water

Review
Microplastic Accumulation and Degradation in Environment
via Biotechnological Approaches
Sonal Thakur 1,2 , Shivangi Mathur 1,3, *, Saumya Patel 4 and Biswaranjan Paital 5, *

1 Department of Microbiology and Biotechnology, School of Science, Gujarat University, Navrangpura,

Ahmedabad 380009, Gujarat, India
2 Department of Biochemistry and Biotechnology, St. Xavier’s College, Surajmal Zaveri Rd, Navrangpura,
Ahmedabad 380009, Gujarat, India
3 Department of Biotechnology, President Science College, Shayona Campus, R.C. Technical Rd, Ghatlodiya,
Chanakyapuri, Ahmedabad 380061, Gujarat, India
4 Department of Botany, Bioinformatics and Climate Change, School of Science Gujarat University,
Navrangpura, Ahmedabad 380009, Gujarat, India
5 Redox Regulation Laboratory, Department of Zoology, College of Basic Science and Humanities,
Odisha University of Agriculture and Technology, Bhubaneswar 751003, Odisha, India
* Correspondence: [email protected] (S.M.); [email protected] (B.P.);
Tel.: +91-674-2397029 (B.P.); Fax: +91-0674-2397970 (B.P.)

Abstract: The extensive use of plastics in daily life has led to the generation of huge amounts of plastic
waste, which causes an enormous burden on the environment. More than half of the plastic waste
ends up in the landfill, and about one-fifth of waste is managed by incineration. Only about one-tenth
of plastic waste is recycled, and the rest, about one-fifth of mismanaged plastic waste, ends up in the
terrestrial and aquatic environment. Here, we review how the deterioration of plastics leads to the
formation of microplastics and nanoplastics, which are now found abundantly and are contaminating
aquatic life and water bodies. It observed that increasing experimental evidence provides data about
the presence of these microplastics in food items, terrestrial environment, and even the human body.
Citation: Thakur, S.; Mathur, S.; Patel, The harmful effects of microplastics on human health still need to be substantiated with more precise
S.; Paital, B. Microplastic experimental studies. However, measures can be taken to reduce the production of microplastics
Accumulation and Degradation in by improving the methods used for plastic degradation. This review focuses on the use of genetic
Environment via Biotechnological engineering, genome editing, synthetic biology, and system biology approaches to increase the
Approaches. Water 2022, 14, 4053. potential of microorganisms to degrade plastics.
https://doi.org/10.3390/
w14244053
Keywords: biodegradation; genetically modified organism; microplastics; polyethylene terephthalate;
Academic Editor: Laura Bulgariu system biology

Received: 23 November 2022

Accepted: 8 December 2022
Published: 12 December 2022
1. Introduction
Publisher’s Note: MDPI stays neutral
Plastics are produced from different natural products following a polymerization
with regard to jurisdictional claims in
or polycondensation process. Plastics are therefore composed of natural and organic
published maps and institutional affil-
materials. For example, cellulose, coal, natural gas, salt, crude oil, etc., act as the raw
iations.
materials for the production of plastics. These are the organic polymers that are derived
from various hydrocarbon and petroleum materials extracted from coal, oil, and natural
gas. They consist of a long chain molecule, connected by strong chemical bonds, arranged
Copyright: © 2022 by the authors.
in repeating units, and exhibiting desirable properties such as strength, flexibility, and a
Licensee MDPI, Basel, Switzerland. lightweight feature [1,2]. The first synthetic plastic, Bakelite, was produced in 1907 by Leo
This article is an open access article Baekeland by mixing phenol and formaldehyde and subjecting it to heat and pressure. He
distributed under the terms and also coined the term plastics after the Greek word “plastikos”, which means moldable [3].
conditions of the Creative Commons The development of new polymers with improved qualities such as transparency and
Attribution (CC BY) license (https:// color-holding ability resulted in a drastic increase in the commercial production of plastics
creativecommons.org/licenses/by/ from the 1930s to the 1950s. Plastic products were extensively used during World War II,
4.0/). and after the war the commercial production of plastic increased by almost 1000-fold [4].

Water 2022, 14, 4053. https://doi.org/10.3390/w14244053 https://www.mdpi.com/journal/water

Water 2022, 14, x FOR PEER REVIEW 2 of 22

Water 2022, 14, 4053 2 of 21

during World War II, and after the war the commercial production of plastic increased by
almost 1000‐fold [4]. Certain properties of plastic such as high flexibility, resistance to
corrosion, high strength‐to‐weight ratios, and low costs in carrying out fabrication work
Certain properties
have resulted in theofhigh
plastic suchofasplastics
usage high flexibility, resistance
in packaging, in the tofield
corrosion, high strength-
of construction and
to-weight ratios, and low costs in carrying out fabrication work have resulted in the high
transportation, and in the production of electrical, medical, and electronic devices [5].
usage of plastics in packaging, in the field of construction and transportation, and in the
The global annual production of plastics reached 359 million tonnes by the end of the
production of electrical, medical, and electronic devices [5]. The global annual production
year 2018 [6]. With the current rate of plastics production and utilization, it is estimated
of plastics reached 359 million tonnes by the end of the year 2018 [6]. With the current rate
that the number of plastics produced annually will double within the next 20 years [7–9].
of plastics production and utilization, it is estimated that the number of plastics produced
Plastics can be classified, according to their size, into three groups. All plastic mate‐
annually will double within the next 20 years [7–9].
rials > 200 mm are considered to be macroplastics. Microplastics have a variety of shapes
Plastics can be classified, according to their size, into three groups. All plastic
such as spheres, fragments, and fibers [10], and have a size range of 1 μm–5 mm and are
materials > 200 mm are considered to be macroplastics. Microplastics have a variety of
present in large quantities in cleaning and cosmetic products, such as facewash/scrubs
shapes such as spheres, fragments, and fibers [10], and have a size range of 1 µm–5 mm and
and toothpaste, and the laundering of synthetic clothes and abrasion of tires through
are present in large quantities in cleaning and cosmetic products, such as facewash/scrubs
driving serve as primary sources of microplastics [11,12]. The secondary microplastics are
and toothpaste, and the laundering of synthetic clothes and abrasion of tires through
produced
driving through
serve the breakdown
as primary sources of of microplastics
large plastic items suchThe
[11,12]. as plastic bags,microplastics
secondary fishing nets,
and bottles. The presence of microplastic particles <1 mm in
are produced through the breakdown of large plastic items such as plastic bags, large amounts in living or‐
fishing
ganisms
nets, andand the The
bottles. environment
presence of is amicroplastic
growing concern.
particlesBecause
<1 mm of in their
large imperceptible
amounts in living na‐
ture they can easily enter in food chains and accumulate in living
organisms and the environment is a growing concern. Because of their imperceptible tissues, as shown in
Figure 1 [13,14]. The presence of microplastics in a sample and their
nature they can easily enter in food chains and accumulate in living tissues, as shown inquantification can be
carried1out
Figure eitherThe
[13,14]. at the individual
presence particle level
of microplastics in abysample
using and
spectroscopic techniquescan
their quantification such
be
as FTIR or RAMAN or by determining the total amount of microplastics
carried out either at the individual particle level by using spectroscopic techniques such present inasa
sample
FTIR or using
RAMAN Py‐GCMS or thermal extraction
or by determining desorption
the total amount GC MS [15].present
of microplastics Nanoplastics are <
in a sample
1 μm in
using diameter,orare
Py-GCMS produced
thermal from the
extraction breakdown
desorption GC ofMS
microplastics and waste
[15]. Nanoplastics areproducts
< 1 µm
of diameter,
in 3D printing, areconstitute
produced afromverytherecent area of environmental
breakdown of microplastics science, and products
and waste are considered
of 3D
to be equally as harmful as microplastics, but their abundance
printing, constitute a very recent area of environmental science, and are considered and effects on the envi‐
to be
ronmentasare
equally yet toasbemicroplastics,
harmful quantified [11,16].
but their Inabundance
this article,andweeffects
reviewedon thethe above aspects
environment are
covering
yet every biodegradable
to be quantified [11,16]. In method for the
this article, degradation
we reviewed theofabove
plastic waste.covering
aspects The review of
every
the literature for
biodegradable this article
method for thewas based onof
degradation extensive peer‐reviewed
plastic waste. The review research articles col‐
of the literature for
lected
this fromwas
article PubMed,
based on Google Scholar,
extensive ScienceDirect,
peer-reviewed and articles
research other trusted sources.
collected The ref‐
from PubMed,
erences Scholar,
Google in this article were organized
ScienceDirect, and other and filtered
trusted with the
sources. Thehelp of Zotero
references software.
in this article were
organized and filtered with the help of Zotero software.

Figure 1.
Figure 1. Life
Life cycle
cycleof
ofmicroplastics.
microplastics.Microplastics
Microplasticsare
are produced
produced from
from various
various sources,
sources, mainly
mainly an‐
anthro-
thropogenic in nature. Some of the examples are household activities, sewage treatments, landfalls,
pogenic in nature. Some of the examples are household activities, sewage treatments, landfalls, etc.
Gradually, they enter into large water bodies and get accumulated in various habitats, which again
come back to the human body via consumption of aquatic products.
Water 2022, 14, 4053 3 of 21

2. Effect of Microplastics on Environment

Plastic waste generated on land enters the marine environment through riverine
runoffs by leaching from open dumpsites or sewage effluents, washing off from beaches,
spilling during transport or accident, or dumping of plastic wastes into the sea, resulting in
plastic debris being found in all major ocean basins across the world [17]. Table 1 shows
the countries in which the maximum amount of mismanaged plastic waste reaches the
ocean [18]. Based on the mismanaged plastic waste in ocean per capita, it can be concluded
that Asian countries need to adopt more efficient strategies to reduce the amount of plastic
waste reaching the ocean.

Table 1. List of countries generating the highest amount of waste that reached the ocean in 2019.

Mismanaged Waste Emitted to Mismanaged Plastic Waste to

Country
the Ocean (Metric tons Year−1 ) Ocean per Capita (kg per Year)
Philippines 356,371 3.296
India 126,513 0.093
Malaysia 73,098 2.288
China 70,707 0.049
Indonesia 56,333 0.208
Brazil 37,799 0.179
Vietnam 28,221 0.293
Bangladesh 24,640 0.151
Thailand 22,806 0.328
Nigeria 18,640 0.093

The numerous effects from ingestion of macroplastics, microplastics, and nanoplastics

and entanglement due to macroplastics have been well-documented in several mammal,
fish, turtles, and bird species [19]. The ingestion of plastics leads to suffocation or blocking
of the digestive tract and ultimately death [20]. Microplastics, due to their small size and
increased surface area, can enter into tissues and cells and react with other chemicals
in the environment (Figure 2). Experimental evidence demonstrates that exposure to
microplastics and nanoplastics in oysters hinders feeding and has negative effects on
fecundity and offspring quality [16,21].
In recent times, it has become increasingly evident that trophic transfer and bioaccu-
mulation of plastic and other associated chemical pollutants is taking place through the
food web. A well-studied example includes the filter feeders, such as mussels, wherein
the microplastics and other pollutants are accumulated and are then transferred to benthic
predators and humans through the consumption of wild or farmed shellfish [22,23]. Studies
on mussels have established that microplastics can enter from the gut into the circulatory
system, and they were found in mussel haemolymph. The presence of microplastic particles
in the gastrointestinal tracts of seals and cetaceans indicates the trophic transfer taking place
from the prey fish to top predators [24,25]. Moreover, studies carried out on fish, seabirds,
and mussels indicate the potential of plastics to cause bioaccumulation of environmental
pollutants [26–28].
Microplastic contamination is present not only in the marine environment, but there is
increasing evidence that microplastics are also abundantly present in the terrestrial envi-
ronment through the physical or chemical decomposition of larger plastic materials [29,30].
Plastic products abundantly used in daily life can generate microplastic particles due to
fragmentation, aging, and deterioration. These microplastics accumulate in soil either by
fragmentation of discarded plastic items or by the leaching of buried wastes present in the
landfills [17].
Disposed plastic waste, when exposed to the natural environment, could be ingested
by terrestrial biota because they mistake it as food [31]. However, very few studies have
been conducted to study the occurrence of microplastics in the terrestrial biota. A survey
of the presence of anthropogenic plastic waste of the size range of 0.5–5 mm in terrestrial
Water 2022, 14, 4053 4 of 21

birds in China was conducted which found that 62.6% of total particles found in birds’
digestive tract were plastics [32]. This research indicates that there is an urgent need to
study the prevalence and effects of microplastics in terrestrial birds. Soil organisms such as
earthworms moved 73.5% of microplastics from the soil surface down into the bulk soil
by burrow formation [30]. Moreover, pollutants such as metals and organic contaminants
Water 2022, 14, x FOR PEER REVIEW 4 of 22
carried by microplastics could affect the soil and groundwater quality due to the leaching
of these chemicals through desorption into the soil [33].

Figure 2. Effects of microplastics pollution on different levels of organizations. The biomagnification

of microplastics takes place via the food chain, and they enter into the population. Gradually,
Figure 2. Effects of microplastics pollution on different levels of organizations. The biomagnifica‐
they reach
tion cell and subcellular
of microplastics levels
takes place in individual
via the food chain,organisms, including
and they enter human
into the beings.Gradually,
population. It has
been predicted that they can also interact with DNA of organisms to alter the gene expression
they reach cell and subcellular levels in individual organisms, including human beings. It has and
been
associated
predictedphysiology.
that they can also interact with DNA of organisms to alter the gene expression and asso‐
ciated physiology.
The innumerable applications of plastics in day-to-day life has resulted in the presence
of plastic wastes in contamination
Microplastic large amounts is inpresent
urban water systems
not only in the [34].
marineMunicipal wastewater
environment, but there
treatment plants are another source for the presence of microplastics in the
is increasing evidence that microplastics are also abundantly present in the terrestrialterrestrial envi-
ronment. The wastewater
environment through the collected from
physical households,
or chemical hospitals, and
decomposition of industries containing
larger plastic materials
microplastics end products
[29,30]. Plastic up in sewage sludge [35,36].
abundantly used inThe usage
daily of sludge
life can in agricultural
generate microplastic lands as
particles
fertilizer has resulted in deposition of microplastics in the soil. A study conducted
due to fragmentation, aging, and deterioration. These microplastics accumulate in soil by [37]
assessed thatfragmentation
either by in European countries, through
of discarded sewage
plastic itemssludge
or by application,
the leaching125–180
of buried tons of
wastes
microplastics per million residents
present in the landfills [17]. have been released to the terrestrial environment. The
challenges faced in
Disposed extraction
plastic waste,and identification
when exposed toofthe
microplastic particles from
natural environment, soil or
could besludge
ingested
could be the reason for the limited availability of data on the presence of microplastics
by terrestrial biota because they mistake it as food [31]. However, very few studies have in
thebeen
terrestrial environment
conducted to study [38] though wastewater
the occurrence treatment
of microplastics in plants which used
the terrestrial biota.primary
A survey
clarification, wherein microplastics are removed based on their densities
of the presence of anthropogenic plastic waste of the size range of 0.5–5 mm either byinsedimen-
terrestrial
tation
birdsorin
floatation
China wasbefore the wastewater
conducted is subjected
which found to biological
that 62.6% of totaltreatment, have proven
particles found in birds’
to be more efficient in the removal of microplastics from wastewater [39].
digestive tract were plastics [32]. This research indicates that there is an urgent need to
study the prevalence and effects of microplastics in terrestrial birds. Soil organisms such
as earthworms moved 73.5% of microplastics from the soil surface down into the bulk
soil by burrow formation [30]. Moreover, pollutants such as metals and organic con‐
taminants carried by microplastics could affect the soil and groundwater quality due to
the leaching of these chemicals through desorption into the soil [33].
Water 2022, 14, 4053 5 of 21

The presence of microplastics is also found in large quantities in drinking water.

The effluents from wastewater treatment plants and stormwater runoff from urban and
agricultural lands are the major sources of microplastics in drinking water [40]. Na et al. [41]
investigated whether drinking water treatment plants, which use different processes such
as coagulation, sand filtration, and disinfection using UV and UV/H2 O2 , can remove
microplastics from drinking water. Through their studies, they concluded that the type
of coagulant used and the pH and organic matter naturally present in water affect the
efficiency of microplastics coagulation. Moreover, after sand filtration, at least 16% of
microplastics which were <10 µm in size were further fragmented by UV/H2 O2 , causing
the leaching of chemicals in water and resulting in increased toxicity of the water sample.

3. Health Risks Associated with the Use of Microplastics

The extensive use of plastics for several decades and their release into the environment
have resulted in wide range of associated problems. All plastics are synthetic polymers
which are synthesized by combining monomer chemicals, some of which are toxic and
carcinogenic in nature, and these monomer residues in plastic products can be hazardous
to humans [42]. Moreover, the presence of additives such as fillers and plasticizers, coloring
agents, flame retardants, and other substances pose health risks for living beings and also
reduce the reuse and recycling potential of plastics [27]. Plasticizers such as phthalates are
widely used to make soft PVC (polyvinyl chloride), and products containing phthalates
include clothing, packaging materials, toys, flooring, and other items used daily. Since the
phthalates are not chemically bound to plastics, they can leach out from the plastics into the
surrounding environment, causing health hazards, as they can influence the endogenous
production of several hormones, hampering reproduction and development. Similarly,
bisphenol A used in polycarbonate baby bottles, water bottles, and protective coating inside
metal food containers has been proven to be “environmental oestrogen” [43].
The presence of primary and secondary microplastics has been proven in aquatic life,
and their entry into the food chain through human consumption leads to their biological
accumulation. The authors of [44,45] designed a study to check the presence of microplastics
in blood using Py-GC/MS. Their experiments demonstrated the presence of PMMA (Poly
(methyl methacrylate), PP (Polypropylene), PS (Polystyrene), PE (Polyethylene) and PET
(Polyethylene Terephthalate)) plastic particles in 77% of donors in a quantifiable amount.
PET, PS, and PE were found in a maximum amount in blood samples in about 2.4, 4.8,
and 7.1 µg/mL, respectively. The plastic particles concentrations could be due to exposure
of humans to personal care products (in toothpaste, face scrubs, and lip gloss), use of
nanoparticles in drug delivery, inhalation through air, or consumption of food and water
containing microplastics. Furthermore, [46] analyzed the presence of microplastics in the
feces of patients with inflammatory bowel disease (IBD) and healthy people. They detected
the presence of microplastics in the feces of patients with IBD at much higher concentrations
than compared to the feces of healthy individuals. Fifteen types of microplastics were
detected in feces, of which PET and polyamide were predominantly present in the form of
sheets and fibers.
According to the researchers, there is a positive link between the presence of microplas-
tics in feces and IBD. Either the exposure to microplastics causes the disease, or the patients
of IBD retain microplastics in feces because of the disease. Though microplastics can have a
negative impact on human health, more experimental evidence is required to prove the
harmful effects of these microplastics in the human body. Moreover, Deng et al. proved the
presence of fluorescent and pristine polystyrene microplastics of 5 µm and 20 µm in mice
liver, kidney, and gut tissue using fluorescence spectroscopy and histological analyses [47].
Moreover, through biochemical biomarkers and metabolomic profiles studies, they con-
cluded that exposure to microplastics affects metabolic pathways and leads to oxidative
stress and neurotoxic responses. Scarcity of proper analytical tools for isolation, detection,
quantification, and characterization of microplastic particles which are <10 µm has resulted
in difficulty in estimating the risk of microplastics to human health. However, it has been
Water 2022, 14, 4053 6 of 21

reported that particulate particles in air which are smaller than 2.5 µm in size and arise
from diesel exhaust have the capacity to enter cells and induce the formation of reactive
oxygen species (ROS) and inflammation and are linked to increased chances of death due
to cardiovascular or respiratory diseases or lung cancer. By comparing these results with
microplastics of smaller size, a parallel can be drawn to estimate the risk of microplastics on
human health [10]. Thus, effective measures need to be taken to reduce the production of
microplastics, and ecofriendly methods need to be used for the biodegradation of plastics,
as the physical and chemical methods used for degradation of plastics have severely harm-
ful effects on environment. Microorganisms have the ability to degrade plastics, but various
strategies need to be adapted to increase the potential of these microorganisms to degrade
plastics. In this review paper, we have discussed the various biotechnological interventions
which can be used to improve the biodegradation of plastics by microorganisms and result
in a reduction in the production of microplastics.

4. Role of Microorganisms in Plastic Degradation

Based on frequency of use, plastics are divided into seven broad groups. (1) Polyethy-
lene Terephthalate (PET or PETE), (2) High-Density Polyethylene (HDPE), (3) Polyvinyl
Chloride (PVC or Vinyl), (4) Low-Density Polyethylene (LDPE), (5) Polypropylene (PP),
(6) Polystyrene (PS or Styrofoam), (7) Other. Out of them, many are degradable. The
particular biodegradable plastics are (acrylonitrile butadiene styrene, acrylic, acetyl cel-
lulose, cellulose triacetate, alkyd, cellophane, epoxy resin, polyamide, polyacrylonitrile,
and poly(butylene adipate-co-teraphthalate)). Therefore, the above plastics are used in
several sectors. Numerous studies carried out in the past few years have reported about
the capability of several microorganisms in the degradation of different synthetic plas-
tics. It is estimated that more than 50% of the plastic waste ends up in the landfill and
19% of the waste is managed by incineration. Only about 9% of plastic waste is recycled
and, the remaining 22% of mismanaged plastic waste ends up in terrestrial and aquatic
environments. The majority of the bacterial species which have the ability to degrade
plastic are Gram-negative bacilli, and, among them, Pseudomonas spp. has the highest
capability in the biodegradation of plastics [48–52]. For polyethylene degradation, Pseu-
domonas spp. was isolated from three different sources. Among them, the Pseudomonas spp.
isolated from sewage sludge dump was most efficient in the biodegradation of natural and
synthetic polyethylene.
Compared to the other species, Pseduomonas spp. formed the most viscous and floc-
culent biofilms on the surface within a period of three weeks [50]. Apart from Pseu-
domonas spp., several other bacterial species capable of carrying out the biodegradation of
plastics include Ideonella, Klebsiella, Streptomyces, Mycobacterium, Flavobacterium, Rhodococcus,
Escherichia, and Azotobacter. Along with bacteria, fungi are also involved in the biodegra-
dation of plastics, as they hasten the biodegradation process by sharing the metabolic
intermediates [53]. Penicillum and Aspergillus spp. are reported to have the capability to de-
grade polyethylene by the formation of biofilms which decrease the hydrophobicity of the
surface [53,54]. Aspergillus spp. is reported to have the ability to degrade LDPE, whereas
Penicillum spp. can degrade both LDPE and HDPE [55]. Among the Aspergillus spp.,
Aspergillus niger has the highest potency in degrading polyethylene (38%), followed by
Aspergillus flavus (31%), in a period of 60 days [56].
Biodegradation of plastics takes place when microorganisms such as bacteria and fungi,
through their enzymatic action, convert them into metabolic products such as methane,
carbon dioxide, water, etc. [57]. Biological deterioration of plastic pollutants depends on
several factors, including surface area, molecular weight, hydrophilicity or hydrophobicity,
crystallinity, functional groups, chemical structure, etc. Increases in molecular weight
leads to decreases in degradation rate, as the solubility also decreases, making the plastics
less susceptible to microbial attack because it becomes difficult for microbes to assimilate
plastics through the cell membrane [58]. Similarly, crystallinity is another important factor
affecting biodegradability, with amorphous-domains-containing polymers being more
 hydrophobicity, crystallinity, functional groups, chemical structure, etc. Increases in
molecular weight leads to decreases in degradation rate, as the solubility also decreases,
making the plastics less susceptible to microbial attack because it becomes difficult for
microbes to assimilate plastics through the cell membrane [58]. Similarly, crystallinity is
Water 2022, 14, 4053 another important factor affecting biodegradability, with amor‐
7 of 21
phous‐domains‐containing polymers being more vulnerable to enzymes produced by
microorganisms [59]. Moreover, the hydrophobic nature of plastics also inhibits the sus‐
ceptibility
vulnerableoftothem to microbial
enzymes produced attack by hindering water
by microorganisms [59].absorption,
Moreover,though this prob‐
the hydrophobic
lem can be solved by biofilm formation on plastics [60]. The process of biodegradation
nature of plastics also inhibits the susceptibility of them to microbial attack by hindering of
plastic is depicted in Figure 3.
water absorption, though this problem can be solved by biofilm formation on plastics [60].
The process of biodegradation of plastic is depicted in Figure 3.

Figure 3. Different stages of plastic degradation by microorganisms. Deciphering different stages

of microplastics
Figure bystages
3. Different microorganisms reveals that
of plastic degradation bydifferent biochemical
microorganisms. processes
Deciphering are involved
different in
stages of
the event. Some of the events are action of extracellular enzymes, assimilation, mineralization,
microplastics by microorganisms reveals that different biochemical processes are involved in the
event. Some of the
bio-deterioration, etc. events are action of extracellular enzymes, assimilation, mineralization,
bio‐deterioration, etc.
5. Enzymes Involved in Biodegradation of Plastic Polymers
Enzymes
5. Enzymes are primarily
Involved responsible forofthe
in Biodegradation degradation
Plastic Polymers of plastics due to their ability to
carryEnzymes
out hydrolysis and oxidation [61]. The enzymatic degradation
are primarily responsible for the degradation of plastics of due
plastic can be
to their mea-
ability
sured by weight loss and addition of functional groups [54]. Hydrolases including
to carry out hydrolysis and oxidation [61]. The enzymatic degradation of plastic can be esterases,
cutinases, and
measured lipasesloss
by weight playand
an instrumental role in plastic
addition of functional degradation
groups [62–65]. including
[54]. Hydrolases Enzymes
from different microorganisms with the ability to degrade different synthetic
esterases, cutinases, and lipases play an instrumental role in plastic degradation [62–65].plastic poly-
mers are shown in Figure 4. Cutinase-like hydrolase isolated from Thermobifida
Enzymes from different microorganisms with the ability to degrade different synthetic fusca had
the ability
plastic to degrade low-crystallinity PET bottles and pellets up to 50% of
polymers are shown in Figure 4. Cutinase‐like hydrolase isolated from Thermo‐its initial weight
at 50 ◦ C in 21 days [66]. LC-cutinase, having the ability to hydrolyze low-crystallinity PET
bifida fusca had the ability to degrade low‐crystallinity PET bottles and pellets up to 50%
films, resulting in 50% of weight loss at 50 ◦ C in 7 days, were identified from a leaf-branch
of its initial weight at 50 °C in 21 days [66]. LC‐cutinase, having the ability to hydrolyze
compost metagenomic library [67]. Yoshida et al. [2] found Ideonella sakaiensis 201-F6, which,
low‐crystallinity PET films, resulting in 50% of weight loss at 50 °C in 7 days, were iden‐
on adherence to PET substrate, produces PETase (hydrolase) and MHETase. These en-
tified from a leaf‐branch compost metagenomic library [67]. Yoshida et al. [2] found Ide‐
zymes have the ability to degrade PET to bis (2-hydroxyethyl) terephthalate (BHET), mono
onella sakaiensis 201‐F6, which, on adherence to PET substrate, produces PETase (hydro‐
(2-hydroxyethyl) terephthalate (MHET), terephthalic acid, and ethylene glycol (Figure 5).
Terephthalic acid is converted to protocatechuic acid (PCA), which, upon cleavage by PCA
3,4 dioxygenase, is converted to 4-carboxy-2-hydroxymuconic acid. On further dehydro-
genation, 4-carboxy-2-hydroxymuconic acid forms 2-pyrone-4, 6-dicarboxylic acid, which
enters the TCA cycle. On the other hand, the glycoaldehyde reductase enzyme oxidized
ethylene glycol to glycoaldehyde, which is further oxidized to glycolate. Enzyme glycolate
oxidase oxidizes glycolate to glyoxylate, which condenses with acetyl CoA to form malate,
which enters the TCA cycle [68].
Santo et al. [69] isolated Rhodococcus ruber C208, which degraded polyethylene by
secretion of extracellular laccase. The laccase enzyme required the presence of copper ions
for its induction and activity. The biodegradation of polyethylene took place due to the
oxidation of amorphous regions of PE films by laccase, which resulted in the formation of
easily accessible carbonyl groups, which was confirmed by FTIR analysis and a decrease in
molecular weight of polyethylene film.
Jeon et al. [70] compared the low molecular weight polyethylene-degrading ability
of two alkane monooxygenases (AlkB1 and AlkB2), which are expressed in Pseudomonas
 lase) and MHETase. These enzymes have the ability to degrade PET to bis
(2‐hydroxyethyl) terephthalate (BHET), mono (2‐hydroxyethyl) terephthalate (MHET),
terephthalic acid, and ethylene glycol (Figure 5). Terephthalic acid is converted to pro‐
tocatechuic acid (PCA), which, upon cleavage by PCA 3,4 dioxygenase, is converted to
4‐carboxy‐2‐hydroxymuconic acid. On further dehydrogenation,
Water 2022, 14, 4053 8 of 21
4‐carboxy‐2‐hydroxymuconic acid forms 2‐pyrone‐4, 6‐dicarboxylic acid, which enters
the TCA cycle. On the other hand, the glycoaldehyde reductase enzyme oxidized eth‐
ylene glycol to glycoaldehyde, which is further oxidized to glycolate. Enzyme glycolate
aeruginosa E7, by measuring
oxidase oxidizes glycolate tothe transcription
glyoxylate, which levels of alkB1with
condenses alkB2 using
and acetyl CoA toquantitative
form mal‐
real-time
ate, whichPCR (qRT-PCR).
enters The transcription
the TCA cycle [68]. levels of alkB2 were higher than compared
to alkB1 when
Santo P. aeruginosa
et al. E7 was
[69] isolated grown inruber
Rhodococcus a medium containing
C208, which low molecular
degraded weight
polyethylene by
polyethylene as the sole source of carbon. Polyurethane esterase isolated from
secretion of extracellular laccase. The laccase enzyme required the presence of copper Comamonas
acidovorans
ions for its TB-35
inductioncomprised a catalytic
and activity. domain and surface-binding
The biodegradation of polyethylenedomain, which
took place duewas to
essential for polyurethane (PU) degradation [71]. Polyurethane was hydrolyzed
the oxidation of amorphous regions of PE films by laccase, which resulted in the for‐ by PU
esterase
mation ofinto polyisocyanate
easily and ethylene
accessible carbonyl groups, glycol.
whichEthylene glycol is
was confirmed byfurther converted
FTIR analysis andtoa
glycoaldehyde and glycolic
decrease in molecular weightacid
of and finally enters
polyethylene film.the TCA cycle (Figure 6).

Figure 4.4. Phylogenetic

Figure Phylogeneticrelationships
relationshipsamong
amongbacteria forfor
bacteria degradation of microplastics.
degradation A phylogenetic
of microplastics. A phyloge‐
tree
neticconstructed based onbased
tree constructed aminoonacid sequence
amino acid homologies of synthetic plastic
sequence homologies polymer-degrading
of synthetic plastic poly‐
mer‐degrading
enzymes enzymes
in bacteria in bacteria
is drawn. The istree
drawn. The tree waswith
was constructed constructed with
Molecular Molecular Evolution‐
Evolutionary Genetics
Analysis v7 (MEGA7, https://www.megasoftware.net/ (accessed on 20 November 2022)).

Schimdt et al. [72] analyzed the ability of four polyester hydrolases, LC Cutinase
(LCC), TfCut2, Tcurl278, and Tcur0390 to degrade polyester PU Elastollan B85A-10 and
C85A-10. After 200 h of incubation at 70 ◦ C, LCC caused weight loss of 4.9% and 4.1%
of Elastollan B85A-10 and C85A-10, respectively. LCC was most efficient because of its
higher thermostability at 70 ◦ C, and FTIR analysis confirmed that the weight losses were
due to cleavage of the ester bonds. Styrene monooxygenase, styrene oxide isomerase,
Water 2022, 14, 4053 9 of 21

Water 2022, 14, x FOR PEER REVIEW 9 of 22

phenylacetaldehyde dehydrogenase, and phenylacetyl coenzyme A ligase are the enzymes

which reported in styrene degradation and are believed to be also responsible for depoly-
ary Genetics Analysis v7 (MEGA7, https://www.megasoftware.net/ (accessed on 20 November
merization of polystyrene to styrene monomers and conversion of styrene monomers to
2022)).
phenylacetate and its incorporation in the TCA cycle [73].

Figure 5. Enzymatic reactions involved in biodegradation of PET. The biodegradation of PET is stated
with the enzyme
Figure PETase,reactions
5. Enzymatic and, subsequently,
involved the by-product enters
in biodegradation of into
PET.TCA
Thecycle to culminate of
biodegradation energy.
PET is
stated with the enzyme PETase, and, subsequently, the by‐product enters into TCA cycle to cul‐
minate energy.
 real‐time PCR (qRT‐PCR). The transcription levels of alkB2 were higher than compared to
alkB1 when P. aeruginosa E7 was grown in a medium containing low molecular weight
polyethylene as the sole source of carbon. Polyurethane esterase isolated from Comamo‐
nas acidovorans TB‐35 comprised a catalytic domain and surface‐binding domain, which
was essential for polyurethane (PU) degradation [71]. Polyurethane was hydrolyzed by
Water 2022, 14, 4053 10 of 21
PU esterase into polyisocyanate and ethylene glycol. Ethylene glycol is further converted
to glycoaldehyde and glycolic acid and finally enters the TCA cycle (Figure 6).

Figure 6. Enzymatic reactions involved in biodegradation of polyurethane (PU). The enzyme PU

Figure 6. Enzymatic reactions involved in biodegradation of polyurethane (PU). The enzyme PU
esterase
esteraseis isthe
theenzyme
enzymethat
thathelps
helpsininbiodegradation
biodegradation of
of PU
PU to
to polyisocyanate and ethylene
polyisocyanate and ethyleneglycol.
glycol.
Finally, the produced glycolic acid enters into TCA cycle.
Finally, the produced glycolic acid enters into TCA cycle.

6. Genetic Engineering Approaches to Enhance Plastic Biodegradation

Schimdt et al. [72] analyzed the ability of four polyester hydrolases, LC Cutinase
Genetic
(LCC), engineering
TfCut2, Tcurl278,approaches
and Tcur0390havetobeen used polyester
degrade by the identification
PU Elastollan of genes
B85A‐10to study
and
the potential of microbes to metabolize xenobiotic compounds by cloning
C85A‐10. After 200 h of incubation at 70 °C, LCC caused weight loss of 4.9% and 4.1% of and transforming
them under appropriate
Elastollan B85A‐10 and host microorganisms
C85A‐10, under
respectively. the control
LCC was mostof promoters [74]. Theofchief
efficient because its
objective of the genetic engineering
higher thermostability approach
at 70 °C, and is to isolate
FTIR analysis the genes
confirmed thatinvolved
the weightin degradation
losses were
pathways, modify of
due to cleavage them, and clone
the ester bonds.them in appropriate
Styrene hosts, such
monooxygenase, as E.
styrene coli, isomerase,
oxide using tech-
niques such as PCR, site-directed
phenylacetaldehyde mutagenesis,
dehydrogenase, antisense coenzyme
and phenylacetyl RNA technology,
A ligase etc.
are[75,76].
the en‐ In
order to be used for recycling processes, the enzymes should be stable and
zymes which reported in styrene degradation and are believed to be also responsible for have the ability
todepolymerization
function at high temperatures.
of polystyrene to A styrene
plant disease
monomers causing
andbacteria
conversion produced
of styreneCutinase,
mon‐
which was present in the outer coat of the plants. This enzyme,
omers to phenylacetate and its incorporation in the TCA cycle [73]. capable of breaking down
polyester linkages, was used in the PET degradation process, but the major limitation was
that the enzyme
6. Genetic was unfolding
Engineering and clumping
Approaches to Enhance with itself Biodegradation
Plastic at higher temperatures.
A Genetic
yeast strain was genetically engineered to
engineering approaches have been used by the produce bacterial Cutinase
identification by incor-
of genes to
porating sugar residues at strategic positions that keep the enzyme
study the potential of microbes to metabolize xenobiotic compounds by cloning folded at elevated
and
temperatures and do not allow the clumping of the enzyme to take place by creating phys-
ical barriers. The genetically engineered enzyme was able to degrade PET at an optimal
temperature and concentration [77]. Oda et al. [78] genetically modified Cutinase (Cut190)
isolated from Saccharomonospora viridis AHK190, which is active when bound to calcium
and inactive in a calcium-free state. Of the three calcium binding sites, introduction of a
disulfide bond at site 2 between Asp250 and Glu296 residues resulted in increased melting
temperature of the mutant enzyme, signifying that disulphide linkage imitates the presence
of a bound calcium effect. Moreover, replacement of surface Asn and Gln residues with
Asp, Glu, and His residues resulted in increased melting temperature of the enzymes.
Engineered mutant enzymes were more efficient in degradation of PET compared to the
native enzyme. Moreover, genetic modifications were also carried out to enhance the plastic
degrading capacity of enzymes. PETase (PET digesting enzyme) has potency to carry out
Water 2022, 14, 4053 11 of 21

biodegradation of polyethylene terephthalate (PET), and the X-ray crystal structure of

the enzyme revealed that PETase has features which are frequently observed in cutinases
and lipases.
Moreover, it was observed that though the enzyme retained the ancestral α/β hydro-
lase fold, the active site of the enzyme had a more open cleft compared to homologous
cutinases. To increase the PET-degrading capability of PETase, two mutations were in-
troduced in the cleft, which resulted in a narrowed opening of the cleft which, in turn,
increased the ability of PETase to degrade PET [79]. Yan et al. [80] isolated a thermophilic
Cutinase which has the ability to degrade PET at high temperatures of around 70 ◦ C from
the plant compost metagenome. The Cutinase was cloned in Clostridium thermocellum,
wherein a strong promoter of a constitutively expressed gene was used to obtain a high
level of gene expression, and signal peptide of exoglucanase Cel48S was used for extra-
cellular secretion of the enzyme. Though the thermostable PET hydrolases have been
expressed in E. coli, B. subtilis, B. megatarium, and Pichia pastoris, only the recombinant C.
thermocellum has been able to express cutinases and carry out biodegradation of PET at
60 ◦ C. Pseudomonas sp.
E4 isolated from beach soil contaminated with crude oil had the ability to degrade
polyethylene in the molecular weight range of 1700–23,000, of which 4.9–28.6% of carbon
was mineralized into CO2 after incubation for 80 days at 37 ◦ C. The alkane hydroxylase
gene (alkB) of Pseudomonas sp. E4 was cloned in E. coli BL21. During the biodegradation
process in the compost for 80 days at 37 ◦ C, the recombinant strain mineralized 19.3%
of carbon into CO2 , indicating that the alkB gene plays a significant role in polyethylene
degradation [81]. By incorporation of a novel biosynthethic pathway using plasmids,
recombinant E. coli acquired the ability to convert terephthalic acid derived from plastic
into vanillin, which is widely used in the food and cosmetic industries. The advantage of
this genetic engineering approach is that no hazardous waste is generated, and the reaction
takes place at room temperature in an aqueous medium with a pH of 5.5–7 and without the
need for the addition of any cofactors or reagents [82]. Thus, using the genetic engineering
approaches, the thermostability and ability of enzymes to degrade plastics can be greatly
enhanced, and plastic waste can be converted into value-added compounds.
The survivability and stability of GMOs is quintessential in their application in biore-
mediation. In order to use GMOs for plastic waste management, the organism should be
able to survive and grow under such environmental conditions. Moreover, factors such
as growth rate, inoculum size, and interactions of GMOs with the indigenous bacterial
community and other components of the ecosystem play a crucial role. Another limitation
of GMOs is the possibility of release of GMOs into natural ecosystems because of horizon-
tal gene transfer. Thus, the introduction of GMOs into field sites and their effect on the
structure and function of the natural ecosystems needs to be taken into consideration [83].
Although several different microorganisms with the ability to degrade plastic polymers
have been identified, most of these studies were carried out in laboratory conditions; hence,
it is too difficult to predict the effectiveness of these microorganisms, in natural conditions,
to biodegrade plastics. So, new methodologies need to be designed which improve the
survival of GMOs in foreign environments, and we must increase our understanding of the
mechanism of biodegradation.

7. Gene Editing Tools for Plastic Waste Degradation

Gene editing uses engineered nucleases, known as molecular scissors, to modify DNA
sequences. CRISPR-Cas, ZFN, and TALENs are the main gene editing tools, and they
work by introducing double strand breaks in the target gene sequence, which is then
repaired by either a homology-directed repair (HRD) or a nonhomologous end joining
(NHEJ) pathway [84,85]. Zinc finger nucleases (ZFN) are artificial restriction enzymes
which have Zinc finger proteins (ZFPs) comprised of alpha-helices, which are 30 amino
acids long and act as DNA binding domains, and Folk1 cleavage domain, which introduces
double strand breaks (DSB) at the target site. ZFPs recognize 18 bp specific sequences on
Water 2022, 14, 4053 12 of 21

DNA, and around four to six ZFPs interact with the cleavage domain, depending upon the
target site, thus allowing target-specific gene editing. Transcription activator-like effector
nucleases (TALENs) use TAL proteins, which consist of two domains: one domain for
specific sequence recognition and one domain for sequence cleavage which results in DSB
in the target DNA [86].
Clustered regularly interspaced short palindromic repeats (CRISPR) and their asso-
ciated proteins (Cas) act as an adaptive immune system of microbes by incorporating
short sequences of invading genomes (spacers) into the CRISPR locus. There are three
types of CRISPR systems and several subtypes which have been identified, of which the
type II system is best characterized. It comprises Cas9 nuclease, the guide crRNA, and
transactivating crRNA, which forms a CRISPR complex and associates with the target
DNA using the mature guide crRNA. Cas9 endonuclease introduces double strand breaks
which are then repaired by the host cell machinery, resulting in either insertion or dele-
tion of genes, thus resulting in disruption of open reading frames of the genes. Thus,
by using these gene-editing tools, knock-in or knock-out mutations can be introduced,
wherein the expression of enzymes which play a crucial role in plastic degradation can
be increased, or such genes coding for the enzymes could be introduced in the host mi-
croorganism [87]. CRISPR-Cas systems have been used by researchers in carrying out gene
editing in Pseudomonas sp. [88,89] and Escherichia coli [90,91]. However, carrying out gene
editing of indigenous microorganisms which are already present at the contaminated site
would be more beneficial, as they have the ability to survive and harbor themselves in
stress conditions.

8. Immobilized Enzymes for Bioremediation of Plastics

Though microorganisms can survive in extreme conditions, it is difficult to maintain
these conditions in the outside environment [92]. Many factors, such as temperature, pH,
moisture content, oxygen, the bioavailability of nutrients, contaminants, and the presence
of other toxic compounds, can influence the enzyme expression in microorganisms [93].
Moreover, the expression of enzymes in microbial cells is regulated by specific inducers,
repressors, and cofactors [94]. This problem can be overcome by using immobilized
enzymes. The advantage of using immobilized enzymes is that it will not lead to inhibition
by inhibitors, can work effectively at low and high concentrations of pollutants, and will be
more mobile compared to microbes [95].
Since the biodegradation of plastics would be more efficient by microbial consortia
rather than single species, an immobilized multienzyme system needs to be designed
to carry out complex biological processes which involve several chemical conversions.
Physical adsorption onto octyl-agarose and octadecyl sepabeads of microbial lipase from
Yarrowia lipolytica resulted in ten-fold-higher stability and better yield than free microbial
lipase [96]. Barth et al. [97] reported a two-fold increase in yield of biodegradation products
by using a dual-enzyme reaction system consisting of polyester hydrolase and immobilized
carboxy esterase from Thermobifida fusca.

9. Designing Synthetic Microbial Consortium for Plastic Degradation

Numerous studies have reported the presence of different types of bacteria, fungi, and
actinomycetes in polluted environments, with the surroundings becoming acclimatized to
plastics and utilizing these waste products as sources for carbon and nitrogen. The limiting
factor affecting the rate and range of hydrocarbon degradation by microorganisms appears
to be the lack of ability of most microbial strains to utilize different components of plastic.
Different strains can degrade different components, but a single strain can usually attack
a limited number of hydrocarbons. Hence, a microbial consortium is more nutritionally
versatile than a single strain and exhibits considerable competence in utilizing a large
number of hydrocarbon components from plastics [98,99].
There are innumerable microorganisms with the potential to degrade plastics in a
consortium, but it is practically difficult to experimentally test all combinations to develop
Water 2022, 14, 4053 13 of 21

the most suitable bacterial consortium. These microbial species can be filtered by simu-
lation to test their compatibility in silico. FLYCOP, which stands for FLexible sYnthetic
Consortium Optimization, is an in silico tool which can be used for designing and opti-
mizing microbial communities. Instead of using a trial-and-error approach for multiple
random consortium configurations, FLYCOP uses a stochastic local search. It takes mul-
tiple consortium configurations as the input and gives back the best configuration as the
output. FLYCOP can be used to carry out simulations and detailed evaluations of diverse
situations before doing actual in vivo experiments. It is a flexible in silico tool applicable to
different microbial communities with diverse objectives to study numerous community
configurations computationally.
Two other tools, which use a hybrid approach, Microbial Community Modeller (MCM)
and COMETs, can also be used for designing the microbial consortium [100]. The use of
in silico tools for designing microbial consortia will circumvent the trial-and-error efforts
and the need for chemical optimization and will save resources and time. A microbial
consortium comprising Mycobacterium spp. PO1 and PO2, Novosphin-gobium pentaroma-
tivorans PY1, Ochrobactrum sp. PW1, and Bacillus sp. FW1 exhibited a three-fold-higher
degradation rate for pyrene compared to individual bacterial strains. The degradation
process was initiated by Mycobacterium spp, and Novosphin-gobium pentaromativorans PY1,
Bacillus sp. FW1, and Ochrobactrum sp. PW1 degraded the intermediates aided by the
release of biosurfactant by Bacillus sp. FW1, which helped in increasing the solubility of
pyrene [101]. A consortium comprising Streptomyces sp. A5, A11, M7, and MC1 removed
86% of Cr(VI), which had an initial concentration of 50 mg/kg in soil and 46% of lindane,
which was present at an initial concentration of 25 mg/kg of soil [102]. Arthrobacter sp.
DNS10, Variovorax sp. DNS12, Arthrobacter sp. DNS9, and Bacillus subtilis DNS4 removed
atrazine present at an initial concentration of 100 mg/L, with 100% efficiency compared to
a single microbial strain when used alone [103].
Similarly, synthetic microbial consortia can be designed for the degradation of dif-
ferent types of plastic polymers. Furthermore, hybrid consortia comprising indigenous
and engineered microorganisms can be designed to have better plastic bioremediation
capabilities. While designing synthetic microbial consortia, the spatial arrangement of
the mixed cultures is important to invigorate the interactions locally and to improve their
ability to survive in environmental stress conditions. Moreover, over a long period, the
microbial strains in the consortia can undergo mutations, which can lead to nonproductive
phenotypes and can decrease population-level performance. Hence, while designing mi-
crobial consortia, care must be taken to decrease mutations along with the competitive and
antagonistic interactions taking place between the native species. However, to ensure that
the engineered microbial communities do not disrupt the natural ecosystem, the biocon-
tainment of synthetic microbial consortia is important, along with population control [104].
In this regard, many specific pathways have been targeted for microbial degradation of
plastics (Table 2).

Table 2. List of biodegradation databases and pathway prediction systems.

Importance in Plastic
Sr. No. Database Link Reference
Degradation
Manually curated database
Biodegradation
https: providing information about
1 Network-Molecular Biology [105]
//bionemo.bioinfo.cnio.es genes and proteins involved in
Database (Bionemo)
biodegradation metabolism
Water 2022, 14, 4053 14 of 21

Table 2. Cont.

Importance in Plastic
Sr. No. Database Link Reference
Degradation
Provides information pertaining
University of Minnesota to metabolic pathways along
2 Biocatalysis/Biodegradation http://eawag-bbd.ethz.ch/ with microbial biocatalytic [106]
Database (UM-BBD) reactions, enzymes, and genes in
the process of biodegradation
Highly curated database
providing information about
3 Metacyc https://metacyc.org/ experimentally elucidated [107]
metabolic pathways and
enzymes for all domains of life
It gives information pertaining
to entire genomes and has
4 Biocyc https://biocyc.org/ [108]
predicted a metabolic network of
an organism
Retrieves information from
KEGG reaction and KEGG
http://www.genome.jp/ RPAIR databases of query
5 PathPred [109,110]
tools/pathpred/ compound and predicts possible
enzyme-catalyzed
reaction pathways
Using the reaction rules based
Biochemical Network on the Enzyme Commission
http://minedatabase.mcs.
6 Integrated Computational classification system, it designs [111]
anl.gov
Explorer (BNICE) novel chemical structures
and pathways
Proposes metabolic pathways
including the information about
enzymes involved and genes
From Metabolite http: and organisms which can be
7 [112]
to Metabolite //FMM.mbc.nctu.edu.tw/ compared between different
species and is based on KEGG
databases and other
integrated databases
Used to design metabolic
http://osslab.ex.ac.uk/ pathways between source
8 Metabolic Tinker [113]
tinker.aspx compounds and
end-products synthetically
Maintains diverse information
http://pdg.cnb.uam.es/ related to bioremediation and
9 MetaRouter [114]
biodeg_net/MetaRouter biodegradation pathways stored
in an integrated framework

10. System Biology Approaches for Plastic Degradation

The Systems biology approach gives insight into the difference in the response of micro-
bial systems under different environmental conditions. Moreover, it helps in understanding
the microbial interactions within the communities and the survival of microorganisms un-
der conditions of extreme temperature and pressure [115,116]. This is possible through the
interdisciplinary field of study combining computational biology and multiomics approach.
Microbial consortia play an important role in the efficient biodegradation of xenobiotic
compounds by cooperative metabolic activities, and hence, the final fate of the compound
by partial or complete degradation to a nontoxic compound can be predicted by in silico
analysis [93,117]. Moreover, several biodegradation databases and pathway prediction
Water 2022, 14, 4053 15 of 21

systems have been designed which assist in implementing microbial consortia to an environ-
mental site for remediation and in hazardous waste management [118]. Since an enormous
number of microbes are present in the natural environment, phylogenetic analysis and
pathway prediction systems can be used to filter, by simulation, the number of possible
combinations which are essential for formulating novel microbial consortia [93]. The dif-
ferent databases which provide data related to biodegradation of chemicals, the genes
and enzymes which are involved in the biodegradation, and the metabolic degradation
pathway are mentioned in Table 2.
Autodock, Schrödinger, Vina, YASARA, Glide, GEMDOCK, PatchDOCK web server,
GOLD, and DARWIN are the different softwares which can be used to carry out molec-
ular docking analysis, wherein the ability of an enzyme to adhere to a pollutant can
potentially be displayed and its functionality in the biodegradation process can be un-
derstood [119–121]. Skariyachan et al. [122] studied the molecular interactions between
lipases from Pseudomonas spp., polyethylene, and polystyrene, which showed that both
polyethylene and polystyrene had good interaction with lipases, and hence, bioinformatic
analysis can be used for hypothesizing degradation mechanisms. In molecular docking
analysis, the protein is held static; this limitation can be overcome by using molecular
dynamic simulations. The stability of a ligand-receptor complex proposed by molecular
docking analysis can be estimated by using molecular dynamic simulations, as it allows
conformational change in both ligand and enzyme in complexes during biological premises.
The programs which can be used to study molecular simulations are Abalone, Amsterdam
Density Functional (ADF), and Desmond [123].
The multiomics approach includes genomics, transcriptomics, proteomics, metage-
nomics, and metabolomics and can be used for identifying uncharacterized biosynthetic
gene clusters within the genome of sequenced organisms (Figure 7). The metagenomics
approach is garnering lot of interest because of its possibility to degrade oil, petroleum,
plastics, and other hydrocarbons by carrying out direct genome analysis of nonculturable
microorganisms. Fang et al. [124] investigated, through metagenomics, the biodegra-
dation genes involved in degradation of DDT (dichlorodiphenyltrichloroethane), HCH
Water 2022, 14, x FOR PEER REVIEW
(hexachlorocyclohexane), and atrazine, where they were able to identify the16genes
of 22 involved

in biodegradation after carrying out genome annotation.

Figure 7. The multiomics approach for plastic degradation. Various approaches such as metagenomic,
Figure 7. The multiomics approach for plastic degradation. Various approaches such as meta‐
genomic, transcriptomic, proteomic, and metabolomic approaches are followed for the degradation
genomic, genomic, transcriptomic, proteomic, and metabolomic approaches are followed for the
of plastics. High-throughput
degradation techniquestechniques
of plastics. High‐throughput can be followed to do so.
can be followed to do so.

Transcriptome‐based studies were conducted on a marine bacterium Bacillus species,

AIIW2, which provided insights into the hydrolytic enzymes involved in the PET degra‐
dation process. The process of PET degradation was confirmed by carrying out weight loss
assay, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy
Water 2022, 14, 4053 16 of 21

Transcriptome-based studies were conducted on a marine bacterium Bacillus species,

AIIW2, which provided insights into the hydrolytic enzymes involved in the PET degrada-
tion process. The process of PET degradation was confirmed by carrying out weight loss
assay, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM)
analysis, and by measuring the hydrolytic products of PET degradation. Aldehyde dehy-
drogenase and carboxylesterase were found to be involved in the PET degradation process.
By observing the changes in the functional groups and HPLC analysis of degraded products,
the degradation pathway was elucidated. Moreover, engineered microbial systems with
higher PET degradation capabilities could be developed using the gene annotations [125].
A similar approach could be used for studying the genes involved in biodegradation of plas-
tics. The genome scale model (GEM) can be used to predict the microorganisms with the
highest potential for bioremediation using the genotype and phenotype data about the par-
ticular microorganism available from proteomics, transcriptomics, and metabolomics [126].

11. Conclusions
This article focuses on the ubiquitous use of plastics in all aspects of life, which has
caused a tremendous increase in the amount of plastic waste generated. The degradation
of this plastic waste into smaller particles gives rise to microplastics, which have proven to
have negative impact on the aquatic and terrestrial environment. Lack of proper analytical
tools to study microplastics < 10 µm has made it difficult to study the detailed effects of
microplastics on human health. Researchers have reported accumulation of microplastics
in animal tissues, and their harmful effects have been found in cell lines. The presence of
microplastics has been reported in human blood, and a positive correlation has been found
between the presence of microplastics in human feces and IBD, but more experimental
evidence is required to prove the deleterious effects of microplastics on human health.
The current methods employed for the degradation of plastic wastes, including in
landfills and incineration, result in the release of more toxic chemicals in the environment.
Microorganisms have the ability to degrade plastics in an ecofriendly manner. In order to
increase the potential of these microorganisms to degrade plastic waste, modifying these
microorganisms using genetic engineering techniques and genome editing tools can be
used. Pseudomonas aeruginosa strains were engineered which were capable of removing
microplastics from seawater samples by secretion of exopolysaccharides using a ‘trap and
release’ mechanism [127]. Modification of bacterial proteins to enhance their ability to
remove microplastics from wastewater can be employed [128]. The major challenge in using
these engineered microorganisms at the industrial scale is the safety concerns associated
with the use of GMOs. By using omics technology and pathway prediction tools, microbial
consortia can be designed, which have more potential to degrade plastics and reduce the
amount of microplastics released in environment.

Author Contributions: Conceptualization, Data curation, Formal analysis, Funding acquisition,

Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation,
Visualization, Roles/Writing—original draft, and Writing—review and editing by S.T., S.M. and B.P.;
Conceptualization, Writing—original draft, Methodology, Writing—review and editing, and Revision
by S.P. All authors have read and agreed to the published version of the manuscript.
Funding: The work was generously supported by the funding to BRP from the Science and Engi-
neering Research Board, Department of Science and Technology, Govt. of India, New Delhi, India
(No. ECR/2016/001984), and by Department of Science and Technology, Government of Odisha
(Grant letter number 1188/ST, Bhubaneswar, dated 01.03.17, ST-(Bio)-02/2017).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated or analyzed during this study are included in this
published article).
Water 2022, 14, 4053 17 of 21

Acknowledgments: The authors duly acknowledge the use of the Central Instrumentation Facility of
Odisha University of Agriculture and Technology, especially from Sashikanta Dash for the analyses
of samples.
Conflicts of Interest: The authors declare no conflict of interest.

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