Utilising organic nitrogen: Can plants produce protease enzymes from their roots?

Ms Lucy Greenfield1, Dr Paul Hill1, Dr Eric Paterson2, Prof Liz Baggs3, Prof Davey Jones1,4

1Bangor University, Bangor, United Kingdom, 2James Hutton Institute, 3University of Edinburgh, 4University of Western Australia, 

Nitrogen (N) is an important macronutrient for plant life. Plants can uptake N in the form of ammonium, nitrate, amino acids and oligopeptides. However, plants do not have direct access to the 40% of total soil N represented by proteins. To utilise proteins they first must be broken down into small peptides and amino acids by protease enzymes. The majority of soil proteases are produced by soil microorganisms to obtain their own carbon and N nutrition. Therefore, the ability of plants to secrete proteases to hydrolyse proteins into small peptides and amino acids would increase the availability of N to plants. In agricultural systems, a decrease in the reliance on inorganic N forms by plants would reduce the use of environmentally detrimental inorganic N fertilisers.

In a laboratory experiment, we investigated whether plant roots release protease enzymes. We also investigated whether protease released from roots were up- or down-regulated by the presence of inorganic N. Seedlings (Zea mays L. and Triticum aestivum L.) were grown in sterile, hydroponic conditions in an inorganic N nutrient solution or a zero N nutrient solution. Each week for one month, the nutrient solutions were analysed for proteolytic activity using a fluorescence aminopeptidase assay. At the end of the experiment, the root tip was excised and its in situ protease activity measured to determine whether root proteases are surface bound. We hypothesise that plants roots will not release exoprotease enzymes but instead remain plasma membrane bound with higher concentrations under the zero N treatment. Consequently, proteases present on the root surface allow the plant to utilise the protein fraction of soil without losing essential nutrients into the soil matrix.


Lucy is a third year PhD student at Bangor University.

Soil carbon and nutrient dynamics in high and low fertility pasture soils following cultivation

Ms Elizabeth C Coonan1,2, Dr Clive A Kirkby2, Dr John A Kirkegaard2, Mr Martin R Amidy1, Dr Craig L Strong1, Dr Alan E Richardson2

1Fenner School of Environment and Society, Australian National University, Canberra, Australia, 2CSIRO Agriculture & Food, Canberra, Australia

Soil organic matter (SOM) can be lost from the soil following cultivation which may affect agricultural productivity. It has previously been shown that supplementary nutrients (nitrogen (N), phosphorus (P) and sulphur (S)) alongside incorporation of carbon (C) rich wheat residue increased SOM after 5 years of cultivation compared to a control without supplementary nutrients. However, SOM-nutrient interactions have mainly been investigated on annually cropped soils. A key point at which SOM is lost from the soil is during the cultivated transition from a pasture to a crop. It is not clear how initial soil fertility or supplementary nutrient addition may affect SOM dynamics during this transition. We investigated the impact of initial soil fertility on the mineralization of soil C and associated changes in soil nutrients in pasture soils following cultivation of a long-term pasture soil with and without lime. The pasture was managed with 20 years of P fertilization compared to a non-P fertilized control. The high P treatment with greater system productivity was shown to have an increase of 12 Mg C ha-¹ soil C to 60 cm depth. Three treatments were applied during the transition from pasture to crop: a cultivated control, cultivated with lime, and cultivated with lime and along with supplementary nutrient addition. Soil C was assessed in both whole soil and in the more stable <0.4 mm soil fraction. Following the cultivated transition to a crop, loss of C from the <0.4 mm fraction was increased in the limed low fertility soils (12.3% loss) and reduced in the limed high fertility soils (9.6% loss). Addition of nutrients to the limed low fertility soils reduced the loss of C (7.0% loss). Addition of nutrients with lime during pasture to crop transitions can reduce SOM loss in low fertility soils which has implications for management of SOM in cropping systems.


Elizabeth Coonan is a PhD student with the Australian National University Fenner School of Environment and Society and CSIRO Agriculture and Food based in Canberra. Her main research interest is soil organic matter in pasture crop rotation systems with a focus on the impact of liming, nutrient addition, and cultivation on soil organic matter when acid pasture soils are converted to cropping. She has an undergraduate degree in Science and Engineering from the Australian National University.

Does soil organic matter stoichiometry varied with agricultural practices on the long-term?

Dr Fabien Ferchaud3, Dr Alain Mollier2, Dr Isabelle Bertrand1

1INRA UMR Eco&Sols, Montpellier, France, 2INRA UMR ISPA, Bordeaux, France, 3INRA UR AGROIMPACT, Laon, France

Carbon (C), nitrogen (N) and phosphorus (P) cycles are intimately linked in ecosystems through key processes such as primary production and litter decomposition. Ecological stoichiometry has become a common approach for exploring relationships between biogeochemical cycles and ecosystems functions in ecological science. In agronomy, the concept of stoichiometry is far less utilized, probably because the addition of fertilizer reduced biotic interactions between the C, N and P cycles. Surprisingly, little is known about the long-term impact of agricultural practices on soil stoichiometry. Within the context of agro-ecology, however, alternative agricultural practices aim to increase nutrient recycling from plant residues, soil organic matter and inorganic reserves (e.g. legacy P), while reducing tillage or mineral fertilizer input. The success of such practices relies on the increase of soil biotic interactions and may impact C storage in soils on the long term, if soil organic matter stoichiometry is constrained.

We aimed at determining the long-term impacts of alternative agricultural practices on soil stoichiometry. To do so, we compiled and completed a dataset of long-term (8-49 yr) field experiments in France in which P or N fertilization rates or tillage intensity was strongly reduced.

The agricultural soils studied presented C:N and C:P ratios ranging from 8 to 14.5 and from 15 to 28 respectively, and N:P ratios ranging from 1.5 to 2.8 (total P). The site effect was significant on the soil CNP contents and ratios (one-way ANOVA, P < 0.05). Interestingly, whereas the soil C:N ratios were constrained and not influenced by the different agricultural practices, the C:P and N:P were more flexible. The C, N and P balance were calculated at each site and related to the soil stoichiometry.


Isabelle BERTRAND is a senior scientist working on soil organic matter dynamic, its interactions with soil microorganisms and fauna. She is working on simple and complex agrosystems such as monocrops and agroforestry systems. Her focussed is in soil functional ecology and the soil C, N and P cycling.

Wheat-derived SOC accumulates more than its maize counterpart in nutrients supplied wheat-maize cropping system

Dr Xinliang Dong1, Prof Hongyong Sun1, Dr Bhupinderpal Singh2

1The Center For Agricultural Resources Research, Institute Of Genetics And Developmental Biology, CAS, Shijiazhuang, China, 2University of New England, Armidale, Australia

Fertilization is the most common way to supply nutrients to the soil and to maintain crop productivity in the agricultural ecosystem, which may further influence soil organic carbon (SOC) accumulation rate. In this study, we set up a long-term fertilization field experiment in the winter wheat-summer maize cropping system. The treatments include no fertilization (Ct), nitrogen (N, 104.5 kg ha-1 N), phosphorous (P, 104.5 kg ha-1 P2O5) and N combined with P (NP, 104.5 kg ha-1 N combined with 104.5 kg ha-1 P2O5) fertilizer application with or without potassium (K, 104.5 kg ha-1 K2O); totally 8 treatments. After 21 years of fertilization, N application did not increase soil total N content, but P application significantly increased soil total P contents by 33.9%. The single application of N or P did not significantly affect SOC content, while the NP combination significantly increased SOC contents by 22.1% and 29.6% compared to Ct in the no K and K treatments, respectively, The natural 13C abundance approach and the SOC contents suggested that the NP combination increased wheat-derived SOC by 37.5% and 49.8% in the no K and K treatments; however, fertilization had no impact on maize-derived SOC content. Wheat-derived SOC was positively correlated to the wheat yield, while maize-derived SOC was not correlated to the maize yield, which indicated that wheat-derived SOC accumulated more than maize-derived SOC in the wheat-maize cropping system. Our results indicate that N combined with P application is more beneficial than N or P alone to enlarge SOC sequestration, especially for the wheat-derived SOC.


Xinliang Dong studied soil science at the China Agriculture University, where he earned his PhD in 2017. Dr. Dong’s research focuses on the dynamics of soil organic matter derived from different plant sources, and biochar effect on soil organic carbon fractionations.

Acceleration in N cycling controlled by aggregate size, moisture, substrate quality and phosphorus fertilization in floodplain soil

Dr Muhammad Riaz1,2, Dr. Pascal A Niklaus3, Dr. Beat  Frey4, Dr. Beat  Stierli4, Dr. Joerg Luster2

1Department of Environmental Sciences & Engineering, Government College University Faisalabad, Pakistan, Faisalabad, Pakistan, 2Forest Soils and Biogeochemistry, Swiss Federal Research Institute WSL, Birmensdorf, Switzerland, 3Department of Evolutionary Biology and Environmental Studies, University of Zurich, Zurich, Switzerland, 4Rhizosphere Processes, Swiss Federal Research Institute WSL,, Birmensdorf, Switzerland

Semi-terrestrial soils, such as floodplain soils, undergo a characteristic changes in their physico-chemical environment which is generally conducive for nitrous oxide (N₂O) production. Being hotspots of N₂O emissions, the floodplain soils can become an important source of terrestrial N₂O flux. Processes of nitrogen (N) cycling are strongly influenced by the availability of water, oxygen and substrate, and these processes may differ between small and large aggregate. We performed a microcosm incubation experiment using soil from restored floodplain section of the Thur River in NE Switzerland to investigate the effects of aggregate size, moisture, carbon (C) sources and phosphorus (P) fertilization on N cycling. Experiment included aggregate size (<250 µm; 250 µm – 4.00 mm), moisture level (60% WHC; submerged conditions), C source (glucose; litter; litter-derived DOC) and P as experimental factors. Head space gas samples were collected for N₂O gaseous analysis and flux calculation. After 28 days incubation, the soil samples were analysed for N species (KCl-extractable NH₄-N, NO₃-N, dissolved organic N total N, water-extractable total N), water-extractable organic C, microbial biomass N,  leucine aminopeptidase activity (LEU), denitrification enzymatic activity (DEA), bacterial abundance (16S-qPCR), fungal abundace (ITS- qPCR) and some N cycling pathway functional genes (nirS, nosZ & nxrB). We found strong effects of experimental factors on N cycling processes. N₂O flux was generally higher in larger aggregates and soil treated with litter-derived DOC and after P addition. Mineral N concentrations were many-fold higher for litter-derived DOC and litter treated soil amended with P under submerged conditions. Dissolved organic N varied dramatically between the treatments. LEU activity was significantly higher in soil treated with litter and P in smaller aggregates. Bacterial and fungal diversity and functional gene abundance varied significantly among the treatments. Results suggested that strong heterogeneity in environmental factors could control hotspots and hot moments of N cycling.


Dr. Muhammad Riaz is working as an Associate Professor in the Department of Environmental Sciences & Engineering, Government College University Faisalabad (GCUF), Pakistan. He earned his PhD in Environmental Sciences from the University of York, UK. His research is mainly focused on soil biogeochemistry, CNP cycling in agroecosystems, biochar as a tool for soil C sequestration and soil quality management, and dynamics of soil organic matter cycling and recycling in semi-arid and arid agroecosystems.

Carbon and nitrogen recycling from microbial necromass to cope with C:N stoichiometric imbalance by priming

Dr Zhenke Zhu1, Dr Jun Cui1, Dr Tida  Ge1, Dr Jinshui Wu1

1Institute of Subtropical Agriculture, Chinese Academy of Sciences, CHANGSHA, China

The relationship between the amount of labile C input and the resulting priming effects (PEs) on soil organic matter (SOM) mineralization remains unclear, particularly under anoxic conditions and at high C input rates common in microbial hotspots. PE intensity and the relevant mechanisms were investigated by incubation of three flooded paddy soils over 60 days after 13C-labeled glucose addition ranging from 50 to 500% of microbial biomass C (MBC). CO2, CH4 and N2O emissions were continuously measured and partitioned for C sources based on 13C-label. PE peaked at moderate glucose addition rates (50-300% of MBC) but decreased, and even became negative, under higher rates. Nitrate production from SOM and N2O emission also peaked at moderate glucose loads in accordance with the N mining from SOM expected for PE. However, low or negative PE at high glucose loads above 300% of MBC posed a paradox of stronger N-acquisition accompanied by decreased SOM mineralization. Particularly at glucose input >3 g kg-1 (corresponding to 300-500% of MBC), strong N immobilization by microorganisms was confirmed by minimum levels of soil mineral N and negligible N2O emission. Concomitantly, microorganisms intensified N acquisition from microbial necromass by increased N-acetyl glucosaminidase and leucine aminopeptidase activities without accelerating SOM decomposition. Several peaks of glucose-derived CO2 and CH4 effluxes were observed between days 13 and 30, confirming decomposition of glucose-derived microbial necromass (contribution to corresponding total CO2: >80%). Contents of glucose-derived mineral-bound C, which originated from microbial biomass turnover was used as a proxy of net necromass accumulation, increased with glucose input levels over 60 days. Labile C input also accelerated the conversion of living microorganisms to necromass. Therefore, necromass recycling was a hypothesized mechanism to alleviate microbial N deficiency without priming SOM (hereafter referred to SOM components other than necromass). Compound-specific 13C-PLFA confirmed redistribution of glucose-derived PLFAs, i.e. 13C recycling, among microbial groups during the 60 days. Initially after labile C input, gram-negative bacteria (presumably r-strategists) rapidly incorporated glucose together with N and used them for growth. After glucose was exhausted and r-strategists died, their necromass became a substrate providing labile C and N for the gram-positive bacteria, actinomycetes and fungi, which were less responsive initially after glucose addition. We conclude that N sources for microorganisms depend on labile C input: to cover N demand under C excess, microorganisms switch from SOM-N mining to the N recycling from microbial necromass.


Zhenke Zhu, an associate professor of the institute of subtropical agriculture, Chinese academy of sciences. His field of study is the carbon and nitrogen cycle process and the microbial mechanism in the subtropical paddy field. The main focus now is the mechanism of C:N:P stoichiometry regulates the organic carbon cycle in the paddy soils.

Nutrient availability drives carbon storage in particulate vs. mineral-associated organic matter in Antarctic soils

MSc. Luís  Almeida2, MSc. Isabel Prater1, MSc. Luis Colocho Hutarte1, Prof. Andreas Richter3, A/Prof. Carsten W. Mueller1

1Chair of Soil Science, Technical University of Munich, Freising, Germany, 2Department of Soil Science, Universidade Federal de Viçosa,  Viçosa, Brazil, 3Department of Microbiology and Ecosystem Science, University of Vienna, Vienna, Austria

Although low in total carbon, Antarctica with its pristine soils offers unique model systems to study soil organic matter cycling unbiased by high carbon inputs of vascular plants or anthropogenic disturbance. Furthermore, seabird rookeries with locally restricted high inputs of marine derived bioavailable C, N and P create natural gradients affecting vegetation distribution and productivity. Deception Island situated at the Antarctic Peninsula offers a unique setting with very young soils due to volcanic eruptions between 1968 and 70. This allows to study soil organic matter accrual, distribution and composition in newly developing soils in relation to soil nutrient availability. To this end we sampled three spatially separated sites on Deception Island distributed along a transect from higher to lower marine nutrient inputs, respectively. At the sampling sites patches below living and dead moss were sampled, to account for possible future effects on plant coverage by a changing climate. Besides bulk soil C, N, pH and EC analysis, we fractionated the top soils according to density and particle size, analysed the C and N contents of all fractions and the POM and clay fraction by 13C-CPMAS-NMR spectroscopy. Beside the clear local effect of the input of C, N and P by seabirds, we were able to also demonstrate distinct effects of dead moss cover on soil organic matter stocks, in which the dying of vegetation promotes increased soil C contents in sites with high primary productivity. Our results evidence that different nutrient availability lead to a clear shift in the dominating C pools from mineral-associated to particulate organic matter. The chemical composition of the input material is not reflected by mineral-associated organic matter in the clay fractions, indicating the microbial transformation prior to association with mineral surfaces.


Carsten W. Mueller is currently an Associate Professor at the Technical University of Munich. Following his graduation in Forest science at the Technical University of Dresden, he did his PhD at the Technical University of Munich. After a PostDoc at Pennsylvania State University he became an Assistant Professor at the Technical University of Munich. He is mainly working on the fate of soil organic matter, from the plant input in rhizosphere and detritusphere over microbial transformations to particulate organic matter and mineral-associated organic matter in the soil. Having worked on soils from all continents, currently he especially focuses on soil structure formation and organic matter allocation in the rhizosphere and pristine environments in the Arctic and in Antarctica. In his research he combines quantitative approaches (e.g. density fractionation, elemental and isotopic analyses, lab incubations) with state of the art chemical (GC-MS, NMR spectroscopy) and spectromicroscopic (SEM, NanoSIMS) techniques.

Resource nutrient stoichiometry controls microbial growth, carbon-use efficiency and soil carbon priming in an organic-amended alkaline sodic-subsoil

Dr Yunying Fang1, Dr Bhupinder Pal Singh1, Dr Mark Farrell2, Dr Roger Armstrong3, Dr Lukas Van Zwieten4, Prof Chengrong Chen5, Dr Ehsan Tavakkoli6

1NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Menangle NSW 2568, Australia , Sydney/Menangle, Australia, 2CSIRO Agriculture & Food, Urrbrae, Australia, 3Agriculture Research, Department of Jobs, Precincts and Resources, Horsham, Australia, 4NSW Department of Primary Industries, Wollongbar Primary Industries Institute, Wollongbar, Australia, 5Australian Rivers Institute, School of Environment and Science, Griffith University, Nathan, Australia, 6NSW Department of Primary Industries, Wagga Wagga Agricultural Institute, Wagga Wagga, Australia

Subsoil sodicity is one of the main constraints on soil functionality, productivity and sustainability, particularly in arid and semi-arid regions. In recent years, the role of organic amendments (OAs) combined with inorganic fertilizers or gypsum has received much attention for ameliorating soil physicochemical constraints and improving carbon (C) storage in sodic-subsoils. However, little is known about the effect of resource nutrient stoichiometry and gypsum on C dynamics in organic-amended sodic-subsoils. Here, we examined the role of OAs (application rate of 6.2 g OA-C kg-1 soil; C4 vegetation-derived: δ¹³C –12 to –15‰) in altering microbial C mineralization, microbial C use efficiency (CUE) and soil C priming, with and without the exogenous supply of nutrients or gypsum, in an alkaline sodic-clay subsoil (5.8 g C kg-1 soil; C3 vegetation-derived: δ¹³C –24‰).

The cumulative OA-C mineralization ranged between 70 and 630 mg CO2-C g-1 OA-C across the treatments over 270 days. The CUE of OAs was the highest in the mill mud-treated subsoil (0.25–0.80) and the lowest in the sorghum stubble-treated subsoil (0.07–0.42). The inherently balanced C-nutrient stoichiometry of OAs (such as mill mud) enhanced CUE, whereas lowering the imbalanced nutrient stoichiometry of other OAs (sorghum stubble, sugarcane bagasse) via exogenous nutrient inputs increased microbial growth but not CUE. Over 270 days, extra 0.7–8.3% of native soil organic carbon (SOC) was lost via priming across the treatments. In the first three months, the positive priming effect by the OAs was the highest in the sorghum stubble-treated subsoil, which was mainly driven by microbial co-metabolism and N mining. At the later stage, the balanced resource nutrient stoichiometry enhanced the PE. This study suggests that balancing the C-nutrient stoichiometry of OAs can increase soil functions and biological processes, such as microbial degradation of OAs, microbial growth and positive SOC priming, which may enhance nutrient availability and improve soil structure during amelioration of sodic-subsoil constraints.

Biography: Adjunct Professor Bhupinder Pal (“BP”) Singh is working as Principal Research Scientist with NSW Department of Primary Industries. His research interests are in the areas of soil science, ecology, biogeochemistry, and understanding the role of soil organic matter functionality, such as carbon and nutrient cycling in agro-ecosystems. Dr Singh disseminates his research outcomes, via invited lectures, field-day participation, and (inter)national conferences to stakeholders, underpinning improved management strategies for sustainable agriculture.

What can isotopes tell us about the controls on the coupling/decoupling of soil nitrogen and carbon?

Dr Naomi Wells1, Ms Kate Summer1, Dr Jeff Baldock2, Dr Mark Farrell2

1Southern Cross University, East Lismore, Australia, 2CSIRO Agriculture & Food, Adelaide, Australia

The isotopic composition of both the nitrogen (δ¹⁵N) and C (δ¹³C) entrained in soil organic matter (SOM) are altered by local biogeochemical reactions. They thus represent a potential integrative measure of SOM function that could bridge the distance between low cost, low sensitivity measures of bulk SOM stoichiometry and highly sensitive, but high cost, emerging SOM characterisation techniques. Here we tested this potential by using archived soil samples to explore δ¹⁵N and δ¹³C variations, 1) between climate and agriculture regions, and, 2) between physically separated SOM fractions. The re-analysed soils were collected at two depths during two previous recent national soil surveys (BASE for soils from natural ecosystems (0-20, 20-40cm), SCaRP for soils from agricultural ecosystems (0-10, 10-20cm)). For (1), we analysed soils from 413 sites across New South Wales. For (2), we analysed the previously separated ‘humic’ and ‘particulate’ components in soils from 300 farm sites across Australia. We found that δ¹³C values were strongly aligned with land-use type (pasture, cropping, or un-cultivated). In contrast, δ¹⁵N values in un-cultivated lands were strongly influenced by climate (aridity index). Cultivated soils deviated from this apparent climate-driven relationship. This suggests that δ¹⁵N could be used to indicate where and when agricultural soils are ‘regenerating’ (moving closer to the expected relationship) or ‘degrading’ (moving farther from the expected relationship) through time. Overall our results suggest that measuring variations in soil δ¹⁵N and δ¹³C can add value to established metrics of SOM functionality.


Naomi Wells is a Lecturer in the Centre for Coastal Biogeochemistry at Southern Cross University. She is a biogeochemist who uses stable isotopes to understand how nitrogen moves through land and water. After realising that most reactive nitrogen in the world is bound in organic matter her research has expanded to try to integrate the carbon and nitrogen worlds. She received her PhD from Lincoln University (NZ) in 2014, an MSc from the University of Aberdeen, and a BA from Wellesley College.

Effects of nutrient enrichment on soil priming effect: a global meta-analysis

Mr Jiguang Feng1, Dr. Biao Zhu1

1Peking University, , China

The inputs of fresh carbon will alter the decomposition of soil organic matter (SOM), which is known as priming effect (PE). Priming effect plays an important role in regulating SOM decomposition and soil carbon storage. Increasing nitrogen (N) and phosphorus (P) inputs and deposition induced by anthropogenic activities have largely increased the availability of soil nutrients, and thus should affect PE. Although many studies have investigated the effects of nutrient enrichment on PE, the general patterns at global scale remain unclear. Here, we compiled available data on PE under nutrient enrichment from primary literatures to examine how nutrient enrichment regulates PE. Results showed that, across all studies, N enrichment and combined N and P enrichment significantly decreased PE (p < 0.05), whereas P enrichment had minor effect on PE (p > 0.05). Specifically, N enrichment effects on PE varied among ecosystem types, with significant negative effect occurring in grassland, boreal, temperate and tropical forest (p < 0.05). Meanwhile, the effects of nutrient enrichment on PE generally decreased with initial soil nutrient status (organic carbon, total nitrogen and phosphorus), indicating that these factors potentially regulate nutrient effects on PE. In addition, the effects of N and combined N and P enrichment on PE were regulated by substrate type, with significant negative nutrient effects in substrates not containing the mineral nutrient added. Combinations of microbial nutrient mining hypothesis and microbial stoichiometric decomposition hypothesis can explain the observed relationships across all ecosystems. Collectively, our findings imply that N deposition might be beneficial to soil carbon storage via suppressing PE, and highlight the need for further field in situ studies investigating nutrient enrichment effects on PE.

Biography: Jiguang Feng is a PhD student of College of Urban and Environmental Sciences, Peking University. His research interests are nutrient limitation in tropical forest, soil carbon cycling under global change, and soil microbial ecology.



7th International Symposium
Soil Organic Matter

6 – 11 October 2019

Hilton Adelaide

Adelaide, South Australia


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