Research advances in deriving renewable energy from biomass in wastewater treatment plants

Abstract

Anaerobic digestion (AD) can be used to derive renewable energy from biomass in wastewater treatment plants, and the produced biogas represents a valuable end-product that can greatly offset operation costs. However, AD is a complex biological process involving many different kinds of microbial communities and biotransformation processes, and it is easily affected by start-up and operation conditions. In order to facilitate improvements in the AD process and enable predictions of biogas production quantities, this review discusses the microbial dynamics, scientific models, and reinforcement strategies of AD biogas production (i.e., sludge pre-treatment, bio-electrochemical and Fe/Fe₃O₄ technologies). During sludge pre-treatment, the focused-pulsed method can be introduced to promote cell lysis, and this requires specific treatment chamber designs. The bio-electrochemical method, especially in regard to the use of microbial electrolysis cells (MECs), can obviously improve biogas production while only consuming small amounts of energy. Mechanisms of AD-MEC systems and associated influencing factors are described in detail in this review. Engineered nanomaterials, which are increasingly being used in commercial products, may have detrimental or even beneficial effects on the sludge AD process. The use of low doses of Fe/Fe₃O₄ nanomaterials (≤0.5% (w/w)) as an economically viable and environmentally sustainable method for improving biogas production is discussed. In addition, removal methods for dissolved methane, which can have a negative environmental impact if not handled properly, in the anaerobic digester effluents are discussed in this review.

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1 Introduction

Wastewater treatment processes consume large amounts of energy and produce unwanted by-products such as sludge,¹ and large amounts of sludge are generated in wastewater treatment plants (WWTPs) every year. In 2014, total sludge production (total solids of 20% or more) amounted to over 3.6 × 10⁷ t in China, and sludge production within each province is shown in Fig. 1.^2,3 Most of the sludge was generated in the eastern region of China, and Guangdong province had the maximum amount of 5.2 × 10⁶ t (Fig. 1). Sludge is a complex material that typically contains about 50% protein, 10% carbohydrates, 10% lipids, and 30% other components such as RNA and fiber,⁴ and treatment and disposal costs for sludge are typically high, i.e., these costs generally amount to 20–45% of the total WWTP capital costs in China.⁵ Common sludge disposal techniques include land applications, sanitary landfill disposals, and incineration. However, these methods are neither economical nor sustainable, and they generate vast amounts of so-called greenhouse gases. Given the energy crisis and increasingly strict sludge disposal laws, cost-effective and sustainable technology for sludge management is urgently needed. Anaerobic digestion (AD) occurs in oxygen-free systems, and this process can utilize organic matter to produce biogas that is an efficient and renewable energy source. Biomass treatment by AD technology is associated with many co-benefits in that this technology can achieve significant reductions in organic matter, pathogens, and odors while imparting the obvious benefits that can be realized by producing energy from biogas to discount WWTP operation costs. Because of this promising potential, AD technology has been attracting significant attention from researchers. As illustrated in Fig. 2(a), AD publications have experienced a dramatic increase over the years 2000–2015 (from 95 in 2000 to 636 in 2015), and nearly all of the annual increments have been positive (the maximum number of 144 was found in 2013) (Fig. 2(a)). These data were based on literature searches in the Thomson Reuters “Web of Science” database with the key words “anaerobic digestion” and “sludge”. Furthermore, through the analysis of all the AD research articles from the year 2000 to 2015, we found that China had the highest counts (773, 17.9%), followed by the USA (489, 11.3%) and Spain (338, 9.0%) (Fig. 2(b)). Obviously, China attaches great importance to the use of AD for sludge treatment.

Fig. 1 Sludge production across provinces of China in 2014.

Fig. 2 (a) Time course for publications on sludge anaerobic digestion from the year 2000 to 2015, and (b) overview of the top 10 most productive countries for sludge anaerobic digestion research from the year 2000 to 2015.

Biogas, which has a caloric value of 21–25 MJ m⁻³, can be used as vehicle fuel or as a fuel to generate heat and electricity; thus, biogas use can lead to reductions in the use of fossil fuels and lower CO₂ emissions to the atmosphere.⁶ On the basis of chemical properties (H/C ratio) and other intrinsic properties of biogas fuel, Bordelanne et al.⁷ estimated that reductions of over 80% in greenhouse gas emissions could be achieved by using the biomethane from AD systems instead of gasoline. However, during the AD process, changes in the pH, temperature, sludge concentration and composition, liquid dynamics, and ionic strength, as well as tank configurations and methods of operation can cause fluctuations in the amount of biogas generated from excess sludge.^8–17 Chen et al.⁸ found that the initial pH had a strong influence on biogas production through its effects on the hydrolysis and acidification stages during the sludge AD process. The maximum methane yield (224 mL CH₄/g VS) was achieved at pH = 9. Moreover, the findings showed that the sludge AD process could be accelerated under proper temperature conditions; soluble chemical oxygen demand (SCOD) yield was usually used to evaluate the efficiency of AD process. Yang et al.⁹ found that the highest SCOD yield (4407 ± 80 mg L⁻¹) was obtained at 60 °C, which was 20-fold higher than that at 75 °C. Recently, sludge concentration was the focus of an international study, especially high sludge concentration AD. Prabhu and Mutnuri¹⁰ compared the performance of high sludge concentration AD (10% < total solid (TS)) with conventional low sludge concentration AD. In their study, high sludge concentration AD achieved highly efficient volatile solid reduction and high CH₄ production because of more numerous total archaea (dominated by Methanosarcina) in a high sludge anaerobic reactor, which enhanced the sludge AD process. However, when the sludge concentration was increased further, the efficiency of sludge AD never increased; it actually decreased because of increasingly bad liquid dynamics.¹¹ Solon et al.¹² reported that the ionic strength could affected AD performance because of significant physico-chemical effects including ion pairing of inorganic and organic substrates with different cations (such as K⁺ and Na⁺) and anions (such as Cl⁻). Wang et al.¹³ reviewed tank configurations (such as continuous stirred tank reactor (CSTR), anaerobic sequencing batch reactor (ASBR), up-flow anaerobic sludge blanket (UASB), expanded granular sludge bed (EGSB), anaerobic packed bed (APB), anaerobic fluidized-bed (AFBR) and hybrid bio-reactor (HBR) configurations) and found that the configuration could influence the AD process performance for different compositions of sludge and operating conditions. Based on specific AD processes, a traditional single-stage process and two-stage process were both developed. The two-stage system was found to both improve the stability and performance of the AD process, and it had a 21% higher specific methane yield than the single-stage system.¹⁴

In order to better understand the mechanisms involved in sludge AD processes, and to provide information that can help to optimize and predict biogas production, this review discusses AD processes in terms of microbial dynamics, scientific models, and reinforcement strategies (i.e., sludge pre-treatment, bio-electrochemical and Fe/Fe₃O₄ technologies). In sludge pre-treatment technologies, the high-efficiency and available focused-pulsed method can promote cell lysis; its mechanisms of cell lysis and specific treatment chamber designs are discussed in detail. State-of-the-art sludge pre-treatment technology, the microbial electrolysis cell (MEC) method, and the mechanisms involved in AD-MEC systems are also introduced in the reinforcement strategies section. Furthermore, because increasing amounts of engineered nanomaterials are being released into wastewater treatment plants, the potential impact of nanomaterials, especially low doses of Fe/Fe₃O₄ nanomaterials (≤0.5% (w/w)), on sludge AD processes and the associated mechanisms are also described in this review. In addition, we also review new technology, namely, anaerobic methane oxidation technology, which can be used to treat dissolved methane in the AD digestate and prevent it from becoming a threat to the environment.

2 Microbial dynamics and scientific models

The energy derived from sludge AD technology in WWTPs has been used to discount up to 33–100% of the electricity costs in some developed countries.¹⁸ To provide information that can be used to improve the operational efficiency, the microbial dynamics and scientific models of sludge AD processes are reviewed here.

2.1 Microbial dynamics

The overall sludge AD process involves several co-dependent biological stages (e.g., hydrolysis, acidogenesis, acetogenesis, and methanogenesis) where biodegradable organics are broken down by different kinds of microbes in an air-tight digester vessel; the final products include biogas (about 40–70% CH₄ and 30–60% CO₂) and residual compounds (AD digestate), which are rich in nutrients and may contain some dissolved CH₄ if the recommended sludge retention time (SRT) values of 10–20 days and temperatures of 0–80 °C are applied (Fig. 3).^19–21 In the hydrolysis stage, complex polymeric organics are broken down to simple organic monomers such as sugars, amino acids, and fatty acids with extracellular enzymes from hydrolytic bacteria. Subsequently, in the acidogenesis stage, these hydrolytic products are fermented by acid-forming bacteria into products such as propionic acid, butyric acid, acetic acid, ethanol, H₂, and CO₂. The products acetic acid, H₂, and CO₂ are then directly in utilized in the fourth stage of the process, whereas the other products from the second stage undergo acetogenesis, whereby more acetic acid, H₂, and CO₂ are produced. Finally, in the methanogenesis stage, methane-forming bacteria such as acetotrophic and hydrogenotrophic methanogens use the acetate, H₂, or CO₂ to produce methane.^22,23

Fig. 3 Sludge anaerobic digestion biochemical pathways for biogas (SRT: sludge residence time).

Methanogens are strictly anaerobic prokaryotes.^24–27 There are three metabolic pathways for methanogenesis, and these include the production of methane from carbon dioxide and hydrogen, methanol and hydrogen, and acetate and water, as shown below (eqn (1)–(3)):^28,29

Methanogenesis from CO₂ + H₂

4H₂ + H₂CO₃ → CH₄↑ + 3H₂O (1)

Methanol to CH₄

CH₃OH + H₂ → CH₄↑ + 3H₂O (2)

Acetate to CH₄

CH₃COOH + H₂O → CH₄↑ + 3H₂CO₃ (3)

The pathway involving methanol to CH₄ is rare and only occurs under specific environments.²⁹ Theoretically, about two-thirds of the total methane production is generated by the pathway involving acetate to CH₄ and the remainder is produced via the CO₂ + H₂ to CH₄ pathway in typical AD systems.³⁰ Temperature plays a key role in AD processes, and these processes are usually classified into psychrophilic (<25 °C), mesophilic (25–40 °C), thermophilic (40–60 °C), and hyperthermophilic (60–80 °C) conditions,^31–33 which can be used to regulate the pathways of methanogenesis. Under psychrophilic conditions, the pathway involving acetate to CH₄ is dominant; the acetate to CH₄ and CO₂ + H₂ to CH₄ pathways both occur at a certain proportion during the AD process under mesophilic conditions, while methane is exclusively generated by methanogenesis from CO₂ + H₂ under thermophilic and hyperthermophilic conditions.^34–36

2.2 Scientific models

Modeling is a useful tool to assess AD process performance, and it can be further used to determine the optimal operating conditions.³⁷ In an attempt to account for the various types of microorganisms involved, the formation of complex intermediate products, differences in the sludge's intrinsic biodegradability, and other types of process influencing factors during the sludge AD process, the ADM1 (Anaerobic Digestion Model No. 1) was formally introduced in March 2002.³⁸ Since that time, great progress has been made in terms of ADM1 modifications. The ADM1 consists of two main reaction types including biochemical reactions (disintegration of complex organics, extracellular hydrolysis, and biomass growth and decay) and physico-chemical reactions (ion association/dissociation and gas–liquid transfers),³⁹ and this work is summarized in Table 1. However, because operating conditions such as the temperature (mesophilic, thermophilic,⁴⁰ and psychrophilic⁴¹) and the presence of high-solid contents (total solid > 10%),⁴² inhibitors (e.g., Na⁺,⁴³ NH₃,⁴⁴ PO₄³⁺,⁴⁵ and ionic strength⁴⁶), and promotion materials (e.g., Fe⁴⁷) can critically impact biochemical reactions, the related parameter values should be revised to improve the ADM1; furthermore, some additional relevant kinetic equations should be added into ADM1. Presently, the lack of a comprehensive understanding of some processes such as anion reduction (NO₃⁻ and SO₄²⁻) and the formation and emission of odorous compounds limits the ability of the ADM1 to predict the outcomes accurately, but new research is being conducted to elucidate the related process mechanisms and evaluate their effect on fermentation and methanogenesis.^48–50 Moreover, operational modes such as the use of co-digestion and two-stage systems, which can also affect the AD process, are being investigated. Sludge co-digestion with carbon-rich wastes, which has been shown to be economically viable and feasible with AD systems, can realize “1 + 1 > 2” effects in regard to biogas production.⁵⁶ However, disintegration and conversion of more complex feed materials into carbohydrates, proteins, and lipids can occur by mechanisms that are different from the conventional digestion pathways, and these related reaction pathways need to be re-modeled with proper kinetics.⁵¹ Two-stage AD systems, which are stable, reliable, and effective systems, consists of two independent phases (i.e., acetogenesis and methanogenesis), and work has been done to optimize the operational conditions in such systems;⁵⁷ thus, a two-stage model would be useful for making improvements in these systems.⁵² While activated sludge models (ASMs) such as the ASM1, ASM2d, and ASM3 can be used to evaluate the efficiency of WWTPs without considering details of the AD process,⁵⁸ overall, performance assessments of WWTPs, especially in regard to effluent quality and biogas production, would benefit greatly from application of coupled ASMs including the ADM1; defining and modeling the interfaces is the difficult part.^53–55

Table 1 Related contributions and advantages for modified ADM1 procedures^a

Modified ADM1	Related contributions	Advantages	Reference
a ADM1: Anaerobic Digestion Model No. 1, HT/MT: mesophilic/thermophilic, PS: psychrophilic, HTS: high total solid, Na: sodium, NH3: ammonia, P: orthophosphate, IS: ionic strength, Fe/ZVI: zero-valent iron, NR: nitrate reduction, SR: sulfate reduction, OC: odorous compounds, CD: co-digestion, TS: two-stage digestion, ASM1: Activated Sludge Model No. 1, ASM2d: Activated Sludge Model No. 2d, ASM3: Activated Sludge Model No. 3, VFAs: volatile fatty acids, SBR: sequencing batch reactor, LBPs: less biodegradable pollutants, CBIM: continuity-based interfacing method, MCN: maximizing the total COD and nitrogen contents, COD: chemical oxygen demand.
HT/MT-ADM1	Studied the dynamic behavior in mesophilic and thermophilic digestion	Provided the accurate kinetic values for ADM1 under mesophilic and thermophilic conditions	40
PS-ADM1	Omitted the hydrolysis step	Simplified ADM1	41
	Studied the dynamic behavior in psychrophilic digestion	Provided the accurate kinetic values for ADM1 under psychrophilic conditions
HTS-ADM1	Studied acetate degradation kinetics during digestion of high-solid contents	Calibrated the kinetic parameters related to acetate uptake	42
Na-ADM1	Added a sodium inhibition function into ADM1	Considered the effect of sodium on acetoclastic methanogens and biogas production and composition	43
NH3-ADM1	Implemented the alternate acetate oxidizing mechanism instead of the standard acetoclastic pathway	Led to more accurate pH predictions	44
		Described metabolic activity of un-ionized species, undissociated acetic acid and un-ionized NH3 used as inhibitors
P-ADM1	Comparison of three modifications of ADM1 with the orthophosphate influence	Introduced an orthophosphate inhibition kinetics equation	45
		Provided insight into the orthophosphate influence on fatty acid dynamics and methane production
IS-ADM1	Davies approach applied instead of molar concentrations for consideration of ionic strength chemical activities	Described impact of the ionic strength on physico-chemical reactions, pH, and biogas	46
Fe-ADM1	Integrated three new processes (electron release and H2 formation from ZVI corrosion, and transformation of LBPs) into ADM1	Described performance of the ZVI-based anaerobic system	47
NR-ADM1	Added nitrate reduction processes into ADM1	Elucidated the effect of nitrate reduction processes on fermentation and methanogenesis	48
SR-ADM1	Added sulfate reducing processes into ADM1	Provided new understanding of substrate competition in anaerobic systems	49
OC-ADM1	Developed a mathematical model based upon ADM1 for the formation and emission of odorous compounds	Gave the general mechanisms for the formation of common odorous sulfur compounds such as CH4S, C2H6S, and H2S, as well as VFAs and NH3	50
CD-ADM1	Added surface-based kinetics (modeling of complex organics disintegration and conversion to carbohydrates, proteins, and lipids) into ADM1	Found that the kinetic constant of the disintegration process just depends on the nature and composition of biomass in co-digestion reactors	51
TS-ADM1	Presented a process model for an anaerobic two-stage digestion system	Predicted the dynamic behavior of a two-stage digestion process	52
ASM1-ADM1	Connected ASM1 and ADM1 with the CBIM and MCN methods, respectively	Evaluated plant-wide control systems and operating strategies	53
ASM2d-ADM1	Combined ASM2d with ADM1 with an interface definition (ADM1 was used to calculate the anaerobic treatment phases and ASM2d was used to calculate the aerobic phases)	Optimized performance of WWTPs	54
		Predicted the SBR potential for detoxification and COD removal
ASM3-ADM1	Coupled ASM3 with ADM1	Optimized performance of WWTPs so that they could meet stricter effluent discharge criteria, especially in regard to N removal	55

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3 Strategies for improving anaerobic digestion

Sludge AD aimed at the production of methane-rich biogas is in widespread use at WWTPs nowadays. However, one major limitation of the AD process is that it cannot efficiently extract the energy from sludge. Productivity of sludge AD systems can be improved with efficient and available strengthening strategies such as pre-treatment technology (especially focused-pulsed technology), bio-electrochemical techniques, and Fe/Fe₃O₄ nanomaterial methods.

3.1 Focused-pulsed technology

During the AD process, microbial cells can account for 70% of the sludge.⁵⁹ Therefore, extracellular polymeric substances and microbial cell walls in the sludge can seriously hinder the release and hydrolysis of intracellular organic matter. Poor sludge AD performance with long SRT values (20–50 days) and low organic degradation efficiencies (20–50%)²¹ can be dramatically improved by pre-treatment technologies, which mainly involve the application of physical, chemical, and biological methods. Physical methods that have been used include stirred ball mills,⁶⁰ high-pressure homogenization techniques,⁶¹ Venturi tubes,⁶² ultrasonic systems,⁶³ freeze/thaw cycles,⁶⁴ and focused-pulsed methods.⁶⁵ Chemical methods that have been used include ozonation,⁶⁶ Fenton reactions,⁶⁷ and biological methods such as enzyme⁶⁸ and enhanced biological techniques;⁶⁹ these pre-treatment methods can increase sludge pyrolysis, which is a limiting step for methanogenesis, through cell lysis. Cell lysis can be initiated by mechanical forces with physical methods (Fig. 4(a)) and by chemical molecules or enzymes with chemical and biological methods, respectively (Fig. 4(b)). Out of all of these pre-treatment methods, the optimal technology is focused-pulsed pre-treatment because of its outstanding performance, low cost, simple operation procedures, use of technology that is easy to retrofit, and application of a physical/non-chemical process.^70,71

Fig. 4 (a) Mechanism of cell lysis initiated by mechanical force during application of the physical method and (b) mechanism of cell lysis initiated by chemical molecules or enzymes during application of the chemical and biological methods, respectively.

In both bench- and pilot-scale studies, focused-pulsed technology has been shown to effectively disrupt and break up sludge flocs and cells, which leads to an obvious increase in SCOD (Table 2). Sludge biodegradation is also greatly improved (larger k_hyd values and fermentation efficiencies) with this technology, and greater biogas production can be achieved. Banaszak et al.⁷² investigated the effect of focused-pulsed technology on improvements in excess sludge biodegradability in a full-scale WWTP (capacity 7.57 × 10⁴ m³ d⁻¹), and a 60% increase in biogas production and a 40% reduction in sludge disposal amounts were obtained, which resulted in a net profit of 5.4 × 10⁵ dollars. Zhang et al.⁷⁵ reported that focused-pulsed pre-treatment not only made the sludge more biodegradable, but it also increased bacterial diversity and the relative abundance of acetoclastic methanogens in the digestate, and this in turn led to very low acetate concentrations in the AD system effluent. Salerno et al.⁷⁸ studied the effect of some factors such as the applied voltage, pulse duration, pulse frequency, sludge conductivity, length of the treatment chamber, and sludge residence time in the treatment chamber on sludge biodegradability and defined the treatment intensity as follows:

where η is the treatment intensity, V is the applied voltage, D is the pulse duration, f is the pulse frequency, σ is the sludge conductivity, T is the sludge residence time in the treatment chamber, L is the length of the treatment chamber, and k is a constant.

Table 2 Effect of focused-pulsed pre-treatment on sludge in bench- and pilot-scale studies^a

Research size	Organic sources	Operation results	Reference
a SCOD: dissolved chemical oxygen demand, khyd: first-order rate constant for hydrolysis, TCOD: total chemical oxygen demand.
Pilot-scale	Excess sludge	158% increase in SCOD	72
		60% increase in biogas production
		40% reduction in disposal sludge
Pilot-scale	Primary + excess sludge	160% increase in SCOD	73
		60% increase in biogas production
		40% reduction in disposal sludge
Pilot-scale	Primary + excess sludge	32% increase in biogas production	74
		17% reduction in disposal sludge
Pilot-scale	Primary + excess sludge	30–40% increase in biogas production	75
Bench-scale	Excess sludge	220% increase in SCOD^a	76
		40% reduction in reactor the size
		33% increase in biogas production
		21% increase in khyd
		18% increase in TCOD removal
Bench-scale	Excess sludge	4.5-fold increase in SCOD/TCOD	77
		2.5-fold increase in biogas production
Bench-scale	Excess sludge	1-fold increase in biogas production	78
Bench-scale	Excess sludge	2.6-fold increase in SCOD	79
		Increase in fermentation efficiency by about 23%

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Recently, a new study showed that focused-pulsed pre-treatment can also remove siloxane in sludge prior to its entry into biogas production.⁸⁰ Siloxane is a problematic material because of its abrasive, non-thermal, and non-electrical properties, and it can seriously damage the heat exchangers and gas engines during the use of biogas.⁸¹

In addition, focused-pulsed technology has the potential to make sludge into a viable electron donor that can drive denitrification processes⁸² or MECs.⁷⁹ Lee et al.⁸² found a 26-fold increase in the sludge's semi-soluble chemical oxygen demand (SSCOD) after focused-pulsed treatment, and the maximum denitrification rate was improved by 5-fold. Ki et al.⁷⁹ reported that the accumulation of desirable volatile fatty acids (VFAs) and acetate could be enhanced by 2.6-fold when focused-pulsed treatment was applied. Correspondingly, there was a 2.4-fold increase in the maximum current density in the MECs compared to MECs prepared without the use of focused-pulsed treated sludge.

3.1.1 Mechanisms of cell lysis. Focused-pulsed technology improves sludge biodegradability mainly by disrupting the cell membranes of cells within the sludge and releasing intracellular organic matter. Generally, two main theories (i.e., electroporation and shockwave) can describe this process.

The sludge cell lysis that occurs when cells are exposed to an electric field is mainly caused by the electromechanical instability of cell membranes. Cell membranes can maintain an effective osmotic boundary and protect microorganisms from surrounding environmental conditions; however, when exposed to an electric field, lipid membranes become charged up and are broken down if the transmembrane potential (TMP) developed across the cell membrane reaches about 1 V.⁸³ For exposure to an external electric field, Sale and Hamilton⁸⁴ established the following equation to describe the TMP:

V(t) = 1.5rE (5)

where V(t) is the transmembrane potential with the same direction of applied electric field strength, r is the sludge cell radius, and E is the applied electric field strength. The normal TMP is about 10 mV because of the dielectric constant difference between dielectric materials in the cell membrane and the environment. When cells are exposed to an electric field, an increase in the TMP can be achieved (eqn (5)). This increase of TMP can lead to a reduction in the cell membrane thickness because the electro-compressive force increases. When moderate electric fields are applied, the viscoelastic restoring force formed by the decreases in cell membrane thickness can oppose the electro-compressive force, but when E increases to a sufficient extent, large pores will form in the cell membrane and an irreversible breakdown occurs. This ultimately results in cell lysis.

After a rapid discharge of high-voltage electricity from an electrode submerged in the liquid medium, tremendously high transient pressure pulses can develop, which produce shockwaves (∼1 × 10³ bars).⁸⁵ These strong shockwaves can cause mechanical oscillations in the cell membranes, which act in concert with the process of electroporation to more effectively breakdown sludge cells.

3.1.2 Treatment chamber design. Treatment chamber design can seriously affect electric fields and the different levels of field uniformity.⁸⁶ In recent years, optimization of the geometry of focused-pulsed treatment chambers has been studied, and designs such as parallel plates and co-axial and co-linear shapes have been modeled with computational fluid dynamic (CFD) equations.⁸⁷ A uniform electric field distribution can be obtained by parallel plate electrode chambers, but unfortunately, the electric field was not found to be strong at the section of the edges of the electrodes because of the limited sizes. Zang et al.⁸⁸ improved the geometry of the parallel plate electrode chamber design by adding baffled flow channels. The co-axial cylindrical configuration can generate a well-defined electric field distribution, but undesirable dielectric breakdown often occurs at strong electric field enhancement points. Qin et al.⁸⁹ optimized the shape of the co-axial cylinder arrangement with numerical electric field computations and by adding a cooling system to overcome the above mentioned disadvantage. By using a similar numerical approach, Meneses et al.⁹⁰ developed a better co-linear chamber structure through considering the treatment parameter electric field strength distribution.

In addition, electrochemical reactions often occur in the treatment chamber, and this can result in corrosion of the electrode.⁹¹ To avoid electrode corrosion, Góngora-Nieto et al.⁹² introduced a new electrode material with a much higher resistance value against corrosion. Morren et al.⁹¹ reported that the use of short pulses could also help to prevent the corrosion.

3.2 Bio-electrochemical technology

Many studies of bio-electrochemical technologies have focused on the interactions between microbial cells and electrodes. Of these technologies, MECs driven by exoelectrogenic bacteria with a small applied voltage (0.2–0.8 V) are commonly used in AD biogas production systems; this voltage is far less than that typically required for water electrolysis (1.8–2.0 V). Chemical production rather than power generation maybe a much more appealing way of utilizing MEC technology because of the current technological implementation limitations and challenges.⁹³ Studies have shown that a pair of electrodes placed into an AD system to form a larger AD-MEC system can accelerate sludge hydrolysis and increase methane production.^94–96 Generally, system designers can choose between the use of two different reactor structures for MEC operations (i.e., single-chamber and two-chamber structures). In two-chamber MECs, the membrane is added between the anode and cathode, thus forming two separate chambers. The membrane materials applied in MECs included proton-exchange membranes (PEMs), cation-exchange membranes (CEMs), and anion-exchange membranes (AEMs).⁹⁷ Although the two-chamber structure better protects the cathode from hazardous substances and reduces the anode consumption during hydrogen diffusion, use of this structure can cause a pH gradient in the two separate chambers, which will not only result in bacterial inactivation, but also increase the reactor resistance and thus produce low current densities, reduced biogas production efficiencies, and high energy consumption rates.⁹⁸ Therefore, many studies of MECs have been carried out with a single-chamber structure. Chen et al.⁹⁹ found that methane production was increased by 76.2% with a 26.6% reduction in volatile suspended solids (VSSs) in an AD-MEC system compared to a single AD system. Guo et al.¹⁰⁰ reported that a significant increase (11.4–13.6-fold) in methane production could be obtained if MEC systems were incorporated into the sludge AD process. An MEC system can accelerate the hydrolysis of sludge flocs, enhance the transformation of SCOD, improve the conversion of VFAs, and maintain an optimal pH range for methane-producing activity. Asztalos and Kim¹⁰¹ found that VSS and chemical oxygen demand (COD) removal in an AD-MEC system was continuously higher by 5–10% than a similar AD system that did not employ MECs under ambient temperatures; this is equivalent to what can be achieved with common AD under mesophilic temperatures. This study indicated that MECs could represent a promising technology for boosting the efficiencies of AD processes under ambient temperatures. Generally, higher methane contents in biogas can be generated by using AD-MEC systems. Song et al.¹⁰² reported that an AD-MEC system that applied 0.3 V between the anode and cathode could generate biogas with a methane content of 76.9%, which is obviously higher than the contents produced by common AD systems. They also found that some operating parameters such as the pH and alkalinity were quite stable and suitable for methane growth, which suggests that MECs can enhance the stability of the AD process. Cusick et al.¹⁰³ reported that the applied voltage could be supported by renewable energy sources, such as solar energy and salinity-gradient energy, which would improve the productivity of AD-MEC systems further.

In addition, MECs have shown great future potential to recover residual energy in AD digesters, and they can minimize environmental impacts because organic matter in the effluent is being changed to less harmful forms. Kondaveeti and Min¹⁰⁴ demonstrated the feasibility of producing (i) biofuels such as butanol, ethanol, and propanol and (ii) biogases such as hydrogen and methane from anaerobic digestion effluents via MECs. Clauwaert et al.¹⁰⁵ discussed how MECs were promising technologies for generating methane from AD digesters based on their analysis of experimental data.

3.2.1 Mechanisms of AD-MEC systems. Sludge consists mostly of proteins and carbohydrates, and sludge hydrolysis during the AD process is commonly considered as the limiting step for methane production. On the basis of many studies, mechanisms of MECs that can accelerate sludge hydrolysis and enhance methane production during the AD process have been elucidated (Fig. 5).^{101,106–109} In AD-MEC systems, organic matter such as VFAs, alcohols, glucose, phenols, and benzene¹¹⁰ are first oxidized by anodic exoelectrogenic bacteria like Shewanella and Geobacter species that play a crucial role in the electron transportation between the anode and cathode; these species breakdown the sludge flocs and cell membranes into simple organics by decomposing proteins and carbohydrates, and then, the sludge hydrolysis efficiency is accelerated.¹⁰⁹ Exoelectrogenic bacteria and anodes with bacteria growing on them are referred to as bio-catalysts and bio-anodes, respectively (Fig. 5).

Fig. 5 Mechanisms of microbial electrolysis cell-based accelerated sludge hydrolysis and increased methane production during anaerobic digestion (AM: acetoclastic methanogenesis, HM: hydrogenotrophic methanogens, MN: Methanosarcina, MT: Methanosaeta, GB: Geobacter, DIET: direct interspecies electron transfer, VFAs: volatile fatty acids).

The pathways used by MECs for increasing biogas production mainly include four different styles (Fig. 5). Of these four styles, pathway I and pathway II are common mechanisms. When MECs are added to the AD process, acetate and CO₂ production efficiency is notably increased by the bio-catalysts. At the same time, H₂ production can be enhanced by the electrons from the processes involving exoelectrogenic bacteria such as Shewanella and Geobacter spp., the decomposition of organic matter, and H⁺ reactions in the mixed solution. Therefore, acetate to CH₄ and methanogenesis from CO₂ + H₂ are both enhanced as a result of the presence of adequate substrate. Sun et al.¹⁰⁷ reported that pathway II involving methanogenesis from CO₂ + H₂ is prominent in AD-MEC systems, and this pathway accounted for 96.01% of the methane generation in their analysis. They also found that exoelectrogenic Geobacter and hydrogen-producing Petrimonas bacteria were the dominant species involved, according to pyrosequencing data. Pathways III and IV are both new mechanisms for increasing methane production in AD-MEC systems. Pathway III involves the situation where the methane is formed by direct cathodic reduction of H₂ (eqn (6)),⁹⁵ which is also referred to as electromethanogenesis:

CO₂ + 8H⁺ = CH₄↑ + 2H₂O E⁰ ≈ −0.44 [vs. the standard hydrogen electrode (SHE) potential] (6)

Geobacter metallireducens can build a direct electron connection with Methanosaeta and Methanosarcina (i.e., through direct interspecies electron transfer, hereafter referred to as DIET) during the production of methane, and this is referred to as pathway IV.^28,111 Zhao et al.¹⁰⁸ found that DIET (pathway IV) played a more important role in the AD process compared to electromethanogenesis (pathway III), which just accounted for a small part of the methane production. In their study, a 12.9% increase in methane production and a 17.2% reduction in sludge production were obtained in AD-MECs.

3.2.2 Influencing factors of AD-MEC systems. In order to further increase AD biogas production with MEC technology, some factors such as the electrode material and structure,^112,113 additions of ferric iron (Fe³⁺),¹¹⁴ the hydraulic retention time (HRT),¹⁰² and applied voltage¹¹⁵ have been explored in depth.

Electrode material and its structure are important factors for the electron exchange between the cathode and microbial cells. Villano et al.¹¹² used anode materials with potentials between +200 mV and −200 mV (versus the SHE potential) to study the effect of anode materials on AD-MEC system performance. Anodes associated with high acetate removal rates and cathodes associated with high methane generation rates both had potentials under +200 mV. However, the sum of the internal potential losses decreased by 52.7% when the anode potential was decreased from +200 mV to −200 mV, which indicates that cathode overpotentials can reduce methane production. The results also clearly show that anode potentials need to be optimized to satisfy both rapid substrate oxidation rates and high energy efficiencies. The optimal electrode potentials for AD-MECs are still under investigation. Zhen et al.¹¹³ improved a bio-anode by using a layer of graphite felt (GF) on a plain carbon stick. The modified bio-cathode was found to enhance the microbial electrocatalysis activity by affording abundant space for exoelectrogenic bacteria growth, and thus, increases in methane production were mainly due to obvious reductions in the cathode overpotential. This study revealed that GF has the substantial potential to upgrade the electromethanogenesis efficiency, which can be attributed to its open structure and high conductivity. Addition of Fe³⁺ has been found to significantly enhance electron exchange through the effect of microbial Fe³⁺ reduction, which further accelerates sludge fermentation.¹¹⁴ Zhang et al.¹¹⁵ found that COD removal could be stably maintained at 88.3% by adding Fe³⁺ into an AD-MEC system, and this value decreased by 5.9% after the applied voltage (0.8 V) was cut off; the study results also indicated that the addition of Fe(III) could enhance anodic oxidation, which was confirmed by pyrosequencing and denaturing gradient gel electrophoresis (DGGE) analyses that showed that richer bacteria and archaea communities were attached on the anode compared to those in other common AD systems. Slow sludge hydrolysis rates resulted in long HRTs for the AD process. Song et al.¹⁰² studied AD-MEC system performance at different HRTs (5 to 20 days), and the VSS removal rate and methane content in the biogas slightly changed as the HRT decreased from 20 to 5 days. The methane recovery efficiency was 69.1–98.7%, and the maximum energy efficiency appeared in under 10 days, which suggests that short HRTs may be profitable for AD-MECs. Feng et al.¹¹⁶ reported that 22.4% and 11% increases in methane production and VSS removal rates, respectively, could be obtained under 0.3 V. However, the methane production decreased and hydrogen appeared in the cathode as the applied voltage increased to 0.6 V, apparently because the excessive utilization of H⁺ in the cathode resulted in a highly alkaline pH that seriously inhibited the methanogens. Moreover, DGGE analysis revealed that archaea and bacteria were both bio-augmented under a low applied voltage (0.3 V), which might be useful information for future improvements in the sludge hydrolysis and methane production processes.

In addition, because of the remaining lack of knowledge in this field, the effects of the design parameters such as electrode size and electrode arrangement, the operational parameters such as temperature, organic loading rate, and pH, and toxicants including organic toxicants and inorganic toxicants on the AD-MECs efficiency and the associated mechanisms should be widely investigated in future research.

3.3 Fe/Fe₃O₄ nanomaterials

Over the past several years, many kinds of nanomaterials have been introduced in commercial consumer products, and these materials have a high probability of being released to WWTPs. The impact of nanomaterials on the AD process is presently a hot topic. Studies that have been conducted to date have found that nanomaterials such as Ag, ZnO, TiO₂, CuO, and CeO₂ can seriously inhibit the AD process because they are toxic to AD microorganisms.^117–119 Nanomaterials often inactivate AD microorganisms through multidimensional effects, which may include direct contact effects (i.e., breaking down of cellular membranes, thereby resulting in losses of membrane-bound enzyme activity) and indirect damage caused by reactive oxygen species (ROS) that are generated by nanomaterials in aqueous solutions; ROS are highly reactive and can damage the double bonds on membrane phospholipids, which will lead to adverse increases in membrane fluidity and membrane permeability.^120–122 However, some studies have shown that relatively low doses of nanoscale Fe/Fe₃O₄ (≤0.5% (w/w)) material could accelerate sludge hydrolysis, thus improving biogas production (30.4–56.0%) and increasing the methane content (5.1–13.2%); this specific type of nanomaterial was also associated with the production of more stabilized sludge and reduced odors during the AD process.^123–126 While Fe/Fe₃O₄ nanomaterials can generate toxic substances such as ROS, this may actually help to improve the sludge biodegradability by breaking down sludge cells.¹²⁷ In addition, Fe/Fe₃O₄ nanomaterials are unstable and can generate Fe²⁺, Fe³⁺, and especially H₂, which may have a beneficial effect on the sludge AD process.^128,129 Specifically, H₂ generated from Fe nanomaterials can ensure that adequate substrate concentrations are present for methanogenesis from CO₂ + H₂ (ref. 128) and this may increase the utilization of CO₂;¹³⁰ furthermore, electrons from Fe nanomaterials can form effective DIET relationships between fermentative bacteria and methanogens, which may enhance the methane production rate.¹²⁹ Viggi et al.¹³¹ found that Fe₃O₄ nanomaterials could indeed enhance DIET between fermentative bacteria and methanogens by acting as electron conduits. Therefore, a metallic iron core could potentially be used as slow-release source for active ions, electron donors, and electron conduits to accelerate methanogenic activity and boost biogas production. Moreover, in the AD process, H₂S is formed during the dissimilatory reduction of sulfate by sulfate reducing bacteria (SRB), and this is the main odor source in AD systems. Since active compounds such as Fe²⁺ and Fe³⁺ can convert H₂S to precipitates such as FeS, FeS₂, and S, the addition of Fe/Fe₃O₄ nanomaterials may be able to greatly reduce the odors associated with the AD process.¹³² Meanwhile, Fe²⁺/Fe³⁺ can also remove the phosphorus that is formed during the degradation of organic matter, as it will form precipitates such as Fe³⁺-hydroxy-P and Fe₃(PO₄)₂.¹³¹ All of the potential behaviors of Fe/Fe₃O₄ nanomaterials in sludge during the AD process are shown clearly in Fig. 6. Because of the above mentioned potential advantages, use of Fe/Fe₃O₄ nanomaterials should be considered in the design of an economically and environmentally sustainable method for improving biogas production.

Fig. 6 All of the behaviors of Fe/Fe₃O₄ nanomaterials during the sludge anaerobic digestion process (ROS: reactive oxygen species, SRB: sulfate reducing bacteria, Fe/Fe₃O₄ NPs: Fe/Fe₃O₄ nano-particles).

4 Removal of dissolved methane

Dissolved methane in anaerobic digestion effluents has great potential to cause environmental harm because it is 25-fold more effective in trapping heat than CO₂ in the atmosphere.¹³³ Therefore, dissolved methane must be removed before the anaerobic digestate is discharged. Some alternatives such as biogas micro-aeration,¹³⁴ de-gassing membranes,^135,136 and increased liquid turbulence¹³⁷ technologies have been proposed for the purpose of removing dissolved methane from effluents. However, none of these technologies have yet been proven to be readily available and economical. Recently, some researchers studied the removal of dissolved methane from anaerobic digestate by coupling the technique with anaerobic methane oxidation (AMO), and the results showed great potential for application of the technology as an AD post-treatment process.

4.1 AMO

Anaerobic methane oxidation, which was first discovered in 1976, efficiently controls atmospheric CH₄ efflux by consuming more than an estimated 90% of all the CH₄ generated on Earth;^138–140 this process and the responsible microorganisms, which are anaerobic methanotrophic archaea (ANME), were identified after about 20 years of research.¹⁴¹ The ANME consist of three distinct clusters with subgroups including ANME-1 (two subgroups: ANME-1a and ANME-1b), ANME-2 (four distinct subgroups: ANME-2a, ANME-2b, ANME-2c, and ANME-2d), and ANME-3.^142–146 So far, depending on the differences in the electron acceptors, AMO models can be categorized into the following four different types: cooperative/un-cooperative SO₄²⁻ dependent anaerobic methane oxidation (CS/US-DAMO) (Fig. 7(I) and (II)), NO₂⁻/NO₃⁻ dependent anaerobic methane oxidation (Nx-DAMO) (Fig. 7(III)), metal ion (Fe³⁺ and Mn⁴⁺) dependent anaerobic methane oxidation (MIs-DAMO) (Fig. 7(IV)), and organic (humic acids) dependent anaerobic methane oxidation (O-DAMO) (Fig. 7(V)). The ANME-1, ANME-2, and ANME-3 archaea can all work with SRB to catalyze CS-DAMO (Fig. 7(I)); the SRB involved are Desulfosarcina, Desulfococcus, and Desulfobulbus.¹⁴⁷ The partner bacteria Desulfosarcina and Desulfococcus often cooperate with ANME-1 and ANME-2 to oxidize CH₄, while Desulfobulbus works with ANME-3 (eqn (7)).^148,149 Furthermore, some studies show that ANME-2 and ANME-3 can also work without SRB present (Fig. 7(II))^150,151 as follows (eqn (7) and (8)):

CH_4(aq) + SO₄²⁻ → HCO₃⁻ + HS⁻ + H₂O ΔG = −34 kJ mol⁻¹ CH₄ (7)

7CH_4(aq) + 8SO₄²⁻ + 5H⁺ → 7HCO₃⁻ + 4H₂S⁻ + 11H₂O ΔG = −26.5 kJ mol⁻¹ CH₄ (8)

Fig. 7 Modes of microbial anaerobic methane oxidation (AMO) depending on the different electron acceptors: (I) and (II) cooperative/un-cooperative SO₄²⁻ dependent anaerobic methane oxidation (CS/US-DAMO), (III) NO₂⁻/NO₃⁻ dependent anaerobic methane oxidation (Nx-DAMO), (IV) metal ions (such as Fe³⁺ and Mn⁴⁺) dependent anaerobic methane oxidation (MIs-DAMO), and (V) organics dependent anaerobic methane oxidation (O-DAMO). ANME: anaerobic methanotrophic archaea (including three distinct clusters, i.e., ANME-1, ANME-2, and ANME-3), ANME-2d: a subgroup of ANME-2, SRB: sulfate reducing bacteria as partner bacteria for ANME, HAs: humic acids.

In the Nx-DAMO model, besides archaea (such as ANME-2d using NO₃⁻)¹⁵² (eqn (9)), the NC10 bacteria may be also capable of independent methane oxidation by using NO₂⁻ (eqn (10)); the relevant reactions are as follows:¹⁵³

CH_4(aq) + 4NO₃⁻ → CO₂ + 4NO₂⁻ + 2H₂O ΔG = −503 kJ mol⁻¹ CH₄ (9)

3CH_4(aq) + 8NO₂⁻ → 3CO₂ + 4N₂ + 10H₂O ΔG = −928 kJ mol⁻¹ CH₄ (10)

In 2009, Beal et al.¹⁵⁴ reported that some metal ions such as Fe³⁺ and Mn⁴⁺ can also be used as electron acceptors by ANME-1 and ANME-3 (eqn (11) and (12)); however, the partner bacteria remain unknown (Fig. 7(IV)):

3CH_4(aq) + 8Fe(OH)₃ + 15H⁺ → HCO₃⁻ + 8Fe²⁺ + 21H₂O ΔG = −270.3 kJ mol⁻¹ CH (11)

3CH_4(aq) + 4MnO₂ + 7H⁺ → HCO₃⁻ + 4Mn²⁺ + 5H₂O ΔG = −556 kJ mol⁻¹ CH₄ (12)

In addition to the use of the above inorganics as electron acceptors, Gupta et al.¹⁵⁵ found that some organics (such as humic acids) can be used as electron acceptors. Unfortunately, the details of this mechanism require further study (i.e., to confirm the involvement of ANME) (Fig. 7(V)).

4.2 Coupled AMO

The research related to AD post-treatment technology coupled to AMO is just at the starting stage, but it has a bright future for application in systems that can remove dissolved methane because of its environmentally friendly and cost-saving advantages. In addition to dissolved methane, high NH₄⁺ concentrations also are contained within the anaerobic digestion effluents. Recently, some studies have shown that Nx-DAMO represents a promising alternative that is potentially capable of achieving the simultaneous removal of dissolved methane and NH₄⁺ efficiently. In 2011, Zhu et al.¹⁵⁶ was the first to achieve a co-culture of (i) anaerobic ammonium oxidation microorganisms (anammox) that could directly convert NO₂⁻ and NH₄⁺ into N₂ in an oxygen-free environment and (ii) ANME that could simultaneously remove dissolved methane and NH₄⁺ in synthetic wastewater containing CH₄, NH₄⁺, and NO₂⁻. The consumption rate of CH₄ was 0.7 mmol L⁻¹ d⁻¹ and the enrichment of methane-oxidizing microorganisms was done with NC10 phylum bacteria. Subsequently, Chen et al.¹⁵⁷ reported that ANME-2d, anammox, and NC10 phylum bacteria could jointly remove dissolved methane. The ANME-2d converted CH₄ into CO₂ by using fed in and/or generated NO₃⁻ by anammox bacteria, and DAMO bacteria also oxidized CH₄ to CO₂ by using the NO₂⁻ produced by anammox bacteria. In order to provide electron acceptors (NO₂⁻/NO₃⁻) for Nx-DAMO, and at the same time limit NO₂⁻ concentrations because high NO₂⁻ concentrations can be toxic or inhibitory to ANME,¹⁵⁸ ammonia-oxidizing bacteria (AOB) were cultured with ANME and anammox species in the anaerobic digestion effluents. Chen et al.¹⁵⁹ simultaneously removed dissolved methane and ammonium from the anaerobic digestion effluents by integrating Nx-DAMO and partial nitritation–anammox processes in a single-stage membrane biofilm reactor (Fig. 8). First, AOB provided NO₂⁻ for anammox by oxidizing NH₄⁺ from bulk liquid and by using O₂ from gas-permeable membranes. Then, ANME-2d converted the NO₃⁻ generated by anammox to NO₂⁻, which was provided to anammox in return or to NC10 phylum bacteria that used CH₄ from bulk liquid as the electron donor; NC10 phylum bacteria converted CH₄ and NO₃⁻ into CO₂ and N₂, respectively. Finally, dissolved methane and NH₄⁺ were simultaneously removed from the system in the form of CO₂ and N₂, respectively. The factors such as oxygen surface loading (L_O2) and HRT that could affect the dissolved methane removal efficiency were studied. The optimal dissolved removal efficiency was over 90% with a HRT > 5.75 days. The suitable L_O2 range for >90% dissolved methane removal efficiency increased with an increase in the HRT.

Fig. 8 Mechanisms present in co-cultures of anaerobic methanotrophic archaea (ANME), anaerobic ammonium oxidation (anammox) bacteria, and ammonia-oxidizing bacteria (AOB). GPMs: gas-permeable membranes, BL: bulk liquid.

Dissolved methane removal by the coupled AMO process in anaerobic digestion effluents has not been studied very much. Therefore, in addition to coupled processes involving Nx-DAMO, other models such as CS/US-DAMO, MIs-DAMO, and O-DAMO should be studied in relation to dissolved methane removal efficiencies in future work.

5 Conclusions and outlook

In this review, in order to sufficiently extract energy from biomass in wastewater treatment plants, microbial dynamics and scientific models involved in the AD process were first presented, which could contribute to future advances in AD systems and optimized performance of the design and operation parameters.

The sludge AD process involves three co-dependent biological stages, and sludge hydrolysis is the limiting step for biogas production. Sludge focused-pulsed pre-treatment technology had been applied in full-scale WWTPs, and this technology can conveniently and effectively breakdown sludge flocs and cells in a way that accelerates sludge hydrolysis. However, treatment chambers used in focused-pulsed technology are easily corroded; thus, they should be designed well with proper materials.

Bioelectrochemical systems such as MECs and Fe/Fe₃O₄ nanomaterial methods have become hot research topics in recent years. While these two methods have only been studied at the bench-scale, they have shown great potential for future applications because of their various advantages. In general, MECs and Fe/Fe₃O₄ nanomaterial methods cannot only improve the AD process, but they can also increase the methane content of the biogas. In the future, a more detailed examination into MECs and Fe/Fe₃O₄ nanomaterial methods for their application potential is urgently needed.

To improve MECs technology for biogas production, the effects of the design parameters such as the electrode size and electrode arrangement, the operational parameters such as temperature, organic loading rate, and pH, and toxicants including organic toxicants and inorganic toxicants on the efficiency of AD-MECs and the associated mechanisms should be further investigated in future studies.

Fe/Fe₃O₄ nanomaterial methods may be capable of simultaneously accelerating sludge hydrolysis, improving biogas production, increasing the methane content, producing more stabilized sludge, and reducing odors during the AD process. However, the dose of Fe/Fe₃O₄ nanomaterials used is an important factor to consider for applications in the sludge AD process. If the dose of Fe/Fe₃O₄ nanomaterials is too large, this could greatly inhibit the AD process.^160,161 Therefore, the optimal dose of Fe/Fe₃O₄ nanomaterials and its effects should be further investigated.

When energy is extracted from biomass in wastewater treatment plants, dissolved methane in the anaerobic digestion effluents can be harmful to the environment if it is released to the atmosphere; therefore, technologies that can effectively remove methane by coupled AMO processes are urgently needed. This area of study is just in the starting stage, but it has a bright future for application because of its environmentally friendly and cost-saving advantages. In addition to coupled processes with Nx-DAMO, other models such as CS/US-DAMO, MIs-DAMO, and O-DAMO should also be studied for dissolved methane removal in future work.

Acknowledgements

This work was supported by the Fundamental Research Funds for the Central Universities, and the Research Funds of Renmin University of China (16XNH040).

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