Carbon Microsphere From Postconsumer Soft Drink Bottles and Their Impact On Plant Growth Study of Cicer Arietinum


 Present work is highlighted on the conversion of waste PET plastics to carbon nanosphere, their characterization by SEM-EDX, XRD and FTIR and finally their application in the field of germination of Cicer arietinum and biochemical analysis. SEM and XRD results revealed that PET plastic are comfortably converted to carbon microsphere with a diameter ranges between 2-8 µm with amorphous nature and FTIR study suggested that the existence of aromatic C-H and aromatic ring along with carbonyl groups. Root morphology suggested that both root length and seminal root number gradually decrease with increasing carbon microsphere dose. Biochemical results revealed that the level of proline, catalase and MDA levels significantly (p < 0.0001) increase with increasing the dose of carbon microsphere. Finally, it can be concluded that lower dose of carbon microsphere could be beneficial for both seed germination and seedling growth, but higher dose may have adverse effect on plant community.

For conversion of plastic materials to a valuable products is the only way to solve the environmental pollution problem as well as economic bene ts (Zhang et al. 2021;Choma et al. 2015). Waste plastic can be converted to various carbonaceous products including carbon based nanoparticles (Kharissova et al. 2021; Liu et al. 2020; Altalhi et al. 2013). This type of waste materials transformation to a valuable products leads to the reduction of pollution burden as well as economic bene ts. Previous researchers (Altalhi et al. 2013; Altalhi et al. 2011) highlighted that non-degradable plastic bags can be connected to multi-walled carbon nanotube (MwCNTs).
Polyethylene terephthalate is a polymeric form of ethylene terephthalate. It is absolutely a nonpolar polymer. With good mechanical barrier and optical properties. Moreover, its chemical inertness is possibly due to presence of high amount of aromatic substance. Annual production rate of this polymer is greater than 56 million tons (Saxena, 2016) and it is also reported that the total production of plastics will reach to 26000 million tons by 2050 (Lennstedt and Eklov, 2016;Geyer et al. 2017). In review of the increasing environmental awareness, recycling remains the most viable option for the treatment of waste PET.
The current practice of plastic waste disposal includes incineration and land lling, including physical and chemical recycling (Chen et al. 2021). However, land lling is the most accepted techniques among the developing countries (Ru et al. 2020). Similarly, incineration is also a reliable technique. But both land lling and incineration of plastic waste leads to environmental pollution. Therefore, recycling of waste plastics through greener technique will be the most viable option. Cursory review of literature revealed that some speci c microorganism have tremendous power to degrade synthetic plastics through secretion of various enzymes (Ru et al. 2020). However, plastic waste can be degrade by using nanoparticles also (David et al. 2020;Maity and kumar, 2016;Mallakpour and Javadpour, 2016). Previous report also highlighted that plastic polymer can be converted to valuable carbon based materials Extensive application of nanomaterials in various elds led to the accumulation of such materials in different compartments of the ecosystem. Therefore, it is extremely important to understand the ultimate fate, migration and impact of such materials on the different compartment of the environment. Keeping in mind the above fact the synthetic plastic waste may be considered as a precursors of carbon source. The ubiquitous polymers such as polyethylene terephthalate is used as a valuable carbon source through thermal cracking and same is used for germination of Cicer arietinum seeds and both morphophysiology and biochemical analysis were performed in laboratory settings. As per our current knowledge, this is the rst report of plastic waste origin carbon microsphere (CMS) on plant community.

Preparation of carbon microsphere
After collection of waste PET plastic bottles from nearby restaurant. Then thoroughly washed with soap water to remove any grease or dirt. Then the bottle was cut into small piceses (2mm). About 2.0 g small pices of plastic was taken in an air-tight stainless steel container without any catalyst. Then the container put into mu e furnace and heat the materials at 800°C for 1h. After overnight cooling, the material was grinded by mechanical grinder and kept in an airtight container.

Characterization of carbon microsphere
The prepared carbon microsphere were characterized by various analytical tools. Initially, small amount of sample was send for SEM-EDX study. SEM analyzer (HITACHI, S-530, Scanning Electron Microscope and ELKO Engineering) at an accelerating voltage of 20.0 kV was used. The Fourier transform infrared (FTIR) study was carried out for identi cation of various functional groups associated with carbon microsphere by using FTIR (Cary 630, Agilent Technology).

Design of experimental set up
Present experiment is conducted in the Environmental Chemistry Laboratory, Department of Environmental science, the University of Burdwan, Burdwan, W.B. Three main concentrations of CMS viz, control (0 mg/L), 100, 200 and 500 mg/L were used for the treatment of C. arietinum seeds. Before adding CMS, seeds were sterilized with 1% sodium hypochlorite solution for one minute followed by washing with double distilled water. Wet Whatman No. 1 lter paper was placed in Petriplate (diameter 90 mm) and sterilized seeds were placed on it and proper cover with lid and incubate at room temperature (28 ± 2˚ C). Carbon microsphere solution (10 mL) was added in each Petriplate after proper sonication in two days interval. Each treatment was replicated thrice following complete randomized design. The entire experimental growth period was 15 days. All the growth and biochemical parameters were measured and presented in graphical and tabular form.
Germination, root and shoot length measurement Percentage germination was calculated in three different incubation period (24, 32 and 40 h) until maximum germination was observed. Following formula was used for calculation of germination: At the end of experimental period, both control and treated plants was sacri ced for the measurement of root and shoot length by using meter scale, white paper and ordinary microscope.

Fresh and dry biomass
Fresh weight of both roots and shoots were measured after through washing of harvested plants and tissue paper was used to soaking the water and weighing by digital balance (Sartorious BSA124 S). Similarly, both roots and shoots were placed in Borosil made Petridish and heated in hot air oven at 70 ˚C for 3 days and nal weight was taken by digital balance.
Estimation of amino acid, protein and carbohydrate Amino acid was measured by following the standard protocol (Moore and William, 1954). Similarly, carbohydrate was estimated by anthrone method (McCready et al. 1950). The protein content in both control and treated plant samples were measured by the method as proposed by Lowry et al. (1951). All these parameters was calculates by using standard curve (Mondal, 2017).

Estimation of enzymatic and non-enzymatic antioxidants
Catalase activity: The enzymatic antioxidant was measured by following the method of Sinha (1972) and the activity was expressed as µm H 2 O 2 / g protein/ min (Tanase et al. 2013).

Ascorbic acid
The non-enzymatic antioxidant, ascorbic acid content was measured according to the protocol described MDA level MDA was measured from leaf sample (0.1 mg) of C. arietinum. Initially leaves were homogenized with 10 ml 0.1 % (w/v) tricholoacetic acid followed by centrifuged at 15000 × g (4˚C) for 10 min. Then 1 ml supernatant was mixed with 4 mL 0.5 % thiobarbutric acid and incubated at 95 ˚C for 30 min. Then the reaction was terminated by cooling (ice) and the absorbance was read at 532 and 600 nm (Heath and Packer, 1968).

Evaluation of cell death
The cell death of C. arietinum roots were examined by Baker and Mock (1994). Measurement of ion leakage from root (Mondal 2017). The percent injury of the membrane was calculated by using the formula (Eq. 2). The electrolytic leakage of control and treated plant roots were used for the measurement of conductivity by following the method Valentoric et al. (2006) with some modi cation. The conductivity was measured by portable conductivity meter (Systronic 304).
Where, RIL = Root ion leakage ; EC 1 and EC 2 are the electrical conductivity at 25 -30 ˚C after shaking for 24 h and autoclaved at 120 ˚C for 20 min and cooled at 25 ˚C, respectively.

Statistical analysis
One way ANOVA followed by Tukey's post hoc test was used to test signi cance (p < 0.05) of data i.e. percentage of germination, root and shoot length, fresh and dry weight of roots and shoots, pigment level (chl 'a', chl 'b', total chl and carotenoid), carbohydrates, protein, amino acid, ascorbic acid, catalase, proline and MDA content. Error bars indicates the standard error at 5 % level of signi cance.

SEM-EDX study
Scanning electron micrograph study along with EDX was used to evaluate the morphology of PET carbon microsphere and it is presented in Figure 1. From the Figure 1, it is clearly demonstrated that PET plastic carbon microsphere are uniformly sphere with diameter 2-8 µm. On the other hand, EDX signature revealed the existence of carbon. Almost similar result was reported by Wei et al. (2011) for the thermal dissociation of PET in a supercritical-CO 2 system at 500°C for 3h. They also reported that perfect spherical carbon with 1-5 µm sized can be achieved at 600°C and these carbon spheres are smoother surface. Present study structurally and morphologically exactly same as reported by Wei et al. (2011). Very minute observation also revealed that the existence of small particles on the surface of the majority of the spheres. These small particles on the spheres are may be the carbon pieces (Wei et al. 2011).

FTIR study
Fourier transform infrared spectroscopy is an important analytical study by which important functional groups associated with nanoparticles can be identi ed (Bhaumik et al. 2017).

XRD study
The XRD diagram of the PET plastic origin carbon microsphere sample is shown in Fig. 2b

Root and shoot length
The variation of root and shoot length was recorded under di cult treatment conditions at 10 and 15 days interval (Fig. S 2 and S 3). After 10 days, root length of all treatments showed signi cantly (p < 0.001) different from control (Fig. 3). Similar, signi cant different (p < 0.039) in root length was also recorded after 15 days (Fig. 3). On the other hand, the variation of shoot length in both time intervals (10 and 15 days) showed non-signi cant variation (Fig. 3). Present results showed good agreement with the

Fresh and dry biomass
Fresh and dry biomass of shoot and root are presented in Table 1. The results revealed that highest fresh weight at treatment T3 compared to control. However, highest root weight was recorded at treatment T 4 with respect to control. Similar non-signi cant results were recorded for dry mass of both root (p < 0.625) and shoot (p < 0.417). Therefore, present nding suggest that fresh weight is accelerated than dry weight. Almost similar improvement in fresh weight under treatment of carbon nanotubes on radish (Haghighi et al. (2014) and switchgress (Pandey et al. 2018) was reported. demonstrated the application of graphene and carbon nanotube on tomato seedlings leads to increase chlorophyll content (chl 'a', chl 'b', total chl). However, they also suggested that the level of chlorophyll is higher in graphene application than carbon nanotube.
Carbohydrate, protein and ascorbic acid level Amino acid, proline and MDA level Present study also assessed the amino acid level under different treatment conditions. From the present nding, it has been found that amino acid level gradually increase with increasing treatment concentration. (Table 2). One way ANOVA analysis revealed that there is signi cant (p < 0.0001) variation among different treatments ( Table 2). Present study results are very much consistent with the earlier ndings as reported by Mehrian et al. (2015). Proline is multifunctional amino acid which is also a good plant stress (biotic or abiotic) indicator (Senthil Kumar and Mysore, 2012). Proline level under treatment of various doses of PET plastic carbon microsphere leads to gradual increase of proline levels ( Table 2). These data clearly suggested that under treatment of carbon microsphere, some sort of stress is generated inside the plant. One-way ANOVA (< 0.0001) clearly endorse the same. During stress condition, the enhancement of proline level is due to proper maintenance of osmotic balance, reduction of ROs, stabilization of membrane (Ahanger and Agarwal, 2017) and protein also proper maintenance of redox potential (Wahid et al. 2007 Antioxidant defense system of plants can function as enzymatic or non-enzymatic activity towards control on reactive oxygen species. Present outcome highlighted in Table 2 where it is clearly revealed that with increasing CMS dose, catalase level increased statistically (p < 0001). Almost opposite results was reported by Shabnam and Kim (2018). They recorded that, nano aluminium has no impact on antioxidant enzyme including catalase.

Cell death
The assessment of cell death was measured through the estimation of root ion leakage (Mishra et al. 2021) and results are depicted in Table 2

Conclusion
Present nding highlighted that commercial PET bottle can be transformed into carbon microsphere which is con rmed from Scanning Electron Micrograph-Energy Dispersive X-ray analysis (SEM-EDX) and Fourier Transform Infrared Spectroscopy (FTIR). Moreover, PET plastic origin carbon microsphere was applied on the germination and seedling growth of Cicer arietinum. The germination result revealed that within 30 h, only lower treatments (T1 and T2) along with control was fully germinated and after 40 h, all treatments exhibited complete germination. The root and shoot length was recorded maximum at minimum dose. On the other hand, biochemical results revealed that maximum chlorophyll at intermediate concentration (T3). Root morphology study suggested that both root length and seminal root number gradually decrease with increasing carbon microsphere dose. Biochemical results revealed that the level of carbohydrate, amino acid and protein are highest in treatment T3 compared to control. On the other hand, proline, catalase and MDA levels signi cantly (p < 0.0001) increase with increasing the dose of carbon microsphere. Therefore, nally, it can be concluded that PET origin carbon microsphere could be potentially applied for priming of seeds including horticultural crops.
Declarations Figure 1 A. SEM and B. EDX image of synthesized carbon microsphere. Biochemical analysis of pigments at treatment variables. Different letters indicate signi cant differences at p < 0.05 according to the Tukey-HSD.