Soil properties and plant growing conditions
The soil used in the experiment was an arable loamy haplic Luvisol developed on loess, collected near Göttingen (Germany, 51°33´36.8´´N, 9°53´46.9´´E) from the upper 10 cm of the Ap-horizon. The basic characteristics of the soil are shown in Table 1.
Table 1 Basic characteristics of the soil sampled from the Ap horizon of a haplic Luvisol originated from loess near Göttingen (Germany). CEC: Cation Exchange Capacity; BS: Base saturation. 1Texture according to the German classification system (KA5 2005; Kramer et al. 2012)
The seedlings of ryegrass (Lolium perenne L.) and alfalfa (Medicago sativa L.) were first germinated on wet filter paper for 5 (M. sativa) and 8 days (L. perenne) and thereafter transferred to the plant pots (inner diameter 7 cm, height 20 cm), each of them filled with 700 g of air-dried, sieved (≤ 2 mm) soil. In each pot, 3 seedlings of M. sativa or 5 seedlings of L. perenne were transferred to achieve a similar biomass for both plant species. The pots were closed with a plastic lid with holes for shoots. The plants were grown at 26 to 28 °C day temperature and at 22 to 23 °C night temperature. At a day length of 14 h the light intensity was approximately 210 μmol m-2 s-1, approximately corresponding to a cumulative daily radiation in the range of field conditions. The soil moisture was maintained at 70 % of the available field capacity by daily watering with distilled water.
13C and 15N labelling
To label the soil of all pots with 15N, 16 mg of K15NO3 (enrichment: 52.7 at. %) were dissolved in water and added to the pots with the watering (28 days after planting).
The 13C labelling was conducted for the first time 50 days after planting (the day of clipping or beginning of shading). One day before 13C labelling, all pots were sealed with silicone paste (NG 3170, Thauer & Co., Dresden). All plants were labelled in a Plexiglas chamber as described by Werth and Kuzyakov (2008). Briefly, 13CO2 was introduced to the chamber by circulating air through a flask containing 150 mg of Na2
13CO3 (13C enrichment: 99.9 atm. %) for labelling of L. perenne or 15 mg of the same Na2
13CO3 for M. sativa solved in 10 ml deionized water. To produce 13CO2, an excess of 5 M H2SO4 was added to the Na2
13CO3 solution. The plants were labelled in the 13CO2 enriched atmosphere for 3 h. Before opening the labelling chamber, the chamber air was pumped through 1 M NaOH solution to remove unassimilated 13CO2. Since the amount of 13C found in the NaOH solution was negligible, it can be assumed, that all 13CO2 was assimilated. Then the chamber was opened and the trapping of CO2 evolved from the soil started. 13C labelling was repeated on day 55 after planting.
Clipping and shading
Three pots of each plant species were used for the clipping procedure or exposed to shading. Additionally, three pots of each plant species were grown under normal conditions as a control treatment. The plants were clipped or shaded 2 h before the first 13CO2 pulse. Lolium perenne shoots were clipped 4 cm above the soil surface, those of M. sativa 8 cm above the surface. Due to the different clipping heights, both plant species achieve similar stubble biomass. The clipped plants continued growth under the conditions described above. For shading, the light intensity was reduced to about 17 μmol m-2 s-1 for 10 days.
Sampling and analysis
Starting after the first labelling, the CO2 evolved from soil was trapped using a closed-circulating system. The air was pumped through tubes containing 15 ml of 1 M NaOH solution. Because of the circulation there were no losses of CO2 due to incomplete absorption by NaOH solution. The NaOH solution was changed 1, 3 and 5 days after each labelling. The pots were destructively harvested at day 60 after planting. Roots were separated from soil by handpicking. Plant and soil material was dried at 65 °C for 3 days.
To estimate total CO2 efflux, the C content of the NaOH solution was determined by titration with 0.01 M HCl against phenolphthalein after adding 1.5 M BaCl2 solution. For 13C measurements the CO2 trapped in NaOH was precipitated as SrCO3 with an excess of 0.5 M SrCl2 solution. The precipitants were centrifuged at 3800 g, washed with deionized water until the pH reached neutral conditions and dried at 65 °C.
Microbial biomass C and N was determined by the chloroform fumigation-extraction-method (CFE) (modified after Vance et al. 1987). For this, the soil was separated into two samples with 5 g each. One of these samples was firstly fumigated with chloroform for 24 h. Both samples were extracted with 20 ml of 0.05 M K2SO4, shaken for 1 h and, thereafter, centrifuged for 10 min at 3800 g. Total C and N contents of fumigated and non-fumigated soil extracts were measured using a N/C analyzer (Multi N/C 2100, AnalytikJena, Germany). The extracts of the non-fumigated samples were used to measure dissolved organic carbon (DOC) and dissolved organic nitrogen (DON). For the determination of 13C and 15N in the microbial biomass, DOC and DON the extracts were oven-dried at 60 °C and measured as described below.
The ground plant and soil material (ball mill), the SrCO3 and the dried extracts of the CFE were analyzed for their 13C and 15N isotope ratios. This was done using an elemental analyzer NC 2500 (CE Instruments, Milano, Italy) linked to a delta plus gas-isotope ratio mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) via a ConFlo III (Thermo Fisher Scientific, Bremen, Germany) interface.
Calculations and statistics
The 13C enrichment of a particular C pool (13
C
excess;p
; μg g-1) was calculated as follows:
$$ ^{13 }{C_{excess;p }}=\left( {{{{^{13}}}_{Cp }}{-^{13}}_{CNA;p }} \right)\cdot {C_p} $$
(1)
where 13
C
NA;p
is the 13C natural abunxdance of the respective pool (atom%), 13
C
p
is the amount of 13C of the pool after labelling (atom%), and C
p
is the total amount of C in this pool (μg g-1).
The 13C recovery in a particular C pool (13
C
rec;p
; %) was calculated by dividing the amount of 13C (mg) of that particular pool (13C enrichment multiplied by the pool mass (mg)) by the sum of the 13C amount (mg) of all pools (shoot, root, soil, DOC. soil microbial biomass and soil CO2):
$$ ^{13 }{C_{rec;p }}=\frac{{^{13 }{C_{excess;p }}\times mas{s_p}}}{{\sum {^{13 }{C_{excess;p }}\times mas{s_p}} }}\times 100 $$
(2)
To determine the δ13C value of microbial biomass (δ13
C
MB
; ‰) a mass balance equation was used:
$$ {\delta^{13 }}{C_{MB }}=\frac{{{\delta^{13 }}{C_{fum }}\cdot {C_{fum }}-{\delta^{13 }}{C_{nf }}\cdot {C_{nf }}}}{{{C_{fum }}-{C_{nf }}}} $$
(3)
where δ13
C
fum
(‰) and δ13
C
nf
(‰) are the δ13C values of the fumigated and non-fumigated samples, respectively, and C
fum
(mg) and C
nf
(mg) are the amounts of C in the fumigated and non-fumigated samples, respectively.
The calculations for 15N correspond to those for 13C.
The experiment was conducted with 3 replicates for all treatments. The values presented in the figures and tables are given as means ± standard errors of the means (±SEM). Significant differences between the treatment and the plant species were obtained by a two-factor analysis of variance (ANOVA) in combination with a post hoc Fisher LSD test.