Book Chapter

Bioremediation Techniques
for Microplastics Removal
Samaneh Hadian-Ghazvini, Fahimeh Hooriabad Saboor,

and Leila Safaee Ardekani
Abstract Plastics’ unique physical and chemical properties made them indispens-
able parts of our everyday life and technology. Due to the mismanagement of plastic
wastes, 10% of global plastic production annually entering the ocean accounts for
60–80% of marine debris. With the current plastic production rate, more plastics will
exist in the oceans than fish by 2050. Plastic waste does not decompose in nature,
or its decomposition takes a long time. Among plastic contaminants, microplas-
tics, which are plastic pieces less than 5 mm in size, have attracted much attention
because of their potential risks to organisms’ lives. This chapter discusses plastic
polymers, their types, and their features that affect plastics’ degradation. Here, we
present the interaction between organisms and microplastics and their hazardous
effects on living organisms. Bioremediation and biodegradation are explained. Also,
new approaches in biodegradation, such as enzyme engineering, are introduced.
Plastic polymers’ chemical and physical features such as molecular weight, molec-
ular backbone’s atoms, chemical bonds, crystallinity, hydrophobicity, and additives
presence are important factors in vulnerability to decomposing agents. Aging and
weathering by abiotic factors including sunlight, heat, moisture, and oxygen decrease
the microplastics’ surface hydrophobicity and facilitate microorganism attachments
and biofilm formation. Microplastics, because of releasing toxic additives, metallic
and organic toxic compounds’ adsorption on their surfaces, threaten organisms’
lives. Microplastics’ harmful effects on marine organisms, especially the primary
S. Hadian-Ghazvini (B)
Laboratory of Bioanalysis, Institute of Biochemistry and Biophysics, University of Tehran,
Tehran, Iran
e-mail: s.hadian@ut.ac.ir
F. Hooriabad Saboor
Department of Chemical Engineering, Faculty of Engineering, University of Mohaghegh Ardabili,
Ardabil, Iran
e-mail: f.saboor@uma.ac.ir
L. Safaee Ardekani
Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University,
Tehran, Iran
e-mail: Leila.safaee@modares.ac.ir
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 327
M. Sillanpää et al. (eds.), Microplastics Pollution in Aquatic Media,
Environmental Footprints and Eco-design of Products and Processes,
https://doi.org/10.1007/978-981-16-8440-1_15
328 S. Hadian-Ghazvini et al.
producers’ food chains such as microalgae, can directly or indirectly influence food
web consumers such as fish, aquatic birds, and even humans. Antibiotic adsorption
on microplastics and, therefore, enrichment of potentially pathogenic and antibiotic-
resistant bacteria and antibiotic-resistance genes through horizontal gene transfer are
other microplastics-related concerns. Following the biofilm formation, microorgan-
isms’ activity and their secreted enzymes and agents deteriorate the microplastics
and lead to molecular fragmentation and depolymerization. Assimilation and miner-
alization of the fragmented molecules are the last biodegradation steps that give
rise to CO2 , H2 O, CH4 , and biomass production. Some genus and species of fungi
and bacteria and their powerful enzymes such as oxidoreductases and hydrolases
are key players in bioremediation by microorganisms. Electron microscopy, spec-
troscopy techniques, weight loss measurements, mechanical properties, molar mass
changes, CO2 evolution/O2 consumption, radiolabeling, clear-zone formation, enzy-
matic degradation, and controlled composting test are employed for biodegradation
evaluation. Since more than 99% of prokaryotes and some eukaryotic microbes
are unculturable, hence, to select plastic-decomposing microorganisms, culture-
independent methods, i.e., metagenomic analysis, are utilized. The metagenome
analysis and in silico mining lead to a deeper investigation of the explored and
unexplored nature to find efficient enzymes and microorganisms for microplastics’
bioremediation. Using microbial consortia and engineered microorganisms and their
enzymes are other promising approaches for plastics bioremediation.
Keywords Microplastics · Bioremediation · Biodegradation · Biodegradable

plastics · Aquatic environment · Bacteria · Fungi · Antibiotic resistance · Enzyme
engineering · In silico and metagenomics analysis
1 An Introduction to Plastics Bioremediation, Types,

and Application
Baekeland, in 1907, discovered the first fully synthetic plastic and called it Bakelit,
which was formed from heating phenol and formaldehyde mixture [156]. Synthetic
plastics revolutionized polymer science and human life [50]. The term plastic origi-
nated from the Greek word plastikos meaning “capable of being molded or formed”
[151]. Plastics are synthetic or semisynthetic high molecular weight polymeric
molecules generally composed of carbon, hydrogen, oxygen, nitrogen, and chlo-
rine as main elements [19, 20, 173]. Due to their physical and chemical properties,
plastics are indispensable parts of our everyday life and technology [20]. In 2018,
plastics’ world production reached 359 million tonnes, which has increased by 11
million tonnes compared to 2017 [1, 2]. Global production and use of plastics are
estimated to increase by 3.8% every year through to 2030 [1, 2]. Therefore, waste
disposal and management of the enormous consumed plastics are crucial, especially
from environmental and human health aspects.
Bioremediation Techniques for Microplastics Removal 329
Since the late eighties, cleaning the environment through bioremediation has
received much attention [114]. Bioremediation refers to the process of detoxifying
contaminants in the air, water, soil, and sediments using living organisms, mainly
microorganisms, plants, microorganisms’ enzymes, or plant enzymes [58, 114]. The
bioremediation purpose is to reduce pollution to undetectable, nontoxic, or levels
below the defined limits [58, 207]. These organisms have physiological capabil-
ities for absorption, detoxifying, transformation, or degrading contaminants [58].
Microorganisms such as bacteria and fungi, due to their enzymes, are the leading
players in bioremediations [58].
Microorganisms exist almost in any place and condition. They can tolerate extreme
conditions such as high temperature, high salt concentrations, low humidity, high
or very low oxygen concentration, and high concentrations of toxic or hazardous
compounds and elements [89]. Microorganisms have different adaptation strategies
that enable them to live in extreme conditions and have diverse catabolic genes
and enzymes. Besides, they can modify their cell membrane to retain the required
biological functions. They can produce certain compounds with surfactant properties
and have biomolecular pumps to efflux the toxic compounds outside of the cell and
reduce the compounds’ inner cell concentration [175]. Therefore, microorganisms
with these fantastic mechanisms and abilities and the possibility to engineer them
for a specific purpose can be a versatile tool for bioremediation.
Two types of bioremediation techniques are in situ and ex situ. In situ bioreme-
diation means treating a contaminated site by adding the proper living organisms,
especially bacteria, bacterial consortia, or fungi, to accelerate the degradation of
desired contaminants. The use of indigenous microorganisms for in situ bioreme-
diation is an ideal solution. However, based on the pollutant type, the laboratory
investigation results, and in some cases, the results of field studies, enriched cultures
of selected microorganisms isolated from other places are added for biodegradation
[58, 131]. In contrast, ex situ bioremediation entails collecting and removing the
contaminants and transferring them to another location for subsequent treatments
such as bioreactors for biological processes and reactions [207].
Bioremediation can be intrinsic or passive or can be performed via human inter-
vention. Natural attenuation is the passive form in which the physical and biolog-
ical process without human mediation can reduce the toxicity, mass, and mobility
of a pollutant [148]. Enhanced bioremediations contain numerous technologies
performed via human intervention. In some of these technologies, indigenous micro-
bial populations are aided by introducing electron donors or electron acceptors or
other sources of nutrients except carbon to stimulate and enhance microbial growth
and degradation efficiency. The pollutants are the carbon source for microorganisms’
energy production. This technology is called biostimulation. In some other technolo-
gies, microbial cultures of natural strains or genetically engineered microorganisms
are added to the desired polluted site, which is named bioaugmentation. The organ-
isms can be indigenous to that site or be isolated from other sites. Besides, they can
be a single microorganism culture or mixed cultures [148]. They can be wild-type
or genetically modified to enhance microbes’ resistivity to the toxic compounds and
the efficiency and the ability of the microbes for degrading contaminants [89].
2 A Brief Introduction to Organisms and Enzymes
Before any other discussion in this chapter, a short introduction about different organ-
isms, especially those critical to biodegradation, is presented. According to their
similarity and as illustrated in Fig. 1, organisms are categorized into three separated
domains: Bacteria, Archaea, and Eukarya [68]. The tree of life, which is supposed
to demonstrate life’s evolutionary processes and genealogical relationships, has a
hierarchical structure [105]. Each domain of the tree, after multiple divisions, ulti-
mately leads to different genus and species. The two first domains, i.e., bacteria and
archaea, are known as prokaryotes and are microscopic, single-cell organisms whose
cells lack a nucleus and other membraneous internal organelles [60, 132].
Bacterial cells, an ancient group of organisms, are diverse almost in all envi-
ronments. They are essential due to their specific roles in decomposing the dead
organisms, converting some atmosphere gas into nutrients consumed by other organ-
isms, and digesting some biomolecules by their enzymes. Some bacteria cause
Fig. 1 Tree of life presenting three domains of organisms, represented from [115]
diseases in humans and other living organisms [132]. Archea are specific bacterial-
like cells with unique biochemistry that differentiate them from bacterial and eukary-
otes. Archea can live in extremely harsh conditions such as the deep sea, hot springs,
and salt marshes [60].
A nucleus in their cells is the primary and common feature in all eukaryotes
that include microscopic organisms or so-called microorganisms and large animal
or plant species. The subdivisions of each domain are called a kingdom. As shown
in Fig. 1, Fungi, Plantae, and Animalia are three kingdoms of the eukaryote domain.
The eukaryote domain also includes Protista, which are unicellular or multicellular
organisms and can be autotroph or heterotroph. Protista comprises multiple kingdoms
and contains a vast collection of eukaryotic, unrelated species that are not fungi,
animals, or plants [66, 132]. Some examples of protists are algae, ameba, slime
molds, and diatoms [132].
Plants are multicellular organisms that are autotroph, meaning that they can
produce organic molecules that store chemical energy in their chemical bonds. Plants
convert CO2 and H2 O to sugar and O2 , so they have an essential role in food chains
[31, 132]. Fungi are unicellular or multicellular organisms that are heterotrophs and
cannot produce their food. Therefore, they are consumers that acquire nutrients by
secreting extracellular enzymes to decompose organic materials and then absorb the
digested molecules [66, 132]. Animals are also multicellular, heterotroph organisms
that obtain their foods by eating other organisms. This kingdom contains the most
advanced creatures on earth [66, 132].
All organisms need food for their life. They use these foods to get energy,
grow, perform their activities, repair their bodies, and reproduce. Nearly all of these
processes are done through chemical reactions. Biological catalysts are necessary
for most biological reactions. These biological catalysts can be proteins or ribonu-
cleic acids, which is called RNA [32]. Like other catalysts, these biological catalytic
molecules increase the chemical reaction rate while they are not consumed for the
reactions. They increase the chemical rate by reducing the reaction’s activation energy
and creation of transition state [32]. Most biological enzymes are protein molecules.
The chemical reaction is done in a region of an enzyme called the active site. For
their functions, enzymes may have one or more groups that help them do catalytic
activity in active sites and convert the substrate to product [78].
3 Plastic Degradation and Biodegradation
Plastics have distinctive properties like low cost, high durability, low density, low
thermal and electrical conductivity, biological inertness, resistance to water and mois-
ture, and corrosion resistance. Therefore, they are versatile materials employed in
almost all aspects of human life [50]. These beneficial properties, which are the
main reasons for plastics’ ubiquitous applications, give rise to today’s environmental
concerns because plastics don’t mainly degrade in the soil and water environments
spontaneously. Natural processes such as photodegradation, thermal degradation,
mechanical stress degradation, and biodegradation can only help, to some extent, the
degradation of plastics [9, 184].
Degradation means any change in polymer properties such as shape, color, tensile
strength, and molecular weight through physical, chemical, and biological processes.
These processes result in bond cleavage and chemical alteration of polymers [151,
161]. In photodegradation, ultraviolet and visible radiations of sunlight can initiate the
degradation of most synthetic polymers. Light absorption creates excited states, and
then polymer radicals that interact with molecular oxygen and subsequent reactions
accelerate photodegradation [161]. Thermal degradation starts with thermal and UV
light absorption. Structural imperfections in the polymer chain are weak points where
depolymerization and degradation can initiate. The temperature directly determines
the rate of thermal degradation [9, 161]. Apart from photo and heat degradation,
mechanical stress accelerates degradation and is morphology-dependent [9].
Biodegradation is the final step of plastic degradation in the environment.
Biodegradation is defined as the biochemical transformation of abiotic degrada-
tion products through microorganisms’ mineralization process [9, 161]. Physical,
thermal, photo-oxidation and abiotic hydrolysis disintegrate plastics and give rise to
their fragmentation, which increases surface areas accessible for microbial attach-
ment and colonization. Mineralization is the decomposition of plastic molecules into
CO2 , CH4 , H2 O, inorganic compounds, and biomass performed by microorganisms’
enzymes. The schematic representation of the plastic degradation and biodegradation
is depicted in Fig. 2.
The chemical structure, morphology, molecular weight, molecular weight distri-
bution, hydrophilic and hydrophobic properties, surface area, melting temperature,
glass transition temperature, elasticity modulus, crystal structure, and crystallinity
are critical factors in polymer biodegradability [173]. Because of microorganisms’
lower affinity to attachment, colonization, and biofilm formation on hydrophobic
surfaces, hydrophobicity is an obstacle for hydrolysis reactions. Hence, polyolefins
and hydrophobic plastics are subjected to lower biodegradation [161]. The polymer’s
molecular weight determining its physical properties is inversely related to plas-
tics’ biodegradation rate [173]. Aging and weathering by sunlight, heat, moisture,
and oxygen create low molecular mass oxidation precursors for microorganisms’
bioassimilation [24]. Looser packing in a polymer’s amorphous domains led to their
easier degradation compared to the more resistant crystalline parts. The microor-
ganisms’ enzymes deteriorate polymers mainly from amorphous domains [173].
Like molecular weight, a polymer’s melting temperature has an inverse effect on its
biodegradability [173].
Many research groups investigate the biodegradability of bioplastics under various
environmental conditions. Microbial diversity in soil and composting condition is
higher than in marine, seawater, and other aquatic environments hence more research
were performed on these two conditions [42]. Researchers use different biodegrad-
ability assessment methods in laboratories. These are visual observations by different
microscopy and spectroscopy techniques, weight loss measurements, changes in
mechanical properties and molar mass, CO2 evolution/O2 consumption, radiola-
beling, clear-zone formation, enzymatic degradation, and controlled composting
test [151]. These techniques allow the researchers to find the degradable polymer,
degradative conditions, and degrading microorganisms or enzymes.
4 Types of Plastics Based on Their Origin, Thermal

Behavior, and Biodegradability
Plastics are classified based on their producing feedstock, polymer-forming

monomers, applications, thermal property, degradability, and other properties. Plas-
tics produced from petrochemical materials originating from oil, natural gas, and coal
are called petrochemical or fossil-based plastics. Another category of plastics, named
bio-based plastics, can be synthesized from renewable and biomass resources such
as sugar cane, starch, lignin, and proteins [20, 173]. Synthetic Plastics are produced
through two main processes. During the first process, additional polymerization on
the broken carbon–carbon double bonds of the original olefins gives rise to new
carbon–carbon bonds and carbon–chain polymers’ production [205]. The long and
linear carbon chains are hydrophobic and resistant to hydrolytic cleavage [134, 205].
The second process for plastic production is a condensation reaction that involves
eliminating water molecules during the formation of an ester or amide bond between
a carboxylic acid and an alcohol or amine group. The polyesters or polyamides with
heteroatoms in their main chain have hydrophilic properties and are susceptible to
hydrolytic cleavage [147, 205].
4.1 Thermoplastics and Thermosets
Two prominent families of plastics based on their behavior after heating and cooling
are thermoplastics and thermosets; they are schematically shown in Fig. 3. Ther-
moplastics synthesized through the first process mentioned above can be heated for
softening and cooled for hardening several times. Thermoplastics can be reprocessed
and reshaped through the heating and cooling process because of their noncova-
lently bound, independent, and linear polymeric molecules. Carbon-chain polymer
of thermoplastics due to hydrolytic cleavage resistance is nonbiodegradable. Ther-
moset plastics are firstly melted by heating then are solidified permanently through
an irreversible process. Thermosets are produced via the above-mentioned second
process. The structure of thermosets is a highly cross-linked network with supe-
rior mechanical properties. Hence, they are non-recyclable such as epoxy resins or
coatings. The existence of heteroatoms such as oxygen and nitrogen in thermosets’
backbone makes them hydrophile and hydrolytically cleavable. Therefore, thermoset
plastics have biodegradability potentials [52, 81, 205]. In Table 1, some examples of
thermoplastics and thermoset plastics are provided.
Fig. 2 Biodegradation steps of a polymer, reproduced from [3, 51, 63]
Fig. 3 Schematic representation of the molecular structure of thermoplastics and thermosets.

Crosslinks of the thermosets are depicted in red. This figure is reprinted from [79]. https://doi.org/
10.13140/RG.2.2.17881.16482. https://www.researchgate.net/publication/329156276_PREDIC
TING_THE_INFLUENCE_OF_WEAVE_ARCHITECTURE_ON_THE_STRESS_RELAXA
TION_BEHAVIOR_OF_WOVEN_COMP
4.2 Bio-based or Fossil-Based Nonbiodegradable Plastics
The term “Nonbiodegradable” describes plastic polymers that do not break down
and decompose to CO2 , CH4 , H2 O, and biomass through a biological process by
microorganisms. Conventional plastics are produced from petrochemical resources
such as crude oil, coal, and natural gas. Based on their practical processing, these
synthetic polymers are divided into four categories: thermoplastics, thermosets, elas-
tomers, and synthetic fibers. Polyethylene (PE), polypropylene (PP), acrylonitrile–
butadiene–styrene (ABS), polyethylene terephthalate (PET), polycarbonate (PC),
polyvinyl chloride (PVC), polystyrene (PS), polyamides (e.g., Nylon 6 and Nylon
66), Teflon (polytetrafluoroethylene), polyurethane (PU, PUR), and poly(methyl
methacrylate) (PMMA, acrylic) are the most prevalent synthetic polymers that are
known as nonbiodegradable. In Table 2, some examples of these plastics are given.
Synthetic polymers are created by three general reactions: polymerization, polyad-
dition, and polycondensation [90]. The main chain of addition polymers like poly-
olefins such as polyethylene, polypropylene, polystyrene, and polyvinyl chloride is
only built from carbons. Therefore, they are resistant to hydrolytic cleavage and
are considered nonbiodegradables [118]. However, condensation polymers such as
polyamides, polyesters, and polyurethanes with oxygen and nitrogen heteroatoms
on their backbone have a hydrophilic main chain. The amide or ester bond of the
polymer backbone makes it vulnerable to hydrolytic cleavage [118].
Table 1 Examples of
Thermoplastics Thermosets
thermoplastics and thermosets
Polyethylene (PE) Polyurethane (PUR or PU)
Polypropylene (PP) Unsaturated polyesters
Polyvinyl-chloride (PVC) Epoxy resins
Polyethylene terephthalate (PET) Melamine resin
Polystyrene (PS) Vinyl esters
Expanded polystyrene (EPS) Silicone
Polycarbonate (PC) Phenol–formaldehyde
resins
Poly methyl methacrylate Urea–formaldehyde resins
(PMMA)
Thermoplastic elastomers (TPE) Phenolic resins
Polyarylsulfone (PSU) Acrylic resins
Fluoropolymers Polytetrafluoroethylene
(PTFE)
Acrylonitrile butadiene styrene Polyvinylidene fluoride
resin (ABS) (PVDF)
Styrene-acrylonitrile copolymer Bismaleimide (BMI)
(SAN)
Polyamides (PA), e.g., Nylon 6 Fluoropolymers
and Nylon 66
Polyetheretherketone (PEEK)
Polyoxymethylene (POM),
acetal, polyacetal
Polybutylene terephthalate (PBT)
Polyacrylonitrile (PAN)
Table 2 Categorization of plastics based on their source and biodegradability [13, 63, 90]
Bio-based plastics Partial Fossil-based
bio-based plastics
Nonbiodegradable (Bio-PE), Polyol-polyurethane, PET, PTT, Bio-PET, Conventional PE,
Polythioester (PTE) PTT, PBT, PP, PET, PS,
SBR, ABS PVC, ABS, PBT,
PA6. PA6.6, PU
Biodegradable Starch-based plastics, polylactic acid-based Starch Polybutylene
plastics (PLA), Polyhydroxyalkanoates blends adipate (PBA),
(PHA), PHA-based plastics (PHB), polybutyrate
poly(hydroxybutyrate-co-hydroxyvalerate) adipate
[PHBV]), cellulose-based plastics terephthalate
(cellophane), protein-based plastics (PBAT),
Polybutylene
succinate (PBS),
Polycaprolactone
(PCL), Polyvinyl
alcohol (PVA or
PVOH)
PE: polyethylene; PP: polypropylene; PET: polyethylene terephthalate; PTT: polytrimethylene
terephthalate; PBT: polybutylene terephthalate; ABS: Acrylonitrile Butadiene Styrene; PVC:
polyvinyl chloride; PS: polystyrene; PU: polyurethane; SBR: Styrene-butadiene; PA6: polyamide
6; PA6.6: polyamide 6.6
Feedstock sources of fossil-based plastics are not renewable, and their production
process is not green and causes the release of greenhouse gas into the atmosphere.
Currently, almost 7% of petroleum is converted to plastic materials [176]. Finding
renewable sources for plastic production due to saving fossil sources and lowering
greenhouse gas emission is needed [176]. Bio-based materials, due to renewability,
were appealing solutions and were utilized for manufacturing plastics. Drop-in plas-
tics are produced from renewable feedstock such as biomaterials but have iden-
tical properties to their fossil-based counterparts [37]. The best example of drop-in
bioplastics is bio-based polyethylene made from ethylene monomers produced from
bioethanol [13, 169]. Corn, sugarcane, and sugar beet are employed as feedstocks for
fermentation by microorganisms and bioethanol production [13]. Drop-in polymers
produced from entirely bio-based materials are nonbiodegradable [37].
The most common commodity polymers currently utilized are nonbiodegradable
or have a prolonged biodegradation rate [3]. Different methods have been used for
developing and industrializing biodegradable plastics [88]. One of these methods
is adding some additives, so-called prodegradant or pro-oxidant, to the conven-
tional nonbiodegradable plastics, e.g., polyethylene, polypropylene, and polyethy-
lene terephthalate, to enhance the oxidation process [88]. These pro-oxidants are
transition metal ion complexes catalyzing plastics oxidation [135]. Abiotic acceler-
ated oxidation and the plastic breakdown cause easier fragmentation and bioassimi-
lation of the oligomer/dimer/monomer molecules released by extracellular enzyme
digestion [9]. It is noteworthy that nonbiodegradability does not mean this kind does
not degrade at all. But the breakdown rate of these plastics is prolonged [90].
4.3 Bio-based or Fossil-Based Biodegradable Plastics
As stated, biodegradable plastics via microbial degradation are broken down and
produce CO2 , CH4 , H2 O, and biomass, i.e., microorganism growth and multiplica-
tion. The molecular structure of the polymers is a crucial factor in their biodegrad-
ability. Therefore, the feedstocks and methods of plastic production necessarily do
not determine their biodegradability. Among bio-based plastics, which are defined as
“plastics produced from biomass”, there are examples of nonbiodegradable plastics
such as bio-polyethylene. Petrochemical or fossil-based plastics can be biodegradable
such as poly(caprolactone) and poly(hydroxybutyrate). It is noteworthy that the word
“bioplastics” is not equal to bio-based plastics and is incorrectly confused with them.
Bioplastics are a large family of plastics, including bio-based and biodegradable
plastics [63, 173]. Figure 4 depicts the bioplastics category and its subgroups.
Bio-based biodegradable plastics are consumed by microorganisms [3]. Poly-
hydroxyalkanoates (PHAs), Polylactic acid (PLA), cellulose, starch, and starch-
based polymers are examples of this group [3]. A group of biodegradable plastics is
starch-based plastics that are complex blends of starch with fossil-based compostable
polyesters such as polylactic acid, poly(caprolactone), polyhydroxyalkanoates, poly-
butylene succinate, and Polybutylene adipate terephthalate. Some additives such as
compatibilizers and plasticizers are added for improving the blending products’ prop-
erties [176]. Examples of these plastics and their chemical structure are provided in
Table 2 and Fig. 5.
Monomers in polyesters are linked via ester bonds, and hydrolysis of these link-
ages is easy. Diversity and abundance of esters in nature are High, and esterases as
esters’ degrading enzymes exist in living organisms [160]. Polyhydroxyalkanoates
are bacterial polyesters and are biodegradable. Polyhydroxybutyrate (PHB), a kind of
bacterial Polyhydroxyalkanoates, and other Polyhydroxyalkanoate copolymers are
Fig. 4 Some examples of

two subgroups of bioplastics
and their interfaces. PBS:
polybutylene succinate;
PCL: poly(caprolactone);
PES: Polyethersulfone; PHB:
poly(hydroxybutyrate); PLA:
polylactic acid; AcC: acetyl
cellulose; NY11: Nylon 11;
PE: polyethylene. The figure
is reproduced from [173]
Fig. 5 Chemical structure of some biodegradable and nonbiodegradable plastics

produced and utilized to make biodegradable plastics [160]. Polylactic acid, a bio-
based polymer, due to its biodegradability in animal and human bodies, has found
many medical applications. Although limited polylactic acid-degrading microorgan-
isms are discovered in the environment, polylactic acid is easily decomposed and
mineralized via composting [160].
Microorganisms can degrade polycaprolactone, which is a synthetic polyester.
The environmental distribution of polycaprolactone-degrading organisms, including
bacteria and some fungi, is wide [160]. Polyvinyl alcohol is a vinyl polymer and
water-soluble with thermoplastic properties. Polyvinyl alcohol is moldable, and its
solubility and biodegradability make it a substitute for starch in industrial proce-
dures. A number of microorganisms are identified that degrade polyvinyl alcohol,
and different enzymes involved in polyvinyl alcohol backbone-cleavage are reported
[160].
During plastic manufacturing, some additives such as pigments, oxidation
inhibitors, lubricants, and fillers are added to reach required mechanical, chemical,
and physical properties [63]. In addition to plastic polymers’ degradation by microor-
ganisms, these additives made concerns about plastics’ biodegradability. For being
biodegradable, all additional ingredients must be biodegradable [63].
5 Microplastics Types
Widespread usage of plastics from the kitchen to industry and the only once usage
culture of the plastics besides the linear economy of “take, make, and disposal”
in which recycling does not make a significant contribution gave rise to producing
a massive volume of plastic waste every year [113, 152]. Incineration, recycling,
road construction, petrol production, landfilling, and bioremediation are different
techniques for managing plastic waste [152]. In the USA, as a developed economy,
only 10% of the plastic waste is recycled, 25% is incinerated, and 65% is landfilled
[142]. The cost and the low quality of recycled plastics are two limiting factors for
plastics recycling [113]. A considerable percent of municipal waste is plastic waste
that, due to mismanagement, finds its way to the aquatic environments such as rivers,
lakes, seas, and oceans [152].
Among hundreds of commercially available plastics, only a few are used in high
volume and produced with relatively low prices, considered commodity plastics.
Polypropylene, polyvinyl chloride, polystyrene, low-density polyethylene, high-
density polyethylene, polyethylene, polyurethane, and polyethylene terephthalate
are nearly 80% of plastics demand worldwide [12]. Most of these plastics are
nonbiodegradable, or their degradable rate is very slow, so their degradation takes
decades [151]. Many of these plastics, such as polyethylene, polyethylene terephtha-
late, polypropylene, and polystyrene, are commonly used in packaging, so they have
a short lifetime and are disposed of after a single use [113]. Though bio-based plastic
production is a very dynamically developing area, their manufacturing’s high cost
prevents them from competing with conventional plastics [169]. Additionally, oxo-
degradable plastics containing additives for accelerating the oxidation process and
facilitating biodegradation are considered biodegradable, and literally don’t degrade
rapidly. Due to accelerated oxidation under outdoor conditions, oxo-degradable plas-
tics’ fragmentation creates tiny fragmented plastics that take a very long time for
complete biodegradation [88].
In the 1970s and 1980s, scientific literature reports small plastic pieces floating
on the ocean’s surface [55]. Ryan and Moloney in 1990 and Thompson in 2004
described plastic fragments and their distributions in South African beaches and
seawater, respectively [139, 171]. In 2008, 5 mm was proposed as an upper limit
for defining the microplastics [108]. Based on their origin, Cole et al. categorized
microplastics into primary and secondary microplastics [30]. According to the defi-
nition of the Joint Group of Experts on the Scientific Aspects of Marine Environ-
mental Protection (GESAMP), microplastics are “plastic particles with less than
5 mm in diameter, which include particles in nano-size range (1 nm)” [55]. These
tiny granules, fragments, and fibers have contaminated marine environments [30].
Primary microplastics are plastic particles manufactured in a microscopic size
that are commonly employed in cosmetics, facial cleansers, toothpaste, air-blasting
media, and as vectors for drugs [30, 39]. Primary microplastics used as exfoliants
in personal care products are polyethylene, polypropylene, and polystyrene [39].
Industrial abrasives used in air-blasting or sandblasting include acrylic, polystyrene,
melamine, thermoset polyester polymer, poly(allyl diglycol carbonate), amino ther-
moset plastic, and urea [43]. Secondary microplastics are tiny plastic fragments
generated via the breakdown of larger plastics [30].
6 Microplastics Toxicity on Living Organisms
Plastics have improved human health by manufacturing different types of medical

equipment, predominantly lightweight and disposable ones. They are widely used in
packaging, especially food packaging that increases food safety and shelf life [129].
Besides these plastics advantages and many others for modern life, there are many
concerns about the future of plastic production and usage. The increasing rate of
plastic production is high, and the produced plastics are estimated to reach two-fold
by the next 20 years [91]. Estimates suggest that by the year 2050, plastic production
and processing are considered reasons for 20% of the consumed petroleum globally
and 14% of the carbon dioxide released into the atmosphere [49].
Approximately, 10% of 280 million tons of global plastic production enter the
ocean annually, accounting for 60–80% of marine debris [77, 170]. The garbage
produced from terrestrial sources are distributed worldwide on shorelines and in the
ocean and increase every year [202]. World Economic Forum and several newspapers
reported that by this rate of plastic pollution, by 2050, more plastics will exist in the
oceans than fish [180]. Primary and secondary microplastics are available in all
marine environments and influence many marine organisms [202]. Microplastics
can be found in samples taken from different settings and geographical areas such
as wastewater, freshwater, marine water, sediments, and even mountain lakes and
polar regions [180]. Microplastics, through different ways, enter the food chain and
transfer along with the trophic level. The food chain interactions can form a food web
and increase the complexity of the organisms’ relationships [180]. Microplastics can
act as vectors for entering other contaminants and toxic compounds and materials
into a marine food web. Therefore, microplastics can create more dangerous effects
in the marine environment [130].
6.1 Microplastics Toxicity on Microalgae
Many studies report the microplastics effects on marine animals from benthic grazer
[123], sea urchins [77], mussels [18], crabs [46], and fishes [21, 104, 122, 181, 183].
But fewer reports on the toxic effects of microplastics on algae, especially microalgae,
were performed. As primary producers in aquatic ecosystems, microalgae do photo-
synthesis, produce organic carbon as an energy source, and release oxygen gas in
the marine environment [99]. Therefore, they have pivotal roles in ecosystems. It is
noteworthy that macroalgae also play essential roles in ecosystems.
The impact of microplastics on micro- and macroalgae was studied from different
aspects. Studies have shown that microplastics can inhibit microalgae’s growth rate,
decrease photosynthesis, increase oxidative stress and gene expression [99, 165, 166,
189]. The impacts of the size, the type of microplastic polymers, surface charge,
exposure time, and concentration of the microplastics were investigated. Besides,
dissolved organic compounds that can cover the microplastics’ surfaces were reported
as an important factor in decreasing microplastics toxicity [99].
Although the mechanisms underlying the microplastics toxicity for microalgae
are not fully understood, some studies were reported in the literature. In the case
of small-sized microplastics, their adsorption on the microalgae can damage the
cell walls, impair cell wall integrity, and ultimately lead to cell death. However,
the shading effect is proposed as the primary reason for toxicity of large-sized
microplastics that decreases photosynthesis. Because microplastics acts as a phys-
ical barrier against light and nutrient transportation and prevents the release of waste
material [99]. Additives leaching from microplastics is another influential agent on
microplastics’ harmfulness [165, 166]. Other environmental pollutants such as persis-
tent organic pollutants and heavy metals can adsorb on microplastics and transfer by
them [96, 97].
6.2 Microplastics Toxicity on Animals and Humans
Microplastics, based on their size, shape, density, and interaction with microorgan-
isms and biofilm formation on their surfaces, can float on the water surface, disperse
in different depths of the water column, or deposit on the sediments [75, 93, 116,
182]. Also, microplastics, because of their similar size and appearance to plank-
tons, are mistaken and consumed as food. A wide range of marine biotas such as
corals, phytoplanktons, zooplanktons, sea urchins, lobsters, shellfish, fish, and even
whales ingest microplastics [29]. Aquatic birds and higher animals via direct or
indirect microplastics consumption are undergone by adverse effects [29]. Ingested
microplastics can give rise to accumulation in the gut of the animals, physical damage
to digestive tracts, oxidative stress, enzyme production alteration, and metabolism
changes [182]. Disintegration into more tiny particles causes their penetration into
the circulatory system and phagocytic cells. Thus, it prolongs microplastics’ resi-
dence in the exposed organisms, increasing the potential harms and helping their
transfer to higher trophic animals [182].
Humans are the final consumers in the aquatic food web, so microplastics can
also contaminate humans. Although some organisms’ digestive tracts containing
the highest amount of microplastics are removed before human consumption, many
kinds of seafood are eaten whole that are reported to carry lots of microplastics
[182]. Additionally, destroyed smaller microplastics with penetration capability into
aquatic organisms’ circulatory systems can introduce into the human body via food
consumption [182].
7 Antibiotic Resistance and Microplastics
Intensive and uncontrolled antibiotic use gives rise to the survival and prolifer-
ation of bacterial cells resistant to single or multi-antibiotics. The evolution of
antibiotic-resistant bacteria and multidrug-resistant bacteria has become a threat to
human health [71, 198]. The discharge of antibiotics into the natural environment
from different sources such as livestock farms, aquaculture systems, pharmaceutical
industry wastes, hospital wastes, and urban wastewater has polluted the groundwa-
ters and other aquatic environments [71, 101]. Due to microplastics’ ecological and
physiological effects, some researchers studied the temporal and spatial distribution
and abundance value in marine environments. The abundance value per volume at
the sea surface, in the water column, and the sediment samples, ranged from 0.022 to
8654, 0.014 to 12.51, and 185 to 80,000 items m−3 , respectively [64]. Adsorption of
pollutants such as polycyclic aromatic hydrocarbons, persistent organic pollutants,
antibiotics, and heavy metals on microplastics and the related toxicological risks on
organisms are under investigation [61, 95, 112]. These pollutants influence microbial
communities colonized the microplastic surface or so-called plastisphere. Research
showed that the plastisphere microbial communities are different from the aquatic
environment encompassing microplastics [181, 183, 190, 201].
There are some reports on the enrichment of antibiotic resistance bacteria and
bacterial pathogens on the microplastic surface [16, 133, 165, 166, 188]. Kirstein
and co-workers collected microplastics and water samples from the North and Baltic
Seas. Due to Vibrio species’ previously reported presence on microplastics, this group
performed selective enrichment experiments for bacterial identification [86, 201].

They used reflectance Fourier transform infrared spectroscopy for plastic polymer
identification. Vibrio bacteria isolation was performed by a specific chromogenic
media and via a kind of mass spectrometry, the species of Vibrio bacteria was assigned
to Vibrio parahaemolyticus. The potentially pathogenic Vibrio species’ presence on
polyethylene, polypropylene, and polystyrene microplastics highlighted the possible
hazardous effect of microplastics on health [86]. Microplastics can act as a vector
for microorganisms’ dispersal.
Li et al. studied five antibiotics’ adsorption, including sulfadiazine, amoxi-
cillin, tetracycline, ciprofloxacin, and trimethoprim, on five types of microplastics,
polyethylene, polystyrene, polypropylene, polyamide, and polyvinyl chloride. They
observed polyamide microplastics have the most remarkable adsorption capacity.
The adsorption capacity attenuated in this decreasing order: ciprofloxacin, amoxi-
cillin, trimethoprim, trimethoprim, and tetracycline, while the first had the highest
adsorption [95]. The salinity of the water and the hydrophilicity of the microplastics
were influential factors in the adsorption. Adsorption increased in freshwater, and
hydrophilic antibiotics were adsorbed to polyamide with higher affinity [61, 95].
Different mechanisms are responsible for antibiotic resistance. This resistance
in bacteria results from genetic factors known as antibiotic resistance genes. These
genetic factors include plasmids, transposons, and integrons present in bacteria [71].
These genetic factors transfer among bacteria via self-replication and horizontal gene
transfer [198]. Microplastics as a new habitat for organisms, especially bacteria with
a high density of cells and nutrients, make possible intense interactions between
microbial cells. This niche is regarded as a hot spot for horizontal gene transfer.
Plasmid transfer via conjugation, a direct connection between two bacteria, is a
way for horizontal gene transfer [16]. Arias-Andres and co-workers demonstrated
that bacterial plasmid transfer frequency is higher in microplastic-resident bacteria
than free-living or natural-surface residents. The genetic material exchange was not
limited to closely related bacteria but also happened in phylogenetically diverse
bacteria [16].
Yang et al. used the metagenomics database of the National Center for Biotech-
nology Information, which contains seawater, macroplastics, and microplastics
microbial communities of North Pacific Gyre. They investigated the abundance,
diversity, and co-occurrence of antibiotic resistance genes and metal resistance genes
in microbial communities between macroplastics, microplastics, and surrounding
aquatic environments. Microbial communities of microplastics and macroplastics
had no significant differences. Metal resistance gene abundance was higher than
antibiotic resistance genes in plastics microbiota. Antibiotic resistance genes were
related to microbial community composition. The average relative abundance was
the highest for bacterial multidrug resistance genes, followed by aminoglycoside
resistance genes and unclassified antibiotic resistance genes [193].
Lu et al. studied microplastics and antibiotic resistance gene abundances in a
recirculating aquaculture system. The abundance of microplastics in the aquaculture
system was 58–72 items m−3 . Total ten antibiotic resistance genes’ abundances were
different on microplastic samples than in water samples. Also, microbial diversity at

phylum and genus levels was higher on microplastics than in water [101].
8 Microorganisms and Microplastics Interaction

and Biofilm Formation
Microplastics are ubiquitous worldwide and accumulate in aquatic environments

such as rivers, lakes, seas, and oceans [194]. Microplastics can interact with and
adsorb organic matters and nutrients and form a conditioning film for the micro-
bial organisms’ attachment and colonization [117]. Microorganisms attached to the
microplastic surface protect themselves from environmental stress, have access to
organic matters and nutrients, and increase their dispersal opportunities [33, 117]. The
hydrophobic character of microplastics helps the biofilm formation [201]. Biofilms
are communities of bacteria and other microbial cells such as cyanobacteria, protists,
fungi, algae, and even viruses attached to the surface and surrounded by extracel-
lular polymeric substances [70, 146, 197]. The thin and three-dimensional layer of the
biopolymers and microorganisms on the microplastics’ surface is called plastisphere
[201].
Biofilm formation is not limited to natural environments [145]. The adaptive
capability of microorganisms, especially bacteria, permits constructing these sessile
communities, biofilm, in even unnatural systems such as water pipes [145]. Biofilm
formation on microparticles affects the particles’ physical properties such as weight,
size, crystallinity, and buoyancy, leading to change in density and accelerating the
particles’ sedimentation. Therefore, biofilm helps in the particles’ vertical disper-
sion, while physical forces such as currents, waves, and wind lead to the horizontal
distribution of the particles [26, 117]. Therefore, microparticles act as a vector in
aquatic systems [117].
The formation of biofilms on microplastics is studied under various environmental
conditions and various microplastic characteristics [194]. The biofilms’ micro-
bial community’s composition is studied in different locations, temporal succes-
sion processes, and even in different seasons [8, 194]. The bacterial community
of microplastic biofilms is investigated vastly, and the main detected phyla are
Proteobacteria, Firmicutes, and Bacteroidetes [194]. The main concern of microor-
ganisms detected in biofilms is pathogenic bacteria and fungi and opportunistic
pathogens [194].
The high density of cells and nutrients in biofilms leads to intense interactions
[16]. So, biofilms are potentially regarded as a hot spot for horizontal gene transfer
(HGT) via mobile genetic elements such as plasmids, transposons, and integrons, and
bacteriophages cause another concern about bacterial biofilms [40, 167]. Through
conjugation between two spatially close bacteria, genetic information can exchange
by plasmid transfer [38]. This genetic exchange can affect bacterial evolution and
cause the spreading of unwanted traits such as antibiotic and heavy metal resistance
genes [16, 27].
By limiting exposure to oxygen, UV light, shear stress, and high temperature,
biofilm creation decreases microplastic abiotic aging. However, microorganisms’
colonization on the microplastic substrates influences their physical and chem-
ical properties to damage the substrate structure [137]. Biofilm microorganisms
exerted biodeterioration and biodegradation through multiple phases. Microorgan-
isms’ attachment and growth, degradation of plasticizers, surface corrosion by
released byproduct metabolites and degrading exoenzymes, hydration, and even
penetration are these phases [48, 137].
9 Biodegradation of Microplastics
The degradation of macro- and microplastics in aquatic mediums starts with nature’s
physical and chemical factors reducing their hydrophobicity. The abiotic degrada-
tions are due to outdoor exposure factors, including physical stress of waves, UV light
or photodegradation, oxygen and heat or thermooxidation, heat or thermal degrada-
tion, and moisture and water or hydrolysis [11, 51, 53]. Due to its eco-friendly
nature, biotic degradation is another way that has attracted much attention in the last
decade. ASTM standard D-5488-94d considers biodegradation as a “process which
is capable of decomposition of materials into carbon dioxide, methane, water, inor-
ganic compounds, or biomass in which the predominant mechanism is the enzymatic
action of microorganisms that can be measured by standard tests, in a specified period
of time, reflecting available disposal conditions” [161].
The first step of biodegradation is plastisphere formation. Microorganisms secrete
extracellular polysaccharides with adhesion properties for more robust biofilm attach-
ment to the microplastics. This step develops rapidly and reduces the plastic buoyancy
and its surface hydrophobicity [100, 164]. The second step or biodeterioration is the
consequence of biofilm formation. Microorganisms secrete biomolecules such as free
radicals, polysaccharides, and degrading enzymes, deteriorating the polymeric parti-
cles. Biodeterioration modifies the physical, mechanical, and chemical properties of
the particles [42, 56, 102].
In the third step, biofragmentation or depolymerization, microbial attack by
degrading enzymes or agents such as free radicals destabilizes the polymer carbon
skeleton [51, 56]. Destabilization leads to backbone breakdown and releases
oligomers, dimers, and monomers, and decreases the polymers’ average molecular
weight [51, 161]. Some of the released molecules bind the microbial cells’ recep-
tors and enter the cells. In the next step, during the assimilation, the internalized
molecules, which have less than 600 Daltons, finally integrate the cell metabolism
and are consumed for producing energy, biomass, and microbial metabolites [73,
102]. In the last step, the integrated molecules of biodegraded polymers eventu-
ally, through microorganisms’ metabolic activity, convert to H2 O, CO2 under suffi-
cient oxygen pressure or so-called aerobic condition, while CH4 , CO2, and H2 O are
produced under anaerobic conditions [3, 151]. Indeed, mineralization which is the
conversion of organic molecules to inorganic molecules rarely reaches 100%. As
mentioned above, a small percentage of the organic molecules is used for microbial
biomass production [151].
In this section, we present some examples of different plastics degraded by three
types of microorganisms. Bacteria and fungi are the most important microorganisms
used so far for the biodegradation of plastics. In selecting the examples, we paid
attention to the aquatic origin of the microorganisms. In Table 3, the reviewed papers
are summarized.
9.1 Biodegradation by Bacteria and Bacteria Consortium
Bacteria are necessary inhabitants of the earth. They play essential roles in all ecosys-
tems and reside in all habitats. Bacteria are involved in many vital activities such
as nitrogen fixation, methane oxidation, phosphate solubilization, and are pivotal
players in most catalytic and crucial transformations [195]. Billion-year exposure of
microorganisms and especially bacteria to a wide variety of organic materials and
molecules has given rise to the evolution of different enzymes with transformation
capability that helps them use organic compounds as carbon and energy sources
[14]. Their powers in the degradation of various natural and synthetic materials such
as natural polymers, antibiotics, organic pollutants, petroleum, metal and organic
compounds, and plastic polymers have attracted much attention for bioremedia-
tion. The plastic degrading ability has been reported for different bacterial species
from bacillus, escherichia, and pseudomonas genera isolated from diverse ecological
niches [10].
Usually, for better characterization and avoiding the complexity in interpreting
the results, a single isolated bacterial culture is utilized in biodegradation studies.
However, due to some limitations of pure bacterial cultures in plastic degradation,
researchers have employed stable microbial communities containing various bacteria
with unique metabolical and physiological capabilities in increasing biodegradation.
Because bacteria have symbiotic and synergistic interactions, this community or
so-called bacterial consortium helps the bacterial growth, increased tolerance, and
biodegradation capability [197]. In the following, we present some examples of
bacterial and fungal species degrading a plastic polymer type.
Polyethylenes
Polyethylene is the most widespread thermoplastic polymer with several applica-
tions in different areas of human life. Low-density polyethylene or LDPE and high-
density polyethylene or HDPE are two forms of polyethylene with various features.
Because of the lack of sensitive chemical groups to degradation in polyethylene,
its high molecular weight, hydrophobic property, and C–C bonds of its molecular
backbone, polyethylene is a very stable biodegradation-resistant plastic polymer.
However, the published research on polyethylene biodegradation show that it is more
Table 3 Some examples of microorganisms with plastic polymer degradation ability
Bacteria
No. Bacteria name Efficient enzyme(s) Microplastics/Plastics Origin of the isolated Special condition References
name strain
1 Bacillus sp. 10 and 40-micron Hospital, Petrol pump, [162]
Staphylococcus sp. polythene (PE) Local area
Pseudomonas sp.
2 Microbulbifer Copper-dependent Low-density polyethylene Wastewater discharged Marine bacterium [96, 97]
hydrolyticus IRE-31 enzymes (LDPE) from a lignin-rich pulp
mill
3 Rhodococcus ruber Polyethylene (PE) Burial site of [119]
(C208), an polyethylene-waste from
actinomycete agricultural use (mainly
films for soil mulching)
4 Pseudomonas Laccase and esterase Di(2-ethylhexyl) phthalate Petrochemical factory [47]
fluorescens FS1 (DEHP), a plasticizer; sludge
Bioremediation Techniques for Microplastics Removal
phthalate esters
5 Bacterial consortium Polyvinyl chloride (PVC) Plastic waste disposal [15]
comprising: sites of North India
Pseudomonas otitidis
strain SPT1, Bacillus
aerius strain SPT2,
Acanthopleuribacter
pedis strain SPT3, and
Bacillus cereus strains
SPK1 strains
(continued)
347
Table 3 (continued)
348
Bacteria
name strain
6 Rhodococcus ruber, an Polystyrene (PS) Previously isolated from [109]
actinomycete burial site of
polyethylene-waste
7 Serratia sp. WSW Unknown PS oxidative Polystyrene (PS) Among the gut flora of [187]
(KCTC 82,146) enzymes (e.g., lignin the larva Plesiophthalmus
peroxidase) davidis
8 Ideonella sakaiensis Extracellular PETase; Polyethylene terephthalate Natural environment [196]
201-F6 strain MHETase, a predicted (PET) exposed to PET
lipoprotein, hydrolyzes
MHET
9 Arthrobacter Polyurethane (PU) [41]
globiformis,
Acinetobacter
calcoaceticus
10 Bacillus subtilis Polyurethane (PU) A soil bacterium [136]
11 Comamonas Esterase secreted in the Polyester-polyurethane [159]
acidovorans culture broth (CBS (PU) and poly(diethylene
esterase), cell-bound glycol adipate), the soft
esterase (PU esterase) segment of the PUR
12 Escherichia and PETase Polyethylene terephthalate – Recombinant strain [158]
Bacillus (PET)
(continued)
S. Hadian-Ghazvini et al.
Table 3 (continued)
Bacteria
name strain
13 Bacillus subtilis 168 PETase Polyethylene terephthalate Recombinant strain, use [67]
(PET) of B. subtilis’s native
signal peptide SPPETase
for secretion
14 Clostridium Cutinase Polyethylene terephthalate – Recombinant strain [191]
thermocellum (PET)
15 Brevibacillus spp. And Protease Polyethylene (PE) – – [82]
Bacilluss spp.
Fungi
No. Fungi name Efficient enzyme(s) Microplastics/Plastics Origin of the isolated Special condition References
name strain
1 Aspergillus versicolor, – Low-density polyethylene Seawater sample [127]

Aspergillus sp. (LDPE) in powder form
2 Bjerkandera adusta Laccase High-density polyethylene Ohgap Mountains, South Under lignocellulose [76]
TBB-03 (HDPE) Korea substrate treatment
3 Aspergillus flavus Laccase-like Polyethylene The gut contents of wax – [203]
multicopper oxidases microplastics moth Galleria mellonella
4 Phanerochaete manganese peroxidase Polyethylenes (PEs) – – [69]
chrysosporium
5 Zalerion maritimum – Microplastic Marine environment – [121]
Polyethylenes (PEs) (Portuguese coastal
waters)
(continued)
349
Table 3 (continued)
350
Bacteria
name strain
6 Chaetomium globosum – Polyvinyl chloride (PVC) – – [178]
(ATCC 16,021) poly (ε-caprolactone)
(PCL)
7 Aureobasidium – Plasticized Polyvinyl – [185]
pullulans chloride (pPVC) film
8 Penicillium – Polyvinyl chloride (PVC) Isolated from 10-month – [140]
janthinellum pPVC buried in soil,
Bulgaria
9 Phanerochaete Polyvinyl chloride (PVC) PVC thin films buried in – [5]
chrysosporium sewage sludge-soaked soil
10 Aspergillus japonicus, – Di 2-ethylhexyl phthalate Plastics-contaminated soil – [126]
Penicillium brocae and as plasticizer of Polyvinyl
Purpureocillium chloride (PVC)
lilacinum
11 Trichoderma viride – Poly lactic acid – – [98]
12 Tritirachium Album Likely protease Poly lactic acid [74]
ATCC 22,563
13 Ascomycota, – Polyester Polyurethane Compost – [200]
Basidiomycota and (PU)
Zygomycota
14 Fusarium solani, – Polyurethane Compost – [199]
Candida ethanolica
(continued)
S. Hadian-Ghazvini et al.
Table 3 (continued)
Bacteria
name strain
15 Pleurotus ostreatus – Disposable diapers – – [44]
cellulose as the main
component
16 Basidiomycotina, PHAs depolymerases Polyhydroxyalkanoates Marine environment – [84]
Deuteromycotina, and (PHAs)
Ascomycotina
17 Phanerocheate Lignin peroxidase Polyvinyl chloride (PVC) A site with long [83]
chrysosporium time-dumped waste
plastic and wood material
18 Fusarium culmorum Laccase di(2-ethyl hexyl) phthalate – – [4]
Esterase (DEHP)
19 Pestalotiopsis Serine hydrolase polyester polyurethane Woody plants of various [138]
microspor E2712A (PU) families

Algae
No. Algae name Efficient enzyme(s) Microplastics/Plastics Origin of the isolated Special condition References
name strain
1 Chlamydomonas Polyethylene Polyethylene terephthalate – Recombinant strain [85]
reinhardtii terephthalate (PET)
2 Phaeodactylum Polyethylene Polyethylene terephthalate Saltwater Recombinant strain [107]
tricornutum terephthalate (PET)
351
vulnerable than other hydrocarbon polymers [87]. From 15 isolated bacteria and after
two screening steps, Singh et al. reported Bacillus sp. has the maximum capability
in polyethylene degradation [162]. Li and co-workers using a strain isolated from
marine pulp mill waste investigated the biodegradation of low-density polyethy-
lene. The natural residence of the isolated Microbulbifer hydrolyticus IRE-31 was
rich in lignin, a complex biopolymer similar to polyethylene, with C–C bonds
in its backbone. Therefore, the ability of the bacterium in LDPE-biodegradation
was to be expected. 30-day cultivation of the IRE-31 bacterium gave rise to
evidence that clearly showed biodegradation of low-density polyethylene [96, 97].
Actinomycetes are filamentous Gram-positive bacteria capable of biodegrada-
tion of recalcitrant biopolymers widely distributed in aquatic and terrestrial ecosys-
tems. With various metabolic and physiological properties, such as extracellular
enzymes, they have an indispensable role in decomposing complex biopolymers of
dead organisms, especially in soil [154, 155]. They have been employed for biotech-
nological applications, for instance, in bioremediation. Gilan (Orr) et al. isolated a
strain of Rhodococcus ruber C208, a kind of actinomycete that formed a biofilm on
polyethylene surface and utilized polyethylene as the sole carbon source [119].
Polyvinyl chloride
Polyvinyl chloride has a broad spectrum of applications in industries, but its
nonbiodegradability creates some issues for its commercial feasibilities [35]. There
are limited reports on the plasticized polyvinyl chloride biodegradation, but Pseu-
domonas aeruginosa and fungi such as Aureobasidium pullulans, Rhodotorula
aurantiaca, and Kluyveromyces spp. were important in colonization and biodeteriora-
tion [35, 185]. Di(2-ethylhexyl)phthalate (DEHP) as an essential additive is used for
polyvinyl chloride and other resin flexibility which also act as a plasticizer. Feng and
co-workers reported Pseudomonas fluorescens FS1 isolated from a petrochemical
factory sludge could consume phthalate esters as the sole energy and carbon source
[47]. For effective degradation of polyvinyl chloride, Anwar et al. used two bacte-
rial consortia, different combinations of bacterial species of Pseudomonas otitidis,
Bacillus cereus, and Acanthopleurobacter pedis. These bacteria were isolated from
a disposal site in North India. The results showed that the consortium-II comprising
four different strains had a superior degradation function than the consortium-I
containing only two strains [15].
Polystyrene
Polystyrene (PS) is a highly consumed thermoplastics and is regarded as
nonbiodegradable. Mor and Sivan monitored the kinetics of biofilm formation on
PS by Rhodococcus ruber, an actinomycete, and reported the partial biodegradation
of PS [109]. Woo et al. have shown the biodegradation of polystyrene in the form of
Styrofoam by larva Plesiophthalmus davidis. The larva ingested 34.27 ± 4.04 mg
of the Styrofoam and survived after 14 days of feeding with the Styrofoam as the
only food. Among the gut flora of the larva, the isolated Serratia sp. WSW (KCTC
82,146) was regarded as the principal biodegrading bacterium. Because the degrada-
tion by the Serratia sp. WSW was less than the flora of the larva gut, it was proposed
that the whole flora of the gut is more efficient than a single bacterium. Due to the
similarity between PS and lignin and cellulose, which are the foods of wood-boring
insects, these animals, and their gut flora can be promising solutions for the world’s
plastic crisis [187].
Polyethylene terephthalate
Polyethylene terephthalate or PET is a synthetic thermoplastic polyester manufac-
tured massively for the packaging and production of synthetic fibers [191]. There are
only a few studies that have reported the biodegradation of polyethylene terephtha-
late, which is degraded by filamentous fungi, Fusarium oxysporum, and F. solani.
Therefore, Yoshida et al., by screening the microbial communities collected from
the natural environment exposed to polyethylene terephthalate, isolated a novel
bacteria that consumes polyethylene terephthalate as the primary carbon and energy
source. Ideonella sakaiensis 201-F6 strain via two hydrolyzing enzymes degraded
and completely assimilated amorphous polyethylene terephthalate. The PETase
enzyme produces mono-(2-hydroxyethyl) terephthalic acid (MHET), TPA, and bis-
2(hydroxyethyl) TPA (BHET) [196]. Han et al. studied catalytic mechanisms of the
PETase [62]. Austin and co-workers engineered this enzyme for improving degrada-
tion and reported this engineered enzyme could degrade a polyethylene terephthalate
replacement [17].
Polyurethane
Unlike popular polymers such as polypropylene or polyethylene, polyurethanes have
heterogeneous structures, but the building block of most polyurethanes is poly-
isocyanates. According to their components, polyurethanes are often classified as
thermoset polymers, but thermoplastic polyurethanes are also produced [87]. They
are versatile polymers for industrial applications employed for thermal insulation,
resistance, conductance, and elasticity. Due to their increasing annual usage and non-
recyclable nature, polyurethane wastes are growing every year [34, 54, 80]. Polyester
polyurethanes are much more biodegradable than polyether polyurethanes. For
biodegradation of polyether polyurethanes, fungal strains exhibited more promising
results, while for biodegradation of polyurethane coating, bacteria species were
mainly employed [80]. Shigeno-Akutsu and co-workers showed that Comamonas
acidovorans using two esterases could degrade the polyurethane. The cell-bound
esterase could degrade polyurethane and its soft segment, poly(diethylene glycol
adipate), while the esterase secreted in the culture broth, the CBS esterase, degraded
only poly(diethylene glycol adipate) [159]. El-Seyed et al. studied the ability of
nine bacterial strains for polyurethane degradation. Arthrobacter globiformis and
Acinetobacter calcoaceticus exhibited the most degradation ability than other species
including Pseudomonas cepacia and Pseudomonas sp. [41]. Bacillus subtilis, a soil
bacterium, was the degrading bacteria in Rowe and co-workers’ study [136]. Graytan
et al. to investigate the degradative ability of a mixed microbial culture used a water
polyurethane dispersion as the culture medium for microbial growth. Polyurethane
dispersion comprising a polyether-polyurethane-acrylate copolymer and xenobiotic
additives including glycol ethers, isopropanol, and N-methylpyrrolidone was the
sole carbon and energy source for the microorganisms. Different techniques such as
nuclear magnetic resonance, mass spectrometry analysis, Fourier-transform infrared
spectroscopy analyses, derivative thermogravimetric analyses, differential scanning
calorimetry, gel permeation chromatography, and proximity ligation-based metage-
nomic analysis were employed. The aim of the research was to perform an inte-
grative study for determining the metabolic pathways involved in the decomposi-
tion of xenobiotics and metagenomics analysis of the selected microbial community
decomposing polyurethane. The founded microorganisms and the over expression
of degrading enzymes and proteins are promising data for creating biotechnological
strategies to diminish the plastic contaminants and other pollutants [54].
It is noteworthy that plastic pollutants are used as the sole carbon and energy
source in many studies. However, some researchers for improving the decomposition
process utilized additional carbon sources. Studies exhibited that a supplementary
carbon source raised the aquatic environment pollutants’ removal rate [204]. Shabir
et al., based on the previous reports and in a relatively innovative approach, investi-
gated three microplastic biodegradation by periphytic biofilm in three carbon-source
backgrounds [150]. Periphyton biofilms are phototrophic, multi-species groups of
organisms including protozoans, metazoans, algae, fungi, bacteria, and even viruses
entangled by secreted extracellular polymeric substances [22, 141]. Shabir et al.
showed that the addition of glucose increased the periphyton biofilm biodegradation
of three structurally different microplastics, including polypropylene, polystyrene,
and polyethylene terephthalate [150].
9.2 Biodegradation by Fungi
Recently, the capability of several microorganisms and enzymes in degrading plastic

has been studied. Aerobic and anaerobic microorganisms in nature take part in the
biodegradation process. In this part, we have summarized the fungi that have been
proven to be capable of degrading plastics [84].
Fungi are eukaryotic, heterotrophic organisms which are morphologically clas-
sified into three groups: filamentous fungi, dimorphic fungi, and yeast. Among
them, filamentous fungi can adapt to changing environments and digest various
environmental pollutants. They catalyze chemical reactions by secreting digestive
enzymes and breaking down organic molecules. Absorbent nutrients from outside are
converted to CO2 , H2 O, and CH4 by fungi under aerobic and anaerobic conditions,
respectively. Generally, biodegradable materials contain linkages in the backbone
of the polymer, which are vulnerable to fungi attack. Several genera of fungi like
Trichoderma, Aspergillus, Pacilomyces, Fusarium, Penicillium, and Phanerochate
have shown plastic degradation activity and can mitigate the environmental impact
of plastics [178].
Polyethylenes
Due to chemical inertness, hydrophobicity, and the lack of functional groups,
polyethylenes are resistant to biodegradation; however, several studies have reported
the biodegradation of polyethylenes by fungi. Pramila and Ramesh have assessed the
ability of isolated Aspergillus versicolor and Aspergillus sp. from seawater for degra-
dation of low-density polyethylene in powder form [127]. High-density polyethy-
lene is a nonbiodegradable plastic polymer, but researchers hope that fungi with
delignifying enzymes help its decomposition. For instance, Lee et al. have studied
the degradation of high-density polyethylene under lignocellulose substrate treat-
ment by Bjerkandera adusta TBB-03. They showed that laccase activity results
in crack formation on the high-density polyethylene surface after 90 days [76].
Liyoshi et al. showed that the manganese peroxidase produced by the lignin-
degrading fungi Phanerochaete chrysosporium could oxidase polyethylene film and
decrease the molecular weight [69]. Zhao et al., in exciting research, have isolated
Aspergillus flavus with the polyethylene microplastics-degrading ability from the
gut contents of wax moth Galleria mellonella. Using molecular techniques, they
observed up-regulated expression of two laccase-like multicopper oxidases (LMCOs)
genes during the biodegradation process and proposed these genes as candidate
polyethylene-degrading enzymes [203]. Paco et al. have demonstrated that the Zale-
rion maritimum, a marine fungus, can use polyethylene as a source of nutrients and
has high potential in combatting aquatic systems’ plastic pollutions [121].
Polyvinyl chloride
Polyvinyl chloride is susceptible to biodegradation, due to it mainly consisting of
plasticizers which are the main source of nutrient carbons for microorganisms.
For instance, the biodegradation of polycaprolactone and polyvinyl chloride by
Chaetomium globosum has been investigated by Attili-Angelis et al. [178]. In another
study, Aureobasidium pullulans exhibited the capability of degrading 3.7% of the
initial weight of tested plasticized polyvinyl chloride film over 42 days [185]. Two
fungi strains, Penicillium janthinellum and Phanerochaete chrysosporium, were
isolated from the polyvinyl chloride film buried in soil, and their polyvinyl chloride-
degrading capability was documented within an incubation period of 300 days based
on the weight loss [5, 140]. In another study, Chen et al. produced mutant Penicil-
lium oxalicum strain DSYD05-1 with high activity efficiency for poly(caprolactone)
biodegradation after four days of cultivation [94]. Three fungi Aspergillus japon-
icus, Penicillium brocae, and Purpureocillium lilacinum were isolated from plastics-
contaminated soil that P. lilacinum showed the highest efficiency for di 2-ethylhexyl
phthalate remediation, which is commonly used as plasticizers in Polyvinyl chloride
plastics construction [126].
Polylactic acid
polylactic acid is a thermoplastic formed by the polymerization of L-lactic acid.
Lipsa et al. examined the breakdown of polylactic acid-based systems by Tricho-
derma viride fungus. They assessed the effect of different components such as
epoxidized soybean oil as a plasticizer, hydrolyzed collagen as biological macro-

molecules, Pluronics F-127 as a compatibilizer, Vitamin E as an antioxidant drug,
and silver nanoparticles on the biodegradation process. Polylactic acid plasticized
with epoxidized soybean oil had the highest biodegradation efficiency showing that
this plasticizer increased the biodegradation rate of polylactic acid [98]. Biodegra-
dation of polylactic acid by other fungi such as Tritirachium Album has also been
reported [74].
Polyurethane
Using commercial composting as a cost-effective method for reducing waste, Robson
et al. in their study on polyurethane biodegradation showed that polyurethane under-
goes substantial physical changes during this process. Pyrosequencing data suggested
that fungal communities on polyester polyurethane’s surface mainly consist of
three phyla: Ascomycota, Basidiomycota, and Zygomycota [200]. In another study,
biodegradation of polyester polyurethane in the presence of compost at different
temperatures was assessed. Results revealed that fungi extensively colonized the
polyester polyurethane surface and deterioration happened at all temperatures.
Pyrosequencing analysis showed that the dominant species at 25 °C was Fusarium
solani and that at both 45 and 50 °C was Candida ethanolica [199].
According to all mentioned studies, fungi have shown specific characteristics
for bioremediation. Their ability to survive in an environment with acidic pH,
low nutrient availability, moisture content and penetration into the substrate, attach
hydrophobic substrate, and pollutant digestion results in their great potential in plas-
tics remediation and makes them a good alternative for mitigating environmental
catastrophes. Like bacteria, fungi combination or fungi consortia can have a more
effective influence on plastics bioremediation by biodegradation.
9.3 Biodegradation by Algae
Algae are considered oxygen-evolving photosynthetic widespread organisms in

different forms, from unicellular and colonial to filament forms and giant kelps
[25, 59]. Besides the prokaryotic cyanobacteria or so-called “blue green algae”,
which are classified in the bacteria domain, algae and plants are producers of the
earth. They, therefore, play essential roles in our planet’s carbon cycle [25]. Algae
are found in aquatic and terrestrial environments [144]. They can even colonize
inside the rocks’ tiny cracks and can be found in the desert soils [25]. Algae are
photoautotrophs, so they do not need organic carbon sources for their growth and are
well adapted to marine habitats. Algae secrete extracellular mucilaginous polymeric
substances that can adhere to and colonize nearly all substances. Thus, algae and
other microorganisms can start biofilm formation which is a primary and critical
step in the biodegradation of many materials such as plastics [154, 155].
These organisms were utilized for the biodegradation of toxic compounds such as
azo dyes. Yan et al. employed a green microalga Chlorella pyrenoidosa for degrada-
tion of dimethyl phthalate (DMP) [192]. In another study, the ability of green alga,
Chlorella fusca, in removing bisphenol A was investigated under different light–
dark conditions. This green alga could almost completely remove bisphenol A in the
concentration range 10–80 μM under 168-h continuous light exposure [65]. Sarmah
and Rout studied the colonization of 20 species of Oscillatoria on sewage water-
submerged polyethylene bags. These species Oscillatoria princeps, O. subbrevis, O.
limosa, O. amoena, O. vizagapatensis, O. okeni, O. limosa, and O. laete-virens are
commonly found on the submerged polyethylene bags [143].
Algae and microalgae’s properties introduce them as new tools in biotechnolog-
ical applications [45, 110, 111]. Moog and his co-workers in their study used a marine
photosynthetic eukaryote, Phaeodactylum tricornutum, which has many biotechno-
logical applications. They employed P. tricornutum for producing a recombinant
protein, polyethylene terephthalate hydrolyzing enzyme, PETase enzyme. Using
proper genetic elements, they successfully converted Phaeodactylum tricornutum
to a factory for the production and secretion of the PETase enzyme [107]. Due to
the amounts and effects of the polyethylene terephthalate pollution, in another study,
two strains of Chlamydomonas reinhardtii were transformed for PETase enzyme
expression and production. Researchers found that the strain C. reinhardtii CC-124
could express active PETase that can hydrolyze polyethylene terephthalate to its fully
degraded form, terephthalic acid (TPA) [85].
9.4 Important Enzymes in Biodegradation
Enzymes have great importance in the biodegradation process by catalyzing chem-

ical reactions, including oxidation, dealkylation, deamination, desulfuration, sulfox-
idation, oxidation, and dehalogenation. Generally, microorganisms secreted exoen-
zymes bind to the substrate and digest polymer into smaller molecules absorbed
and used as nutrients by microorganisms [3]. Fungi, with their active exoenzymes
such as ligninolytic enzymes like laccase, manganese peroxidase, and lignin perox-
idase that are secreted from their cells into the environment, have an indispensable
role in bioremediation [28]. So far, several plastic-degrading enzymes such as Poly-
3-hydroxybutyrate depolymerase, Lipase B, Styrene monooxygenases, Oxidase,
Hydrolase, Dehydrogenase, Polyurethanases, and Lipase have been detected [51].
Recently, biodegradation by enzymes has attracted significant attention compared
to bioremediation based on microorganisms because it is a fast and more specific
process. Here, we will introduce different kinds of plastic-degrading enzymes from
two main groups and describe enzyme activity characterization methods.
9.4.1 Oxidoreductases
These enzymes catalyze an oxidative reaction by transferring an electron from reduc-

tants to oxidants produced by various plants, fungi, and bacteria to detoxify and
degrade compounds. This category includes oxygenases, laccases, and peroxidases.
The principal enzymes that can aerobically catalyze the ring cleavage in aromatic
compounds are oxygenases. They consume one or two oxygen molecules during their
catalytic activity and accordingly are divided into two groups: monooxygenase and
dioxygenase, respectively [153]. Laccases that contain multicopper atoms in their
active sites perform the oxidation of many phenolic and aromatic compounds. Perox-
idases use hydrogen peroxide for their oxidase activity [153]. Here, we bring some
examples of laccases and peroxidases in the biodegradation of plastic contaminants.
Laccase
Most laccases are extracellular enzymes secreted to the outside of the cell and oxidase
a wide range of substrates from phenolic to aromatic compounds. The immobilized
form of enzyme has been used in various industries such as textile bleaching and
pollutant bioremediation [153]. The typical method for enzyme activity measurement
is monitoring the change in absorbance of the related substrate.
Lee et al. demonstrated laccase and peroxidase production in high-density
polyethylene biodegradation with Bjerkandera adusta TBB-03. Oxidative activity
of laccase and peroxidase enzymes was evaluated by oxidation of 2,2’-azino-bis(3-
ethylbenzthiazoline-6-sulfonic acid) (ABTS) as an indicator. ABTS oxidation creates
green color with absorbance at 420 nm. According to this color change, B. adusta
TBB-03 growth in the presence of high-density polyethylene was confirmed [76].
Zhang et al. found that Aspergillus flavus had catalytic activities of two laccase-like
multicopper oxidases (LMCOs) when polyethylene was used as substrate. They used
reverse transcription-polymerase chain reaction (RT-PCR) to identify enzymes that
take participants in the degradation process. The results revealed that two laccase-
like multicopper oxidases (LMCOs) genes, AFLA_006190 and AFLA_053930, had
up-regulated expression during the degradation process [203].
For evaluating the biodegradation of di(2-ethyl hexyl) phthalate (DEHP), a plasti-
cizer, Ahuactzin-Perez et al. have studied laccase activity of Fusarium culmorum via
measuring the absorbance of 2,6-dimethoxyphenol as a laccase substrate at 468 nm.
The results showed that laccase activity was increased in media supplemented with
di(2-ethyl hexyl) phthalate. They reported a novel pathway for biodegradation of di(2-
ethyl hexyl) phthalate by F. culmorum that completely metabolized di(2-ethyl hexyl)
phthalate, and the final product was butanediol. They also observed that esterase
activity, a hydrolyzing enzyme, was higher than laccase activity [4].
Peroxidase
These kinds of enzymes catalyze reactions in the presence of hydrogen peroxidase

and can be divided into two groups: heme-containing and non-heme-containing
peroxidases. The first group, heme-containing peroxidase, is subdivided into two
superfamilies. The first superfamily consists of animal peroxidases and the second
superfamily is found in fungi, bacteria, and plants. Lignin peroxidase and manganese
peroxidase are parts of the second superfamily and are well-known for their plastic
biodegradation capability [174].
Lignin peroxidase:
Lignin peroxidase is a heme-containing extracellular enzyme that belongs to oxidore-
ductase. This enzyme is usually found in white-rot basidiomycetes such as Phane-
rocheate chrysosporium, Pleurotus ostreatus [56, 83]. Lignin is similar to certain
plastics; therefore, lignin-degrading fungi can degrade these plastics [87]. Lignin
peroxidase’s capability in oxidative degradation of phenolic and non-phenolic
aromatic moieties of lignin causes its wide application in different substrates’
bioremediation [78].
Manganese peroxidase:
Like lignin peroxidase, manganese peroxidase belongs to the oxidoreductase family
and breaks phenolic and non-phenolic substrates during oxidation of Mn(II) to
Mn(III) by a multistep reaction. These are extracellular enzymes produced by fungi
and contain the heme group [153]. Iiyoshi et al. found that manganese peroxidase
plays a crucial role in polyethylene degradation by lignin-degrading fungi [69]. In
another study to enhance the bioremediation efficiency and rate, the gene encoding
manganese peroxidase was also expressed in Saccharomyces cerevisiae [92]. Addi-
tionally, to improve the enzyme’s half-life, manganese peroxidase was immobilized
on a sol–gel matrix [153].
9.4.2 Hydrolase
Hydrolytic enzymes degrade a wide range of substrates such as esters and peptide
bonds and are widely utilized for biodegradation. They use a water molecule for
breaking a chemical bond and therefore splitting a molecule into two molecules.
Esterases and proteases are two important hydrolases. Esterases divide an ester
into an acid and an alcohol molecule and include many necessary enzymes such
as lipases, glycosidases, and phosphatase. Esterases are very common hydrolases.
In this section, three examples of hydrolyses are provided. Because of their low
substrate specificity, hydrolyses may be essential players in the bioremediation of
various pollutants [58].
Serin Hydrolase
Russell et al. found that a Pestalotiopsis microspora E2712A was able to decompose
the synthetic polymer polyester polyurethane, showing serine hydrolase activity.
In this study, mechanism-based inhibitors were employed to characterize the
enzyme responsible for polyurethane biodegradation. Accordingly, a serine hydro-
lase inhibitor, a cysteine hydrolase inhibitor, and metallohydrolase inhibitors were
separately added to the pre-incubated crude extracellular extract and Impranil DLN,
which contains polyurethane as substrate. The addition of a serine hydrolase inhibitor
prevented the biodegradation activity, while other inhibitors did not affect the degra-
dation activity [138]. Polyhydroxyalkanoates depolymerases are a class of serine
hydrolases with a fungal and bacterial origin. Different fungal genera with Polyhy-
droxyalkanoates degenerative abilities such as Basidiomycotina, Deuteromycotina,
and Ascomycotina were isolated from marine sources [128].
Esterase
Zafar et al. reported the fungal community’s enhanced esterase activity when they
were grown on polyester polyurethane [200]. In another study, p-nitrophenyl butyrate
(pNPB) was used as an indicator for measuring the esterase activity of Fusarium
culmorum. The absorbance intensity at 405 nm showed that esterase activities were
increased in media supplemented with di(2-ethyl hexyl) phthalate [4].
Protease
Proteases are a kind of hydrolases that play a role in plastic biodegradation. The
detection of protease activity is done using methods such as staining the solid culture
medium with a 0.25% coomassie blue. Hydrolysis of the substrate forms transparent
holes around the culture, which shows protease production. The transparent hole
width is proportional to protease activity [177].
In a study, Jarerat and Tokiwa investigated the degradation of poly(l-lactide) (PLA)
by fungus Tritirachium album ATCC 22,563. They reported about 76% degradation
of PLA film is done only when gelatin is added to the basal medium. They observed
that the crude enzyme obtained after filtration of T. album ATCC 22,563 culture
medium could degrade a synthetic tripeptide substrate and silk fibroin. These results
confirmed that the polylactic acid-degrading enzyme in T. album ATCC 22,563 is a
protease and has similar substrate specificity to commercial proteinase K [74].
Phosphatase, phytase, lipase, and carbohydratase such as cellulases, amylases, and
xylanases are other types of hydrolase involved in pollutants degradation [58]. But in
this chapter, we limited the examples and introduced some important and interesting
ones. However, this research area is growing, and flourishing and new experimental
and computer-based methods are used for better understanding. In Sects. 11 and 12,
the new approaches are discussed.
10 How to Find Proper Microorganisms for Biodegradation
Plastic degrading microorganisms are present in different places, from terrestrial to

marine environments. Therefore, to find the suitable microorganism for biodegra-
dation, sampling from other sites is performed. For selecting the microorganisms
with decomposing activity, samples are isolated using sterile swabs and trans-
ferred onto cultivation media to obtain a pure culture. Several analytical methods
assess the biodegradation activity of microorganisms. These methods consist of a
broad range from simple techniques such as morphological, color change, mass
loss, and optical microscopy to complicated approaches like scanning electron
microscopy, mass spectroscopy, Fourier-transform infrared spectroscopy, thermo-
gravimetry, Raman spectroscopy, nuclear magnetic resonance, biochemical assays,
gel permeation chromatography, and differential scanning calorimetry [57, 76, 94,
98, 168, 178, 179, 203]. After that, genomic DNA is extracted, amplified, sequenced,
and the phylogenetic tree is generated by applying the neighbor-joining method [76].
As mentioned, plastic-degrading microorganisms are present in various areas,
like landfills, plastic-dumping sites, waste of mulch film, sewage sludge, and the
gut of plastic-eating worms and marine water. For instance, endophytic fungi, which
contribute to lignocellulose polymers’ decomposition, live within the plant’s inner
tissues. Strobel et al. isolated endophytes from plant stem and screened their capa-
bility in polyurethane degradation. For this purpose, the outer layer of sterilized plant
stems was removed, and inner tissue was plated on potato dextrose agar. Isolated fungi
are grown in the presence of polyurethane as the sole source of carbon. Colonies that
produced a clear zone in the opaque medium were isolated as polyurethane-degrading
fungi [138].
Polyethylene has unique features such as portability, ductility, and stability which
causes its wide application in plastic synthesis. Several polyethylene-degrading
fungi such as Aspergillus, Acremonium, Fusarium, Penicillium, and Phanerochaete
have been detected in our environment, such as soil landfill, industrial waste,
and compost. In addition to solid matrices, the marine environment is a habitat
of polyethylene-degrading microorganisms such as Zalerion maritimum [121].
Besides our surrounding environment, some insects provide resources for plastic-
biodegrading microorganisms. For example, the larvae of Plodia interpunctella and
the larvae of the wax moth Galleria mellonella have shown the ability of plastic
degradation. Aspergillus flavu is a kind of polyethylene-degrading fungi isolated
from the gut content of Galleria mellonella [203]. Several assays are used to eval-
uate the plastic digestion process after the isolation of appropriate microorganisms
for plastic remediation. The effects of microorganisms on plastics’ physicochemical
properties include changes in the morphology, weight, and structural features that
we will explain in this chapter.
10.1 Weight Measurement
Weight measurement is a standard method for microorganism attack examination.

For this aim, after degradation, the sample is washed and dried to remove the microor-
ganisms. Then the difference of sample mass before and after biodegradation is calcu-
lated and divided by sample mass before degradation. Angelis et al. have compared
polycaprolactone and polyvinyl chloride biodegradation by measuring mass loss.
Their results revealed that polycaprolactone showed significant mass loss compared
to polyvinyl chloride due to polycaprolactone structure and its hydrophilicity [178].
Comparing the sample’s molecular weight between before and after degradation
is another method applied for the investigation of the biodegradation process. Gel
permeation chromatography is a technique that separates analytes based on molecular
weight. The larger analytes are eluted quicker than smaller analytes which enter
the pores and spend more time in the column. Biodegradation efficiency achieved
by Chaetomium globosum and Phanerochaete chrysosporium is measured by gel
permeation chromatography. The chromatogram showed that the molecular weight
of the polylactic acid sample decreased after fungal degradation [168].
10.2 Morphology Assessment
Scanning electron microscopy is used for the investigation of the morphological

changes in the surface of samples. The produced organic acid metabolite due to
microorganism activity causes sample weakness and forms pits and cracks on the
surface. Lee et al. have used scanning electron microscopy for analyzing the degra-
dation of high-density polyethylene using Bjerkandera adusta. The results have
revealed that degradation formed on the surface of high-density polyethylene after
90 days [76].
10.3 Structural Changes
Raman spectroscopy has been widely applied to investigate structural changes.

Raman bands of a sample before and after degradation give precious data about
the remediation process. Generally, enzyme activity on a substrate is associated with
changes in interaction links. Raman analysis of high-density polyethylene degraded
by Bjerkandera adusta TBB-03 showed no band disappearance or formation in the
high-density polyethylene spectrum, although the intensity of peaks corresponding to
C–C symmetric stretching, CH2 twisting, and CH2 bending increased. These trends
are due to an increase in crystallinity and a decrease in amorphous changes [76].
Fourier-transform infrared spectroscopy is a technique to identify functional
groups in a sample. FTIR spectroscopy was used to analyze polyhydroxyalkanoate
film following the biodegradation activity of Bacillus circulans. The results showed
the decrease in the ester carbonyl bond’s peak intensity, which was related to the
ester bond’s breakdown in polymer films after degradation. Besides, the degradation
of minor bands’ effect on the degree of crystallinity can be detected by FTIR spectra
[124]. FTIR spectroscopy is also used for analyzing polylactic acid biodegradation.
The Trichoderma viride degrades the ester group, resulting in more polylactic acid
oligomer and higher intensity of the C=O stretching vibrations of the COOR group in
FTIR spectra. In this study, the thermal stability of polylactic acid was also evaluated
by TGA analysis [98].
Mass spectrometry is an analytical technique that measures the mass-to-charge
ratio of ions and has been used to understand the chemical identity or structure of
analytes. Chen et al. have applied this technique to investigate poly(ε-caprolactone)
(PCL) degradation. Mass spectra indicated the presence of 6-hydroxyhexanoic acid,
which is due to the polycaprolactone-degrading activity of Penicillium oxalicum
fungus [94].
In addition to plastic, microorganisms’ biomass is analyzed through Nuclear
magnetic resonance (1H NMR) spectroscopy to investigate biodegradation. In this
method, the absorption of radiofrequency signals by the nuclei of different atoms is
measured and used to determine the analyte’s overall structure. Microplastics with
reduced carbon or nitrogen resource cause changes in microorganism metabolisms.
The NMR result showed that a long exposure of Zalerion maritimum to polyethylene
as the sole source of nutrients caused a decreasing concentration of lipid and protein
content of biomass [121].
10.4 Thermal Behavior
Thermal gravimetric analysis, TGA, is a method for evaluating thermal stability

by measuring the sample’s mass over time as the temperature changes. The result
showed that polylactic acid samples exposed to the fungi environment had lower T10
and T20 values, a temperature for ten wt.% and 20 wt.% weight loss, respectively.
The lower T10 and T20 values result from decomposed macromolecular chains with
decreased molecular weight [98].
Differential scanning calorimetry is another thermoanalytical method that several
different parameters such as glass transition temperature or Tg , melt temperature
or Tm , crystallization temperature or Tcc , endothermal melting enthalpy or Hm,
and degree of crystallization or Xc of samples are obtained by this technique [106,
179]. In the study by Yilmaz et al., the differential scanning calorimetry curves of the
polylactic acid sample were measured before and after biodegradation in the presence
of fungi. The results showed that after biodegradation, the polylactic acid melting
peak is split into two extremes consistent with gel permeation chromatography results
that showed the decrease of average molecular weight. The Tcc was not present in the
differential scanning calorimetry curve, and Tg value decreased after biodegradation
[168].
All in all, in this chapter, we discussed how we could select good microor-
ganisms for plastic degradation and assess the remediation process. Biodegrada-
tion is evaluated through several methods such as structural analysis, changes in
morphology, thermal behavior, and biochemical assays. The study of enzyme activity
and biochemical assays will be thoroughly explained in the enzyme section.
11 Engineering of Microorganisms for Bioremediation
The role of several microorganisms such as bacteria, fungi, and algae and their
enzymes in bioremediation has been identified. However, the identified enzymes
suffer from limitations such as low stability and activity. Also, the production level
of the enzyme is inadequate in its native hosts. Therefore, some techniques such as
genetic engineering have been applied to overcome the above limitations. Genetic
engineering provides ways to increase an enzyme’s production level, i.e., plasmid
transferring gene, or to improve enzyme’s properties, i.e., enzyme engineering.
11.1 Enzyme Engineering
Since nature did not have time to evolve microbes and their enzymes to depolymerize
human-made material, identifying and modifying the genes that control plastic
metabolism can allow recycling plastic wastes. Amino acid sequence modifications
change an enzyme’s different properties, including its activity, temperature or pH
resistance, and stress tolerance. Gene manipulation is a popular method for enzyme
engineering and is classified into four different categories: endonuclease-based
editing, recombinase-based editing, post-transcriptional gene silencing by RNA
interference (RNAi), and random or site-directed mutagenesis. These targeted tech-
niques create changes in site-specific locations of host genomes, unlike early and
traditional genetic engineering methods that genetic material randomly inserted
[128].
Site-directed mutagenesis aims to create targeted changes in a specific gene with
a copy containing the desired mutation. This method is used to evolve different
enzymes with the capability to degrade plastic material with higher efficiency.
For example, Wei et al. have used an engineered bacterial hydrolase to enhance
polyethylene terephthalate film degradation [186]. In another study, rational protein
engineering has been employed to improve poly (ethylene terephthalate) hydrolase
activity. Six key residues around the substrate-binding groove have been mutated,
and an increase of 2.5-fold degradation rate against poly (ethylene terephthalate)
has been observed [103]. Islam et al. have established the engineered cutinase that
degraded the polyurethane faster than wild type [72].
11.2 Transgenesis
In the plasmid-transferring gene method, the gene encoding enzyme is isolated

and transferred into another expression host. By using this method, the engineered
enzyme is produced in large amounts with higher stability and activity. Bacteria are
usually selected as host cells due to their low cost, high growth rate, and easiness of
manipulation. Recombinant PETase has been generated in Escherichia and Bacillus
for recycling polyethylene terephthalate [67, 149, 196].
In another study, Clostridium thermocellum was engineered to degrade polyethy-
lene terephthalate. Liu et al. engineered Clostridium thermocellum, a thermophilic
anaerobic bacterium, to express a thermophilic cutinase. For this purpose, gene
encoding cutinase is inserted into PHK plasmid and transformed into C. thermo-
cellum. The enzyme’s expression was examined by sodium dodecyl sulfate–poly-
acrylamide gel electrophoresis and western blotting. The activity of the enzyme was
evaluated by amorphous polyethylene terephthalate film. The engineered microor-
ganism showed high degradation efficiency at 60 °C, and more than 60% weight loss
happened after 14 days [191].
Bacterial systems require a rich carbon source for growth that has to be overcome.
However, microalgae have attracted great interest for genetic material transforma-
tion because they do not need organic carbon sources and are well adapted to marine
habitats. For example, Kim et al. examined two strains of Chlamydomonas rein-
hardtii, green algae, for transformation and expression of polyethylene terephthalate
degrading enzyme encoding gene. Expression of PETase was confirmed by western
blot analysis, and PETase activity was investigated against commercial polyethylene
terephthalate bottles. The result showed that CC-124 strain could degrade polyethy-
lene terephthalate with high efficiency [85]. Maier et al. worked on the decomposi-
tion of polyethylene terephthalate waste in a marine environment. They generated
an engineered photosynthetic microalga, Phaeodactylum tricornutum, to produce
and secrete PETase. The produced PETase has been shown to be able to degrade
industrially shredded polyethylene terephthalate particles in saltwater as verified by
scanning electron microscopy (SEM) and high-performance liquid chromatography
(HPLC) [107]. All in all, recombinant DNA technology is a promising strategy to
enhance production level, thermostability, substrate specificity, and improve kinetic
properties of enzymes.
12 Metagenomics and in Silico Studies on Microplastics

Bioremediation
According to the estimations, researchers have reported only in marine environment

3.6 × 1029 bacterial cells, 1.3 × 1028 archeal cells exist [36]. Traditional microbi-
ology and microbial genomic study are based on microbial cultures and their clonal
cultures. But new studies on environmental DNA, eDNA, have estimated that only
0.1–1% of the prokaryotes are culturable. Also, some eukaryotic microbes are uncul-
turable [23]. Therefore, culture-independent methods for studying and employing
all microbial capabilities are necessary [23]. In 1986, for the first time, Pace et al.
presented the new idea of direct DNA cloning from environmental microbial samples
without cultivating them to analyze better the diversity and complexity of microbial
populations [120]. In 1998, Handelsman et al. have used the “metagenome” term
to describe soil microorganisms as sources for unfounded natural compounds [7].
Therefore, metagenome is defined as mining the unculturable microbial populations
to discover novel organisms, enzymes, chemical compounds, metabolic pathways,
and active molecules with better or new functions [7, 172].
Metagenomics reveals hidden genetic features for biotechnological applications
in demanding eras like bioremediation [172]. Structural and functional genomics
are two aspects of metagenomics that seek crucial ecological and biotechnological
characteristics in environmental microbial samples, respectively [7]. A new metage-
nomics study by shotgun sequencing of coastal lagoon microorganisms creating
biofilms on plastics and bioplastics revealed that in bioplastic biofilms, sulfate-
reducing microorganisms (SRM) were dominant. Gene pool analysis of the bioplas-
tics biofilms manifested the enrichment of some genes, including depolymerases,
adenylyl sulfate reductases (aprBA), esterases, and dissimilatory sulfite reductases
(dsrAB). Also, metagenomic genomes reconstruction led to identifying new species
of Desulfovibrio, Desulfobacteraceae, and Desulfobulbaceae among the abundant
SRM [125].
Today, in silico studies help accelerate the in vitro studies by simplifying the
problems [163]. Genome and protein databases make possible mining of unidentified
plastic degrading enzymes with strategies such as homology alignment. Computa-
tional technologies employed in systems biology and synthetic biology, together with
omics data, especially genomics, transcriptomics, and metabolomics, help to find the
expected molecules [206]. Almeida et al., using the most efficient bacterial polyethy-
lene terephthalate hydrolase derived from Ideonella sakaiensis 201-F6 (IsPETase),
performed an in silico-based screening method on terrestrial- and marine-isolated
relevant genus Streptomyces. They found three potential enzymes with PETase-like
activity. The protein sequence and 3D structure of a PETase-like gene identified
in Streptomyces sp. SM14 (SM14est) was determined and predicted by in silico
approaches [6]. Protein modeling, structural analysis, and molecular docking were
done for SM14est enzymatic activity. Heterologous expression of SM14est enzyme
and enzymatic activity tests showed degrading activity of the isolated enzyme [6].
Investigating the new or minimally explored ecosystems such as rainforests and
deep-sea due to these locations’ environmental conditions and biodiversity will be
promising for finding new species with fantastic enzymes [157]. The metagenome
analysis and in silico mining lead to a deeper investigation of the explored and
unexplored nature to find efficient enzymes and microorganisms for microplas-
tics’ bioremediation. Biotechnological and bioengineering methods pave the way for
developing more efficient and stable enzymes catalyzing the degradation of plastics.
13 Noteworthy Points on Biodegradable Plastics
The biodegradability of a plastic polymer is tested under defined protocols in a

certified laboratory. Based on the plastics’ destination for biodegradation, such as soil
or marine environment, the standard organization has determined different standards.
For example, ASTM D6691, ASTM D7473, and ISO 16221 are three standards for
plastic biodegradability in the marine environment, and ISO 14851 is the plastic
material biodegradability standard in an aqueous medium [63]. A list of standards is
provided in the Ammala et al. paper [9].
Biodegradable plastics are certified based on the standards criteria that are
following the conditions of composting facilities. For this reason, a biodegradable
plastic product does not necessarily biodegrade in natural environmental conditions
in the time frame of tested protocols [63, 88]. In natural environments, the required
time for complete biodegradation of biodegradable products will be much longer,
e.g., several decades [88]. Additionally, during disintegration, fragmentation results
in many tiny plastic parts harmful to the environment. These small fragments, such as
microplastics, create health risks for humans and other organisms. Therefore, more
obvious terminology to help companies or individual consumers make a conscious
selection seems essential [88].
Recycling biodegradable plastics is also challenging because of their structural
and physicochemical properties. Their isolation from plastic wastes containing non-
recyclable and recyclable plastics is difficult. They can be regarded as a contaminant
for recycling streams of conventional plastics [88]. It is needed to separate biodegrad-
able plastics from all waste materials, even conventional plastics, and guide them to
industrial composting facilities. Composting biodegradable plastics and their miner-
alization cause these potentially valuable materials to be eliminated from the produc-
tion and consumption cycle. In a circular economy with continual use of resources
as the primary goal, finding recycling approaches for biodegradable materials will
be necessary, mainly when produced in high volume [88].
Acknowledgements This chapter is written by a team of writers who have tried their best even
in difficult circumstances and hope to do well. We all thank our families for their love, kindness,
patience, and encouraging support.
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Bioremediation Techniques

for Microplastics Removal

Samaneh Hadian-Ghazvini, Fahimeh Hooriabad Saboor,

Keywords Microplastics · Bioremediation · Biodegradation · Biodegradable

1 An Introduction to Plastics Bioremediation, Types,

2 A Brief Introduction to Organisms and Enzymes

3 Plastic Degradation and Biodegradation

4 Types of Plastics Based on Their Origin, Thermal

Plastics are classified based on their producing feedstock, polymer-forming

4.1 Thermoplastics and Thermosets

Fig. 2 Biodegradation steps of a polymer, reproduced from [3, 51, 63]

Fig. 3 Schematic representation of the molecular structure of thermoplastics and thermosets.

4.2 Bio-based or Fossil-Based Nonbiodegradable Plastics

4.3 Bio-based or Fossil-Based Biodegradable Plastics

Fig. 4 Some examples of

Fig. 5 Chemical structure of some biodegradable and nonbiodegradable plastics

6 Microplastics Toxicity on Living Organisms

Plastics have improved human health by manufacturing different types of medical

6.1 Microplastics Toxicity on Microalgae

6.2 Microplastics Toxicity on Animals and Humans

7 Antibiotic Resistance and Microplastics

performed selective enrichment experiments for bacterial identification [86, 201].

different on microplastic samples than in water samples. Also, microbial diversity at

8 Microorganisms and Microplastics Interaction

Microplastics are ubiquitous worldwide and accumulate in aquatic environments

9.1 Biodegradation by Bacteria and Bacteria Consortium

1 Aspergillus versicolor, – Low-density polyethylene Seawater sample [127]

microspor E2712A (PU) families

9.2 Biodegradation by Fungi

Recently, the capability of several microorganisms and enzymes in degrading plastic

epoxidized soybean oil as a plasticizer, hydrolyzed collagen as biological macro-

9.3 Biodegradation by Algae

Algae are considered oxygen-evolving photosynthetic widespread organisms in

9.4 Important Enzymes in Biodegradation

Enzymes have great importance in the biodegradation process by catalyzing chem-

These enzymes catalyze an oxidative reaction by transferring an electron from reduc-

These kinds of enzymes catalyze reactions in the presence of hydrogen peroxidase

10 How to Find Proper Microorganisms for Biodegradation

Plastic degrading microorganisms are present in different places, from terrestrial to

10.1 Weight Measurement

Weight measurement is a standard method for microorganism attack examination.

10.2 Morphology Assessment

Scanning electron microscopy is used for the investigation of the morphological

10.3 Structural Changes

Raman spectroscopy has been widely applied to investigate structural changes.

10.4 Thermal Behavior

Thermal gravimetric analysis, TGA, is a method for evaluating thermal stability

11 Engineering of Microorganisms for Bioremediation

11.1 Enzyme Engineering

In the plasmid-transferring gene method, the gene encoding enzyme is isolated

12 Metagenomics and in Silico Studies on Microplastics

According to the estimations, researchers have reported only in marine environment

13 Noteworthy Points on Biodegradable Plastics

The biodegradability of a plastic polymer is tested under defined protocols in a

4. Ahuactzin-Perez M, Tlecuitl-Beristain S, Garcia-Davila J, Gonzalez-Perez M, Gutierrez-

69. Iiyoshi Y, Tsutsumi Y, Nishida T (1998) Polyethylene degradation by lignin-degrading fungi

160. Shimao M (2001) Biodegradation of plastics. Curr Opin Biotechnol 12(3):242–247

182. Wang W, Gao H, Jin S, Li R, Na G (2019) The ecotoxicological effects of microplastics

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