MICROBAL DETERIORATION OF RUBBER LATEX

ABSTRACT

The aim of the study to was to assess the microbial deterioration of rubber latex gotten from rubber tree in Umuahia, Abia State. The microbial evaluation of these products exhibited high bacteria count ranging between 4.7 x 104 to 8.2 x 104 while total coliform plate count ranged from 4.6 x 104 to 7.8x 104 and total fungal plate count has 2.0 x 104 5.0 x 104 from used rubber latex samples while the Unused rubber latex ranged from 3.6 x 104 to 3.6 x 104 while total coliform plate count ranged from 4.5 x 104 to 9.9x 104 and total fungal plate count has 3.4 x 104 to 4.0 x 104. The bacterial isolates from rubber latex sample identified by morphological characteristics. The table reveals the major bacterial isolates to belong to Bacillus specie, E. coli, Staphylococcus aureus, Pseudomonas aeruginosa, Proteus and Micrococcus sp respectively. Three (3) fungal isolates from rubber latex which were identified by their morphological characteristics. The table revealed the fungal isolates to belong to Aspergillus, Aspergillus flavus, and Rhodotorula sp. The percentage occurrence of bacterial isolates from different rubber latex Staphylococcus aureus (28%) was predominant among the samples used in this study followed by E. coli (20%), Micrococcus sp (20%), Bacillus sp (16%), Proteus (12%) while Pseudomonas aeruginosa (4%) was least predominant. The percentage occurrence of fungal isolates from different rubber latex. Aspergillus 46%, was predominant followed by Aspergillus flavus (30.8%) while Rhodotula sp (23%) was less predominant. Natural rubber latex serves as a nutritious medium for the growth and proliferation of rubber degrading microorganisms. This is as a result of the components found in natural rubber latex. Microorganisms specifically gain access to latex mostly as a result of poor technical skill by personnel during tapping and processing in the factory. Microbial degradation of natural rubber is mainly carried out by microorganisms such as bacteria and fungi.

TABLE OF CONTENTS

Title page                                                                                                                                i

Certification                                                                                                                           ii

Dedication                                                                                                                              iii

Acknowledgments                                                                                                                  iv

Table of Contents                                                                                                                   v

List of Tables                                                                                                                          vii

Abstract                                                                                                                                  viii

1.0 CHAPTER ONE                                                                                                             1

1.1 INTRODUCTION                                                                                                          1

1.1.1    Rubber latex                                                                                                               1

1.2       Microbial deterioration of latex                                                                                4

1.3       Uses of Rubber Latex                                                                                                             4

1.4       Advantage of Rubber latex                                                                                         5

1.4.1    Disadvantage of Rubber latex                                                                                    6

1.5       Factor that Affect Rubber latex                                                                                  6

1.5.1    pH and Acidity                                                                                                           6

1.5.2    Nutrient Content                                                                                                         8

1.6       Where Rubber is Gotten from                                                                                    9

1.7       Type of Rubber latex                                                                                                  9

1.7.1    Natural Rubber                                                                                                          9 

1.7.2    Synthetic Rubber                                                                                                        10

1.8       Source of Rubber Latex                                                                                              11

1.8.1    Articulated Laticifers                                                                                                  11

1.8.2    Non- articulated Laticifers                                                                                          11

1.9       Aim and Objectives                                                                                                    12

1.9.1    Objectives                                                                                                                  12

2.0       CHAPTER TWO                                                                                                      13

2.1       LITERATURE REVIEW                                                                                        13

2.1.1    Biodegradation of Different types of Rubber latex                                                    13

2.1.1.1 Biodegradation of Natural rubber Latex                                                                    13

2.1.1.2 Natural Rubber Degrading Bacteria                                                                           14

2.1.2    Natural Rubber Degrading Fungi                                                                               16

2.2       Biodegradation of Synthetic Rubbers                                                                         17

2.2.1    Synthetic Rubber Degrading Bacteria                                                                        17

2.2.2    Synthetic Rubber Latex Degrading Fungi                                                                  19

2.3       Isolation of Microorganisms from Rubber Latex                                                       21

3.0       CHAPTER THREE                                                                                                  23

3.1       MATERIALS AND METHODS

3.1.1    The Study Area                                                                                                           23

3.2       Sample Collection                                                                                                      23

3.3       Media Used                                                                                                                 24

            3.4       Sterilization                                                                                                                24

3.5       Isolation of Microorganism from Rubber Latex                                                        24

3.6       Identification and Characterization of Isolates                                                           25

3.6.1    Gram Staining                                                                                                            25

3.6.2    Motility Test                                                                                                               25

3.7       Biochemical Test                                                                                                        26

3.7.1    Catalase Test                                                                                                               26

3.7.2    Coagulase Test                                                                                                           26

3.7.3    Citrate Test                                                                                                                 26

3.7.4    Oxidase Test                                                                                                               26

3.7.5    Indole Test                                                                                                                  27

3.7.9    Sugar Fermentation Test                                                                                            27

3.8       Identification of Fungi                                                                                                28

3.9       Statistical Analysis                                                                                                     28

4.0       CHAPTER FOUR                                                                                                    29

4.1       RESULTS                                                                                                                 29

5.0       CHAPTER FIVE

5.1 DICUSSION, CONCLUSION AND RECOMMEDNATION                                      35

5.1.1    Discussion                                                                                                                   39

5.2       Conclusion                                                                                                                  39

5.3       Recommendation                                                                                                       39

Reference                                                                                                                    40

Appendix                                                                                                                    43                                                                                                       

LIST OF TABLES

CHAPTER ONE

1.1     INTRODUCTION               

1.1.1 Rubber Latex

The natural rubber which is derived from an Indian word “caoutchouc” can be defined as a coagulated or precipitated product from the latex of rubber tree (Hevea brasiliensis). The rubber

plant which is a native of Brazil was introduced to Nigeria around 1895. It is a variety of plant

belonging to the genus Hevea and the family Euphoribiaceae (Rose et al., 2015). The natural rubber is made from runny, milky liquid called latex that oozes from rubber plants when they are cut. Natural rubber latex refers to the white sap coming out from the Hevea brasiliensis tree and contains minority but relevant components, especially proteins, carbohydrate, phospholipids and inorganic compounds in variable amounts. However, based on the seasonal effects, clone and the state of the soil, the average composition of latex has been given as 25 to 3 polyisoprene; 1 to 1.8% (wt/wt) protein; 1 to 2% (wt/wt) carbohydrates; 0.4 to 1.1% (wt/wt) neutral lipids; 0.5 to 0.6% (wt/wt) polar lipids; 0.4 to 0.6% (wt/wt) inorganic components; 0.4% (wt/wt) amino acids, amides, etc.; and 50 to 70% (wt/wt) water  (Subramaniam, 2015).

According to Koyoma and Steinbuchel (2010) particles are formed specifically in the cytoplasm

of specialized cells called latifiers which are found in the rubber plant. Thus, latex is an endogenous milky fluid synthesized and accumulated under pressure in a net laticifer cells (Marcio et al., 2011). Rubber latex contains a large number of chemical compounds from P, C, N, O, S, Ca, K, Mg, Co, and Fe, either due to their role in latex biosynthesis or just because they are absorbed from the soil. Natural rubber is used in a large variety of products due to its flexibility, resistance, impermeability and insulating properties (Mooibroek and Cornish, 2010).

The latex from rubber is a vital material in the automobile industry as it is used in the manufacture of tyre, car bumpers, seats etc. It takes several distinct steps to make a product out of natural rubber. First, the latex is collected from the rubber trees using a traditional process called rubber tapping. This involves making a wide U-shaped cut in the tree bark. As the latex drips out, it is then collected in a cup. The collected latex from many trees is then filtered, washed and reacted with acid to bring about the coagulation of the rubber particles Natural rubber consists of C₅H₈ units (Isoprene), each of which contains one double bond in the cis configuration with poly-isoprene of brasiliensis containing two additional trans isoprene units in the terminal region. Although dehydrated natural rubber of H. brasiliensis been reported to contain approximately 6% nonpolyisoprene constituents, Rose et al. (2015) stated that out of approximately 2,000 plants that synthesize poly (cis – 1, 4 – isoprene), only natural rubber of Hevea brasiliensis  (99% of the world market) and guayule rubber of argentatum (1% of the world market) are produced commercially (Rose et al., 2015).

Rubber latex contains a large number of microorganisms. Microorganisms such as fungi, bacteria and actinomycetes are capable of degrading natural rubber by producing extra cellular enzymes. Actinomycetes such as Streptomyces spp. are capable of degrading natural rubber as they produce variety of enzymes. Microorganisms gain access to the latex mostly as a result of poor technical skill by personnel during tapping and processing in the factory (Omorusi, 2013). The commonest microorganisms are bacteria such as Streptococcus, Escherichia coli and other related coliforms (Atagana et al., 2009). The fungus (Schizosaccharomyces) also affects the latex by degrading it. According to Rose and Steinbuchel (2013), fungi degrading natural rubber have been isolated from soil and deteriorated tyres. It has been documented that the crumb and matrix of virgin rubber material form interfacial sulphur crosslinks. This therefore causes a problem in the recycling of old tyres by blending ground spent rubber and the virgin rubber followed by vulcanization. Thus, microorganisms capable of breaking sulphur-sulphur and sulphurcarbon bonds are been used to devulcanize waste rubber so as to make the surface polymer chains more flexible and increase their binding upon vulcanization (Atagana, 2009).

 Holst et al. (2016) have studied many sulphur oxidizing species for this purpose and Borel et al. (2017) reported attempts by Faber to grow Fusarium solani upon vulcanized rubber tyres. For microorganisms to thrive in latex, certain factors are taken into consideration such as temperature, nutrient availability, pH, moisture content and aeration. The survival of these organisms through the subsequent stages of processing leads to mechanical instability of the latex due to breakdown of the constituent materials of the latex and depletion of oxygen level. This mechanical instability could lead to the destruction of the refined product of rubber due to the loss of flexibility. Tree tapping creates room for microbial infection as the cambium of the tree will be affected during the process and so is exposed to infections by microbes (Omorusi, 2013). Poor hygienic conditions also lead to the introduction of microbes into the latex. For example when buckets used for collection of latex from the field are not clean, it results to enzyme accumulation which contaminates the newly collected latex by precoagulating it and thereby leading to inferior quality of coagulum. Enzyme accumulation is as a result of the presence of organisms in the bucket, utilizing a substrate of its choice to produce the enzyme. Production of amylase could be as a result of utilization of carbohydrate (starch), or protease production as a result of utilization of protein in the latex present in the bucket. However, preservatives such as phenolic compounds and simple inorganic compounds can be used to preserve rubber latex from putrefaction and coagulation. Some of these could also serve as anti-coagulants (Omorusi, 2013).

1.2 MICROBIAL DETERIORATION OF LATEX

Rubber latex within the tree is sterile (John, 2018), but it is heavily contaminated by microorganisms after tapping as the latex flows along the tapping cut and spout into the cup. In the tropics, the high nutrient level of the latex and the permissible temperature conducive for the growth of microbes enhance the proliferation of the microbial contaminants at the expense of the non-rubber producing substrate. This destabilizes the latex as it thickens and finally coagulates. This has resulted to the addition of preservatives such as ammonia, sodium hypochlorite, sodium sulphite as anti-coagulant. Studies have shown that latex is subject to microbial deterioration even in the presence of these preservatives (Buirke 2000). John and Verstracte (2009) report Azobacter sp, Bacillus chromobacter, Escherichia coli, Klebsiella, Listeria, Micrococcus, Nocardia, Sarcina and Streptococcus from fresh and ammoniorated latex. Bealings and Chua (2012) reported isolation of Escherichia coli and Streptococcus from rubber effluents. Although rubber related business and factories abound in Nigeria, there is obvious paucity of literature, a suppressing fact when there are rubber plantations in virtually all the Southern States.

1.3 USES OF RUBBER LATEX

The most important use of rubber is in vehicle tires, condoms; about half of all the world's rubber ends up wrapped around the wheels of cars, bicycles, and trucks you’ll find rubber in the hard, black vulcanized outsides of tires and (where they have them) in their inner tubes and liners. The inner parts of tires are usually made from a slightly different, very flexible butyl rubber, which is highly impermeable to gases (traps them very effectively), so tires (generally) stay inflated for long periods of time. The fact that rubber can be made either soft or hard greatly increases the range of things we can use it for. Soft and stretchy latex is used in all kinds of everyday things, from pencil erasers, birthday balloons, and condoms to protective gloves, adhesives (such as sticky white Polyvinyl alchol), and paints. Harder rubbers are needed for tougher applications like roofing membranes, waterproof butyl liners in garden ponds, and those rigid inflatable boats (RIBs) used by scuba divers. Because rubber is strong, flexible, and a very poor conductor of heat and electricity, it's often used as a strong, thin, jacketing material for electrical cables, fiber-optic cables, and heat pipes. But the range of applications is truly vast: you'll find it in everything from artificial hearts (in the rubber diaphragms that pump blood) to the waterproof gaskets that seal the doors on washing machines. Neoprene (polychloroprene) is best known as the heat-insulating, outer covering of wetsuits but it has far more applications than most people are aware of. Medical supports of various kind use it because, tightly fitted, it compresses and warms injured bits of your body, promoting faster healing. Since it's flexible and waterproof, it's also widely used as a building material, for example, as a roof and floor sealant, and as a spongy absorber of sound and vibration in door and window linings (Omorusi, 2013).

1.4 ADVANTAGES OF RUBBER LATEX

Ø  Latex is ready to use right out of the container. Latex is inexpensive, it exhibits good abrasion resistance, and is an elastic mold rubber.  Because of its high elasticity, a feature unique to latex is its ability to be removed from a model like a glove. Latex molds are also good for casting wax and gypsum.

Ø  Polysulfide rubber:  Polysulfide molds are very soft, “stretchy” and long lasting, some ven lasting 40 years old. It is good for making molds with severe undercuts and/or very fine detail.

Ø   Silicone rubbers: Silicone rubber has the best release properties of all the mold rubbers. The combination of good release properties, chemical resistance and heat resistance makes silicone the best choice for production casting of resins.

Ø  Polyurethane rubbers: Polyurethanes are easy to use, with many having a simple mix ratio by volume. They are less expensive than silicones and polysulfide.

1.4.1      Disadvantages of Rubber Latex: Low-cost latex products generally shrink. Making molds with latex rubber is slow and time-consuming. Making a brush-on latex mold takes ten days or more. Latex molds are generally not suitable for casting resins.

Ø  Polysulfide rubber: Has offensive odour. polysulfide must be mixed accurately by weight or they will not work. Polysulfide rubber costs higher than latex.

Ø  Silicone rubbers: Silicones are generally high in cost. They are also sensitive to substances, and do not have a long library life.

Ø  Polyurethane rubbers: As silicone rubber has the best release properties, urethane rubber has the worst release properties and will adhere to just about anything. Limited shelf life after opening remaining product may be affected by ambient moisture in the air.

1.5 FACTORS THAT AFFECT RUBBER LATEX

1.5.1 pH and Acidity

Increasing the acidity of latex, either through fermentation or the addition of weak acids, has been used as a preservation method since ancient times. In their natural state, most latexs such as meat, fish, and vegetables are slightly acidic while most fruits are moderately acidic. A few latexs such as egg white are alkaline. The pH is a function of the hydrogen ion concentration in the latex: Another useful term relevant to the pH of fluids is the pKa. The term pKa describes the state of dissociation of an acid. At equilibrium, pKa is the pH at which the concentrations of dissociated and undissociated acid are equal. Strong acids have a very low pKa, meaning that they are almost entirely dissociated in solution (ICMSF, 2012).  For example, the pH (at 25 °C [77 °F]) of a 0.1M solution of HCl is 1.08 compared to the pH of 0.1 M solution of acetic acid, which is 2.6. This characteristic is extremely important when using acidity as a preservation method for latexs. Organic acids are more effective as preservatives in the undissociated state. Lowering the pH of a latex increases the effectiveness of an organic acid as a preservative.

It is well known that groups of microorganisms have pH optimum, minimum, and maximum for growth in latexs. As with other factors, pH usually interacts with other parameters in the latex to inhibit growth. The pH can interact with factors such as a_w, salt, temperature, redox potential, and preservatives to inhibit growth of pathogens and other organisms. The pH of the latex also significantly impacts the lethality of heat treatment of the latex. Less heat is needed to inactivate microbes as the pH is reduced (Mossel et al., 2015). 

Another important characteristic of a latex to consider when using acidity as a control mechanism is its buffering capacity. The buffering capacity of a latex is its ability to resist changes in pH.  Latexs with a low buffering capacity will change pH quickly in response to acidic or alkaline compounds produced by microorganisms as they grow. Titratable acidity (TA) is a better indicator of the microbiological stability of certain latexs, such as salad dressings, than is pH. Titratable acidity is a measure of the quantity of standard alkali (usually 0.1 M NaOH) required to neutralize an acid solution (ICMSF, 2012). It measures the amount of hydrogen ions released from undissociated acid during titration. Titratable acidity is a particularly useful measure for highly buffered or highly acidic latexs. Weak acids (such as organic acids) are usually undissociated and, therefore, do not directly contribute to pH. Titratable acidity yields a measure of the total acid concentration, while pH does not, for these types of latexs.

1.5.2 Nutrient Content

Microorganisms require certain basic nutrients for growth and maintenance of metabolic functions. The amount and type of nutrients required range widely depending on the microorganism. These nutrients include water, a source of energy, nitrogen, vitamins, and minerals (Mossel et al., 2013).  Varying amounts of these nutrients are present in latexs. Meats have abundant protein, lipids, minerals, and vitamins. Most muscle latexs have low levels of carbohydrates. Animal latexs have high concentrations of different types of carbohydrates and varying levels of proteins, minerals, and vitamins. Latexs such as milk and milk products and eggs are rich in nutrient.  Microorganisms found in latex can derive energy from carbohydrates, alcohols, and amino acids. Most microorganisms will metabolize simple sugars such as glucose. Others can metabolize more complex carbohydrates, such as starch or cellulose found in plant latexs, or glycogen found in muscle latexs. Some microorganisms can use latexs as an energy source.

Amino acids serve as a source of nitrogen and energy and are utilized by most microorganisms. Some microorganisms are able to metabolize peptides and more complex proteins. Other sources of nitrogen include, for example, urea, ammonia, creatinine, and methylamines.  Examples of minerals required for microbial growth include phosphorus, iron, magnesium, sulfur, manganese, calcium, and potassium. In general, small amounts of these minerals are required; thus a wide range of latexs can serve as good sources of minerals (Struchtemayer, 2012).  In general, the Gram-positive bacteria are more fastidious in their nutritional requirements and thus are not able to synthesize certain nutrients required for growth (Jay 2010). For example, the Gram-positve organisms, S. aureus requires amino acids, thiamine, and nicotinic acid for growth (Jay 2010).

1.6 WHERE RUBBER IS GOTTEN FROM

According to Martin (2008), latex was known to be the basic raw materials from rubber tree which goes into the processing of all rubber. Latex is obtained by tapping the rubber tree, the most common system being the half spiral type which starts as high on the tree as possible and extends downwards around the tree at an angle of 30⁰C to sever the maximum number of latex vessels (John and Verstracte, 2009).

1.7 TYPES OF RUBBER LATEX

1.7.1 Natural Rubber

 Natural rubber is made from a runny, milky white liquid called latex that oozes from certain plants when you cut into them. (Common dandelions, for example, produce latex; if you snap off their stems, you can see the latex dripping out from them. In theory, there's no reason why we couldn't make rubber by growing dandelions, though we'd need an awful lot of them.) Although there are something like 200 plants in the world that produce latex, over 99 percent of the world's natural rubber is made from the latex that comes from a tree species called Hevea brasiliensis, widely known as the rubber tree. This latex is about one third water and one third rubber particles held in a form known as a colloidal suspension. Natural rubber is a polymer of isoprene (also known as 2-methylbuta-1,3-diene) with the chemical formula (C₅H₈)n. To put it more simply, it's made of many thousands of basic C₅H₈ units (the monomer of isoprene) loosely joined to make long, tangled chains. These chains of molecules can be pulled apart and untangled fairly easily, but they spring straight back together if you release them and that's what makes rubber elastic (Omorusi, 2013).

1.7.2   Synthetic Rubbers

Synthetic rubbers are made in chemical plants using petrochemicals as their starting point. One of the first (and still one of the best known) is neoprene (the brand name for polychloroprene), made by reacting together acetylene and hydrochloric acid. Emulsion styrene-butadiene rubber (E-SBR), another synthetic rubber, is widely used for making vehicle tires. It takes several quite distinct steps to make a product out of natural rubber. First, you have to gather your latex from the rubber trees using a traditional process called rubber tapping. That involves making a wide, V-shaped cut in the tree's bark. As the latex drips out, it's collected in a cup. The latex from many trees is then filtered, washed, and reacted with acid to make the particles of rubber coagulate (stick together). The rubber made this way is pressed into slabs or sheets and then dried, ready for the next stages of production. By itself, unprocessed rubber is not all that useful. It tends to be brittle when cold and smelly and sticky when it warms up. Further processes are used to turn it into a much more versatile material. The first one is known as mastication (a word we typically use to describe how animals chew food). Masticating machines "chew up" raw rubber using mechanical rollers and presses to make it softer, easier to work, and more sticky. After the rubber has been masticated, extra chemical ingredients are mixed in to improve its properties (for example, to make it more hardwearing). Next, the rubber is squashed into shape by rollers (a process called calendering) or squeezed through specially shaped holes to make hollow tubes (a process known as extrusion). Finally, the rubber is vulcanized (cooked): sulfur is added and the rubber is heated to about 140°C (280°F) in an autoclave (a kind of industrial pressure cooker) (Omorusi, 2013).

1.8 SOURCES OF RUBBER LATEX

1.8.1 Articulated Laticifers 

The cells (laticifers) in which latex is found make up the laticiferous system, which can form in two very different ways. In many plants, the laticiferous system is formed from rows of cells laid down in the meristem of the stem or root. The cell walls between these cells are dissolved so that continuous tubes, called latex vessels, are formed. Since these vessels are made of many cells, they are known as articulated laticifers. This method of formation is found in the poppy family and in the rubber trees (Para rubber tree, members of the family Euphorbiaceae, members of the mulberry and fig family, such as the Panama rubber tree Castilla elastica), and members of the family Asteraceae. For instance, Parthenium argentatum the guayule plant, is in the tribe Heliantheae; other latex-bearing Asteraceae with articulated laticifers include members of the Cichorieae, a clade whose members produce latex, some of them in commercially interesting amounts. This includes Taraxacum kok-saghyz, a species cultivated for latex production (Omorusi, 2013).

1.8.2 Non-Articulated Laticifers    

In the milkweed and spurge families, on the other hand, the laticiferous system is formed quite differently. Early in the development of the seedling, latex cells differentiate, and as the plant grows these latex cells grow into a branching system extending throughout the plant. In many euphorbs, the entire structure is made from a single cell – this type of system is known as a non-articulated laticifer, to distinguish it from the multi-cellular structures discussed above. In the mature plant, the entire laticiferous system is descended from a single cell or group of cells present in the embryo. The laticiferous system is present in all parts of the mature plant, including roots, stems, leaves, and sometimes the fruits. It is particularly noticeable in the cortical tissues.

Latex is usually exuded as a white liquid, but is some cases it can be clear, yellow or red, as in Cannabaceae (Omorusi, 2013).

1.9 AIM AND OBJECTIVES

The aim of this study is to assess the microbial deterioration of rubber latex gotten from rubber tree in Umuahia, Abia State.

1.9.1 Objectives

1. To determine the bacterial and fungal load from rubber latex.

2. To characterize microorganisms isolated from rubber latex.

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