Effect of gradual increase of salt on performance and microbial community during granulation process.

Salinity was considered to have effects on the characteristics, performance microbial communities of aerobic granular sludge. This study investigated granulation process with gradual increase of salt under different gradients. Two identical sequencing batch reactors were operated, while the influent of Ra and Rb was subjected to stepwise increments of NaCl concentrations (0-4 g/L and 0-10 g/L). The presence of filamentous bacteria may contribute to granules formed under lower salinity conditions, potentially leading to granules fragmentation. Excellent removal efficiency achieved in both reactors although there was a small accumulation of nitrite in Rb at later stages. The removal efficiencies of chemical oxygen demand (COD), total nitrogen (TN), and total phosphorus (TP) in Ra were 95.31%, 93.70% and 88.66%, while the corresponding removal efficiencies in Rb were 94.19%, 89.79% and 80.74%. Salinity stimulated extracellular polymeric substances (EPS) secretion and enriched EPS producing bacteria to help maintain the integrity and stability of the aerobic granules. Heterotrophic nitrifying bacteria were responsible for NH4+-N and NO2--N oxidation of salinity systems and large number of denitrifying bacteria were detected, which ensure the high removal efficiency of TN in the systems.

Nitrate and nitrite utilization during denitrifying phosphorus removal: Electron acceptor preference and feasible process combinations.

Denitrifying phosphorus removal (DPR), which is dominated by denitrifying polyphosphate-accumulating organisms (DPAOs), is a promising process for nitrogen and phosphorus removal. Denitrifying glycogen-accumulating organisms (DGAOs) and DPAOs typically coexist in the DPR sludge, complicating the study of DPAOs' denitrification capacity. In this study, two reactors were fed with nitrate and nitrite during the anoxic phase to cultivate nitrate-DPR and nitrite-DPR sludge. Both reactors yielded high and low DGAO abundance sludges, enabling the evaluation of the denitrification capacity of DPAOs. For the nitrate-DPR sludge, the nitrite reduction rate was 1.63 times higher than the nitrate reduction rate when DPAOs were the primary denitrifiers. For the nitrite-DPR sludge, the reduction rate of nitrite was more than three times that of nitrate, irrespective of DGAO abundance. These findings indicated that DPAOs preferred nitrite to nitrate and were well suited to reduce nitrite rather than reduce nitrate to supply nitrite.

Enhanced anaerobic digestion performance and sludge filterability by membrane microaeration for anaerobic membrane bioreactor application.

Slow dissolution/hydrolysis of insoluble/macromolecular organics and poor sludge filterability restrict the application potential of anaerobic membrane bioreactor (AnMBR). Bubble-free membrane microaeration was firstly proposed to overcome these obstacles in this study. The batch anaerobic digestion tests feeding insoluble starch and soluble peptone with and without microaeration showed that microaeration led to a 65.7-144.8% increase in methane production and increased critical flux of microfiltration membrane via driving the formation of large sludge flocs and the resultant improvement of sludge settleability. The metagenomic and bioinformatic analyses showed that microaeration significantly enriched the functional genes and bacteria for polysaccharide and protein hydrolysis, microaeration showed little negative effects on the functional genes involved in anaerobic metabolisms, and substrate transfer from starch to peptone significantly affected the functional genes and microbial community. This study demonstrates the dual synergism of microaeration to enhance the dissolution/hydrolysis/acidification of insoluble/macromolecular organics and sludge filterability for AnMBR application.

Effects of exogenous N-acyl homoserine lactones (AHLs) on methanogenic activities and microbial community differences during anaerobic digestion.

N-acyl homoserine lactones (AHLs) function as signaling molecules influencing microbial community dynamics. This study investigates the impact of exogenously applied AHLs on methane production during waste-activated sludge (WAS) anaerobic digestion (AD). Nine AHL types, ranging from 10-4 to 10 µg/g VSS, were applied, comparing microbial community composition under optimal AHL concentrations. Firmicutes, Bacteroidetes, Chloroflexi, and Proteobacteria were identified in anaerobic digesters with C4-HSL, C6-HSL, and C8-HSL. Compared to the control, Halobacterota increased by 19.25%, 20.87%, and 9.33% with C7-HSL, C10-HSL, and C12-HSL. Exogenous C7-HSL enhanced the relative abundance of Methanosarcina, Romboutsia, Sedimentibacter, Proteiniclasticum, Christensenellaceae_R-7_group. C10-HSL increased Methanosarcina abundance. C4-HSL, C6-HSL, C8-HSL, C10-HSL, and C12-HSL showed potential to increase unclassified_Firmicutes. Functional Annotation of Prokaryotic Taxa (FAPROTAX) predicted AHLs' impact on related functional genes, providing insights into their role in AD methanogenesis regulation. This study aimed to enhance the understanding of the influence of different types of exogenous AHLs on AD and provide technical support for regulating the methanogenesis efficiency of AD by exogenous AHLs.

Effect of zero-valent iron (ZVI) and biogas slurry reflux on methane production by anaerobic digestion of waste activated sludge.

This study aimed to improve anaerobic digestion (AD) efficiency through the addition of zero-valent iron (ZVI) and biogas slurry. This paper demonstrated that methane production was most effectively promoted at a biogas slurry reflux ratio of 60%. The introduction of ZVI into anaerobic systems does not enhance its bioavailability. However, both biogas slurry reflux and the combination of ZVI with biogas slurry reflux increase the relative abundance of microorganisms involved in the direct interspecific electron transfer (DIET) process. Among them, the dominant microorganisms Methanosaeta, Methanobacterium, Methanobrevibacter, and Methanolinea accounted for over 60% of the total methanogenic archaea. The Tax4Fun function prediction results indicate that biogas slurry reflux and the combination of ZVI with biogas slurry reflux can increase the content of key enzymes in the acetotrophic and hydrotrophic methanogenesis pathways, thereby strengthening these pathways. The corrosion of ZVI promotes hydrogen production, and the biogas slurry reflux provided additional alkaline and anaerobic microorganisms for the anaerobic system. Their synergistic effect promoted the growth of hydrotrophic methanogens and improved the activities of various enzymes in the hydrolysis and acidification phases, enhanced the system's buffer capacity, and prevented secondary environmental pollution. PRACTITIONER POINTS: Optimal methane production was achieved at a biogas slurry reflux ratio of 60%. Biogas slurry reflux in anaerobic digestion substantially reduced discharge. ZVI addition in combination with biogas slurry reflux facilitates the DIET process.

Innovative cation exchange-driven carbon migration and recovery patterns in anaerobic fermentation of waste activated sludge.

Despite numerous treatments have been developed to enhance anaerobic fermentation of waste activated sludge, the innovative cation exchange (CE) approach has been rarely reported, little attempt was conducted to revealcarbon source fate. The interphase carbon balance was illustrated to clarify endogenous carbon dissolution, biotransformation,and recovery patterns. By CE-mediated divalent cation removal, almost 34.72 % of particulate carbon sources were dissolved in 2-day treatment, corresponding to soluble carbon content of 1165.58 mg C/L. Most of the originally dissolved carbon sources (58.01-66.81 %) were bio-transformed to volatile fatty acids with high bioavailability, while the further transformation to biogas was inhibited, contributing to recoverable carbon source accumulation. Overall, 21.38 % of total solid carbon sources were recovered through 8-day fermentation, the carbon extraction was implemented by solid-liquid separation with carbon loss of 14.21-22.91 %, manifesting the valid carbon recovery of 85.05-87.96 mg C/g VSS. Such CE-driven carbon recovery provided negentropy benefits in sustainable cycle economy.

Effect of thermal hydrolysis pretreatment on the occurrence of N-acyl homoserine lactone during anaerobic digestion of waste activated sludge.

This study sought to investigate the relationship between N-acyl homoserine lactones (AHLs) and methanogenic microorganisms, focusing on endogenous AHLs in the anaerobic digestion (AD) process. By analyzing waste activated sludge (WAS) samples, we examine the changes in microbial communities and the AHLs-methanogens connection. The Mantel test and Spearman correlation analysis were conducted to gain novel insights into the AD process. Our findings demonstrate that thermal hydrolysis pretreatment (THP) modifies AHL concentrations during AD, thereby enhancing methanogenic bacteria activity and regulating social interactions among microorganisms. In the Eth group (AD of THP samples labeled Eth), Methanobacterium and Methanosarcina accounted for over 80% of the methanogenic bacteria, with correlation coefficients greater than 0.5 between these bacterial taxa and N-hexyl-l-homoserine lactone (C6-HSL) and N-enanthyl-l-homoserine lactone (C7-HSL).

Characteristics of digested sludge-derived biochar for promoting methane production during anaerobic digestion of waste activated sludge.

This study investigated a novel method for enhancing methane production during anaerobic digestion of waste activated sludge with digested sludge-derived biochar (DSBC). Using response surface methodology, the following process conditions for DSBC synthesis were optimized: heating rate = 13.23 °C/min, pyrolysis temperature = 516 °C, and heating time = 192 min. DSBC significantly enhanced the methane production by 48 % and improved key coenzyme activity that accelerated the bioconversion of organic matter while promoting the decomposition and transformation of volatile fatty acids. Consequently, the lag period of methane production was shortened to 4.89 days, while the average proportion of methane greatly increased to 73.22%. Thus, DSBC could facilitate efficient methanogenesis in the anaerobic system by promoting electron transfer between syntrophic partners through the charge-discharge cycle of surface oxygen-containing functional groups. The study provides a reference for the resource utilization of anaerobic sludge residues and efficient anaerobic methanogenesis from sludge.

Investigating the synergic role of asynchronous dosed protease and lysozyme for facilitating excess sludge solubilization and acidogenic fermentation.

Improving the anaerobic fermentation (AF) efficiency of excess sludge (ES) is essential for attaining biosolid minimization, stabilization, resource recovery, and carbon-emission reduction. Along these lines, here, the synergistic mechanism of protease and lysozyme for enhancing hydrolysis and AF efficiency with better recovery of volatile fatty acids (VFAs) was thoroughly investigated. Single lysozyme was capable of reducing the zeta potential and fractal dimension when dosed into the ES-AF system, which was beneficial for increasing the contact probability between proteases and extracellular proteins. Moreover, the weight-averaged molecular weight of the loosely-bound extracellular polymeric substance (LB-EPS) reduced from 1867 to 1490 in the protease-AF group, which facilitated the penetration of EPS by the lysozyme. The soluble DNA and extracellular DNA (eDNA) of the enzyme cocktail pretreated group increased by 23.24 % and 77.09 %, and the cell viability decreased after 6-hour hydrolysis, demonstrating a better hydrolysis efficiency. Remarkably, the asynchronous dosed enzyme cocktail pretreatment was proven a better strategy to enhance both the solubilization and hydrolysis processes since the synergistic effect of these two enzymes can exclude the mutual interference. As a result, the VFAs were increased by 1.26 times higher than the blank group. The underlying mechanism of an environmental-friendly and effective strategy was examined to promote ES hydrolysis and acidogenic fermentation, which was beneficial for the recovery of VFAs and carbon-emission reduction.

Sludge source-redox mediators obtainment and availability for enhancing bioelectrogenesis and acidogenesis: Deciphering characteristics and mechanisms.

Anaerobic biological treatment was regarded as one of promising options for realizing concurrent WAS reduction, stabilization and bioenergy/bioresource recycle. But the relatively low treatment efficiency limited its spreading application toward larger scale considerably in China. Aimed at such barrier, this study offered a novel enhancing strategy for achieving high-efficiency of bioenergy/bioresource recycle from WAS anaerobic treatment via improving bioelectrogenesis/acidogenesis using sludge source-redox mediators (SSRMs). SSRMs not only facilitated bioeletrogenesis with an increasing efficiency of 36% for voltage output and 39% for bioelectricity bioconversion, but also enhanced acidogenesis of WAS with a mean elevating efficiency of 37.5% of volatile fatty acids (VFAs) production within 5 d Mechanistic investigations indicated that SSRMs had a potential influence on improving the protein and carbohydrate metabolisms-related genes' expression for enhancing bioelectrogenesis and acidogenesis. Moreover, SSRMs exerted roles of electrochemical "catalysts" or as terminal electron acceptors with affecting functional proteins of complexes of Ⅰ and Ⅳ in electron transfer chains for improving electron transfer efficiency. Meanwhile, the core members' abundance, microbial diversity and community distributive evenness were prompted concurrently for carrying out superior bioelectrogenesis and acidogenesis. A schematic illustration was established for demonstrating the mechanism of SSRMs for enhancing bioelectrogenesis and acidogenesis via changing microbial metabolism functions, enhancing electron transfer efficiency, and regulating functional genes' expression of functional proteins (up-regulating cytochrome c oxidase and down-regulating-NADH dehydrogenase). This study provided an effective enhancing strategy for facilitating WAS bioconversion to bioenergy/bioresource with well-process sustainability.

Revealing the mechanism of novel nitrogen-doped biochar supported magnetite (NBM) enhancing anaerobic digestion of waste-activated sludge by sludge characteristics.

Anaerobic digestion (AD) is a promising technology in waste treatment and energy recovery. However, it suffers from long retention time and low biogas yield. In this study, novel nitrogen-doped biochar supported magnetite (NBM) was synthesized and applied to enhance the AD of waste-activated sludge. Results showed that NBM increased cumulative methane production and SCOD removal efficiency by up to 1.75 times and 15% respectively at 5 g/L compared with the blank. NBM enhanced both hydrolysis and methanogenesis process during AD and the activities of α-glucosidase, protease, coenzyme F420 and electron transport system were increased by 19%, 163%, 104% and 160% respectively at 5 g/L NBM compared with the blank. NBM also facilitated the secretion of conductive protein in extracellular polymeric substances as well as the formation of conductive pili, leading to 3.18-7.59 times higher sludge electrical conductivity. Microbial community analysis revealed that bacteria Clostridia and archaea Methanosarcina and Methanosaeta were enriched by the addition of NBM, and direct interspecies electron transfer might be promoted between them. This study provides a practical reference for future material synthesis and its application.

Acclimation of anaerobic fermentation microbiome with acetate and ethanol for chain elongation and the biochemical response.

Medium chain fatty acids (MCFAs) production is a promising method for resource recovery from organic wastes. In this study, the microbial community structure shift along the long-term acclimation experiment and the concomitant effect of H2 level on chain elongation performance was investigated. Chain elongation microbiome could be rapidly acclimated from traditional anaerobic fermentation consortia. Genera Caproiciproducens, Clostridium sensu stricto 12, Rummeliibacillus and Oscillibacter was found to be dominant during the operation. The H2 was accumulated in the headspace by increasing the ethanol input, which inhibited oxidation of caproate and butyrate immediately, while its inhibition effect on chain elongation was delayed. H2 level in the headspace was positively correlated to the MCFAs production related bacteria. However, too much H2 accumulated might be suppressive for MCFAs production in the long term. It might result from the thermodynamic barrier for discarding excess reducing equivalents under high H2 level, which further gave rise to ethanol accumulation in this system.

Effect of granular activated carbon and chloroform on chain elongation with simple substrate ethanol and acetate.

Chain elongation is a promising technology for production of medium-chain fatty acids (MCFAs). Granular activated carbon (GAC) is commonly used in anaerobic fermentation. Low level CHCl3 can inhibit methanogenesis and homoacetogenesis at the same time. However, the effect of them on chain elongation performance with highly enriched consortia and simple substrate (i.e., ethanol and acetate) was still unclear. Hence, the effects of CHCl3 and on MCFAs production and the microbial community was studied here. CHCl3 displayed fatal effect on chain elongation system when its concentration was higher than 0.1% v/v. 0.05% v/v CHCl3 was enough to inhibit homoacetogens and further decreased the caproate production efficiency without altering the core bacteria tremendously. GAC was found to be adverse for chain elongation with simple substrate (i.e., ethanol and acetate) and highly enriched microbial consortia dominated by Clostridium sensu stricto, less than 20% electrons were finally distributed in caproate. It might be attributed to other electron consuming activities induced by GAC.

Novel nitrogen-doped biochar supported magnetite promotes anaerobic digestion: Material characterization and metagenomic analysis.

Although different conductive materials have been applied to anaerobic digestion, there has not been a material that can really combine their merits and make up their shortcoming from each other. In this study, a novel nitrogen-doped biochar supported magnetite (Fe3O4@N-BC) was synthesized. Various material characterizations confirmed that nitrogen atoms were successful doped into the biochar and magnetite precipitated on its surface. 5 g/L Fe3O4@N-BC achieved the highest promotion of cumulative CH4 production by 1.75 times compared with the blank group. Further metagenomic analysis revealed that Fe3O4@N-BC could increase the gene abundances of pilA, MmcA, Fpo, Rnf and HdrEd in bacteria Clotridium, Pseudomonas and Syntrophomonas and archaea Methanosarcina. Redundancy analysis showed that it was electrical conductivity and electron exchange capacity that were the key physicochemical characteristics for Fe3O4@N-BC to facilitating direct interspecies electron transfer. This study provides a reference for future conductive material synthesis and its application for anaerobic digestion.

Enhanced acidogenic fermentation from Al-rich waste activated sludge by combining lysozyme and sodium citrate pretreatment: Perspectives of Al stabilization and enzyme activity.

The accumulation of poly aluminum chloride (PAC) in dewatered waste activated sludge (WAS) can cause severe Al pollution and significantly reduce the production of volatile fatty acids (VFAs) from anaerobic fermentation. Herein, the combination of lysozyme and sodium citrate pretreatment was applied to stabilize the aluminum and enhance the VFAs production via anaerobic fermentation. The complexation and stabilization of aluminum by the citrate was efficient, which is conducive to relieving the inhibition of aluminum on lysozymes and other extracellular hydrolases. Compared with the control group, the lysozyme, protease and α-glucosidase activities were obtained at 1.86, 1.72, and 1.15 times, respectively, following the pretreatment. 129.71 mg/g volatile suspended solids (VSS) of soluble proteins and 26.3 mg/g VSS of polysaccharides were obtained within 4 h, together with the degradation of 124 % more proteins and 75 % more polysaccharides within three days. This provided a sufficient number of substrates for VFA production. 588.4 mg COD/g VSS of total VFAs were obtained after the six-day anaerobic fermentation from Al-rich WAS following the combination of lysozyme and sodium citrate pretreatment, which was 7.3 times higher than that of the control group. This study presents a novel approach for enhancing VFA production in anaerobic fermentation as well as reducing risk of Al hazards from Al-rich WAS.

Effect of activated carbon/graphite on enhancing anaerobic digestion of waste activated sludge.

The conductive media was capable to enhance anaerobic digestion and promote direct interspecific electron transfer (DIET). In this study, the effects of activated carbon- and graphite-conductive media on promoting anaerobic digestion efficiency of waste activated sludge were experimentally studied. The results show that the 100 mesh-activated carbon group reactor produced a largest biogas yield of 468.2 mL/g VSS, which was 13.8% higher than the blank test. The graphite group reactor with 400-grain size produced a largest biogas yield of 462.9 mL/g VSS, which was 12.5% higher than the blank test. Moreover, the optimal particle size of such two carbon- conductive mediators were optimized for enhancing degradation efficiency of VSS, TCOD, total protein and total polysaccharide of waste sludge. Activated carbon was capable to promote the hydrolytic acidification stage in anaerobic digestion of waste sludge. When the particle size reduced to the optimal particle size, the promoting effect could be strengthened for producing more hydrolytic acidification products for methanogenesis. However, in the graphite group, the methane production is increased by promoting the consumption of hydrolysis and acidification products and is enhanced with the particle size reduction, thus promoting the methanogenesis process, and improving the anaerobic digestion efficiency. Microbial community analysis showed that both activated carbon and graphite cultivated the genera of Methanosaeta, Methanobacterium, Nitrososphaeraceae, which promoted the improvement of methane production through the acetate debris methanogenesis pathway.

Integration of a pure moving bed biofilm reactor process into a large micro-polluted water treatment plant.

The pure-MBBR process was applied to remove ammonia in a full-scale micro-polluted-water treatment plant with a daily treatment capacity of 260 × 104 m3/d, Guangdong, China. The relationship between treatment efficiency, physical and chemical properties and microbial diversity in the process of biofilm growth was explored, and the oxygen transfer model of biofilm was established. The results show that the effluent of two-stage pure MBBR process is stable and up to standard after 10 days' incubation. The nitrification loads of two-stage biofilm was stable on the 14th day. The biomass and biofilm thickness lagged behind the nitrification load, and reached a relatively stable level on the 28th day. The species richness of biofilm basically reached a stable level on the 21st day, and the microbial diversity of primary biofilm was higher. In the primary and secondary stage at different periods, the relative abundance of dominant nitrifying bacteria Nitrospira reaches 8.48-13.60%, 6.48-9.27%, and Nitrosomonas reaches 2.89-5.64%, 0.00-3.48%. The pure MBBR system mainly adopts perforated aeration. Through the cutting and blocking of bubbles by suspended carriers, the oxygen transfer rate of the system was greatly improved.

[Analysis of Microbial Interaction Law of Mud Membrane in IFAS Process for Treating Low Carbon Source Sewage in South China].

To assess the problem of sewage treatment under the condition of low carbon sources, we carried out a study of activated sludge and a biofilm symbiosis system (IFAS). The occurrence characteristics and interaction law of microorganisms in two phases of sludge membrane under low carbon source conditions were discussed, and their niche and influence on treatment efficiency were clarified. Through a pilot-scale experiment in actual water plants, the biofilm characteristics, sludge membrane activity, and succession law of flora were analyzed, and the microbial structure and interaction in sludge membrane in two phases under the control of different activated sludge ages were compared. The results showed that the sludge concentration in the reactor increased with the increase in SRT under variable SRT. Because the microbial concentration in SRT-H was much higher than that in SRT-L, the competition between mud films in SRT-H was more intense than that in SRT-L, and the pollutant removal efficiency in SRT-H was lower than that in SRT-L. Under the condition of low-carbon feed water, the sludge activity in the IFAS process decreased with the increase in SRT. Under the condition of low SRT(5 d), the nitrification, denitrification, phosphorus accumulation, and phosphorus absorption rate of activated sludge increased by 122%, 88%, 34%, and 44%, respectively, compared with that of high SRT (25 d). However, SRT had little effect on biofilm activity, and there was little difference in nitrification activity and denitrification activity between the two SRTs. Microbial sequencing analysis showed that the functional bacteria of the IFAS process were enriched and transferred with the change in SRT between the two phases of mud membrane. In SRT-L, the functional bacteria that were enriched and transferred between the two phases of mud film owing to the "seeding" effect were mainly unclassified_g__Enterobacteriaceae, whereas in SRT-H, Acinetobacter was mainly used. At the same time, by analyzing the distribution of dominant functional bacteria, it was found that there was some competition between denitrifying bacteria and phosphorus-accumulating bacteria in activated sludge. Under the condition of a lack of organic substrate in the influent, the relative abundance of denitrifying bacteria was obviously higher than that of phosphorus-accumulating bacteria, which indicated that denitrifying bacteria could better adapt to low-carbon source conditions. Thus, they could occupy a dominant competition position, which was mainly reflected in the increase in the relative abundance of aerobic denitrifying bacteria. In addition, the SRT change in the mud phase reacted in the membrane phase, making the residence time of biofilm change correspondingly, thus changing the flora structure, screening out different dominant bacteria genera, and further increasing the difference.

Metagenomic analysis reveals the size effect of magnetite on anaerobic digestion of waste activated sludge after thermal hydrolysis pretreatment.

Although magnetite has been widely investigated in anaerobic digestion (AD), its role in the practical AD of waste-activated sludge (WAS) after thermal hydrolysis pretreatment (THP) and its size effect remain unclear. In this study, magnetite with four different particle sizes was added during the AD of WAS after THP. With the reduction of magnetite particle size, cumulative methane production was increased, while the optimal dosage of magnetite decreased, with 0.1 µm magnetite at an optimal dosage of 2 g/L achieving the highest cumulative methane production increase of 111.97 % compared with the blank group (without magnetite). Smaller magnetite particles increased α-glucosidase and protease activities, coenzyme F420 concentration, and electron-transport system activity (20.30 %, 173.02 %, 60.39 % and 158.08 % higher respectively than the blank group). The size of magnetite also influenced the establishment of direct interspecies electron transfer (DIET) during AD. Based on the analysis of the pilA gene abundance, magnetite with a large particle size could promote the formation of e-pili in syntrophic electroactive bacteria (Clostridium, Syntrophomonas, and Pseudomonas) and methanogens (Methanospirillum), thereby completing electron transfer. However, small-sized magnetite particles stimulated DIET by enhancing the secretion of conductive proteins in extracellular polymeric substances and membrane-bound enzymes (Fpo) in Methanosarcina.

New insight into electrochemical denitrification using a self-organized nanoporous VO-Co3O4/Co cathode: Plasma-assistant oxygen vacancies catalyzed efficient nitrate reduction.

A novel self-organized nanoporous VO-Co3O4/Co cathode was prepared via anodization and plasma treatment and obtained a significant nitrate reduction efficiency. In the anodization, an oxide layer with the nano-sized pore structure initially grew in-situ on the Co substrate and showed a better surface area. Subsequently, He-plasma increased surface oxygen vacancies (VO) from 24 % to 57 %. Electrons in vacancies were charged into empty eg orbital of low-spin Co3+(Oh, octahedral) and firstly generated high-spin Co2+(Oh) with the configuration of t2g6eg1, accounting for 71.7 % of cobalt species. Accordingly, two original mechanisms (Vo-catalyzed and Co2+(Oh)-catalyzed) were concluded in this study. Oxygen vacancies increased the charge intensity and served as absorption sites in nitrate reduction. Meanwhile, massive Co2+(Oh) provided electrons in the eg orbital with a higher energy state and mediated the faster electron transfer through a Co2+-Co3+-Co2+ redox cycle, compared with Co2+ (Td, tetrahedral). Ultimately, a faster reaction kinetic of 0.0220 min-1 was achieved by VO-Co3O4 than other cathodes e.g., Co3O4 (0.0150 min-1). Such VO-Co3O4/Co cathode-based denitrification strategy displayed great advantages in engineering application and completely removed 90 % of TN from actual wastewater.