Proposal

An integrated Approach for Biodegradation of Plastic Waste

Background
Since from the last few decades the environmental crises due to plastic pollution has increased to
such an extent that worldwide environmentalist groups and different regulatory authorities has
put ban on the use of plastics. Globally, the amount of waste plastics flowing into the different
environment is estimated to be in the range of 5-12 million tonnes per year (Mt/y) [1, 2].
However, there is no dispute about the fact that plastic has become an indispensible part of our
lives as in certain application plastic has an edge over to conventional materials. Thus, it is
impossible to prevent even in part the release of this material into the environment.
Consequently, it is important to find out different ways to degrade this material by employing
various means including physical, chemical, mechanical, biological or an integrated approach of
combining different methods to get enhance degradation in a short period of time.
Incineration, de-polymerization, recycling, use of biodegradable polymers and land-filling are
some of the traditional ways of handling post consumer plastic waste. However, these methods
are costly and often create environmental difficulties. Biodegradation of plastic waste could be
used as an alternative to the traditional waste disposal method mainly for plastic which is already
dumped into the environment and by any means not possible to segregate for re-use, recycle or
burn. Widespread studies has been carried out on biodegradation of plastics which includes
isolation of plastic degrading microorganisms from different natural and artificially stimulated
environment, identification of different degradative enzymes and cloning of genes for synthetic
plastic degradation.
The main problem of using biodegradation as solution for plastic waste is the presence of
different types of polymeric constitutes in the plastic waste as plastic can be made from different
types of polymers such as polypropylene, polyethylene, polyvinyl chloride etc. Microbial strains
that can degrade many kinds of polymers are desirable for their application in plastic
degradation. On the contrary degradative capability of microbial strain is often restricted to a
single class of polymer and microorganisms with such ability are itself rare in nature. In nature
complete mineralization of the plastic needs a synergistic association between different groups of
microorganisms and thus a consortium of different groups of microorganisms is required for
complete mineralization of polymer. The major steps associated with plastic biodegradation
include;
Bio-deterioration
Biodegradation of plastic was initiated with the process of bio-deterioration which involved
superficial degradation at the surface level and leads to modification of mechanical, physical and
chemical properties of the plastic. Different non-biological parameters like temperature, solar

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radiation, UV radiation etc. are also reported to contribute in weakening of the polymeric
structure and thus are useful as a synergistic factor to initiate the biodegradation process.

Physical deterioration
The physical deterioration is associated with formation of the microbial biofilm due to secretion
of extracellular polymeric substances (EPS). This EPS enters the pores of polymer and helps the
microorganisms to grow inside forming a strong bioflim structure. This results in increasing the
pore size and initiate cracks within the polymer making the plastic physically week.
Chemical deterioration
A bioflim formed on the polymer surface comprising bacterial community of different species
mainly from the group of chemolithotrophs (e.g. Nitrobacter spp.) releases different compounds
which are mainly acidic in nature such as nitrous and nitric acid. Organic acids such as oxalic,
citric, fumaric, gluconic, glutaric, glyoxalic, oxalic and oxaloacetic acids may also be released by
chemoorganotrophic communities. The change in pH inside the polymer results in change in the
microstructure of the plastic matrix leading to its deterioration.
Bio-fragmentation
Followed by chemical deterioration the plastic polymer undergoes into fragmentation resulting in
formation of oligomers or monomers. The fragmentation can be due to a combine physical,
chemical or biological phenomenon. However, the fragments forms are of high molecular weight
and hence cannot be directly assimilated by microorganisms as a source of nutrients.
Microorganisms secrete extracellular enzymes (exoenzymes) that can catalyze reactions
principally at the boundaries of the plastic polymer. The other different enzymes involved to
convert the high molecular weight fragments into the form that can be easily assimilated by
microorganisms are oxygenases, lipases and esterase.
Bacillus sp., Staphylococcus sp., Streptococcus sp., Diplococcus sp., Micrococcus sp., Pseudomo
nas sp. and Moraxella sp. are some of the microbes reported in the literature, which have the
capacity to degrade plastic polymers into their respective simple monomers. Apart from this
different groups of microorganisms reported in literature known for their ability to degrade
different types of polymer is given in Table 1.

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Table 1: Microorganisms showing ability to degrade different types of plastic constituents

Type of Enzyme Microorganisms Plastic as a source of References

microorganism carbon

Fungal Glucosidae Aspergillus flavus Polycaprolactone 5

(PCL)

Unknown Penicillium Polyhydroxybutyrate 5

fimiculosum (PHB)

Catalase, Aspergillus niger PCL 5

proteases

Unknown Sterptomyces PHB, PCL 5

Cutinase Fusarium PCL 4

Unknown Amycolaptosis sp. Polylactic acid (PLA) 4

Syrine Pestalotiopsis Polyurethane 6

hydrolase microspora

Manganese Phanerochaete Polyethylene 4

peroxidase chrysosporium

Bacteria Lipase Rhizopus delemar PCL 5

Unknown Proteobacteria PHB, PCL and PBS 5

Unknown Firmicutes PHB, PCL and PBS 5

Lipase Rhizopus arrizus Polyethylene Adipate 5

(PEA, PBS, PCL)

Serine Pseudomonas Polyhydroxyalkanoate 4

hydrolase stutzeri

Note: Information in the table is adopted mainly from reference [3]

Assimilation and mineralization

The assimilation involves integration of atoms inside microbial cells which may result in
numerous secondary metabolites that can be extracted outside by the cells. The secondary
metabolites excreted may be used by another cell that can perform further degradation. The
mineralization refers to the complete degradation of primary and secondary metabolites that

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resulted in the excretion of completely oxidized metabolites (CO2, N2, CH4, H2O etc). Figure 1
summarizes the biodegradation process.

Plastic
Polymer
Degradation Steps

Bio- Assimilation and

Bio- Physical Chemical
fragmentation mineralization
deterioration deterioration deterioration

Figure 1: Summary of biodegradation process

Our Proposal
The biodegradation of synthetic plastics is a complex phenomenon. To date, most of the
knowledge on the microbial ability to degrade synthetic plastics is based on few bacteria able to
grow on culture media, that represent <0.1% of the total bacteria. Hence, there is a huge
opportunity to exploit different natural source for isolation of plastic degrading microorganisms
and understanding complex process of synergistic mechanisms of plastic degradation by
different microbial communities using metagenomics and bioinformatics approach. With this
approach is it is possible to discover new non-culturable microorganisms involved in plastic
colonization and degradation.In a view of this background the research is proposed on isolation
of different microbial strains from natural environment and by using application of molecular
biology technologies such as genomic, transcriptomic, proteomic, metabolomics).
The study is mainly to discover new non-culturable microorganisms involved in the plastic
colonization and degradation along with conventional isolation method. We believe that such a
research will be useful in exploring the new properties of microorganisms that arise from the
interplay of genes, enzymes, and different environmental parameters. The other major goal of the
study is to understand synergistic effect of different physical, chemical and mechanical process
such as solar radiation, UV radiation, ultrasound, temperature, pH, chemical treatment etc. in the
biodegradation of plastic. The optimized process then can be use to grow microorganism in
different conditions which direct them to grow and use the plastic as a source of carbon and
energy. Such research will be highly helpful in developing low cost, efficient, eco-friendly
technology capable of reducing plastic pollution from the environment.

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Experimental approach
 Collection of samples from different environmental locations where possibility of
obtaining plastic degrading microbial strains are very high
 Developing an artificially stimulated environment for facilitating growth of plastic
degrading microorganisms. Sample from this can be directly use for isolation of plastic
degrading microbial strains
 Isolation and identification of different microbial strains from natural environment using
conventional culturing approach
 Use of metagenomics approach to identify overall population of microorganisms from
different environmental sources.
 Optimization of biodegradation conditions with respect to different physical, chemical and
mechanical parameters.
 Determination of biodegradation efficiency
 Identification of the biodegradation products
 Enzyme identification
 Developing a model system for bioremediation of plastic waste using combined approach
of non-biological and biological parameters under optimized conditions.
Test Method approach
Degradation of plastic polymer under different test conditions can be assessed by measuring the
decay of relevant physical properties and change in chemical composition of growth
environment or polymer itself. The physical changes such as: changes in molecular weight mass
and molecular mass distribution, tensile properties, mass loss, morphological changes required
availability of different instruments like analytical balance, tensile strength measuring
instrument, SEM etc. The change in chemical composition can be monitor by using FTIR, Mass
spectrometry, HPLC etc. A detailed approach that can be used for monitoring plastic
biodegradation during the course of work is given in Table 2.

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Table 2: Different test parameters for monitoring plastic biodegradation

Level of Test parameter Analytical techniques

biodegradation

Changes in polymer Mechanical Properties Instron (measuring tensile strength) DMA

properties (Macroscopic) (Dynamic Mechanical analysis)

Surface properties Electron spectroscopy for chemical analysis

(Microscopic) (ESCA), SEM (Scanning electron
microscopy)

Physical properties X-ray diffraction (XRD), Differential

(Macromolecular) scanning Calorimetry (DSC),
Thermogravimetric analysis (TGA), Melt
flow index (MFI), Gel permeation
chromatography (GPC)

Chemical Properties Fouirer Transform Infrared Spectroscopy

(FTIR), Nuclear magnetic resonance
spectroscopy (NMR), spectroscopy (UV),
Chemilluminous (CL), Phosphorences

Reactive Intermediates Electron Spin Resonance (ESR), CL

Breakdown of Characterization of Thin Layer Chromatography (TLC), GC (Gas

polymer into oligo intermediate by-product Chromatography), Gas Chromatography-
and monomer Mass Spectrometry (GC-MS), High
performance liquid chromatography (HPLC),
Matrix assisted Laser Desorption/Ionization-
Time of flight (MALDI-TOF)

Ultimate Mineralization to final Respirometer, CO2 analyser, GC-MS, Total

degradation products, CO2, CH4 or H2 or organic carbon (TOC)
consumption of O2 and
water

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 How vision will be achieved (Roadmap of the Project)

Step 3 Step 4
Step 1 Step 2
12 – 18 months 18-36 months
0 to 6 months 6-12 months

Isolation of plastic Screening of Enhancement of Developing an Ex-situ

degrading potential plastic
microorganisms degraders
• Sample collection from • Standardizing different
natural habitat assay procedure for
• Enrichment of plastic testing plastic
degrader biodegradation
• Metagenomics to study • Screening of strains
plastic degraders showing high plastic
directly from soil degradation potential
samples • Identification of strains
• Isolation of microbial • Identification of plastic
strain s from different degradation products
groups e.g. bacteria, • Identifying sensors for real
fungus, algae time monitoring of plastic
degradation
biodegradation using model system for plastic
integrated approach biodegradation
• Creating an artificial
• Studying effect of different environmental system for
physical, chemical and stimulating plastic
mechanical process on biodegradation process in a
rate of plastic short period of time
biodegradation • The system will comprise
• Different parameters – - A unit for pre-processing and
- Solar radiation pre-treating plastic waste
- Temperature - Composite media essential for
- Ultrasonication biodegradation
- Chemical parameters - Consortia of different
• Optimizing the process microorganisms showing
• Developing a process potential for plastic
protocol for plastic degradation
biodegradation based on - Sensors for analysing real
optimized parameters. time biodegradation of plastic
waste

References
1. Billard, G. and Boucher, J., 2019. The challenges of measuring plastic pollution (No.
ARTICLE).
2. Boucher, Julien, and Damien Friot. 2017. “Primary Microplastics in the Oceans: A Global
Evaluation of Sources.” IUCN
3. Muhamad, W.N.A.W., Othman, R., Shaharuddin, R.I. and Irani, M.S., 2015.
Microorganism as plastic biodegradation agent towards sustainable environment. Adv
Environ Biol, 9, pp.8-14.
4. Shimao, M., 2001. Biodegradation of plastics. Curr Opinion Biotechnol, 12: 242–7.
5. Tokiwa, Y., B.P. Calabia, C.U. Ugwu and S. Aiba, 2009. Biodegradability of plastics. Int J
Mol Sci., 10: 3722-3742.
6. Russel, J.R., J. Huang, P. Anand, K. Kucera and A.G. Sandoval, 2001. Biodegradation of
polyester polyurethane by endophytic fungi. Appl Environ Microbiol. 77: 6076-6084.