Wastewater Denitrification with Solid-Phase Carbon: A Sustainable Alternative to Conventional Electron Donors

Abstract

Nitrate pollution in aquatic environments poses significant environmental and public health issues, mostly due to industrial activities and agricultural runoff. Biological denitrification, the favored method for removing nitrates, typically needs an external carbon source to support microbial processes. Traditional electron donors like methanol, ethanol, and acetate are effective but introduce economic, environmental, and operational challenges such as cost variability, flammability hazards, and excessive residual organic material. Recently, solid-phase carbon sources—like biodegradable polymers and organic agricultural waste—have shown promise as alternatives because they allow for controlled carbon release, improved safety, and enhanced long-term sustainability. This review systematically examines the performance of solid-phase carbon in wastewater denitrification by analyzing peer-reviewed studies and experimental data. The findings suggest that solid-phase carbon sources, including polycaprolactone (PCL) and polyhydroxyalkanoates (PHA), offer stable and extended carbon release, ensuring consistent denitrification effectiveness. Nonetheless, challenges remain, including optimizing biofilm development, balancing carbon availability, and reducing operational costs. Furthermore, the review emphasizes the potential for integrating machine learning in process optimization and highlights the need for more research to enhance the economic viability of these materials. The findings confirm the practicality of solid-phase carbon sources for extensive wastewater treatment and their capability to sustainably address nitrate contamination.

1. Introduction

Since 1970, the increase in the global population, industrialization, and the extensive use of fertilizers [1] have led to the entry of nitrates into various water and wastewater bodies, which has raised significant concerns about eutrophication and its severe impact on human health and aquatic life [2,3,4].

Nitrate contamination in drinking water is often linked to sources such as agricultural runoff, industrial discharge, and wastewater effluent [5]. Inadequate wastewater treatment can lead to elevated nitrate levels in receiving water bodies, which may then infiltrate groundwater and affect drinking water supplies. Consequently, wastewater treatment plays a vital role in preventing nitrate pollution and protecting drinking water quality. High levels of nitrates in drinking water are known to have detrimental effects on human well-being, especially for infants and pregnant women, resulting in birth defects and miscarriages [6]. Furthermore, a prevalent illness linked to nitrate contamination is the blue baby syndrome [7], which primarily affects infants under six months old. This condition occurs when hemoglobin in red blood cells is oxidized to methemoglobin, and due to the limited presence of the enzyme methemoglobin reductase in infants, this condition can have fatal consequences [8]. According to the World Health Organization (WHO), 785 million people do not have access to clean water [9]. Drinking contaminated water can also lead to the spread of diseases such as hepatitis, diarrhea, and, in severe cases, various types of cancer [10].

As discussed earlier, the quality of water significantly impacts human well-being and the health of our environment, influencing the economies of both developing and industrialized nations. To address these challenges, various approaches have been proposed, including the use of chemicals to remove contaminants. However, in many countries, the lack of adequate infrastructure makes these chemically intensive treatment methods less desirable. In addition, in recent years, stricter effluent requirements have been implemented alongside decreasing plant volumes, even as their loads continue to increase. Therefore, there is a need for more effective and innovative solutions to cleanse water from contamination while minimizing environmental stress and protecting human health during the treatment process [11,12].

The treatment of wastewater is a very complex process due to the presence of various pollutants, such as organic (pharmaceuticals, pesticides, and food) and inorganic (nutrients and heavy metals) substances and pathogens, which react together to create even more complex products [13,14,15]. Inorganic substances that are found in wastewater can have some advantages, such as facilitating the growth of microorganisms at low concentrations. However, a slight excess of these materials can pose significant risks. For instance, the WHO sets limits on nitrate–nitrogen levels to 11.3 mg/L (=50.059 mg/L nitrate), Cu concentrations to less than 2 mg/L, and Pb concentrations to not more than 0.01 mg/L in drinking water [13,16,17]

2. Denitrification

2.1. Overview of Biological Denitrification

Denitrification is a biological process in which nitrate or nitrite is converted to inert nitrogen gas [17]. Nitrate reduction reaction occurs in a five-stage stepwise process outlined below [18]:

NO₃⁻ + 2e⁻ + 2H⁺ → NO₂⁻ + H₂O

(1)

NO₂⁻ + e⁻ + 2H⁺ → NO + H₂O

(2)

NO + e⁻ + H⁺ → 0.5N₂O + 0.5H₂O

(3)

0.5N₂O + e^– + H⁺ → 0.5N₂ + 0.5H₂O

(4)

2NO₃^– + 12H⁺ + 10e⁻ → N₂ + 6H₂O

(5)

The theoretical stoichiometric equations for denitrification using ethanol (Equation (6)) and acetic acid (Equation (7)) are as follows [19]:

5C₂H₅OH + 12NO₃⁻ + 12H⁺ → 10CO₂ + 21H₂O + 6N₂

(6)

5CH₃COOH + 8NO₃⁻ + 8H⁺ → 10CO₂ + 14H₂O + 4N₂

(7)

The C/N ratio for Equations (6) and (7) are 1.38 (mol/mol) and 1.46 (mol/mol), respectively. These values exceed the theoretical values because some carbon is consumed in the formation of new biomass cells.

Depending on the source and nature of the wastewater, several remediation techniques have been developed to treat polluted water for various purposes [20,21,22], classified into three categories: chemical, physical, and biological. To be more precise, various methods have been developed for denitrifying wastewater, including chemical denitrification, biological denitrification, reverse osmosis, and adsorption/ion exchange, with adsorption/ion exchange being regarded as the easiest and most efficient among them. However, ion exchange comes with two main challenges: first, there is no resin available with high selectivity for nitrates over other ions commonly found in groundwater, and second, finding a suitable resin regenerant is difficult, as its disposal can create additional problems [23]. Overall, biological denitrification is the preferred method for removing nitrogen from domestic and industrial wastewater. In addition to these techniques, other materials, including natural ones, like wood chips, corn cobs, wheat straw, and cotton [7], as well as synthetic materials, like biodegradable plastics, can also be used. However, their nitrate removal efficiency is not as high as that of other sources [24,25]. Figure 1 shows the nitrogen transformations in biological treatment processes [26].

In the biological denitrification process, denitrifying bacteria use a carbon source available in the water as an electron donor for growth. These bacteria thrive in an environment with low levels of oxygen, a pH between 5.5 and 8.0, and a temperature of 25–35 °C [27].

Based on the type of electron donors in the denitrification process, nitrate reduction can be categorized into two main types: heterotrophic and autotrophic denitrification [28].

2.2. Heterotrophic Denitrification Processes

Heterotrophic denitrification (HD), a key term in biological denitrification, involves heterotrophic nitrifying bacteria (such as Paracoccus pantotropha), which depend on the source of organic carbon (methanol, glycerol, acetate, or ethanol) as an electron donor to transform nitrate (the electron acceptor) into nitrite and eventually into nitrogen gas. The equation below shows the process of consuming organic matter (acetate ion) and reducing nitrate to nitrogen gas in a heterotrophic manner [29].

5CH₃COO^– + 13H⁺ + 8NO₃^– → 4N₂ + 10CO₂ + 14H₂O

(8)

Although this process generally occurs in the absence of oxygen, bacteria such as Paracoccus pantotropha are capable of functioning under aerobic conditions as well. The process begins with the reduction of ammonia to nitrate, which is then further reduced to gaseous nitrogen [26], positioning this method as the most economically viable technique for nitrate removal when compared to alternatives like electrodialysis or reverse osmosis [1].

Table 1. Stoichiometric correlations for heterotrophic denitrification using different carbon sources.

Substrate	Stoichiometric Equation
Ethanol	5C2H5OH + 12NO3− → 10HCO3− + 2OH− + 9H2O + 6N2
	0.613C2H5OH + NO3− → 0.102C5H7NO2 + 0.714CO2 + 0.286OH− + 0.980H2O + 0.449N2
Acetic acid	5CH3COOH + 8NO3− → 8HCO3− + 2CO2 + 6H2O + N2
	0.819CH3COOH + NO3− → 0.068C5H7NO2 + HCO3− + 0.301CO2 + 0.902H2O + 0.466N2
Glucose	C6H12O6 + 2.8NO3− + 0.5NH4+ → 2.3H+ + 0.5C5H7NO2 + 1.4N2 + 3.5CO2 + 6.4H2O
Cellulose	5(C6H10O5)n + 24n NO3− → 6n CO2 + 13n H2O + 12n N2 + 24n HCO3−

presents stoichiometric relationships for heterotrophic denitrification, emphasizing variations in substrate utilization and nitrogen removal efficiency with different carbon-based electron donors [23]. As shown in Table 1, the first reactions for each carbon source represent the overall stoichiometric equations, which illustrate the total number of moles of substrate and nitrate involved in the reaction. Conversely, the second reactions show the reactions normalized to 1 mole of nitrate, providing a more detailed representation of the stoichiometric amount of substrate consumed per mole of nitrate. For example, approximately 0.613 moles of ethanol are needed to reduce 1 mole of nitrate. These normalized reactions are more useful for determining the required doses since they directly indicate the amount of substrate needed for each mole of nitrate.

2.3. Autotrophic Denitrification Processes

In autotrophic denitrification (AD), the denitrifying bacteria use inorganic substances such as sulfide, sulfur, and hydrogen, as well as bicarbonates, as a source of carbon to reduce the nitrate [30]. These bacteria have a slower growth rate, making nitrate removal in this method slower than in heterotrophic denitrification, where the bacteria grow faster [18]. This method is preferable for wastewater with low organic carbon concentrations, but it requires a longer start-up time and may result in accumulation of sulfate in the system [29].

The autotrophic denitrification can be divided into three main categories [31].

2.3.1. Hydrogen-Driven Denitrification Process

In this process, hydrogen gas serves as the primary electron donor. However, using hydrogen in this role has several drawbacks. These drawbacks include high operational and maintenance costs, complex bioreactor requirements, and the risk of sulfate reduction. Furthermore, hydrogen gas has low solubility in water (1.6 mg/L at 20 °C), making it difficult to manage and handle safely.

2.3.2. Anaerobic Ammonium Oxidation (ANAMMOX) Process

During this process, nitrite serves as an electron donor, while ammonia is converted to nitrogen gas [32]. This method can reduce operational costs by up to 90% by decreasing aeration by as much as 63% [33]. However, it has been reported that anammox bacteria have a relatively slow growth rate [34], which leads to a long startup time for the reactor.

2.3.3. Sulfur-Based Denitrification SPD Process

Elemental sulfur is frequently used in autotrophic solid-phase denitrification as an economical energy source for biological denitrification due to its non-toxic nature, insolubility in water, and stability under normal conditions [35,36]. One significant drawback of the sulfur-based denitrification process is the release of sulfate in the effluent water, which, at high concentrations and especially if magnesium is present, can cause laxative effects upon consumption [36,37].

3. Conventional Carbon Sources for Denitrification

3.1. Overview of the Conventional Carbon Sources

As previously stated, a carbon source is essential as an electron donor in the process of heterotrophic denitrification. When there is a lack of organic carbon in the system, it becomes necessary to introduce an external carbon source to improve denitrification efficiency [38]. Methanol, ethanol, acetic acid, and glucose, among others, are the main carbon sources used in the denitrification process [39], with methanol (also known as methyl alcohol, wood alcohol, and carbinol) being the most commonly used alcohol carbon source [40,41], due to its affordability, availability, and effectiveness in enhancing denitrification [42]. Denitrification efficiency greater than 95% has been achieved using methanol and acetate to treat industrial wastewater within six hours [43]. However, it should be acknowledged that using this product may lead to increased sludge production [44]. Notably, sucrose, ethanol, and methanol testing showed that ethanol is the most suitable electron donor based on the experimental results. A study by Constantin and Fick [19] indicated that ethanol exhibited a higher overall denitrification rate in comparison to acetic acid, and a denitrification efficiency of over 90% could also be achieved using ethanol if the HRT is longer than 2 h [45]. However, acetic acid showed superior performance in terms of its specific denitrification rate.

A study conducted by Peng et al. [11] examined the nitrate utilization rate during batch tests in a pre-denitrification system consisting of five parallel sequencing batch reactors (SBRs) operating in pulse mode at 20 °C. The stirred mixed liquor suspended solid (MLSS) concentration in the reactors was approximately 2400 mg/L, with a mixed liquor volatile suspended solid (MLVSS)-to-MLSS ratio of 0.80 and a solids retention time (SRT) of 15 days. The study found that ethanol was the most effective carbon source, achieving a higher denitrification rate and a shorter response and adaptation time compared to methanol and acetate. An extended lag period is a significant issue in the wastewater industry because, during this time, the plant experiences reduced denitrification capacity and generally moderate process performance [41]. Therefore, ethanol is an excellent external carbon source, particularly compared to methanol.

Table 2. Denitrification performance comparison of three carbon sources.

Carbon Source	Denitrification Rate (mg NO3-N/(g VSS/h))	Sludge Yield (g MLSS/g COD)	Adaptation Time	Response Time
Methanol	3.2	0.40	Long (40 d)	Slow (several days)
Ethanol	9.6	0.42	Short	Fast (several hours)
Acetate	12	0.65	Short	Fast (several hours)

summarizes the denitrification rates, sludge yields, adaptation times, and response times of the three carbon sources used in the study.

According to the findings of this study, the denitrification rate of methanol is one-third that of ethanol and nearly one-fourth that of acetate [11]. The prolonged lag period in methanol-utilizing systems is due to shifts in denitrifying populations, which represents a significant limitation of using methanol as an external carbon source [46].

The reason for the shorter adaptation time with systems using ethanol as the carbon source is the presence of some well-known microbial communities such as Azoarcus, Dechloromonas, Thauera, and Acidovorax denitrifiers, which have a natural preference for ethanol in activated sludge processes [41].

Considering the extensive use of these sources as an additional carbon source for denitrification, it is important to address potential drawbacks. This highlights the need for effective process control strategies to ensure safety while optimizing system performance.

3.2. Process Control Strategies

One of the major concerns with using conventional substances is their flammability [47]. The flammability level of the carbon source utilized in the system affects overall costs, including both capital and maintenance expenses, due to some required additional safety measures for mitigating the risk of fire happening in the system. For instance, two effective technologies that could be used for mitigating the risk of fire in methanol utilizing systems are vapor detection and thermal imaging. Vapor detection is used to identify early vapor emissions, which helps prevent ignition, while thermal imaging is useful for locating hot spots and identifying loose connections in electrical systems [48]. Furthermore, the plant operation staff should receive proper training and regular assessments, as handling hazards requires precise knowledge of the substances and the associated risks [47].

It was discovered that methanol can be toxic if not properly removed from the treated effluent [40]. Due to the risk of overdosing and increased dissolved organic carbon (DOC) amounts in the effluent, properly integrating it into the system is essential [49]. A study has been conducted by Puznava et al. [50] investigated two control strategies known as feedback (FB) and feedforward (FF) control. Additionally, a third approach was implemented, which combined both strategies. In the case of FB, control is based on the online measurement of nitrate outlet concentrations, while in FF, control relies on the online measurement of nitrate inlet concentrations. Each method presented an effective solution for mitigating the risks associated with overdosing the system while using methanol as the carbon source in the system. For example, the FF approach was the most cost-effective of the three, reducing methanol consumption by 20% compared to stepwise addition while still maintaining high-quality effluent.

Table 3. Comparison of biological denitrification techniques and their performance.

Denitrification Type	Carbon Source	Key Characteristics	Performance and Efficiency	Limitations
Heterotrophic Denitrification (Soluble Carbon Source)	External liquid carbon (e.g., methanol, ethanol, acetate)	Requires frequent dosing of organic carbon for microbial growth. Rapid nitrate removal under optimal conditions.	High denitrification efficiency (≥95%) when sufficient carbon is available. Acetate shows a higher removal rate than methanol, which requires a longer adaptation time.	Safety concerns due to flammability, risk of overdosing, and requires continuous carbon supply and monitoring.
Heterotrophic Denitrification (Solid-Phase Carbon Source)	Biodegradable polymers (e.g., PCL, PLA, PHBV)	Uses slow-release solid carbon sources, reducing the need for continuous dosing. Denitrification occurs as the solid material degrades.	Provides long-term denitrification with sustained carbon release. PCL and PHA blends achieve nitrate removal > 90%.	Higher initial material cost. Biofilm formation and degradation rate depend on microbial activity and environmental conditions.
Autotrophic Denitrification	Inorganic electron donors (e.g., sulfur, hydrogen, ammonia)	Suitable for wastewater with low organic carbon content. Bacteria oxidize inorganic compounds instead of using an organic carbon source.	Anammox can reduce aeration costs by 63%. Can be effective in treating nitrate-rich industrial wastewater.	Slow bacterial growth, long start-up time, and potential sulfate accumulation.

presents a comparative analysis of various biological denitrification techniques, emphasizing their carbon sources, key characteristics, performance efficiency, and limitations. This comparison enhances understanding of how different approaches work before assessing their economic feasibility in the next section.

4. Comparative Cost Analysis of Common Carbon Sources

Table 4. Denitrification rates and the costs of nitrogen removal using various carbon sources.

Reference	Carbon Source	Temperature (°C)	Price (CAD/kg Substrate)	Substance Utilization (kg/kg NO3−-N)	Cost (CAD/kg NO3−-N Removed)
[51]	PCL	20–25	7.45	1.33–1.77	10–13.1
	Acetic acid		3.6	3.5	12.6
	Methanol		1.5	2.08–3.98	3.1–6
	Ethanol		1.8	2.0	3.6
[52]	PCL	18–20	13.1	2.34	30.6
	Methanol	25	0.5	2.46 *	1.2
	Sodium acetate	20	0.87	5.78	5

* In practical applications, approximately 25 to 30% of methanol is utilized for the synthesis of bacterial cells. The presence of dissolved oxygen leads to a corresponding increase in methanol demand. Consequently, a widely accepted estimate is that 3.0 kg of methanol is needed for the removal of each kilogram of NO₃⁻-N, as opposed to the stoichiometric requirement of 2.46 kg of methanol [23].

shows the cost of denitrification based on the amount of substrate needed for the process and the price of each substrate per unit kilogram. Using polycaprolactone (PCL) was about four times more expensive than methanol and ethanol, but it was similar to the cost of acetic acid. However, it is important to note that the price of methanol, for instance, is closely tied to fluctuations in the fossil fuel market, making it vulnerable to volatility.

It is noteworthy that this cost estimation only reflects the production cost of the substrates and does not include additional expenses associated with conventional systems, such as process control costs. Considering the high efficiency of denitrification, biodegradable polymers could become a competitive carbon source for solid-phase denitrification in practice, provided that their prices are reduced to a more reasonable level [7].

Natural organic substances are significantly less expensive than synthetic biodegradable polymers. Research shows that using sugarcane as a substrate is more effective than methanol for biological denitrification in groundwater as it can achieve an impressive nitrate removal rate of 98% without requiring deoxygenation, which could help decrease operational costs. The low cost and widespread availability of sugarcane make it a favorable option, potentially reducing both investment and operational expenses for biological denitrification facilities. However, challenges such as the potential for bacteriological degradation of water quality and increased turbidity in the treated water compared to that treated with methanol remain when using sugarcane. Consequently, additional steps such as disinfection and softening treatments are necessary before the water can be distributed [1,27].

Moreover, using biodegradable polymers leads to a more stable effluent quality, which is reflected in lower total organic carbon (TOC) and color values as well as requiring less management of the system. Additionally, as production increases, the decrease in prices makes biodegradable polymers more attractive for denitrification [1]. Furthermore, while producing bioplastics from plants, such as corn and maize, repurposing agricultural land that could be used for food production becomes necessary. A recent study showed that nearly a quarter of the land dedicated to grain cultivation is now being used to produce biofuels and bioplastics. As more agricultural land is used for these purposes, food prices may rise significantly, which could negatively impact economically vulnerable populations [53].

5. The Environmental Impact of Various Carbon Sources

Conventional wastewater treatment processes are energy-intensive and produce a significant amount of sludge. It is estimated that approximately 580,000 metric tons of sludge (dry basis) are generated annually in Turkey, while the figures for the European Union (EU), Canada, the USA, Brazil, and China are 8910, 550, 6510, 370, and 6510 thousand metric tons, respectively. This sludge must be disposed of properly, as it primarily consists of biodegradable bacterial biomass. There is a growing interest in processes that combine waste treatment with energy generation. Many researchers are actively working on developing effective methods for harnessing energy from waste [18].

Released TOC is a crucial factor in assessing carbon sources, as it significantly impacts the quality of effluent water. One advantage of biodegradable polymers over liquid organics is their low and stable TOC release [49].

Organic carbon media, such as wood and straw, can promote denitrification with variable removal efficiencies. However, these materials may also leach certain unwanted substances, including phosphorus, which can contribute to eutrophication, and greenhouse gases like CO₂, CH₄, and N₂O [6].

Additionally, the use of bioplastics has also been linked to the emission of greenhouse gases [54]. Conventional petroleum-based synthetic plastics are produced through several steps, starting with the distillation of crude oil. This process separates and breaks down the heavy crude oil into lighter components called segments. Each segment is made up of a mixture of polymeric hydrocarbon chains that vary in size and structure. One important fraction, which is called naphtha, is crucial for producing monomers such as ethylene, propylene, and styrene, which are essential in making plastics [55,56]. These monomers undergo processes called polyaddition and/or polycondensation, facilitated by specific catalysts, to form plastics. However, this conversion process generates pollutants and greenhouse gases, such as CO₂, which contribute to the pollution of the environment and global warming [54]. Furthermore, many petroleum-based plastics are non-biodegradable, which allows them to persist in disposal sites and harm the environment. A study has been carried out by Walker and Rothman [57], who compared seven traditional plastics, four bioplastics, and one type of plastic made from both fossil fuel and renewable sources and found that the production of bioplastics contributes to increased pollution from fertilizers and pesticides used in crop cultivation, as well as the chemical processes required to convert organic materials into plastic. Additionally, it was discovered that bioplastics contribute more to ozone depletion than conventional plastics made from fossil fuels. Considering all these, bioplastics have eco-friendly characteristics that make their use promising. For instance, studies have shown that the biodegradation of polylactic acid (PLA) does not lead to a net increase in carbon dioxide emissions [58]. This is because the plants used in the production of PLA absorb the same amount of carbon dioxide during cultivation as is released during the biodegradation process [58,59]. Notably, when PLA degrades in landfills, it emits 70% less greenhouse gas compared to traditional plastics. These examples demonstrate that future bioplastic production can utilize renewable energy sources while significantly reducing greenhouse gas emissions [53]. Taking all of this into account, biodegradable polymers are still preferred over liquid materials in some cases despite being more expensive; for example, in the treatment of sensitive aquaculture wastewater, using methanol is not preferred due to its toxicity and overdosing risks. Considering the benefits and relatively higher costs, we remain optimistic about the potential of a biodegradable solid substrate [7].

6. Emerging Alternatives: Solid-Phase Carbon Sources

6.1. Solid-Phase Denitrification: Principles and Applications

Solid-phase denitrification (SPD) is a new technology in heterotrophic biological denitrification that employs insoluble organic materials as solid carbon sources [60], and it typically involves two steps: first, the hydrolysis of carbon sources by extracellular enzymes secreted by biofilm microbes; second, the utilization of the resulting small molecular substrates by denitrifying microbes [7]. Organic materials function both as electron donors and as carriers for biofilms. This method stands in contrast to conventional techniques that depend on low-molecular-weight organic compounds and saccharides, which are generally in liquid form [61,62].

Solid carbon sources can be categorized into two main types: natural sources (particularly materials that are rich in cellulose [60]) and artificial sources. Natural sources include agricultural by-products such as peanut shells, wood chips, and corn cobs. On the other hand, artificial sources consist of biodegradable polymers (BDPs) like polyhydroxyalkanoates (PHA) and PCL. These carbon sources have proven to be effective in the process of denitrifying water with a low carbon-to-nitrogen (C/N) ratio. Additionally, they address concerns related to flammability and allow for precise dosing within the system [7,63,64,65,66]. Moreover, denitrifying microbes can access solid carbon sources only after they have been decomposed by extracellular enzymes. The amount of carbon released is regulated by bacteria in response to nitrate loading to prevent overdosing [67].

PHAs, PCL, PLA, and polybutylene succinate (PBS) are commonly used for denitrification due to their effectiveness and large availability [7], but their addition influences the operational costs of biosystems and determines the denitrification capabilities of the bacterial community [38]. Therefore, even though PHAs, including poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV), are easily degraded and have a high nitrogen removal rate, their prohibitive cost prevents practical application. From a financial perspective, PCL is preferred and has shown a strong performance in denitrification [49].

Table 5. Denitrification efficiency and operating conditions of different carbon sources.

Reference	Carbon Source	Influent	Influent Nitrate Conc. (mg/L)	Temperature (°C)	Days of Operation (d)	Reactor Type	Denitrification Rate (gN/Ld)	Nitrate Removal (%)
[67]	PCL/PS (peanut shell)	synthetic water	20	25	162	Continuous up-flow SPD reactor		87.60 ± 0.06
	PCL/SB (sugarcane bagasse)							87.93 ± 0.05
	PCL/TPS (thermal plastic starch)							81.83 ± 0.05
	PCL							83.28 ± 0.07
[52]	PCL	Secondary effluent	25–35	8	249	SPD biofilter	1.23–1.67	20–31
				18	249	SPD biofilter	1.23–3.80	88–99
[68]	PCL	Synthetic water	55	25	70	Continuous-flow reactor	0.64	70
[1]	PCL	Groundwater	60–80	20–30	561	Fixed-bed bioreactor	0.19–0.56	92–96
[49]	PCL	Tap water with nitrate	15–52	25	184	Packed-bed bioreactor	0.59–0.66	93
[60]	PCL/Starch	Synthetic water	15–50	15–25	280	Packed-bed bioreactor	0.54–0.64	90
[69]	PCL, PLA and PHBV	Synthetic water	3–4.2	30	72	Batch	0.16	82–95

summarizes the denitrification efficiency of various solid carbon sources discussed earlier, with a particular emphasis on pure PCL and PCL blends under different conditions. In every case, nitrate removal exceeded 70%, demonstrating the suitability of PCL as an effective carbon source for denitrification.

Notably, one major challenge of solid-phase denitrification is developing new and cost-effective solid substrates that ensure the quality of effluent water. In contrast to costly synthetic polymers, natural materials like wheat straw, cotton, discarded newspapers, pine bark, and chitin from crab shells are significantly more affordable. One approach could be using natural solid substrates such as wheat straw, tree bark, and cotton. Among the various natural plant-like materials used, woodchips are the most popular. They are considered attractive for practical applications due to their lower cost, higher carbon-to-nitrogen ratio, longer effectiveness, and moderate availability [1]. However, another effective approach could be blending organic materials to reduce product costs, particularly in the production of biodegradable plastics [60].

6.2. Factors Affecting Denitrification Efficiency

6.2.1. Concentration of Nitrate in the Influent

The concentration of nitrate in the input significantly affects the efficiency of the denitrification process. It is observed that high nitrate concentrations in the influent tend to increase the denitrification rate; however, this can lead to reduced nitrate removal efficiency due to a lack of soluble carbon substrates in comparison to high nitrate loading. Furthermore, according to the investigation conducted by [31], higher concentrations of nitrate and nitrite in the influent resulted in longer hydraulic retention times (HRTs). Additionally, increased concentrations in the influent may lead to the inhibition of N₂ gas production resulting from the reduction of N₂O. At the same time, low nitrate inputs can result in a situation where denitrification is limited by the availability of nitrate [6,7,70].

6.2.2. Temperature

Many studies have reported that temperature plays a significant role in the denitrification process by affecting the effectiveness of enzymes involved in substrate hydrolysis and nitrate reduction [7,71,72]. Denitrification efficiency is generally improved at higher temperatures [31]; nonetheless, elevated temperatures may also accelerate the microbial decomposition of solid carbon sources, resulting in higher releases of ammonia and DOC [73]. Conversely, the denitrification rate drops significantly when the temperature falls below optimal. For instance, a 50% reduction in the denitrification rate was observed with just a 5 °C decrease in temperature while using PCL as the external carbon source for denitrifying groundwater [7]. In another study, denitrification rates for PCL at 10 °C and 15 °C were observed to be 5% and 10% of the rates at 30 °C, respectively [74]. Additionally, a study by Dold et al. [75] demonstrated that lower temperatures complicate the denitrification process by negatively impacting microbial growth. This finding aligns with the study conducted by Canziani et al. [71], which reported that decreased temperatures not only limit the hydrolysis of the carbon source but also reduce the activity of denitrifying bacteria.

6.2.3. pH

A pH range of 6.5 to 8.5 is generally suitable for solid-phase denitrification, as it aligns with the optimal range for denitrifying bacteria. pH levels outside this range can lead to a significant decline in denitrification efficiency by disrupting bacterial activity. Redundancy analysis (RDA) further highlights pH as a crucial environmental factor influencing the distribution of these microbes [6,7,76].

6.2.4. Hydraulic Retention Time

Hydraulic retention time (HRT) plays a crucial role in denitrification and affects operational costs [77]. Furthermore, its influence surpasses that of the influent nitrate concentration. When designing a denitrification reactor, optimizing the HRT is crucial because it directly impacts nitrate removal efficiency and microbial diversity. Different HRTs generate varying shear forces, which subsequently affect biofilm thickness and mass transfer.

Additionally, research indicates that reducing HRT to specific levels can lead to increased nitrite accumulation. A study conducted by Wang and Wang [78] using a synthetic influent at room temperature was in a 40 cm long cylindrical plexiglass biodenitrification unit and applied the following characteristics: 50 mg/L of nitrogen and 10 mg/L of phosphorus, NO₃-N of 6.5–9.6 mg/L, NO₂-N of 0.0 mg/L, DOC of 0.0–1.3 mg/L, and HRT of 2.1 h. When HRT dropped from 2.1 h to 1.4 h, nitrite accumulation was observed with a maximum value of 0.09 mg/L. As the HRT decreased from 2.1 to 1.5 h, the denitrification rate steadily increased from approximately 23.9 mg NO₃-N/(L/h) to 27.7 mg NO₃-N/(L/h). However, further reductions in HRT to 1.4 h led to a slight decline in the denitrification rate to 27 mg NO₃-N/(L/h).

Moreover, HRT is a crucial factor in regulating the release and utilization of DOC, which can also change the structure of microbial communities. Extended HRT may enhance the degradation of organic substrates, leading to improved nitrate removal efficiency. However, it can also lead to a higher release of DOC and ammonia [79].

6.2.5. Surface Area

As previously mentioned, solid carbon sources provide a surface for microorganism attachment. Consequently, surface area is crucial for the growth of these microorganisms. Reducing the size of the medium particle increases its specific surface area, leading to more biofilm attachment and enhancing pollutant degradation. However, smaller particle sizes also reduce porosity, resulting in shorter operational cycles, difficulties in cleaning the filter, and a greater demand for backwash water [77].

6.2.6. Trace Metal Elements

The presence of trace metals can influence the efficiency of denitrification in both positive and negative ways, depending on their concentration and interaction with other elements. Certain micronutrients, such as boron (B), cobalt (Co), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), and zinc (Zn), along with carbon (C), sulfur (S), and phosphorus (P), are essential for microbial growth. However, some of these micronutrients may also have inhibitory effects. Furthermore, minerals like sodium chloride and calcium ions play a crucial role in sludge granulation, which helps accelerate the nitrate removal process. On the other hand, certain sulfur compounds, such as sulfate, sulfide, and thiosulfate, have been shown to inhibit denitrification rates. Additionally, heavy metals, pesticides, and their by-products can also hinder the denitrification process [6].

6.2.7. Dissolved Oxygen

The denitrification process is typically carried out under anaerobic conditions. Oxygen, being a more energetically favorable electron acceptor, can significantly prevent the activity of denitrification enzymes, resulting in the accumulation of nitrite or nitrous oxide [80].

Most denitrifying bacteria are facultative anaerobes that use nitrate as a terminal electron acceptor in the absence of oxygen. Consequently, the presence of dissolved oxygen (DO) may hinder the denitrification process through direct competition or enzyme inhibition.

6.2.8. Salinity

The research on the impact of salinity is still limited, but it concluded that the denitrification ability of PHB in a recirculating system was similar across all tested salinities, which ranged from 0 to 30 g/L [63]. Additionally, salinity plays a crucial role in shaping microbial communities at both the taxonomic and functional gene levels [7,76].

6.2.9. Type of Carbon Source

The selection of a carbon source is essential in denitrification systems, as it greatly affects the efficiency of the process, cost, and environmental impact. The primary categories of pure chemicals used as carbon sources are alcohols, organic acids, and saccharides [27].

A detailed analysis of sequencing data reveals that different carbon sources are associated with specific dominant denitrifying bacteria. In addition to enriched denitrifiers, a significant number of general bacteria with unclear functions were present. The choice of carbon source influences the reproduction and survival of neighboring bacteria, affecting the biodiversity and network complexity of the denitrifying consortia (DNCs). In acetate/glucose-fed and glucose-fed DNCs, dominant species outcompeted rarer ones, likely due to faster growth rates with glucose. Some bacteria may act as scavengers, decomposing by-products from denitrifiers or dead cells. The acetate-fed DNC showed a higher specific denitrification rate than those fed with acetate/glucose mixtures or glucose alone, likely because (i) acetate can be directly metabolized by denitrifiers, while glucose must be broken down into simpler compounds first [38], and (ii) glucose transport into cells may occur through group translocation, unlike the transmembrane transport of acetate [81,82]. From an engineering perspective, using glucose as a carbon source resulted in higher biomass concentrations and more non-settleable flocs compared to acetate-fed DNCs. Thus, low-molecular-weight, biodegradable compounds (e.g., methanol and sodium acetate) may support the long-term stability of the denitrification system [83].

6.2.10. Yield

While controlling conditions such as pH, DO, and salinity is necessary to achieve higher denitrification efficiency, understanding the role of another critical parameter known as biomass yield (Y) is equally essential.

Biomass yield represents the amount of biomass produced per unit of substrate consumed (g biomass/g substrate) and is typically defined in relation to the electron donor used. Biomass yield can be expressed in various ways. For example, in aerobic or anaerobic treatment of municipal and industrial wastewater containing a wide range of organic compounds, yield is often based on a measurable parameter that reflects overall organic compound consumption, such as COD or BOD. In these cases, yield is expressed as g biomass/g COD removed or g biomass/g BOD removed. When evaluating and modeling biological treatment systems, yield is generally categorized into two main types: observed yield and synthesis yield (also known as true yield). The observed biomass yield is derived from actual measurements of net biomass production and substrate consumption but is lower than the synthesis yield, due to biomass decay occurring alongside cell growth. The relation between the observed yield and true yield is illustrated in Equation (9). In contrast, the synthesis yield refers to the biomass produced directly upon the consumption of the growth substrate or the oxidation of the electron donor, particularly in autotrophic bacteria. This value can be estimated when the stoichiometry or the energy produced in the oxidation–reduction reaction is known [26].

$${\mathrm{O}\mathrm{b}\mathrm{s}\mathrm{e}\mathrm{r}\mathrm{v}\mathrm{e}\mathrm{d} \mathrm{y}\mathrm{i}\mathrm{e}\mathrm{l}\mathrm{d} \mathrm{Y}}_{\mathrm{o}\mathrm{b}\mathrm{s}}\left(\mathrm{g}{\displaystyle \raisebox{1ex}{$\mathrm{V}\mathrm{S}\mathrm{S}$}\!\left/ \!\raisebox{-1ex}{$\mathrm{g}\mathrm{C}\mathrm{O}\mathrm{D}$}\right.}\right)={\displaystyle \frac{{\mathrm{Y}}_{\mathrm{t}\mathrm{r}\mathrm{u}\mathrm{e}}}{1+\left(\mathrm{b}\ast {\mathrm{ϴ}}_{\mathrm{c}}\right)}}$$

(9)

6.2.11. Maximum Specific Growth Rate (µ_max)

The maximum specific growth rate (µ_max) indicates the highest rate at which a microorganism population can grow per unit time under optimal substrate conditions. This parameter is crucial in microbial growth kinetics. One commonly used model to describe this relationship is the Monod model (Equation (10)), which presents an empirical equation linking the specific growth rate (µ), the substrate concentration in the bulk (S_bulk), the maximum specific growth rate (µ_max), and the half-saturation coefficient (K_s). This model helps to better understand and predict the behavior of microbial populations in systems such as activated sludge and biofilms, thereby facilitating the design and optimization of treatment processes [84].

$$\mathrm{\mu }={\mathrm{\mu }}_{\max } {\displaystyle \frac{{\mathrm{S}}_{\mathrm{b}\mathrm{u}\mathrm{l}\mathrm{k}}}{{\mathrm{K}}_{\mathrm{s}}+{\mathrm{S}}_{\mathrm{b}\mathrm{u}\mathrm{l}\mathrm{k}}}}$$

(10)

The maximum specific growth rate is an important parameter since the anoxic capacity requirements depend on how fast substrate-specific bacteria can grow and how many of them are actively functioning in the system [85]. This study found that a decrease in µ_max decreases the extent of nitrate removal. At 13 °C (winter conditions) µ_max values were approximately 0.5 d⁻¹ for methanol utilizers, 1.2 d⁻¹ for acetate utilizers, and 1.3 d⁻¹ or corn syrup utilizers. At 19 °C, these values increased to 1 d⁻¹, 3.7 d⁻¹, and 3.5 d⁻¹ for methanol, acetate, and corn syrup utilizers, respectively indicating that acetate utilizers consistently show the highest growth rates, followed by corn syrup utilizers, with methanol utilizers having the lowest rates. Moreover, all growth rates increase significantly at higher temperatures, highlighting the influence of temperature on bacterial activity and substrate utilization efficiency.

6.3. Types of Biodegradable Plastics

As the demand for sustainable materials increases, understanding the different types of biodegradable and bio-based plastics has become more important. The environmental issues stemming from discarded synthetic plastics have driven efforts to find suitable alternatives. Bioplastics, which offer both functional similarity to synthetic plastics and greater environmental sustainability, are seen as promising materials to tackle these challenges. The term “bioplastics” refers to plastics that either (1) are biodegradable, like PCL or PBS, or (2) may or may not be biodegradable but are derived from biological sources or renewable feedstocks, such as starch, cellulose, vegetable oils, and fats. Like other polymeric materials, the degradability of bioplastics depends on factors such as their composition, crystallinity, and environmental conditions, resulting in degradation times that can range from a few days to several years. Due to this variability, the development of biodegradable bioplastics has gained significant interest in recent years [86,87,88,89]. Based on their degradation mechanisms, biodegradable bioplastics are classified into two main types: oxo-biodegradable and hydro-biodegradable. Oxo-biodegradable plastics consist of petroleum-based polymers combined with a pro-degradant additive, typically a metal salt (such as manganese or iron salts), which accelerates degradation in the presence of oxygen through abiotic processes. These plastics are primarily derived from naphtha, a by-product of oil or natural gas, and their degradation timeframe can be “programmed” during manufacturing, similar to industrial processes for methane or nitrous oxide. Oxo-biodegradable plastics typically degrade over months to years. In contrast, hydro-biodegradable plastics decompose through hydrolysis at a faster rate than oxo-degradable plastics and can be converted into synthetic fertilizers. Examples of hydro-biodegradable plastics include those made from plant sources, like starch and PLA [53].

Table 6. Overview of terms related to biodegradable and bio-based plastics.

Term	Definition
Bioplastics	Plastics that (1) can be degraded naturally or (2) may or may not decompose but are made from biological substances or renewable resources.
Biodegradable plastics	Biodegradable materials can be broken down by microorganisms into monomeric or polymeric substances, such as biomass, water, and carbon dioxide or methane. In an industrial context, biodegradable materials are referred to as “compostable” and can be almost entirely converted into non-toxic waste within a few months when situated in a composter.
Bio-based plastics	Plastics classified as partially bio-based, or hybrid plastics, incorporate renewable carbon materials, such as plant matter. These materials possess a dual composition that includes both renewable resources and carbon derived from conventional fossil fuels. This combination allows for a more sustainable approach to plastic production, as it utilizes renewable inputs while still relying on established fossil fuel sources.

presents an overview of terms related to biodegradable and bio-based plastics and their definitions, helping to clarify the distinctions between commonly used terms in the context of biodegradable materials [53].

6.3.1. Polycaprolactone (PCL)

PCL is a biodegradable, biocompatible, semi-crystalline, and non-toxic aliphatic polyester with water, oil, and solvent resistance, attributed to the hydrophobic nature of its lactone monomer (See Figure 2 for the chemical structure of PCL.). Its favorable biological, rheological, and viscoelastic properties, along with its compatibility with numerous other polymers such as poly (vinyl chloride), poly (vinyl acetate), and poly (styrene-acrylonitrile), make it suitable for use in blends, composites, packaging, and tissue engineering scaffolds. The physical, thermal, and mechanical properties of PCL are influenced by its molecular weight and degree of crystallinity. Its large-scale application is still limited by its high cost relative to other traditional polymers. Additionally, for use in advanced applications, PCL often needs to be combined or blended with more affordable materials. Depending on its molecular weight, crystallinity, and degradation conditions, PCL can take several months to several years to fully degrade [90].

PCL is often used as a solid carbon source in solid-phase denitrification processes. Its insolubility allows PCL to undergo microbial degradation, releasing carbon for denitrification. When contact times are shorter, there is less decomposition of the substrate, resulting in lower TOC levels in the effluent. This feature makes PCL more advantageous than other liquid organic substrates. Moreover, studies have shown that PCL can enable simultaneous denitrification and pesticide removal when used as a substrate [49].

6.3.2. Polylactic Acid (PLA)

PLA is a biodegradable, biocompatible, and bioabsorbable aliphatic polyester that is derived from lactic acid (See Figure 3 for the chemical structure of PLA). It is known for its high mechanical strength and transparency, and it is made from renewable materials (such as corn starch, tapioca roots, chips or starch, and sugarcane), making it a compelling alternative to petrochemical plastics. PLA-based plastics can be molded and fabricated using conventional techniques, and their mechanical properties can be improved by adding compatibilizers and plasticizers. Due to its similar characteristics to traditional plastics like polyethylene (PE), PLA-based bioplastics offer a promising long-term solution to the environmental pollution caused by petrochemical-based plastics [53,90].

PLA is challenging to degrade in aqueous environments, rendering it unsuitable as a carbon source for denitrification. A study by Xu et al. [69] observed that PLA did not release COD throughout the experiment, further indicating its ineffectiveness in supporting denitrification processes. It is predominantly utilized in the food industry for producing disposable items such as drinking cups, cutlery, trays, plates, containers, and packaging for delicate food products. However, due to its inherent fragility, PLA bioplastics require additives to enhance their durability for broader packaging applications. Beyond food-related uses, PLA serves in agricultural applications like soil retention sheaths and films, as well as in manufacturing waste bags and other packaging materials. Additionally, this bioplastic can be spun into fibers to create woven, disposable, and biodegradable textile products, including garments, feminine hygiene items, and diapers [53].

6.3.3. Polyhydroxyalkanoates (PHA)

PHAs are a group of bio-based plastics categorized under the polyhydroxyester family, derived from 3-, 4-, 5-, and 6-hydroxy alkanoic acids. These biocompatible, biodegradable, and non-toxic polyesters are synthesized by specific bacteria and plants from renewable resources (See Figure 4). Methane emissions from sources such as wastewater treatment plants, landfills, composting facilities, farms, food processors, and bio-refinery operations can serve as a low-cost feedstock for commercial PHA production. Additionally, PHA can be generated from non-edible biomass sources, including wood, grass, and agricultural residues, providing an economical alternative to biomass from edible crops. PHAs have diverse commercial applications, including bioplastic wraps, shampoo bottles, and polyester fibers blended with natural materials for textiles.

Notably, PHA bioplastics are environmentally friendly, capable of natural decomposition by marine microorganisms into methane upon entering the ocean. Furthermore, at the end of their lifecycle, these compostable and marine-degradable bioplastics can break down into virgin plastic. Polyhydroxybutyrate (PHB) is a prevalent type of PHA synthesized by various microorganisms, including Cupriavidus necator, Methylobacterium rhodesianum, and Bacillus megaterium, often utilizing methane as a carbon source. As a biodegradable bioplastic, PHB offers an eco-friendly alternative to traditional fossil-based thermoplastics. It can be melt-processed into semi-crystalline thermoplastic forms through the fermentation of renewable carbohydrate feedstocks. Commercially available PHB grades exhibit properties closely resembling those of polypropylene (PP) derived from fossil fuels. PHB is commonly used in manufacturing disposable tableware, soil retention sheaths, waste wraps, and packaging materials. In biomedical engineering, PHB is utilized for producing surgical sutures and as a component in drug delivery systems [53].

The findings from the literature review highlight key aspects of wastewater denitrification, particularly the roles of conventional and solid-phase carbon sources, factors that influence denitrification efficiency, and emerging research directions. To provide a clear and structured overview,

Table 7. Key insights from the literature review about various carbon sources used in the denitrification process.

References	Aspect	Key Findings	Challenges and Research Gaps
[11,23,41]	Conventional Carbon Sources (Methanol, Ethanol, Acetate, etc.)	Effective electron donors for denitrification demonstrate high removal efficiencies. Ethanol and acetate outperform methanol in terms of adaptation time and denitrification rate.	Methanol is characterized by high costs, toxicity, flammability, and inconsistent residual organic loads. Additionally, it has a long adaptation time.
[1,49,60]	Solid-Phase Carbon Sources (PCL, PLA, PHA, etc.)	Facilitate ongoing carbon release to support long-term denitrification. PCL presents a promising, cost-effective option.	High production costs indicate a need for optimization in biofilm formation and degradation rate control.
[7,31,74]	Factors Affecting Denitrification Efficiency (Temperature, pH, HRT, etc.)	Higher temperatures improve enzyme activity and denitrification rates. HRT optimization is critical for efficiency.	Low temperatures significantly reduce performance. Short HRT can lead to nitrite accumulation.
[6,53,57]	Environmental Impact of Carbon Sources	Solid-phase carbon sources reduce flammability risks and offer controlled release, improving effluent quality. Bioplastics degrade with lower greenhouse gas emissions than traditional plastics.	Certain bioplastics have environmental impacts through land use changes and chemical processing.
[33,91]	Machine Learning and AI for Process Optimization	Potential to predict process efficiencies, optimize dosing, and improve decision-making in denitrification.	Limited real-world applications; high computational demands.
[7,49,92]	Research Gaps	There is a need to reduce costs in biodegradable plastics, optimize biofilm dynamics, and integrate machine learning. Further studies are required on nitrogen recovery and the removal of emerging contaminants.	Cost remains a significant barrier; further studies are needed on biofilm interactions and post-treatment challenges.

summarizes these findings by outlining the benefits and limitations of various carbon sources, the impact of different operational parameters, and the remaining research gaps.

Several review articles have examined solid-phase denitrification and its influencing parameters, such as temperature, HRT, and cost considerations. However, these studies have not comprehensively and systematically compared multiple biodegradable polymers. Furthermore, research gaps related to biofilm dynamics, material degradation rates, and the potential role of machine learning in optimizing denitrification efficiency remain largely unexplored. This review aims to expand on existing knowledge by offering a more in-depth analysis of SPD performance across various conditions, addressing critical operational challenges, and evaluating the practical feasibility of SPD for large-scale wastewater treatment.

7. Research Gaps

7.1. Mass Reduction and Post-Treatment Challenges

Solid-phase materials serve as both carbon sources and carriers for biofilms, which are essential for microbial growth. Understanding the mass reduction rate of these materials during the SPD process is crucial for predicting their renewal period. For example, if a material degrades quickly, it must be replaced more often, resulting in higher operational costs. On the other hand, if the material has a slow degradation rate, it may not adequately meet the needs of the microorganisms. To address the differences in degradation rates, it may be beneficial to create blends of different materials. By varying the proportions of each material in these blends, it is possible to achieve different denitrification rates that can better align with the requirements of the process [7].

7.2. Post-Treatment Challenges

More research should focus on developing precise methods for removing DOC to enhance practical applications in drinking water treatment. Additionally, it is essential to design more effective strategies for the simultaneous removal of nitrate and emerging contaminants, such as pharmaceuticals and personal care products (PPCPs), which may pose a threat to human health [7]. A study by Suarez et al. [91] evaluated the efficiency of nitrifying and denitrifying conditions in eliminating various PPCPs from synthetic wastewater. Understanding the behavior of these contaminants can provide valuable insights for managing them under different conditions.

7.3. Biofilm Characteristics on Biodegradable Carriers

Characteristics of the biofilm, such as its thickness or structure, can significantly impact the efficiency of the denitrification process, but there is limited literature on the biofilm characteristics of biodegradable carriers and the mechanisms for utilizing solid carbon sources. More research is needed to understand the role of solid carbon sources in bioreactors, especially in relation to biofilm dynamics [1].

7.4. PCL Modification and Cost Reduction

Current research is focused on modifying PCL to reduce costs and enhance its effectiveness in wastewater treatment [49]. As noted earlier, PCL is considered one of the most cost-effective options among synthetic biopolymers, offering relatively good denitrification performance compared to other synthetic materials. However, future studies should also investigate the potential of creating blends with other materials to further optimize its performance.

7.5. Machine Learning for Operational Optimization

The interaction between operating parameters and environmental conditions significantly affects the performance of nitrification and denitrification processes. Current analytical techniques are often time-consuming. However, by incorporating machine learning (ML), research efforts can be accelerated through data analysis, predicting processing efficiencies, and identifying optimal system settings for various environmental scenarios [33]. The combination of artificial intelligence (AI) and machine learning models has the potential to significantly transform water treatment by optimizing and improving the control of process parameters. These advanced technologies are particularly effective at removing pharmaceutical contaminants and reducing the risk of pathogens and viruses in various water sources, thereby minimizing health risks associated with them. Additionally, blockchain technology could play a crucial role in wastewater management and promoting sustainable energy practices in both urban and rural areas. However, further research is necessary to assess the feasibility of this technology and estimate its operational costs. Furthermore, the integration of the Internet of Things (IoT) and cyber-physical systems can improve decision-making processes, enhance waste transportation systems, and aid in reducing the carbon footprint [92].

7.6. Nitrogen Recovery for Enhanced Nitrogen Removal

While nitrogen removal has been a primary focus, nitrogen recovery has received minimal attention. Future studies on nitrification and denitrification should explore strategies to recover N₂O to improve nitrogen removal. However, it is important to note that the production of N₂O, which is a by-product of the nitrification and denitrification processes, can contribute to global warming and ozone layer depletion. Thus, it is crucial to investigate the limitations of this recovery [33].

7.7. Environmental Impact Assessments for Bioplastics

A study found that while bioplastic production increases greenhouse gas emissions, the overall impact is still lower than that of fossil-based counterparts. As new-generation bioplastics are developed, life cycle assessments (LCAs) and land use change (LUC) analysis become crucial for confirming their environmental benefits. Comprehensive studies will assist policymakers in evaluating the sustainability and eco-friendliness of these bioplastics [93].

8. Conclusions

This review focuses on various denitrification technologies, emphasizing biological denitrification as the most effective and sustainable method for removing nitrates from wastewater. This study analyzes different external carbon sources, including conventional liquid options (such as methanol, ethanol, and acetate) and solid-phase carbon sources (like PCL, PLA, and PHA). Moreover, the performance, efficiency, and economic feasibility of each approach are explored, highlighting their advantages and challenges. Additionally, this review identifies research gaps in areas like biofilm formation, carbon release dynamics, and process optimization.

The findings indicate that while methanol is the most commonly used carbon source because of its cost-effectiveness and high denitrification efficiency, its toxicity and handling risks pose significant challenges. Solid-phase carbon sources, especially biodegradable polymers, have shown promising results in maintaining long-term denitrification and provide a safer alternative to liquid carbon sources.

Future efforts should also focus on optimizing the performance of solid carbon sources, exploring innovative blends to reduce costs, and leveraging machine learning for system optimization. Additionally, addressing research gaps in biofilm dynamics, nitrogen recovery, and simultaneous removal of emerging contaminants will be crucial for advancing the field. Through these developments, solid-phase carbon sources have the potential to transform wastewater treatment into a more sustainable and efficient process, contributing to global efforts in environmental preservation and public health protection.