Publications

The concept of microbial biomass growth is central to microbial carbon (C) cycling and ecosystem nutrient turnover. Growth is usually assumed to occur by cellular replication, despite microorganisms’ capacity to increase biomass by synthesizing storage compounds. Here we examined whether C storage in triacylglycerides (TAGs) and polyhydroxybutyrate (PHB) contribute significantly to microbial biomass growth, under contrasting conditions of C availability and complementary nutrient supply. Together these compounds accounted for 19.1 ± 1.7% to 46.4 ± 8.0% of extractable soil microbial biomass, and revealed up to 279 ± 72% more biomass growth than observed by a DNA-based method alone. Even under C limitation, storage represented an additional 16 – 96% incorporation of added C into microbial biomass. These findings encourage greater recognition of storage synthesis and degradation as key pathways of biomass change and as mechanisms underlying resistance and resilience of microbial communities.

Mucilage is a gelatinous high-molecular-weight substance produced by almost all plants, serving numerous functions for plant and soil. To date, research has mainly focused on hydraulic and physical functions of mucilage in the rhizosphere. Studies on the relevance of mucilage as a microbial habitat are scarce. Extracellular polymeric substances (EPS) are similarly gelatinous high-molecular-weight substances produced by microorganisms. EPS support the establishment of microbial assemblages in soils, mainly through providing a moist environment, a protective barrier, and serving as carbon and nutrient sources. We propose that mucilage shares physical and chemical properties with EPS, functioning similarly as a biofilm matrix covering a large extent of the rhizosphere. Our analyses found no evidence of consistent differences in viscosity and surface tension between EPS and mucilage, these being important physical properties. With regard to chemical composition, polysaccharide, protein, neutral monosaccharide, and uronic acid composition also showed no consistent differences between these biogels. Our analyses and literature review suggest that all major functions known for EPS and required for biofilm formation are also provided by mucilage, offering a protected habitat optimized for nutrient mobilization. Mucilage enables high rhizo-microbial abundance and activity by functioning as carbon and nutrient source. We suggest that the role of mucilage as a biofilm matrix has been underestimated, and should be considered in conceptual models of the rhizosphere.

Soils, paleosols and terrestrial sediments serve as archives for studying climate change, and represent important terrestrial carbon pools. Archive functioning relies on the chronological integrity of the respective units. Incorporation of younger organic matter (OM) e.g. by plant roots and associated microorganisms into deep subsoil and underlying soil parent material may reduce reliability of paleoenvironmental records and stability of buried OM. Long-term effects of sedimentary characteristics and deep rooting on deep subsoil microbial communities remain largely unknown. We characterized fossil and living microbial communities based on molecular markers in a Central European Late Pleistocene loess-paleosol sequence containing recent and ancient roots with ages of several millenia. The molecular approach, comprising free and phospholipid fatty acids (FAs), core and intact polar glycerol dialkyl glycerol tetraethers (GDGTs), as well as 16S rRNA genes from bacterial DNA, revealed the presence of living microorganisms along the sequence, with bacterial community composition comparable to that of modern topsoils. Up to 88% redundancy between bacterial genetic fingerprint and molecular signature of fossil microorganisms suggested a time-integrated signal of the molecular markers accumulated over a time span potentially lasting from sedimentation over one or more rooting phases until today. Free FAs, core GDGTs and DNA, considered as remains of fossil microorganisms, corresponded with ancient and recent root quantities, whereas phospholipid FAs and intact polar GDGTs, presumably derived from living microorganisms, correlated only with living roots. The biogeochemical and ecological disequilibrium induced by postsedimentary rooting may entail long-term microbial processes like OM mineralization, which may continue even millenia after the lifetime of the root. Deep roots and their fossil remains have been observed in various terrestrial settings, and roots as well as associated microorganisms cause both, OM incorporation and mineralization. Therefore, these findings are crucial for improved understanding of OM dynamics and carbon sequestration potential in deep subsoils.

The transformation and turnover time of medium- to long-chain dicarboxylic acids (DCA) in soil is regulated by microbial uptake and mineralization. However, the chain length of n-alkyl lipids may have a remarkable influence on its microbial utilization and mineralization and therefore on the formation of stable soil organic carbon from e.g. leave- needle- and root-derived organic matter during decomposition. To investigate their size dependent mineralization and microbial incorporation, four DCA of different chain lengths (12–30 carbon atoms), that were 13 C labeled at each of their terminal carboxylic groups, were applied to the Ah horizon of a Fluvic Gleysol. Incorporation of 13 C into CO 2 and in distinct microbial groups classified by phospholipid fatty acid (PLFA) analysis was investigated. Mineralization of DCA and incorporation into PLFA decreased with increasing chain length, and the mineralization rate was highest during the first days of incubation. Half-life time of DCA carbon in soil increased from 7.6 days for C 12 DCA to 86.6 days for C 18 DCA and decreased again to 46.2 days for C 22 DCA, whereas C 30 DCA had the longest half-life time. Rapid and efficient uptake of C 12 DCA as an intact molecule was observable. Gram-negative bacteria incorporated higher amounts of DCA-derived 13 C compared to other microbial groups, especially compared to actinomycetes and fungi during the first phase of incubation. However, the incorporation of C 12 DCA derived 13 C into the PLFA of actinomycetes, and fungi increased steadily during the entire incubation time, suggesting that those groups take up the 13 C label from necromass of bacteria that used the C 12 DCA for formation of their lipids before.

<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.

&amp;lt;p&amp;gt;Root morphology and the composition of root exudates shape the spatial organization and various processes in the rhizosphere. For instance, root hairs are essential for plant nutrition, while secondary plant metabolites (i.e. benzoxazinoids) ensure plant defence from herbivore and fungal infection. Nevertheless, it is still unknown to which extent root hairs and benzoxazinoids may change the microbiome and enzymatic activities, as well as formation of rhizosphere hot- and coldspots.&amp;lt;/p&amp;gt;&amp;lt;p&amp;gt;To study the effect of root hairs and benzoxasinoids on the rhizosphere microbiome structure and its enzymatic activities we compared mutants with defective root hairs &amp;lt;em&amp;gt;rth3 &amp;lt;/em&amp;gt;or with reduced benzoxazinoids &amp;lt;em&amp;gt;bx1&amp;lt;/em&amp;gt; with the corresponding wild-type (WT) maize.&amp;lt;/p&amp;gt;&amp;lt;p&amp;gt;Root hairs increased acid phosphatase activity by 80 % promoting mineralization of organic phosphorus sources to available forms in the hotspots. In the coldspots, broken root hairs in WT facilitated the intensive microbial hotspots with up to two times higher &amp;amp;#946;-glucosidase and chitinase activities, compared to &amp;lt;em&amp;gt;rth3&amp;lt;/em&amp;gt;.&amp;lt;/p&amp;gt;&amp;lt;p&amp;gt;The presence of benzoxazinoids in root exudates strongly supported plant defence against pathogenic fungi (i.e., genus &amp;lt;em&amp;gt;Fusarium&amp;lt;/em&amp;gt; and &amp;lt;em&amp;gt;Gibberella&amp;lt;/em&amp;gt;) while the total microbial biomass remained unaffected. In response to the presence of pathogenic fungi, &amp;lt;em&amp;gt;bx1&amp;lt;/em&amp;gt; exuded 70 % more chitinase for defence purpose to partly compensate for benzoxazinoids deficiency, which was however, less efficient against pathogens than the presence of benzoxazinoids.&amp;lt;/p&amp;gt;&amp;lt;p&amp;gt;Overall, we conclude that: i) root hairs facilitate better plant nutrition at the shortage of available nutrients (i.e., coldspots), while; ii) the presence of benzoxazinoids in exudates protect plant from pathogenic microorganisms. This two root traits are promising for plant breeding of genotypes suitable for sustainable agriculture and organic farming.&amp;lt;/p&amp;gt;

The increasing input of anthropogenically-derived nitrogen (N) to ecosystems raises a crucial question: how do N inputs modify the soil microbial stability

Biochar (BC) and nitrogen (N) fertilizers are frequently applied to improve soil properties and increase crop productivity. Nonetheless, our mechanistic understanding of plant-soil interactions under single or combined application of BC and N remains incomplete. For the first time, we applied a split-root system to evaluate how BC or N contributes to the changes in soil enzyme activities, N and phosphorus (P) cycling as well as root plasticity. Left and right parts of rhizoboxes were filled with silty-clay loamy soil amended with BC (15 g kg−1 soil, from wheat straw, 300 °C), N (0.05 g KNO3-N kg−1 soil) or a control (no amendments), resulting in the following combinations: BC/Control, N/Control, BC/N. Soil enzyme activities, available N and P, root morphology and plant biomass were analyzed after plant harvest. Plant biomass (shoot + root) ranged from 0.56 g pot−1 (BC/Control) to 0.91 g pot−1(BC/N). The decreased soil bulk density and increased P availability in the BC compartment (BC/Control and BC/N) stimulated root length by 1.4–1.8 times – an effect that was independent of N availability in the same rhizobox. Biochar stimulated activities of β-glucosidase and leucine aminopeptidase (by 33–39%) compared to N due to the coupling of C, N and P cycles in BC/N treated soil. Nitrogen fertilization also increased β-glucosidase activity compared to the unfertilized control, whereas root elongation remained unaffected. Thus, the combined application of BC/N had more efficient benefits for plant growth than BC or N alone. This is linked with i) the stimulation of enzyme activities at the BC locations to reduce N limitation for both microorganisms and plants, and ii) an increase of fine root production to improve N uptake efficiency. Thus, combined BC/N application is potentially especially sustainable to overcome nutrient limitation as well as to maintain crop productivity because it accelerates root-microbial interactions.

Besides environmental and soil physical drivers, the functional properties of microbial populations, i.e. growth rate, enzyme production and maintenance requirements are dependent on the microbes' environment. The soil nutrition status and the quantity and quality of the substrate input, both infer different growth strategies of microorganisms. It is uncertain, how enzyme systems respond during the different phases of microbial growth and retardation in soil. The objective of this study was to uncover the changes of microbial functioning and their related enzyme systems in nutrient-poor and nutrient-rich beech forest soil during the phases of microbial growth. We determined microbial growth via kinetic approach by substrate-induced respiratory response of microorganisms, enabling the estimation of total and growing biomass of the microbial community. To induce microbial growth we used glucose, while yeast extract simulated additional input of nutrients and factors indicating microbial residues (i.e. necromass compounds). Microbial growth on glucose showed a 12–18 h delay in associated enzyme activity increase or the absence of distinct activity responses (Vmax). β-glucosidase and chitinase (NAG) demonstrated clear differences of Vmax in time and between P-rich and P-poor soils. However, during microbial growth on glucose + yeast extract, the exponential increase in enzymatic activity was clearly stimulated accompanied by a delay of 8–12 h, smoothing the differences in nutrient-acquisition dynamics between the two soils. Furthermore, cross-correlation of β-glucosidase and acid phosphatase between the two sites demonstrated harmonized time constraints, which reflected the establishment of comparable and balanced enzymatic systems within the decomposition network. The network accelerated nutrient acquisition to maintain microbial growth, irrespective of the contrasting soil properties and initial nutrient stocks, indicating similar tradeoffs of C- and P- cycling enzymes in both soils. This reflects comparable temporal dynamics of activities in nutrient-poor and nutrient-rich soil when the glucose + yeast extract was added. During lag phase and phase of exponential microbial growth, substrate turnover time of all enzymes was shortened in nutrient-poor forest soil exclusively, reflecting that the quality of the added substrate strongly matters during all stages of microbial growth in soil.

Climate change impacts soil microbial communities, activities and functionality. Nonetheless, responses of the microbiome in soil microenvironments with contrasting substrate availability in the rhizosphere to climatic stresses such as drought are largely unknown. To fill this knowledge gap, we coupled soil zymography with site-specific micro-sampling of the soil and subsequent high-throughput sequencing. This helped identify how the bacterial community structure and the genes encoding N-cycling enzymes (leucine aminopeptidase and chitinase) in rhizosphere hotspots and coldspots (microsites with activities in the range of bulk soil but localized within the rhizosphere) of maize respond to drought (20% WHC, two weeks). The elevated activities of leucine aminopeptidase and chitinase in rhizosphere hotspots were caused by the tight collaborative relationships between bacteria and their stable network structure rather than by any significant shift in bacterial community structure or enzyme-encoding gene copies. Despite the similarity in bacterial community structure in soil under drought and optimal moisture, functional predictions indicated the increased relative abundance of genera belonging to Actinobacteria capable of leucine aminopeptidase and chitinase production, especially Streptomyces, Nocardioides, Marmoricola, and Knoellia. Accordingly, the number of gene copies encoded by Actinobacteria for these two enzymes increased by 5.0–17% under drought. Among the bacteria with increased relative abundance under drought, Luedemannella played a crucial role in mediating nutrients and energy fluxes between bacteria. This was reflected in a 35–70% increase in leucine aminopeptidase and chitinase activities under drought. The resistance of enzyme activities to drought was higher in hotpots than that in coldspots. These results revealed that rhizosphere bacterial community composition remained stable, and that the number of gene copies encoded by Actinobacteria responsible for N-cycling enzymes increased under drought. The expected reduction of processes of N cycle was absent. Instead, bacteria increased N mining rate in those hotspots remaining active despite water scarcity.