Species that first colonize litter and are involved in its digestion are called primary saprotrophs. Most of these are prokaryote and fungal species that release extracellular enzymes. The digestion is extracellular, and complex molecules and polymers are hydrolysed to smaller molecules which can be absorbed through cell membranes. The transfer of nutrient molecules into cells through the cell membrane is called osmotrophy.
Yeasts and testate amoebae are also important early colonizers of litter and contribute to its decomposition and soil nutrient absorption.
Saprotrophic bacteria
Bacteria obtain their nutrients from the soil solution by osmotrophy.
They rely on nutrients and minerals already dissolved from litter. Most species secrete small enzymes that can pass through the bacterial mem-
branes, or that assemble on the outer membrane (White, 1995; Van Wely et al., 2001). These enzymes digest specific chemical bonds of sub- strate compounds in the soil environment. The soluble molecules released into the soil solution become accessible for uptake by the bacte- rial cell membranes, and other osmotrophs. The secretion of specific enzymes, and the activation of substrate molecule transport mecha- nisms, depend on chemical detection of the substrate. In the presence of sufficient nutrient molecules, nutrient intake can be sufficient to main- tain cell growth to division. This condition also requires adequate pH, salt balance, temperature and moisture. Some species have single sub- strate requirements for growth. Other species require more than one source of nutrients or may need cofactors in the soil solution. Most species can grow on a variety of substrate molecules, such as the Pseudomonads which are particularly versatile in their substrate utiliza- tion. In these species, when a preferred substrate is absent or at low con- centration in the soil solution, an alternative substrate is obtained for growth. The switch to new substrate usually involves synthesis of new enzymes by the bacterium. Availability of a preferred substrate inhibits the uptake of alternative substrates. In order to understand substrate use and regulation of nutrient intake by bacteria, one must understand the metabolic regulation of their operons (Lengeler et al., 1999). The substrate preferences of bacteria are key in assigning their taxonomic species name, genus and family. Their main sources of organic nutrients in the soil are indicated below.
Cellulose
The ability to digest cellulose is significant, because it is about half of the total biomass synthesized by plants, which returns to soil as litter. The plant cellulose cell wall holds the more nutritious cytoplasm remaining inside. Therefore, breaking into these cellulose boxes provides access to a better source of nutrients. It consists of chains of β-D-glucopyranose in 1,4-glycosidic linkage that are polymerized into long fibrils, about 80 units in length (Fig. 4.6). The fibrils are held together in a three-dimen- sional matrix by inter- and intramolecular hydrogen bonds. The enzyme subunits assemble on the external cell membrane surface into cellulosomes. Aerobic digestion of cellulose occurs through the action of several genera, such as Archangium, Cellovibrio, Cellulomonas, Cytophaga, Polyangium, Sorangium (Myxobacteria), Sporocytophaga, Thermomonospora, the ActinomycetesMicromonospora and severalStreptomycetes.The anaero- bic digestion is also possible by species in the genera Acetovibrio, Bacteroides, Clostridium (C. thermocellum and C. cellubioparum) and animal rumen bacteria such asRuminococcus.
Digestion of the lignin component of plant cell walls is discussed below with the fungi. Some bacteria have been implicated in lignin decomposition, but they are poorly studied. Usually, mixed species are
required to provide the adequate combination of enzymes by syntrophy (Blanchette, 1995; Boddy and Watkinson, 1995). Only moderate diges- tion of the lignin occurs. These species occur more commonly in water- saturated near-anaerobic conditions, or where fungi are inhibited by wood chemicals. They have been recognized in high lignin hardwoods which are difficult for hyphae to access. For example, they occur in Eusideroxylon zwagei wood which is very resistant to decomposition because it contains fungal inhibitors. In general, a good working hypothesis is that bacteria will be out-competed by fungal hyphae in lignin decomposition.
Xylan
Xylans are part of the heterogeneous group of molecules known as hemicelluloses. These are the next most abundant after cellulose in plant cell walls (Table 4.7). These fibrils consist of 1,4-glycosidic links of β-D-xylose, but also of a variety of other pentose and hexose sugars. The polymer forms branched structures, 30–100 units long and partially sol- uble in water. The variety of xylanase enzymes synthesized by species have preferences for different specific bonds of the fibrils. In Clostridium, secretion of xylanases is constitutive, although it is under inducible reg- ulation in other bacteria. Similar branched structures of other sugar polymers occur and are collectively known as hemicelluloses. Mannans, for instance, contribute <11% of conifer wood dry weight, and are also produced by some yeasts in their cell walls. Fructans occur as storage
H
HOCH2 H HO
H OH H
O O
H
H HOCH2
HO H
OH H
O O
H
H OH
H HOCH2
H HO H
OH H
O O
H C
H HOCH2
H HO H
OH H
O OH
H
OH
A B
HNCOCH3 OH
H HOCH2
H HO H
H O
H
OH D
Fig. 4.6. Common saccharide and polysaccharide molecules in litter. (A) Glucose monomer of cellulose, starch and glycogen polymers. (B) Starch or glycogen branching structure of glucose polymer. (C) End of a cellulose chain of glucose monomers. (D) Monomer of chitin polymer found in fungi, other protists and invertebrates.
compounds in some plants such as Phleum pratense (Timothy grass).
They are also excreted by some bacteria when grown on sucrose. In some cases, it was demonstrated that the fructans are then used as sub- strate when sucrose runs out (in Azotobacter, Bacillus and Streptococcus) (Schlegel, 1993). The genus Erwinia belongs to the soft rot (see below) and, when present, is an important contributor to hydrolysis of hemicel- luloses, pectins and cellulose.
Starch
This is the primary plant storage compound, and therefore a rich source of energy for organisms that can metabolize it. However, since plants translocate nutrients from senescing organs before shedding them, much less starch becomes litter than one would anticipate. Starch consists of aggregates of two polymers, amylose and amylopectin.
Amylose consists of 1,4 α-glycosidic links of D-glucose, forming unbranched, water-soluble chains of hundreds of glucose units in length. Amylopectin consists of the same polymerized units, but with a 1,6-branching approximately every 25 units, with PO43, Mg2+and Ca2+
ions incorporated. Numerous bacteria synthesize glycosidases to dissolve amylose and amylopectin. α-Amylases are endoglycosidases and release glucose, maltose and various soluble oligomers. β-Amylases are exogly- cosidases and release monomers until the branch points.
Chitin
Chitin is the most abundant polysaccharide in the soil after cellulose. It is one of the main components of the cell wall of fungi and of inverte- brate cuticle, and has been reported from numerous protozoa in cyst walls and tests, as well as in the zoosporic fungi and chromista. It con- sists of the polymer of N-acetylglucosamine, linked in 1,4-β-glycosidic linkage (Fig. 4.6). The chitin polymer is usually associated with other fibrils, which hold the matrix together. Therefore, it is important to note that as for plant cell walls, digestion of one wall component alone, although it may loosen the matrix, will not dissolve or remove it. For example, invertebrate cuticles also contain fibrous proteins and fungal Table 4.7. Example of plant and an Ascomycetes cell wall composition for selected molecules (% dry weight) in several litter substrates.
Substrate Lignin Cellulose Hemicelluloses Protein Sugars Chitin
Lucerne stem 6.0–16 13–33 13–33 15–18 0 0
Wheat straw 18–21 27–33 21–26 3 0 0
Beech wood 18–21 45–51 45–51 0.6–1.0 0 0
Aspergillus nidulans 0 0 0 10.5 78.4 19.1
Data from Paul and Clark (1996).
cell walls may contain β-glucans. Many bacteria have the ability to digest chitin polymers, such as species in the genera Bacillus, Cytophaga, Flavobacterium,Micromonospora,Nocardia,Pseudomonas andStreptomyces.
Murein
This component of bacterial cell wall consists of repeated units of two amino sugars, N-acetylglucosamine and N-acetylmuramic acid dimer, with amino acid side chains which cross-link the chains together. They are digested at the glycosidic linkage, or at the peptide linkage, by gly- cosidases or peptidases. Murein contributes <10% of Gram-negative bac- teria dry weight, but that of Gram-positive bacteria is 30–70% murein.
Proteins, lipids, nucleic acids and other cell components
These are the richest source of nutrient input into the soil and are most readily digested, because most species have enzymes to digest them.
Therefore, their half-life in the soil is short, in the order of minutes to hours. However, a portion of peptides cross-react with other compo- nents of the soil organic matter to produce less accessible compounds.
Furthermore, even some labile and soluble DNA can be stabilized from enzyme digestion by binding to clays (Stotzky, 2000). These enzymes are part of the metabolic pathways of most prokaryote and eukaryotes.
Proteins are digested by endo- and exoproteases at the peptide bonds between amino acids, releasing more soluble and less hydrophobic pep- tides, or soluble amino acid monomers. Deamination reactions release NH3+ from amino acids or amino sugars. Lipases have low substrate specificity and digest lipids (sterols, phospholipids and other membrane lipids), releasing the fatty acids from the alcohol moiety. Nucleases digest nucleic acids into nucleotide monomers. These are a rich source of soluble phosphates that are easily absorbed by living cells. RNAs are naturally unstable and denature rapidly outside the cytoplasm.
Intracellular decarboxylation through cell autolysis or bacterial diges- tion, especially from animal cadavers which are rich in proteins, releases a variety of nauseous amines (such as cadaverine from lysine, putrescine from ornithine and agmatine from arginine).
Hydrocarbons and phenolics
A variety of alkanes occur naturally as a by-product of metabolism, or from digestion of other molecules. Aromatic compounds accumulate from plant cell wall lignins. Other sources of alkanes and polyphenolic compounds are petroleum (naturally occurring or from pollution), and anthracene, asphalt, graphite or naphthalene. There are bacteria that can grow on these substances, usually through aerobic respiration. They tend to be enriched in chronically polluted soils or sediments. For exam- ple, a mixture of xylene, naphthalene and straight chain aliphatic hydrocarbons (C14–C17) was decomposed by natural soil microorganisms
to low levels in 20 days (xylene), 12 days (naphthalene) and within 5 days (aliphatic hydrocarbons), under aerobic conditions (Eriksson et al., 1999). The decomposition of substrates occurred above threshold tem- peratures. This may be due to inactivation of particular species or too slow metabolism of one or more participating species. For example, a study conducted on biodegradable plastics showed that both tempera- ture and O2 availability modified species that digested the plastics (Nishide et al., 1999). Interestingly, only a small number of isolates could decompose the plastics in the laboratory. This emphasizes the cooperat- ing role of multiple species co-occurring naturally, in providing a combi- nation of enzymes to digest substrates. For example, in nitrogen-poor substrates, the role of nitrogen-fixing bacteria in the soil is important to provide an input of fixed nitrogen. Sometimes both functions can occur in the same species (Perez-Vargas et al., 2000).
Overall, a useful working hypothesis is that one can always find a bacterium to digest almost any compound, under suitable conditions.
There are even unpublished reports of bacterial growth in old glu- taraldehyde, a rapid penetrating biological fixative used in light and electron microscopy. An area of bacterial nutrient acquisition in soil which remains understudied is the correlation of species that are active together on one substrate, and cooperate by syntrophy to digest litter.
Activity of several species on one substrate provides a diversity of enzyme functions, that together more effectively digest complex mole- cules into soluble nutrients. This may be the prime mechanism of bacte- rial function in natural systems.
However, one must not ignore the role of other soil species in the decomposition of hydrocarbons and phenolic molecules. Most inverte- brates and protists ingest the soil solution when feeding and are in con- tact with contaminated soil. Bacterivores and osmotrophs in particular can be severely affected as they ingest bacterial cells and the soil solu- tion. These higher order consumers will accumulate the organic mole- cules to toxic levels if undigested. Many species of protozoa and invertebrates can be isolated that tolerate or digest hydrocarbons to var- ious levels (Rogerson and Berger, 1981, 1982; Rogerson et al., 1983).
Saprotrophic fungi
In terms of function (enzymatic activity on substrate), many of the enzymes found in primary saprotrophic fungi are similar to those described above for bacteria, and they are not repeated here. Several points are made below simply to distinguish between bacteria and fungi.
Most notable is that as eukaryotes, fungi can synthesize larger proteins for secretion, in much greater quantities (see Chapter 1). The impact on the immediate microenvironment is far greater. Unless bacterial cells are
protected from fungal proteases and other enzymes (through capsules or inhibitors), they will be partially digested. Unlike many bacteria which tend to focus on one substrate at a time, fungal cells can secrete enzymes for several substrates at the same time. This is advantageous because usually many compounds are mixed together. For example, plant cell walls contain cellulose, proteins, lignins, hemicelluloses and pectins mixed into a matrix (Table 4.7), and sometimes with additional components that can be defensive. To disentangle the cell wall and obtain soluble substrates, the whole structure needs to be dissolved. This can be achieved by bacteria if multiple species contribute their panoply of enzymes (syntrophy). Fewer species are required for simpler sub- strates or if a species can produce diverse enzymes.
Based on their ability to digest plant cell walls, these fungi are recog- nized as three groups. The white rotinclude about 2000 species of mostly Basidiomycetes, which can digest cellulose and lignin components of the plant cell wall. The brown rot include about 200 species of mostly Basidiomycetes which are unable to digest the lignin component, but only cellulose and hemicelluloses. The soft rot consist of those Ascomycetes and mitosporic species (deuteromycetes) which are efficient on cellulose and hemicelluloses, but digest lignin slowly or incompletely. Another important group of species is involved in the invasion and digestion of plant seeds (Watanabe, 1994). It is estimated that up to 80% of seeds are naturally decomposed by fungal digestion. These species need to break into the seed protective coats but, once penetrated, obtain a rich supply of starch, protein and oils that are common seed storage molecules.
Cellulose, xylan and pectin
Cellulase activity is from a complex of several enzymes which assemble into a cellulosome (Lemaire, 1996) similar to that in bacteria. It consists of several endoglucanases, exoglucanases and β-glucosidases (or cellubiases) which attack the polymer at multiple sites. The brown rot cellulase activity generally results in more complete digestion of the cellulose polymer than white rot fungi. The reason is believed to be the synthesis of H2O2and ferrous ions which reach fibrils embedded in lignin and contribute to hydrolysis of cellulose (Moore, 1998). The accumulation of ferrous ions from ferric ions is through oxalate. This non-enzymatic process occurs through the Fenton–Haber–Weiss reaction and involves siderophores and lignin, which becomes chemically modified but not degraded. It helps loosen the cellulose fibrils, which are otherwise packed too tightly to allow the large cellulosome enzyme complex into the fibrils. Brown rot are com- mon on coniferous wood, which is decomposed almost fully, leaving the lignin matrix on the forest floor. This chemically altered but undegraded lignin which remains is very recalcitrant to digestion. The reticulum of tunnels that remain functions as a sponge. It is an important sink for water and nutrients, and a refuge for microorganisms.
Xylanases are also complex enzymes formed from two endoxy- lanases and one β-xylosidase. Pectinases are particularly important in tis- sue invasion by parasitic fungi (and bacteria), as they involve loosening the pectin which holds adjacent plant cells together. They consist of polygalacturonases and pectin lyases, with arabanase and galactanase to hydrolyse pectin-associated sugars.
Lignin
The next most abundant plant cell wall component after cellulose and hemicellulose is lignin. It is particularly abundant in woody tissues, and becomes more abundant in older tissues, late season cells and other stressed conditions (Table 4.8). It provides reinforcement to the cellulose fibrils in the secondary cell wall deposition. It represents 18–30% of the dry weight of wood. Lignin is a complex compound derived from phenyl propanoid units, but polymerized variously through chemical reactions, not through precise enzymatic activity, so that its structure is variable (Fig. 4.7). The proportions of the lignin precursors coniferyl, sinapyl and
Table 4.8. Estimates of lignified material in roots and aerial parts of plants, from various ecosystems.
Biome Roots (%) Above-ground perennial parts (%)
Tundra, alpine 75 12
Desert, semi-desert scrub 57–87 2–40
Temperate grassland 83 0
Tropical savannah 28 60
Temperate deciduous forest 25 74
Northern coniferous forest 22 71
Tropical rainforest 18 74
Data from various sources (see Boddy and Watkinson, 1995).
CH CH CH2OH
OH
CH CH CH2OH
OH
OCH3
CH CH CH2OH
OH
OCH3 CH3O
A B C
Fig. 4.7. Several lignin precursors. (A) Coumaryl alcohol. (B) Coniferyl alcohol. (C) Sinapyl alcohol.
coumaryl alcohols in lignin vary between angiosperms and gymnosperms (Moore, 1998). The reason for slow decomposition of lignified tissues is the relative scarcity of primary saprotrophs that are able to digest it. This implicates some Basidiomycetes, fewer Ascomycetes and some bacteria (Table 4.9). Most of the data on lignin digestion are based on two species, the Basidiomycetes (fungi) Phanerochaete chrysoporium which can com- pletely digest lignin to CO2 and H2O, and the Actinobacterium Streptomyces viridosporus. The fungal enzyme is a complex of up to 15 lignin peroxidases (ligninase), Mn-dependent peroxidases and Cu-oxyge- nases (laccase) which oxidize o- and p-phenols. The enzyme activity has been reported to occur when available nitrogen sources are depleted, through a cAMP-mediated activation pathway. It is unclear if this is a generalization, as too few species have been investigated.
The white rot fungi which digest lignin are grouped in two func- tional groups (Blanchette, 1991, 1995). One group is non-selective and will digest both lignin and cellulose in varying proportions. Examples are Trametes versicolor, Ganodermer applanatum, G. tsugea and Heterobasidion annosum. The other group is more selective and will digest lignin more completely. Examples are Phlebia tremellosa, Inonotus dryophyllus, Phenillus pini and P. nigrolimitatus. There is variability between the proportions of cellulose, hemicellulose and lignin which are digested by different strains of one species, and even by the same strain through various regions of the substrate. The white rot also digest the middle lamella of pectins to loosen cells, and deposit MnO2and calcium oxalate at zones of digestion, and siderophores are involved. The white rot ligninases have a substrate preference for syringylpropyl against guaiacylpropyl. They predominate in angiosperm wood decomposition, but also occur in gymnosperms.
Table 4.9. Amount of remaining molecules in well-decomposed wood (% remaining) after decomposition by different types of fungi, compared with average initial plant cell wall content (g/kg).
Initial composition (g/kg) Lignin Cellulose Hemicelluloses Protein
Plant cell wall 1 41 386 463 110
Plant cell wall 2 117 352 477 64
In decomposed litter (%) Lignin Glucose Xylose Mannose
White rot (selective) 30 47 6 13
White rot (non-selective) 1 97 1 1
Brown rot 60 20 1 2
Soft rot 61 13 2 2
Bacteria 80 6 1 9
Data for lignin from Kleson analysis, and from HPLC for sugars: glucose from cellulose polymer; and xylose and mannose from xylan and mannan hemicelluloses. Data from Blanchette (1995) and Cadish and Giller (1997).