Soil amendment with sewage sludge-derived chars increases C-sequestration potential and provides N and P for plant growth during a second cropping period with Lolium perenne

Soil amendment with sewage sludge-derived chars increases C-sequestration potential and provides N and P for plant growth during a second cropping period with Lolium perenne Blanca Leiva-Suáreza, Marina Panequea*, José María de la Rosaa, José A. González-Péreza, María José Leivab, Heike Knickera a Institute of Natural Resources and Agrobiology of Seville (IRNAS-CSIC), Reina Mercedes 10, 41012, Seville, Spain b Department of Vegetal Biology and Ecology, University of Seville, Reina Mercedes, 41012 Seville, Spain *present address: Department of Medical Biochemistry and Biophysics, Umeå University, Umeå SE-901 87, Sweden Correspondence: Heike Knicker, email: kn[email protected], Tel: +34 954 624711 Running Title: Sewage sludge chars as slow-release fertilizers Abstract Hydrothermal carbonization and dry pyrolysis transform sewage sludge (SS) into nitrogen- (N) and phosphorus- (P) rich hydrochars (Hyd) and pyrochars (Py), respectively, which may This article is protected by copyright. All rights reserved. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/ejss.13000 Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press 2 act as slow-release fertilizers with carbon (C) sequestration potential. Whereas this has been mostly studied with short-term experiments, this study focussed on the cycling of charderived N, P and C after aging during a second grass cropping cycle. Lolium perenne was grown for 3 months in pots on soil mixed with 13C and 15N-enriched SS, Hyd or Py and allowed to age during a first cropping period of ten-month incubation. The δ15N in the plants confirmed that even during the second cropping, N derived from the amendments was plant accessible. Higher uptake of N from Hyd than from Py is explained by the lower biodegradability of the latter. Plant growth during the second cropping period was associated with a decrease of total P in all treatments, but only the soils with Hyd and Py evidenced an increase of Olsen P. Thus, during the second cropping, more insoluble P was mobilized from the carbonized residues than P needed for plant growth. Compared to control soils prepared with and without KNO3, higher biomass production was yielded with the amended soils. Hyd proved to have the highest long-term N mobilization potential. Following the change of δ13C in the soil, we observed that during the second incubation, independently of their aromaticity, all amendments and the native SOM had comparable turnover rates, although the amount of slow-turning organic matter added with the amendment, increased with aromaticity. A rough estimation of the impact of thermal treatment of SS on its C-sequestration potential revealed no major differences for both char types. The higher fertilization capacity of Hyd, however, turns it into a good candidate for soil amendment if long-term fertilization should be combined with a long-term increase of the soil organic C pool. Keywords: biochar, carbon sequestration, long-term fertilization, pot experiments, soil amendments, 13C and 15N-labeling This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press 3 Highlights  Medium-term fertilization and C-sequestration potential of chars from sewage sludge were tested  Hydrochar (Hyd) and pyrochar (Py) provide N and P for plant growth during a second cropping period  Compared to soils with and without KNO3, Hyd and Py increase plant productivity of the second crop  On a long term scale, native SOM, amended SS, its aged Hyd and Py show comparable turnover rates 1. Introduction In the European Union (EU), the major source of nitrogen (N) and phosphorous (P) input into agricultural soils are mineral fertilizers. Although the use of synthetic fertilizers has dramatically increased food production worldwide, the unintended costs to the environment and human health due to surplus and inefficient applications have also been substantial. Nitrogen and P runoff from farms has contaminated surface and groundwater. Ammonia from fertilized cropland has become a major source of air pollution, whereas emissions of nitrous oxide contribute to the greenhouse effect. These and other negative environmental impacts still lead policymakers to call for further reductions of the use of synthetic fertilizers. In addition, it is estimated that the remaining phosphate rock resources may be exhausted within the next 50 to 100 years (Cordell et al., 2009). Consequently, the search for alternative N and P sources has increased during the recent years. Sewage sludge (SS) may be one of those This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press 4 alternatives since it is rich in organic N and P, which could be recycled in agriculture (Paneque et al., 2019). According to Eurostat (2019), 9.36 x 106 t of SS were produced in the EU during 2015, a considerable part of which is still sent to landfill. However, before applying it to the field, the environmental sustainability of this approach has to be ensured. This implies that heavy metal contents are below the allowed legal threshold. If this prerequisite is fulfilled, pathogenic organisms, as well as potential organic contaminants present in the wastewater still have to be eliminated. Such a hygienization of SS can be achieved for example by a thermal treatment such as hydrothermal carbonization (HTC) or dry pyrolysis. HTC is performed under pressure at temperatures up to 200-320 ºC in the presence of water and producds hydrochars (Hyd) (Libra et al., 2011). Dry pyrolysis occurs in the absence of water and at higher temperatures of 300-700º C. In the following, the products of the latter derived from SS are termed pyrochars (Py). This is based on the high mineral content of this feedstock leading to pyrolysed residues with organic C (Corg) contents below the threshold established by the European Biochar Foundation (Corg > 50%) to be certificated as biochars (EBC, 2019). During the thermal treatment of SS, its organic N is transformed into heterocyclic N compounds (Paneque et al., 2017). Former studies demonstrated that, during the microbial degradation of charcoal produced from grass at 350°C in the presence of oxygen, at least some of the N of such heterocyclic N structures were mobilized and transformed into plantavailable forms (De la Rosa & Knicker, 2011). However, other experiments indicated that this N is less available for plant growth than inorganic N or peptides (Lopez-Martin et al., 2017; Paneque et al., 2019). Based on such observations, it was suggested that N-rich charcoal can act as a slow-release fertilizer (De la Rosa & Knicker, 2011). To test this hypothesis, a pot experiment was performed in which Lolium perenne was grown on soils amended with Hyd and Py for 80 days (Paneque et al., 2019). In these experiments, greater plant biomass was This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press 5 produced on soils amended with Hyd, than with Py or the unamended soil. This was attributed to the presence of easily available inorganic N (Ni) (2% of the total N) and amide N in Hyd, whereas no Ni was detected in Py. It was further demonstrated that although SS derived chars show large total P contents, the plant growth depended mainly on the amount of N. Studies about the ability of chars to release P into soils or the processes involved in its transformation into available forms, are scarce. Qian and Jiang (2014) reported that during carbonization, P is concentrated and immobilized in the chars. Uchimiya et al. (2015) suggested that Py of SS could stabilize pyrophosphate with bridging cations (Al3+, Fe3+, and Mg2+) to form stable six-membered ring complexes, or through direct hydrogen bonding. Jin et al. (2019) found that after the amendment of soils, chars can slowly release P into the soils. Qian & Jiang (2014) observed that the release of P contained in the chars is promoted at low pH, high environmental temperature and in the presence of coexisting anions. It seems that once applied to soil, the release of P from char is complex and both, biotic and abiotic factors must be taken into account. However, comparable to the fate of N derived from Hyd and Py in soils or the impact of SS derived Hyd or Py on the C sequestration potential of soils, our knowledge about their P-cycling derives mainly from short term studies, focused on the first growing season. Little is understood about the longer term effects of sewage-sludge derived char on the soil C, P an N cycles, and, specifically, the potential to increase SOC and to provide sustained sources of N and P to crops in the second crop season after application. To fill this knowledge gap, we designed a greenhouse pot experiment in which we followed the turnover of C, N and P derived from aged Hyd and Py of SS in a soil during a second cropping period with Lolium perenne. The used soils with the aged chars had been prepared previously by planting Lolium perenne for ten months after the initial amendment using 15N/13C-labelled Hyd and Py from SS. With this we wanted to test the hypothesis that 1) SS-derived Hyd and Py provide P and N to plants beyond the first crop cycle and 2) aging of Hyd and Py in soils increases the C-sequestration potential of amended soils. This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press 6 2. Materials and methods 2.1. Production of 15N/13C-enriched sewage sludge, hydrochar and pyrochar Isotopically enriched SS was produced in a sequencing batch reactor (SBR) of 10 L with wastewater subjected to an enhanced biological phosphorus removal (EBPR) system of the wastewater treatment plant CENTA located close to the small village of Carrión de los Céspedes, approximately 20 km from the city of Seville, Southern Spain. Every 8 hours, part of the residual water in the reactor was renewed to create a flow-through system. Using this approach, it was considered that during waste water treatment, microbial activity in the residual waters is associated with a constant turnover of labile organic fractions into more biochemically stable forms that subsequently form the SS. Thus, for the 13C and 15N enrichment of the SS we supplied the microbial community with a daily addition of 100 mg of glucose (13C, 99% abundance), 15 mg of ammonium chloride (15N, 99% of abundance) and 20 mg of potassium nitrate (15N, 99% abundance) to the reactor. The generated isotopically enriched SS was periodically removed, dried in an oven (40°C) and stored in sealed bags at 4°C. Analysis of its chemical composition by solid-state nuclear magnetic resonance (NMR) spectroscopy indicated an alkyl C/carboxyl C ratio of 2.5, which is smaller than the respective values determined for the original primary (4.7) and secondary SS (3.5) derived from the same waste water treatment plant (Paneque et al., 2017). This indicates a higher contribution of short-chain alkyl C. Together with the narrow Corg/N of 7 (Table 1), it can be concluded that the isotopically enriched SS contains a higher amount of peptide-like compounds than the primary and secondary SS with Corg/N values of 12 and 8, respectively (Paneque et al., 2017). Most tentatively this is due to a higher contribution of residues from the microbial biomass, which are considered typical ingredients of SS. An aliquot of the dried product was mixed with water (1:20) and subsequently carbonized in a 1 L pressure reactor (Parr reactor 4520 This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press 7 series, IL, USA) at 200 ºC for 30 minutes under autogenous pressure (Reza et al., 2014) to yield the 15N/13C-enriched Hyd. Respectively, the 15N/13C-enriched Py was obtained by dry pyrolysis of the isotopically enriched SS in a closed steel container which was heated in a muffler at 600 °C for 1 h (Paneque et al., 2017). The respective total C and N contents and the obtained 13C and 15N enrichments are given in Table 1. 2.2. Preparation of the soils with aged and 13C/15N-enriched amendments Soils containing aged 15N/13C-enriched SS and its Hyd and Py, as well as soil fertilized with K15NO3 and unamended soil were prepared using topsoil treated with each amendment and used to grow Lolium perenne for 10 months in a greenhouse to simulate the first growing season of common forage grass. The soil derived from the Ah horizon of a Dystric Cambisol (IUSS Working Group WRB 2014) (N 37º 32' W 6º 15') sampled in the Sierra of Aznalcóllar (Seville).A brief description of the soil is given in (Lopéz-Martín et al., 2016). The C and N contents are listed in Table 1. The pH of the soil was 5.7 and addition of the chars did not affect the pH of the mixtures. At the end of the soil preparation period, the soil of the top 4 cm of the three replicates was mixed, homogenized, dried and stored for approximately one year in the dark. Full details of the aging process are given in the Supplementary Material. In the following soils containing aged 15N/13C-enriched SS, Hyd and Py are named S-SS, S-Hyd and S-Py, respectively, whereas the soil fertilized with K15NO3 and the unamended soils are refered to as S-KNO3 and S-control. 2.3. Greenhouse experiment Three replicates of 35 g of each soil amended with the different treatments were put into 40 mL plant pots (height of 7 cm) at a soil moisture of 60% of the maximal water holding capacity (WHC) into which 10 seeds of Lolium perenne (ILURO seeds company, Barcelona, This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press 8 Spain) were sown. After keeping the pots for 24 hours at 4 °C to induce germination, they were placed into a greenhouse (25°C, 14 h light per day) for 3-months. Their position in the greenhouse was changed regularly to ensure comparable growth and light conditions for the plants in all pots. The irrigation was controlled to maintain soil moisture at 60% of WHC during the entire experiment and to avoid loss of soluble organic matter and nutrients by leaching. No nutrients were added. Germination and survival of plants were measured by counting the number of living plants every 7 to 10 days. Leaf length was determined after 12 and 83 days of incubation, considering only the five largest leaves. After three months, leaves, soil and roots were separated manually and dried in an oven at 60 °C for 72 hours before further analysis. 2.4. Plant and soil analysis The total P (TP) and Olsen P contents were determined in each replicate of the prepared soils with the aged amendments to obtain the amount of P which is potentially available for plant growth during our experiment. Since there was no labelling of the P in the amendments, distinguishing between soil-born P and amendment derived P was not possible. The P concentrations in the soil were measured again at the end of the greenhouse pot experiment (Analytical Service of the IRNAS-CSIC, Seville). In brief, TP was determined after digestion with aqua regia using an inductively coupled plasma-optical emission spectrometer (ICPOES) 150 spectrophotometer (Varian ICP720-ES, Varian Inc. Palo Alto, CA, USA). In this extract, other elements such as calcium (Ca), aluminium (Al), iron (Fe) and magnesium (Mg) were also determined. The Olsen P was quantified after extraction with sodium carbonate at pH 8.5 (Olsen et al., 1954). NO3- and NH4+ concentrations in the soils were measured in 2.5 g of soil after extraction with 25 mL 1 M KCl solution and measured colorimetrically as described in Benton-Jones (2000) by the Analytical Service of the IRNAS-CSIC, Seville. This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press 9 2.5. Isotope ratio mass spectrometry (IRMS) Aprroximately, 1.5 g soil from each pot was ground to a fine powder using a mixer ball mill (Model MM400, Retsch GmBH, Haan, Germany). Plant leaves were cut into small pieces and also milled. Due to the high 15N-enrichment in the leaves of the plants grown in the presence of K15NO3, they were diluted with non-enriched leave biomass (1:12 w:w) prior to their analysis. The 13C and 15N contents of the soils and the harvested plant leaves were measured with a Delta-V advantage isotope ratio mass spectrometer (IRMS) coupled to a Flash 2000 HT combustion elemental microanalyser via a ConFlo IV interface (Thermo Scientific, Bremen, Germany) in duplicates. The analytical error of the elemental microanalyser, reported by the manufacturer, is ± 0.2%. The stable isotope results are expressed in the conventional delta notation (δ), as parts per thousand deviations (‰) from corresponding international standards recognized by the International Atomic Energy Agency (IAEA) [Eq. (1)]. δ (‰) = (1) In [Eq. (1)], R means the ratio of heavy-to-light isotopes. Rsample is this ratio in the sample and Rstandard is that of the standard. The δ of 15N and 13C were used to calculate the proportion of the C (Ctsoil) and N (Ntsoil) of the soil which derived from the C and N of the aged amendments (Camend of Ctsoil (%) and Namend of Ntsoil (%)) by applying a two components isotopic mixing model (Dignac et al., 2005) [Eq. (2)]. Rstandard Rsample -1 x 1000 This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press 16 decrease of its extractability (Christel et al., 2014; Dai et al., 2015; Huang & Tang, 2015). Huang & Tang (2015) suggested that in SS, P-forms are dominated by organic P, orthophosphate and long-chain polyphosphates. Whereas pyrolysis increases the amount of pyrophosphate and short chain polyphosphates, hydrothermal treatment results in the production of only inorganic orthophosphate (Huang & Tang, 2015). It was suggested that the latter reacts with Al, Mn, Fe or Ca to form insoluble P-forms which are trapped within the organic network of hydrochars (Heilmann et al., 2014). A comparable mechanism was suggested for the stabilization of P in pyrochars (Frišták et al., 2018). Considering that both in the soil at the beginning and the end of the incubation, we found higher Ca and Al concentrations in the S-Hyd and S-Py than in the control soil (Table 8), some of the TP that could not be extracted as Olsen P may still have been stabilized by this mechanism. Having a closer look at the concentrations of Olsen P before and after the second growth period, it becomes evident that in contrast to the control soils and S-SS, in the char amended soils more non-extractable P was mobilized than was needed for plant growth. Thus, although SS amendment demonstrated a high potential as a P-fertilizer, it is likely that without additional fertilization, plant-available P concentrations will constantly decrease with each cropping period. It seems that charring sequesters some of the P in its residues in forms that can be slowly but constantly mobilized during plant growth. Bearing in mind that the majority of P used in agriculture derives from mined phosphate rock, which is a non-renewable resource, recycling P from SS with low heavy metal contents via its transformation into Hyd or Py certainly represents a sustainable alternative approach with a positive long-term impact to common fertilization practices. 4.1.1. Fate of the N derived from the amendments In spite of high addition of Ni with KNO3 and the amendment of SS and Hyd, almost comparable Ni contents were observed after the soil preparation phase (Table 3). Bearing in This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press 17 mind that keeping the WHC at 60% prevents leaching of water, we must assume that aside from being incorporated into microbial and plant biomass, a considerable part of the Ni added with those amendments were lost most likely by volatilization. Note that this N loss is in line with the decreasing Ntsoil content of S-KNO3 during the second growing period and the fact that here only 3 and 2% of Ntadd was recovered at the beginning and the end of the pot experiment. Loss of Ntadd was also evidenced for the soils with aged chars, but it was certainly much lower, which allows the conclusion that part of the N-structures in the char were microbially attacked and thus degraded. Comparable conclusion were obtained in earlier studies with pyrogenic organic matter produced in the presence of oxygen (De la Rosa & Knicker, 2011; Lopez-Martin et al., 2017). Having a look at the proportion of Namend to Ntsoil during our experiment (Fig. 2B), the degradation of this Namend occurred either at the same rate as or slightly faster than the native soil N. 4.2. Potential of hydrochars and pyrochars from sewage sludge to increase the soil C sink Our study revealed that, one year after soil amendment, irrespective of the amendment, the relative contribution of Camend to Ctsoil, did not change with incubation time (Fig. 2A). This confirms that, at this stage of the humification process, native SOM and amendment had comparable turnover times. A different situation is evidenced for the soil preparation period. As expected, a considerably faster turnover of Cadd from SS than of native soil C caused a decrease of Camend from 11% to 9% lowering the recovery of Ctadd to 26% (Table 4, Fig. 2A). Our results suggest that after the decomposition of the labile fraction of SS, its more stable organic material (OM) exhibits a turnover rate comparable to that of mature native SOM. For S-Hyd, the relative contribution of Camend to Ctsoil remained relatively constant, both throughout the soil preparation phase and the second cropping period, confirming that the This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press 18 HTC treatment increased the biochemical recalcitrance of the feedstock to turnover rates comparable to the native SOM. At the end of the experiment, half of Ctadd of Hyd was recovered which corresponds to twice the percentage of the Ctadd-recovery with SS (Table 4). This confirms that in spite of the mild production conditions, Hyd can increase the pool size of the slow turning SOM pool. Pyrochars are commonly considered to exhibit a high biochemical recalcitrance (Malghani et al., 2013). Compared with the other amendments of our study, this view is supported by the obtained results. Only 7 % of its Ctadd was lost during its aging in the soil which went along with an enhancement of its relative contribution to Ctsoil until the end of the soil preparation phase. However, this increase did not continue during the second growing period, pointing towards a change of the microbial accessibility of its carbon. It seems that microbial reworking and aging decreased the biochemical recalcitrance of the highly aromatic material. This decrease is related to the loss of the potential of being selectively preserved with respect to humified SOM. One objective of using charred organic residues as soil amendments is increasing the amount of C sequestered in soils in order to reduce the emission of greenhouse gases (Jung et al., 2019). Using the recoveries of Ctadd at the end of the soil preparation phase as a rough estimate for the potential of an amendment to increase the soil carbon pool with slow turnover, SS can contribute with 20 to 30% of its Ct to this fraction. HTC treatment doubles this number, whereas pyrolysis at 600°C increases it to 80 to 90%. However, carbon is also released during the thermal treatment. A former study evidenced that the application of HTC on SS as it was applied in the present investigation leads to a C-loss of approximately 15- 20%, whereas between 50 and 60 % of the carbon in the feedstock were reported to be released during dry pyrolysis at 600°C (Paneque et al., 2017). Taking those numbers, it can be estimated that for either Hyd or Py only 30 and 45% of the carbon of the original feedstock remained in the soil as aged amendment. Considering the higher energy need for the This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press 19 production of Py, the application of Hyd as soil additive may be a more efficient tool for C- sequestration than Py amendment, if neither energy nor by-products emitted during pyrolysis can be reused. 4.3. Impact of the amendment on plant development In general, the plants showed similar germination and plant survival rates in all the treatments (Table 4/5). Thus our amendments neither promoted nor decreased to a larger extent the health of the plants compared to the control. Only at the end of the incubation, the S-Hyd treatment slightly decreased the number of living plants. In fact, Hyd may decrease plant germination due to the presence of phytotoxic volatile compounds (Bargmann et al., 2013). However, these deleterious compounds are usually easily degraded which may question their real impact. Thus, more research is needed to unveil the reason of this observation. Among the different experiments, chars and SS amended pots produced more leave biomass than the control and S-KNO3 pots (Fig. 3B). This suggest that the amendments preserved their fertilisation potential even after aging. Comparable results have been previously reported elsewhere (Rajkovich et al., 2012; Bargmann et al., 2014; Dong et al., 2017; Tan et al., 2018). This is not necessarily due to fertilization with N and P, since deficiency of those nutrients is not evident in any of the trials. At least, comparable C/N ratios were obtained for the plants of all treatments, even for those growing on S-Py. Here, only 2% of their N derived from the amendment (Table 7). The higher biomass production could also have been caused by the addition of other micro-nutrients with the organic amendments. Likewise, improvement of other soil parameters such as soil density, water holding capacity or soil microbiota due to the presence of the amendments may also have contributed to the increase of biomass production. This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press 20 Finally, the root biomass was comparable among the treatments (Table 5), supporting the lack of nutrient deficiency, which had forced the plants to increase their root development. Alternatively, the small size of the pots may have prevented normal root growth. 4.4. Potential of the amendments to act as slow-release N fertilizer At the beginning of the second cropping period, the contribution of Ntadd to Ntsoil of S-KNO3, was already very low with respect to S-SS or the soils with aged chars (Fig. 2B). This is relevant because it underlines the short availability of mineral fertilizers in soils and explains the low Ntadd-plant to Ntplant in the plants harvested from S-KNO3 at the end of the experiment (Table 7). Assuming that N availability was not a limiting plant-growth factor, it can be concluded that the amount of Ntadd-plant was controlled by the portion of available Ntadd in the soil rather than by N deficiency or the lack of alternative N sources. As a consequence, the amount of Ntadd-plant must be related to the rate of N mobilization during amendment turnover. This consideration explains the low incorporation of Ntadd derived from the slowly degrading Py into the plants harvested at the end of the experiment. Higher amounts N-uptake rates were observed for Ntadd from Hyd. Paneque et al. (2019), Tan et al. (2018) and Liu et al. (2018) also found better assimilation of N by plants when the biochar was produced at lower, thus milder temperature conditions. Thuille et al. (2015) observed that in the presence of Hyd, plants were able to use N from Hyd instead of N from synthetic fertilizers. On the other hand, the comparable contribution of Ntadd-plant to Ntplant in the plants grown on S-Hyd and S-SS can be misleading. Here one has to bear in mind that although the same amount of amendment was used, twice the amount of N added with Hyd was applied with SS. Consequently, relative to SS, Hyd exceeds the potential to provide N for plant growth by a factor 2. This is interesting to mention, since it is commonly assumed that peptide structures in organic residues are easily available for microbial reworking and N mobilization. However, This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press 21 those structures are likely to have been already attacked during the soil preparation phase, leaving only more biochemically recalcitrant N-containing remains in the soil. Alternatively, incorporation of SS-derived N into microbial biomass or matured SOM during its turnover may have decreased its remobilization potential. As a consequence, we assume that among the tested amendments, Hyd provides the best N mobilization potential during a second growing period. However, to which extend this is also valid for even longer time frames has still to be tested. 5. Conclusions The present research confirmed that both Hyd and Py derived from SS can act as slow-release P fertilizer, since even during the second cropping period after their addition to the soils, part of their insoluble P, produced during their carbonization, was transformed into extractable, thus plant-available P. With respect to N, Hyd provided more plant available N than Py during the second cropping experiment. However, it may be assumed that although at a slow rate, Py may release N over a longer time range than Hyd. The high medium-term fertilization potential of Hyd combined with the observation that Py and Hyd provide a comparable proportion of the organic C of their feedstock to the slow SOM pool suggest that application of Hyd may be more efficient than Py for increasing soil resilience on a long term scale. Considering the fact that in spite of different aromaticity, comparable C turnover was evidence for the aged amendments, it may be concluded further that at an advanced aging state, aromaticity loses its importance for their degradability. Most likely, factors and mechanisms, commonly controlling the turnover of humified native SOM determine and control their future biochemical stability. This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press 22 Author’s contribution The experimental part, the data analysis and writing of this manuscript was performed by the first author BLS. MP provided the soil samples and was involved in the design and performance of the experiment. JAGP assisted with the measurement and interpretation of the stable isotope data. HK developed the original idea, led the project and together with MP, JMR and MJL, supervised the experiment and contributed to the discussion and interpretation of the data. All authors were active in correcting and editing the manuscript. Conflicts of interest There have not been any conflicts of interest to declare. Acknowledgements The Spanish Ministry of Education is acknowledged for the Collaboration Scholarship for the author of this study. Desiré Monis Carrere is thanked for technical assistance in performing the IRMS measurements. Funding This research was funded by the Spanish Ministry of Science, Innovation and Universities and the European Regional Development Fund (CGL2012-37041, CGL2015-64811-P, CGL2016- 78937-R). Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press 23 References Bargmann, I., Rillig, M.C., Buss, W., Kruse, A. & Kuecke, M. (2013). Hydrochar and biochar effects on germination of spring barley. Journal of Agronomy and Crop Science, 199, 360–373. DOI:10.1111/jac.12024 Bargmann, I., Rillig, M.C., Kruse, A., Greef, J.-M. & Kücke, M. (2014). Effects of hydrochar application on the dynamics of soluble nitrogen in soils and on plant availability. Journal of Plant Nutrition and Soil Science, 177, 48–58. DOI:10.1002/jpln.201300069 Benton-Jones, J. (2000). Soil Analysis: Handbook of Reference Methods. CRC Press, Boca Raton, FL. Christel, W., Bruun, S., Magid, J. & Jensen, L.S. (2014). 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Values were calculated from Figure 2 and the respective carbon (Ctsoil) and N content (Ntsoil, Table 3) of the samples: Ctsoil (g kg-1) Camend (% Ctsoil) Camend (% of Ctadd) Namend (% of Ntadd) T0* T1 T2 T0* T1 T2 T1 T2 S-KNO3 39 37 ± 4 41 ± 3 - 3 2 S-SS 45 43 ± 1 43 ± 3 17 26 22 18 13 S-Hyd 44 41 ± 2 41 ± 2 14 43 43 64 47 S-Py 42 42 ± 3 40 ± 2 11 93 84 66 45 * calculated from Table 1 and 2 This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press Table 5. Number of living plants grown on the soil without (S-control) and with amendments (S-KNO3, S-SS, S-Hyd or S-Py) during the second growing season in the greenhouse. Values in the columns are means ± standard deviation (n=3). Number of living plants Days 6 11 18 27 36 43 50 57 64 76 85 S-Control 6.0±2.6 9.0±1.2 11.0±1.2 9.0±1.2 9.0±1.2 10.0±0.6 10.0±0.6 10.0±0.6 10.0±0.6 10.0±1.0 10.0±1.5 S-KNO3 6.0±3.2 10.0±2.5 11.0±2.7 10.0±0.6 10.0±1.5 10.0±2.0 10.0±1.5 11.0±2.7 11.0±2.5 11.0±2.0 11.0±2.1 S-SS 10.0±1.5 10.0±2.0 11.0±2.7 11.0±1.2 11.0±2.3 11.0±2.3 11.0±2.3 11.0±1.7 11.0±1.5 11.0±1.5 11.0±1.5 S-Hyd 9.0±2.6 9.0±1.8 10.0±1.7 10.0±1.7 10.0±1.0 10.0±1.0 9.3±1.5 9.0±1.0 9.0±1.0 8.0±1.5 8.0±1.5 S-Py 8.0±3.2 10.0±2.7 10.0±1.2 11.0±1.5 10.0±1.2 10.0±1.7 10.0±1.7 10.0±2.1 10.0±2.1 11.0±1.5 11.0±1.5 This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press Table 6. Root biomass production of Lolium perenne grown on soils without (S-control) and with amendments (S-KNO3, S-KNO3, S-SS, S-Hyd or S-Py) during a second cropping period after soil amendment. Values in the columns are means ± standard deviation (n=3). Root biomass production (g) S-Control 1.5 ± 0.2 S-KNO3 1.4 ± 0.1 S-SS 1.5 ± 0.3 S-Hyd 1.3 ± 0.2 S-Py 1.5 ± 0.2 This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press Table 7: Recovery of added N (Ntadd) in the plants grown on the soils amended with KNO3, sewage sludge and its hydro- and pyrochar (S-KNO3, S-SS, S-Hyd, S-Py) during a the second growing period of 3 months. The Ntadd in the plants (Ntadd-plant) was calculated using the stable 15N/14N ratios of the plants, their N content (Ntplant), dry weight and the amount of Ntadd provided at the beginning of the soil preparation. Ntplant (g kg-1) C/N (w: w) Ntaddplant /Ntplant (%) Ntaddplant % of Ntadd Ntadd-plant/plant dry mass (mg g-1) Plant dry mass /Ntadd (g/g) S- KNO3 7. 7 ± 0.9 52 7.7 ± 1. 0 0.26 0.6 3.1 S-SS 7. 7 ± 1.2 55 13. 4 ± 0. 5 0.23 1.0 2.8 S- Hyd 7. 6 ± 0.9 53 12. 2 ± 0. 2 0.50 0.9 5.4 S-Py 6. 9 ± 0.5 55 1.8 ± 0. 5 0.04 0.1 4.2 This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press Table 8. Al, Ca, Fe and Mg concentrations in the soils without (S-control) and with amendments (S-SS, S-Hyd or S-Py) at the end of the first (T1) and second growing season (T2). Values in the columns are means ± standard deviation (n=3). Treatments T1 T2 Al Ca Fe Mg Al Ca Fe Mg (g kg-1) (g kg-1) (g kg-1) (g kg-1) (g kg-1) (g kg-1) (g kg-1) (g kg-1) S-Control 23.9 ± 2.2 2.3 ± 0.1 41.3 ± 3.1 2.8 ± 0.1 15.7 ± 0.2 2.2 ± 0.1 44.4 ± 1.8 3.0 ± 0.1 S-SS 36.8 ± 3.7 3.7 ± 0.1 40.7 ± 2.0 3.2 ± 0.1 15.9 ± 0.1 3.5 ± 0.2 41.6 ± 0.7 3.1 ± 0.1 S-Hyd 24.2 ± 2.8 4.7 ± 0.4 41.3 ± 0.9 3.1 ± 0.1 23.7 ± 6.8 4.3 ± 0.3 42.2 ± 0.8 3.3 ± 0.2 S-Py 56.6 ± 7.9 5.2 ± 0.2 39.7 ± 0.5 3.5 ± 0.2 30.8 ± 2.5 4.7 ± 0.1 43.3 ± 2.3 3.5 ± 0.1 This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press TP (mg kg-1) Olsen P (mg kg-1) Figure 1 1600 1400 1200 1000 T1 T2 C * c B b S-SS S-Hyd Treatments D d A 80 70 60 50 * B b * c C S-Hyd Treatments d D B 800 40 600 A a 30 400 20 200 10 A a 0 0 S-Control S-Py S-Control S-SS S-Py This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press Camend of Ct (%) Namend of Nt (%) Figure 2 25 T1 20.7 19.8 NS A 25 16.8 14.2 20.5 16.3 B 20 T2 20 * 15 12.6 12.6 15 10.2 8.2 10 8.8 7.5 10 1.3 0.9 5 5 * 0 S-SS S-Hyd S-Py 0 S-KNO3 S-SS S-Hyd S-Py This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press cm g per pot Figure 3 18 16 14 12 10 8 6 4 2 0 12 d 83 d S-Control S-KNO3 S-SS Treatments S-Hyd S-Py NS A 0.70 B 0.60 0.50 A A 0.40 0.30 b a a 0.20 0.10 0.00 S-Control S-KNO3 S-SS Treatments B b S-Hyd B b S-Py B Fresh biomass Dry biomass This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press Figure 4 Treatments S-Py S-Hyd S-SS S-KNO3 0 d 100 B 1st 2nd 300 200 A b 400 A * c a 500 600 δ15Nnorm (‰ mg-1 15Nadd) This article is protected by copyright. All rights reserved. Postprint of European Journal of Soil Science (2020) DOI: 10.1111/ejss.13000 In Press