MASTER’S THESIS ABSTRACT Name in alphabet: NGUYEN, Thi Chung Thesis title: Simultaneous nitrification and denitrification using ethanol-packed membrane biofilm system for drinking water
LITERATURE REVIEW
Environment concerns about nitrate contamination in drinking water
Nitrate contamination originates from both anthropogenic and geogenic sources, significantly impacting water quality in surface and groundwater systems The complexity of nitrate pollution stems from multiple factors, including agricultural runoff, industrial discharges, urban wastewater, atmospheric deposition, and natural geological processes, with each source contributing nitrate at different levels that may exceed regulatory thresholds This contamination poses substantial environmental and health risks, underscoring the need for comprehensive monitoring, source control, and remediation to protect drinking water resources and aquatic ecosystems.
Agricultural practices are the main source of nitrate contamination in water resources, driven by the widespread use of nitrogen-rich fertilizers and poor management of animal waste While synthetic and organic fertilizers are essential for boosting agricultural productivity, their overuse leads to nitrogen loss through leaching into groundwater, surface runoff, volatilization, and microbial denitrification Nitrate levels in agricultural runoff typically range from 50 to 200 mg/L, influenced by soil characteristics, irrigation methods, and local climatic conditions (Ayub et al., 2019) In regions with intensive farming, groundwater nitrate concentrations can reach 300 to 400 mg/L, far exceeding permissible drinking-water limits.
Livestock operations are a major source of nitrate pollution, with animal manure representing a key nitrogen input Mismanagement of manure leads to nitrate leaching into groundwater, and large-scale cattle farms and poultry facilities generate substantial nitrogen waste that, if not properly treated, can contaminate nearby water bodies Groundwater samples near waste lagoons and septic systems have shown nitrate concentrations exceeding 100 mg/L NO3-, particularly in areas with shallow water tables and permeable soils Moreover, the mobile ion effect, driven by high calcium and chloride levels, enhances nitrate solubility and transport in agroecosystems, intensifying contamination risks.
The prolonged buildup of nitrates in soil disrupts nitrogen cycling and creates a legacy pollution problem, as past fertilizer applications continue to leach into groundwater and surface water for decades This enduring contamination highlights the need for long-term monitoring, preventive management, and targeted remediation strategies to safeguard water quality and agricultural productivity.
Industrial activities are a major source of nitrate pollution, contributing through direct wastewater discharges and indirect pathways such as atmospheric emissions and leachate from industrial landfills Sectors including fertilizers, explosives, dyes, pharmaceuticals, food processing, and metal finishing release nitrate-rich effluents, with untreated wastewater containing nitrate concentrations up to 500 mg/L (NO3−), as reported by Rizeei et al (2018) Inadequate regulatory control of these discharges drives widespread nitrate accumulation in surface waters and regional aquifers.
Urbanization and wastewater mismanagement drive nitrate contamination in water bodies Stormwater runoff from densely populated areas picks up nitrogen from vehicular emissions, sewage overflows, and construction sites, delivering it to urban waterways and raising nitrate concentrations In many cities, inadequate stormwater infrastructure compounds the problem, leading to elevated nitrate levels in lakes and rivers Addressing stormwater and wastewater management is crucial to reduce nitrate pollution and protect aquatic ecosystems.
Municipal wastewater treatment plants (WWTPs) contribute to nitrate pollution when denitrification is incomplete or treatment capacity is exceeded, yielding treated effluent with nitrate concentrations commonly in the 10–50 mg/L NO3⁻ range (Delgadillo-Mirquez et al., 2016) Although modern WWTPs employ biological nutrient removal strategies, operational gaps can still produce elevated nitrate in discharges In many developing regions, aging septic systems and outdated sewage infrastructure substantially drive groundwater nitrate contamination, particularly where leaky septic tanks release untreated wastewater into the groundwater.
Atmospheric Deposition and Natural Sources
Atmospheric chemistry drives a significant portion of nitrate pollution alongside direct emissions NOx from vehicles, industrial facilities, and biomass burning reacts in the atmosphere to form nitrates that are transported and deposited on land and water via precipitation In urban and industrial regions, rainwater nitrate levels can reach 5–25 mg/L, greatly increasing nitrogen inputs to surface waters (Panno et al., 2006).
Geogenic nitrate pollution, though less common than human-induced sources, still contributes to contamination in certain regions Lightning-induced nitrogen fixation converts atmospheric nitrogen into reactive nitrates that enter water bodies via rainfall Additionally, natural weathering of nitrogen-bearing minerals in nitrate-rich formations—such as niter and nitratine—releases nitrates into groundwater In some aquifers underlain by nitrate-bearing bedrock, nitrate concentrations have been recorded as high as 250 mg/L, well above safe drinking water limits (Ayub et al., 2019).
Saltwater intrusion into coastal freshwater aquifers contributes to groundwater nitrate pollution The ion exchange between saltwater and freshwater triggers the release of nitrates from sediments, elevating nitrate concentrations in groundwater (Carroll et al., 2014) This issue is particularly acute in regions with groundwater over-extraction, where falling water tables mobilize nitrates more readily.
Combined Effects and Long-Term Trends
Nitrate pollution arises from a mix of agricultural, industrial, and urban activities, producing a cumulative and long-term contamination challenge that often defies a single dominant source in many watersheds Because nitrates persist in groundwater for extended periods, reducing inputs today may not immediately restore water quality, as legacy contamination can linger for decades Addressing this issue requires integrated watershed management that blends source control, targeted remediation, and sustainable land-use practices to effectively mitigate nitrate pollution and safeguard drinking water and ecosystem health.
Climate change is likely to intensify nitrate pollution by changing precipitation patterns, increasing the frequency of extreme weather events, and prolonging droughts, all of which influence nitrate transport and accumulation in water bodies (Taneja et al., 2019) Understanding the spatial and temporal variability of nitrate sources is essential for developing adaptive management approaches that safeguard long-term water security.
1.1.2 Current status of nitrate pollution in the world and Vietnam
Nitrate contamination of surface and groundwater is a global concern, with numerous regions reporting elevated levels A U.S study conducted from 1991 to 2003 across 5,101 wells in 51 locations found that more than 4% of samples exceeded the EPA’s regulatory limit of 10 mg NO₃⁻/L In Europe, many local and regional water sources are approaching the World Health Organization guideline of 50 mg NO₃⁻/L In South Korea, where the national standard is 44.3 mg NO₃⁻/L, agricultural areas show average nitrate levels of 79.4 mg NO₃⁻/L Serbia has experienced fluctuating nitrate levels that have substantially degraded groundwater quality, making it unsuitable for drinking German data show that over 28% of analyzed wells exceed the 50 mg NO₃⁻/L threshold In India, nitrate pollution is widespread, with nearly one-third of states above permissible limits; more than 108 million people drink water with nitrate concentrations above 100 mg NO₃⁻/L, and about 118 million rely on sources containing 45–100 mg NO₃⁻/L.
Nitrate contamination of surface water in Vietnam has become a growing environmental concern, particularly in areas with intensive agricultural and industrial activity The Red River Delta, in particular, has experienced heavy application of nitrogen-based fertilizers Studies have shown that nitrate concentrations in many surface water bodies across the country exceed national water quality regulations For example, in the Nhue River basin, nitrate levels ranged from 2.4 to 48.5 mg NO3−/L, with numerous monitoring points above the 15 mg NO3−/L standard for domestic water use (Nguyen et al., 2019) Likewise, monitoring of the To Lich River in Hanoi recorded nitrate concentrations as high as 52 mg NO3−/L, mainly due to direct discharge of untreated wastewater and runoff from nearby agricultural land (Pham et al., 2021).
The Cau River, one of the longest rivers in northern Vietnam, is increasingly affected by nitrate contamination from human activities, raising concerns about water quality and ecosystem health Spanning 290 km, it flows through Bac Ninh province for about 70 km before merging with the Thai Binh River and serves as a vital water source for irrigation, domestic use, and regional economic activities The river exhibits significant seasonal fluctuations in water level, ranging from about 0.5 meters during dry periods to around 8 meters during peak flooding, with an estimated annual discharge of 5 billion cubic meters Effective management of nitrate pollution and water resources in the Cau River basin is essential to protect agricultural productivity and public health.
Methods for nitrate treatment in drinking water
Nitrate treatment technologies for water sources are generally categorized into two main groups: separation-based and destruction-based approaches Separation-based methods, such as ion exchange, electrodialysis, and reverse osmosis, operate by physically removing nitrate ions from water However, these techniques typically produce highly concentrated brine as a byproduct, which presents significant environmental management and disposal challenges.
2012 highlights a contrast: elimination-based methods, including biological and chemical denitrification, convert nitrate into nitrogen gas, offering complete removal However, these processes require further optimization to be viable at large scales (Samatya et al., 2006; Jensen et al., 2014).
1.2.1 Separation- based technologies for nitrate reduction
Reverse osmosis (RO) is a widely used membrane filtration technology capable of removing a broad range of pollutants from water It removes dissolved ions such as nitrate, arsenic, sodium, chloride, and fluoride, as well as suspended particles like protozoan cysts and asbestos, and certain organic substances including pesticides The RO system operates by applying pressure to push water through a semipermeable membrane, separating impurities based on size and charge rather than selective chemical affinity The required pressure increases with the concentration of solutes in the feed water A major drawback is the production of a concentrated reject stream containing the removed contaminants, including nitrates, creating important environmental and operational disposal concerns that must be managed.
Reverse osmosis (RO) for nitrate removal typically requires operating pressures of about 2–10 MPa (2000–10000 kPa) to overcome osmotic pressure and drive water across the membrane Critical operating factors—membrane configuration, permeate flux, system staging, flow dynamics, and routine maintenance such as chemical cleaning and anti-scallant dosing—are essential for sustaining optimal performance and delivering consistent water quality RO membranes are usually made from cellulose acetate, polyamide, or advanced composite materials, with durability and efficiency closely tied to feedwater quality and the effectiveness of pretreatment Despite these controls, membrane fouling, structural compaction, and progressive degradation remain persistent challenges that can reduce treatment performance over time.
One of the primary challenges in reverse osmosis (RO) applications is managing the nitrate-rich concentrate, which has driven extensive research into reuse options and more sustainable treatment of reject water Among proposals, integrating RO with subsequent biological, chemical, or catalytic denitrification steps shows promise for converting residual nitrate in the concentrate to nitrogen gas, thereby reducing environmental impact and improving overall treatment efficiency Although RO achieves nitrate removal of about 59% to 95%, it is inherently non-selective and removes a broad range of dissolved minerals along with nitrate, resulting in permeate that is low in minerals and can be softened and corrosive, which necessitates post-treatment remineralization to restore essential ions and meet drinking water quality standards.
Electrodialysis (ED) is a membrane-driven separation technology that uses an electric field to selectively transport ions through alternating cation and anion exchange membranes During operation, nitrate and other charged species migrate from the feed solution toward designated concentrate compartments under an applied electrical potential, enabling efficient nitrate removal The effectiveness of nitrate separation depends on the voltage applied across the membrane stack, with higher voltages enhancing transport Compared with reverse osmosis (RO), ED reduces chemical reagent requirements and achieves high water recovery, making it a competitive option for nitrate removal in water treatment applications.
Electrodialysis (ED) shares a key limitation with reverse osmosis: it cannot selectively remove nitrate ions, often necessitating a remineralization step to replenish minerals lost during treatment While ED can purify drinking water from nitrate-contaminated sources, it also generates a nitrate-rich byproduct stream that creates disposal challenges Additionally, the operational complexity and relatively high cost of ED systems make them less attractive for wide-scale deployment when compared with other nitrate removal technologies.
2015) Large-scale applications of ED in denitrification remain limited, with only a few full-scale systems reported One such system, developed by Austrian Energy in
In 1997, an electrodialysis system using anion-exchange membranes achieved a nitrate removal efficiency of 66% However, it was decommissioned shortly after commissioning due to unresolved waste-stream management issues (Jensen et al., 2014) These limitations underscore the need for further refinement of ED technology to improve its practical applicability, cost-effectiveness, and environmental sustainability in nitrate treatment applications.
1.2.2 Elimination-based technologies for nitrate removal
Chemical denitrification (CD) involves the reduction of nitrate via reactions with metals such as elemental aluminum and iron in zero-valent (Fe0) and ferrous (Fe2+) forms The use of catalytic agents, including copper, palladium, and rhodium, can substantially enhance these redox processes (Jensen et al., 2014) Despite extensive research, a straightforward chemical approach that fully converts nitrate to nitrogen gas under ambient temperature and pressure has not yet been achieved The reaction typically proceeds through multiple intermediate stages toward dinitrogen (N2); however, incomplete reduction often yields ammonium as a byproduct, indicating incomplete denitrification (Hao et al., 2005).
CD technology holds significant commercialization potential, yet it is hindered by two key challenges: controlling catalytic activity to prevent ammonia formation, and selecting catalyst forms that are practical for large-scale use While powdered or fiber catalysts can help suppress ammonia formation, they bring operational hurdles such as high-pressure drops in fixed-bed reactors and separation difficulties, complicating scale-up and routine processing Overcoming these trade-offs is essential for translating CD technology from lab demonstrations to market-ready, industrial deployment.
Chemical denitrification (CD) offers a potential advantage over separation-based methods like reverse osmosis and ion exchange by transforming nitrate into alternative nitrogen species rather than simply concentrating it in waste streams (Jensen et al., 2014) However, CD’s application in potable water treatment remains limited due to practical concerns, including the risk of over-reduction where nitrate is converted not only to nitrogen gas but also to ammonia, with its own health and environmental hazards Moreover, the process can introduce residual chemicals that may threaten drinking water safety To date, there have been no documented full-scale CD systems successfully deployed for nitrate removal in drinking water applications (Jensen et al., 2014).
Recent studies have explored hydrogen as an electron donor in combination with palladium-based catalysts to enhance nitrate reduction in water and brine treatment applications (Jensen et al., 2014) While promising in laboratory settings, large-scale deployment remains hampered by technical limitations (Chaplin et al.).
2012) Further research is required to optimize CD technology and address challenges related to selectivity, reaction control, and catalyst stability to make it a viable alternative for drinking water treatment
Biological denitrification (BD) is among the most effective methods for converting nitrate to harmless nitrogen gas, eliminating the need for waste disposal or remineralization steps It operates under anoxic conditions, with denitrifying bacteria using nitrate as the electron acceptor while metabolizing an external carbon source BD yields minimal biomass, reducing sludge management needs However, its main drawbacks include potential microbial contamination of treated water, accumulation of metabolic by-products, and the requirement for long hydraulic retention time (HRT) to achieve complete denitrification.
Biological denitrification (BD) occurs in groundwater aquifers either in situ using injection wells or ex situ in bioreactors In situ BD benefits from stable temperatures but is limited by slow reaction rates, clogging, and uneven substrate distribution Ex situ BD uses fixed-film or suspended-growth bioreactors, enhancing bacterial activity with support materials such as sand, activated carbon, and sulfur Bioreactor designs include fluidized bed reactors, packed bed reactors, membrane bioreactors, and biofilters, with fluidized bed reactors offering high denitrification rates, minimal clogging, and efficient biomass control.
Numerous pilot-scale and operational biological denitrification (BD) systems have been deployed across Europe and the United States, highlighting BD as a viable approach for potable water treatment The first above-ground BD reactor was installed in France in 1983, with subsequent implementations reported in Germany and Italy, illustrating early international adoption A broad range of carbon substrates—including methanol, ethanol, acetate, hydrogen, elemental sulfur, and even natural gas—have been explored to drive microbial denitrification, often with phosphate added to meet microbial nutrient requirements The overall effectiveness of BD reactors depends on factors such as hydraulic retention time, the nature and availability of the carbon source, the initial nitrate load, ambient temperature, and pH Ongoing research remains essential to refine reactor design and enhance the efficiency of BD for potable water treatment systems.
1.2.3 Comparison of different nitrate treatment technologies
Nitrification & denitrification: Challenges and Performance Optimization
The historical progression of nitrification and denitrification as biological mechanisms for nitrate removal from drinking water marks a pivotal chapter in environmental microbiology and water treatment engineering Beginning with early empirical observations, these processes evolved from theoretical concepts into proven treatment methods now integrated into modern drinking-water systems As scientific understanding and public health awareness advanced, nitrification-denitrification schemes became essential components, driving innovations in technology and ensuring safer, nitrate-controlled drinking water worldwide.
Early Discoveries and Conceptual Shifts
Prior to the late 19 th century, nitrate formation in soils and waters was largely attributed to abiotic chemical reactions However, a major paradigm shift occurred in
In 1877, Schloesing and Müntz conclusively demonstrated that the oxidation of ammonia to nitrate is a biological process mediated by soil microorganisms, a finding that marked a turning point in our understanding of nitrogen transformations This pivotal discovery laid the groundwork for recognizing the microbial nitrogen cycle and the crucial role of microorganisms in soil nitrogen cycling.
Building on earlier findings, Sergei Winogradsky’s landmark work from the 1890s clarified the nitrification process He identified two key nitrifying bacteria genera: Nitrosomonas, which oxidizes ammonia to nitrite, and Nitrobacter, which completes the oxidation to nitrate (Winogradsky, 1890).
Emergence of Denitrification and Early Applications
Denitrification is an anaerobic microbial pathway in which nitrate is reduced step by step to nitrogen gas, with nitrite, nitric oxide, and nitrous oxide acting as intermediates Although the process was identified soon after early nitrate studies, it had already been widely observed in natural environments such as soils and wetlands by the beginning of the 20th century Only in the mid‑twentieth century did engineered systems begin to employ denitrifying microorganisms to remove nitrate from drinking water, as documented by Knowles (1982).
A major challenge in applying biological denitrification to drinking water is precisely controlling environmental conditions, especially the supply of electron donors (typically organic carbon) and the maintenance of anoxic environments, to prevent incomplete denitrification and the accumulation of by-products (Mateju et al., 1992).
Adoption in Drinking Water Treatment
Growing awareness of nitrate contamination in groundwater, especially in the post-World War II era, spurred the integration of biological nitrogen removal techniques into potable water treatment Excessive nitrate concentrations threaten public health by causing methemoglobinemia, also known as blue baby syndrome, underscoring the need for safe drinking water To meet this need, water utilities adopted scalable biological processes that remove nitrogen compounds before distribution These solutions provide effective, cost-efficient protection against nitrate-related health risks and help ensure a reliable supply of clean drinking water.
Innovations and Microbial Ecology Insights:
Recent decades have brought significant advancements in both the microbiological understanding and technological application of these processes The identification of heterotrophic nitrification-aerobic denitrification bacteria, such as
Bacillus licheniformis and other facultative strains offer new opportunities to simplify water treatment by enabling nitrogen removal under fully aerobic conditions, reducing the need for strict redox management and potentially lowering operating costs This approach challenges the traditional two-step nitrification model, with implications for evolving nitrification dynamics Comammox organisms are capable of oxidizing ammonia to nitrate within a single metabolic pathway, presenting new avenues to optimize biological treatment processes for both wastewater and drinking water applications.
1.3.2 Working principle and treatment performance
Nitrification is an aerobic microbial process that occurs in two distinct steps, where ammonia is biologically converted into nitrate This transformation involves two main groups of autotrophic microorganisms:
1 Ammonia-oxidizing bacteria (AOB), particularly those belonging to the
Nitrosomonas genus, are responsible for converting ammonium ions (NH₄⁺) into nitrite (NO₂⁻)
2 Following this, nitrite-oxidizing bacteria (NOB), such as Nitrobacter species, further oxidize nitrite into nitrate (NO₃⁻)
The fundamental biochemical reaction can be expressed as follows:
This oxidative conversion not only mitigated ammonia toxicity but also prepares nitrate for downstream removal via denitrification
Denitrification is a microbial process that occurs under anaerobic or anoxic conditions, during which nitrate (NO3−) is sequentially reduced to nitrogen gas (N2) through intermediate compounds such as nitrite (NO2−), nitric oxide (NO), and nitrous oxide (N2O) In heterotrophic denitrification, microorganisms use organic carbon sources—commonly methanol, ethanol, or acetate—as electron donors to drive the reduction steps By contrast, autotrophic denitrification relies on inorganic electron donors, including hydrogen gas, reduced sulfur compounds, or ferrous iron, to power the reduction of nitrate to N2.
Nitrification is commonly used as a pretreatment step to lower ammonia concentrations, reducing chlorine demand and limiting disinfection by-product (DBP) formation Empirical data indicate that nitrification efficiencies can exceed 90% under optimal conditions, though performance is sensitive to factors such as temperature (20–30°C), pH (7.5–8.5), and dissolved oxygen levels, as reported by Rezvani et al.
Heterotrophic denitrification consistently achieves nitrate removal efficiencies above 95% when carbon dosing and redox conditions are properly regulated, as demonstrated by Matějů et al (1992) However, excess organic carbon may lead to elevated concentrations of residual organics, necessitating additional post-treatment to achieve desired water quality standards.
Autotrophic denitrification pathways, including hydrogenotrophic and sulfur-based systems, have attracted attention for their lower biomass yield and reduced risk of secondary pollution Hydrogenotrophic reactors can achieve nitrate removal rates up to about 96%, while sulfur-based denitrification typically surpasses 90% removal but may generate sulfate requiring additional mitigation Although heterotrophic denitrification remains widely implemented, autotrophic processes—especially hydrogen-driven systems—offer clear benefits in sustainability and operational simplicity Future research should prioritize refining reactor configurations, developing efficient electron-donor delivery systems, and integrating hybrid biological–physicochemical treatment strategies to enhance performance and practicality.
1.3.3 Advantages and Disadvantages of traditional nitrification & denitrification processes
The traditional nitrification- denitrification sequence offers several operational and environmental benefits as a biological treatment process for nitrate removal in drinking water
Biological denitrification, including heterotrophic and autotrophic processes, offers a more sustainable and cost-effective alternative to conventional physicochemical methods such as reverse osmosis (RO) and ion exchange (IX) These bioprocesses typically incur lower operating costs and require fewer chemicals, enhancing overall efficiency and environmental performance, as noted by Rezvani et al (2019).
Complete nitrogen removal: Denitrification effectively reduces nitrate to nitrogen gas, eliminating nitrate without generating secondary waste streams such as brines typical of RO or IX system ((Matějů et al., 1992)
Multi-contaminant treatment: Denitrification systems are capable of simultaneously removing other contaminants (e.g organic carbon, some heavy metals) under optimized conditions (Vasiliadou et al., 2006)
Low sludge production: Autotrophic denitrification, particularly sulfur- and hydrogen-based systems, yields lower biomass compared to heterotrophic process, minimizing sludge management requirements (Sahinkaya et al., 2015)
Despite their advantages, traditional nitrification and denitrification processes exhibit several limitations that impact their performance and applicability
Nitrification and denitrification are highly sensitive to environmental parameters, including temperature, pH, dissolved oxygen, and the carbon-to-nitrate ratio When these conditions are suboptimal, nitrate accumulates, denitrification becomes incomplete, and undesirable intermediates such as nitrous oxide (N2O) can be produced.
Heterotrophic denitrification relies on an external organic carbon source—such as methanol, ethanol, or acetate—and excessive dosing can lead to secondary pollution Residual dissolved organic carbon (DOC) remaining in treated effluent may fuel microbial regrowth in distribution networks, posing a risk to water quality (Robertson et al., 2000).
Hydraulic retention time (HRT) and start-up periods are critical determinants of denitrification performance Denitrification processes generally require longer HRTs and longer start-up phases, with autotrophic systems showing the most pronounced needs, which can constrain their adaptability in high-throughput wastewater treatment facilities (Matějů et al., 1992).
By-product generation: autotrophic sulphur- based denitrification produces sulphate as a byproduct, which may exceed regulatory limits of drinking water quality and necessitate further post-treatment (Sahinkaya et al., 2015)
Identify Research Gaps
Recent studies show promising advances in biological nitrogen removal, particularly with the ethanol-packed membrane biofilm reactor (MBfR) for achieving simultaneous nitrification and denitrification (SND) Despite these positive findings, several critical research gaps remain, including limited understanding of long-term performance, process stability under variable wastewater compositions, and optimal operational parameters for scaling MBfR-based SND systems Addressing these gaps will be essential to fully exploit the environmental and economic benefits of MBfR technology for sustainable nitrogen removal.
1.4.1 Limited Full-Scale Application and Long-Term Performance Data
Although experimental and pilot-scale investigations such as those by Shoji et al
Studies by (2014) and Uemoto et al (2014) confirm the technical viability of ethanol-packed membrane biofilm reactors (MBfRs), yet data on their sustained operation in full-scale drinking-water treatment facilities remain limited Most research to date has focused on niche applications such as aquaculture or closed-loop systems in space-constrained environments, creating a gap in field evidence The lack of field studies that evaluate MBfR robustness under varying operational conditions—such as fluctuating nitrate loads and temperature variations—restricts confidence in its broader applicability to municipal water treatment facilities.
1.4.2 Ethanol Diffusion Optimization and Membrane Material Constraints
Current ethanol-packed MBfR designs rely on polyethylene membranes that enable ethanol diffusion while allowing water vapor ingress, leading to ethanol dilution over time Although ethylene-vinyl acetate (EVA) membranes have been proposed as alternatives, systematic investigations into essential membrane material properties—such as permeability, durability, and biofilm compatibility—are still lacking More research is needed to develop membrane materials that enhance ethanol retention without compromising diffusion rates required for effective denitrification in MBfRs.
1.4.3 Incomplete Understanding of Biofilm Stratification and Microbial Interactions
Achieving effective simultaneous nitrification-denitrification (SND) in ethanol-packed MBfRs hinges on establishing distinct aerobic and anoxic microenvironments within the biofilm matrix However, the detailed understanding of biofilm architecture, microbial community dynamics, and the interactions between autotrophic nitrifiers and heterotrophic denitrifiers remains limited To advance knowledge in this area, advanced molecular techniques such as metagenomics and fluorescence in situ hybridization (FISH), along with real-time monitoring tools, are necessary to reveal how microbial populations adapt to ethanol-based carbon delivery and oxygen gradients within the biofilm.
1.4.4 Ethanol Supply and Operational Control
Ethanol membrane diffusion provides a controlled carbon source for denitrification in wastewater treatment, but optimizing the release rate to balance denitrification without impeding nitrification remains challenging While several studies have identified an optimal ethanol release rate (for example, around 48 mg COD/L/day), knowledge is still limited on how variations in membrane surface area, ethanol concentration, or replenishment frequency influence system stability and nitrogen removal efficiency under real-world conditions.
Although ethanol-packed MBfRs show potential for energy and space savings, few studies have conducted life cycle assessments (LCA) or cost-benefit analyses comparing these systems with conventional nitrification-denitrification setups, reverse osmosis, or ion exchange systems Additionally, the environmental impact of ethanol leakage or incomplete ethanol utilization in the treated water stream has not been thoroughly assessed
1.4.6 Adaptability to High-Strength Nitrogen Loads and Broader Contaminant Profiles
Most research on drinking water treatment has focused on moderate nitrate concentrations, but the ethanol-packed MBfR's performance under high-strength nitrogen loading or in waters with complex contaminant matrices, such as coexisting sulfate, phosphate, or heavy metals, remains poorly understood Understanding how the ethanol-packed MBfR operates under these challenging conditions is essential for broadening its application scope and improving nitrate removal across diverse water sources.
MATERIALS AND METHODOLOGIES
Materials
This chapter provides a comprehensive overview of the materials and methodologies employed in this study to evaluate the feasibility of simultaneous nitrification and denitrification (SND) using an ethanol-packed membrane biofilm system It details the experimental setup, including the ethanol-packed membrane biofilm reactor configuration, materials selection, operational conditions, and the analytical techniques used to monitor nitrogen species, biofilm development, and overall process performance By defining the feasibility criteria and key performance metrics—such as nitrification and denitrification rates, nitrogen removal efficiency, system stability, and biofilm characteristics—the chapter establishes a clear framework for assessing how the ethanol-packed membrane biofilm approach enables effective SND.
The biocarriers used in this study were sourced from an operational water treatment facility in Bac Ninh, Vietnam in September 2024 (Appendix III)
Biofilm-attached carriers are MBBR K1-type media (HDPE, size 11 x 10 mm, effective surface area > 1,200 m2/m3, 140 kg/m3), produced by Lamela Co., Ltd (Vietnam), and collected from the Aerobic tank of the Bac Ninh water treatment plant As shown in the system schematic, the nitrogen removal treatment train consists of multiple biological treatment units, including Aeration Tanks 1–3 and an Anoxic Tank, all equipped with fine-bubble diffusers and ethanol dosing lines to support aerobic nitrification and anoxic denitrification, respectively.
Figure 2.1 Biocarriers (MBBR K1) in anoxic tank- Bac Ninh plant
These carriers were pre-colonized with microbial biofilms that included ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), and denitrifying bacteria To preserve microbial viability before use in the experimental reactors, the biocarriers were stored under controlled conditions.
DNA sequencing is used to analyze biofilm thickness and microbial community diversity, confirming the presence of key functional bacteria involved in nitrogen transformations By optimizing the biocarrier size, shape, and material composition, mass transfer efficiency is enhanced and clogging is minimized during long-term operation.
A critical component of this study was the ethanol-packed polyethylene (PE) membrane, which served as a controlled carbon source for denitrification Two types of PE membranes were employed:
Miporon film (0.1 mm thickness, Mizwa Co., Ltd., Japan)
These membranes were specifically designed to regulate ethanol diffusion, providing a continuous and stable carbon supply to denitrifying bacteria while preventing excessive ethanol release that could inhibit microbial activity or cause secondary contamination The ethanol concentration within the membranes was maintained at 99.5%, and the release characteristics were evaluated under defined environmental conditions, including a temperature of 23°C and membrane thicknesses of 0.1 mm and 0.08 mm.
Figure 2.2 An ethanol-packed membrane with non-woven fabric biofilm
Effective ethanol diffusion is ensured by characterizing membranes in terms of permeability, porosity, and mechanical stability The diffusion rates of ethanol through membranes of varying thicknesses are modeled with Fick’s law of diffusion, and these theoretical results are validated against experimental data collected via total organic carbon (TOC) analysis.
The selection of polyethylene (PE) membrane thicknesses of 0.08 mm and 0.1 mm in this study reflects the established inverse relationship between membrane thickness and ethanol release rate reported by Shoji (2014) and Uemoto (2014) Prior findings indicate that a 0.05 mm PE film releases ethanol about twice as fast as a 0.1 mm film, implying a rapid yet potentially excessive ethanol release, while a thicker 0.3 mm film shows insufficient ethanol diffusion that can impair denitrification efficiency Consequently, 0.08 mm and 0.1 mm membranes were chosen to probe subtle variations in ethanol permeation and to enable a detailed assessment of membrane sensitivity and overall system efficiency.
The experimental procedures employed various high- quality analytical- grade chemicals and reagents to ensure accuracy and reproducibility The chemicals employed in this study included:
• NH₄Cl (Ammonium chloride) is used as the ammonium (NH₄⁺) source in nitrification studies, providing a readily available substrate for ammonia- oxidizing bacteria (AOB)
• KNO₃ (Potassium nitrate) is used in denitrification studies as a source of nitrate (NO₃⁻) for denitrifying bacteria, facilitating the evaluation of nitrate reduction processes
• K2HPO₄ (Potassium phosphate) serves as both a buffering agent to help stabilize pH and a source of phosphorus, an essential nutrient for microbial growth and metabolism
To protect microbial activity in biological wastewater treatment systems, residual chlorine such as free chlorine and chloramines must be quenched before treatment This is achieved by adding chemicals to the influent water, notably sodium azide (NaN3) and sodium thiosulfate pentahydrate (Na2S2O3·5H2O), which neutralize residual chlorine and prevent inhibition of essential microbial processes.
All reagents are meticulously verified upon delivery from certified suppliers
A portable dissolved oxygen (DO) meter—the HQ40D multiparameter instrument by Hach, equipped with an LDO100 luminescent DO probe—is used to continuously monitor and regulate DO concentrations inside the reactors This real-time DO control creates optimal conditions for both aerobic and anoxic zones, supporting the activity of nitrifying bacteria for nitrification and denitrifying bacteria for denitrification Maintaining appropriate DO levels is essential to enhance nitrification efficiency while avoiding inhibitory effects on microbial metabolism during denitrification.
Nitrate Nitrogen (NO3-N) is measured using the Kyoritsu Digital Pack Test (Model: DPM2-NO3, Kyoritsu Chemical-Check Lab Corp., Japan) with a detection range of 0.20–5.80 mgN/L To perform the test, select the NO3-N mode and add 1.5 mL of the water sample to the test cup Open the chemical reagent pack, insert the sample into the device, and initiate the measurement The nitrate concentration is displayed after 5 minutes All measurements are performed in triplicate to ensure accuracy.
Ammonium nitrogen (NH4+-N) is quantified using the Kyoritsu Digital Pack Test (Model DPM2–NH4; Kyoritsu Chemical-Check Lab Corp., Japan) with a detection range of 0.20–3.00 mgN/L A 1.5 mL water sample is added to the test cup, then transferred to a reagent-containing chemical pack designed for ammonium detection After expelling air from the pack, the sample is agitated for 30 seconds to ensure complete reaction, and the reacted solution is returned to the test cup and inserted into the device The ammonium concentration is displayed after 10 minutes This method enables rapid and user-friendly quantification of ammonium nitrogen in experimental samples.
Figure 2.3 Kyoritsu Digital Pack Test for Nitrogen Species monitoring
Nitrite nitrogen (NO₂⁻-N) was determined using the Kyoritsu Digital Pack Test system (DPM2–NO₂ model, Kyoritsu Chemical-Check Lab Corp., Japan) A 1.5 mL water sample was transferred into the test cup, then the pre-filled reagent pack specific for nitrite analysis was added, followed by removing trapped air and shaking the pack about 5–6 times to ensure complete mixing and reaction The solution was returned to the cup and placed into the measuring device, which automatically measured and displayed the NO₂⁻-N concentration after a 3-minute reaction period This method provides a convenient and efficient approach for nitrite monitoring in water samples.
To assess the presence of microbial populations involved in nitrification and denitrification, we used PCR to amplify key functional genes: amoA genes encoding ammonia monooxygenase in ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and comammox organisms, along with narG genes encoding the catalytic subunit of the membrane-bound nitrate reductase This confirms the occurrence of nitrifiers and denitrifiers in the samples Amplifications were performed on a Biometra T3 Thermal Cycler (Analytik Jena, Germany) using standard PCR conditions, and the products were verified by gel electrophoresis on a 1.5% agarose gel.
Experimental Methodologies
Experimental Design and Objective: The primary objective was to evaluate the feasibility and performance of an ethanol-packed membrane biofilm system for simultaneous nitrification and denitrification Specific goals included:
Quantifying ethanol diffusion kinetics from membranes
Determining efficiencies of simultaneous nitrification and denitrification processes
To evaluate the controlled release behaviour of ethanol from polyethylene (PE) membranes, a batch experiment was conducted under the following conditions:
- Stirring: Continuous at 60 rpm using a magnetic stirrer
- Volume of water: each tank contained 1 liter of Milli- Q water
Milli-Q ultra-pure water, produced by the Milli-Q water filtration system from Merck Millipore, delivers extremely high purity for biological, chemical and analytical experiments, with a conductivity of about 0.055 μS/cm.
- Additive: 0.02 sodium azide (NaN3) was added to each tank to inhibit bacterial growth throughout the experiment
- Lighting condition: The experiment was conducted in a dark environment to avoid any photodegradation or unwanted reactions due to light exposure
- Container setup: Beakers were covered with aluminium foil to minimize ethanol evaporation and further protect against light exposure
Three experiment setups were prepared as follows:
- Control beaker: Milli- Q water (01 liter) + 0.02% sodium azide (no membrane, no ethanol)
- Beaker 1: Milli- Q water (01 liter) + 0.02% sodium azide + YUNIPACK membrane (0.08 mm thickness) filled with 5 mL of 99.5% ethanol
- Beaker 2: Milli- Q water (01 liter) + 0.02% sodium azide + Miporon membrane (0.1 mm thickness) filled with 5 mL of 99.5% ethanol
Figure 2.4 Ethanol diffusion experiment with different PE thicknesses
Sampling Schedule: Water samples were collected on days 1, 2, 5, 6, and 7 for Total Organic Carbon (TOC) analysis:
The kinetic data for ethanol release were analyzed using diffusion coefficients and membrane permeability to quantify transport dynamics These results guided the optimization of membrane thickness and ethanol dosage, ensuring stable long-term performance in future applications.
2.2.2 Nitrification and Denitrification Potential Tests
Nitrification and denitrification tests were performed to assess microbial activity within the biofilm The removal rates of NH₄⁺ and NO₃⁻ were evaluated under aerobic and anoxic conditions:
During September 2024, a nitrification test was conducted to determine the nitrification capacity of biocarriers by measuring ammonium (NH4+) removal under controlled aerobic conditions The detailed experimental conditions are outlined below.
- Objective: Evaluate the nitrification potential of the biocarrier by measuring the rate of NH4+ oxidation under aerobic conditions
The biocarrier source comprises biofilm-attached MBBR K1-type media (HDPE), sized 11 × 10 mm, with an effective surface area exceeding 1,200 m²/m³ and a density of 140 kg/m³ These media are produced by Lamela Co., Ltd., Vietnam, and were collected from the aerobic tank of the water treatment plant in Bac Ninh.
+ 10 mL of NH4Cl 1000 mgN/L stock solution in order to achieve influent ammonium concentration: 10 mgN/L
- Biocarrier dosage: 100 grams of biocarrier (wet weight)
+ Continuous aeration to maintain aerobic conditions (Dissolved oxygen value is higher than 2 mg/L) and ensure adequate oxygen supply for nitrifiers
Figure 2.5 Nitrification potential test using biocarriers from the aerobic tank at the
- Sampling interval: 0, 1, 2, 4, 6, and 24 hours after the start of the experiment operation
Samples were collected at specific time points, extracted, and filtered through 0.2 μm disposable membrane filters to remove suspended solids, then analyzed for ammonium (NH4+) and nitrate (NO3-) concentrations Kyoritsu Digital Pack Test devices were used to quantify these nitrogen species with high accuracy.
An observed decrease in NH4+ concentration over time reflects the activity of nitrifying microorganisms within the biofilm and demonstrates that these carriers can effectively support aerobic ammonia oxidation in both engineered wastewater treatment systems and natural treatment environments.
Denitrification test was conducted by placing biocarriers in an anoxic reactor with ethanol supplied as the controlled carbon source, and NO3- removal rates were recorded using the Kyoritsu Digital Pack Test device The experimental setup maintained strict anoxic conditions and precise carbon dosing to enable accurate measurement of nitrate reduction kinetics By monitoring NO3- removal over time, the study evaluated the denitrification performance of the biocarriers under ethanol-driven conditions, providing data on removal rates and overall denitrification efficiency.
- Nitrate solution: 10 mL of KNO 3 1000 mg/L, achieving 10 mgN/L nitrate initial concentration
- Test volume: total 1000 mL (990 mL filtered water + 0.034 mL ethanol at 99.5% purity)
- Biocarrier dosage: 100 grams of Biofilm- attached carriers is MBBR K1 -type media and are collected from the Anoxic tank- the water treatment plant in Bac Ninh
- Anoxic conditions initiation: Nitrogen gas (N 2 ) purged continuously for 10 minutes to eliminate dissolved oxygen Dissolved oxygen value is lower than 2 mg/L)
- Stirring condition: The mixture was subsequently stirred gently using a magnetic stirrer to ensure consistent dispersion of biocarriers and nutrients
Figure 2.6 Denitrification potential test using biocarriers from the anoxic tank at the
- Sampling intervals: liquid samples were drawn from the reactor at predetermined intervals (0, 1, 2, 3 and 24 hours) to monitor changes in nitrate concentrations over time
- Sample preparation: Filtration was performed using 0.2 àm disposable membrane filters prior to chemical analysis
- Analytical method: Nitrate concentrations were analyzed by the Kyoritsu Digital Pack Test device
The observed decline in nitrate (NO3−) concentration during the study indicates denitrification driven by biofilm-embedded microorganisms This finding confirms that biocarriers effectively support microbial nitrate reduction under anoxic conditions, underscoring their applicability in engineered and natural wastewater treatment systems for enhanced biological nitrogen removal.
Microbial communities associated with the biofilm were characterized using molecular biological methods to confirm the presence and functional roles of nitrifying and denitrifying microorganisms The study was conducted with a meticulously followed experimental protocol, employing clearly defined steps to ensure precise detection and interpretation of nitrification and denitrification activities within the biofilm.
- Sample collection: In September 2024, biocarriers (MBBR K1- type media) with biofilm were collected from the water treatment plant in Bac Ninh, Vietnam
Sample preparation involved collecting biocarrier samples, transporting them to the Laboratory at the University of Tokyo, and freezing them until further analysis The PCR-based microbial community analysis was subsequently conducted in October to characterize the microbial populations in the samples.
Biofilms were gently detached from biocarriers and subjected to genomic DNA extraction using the FastDNA Spin Kit for Soil The extracted DNA underwent PCR amplification with TaKaRa EX Taq HS polymerase, employing gene-specific primers to target critical functional genes for downstream analyses.
+ amoA genes specific to ammonia- oxidizing archaea (AOA), ammonia- oxidizing bacteria (AOB), and Comammox clade A, Comammox Clade B, are responsible for ammonia oxidation
+ narG genes are associated with nitrate reduction performed by denitrifying bacteria
- Gel Electrophoresis and Visualization: The amplified PCR products were subjected to electrophoretic separation on agarose gels to confirm the presence and approximate size of targeted functional genes
Figure 2.7 Ammonia oxidizing microorganism & denitrifying microorganisms by
These procedures were conducted within approximately 02 hours
Electrophoresis showed distinct bands for amoA genes of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), as well as Comammox clade A and clade B in the nitrification tank samples, with narG detected in the denitrification reactor, clearly indicating their roles in ammonia oxidation and nitrate reduction within the engineered biofilm-based water treatment system These molecular findings provide fresh insights into microbial ecology by revealing functional specificity and habitat preferences among nitrifying and denitrifying communities in this controlled treatment setup.
2.2.4 Batch Reactor Experiment for Nitrate Removal Evaluation:
A laboratory- scale batch reactor experiment was conducted to examine the efficiency of nitrate removal from November to December 2024 under carefully controlled conditions Specific experimental conditions were established as follows:
- Reactor specifications: A small batch reactor with a total effective working volume of 2.5 liters was employed
- Membrane composition: Polyethylene (PE) membranes measuring 10 x 10 cm with a thickness of 0.1 mm were setup in the reactor
- To fabricate the polyethylene packs containing ethanol, a manual bag sealing machine (dimension: 28 x 31 x 10 mm) was utilized by manually pressing the sealing arm along the bag edges
Three membrane packs were infused with ethanol (99.5% purity), providing 20 mL per pack as the organic carbon source for microbial denitrification In the reactor configuration, Reactor 1 used one pack while Reactor 2 used two packs to assess performance differences.
- Initial Nitrate condition: The experimental solution comprised river water enriched with potassium nitrate (KNO3) to achieve a consistent initial nitrate concentration of 30 mgN/L
- Phosphorus supplementation: To support microbial growth and activity, the enriched water solution was supplemented with potassium dihydrogen phosphate (KH₂PO₄) at a concentration of 1.35 mgP/L
- Mixing conditions: Continuous and gentle mixing was ensured using a magnetic stirrer operating at a speed of 1000 rpm to maintain homogeneous conditions within the reactor
- Temperature Control: All tests were conducted at a stable laboratory temperature of 25 o C to minimize temperature-induced variability
- Dissolved Oxygen (DO): DO levels is stringently monitored and consistently maintained below 2 mg/L to ensure strict anoxic condition conductive to denitrification
Figure 2.8 Batch Reactor Experiment with Reactor 1 (01 ethanol pack) and Reactor 2
During the experimental period, daily sampling at predetermined time intervals tracked fluctuations in nitrate concentrations The collected effluents were analyzed to quantify nitrate removal efficiency and to enable a comprehensive assessment of the reaction kinetics governing the process.
2.2.5 Continuous Flow Reactor Experiment for Evaluation of Simultaneous Removal of Nitrate and Ammonium in One Reactor:
Experiment I: Enrichment of Denitrifying Bacteria (0-7 days) from March to April 2025:
The primary objective of the initial experiment was to promote colonization and to employ ethanol as an organic carbon substrate to fuel microbial growth and nitrate reduction This preliminary stage was essential for establishing a robust microbial biofilm capable of sustained denitrification under controlled laboratory conditions.
Data Analysis
Ethanol diffusion from polyethylene (PE) membranes was quantified using total organic carbon (TOC) analysis, with the release rate modeled to ensure a stable carbon supply The results were described by mathematical models to optimize ethanol delivery over extended operational periods, enabling predictable, long‑term performance and improved process efficiency.
NH₄⁺, NO₂⁻, and NO₃⁻ concentrations were analysed statistically to evaluate nitrification and denitrification efficiency
The nitrogen removal rate was calculated based on initial and final concentrations in the reactor, considering mass balance and reaction kinetics
PCR and sequencing analyses were used to determine microbial diversity, confirming the presence of ammonia-oxidizing and denitrifying bacteria contributing to nitrogen transformation.