4.2.1 Incubation experiment and experimental design
For the laboratory incubation experiment, ten different soil mixtures with four replicates each were placed in PVC tubes (21.5 cm long and 4.5 cm diameter) inside an incubator at temperatures ranging from 23.5−26.5 °C for six months (Appendix C: Figure C.1). The treatments consisted of a control containing 350 g (dry weight) of sandy soil, and nine more soil mixtures containing the same amount of soil with an addition of a mineral fertilizer or four different types of compost with two different application rates (Table 4.1; Appendix C: Table C.1). The height of the different soil mixtures filled inside the PVC tubes ranged from 13.7−14.9 cm. The sandy soil was prepared by mixing a standard silty sand agricultural soil (uS, No.
2.1, LUFA Speyer, Germany) and quartz sand (Quarzwerke, Germany) on a 1:5 ratio (dry basis). The mixture had a moisture content of 9.9 %, water holding capacity of 19.3 %, bulk density of 1.3 g cm-3, and classified as class A soil with an available fraction of P and K of <15 mg kg-1 and <30 mg kg-1 for sandy soil, respectively (Landwirtschaftskammer Niedersachsen 2018; VDLUFA 1999, 2018). The mineral fertilizer used was a NPK 15-15-15 fertilizer with 6.3 % NO3--N, 8.7 % NH4+-N, 15 % P2O5, 15 % K2O and 3.7 % SO3 (Sulfan Mila Universal, YARA GmbH & Co. KG, Dülmen, Germany). The four types of compost used were human excreta (from now on called “humanure”), cattle manure compost, and each of these composts with biochar addition. We produced these organic fertilizers by thermophilic composting of humanure or cattle manure, vegetable scraps, teff straw, sawdust with and without biochar (18.5 % of compost dry weight) as described in chapter 2 (section 2.2.2). The main physical and chemical properties of the soil and compost treatments are listed in Table 4.1. The biochar in the amended composts was produced from Eucalyptus wood (Eucalyptus camaldulensis) with a top-lit up-draft micro-gasifier at pyrolysis temperatures between 500−600 °C and a residence time of 40−50 min. The biochar fulfilled the
“premium” requirements of the European Biochar Certificate, except for the polycyclic aromatic hydrocarbons (PAHs) concentration, which exceeded the premium threshold value, but complied with the
“basic” criteria (EBC 2012).
The amount of mineral fertilizer and compost for the lower application rate (Table 4.1) followed the regulations of the German Fertilizer Ordinance ("Düngeverordnung"; Bundesministerium der Justiz und für
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Verbraucherschutz and Bundesamts für Justiz 2017) and of the Chamber of Agriculture of Nordrhein-Westfalen (Landwirtschaftskammer Nordrhein-Nordrhein-Westfalen 2015) for N application for maize. According to these regulations, the N requirement of maize is 180−200 kg ha-1 and the application of compost in agricultural fields must not exceed an average amount of total N of 510 kg ha-1 over a period of three years.
To calculate the amount of mineral fertilizer and compost for the lower application rate, we considered a N requirement of maize of 200 kg ha-1 and from this, we subtracted: 1) the amount of available N (sum of NH4+-N and NO3--N) in the soil (14 kg ha-1 in 13.7 cm), and 2) a soil N mineralization potential during the first two months of the cultivation period of 10 kg ha-1 (Landwirtschaftskammer Nordrhein-Westfalen 2015). The higher compost application rate was set three times higher than that of the lower compost application rate (Table 4.1).
The amount of water in the soil mixtures was adjusted to 60% of the water holding capacity of the control treatment and was gravimetrically maintained during the incubation period by irrigating each tube once a week according to the evaporative weight loss.
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Table 4.1. Description and nutrient application rates per treatment and amount of available nutrients and dissolved organic C and N on day 0. Values provided as means with n=4 and in dry weight basis. Different superscript letters indicate significant differences at p<0.05 among treatments.
Treatment Description
Amount of fertilizer (kg ha-1) Amount extracted from soil mixtures on day 0 (mg kg-1)
pH on day 0
Total Available Dissolved
N P K N P K organic C N
Control Soil - - - 6.2 a 6.0 a 10.0 a 3.4 a 2.2 a 4.9 a
Mineral fertilizer and lower compost application rate (according to the German Fertilizer Ordinance)
MF Soil + mineral fertilizer (NPK 15-15-15) 176 76 146 81.6 e 44.7 bcd 63.0 ab 4.9 ab 18.7 c 4.9 a
HM1 Soil + humanure compost 177 73 347 20.3 abc 35.3 bc 106.7 b 10.7 bcd 6.8 ab 6.1 b
HM+BC1 Soil + humanure with biochar compost 175 73 404 20.8 abc 25.3 abc 113.3 b 10.5 abc 5.0 a 6.4 b
CM1 Soil + cattle manure compost 176 68 295 13.5 abc 33.3 bc 101.3 b 8.4 abc 3.3 a 6.3 b
CM+BC1 Soil + cattle manure with biochar compost 176 56 296 11.8 ab 23.0 ab 80.7 b 13.0 cd 5.2 a 6.4 b Higher compost application rate (three times more)
HM2 Soil + humanure compost 531 220 1041 56.0 d 70.0 de 314.0 de 22.3 de 17.1 c 6.9 c
HM+BC2 Soil + humanure with biochar compost 526 220 1212 22.5 bc 68.3 de 324.0 e 23.9 e 12.9 bc 7.0 c
CM2 Soil + cattle manure compost 527 203 886 26.7 c 75.3 e 256.0 cd 17.8 de 5.9 a 7.0 c
CM+BC2 Soil + cattle manure with biochar compost 527 168 889 42.0 d 50.7 cde 228.0 c 20.9 de 7.5 b 6.9 c
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We measured fluxes of CO2, CH4, and N2O from the PVC tubes containing the incubated soil mixtures with the dynamic chamber method. The chamber consisted of a PVC lid with an inner diameter of 4.5 cm and a height of 10.6 cm, and was equipped with a rubber seal which fit gas-tight on the PVC soil columns. The chamber lid was equipped with two stainless steel tube fittings (¼ in) as sampling ports. The sampling ports were connected with 2.37 m long tubing in closed-loop mode to an infrared laser absorption analyzer (G2508, Picarro, Inc., Santa Clara, CA, USA; Appendix C: Figure C.2). A 1-m long open tubing with an inner diameter of 4 mm was connected to a third gas-tight stainless steel tube fitting (¼ in) as vent tube to minimize pressure perturbations during chamber deployment. The headspace comprised a soil surface area of 0.0016 m2, and, depending on the treatment. a height of 13.7−14.9 cm and a volume of 0.00019−0.00021 m3.
We carried out GHG flux measurements on days 0, 1, 2, 3, 5, 8, 15, 21, 29, 36, 44, 50, 57, 67, 71, 78, 86, 99, 113, 127, 141, 155, 169, and 180 with a deployment time of the analyzer of 10 min.
We calculated the GHG fluxes and expressed them as mg of CO2-C m-2 d-1, µg of CH4-C m-2 d-1, or µg of N2O-N m-2 d-1 by using Eq. 4.1.
FluxGHG = 1000000S × R TP × VA × M × 60 (Equation 4.1)
where S is the slope of the linear equation fitted to the change of the gas concentrations in the chamber during the measurements (ppm min-1); 1,000,000 is used to convert ppm (µL L-1) into the unit L L-1; P is the air pressure (atm) at 80 m a.s.l.; R is the universal ideal gas constant (m3 atm mol-1 K-1); T is the average temperature (K) of the chamber during the deployment time; M is the molar mass of C or N (g mol-1); and 60 the value used to convert the unit time from min to hours. The concentration values obtained during the first 2 min were not used for the calculation of the slope due to the fluctuations produced by pressure disturbances during the closure of the chamber. Fluxes with linear fits of R2 < 0.81 were considered below the detection limit and therefore, we reported them as zero. We estimated the cumulative emissions by calculating the emission average rate of two consecutive measurement days and multiplying it by the time between these two measurements. Thereafter, we summed up all the resulting values and expressed the cumulative fluxes as g C m-2 for CO2and CH4, and mg N m-2 for N2O, and also as % of initial C or N.
Compost degradation rates after 180 days of incubation were expressed as % of organic C added with the compost and calculated based on the cumulative CO2-C emissions attributed only to compost decomposition, i.e. by subtracting the cumulative CO2-C emissions of the control from the compost treatments, by using Eq. 4.2.
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Compost decomposition rate (%) = TOC - ( COTOC2-C × A) × 100 (Equation 4.2)
where TOC is the total organic C added through compost (mg); CO2-C the cumulative CO2-Cemissions attributed only to compost decomposition (mg m-2); and A the area of the PVC tubes where the soil mixtures were placed (0.0016 m2).
To determine expected percentages of compost degradation after one year of compost application, we fitted logistic decay functions to the degree of decomposition at each measuring day with time for each treatment.
4.2.3 Soil sampling and sample preparation
Soil samples were collected on days 0, 30, 60, 90, 120, 150, and 180 by emptying the soil contained in each PVC tube and mixing it thoroughly. At each sampling day, we destructively sampled 40 samples (four replicates of each of the 10 treatments) to follow the nutrient dynamics throughout the incubation process.
Fresh soil samples were used for determining NH4+, NO3–, and plant-available P and K. Dry soil samples (at 105 °C) were used for measuring pH. Total C, total N, total P, and total K were determined from dried (at 105 °C) and ball-milled samples.
4.2.4 Chemical analysis
Ammonium and nitrate were determined by ion chromatography (Dionex ICS-3000, USA) after extracting fresh soil samples with 0.01 mol L-1 CaCl2 (1:10 w/v ratio for 2 h; VDLUFA 2014). Available N was obtained by summing up NH4+-N and NO3–-N. Plant-available P and K were determined by inductively coupled plasma optical emission spectroscopy (ICP-OES, iCAP 6500, Thermo Fisher Scientific, Oberhausen, Germany) after extracting fresh soil samples with 0.05 mol L-1 calcium-acetate-lactate solution (CAL extraction, 1:20 w/v, shaking for 90 min at 200 rpm; VDLUFA 2014). Total organic C and total N were measured with a CHN elemental analyzer (vario EL cube, Elementar). Total contents of P and K were determined by inductively coupled plasma optical emission spectroscopy (ICP-OES, iCAP 6500, Thermo Fisher Scientific) after an aqua regia (nitric and hydrochloric acid) microwave extraction/digestion. The pH was measured with a glass electrode (WTW multi 340i, Xylem Analytics, Weilheim, Germany) in a CaCl2
0.01 mol L-1 extract (1:2.5, w/v) according to ISO 10390 (ISO 2005). Dissolved organic C and dissolved N were measured with a Shimadzu TOC/TN analyzer in a CaCl2 0.01 mol L-1 extract (1:4, w/v ratio for 0.5 h,
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centrifugation at 3500 rpm for 10 min, and filtrating with a 0.45 µm cellulose membrane filter (PORAFIL MV, Macherey-Nagel, Düren, Germany).
4.2.5 Statistical analysis
All the statistical analyses were conducted with the SAS software University Edition (SAS Studio, version 5.1). Data are reported as mean values ± standard error (SE) of each treatment (n=4) and we considered a statistical significance level of α = 0.05.
A one-way repeated-measures ANOVA by using a mixed model with the Kenward-Roger method was conducted to test differences in nutrient, total C, and greenhouse gas dynamics among the soil treatments over the 180 days of incubation. In this mixed model, we considered treatment, time, and time–treatment interaction effects, and we accounted for the correlation among observations on the same treatment by adding two random effects, replicate–treatment, and replicate–treatment–time effects.
To compare chemical parameters measured on day 0 and cumulative GHG emissions among the soil treatments, we conducted a one-way ANOVA followed by the general linear model procedure for least squares means with the Tukey method for the adjustment for multiple comparisons.
Cumulative CO2-C emissions from the compost treatments were linearly correlated with the corresponding dissolved N values and dissolved organic C to dissolved N ratios to obtain Pearson correlation coefficients.
A one-way ANOVA by using the general linear model procedure for least squares means with the Tukey method was used to analyze if NH4+, NO3- and available N, P and K final values were significantly increased or decreased at the end of the incubation period.