Hydrolysis

Acid hydrolysis (usually with 6 M HCl), used to release amino acids from proteins, is also often used to liberate N compounds from soil (Bremner 1949, 1952). Bremner (1965) and Kelly and Stevenson (1996) give a detailed description of the procedures and difficulties associated with this method. The hydrolysis is followed either by a chromatographic separation of the individual amino acids released [usually by high-performance liquid chromatography (HPLC), or gas chromatography) or by photometric determination of the total concentration of a-amino groups in the hydrolysate (Stevenson and Cheng 1970; Kogel-Knabner 1995). This method leaves about 20-35% of the total SON unattacked (Sowden et al. 1977; Leinweber and Schulten 2000). Due to its insolubility, this so-called non-hydrolysable residue is excluded from the investigation with common chemolytic approaches.

With common analytical methods between about 30-50% (Stevenson and Cole 1999) of the total SON was identified, mostly as amino acids and amino sugars. Besides amino acids and amino sugars, the hydrolysate contains nucleic acids and other N biomolecules. For their separation and identification specified techniques are required (Kelly and Stevenson 1996).

Ammonia, produced during hydrolysis, can be recovered by steam distillation with MgO and amounts to 20-25% of the N in surface soils (Kelly and Stevenson 1996). Because in most cases an unbiased assignment to the originating compounds is not possible, this fraction is commonly termed the hydrolysable unknown-N (HUN) fraction (Stevenson 1994). It partly derives from soil NH+ and NH3 liberated after degradation of amino acid amides, such as asparagine and glutamine. Another part of this N may originate from partial destruction of amino sugars, but also amino acids such as serine, threonine or tryptophan. Threonine and serine are slowly degraded to NH+ and carbonyl. Tryptophan is stable if presence of air is avoided. However, it was shown that in the presence of iron(III) and copper(II) even degassing prior to hydrolysis gives no protection. An accounting of all known potential sources of NH3 in soil hydrolysates shows that about one-half of the NH3-N, equivalent to 10-12% of the total organic N, is still obscure (Kelly and Stevenson 1996). Some of this unknown hydrolysable N could originate from pseudoamines, such as iminoquinones, Schiff bases and enamines, hydroxyamino acids, amino alcohols and sugars. Purines and pyrimidines were also suggested as possible origins ofthis unidentified hydrolysed N.

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