A survey of soil enzyme dehydrogenase activity in soil environment.

A Survey of Soil Enzyme Dehydrogenase Activity in Soil Environment.

Submitted to Department of Biotechnology Sant Gadge Baba Amravati University, Amravati.

As a part of curriculum activity for the Master's Degree in Biotechnology (2016-2017) Submitted by Ms. Pooja R. Agrawal (M.Sc.-II year)

Under the guidance of Mr. Sudarshan D. Kove Department of Biotechnology Sant Gadge Baba Amravati University, Amravati (M.S).

CERTIFICAT This is to certify that the entitled "A Survey of Soil Enzyme Dehydrogenase Activity in Soil Environment" submitted to the Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati by Ms. Pooja R. Agrawal for partial fulfillment of master's degree in biotechnology for academic year 2016-2017 has been carried out under the guidance of

Mr. S.

D. Kove, Assistant Professor, Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati.

Date :

Prof. M. K. Rai (Head of Dept. of Biotechnology)

Place :Amravati

Department of Biotechnology Sant Gadge Baba Amravati University Amravati- 444602

CERTIFICATE

This is to certify that the entitled "A Survey of Soil Enzyme Dehydrogenase Activity in Soil Environment " submitted to the Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati in partial fulfillment of master's degree in Biotechnology, is record bona fide work carried out by Ms. Pooja R. Agrawal. She have worked under my guidance and fulfilled the entire requirement for the project work during year 2016-2017.

Date: Place: Amravati

Mr. S. D. Kove Assistant Professor Department of Biotechnology.

Sant Gadge Baba Amravati University.

Amravati- 444602

DECLARATION

I hereby declared the dissertation entitled "A Survey of Soil Enzyme Dehydrogenase Activity in Soil Environment." submitted by Ms. Pooja R. Agrawal for partial fulfillment of Master's degree in Biotechnology for academic year 2016-2017 is done under the guidance of Mr. S. D. Kove, Assistant Professor, Department of Biotechnology, Sant Gadge Baba Amravati University , Amravati.

I further declare that this dissertation work or any part of this has not been previously submitted for any degree in any other university.

Date: Place: Amravati

Submitted by Ms. Pooja R. Agrawal.

ACKNOWLEDGEMENT Any work is not done single handedly, it is done by the cooperation of many hands. It is a collective effort. Words cannot explain the feelings of gratitude and respect I have for Mr. S. D. Kove, Assistant Professor, Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati. Whose encouraging guidance and valuable suggestions have contributed largely in formulation and successful completion of the project. I consider myself extremely fortunate to work under his supervision. My deepest thanks to him for taking keen interest in my work and guiding me by adding valuable inputs to improve my work.

I am highly indebted to Prof. M. K. Rai, Head of the Department of Biotechnology, SGB Amravati University, Amravati for his kind and helpful suggestions.

I express my sincere thanks to Prof. M. K. Rai, Dr. P. A. Wadegaonkar, Dr. P. V. Thakre, Dr. (Mrs.) A. S. Patil, Dr. A. K. Gade and Dr. V. P. Wadegaonkar for their guidance and words of blessing.

I acknowledge my sincere thanks to Ms. Rubina Sheik, Mr. Gaurav Ajane and Mr. Roshan Godse. I am also thankful to all my colleagues for their co- operation and constant encouragement. I also want to thank all members of non- teaching staff for their support and suggestions.

Finally, I give lot of thanks to my parents for their moral support. I wish to thank all those who have helped me directly or indirectly in completion of this work.

(Ms. Pooja R. Agrawal.)

CONTENT

Sr. No.

Chapter

1).

Introduction

2).

Review of Literature

3).

Materials and Methods

4).

Results and Discussion

5).

Conclusion

6).

Reference

Page No.

INTRODUCTION

INTRODUCTION

INTRODUCTION The quality and fertility of soils play an important role in the sustainable development of the terrestrial ecosystem. Soil quality has been defined as ‘the continued capacity of soil to function as a vital living system, within ecosystem and land use boundaries, to sustain biological productivity, promote the quality of air and water environments and maintain plant, animal and human health. Soil fertility is an integral part of soil quality that focuses more on the productivity of the soil, which is a measure of the soil’s ability to produce a particular crop under a specific management system. All productive soils are fertile for the plant being grown, but many fertile soils are unproductive because they are subjected to same unbeneficial natural factors (e.g., drought) or management practices.

Interest in evaluating the quality and health of our soil resources has been stimulated by increasing awareness that soil is a critically important component of the earth’s biosphere, functioning not only in the production of food and fiber but also in the maintenance of local, regional, and global environmental quality. Soil is also the basis of agricultural and of natural plant communities. Thus, the thin layer of soil covering the surface of the earth represents the difference between survival and extinction for most land-based life. (Doran and Zeiss, 2000).

Enzymes are the vital activators in life processes, likewise in the soil they are known to play a substantial role in maintaining soil health and its environment. The enzymatic activity in the soil is mainly of microbial origin, being derived from intracellular, cell-associated or free enzymes. A unique balance of chemical, physical, and biological (including microbial especially enzyme activities) components contribute to maintaining soil health. Evaluation of soil health therefore requires indicators of all these components (Ellert et al., 1997). Reactions in the environment involve chemical, biochemical, and physical processes. It is well known that most biochemical reactions are catalyzed by enzymes, which are proteins with catalytic properties.

Catalysts are substances that, without undergoing permanent alteration, cause chemical reaction to proceed at faster rates. In addition, enzymes are specific for the type of chemical reactions in which they participate.

All living systems, ranging from bacteria to the animal kingdom, from algae and molds to the higher plants, contain a vast number of enzymes catalyzing both simple and complex networks of chemical reactions. Enzymes also are found in ponds, lakes, rivers, water treatment plants, animal manures, and soils and exist either as extracellular forms separated from their origins or as intracellular forms as part of the living biomass. These enzymes are involved in the synthesis of proteins, carbohydrates, nucleic acids, and other components of living systems and also in the degradation and essential cycling of carbon, nitrogen, phosphorus, sulfur, and other nutrients. (Tabatabai et al., 2002).

The role of soil enzymes and their activities are defined by their relationships with soil and other environmental factors (e.g., acid rain, heavy metals, pesticides, and other industrial chemicals) that affect their activities (Burns, 1982; Hussain et al., 2009). Soil enzymes are the mediators and catalysts of important soil functions that include: decomposition of organic inputs; transformation of native soil organic matter; release of inorganic nutrients for plant growth; N2 fixation; nitrification; denitrification; and detoxification of xenobiotics (Dick, et al., 1997). In addition, soil enzymes have a crucial role in C (β-glucosidase and β-galactosidase), N (urease), P (phosphatase), and S (sulphatase) cycle (Karaca et al., 2011). Soil enzymology is nowadays of practical importance because the influence of agro-chemicals, industrial waste, heavy metals, as well as soil fertility management can be measured.

Soil Enzymes:

Soil enzymes are a group of enzymes whose usual inhabitants are the soil and are continuously playing an important role in maintaining soil ecology, physical and chemical properties, fertility, and soil health. These enzymes play key biochemical functions in the overall process of organic matter decomposition in the soil system (Sinsabaugh et al., 1991). Enzymes are the direct mediators for biological catabolism of soil organic and mineral components. Thus, these catalysts provide a meaningful assessment of reaction rates for important soil processes.

Soil enzyme activities are often closely related to soil organic matter, soil physical properties and microbial activity or biomass, changes much sooner than other parameters, thus providing early indications of changes in soil health, and involve simple procedures (Dick et al.,

1996). In addition, soil enzyme activities can be used as measures of microbial activity, soil productivity, and inhibiting effects of pollutants (Tate, 1995). Easy, well-documented assays are available for a large number of soil enzyme activities (Dick et al., 1996; Tabatabai, 1994).

Urease:

Urease enzymes can exist in two possible states in soil. They may be intracellular, that is present within the cells of micro-organisms, or alternatively, extracellular, having been released from disrupted plant and microbial cell. (Lloyd and Shaffer, 1973). Urease is an enzyme that catalyses the hydrolysis of urea into CO2 and NH3 with a reaction mechanism based on the formation of carbamate as an intermediate (Tabatabai, 1982).

H2NCONH2 + H2O

2NH3 + CO2

Since urease plays a vital role in the hydrolysis of urea fertilizer, it is important to uncover other unknown factors that may reduce the efficiency of this enzyme in the ecosystem.

Generally, urease activity increases with increasing temperature. Consequently the understanding of urease activity should provide better ways to manage urea fertilizer, especially in warm high rainfall areas, flooded soils and irrigated conditions (Makoi and Ndakidemi, 2008).

Invertase:

Invertase (beta-D-fructofuranoside fructohydrolase, EC [Enzyme Commission] 3.2. 1 .26) is the enzyme that catalyzes the hydrolysis of sucrose and yields glucose and fructose.

C12H22O11 + H2O Sucrose

C6H12O6 + C6H12O glucose

fructose

The activity of this enzyme was monitored by systematically developing a sensitive and rapid method to detect reducing sugars with the precision of 1.4 to 6.1% C.V. The method involves the colorimetric determination of reducing sugars which react with 3, 5-dinitrosalicylic

acid when soil is incubated with buffered sucrose solution and toluene at 37°C for 24 h. The detection limit for the method described is 100 microgram of reducing sugar per ml of soil extract. The color intensity remained constant up to 24 hours. (Frankenberger and Johanson,1983).

Dehydrogenase:

The dehydrogenase enzyme activity is commonly used as an indicator of biological activity in soils (Burns, 1978). This enzyme is considered to exist as an integral part of intact cells but does not accumulate extracellularly in the soil. Dehydrogenase enzyme is known to oxidize soil organic matter by transferring protons and electrons from substrates to acceptors.

These processes are the part of respiration pathways of soil microorganisms and are closely related to the type of soil and soil air-water conditions (Kandeler 1996; Glinski and Stepniewski 1985). Since these processes are the part of respiration pathways of soil microorganisms, studies on the activities of dehydrogenase enzyme in the soil is very important as it may give indications of the potential of the soil to support biochemical processes which are essential for maintaining soil fertility as well as soil health.

They may serve as indicators of the microbiological redox systems in soils and can be considered a possible measure of microbial oxidative activity (Tabatabai 1982; Trevor's 1984).

Additionally, dehydrogenase enzyme is often used as a measure of any disruption caused by pesticides, trace elements or management practices to the soil (Reddy and Faza 1989; Wilkes 1991; Frank and Malkomes 1993), as well as a direct measure of soil microbial activity (Trevor's 1984; Garcia and Herna´ndez 1997). When pesticides are applied at recommended field rates, short-term studies often show an initial stimulatory, but small, effect on dehydrogenase activity: this may or may not occur with other enzymes (Dick, 1997).

Soil enzyme as bioindicator of soil health:

Soil enzymes have been reported as useful soil quality biological indicators due to their relationship to soil biology, being operationally practical, sensitive, integrative, ease to measure and described as "biological fingerprints" of past soil management, and relate to soil tillage and

structure (Bandick and Dick, 1999). Easy and inexpensive to measure because, the ultimate determinant of soil quality and health is the land manager, indicators of soil quality and sustainability should be both accessible to them and economic in terms of both time and money.

(Doran and Zeiss, 2000). Pesticides, which include herbicides, fungicides, and insecticides etc., introduced into the environment, have potential to affect non-target organisms and soil biochemical processes. (Das and Varma, 2010). Pesticides reaching the soil may disturb local metabolism or enzymatic activities (Engle et al., 1998; Liu et al., 2008).

Soil Enzymes as Bioindicators of Change in Agricultural Practices:

Fertilization of soils is conducted in soils by using different fertilizers such as mineral, manure, green manure, compost, and vermicompost. (Kandeler et al., 1999) showed that farmyard manure enhanced microbial biomass, urease, deaminase and alkaline phophatase activities in soils compared with other treatments (mineral fertilizers) under rotations. Also, the type of mineral fertilizer used influence soil enzyme activity depending on the soil enzymes involved, in which there is, nutrient cycling (N, P, C, and S). Soil enzyme activities were inhibited with N fertilizer while they were promoted by P and K fertilizers. A decrease of urease activity could be explained by the activation of nitrification and denitrification causing suppression in urease production (Aon et al., 2001).

Organic fertilizers are used in agricultural systems, especially organic farming. Compost application is important in establishing and maintaining soil organic matter to a certain level in organic farming. Chang et al., (2007) found that soil enzyme activities (dehydrogenase, cellulase, protease, arylsulphatase, β-glucosidase, urease, arylsulphatase, and acid and alkaline phosphatases), as well as other microbial properties increased significantly in compost-treated soil compared with chemical-fertilizer soils. Saha et al., (2008) observed that dehydrogenase activity is higher in composted cattle manure (44-200%) and vermi-compost (22-108%) than in control. They concluded that: (I) organic applications, enhanced organic matter contents and microbial biomass and thus provide better potential for higher enzyme production and greater enzyme activities. (ii) additions of organic amendments showed different responses on soil enzyme activities depending on the organic matter type, and (iii) addition of organic amendments

(cattle manure, compost or vermicompost) improve soil quality, increase soil organic matter content and stimulate biological and biochemical properties (Saha et al., 2008).

Soil Enzymes as Bioindicators of Xenobiotic Pollution:

Xenobiotics are by definition unnatural compounds (e.g. pesticides, industrial wastes) but the wider definition include naturally occurring compounds (e.g. heavy metals) that are synthesized or are present in unnaturally high concentrations in the environment (Skladany et al., 1993). Such compounds are of crucial concern in the soil environment as they could affect many biological and biochemical reactions in soils (Dick, 1997). Pesticides, which include herbicides, fungicides, and insecticides etc., introduced into the environment, have potential to affect nontarget organisms and soil biochemical processes. The negative impact of pesticides on soil enzymes (hydrolases, oxidoreductases, and dehydrogenase) activities has been widely reported in the literature (Ismail et al., 1998; Monkiedje et al., 2002; Menon et al., 2005). There is also evidence that soil enzyme activities and ATP contents are increased by some pesticides. (Shukla, 1997; Megara et al., 1999).

In relation to the effects of pesticides in soils, the two most widely-studied enzymes other than deydrogenase are phosphatase and urease. Again, short-term studies involving applications of pesticides to soils at recommended dosages for periods ranging from a few days up to 8 weeks have shown slight increases or no significant effect on the activity of these two enzymes (Baruah and Mishra, 1986; Tu 1993).

Importance of Dehydrogenase Enzymes Activity in Soil:

Dehydrogenase enzyme is often used as a measure of any disruption caused by pesticides, trace elements or management practices to the soil, as well as a direct measure of soil microbial activity. It can also indicate the type and significance of pollution in soils. It has been found that dehydrogenase enzyme is high in soils polluted with pulp and paper mill effluents ,but low in soils polluted with fly ash . Similarly, higher activities of dehydrogenases have been reported at low doses of pesticides and, lower activities of the enzyme at higher doses of pesticides. As most areas of the world are often polluted by different industrial bio-chemical products, better

understanding of the role of this enzyme in environmental science will open greater possibilities of using it as a diagnostic tool for better ecosystem assessment and amelioration.

Importance of Urease Enzymes Activity in Soil: Urease enzyme is responsible for the hydrolysis of urea fertilizers applied to the soil into NH3 and CO2 with the concomitant rise in soil pH (Andrews et al. 1989; Byrnes and Amberger 1989). This, in turn, results in a rapid N loss to the atmosphere through ammonia volatilization (Simpson et al. 1984; Simpson and Frenzy 1988). Due to this role, urease activities in soils have received a lot of attention since it was first reported by Rotini (in 1935), a process considered vital in the regulation of N supply to plants after urea fertilization. For example, studies have shown that urease was very sensitive to toxic concentrations of heavy metals (Yang et al. 2006).

Generally, urease activity increases with increasing temperature. It is suggested that higher temperatures increase the activity coefficient of this enzyme. Therefore, it is recommended that urea be applied at times of the day when temperatures are low. Since urease plays a vital role in the hydrolysis of urea fertilizer, it is important to uncover other unknown factors that may reduce the efficiency of this enzyme in the ecosystem.

Enzyme activity of soil is under the influence of many factors such as soil structure, plant variety, water quantity, and climatic conditions. However, the relationship between the aforementioned factors and enzyme activity is not revealed thoroughly as the studies regarding soil enzymes are not adequate. Detailed studies in the future should dwell on the relationships between enzyme types and functions, enzymatic activities, and soil fertility as well as basic subjects such as the influence of climatic and edaphic factors on enzyme activity. In addition, the determination of the influence of culture maintenance operations such as fertilization, weeding, and irrigation on enzymatic activities is important for better definitions of soil enzymes, and their use to increase soil fertility.

Importance of enzymes present in soil environment: 1. The soil enzymes are very important as they release the nutrients into the soil by degrading the organic matter degradation present in the soil.

2. They help in the identification of soils, whether the soil is polluted by heavy metals or ecessive pesticides.

3. Large number of diverse micro organisms are present in the soil. Microorganisms are very important because, they degrade the organic matter in soil and provide nutrient to plants. If, micro organisms are healthy in soil then, soil is healthy. (Identification of microbial activity) 4. Importance of soil enzymes as sensitive indicators of ecological change

Application of Soil Enzymes: ❖ Correlation with soil fertility ❖ Correlation with microbial activity ❖ Correlation with biochemical cycling of various elements in soil (C, N, S) ❖ Degree of pollution (heavy metals, SO4) ❖ To assess the successional stages of an ecosystem ❖ Forensic purposes ❖ Rapid degradation of pesticides ❖ Disease studies ❖ Enzyme activity in soil fluctuates with environment.

It is very essential to understand the possible roles of soil enzymes in order to maintain soil health and its fertility management in ecosystems. These enzymes, usually found in the soil, may have significant effects on soil biology, environmental management, growth and nutrient uptake in plants growing in ecosystems. Their activities may, however, be influenced by unknown cultural management practices either in a major or minor amount. Studies focusing the discovery of new enzymes from microbial diversity in the soil might be the most suitable practices that may positively influence their activities for improved plant growth as well as rendering the friendly biological environments in order to sustain other living beings.

REVIEW OF LITERATURE

REVIEW OF LITERATURE A variety of methods were developed to measure soil biological activity. All these methods are not suited to produce generally accepted results, but they give relative information about the ecological status of soil. Soil enzymatic activity assays is only one way to measure the ecosystem status of soils. The technique is quite simple and produces reproducible results, and is nowadays of practical importance because the influence of agro-chemicals, industrial waste, heavy metals, as well as soil fertility management can be measured. Especially the search for urease inhibitor is of particular interest in order to reduce ammonia losses from soils. Soil enzymes have been reported as useful soil quality indicators due to their relationship to soil biology, being operationally practical, sensitive, integrative, ease to measure and described as "biological fingerprints" of past soil management, and relate to soil tillage and structure. The focus of this article is to provide a review of soil enzyme activity as a biological, process-level indicator for impacts of natural and anthropogenic activities on soils. This knowledge of soil enzymology can be applicable as bioindicator to human Endeavour of ecosystem perturbation, agricultural practices and xenobiotic pollution. (Ubtobo and Teary, 2015).

Bacteria which can hydrolyse urea are common in soils. Llyod et al., have worked on the urease enzyme activity in soil. Of six soils mined, some 17-30 per cent of the total bacterial populations, including bees, micro-aerophiles and anaerobes, could hydrolyse urea. One of the had been enriched with urea for at least ten years, yet the proportion ureolytic bacteria (24 per cent) was similar to that of normal soils. Addition of urea to a red-yellow podzolic soil low in available carbon under different moisture conditions did not increase the total urease activity, the size of the bacterial population or the ratio of ureolytic to non-ureolytic bacteria. However, when available carbon as glucose was added with urea this soil, urease activity and size of the bacterial population both increased, but the ratio of ureolytic to non-ureolytic bacteria in the population remained unchanged. (Lloyd and Shaffer, 1973) These include cropping history, organic matter content of the soil, soil depth, soil amendments, heavy metals, and environmental factors such as temperatures (Tabatabai 1977; Yang et al. 2006).

Frankenberger et al., have observed inhibition of soil urease when sewage sledges were applied at the lower loading rates. Such an inhibition has been attributed to the extremely high concentration of heavy metals present in the sledges. When greater amounts of sewage sledges were applied urease activity was enhanced substantially.

Soil dehydrogenase activity determination in soil was first initiated by Leonhard in 1956, since then it has widely been used because of its simplicity as compared to other quantitative methods. Dehydrogenase assay in based on the reduction of 2, 3, 5- triphenyl tetrazolium chloride (TTC) to the creaming red-colored Formosan (TPF), have been used to determine microbial activity in soil. Different biotic and biotic factors such as incubation time and temperature, pre-incubation, soil aeration and moisture content have significant effect on dehydrogenase activity in soil. Highest dehydrogenase activity is reported from forest soil in autumn seasons while the disturbed soil from coal mines soils containing lowest dehydrogenase activities along the soil erosion gradient of experimental slopes. Least value of enzymes activity is reported from polluted sites than restored and undisturbed sites. Dehydrogenase enzyme is often used as a measure of any disruption caused by pesticides, trace elements or management practices to the soil, as well as a direct measure of soil microbial activity. (Kumar et.al., 2013)

Casida et al., have worked on the dehydrogenase enzyme activity in soil. The objective of the study was to revise the TTC dehydrogenase technique so that many samples of quite varying soil could be easily be examined and so, that formazan production by a given soil sample could be more reproducible. (Casida et al., 1964).

Soil enzyme activities are very sensitive to both natural and anthropogenic disturbances and show a quick response to the induced changes. Soil dehydrogenase enzymes are one of the main components of soil enzymatic activities participating in and assuring the correct sequence of all the biochemical routes in soil biogeochemical cycles. Dehydrogenase activity is measured by two methods using the TTC and INT substrate; however, various authors reported poor results when TTC is used as substrate. Different biotic and biotic factors such as incubation time and temperature, pre-incubation, soil aeration and moisture content have significant effect on dehydrogenase activity in soil. Highest dehydrogenase activity is reported from forest soil in

autumn seasons while the disturbed soil from coal mines soils containing lowest dehydrogenase activities along the soil erosion gradient of experimental slopes. Least value of enzymes activity is reported from polluted sites than restored and undisturbed sites. Dehydrogenase enzyme is often used as a measure of any disruption caused by pesticides, trace elements or management practices to the soil, as well as a direct measure of soil microbial activity.

Frankenberger et al., have worked on the invertase enzyme activity in soil. A total of 19 California surface soil samples, were selected to obtain a wide range in pH (5.91 to 8.55), organic C (0.28 to 2.61%), total N (0.048 to 0.240%), and texture (16 to 64% clay and 2 to 76% sand) were used. The samples were sieved (2-mm screen) in the field-moist condition, and subsamples of nine of the sieved soils were subjected to air-drying. The soils were air-dried by spreading subsamples of sieved field-moist soils in a thin ( < 2-cm) layer on clean paper and left to dry at laboratory temperature (23°C) for 2 days. The profile samples selected in this study had a wide range in organic C and invertase activity. Before use, each sample was air-dried and crushed to pass a 2-mm screen. A subsample of each soil was ground (<100 mesh) for determination of organic C and total N. The chemical and physical properties of the soils used were determined as described by Frankenberger and the method for determination of invertase enzyme activity in soil is colorimetric determination of reducing sugars which react with 3, 5dinitrosalicylic acid when soil is incubated with buffered sucrose solution and toluene at 37°C for 24 h. The color intensity remained constant up to 24 h. The activity of this soil enzyme was measured by the release and quantitative determination of reducing sugars in the reaction mixture when soil samples were incubated with sucrose in the presence of toluene and modified universal buffer (0.17M, pH 5.0). The method developed employs the use of 3, 5-dinitrosalicylic acid as a color reagent which is reduced in the presence of reducing sugars to 3-amino-5nitrosalicylic acid. (Frankenberger and Johansson, 1983)

Junli Hu et al., have worked on the invertase enzyme in sandy loam soil. The objective of this study was to investigate the effects of long term fertilization regimes on soil microbial community functional diversity, metabolic activity, and metabolic quotient and to find out the main factors that influence these parameters. Top soil samples (0â&#x20AC;&#x201C;15 cm) from four individual plots per treatment were collected for the analysis of chemical properties and microbial

parameters. Invertase activity and basal respiration were determined based on incubation method. Microbial biomass C was analyzed using the fumigationâ&#x20AC;&#x201C;extraction method. Invertase activity and basal respiration were determined based on incubation method. Then, the microbial metabolic quotient was calculated as the ratio of basal respiration to microbial biomass C. To this end, microbial functional diversity was evaluated using the community level physiological profile method by Biology Eco-microplate. They found that both the sole-carbon-source utilization activity and the functional diversity of soil microbial community were significantly increased.

The implementation of increasingly stringent standards for the discharge of wastes into the environment has necessitated the need for the development of alternative waste treatment processes. A review of research directed toward developing enzymatic treatment systems for solid, liquid and hazardous waste is presented. A large number of enzymes from a variety of different plants and microorganisms have been reported to play an important role in an array of waste treatment applications. Enzymes can act on specific recalcitrant pollutants to remove them by precipitation or transformation to other products. They also can change the characteristics of a given waste to render it more amenable to treatment or aid in converting waste material to valueadded products. Before the full potential of enzymes may be realized, it is recommended that a number of issues be addressed in future research endeavors including the identification and characterization of reaction by-products, the disposal of reaction products and reduction of the cost of enzymatic treatment. (Karma and James, 1997)

Recent studies have revealed that soil enzymes influence soil fertility and plant growth to a great extent. The findings obtained from soil enzyme measurements are evaluated periodically as an indicator of soil fertility (Kiss et al., 1978). Soil enzyme activities have good potential to determine the health of living elements of soil, and total biological activity of soils. Soil enzyme trials may offer information regarding the soil potential in terms of conducting certain biochemical processes.

MATERIAL AND METHODS

MATERIALS AND METHODS 1. Selection and collection of soil sample from SGBAU campus : Soil samples were collected from various location (Lake side soil; lake side garden; department of Zoology garden; department of Biotechnology garden- front side; department of Biotechnology garden- back side; University main garden) of University during, the month of April. Wet soil sample was taken from University. The soil sample was taken at depth of 15 cm by removing upper layer of soil. The sample bottles (Himedia) were closed immediately after taking sample and bottles were labeled properly and the soil samples were stored in refrigerator.

2. To study the biophysical properties of soil of samples: Only selected soil sample was taken for to check the biophysical properties (pH, Temperature, soil colour and texture of soil etc.).

i). pH: Soil pH is a measure of the acidity and alkalinity in soils. The effect of soil pH is great on the solubility of nutrients or minerals. Before the nutrient is utilized by plants it must be dissolve in soil solution. Most of the minerals or nutrients are soluble or available in acidic soils.

Method: The pH of the selected soil sample was detected by using pH strip. Following steps was used for check the pH: • 0.5 gm of soil was weighed.

• In clean test tubes 5 ml of auto-calved distilled water was taken.

• Soil was added to test tubes with auto-calved distilled water and was mixed well.

• It was allowed to stand for 5 - 10 min.

• Then, the pH strip was immersed in the soil suspension and pH of the soil was noted.

ii). Temperature: Temperature is the critical factor as well as important which, affects the activity of enzyme in soil.

Method: The temperature was measured by using thermometer.

•

The thermometer was cleaned by using 70% alcohol.

•

Then, the thermometer was directly inserted in the soil sample to measure the temperature of soil.

•

Lastly, the temperatures of each soil sample were noted.

iii). Soil colour: Colour can be a useful indicator of some of the general properties of a soil, as well as some of the chemical processes that are occurring beneath the surface. The color of the soil sample was blackish brown.

iv). Texture of soil: It is the relative content or percent of sand, silt and clay present in the soil. These particles differ amongst each other in terms of their size. Particle that range in between 0.05-2mm are sand. Particles that follows between 0.002-0.05mm are silt. The smallest particles are clay, which are less than 0.002mm in size.

Method: The bottle test- This is the simple test which give the information about the proportion of sand, silt and clay present in the soil.

•

1 gm of soil was taken in clean and sterile test tube.

•

Then, 10 ml of auto calved distilled water was added to the soil sample. The mouth of test tube was covered by paraffin wax.

•

The soil and distilled water was mixed properly by shaking.

•

It was allowed to stand undisturbed for overnight.

•

Next day the layers of clay, silt and sand was measured by ruler.

•

The results were noted down in notebook.

•

The percent of clay, silt and sand was calculated.

3. To study the chemical properties of soil samples: Soil chemistry is the interaction of various chemical constituents that takes place among soil particles and in the soil solution—the water retained by soil. The chemical interactions that occur in soil are highly complex, but understanding certain basic concepts will better help you manage turf and ornamentals. In this chemical properties we determine the Organic carbon content only.

i). Determination of Organic carbon content in selected soil sample: Soil organic matter (OM) has important effects not only on soil enzymes activities but also on microorganisms activities. Soil OM has been considered as an indicator of soil quality (similarly like dehydrogenases,) because of its character of nutrient sink and source that can enhance soil physical and chemical properties, and also promote biological activity Interestingly, not only amount of OM in the soil is important but most of all its quality, as OM affects the supply of energy for microbial growth and enzyme production.

Organic carbon is a measure of soil organic matter (SOM), which impacts on soil fertility, and nutrient cycling, soil structure, buffering and sorption capacity, biological diversity and water retention. Additionally organic matter affects mobility of pollutants. Measurements of soil organic carbon (SOC) are important for monitoring of soil quality, impact of agriculture on SOC levels and trends in carbon sequestration in or release from soil. The determination of soil organic carbon is based on the Walleye-Black chromic acid method.

Material :

1 N Potassium Dichromate: Dissolve 49.040 g K2Cr2O7 AR (dried at 105 °C) in distilled water, transfer to a 1 L volumetric flask and make to volume with distilled water.

Sulphuric Acid 98%: This should be used fresh from the bottle and not left standing in a burette or beaker, as it rapidly picks up moisture from the air. It is satisfactory until the strength falls to <96%.

0.4 N Ferrous Sulphate: Dissolve 112 g FeSO4.7H2O in 800 mL distilled water containing 15 mL concentrated H2SO4. Dilute to 1 L with distilled water and store in a dark bottle.

Method: •

0.5 gm of soil was weighed and taken into a dry 250 ml of conical flask.

•

5 ml of 1 N potassium dichromate was added to the conical containing soil and flask was swirl to mix.

•

Then, 10 ml of sulphuric acid was added to it and mixed.

•

Flask was allowed to stand for 30 min. in room temperature.

•

50 ml of distilled water was added to it.

•

5 ml of 85 % phosphoric acid was added to it and mixed well.

•

Then, acetaldehyde was added as an indicator and it was titrated against 0.5 N of ferrous ammonium sulphate, till the color changes from violet through blue to green.

Calculation: The following formula was used to calculate the Organic Carbon content.

Percent of carbon content in soil = 0.003 x N x (V1 - V2) x 100 x 10 W

Where, W - Weight of sample ; V1 - Blank titer value ; V2 - Sample titer value ; N - Normality of K2Cr2O7.

Urease enzyme assay: Urease is the common name for enzymes that decompose urea into carbon dioxide and ammonium. The enzyme is considered important in soil nitrogen cycling, particularly in agricultural systems.

Material: 40 mM urea: 0.240 g urea 100 ml sodium acetate buffer, pH 5 0.3 M NaOH 12 g NaOH 1000 ml DI water Na salicylate solution: 17 g sodium salicylate 120 mg sodium nitroprusside 100 ml DI water Na salicylate/NaOH solution (make fresh daily) 1 part 0.3 M NaOH 1 part Na salicylate solution 1 part DI water Na dichloroisocyanide solution (make fresh daily) 0.1 g sodium dichloroisocyanide 100 ml DI water Method:

0.5 gm of wet soil was taken inn sterile conical flask. The samples were incubated with 0.1 M of citrate buffer for 30 min at 37˚C. Then, 1 ml of 10 % of urea solution was added to each flask containing sample and mixed well by shaking. Again, the flask was incubated for 3 hours at 37˚ C. After incubation 10 ml of 2M of KCl was added to it and was mixed in rotary shaker for 30 minutes. The mixture was then filtered and filtrate was collected in clean conical flask. 1 ml of the filtrate was added to 9 ml of distilled water and, successively, 5 ml phenol- nitroprussiate (5g of phenol and 25 mg of sodium nitroprussiate in 500 ml of distilled water and 5 ml of alkaline hypochlorite (2.5 g of NaOH and 4.2 ml of sodium hypochlorite containing 5% of active Cl2 were dissolved in distilled water and the volume brought to 500 ml were added. It was mixed properly and was allowed to stand for 30 minutes. Then, the absorbance was taken at 690 nm.

Invertase enzyme assay: Invertase assay detects the invertase enzyme in the soil.

Material:

Toluene.

Sucrose solution (10%) (0.29 M) Add 10 g of sucrose into a 100-ml volumetric flask. The volume by adding distilled water, and was mixed.

Sodium hydroxide (2 M) Dissolve 8.0 g of NaOH in 100 ml of water.

Color reagent: Prepare this reagent by adding 0.2 g of 3,5-dinitrosalicylic acid monohydrate ,0.025 g of sodium carbonate, and 0.005 g of EDTA (ethylenedinitrilo)-tetraacetic acid disodium salt to a 50-ml volumetric flask and the mixture was diluted with water.

Method: 0.5 gm of soil was taken in clean conical flask.0.2 ml of toluene and 5 ml of modified universal buffer was added to it. Then, 5 ml of 10 % sucrose was added and swirled. Then stopper the flask and place it in an incubator at 37°C for 24 hours. After incubation, remove the stopper and filter the contents through Whatman's No. 42 filter. Then treat the reaction mixture with 5 ml of deionized water, 2 ml of 2M NaOH, and 2 ml of the color reagent.( 0.2 g of 3,5-

dinitrosalicylic acid monohydrate ,0.025 g of sodium carbonate , and 0.005 g of EDTA (ethylenedinitrilo)-tetraacetic acid disodium salt to a 50-ml volumetric flask and dilute the mixture with water. Then, a stream of nitrogen gas was passed through the reaction mixture for 10 minutes. With inverted 50-ml beakers on top of each test tube, place all of the tubes into a boiling water bath for 5 min and then allow them to cool to room temperature. Measure the color intensity (O.D.) with a spectrophotometer at a wavelength of 540 nm.

4.Determination of dehydrogenase enzyme activity in soil sample: Soil enzyme activities are very sensitive to both natural and anthropogenic disturbances and show a quick response to the induced changes. Soil dehydrogenase enzymes are one of the main components of soil enzymatic activities participating in and assuring the correct sequence of all the biochemical routes in soil biogeochemical cycles. Dehydrogenase activity is measured by methods using the TTC/MTT substrate; Different biotic and abiotic factors such as incubation time and temperature, pre-incubation, soil aeration and moisture content have significant effect on dehydrogenase activity in soil. Dehydrogenase enzyme is often used as a measure of any disruption caused by pesticides, trace elements or management practices to the soil, as well as a direct measure of soil microbial activity.

All selected soil sample was used to dermine the dehydrogenase enzyme activity.

Dehydrogenase assay: The method allows to determine the effect of exogenous organic matter on activity of soil dehydrogenases. Soil dehydrogenase activity (DHA) is the result of the activity of different dehydrogenase enzymes involved in the oxidative metabolism of viable microbial cells. Since dehydrogenases occur in soil in all living microbial cells, the DHA test is used as an indicator of the overall soil microbial activity, or a measure of the general soil metabolic activity.

Instruments and equipment: • Thermostatic incubator (30°C) • Microplate reader / Spectrophotometer (wavelength of λ=485 nm)

Chemicals, solutions: • 0.1 M Tris-HCl buffer, pH 7.4 • 1% solution of 2, 3, 5-triphenyltetrazolium chloride (TTC/MTT) in 0.1 M Tris-HCl buffer.

• Methanol Method: 0.5 gm of moist soil was weighed and was taken in well of 24 micro plate. 1ml of 1 % TTC (triphenyl tetrazolium chloride salt)/MTT was added to each well with soil sample and mixed. The plates with suspension was incubated at 37 ˚C for 96 hours in dark. After, incubation 2 ml of methanol was added to each well and mixed. The control consist of 3 ml of Tris buffer without soil sample. The microplates were centrifuged at 4000 rpm for 2 minutes. Then, supernatant was taken into sterile micro plate and was read in micro plate reader at absorbance 485 nm.

RESULT AND DISCUSSION RESULT AND DISCUSSION

RESULT AND DISCUSSION

RESULT AND DISCUSSION 1). Selection and collection of soil samples from SGBAU campus: Total 6 soil samples were collected from different regions of Sant Gadge Baba Amravati University. The pH, organic carbon content, soil texture, soil color and temperature of collected soil samples were determined. It is very important to check the physical and chemical properties of soil, as the enzyme activity depends on it. The pH of the six soil sample collected was in the range of 6- 6.8.

Samples were collected from various regions of Sant Gadge Baba Amravati University, Amravati.

4

1 3

6 5

2

Figure no.1: University map showing the location of soil sample collected.

Soil sample

Sample Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 1 Regions/Site Lake Department University University Department of Department of area side soil of Zoology Lake side main Biotechnology Biotechnology garden. garden garden garden back garden front side side Table no. 1: Showing the sampling site, where the soil samples were collected.

Figure no. 2. : Sample bottles containing soil sample collected from University Campus.

2). To study the Biophysical properties of soil sample: The pH, soil texture, soil colour and temperature of collected soil samples were determined.

i). pH: Soil pH is a measure of the acidity or basicity of a soil. In soils, it is measured in a slurry of soil mixed with water (or a salt solution), and normally falls between 3 and 10, with 7 being neutral. Acid soils have a pH below 7 and alkaline soils have a pH above 7. Ultra-acidic soils (pH<3.5) and very strongly alkaline soils (pH>9) are rare.

Soil sample

Sample 1

pH

6

Sample 2

6

Sample 3

6.5

Sample 4

Sample 5

7

6

Sample 6

6

Table no.2: Showing the pH of soil samples.

The variation in the pH of the soil may be occurring due to the presence of minerals in soil.

Graph no. 1: The graph showing variation in pH vs. soil samples.

ii). Temperature: Soil temperature is the measure of how hot or cold the soil is. If you want to get more technical, itâ&#x20AC;&#x2122;s the detection of the internal energy of the soil, or its heat. Soil scientists consider temperature a very important soil physical property and it controls many chemical and biological processes within the soil.

Soil sample

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Sample 6

Temperature(Ë&#x161;C)

26

27

27

29

27

26

Table no. 3: Showing the temperature of soil samples.

The change in temperature of soil may be due to the different environmental factors.

Graph no. 2: The graph showing the temperature of the soil sample vs. Soil samples.

iii). Soil colour: Colour is an obvious characteristic of soil. It can provide a valuable insight into the soil environment. Thus it can be very important in assessment and classification. The most influential colours in a well drained soil are white, red, brown and black.

The colour of the soil samples were blackish brown.

iv). Soil Texture: Soil texture refers to the weight proportion (relative proportion by weight percentage of sand, silt, and clay) of the mineral soil separates for particles less than two millimeters (mm) as determined from a laboratory particle-size distribution.

Figure no. 3: Image showing the soil texture of different soil sample.

Soil Sample

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Sample 6

Sand

14 %

8%

16 %

23 %

11 %

21 %

Silt

20 %

35 %

66 %

36 %

67 %

35 %

Clay

15 %

10 %

18 %

41 %

22 %

44 %

Table no. 4: Showing the soil texture of different soil samples.

Graph no. 3: The graph showing in the % of Sand, Silt and Clay present in the soil vs. Soil samples.

3). To study the Chemical properties of soil sample: i). Organic Carbon content: Soil organic matter (SOM) is mainly composed of carbon, hydrogen and oxygen but also has small amounts of nutrients such as nitrogen, phosphorous, sulphur, potassium, calcium and magnesium contained within organic residues.

Soil Sample

Organic Carbon (%)

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Sample 6

0.8 %

1.6%

0.4%

0.64%

0.16%

2.0%

Table no. 5: Showing the Organic Carbon content of soil sample.

Graph no.4: Showing the Organic carbon content vs. Soil sample

Part 1: (Soil sapmle 1, 2 and 3)

Part 2: (Soil sapmle 4, 5, 6 and 7) Figure no. 4: Organic Carbon content of soil sample.

4). Determination of dehydrogenase enzyme activity in soil sample: There are lots of enzymes in soil the environment, such as Oxidoreductases, Hydrolases, Isomerases, Lyases and Ligases. Each of them play key biochemical functions in the overall process of material and energy conversion.

Soil dehydrogenases (EC 1.1.1.) are the major representatives of the Oxidoreductase enzymes class. Among all enzymes in the soil environment, dehydrogenases are one of the most important, and are used as an indicator of overall soil microbial activity because they occur intracellular in all living microbial cells Moreover, they are tightly linked with microbial oxidoreduction processes Determination of soil dehydrogenase activity (DHA) in the soil samples gives us large amount of information about biological characteristic of the soil. It was confirmed that although oxygen and other electron acceptors can be utilized by dehydrogenases, most of the enzyme is

produced by anaerobic microorganisms. In other words, soil DHA strongly increases under anaerobic conditions.

S

C

1 1

1 1

2 1

3 1

4 1

2 1

3

4 1

5 1

6 1

5 1

6 1

Figure no. 5: Showing Dehydrogenase activity/assay in soil sample.

(S-sample; C-control)

In this Titer plate Dehydrogenase activity (DHA) was observed qualitatively only in sample no.

3, 4 and 6.

Microorganisms respond quickly to changes; hence they rapidly adapt to environmental conditions, and thus they can be used for soil health assessment, and changes in microbial populations and activities may therefore function as an excellent indicator of change in soil health (Kennedy and Papendick 1995; Pankhurst et al., 1995).

The soil samples were collected from various regions of university. The regions are university lake side soil, lake side garden soil, department of zoology garden soil, department of biotechnology back and front side garden soil, university main garden. The pH of the soil was found in the range of 6-7. The pH of samples(1,2,5,6) were found to be 6, and the result of

sample(3,4) were found to be 7. The change in pH may be take place due to environmental factors.

The temperature of the soil samples were in the range of 26-29Ë&#x161;C. The temperature of the soil sample 4 was highest among all i.e. 29Ë&#x161;C.

The colour of soil was brownish black due to because of degrade the organic matter by microorganism.

The soil texture of the soil samples were found to be sample 1(sand:14 %,silt:20% clay:15% ) sample 2(sand: 8%, silt:35% , clay:10 %), sample 3(sand:16%,silt:66% ,clay:18% ), sample 4(sand:23 %,silt: 36%,clay:41% ), sample 5(sand:11% ,silt:67% ,clay:22% ) and sample 6(sand:21% ,silt:35% ,clay:44% ).

Soil organic matter (OM) has important effects not only on soil enzymes activities but first of all on microorganisms activities. Soil OM has been considered as an indicator of soil quality (similarly like dehydrogenases,) because of its character of nutrient sink and source that can enhance soil physical and chemical properties, and also promote biological activity (Salazar et al., 2011). The organic carbon content of the soil samples was in range of 0.4-2%. The 0.4% of organic carbon was present in soil sample 3 and 2% was present in soil sample 6.

Soil quality can be evaluated by soil enzyme activities, which show quick responses to both natural and anthropogenic disturbances. In this study, we investigated dehydrogenase soil enzyme activity. The dehydrogenase enzyme activity was found positive for soil samples (3, 4 and 6).

The dehydrogenase enzyme activity is commonly used as an indicator of biological activity in soils (Burns 1978). The DHA is considered a direct measure of soil microbial activities because only the viable cells contain the DHA. The DHA activity affects the rate of soil nutrient availability for plants (Makoi et el., 2008). The lower DHA activity in urban soils indicates the lower rate of soil respiration, which suggests less microbial activity. (Chelikani et el., 2004)

REFERENCES

REFERENCES Alef K. and Nannipieri P., (1995). Dehydrogenase activity. In: Methods in Applied Soil Microbiology and Biochemistry (Eds K. Alef and P. Nannipieri). Academic Press, London, 228231.

Andrews RK, Blakeley RL, Zerner B, (1989) .Urease: a Ni (II) metalloenzyme. In: Lancaster JR (ed) The bioinorganic chemistry of nickel. VCH, New York, 141–166.

Aon, M.A., Colaneri, A.C. (2001). Temporal and Spatial Evolution of Enzymatic Activities and Physico-Chemical Properties in an Agricultural soil. - Appl. Soil Ecol. 18: 255–270.

Bandick, A.K., Dick, R.P.,(1999). Field Management Effects on Soil Enzyme Activities. - Soil Biol Biochem. 31:1471–1479.

Baruah, M., Mishra, R.R.,(1986). Effect of Herbicides Butachlor, 2,4-d and Oxyfluorfen on Enzyme Activities and CO2 Evolution in Submerged Paddy Field Soil. - Plant Soil 96: 287-291.

Burns, Richard G.,(1982). "Enzyme activity in soil: location and a possible role in microbial ecology." Soil Biology and Biochemistry 14(5): 423-427.

Burns RG (1978). Enzyme activity in soil: some theoretical and practical considerations. In: Bums RG (ed) Soil enzymes. Academic, London, 295–340.

Byrnes BH, Amberger A ,(1989) .Fate of broadcast urea in a flooded soil when treated with N(nbutyl) thiophospheric triamide, a urease inhibitor. Fertil Res 18:221–231.

Casida Jr, L. E., Klein, D. A., & Santoro, T. ,(1964). Soil dehydrogenase activity. Soil science, 98(6), 371-376.

Chang, E.H., Chung, R.S., Tsai, Y.H. (2007). Effect of Different Application Rates of Organic Fertilizer on Soil Enzyme Activity and Microbial Population. - Soil Sci Plant Nutr. 53:132–140.

Das, Shonkor Kumar, and Ajit Varma,(2010). "Role of enzymes in maintaining soil health." Soil enzymology. Springer Berlin Heidelberg,. 25-42.

Chelikani, P.; Fita, I.; Loewen, P.C(2004). Diversity of structures and properties among catalases. Cell. Mol. Life Sci..61. 192–208.

Dick RP, Break well DP, Turco RF (1996). Soil enzyme activities and biodiversity measurements as integrative microbiological indicators. In: Doran JW, Jones AJ (eds) Methods of assessing soil quality. Soil Science Society of America, Madison, WI, 247–271.

Dick RP, (1997) .Soil enzyme activities as integrative indicators of soil health. In: Pankhurst CE, Doube BM, Gupta VVSR (eds) Biological indicators of soil health. CABI, Wellingford, 121–156

Doran, John W., and Michael R. Zeiss,(2000). "Soil health and sustainability: managing the biotic component of soil quality." Applied soil ecology 15(1):3-11.

Ellert, B. H., M. J. Clapperton, and D. W. Anderson(1997) . "An ecosystem perspective of soil quality." Developments in Soil Science. 25: 115-141.

Engle, B., Menken, K., von Wintzingerode, F., Heuer, H., Malkomes, H. P., Backhaus, H. (1998). Monitoring Impacts of a Pesticide Treatment on Bacterial Soil Communities by Metabolic and Genetic Fingerprinting in Addition to Conventional Testing Procedures. - Appl.

Environ. Microbiol. 64: 2814–2821.

Frank T, Malkomes HP (1993). Influence of temperature on microbial activities and their reaction to the herbicide Glottis in different soils under laboratory conditions. Zentralblatt fur Mikrobiol 148:403–412.

Frankenberger, W. T., and J. B. Johansson(1983). "Method of measuring invertase activity in soils." Plant and Soil." 74(3): 301-311.

Frankenberger W T, Johanson J B and Nelson C O (1983). Urease activity in sewage sludgeamended soils. Soil Biol. Biochem. 15:543-549.

Garcia C, Herna´ndez T, (1997). Biological and biochemical indicators in derelict soils subject to erosion. Soil Biol Biochem. 29:171–177.

Humid, Rifat, et al.(2013)"Chitinases: an update." Journal of Pharmacy and Bioallied Sciences ".5.1: 21.

Ismail, B. S., Yapp, K. F., Omar, O. (1998). Effects of Metsulfuron-Methyl on Amylase, Urease, and Protease Activities in two Soils. - Aust. J. Soil Res. 36: 449-456.

Kandeler E (1996) Nitrate. In: Shiner F, O ¨ linger R, Kandeler E, Marge sin R (eds) Methods insole biology. Springer, Berlin, 408–410.

Kandeler, E., Tscherko, D., Spiegel, H. ,(1999). Long-term Monitoring of Microbial Biomass, N Mineralization and Enzyme Activities of a Chernozem under Different Tillage Management. Biol Fertil Soils 28:343–351.

Karma, Jean, and James A. Nicell,(1997). "Potential applications of enzymes in waste treatment." Journal of Chemical Technology & Biotechnology 69(2): 141-153.

Kumar, S., S. Chaudhuri, and S. K. Maiti. (2013)."Soil dehydrogenase enzyme activity in natural and mine soil-a review." Middle-East J Sci Res 13(7): 898-906.

Leirós, M.C., C. Trasar-Cepeda, S. Seoane and F. GilSotres, (2000). Biochemical properties of acid soils under climax vegetation (Atlantic oakwood) in an area of the European temperatehumid zone (Galicia, NW Spain): general parameters. Soil Biol Biochem., 32: 733-745.

Liu, J., Xie, J., Chu, Y., Sun, C., Chen, C., Wang, Q. (2008). Combined Effect of Cypermethrin and Copper on Catalase Activity in Soil. - J. Soils Sed. 8: 327-332.

Lloyd, A. B., and M. Jane Shaffer(1973) . "Urease activity in soils." Plant and Soil 39(1): 71-80.

Makoi, J., Ndakidemi, P. (2008).Selected Soil Enzymes: Examples of their Potential Roles in the Ecosystem. - African Journal of Biotechnology 7:181-91.

McCarthy, G.W., R. Siddaramappa, R.J. Reight, E.E. Coddling and G. Gao, (1994). Evaluation of coal combustion by products as soil liming materials: their influence on soil pH and enzyme activities. Biol. Fert. Soils, 17: 167-172.

Megara, M., Boul, H.L., Thiele, J.H. (1999). Effects of DDT and its Metabolites on Soil Algae and Enzymatic Activity. - Biol. Fertil. Soils. 29: 130-134.

Menon, P., Goal, M., Parsed, R. (2005). Effects of Chlorpyrifos and Quinalphos on Dehydrogenase Activities and Reduction of Fe3þ in the Soils of two Semiarid Fields of Tropical India. - Agric. Ecosyst. Environ. 108: 73–83.

Monkiedje, A., Ilori, M. O., Spiteller, M. (2002). Soil Quality Changes Resulting from the Application of the Fungicides Mefenoxam and Metalaxyl to a Sandy Loam Soil. - Soil Biol.

Biochem. 34: 1939-1948.

Pitchel, J.R. and J.M. Hayes, (1990). Influence of fly ash on soil microbial activity and populations. J. Environ. Qual., 19: 593-597.

Reddy GB, Faza A, (1989). Dehydrogenase activity in sludge amended soil. Soil Biol Biochem 21:327.

Saha, S., Mina, B. L., Gopinath, K. Kudu, A.S., Gupta, H. S. (2008): Organic Amendments Affect Biochemical Properties of a Sub temperate Soil of the Indian Himalayas. - Nutr Cycle Agroecosystem. 80:233-242.

Shula, A. K. (1997): Effect of Herbicides Butachlor, Fluchloralin, 2, 4-d and Oxyfluorfen on Microbial Population and Enzyme Activities of Rice Field Soil. - Indian J. Ecol. 24: 189–192.

Simpson JR, FFrenzyJR, Wetselaar R, Muirhead WA, Leuning R, Denmead OT (1984) Transformations and losses of urea nitrogen after application to flooded rice. Aust J Agric Res 35:189–200.

Simpson JR, Frenzy JR (1988) .Interacting processes in gaseous nitrogen loss from urea applied to flooded rice fields. In: Pushparajah E, Husin A, Bachik AT (eds) Proceedings of international symposium on urea technology and utilization. Malaysian Society of Soil Science, Kuala Lumpur, 281–290.

Sinsabaugh, Robert L., Robert K. Antibes, and Arthur E. Linins(1991) . "An enzyme approach to the analysis of microbial activity during plant litter decomposition." Agriculture, Ecosystems & Environment. 34(1)-4: 43-54.

Skladany, G.J., Metting, F.B. Jr. (1993). Bioremediation of Contaminated Soil. In: Metting, F.B., Jr (ed.) Soil Microbial Ecology, Marcel Dekker, New York.

Tabatabai MA (1994). Soil enzymes. In: Weaver RW, Angle JS, Bottomley PS (eds) Methods of soil analysis, part 2. Microbiological and biochemical properties. SSSA Book Series No. 5. Soil Science Society of America, Madison, WI, 775–833.

Tabatabai MA (1977). Effect of trace elements on urease activity in soils. Soil Biol Biochem.

9:9–13.

Tabatabai MA (1982) Soil enzyme. In: Page AL (ed) Methods of soil analysis, Part 2. American Society of Agronomy, Madison, WI, 903–948.

Tabatabai, M. Ali, and Warren A. Dick; (2002). "Enzymes in soil: research and developments in measuring activities." Enzymes in the environment. Marcel Dekker, New York : 567-596.

Tate RL (1995) Soil microbiology. John Wiley, New York.

Trevor's JT (1984) Dehydrogenase activity in soil: a comparison between the INT and TTC assay. Soil Biol Biochem. 16:673–674.

Wilkes BM (1991). Effect of single and successive additions of cadmium, nickel and zinc on carbon dioxide evolution and dehydrogenase activity in a sandy Lavishly. Biol Fertil Soils .11:34–37.

Ubtobo, E. B., and L. Teary (2015). "Soil enzymes as bioindicators of soil ecosystem status." Appl. Ecol. Environ. Res. 13: 147-169.

Yang Z, Liu S, Zhen D, Fang S (2006) Effects of cadmium, zinc and lead on soil enzyme activities. J Environ Sci .18:1135–1141.