Sludge reduction based on microbial metabolism for sustainable wastewater treatment

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Review

Sludge reduction based on microbial metabolism for sustainable wastewater treatment ⁎

Jin-Song Guo, Fang Fang, Peng Yan , You-Peng Chen Key Laboratory of the Three Gorges Reservoir Region’s Eco-Environment, Ministry of Education, Chongqing University, Chongqing 400045, China

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO

A B S T R A C T

Keywords: Sludge reduction Microbial metabolism Substrate allocation Energy conversion Thermodynamics Stoichiometry

Sludge reduction via microbial metabolism does not require extra energy and resource inputs and thus merits attention as an alternative approach for sustainable wastewater treatment. This review presents a summary and analysis of the existing literatures on sludge reduction based on microbial metabolism, as well as interprets these sludge reduction mechanisms using bacterial thermodynamics and stoichiometry. Future efforts should be directed toward using advanced analytical techniques to further reveal sludge reduction mechanisms. The feasibility of coupling sludge reduction and nutrient removal by microorganism metabolism needs to be further evaluated to minimize the effect of sludge reduction on nutrient removal. A comprehensive life cycle assessment of sludge reduction strategies is recommended to effectively confirm their sustainability. Full-scale research is needed to verify the results obtained from bench- and pilot-scale experiments. This review presents the future opportunities and challenges for sludge reduction based on microbial metabolism in the excess sludge disposal.

1. Introduction In the conventional activated sludge process (CAS), microbes degrade pollutants to sustain growth and propagate, which inevitably generates large amounts of excess sludge as a byproduct (Li et al., 2015). The treatment and disposal of excess sludge has become a difficult problem due to its high proportion of volatile solids (VS), large amounts of water, and harmful elements such as heavy metals,

⁎

pathogens, and persistent organic pollutants (Cieślik et al., 2015; Yan et al., 2013b). More importantly, treatment and disposal of excess sludge requires significant amounts of energy and chemical agents, which result in significant increases in the carbon footprint and resource consumption of the wastewater treatment process (Wang et al., 2015). Thus, the disposal of excess sludge has become an obstacle to the activated sludge process as a green and sustainable wastewater treatment technology (Foladori et al., 2010).

Corresponding author. E-mail address: [email protected] (P. Yan).

https://doi.org/10.1016/j.biortech.2019.122506 Received 30 September 2019; Received in revised form 21 November 2019; Accepted 25 November 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Jin-Song Guo, et al., Bioresource Technology, https://doi.org/10.1016/j.biortech.2019.122506

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Sludge reduction technologies developed to decrease sludge production within wastewater treatment plants (WWTPs) have attracted considerable attention in recent years. However, most sludge minimization technologies, including mechanical methods (freezing or mill), chemical methods (alkaline hydrolysis or ozonation), thermal methods (microwave irradiation or pyrolysis), and biological methods (enzymolysis) require consumption of extra energy and chemical reagents (Wei et al., 2003; Yan et al., 2013a; Yan et al., 2015). Therefore, these technologies have been widely questioned as environmentally sustainable wastewater treatment strategies. Sludge is a carrier of energy and nutrients, and future-oriented wastewater treatment strategies are focused on the recovery of biomass energy and nutrients from sludge (Logan and Rabaey, 2012). Greater sludge production has the obvious benefit of promoting energy self-sufficiency during wastewater treatment. Therefore, there is a prominent contradiction between the goals of biomass energy recovery and sludge minimization during wastewater treatment. Clearly this contradiction does not mean that sludge reduction should be abandoned. Although energy recovery from sludge has received increased attention and has been achieved in several practical projects, it cannot be used as an omnipotent strategy for all WWTP scenarios. Some adverse conditions, including lower sludge organic matter content, cold climate, and smaller treatment scale, may lead to situations where the energy recovered from sludge is less than the energy consumed by the energy-yielding process (Gu et al., 2017; Yan et al., 2017). In addition, CapEx assessed the energy recovery system to be very expensive (Vishwanathan et al., 2018). Therefore, technical and economic applicability will affect a stakeholder’s decision of whether to adopt the strategy. In order to adapt to different complex situations in wastewater treatment (climatic conditions, scale, wastewater characteristics, etc.), sludge treatment and disposal should be diversified and multi-selective. Therefore, sludge reduction can continue to be used as an alternative plan to dispose of excess sludge, but the sludge reduction strategy must be green to achieve sustainable wastewater treatment. The development of sustainable sludge reduction strategy needs back to the essence of sludge production-microbial metabolism. Future efforts and technology development are aimed at reducing cell synthesis within the microbial metabolism through environmentally friendly and green measures. Currently, there are some sludge reduction technologies based on microbial metabolism that do not require extra energy and resource inputs, such as electron acceptor, endogenous metabolism, maintenance metabolism, and biological predation approaches (Khursheed and Kazmi, 2011; Semblante et al., 2017). There are numerous reviews on sludge reduction and articles that present different perspectives, but the field lacks a comprehensive review of sludge reduction based on natural microbial metabolism; therefore, special emphasis is given here to highlight the efforts from the past few years. This review aims to critically summarize recent developments in sludge reduction based on microbial metabolism (Fig. 1, sludge reduction technologies that require input of energy and resources are not included in the scope of this review) and to interpret these sludge reduction mechanism based on the bacterial thermodynamics and stoichiometry. We provide a critical analysis of the existing literature, identify gaps in knowledge, and highlight areas for future research in order to promote the application and extension of sustainable sludge reduction technologies and address the social and environmental challenges facing current sludge disposal methods.

Fig. 1. Logical tree of the comprehensive review of sludge reduction based on microbial metabolism.

for understanding the dynamics of biomass production in microbial metabolism (Supplementary information) (Rittmann and McCarty, 2012). These principles have been used to develop steady-state and dynamic models for microbial growth (Supplementary information). The stoichiometry of sludge growth regarding CAS process with different metabolism are described in Supplementary information. In a CAS system, microorganisms completely oxidize and decompose a portion of substrates as electron donors through a series of biochemical reactions with a terminal electron acceptor (catabolism) (Fig. 2A). Cell synthesis depends on substrate allocation and energy conversion during this microbial metabolic process (Henze et al., 2008). Therefore, biomass can be reduced by modulating the substrate allocation and energy conversion in microbial metabolism. More substrates are decomposed instead of used for cell synthesis, which increases the amount of energy that dissipates as heat and decreases cell synthesis. Regulation of the factors that influence substrate allocation and energy conversion represent potential mechanisms to reduce biomass production in microbial metabolism. The bacterial yield is closely related to the fs (the observed fraction of electron-donor electrons utilized for cell synthesis, Supplementary information). Furthermore, fs is the only influenced parameter in specific functional metabolic processes (aerobic decomposition, nitrification, and denitrification), as shown in Supplementary information. The fs is closely related to cell decay, endogenous respiration, and energy generation and transfer, as described in Supplementary information. Simultaneously, free energy associated with electrons and protons is released and conserved by the microorganism in the form of ATP in the electron transport pathway (Mitchell, 1961). Therefore, regulating bacterial behaviors involved in cell decay, endogenous respiration, energy generation and transfer, and electron transport to reduce cell synthesis is a widely used strategy to decrease sludge production during

2. Sludge reduction mechanism based on bacterial metabolism Thermodynamic and stoichiometry are useful for evaluating bacterial yield and microbial growth characteristics (Von Stockar et al., 2006). McCarty established a thermodynamic electronic equivalent model to evaluate bacterial yield during the growth process (Supplementary information) (McCarty, 2007). The elemental stoichiometry and substrate allocation of microorganisms provide a basis 2

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Fig. 2. Diagram of microorganism metabolism in wastewater treatment system (A) and the proposed sludge reduction strategies based on bacterial metabolism (B).

energy metabolism. Therefore, sludge minimization is successfully achieved based on ATP yield in electron transport pathways with different terminal electron acceptors. The abovementioned sludge reduction strategies can be divided into three major categories based on their mechanism: thermodynamics, substance metabolism, and electron transport. The following review of sludge reduction strategies based on the microbial metabolism focuses on these three perspectives.

wastewater treatment (Fig. 2B). In addition, biomass reduction based on energy loss during trophic transfer in the microbial food web has also attracted considerable attention for wastewater treatment (Elissen et al., 2006). The abovementioned sludge reduction strategies regulate bacterial metabolism to achieve biomass reduction without input of extra energy and resources. For example, enhanced endogenous metabolism and cell decay can reduce substrate availability for cell synthesis to decrease cell generation; in addition, the lysis-cryptic growth can drive repeated metabolism of the cell matrix to generate more organic carbon for conversion into inorganic carbon instead of cell components. Energy uncoupling and spilling as well as biological predation aggravate energy dissipation in microbial individuals and the food network, respectively, and as a result, decrease the energy used by cell synthesis. Similarly, the same phenomenon can be achieved by enhancing the maintenance energy in the cell. Synthesis of bacterial cells depends on the energy yield in metabolism. The electron transport pathway displays significant differences between different electron acceptors, which leads to differences in energy transfer efficiency in

3. Sludge reduction based on thermodynamics 3.1. Maintenance metabolism 3.1.1. Thermodynamics of maintenance metabolism A thermodynamic model has been developed to describe the maintenance energy of microorganisms (Tijhuis et al., 1993). The maintenance energy mE (kJ/C-mol biomass·h) under aerobic and anaerobic conditions can be calculated by Eqs. (1) and (2), respectively:

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Aerobic: mE = 5.7exp ⎧ ⎨ ⎩

−6.94 × 10 4 1 1 ⎫ ( − ) R T 298 ⎬ ⎭

Anaerobic: mE = 3.3exp ⎧ ⎨ ⎩

−6.94 × 10 4 1 1 ⎫ ( − ) R T 298 ⎬ ⎭

(2004) found that under the most economical MBR operational conditions of MBR, the cost decrease resulting from sludge reduction can offset the cost increase due to decreased oxygen transfer efficiency at high MLSS. When the coarse aeration flow is introduced into the submerged MBR, the effect of filtration on the total energy consumption can be reduced (Fenu et al., 2010). Regrettably, there is little information regarding the energy consumption and cost analysis of MBRs in the scientific literature. The sludge treatment and the aeration cost are two important economic factors that influence the economic efficiency of MBRs. Of course, MBR with complete sludge retention and zero sludge production is not always feasible in practice, and there must be a minimum waste rate of excess sludge in order to maintain an optimal sludge concentration range in the MBR. An appropriate SRT range should be adopted in MBRs to simultaneously achieve effective sludge reduction and excellent filtration performance. More studies on cost benefit analysis should been conducted to determine the proper SRT range in MBR applications.

(1)

(2)

where R is 8.314 J/(mol·°C), and T is the thermodynamic temperature. The mE value of an anaerobic growth system is less than that of an aerobic growth system. The maintenance energies of bacteria in activated sludge are summarized in Supplementary information. In this model, temperature is a key factor that influences the bacterial maintenance energy, an assertion which has generated widespread controversy. Temperature is not a process parameter in wastewater treatment plants, which impedes application of the model to sludge reduction based on maintenance metabolism in an activated sludge system. A long sludge retention time (SRT) or a low food-to-microorganism (F/M) ratio results in increased demand for maintenance energy in bacterial metabolism, which leads to reduced biomass production (van Loosdrecht and Henze, 1999). The relationship between substrate utilization for maintenance and biomass production was established by Low and Chase (1999a).

−rSG =

−1 rX = −rS + qm X YG

3.1.1.2. Sludge reduction by enhancing maintenance metabolism in bioreactors with immobilized biomass. Moving bed biofilm reactors (MBBRs) were developed based on biofilm technology. In MBBRs, biomass grows as biofilms on small carrier elements that move freely in the reactor, allowing for higher biomass concentrations and SRT (Revilla et al., 2016a). As a result, MBBRs usually achieve lower sludge production when compared with the conventional activated sludge process (Kawan et al., 2016). The average sludge yield was 0.0538 gVSS/gCOD in an anaerobic-aerobic MBBR system (Chen et al., 2008). In an MBBR with an aerobic-anaerobic coupled process, the sludge yield was reduced to 20.2% of that of the conventional activated sludge process (Li et al., 2014). The average efficiencies of COD, NH4+N, and TN removal reached 98.6%, 99.4%, and 72.8%, respectively, in the MBBR process. In a continuous MBBR with an aerobic-anaerobic coupled process, the sludge production rate was 0.17 g MLSS/g COD, which was only 34.5% of the rate of the conventional activated sludge process (Wang et al., 2012). Aerobic granules are considered a special form of biofilm-producing self-immobilized cells (Wagner et al., 2015). Application of aerobic granular sludge is regarded as a promising biotechnology in wastewater treatment (Sarma et al., 2017) and has the advantage of reducing sludge production. The sludge production in a sequencing batch aerobic granular sludge system was only 17.8% of that in a conventional activated sludge plant with a capacity of 8 million m3/year (Lotito et al., 2014). Low sludge production (0.1 g VSS/g COD) was obtained using the sequencing batch biofilter granular reactor (SBBGR), which achieved good treatment efficiencies in treatment of real wastewater from dyeing textiles (Lotito et al., 2012). Net sludge production was almost zero in a full-scale sewage treatment plant based on granular sludge sequencing batch reactors (De Bruin et al., 2004). Although excellent sludge reduction potential can be obtained by using an aerobic granular process, the mechanisms responsible for sludge reduction in this process require more in-depth investigation.

(3)

where rs and rx are the substrate uptake rate and biomass production per unit volume, respectively. YG and qm are true yield and maintenance requirement, respectively. X is biomass concentration. −rSG is the substrate uptake rate specifically for cell synthesis per unit volume. Low and Chase’s model has solved this problem by introducing the biomass concentration (X), which provides a more appropriate description of the activated sludge process with partial biomass recycling. The biomass concentration is an accessible control parameter, as it is a function of the sludge return rate in the activated sludge system. 3.1.1.1. Sludge reduction by enhancing maintenance metabolism in a membrane bioreactor. High sludge concentrations tend to result in low conversion yield (Eq. (3)), as less energy is available for cell synthesis. Sludge concentration can be significantly increased by using a membrane filtration device instead of a settling tank in CAS processes. Therefore, effective sludge reduction has been achieved in membrane bioreactors (MBRs) with a high mixed liquid suspended solids (MLSS) concentration and a long SRT based on the bacterial maintenance metabolism (Kim et al., 2010). Sludge reduction potentials in MBRs based on microbial maintenance metabolism are summarized in Supplementary information. Previous studies indicate that the sludge yield is negatively related to the SRT used in MBRs. The SRT was longer than 300 d, which led to a 90% reduction in the sludge yield. Ultimately, sludge production was completely inhibited when SRT was increased to 1000 d in the MBR (Yoon et al., 2004). The net sludge yield in an MBR with complete sludge retention reached zero after approximately 180 days of reactor operation (Pollice et al., 2008). Three submerged MBRs with prolonged SRT values achieved minimal sludge and excellent effluent quality (Teck et al., 2009). In three MBRs, TN and COD were reduced by more than 97% and 98%, respectively. Teck et al. (2009) suggested that most of the energy from the substrate was utilized for endogenous respiration, and net growth was limited as time progressed. The sludge concentration had been stabilized at a level at which the energy supply is mainly used for maintenance. Maintenance of a higher MLSS at a high or prolonged SRT was not an economic operation strategy in the MBRs due to the energy demand (Judd, 2007). Due to the energy consumption from sludge mixing in the MBR, the increase in MLSS from 3 to 30 g TSS/L resulted in energy requirements increasing by 25%-30% (Pollice et al., 2007). Although a high SRT led to an increased energy demand, it reduced sludge treatment and disposal costs when compared with CAS processes. Yoon et al.

3.2. Energy dissipation in the food chain 3.2.1. Sludge reduction by predation The biota present in activated sludge consist of bacteria, archaea, protozoa, and metazoa as well as viruses (Rittmann and McCarty, 2012). Of these, bacteria represent the main components of the activated sludge community. These bacteria mainly include nitrifiers [ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)], denitrifiers, and phosphorus-accumulating organisms (PAOs), all of which play important roles in nutrient removal (Rittmann and McCarty, 2012). Filamentous bacteria function as a backbone in the structure of the floc (Andreadakis, 1993). Through predation, protozoan and metazoan species participate in balancing the ecosystem in activated sludge systems; these species are quite sensitive to physical, 4

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are balanced between biological nutrient removal and sludge reduction by predation is essential to achieving simultaneous nutrient removal and sludge reduction in a biological wastewater treatment system. The selected conditions should not only be suitable for functional bacteria, but also can not affect the stability of the sludge predation system. Hence, despite the higher sludge reduction and nutrient removal efficiencies achieved in several previous studies, the optimal technological conditions for a biological wastewater treatment system with sludge predation require further investigation. Advances in mathematical modeling of biological wastewater treatment will provide a reliable method for uncovering these optimal technological conditions to balance biological nutrient removal and predation by protozoans and metazoans in the biological wastewater treatment with predation on bacteria (Revilla et al., 2016b).

chemical, and operational processes (Ginoris et al., 2007). Substances and energy are transferred from substrates to bacteria and then to protozoans or metazoans through the food chain in the wastewater treatment system (Elissen et al., 2006). Energy is lost due to inefficient biomass conversion during energy transfer from low to high trophic levels (Rittmann and McCarty, 2012). Under the optimal conditions, the total energy loss will be maximal and the total biomass production will thus be minimal. Therefore, the predation of microfauna on bacteria can reduce sludge production. Although the density of protozoans is high, each protozoa is tiny, and most are filter feeders that consume only dispersed bacteria. Therefore, protozoans do not represent an effective method for sludge reduction. In addition, the application and distribution of predators in biological wastewater treatment system are uncontrollable. This problem was addressed by the development of a two-stage sludge predation system. The first stage in the predation system is defined as the bacterial stage and uses a completely mixed reactor without sludge retention to promote growth of the dispersed bacteria; the second stage, described as the predator stage, uses an activated sludge system with a long SRT for the growth of protozoans and metazoans (Ratsak et al., 1994). However, there remains significant debate regarding the impacts of predation on effluent quality in the two-stage system. The sludge production of the two-stage submerged MBR system was 20–30% less than that of the two-stage CAS system with a similar SRT and F/M ratio; however, increased grazing on nitrifiers decreased the nitrification performance, which then deteriorated the effluent quality (Ghyoot and Verstraete, 2000). Wang et al. (2011) reported that predation in an MBR had negative effects on TP removal efficiency. The potential of worm-induced sludge reduction was investigated using an integrated oxidation ditch cycle (IODVC) reactor (Wei et al., 2009). The average sludge yield reached 0.33 kg SS/kg COD in the IODVC. However, the presence of the worms resulted in release of phosphorus into the effluent, which may hinder further full-scale application of this approach. Improved and novel processes have been developed for sludge reduction by predation to achieve nutrient removal simultaneous with sludge reduction. Effective nitrification and denitrification were obtained in a static sequencing batch worm reactor (SSBWR), which also simultaneously achieved nutrient removal and sludge reduction (Tian and Lu, 2010). The soluble COD released through sludge predation as a carbon source was used for denitrification, which led to reduction of carbon emissions as well as cost savings (Tian and Lu, 2010). In a fullscale wastewater treatment plant, the presence of Tubificidae resulted in a 75% reduction of sludge compared to the control system and improved the removal of COD and SS by 8.7% and 13.6%, respectively (Lou et al., 2011). An A2O-MBR-worm reactor (WR)-phosphorus removal reactor (PRR) combined system simultaneously achieved excellent effluent quality and low sludge production. The sludge reduction rate reached 49.8% compared with the conventional A2O-MBR, and the TN and TP removal rates increased by 11.6% and 100.8%, respectively, in the wastewater treatment process (Li et al., 2017). Although nutrient removal and sludge reduction can be simultaneously achieved through these novel predation processes, considerable difficulties remain in maintaining a stable bio-predation system, due to unstable protozoan and metazoan growth and high variation in worm density. Environmental factors (pH, temperature, oxygen, etc.), biological factors (life cycle and population dynamics, bacteria preference, etc.) and process parameters (HRT, SRT, electron acceptor, organic loading rate, etc.) can markedly influence the predatory activity of protozoans and metazoans (Ratsak and Verkuijlen, 2006). Therefore, a better understanding of the effects of environmental factors, biological factors, and process parameters on protozoans and metazoans will facilitate stability of the sludge reduction system. Similarly, biological nutrient removal depends on the natural metabolism of functional bacteria in the CAS system. Efficient and stable nutrient removal is required to maintain an appropriate living environment for functional bacteria. Therefore, ensuring that the optimal technological conditions

3.3. Sludge reduction by energy uncoupling and spilling Uncoupling short-circuits the proton motive force (PMF) to inhibit ATP formation with simultaneous substrate oxidation, indicating part of the energy is consumed by non-growth–associated reactions. Therefore, a reduction in microbial synthesis is induced when the microorganism is maintained under energy-uncoupling metabolic conditions. Low et al. (2000) reported that futile energy cycles were driven using energy generated by respiration, but the substrate removal rate was not significantly affected. Numerous energy-uncoupling conditions were observed, including abnormal temperatures, nutrient limitation, heavy metals, excess energy sources, inhibitory compounds, and exposure of sludge to cyclic changes in ATP content (Liu and Tay, 2001). However, in this review, we focus only on energy uncoupling conditions without input of extra energy or chemicals. 3.3.1. Oxic-settling-anaerobic process The oxic-settling-anaerobic (OSA) process introduced an anaerobic sludge-holding tank in the sludge return line of the CAS system, which created fasting/feasting conditions to eventually achieve a significant biomass reduction (Westgarth et al., 1964). Currently, Novel OSA and similar processes were developed to achieve excellent nutrient removal, easy improvement, flexible operation, cost savings, and environmental friendliness. These processes are already considered promising and attractive processes for future research. In future investigations, OSA processes with enhanced biological phosphorus removal (EBPR) will be developed to satisfy the more stringent effluent standards. A + OSA system was established to reduce sludge and remove nitrogen (Zhou et al., 2015). In this process, the OAS system is modified by placing an anoxic tank prior to the oxic tank, which achieved excellent nitrogen removal due to the enhanced denitrification caused by the release of soluble organic matter as carbon source in the sludge holding tank. As an improved OSA system, the stage-aerated anaerobic, anoxic, microaerobic, and aerobic system combined with a micro-aerobic starvation pool (A2MO-M) process achieved 16.3% less sludge, and 87.3% and 91.9% TN and TP removal efficiencies, respectively (Yang et al., 2016). Energy uncoupling and sludge decay contributed 16.7% and 21.2% to sludge reduction, respectively, in the A2MO-M system. In addition, lysis-cryptic growth and enrichment of slow-growing bacteria also influenced sludge reduction in the process. A micro-aerobic tank and a settler positioned prior to the CAS process achieved excellent nutrient removal and sludge reduction. The sludge reduction rate increased from 42.9% to 68.3%, while the DO decreased from 2.5 to 0.5 mg/L (Niu et al., 2016). In the OSA process, remarkable variation in sludge reduction performance was observed with changes in feed, ORP, sludge return ratio, and SRT. Therefore, it is difficult to optimize these factors to further reduce sludge production, which limits widespread use of the process. A better understanding of sludge reduction mechanisms in the OSA process is required. The current findings indicate that in the OSA process, various mechanisms occur simultaneously to lead to sludge reduction. 5

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conversion of solids into liquid and gas. This study established a method to determine the sludge reduction rate caused by D. magna, A. hemprichi, P. acuta, and T. tubifex based on the variation in carbon forms. Tian et al. (2012) reported that 8.0% of the influent COD was transformed into excess sludge, and 6.4% of the influent COD was discharged within the effluent. The sludge reduction was the primary driver of the significant decrease of the total COD discharge rate in an integrated system comprised of a membrane bioreactor and a worm reactor. Bacterial metabolism and the assimilation of worms were the primary factors influencing sludge consumption, contributing 36% to the total sludge reduction. Energy-saving operation and sludge minimization were achieved in an integrated mainstream anammox into an A-2B process treating municipal wastewater (Gu et al., 2018). At the Astage, 58% of influent COD was converted to methane gas, and 87% of the total inorganic nitrogen (TIN) was removed. The total process obtained at least 75% sludge reduction because a large amount of the influent COD was captured at the A-stage.

These include energy uncoupling, biomass decay, and EPS destruction (Semblante et al., 2014). Wang et al. (2008) suggested that in the OSA process, sludge reduction was caused by energy uncoupling metabolism, sludge decay, and anaerobic oxidation. Cell decay, uncoupling mechanisms, and anaerobic oxidation contribute to 66.7%, 7.5%, and 23% of sludge reduction, respectively. Therefore, the relationships between these specific mechanisms should be clarified to improve process performance. 3.3.2. Energy uncoupling and spilling by high S0/X0 The catabolic ATP synthesis rate is greater than the anabolic consumption rate under conditions where there is a high ratio of initial substrate concentration to the initial biomass concentration (S0/X0 as COD/biomass). The accumulation of ATP leads to energy uncoupling between catabolism and anabolism. A significant decrease in growth yield was observed in high S0/X0 ratio conditions in a substrate-sufficient batch culture (Chudoba et al., 1991). There are two possible mechanisms that may explain the sludge reduction induced by a high S0/X0 ratio (Low and Chase, 1999b). First, energy dissipation by leakage of ions (H+ or K+) through the cytoplasmic membrane weakens the potential across it and thus uncouples oxidative phosphorylation. Second, microorganisms upregulate a metabolic pathway that bypasses the energy-conserving steps of glycolysis. Biomass can be reduced due to energy-uncoupling metabolism when the S0/X0 ratio is ≥5 mg COD/mg MLSS under substrate-sufficient conditions (Chudoba et al., 1991). A kinetic model was proposed to quantify the dependence of the degree of energy uncoupling on the S0/X0 ratio for substrate-rich batch cultures in biomass production (Liu et al. 1998). The model indicated that a significant dissociation of catabolism from anabolism occurs when the S0/X0 ratio is > 5 mg COD/mg MLSS, respectively. Although sludge reduction can be achieved by increasing the food-tomicroorganism ratio (high S0/X0 ratio) in engineering, the actual S0/X0 values are usually 0.01–0.13 mg COD/mg MLSS in domestic WWTPs (Chudoba et al., 1992). This sludge reduction strategy may only be suitable for the biological treatment of high-strength organic wastewaters. There has been no further research progress on this sludge reduction strategy in the last few decades.

4.2. Endogenous metabolism Intracellular substances can be thoroughly metabolized to water and carbon dioxide via endogenous metabolic processes, thereby reducing biomass production (Foladori et al., 2010). Liu and Tay (2001) claimed that the balance between microbial growth and nutrient removal by endogenous metabolism has much practical significance in wastewater treatment processes. The total effect of endogenous metabolism can be combined and simplified into a single decay rate for more convenient and practical application to wastewater treatment. The decay rate is closely related to bacterial population, temperature, electron acceptors, and pH (Rittmann and McCarty, 2012). Lawrence and McCarty (1970) established the relationship among decay rate, observed sludge yield (Yobs), and SRT (θc) to intuitively describe the effect of endogenous metabolism on sludge yield in wastewater treatment processes, as shown in Eq. (5):

1 θK 1 = c d + Yobs Ymax Ymax

4. Sludge reduction based on carbon metabolism 4.1. Carbon balance in sludge reduction During wastewater treatment, the carbon balance in a conventional wastewater treatment system can be described by Eq. (4):

Ci = Cg + Ce + Cb

(5)

where Ymax and Kd are the true growth yield and specific endogenous rate, respectively. Based on the theory of endogenous metabolism, sludge can be minimized by controlling the decay rate and SRT. Excess sludge was decreased by 60% when the SRT increased from 2 to 18 days, which had no effect on COD removal in the reactor (Stall and Sherrard, 1976). Sludge reduction can be achieved by excess oxidation of cells in extended aeration or high dissolved oxygen conditions. The sludge yield was 0.19 mg VSS/mg COD in an SBR reactor with an extended aeration process (Goel and Noguera, 2006). External aeration achieved a volatile suspended solids reduction of 68%, due to the relatively higher hydrolysis rate for the accumulated particulate metabolic products (Özdemir et al., 2014). Sludge reduction reached 25% when the dissolved oxygen (DO) increased from 2 to 6 mg/L with a sludge loading of 1.7 mg BOD5/mg MLSS (Abbassi et al. 2000). The high DO enhanced the diffusion of oxygen inside the flocs, which led to hydrolysis of microorganisms and decreased biomass in the floc matrix. A high-oxygen process can easily be used for sludge reduction in practical engineering. Economic feasibility and energy consumption should be taken into consideration as important factors for the implementation of the highoxygen process.

(4)

where Ci and Ce represent the COD in the influent and effluent per day, respectively, in mg/day; Cg represents the carbon converted into carbon dioxide or methane per day, in mg/day; and Cb represents the carbon converted into biomass per day, in mg/day. The desirable levels of effluent organic matter meet the stringent effluent quality regulations, which is the primary objective of wastewater treatment systems. Therefore, biomass can only be reduced by enhancing the carbon converted into gas based on Eq. (4). Investigation of the fate and conversion of carbon in wastewater treatment systems with sludge reduction will help illuminate the sludge reduction mechanism. Carbon fate was investigated using a stable isotope of carbon in a laboratory-scale reactor with simultaneous nutrient removal and sludge reduction (Huang et al., 2014). Based on the mass balance of the spiked 13C, almost 41.5% of the total spiked 13C mass flowed to new solids in the control SBR; however, it was about 29.5% for the modified SBR. Approximately 56.5% and 74% of the total spiked 13C was embodied in CO2 gas for the control and modified SBRs, respectively. The abovementioned findings further support the viewpoint that the modified SBR achieved low solids yield. Liang et al. (2006) indicated that the sludge reduction rate was closely related to the rate of

4.3. Cryptic growth Biomass growth that utilizes autochthonous substrates is called cryptic growth (Mason and Hamer, 1987) and can lead to a reduction in total biomass yield. Although cryptic growth strategies have already been widely researched in association with sludge reduction, these processes have only been implemented in a few bench- or pilot-scale 6

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the Cannibal process, and that 10% is optimal interchange rate for destruction of solids. Goel and Noguera (2006) investigated the feasibility of simultaneous sludge reduction and enhanced biological phosphorus removal (EBPR) in the Cannibal process. Sludge production was reduced by 16–33% compared to a control system with a 10-day SRT. Greater than 98% phosphorus removal was achieved in the Cannibal-EBPR system. Activated sludge is composed of organic and inorganic matter (grit and other inert solids from the influent). Previous studies of the Cannibal process were not concerned that the contribution of inorganic matter removal by solids removal module to sludge reduction. Of course, in a wastewater treatment plant with real wastewater, inorganic matter can contribute to sludge yield. Currently, there is lack of a thorough assessment of the effect of physical pre-treatment on overall sludge reduction.

experiments. Application of the cryptic growth strategy has been impeded in large- and full-scale WWTPs due to consumption of additional energy and chemicals (ozonation, ultrasonication, mill, pyrolysis, and microwave) in most of these processes. Of course, this drawback impedes the environmental sustainability of a cryptic growth sludge reduction strategy. As a result, there is sufficient need to review to cryptic growth technologies that do not require any external energy and chemical inputs. 4.3.1. Anaerobic side-stream reactor The anaerobic side-stream reactor (ASSR) process has received extensive attention due to its excellent sludge reduction, nutrient removal and sludge settling property. The main difference between the ASSR and OSA configurations is the reduced space requirement in the mainstream due to absence of secondary settling and an alternate, rather than continuous, loading mode (Ferrentino et al., 2016a). There is debate regarding the mechanism of achieved sludge reduction when using activated sludge with an ASSR. Different mechanisms have been proposed to be occurring in the ASSR, including cryptic growth, metabolism of slow-growing microorganisms, uncoupling metabolism, and destruction of EPS (Ferrentino et al., 2016b). In particular, cryptic growth has received widespread attention. Previous studies have highlighted its contributions to sludge reduction in ASSR processes and thus are the main mechanisms discussed in this review. Activated sludge exists as flocs, which are aggregates of microorganisms. Flocs aggregate by secreting EPS, which include proteins, polysaccharides, lipids, DNA, and other organic macromolecules from bacteria that act as the substrates for intercellular adhesion and communication (Sheng et al., 2010). Novak et al. (2003) proposed an EPS destruction mechanism about sludge reduction. In aerobic conditions, calcium (Ca2+) and magnesium (Mg2+) in the flocs are released into solution, resulting in floc destruction and polysaccharide accumulation. In anaerobic conditions, considerable protein was released, but divalent cations were not. Protein release is attributed to the destruction of selective binding between protein and iron (Fe3+) due to reduction of Fe3+ to Fe2+ in anaerobic conditions. The released EPS (proteins and polysaccharides) are recycled back to the aerobic reactor during sludge cycling and are then degraded. Therefore, sludge reduction is achieved by anaerobically-driven degradation of EPS in the ASSR (Chon et al., 2011). The role of cell lysis-cryptic growth in ASSRs has shed new light on sludge minimization (Foladori et al., 2015). Significant cell lysis is not observed under anaerobiosis at ambient temperature. Bacterial decay and lysis occurred principally under aerobic conditions. Under anaerobic conditions, significant solubilization of COD and NH4+-N led to hydrolysis of organic matter instead of cell lysis. These findings support the model that two independent mechanisms contribute equally to sludge reduction: (1) sludge hydrolysis under anaerobic conditions, and (2) bacterial cell lysis and oxidation of released biodegradable compounds under aerobic conditions.

4.3.1.2. Other processes. Coma et al. (2013) developed a BIMINEX process (an anoxic side-stream reactor in the sludge line within a University of Cape Town [UCT] system) to reduce biomass production. The most obvious distinction between the BIMINEX process and the more common format of the ASSR process is a continuous loading of settled sludge into the SSR. Sludge yield was reduced by 18.3% in the BIMINEX pilot plant treating the total sludge return line. The effect of the side-stream ratio on sludge reduction was investigated in ASSRcoupled MBRs (Cheng et al., 2017). The results indicated that sludge reduction increased from 6.0% to 49.7% when the side-stream ratio (SR) increased from 0.2 to 1.0, and nitrogen removal was enhanced. A higher side-stream ratio enriched the slow-growing microbes, while a lower SR favored the growth of hydrolytic and predatory bacteria. In addition, the hydraulic retention time of ASSR was also considered an important parameter for sludge reduction in the ASSR-coupled MBRs (Cheng et al., 2017; Jiang et al., 2018a). In an ASSR-coupled MBRs with carriers, sludge reduction reached to 46.4%, because packing carriers greatly enhanced the hydrolysis of the sludge (Zheng et al., 2019). 4.3.1.3. Effects of the side-stream sludge line on the main-stream system. Although the ASSR process has displayed significant sludge reduction potential without deteriorated sludge settling and effluent quality (Novak et al., 2007; Chon et al., 2011), many controversies remain regarding the effects of the side-stream line on the main-stream performance, especially, effluent quality. Efficient and stable nutrient removal is required to maintain an appropriate living environment for functional bacteria. However, in existing anaerobic side-stream reactor technologies, sludge reduction is achieved based on biotic stress, cell lysis, and predation on bacteria rather than on natural microbial metabolic processes (Foladori et al., 2010). Therefore, the optimal technological conditions for biological nutrient removal and sludge reduction are different. Introduction of a side-stream sludge line into the activated sludge process changes the environment within the mainstream system so that it is less suitable for functional bacteria, which affects the performance and stability of the total process (Datta et al., 2009; Yan et al., 2015). In addition, inert solids may gradually accumulate in the system due to the continuous sludge recirculation between the main-stream and the side-stream reactors and the reduced discharge of excess sludge due to sludge reduction. Therefore, the potential effects of a side-stream sludge line on the main-stream system require further investigation.

4.3.1.1. Cannibal process. The Cannibal process is a typical anaerobic side-stream reactor and has been applied in practical engineering. It is based on ASSR but includes a physical treatment for return sludge. The sludge line of the Cannibal process is composed of a drum screen, a hydrocyclone, and an intermediate tank (Sheridan and Curtis, 2004). The hydrocyclone can remove grit and other inert solids from sludge. In a laboratory study, the sludge generated by the Cannibal process contained 60% less solids than that generated by the CAS system. Readily biodegradable organic matter was released from the recycled biomass in the intermediate tank, and the released organic matter then degraded rapidly when the Cannibal biomass was recirculated back to the aeration tank (Novak et al., 2007). Easwaran et al. (2009) investigated the effect of the HRT and interchange rate in the Cannibal intermediate tank on the solids crack in the Cannibal system and found that an HRT of 7 days is the minimum retention period for

5. Sludge reduction induced by electron transport 5.1. Sludge reduction by heterotrophic microorganisms 5.1.1. The electron transport mechanism in heterotrophic facultative microorganisms The electron transport process (ETP) for a typical heterotrophic organism that uses O2 and NO3− as its terminal electron acceptor is 7

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Fig. 3. The ETP for a typical heterotrophic organism. (A) O2 as electron acceptor, and (B) NOx− as electron acceptor.

Functional bacteria select the appropriate environment (aerobic, anoxic, and anaerobic) to satisfy the survival needs, which essentially is the choice of appropriate electron acceptors to themselves. Orhon et al. (1996) observed that the sludge yields under aerobic conditions were 0.79–0.85 g cell COD/g COD, while the sludge yield was 0.53 g cell COD/g COD under anoxic conditions. Sludge production was approximately 40% less in anoxic conditions than in aerobic conditions in a bench-scale activated sludge system (McClintock et al., 1988). Copp and Dold (1998) observed a significant difference in activated sludge yield under anoxic and aerobic conditions using a variety of soluble substrates based on 32 batch tests. The anoxic yield was 0.402 g particulate COD/g consumed COD, which was 62% of the corresponding aerobic yield of 0.645 g particulate COD/g consumed COD. Sperandio et al. (1999) claimed that anoxic biomass yields were lower than those obtained using aerobic conditions, and the observed ratios between them ranged from 0.66 to 0.85. Muller et al. (2004) reported that the anoxic yield of heterotrophic organism is approximately 81% of the aerobic yield value, and the anoxic and aerobic yields were 0.54 mg COD/mg COD and 0.67 mg COD/mg COD, respectively. The theoretical sludge yields using SO42−, NO3−, and O2 as electron acceptors were calculated as 0.04 g VSS/g COD, 0.17 g VSS/g COD, and 0.22 g VSS/g COD, respectively, and were based on a thermodynamic model (Yan et al., 2018). Yan et al. (2018) also evaluated the feasibility of simultaneous sludge reduction by different electron acceptor and

shown in Fig. 3A and B, respectively. Electron transport can be described in Supplementary information. The difference in the electron transport chains between aerobic and anoxic respiration becomes apparent only after the ubiquinone complex. There are three proton pumps in the ETC that use O2 as a terminal electron acceptor (NADH dehydrogenase, ubiquinone, and cytochrome aa3), but only two proton pumps that use NOx− as a terminal electron acceptor (NADH dehydrogenase and ubiquinone). When cytochrome aa3 is the terminal oxidase, electrons pass through three proton pumping sites to generate 3 molecules of ATP. Under anoxic conditions, electrons pass through only the first two of the three proton pumping sites, generating 2 mol of ATP. Thus, the energy transfer efficiency associated with electron flow to NOx− would be about 2/3 of that associated with electron flow to oxygen via cytochrome aa3 (Fig. 3B). Less energy (ATP) is captured when nitrogen oxides are used as electron acceptors instead of oxygen, and anoxic respiration is a less efficient mechanism of supporting microbial growth when compared to aerobic respiration (Yan et al., 2017). 5.1.2. Sludge reduction induced by different terminal electron acceptors O2, NO2−, NO3−, S2−, and SO42− are common electron acceptors to bacteria in activated sludge process. There are significant differences in energy release among different electron acceptors due to differences in oxidation–reduction potential, which provides a theoretical basis for sludge reduction induced by different terminal electron acceptors. 8

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nutrient removal (NOx− replaces O2 as the electron acceptor) in the activated sludge process with low dissolved oxygen (LDO). The Yobs values obtained in the LDO and control systems were 0.22 g VSS/g COD and 0.26 g VSS/g COD, respectively, during steady-state operation. The sludge reduction of the LDO system (DO 0.4 mg/L) reached 14.8% of that of the control system (DO 2.5 mg/L). The rates of TN, TP, and COD removal were 81.4%, 95.3%, and 93.8%, respectively, in the LDO process, with a DO of 0.4 mg/L. Replacing NOx− with O2 as the electron acceptor can stimulate catabolism, resulting in substrate decomposition instead of utilization for cell synthesis (Yan et al., 2018). Therefore, sludge reduction was induced by the change in electron acceptor. Currently, reports of sludge reduction rates using different electron acceptors are limited, and the potential for sludge reduction based on this strategy requires in-depth investigation.

alternate cycles process in sludge line (ACSL) process was applied to reduce sludge production in a municipal WWTP of 5000 m3/d, which obtained a low Yobs of 0.09 g VSS/g COD at an HRT of 10 days (Troiani et al., 2011). A full-scale modified sequencing bioreactor (20000 m3/d) with a micro-aerobic tank achieved efficient pollutant removal from industrial park wastewater and a low observed sludge yield of 0.074 g VSS/g COD using a process based on cryptic growth (Jiang et al., 2018b). An activated sludge process with a worm reactor (10000 m3/d) was operated over a period of 4 years at WWTP Wolvega. Secondary sludge degradation by the aquatic oligochaete worm Aulophorus furcatus led a sludge reduction of at least 65% on TSS basis (Tamis et al., 2011). As a landmark sludge reduction process, the Cannibal process has spread widely in the United States, and the process was selected as the best future sludge management alternative to the current cost-effective sludge disposal practice. An implementation of the Cannibal process was completed at Oak Lodge Sanitary District, and the plant with a capacity of 40000 m3/d reduced sludge production by 65% (Walter et al., 2014). In the village of Carol Stream, a wastewater treatment plant (24600 m3/d) used the Cannibal process to reduce sludge production. A low observed sludge yield of 0.1 g VSS/g COD was obtained with a 10-day SRT in the interchange reactor (Rickermann and Johnson, 2007). The Cannibal system was selected to reduce sludge yield in a plant expansion at the Rock Springs WWTP (9000 m3/d), and the volume of waste-activated sludge was reduced by 80 percent (Mesloh et al., 2007). An MBR system coupled with a Cannibal process was adopted in the wastewater plant in Clovis, CA and resulted in a reduction in observed sludge from 0.9 g VSS/g COD in the CAS process to 0.1 g VSS/g COD (Johnson et al., 2007). The process was capable of routinely meeting a 0.3 mg/L effluent TP. Jiang et al. (2018b) reported that the total electricity consumption was 0.354 kWh/m3 in a full-scale activated sludge system with sludge reduction, and the sludge reduction module accounted for 21.9% of the total electricity consumption. The operating cost for the sludge reduction process was 102 €/tDS, indicating net savings of 77 and 156€/tDS compared to TADL (sludge thickening, anaerobic digestion, dewatering, and landfill) and TDCL (sludge thickening, dewatering, combustion, and landfill), respectively. In the Marche WWTP with alternate cycles process in the sludge line, the sludge disposal cost decreased by 11,270€ for the entire experimental period (230€/tDS), given a specific disposal price in an urban landfill of 115€/tDS, and a net saving on tons of unproduced sludge evaluated to be 96€/ tDS (Troiani et al., 2011). Mesloh et al. (2007) announced that a Cannibal system was estimated to cost $870,000 more to construct than an aerobic digestion system, and there should be an approximate savings of $4,091,000 in net present worth over 20 years in the Rock Springs WWTP. Considering CapEx and OpEx, sludge reduction technologies based on microbial metabolism are economically effective approaches for sludge disposal.

5.2. Sludge reduction by autotrophic microorganisms Autotrophic growth leads to a relatively low biomass yield due to the resulting slow growth rate and high maintenance energy requirement (Strous et al., 1998). The low biomass yield associated with autotrophic growth is beneficial to sludge reduction during wastewater treatment. Therefore, a wastewater treatment process based on autotrophic bacteria, such as anaerobic ammonium oxidizing (ANAMMOX), has received widespread attention for sludge reduction. NH4+-N and NO2−-N act as the electron donor and acceptor in the ANAMMOX reaction, respectively. The ANAMMOX process is generally acknowledged as a wastewater treatment process with a low sludge yield (Strous et al., 1998). The ANAMMOX biomass yields observed in previous studies are detailed in Supplementary information. The ANAMMOX yield was 0.07 g VSS·g NH4+-N−1 in a fluidized bed reactor (van de Graaf et al., 1996). The ANAMMOX biomass yield was 0.22 g VSS·g NH4+-N−1 in a high-loaded ANAMMOX upflow anaerobic sludge bed (UASB) reactor (Tang et al., 2011). The ANAMMOX granular sludge SBR-process was used to treat ammonium-rich wastewater. Greater than 80% of the ammonia was converted into dinitrogen gas at a load of 1.2 kgN/m3 per day, and the biomass yield was 0.11 g VSS·g NH4+-N−1 (van Dongen et al., 2001). The sludge reduction potential of an ANAMMOX-based mainstream wastewater treatment process has also received considerable attention due to the resulting low sludge biomass yield reported in recent years. A novel A-2B process integrating ANAMMOX was developed to achieve energy-saving operation and sludge reduction. Greater than 75% sludge reduction was obtained in the A-2B process (Gu et al., 2018). A novel hybrid upflow anaerobic sludge blanket-moving bed biofilm reactor followed by rope bed biofilm reactor (UASB-MBBR-RBBR) was designed and operated to achieve COD and nitrogen removal. The sludge yield in the reactor was in the range of 0.06 kg VSS/kg COD, and the SRT ranged from 120 to 230 days with higher SRTs at lower OLRs (Chatterjee et al., 2016). Conventional biological processes for municipal wastewater treatment are facing the challenges of excessive sludge production, high energy consumption, and nitrogen removal. ANAMOXX-based processes represent an alternative approach to address these challenges.

7. Future research prospects The extensive knowledge gap in the understanding of sludge reduction technologies based on microbial metabolism limits their widespread use, which has increasingly prompted efforts to thoroughly understand the metabolic mechanisms behind these sludge reduction strategies. Some sludge reduction technologies, such as ASSR, OSA, and MBR, are subject to debate due to the various mechanisms that may simultaneously occur in these sludge reduction technologies. Therefore, the microbial metabolic pathways and substrate and energy conversion of microorganism within sludge reduction systems should be thoroughly investigated. The substrate decomposition and energy dissipation are of particular importance and should be quantified and expressed to evaluate sludge reduction potential in the implementation of these sludge reduction strategies. Advanced analytical techniques, such as isotope analysis, molecular biology techniques, and imaging technologies, are needed to determine the contribution of these mechanisms

6. Engineering application and economic analysis Many attempts have been made apply sludge reduction strategies in practical engineering. Several engineering cases addressing sludge reduction based on microbial metabolism are summarized in Supplementary information. The first full-scale ASSR (Cannibal process) in Europe was operated in 2008 in the Levico WWTP (Italy) with a capacity of 10000 m3/d, and the process reduced the observed sludge yield from 0.44 g VSS/g COD to 0.35 g VSS/g COD (Velho et al., 2016). The plant operated for more than 5 years and had no negative impact on effluent quality in the ASSR configuration. The removal efficiencies for TN, TP, and COD were 77%, 71%, and 97%, respectively. The 9

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increase of oxygen concentration in activated sludge flocs: experimental and theoretical approach. Water Res. 34 (1), 139–146. Andreadakis, A.D., 1993. Physical and chemical properties of activated sludge floc. Water Res. 27 (12), 1707–1714. Cieślik, B.M., Namieśnik, J., Konieczka, P., 2015. Review of sewage sludge management: standards, regulations and analytical methods. J. Cleaner Prod. 90, 1–15. Coma, M., Rovira, S., Canals, J., Colprim, J., 2013. Minimization of sludge production by a side-stream reactor under anoxic conditions in a pilot plant. Bioresour. Technol. 129, 229–235. Copp, J.B., Dold, P.L., 1998. Comparing sludge production under aerobic and anoxic conditions. Water Sci. Technol. 38 (1), 285–294. Chatterjee, P., Ghangrekar, M.M., Rao, S., 2016. Organic matter and nitrogen removal in a hybrid upflow anaerobic sludge blanket—moving bed biofilm and rope bed biofilm reactor. J. Environ. Chem. Eng. 4 (3), 3240–3245. Chen, S., Sun, D., Chung, J.S., 2008. 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to sludge reduction and to optimize sludge reduction performance. The effects of various sludge reduction strategies on nutrient removal in the activated sludge system remain unclear. Efficient and stable nutrient removal relies on maintenance of appropriate living conditions for functional bacteria. Implementation of a sludge reduction strategy within the activated sludge process changes the living environment for functional bacteria and thus affects the performance and stability of the total process. Minimizing this impact is key to simultaneously achieving excellent BNR and sludge reduction in the wastewater treatment process. Coupling sludge reduction and nutrient removal by microbial metabolism is an alternative approach for solving the above problem. Similarly, effective sludge reduction and nitrogen removal can be simultaneously achieved by microbial metabolism when O2 is replaced by NOx- as the electron acceptor in bacteria within the activated sludge process. However, considerable difficulties remain in simultaneously achieving optimal phosphorus removal and sludge reduction by microbial metabolism in the wastewater treatment process. A comprehensive life cycle assessment (LCA) of sludge reduction strategies based on microbial metabolism is recommended to assess the sustainability of these sludge reduction strategies and identify an ideal strategy for achieving overall sustainability of WWTPs. Additionally, mathematical models of sludge reduction should be developed to understand the BNR, and an in situ sludge reduction mechanism should be a topic of future research to fine-tune operating conditions such as HRT, SRT, and temperature, in order to balance the appropriate process conditions between biological nutrient removal and sludge reduction. Full-scale investigations are still required to verify the results obtained by bench- and pilot-scale experiments. 8. Conclusion Recent developments of sludge reduction via microbial metabolism were reviewed from the perspectives of thermodynamics, carbon metabolism, and electron transport. An improved understanding of these sludge reduction mechanisms should be the goal of future research efforts. Further investigations of the effect of these sludge reduction strategies on the performance of an activated sludge system are necessary. Mathematical models of sludge reduction should be developed to balance the appropriate process parameters between sludge reduction and biological nutrient removal to simultaneously achieve excellent BNR and sludge reduction. This review provides new insights into sludge reduction within the activated sludge process. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was supported by the National Natural Science Foundation of China (51508546 and 51878091), the Chongqing Science and Technology Commission (cstc2018jcyjAX0610) and the Fundamental Research Funds for the Central Universities (2019CDQYCH036 and 2019CDXYCH0026). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.122506. References Abbassi, B., Dullstein, S., Rabiger, N., 2000. Minimization of excess sludge production by

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