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Differences in degradability of plant species

3.2.1 Differences in anatomy

Plant species differ in their anatomical arrangement. Roughly, differences in stem anatomy between monocots and dicots can be distinguished by the distribution of the vascular bundles (Mauseth, 1988). However, the distribution of the ground tissues also varies between species of both monocots and dicots and the monocots tend to have more cells with lignified cell walls outside the vascular tissue than do dicots. Besides being dependent on the chemical composition of the material which differs among plant materials, decomposition of plant tissue is dependent on the anatomical arrangement of the tissues. This is because the plant anatomy determines the accessibility for the microorganisms. The cuticle impedes access to underlying tissues and microorganisms can only penetrate through the stomata and lenticels or where the cuticle has been damaged (Chesson, 1997). Initial mastication by animals or shredding of material before composting, increases the microbial accessibility and thereby decomposition rate, whereas decomposition in soil often proceeds at a much slower rate as fallen plant litter is far more intact and microbial attacks thus impeded (Chesson, 1997).

Large differences in degradability of different cell types have been observed to rely on the chemical composition of cell walls. Nonlignified primary cell types such as mesophyll or phloem cells are clearly degraded in preference to the lignified xylem and sclerenchyma (Chesson, 1997; Jung and Engels, 2001). Xylem fibres and primary and secondary xylem vessels appears to be very recalcitrant towards degradation, although these tissues can exhibit differential patterns of degradation depending on the distribution of lignin in their wall structures (Jung and Engels, 2001). Thin walled cells like parenchyma and epidermis occupy an intermediate position between the former. Epidermal cells are often highly degradable in isolation but little attacked in situ because they are sandwiched between the inert cuticle and resistant lignified sclerenchyma (Chesson, 1997).

In some cell types, wall material is laid down over the primary wall after cell expansion has stopped. This by definition is secondary wall formation (Harris, 1990) and with only few exceptions, secondary thickening is accompanied by lignification. Unlike sclerenchyma fibers, the secondary wall in the tracheary elements of the xylem is often not laid down uniformly over the primary wall. The protoxylem, which is the first formed tracheary elements and differentiates in organs that are elongating, have lignified secondary wall thickenings laid down on the primary wall in the form of rings and helices (Harris, 1990).

Differences between cell types in presence and form of secondary walls, as well as degree of lignification affect the degradability of the cells. In addition, the arrangement of these cells is a key factor in regulation of the microbial breakdown. Monocotyledonous and dicotyledonous plants differ significantly in the arrangement of cells (Fig.5) and the effect of the arrangement has been examined extensively using grasses and legumes as test plants, as both are used as ruminant feed. Grasses have vascular bundles distributed throughout the parenchyma of stem cross-sections, and lignified cells are not exclusively found in connection with the vascular bundles but are spread across many types of tissues. As a result, degradation of cell walls in most tissues is affected to some extent (Wilson and Hatfield, 1997).

Figure 5. Stem cross-sections of A) Michanthus straw, arrows show the vascular bundles distributed throughout the stem. B) Hemp straw, arrows indicated the solid ring of lignified xylem tissue.

During the maturation of legume stems, isolated vascular bundles soon become unified into a complete lignified xylary ring around the stem that expands through cambial activity (Wilson,

Figure 5. Stem cross-sections of A) Michanthus straw, arrows show the vascular bundles distributed throughout the stem. B) Hemp straw, arrows indicated the solid ring of lignified xylem tissue.

During the maturation of legume stems, isolated vascular bundles soon become unified into a complete lignified xylary ring around the stem that expands through cambial activity (Wilson,

1991; Wilson and Hatfield, 1997). Thus legume stems, from a wall degradability viewpoint, are essentially composed of two populations of cells. The highly lignified xylem ring that appears to be difficult to decompose even though the cells do not have limited microbial access, and the remainder that appears to be easily degraded with no anatomical restrictions to wall degradation because few cortical and central pith cells appear to be lignified, and the cells have a weak structure with a degradable middle lamella-primary wall (Wilson and Hatfield, 1997). The anatomy of many dicots is comparable to that of the legumes and a similar anatomical arrangement was observed in hemp straw (Dresboll and Magid, submitted). During incubation of clover and grass in soil, these anatomical differences were reflected in the degradability of the material. The clover had an initial high mineralisation rate but the rate decreased after 30-40 days. In contrast, the grass had an initial lower mineralisation rate but the rate did not decrease as fast as for the clover (de Neergaard et al., 2001).

In maturing grass stems, anatomical limitations to wall degradation appear to be of major importance. Secondary walls of all major cell types progressively thicken as the stem matures, and the development of a recalcitrant middle lamella primary wall creates a structurally strong tissue composed of large blocks of cells with thick secondary walls. Microbial degradation is confined to the narrow lumen surface of only those cells with an open end (Wilson and Hatfield, 1997). When microbes have ready access to the surface of cell walls as when digesting thin slices (<100 ^m) of material in rumen fluid or when the anatomical structure of lignified tissues is destroyed by isolating and grinding specific cells, then degradation of most of the secondary wall is not prevented by lignification (Wilson and Mertens, 1995). The situation is different for legumes: first, their lignin content is higher than that of grasses at comparable levels of dry matter degradability, and second, the lignin is confined to xylem and tracheary cells only and not spread across many types of tissues as in grasses. This means that in the lignified tissue of legumes, the lignin concentration per unit cell wall will be very high and appears to prevent degradation of secondary walls even when thin sections allow easy microbial access to the walls. Thus, compositional differences between legume lignin and grass lignin may not be as significant as differences in its localisation or concentration in specific wall types, when analysing the contrast in degradation kinetics between legume and grass stems. The poor degradation of xylem elements in the legume stem is almost certainly the effect of their extremely high lignin concentration (Wilson and Mertens, 1995).

The anatomical architecture of the thick-walled lignified fibres of grasses may be as significant a limitation to digestion of secondary cell walls as is the chemical structure (Wilson and Mertens, 1995). This does not mean that lignification of walls and chemical bonding of lignin and phenolics to polysaccharides do not add a further limitation to the rate and extent of wall degradation. Wilson and Mertens (1995) suggested, however, that this effect is largely expressed in the primary wall middle lamella region, where lignin is at a much higher concentration than in the secondary wall. Such an anatomical arrangement influences the accessibility for the microorganisms, as was shown in the degradation of the Mischanthus straw (Dresboll and Magid, submitted). Degradation of the exposed surfaces of the straw pieces was observed after three weeks of composting whereas a freshly cut surface in the middle of a straw piece did not show degradation until after eight weeks (Fig.6).

Differences were also observed between the dicotyledonous hemp and the monocotyledonous Mischanthus and wheat straw (Dresboll and Magid, submitted). Although the easily degradable tissues in the hemp straw already had been degraded during the retting process, where the hemp straw is left in the field to initiate decomposition in order to release the bast fibres from the stem, it was obvious that the lignified secondary walls of the xylem were not degraded during the 8 weeks of composting (Dresboll and Magid, submitted).

Figure 6. Mischanthus straw after 3 weeks of composting. A) Freshly cut surface in the middle of the straw piece. No visible signs of degradation were observed. B) Exposed surface showing signs of degradation of the phloem (indicated by arrow). After Dresboll and Magid (submitted).

3.2.2. Degradation of lignin

Anatomical arrangement of the cells within the various tissues is very important in determining the extent and rate of degradation. Nonetheless, a broad inverse correlation exists between cell wall degradability and lignin content. The variation found within any one group of closely related plants also suggests that the nature of the lignin-carbohydrate association can be as important as the total amount of lignin present. Lignin is one of the most recalcitrant compounds in nature and acts to protect the structural polysaccharide to which it is covalently linked. This is consistent with its biological functions, which are to protect structural polysaccharides and ensure rigidity of vascular plants (Hammel, 1997). Lignin can limit cellulose and hemicellulose degradation by impeding enzymatic access to polysaccharides, acting as a physical or chemical barrier (Agosin et al., 1985). The most probable mechanism is a steric hindrance but the hydrophobicity of the complex also moderate hydrolysis. Thus lignin-carbohydrate-complexes (LCC) exposed at the surface of the cell wall are almost resistant to microbial attack, and are either not degraded or are degraded at a rate substantially lower than any surrounding polysaccharide free from any association with lignin (Fig.7). There is a tendency for most LCC to be undermined by the breakdown of surrounding polysaccharides and released from the wall with time, although some LCC accumulate at the wall surface at the expense of non-associated polysaccharides. This changes the surface layer significantly, resulting in slowing the further degradation. The rate at which this inert LCC surface develops is dependent on the initial concentration of lignin in the wall (Chesson, 1997).

a ii_LB

Middle lamella

Middle lamella

Figure 7. A model of the decomposition of a lignified cell wall seen in cross-section. Microbial attack occurs from the luminal side of the wall. The blue boxes represents regions of polysaccharides closely associated with lignin, whereas open boxes represent free polysaccharides. At the initiation of decomposition the surface (top layer) consists of both lignified and non-lignified polysaccharides. As decomposition proceeds the free polysaccharides are preferentially decomposed leaving a more extensively lignified surface which impedes further decomposition. Some lignin-carbohydrate complex (A and B) is undermined by the degradation of the surrounding polysaccharides and released (Redrawn from Chesson, 1997).

Middle lamella

Figure 7. A model of the decomposition of a lignified cell wall seen in cross-section. Microbial attack occurs from the luminal side of the wall. The blue boxes represents regions of polysaccharides closely associated with lignin, whereas open boxes represent free polysaccharides. At the initiation of decomposition the surface (top layer) consists of both lignified and non-lignified polysaccharides. As decomposition proceeds the free polysaccharides are preferentially decomposed leaving a more extensively lignified surface which impedes further decomposition. Some lignin-carbohydrate complex (A and B) is undermined by the degradation of the surrounding polysaccharides and released (Redrawn from Chesson, 1997).

Substrate quality is defined by chemical composition of the decomposing material and has often been considered a critical factor in determining the rate of decay. Nitrogen as well as lignin content of plant material is important in controlling the rate of decomposition. Lignin is an interfering factor in the enzymatic degradation of cellulose and other carbohydrates as well as proteins. High initial levels of lignin may thus slow decomposition rates (Melillo et al., 1982). However, spatial distribution of the lignified cells is a key factor regulating decomposition, as mature lignified secondary walls can be degraded when anatomical factors, i.e. tissue organisation, have been eliminated (Wilson and Hatfield, 1997).

Lignin decomposition is conducted by different groups of microorganisms, depending on the physiochemical conditions of the media. The organisms primarily responsible for lignocellulose degradation are aerobic filamentous fungi (Tuomela et al., 2000). The most rapid degraders in this group are basidiomycetes, especially white-rot fungi. The ability to degrade lignocellulose efficiently is thought to be associated with a mycelial growth habit which allows the fungi to transport scarce nutrients, e.g. nitrogen into the nutrient-poor lignocellulosic substrate that constitutes its carbon source (Hammel, 1997). However, the white-rot fungi do not survive the thermophilic phase of composting, and thus cannot play any significant role in lignin degradation in this environment. Other microorganisms in compost, mainly thermophilic microfungi, are probably the most important lignin degraders. The mineralisation of lignin by compost microorganisms is probably of the same order of magnitude as of a mixed population of soil microorganisms. Lignin degradation in composts is regulated by temperature, the original lignin content and the thickness of the material (Tuomela et al., 2000). Since lignin is degraded by the aerobic fungi of the white-rot and brown-rot species as well as by bacteria to a much smaller extent, the degradation of lignin in aerobic environments is higher compared to anaerobic environments (Komilis and Ham, 2004). Deuteromycetes and other microfungi, which - in contrast to basidiomycetes - are always present in compost, may be involved in the conversion of lignin in compost environments. Ligninolytic peroxidases are considered the most important enzymes in the lignin degradation. The laccases of deuteromycetous fungi seem to be the key enzymes responsible for the low but steady mineralisation of lignin (Kluczek-Turpeinen et al., 2003). In some cases bacteria was also seen to be attached to lignified cell walls (Davis et al., 1992).

In a leaching tube experiment, lignin degradation in composted plant residues was rarely seen (Dresboll et al., in prep). After almost 8 month of degradation, a small decline in the total lignin mass was observed. During the actual composting process of 8-10 weeks, lignin was not degraded. On scanning electron micrographs it was obvious how thin-walled non-lignified cells were degraded, whereas the lignified cells did not show any degradation (Dresboll and Magid, submitted). As the hemp straw practically only consisted of the xylem core, the degradation of the non-lignified cells could not be observed. However, the degradation of primary cell walls in the xylem tracheary tissue was observed, revealing the helical secondary reinforcements (Fig.8).

When producing compost of plant residues to be used as a growing medium, knowledge of plant anatomy is of immense importance, as the remaining tissues after degradation determines the overall physical properties of the growing medium.

Figure 8. Helical secondary reinforcements of hemp (Cannabis sativa) after 8 weeks of composting.

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