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Utilization of Starch to Accelerate the Growth of Degrading Microorganisms

on the Surface of Natural Rubber Latex Films

Article  in  JOURNAL OF CHEMISTRY AND CHEMICAL ENGINEERING · February 2013

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J. Chem. Chem. Eng. 7 (2013) 137-144 D DAVID PUBLISHING

Utilization of Starch to Accelerate the Growth of

Degrading Microorganisms on the Surface of
Natural Rubber Latex Films

M. M. Afiq and Azura A. Rashid*

School of Materials and Mineral Resources Engineering (SMMRE), Engineering Campus, Universiti Sains Malaysia, Pulau Pinang
14300, Malaysia

Received: November 27, 2012 / Accepted: December 15, 2012 / Published: February 25, 2013.

Abstract: NRL (natural rubber latex) films with 0 (control) and 10 phr sago starch loading were buried in compost soil for 4 weeks.
The biodegradation assessments were carried out through films WVT (water vapor transmission) and mass loss. Scanning electron
microscopy was carried out to identify and monitor the properties of rubber degrading organism colonies. Results showed that
incorporation of sago starch increased the formation and rates of propagation for microorganism colonies on NRL films with duration
of biodegradation. The results also indicate the mechanism of sago starch granules utilization as sole source of energy for microbial
growth. The behavior and characteristics of microorganisms involved in NRL films degradation also successfully discussed.

Key words: NRL, sago starch, microorganism, biodegradation.

1. Introduction several enzymes such as amylase and glucosidases

which were able to break the glucosidic linkages in
NRL is a colloidal dispersion of polyisoprene
starch [5]. According to Rose et al. (2005), bacteria as
macromolecules in a form of milky sap obtained from
well as fungi have the ability to degrade rubber and
incised bark of Havea Brasiliensis tree [1, 2]. Due to
the process is reported to be slowed.
the presence of hydrocarbon chain in cis-1,
Conventionally, researcher is using latex overlay
4-polyisoprene backbone chain, NRL usually takes
agar plate to observe the effect of biodegradation on
longer time to degrade upon disposal. In attempt to
rubber. As a result, out of 1,220 of rubber degrading
cater these problems, several researchers introduce a
bacteria, 50 forms a clear zone on latex overlay agar
combination of natural biodegradable fillers in NRL
plates. All of these clear forming zone bacteria were
compounding [3]. One of the natural biodegradable
identified as mycelium-forming-actinomyces [6].
fillers which extensively study to be incorporated with
These Microorganisms secrete enzymes such as
NRL is starch base materials [4]. Thus in this study,
Laccase, Lipoxygenase, and Peroxidase, and lead into
sago starch from Metroxylon Sago palm tree was used
formation of fatty acids, such as lineloic acids and
as a fillers in NRL compounding.
1-hydroxybenzathiazole radicals. These radicals were
During microbial degradation of SSNRL (sago
able to break the polyisoprene backbone chain via
starch-NRL) films, microbial will consume the
β-scission due to interaction of polyisoprene backbone
polyisoprene chain in NRL colloidal particles secretes
chain with lipoxygenase-lineloic acid or
*
Corresponding authors: Azura A. Rashid, Associate peroxidase-1-hydroxybenzathiazole radicals and
Professor, research fields: rubber and latex. E-mail: continued further by alkoxyl radicals generated from
azura@eng.usm.my.
138 Utilization of Starch to Accelerate the Growth of Degrading Microorganisms
on the Surface of Natural Rubber Latex Films
the reactions. Even though recent studies provide an
extensive data regarding the subject, the use of latex
agar overlay has limitations such as, the results
obtained do not represent biodegradation process in
actual environment, and for some researchers, these
methods are only suitable to understand the resistance
of materials towards microbial infection [5]. Rubber
waste usually disposed in the landfill [7]. Thus the
degradation of rubber normally slowed as it
degradation only at the surface of the rubber waste
and involved more than one types of colonies. Hence,
it is difficult to determine the most significant
microorganism in rubber degradation process. Thus, in
this study, SS/NRL composite films were buried in a
controlled compost soil and the progress of
biodegradation process was investigated via assessing
SS/NRL films mass loss retention and WVT (water
vapor transmission) rates. The characteristics of
microorganism involved in biodegradation process of
NRL and SS/NRL films were observed through SEM
(scanning electron microscopy) analysis.
2. Methodology
SS/NRL (sago starch filled natural rubber latex)
compounds were prepared by addition of
compounding ingredients including vulcanization
agent as shown in Table 1. The compound was
prevulcanized at 80 °C until the desired state of
vulcanization is achieved.
15 cm2 of SS/NRL and control films were prepared
via dipping process and with the thickness of
0.01-0.02 mm. The films were prepared in 5 different
series and soil burial in compost soil for 4 weeks. The
Table 1 NRL compounding ingredients.
Chemical Dry weight, phr
61.0% NR Latex 100
10.0% KOH* 0.30
51.3% Sulfur 0.50
53.4% ZDEC** 0.75
33.1% Zinc oxide 0.25
52.2% Antioxidant 0.50
% Sago Starch. 10
*Potassium Hydroxide;
** Zinc Diethyldithiocarbamate.
tensile and tear strength of the films were measured
weekly according to ASTM D412 using Instron
machine. The reduction in sample mass loss after the
films were leached in distilled water and dried at room
temperature for 12 h were recorded weekly (including
initial weight before soil burial) to assess the progress
of biodegradation. The biodegradation rates were
calculated according to Eq. (1).
, ,
(1)
The films also subjected to WVT (water vapor
transmission) test according to ASTM E96, where a
cup filled with silica gel covered with the samples at
the top. The cups are then put in desiccators with a
bowl of water at the bottom to ensure high humidity
environment. The desiccator is then sealed to prevent
vapor loss except through the test sample. An initial
weight is measured from the apparatus and then
periodically weighed over time until the linear results
is obtained. SS/NRL films Water vapor transmission
were calculated by using Eq. (2).
, ,
,
, (2)
After mass loss and WVT rates measurements, the
samples were subjected to SEM analysis by Zeiss
Supra 35VP SEM machine after coated with gold
palladium.
3. Results and Discussion
3.1 Tensile Strength
Referring to Fig. 1, addition of sago starch in NRL
compound decreased the tensile properties of SS/NRL
films. This is due to the incompatibility of
hydrophobic NRL and hydrophilic sago starch system
[1]. The tensile strength for both films are further
reduced with the progress of biodegradation process.
For control films, the tensile strength shows the
highest value until 4 week of biodegradation
periods compared to SS/NRL films. This is due to
the microorganism activities which is low compared to
 Utilization of Starch to Accelerate the Growth of Degrading Microorganisms 139
on the Surface of Natural Rubber Latex Films

25 reduced the NRL films tensile strength [11].

Tensile Strength, (MPa)

20
3.2 Tear Strength
15
As depicted in Fig. 2, addition of sago starch
10
increased the tear strength of the films. During tear
5 o phr 10 phr
test, polyisoprene chain is aligned, and acts as crack
0 stopper during crack propagation [12]. Moreover, the
0 1 2 3 4 starch induced crystal region also helps to prevent the
Week of Biodegradation, Weeks
Fig. 1 Tensile strength for NRL films with respect to
tearing forces and increased the tearing strength of
biodegradation weeks. SS/NRL film.
As the sago starch is introduced in SS/NRL films,
sago starch filled NRL films.
the cementing mechanisms of rubber chain also
Control films are likely to degenerate due to
improved and produced the films which are more
oxidation process which takes place in rubber
resistance to tearing. Both films showed decreased in
backbone chain. During the process, doubly bonded
tearing resistance as the biodegradation test elapses.
carbon in unsaturated hydrocarbon chain in
During biodegradation process, the utilization of NRL
polyisoprene was attacked by primary oxygen which
films by microbial resulted with an empty holes which
first occurred and takes place at α-methalynic position
act as stress concentration point in SS/NRL films. As
in polyisoprene backbone. The process continued to
the forces either in plane shearing or uniaxial applied
be an auto-oxidation process [1].
on the film, the stress concentration point increased
Although the role of microorganism is not
the propagation and formed an active crack that
significant for control films, the microorganism
further increased the susceptibility of the films to
contribution is limited on the surface only. This
failure.
minimum contribution of microorganism is due to
introduction of various compounding ingredients 3.3 Mass Loss Retention
especially anti-oxidant in NRL compounding which
According to Fig. 3, as the week of biodegradation
retards the penetration of microbial colonies on the
elapses, the mass loss retention for both films
films to accelerate the process of biodegradation.
increased from week 2 to week 4. At the last week of
For sago starch filled films, due to the addition of
biodegradation periods, the trends are entering steady
polysaccharide materials, these organic substances is
state as the microorganism enters its death phase. This
able to be utilized as source of energy for
microorganism during biodegradation process. At the 65
Tear strength, (N/mm)

same time, microorganism metabolisms process 60

induces the formation of potential rubber degrading
55
enzymes and these enzymes are able to hydrolyze
polyisoprene chain and lead into further chain rupture 50
and films failure [5, 10]. 45 0 phr 10 phr
The hydrolysis of amylose and amylopectin
40
glucosidic linkages also leads into formation of low
0 1 2 3 4
molecular weight product of amylose and amylopectin
Week of Biodegradation, Weeks
structure. These degenerative processes usually Fig. 2 Tear strength for NRL films with respect to
increase the molecular chain mobility and further biodegradation weeks.
140 Utilization of Starch to Accelerate the Growth of Degrading Microorganisms
on the Surface of Natural Rubber Latex Films
0.1
Films mass loss retention
0.08
0.06
0.04
Control
0.02 10 phr
0 0.E+00
0 2 4 6
Biodegradation weeks, week
Fig. 3 Mass loss retention for control and SSNRL films.
is due to the increasing of holes formation on the NRL
films.
For SS/NRL films, the films showed higher mass
loss retention compared to control films throughout
entire biodegradation periods. During biodegradation
periods, microorganisms which favor to consume the
simple glucosidic linkages in starch formed a
bio-films for their colonies for further growth. As for
control films, the attachment of microorganism on
films surfaces are tough due to presence of
hydrocarbon chain and several other compounding
ingredients which are more complex to metabolize by
microbes.
3.4 Water Vapor Transmission (WVT) Rates
Referring to Fig. 4, as sago starch loading
introduced, the WVT properties are also increased.
This is due to the formation of holes on NRL films is
higher compared to low or non sago starch films. The
formation of various hydrolysis enzymes also detected
[8] together with microbial consumption
polyisoprene chain during biodegradation. This
hydrolysis enzyme further breaks the glucosidic
linkages in amylase and amylopectin structure in
starch [5].
These hydrolysis processes resulting in further
enlarge the holes formation on the NRL film surface.
As the volume of holes increases, the water vapors are
more feasible to transfer from high to the lower
humidity regions.
6.E-04
Water vapor transmission
5.E-04
4.E-04
gday-1cm-2
rate,
3.E-04
2.E-04
1.E-04
0 10
Sago starch filler loading
Fig. 4 WVT rates for both films at week three of
biodegradation process.
3.5 Scanning Electron Microscopy (SEM) Analysis
Fig. 5 shows the respective shape of sago starch
granules. The average size of the starch granules is
17.5 µm as determined by Malvern ™ Mastersizer
particle analyzer. This size is considered as standard
size of sago granules which usually found in a range
of 10-50 µm [9]. The shape of the granules generally
asymmetric oval with a little pitting observed on the
granular surface [10]. The granules are made of
amylose and amylopectin chain which packed layer by
layer [11] as indicated in Fig. 6. During preparation on
sago starch dispersion, the gelatinization process of
sago starch occured. Due to the syneresis process, the
amylose chains in sago starch granules were released
and stabilized the water around the granules [12]. The
amylose chains are then formed complexes with NRL
colloidal particles and formed an interparticle
interaction [1, 13, 14].
Fig. 7 shows the morphological analysis for
of control (0 phr) NRL films and SS/NRL films. As the
Fig. 5 SEM images of sago starch granules [15].
 Utilization of Starch to Accelerate the Growth of Degrading Microorganisms 141
on the Surface of Natural Rubber Latex Films

biodegradation weeks elapses, for control NRL films,

formation of microbial colonies is difficult to
determine compared to SS/NRL films. For SS/NRL
films, the formations of microorganism hyphea were
detected from week 1 of biodegradation periods. The
microbial colonies continued to grow in week 2 by
formation of mycelia structures. The growth continues
until the microorganism enters its death phase at week
3 to week 4 (Figs. 3d and 3e).
Fig. 6 Schematic diagram of amylose and amylopectin The infectivity is highest on the week 2 of
chain in sago starch granules [16]. biodegradation periods due to the spreading of
Control NRL films 10 phr SS/NRL films microbial colonies throughout the entire films. The
growth of microbial is reported to be the highest in a
first stage where the multiplication is in exponential
terms [17]. At the second stage, due to the colonies
saturation, the growth stop and the number are
constant. After a while, due to short of energy sources
or others factors, the microbial growth are decreased
and the phase are likely to be referred as death phase.
As shown in Fig. 8, during microbial consumption
process, the microorganisms formed a biofilms on the
NRL films surfaces. The microorganism are then
growing its hyphea through the entire NRL films
surfaces as well as going deep into latex substrates to
absorb nutrients. As the biodegradation week
progressed, the hyphea started to form branches and
the growth of colonies usually determined by the
exponential growth of these branches. The primary
branches of hyphea are usually thick compared to the
branches. The branches could takes in about 12.4 µm
in diameter [17].

Fig. 7 Microbial growth on 10 phr sago starch filled NRL

films at (a) control, (b) first, (c) second, (d) third, and (e)
fourth weeks of biodegradation periods. Fig. 8 Fungus hyphea under SEM.
142 Utilization of Starch to Accelerate the Growth of Degrading Microorganisms
on the Surface of Natural Rubber Latex Films
Fig. 9 indicates the formation of
mycelium-forming-actinomyces on the SS/NRL films
surfaces after 7 weeks of incubation with SS/NRL
films [6]. From the image obtained, the mycelial
hyphal structures surround the sago starch granules
and utilized it as the source of nutrients for growth.
The mechanisms of absorption of nutrients are by
secretion of an enzyme on or into the granules and
break the long chain of amylose and amylopectin
backbone chain in sago granules into smaller and
simpler compound. The small monomers are then
being transferred into the mycelium via facilitated
diffusion or active transport from the hyphea [17].
The formation of Mycelium structure on the
SS/NRL films indicates that these microbes are able to
utilize natural compound as their source of energy.
According to Bode et al. [18], most of the
microorganism which could utilize polyisoprene chain
of rubber lies within gram positive bacteria. Most of
gram positive microorganism could survive in low pH
environment and considered as acidophiles organism
[19]. The mechanism of resistance is in several types.
As reported; (i) Proton pump, (ii) Protection or repair
of macromolecules, (iii) Changes of cell membranes,
(iv) Production of Alkali, (v) Regulators, (vi) Cell
density and biofilms [20].
As the biodegradation process progressed, the
consumption of polyisoprene chain of rubber and
amylose-lipids complexes between sago and NRL
particles leave sago starch granules at the end due to
Fig. 9 Mycelium-forming-actinomyces on the surface of
degraded 10 phr SS/NRL films with its hyphea surrounding
sago starch granules.
starch retrogradation process. As the sago granules
only consist of amylopectin structures, it formed hard
surfaces to be penetrated by the microorganism
hyphea. As the nutrients depleted, the hyphea extends
its soft tips and surrounds the sago starch granules to
find a pit or hole which makes the penetration process
become easier. This phenomenon could be observed in
Fig. 10.
Fig. 11a shows the densities of microorganism
colonies on NRL surfaces. As the NRL substrates
depleted and formed a hole, an extensive network of
hyphea as well as mycelia structure was observed.
Figs. 11b and 11c indicate the phenomenon. From the
images, it is observed that at the depleted nutrients
regions on NRL films formed a hole. These are due to
the utilization of the substrates as the growing
nutrients for the fungi colonies.
When nutrients depleted, mycelium will act as
regulator for distributing newly synthesized biomass
to support hyphal extension and new branch formation.
The branching are reduced and maintained the hyphal
growth hence maximize the spread of mycelium. As
the spreading maximized, it increased the chances of
reaching fresh resources or nutrients. This
phenomenon could be observed in Figs. 11a, 11b and
11c where at the fresh region (Un-hollowed region),
small formation of mycelia was formed in order to
explore fresh nutrient-rich area compared to saturated
(hollow) regions.
Fig. 10 Penetration of fungi hyphea into sago starch
granules via pit on granule surfaces.
 Utilization of Starch to Accelerate the Growth of Degrading Microorganisms 143
on the Surface of Natural Rubber Latex Films

(a) utilization in SS/NRL films is observed through the

extend of the microorganism hyphea structure to
explore the nutrients and growing of mycelium to
regulate the production of biomass and active
branching. As the nutrients depleted, the mycelium
growth were maximized and branching of hyphea is
controlled in order to search for more nutrients rich
sites in NRL films substrates.

Acknowledgments
This research was supported under University Sains
(b)
Malaysia-Research University (USM-RU No: 814093)
and Post Graduate Research Scheme (PRGS-USM No:
8034034) grant.

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