Methods in Soil Biology

The degradation of plant and animal matter, i.e. the release and binding of nutrients and trace elements, is one of the most important functions of soil organisms. The task of the soil fauna is to loosen the soil, and to diminish and to mix the litter. The microorganisms are important for the enzymatic degradation of the complex organic substances to nutrients and for the release of nutrients and trace elements from the mineral soil fraction. Soil microorganisms include bacteria, fungi and algae, as well as the mycorrhizae, which contribute to better plant development. Further, there are many pathogenic plant and animal fungi in the soil. The sensitive reaction of the soil microflora to external influences results in a quick adaptation to the new conditions, but also in a changed reaction towards the plants.

Each mechanical disturbance of the soil causes changes in the soil biota and its activity. Soil sampling removes part of the soil from its natural environment. Important biological and physico-chemical processes are disrupted. The extent of the activity change depends on the size of the soil sample and the subsequent sample treatment (sieving, drying, or cooling of field-moist samples). An air-dried and rewetted soil sample usually does not regain its original activity. Activity measurements are therefore done with field-moist soil samples, which should not have been dried prior to analysis.

Among soil microorganisms, bacteria are particularly suitable for quantification by counting. It should be considered, however, that bacteria are never uniformly distributed in the soil and that their spatial arrangement varies even in neighbouring microsites. Furthermore, the bacterial biomass may fluctuate drastically within short periods. Counting of soil bacteria can be achieved by direct microscopy as well as by cultural methods. The most widespread cultural method, the plate count technique, is based on the development of colonies from individual propagules. Microscopic bacteria counts of soil samples are often up to 1000 times greater than viable counts obtained by cultural techniques. The reasons for the greater values obtained by microscopy are the inclusion of dead cells and organisms which fail to grow in conventional culture media under the conditions of incubation provided. Groups of bacteria generally not included in viable counts are e.g. nitrogen-fixing bacteria, nitrifiers, cellulolytic organisms and anaerobic bacteria, as they depend on selective media and/or special growth conditions.

In soils, microorganisms occur in great density and variety. Bacteria and fungi are the most abundant microorganisms, protozoans and algae occur in smaller numbers. The proportion of carbon biomass in the soil has been found to be 1–3% of the organic carbon (Sparling 1985). Investigations on cultivated soils showed that the proportion of the metabolically active microbial biomass is 1–5 and 2–8% of the organic matter in arable and grassland soils, respectively (Beck et al. 1992). Biomass determinations based on mathematical analysis of the respiration curves suggest that only 2–30% of the total biomass is metabolically active (Van de Werf and Verstraete 1987a,b).

The structural analyses of microorganisms in soil present a far more complex problem than their functional analyses (C0₂ determination, ATP measurement etc.). Microbial communities of soils are extremely diverse, and it is assumed that with conventional microbiological cultivation techniques only about 1% of the indigenous species are recovered (Torsvik et al. 1990). Therefore, methods that require neither growth nor removal of cells from the soil matrix are needed. Microbial diversity and community structure may be best estimated by RNA analysis (Ward et al. 1990) or DNA extraction (Torsvik et al. 1990). Besides these techniques, the extraction and analysis of the fatty acids derived from phospholipids (PLFAs) and lipopolysaccharides (LPS; Vestal and White 1989; Tunlid and White 1992) are a promising approach to classify the structure of the microbial communities in soils. The measurement of the content of the phospholipids has also been used to estimate microbial biomass in sediments and soils (Smith et al. 1986; Baath et al. 1992; Korner and Laczko 1992; Zelles et al. 1992; Frostegard et al. 1993; Hill et al. 1993).

Microbial respiration (soil respiration) is defined as oxygen uptake or carbon dioxide evolution by bacteria, fungi, algae and protozoans, and includes the gas exchange of aerobic and anaerobic metabolism (Anderson 1982). Soil respiration results from the degradation of organic matter (e.g. mineralization of harvest residues). This soil biological activity consists of numerous individual activities; the formation of C0₂ is the last step of carbon mineralization. In undisturbed soils (no nutrient addition etc.), there will be an ecological balance between the organisms and their activities. The respiration is then called “basal respiration”. Upon a disturbance, e.g. through addition of organic matter, one can observe a change in the soil respiration due to more rapid growth and a higher mineralization of the microorganisms. This increased respiration is characterized by an initial, an acceleration, an exponential, a delay, a stationary and a decreasing phase (Freytag 1977). C0₂ evolution from a soil is thus a measure of the total soil biological activity.

Soil organic matter is defined as the dead material from plants and animals and the respective degradation and transformation products in and on mineral soil (Schachtschabel et al. 1989). The organic matter subjected to microbial decay in soils comes from several sources. The decomposition of organic matter provides energy for the growth of microorganisms and supplies carbon for the formation of new cell material.

A range of microorganisms is able to reduce dinitrogen from the atmosphere and use it as a source of nitrogen for cell growth. Besides the genera Clostridium and Azotobacter, nitrogen fixing abilities have also been revealed in anaerobic phototrophic bacteria as well as in many cyanobacteria, facultative anaerobes, autotrophic and methylotrophic bacteria, and desulfurizing and methanogenic bacteria in the last decades (Schlegel 1992).

The conversion of organic nitrogen to the more mobile, inorganic state is known as nitrogen mineralization. Microorganisms with different physiological properties take part in this process. In the first step, ammonium is formed from organic compounds (ammonification); in the second step, ammonium is oxidized to nitrate (nitrification).

Nitrification is the conversion of inorganic or organic nitrogen from a reduced to a more oxidized state. Chemoautotrophic bacteria are largely or solely responsible for nitrification in soil with a pH above 5.5 (Focht and Verstraete 1977), at a lower pH there is evidence for the presence of acid-tolerant heterotrophic nitrifiers (Schimel et al. 1984). The heterotrophic nitrifiers (e.g. few bacteria, such as strains of Arthrobacter, and fungi, such as Aspergillus) do not derive energy from the oxidation of NH₄ ⁺. In arable soils the production of nitrate by heterotrophs appears to be insignificant in relation to that brought about by the chemoautotrophs (Paul and Clark 1989). In contrast, 90% of the potential nitrification in acid forest soils is produced by heterotrophs (Kilham 1987).

Proteins are important constituents of both seed and root exudates, they are a primary structural and functional component of plant cell walls, and represent a third of the total nitrogen in soil (O’Sullivian et al. 1991). Proteins are supplied to soil from all dead organisms, plants and animals. Proteins in soil are decomposed readily by many bacteria and fungi. During degradation, extracellular proteases produce oligopeptides from proteins, resulting in the subsequent release of low molecular weight compounds which are assimilated by microorganisms. Upon the release of proteases from microbial cells these soil enzymes become physically adsorbed onto soil colloids or covalently bound to soil organic matter. These immobilized enzymes show a high degree of resistance to proteolysis. In contrast, proteases are inhibited by desiccation.

Cellulose is, as far as quantity is concerned, the most important natural organic compound. Plants consist of 40–70% cellulose. The microbial degradation of cellulose utilizes at least three different synergistic enzymes (Enari and Markkanen 1977). The endo-β-1,4-glucanases (C_x-cellulase or carboxymethylcellulase) split the β-1,4- bond within the cellulose molecule. The exo-β-l,4-glucanases attack the free ends of the chains, and the disaccharide cellobiose is formed which is further degraded by the β-1,4-glucosidases (cellobiases), producing glucose. Fungi (e.g. Chaetomium, Fusarium, Polyporaceae, Poriaceae), mycobacteria, and some eubacteria (e.g. pseudomonads, actinomycetes) are the most important aerobic cellulose degraders. Under anaerobic conditions, cellulose is hydrolyzed by bacteria of the genus Clostridium. The extracellular degradation of cellulose presumably ends with cellobiose (Schlegel 1992).

The importance of phosphatases for plant nutrition has repeatedly been pointed out (Cosgrove 1967; Ramirez-Martinez 1968; Halstead and McKercher 1975; Hayman 1975; Kiss et al. 1975; Cosgrove 1977; Dalai 1977; Speir and Ross 1978, Dick and Tabatabai 1987). In most soils, the organically bound P-fraction is higher than the inorganic. Among the organic phosphoric acid esters, the largest fraction in the soil is phytanic acid or phytin (Halstead and McKercher 1975; Speir and Ross 1978). Phophorus uptake by plants requires mineralization of the organic P-component by phosphatases to orthophosphate (Speir and Ross 1978; Malcolm 1983). Phosphatases are inducible enzymes that are produced predominantly under conditions of low phosphorus availability. Phosphatases are excreted by plant roots and by microorganisms. Microbial phosphatases dominate in soils.

Sulfatases are important for the mineralization of sulfur-containing compounds in the soils. They hydrolyze organic sulfates, and thus provide plants with available sulfur (Freney et al. 1975). Sulfatases are predominantly of microbial origin. In the soil, they also occur as exoenzymes, and have a close relationship to organic matter. In nature, different types of sulfatases occur (Tabatabai 1982): arylsulfatases, alkylsulfatases, steroidsulfatases, glucosesulfatases, chondrosulfatases, myrosulfatases. Arylsulfatase was the first enzyme group to be discovered and has thus predominantly been investigated. It catalyzes the hydrolysis of an arylsulfate anion by splitting the O-S-bond. In soils, arylsulfatase was first determined by Tabatabai and Bremner (1970). Methodological and applied studies were performed by Speir and Ross (1975, 1978), Speir (1977), Al Khafaji and Tabatabai (1979), and Speir et al. (1980).

The catalase of aerobic organisms splits the toxic H₂O₂ produced from the mitochondrial electron transport and from various hydroxylation and oxygenation reactions into water and oxygen. Since aerobic organisms predominate in non-compacted and non-waterlogged soils, catalase activity was used to characterize soil microbial activities (Beck 1971). Catalase was also the first enzyme that was investigated in soils (Woods 1899, cited in Skujins 1978). While Kuprevich and Shcherbakova (1956, 1971) and Beck (1971) determined the activity via the produced oxygen, Baroccio (1958) and Johnson and Temple (1964) measured the unused H₂O₂. Soil catalase is characterized by its high persistence. Fresh root material distorts microbial catalase activity measurements since plants also contain catalase.

The adenine nucleotides AMP (adenosine 5’monophosphate), ADP (adenosine 5’diphosphate), and especially ATP (adenosine 5’triphosphate) serve as energy carriers that are generated during exergonic reactions and used to drive endergonic reactions. At the substrate-level, phosphorylation, and in the electron transport, chain oxidation reactions, supply the energy for the phosphorylation of AMP and ADP to ATP. The ratios of these three adenylates are a measure of the energy status of cells. As ATP is very sensitive to environmental factors and to phosphatases, it does not persist in soils in a free state. Assuming that the ATP content of all living cells is constant under standard conditions, it can be used as a measure for the biomass content of soils (Holm-Hansen and Booth 1966; Sparling and Eiland 1983). There is no general agreement about the correlation between the amount of ATP and the microbial biomass (Ausmus 1973; Karl 1980; Sparling and Eiland 1983). The ATP content of microorganisms can be widely divergent depending on their metabolic activity (Fairbanks et al. 1984). Jenkinson (1988) pointed out that ATP contents of different soils can only be compared if such parameters as water content and temperature are standardized.

Soil microbial production of indolic and phenolic compounds derived from aromatic amino acids is regarded as an important factor in plant development. Plant growth was promoted upon the addition of L-tryptophan (Trp) to soil (Frankenberger et al. 1990; Frankenberger and Arshad 1991a,b). Environmental fluctuations cause increased Trp contents in soil and regulate the microbial production of indole-3-acetic acid (auxin), of its storage product indole-3-ethanol, and of anthranilic acid from the precursor Trp (Lebuhn et al. 1994). Microbial auxin and indole-3-ethanol exert auxin-phytohormone effects on plants (Martin et al. 1989; Müller et al. 1989; Selvadurai et al. 1991). Auxin also seems to control the induction of plant resistance mechanisms (Jouanneau et al. 1991).

“Mycorrhiza” describes the symbiontic relationship between plant roots and fungi. Depending on the type and intensity of the contact between the two partners, one can differentiate between ecto- and endotrophic mycorrhizae. A detailed survey is given by Harley (1991).

Eukaryotic algae and terrestrial cyanobacteria compose, together with bacteria and fungi, the main part of the plant mass in the soil. According to Shtina (1974), the algal biomass in soils of temperate regions varies from 150 to 500 kg · ha^-1. As phototrophic organisms, algae accumulate organic matter, they stabilize soil aggregates, have inducing effects on the development of soil-fungi and bacteria, and serve as sources of energy for the soil-microfauna.

In addition to the quantification of the microbial biomass (and its carbon and nutrient pools), the characterization of the physiological state of the microbial community is gaining more importance. The physiological state is determined by the nutritional status as well as by factors like soil type, climate, influence of pollutants etc. The methods mentioned below are described in detail by Martens (1991).

The soil contains a rich variety of animals of very different sizes and life forms. The most important groups are the Protozoa, Nematoda, Annelida and Arthropoda. In interaction with the microorganisms, the predominant role of the animals is shredding, inoculation and chemical changes in the plant litter. Digging and burrowing animals increase the pore volume and improve aeration and mixing of the soil. For such functional descriptions, the size, life form and type of nutrition of an organism are more important than its taxonomy. Based on the size of an organism, one can differentiate between micro-, meso- and macrofauna. The microfauna (e.g. Testacea, Ciliata, Nematoda) utilizes pores with a diameter of less than 100 µm; the mesofauna (Acari, Collembola, Enchytraeidae) occurs predominantly in the larger pore space (macropores, <2mm). The macrofauna (2–20 mm) utilizes the existing cracks and root canals, but its digging and burrowing activity also contributes considerably to the loosening and aeration of the soil. The classification according to the life form is based on morphological and ecophysiological differences depending on the soil depth. If one considers the type of nutrition of the soil animals as well as the kind of interactions with other soil organisms, one can finally evaluate the role of the animals in nutrient mineralization in the soil.

The microfauna consists of eukaryotic, single-celled protozoans (naked and testate amoebae, flagellates, ciliates) and multicellular organisms (rotifers, tardigrades, nematodes) too small to be studied and identified without the help of a microscope.

Enchytraeidae are small oligochaetes of large soil-biological importance. They feed on substrate like plant litter, fungi, mineral partiles, or faeces from other soil animals. They are very adaptable, occur everywhere from mineral soils to deciduous litter, and are mostly aggregated. Potworms establish partially high seasonally variable abundance and biomass (in coniferous forests up to 10⁵ individuals (ind.) · m^-2= ca. 2.5 g fresh mass (fm) · m^-2. When compared to earthworms, they show higher metabolic activity for equal mass. Potworms occur in almost all soils with sufficient moisture.

Earthworms are medium-sized to large oligochaetes and very important for decomposition (substrate feeders). Their activity increases the soil fertility (transport of organic matter to deeper soil layers, aeration, spreading of microorganisms). Their distribution is strongly dependent on water content, soil type, vegetation (palatability of the litter), and pH. Buché (1977) distinguished three ecological groups: litter dwellers (epigeic species), horizontal burrowers (endogeic), deep burrowers (anecic). Due to their size, earthworms contribute a large fraction to the biomass in loamy meadows. Few or no earthworms occur in shallow and acid soils.

Animals on the soil surface are in continuous contact with the the soil interior and can thus give valuable information on the condition of the soil.

In this chapter, some actual methods to determine functional activity of soil animals are summarized without extensive descriptions.

Interactions among bacteria, fungi and invertebrates are important for many soil processes. Numerous review articles and books give a comprehensive survey of the wide range of possible mechanisms of interaction (Anderson et al. 1984; Fitter et al. 1985; Edwards et al. 1988; Verhoef and Brussaard 1990; Lussenhop 1992). These include mainly the grazing of bacteria by protozoans and nematodes, the grazing of fungi and the transport of fungal propagules by microarthropods and enchytraeids, and the comminution of litter and the physical alteration of the soil substrate by macroarthropods and earthworms.

Soil samples are dried at 105°C, and dry matter and water content are determined from the weight loss (Schlichting and Blume 1966).

The potential acidity corresponds to the sum of the protons in the soil solution which can be desorbed with a 0.01 M CaCl₂ solution on a short-term basis (ÖNORM L1083).