Anaerobic ammonium oxidation (anammox) by anammox bacteria contributes significantly to the global nitrogen cycle and plays a major role in sustainable wastewater treatment. To date, autotrophic nitrogen removal by anammox bacteria is the most efficient and environmentally friendly process for the treatment of ammonium in wastewaters; its application can save up to 60% of the energy input, nearly 100% elimination of carbon demand and 80% decrease in excess sludge compared to conventional nitrification/denitrification process. In the anammox process, ammonium (NH4+) is directly oxidized to dinitrogen gas (N2) using intracellular electron acceptors such as nitrite (NO2–) or nitric oxide (NO). In the absence of NO2– or NO, anammox bacteria can couple formate oxidation to the reduction of metal oxides such as Fe(III) or Mn(IV). Their genomes contain homologs of Geobacter and Shewanella cytochromes involved in extracellular electron transfer (EET). However, it is still unknown whether anammox bacteria have EET capability and can couple the oxidation of NH4+ with transfer of electrons to extracellular electron acceptors. In this dissertation, I discovered by using complementary approaches that in the absence of NO2–, freshwater and marine anammox bacteria couple the oxidation of NH4+ with transfer of electrons to carbon-based insoluble extracellular electron acceptors such as graphene oxide (GO) or electrodes poised at a certain potential in microbial electrolysis cells (MECs). Metagenomics, fluorescence in-situ hybridization and electrochemical analyses coupled with MEC performance confirmed that anammox electrode biofilms were responsible for current generation through EET-dependent oxidation of NH4+. 15N-labelling experiments revealed the molecular mechanism of the EET-dependent anammox process. NH4+ was oxidized to N2 via hydroxylamine (NH2OH) as intermediate when electrode was used as the terminal electron acceptor. Comparative transcriptomics analysis supported isotope labelling experiments and revealed an alternative pathway for NH4+ oxidation coupled to EET when electrode was used as electron acceptor. The results presented in my dissertation provide the first experimental evidence that marine and freshwater anammox bacteria can couple NH4+ oxidation with EET, which is a significant breakthrough that is promising in the context of implementing EET-dependent anammox process for energy-efficient treatment of nitrogen using bioelectrochemical systems.

Heavy metal contamination in water is a serious environmental and human health issue. Lead (Pb2+) and cadmium (Cd2+) are strictly regulated in wastewater effluent due to their high toxicity at low concentrations. Heavy metals are difficult to remove in conventional biological wastewater treatment because they are water soluble and non-biodegradable. Advanced treatment, such as tight membrane filtration and ion exchange, can be applied but they often require a high electrical energy input and a large amount of chemicals for pre- or post-treatment. Microbial electrolysis cells (MECs) can be used to treat wastewater while simultaneously recovering energy in the form of hydrogen gas. Additionally, MECs were proven to be effective for heavy metal removal. The commonly investigated removal mechanism for heavy metals in MECs is reduction at the cathode where heavy metal ions are reduced to metallic solids. The research presented in this thesis examined the effectiveness of cathodic reduction and other heavy metal removal mechanisms in MECs over a wide range of metal concentrations (10 μg/L-12 mg/L). Lab-scale MEC operation demonstrated successful removal of both Pb2+ and Cd2+ under different electric conditions, operation times, and initial metal concentrations. In addition to cathodic reduction, heavy metal removal in MECs was demonstrated through chemical precipitation at the cathode and electrochemical reduction and biosorption at the bioanode. The results of this research also confirmed the importance of microbial activity at the bioanode to efficiently drive the removal mechanisms in MECs. / Thesis / Master of Applied Science (MASc)

The treatment of water and wastewater is energy intensive, and there is an urgent need to develop new approaches to address the water-energy challenges. Bioelectrochemical systems (BES) are energy-efficient technologies that can treat wastewater and simultaneously achieve multiple functions such as energy generation, hydrogen production and/or desalination. The objectives of this dissertation are to understand the fundamental microbiology of BES, develop cost-effective cathode catalysts, optimize the process engineering and identify the application niches. It has been shown in Chapter 2 that electrochemically active bacteria can take advantage of shuttle-mediated EET and create optimal anode salinities for their dominance. A novel statistical model has been developed based on the taxonomic data to understand and predict functional dynamics and current production. In Chapter 3, 4 and 5, three cathode catalyst (i.e., N- and S- co-doped porous carbon nanosheets, N-doped bamboo-like CNTs and MoS2 coated on CNTs) have been synthesized and showed effective catalysis of oxygen reduction reaction or hydrogen evolution reaction in BES. Chapter 6, 7 and 8 have demonstrated how BES can be combined with forward osmosis to enhance desalination or achieve self-powered hydrogen production. Mathematical models have been developed to predict the performance of the integrated systems. In Chapter 9, BES have been used as a research platform to understand the fate and removal of antibiotic resistant genes under anaerobic conditions. The studies in this dissertation have collectively demonstrated that BES may hold great promise for energy-efficient water and wastewater treatment. / Ph. D.

Bioelectrochemical systems (BES) is a platform technology that is able to realize versatile engineering functions and recover valuable resources in an energy-efficient manner. One of the potential applications of BES is to remove and recover nutrients simultaneously from nutrient-rich wastewater, such as digested manure from livestock. A four-chamber BES was developed and used in this study to explore the potential to remove and recover hexavalent Chromium from synthetic wastewater, and ammonia and phosphate from digested manure. The BES was able to achieve 99.6% removal of Chromium by membrane adsorption in 5 days but failed to recover in the concentration chamber. Nutrients were removed from the waste stream and recovered in the recirculated catholyte by the electrical field generated from the waste. The BES was demonstrated to achieve substantial COD removal, nutrients removal and recovery. On average, the removal efficiencies were about 50% for COD, 85% for NH4-N and 40% for PO4-P, and the concentration of NH4-N recovered in the catholyte was 670 mg/L after 5 cycles under an applied voltage of 0.8 V. PO4-P was not recovered in solution, probably because it has precipitated under the alkaline condition together with Mg2+ and Ca2+ concentrated in the catholyte. It was also demonstrated that nutrients removal and recovery depended on the current generation and were mostly completed at high current. To sum up, the BES was proven to be an effective and sustainable approach to remove and recover nutrients from digested manure. / Master of Science

Wastewater treatment has developed as a mature technology over time. However, conventional wastewater treatment is a very energy-intensive process. Bioelectrochemical system (BES) is an emerging technology that can treat wastewater and also recover resources such as energy in the form of electricity/hydrogen gas and nutrients such as nitrogen and phosphorus compounds. Microbial electrolysis cell (MEC) is a type of BES that, in the presence of an additional voltage, can treat wastewater and generate hydrogen gas. This is a promising approach for wastewater treatment and value-added product generation, though it may not be sustainable in the long run, as it relies on fossil fuels to provide that additional energy. Thus, it is important to explore alternative renewable resources that can provide energy to power MEC. Waste heat is one such resource that has not been researched extensively, particularly at the low-temperature spectrum. This was utilized as a renewable resource by converting waste heat to electricity using a device called thermoelectric generator (TEG). TEG converted simulated waste heat from an anaerobic digester to power an MEC. The feasibility of TEG to act as a power source for an MEC was investigated and its performance compared to the external power source. Various cold sources were analyzed to characterize TEG performance. To explore this integrated TEG-MEC system further, a hydraulic connection was added between the two systems. Wastewater was used as a cold source for TEG and it was recirculated to the anode of the MEC. This system showed improved performance with both systems mutually benefitting each other. The operational parameters were analyzed for the optimization of the system. The integrated system could generate hydrogen at a rate of 0.36 ± 0.05 m3 m-3 d-1 for synthetic domestic wastewater treatment. For the practical application, it is necessary to estimate the cost and narrow the focus on the functions of the system. Techno-economic analysis was performed for MEC with cost estimation and net present value model to understand the economic viability of the technology. The application niche of the BES was described and directions for addressing the challenges towards a full-scale operation were discussed. The present system provides a sustainable method for wastewater treatment and resource recovery which can play an important role in human health, social and economic development and a strong ecosystem. / Doctor of Philosophy / An average person produces about 50-75 gallons of wastewater every day. In addition to the households, wastewater is generated from industries and agricultural practices. As the population increases, the quantity of wastewater production will inevitably increase. To keep our rivers and oceans clean and safe, it is essential to treat the wastewater before it is discharged to the water bodies. However, the conventional wastewater treatment is a very energy (and thus cost) intensive process. For low-income and developing parts of the world, it is difficult to adapt the technology everywhere in its present form. Furthermore, as the energy is provided mostly by fossil fuels, their limited reserves and harmful environmental effects make it critical to find alternative methods that can treat the wastewater at a much lower energy input. For a circular and sustainable economy, it is important to realize wastewater as a resource which can provide us energy, nutrients, and water, rather than discard it as a waste. Bioelectrochemical systems (BES) is an emerging technology that can simultaneously treat wastewater and recover resources in the form of electricity/hydrogen gas, and nitrogen and phosphorus compounds. Microbial electrolysis cell (MEC) is a type of BES that is used to treat wastewater and generate hydrogen gas. An additional voltage is supplied to the MEC for producing hydrogen. In the long run, this may not be sustainable as it relies on fossil fuels to provide that additional energy. Thus, it is important to explore alternative renewable resources that can provide energy to power MEC. Waste heat is a byproduct of many industrial processes and widely available. This was utilized as a renewable resource by converting waste heat to electricity using a device called thermoelectric generator (TEG). TEG converted simulated waste heat from an anaerobic digester to power an MEC. The mutual benefit for MEC and TEG was also explored by connecting the system electrically and hydraulically. Cost-estimation of the system was performed to understand the economic viability and functions of the system were developed. The present system provides a sustainable method for wastewater treatment and resource recovery which can play an important role in human health, social and economic development and a strong ecosystem.

O nitrato nas águas subterrâneas é considerado um dos principais problemas com relação aos padrões de potabilidade estabelecidos pela Portaria MS nº 2914/2011, o limite é de 10 mg NNO3-/L, tendo em vista que a ingestão de altas concentrações de nitrato está associada a doenças como câncer e a metahemoglobinemia. As preocupações com o nitrato devem-se as concentrações insidiosas e persistentes deste íon registradas pela CETESB desde o início do monitoramento das águas subterrâneas em 1990. O presente trabalho propôs a avaliação de um processo alternativo para remoção de nitrato, trata-se de um sistema bioeletroquímico, também conhecido como MFC (Microbial Fuel Cells), que utiliza microrganismos para desnitrificação. Esse tratamento consiste no uso de processos biológicos potencializados pelo processo de eletrólise, aproveitando desta forma, os principais pontos característicos de cada processo de maneira combinada. O sistema foi testado em escala de bancada, e consiste basicamente em uma câmara anódica onde ocorre a oxidação da matéria orgânica e uma câmara catódica onde ocorre o processo de redução do nitrato a N2. Entre as câmaras é utilizada uma membrana de troca iônica de forma a permitir somente a passagem de prótons da câmara anódica para a catódica, além de impedir a difusão de oxigênio para a câmara catódica. O experimento realizou 8 testes variando a taxa de aplicação total de 2,88 a 11,52 L/dia com uma concentração 15 mg N-NO3-/L. Nos últimos dois testes ainda foi aplicada uma tensão externa. O sistema atingiu uma eficiência média de remoção de nitrato de 80,84 ± 16,73 %. As concentrações finais de nitrato permaneceram dentro dos padrões de potabilidade, com valor médio de 1,88 ± 2,03 mg N-NO3-/L, obtendo-se uma taxa de desnitrificação de 0,0498 ± 0,03 kg/m³.dia. O acúmulo de nitrito no sistema teve valor médio de 0,36 ± 0,37 mg N-NO2-/L. / Nitrate in groundwater is considered to be one of the main problems with regard to the potability standards established by Ordinance MS nº 2914/2011, the limit is 10 mg N-NO3-/L, considering that the intake of high concentrations of nitrate Is associated with diseases such as cancer and methemoglobinemia. Concerns with nitrate are due to the insidious and persistent concentrations of this ion recorded by CETESB since the beginning of groundwater monitoring in 1990. The present work proposed the evaluation of an alternative process for nitrate removal. This is a bioelectrochemical system, also known as MFC (Microbial Fuel Cells), which uses microorganisms for denitrification. This treatment consists in the use of biological processes potentiated by the electrolysis process, thus taking advantage of the main characteristic points of each process in a combined manner. The system was tested on a bench scale, and basically consists of an anodic chamber where the oxidation of organic matter occurs and a cathodic chamber where the process of nitrate reduction to N2 occurs. Between the chambers an ion exchange membrane is used in order to allow only the passage of protons from the anode chamber to the cathodic, in addition to preventing the diffusion of oxygen to the cathodic chamber. The experiment performed 8 tests varying the total application rate from 2.88 to 11.52 L/d with a concentration of 15 mg N-NO3-/L. In the last two tests an external voltage was still applied. The system achieved an average nitrate removal efficiency of 80.84 ± 16.73%. The final concentrations of nitrate remained within the potability standards, with an average value of 1.88 ± 2.03 mg N-NO3-/L, obtaining a denitrification rate of 0.0498 ± 0.03 kg/m³.d. The accumulation of nitrite in the system had an average value of 0.36 ± 0.37 mg N-NO2-/L.

A sustainable and low-cost system, namely a photo-bioelectrochemical system (photo-BES), based on the natural blueprint of photosynthetic microorganisms was studied. The aim of this research work is to improve the efficiency of electron transfer of the microorganisms for bioelectricity generation. The first strategy adopted was the evaluation of the exoelectrogenic activity of oxygenic photosynthetic cyanobaterium, Synechococcus elongatus PCC 7942, in biophotovoltaic (BPV) platforms through a comparative performance analysis of bioanode materials. The second approach involved improving the performance of anoxygenic photosynthetic bacterium, Rhodopseudomonas palustris ATCC® 17001™, by varying the ratio of nitrogen to carbon sources (N:C) to maximise both biohydrogen production and exoelectrogenesis for conversion into bioelectricity in photosynthetic microbial fuel cells (photoMFCs). A linear correlation was obtained between average surface roughness/surface area and maximum power density of ITO-coated and graphene/ITO-coated substrates. Graphene/ITO-coated PET bioanodes produced the highest maximum power output of 29±4 μW m-2 in a single chamber BPV device due to improved biofilm formation and improved electrochemical activity. XG Leaf®, also known as graphene paper, helped to bridge the shortcomings of carbon fibres in terms of wettability. The most hydrophilic, 240 μm thick graphene paper, produced the highest maximum power output of 393±20 μW m-2 in a membrane electrode assembly (MEA)-type BPV device, mainly due to reduced electrochemical polarisation. A proof of concept study compared the performance of screen-printed graphene onto a membrane separator against 3D-printed bioanodes coated with carbon nanotubes. One mm thick 3D-printed bioanode was better performing as its structures promoted a much denser biofilm with extensive fibrous extracellular matrix. Using a ratio of N:C=0.20 resulted in higher biohydrogen production and higher exoelectrogenic activity, generating a maximum power output of 361±157 mW m-2 and 2.39±0.13 mW m-2, respectively. This study provided additional insight in improving the electron transfer efficiency, which could be used to further optimise photo-BESs as part of future research and development for sustainable technologies.

The aims of this project were to investigate a range of limitations affecting the electrical performance of bioelectrochemical systems (BES) and their use as analytical tools. The model cyanobacterium Synechocystis sp. PCC6803 was used to characterize light-driven BESs, or biophotovoltaic (BPV) devices. The phycobilisome (PBS) antenna size was altered to modify light absorption. At low to medium light intensities the optimum PBS antenna size was found to consist of one phycocyanin (PC) disc. Incorporating pulsed amplitude fluorescence (PAM) measurements into the BPV characterization allowed simultaneous comparison of photosynthetic efficiency to EET in Synechocystis. Non-photochemical quenching (NPQ) was investigated as a limiting factor in biophotovoltaic efficiency and was found to be reduced in the PBS antenna-truncated mutants. Fluorescence and electrochemical data were combined to develop a framework for quantifying the efficiency of light to bioelectricity conversion. This approach is a first step towards a more comprehensive and detailed set of analytical tools to monitor EET in direct relation to the underlying photosynthetic biology. A set of metabolic electron sinks were deleted to remove a selection of pathways that might compete with extracellular electron transfer (EET). The combined deletion of a bi-directional hydrogenase - HoxH, nitric oxide reductase - NorB, cytochrome-c oxidase - COX, bd-quinol oxidase - cyd, and the respiratory terminal oxidase - ARTO, roughly doubled light driven electron flux to EET. Deletion of nitrate reductase - NarB, and nitrite reductase - NirA, increased EET to a similar degree, but combination with the other knockouts compromised cell viability and did not increase output further. In addition to Synechocystis, the purple non-sulphur α-proteobacterium Rhodopseudomonas palustris CGA009 was used to test the effect of storage molecule synthesis knockout in a more industrially relevant organic carbon source driven BES, or microbial fuel cell (MFC). However, the removal of glycogen and poly-ß-hydroxybutyrate (PHB) did not have a significant effect on electrical output. Finally, the importance of electrode material and design for cell to anode connections in an MFC was investigated. EET from R. palustris was greatly enhanced using custom designed graphene and poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) aerogels. Pristine graphene is also shown for the first time to be a viable, low cost alternative to platinum as a cathodic catalyst. Together, these results present a holistic view of major limitations on electrical output from BESs that may contribute to enhancing EET for power generation from MFCs in the long term, and optimization of BPV devices as reliable analytical tools in the short term.

Les dispositifs bioélectrochimiques utilisent souvent le co-facteur NADH (nicotinamide adénine dinucléotide) comme biomolécule impliquée dans les réactions d’oxydo-réduction avec des enzymes de grand intérêt biochimique, comme par exemple les glucose oxydases ou les déshydrogénases. Il est nécessaire d’utiliser de nouveaux matériaux d’électrode afin de diminuer les sur-potentiels nécessaires au transfert d’électrons avec le système NADH/NAD+ et éviter l’adsorption des produits de la réaction à la surface de l’électrode (biofouling). Les nanotubes de carbone (NTCs) constituent un matériau conducteur de grande aire spécifique qui semble prometteur pour modifier ainsi la surface des électrodes. Ce travail de thèse a consisté à développer de nouvelles méthodes de greffage covalent de groupements fonctionnels électro-actifs vis-à-vis du système NADH/NAD+ en contrôlant les différentes étapes du procédé avec un protocole particulièrement poussé d’analyses physico-chimiques impliquant les spectroscopies de diffusion Raman, d’absorption infrarouge, de photo-électrons X, les microscopies électroniques à transmission, l’ellipsométrie spectroscopique et les analyses thermogravimétriques et de volumétrie d’adsorption. Nous avons développé un procédé reposant sur une première étape d’oxydation des NTCs par assistance micro-ondes dans des milieux acides dilués. Ceci permet de transformer les défauts existant à la paroi des nanotubes (atomes de carbone en hybridation sp3) pour les convertir en fonction acides carboxyliques, qui serviront dans les étapes ultérieures du procédé au greffage covalent des groupements électro-actifs. Ainsi l’intégrité structurale des NTCs, et donc leurs excellentes propriétés électroniques et mécaniques, sont préservées. Le succès de cette approche est pleinement démontré dans ce travail aussi bien en utilisant des nanotubes monoparois purifiés que des nanotubes multiparois. Un net effet électrocatalytique est obtenu avec les groupes fonctionnels dérivés du ferrocène. On montre également le rôle crucial de la nature du bras espaceur reliant les groupes électro-actifs à la paroi des NTCs. Ce travail a permis de mettre au point une méthode générale de greffage covalent des NTCs et son contrôle étape par étape. On montre enfin en perspective de ce travail qu’il est possible de greffer directement la molécule de NAD+ à la surface des NTCs. / Bioelectrochemical devices often use the NADH co-factor (nicotinamide adenine dinucleotide) as a biomolecule involved in oxidation-reduction reactions with enzymes of high biochemical interest, such as glucose oxidases or dehydrogenases. It is necessary to use new electrode materials to reduce the over-potentials required for electron transfer with the NADH/NAD+ system and avoid adsorption of the reaction products to the electrode surface (biofouling). Carbon nanotubes (CNTs) are a conductive material with a large specific surface area that seems promising for modifying the surface of electrodes. This thesis work consisted in developing new methods for covalent grafting of electro-active functional groups with respect to the NADH/NAD+ system by controlling the various stages of the process with a particularly advanced physico-chemical analysis protocol involving Raman scattering spectroscopy, infrared absorption, X-ray photoelectron spectroscopy, transmission electron microscopy, spectroscopic ellipsometry and thermogravimetric and volumetric adsorption analyses. We have developed a process based on a first step of oxidation of the CNTs by microwave assistance in diluted acid media. This makes it possible to transform existing defects in the wall of the nanotubes (carbon atoms in sp3 hybridization) into carboxylic acid functions, which will be used in the subsequent steps of the process for covalent grafting of electro-active groups. Thus, the structural integrity of the CNTs, and therefore their excellent electronic and mechanical properties, are preserved. The success of this approach is fully demonstrated in this work both by using purified single-walled nanotubes and multi-walled nanotubes. A clear electrocatalytic effect is obtained with the functional groups derived from ferrocene. The crucial role of the nature of the spacer arm connecting the electro-active units to the wall of the CNTs is also shown. This work made it possible to develop a general method for covalent grafting of CNTs and its step-by-step control. Finally, we show in perspective of this work that it is possible to directly graft the NAD+ molecule onto the surface of the CNTs.

O nitrato nas águas subterrâneas é considerado um dos principais problemas com relação aos padrões de potabilidade estabelecidos pela Portaria MS nº 2914/2011, o limite é de 10 mg NNO3-/L, tendo em vista que a ingestão de altas concentrações de nitrato está associada a doenças como câncer e a metahemoglobinemia. As preocupações com o nitrato devem-se as concentrações insidiosas e persistentes deste íon registradas pela CETESB desde o início do monitoramento das águas subterrâneas em 1990. O presente trabalho propôs a avaliação de um processo alternativo para remoção de nitrato, trata-se de um sistema bioeletroquímico, também conhecido como MFC (Microbial Fuel Cells), que utiliza microrganismos para desnitrificação. Esse tratamento consiste no uso de processos biológicos potencializados pelo processo de eletrólise, aproveitando desta forma, os principais pontos característicos de cada processo de maneira combinada. O sistema foi testado em escala de bancada, e consiste basicamente em uma câmara anódica onde ocorre a oxidação da matéria orgânica e uma câmara catódica onde ocorre o processo de redução do nitrato a N2. Entre as câmaras é utilizada uma membrana de troca iônica de forma a permitir somente a passagem de prótons da câmara anódica para a catódica, além de impedir a difusão de oxigênio para a câmara catódica. O experimento realizou 8 testes variando a taxa de aplicação total de 2,88 a 11,52 L/dia com uma concentração 15 mg N-NO3-/L. Nos últimos dois testes ainda foi aplicada uma tensão externa. O sistema atingiu uma eficiência média de remoção de nitrato de 80,84 ± 16,73 %. As concentrações finais de nitrato permaneceram dentro dos padrões de potabilidade, com valor médio de 1,88 ± 2,03 mg N-NO3-/L, obtendo-se uma taxa de desnitrificação de 0,0498 ± 0,03 kg/m³.dia. O acúmulo de nitrito no sistema teve valor médio de 0,36 ± 0,37 mg N-NO2-/L. / Nitrate in groundwater is considered to be one of the main problems with regard to the potability standards established by Ordinance MS nº 2914/2011, the limit is 10 mg N-NO3-/L, considering that the intake of high concentrations of nitrate Is associated with diseases such as cancer and methemoglobinemia. Concerns with nitrate are due to the insidious and persistent concentrations of this ion recorded by CETESB since the beginning of groundwater monitoring in 1990. The present work proposed the evaluation of an alternative process for nitrate removal. This is a bioelectrochemical system, also known as MFC (Microbial Fuel Cells), which uses microorganisms for denitrification. This treatment consists in the use of biological processes potentiated by the electrolysis process, thus taking advantage of the main characteristic points of each process in a combined manner. The system was tested on a bench scale, and basically consists of an anodic chamber where the oxidation of organic matter occurs and a cathodic chamber where the process of nitrate reduction to N2 occurs. Between the chambers an ion exchange membrane is used in order to allow only the passage of protons from the anode chamber to the cathodic, in addition to preventing the diffusion of oxygen to the cathodic chamber. The experiment performed 8 tests varying the total application rate from 2.88 to 11.52 L/d with a concentration of 15 mg N-NO3-/L. In the last two tests an external voltage was still applied. The system achieved an average nitrate removal efficiency of 80.84 ± 16.73%. The final concentrations of nitrate remained within the potability standards, with an average value of 1.88 ± 2.03 mg N-NO3-/L, obtaining a denitrification rate of 0.0498 ± 0.03 kg/m³.d. The accumulation of nitrite in the system had an average value of 0.36 ± 0.37 mg N-NO2-/L.