Reducing Soil-Emitted Nitrous Acid as a Feasible Strategy for Tackling Ozone Pollution.

Severe ozone (O3) pollution has been a major air quality issue and affects environmental sustainability in China. Conventional mitigation strategies focusing on reducing volatile organic compounds and nitrogen oxides (NOx) remain complex and challenging. Here, through field flux measurements and laboratory simulations, we observe substantial nitrous acid (HONO) emissions (FHONO) enhanced by nitrogen fertilizer application at an agricultural site. The observed FHONO significantly improves model performance in predicting atmospheric HONO and leads to regional O3 increases by 37%. We also demonstrate the significant potential of nitrification inhibitors in reducing emissions of reactive nitrogen, including HONO and NOx, by as much as 90%, as well as greenhouse gases like nitrous oxide by up to 60%. Our findings introduce a feasible concept for mitigating O3 pollution: reducing soil HONO emissions. Hence, this study has important implications for policy decisions related to the control of O3 pollution and climate change.

Impacts of free nitrous acid on stabilizing food waste and sewage sludge for anaerobic digestion.

This work investigated the effectiveness of free nitrous acid (FNA) in enhancing organic waste solubilization to improve biogas production in anaerobic digestion (AD). The results indicated that FNA pretreatment can enhance soluble organic content and control H2S odor in tested organic wastes, including food waste, sewage sludge, and their combination. However, a significant decrease (>50 %) in FNA concentration was found in the reactors, possibly due to denitrifier-driven NO2- consumption. Biochemical methane potential (BMP) tests showed a 25 ± 8 % enhancement in CH4 production in the reactors fed with mixed substrate pretreated with 2.9 mg FNA-N/L. However, the presence of NO2- (325.6-2368.0 mg N/L) in some BMP reactors, due to carryover from FNA pretreatment, adversely affected CH4 production (>55 %) and prolonged lag time (>4.2 times). These findings are valuable for researchers and practitioners in waste management, offering insights for implementing FNA pretreatment to enhance the biodegradability of organic wastes in AD.

Potential of free nitrous acid (FNA) for sludge treatment and resource recovery from waste activated sludge: A review.

The escalating production of waste activated sludge (WAS) presents significant challenges to wastewater treatment plants (WWTPs). Free nitrous acid (FNA), known for its biocidal effect, has gained a growing focus on sludge dewatering, sludge reduction, and resource recovery from WAS due to its eco-friendly and cost-effective properties. Nevertheless, there have been no attempts made to systematically summarize or critically analyze the application of FNA in enhancing treatment and resource utilization of sludge. In this paper, we provided an overview of the current understanding regarding the application potential and influencing factors of FNA in sludge treatment, with a specific focus on enhancing sludge dewatering efficiency and reducing volume. To foster resource development from sludge, various techniques based on FNA have recently been proposed, which were comprehensively reviewed with the corresponding mechanisms meticulously discussed. The results showed that the chemical oxidation and interaction with microorganisms of FNA played the core role in improving resource utilization. Furthermore, current challenges and future prospects of the FNA-based applications were outlined. It is expected that this review can refine the theoretical framework of FNA-based processes, providing a theoretical foundation and technical guidance for the large-scale demonstration of FNA.

Phase State Regulates Photochemical HONO Production from NaNO3/Dicarboxylic Acid Mixtures.

Field observations of daytime HONO source strengths have not been well explained by laboratory measurements and model predictions up until now. More efforts are urgently needed to fill the knowledge gaps concerning how environmental factors, especially relative humidity (RH), affect particulate nitrate photolysis. In this work, two critical attributes for atmospheric particles, i.e., phase state and bulk-phase acidity, both influenced by ambient RH, were focused to illuminate the key regulators for reactive nitrogen production from typical internally mixed systems, i.e., NaNO3 and dicarboxylic acid (DCA) mixtures. The dissolution of only few oxalic acid (OA) crystals resulted in a remarkable 50-fold increase in HONO production compared to pure nitrate photolysis at 85% RH. Furthermore, the HONO production rates (PHONO) increased by about 1 order of magnitude as RH rose from <5% to 95%, initially exhibiting an almost linear dependence on the amount of surface absorbed water and subsequently showing a substantial increase in PHONO once nitrate deliquescence occurred at approximately 75% RH. NaNO3/malonic acid (MA) and NaNO3/succinic acid (SA) mixtures exhibited similar phase state effects on the photochemical HONO production. These results offer a new perspective on how aerosol physicochemical properties influence particulate nitrate photolysis in the atmosphere.

Favipiravir biotransformation by a side-stream partial nitritation sludge: Transformation mechanisms, pathways and toxicity evaluation.

Information on biotransformation of antivirals in the side-stream partial nitritation (PN) process was limited. In this study, a side-stream PN sludge was adopted to investigate favipiravir biotransformation under controlled ammonium and pH levels. Results showed that free nitrous acid (FNA) was an important factor that inhibited ammonia oxidation and the cometabolic biodegradation of favipiravir induced by ammonia oxidizing bacteria (AOB). The removal efficiency of favipiravir reached 12.6% and 35.0% within 6 days at the average FNA concentrations of 0.07 and 0.02 mg-N L-1, respectively. AOB-induced cometabolism was the sole contributing mechanism to favipiravir removal, excluding AOB-induced metabolism and heterotrophic bacteria-induced biodegradation. The growth of Escherichia coli was inhibited by favipiravir, while the AOB-induced cometabolism facilitated the alleviation of the antimicrobial activities with the formed transformation products. The biotransformation pathways were proposed based on the roughly identified structures of transformation products, which mainly involved hydroxylation, nitration, dehydrogenation and covalent bond breaking under enzymatic conditions. The findings would provide insights on enriching AOB abundance and enhancing AOB-induced cometabolism under FNA stress when targeting higher removal of antivirals during the side-stream wastewater treatment processes.

Additional HONO and OH Generation from Photoexcited Phenyl Organic Nitrates in the Photoreaction of Aromatics and NOx.

HONO acts as a major OH source, playing a vital role in secondary pollutant formation to deteriorate regional air quality. Strong unknown sources of daytime HONO have been widely reported, which significantly limit our understanding of radical cycling and atmospheric oxidation capacity. Here, we identify a potential daytime HONO and OH source originating from photoexcited phenyl organic nitrates formed during the photoreaction of aromatics and NOx. Significant HONO (1.56-4.52 ppb) and OH production is observed during the photoreaction of different kinds of aromatics with NOx (18.1-242.3 ppb). We propose an additional mechanism involving photoexcited phenyl organic nitrates (RONO2) reacting with water vapor to account for the higher levels of measured HONO and OH than the model prediction. The proposed HONO formation mechanism was evidenced directly by photolysis experiments using typical RONO2 under UV irradiation conditions, during which HONO formation was enhanced by relative humidity. The 0-D box model incorporated in this mechanism accurately reproduced the evolution of HONO and aromatic. The proposed mechanism contributes 5.9-36.6% of HONO formation as the NOx concentration increased in the photoreaction of aromatics and NOx. Our study implies that photoexcited phenyl organic nitrates are an important source of atmospheric HONO and OH that contributes significantly to atmospheric oxidation capacity.

Modeling Free Nitrous Acid Inhibition on the Removal of Nitrogen and Atenolol during Sidestream Partial Nitritation Processes.

Sidestream serves as an important reservoir collecting pharmaceuticals from sludge. However, the knowledge on sidestream pharmaceutical removal is still insufficient. In this work, atenolol biodegradation during sidestream partial nitritation (PN) processes characterized by high free nitrous acid (FNA) accumulation was modeled. To describe the FNA inhibition on ammonia oxidation and atenolol removal, Vadivelu-type and Hellinga-type inhibition kinetics were introduced into the model framework. Four inhibitory parameters along with four biodegradation kinetic parameters were calibrated and validated separately with eight sets of batch experimental data and 60 days' PN reactor operational data. The developed model could accurately reproduce the dynamics of nitrogen and atenolol. The model prediction further revealed that atenolol biodegradation efficiencies by ammonia-oxidizing bacteria (AOB)-induced cometabolism, AOB-induced metabolism, and heterotrophic bacteria-induced biodegradation were 0, ∼ 60, and ∼35% in the absence of ammonium and FNA; ∼ 14, ∼ 29, and ∼28% at 0.03 mg-N L-1 FNA; and 7, 15, and 5% at 0.19 mg-N L-1 FNA. Model simulation showed that the nitritation efficiency of ∼99% and atenolol removal efficiency of 57.5% in the PN process could be achieved simultaneously by controlling pH at 8.5, while 89.2% total nitrogen and 57.1% atenolol were removed to the maximum at pH of 7.0 in PN coupling with the anammox process. The pH-based operational strategy to regulate FNA levels was mathematically demonstrated to be effective for achieving the simultaneous removal of nitrogen and atenolol in PN-based sidestream processes.

Low alkalinity, free ammonia, and free nitrous acid cooperatively stabilize partial nitrification under excessive aeration condition.

In this study, a lab-scale sequencing batch reactor (SBR) was established to assess the feasibility of maintaining partial nitrification in treating high-ammonium wastewater under the condition of excessive aeration (1 L/min, 1289 min). Results showed that the average ammonia (NH4+-N) removal efficiency (ARE) was 93.4 ± 2.7% and the average nitrite accumulation ratio (NAR) was 90.7 ± 2.8% during 15-50 d. In a typical cycle, free ammonia (FA) and free nitrous acid (FNA) alternately inhibited the activity of nitrite oxidizing bacteria (NOB), while low alkalinity limited further nitrification at even excessive aeration. Metagenomic analysis revealed that the relative abundance of Nitrosomonas, as the ammonia oxidizing bacteria (AOB), was up to 1.61%, while NOB were not detected. Taken together, this study indicated partial nitrification was almost not affected by excessive aeration, demonstrating its robustness depending on the cooperative strategy of the low alkalinity limitation and the inhibition of FA and FNA.

Revisiting the Ultraviolet Absorption Cross Section of Gaseous Nitrous Acid (HONO): New Insights for Atmospheric HONO Budget.

Nitrous acid (HONO) is an important source of hydroxyl radicals (OH) in the atmosphere. Precise determination of the absolute ultraviolet (UV) absorption cross section of gaseous HONO lays the basis for the accurate measurement of its concentration by optical methods and the estimation of HONO loss rate through photolysis. In this study, we performed a series of laboratory and field intercomparison experiments for HONO measurement between striping coil-liquid waveguide capillary cell (SC-LWCC) photometry and incoherent broadband cavity-enhanced absorption spectroscopy (IBBCEAS). Specified HONO concentrations prepared by an ultrapure standard HONO source were utilized for laboratory intercomparisons. Results show a consistent ∼22% negative bias in measurements of the IBBCEAS compared with a SC-LWCC photometer. It is confirmed that the discrepancies occurring between these techniques are associated with the overestimation of the absolute UV absorption cross sections through careful analysis of possible uncertainties. We quantified the absorption cross section of gaseous HONO (360-390 nm) utilizing a custom-built IBBCEAS instrument, and the results were found to be 22-34% lower than the previously published absorption cross sections widely used in HONO concentration retrieval and atmospheric chemical transport models (CTMs). This suggests that the HONO concentrations retrieved by optical methods based on absolute absorption cross sections may have been underestimated by over 20%. Plus, the daytime loss rate and unidentified sources of HONO may also have evidently been overestimated in pre-existing studies. In summary, our findings underscore the significance of revisiting the absolute absorption cross section of HONO and the re-evaluation of the previously reported HONO budgets.

Limitations of a biokinetic model to predict the seasonal variations of nitrous oxide emissions from a full-scale wastewater treatment plant.

A biokinetic model based on BioWin's Activated Sludge Digestion Model (ASDM) coupled with a nitrous oxide (N2O) model was setup and calibrated for a full-scale wastewater treatment plant (WWTP) Amsterdam West, in the Netherlands. The model was calibrated using one year of continuous data to predict the seasonal variations of N2O emissions in the gaseous phase. This, according to our best knowledge, is the most complete full-scale data set used to date for this purpose. The results obtained suggest that the currently available biokinetic model predicted the winter, summer, and autumn N2O emissions well but failed to satisfactorily simulate the spring peak. During the calibration process, it was found that the nitrifier denitrification pathway could explain the observed emissions during all seasons while a combination of the nitrifier denitrification and incomplete heterotrophic denitrification pathways seemed to be dominant during the emissions peak observed during the spring season. Specifically, kinetic parameters related to free nitrous acid (FNA) displayed significant sensitivity leading to increased N2O production. The obtained values of two kinetic parameters, i.e., the FNA half-saturation during ammonia oxidising bacteria (AOB) denitrification and the FNA inhibition concentration related to heterotrophic denitrification, suggested a strong influence of the FNA bulk concentration on the N2O emissions and the observed seasonal variations. Based on the suboptimal performance and limitations of the biokinetic model, further research is needed to better understand the biochemical processes behind the seasonal peak and the influence of FNA.

Atmospheric HONO emissions in China: Unraveling the spatiotemporal patterns and their key influencing factors.

Nitrous acid (HONO) can be photolyzed to produce hydroxyl radicals (OH) in the atmosphere. OH plays a critical role in the formation of secondary pollutants like ozone (O3) and secondary organic aerosols (SOA) via various oxidation reactions. Despite the abundance of recent HONO studies, research on national HONO emissions in China remains relatively limited. Therefore, this study employed a "wetting-drying" model and bottom-up approach to develop a high-resolution gridded inventory of HONO emissions for mainland China using multiple data. We used the Monte Carlo method to estimate the uncertainty in HONO emissions. In addition, the primary sources of HONO emissions were identified and their spatiotemporal distribution and main influencing factors were studied. The results indicated that the total HONO emissions in mainland China in 2016 were 0.77 Tg N (R50: 0.28-1.42 Tg N), with soil (0.42 Tg N) and fertilization (0.26 Tg N) as the primary sources, jointly contributing to over 87% of the total. Notably, the North China Plain (NCP) had the highest HONO emission density (3.51 kg N/ha/yr). Seasonal HONO emissions followed the order: summer (0.38 kg N/ha) > spring (0.19 kg N/ha) > autumn (0.17 kg N/ha) > winter (0.06 kg N/ha). Moreover, HONO emissions were strongly correlated with fertilization, cropland, temperature, and precipitation. This study provides vital scientific groundwork for the atmospheric nitrogen cycle and the formation of secondary pollutants.

Unveiling the effect of O2 on the photochemical reaction of NO2 with polycyclic aromatic hydrocarbons.

The photochemical reaction of NO2 with organics may be a source of atmospheric HONO during the daytime. Here, the conversion of NO2 to HONO on polycyclic aromatic hydrocarbons (PAHs) under solar irradiation under aerobic and anaerobic conditions was investigated using a flow tube reactor coupled to a NOx analyzer. O2 played an inhibition role in NO2 uptake and HONO formation on PAHs, as shown by 7%-45% and 15%-52% decrease in NO2 uptake coefficient (γ) and HONO yield (YHONO), respectively. The negative effect of O2 on the reaction between NO2 and PAHs should be attributed to three reasons. First, O2 could compete with NO2 for the available sites on PAHs. Second, the quenching of the triple excited state of PAHs (3PAHs*) by O2 inhibited the NO2 uptake. Third, NO3- formed under aerobic conditions reduced the conversion efficiency of NO2 to HONO. The environmental implications suggested that the NO2 uptake on PAHs could contribute to a HONO source strength of 10-120 ppt h-1 in the atmosphere.

Quantifying the Nitrogen Sources and Secondary Formation of Ambient HONO with a Stable Isotopic Method.

Nitrous acid (HONO) is a reactive gas that plays an important role in atmospheric chemistry. However, accurately quantifying its direct emissions and secondary formation in the atmosphere as well as attributing it to specific nitrogen sources remains a significant challenge. In this study, we developed a novel method using stable nitrogen and oxygen isotopes (δ15N; δ18O) for apportioning ambient HONO in an urban area in North China. The results show that secondary formation was the dominant HONO formation processes during both day and night, with the NO2 heterogeneous reaction contributing 59.0 ± 14.6% in daytime and 64.4 ± 10.8% at nighttime. A Bayesian simulation demonstrated that the average contributions of coal combustion, biomass burning, vehicle exhaust, and soil emissions to HONO were 22.2 ± 13.1, 26.0 ± 5.7, 28.6 ± 6.7, and 23.2 ± 8.1%, respectively. We propose that the isotopic method presents a promising approach for identifying nitrogen sources and the secondary formation of HONO, which could contribute to mitigating HONO and its adverse effects on air quality.

Comparison of typical nitrite oxidizing bacteria suppression strategies and the effect on nitrous oxide emissions in a biofilm reactor.

In mainstream partial nitritation/anammox (PN/A), suppression of nitrite oxidizing bacteria (NOB) and mitigation of N2O emissions are two essential operational goals. The N2O emissions linked to three typical NOB suppression strategies were tested in a covered rotating biological contactor (RBC) biofilm system at 21 °C: (i) low dissolved oxygen (DO) concentrations, and treatments with (ii) free ammonia (FA), and (iii) free nitrous acids (FNA). Low emerged DO levels effectively minimized NOB activity and decreased N2O emissions, but NOB adaptation appeared after 200 days of operation. Further NOB suppression was successfully achieved by periodic (3 h per week) treatments with FA (29.3 ± 2.6 mg NH3-N L-1) or FNA (3.1 ± 0.3 mg HNO2-N L-1). FA treatment, however, promoted N2O emissions, while FNA did not affect these. Hence, biofilm PN/A should be operated at relatively low DO levels with periodic FNA treatment to maximize nitrogen removal efficiency while avoiding high greenhouse gas emissions.

Effect of free nitrous acid on extracellular polymeric substances production and membrane fouling in a nitritation membrane bioreactor.

The membrane bioreactor (MBR) with nitritation based nitrogen removal processes has attracted growing interest in recent years, although membrane fouling in the nitritation MBR is a challenging issue. In this study, the inhibitory effect of free nitrous acid (FNA) on microbial extracellular polymeric substances (EPS) production and membrane fouling in a nitritation MBR was investigated. Results showed that EPS played a critical role in the biofouling process, and EPS production was affected by FNA concentration. As FNA concentration increased from 5.10 × 10-3 mg N/L to 1.34 × 10-2 mg N/L, protein (PN) and polysaccharide (PS) contents increased from 8.20 to 60.28 mg/g VSS and 4.74-30.46 mg/g VSS, respectively. However, when FNA concentration was 1.48 × 10-2 mg N/L, PN and PS reduced by 20.0% and 10.9%, respectively, indicating that the higher FNA concentration could reduce EPS production. The EPS reduction could be attributed to reduction in the loosely bound (LB) and tightly bound (TB) EPS but not the soluble microbial products (SMP). It was further revealed that higher FNA concentrations up to 1.48 × 10-2 mg N/L consequently mitigate trans-membrane pressure (TMP) rate in terms of dTMP/dt by 25.5% in the nitritation MBR. High throughput sequencing analysis revealed that the increase in FNA led to enrichment of Nitrosomonas but reduction in heterotrophic bacteria. This study showed that the appropriate FNA concentration affected EPS production and hence membrane fouling, leading to the possibility of membrane fouling mitigation by in-situ generated FNA in the nitritation MBR.

A novel free nitrous acid (FNA)-generation pathway via ferric salts hydrolysis to mitigate sulfide and methane production in sewer: Insights into the performance and microbial mechanisms.

Ferric chloride (FeCl3) served as a solid acid has attracted attention recently. However, the feasibility of FeCl3 combined with nitrite for free nitrous acid (FNA) generation in controlling sulfide and methane as well as the triggering mechanisms in the complex syntrophic consortium (i.e., sewer biofilm) remain largely unknown. This work disclosed FeCl3 as an alternative acid source could obtain comparable sulfide and methane mitigations at a low FNA dose (i.e., 0.26 mg N/L), compared to that of HCl acid source. Whereas, a faster recovery rate of sulfide production was observed using FeCl3 under a higher FNA dose (i.e., 0.81 mg N/L) despite the methane control still being comparable. The toxicological mechanisms revealed FNA reacted with proteins amide Ⅰ in extracellular polymeric substances and destroyed protein hydrogen bond. Enzymatic and genic analysis unveiled the overall suppression of hydrolysis, acidogenesis, acetogenesis, sulfidogenesis and methanogenesis steps due to the inactivation of viable cells by reactive nitrogen species. Economic and environmental assessments demonstrated that the ferric-based FNA strategy reduced chemical costs and N2O emission (ca. 26.5% decrease) compared to the traditional HCl-based FNA method. This work broadens the application of iron salt-based technology in urban water system, together with understanding the biological mechanisms of FNA-based technology.

Free nitrous acid prediction in ANAMMOX process using hybrid deep neural network model.

Free nitrous acid (FNA) is a critical metric for stabilization of ANAMMOX but can not be directly and immediately measured by sensors or chemical measurement method, which hinders the effective management and operation for ANAMMOX. This study focuses on FNA prediction using hybrid model based on temporal convolutional network (TCN) combined with attention mechanism (AM) optimized by multiobjective tree-structured parzen estimator (MOTPE), called MOTPE-TCNA. A case study in an ANAMMOX reactor is carried out. Results show that nitrogen removal rate (NRR) is highly correlated with FNA concentration, indicating that it can forecast the operational status by predicting FNA. Then, MOTPE successfully optimizes the hyperparameters of TCN, helping TCN achieve a high prediction accuracy, and AM furtherly improves model accuracy. MOTPE-TCNA obtains the highest prediction accuracy, whose R2 value gets 0.992, increasing 1.71-11.80% compared to other models. As a deep neural network model, MOTPE-TCNA has more advantages than traditional machine learning methods in FNA prediction, which is beneficial to maintain the stable operation and easy control for ANAMMOX process.

Direct Formation of Electronic Excited NO2 Contributes to the High Yield of HONO during Photosensitized Renoxification.

Photosensitized renoxification of HNO3 is found to produce HONO in an unexpectedly high yield, which has been considered an important source for atmospheric HONO. Conventionally, the production of HONO is ascribed to the secondary photolysis of the primarily formed NO2. In this study, by using humic acid (HA) as a model environmental photosensitizer, we provide evidence of the direct formation of NO2 in its electronic excited state (NO2*) as a key intermediate during the photosensitizing renoxification of HNO3. Moreover, the high HONO yield originates from the heterogeneous reaction of the primarily formed NO2* with the co-adsorbed water molecules on HA. Such a mechanism is supported by the increase of the product selectivity of HONO with relative humidity. Further luminescence measurements demonstrate clearly the occurrence of an electronic excited state (NO2*) from photolysis of adsorbed HNO3 on HA. This work deepens our understanding of the formation of atmospheric HONO and gives insight into the transformation of RNS.

Identification and Analysis of the Biosynthetic Gene Cluster for the Hydrazide-Containing Aryl Polyene Spinamycin.

Natural products containing nitrogen-nitrogen (N-N) bonds have attracted much attention because of their bioactivities and chemical features. Several recent studies have revealed the nitrous acid-dependent N-N bond-forming machinery. However, the catalytic mechanisms of hydrazide synthesis using nitrous acid remain unknown. Herein, we focused on spinamycin, a hydrazide-containing aryl polyene produced by Streptomyces albospinus JCM3399. In the S. albospinus genome, we discovered a putative spinamycin biosynthetic gene (spi) cluster containing genes that encode a type II polyketide synthase and genes for the secondary metabolism-specific nitrous acid biosynthesis pathway. A gene inactivation experiment showed that this cluster was responsible for spinamycin biosynthesis. A feeding experiment using stable isotope-labeled sodium nitrite and analysis of nitrous acid-synthesizing enzymes in vitro strongly indicated that one of the nitrogen atoms of the hydrazide group was derived from nitrous acid. In vitro substrate specificity analysis of SpiA3, which is responsible for loading a starter substrate onto polyketide synthase, indicated that N-N bond formation occurs after starter substrate loading. In vitro analysis showed that the AMP-dependent ligase SpiA7 catalyzes the diazotization of an amino group on a benzene ring without a hydroxy group, resulting in a highly reactive diazo intermediate, which may be the key step in hydrazide group formation. Therefore, we propose the overall biosynthetic pathway of spinamycin. This study expands our knowledge of N-N bond formation in microbial secondary metabolism.

Pretreatment of free nitrous acid combined with calcium hypochlorite for enhancement of hydrogen production in waste activated sludge.

A variety of variables limit the recovery of resources from anaerobic fermentation of waste activated sludge (WAS), hence pretreatment strategies are necessary to be investigated to increase its efficiency. A combination of free nitrous acid (FNA) and calcium hypochlorite [Ca(ClO)2] was employed in this investigation to significantly improve sludge fermentation performance. The yields of cumulative hydrogen for the blank and FNA treatment group were 1.09 ± 0.16 and 7.36 ± 0.21 mL/g VSS, respectively, and 6.59 ± 0.24 [0.03 g Ca(ClO)2/g TSS], 7.75 ± 0.20 (0.06), and 8.58 ± 0.22 (0.09) mL/g VSS for the Ca(ClO)2 groups. The co-treatment greatly boosted hydrogen generation, ranging from 39.97 ± 2.26 to 76.20 ± 4.78 % as compared to the solo treatment. Mechanism analysis demonstrated that the combined treatment disturbed sludge structure and cell membrane permeability even more, which released more organic substrates and enhanced biodegradability of fermentation broth. This paper describes a unique strategy to sludge pretreatment that expands the use of Ca(ClO)2 and FNA in anaerobic fermentation, with implications for sludge disposal and energy recovery.