Biodegradation of Polypropylene By Pseudomonas Aeruginosaisolated From Wastewater Associated Soil; A Potential Method To Eliminate The Plastic Pollution To Save Ecosystem

The face mask introduction in SARS-CoV-2 pandemic situation, one of the precautionary measure to reduce the rate of transmission of infection from person to person. There are many type of of face masks enter into our global market with various size and designs. Among those, surgical mask belongs to secure an important place and this aimed to evaluate the degrading eciency disposable face masks (single use face masks). The present study dealt with, biodegradation of face masks which is made of polypropylene with the help of bacterial strainPesudomonasaeruginosaisolated from the plastic waste dumping sites in an around the Tiruchirappalli, India. The bacteriacould degrade PP mask via the formation of biolm on a solid medium. To degrade the PP, the mask pieces were incubated with the culture of P. aeruginosain three different solid and liquid medium (nutrient agar, Bushnell Haas agar and mineral salt medium) for 30 days at 37(cid:0)C. The microbial degradation(up to 33% of weight reduction of PP lms within 30 days) was proved by surface changes along with the variation in the intensity of functional groups as well as carbonyl index variations using Field-Emission Scanning Electron Microscopy (FE-SEM) and Fourier Transform Infra-Red Spectroscopy (FTIR) analysis. These results suggested that P.aeruginosa strain can prove to be a suitable candidate for polypropylene mask biodegradation without causing any impairment to our health or environment. that P.aeruginosa cells better colonization, of PP lm in the three media. These only specify cells for the PP lm but also increase the possibilities of P.aeruginosa biolms interactions at low levels of carbon availability. In model, the course of action of biolm formation by microbes when cells density and help cells


Introduction
The ongoing quick universal spread of SARS-CoV-2 pandemic situation has led to the critical efforts to diminish transmission, leading to signi cant and extensive socioeconomic disruption(Sohrabiet al, 2020). On or after 3rd March 2020, the con rmed COVID-19 cases are about 90,870. From that con rmed cases 80,304 were con ned to China. The remaining 10,566 cases were belongs to other 72 countries including Japan, United Nations and Australia of these cases 166 were fatal (the Japan, Philippines, Italy, France, Iran, Australia, Korea, Thailand and the United Nations). It is important to note that these data's were depicted laboratory -con rmed results only(WHO, 2020).Near July 2020, over 10 million cases have been reported globally, as well as around 50,000 death cases were recorded with going on spread in most of the parts of our earth(COVID, 2020). The corona virus (COVID-19) pandemic has caused the development of emergency responses and progressive moves were aimed to deal with and overcoming COVID-19 virus while reduces the exposures and supporting healthy life of all people around the world. One of such precautionary measure designed to minimize the community spread of COVID-19 transmitted via droplet is the compulsory wearing of plastic-based personal protective equipment (PPE) by the healthcare workers and face masks for the public leaving their homes for important necessary reasons (Benson et al, 2021).
The use of face masks by public people can minimizes the community spread of COVID-19, but it also develops severe troubles for our ecosystem. The number of face masks used in 49 countries of Asia was estimated from the COVID-19 pandemic database on July 31, 2020. The result of estimated value of the daily face mask use in Asia is 2.2 billion (Sangkham, 2020). There are three types of face masks can use in current situation i) Respirator Mask (N95, FFP2), ii) Surgical or Medical mask and iii) Non-certi ed disposable-mask (cloth mask)(Dharmarajet al, 2021). Top and bottom layers of disposable (single use) face masks were made up of woven fabric and the middle layer were meltdown polypropylene polymers.
It was estimated that the authors models were generated 6.88 billion (approximately 206,470 t) around the world each day, which are eventually sent to land lling or incinerated after use (Nzediegwu and Chang, 2020). Also, the used face masks can be inappropriately spotted everywhere from the city streets to local parks, due to its lightweight, the rain water and wind can move this mask freely into city streets or rivers and oceans, where it was fragmented into microplastics(Prataet al, 2020; Saberianet al, 2021). In addition, the face masks used as single purpose are made up of non-biodegradable and take more than hundreds of years to break down in the environment(Saberianet al, 2021; Dhawanet al, 2019). As a result, a multidisciplinary approach is needed immediately to ght against the COVID-19 pandemic as well as to reduce the environmental troubles associated with the disposal of used personal protective equipment's.
In order to overcome the negative environmental issues due to the pandemic situation many authors works to reduce the pandemic-generated waste management in an innovative way. One of the best and eco-friendly approaches is the use of microorganisms for waste management. The investigation of microorganisms towards polypropylene and polyethylene degradation is well studied by several authors.
Biodegradation is one of the best ability of microorganisms to persuade abiotic degradation by physical, chemical of enzymatic action(Albertssonet al, 1987). For biodegradation process microorganisms were well suited for their ability to possess enzymes and the small size make them able to contact with the surface of the plastic (Devi et al, 2016). The microbial exo-enzymes can breaks the complex polymers to simpler one which was then easily pass through the semi-permeable outer membranes and then it can be used as carbon and energy sources of the microbes (Gu, 2003). A number of bacterial and fungal genera were reported by several researchers for microplastic degradation process including Pseudomonas sp(Gupta and Devi, 2020; Vague et al, 2019; Muniret al, 2018; Roberts et al, 2020). The ability of microbes to mediate plastic degradation directly there are several gut bacterial species were isolated from worms and veri ed. In vitro depolymerization of PE can be done by the bacterial strains namely Enterobacterasburiae YT1 and Bacillus sp. YP1 isolated from the waxworms (Yang et al, 2014). P.aeruginosa isolated from the guts of superworms can have extradinarybiodegradative ability on four types of plastics (PE, PP, PPS and PP). On the other hand the e ciency of biodegradation varied from one plastic to another, the fastest biodegradation occurs on PE (Lee et al, 2020). The present study was aimed to evaluate the biodegradation ability of P.aeruginosa on surgical face mask with eco-friendly manner and to save our environment from pandemic-generated wastes.

Chemicals and Reagents
The soil sample was collected from the plastic waste dumping sites around the Tiruchirappalli district, Tamil Nadu, India. Then the sample was carefully transported for our laboratory in order to isolate the plastic degrading Pseudomonas sp. The used face masks were not allowed to use in our laboratory test due to the laboratory rules and regulations during this covid-19 pandemic situation. Therefore, clean surgical face masks were used in the experiments (ear loops were removed from the masks). The chemicals used in our study were purchased from Hi-media laboratory, India.

Isolation of bacteria from soil sample
Serially diluted soil sample was inoculated onto the previously prepared sterile nutrient agar medium (NAM) and incubated at 37 C for 24-48 hours in order to isolating bacteria from the sample. Individual colonies obtained after incubation were used for the preparation of pure culture. The pure colonies were subjected to Gram's staining for primary identi cation. Then the isolated bacterium was maintained on Nutrient agar at 4 C in our laboratory for performing the biodegradation experiment in our present study.

Biochemical identi cation of the bacterium
The isolated bacterial strain was identi ed based on the basic biochemical tests. The biochemical tests were conducted on 24 hours old cultures grown on Nutrient agar medium at 37 C. Indole test was performed in order to determine their tryptophan utilization ability according to the supplier protocol. Catalase analysis was conducted according to Kumariet al, 2013.Citrate metabolism was studied by inoculating bacterial colonies on Simmon's Citrate agar (According to supplier protocol).Oxidase test was conducted in order to check the bacteria that possess the enzyme cytochrome oxidase. Sugar fermentation test (Glucose, Lactose, and Sucrose) was studied by inoculating bacteria onto the TSI agar medium (According to supplier protocol).Motility and H2S production test were conducted using SIM medium (According to supplier protocol). The MR-VP test was done to identify the isolate as facultative anaerobe by their sugar fermentation pro les. Also the bacterium was streaked onto the Cetrimide agar for further clari cation of the bacterial identi cation processes.

Pretreatment of Polypropylene mask pieces
The metal strips and ear loops were removed from the masks. Then the masks were pretreated by the method previously described by Gupta and Devi (2020). For this process the masks were cut into (3×3 cm) pieces and soaked into the solution containing 7ml of Tween-80, 10ml of bleach and 983ml of sterile water for 30-60 minutes with continuous stirring. Then the mask pieces were washed with distilled water at room temperature. Then the pieces were surface sterilized with 70% ethanol for 30 minutes and allowed to dry at 45 C. After drying the mask pieces were weighed using the weighing balance and initial weight of the pieces was noted.

Biodegradation Experiment
The bacterium P.aeruginosa was aseptically inoculated by carpet culture method on to Nutrient agar, BHM agar and MSM agar plates. After inoculation the pretreated polypropylene mask pieces (0.1 g) were aseptically placed over the inoculated plates using sterile forceps and incubated at 30 C and 37 C separately for one month (30 days). The agar plates containing the same quantity of mask pieces without bacterial inoculation were maintained at the same temperature as negative control. Also the pretreated mask pieces were incubated with P.aeruginosa in NB, BHM broth and MSM broth (each media contain 0.1g of mask pieces) and the liquid media containing same quantity of mask pieces without culture maintained at the same duration as control. Monitoring the planktonic growth of P.aeruginosa strain The growth of the P.aeruginosa in the liquid media such as NB, BHM and MSM owing to the presence of PP lm and the characteristics of the bio lm produced on the surface of the polymer throughout the period of 30 days (1 month) with 15 days interval was scrutinized as explained below. Before analyzing the bio lm, polymer samples were taken away from the media by means of sterile forceps and cautiously rinsed with sterilized distilled water in order to con scate the slackly adhered bacteria. Then the bio lm was removed from the surface of the polymer by subjecting the sample to a mild water bath sonication in 1 ml of 0.85% saline solution, for a total of 4 min at 1 min intervals. Thus obtained saline solution was serially diluted up to 10 −7 and the aliquots were spreaded on Nutrient agar and the number colonies were calculated as CFU/ml (Arkatkaret al, 2010).

Viability testing of Surface attached bacteria
The viability of bacterial strains attached on the surface of the PP lms was tested as per the method described by Andes et al (2004). PP lms were removed from the media at 10 days interval and washed with sterile distilled water then it was subjected to mild bath sonication with 0.85% saline solution. The obtained solution was serially diluted and plated on NA and incubated at 35°C for 48 h. Number of viable bacteria species were obtained as CFU/ml.

Assessment of cell surface hydrophobicity of P.aeruginosa
The hydrophobicity of the bacterial cell surface was determined using the BATH test by little modi cations (Harshvardhan and Jha, 2013) was explained as follows: the bacteria were cultured in Nb medium until the growth of bacteria reach the mid-log stage. Then it was centrifuged and washed twice with phosphate urea buffer (PUM containing 17g K2HPO4, 7.26g KH2PO4, 1.8g Urea and 0.2g MgSO4.7H2O per liter). After washing the cells were resuspended in PUM buffer to an OD at 400nm (OD400) value of 1.0-1.2. Aliquots of 1.2 ml of above obtained suspension were added to a series of test tubes containing increased volumes of xylene (ranges from 0-0.2ml) and shake well for 10 min then allowed to stand for 2 min in order to facilitate phase separation. The OD400nm of the lower aqueous phase was then measured and noted as OD1. The cell surface hydrophobicity was stated as the percentage of the xylene -bound cells described as the fraction of the cells expelled from the aqueous phase. Plain PUM buffer served as the blank. Therefore, the percentage of adhering cells are represented using the following formula: Cell Surface Hydrophobicity (%) = ((OD 0 -OD 10 )/ OD 0 ) ×100 (OD 0 is the initial OD of the aqueous phase).

Determination of Dry weight of the recovered Mask pieces
The residual mask pieces were recovered from the culture plates after the completion of one month incubation. The bacterial biomass adhering on the polypropylene mask surface was washed with the sodium dodecyl sulfate (SDS) solution (2%v/v) for 2 hours. After the pieces were rinsed with distilled water in order to remove any impurities on the surface and dried overnight at 45 C. Then the degraded mask pieces were weighed using weighing balance and the percentage of bio degradation (weight loss) was calculated using the following formula Surfaceanalysis of mask pieces

Scanning electron microscopy
The mask pieces treated with the bacterium P.aeruginosa for 30 days were removed from the medium and subjected to FE-SEM for the observing bio lm formation and surface erosion. Bacterial morphology of bio lm on PP mask surface was observed. Before the observation, the treated mask pieces were washed with 0.01M phosphate buffer solution for 2 min in order to remove the excess medium adheres with the bacterial colonies. For observing the surface alteration of treated mask pieces, the pieces were washed with 2% SDS along with warm water for 10-20 min to facilitate the complete removal of bacterial biomass. After the process was completed, the mask pieces were xed in 4% glutaraldehyde at 4 C for 2 hours and dehydrated with 50% ethanol for 30 min. Then the recovered mask pieces were incubated

Results And Discussion
Isolation and Identi cation of Pseudomonas sp.
Typical colonies of bacterial isolates were sub-cultured on nutrient agar and incubated at 37°C for 24 h.
Isolated identi cation was performed according to the morphological, staining reaction, cultural and various biochemical characteristics by following the Bergey'sMannual of Systematic Bacteriology. Based on presumptive identi cation of bacteria by Gram's staining protocol Pseudomonas sp. was identi ed as gram negative rod. Depending upon the different biochemical characterization the isolated bacteria was identi ed as P. aeruginosa strain (Fig. 1). It was further con rmed by streaking the strain onto the Cetrimide agar which is selective media for isolating P.aeruginosa. The isolates were showed green pigmented, circular and opaque colony morphology after incubation. Similar to ours study, the another author reported that the isolated bacteria were also grown in milk agar with cetrimide for the preliminary detection of Pseudomonas spp. (Szitaetal, 1998). Based on the results it was reasonable, to identify the isolated bacterium as P. aeruginosa. This identi ed strain was used for the biodegradation of pre-treated polypropylene mask pieces. The bacterium P.aeruginosa isolated from Sisdol land ll site and Sanothimi household garbge site soil in Nepal has similar biochemical properties(Badahitet al, 2018). It has been reported that P. aeruginosa ISJ14 showed tremendous e ciency to degrade low density polyethylene (LDPE) in BHM(Gupta and Devi, 2020).In contrast, the present study reports for the rst time that P.aeruginosastrain is capable of biodegradation of surgical face mask made up of hardy PP like substance.

Cell surface hydrophobicity of bacteria
The capability of bacteria to use any substrate depends upon its development on and adherence to that substrate. The adhesion ability of bacteria to either hydrophilic or hydrophobic surfaces is addressed by a number of physical factors, together with the forces which help the bacterium to adhere to solid substrates, properties of that substrate and the nature of bacteria. For the most part, a hydrophobic bacterium favors a hydrophobic surface for adhesion, though the inverse is substantial for bacterium with hydrophilic properties (Gupta and Devi, 2020). In the current investigation, the hydrophobicity of midlog phase cells of P.aeruginosa at 0.2ml concentration of xylene, we showed a signi cance increase in hydrophobicity (30.39%) ( Table 1 and Fig. 2). These results are in agreement with Gupta and Devi (2020), who observed that bacterial cells in log phase are more hydrophobic in nature. Previous study also documented similar ndings, where, maximum increase in hydrophobicity i.e, the isolates KocuriapalustrisM16 andBacillus subtilis H1584 showed approximately 24% turbidity reduction at 0.25 µl and a maximum reduction of turbidity 32% at 150µl concentration of hydrocarbon like hexadecane (Harshvardhan and Jha, 2013). Another recent research also reported that the hydrophobobicity of L.monocytogenes strain CICC 21332 showed lowest hydrophobicity (12.5%) and the strain FSIS 57034 displayed highest percentage of CSH (74.81%) at 1ml of xylene concentration(Fun et al, 2020).  Figures 3 and 4 show a surface attachment pattern of bacteria on the PP lm. The bacterial cell growth was characterized by a precipitous raise in planktonic cells after 15 and 30 days of incubation and is also revealed by a raise in the surface-attached bacterial mass. After 15-20 days of incubation the growth of P.aeruginosa was able to reach a steady, almost 10 7 CFU/ml in all the liquid media used. In all the three media, bio lm formation patterns exhibited analogous to that of the growth of planktonic cells.
The results recommend that P.aeruginosa cells exhibit better colonization, formation of bio lm and fractional biodegradation of PP lm in all the three media. These observations not only specify high a nity of P.aeruginosa cells for the PP lm but also increase the possibilities of P.aeruginosa cultures to form bio lms by hydrophobic interactions at low levels of carbon availability. In considering of concurrence model, the course of action of bio lm formation by microbes is began when the planktonic growth of cells achieves high density and help for the attachment of bacterial cells to a surface through cell signaling, likewise bringing about the development of microcolonies that will eventually frame the mature bio lms (Costertonet al, 1999). This bio lm population is diverse and comparativelysteadyintendedforextensiveperiod of time (Bodtkeret al, 2008). Conversely, this circumstance is not inevitably familiar to the entire bio lm forming bacterial species. In our investigation, PP lms provided as a substrate for the attachment and bio lms formation as well as a source of carbon for P.aeruginosa strain. The extended incubation of P.aeruginosa with PP lms resulted in a solid bio lm on PP surface which may lead to the fractional deduction of this polymer. Likewise, the formation and maintenance of active bio lm throughout the 30 days of incubation possibly will perhaps by the utilization of low molecular massmaterials in the polymer. Similar ndings were recorded in earlier studies such as biodegradation of untreated lms of polyethylene by P.putita IRN22, Micrococcus luteus IRN20, Acinetobacterpittii IRN19 (Montaveret al, 2019) and other bacterial genera including Delftia, Stenotrophomonas and Comamonas(Peixotoet al, 2017) and Galleria melonella isolated from the gut of the wax worm also have been establish the capabilities of PE degradation (Cassoneet al, 2020).

Dry Weight Determination of recovered Polypropylene Mask pieces
The residual polypropylene mask strips were recovered from the media after one month (30 days) of incubation. The adhered media and bacterial biomass were washed with appropriate solutions and allowed to air dry. The air dried lms were weighed and the nal weight loss for P.aeruginosa in respective culture media (NA, NB, BHM agar, BHM broth, MSM agar and MSM broth) has been provided in table 4 and Fig. 5a and 5b. The growth kinetics of the P.aeruginosastrain in media demonstrated its colonization on PP mask pieces surface consequently, reduction in weight was observed as a result of utilization of pp mask lm as nutrient source. Our study describes the potency of the P.aeruginosastrain was very high because it shows 26 and 33 % of weight reduction in case of PP lms placed in NB and NA, 28 and 31% weight reduction observed in BHM agar and BHM broth and 24 and 23% weight reduction was noted in MSM agar and MSM broth after one month duration (30 days (Howard and Hilliard, 1999). Our results were supported by various previous research studies noticed the formation of functional groups and disappearance of these groups in the LDPE degradation using the strainBacillus amyloliquefaciens (Das etal, 2015). In relation to our study, (Gajendiranet al, 2016) have also noticed visible modi cations in the synthetic polymers undergo biodegradation, before and after exposure to microbes by FTIR analysis. The conformational changes on PP mask lm were supported by the changes in the peak values of almost all functional groups (Fig .7a-7l).

Conclusion
In sum, this work offered a versatile biological process to evaluate the degradation of disposable face mask used in this SARS-CoV-2 pandemic situation. The in vitro biodegradation of P.aeruginosastrainin three different solid and liquid medium re ects the eco friendly approach. We observed a tremendous biodegradation e ciency of our isolates towards the PP based disposable face mask whose molecular weight was as high as 228,000. The isolate has a capability to forming bio lm on PP surface and utilized it as a sole nutrient source for growth was re ected by weight reduction of PP mask lms in comparison with un-treated control lms (up to 33% of weight reduction within 30 days). Based on the results of FE-SEM as well as FTIR analysis, the P.aeruginosastrain is a best suited candidate for PP degradation without an UV treatment. However, further studies on enzyme based metabolic passages of P.aeruginosastrain are also recommended to better understanding of its tremendous role in the process of biodegradation. Declarations