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It is uncertain how the predicted further rise of atmospheric carbon dioxide (CO2) concentration will affect plant nutrient availability in the future through indirect effects on the gross rates of nitrogen (N) mineralization (production of ammonium) and depolymerization (production of free amino acids) in soil. The response of soil nutrient availability to increasing atmospheric CO2 is particularly important for nutrient poor ecosystems. Within a FACE (Free-Air Carbon dioxide Enrichment) experiment in a native, nutrient poor Eucalyptus woodland (EucFACE) with low soil organic matter (≤ 3%), our results suggested there was no shortage of N. Despite this, microbial N use efficiency was high (c. 90%). The free amino acid (FAA) pool had a fast turnover time (4 h) compared to that of ammonium (NH4+) which was 11 h. Both NH4-N and FAA-N were important N pools; however, protein depolymerization rate was three times faster than gross N mineralization rates, indicating that organic N is directly important in the internal ecosystem N cycle. Hence, the depolymerization was the major provider of plant available N, while the gross N mineralization rate was the constraining factor for inorganic N. After two years of elevated CO2, no major effects on the pools and rates of the soil N cycle were found in spring (November) or at the end of summer (March). The limited response of N pools or N transformation rates to elevated CO2 suggest that N availability was not the limiting factor behind the lack of plant growth response to elevated CO2, previously observed at the site.

The billion tons of synthetic-polymer-based materials (i.e. plastics) produced yearly are a great challenge for humanity. Nature produces even more natural polymers, yet they are sustainable. Proteins are sequence-defined natural polymers that are constantly recycled when living systems feed. Digestion is the protein depolymerization into amino acids (the monomers) followed by their re-assembly into new proteins of arbitrarily different sequence and function. This breaks a common recycling paradigm where a material is recycled into itself. Organisms feed off of random protein mixtures that are "recycled" into new proteins whose identity depends on the cell's specific needs. In this study, mixtures of several peptides and/or proteins are depolymerized into their amino acid constituents, and these amino acids are used to synthesize new fluorescent, and bioactive proteins extracellularly by using an amino-acid-free, cell-free transcription-translation (TX-TL) system. Specifically, three peptides (magainin II, glucagon, and somatostatin 28) are digested using thermolysin first and then using leucine aminopeptidase. The amino acids so produced are added to a commercial TX-TL system to produce fluorescent proteins. Furthermore, proteins with high relevance in materials engineering (beta-lactoglobulin films, used for water filtration, or silk fibroin solutions) are successfully recycled into biotechnologically relevant proteins (fluorescent proteins, catechol 2,3-dioxygenase)., Advanced Materials, 33 (44), ISSN:0935-9648, ISSN:1521-4095

The billion tons of synthetic polymer-based materials (i.e. plastics) produced every year are one of the greatest challenges that humanity has to face. Nature produces even more natural polymers, yet they are sustainable. For example, proteins are sequence-defined natural polymers, that are constantly recycled when living systems feed. Indeed, digestion is the protein depolymerization into amino acids (i.e. the monomers) followed by their re-assembly into new proteins of arbitrarily different sequence and function. This process breaks a common recycling paradigm where a material is recycled into itself. Organisms feed of random protein mixtures that are ‘recycled’ into new proteins whose identity depends on the cell’s needs at the time of protein synthesis. Currently, advanced materials are increasingly made of proteins, but the abovementioned ideal recyclability of such materials has yet to be recognized and established. In this study mixtures of several (up to >30) peptides and/or proteins were depolymerized into their amino acid constituents, and these amino acids were used to synthesize new fluorescent, and bio-active proteins extra-cellularly by using an amino acid-free cell-free transcription-translation system. Proteins with high relevance in materials engineering (β-lactoglobulin films, used for water filtration, or silk fibroin solutions) were successfully recycled into biotechnologically relevant proteins (green, and red fluorescent proteins, catechol 2,3-dioxygenase). The potential long-term impact of this approach to recycling lies in its compatibility with circular-economy models where raw materials remain in use as long as possible, thus reducing the burden on the planet.

Proteins comprise the largest soil N reservoir but cannot be taken up directly by microorganisms and plants due to size constraints and stabilization of proteins in organo-mineral associations. Therefore the cleavage of this high molecular weight organic N to smaller soluble compounds as amino acids is a key step in the terrestrial N cycle. In the last years two isotope pool dilution approaches have been successfully established to measure gross rates of protein depolymerization and microbial amino acid uptake in soils. However, both require laborious sample preparation and analyses, which limits sample throughput. Therefore, we here present a novel isotope pool dilution approach based on the addition of 15N-labeled amino acids to soils and subsequent concentration and 15N analysis by the oxidation of α-amino groups to NO2− and further reduction to N2O, followed by purge-and-trap isotope ratio mass spectrometry (PT-IRMS). We applied this method in mesocosm experiments with forest and meadow soils as well as with a cropland soil amended with either organic C (cellulose) or organic N (bovine serum albumin). To measure direct organic N mineralization to NH4+, the latter was captured in acid traps and analyzed by an elemental analyzer coupled to an isotope ratio mass spectrometer (EA-IRMS). Our results demonstrate that the proposed method provides fast and precise measurements of at%15N even at low amino acid concentrations, allows high sample throughput and enables parallel estimations of instantaneous organic N mineralization rates.

The common spread pattern of ovarian cancer is peritoneal implantation. The growth of the shed ovarian cancer cells in the peritoneal cavity is closely related to the tumor microenvironment. Cancer-associated fibroblasts are vital in the tumor microenvironment. It is not clearly defined that the protein expression alters during the activating process of fibroblasts. This study detected the protein alterations in fibroblasts induced by ovarian cancer cells and explored the potential biological relevance through two-dimensional gel electrophoresis and mass spectrometry. Our data showed that the level of CENPE, BAG2, SOD2, GDI2, CORO1C, CFL1, DSTN, CALD1, PHGDH, PDHA1, AKR1B1, TST and TBCA proteins were significantly up-regulated in the fibroblasts co-cultured with ovarian cancer cells, whereas HSPB1, P4HB and VIM were significantlydown-regulated. However, only BAG2, SOD2 and CORO1C proteins were confirmed to be significantly increased by western blot analysis. The differentially expressed proteins were mainly involved in metabolic processes, cellular component organization, responses to stimulus, multicellular organismal processes, localization, protein depolymerization, cellular senescence and the mitotic pathway. These data demonstrated that fibroblasts had an altered protein expression pattern after being induced by ovarian cancer cells, and participated in multiple cell processes resulting in tumor progression. The differentially expressed proteins should be considered as targets for cancer treatment.

Rising carbon dioxide (CO2) concentrations and temperatures are expected to stimulate plant productivity and ecosystem C sequestration, but these effects require a concurrent increase in N availability for plants. Plants might indirectly promote N availability as they release organic C into the soil (e.g., by root exudation) that can increase microbial soil organic matter (SOM) decomposition ("priming effect"), and possibly the enzymatic breakdown of N-rich polymers, such as proteins, into bio-available units ("N mining"). We tested the adjustment of protein depolymerization to changing soil C and N availability in a laboratory experiment. We added easily available C or N sources to six boreal forest soils, and determined soil organic C mineralization, gross protein depolymerization and gross ammonification rates (using N-15 pool dilution assays), and potential extracellular enzyme activities after 1 week of incubation. Added C sources were C-13-labelled to distinguish substrate from soil derived C mineralization. Observed effects reflect short-term adaptations of non-symbiotic soil microorganisms to increased C or N availability. Although C input promoted microbial growth and N demand, we did not find indicators of increased N mobilization from SOM polymers, given that none of the soils showed a significant increase in protein depolymerization, and only one soil showed a significant increase in N-targeting enzymes. Instead, our findings suggest that microorganisms immobilized the already available N more efficiently, as indicated by decreased ammonification and inorganic N concentrations. Likewise, although N input stimulated ammonification, we found no significant effect on protein depolymerization. Although our findings do not rule out in general that higher plant-soil C allocation can promote microbial N mining, they suggest that such an effect can be counteracted, at least in the short term, by increased microbial N immobilization, further aggravating plant N limitation.

Nitrogen (N) dynamics in Phragmites australis litter due to anaerobic decomposition in three anoxic wetland substrates were analyzed by elemental analyses and infrared spectroscopy (FTIR). After 75 days of decomposition, a relative accumulation of bulk N was detected in most litters, but N accumulated less when decomposition took place in a more N-poor environment. FTIR was used to quantify the relative content of proteins in litter tissue and revealed a highly linear relationship between bulk N content and protein content. Changes in bulk N content thus paralleled and probably were governed by changes in litter protein content. Such changes are the result of two competing processes within decomposing litter: enzymatic protein depolymerization as a part of the litter breakdown process and microbial protein synthesis as a part of microbial biomass growth within the litter. Assuming microbial homeostasis, DNA signals in FTIR spectra were used to calculate the amount of microbial N in decomposed litter which ranged from 14 to 42 % of the total litter N for all leaf samples. Microbial carbon (C) content and resultant calculated carbon-use efficiencies (CUEs) indicate that microbial N in litter accumulated according to predictions of the stoichiometric decomposition theory. Subtracting microbial C- and N-contributions from litter, however, revealed decomposition site dependent variations in the percentual amount of remaining, still unprocessed plant N compared to remaining plant C, an indicator for preferential protein depolymerization. For all leaf litters, the coefficient of preferential protein depolymerization (α), which relates N-compound depolymerization to C-compound depolymerization, ranged from 0.74–0.88 in a nutrient-rich detritus mud to 1.38–1.82 in Sphagnum peat, the most nutrient-poor substrate in this experiment. Preferential protein depolymerization leads to a gradual N depletion of decomposing litter which we propose as a preservation mechanism for vascular litter decomposing in Sphagnum peat.

Proteinaceous compounds are abundant forms of organic nitrogen in soil and aquatic ecosystems, and the rate of protein depolymerization, which is accomplished by a diverse range of microbial secreted peptidases, often limits nitrogen turnover in the environment. To determine if the distribution of secreted peptidases reflects the ecological and evolutionary histories of different taxa, we analyzed their distribution across prokaryotic lineages. Peptidase gene sequences of 147 archaeal and 2,191 bacterial genomes from the MEROPS database were screened for secretion signals, resulting in 55,072 secreted peptidases belonging to 148 peptidase families. These data, along with their corresponding 16S rRNA sequences, were used in our analysis. Overall, Bacteria had a much wider collection of secreted peptidases, higher average numbers of secreted peptidases per genome, and more unique peptidase families than Archaea. We found that the distribution of secreted peptidases corresponded to phylogenetic relationships among Bacteria and Archaea and often segregated according to microbial lifestyles, suggesting that the secreted peptidase complements of microbial taxa are optimized for the environmental microhabitats they occupy. Our analyses provide the groundwork for examining the specific functional role of families of secreted peptidases in relationship to the organisms and the corresponding environments in which they function.

Soil nitrogen (N) mineralization is generally limited by amino acid production by proteases (depolymerization). Protease activities can be stimulated under N limitation, but it cannot directly increase microbial N release (ammonification) in temperate forest soils. To analyze factors determining rate-limiting steps of soil N mineralization, we compared microbial potentials of depolymerization and ammonification by measuring casein degradation and arginine ammonification rates in casein- or arginine-amended and control soils, respectively. The microbial potentials of casein depolymerization were positively correlated with soil carbon (C)/N ratios, whereas arginine ammonification exhibited the opposite pattern. The soil N mineralization was limited by depolymerization in N-rich broad-leaved forest and cropland soils, while N mineralization could be limited by the low microbial potentials of ammonification for microbial N conservation in coniferous forest soils with C/N ratios > 20. Due to the increased depolymerization and microbial N conservation under N limitation, ammonification as well as depolymerization can be a rate-limiting step of N mineralization in fungi-dominated acidic forest soils. Contrasting responses of depolymerization and ammonification to N limitation and low pH can induce a shift in rate-limiting steps in soil N mineralization.

Reactive nitrogen (N) input can profoundly alter soil N transformations and long-term productivity of forest ecosystems. However, critical knowledge gaps exist in our understanding of N deposition effects on internal soil N cycling in forest ecosystems. It is well established that N addition enhances soil N availability based on traditional net mineralization rate assays. Yet, experimental additions of inorganic N to soils broadly show a suppression of microbial activity and protein depolymerization. Here we show, from a global meta-analysis of 15N-labelled studies that gross N transformation rates in forest soil organic and mineral horizons differentially respond to N addition. In carbon (C)-rich organic horizons, N addition significantly enhanced soil gross rates of N mineralization, nitrification and microbial NO3¯ immobilization rates, but decreased gross microbial NH4+ immobilization rates. In C-poor mineral soils, in contrast, N addition did not change gross N transformation rates except for increasing gross nitrification rates. An initial soil C/N threshold of approx. 14.6, above which N addition enhanced gross N mineralization rates, could explain why gross N mineralization was increased by N deposition in organic horizons alone. Enhancement of gross N mineralization by N deposition was also largely attributed to enhanced N mineralization activity per unit microbial biomass. Our results indicate that the net effect of N input on forest soil gross N transformations are highly stratified by soil C distribution along the soil profile, and thus challenge the perception that N availability ubiquitously limits N mineralization. These findings suggest that these differences should be integrated into models to better predict forest ecosystem N cycle and C sequestration potential under future N deposition scenarios.

The aim of the study was the selection of food grade, an inexpensive and commercially available enzyme that would allow to obtain a hydrolysate from carp (Cyprinus carpio) skin gelatin with the highest antioxidant activity. Moreover, the objective of the study was to investigate the effects of pH temperature and incubation duration of the hydrolysis on protein degradation, free amino acids and antioxidant properties of the obtained hydrolysates. Protein hydrolysates obtained from carp skin gelatin using Protamex show potent antioxidant properties, which differed depending on the hydrolysis parameters. The hydrolysis parameters significantly influenced the free amino acid profiles as well as protein depolymerization and antioxidant properties of the hydrolysates. The acquired hydrolysates showed very potent antioxidant properties (FRAP value of 5.12 μM Trolox/mg) and a high degree of the depolymerized protein (27.73%). Furthermore, there was a clear positive correlation between the pH of the hydrolysis and the metal chelating activity of the hydrolysate, with all the hydrolysates produced at pH 6 exhibiting no metal chelating activity, while hydrolysates produced at pH 8 showed metal chelating activity. The carp skin gelatin hydrolysates with the highest antioxidant properties were obtained using Protamex at pH 7 and a temperature of 50 °C for 3 h.

Soil N availability for plants and microorganisms depends on the breakdown of soil polymers such as proteins into smaller, assimilable units by microbial extracellular enzymes. Changing climatic conditions are expected to alter protein depolymerization rates over the next decades, and thereby affect the potential for plant productivity. We here tested the effect of increased CO2 concentration, temperature, and drought frequency on gross rates of protein depolymerization, N mineralization, microbial amino acid and ammonium uptake using 15N pool dilution assays. Soils were sampled in fall 2013 from the multifactorial climate change experiment CLIMAITE that simulates increased CO2 concentration, temperature, and drought frequency in a fully factorial design in a temperate heathland. Eight years after treatment initiation, we found no significant effect of any climate manipulation treatment, alone or in combination, on protein depolymerization rates. Nitrogen mineralization, amino acid and ammonium uptake showed no significant individual treatment effects, but significant interactive effects of warming and drought. Combined effects of all three treatments were not significant for any of the measured parameters. Our findings therefore do not suggest an accelerated release of amino acids from soil proteins in a future climate at this site that could sustain higher plant productivity.

Ultra-high temperature (UHT) treatment of milk forms a deposit or fouling in the processing equipment that is mineral-based with an enclosed protein network. This study addresses the fundamental mechanisms that control the removal of this deposit. For this purpose, the structural and compositional changes during the cleaning process have been studied. The structure analysis was performed with scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) on samples that were quenched at different stages of the cleaning process. It was found for acid cleaning that the mineral content is rapidly decreasing in the fouling layer as the cleaning continues, but there is still an intact protein structure with the similar thickness as the original fouling. For alkali cleaning, part of the protein structure was subsequently removed from the outside towards the stain-less steel as a function of time, while the mineral structure was mostly remaining. The break-up of the organic network structure, which likely involves depolymerization of protein aggregates, were found to control the cleaning efficiency. The weakening of the protein network facilitates the removal of the UHT fouling layer during the acid cleaning step and allow for an efficient cleaning cycle. The chemical reactions that occur within the fouling layer between the hydroxyl ions and the protein network was modeled according to a depolymerization reaction and a mechanistic model of the cleaning process is presented.

Although the rates of gross protein depolymerization are central in litter decomposition, they cannot be measured directly by any existing method. Here we report the development of a novel assay to quantify gross protein depolymerization (i.e. gross amino acid production) based on a 15N isotope pool dilution technique. The assay is based on the concurrent labeling of the pool of 18 proteinogenic amino acids, which are present in a free form in litter, and the measurement of 15N:14N ratios in the individual amino acids by GC–MS over time. The method proved to be highly linear, precise and sufficiently sensitive. We tested the applicability of the novel method on a litter decomposition experiment, in which we could demonstrate that gross protein depolymerization exceeded gross N mineralization by >8 fold indicating that only a small fraction of amino acids released by extracellular enzymes was actually mineralized to ammonium. Moreover, the results provide evidence that protein depolymerization was limited by protein availability or accessibility, not by the size of protease pool.

In this study, the application of high-pressure processing of sorghum batters was investigated in order to evaluate the potential of pressure-treated sorghum as a gluten replacement in the production of sorghum breads. For this purpose, sorghum batters were treated at pressures from 200 to 600 MPa at 20 °C, and the microstructure was investigated using scanning electron microscopy. Furthermore, the rheological properties of the control and pressure-treated batters were determined. The results revealed weakening of the batter structure at pressures ≤300 MPa. Addition of a blocker of free thiol groups indicated that protein depolymerization played a role in this strength decrease. At pressures >300 MPa, the batter consistency increased, mainly due to pressure-induced gelatinization of starch. Furthermore, freeze-dried sorghum batters treated at 200 MPa (weakest batter) and at 600 MPa (strongest batter) were added to a sorghum bread recipe, replacing 2 and 10% of untreated sorghum flour. The results showed a delayed staling for breads containing 2% of sorghum treated at 600 MPa. However, adding 10% resulted in a low specific volume and poor bread quality. The quality of breads containing different amounts of sorghum treated at 200 MPa was not significantly different from the control bread.

Arctic plant productivity is often limited by low soil N availability. This has been attributed to slow breakdown of N-containing polymers in litter and soil organic matter (SOM) into smaller, available units, and to shallow plant rooting constrained by permafrost and high soil moisture. Using N-15 pool dilution assays, we here quantified gross amino acid and ammonium production rates in 97 active layer samples from four sites across the Siberian Arctic. We found that amino acid production in organic layers alone exceeded literature-based estimates of maximum plant N uptake 17-fold and therefore reject the hypothesis that arctic plant N limitation results from slow SOM breakdown. High microbial N use efficiency in organic layers rather suggests strong competition of microorganisms and plants in the dominant rooting zone. Deeper horizons showed lower amino acid production rates per volume, but also lower microbial N use efficiency. Permafrost thaw together with soil drainage might facilitate deeper plant rooting and uptake of previously inaccessible subsoil N, and thereby promote plant productivity in arctic ecosystems. We conclude that changes in microbial decomposer activity, microbial N utilization and plant root density with soil depth interactively control N availability for plants in the Arctic.

Proteins represent the dominant input of organic N into most ecosystems and they also constitute the largest store of N in soil organic matter. The extracellular protease mediated breakdown of proteins to amino acids therefore represents a key step regulating N cycling in soil. In this study we investigated the influence of a range of environmental factors on the rate of protein mineralization in a grazed grassland and fallow agricultural soil. The protein turnover rates were directly compared to the rates of amino acid mineralization under the same conditions. Uniformly 14 C-labelled soluble protein and amino acids were added to soil and the rate of 14 CO 2 evolution determined over 30 d. Our results indicate that the primary phase of protein mineralization was approximately 20 ± 3 fold slower that the rate of amino acid mineralization. The addition of large amounts of inorganic NO 3 − and NH 4 + to the soil did not repress the rate of protein mineralization suggesting that available N does not directly affect protease activity in the short term. Whilst protein mineralization was strongly temperature sensitive, the presence of plants and the addition of humic and tannic acids had relatively little influence on the rate of soluble protein degradation in this fertile grassland soil. Our results suggests that the extracellular protease mediated cleavage of proteins to amino acids rather than breakdown of amino acids to NH 4 + represents the limiting step in soil N cycling.

Ectomycorrhizal (EM) fungi are abundant in temperate and boreal ecosystems and are understood to be an important means whereby plants can fulfill their nutrition requirements. The extent of the EM fungal involvement in accessing organic sources of N, however, remains unknown. Some EM fungi have been found to produce lignolytic and proteolytic enzymes which are necessary to depolymerize organic substrates, but this ability varies by species. Both EM fungal communities and the activities of lignolytic and proteolytic enzymes may be sensitive to changes in inorganic N availability such as through increased atmospheric deposition. Our objectives were to simulate an ecologically relevant increase in atmospheric N deposition in areas currently receiving very little exogenous N and examine changes in EM community composition, lignin degrading enzyme activity, and soil protein depolymerization. We found a distinct shift in the EM community composition following simulated atmospheric N deposition. Likewise, we found a significant decrease in the activity of lignin degrading enzymes, which could have important implications on ecosystem N and C cycling. Contrary to our hypotheses, proteolysis increased following N addition. The fact that lignolytic and proteolytic enzymes exhibit opposite responses is counterintuitive and suggests much is yet to be learned about how N addition affects global C storage by affecting the decomposition of organic matter. Our data suggest small increases in atmospheric N deposition could produce significant changes in communities of EM fungi and N and C cycles.

Until recently, the common view of the terrestrial nitrogen cycle had been driven by two core assumptions—plants use only inorganic N and they compete poorly against soil microbes for N. Thus, plants were thought to use N that microbes “left over,” allowing the N cycle to be divided cleanly into two pieces—the microbial decomposition side and the plant uptake and use side. These were linked by the process of net mineralization. Over the last decade, research has changed these views. N cycling is now seen as being driven by the depolymerization of N-containing polymers by microbial (including mycorrhizal) extracellular enzymes. This releases organic N-containing monomers that may be used by either plants or microbes. However, a complete new conceptual model of the soil N cycle needs to incorporate recent research on plant–microbe competition and microsite processes to explain the dynamics of N across the wide range of N availability found in terrestrial ecosystems. We discuss the evolution of thinking abou...

The effects of a laccase from the fungus Pycnoporus cinnabarinus on the mixing of a wheat flour dough with or without added ferulic acid (FA) were studied. Laccase reduced dough time-to-peak and accelerated dough breakdown in comparison with the control. Its effect was enhanced with FA. The water extractability of arabinoxylans (AX) increased during mixing of a dough free of added laccase, especially with exogenous FA. At the same time, the extractability of FA decreased during mixing. Added FA may have competed with endogenous AX feruloyl esters, inhibiting partly oxidative gelation. Laccase decreased AX extractability by chain cross-linking through oxidative dimerization of feruloyl esters. FA and, moreover, FA plus laccase, increased the oxidation of sulfhydryl (SH) groups. FA and, even more, FA in combination with laccase, increased the rate of protein depolymerization during mixing. FA and the products of FA laccase oxidation participated in a redox reaction involving SH groups. A coupling r...

The Wareham Experimental Earthwork was constructed in 1963 in an area of heathland in the south of England with acidic sandy soils to investigate the processes that occurred early in the establishment of the archaeological record. Amongst its objectives was monitoring the changes to various archaeological materials that were buried in the earthwork. In this paper we present data on the interaction of soil micro-organisms with linen, flax, goatskin and hemp buried in 1963 and recovered during the 1996 excavation of the earthwork. These materials were originally buried on the old land surface beneath a stack of freshly cut turves (the turf environment), where the total C and N contents were relatively high, and higher up in the earthwork (the sand environment). In the 33 years since the earthwork was constructed all the visible remains of the linen, flax and hemp had been lost except where preservation had been aided by the presence of copper alloy or steel discs. The size of the soil microbial community (microbial biomass) and microbial activity (respiration rate) were greater in the turf environment than in the sand environment, but all the values were very small compared with typical ranges for soils. There was some evidence, in the form of increased respiratory responses to added glucose and greater respiration rates, that a legacy from the flax and the hemp persisted after they ceased to be visible. However, the results from adenosine triphosphate (ATP) analysis, which provides a measure of physiologically active micro-organisms, and from substrate utilization profiles (which provide an indication of the metabolic capability of the microbial community) did not support this. A short-term (7 months) laboratory experiment using samples of the original materials from the experimental archive and soil samples from the earthwork was conducted to attempt to simulate the conditions at the outset of the experiment. The rate of decomposition of the materials during this experiment was initially rapid and after 7 months between about 10 and 20% of the carbon in the buried materials had been lost. The substrate utilization profiles of the microbial communities associated with the decomposing materials could be related to their chemical composition, with the communities associated with plant-derived (carbohydrate-rich) materials giving large responses to carbohydrates, and the goatskin (mostly proteinaceous tissues) giving large responses to amino acids (components of proteins). Solid-state nuclear magnetic resonance (NMR) spectroscopy was used to investigate changes in the distribution of carbon between differentfunctional groups indicative of some of the main types of biochemicals in the materials during the short term decomposition experiment. Only small changes were observed in the NMR spectra of the plant-derived materials. Consistent with the substrate utilization profiles, there was evidence of protein depolymerization to amino acids in the NMR spectra for the decomposing goatskin. When the materials were incubated in a reference soil that contained more N and a larger community of active micro-organisms than either of the soil types from the earthwork, the short term decomposition rate of all the materials was more rapid than in the earthwork soils, with the increase in decomposition rate being more marked for the plant-derived materials. It is hypothesized that this observation is due to a lack of available N restricting decomposition of the plant-derived materials in the earthwork soils.

Turbic Cryosols (permafrost soils characterized by cryoturbation, i.e., by mixing of soil layers due to freezing and thawing) are widespread across the Arctic, and contain large amounts of poorly decomposed organic material buried in the subsoil. This cryoturbated organic matter exhibits retarded decomposition compared to organic material in the topsoil. Since soil organic matter (SOM) decomposition is known to be tightly linked to N availability, we investigated N transformation rates in different soil horizons of three tundra sites in north-eastern Siberia and Greenland. We measured gross rates of protein depolymerization, N mineralization (ammonification) and nitrification, as well as microbial uptake of amino acids and NH4+ using an array of 15N pool dilution approaches. We found that all sites and horizons were characterized by low N availability, as indicated by low N mineralization compared to protein depolymerization rates (with gross N mineralization accounting on average for 14% of gross protein depolymerization). The proportion of organic N mineralized was significantly higher at the Greenland than at the Siberian sites, suggesting differences in N limitation. The proportion of organic N mineralized, however, did not differ significantly between soil horizons, pointing to a similar N demand of the microbial community of each horizon. In contrast, absolute N transformation rates were significantly lower in cryoturbated than in organic horizons, with cryoturbated horizons reaching not more than 32% of the transformation rates in organic horizons. Our results thus indicate a deceleration of the entire N cycle in cryoturbated soil horizons, especially strongly reduced rates of protein depolymerization (16% of organic horizons) which is considered the rate-limiting step in soil N cycling., Highlights • N turnover in cryoturbated Arctic soils measured by isotope pool dilution assays. • Protein depolymerization strongly exceeds NH4+ and NO3− production rates. • Microbial N use efficiency indicates N limitation in Siberian sampling sites. • Deceleration of microbial N transformation rates by cryoturbation.

Microbial nitrogen use efficiency (NUE) describes the partitioning of organic N taken up between growth and the release of inorganic N to the environment (that is, N mineralization), and is thus central to our understanding of N cycling. Here we report empirical evidence that microbial decomposer communities in soil and plant litter regulate their NUE. We find that microbes retain most immobilized organic N (high NUE), when they are N limited, resulting in low N mineralization. However, when the metabolic control of microbial decomposers switches from N to C limitation, they release an increasing fraction of organic N as ammonium (low NUE). We conclude that the regulation of NUE is an essential strategy of microbial communities to cope with resource imbalances, independent of the regulation of microbial carbon use efficiency, with significant effects on terrestrial N cycling.

Soil carbon (C) and nitrogen (N) availability depend on the breakdown of soil polymers such as lignin, chitin, and protein that represent the major fraction of soil C and N but are too large for immediate uptake by plants and microorganisms. Microorganisms may adjust the production of enzymes targeting different polymers to optimize the balance between C and N availability and demand, and for instance increase the depolymerization of N-rich compounds when C availability is high and N availability low (“microbial N mining”). Such a mechanism could mitigate plant N limitation but also lie behind a stimulation of soil respiration frequently observed in the vicinity of plant roots (“priming effect”). We here compared the effect of increased C and N availability on the depolymerization of native bulk soil organic matter (SOM), and of 13C-enriched lignin, chitin, and protein added to the same soil in two complementary ten day microcosm incubation experiments. A significant reduction of chitin depolymerization (described by the recovery of chitin-derived C in the sum of dissolved organic, microbial and respired C) upon N addition indicated that chitin was degraded to serve as a microbial N source under low-N conditions and replaced in the presence of an immediately available alternative. Protein and lignin depolymerization in contrast were not affected by N addition. Carbon addition enhanced microbial N demand and SOM decomposition rates, but significantly reduced lignin, chitin, and protein depolymerization. Our findings contrast the hypothesis of increased microbial N mining as a key driver behind the priming effect and rather suggest that C addition promoted the mobilization of other soil C pools that replaced lignin, chitin, and protein as microbial C sources, for instance by releasing soil compounds from mineral bonds. We conclude that SOM decomposition is interactively controlled by multiple mechanisms including the balance between C vs N availability. Disentangling these controls will be crucial for understanding C and N cycling on an ecosystem scale.

Turbic Cryosols (permafrost soils characterized by cryoturbation, i.e., by mixing of soil layers due to freezing and thawing) are widespread across the Arctic, and contain large amounts of poorly decomposed organic material buried in the subsoil. This cryoturbated organic matter exhibits retarded decomposition compared to organic material in the topsoil. Since soil organic matter (SOM) decomposition is known to be tightly linked to N availability, we investigated N transformation rates in different soil horizons of three tundra sites in north-eastern Siberia and Greenland. We measured gross rates of protein depolymerization, N mineralization (ammonification) and nitrification, as well as microbial uptake of amino acids and NH4+ using an array of 15N pool dilution approaches. We found that all sites and horizons were characterized by low N availability, as indicated by low N mineralization compared to protein depolymerization rates (with gross N mineralization accounting on average for 14% of gross protein depolymerization). The proportion of organic N mineralized was significantly higher at the Greenland than at the Siberian sites, suggesting differences in N limitation. The proportion of organic N mineralized, however, did not differ significantly between soil horizons, pointing to a similar N demand of the microbial community of each horizon. In contrast, absolute N transformation rates were significantly lower in cryoturbated than in organic horizons, with cryoturbated horizons reaching not more than 32% of the transformation rates in organic horizons. Our results thus indicate a deceleration of the entire N cycle in cryoturbated soil horizons, especially strongly reduced rates of protein depolymerization (16% of organic horizons) which is considered the rate-limiting step in soil N cycling.

Proteins constitute the single largest soil organic nitrogen (SON) reservoir and its decomposition drives terrestrial N availability. Protein cleavage by extracellular enzymes is the rate limiting step in the soil organic N cycle and can be controlled by extracellular enzyme production or protein availability/stabilization in soil. Both controls can be affected by geology and land use, as well as be vulnerable to changes in soil temperature and moisture/O2. To explore major controls of soil gross protein depolymerization we sampled six soils from two soil parent materials (calcareous and silicate), where each soil type included three land uses (cropland, pasture and forest). Soil samples were subjected to three temperature treatments (5, 15, 25 °C at 60% water-holding capacity (WHC) and aerobic conditions) or three soil moisture/O2 treatments (30 and 60% WHC at 21% O2, 90% WHC at 1% O2, at 20 °C) in short-term experiments. Samples were incubated for one day in the temperature experiment and for one week in the moisture/O2 experiment. Gross protein depolymerization rates were measured by a novel 15N isotope pool dilution approach. The low temperature sensitivity of gross protein depolymerization, the lack of relationship with protease activity and strong effects of soil texture and pH demonstrate that this process is constrained by organo-mineral associations and not by soil enzyme content. This also became apparent from the inverse effects in calcareous and silicate soils caused by water saturation/O2 limitation. We highlight that the specific soil mineralogy influenced the response of gross depolymerization rates to water saturation/O2 limitation, causing (I) increasing gross depolymerization rates due to release of adsorbed proteins by reductive dissolution of Fe- and Mn-oxyhydroxides in calcareous soils and (II) decreasing gross depolymerization rates due to mobilization of coagulating and toxic Al3+ compounds in silicate soils.

Resource stoichiometry (C:N:P) is an important determinant of litter decomposition. However, the effect of elemental stoichiometry on the gross rates of microbial N and P cycling processes during litter decomposition is unknown. In a mesocosm experiment, beech (Fagus sylvatica L.) litter with natural differences in elemental stoichiometry (C:N:P) was incubated under constant environmental conditions. After three and six months, we measured various aspects of nitrogen and phosphorus cycling. We found that gross protein depolymerization, N mineralization (ammonification), and nitrification rates were negatively related to litter C:N. Rates of P mineralization were negatively correlated with litter C:P. The negative correlations with litter C:N were stronger for inorganic N cycling processes than for gross protein depolymerization, indicating that the effect of resource stoichiometry on intracellular processes was stronger than on processes catalyzed by extracellular enzymes. Consistent with this, extracellular protein depolymerization was mainly limited by substrate availability and less so by the amount of protease. Strong positive correlations between the interconnected N and P pools and the respective production and consumption processes pointed to feed-forward control of microbial litter N and P cycling. A negative relationship between litter C:N and phosphatase activity (and between litter C:P and protease activity) demonstrated that microbes tended to allocate carbon and nutrients in ample supply into the production of extracellular enzymes to mine for the nutrient that is more limiting. Overall, the study demonstrated a strong effect of litter stoichiometry (C:N:P) on gross processes of microbial N and P cycling in decomposing litter; mineralization of N and P were tightly coupled to assist in maintaining cellular homeostasis of litter microbial communities.

Turbic Cryosols (permafrost soils characterized by cryoturbation, i.e., by mixing of soil layers due to freezing and thawing) are widespread across the Arctic, and contain large amounts of poorly decomposed organic material buried in the subsoil. This cryoturbated organic matter exhibits retarded decomposition compared to organic material in the topsoil. Since soil organic matter (SOM) decomposition is known to be tightly linked to N availability, we investigated N transformation rates in different soil horizons of three tundra sites in north-eastern Siberia and Greenland. We measured gross rates of protein depolymerization, N mineralization (ammonification) and nitrification, as well as microbial uptake of amino acids and NH4 + using an array of 15N pool dilution approaches. We found that all sites and horizons were characterized by low N availability, as indicated by low N mineralization compared to protein depolymerization rates (with gross N mineralization accounting on average for 14% of gross protein depolymerization). The proportion of organic N mineralized was significantly higher at the Greenland than at the Siberian sites, suggesting differences in N limitation. The proportion of organic N mineralized, however, did not differ significantly between soil horizons, pointing to a similar N demand of the microbial community of each horizon. In contrast, absolute N transformation rates were significantly lower in cryoturbated than in organic horizons, with cryoturbated horizons reaching not more than 32% of the transformation rates in organic horizons. Our results thus indicate a deceleration of the entire N cycle in cryoturbated soil horizons, especially strongly reduced rates of protein depolymerization (16% of organic horizons) which is considered the rate-limiting step in soil N cycling.

Phragmites australis litters were incubated in three waterlogged anoxic wetland soils of different nutrient status for 75 d, and litter nitrogen (N) dynamics were analyzed by elemental analyses and Fourier transform infrared spectroscopy (FTIR). At the end of the incubation time, the N content in the remaining litter tissue had increased in most samples. Yet, the increase in N content was less pronounced when litters had been decomposed in a more-N-poor environment. FTIR was used to quantify the relative content of proteins in litter tissue and revealed a highly linear relationship between bulk N content and protein content. Changes in bulk N content thus paralleled and probably were governed by changes in litter protein content. Such changes are the result of two competing processes within decomposing litter: enzymatic protein depolymerization as a part of the litter breakdown process and microbial protein synthesis as a part of microbial biomass growth within the litter. Assuming microbial homeostasis, DNA signals in FTIR spectra were used to calculate the amount of microbial N in decomposed litter which ranged from 14 % to 42 % of the total litter N for all leaf samples. Microbial carbon (C) content and resultant calculated carbon use efficiencies (CUEs) indicate that microbial N in litter accumulated according to predictions of the stoichiometric decomposition theory. Subtracting microbial C and N contributions from litter, however, revealed site-dependent variations in the percentual amount of the remaining still-unprocessed plant N in litter compared to remaining plant C, an indicator for preferential protein depolymerization. For all leaf litters, the coefficient of preferential protein depolymerization (α), which relates N-compound depolymerization to C-compound depolymerization, ranged from 0.74–0.88 in a nutrient-rich detritus mud to 1.38–1.82 in Sphagnum peat, the most nutrient-poor substrate in this experiment. Preferential protein depolymerization from litter decomposing in Sphagnum peat leads to a gradual N depletion in the early phase of litter decomposition, which we propose as a preservation mechanism for vascular litter in Sphagnum peatlands.

Proteins comprise the largest soil N reservoir but cannot be taken up directly by microorganisms and plants due to size constraints and stabilization of proteins in organo-mineral associations. Therefore the cleavage of this high molecular weight organic N to smaller soluble compounds as amino acids is a key step in the terrestrial N cycle. In the last years two isotope pool dilution approaches have been successfully established to measure gross rates of protein depolymerization and microbial amino acid uptake in soils. However, both require laborious sample preparation and analyses, which limits sample throughput. Therefore, we here present a novel isotope pool dilution approach based on the addition of 15N-labeled amino acids to soils and subsequent concentration and 15N analysis by the oxidation of α-amino groups to NO2− and further reduction to N2O, followed by purge-and-trap isotope ratio mass spectrometry (PT-IRMS). We applied this method in mesocosm experiments with forest and meadow soils as well as with a cropland soil amended with either organic C (cellulose) or organic N (bovine serum albumin). To measure direct organic N mineralization to NH4+, the latter was captured in acid traps and analyzed by an elemental analyzer coupled to an isotope ratio mass spectrometer (EA-IRMS). Our results demonstrate that the proposed method provides fast and precise measurements of at%15N even at low amino acid concentrations, allows high sample throughput and enables parallel estimations of instantaneous organic N mineralization rates.