Potential accelerated biotransformation of petrochemical plastic surfaces by anaerobic digester sludge microorganisms

doi:10.21203/rs.3.rs-7632822/v1

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Potential accelerated biotransformation of petrochemical plastic surfaces by anaerobic digester sludge microorganisms

https://doi.org/10.21203/rs.3.rs-7632822/v1

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This study investigated the biotransformation of three different plastic surfaces, polypropylene (PP), polyvinyl chloride (PVC) and polyethylene (PE), by anaerobic digestion (AD) system microorganisms under mesophilic conditions. For that, a laboratory-scale AD system was established and plastics were immersed in the sludge for a 50-day incubation period, measured for any significant mass loss. Statistical analysis showed a significant mass loss (p < 0.05) in PVC pieces with a 1.1 ± 0.16 mg average reduction, while PP and PE didn’t show any significant mass loss. Raman spectroscopy analysis revealed temporarily increasing novel peaks in PVC at 1729 cm^− 1 corresponding to C = O stretching vibrations. This was considered significant and compared against an unchanged marker of PVC, revealing a newly identified peak that has not been documented in prior studies of this material. PP also revealed temporarily increasing novel peaks in the spectral range of C = C stretching vibrations in the region of 1512 cm^− 1. Atomic force microscopy (AFM) analysis of PVC showed a reduction in average roughness amplitude from 100 nm to 90 nm in 30 days and 65nm to 50 days revealing surface biotransformation. Phase-contrast microscopy further confirmed surface embrittlement across all plastics. Four bacterial species that were associated with plastic biotransformation were isolated and characterized using 16s rRNA molecular marker gene based identifications as Pseudomonas fluvialis, Bacillus cereus, Proteus mirabilis and Gottfriedia luciferensis. In conclusion, this study suggests that, the changes to overall surface of the plastics and newly observed biodegradation of PVC suggesting a biotransformation leading to biodeterioration, by AD system microorganisms.

Biodeterioration

Synthetic polymers

Mesophilic

anaerobic digester

Vibrational spectroscopy

Plastic has been a dynamic part in human lives for many years, providing inventive solutions to erratic necessities. To the same extent that fossils themselves record the emergence of extinct living forms, plastics tends to act as geological witness to humanity’s rise to global supremacy (Zalasiewicz et al., 2016).They are so important to human activity that 140 million tons of plastics are produced globally each year (Shimao, 2001).

Plastics are, synthetic or semi-synthetic materials composed of polymers, which are extended chains of molecules formed from repeating units known as monomers (Shah et al., 2008). These versatile materials are vital to modern life because they can be shaped into a vast array of forms (Zheng et al., 2005). Plastics are used extensively in a variety of residential, agricultural, and industrial applications due to their strength, affordability, and versatility. These applications ranged from building and packaging household to automotive parts (Rosato, 2011). The key benefits of these macromolecules are their exceptional thermo-mechanical properties, corrosion resistance, inexpensiveness, and adaptability (Desidery and Lanotte, 2022).

However, the environment is now becoming diminished by the widespread use of plastics. Regardless of the environment, the majority of plastics are non-biodegradable, which signifies they do not break down naturally and can persist in the environment for centuries (Chamas et al., 2020). Plastics pose serious environmental and public health risks despite its advantages. They break down into micro and nano-particles, which move through the biosphere and impact human and animals on land and in water (Pilapitiya and Ratnayake, 2024). Moreover, earth’s elemental cycles are being significantly impacted by manufacturing and disposal of plastics which results in carbon cycle being impacted by them, a novel type of carbon-based geo-material (Stubbins et al., 2021).

Polypropylene (PP), Polyethylene (PE), Polystyrene (PS), and Polyvinyl chloride (PVC) are among the most prevalent petrochemical plastics that are not biodegradable (Dussud and Ghiglione, 2014;Ziajahromi et al., 2017) This accumulation is acknowledged as a key human caused matter through coastal and marine ecosystems globally, causing pollution in landfills and oceans, which endangers ecosystems as well as wildlife. The unparalleled and continuous accumulation of plastic contaminants from anthropogenic activities destabilizes the structural and functional characteristics of these aquatic ecosystems, impacting their ecosystems’ parameters (Thushari and Senevirathna, 2020).

Existing plastic-related matters are now being discussed via reduce, reuse, and recycling processes. However, recycling post-consumer plastic poses significant challenges attributed to a variety of aspects. One of the critical issues is that plastic transmits hazardous emissions into the atmosphere and requires more space for the procedure (Vimal Kumar et al., 2017). Correspondingly, there is a diverse range of plastic types that are recurrently consolidated during the process of manufacturing that are often more complicated than others. Due to their inconsistent molecular patterns, this blending makes it harder to figure materials efficiently, which results in inefficient recycling processes (Vogt et al., 2021). Thus, all such methods are unusable for mixed plastic waste (Drzyzga and Prieto, 2019)

Plastics, despite their tendency to persist in the environment for many years, nonetheless show abiotic or environmental degradation pathways. Physical degradation that is supported by agents such as Ultra Violet (UV) light supplied by sunlight, causes changes in the structure such as cracks, embrittlement and flaking and chemical degradation causes changes happening at the molecular level. However, these pathways require hydrolysis mechanisms requiring water (H₂O) or oxidation (O₂), both of which that can be accelerated by microbial action (Chamas et al., 2020).

Since plastic pollution has become a significant ecological problem, it requires the development of long-term strategies to mitigate its impacts. One viable approach is the use of microorganisms to biodegrade plastic, which may decompose complex plastic polymers into simpler, non-toxic monomers via enzymatic processes (Fachrul et al., 2021). Enzymatic degradation then breaks down these materials into oligomer, dimers and monomers, leading to changes in mechanical properties, alterations in chemical bonds and the emergence of new chemical bonds (Arutchelvi et al., 2008). This process involves four stages: bio deterioration, depolymerization, assimilation and mineralization. Microbial biofilms accelerate the mechanical and physiochemical changes that result from bio-deterioration, which is broke down by combined actions of micro-organisms and abiotic stimuli. Subsequently polymers are broken down into smaller molecules by the enzymatic activity of microbes and free radicals. These molecules are then absorbed by microorganisms and undergo internal metabolism resulting in the production of metabolites and energy. Finally mineralization completes the process by completely turning the carbon (C) atoms into substances like Carbon dioxide (CO2), Methane (CH4), and water (H2O).

Given that, aerobic biodegradation process is proven to be efficient and ecologically beneficial than other approaches, when it comes to biotransformation of certain pollutants such as different polymers and plastics, the present state of the research using anaerobic digestion highlights their significance. According to study done by Cazaudehore et al. (2023), it has shown that bio plastics like Polyhydroxubutyrate (PHB) and Thermo-plastic starch (TPS) has undergone rapid biodegradation when treated under mesophilic anaerobic digester conditions at 38⁰C. Moreover by contrast, Polycaprolactone (PCL) and Polylactic acid (PLA) has undergone biodegradation at 500 days. Also a research conducted by El-Mashad et al. (2012) stated that, anaerobic biodegradability of six commercial plastics and two cellulose-based products were seemingly low in a time of small time period but only after 43 days of digestion time, Polyhydroxyalkanoates (PHA) were almost digested leading to the conclusion that other technologies and methods should be used for the disposal of non-recycled plastics. According to Itävaara et al. (2002), results of their study demonstrated that Polylactic acid (PLA), which is biodegradable in both aerobic and anaerobic conditions had shown more significant biodegradation in anaerobic thermophilic conditions due to the fact that lactic acid being more favorable substrate for anaerobic micro-organisms rather than aerobic microbes. Yagi et al. (2012) performed an anaerobic biodegradation for Polylactic acid (PLA) in different sizes and have successfully conducted a biotransformation in the plastics where significant degradation was observed in the Polylactic acid (PLA) films rather than crushed materials. This suggests that in anaerobic biodegradation test, there is a possibility that a plastic film should not be crushed or cut into too small pieces.

Alassali et al. (2018), evaluated the quality of Low-density polyethylene (LDPE) in the anaerobic treatments supported by composting methods, showing signs of alteration in microscopic level. Further, Fourier Transform Infrared Spectroscopy (FTIR) revealed slight transmission spectrum changes in areas representing C-O and O-H bonds, suggesting the presence of oxidized products which the Energy Dispersive X-ray (EDS) analysis confirmed.

Also, Belone et al. (2024) analysed the visual, chemical, mechanical and thermal changes brought by anaerobic digestion using a variety of characterization techniques. The findings demonstrated signs of polymer deterioration, including surface fracturing of styrene-butadiene rubber (SBR), breakdown of Polystyrene (PS) and uPVC (unplasticized Polyvinyl chloride), hydrolysis of Polyethylene terephthalate (PET), and surface smoothening of liner low density Polyethylene (LLDPE), high density Polyethylene (HDPE) and Polypropylene (PP).

Moreover, a study conducted by Cazaudehore et al. (2021) showed that anaerobic biodegradation of three biodegradable coffee capsules made out of plastics were significant and approximately three times higher at thermophilic conditions (58⁰C) than the mesophilic conditions (38⁰C). Lera et al. (2025) evaluated the impact of mesophilic anaerobic digestion on the fate of Microplastics (MPs) in waste water treatment plant sludge using both continuous stirred tank reactor (CSTR) and anaerobic membrane bioreactor (AnMBR). The Microplastic concentration was significantly reduced, according to data AnMBR achieved a higher microplastic removal efficiency.

According to Zafiu et al. (2023), the results of their study showed that packing products composed of plastics like Polyethylene terephthalate (PET) or Polypropylene (PP) that were treated with additives to promote oxidative or enzymatic degradation exhibit molecular degradation characteristics in a microscopic level. Further, Lund Nielsen et al. (2019) showed, in comparison to all other environments previously studied, the biodegradation of polypropylene in thermophilic conditions was to be several orders of magnitude higher in this study. While Fourier Transform Infrared spectroscopy (FTIR), Raman spectroscopy, and Nuclear magnetic resonance (NMR) were used to analyse the extracted and concentrated products spectroscopically, a series of imaging AFM and SEM tests showed the existence of oxidation products collected on the surface. Important information on the composition and a model of polymer breaking including the formation of integrated cis-configured double bonds (olefins) were provided by the spectroscopic analysis. The results indicate that conventional ADs have a strong potential for (micro) plastic biodegradation. The findings also lend credence to the application of anaerobic digesters for the treatment of domestic waste that is highly energetic and contains a significant amount of plastics. Henceforth, weight loss and even destruction of certain petrochemical plastics, including polypropylene and polyvinyl chloride, were observed. (Malakhova et al., 2023).

Utilization of biotransformation strategies face many challenges, and this is one such area where current research on plastic biodegradation reveals a number of gaps. This primarily includes using the aforementioned strategies mainly in biodegradable, bio-based plastics, and very few research indicate that anaerobic digestion can lead to substantial weight loss of petrochemical plastic materials in a relatively short period, underscoring its potential as a sustainable waste management solution. Recently, only a few studies with regards to petrochemical plastic biodegradation has been done worldwide to see if these problems can be overcome and if this technology can be used in practical applications. Despite a few previous studies suggesting surface biotransformation of petrochemical plastics in anaerobic digestion (AD) systems, the underlying mechanisms of plastic surface chemical transformations are poorly understood.

Therefore, this research aimed to assess the potential biotransformation of anaerobic digester sludge microorganisms on selected petrochemical plastic surfaces; Polypropylene (PP), Polyvinyl chloride (PVC), and Polyethylene (PE) under controlled laboratory conditions over a defined period of time. The main objectives of this study was to design and create a functioning anaerobic mesophilic digester microbial community in a lab-scale Anaerobic Digestion (AD) reactor and evaluate biotransformation of different types of petrochemical plastics surfaces under controlled anaerobic conditions, and to isolate, identify and characterize potential microbial communities from different plastic surfaces that exhibit biotransformation capability.

2.1. Sample collection

The collection of sewage sludge samples from a well-functioning anaerobic reactor at Negombo municipal wastewater treatment plant, Sri Lanka were conducted. Approximately 4 liters of anaerobic sludge were collected from these reactors, which were chosen for their optimum operating conditions with an active and representative microbial community. The samples were then transported to the laboratory with containers kept upright and secure to prevent spills. Since the immediate biodegradation study was not feasible, the samples were stored in the same containers and homogenized to ensure uniform distribution of the community and fed with 1g of glucose for each container as the substrates, followed by measuring the initial pH of the sludge, until dividing into smaller portions in smaller sub- bioreactors for plastic biodegradation study.

2.2. Laboratory scale Anaerobic Digester (AD) setup construction

A laboratory-scale Anaerobic Digestion (AD) reactor system was prepared in a 5-liter glass jar equipped with an inlet valve and outlet valve. The collected sludge sample was poured into the jar, filling it to 75% of the jar’s total volume. To enhance the functionality of the bioreactor system, it was supplemented with cow manure and food waste at a ratio of 1:4, De-ionized (DI) water was then added to dilute the mixture, bringing the final volume up to the 5-liter capacity. The system was initially fed with 2g/L sodium acetate (CH3COO ^–Na⁺) solution once and glucose every day, during which the contents were gently swirled to ensure uniform distribution and optimal microbial activity. A 3 Molar NaOH solution was prepared in a 1 liter conical flask and utilized as the NaOH compartment. The primary function of this compartment was to strip off carbon dioxide (CO₂) and other acidic gases emanating from the main digester ensuring that there is only a methane-rich gas stream obtained and utilized for assessment of the health and the active status of the bioreactor. The gas source coming out from the conical compartment was connected to the inverted cylinder using a delivery tube. The volume of biogas collected were measured with water displacement method.

2.3. AD system operation

Upon setting up the bioreactor, 20 mL of 2 g/L high concentrated glucose solution was provided as the sole substrate daily, spanning a total duration of 30 days. By the time of the 15th day, a small amount of sludge was carefully recovered from the bioreactor to measure the pH value. Moreover, cumulative and daily gas volumes were measured and recorded. Once the gas volume reached 4 ½ times the working volume of the bioreactor, the sludge was considered active and served as the inoculum for the small sub-bioreactors. This was followed by measuring the final pH of the sludge.

2.4. Petrochemical plastic biodegradation study

2.4.1. Plastic piece preparation

Three types of petrochemical plastics – Polyvinyl Chloride (PVC), Propylene (PP), and Polyethylene (PE) were selected as the potential plastic polymers. Each of the plastics were selected with similar dimensions 2.4 cm x 1.2 cm and carefully weighed to ensure a known finite mass.

2.4.2. Sub-bioreactor preparation

The preparation of sub-bioreactors involved using 45 glass sub-bioreactors throughout the study, each with a volume of 50 mL.15 sub-bioreactors were allocated for each plastic polymer type, and all were filled with 40 mL of active sludge. This was also followed by measuring and recording the final pH of the sludge mixture. Then the study was carried out as triplicates, with a total of 5 sampling events where 15 reactors were used over 50 days’ time. The plastic pieces that were cut into same dimensions 2.4 cm x 1.2 cm with a known finite mass were immersed in reactors with 2 pieces in a one replicate altogether 6 pieces per one sampling event. Then the glass tubes were sealed tightly to ensure anaerobic conditions. Every 5 days these were fed with 2g/L of glucose and gently swirled to ensure the even distribution of the nutrients. Once every 10 days, plastics were recovered from the sub-bioreactors, six pieces for each polymer type for a total of 5 sampling events over 50 days. Parallel control experiments involved immersing plastic pieces in distilled water to compare biodegradation rates and activity in the absence of active sludge.

2.5. Plastic biodegradation assessment.

2.5.1. Mass loss analysis

Once the plastic pieces were recovered, one piece of plastic from each replicate of every sampling events of all three-polymer type was subjected to Wet Peroxide Oxidation (WPO) as described by Rodrigues et al. (2018). Afterwards, the biodegradation of each plastic material was assessed by calculating mass loss as noted by Lund Nielson et al. (2019) and Belone et al. (2024) as follows.

$$\:biotransformation=\:mass\:loss\:percentage\left(\%\right)\frac{initial\:mass-final\:mass}{initial\:mass}\:\text{x}\:100$$

2.5.2. Statistical analysis

Then the mass loss data was analysed using Kruskal-Wallis’s test using Python (version 3.11.12) libraries in Google colaboratory environment to compare mass loss differences across each treatment and plastic type. Further, to analyse whether the types of plastics and treatments show a significant difference, a post-hoc test: Dunn’s test with Bonferroni correction was performed. All data were tested for normality using the Shapiro-Wilk test before analysis.

2.5.3. Surface characterization by Phase contrast microscopy

From the plastics that were not subjected to wet peroxide oxidation (WPO), a set of total five plastics from each sampling event were recovered and cleaned with 70% ethanol followed by deionized water cleaning to remove residual junks in the plastic surfaces. Subsequently these were placed in a desiccator to absorb moisture of the plastics allowing it to dry out. Then these plastic polymers were observed under phase contrast microscope (OPTIKA-B500 TPL, Italy) for surface characterization.

2.5.4. Surface characterization by Raman analysis

From the plastics that were not subjected to wet peroxide oxidation, another set of plastics from each sampling event were recovered and cleaned with 70% ethanol followed by deionized water cleaning to remove residual junks in the plastic surfaces. Subsequently these were placed in a desiccator to absorb moisture of the plastics allowing it to dry out. Then these plastic polymers were subjected to vibrational spectroscopy by Raman analysis.

2.5.5. Surface characterization by atomic force microscopy (AFM)

Another set plastic polymers that were not subjected to wet peroxide oxidation, were subjected to surface characterization by Atomic Force Microscopy where topography of the plastics pieces was analysed using a Multimode Atomic Force Microscope in a Nanoscope IIIa controller (Bruker Optik GmbH, Germany), operating in intermittent contact mode. Plastic coupons were washed with 2% w/v aqueous sodium dodecyl sulfate (SDS) for 30 minutes and subsequently rinsed in deionized water and air-dried prior to analysis to clean the polymer surface. Roughness analysis was processed using the WSXM software package (Horcas et al., 2007).

2.6. Molecular characterization of plastic degrading microorganisms

2.6.1. Isolation and identification

Microorganisms from the bioreactor that have the ability to biodegrade plastics were isolated on a Nutrient Agar (NA) mediun.1g/L Cysteine was used in the growth medium to scavenge residual oxygen and to act as a reducing agent that will exhaust all other potential terminal electron acceptors. The bacteria that appeared as single pure colonies was then introduced into solidified medium by streak plate method and plates were incubated under strict anaerobic conditions in an anaerobic jar (HiMedia, India). The anaerobic atmosphere inside was generated by AnaeroGas pack sachets which was confirmed by using Anaero indicator tablet.

2.6.2. DNA extraction and molecular characterization

Molecular microbial characterization of the plastic degrading bacterial isolates was conducted using the 16s ribosomal RNA (16s rRNA) marker gene. Initially single colonies of the bacterial isolates were suspended in the same broth medium described in section 2.6.1. Subsequently the broth media were allowed to grow overnight and total genomic DNA extraction from single colonies was conducted using a Wizard TM genomic DNA extraction kit (Promega Corporation, USA) as per the manufacturer instructions. Then the 16s ribosomal RNA gene was amplified from the extracted genomic DNA using 27F (AGA GTT TGA TCM TGG CTC AG) and 1492R (CGG TTA CCT TGT TAC GAC TT) bacterial universal primer pairs. 16s ribosomal RNA marker gene was amplified using PCR master mix (Promega, USA) in a PCR thermocycler (Thermo-Fisher, UK) under following set of conditions; initial denaturation at 95⁰C for 4 min, followed by 30 cycles of 95⁰C for 0.5 min, 58⁰C for 1 min, 72⁰C for 0.5 min, and finally at 72⁰C for 7 min. After, both raw DNA and PCR products were quantified using Nanodrop micro-volume spectrophotometer at the technology faculty Rajarata University of Sri Lanka. Subsequently, the products were confirmed and verified on 1% agarose gel before being sequenced using Sanger DNA sequencing at Macrogen, Republic of Korea. The phylogenetic analysis were conducted using Molecular Evolutionary Genetics Analysis (MEGA 12) software.

3.1. Performance of the bioreactor

The pH of an AD reactor plays a critical role in maintaining microbial activity and in optimizing biogas production which helps in assessing the health of the bioreactor as an indirect measurement. Initially, the pH of the sludge purely obtained from the anaerobic treatment unit showed 4.9 (26.1⁰ C) where, addition of substrates (such as glucose, cow manure and sodium acetate) in the initial run increased the pH up to the level of 5.3 (26.1⁰ C). Throughout the 30 days’ time the reactor was operated, small amount of sludge was recovered from the 15th day where the pH was recorded as 6.01 (26.4 ⁰C). By the end of the 30th day all the sludge was recovered where the pH was recorded as 6.84 (26.2⁰C).

The AD reactor produced a total of 522 cm³ of biogas over the reporting 30 days’ time period, with an average daily production of 17.4 cm³. The yield coefficient (Yx/s (methane)) for the reactor averaged 1.74 cm³/g, indicating an efficient conversion of substrates into methane. The biogas composition was approximately, consistent with typical anaerobic digestion outputs. Composition and relative quantity of the headspace gases (CH₄, CO₂, H₂S and O₂) during AD operation experiments were measured using a GasBoard 3200 Plus handheld biogas analyzer (Cubic-Ruiyi Instrument Co., Ltd, China). The gas composition and relative quantity of the headspace gases produced by the anaerobic digestion (AD) reactor are given below in the table 4.2, and the emanated methane volumes and their yield co-efficient are denoted by the Figs. 5, 6, 7 and 8 respectively.

Table 1
The gas composition and relative quantity of the headspace gases produced by anaerobic digestion (AD) reactor.
Gas	Percentage composition (%)
CH₄	89.2
H₂	7.1
CO₂	3.0
O₂	0.6
Water vapor	nil

3.2. Mass loss analysis

The biotransformation study of Polypropylene (PP) samples over 50-day period revealed a minimal mass loss, indicating a strong material stability against microbial attack and corrosive gases. Across five sampling events in 10- day intervals, only few (4) showed a promising mass loss and average mass loss across all samples ,corresponding to approximately 0.04% of the initial mass with an average of 0.04 mg, indicating minimal degradation over 50 day period (Fig. 3.a) .

During the biotransformation study of 50-day time period, all the Polyvinyl chloride (PVC) samples showed a visible mass loss with an average of 1.5%. The standard deviation showed a moderate variability in mass loss among samples which indicates that some samples degrade more than other, possibly due to differences in sample composition, additives, or experimental conditions. Compared to materials with near zero or zero as PP, PVC was less stable and showed a prominent amount of mass loss. This pattern suggests that almost all samples have undergone biotransformation under the conditions tested with an average mass loss of 1.11 mg which is the highest among all plastic types (Fig. 3.b).

During the biotransformation study of 50-day period. 7 out of 15 Polyethylene samples showed a visible mass loss with an average reduction of 0.15 mg This few samples with mass loss show small to moderate deterioration up to 0.7 units over 50 day time period (Fig. 3.c)

3.3. Statistical analysis

Initially the raw data were tested under Shapiro-Wilk test for normality and it indicated a significant departure from normality where, W (45) = 0.74 and p < 0.001.Thus, non-parametric statistical methods was conducted and by the Kruskal-Wallis H test indicated that there is a significant difference in the mass loss between the different plastic types, χ²(2) = 28.61, p < 0.001, η² = 0.63 with a mean rank score of 13.7 for PP, 36.97 for PVC, 18.33 for PE. The Post-Hoc Dunn's test using a Bonferroni corrected alpha of 0.017 indicated that the mean ranks of the following pairs are significantly different: Polypropylene- Polyvinyl chloride (Z= -4.851, Adjusted p = 0.00031, p < 0.05) Polyvinyl chloride- Polyethylene. (Z=-3.885, Adjusted p = 0.00000368, p < 0.05). Moreover, the same test indicated that the mean ranks of the pair of Polypropylene-Polyethylene is not significantly different with a Z = 0.966, and an Adjusted p = 1.0.The statistical analysis by the Kruskal-Wallis H test indicated that there is a non-significant difference in the mass loss between the different sampling events of all three plastic types. It was indicated that, Polypropylene shows a non-significant difference in the mass loss between sampling events having χ² (4) = 11.06, p = 0.026, η² = 0.0028 with a mean rank score of 8.33 for sampling event 1, 13.67 for sampling event 2, 6 for sampling event 3, 6 for sampling event 4, and 6 for sampling event 5. Further, in PVC it indicated there is a non-significant difference in the mass loss between the sampling events of PVC, χ²⁽4) = 8.3, p = 0 .081, with a mean rank score of 11.33 for Group1, 6.5 for Group2, 11.33 for Group3, 8.33 for Group4, 2.5 for Group5. Moreover in PE it also indicated a non-significant difference in mass loss between different sampling events, χ²(4) = 7.56, p = 0.109, with a mean rank score of 8.5 for sampling event 1, 7.67 for sampling event 2, 13.17 for sampling event 3, 4.5 for sampling event 4, and 6.17 for sampling event 5.

3.4. Surface characterization by phase-contrast microscopy

Surface analysis by phase-contrast microscopy of three different plastic pieces showed evidences for polymer biodeterioration, including surface smoothening, embrittlement for all plastic types.

3.5. Surface characterization by Raman analysis.

Raman spectra overlaid from average single spectra from Polypropylene samples, showed a distinct spectral signatures featuring prominent peaks at positions, 808 cm^− 1, 840 cm^− 1, 1167 cm^− 1 and 1512 cm^− 1. The Raman shift in the spectral range of C = C stretching vibrations in the region of 1512 cm^− 1 was considered significant and compared against an unchanged marker of PP revealed a new peak with an increasing intensity.

Furthermore Raman spectra overlaid from average single spectra from Polyvinyl chloride (PVC) samples on different days showed distinct spectral signatures featuring prominent peaks at positions 636.5 cm^− 1, 696 cm^− 1, 1429 cm^− 1, 1729 cm^− 1 and 2916 cm^− 1. The Raman shift in the spectral range of C = O stretching vibrations in the region of 1729 cm^− 1 was considered significant and compared against an unchanged marker of PVC revealed a newly identified peak which has not been documented in prior studies of this material.

Table 1
Raman shift markers, their respective peaks observed from Polypropylene (PP) samples.
Raman shift marker (cm^− 1)	Peak assignment	Reference
808	-C-H stretching vibrations	Furukawa et al. (2006)
840	-C-H3 rocking vibrations	Furukawa et al. (2006)
1167	C–C stretching vibrations	Furukawa et al. (2006)
1512	-C = C- stretching vibrations of carbon double bonds	Marshall and Olcott Marshall (2010)

Table 2
Raman shift markers, their respective peaks observed from Polyvinyl chloride (PVC) samples.
Raman shift marker (cm^− 1)	Peak assignment	Reference
636.5	-C-Cl stretching vibrations	Solodovnichenko et al. (2016)
696	-C-Cl stretching vibrations	Solodovnichenko et al. (2016)
1429	-C-H stretching vibrations	Solodovnichenko et al. (2016)
1729	-C = O stretching vibrations of the carbonyl carbon	França De Sá et al. (2021)
2916	-C-H stretching vibrations of the –CH₂ fragment	Solodovnichenko et al. (2016)

3.6. Surface characterization by atomic force microscopy (AFM)

The control samples of Polyvinyl chloride (PVC) showed a relatively broad distribution of topographical variation, with an average roughness amplitude of ~ 100 nm, this surface roughness was assumed to be associated with the manufacturing process. The roughness measurements recorded after 30 and 50 days of incubation in digester sludge (Figure C-D) remained within a similar range (0–205 nm), but with a time dependent decrease in the average roughness amplitude. After 30 days, the average roughness amplitude was approximately 90 nm, while after 50 days the average roughness amplitude had decreased to around 65 nm.

3.7. Molecular characterization of plastic degrading microorganisms.

Microorganisms that were isolated from the active sludge in the bioreactor that were associated with plastic biotransformation were observed in Nutrient agar with different colony morphologies.

Four bacterial species that were associated with plastic biotransformation under anaerobic conditions were isolated and characterized using 16s rRNA molecular marker gene based identifications.(Nazeer and Fernando, 2022). Their taxonomic assignments were done based on the 16s rRNA gene sequences available in NCBI GenBank.

Table 3
Bacterial isolates associated with plastic biotransformation and their taxonomic assignment
Isolate	NCBI accession number	Scientific name	Percent identity.
S01_01	NR_181729.1	Pseudomonas glycinis strain PI111	98.35%
S03_03	NR_115714.1	Bacillus cereus strain CCM 2010	100%
S04_01	NR_025511.1	Gottfriedia luciferensis strain LMG 18422	100%
S04_02	NR_113344.1	Proteus mirabilis strain JCM 1669	100%

Using the Maximum Likelihood (ML) approach included in the MEGA software, a phylogenetic analysis was carried out to further clarify the evolutionary relationships between these isolates and related species. The ML tree was built using bootstrap analysis (2500 replicates) to evaluate the robustness of the inferred clades, and the best-fit nucleotide substitution model was chosen based on model testing criteria. The phylogenetic tree revealed that (Fig. 4.19) the isolates: S01_01, S03_03, S04_01, S04_02 clusters with Pseudomonas glycinis strain PI111, Bacillus cereus strain CCM 2010, Gottfriedia luciferensis strain LMG 18422 and Proteus mirabilis strain JCM 1669 with bootstrap values of 87%, 93%, 98 and 87% respectively The taxonomic placement and evolutionary divergence of the anaerobic bacterial isolates involved in plastic biotransformation were shown by the generated phylogenetic tree.

The daily methane volume curve for the reactor showed an increasing pattern of gas production over 24 hour period reaching 0.725 cm³/hour. The daily measurements recorded over a 30-day periods exhinits a significant variability ranging from a minimal 1 mL to maximun of 38 mL producing 17.4 mL avaergae daily volume directly attributing to the metabolic activity of biogas-producing microorganims within the system. Initially there is only low volume of gas recorded in first few date reflecting the lag phase, where microbial communities are adapting and beginning to establish themselves.There’s a sudden increase followed by decrease in day 5 and 6 respectively, suggesting an anomaly of the beginning of the increased microbial activity that corresponds with exponential growth phase. Then onwards the volumes stabilize, possibly because of substrate limitations or enviromental factors within the AD reactor. As the microorganisms continue to metabolize substrates, their population activity intesifies, leading to more rapid increase in biogas volume observed in the latter part of the peiod. The data revelaed a general upward trend in volume over time, with lower values observed only in the

early days and higher volumes recording at the later days, reaching upto 38 mL. Overall, the data demostrated a dynamic but steadilly increasing daily volume trend.

The cumulative methane volume curve for AD reactor shows a steady increas in methane production over 30 days time period(Fig. 4.3). Initially the curve rises sharply with 20% of the total methane produced within first 15 days. By the end of the 20th day nearly 41% of the total methane is produced and after the 25th day, during just after a 5 day timespan it completes the prodction of nearly 70% of the total volume. In between the day 20–25 the rapid production is likely to be attributed to the digestion of easily accessible organic matter in the substrates. By day 30, the cumulative methane volume reaches 522 cm³, with a specific methane yield coeffcient (Yx/s (methane)) of 1.74 mL CH₄/g of substrate provided.

Furthermore, data showed a direct correlation between methane production and microbial activity which is fueled by the microorganisms that produce biogas as they breakdown the substrates. The daily methane volume is initially quite low during which bacteria adjust to substrate and hence indicative of the microbial lag period. As shown by the increase in daily quantity beginning around 5th day, methane production rises when the microbial population adjusts and starts rapidly breaking down the substrates. This rise is consistent with the microbial populations’ exponential development phase, where improved methanogenesis results from substrate breakdown. A transient equilibrium between substrate supply and microbial consumption rate is suggested by the plateau seen between days 7 and 12. Methane quantities gradually rise after this stage, peaking at 38 mL on day 30, a sign of ongoing microbial activity and effective substrate conversion. The steady 10g intake of substrate promotes ongoing microbial metabolism, yet the rise in volume indicates the microbial biomass and enzymatic activity growth within time, increasing the efficiency of substrate consumption.

The relationship between yield coefficient (Yx/s (methane)) and substrate is a critical aspect of biogas production systems, in the understanding how substrates affect the efficiency of the AD reactor. According to Fig. 4.5, this experiment showed a significant increase in the yield coefficient of microbial efficiency in converting substrates into biogas. Initial low yield coefficients indicate microbial population’ adaptation phase, as they progress, metabolic pathways become more efficient, leading to higher biogas yields per unit of substrates. This highlights that efficiency is not solely dependent on substrate quantity but combined influenced with microbial activity. The gases produced in the bioreactor with the action complex microbial community helps in the process of the biotransformation.

Throughout the study PP showed a minimal mass loss with an average reduction of 0.04 mg over 50-day incubation period, indicating a strong material stability against microbial attack and corrosive gases. Although the standard deviation is twice the mean, suggesting some variability in mass loss, overall mass loss still remains negligible. At 10 and 20 days, some samples show minor mass loss but from 30 day onwards no measurable mass loss is observed in any samples. Replicates within each sampling event mostly show consistent results, with zero or near zero mass losses. This pattern suggests that only few samples have undergone minimal biotransformation under the conditions tested. The low average mass loss and high number of zero values indicate good overall stability of the plastic type.

PE also showed a mass loss indicating a moderate biotransformation under the conditions tested. The low average mass loss and high number of zero values indicate good overall stability over microbial attack and corrosive gases compared to Polyvinyl chloride (PVC). PVC, however, showed a notable mass loss for all pieces compared to PP and PE, showing the highest mass loss among all polymer types with an average of 1.11 mg. However, the mass loss did not exhibit any trend or temporal increment during the study period. The maximum mass loss observed to be 3.49%, which is also observed to be the highest of all plastic types. The higher average mass loss and minimal of zero values indicate poor overall stability of the plastic type within condition tested.

Chemical alterations brought on by microbial attack and the presence of corrosive gases during the sampling time are reflected in the various spectrum changes that were seen, particularly the emergence of the carbonyl peak when compared with the control samples. The Raman shift in the spectral range of C = O stretching vibrations in the region of 1729 cm-1 was considered significant and compared against an unchanged marker of PVC revealed a newly identified peak which has not been documented in prior studies of this material This suggests a novel information where chemical transformation and supports the evidence of surface changes, significant mass loss in the PVC compared to other polymer types as well as the appearance of degradation products. This can be further confirmed through the AFM analysis which showed a time-dependent decrease in the samples over 50 days.

Furthermore, the other prominent peaks at 636.5 cm⁻¹ and 696 cm⁻¹ correspond well with known C–Cl stretching and bending modes characteristic of PVC’s chlorinated backbone, consistent with literature reports. The peak at 1429 cm⁻¹ is associated with CH₂ bending vibrations, while the band at 2916 cm⁻¹ relates to C–H stretching modes, both typical of PVC’s hydrocarbon chains.

Polypropylene samples also showed a distinct spectral signatures featuring prominent peaks at positions, 808 cm^− 1, 840 cm^− 1, 1167 cm^− 1 and 1512 cm^− 1. The Raman shift in the spectral range of C = C stretching vibrations in the region of 1512 cm^− 1 was considered significant and compared against an unchanged marker of PP revealed a new peak with an increasing intensity supporting the evidence of surface changes as well as the appearance of degradation products over the course of 50 days. Similar response was reported by Lund Nielsen et al. (2019).

Among the isolated bacteria that associated with plastic biotransformation, organisms belonging to genus Pseudomonas are famously documented for their plastic biotransformation potential that shows the ability to biodeteriorate plastics such as PP, PE and PS through biofilm formation and enzymatic breakdown. Furthermore, species belonging to genus Bacillus including Bacillus cereus have been reported to transform plastic surfaces such as PE, through enzymatic action contributing to surface changes or mass loss under anaerobic conditions. Furthermore, few studies have shown that species belonging to genus Proteus exhibit the biodeterioration ability of plastics such as PE and PET (Ojiego et al., 2022)

However, other isolated bacteria from plastic surfaces; Gottfriedia luciferensis show limited or no direct evidence and were not previously known in studies to exhibit plastic biotransformation ability. Related species of Genus Gottfriedia which was formally part of genus Bacillus may possess the ability of biodeterioration which explains the potential of Gottfriedia luciferensis in plastic biotransformation in this study. Therefore, it could be concluded that the biotransformation properties of aforementioned bacterial isolate were unknown until now and have been amply proven by this study's findings.

The widespread use of petrochemical plastics poses significant environmental issues, including pollution, long term ecological risks, and challenges in recycling due to their non-degradable nature. Although research indicates that biotransformation occurs during anaerobic digestion (AD), the mechanisms of these transformations are poorly understood. Therefore, this study investigated the possibility of using AD system microorganisms in biotransforming polymers as a low-cost alternative method. Among the plastics tested, PVC exhibited a significant mass loss in which Raman spectroscopy revealed the emergence of newly identified peaks at 1729 cm^− 1, corresponding to C = O stretching vibrations. This spectral change indicates chemical changes on plastic surfaces, supporting the microbial driven transformation process. Although the initiation of biodegradation of polymers with only carbon backbone in the main chain like PP are very scarce under AD condition, a significant Raman peak at 1512 cm^− 1 related to C = C stretching vibrations of this polymer showed that could be regarded as the early stages of their degradation. Collectively, these findings highlight that, while AD systems can facilitate the biotransformation of plastics like PVC, few other plastics remain largely resistant under the tested conditions. Therefore, further research is needed to elucidate the detailed mechanisms, optimize conditions for broader plastic degradation, and assess the environmental fate of transformation byproducts.

Author Contribution

AM, SR wrote the original manuscript, CB, MS and AM collected anaerobic sludge samples and constructed the digester, AM and SR conducted experiments, PNY, CB, and MS analyzed data and EYF conceptualized the study and conducted molecular phylogeny work.

Acknowledgement

The academic support staff of the Botany and Zoology labs of the Rajarata University, Faculty of Applied Sciences are thankfully acknowledged for their efforts in setting up the anaerobic digestion system used in this study.

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No competing interests reported.

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Potential accelerated biotransformation of petrochemical plastic surfaces by anaerobic digester sludge microorganisms

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Abstract

Figures

1. Introduction

2. Material and methods

2.1. Sample collection

2.2. Laboratory scale Anaerobic Digester (AD) setup construction

2.3. AD system operation

2.4. Petrochemical plastic biodegradation study

2.4.1. Plastic piece preparation

2.4.2. Sub-bioreactor preparation

2.5. Plastic biodegradation assessment.

2.5.1. Mass loss analysis

2.5.2. Statistical analysis

2.5.3. Surface characterization by Phase contrast microscopy

2.5.4. Surface characterization by Raman analysis

2.5.5. Surface characterization by atomic force microscopy (AFM)

2.6. Molecular characterization of plastic degrading microorganisms

2.6.1. Isolation and identification

2.6.2. DNA extraction and molecular characterization

3. Results

3.1. Performance of the bioreactor

3.2. Mass loss analysis

3.3. Statistical analysis

3.4. Surface characterization by phase-contrast microscopy

3.5. Surface characterization by Raman analysis.

3.6. Surface characterization by atomic force microscopy (AFM)

3.7. Molecular characterization of plastic degrading microorganisms.

4. Discussion

5. Conclusion

Declarations

Author Contribution

Acknowledgement

References

Additional Declarations

Supplementary Files

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