PHOSPHATE SOLUBILIZING BACILLUS SPECIES FROM A RHIZOSPHERE SOIL

ASBTRACT

The aims of this research work is to isolate and identify phosphate solubilizing Bacillus from soil. Soil samples were collected and evaluated for phosphate solubilizing Bacillus in piskovskaya agar plates. A total of eight (8) phosphate solubilizing Bacillus colonies were isolated from the rhizosphere soils on pikovskaya agar (PVK) medium containing tricalcium phosphate. Out of the 8 bacterial isolates three showed clear halo zones around the colony. Phosphate solubilizing index (PSI) of each isolate was calculated. The three isolates include Bacillus species, streptomyces species and Lysini bacillus. From the results it was revealed that isolate Lysini bacillus had the highest phosphate solubilizing ability with a PSI value of 4.00 followed by Lysini bacillus (3.60) while the lowest was found in Streptomyces with 3.42. The halo zone of inhibition was found in Bacillus species with 13.0mm while the lowest was found in Lysini bacillus. from the result also it was shown that Streptomyces spp has phosphate solubilizing efficiency (μg/ml) in Pikovskaya broth. Eight phosphate solubilizing actinomycete isolates were identified on the basis of colony characteristics, microscopic observation and biochemical test. It is clear from the present study that the rhizosphere actionomycete are efficient in solubilization of phosphate in soil by producing organic acids. The strain M6 belongings to genera Bacillus spp showed significant potential in solubilizing the phosphate may be due to its capability to produce high amount of organic acids which release phosphate to soil solution from phosphate bearing minerals. The release of phosphate from the muscovite mica increased with the inoculation period of specific strain of streptomycete and Bacillus. As the strains of M2 and M6 gave very good results, they can be now tested on the field to rate its performance as biofertilizer for the particular location.

TABLE OF CONTENT

TITLE                                                                                             PAGES

     Title page                                                                                                                                  i

    Certification                                                                                                                             ii

    Dedication                                                                                                                                 iii

    Acknowledgements                                                                                                                   iv

    Table of contents                                                                                                                        v

     Lists of tables                                                                                                                           ix

    Abstract                                                                                                                                     x

  CHAPTER ONE                                             1

1.1 Introduction                                                                                                                      1

1.2 Objectives the Study                                2

CHAPTER TWO                                                                                                                  3

2.0    Literature Review                                                                                                          3

2.1      Soil                                                                                                  4

2.2      Function of soil                                                                                                            5

2.3       Soil organic matter                                                                                                     6

2.4       Microbial diversity of the soil                     6

2.5      Phosphate solubilizing microorganism                                                                       8

2.5.1      Mechanism of phosphate solubilization                                                                   8

2.5.2    Inorganic phosphate solubilization                                                                           8

2.5.3    Organic phosphate solubilization                                                                               9

2.5.4    Phosphate solubilizing bacteria as plant growth promoters                                       11

2.4.1    Genetic engineering of phosphate solubilizing microorganisms                                    12

CHAPTER THREE                                                                                                    13

3.0 Materials and Methods                                                                                                     13

3.1 Materials                                                                                                                           13

3.2 Method                                                                                                                              13

3.2.1 Sample Collection and preparation                                                                               13

3.2.2  Microbiology analysis serial dilution                                                                           13

3.2.3 Identification of phosphate solubilizing bacteria                                                          14

3.2.3.1 Gram Staining                                                                                                            14

3.2.4   Biochemical Reaction Test                                                                                          27

3.2.4.1 Catalase Test                                                                                                               27

3.2.4.2 Methyl Red Test                                                                                                         27                                                                    

 3.2.4.3 Voges- Proskauer (Vp) Test                                                                                      28

3.2.4.4  Indole Test                                                                                                                 28

3.2.4.5 Citrate Utilization Test                                                                                               28

3.2.4.6 Sugar Utilization Tests                                                                                               28

3.2.5 Identification of Isolate                                                                                                 29

3.2.6 Optimal Growth Condition of the Isolates                                                                    29

CHAPTER FOUR                                                                                                                30

4.0 Results                                                                                                                              30

CHAPTER FIVE                                                                                                                  38

5.0   Discussion and Conclusion                                                                                            38

5.1       Discussion                                                                                                                   38

5.2       Conclusion                                                                                                                  39

References                                                                                                                              40

LIST OF TABLES

TABLES                                                   TITLES                        PAGES

4.1:   Phosphate solubilization efficiency of the isolates          19

4.2:  Colonial characteristic and Biochemical features of the Bacterial isolates            20

CHAPTER ONE

            1.0.         INTRODUCTION

Phosphorous is a one of the most abundant metallic elements found in the earth’s crust and is present in the soils in both inorganic and organic forms (Gyaneshwar et al., 2002). It is utilized or absorbed by the plants in inorganic form i.e. in orthophosphate (H2PO4-and HPO42-) (Hinsinger, 2001). It has a key role in metabolic processes such as photosynthesis, energy transfer, signal transduction, nitrogen fixation in legumes, crop quality and resistance to plant diseases are the main features associated with phosphorous nutrition ( Khan et al, 2014).

The solubilization of phosphate-bearing inorganic materials by microorganisms would seem to be an attractive solution that has been actively studied during the last decade. Several mechanisms, such as lowering the pH by acid production, ion chelation and exchange reaction in the growth environment, have been reported to play a role in solubilization by phosphate solubilizing microorganisms (PSM) (Rajankar et al., 2007). Among these PSM, Actinomycetes are of special interest since these filamentous sporulating bacteria are able to develop in extremely different soils (Jiang et al., 2005; Pathom-Aree et al., 2006) and produce various substances (anti-fungi, insecticides, anthelminthics, phytohormone-like compounds etc.) that could benefit plant growth (Jain and Jain, 2007) showed that the abundance of actinomycetes solublizing Moroccan rock phosphate, from a Togolese phosphate mine, was approximately 19%. Reports in the literature suggested that microbial solubilization of mineral phosphate might be either due to the excretion of organic acids causing acidification of the external medium (Whitelaw, 2000), or to the excretion of chelating substances (such as sidero-phores) that form stable complexes with phosphorus adsorbents (aluminium, iron and calcium) (Welch et al.,2002), and thus increase phosphate solubilization.

A number of heterotrophic microorganisms excreting organic acids which solubalized P that chelate cationic partners of P ions and release the P directly into solution (He et al., 2002). These phosphate solubilizing bacteria (PSB) are being used as biofertilizer since 1950s. Microbial inoculants assimilate soluble P, and prevents it from adsorption or fixation (Khan and Joergesen, 2009). These microorganisms influences soil fertility through various processes viz. decomposition, mineralization and release of nutrients. Microorganisms enhance the P availability to plants through solubilization of inorganic form of P to in available form (Chen et al., 2006). Hence, microbial inoculants are used as an alternate source, which are both economic as well as eco-friendly. A continued exploration of the natural biodiversity of soil microorganisms and the optimization of microbial interactions in the rhizosphere represents a prerequisite step to develop the more efficient microbial inoculants with phosphorus-solubilizing ability.

Microorganisms constitute an inexhaustible reservoir of compounds with pharmacological, physiological, medical or agricultural applications (Nanjwade et al., 2010). Soil is not only important for agriculture but also very useful for living organism (Jain and Singh, 2013). The diversity of physical characteristics of soil associate with aggregation at small scales means that soil can contain a large diversity of microorganisms in close proximity, and the chemical composition of soil is highly heterogeneous in both vertical and horizontal dimensions (Kang and Mills, 2006). Jain and Singh (2013) suggested that different variety of soil contains different properties, which support many organisms of the soil. Soil in particular is an extensively exploited ecological niche, the inhabitants of which produce many biologically active natural products including clinically important antibiotics (Kumar, Durapanduyan and Ignacimuthu, 2014).

1.2. AIM AND OBJECTIVES

This research aims is to isolate and identify phosphate solubilizing bacteria from soil

1.2.1    OBJECTIVES

The specific objectives of this study are, namely;                     

·       To isolate bacteria from soil

·       To characterize and identify bacteria from soil.

·       To determine phosphate solubilizing bacteria isolates from soil.

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