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• MPs' specific surface area increased while relative crystallinity decreased after HTP. • Poor thermal stability groups of MPs were converted to ketone or carboxyl groups. • HTP resulted in blocking the adsorption of a variety of MPs for pollutants. • HTP enhanced the inhibitory effect of MPs on AD by accelerating TOC release. • Leachate of MPs reduced the abundance of vital anaerobes. Waste activated sludge (WAS) contains high concentrations of microplastics (MPs), which could serve as vectors of various organic pollutants and heavy metals, causing synergistic transportation and pollution. The application of combined hydrothermal pretreatment (HTP) and anaerobic digestion (AD) has raised growing concerns since the low-temperature hydrothermal treatment could enhance the biogas production of WAS. However, the changes in physicochemical properties, adsorption performances, and effects on AD of MPs by HTP have not been studied. The study used three typical MPs in WAS, and it was found that the HTP (170°C & 30min) increased MPs' specific surface area and carbonyl index (CI) while decreasing the relative crystallinity. The adsorption capacity to Cd increased through the carbonylation for polyethylene microplastic (PE-MP) and polystyrene microplastic (PS-MP) while decreasing by the dechlorination for polyvinyl chloride microplastic (PVC-MP). Meanwhile, increased hydrophilicity reduced the adsorption capacities of all three typical MPs for ofloxacin. The above results indicated that the HTP could be worth blocking the adsorption of polar MPs for polar pollutants. For the pristine MPs, only PVC-MP at the highest concentration (0.5 g kg−1 VS) significantly (p < 0.05) reduced methane production by 16.2 ± 3.3% of WAS without the HTP. However, the HTP resulted in significant (p < 0.05) inhibition of methane production of WAS at high concentrations of PE-MP and PVC-MP (e.g., 0.1 and 0.5 g kg−1 VS), which was due to the acceleration of the released toxic plastic additives (dibutyl phthalate, dimethyl phthalate, and bisphenol-A). Microbial analysis showed the abundances of vital anaerobes, such as acid-producing bacteria (Acetoanerrobium and Mesotoga), proteolytic bacteria (Proteiniborus), and methanogens (Methanosaeta) clearly decreased with the PE-MP and PVC-MP after the HTP, which might result in the decreased methane production. The study provided deep-insight of MPs' behaviors during the combined HTP-AD process. [Display omitted] [ABSTRACT FROM AUTHOR]

• Roxithromycin decreased the methane production from WAS anaerobic digestion. • Roxithromycin particularly inhibited acidogenesis and methanogenesis. • Roxithromycin increased antibiotic resistance genes in digested sludge. • Roxithromycin might affect sludge treatment in practical applications. The macrolide antibiotic roxithromycin is widely detected in varying aquatic environments, especially in the wastewater systems, as an emerging contaminant and leads to significant impacts on the microorganisms involved. In this study, the impact of a shock load of roxithromycin on waste activated sludge (WAS) anaerobic digestion was comprehensively investigated. The biochemical methane potential tests showed that the methane production from WAS anaerobic digestion was significantly inhibited by roxithromycin. With the dosage of roxithromycin increasing from 0 to 1000 μg/L, the maximum cumulative methane production decreased from 163.5 ± 2.6 mL/g VS to 150.9 ± 4.5 mL/g VS. In particular, roxithromycin inhibited the acidogenesis and methanogenesis in WAS anaerobic digestion, leading to the decreased methane production. The methanogenic archaea in the studied system mainly belonged to the genera of Methanoseata, Candidatus Methanofastidiosum and Methanolinea and their relative abundances also decreased with roxithromycin addition. The analysis of antibiotic resistance genes (ARGs) in the digested sludge indicated that the abundances of most ARGs detected in this study were increased with roxithromycin exposure, suggesting the potential of growing antibiotic resistance, which was probably caused by enhancing the effect of esterases, methylases and phosphorylases. This work reveals how roxithromycin affects the WAS anaerobic digestion and the change of ARGs in the anaerobic digestion with roxithromycin exposure, and provides useful information for practical operation. [ABSTRACT FROM AUTHOR]

Heterotrophic-sulfur autotrophic denitrification (HAD) has been proposed to be a prospective nitrogen removal process. In this work, the potential of fermentation liquid (FL) from waste-activated sludge (WAS) as the electron donor for denitrification in the HAD system was explored and compared with other conventional carbon sources. Results showed that when FL was used as a carbon source, over 99% of NO 3 −-N was removed and its removal rate exceeded 14.00 mg N/g MLSS/h, which was significantly higher than that of methanol and propionic acid. The produced sulfate was below the limit value and the emission of N 2 O was low (1.38% of the NO 3 −-N). Microbial community analysis showed that autotrophic denitrifiers were predominated in the HAD system, in which Thiobacillus (16.4%) was the dominant genus. The economic analysis showed the cost of the FL was 0.062 €/m3, which was 30% lower than that in the group dosed with methanol. Our results demonstrated the FL was a promising carbon source for the HAD system, which could reduce carbon emission and cost, and offer a creative approach for waste-activated sludge resource reuse. [Display omitted] • Waste activated sludge FL could be an alternative carbon source for HAD systems. • FL achieved higher NO 3 −-N removal rate compared to methanol and propionic acid. • Low N 2 O emissions were achieved in the HAD systems used FL as carbon source. • The cost of the FL as carbon source was 30% lower than that of methanol. [ABSTRACT FROM AUTHOR]

Zinc oxide nanoparticles (ZnO NPs) has been documented to accumulate in waste activated sludge (WAS). Nevertheless, its potential effect on hydrogen accumulation during anaerobic fermentation has never been reported. Such impact exists in anaerobic sludge treatment systems but is unrecognized before. Therefore, this work aims to explore the effect of ZnO NPs on hydrogen accumulation during WAS fermentation while avoiding hydrogen consumption by maintaining pH around 10. Deep insights into the mechanisms of the enhanced hydrogen production after dosing ZnO NPs can then be provided. The 21.1 to 74.0 mL-H 2 /g-VS hydrogen yields were obtained after dosing 0–150 mg/g-TS ZnO NPs into the system, which is 2.5 times higher than the control. The zinc ions released from ZnO NPs did not pose obvious cytotoxic effect on organisms based on the mechanism exploration of this study. The reactive oxygen species (ROS) induced by ZnO NPs facilitated sludge disintegration and improved the biodegradability of released organics, but restrained the bio-processes of hydrolysis and acetogenesis simultaneously. This was further verified by the microbial community analysis, as the abundance of key microbes related to hydrolysis and acetogenesis was reduced evidently in the presence of 150 mg/g-TS ZnO NPs. This study would provide a green and economic strategy for the sustainable engineering application of WAS containing ZnO NPs. [Display omitted] • ZnO NPs at 150 mg/g-TS enhanced hydrogen production by 3.5 folds of control. • ZnO NPs improved sludge disintegration and organics biodegradability. • ZnO NPs reduced the abundance of key hydrolytic microbes and acetogens. • ROS played an essential role in the cytotoxicity rather than the released Zn2+. [ABSTRACT FROM AUTHOR]

Anaerobic digestion (AD) is a promising technique for waste management, which can achieve sludge stabilization and energy recovery. This study successfully prepared Fe 3 O 4 @ceramsite from WAS and applied it as an additive in sludge digestion, aiming to improve the conversion of organics to biomethane efficiency. Results showed that after adding the Fe 3 O 4 @ceramsite, the methane production was enhanced by 34.7% compared with the control group (88.0 ± 0.1 mL/g VS). Further mechanisms investigation revealed that Fe 3 O 4 @ceramsite enhanced digesta stability by strong buffering capacity, improved sludge conductivity, and promoted Fe (III) reduction. Moreover, Fe 3 O 4 @ceramsite has a larger surface area and better porous structure, which also facilitated AD performance. Microbial community analysis showed that some functional anaerobes related to AD such as Spirochaeta and Smithella were enriched with Fe 3 O 4 @ceramsite treatment. Potential syntrophic metabolisms between syntrophic bacteria (Syntrophomonas , associated with DIET) and methanogens were also detected in the Fe 3 O 4 @ceramsite treatment AD system. • Fe 3 O 4 @ceramsite promoted methane production from WAS digestion. • Fe 3 O 4 @ceramsite enhanced digesta stability and sludge organics dissolution. • Larger surface area and better pore structure of Fe 3 O 4 @ceramsite facilitated AD performance. • Some functional anaerobes related to AD were enriched. [ABSTRACT FROM AUTHOR]

Methane recovery from waste activated sludge (WAS) through anaerobic digestion is generally restricted by the poor degradability of WAS. Herein, a novel sludge pretreatment technology by using the calcium hypochlorite (Ca(ClO) 2) in enhancing the methane production from WAS anaerobic digestion was reported. The solubilization of WAS was significantly increased after 10–240 mg Ca(ClO) 2 /g VS (VS: volatile solids) pretreatment for 48 h, under which the solubilization was 1.7–3.4 folds (i.e., 0.17–0.34 mg SCOD/mg VS; SCOD: soluble chemical oxygen demand) higher than that without Ca(ClO) 2 pretreatment (i.e., 0.1 mg SCOD/mg VS). Correspondingly, the methane production was increased from 250.0 ± 5.3 mL/g VS to 385.1 ± 3.3 mL/g VS with the doses of Ca(ClO) 2 increasing from 10 mg/g VS to 240 mg/g VS, resulted in an increasing methane production of 3.6%–59.7% than that without Ca(ClO) 2 pretreatment. The microbial community composition results exhibited that the populations of key acidogens (e.g., Longilinea sp.) and methanogens (e.g., Methanosaeta sp.) were both reduced significantly. Moreover, Ca(ClO) 2 decreased the cells viability, leading to a 76.2% reduction of living cells fraction. Accordingly, it was further confirmed that high dosage of Ca(ClO) 2 could inhibit three microbial-related processes relevant to methane production, i.e., acidification, hydrolysis and methanogenesis. [Display omitted] • Ca(ClO) 2 pretreatment enhanced methane production from WAS digestion. • Methane yield is positively related to pretreatment level of Ca(ClO) 2. • High dosage of Ca(ClO) 2 decreased the abundance of acidogens and methanogens. • High dosage of Ca(ClO) 2 inhibited hydrolysis, acidification, and methanogenesis. [ABSTRACT FROM AUTHOR]

In this work, a low-cost alternative method (i.e., adding nitrate into WAS) to significantly enhance hydrogen production was reported. Experimental results showed that with an increase of nitrate addition from 0 to 362 mg/L, the maximal hydrogen production from acidic (pH 5.5) fermentation of WAS obviously increased from 12.6 ± 0.5 to 19.3 ± 0.9 mL per gram volatile suspended solids (VSS). The mechanism investigations illustrated more substrates were provided for subsequent hydrogen production. Although the nitrate added inhibited all the biological processes, its inhibition to the hydrogen consumption processes was much severer than that to the hydrogen production processes. The enzyme analyses on the long-term semi-continuous fermenters showed that the nitrate addition slightly inhibited the relative activities of protease, butyrate kinase, acetate kinase, CoA-transferase, and [FeFe] hydrogenase but largely suppressed the relative activities of coenzyme F 420 , carbon monoxide dehydrogenase, and adenylyl sulfate reductase. Image 1 •The presence of nitrate enhanced hydrogen production from waste activated sludge. •Nitrate accelerated the disruption of extracellular polymeric substances. •Nitrate inhibited all the biological processes in anaerobic fermentation. •Inhibition to hydrogen consumption was severer than that to hydrogen production. •A new "closed-loop" concept for the energy cycle of WWTPs was proposed. [ABSTRACT FROM AUTHOR]

Medium-chain fatty acids (MCFAs) have drawn great attention due to their high energy density and superior hydrophobicity. Waste activated sludge (WAS) has been documented as a renewable feedstock for MCFAs production via anaerobic fermentation. However, MCFAs production from WAS depends on exogenous addition of electron donor (ED, e.g., lactate) for chain elongation (CE) bioprocess, which results in increased economic cost and limited practical application. In this study, a novel biotechnology was proposed to produce MCFAs from WAS with in-situ self-formed lactate by inoculating Yoghurt starter powder containing with Lactobacillales cultures. The batch experimental results revealed that the lactate was in-situ generated from WAS and the maximum production of MCFAs increased from 1.17 to 3.99 g COD/L with the increased addition of Lactobacillales cultures from 6✕107 to 2.3✕108 CFU/mL WAS. In continuous long-term test over 97 days, average MCFA production reached up to 3.94 g COD/L with a caproate yield of 82.74% at sludge retention time (SRT) 12 days, and the average MCFA production increased to 5.87 g COD/L with 69.28% caproate and 25.18% caprylate at SRT 15 days. A comprehensive analysis of the metagenome and metatranscriptome demonstrated that the genus of Lactobacillus and Streptococcus were capable of producing lactate from WAS and upgrading to MCFAs. Moreover, another genus, i.e., Candidatus Promineofilum , was firstly revealed that it might be responsible for lactate and MCFAs production. Further investigation of related microbial pathways and enzyme expression suggested that D-lactate dehydrogenase and pyruvate ferredoxin oxidoreductase contributed to lactate and acetyl-CoA production, which were the crucial steps for MCFAs generation and were most actively expressed. This study provides a conceptual framework of MCFAs from WAS with endogenous ED, potentially enhancing the energy recovery from WAS treatment. [Display omitted] • MCFA production from WAS with in-situ self-formed lactate was achieved. • MCFA production was positively related to inoculated Lactobacillales cultures level. • The SRT of 15 days was preferable for MCFA production than the SRT of 12 days. • Key microorganisms were revealed to contribute to lactate and MCFA productions. [ABSTRACT FROM AUTHOR]

Energy conversion of waste activated sludge alkaline fermentation liquor (WASAFL) to medium-chain fatty acids (MCFAs) is promising for sludge treatment and carbon recovery. However, the single electron donor (ED) fermentation for MCFAs production has irreparable defects. To resolve the respective shortcomings of single electron donor (ED) and improve the MCFAs production efficiency from WASAFL, a novel biotechnical process utilizing ethanol and lactate as co-EDs within different combination ratios were investigated. The results verified that MCFAs production was highest with ethanol to lactate ratio of 1:3 (6988.54 ± 208.18 mg COD/L), being 1.46 and 1.87 times of that with ethanol and lactate as single ED. The kinetic analysis results confirmed that ethanol to lactate ratio of 1:3 resulted in the highest MCFAs yield and formation rate. The microbial taxa results uncovered that the relative abundance of Sphaerochaeta and Haloimpatiens showed positive correlation with MCFAs production. The metabolic pathway analysis indicated that the ethanol oxidization, lactate oxidization, acrylate pathway, reverse β oxidization and fatty acid biosynthesis pathway might take place in the WASAFL fermentation system, contributing to the WASAFL-to-MCFAs conversion. [Display omitted] • Lower ratio of ethanol: lactate promoted MCFAs production from WASAFL. • Higher or equal ratio of ethanol: lactate reduced MCFA production and selectivity. • Under sole ED condition, ethanol is more efficient for MCFA production than lactate with WASAFL as substrate. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Sophorolipid enhanced the SCFA production from anaerobic fermentation of WAS. • Sophorolipid facilitated the WAS solubilization. • Sophorolipid enhanced release of biodegradable organics. • Inhibition of methanogens by sophorolipid led to accumulation of SCFA. • Sophorolipid increased the abundance of hydrolytic microbes and SCFA producers. Abstract It was found in this study that the presence of sophorolipid (SL) enhanced the production of short-chain fatty acid (SCFA) from anaerobic fermentation of waste activated sludge (WAS). Experimental results showed that with an increase of SL addition from 0 to 0.1 g/g TSS, the maximal SCFA yield increased from 50.5 ± 4.9 to 246.2 ± 7.5 mg COD/g VSS. The presence of SL reduced the surface tension between hydrophobic organics and fermentation liquid, which thereby accelerated the disintegration of WAS and improved the biodegradability of the released organics. SL promoted the carbon/nitrogen ratio of the fermentation system, enhancing the conversion of proteins in WAS. Moreover, SL suppressed severely the activities of methanogens, probably due to the drop of pH caused by SL addition. Amplicon sequencing analyses revealed that SL increased the abundance of hydrolytic microbes such as Bacteroides sp. and Macellibacteroides sp., and SCFA producers (e.g., Acinetobacter sp.). [ABSTRACT FROM AUTHOR]

• The combination of heat pretreatment with FA promoted hydrogen yield from WAS. • The accumulative hydrogen yield was in the order of blank < heat < FA < heat + FA. • The combination of heat pretreatment with FA accelerated WAS disintegration. • The combined heat pretreatment with FA severely inhibited hydrogen consumers. Controlling free ammonia in an anaerobic fermenter at pertinent levels is reported recently to be an economically attractive and practically feasible approach to enhance hydrogen yield from waste activated sludge (WAS). This paper reports a new technology for WAS dark fermentation, i.e., using heat pretreatment (70 °C for 60 min) to assist free ammonia for further improving hydrogen yield. The experimental results showed that the accumulative hydrogen production from combined reactors was promoted from 12.3 to 19.2 mL/g VSS (volatile suspended solids), the maximum of which was 1.8, 2.7, and 7.1 times of that from sole free ammonia (131.9 mg NH 3 -N/L), sole heat, and blank reactors, respectively. Mechanism explorations showed that the combination strategy significantly enhanced WAS disintegration, providing more substrates for hydrogen production. Moreover, the combination suppressed activities of all microbes associated with anaerobic fermentation, but its inhibition to hydrogen consumers was much severer than that to other microbes. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Pretreatment of polyacrylamide-flocculated WAS (PF-WAS) was conducted. • Hydrolysis rate of PF-WAS was improved through thermal-alkaline pretreatment (TAP) • Biochemical methane potential of PF-WAS was improved through TAP. • Optimal TAP for methane yield of PF-WAS was 75 °C and pH 11.0 for 17.5 h. • Optimal TAP improved PAM's degradation and avoided acrylamide's accumulation. Abstract Deterioration of anaerobic digestion can occur with the presence of polyacrylamide (PAM) in waste activated sludge, and little information on mitigating this deterioration is currently available. In this study, simultaneous mitigation of PAM negative effects and improvement of methane production was accomplished by thermal-alkaline pretreatment. Under the optimized pretreatment conditions (i.e., 75 °C, pH 11.0 for 17.5 h), the biochemical methane potential of PAM-flocculated sludge increased from 100.5 to 210.8 mL/g VS and the hydrolysis rate increased from 0.122 to 0.187 d−1. Mechanism investigations revealed that the pretreatment not only broke the large firm floccules, improved the degradation of PAM, but also facilitated the release of biodegradable organics from sludge, which thereby provided better growth environment and enough nutrients to anaerobic microbes for methane production. The activities of key enzymes responsible for methane production and PAM degradation were greatly improved in pretreated reactor, with the accumulation of acrylamide being avoided. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Calcium peroxide affected hydrogen production in fermentation system. • Calcium peroxide facilitated solubilization and release of biodegradable organics. • Calcium peroxide inhibited methanogenesis process. • Contributions of ingredients were in the order of alkali > O 2 − > OH. Abstract Calcium peroxide (CaO 2), one of versatile and harmless inorganic peroxy compounds, has been recently used as a highly effective oxidant to degrade pollutants in sludge and a method to achieve sludge resource utilization. However, its impact on hydrogen production has never been studied before. This investigation therefore aims to fill this knowledge gap. Results indicated that with CaO 2 increased from 0.05 to 0.25 g/g VSS (volatile suspended solids), the maximum hydrogen yield increased from 0.77 to 10.55 mL/g VSS. The mechanism studies revealed that CaO 2 accelerated the breakage and death of sludge cells. Excitation emission matrix (EEM) analyses further indicated that CaO 2 increased the biodegradability of the released substances, which could provide more biodegradable organics for the following reactions. Although CaO 2 caused inhibitions to some extents to all the tested microbes, its inhibition to hydrogen consumers was much severer than that to hydrolytic microbes and hydrogen producers. The enzyme analyses also demonstrated that the suppression of calcium peroxide to the activities of enzymes related to hydrogen consumption process was much severer than that to the activities of the activities of enzymes related to hydrogen production process. Further investigations exhibited that alkali, O 2 − and OH radicals, were the major intermediate products of CaO 2 decomposition. All of them were verified to contribute the increased hydrogen production, and their contributions were in the order of alkali > O 2 − > OH. This is the first work demonstrating that CaO 2 can enhance hydrogen production from WAS. The findings reported in our paper not only expand the application field of CaO 2 but also deepen the understanding of the CaO 2 -involved sludge fermentation process. [ABSTRACT FROM AUTHOR]

Abstract The wide application of graphene oxide nanoparticles inevitably leads to their discharge into wastewater treatment plants and combination with the activated sludge. However, to date, it is largely unknown if the nano-graphene oxide (NGO) has potential impacts on the anaerobic digestion of waste activated sludge (WAS). Therefore, this work aims to fill the knowledge gap through comprehensively investigating the effects of NGO on carbon transformation and methane production in the anaerobic digestion of WAS. Biochemical methane potential tests demonstrated the methane production dropped with increasing NGO additions, the cumulative methane production decreasing by 7.6% and 12.6% at the NGO dosing rates of 0.054 mg/mg-VS and 0.108 mg/mg-VS, respectively. Model-based analysis indicated NGO significantly reduced biochemical methane potential, with the highest biochemical methane potential decrease being approximately 10% at the highest NGO dosing rate. Further experimental analysis suggested that the decreased methane production was firstly related to a decrease in soluble organic substrates availability during the process of sludge disintegration, potentially attributing to the strong absorption of organic substrates by NGO. Secondly, NGO significantly inhibited the methanogenesis by negatively affecting the corresponding enzyme activity (i.e. coenzyme F420), which could also resulted in a decreased methane production. Graphical abstract Unlabelled Image Highlights • The methane production from WAS anaerobic digestion deteriorated with NGO exposure. • NGO decreased the release of soluble organics during WAS disintegration due to adsorption. • NGO negatively affected the methanogenesis through inhibiting coenzyme F420 activities. • Hydrolysis and acidogensis were not affected by NGO during WAS anaerobic digestion. [ABSTRACT FROM AUTHOR]

Abstract Simultaneous sludge fermentation and nitrite removal is an effective approach to enhance nutrient removal from low carbon-wastewater. It was found in this work that the presence of nitrite largely promoted hydrogen production from acidic fermentation of waste activated sludge (WAS). The results showed that with an increase of nitrite from 0 to 250 mg/L, the maximal hydrogen yield increased from 8.5 to 15.0 mL/g VSS at pH 5.5 fermentation and 8.1–13.0 mL/g VSS at pH 6 fermentation. However, the maximal hydrogen yield from WAS fermentation at pH 8 remained almost constant (2.9–3.7 mL/g VSS) when nitrite was in the range of 0–250 mg/L. Further analyses revealed that free nitrous acid (FNA) rather than nitrite was the major contributor to the promotion of hydrogen yield. The mechanism investigations showed that FNA not only accelerated the disruption of sludge cells but also promoted the biodegradability of organics released, thereby provided more biodegradable substrates for subsequent hydrogen production. Although FNA inhibited activities of all microbes involved in the anaerobic fermentation, its inhibitions to hydrogen consumers were much severer than those to hydrolytic microorganisms and hydrogen producers. Further investigations with microbial community showed that FNA increased the abundances of hydrogen producers (e.g., Citrobacter sp.) and denitrifiers (e.g., Dechloromonas sp.), but reduced the abundances of hydrogen consumers (e.g., Clostridium_aceticum). This work demonstrated for the first time that FNA in WAS fermentation systems enhanced hydrogen production. The findings obtained expand the application field of FNA and may provide supports for sustainable operation of wastewater treatment plants. Graphical abstract Image 1 Highlights • Nitrate addition promoted hydrogen production from anaerobic fermentation of sludge. • Free nitrous acid (FNA) rather than nitrite was the major contributor to the promotion of hydrogen yield. • FNA not only accelerated the disruption of sludge cells but also promoted the biodegradability of organics released. • FNA increased the abundances of hydrogen producers and denitrifiers, but reduced the abundances of hydrogen consumers. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • CFHP was applied to improve methane production from WAS for the first time. • Hydrolysis rate of WAS are improved through CFHP. • Biochemical methane potential of WAS are improved through CFHP. • CFHP is an attractive technology for the application in real-world situations. Abstract Anaerobic digestion of waste activated sludge (WAS) is often limited by low hydrolysis efficiencies and poor methane potentials. This work presents a novel pretreatment technology for WAS anaerobic digestion, i.e., combining free ammonia with heat pretreatment (CFHP). Experimental results showed that compared with control, solo free ammonia (135.4 mg NH 3 -N/L) and solo heat (70 °C) pretreatment, the combined free ammonia and heat (135.4 mg NH 3 -N/L with 70 °C) obtained 52.2%, 25.5% and 30.2% faster in hydrolysis rate and 25.2%, 17.9% and 16.5% higher in biochemical methane potential, respectively. Mechanism investigations showed that the combined pretreatment not only largely facilitated the disintegration of WAS but also increased the proportion of biodegradable organic matters, thereby providing better contract between biodegradable organics and the anaerobic microbes for methane production. Considering its effectiveness and renewability, the combined pretreatment is an attractive technology for the application in real-world situations. [ABSTRACT FROM AUTHOR]

Ammonium and/or free ammonia (the unionized form of ammonium) are generally thought to inhibit the activities of microbes involved in anaerobic digestion of waste activated sludge. It was found in this work, however, that the presence of ammonium (NH 4 + -N) largely enhanced dark fermentative hydrogen production from alkaline pretreated-sludge. With the increase of initial NH 4 + -N level from 36 to 266 mg/L, the maximal hydrogen production from alkaline (pH 9.5) pretreated-sludge increased from 7.3 to 15.6 mL per gram volatile suspended solids (VSS) under the standard condition. Further increase of NH 4 + -N to 308 mg/L caused a slight decrease of hydrogen yield (15.0 mL/g VSS). Experimental results demonstrated that free ammonia instead of NH 4 + -N was the true contributor to the enhancement of hydrogen production. It was found that the presence of free ammonia facilitated the releases of both extracellular and intracellular constituents, which thereby provided more substrates for subsequent hydrogen production. The free ammonia at the tested levels (i.e., 0–444 mg/L) did not affect acetogenesis significantly. Although free ammonia inhibited all other bio-processes, its inhibition to the hydrogen consumption processes (i.e., homoacetogenesis, methanogenesis, and sulfate-reducing process) was much severer than that to the hydrolysis and acidogenesis processes. Further investigations with enzyme analyses showed that free ammonia posed slight impacts on protease, butyrate kinase, acetate kinase, CoA-transferase, and [FeFe] hydrogenase activities but largely suppressed the activities of coenzyme F420, carbon monoxide dehydrogenase, and adenylyl sulfate reductase, which were consistent with the chemical analyses performed above. [ABSTRACT FROM AUTHOR]

In this work, a low-cost alternative approach (i.e., adding aged refuse (AR) into waste activated sludge) to significantly enhance anaerobic digestion of sludge was reported. Experimental results showed that with the addition dosage of AR increasing from 0 to 400 mg/g dry sludge soluble chemical oxygen demand (COD) increased from 1150 to 5240 mg/L at the digestion time of 5 d, while the maximal production of volatile fatty acids (VFA) increased from 82.6 to 183.9 mg COD/g volatile suspended solids. Although further increase of AR addition decreased the concentrations of both soluble COD and VFA, their contents in these systems with AR addition at any concentration investigated were still higher than those in the blank, which resulted in higher methane yields in these systems. Mechanism studies revealed that pertinent addition of AR promoted solubilization, hydrolysis, and acidogenesis processes and did not affect methanogenesis significantly. It was found that varieties of enzymes and anaerobes in AR were primary reason for the enhancement of anaerobic digestion. Humic substances in AR benefited hydrolysis and acidogenesis but inhibited methanogenesis. The effect of heavy metals in AR on sludge anaerobic digestion was dosage dependent. Sludge anaerobic digestion was enhanced by appropriate amounts of heavy metals but inhibited by excessive amounts of heavy metals. The relative abundances of microorganisms responsible for sludge hydrolysis and acidogenesis were also observed to be improved in the system with AR addition, which was consistent with the performance of anaerobic digestion. [ABSTRACT FROM AUTHOR]

[Display omitted] • Refractory substances are primary but often ignored factor related to WAS biodegradability. • The presence and role of refractory substances in WAS biodegradability are reviewed. • Inhibition mechanisms of refractory substances in WAS are analyzed. • Mechanisms of pretreatment strategies to enhance WAS biodegradability are discussed. • Challenges of WAS enhanced biodegradability technology are prospected. Anaerobic digestion (AD) is a low-cost technology widely used to divert waste activated sludge (WAS) to renewable energy production, but is generally restricted by its poor biodegradability which mainly caused by the endogenous and exogenous refractory substances present in WAS. Several conventional methods such as thermal-, chemical-, and mechanical-based pretreatment have been demonstrated to be effective on organics release, but their functions on refractory substances conversion are overlooked. This paper firstly reviewed the presence and role of endogenous and exogenous refractory substances in anaerobic biodegradability of WAS, especially on their inhibition mechanisms. Then, the pretreatment strategies developed for enhancing WAS biodegradability by facilitating refractory substances conversion were comprehensively reviewed, with the conversion pathways and underlying mechanisms being emphasized. Finally, the future research needs were directed, which are supposed to improve the circular bioeconomy of WAS management from the point of removing the hindering barrier of refractory substances on WAS biodegradability. [ABSTRACT FROM AUTHOR]

[Display omitted] • CaO 2 favoured the aerobic digestion sludge for improving sludge treatment. • CaO 2 contributed to the disintegration of sludge for providing more substrates. • CaO 2 promoted the biodegradability and subsequent utilization of substrate. This work put up a novel strategy of applying calcium peroxide (CaO 2) in aerobic sludge digestion and provided insights into such system. The degradation percentage of sludge and total inorganic nitrogen production in the digesters with CaO 2 at 0.02 g/g-VS-WAS increased by 25.8% and 18.8% of control. CaO 2 addition allowed various key microbes related to organics degradation to accumulate in the system. Moreover, the modelling and chemical (i.e., excitation emission matrix (EEM) fluorescence and fourier transformation spectroscopy (FTIR)) analyses revealed that CaO 2 addition enhanced sludge biodegradability with more release of biodegradable organics and increased degradation of recalcitrant organics, which can be transformed into biodegradable organics with the action of CaO 2. Subsequent transformation test indicated that CaO 2 enabled to promote hydrolysis and catabolism of biodegradable substrates in sludge. Further investigations on function mechanism suggested that CaO 2 carried on positive action for sludge aerobic digestion mainly through the enhancement by ·OH. [ABSTRACT FROM AUTHOR]

Bioenergy recovery in the form of medium-chain fatty acids (MCFAs) from waste activated sludge (WAS) is increasingly attractive, which are valuable building blocks for fuel production. This study experimentally demonstrated the long-term MCFAs (C6–C8) production from WAS in two-stage anaerobic sludge fermentation at different pH conditions, using continuously operated bench-scale anaerobic reactors. The WAS was continuously converted to short chain fatty acids (SCFAs, 3500–3800 mg chemical oxygen demand (COD)/L) at the first stage via alkaline anaerobic fermentation, which was directly fed into the second stage as both substrates and inoculum for MCFAs production through chain elongation (CE). The productions of MCFAs at the second stage were continuously studied under three different pH conditions (i.e., 10, 7 and 5.5). The results demonstrated that there was no significant MCFAs production at pH 10 during the steady state, whereas the MCFAs productions were clearly observed at both pH 7 and pH 5.5, with much higher MCFAs production from WAS at pH 7 (i.e., 10.32 g COD/L MCFAs) than that at pH 5.5 (i.e., 8.73 g COD/L MCFAs) during the steady state. A higher MCFAs selectivity of 62.3% was also achieved at pH 7. The relatively lower MCFAs production and selectivity at pH 5.5 was likely due to the higher undissociated MCFAs generated at pH 5.5, which would pose toxicity impact on CE microbes and thus inhibit the CE process. Microbial community analysis confirmed that the relative abundances of CE related microbes (e.g., Clostridium sensu stricto 12 sp. and Clostridium sensu stricto 1) increased at pH 7 compared to those at pH 5.5, which enabled more efficient MCFAs production from WAS. [Display omitted] • MCFAs production from sludge was achieved in continuous two-stage system. • MCFAs production was completely suppressed at pH 10 at s stage. • Higher MCFAs production was observed at pH 7 than at pH 5.5. • Higher undissociated MCFAs at pH 5.5 might inhibit the CE microbes. • Key CE related microbes were enriched more at pH 7 than those at pH 5.5. [ABSTRACT FROM AUTHOR]

Bioenergy recovery in the form of medium-chain fatty acids (MCFAs) from waste activated sludge (WAS) is increasingly attractive, which are valuable building blocks for fuel production. This study experimentally demonstrated the long-term MCFAs (C6–C8) production from WAS in two-stage anaerobic sludge fermentation at different pH conditions, using continuously operated bench-scale anaerobic reactors. The WAS was continuously converted to short chain fatty acids (SCFAs, 3500–3800 mg chemical oxygen demand (COD)/L) at the first stage via alkaline anaerobic fermentation, which was directly fed into the second stage as both substrates and inoculum for MCFAs production through chain elongation (CE). The productions of MCFAs at the second stage were continuously studied under three different pH conditions (i.e., 10, 7 and 5.5). The results demonstrated that there was no significant MCFAs production at pH 10 during the steady state, whereas the MCFAs productions were clearly observed at both pH 7 and pH 5.5, with much higher MCFAs production from WAS at pH 7 (i.e., 10.32 g COD/L MCFAs) than that at pH 5.5 (i.e., 8.73 g COD/L MCFAs) during the steady state. A higher MCFAs selectivity of 62.3% was also achieved at pH 7. The relatively lower MCFAs production and selectivity at pH 5.5 was likely due to the higher undissociated MCFAs generated at pH 5.5, which would pose toxicity impact on CE microbes and thus inhibit the CE process. Microbial community analysis confirmed that the relative abundances of CE related microbes (e.g., Clostridium sensu stricto 12 sp. and Clostridium sensu stricto 1) increased at pH 7 compared to those at pH 5.5, which enabled more efficient MCFAs production from WAS. [Display omitted] • MCFAs production from sludge was achieved in continuous two-stage system. • MCFAs production was completely suppressed at pH 10 at s stage. • Higher MCFAs production was observed at pH 7 than at pH 5.5. • Higher undissociated MCFAs at pH 5.5 might inhibit the CE microbes. • Key CE related microbes were enriched more at pH 7 than those at pH 5.5. [ABSTRACT FROM AUTHOR]

Anaerobic digestion is generally restricted by the low degradability of waste activated sludge (WAS) and slow hydrolysis rate. This work provided an innovative technique to promote continuous anaerobic digestion performance mediated by natural diatomite. In continuous tests over 125 days, 2 g/g-Total solids (TS) of natural diatomite addition increased daily methane production and volatile solids (VS) destruction by 23.3% and 23.5%, respectively, leading to a 5.6% decrease in digestate volume for disposal. Microbial community in the natural diatomite mediated digester changed toward a favorable direction for anaerobic sludge digestion. Process modelling indicated that the increased natural diatomite presence increased hydrolysis rate, biomethane potential and degradation extent of the rapidly biodegradable substrates in WAS, with the highest enhancement being approximately 78.9%, 44.5% and 45.0%, respectively, achieved at 2 g/g- TS diatomite. However, the slowly biodegradable substrates in WAS stayed relatively stable. The mechanism studies revealed that natural diatomite mitigated ammonia inhibition and enhanced electron transfer for WAS-to-methane conversion mediated by its strong adsorption capacity and conductivity. The versatility of the technology proposed is also verified by the diatomite mediated experiments using different natural diatomite from different sources. Compared to the most natural minerals, diatomite exhibited either a greater anaerobic digestion performance or a lower dosage, bringing strong benefits in economy, environment and technology. [Display omitted] • An innovative diatomite mediated continuous anaerobic digestion was proposed. • Diatomite enhanced VS destruction and methane production continuously. • Diatomite shifted microbial community toward favorable direction for digestion. • Diatomite changed process kinetics and digestate characteristics. • Diatomite significantly reduced the digestate volume for disposal. [ABSTRACT FROM AUTHOR]

[Display omitted] • TCS was partially degraded in anaerobic fermentation process. • TCS inhibited hydrogen production from anaerobic fermentation of sludge. • The presence of TCS inhibited acidogenesis process. • High level of TCS changed the relative abundances of functional microbes. Triclosan (TCS), a widely used antibacterial agent, was accumulated at significant levels in waste activated sludge (WAS). To date, however, the interaction between TCS and sludge anaerobic fermentation was rarely reported. Hence, this work aimed to deeply understanding the degradation of TCS in sludge anaerobic fermentation and its impact on hydrogen production. Experimental results showed that ~ 45% of TCS was degraded in long-term anaerobic fermentation, with 2,4-dichlorophenol as its main intermediate. Based on the information from high performance liquid chromatography–mass spectrometry analysis, three pathways i.e., dechlorination, hydroxylation, and cleavage of ether bonds, were proposed for TCS degradation. It was found that the maximum hydrogen yield decreased from 18.6 to 12.8 mL/g VSS with the increase of TCS from 12 to 487 mg/kg TSS. One possible reason for the decreased hydrogen yield was that a part of hydrogen generated might serve as electron donors for TCS dechlorination. Besides, the presence of TCS significantly suppressed acidogenesis (an important step responsible for hydrogen generation). This inhibition to acidogenesis is likely due to that the high-affinity functional groups of TCS such as hydroxyl groups could bind to the active sites of acetate kinase (AK, a key enzyme in acidogenesis), which reduced the active sites available for original fermentation substrates. Microbial analysis revealed that TCS increased the relative abundances of potential contaminant decomposers such as Guggeheimella but inhibited the populations of hydrogen producers such as Proteiniborus , which was consistent with the results obtained by chemical analyses. [ABSTRACT FROM AUTHOR]

Tetracycline (TC), a widely used antibiotic, was enriched in waste activated sludge (WAS) at significant levels. However, the TC impact on WAS anaerobic fermentation are still poorly understood. This work aims to analyze the effect of TC to the WAS anaerobic fermentation by investigating the differences of sludge properties, short-chain fatty acids (SCFAs) production and microbial community abundance. The results showed that the environmental level of TC had no effect on the SCFAs production, but with the further increase of the content of TC to 60 mg/kg TSS, the maximum SCFAs yield decreased from 125.1 ± 3.2 to 90.8 ± 1.7 mg COD/g VSS. Mechanism exploration indicated that TC had no significant effect on solubilization, hydrolysis and homoacetogenesis processes, but severely inhibited acidogenesis, acetogenesis and methanogenesis processes. Microbial analysis showed that the presence of TC reduced the diversity of microbial communities and the abundance of functional microorganisms relevant to SCFAs production and complex organic degradation, such as Proteiniclasticun and Novosphingobium. This negative effect was persistent because only a small amount of TC can be degraded in the anaerobic fermentation process. Hence, CaO 2 was proposed and studied as a regulation strategy that can reduce the toxicity of TC on anaerobic fermentation. [Display omitted] • When the TC level were 5, 30 and 60 mg/kg TSS, the SCFAs production decreased by 11.0%, 18.2%, and 27.4%, respectively. • TC severely inhibited acidogenesis, acetogenesis and methanogenesis processes. • The most of TC was stuck after anaerobic fermentation of WAS. • CaO 2 improved the SCFAs production by degrading TC and disintegrating sludge. [ABSTRACT FROM AUTHOR]

l RL promoted sludge solubilization and SCFA production in the acidogenic phase. l RL increases biochemical methane potential of sludge in the methanogenic phase. l The enhancement of the acidogenic phase by RL is a contributor to the promotion of methane yield. l RL could be applied to anaerobic digestion of waste activated sludge. In this work, a rhamnolipid (RL) pretreatment technology was proposed to promote methane production from two-phase anaerobic digestion of waste activated sludge. In the first phase (i.e., acidogenic phase), the WAS hydrolysis and acidogenesis were significantly enhanced after RL pretreatment for 4 day, under which the concentration of soluble protein and the short-chain fatty acids (SCFA) in the presence of RL at 0.04 g/g TSS was respectively 2.50 and 5.02 times higher than that without RL pretreatment. However, methane production was inhibited in the presence of RL. In the second phase (i.e., methanogenic phase), batch biochemical methane potential tests suggested that the addition of RL is effective in promoting anaerobic methane production. With an increase of RL dosage from 0 to 0.04 g/g TSS, the cumulative methane yield increased from 100.42 ± 3.01 to 168.90 ± 5.42 mL. Although the added RL could be utilized to produce methane, it was not the major contributor to the enhancement of methane yield. Further analysis revealed that total cumulative yield from the entire two-phase anaerobic digestion (sum of the yield of the acidogenic phase and methanogenic phase) increased from 113.42 ± 3.56 to 164.18 ± 5.20 mL when RL dosage increased from 0 to 0.03 g/g TSS, indicating that the addition of RL induced positive effect on the methane production of the entire two-phase anaerobic digestion. The enzyme activity analysis showed that although higher dosages of RL still inhibited the microorganisms related to methanogenesis to some extends in the methanogenic phase, the inhibitory effect was significantly weakened compared to the acidogenic phase. Microbial analysis revealed that RL reduced the abundance of Candidatus_Methanofastidiosum sp. while increased the abundance of Methanosaeta sp., causing the major methanogenesis pathway to change from hydrogenotrophic to aceticlastic. Moreover, the community of hydrolytic microbes and acidogens was shifted in the direction that is conducive to hydrolysis-acidogenesis. The findings reported not only expand the application field of RL, but also may provide supports for sustainable operation of wastewater treatment plants (WWTPs). [Display omitted] [ABSTRACT FROM AUTHOR]

• Pretreatment with 0.1 g/g TSS KMnO 4 increased SCFAs yield from WAS to 7.4 times. • KMnO 4 pretreatment promoted the degradation of humus and lignocellulose. • KMnO 4 reduced the species and total detection frequency of organic contaminants. • KMnO 4 pretreatment enhanced the inactivation of the fecal coliforms. Potassium permanganate (KMnO 4), one typical strong oxidant, is used extensively in various applications. However, its impact on anaerobic fermentation of waste activated sludge is unknown. This paper therefore aims to investigate its effect on generation of short-chain fatty acids (SCFAs) and qualities of fermentation liquor and fermented solid sludge. Experimental results showed that when KMnO 4 increased from 0 to 0.1 g/TSS, the highest SCFAs production enhanced from 33.9 to 251.8 mg COD/g VSS. The mechanism analysis revealed that KMnO 4 addition increased the disintegration of sludge cells and the degradation of substantial recalcitrant organics in sludge such as humus and lignocellulose, thereby provided more substances for SCFAs generation. Ultraviolet absorption spectroscopy analysis showed that KMnO 4 addition destroyed unsaturated conjugated bonds and decreased organics aromaticity, thereby promoting humus and lignocellulose degradation. GC/MS analyses showed that several micro-molecular substances (e.g., acid-like, alkane-like, and alcohol-like organics) were generated from KMnO 4 pretreated humus and lignocellulose, and some of them were demonstrated to serve as substrates to produce SCFAs. The enzyme activity and model-based analyses showed that although KMnO 4 restrained all the microorganisms to some extends, the activities of SCFAs generators were much higher than those of SCFAs consumers. In addition, the sludge mixture was separated into liquid and solid fraction after anaerobic fermentation, and the components in fermentation liquid and solid sludge were tested. It was found that the addition of KMnO 4 effectively reduced the categories and total detected frequency of refractory organic pollutants in fermentation liquid, which improved the quality of the produced SCFAs. KMnO 4 addition also increased the inactivation of fecal coliforms and degradation of emerging contaminants in fermented sludge, which were helpful to its final disposal. [ABSTRACT FROM AUTHOR]

• The influences of PET microplastics on WAS aerobic digestion is firstly explored; • PET microplastics of different entry paths affect WAS aerobic digestion differently; • PET microplastics directly entered inhibit WAS aerobic digestion more seriously; • PET microplastics reduce populations of key bacteria involved in aerobic digestion; • Function mechanisms of PET microplastics with different entry paths are disclosed. The entering of the widespread polyethylene terephthalate (PET) microplastics into biological wastewater treatment system results in their retention in sewage sludge, which inevitably enters the sludge treatment system. However, all previous studies regarding the impact of microplastics on sludge treatment system were conducted by directly adding microplastics to system and focusing on anaerobic sludge digestion, although PET microplastics commonly enter into the biological wastewater treatment system first before sludge being subsequently treated. The potential impact of the microplastics on waste activated sludge (WAS) aerobic digestion is also completely missing. Therefore, herein the influences of PET microplastics with different entry paths on WAS aerobic digestion as well as the key mechanisms involved was firstly explored. Experimental results demonstrated that compared to the control test, the entering of PET microplastics to biological wastewater treatment system inhibited WAS aerobic digestion by 10.9 ± 0.1% through the decreased hydrolysis, although WAS solubilization during aerobic digestion was improved due to the change of generated WAS characteristics. In contrast, when PET microplastics was directly added to the sludge aerobic digester, there was little impact on solubilization, while the hydrolysis were inhibited seriously, thereby suppressing WAS aerobic digestion more severely by 28.9 ± 0.1%. Further investigation revealed that PET microplastics reduced the populations of key bacteria (e.g., Saprospiraceae, Chitinophagaceae and Xanthomonadaceae) involved in aerobic digestion via induced oxidative stress or/and releasing toxic chemical. This study provided a more accurate approach to assessing the real situation regarding the influences of PET microplastics on aerobic sludge digestion. Image, graphical abstract [ABSTRACT FROM AUTHOR]

• PFS's inhibition on anaerobic WAS digestion was firstly evaluated • PFS restrained bioprocess of solubilization, hydrolysis, acidogenesis and methanogenesis • PFS decreased microbial diversity and caused a shift of microbial structure • Hydroxyl polymers and sulfate from PFS contributed most to the decreased biomethane production • Hydroxyl polymers were the chief buster to the slowed digestion rate and extended lag time Poly ferric sulfate (PFS), one of the typical inorganic flocculants widely used in wastewater management and waste activated sludge (WAS) dewatering, could be accumulated in WAS and inevitably entered in anaerobic digestion system at high levels. However, knowledge about its impact on methane production is virtually absent. This study therefore aims to fill this gap and provide insights into the mechanisms involved through both batch and long-term tests using either real WAS or synthetic wastewaters as the digestion substrates. Experimental results showed that the maximum methane potential and production rate of WAS was respectively retarded by 39.0% and 66.4%, whereas the lag phase was extended by 237.0% at PFS of 40 g per kg of total solids. Mechanism explorations exhibited that PFS induced the physical enmeshment and disrupted the enzyme activity involved in anaerobic digestion, resulting in an inhibitory state of the bioprocess of hydrolysis, acidogenesis, and methanogenesis. Furthermore, PFS's inhibition to hydrogenotrophic methanogenesis was much severer than that to acetotrophic methanogenesis, which could be supported by the elevated abundances of Methanosaeta sp and the dropped abundances of Methanobacterium sp in PFS-present digester, and probably due to the severe mass transfer resistance of hydrogen between the syntrophic bacteria and methanogens, as well as the higher hydrogen appetency of PFS-induced sulfate reducing bacteria. Among the derivatives of PFS, "multinucleate and multichain-hydroxyl polymers" and sulfate were unveiled to be the major contributors to the decreased methane potential, while the "multinucleate and multichain-hydroxyl polymers" were identified to be the chief buster to the slowed methane-producing rate and the extended lag time. Image, graphical abstract [ABSTRACT FROM AUTHOR]

The utilization of waste activated sludge (WAS) to recover energy in the form of methane or short-chain fatty acids (SCFAs) is generally restricted by the low energy density of products and poor degradability of WAS. Herein, this study reported a novel alternative WAS fermentation technology to produce high-energy medium-chain fatty acids (MCFAs) from WAS in a one-stage anaerobic fermentation system using ethanol as electron donor. The MCFAs production and WAS degradation at different ethanol levels were investigated. The increased ethanol levels resulted in increasing MCFAs production (from 1875 to 6115 mg chemical oxygen demand (COD)/L) and selectivity (from 30.3 to 56.2%). The main MCFAs products were n-caproate and n-caprylate at lower level of ethanol, while n-caproate was the sole MCFA product at higher level of ethanol with longer chain alcohol (i.e., n-hexanol) produced as well. The ethanol markedly increased WAS degradation, with the greatest degradation (0.72 g COD/g volatile solids (VS)) being 1.9 times of that without ethanol (0.38 mg COD/mg VS, at 0 mmol/L), which was ascribed to the advancement of sludge solubilization, hydrolysis and acidification. Microbial community revealed that the ethanol participation induced the community shift to the favorable direction for hydrolysis-acidification and chain elongation in anaerobic WAS fermentation. Image 1 • MCFAs production from WAS and enhanced WAS degradation were simultaneously achieved. • Sludge solubilization, hydrolysis and acidification were enhanced by ethanol. • MCFAs production and selectivity were positively related to the ethanol addition. • Hydrolytic, acidification and CE microbes were enriched for MCFAs production. [ABSTRACT FROM AUTHOR]

Hydrogen production from waste activated sludge (WAS) was widely considered and intensively investigated as a promising technology to recover energy from wastewater treatment plants. To date, no efforts have been made on either systematic summarization or critical thinking of the application niche of hydrogen production from WAS treatment. It is therefore time to evaluate whether and how to recover hydrogen in a future paradigm of WAS treatment. In this critical review, the principles and potentials, microorganisms, possible technologies, and process parameters of hydrogen generation were analyzed. Microbial electrolysis cell shows high theoretical hydrogen yield and could utilize a variety of organic compounds as substrates, which is regarded as a prospective technology for hydrogen production. However, the poor organics utilization and rapid consumptions of produced hydrogen hindered hydrogen recovery from WAS. Based on the analysis of the current state of the literatures, the opportunities and challenges of hydrogen production from WAS are rethought, the detailed knowledge gaps and perspective of hydrogen production from WAS were discussed, and the probable solutions of hydrogen recovery from WAS treatment are figured out. To guide the application and development of hydrogen recovery, a more promising avenue through rational integration of the available technologies to form a hybrid process is finally proposed. The integrated operational paradigm of WWTPs could achieve substantial technical, environmental and economic benefits. In addition, how this hybrid process works is illustrated, the challenges of this hybrid process and future efforts to be made in the future are put forward. Image 1 • H 2 is mainly produced from the acidogenesis process of WAS dark fermentation. • Microbial electrolysis cell is a prospective technology for H 2 production. • The poor organics utilization and rapid H 2 consumption hinder hydrogen recovery. • A promising avenue through integration of the available technologies was proposed. • The hybrid process could achieve substantial technical and economic benefits. [ABSTRACT FROM AUTHOR]

• Underlying mechanisms of CaO 2 disintegrating EPS and cell membrane are reviewed. • Sludge dewatering and minimization can be achieved effectively by CaO 2. • Methane production from sludge anaerobic digestion can be enhanced significantly by CaO 2 -based treatment. • SCFA and hydrogen yield in sludge anaerobic fermentation can be improved prominently with CaO 2 addition. • CaO 2 can promote the removal of emerging contaminants via the generated free radicals oxidation and alkali. The treatment and disposal of waste activated sludge (WAS) has become one of the major challenges for the wastewater treatment plants (WWTPs) due to large output, high treatment costs and enriched substantial emerging contaminants (ECs). Therefore, reducing sludge volume, recovering energy and resource from WAS, and removing ECs and decreasing environmental risk have gained increasing attentions. Calcium peroxide (CaO 2), a versatile and safe peroxide, has been widely applied in terms of WAS treatment including sludge dewatering, anaerobic sludge digestion and anaerobic sludge fermentation due to its specific properties such as generating free radicals and alkali, etc., providing supports for sludge reduction, recycling, and risk mitigation. This review outlines comprehensively the recent progresses and breakthroughs of CaO 2 in the fields of sludge treatment. In particular, the relevant mechanisms of CaO 2 enhancing WAS dewaterability, methane production from anaerobic digestion, short-chain fatty acids (SCFA) and hydrogen production from anaerobic fermentation, and the removal of ECs in WAS and role of experiment parameters are systematically elucidated and discussed, respectively. Finally, the knowledge gaps and opportunities in CaO 2 -based sludge treatment technologies that need to be focused in the future are prospected. The review presented can supply a theoretical basis and technical reference for the application of CaO 2 for improving the treatment of WAS. Image, graphical abstract [ABSTRACT FROM AUTHOR]

• Medium-chain fatty acid (MCFA) and Long-chain alcohol (LCA) co-production was gained. • MCFA and LCA co-production is positively related to ethanol content. • High level of ethanol could improve the selectivity of MCFA but reduce that of LCA. • Key functional microbes for co-production of MCFA and LCA were enriched. Traditional bioenergy recovery in the form of short chain fatty acids (SCFAs) from waste activated sludge (WAS) is generally limited by economic unattractiveness and complexity of products separation. Herein, a novel biotechnology process of two-stage anaerobic fermentation for converting the WAS into high energy density, easy-separated medium chain fatty acids (MCFAs) and long-chain alcohols (LCAs) was evaluated. In this process, the WAS was first converted to WAS alkaline fermentation liquid (WASAFL), serving as electron acceptors (EAs) and inoculum, then adding ethanol as electron donor (ED) for chain elongation (CE). The co-production of MCFAs and LCAs during CE were studied under three different ED to EA ratios, i.e., 3:1, 4:1 and 5:1. Experimental results demonstrated that when the ratio of ED to EA increased from 3:1 to 5:1, the production of MCFA and LCAs respectively increased from 5.57 ± 0.17 and 2.58 ± 0.18 to7.67 ± 0.48 and 4.21 ± 0.19 g COD/L. A similar observation was made in the total product electron efficiency, increasing from 59.9% to 72.1%. However, the highest total product selectivity (i.e., 68.0%) and highest products production yield (i.e., 59.77%) were not achieved at the ED to EA ratio of 5:1 due to toxicity caused by higher accumulation of n-caproate. The kinetic analysis further confirmed that high ratio of ED to EA induced improvement in product maximum yield, production rate for both MCFAs and LCAs. Microbial community analysis indicated that Clostridium, Caproiciproducens, Acinetobacter, Exilispira , and Oscillibacter were clearly enriched in the CE reactor and had positive correlation with MCFAs and LCAs production. Image, graphical abstract [ABSTRACT FROM AUTHOR]

• Freezing activated nitrite pretreatment (FN) enhanced anaerobic digestion from WAS. • FN benefited WAS disintegration and the biodegradable of organics released. • FN benefited the dewaterability or pathogens inactivation of digested sludge. • Freezing induced reactive derivatives from nitrite were the major contributors. • FN was economically and environmentally attractive. Nitrite-based pretreatment was demonstrated to effectively improve anaerobic digestion of waste activated sludge. It was found in this work that the freezing activated nitrite pretreatment could further enhance the performances. With the increase of nitrite addition from 0 to 600 mg NO 2 −-N/L during freezing process, the biochemical methane potential of pretreated-sludge gradually increased from 191.3 ± 8.0 to 233.2 ± 10.6 mL per gram volatile solid (VS), while only 178.6 ± 7.3 mL/g VS was obtained in the raw sludge. Mechanism explorations revealed that the freezing activated nitrite pretreatment remarkably facilitated the disintegration of sludge. Excitation emission matrix and fluorescence regional integration analyses further revealed that nitrite addition during freezing process promoted the proportion of biodegradable organics released, thereby providing more bio-available substrates for subsequent anaerobic digestion. Freezing condition induced reactive derivatives from nitrite (e.g., free nitrite acid, NO 2 , N 2 O 3) were assumed to be the major contributors to the enhanced sludge disintegration and recalcitrant organics (e.g., humic acid-like substances) degradation. It was also found that 600 mg NO 2 −-N/L addition activated by freezing pretreatment produced an anaerobically digested sludge with an improved dewaterability, as indicated by the decrease of the specific resistance to filterability and moisture content of dewatered cake. Moreover, 600 mg NO 2 −-N/L addition activated by freezing pretreatment and subsequent anaerobic digestion largely inactivated the pathogens to the levels below Class A biosolids requirements. Considering that nitrite can be in-situ produced in wastewater treatment plants through nitritation of the digestion liquid, this nitrite-based freezing process for sludge pretreatment was environmental-friendly and economically attractive. [ABSTRACT FROM AUTHOR]

The increasing use of synthetic musks has led to a large amount of synthetic musks retaining in waste activated sludge (WAS) via wastewater treatment, thereby entering anaerobic digester. However, the potential effects of synthetic musks on WAS anaerobic digestion remain unknown. Herein, this study selected the dominant galaxolide (HHCB) in WAS as the typical synthetic musks and experimentally evaluated the long-term effects on WAS anaerobic digestion using continuous lab-scale anaerobic digesters as well as the mechanisms involved. The results demonstrated that the increased HHCB levels (i.e., 90, 150 and 200 mg/kg-dw) resulted in the decreased methane production, with the methane production at 200 mg/kg-dw being only 80.5 ± 0.1% of the control. Supporting the methane production data, volatile solids (VS) destruction decreased by 18.6 ± 0.9%, which increased 6.8% of volume waste sludge for transfer and disposal. Correspondingly, the microbial community was shifted in the direction against anaerobic digestion. By modeling based on biochemical methane potential tests and investigating the key stages involved in anaerobic digestion, it was found that although the HHCB showed little impacts on the solubilization, WAS hydrolysis-acidification steps was inhibited by HHCB with the decreased hydrolysis rate and methane production potential, thereby causing the deteriorated performance of WAS anaerobic digestion. Unlabelled Image • Inhibitory effect of HHCB on WAS during anaerobic digestion is revealed. • Decreased k and Y with HHCB result in the deteriorative digestion performance. • HHCB would cause a 6.8% increase in the volume of waste sludge for final disposal. • Quantities of key anaerobic microorganisms reduced in the presence of HHCB. [ABSTRACT FROM AUTHOR]

Alkaline pretreatment was demonstrated to be effective in the enhancement of hydrogen production. However, the sludge solubilization rate of alkaline pretreatment is still limited. This study reports a new strategy of K 2 FeO 4 + pH 9.5 for sludge mesophilic anaerobic fermentation. Experimental results showed that the combination of K 2 FeO 4 /pH 9.5 pretreatment had a greater hydrogen yield than the individual K 2 FeO 4 and pH 9.5. The maximum hydrogen yield was 19.2 mL per gram volatile suspended solids (VSS) under the optimal condition (0.02 g per gram total suspended solids K 2 FeO 4 + pH 9.5). Kinetic analysis showed that the highest hydrogen production potential of 19.9 mL/g VSS was obtained in the combined reactor, which well fitted the first-order kinetic model (R2 = 0.9925). Besides, the fermentation type was mainly acetic and butyric in the combined reactor, which contributed to hydrogen production. Further analyses showed that the combined pretreatment reduced hydrogen sulfide yield, providing an environmentally friendly method to sludge treatment. Unlabelled Image • pH 9.5 + K 2 FeO 4 treatment promoted hydrogen yield from waste activated sludge. • pH 9.5 + K 2 FeO 4 treatment benefited the lysis of sludge cells. • The fermentation type in the combined reactor was mainly acetic and butyric. • pH 9.5 + K 2 FeO 4 treatment inhibited all the microbes in anaerobic fermentation. [ABSTRACT FROM AUTHOR]

Due to the poor biodegradability of released organics and the rapid consumption of hydrogen, hydrogen production from the untreated waste activated sludge (WAS) and/or inocula is still limited. In this study, it was found that the dark fermentative hydrogen production was largely enhanced from WAS pretreated by freezing in the presence of nitrite. With an increase of nitrite addition from 100 to 400 mg NO 2 −-N/L during freezing pretreatment (−5 °C for 4 h), the maximal hydrogen yield increased from 7.96 to 19.40 mL/g VS (volatile solids), which was 5.5–13.4 times of that in the control (without freezing and nitrite addition). Mechanism explorations revealed that the proposed pretreatment not only accelerated the disintegration of sludge but also promoted the proportion of biodegradable organics released, thereby provided more bio-available substrates for subsequent hydrogen production. Proposed pretreatment severely suppressed the sludge microorganisms responding to homoacetogenesis (−32.1%), methanogenesis (−58.4%), and sulfate-reducing process (−51.5%), inhibited the consumption of hydrogen. Moreover, there was more acetic and butyric (76% versus 57.5%) but less propionic acid (22.6% versus 13.4%) in this pretreated fermenter, which was in correspondence with the theory of fermentation type affecting hydrogen production. Long-term fermentation experiments indicated that the proposed pretreatment boosted the [FeFe]-hydrogenase activities while suppressed the activities of carbon monoxide dehydrogenase, coenzyme F420, and adenylyl sulfate reductase. Image 1 • FNP enhanced dark fermentative hydrogen production from WAS by 5.5–13.4 times. • FNP accelerated sludge disintegration and promoted organics biodegradability. • FNP suppressed the hydrogen-consuming bacteria by 32.1–58.4%. • FNP induced the shift of fermentation to butyrate-type. [ABSTRACT FROM AUTHOR]

Effective sludge treatment with bioenergy production is attracting increasing interests as large quantities of waste activated sludge (WAS) are produced during the wastewater treatment. In this study, a new biotechnical process for converting the WAS alkaline fermentation liquor (WASAFL) into valuable, easy-separated medium chain fatty acids (MCFAs) through chain elongation (CE) was investigated, which may provide a new insight into sludge treatment. In the process, ethanol was served as the electron donor (EDs) and WASAFL were main electron acceptors (EAs). The MCFAs productions were investigated under three different ED to EA ratios (i.e., 1:2, 1:1 and 2:1). The result showed that MCFAs production was increased from 2.88 ± 0.01 to 5.28 ± 0.18 g COD/L with the increase of ED to EA ratio. However, the highest MCFA selectivity was achieved at 72.9% when the ED to EA ratio was 1:1. The decrease in the selectivity at high ED:EA ratio is mainly due to the production of higher alcohol (i.e., n-butanol and n-hexanol). The thermodynamic analysis confirmed all CE processes for MCFAs production from WASAFL were exothermic reactions, with the spontaneity and energy release of the reactions increased with the ethanol level. The microbial community analysis showed that the relative abundances of Clostridium, Oscillibacter , Leptolinea and Exilispira were positively correlated with the MCFAs production. The metagenomic analysis suggested that both the reverse β-oxidization pathway and fatty acid biosynthesis pathway contributed to the CE process in the studied system. The functional enzymes were mainly associated within Clostridium, with Clostridium Kluyveri , Clostridium botulinum and Clostridium magnum being likely the key species responsible for the CE process. Image 1 • MCFAs production from WAS alkaline fermentation liquid was achieved. • MCFA production is positively related to the level of ethanol as electron donor. • A high level of ethanol could reduce the selectivity of MCFA production. • Key functional microbes involved C. Kluyveri , C. botulinum and C. magnum. • Production pathways included reverse β-oxidation and fatty acid biosynthesis. [ABSTRACT FROM AUTHOR]

Recent investigations verified that calcium peroxide (CaO 2) could be used to pretreat waste activated sludge to promote methane yield from anaerobic digestion. However, the underlying mechanism of how CaO 2 pretreatment promotes methane production is unclear. This work therefore aims to provide insights into such systems. Experimental results showed that with an increase of CaO 2 dosage from 0 to 0.14 g/g VSS (volatile suspended solids) the methane yield increased linearly from 146.3 to 215.9 mL/g VSS. Further increases of CaO 2 resulted in decreases in methane yield. CaO 2 pretreatment promoted the disintegration of sludge and the degradation of sludge recalcitrant organics (especially humus and lignocellulose), thereby providing more substrates for subsequent methane production. Ultraviolet absorption spectroscopy indicated that CaO 2 enhanced the cleavage of unsaturated conjugated bonds and reduced the aromaticity of humus and lignocellulose. Fourier transform infrared spectroscopy showed that CaO 2 changed the structures and functional groups of humus and lignocellulose, making them transform to be biodegradable. GC/MS analyses exhibited that the degradation products of humus and lignocellulose included several types of small molecular organics such as ester-like, acid-like, and alcohol-like substances. Further investigation demonstrated that substantial methane could be produced from these degradation products. It was also found that the presence of recalcitrant organics was detrimental to anaerobes relevant to anaerobic digestion, and the degradation of such recalcitrant organics mitigated their inhibitions to the anaerobes. Model-based analysis suggested that CaO 2 pretreatment increased the maximum methane yield and methane production rate, which were consistent with the analysis above. Image 1 • Pretreatment with 0.14 g/g VSS CaO 2 increased methane yield to 1.3 times from WAS. • CaO 2 pretreatment promoted the degradation of humus and lignocellulose. • CaO 2 could mitigate inhibitions of recalcitrant organics to the anaerobes. • The CaO2 reaction residues inhibited all the microbes relevant to anaerobic digestion. [ABSTRACT FROM AUTHOR]

Alkaline (especially pH 10) anaerobic fermentation of waste activated sludge (WAS) has been reported to be an effective approach for hydrogen production through inhibiting the homoacetogenesis and methanogenesis. However, the potential effect of the widespread microplastics in sludge on the performance of hydrogen production has never been reported. To fill this knowledge gap, the dominant polyethylene terephthalate (PET) microplastics in WAS were selected as the model microplastics to evaluate their influences on hydrogen production during alkaline anaerobic fermentation of WAS as well as the key mechanisms involved. Experimental results demonstrated that hydrogen production from WAS decreased in the presence of PET microplastics (i.e., 10, 30 and 60 particles/g-TS) compared to the control, with the hydrogen yield at 60 particles/g-TS being only 70.7 ± 0.9% of the control. Although the hydrogen consumption (i.e., homoacetogenesis and methanogenesis) was restrained under alkaline (pH 10) condition, PET microplastics inhibited hydrolysis, acidogenesis and acetogenesis in alkaline WAS anaerobic fermentation, leading to the inhibitory effect on hydrogen production. This was further confirmed by the microbial analysis, which clearly showed PET microplastics caused the shift of the microbial community toward the direction against hydrolysis-acidification. Mechanism studies revealed that PET microplastics carried on their negative influence mainly through leaching the toxic di-n-butyl phthalate (DBP). The reactive oxygen species (ROS) and live/dead staining tests indicated that the increased ROS was induced by PET microplastics, causing more cells dead, which further resulted in the decreased production of hydrogen. Image 1 • PET microplastics decreased hydrogen production from alkaline (pH 10) anaerobic fermentation of WAS. • PET microplastics inhibited sludge hydrolysis, acidogenesis and acetogenesis processes. • PET microplastics caused the shift of the microbial community toward the direction against hydrolysis-acidification. • PET microplastics carried on their negative effect mainly through the toxic DBP leached and ROS induced by PET microplastics. [ABSTRACT FROM AUTHOR]

• Microwave pretreatment (MP) benefited the degradation of PAM. • MP accelerated the release and hydrolysis of organics from PF-WAS. • MP facilitated the breaking of large firm "PAM-sludge" floccules. • Hydrolysis rate of PF-WAS was accelerated through MP. • Biochemical methane potential of PF-WAS was improved through MP. Deterioration of anaerobic digestion can occur with the presence of polyacrylamide (PAM) in waste activated sludge, but the information on alleviating this deterioration is still limited. In this study, the simultaneous alleviation of negative effect of PAM and improvement of methane production during anaerobic digestion was accomplished by microwave pretreatment. Experimental results showed that with the microwave pretreatment times increased from 0 to 12 min, the biochemical methane potential of PAM-flocculated sludge (12 g PAM/kg total solids) asymptotically increased from 123.1 to 242.5 mL/g volatile solids, hydrolysis rate increased from 0.06 to 0.13 d−1. Mechanism analysis indicated that the microwave pretreatment accelerated the release and hydrolysis of organic substrates from PAM-flocculated sludge, facilitated the breaking of large firm "PAM-sludge" floccules, and benefited the degradation of PAM, which alleviated the PAM inhibitory impacts on digestion and meanwhile provided better contact between the released organic substrates and anaerobic bacteria for methane production. [ABSTRACT FROM AUTHOR]