Studies on Microbial Degradation of Natural Rubber Using Dilute Solution Viscosity Measurement and Weight Loss Techniques

INTERNATIONAL JOURNAL OF BASIC AND APPLIED SCIENCE

Insan Akademika Publications

P-ISSN: 2301-4458 E-ISSN: 2301-8038 Vol. 01, No. 02 Oct 2012

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Studies on Microbial Degradation of Natural Rubber Using Dilute Solution Viscosity Measurement and Weight Loss Techniques
G. N. Onyeagoro 1, E. G. Ohaeri 2, U. J. Timothy 3 Department of Polymer and Textile Engineering, Federal University of Technology, Owerri, Imo State, NIGERIA. nkemgoddi@yahoo.com Department of Polymer and Textile Engineering, Federal University of Technology, Owerri, Imo State, NIGERIA. enyi.ohaeri@yahoo.com Department of Polymer and Textile Engineering, Nnamdi Azikiwe University, Awka, Anambra State, NIGERIA. ukemejames22@yahoo.com
3 2 1

Key words
Dilute solution viscosity, Microbial degradation, Natural rubber, Nocardia sp, Weight loss

Abstract
Reduction of molecular weight of rubber polymers for easy absorption of compounding ingredients is critical in every rubber compounding operation. Yet, rubber mastication which is the current practice of achieving this is expensive, requiring high energy and equipment cost. To address this problem, microbial degradation of natural rubber (NR) and waste rubber tire (WRT) by Nocardia sp. strain 385A was studied using dilute solution viscosity measurement and weight loss methods. Solutions of NR and WRT in toluene were inoculated with the microbes (Nocardia sp. strain 385A) and kept in incubator. Incubation period varied between 0 to 10 weeks. The results obtained show that NR and WRT were mineralized and degraded by the microbes. Intrinsic viscosity values of both NR and WRT decreased with increasing period of incubation, indicating that degradation increases with increase in the incubation period. For the incubation periods investigated, WRT produced higher intrinsic viscosity values than NR due to the inhibitory effect of additives present in WRT to microbial degradation. Equivalent reduction in molecular weight obtained by rubber mastication technique was achieved by microbial degradation after 10 weeks incubation period. Rubber degrading bacteria can be useful for the disposal of discarded rubber products
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Introduction

Microbial degradation is a natural process by which organic compounds, including rubber polymers are converted by the action of bacteria to simpler compounds, mineralized and redistributed through the elemental cycles (Enoki et al, 2003; Cui et al, 2005). Over the years, there have been concerted efforts to investigate microbial rubber degradation. It became obvious that bacteria as well as fungi, are capable of degrading rubber and that rubber biodegradation is a slow process (Gallert, 2000; Jendrossek et al, 1997; Nette et al, 1959; Ibrahim et al, 2006). Natural rubber (NR) is a biopolymer (polyisoprene) that is synthesized by many plants and some fungi. It has been commercially produced from Hevea brasiliensis trees at a level of several million tons per year. NR contains a minimum of 90% rubber hydrocarbon together with small amounts of proteins, resins, fatty acids, sugars, and minerals (Rose and Steinbuchel, 2005). Organic impurities in the rubber can support microbial growth, and many reports on its biodegradability have been published (Tsuchii, 1995; Jendrossek et al, 1997; Linos and Steinbuchel, 1998; Arenskotter et al, 2001; Arenskotter et al, 2004; Linos et al, 2002; Rose and Steinbuchel, 2005). It is assumed that degradation of the rubber backbone is initiated by oxidative cleavage of the double bond (Tsuchii et al, 1985; Tsuchii and Takeda, 1990; Linos et al, 2000; Bode et al, 2001; Yamamura et al, 2005; Rose and Steinbuchel, 2005). In its raw state, NR has a high molecular weight (> 1 million) and, because of molecular entanglements, it is quite elastic and incapable of shaping by melt processing. It is therefore necessary to subject the rubber to an intensive shearing process technically known as mastication in order to break the molecular weight to a state in which the material may be more easily blended with additives, shaped and then vulcanized. Rubber mastication is an important industrial practice, and has remained the most commonly practiced method of breaking down the molecular weight of rubbers. It requires the use of heavy-duty equipment that are procured at the expense of huge foreign exchange. In addition, these equipments are run at very high energy cost, resulting to high overall cost of production and rising cost of rubber products. Furthermore, waste rubber is becoming a global waste disposal problem (Bode et al, 2000). A major concern is the huge number of natural rubber products manufactured and discarded annually and the potential environmental hazards should a NR stock pile catch fire. Consequently, it is very important to develop a microbial process for waste NR disposal (Sato et al, 2003; Barekaa et al, 2000). The present study seeks to address these problems by investigating a natural process (microbial degradation) of breaking down the rubber molecular weight, which is more cost effective in terms of equipment procurement and energy consumption. This is expected to reduce the price of rubber products. It also suggests methods of investigation (dilute solution viscosity measurement and weight loss techniques) that are simple, cheap and accurate. It also suggests that rubber- degrading bacteria might be useful for the disposal of discarded rubber products. Biodegradation of NR and waste rubber tire in the presence of Nocardia sp. strain 835A as the rubber degradant was investigated. The microbe was first isolated and cultured followed by addition of the investigated rubber granules. All cultures were inoculated and incubated. Biodegradation at selected periods of exposure to the microbes was monitored by graphical determination of the intrinsic viscosity through dilute solution viscosity measurements, as well as by conventional weight loss method. The intrinsic viscosity of masticated NR was also determined and compared with that of the biodegraded NR.

Theoritical Background

Nocardia sp. strain 835A, which exhibited reasonable growth on natural and synthetic rubber, was one of the first strains that was investigated in detail with regard to rubber biodegradation, and it was postulated that there was oxidative cleavage of poly (cis-1, 4-isoprene) at the double bond position (Tsuchii et al, 1985). Weight losses of the rubber material used of 75 and 100% (w/w) after 2 and 8 weeks of incubation,

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respectively, and of the latex glove material used of 90% (w/w) after 8 weeks were obtained. Gel permeation chromatography (GPC) of the chloroform-soluble fraction of degraded glove material revealed two fractions of fragments with molecular weight of 1 x 104 and 1.6 x 103, comprising 114 and 19 isoprene molecules, respectively. Both fractions exhibited infra-red spectra identical to those of aldehyde derivatives of dolichol. A method widely used for routine molecular-weight determination is based upon the determination of the intrinsic viscosity, [], of a polymer in solution through measurements of solution viscosity. Molecular weight is related to [] by the Mark-Houwink-Sakurada equation (Fried, 2005) given as: [] = KMva ...(1)

where Mv is the viscosity-average molecular weight. Both K and a are empirical (Mark-Houwink) constants that are specific for a given polymer, solvent, and temperature. The exponent a normally lies between the values of 0.5 for a solvent and 1.0 for a thermodynamically good solvent. Intrinsic viscosity can be expressed, in the region of low solute concentration c, in the form: [] = []0 { 1 + []c + Kc2 + higher term of c3} ...(2)

where K is a concentration-independent parameter and [] is the limiting viscosity number (or intrinsic viscosity) defined by eq. (3) or (4): [] = lim sp/c c0 or [] = lim (ln r)/c c0 where sp = (-0)/0 = r-1 where sp is the specific viscosity and r ( = /0) is the viscosity ratio (or relative viscosity. The sp and r values of a dilute solution are represented by a polynomial approximation (neglecting c3 and higher terms in eq. 2 as follows: sp/c = [] + k[]2c (ln r)/c = [] + k[]2c (Huggins equation) (Kraemers equation) ...(6) ...(7) ...(5) ...(4) ...(3)

where the coefficients k (referred to as the Huggins constant) and k are characteristic of the polymerpolymer interaction in the solvent. The probability of polymer molecules contacting another is large in a poor solvent, resulting in larger k. For polymers in a poor solvent (Flory -solvent) k is about 0.5 or slightly higher, and in a good solvent k frequently lies in the range 0.2-0.5. A plot of sp/c versus c is a Huggins plot (Huggins, 1942), and a plot of (ln r)/c versus c is a kraemer plot (Kraemer, 1938). The constant k is related to k (Hunt and James, 1993) by: k = k- ...(8)

According to eqs. 6 and 7, [] can be obtained from the intercept of a plot of sp/c (reduced viscosity) or (ln r)/c (inherent viscosity) versus c. In practice, reduced viscosity is obtained at different concentrations not by direct measurement of solution and solvent viscosities but by measurement of the time required for a dilute solution (t) and pure solvent (ts) to fall from one etched mark to another in a small glass capillary viscometer.
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If these efflux times are sufficiently long (> 100 s), the relative viscosity increment (specific viscosity, sp) can be obtained as tts sp = ts

...(9)

Efflux times may be noted visually or more precisely by means of commercially available photocell devices. Capillary viscometers may be either Ostwald-Fenske or Ubbelohde types as shown in Figure 1. The latter have the advantage that different solution concentrations can be made directly in the viscometer by successive dilutions with pure solvent. During measurement, the viscometer is immersed in a constant temperature bath controlled to within 0.020C of the set temperature. Numerous experimental versus c relations other than Huggins and Kraemers have been proposed (Kamide and Saito, 1989). They are special cases or equivalent to Huggins equation (eq. 6).

Fig. 1: (A) Ostwald-Fenske and (B) Ubbelohde capillary viscometers.

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3.1

Materials and Method

Preparation of Culture Medium

The culture medium was prepared following procedures described by Berekaa, 2006. Cultivation was carried out in screw-capped Erlenmeyer flasks, with internal glass container, containing Mineral Salts Medium (MSM) and rubber as sole carbon source (Linos and Steinbuchel, 1998). The rubber materials were added in concentration of 0.5% (w/v). Rubber substrate (NR or waste rubber tire granules) were added directly and the entire media was autoclaved. All cultures were inoculated with cells obtained from 4-6 days pre-culture in Luria-Bertani complex medium which were washed twice with saline before use. The inoculated plates were incubated in an inverted position with the lid at the bottom at 300C separately and in closed containers, and left for 2, 4, 6, and 8 weeks.

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3.2

Mineralization of Rubber Substrate

Mineralization of rubber substrate was estimated by determination of the amount of CO2 released during cultivation of cells on the rubber substrate. The released CO2 was trapped in Ba(OH)2 solution resulting in precipitation of CO2 as BaCO3. The decrease in alkalinity was determined by titration with 0.25 N HCl at different time intervals of incubation (Linos and Steinbuchel, 1998).

3.3

Measurement of Weight Loss of Rubber Substrate

Equal amount of each of the rubber substrates, NR and waste rubber tire was dissolved in toluene and left in 250ml Erlenmeyer flask till evaporation and 50ml MSM were added and autoclaved. The flasks were inoculated with 2ml of a 6 days old pre-culture of Nocardia sp. strain 835A cells that were washed with sterile saline solution 0.99 (w/v). At the end of 10 weeks incubation period the % reduction in weight of the rubber substrates was determined (Berekaa, 2006).

3.4

Measurement of Solution Viscosity of Rubber Substrate

The inoculated rubber substrate was sterilized in an autoclave to terminate the activity of the Nocardia sp. strain 835A before conducting the viscosity test. 3.5g of rubber substrate was dissolved in 50ml of toluene to obtain a standard solution of 0.175g/ml. This standard solution was then diluted to various concentrations in a 10ml volumetric flask. The concentrations of the diluted solutions were 0.2, 0.4, 0.6, and 0.8 g/dl. The viscosity bath was equilibrated at 320C for 45 minutes. After equilibration, 10ml of pure solvent was filtered into reservoir of the Ubbelohde viscometer using a sintered glass funnel. The filtration was done to remove any dust particles or suspension in the solvent. The viscometer containing the solvent was then immersed in the water bath and equilibrated for 15 minutes. This was followed by measurement of flow time of the solvent, which was obtained by timing the flow between the two etched marks on the viscometer (Figure 1). An average of three timings which agree within 0.20 seconds was recorded as the flow time of the solvent. The solvent was removed and the viscometer rinsed with little amount of the filtered solution before sufficient amount of the filtered solution was transferred into the reservoir of the viscometer. The flow times of the solution at incubation periods of 2, 4, 6, and 8 weeks were measured as described above. Flow times of the masticated NR solution at the concentrations investigated were also determined. After each measurement, the viscometer was rinsed with small amount of the pure solvent, followed by rinsing with the test solution before the next flow time measurement.

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4.1

Results and Discussion

Mineralization of NR and Waste rubber tire Substrates

The ability of Nocardia sp. strain 385A to degrade NR and waste rubber tire was studied. The results presented in Figure 2 showed that Nocardia sp. strain 385A were able to mineralize and degrade NR and waste rubber tire . Optimum mineralization levels of NR and waste rubber tire were 7.7 and 5.6, respectively, giving approximately 2.1% increase in % CO2 release, reached after 10 weeks of incubation. The result also showed that mineralization was rapid at the initial stage of incubation, from zero to 6 weeks and thereafter, gradually slowed down with further increase in incubation period. This shows that the activity profile of the microbe (Nocardia sp. strain 385A) reduces as mineralization and degradation progresses.

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7 Mineralization (% Carbon dioxide release 6 5 4 3 2 1 0 0 2 4 6 8 10 12 Incubation period (weeks) NR substrate WRT substrate

Fig. 2:

Mineralization (% Carbon dioxide release) of NR and Waste rubber tire (WRT) by Nocardia sp. strain 385A

Weight Loss (%)

NR substrate WRT substrate

0 0 2 4 6 8 10 12 Incubation period (weeks)

Fig. 3:

Plot of weight loss versus incubation period for NR and Waste rubber tire (WRT)

4.2

Determination of Weight Loss of NR and Waste Rubber Tire (WRT)

The activity profile of Nocardia sp. strain 385A on NR and WRT was also assessed after 10 weeks of incubation by the conventional weight loss method. The results presented in Figure 3 showed that NR was the most susceptible to degradation by the Nocardia sp. strain 385A, while waste rubber tire (WRT) was the most resistant. This is attributed to the inhibitory effect of accelerators, antioxidants and other additives present in waste rubber tire to microbial degradation. This observation is consistent with the report of Berekaa et al, 2000. The authors revealed an enhancement in the degradation of natural rubber latex gloves

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by the removal of antioxidants and other toxic compounds prior to introducing the latex to microbial degradation. For both NR and WRT, there was a rapid increase in weight loss at the initial stage of incubation up to 6 weeks and thereafter slowed down with further increase in incubation period. This indicates a reduction in the activity profile of the Nocardia sp. strain 385A as mineralization and degradation progressed.

4.3

Determination of Solution Viscosity of Rubber Substrate

Plots of reduced viscosity versus concentration (Huggins plot) and inherent viscosity versus concentration (Kraemers plot) for NR and WRT are presented in Figures 4-10. The intrinsic viscosities [] of NR and WRT in toluene at 250C at the concentrations investigated were obtained as the intercepts on the concentration axis. The results show linearity in the relationship between reduced viscosity versus concentration and inherent viscosity versus concentration as predicted by Huggins and Kraemers equations, respectively. This observation is

Reduced viscosity x 10 , Inherent viscosity x 10 (dl/g)

35 30 25 20 15 10 5 0 0 0,2 0,4 0,6 0,8 1 Concentration (g/dl)

NR (Huggins plot) NR (Kraemer's plot) WRT (Huggins plot) WRT (Kraemer's plot)

Fig. 4:

Plots of Reduced viscosity versus concentration (Huggins plot) and Inherent viscosity versus concentration (Kraemer's plot) of NR and WRT in toluene at 25C for 0 week exposure to microbes (Nocardia sp. strain 385A).

Consistent with the report of Du et al, 2006. Working with solution of polystyrene in toluene at room temperature the authors reported a linear relation in the plots of reduced viscosity versus concentration and inherent viscosity versus concentration in the extremely dilute concentration region (concentration < 0.075 g/dl). The results also show that degradation of NR and WRT increased with increasing time of exposure to the microbes as shown by the decrease in intrinsic viscosity with increase in the time of exposure. For both Huggins and Kraemers plots, intrinsic viscosity is in the order: 0.1 dl/g (2 weeks) > 0.082 dl/g (4 weeks) > 0.062 dl/g (6 weeks) > 0.056 dl/g (8 weeks) > 0.054 dl/g (10 weeks) for NR, and 0.112 dl/g (2 weeks) >

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0.091 dl/g (4 weeks) > 0.074 dl/g (6 weeks) > 0.068 dl/g (8 weeks) > 0.066 dl/g (10 weeks) for WRT. These results clearly show that degradation was rapid at the initial stage up to 8 weeks and then slowed down thereafter. This is due to high concentration of microbes at the commencement of degradation which become exhausted with low activity as degradation of NR and WRT progressed. WRT produced higher intrinsic viscosity values than NR for the periods of exposure investigated. This is attributed to the inhibitory effect of accelerators, antioxidants and other additives present in WRT to microbial degradation. This observation is consistent with the report of Berekaa et al, 2000. The authors revealed that the extraction of latex gloves with organic solvents before incubation enhanced the growth of some rubber-degrading strains which resulted to increase in the rate of degradation.

Reduced viscosity x 10 , Inherent viscosity x 10 (dl/g)

18 16 14 12 10 8 6 4 2 0 0 0,2 0,4 0,6 0,8 1 Concentration (g/dl) NR (Huggins plot) WRT (Huggins plot) NR (Kraemer's plot) WRT (Kraemer's plot)

Fig. 5:

Plots of Reduced viscosity versus concentration (Huggins plot) and Inherent viscosity versus concentration (Kraemer's plot) of NR and WRT in toluene at 25C for 0 week exposure to microbes (Nocardia sp. strain 385A).

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16 Reduced viscosity x 10 , Inherent viscosity x 10 (dl/g) 14 12 10 NR (Huggins plot) 8 6 4 2 0 0 0,2 0,4 0,6 0,8 1 Concentration (g/dl) WRT (Huggins plot) NR (Kraemer's plot) WRT (Kraemer's plot)

Fig. 6:

Plots of Reduced viscosity versus concentration (Huggins plot) and Inherent viscosity versus concentration (Kraemer's plot) of NR and WRT in toluene at 25C for 2 weeks exposure to microbes (Nocardia sp. strain 385A).

14 Reduced viscosity x 10 , Inherent viscosity x 10 (dl/g) 12 10 8 6 4 2 0 0 0,5 Concentration (g/dl) 1 NR (Huggins plot) WRT (Huggins plot) NR (Kraemer's plot) WRT (Kraemer's plot)

Fig. 7:

Plots of Reduced viscosity versus concentration (Huggins plot) and Inherent viscosity versus concentration (Kraemer's plot) of NR and WRT in toluene at 25C for 4 weeks exposure to microbes (Nocardia sp. strain 385A).

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16 Reduced viscosity x 10 , Inherent viscosity x 10 (dl/g) 14 12 10 NR (Huggins plot) 8 6 4 2 0 0 0,2 0,4 0,6 0,8 1 Concentration (g/dl) WRT (Huggins plot) NR (Kraemer's plot) WRT (Kraemer's plot)

Fig. 8:

Plots of Reduced viscosity versus concentration (Huggins plot) and Inherent viscosity versus concentration (Kraemer's plot) of NR and WRT in toluene at 25C for 6 weeks exposure to microbes (Nocardia sp. strain 385A).

Reduced viscosity x 10 , Inherent viscosity x 10 (dl/g)

14 12 10 8 6 4 2 0 0 0,2 0,4 0,6 0,8 1 Concentration (g/dl)

NR (Huggins plot) WRT (Huggins plot) NR (Kraemer's plot) WRT (Kraemer's plot)

Fig. 9:

Plots of Reduced viscosity versus concentration (Huggins plot) and Inherent viscosity versus concentration (Kraemer's plot) of NR and WRT in toluene at 25C for 8 weeks exposure to microbes (Nocardia sp. strain 385A).

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14 Reduced viscosity x 10 , Inherent viscosity x 10 (dl/g) 12 10 8 6 4 2 0 0 0,5 Concentration (g/dl) 1 NR (Huggins plot) NR (Kraemer's plot) WRT (Huggins plot) WRT (Kraemer's plot)

Fig. 10: Plots of Reduced viscosity versus concentration (Huggins plot) and Inherent viscosity versus concentration (Kraemer's plot) of NR and WRT in toluene at 25C for 10 weeks exposure to microbes (Nocardia sp. strain 385A).

Reduced viscosity x 10 , Inherent viscosity x 10 (dl/g)

16 14 12 10 8 6 4 2 0 0 0,5 Concentration (g/dl) 1 NR (Huggins plot) WRT (Huggins plot) NR (Kraemer's plot) WRT (Kraemer's plot) FMR (Huggins plot) FMR (Kraemer's plot)

Fig. 11: Comparison of Intrinsic viscosity [] of fresh masticated rubber (FMR) and Intrinsic viscosity [] of NR (10 weeks exposure to microbes) and WRT (10 weeks exposure to microbes) in toluene at 25C.

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Intrinsic viscosities of NR and WRT in toluene at 25C obtained at 10 weeks exposure to the microbes were compared with the intrinsic viscosity of fresh masticated natural rubber (FMR) as shown in Figure 11. The result shows that the order in the value of intrinsic viscosity is: FMR (0.058 dl/g) > WRT (0.049 dl/g) > NR (0.044 dl/g). The decrease in intrinsic viscosity values of NR from 0.21 dl/g at o week exposure to 0.044 dl/g after 10 weeks exposure to the microbes is an indication of progressive decrease in molecular weight of NR. In comparison with FMR ([] = 0.058 dl/g), it is clear that appreciable reduction in molecular weight of NR ([] =0.044 dl/g) can be achieved after 10 weeks exposure to the microbes.

Conclusion

The following conclusion can be drawn from the work: a. For easy absorption of compounding ingredients during rubber processing, molecular weight of rubber polymers are usually broken down into smaller units by mechanical shearing action in a process technically known as mastication. This process is expensive, both in terms of energy consumption and equipment procurement, thus impacting adversely on the price of rubber products. To address this problem, microbial degradation of natural rubber by Nocardia sp. strain 385A was investigated. The present study suggests a method of achieving molecular weight reduction that is not only cost effective but also employs techniques that are cheap, simple and accurate. b. Solutions of natural rubber (NR) and WRT were inoculated with Nocardia sp. strain 385A and subjected to various periods of incubation. It was found that NR and WRT were mineralized and degraded by the microbes (Nocardia sp. strain 385A). c. Degradation of NR and WRT increased with increase in incubation period as revealed by the decrease in intrinsic viscosity with increasing period of incubation. However, WRT produced higher intrinsic viscosity values than NR due to the inhibitory effect of additives present in WRT to microbial degradation. Equivalent reduction in molecular weight obtained by rubber mastication technique was achieved by microbial degradation after 10 weeks incubation period.

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