1Geomicrobiology & Microbial Ecology, Center for Applied Geosciences, University of Tübingen

Linking N2O emissions from biochar-amended soil to the structure and function of the N-cycling microbial community

Johannes Harter1§, Hans-Martin Krause1§, Stefanie Schuettler1, Reiner Ruser2, Markus Fromme3, Thomas Scholten3, Andreas Kappler1, Sebastian Behrens1*

1Geomicrobiology & Microbial Ecology, Center for Applied Geosciences, University of Tübingen, Germany

2Fertilisation and Soil Matter Dynamics, Institute of Crop Science, University of Hohenheim, Germany

3Soil Science and Geomorphology, Department of Geography, University of Tübingen, Germany

§Both authors contributed equally to this study.

Supplementary Information

*To whom correspondence should be sent:

Sebastian Behrens, Geomicrobiology & Microbial Ecology, Center for Applied Geosciences

University of Tübingen, Sigwartstraße 10, D-72076 Tübingen, Germany

Phone: +49-7071-2975496, Fax: +49-7071-295059

Email:

Geochemical analyses

Total and organic carbon, sulfur and nitrogen were quantified using a Vario EL elemental analyzer (Elementar, Hanau, Germany) according to ISO 10694, 13878 and 15178. Particle size distribution of the soil was determined according to ISO 11277 by sieving and sedimentation. CEC was determined according to ISO 13536. Surface area of the biochar was determined after Brunauer, Emmett and Teller (BET) according to ISO 9277 with a ASAP 2000 (Micromeritics, Norcross, GA). Ash and moisture content were determined according to ASTM D1762. EC and pH for characterization of the biochar were determined in a 1:20 biochar/water solution according to Rajkovich et al (2012) with a TetraCon® 925 conductivity meter (WTW, Weilheim, Germany) and a SenTix® S940 pH meter (WTW, Weilheim, Germany), respectively (Rajkovich et al 2012). Soil pH was determined in a 1:5 solution (deionized water) according to ISO 10390. Particle density of the biochar was determined with a pycnometer according to DIN 18124.

Table S1: Elemental composition of the soil (calcaric leptosol) and the biochar as determined by X-Ray Fluorescence (XRF). Values are given in % of total mass or in mg kg-1.

Fig. S1: Soil microcosms. From left to right: biochar-free control, 2% (w/w) biochar, and 10% (w/w) biochar-containing microcosms. The water-filled pore space (WFPS) was adjusted to 95%.

Table S2: Bacterial strains and primers used for the construction of qPCR-standards.

qPCR-standards for archaeal and bacterial amoA genes have been commercially synthesized.

a vectors obtained from Invitrogen™, Life Technologies Ltd, Paisley, UK.

Table S3: Quantitative PCR reaction mixtures and thermal profiles for the different target genes.

a Same protocol was used for the quantification of nosZ gene copy numbers and nosZ transcript copy numbers.

Table S4: Primers used for quantitative PCR.

a Insertion of a cytosine (underlined) in order to increase target gene coverage.

Table S5: qPCR parameters (efficiency, slope and R2) for evaluation of the different target gene assays.

Fig. S2: Change in nitrogen (a, b) and carbon (c) geochemical parameters in the 2% (w/w) biochar-containing and control soil microcosms over time. Panel a and b show changes in the concentrations of nitrate, nitrite, ammonium and nitrous oxide, while panel c shows the DOC and carbon dioxide data. The small inserted graphs show a magnified view of the data for the first 8 days. Open symbols with dashed lines represent data of the control microcosms without biochar. Filled symbols with solid lines represent data of the soil microcosms with 2% (w/w) biochar. Statistically significant differences (univariate ANOVA, post-hoc: LSD) between control and 2% (w/w) biochar microcosms at a certain time point are indicated by lower-case letters above the individual data points (a = NO3-, b = NH4+, c = N2O, d = NO2-, e = DOC, f = CO2).

Fig. S3: Gene copy numbers per gram dry soil over time for various key genes of microbial nitrogen transformation processes in the 2% (w/w) biochar-containing and control microcosms. Panel a shows changes in total bacterial 16S rRNA and nifH genes copy numbers. In panel b archaeal and bacterial amoA gene copy numbers are shown. Panel c summarizes the gene copy number data for nirS, nirK, and nosZ. The small inserted graphs show a magnified view of the data for the first 8 days. Open symbols with dashed lines represent data measured in the control microcosms without biochar. Filled symbols with solid lines represent data of the soil microcosms with 2% (w/w) biochar. Statistically significant differences (univariate ANOVA, post-hoc: LSD) between control and 2% (w/w) biochar microcosms at a certain time point are indicated by lower-case letters above the individual data points (a = nifH, b = nosZ, c = nirS).

Fig. S4: Ratio of water- to K2SO4-extractable (0.5 N) NO3- (black bars) and NH4+ (gray bars) in the water-saturated (95% water-filled pore space) soil microcosms with different amounts (w/w) of biochar after 24h of incubation at room temperature. Data has been normalized to the K2SO4-extractable NO3- and NH4+. The amount of water-extractable NO3- and NH4+ is expressed as fraction of the K2SO4-extractable NO3- and NH4+. % BC refers to the amount (w/w) of biochar added to the individual soil microcosms.

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