Publications

<p>It is well known, that phospholipid fatty acids (PLFAs) are very dynamic, and reflect the living microbial community. The vast majority of previous studies limited its turnover determination to the lipid moiety (“the tail”) of the phospholipids. Thus, it remains unclear how dynamic the head groups of phospholipids are, and whether environmental conditions, i.e. amount of available carbon (C) have an effect on the dynamics of parts of the phospholipid molecule. To answer these questions, the double-labeling <sup>14</sup>C/<sup>33</sup>P-was used in the present experiment. </p><p>The soil was collected from a 45-75 cm depth at the Klein-Altendorf experimental research station Bonn, Germany. The site is an agricultural field for more than 100 years. Formation of PLFA was traced for the two conditions: C limited (2.5 mg glucose-C kg<sup>-1</sup> soil added, 1% from microbial biomass C) and C rich (250 glucose-C kg<sup>-1</sup> soil added, 100% of MBC). For both conditions, <sup>14</sup>C labeled glucose and <sup>33</sup>P-K<sub>2</sub>HPO<sub>4</sub> (12.5 mg P kg<sup>-1</sup> soil) were added with 1 mL of water and supplemented with (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> (25 mg N kg<sup>-1</sup> soil). These ratios of C/P and C/N were chosen relative to a 100% glucose-C application to reach a ratio of C:P=20:1 and C:N=10:1. The soil was incubated for 10 d, and destructive samplings were performed after 5 h, 19 h, 1, 3, 5, 7 and 10 days, and each time four replicates were harvested. Soils were extracted for PLFAs in 2 steps: first PLFAs were obtained following the standard procedure, but both phospholipid tails and head groups were collected for further <sup>14</sup>C and <sup>33</sup>P counting. The second step included separate extraction and compound-specific PLFA analysis by GC-MS to reveal changes in the community composition induced by C, N, P addition that might explain de-novo formation of phospholipids.</p><p>The peak of <sup>33</sup>P incorporation into headgroups under high glucose addition was after 19 h, and accounted 0.3% from the applied tracer, whereas it was up to 1% after low glucose addition and peaked in the middle of incubation time. Incorporation of <sup>14</sup>C into the head groups and tails after high glucose addition showed an identical temporal dynamic and was 1.5 times higher in heads than in tails. Both, <sup>33</sup>P and <sup>14</sup>C incorporation into head and tail had a temporal minimum at day 3 and increased afterwards suggesting two different underlying processes: direct incorporation versus C recycling. After low glucose addition, <sup>14</sup>C incorporation was maximum on day 5 but was 3 times lower compared to growth conditions. This shows that even under limited C supply microorganisms construct new phospholipids from available glucose. Irrespective of the C supply, the ratio of head to tail incorporation relative to the ratio of head-to-tail C atoms demonstrates a significantly higher turnover of headgroup C than lipid C, suggesting recycling as an important process to cover microbial lipid demand. Thus, for the first time the different dynamics of phospholipid heads and tails was found and suggest recycling as an important process for growth and maintenance lipid formation.</p>

The pivotal role of the soil microbiome in global biogeochemical cycles is undisputed. The subsequent demand for simplified quantitative descriptions of its functions in modelling approaches resulted in transferring the pure-culture based microbial yield concept into microbial carbon use efficiency (CUE) – a “one-number” approach to partition C input to soils and to describe the physiological efficiency of the microbiome.The holy grail lost its sanctity once our challenges to reliably determine it became evident. The method comparison of Geyer et al. (2019) identified which critical assumptions underly the contrasting outcomes in CUEs derived from these methods. Our own data just underline this: While substrate-based CUE has a temporal and substrate dependency, 18O-based and metabolic CUE remain often unaffected by substrate addition but cover, with either DNA-replication or anabolic precursor-based upscaling of biomass C formation contrasting physiological processes of microbial cells.Such divergent findings highlight that despite decades of research, current methods do not allow an unambiguous quantification of microbial substrate use in soils, owing to two overlapping methodological challenges: 1) Neither extracting microbial biomass nor predicting it from de-novo formed DNA can deliver a reliable quantitative estimation of the newly formed microbial biomass carbon; and 2) Whatever we add as substrate to soils does not reflect what microbes use for growth under native conditions. Our progress in quantitatively covering an increasing number of cellular pools (e.g. also considering cell walls and membranes), the increased consideration of storage, and first concepts on how to integrate secreted extracellular carbon offer perspectives to tackle the first of the two challenges. However, experimentally representing the incredible diversity of organic molecules accessible to microbes for consumption in soils is yet rather avoided, although Lehmann et al (2020) postulated compound diversity as a central factor determining the fate of carbon in soils. Comparing incubations with individual compounds to those of complex monomer mixture revealed that the microbial use of an individual compounds is significantly affected by the presence or absence of other compounds, i.e. the molecular diversity in soil solution. This can readily be explained by viewing microbes through the lens of their metabolic capacities, which impose fundamental constraints on their functioning. Formation of microbial biomass requires a defined ratio of precursor building blocks, which are products of distinct pathways of the basic carbon metabolism. De-novo production requires expression and formation of all pathway-related enzymes, while direct precursor uptake from soil solution allows for “saving” this energy. Therefore, we postulate that monomer diversity would positively affect microbial efficiency. This may be contrasting for polymer diversity, where extracellular enzyme costs exceed those of intracellular de-novo formation and thus a low diversity may be bioenergetically favorable. Thus, substrate diversity-efficiency relationships may centrally underlie deviations between our current CUE approaches. We recommend microbial ecologists to whenever possible replace CUE by the actual processes of interest, i.e. the ecophysiological response and subsequent changes in microbial pools (metabolome, growth) and fluxes (fluxome). This would provide parameters allowing for quantitative upscaling to pools and fluxes required for higher scale soil system models.

Fatty acids have been used as plant and microbial biomarkers, and knowledge about their transformation pathways in soils and sediments is crucial for interpreting fatty acid signatures, especially because the formation, recycling and decomposition processes are concurrent. We analyzed the incorporation of free fatty acids into microbial fatty acids in soil by coupling position-specific 13C labeling with compound-specific 13C analysis. Position-specifically and uniformly 13C labeled palmitate were applied in an agricultural Luvisol. Pathways of fatty acids were traced by analyzing microbial utilization of 13C from individual molecule positions of palmitate and their incorporation into phospholipid fatty acids (PLFA). The fate of palmitate 13C in the soil was characterized by the main pathways of microbial fatty acid metabolism: Odd positions (C-1) were preferentially oxidized to CO2 in the citric acid cycle, whereas even positions (C-2) were preferentially incorporated into microbial biomass. This pattern is a result of palmitate cleavage to acetyl-CoA and its further use in the main pathways of C metabolism. We observed a direct, intact incorporation of more than 4% of the added palmitate into the PLFA of microbial cell membranes, indicating the important role of palmitate as direct precursor for microbial fatty acids. Palmitate 13C was incorporated into PLFA as intact alkyl chain, i.e. the C backbone of palmitate was not cleaved, but palmitate was incorporated either intact or modified (e.g. desaturated, elongated or branched) according to the fatty acid demand of the microbial community. These modifications of the incorporated palmitate increased with time. Future PLFA studies must therefore consider the recycling of existing plant and microbial-derived fatty acids. This study demonstrates the intact uptake and recycling of free fatty acids such as palmitate in soils, as well as the high turnover and transformation of cellular PLFA. Knowledge about the intact uptake and use of soil-derived free fatty acids is crucial for interpreting microbial fatty acid fingerprints and their isotopic composition.

. Microorganisms regulate the carbon (C) cycle in soil, controlling the utilization and recycling of organic substances. To reveal the contribution of particular microbial groups to C utilization and turnover within the microbial cells, the fate of 13C-labelled glucose was studied under field conditions. Glucose-derived 13C was traced in cytosol, amino sugars and phospholipid fatty acid (PLFA) pools at intervals of 3, 10 and 50 days after glucose addition into the soil. 13C enrichment in PLFAs ( ∼ 1.5 % of PLFA C at day 3) was an order of magnitude greater than in cytosol, showing the importance of cell membranes for initial C utilization. The 13C enrichment in amino sugars of living microorganisms at day 3 accounted for 0.57 % of total C pool; as a result, we infer that the replacement of C in cell wall components is 3 times slower than that of cell membranes. The C turnover time in the cytosol (150 days) was 3 times longer than in PLFAs (47 days). Consequently, even though the cytosol pool has the fastest processing rates compared to other cellular compartments, intensive recycling of components here leads to a long C turnover time. Both PLFA and amino-sugar profiles indicated that bacteria dominated in glucose utilization. 13C enrichment decreased with time for bacterial cell membrane components, but it remained constant or even increased for filamentous microorganisms. 13C enrichment of muramic acid was the 3.5 times greater than for galactosamine, showing a more rapid turnover of bacterial cell wall components compared to fungal. Thus, bacteria utilize a greater proportion of low-molecular-weight organic substances, whereas filamentous microorganisms are responsible for further C transformations. Thus, tracing 13C in cellular compounds with contrasting turnover rates elucidated the role of microbial groups and their cellular compartments in C utilization and recycling in soil. The results also reflect that microbial C turnover is not restricted to the death or growth of new cells. Indeed, even within living cells, highly polymeric cell compounds are constantly replaced and renewed. This is especially important for assessing C fluxes in soil and the contribution of C from microbial residues to soil organic matter.

The air we breathe, the water we drink, the food we eat, and other resources we use, have resulted from interactions between the Earth’s geosphere and biosphere. Understanding these interactions is thus essential for human wellbeing, which is endangered by anthropogenically induced climate and land-use change. While contemporary anthropogenic pressures are unprecedented, the processes and natural laws governing the Earth System remain universal. Interactions between the geosphere (rocks, soils, water, atmosphere and the Earth’s surface) and the biosphere (microorganisms, fungi, plants, and animals) determine how the Earth System responds to change. Past research has largely considered geosphere and biosphere responses to Earth-System change separately. The new cluster of excellence TERRA at the Univesity of Tübingen develops an integrated understanding of how geo-biosphere interactions in terrestrial systems induce and respond to environmental changes, using evidence from both the geological past and the present to improve projections of future global change impacts and assess the effectiveness of mitigation and adaptation strategies. Understanding feedbacks between diversity and stability in the geosphere and the biosphere lies at the heart of TERRA. In particular, we hypothesize that diversity in the geosphere stabilizes the biosphere, and that vice versa biodiversity is key to stabilizing the geosphere.TERRA represents an interdisciplinary Earth-System-Science approach. We will integrate observational, experimental, and modeling approaches spanning different periods of Earth history, incorporating the full spectrum of geological and biological sciences. We will analyze past geo-biosphere interactions preserved in geological records to elucidate how the Earth System responded to conditions that have not yet been encountered in historical times but may be encountered in the future. A mechanistic understanding of processes will be achieved by studying contemporary geo-biosphere interactions on different spatial scales. The newly established Diversitorium will facilitate field and laboratory experiments where the diversity of one sphere is selectively manipulated to study effects on the other sphere. Synthesis across spatio-temporal scales will be provided by developing and advancing integrative models merging machine learning and process-based approaches. These models will be used to evaluate the effectiveness of mitigation and adaptation measures to cope with global change.

As one of the most important forage species in Europe, white clover ( Trifolium repens ) is a legume that is well recognized for its potential to increase productivity especially under reduced N input. It is hypothesized that legumes have the potential to decrease overwinter soil greenhouse gas (GHG) emissions due to more efficient N recycling as compared to non-legume forbs. We conducted a field experiment recording high-resolution soil nitrous oxide (N 2 O) and methane (CH 4 ) fluxes during the winter months (December 2019 to March 2020) on a five-year-old grassland in central Germany with white clover, fertilized and unfertilized perennial ryegrass ( Lolium perenne ), and bare soil. White clover and fertilized ryegrass stimulated soil N 2 O emissions by 174% and 212% as compared to bare soil, and by 36% and 56% as compared to unfertilized ryegrass, respectively, due to their greater N availability and higher water-filled pore space (WFPS). The estimated cumulative CH 4 fluxes under white clover were a net CH 4 sink, whereas ryegrass and bare soil were net CH 4 sources. Soil N 2 O fluxes were predominantly regulated by both mineral N and WFPS, while CH 4 fluxes were mainly explained by WFPS. N-fertilization during the growing season did not affect off-season N 2 O and CH 4 fluxes in perennial ryegrass plots. The combined non-CO 2 global warming potential highlighted the possible mitigation effect of white clover on overwinter GHG emissions. Our findings suggest that GHG emissions from legumes are not offsetting their productive benefits during the non-frozen winter seasons.

Many bacteria synthesize carbon (C) and energy storage compounds, including water-insoluble polyester lipids composed mainly or entirely of poly(3-hydroxybutyrate) (PHB). Despite the potential significance of C and energy storage for microbial life and C cycling, few measurements of PHB in soil have been reported.A new protocol was implemented, based on an earlier sediment extraction and derivatization procedure, with quantification by gas chromatography/mass spectrometry (GC/MS) and 13 C-isotopic analysis by GC/combustion/isotope ratio mass spectrometry (GC/C/IRMS).The PHB content was 4.3 μg C g-1 in an agricultural soil and 1.2 μg C g-1 in a forest topsoil. This was an order of magnitude more PHB than obtained by the existing extraction method, suggesting that native PHB in soil has been previously underestimated. Addition of glucose increased the PHB content by 135% and 1,215% over 5 days, with the largest increase in the relatively nutrient-poor forest soil. In the agricultural soil, 68% of the increase was derived from added 13 C-labeled glucose, confirming synthesis of PHB from glucose for the first time in soil.The presence and responsiveness of PHB in both these contrasting soils show that PHB could provide a useful indicator of bacterial nutritional status and unbalanced growth. Microbial storage could be important to C and nutrient cycling and be a widespread strategy in the life of soil bacteria. The presented method offers new insight into the significance of this compound in soil.

Organo-mineral interactions are the most important mechanisms of long-term C stabilization in soils. Nevertheless, a part of the sorbed low molecular weight organic substances (LMWOS) remains bioavailable. Uniformly labeling of substances by 14C or 13C reflects only the average fate of C atoms of a LMWOS molecule. The submolecular tool of position-specific labeling allows to analyze metabolic pathways of individual functional groups and thus reveals deeper insight into mechanisms of sorption and microbial utilization. Alanine labeled with 14C in the 1st, 2nd or 3rd position was adsorbed to five sorbents: two iron oxides with different crystalline structure: goethite and haematite; two clay minerals with 2:1 layers – smectite, and 1:1 layers – kaolinite; and activated charcoal. After subsequent addition of these sorbents to a loamy haplic Luvisol, we analyzed 14C release into the soil solution, its microbial utilization and 14CO2 efflux from individual C positions of alanine. All sorbents bound alanine as an intact molecule (identical sorption of 1st, 2nd or 3rd positions). The bioavailability of sorbed alanine and its microbial transformation pathways depended strongly on the sorbent. Goethite and activated charcoal sorbed the highest amount of alanine (∼45% of the input), and the lowest portion of the sorbed alanine C was microbially utilized (26 and 22%, respectively). Mineralization of the desorbed alanine peaked within the first 5 h and was most pronounced for alanine bound to clay minerals. The initial mineralization to CO2 of bound alanine was always highest for the C-1 position (–COOH group). Mineralization rates of C-2 and C-3 exceeded the C-1 oxidation after 10–50 h, reflecting the classical biochemical pathways: 1) deamination, 2) decarboxylation of C-1 within glycolysis, and further 3) oxidation of C-2 and C-3 in the citric acid cycle. The ratio between two metabolic pathways – glycolysis (C-1 oxidation) versus citric-acid cycle (oxidation of C-2 and C-3) – was dependent on the microbial availability of sorbed alanine. High availability causes a peak in glycolysis C-1 oxidation followed by an abrupt shift to oxidation via the citric acid cycle. Low microbial availability of sorbed alanine, in turn, leads to a less pronounced, parallel oxidation of all three positions and to a higher relative incorporation of alanine C into microbial compounds. Modeling of C fluxes revealed that a significant portion of the sorbed alanine was incorporated in microbial biomass after 78 h and was further stabilized at the sorbents' surfaces. Position-specific labeling enabled determination of pathways and rates of C utilization from individual molecule positions and its dependence on various sorption mechanisms. We conclude that position-specific labeling is a unique tool for detailed insights into the submolecular transformation processes, mechanisms and rates of C stabilization in soil.

Summary The present study aimed to reveal the steps in the formation of fungal and bacterial amino sugars (AS) from glucose. Glucose labelled uniformly and position‐specifically at C‐1, C‐2, C‐4 and C‐6 was applied to an agricultural soil. Fungal and bacterial pathways of AS formation were reconstructed by a new approach: 13 C recovery from individual C positions of glucose in AS by IC‐O‐IRMS. Only 0.75% of 13 C from initially applied glucose was incorporated into AS within 10 days and followed the order: glucosamine > galactosamine > muramic acid. Relative incorporation of 13 C (% of AS‐C) had the largest values for bacterial muramic acid and the smallest for fungal galactosamine. This reflects faster turnover of bacterial than fungal cell walls. A maximum of 55% ± 19% of the 13 C incorporated in glucosamine was derived from intact, untransformed glucose. The smallest recovery was observed for C‐1 and C‐4. Recovery of C‐1 even decreased from day 3 to day 10, reflecting the metabolization by the pentose phosphate pathway. To be incorporated into AS, metabolites resulting from either the pentose phosphate pathway or glycolysis needed to be reconstructed to glucose by the reversible pathways of gluconeogenesis. The direct formation of AS (e.g. intact recovery of the glucose precursor), as well as amino sugar formation from recycled metabolites allocated to the hexose pool by back flux, occurred in parallel. Bacterial muramic acid showed the most variation in the recovery of individual C positions during 10 days, which reflects its intensive transformation by glycolysis, pentose phosphate pathway and gluconeogenesis. It is explained by the lower metabolic activity of fungi than bacteria at steady‐state conditions. The present approach enabled us to analyse the biochemical pathways in bacteria and fungi, which will considerably improve our understanding of the formation and stabilization of microbially derived SOM compounds. Highlights Fungal and bacterial pathways of the formation of amino sugars from glucose are reconstructed Intact use of glucose is distinguished from recycling by a metabolic tracing approach Half of the precursor (glucose) is metabolized prior to incorporation into amino sugars Intracellular recycling plays a crucial role in microbial SOM formation

Microbial acquisition and utilization of organic and mineral phosphorus (P) sources in paddy soils are strongly dependent on redox environment and remain the key to understand P turnover and allocation for cell compound synthesis. Using double 32/33P labeling, we traced the P from three sources in a P-limited paddy soil: ferric iron-bound phosphate (Fe-P), wheat straw P (Straw-P), and soil P (Soil-P) in microbial biomass P (MBP) and phospholipids (Phospholipid-P) of individual microbial groups depending on water regimes: (i) continuous flooding or (ii) alternate wetting and drying. 32/33P labeling combined with phospholipid fatty acid analysis allowed to trace P utilization by functional microbial groups. Microbial P nutrition was mainly covered by Soil-P, whereas microorganisms preferred to take up P from mineralized Straw-P than from Fe-P dissolution. The main Straw-P mobilizing agents were Actinobacteria under alternating wetting and drying and other Gram-positive bacteria under continuous flooding. Actinobacteria and arbuscular mycorrhiza increased P incorporation into cell membranes by 1.4–5.8 times under alternate wetting and drying compared to continuous flooding. The Fe-P contribution to MBP was 4–5 times larger in bulk than in rooted soil because (i) rice roots outcompeted microorganisms for P uptake from Fe-P and (ii) rhizodeposits stimulated microbial activity, e.g. phosphomonoesterase production and Straw-P mineralization. Higher phosphomonoesterase activities during slow soil drying compensated for the decreased reductive dissolution of Fe-P. Concluding, microbial P acquisition strategies depend on (i) Soil-P, especially organic P, availability, (ii) the activity of phosphomonoesterases produced by microorganisms and roots, and (iii) P sources – all of which depend on the redox conditions. Maximizing legacy P utilization in the soil as a function of the water regime is one potential way to reduce competition between roots and microbes for P in rice cultivation.

A deeper understanding of the mechanisms underlying the impacts of multiple stresses in crops is direly needed given the climate change-induced risks to achieving food security for a growing world population. Global warming has already led to a higher frequency of multiple stresses occurring concurrently or subsequently and will continue to do so for the next decades. Plant-stress interactions are commonly subdivided into abiotic and biotic stresses and studied separately. Under field conditions, these stress interactions are usually multiple and interactive in character.To date, the mechanisms determining interactions between abiotic and biotic stresses and their effects on crop performance are unknown for most crops and stress combinations. Field data are particularly scarce as most studies have focused on laboratory model systems using few environmental parameters in controlled conditions, which cannot reflect the dynamics in the field. Adequate modelling approaches capable of describing basic crop growth processes and simultaneously capturing response to abiotic and biotic stress interactions and their impacts on crop yield and quality do not exist so far.The aim of this paper is to present the design of a joint experimental and modelling platform (MultiStress) capable of creating a deeper understanding of the overall impact of combined (abiotic+biotic) stresses on crop physiology and productivity (grain yield, biomass, grain and stover quality, nutrient/water use efficiency, etc.) using the cereal maize as one of the most important crops globally as a model.The empirical knowledge gained from the experimental set-up and formalized in an associated modelling platform is utilized to define traits for stress tolerant breeding to be considered in ideotyping cereal cultivars for future target environments. In our example, in a research Pillar I, we describe a field experimental platform (with rainout shelters) applicable under temperate and tropical climate conditions to investigate the interactions of drought and nitrogen deficiency with the foliar disease Northern Corn Leaf Blight caused by Setosphaeria turcica on the one hand, and stem borer caterpillars on the other.  Pillar II is an associated process-based modelling platform enabling integration of new genetic and ecophysiological knowledge and extrapolate the findings in time and space.Applying a systems approach in conjunction with this platform we can test the following hypotheses: (i) the impact of combined abiotic and biotic stress interactions on crop growth and yield formation and quality is non-additive and thus differs from the sum of individual stress impacts; (ii) while the mechanisms underlying the abiotic and biotic stress interactions are of universal validity, their impacts are modulated by certain environmental conditions (such as temperature, light conditions and soil properties).Realization and evaluation of such platform will allow consideration of interactions between abiotic and biotic stresses and hence improve the predictive skill of crop growth models.