**2. Methods and Results**

The degradation of two plastic polymers used in the production of supermarket plastic bags was evaluated (**Figure 1**, I). The oxo-biodegradable and green polyethylene polymers were submitted the abiotic and biotic degradation (**Figure 1**, II). The oxo-biodegradable bags contain titanium oxide as pro-oxidant additive and low-density polyethylene [14].

The abiotic degradation of the plastic bags was the exposure to sunlight up to 120 days (**Figure 1**, III). This exposure was in the summer time in a green house. In this season, the sunlight is from 6:00 am to 5:00 pm.

**263**

ments of 5 cm2

**Figure 1.**

towel fragments (5–10 cm<sup>2</sup>

*green polyethylene plastics bags.*

days (**Figure 1**, VIII).

(**Figure 1**, IX a3).

diffraction.

plastics and paper towel was of 99:1.

analysis done before of the exposure to sunlight.

lulolytic enzymes activity (**Figure 1**, IX c5).

*Plastics Polymers Degradation by Fungi DOI: http://dx.doi.org/10.5772/intechopen.88608*

For the biotic degradation (**Figure 1**, IV at VIII) the plastic polymers without (**Figure 1**, II) or with the exposure to sunlight (**Figure 1**, IV) was used *P. ostreatus* PLO6 (GenBank accession number KC782771). These polymers were cut in frag-

*Steps and technics used for monitoring the abiotic and biotic (fungal) degradation of oxo-biodegradable e* 

In each glass flask fours discs of agar (6–8 mm) containing the mycelium of *P. ostreatus* PLO6 were inoculated (**Figure 1**, VII). This fungus was cultivated in 20 mL of potato dextrose lignin (0.1%) agar (PDLA) for 15 days. The initial inoculum was obtained from the collection of the Department of Microbiology of Universidade

After inoculation the glass flask were incubated at 25°C for 30, 60, 90 and 120

The alterations in plastic polymers (**Figure 1**, IX) after each time of incubation were performed (**Figure 1**, III, IV at VIII). These alterations were compared with

Physical alterations (**Figure 1**, IX a), such as wrinkles on the surface, formation of holes and cracks, crumbling, discoloration, were performed by digital photograph and scanning electron microscopy (SEM) with a magnification of 50,000 (**Figure 1**, IX a2). Mechanical properties, such as, energy at break and load at tensile strength were made in universal testing equipment (Instron model 3367)

Chemical changes (**Figure 1**, IX b) by Fourier transform infrared spectroscopy (FTIR) (**Figure 1**, IX b1) and SEM coupled with X-ray diffraction (**Figure 1**, IX b2) were determined. These alterations were the disappearance or formation of new functional groups in spectrum of FTIR with scanning of 500 at 4000 cm<sup>−</sup><sup>1</sup>

numbers and the decrease in-oxidant additive concentration by spectrum of X-ray

The mycelial growth (**Figure 1**, IX c), the main agent of the biological alterations, was evaluated by dry mass (**Figure 1**, IX c1), respiratory activity (**Figure 1**, IX c2) determined without interruption for 120 days of incubation, electronic micrograph (**Figure 1**, IX c3), digital photography (**Figure 1**, IX c3) and lignocel-

Federal de Viçosa. The stock culture is maintained on PDLA at 4°C.

(**Figure 1**, V) and placed in a glass flask (100 mL) containing paper

) and mineral medium (**Figure 1**, VI). The proportion of

wave-

*Plastics Polymers Degradation by Fungi DOI: http://dx.doi.org/10.5772/intechopen.88608*

#### **Figure 1.**

*Microorganisms*

fermentation processes.

degradation process [6].

degraded by action of microbial enzymes [7].

heat, by bacteria and fungi [10–16].

waste [20–22].

**2. Methods and Results**

low-density polyethylene [14].

this season, the sunlight is from 6:00 am to 5:00 pm.

results only in selective collection. In 2014, with the deadline for replacing dumps to landfills, new deadlines for 2021 are in discussion in the National Congress [5]. Our results of biotic and fungal degradation of oxo-biodegradable plastics and green polyethylene could contribute for development of a process of degradation of these residues using white rot fungi. These microorganisms can grow under adverse temperature, nutrient and moisture conditions that facilitate composting and

The plastics polymers degradation is analyzed by alterations in mechanical, optical or electrical characteristics, cracking, fission, corrosion, discoloration, phase separation, chemical transformations and formation of new functional groups after

Unlike of the petroleum-derived synthetic polymers, the biodegradable plastics polymers, when discarded in the environment, can be degraded by non-biological and biological processes [7]. Exposure to ultraviolet light, thermal heating, and treatment with acidic or basic substances function as term initiators or photooxidation of polyethylene [6]. After this oxidation fragments of polyethylene are

Oxo-biodegradable or d2W plastics are polymers that contain a pro-oxidant additive to accelerate photo or thermo-oxidation [8, 9]. So, these polymers when exposed to ultraviolet light or at high temperatures are cleaved in low molecular mass compounds that are assimilated by microorganisms [8]. Several studies have shown the biodegradable plastics degradation, after exposed to ultraviolet light or

The plastic bags of green polyethylene are produced using low-density polyethylene (LDPE) and green polymers obtain of sugarcane [17]. We have showed the green polyethylene degradation by *Pleurotus ostreatus* PLO6 [15]. However, little

The microbial enzymes, such as depolymerase, esterase and lignolytic ones, that cleave the polymers in small chain compounds, may be involved in the plastics degradation [6, 13, 18, 19]. Thus, white rot fungi have a great potential, because they are enzymes producers and have shown their ability for treatment of industrial

The white rot, *P. ostreatus*, is a potent degrader of lignin, cellulose and hemicellulose, which lives as saprophyte in wood. This fungus has also been used in the bioconversion of agricultural residues, in biodegradation of organic pollutants, xenobiotics, and industrial effluents, in the cellulose bleaching and production of food and enzymes [23–25]. We showed that *P. ostreatus* PLO6 are capable to degrade oxo-biodegradable plastics and green polyethylene [13–15]. Furthermore, this fungus form edible mushroom that is source of proteins, fibers, minerals and carbohydrates. Thus, in this chapter described the plastics bags degradation, by abiotic and

The degradation of two plastic polymers used in the production of supermarket plastic bags was evaluated (**Figure 1**, I). The oxo-biodegradable and green polyethylene polymers were submitted the abiotic and biotic degradation (**Figure 1**, II). The oxo-biodegradable bags contain titanium oxide as pro-oxidant additive and

The abiotic degradation of the plastic bags was the exposure to sunlight up to 120 days (**Figure 1**, III). This exposure was in the summer time in a green house. In

information regarding to the biodegradation of these bags is available.

microbial process, using the exposure to sunlight and *P. ostreatus*.

**262**

*Steps and technics used for monitoring the abiotic and biotic (fungal) degradation of oxo-biodegradable e green polyethylene plastics bags.*

For the biotic degradation (**Figure 1**, IV at VIII) the plastic polymers without (**Figure 1**, II) or with the exposure to sunlight (**Figure 1**, IV) was used *P. ostreatus* PLO6 (GenBank accession number KC782771). These polymers were cut in fragments of 5 cm2 (**Figure 1**, V) and placed in a glass flask (100 mL) containing paper towel fragments (5–10 cm<sup>2</sup> ) and mineral medium (**Figure 1**, VI). The proportion of plastics and paper towel was of 99:1.

In each glass flask fours discs of agar (6–8 mm) containing the mycelium of *P. ostreatus* PLO6 were inoculated (**Figure 1**, VII). This fungus was cultivated in 20 mL of potato dextrose lignin (0.1%) agar (PDLA) for 15 days. The initial inoculum was obtained from the collection of the Department of Microbiology of Universidade Federal de Viçosa. The stock culture is maintained on PDLA at 4°C.

After inoculation the glass flask were incubated at 25°C for 30, 60, 90 and 120 days (**Figure 1**, VIII).

The alterations in plastic polymers (**Figure 1**, IX) after each time of incubation were performed (**Figure 1**, III, IV at VIII). These alterations were compared with analysis done before of the exposure to sunlight.

Physical alterations (**Figure 1**, IX a), such as wrinkles on the surface, formation of holes and cracks, crumbling, discoloration, were performed by digital photograph and scanning electron microscopy (SEM) with a magnification of 50,000 (**Figure 1**, IX a2). Mechanical properties, such as, energy at break and load at tensile strength were made in universal testing equipment (Instron model 3367) (**Figure 1**, IX a3).

Chemical changes (**Figure 1**, IX b) by Fourier transform infrared spectroscopy (FTIR) (**Figure 1**, IX b1) and SEM coupled with X-ray diffraction (**Figure 1**, IX b2) were determined. These alterations were the disappearance or formation of new functional groups in spectrum of FTIR with scanning of 500 at 4000 cm<sup>−</sup><sup>1</sup> wavenumbers and the decrease in-oxidant additive concentration by spectrum of X-ray diffraction.

The mycelial growth (**Figure 1**, IX c), the main agent of the biological alterations, was evaluated by dry mass (**Figure 1**, IX c1), respiratory activity (**Figure 1**, IX c2) determined without interruption for 120 days of incubation, electronic micrograph (**Figure 1**, IX c3), digital photography (**Figure 1**, IX c3) and lignocellulolytic enzymes activity (**Figure 1**, IX c5).

The capacity of *P. ostreatus* to produce mushrooms in plastics waste may be evaluated under the same growing conditions, steps and procedures shown in **Figure 1**. However, for mushrooms formation it is need, after the mycelial growth (about 20 days), a thermal shock that can be performed by reducing the incubation temperature to 4°C for 24 h and returning to 25°C. During the mushrooms growth, the flasks should be kept in a place at 18 ± 2°C and a relative humidity of 80%.

A total of 240 days were the time applied to degradation of the plastic bags, being 120 days of exposure to sunlight and 120 days of fungal incubation. According to the manufacturer, depending on environmental conditions, for example, the exposure to oxygen and outdoor element, oxo-biodegradable plastic bags decompose within a maximum period of 18 months after disposal [26, 27]. They also add that in only 121 days the biodegradability index of d2W plastics was 88.86% [28]. Our time of abiotic degradation is the same those used for calculating the biodegradability index and corresponds to ¼ of the required time for the decomposition of these bags. However, after 4 months of exposure to sunlight we did not observe any fragmentation of the plastic bags, only the appearance of small cracks and the bleaching of the film were observed (**Figure 2**). Da Luz et al. [14, 15] also showed changes in mechanical properties of oxo-biodegradable and green polyethylene after 120 days of exposure to sunlight. According to them, this time of exposure is insufficient for other physical or chemical changes, concluding that the mechanical properties alterations, such as the reduction of breaking energy and elasticity facilitated the fungal colonization of plastic waste. The chemical and physical changes in the low-density polyethylene (LDPE) was observed after pretreated of the LDPE sheets with low discharge plasma (O2, 3.0 × 10<sup>−</sup><sup>2</sup> mbar, 600 V) for 6 minutes [29, 30]. According to authors, this pretreated was important by plastics biodeterioration by *P. ostreatus*.

In a new experiment, we observed a fragmentation of oxo-biodegradable plastics bags after 21 months of exposure to sunlight (**Figure 3**). The control samples were cut with a scissors (**Figure 3A**), but after that exposure to sunlight, it was no longer possible to cut the bags. These plastics were easily fragmented using the hand, resulting in a powder (**Figure 3B**, **C**). This result shows that there are needed more than 18 months of exposure to sunlight to completely degradation of the plastics. However, this result is promising, shows the ability of abiotic degradation of these bags and enables new testing using these bags with exposure to sunlight in a period equal to or greater than 18 months and inoculation of microorganisms to complete degradation of the remaining polymers. Degradation analysis with *P. ostreatus* has not been performed in this experiment.

#### **Figure 2.**

*Scanning electron micrograph of oxy-biodegradable plastics before (A) and after 120 days of exposure to sunlight (B).*

**265**

plastic bags.

*Plastics Polymers Degradation by Fungi DOI: http://dx.doi.org/10.5772/intechopen.88608*

**Figure 3.**

**Figure 4.**

*shows the fragmented plastic.*

The oxo-biodegradable polyethylene degradation, assessed by carbonyl index, was observed through exposure to sunlight, up to 90 days, in soil with of moisture and pH control [28]. However, these authors concluded that the polyethylene films without pro-oxidant additive had greater structural and superficial modifications, than the films with the additive. Thus, action of the pro-oxidants by the effect of

*Spatial distribution and relative concentration of the main elements found in oxo-biodegradable bags. Analysis* 

*Oxo-biodegradable plastic bags before (A) and after 21 months of exposure to sunlight (B and C). The letter C* 

In the plastic bags the presence of titanium was identified, a component of the pro-oxidant additive (**Figure 4**). This element presents a higher relative concentration than the other elements analyzed and it is uniformly distributed on the surface of the bags. This homogeneous distribution was also observed to manganese, iron and cobalt (**Figure 4**). Furthermore, with the exception of titanium and cadmium, the other elements analyzed are important for fungal metabolism (**Figure 4**). These micronutrients may be elicitors or enzyme cofactors. Thus, the presence of these elements may also have contributed to the *P. ostreatus* growth on the surface of

Mycelial growth of *P. ostreatus* was observed on the surface of the paper towel (**Figure 5A**) and the plastic waste (**Figure 5B**). This figure shows an example of mycelial growth in oxo-biodegradable plastics after 30 (**Figure 5A**) and 90 days

sunlight depends on e conditions and time of exposure to sunlight.

*carried out by a scanning electron microscope coupled to the X-ray diffraction detector.*

#### **Figure 3.**

*Microorganisms*

The capacity of *P. ostreatus* to produce mushrooms in plastics waste may be evaluated under the same growing conditions, steps and procedures shown in **Figure 1**. However, for mushrooms formation it is need, after the mycelial growth (about 20 days), a thermal shock that can be performed by reducing the incubation temperature to 4°C for 24 h and returning to 25°C. During the mushrooms growth, the flasks should be kept in a place at 18 ± 2°C and a relative humidity of 80%. A total of 240 days were the time applied to degradation of the plastic bags,

being 120 days of exposure to sunlight and 120 days of fungal incubation. According to the manufacturer, depending on environmental conditions, for example, the exposure to oxygen and outdoor element, oxo-biodegradable plastic bags decompose within a maximum period of 18 months after disposal [26, 27]. They also add that in only 121 days the biodegradability index of d2W plastics was 88.86% [28]. Our time of abiotic degradation is the same those used for calculating the biodegradability index and corresponds to ¼ of the required time for the decomposition of these bags. However, after 4 months of exposure to sunlight we did not observe any fragmentation of the plastic bags, only the appearance of small cracks and the bleaching of the film were observed (**Figure 2**). Da Luz et al. [14, 15] also showed changes in mechanical properties of oxo-biodegradable and green polyethylene after 120 days of exposure to sunlight. According to them, this time of exposure is insufficient for other physical or chemical changes, concluding that the mechanical properties alterations, such as the reduction of breaking energy and elasticity facilitated the fungal colonization of plastic waste. The chemical and physical changes in the low-density polyethylene (LDPE) was observed after

pretreated of the LDPE sheets with low discharge plasma (O2, 3.0 × 10<sup>−</sup><sup>2</sup>

by plastics biodeterioration by *P. ostreatus*.

not been performed in this experiment.

600 V) for 6 minutes [29, 30]. According to authors, this pretreated was important

*Scanning electron micrograph of oxy-biodegradable plastics before (A) and after 120 days of exposure to* 

In a new experiment, we observed a fragmentation of oxo-biodegradable plastics bags after 21 months of exposure to sunlight (**Figure 3**). The control samples were cut with a scissors (**Figure 3A**), but after that exposure to sunlight, it was no longer possible to cut the bags. These plastics were easily fragmented using the hand, resulting in a powder (**Figure 3B**, **C**). This result shows that there are needed more than 18 months of exposure to sunlight to completely degradation of the plastics. However, this result is promising, shows the ability of abiotic degradation of these bags and enables new testing using these bags with exposure to sunlight in a period equal to or greater than 18 months and inoculation of microorganisms to complete degradation of the remaining polymers. Degradation analysis with *P. ostreatus* has

mbar,

**264**

**Figure 2.**

*sunlight (B).*

*Oxo-biodegradable plastic bags before (A) and after 21 months of exposure to sunlight (B and C). The letter C shows the fragmented plastic.*


#### **Figure 4.**

*Spatial distribution and relative concentration of the main elements found in oxo-biodegradable bags. Analysis carried out by a scanning electron microscope coupled to the X-ray diffraction detector.*

The oxo-biodegradable polyethylene degradation, assessed by carbonyl index, was observed through exposure to sunlight, up to 90 days, in soil with of moisture and pH control [28]. However, these authors concluded that the polyethylene films without pro-oxidant additive had greater structural and superficial modifications, than the films with the additive. Thus, action of the pro-oxidants by the effect of sunlight depends on e conditions and time of exposure to sunlight.

In the plastic bags the presence of titanium was identified, a component of the pro-oxidant additive (**Figure 4**). This element presents a higher relative concentration than the other elements analyzed and it is uniformly distributed on the surface of the bags. This homogeneous distribution was also observed to manganese, iron and cobalt (**Figure 4**). Furthermore, with the exception of titanium and cadmium, the other elements analyzed are important for fungal metabolism (**Figure 4**). These micronutrients may be elicitors or enzyme cofactors. Thus, the presence of these elements may also have contributed to the *P. ostreatus* growth on the surface of plastic bags.

Mycelial growth of *P. ostreatus* was observed on the surface of the paper towel (**Figure 5A**) and the plastic waste (**Figure 5B**). This figure shows an example of mycelial growth in oxo-biodegradable plastics after 30 (**Figure 5A**) and 90 days

**Figure 5.**

*Mycelial growth of* Pleurotus ostreatus *after 30 days of incubation in paper towel and oxo-biodegradable plastic bags after 30 (A) and 90 (B) days of exposure to sunlight. The arrow and circle show mycelial growth.*

(**Figure 5B**) of exposure to sunlight and 30 days incubation with the fungus. The yellow circle and the red arrow show, respectively, the mycelial growth on the paper towel and plastic. This paper was added in the culture medium to retain moisture and to be an inducer of fungal growth and stimulates the synthesis of lignocellulolytic enzymes that degrades the paper itself and the plastic (**Figure 5**).

The respiratory activity of *P. ostreatus* was influenced by time exposure to sunlight. This result confirms that the physical changes caused by sunlight contributed to the fungal growth in plastic bags. Furthermore, we did not observe reduction of this activity until 90 days of incubation showing a cellular activity for a long period. The activity of lignocellulolytic enzymes, like laccase, cellulase and xylanase, during 45 days of incubation of *P. ostreatus* in oxo-biodegradable plastics was observed [13].

The *P. ostreatus* growth on the surface of oxo-biodegradable plastic was also observed by SEM (**Figure 6**). In this micrograph, the red arrows show hyphae in plastic waste after 60 days of exposure to sunlight and 30 days of incubation. Da Luz et al. [13–15] showed the formation of mycelium on the surface of d2w plastic and green polyethylene with different time of exposure to sunlight and of fungal incubation. They also reported the morphological characteristics of the mycelia of *P. ostreatus* PLO6.

#### **Figure 6.**

*Scanning electron micrograph of oxo-biodegradable plastics after 60 days of exposure to sunlight and 30 days of incubation with* Pleurotus ostreatus*. The arrows show the hyphae.*

**267**

**Figure 7.**

**Figure 8.**

days of incubation with benthic microbes [3].

*(0uv) or after 30, 60, 90 and 120 days of exposure to sunlight.*

The loss of plastic dry mass was influenced by the time of exposure to sunlight and fungal incubation (**Figure 7**). Fungal growth was lower in plastic polymers without exposure to sunlight than in others with different time of exposure to sunlight. This result shows that *P. ostreatus* can grow in plastic waste without or with exposure to sunlight. However, this exposure facilitates the fungal growth, as shown by Da Luz et al. [14, 15]. Thus, the combination of abiotic and biotic processes shows to be more efficient in the oxo-biodegradable plastic and green polyethylene degradation. In addition, the presence of other carbon sources from marine sediments and lack of abiotic degradation as the initiator were the main factors of the lack of biodegradation of polyethylene and biodegradable plastic bags after 100

*Scanning electron micrograph of oxo-biodegradable plastics bags after 30 days of exposure to sunlight and 30 days of incubation with* Pleurotus ostreatus*. Micrograph without (A) and with a scale of 100 folds (B).*

*Loss of dry mass plastic after inoculated with* Pleurotus ostreatus *in oxo-biodegradable plastic bags before* 

In this study, we observed the formation of cracks and holes in oxo-biodegradable plastics and green polyethylene after fungal growth (**Figures 8** and **9**).

*Plastics Polymers Degradation by Fungi DOI: http://dx.doi.org/10.5772/intechopen.88608*
