Keywords

10.1 Introduction

Soil is a crucial part of the terrestrial ecosystem and provides fundamental support to all terrestrial life forms. Therefore, proper soil protection programmes are indispensable to avoid problems of soil erosion, infertility, contamination of groundwater and poor water holding capacity and most importantly to avoid the loss of biodiversity. Soil quality is directly related to biological properties of the soil, which are very sensitive to any environmental disturbances. Soil microbiota is equally sensitive and changes quickly in response to environmental perturbations. Profile of soil microbiota and enzymes is interrelated and an important indicator of soil health and quality (Pajares et al. 2011). Soil enzymes which are mainly produced by soil-inhabiting microbes play a crucial role in nutrient cycling and reflect soil microbial activity and fertility (Bentez et al. 2000). A fine balance of biological (which includes enzymatic activity), chemical and physical components is essential to maintain soil health. Soil enzymes play a key role in the overall process of decomposition of organic matter in the soil (Sinsabaugh et al. 1991). Thus, it is clear that a balanced soil enzyme system is imperative to maintain soil processes.

Rhizosphere region of the soil is biologically very active and rich in soil enzymes as compared to rest of the soil. Functions of the plant roots modify biogeochemical parameters of the soil and bring changes in nutrient levels, pollutants, concentrations of various chelating compound, pH and redox potential, partial pressures of oxygen and carbon dioxide [pO2 and pCO2], etc. (Gianfreda 2015). Production of soil enzymes in the rhizosphere region and their activity depends on several factors. A higher rhizosphere enzyme activity is correlated to greater functional diversity of resident microbial population. Absence or inhibition of soil enzyme activities reduces processes that can result in poor nutrition of the plants. Suppression of certain enzyme activity (e.g. pesticide-degrading enzymes) can result in pile-up of harmful chemicals in the soil; some of these chemicals may further inhibit soil enzyme and deteriorate soil quality. Although there are several publications on soil enzymes piling up day by day, several key questions are yet not answered. In this chapter, we try to shed light on key aspects related to soil enzymology, in situ and ex situ estimation of soil enzymes, factors affecting their activity and molecular approach to mine soil enzyme-encoding genes and significance of soil enzymes.

10.2 Sources of Soil Enzymes

Sources of soil enzyme mainly include microbes, plant roots and soil animals. Since long soil enzymes have been categorized in two main groups, extracellular soil enzymes and intracellular soil enzymes (Burns et al. 2013), enzymes that are present and function inside the living cell are grouped under the latter category, while those produced by living cells but secreted outside comprise the latter. In long terms, soil enzymes get stabilized by accumulation and complexation with humus (organic matter) in the soil. These stabilized soil enzymes, which are no longer associated with viable cells, contribute to about 40–60% activity of soil enzymes (http://soilquality.org/home.html). An exception to this is dehydrogenases, which can only be produced by living cells, thus contributed to the pool of soil enzymes by viable cells only (Yuan and Yue 2012). Various biochemical, chemical and physiochemical reactions play their role in carrying out nutrient cycles in soil. Extracellular enzymes help decay of organic matter of the soil and aid in mineralization of soil organic carbon (C), phosphorus (P) and nitrogen (N) (Bandick and Dick 1999; Finzi et al. 2006). Forest soils play a significant role in the global carbon cycle (Jobággy and Jackson 2000).

Soil microbe mediates biochemical processes along with plant roots and soil animals. Biochemical processes are carried out by a host of enzymes (β-glucosidase, α-glucosidase, β-xylosidase, amylase, chitinase, dehydrogenase, urease, protease, phenol oxidase, L-leucine aminopeptidase, N-acetyl-glucosaminidase, phosphatase, arylsulphatases) that are found in soil (Miwa et al. 1937; Zahir et al. 2001; Ji et al. 2014; Herold et al. 2014). Bulk of the enzyme activity is contributed by microbes that can be rationalized by their large biomass, comparatively higher metabolic activity and larger quantities of secretion of extracellular enzymes into the soil solution (Spier and Ross 1978). Production of several polymer degrading enzymes is commonly ascribed to fungi (Hättenschwiler et al. 2005; Baldrian and Valášková 2008). Saprotrophic species of Basidiomycota are known to be the exclusive producers of ligninolytic enzymes such as Mn-peroxidase and lignin peroxidase (Hofrichter 2002; Baldrian and Valášková 2008). Arylsulphatases are widespread in soils (Dodgson et al. 1982; Gupta et al. 1993; Ganeshamurthy et al. 1995). Primarily they are secreted by bacteria into the external environment in response to sulphur limitation and hydrolyze sulphate esters in soil (McGill and Colle 1981; Kertesz and Mirleau 2004). So far, very limited information is available regarding arylsulphatases synthesizing specific microbial genera that play significant role in the soil organic sulphur cycle (Kertesz and Mirleau 2004). Chitinase, which hydrolyze chitin (poly β-1-4-(2-ncetamido-2-deoxy)-D-glucoside) is an agriculturally important class of soil enzymes. These are produced by both microbes and plants and have been reported to control various soil-borne diseases by hydrolysing the cell wall of phytopathogenic fungi such as Sclerotium rolfsii and Rhizoctonia solani (Ordentlich et al. 1988; Shapira et al. 1989). Supplementation of chitinase to frequently applied chemical fungicides will not only make them effective but also minimize the use of otherwise harmful chemical insecticides and fungicide, contributing to sustainable agriculture (Gunaratna and Balasubramanian 1994; Wang et al. 2002). Phosphatases are abundant in rhizospheric region as compared to the bulk soil and exhibit a very good relationship with mycorrhizal association (Kumar et al. 2011). Wu et al. (2012) studied protease and β-glucosidase in the rhizosphere region of Citrus unshiu and established a correlation of these enzymes with root mycorrhiza, spatial distribution of glomalin-related soil proteins (GRSP) and carbohydrates. Similarly, in a fire chronosequence in Alaska, Gartner et al. (2012) found a correlation between five enzymes involved in the transformation of C, P and N substrates and in the presence of mycorrhiza. Thus, mycorrhiza has also been an important source of soil enzymes along with other microbial sources.

10.3 Methods to Determine Soil Enzyme Activities

Enzyme activities in soil affect various aspects of soil biology and are very useful for gauging soil fertility, functional diversity of soil microbiota and overall turnover of organic compounds in soil systems at different geographical locations (Kandeler et al. 1999). Estimation of the soil enzymes remained a challenge for several years due to want of appropriate quantitative and qualitative techniques. However, recent advances in the field of soil enzymology have enabled us to measure the soil enzyme activity both in situ and ex situ assay. Assay methods that provide reliable results on soil enzyme concentration and rate of the reaction have also been developed (Baldrian 2009). Ndiaye et al. (2000) observed that any change in soil management approach and land use technique results in corresponding changes in the soil enzyme activities and suggested that alterations in soil quality can be anticipated by recording changes in soil enzyme profile, before they are detected by any other soil analyses methods. As discussed by Rao et al. (2014), currently available methods to assay soil enzyme activity suffer with several limitations:

  1. (a)

    These methods do not provide adequate information on real enzyme activities but measure the potential enzyme activities.

  2. (b)

    They take into consideration and provide information on stabilized enzymes which might not be active at conditions prevailing under in situ soil environment.

  3. (c)

    They do not furnish any information related to production and origin of the soil enzyme

  4. (d)

    They do not provide information on changes occurring in enzyme activity that occur in continuously changing in situ environment conditions.

  5. (e)

    As soil enzymes are part of complex and dynamic processes, estimation of single enzyme activity provides no clue about their role in such dynamics.

  6. (f)

    In soil, enzymes are exposed to several environmental, physicochemical, anthropogenic activities, and laboratory assays do not allow correct interpretation of the effect of such disturbances on soil enzyme activity.

In case of in situ assay of soil enzymes, a different set of technological challenges exists including hindrances by electron-dense humic substances and soil minerals and reduced rate of diffusion which reduces overall on-site interaction of enzymes and substrate (Steinweg et al. 2012). Methods including electron microscopic observation (Ladd et al. 1996), zymography (Spohn et al. 2013) and near-infrared spectroscopy (Dick et al. 2013) have been employed to estimate the enzyme activity directly on site. Majority of researchers opt for assays which are carried out under laboratory conditions. In such assays it is imperative to give careful details of soil sampling, handling, storage and enzyme assay so that the method can be reproduced and compared with other studies. Also, extraction of the enzymes is carried out before performing the biochemical assay. A considerable amount of the enzymes is bound to soil components or microbial biomass and is not extractable and thus remains out of the estimation (Claus and Filip 1990; Valásková and Baldrian 2006).

As compared to organic matter-rich forest soils, enzyme extraction from high clay-containing soils is found to be poor (Vepsäläinen 2001; Šnajdr et al. 2008b). Further processing of the extracts is required to get rid of inhibitory compounds such as heavy metals and humic acid (Baldrian and Gabriel 2002; Zavarzina et al. 2004). Vancov and Keen (2009) developed a rapid and high-throughput method of enzyme extraction from soil. They reported that their 1-day extraction protocol included physical disruption of the soil samples with bead beating and was reproducible. In a study carried out by DeForest (2009), it was clearly demonstrated that soil storage conditions and processing method significantly affect the estimation of enzymatic activity in acidic soil of forest. In this study, six extracellular enzymes were measured [employing 4-4-methylumbelliferone (MUF)-linked substrates and L-dihydroxyphenylalanine (L-DOPA)] from soil samples stored for varying time duration at different temperatures. Results of this study revealed that in contrast to storage temperature, enzyme activity values were affected by extended time in buffer. It has been observed that freezing of the soil sample affect soil enzyme activity more than air-drying of the sample (Wallenius et al. 2010; Peoples and Koide 2012). Fluorimetric and spectrophotometric assays [which employ p-nitrophenol (pNP)- and MUF(4-4-methylumbelliferone)-based substrates] are very popular and routinely used for measuring activity of soil hydrolases such as glucosaminidase, glucosidase, galactosidase, etc. (Moscatelli et al. 2012; Trap et al. 2012; Dick et al. 2013). For high-throughput results, these assays are also being carried out by using microplate methods (Trap et al. 2012).

10.4 Factors Affecting Soil Enzymes

Soil enzyme activities are very sensitive to any external disturbances including both anthropogenic and climatic perturbations (Vepsäläinen 2001). Several physicochemical and biological factors affect either enzyme quantities or their activity levels. For example, enzyme activity in soils changes with seasonal variables in moisture, temperature and addition of fresh litter. Like other enzymes, soil enzymes also exhibit varying optimum pH and temperature at which they are most active. For instance, activity of arylsulphatase, phosphatase and amidase involved in sulphur, phosphorus and nitrogen cycling, respectively, is strongly correlated to alteration in pH of the soil (Tabatabai 1994; Kertesz and Mirleau 2004; Chaudhari and Bhatt 2014). Temperature affects several aspects of soil enzymes such as soil enzyme activities, stability and enzyme kinetics, substrate affinity and production levels of enzyme as it also influences the activity and population of soil microbes (Wallenstein et al. 2009; Baldrian et al. 2013). Heat and extreme cold temperature can alter enzyme structure and substrate binding site and therefore, can decrease the enzyme activity above and below the temperature optimum. In a study carried out by McClaugherty and Linkins (1990), it was observed that there was 33–80% decline in the chitinase, peroxidase and laccase activities in winter samples where temperature remains at 0 °C, as compared to those in autumn samples where temperature reaches 15 °C. This is evidence that the seasonal patterns of temperature of ecosystems can affect activity of soil enzymes. The activity of many enzymes often correlates with soil moisture content, as well. Drought may suppress enzyme activity (Sardans and Penuelas 2005; Gömöryová et al. 2006; Baldrian et al. 2010). Upon reduction of 21 % of soil moisture, a corresponding reduction in urease, protease, β-glucosidase and acid phosphatase activity was recorded by Sardans and Penuelas (2005).

Levels of organic matter, nitrogen content and various macronutrients appear to regulate the production of enzymes in soil. It has been reported that with increase in organic matter content, activity of several hydrolytic enzymes including cellobiohydrolase, β-glucosidase, phosphatase and N-acetylglucosaminidase increases (Nsabimana et al. 2004; Sinsabaugh et al. 2008). Decrease in the levels of N-acetylglucosamine liberating enzyme chitinase has been associated with increasing nitrogen content in the soil environment (Olander and Vitousek 2000; Andersson et al. 2004). According to Prietzel (2001), addition of (NH4)2SO4 reduces activity of arylsulphatase. Addition of nitrogen can significantly affect the kinetics of the soil enzyme as observed in case of β-glucosidase, β-xylosidase, cellobiohydrolase and β-N-acetylglucosaminidase involved in soil organic matter degradation in forest soils (Stone et al. 2012). In general, the presence of available phosphorous (P) in soil is related to decreases in phosphatase activity (Venkatesan and Senthurpandian 2006). It has also been confirmed by many researchers across the globe that the presence of pollutants such as heavy metals and organic xenobiotic changes the enzyme profile of the soil (Burns and Dick 2002; Effron et al. 2004).

Although soil enzymes of microbial origin are produced by a diverse array of microbes, production of certain enzymes are limited to certain taxa. Fungi are the most common producer of lignocellulose-hydrolysing enzymes (Moller et al. 1999; Caldwell 2005; Baldrian and Valášková 2008). Chitinase activity is also associated to fungal biomass (Miller et al. 1998; Sinsabaugh et al. 2008). Through microcosm studies it has been demonstrated that introduction of saprophytic fungi in soil increases the activity of different oxidative and hydrolytic enzymes (Šnajdr et al. 2008a, 2011). In forests, soil enzyme activities vary with the change in the dominant tree species of the forest as it changes the litter input (Weand et al. 2010). At the harvesting time, reduction in microbial population, litter input and alteration in soil microenvironment causes decline in enzyme activity (Hassett and Zak 2005)

Spatial heterogeneity is one of the key attributes of the soil environment (Paul 2007). Changes in enzyme activity have been observed with changes in depth. In forest soil, vertical gradient of enzyme activity is more prominent than any other ecosystem. Fresh carbon input in the form of leaf litter and root exudates makes the surface soil horizons (~ 10 cm thick) carbon rich. Organic compounds thus entering the soil accelerate the growth of microorganisms, which in turn produces extracellular enzymes. However, despite smaller carbon inputs, a significant amount of carbon is also stored in subsoil horizons because of its larger thickness (Wang et al. 2010). In case of grasslands where extensive root system of trees is absent and agricultural soils where soil homogenization is a routine practice, considerable level of spatial variability with respect to activity of extracellular enzymes and soil chemistry has been observed (Štursová and Baldrian 2011).

Soil type and texture also influence the enzymatic activities. According to Burns (1982), soil texture plays a significant role in stabilizing soil enzymes; importantly the interactions with soil organic matter and clay minerals affect the stability of the enzymes. Studies on soils from different regions have shown that activities of soil enzyme are sensitive to changes in occurring to the soil because of tillage, cropping system and land use (Staben et al. 1997; Gewin et al. 1999; Ndiaye et al. 2000; Acosta-Martinez and Tabatabai 2001; Ekenler and Tabatabai 2002; Ji et al. 2014).

10.5 Mining of Soil Enzymes Encoding Genes Through Metagenomics and Metatranscriptomics

Among soil-inhabiting microbes, a large number of them remain unculturable, but they do contribute to enzyme repertoire of the soil (Lorenz and Eck 2005). The soil metagenome, the collective microbial genome, could be cloned and sequenced directly from soils to search for novel microbial resources. Metagenome analysis has become a remarkable tool to tap yet uncultured microbial diversity present in soil. Recent advances in molecular methods have enabled us to target the abundance of genes encoding enzymes using metagenome or metatranscriptome analysis. These techniques have very high theoretical potential and been employed for assigning gene sequences to specific groups of soil microorganisms along with specifically targeting exocellulase (Baldrian et al. 2012), laccase (Luis et al. 2005; Hassett et al. 2009; Lauber et al. 2009) or a range of various oxidases and glycosyl hydrolases (GH) present in forest soils (Kellner and Vandenbol 2010). The limitations of single-gene surveys (which are applicable for only highly similar gene sequences) might be overcome by sequencing whole transcriptomes that would help the analysis of the entire spectrum of expressed genes. In a recent study by Damon et al. (2012), several families of GHs and other hydrolytic enzymes were detected upon analysis of eukaryotic gene expression in forest soils. Cellulolytic and cell wall-degrading enzymes are of special interest owing to their application in biotechnology sector for bioenergy production. Liu et al. (2011) isolated a low-temperature active, thermostable, halotolerant cellulase from red soil metagenome. Similarly, Verma et al. (2013) fished out a novel thermo-alkali-stable xylanase from compost soil metagenome. Faoro et al. (2012) reported isolation of lipolytic enzyme from forest soil (Paraná state, Brazil). From the mountain soil of north-western Himalayas, Sharma and co-workers (2010) reported the recovery of a cold-adapted amylolytic enzyme. Various other enzymes including oxidases, reductase, racemase, lactonase, esterase, glucosidase, etc. have been isolated from soil metagenome as reviewed by Lee and Lee (2013). Along with metagenomics and metatranscriptomics, a more challenging environmental proteomics approach has demonstrated its potential for analysis of protein pool in soil environment (Schneider et al. 2012). In the near future, it may be one of the most powerful approaches in soil enzyme research.

10.6 Importance of Soil Enzymes

Soil enzymes are of immense significance in maintaining ecophysiological life of soil. Enzyme activity ratios have been employed to analyse ecoenzymatic stoichiometry of freshwater sediments and terrestrial soils (Sinsabaugh et al. 2009), along with the studies on the effects of climate and soil properties of different ecosystems (Sinsabaugh et al. 2008; McDaniel et al. 2013). The ratio of activities of extracellular enzymes, which are related to energy and nutrient acquisition, i.e. ratio of β-glucosidase activity/phosphatase activity (an indicator of potential C/P utilization activity), can be utilized to follow the shifts and pattern of energy supply and demand (Sinsabaugh et al. 2008; McDaniel et al. 2013).

By assessing activities of hydrolases, valuable information can be obtained on the status of key reactions involved in the rate-limiting steps of organic matter decomposition along with those of nutrient transformation. Thus, information on the soil degradation potential can be obtained by knowing soil enzyme activities (Trasar-Cepeda et al. 2000). Bolton et al. (1985) proposed that concomitant estimation of different enzyme activities can be used as an effective indicator of soil microbial activity.

There is an exponentially growing interest in finding and developing green technologies for partial or total recovery of sites with polluted soil. Co-occurrence of different types of polluting compounds (both inorganic and organic) makes the remediation of such sites very problematic. Enzymes can be applied to a large array of different compounds, as enzymes with both narrow (chemo-, region- and stereoselectivity) and broad specificity are known, and therefore can be used in a case-specific manner for transformation of innocuous compounds. Enzymes either released by the plants or by soil microorganisms in rhizosphere and the bulk part of the soil are capable of degrading pollutant reaching to the soil. The representative enzymatic classes in the restoration of polluted environments are hydrolases, dehalogenases, oxidoreductases and transferases. Primarily oxidoreductases and hydrolases effectively degrade and transform phenols, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and estrogenic chemicals (Gianfreda and Rao 2004; Gianfreda and Ruggiero 2006). Exploiting the fact that soil enzymes are capable of biodegradation and remediation of xenobiotic compounds, a number of transgenic plants expressing/secreting relevant enzymes have been generated and have been used for restoration of highly polluted soils in various locations (Abhilash et al. 2009). A surge in the release of superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) activities was recorded by the plants when subjected to heavy metal (Hg) pollution of the soil (Li et al. 2013). This observation indicates towards the adaptation of the plant to Hg stress by means of enhanced release of enzymes to deal with the metal stress. Several oxidative enzymes, laccases, catechol dioxygenase, tyrosinase, manganese peroxidase, chloroperoxidase, etc., have been employed in remediation of contaminated soil environments (Duran and Esposito 2000).

10.7 Conclusions

Soil enzymes are of paramount importance for achieving and maintaining physicochemical and biological balance for soil health. Despite several studies, a universal and accurate methodology is still needed to quantify soil enzymes. While substantial progress has been made towards unravelling soil enzymes, applications of enzymes in soil management programmes are still in its infancy. An integrated approach of discovery, quantification and application has to be developed, so that the potential of soil enzymes can be fully utilized for both environment restoration and human welfare.