Enzymatic activities in crop and grassland
soils - Impact of copper addition
Isabelle Trinsoutrot-Gattin, Marthe Vinceslas-Akpa,
Caroline Bailleul, Agathe Brault, Christian Mougin and
Karine Laval
Introduction
• Soil : non-renewable resource essential for life
• Several functions :
•
•
•
•
Physical and cultural environment for humankind
Food and biomass production
Storing, filtering and tranformation of organic matter
Habitat and gene pool of living organisms...
• Pressures : agricultural production, urban and
industrial development, transport networks, leisure
activities…
• Necessity to guaranty the protection and sustainable
use of the soil
!Project of European policy 2006.
Context
• Current tools in agronomic practices : physical and
chemical properties
• Soil organisms responsible for biogeochemical cycles, ...
• This study is a part of a vast project intending to elaborate
an index of the biological state of soil.
! microbial, fungal and bacterial biomass
! Enzymatic activities
• Enzymatic activities :
• Soil functioning
• Scientifically recognized
• Integrative tool : pedological characteristics, climate,
soil management
Objectives
" Impact of copper on soil enzymatic activites
" Considering the natural variations of the
enzymatic activities in our referenced agricultural
systems (grassland and crop plots)
Material and Methods
Enzymatic activities assessed
Enzymes
Dehydrogenase
Substrate
1,3,5-triphenyltetrazolium chloride (TTC)
pH
7
Acid Phosphatase
p-nitrophenyl phosphate (PNP)
6,5
Alkaline Phosphatase
p-nitrophenyl phosphate (PNP)
11
β-glucosidase
p-nitrophenyl b-glucoside (PNP)
6
p-nitrophenyl acetyl glucosaminide (PNP)
6
urea
7
N-acetyl glucosaminidase
Urease
Dick et al. (1996)
Material and Methods - Field plots
Two sites, located in the North West of France
20 samples per plot
1m
0-10cm, 4-kg
Yvetot
Meadow pH 6,5
18.2% clay
62.1% silt
19.7 sand
Crop pH 6,8
April, June, October
Meadow pH 5,8
Saint Georges sur
Fontaine
18.0% clay
67.2% silt
14.8% san
Crop pH 6,8
Material and Methods
Cosme experimentation
Undisturbed cosmes sampled from
Yvetot grassland and crop plots.
5 cosmes for each model
4 dates: 0, 7, 35 and 70 days
Incubation in field conditions
12cm
Models : Control
2 ppm Cu
200 ppm Cu
15 cm
Results
Acidic, alkaline phosphatases
and b-glucosidase activities
exhibit a great variability
3500
enzymatic
activites
nmol
Enzymatic
activity
in nmolinhydrolysed
-1 -1
-1
-1
hydrolysed
product
product
g h g h
Temporal and spatial
variations for enzymatic
activities
Spatio temporal variability in Yvetot meadow soils
3022.4
3000
2500
2508.8
1959.8
2000
1500
1000
551.7
500
0
PAC
enzymatic
in nmol
Enzymatic
activityactivity
in nmol hydrolysed
-1 -1
h
product
g
hydrolysed product g-1 h -1
B-glu
NAG
DH
URE
Spatio temporal variability in Yvetot crop soils
3500
The level of activities is
higher in grassland than in
crop plots
PAL
3000
2500
2000
1842.5
1500
1291.4
1000
640.8
500
263.0
0
Yvetot site
PAC
PAL
B-glu
NAG
DH
URE
Enzymatic activities in Yvetot plots and in cosmes
cosmes versus plots - Yvetot Meadow
900
day 0
nmol PNP g-1 h-1
800
700
600
day 70
500
400
300
200
100
0
acid-P
cosme J0
alkaline-P
Field plot J0
b-glu
cosme J70
NAG
Field plot J70
Enzymatic activities are similar in plots and control cosmes
! Validation of our undisturbed cosmes approach
Impact of the two copper concentrations on
enzymatic activities in crop cosmes
Acid phosphatase - crop
b-glucosidase - crop
800
600,00
12
500,00
10
500
400,00
8
400
300,00
6
200,00
4
100,00
2
nmol PNP g-1 h-1
700
600
300
200
100
0
0,00
0
0
7
35
0
70
600
250
0
7
35
70
200
150
100
2 ppm Cu
200 ppm Cu
7
35
70
15,00
10,00
5,00
0,00
0
0
35
days after Cu inoculation
0
days after Cu inoculation
control
7
Urease - crop
50
0
0
70
n m o l N - N H 4+ g -1 h -
300
nmol PNP g-1 h-1
800
200
35
N Acetyl Glucosaminidase - crop
Alkaline phosphatase - crop
400
7
days after Cu inoculation
days after Cu inoculation
n m o l PN P g -1 h -1
dehydrogenase - crop
70
7
35
70
days after Cu inoculation
days after Cu inoculation
control
2 ppm Cu
200 ppm Cu
control
2 ppm Cu
200 ppm Cu
Impact of Cu on enzymatic activities :
Literature controversy
" Differences in experimental conditions
! Sieved soil vs undisturbed cosmes
. microbial physiological status
. physical protection on soil microorganisms
. availability of copper
! High Cu concentration vs agronomic reality
. Status of copper
. Distribution of copper
Distribution of inoculated copper in
microcosms
Content (mg) after equilibration
Cu 330mg/25mL
30
15cm
35
6
32
35
19
13
12cm diameter
13
16
Cu distribution in cosmes
Copper distribution according height
160,00
140,00
amount of Cu (ppm) mg
120,00
100,00
CP Cu 200ppm
80,00
60,00
40,00
20,00
0,00
0 - 51cm
2
3
5 - 10
4 cm
5
6
10 - 7
15 cm
Evaluation of the copper impact on the descriptors of the
microbial communities
C16:1w5
Fungal DNA
1
ADN Fong.
N-acétyl-β-D-Glucosaminidase
Meadow (reference)
Meadow (200ppm Cu)
Crop
Crop (200ppm Cu)
Ergostérol
β-D-Glucosidase
0
Microbial DNA
ADN total
Phosphatase Acide
Cultivable R2A
Bactéries Totales
Bacterial DNA
ADN Bact.
Results at T1
Conclusion
"Spatiotemporal variability of enzymatic activities ranged
from 6 to 95 % in referenced systems
"Total enzymatic activities are higher in meadow related
to soil biomass increase
" Anova analysis shows :
! Agricultural practices
! Sampling date (including season and plant cover)
! No copper effect
Conclusion
"Enzymatic activities as indicator of soil « quality »?
"Links between microbial communities structure and
functional diversity?
"Enzymatic activity of interest ?
!stage of organic matter decomposition ?
"Methodological strategies for Soil Science :
!pH?
!Substrate?
!spectrophotometry versus fluorimetry?
!Units?
" Agronomic decision tools?
Thank you for your attention