Resilience and Adaptability of Biofilms in Membrane Aerated Biofilm Reactors Under Salinity Stress: Impact on EPS Production and Nitrogen Removal

Resilience and Adaptability of Biofilms in Membrane Aerated Biofilm Reactors Under Salinity Stress: Impact on EPS Production and Nitrogen Removal N. Hernandez-Alcayaga*, G. Campo*, A. Cerutti*, M. Zanetti* and B. Ruffino* * Department of Environment, Land and Infrastructure Engineering, Politecnico di Torino, Torino, Italy (E-mail: [email protected]) Abstract This study investigates the adaptive response of a Membrane Aerated Biofilm Reactor (MABR) to increasing salinity, focusing on extracellular polymeric substances (EPS) production and nitrogen removal performance. A lab-scale MABR was operated under sequential NaCl concentrations (0– 10 g/L), and EPS were analyzed by 3D excitation–emission matrix (3D-EEM) fluorescence and parallel factor analysis (PARAFAC). Three major EPS components—tyrosine-like, tryptophanlike, and humic-like substances—were identified. A strong increase in EPS occurred after the first salinity shock (2 g/L), indicating a protective stress response that enhanced biofilm cohesion and activity. Further salinity increases did not promote additional EPS accumulation, suggesting biofilm adaptation and tolerance. Ammonium removal efficiency showed a similar trend, reaching 100% after the first shock and stabilizing at 78–85% at higher salinities. Significant correlations (r = 0.63–0.70, p < 0.01) between EPS components and nitrogen removal highlight EPS as a key factor supporting microbial resilience. The results demonstrate that MABR performance under saline stress depends on both environmental and biological interactions. Keywords Extracellular polymeric substances (EPS), simultaneous nitrification-denitrification, wastewater treatment, biofilm adaptation. INTRODUCTION Membrane Aerated Biofilm Reactor (MABRs), although it is not a recent technology, has recently elicited considerable interest owing to its ability to augment energy efficiency in biological wastewater treatment (Campo et al., 2024). Within MABRs, the biofilm adhered to the gaspermeable membrane surface creates a partition, establishing distinct aerobic (membrane-biofilm) and anaerobic (biofilm-bulk liquid) sections. The co-existence of the two above-mentioned sections allows the simultaneous nitrification and denitrification of ammonia nitrogen (NH4-N). Therefore, due to the effect of using the membrane together with the biofilm, the concentration of pollutants such as NH4 and Chemical Oxygen Demand (COD) in the biofilm decreases in an opposite direction than O2, generating what is called a counter-diffusion system (Martin and Nerenberg, 2012). Stress factors such as pH, inadequate dissolved oxygen level, toxic organic compounds or high salinity can significantly affect biological wastewater treatment performance (Song, 2023). These stressors challenge microbial activity and pollutant removal efficiency, making it crucial to understand how biofilms adapt to maintain system functionality. The production of Extracellular Polymeric Substances (EPS) plays a vital role in biofilm integrity, serving as a protective mechanism against environmental stresses. EPS are complex polymers secreted by microorganisms that form a matrix around the biofilm, providing structural support and helping to retain water, nutrients, and microorganisms within the biofilm (Salama et al., 2016). Understanding how EPS production is affected by varying salinity levels can provide valuable insights into the adaptive strategies of biofilms and improve the performance of MABRs in complex wastewater treatment. MATERIALS AND METHODS Experimental Set-Up The set-up used for the experiments consisted of a Zeelung lab-scale module (SUEZ Water Technologies and Solutions) with a surface area of 0.25 m2, installed in a plexiglass rectangularsection reactor with a working volume of 3.2 L. The air was supplied to the membrane by a 25 L oil-free compressor (AirCLEAN), and regulated by a gas mass-flow meter and controller (0-2 NLh, red-y, Vogtlin instruments GmbH), with the out airflow maintained at 80 Nml/h , a value previously reported as optimal for ensuring efficient oxygen transfer (Campo et al., 2024). The reactor was inoculated with activated sludge obtained from the SMAT WWTP (Castiglione Torinese, Italy) and operated in continuous mode using a peristaltic pump to feed 0.2 L/h of a synthetic wastewater solution. The solution was prepared to simulate the influent wastewater from the SMAT WWTP, using approximately 230 mg sCOD/L and 33 mg NH₄⁺-N/L. The carbon and nitrogen sources were sodium acetate trihydrate and diammonium hydrogen phosphate, respectively. The mixed liquor was recirculated to ensure homogeneity. The experiment was divided into four operational phases, each corresponding to a different NaCl concentration: 0 g/L (control), 2 g/L, 5 g/L, 10 g/L. Samples of inlet and outlet wastewater were collected every working day during each phase to monitor nitrogen species. Biofilm samples were collected four times per phase to assess EPS production and composition. Analytical Methods Liquid sampling and Analysis. Liquid samples were collected daily directly from the influent and effluent pipe, and were analyzed for total nitrogen (TN), nitrite (N-NO2) and nitrate (N-NO3) nitrogen. TN were measured using a Shimadzu Total Organic Carbon Analyzer (TOC-L) with a Total Nitrogen Measuring Unit (TNM-L). N-NO2 and N-NO3 were analyzed using a Metrohm ECO IC ion chromatograph fitted with a guard column (Metrosep A Supp 17 guard/4.0) and an analytical column (Metrosep A Supp 17-250 mm length, 4 mm diameter). Ammonium Nitrogen (NNH4) was obtained as the difference between TN and N-NO2 and N-NO3. Biofilm sampling and Analysis. EPS extraction from the biofilm was made following an adapted multi-step procedure based on (Yu et al., 2015). Biofilm samples were centrifuged at 3,000g for 5 minutes to isolate the pellet. A 0.5 ml portion of the pellet was then re-suspended in 20 ml of phosphate buffer, mixed on a shaker for 15 minutes, and treated with ultrasound for 3 minutes to disrupt cell aggregates and release loosely bound EPS. Later on, the samples were heated at 80°C for 30 minutes to facilitate the extraction of tightly bound EPS. The mixture was then centrifuged at 10,000 g for 15 minutes, and the resulting supernatant was filtered using a 0.45 μm pore acetate membrane syringe filter. The filtered supernatant is composed of both tightly and loosely bound EPS, and from now on will be called EPS sample. The remaining pellet was used to determine the total solids content of the biofilm sample according to Standard Methods (American Public Health Association (APHA) et al., 2017). A 3D-EEM spectra of all EPS samples were measured using a fluorescence spectrophotometer (Agilent Cary Eclipse), scanning emission spectra from 250 to 550 nm by varying the excitation wavelength from 200 to 450 nm. The 3D-EEM data were processed using parallel factor analysis (PARAFAC) to identify and quantify the EPS components. Fluorescence intensity was normalized by total suspended solids (TSS) to obtain EPS values (a.u./g TSS). The correlation between measured parameters (e.g., N-NH4, and EPS components) was evaluated using Pearson correlation coefficients (r). RESULTS AND DISCUSSION MABR has demonstrated distinct responses to increasing salinity levels in terms of biofilm’s EPS production. The PARAFAC of the 3D-EEM spectra revealed three distinct peaks: Peak 1 corresponded to tyrosine-like proteins Peak 2 to tryptophan-like proteins, and Peak 3 to humic-like substances, consistent with previous assignments in EPS studies (W. Chen et al., 2003; Jacquin et al., 2017). As can be observed in Figure 1, after the first salinity shock (stage 2), an important increase of EPS production was detected. The increase in EPS production is likely the biofilm’s response to environmental stress, as EPS enhances mechanical cohesion, water retention, and nutrient accumulation (Flemming, 2016). However, after the second increase of the salinity in the medium there was not an increase of the EPS component compared to the first salinity increase, which demonstrates a resistance of the biofilm to changes in environmental salinity. Several studies have demonstrated an increase in the production of EPS in response to elevated salinity levels in the environment (Zeng, 2016). However, the adaptive capacity of biofilms to continuous salinity increases is equally compelling to investigate, as it provides insights into the biofilm's resilience to sustained and fluctuating stress conditions. Figure 1. 3D-EEM fluorescence spectra (left) and fluorescence components concentration (right) of EPS extracted from the biofilm during the experiment. Figure 2 illustrates the NH₄ removal performance of the reactor across the different weeks of the experiment. During Stage 1, the system demonstrated stable behavior, achieving an average total NH₄⁺-N removal efficiency of 61.5%. When the salinity was increased to 2 g NaCl/L in Stage 2, the removal efficiency of NH₄⁺-N rose sharply, reaching a maximum of 100% after two days and averaging 84.7% throughout the stage. Upon moving to Stage 3, the average total NH₄⁺-N removal remained stable, showing no significant variation under the salinity shock. Nevertheless, although the average removal efficiency in Stage 4 (78.5%) was nearly the same as in Stage 3 (78.8%), the system experienced a sudden drop in performance immediately following the shock, unlike the trend seen in earlier stages. Taken together, despite higher performance in all later stages compared to Stage 1, three distinct responses to salinity shock can be distinguished according to salinity shock applied to the system: stimulatory (Stage 1 to Stage 2), stable (Stage 2 to Stage 3), and inhibitory (Stage 3 to Stage 4). Figure 2. N-NH4 Removal performance during the experiments. Correlation analysis indicates a significant relationship between the composition of fluorescent EPS in the biofilm and the nitrogen removal performance of the reactor. Tyrosine-like proteins (r = 0.63, p = 0.008), tryptophan-like proteins (r = 0.70, p = 0.002), and humic-like substances (r = 0.69, p = 0.003) all showed statistically significant correlations with nitrogen removal efficiency. Additionally, the total fluorescence intensity of EPS exhibited a similar strong positive correlation (r = 0.69, p = 0.003), suggesting that higher overall EPS production is associated with improved reactor performance. Due the response of the biofilm under the external stress, manifested as an increase of the EPS production, the nitrogen removal can be enhanced due the increase of enzymatic activity, resource capture and redox activity in the biofilm (Flemming, 2016). However, neither the EPS content nor the nitrogen removal performance exhibited a clear correlation with the salt concentration in the medium, indicating that their trends do not directly mirror changes in salinity. Although the observed dynamics appear to be triggered by the increase in salinity, the results suggest that this response is not solely influenced by salt concentration. This implies that other factors are playing a significant role in modulating EPS production and nitrogen removal under saline conditions. CONCLUSIONS This study demonstrates that the MABR system exhibits distinct adaptive responses to salinity stress. The first salinity shock triggered a significant increase in EPS production, reflecting a protective mechanism that enhances the biofilm’s structural stability and resistance to osmotic stress. However, further salinity increases did not induce additional EPS accumulation, indicating that the biofilm achieved physiological adaptation and tolerance to the saline environment. Nitrogen removal performance followed a similar trend: an initial stimulation under moderate salinity, followed by stable or slightly reduced efficiency at higher concentrations. Strong positive correlations between EPS components and nitrogen removal confirm that EPS plays a central role in sustaining biofilm activity by supporting enzymatic function, resource capture, and redox stability. Overall, the results reveal that while salinity acts as a key trigger for biofilm adaptation, its effects are not solely concentration-dependent. Other environmental and biological factors contribute to regulating EPS synthesis and reactor performance, emphasizing the importance of understanding these interactions to enhance the resilience of MABR systems under saline conditions. REFERENCES Campo, G., Cerutti, A., Zanetti, M., & Ruffino, B. (2024). Membrane aerated biological reactors (MABRs) to enhance the biological treatment process at a WWTP. Journal of Environmental Management, 371. Chen, W., Westerhoff, P., Leenheer, J. A., & Booksh, K. (2003). Fluorescence Excitation-Emission Matrix Regional Integration to Quantify Spectra for Dissolved Organic Matter. Environmental Science and Technology, 37(24), 5701–5710. https://doi.org/10.1021/es034354c Martin, K. J., & Nerenberg, R. (2012). The membrane biofilm reactor (MBfR) for water and wastewater treatment: Principles, applications, and recent developments. Bioresource Technology, 122, 83–94. Yu, W., Graham, N., Yang, Y., Zhou, Z., & Campos, L. C. (2015). Effect of sludge retention on UF membrane fouling: The significance of sludge crystallization and EPS increase. Water Research, 83, 319–328. Salama, Y., Chennaoui, M., Sylla, A., Mountadar, M., Rihani, M., & Assobhei, O. (2016). Characterization, structure, and function of extracellular polymeric substances (EPS) of microbial biofilm in biological wastewater treatment systems: a review. Desalination and Water Treatment, 57(35), 16220–16237. Song, Q., Chen, X., Hua, Y., Chen, S., Ren, L., & Dai, X. (2023). Biological treatment processes for saline organic wastewater and related inhibition mechanisms and facilitation techniques: A comprehensive review. In Environmental Research (Vol. 239). Yu, J., Xiao, K., Xue, W., Shen, Y. xiao, Tan, J., Liang, S., Wang, Y., & Huang, X. (2020). Excitation-emission matrix (EEM) fluorescence spectroscopy for characterization of organic matter in membrane bioreactors: Principles, methods and applications. In Frontiers of Environmental Science and Engineering (Vol. 14, Issue 2). Flemming, H. C. (2016). Eps—then and now. In Microorganisms (Vol. 4, Issue 4). MDPI AG. https://doi.org/10.3390/microorganisms4040041 Zeng, J., Gao, J. M., Chen, Y. P., Yan, P., Dong, Y., Shen, Y., Guo, J. S., Zeng, N., & Zhang, P. (2016). Composition and aggregation of extracellular polymeric substances (EPS) in hyperhaline and municipal wastewater treatment plants. Scientific Reports, 6. Jacquin, C., Lesage, G., Traber, J., Pronk, W., & Heran, M. (2017). Three-dimensional excitation and emission matrix fluorescence (3DEEM) for quick and pseudo-quantitative determination of proteinand humic-like substances in full-scale membrane bioreactor (MBR). Water Research, 118, 82–92. https://doi.org/10.1016/j.watres.2017.04.009