ISOLATION AND IDENTIFICATION OF PROTEOLYTIC AND LIPOLYTIC MICROORGANISMS FROM SOIL RECEIVING ABATTOIR EFFLUENTS.

Abstract

The isolation and identification of proteolytic and lipolytic microorganisms from soil receiving abattoir effluents were carried out. Soil samples contaminated with abattoir effluent were collected. The total aerobic plate count ranged from 1.1x107cfu/ml - 1.46x107cfu/ml; proteolytic count ranged from 1.01x106cfu/ml - 9.4x106cfu/ml; lipolytic count ranged from 0 to 6.1x106cfu/ml and fungal count ranged from 4.2x106cfu/ml - 6.6x106cfu/ml. The microorganisms isolated and their percentage occurrence were Staphylococcus aureus, 20%; Pseudomonas species,16%; Streptococcus species, 16%; Bacillus species, 14%; Micrococcus species, 6%; Escherichia coli, 12%; Klebseilla species, 16%; Aspergillus species, 57% and Rhizobus species, 42%. The results showed that the soil receiving abattoir effluent was contaminated heavily with known pathogenic microorganisms. The abattoir effluent should therefore be treated before discharge and good hygiene practices should also be maintained.

Table of contents

Title i

Certification ii

Dedication iii

Acknowledgement iv

Table of contents v

List of tables vii

Abstract viii

Chapter One

1.1 Introduction 1

1.2 Aim and Objectives 5

Chapter Two

2.1 Literature Review 6

2.2 Characteristics of proteases 6

2.3 Effect of Temperature on Protease and Lipases Production 7

2.4 Lipase producing Microorganisms 7

2.5 Bacteria Lipases 8

2.6 Sources of Lipases 9

2.7 Application of Microbial Lipases 10

2.7.1 Effluent Treatment 10

2.7.2 Medical Application 11

2.7.3 Lipases as a biosensor 11

2.7.4 Production of Biodegradable Polymers 12

Chapter Three

3.0 Materials and Methods 13

3.1 Study Site. 13

3.2 Collection of Samples 13

3.3 Chemical Reagents 13

3.4 Sterilization 13

3.5 Enumeration of Total Heterotrophic Bacteria and Fungi 14

3.6 Enumeration of Proteolytic and Lipolytic Bacteria. 14

3.7 Microbial Characterization/Identification 15

3.7.1 Gram Staining 15

3.7.2 Motility Test 16

3.7.3 Coagulase Test 16

3.7.4 Indole Test. 16

3.7.5 Catalase Test 16

3.7.6 Citrate Utilization 17

3.7.7 Oxidase Test. 17

Chapter Four

4.1 Results 18

5.1 Discussion 23

5.2 Conclusion 25

5.3 Recommendation 25

References 22

Appendix 26

List of tables

Table Title Page

Table 1: The mean counts of the microorganism isolated from

soil samples containing abattoir effluents 19

Table 2: Microorganism isolated from soil abattoir effluent and

their percentage occurrence 20

CHAPTER ONE

1.1 INTRODUCTION

Proteolytic is defined as an enzymes (proteases) that promote proteolysis, the breaking down of protein into simpler compound as in digestion. Proteases are the most important industrial enzymes that execute a wide variety of functions and have various important biotechnological applications (Mohen and Dilee, 2005). They constitute two thirds of the total enzymes used in various industries and account for at least a quarter of the total global enzymes production, which represent about 60 % of all the industrial enzyme’s sales in the world, due to their applications in several industrial sectors (Kumar et al., 2002; Gupta et al., 2002). A number of bacteria and fungi have been reported for protease production. Properties of this protease such as alkaline pH, thermo stability in solvents and detergent resistance make the enzyme useful for different applications. Proteolytic enzyme producers are also helpful for the health of the ecosystems of this earth as these microbes decompose the dead and decaying animal or plant tissues in water or land. They can create pollution free environment and they are responsible for the recycling of nutrients (Gupta et al., 2007).

The induction of protease requires a substrate having peptide bonds including substrates like peptone, casein and other proteins. The ammonia, as final product of enzymatic reaction of substrate hydrolysis, represses enzyme synthesis by a well-known mechanism of catabolite repression. This extracellular protease has also been commercially exploited to assist protein degradation in various industrial processes (Srinubabu et al., 2007). The great advantages offered by microbial enzymes are low material costs coupled with high and faster productivity and the ease with which the enzymes can be modified (Sharma et al., 2007). At present, due to high cost of substrates and mediums used, the overall cost of enzyme production is very high and therefore, development of novel processes to increase the yield of proteases with respect to their industrial requirements coupled with lowering down the production cost is highly appreciable from the commercial point of view (Kammoun et al., 2008).

Proteases show variety of characteristics under different conditions. Microorganisms, which produced extracellular acid proteases, often acidify the medium in which they grow (Shumi et al., 2004), and the ability to produce alkaline proteinases has been correlated with growth of organisms at neutral to alkaline pH (Shumi et al., 2004). Formation of proteinases varies in the presence of different carbon and nitrogen sources (Shumiet al., 2004), medium pH (Hossain et al, 1999.), and also the incubation temperature and time (Marzan et al., 2004; Shumi et al., 2004). Heat stable alkaline proteases, reported by many workers (Thangam and Rajkumar, 2002), have potential for industrial use.

Proteases are complex multi-enzyme system which catalyzes the hydrolysis of amide bond in a protein molecular hence it has been used in the field of textile processing for degumming of silk and processing of wool (Ravel and Banerjee, 2003; Adinarayana et al., 2005). With the advent of new frontiers in biotechnology, the spectrum of protease application has expanded into many new fields, such as clinical, medicinal and analytical chemistry. To meet the current largely increased demand, studies on the cost-effective production of industrially important enzymes have become the need of this day.

Microorganisms are the most important sources for enzyme production. Selection of the right organism plays a key role in high yield of desirable enzymes. For production of enzymes for industrial use, isolation and characterization of new promising strains using cheap carbon and nitrogen source is a continuous process. Habitats that contain protein are the best sources to isolate proteolytic microorganisms. Waste products of meat, poultry and fish processing industries can supply a large amount of protein rich materials for bioconversion to recoverable products (Dalev, 1994). The types of proteolytic enzymes formed may depend on the composition of the medium. Culture conditions plays significant roles on growth and production of proteolytic bacteria.

Lipolytic is defined as an enzyme (Lipases) that breaks down of lipids and involves hydrolysis of triglyceride into glycerol and free fatty acids. Hydrolytic enzymes like lipases furnish the greatest share in the industrial enzyme market. Lipases (triacylglycerol, EC.3.1.1.3) are a major group of biocatalyst that catalyzed the hydrolysis of triacylglycerol to glycerol and fatty acid (Fujii et al., 1986). In wake of recent advancements in microbiology and biotechnology, lipases have emerged as key enzymes owing to their multifaceted properties which find use in a wide array of industrial application. Lipases have been isolated and purified from fungi, bacteria, plant and animal sources. But bacteria lipases are more economical and stable. Bacteria lipases are extensively used in food diary industry, cheese ripening, flavor enhancement, detergent industries, textile industries, for the synthesis of biodegradable polymers or compounds, different trans-esterification, cosmetic industries, in pulp and paper industries in synthesis of biodiesel and pharmaceutical industries (Hasan et. al., 2006).

An abattoir has been defined as a premise approved and registered by the controlling authority for hygienic slaughtering and inspection of animals, processing and effective preservation and storage of meat products for human consumption. Abattoir operations like slaughtering, processing and meat packaging produce a characteristic highly organic waste into the environment (Alonge, 1991; Eze and Ikeri, 2010). Livestock waste spills can introduce enteric, pathogens and excess nutrients into surface waters (Meadow, 1995). Livestock production, which is perceived by the public to be potential food for the world’s needy people, is a major pollutant of the countryside, where the animals are raised and cities, if processors do not manage slaughter wastes properly with dung and slurry washed into water ways. Other environmental problems include pollution of soil with dung and the atmosphere with methane (a greenhouse gas) from decomposing waste (Chukwu et al., 2008). Wastes could be hazardous or non hazardous. Hazardous waste is defined as the wastes that possess substantial harm to human health or the environment when not properly treated, stored, transported or disposed off or otherwise managed. While non hazardous waste refers to the waste that is converted into economical use either by analysis or treatment (Gilbert, 1998). Abattoir waste are hazardous waste and is another form of agricultural waste which includes intestinal content, rumen, scraps of tissues, horns, bones, blood, faecal matter, fatty and proteinous materials. Abattoir waste contains small quantities of components which are dangerous or potentially dangerous to the environment. It is not pleasant statistics that a 100 cow dairy herd can produce as much waste as 2400 people. But it is not the only unpleasant fact, in certain types of soil this waste can seep through the ground and reach groundwater, polluting it with nitrate and bacteria. Meat processing industries (abattoirs) are generally less developed in developing countries like Nigeria unlike advanced countries where waste generation, analysis and treatment are considered before constructing the abattoir (Ogbonnaya, 2008). The elimination of a wide range of pollutants and wastes from this environment is an absolute requirement to promote a sustainable development of our society with low environmental impact. Biological processes play a major role in the removal of contaminants and they take advantage of the astonishing catabolic versality of microorganisms to degrade or convert such compounds (Madigan and Markinko, 2008). The process for the microbial degradation of waste comprises determining the significant constituents of a waste, providing one or more microorganisms able at least partially to degrade each determined constituent of the waste, optionally providing one or more other microorganisms capable of partial degradation of products, growing one or more mixed cultures of at least some of the microorganisms on a synthetic mixture of at least some of the determined constituents of the waste and utilizing the adapted population of microorganisms substantially to degrade the actual waste (Ryan and Ray, 2004). Microorganisms are excellent recyclers, breaking down animal and plant matter into molecules that can be re-used by other organisms. These organisms produce enzymes that allow them to break up complex compounds into pieces that can enter the cell to be used for growth and reproduction (Willey et al., 2008).  

1.2 Aim and Objectives

1. Isolation and identification of proteolytic and lipolytic microorganism from soil receiving abattoir effluents.

To isolate and identify possible pathogens from soil receiving abattoir effluents

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