sand (8:2)

Sand Microcosm Experiments

• Liquid medium: MCPA stock solution (750 mg/L) + R2B medium + Sterile water.

-constant mass of substrate in each microcosm

• Estimate volume of liquid medium for microcosms - construction of Soil Water Characteristics Curve and fitting to parametric model

(e.g., van Genuchten ) for Soil Water Characteristics Curve.

• Embedding erg5 in alginated beads - 4 beads per microcosm

Parameters; m, n, α

Calculation of Liquid film thickness

Fine sand matrix

Mixed Sand Matrix

Schaefer et al. (1995)

Sand Microcosm Experiments

Mixed Sand Fine Sand

2 types of microcosms

3 different water contents

Total : 60 microcosms

Experimental Setup

Sterilize sand

(6 batchs= 3 mixed + 3 fine)

Mix the liquid medium ( leave for

equilibration)

Construct microcosms (sand +

erg5 embedded alginate beads)

Every week sacrificial slicing of

microcosms (8-10 slice)

Weigh and transfer each

slice into MSN +MCPA

liquid medium

Incubation for 2-3 days

Plate and incubate (2-3 days) on R2A

solid medium

Erg5 verification in individual

slice by fluorescence microscopy

Erg5 dispersion in microcosms

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 2 4

Dis

tan

ce f

rom

ino

cula

tio

n p

oin

t (c

m)

Time (weeks)

Fine Sand Matrix

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6 0 1 2 3 4

Time (weeks)

Mixed Sand Matrix

0.266 cm3.cm-3

0.106 cm3.cm-3

0.026 cm3.cm-3

Erg5 Dispersion rate 16.28 μm/hr 10.86 μm/hr 9.10 μm/hr

Erg5 Dispersion Rate 20.35 μm/hr 12.67 μm/hr 8.66 μm/hr

Effect of particle size distributions on erg dispersion

0

2

4

6

8

10

12

14

16

18

0

5

10

15

20

25

0 0.1 0.2 0.3

Liq

uid

film

th

ickn

ess

(µ

m)

Dis

pe

rsio

n r

ate

(µ

m/h

r)

Volumetric water content (cm3.cm-3)

Dispersal rate (fine sand matrix)

Dispersal rate (mixed sand matrix)

Liquid film thickness (fine sand matrix)

Liquid film thickness (mixed sand matrix)

Erg5 dispersion in PSM and Sand Microcosms

0

5

10

15

20

25

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6

Erg5

dis

pe

rsio

n r

ate

(μ

m/h

r)

Matric Potentail (-kPa)

Sand Microcosm PSM

•Difference in geometry of liquid film thickness •Substrate diffusion

t = 16.47 μm

t ~ 1.4 μm

t=1.71μm

t = 1.2 μm

MCPA mineralization

•About 25% reduction in initial mass •Hydration status of microcosms exerted no significant effect on MCPA biodegradation.

0

0.4

0.8

1.2

1.6

2

2.4

0.266 0.106 0.026

MC

PA lo

ad (

mg/

Kg)

Volumetric Water Content (cm3.cm-3)

Mixed Sand Matrix

Initial

Final R1

Final R2

0

0.4

0.8

1.2

1.6

2

2.4

0.266 0.106 0.026

MC

PA L

oad

(m

g/kg

) Volumetric Water content (cm3.cm-3)

Fine sand Matrix

Initial

Final R1

Final R2

Conclusion

• Hydration status and particle size distributions both influenced spatial dynamics of degraders in unsaturated porous medium. However dispersion of pesticide degraders is not limiting factor for pesticide mineralization.

• Almost similar final residual concentration of MCPA within microcosms revealed that the water content soely doesn´t influence the pesticide biodegradation.

• The estimated dispersion rate of erg5 with 2-D and 3-D system showed no correlation. However, the dimension of liquid film created on the surface of both systems controlled the degrader dispersion rate.

• Dispersion rate of pesticide degraders at varied saturation conditions can be estimated successfully with simple microcosm experiment.

Acknowledgements

At DTU Arnaud Dechesne

Uli Klümper Barth F smeths

Lene Kirstejn Jensen

At UDE Wolfgang Sand

Funding CREAM

At GEUS Nora Badawi