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

7/29/2019 Studies on Microbial Degradation of Natural Rubber Using Dilute Solution Viscosity Measurement and Weight Loss

1/13

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

Insan AkademikaPublications

INTERNATIONAL JOURNAL

OF BASIC AND APPLIED SCIENCE

P-ISSN: 2301-4458E-ISSN: 2301-8038

Vol. 01, No. 02Oct 2012

www. insikapub. com

448

Studies on Microbial Degradation of Natural Rubber Using Dilute Solution

Viscosity Measurement and Weight Loss Techniques

G. N. Onyeagoro1, E. G. Ohaeri

2, U. J. Timothy

3

1Department of Polymer and Textile Engineering,Federal University of Technology, Owerri, Imo State, NIGERIA.

[email protected]

Department of Polymer and Textile Engineering,Federal University of Technology, Owerri, Imo State, NIGERIA.

[email protected] Department of Polymer and Textile Engineering,

Nnamdi Azikiwe University, Awka, Anambra State, [email protected]

Key words Abstract

Dilute solution viscosity,Microbial degradation,

Natural rubber,

Nocardia sp,

Weight loss

Reduction of molecular weight of rubber polymers for easy absorption ofcompounding 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

2012 Insan Akademika All Rights Reserved


2/13

Onyeagoro, et. al. International Journal of Basic and Applied Science,Vol 01, No. 02, Oct 2012, pp. 448-460

www. insikapub. com 449

1 Introduction

Microbial degradation is a natural process by which organic compounds, including rubber polymers areconverted 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 toinvestigate microbial rubber degradation. It became obvious that bacteria as well as fungi, are capable ofdegrading 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 hasbeen commercially produced from Hevea brasiliensis trees at a level of several million tons per year. NRcontains 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 microbialgrowth, 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 andSteinbuchel, 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 isquite elastic and incapable of shaping by melt processing. It is therefore necessary to subject the rubber to anintensive shearing process technically known as mastication in order to break the molecular weight to a statein 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 practicedmethod of breaking down the molecular weight of rubbers. It requires the use of heavy-duty equipment thatare procured at the expense of huge foreign exchange. In addition, these equipments are run at very highenergy 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 hugenumber of natural rubber products manufactured and discarded annually and the potential environmentalhazards should a NR stock pile catch fire. Consequently, it is very important to develop a microbial processfor 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 equipmentprocurement and energy consumption. This is expected to reduce the price of rubber products. It alsosuggests methods of investigation (dilute solution viscosity measurement and weight loss techniques) that aresimple, cheap and accurate. It also suggests that rubber- degrading bacteria might be useful for the disposalof discarded rubber products. Biodegradation of NR and waste rubber tire in the presence ofNocardia sp.strain 835A as the rubber degradant was investigated. The microbe was first isolated and cultured followedby addition of the investigated rubber granules. All cultures were inoculated and incubated. Biodegradationat selected periods of exposure to the microbes was monitored by graphical determination of the intrinsicviscosity 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 biodegradedNR.

2 Theoritical Background

Nocardia sp. strain 835A, which exhibited reasonable growth on natural and synthetic rubber, was one of thefirst 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,


3/13

International Journal of Basic and Applied Science,Vol 01, No. 02, Oct 2012, pp. 448-460

Onyeagoro, et. al..

450 Insan Akademika Publications

respectively, and of the latex glove material used of 90% (w/w) after 8 weeks were obtained. Gel permeationchromatography (GPC) of the chloroform-soluble fraction of degraded glove material revealed two fractionsof 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 theintrinsic viscosity, [], of a polymer in solution through measurements of solution viscosity. Molecularweight 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) constantsthat are specific for a given polymer, solvent, and temperature. The exponent a normally lies between thevalues of 0.5 for a solvent and 1.0 for a thermodynamically good solvent. Intrinsic viscosity can beexpressed, 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 intrinsicviscosity) defined by eq. (3) or (4):

[] = lim sp/c ...(3)c0

or

[] = lim (ln r)/c ...(4)c0

where

sp = (-0)/0 = r-1 ...(5)

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 (Huggins equation) ...(6)

(ln r)/c = [] + k[]2c (Kraemers equation) ...(7)

where the coefficients k (referred to as the Huggins constant) and k are characteristic of the polymer-polymer interaction in the solvent. The probability of polymer molecules contacting another is large in a poorsolvent, resulting in larger k. For polymers in a poor solvent (Flory -solvent) k is about 0.5 or slightlyhigher, and in a good solvent k

frequently lies in the range 0.2-0.5. A plot ofsp/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 ofsp/c (reduced viscosity) or (ln

r)/c (inherent viscosity) versus c. In practice, reduced viscosity is obtained at different concentrations not bydirect measurement of solution and solvent viscosities but by measurement of the time required for a dilutesolution (t) and pure solvent (ts) to fall from one etched mark to another in a small glass capillary viscometer.


4/13



If these efflux times are sufficiently long (> 100 s), the relative viscosity increment (specific viscosity, sp)can be obtained as

tts

sp = ...(9)ts

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 latterhave the advantage that different solution concentrations can be made directly in the viscometer bysuccessive dilutions with pure solvent. During measurement, the viscometer is immersed in a constanttemperature bath controlled to within 0.02

0C of the set temperature. Numerous experimental versus c

relations other than Huggins and Kraemers have been proposed (Kamide and Saito, 1989). They are specialcases or equivalent to Huggins equation (eq. 6).

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

3 Materials and Method

3.1 Preparation of Culture Medium

The culture medium was prepared following procedures described by Berekaa, 2006. Cultivation was carriedout 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 inconcentration of 0.5% (w/v). Rubber substrate (NR or waste rubber tire granules) were added directly andthe entire media was autoclaved. All cultures were inoculated with cells obtained from 4-6 days pre-culturein Luria-Bertani complex medium which were washed twice with saline before use. The inoculated plateswere incubated in an inverted position with the lid at the bottom at 30

0C separately and in closed containers,

and left for 2, 4, 6, and 8 weeks.


5/13


Onyeagoro, et. al..


3.2 Mineralization of Rubber Substrate

Mineralization of rubber substrate was estimated by determination of the amount of CO2 released duringcultivation 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 atdifferent 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 in250ml Erlenmeyer flask till evaporation and 50ml MSM were added and autoclaved. The flasks wereinoculated with 2ml of a 6 days old pre-culture of Nocardia sp. strain 835A cells that were washed withsterile saline solution 0.99 (w/v). At the end of 10 weeks incubation period the % reduction in weight of therubber 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 toobtain a standard solution of 0.175g/ml. This standard solution was then diluted to various concentrations ina 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 wasfiltered into reservoir of the Ubbelohde viscometer using a sintered glass funnel. The filtration was done toremove any dust particles or suspension in the solvent. The viscometer containing the solvent was thenimmersed 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 thesolvent. The solvent was removed and the viscometer rinsed with little amount of the filtered solution beforesufficient amount of the filtered solution was transferred into the reservoir of the viscometer. The flow timesof the solution at incubation periods of 2, 4, 6, and 8 weeks were measured as described above. Flow timesof the masticated NR solution at the concentrations investigated were also determined. After eachmeasurement, the viscometer was rinsed with small amount of the pure solvent, followed by rinsing with thetest solution before the next flow time measurement.

4 Results and Discussion

4.1 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 resultspresented in Figure 2 showed that Nocardia sp. strain 385A were able to mineralize and degrade NR andwaste 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 alsoshowed 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 themicrobe (Nocardia sp. strain 385A) reduces as mineralization and degradation progresses.


6/13



Fig. 2: Mineralization (% Carbon dioxide release) of NR and Waste rubber tire(WRT) byNocardia sp. strain 385A

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 ofincubation by the conventional weight loss method. The results presented in Figure 3 showed that NR wasthe most susceptible to degradation by the Nocardia sp. strain 385A, while waste rubber tire (WRT) was themost 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 ofBerekaa et al, 2000. The authors revealed an enhancement in the degradation of natural rubber latex gloves

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12

Mineralization(%Carbondioxid

erelease

Incubation period (weeks)

NR substrate

WRT substrate

0

1

2

3

4

5

6

0 2 4 6 8 10 12

WeightLoss(%)

Incubation period (weeks)

NR substrate

WRT substrate


7/13


Onyeagoro, et. al..


by the removal of antioxidants and other toxic compounds prior to introducing the latex to microbialdegradation. For both NR and WRT, there was a rapid increase in weight loss at the initial stage ofincubation up to 6 weeks and thereafter slowed down with further increase in incubation period. Thisindicates 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 andWRT in toluene at 250C at the concentrations investigated were obtained as the intercepts on theconcentration axis. The results show linearity in the relationship between reduced viscosity versusconcentration and inherent viscosity versus concentration as predicted by Huggins and Kraemers equations,respectively. This observation is

Fig. 4: Plots of Reduced viscosity versus concentration (Huggins plot) and Inherentviscosity versus concentration (Kraemer's plot) of NR and WRT in toluene at25C 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 roomtemperature the authors reported a linear relation in the plots of reduced viscosity versus concentration andinherent viscosity versus concentration in the extremely dilute concentration region (concentration < 0.075g/dl).

The results also show that degradation of NR and WRT increased with increasing time of exposure to themicrobes 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) >

0

5

10

15

20

25

30

35

0 0,2 0,4 0,6 0,8 1

Reducedviscosityx10,

Inherentviscosityx10

(dl/g)

Concentration (g/dl)

NR (Huggins plot)

NR (Kraemer's plot)

WRT (Huggins plot)

WRT (Kraemer's plot)


8/13



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. Theseresults clearly show that degradation was rapid at the initial stage up to 8 weeks and then slowed downthereafter. This is due to high concentration of microbes at the commencement of degradation which becomeexhausted 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 ofaccelerators, antioxidants and other additives present in WRT to microbial degradation. This observation isconsistent with the report of Berekaa et al, 2000. The authors revealed that the extraction of latex gloves withorganic solvents before incubation enhanced the growth of some rubber-degrading strains which resulted toincrease in the rate of degradation.

Fig. 5: Plots of Reduced viscosity versus concentration (Huggins plot) and Inherentviscosity versus concentration (Kraemer's plot) of NR and WRT in toluene at25C for 0 week exposure to microbes (Nocardia sp. strain 385A).

0

2

4

6

8

10

12

14

16

18

0 0,2 0,4 0,6 0,8 1


Inherentvi

scosityx10

(dl/g)


NR (Huggins plot)

WRT (Huggins plot)

NR (Kraemer's plot)



9/13


Onyeagoro, et. al..


Fig. 6: Plots of Reduced viscosity versus concentration (Huggins plot) and Inherentviscosity versus concentration (Kraemer's plot) of NR and WRT in toluene at25C for 2 weeks exposure to microbes (Nocardia sp. strain 385A).


0

2

4

6

8

10

12

14

16

0 0,2 0,4 0,6 0,8 1


In

herent

viscosityx10(dl/g)


NR (Huggins plot)

WRT (Huggins plot)

NR (Kraemer's plot)


0

2

4

6

8

10

12

14

0 0,5 1


Inherentviscos

ityx

10(dl/g)


NR (Huggins plot)

WRT (Huggins plot)

NR (Kraemer's plot)



10/13




Fig. 9: Plots of Reduced viscosity versus concentration (Huggins plot) and Inherentviscosity versus concentration (Kraemer's plot) of NR and WRT in toluene at

25C for 8 weeks exposure to microbes (Nocardia sp. strain 385A).

0

2

4

6

8

10

12

14

16

0 0,2 0,4 0,6 0,8 1


Inherentv

iscosityx

10(dl/g)


NR (Huggins plot)

WRT (Huggins plot)

NR (Kraemer's plot)


0

2

4

6

8

10

12

14

0 0,2 0,4 0,6 0,8 1

Reducedviscos

ityx10,

Inherentviscosityx10

(dl/g)


NR (Huggins plot)

WRT (Huggins plot)

NR (Kraemer's plot)



11/13


Onyeagoro, et. al..



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

0

2

4

6

8

10

12

14

0 0,5 1


Inherentvisc

osityx

10(dl/g)


NR (Huggins plot)

NR (Kraemer's plot)

WRT (Huggins plot)


0

2

4

6

8

10

12

14

16

0 0,5 1


Inherentviscosity

x10(dl/g)


NR (Huggins plot)

WRT (Huggins plot)

NR (Kraemer's plot)


FMR (Huggins plot)

FMR (Kraemer's plot)


12/13



Intrinsic viscosities of NR and WRT in toluene at 25C obtained at 10 weeks exposure to the microbes werecompared with the intrinsic viscosity of fresh masticated natural rubber (FMR) as shown in Figure 11. Theresult 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.

5 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 processtechnically known as mastication. This process is expensive, both in terms of energy consumption andequipment 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. Thepresent study suggests a method of achieving molecular weight reduction that is not only cost effectivebut also employs techniques that are cheap, simple and accurate.

b. Solutions of natural rubber (NR) and WRT were inoculated with Nocardia sp. strain 385A andsubjected to various periods of incubation. It was found that NR and WRT were mineralized anddegraded by the microbes (Nocardia sp. strain 385A).

c. Degradation of NR and WRT increased with increase in incubation period as revealed by the decreasein intrinsic viscosity with increasing period of incubation. However, WRT produced higher intrinsicviscosity values than NR due to the inhibitory effect of additives present in WRT to microbialdegradation. Equivalent reduction in molecular weight obtained by rubber mastication technique wasachieved by microbial degradation after 10 weeks incubation period.

References

Arenskotter, M; Baumeister, D; Berekaa, M.M; Potter, G; Kroppenstedt, R.M; Linos, A, and Steinbuchel, A,(2001), Taxonomic characterization of two rubber degrading bacteria belonging to the speciesGordonia Polyisoprenivorans and analysis of hyper-variable region of 16s rDNA sequences, FEMSMicrobiol. Letter, 205: 277-282.

Arenskotter, M; Broeker, D, and Steinbuchel, A, (2004), Biology of metabolically diverse genusGordonia, Applied Environ. Microbiol, 70: 3195-3204.

Berekaa, M.M, (2006), Colonization and Microbial degradation of polyisoprene rubber by Nocardioform

ActinomyceteNocardia sp

. strain-MBR, Biotechnology 5 (3): 234-239.Berekaa, M.M; Linos, A; Reichelt, R; Keller, U, and Steinbuchel, A, (2000), Effect of pre-treatment of

rubber material on its biodegradability by various rubber degrading bacteria, FEMS Microbiol. Lett,184: 199-206.

Bode, H; Kerkhoff, K, and Jendrossek, D, (2001), Bacterial degradation of natural and synthetic rubber,Biomacromolocules, 2: 295-303.

Bode, H.B; Zeeck, A; Pluckhahn, K, and Jendrossek, (2000), Physiological and chemical investigations intoMicrobial degradation of synthetic poly (cis-1,4-isoprene), Applied Environ. Microbiol, 66: 3680-3685.

Cui, Q; Wang, L; Huag, Y; Liu, Z, and Goodfellow, M, (2005), Nocardia jiangxiensis sp. nov. and Nocardia

miyunensis sp. nov., isolated from acidic soils, Int. J. Syst. Evol. Microbiol, 55: 1921-1925.Du, D; Zuo, J; An, Y; Zhou, L, and Liu, Y (2006),Journal of Applied Polymer Science, 102: 4440-4450.


13/13


Onyeagoro, et. al..


Enoki, M; Doi, Y, and Iwata, T, (2003), Oxidative degradation of cis-and trans-1,4-polyisoprene andvulcanized natural rubber with enzyme-mediator systems, Biomacromolecules, 4: 314-320.

Fried, J.R, (2005, Polymer Science and Technology, 2nd ed, Prentice-Hall of India, New Delhi, 139-141.

Gallart, C, (2000), Degradation of latex and of natural rubber by Streptomyces strain La7, Syst. Appl.Microbiol, 23: 433-441.

Huggins, M.L, (1942), J. Am. Chem. Soc, 64, 2716.

Hunt, B.J and James, M.I, (1993), Polymer characterization, 1st

ed, Chapman and Hall, London, UK, 135-140.

Ibrahim, E.M.A; Arenskotter, M; Luftmann, H, and Steinbuchel, A, (2006), Identification of poly (cis-1,4-isoprene) degradation intermediates during growth of moderately thermophilic actimycetes on rubberand cloning of a functional ICP homologue from Nocardia farcinica strain EI, Applied Environ.Microbiol, 72: 3375-3382.

Jendrossek, D; Tomasi, G, and Kroppenstedt, R, (1997), Bacterial degradation of natural rubber: a privilege

of actinomycetes, FEMS Microbiol. Lett, 150: 179-188.Kamide, K and Saito, M, (1989), Viscometric determination of molecular weight in Determination of

Molecular Weight, A Cooper (ed.), John Wiley & Sons, In; New York, 8: 234-246.

Kraemer, E.O, (1938), Ind. Eng. Chem., 30, 1200.

Linos, A; Berekaa, M.M; Reichelt, R; Keller, U; Schmitt, J; Flemming, H.C; Kroppenstedt, R.M, andSteinbuchel, A, (2000), Biodegradation of cis-1,4-polyisoprene rubbers by distinct actinomycetes:Microbial Stratigies and detailed surface analysis, Applied Environ. Microbiol, 66: 1639-1645.

Linos, A; Berekaa, M.M; Steinbuchel, A; Kim, K.K; Sproer, C, and Kroppenstedt, R.M, (2002), Gordoniawestfolica Sp. nov, a novel rubber-degrading actinomycete, Intl. J. Suyst. Evol. Microbiol, 52: 1133-1139.

Linos, A and Steinbuchel, A, (1998), Microbial degradation of natural and synthetic rubbers by novelbacteria belonging to the genus Gordonia, Kautsch. Gummi Kunstst, 51: 496-499.

Nette, I.T; Pomortseva, N.V, and Kozlova, E.I, (1959), Destruction of rubber by microorganism,Microbiologiya (USSR), 28: 821-827.

Rose, K and Steinbuchel, A, (2005), Biodegradation of natural rubber and related compounds: Recentinsights into a hardly understood catabolic capability of microorganisms, Applied Environ.Microbiol, 71: 2803-2812.

Sato, S; Honda, Y; Kuwahara, M, and Watanabe, T, (2003), Degradation of vulcanized and non-vulcanizedpolyisoprene rubbers by lipid peroxidation catalyzed by oxidative enzymes and transition metals,Biomacromolecules, 4: 321-329.

Tsuchii, A, (1995), Microbial degradation of natural rubber. In: Biotransformation: Microbial Degradationof Health Risk Compounds (Singh, Ved Pal. Ed.), Progress Ind. Microbiol, 32: 177-187.

Tsuchii, A; Suzuki, T, and Takeda, K, (1985), Microbial degradation of natural rubber vulcanizates,Applied Environ. Microbiol, 50: 965-970.

Tsuchii, A and Takeda, K, (1990), Rubber-degrading enzyme from bacterial culture, Applied Environ.Microbiol, 56: 269-274.

Yamamura, H; Hayakawa, M; Nakagawa, Y; Tamura, T; Kohno, T; Komatsu, F, and Limura, Y, (2005),Nocardia takedensis sp. nov., isolated from moat sediment and scumming activated sludge, Int. J.Syst. Evol. Microbiol, 55: 433-436.

Documents

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