High-efficient nutrient removal in a single-stage electrolysis-integrated sequencing batch biofilm reactor (E-SBBR) for low C/N sanitary sewage treatment.

To efficiently remove nutrients from low C/N sanitary sewage by conventional biological process is challenging due to the lack of sufficient electron donors. A novel electrolysis-integrated sequencing batch biofilm reactor (E-SBBR) was established to promote nitrogen and phosphorus removal for sanitary sewage with low C/N ratios (3.5-1.5). Highly efficient removal of nitrogen (>79%) and phosphorus (>97%) was achieved in the E-SBBR operating under alternating anoxic/electrolysis-anoxic/aerobic conditions. The coexistence of autotrophic nitrifiers, electron transfer-related bacteria, and heterotrophic and autohydrogenotrophic denitrifiers indicated synergistic nitrogen removal via multiple nitrogen-removing pathways. Electrolysis application induced microbial anoxic ammonia oxidation, autohydrogenotrophic denitrification and electrocoagulation processes. Deinococcus enriched on the electrodes were likely to mediate the electricity-driven ammonia oxidation which promoted ammonia removal. PICRUSt2 indicated that the relative abundances of key genes (hyaA and hyaB) associated with hydrogen oxidation significantly increased with the decreasing C/N ratios. The high autohydrogenotrophic denitrification rates during the electrolysis-anoxic period could compensate for the decreased heterotrophic rates resulting from insufficient carbon sources and nitrate removal was dramatically enhanced. Electrocoagulation with iron anode was responsible for phosphorus removal. This study provides insights into mechanisms by which electrochemically assisted biological systems enhance nutrient removal for low C/N sanitary sewage.

Response of nutrients removal efficiency, enzyme activities and microbial community to current and voltage in a bio-electrical anammox system.

The effects of different current intensities and voltage levels on nutrient removal performance and microbial community evolution in a Bio-Electrical Anammox (BEA) membrane bioreactor (MBR) were evaluated. The nitrogen removal efficiency increased with the current intensity within the range of 64-83 mA, but this improvement was limited at the current further increased. The phosphorus removal in the BEA MBR was attributed to the release of Fe2+, which was closely associated with the applied current to the electrodes. Heme c concentration, enzyme activities, and specific anammox activity exhibited a decreasing trend, while the functional denitrification genes showed a positive correlation with rising voltage. The nitrogen removal efficiency of the BEA system initially increased and then decreased with the voltage rose from 1.5V to 3.5V, peaking at 2.0V of 94.02% ± 1.19%. Transmission electron microscopy and flow cytometry results indicated that accelerated cell apoptosis/lysis led to an irreversible collapse of the biological nitrogen removal system at 3.5V. Candidatus Brocadia was the predominant anammox bacteria in the BEA system. In contrast, closely related Candidatus Kuenenia and Chloroflexi bacteria were gradually eliminated in electrolytic environment. The abundances of Proteobacteria-affiliated denitrifiers were increased with the voltage rising since the organic matter released by the cell apoptosis/lysis was accelerated at a high voltage level.

The performance and microbial response of zero valent iron alleviating the thermal-alkaline stress and enhancing hydrolysis-acidification of primary sludge.

The biological thermal-alkaline hydrolysis-acidification (BTAHA) could promote sludge disintegration, which was conducive to producing volatile fatty acids (VFAs). However, high temperature and strong alkali could reduce the BTAHA effluent quality. Because high temperature denatures proteins and significantly changes the material and energy metabolism of bacteria, while strong alkali inhibits fermentation microorganisms (especially acid-producing microorganisms). This study investigated the internal mechanism of zero valent iron (ZVI) and magnetite (Mag.) alleviating temperature and alkali stress and improving the quality of hydrolysis-acidification effluent. At pH 7-10, compared with the control and magnetite, ZVI increased the average effluent VFAs by 24.0%-40.1% and 11.6%-18.1%, respectively. At pH 9, ZVI could provide an ecological niche for acidifying bacteria that preferred neutral and weakly alkaline conditions, with a 49.8% proportion of VFAs to soluble chemical oxygen demand (SCOD). At pH 12, the fluorescence intensity ratio of easy to difficult biodegradable organic matter in control, RMag., and RZVI were 0.63, 0.62, and 1.31, respectively. It indicated ZVI effectively alleviated high temperature and strong alkali stress. This study provides a reference for improving the quality of BTAHA effluent.

Organic matter removal performance, pathway and microbial community succession during the construction of high-ammonia anaerobic biosystems treating anaerobic digestate food waste effluent.

This study aimed to establish anaerobic biosystems which could tolerate high ammonia, and investigate the microbial community structure in these reactors. High-ammonia anaerobic biosystems that could tolerate 3600 mg L-1 total ammonia nitrogen (TAN) and 1000 mg L-1 free ammonia nitrogen (FAN) were successfully established. The removal efficiencies of COD and total volatile fatty acids (TVFAs) in R1 with dewatered sludge as inoculum were 68.8% and 69.2%, respectively. The maximum methane production rate reached 71.7 ± 1.0 mL CH4 L-1 d-1 at a TAN concentration of 3600 mg L-1. The three-dimension excitation-emission matrix analysis indicated that both easily degradable organics and refractory organics were removed from ADFE in R1 and R2. Functional microorganisms which could bear high ammonia were gradually enriched as TAN stress was elevated. Lysinibacillus, Coprothermobacter and Sporosarcina dominated the final bacterial community. Archaeal community transformed to hydrogenotrophic methanogen. The synergy of Coprothermobacter and Methanothermobacter undertook the organic matter degradation, and was enhanced by increasing TAN stress. This study offers new insights into anaerobic bioremediation of ammonia-rich wastewater.

Iron-electrolysis assisted anammox/denitrification system for intensified nitrate removal and phosphorus recovery in low-strength wastewater treatment.

Two iron-electrolysis assisted anammox/denitrification (EAD) systems, including the suspended sludge reactor (ESR) and biofilm reactor (EMR) were constructed for mainstream wastewater treatment, achieving 84.51±4.38 % and 87.23±3.31 % of TN removal efficiencies, respectively. Sludge extracellular polymeric substances (EPS) analysis, cell apoptosis detection and microbial analysis demonstrated that the strengthened cell lysate/apoptosis and EPS production acted as supplemental carbon sources to provide new ecological niches for heterotrophic bacteria. Therefore, NO3--N accumulated intrinsically during anammox reaction was reduced. The rising cell lysis and apoptosis in the ESR induced the decline of anammox and enzyme activities. In contrast, this inhibition was scavenged in EMR because of the more favorable environment and the significant increase in EPS. Moreover, ESR and EMR achieved efficient phosphorus removal (96.98±5.24 % and 96.98±4.35 %) due to the continued release of Fe2+ by the in-situ corrosion of iron anodes. The X-ray diffraction (XRD) indicated that vivianite was the dominant P recovery product in EAD systems. The anaerobic microenvironment and the abundant EPS in the biofilm system showed essential benefits in the mineralization of vivianite.

Overlooked pathways of endogenous simultaneous nitrification and denitrification in anaerobic/aerobic/anoxic sequencing batch reactors with organic supplementation.

The anaerobic/aerobic/anoxic (A/O/A) process is a promising biotechnology to intensify denitrification in low carbon/nitrogen (C/N) wastewater treatment, but the neglected typical rate-limiting step-nitrification-would hinder its wider application. Heterotrophic nitrification driven by intracellular carbon (PHAs) could enhance nitrification and achieve endogenous simultaneous nitrification and denitrification (ESND) in the A/O/A process, but its feasibility remains unexamined. Here we established four A/O/A-SBRs at different C/N ratios (3, 7.5, 12, and 16.5) to address the above-mentioned knowledge gaps. The results showed that organic supplementation promoted both nitrification and denitrification (performance and relevant enzymatic activities) until organic overdose (C/N = 16.5) exacerbated niche competitions from other non-functional heterotrophs. qPCR and batch tests indicated that high C/N ratios inhibited autotrophic nitrifiers, and heterotrophic nitrifiers (HNB) dominated in the enhanced nitrification. Given the high HNB contribution (43.7%) and low COD variation (< 10 mg L-1) in the SND (76.4%) of CN12, we proposed a potential SND pathway based on heterotrophic nitrification and denitrification driven by PHAs and verified it with batch tests. Microbial and functional analyses suggested that CN12 favored the intracellular carbon transformation and harbored the minimum autotrophic nitrifiers, supporting the dominance of ESND in the enhanced SND. Our findings expand the understanding of the relationships between intracellular carbon transformation and SND and provide a novel nitrogen removal pathway for the practical application of the A/O/A process.

Accelerating denitrification and mitigating nitrite accumulation by multiple electron transfer pathways between Shewanella oneidensis MR-1 and denitrifying microbial community.

The bio-denitrification was usually retarded by the unbalance of electron generation and consumption. In this study, mixing S. oneidensis MR-1 with denitrifying microbial community increased the nitrogen removal rate by 74.74 % via the interspecies electron transfer (IET), and reduced the accumulated nitrite from 9.90 ± 0.81 to 0.02 ± 0.03 mg/L. Enhanced denitrification still appeared but relatively decreased, when S. oneidensis MR-1 was separated by a dialysis bag (MW < 3000), indicating mediated interspecies electron transfer (MIET) counted in IET. The results of electron transfer activity and sludge conductivity suggested DIET and MIET jointly transfer electrons from MR-1 to electroactive denitrifying bacteria (EDB), improving denitrifying reductase activities. Electron distribution among denitrifying reductases was found to be associated with the IET rate. Microbial insights showed the total abundance of EDB was increased, and denitrifying genes were correspondingly enriched. Pseudomonas was found to cooperate with exoelectrogens in a complicated microbial community.

The coupling system of magnetite-enhanced thermophilic hydrolysis-acidification and denitrification for refractory organics removal from anaerobic digestate food waste effluent (ADFE).

The aim of this study was to remove the refractory organics from high-temperature anaerobic digestate food waste effluent by the coupling system of hydrolysis-acidification and denitrification. Iron-based materials (magnetite, zero-valent iron, and iron-carbon) were used to enhance the performance of thermophilic hydrolysis-acidification. Compared with the control group, magnetite had the best strengthening effect, increasing volatile fatty acids concentration and fluorescence intensity of easily biodegradable organics in the effluent by 47.6 % and 108.4 %, respectively. The coupling system of magnetite-enhanced thermophilic hydrolysis-acidification and denitrification achieved a nitrate removal efficiency of 91.2 % (influent NO3--N was 150 mg L-1), and reduced the fluorescence intensity of refractory organics by 33.8 %, compared with influent. Microbiological analysis indicated that magnetite increased the relative abundance of thermophilic hydrolytic acidifying bacteria, and coupling system enriched some genera simultaneously removing nitrate and refractory organics. This study provided fresh information on refractory organics and nitrogen removal of thermophilic wastewater biologically.

Enhanced nitritation/denitritation and potential mechanism in an electrochemically assisted sequencing batch biofilm reactor treating sludge digester liquor with extremely low C/N ratios.

Nitritation/denitritation is a promising strategy to treat sludge digester liquor but would be unstable and inefficient at extremely low C/N ratios. Here, a novel electrochemically assisted sequencing batch biofilm reactor (E-SBBR) was established to treat synthetic/real sludge digester liquor with decreasing C/N ratios. The results showed that the E-SBBR achieved stable nitritation and appreciable TN removal (>70 %) even at C/N < 0.5. The high-strength free ammonium (FA) (91.1-132.8 mg NH3-N/L) and long inhibition time (>9h) magnified by electrolysis promoted the robustness of nitritation through efficient nitrite-oxidizing bacteria elimination. Meanwhile, mass balance denoted that heterotrophic denitritation dominated in the enhanced TN removal and relied on carbon supplementation from cell apoptosis/lysis stimulated by electrolysis and high-strength FA, further supported by the recovery of heterotrophic denitrifiers, fermentation bacteria, and relevant functional genes at extremely low C/N ratios. This study provides a novel nitrogen removal approach for the sludge digester liquor treatment.

Biologically induced phosphate precipitation in heterotrophic nitrification processes of different microbial aggregates: Influences of nitrogen removal metabolisms and extracellular polymeric substances.

Phosphorus (P) removal occurred in heterotrophic nitrification process, but its mechanism has not been fully explored. In this study, the P removal performances, pathways, and mechanisms in heterotrophic nitrification processes of different microbial aggregates (activated sludge and biofilm) were investigated. The results showed that the biofilm reactor had more efficient total nitrogen removal (98.65%) and phosphate removal (94.17%). Heterotrophic nitrification and denitrification processes generated alkalinity for biologically induced phosphate precipitation (BIPP), which contributed to 64.12%-78.81% of the overall P removal. The solid phase P content reached 48.03 mg/gSS with hydroxyapatite and calcium phosphate formation. The study clarified that biofilm was beneficial to BIPP because of the nitrogen removal metabolism and extracellular polymeric substance (EPS). Heterotrophic nitrogen removal metabolism was the driving force of BIPP, while EPS with abundant carboxyl and amide groups promoted the precipitation. The study would provide new insights into simultaneous nutrients removal and P recovery from wastewater.

High-temperature biofilm system based on heterotrophic nitrification and aerobic denitrification treating high-strength ammonia wastewater: Nitrogen removal performances and temperature-regulated metabolic pathways.

Conventional autotrophic nitrification process is difficult to treat high-temperature wastewater with high-strength ammonia. In this study, a high-temperature (50 °C) biofilm system based on heterotrophic nitrification and aerobic denitrification (HNAD) was established. The results showed that the HNAD process was high temperature resistant, and the nitrogen removal performance, pathway and microbial mechanism varied remarkably at different temperatures. The high-temperature system showed excellent nitrogen and COD removal capacities at 50 °C. Ammonia oxidation was mainly undertaken by heterotrophic nitrification, while anoxic and aerobic pathways worked in concert for denitrification. High-throughput sequencing indicated that heterotrophic nitrifying bacteria (8.58%) and denitrifying bacteria (52.88%) were dominant at 50 °C. Metagenomic analysis further suggested that the carbon metabolism was up-regulated in response to the increasing temperature, so more energy was generated, thereby promoting the HNAD-related nitrogen removal pathways. The study revealed the microbial mechanism of HNAD at high temperature and provided new insights into high-temperature biological nitrogen removal.

Deep dewatering of activated sludge using composite conditioners of surfactant, acid and flocculant: The mechanism and dosage model.

To address the problem of difficult disposal caused by poor dewaterability of high-organic sludge in wastewater treatment plant, this study developed a sludge composite conditioner (SCC) consisting of sodium dodecyl sulfate (SDS), HCl and FeCl3. It has the potential to significantly improve the dewaterability of the high-organic sludge with the VSS/MLSS of 80%. The moisture content (MC) and bound water content of sludge were reduced from 98.00 to 59.65% and from 3.42 to 0.91 g/g dry sludge (DS) after being conditioned, respectively. The surfactant (SDS) promoted the dissolution of extracellular polymeric substances (EPS). The acid (HCl) enhanced the decomposition of the sludge flocs, making the insoluble EPS peel off and turn into the liquid phase. As a result, total EPS decreased by 52.70% compared to the original sludge. In addition, due to the neutralization effect of protons and FeCl3, the Zeta potential increased remarkably from -13.80 mV to -1.72 mV and the dispersed sludge particles formed during EPS dissolution process were re-flocculated, which increased the average size of the sludge particles. The ratio of proteins (PN)/polysaccharides (PS) also increased from 1.69 to 3.81. And a quantitative model of optimum dosage of SCC agents based on the influence of the sludge PS, PN and EPS content has been established, aiming to determine the dosage of each conditioner according to the properties of target sludge. In general, the SCC provided an effective pathway for sludge deep dewatering.

Mesophilic condition favors simultaneous partial nitrification and denitrification (SPND) and anammox for carbon and nitrogen removal from anaerobic digestate food waste effluent.

Three simultaneous partial nitrification and denitrification (SPND) bioreactors were established on ambient (30 °C), mesophilic (40 °C) and thermophilic condition (50 °C) at high dissolved oxygen levels (2-7 mg L-1) to remove nitrogen and carbon from anaerobic digestate food waste effluent (ADFE). The bioreactor performed best under mesophilic condition, with TN and COD removal efficiency of 96.3 ± 0.1% and 91.7 ± 0.1%, respectively. Free ammonia (FA) and free nitrous acid (FNA) alternately ensured selective inhibition of nitrite-oxidizing bacteria (NOB) in long-term operation of SPND systems. Candidatus Brocadia, known as anammox bacteria, was observed unexpectedly in the bioreactors. The analysis of microbial community and metabolic pathways revealed that mesophilic strategy stimulated SPND and anammox process. Mesophilic condition helped autotropic microbes resist the competitive pressure from heterotrophic bacteria, improving the balance between nitrifiers, anammox bacteria and other co-existing heterotrophs. Overall, this study offers new insights into the linkage among temperature, pollutant removals (carbon and nitrogen) and metabolic potential in the SPND bioreactors.

Response of nitrogen removal performance and microbial community to a wide range of pH in thermophilic denitrification system.

Thermophilic biological nitrogen removal would be a promising alternative to conventional approaches for the treatment of high-temperature wastewater. In this study, the response of thermophilic denitrification system (50 °C) to a wide range of pH (3-11) was investigated. The results showed that thermophilic denitrification could adapt to pH 5-11, but suffered from obvious nitrite and ammonia accumulation at pH 3. Microbial insights indicated that the enrichment of specific functional thermophiles has contributed to the tolerance towards unfavorable pH. Besides, the potential selecting advantage of nitrate reducing bacteria over nitrite reducing bacteria and the enrichment of dissimilatory nitrate reduction to ammonium (DNRA) bacteria could be responsible for the nitrite and ammonia accumulation at pH 3. Moreover, the functional gene prediction denoted higher narG/(nirK + nirS) and nrfA at pH 3, which could facilitate partial denitrification and DNRA. These findings could provide new insight into the application of thermophilic biological nitrogen removal.

Simultaneous nitrogen and phosphorus removal from simulated digested piggery wastewater in a single-stage biofilm process coupling anammox and intracellular carbon metabolism.

A Single-stage biofilm process coupling Anammox and Intracellular Carbon metabolism (SAIC) was constructed for treating simulated digested piggery wastewater with low carbon/nitrogen ratio (C/N) in this study. TN removal in SAIC system increased by more than 12.77% compared to the reference, and the maximum total phosphorus (TP) removal efficiency reached to 83.70% (C/N = 1.5). Denitrification driven by intracellular carbon, mainly poly-ß-hydroxybutyrate (PHB, 78.57%), contributed 32.60% of TN elimination at most, and at least 67.40% should be attributed to anammox. Phosphorus was thought to be mainly removed through biological route, while chemical precipitation also explained around 10% of removed TP. Furthermore, commensalism of glycogen accumulating organisms (GAOs), phosphate accumulating organisms (PAOs), nitrifiers and anammox bacteria was revealed by combining 16S rRNA amplicon sequencing and metagenomics. As a result, multiple metabolic pathways including anammox, (partial) nitrification, endogenous (partial) denitrification and biological P-removal played synergistic effect in SAIC system.

Hydraulic flow direction alters nutrients removal performance and microbial mechanisms in electrolysis-assisted constructed wetlands.

In this study, an electrolysis-assisted down-flow constructed wetland (E-DFCW) was successfully established, and achieved simultaneously efficient removal of PO43--P (93.6% ± 3.2%), NO3--N (97.1% ± 2.0%) and TN (80.6% ± 5.4%). When compared with electrolysis-assisted up-flow constructed wetland (E-UFCW), E-DFCW allowed significantly lower concentrations of PO43--P, NO3--N, total Fe and SO42--S in effluents. In addition, microbial community and functional genes prediction results indicated that hydraulic flow direction significantly altered microbial nitrogen, sulfur and carbon metabolisms in electrolysis-assisted constructed wetlands (E-CWs). Specifically, multi-path denitrification facilitated NO3--N reduction in cathodic chamber of E-DFCW, whereas autohydrogenotrophic denitrification might dominate NO3--N reduction in cathodic chamber of E-UFCW. More abundant and diverse denitrifiers in cathodic chamber of E-DFCW contributed to enhanced denitrification performance. Overall, this work provides microbial insights into multi-path nitrogen metabolisms in electrolysis-assisted denitrification systems in response to hydraulic flow direction.

Facilitating harmful algae removal in fresh water via joint effects of multi-species algicidal bacteria.

Harmful algae blooms posing serious threats to the ecological environment occur frequently across the world. Multi-species algicidal bacteria were enriched by utilizing immobilized carriers in a pilot scale experiment, which significantly promoted the effect of algal control in the reactors. Under the optimal condition, the algicidal ratio and chlorophyll a degradation rate reached 87.69% and 47.00 µg/(L·d), respectively. The growth of Cyanophyta, diatom, Dinoflagellate and Cryptophyta was inhibited significantly by the joint action of algicidal bacteria and light shading of fillers, accounting for 53.74% and 36.47%, respectively. The results of 16S rRNA high-throughput sequencing suggested algicidal bacteria (10.17%) belonging to 13 genera were enriched. Among the algicidal process, Bacillus and Pseudomonas played crucial roles. Fluorescence spectroscopy and UV254 were adopted to assess the release of dissolved organic matter (DOM) and the precursors of disinfection by-products (DBPs). Two efficient algicidal strains (C1, C4) were isolated which showed high homology with Enterobacter asburiae JCM6051(T) and Pseudomonas simiae oli(T), respectively. This study provided new insights into the in-situ bioremediation of eutrophication in fresh water.

The variation on nitrogen removal mechanisms and the succession of ammonia oxidizing archaea and ammonia oxidizing bacteria with temperature in biofilm reactors treating saline wastewater.

To reveal nitrogen removal mechanisms under environmental stresses, biofilm reactors were operated at different temperatures (10 °C-35 °C) treating saline wastewater (salinity 3%). The results showed nitrogen removal efficiency was 98.46% at 30 °C and 60.85% at 10 °C, respectively. Both ammonia oxidizing archaea (AOA) and ammonia oxidizing bacteria (AOB) participated in nitrification. 94.9% of the overall ammonia oxidation was attributed to AOA at 10 °C, but only 48.2% of that was undertaken by AOA at 35 °C. AOA had a greater contribution at low temperature, which demonstrated that nitrogen removal pathway varied with temperature. Aerobic denitrification was more stable than anoxic denitrification. High-throughput sequencing showed Crenarchaeota was the dominant AOA (97.02-34.47%), cooperating with various heterotrophic AOB. Real-time PCR indicated that AOA was three orders of magnitude more abundant than AOB. AOA was more resistant to low temperature and high-saline stresses. Ammonia oxidizers had distinct responses to temperature change and showed diverse relationships at different temperatures.

Effects of COD/TN ratio on nitrogen removal efficiency, microbial community for high saline wastewater treatment based on heterotrophic nitrification-aerobic denitrification process.

High salinity and high organic concentration impose negative impacts on autotrophic nitrification, which hinders efficient nitrogen removal. To achieve efficient nitrogen removal, high saline wastewater nitrogen removal systems with different COD/TN ratios based on heterotrophic nitrification-aerobic denitrification (HN-AD) process were established in this study. Results demonstrated that the COD/TN ratio had significant effects on nitrogen removal efficiency, microbial community structures and metabolic pathways. The optimal COD/TN ratio was 25 for nitrogen removal, with NH4+-N and TN removal rates of 11.86 mg·L-1·h-1 and 11.50 mg·L-1·h-1, respectively (3.65 and 3.31 times higher than those COD/TN ratio of 10). 16S rRNA high-throughput sequencing revealed that HN-AD functional bacteria (Pseudomonas, Bacillus, Paracoccus) with highest abundance (7.61%) played a key role in high saline wastewater treatment. And Halomonas, Nitrincola and Oceanimonas participated in the denitrification process. Moreover, the abundance of genes related to nitrogen removal was the highest (1.90%) revealed by functional genes prediction.