Thermophilic Digestion is Enhanced in The Presence of Polyamide Microplastics

INTRODUCTION Despite the fact that MPs can be removed from wastewater with an efficiency of up to 90 %, still 105108 microplastics (MPs) are released from WWTPs each day (HatinoÄŸlu & Sanin, 2021). Indeed, more than 90% of the removed MPs accumulate in sludge during treatment (Okoffo et al., 2019) reaching an abundance varying from 510 to 495,000 MP particles/g DS (HatinoÄŸlu & Sanin, 2021). Studies reveal that MPs have harmful impacts on organisms; they may act as a vector of hazardous pollutants such as pharmaceuticals and heavy metals with an absorption mechanism onto their surfaces. Moreover, they provide sustainable surfaces for pathogens to colonize and form biofilm. Even though pathogens can be removed by WWTPs to a great extent, MPs can offer a safe environment for them to survive during the treatment process. MPs themselves, together with the hazardous chemicals and pathogens they can accumulate on their surfaces, pose a new concern for the land application of biosolids. Anaerobic digestion (AD) is a sustainable stabilization system with renewable energy generation and good-quality biosolids production for land application. Thermophilic digestion (TAD) is well known to be more effective in stabilization compared to mesophilic AD (MAD) due to its effective pathogen inactivation and Class A biosolids generation. Recent studies have shown that the presence of MPs can affect AD, and in addition, MPs themselves seem to be affected during the process. Yet, all these studies investigating MPs in AD are conducted under mesophilic conditions. TAD has not been investigated so far to reveal the fate and effect of MPs during the process. Polyamide (PA) (nylon) with amide groups (-CO-NH-) having the strength and fatigue resistance is one of most commonly encountered type of MP in sludge (El Hayany et al., 2022). Its high abundance and leaking potential of its monomer caprolactam (CPL) during MAD (Chen et al. 2021) creates interest for PA6. This study aims to comparatively investigate the effect of PA6 on methane production with MAD and TAD while evaluating this MPs' fate during these processes. This work is unique for identifying the behavior of MPs in TAD and specifically evaluating the fate and effects of PA6 comparatively in MAD and TAD processes. MATERIAL & METHOD For the simulation of MAD and TAD, biochemical methane potential (BMP) test was conducted at 35oC and 55oC, respectively. Waste activated sludge (WAS) (substrate) and anaerobically digested sludge (ADS) (inoculum) were sampled from a conventional WWTP designed for a population equivalent of 4 million with a capacity of 765,000 m3/day. The seed was used as sampled for MAD BMPs but was acclimated for the TAD study. As PA6, a fishline with a diameter of 0.5 mm was cut into sizes in the 425-500 µm range and used. Each mesophilic and thermophilic BMP tests had 13 sets of triplicate reactors comprising biotic, abiotic control and seed control groups. Each biotic and abiotic set included six different PA6 doses in reactors, 0, 10, 30, 60, 100, and 200 MPs/g TS. Reactor TS values were adjusted to 2% with an F/M of 1 (g VS of WAS/g VSS of seed). All reactors were purged with 99% nitrogen gas, and were incubated for 60 days. Biogas volume was measured routinely with water displacement and its composition was determined by Gas Chromatography (GC) with a thermal conductivity detector (TCD). Solids were measured using Standard Methods and COD was measured with the US EPA-approved digestion method. Mass, size, and morphology of PA6 before and after digestion were evaluated with scanning electron microscope (SEM), fourier transform infrared (FTIR) spectroscopy, and differential scanning calorimetry (DSC) to comparatively assess the changes in MPs during digestion. RESULTS & DISCUSSION The cumulative methane productions of MAD and TAD reactors are shown in Figure 1 and Figure 2, respectively. In both groups, methane production started immediately and increased steadily as time went on. In the thermophilic reactors different than the mesophilic reactors, methane volume increased in two distinct steps; second step coming about 10 days after the first. In both mesophilic and thermophilic reactors, the highest methane production has been observed in the reactors containing the highest dose (200 PA6/g TS), with the lowest dose, 10 PA6/g TS, following. However, for the mesophilic group, reactors with all other doses of PA6 produced lower methane than the control set (R0M). Even though one-way ANOVA test showed a significant difference between the sets (p<0.05), a further test of post hoc (TUKEY test) demonstrated that the significant difference was only between the R30M and R200M sets. As a consequence, when compared to the control set, PA6 addition seemed to have no effect on methane production. In TAD reactors however, while no pattern between dose and methane production is seen (Figure 2), the outstanding results reveal that all PA6-added reactors produced significantly higher methane than the control set (R0T), as validated by One-Way ANOVA results (p<0.05). Additionally, the post hoc test (TUKEY test) found that except R100T, all the PA6 added sets have significantly different methane production compared to the control set (R0T), indicating PA6 affected methane production positively in TAD. This is thought to be due to leaching of monomer CPL. Chen et al., (2021) claims that CPL attaches to enzyme molecules, changing the enzyme's active site and other conformations, increasing the enzyme's affinity for the substrate and catalytic activity. The distinct second step of increase in methane production may hint the leaching of CPL providing additional affinity for enzyme-substrate system, which is currently under investigation. SEM images were captured to examine the changes in the shape and surface of PA6 during the MAD and TAD processes. Figure 3 depicts the SEM results of biotic and abiotic controls from the mesophilic BMP test. Images exhibit minor changes on the borders of PA6 after digestion. In order to better understand the changes experienced by PA6, FTIR and DSC analyses for both mesophilic and thermophilic reactors are currently underway. ACKNOWLEDGEMENT This study is supported by METU-BAP with a grant number of BAP-GAP-311-2021-10613.