Novel fluidized-bed bioreactors with density-graded carriers for the bioremediation of nitrate in uranium industry effluents

Mariano Venturini ^a, Paula Bucci *^b and Raúl Muñoz ^b
aBiomining and Environmental Biotechnology Laboratory – National Commission of Atomic Energy, Av. Ptero González y Aragón No. 15, B1802AYA, Buenos Aires, Argentina
^bInstitute of Sustainable Processes, University of Valladolid, Doctor Mergelina s/n, 47011, Valladolid, Spain. E-mail: paulalorena.bucci@uva.es

First published on 31st March 2025

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Water impact Water is an essential resource, and its quality is directly linked to the health of ecosystems and human communities. Our manuscript, “Novel Fluidized-Bed Bioreactors with Density-Graded Carriers for the Bioremediation of Nitrate in Uranium Industry Effluents”, highlights the crucial role that advanced wastewater treatment technologies can play in addressing current water sustainability challenges, particularly in the nuclear industry. High-nitrate effluents from nuclear processes pose significant risks to both environmental and public health, contaminating water sources and disrupting ecosystems. Our research presents a novel approach to treating these complex wastewater streams by using density-graded floating carriers to optimize bioreactor performance. The impact of our research on water sustainability is multifaceted: 1. Water quality improvement: our bioreactor system offers a sustainable and efficient solution for reducing nitrate levels in high-nitrate wastewater, ensuring cleaner and safer water for both environmental and human use. 2. Ecosystem protection: by efficiently removing harmful contaminants such as nitrates, our system helps preserve the integrity of aquatic ecosystems, preventing eutrophication and supporting biodiversity in water bodies. 3. Environmental sustainability: the use of floating carriers in a fluidized bed bioreactor minimizes energy consumption and chemical additives, contributing to the development of low-impact, resource-efficient wastewater treatment technologies. 4. Innovation in water treatment: our research demonstrates the innovative potential of density-graded floating carriers in wastewater treatment, setting a new precedent for the design of high-performance, sustainable bioreactor systems capable of handling complex industrial effluents. In conclusion, our manuscript underscores the importance of innovative and sustainable solutions for treating high-nitrate wastewater, offering a path forward for improving water quality, protecting ecosystems, and advancing the goal of water sustainability. We believe that sharing our findings in Environmental Science: Water Research & Technology will catalyze further research and inspire the development of advanced, eco-friendly water treatment technologies for industrial applications.

1 Introduction

To produce nuclear fuels, uranium ore must be converted into ceramic-grade UO₂, and the process involves the use of various substances such as kerosene, methanol, nitric acid, ammonia, and to a lesser extent, tributyl phosphate (TBP). The characterization of the effluents generated reveals the presence of uranium typically ranging from 300–600 mg L⁻¹ as U–U₃O₈, kerosene and methanol at levels of 900–1600 mg L⁻¹ as COD, and nitrogen compounds up to 1500 mg L⁻¹ as N–NO₃⁻. High-nitrate concentrations contribute to eutrophication, while uranium poses risks of radioactive contamination. Strict environmental regulations mandate effective treatment to minimize emissions and ensure compliance.

Conventional treatment methods, such as reverse osmosis, evaporation, and electrodialysis, can achieve high-nitrate removal efficiencies above 82% at 1000 mg L⁻¹ N–NO₃⁻. Uranium-contaminated effluents pose a significant challenge in the nuclear industry. Treatment methods such as zero liquid discharge (ZLD) processes rely on energy-intensive thermal operations such as evaporation and crystallization, resulting in high operational costs. In contrast, the system proposed in this study employs a biological approach that avoids thermal operations, suggesting a potential advantage in terms of energy efficiency and operational costs. In addition, ZLD requires the use of corrosion-resistant materials, such as high-grade stainless steels, due to the highly corrosive nature of the effluents. This work focuses on the kinetics of nitrate removal from nuclear effluents, providing a sustainable alternative to conventional methods.^1–4

Other methods often require extensive pretreatment stages (ultrafiltration, microfiltration, and sand filtration) and incur high operating costs, limiting their sustainability for e.g. the use of IONAC SR-7 coupled with reverse osmosis.^5,6 Additional approaches such as supercritical water treatment at 374 °C, electrochemical methods, and electrodialysis are effective yet associated with high energy demands and potential NOx emissions.^7,8

Biological treatment methods provide promising and economically viable alternatives, particularly for high-nitrate and COD effluents in the uranium industry. Anaerobic biotechnologies excel at nitrate and COD removal under controlled conditions.^9,10 Biofilm-based reactors provide advantages such as robustness and tolerance to environmental variations. However, conventional biofilm reactors are susceptible to clogging, which limits mass transfer and hydrodynamic efficiency. To address these challenges, this study proposes a hybrid bioreactor system that combines the benefits of packed-bed and fluidized-bed reactors using density-graded hydrogel carriers.^11–13 These issues necessitate bioreactor designs that minimize clogging and maximize mass transfer, ensuring effective treatment of uranium-contaminated effluents with high nitrate and COD concentrations.

Two major types of anaerobic biofilm bioreactors are widely used for industrial wastewater treatment: packed-bed bioreactors (PBBRs) and fluidized-bed bioreactors (FBBRs). PBBRs immobilize cells within stationary packing materials such as plastic or ceramic rings, facilitating biofilm formation and enhancing mass transfer. PBBRs typically operate under laminar flow conditions, characterized by smooth, orderly fluid motion that maximizes contact between the fluid and the biofilm. However, laminar flow can also lead to clogging and dead zones, reducing treatment efficiency. In contrast, FBBRs suspend biofilm-attached carriers in a fluid, maintaining biofilm suspension through fluid velocity. Turbulence is quantified using the Reynolds number, which must exceed a minimum threshold to ensure fluidization of the carriers and the liquid phase.

This configuration enables uniform particle mixing and continuous operation, reducing clogging due to biofilm accumulation. FBBRs have been shown to offer higher resistance to system disturbances and greater resilience to load fluctuations, enhancing long-term hydrodynamic stability and biofilm growth even under varying influent conditions.^14–16 FBBRs generally require larger reactor volumes, higher pumping capacity, and greater energy inputs due to the fluidization requirements. Additionally, particle entrainment can affect their efficiency and scalability.

To overcome these limitations, FBBRs can be modified with specific biofilm carriers that optimize treatment efficiency while maintaining fluidization. Numerous commercially available carriers, including polyethylene, polypropylene, and biopolymer-based options, are designed with variations in density, shape, and buoyancy to suit specific applications. Methacrylate hydrogels are particularly promising carriers due to their ability to copolymerize with amides, creating carriers with variable densities and buoyancy characteristics.^17–20 This flexibility supports hybrid bioreactor configurations that combine the mass transfer benefits of FBBRs with the surface area advantages of PBBRs, addressing limitations related to clogging and hydrodynamics.

We hypothesize that by employing density-graded hydrogel carriers within a fluidized-bed reactor, we can create a stratified biofilm environment, aimed at the advantages of a packed bed while mitigating the risk of clogging. This hybrid approach achieves high biomass retention and efficient mass transfer, characteristic of PBBRs, while maintaining the fluidization benefits and minimizing energy consumption associated with FBBR.

This study introduces a novel approach to bioreactor design by utilizing density-graded hydrogel carriers within a fluidized-bed configuration. This approach differs from conventional FBBRs by creating a stratified biofilm environment, giving the advantages of packed-bed reactors while maintaining the benefits of fluidization. The high nitrate and COD concentrations, combined with the presence of potentially inhibitory compounds, present significant challenges for the effective treatment of uranium-contaminated effluents.

Packed-bed and fluidized-bed reactors are widely used in chemical and biotechnology industries due to their advantages over suspended cell systems, particularly in wastewater treatment, where they demonstrate high resistance to disturbances such as inhibitors, pH fluctuations, and biomass loss.²¹ While PBBRs typically operate under laminar flow, FBBRs predominantly function under turbulent conditions. This study contributes fundamental insights into laminar-regime FBBR hydrodynamics for nuclear wastewater treatment, emphasizing shear forces' role in balancing biofilm growth and detachment to prevent clogging. Strategic carrier selection and placement further enhance the reactor's performance by optimizing biofilm growth and fouling control.^22,23

Successful development of this hybrid bioreactor system has the potential to significantly advance the field of wastewater treatment, providing a more sustainable and cost-effective solution for the remediation of uranium-contaminated effluents and other challenging industrial waste streams. The proposed biological system operates at ambient temperature and pressure, eliminating the need for energy-intensive thermal operations. Additionally, the system can be constructed using durable plastics, which are less expensive and more resistant to corrosion than stainless steels. This represents a potential advantage in terms of both energy efficiency and capital costs.

2 Materials and methods

2.1 Blended real nuclear wastewater (BRNW)

The production of ceramic-grade UO₂ fuel begins with yellow cake, which is dissolved in nitric acid, purified, and precipitated using methanol and kerosene at uranium concentrations of up to 200 g L⁻¹. The final effluent from this process, known as blended real nuclear wastewater (BRNW), contains residual uranium (300–600 mg L⁻¹ as U–U₃O₈), methanol, kerosene, and high-nitrate concentrations (1000–1300 mg L⁻¹ as N–NO₃⁻). This effluent was used in our experiments without additional pretreatment, except for pH adjustment and equalization to stabilize COD levels.^24,25 Real nuclear wastewater samples used in this study were obtained from an Argentinian uranium conversion facility. This blended real nuclear wastewater (BRNW) was characterized by the composition shown in Table 1. Prior to use, the effluent underwent an equalization process to stabilize the pH and COD levels, after which the resulting precipitate was discarded. The BRNW also contains methanol and kerosene as major organic contaminants. This industrial effluent, which includes contributions from domestic wastewater, is further treated with surfactant compounds to adjust the pH to 8.

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2.2 Synthesis of hydrogel carriers

A range of carrier materials including biodegradable polymers (BDP), high-density polyethylene (HDPE), polyvinyl alcohol (PVA), polyurethane sponge (PS), and granular activated carbon (GAC) are currently employed in various types of bioreactors, such as moving-bed biofilm reactors (MBBRs) and fluidized-bed biofilm reactors (FBBRs). Commercial polymeric carriers such as Kaldness 5 and Saddle-Chips SC exhibit properties including a density of 0.96 kg m⁻³, a surface area ranging from 800 to 700 m³ m⁻³, and varying porosity levels (65–87%).²⁶

In the present study, acrylic materials (HEMA and AA) were chosen according to the properties of swelling (hydrogel) induced by gamma-radiation synthesis. The concept behind synthesizing hydrogels via gamma polymerization is to achieve carrier polymerization and sterilization simultaneously in a single step, eliminating the need for elevated temperatures and crosslinking agents that may exert a toxic influence on microorganisms. Additionally, the gradual addition of acrylamide imparts primary amino groups to the polymer, enhancing its swelling properties and consequently reducing density through volume expansion. HEMA was polymerized by gamma radiation and copolymerized with AA, which were purchased from Sigma (Aldrich, WI, USA). Following polymerization, the sample was homogenized and air was removed by bubbling with nitrogen. The samples were irradiated at 25 kgy (5 kgy h⁻¹) by ⁶⁰Co gamma-irradiation source at PISI – Centro Atómico Ezeiza (Argentina). The concentrations of HEMA (50%) and AA (25% and 50%) were chosen to optimize hydrogel properties. A 50% HEMA concentration balances the mechanical strength and flexibility, while AA concentrations ensure optimal swelling without compromising structural integrity. These choices enable the hydrogels to support biofilm growth and withstand fluidized-bed reactor conditions.

The swelling dynamic and equilibrium were determined by the Cuggino methodology.²⁷ The swelling rate (qw) was determined gravimetrically at different times using eqn (1a) and during the equilibrium using eqn (1b):

qw = mh/ms(1)	(1a)

qw = me/ms	(1b)

where mh is the swelled hydrogel (HG) mass at each time, me is the HG mass during the equilibrium, and ms is the dry HG mass. The HG carriers were hydrated, previously dried at 35 °C, and then, they are weighed in determined periods of time. This test finished when the carrier weight was stable in time (water equilibrium). This essay allowed determining the maximum weight and volume increase. The carriers were constructed with a cylindrical shape with 15 mm external diameter and 5 mm internal diameter with 20 mm length. Different concentrations of HEMA were tested: 50.0% v/v in water and the addition of AA 0, 25 and 50.0% (% V/V HEMA). The densities were decreased according to AA incorporation.

2.3 Bioreactor configuration and operational mode

This study utilized a comparative approach to evaluate the performance of two bioreactor configurations: a packed-bed bioreactor (PBBR) and a fluidized-bed bioreactor (FBBR),²⁸ with the aim of high biomass retention while minimizing energy consumption and hydrodynamic properties.

The PBBR was a single-unit, multi-layer fixed-bed reactor fabricated using polypropylene sheets. This configuration offers several advantages such as high volumetric organic removal rates and efficient liquid–solid separation. The FBBR was designed to optimize fluidization characteristics and minimize clogging.^15,16 This study comprehensively reviewed the fundamental aspects of FBBRs including their applications, configurations, and recent advancements in reactor performance.^29–31

The FBBR was designed to optimize the fluidization characteristics and minimize clogging. To achieve this, the reactor was loaded with hydrogel carriers, exhibiting varying densities (50% p/p HEMA/AA, 25% p/p HEMA/AA, and HEMA only). This density gradient facilitated a stratified arrangement of carriers, promoting optimal biofilm growth while preventing clogging.

To facilitate comparison between systems of different scales, the hydraulic retention time (HRT) values were converted into dimensionless residence time units (RTUs) using the following equation, where the actual residence time (RT) was divided by the HRT. The reference time unit (t₀) can be chosen based on the characteristic time scale of the system, such as the average particle size or the reactor volume. The output values and the resulting plotted curves followed a logarithmic-type function of the form Cs − logCo·e^−a.trC, where Cs is the output nitrate concentration, Co is the initial nitrate concentration, tr is the residence time, r is the radial position, and a is an experimental coefficient obtained using appropriate statistical methods.

The Peclet number (Pe) was calculated using the following standard equation for tubular systems:

Pe = (v × L)/Dz

where v is the superficial velocity, L is the reactor length, and Dz is the axial dispersion coefficient. The dispersion coefficient was estimated from the response curve using the method of moments. The obtained Pe values indicated a predominant plug flow regime, with relatively low axial dispersion. This suggests that the reactor design promotes efficient conversion and minimal backmixing.

2.4 Control of environmental conditions to monitor ORP

Nitrate concentrations were determined according to standardized methods (refer to the test analysis section for details).

The oxidation–reduction potential (ORP) in the system was modeled using the Nernst equation, as proposed by Chang and Venturini.^32,33 This model, based on the Nernst equation, describes the relationship between ORP and the concentrations of redox couples in the system, assuming a 1/1 stoichiometric ratio for each chemical reaction

The standard potential (E°) used in this work was 154 mV for ammonium oxidation to nitrite (NO₂⁻) and −340 mV for ammonium oxidation to nitrate E⁰ (NO₃⁻). The original equation was modified by replacing ln(1/[H⁺]) for 2.3026 × 4 pH according to the author:

where E is the potential activity of the reduced and oxidized species, a_Ox and a_Red. E₀ is the standard potential: E₀ (NO₂⁻) = 154 mV or E₀ (NO₃⁻) = −340 mV. In applying the Nernst equation, it was assumed that the substrate concentration remained constant and in excess. Thus, the modified Nernst equation used in this work is expressed as follows:

2.5 Test analysis

The system was monitored using chemical and physical analyses of influent and effluent samples according to Standard Methods for the Examination of Water and Wastewater. The parameters analyzed include: pH and ORP measurements, chemical oxygen demand (COD), nitrate (N–NO₃⁻) and ammonium (NH₄⁺) analyses. pH and oxidation–reduction potential (ORP) were monitored using a Mettler Toledo pH 2100 sensor. The pH measurement sensitivity is ±0.01 units, and the ORP detection range is ±1200 mV, enabling accurate environmental monitoring during anaerobic processes. The COD was analyzed using Method 5220 D, with detection ranges adaptable for high (0–1500 mg L⁻¹) and low (0–150 mg L⁻¹) concentrations. This dual range allowed the precise monitoring of organic load variations in the reactor. Nitrate concentrations were measured using the 4500 A Standard Method with UV absorption at 220/270 nm, providing a detection limit of approximately 0.1 mg L⁻¹. The ammonium levels were determined via the phenate method at 640 nm with a sensitivity threshold of 0.02 mg L⁻¹, which ensured accurate detection under fluctuating influent characteristics. These specific detection limits and equipment sensitivities ensure a high level of accuracy in measuring environmental conditions and pollutant levels throughout the experimental setup, supporting reliable monitoring and reproducibility.

3 Results and discussion

3.1 Synthesis of hydrogel carrier

The HG carrier was obtained by the radio-induced polymerization of hydroxyethyl methacrylate (HEMA) and copolymerized with acrylamide (AA). The use of different ratios of HEMA and AA resulted in carriers with varying densities and swelling grades, depending on the percentage weight/weight of AA present in the dry HEMA. The results of the swelling of different compositions of HEMA/AA at 25 kGy are shown in Table 2.

The samples swelled in proportion to the increased AA content. The HEMA sample swelled up to double its weight, while the incorporation of 50% w/w of AA increased the weight by several times. This was due to the high formation and superficial charge of ammonium incorporated, which promoted water interaction. Swelling kinetics and diffusion mechanisms indicated that the water penetration followed first-order kinetics for the initial periods. These results are similar to those obtained by Karadağ and Turan.^19,34

The HG 50/50 AA materials showed that they could be operated in the upper zone due to their low density. However, carriers obtained with high densities showed that this composition could be used in a bioreactor with a high fluidized regime, such as a fluidized bed. This is because this material should stay in the lower zone to avoid recirculation by tubes and pumps, and media with low density could be stratified by the same liquid, establishing three definite zones and behaving like a packed bed.

Microphotographs reveal that the dense materials have an elastomeric consistency when swelled in water, as shown in the additional figure. In this figure, it is possible to see the biofilm in contact with the hydrogel, which could be due to a biocompatibility process.

The microorganisms were mainly Pseudomonas (1–2 μm), which adhered to the carrier surface. Culture samples were cultivated in nutritive agar and EMB (Levine). The colonies were stained by Gram Tinction and then, were analyzed using API strips. The API-20E multitest system identified Pseudomonas aeruginosa (CODE: 1-353-575) with a high identification accuracy, and (CODE: 1-000-477) was compatible with Xylosoxidans denitrificans. Additional tests, such as the acetamide reaction, growth at 42 °C, the oxidative (OF) glucose test, and the production of pyocyanin pigment, support the identification of P. aeruginosa.

Microbial processes driving COD and nitrate removal involve aerobic and anaerobic degradation for COD, with aerobic heterotrophs oxidizing organic compounds under aerobic conditions, and anaerobic bacteria utilizing alternative electron acceptors in anaerobic environments. For nitrate removal, denitrifying bacteria sequentially reduce nitrate to nitrogen gas under anaerobic conditions.

3.2 Bioreactor configuration and operation modes

4 Conclusions

When compared with other systems, HG carriers emerge as highly productive, cost-effective options with favorable hydrodynamic characteristics. Based on the results, we conclude that the fluidized-bed bioreactor (FBBR) with HG biofilm carriers demonstrated superior performance to the packed-bed bioreactor (PBBR) in terms of nitrate and COD removal efficiencies. The FBBR's fluidized design provided enhanced mass transfer, minimized clogging through uniform biofilm distribution, and showed greater resilience to variable influent conditions. These results highlight the FBBR with HG carriers as a robust option for industrial applications, particularly in treating high-contaminant and fluctuating wastewater sources. Comparing fluidized-bed bioreactors with other systems, the FBBR is suitable for environmental bioremediation. Successful scale-up can be achieved by maintaining similar chemical and physical conditions in both scales while using carriers with different densities. Further research is needed to assess pilot-scale feasibility and improve the anaerobic digestion system for bacterial adhesion.

Future research should focus on several key areas to advance the pilot-scale feasibility of these bioreactor systems. First, there is a need for comprehensive studies to optimize the operating parameters and design configurations of PBBRs and FBBRs to ensure efficient performance and scalability into the long-term stability and robustness of these systems, which are essential to assess their performance under varying environmental conditions and fluctuating influent characteristics. Understanding the dynamics of FBBRs over extended operational periods is crucial for reliable and sustainable operation at the pilot scale. Additionally, further exploration is needed to improve bacterial adhesion. Strategies such as surface modification of carrier materials may be effective. Furthermore, interdisciplinary research efforts integrating bioreactor engineering, microbiology, and environmental science will be instrumental in addressing the complex challenges associated with scaling up FBBRs for practical applications.

Data availability

The data supporting the findings of this study, including the synthesis parameters of floating carriers, hydrodynamic properties, and bioreactor performance metrics, are available within the manuscript and materials. Additional raw data, experimental protocols, and detailed performance results for nitrate and COD removal, as well as uranium-contaminated wastewater treatment, can be obtained from the corresponding author upon reasonable request. This ensures transparency and facilitates further research to build upon our findings in the field of sustainable water treatment.

Author contributions

Conceptualization: V. M. and P. B.; methodology: V. M. and P. B.; investigation: V. M. and P. B.; writing – original draft preparation: V. M. and P. B.; writing – review and editing: V. M., P. B. and R. M. All authors have read and agreed to the published version of the manuscript.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This research received no external funding from the National Commission of Atomic Energy.

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