Metagenomic insights into microbial metabolic mechanisms of a combined solid-phase denitrification and anammox process for nitrogen removal in mainstream wastewater treatment.

To overcome the significant challenges associated with nitrite supply and nitrate residues in mainstream anaerobic ammonium oxidation (anammox)-based processes, this study developed a combined solid-phase denitrification (SPD) and anammox process for low-strength nitrogen removal without the addition of nitrite. The SPD step was performed in a packed-bed reactor containing poly-3-hydroxybutyrate-co-3-hyroxyvelate (PHBV) prior to employing the anammox granular sludge reactor in the continuous-flow mode. The removal efficiency of total inorganic nitrogen reached 95.7 ± 1.2% under a nitrogen loading rate of 0.18 ± 0.01 kg N·m3·d-1, and it required 1.02 mol of nitrate to remove 1 mol of ammonium nitrogen. The PHBV particles not only served as biofilm carriers for the symbiosis of hydrolytic bacteria (HB) and denitrifying bacteria (DB), but also carbon sources that facilitated the coupling of partial denitrification and anammox in the granules. Metagenomic sequencing analysis indicated that Burkholderiales was the most abundant HB genus in SPD. The metabolic correlations between DB (Betaproteobacteria, Rhodocyclaceae, and Anaerolineae) and anammox bacteria (Candidatus Brocadiac and Kuenenia) in the granules were confirmed through microbial co-occurrence networks analysis and functional gene annotations. Additionally, the genes encoding nitrate reductase (Nap) and nitrite reductase (Nir) in DB primarily facilitated nitrate reduction, thereby supplying nitric oxide to anammox bacteria for subsequent nitrogen removal with hydrazine synthase (Hzs) and hydrazine dehydrogenase (Hdh). The findings provide insights into microbial metabolism within combined SPD and anammox processes, thus advancing the development of mainstream anammox-based processes in engineering applications.

Successful operation of nitrifying granules at low pH in a continuous-flow reactor: Nitrification performance, granule stability, and microbial community.

Acidic nitrification, as a novel process for treating wastewater without sufficient alkalinity, has received increasing attention over the years. In this study, a continuous-flow reactor with aerobic granular sludge was successful operated at low pH (<6.5) performing high-rate acidic nitrification. Volumetric ammonium oxidation rate of 0.4-1.2 kg/(m3·d) were achieved with the specific biomass activities of 5.8-13.9 mg N/(gVSS·h). Stable partial nitritation with nitrite accumulation efficiency over 85% could be maintained at pH above 6 with the aid of residual ammonium, whereas the nitrite accumulation disappeared when pH was below 6. Interestingly, the granule morphology significantly improved during the acidic operation. The increased secretion of extracellular polymeric substances (especially polysaccharides) suggested a self-protective behavior of microbes in the aerobic granules against acidic stress. 16S rRNA gene sequencing analyses indicated that Candidatus Nitrospira defluvii was always the dominant nitrite-oxidizing bacteria, while the dominant ammonia-oxidizing bacteria shifted from Nitrosomonas europaea to Nitrosomonas mobilis. This study, for the first time, demonstrated the improved stability of aerobic granules under acidic conditions, and also highlighted aerobic granules as a useful solution to achieve high-rate acidic nitrification.

Multi-omics analysis reveals the collaboration and metabolisms of the anammox consortia driven by soluble/non-soluble Fe(III) as the sole iron element.

Iron is recognized as a physiological requirement for anammox bacteria (AnAOB), with Fe(II) considered to be the most effective form. However, Fe(III), instead of Fe(II) is the common iron form in natural and artificial ecosystems. In this study, the nitrogen removal performance and metabolic mechanisms in anammox consortia with soluble and non-soluble Fe(III) as the sole iron element were investigated. After the 150-day operation, the soluble (FeCl3) and insoluble (Fe2O3) Fe(III)-fed anammox systems reached nitrogen removal rates of 71.84 ± 0.80% and 50.20 ± 0.98%, respectively. AnAOB could survive with soluble (FeCl3) or insoluble (Fe2O3) Fe(III) as the sole iron element, reaching relative abundances of 18.49% and 13.16%, respectively. The results show that the formation of anammox core consortia can enable AnAOB's survival to adverse external conditions of Fe(II) deficiency. Metagenomic and metatranscriptomic analysis reveal that Ca. Kuenenia can only uptake Fe(II) into the cell for metabolisms either independently through the extracellular electron transfer or with the cross-feeding of symbiotic microbes. This study provides insight into the utilization and metabolic mechanisms of Fe(III) in Ca. Kuenenia-dominated consortia, and deepens the understanding of anammox core consortia in the nitrogen, carbon, and iron cycling, further promoting the practical applications of anammox processes.

Achieving robust nitritation in a modified continuous-flow reactor: From micro-granule cultivation to nitrite-oxidizing bacteria elimination.

In this study, a modified continuous-flow nitrifying reactor was successfully operated for rapid cultivation of micro-granules and achieving robust nitritation. Results showed that sludge granulation with mean size of ca. 100 µm was achieved within three weeks by gradually increasing settling velocity-based selection pressure from 0.48 to 0.9 m/hr. Though Nitrospira like nitrite-oxidizing bacteria (NOB) were enriched in the micro-granules with a ratio between ammonia-oxidizing bacteria (AOB) and NOB of 5.7%/6.5% on day 21, fast nitritation was achieved within one-week by gradually increasing of influent ammonium concentration (from 50 to 200 mg/L). Maintaining ammonium in-excess was the key for repressing NOB in the micro-granules. Interestingly, when the influent ammonium concentration switched back to 50 mg/L still with the residual ammonium of 15-25 mg/L, the nitrite accumulation efficiency increased from 90% to 98%. Experimental results suggested that the NOB repression was intensified by both oxygen and nitrite unavailability in the inner layers of micro-granules. Unexpectedly, continuous operation with ammonium in excess resulted in overproduction of extracellular polysaccharides and overgrowth of some bacteria (e.g., Nitrosomonas, Arenimonas, and Flavobacterium), which deteriorated the micro-granule stability and drove the micro-granules aggregation into larger ones with irregular morphology. However, efficient nitritation was stably maintained with extremely high ammonium oxidation potential (> 50 mg/g VSS/hr) and nearly complete washout of NOB was obtained. This suggested that smooth and spherical granule was not a prerequisite for achieving NOB wash-out and maintaining effective nitritation in the granular reactor. Overall, the micro-granules exhibited a great practical potential for high-rate nitritation.

Microbial dynamics reveal the adaptation strategies of ecological niche in distinct anammox consortia under mainstream conditions.

The feasibility of anammox-based processes for nitrogen-contained wastewater treatment has been verified with different anammox bacteria, however, the ecological niche of anammox bacteria under mainstream conditions is still elusive. In this study, six sludge samples collected from different habitats were utilized to culture anammox bacteria under mainstream conditions, and two distinct anammox genera (Ca. Kuenenia and Ca. Brocadia) with a relative abundance of 6.31% (C1) and 3.09% (C3), respectively, were identified. Notably, the microbial dynamics revealed that anammox bacteria (AMX), ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), Chloroflexi bacteria (CFX), and heterotrophic denitrification bacteria (HDB) were the core members in anammox consortia. However, Ca. Kuenenia and Ca. Brocadia occupied different ecological niches in anammox consortia. The dissolved oxygen and microbial structures of the anammox-continuous stirred tank reactor systems were the main factors to affect their niche differentiation. Meanwhile, comammox might exist in the systems and occupy the ecological niche of AOB in nitrogen cycling. The network analysis suggested that Ignavibacterium could be the associated bacteria in Ca. Kuenenia-dominated consortia, while Ca. Nitrotoga was that in the Ca. Brocadia-dominated consortia. Our findings reveal a valuable reference for the observation of distinct anammox genera under mainstream conditions, which provides theoretical guidance for the engineering application of mainstream anammox-based processes.

Achieving high-rate partial nitritation with aerobic granular sludge at low temperatures.

Partial nitritation is necessary for the implementation of the mainstream anammox (anaerobic ammonium oxidation) process in wastewater treatment plants. However, the difficulty in outcompeting nitrite-oxidizing bacteria (NOB) at mainstream conditions hinders the performance of partial nitritation. The present work aimed to develop a high-rate partial nitritation process for low-ammonium wastewater treatment at low temperatures by seeding aerobic granules. Experimental results suggested that both stratified structure of nitrifiers developed in the granules and sufficient residual ammonium concentration (18-35 mg N L-1) in the bulk liquid contributed to efficient NOB repression. With the hydraulic retention time progressively shortened from 1.0 to 0.17 h, the influent nitrogen loading rate of the partial nitritation process reached 6.8 ± 0.4 kg N m-3 d-1 even at 10-15 °C. The high concentration (7.5 gVSS L-1) and activity (0.48 g N g-1 VSS d-1 at 11 °C) of granular sludge made the reactor possess an overcapacity evaluated by the ratio between the actual ammonium oxidation rate of the granules and their maximum potential. The overcapacity helped the reactor to face the adverse effect of decreasing temperatures. Overall, this work indicated the great potential of applying aerobic granules to achieve high-rate partial nitritation at mainstream conditions. Moreover, anammox bacteria with a relative abundance of 2.8% was also identified in the partial nitritation granules at the end of this study, suggesting that the granules provided a habitable niche for anammox bacteria growth. Note that these results cannot fully relate to the treatment of real domestic/municipal wastewater, they are a source of important information increasing the knowledge about low temperature partial nitrification.

The organics-mediated microbial dynamics and mixotrophic metabolisms in anammox consortia under micro-aerobic conditions.

The engineering applications of mainstream anaerobic ammonium oxidation (anammox) have raised increasing attention due to its energy-efficient, however, the organics-mediated microbial dynamics and mixotrophic metabolisms in anammox consortia under micro-aerobic conditions are still elusive. Here, the response of the anammox process to sodium acetate and glucose at a C/N ratio ranging from 0 to 0.5 was investigated under micro-aerobic conditions, respectively. Results showed that the additional glucose could promote the nitrogen removal efficiency (NRE) and nitrogen removal rate (NRR) of anammox processes at a low C/N ratio (0.3), representing 84.00% and 0.53 N kg·m-3·d-1. The introduced organics could regulate the diversity of the microbial community and simplify the microbial relationship in anammox consortia. Anammox could not benefit from the introduced sodium acetate, while glucose could effectively enhance the anammox activity and microbial interactions in anammox consortia. Glucose might also stimulate the mixotrophic mechanism of Ca. Kuenenia, further promotes the proliferation of anammox sludge under micro-aerobic conditions. This study reveals that glucose could positively mediate microbial interactions and mixotrophic metabolism in anammox consortia under micro-aerobic conditions, which raises a new horizon for the proliferation of anammox sludge for mainstream engineering applications.

Quantitative Analysis of Cold Stress Inducing Lipidomic Changes in Shewanella putrefaciens Using UHPLC-ESI-MS/MS.

Shewanella putrefaciens is a well-known specific spoilage organism (SSO) and cold-tolerant microorganism in refrigerated fresh marine fish. Cold-adapted mechanism includes increased fluidity of lipid membranes by the ability to finely adjust lipids composition. In the present study, the lipid profile of S. putrefaciens cultivated at 30, 20, 10, 4, and 0 °C was explored using ultra-high-pressure liquid chromatography/electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) to discuss the effect of lipid composition on cold-adapted tolerance. Lipidomic analysis detected a total of 27 lipid classes and 606 lipid molecular species in S. putrefaciens cultivated at 30, 20, 10, 4, and 0 °C. S. putrefaciens cultivated at 30 °C (SP-30) had significantly higher content of glycerolipids, sphingolipids, saccharolipids, and fatty acids compared with that at 0 °C (SP-0); however, the lower content of phospholipids (13.97%) was also found in SP-30. PE (30:0), PE (15:0/15:0), PE (31:0), PA (33:1), PE (32:1), PE (33:1), PE (25:0), PC (22:0), PE (29:0), PE (34:1), dMePE (15:0/16:1), PE (31:1), dMePE (15:1/15:0), PG (34:2), and PC (11:0/11:0) were identified as the most abundant lipid molecular species in S. putrefaciens cultivated at 30, 20, 10, 4, and 0 °C. The increase of PG content contributes to the construction of membrane lipid bilayer and successfully maintains membrane integrity under cold stress. S. putrefaciens cultivated at low temperature significantly increased the total unsaturated liquid contents but decreased the content of saturated liquid contents.

Two-stage partial nitritation-anammox process for high-rate mainstream deammonification.

Increasing information supported that achieving high-rate mainstream deammonification through two-stage partial nitritation (PN)-anammox process should be a better option than through single-stage process. However, direct experimental evidence was limited so far. Herein, a two-stage PN-anammox process was successfully operated for nitrogen removal from low-strength wastewater in winter. Influent shift from synthetic wastewater to actual anaerobically pretreated sewage had little impact on the process performance. Promising nitrogen removal rates (NRRs) of 0.28-0.07 kg N m-3 d-1 with an average effluent concentration of 5.2 mg TN L-1 were achieved for the anaerobically pretreated sewage treatment at 15-7 °C. Moreover, nearly all the degradable COD in the pretreated sewage was steadily removed in the first-stage PN reactor, despite the varied influent COD concentrations of 22-78 mg L-1 and the operating temperature decrease, suggesting the positive role of the first-stage PN in protecting anammox bacteria. The low temperature seemingly was the only deterministic factor inhibiting the anammox activity, and hence made the anammox reaction to be the rate-limiting step for nitrogen removal in the two-stage PN-anammox process. Unexpectedly, nearly all the anammox bacteria remained active at low temperatures with the process actual anammox activity reached about 76-85% of their maximum potential, implying that higher NRRs would be easily realized through bioaugmentation or enrichment of anammox bacteria. Overall, the present investigation provides direct and valuable information for implementing the two-stage PN-anammox process to treat mainstream municipal wastewater. A control strategy was also proposed to optimize the operation of the two-stage mainstream deammonification process.

Single-stage autotrophic nitrogen removal process at high loading rate: granular reactor performance, kinetics, and microbial characterization.

For the possible highest performance of single-stage combined partial nitritation/anammox (PNA) process, a continuous complete-mix granular reactor was operated at progressively higher nitrogen loading rate. The variations in bacterial community structure of granules were also characterized using high-throughput pyrosequencing, to give a detail insight to the relationship between reactor performance and functional organism abundance within completely autotrophic nitrogen removal system. In 172 days of operation, a superior total nitrogen (TN) removal rate over 3.9 kg N/(m3/day) was stable implemented at a fixed dissolved oxygen concentration of 1.9 mg/L, corresponding to the maximum specific substrate utilization rate of 0.36/day for TN based on the related kinetics modeling. Pyrosequencing results revealed that the genus Nitrosomonas responsible for aerobic ammonium oxidation was dominated on the granule surface, which was essential to offer the required niche for the selective enrichment of anammox bacteria (genus Candidatus Kuenenia) in the inner layer. And the present of various heterotrophic organisms with general functions, known as fermentation and denitrification, could not be overlooked. In addition, it was believed that an adequate excess of ammonium in the bulk liquid played a key role in maintaining process stability, by suppressing the growth of nitrite-oxidizing bacteria through dual-substrate competitions.

Cultivation and characteristics of partial nitrification granular sludge in a sequencing batch reactor inoculated with heterotrophic granules.

The aim of this study was to develop a simple operation strategy for the cultivation of partial nitrification granules (PNGs) treating an autotrophic medium. For this strategy, aerobic granular sludge adapted to high concentration organics removal was seeded in a sequencing batch reactor (SBR) with a height/diameter ratio of 3.8, and the ratio of organics to the ammonia nitrogen-loading rate (C/N ratio) in the influent was employed as the main control parameter to start up the partial nitrification process. After 86 days of operation, the nitrite accumulation rate reached 1.44 kg/(m3 day) in the SBR, and the removal efficiency of ammonia nitrogen (NH4+-N) was over 95 %. The PNGs showed a dense and compact structure, with an excellent settling ability, a typical extracellular polymeric substance (EPS) composition, and a high ammonia oxidation activity. The high-throughput pyrosequencing results indicated that the microbial community structure in the granules was significantly influenced by the C/N ratio, and ammonia-oxidizing bacteria (AOB), including the r-strategist Nitrosomonas and k-strategist Nitrosospira genre, which accounted for approximately 40 % of the total biomass at the end of operation. The effective suppression of nitrite-oxidizing bacteria (NOB) growth was attributed to oxygen competition on the granular surface among functional bacteria, as well as the high free ammonia or free nitrous acid concentrations during the aeration period.

Metabolic evolution and bottleneck insights into simultaneous autotroph-heterotroph anammox system for real municipal wastewater nitrogen removal.

When biological nitrogen removal (BNR) systems shifted from treating simulated wastewater to real wastewater, a microbial succession occurred, often resulting in a decline in efficacy. Notably, despite their high nitrogen removal efficiency for real wastewater, anammox coupled systems operating without or with minimal carbon sources also exhibited a certain degree of performance reduction. The underlying reasons and metabolic shifts within these systems remained elusive. In this study, the simultaneous autotrophic/heterotrophic anammox system demonstrated remarkable metabolic resilience upon exposure to real municipal wastewater, achieving a nitrogen removal efficiency (NRE) of 82.83 ± 2.29 %. This resilience was attributed to the successful microbial succession and the complementary metabolic functions of heterotrophic microorganisms, which fostered a resilient microbial community. The system's ability to harness multiple electron sources, including NADH oxidation, the TCA cycle, and organics metabolism, allowed it to establish a stable and efficient electron transfer chain, ensuring effective nitrogen removal. Despite the denitrification channel's nitrite supply capability, the analysis of the interspecies correlation network revealed that the synergistic metabolism between AOB and AnAOB was not fully restored, resulting in selective functional bacterial and genetic interactions and the system's PN/A performance declined. Additionally, the enhanced electron affinity of PD increased interconversion of NO3--N and NO2--N, limiting the efficient utilization of electrons and thereby constraining nitrogen removal performance. This study elucidated the metabolic mechanism of nitrogen removal limitations in anammox-based systems treating real municipal wastewater, enhancing our understanding of the metabolic functions and electron transfer within the symbiotic bacterial community.

Waste iron scraps promote anammox bacteria to resist inorganic carbon limitation.

The anaerobic ammonium oxidation (anammox) process is adversely affected by the limitation of inorganic carbon (IC). In this research, a new technique was introduced to assist anammox biomass in counteracting the adverse effects of IC limitation by incorporating waste iron scraps (WIS), a cheap and easily accessible byproduct of lathe cutting. Results demonstrated that reducing the influent IC/TN ratio from 0.08-0.09 to 0.04 resulted in a 20 % decrease in the nitrogen removal rate (NRR) for the control reactor, with an average specific anammox activity (SAA) of 0.65 g N/g VSS/day. Nevertheless, the performance of the WIS-assisted anammox reactor remained robust despite the reduction in IC supply. In fact, the NRR and SAA of the WIS-assisted reactor exhibited substantial improvements, reaching approximately 1.86 kg/(m3·day) and 0.98 g N/g VSS/day, respectively. These values surpassed those achieved by the control reactor by approximately 39 % and 51 %, respectively. The microbial analysis confirmed that the WIS addition significantly stimulated the proliferation of anammox bacteria (dominated by Candidatus Kuenenia) under IC limitation. The anammox gene abundances in the WIS-assisted anammox reactor were 3-4 times higher than those in the control reactor. Functional genes prediction based on the KEGG database revealed that the addition of WIS significantly enhanced the relative abundances of genes associated with nitrogen metabolism, IC fixation, and central carbon metabolism. Together, the results suggested that WIS promoted carbon dioxide fixation of anammox species to resist IC limitation. This study provided a promising approach for effectively treating high ammonium-strength wastewater using anammox under IC limitation.

[Rapid Start-up of Continuous Autotrophic Nitrogen Removal Reactor by Hybrid-inoculating PN and PN/A Granular Sludges].

The rapid cultivation of partial nitritation/ANAMMOX （PN/A） granular sludge in a continuous-flow mode is one of the key technologies for efficient biological nitrogen removal in domestic wastewater treatment. Compared with that in PN/A granular sludge, PN granular sludge demonstrates a shorter incubation period and suitability for batch culture. It is also a good carrier for enriching ANAMMOX （AMX） bacteria. In this study, we established a continuous-flow autotrophic nitrogen removal process in three continuously stirred tank reactors （CSTR） （R1-R3） by hybrid-inoculating PN/A and PN granular sludge at the mass ratios of 3∶1, 1∶1, and 1∶3, respectively. By implementing high ammonium nitrogen loading and short hydraulic retention time, continuous autotrophic nitrogen removal processes were successfully started up in the three CSTRs. The results showed that compared with that of R1 and R2, R3 had a longer start-up time but a similar steady-state nitrogen removal performance. The total nitrogen removal load of R3 could be more than 2.6 kg·ï¼ˆm3·d）-1. Intriguingly, the inoculated PN granular sludge served as a precursor for PN/A granular sludge cultivation. This approach facilitated the enrichment of anaerobic ammonia-oxidizing bacteria （AMX） by introducing abundant ammonium-oxidizing bacteria （AOB） and nitrite nitrogen substrates into the CSTR. According to the results of high-throughput sequencing, the microbial abundance and diversity of the mature granules in R1-R3 were significantly higher than those of the inoculation sludge. AOB （genus Nitrosomonas）, AMX （genera Candidatus Kuenenia and Candidatus Brocadia）, and symbiotic heterotrophs, such as Chloroflexi, Bacteroidetes, and Chlorobi, drove the autotrophic nitrogen removal process and maintained the stability of the granular structure. In summary, a novel start-up strategy of hybrid-inoculating granular sludge was provided for a continuous-flow autotrophic nitrogen removal in engineering application.

Microbial interactions play a keystone role in rapid anaerobic ammonium oxidation sludge proliferation and biofilm formation.

Two mature anaerobic ammonium oxidation (anammox) consortia with high/low relative abundance of anammox bacteria were inoculated for the rapid sludge proliferation and biofilm formation in this study, named up-flow anaerobic sludge blanket reactor (UASB1) (high) and UASB2 (low), respectively. Results showed that the nitrogen removal efficiency of UASB2 reached 90.94% after the 120-day operation, which was 13% higher than that of UASB1. Moreover, its biomass amounts were 22.18% (biofilm) and 40.96% (flocs) higher than that of UASB1, respectively. Ca. Kuenenia possessed relative abundances of 29.32% (flocs), 27.42% (biofilm) and 31.56% (flocs), 35.20% (biofilm) in the UASB1 and UASB2, respectively. The relative abundances of genes involved in anammox transformation (hzs, nir) and carbon metabolism (fdh, lgA/B/C, acs) were higher in the UASB2, indicating that Ca. Kuenenia might produce acetate and glycogen to enhance microbial interactions. These findings emphasized the importance of microbial interactions in anammox sludge proliferation and biofilm formation.

Succession of microbial communities reveals the inevitability of anammox core in the development of anammox processes.

The lack of anammox seeds is regarded as the bottleneck of anammox-based processes. Although the interactions in anammox consortia have attracted increasing attention, little is known about the influence of inoculated sludge populations on the growth of anammox bacteria. In this study, four sludge of distinct communities mixed with anammox sludge (the relative abundance of Ca. Kuenenia was 1.96 %) were used as the seeds, respectively for the start-up of anammox processes. Notably, all these mixed microbial communities tend to form a similar microbial community, defined as the anammox core, containing anammox-bacteria (22.9 ± 5.9 %), ammonia-oxidizing-bacteria (0.8 ± 0.7 %), nitrite-oxidizing-bacteria (0.2 ± 0.2 %), Chloroflexi-bacteria (0.7 ± 0.4 %), and heterotrophic-denitrification-bacteria (0.3 ± 0.2 %). It also elucidated that the communities of Nitrosomonas-dominated sludge were the closest to the anammox core, and achieved the highest nitrogen-removal rate of 0.73 kg-N m-3 d-1. This study sheds light on the solution to the shortage of anammox seeds in the full-scale wastewater treatment application.

Characterization of Sodium Alginate-Locust Bean Gum Films Reinforced with Daphnetin Emulsions for the Development of Active Packaging.

In this study, we characterized an active film made of sodium alginate (SA)-locust bean gum (LBG) containing daphnetin-based film. Physicochemical characteristics, as well as antioxidant and antibacterial properties, were investigated. The results showed that the addition of a low concentration of daphnetin increased the flexibility of SA-LBG cling film, leading to an improvement in elongation at break and tensile strength. As the daphnetin content increased, solubility, brightness and transparency of the cling film decreased, and the moisture permeability increased. The antioxidant capacity and antibacterial activity of films with daphnetin were improved compared to those of the basal film. In addition, the cling film formed by adsorption had higher bacterial (Shewanella putrefaciens and Pseudomonas fluorescens) inhibition and antioxidant activity rates than direct film formation. The results indicate that the combination of daphnetin in SA-LBG film provides an active film with antioxidant and antibacterial properties, with potential for the development of food-grade packaging material.

Response of nitritation granules to anaerobically pre-treated municipal wastewater at low temperatures in a continuous-flow reactor.

Achieving mainstream nitritation with aerobic granules is attractive based on increasing evidence but generally treating artificial low-ammonium wastewater. Real municipal wastewater is much more complex in composition, the behavior of the nitritation granules would be different when treating real municipal wastewater. Herein, the response of nitritation granules to influent shift from artificial low-ammonium (35-40 mg/L) wastewater to anaerobically pre-treated municipal wastewater (MWWpre-treated) was investigated at low temperatures. Results showed that MWWpre-treated caused the outgrowth of filamentous bacteria on the granule surface and developed into finger-like structures, which in turn resulted in the decrease of the overall granular sludge settleability. Batch-tests and microbial analysis indicated the functional and microbial differentiation between the newly formed fluffy exterior and the original compact granule. The fluffy exterior was dominated by genus Flavobacterium (66.6%) and primarily functioned as COD removal, whereas the nitrifiers (mainly Nitrosomonas) were still located in the compact core and performed nitritation. Moreover, the heterotrophs-dominated fluffy exterior hindered the oxygen transfer towards nitrifiers located in the compact granule and thereby facilitated the stable NOB repression in the granule particularly at low temperatures (<10 °C). Finally, gradual recovery of the granular sludge morphology and settleability occurred after the influent reverted to synthetic low-ammonium wastewater. Overall, this work demonstrated that the feeding of MWWpre-treated only caused morphological changes of the nitritation granules, but its structural and functional stability could be maintained stably.

A new concept of waste iron recycling for the enhancement of the anammox process.

As a by-product of industry, waste iron scraps (WIS) are low-cost and widely available, which was potential for the development of iron-assisted anammox. In this study, the feasibility of adding WIS to enhance the nitrogen removal of the anammox process (also called WIS-assisted anammox) was demonstrated. Results indicated that the WIS-assisted anammox reactors performed a 15-35% higher nitrogen removal efficiency than that of the control. Compared to the sludge from the control, the sludge from the WIS-assisted anammox reactors had a higher iron content (78-113 g kg-1 SS) and a better specific anammox activity (10.8-15.5 mg N g-1 VSS h-1). The enhanced growth of the anammox bacteria (related to Ca. Kuenenia stuttgartiensis with 99% similarity) in the WIS-assisted anammox reactors was also confirmed by high-throughput sequencing and qPCR. Furthermore, the functional genes predicted by PICRUSt2 revealed a higher level of hydroxylamine oxidoreductase (hao)-like proteins expression of the biomass from the WIS-assisted anammox reactors, implying that the hydroxylamine-related anammox pathway was promoted. Additionally, the observation of cytoplasmic nitrate reductase (narG), copper-containing nitrite reductase (nirK), and nitric oxide reductase (norB) suggested that the introduction of WIS might promote the denitrification ability. This was correlated to the lower ΔNO3-/ΔNH4+ ratio observed in these WIS-assisted anammox reactors. Overall, the WIS-assisted anammox offers a sustainable nitrogen removal process for wastewater treatment with waste iron recycling.

Nitrogen contribution and microbial community of size-fractionated anammox sludge in continuous stirred-tank reactors.

In this study, the microbial diversity of size-fractionated anammox sludge in a well-mixed system and their contribution to nitrogen transformation were investigated. Results showed that small granules (0.2-1.0 mm) contributed to the major part of the nitrogen removal rate (56 %) due to its largest mixed liquor volatile suspended solids (1240 ± 80 mg·L-1). However, large granules (>1.0 mm) possessed the highest relative abundances of Ca. Kuenenia stuttgartiensis and specific anammox activity, representing 49.34 % and 24.45 ± 0.01 mg-N·g-1-mixed liquor volatile suspended solids·h-1, respectively. The microbial diversity decreased as the increase of granular size, resulting in microbial community shifting to a simpler model. Metagenomic analysis showed that fine sludge might be the potential major for NO/N2O production in the mature well-mixed system under inorganic conditions. This study provides guidance for the evaluation of nitrogen contribution by anammox size-fractionated sludge and the inhibition of the potential NO/N2O emission in anammox processes.