Soil moisture effects on soil carbon cycling –
a 13CO2 pulse labelling experiment in a sandy
heathland ecosystem
Sabine Reinsch, Per Ambus, Iver Jakobsen
Risø, Technical University of Denmark
NordSIR | Holbæk | 05 October 2011
1
Carbon, Microbes and Climate Change
Amundson, 2001
new carbon
a) Temperature effect
old carbon
Briones et al., 2009
c) Effects of increasing [CO2]
b) Effects of water stress
decreasing C
storage
as a result of a changed
microbial community
Carney et al., 2007
slower C mineralization
Andresen et al., 2010
2
Microbes turn the wheel of carbon and nutrient turnover in soils
13
CO
CO22uptake
uptakevia
via
photosynthesis
C losses
C inputs to the soil
Manipulation
13C
12C
= 1.11 %
= 98.89 %
50 %
50 %
Plant
respiration
Soil respiration (SR)
 Roots
 Microbes
 Fauna
Leaf litter
Ecosystem
respiration
(ER)
Rhizodeposition
Delayed C
transport into
roots
SOM
 Rhizosphere bacteria
 Fungi
Roots directly colonized/consumed by
 Rhizoplane bacteria
 Mycorrhizal fungi
 Soil fauna
Microbial functional
groups
 AM fungi
 Saprophytic fungi
 Gram negative bacteria
 Gram positive bacteria
 Actinomycetes
3
In-situ climate manipulations to study future C turnover
Pilot
experiment
Climate
experiment
Treatments
C, D, I
D, T, eCO2
in full combination
Replicates
5
6
September 2010
May 2011
7h
4h
Labell (aim)
Labell (achieved)
45 atom% 13C
13 atom% 13C
50 atom% 13C
45 atom% 13C
CO2 concentration
390 ppm
390 and 510 ppm
Time
Labelling time
Measurements
Soil and ecosystem respiration,
microbial fatty acids, microbial
biomass, plant labell
CO2
CO2
+T
CO2
+D
CO2 +
D+ T
www.climaite.dk
4
In-situ
13CO
2
pulse-labelling
Flow-Through-System
Air reservoir
(air with
~45 atom% 13CO2;
~390 ppm [CO2])
Air flow rate: 12.3 l*min-1 to achieve a gas
exchange in the chamber ~every 10 min
chamber
pump
5
Constant Labell over time in the air reservoir :)
13C atom% over time
Pilot experiment
13C atom% over time
Climate experiment
50
balloon
chamber
50
50
40
40
40
reservoir
chamber
30
30
30
30
20
20
20
10
10
10
40
13C atom%
atom%
13C atom%
40
13C
50
50
30
20
20
10
10
balloon
chamber
0
0
11
22
33
44
55
66
Sampling time points [h]
77
0
0
0
11
22
Sampling over time [hours]
33
44
55
Sampling time points [h]
6
Stable Carbon Dioxide concentration in the air reservoir
CO2 concentration
Ambient (390
ppm) over time in non
[CO2] in ppm
CO2concentration
concentration[ppm]
[ppm]
CO
2
600 600
reservoir
600
CO2 concentration
Elevated (510
ppm) over time in CO
600
600
500
500
500 500
500
400
400
400 400
400
300
300
300 300
300
200
200
200 200
200
chamber
11
22
33
44
Sampling time points [h]
55
11
22
Sampling over time [hours]
33
44
55
Sampling time points [h]
7
Chamber technique – CO2 flux measurements
13Catom%
1) Keeling plots (Keeling, 1958)
intercept = source C
13Catom% ”signal”
2) Two-endmember mixing model
source1
Source2 = recently fixed C
SOM δ13C
chamber δ13C
mix
1/[CO2]
”Keeling” δ13C
(source1 – mix)
(source1 – source2)
= % Source2
3) CO2 flux calculations with the new HMR package in R
 for closed chamber measurements
 linear and non-linear estimates of carbon (and N2O) efflux (Pedersen et al. 2010)
8
Short term carbon flow affected by water stress
Tendency towards a slower recent C turnover under drought – reasons?
13
4 0.04
0.08C0.08
W
0.060.06
D
0.040.04
3 0.03
2 0.02
C
D
C
D
320320
300
280280
260
240240
220
200200
C
Treatment
CC.1 DD.1
D
I
I
Treatment
day 6
day 12
day 1
D
C
I
I
0.01
0 0.00
Microbial C (ug C g-1 dry soil)
deDato, 2010
0.100.10
5 0.05
1
Microbial C (μg C g-1 dry soil)
-1
Respired KgCorrected
CO2 m-2plant
periodCatom%
Recently fixed carbon Recently
(%) released
fixed carbon (%) by SR
6 0.06
Corrected plant 13Catom% excess
Treatments effects on recently fixed carbon (Soil respiration)
Treatment influence on microbia
Treatment effects on 13C incorpora
Microbial biomass
C
Plant 13Catom%
excess
day 29
day 30
day 2
II.1
C.2
C
DD.2
II.2
C.6
C
DD.6
II.6
C.12
C
DD.12
II.12
C.29
D.29
C
D
I.29
I
CC.30 DD.30 II.30
Treatments over time
Treatments over time (days after labelling)
9
CO2 flux [umol CO2/m2*sec]
CO2 flux [μmol CO2/m2 sec]
CO2 flux [umol CO2/m2*sec]
Carbon dioxide fluxes under future climatic conditions
Soil respiration fluxes over time
Soil respiration
4
A
CO2
A
CO2
D
D
DCO2
DCO2
T
TCO2
T
TCO2
TD
TDCO2
TD
TDCO2
4
4
2
2
2
0
0
0
-2
-2
-2
11
00
22
88
Days after labelling
respiration fluxes over time
EcosystemEcosystem
respiration
Plant respiration fluxes over time
Plant respiration
5
5
5
5
5
4
4
4
4
4
3
3
3
3
2
2
2
2
1
1
3
2
1
1
1
0
0
0
0
0
-1
-1
-1
-1
22
88
2
Days after labelling
2
8
8
Days after labelling
Time after labelling (days)
10
Effects of climate on recently fixed carbon [%]
Treatments effects on recently fixed carbon (Soil respiration)
Recently fixed carbon (%)
0.02
Day 1
0.020
Day 8
Day 2
0.015 0.015
A
TD
TDCO2
T
TCO2
D
CO2
T
DCO2
TD
TDCO2
A
0.0050.005
0
CO2
CO2
TCO2
A
TDCO2
T
TCO2
D
0.010
DCO2
0.01
DCO2
D
TD
0.000
A.11
D.11
T.11
TD.11
A.12
D.12
T.12
TD.12
A.13
D.13
T.13
TD.13
Treatments over
time
Treatments
over(days)
time
11
Conclusions and outlook
1) The heathland was probably not water limited during autumn (anymore) 
microbes not water stressed
 because we lack a faster C turnover in irrigated plots
2) Drought stressed plots stored more recent carbon aboveground probably due to a
time lag in plant development.
3) Future contribution of soils to atmospheric CO2 concentrations might not be
different from todays. Drought and high temperature periods might influence the
global carbon balance…
 PLFA and NLFA analyses are pending
 will give information about fungal:bacterial ratios and microbial community
activity according to treatments
12
Thanks to…
Special thanks to:
 Per Ambus
 Helge Egsgaard (FA)
 Marie Merrild (FA)
 Climaite PhDs and researchers
 Risø gasification group
 Risø eco group
 technicians and student helpers
13
Microbial fatty acids (FA) as biomarkers coupled with GC-c-IRMS
PLFAs and NLFAs
roots/soil
CHCl3MeOHbuffer mix
prepacked
columns
C17:0
13C
13C
13C
GC-c-IRMS
i15:0
13C
13C
13C
Intensity [mV]
a15:0
16:0
Mild alkaline
methanolysis
C19:0
C18:1
i15:0
13C
FA coupled with stable isotopes
- culture independent
Time [sec]
14
Microbial community composition and C cycling activity
Ambient
Elevated
CO2double bond position:
 pending
fatty acid identification:
determination
of the
Plant δ13C

C16:1 can be
C16:1ω5 – AM fungi
C16:1ω7 – gram negative bacteria
C16:1ω9 – bacteria in general
 rhizosphere soil is not sufficiently labelled for microbial activity analysis

root samples are the next step - definitely labelled :)
Jin & Evans 2010
15
Amundson (2001)
Andresen LC, Michelsen A, Jonasson S, Schmidt IK, Mikkelsen TN, Ambus P and Beier C (2010) Plant nutrient mobilization in temperate heathland
responds to elevaleted CO2, temperature and drought. Plant Soil 328: 381-396.
Keeling CD (1958) The concentration and isotopic abundances of atmospheric carbon dioxide in rural areas. GeochimicaCosmochimicaActa 13: 322334.
Selsted MB (2010) Ecosystem-atmosphere exchange of carbon in a heathland under future climatic conditions. PhD thesis, Risø DTU.
Carney KM, Hungate BA, Drake BG and Megonigal P (2007) Altered soil microbial community at elevated CO2 leads to loss of soil carbon. PNAS 104:
4990-4995.
Mikkelsen TN, Beier C, Jonasson S, Holmstrup M, Schmidt IK, Ambus P, Pilegaard K, Michelsen A, Albert K, Arndal MF, Bruun N, Christensen S,
Danbæk S, Gundersen P, Jørgensen P, Linden LG, Kongstad J, Maraldo K, Priemè A, Riis-Nielsen T, Ro-Poulsen H, Stevnbak K, Selsted MB, Sørensen
P, Larsen KS, Carter MS, Ibrom A, Martinussen I, Miglietta F and Sverdrup H (2008) Experimental design of multifactor climate change experiments
with elevated CO2, warming and drought: the CLIMAITE project
Pedersen AR, Petersen SO and Schelde K (2010) A comprehensive approach to soil-atmosphere trace-gas flux estimation with static chambers.
Europ J Soil Sci61: 888-902.
Good review on pulse and long-term labelling: Meharg A (1994) A critical review of labelling techniques used to quantify rhizosphere carbon flow.
Plant Soil 166:55-62
Denmark map (slide2): http://www.google.dk/imgres?imgurl=http://mappery.com/maps/DenmarkMap.mediumthumb.jpg&imgrefurl=http://metagini.com/location/Denmark/Gistrup/70663/Pictures/&usg=__ucT6UYpP83YPsIvkSZVcPyNnJ8=&h=492&w=600&sz=104&hl=da&start=21&zoom=1&tbnid=w5Mm75bCEKpiFM:&tbnh=151&tbnw=184&ei=4I8JTo7dCMvrObm1zLIB&prev=/search%3Fq%3Dden
mark%26um%3D1%26hl%3Dda%26client%3Dfirefox-a%26sa%3DN%26rls%3Dorg.mozilla:enGB:official%26biw%3D1274%26bih%3D841%26tbm%3Disch&um=1&itbs=1&iact=rc&dur=506&page=2&ndsp=20&ved=1t:429,r:17,s:21&tx=136&ty=83&biw=1274&bih=841
Gram positive cell : http://www.ncbi.nlm.nih.gov/books/NBK1945/