Characterization of the amino acid composition of

Characterization of the amino acid composition of soils under organic and conventional management after addition of different fertilizers Pablo Gonzalez Perez, Rui Zhang, Xiaoli Wang, Jun Ye & Danfeng Huang

Journal of Soils and Sediments ISSN 1439-0108 J Soils Sediments DOI 10.1007/s11368-014-1049-3

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Author's personal copy J Soils Sediments DOI 10.1007/s11368-014-1049-3

SOILS, SEC 2 • GLOBAL CHANGE, ENVIRON RISK ASSESS, SUSTAINABLE LAND USE • RESEARCH ARTICLE

Characterization of the amino acid composition of soils under organic and conventional management after addition of different fertilizers Pablo Gonzalez Perez & Rui Zhang & Xiaoli Wang & Jun Ye & Danfeng Huang

Received: 11 July 2014 / Accepted: 6 December 2014 # Springer-Verlag Berlin Heidelberg 2015

Abstract Purpose The classical nitrogen (N) cycling model has provided good understanding of inorganic N dynamics in agricultural soils, but largely ignores organic N available to plants. The ability of numerous crop plant species to take up and use amino acids underlines the importance of this N pool in agricultural systems; therefore, the soil free amino acids (FAA) pool was quantified in soils under organic (organic soil) and conventional (conventional soil) management after addition of different types of fertilizer. Materials and methods After application of the same amount of N as urea, alfalfa, rice straw, or compost in the organic soils and urea or alfalfa in the conventional soils, water-extractable amino acid composition and concentrations, and inorganic and microbial N were measured during a 56 day soil incubation. Results and discussion Alanine, glutamic acid, glycine, isoleucine, leucine, phenylalanine, serine, tryptophan, and valine were the most abundant soil FAA. Organic and conventional soils did not significantly differ in their soil FAA composition Responsible editor: Weijin Wang Electronic supplementary material The online version of this article (doi:10.1007/s11368-014-1049-3) contains supplementary material, which is available to authorized users. P. Gonzalez Perez : R. Zhang : X. Wang : J. Ye : D. Huang (*) Department of Horticulture, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China e-mail: [email protected] D. Huang Key Laboratory of Urban Agriculture (South), Ministry of Agriculture, Shanghai, People’s Republic of China Present Address: J. Ye School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney 2052, Australia

and concentrations. Urea significantly modified FAA composition, but only in organic soils, suggesting that urea disrupts microbial structure and/or metabolic pathways in organic soils. Alfalfa and compost did not alter FAA composition and concentrations, indicating that any pulses of amino acids from these materials are short lived. On the contrary, straw significantly increased FAA concentrations after 15 days, coinciding with an increase in microbial biomass N. Conclusions FAA concentrations remain low and have a largely constant composition in both organic and conventional soils; however, the addition of some fertilizers can significantly alter FAA composition and concentrations, which may affect the importance of amino acid N in the total N budget of plants. These findings warrant further research into the mechanisms controlling soil FAA composition and concentration in agricultural soils. Keywords Agricultural soil . Amino acids . Fertilizer . Organic cultivation . Organic fertilizer . Urea

1 Introduction Recent research on the role of organic N in soil biogeochemical cycling of C and N has increased our understanding of the use of amino acids as an N source by both plants and soil microbes (Kielland 1994; Näsholm et al. 1998; Jones and Hodge 1999; Berthrong and Finzi 2006; Geisseler et al. 2009). The ability to take up amino acids is widespread among crop plants and has been demonstrated in laboratory experiments and in the field (Shobert and Komor 1987; Jones and Darrah 1994; Näsholm et al. 2000, 2001; El-Naggar et al. 2009), and some plants even use amino acids preferentially over inorganic N (Näsholm et al. 1998; Lipson and Näsholm 2001). Such use of amino acids by plants is largely dependent on the free amino acid (FAA) pool size as well as its

Author's personal copy J Soils Sediments

proportion within the N available pool (Owen and Jones 2001; Jones et al. 2005); a number of studies have measured the various N forms in the soil though the majority have focused on natural ecosystems (Jämtgård et al. 2010). Given the ability of plants to acquire N in an organic form, making more effective use of the N resources in agricultural systems requires a better understanding of the effect of management practices on both organic and inorganic forms of soil N. Fertilizers are critical in order to maintain the levels of available N and sustain crop production in agricultural systems. However, following the classical N cycling paradigm (Schimel and Bennett 2004), studies of the effects of fertilization on N availability have mostly focused on inorganic N and have largely ignored the organic N pool. Organic systems rely on organic fertilizers (i.e., green manures, compost, and animal manure) to meet crop N demands; therefore, amino acid N may be present at higher concentrations in these systems compared to conventionally managed soils. Besides, a fertilization event can also influence the soil FAA pool, as organic fertilizers of different chemical composition and quality will have varying pathways and timings of N release into the soil, which to a large extent depend on the C/N ratio of the organic material (Mafongoya et al. 2000; Geisseler et al. 2009). Plant residues can release amino acids and easily degradable proteins from lysed cells in fresh plant residues or via depolymerization of proteins in older vegetable tissue. For example, in a 10-day aerobic incubation of fresh alfalfa residue extract, significant proportions (29–100 % depending on the incubation temperature) of soluble N containing diverse amino acids can be released (El-Naggar et al. 2010). On the contrary, the proteins contained in poor quality residues, such as straw, must be enzymatically digested to release the amino acids contained therein (Geisseler and Horwath 2008). Organic fertilizers, such as composted organic matter, also contain high concentrations of low molecular weight organic nitrogen (Chantigny 2003) and constitute a source of available amino acids in soil (He et al. 2014). Synthetic fertilizers contain mineral N, which dissolves directly in the soil matrix and rapidly alters soil N status, with impacts on soil carbon (C) cycling (Craine et al. 2007). In addition, fertilization can alter soil microbial biomass (Crecchio et al. 2001; Ge et al. 2010), which is an essential component of nutrient cycling, by either actively mediating soil processes or as a direct sink-source nutrient pool, and it has been suggested that the pool of microbial biomass is intimately related to the FAA pool (Weintraub and Schimel 2005). Despite the increasing importance that organic N is receiving in current models explaining global N cycling and its potential role as plant N source, little is known about the FAA pool in agricultural soils. The individual properties of amino acids play a crucial role in soil C and N cycles. For example, nitrous oxide flux from non-fertilized soil could be predicted and correlated highly with the soil concentration of individual amino acids and the

N2O–N conversion rate for each amino acid (McLain and Martens 2005). Weigelt et al. (2005) and Harrison et al. (2007) reported preferential use of specific amino acids by different plant species. To a large extent, abiotic processes, such as sorption of amino acids to the soil phase, are highly dependent on the properties of individual amino acids, which in turn determine how different amino acids diffuse in the soil matrix and their bioavailability (Owen and Jones 2001; Rothstein 2010). The variety of soil processes that involve amino acids necessitates identification of amino acid compositions in order to elucidate soil N dynamics, particularly as the pool of amino acid species available for plant nutrition may be influenced by the properties of individual amino acids (Weigelt et al. 2005); yet, few studies have quantified the concentrations of individual amino acids in agricultural soils. The aim of the present study was to quantify and characterize the FAA pool in organically and conventionally managed soils, and investigate the effect of a fertilization event on the FAA pool during incubation experiments. We hypothesized that (1) FAA concentrations will alter as a result of agricultural management; as N supply to crops relies solely on organic fertilizers in organic soil, the fluxes of amino acids in these soils may potentially be greater than in conventional soil. Scheller and Raupp (2005) observed substantially different hydrolysable amino acid pools (i.e., peptides and proteins) in conventional and organic soil; therefore, we also expected that the composition of FAA would differ by management type. (2) Addition of alfalfa, compost, and straw will increase soil FAA at different times during incubation; we speculated that alfalfa and compost would increase the concentration of soil FAA at the beginning of the incubation as a result of early leaching of amino acids from alfalfa tissue cells and amino acids in compost, whereas straw would likely increase soil FAA at a later stage of incubation after partial decomposition and depolymerization of the straw proteins. As the fertilizers have different chemical properties, we expected fertilizers would also modify the soil FAA composition. In order to assess the interactions between management type and fertilizer treatments, urea and alfalfa were added to both organic and conventional soil.

2 Material and methods 2.1 Soil and fertilizer materials Soil was collected from the Chongbentang experimental horticultural farm, Shanghai, China (30°57′ N, 121°21′ E). The climate is humid subtropical, and 70 % of annual precipitation (1255 mm) occurs between May and September. The mean annual air temperature is 17.5 °C, and the average total annual sunshine is 1778 h. The soil is approximately 8.3 % sand, 70.7 % silt, and 21 % clay to a soil depth of 40 cm. The


organically and conventionally managed fields are spatially separated within the same farm. The site has been farmed under contrasting organic and conventional management systems for a substantial period of time: The organic fields were established in 2002; the conventional fields have been maintained for over 30 years. The organically managed fields are fertilized with composted chicken manure at an average rate of 12 ton ha−1 crop−1. The conventional fields use an NPK compound fertilizer (14/16/15) at an average rate of 750 kg ha−1 crop−1 and urea at a rate of 225 kg ha−1 crop−1. Three crops are planted in the organic and conventional fields per year. The main crops cultivated are spinach (Spinacia oleracea), pepper (Capsicum annuum), onion (Allium cepa), celery (Apium graveolens var. dulce), Chinese cabbage (Brassica rapa pekinensis), coriander (Coriandrum sativum), bean (Glycine max and Vicia faba), bok choi (Brassica rapa chinensis), and garlic (Allium sativum). Legumes are included in the rotation of both the organic and conventional fields. Weeds are controlled manually in the organic fields and chemically in the conventional fields (the most common herbicides used are glyphosate and linuron, in dosages recommended by the vendor). Cultural practices (e.g., rotation and intercropping) are sufficient to control pests and diseases in the organic plots, and synthetic pesticides are used in the conventional plots (the most common pesticides used are tebuconazole, mandipropamid, chlorpyrifos, and spinosad in dosages as recommended by the vendor). Furrow irrigation is employed with the aim of maintaining adequate soil moisture for the needs of the crop; The volume and frequency of water application differs in the open field and greenhouse due to variations in evapotranspiration. In March 2013, representative soil samples were collected and pooled at a depth of 0– 20 cm over an area of 15×50 m from both the organic and conventional systems. The soil was sieved (2 mm mesh), cleared of root fragments and earthworms, and homogenized. The sieved soil was used to calculate gravimetric moisture content and water-holding capacity. Fresh alfalfa and 6-month-old rice straw from rice plants that did not reach commercial maturity were collected in the spring of 2013 from Chongming Island, Shanghai, China (31°38′ N, 121°39′ E) and ground (10–5 mm) to increase surface area and minimize the effects of tissue structure. Urea and compost were provided by Shanghai Jiaotong University greenhouse experimental facility. The main ingredients of the compost were chicken manure and various crop residues. 2.2 Soil incubation Exactly 250 g of soil (dry basis) was placed into 580-ml Mason jars, covered with plastic film in which holes were punched to allow aeration, and incubated in a dark, climatecontrolled chamber at 20±1 °C. The soils were incubated at 50 % water-holding capacity to maintain aerobic conditions

while keeping the soils moist. Soil moisture content was adjusted with distilled water before gravimetrically determined water-holding capacity dropped below 48 % (one to two times a week). Treatments were applied to both soils after 10 days of pre-incubation at 20 °C to minimize the impact of variations in antecedent moisture conditions, and to minimize the pulse of microbial activity associated with the disturbance of sieving and manipulating moisture (Hofmockel et al. 2010). The organic control and conventional control treatments did not receive any fertilization. The same amount of nitrogen (0.1 mg N g−1 dry soil) was added to the jars containing organic soil as urea (45.5 % N, 20.0 % C), alfalfa (3.8 % N, 43.3 % C), rice straw (1.4 % N, 40.5 % C), or compost (2.0 % N, 31.6 % C). The same amounts of N were added as urea or alfalfa to the jars containing conventional soil. For each treatment, three replicate jars were prepared and destructively harvested on days 1, 3, 7, 15, 28, 42, and 56 after the addition of fertilizer (i.e., a total of 21 jars for each of the six treatments and two controls). For each sample, distilled water-extracted soil FAA were determined, microbial biomass C and N were estimated using chloroform fumigation, soil humidity was measured, and the soil N nutrients ammonium (NH4+) and nitrate (NO3−) were determined after extraction with water. The samples were sieved (2 mm) before analysis. 2.3 FAA analysis FAA were extracted as described by Wang et al. (2013) with slight modifications. In brief, immediately after sampling 6 g (dry weight) of soil was mixed in a 1:5w/v ratio with distilled water in 50 ml centrifuge tubes, shaken at 150 rpm for 1 h at 4 °C, centrifuged at 12,000 rpm for 10 min, and the supernatant was vacuum filtered using glass fiber filters (Membrane Solutions, LLC., Plano, TX, USA). Exactly 45 ml of the filtrate was frozen at −20 °C, freeze-dried, and resuspended in 1.5 ml of 0.02 M HCl. The resulting solution was centrifuged at 15,000 rpm for 5 min at 4 °C, and filtered through a 0.22-μm polyethersulfone (PES) filter (Anpel Ltd., Shanghai, China) before analysis on an L-8900 Amino Acid Analyzer (Hitachi Ltd., Tokyo, Japan). The amino acid concentrations were calculated from the peak areas with reference to amino acid standards (amino acids mixture standard solution; Wako Pure Chemical Industries, Ltd., Osaka, Japan). PVC gloves were used throughout each step, and laboratory equipment was washed in soap and water, acid-washed (10 % HCl), and rinsed in nanopure water before use to ensure that amino acid contamination did not occur during extraction, sample preparation, and measurement. Total FAA (TFAA) concentration was calculated as the sum of the concentrations of all amino acids measured in the sample. The composition of the amino acid pool was expressed as a proportion of the total amino acid pool, by dividing each individual amino acid concentration by the TFAA concentration.


The L-8900 Amino Acid Analyzer can analyze 17 of the 20 primary amino acids (excluding proline, asparagines, and glutamine). This analyzer can also detect some dipeptides, organic acid derivatives of amino acids, and amines including taurine, phosphoethanolamine, theanine, sarcosine, alphaaminoadipic acid (a-AAA), citrulline, alpha-aminobutyric acid (a-ABA), cystathionine, beta-aminoisobutyric acid (BAIBA), gamma-aminobutyric acid (GABA), h y d r o x y l y s i n e , o r n i t h i n e , 1 - m e t h y l h is ti d i n e , 3 methylhistidine, anserine, ethanolamine, and carnosine. Of these molecules, taurine, theanine, sarcosine, 3methylhistidine, anserine, and ethanolamine were not detected in any soils.

2.4 Soil microbial biomass and nutrient analyses Microbial biomass C (Mic.C) and N (Mic.N) were determined in field-moist soil samples using chloroformfumigation extraction as described by Weintraub et al. (2007) with slight modifications. In brief, 5 g of soil was combined with 2 ml of ethanol-free chloroform, incubated in the dark at room temperature for 24 h in a 250 ml Schott flask, then the flasks were opened and aerated in a fume hood for 30 min, the contents were extracted with 25 ml of 0.5 M potassium sulfate in an orbital shaker for 1 h at 250 rpm, and total dissolved organic carbon (DOC) and total soluble nitrogen (TSN) were analyzed in the extracts using a total organic carbon and nitrogen (TOC/TN) analyzer (Multi N/C 3100; Analytik Jena AG, Jena, Germany). Mic.C and Mic.N were calculated as the differences between the DOC and TSN extracted from fumigated and non-fumigated samples. No recovery coefficient was applied; therefore, the values presented only represent extractable C and N; Mic.C and Mic.N were expressed as μg C or N g−1 dry soil. Soil moisture content was determined by drying the soils at 120 °C for 48 h. Total soluble N was extracted from the field-moist samples using distilled water (1:5 w/v) by shaking on an orbital shaker for 1 h at 250 rpm. Samples were vacuum-filtered using glass fiber filters (Membrane Solutions, LLC) and frozen at −20 °C until analysis. Nitrate and ammonium concentrations were determined using an automated flow injection analyzer (Smartchem 200; Westco, Frepillon, France). Soil pH was measured in 1:2 (w/v) air-dried soil/water extracts using standard electrodes (Mettler Toledo Delta 320; Mettler-Toledo, Greifensee, Switzerland). Total C and N from the organic and conventional control soils and the fertilizer materials were measured using a Vario ELIII/ Isoprime elemental analyzer (Elementar Analysensysteme GmbH, Hanau, Germany).

2.5 Statistical analysis The effects of management and fertilization on FAA composition and concentrations were tested using one-way ANOVA with PASW Statistics 20.0.0 (PASW, Chicago, IL, USA). Soil amino acid data were transformed when necessary to meet the assumptions of normality and homoscedasticity; where transformation did not improve heterogeneity of variances, Games–Howell post hoc tests were used. Comparisons were deemed statistically significant if P