Effects of Different Polymeric Materials on the Bacterial Attachment and Biofilm Formation in Anoxic Fixed-Bed Biofilm Reactors

Six biofilm carriers with different polymer were studied in fixed-film systems under anoxic conditions. Different media of polymers influence wastewater treatment performance. The aim of this study was to investigate different polymeric materials, polyethylene terephthalate (PET), polyvinyl chloride (PVC), polypropylene (PP), high-density polyethylene (HDPE), and polymethyl methacrylate (acrylic), that affect bacterial attachment and biofilm formation in biofilm-based wastewater treatment technologies. Water contact angle (WCA) measurement was employed to analyze the role of wetting (hydrophilic/hydrophobic) of polymeric material surfaces in the initial phase of bacterial attachment. The increase of biofilm formation during the observation was determined by gravimetric (total attached solid) and microscopic (SEM and CLSM) analysis. The results showed the value for WCA of PET < HDPE < PVC < PP < acrylic, which indicated that a higher hydrophilicity surface leads to a higher total attached solid (TAS), biofilm formation rate, and biofilm thickness on the surface of media. The hydrophilic material (i.e., PET and HDPE) demonstrated wastewater treatment performance better than slightly hydrophilic material (i.e., PVC, PP, and acrylic) under a steady-state period (over an 80-day operation). The data showed a positive correlation between hydrophilic material and COD, NH4+-N, and TP removal. Hydrophilic material was beneficial for a fast start-up and stable biofilm formation of a fixed-bed biofilm reactor. PET media showed feasible polymer types compared to HDPE, PVC, and PP; thus, it can be used as an alternative biofilm carrier media in a larger-scale application. The findings of this study highlighted the polymeric material type has a significant effect on the performance of fixed-bed wastewater treatment.


Introduction
Biofilm-based systems such as moving bed biofilm reactors (MBBR) and fixed-bed biofilm reactors (FBBR) are widely applied in the integrated nitrogen and phosphorous removal of nitrogen and phosphorus in wastewater treatment plants (WWTPs) (Ahmad et al., 2014;Al-amshawee et al., 2021). This promising technology offers several benefits over the conventional activated sludge (CAS) method, e.g., increased biomass retention and performance stability, low sludge production, and resistance to toxic shock load (Huang et al., 2017;Shao et al., 2017). Surface-immobilized cells (fixed biofilms) on biofilm carriers provide several benefits over to suspension culture, including greater microorganism population diversity, lower sensitivity to environmental variations (temperature, pH, and toxicants), higher growth rates, and rapid substrate utilization. The key process in the initial attachment and biofilm development within biofilm systems is microbial adhesion to the surface carrier (Huang et al., 2017;Mahto & Das, 2021).
Polymeric media, for instance, polypropylene (PP), polyethylene (PE), polystyrene (PS), polyurethane (PU), high-density polyethylene (HDPE), polyvinyl chloride (PVC), are widely applied in biological wastewater treatment plants, due to their excellent mechanical strength, lightweight, chemically inert, and durability (Deng et al., 2016;Yuan et al., 2015). The physicochemical properties of polymer media have a significant effect on biomass adhesion (Mao et al., 2017). Bacterial adhesion or the initial attachment of microorganisms to the surface of the media is affected by the wettability (hydrophobic or hydrophilic) of a biofilm carrier Zhang et al., 2018). Wettability strongly influenced the rate of initial microbial adherence and interaction with a carrier surface. In wastewater treatment, the term wettability is the degree of contact possible between wastewater with a biofilm carrier surface. Water contact angle (WCA) measurements are commonly used to characterize the wettability of a material's surface (Al-Amshawee et al., 2021) and are the best method for determining bacterial hydrophobicity (Doyle, 2000).
Surface hydrophobicity/hydrophilicity is a critical factor for bacterial adhesion in surface materials. Materials with a WCA below 90° are hydrophilic, while materials with a WCA above 90 0 are hydrophobic (Chu et al., 2014;Feng et al., 2015;Liu et al., 2022). Chavant et al. (2002) showed biofilm formation tends to be faster on hydrophilic surfaces. This is in line with other studies, where materials with water contact angles above 90° exhibited lower microbial adhesion (Chu et al., 2014;Feng et al., 2015;Nguyen et al., 2019;Yuan et al., 2017). For this reason, material surfaces are often modified to enhance bacterial adhesion Mao et al., 2017;Yao et al., 2013). Hydrophilic carriers typically have a higher energy surface than hydrophobic carriers (Nguyen et al., 2019), implying bacteria in the water attach and develop easier on biofilm carriers with hydrophilic surfaces (Renner & Weibel, 2011). However, several reports have debated the influence of wettability on bacterial adhesion. Peng et al. (2018) and Liu et al. (2017) found no consistent relationship between adhesion and the hydrophobicity of a polymer, while Lackner et al. (2009) highlighted the inconsistencies among literature in favoring wettability for bacterial adhesion. This led to the emergence of several fundamental questions which need to be answered. Zhou et al. (2021) showed a positive correlation between seven types of polymers with biofilm growth, bacterial population, and wastewater treatment performance in fixed-bed systems under anaerobic conditions. The use of waste polyethylene terephthalate (PET) plastic bottles as a fixed biofilm carrier has been studied. PET possesses ideal surface properties as a novel biofilm carrier (Dorji et al., 2021). In Indonesia, waste PET plastic bottles are widely applied within residential-scale WWTPs. As a low-cost fixed biofilm carrier, PET can improve the performance of conventional on-site sanitation systems. These polymers achieve removal efficiency of organic pollutants between 70 and 90%, and some of them meet the discharge standard of Indonesian domestic wastewater (Nur et al., 2020). Indonesia is the world's 4th highest consumer of PET bottles and this consumption is constantly increasing (PT Chandra Astri Petrochemical, 2017), therefore, the use of waste PET plastic bottles is one of the effective solutions for the local government to reduce the environmental impacts. The use of waste PET bottles as biofilm-carrier media has also been adopted in other developing countries because the use of locally available materials helps the cost of wastewater treatment affordable. The cost of treatment is a concern for developing countries. Previous studies showed that the use of shredded waste PET bottles as biofilm carriers achieved a 70 to 90% COD removal, which could meet the Bhutan discharge standard (Dorji et al., 2021(Dorji et al., , 2022. In other studies, waste PET plastic bottles removed 67% of ammonia and 98% of E. coli (Wąsik & Chmielowski, 2017), and achieved 84%, 66%, and 90% removal efficiency of COD, total nitrogen (TN), and NH 4 + -N, respectively (Kottatep et al., 2022). Therefore, investigating the influence of PET on the initial phase of biofilm formation and growth is expected to provide a better understanding of its use in the improvement of on-site sanitation WWTPs in Indonesia. The fast biofilm formation rate may even be of benefit for operating conditions (Zhao et al., 2019).
Given the important role of the influence of wettability on the surface of carrier, there have been limited studies on the effect of PET polymer on biofilm adhesion, biofilm formation, biofilm thickness, biofilm growth rate, and wastewater treatment performance. Therefore, the comparison of PET with four other polymers commonly utilized in full-scale wastewater treatment plants in Indonesia: PVC, PP, HDPE, and polymethyl methacrylate (acrylic), is bound to contribute to the scientific understanding of the effect of different polymeric materials on the initial phase of biofilm formation in fixed-bed biofilm reactors under anoxic condition. The findings of this study are expected to provide information for the design and startup of fixed-bed reactors, moreover the possibility of using PET as a novel carrier for Indonesian public sewage infrastructure and other developing countries that will offer similar effectiveness as widely available commercial carriers.

Experimental Set-up
The 6 polymer materials used as biofilm carriers in this study were PET brand 1 (PET-1), PET brand 2 (PET-2), PVC, PP, HDPE, and acrylic. PET-1 and PET-2 are the most prevalent local mineral bottle brands applied as biofilm carriers in residential WWTPs across Indonesia, while PVC, PP, and HDPE are widely applied in fixed-bed wastewater treatment systems. Two replicates of each polymer media (5 polymers were in the form of 10 × 2 cm 2 coupons, while 1 PP media was still in the form of a bio-ball) were vertically placed inside the reactor. In total, each media was made in 16 pieces for analysis on days 10, 20, 30, 40, 50, 60, 70, and 80. These eight-time points were selected to explore the evolution of biofilm growth from the initial phase to the mature phase. Previous studies investigated the maturation of biofilm between weeks 1 and 6 (Bassin et al., 2016;Tang et al., 2016), short-time biofilm formation between weeks 0 and 8, (Ganesan et al, 2022), while Zhou et al. (2021) used three-time to illustrated biofilm formation phases in anaerobic fixed-film systems.
The 6 laboratory-scale acrylic circulating batch reactors (CBRs) with a working volume of 900 ml (15 cm diameter and 10 cm height) (see Fig. 1) were designed to study the initial phase of biofilm development under anoxic conditions. The recirculation pump was installed to ensure that hydraulic flow was distributed uniformly throughout the system. Before starting the operation, N 2 gas was injected from the bottom of the reactor for 10 to 15 min to generate anoxic conditions, forcing the oxygen in the reactor to exit through the liquid airlock. After the injection was complete, the liquid airlock was filled with distilled water to ensure no oxygen entered or exited the reactor. The recirculation pump was used to pump back the gas in the CBR freeboard into the reactor through the perforated plate at the bottom, on which the media was placed vertically. As the gas enters the reactor, the lifting force completely stirs the substrate and inoculum, facilitating the attachment of microorganisms to the media and preventing the inoculum from settling at the bottom of the reactor. The pH, temperature, and DO of each reactor were monitored periodically to ensure optimal bacterial growth. The reactor was operated at room temperature and DO concentration was kept below 0.5 mg O 2 /L in anoxic conditions.

Inoculum and Feed Wastewater
The inoculum of the reactor was fecal sludge collected from the sedimentation zone of a septic tank in a residential area of Bandung, Indonesia. The reactor fed with synthetic domestic wastewater consists of glucose as the primary substrate, NH 4 Cl and KH 2 PO 4 as sources of nitrogen and phosphate, respectively. Synthetic wastewater has characteristics that are similar to domestic wastewater in one of Bandung City's residential WWTPs (Nur et al., 2020). The main characteristics were as follows: COD 279.14 mg/L, NH 4 + -N 12.88 mg/L, TN 15.04 mg/L, and TP 2.9 mg/L. The C:N:P ratios were 100:5.38:1.04 and it is the C:N:P ratios for optimum bacterial growth conditions for anoxic 100:5:1 (Metcalf & Eddy, 2014). The characteristics of synthetic domestic wastewater have the same values when fed into the six reactors.

Biofilm Attachment and Development
In this study, the attached biofilm quantity was presented as Total Attached Solid (TAS). Several studies used the TAS parameter to observe the increase in biomass attached to the media (Dias et al., 2018;Zan et al., 2021;Zhao et al., 2019;Zhou et al., 2021). Biofilm attachment and development were illustrated through biofilm formation, biofilm thickness, biofilm growth rate, and wastewater treatment performance, which were obtained on days 10, 20, 30, 40, 50, 60, 70, and 80. On each of these days, two polymer materials were selected randomly from each reactor; one for TAS analysis, while the other for biofilm thickness analysis and CLSM visualization. Dry-weight biofilms were used for gravimetric and SEM thickness measurements.
TAS analysis used gravimetric methods and it measured following the Standard Methods 2540 B (APHA, 2017). The biofilm attached to material polymers were dried at 65 °C for 24 h and weighed. The dried biofilm was gently scraped off with a sterilized razor blade until it was completely removed. The rate of biofilm growth was calculated using Eq. 1, where CB i and CB t are the initial and final TAS values, while t i and t f are the time required for CB i to reach CB t .

Wetting Test
The wettability test was performed using a contact angle meter (tantec V, Half-Angle™ Technique, US Patent #5.268.723). The hydrophilicity of polymer material was determined by measuring the WCA between droplets of water and its surface. For this analysis, 25 µL drop of distilled water on horizontal polymer materials using a 500-µL syringe with narrow-gauge stainless steel (Mitutoyo). The needle has a very small diameter to avoid distorting the drop profile shape. The imaging of the water droplet was captured with a micro camera and measured the water contact angle with software image J. There were five angle tests at five different locations on each type of polymer, and at each location there were three measurements per droplet of water.

Analytics
Daily measurements of DO, temperature, and pH were taken with a portable DO meter (LT Lutron PDO-519) and a portable pH meter (Kedida CT-6022), respectively. Meanwhile, the concentration of COD was evaluated every ten days, following Standard Methods 5220-B. Ammonia and total phosphate (TP) were measured at the start and the end (steady-state condition) of the experimental process, Vol.: (0123456789) based on Standard Methods 4500-NH 3 -B and 4500-P-B-D (APHA, 2017).

Microscopy
A scanning electron microscope (SEM) Hitachi SU3500 was used to visualize the surface morphology of biofilm-covered polymer media and characterized every 10 days of growth. The thickness of the biofilm adhered to the media was measured using SEM through the cross-section of each media. The measurement was repeated five times to provide an average value representing biofilm thickness. Furthermore, the 2D structure of the biofilm was visualized using a Confocal Laser Scanning Microscope (CLSM, Olympus FluoView FVI 200). Samples were previously washed in sterile Deionized (DI) water to eliminate non-adherent cells, scraped off with a sterile razor, a protocol adapted from Zhou et al. (2021), and placed on a micro-well plate. The samples were stained using SYTO9 and Propidium Iodide (PI), each of 100 ml. SYTO9 and PI were dissolved in 10 mL of phosphate buffer saline (PBS) each before being employed as staining in a sterile room. All preparations, from staining to CLSM visualization, were executed in low light. Under fluorescence microscopy, live bacterial cells were stained with SYTO9 fluoresced green, while dead cells were stained with PI fluoresced red (Zhou et al., 2021). SYTO9 and PI staining is a test for live/dead cell viability.

Statistical Analysis
The results were expressed as mean data with error bars referring to standard deviation. One-way analysis of variance (ANOVA) test was used for statistical comparisons at 95% confidence interval (p < 0.05) by using the software IBM SPSS Statistics 21.

Wettability Analysis
The WCA values were used to determine the wettability of sample surfaces. Figure 2 shows the WCA phenomenon of PET, PVC, PP, HDPE, and acrylic polymer which showed different surface hydrophilicity (WCA < 90 0 ). Polymers ranked in order of wettability based on WCA measurement were PET > HDPE > PVC > PP > acrylic. As a result, PET having the lowest WCA (most hydrophilic) of 70.67 0 ± 0.15 and 70.68 0 ± 0.05, followed by HDPE, PVC, PP and acrylic (least hydrophilic) with a WCA of 77.20 0 ± 0.15, 77.77 0 ± 0.14, 81.08 0 ± 0.06 and 84.81 0 ± 0.13, respectively. The hydrophilic materials are favorable for accelerating microorganism adhesion (Habouzit et al., 2011), reducing the startup duration and stabilizing biofilm formation (Zhu et al., 2015). The different in hydrophilicity properties of each type of polymer due to polymer functional groups on hydrocarbon backbones represented by methyl groups (Ganesan et al., 2022).
Bacterial adhesion tends to occur faster on hydrophilic surfaces because the hydrophilic properties of the polymer surface help promote the fast adhesion of bacterial cells (Jones & Buie, 2019;Zhao et al., 2019). Therefore, PET is bound to facilitate faster bacterial attachment, compared to HDPE, PVC, PP, and acrylic. Bacterial adhesion to the surface begins with cells adhering to the surface, followed by Water contact angle ( 0 ) hydrophilic adsorption and attachment (Boks et al., 2008). The first attachment is rapid, reversible, and characterized by hydrodynamic and electrostatic interactions. In addition, the adhesion force between bacteria and surfaces of polymer grows considerably throughout the period. Extracellular polymeric substance (EPS) is responsible for biofilm adhesion, as well as development, and the most important component of biofilm (Ganesan et al., 2022). Table 1 shows the WCA of PET, PVC, HDPE, PP, acrylic, reported by previous studies. In line with these studies, the WCA of PET (Ahmad et al., 2014;Peng et al., 2018), HDPE (Lakshmi et al., 2012), acrylic (Zisman, 1964), PVC (Lakshmi et al., 2012;Zhao et al., 2019) and PP (Hadjiev et al., 2007;Lakshmi et al., 2012;Zhao et al., 2019;Youli Zhu, 2017) are hydrophilic. However, some studies reported the WCA of PP to be above 90 0 (Jurecska et al., 2013;Lackner et al., 2009). The differences in the WCA values reported by other studies are due to the different types of wastewater. In cases where wettability is the most important factor, biofilm detachment is critical because detached biofilms influence the mixed liquid's hydrophobicity/hydrophilicity. Therefore, irreversible deposition is often preferred to biomass immobilization. Rather than aiming to achieve zero detachment, investigations ought to be carried out to account for the rate of biofilm detachment.
According to the references, PET and acrylic possess the highest and least surface energy values of the 6 polymers. The surface energy of a substance increases with increasing hydrophilicity, thereby influencing the rate of bacterial adhesion and biofilm formation. Liu and Zhao (2005) showed surfaces with high energy exhibited high bacterial adhesion and the biofilm was difficult to remove with moderate brushing. Conversely, hydrophobic surfaces are believed to possess low surface energy (Bae et al., 2013;Zhao et al., 2019). Liu and Zhao (2005) showed that low surface energy materials tend to attract fewer bacteria which are more loosely attached and easily detachable, compared to high surface energy counterparts. Bacterial adhesion to surfaces with varying surface energy has been studied for the past two decades. The extended Derjaguin, Landau, Verwey, and Overbeek (DLVO) model was used to explain the effect of surface energy on bacterial adhesion, as well as the interaction energy between bacteria and substrates in water. 3.2 Biofilm Attachment and Development Figure 3a shows the trend in the Total Attached Solid (TAS) values for 6 different polymers over 80 days of observation, where the values increased during the observation period, indicating increasing biomass growth and adhesion for the 6 media surfaces. This was supported by microorganism growth-promoting environmental factors (temperature, pH, and DO). The temperature, pH, and DO were maintained ranging from 23.67 to 25.27 °C, 6.05 to 7.11, and 0.2 to 0.45 mg O 2 /L, respectively. However, the rate of attachment and biomass growth varied for each polymer material. Biomass adheres and grows faster on PET than on HDPE, acrylic, PVC, and PP. PVC exhibited a higher adhesion than acrylic until day 50, however, by day 60, the biomass adhesion was higher for the acrylic polymer. Despite differences in TAS values between PET, PVC, PP, HDPE, and acrylic, the overall trend of increasing TAS values in all polymers has remained consistent throughout the observations. A sloping graph indicated that microorganism growth was slow up to the 20th day, indicating that the microorganisms were adapting to their new environment. The fastest growth occurred between days 20 and 60. In this condition, the microorganism became accustomed to its new environment, allowing it to multiply rapidly, the mass and density of the number of cells increased exponentially, then began to slow until the final day of observation, when they reached a steady state condition.
There was a positive correlation between the TAS and biofilm thickness for the different polymer materials during the operational period of the anoxic fixbed biofilm reactor (r values ranging from 0,0984 to 0,9948). Figure 3b illustrates the same trend of increasing biofilm thickness as TAS. At the end of day 80, the biofilm thickness for the polymer materials was PET > HDPE > PVC > acrylic > PP. The statistical analysis showed significant differences (p < 0.05) in the average biofilm thickness of the PET, PVC, PP HDPE, and acrylic (see Table 2).
The surface of the polymer influences the difference in initial biofilm attachment rate and biofilm adhesion resistance on an adhesive medium (Dias et al., 2018) (Gomes et al., 2013). Materials with a water contact angle below 90 0 are hydrophilic, while materials with a water contact angle above 90 0 are hydrophobic. This method has been proven to be the most accurate for describing surface hydrophobicity/hydrophilicity (Chu et al., 2014;Feng et al., 2015;Yue Yuan et al., 2017). According to Zhao et al. (2019), well-wetted carriers tend to enhance initial adhesion and accelerate the start-up of biofilm-based wastewater treatment. PET has a smaller θ w 0 (70.67 ± 0.15 and 70.70 ± 0.06), as well as higher hydrophilicity and surface energy, compared to PVC and PP, indicating microorganisms present in water are more easily absorbed and grow better on hydrophilic surfaces. The attachment of microorganisms to a material surface increases with reducing θ w 0 (Dias et al., 2018;Peng et al., 2018;Zhou et al., 2021). The anoxic TAS of PVC in this study is similar to Zhou et al. (Zhou et al., 2021), where the adhesion and growth of microorganisms on 7 types of polymers (HDPE, ABS, polycarbonate: PC, PVC, PP, PVDF, and Acrylic) were investigated using artificial domestic wastewater and the TAS of HDPE, PC, PVC, PP, and acrylic polymers were determined on the 81 st day. Trinh et al. (Trinh et al., 2020) compared the adhesion of biofilm on PVC and HDPE pipes under aerobic conditions and discovered HDPE exhibited better adhesion, compared to PVC. PP is highly hydrophobic, possesses low wettability, and consequently, takes months to get completely wet (Lacker et al., 2009). Polymers with high surface energy are commonly used to create hydrophilic products. Several studies showed PET possesses higher surface energy, compared to PVC (Krevelen & Nijenhuis, 2009;Lakshmi et al., 2012), PP (Krevelen & Nijenhuis, 2009); (Lakshmi et al., 2012) and HDPE (Lakshmi et al., 2012).
In addition to surface energy (hydrophilicity), the electrophilic property of the biofilm carrier influences initial attachment and biofilm formation (Yuan & Lee, 2013). Microorganism surfaces tend to have a negative charge due to the presence of carboxylic acid and phosphoric acid groups in cell membranes. Therefore, negatively charged surfaces impede biofilm formation due to repulsions between bacteria and carrier surfaces. This challenge is often overcome by modifying biofilm carrier materials to generate positively charged surfaces or change the surface electronegativity of biofilm carriers. Modified carriers with positively charged surfaces that have a lower water contact angles tend to exhibit higher biofilm adhesion, compared to unmodified carriers with negatively charged surfaces Mao et al., 2017).
Previous studies described surface modification as an effort to boost positive charge, hydrophilicity, and surface energy to improve the rapid start-up of microbial initial attachment and biofilm development and achieve excellent performance (Khan et al., 2013). Several physical and chemical surface modifications have been carried out to facilitate bacterial attachment, as well as biofilm formation, thereby accelerating the reactor start-up. Electrophilic modified carriers are expected to have higher removal efficiencies of target pollutants in the biofilm reactor, compared to unmodified carriers (Mao et al., 2017;Quan et al., 2017;Song et al., 2019). Liu et al. (2021) investigated the effectiveness of modified PE carriers in the treatment of synthetic wastewaters and discovered the carriers performed commendably, due to the increased hydrophilicity and positively charged surface. Meanwhile, Song et al. (2019) conducted a comparative study between modified and unmodified sponge bio-carriers and discovered modified sponge removed more total nitrogen (TN), compared to the unmodified counterpart. According to Liu et al. (Liu et al., 2017), modified carriers (PQAS-10 and Fe 2 O 3 modified PE) have more associated biomass, compared to unmodified carriers and consequently, produce higher TN, COD, and NH 4 -N reduction from the biofilm reactor. A recent study also reported that the addition of ferrous magnetic elements to bio-carrier surfaces increased biomass density and ammonia removal efficiency, but shortened the bioreactor startup time. Ferrous oxalate (C 2 FeO 4 ) has strong adsorption potentials, and was selected as a suitable nanoparticle (Zhao et al., 2019). Figure 4 shows the biofilm growth rate of anoxic biofilms during the 80-day observation period. The fastest biofilm growth rate occurred on day 20 to 50 on PET-1 and PP, which were 0.0146 ± 0.006 mg/ cm 2 .day and 0.0075 ± 0.004 mg/cm 2 .day, respectively. However, these rates slowed down to 0.0150 ± 0.009 mg/cm 2 .day and 0.0062 ± 0.005 mg/ cm 2 .day, respectively, after day 50. The fastest biofilm growth rate of 0.0107 ± 0,005 mg/ cm 2 .day on day 25 for PVC. This slowed down to 0.0074 ± 0.007 mg/cm 2 .day by day 80. PET-2, HDPE, and acrylic showed the fastest biofilm growth rates of 0.015 ± 0.006, 0.0134 ± 0.005, and 0.009 ± 0.004 mg/ cm 2 .day, respectively, between days 20 to 60. The rates began to slow down after day 60, until day 80, where values of 0.0102 ± 0.012, 0.0081 ± 0.010, and 0.0064 ± 0.007 mg/cm 2 .day, respectively.

Biofilm Growth Rate
PET exhibited the highest biofilm growth rate among all the polymer samples. Figures 3 and 4 show the biofilm thickness on each media is proportional to the growth rate of the biofilm. This is in line with previous research (Dias et al., 2018;Trinh et al., 2020;Zan et al., 2021); however, Zhou et al. (2021) showed no clear relationship between hydrophilic surfaces and biofilm thickness or the percentage of live dead after 80 days.
During the phase of the highest biofilm growth rate, the surface biofilm layer developed due to continuous growth and adsorption, leading to the accumulation of biomass in the form of a slime layer, which continues to develop and becomes thicker. Subsequently, food diffusion continues until the maximum thickness is reached, at which point food diffusion will no longer reach the solid inner layer. In this condition, exfoliation of the biomass layer begins, leading to the immediate formation of new microorganism colonies to continue the biofilm formation. The growth rate reduces, due to flaking which becomes lysed biomass and tends to form new colonies.

Microscopy
SEM and CLSM were used to determine the surface structure and thickness of the biofilm. Figure 5 illustrates the SEM image of the surface morphology of the biofilms growing on all 6-polymer media from day 0 to day 80. Based on the visualization, the biofilm occupies the surface area and increases in thickness with time, however, this accumulation was not uniform on each side of the polymer surfaces. Therefore, the biofilm thickness was measured in quintuplicates and averaged to obtain a representative value for each sample.
The surface morphology of the 2D biofilm attachment on the PET coupon samples, as well as the crosssection of biofilm thickness, were visualized using CLSM. The image of biofilm growth using CLSM at a frame area of 1260 × 1260 m (Fig. 6) shows the 2D structure of the biofilm in the form of live bacterial cells (green color) and dead bacteria (red color). The biofilm thickness on the PET medium increased with time, however, by day 80, some dead bacteria were observed, even though in very small numbers. The accumulation of microorganisms or biofilm thickness on the media surface prevents food and oxygen diffusion to the media surface, which then leads the biofilm layer less adherent and allows the biomass to detach (Dias et al., 2018). Biomass thickness was measured from the 2D structure of the CLSM image; however, this method produces slightly lower values, compared to SEM measurements. This is because biofilm preparations were carried out before CLSM observations while the SEM method directly counted the biofilm attached to the media.

Wastewater Treatment Performance
The metabolic activity of microorganisms attached to each polymer media influenced the performance of each treatment in each reactor, as evidenced by the removal efficiency of COD, ammonia (NH 4 + -N), and total phosphate (TP) (Fig. 7). The removal efficiency was calculated from the moment steady-state conditions were reached until day 80. Based on the calculations, all the polymer samples successfully removed organic pollutants and nutrients from wastewater, however, the removal efficiencies varied for each media sample. The highest COD removal efficiencies was 72.75% and 72%, respectively, for PET-1 and PET-2, followed by 67%, 65%, 60.52%, and 64.96%, for HDPE, acrylic, PVC, and PP, respectively. Similar results were recorded for the nutrient (ammonia and phosphate) removal efficiency. In all reactors,  Fig. 4 The biofilm growth rate for different polymeric materials in the anoxic fixed-bed biofilm reactor the ammonia removal efficiency ranged from 54.35 to 69.67%, and was higher, compared to phosphate removal which ranged from 40.56 to 63.86%. This reinforces the hypothesis that hydrophilic polymers exhibit higher biofilm growth rates and more stable biofilm formation, which significantly influences the performance of wastewater treatment. A strong positive correlation was found in this study between the TAS value and biofilm thickness for different polymers by using the Pearson Correlation test (see Table 3), with r-values ranging from 0.984 to 0.998. Material of polymers plays important role in wastewater treatment performance. Based on the results, substrate removal by the microbial layer increases linearly with increasing film thickness up to the maximum thickness, after which it remains constant with further increase in thickness (Trinh et al., 2020;Yuan et al., 2020;Zhou et al., 2021).
Previously, modified bio-carrier received wide attention because these surfaces were expected to promote metabolism, biofilm growth, and improve biodegradation in wastewater treatment technologies  (Mao et al., 2017). In addition, unmodified PP (poor hydrophilic surface) has an adsorption capacity of 8.4 × 10 4 (CFU/gh) on biofilm growth and removed 80% COD, as well as 63.70% NH 4 + -N. Surfaces modified with Barium ferrite or diatomite tend to exhibit hydrophilic surface properties (58.5 0 ), possess higher bigger adsorption capacity, require shorter time for biofilm formation, which increase from days 12 to 15, and produce COD and NH 4 + -N removal efficiencies above 90% and 85.40%, respectively (Zhu, 2017).
PET is receiving new attention and providing new expectation as a feasible bio-carrier on wastewater treatment facilities across developing countries, including Indonesia. Based on this study's findings,  PET is comparable to other manufactured bio-carrier materials in wastewater treatment systems and offers significant benefits of reduced costs, as well as increased performance for anoxic fixed-film wastewater treatment systems. The application of PET as a biofilm-carrier media applied in some residentialscale WWTPS across Bandung, West Java Province, Indonesia, successfully removed organic and nutrient pollutants from domestic wastewater, thereby improving the performance of domestic wastewater treatment (Nur et al., 2020). Therefore, waste PET bottles not only have the potential for application as biofilm-carriers for public sewerage infrastructure but also offer an efficient waste management strategy for Indonesia.

Conclusion
This study demonstrated the influence of six polymeric materials (PET, PVC, HDPE, PP, acrylic) on biofilm formation, biofilm growth rate, and wastewater treatment performance during the initial phase of biofilm formation under anoxic conditions. Based on the findings, the type of polymers influences the initial attachment of microorganisms. Under anoxic conditions for 80 days of operation, microorganisms in the wastewater adhered quickest to PET, which has the lowest WCA (most hydrophilic) among the media materials. The media materials were arranged in higher order of bacterial adhesion as: PET > H DPE > PVC > PP > acrylic. In addition, the quickest biofilm growth rate occurred from day 20 to day 60 and subsequently began to slow down until day 80, when steady-state conditions were reached. The COD, NH 4 + -N, and TP removal efficiencies ranged from 60.52 to 72.75%, 54.35 to 69.67%, and 39.35 to 63.86%, respectively. In wastewater treatment systems based on the biofilm process, the type of polymer media in a fixed-bed system influences the initial attachment of microorganisms, biofilm thickness, biofilm growth rate, and domestic wastewater treatment performance. The results showed PET produced desirable wastewater treatment efficiency, compared to HDPE, PVC, PP, and acrylic, thus it can be used as an alternative biofilm carrier media for large-scale application. This is bound to facilitate an increase in the economic competitiveness of fixed attached growth system technologies.
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