OPTIMIZATION OF GROWTH FACTORS OF BACILLUS MEGATERIUM ISOLATED FROM A RHIZOSPHERIC SOIL OF TUMERIC PLANT

ABSTRACT

Growth factors of Bacillus megaterium isolates from rhizospheric of turmeric plants were optimized. Five soil samples were obtained from rhizosphere of five different turmeric plants were screened for the presence of Bacillus megaterium and the isolates were culture under varying cultural conditions of pH, incubation temperature and time. Result showed a 60% occurrence of Bacillus megaterium in the turmeric rhizospheric soils and variation in the optimal of different cultural condition studied. An optimum culture pH of 6.0 was needed for each of the three Bacillus megaterium isolates while the highest bacterial load was obtained when incubated at 40oC for all the isolates. Similarly the optimum incubation time of 72hours was the same for each of the isolate. Peak population of Bacillus megaterium at the optimum conditions varied among the three Bacillus megaterium with values of 5.2 ×107, 5.77×107 and 6.01×107cfu/ml. At optimum temperature and corresponding biomasses of 32.56mg/ml to 35.64mg/ml. At the optimum pH (6.0) the bacterial loads were 7.77×107cfu/ml, 7.78 ×107cfu/ml and 7.87×107cfu/ml for the three isolates and with corresponding biomasses of 34.30mg/ml to 36.10mg/ml. incubation time optimum recorded peak loads of 6.77×107cfu/ml, 7.75×107cfu/ml and 8.03×107cfu/ml. incubation time was found to have the highest positive effect on the test isolates.

TABLE OF CONTENTS

Title page i

Certification ii

Dedication iii

Acknowledgments iv

Table of contents v

Lists of tables’ ix

List of figures x

Abstract xi

CHAPTER ONE

1.0 Introduction 1

1.1 Aims 3

1.2 Objectives 3

CHAPTER TW0

2.0 Literature review 5

2.1 Bacillus subtilis 5

2.2 Bacillus thurengeinsis 6

2.3 Bacillus cereus 6

2.3.1 History 7

2.3.2 Ecology 7

2.3.3 Identification through testing 8

2.4 Bacillus megaterium 9

2.4.1 History 9

2.4.2 Characteristics 10

2.4.3 Importance 10

2.4.4 Isolation 11

2.5 Tumeric 12

2.5.1 Dye 14

2.5.2 Indicator 15

2.5.3 Tradition use 15

CHAPTER THREE

3.0 Materials and methods 17

3.1 Materials 17

3.2 Methods 17

3.2.1 Samples and media operation 17

3.2.2 Processing of soil for bacterial isolation 17

3.2.3 Media preparation 18

3.3.0 Isolation of Bacillus from soil 18

3.3.1 Characterization of Bacillus isolates 19

3.3.1.1 Cultural morphology tests 19

3.3.1.2 Microscopy characteristics test 19

3.3.1.3 Biochemical tests 20

3.3.1.4 Sugar utilization 20

3.3.2 Identification of Bacillus isolates 20

3.4.0 Occurrence of Bacillus megaterium 21

3.5.0 Optimization of growth factors 21

3.5.1 Optimization of pH 21

3.5.2 Optimization of incubation temperature 22

3.5.3 Optimization of incubation time 23

3.6.0 Determination of growth performances 23

3.6.1 Determination of TVC (population) 24

3.6.2 Determination of biomass 24

CHAPTER FOUR

4.0 Results 26

CHAPTER FIVE

5.0 Discussion, Conclusion and Recommendation

5.1 Discussion 36

5.2 Conclusion 38

5.3 Recommendation 39

References 40

Appendix 4

LIST OF TABLES

TABLE                       TITLES                                               PAGES

1                                           Occurrences of Bacillus megaterium              29

                                                  LIST OF FIGURES

FIGURES                                                    TITLES                         PAGES

Effect of incubation temperature in the

growth of Bacillus megaterium isolates  30

Effect of temperature in the biomass of

Bacillus megaterium   31

Effect of PH in the growth of Bacillus megaterium

isolates       32

Effect of PH in the biomass of Bacillus megaterium

isolates    33

Effect of incubation time on the growth of Bacillus

megaterium isolates                                                                          34

Effect of incubation time on the biomass of Bacillus

megaterium isolates                                                                          35

CHAPTER ONE

1.0 INTRODUCTION

Microorganisms in the rhizophere contribute to chemical and physical modifications that directly affect plants. Chemical changes occur as a result of the conversion of organic matter and media biomass to mineral compounds that promotes plants with a form of nutrients that are readily available for uptake. Physical changes occur primarily through the production of extracellular polymer substances such as polysaccharides and glomalin which improve soil aggregation and soil texture

Plants require some nutrients to grow but some of these nutrients are not made readily available for plant growth if some microorganisms are absent in the rhizophere. In fact, some of these microorganisms have been used for pharmaceutical application (Salas et al 2007).

In microorganisms, several species have been studied has been helpful and others have been harmful to both plants and animals. Research have been made over 2 decades that environmental friendly plants protecting agents of biological origin has been greatly emphasized as main subjects s of research especially in developed countries(whips and Nihorimbere et al, 2011)

Plants secrets secondary metabolites that inhibit certain effective microbes, which can either be beneficial to the plants by pathogen suppression or be detrimental to the plants by competing for nutrients. One the other hand, microorganisms can be beneficial to the plants by either providing essential macronutrients (nitrogen, phosphorus), or by inhibiting the activity of pathogenic microorganisms; nevertheless, pathogenic microorganisms can be harmful to the plants by either directly infecting the plants through releasing toxins or by infecting the beneficial microorganisms around them. Several species of Bacillus genus are able to produce biologically active substances thatare capable of disintegrating the fungal cells walls (Tserrkovniak et al, 2009). Amidst these substances, are some with increasing scientific, pharmaceutical and biotechnological interest (Ongena, and Jacques, 2008).

The use of beneficial plant bacteria, like Bacillus megaterium and Bacillus subtiliswhose biomass are largest than other Bacillus species, has a very positive effect on plants growth, root mass and crop yield. Specifically, beneficial plant bacteria produce plants with better growth response, biomass yield and nutrient content when compared to plants without these important beneficial soil bacteria.

Bacillus subtilis is a key beneficial plant bacterium which plays a role of replenishing soil nutrients through both the carbon cycle and nitrogen cycle. To aid the carbon cycle, Bacillus subtilis degrades the thatch, pectin, cellulose and other organic materials. They produce biofilms which are dense population of organisms. These biofilms provide a protective colonization which prohibits other microorganisms from Colonies around the plants roots (Kai et al., 2007).

Bacillus megaterium could be distinguished from other Bacillus species because of its size which is 2times larger compared to the size of E. coli when viewed with the aid of a light microscope. Strains of Bacillus megaterium has helped in the study of protein localization of bacteria. Bacillus megaterium is also used as an industrial organism that is able to produce a variety of proteins and sources of bioremediation. Been an industrial organisms for protein production, it is bit a desirable cloning host and produces a large variation of enzymes. Scientist have developed numerous protons that are commonly used in the medical and the penicillin amidase in the bacteria; harvested glucose dehydrogenase is used in glucose blood tests. B-Amylase which are often used in the bread industry (Pratricia et al., 2007).

Speaking of more benefits conferred by Bacillus subtilis, Bacillus megaterium is known to be a phosphate solubilizing Bacteria (PSB). PSB have some important benefits which include provision ofphosphrous more available to the plant, reduce need for tradition fertilizer application and reduce environmental problems downstream

Scientists have studied strains of Bacillus megaterium which involves growing its strain in a conducive physical and chemical conditions similar to the natural habitat. Its growth factor is essential and makes studies and production of groups of protein possible. Optimization of the growth factors which include incubation time, pH and incubation temperature, begins with Isolation and characterization of different strains of Bacillus species and subjecting them to growth factors variations mentioned above, of different ranges so as to provide its strain with varied environments yet similar to its natural habitat.

1.1 AIMS

The aim of this study is to optimize the growth factors of Bacillus megaterium from rhizopheric soil of turmeric plant.

1.2 OBJECTIVES

1. To isolate, characterize and identify Bacillus megateriumfrom rhizopheric soil of turmeric plants.

2. Optimize the growth factors which encourage the growth of Bacillus megaterium as follows:

i) Determination of the effect of temperature of incubation on the growth of the isolates

ii) Determination of the effect of pH of medium on the growth of the isolates.

iii) Determination of the effect of incubation time on the growth if the isolates.

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