Urease, Amidase activity in semi-arid soil of Patan District. SUBMISSION OF DISSERTATION REPORT FOR PARTIAL FULFILMENT OF

MASTER OF SCIENCE IN

BIOTECHNOLOGY

SUBMITTED TO

HEMCHANDRACHARYA NORTH GUJARAT UNIVERSITY PATAN - 384265 (GUJARAT)

BY

MS. VIJETA SHANKARBHAI PATEL SEAT NO. 21

UNDER THE SUPERVISION OF

DR. SHREYAS BHATT DEPARTMENT OF LIFE SCIENCES HEMCHANDRACHARYA NORTH GUJARAT UNIVERSITY, PATAN - 384265 GUJARAT

APRIL-MAY, 2008

DEPARTMENT OF LIFE SCIENCE HEMCHANDRACHARYA NORTH GUJARAT UNIVERSITY PATAN-384 265

CERTIFICATE

This is to certify that the dissertation entitled

“Urease, Amidase activity in

SemiSemi-arid Soil of Patan District” is submitted by Ms. Vijeta Shankarbahi Patel in partial fulfilment of the requirement for the award of the degree of Master of Science in Subject of BIOTECHNOLOGY to the Department of Life Sciences at the Hemchandracharya North Gujarat University, Patan. This is record of bonafide research work carried out by her under my guidance and supervision during the academic year 2008. In this department.

Place: Patan Date:

Dr.S.A.Bhatt

Dr.S.A.Bhatt

(Guide)

I/C. Head of the Department

Department of Life sciences

Department of Life sciences

H.N.G.U,Patan.

H.N.G.U,Patan.

ACKNOWLEDGEMENT

First off all I thank my teacher and Guide. Dr.Shreyas A. Bhatt, I/C Head, Department of life science (including Enviornmentel Scienceand Microbiology) Hemchadracharaya North Gujarat University; Who helped me with his suggestion regarding a dissertation. He remained a constant source of inspi ration with his through Knowledge . He has been a wonderful guide and teacher.

Special thanks to Ms. Payal Patel

for helping me through my queries and

suggesting me some important tips to carry out the work.

I am also very thankful to Mr.Rajesh Chaudhary for helping me through the dissertation work.

I would like to thank my colleague Kavita Modi,Krunal Patel and Hardik Gothi for constant support and help during dissertation work.

My thanks to Darshan Patel who remain behind me as a great encouragement and give help to me.

I would like to thank the non-teaching staff of lab vinubhai for help me during the work.

I would also like to extend my heartfelt thanks to my beloved father and my mother and my family members without whom this study would not have been succeeded.

Last but not the least thanks to all who have directly or indirectly been helpful to me.

INDEX

TOPIC

PAGE NO.

1. Introduction

1-9

2. Review of literature

1010-12

3. Material and Method

13-18

4. Result And Discussion

19-32

i. Collection sample

19

ii. Physical Characterization of soil

19

iii. Chemical Characterization of soil

22

iv . Enzyme Activities

26

Discussion

29

5. Conclusion

31

6. References

3333-36

INTRODUCTION INTRODUCTION:-

Enzymes are bimolecular that catalyze (i.e. increase the rates of) chemical reactions. Almost all enzymes are proteins. In enzymatic reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, the products. Almost all processes in a biological cell need enzymes in order to occur at significant rates. Since enzymes are extremely selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell. Like all catalysts, enzymes work by lowering the activation energy (Ea or ∆G‡) for a reaction, thus dramatically increasing the rate of the reaction. Most enzyme reaction rates are millions of times faster than those of comparable unanalyzed reactions. As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions. However, enzymes do differ from most other catalysts by being much more specific. Enzymes are known to catalyze about 4,000 biochemical reactions. A few RNA molecules called ribozymes catalyze reactions, with an important example being some parts of the ribosome. Synthetic molecules called artificial enzymes also display enzyme-like catalysis. Enzyme activity can be affected by other molecules. Inhibitors are molecules that decrease enzyme activity; activators are molecules that increase activity. Many drugs and poisons are enzyme inhibitors. Activity is also affected by temperature, chemical environment (e.g. pH), and the concentration of substrate. Some enzymes are used commercially, for example, in the synthesis of antibiotics. In addition, some household products use enzymes to speed up biochemical reactions (e.g., enzymes in biological washing powders break down protein or fat stains on clothes; enzymes in meat tenderizers break down proteins, making the meat easier to chew).

1

INTRODUCTION soil enzymes play an essential role in catalyzing reactions necessary for organic matter decomposition and nutrient cycling in ecosystem E~?. They are involved in energy transfer, environmental quality and crop productivity under the influence of edaphic factors Forest management activities have diverse effects on various enzymes activities of soil (e. g. burning, fertilization) . Changes in enzyme activities could alter the availability of nutrients for plant uptake, and these changes are potentially sensitive indicators of soil quality Es]. So, enzymatic activities are candidate "sensor" of soil stress to management practice that may sensitively warn us about soil degradation on Organic carbon content in the soil fraction, which consists of cell of microorganism,plant and residues as various stages of decomposition.Stable humus synthesized form residus,highly carbonized compound such as charcoal ,graphite. Our study indicated that the soil enzyme activities were associated with surface soils and decreased with depth. The distribution pattern of soil enzyme activities suggested that the transfer rate of organic matter decomposition and nutrient cycling depended on its depth in soil.soil enzyme activities in restoration process and old-growth forests ecosystem would allow greater understanding of ecosystem functions and the effects of disturbance on forest ecosystem, which can aid in developing sustainable forest management practices

Although enzymes undoubtedly perform functions critical to the cycling of nutrients in the soil, the role these assays can play in determining the "soil" health is less clear. They may be most useful for monitoring trends (positive of negative) in a soil over time. This would eliminate problems such as seasonal changes and inherent differences on activities to be early indicators of management- induced changes in the soil.

2

INTRODUCTION There was no apparent pattern that can be applied to all enzyme activities.enzyme activities significantly correlated with available soil N, with a stronger relationship. There were few significant linear correlations of enzyme activities with net N mineralization potentials. Soil enzyme activities are often used as indices of microbial growth and activity in soil soil enzyme activities like urease, amidase, asparaginase, glutaminase, histidine, ammonia lyase, protease were measured in soil profile of clay loaf from jan-may 2008.collection of 14 soil sample from different site so semi arid region relation between activity of six enzymes.viable plate count and soil properties were determinate for 14 sample of diverse soil. Various parameter have been used as indices of microbial growth and activities in soil no one parameter has been found to be acceptable because of complex growth and activity of microorganism of soil.several method have been proposed that would serve as specific indices of microbial activities i.e. of activities indices include microcolorimetry inspiration rates, measurement of ATP in soil extract and soil enzyme activities. The important of soil enzyme assays in estimating the relative microbial activity and carbon, nitrogen biomass because (1) previous work has shown that some soil enzyme may be useful in evaluating activities and biomass soil microbial population and (2) soil enzyme assay simple and convenient so, it is chosen by many investigators in soil microbial study. Drying and remoistening of soils increase the amount of carbon and nitrogen mineralized during subsequent incubation when compared with mineralization from soils that are kept moist (Stevenson).Ladd has described direct correlations between soil texture,cation exchange capacity and percentage of added substrate residual in biomass and in soil organic carbon .

3

INTRODUCTION

Soil exhaustion which implies a detoariation in soil properties at the chemical physical as well as biological levels. significant reduction in C:N,C:P ratios following cultivation suggest that mineralization losses of carbon are higher than loss of nitrogen wher as P is lowest. Lack o significant correlation between organic carbon and P levels in alll types of soils suggest that the bulk o f P is in the inorganic form.

Nitrogen is a different essential constituent of biologically significant organic molecules such as amino acids and proteins, pigments, nucleic acids and vitamins. It is also the major constituent of the atmosphere, comprising about 79 percent of it. The paradox is that in its gaseous state, N2 is abundant but is unavailable to most life. Before it can be utilized it must be converted to some chemically usable form. To be used biologically, the free molecular nitrogen has to be fixed and fixation requires an input of energy. In the first step molecular nitrogen, N2

2N. The

free nitrogen atoms then must be combined with hydrogen to form ammonia, with the release of some energy: 2N+3H2

2NH3

Gottschalk (1986) indicates that nitrogen comprises about 10% of the dry weight of bacteria, thus it is required in large quantities for microbial growth. In comparison, the dry weight of a bacterial cell is about 50% carbon, 20% oxygen, 9% hydrogen, and 2% phosphorus (Metcalf and Eddy, 2003). Ammonia is the preferred source of nitrogen for bacteria. Practically all organisms can and do utilize ammonia through assimilatory nitrate reduction. Nitrate is also taken up and utilized by many organisms but not all. Before nitrate can be incorporated into cellular material, it must be reduced to ammonia. Nitrite is the product of nitrate-nitrite respiration and of the metabolic activities of bacteria such as

4

INTRODUCTION Nitrosomonas. Nitrite is used by an important group of bacteria, the anaerobic ammonia oxidizing bacteria (Anammox), as their primary electron acceptor (Strous and Jetten, 2004). Nitrogen metabolism in bacteria is very diverse among the many species. Three mechanisms of particular interest to soil heterotrophic nitrification, aerobic denitrification, and anaerobic ammonia oxidation (Anammox) (Richardson and Watmouth, 1999). Oxygen concentration (Stenstrom and Poduska 1980; Triska et al. 1990), competitionfor NH (Verhagen Laanbroek 1991; Strauss and 14Dodds 1997), and organic carbon availability (Verhagen andLaanbroek 1991; Strauss and Dodds 1997). Among these factors, pH, temperature, and oxygen concentration serve the primary role of defining the physical and chemical environmental conditions that allow nitrification to occur and set the maximum and minimum nitrification potentials. Within these limits, however, the regulation of nitrification rates is not understood. And organic carbon availability in determining nitrification rates in soil. These three factors may be intimately linked via the C:N (total organic carbon: total nitrogen) ratio of the available substrate. The amount of phosphorus available plant is genrally not exceed 0.01% of the P.Organic p Is 20 to 28% and inorganic p.only on small fraction of the totl amount present may be available plants,which is direct relevance in assay in the phosphrous in soi. Sulfer is the most abundant elements in the earth crust,average between 0.06 and 0.10% in soil containing 5ppm and sulfate concentration of 3 to 5 ppm in soil solution are adequate per growth of plant.it is presenting soil in both organic and inorganic form.

5

INTRODUCTION Chloride is ubiquitous in the soil environment it is added soil form irrigation water.It is an essential micro nutrient it can range form 35 ppm as much as several percent.it is determined it neutrals or slightly alkaline pH. Often ignored by those unfamiliar with the metabolic capabilities of bacteria is the fact that many heterotrophic bacteria are able to oxidize nitrogen compounds. Not only do these organisms oxidize ammonium, enzymes are biological catalysts, which initiate and accelerate thousands of biochemical reactions in living cells. Major sources of enzymes are the biological organisms, Plants, animals and microorganisms (bacteria and fungi). These sources the microbial enzymes account for the major volume. However, only about less than 50 species are actually used to produce all of the microbial enzymes. The potential obviously exits to search for the species producing novel enzyme or enzymes with better properties and yield. Enzyme produce by microorganisms are either inducible (adaptive) or constitutive. The constitutive enzyme is always produces by cell regardless of weather its substrate is present in the medium or not, while the inducible enzymes are produced in response to the presence of its substrate. Enzyme may either be extracellular or end enzymes.

6

INTRODUCTION Urease activity

The enzyme urease catalyses the hydrolyses of urea to CO2 and ammonia with reaction mechanism based on the formation of carbamate as an intermediate (tabatabai,1982).this enzyme is very widely distributed in nature ,bing presenting microbial plant and animal cells.itaso catalyses the hydrolyses of hydroxyl urea,dihydroxy urea and semi carabazid ; it contains nikal and it’s molecular weight it many ranger from 151,000 - 480,000 Da most of methods involve determination of ammonia liberated on the incubation of toluene treated soil and with buffered urea solution. There are controversial reports regarding the pH optimum of urease activity in soil.urease in soil is totally bound to soil organic matter and soil minerals and km values have been found tob range between 1.32 to 2.13 mM.Temperature optimum as high as 60 has been observed and urease is usually denatured as 70. Urease is extracted from soil have been found to be resistant to thermal and proteolysis denaturation.the urease activity in soil is very stable and rarely infused by air drying, radiation or storage air tem between 60'C.it was not significantly correlated with microbial biomass and was affected differently by heave metals ,oxygen concentration and N avability in different type of soils.urease is an extra cellular enzyme in soil.oreganed from plant.in free from it is well protected by humans within the soil and they are for persists longer in soil. The activity of an enzyme is composites activity associated with different biotic and a biotic factor.other factor such as method for handling ,storage conditions and pre treatment prior to assay also affects enzyme assay. Uresae inhibitors have been used frequently to prevent a repaid hydolysis of urea an agriculture soil.soil are generally inqbator at 30'C.the leaching techniques can also be used to estimated the urease activity in soil. 7

INTRODUCTION Amidase Activity :-

Activities of soil enzymes are affected by methods of handling, storing, and pretreating the sample before enzyme assay. Amidase was recently detected in soils, and this study was carried out to assess the factors affecting its stability and distribution. Results showed that storage of field-moist samples at 5°C for 3 months decreased the activity in five soils by an average of 4%. Air-drying fieldmoist samples resulted in decreases of amidase activity ranging from 14 to 33% (avg = 21%). Freezing of field-moist samples at –20°C for 3 months resulted in activity increases ranging from 3 to 16% (avg = 9%). Heating of field-moist and air-dried samples for 2 hours before assay of amidase activity showed that this enzyme is inactivated at temperatures above 50°C. The effects observed were similar for the three substrates (formamide, acetamide, and propionamide). Studies of distribution of amidase in soils showed that it is concentrated in surface soils and decreases with depth. Statistical analysis indicated that the activity of this enzyme is significantly correlated with organic C in surface soils (r = 0.74***) and in soil profiles (r = 0.89**). Amidase activity also was significantly correlated with percentage N (r = 0.74***), percentage clay (r = 0.69***), and urease activity (r = 0.73***) in the 21 surface soil samples studied. There was no significant relationship between amidase activity and soil pH or percentage sand. Amidase activity and microbial counts obtained with acetamide or propionamide as a substrate in the absence of toluene indicated that these substrate Induce production of this enzyme by soil microorganisms amidase in enzyme catalyze the hydrolyze of amides and produce ammonia and the carboxylic acid amidase on C-N bond other than peptide bond in liner amides.spesific for aliphatic amides, and aryl amides can’t at as substrate.it is 8

INTRODUCTION present in the leaves of corn, alfalfa an soybean. Microorgamisms shown to possess amidse actvity include bacteria, yeast, and fungi. Air drying field moist samples result in a decrease of amidase activity ranging form 14 to 33%.the optimum pH observed for amidse activity it some what higher then that reported amidse activity of P pseudomonas isolated for garden soil.

9

REVIEW OF LITERATURE REVIEW OF LITERATURE :-

Enzymes are among the most remarkable bimolecules because they show extraordinary specificity in catalyzing biological reactions. Research into soil enzymes has increased steadily over the last 30 years; new theoretical approaches and methods have been introduced through which a wealth of information on various enzyme reactions in soil has been collected. Enzymes may also be present as extra cellular soluble molecules, temporarily associated in enzyme - substrate complexes, adsorbed to clay minerals or associated with humic colloids (Nannipieri., 1988).

The concentration of substrates is in excess and optimal value of pH and temperature are selected as to permit the highest rate of enzyme activity, and the volume of the reaction mixture is such that it allows free diffusion of substrate (Nannipieri., 1988).

Soil enzyme are of great significance as their activities are often used as indices of microbial growth and activity in soils. Quantitative information concerning which soil enzymes most accurately reflect microbial growth and activity is lacking. Various parameters have been used as indices of microbial growth and activity in soil (Parkinson et al., 1971). The activities of the various soil enzymes were based on the released and quantitative determination of the product in the reaction mixture when soil samples are those microorganisms activites which determine the total nitrogen cycling in the soil and these affecting fertility of soil. incubated with substrate and buffer solution. The functional diversity with Diazotrophs mainly concern with Urease, and Amidase. The activites of these enzyme results in the most simple ,inorganic form of nitrogen.e.g. ammonia. Individual enzyme have been studied

10

REVIEW OF LITERATURE by different workers with a reageant to different soil types and also the effect of various environmental parameter.

Urease

The enzyme Urease catalyses the hydrolysis of urea to CO2 and NH3 with a reaction mechanism based on the formation of carbamate as in intermediate (Tabatabai., 1972). H2NCONH2 +

H2O

___

2NH3 +

CO2

This enzyme is very widely distributed in nature, being present in microbial, plant and animal cells. A variety of methods have been used to assay the urease activity of soil. Most of these methods involve determination of ammonia liberated on the incubation of toluene-treated soil with buffered urea solution (Tabatabai., 1982; Misra., 1968).

However, soils are generally incubated at 30 0C. The Urease activity in soils is very stable and rarely influenced by air drying, irradiation or storage at temperatures between –60 0C and 22 0C (Zunatua. 1975b; Zunatua. 1977).

11

REVIEW OF LITERATURE Amidase Amidase is the enzyme that catalyses the hydrolysis of amides, and produce ammonia and the corresponding carboxylic acid R: CONH2

+

H2O

NH3

+

R: COOH

Amidase acts on C-N bonds other than peptide bonds in linear amides. It is specific for aliphatic amides, and aryl amides cannot act as substrates. This enzyme is widely distributed in nature. It has been detected in animals and microorganisms. Amidase is also present in the leaves of corn, alfalfa and soybeans. Microorganisms shown to possess amidase activity include bacteria, yeast and fungi. Amidase activity and microbial counts obtained with acetamide or propionamide as a substrate in the absence of toluene indicated that these substrates induced production of this enzyme by soil microorganisms (Frankenberger, 1991c) . Activity of the enzyme have been reported from various parts of the world but very few or no reports are available from semi-arid soils of the entire north Gujarat and therefore these work has been planned to make it.

12

MATERIAL AND METHOD MATERIAL AND METHOD :-

Description of site: Patan: This district is located between 24.41 to 23.55 latitude and 71.31-to 72.20longitute. The climate of this district is dry and hot. On its Northern side, there is Banaskantha district, in Southern side; there is the famous desert of Kachchh and some parts of Surendranagar district. On the Eastern side there is Mehsana district. The diurnal temperature range between 450C and minimum 70C.

Habitat The soil samples were collected from Patan district in North Gujarat. Nature of the soil is mainly semi arid to dry; which was characterized by lack of moisture and neutral to alkaline pH, which influences the activities of soil organisms. The soil samples were collected from January 2008 in early stage of winter seasons. For studying biodiversity it is very important to collect the samples from all representative site to get the overall idea of microbial population. As per practical requirements the material and methods are describes bellow.

Sampling Collection of soil samples To understood microbial diversity; total 14 samples of 7 sites from depth 0-10 cm and 20-30 cm were collected in triplicates from winter. Out of them 6 sample were collected from cultivated soil and 1 from uncultivated soil. Samples were

13

MATERIAL AND METHOD collected in sterilized plastic bags by a sterile PVC sampler. All soil samples were mixture of at least three representative soils of the same area.

Storage of the soil samples For biological analysis like plate count and various enzyme activities transportation and storage time should be kept to a minimum, preferably 24 to 48 hours, after the sample collection. The collected soil samples were immediately brought to laboratory and stored at 4 0C temperature. Soil and its characteristics Soil in this area is dry, clayey loam and consisting low density.

Soil Physical characters :-

Soil texture Soil

particle

size

was

determined

by

Standard British sieve method

(Alexander,1958). Soil water paste was made from 100 g oven dry soil and passed through the set of the standard sieves and each fraction thus obtained was oven dried and weighed. It has been expressed in percent of oven dry soil.

Water holding capacity ( % WHC) It is defined as the water held by the soil particles against gravity force. WHC was determined as described by Misra (1968). Air dry soil was taken in Gouch crucible and then kept in a tray filled with water. Water was absorbed by the soil by capillary action and when thin film of water was observed on the top of the

14

MATERIAL AND METHOD soil, the crucible was taken out and the wet weight of the soil was taken. The crucible was then kept in oven at 105 0C. After soil had dried, the dry weight of soil was taken and the amount of water held by the soil was measured as the difference of wet and dry weight. It is expressed as percent of oven dry soil.

% Soil moisture Soil moisture was determined from the weight loss after drying the soil sample oven at 105 °C for 24 hrs by following formula and expressed as percent oven dry weight of the soil.

% Soil moisture =

loss in weight upon drying

x 100

fresh weight of soil

Soil Chemical characters :pH A soil suspension was prepared using 1:4 soil water ratio, kept over night and pH was measured by using pH meter. (Orion, )

Total organic carbon Organic carbon was estimated by Walkley and Black method(APHA,1994). Organic metter is oxidized by H2SO4 and orthophospharic acid .Fe+2 is converted to fe+3 it gives higher content of carbon in soil. Here we are using 1n potassium dichromate and 0.5M of ferrous ammonium sulfate diephenyl amine is used as indicator green colour is obased take is final reading.

15

MATERIAL AND METHOD

Total Nitrogen Total nitrogen was estimated by micro-kjeldahal method (APHA, 1994). Total nitrogen is estimated by two method kjeldahal and Dums method.it is a two step prosses digestion of soil and to convert to ammonia. Here we are using saturated NaoH and boric acid indicator.for titration 0.1n HCl is used. After digestion of soil to measured the ammonia run the stem distillation jeldar assembly.take a blank which will be less than test sample.

Available Phosphorus Available phosphorous measured by brays method. (S.K.Maiti,2003) It is also one of the

nutrient element found in soil.It is in Ortho phosphate form it is

estimated by Brays method it is two step process.extration suitable reaction according

to

specified

soil

colorimetrically

ammonium

measured.

(S.K.Maiti,2003)

Plant Available sulfur Plant available sulphur is measured by Turbidometric method. It is the13 th most abundant element in earth It is absorbed as sulfate a ions two extractable method

are

used.

phosphate

extractable

and

CaCl2

extractable

are

used.(S.K.Maiti,2003). Chloride It is added to soil from water,fertilizer, rain water and other forms. It is used in estimated leaching fraction and salt balance.Cholride is estimated by silver nitrat treatment. (S.K.Maiti,2003)

16

MATERIAL AND METHOD Determination of enzyme activities Determination of different enzyme activities which are involved in nitrogen transformation viz., Urease (Tabatabai., 1982; Misara 1968), and Amidase (Frangnerger., 1991c) estimated as per standard protocol (Nannipieri, P.; Alef, K., 1998).

Urease The method is based on the determination of ammonia released after the incubation of the soil sample with urea solution for 2 h at 37˚c.place 5g .moist soil in a volumetric flask and adds 0.2 ml. Of toluene and 9ml. tries buffer, mix the contents, add 1ml of urea solution and mix the contents for a few second. .

Then incubate the tubes at 37˚c. Then add 37ml of kcl-Ag2SO4 solution, swirl the flask to stand the flask until the contents have cooled to room temperature (about five minute). Bring up the contents to 50 ml by addition of kcl-Ag2SO4 and mix contents thoroughly .to perform control follow the procedure described for assay for urease activity .

There are mainly two method are involved two method buffered method and nonbuffered method. Then ammonia is released which is determined by boric acid indicater. (Bremner and Edward) then Steam distillate the contents until 30 ml of the distillate are collected are in flask .then titrate it with 1N HCl .

17

MATERIAL AND METHOD Amidase

This method involve determination of the ammonia realsed by amidase activity main

soil

is

incubated

with

buffer.amine

solution

and

toluene

at

37˚c(Frankenberger., M.A.Tabatabai,1982)..The ammonium realsed is determine by a rapid procedure involving treatment of the incubated soil sample with Kcl contain and amidase inhibitor (uranyal acetate) n steam distillation of and aliquot of the resulting suspension with MgO. 5 g soil is is treated with 0.2 ml toluene and 9 ml of tris buffer.then add 1 ml 0.5 M amide solution and incubate at 37˚c. There are two hydrolysis of formamide and acetamide is occur time duration in different in both after incubation add 35 ml Kcl – Uo2 solution. To determine ammonia in the resulting suspension take a 20 ml aliquot of flask in determine by steam distillation with o.2 g Mgo (Bremner).the optimum pH oberseved for amidase activity is somewhat higher then that reported for Pseudomonas. Control should be performed each series of ammonia make the addition of 1ml of solution. Here 0.05M concentration is satisfactory for assay of amidase activity because the enzyme is saturated with substrate and the reaction rate of follows zero order kinetics formation of ammonia with formamide incubation with 4 H.

18

RESULT AND DISCUSSION RESULT AND DISCUSSION :-

Collection sample Soil samples were collected from 7 different locations

with and without

vegetation and at two-depth viz., 0-10 cm and 20-30 cm. Sites of Soil collection are listed in Table.1.

Physical Characterization of soil Soil is the three-dimensional matrix of solid, liquid and gaseous phase that receives pulsed inputs of water through rainfall, nitrogen compound through fertilizer and organic carbon through plant residue. It is also characterized by physical factors such as moisture content. The conditions within the soil aggregates therefore vary markedly in space and time and also the soil organisms participate in the genesis of habitat wherein they live. Microbial diversity and their activities increase with increase in moisture content, temperature variable organic carbon and decrease of the available carbon / available nitrogen ration in plant litter. It is difficult to pinpoint individual influence of specific physical factors on organisms because the factors interact and produce complex inter-related effect. Even then study of physicochemical characteristics of the semi-arid environment becomes a requirement for understanding the ecological interactions and microbial diversity. Soil physical characteristies are depicted in Table 2.

19

RESULT AND DISCUSSION

Table 1:Sampling sites and locations Sample No

Location

Soil Type

Depth in cm

1

Chansma 1

Cultivated

0-10

2

Chansma 2

Cultivated

20-30

3

Balisana 1

Cultivated

0-10

4

Balisana 2

Cultivated

20-30

5

Vadu 1

Cultivated

0-10

6

Vadu 2

Cultivated

20-30

7

Kharivavdi 1

Cultivated

0-10

8

Kharivavdi 2

Cultivated

20-30

9

Dhudharampura 1

Cultivated

0-10

10

Dhudharampura 2

Cultivated

20-30

11

Patan 1

Cultivated

0-10

12

Patan 2

Cultivated

20-30

13

Sujnipur 1

Un cultivated

0-10

14

Sujnipur 2

Un cultivated

20-30

20

RESULT AND DISCUSSION

Table 2:Soil Physical Characters and Major Soil Types

zone

Location

MC* %

PH

Semi arid

Chansma 1

13.26

7.4

Semi arid

Chansma 2

16.27

7.4

Semi arid

Balisna 1

11.11

7.6

Semi arid

Balisna 2

12.35

7.2

Semi arid

Vadu 1

6.38

7.3

Semi arid

Vadu 2

9.89

7.4

Semi arid

Kharivavdi 1

8.69

7.5

Semi arid

Kharivavdi 2

5.26

7.6

Semi arid

Dudharampura 1

8.69

7.2

Semi arid

Dudharampura 2

6.38

8.4

Semi arid

Patan 1

12.35

8.4

Semi arid

Patan 2

11.0

8.4

Semi arid

Sujanipur 1

1.41

8.20

Semi arid

Sujanipur 2

11.1

8.30

*MC=moisture content

21

RESULT AND DISCUSSION

Soil Moisture content Soil water has profound effect on the plant growth and to lesser extent on the other soil factors. Gravity and metric forces mainly affect the water entering into the soil. Therefore, water holding capacity (WHC )has been worked out and the same are presented in (Table.2).The average soil pH ranges between 7.2 and 8.4.

Chemical Characterization of soil The average value of pH(Table.2), chloride, nitrite-nitrogen, Organic carbon and total nitrogen of each location are represented with seasonal variations. The chemical characteristics play important role in microbial diversity because of concentration of electrolytes play important role in microbial activities.

Soil pH Seasonal variation in all the soils observed with increased alkalinity. The concentration of chloride was play important role in pH variations. During the Monsoon seasons where though there were low rain fall pH of the samples were slightly decreased in reached to neutral where during Post Monsoon Seasons samples having highest alkaline pH. Alkaline pH of the samples was favorable for the enzyme activities, which reflected in the results. Because of over exploitation of Ground recumces of water,the water in these region has some down.this has results in precipitation of salts increasity pH of soil.

22

RESULT AND DISCUSSION

Table 3 :Soil Chemical Properties

Location

%OC

Total Nitogen

C:N Ratio

Chansma 1

2.48

0.003

82.6

Chansma 2

4.13

0.002

206

Balisana 1

0.76

0.003

25.3

Balisna 2

1.11

0.0005

62.0

Vadu 1

0.62

0.0014

44.2

Vadu 2

1.44

0.0022

72.0

Kharivavdi 1

2.56

0.008

32.0

Kharivavdi 2

2.06

0.002

103

Dudharampura 1

2.89

0.0005

57.8

Dudarampura 2

0.86

0.003

28.6

Patan 1

3.96

0.0056

70.7

Patan 2

2.76

0.0058

46.5

Sujanipur 1

2.0

0.014

14.2

Sujanipur 2

0.6

0.015

4.0

* OC =Oraganic carbon : TN=Total nitrogen. C:N =Carbon Nitrogen ratio

23

RESULT AND DISCUSSION Soil Organic Carbon: Vriation in soil organic carbon was observed but which was remaining constant through out the study because use of manure. The soil organic carbon content was found higher in the all the soils and trends was decreasing during low moisture content during summer season. Enzyme activities like L-Asparaginase and L-Glutaminase are significantly correlated with organic carbon.(Table 3).Samples from Balisana and dhutharampura containd very organic carbon where as, it was repotaed higher.

Soil Total Nitrogen: Variation has been reported in soil total nitrogen in all samples. which was found to be very low.It ranged between 0.0005 to 0.015. The soil nitrogen content was found very low in the all the soils and trend was decreasing towards low moisture content during summer season. Enzyme activities were significantly correlated with nitrogen contents. Low concentration of nitrogen may be due to the higher concentration of salts and water-stress condition play important role in nitrogen releasing.Reaction catalysed by different enzyme involved in nitrogen transformation.The C:N ratio was very high in almost all the soil sample.This has positive inpect on soil sample.The development of diazotrophas whose activity increase available nitrogen in soil (Table 3).

Available Phosphorous

ranges from 0.001 to 0.009%.Sulfur content ranges

between 0.001 and 0.007. While chloride varies from 0.111 to 0.49%. Which is higher than more of values.

24

RESULT AND DISCUSSION

Table 4 :Soil Chemical Properties

Location

%P

%S

%Cl

Chansma 1

0.006

0.0035

0.11

Chansma 2

0.001

0.0009

0.14

Balisana 1

0.001

0.0008

0.19

Balisana 2

0.002

0.002

0.26

Vadu 1

0.001

0.004

0.26

Vadu 2

0.0009

0.0002

0.15

Kharivavdi 1

0.009

0.001

0.38

Kharivavdi 2

0.008

0.0006

0.32

Dudharampura 1

0.006

0.0002

0.13

Dudharampura 2

0.001

0.0002

0.46

Patan 1

0.001

0.0007

0.49

Patan 2

0.001

0.0003

0.37

Sujanipur 1

0.006

0.0001

0.31

Sujanipur 2

0.0004

0.0001

0.26

*

P=Phosphorus, S=Sulfur, Cl=chloride

25

RESULT AND DISCUSSION Enzyme Activities

Enzymes involved in the transformation of nitrogen have been studied at every site. Enzyme activities of consortial samples also used for studying microbial metabolic diversity study. It is important to assess functional diversity in microbial communities by microbial activities. Functional diversity has been studied at substrate level utilization at producing of different enzymes viz., Urease and Amidase. Effect of temperature and moisture content remarkable observed in all locations. The presence of a number of microorganisms also have effect on enzyme activity in soil. Table number 5 and 6 show enzyme activities.

26

RESULT AND DISCUSSION

Table 5 :Urease activity (µg NH3 releasded g‐1 Soil)

Zone

Location

Urease µg NH3 g‐1 Soil

27

SemiArid

Chansma:1

23.86

SemiArid

Chansma:2

20.81

SemiArid

Balisana:1

25.86

SemiArid

Balisana:2

28.63

SemiArid

Vadu:1

22.34

SemiArid

Vadu:2

23.87

SemiArid

Kharivavadi:1

29.67

SemiArid

Kharivavadi:2

23.87

SemiArid

Dudharampura:1

22.34

SemiArid

Dudharampura:2

22.10

SemiArid

Patan:1

23.59

SemiArid

Patan:2

23.33

SemiArid

Sujanipur:1

21.29

SemiArid

Sujanipur:2

21.21

RESULT AND DISCUSSION

Table 6 :Amidase activity (µg NH3 releasded g‐1 Soil )

Zone

Location

Amidase µg NH3 g‐1 Soil

SemiArid

Chansma:1

300

SemiArid

Chansma:2

238

SemiArid

Balisana:1

18

SemiArid

Balisana:2

11

SemiArid

Vadu:1

24

SemiArid

Vadu:2

24

SemiArid

Kharivavadi:1

224

SemiArid

Kharivavadi:2

214

SemiArid

Dudharampura:1

238

SemiArid

Dudharampura:2

40

SemiArid

Patan:1

20

SemiArid

Patan:2

154

SemiArid

Sujanipur:1

200

SemiArid

Sujanipur:2

238

28

RESULT AND DISCUSSION

DISCUSSION

Urease Activity

The tris buffer is used in the assay involving the ammonium determination because it prevents ammonias fixation by soil. The urease activity has been repotated higher in buffer than in without buffer soil suspension (Nannipieri, P.; Alef, K., 1998).

The sample dilution by Kcl may considerably slow down the enzymatic activity raters; tomes for ammonium extraction and filteration should not different among different measurements. The color developed in the ammonium analysis is stable for at least 8 h at room temperature. The filteration of soil suspension should be carried out with nitrogen free filter paper to avoid nitrogen contamination of the filtrate.

urease inhibitiors are frequently used to prevent a rapid chydrolysis of urea. it was affected by air drying,oxygen concentration, biomass. One observation made urease activity was that enzyme activity was slightly higher at 10 to 20 cm depth then 0 to 10 cm.This may perhaps due to move availability of carbon contain at slighty depth layer of soil were microbs may be higher that are involved in urea hydolysis to prefer that environmental conditions.

29

RESULT AND DISCUSSION Amidase Activity

The use of tris buffer is based on the finding that the ammonia release by amidase activity is considerably higher in the presence of tris than in the presence of phosphate, citrate or modified universal buffer. The other reason for choosing this buffer is that no ammonium fixation by soil is observed in the presence of tris buffer. The purpose of adding the kcl-uranyl acetate solution to the sample after incubation is to inactivate amidase and to allow quantitative determination of ammonia released. The effects of several salts selected to provide of variety of cation of and anion showed that soil amidase activity is not affected when the tris buffer contain five ions. The data for amidase activity are depicted in Table 6. Data show that the amidase activity were high in at Chansma,Kharivavdi,Dudharampura,sujnipur etc.The soil texture is almost silty sand.Which have good WHC. Chansma soil show highest activity at 0 to 10 cm depth.While Kharivavdi recoded comparitaliy very low amidase activity at 10 to 20 cm depth. At oter location the varation is not to range which may be because some of the enzyme may be contributed by plant also.

The treand of enzyme activity in general was decrease with depth and oraganic carbon soil texture also have profound effect on soil enzyme activity (Bhatt,1997) and there for futher research is riqird to study seasionan variation in enzyme activity.in the soil.as these enzymes are importance in maintaining soil fertility.

30

RESULT AND DISCUSSION CONCLUSION

Soil in most locations show sandy loamy nature except one location in semiarid.soil. Moisture content was 16.27%. Organic carbon recorded was maximum 4.13%, soil pH in most locations was either or near neutral slightly alkaline. The C/N ratio in most soil was higher.(table 3).Highest carbon was recorded at site 2 which is chansma 2 at depth 10 to 20 cm. Physical properties of the soil varied in the most of the soil layers because of humification.Some even to deep layers.Some site possessed lower biomass ratio may be perhapeps the reason for higher microbial population. The highest total nitrogen was recorded at site 6 at vadu 2.and lowest nitrogen was was at site 2.it is chansma 2.There are reasons for beliving that the differences not large except with soil were the sample become dry. Total phosphorus at maximum measueed maximum at site 6 at vadu 2 and lowest phophorus site 14 at sujanipur 2 at depth 10 to 20 cm. biomass phosporus ranged to 0.00042 to 0.0092 gm℅. Maximum Sulfur measured site 1 which a chansma 1 depth form 0 to 10 cm.and lowest sulfur in gm% is at site 14 sujnipur 2 at a depth 10 to 20 cm and it is a uncultivated. The Values of chloride is 0.265 gm% at site 4 and Balisana 2 to it is depth form 0 to 20 cm.The lowest chloride content in soil is site 9 of Dhudharampura 1 at depth 0 to 10 cm. The higher urease activity is seen in sample no 7 it is form kharivavdi 1 at depth 0 to 10 cm. it is 29.67µg.and lowest activity is measured at sample 2 which is a chansma 2. it is 20.81µg.that activity of soil urease two differing site differ in topographically and to depth study during jan ’08 to April ’08.During winter season there are higher value of carbon and nitrogen soil.

31

RESULT AND DISCUSSION Site 1 and site 2of kharivavdi village. rocoded higher activity indicating soil texture affects of activity of urease in this soil. Soil texture it may have positive effect on urease activity as higher clay content it may protect enzyme from inhibitory effects of soil edalphic factors. The higher amidase activity in soil each shown in sample 1 at chansma 1 0 to 10 cm. it is 238 µg. and lowest activity is measured at sample 4 which balisana 2 it is 11µ g. the activity of soil amidase is differ in two site during Jan ’08 to April ’08. winter there are higher value of carbon and nitrogen content. Highest amidase activity shows lowest organic carbon content. And lowest activity content higher value of carbon content Effect of temperature highest observed during Urease & Amidase activity. Where enzymes were inactivated at higher temperature. Amidase in soils showed that it was concentrated in surface soils & decreased with depth.

Urease activity in soils was stable & affected by higher temperature. Urease was not significantly correlated with microbial Biomass but affected by nitrogen availability in different types of soils. Statistical analysis indicated that the activity of this enzyme is significantaly related with organic C content in sub surface soils and in soil profiles. Amidase activity also was significantly correlated with 0total nitrogen, percentage clay .

32

REFERENCES REFERENCES 1.

Tabatabai MA 1994. “Soil enzymes”. In: Weaver RW, Angle JS,Bottomley PS (eds) Methods of soil analysis, part 2. Microbiologica and biochemical properties. SSSA Book In: Standered methods for the examination of water and waste water, Lerores.Clesceri, Arnold.E. Greenberg, Andrew.D. Eaton, 1998, 20th edition, P.P.5.18.

2.

Gottschalk, G. 1986. Bacterial Metabolism, 2nd edition. Springer-Verlag, Inc. NY.Metcalf and Eddy, 2003).pp 39 – 67.

3.

Strous M and Jetten MSM. 2004. Anaerobic Oxidation of Methane and Ammonium. Annu Rev. Microbiol. 58: 99-117.

4.

Richardson DJ, Watmough NJ. 1999. Inorganic Nitrogen Metabolism in Bacteria. Curr Opin Chem Biol. 3:207-219.

5.

Molin, J., Molin, S., 1997. CASE: complex adaptive systems ecology. In: Jones, J.G. (Ed.), Advances in Microbial Ecology,vol. 15. Plenum, New York, pp. 27– 79.

6.

Trevors, J.T., 1998b. Bacterial biodiversity in soil with an emphasis on chemically-contaminated soils. Water Air Soil Pollut. 101, 45– 67.

7.

Wall, D.H., Virginia, R.A., 1999. Controls on soil biodiversity: insights from extreme environments. Appl. Soil Ecol. 13, 137– 150.

8.

George, E., Marschner and H., Jakobsen, I., 1995. Role of arbuscularmycorrhizal fungi in uptake of phosphorous and nitrogen from soil. Crit. Rev. Biotechnol. 15, 257–270.

9.

Timonen, S., Finlay, R.D., Olsson, S. and Soderstrom, B., 1996. Dynamics of phosphorous translocation in intact ectomycorrhizal systems: non-destructive monitoring using a B-scanner. FEMS Microbiol. Ecol. 19, 171– 180.

10. Srivastava, D., Kapoor, R., Srivastava, S.K. and Mukerji, K.G., 1996.Vesicular arbuscular mycorrhiza—an overview. In: Mukerji,K.G. (Ed.), Concepts in Mycorrhizal Research. Kluwer AcademicPublishing, Netherlands, pp. 1 –39. 11. Filion, M., St-Arnaud, M. and Fortin, J.A., 1999. Direct interaction between the arbuscular mycorrhizal fungus Glomus intraradices and different rhizosphere microorganisms. New Phytol. 141, 525–533.

33

REFERENCES

12. Wright, S.F., Upadhyaya, A., 1998. A survey of soils for aggregate stability and glomalin, a glycoprotein produced by hyphae of arbuscular mycorrhizal fungi. Plant and Soil 198, 97–107. 13. Dodd, J.C., Boddington, C.L., Rodriguez, A., Gonzalez-Chavez, C. and Mansur, I., 2000. Mycelium of arbuscular mycorrhizal fungi (AMF) from different genera: form, function and detection. Plant and Soil 226, 131–151. 14. Yao, H., He, Z., Wilson, M.J. and Campbell, C.D., 2000. Microbial biomass and community structure in a sequence of soils with increasing fertility and changing land use. Microb. Ecol. 40, 223– 237. 15. O’Donnell, A.G., Seasman, M., Macrae, A., Waite, I. and Davies, J.T.,2001. Plants and fertilisers as drivers of change in microbial community structure and function in soils. Plant and Soil 232, 135– 145. 16. Torsvik, V., Goksoyr, J. and Daae, F.L., 1990. High diversity in DNA of soil bacteria. Appl. Environ. Microbiol. 56, 782–787. 17. Torsvik, V., Salte, K., Soerheim, R. and Goksoeyr, J., 1990. Comparison of phenotypic diversity and DNA heterogeneity in a population of soil bacteria. Appl. Environ. Microbiol. 56,776– 781. 18. Pace, N.R., 1997. A molecular view of microbial diversity and the biosphere. Science 276, 734– 740. 19. Pace, N.R., 1999. Microbial ecology and diversity. ASM News 65, 328– 333. 20. Torsvik, V., Daae, F.L., Sandaa, R.-A. and Ovreas, L., 1998. Reviewarticle: novel techniques for analysing microbial diversity in natural and perturbed environments. J. Biotechnol. 64, 53–62.137– 150. 21. Giller, K.E., Beare, M.H., Lavelle, P., Izac, A.-M.N. and Swift, M.J.,1997. Agricultural intensification, soil biodiversity and agroecosystem function. Appl. Soil Ecol. 6, 3 –16. 22. Thorn, G., 1997. The fungi in soil. In: van Elsas, J.D., Trevors J.T., Wellington, E.M.H. (Eds.), Modern Soil Microbiology.Marcel Dekker, New York, pp. 63– 127. 34

REFERENCES 23. van Elsas, J.D., Frois-Duarte, G., Keijzer-Wolters, A. and Smit, E., 2000.Analysis of the dynamics of fungal communities in soil via fungal-specific PCR of soil DNA followed by denaturing gradient gel electrophoresis. J. Microbiol. Methods 43, 133–151. 24. Pace, N.R., 1997. A molecular view of microbial diversity and the biosphere. Science 276, 734– 740. 25. Van der Heijden, M.G.A., Klironomos, J.N., Ursic, M., Moutoglis,P., Streitwolf-Engel, R., Boller, T., Wiemken, A. and Sanders, I.R.,1998. Mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity. Nature 396, 69–72. 26. Wall, D.H., Virginia, R.A., 1999. Controls on soil biodiversity: insights from extreme environments. Appl. Soil Ecol. 13,137– 150 Curl, E.A., Truelove, B., 1986. The Rhizosphere. Springer-Verlag, Berlin. 27. Verstraete W , Alexander M. 1972. Heterotrophic Nitrification by Arthrobacter sp. J Bacteriol. 110(3): 955-961. 28. Robertson LA, Cornelisse R, Zeng R and Kuenen JG. 1989. The effect of thiosulfate and other inhibitors of autotrophic nitrification on heterotrophic nitrifiers. Antonie van Leeuwen. 56:301- 309. 29. Castignetti, D. , Hollocher T. 1982. Nitrogen Redox Metabolism of a Heterotrophic, Nitrifying-Denitrifying Alcaligenes sp. from Soil. Appl Environ Microbiol. 44(4): 923-928 30. Acosta-Martínez V, Tabatabai MA 1999 Arylamidase activity of soils. Soil Sci Soc AmJ (in press)Ajwa HA, Tabatabai MA 1994 Decomposition of different organicmaterials in soils. Biol Fertil Soils 18 :175–182. 31. Bremner JM, Mulvaney CS 1982 Nitrogen-total. In: Page AL, Miller RH, Keeney DR (eds) Methods of soil analysis, part 2,2nd edn. ASA and SSSA, Madison, Wis., pp 595–641Martinez CE, Tabatabai MA 1997 Decomposition of biotechnology by-products in soils. J Environ Qual 26:625–632 32. Skujins J 1976 Enzymes in soil. In: McLaren AD, Peterson GH (eds) Soil biochemistry, vol 1. Dekker, New York, pp371–414. 33. Series No. 5.Dick W A, Chcng I. and Wang P. Soil Acid and Alkaline Phosphatase Activity as pH Adjustment Indicators. Soil Boil Biochem, 2000, 32: 1915-1919.

35

REFERENCES 34. Institute of Soil .Science, Chinese Academy of Sciences.PhysicabChemical Analysis o/ Soil. Shanghai: ShanghaiScience and Technology Publishing House, 1978(Ch).Taylor J P, Wilson B and Mills M S, el al. Comparison of MicrobialNumbers and Enzymatic Activities in Surface and SubsoilsUsing Various Techniques. Soil Biol Biochem, 2002,34:387 401. 35. Zhang Y M, Zhou G Y and Wu N, et al. Soil Enzyme ActivityChanges in Different-aged Spruce Forests of the EasternQinghai-Tibet Plateau. Pedosphere, 2004, 14(3) : 305-312. 36. Guan S Y. Soil Enzymes and Its Methodology. Beijing: AgriculturalPress, 1986(Ch).Aksoy, A., Sahin, U., & Duman, F. 2000. Robinia pseudoacaciaL. as a possible biomonitor of heavy metal pollution inKayseri. Turkish Journal of Botany, 24(5), 279–284. 37. Taylor, J. P., Wilson, B., Mills, M. S., and Burns, R. G. 2002.Comparison of microbial nnumbers and enzymatic activities in surface soils and subsoils using various techniques. Soil Biology and Biochemistry, 34, 387–401. 38. Ward, N. I. 1990. Multielement contamination of British motorway environments. Science of the Total Environment, 93, 393–401. 39. Wardle, D. A., Ghani, A. 1995. A critique of the microbial metabolic quotient (qCO2) as a bioindicator of disturbance and ecosystem development. Soil Biology and Biochemistry, 27, 1601–1610. 40. In: Hand book of methods in environmental studies, S.k. Maiti, 2003, vol-2, P.P.153, 163, 167-183. 41. In: Handbook of Microbiological Media, Lawrence.C. parks, 1997, Second edition, P.P.839.

36