CHAPTER ONE 1. 0 INTRODUCTION Natural rubber is produced by over 2000 plants species and its main constituent is poly (cis-I,4-isoprene). a highly unsaturated hydrocarbon. Since 1914 there have been efforts to investigate microbial rubber degradation: However, only recently have the first proteins involved in this process have been identified and characterized and have the corresponding genes cloned. Analysis of the degradation product of natural rubber and synthetic rubbers isolated from various bacterial cultures indicated without expectation that there was oxidative cleavage of the double bond in the polymer backbone.
A similar degradation mechanism was postulated for the cleavage of squalene, which is a triterpene intermediate and precursor of steroids and triterpeniods, aldehydes and /or carbonyl groups were detected in most of the analyzed degradation products isolated from cultures of various rubber degrading strains. Knowledge of the degradation at the protein and genes levels and detailed analysis of detectable degradation products should result in a detailed understanding of these obviously new enzymatic reactions. 1. 1 PROBLEM STATEMENT
Some difficulties arise while investigating microbial degradation of natural rubber. a. Microbial degradation of natural rubber is a slow process. The growth of bacterial which utilizes the rubber (i. e. the carbon source) is slow also. Therefore time factor for incubation of strain is a problem because to obtain enough cell mass or degradation product of the polymers the strain period of incubation is extended over weeks. b. Additional problems aroused from the presence of other natural biodegrading compounds in natural rubber and latex, even from additives which are required for vulcanization.
The latex coagulates spontaneously after collection so the strain was inoculated into its whilst in the liquid phase. Because working with it in its cuplum form is very tedious. However fillers and stoppers are added to inhibit biodegradation of the rubber strain. c. Preserving the life of the micro-organism was also a problem because continual degradation requires sustaining the life of the microbe. 1. 2 OBJECTIVE OF STUDY The aim of this study simply put is as follows; a. Clarifying the taxonomic position of strain. . Reduce the effect done by rubber degradation in the environment (i. e. environmental hazard). c. To determine the extent and time to achieve complete degradation of the rubber by strain (Gordonia specie). 1. 3 JUSTIFICATION OF STUDY The degradation of natural rubber by Gordonia sp. Involves chemical changes and therefore requires some chemical means of demonstration. For instance, the occurrence of isoprene oligomers containing aldehydes and ketones after incubation of the latex glove with G. oly-isoprenivorans and other bacterial and the reduction in the numbers of double bonds in the polyisoprene chain were demonstrated by straining with Schiff’s reagents using Fourier transform infrared spectroscopy with attenuated total reflectance. This project tries to justify the degrading characteristic of “Gordonia specie” on natural rubber by a suitable physical means viscosity measurement using a simple ubbelohde viscometer. It is suitably the most reliable test for degradation as it’s requires simple equipments for optimum yield, because the use of enzymes is very economical, requires fewer chemicals.
Only a suitable solvent for the rubber is necessary and it is very efficient in evaluating the degree of degradation. 1. 4 SCOPE OF STUDY The project deals with the degradation of natural rubber latex by specie of microbe known as Gordonia specie, using dilute solution viscosity measurement. Hevea brasilienses is the specie of rubber tree used and all measurements were carried out using an ubbelohde viscometer. This project is organized carefully to obtain a positive and commendable result in the following ways; Five sections (chapters) were used to organize this research work.
The first section gives precise information on the research study. The second section deals with the general background information on the study; the literature review and also it contains contributions from authors of similar works. Section three covers the methods and procedures used to obtain the research results while the fourth section contains observations, data collections and graphical representations for the research work. The final section contains the summary, and conclusion drawn from the project work with recommendations.
CHAPTER TWO 2. 0 LITERATURE REVIEW Microbial degradation of natural rubber has been investigated for 100years (48) and it’s obvious that bacterial as well as fungi are capable of degrading rubber and the rubber degradation is a slow process (14,19,21,23,34,50). The introduction of latex plate which consisted of a bottom agar layer of mineral salt medium and a layer of agar on top, for isolation and cultivation of rubber degrading micro-organism was an achievement (50).
Micro-organisms growing on such plates formed clear zone around when 1,220 different bacteria were investigated for the ability to degrade rubber elastomer overlay agar technique, 50 clear-zones rubber degrading strains all bacteria mycelium-forming actinomycetes were identified. Formations of cleavage inhibited by addition of glucose, indicating that there was regulation of the expression in degrading enzymes. Growth of some of the strains on natural rubber led to significance in (10-30%, wt/wt) of the mineral used and to a decrease in the average molecular weight of the polymer from 640,000 to about 25,000.
One major disadvantage of latex overlay agar plate is that not all rubber-degrading bacteria is cultivated in this way, because many do not form halos on such plates and because polyisoprene is locally available to allow formation of visible colonies by these, the degrading bacteria were therefore divided into two groups according to the growth characteristics. With one exception, representative of the first group belongs to the forming actinomycetes mentioned above and metabolize the polyisoprene by secretion of several enzymes.
Most representatives of this group show weak growth in synthetic rubbers. Members of this second group do not form halos and do not grow, rather they require direct contact with the polymer and growth in rubber is adhesive in all the members of this group showing relatively strong growth on polyisoprene and belongs to corynebacterium-Nocardia-mycobacterium group. Some new rubber degrading species, the corynebacterium-Nocardia-mycobacterium group such as Gordonia polyisoprene VH2 and Y2K, G. westfalica strain kb1 and mycobacterium fortuitum strain NF4 re recently species of the genus Gordonia. 2. 1 OCCURRENCE AND CHEMICAL STRUCTURE OF NATURAL RUBBER. Natural rubber is an elastic hydrocarbon polymer which occurs as a milky colloidal suspension, or latex. The term natural or caoutchouc (from Indian: caa=tears; ochu=tree; caoutchouc=weeping tree) refers to a coagulated or precipitated product obtained from latex of rubber plant (hevea brasilienses) which forms nonlinked but partially vulcanizable polymer chains having molecular masses of about 106 Da with elastic properties.
At higher temperatures natural rubber is plastically ductile and useful for production of elastomers. Latex acts as clogging material during healing of wounds caused by mechanical injury in plants. Natural rubbers consist of C5H8 unit (isoprene), each containing one double bond in the cis configuration. Although approximately 2,000 plants synthesizes poly (cis-1, 4-isoprene). Only natural rubber of Hevea brasilienses (99% of the world market) and guayule rubber of parthenium argentatum (1% of the world market) are produced commercially (52).
Latex of Hevea plants contains about 30% ploy (cis-1,4-isoprene) and is harvested by a “tapping” procedure after the back of the plant is notched diagonally, which yield 100-200ml latex resin within 3hours. Such tapping is usually carried out every 2 to 3 days, yielding up to 2,500kg of natural rubber per year per ba. In 1998, the world production of natural rubber was about 6. 6 million tons; more than 70% of this rubber was produced in only three countries (Malaysia, Thailand, and Indonesia), and about 40% was purchased by only three countries (United State, China, and Japan).
Most of the natural rubber (75%) is used for production of automobile tires. CH2 CH CH CH2 CH3 n poly (1,4-isoprene) CH3 H CH3 CH2 C C C C CH2 CH2 n CH2 H n Cis-isomer. Trans-isomer Fig 2. 1 structure of natural rubber COMPOSITION Depending on the clone, seasonal effect, and the state of the soil, the average composition of latex is as follows; 25 to 35% (wt/wt) polyisoprene; 1 to 1. % (wt/wt) protein; 1 to 2% (wt/wt) carbohydrate; 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 e. t. c and 50 to 70% (wt/wt) water. The polymer is present in 3 to 5µm called rubber particles. Which are covered by a layer of protein and lipids, which separates the hydrophobic rubber molecules from the hydrophilic environment? PROPERTIES Rubber exhibits unique physical and chemical properties.
Rubber stress-strain behavior exhibits the Mullins effect, the Payne effect and is often modeled as hyper elastic. Natural rubber is capable of crystallizing on stretching which shows high tensile strength and modulus. Owing to the presence of double bond in each and every repeat unit, natural rubber is sensitive to ozone attack. Natural rubber is water repellant and resistant to alkalis and weak acids. It has good elasticity capable of stretching up to 1000% of its original size when vulcanized; it has toughness, its impermeable, and has good adhesiveness and good electrical resistance.
However, natural rubber has poor aging and weathering properties and its less resistant to oil, solvents, oxygen, ozone and certain chemicals and resilient over a wide temperature range, when compared to synthetic rubbers. USES The use of natural rubber is widespread, ranging from house-hold to industrial products. Tires and tubes are the largest consumers of rubber, accounting for around 56% total consumption in 2005. The remaining 44% are taken up by the general rubber goods (GRG) sector, which includes all products except tires and tubes.
Other important uses of natural rubber are in door and window profiles, belts, matting, hoses, flooring and dampeners (anti-vibration mounts) for automotive industry. Gloves (medical, household and industrial) are also large consumers of rubber and toy balloons. Though the type of rubbers used is that of the concentrated latex. Significant tonnage of rubber is used as adhesives in many manufacturing industries and products and the two most noticeable are the paper and carpet industry. Rubber is also used in making rubber bands and pencil erasers. Furthermore, ubber produced as a fiber sometimes called elastic, has significant value for use in the textile industry because of its excellent elongation and recovery properties. Because of its low dye acceptance, feel and appearance, the rubber fiber is either covered by yarn of another fiber or directly woven with other yarns into the fabric. Seeking a way to address this short comings, the textile industry has turned into neoprene (polymer form of chloroprene), a type of synthetic rubber and another more commonly used elastomer fiber, spandex (also known as elastane) because of their superiority to rubber in both strength and durability. . 2 BIOLOGY OF THE METABOLICALLY DIVERSE GENUS GORDONIA The actinomycete genus GORDONIA has attracted much interest in recent years for variety of reasons. Most specie were isolated due to their abilities to degrade xenobiotics, environmental pollutant, or otherwise slowly biodegradable. Natural polymers as well as to transform or synthesize possibly useful compounds. The varieties of chemical compounds being transformed, biodegraded and synthesize by gordoniae makes these bacteria potentially useful environmental and industrial biotechnology.
Gordonia specie have been isolated from various native biotopes such as soil and mangrove rhizosphere, from extensively industrially influenced habitats such as oil-producing wells or hydrocarbon contaminated soil, and from diseased humans. The genus gordonia belongs phylogenetically to the suborder corynebacterineae, the mycolic acid group within the order Actinomycetales, and its classification has changed drastically in recent years, with several species being reclassified and many novel species being described. Fig 2. 2 Degradation of N. R by Gordonia polyisoprenivorans VH2.
Left: Noninoculated control. Right: Complete disintegration of the inoculated sample after 6 weeks. 2. 3 TAXONOMY OF THE GENUS GORDONIA In 1971, Tsukamura proposed Gordonia as a new genus for coryneform bacteria isolated from sputa of patients with pulmonary disease or from soil. The name from this novel genus was chosen to pay tribute to the American bacteriologist Ruth E. Gordon. Members of this genus are distinguished from fast growing mycobacterial by their slight acid fastness and the absence of the arylsulfatase, and the genus Nocardia by their ability to reduce nitrate and the absence of a mycelium.
In 1997, the etymologically correct name Gordonia instead of gordona was proposed by Stackebandt et al (86). According to their newly proposed hiercharchic classification system for the actinomycete line, Gordonia is the type genus of Gordoniaceae (the Gordonia family) within the suborder includes also the families’ corynebacteriaceae, Dietziaceae, mycobacteriaceae, Nocardiaceae, Tsukamureuaceae, and Williamsiaceae. At present the genus gordonia comprises 19 validly published species, and at least two further species are in the process of classification.
Meanwhile, gordoniae have been shown to be ubiquitously distributed in nature. 2. 4 MORPHOLOGICAL AND CHEMOTAXONOMIC PROPERTIES Morphological and structural aspects: members of the genus gordonia are aerobic, catalase-positive, gram-positive to gram-variable, slightly acid-fast, nonmotile nocardioform actinomycetes. They have an oxidative carbohydrate metabolism and are arylsulfatase negative; susceptibility to lyzosome has been shown. The term “Nocardioform” is morphologically descriptive and refers to mycelia growth with fragmentation into rod-shaped or cocciod elements. Gordoniae do not generate spores.
The colony morphology of gordonia species varies from slimy, smooth, and glossy to irregular and rough; it may even differ within one species depending on the medium used for growth. Some strains such as Gordonia alkanivorans DMS 44369 and Gordonia westfalica DSM 44215 are also able to form both smooth rough colonies usually cultured with smooth phenotypes can generate rough colonies, this alteration seems to be irreversible and could be due to mutations in the genes encoding. The importance of glycosylated peptidolipids for colony morphology has also been demonstrated for G. ydrophobica. The colors of the colonies cover a broad range include white, yellow, tannish, orange, red, and pink. The cell walls belong to the wall chemo type IV sensu lechevalier and lechevalier. The peptidoglycanis of the A1 r type containing mesodiaminopimelic acid as the only diamino acid muramic acid with N-glycolyl residues. The major cell wall sugars are arabinose and galactose. 2. 5 PATHOGENIC GORDONIA SPECIES Most gordonia species have been isolated from environmental source. However, a few are also sporadically associated with human infections.
In almost all cases, patients were immunosupressed after underlying diseases, and infections by Gordonia species occurred only secondarily. Altogether, approximately 20 cases reports of gordonial infections can be found in the literature. Most of these infections are caused by G. bronchialis and were associated with stenal wounds resulting from surgery. A few infections also occur after coronary artery bypass surgery and were associated with heart-lung machines. Two additional infections with gordonia species have been reported in the literature. Interestingly one of this reports described an infection by a Gordonia specie.
Taxonomically closely related to G. sputa. Surprisingly, DNA-DNA relatedness to G. sputa, as analyzed by the stringent nuclease S1 method, did not meet the genetic criterion of more than 70% similarity to belong to the same species. Consequently, one must suggest that this strain could belong to the novel gordonial specie; so far, reports of human gordonial infections are rather rare in comparism to report on infection caused by other opportunistically pathogenic bacteria belonging to taxonomically related genera such as Rhodococcus and Nocardia. . 6 CAPABILITIES FOR BIODEGRADATION AND BIOREMEDIATION. Gordoniae are probably important in natural environments and are powerful candidate for bioremediation processes because of their capacity to degrade substituted and non-substituted hydrocarbons. Widespread environmental pollutants, other xenobioties and natural compounds that is not readily biodegradable. Gordonia species may play an important role during waste water treatment and in boifilters. Several Gordonia sp.
Were isolated due to their capabilities to degrade or modify aliphatic and aromatic hydrocarbons, halogenated aromatic compounds, benzothiophene, nitrile polyisoprene, xylene and so forth. Incorporation of the long chain of the mycolic acids into the cell walls is associated with hydrophobicity and surface adhesion and may play a role in the degradation of hydrophobic pollutants. Examples of this growth of several gordonia strains during the biodegradation of rubber materials (2,63,65) and the utilization of hydrophobic hydrocarbon by many species of this genus. 2. ANABOLIC CAPABILITIES. Biosurfactants: The capability for biodegradation of water-insoluble and hydrophobic compounds is often associated with the production of biogenic surface-active amphiphilic compounds by a bacterium. The relevance of biosurfactant to the biodegradation of pollutants is three fold. i. Cellular biosurfactant such as mycolic acid causes adherence of the microbial cells to hydrophobic phases in two phase system. ii. Surfactants promote the access of hydrophobic compounds to microbial cells by decreasing the interfacial tension between the phases. ii. Surfactants dispersed hydrophobic compounds, leading to an increased surface area accessible for microbial attacks. On the other hand, biosurfactants may cause undesired mobilization and movement of hydrophobic pollutants in natural environments, resulting in the release of the pollutants into groundwater. In general, biosurfactants can be sub-divided into low molecular weight compounds such as glycolipids and lipopeptides and high molecular weight polymeric compounds such as polysaccharides, lipoproteins, and lipo-polysaccharides. . 8 GENETIC MANIPULATION OF GORDONIA STRAINS. An investigation of the interesting metabolic pathways existing in gordoniae and the genes encoding the respective enzymes was hampered until recently by the unavailability of suitable genetic tools. The first vectors appropriate for gene transfer to Gordonia sp. Have been described only recently by Arenskotter et al. These are based on an origin of replication (oriv) of a native Rhodococcus plasmid, and genes can be transferred either by electro-transformation or by conjugational transfer.
Evidence was obtained that pKB1 encodes genes essential for cadmium resistance and rubber biodegradation based on the oriv region of this megaplasmid, E. coli-gordonia shuttle vectors suitable for genes cloning and expression in several gordonia species and members of related taxa were constructed. 2. 9 UBBELOHDE VISCOMETER. An ubbelohde viscometer is a measuring instrument which uses a capillary based method of measuring viscosity. It is recommended for higher viscosity cellulosic solutions. The advantage of this instrument is that the values obtained are independent of the concentration.
The device was invented by the German chemist Leo Ubbelohde (1877-1964). The ubbelohde viscometer is closely related to the Ostwald viscometer. Both are U-shaped pieces of glassware with a reservoir on the right and a measuring bulb with a capillary on the left. A liquid is introduced and a pressure head forces this liquid from the bulb through the capillary to the reservoir. The time it takes for the liquid to pass through two calibrated marks is a measure for viscosity. The ubbelohde device a third arm extending from the end of the capillary and open to the atmosphere.
In this way the pressure head only depends on a fixed height and no longer on the total volume of the liquid. Fig 2. 3 A pictorial diagram of an ubbelohde viscometer This kind of viscometer cannot operate at a very low concentration and must therefore use several concentrations for Huggins plot to derive intrinsic viscosity by extrapolation to zero concentration. ?red ?sp/c ? intr C Fig 2. 4 Huggins/Kraemer plot CHAPTER THREE 3. 0 MATERIALS AND METHODS 3. 1 MATERIALS a. Culture materials for Gordonia sp.
And functions * Culture medium; this is the basic growth medium for Gordonia sp. * Petri dish; a round flat bottom plate in which the strain culture is prepared. * Flame burner; used to sterilize the wire loop and glass hockey. * Incubator; equipment which provides a suitable environment for the growth of the strain. It regulates the temperature within it. * Wire loop; a metal-like tool used for inoculation. * Glass hockey; the strain is prepared over the culture medium using this device. * Microscope; micro features of organisms are observed under the microscope. Inoculation chamber; a partially confined chamber for inoculation to prevent contamination * Masking tape; used to distinguish similar items. * Latex coagulum; a possible source of the strain. * Autoclave; for sterilization. * Distilled water; for preparation of medium and dilution. b. Materials used for viscometric analysis * Ubbelohde viscometer; a measuring instrument which uses a capillary based method of measuring viscosity. * Retort stand; used to suspend the viscometer. * Latex (coagulum); based material on which degradation is quantified. * Solvent (xylene); for latex dissolution. Measuring cylinder; an apparatus used to measure required quantities of fluids. * Digital weighing balance; this is an electronic device used to determine weight of objects and substances. * Stirrer rod; for the homogenization of solutions. * Test tubes; transparent glassware used to store fluid samples for observation and tests. * Suction pump; to aid the flow of fluid through the capillary in the viscometer against gravity. * Test tube rack; where the test tubes are held / kept. * Stop watch; this electronic device helps to measure the efflux time of fluids in the viscometer. 3. 2 METHODS a.
Isolation of the rubber-assimilating organism A mineral agar medium without a carbon source was prepared. This medium contains 10g of casein peptone dissolved in 850ml of distilled water, 5g/850ml glucose, 5g/850ml yeast extract, 5g/850ml sodium chloride, and 15g agar. The PH of the mixture was adjusted to 7. 3 and then diluted to 1000ml with distilled water. Appropriate amount of organic soil (enriched with long standing latex coagulum) were soaked in distilled water for about 168 hours (1 week) and then filtered. 10-15ml of the filtrate were introduced into the agar media and incubated at 25oC until colonies grew.
The colonies were spread onto new mineral agar plates for purification and incubated as described above. b. Cultivation of Gordonia sp. Bacterium medium was used to cultivate the newly isolated-degrading organism. 1g inoculums specie was collected from the composite sample and diluted serially in a 10-fold dilution. 0. 1ml and 1. 0ml sob-inoculums were separated cultured using the plate and pour plate techniques respectively. In the spread plate, a glass hockey was used to spread the 0. 1ml inoculums evenly over the surface of the plate containing solid agar.
It was then incubated at 35oC and monitored daily for growth. 1. 0ml was carefully spread in an empty sterile Petri dish and 15ml of the culture medium were added to the inoculums. The plates were then incubated at 35-37oC for 48 hours and later observed for certain characteristic features such as colonies, distinct colors, biochemical reactions, carbohydrate utilization and gram stained for microscopy. Gordonia sp. On identification and isolation, pure sub-cultures were prepared as described for use. c. Inoculation Freshly taped latex was poured in two beakers, both contained equal amounts.
The beakers were then transferred to the inoculation chamber. A wire loop was used to inoculate the strain into one of the latex-contained beakers and was stirred vigorously. The entire content was incubated at 30oC for about 120 hour Fig 3. 1 A: Solubilized material recovered from a 5-day-old NR latex culture of G. polyisoprenivorans VH2 after ultracentrifugation and chloroform extraction of the solid-free supernatant. B: Demonstration of the solubilizing properties of the supernatant of a 5-day old NR latex culture of G. polyisoprenivorans VH2. Left: Latex coagulate.
Middle: Prevention of latex coagulation after addition of 3mL of filter-sterilized supernatant. Right: Total inhibition of latex coagulation. d. Viscometric analysis. The prepared sample was sterilized in an autoclave to terminate the activity of the Gordonia sp. Strain before conducting the volumetric test. 1g of latex was weighed and dissolved in 50ml of xylene and then stirred until a homogenous was obtained. The solution was diluted in turn to obtain the following concentrations; 0. 2, 0. 4, 0. 6, 0. 8 grams-latex/50ml-xylene. The water bath was equilibrated at 60oC for about 20minutes. 0ml of xylene (pure solvent) was introduced into the viscometer suspended by a retort stand and allowed to equilibrate for about 5 minutes in the constant temperature bath. The efflux time of the solvent was measured. The solvent was then removed and the viscometer rinsed with distilled water, dried at 50oC in an oven and allowed to cool to room temperature. The procedure was repeated at 5-days intervals for the dilute solutions prepared from the two beakers until 40-days. The results are presented in chapter four of the project. 3. 3 DETERMINATION OF VISCOSITY The determination of viscosity is based on poiseuille’s law. Vdt=v? R2 =? R48? -? P? x= ? R48? ?? P? L Where “t” is the time it takes for a volume V to elute. The ratio dVdt depends on R as the capillary radius, on the average applied pressure P, on its length L, and on the dynamic viscosity ?. The average pressure head is given by: ?P=? g? H With ? the density of the liquid, g the standard gravity and h the average head of the liquid. In this way the viscosity of a fluid can be determined. Usually the viscosity of a liquid is compared to a liquid with an analyte for example, if a polymer dissolves in it. The relative viscosity is given as; ? r = ?? o= t? to? o
Where to and po are the elution time and density of the pure liquid. When the solution is very dilute. p ? po Then, the specific viscosity becomes: ?sp = ? r – 1= t-toto This specific viscosity is related to the concentration of the analyte through the intrinsic viscosity [? ] by the power series: ? sp = [? ]C + k[? ]2 C2 + …….. Or ?spC=? + K[? ]2C+… Where ? sp/C is called the viscosity number or the reduced viscosity ? red. The intrinsic viscosity can be determined experimentally by measuring the viscosity number as a function of concentration as the Y-axis intercept. INSTRUCTION FOR THE USE OF THE UBBELOHDE VISCOMETER . Clean the viscometer using suitable solvents, and by passing clean, dry, filtered air through the instrument to remove the final traces of solvents. Periodically, traces of organic deposits should be removed with chromic acid or non-chromium cleaning solution. 2. If there is a possibility of lint, dust, or other solid material in the liquid sample, filter the sample through a fritted glass filter or fine mesh screen. 3. Charge the viscometer by introducing sample through tube L into the lower reservoir; introduce enough samples to bring the level between lines G and H. 4.
Place the viscometer into the holder, and insert it into the constant temperature bath. Vertically align the viscometer in the bath if a self dydx aligning holder has not been used. 5. Allow approximately 20 minutes for the sample to come to the bath temperature. 6. Place a finger over tube M and apply suction to tube N until the liquid reaches the center of bulb D. Remove suction from tube N. Remove finger from tube M, and immediately place it over tube N until the sample drops away from the lower end of the capillary into bulb B.
Then remove finger and measure the efflux time. 7. To measure the efflux time, allow the liquid sample to flow freely down past mark E, measuring the time for the meniscus to pass from mark E to mark F . 8. Calculate the kinematic viscosity of the sample by multiplying the efflux time by the viscometer constant. 9. Without recharging the viscometer, make check determinations by repeating steps 6 to 8. Fig 3. 2 Ubbelohde viscometer CHAPTER FOUR 4. 0 RESULTS AND DISCUSSION 4. 1 Isolation and Characterization of Gordonia sp. Gordonia sp. as isolated from an organic soil composites enriched with long exposed latex coagulum and it was able to utilize natural rubber as a sole source of carbon and energy. Colonies of the spores were located at various spores formed on the latex coagulum. It emits a fermented starch-like odour, produced a variety of colored spots and exhibited surface irregularities. The cells were gram positive to corynebacterium media, rod shaped of approximately 0. 5-0. 8µm in the bacterium agar for growth. The strain was cultivated in liquid medium supplemented with the organism of xylose and D-frutose and were also identified as a sole carbon sources.
The dilute solution viscosity measurement of Gordonia sp. Degrades natural rubber was carried out at 60oC using xylene as the solvent. The efflux time (to) of the solvent was measured and that of the dilute solution are presented below. 4. 2 RESULTS Efflux time of solvent, to =15. 25sec Temperature T =60oC Efflux time of various solutions of natural rubber. a. Without the strain (control) CONCENTRATION,C (g/dl)| EFFLUX TIME, t(s)| 0. 2| 181. 91| 0. 4| 206. 68| 0. 6| 240. 26| 0. 8| 277. 75| 1. 0| 322. 74| b. After 5 days activity of the strain.
CONCENTRATION,C (g/dl)| EFFLUX TIME, t(s)| 0. 2| 181. 58| 0. 4| 206. 00| 0. 6| 236. 35| 0. 8| 272. 54| 1. 0| 314. 59| c. After 10 days activity of the strain. CONCENTRATION,C (g/dl)| EFFLUX TIME, t(s)| 0. 2| 181. 26| 0. 4| 205. 38| 0. 6| 235. 37| 0. 8| 271. 23| 1. 0| 312. 96| d. After 15 days activity of the strain. CONCENTRATION,C (g/dl)| EFFLUX TIME, t(s)| 0. 2| 180. 60| 0. 4| 204. 00| 0. 6| 233. 42| 0. 8| 265. 2| 1. 0| 309. 70| Corresponding viscosity numbers of various dilute solutions of natural rubber. CONCENTRATION, C (g/dl)| VISCOSITY NUMBER, ? red (dl/g)| | Control| Day 5| Day 10| Day 15| 0. 2| 0. 58| 0. 57| 0. 56| 0. 54| 0. 4| 0. 67| 0. 66| 0. 65| 0. 63| 0. 6| 0. 79| 0. 75| 0. 74| 0. 72| 0. 8| 0. 88| 0. 84| 0. 83| 0. 79| 1. 0| 0. 98| 0. 93| 0. 92| 0. 90| Viscosity number, (? ) (dl/g) Concentration, C (g/dl) Fig 4. 1 Extrapolated values of Huggins plots HUGGINS PLOTS| INTRINSIC VISCOSITY (? intr)| Control| 0. 480| 5 days incubation| 0. 480| 0 days incubation| 0. 470| 15 days incubation| 0. 450| Efflux time of solvent to = 15. 25sec Temperature T = 60oC. Efflux time of various solutions of natural rubber from 20 days. After 20 days of activity. CONCENTRATION, C (g/dl)| EFFLUX TIME, t (s)| 0. 2| 22. 500| 0. 4| 30. 256| 0. 6| 38. 308| 0. 8| 46. 726| After 25 days of activity CONCENTRATION, C (g/dl)| EFFLUX TIME, t (s)| 0. 2| 22. 326| 0. 4| 29. 768| 0. 6| 37. 393| 0. 8| 45. 263|
After 30 days of activity CONCENTRATION, C (g/dl)| EFFLUX TIME, t (s)| 0. 2| 22. 204| 0. 4| 29. 402| 0. 6| 36. 844| 0. 8| 44. 042| After 35 days of activity CONCENTRATION, C (g/dl)| EFFLUX TIME, t (s)| 0. 2| 22. 082| 0. 4| 29. 036| 0. 6| 36. 112| 0. 8| 42. 822| After 40 days of activity. CONCENTRATION, C (g/dl)| EFFLUX TIME, t (s)| 0. 2| 21. 960| 0. 4| 28. 792| 0. 6| 35. 380| 0. 8| 41. 602| Viscosity numbers of each period of culture of strain.
At 20 days CONC, C (g/dl)| EFFLUX TIME| ? SP =t-toto| ? red = ? spC| 0. 2| 22. 500| 0. 475| 2. 380| 0. 4| 30. 256| 0. 984| 2. 460| 0. 6| 38. 308| 1. 512| 2. 520| 0. 8| 46. 726| 2. 064| 2. 540| At 25 days CONC, C (g/dl)| EFFLUX TIME| ? SP =t-toto| ? red = ? spC| 0. 2| 22. 326| 0. 464| 2. 320| 0. 4| 29. 768| 0. 952| 2. 380| 0. 6| 37. 393| 1. 452| 2. 420| 0. 8| 45. 262| 1. 968| 2. 460| At 30 days CONC, C (g/dl)| EFFLUX TIME| ? SP =t-toto| ? red = ? spC| 0. 2| 22. 204| 0. 456| 2. 280| 0. 4| 29. 402| 0. 928| 2. 320| 0. 6| 36. 844| 1. 416| 2. 360| 0. 8| 44. 042| 1. 888| 2. 60| At 35 days CONC, C (g/dl)| EFFLUX TIME| ? SP =t-toto| ? red = ? spC| 0. 2| 22. 082| 0. 456| 2. 280| 0. 4| 29. 036| 0. 928| 2. 320| 0. 6| 36. 112| 1. 416| 2. 360| 0. 8| 42. 822| 1. 808| 2. 360| At 40 days CONC, C (g/dl)| EFFLUX TIME| ? SP =t-toto| ? red = ? spC| 0. 2| 21. 960| 0. 440| 2. 200| 0. 4| 28. 792| 0. 904| 2. 260| 0. 6| 35. 380| 1. 320| 2. 200| 0. 8| 41. 602| 1. 728| 2. 160| Corresponding viscosity numbers of the various solutions of natural rubber. CONCENTRATION, C (g/dl)| VISCOSITY NUMBER, (? red) (dl/g)| | 20 DAYS | 25 DAYS| 30 DAYS| 35DAYS| 40DAYS| . 2| 2. 380| 2. 320| 2. 280| 2. 240| 2. 200| 0. 4| 2. 460| 2. 380| 2. 320| 2. 260| 2. 260| 0. 6| 2. 520| 2. 420| 2. 360| 2. 280| 2. 200| 0. 8| 2. 540| 2. 460| 2. 360| 2. 260| 2. 160| NOTE: The viscosity was calculated based on poiseulle’s law. The intrinsic viscosity (? ) is derived by extrapolation of the Huggins plots to zero concentration. The values of the intercept (? ) are presented from the plot. Extrapolated Values of Huggins plots from 20 days. HUGGINS PLOTS | INTRINSIC VISCOSITY, (?
Intr )| 20 days incubation| 2. 335| 25 days incubation| 2. 300| 30 days incubation| 2. 240| 35 days incubation| 2. 220| 40 days incubation| 2. 200| Concentration, C (g/dl) Viscosity number, (dl/g) Fig 4. 2 4. 3 DISCUSSIONS On incubation, the latex coagulated and the following observations were noted. * Spores were formed on the coagulated latex. * There are surface irregularities on the coagulated latex. * Rod-like clustering on the surface of the latex crumb when viewed under a microscope.
From the experiment carried out, the result shows a gradual decrease in intrinsic viscosity with increasing time of exposure to the microbe. This shows that the degradation increase with increasing time of exposure. The continuous decreases in intrinsic viscosity suggest a corresponding decrease in molecular weight since intrinsic viscosity is empirically related to molecular weight according to Mark-Houwinks equation. [? ] = KMva. CHAPTER FIVE 5. 0 CONCLUSION AND RECOMMENDATIONS. 5. 1 CONCLUSION. From this research of degradation of natural rubber by Actinomycete Genus Gordonia sp.
After 40 days of exposure, its intrinsic viscosity reduced from 2. 335 to 2. 200 which signifies that the molecular weight of the rubber have been reduced with reference to Mark-Houwinks equation. [? ] = KMva. On employing the viscosity measurement technique to study the degradation of natural rubber, I obtained the following; * The intrinsic viscosity of the rubber latex upon incubation with the genus for 20 days is 2. 335dl/g. * The intrinsic viscosity of the rubber latex upon incubation with the strain for 25 days is 2. 300dl/g. * The intrinsic viscosity of the rubber latex upon incubation with the strain for 30 days is 2. 240dl/g. The intrinsic viscosity of the rubber latex upon incubation with the strain foe 35 days is 2. 220dl/g. * The intrinsic viscosity of the rubber latex upon incubation with the strain for 40 days is 2. 200dl/g. It’s obvious that lower intrinsic viscosity for natural rubber latex would be obtained for longer period of incubation with genus Gordonia sp. This shows that the rubber is degraded. 5. 2 RECOMMENDATION Viscosity analysis of the degradation of natural rubber latex is a physical means of evaluating degradation. The viscosity of the rubber before and after activity of the genus was determined using a simple Ubbelohde viscometer.
I recommend the use of viscometer in analyzing degradation as it requires simple equipment for optimum yield. It is economical, requires fewer chemicals, and only a suitable solvent is necessary for the rubber. This is very efficient in evaluating the degree of degradation. Furthermore, I recommend that more work should be carried out on microbial degradation of rubber but with varying concentrations. It is an interesting research and u will always be sure of concrete results. REFERENCE 1. Arenskotter, M. , D. Baumeister, M. M. Berekaa, G. Potter, R. M. Kroppenstedt, A. Linos, and A. Steinbuchel. 2001.
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