This document summarizes the results of a study examining the effects of experimental acidification and nitrogen addition treatments on carbon cycling in mature broadleaved beech and coniferous spruce forests in the Czech Republic. The key findings are: 1) Acidification treatments significantly reduced dissolved organic carbon losses and soil respiration in both forest types. Nitrogen addition alone did not significantly affect carbon fluxes. 2) The suppression of soil respiration was detected earlier in the spruce stand than in the beech stand. 3) Differences in soil pH, microbial community interactions, and fungal strategies between the two stands help explain their varying responses to acidification treatments over time.

1. Filip Oulehle, Karolina Tahovská, Tomáš Chuman,
Chris Evans, Jakub Hruška, Michal Růžek, Jiří Bárta

2. Some unexpected effects of
declining acid deposition on C
cycling in Czech forests.
2

3. 1. Forest floor carbon pool
5.80 5.33 4.18 3.74
0
5
10
15
20
1994 1997 2003 2010
ForestfloorOM
pool(kgm-2)
Forest floor mass Forest floor C pool
35%
Oulehle, F. Major changes in forest carbon and nitrogen cycling
caused by declining sulphur deposition (2011) Global Change
Biology, 17 (10), pp. 3115-3129.
0
10
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30
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50
1993
1994
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1998
1999
2000
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2003
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2009
2010
2011
2012
DOC(mg/l)
Lysina watershed
2. Dissolved organic carbon (DOC) leaching from forested watersheds
Hruška, J. et al. Changes in soil dissolved organic carbon affect
reconstructed history and projected future trends in surface
water acidification (2014) Water, Air, and Soil Pollution, 225 (7),
art. no. 2015,
30%
Changes in C cycling inferred from long-term monitoring
- integrated understanding of soil C responses to changing
inputs of both S and N is needed to predict whether soils will in
future act as C sinks or C sources as atmospheric pollutant
loadings change 3

4.  a set of replicated acidity/N availability manipulation
experiments in adjacent mature broadleaved (beech forest)
and coniferous (spruce forest) sites
Ctrl
Acid treatment
(H2SO4; 50kg S/ha/yr)
Nitrogen treatment
(NH4NO3; 50kg N/ha/yr)
Combined treatment
(N+S)
Monthly addition
4
2013
Pre-
treatment
2014
Treatment
year
2015
Treatment
year
2016
Treatment
year
2017
Treatment
year

5.  a set of replicated acidity/N availability manipulation
experiments in adjacent mature broadleaved (beech forest)
and coniferous (spruce forest) sites
European beech(ca 140 yr)Norway spruce (ca. 85 yr)
780 m a.s.l.; 1000 mm; 6°C
5

6.  Soil pH (forest floor soil solution)
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3.0
3.5
4.0
4.5
Pre-13 2014 2015 2016 2017
pHunits
CTRL N S S/N
*** *** *** ***
3.0
3.5
4.0
4.5
pre-13 2014 2015 2016 2017
pHunits
CTRL N S S/N
*** ***
***
**

7.  Soil DOC (forest floor soil solution)
Consistent reductions of mean annual DOC concentrations in response to pH manipulation were detected
at both forest stands. DOC concentrations were reduced in acid treatments by 30% - 39% at the spruce
stand, and by 45% - 53% at the beech stand. Nitrogen addition treatments did not lead to significant
changes in forest floor soil water DOC concentrations.
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pre-13 2014 2015 2016 2017
DOC(mg/l)
CTRL N S S/N
* **
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***
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80
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pre-13 2014 2015 2016 2017
DOC(mg/l)
pre-13 CTRL N S
*** *** **

8.  Soil respiration (LiCOR system attached to survey chamber ø 20 cm)
Consistent reductions of mean annual soil respiration were observed under the acid treatments in the spruce
stand in 2015, 2016 and 2017 (p < 0.05), by 6 % - 10 %. No significant effect of N addition on soil respiration
was detected in the spruce stand. In the beech stand, acidity effect on soil respiration was detected only in
2017 (p < 0.05), by 15 %. No significant effect of N addition on soil respiration was detected in the beech
stand.
Reduced soil C efflux by 6 % - 10 % in spruce stand accounted for ca. 530 – 950 kg C ha-1 yr-1 (litterfall = 1937 kg C ha-1 yr-1).
Reduced soil C efflux by 15% in beech stand accounted for ca. 1360 kg C ha-1 yr-1 (litterfall = 3020 kg C ha-1 yr-1).
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pre-13 2014 2015 2016 2017
Rs(µmolCO2/m2/s)
CTRL N S S/N
* * *
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pre-13 2014 2015 2016 2017
Rs(µmolCO2/m2/s)
CTRL N S S/N
*

9.  Microbial characteristics
Significantly lower bacterial gene copies were measured under acid treatments in spruce forest resulting in
higher fungi to bacteria ratio in 2016 and 2017. Therefore, consistent increases of fungi/bacteria ratio
under acid treatments appear to be attributable to decreases in bacterial abundance rather than increases
in fungi. In the beech stand, a significant treatment effect on fungi/bacteria ratio was detected in 2016 only.
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pre-13 2014 2015 2016 2017
Cmic(mmol/g)
ctrl N S SN
**
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pre-13 2014 2015 2016 2017
Cmic(mmol/g)
ctrl N S SN
0.00
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pre-13 2014 2015 2016 2017
F/Bratio
ctrl N S S/N
**
0.00
0.05
0.10
pre-13 2014 2015 2016 2017
F/Bratio
ctrl N S S/N
*
In spruce stand, lower microbial biomass was measured in acid treated plots in 2016 and 2017, which is
consistent with soil respiration measurements. No change in microbial biomass in N treated plots were
detected. In the beech stand neither acidity nor N manipulations affected microbial biomass yet.

10.  Our results provide evidence that soil acidification alters C fluxes
in both deciduous and conifer forests. Dissolved organic carbon
losses were suppressed by acidification in both forest types.
Suppression of soil respiration was detected in the spruce
stand since onset of manipulation, whilst suppression of soil
respiration was detected in beech stand in 2017.
 The two forest ecosystems can by characterized by:
 1) higher soil pH and base saturation in the beech stand;
 2) an apparently higher degree of interactions among the microbial
community in the spruce stand (Bárta et al., 2017);
 3) contrasting fungal life strategies, with saprotrophic fungi more
abundant in the beech soil, and ectomycorrhizal fungi more
abundant in the spruce soil (Bárta et al., 2017).
 Taken together, these features suggest a much stronger
connection of soil metabolism to C acquisition in the spruce
stand compared to beech stand.
10

11.  Nitrogen addition alone did not affect either dissolved or
gaseous C fluxes in either forest type.
 The lack of pronounced effects of N addition on the C cycle at
our sites may be attributable to the high pre-existing levels of
available N in these systems; both forests have been subjected
to elevated N deposition since the 1950s.
 Microbial communities may therefore already be adapted to
increased N availability at our sites, whereas systems receiving
lower levels of ambient N deposition may be more responsive to
additional N inputs
 With regard to dissolved carbon losses (and to a lesser extent
also gaseous C fluxes) it is also possible that the absence of
responses to N addition also reflects the lack of resulting
changes in soil acidity.
11

12. 12
OTU (operational taxonomic units) network analyses of the beech and spruce
prokaryotic communities