SOIL ENZYME ESTIMATION

ABSTRACT

Soil enzymes play essential roles in catalyzing reactions necessary for nutrient cycling in the biosphere. The purpose of this study is to estimate soil microbial enzymes activities of collected soil samples using fluorimetric assays. The general concept of the fluorescence enzyme assay is that synthetic Carbon, Nitrogen or Phosphorus rich substrates bound with a fluorescent dye are cleaved from their substrates, which allows them to Florence. Potential Carbon, Nitrogen and Phosphorus enzyme acquisition activities assayed at 0-5cm soil depths did not differ by experimental treatment. Calculating and plotting the sum of all Carbon, Nitrogen and Phosphorus cycling enzyme activities was a useful approach to observe broader patterns regarding their potential cycle. Enzymes activities for total Carbon, Nitrogen and Phosphorus cycling trended lower in the plot exposed to Elevated Heating Condition and CO2  (EHC) compared to the plots exposed to Ambient Climatic Conditions (ACC) at the 5-15cm soil depths. This trend was on significant for total N and P cycling activities (p< 0.046). Potential enzyme C: P and N : P ratios were higher in the plots exposed to Elevated Heating Condition and CO2 (EHC) compared to the plots exposed to Ambient Climatic Condition (ACC) at 5-15cm soil depths (p= 0.05). This observation suggests that there is a relatively higher P mineralization enzyme activity at lower soil depth (5-15cm). For the estimation of soil enzymes activities, Fluorescence-based assays are a useful tool that is widely used to examine potential Enzyme activities among treatment plots.

TABLE OF CONTENTS

Title page                                                                                                                    i

Certification                                                                                                               ii

Dedication                                                                                                                  iii

Acknowledgement                                                                                                      iv

Table of contents                                                                                                        v

List of tables                                                                                                               vi        

List of figures                                                                                                             vii

Abstract                                                                                                                      viii                                                                                          

CHAPTER ONE

1.0       Introduction                                                                                                    1                                                                                             

1.1       Objective of the Study                                                                                    3

CHAPTER TWO

2.0       Literature Review                                                                                           4

2.1       Soil Enzymes                                                                                                  4

2.2       Microorganism as Indicators of Soil Health                                                  5

2.3       Types of Soil Enzymes                                                                                   6

2.4       Origin and State of Soil Enzymes                                                                  9

2.5       Importance of Enzymes to Soil Management                                    9

2.6       Soil Enzyme Activities; A Component of Soil Biodiversity                         11

2.7       Environmental Factors Affecting the Distribution of Soil Enzymes            12

2.8       Sources of Soil Enzymes Activities                                                               15

2.9       Methodological Consideration for Estimating/Measuring Enzyme

Activities in Soils                                                                                           16                                                                                                                               

CHAPTER THREE

3.0       Materials and Methods                                                                                   20

3.1       Materials/Equipment                                                                                      20

3.2       Enzyme Assay Set-up                                                                                     20

3.3       Incubation of Assay Plates                                                                            22

3.4       Florescent Measurements on a Microplate Fluorometer                                23

3.5       Data Analysis                                                                                                  23

CHAPTER FOUR

4.0       Results                                                                                                            26

CHAPTER FIVE

5.0       Discussion, Conclusion and Recommendation                                              37

5.1       Discussion                                                                                                       37

5.2       Conclusion                                                                                                      38

5.3       Recommendation                                                                                           39

REFERENCES                                                                                             40

LIST OF TABLES

Table 1:  Important Classes of Soil Enzymes                                                                        8

Table 2: Roles of Soil Enzymes                                                                                             10

Table 3: Deep well plate design for fluorescence standard concentrations

   with soil samples                                                                                                     28

Table 4: Deep well palate design for soil samples with substrate                                          29

Table 5: Incubator temperatures required for corresponding incubation time periods    29

Table 6: MUB and MUC standard curve calculations                                                           30

Table 7: Enzyme activity calculations                                                                                   31

LIST OF FIGURES

Figure 1:  MUB standard curve plot                                                                                       32

Figure 2: C, N and P cycling enzyme activities                                                                     33

Figure 3: Total C, N and P cycling enzyme stiochiometric ratios                                         34

Figure 4: Enzyme stoichiometry for total C, N and P cycling enzyme activities                     35

CHAPTER ONE

1.0       INTRODUCTION

Enzymes are defined as biological catalysts for specific reactions, which depend on several biotic and abiotic factors such as: pH, temperature, presence or absence of inhibitors, soil organic matter composition, cultivation technique, and other factors, which can directly influence the chemical reactions of these molecules in the soil (Shukla et al., 2006).  Enzymes are released in soils by microbes and plant roots. They can degrade complex substrates into low molecular weight compounds in soils (Schimel and Bennett, 2004).  The chemical and physical processes related to organic matter decomposition may be better understood when accompanied by the activity of several enzymes, not only one, because organic substrates contain a great diversity of organic compounds and ions with active participation in the soil biogeochemical cycles (Caldwell et al., 2005).

The origin of soil enzymes is mainly microbial. They can be found within microbial cells as well as extracellularly in the soil, bound to clay minerals and humic substances.  Extracellular enzymes are important for the breakdown of macromolecules such as celluloses, hemicelluloses, and lignin, while intracellular enzymes are the disrupting agents of smaller molecules, e.g., sugars or amino acids (Dick and Kandeler, 2004).  Microbial enzymes can participate in adsorption, oxidation, reduction, hydrolysis, and complexation reactions, converting organic substances into other products to maintain the balance in the soil environment (Dinesh et al., 2004; Caldwell et al., 2005).

Enzymes can be affected by many factors, biological (microbial populations, fauna, etc.), chemical (pH, organic matter contents, etc.) and, human activities (fire treatment, pesticides, heavy metals, etc.) (Sannino and Gianfreda, 2001; Shen et al., 2005; kicker, 2007), all of which change the biogeochemical cycles in an ecosystem. Therefore, the evaluation of enzyme activity in the soil is very important in order to assess the biochemical function, organic matter fraction, nutrient cycle, and decomposition of xenobiotics.

Enzyme activities are involved in processes important to soil function such as organic matter decomposition and synthesis, nutrient cycling, and decomposition. Enzyme activity stoichiometry has been more recently adopted as an index to assess soil biochemical nutrient cycling by intersecting ecological stoichiometric theory and metabolic theory of ecology to assess potential microbial nutrient imbalances corresponding to environmental conditions (Sinsabaugh et al., 2009). Numerous studies have suggested that wide stoichiometric ratios are indicative of nutrient growth limitations (Fujita et al., 2010) and as soil nutrients become limited, microbes respond by allocating metabolic resources to produce specific enzymes to acquire deficient nutrients (Allison et al., 2007).

The aim of this study is to estimate the activities of soil microbial enzymes in collected soil samples using fluorometric assays. The general concept of the fluorescence enzyme assay is that synthetic Carbon, Nitrogen, or Phosphorus rich substrates bound with a fluorogenic moiety (fluorescent dye) are added to soil samples. When intact, the labeled substrates do not fluoresce. During enzyme-catalyzed substrate degradation, the bond breaks between the fluorescent dye and the substrate. The fluorescent dye liberated from the substrate is consequently used as an indirect estimation of enzyme activity, and can be quantified using a microplate reader to detect the fluorescence intensity of the dye. In brief, fluorescence quantification is accomplished as the liberated dye emits light of one wavelength after absorbing light of a different wavelength.

Enzyme activity can be subsequently quantified based on the known fluorescent-dye concentrations of the substrate (i.e. known quantities of synthetic substrate added to soil samples) along with referencing a standard dilution curve of fluorescence intensities for the specific fluorogenic moiety of the substrate used in the assay (i.e. 4-methylumbelliferone (MUB) or 7-amino-4-methylcoumarin (MUC)).

The two most commonly used synthetic fluorescent indicators are 4-methylumbelliferone (MUB) and 7-amino-4-methylcoumarin (MUC) (Jasinski and Woudenberg, 1994). MUC-linked substrates are commonly associated with N-rich synthetic substrates such as proteins and/or amino acids.

1.1       OBJECTIVES OF THE STUDY

The broad objective of this study is to estimate soil enzyme activities in collected soil samples of different depth, using fluorometric enzyme assays.

The specific objectives of the study are to;

      i.         determine the overall soil enzyme activity in the collected soil sample

     ii.         determine the Carbon, Nitrogen, and Phosphorous enzyme acquisition activities

   iii.         determine the soil Enzyme activities stoichiometry for total Carbon, Nitrogen, and Phosphorus cycling 

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