master thesis ppt slide
TRANSCRIPT
ACTIVITY AND SPATIAL DYNAMICS OF PESTICIDE DEGRADERS IN
UNSATURATED POROUS MEDIA By
Kishor Acharya Master in Water Science
University of Duisburg-Essen, Germany [email protected]
Supervisor: Arnaud Dechesne DTU Environment
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