Biodegradation of Polyethylene Using Bacillus tropicus Isolated from Sewage Wastewater Treatment Plant

Abstract

One of the most pressing environmental problems contemporary civilizations confront is the ever-increasing amount of plastic waste. Because of their impact on every living thing, these wastes are seen as a major issue on a global scale. To counteract the harmful environmental effects caused by conventional disposal methods, it is critical to show that eco-friendly alternatives are viable. Biodegradation is one of the best eco-friendly methods for removing plastic waste. In this study, we aimed to identify bacteria from sewage wastewater treatment plants (SWWs) that could degrade low-density polyethylene (LDPE). Bacterial strains isolated from sewerage wastewater were incubated for 120 days in 50 mL of minimal salt media (MSM) containing 60 mg of low-density polyethylene (LDPE). After four months, our research revealed that Bacillus tropicus (SH4) demonstrated significant potential, degrading the LDPE up to 21.6%. We observed the changes after biodegradation using FTIR, GC-MS, and SEM analysis. In conclusion, microorganisms extracted from sewage wastewater possess the ability to mitigate plastic contamination in aquatic ecosystems. Future proteomics and genome investigations are necessary to elucidate the enzymes and metabolic processes implicated in plastic breakdown.

1. Introduction

Plastic pollution has become one of our most urgent environmental issues. Due to its escalating manufacturing and widespread application in numerous industries, plastic waste has permeated practically every region of our world, from the deepest oceans to the most isolated wilderness areas. The ramifications of this development are extensive, profoundly affecting ecosystems, human health, and the overall welfare of our planet [1]. Recognizing the necessity of solving this global issue, academics and environmentalists have been aggressively pursuing creative solutions to mitigate plastic pollution.

Plastics also referred to as organic polymers, consist of elongated carbon chains that form the foundation of their molecular architecture. These synthetic materials are mostly sourced from fossil fuels and comprise carbon, hydrogen, nitrogen, and sulfur, along with different inorganic and organic chemicals [2].

Plastics are categorized into various types: natural, semi-synthetic, synthetic, thermoplastics, and thermosetting plastics. Plastic mass production commenced in the 1950s, with the majority of polymers first engineered for single-use purposes [3].

Although beneficial in numerous industrial and consumer applications, the durability and versatility of plastics have prompted environmental concerns. Non-biodegradable plastics can endure in the environment for millennia, substantially exacerbating global garbage accumulation. In 2010, China generated 8.8 million tons of plastic garbage, representing 27% of global production [4]. Indonesia generated 3.2 million tons of plastic garbage annually, accounting for 10% of the global total [5]. Plastic has emerged as a substantial element of Indonesia’s everyday refuse, comprising roughly 15% of its municipal waste [6]. In 2018, the European plastics industry indicated that global plastic production reached 335 million tons, with Europe accounting for 60 million tons of this significant figure. Furthermore, these output figures are anticipated to rise significantly in the forthcoming decades [7].

Although plastic is now essential to our daily existence, its durability and prevalence provide a significant environmental hazard. Due to their remarkable endurance, plastics remain insoluble for years, causing significant environmental damage as they gradually decompose into tiny particles. This persistent problem has led to an increase in plastic trash that jeopardizes numerous animals, including humans. In addition to contaminating our landscapes and waterways, plastic garbage imposes considerable environmental expenses and public health risks through incineration. Incinerating plastic trash emits toxic chemicals, such as carbon dioxide and dioxins, which are associated with respiratory disorders and cancer [8].

Plastic pollution, a widespread problem that crosses borders and cultures, requires efficient waste management and mitigation techniques. Although minimizing, recycling, and reusing plastics have become prevalent strategies for addressing the issue, there is still a necessity for more effective techniques, especially for mixed plastic trash. Plastic garbage disposal in landfills or incinerators occupies considerable space and poses the risk of releasing toxic gases into the environment. Consequently, it is essential to devise recycling technologies that are both efficient and environmentally sustainable. Biodegradation has surfaced as a viable and economical solution to this global issue [9].

Recent years have seen the discovery of several microorganisms with the ability to break down polymers. A combination of novel Enterobacter and Pseudomonas spp. isolated from cow manure, for instance, has been shown by Skariyachan et al. (2021) to hasten the breakdown of polyethylene and polypropylene. The weight of low-density polyethylene (LDPE) decreased by 64% after 160 days in the lab [10]. Maroof et al. (2021) found that soil samples taken from garbage disposal sites contained microorganisms that break down plastic, including Bacillus siamensis, Bacillus cereus, and Bacillus wiedmannii. From 5.39 to 8.46% of the LDPE’s weight could be reduced by the bacteria [11].

LDPE, or low-density polyethylene, is a common ingredient in plastic production. The weight reduction test results for 23 Rhodococcus isolates from Malaysia were studied. The test looked at the strains’ degrading capabilities. We used low-density polyethylene (LDPE) as our test material. Shake flask incubation was utilized in the experiment. All of the isolates were able to degrade the LDPE that was added to the culture media, even though their degradation rates varied. The most rapid degradation rate among the 23 isolates was observed in RhodococcusUCC0018, with an 8.69% reduction in LDPE weight [12]. Compared to all these studies, our strain, Bacillus tropicus SH4, isolated from sewage wastewater, also showed a significant reduction in LDPE.

However, despite extensive research into LDPE biodegradation, few studies have focused on isolating LDPE-degrading bacteria from sewage wastewater. As almost all waste materials flow through the sewage, sewage wastewater contains bacterial strains that have the potential to degrade plastic; the current study aimed to isolate polyethylene-degrading bacteria from a sewage wastewater treatment plant. Sewage was selected as the source for isolating polyethylene-degrading bacteria due to its diverse microbial community, high organic load, and exposure to pollutants, including plastics. Polyethylene was chosen as the specific material to be biodegraded because of its ubiquity, recalcitrance, and significant environmental impact, making it a key target for bioremediation strategies to mitigate plastic pollution. By exploring sewage, we aimed to discover novel microbial strains and enzymes that can break down polyethylene, contributing to sustainable solutions for plastic waste management.

2. Materials and Methods

2.1. Sample Collection

We collected bacterial samples from a sewage wastewater treatment plant (Township, Lahore). The samples were stored in a bottle and taken to the laboratory. Wastewater was spread on Petri plates containing nutrient agar to obtain colonies.

2.2. Applying Stress to Bacteria to Use Plastic as a Carbon Source

We incubated the obtained bacteria colonies with a total of 60 mg commercially available polyethylene balloon pieces (brand: Kuber Polyfilms, Dehli, India, ten pieces in all, for 120 days in 50 mL minimal salt media (MSM). The flasks were placed in a shaking incubator (100 RPM) at 37 °C. To assess the effect of environmental factors, we incubated a control flask containing 60 mg of LDPE pieces in 50 mL MSM media at 37 °C, without any bacteria. After 120 days, we compared the weight of the treated plastic pieces against those in the control flask.

Minimal Salt Media (MSM) Preparation

A total of 500 mL minimal salt media was prepared in an autoclaved flask by adding salts to 500 mL distilled water (

Table 1. indicates the ingredients and quantities used to make MSM media.

Serial No.	Compound	Quantity/500 mL
1	K2HPO4	2.27 g
2	Na2HPO4	5.97 g
3	NH4Cl	0.5 g
4	MgSO4	0.25 g
5	CaCl2	0.0025 g
6	FeSO4	0.001 g
7	MnSO4	0.0005 g
8	ZnSO4	0.001 g

).

2.3. Molecular Identification of Bacteria

The sample from the trial flask was streaked onto a nutrient agar plate for further screening after 120 days. The molecular-level characterization of the strain was preceded by Gram staining. Thermo Scientific’s geneJET Genomic DNA purification kit (#K0721, #K0722) (Thermo Scientific, Vilnius, Lithuania) was used to isolate bacterial genomic DNA. The 16S rRNA gene was amplified using PCR with universal primers RS1 (5′AAACTCAATGAATTGACGG 3′) and RS3 (5′ACGGGCGGTGTGTA 3′). The ingredients for the 50 µL reaction mixture were 25 µL master mix, 1.5 µL primers, 10 µL template, and 12 µL nuclease-free water. Denaturation at 94 °C for 5 min was the first step in the PCR process. There were 35 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 45 s, and extension at 72 °C for 5 min. Following that, 1% of agarose gel was used to visualize the final products [13].

To purify the 16S rRNA gene, we employed the Gene JETTM PCR purification kit. The DNA and binding buffer were added to a reaction mixture before being passed through a purification column. Eliminating contaminants is as easy as washing. Following the removal of the DNA from the column by means of the elution buffer, it was submitted for sequencing [10]. After gene cleanup, the isolated bacteria’s nucleotide sequences were analyzed using the NCBI BLAST database as part of the ribotyping procedure. Multiple sequence alignments were conducted using CLUSTAL W (Version 1.81), which uncovered their commonalities [14]. The sequencing of all the samples identified a novel strain, SH4.

Saitou and Nei (1987) utilized the Neighbor-Joining method to infer an evolutionary history. Each branch is accompanied by the frequency of occurrences of the associated species clustered in the bootstrap test (1000 replicates) [15,16]. The Maximum Composite Likelihood method was employed to compute the evolutionary distances, represented as the mean number of base substitutions per site. MEGA Version 11.0.10 software was utilized to perform the evolutionary studies [17,18].

2.4. Weight Loss Experiment

The plastic pieces were removed, washed with 70% ethanol, dried, and weighed. The % weight loss was measured using the following formula:

$$Weight loss={\displaystyle \frac{{\mathrm{I}}_{\mathrm{w}}-{\mathrm{F}}_{\mathrm{w}}}{{\mathrm{I}}_{\mathrm{w}}}}\times 100$$

I_W = Initial weight

F_W = Final weight

2.5. Scanning Electron Microscopy (SEM) of Degraded Plastic Pieces

To obtain a highly magnified pictorial view, the physical change that appeared in the plastic pieces was further examined under SEM. Samples were sent to LCWU (Lahore College for Women University) for analysis.

2.6. Gas Chromatography–Mass Spectrometry Analysis (GC-MS)

Following a four-month period in the incubator, the flasks were extracted. The plastic components were extracted from the flask. The culture was centrifuged at 6500 RPM for 10 min to eliminate cell debris, and GC-MS analyzed the supernatant in the TTI (Textile Testing) Lab in Lahore.

2.7. Fourier-Transform Infrared Spectroscopy (FTIR)

The plastic fragments were dispatched to LCWU (Lahore College for Women University) for FTIR analysis to assess any alterations in chemical bonds resulting from the enzymatic activity of bacteria.

3. Results

3.1. Molecular Identification

Polyethylene waste has emerged as a significant environmental concern due to its inert chemical properties. Biodegrading and eradicating plastic waste from the ecosystem is a significant problem. Microbes are crucial for the biodegradation of synthetic polymers, including low-density polyethylene. The isolation of pure plastic-degrading strains requires the careful cultivation and sub-culturing of the strain on media where plastic is the sole carbon and energy source [19].

In the present study, bacterial strains for polyethylene were collected from sewage wastewater treatment plants. After 16S rRNA gene sequencing, sample SH4 was identified as Bacillus tropicus, which showed 21.6% biodegradation. The PE-degrading bacteria that have been reported so far belong to Pseudomonas sp. [20], Bacillus sp. [21], Mycobacterium sp. [22], and Nocardia sp. [23].

3.2. Weight Reduction

The present study utilized 60mg of plastic pieces to examine the degradation capacity of B. tropicus obtained from a sewage wastewater treatment plant. B. tropicus exhibited a 21.6% reduction in the weight of polyethylene fragments from the original mass. LDPE films subjected to treatment with Pseudomonas knackmussii N1-2 and Pseudomonas aeruginosa RD1 for a duration of 8 weeks exhibited weight reductions of 5.95% and 3.62%, respectively [24]. In order to study the breakdown of polymers, Das and Kumar (2015) collected two bacterial isolates from municipal solid soil: Bacillus amyloliquefaciens (BSM-1) and Bacillus amyloliquefaciens (BSM-2). The degradation of LDPE was 16% by Bacillus amyloliquefaciens (BSM-1) and 11% by Bacillus amyloliquefaciens (BSM-2) [25].

3.3. GC_MS Analysis

The current study performed a GC-MS analysis to determine which low-density polyethylene (LDPE) products had deteriorated. They were found when the compounds that had the strongest correlation with the Wiley Library database and the MS fragmentation pattern were compared [24,26] [Figure 1].

As a result of the activity of the strain B. tropicus SH4, we detected the compounds in the supernatant given in [

Table 2. In a GC-MS analysis, the biodegradation of B. tropicus SH4 results in the production of the chemicals listed.

Bacteria	Compounds
Bacillus tropicus	Pentasiloxane, 1,1,3,3,5,5,7,7,9,9-decamethyl
	1,3,5-Benzetriol, 3TMS derivative
	Cyclotrisiloxane, hexamethyl
	Arsenous acid, tris(trimethylsilyl) ester
	Tris(tert-butyldimethylsilyloxy)arsane
	Cyclopentasiloxane, decamethyl-
	Cyclohexasiloxane, dodecamethyl
	3-Amino-2-phenazinol ditms
	Benzeneethanamine, N-[(pentafluorophenyl)methylene]-.beta.,3,4-tris[(trimethylsilyl)oxy]
	Benzamide, 4-ethyl-N-benzyl-N-propyl
	trans-(2-Chlorovinyl)dimethylethoxysilane
	Ethanone, 2-(4-hydroxy-5,6-dimethylthieno[2,3-d]pyrimidin-2-ylthio)-1-(4-ethylphenyl)
	Cyclotetrasiloxane, octamethyl
	1,4-Benzenedimethanethiol, 2TBDM
	2,6-Dihydroxybenzoic acid, 3TMS
	Octasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-hexadecamethyl

]. Research conducted by Kavitha and Bhuvaneswari (2021) found that following 90 days of exposure to Bacillus sp. (PE3), the following compounds were biodegraded: benzoic acid, eicosamethyl cytodecane (with a retention time of 15.96), stearic acid, octinoic acid, decamethyl pentane, and hexa decamethyl heptane (with a retention time of 15.79). Acetic acid, 1,2 propane diol, propane 1,3 hexadiene, decamethyl hexane, propane, and 2,3 hexadiene were the degradation products after 120 days of contact with PE3. The retention times for these compounds were 4.38 and 5.01, respectively [27]. Using gas chromatography–mass spectrometry (GC–MS), Sangale et al. (2019) isolated two fungal strains that break down polythene: Aspergillus terreus strain MANF1/WL and Aspergillus sydowii strain PNPF15/TS. 7-Methylenebicyclo [3.2.0] hept-3-en-2-one, dibutyl phthalate, 1,4-benzenediol, and dodecahydropyrido [1,2-b] isoquinolin-6-one were discovered as the degradation products following 60 days of incubation with Aspergillus sydowii strain PNPF15/TS. 2-Naphthalene carboxylic acid, 2-butyl phthalate, 2-cyclohexen, 1,2-bis (trimethylsilyl) benzene, hexasiloxane, and hexadecanoic acid were the products of degradation with the MANF1/WL strain of Aspergillus terreus [28].

3.4. FTIR Analysis

Our study also incorporated an FTIR analysis to observe the changes in plastic chemical structure after degradation. We found peaks at 3270, 3016, 2946, 2910, 2840, 1729, 1642, 1536, 1365, 1209, 1088, 1027, and 833. The FTIR spectra showed bond bending and bond stretching of the C-C bond of alkane, C=C of alkene, and N-O of the nitro compounds. According to Samanta et al. (2020), the ester group’s C-O stretching and the alkene group’s C=C bending are indicated by peaks at 1027 and 960, respectively [28].

3.5. SEM Analysis of Physical Changes in Plastic Pieces

After 120 days, significant changes were observed in the physical characteristics of the plastic pieces. In comparison with the control, the treated pieces were thin and porous. SEM analysis assessed the LDPE surface alterations with analogous investigations documented in the prior literature. These findings robustly corroborate the data given in the current study [24,29,30].

4. Discussion

When plastic pollution entering an area surpasses the rate of natural elimination processes or cleanup operations, plastic accumulates in the ecosystem. Natural degradation processes for plastics can take decades or even millennia [31]. There is an increasing problem with marine contamination due to solid waste on a global scale. This problem could affect generations to come [32].

Marine litter is defined as any human-made solid waste in the ocean, whether on land or at sea. This includes materials transported to the ocean via rivers, drains, sewage, wind, or water systems but does not include organic materials like food and vegetable scraps [33].

The current study aimed to isolate and characterize low-density polyethylene (LDPE)-degrading bacteria in sewage wastewater. This is because sewage is one of the biggest sources of natural water pollution and potential microbes for removing toxicants. We collected sewage samples from a wastewater treatment plant to isolate plastic-degrading bacteria. After an initial screening and 120 days of incubation with polyethylene, we found that sample SH4 reduced the LPDE weight. After 16S rRNA gene sequencing, this strain was identified as Bacillus tropicus [Figure 2].

To observe the potential of our isolated strain, the weight loss of the polyethylene was observed over a period of 120 days. With the passing days, the polyethylene weight decreased, and after 120 days, we observed a maximum weight loss of 21.6% [Figure 3]. The study by Mukhaifi et al. (2021) sought to isolate and characterize bacteria capable of degrading polyethylene terephthalate (PET) from Shatt al-Arab water and sewage in Basra, identifying the bacteria as Klebsiella pneumoniae. The results indicated a statistically significant difference in PET degradation, with a 24% reduction over 7 days, which grew to 46% after 4 weeks in comparison to the control group. These results correlate with the current study [34]. Meng et al. (2024) identified a novel marine strain of Pseudalkalibacillus sp. MQ-1 capable of degrading polyethylene (PE) up to 6.37% in 60 days. Scanning electron microscopy and water contact angle analyses demonstrated that MQ-1 may cling to polyethylene films, rendering them hydrophilic [35].

We performed a GC-MS analysis to detect compounds in the MS media produced due to the biodegradation of LDPE [Figure 1]. Many new compounds were produced by B. tropicus activity. We neglected the compounds that were in common with the control. We found Pentasiloxane, 1,1,3,3,5,5,7,7,9,9-decamethyl, 1,3,5-Benzetriol, 3TMS derivative, Cyclotrisiloxane, hexamethyl, Arsenous acid, tris(trimethylsilyl) ester, tris(tert-butyldimethylsilyloxy)arsane, Cyclopentasiloxane, decamethyl-, Cyclohexasiloxane, dodecamethyl, 3-Amino-2-phenazinol ditms, Benzeneethanamine, N-[(pentafluorophenyl)methylene]-.beta.,3,4-tris[(trimethylsilyl)oxy], Benzamide, 4-ethyl-N-benzyl-N-propyl, trans-(2-Chlorovinyl)dimethylethoxysilane, Ethanone, 2-(4-hydroxy-5,6-dimethylthieno[2,3-d]pyrimidin-2-ylthio)-1-(4-ethylphenyl), Cyclotetrasiloxane, octamethyl, 1,4-Benzenedimethanethiol, 2TBDM, 2,6-Dihydroxybenzoic acid, 3TMS, Octasiloxane, and 1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-hexadecamethyl by GC-MS analysis [Table 2]. The plastic polymers’ biodegradation byproducts following 140 days of incubation were investigated using gas chromatography–mass spectrometry. Three compounds were identified in this study: cis-2-chlorovinyl acetate (7.11 min), tri-decanoic acid (21.43 min), and octa-decanoic acid (22.46 min). The presence of tri-decanoic and octa-decanoic acids in the material under investigation implies the biodegradation process involves the creation of carbonyl groups. These groups are further oxidized to produce ketones and aldehydes, as confirmed by nuclear magnetic resonance (NMR) analysis. Thus, the current research shows that fatty acids and other metabolic intermediates are important for microbial consortiums to biodegrade plastic. It was determined that the end products in this investigation did not pose any health risks [36].

Shahnawaz et al. (2016) identified 1-trimethylsilylmethanol, 1,2,3-trimethylbenzene, ethyl-3,5-dimethylbenzene, hexadecanoic acid, 1,4-dimethyl-2-ethylbenzene, and 1,2,3,4-tetramethylbenzene [37]. Roy et al. (2008) cultivated a consortium of Bacillus pumilus, Bacillus halodenitrificans, and Bacillus cereus on polyethylene particles, revealing the presence of both oxygenated chemicals and unoxidized low-molecular-weight hydrocarbons [38]. Roy et al. (2008) identified alkanes, fatty acids, and ester-containing compounds as products of biodegradation by Pseudomonas putida, Pseudomonas syringae, and Pseudomonas aeruginosa [38].

Through an FTIR analysis, we observed the changes in chemical bonds. We compared the FTIR of treated plastic pieces (^____T) with the FTIR of control plastic pieces (^____C) [Figure 4]. We found that the O-H stretching of carboxylic acid caused a signal at 3270 cm⁻¹ in the FTIR spectra of Bacillus tropicus SH4-treated polythene. At 3016, you can see the peak that represents the alkene’s C-H stretching. The C-H stretching of the alkane group is shown by peaks at 2910, 2946, and 2840. At the same time, the peak at 1729 corresponds to the C-H bending of the aromatic compound. The C=C stretching of conjugated alkene is demonstrated by the peak at 1642. The peak at 1536 shows the N-O bond stretching of the nitro compound. The peak at 1365 is attributed to the C-H bond bending of alkane (gem dimethyl). The C-O stretching of the alkyl aryl ether is seen by the peak at 1209. Peaks at 1088 and 1027 indicate C-N stretching of the amine group [Figure 4].

In the study of Khandare et al. (2021), chemical changes in the LDPE structure were investigated using FTIR. If you want to know what functional groups are in LDPE or any other biodegradable molecules, you can use the FTIR analysis to see changes in the carbon backbone [39]. Previous research has made heavy use of this method, as seen by the work of Nadeem et al. (2021), who demonstrated a decrease in transmittance between 1100 and 1150 cm⁻¹, suggesting the formation of new (-C-O-C) bonds due to the weakening of C-C bonds. Except for the untreated control, all LDPE film FTIR spectra exhibited the production of a characteristic carbonyl peak at 1712 cm⁻¹, which was significantly diminished following 90 days of bacterial incubation. The process of plastic biodegradation can be better understood by observing the presence and disappearance of carbonyl peaks [40]. In another investigation, Selke et al. (2015) also noticed a reduction in the creation of a carbonyl peak at 1712 cm⁻¹ after 90 days of treatment with bacteria [41]. In order to understand the basic process of biodegradation, previous studies have shown that new functional groups can appear and disappear in both treated and control LDPE films, which supports the current study’s conclusions [30,42,43].

We also performed scanning electron microscopy to observe the physical changes after biodegradation. The SEM examination allowed us to observe the surface morphological changes on the LDPE film following 120 days of treatment with sewage bacteria. The scanning electron micrographs (SEMs) revealed surface degradation, fragility, damaged layers, cracks, and scratching in the LDPE film treated with any of the four marine bacteria compared to the control film, which remained smooth, undamaged, and clear [Figure 5]. Sanin et al. (2003) found that FE-SEM images of synthetic polymer (LDPE) fragmented into monomeric forms when incubated with certain bacterial strains. These strains included Rhodococcus corallinus strain 11, Pseudomonas sp. strain A, and Pseudomonas sp. strain D. Using AFM images, it was also noted that all LDPE films treated with bacterial isolates developed cracks, grooves, and roughness after the same incubation conditions, whereas the control film, which was not supplemented with any bacteria, remained smooth and intact. This confirms that the degradation is caused by enzyme activity. Results from scanning electron microscopy (SEM) and atomic force microscopy (AFM) demonstrate that, under low-nutrient conditions, bacterial isolates cling to the surfaces and use the C-source from LDPE substrates [39,44].

5. Conclusions

In conclusion, plastic pollution is an emerging global problem that needs some sustainable, economical, and eco-friendly approaches for mitigation. Conventional methods, such as the incineration of plastic, can be toxic to the environment. Biodegradation is an environmentally friendly method for plastic removal. This study found Bacillus tropicus from a sewage wastewater treatment plant that degraded up to 21.3% of polyethylene. Therefore, sewage wastewater can be a good source of potential bacteria that can degrade plastic significantly. An FTIR analysis indicated significant changes in the plastic structure after biodegradation. A GC-MS analysis indicated new compounds produced as a result of B. tropicus activity. A SEM analysis showed the deterioration of the plastic surface. So, we can infer that this bacterium has significant potential in polyethylene reduction in sewage. However, we tested the bacteria in controlled settings; it is very crucial to assess its role in natural settings to apply it on a large scale. Applying Bacillus tropicus on a large scale for polyethylene biodegradation is theoretically feasible but would require significant modifications to existing infrastructure and careful consideration of environmental factors. Scalability, nutrient supply, pH and temperature control, oxygen levels, and contamination risks are challenges that need to be addressed. Bioreactors, nutrient management, temperature and pH control systems, aeration and mixing, and monitoring and control systems would be necessary to support bacterial proliferation. Further proteomics and metagenomics studies are needed to learn more about the plastic breakdown pathways these bacteria use. Future studies should focus on finding a role for the bacterial community of sewage in environmental sustainability and the world economy.