ISOLATION AND CHARACTERIZATION OF LOW-DENSITY

POLYETHYLENE DEGRADING AND BIOSURFACTANT-

PRODUCING BACTERIA FROM SOILS

This thesis is submitted to BRAC University in partial fulfillment of the requirements for the
degree of Bachelor of Science in Biotechnology

ROMI MARINA GEORGE

Student ID # 14236008

Biotechnology Program
Department of Mathematics and Natural Sciences
BRAC University
66, Mohakhali, Dhaka-1212
Bangladesh
March, 2019
 IN DEDICATION OF MY PARENTS & SISTERS
GEORGE P. BAROI, LUCKY DAS, CLARA CAMELIA GEORGE & FABIAN
PARSIA GEORGE
&
MY BEST FRIEND, MOHAMMED NIMEREE MUNTASIR
 DECLARATION OF AUTHENTICITY

I, the undersigned, declare that the research work embodying the results reported in this thesis
entitled “Isolation and characterization of Low-Density Polyethylene degrading bacteria from
locally collected soils” is my original work, gathered and utilized for the sole purpose of
fulfilling the objectives of this study. I confirm that the work has not been previously submitted
to any other institution, in whole or in part, for a higher degree or diploma. I further declare
that the thesis has been composed entirely by me under the supervision of Dr. Mahboob
Hossain, Professor, Microbiology Programme, Department of Mathematics and Natural
Sciences, BRAC University, Dhaka, except where stated otherwise by reference or
acknowledgment.

__________________

Romi Marina George

Certified

_______________________

Dr. Mahboob Hossain,

Supervisor.
Professor, Microbiology Programme,
Department of Mathematics and Natural Sciences,
BRAC University, Dhaka
 ACKNOWLEDGMENT

I extend my sincerest gratitude to the Almighty, the Knower of the unseen and the witnessed
for granting me health, wisdom, and patience in delivering this piece of work. First and
foremost, I would like to thank my parents and my sisters without whose constant support and
encouragement this thesis would not have been possible.

My deepest regards, gratefulness, and appreciation goes to my supervisor Dr. Mahboob

Hossain, Professor, Microbiology Program, Department of Mathematics and Natural Sciences,
BRAC University for his constant guidance, constructive critique and constant encouragement
to pursue novel ideas throughout the course of this work.

I am grateful to Professor A F M Yusuf Haider, Professor, and Chairperson, Department of

Mathematics and Natural Sciences, BRAC University for his benevolent guidance and
encouragement.

My regards and gratitude go to Aparna Islam, Professor, Jebennesa Chowdhury, Assistant

Professor, and Romana Siddique, Senior Lecturer, Department of Mathematics and Natural
Sciences, BRAC University, for all their support and encouragement throughout the duration
of my undergraduate degree.

I would like to extend my deepest gratitude and regards to Asma Binte Afzal for her constant
guidance, support and wise words throughout the work in the lab.

I would like to thank Akash Ahmed, Rezwanul Kabir, Mahmudul Hassan, Shabnam Syeed,
Raghib Mubassir Qazi, Shaeri Nawar, and Maliha Tabassum Rashid, Olia Najon Tuson,
Phoebe A. William, Anika Nawar, Abu Nowroze Rafin, Saiful Islam Shanto and Nowrin
Hosain for their words of encouragement and support and valuable company.

My regards to Ashiqe-E-Khuda, Tanzila Ahmmed Bonna, Shilpi Akter, Nadira Begum, Md.
Furkan Mia, Md. Morshed-Al-Mamun for helping me out each time I asked.

Last but not the least, I would like to express my heartiest and warmest thanks to my biggest
well-wisher and my best friend, Mohammed Nimeree Muntasir, without whose belief,
motivation and hard-work, completion of this thesis would have been impossible.

Author
Table of Contents

DECLARATION OF AUTHENTICITY ....................................................................................... 3

ACKNOWLEDGEMENT .............................................................................................................. 4
ABSTRACT .................................................................................................................................... 8
1. INTRODUCTION ................................................................................................................... 9
1.1 Background ...................................................................................................................... 9
1.2 LDPE .............................................................................................................................. 11
1.2.1 Properties and Characteristics ...................................................................................... 12
1.2.3 Business market of LDPE............................................................................................. 13
1.2.3 Disadvantages ............................................................................................................... 14
1.2.4 Extent of Inertness of LDPE ......................................................................................... 15
1.2.5 Factors contributing to its inertness .............................................................................. 15
1.2.6 Enhancement of Biodegradation of LDPE ................................................................... 16
1.3 Microorganisms involved in polyethylene degradation: ..................................................... 16
1.3.1 Consumption of the polymer ........................................................................................ 19
1.3.2 Mechanism of microbial biodegradation of polyethylene: ........................................... 19
1.3.4 Enzymes associated with degradation .......................................................................... 21
13.5 Effect of microbial activity on the degradation of polyethylene ................................... 21
2. MATERIALS AND METHODS .............................................................................................. 24
2.1 Working place for the study ................................................................................................ 24
2.2 Media, Solutions, and Reagents: ......................................................................................... 24
2.3 Handling of Laboratory Apparatus, Glassware and Analysis Equipment .......................... 24
2.4 Sample Collection: .............................................................................................................. 24
2.4.1 Soil samples: ................................................................................................................. 24
2.4.2 LDPE films: .................................................................................................................. 25
2.5 Media and LDPE emulsion/films preparation:.................................................................... 25
2.5.1 Agar media and broth: .................................................................................................. 25
2.6 LDPE films preparation: ..................................................................................................... 27
2.7 LDPE emulsion preparation ................................................................................................ 27
2.8 Staining and Destaining solutions: ...................................................................................... 28

5
 2.9 Starter inoculum preparation ............................................................................................... 28
2.10 Isolation of LDPE degrading organisms: .......................................................................... 28
2.10.1 Primary screening ....................................................................................................... 28
2.10.2 Identification of PEG degrading isolates: ...................... Error! Bookmark not defined.
2.10.3 Petri plate method- secondary screening: ................................................................... 29
2.10.4 Clear zone method: Tertiary screening ....................................................................... 29
2.10.5 Screening for bacterial survival .................................................................................. 29
2.10.6 Screening of Biosurfactant producing bacteria: ................................................................. 30
2.11 Gram’s staining ................................................................................................................. 30
2.12 Biochemical tests............................................................................................................... 31
2.12.1 Methyl Red (MR): ...................................................................................................... 31
2.12.2 Voges Proskauer (VP): ............................................................................................... 31
2.12.3 Indole: ......................................................................................................................... 32
2.12.4 Citrate: ........................................................................................................................ 32
2.12.5 Catalase:...................................................................................................................... 32
2.12.6 Oxidase: ...................................................................................................................... 32
2.12.7 Motility and Urea: ...................................................................................................... 32
2.12.8 Triple sugar iron test ................................................................................................... 32
2.12.9 Nitrate reduction test: ................................................................................................. 33
2.12.10 Starch Hydrolysis: .................................................................................................... 33
3. RESULTS ................................................................................................................................. 34
3.1 Result of Primary screening ................................................................................................ 34
3.2 Result of PEG degradation: .................................................... Error! Bookmark not defined.
3.3 Result of Petri plate screening ............................................................................................. 37
3.4 Result of Clear zone method: Tertiary screening ................................................................ 38
3.5 Result of bacterial survival screening: ................................... Error! Bookmark not defined.
3.6 Result of biosurfactant production: ..................................................................................... 41
3.7 Result of Gram’s staining.................................................................................................... 42
3.8 Results of Endospore staining: ............................................................................................ 43
3.9 Biochemical tests................................................................................................................. 44
4. DISCUSSION ........................................................................................................................... 47

6
4. CONCLUSION ......................................................................................................................... 51
REFERENCES ............................................................................................................................. 52

7
 ABSTRACT

Microorganisms play a very important role in the biological decomposition of various materials in
the natural environment, this is called biodegradation. Synthetic materials including plastic and
polyethylene waste accumulate in the environment and pose an ever-increasing ecological threat
to humankind and the overall ecosystem on earth. Biodegradation of these plastic wastes using
potent microbial strains could provide a solution to the problem. In the present study, the
microorganisms responsible for biodegradation were isolated and characterized from local samples
contaminated with hydrocarbon polymers. The experiment was conducted over a series of
screening methods. The primary screening involved culturing collected samples for over 40 days
to screen out low-density polyethylene (LDPE) non-degrading bacteria, followed by observation
of polyethylene glycol (PEG) utilization by the formation of a clear zone. This led to the isolation
of 43 different colonies. Secondary screening further allowed for selection of 14 different isolates
that were able to form biofilm over the polymer surface. Finally, the tertiary screening allowed for
the selection of 10 different isolates that are confirmed LDPE-degraders. From continuous
culturing for 36 days, 8 out of 10 isolates were able to survive and propagate in minimal salt broth,
namely, Staphylococcus sp., Acinetobacter sp., Clostridium sp., Bacillus sp., and Lysinibacillus
sp. Finally, organism able to produce anionic and cationic biosurfactant were identified as
Clostridium novyitype A and Staphylococcus massiliensis respectively.

8
 1. INTRODUCTION

1.1 Background

Plastics are organic polymers of high molecular mass that have adapted their name from the Greek
word ‘plastikos’ which means ‘capable of being shaped or molded’ (Restrepo-Flórez, et al., 2014).
Plastics have adopted widespread use in packaging industries such as food, pharmaceuticals, and
cosmetics (Sharma, et al., 2014). As much as one-third of the plastics produced are being for
packaging purposes (Raaman, et al., 2012). Piping, plumbing, automobiles, furniture, toys, etc.
have all taken on the trend of using plastics in replacement of wood, stone, leather, horn, paper,
metal, glass, ceramics, etc (Divyalakshmi, et al., 2016).

The most commonly used plastics are polyethylene (PE), polypropylene (PP), polystyrene (PS),
polyvinyl chloride (PVC), polybutyrene tetraphthalate (PBT) and nylon (Sharma, et al., 2014). The
most common types of polyethylene are Low-Density Polyethylene (LDPE), High-Density
Polyethylene (HDPE), Linear Low-Density Polyethylene (LLDPE) and Cross-Linked
Polyethylene (XLPE). All these different subgroups of polyethylene differ in terms of the
extensiveness of branching, the presence of functional groups on the surface and their density
(Restrepo-Flórez, et al., 2014). Some may even have pro-oxidants and starch integrated into them
as additives in order to facilitate the biodegradation of the substrate by microorganisms (Zheng, et
al., 2008) (Koutny, et al., 2006).

9
Figure 1 Different plastics, their resin identification code (RIC) numbers and recycled
reuses by (Pūtaiao, 2017)

10
1.2 LDPE

The most commonly used type of polyethylene is the Low-Density Polyethylene (LDPE) that
comprises of approximately 60% of the total plastic production (Raaman, et al., 2012). It is a
thermoplastic made from repeating units of the monomer ethylene and has SPI resin ID code 4
(Mahdiyah, et al., 2006). LDPE is widely used in the manufacture of containers, dispensing bottles,
wash bottles, tubing, plastic bags for computer components and various molded laboratory
equipment. Other common uses may include the making of trays and general purpose containers,
corrosion-resistant work surfaces, very soft and pliable parts such as snap-on lids, six-pack rings,
the inner and outer layer of juice and milk cartons, playground slides and plastic wraps.

Figure 2 A piece of packaging foam made Figure 3 LDPE packaging roll

from LDPE

In Bangladesh, the most common use of LDPE goes into the making of transparent plastic bags by
vendors in grocery stores and street markets. A report in The Daily Star informed mentioned that
single family uses four polythene bags every day and around 2 crore polythene bags are being used
in Dhaka on a daily basis. The news article also stated that Prof Ahmad Kameuzzaman Majumder,
chairman of Environment Science department of Stamford University Bangladesh, cited
information in his keynote of Waste Concern to say that 3.5 kg plastic items were used by a person

11
in the country in 2014. The use of plastics in Bangladesh has increased by 80 times in the
past 28 years, says the green group, Save the Environment Movement (POBA). Quoting a
recent survey of POBA, the use of plastics in Bangladesh has grown to 1,200,000 metric
tons in 2018 compared to just 15 thousand metric tons in 1990 (Tribune, 2018).

Figure 4 Plastic pollution in remote areas in Bangladesh by (Tribune, 2018)

1.2.1 Properties and Characteristics

The general properties of LDPE include the following. It has a density range of 0.917-0.930 g/cm3.
It is unreactive at room temperature and pressure (r.t.p.) with the exception of strong oxidizing
agents, and some solvents cause swelling. It can withstand temperatures as high as 80°C
continuously or 90°C temporarily. (n.d.). Also, the degree of branching in LDPE is a lot higher
than HDPE, making former have fewer intermolecular forces as compared to the latter. This, in
turn, coincides with the observation that LDPE has a lower tensile strength and higher resilience
than HDPE, as is expected of it. It also further demonstrates that because of this extensive side

12
branching, LDPE is less tightly packed and contain fewer crystalline structure, resulting in a lower
density as compared to that of HDPE (Royer, et al., 2018).

1.2.3 Business market of LDPE

Apart from plasticity, their other advantages involve ease of manufacture, low cost, versatility,
high hydrophobicity (imperviousness to water), durability, etc (Divyalakshmi, et al., 2016). As a
result, as has been observed, the worldwide LDPE market had reached a volume of about US$33
billion in 2013. The use of this synthetic polymer is growing at a rate of 12% per year, and while
140 million tons are produced worldwide, only 5.7% of this is being recycled each year according
to EPA estimation (Mahdiyah, et al., 2006).

Figure 5 World consumption of LDPE in the year 2016 by (Hakkarainen, et al., 2004)

13
 Figure 6 Total Polyethylene demand by region in 2016 by (Hakkarainen, et al., 2004)

1.2.3 Disadvantages

As for the remaining polyethylene, it takes thousands of years for their efficient degradation, which
leaves with only two other options: incineration and landfilling. Incineration produces a massive
amount of harmful greenhouse gases such as carbon dioxide and methane and requires the input
of enormous energy to carry out such large projects, making the process both hazardous to the
environment and expensive. On the other hand, landfilling requires the closing of landfill sites,
requiring a lot of land space. It is also a major contributor of environmental pollution, as
improperly disposed of plastic materials do not allow water and air to enter the earth, causing soil
infertility (Restrepo-Flórez, et al., 2014). The leachate from the dumping site may seep into the
soil and contaminate the groundwater, which could be the only source of water for drinking and
other day to day purposes in some places. In the sea, the polythene may also cause blockage in
intestines of fish, birds and marine mammals (Secchi, et al., 1999) (Spear, et al., 1996) (Starnecker,

14
et al., 1996). Plastics have been found to cover the sea-bed during deep-sea investigation using
research submersibles (Fugikura, et al., 2008).

1.2.4 Extent of Inertness of LDPE

Compared to other methods, biodegradation ultimately remains as the only means of dealing with
accumulating polythene. Albeit slow, the process is completely eco-friendly. The inertness of the
polymer is demonstrated in the long-term study conducted by Albertsson and Karlson (1990). In
the study, upon observation of the biodegradation of 14C-labeled polyethylene, it was found that
less than 0.5% of carbon (as CO2) by weight was given off from an u.v.-irradiated polyethylene
sheet after 10 years of incubation. Whereas, for nonirradiated polyethylene, only less than 0.2% of
carbon was emitted for the same incubation period. Additionally, after incubation in moist soil for
a decade, no further degradation was observed, and finally, after 32, partial degradation was
observed for the same sheet of polyethylene in the soil burial (Otake, et al., 1995)

1.2.5 Factors contributing to its inertness

From a chemical perspective, one would expect polyethylene to be biodegradable since it is

composed of linear alkene groups stacked side by side. However, in the case of polyethylene, the
reason for its least susceptibility to biodegradation is the larger the molecular weight of the
polymer. Macromolecules with molecular weight lower than 620 do support microbial growth,
while those larger than 620 have low chances of supporting microbial growth (Haines, et al., 1974)
(Potts, 1978). Furthermore, the polyethylene surface is hydrophobic, which make it impermissive
and immiscible to hydrophilic solvents such as water. Due to its chemical nature, the polyethylene
is unable to allow the microbial population to stick to its surface as most microorganisms have a
hydrophilic surface. Therefore, it is widely accepted that the resistance of polyethylene to
biodegradation stems from its high molecular weight, its hydrophobic nature, and its three-
dimensional structure, all of which interfere with its availability to microorganisms (Arutchelvi, et
al., 2008).

15
1.2.6 Enhancement of Biodegradation of LDPE

Given the circumstances of its inert nature, biodegradation can still be enhanced on exposure to
biological agents like bacteria, fungi, enzymes etc., aided by initial pretreatment with abiotic
factors such as u.v. irradiation (Cornell, et al., 1984), thermal treatment (Albertsson, et al., 1998)
or oxidation with nitric acid (Brown, et al., 1974). All these abiotic factors work synergistically to
increase the surface hydrophilicity of the polymer by introducing carbonyl groups by the process
of oxidation which can be easily targeted by microorganisms (Albertsson, 1978) (Albertsson,
1980) (Cornell, et al., 1984). The biodegradability can be further improved by blending it with
biodegradable additives, photo-initiators or copolymerization or using surfactants to reduce
surface tension between hydrophilic and hydrophobic parts (G.J.L., 2007) (Hakkarainen, et al.,
2004).

1.3 Microorganisms involved in polyethylene degradation:

Microorganisms able to degrade polyethylene has so far been limited to 17 genera of bacteria and
9 genera of fungi (Restrepo-Flórez, et al., 2014). Table 1 and 2 summarize the list of
microorganisms responsible for colonization or degradation of polyethylene, or both. The more
common microorganisms are Bacillus megaterium, Pseudomonas sp., Azotobacter, Ralstonia
eutropha, Halomonas sp (Chee, et al., 2010).

Brevibaccillus borstelensis (30 days, 50℃) reduced its gravimetric and molecular weights by 11%
and 30% respectively (Hadad, et al., 2005). Bacillus sp. showed 42.5% followed by
Staphylococcus sp. 20%, Pseudomonas sp. 7.5% and consortium 5% degradation by weight loss
in 40 days (Singh, et al., 2016). Staphylococcus arlettae, after 30 days of incubation at 37℃,
caused loss of 13.6% of maximum weight loss (Divyalakshmi, et al., 2016). Bacillus subtilis, with
the addition of its biosurfactant (surfactin), proved to cause a weight loss percentage of 9.26% in
30 days (Vimala, et al., 2016).

16
Table 1 Bacterial strains associated with polyethylene degradation by (Restrepo-Flórez, et al.,
2014)

17
Table 2 Fungal strains associated with polyethylene degradation by (Restrepo-Flórez, et al.,
2014)

Table 3 Weight loss percentage due to biological action for different types of polyethylene in
various environments with pre-oxidative treatments by (Restrepo-Flórez, et al., 2014)

18
1.3.1 Consumption of the polymer

The consumption of the polymer can be detected, although the process of its assimilation is very
slow and difficult to detect. Some studies have used a reduction in the weight of samples
determined by gravimetric analysis to monitor the corresponding degradation of the material
(Hadad, et al., 2005) (Sivan, et al., 2006) (Sudhakar, et al., 2008) . However, degradation can also
be monitors by the evolution of CO2 from the samples, which, out of the two techniques, is more
widely accepted and used (Albertsson, 1980) (Karlsson, et al., 1988) (Pramila, et al., 2011).
Although the latter is an indirect measurement, it does provide greater insight than the former. CO2
evolution has the added benefit of not only monitoring the net degradation but also the rate of
degradation over a given period of time (Albertsson, 1980) (Karlsson, et al., 1988).

1.3.2 Mechanism of microbial biodegradation of polyethylene:

Although there is enough evidence that proves biodegradation of polyethylene, there is still a lack
of knowledge on the complete metabolic pathways involved in the process and the structure and
identity of all the enzymes involved. The mechanism comprises of roughly three different steps:

1) microbial colonization of polymer

2) reduction of its molecular weight
3) oxidation of the molecules into CO2 and H2O.

Polyethylene is a hydrophobic, high molecular weight molecule, and therefore it is commonly

accepted that biofilm colonization is the first step for degradation of this polymer (Gilan, et al.,
2004). Biofilms are sessile communities of microorganisms developed on a surface that can be
composed of individuals from the same or different species (Donlan, 2002). Studies on
microorganisms’ attachment to polyethylene have identified that the main limitation of the
colonization process is the relatively high hydrophobicity of the polymer in contrast to the
regularly hydrophilic surfaces of most microorganisms (Gilan, et al., 2004) (Tribedi, et al., 2013).
It has been proposed that strains with more hydrophobic surfaces can play an important role in the
initial colonization of the polymer. The other metabolic adaptation that can be important in

19
polymer colonization is the production of surfactants, molecules that can mediate the attachment
process of microorganisms to the hydrophobic surface (Karlsson, et al., 1988) (Tribedi, et al.,
2013). Theoretically, polyethylene can be used as a carbon source for microorganisms similar to
many other hydrocarbons; however, its high molecular weight limits its use as a substrate for
enzymatic reactions to take place.

Once colonization is done, there are two key reactions that follow, the first one being the reduction
of its molecular weight and the second being the oxidation of the molecules. (Restrepo-Flórez, et
al., 2014). Reduction of molecular weight is required for two reasons, firstly to enable transport of
molecules through the cell membrane, and secondly because enzymatic systems present in the
microorganisms are only able to attack certain molecular weights, usually in the range of 10-50
carbons, though there has been a report of enzymatic activity up to 2000 carbons (Yoon, et al.,
2012). Once the size of the molecule is reduced, oxidation is required in order to transform the
hydrocarbon into a carboxylic acid that can be metabolized by means of b-oxidation and the Krebs
cycle (Albertsson, et al., 1987). Fig. 7 presents the proposed mechanisms of biodegradation for
polyethylene.

Figure 7 Hypothetical mechanisms of polyethylene degradation. This mechanism is the

result of the adaptation of the works presented by different authors by (Albertsson, et al.,
1998) (Rojo, 2010) (Santo, et al., 2012)

20
1.3.4 Enzymes associated with degradation

There are very few works devoted to studying the enzymes involved in these processes. Breaking
down large polyethylene molecules can be accomplished by enzymatic action, as proven by (Santo,
et al., 2012), who found that by incubation with the enzyme laccase the molecular weight of
polyethylene was reduced and its keto-carbonyl index increased. These two factors were felt to
indicate that both scission and oxidation reactions were taking place by the same enzyme. In
regards to the oxidation process, there was another important work, this one by (Yoon, et al., 2012)
that isolated an alkane hydroxylase from the AlkB family that was active to polyethylene samples
with molecular weights up to 27,000 Da. In general, it is accepted that alkane hydroxylase performs
the first oxidation that leads to the subsequent degradation of a hydrocarbon (Rojo, 2010)

13.5 Effect of microbial activity on the degradation of polyethylene:

One may wonder what happens to the LDPE films during and after the microbial attack. Upon
colonization by microorganisms on polyethylene surfaces, the properties of the synthetic polymer
undergo diverse changes. The extent of biodegradation takes a toll on seven different
characteristics of the polymer: functional groups on the surface, hydrophobicity/hydrophilicity,
crystallinity, surface topography, mechanical properties, molecular weight distribution and mass
balance (Restrepo-Flórez, et al., 2014). The modifications to the surface chemistry of LDPE are
evidence of interactions by microorganisms with the surface.

 1.3.5.1 Functional groups on the surface

The presence and nature of functional groups on the surface of polyethylene are determined
by FTIR spectroscopy. Whenever there are biological activities occurring on the surface of
a synthetic polymer, changes in following functional groups are observed: carbonyls (1715
cm-1) esters (1740 cm-1), vinyls (1650 cm-1) and double bonds (908cm-1) (Albertsson, et
al., 1987) (Gilan, et al., 2004) (Hadad et al., 2005) (Santo, et al., 2012) (Sudhakar, et al.,
2008). Some studies claim that there is a corresponding decrease in the concentration of
carbonyl indices and increase in double bonds (Albertsson, et al., 1987) (Raghavan, et al.,

21
 1992) (Gilan, et al., 2004) (Hadad, et al.) while others claim an increase in the concentration
of carbonyl group and decrease in double bonds during biodegradation (Gilan, et al., 2004)
(Raghavan, et al., 1992) (Manzur, et al., 2004). The indifference in these reports suggests
that the biodegradation is rather a complex process that can differ for different
microorganisms and what holds true is the fact that the surface of LDPE most certainly
undergoes a change in surface chemistry.

 1.3.5.2 Hydrophobicity/Hydrophilicity
The hydrophobicity/hydrophilicity of a surface depends on the nature, concentration, and
exposition of the functional groups present in the material. If the degree of oxidation by
the abiotic factors such as UV or activity of enzymes is higher than the degree of
consumption of functional groups, then there will be an increase in the net concentration
of hydrophilic functional groups, hence the hydrophilicity of the material will increase. On
the contrary, if the degree of oxidation by the abiotic factors is lower than the degree of
consumption of functional groups, then the net concentration of hydrophilic functional
groups will tip to hydrophobicity. Therefore, the material will become more hydrophobic
(Restrepo-Flórez, et al., 2014). It is important to maintain this ratio of oxidation versus
consumption as hydrophilic surfaces facilitate degradation but hydrophobic surfaces do not
unless the microbial colony has hydrophobic surface too.

 1.3.5.3 Crystallinity:
Comprising of crystalline microstructures surrounded by amorphous regions, polyethylene
is a semi-crystalline polymer. Experimentally, it has been corroborated that amorphous
regions are consumed first due to their accessibility before they move on to consume
smaller crystals (Manzur, et al., 2004), resulting in an increase in the proportion of larger
crystals (Albertsson, et al., 1998) (Sudhakar, et al., 2008).

22
 1.3.5.4. Molecular weight distribution
Just as was with crystallinity, the average molecular weight seems to increase after a
microbial attack as a result of consumption of lower molecular weight chains. (Hadad et
al., 2005) (Santo, et al., 2012).

 1.3.5.5 Surface topography

Surface colonization by microorganisms causes substantial changes in the surface
topography. For fungi, hyphal structures may penetrate the surface (Gilan, et al., 2004)
(Sivan, et al., 2006) (Koutny, et al., 2006) (Pramila, et al., 2011) (Tribedi, et al., 2013).
Cracking and pitting in the polymer surface after biodegradation process can be observed,
causing superficial damage to the film.

23
 2. MATERIALS AND METHODS

2.1 Working place for the study

The present research work was performed in the Biotechnology and Microbiology laboratory of
the Department of Mathematics and Natural Sciences, BRAC University, Mohakhali, Dhaka 1212,
Bangladesh.

2.2 Media, Solutions, and Reagents:

Most of the required solutions, reagents and media available in the laboratory were of reagent
grade and were used without further purification.

2.3 Handling of Laboratory Apparatus, Glassware and Analysis Equipment

All glassware was washed with detergents, rinsed 4-5 times with tap water and sterilized in hot air
oven (Binder ED23, Germany) at 160 for 2 hours. McCartney bottles, Durham bottles, conical
flasks, micropipette tips, glass spreader, glass test tubes, falcon tubes, and microfuge tubes were
sterilized by autoclaving at 121 for 15 minutes at 15 psi (Sturdy, SA-300VF, Taiwan). All the
microbiological works were done inside the Biological Safety Cabinet (LabTech, LCB-1803B-A2,
Korea). For the measurement of absorption in the UV-Visible region, a high-performance compact
split beam spectrophotometer (PG Instruments, T60, UK) was used.

2.4 Sample Collection:

2.4.1 Soil samples: Samples were collected in sterile test tubes from 7 different sources listed
below. The soil collected was dug out from approximately 10 centimeters deep from the surface

24
using sterilized forceps and spatula. Marine water was collected from an approximate 10
centimeter deep from surface water. The soils collected from petrol pump stations were
hypothesized to harbor bacteria able to breakdown larger and complex hydrocarbons like gasoline
and diesel. Garbage dump sites were ravaged for soil sample as the majority of plastic wastes
would be piled up at the sites. Marine water from sea-shore was nearer to the large dump sites of
plastics and polythenes, and one would expect the leachates from the dump site to wash into the
water near the shore with tidal waves. Table 4 lists the details of sample collection.

2.4.2 LDPE films: Low-Density polyethylene bags were collected from local markets

Table 4 Tabulation describing the sample number, material and location of sample collection
throughout Bangladesh

Sample Material Location

number collected
S1 Soil Shyamoli petrol pump station
S2 Soil Shyamoli garbage dump site
S3 Soil Farmgate garbage dump site under
City Corporation
S4 Soil Mirpur petrol pump station
S5 Soil Gabtoli petrol pump station
S6 Marine Cox’s Bazar sea-shore
water
S7 Marine Moheshkhali sea-shore
water

2.5 Media and LDPE emulsion/films preparation:

2.5.1 Agar media and broth:

25
 Minimal salt broth (MS 1) was used for the primary screening of LDPE degrading bacteria
where only pieces of LDPE films were used as carbon source along with inorganic. The
composition follows: KH2PO4 (3.0 g/L), K2HPO4 (0.1 g/L), NaCl (5.0 g/L), NH4Cl (2.0
g/L), MgSO4 (0.2 g/L) and CaCl2.2H2O (0.15 g/L). The medium was supplemented with
0.3% LDPE films as a carbon source.

 Nutrient agar was used as a common bacteriological growth media. As an alteration, LDPE
strips were placed on the media after the spread plate method, as a method of secondary
screening
 Minimal salt agar (MSA) media was used for the tertiary screening of LDPE degrading
bacteria supplemented with LDPE powder as carbon source made from scratch. The media
composition follows: (per liter of distilled water) K2HPO4 , 1g; KH2PO4, 0.2g; NaCl, 1g;
CaCl2.2H2O, 0.002g; boric acid, 0.005g; (NH4)2SO4, 1g; MgSO4.7H2O, 0.5g;
CuSO4.5H2O, 0.001g; ZnSO4.7H2O, 0.001g; MnSO4.H2O, 0.001g and FeSO4.7H2O, 0.01g.
Polyethylene (LDPE) emulsion was added to the mineral salt medium at a final
concentration of 0.1% (w/v) respectively.

 Minimal salt broth (MS 2) was used to observe the colony-forming unit (CFU) of those
bacteria that tested positive for LDPE degradation in order to evaluate whether the specific
species will be able to sustain in MS 2 broth on its own without needing to add additional
inoculum periodically. This adaptation was employed to study the degradation of LDPE
films over 30 days’ incubation which is not covered in this dissertation. The MS 2 broth
has the same composition as above, only that the instead of emulsified LDPE, LDPE strips
were used at a final concentration of 0.1% (w/v) respectively. The pH of the broth was
adjusted to pH 6, pH 7 and pH 8 to check for optimal conditions for growth.

 Bushnell-Haas supplemented CTAB/SDS-methylene blue agar was used to screen

anionic/cationic biosurfactant producing bacteria. The media composition follows: MgSO4
(0.200 g/L) CaCl2.2H2O (0.020 g/L), KH2PO4 (1.000 g/L), K2HPO4 (1.000 g/L), NH2NO3
(1.000 g/L), FeCl2 (0.050 g/L), glucose (20 g/L), methylene blue (0.2 g/L) and Agar 20.000
(g/L). To screen for anionic biosurfactant, 0.5 mg/mL CTAB was added to the media.

26
 Alternatively, for screening of cationic biosurfactant, 0.5 mg/mL SDS was added to the
media.

 Peptone water used as enrichment broth for all microorganisms. It is composed of peptone
(10 g/L) and NaCl (5 g/L).

 T1N1 agar was used for preservation or storage of organisms for over 6 months in RTP. It
is composed of trypticase or tryptone, 10g/L; NaCl, 10g/L, and Agar, 20g/L.

2.6 LDPE films preparation:

Polyethylene sheets were cut into 2x2 cm. Pre-weighed strips were transferred to a fresh solution
having 70 ml Tween 20, 10 ml disinfectant (Detol) and 983 ml distilled water and stirred for 60
minutes. The strips were transferred into a beaker with distilled water and stirred for another 60
minutes. This step was repeated 4 or 5 times until the LDPE films were ridden of any residual
disinfectant or tween. Finally, the strips were aseptically placed in 70% ethanol solution overnight.
Finally, the disinfected strips were transferred to a sterile petri dish and dried in the laminar hood
and put away for further use. The whole process was carried out inside laminar hood using
autoclaved distilled water, autoclaved beaker, and sterile forceps/spatula.

2.7 LDPE emulsion preparation

One gram of packaging LDPE foam was dissolved in 100 ml of benzene. The solution was
emulsified in 1000 ml of M2 broth using a sonicator. 20 g of agar was added to the emulsion in 1L
Erlenmeyer flask and stirred continuously while heating for at least 30 minutes to evaporate the
benzene completely. The medium was autoclaved resulting in homogenous opaque plates before
being plated.

27
2.8 Staining and Destaining solutions:

Staining solutions were prepared by mixing 40% methanol, 10% acetic acid and 0.1% Coomassie
blue R250 mixed in 50% distilled water. Destaining solutions were prepared the same way,
omitting the addition of coomassie blue.

2.9 Starter inoculum preparation

1 gram or mL of soil/water from sample was added to 9 ml peptone water in test tubes, vortexed
and incubated overnight.1 ml of the solution was transferred to 9 ml nutrient broth (NB), vortexed
and incubated for 24 hours. The solution was transferred to falcon tubes and centrifuged at 15,000
rpm for 10 minutes to obtain a cell pellet. The supernatant was discarded and the pellet was
resuspended in 0.9% NaCl solution. The above steps were done for S1 through S7.

2.10 Isolation of LDPE degrading organisms:

2.10.1 Primary screening

For primary screening, 1 ml of initial inoculum was added to 100 ml of MS 1 broth in 250 ml
Erlenmeyer flask containing 0.3% (w/v) LDPE strips. The inoculated broth was incubated at 37℃
at 150 rpm for 1 week. After 1 week of incubation, 1 ml of the old broth was into a fresh MS 1
broth supplemented with polyethylene films as a carbon source. Every 1st and 7th day of each week,
the OD of the broth was taken using a spectrophotometer, with an increase in OD corresponding
to an increase in growth. Positive and negative controls were maintained at all times where the
positive control had 1 g/L of glucose supplemented as a carbon source instead of polyethylene
strips and the negative control was maintained where no carbon source was present at all. The
above steps were repeated for samples S1 through S7. The surviving bacteria were obtained as
single colonies were obtained by streaking on Nutrient Agar media.

28
2.10.2 Petri plate method- secondary screening:

A single colony of each isolate was resuspended in 9 ml of 0.9% NaCl solution and 100 µl of the
solution was spread onto NA. Sterilized LDPE strips were placed aseptically onto the NA plates
using sterilized forceps. The NA plates were incubated for 1- 2 weeks at 37℃ (Kowalczyk, et al.,
2016) (Urbanek, et al., 2017) (Singh, et al., 2016).

2.10.3 Clear zone method: Tertiary screening

For the final step of screening, the 24hr old culture of each isolate was streaked onto the MSA
medium. The medium was incubated for 7 days at 37℃. After incubation, the plates were stained
with a staining solution for 15 minutes. The stain was decanted and destained with a destaining
solution. Isolates that gave a clear zone were positive for LDPE degradation. Isolates that rendered
positive for clear zone method were preserved on T1N1 agar in small vials.

2.10.4 Screening for bacterial survival

For the final part of the experiment, the 24-hr culture of LDPE degrading organisms (obtained
after tertiary screening) were inoculated in 3 ml of MS 2 (pH7) broth in vials. The turbidity of the
culture was referenced against McFarland 2.0 standards. The 3ml of the culture was added to 50
ml of MS 2 broth in 100 ml Erlenmeyer flask containing 0.1% (w/v) sterile LDPE films. The broth
was incubated at 37℃ at 150 rpm in shaker-incubator. After every 9 days, 1 ml of fresh MS 2
broth was added to the existing broth and 100 µl of the culture was spread onto NA and incubated
for 24 hours at 37℃. The CFU was measured to monitor the survival and reproduction of each
isolate in extreme conditions. The above steps were repeated again for MS2 broths of lower pH,
i.e., pH 6 and added 250 µl of Tween 20. This was done to see if lowering the pH and adding a
little bit of biosurfactant would enhance or aid the survival of specific bacteria. The whole process

29
took place over a span of 36 days in total in continuous incubation in the shaker-incubator. Isolates
that were still TNTC (too numerous to count) were preserved in T1N1 for storage.

2.10.5 Screening of Biosurfactant producing bacteria:

Isolates obtained after the primary screening were tested for their ability to produce biosurfactants.
A single colony of each isolate was picked up and inoculated in 0.9% saline solution. To test for
production of anionic surfactants, Bushnell-Haas mediated CTAB-methylene blue agar was used.
Well was cut into the agar using a cork-borer and 50 µl of the liquid inoculum was micropipette
into the wells. The plate was then incubated at 37℃ for 72 hours. To test for production of cationic
surfactants, Bushnell-Haas mediated SDS-methylene blue agar was used and step 4 was repeated
for this medium as well. Formation of a dark blue ring around the wells signified formation of
cationic or anionic surfactant by the concerned organism.

2.11 Gram’s staining

Gram staining is a common technique that is used to differentiate two large groups of bacteria
based on their different cell wall constituents. The Gram stain procedure distinguishes between
gram-positive and Gram-negative groups. The morphology of the bacteria can also be checked
using this method.

Endospore staining Endospores were stained via modified Schaeffer-Fulton method using
malachite green and safranin dyes. A loopful of the culture was smeared onto a glass slide with a
few drops of distilled water. The smear was heat fixed before adding 5-6 drops of malachite green
over top. The slide was carefully held over a flame until the dye started to steam and bubble, at
which point it was removed from the flame and allowed to cool. Cooled slides were washed
thoroughly with distilled water before the addition of 5-6 drops of safranin. The dye was allowed
to sit for 3 minutes before the slides were again washed with distilled water and allowed to air dry.
The dried stained slides were then observed 19 under 100x magnification of microscope with
immersion oil to confirm presence or absence of endospores. Isolates were only designated

30
negative for spore formation if there were no visible spores under the microscope after 7 days of
incubation

2.12 Biochemical tests:

A range of biochemical tests was carried out in order to further classify the presumptive species of
isolates bacteria. All tests were carried out according to Bergey’s Manual of Systematic
Bacteriology [21] using fresh, 24 hour cultures from nutrient agar plates. The following tests were
performed:

 MR
 VP
 Indole
 Citrate
 Catalase
 Oxidase
 Motility
 Urea
 TSI
 Nitrate Reduction
 Starch hydrolysis

2.12.1 Methyl Red (MR): Half a loopful of culture was inoculated into a test tube containing 5ml
of MRVP broth, and the tube was incubated at 37°C for 24 hours. For observation, 5 drops of
methyl red dye were added to the tube without shaking. A cherry red color indicated positive for
mixed pathway fermentation of glucose, while orange indicated inconclusively, and yellow
indicated negative results.

2.12.2 Voges Proskauer (VP): Half a loopful of culture was inoculated into a test tube containing
5ml of MRVP broth, and the tube was incubated at 37°C for 24 hours. After incubation, 6 drops
of Barritt’s reagent A were added and the tube was shaken. After that, 6 drops of Barritt's reagent

31
B were added to the tube and observed up to 1 hour. Formation of a pink ring indicated the presence
of acetoin, 2while a brownish ring indicated negative results.

2.12.3 Indole: A loopful of culture was inoculated into a test tube containing 5ml of tryptophan
broth, and the tube was incubated at 37°C for 24 hours. After addition of 5 drops of Kovac’s
reagent to the broth, a red color indicated positive tryptophan hydrolysis while a yellow color was
taken as negative.

2.12.4 Citrate: Sterile slants of Simmon’s Citrate Agar were prepared in vials and the surface was
streaked heavily with a loopful of culture. The vials were incubated at 37°C for 48 hours and
observed for bacterial growth and color change. Growth on the slant with the color change of the
medium to blue indicated positive for utiliza5tion of citrate as a carbon source, while growth
without color change was taken as a negative result.

2.12.5 Catalase: 3 drops of 3% hydrogen peroxide were taken on a clean glass slide. Half a loopful
of bacterial culture was mixed with the hydrogen peroxide on the slide and observed for bubble
formation. Immediate, sustained formation of bubbles was taken as a positive indicator of catalase
production by the sample bacterium, while the slow or delayed formation of a small number of
bubbles was taken as negative.

2.12.6 Oxidase: On a sterile petri dish lid, the filter paper was soaked in oxidase reagent. Using a
sterile inoculating loop, a heavy inoculum of bacteria was smeared onto a section of reagent soaked
filter paper and observed for color change. Changing of the smear to a pink color indicates a
positive test for the presence of cytochrome c oxidase enzyme, while no color change indicates a
negative result.

2.12.7 Motility and Urea: Both motility and urease tests were carried out using MIU agar. The
agar was prepared first, sterilized in test tubes, and then cooled to approximately 50°C before
adding 5% (v/v) of 40% syringe filtered urea solution. Once solidified, the agar was inoculated via
a single stab of heavy inoculum using a sterile inoculating needle and incubated at 37°C for 24
hours. Change of the orange media to pink indicated positive for the presence of urease enzyme
while spiraling growth away from the stab line was indicative of motility.

2.12.8 Triple sugar iron test: Triple sugar iron agar is a differential medium used to determine
H2S production and the type of carbohydrate fermentation from the discoloration of butt and slant.

32
Gas from carbohydrate metabolism can also be detected. To conduct the test, an isolated colony
was inoculated in the TSI medium. The results were observed after 24 hours of incubation at 37°C.

2.12.9 Nitrate reduction test: Many gram-negative bacteria use nitrate as the final electron
acceptor. Nitrate reduction test is a test that determines the production of an enzyme called nitrate
reductase, which results in the reduction of nitrate (NO3). Bacterial species may be differentiated
on the basis of their ability to reduce nitrate to nitrite or nitrogenous gases. Positive Test
corresponds to the development of a cherry red coloration on the addition of reagent A and B and
the absence of red color development on adding Zn powder. Negative Test corresponds to the
development of red color on the addition of Zn powder

2.12.10 Starch Hydrolysis: Pure cultures were streaked onto starch agar plates and incubated at
37°C for 24 hours. After incubation, the plates were flooded with Gram's iodine. Clear zones
around bacterial colonies against a now dark blue medium indicated positive results for hydrolysis
of starch.

33
 3. RESULTS

The main objective of the experiment was to isolate bacteria that were able to degrade a type of
polyethylene known as Low-Density polyethylene or LDPE. For that purpose, samples were taken
from multiple oil or plastic contaminated soil and water from places all over within and outside
Dhaka city. In total of 7 different samples were collected from petrol pump stations and garbage
dumping sites that have a high probability of harboring high molecular weight polymer degrading
bacteria. The samples were collected in autoclaved sterile tubes from a depth of no less than 10
centimeters from the surface as polymer degrading bacteria mainly favor higher temperatures for
optimum growth. The samples were subjected to a primary screening where they were incubated
in minimal salt broth supplemented with LDPE films as a carbon source for over 40 days, which
was followed by growth of isolates on 1% PEG media and looking for a subsequent zone of the
clearance formed by them. Next, the isolates were subjected to secondary screening or the Petri
plate method where pure colonies of bacteria were spread on NA agar plate and were checked to
see if their growth extended onto the hydrophobic surface of LDPE. For the final part of the
screening method, LDPE emulsion plates were used for clear zone formation method by organisms
that degrade LDPE. The experiment was concluded by growing positive isolates in 36 days’ of
continuous incubation in minimal salt broth (pH 7 and 6) supplemented in 0.3% LDPE film as a
carbon source to observe CFU throughout the incubation period. Last but not the least, to check
for biosurfactant producing ability by isolates, Bushnell-Haas media SDS/CTAB methylene blue
agar plates were inoculated with isolates and left for incubation for 48 hours to observe the
formation of the dark blue ring or precipitate around wells.

3.1 Primary screening

Primary screening involved the inoculation of bacterial pellet isolated from S1, S2, S3, S4, S5, S6
and S7 into minimal salt broth (MS 1 broth) and incubating for 7 days at 37℃ at 150 rpm before
transferring the old inoculum into the fresh broth. During each periodic transfer of inoculum from
old to fresh MS1 broth, the growth was monitored by taking the OD immediately after inoculation
and 7 days later (Table 5). The interpretation of the rising/falling OD reading follows in the

34
Discussion section (figure 11 & 12). It was an indirect method of monitoring the increase in
bacterial growth alongside observing the changing of the color of broth from transparent to milky
white (Fig 8). A negative and positive control was taken at all times for each sample. The positive
control had glucose as carbon source and the negative control was transparent colorless (Fig 9 and
10). The tabulation (table 5) below lists the recorded OD.

Figure 8 Semi-milky color of broth containing LDPE films after 7 days incubation

Figure 9 Milky color of broth supplemented with 1% glucose after 7 days incubation
(positive control)

Figure 10 Transparent color of broth containing inoculum but no carbon source after 7
days incubation (negative control)

35
 Table 5 The initial and final OD recorded periodically after every 7 days in MS broth
supplemented with LDPE films

Sa Day 1 Day 7 Day 14 Day 21 Day 27 Day 35 Day 42

mpl I1/nm F7/nm I7/nm F14/nm I14/nm F21/nm I21/nm F27/nm I27/nm F35/nm I35/nm F42/nm
e
1 0.315 0.572 0.120 0.275 0.100 0.118 0.000 0.052 0.000 0.048 0.000 0.045
2 0.302 0.505 0.100 0.198 0.056 0.068 0.000 0.020 0.000 0.012 0.000 0.010
3 0.276 0.455 0.089 0.180 0.054 0.066 0.000 0.020 0.000 0.010 0.000 0.010
4 0.303 0.512 0.112 0.252 0.076 0.090 0.000 0.037 0.000 0.026 0.000 0.022
5 0.299 0.499 0.095 0.200 0.080 0.102 0.000 0.040 0.000 0.027 0.000 0.024
6 0.310 0.568 0.118 0.262 0.089 0.111 0.000 0.050 0.000 0.023 0.000 0.019
7 0.250 0.423 0.070 0.160 0.042 0.056 0.000 0.017 0.000 0.009 0.000 0.005

0.7

0.6
Final Optical Density/nm

0.5
Sample 01

0.4 Sample 02
Sample 03
0.3 Sample 04
Sample 05
0.2
Sample 06

0.1 Sample 07

0
Day 7 Day 14 Day 21 Day 27 Day 35 Day 42
Time/day

Figure 11 A graphical representation of the declining microbial population in primary

screening as a result of the absence of simple sugars as a carbon source

36
 Figure 12 Graphical representation of increasing differences between final OD and Initial
OD

3.3 Secondary Screening: Petri plate screening

After obtaining 43 different isolates from primary screening, the isolates were observed after 14
days incubation to check for biofilm formation over the hydrophilic polyethylene surface placed
on top of agar surface. The 14 isolates that did were considered positive for the screening were
A.a, B.a, B.b, B.c, E1, E2, F, G, H, J.a, J.b, L, N9, N53a. Escherichia coli was taken as the negative
control.

37
Figure 14 Growth of bacteria on the surface of the polyethylene. Negative control (top left)
was maintained by E. coli

3.4 Clear zone method: Tertiary screening

For the final screening, the 14 positive isolates were streaked on LDPE turbid agar where the
LDPE foam was boiled in benzene, emulsified in MS2 broth by sonication and boiled till
evaporation of benzene. After streaking and 7 days’ incubation, the plates were stained with
Coomassie blue staining dye and destained with the destaining dye. A zone of clearance around
isolates was taken positively for LDPE degradation. The isolates that gave clear zones were: B.c,
E1, E2, F, G, H, J.a, J.b and N53a (figure 13). The zone of clearance was ranked in order of
prominence with ‘+++++’ being the biggest zone while ‘+’ being the smallest. Table 6 lists the
results obtained from the tertiary screening.

38
Figure 15 Clear zone formed by isolates on LDPE emulsified turbid agar plates

Table 6 Clear zones formed by isolates

Serial Number Isolate number Zone of clearance

1 B.c ++++
2 F ++++
3 G ++++
4 A.a +++
5 E1 +++
6 E2 +++
7 J.b ++
8 J.a +
9 H +
10 N53a +
11 B.b -
12 B.a -
13 L -
14 N9 -

39
3.5 Bacterial survival screening:

The 10 different isolates found positive in the tertiary screening were further subjected to survival
in MS2 broth supplemented with LDPE films yet again. The isolates were grown in 50 ml broth
in 100 ml conical flasks grown at 37℃ at 150 rpm (Figure 16). After every 9 days, 100 µl of broth
is spread on Nutrient Agar (NA) and the colony count is observed after 24-hour incubation.
Furthermore, 1 ml of fresh broth was added to each flask. After 36 days of continued incubation,
out of 15 different isolates, 8 out of 10 isolates resulted in TNTC (too numerous to count) colonies
namely: A.a, B.c, E1, E2, G, H, J.a and N53a (figure 18). These colonies were also observed to
form biofilm over the film surface. The remaining isolates (J.b) and (F) ultimately gave lesser
colonies until none over the incubation period (figure 19). A negative control was maintained at
all times. For the same isolates, the experiment was conducted for pH 6 adjusted MS2 broth
supplemented with 250 µl of Tween 20 to reduce surface tension between two phases (figure 17).
The result did not vary from that obtained in pH 7.

Figure 16 Growth of isolates in MS2 broth in presence of 0.1% LDPE strips

Figure 17 Growth of isolates in MS 2 broth (pH 6 with 250 ul Tween 20) with 0.1% LDPE

40
 Figure 18 CFU of isolates after 36 days of continued growth in minimal media

Figure 19 Minimal or no growth is observed for some isolates after 36 days of incubation;
negative control at left

3.6 Biosurfactant production:

41
43 isolates obtained from primary screening were subjected to testing for biosurfactant production.
2 different media were used. Both the media had similar composition, except that CTAB media
had CTAB supplemented in it while SDS media had SDS supplemented in it (figure 17). The zone
observation can be aided by making a well in the agar and pipetting liquid inoculum into the well.
A blue halo around the well or blue precipitate is an indication of positive biosurfactant production.
The plate was then incubated for 48 hours at 37℃. The isolates that produced anionic
biosurfactants were B76a, E133, and B103. The organisms that produced cationic biosurfactants
were G159 which is also LDPE degrader.

Figure 20 SDS agar giving dark blue halo Fig 21 CTAB agar giving dark blue precipitate
around isolate G159 (E1) showing around well for B76a, E133 & B103 showing
cationic biosurfactant production anionic biosurfactant production

3.7 Gram’s staining

The Gram’s staining was done twice for the same isolates, once before and once after 36 days of
checking for bacterial survival in MS2 broth. The results were the same both times. The smears
were observed under 100x oil immersion lens. The color and morphology of the isolates were

42
observed as seen in figure 22. The chart below lists the color, morphology, and structure of all
the isolates (table 7).

Figure 22 Gram-negative (left) and Gram-positive (right) isolates

3.8 Endospore staining:

The Endospore staining was done twice for the same isolates, once before and once after 36 days
of checking for bacterial survival in MS2 broth. The results were the same both times. The
smears were observed under 100x oil immersion lens. The spore was observed as green
structures as seen in figure 9. The chart below lists the presence or absence of all the isolates
(table 7).

Figure 23 Non-sporing bacteria (left) and Sporing bacteria (right)

43
3.9 Biochemical tests

The biochemical tests were done twice for the same isolates, once before and once after 36 days
of checking for bacterial survival in MS2 broth. The results of biochemical tests were the same
for both times (figure 24) (Table 7). The probable organism was identified using ABIS Bacterial
Identification online software.

(A) (B)

(C) (D)

44
 (E) (G)
Y

(F)

Figure 24: (A) Oxidase test: pink color for oxidase positive reaction and no color change for
negative reaction, (B) Starch hydrolysis test: presence of amylase detected by clear zone
around colony, (C) Citrate test: Citrate fermention positive gives green color while negative
reaction gives no color change, (D) Nitrate Reduction: Nitrate reduction by formation of red
color while nitrate reduction negative gives no color change, (E) TSI: for glucose, fructose
sugar fermentation, (F) MIU: for motility and indole reaction, (G) MR: MR positive gives
red ring on the surface

45
 Table 7 List of Gram staining, Endospore staining and Biochemical Test

46
 4. DISCUSSION

The soil samples were mainly collected from petrol pumps and garbage sites (Albertsson, et al.,
1998). Garbage sites have greater chances of harboring polyethylene degrading bacteria as these
sites contain huge dumps of plastic wastes. On the other hand, petrol pumps deal with diesel,
kerosene, petrol, etc, a large amount of which is spilled when oil trucks are emptied into reservoir
tanks. These oils seep into the soil, for which reason, these soils are thought to be rich in
hydrocarbon-degrading bacteria. As both oil and polyethylene are both macromolecular
hydrocarbons, bacteria able to degrade oil are also suspected to be able to release a complex array
of enzymes that may degrade polyethylene too (Albertsson, et al., 1987). That is why petrol pump
sites are targeted in this experiment. Furthermore, water bodies around garbage dump sites have
also been targeted in this experiment. These water bodies are along the shoreline where huge piles
of plastic wastes stand. Rainfall causes leachates from these sites to trickle down into a water body.
This water then, too, becomes a reservoir for polyethylene degrading bacteria. The samples (soil
and water) have been collected from a depth of nearly 10 centimeters because hydrocarbon
degrading bacteria usually have a higher optimum temperature, therefore, chances are these
bacteria would be found in layers deeper than the surface where the temperatures are a bit higher
(Brown, et al., 1974).

Primary screening screens out bacteria unable to survive on a synthetic polymer such as LDPE
from the ones that can. The broth that has been used in the screening procedure is selective in the
sense that it only has LDPE films as a carbon source. Periodic inoculation of the previous inoculum
into fresh broth ensures that the organisms do not run out of nutrients to survive (Mahdiyah, et al.,
2006). In other words, the only component that is limiting in the broth is the carbon source itself.
Nevertheless, some bacteria unable to degrade polyethylene may still survive in this selective
broth, the reason being that breakdown of polyethylene produces by-products that include ketones,
aldehydes, acids, alkalis, etc (Raaman, et al., 2012). These by-products are fed upon by non-
degraders that ensure their survival even in selective broth. Therefore, primary screening is not
enough alone (Tribedi, et al., 2013).

The spectrophotometer readings that were taken throughout the incubation period is an indirect
measure of the falling bacterial population in the broth. The graph (figure 11) below is a visual

47
demonstration of the falling final OD with increasing time, i.e., the final OD is inversely
proportional to time. The final OD is indicative of the total microbial population in the culture,
which suggests that over the increasing period of time, the organisms are dying for not being able
to breakdown LDPE (Mahdiyah, et al., 2006). On the other hand, Figure 12 shows a graph that
demonstrates the increasing OD between the initial OD and final OD. The positive difference
between the two ODs is indicative of the presence of bacteria that are able to degrade LDPE as a
carbon source, hence, their numbers are rising. The difference between the two values keeps
decreasing over time, which further signifies that undesired organisms are dying off from lack of
food source (Mahdiyah, et al., 2006).

Secondary screening is targeted towards organisms that can perform bacterial colonization over
the surface of LDPE films because the first step in the mechanism of LDPE degradation is biofilm
formation. Without proper attachment of bacteria to the surface of polyethylene, degradation will
not occur. Therefore, the experiment set out to observe and isolate organisms that were able to
grow over the polyethylene films placed on Nutrient Agar (NA). Furthermore, those that grow on
polyethylene surfaces were most likely to secrete the complex array of enzymes that can degrade
the polymer (Gilan, et al., 2007). One drawback to this technique is that some non-degrading
organisms may overgrow on the far end of the plastic corners. Therefore, these organisms were
further analyzed in the tertiary screening. Out of 43 isolates obtained as PEG degraders, only 14
isolates were able to form microbial colonization or biofilm over LDPE surface.

Tertiary screening of organisms is a final method of interpretation of polymer-degrading

organisms. Here, the LDPE (low-density polyethylene collected from the market) is incorporated
into the solid agar media, resulting in a turbid agar plate. Organisms that are able to breakdown
the LDPE in the media will secrete LDPE degrading enzymes around it. As the enzymes use the
LDPE as a carbon source, they cause a clearing of the turbidity around the colony, giving a clear
zone in its surrounding area (Pramila, et al., 2011). To make a clear zone prominent, a staining
solution followed by the destaining solution is added to the media (Tribedi, et al., 2013). This
causes the zone to become more prominent. The media is also selective and the growth on the
media is very slow. However, very few organisms are able to grow on the media, though they do
not give a clear zone. It is because these bacteria are able to use the agar as a carbon source and
survive on the media. Nevertheless, this method is an accurate screening method as it gives visual

48
confirmation of the degrading organisms. Out of the 14 isolates retrieved from secondary
screening, 10 were able to form a zone of clearance in the tertiary screening.

For the final part, the isolates were grown in MS2 broth supplemented with 0.1% LDPE as a carbon
source. The purpose of growing isolates in a continuous incubation was to observe whether the
isolates were able to sustain growth in salt broth by itself by visually witnessing the CFU (Colony
Forming Units) after every 9 days in a condition with a limited carbon source. The broth culture
was replenished with 1 or 2 milliliters of salt media after every 9 days so that organisms do not
run out of nutrients to grow and divide. Out of a total of 11 isolates retrieved from primary
screening, 8 isolates were able to survive in continuous culture method over 36 days period,
namely Staphylococcus massiliensis (A.a), Acinetobacter baumannii (B.c), Clostridium novyitype
A (E1), Clostridium novyitype A (E2), Lysinibacillus sphaericus (G), Bacillus
psychrosaccharolyticus (H), Acinetobacter iwoffii (J.a) and Bacillus psychrosaccharolyticus
(N53a). Theoretically, all the 10 isolates should have been able to survive in this limiting
conditions as all the 10 isolates were able to form clear zones in LDPE media. The slight derivation
by two isolates, namely, organism Staphylococcus arlettae (F) and Citrobacter rodentium (J.b),
may be due to the fact that these organisms survive in SM (Synthetic Media) rather than MSM
(Minimal Salt Media) (Sen, et al., 2015). MSM media are composed of all salts whereas, SM media
are composed of fewer salts and yeast or peptone extracts (Gilan, et al., 2007). The added yeast or
peptone acts as a co-substrate that act as electron donors and enhance the reductive cleavage of the
polymer (Santo, et al., 2012). In recent literature, (Brandon, et al., 2018), mealworms that were
fed polyethylene were found to degrade the polymer by 60.1% of weight loss. It was found that
the gut microbiome consisted of the bacteria Citrobacter sp and one other organism. This shows
that Citrobacter propagates in rather complex media than simple minimal salt media, which further
goes on to explain why the organism did not grow in MSM2 broth. The experiment was repeated
in pH 6 and the addition of Tween 20 as a surfactant. The purpose of this method was to see if the
bacterial count of isolates F and J.b increased with lower pH. A lower pH is often time the case
found in liquid media as the CO2 emitted mixes with the aqueous solution to cause a drop in the
pH (Tribedi, et al., 2013). Addition of Tween 20 was carried out to enhance to jump-start the
degradation and increase CFU number. However, all these factors did not bring any change to the
final result and isolates F and J.b showed decreasing CFU number with increasing time.

49
Finally, in the case of biosurfactant production screening, the CTAB media were used for the
screening of anionic biosurfactant producing organisms while SDS media were used for screening
of cationic biosurfactant producing organisms (Karlsson, et al., 1988). The basic mechanism
follows. CTAB itself is a cationic surfactant. When the CTAB (cationic) is incorporated into the
media, and an organism produces an anionic biosurfactant, the CTAB (cationic) and anionic
biosurfactant produced by the microorganism form an insoluble complex with the methylene blue
in the media giving a dark blue halo zone around the colony (Aouseoud, et al., 2011). The opposite
is true for SDS media, however, the mechanism remains the same. The 3 isolates that produced
anionic surfactants namely B76a, E133, and F103 were identified to be Staphylococcus
massiliensis in ABIS. The G159 or E1 isolate was identified to be Clostridium novyitype A as
mentioned earlier.

Gram’s staining, endospore staining, biochemical tests, and clear zone screening were done twice,
once before the 36 days incubation in continuous culture, and once after the culture. This was done
so as to ensure that the organisms that were present in 36 days culture were the same organisms
that were identified after the primary, secondary and tertiary screening, and not contaminants. This
step was a necessary precaution as it is very likely that conical flasks may easily get contaminated
when cultured in continuity for such a long period of time.

50
 4. CONCLUSION

In conclusion, the microbial strains able to degrade synthetic market-grade low-density

polyethylene were successfully isolated from locally collected soil samples. After the primary,
secondary and tertiary screening, the microorganisms that were discovered to as potential
degraders are Staphylococcus massiliensis, Acinetobacter baumannii Clostridium novitype A,
Staphylococcus arlettae, Lysinibacillus sphaericus, Bacillus psychrosaccharolyticus,
Acinetobacter iwoffii, Citrobacter rodentium. The process of degradation is, as of now, a slow
process, hence, the isolated biosurfactant producing bacteria Staphylococcus massiliensis and
Clostridium novyitype Acould be used to enhance the degradation. Nevertheless, degradation of up
to 60.1% has been achieved in earlier experiments, albeit over a 30 days’ incubation period. The
process can be paced by performing the pretreatment of polyethylene before subjecting them to
biodegradation, using microbial consortium to aid in the process, or a more modern approach
would be to perform protein engineering in order to increase the efficiency of enzymes responsible
for degradation. The isolation is a part of an even bigger experiment, where the next step is the
determination of the rate of polyethylene degradation, observation of the presence of enzyme gene
responsible for degradation, isolation of crude enzyme and determines its effect on degradation.
These findings have important applications in solving the plastic waste problem through
bioremediation where modern approach developed for remediation can be combined and applied
with these organisms.

51
 REFERENCES

Albertsson A. -C., Andersson S. O. and Karlsson S. The mechanism of biodegradation of

polyethylene [Journal] // Polym. Degrad. Stab.. - 1987. - pp. 73-87.

Albertsson A.C. [et al.] Molecular weight changes and polymeric matrix changes correlated with
the formation of degradation products in biodegraded polyethylene [Journal] // Journal of
Environmental Polymer Degradation. - 1998. - pp. 187-195.

Albertsson A.C. Biodegradation of synthetic polymers: Limited microbial conversion of C-14 in

polyethylene to (CO-2)-C-14 by some soil fungi [Journal] // Journal of Applied Polymer Science. -
1978. - pp. 3419–3433.

Albertsson A.C. The shape of the biodegradation curve for low and high density polyethylenes in
prolonged series of experiments [Journal] // European Polymer Journal. - 1980. - pp. 623-630.

Aouseoud M, Maachi R. and Amrane A Biosurfactant Production from olive oil by

Pseudomonas fluorescens [Journal] // Applied Microbiol Biotechnol. - 2011.

Arutchelvi J. [et al.] Biodegradation of polyethylene and polypropylene [Journal] // Indian J.

Biotechnol. - 2008. - pp. 9-22.

Ban on polythene: Punish rule violators, demand environmentalists [Online] // The Daily Star. -
2018.

Brandon A. M. [et al.] Biodegradation of Polyethylene and Plastic Mixtures in Mealworms

(Larvae of Tenebrio molitor) and Effects on the Gut Microbiome. [Journal]. - China : ACS
Publications, 2018.

Brown B.S., Mills J. and Hulse J.M. Chemical and biological degradation of plastics [Journal] //
Nature. - 1974. - pp. 161-163.

52
Chee J. Y. [et al.] Bacterially Produced Polyhydroxyalkanoate (PHA): Converting Renewable
Resources into Bioplastics [Journal] // Appl Microbiol & Microbiol Biotech A Mendez Vilas
(Ed). - 2010.

Cornell J.H., Kaplan A.M and Rogers M.R. Biodegradation of photooxidized polyalkylenes
[Journal] // Journal of Applied Polymer Science. - 1984. - pp. 2581–2597.

Divyalakshmi S and Subhashini A Sreening and Isolation of Polyethylene Degrading Bateria

from Various Soil Environments [Journal] // IOSR Journal of Environmental Science, Toxicology
and Food Tehnology. - 2016.

Donlan R. Biofilms: microbial life on surfaces [Journal] // Emerg. Infect. Dis. - 2002. - pp. 881-
890.

Fugikura K., Okutani S. and Maruyama T. Deep-sea Life-Biological observations using

research submersibles [Journal] // Tokai University Press. - 2008. - pp. 57-80.

G.J.L. Griffin Degradation of polyethylene in compost burial [Journal] // J. Polym. Sci. Polym.
Symp. - 2007. - pp. 281–286.

Gilan I, Hadar Y and Sivan A Colonization, biofilm formation and biodegradation of

polyethylene by a strain of Rhodococcus ruber [Journal] // Applied Microbiol Biotechnol. - 2007. -
pp. 97-104.

Gilan I., Hadar Y. and Sivan A Colonization, biofilm formation and biodegradation of
polyethylene by a strain of Rhodococcus ruber [Journal] // Appl. Microbiol.Biotechnol. - 2004. -
pp. 97-104.

Hadad D., Geresh S. and Sivan A. Biodegradation of polyethylene by the thermophilic bacterium
Brevibacillus borstelensis [Journal] // Journal of Applied Microbiology. - 2005. - pp. 1093–1100.

Hadad D., Geresh S. and Sivan S. [Journal].

Haines J.R. and Alexander M. Microbial degradation of high molecular-weight alkanes

[Journal] // Applied Microbiology. - 1974.

Hakkarainen M and Albertsson A.C. Environmental degradation of polyethylene [Journal] //

Adv. Polym. Sci. . - 2004. - pp. 177–199.

53
Hussain Amal A., Al-Mayaly Ithar K. and Khudeir Saad H. Isolation, Screening and
Identification of Low Density [Journal] // Mesopotamia Environmental Journal . - 2015. - pp. 1-
14.

Karlsson S., Ljungquist O. and Albertsson A Biodegradation of polyethylene and the influence
of surfactants [Journal] // Polym. Degrad. Stab. - 1988. - pp. 237-250.

Koutny Marek, Lemairea Jacques and Delort Anne-Marie Biodegradation of polyethylene

films with prooxidant additives [Journal] // Chemosphere. - 2006.

Kowalczyk Anna [et al.] Achromobacter xylosoxidans as a new microorganism strain colonizing
high-density polyethylene as a key step to its biodegradation [Journal]. - [s.l.] : Environmental
Science and Pollution Research, 2016. - 11.

Mahdiyah Dede, Elpawati and Mukti Bayu Hari ISOLATION OF POLYETHYLENE

PLASTIC DEGRADING-BACTERIA [Journal]. - 2006.

Manzur A., Limón-González M. and Favela-Torres E. Biodegradation of physicochemically

treated LDPE by a consortium of filamentous fungi [Journal] // J. Appl. Polym. Sci. - 2004. - pp.
105-111.

Otake Y. [et al.] Biodegradation of low-density polyethylene, polystyrene, polyvinyl-chloride,

and urea-formaldehyde resin buried under soil for over 32 years [Journal] // Journal of Applied
Polymer Science. - 1995. - pp. 1789-1796.

Polyethylene (Low Density) LDPE, LLDPE [Online] // British Plastics Federation. - n.d..

Potts J. E. Biodegradation in aspects of degradation and stabilization of polymers [Journal] //

H.H.G. Jellinek. - 1978. - pp. 617-658.

Pramila R. and Ramesh K. Biodegradation of low density polyethylene (LDPE) by fungi isolated
from marine water- a SEM analysis [Journal] // Afr. J. Micriobiol. - 2011. - pp. 5013-5018.

Pūtaiao Science Learning Hub – Pokapū Akoranga [Journal]. - 2017.

Raaman N [et al.] Biodegradation of plastic by Aspergillus spp. Isolated from polythene polluted
sites around Chennai [Journal] // Journal of Academia and Industrial Research. - 2012.

54
Raghavan D. and Torma A. E. DSC and FTIR characterization of biodegradation of polyethylene
[Journal] // :olym. Eng. Sci. - 1992. - pp. 438-442.

Restrepo-Flórez Juan-Manuel , Bassi Amarjeet and Thompson Michael R. Microbial

degradation and deterioration of polyethylene- A review [Journal] // International Biodeterioration
& Biodegradation. - 2014. - pp. 83-90.

Rojo F. Handbook of Hydrocarbon and Lipid Microbiology [Journal] // Springer. - 2010.

Rosario Ligi Lambert D. and Baburaj Saya Isolation and Screening of Plastic Degrading
Bacteria from Polythene Dumped Garbage Soil [Journal] // International Journal for Research in
Applied Sciene & Engineering Technology (IJRASET). - 2017. - pp. 1028-1032.

Royer Sarah-Jeanne [et al.] Production of methane and ethylene from plastic in the environment
[Journal] // Plos One. - 2018.

Santo M., Weitsman R. and Sivan A. The role of copper-binding enzyme laccase- in the
biodegradation of polyethylene by the actinomycete Rhodococcus ruber [Journal] // Int.
Biodeterior. Biodegrad.. - 2012. - pp. 1-7.

Secchi E. R. and Zarzur S. Plastic debris ingested by a Blainville‟s beaked whale, Mesoplodon
densirostris, Washed ashore in Brazil [Journal] // Journal of Aquatic Mammals. - 1999. - pp. 21-
24.

Sen Sudip Kumar and Raut Sangeeta Microbial Degradation of Low Density Polyethylene: A
review [Journal]. - India : Elsevier, 2015.

Sharma Juhi [et al.] Isolation and Characterization of Plastic Degrading Bacteria from Soil
Collected from the Dumping Grounds of an Industrial Area [Journal] // International Journal of
Advanced and Innovative Research. - 2014.

Singh Gauri, Singh Ashok Kumar and Bhatt Kalpana Biodegradation of polythenes by bacteria
isolated from soil [Journal] // International Journal of Research and Development in Pharmacy and
Life Sciences. - 2016. - pp. 2056-2062.

Sivan A., Szanto M. and Pavlov V. Biofilm development of the polyethylene degrading
bacterium Rhodococcus ruber [Journal] // Applied Microbiol. Biotechnol.. - 2006. - pp. 346-352.

55
Spear L. B., Ainley D. G. and Ribic C. A. Incidence of plastic in seabirds from the tropical
Pacific 1984-91, Relation with distribution of species, sex, age, season, year and body weight
[Journal] // Marine Environmental Research. - 1996. - pp. 123-141.

Sreelekshmi S, Lekshmi M and Jayadev Ayona Effects of Co-substrates on Biodegradation of

Azo Dyes: A case study of Marina Bacteria [Journal] // Society for Development and
Environment. - 2016. - pp. 9-16.

Starnecker A. and Menner M. Assessment of biodegradability of plastics under stimulated

composting conditions in a laboratory test system [Journal] // International Biodeterioration &
Biodegradation. - 1996. - pp. 85-92.

Sudhakar M. [et al.] Marine microbe mediated biodegradation of low and high density
polyethylenes [Journal] // Int Biodeterior. Biograd.. - 2008. - pp. 203-213.

Tribedi P. and Sil A. K. Low-densisty polyethylene degradation by Pseudomonas sp. AKS2

biofilm [Journal] // Environ. Sci. Pollut. Res. Int.. - 2013. - pp. 141-151.

Tribune Dhaka POBA: Bangladesh witnesses an 80 fold increase in plastic use since 1990
[Online] // Dhaka Tribune. - April 22, 2018. -
https://www.dhakatribune.com/bangladesh/environment/2018/04/22/poba-bangladesh-witnesses-
80-fold-increase-plastic-use-since-1990.

Urbanek [et al.] Isolation and characterization of Arctic microorganisms decomposing bioplastics
[Journal]. - [s.l.] : Springer, 2017.

Vimala P. P. and Mathew Lea' Biodegradation of Polyethylene using Bacillus subtilis [Journal] //
Procedia Technology. - 2016. - pp. 232-239.

www.sciencelearn.org.nz/resources/142-adaptations-of-marine-organisms Science Learning

Hub – Pokapū Akoranga Pūtaiao. (2014). Adaptations of marine organisms. Retrieved from
[Journal].

Yoon M. G., Jeon J. H. and Kim M. N. Biodegradation of polyethylene by a soil bacterium and
AlkB cloned recombinant cell [Journal] // J. Bioremed Biodegr.. - 2012.

56
Zheng Ying, Yanful Ernest K. and Bassi Amarjeet S. A Review of Plastic Waste
Biodegradation [Journal] // Critical Reviews in Biotechnology. - 2008.

57