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<p>Biochar and nitrogen (N) fertilizers are frequently applied to improve soil properties and increase crop productivity. However, it remains unclear how root plasticity, soil enzyme activities, N and (phosphorus) P cycling in plant-soil system are changed after application of biochar, N or their combination. To address these questions, left and right parts of rhizoboxes were filled with silty-clay loam subsoil amended with biochar (15 g kg<sup>-1</sup> soil, wheat straw, 300 °C), N (0.05 g KNO<sub>3</sub>-N kg<sup>-1</sup> soil) or a control (no amendments), resulting the following combinations (Cm): biochar/control (Cm1), N/control (Cm2) and biochar/N (Cm3). One seed of maize (Zea mays L.) was planted in the middle of each rhizobox, thus allowing roots to choose freely the growth direction. Root growth was quantified by a photographic approach constantly during the experiment (30 d), and soil enzyme activities, available N and P, root morphology and plant biomass were analyzed after plant harvest.</p><p>Maximum plant biomass was found for biochar/N application (0.91 g), whereas minimal values was for biochar/control (0.56 g). At the same time, decreased soil bulk density and increased availability of P in the biochar compartment (Cm1 and Cm3) stimulated root length by 1.4-1.8 times – an effect which was independent from the presence of N in the same rhizobox. Together with stimulated activities of ß-glucosidase and leucine aminopeptidase (by 33%-39%) in presence of biochar (Cm3) compared to N, this shows the coupling of C, N and P cycles in biochar/N treated soils. Application of N (Cm2) also increased ß-glucosidase activity compared to control soil, whereas root elongation stayed unaffected. Thus, combined application of biochar/N over-win benefits of biochar or N alone for plant growth, which is linked with i) the stimulation of microbial enzyme activity at the biochar locations to reduce C and N limitation for both plant and microorganisms, and ii) increasing of fine root proportion to improve N utilization efficiency in the N-treated compartment. Thus, strategy of combined biochar/N application can not only improve the above-ground biomass production, but also increase root-microorganism interactions to overcome nutrient limitation in low fertile agricultural soils.</p>

Large scale oil palm monocultures are continuously replacing Indonesia's tropical rainforests. The impact of intensive nitrogen (N) fertilization and herbicide application on biogeochemical processes in these monocultures remains unknown. Thus, we aim to understand NPK and herbicide effects on soil carbon (C) cycle. We hypothesize that (1) increased microbial activity due to N fertilization leads to faster soil organic matter (SOM) decomposition and to increased CO2 emissions from soil. Despite this, we expect (2) that herbicide application increases soil organic carbon (SOC) sequestration, as it counteracts the fertilizer's effects by hampering soil microbial activity, by deactivating the same synthesizing pathway as in plants, leading to decreased microbial respiration. We investigated the effects of fertilization (conventional and reduced NPK) and weed control (herbicide or mechanical weeding) on SOC decomposition in the rows between the palms (interrows) and around the palm stems (weeding circles). 14C labelled glucose was added to soil to analyze the response of microbial activity and SOC mineralization during 30-day incubation. Conventional fertilization caused microbial activation and destabilized SOC by priming, resulting in increased CO2 efflux. Glyphosate application plus fertilization further increased microbial activity contradicting our hypothesis. In combination with high fertilizer amounts, glyphosate strongly increased microbial activity. NPK might occupy a greater share of sorption capacities; hence, glyphosate is still available and can act as an additional source of C and possibly N and P. Therefore, SOC decomposition was 1.5 accelerated compared to the control when glyphosate and fertilizer application were combined. In contrast, reduced SOC decomposition, i.e., a strong negative priming effect (−47.9 μg g −1), occurred under reduced NPK fertilization without herbicides suggesting positive effects on C accumulation and storage. Our results emphasize the risk of management intensification and the need for a low-impact management strategy to maintain soil fertility and the function as a C reservoir.

<strong>VOCs and CO2</strong> In order to capture any rapid changes in gas fluxes due to changes in microbial activity after pyruvate addition, measurement intervals were increased to high frequency (30 minutes) directly prior to labeling. After gas fluxes were expected to equilibrate, approximately 8 hr post pyruvate labeling (~ 6 PM), measurement intervals were decreased to low frequency (50 minutes) and remained at this frequency until measurements were stopped at 48 hr post labeling. For each measurement, the automatic chamber closed over the collar for a total of 10 min (pre-purge, 2.5 min; measurements, 6.5 min; post-purge, 1 min). Fluxes were measured using an automated, multiplexed Licor soil flux system (Licor 8100, Li-8150 16-port multiplexer and Lic 8100-104 Long-Term Chambers with opaque lids, Licor Inc.). The system was coupled to a Picarro G2201-i analyzer (Picarro Inc., Santa Clara, US) to measure CO2 and isotopic composition and a proton-transfer-reaction time-of-flight mass spectrometer (PTR-TOF 8000, Ionicon Inc. Innsbruck, Austria) for VOCs (including 13C-VOCs). The PTR-ToF-MS sampled the sub-flow from the soil flux system at 30 sccm <em>via</em> fluorinated ethylene propylene tubing heated at 60 °C. The drift voltage was 600 V, the drift temperature was 60°C and the drift pressure was 2.2 mbar, resulting in an E/N ratio of 137 Td. The time resolution was 10 ms and the mass range was up to 500 amu. The PTR-ToF was operated in the H3O+ mode, therefore only compounds having proton affinity higher than water (697 kJ/mol) underwent proton-transfer reactions and could be detected on their protonated mass to charge ratio (<em>m/z</em>), which includes the vast majority of VOCs . Measured ions were attributed to chemical formulas and specific chemical species based on the exact protonated <em>m/z</em>. PTR-TOF data were processed using the software PTRwid. To account for possible variations of the reagent ion signals, measured ion intensities were normalized to the H3O+ counts in combination with the water-cluster ion counts. At midnight, automatic calibrations were performed using standard gas cylinders containing different multi-VOC component calibration mixtures in Ultra-High Purity (UHP) nitrogen (Apel-Riemer Environmental, Inc., Colorado, USA). For a detailed description of the calibration setup see Werner et al. (2021). The concentrations of compounds included in the standard were calculated with an uncertainty of ≤23%. Concentrations of compounds not included in the calibration standard cylinders were calculated by applying the kinetic theory of proton transfer reaction with an uncertainty of ≤50%. Select additional soil experiments were conducted with a Vocus proton transfer reaction time-of-flight mass spectrometer (PTR-TOF; TOFWERK, AG, Thun, Switzerland) coupled to a custom-built gas chromatograph (GC; Aerodyne Research, Inc., Billerica, MA, USA). The GC contains an integrated two-stage adsorbent-based thermal desorption preconcentration system for <em> in situ</em> collection of VOCs prior to separation on the chromatographic column. For preconcentration, a multi-bed (Tenax TA/Graphitized Carbon/Carboxen 1000; Markes International) sorbent tube was used for the first stage of sample collection, this tube is then subjected to a post-collection water purge before the sample was thermally desorbed to a multi-bed focusing trap (Tenax, Carbopack X, Carboxen 1003; Markes International) prior to injection onto the GC column. For this study, the GC was equipped with a 30-m Rxi-624 column (Restek, 0.25 mm ID, 1.4 µm film thickness) which resolves non- to mid-polarity VOCs in the C5 - C12 volatility range prior to ionization in the PTR detector. The GC-PTR sampled from <em>in-situ </em>soil gas probes on an alternating timed schedule. The GC-PTR can speciate structural isomers and help identify some unknowns by matching to calibrated retention times. <br> <strong>NMR </strong> 1H-NMR bulk metabolite characterization was performed on soil water extracts. Samples (180 µL) were combined with 2,2-dimethyl-2-silapentane- 5-sulfonate-d6 (DSS-d6) in D2O (20 µL, 5 mM) and thoroughly mixed prior to transfer to 3 mm NMR tubes. Resonances corresponding to 13C labeling were identified by visual inspection, comparing labeled spectra to unlabeled spectra. Once differences were identified, the molecular location and quantification of 13C incorporation was determined by J-coupling pattern analysis and the ‘linefitting’ tool in Mestrenova, respectively. NMR spectra were acquired on a Bruker Avance III spectrometer operating at a field strength of 17.6 T (1H ν0 of 750.24 MHz) and equipped with a 5 mm Bruker TCI/CP HCN (inverse) cryoprobe with Z-gradient and at a regulated temperature of 298 K. The one-dimensional 1H spectra were acquired using a nuclear Overhauser effect spectroscopy (noesypr1d) pulse sequence. The 90° H pulse was calibrated prior to the measurement of each sample with a spectral width of 12 ppm and 1024 transients. The NOESY mixing time was 100 ms and the acquisition time was 4 s followed by a relaxation delay of 1.5 s during which presaturation of the water signal was applied. Time domain free induction decays (72114 total points) were zero filled to 131072 total points prior to Fourier transform, followed by exponential multiplication (0.3 Hz line-broadening), and semi-automatic multipoint smooth segments baseline correction. Chemical shifts were referenced to the 11H methyl or 13C signal in DSS-d6 at 0 ppm. The 1D 11H NMR spectra of all samples were processed, assigned and analyzed using Chenomx NMR suite 9.2 (Chenomx Inc.; Edmonton, AB, Canada) with quantification of spectral intensities of compounds in the Chenomx library relative to the internal standard. Candidate metabolites present in each of the complex mixtures were determined by matching chemical shift, J-coupling and intensity information of the experimental signals against signals of the standard metabolites in the Chenomx library. Signal to noise ratios (S/N) were measured using MestReNova v 14.2.014 with the limit of quantification equal to a S/N of 10 and the limit of detection equal to a S/N of 3. Standard 2-D experiments such as 11H / 13C - heteronuclear correlation (HSQC) experiments or 2-D 11H/ 11H Total Correlation spectroscopy (TOCSY) experiments further aided corroboration of several metabolite identifications where there was sufficient S/N. <strong>FTICR </strong> A 12 Tesla Bruker FTICR mass spectrometer (MS) was used to collect high-resolution mass spectra of WEOC by direct injection for secondary metabolite characterization. Approximately 100 µL of water extract was mixed with methanol (1:2) before injection onto the mass spectrometer to enhance ionization. A standard Bruker electrospray ionization (ESI) source was used to generate negatively charged molecular ions. Samples were introduced directly to the ESI source. The instrument settings were optimized by tuning on a Suwannee River fulvic acid standard, purchased from the International Humic Substances Society. Blanks (HPLC grade methanol) were analyzed at the beginning and end of the day to monitor potential carry over from one sample to another. The instrument was flushed between samples using a mixture of water and methanol. The ion accumulation time varied to account for differences in C concentration between samples. One hundred and forty-four individual scans were averaged for each sample and internally calibrated using an OM homologous series separated by 14 Da (CH2 groups). The mass measurement accuracy was &lt;1 ppm for singly charged ions across a broad m/z range (100–1,000 m/z). The mass resolution was ∼240 K at 341 m/z. The transient was 0.8 s. Data Analysis software (BrukerDaltonik version 4.2) was used to convert raw spectra to a list of m/z values applying FTICR–MS peak picker module with a signal-to-noise ratio (S/N) threshold set to 7 and absolute intensity threshold to the default value of 100. Putative chemical formulae were then assigned using Formularity software, as previously described. Chemical formulae were assigned based on the following criteria: S/N &gt; 7, mass measurement error &lt;1 ppm, and taking into consideration the presence of C, H, O, N, S, and P and excluding other elements. To ensure consistent formula assignment and eliminate mass shifts that could impact formula assignment, all sample peak lists were aligned to each other. The following rules were implemented to further ensure consistent formula assignment: (a) picking formulae with the lowest error between predicted and observed m/z, and with the lowest number of heteroatoms, and (b) the assignment of one phosphorus atom requires the presence of at least four oxygen atoms. The chemical character of thousands of peaks in each sample's ESI FTICR–MS spectrum was evaluated using van Krevelen diagrams.

Plant litter decomposition is a key ecosystem process with significant implications for global carbon cycling, soil fertility and plant productivity. Given that microbial decomposers are the main players in the decomposition process, it is surprising how little is known about their functional diversity in different habitats or their ability to respond to environmental changes. To fill this knowledge gap, we conducted a litterbag decomposition experiment along a pronounced climate and vegetation gradient in the Chilean Coastal Cordillera (26°S to 38°S), ranging from hyper-arid to temperate, using mixtures of four plant species, native to the respective ecosystems. We analyzed potential decomposition functions of bacterial and fungal litter communities along with their biotic (litter traits) and abiotic (meteorological conditions and soil properties) environments, to determine the relative importance of these environmental factors for microbial community functioning. We also tested the impact of the functional diversity of the decomposer communities (i.e., the diversity of decomposition related functions) on litter mass loss. Functional composition was related most strongly to the temporal variation of precipitation and radiation, explaining about 19 % and 6 % of variation in bacteria and fungi, respectively. In contrast, functional diversity was quite strongly related to litter chemical traits (C:N, C:P, tannins, phenols). Litter mass loss after six months of decomposition was not correlated to the functional diversity of decomposer communities but increased with the presence of habitat generalists like Proteobacteria , Actinobacteria , Firmicutes , and Bacteroidetes . Taken together, these results highlight i) the interplay between abiotic factors and chemical litter traits on litter microbial functions and functional diversity and ii) the importance of microbial generalists for litter decomposition across different ecosystems. These results enhance our ability to predict changes in microbial decomposer communities and litter decomposition under future climate-change scenarios.

No abstract is provided for this article.

Metabolic flux analysis is an integrated experimental and computational approach for quantitative understanding of biochemical reaction networks with particular relevance in systems biology. Mass and energy flows through soil microbial metabolism are subject to the laws of thermodynamics. Carbon (C) allocation through central metabolic pathways (e.g. glycolysis, pentose phosphate, and Entner-Doudoroff) can be reconstructed by 13C-labelling coupled to metabolic flux analysis (13C-MFA) by tracing specific C atoms from within substrate molecules into metabolic products such as carbon dioxide (CO2) or fatty acids. However, mass flow calculated via 13C-MFA alone cannot fully characterise microbial carbon use. Here, we took the novel approach of coupling MFA with microcalorimetry, to also take bioenergetic constraints into account. We coupled energetics and mass flow on a metabolic level by selecting optimal sets of isotopomer tracers. Fifteen position-specific or uniformly 13C-labelled isotopomers - four alanine, seven glucose, and four glutamic acid ones &amp;#8211; were added to a Luvisol (in total 4 folds of the microbial biomass C), and we analyzed substrate-derived 13CO2 fluxes as well as heat dissipation via isothermal microcalorimetry. Our results demonstrate that the temporal dynamics of catabolic CO2 release resembles that of the heat dissipation, i.e. peak respiration and peak heat dissipation were reached approximately 18 h after substrate addition, irrespective of whether the substance entered the central metabolic pathway at the monosaccharide level (glucose), at the pyruvate level (alanine) or in the citric acid cycle (glutamic acid). This indicates that heat dissipation in the initial growth period was strongly dominated by catabolic processes. However, whereas 13CO2 release leveled off during the 36 hours of incubation, the heat dissipation remained above its original level, suggesting that anabolic processes increasingly contribute to the heat dissipation in the later phases of incubation. Glucose isotopomer utilization indicated dominance of the pentose phosphate and Entner Douderoff pathways over glycolysis, suggesting a high activity of fast-growing organisms with considerable C allocation to anabolism. The dominance of this anabolic C use in the later stage of the incubation was confirmed by the isotopomer utilization of alanine and glutamic acid. This study shows that the heat dissipation of growing microbial communities under high C supply is closely linked to their catabolic CO2 release, whereas slow, potentially recycling-based growth after resource depletion releases energy more via anabolic reactions. We furthermore demonstrated that coupled MFA and calorespirometry provides a powerful tool to differentiate among metabolic contributions to the energy use of soil microbial communities in different growth phases.

Abstract Biochar contributes to long-term soil carbon stabilization (C) by acting both as a stable carbon pool and as a sorbent for labile compounds. Biogels from roots and microbes are known to form persistent surface coatings with soil sorbents, where they host microbial hotspots. Yet research has mainly focused on their interactions with minerals. The interactive effects of biogels and biochar on soil carbon dynamics remain largely unexplored. This study aimed to assess the effects of biogel coatings on fresh and aged biochar, particularly under drought, with a focus on biochar degradation and microbial responses. We conducted a three-factorial soil incubation study to examine the effects of fresh and aged biochar, with or without biogel amendment, under two moisture levels (30% and 70% water holding capacity, WHC). Using 14 C labelled biochar, we quantified biochar degradation and biochar- induced priming effects by measuring 14 CO 2 respiration, microbial biomass carbon (MBC) and its 14 C incorporation, hydrolytic and oxidative enzyme activities, and changes in biochar surface area. Mucilage strongly enhanced microbial incorporation of biochar-derived C, specifically from aged biochar under drought conditions, while the opposite effect was observed in soils amended with fresh biochar. This demonstrates the greater microbial accessibility of aged biochar surfaces and limited use of fresh biochar due to inaccessibility of the hydrophobic surfaces, and in consequence the preference for more easily accessible C sources. Additionally, mucilage significantly reduced the Michaelis Menten constant ( K m ) of ß-glucosidase by up to 58% in soils amended with aged biochar under drought. This indicates that under these conditions, the interaction of the aged biochar with the biogel may have created a unique habitat requiring specific enzyme systems with higher affinity. Our findings highlight the role of mucilage in regulating microbial surface access and thus the decomposition of biochar, particularly under moisture- limited conditions. The synergy between aged biochar and biogels provides a promising new perspective to further enhance biochar-based drought mitigation, specifically for managing microbial activity in drought-prone soils.

Despite the importance of subsoil carbon (C) deposition by deep-rooted crops in mitigating climate change and maintaining soil health, the quantification of root C input and its microbial utilization and stabilization below 1 m depth remains unexplored. We studied C input by three perennial deep-rooted plants (lucerne, kernza, and rosinweed) grown in a unique 4-m deep RootTower facility. 13C multiple pulse labeling was applied to trace C flows in roots, rhizodeposition, and soil as well as 13C incorporation into microbial groups by phospholipid fatty acids and the long-term stabilization of microbial residues by amino sugars. The ratio of rhizodeposited 13C in the PLFA and amino sugar pools was used to compare the relative microbial stability of rhizodeposited C across depths and plant species. Belowground C allocation between roots, rhizodeposits, and living and dead microorganisms indicated depth dependent plant investment. Rhizodeposition as a fraction of the total belowground C input declined from the topsoil (0–25 cm) to the deepest layer (360 cm), i.e., from 35%, 45%, and 36%–8.0%, 2.5%, and 2.7% for lucerne, kernza, and rosinweed, respectively, where lucerne had greater C input than the other species between 340 and 360 cm. The relative microbial stabilization of rhizodeposits in the subsoil across all species showed a dominance of recently assimilated C in microbial necromass, thus indicating a higher microbial stabilization of rhizodeposited C with depth. In conclusion, we traced photosynthates down to 3.6 m soil depth and showed that even relatively small C amounts allocated to deep soil layers will become microbially stabilized. Thus, deep-rooted crops, in particular lucerne are important for stabilization and storage of C over long time scales in deep soil.

Although root mucilage plays a prominent role in soil-plant-water relations, especially under drought, its persistence in soil and its microbial decomposition remain unknown. The aim of this study was to investigate: 1) the effect of soil moisture on mucilage decomposition, 2) the effect of mucilage on enzyme activities, and 3) the effect of mucilage on soil organic matter (SOM) decomposition. We hypothesized that mucilage benefits soil microorganisms by compensating for the detrimental effects of drought. Consequently, low water content was expected to reduce SOM mineralization and enzyme activities only in soil without mucilage. High moisture was predicted to support high microbial activities and therefore rapid decomposition of the mucilage. Two doses of maize root mucilage (40 and 200 μg C g−1 soil; C4 plant derived) were added to a C3 soil at optimum moisture (80% WHC) and under drought (30% WHC) to test these hypotheses. Under optimum moisture conditions, CO2 efflux from soil increased in proportion to mucilage addition. In contrast, there was no effect of mucilage on CO2 efflux under drought. At 80% WHC, mucilage was nearly completely decomposed (98% and 88% for low and high dose, respectively) after 15 days. Drought significantly suppressed mucilage mineralization. Microbial uptake of mucilage C was independent of soil moisture, suggesting that its bioavailability is regulated not by the water content of the whole soil, but by the water within the swollen mucilage. The high mucilage dose increased microbial biomass at both moisture levels compared to the soil without mucilage. Positive priming of soil organic matter decomposition was induced by mucilage at 80% WHC, whereas at 30% WHC, mucilage caused slightly negative priming. Mucilage addition counteracted the decrease of enzyme activities at 30% WHC, and so, stabilized the catalytic activity irrespective of soil moisture content. We conclude that mucilage provides biofilm-like properties that maintain microbial and exoenzymatic activities, even under drought. The slow decomposition of mucilage in drying soils suggests that mucilage maintains moist conditions around the roots for a long period, supporting beneficial root-microbial interactions at low water availability. This would result in a positive ecological feedback for microbial life in the rhizosphere and enhance nutrient release for roots under water scarcity.

No abstract is provided for this article.