This section provides an overview of the chemically defined Norg
compounds identified using the analytical methods described in Section 2, as well as a discussion of their ecological significance in soil. Classes of Norg compounds that are important constituents of SOM, and the analytical methods used for their detection, are highlighted in Table 2.3.
Noncyclic compounds comprise the majority (60–90%) of the Norg with amides—including peptides, proteins and amino sugars—being the predominate compounds. Peptides and proteins are usually combinations of the 20 most common amino acids, which form oligo-, poly- and mac- ropeptides.
Ecologically, soil proteins can be subdivided into (1) detrital proteins released upon cell death and (2) functional proteins actively released into the soil for specific functions (Rillig, 2004). Detrital proteins originate mainly from vascular plants (>95%), but also from animals and microorganisms
inUnderstandingOrganicNitrogenChemistryinSoilsUsingState-of-the-art123 Table 2.3 An Overview of Important Classes of Norg Compounds in Soils, Ranges of their Proportions in Mineral Top Soils, and Indications of The Analytical Methods by Which these Ranges of Proportions Were Determined
Class of Norg compounds Proportions (% Norg)
Research methods
Wet-chemical Chromatography NMR Py-MS XPS XANES
Noncyclic
Amides 60–90 X X X X X X
Peptides/proteins 50–80 X X X
Amino sugars 5–10 X X
Amines
Free amino acids 1–5 X X X
Aliphatic nitriles 1–5 X X X*
Cyclic
Aromatics 5–35 X X X X X
Heterocyclics 5–25 X X X X X
Nitriles 1–5 X X X*
Anilides <10–25† X X X
Nonaromatic
Nitriles <1 X X X*
*No analytical separation of aliphatic and aromatic nitriles.
†Schmidt-Rohr et al. (2004).
(Stevenson, 1994). Functional proteins include extracellular enzymes and surface-active proteins of microbial origin (e.g. glomalin, hydrophobins) (Rillig, 2004). Among functional proteins, the extracellular enzymes are the most extensively investigated. Caldwell (2005) compiled soil enzyme data that could be used to distinguish inter- and extra-cellular enzyme sources and substrate specificity of single enzymes within (e.g. N cycle) and between (e.g. N and P or N and C cycles) major nutrient cycles. The functional diversity of soil enzymes links resource availability, microbial community structure and nutrient turnover in soils. Extracellular enzymes contribute to the stabilization of soil aggregates, decomposition of organic matter, for- mation of humified SOM, and nutrient cycling (Bakshi and Varma, 2011).
Their activities are often used as indices of microbial growth and activity in soils, though quantitative information regarding which enzymes are asso- ciated with a particular microbial process is generally lacking (Bakshi and Varma, 2011). The same type of enzyme can be found in multiple locations including both intracellular (e.g. in the cytoplasm, periplasm, or attached to the outer surface of active cells, resting cells, dead cells or cell debris) and extracellular (in the soil solution, or adsorbed on substrates or soil colloids) locations (Nannipieri, 2006). Mass spectrometric protein analy- sis from DOM can distinguish the phylogenetic origin of proteins in soil leachates. Schulze (2004) suggested that a proteomics approach could be taken to obtain a “proteomic fingerprint” of the presence and activity of soil organisms. Nannipieri (2006) suggested two approaches: (1) functional proteomics (which considers the ca 4% of soil N occurring in microbial biomass) and (2) structural proteomics (which considers the ca 30–45% of soil N occurring in extracellular proteins stabilized by soil colloids).
Recently, Gillespie et al. (2011b) used structural proteomics to investi- gate the extracellular protein glomalin. Glomalin is a glycoprotein associated with carbohydrates, contains 30–40% (w/w) C (González-Chávez et al., 2004), is assumed to be stable and persistent in soil, and is thought to be pro- duced in copious quantities by arbuscular mycorrhizal fungi (Glomeromy- cota). Glomalin-related soil protein (GRSP) is operationally defined by the extraction method (high-temperature sodium citrate extraction followed by either trichloroacetic acid or hydrochloric acid precipitation). Synchrotron- based N-XANES spectroscopy and Py-FIMS revealed that GRSP extracts contain a consortium of proteins along with many impurities (i.e. phenolics, lipids and humic substances) (Gillespie et al., 2011b). Taking a proteomics approach, the authors found that glomalin itself may be a thioredoxin- containing chaperone, but that no homologies with proteins or DNA of
mycorrhizal origin were detected. Proteomics also revealed that the extracts contained large amounts of soil-related, heat-stable proteins and proteins of nonmycorrhizal origin. Despite its obvious advantages, the applicability of proteomics to soil systems suffers, in part, from poor protein extraction efficiency caused by the impact of clay minerals and SOM (Giagnoni et al., 2011). For this reason, research progress will depend greatly on improved extraction methods (e.g. Chourey et al., 2010; Taylor and Williams, 2010).
Hydrophobins constitute another group of structural proteins and are similar to glomalin in their suspected functionality (Nichols, 2003). They are small (ca 100 amino acids), cysteine-rich proteins unique to filamentous fungi (Linder et al., 2005; Wessels, 1997) and are thought to contribute to SOM stability and contribute to the water repellency of soils (Rillig, 2005). For this reason, structural proteins were suspected to reside longer in soil than the majority of other proteins. However, all proteins in soil can be digested by proteolytic enzymes and taken up as ammonium or nitrate by plants and microorganisms; taken up directly by plants into root cells, most likely via endocytosis; or taken up directly by microorganisms (Nọsholm et al., 2009). Plants colonized by mycorrhizal fungi are predicted to have greater access to Norg than noncolonized plants (Schimel and Ben- nett, 2004).
Amino sugars, which account for 5–10% of the soil Norg (Table 2.3), are mainly of microbial origin (Amelung, 2003). The most important amino sugars in the soil are glucosamine, galactosamine, muramic acid and man- nosamine (Appuhn et al., 2004). However, free amino sugars and amino acids comprise only a small proportion of the dissolved Norg in soils (Rob- erts et al., 2007). For example, Roberts et al. (2007) showed that glucos- amine had a half-life of only 1–3 h in soil solution—its removal from solution being a predominantly biotic process—and that glucosamine was only weakly sorbed to the solid phase of the soil (Kd = 6.4 ± 1.0). Based on these results, Roberts et al. (2007) suggested that free amino sugars turn over rapidly in soil. Glucosinolates are another group of amino sugars that enter the soil through plant litter. These compounds, which are secondary metabolites produced mainly by plants of the order Brassicales, can have diverse fungicidal, bacteriocidal, nematocidal and allelopathic effects on soil ecological functioning (Fahey et al., 2001). To date, more than 120 different glucosinolate structures have been described.
Several thousands of amines originating from plants, animals and micro- organisms can be detected in soil. Ecologically they can exert antimicrobial and allelopathic effects and can be toxic to both invertebrates and vertebrates
(Vranova et al., 2011). Some amines are toxic, odorous, volatile compounds that contribute to soil fungistasis (Fekete et al., 2010; Zou et al., 2007). Free amino acids constitute a very diverse group of amines in the soil and occur in smaller quantities than amino acids bound in peptides and proteins. Plants release free amino acids (e.g. arginine, asparagine, aspartate, cysteine, cystine, glutamine) via diffusion into the rhizosphere. However, exudation of amino acids is a function of plant genotype and environmental conditions, and is enhanced under stress (Bertin et al., 2003). Free amino acids represent a sig- nificant source of available N for soil microorganisms and some plants. Plant uptake of free amino acids is maximized when soil amino acid concentrations are high; a condition caused by a slow microbial utilization ( Jones et al., 2005a).
Cyclic Norg compounds in soil consist of aromatic heterocyclics, nitriles and anilides, and nonaromatic nitriles (Table 2.3). Quantitatively, this group of compounds accounts for about 5–35% of the soil Norg and originates from a variety of biological sources, including chromatophores (porphy- rin structures in chlorophyll) from microorganisms and plants; purine and pyrimidine bases of RNA and DNA; and secondary metabolites such as alkaloids from most organisms. Inputs of chlorophyll at the surface of the soil are significant during primary succession—reflecting high densities of cyanobacteria, algae and mosses (Castle et al., 2011). DNA and RNA are present in all habitats occupied by prokaryotic and eukaryotic organisms, and contribute to the soil Norg pool in the form of both living and dead cells. DNA also can exist extracellularly upon its release from organisms into the soil (Wackernagel, 2006). Extracellular DNA in the soil can be protected against DNase degradation by interactions with clay minerals and soil colloidal particles (Cai et al., 2006).
A tremendous diversity of cyclic Norg compounds originate from sec- ondary metabolites produced by plants, animals, and microorganisms. An evaluation of Natural Product Reports (RSC publishing) indicates that more than 25,000 cyclic N compounds from the biogeosphere were newly detected and reported during the period from 2000 to 2011 (Fig. 2.13).
Evidence for these structures was provided by isolation, purification and analysis, as well as through synthesis.
The majority of alkaloids are N heterocyclics and, at present, about 10,000 alkaloid structures have been described (Hesse, 2002; Joosten and van Veen, 2011). Alkaloids are produced mainly by plants, but also by bacteria, fungi and animals. They are often toxic, and many (e.g. berberine) exhibit antibi- otic effects to soil microorganisms. Alkaloids can also affect the litter feeding preferences of soil fauna because of their generally bitter taste (Hesse, 2002).
Although the ecological importance of N heterocycles in general remains controversial, the ecological effects of a variety of specific cyclic Norg com- pounds in the soil have been described. These include antibiotics (e.g.
calvulinic acid and β-lactam calvam from streptomycetes and pyridone har- zianopyridone from Trichoderma spp.) and metal-chelating agents (e.g. the sid- erophore desferrioxamine from streptomycetes) (Hanson, 2008; Tarkka and Hampp, 2008). Furthermore, a diverse group of fungal pigments (e.g. azaqui- none agaricone from Agaricus spp.), toxins (e.g. amanitins from Amanita spp.) and volatile compounds (e.g. methyl pyrazine from Paecilomyces spp.) found in the soil contain N heterocyclic compounds (Hanson, 2008). Yet, due to their high specificity (often strain-specific), low concentration, and heterogeneous distribution—and despite their considerable ecological significance—these Norg compounds are only rarely quantified in soils. Nevertheless, cyclic Norg compounds have been identified as soil fungicides (Chuankun et al., 2004), biocontrol agents against soil-borne pathogens (e.g. Paula and Hau, 2007) and as controls on feeding preferences of soil animals (e.g. Bửllmann et al., 2010).
A final group of cyclic aromatic Norg compounds are the anilides.
Schmidt-Rohr et al. (2004) reported finding significant amounts of amide N bound directly to aromatic rings in a humic acid fraction from a sub- merged rice soil. They also reported that most of aromatic-bound-N was
Figure 2.13 Cumulative number of described natural occurring cyclic Norg compounds in the journal “Natural Products Reports” (RSC publishing) within the period 2000–2011.
Numbers within the bars indicate the total number of publications in the year describ- ing at least one cyclic organic N compound. On average, 76 new compounds were described per publication with a maximum number of 791 in a single publication.
anilide N and suggested that this represented an agronomically significant fraction of the soil N. Indeed, the large amount of anilide N in the continu- ally submerged soil was thought to contribute to a decline in yield, relative to a comparable aerobic rice soil.
The cyclic nonaromatic nitrile N compounds, including cyanogenic glucosides, comprise a minor (<1%) class of Norg in soil (Table 2.2). They are toxic compounds, which can affect the feeding preference of the soil fauna and the microbial colonization of the rhizosphere (Ubalua, 2010). These compounds are produced by plants, arthropods and microorganisms. At present, about 60 cyanogenic glycosides have been described in the literature (Ubalua, 2010).
In addition to biological sources, Norg compounds can enter soils as xeno- biotics; e.g. through the application of pesticides or as contaminants from dyes, pharmaceuticals and petroleum products. The primary N-containing xenobiotic species that undergo biological degradation in soils are nitroaro- matics, nitrate esters, and compounds containing N-ring heterocycles (Ye et al., 2004). Most herbicides that target photosynthesis or amino acid or lipid biosynthesis are biodegradable N heterocycles. For example, the herbicide atrazine can be biodegraded in the soil via a series of biochemical processes including N-dealkylation, dechlorination and ring cleavage (Ye et al., 2004).
Regardless of the processes involved, the use of xenobiotic N by soil micro- organisms as an N source is controlled by the general availability of N in the soil, and increases in times of N starvation (Sims, 2006).
In summary, soils contain an incredible diversity of biogenic, pyrogenic and xenobiotic cyclic Norg compounds. A particular challenge for inves- tigators originates from similarities in the molecular structures of Norg compounds, which are often present only in low concentrations and are heterogeneous in distribution, but vary fundamentally in their ecological effects. The method overview in Table 2.3 illustrates the toolbox of analyti- cal methods that can be applied to detect and quantify the Norg species or compound classes. Moreover, it illustrates the versatility and limitations of methods which, at best, are applied in combination to provide insights into the composition and turnover of soil Norg.