Combined methods for thesimultanous estimation

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Combined methods for the determination of lignin and cellulose in leaf litter Article in Sciences of Soils · July 1999 DOI: 10.1007/s10112-999-0002-x

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Springer LINK: Sciences of Soils (1999) 4:2

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Sciences of Soils (1999) 4:2

Combined methods for the determination of lignin and cellulose in leaf litter M. Zimmer Institut für Neurobiologie, AG Zoologie und Didaktik der Biologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany Correspondence to: M. Zimmer (email: [email protected], Fax: +49-211-8111971) Received: 26 September 1998 / Accepted: 22 June 1999

Abstract. Precise and specific methods for the simultaneous quantitative determination of lignin and cellulose are discussed in this paper, enabling the monitoring of even slight changes in the content of lignocellulose in dead plant material during decomposition processes. Results from different leaf litter samples as well as a comparison of the lignocellulose content of freshly fallen leaf litter, leached and microbially inoculated leaf litter, and feces of saprophagous soil animals fed on this food source are presented. The suitability of the described methods for investigating decomposition processes is discussed. Key words: Decomposition, Lignocellulose, Litter degradation, Monitoring · · · ·

Introduction Materials and Methods · Principle · Implementation Results and Discussion References



Introduction Leaf litter mainly consists of cellulose (e.g. [47]) that is complexed with lignin (e.g. [25]; [7]). Lignocellulose is highly recalcitrant, and thus its degradation is a limiting factor in decomposition processes of dead plant material ([7]). Consequently, investigations on decomposition processes require the determination of lignin and cellulose contents: for example, Meentemeyer ([27]) stated that many studies on decomposition of leaf litter lack information on the lignin content of the decomposing material. Several more recent studies have determined the lignin or cellulose content in decomposition processes (e.g., [4]; [28]; [26]; [20]; [10]). Several methods for the determination of lignin can be found in the literature (summarized in [39]; [14]). However, most of these methods are relatively unspecific or are characterized by low recoveries and potentially interfering substances (cf. [1]; [14]), resulting in over- or underestimation of the compound concentrations, respectively. Furthermore, these methods were mostly developed for woody tissues and may not be applicable to leaf litter samples ([1]). Lignin is a mixture of highly methoxylated phenolic polymers. Due to their biosynthesis via random free-radical condensation, no unique structure can be established. Since lignin and cellulose are intimately associated, lignin phenolics cannot be isolated from lignocellulose without partial denaturation ([26]; [7]). Due to their complex structure, these polymers must be degraded prior to quantitative determination, but the distinction between true lignin and nonlignin phenolic compounds in chemical degradation residues is very difficult (cf. [14]). The nonlignin phenolic fraction includes flavonoids ([8]), condensed tannins ([42]), cinnamic acid esters ([14]), and constituents of suberin and cutin ([23]). However, many phenolic compounds can be removed from litter via methanolic extraction ([36]; [21]; [45]). Lignin degradation in hot acidic ethanol results in a characteristic mixture of simple compounds that react with phloroglucinol, commonly used for specifically staining lignin in histochemistry, by forming colored complexes. Due to the destruction of the complex lignin molecule, ethanolysis of lignocellulosic material provides access to the cellulose moiety. The determination of cellulose was often performed by delignifying plant material (sodium chlorite: [22]) and subsequent weighing of residuals (described in [1]). The delignification procedure implies that mainly woody tissue was intended to be observed, while most of the utilized methods may not be suitable for leaf litter ([1]). The polymeric structure of a-cellulose, consisting of b(1,4)-linked chains of glucose monomers, can be degraded by acidic hydrolysis, resulting in a mixture of monomers and a few oligomers of glucose ([51]; [49]; [40]; [50]). The released glucose is usually determined as a reducing sugars. For this purpose, several assays have been developed (Nelson-Somogyi method: [31], [44]; the Hagedorn-Jensen method: [18]; Anthron staining: [50]). However, these methods are unspecific and sensitive to interfering substances (cf. [43]). Overall, the methods available for the determination of lignin and cellulose in decomposing leaf litter appear to be not sufficiently satisfactory. The need in suitable methods when using small samples of leaf litter initiated the development of techniques for the simultaneous estimation of lignin and cellulose in lignocellulosic material.



Material and Methods Principle Both phenolic products of lignin degradation and glucosic products of cellulose hydrolysis are determined quantitatively with the introduced methods. Monomers and oligomers of lignin derived from ethanolysis are specifically stained with acidic solutions of phloroglucinol in histological studies. The intensity of the color reaction depends on the concentration of either the lignin compounds or the phloroglucinol. Thus, the absorbance of samples (l = 488 nm) is proportional to the lignin concentration when the concentration of phloroglucinol is constant. Glucose, the product of cellulose hydrolysis, can be determined very precisely and specifically by the enzymatic oxidation to glucose-6-phosphate, including the reduction of NADP+ to NADPH + H+. The content of NADPH + H+ (absorbance at l = 340 nm) is a function of the amount of oxidized glucose, and thus can be used as a measure of the content of hydrolytically released glucose that is proportional to the cellulose content. Implementation The introduced method was tested by comparing alder (Alnus glutinosa) litter with litter samples of birch (Betula pendula) and oak (Quercus robur). Freshly fallen litter was collected in early autumn 1997 in the vicinity of Cologne (Germany) and air dried (cf. [53]). Furthermore, the changes in lignin and cellulose content of alder (Alnus glutinosa) litter during decomposition through leaching, microbial processing, and digestion by saprophagous soil animals were monitored. For the decomposition screening, the air-dried alder litter ("fresh") was soaked in water for 1 week ("leached"), and subsequently inoculated in a pool of freshly collected mixed leaf litter ("processed"; cf.[53]). The processed leaf litter served as food for the terrestrial isopods Porcellio scaber Latreille 1804 and Oniscus asellus Linnaeus 1758 (Isopoda: Oniscidea). Feces were collected immediately after their egestion (processed by the woodlice: "digested") and after a microbial inoculation of 3 weeks (cf. [16]; [48]; [53]; additionally processed by microbiota: "colonized"). These samples represent different stages of the decomposition process. For the measurement of lignocellulose, samples of 50–100 mg (dry wt.) may be used. Samples were homogenized and extracted twice in 1000 µl of 50% methanol at 60 °C to get rid of phenolic compounds of the leaf litter that might interfere with the subsequent enzymatic reaction (cellulose determination). After centrifuging (25,500 g for 5 min at 4 °C), the supernatant solution may be used for the determination of hydrolyzable tannins ([52]; modified after [3]), condensed tannins ([52]; modified after [34]), and the content of total phenolics ([52]; modified after [21]). Samples were resuspended in 1000 µl ethanolic HCl (absolute ethanol : 1 M HCl, 1 : 1) and boiled for 3 h. After cooling and centrifuging (25,500 g for 5 min at 4 °C), 100 µl of the supernatant was diluted in 900 µl ethanolic HCl. Immediately before adding 100 µl of 10 mM phloroglucinol (in 1 M HCl), the absorbance at l = 488 nm was determined. Optimal and stable staining was observed after 4-h storage in darkness and at room temperature. Thus, after 4 h , the absorbance (l = 488 nm) was determined again. The difference in absorbance served as a measure of the lignin content of the sample. Standards were determined using a mixture (1:1, v:v) of lignins from basic and organic extraction (Sigma, St. Louis, MO, USA). The remaining samples were reduced at 60 °C and resuspended in 1000 µl of 0.75 M aqueous tri-ethanolamine buffer (Boehringer, Mannheim, Germany) (pH 7.6). The determination of glucose followed the



instructions of the commercially available enzymatic test (Boehringer). Crystalline a-cellulose (Sigma) served as a standard. In the present study, the slopes of the standard curves for lignin and cellulose were 379 (±13) (r = 0.986; n = 11; p