Regulatory Mechanisms of Quorum Sensing System of Bacteria in Response to Chlorine and Ozone Disinfection.

Waterborne pathogens invariably present considerable threats to public health. The quorum sensing (QS) system is instrumental in coordinating bacterial growth and metabolisms. However, the responses and regulatory mechanisms of bacteria to various disinfection technologies through quorum sensing are still unclear. This study examines the inactivation effect of chlorination and ozonation on biofilms and planktonic cells of QS signaling-deficient mutants of Pseudomonas aeruginosa. Cell counting and viability assessment revealed that the combined disinfection of chlorine and ozone was the most effective for inactivating planktonic P. aeruginosa within 10 min of exposure. Additionally, microfluidic chip culture demonstrated that the secretion of quinolone signals escalated biofilms' disinfection resistance. Disinfection exposure significantly altered the gene expression of wild-type strains and QS signaling-deficient mutants. Moreover, the QS system triggered multilayered gene expression programs as a responsive protection to disinfectant exposure, including oxidative stress, ribosome synthesis, and the nutrient absorption of bacteria. These insights broaden our understanding of bacterial QS in response to disinfection, promising potential strategies toward efficient disinfection processes.

Deciphering the formation of granules by n-DAMO and Anammox microorganisms.

Nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) process is a promising wastewater treatment technology, but the slow microbial growth rate greatly hinders its practical application. Although high-level nitrogen removal and excellent biomass accumulation have been achieved in n-DAMO granule process, the formation mechanism of n-DAMO granules remains unresolved. To elucidate the role of functional microbes in granulation, this study attempted to cultivate granules dominated by n-DAMO microorganisms and granules coupling n-DAMO with anaerobic ammonium oxidation (Anammox). After long-term operation, dense granules were developed in the two systems where both n-DAMO archaea and n-DAMO bacteria were enriched, whereas granulation did not occur in the other system dominated by n-DAMO bacteria. Extracellular polymeric substances (EPS) measurement indicated the critical role of EPS production in the granulation of n-DAMO process. Metagenomic and metatranscriptomic analyses revealed that n-DAMO archaea and Anammox bacteria were active in EPS biosynthesis, while n-DAMO bacteria were inactive. Consequently, more EPS were produced in the systems containing n-DAMO archaea and Anammox bacteria, leading to the successful development of n-DAMO granules. Furthermore, EPS biosynthesis in n-DAMO systems is potentially regulated by acyl-homoserine lactones and c-di-GMP. These findings not only provide new insights into the mechanism of granule formation in n-DAMO systems, but also hint at potential strategies for management of the granule-based n-DAMO process.

Deep insights into the biofilm formation mechanism and nitrogen-transformation network in a nitrate-dependent anaerobic methane oxidation biofilm.

Nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) process offers a promising solution for simultaneously achieving methane emissions reduction and efficient nitrogen removal in wastewater treatment. Although nitrogen removal at a practical rate has been achieved by n-DAMO biofilm process, the mechanisms of biofilm formation and nitrogen transformation remain to be elucidated. In this study, n-DAMO biofilms were successfully developed in the membrane aerated moving bed biofilm reactor (MAMBBR) and removed nitrate at a rate of 159 mg NO3--N L-1 d-1. The obvious increase in the content of extracellular polymeric substances (EPS) indicated that EPS production was important for biofilm development. n-DAMO microorganisms dominated the microbial community, and n-DAMO bacteria were the most abundant microorganisms. However, the expression of biosynthesis genes for proteins and polysaccharides encoded by n-DAMO archaea was significantly more active compared to other microorganisms, suggesting the central role of n-DAMO archaea in EPS production and biofilm formation. In addition to nitrate reduction, n-DAMO archaea were revealed to actively express dissimilatory nitrate reduction to ammonium and nitrogen fixation. The produced ammonium was putatively converted to dinitrogen gas through the joint function of n-DAMO archaea and n-DAMO bacteria. This study revealed the biofilm formation mechanism and nitrogen-transformation network in n-DAMO biofilm systems, shedding new light on promoting the application of n-DAMO process.

Chemical Pretreatments and Anaerobic Digestion Shape the Virome and Functional Microbiome in Fecal Sludge.

The decomposition and pathogen inactivation of fecal sludge (FS) are vitally important for safely managing onsite sanitation and protecting public and environmental health. However, the microbiome and virome assemblages in FS after chemical and biological treatments remain unclear. Here, we reported the differences in the solid reduction and microbiomes of FS subjected to potassium ferrate (PF), alkali (ALK), and sodium hypochlorite (NaClO) pretreatments and anaerobic digestion (AD). The PF and NaClO pretreatments enhanced FS hydrolysis and pathogen suppression, respectively; AD suppressed Gram-positive bacteria. Most of the viromes were those of bacteriophages, which were also shaped by chemical pretreatments and AD. Metatranscriptome analysis revealed distinct gene expression patterns between the PF- and ALK-pretreated FS and the subsequent AD. Differentially expressed gene profiles indicated that genes related to biological processes, molecular functions, and transcriptional regulators were upregulated in ALK-AD and PF-AD samples. These findings suggested that the effect of different treatment technologies on the viral diversity, pathogen abundance, and metabolic function of the core microbiome extends beyond FS decomposition and that the use of combined processes would provide possible alternatives for FS management in pandemic emergencies.

Microbial Niche Differentiation during Nitrite-Dependent Anaerobic Methane Oxidation.

Nitrite-dependent anaerobic methane oxidation (n-DAMO) has been demonstrated to play important roles in the global methane and nitrogen cycle. However, despite diverse n-DAMO bacteria widely detected in environments, little is known about their physiology for microbial niche differentiation. Here, we show the microbial niche differentiation of n-DAMO bacteria through long-term reactor operations combining genome-centered omics and kinetic analysis. With the same inoculum dominated by both species "Candidatus Methylomirabilis oxyfera" and "Candidatus Methylomirabilis sinica", n-DAMO bacterial population was shifted to "Ca. M. oxyfera" in a reactor fed with low-strength nitrite, but shifted to "Ca. M. sinica" with high-strength nitrite. Metatranscriptomic analysis showed that "Ca. M. oxyfera" harbored more complete function in cell chemotaxis, flagellar assembly, and two-component system for better uptake of nitrite, while "Ca. M. sinica" had a more active ion transport and stress response system, and more redundant function in nitrite reduction to mitigate nitrite inhibition. Importantly, the half-saturation constant of nitrite (0.057 mM vs 0.334 mM NO2-) and inhibition thresholds (0.932 mM vs 2.450 mM NO2-) for "Ca. M. oxyfera" vs "Ca. M. sinica", respectively, were highly consistent with genomic results. Integrating these findings demonstrated biochemical characteristics, especially the kinetics of nitrite affinity and inhibition determine niche differentiation of n-DAMO bacteria.

Evidence for Nitrous Oxide Emissions by Nitrite-Dependent Anaerobic Methane Oxidizing Bacteria.

Nitrite-dependent anaerobic methane oxidizing (n-DAMO) bacteria generally convert nitrite to dinitrogen and bypass the nitrous oxide (N2O) formation step. However, N2O is often detected in n-DAMO bacteria dominated cultures and it remains an open question as to the microbial origin of N2O in these enrichments. Using a stable nitrite consuming microbial community enriched for n-DAMO bacteria, we demonstrated that N2O production was coupled to methane oxidation and the higher initial nitrite concentrations led to increased quantities of N2O being formed. Moreover, continuous exposure of the enrichment culture to about 5 mg of N L-1 nitrite resulted in constant N2O being produced (12.5% of nitrite was reduced to N2O). Metatranscriptomic analyses revealed that nitrite reductase (nirS) and nitric oxide reductase (norZ) transcripts from n-DAMO bacteria increased in response to nitrite exposure. No other bacteria significantly expressed nor genes under these conditions, suggesting n-DAMO bacteria are responsible for N2O being produced. In a 35-day bioreactor experiment, N2O produced by the n-DAMO bacteria accumulated when nitrite was in excess; this was found to be up to 3.2% of the nitrogen that resulted from nitrite removal. Together, these results suggested that excess nitrite is an important driver of N2O production by n-DAMO bacteria. To this end, proper monitoring and control of nitrite levels in wastewater treatment plants would be effective strategies for mitigating N2O emissions to the atmosphere.

Revisiting the Engineering Roadmap of Nitrate/Nitrite-Dependent Anaerobic Methane Oxidation.

Nitrate/nitrite-dependent anaerobic oxidation of methane (n-DAMO) is a recently discovered process, which provides a sustainable perspective for simultaneous nitrogen removal and greenhouse gas emission (GHG) mitigation by using methane as an electron donor for denitrification. However, the engineering roadmap of the n-DAMO process is still unclear. This work constitutes a state-of-the-art review on the classical and most recently discovered metabolic mechanisms of the n-DAMO process. The versatile combinations of the n-DAMO process with nitrification, nitritation, and partial nitritation for nitrogen removal are also clearly presented and discussed. Additionally, the recent advances in bioreactor development are systematically reviewed and evaluated comprehensively in terms of methane supply, biomass retention, membrane requirement, startup time, reactor performance, and limitations. The key issues including enrichment and operation strategy for the scaling up of n-DAMO-based processes are also critically addressed. Moreover, the challenges inherent to implementing the n-DAMO process in practical applications, including application scenario recognition, GHG emission mitigation, and operation under realistic conditions, are highlighted. Finally, prospects as well as opportunities for future research are proposed. Overall, this review provides a roadmap for potential applications and further development of the n-DAMO process in the field of wastewater treatment.

Reactivated biofilm coupling n-DAMO with anammox achieved high-rate nitrogen removal in membrane aerated moving bed biofilm reactor.

As a promising technology, the combination of nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) with Anammox offers a solution to achieve effective and sustainable wastewater treatment. However, this sustainable process faces challenges to accumulate sufficient biomass for reaching practical nitrogen removal performance. This study developed an innovative membrane aerated moving bed biofilm reactor (MAMBBR), which supported sufficient methane supply and excellent biofilm attachment, for cultivating biofilms coupling n-DAMO with Anammox. Biofilms were developed rapidly on the polyurethane foam with the supply of ammonium and nitrate, achieving the bioreactor performance of 275 g N m-3 d-1 within 102 days. After the preservation at -20 °C for 8 months, the biofilm was successfully reactivated and achieved 315 g N m-3 d-1 after 188 days. After reactivation, MAMBBR was applied to treat synthetic sidestream wastewater. Up to 99.9% of total nitrogen was removed with the bioreactor performance of 4.0 kg N m-3 d-1. Microbial community analysis and mass balance calculation demonstrated that n-DAMO microorganisms and Anammox bacteria collectively contributed to nitrogen removal in MAMBBR. The MAMBBR developed in this study provides an ideal system of integrating n-DAMO with Anammox for sustainable wastewater treatment.

Deciphering the Inhibition of Ethane on Anaerobic Ammonium Oxidation.

Anaerobic ammonium oxidation (anammox) and nitrification, two common biological ammonium oxidation pathways, are critical for the microbial nitrogen cycle. Short chain alkanes (C2-C8) have been well-known as inhibitors for nitrification through interaction with the ammonia monooxygenase, while whether these alkanes affect anammox is an open question. Here, this work demonstrated significant inhibition of ethane on anammox and revealed the inhibitory mechanism. The acute inhibition of ethane on anammox was concentration-dependent and reversible; 0.86 mM dissolved ethane caused 50% inhibition (IC50), and 1.72 mM ethane almost completely inhibited anammox. After long-term exposure to 0.09 mM ethane for 30 days, the ammonium (nitrite) removal rate dropped from 202 (267) mg N L-1 d-1 to 1 (1) mg N L-1 d-1, and the abundance of anammox bacteria decreased from 61.9% to 9.5%. The intercellular ammonium concentration of anammox bacteria decreased after ethane exposure, while metatranscriptome analysis showed significant upregulation of genes for ammonium transport of anammox bacteria. Thus, ethane could suppress ammonium uptake resulting in the inhibition of anammox activities. As ethane is the second most prevalent alkane after methane in various anoxic environments, ethane may have an important effect on the nitrogen cycle driven by anammox that should be investigated in future research.

Mainstream Nitrogen and Dissolved Methane Removal through Coupling n-DAMO with Anammox in Granular Sludge at Low Temperature.

Mainstream anaerobic wastewater treatment has received increasing attention for the recovery of methane-rich biogas from biodegradable organics, but subsequent mainstream nitrogen and dissolved methane removal at low temperatures remains a critical challenge in practical applications. In this study, granular sludge coupling n-DAMO with Anammox was employed for mainstream nitrogen removal, and the dissolved methane removal potential of granular sludge at low temperatures was investigated. A stable nitrogen removal rate (0.94 kg N m-3 d-1 at 20 °C) was achieved with a high-level effluent quality (<3.0 mg TN L-1) in a lab-scale membrane granular sludge reactor (MGSR). With decreasing temperature, the nitrogen removal rate dropped to 0.55 kg N m-3 d-1 at 10 °C, while the effluent concentration remained <1.0 mg TN L-1. The granular sludge with an average diameter of 1.8 mm proved to retain sufficient biomass (27 g VSS L-1), which enabled n-DAMO and Anammox activity at a hydraulic retention time as low as 2.16 h even at 10 °C. 16S rRNA gene sequencing and scanning electron microscopy revealed a stable community composition and compact structure of granular sludge during long-term operation. Energy recovery could be maximized by recovering most of the dissolved methane in mainstream anaerobic effluent, as only a small amount of dissolved methane was capable of supporting denitrifying methanotrophs in granular sludge, which enabled high-level nitrogen removal.

Anaerobic Oxidation of Methane Coupled with Dissimilatory Nitrate Reduction to Ammonium Fuels Anaerobic Ammonium Oxidation.

Nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) is critical for mitigating methane emission and returning reactive nitrogen to the atmosphere. The genomes of n-DAMO archaea show that they have the potential to couple anaerobic oxidation of methane to dissimilatory nitrate reduction to ammonium (DNRA). However, physiological details of DNRA for n-DAMO archaea were not reported yet. This work demonstrated n-DAMO archaea coupling the anaerobic oxidation of methane to DNRA, which fueled Anammox in a methane-fed membrane biofilm reactor with nitrate as only electron acceptor. Microelectrode analysis revealed that ammonium accumulated where nitrite built up in the biofilm. Ammonium production and significant upregulation of gene expression for DNRA were detected in suspended n-DAMO culture with nitrite exposure, indicating that nitrite triggered DNRA by n-DAMO archaea. 15N-labeling batch experiments revealed that n-DAMO archaea produced ammonium from nitrate rather than from external nitrite. Localized gradients of nitrite produced by n-DAMO archaea in biofilms induced ammonium production via the DNRA process, which promoted nitrite consumption by Anammox bacteria and in turn helped n-DAMO archaea resist stress from nitrite. As biofilms predominate in various ecosystems, anaerobic oxidation of methane coupled with DNRA could be an important link between the global carbon and nitrogen cycles that should be investigated in future research.

Accelerated start-up for photo-fermentative hydrogen production in biofilm reactor by adding waste effluent.

The difficulty and long duration of start-up wastes numerous costs, labors and time and a little fluctuate during the process might fail it. However, studies dealing with the problem hindering accelerated start-up are still insufficient. Current research focused to develop a method for accelerated start-up in an efficient way. This work outlined a novel alternative for accelerated start-up. This joint method, adding waste effluent with applying biofilm reactor, could successfully start up hydrogen production in the first 24 h via increasing ability of hydrogen producers while the control group produced little hydrogen. The two factors, biofilm formation and addition of waste effluent, expressed the combined effects on accelerated start-up. This study suggested that little molecules like quorum sensing system factors and indoles might be the crucial regulating and stimulating factors and express the accelerated start-up ability only in biofilm reactors.

Nitrate/nitrite dependent anaerobic methane oxidation coupling with anammox in membrane biotrickling filter for nitrogen removal.

Combining nitrate/nitrite dependent anaerobic methane oxidation (n-DAMO) and anaerobic ammonium oxidation (Anammox) is a promising sustainable wastewater treatment technology, which simultaneously achieve nitrogen removal and methane emission mitigation. However, the practical application of n-DAMO has been greatly limited by its extremely slow growth-rate and low reaction rate. This work proposes an innovative Membrane BioTrickling Filter (MBTF), which consist of hollow fiber membrane for effective methane supplementation and polyurethane sponge as support media for the attachment and growth of biofilm coupling n-DAMO with Anammox. When steady state with a hydraulic retention time (HRT) of 6.00 h was reached, above 99.9% of nitrogen was removed from synthetic sidestream wastewater at a rate of 3.99 g N L-1 d-1. This system presented robust capacity to withstand unstable partial nitritation effluent, achieving complete nitrogen removal at a varied nitrite to ammonium ratio in the range of 1.10-1.40. It is confirmed that n-DAMO and Anammox microorganisms jointly dominated the microbial community by pyrosequencing technology. The complete nitrogen removal potential at high-rate and efficient biomass retention (12.4 g VSS L-1) of MBTF offers promising alternative for sustainable wastewater treatment by the combination of n-DAMO and Anammox.

Sulfate dependent ammonium oxidation: A microbial process linked nitrogen with sulfur cycle and potential application.

Sulfate dependent ammonium oxidation (Sulfammox) is a potential microbial process coupling ammonium oxidation with sulfate reduction under anaerobic conditions, which provides a novel link between nitrogen and sulfur cycle. Recently, Sulfammox was detected in wastewater treatments and was confirmed to occur in natural environments, especially in marine sediments. However, knowledge gaps in the mechanism of Sulfammox, functional bacteria, and their metabolic pathway, make it challenging to estimate its environmental significance and potential applications. This review provides an overview of recent advances in Sulfammox, including possible mechanisms, functional bacteria, and main influential factors, and discusses future challenges and opportunities. Future perspectives are outlined and discussed, such as exploration of microbial community structure and metabolic pathways, possible interactions with other microbes, environmental significance, and potential applications for nitrogen and sulfate removal, to inspire more researches on the Sulfammox process.

Denitrifying Anaerobic Methane Oxidation and Anammox Process in a Membrane Aerated Membrane Bioreactor: Kinetic Evaluation and Optimization.

Denitrifying anaerobic methane oxidation (DAMO) coupled to anaerobic ammonium oxidation (anammox) is a promising technology for complete nitrogen removal with economic and environmental benefit. In this work, a model framework integrating DAMO and anammox process was constructed based on suspended-growth systems. The proposed model was calibrated and validated using experimental data from a sequencing batch reactor and a membrane aerated membrane bioreactor (MAMBR). The model managed to describe removal rates of ammonium (NH4+), nitrite (NO2-), and total nitrogen, as well as biomass changes of DAMO archaea, DAMO bacteria, and anaerobic ammonium oxidizing bacteria (AnAOB) in both reactors. The estimated parameter values revealed that DAMO archaea possessed properties of faster growth and higher biomass yield in suspended-growth systems compared to those in attached-growth systems (e.g., biofilm). Model simulation demonstrated that solid retention time (SRT) was effective in washing out DAMO bacteria, but retaining DAMO archaea and AnAOB in the MAMBR. The optimal SRT and nitritation efficiency (the ratio of the NO2- to the sum of NH4+ and NO2- in the MAMBR influent) were simulated so that 99% of total nitrogen was removed to meet the discharge standard. MAMBR further suggested to be operated with SRT between 15 and 30 days so that the optimal nitritation efficiency could be minimized to 49% for cost savings.

Granular Sludge Coupling Nitrate/Nitrite Dependent Anaerobic Methane Oxidation with Anammox: from Proof-of-Concept to High Rate Nitrogen Removal.

This work developed a novel Membrane Granular Sludge Reactor (MGSR) equipped with a gas permeable membrane module for efficient methane delivery to cultivate nitrate/nitrite dependent anaerobic methane oxidation (n-DAMO) microorganisms in granular sludge. As proof of concept, the MGSR was fed with synthetic wastewater containing nitrate and ammonium to facilitate the growth of n-DAMO microorganisms. The granular sludge of n-DAMO and Anammox was gradually developed and achieved a nitrogen removal rate of 1.08 g NO3--N L-1 d-1 and 0.81 g NH4+-N L-1 d-1. Finally, enriched granular sludge was successfully applied for nitrogen removal from the synthetic partial nitritation effluent. The combined dominance of n-DAMO archaea, Anammox bacteria, and n-DAMO bacteria in the microbial community was confirmed by 16S rRNA amplicon sequencing. Fluorescence in situ hybridization revealed that a layered structure was formed in the granular sludge with Anammox bacteria in the outer layer and n-DAMO microorganisms in the inner layer when granules were fed with nitrite and ammonium. The high performance of nitrogen removal (16.53 kg N m-3 d-1) with satisfactory effluent quality (∼8 mg N L-1) and excellent biomass retention capacity (43 g VSS L-1) make the MGSR promising for the practical application of n-DAMO and Anammox in wastewater treatment.

Development of granular sludge coupling n-DAMO and Anammox in membrane granular sludge reactor for high rate nitrogen removal.

The integration of nitrate/nitrite dependent anaerobic methane oxidation (n-DAMO) and anaerobic ammonium oxidation (Anammox) provides sustainable solution to simultaneously remove nitrate, nitrite and ammonium. This study demonstrated the sludge granulation process coupling n-DAMO and Anammox from mixed inoculum including river sediment, return activated sludge and crushed anaerobic granule sludge in a novel membrane granular sludge reactor (MGSR). Flocculent biomass gradually turned into compact aggregates and retained as granular sludge with an average diameter of 2.2 mm in MGSR after 684 days' operation. When steady state with a hydraulic retention time of 1.19 days was reached, the MGSR achieved a nitrogen removal rate of 1.77 g N L-1 d-1. Granules with density of 1.043 g mL-1, settling velocity of 72 m h-1 and sludge volume index of 22 mL g-1 leaded to excellent biomass retention (42 g VSS L-1). Pyrosequencing analysis revealed that two dominant microbial groups, n-DAMO archaea and Anammox bacteria, in the microbial community of the granule were enriched to 31.09% and 12.45%. Fluorescence in situ hybridization revealed a homogenous distribution of n-DAMO archaea and Anammox bacteria throughout the granule. The granular sludge coupling n-DAMO and Anammox microorganisms provides significant potential for high rate nitrogen removal from wastewater.

Subcritical n-hexane/isopropanol extraction of lipid from wet microalgal pastes of Scenedesmus obliquus.

Subcritical co-solvents of n-hexane/isopropanol were primarily utilized to extract lipid from wet microalgal pastes of Scenedesmus obliquus. The effects of key operational parameters were investigated, and the optimal parameters were obtained: solvent ratio of n-hexane to isopropanol was 3:2 (V:V), phase ratio of co-solvents to microalgal biomass was 35:1 (mL:g), reactor stirring speed was 900 rpm, extraction time was 60 min. Additional pretreatment with acid, ultrasonic and microwave as well as enhanced subcritical pressure/heating treatments were also applied to further study their effects on lipid extraction. The results showed that the lipid recovery rate with acid pretreatment was 8.6 and 6.2% higher than ultrasonic and microwave pretreatment; the optimum enhanced subcritical condition was 55 °C with atmospheric pressure. Under optimal operating conditions, the lipid and FAME yield were 13.5 and 7.2%, which was 82.6 and 135.1% higher than the traditional method. The results indicated that the subcritical n-hexane/isopropanol extraction process had promising application potential.

Combined zero valent iron and hydrogen peroxide conditioning significantly enhances the dewaterability of anaerobic digestate.

The importance of enhancing sludge dewaterability is increasing due to the considerable impact of excess sludge volume on disposal costs and on overall sludge management. This study presents an innovative approach to enhance dewaterability of anaerobic digestate (AD) harvested from a wastewater treatment plant. The combination of zero valent iron (ZVI, 0-4.0g/g total solids (TS)) and hydrogen peroxide (HP, 0-90mg/g TS) under pH3.0 significantly enhanced the AD dewaterability. The largest enhancement of AD dewaterability was achieved at 18mg HP/g TS and 2.0g ZVI/g TS, with the capillary suction time reduced by up to 90%. Economic analysis suggested that the proposed HP and ZVI treatment has more economic benefits in comparison with the classical Fenton reaction process. The destruction of extracellular polymeric substances and cells as well as the decrease of particle size were supposed to contribute to the enhanced AD dewaterability by HP+ZVI conditioning.

A novel free ammonia based pretreatment technology to enhance anaerobic methane production from primary sludge.

This study proposed a novel free ammonia (FA, i.e., NH3 ) pretreatment technology to enhance anaerobic methane production from primary sludge for the first time. The solubilization of primary sludge was substantially enhanced following 24 h FA pretreatment (250-680 mg NH3 -N/L), by which the release of soluble chemical oxygen demand (SCOD) (i.e., 0.4 mg SCOD/mg VS added; VS: volatile solids) was approximately 10 times as much as that without pretreatment (i.e., 0.03 mg SCOD/mg VS added). Then, biochemical methane potential (BMP) tests demonstrated that FA pretreatment of 250-680 mg NH3 -N/L was capable of enhancing anaerobic methane production while the digestion time was more than 7 days. Model based analysis indicated that the improved anaerobic methane production was due to an increased biochemical methane potential (B0 ) of 8-17% (i.e., from 331 to 357-387 L CH4 /kg VS added), with the highest B0 achieved at 420 mg NH3 -N/L pretreatment. However, FA pretreatment of 250-680 mg NH3 -N/L decreased hydrolysis rate (k) by 24-38% compared with control (i.e., from 0.29 d-1 to 0.18-0.22 d-1 ), which explained the lower methane production over the first 7 days' digestion period. Economic analysis and environmental evaluation demonstrated that FA pretreatment technology was environmentally friendly and economically favorable. Biotechnol. Bioeng. 2017;114: 2245-2252. © 2017 Wiley Periodicals, Inc.